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1
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Hu X, Li H, Chen M, Qian J, Jiang H. Reference-informed evaluation of batch correction for single-cell omics data with overcorrection awareness. Commun Biol 2025; 8:521. [PMID: 40158033 PMCID: PMC11954866 DOI: 10.1038/s42003-025-07947-7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/06/2024] [Accepted: 03/18/2025] [Indexed: 04/01/2025] Open
Abstract
Batch effect correction (BEC) is fundamental to integrate multiple single-cell RNA sequencing datasets, and its success is critical to empower in-depth interrogation for biological insights. However, no simple metric is available to evaluate BEC performance with sensitivity to data overcorrection, which erases true biological variations and leads to false biological discoveries. Here, we propose RBET, a reference-informed statistical framework for evaluating the success of BEC. Using extensive simulations and six real data examples including scRNA-seq and scATAC-seq datasets with different numbers of batches, batch effect sizes and numbers of cell types, we demonstrate that RBET evaluates the performance of BEC methods more fairly with biologically meaningful insights from data, while other methods may lead to false results. Moreover, RBET is computationally efficient, sensitive to overcorrection and robust to large batch effect sizes. Thus, RBET provides a robust guideline on selecting case-specific BEC method, and the concept of RBET is extendable to other modalities.
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Affiliation(s)
- Xiaoyue Hu
- Center for Data Science, Zhejiang University, Hangzhou, China
- School of Mathematical Sciences, Zhejiang University, Hangzhou, China
| | - He Li
- Center for Data Science, Zhejiang University, Hangzhou, China
| | - Ming Chen
- College of Life Sciences, Zhejiang University, Hangzhou, China
| | - Junbin Qian
- Zhejiang Key Laboratory of Precision Diagnosis and Therapy for Major Gynecological Diseases, Women's Hospital, Zhejiang University School of Medicine, Hangzhou, China.
- Institute of Genetics, Zhejiang University School of Medicine, Hangzhou, China.
- Cancer Center, Zhejiang University, Hangzhou, China.
- Zhejiang Provincial Clinical Research Center for Child Health, Hangzhou, China.
| | - Hangjin Jiang
- Center for Data Science, Zhejiang University, Hangzhou, China.
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2
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Eldesoky SMM, Hussein MM, Abdel-Maksoud FM. Dynamics of the Posthatching Testicular Development in Japanese Quail (Coturnix coturnix japonica): Histological and Ultrastructural Study. MICROSCOPY AND MICROANALYSIS : THE OFFICIAL JOURNAL OF MICROSCOPY SOCIETY OF AMERICA, MICROBEAM ANALYSIS SOCIETY, MICROSCOPICAL SOCIETY OF CANADA 2025; 31:ozaf012. [PMID: 40233282 DOI: 10.1093/mam/ozaf012] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/18/2024] [Revised: 11/21/2024] [Accepted: 02/23/2025] [Indexed: 04/17/2025]
Abstract
The posthatching development of the testis is a well-organized process comprising the maturation of Sertoli cells, the development of Leydig cells, and the differentiation of germ cells. This study aimed to investigate the posthatching testicular development in the Japanese quail, using light and electron microscope. The current study was performed on 25 healthy Japanese quail chicks at 0, 7, 21, 40, and 50 posthatching days. The results revealed that the testis consists of solid seminiferous cords, and their lining epithelium is composed of two types of cells; immature Sertoli cells and gonocyte or spermatogonia at the early stage, which begins from the day of hatching till 21 days posthatching. The interstitium during this period consisted of different developmental stages of the Leydig cells. However, at the late posthatching developmental stage the testis is characterized by the presence of round and elongated spermatids as well as the initiation of spermiogenesis. The interstitial compartment also showed an increase in the number and size of Leydig cells. The findings of the current study provide comprehensive insights into the posthatching development of the Japanese quail testis, contributing to the understanding of avian reproduction.
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Affiliation(s)
- Sara M M Eldesoky
- Department of Anatomy and Embryology, Faculty of Veterinary Medicine, Assiut University, Assiut 71526, Egypt
| | - Marwa M Hussein
- Department of Cell and Tissues, Faculty of Veterinary Medicine, Assiut University, Assiut 71526, Egypt
| | - Fatma M Abdel-Maksoud
- Department of Anatomy and Embryology, Faculty of Veterinary Medicine, Assiut University, Assiut 71526, Egypt
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3
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Fomichova O, Oliveira PF, Bernardino RL. Exploring the interplay between inflammation and male fertility. FEBS J 2024. [PMID: 39702986 DOI: 10.1111/febs.17366] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/02/2024] [Revised: 10/02/2024] [Accepted: 12/10/2024] [Indexed: 12/21/2024]
Abstract
Male fertility results from a complex interplay of physiological, environmental, and genetic factors. It is conditioned by the properly developed anatomy of the reproductive system, hormonal regulation balance, and the interplay between different cell populations that sustain an appropriate and functional environment in the testes. Unfortunately, the mechanisms sustaining male fertility are not flawless and their perturbation can lead to infertility. Inflammation is one of the factors that contribute to male infertility. In the testes, it can be brought on by varicocele, obesity, gonadal infections, leukocytospermia, physical obstructions or traumas, and consumption of toxic substances. As a result of prolonged or untreated inflammation, the testicular resident cells that sustain spermatogenesis can suffer DNA damage, lipid and protein oxidation, and mitochondrial dysfunction consequently leading to loss of function in affected Sertoli cells (SCs) and Leydig cells (LCs), and the formation of morphologically abnormal dysfunctional sperm cells that lay in the basis of male infertility and subfertility. This is due mainly to the production and secretion of pro-inflammatory mediators, including cytokines, chemokines, and reactive oxygen species (ROS) by local immune cells (macrophages, lymphocytes T, mast cells) and tissue-specific cells [SCs, LCs, peritubular myoid cells (PMCs) and germ cells (GCs)]. Depending on the location, duration, and intensity of inflammation, these mediators can exert their toxic effect on different elements of the testes. In this review, we discuss the most prevalent inflammatory factors that negatively affect male fertility and describe the different ways inflammation can impair male reproductive function.
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Affiliation(s)
- Oleksandra Fomichova
- UMIB - Unit for Multidisciplinary Research in Biomedicine, ICBAS - School of Medicine and Biomedical Sciences, University of Porto, Portugal
| | - Pedro F Oliveira
- LAQV-REQUIMTE and Department of Chemistry, University of Aveiro, Portugal
| | - Raquel L Bernardino
- UMIB - Unit for Multidisciplinary Research in Biomedicine, ICBAS - School of Medicine and Biomedical Sciences, University of Porto, Portugal
- Laboratory for Integrative and Translational Research in Population Health (ITR), University of Porto, Portugal
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4
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Li SY, Kumar S, Gu X, DeFalco T. Testicular immunity. Mol Aspects Med 2024; 100:101323. [PMID: 39591799 PMCID: PMC11624985 DOI: 10.1016/j.mam.2024.101323] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/02/2024] [Revised: 11/13/2024] [Accepted: 11/16/2024] [Indexed: 11/28/2024]
Abstract
The testis is a unique environment where immune responses are suppressed to allow the development of sperm that possess autoimmunogenic antigens. There are several contributors responsible for testicular immune privilege, including the blood-testis barrier, testicular immune cells, immunomodulation by Sertoli cells, and high levels of steroid hormones. Despite multiple mechanisms in place to regulate the testicular immune environment, pathogens that disrupt testicular immunity can lead to long-term effects such as infertility. If testicular immunity is disturbed, autoimmune reactions can also occur, leading to aberrant immune cell infiltration and subsequent attack of autoimmunogenic germ cells. Here we discuss cellular and molecular factors underlying testicular immunity and how testicular infection or autoimmunity compromise immune privilege. We also describe infections and autoimmune diseases that impact the testis. Further research into testicular immunity will reveal how male fertility is maintained and will help update therapeutic strategies for infertility and other testicular disorders.
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Affiliation(s)
- Shu-Yun Li
- Reproductive Sciences Center, Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, 45229, USA
| | - Sudeep Kumar
- Reproductive Sciences Center, Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, 45229, USA
| | - Xiaowei Gu
- Reproductive Sciences Center, Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, 45229, USA
| | - Tony DeFalco
- Reproductive Sciences Center, Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, 45229, USA; Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH, 45267, USA.
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5
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Porubska B, Plevakova M, Fikarova N, Vasek D, Somova V, Sanovec O, Simonik O, Komrskova K, Krylov V, Tlapakova T, Krulova M, Krulova M. Therapeutic potential of Sertoli cells in vivo: alleviation of acute inflammation and improvement of sperm quality. Stem Cell Res Ther 2024; 15:282. [PMID: 39227878 PMCID: PMC11373210 DOI: 10.1186/s13287-024-03897-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/29/2024] [Accepted: 08/26/2024] [Indexed: 09/05/2024] Open
Abstract
BACKGROUND Inflammation-induced testicular damage is a significant contributing factor to the increasing incidence of infertility. Traditional treatments during the inflammatory phase often fail to achieve the desired fertility outcomes, necessitating innovative interventions such as cell therapy. METHODS We explored the in vivo properties of intravenously administered Sertoli cells (SCs) in an acute lipopolysaccharide (LPS)-induced inflammatory mouse model. Infiltrating and resident myeloid cell phenotypes were assessed using flow cytometry. The impact of SC administration on testis morphology and germ cell quality was evaluated using computer-assisted sperm analysis (CASA) and immunohistochemistry. RESULTS SCs demonstrated a distinctive migration pattern, importantly they preferentially concentrated in the testes and liver. SC application significantly reduced neutrophil infiltration as well as preserved the resident macrophage subpopulations. SCs upregulated MerTK expression in both interstitial and peritubular macrophages. Applied SC treatment exhibited protective effects on sperm including their motility and kinematic parameters, and maintained the physiological testicular morphology. CONCLUSION Our study provides compelling evidence of the therapeutic efficacy of SC transplantation in alleviating acute inflammation-induced testicular damage. These findings contribute to the expanding knowledge on the potential applications of cell-based therapies for addressing reproductive health challenges and offer a promising approach for targeted interventions in male infertility.
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Affiliation(s)
- Bianka Porubska
- Department of Cell Biology, Faculty of Science, Charles University, Vinicna 7, Prague, 2, 128 00, Czech Republic
| | - Marie Plevakova
- Department of Cell Biology, Faculty of Science, Charles University, Vinicna 7, Prague, 2, 128 00, Czech Republic
| | - Natalie Fikarova
- Department of Cell Biology, Faculty of Science, Charles University, Vinicna 7, Prague, 2, 128 00, Czech Republic
| | - Daniel Vasek
- Department of Cell Biology, Faculty of Science, Charles University, Vinicna 7, Prague, 2, 128 00, Czech Republic
| | - Veronika Somova
- Department of Cell Biology, Faculty of Science, Charles University, Vinicna 7, Prague, 2, 128 00, Czech Republic
| | - Ondrej Sanovec
- Laboratory of Reproductive Biology, Institute of Biotechnology of the Czech Academy of Sciences, BIOCEV, Vestec, Prumyslova 595, Prague, 252 50, Czech Republic
- Department of Physiology, Faculty of Science, Charles University, Vinicna 7, Prague, 2, 128 00, Czech Republic
| | - Ondrej Simonik
- Laboratory of Reproductive Biology, Institute of Biotechnology of the Czech Academy of Sciences, BIOCEV, Vestec, Prumyslova 595, Prague, 252 50, Czech Republic
| | - Katerina Komrskova
- Laboratory of Reproductive Biology, Institute of Biotechnology of the Czech Academy of Sciences, BIOCEV, Vestec, Prumyslova 595, Prague, 252 50, Czech Republic
- Department of Zoology, Faculty of Science, Charles University, Vinicna 7, Prague, 2, 128 00, Czech Republic
| | - Vladimir Krylov
- Department of Cell Biology, Faculty of Science, Charles University, Vinicna 7, Prague, 2, 128 00, Czech Republic
| | - Tereza Tlapakova
- Department of Cell Biology, Faculty of Science, Charles University, Vinicna 7, Prague, 2, 128 00, Czech Republic
| | - Magdalena Krulova
- Department of Cell Biology, Faculty of Science, Charles University, Vinicna 7, Prague, 2, 128 00, Czech Republic.
| | - Magdalena Krulova
- Department of Cell Biology, Faculty of Science, Charles University, Vinicna 7, Prague, 2, 128 00, Czech Republic
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6
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Khanmohammadi N, Malek F, Takzaree N, Malekzadeh M, Khanehzad M, Akanji OD, Rastegar T. Sertoli Cell-Conditioned Medium Induces Differentiation of Bone Marrow-Derived Mesenchymal Stem Cells to Male Germ-Like Cells in Busulfan-Induced Azoospermic Mouse Model. Reprod Sci 2024; 31:375-392. [PMID: 37737972 DOI: 10.1007/s43032-023-01332-7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/10/2023] [Accepted: 08/15/2023] [Indexed: 09/23/2023]
Abstract
Non-obstructive azoospermia is a severe form of male infertility, with limited effective treatments. Bone marrow mesenchymal stem cells (BMSCs) can differentiate to different cell lines; therefore, transplantation of these cells is used for treatment of several diseases. Since these cells require induction factors to differentiate into germ cells, we co-transplanted bone marrow stem cells (BMSCs) with Sertoli cell-conditioned medium (SCCM) into the testis of azoospermic mice. This study was carried out in two sections, in vitro and in vivo. For in vitro study, differentiating factors (c-kit and ID4) were examined after 15 days of co-culture of bone marrow cells with Sertoli cell-conditioned medium, while for in vivo study, the azoospermia model was first created by intraperitoneal administration of a single-dose busulfan (40 mg/kg) followed by single-dose CdCl2 (2 mg/kg) after 4 weeks. Mice were divided into 4 groups including control (azoospermia), BMSC, SCCM, and BMSC + SCCM. Eight weeks after transplantation, samples were assessed for proliferation and differentiation via the expression level of MVH, ID4, SCP3, Tp1, Tp2, and Prm1 differentiation markers. The results showed that BMSC co-cultured with SCCM in vitro differentiated BMSC to germ-like cells. Similarly, in vivo studies revealed a higher level of BMSC differentiation into germ-like cells with significant higher expression of differentiation markers in transplanted groups compared to the control. This study confirmed the role of SCCM as an inductive factor for BMSC differentiation to germ cells both in vivo and in vitro conditions.
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Affiliation(s)
- Nasrin Khanmohammadi
- Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
| | - Fatemeh Malek
- Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
| | - Nasrin Takzaree
- Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
| | - Mehrnoush Malekzadeh
- Department of Anatomy, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
| | - Maryam Khanehzad
- Department of Anatomy, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
| | | | - Tayebeh Rastegar
- Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
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7
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Zhang XN, Tao HP, Li S, Wang YJ, Wu SX, Pan B, Yang QE. Ldha-Dependent Metabolic Programs in Sertoli Cells Regulate Spermiogenesis in Mouse Testis. BIOLOGY 2022; 11:1791. [PMID: 36552300 PMCID: PMC9775226 DOI: 10.3390/biology11121791] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 11/07/2022] [Revised: 11/30/2022] [Accepted: 12/07/2022] [Indexed: 12/13/2022]
Abstract
Sertoli cells play indispensable roles in spermatogenesis by providing the advanced germ cells with structural, nutritional, and regulatory support. Lactate is regarded as an essential Sertoli-cell-derived energy metabolite that nurses various types of spermatogenic cells; however, this assumption has not been tested using genetic approaches. Here, we have reported that the depletion of lactate production in Sertoli cells by conditionally deleting lactate dehydrogenase A (Ldha) greatly affected spermatogenesis. Ldha deletion in Sertoli cells significantly reduced the lactate production and resulted in severe defects in spermatogenesis. Spermatogonia and spermatocytes did not show even mild impairments, but the spermiogenesis of Ldha conditional knockout males was severely disrupted. Further analysis revealed that 2456 metabolites were altered in the sperm of the knockout animals, and specifically, lipid metabolism was dysregulated, including choline, oleic acid, and myristic acid. Surprisingly, choline supplementation completely rescued the spermiogenesis disorder that was caused by the loss of Ldha activities. Collectively, these data have demonstrated that the interruption of Sertoli-cell-derived lactate impacted sperm development through a choline-mediated mechanism. The outcomes of these findings have revealed a novel function of lactate in spermatogenesis and have therapeutic applications in treating human infertility.
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Affiliation(s)
- Xiao-Na Zhang
- Key Laboratory of Adaptation and Evolution of Plateau Biota, Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining 810008, China
- University of Chinese Academy of Sciences, Beijing 100049, China
| | - Hai-Ping Tao
- Key Laboratory of Adaptation and Evolution of Plateau Biota, Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining 810008, China
- University of Chinese Academy of Sciences, Beijing 100049, China
| | - Shuang Li
- Key Laboratory of Adaptation and Evolution of Plateau Biota, Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining 810008, China
- University of Chinese Academy of Sciences, Beijing 100049, China
| | - Yu-Jun Wang
- Key Laboratory of Adaptation and Evolution of Plateau Biota, Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining 810008, China
- University of Chinese Academy of Sciences, Beijing 100049, China
| | - Shi-Xin Wu
- Key Laboratory of Adaptation and Evolution of Plateau Biota, Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining 810008, China
- University of Chinese Academy of Sciences, Beijing 100049, China
| | - Bo Pan
- Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, College of Animal Science and Technology, Sichuan Agricultural University, Chengdu 611130, China
| | - Qi-En Yang
- Key Laboratory of Adaptation and Evolution of Plateau Biota, Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining 810008, China
- University of Chinese Academy of Sciences, Beijing 100049, China
- Qinghai Key Laboratory of Animal Ecological Genomics, Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining 810001, China
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8
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Washburn RL, Hibler T, Kaur G, Dufour JM. Sertoli Cell Immune Regulation: A Double-Edged Sword. Front Immunol 2022; 13:913502. [PMID: 35757731 PMCID: PMC9218077 DOI: 10.3389/fimmu.2022.913502] [Citation(s) in RCA: 37] [Impact Index Per Article: 12.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/05/2022] [Accepted: 04/29/2022] [Indexed: 12/18/2022] Open
Abstract
The testis must create and maintain an immune privileged environment to protect maturing germ cells from autoimmune destruction. The establishment of this protective environment is due, at least in part, to Sertoli cells. Sertoli cells line the seminiferous tubules and form the blood-testis barrier (BTB), a barrier between advanced germ cells and the immune system. The BTB compartmentalizes the germ cells and facilitates the appropriate microenvironment necessary for spermatogenesis. Further, Sertoli cells modulate innate and adaptive immune processes through production of immunoregulatory compounds. Sertoli cells, when transplanted ectopically (outside the testis), can also protect transplanted tissue from the recipient’s immune system and reduce immune complications in autoimmune diseases primarily by immune regulation. These properties make Sertoli cells an attractive candidate for inflammatory disease treatments and cell-based therapies. Conversely, the same properties that protect the germ cells also allow the testis to act as a reservoir site for infections. Interestingly, Sertoli cells also have the ability to mount an antimicrobial response, if necessary, as in the case of infections. This review aims to explore how Sertoli cells act as a double-edged sword to both protect germ cells from an autoimmune response and activate innate and adaptive immune responses to fight off infections.
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Affiliation(s)
- Rachel L Washburn
- Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, TX, United States.,Immunology and Infectious Disease Concentration, Texas Tech University Health Sciences Center, Lubbock, TX, United States
| | - Taylor Hibler
- Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, TX, United States.,Immunology and Infectious Disease Concentration, Texas Tech University Health Sciences Center, Lubbock, TX, United States
| | - Gurvinder Kaur
- Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, TX, United States.,Department of Medical Education, Texas Tech University Health Sciences Center, Lubbock, TX, United States
| | - Jannette M Dufour
- Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, TX, United States.,Immunology and Infectious Disease Concentration, Texas Tech University Health Sciences Center, Lubbock, TX, United States.,Department of Medical Education, Texas Tech University Health Sciences Center, Lubbock, TX, United States
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9
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Liu Z, Wang H, Larsen M, Gunewardana S, Cendali FI, Reisz JA, Akiyama H, Behringer RR, Ma Q, Hammoud SS, Kumar TR. The solute carrier family 7 member 11 (SLC7A11) is regulated by LH/androgen and required for cystine/glutathione homeostasis in mouse Sertoli cells. Mol Cell Endocrinol 2022; 549:111641. [PMID: 35398053 DOI: 10.1016/j.mce.2022.111641] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/16/2022] [Revised: 03/30/2022] [Accepted: 04/02/2022] [Indexed: 01/19/2023]
Abstract
Luteinizing hormone (LH) stimulates testosterone production from Leydig cells. Both LH and testosterone play important roles in spermatogenesis and male fertility. To identify LH - and testosterone - responsive transporter genes that play key roles in spermatogenesis, we performed large-scale gene expression analyses on testes obtained from adult control and Lhb knockout mice. We found a significant reduction in cystine/glutamate transporter encoding Slc7a11 mRNA in testes of Lhb null mice. We observed that Slc7a11/SLC7A11 expression was initiated pre-pubertally and developmentally regulated in mouse testis. Immunolocalization studies confirmed that SLC7A11 was mostly expressed in Sertoli cells in testes of control and germ cell-deficient mice. Western blot analyses indicated that SLC7A11 was significantly reduced in testes of mutant mice lacking either LH or androgen receptor selectively in Sertoli cells. Genetic and pharmacological rescue of Lhb knockout mice restored the testicular expression of Slc7a11 comparable to that observed in controls. Additionally, Slc7a11 mRNA was significantly suppressed upon Sertoli cell/testicular damage induced in mice by cadmium treatment. Knockdown of Slc7a11 in vitro in TM4 Sertoli cells or treatment of mice with sulfasalazine, a SLC7A11 inhibitor caused a significant reduction in intracellular cysteine and glutathione levels but glutamate content remained unchanged as determined by metabolomic analysis. Knockdown of Slc7a11 resulted in compensatory upregulation of other glutamate transporters belonging to the Slc1a family presumably to maintain intracellular glutamate levels. Collectively, our studies identified that SLC7A11 is an LH/testosterone-regulated transporter that is required for cysteine/glutathione but not glutamate homeostasis in mouse Sertoli cells.
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Affiliation(s)
- Zhenghui Liu
- Division of Reproductive Sciences, Department of Obstetrics and Gynecology, University of Colorado Anschutz Medical Campus, Aurora, CO, 80045, USA
| | - Huizen Wang
- Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, KS, 66160, USA
| | - Mark Larsen
- Division of Reproductive Sciences, Department of Obstetrics and Gynecology, University of Colorado Anschutz Medical Campus, Aurora, CO, 80045, USA
| | - Sumedha Gunewardana
- Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, KS, 66160, USA
| | - Francesca I Cendali
- Department of Biochemistry and Molecular Genetics, University of Colorado Anschutz Medical Campus, Aurora, CO, 80045, USA
| | - Julie A Reisz
- Department of Biochemistry and Molecular Genetics, University of Colorado Anschutz Medical Campus, Aurora, CO, 80045, USA
| | - Haruhiko Akiyama
- Department of Orthopedic Surgery, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan
| | - Richard R Behringer
- Department of Molecular Genetics, MD Anderson Cancer Center, Houston, TX, 77030, USA
| | - Qianyi Ma
- Department of Human Genetics, University of Michigan Medical School, Ann Arbor, MI, 48109, USA
| | - S Sue Hammoud
- Department of Human Genetics, University of Michigan Medical School, Ann Arbor, MI, 48109, USA
| | - T Rajendra Kumar
- Division of Reproductive Sciences, Department of Obstetrics and Gynecology, University of Colorado Anschutz Medical Campus, Aurora, CO, 80045, USA.
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10
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Yokonishi T. [Reconstruction of spermatogonial niche for male fertility preservation]. Nihon Yakurigaku Zasshi 2022; 157:168-171. [PMID: 35491111 DOI: 10.1254/fpj.21106] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/18/2022]
Abstract
Infertility is one of the late side effects of cancer treatment. Expansion of anti-cancer treatment allow patients to have more life time, however infertility is becoming a matter damaging QOL during the young cancer survivors. The passive strategy such as avoiding the gonad-toxic drug or decreasing the total volume of them and shielding the gonads against cancer therapy has been conducted. To preserve the fertility of young female, ovary tissue cryopreservation is becoming a standard over the world after the success of offspring from cryopreserved ovary tissue autograft was reported. Sperm preservation method is established for the male fertility preservation method, however this is only applicable for sexually matured male patients. For the sake of preserving fertility of sexually immature male patients, many trials using cryopreserved testis tissues or testicular cells have been undergone. Recently, in vitro gametogenesis from stem cell of the human and the mouse to primordial germ cell like cell has been achieved. Here the previous challenges and the latest reports for obtaining functional sperm from immature testis and the reconstruction of spermatogonial niche as a potential approach for preserving fertility procedure are described.
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11
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Yang Y, Li Q, Huang R, Xia H, Tang Y, Mai W, Liang J, Ma S, Chen D, Feng Y, Lei Y, Zhang Q, Huang Y. Small-Molecule-Driven Direct Reprogramming of Fibroblasts into Functional Sertoli-Like Cells as a Model for Male Reproductive Toxicology. Adv Biol (Weinh) 2022; 6:e2101184. [PMID: 35212192 DOI: 10.1002/adbi.202101184] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/15/2021] [Revised: 01/13/2022] [Indexed: 01/27/2023]
Abstract
Sertoli cells (SCs) are vital to providing morphological and nutritional support for spermatogenesis. Defects in SCs often lead to infertility. SCs transplantation is a promising potential strategy to compensate for SC dysfunction. However, isolation of SCs from testes is impractical due to obvious and ethical limitations. Here, a molecular cocktail is identified comprising of pan-BET family inhibitor (I-BET151), retinoic acid, and riluzole that enables the efficient conversion of fibroblasts into functional Sertoli-like cells (CiSCs). The gene expression profiles of CiSCs resemble those of mature SCs and exhibit functional properties such as the formation of testicular seminiferous tubules, engulfment of apoptotic sperms, supporting the survival of germ cells, and suppressing proliferation of primary lymphocytes in vitro. Moreover, CiSCs are sensitive to toxic substances, making them an alternative model to study the deleterious effects of toxicants on SCs. The study provides an efficient approach to reprogram fibroblasts into functional SCs by using pure chemical compounds.
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Affiliation(s)
- Yan Yang
- Department of Cell Biology, Jinan University, Guangzhou, 510632, China.,Guangdong Province Key Laboratory of Bioengineering Medicine, Jinan University, Guangzhou, 510632, China
| | - Quan Li
- Department of Cell Biology, Jinan University, Guangzhou, 510632, China
| | - Rufei Huang
- Department of Pharmacology, Jinan University, Guangzhou, 510632, China
| | - Huan Xia
- Department of Cell Biology, Jinan University, Guangzhou, 510632, China
| | - Yan Tang
- Department of Cell Biology, Jinan University, Guangzhou, 510632, China
| | - Wanwen Mai
- Department of Cell Biology, Jinan University, Guangzhou, 510632, China
| | - Jinlian Liang
- Department of Cell Biology, Jinan University, Guangzhou, 510632, China
| | - Siying Ma
- Department of Cell Biology, Jinan University, Guangzhou, 510632, China
| | - Derong Chen
- Department of Cell Biology, Jinan University, Guangzhou, 510632, China
| | - Yuqing Feng
- Department of Cell Biology, Jinan University, Guangzhou, 510632, China.,Department of Pharmacology, Jinan University, Guangzhou, 510632, China
| | - Yaling Lei
- Department of Cell Biology, Jinan University, Guangzhou, 510632, China
| | - Qihao Zhang
- Department of Cell Biology, Jinan University, Guangzhou, 510632, China.,Guangdong Province Key Laboratory of Bioengineering Medicine, Jinan University, Guangzhou, 510632, China
| | - Yadong Huang
- Department of Cell Biology, Jinan University, Guangzhou, 510632, China.,Guangdong Province Key Laboratory of Bioengineering Medicine, Jinan University, Guangzhou, 510632, China
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12
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Zhang W, Nie R, Cai Y, Xie W, Zou K. Progress in germline stem cell transplantation in mammals and the potential usage. Reprod Biol Endocrinol 2022; 20:59. [PMID: 35361229 PMCID: PMC8969385 DOI: 10.1186/s12958-022-00930-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/16/2021] [Accepted: 03/10/2022] [Indexed: 11/10/2022] Open
Abstract
Germline stem cells (GSCs) are germ cells with the capacities of self-renewal and differentiation into functional gametes, and are able to migrate to their niche and reconstitute the fertility of recipients after transplantation. Therefore, GSCs transplantation is a promising technique for fertility recovery in the clinic, protection of rare animals and livestock breeding. Though this novel technique faces tremendous challenges, numerous achievements have been made after several decades' endeavor. This review summarizes the current knowledge of GSCs transplantation and its utilization in mammals, and discusses the application prospect in reproductive medicine and animal science.
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Affiliation(s)
- Wen Zhang
- Germline Stem Cells and Microenvironment Lab, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, 210095, China
| | - Ruotian Nie
- College of Life Science, Nanjing Agricultural University, Nanjing, 210095, China
| | - Yihui Cai
- Germline Stem Cells and Microenvironment Lab, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, 210095, China
| | - Wenhai Xie
- School of Life Sciences, Shandong University of Technology, NO. 266 Xincun Road, Zibo, 255000, Shandong, China.
| | - Kang Zou
- Germline Stem Cells and Microenvironment Lab, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, 210095, China.
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13
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Kanatsu-Shinohara M, Ogonuki N, Matoba S, Morimoto H, Shiromoto Y, Ogura A, Shinohara T. Regeneration of spermatogenesis by mouse germ cell transplantation into allogeneic and xenogeneic testis primordia or organoids. Stem Cell Reports 2022; 17:924-935. [PMID: 35334214 PMCID: PMC9023780 DOI: 10.1016/j.stemcr.2022.02.013] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/01/2021] [Revised: 02/19/2022] [Accepted: 02/21/2022] [Indexed: 12/30/2022] Open
Abstract
Gametogenesis requires close interactions between germ cells and somatic cells. Derivation of sperm from spermatogonial stem cells (SSCs) is hampered by the inefficiency of spermatogonial transplantation technique in many animal species because it requires a large number of SSCs and depletion of endogenous spermatogenesis. Here we used mouse testis primordia and organoids to induce spermatogenesis from SSCs. We microinjected mouse SSCs into embryonic gonads or reaggregated neonatal testis organoids, which were transplanted under the tunica albuginea of mature testes. As few as 1 × 104 donor cells colonized both types of transplants and produced sperm. Moreover, rat embryonic gonads supported xenogeneic spermatogenesis from mouse SSCs when transplanted in testes of immunodeficient mice. Offspring with normal genomic imprinting patterns were born after microinsemination. These results demonstrate remarkable flexibility of the germ cell-somatic cell interaction and raise new strategies of SSC manipulation for animal transgenesis and analysis of male infertility.
SSCs can be injected into embryonic gonads or reaggregated neonatal testes Spermatogenesis occurs in the gonads or reaggregated testes after transplantation Offspring are born from SSC-derived sperm using microinsemination Offspring show normal DNA methylation in imprinted genes
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Affiliation(s)
- Mito Kanatsu-Shinohara
- Department of Molecular Genetics, Graduate School of Medicine, Kyoto University, Yoshida Konoe, Sakyo-ku, Kyoto 606-8501, Japan; AMED-CREST, AMED 1-7-1 Otemachi, Chiyodaku, Tokyo 100-0004, Japan
| | - Narumi Ogonuki
- RIKEN, BioResource Research Center, Tsukuba 305-0074, Japan
| | - Shogo Matoba
- RIKEN, BioResource Research Center, Tsukuba 305-0074, Japan
| | - Hiroko Morimoto
- Department of Molecular Genetics, Graduate School of Medicine, Kyoto University, Yoshida Konoe, Sakyo-ku, Kyoto 606-8501, Japan
| | - Yusuke Shiromoto
- Department of Molecular Genetics, Graduate School of Medicine, Kyoto University, Yoshida Konoe, Sakyo-ku, Kyoto 606-8501, Japan
| | - Atsuo Ogura
- RIKEN, BioResource Research Center, Tsukuba 305-0074, Japan
| | - Takashi Shinohara
- Department of Molecular Genetics, Graduate School of Medicine, Kyoto University, Yoshida Konoe, Sakyo-ku, Kyoto 606-8501, Japan.
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14
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Wang D, Hildorf S, Ntemou E, Dong L, Pors SE, Mamsen LS, Fedder J, Hoffmann ER, Clasen-Linde E, Cortes D, Thorup J, Andersen CY. Characterization and Survival of Human Infant Testicular Cells After Direct Xenotransplantation. Front Endocrinol (Lausanne) 2022; 13:853482. [PMID: 35360067 PMCID: PMC8960121 DOI: 10.3389/fendo.2022.853482] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/12/2022] [Accepted: 02/11/2022] [Indexed: 11/30/2022] Open
Abstract
BACKGROUND Cryopreservation of prepubertal testicular tissue preserves spermatogonial stem cells (SSCs) that may be used to restore fertility in men at risk of infertility due to gonadotoxic treatments for either a malignant or non-malignant disease. Spermatogonial stem cell-based transplantation is a promising fertility restoration technique. Previously, we performed xenotransplantation of propagated SSCs from prepubertal testis and found human SSCs colonies within the recipient testes six weeks post-transplantation. In order to avoid the propagation step of SSCs in vitro that may cause genetic and epigenetic changes, we performed direct injection of single cell suspension in this study, which potentially may be safer and easier to be applied in future clinical applications. METHODS Testis biopsies were obtained from 11 infant boys (median age 1.3 years, range 0.5-3.5) with cryptorchidism. Following enzymatic digestion, dissociated single-cell suspensions were prelabeled with green fluorescent dye and directly transplanted into seminiferous tubules of busulfan-treated mice. Six to nine weeks post-transplantation, the presence of gonocytes and SSCs was determined by whole-mount immunofluorescence for a number of germ cell markers (MAGEA, GAGE, UCHL1, SALL4, UTF1, and LIN28), somatic cell markers (SOX9, CYP17A1). RESULTS Following xenotransplantation human infant germ cells, consisting of gonocytes and SSCs, were shown to settle on the basal membrane of the recipient seminiferous tubules and form SSC colonies with expression of MAGEA, GAGE, UCHL1, SALL4, UTF1, and LIN28. The colonization efficiency was approximately 6%. No human Sertoli cells were detected in the recipient mouse testes. CONCLUSION Xenotransplantation, without in vitro propagation, of testicular cell suspensions from infant boys with cryptorchidism resulted in colonization of mouse seminiferous tubules six to nine weeks post-transplantation. Spermatogonial stem cell-based transplantation could be a therapeutic treatment for infertility of prepubertal boys with cryptorchidism and boys diagnosed with cancer. However, more studies are required to investigate whether the low number of the transplanted SSC is sufficient to secure the presence of sperm in the ejaculate of those patients over time.
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Affiliation(s)
- Danyang Wang
- Laboratory of Reproductive Biology, University Hospital of Copenhagen, Rigshospitalet, Copenhagen, Denmark
- Department of Clinical Medicine, University of Copenhagen, Copenhagen, Denmark
- *Correspondence: Danyang Wang,
| | - Simone Hildorf
- Department of Clinical Medicine, University of Copenhagen, Copenhagen, Denmark
- Department of Pediatric Surgery, University Hospital of Copenhagen, Rigshospitalet, Copenhagen, Denmark
| | - Elissavet Ntemou
- Laboratory of Reproductive Biology, University Hospital of Copenhagen, Rigshospitalet, Copenhagen, Denmark
| | - Lihua Dong
- Laboratory of Reproductive Biology, University Hospital of Copenhagen, Rigshospitalet, Copenhagen, Denmark
| | - Susanne Elisabeth Pors
- Laboratory of Reproductive Biology, University Hospital of Copenhagen, Rigshospitalet, Copenhagen, Denmark
| | - Linn Salto Mamsen
- Laboratory of Reproductive Biology, University Hospital of Copenhagen, Rigshospitalet, Copenhagen, Denmark
| | - Jens Fedder
- Centre of Andrology & Fertility Clinic, Department D, Odense University Hospital, Odense C, Denmark
- Research Unit of Human Reproduction, Institute of Clinical Research, University of Southern Denmark, Odense, Denmark
| | - Eva R. Hoffmann
- Danish National Research Foundation (DNRF) Center for Chromosome Stability, Department of Cellular and Molecular Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Erik Clasen-Linde
- Department of Pathology, University Hospital of Copenhagen, Rigshospitalet, Copenhagen, Denmark
| | - Dina Cortes
- Department of Clinical Medicine, University of Copenhagen, Copenhagen, Denmark
- Department of Pediatrics and Adolescent Medicine, Copenhagen University Hospital Hvidovre, Copenhagen, Denmark
| | - Jørgen Thorup
- Department of Clinical Medicine, University of Copenhagen, Copenhagen, Denmark
- Department of Pediatric Surgery, University Hospital of Copenhagen, Rigshospitalet, Copenhagen, Denmark
| | - Claus Yding Andersen
- Laboratory of Reproductive Biology, University Hospital of Copenhagen, Rigshospitalet, Copenhagen, Denmark
- Department of Clinical Medicine, University of Copenhagen, Copenhagen, Denmark
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15
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Ruthig VA, Lamb DJ. Updates in Sertoli Cell-Mediated Signaling During Spermatogenesis and Advances in Restoring Sertoli Cell Function. Front Endocrinol (Lausanne) 2022; 13:897196. [PMID: 35600584 PMCID: PMC9114725 DOI: 10.3389/fendo.2022.897196] [Citation(s) in RCA: 16] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/15/2022] [Accepted: 03/31/2022] [Indexed: 01/16/2023] Open
Abstract
Since their initial description by Enrico Sertoli in 1865, Sertoli cells have continued to enchant testis biologists. Testis size and germ cell carrying capacity are intimately tied to Sertoli cell number and function. One critical Sertoli cell function is signaling from Sertoli cells to germ cells as part of regulation of the spermatogenic cycle. Sertoli cell signals can be endocrine or paracrine in nature. Here we review recent advances in understanding the interplay of Sertoli cell endocrine and paracrine signals that regulate germ cell state. Although these findings have long-term implications for treating male infertility, recent breakthroughs in Sertoli cell transplantation have more immediate implications. We summarize the surge of advances in Sertoli cell ablation and transplantation, both of which are wedded to a growing understanding of the unique Sertoli cell niche in the transitional zone of the testis.
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Affiliation(s)
- Victor A. Ruthig
- Department of Urology, Weill Cornell Medicine, New York, NY, United States
- Sexual Medicine Lab, Weill Cornell Medicine, New York, NY, United States
| | - Dolores J. Lamb
- Department of Urology, Weill Cornell Medicine, New York, NY, United States
- Center for Reproductive Genomics, Weill Cornell Medicine, New York, NY, United States
- *Correspondence: Dolores J. Lamb,
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16
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Gewiss RL, Law NC, Helsel AR, Shelden EA, Griswold MD. Two distinct Sertoli cell states are regulated via germ cell crosstalk. Biol Reprod 2021; 105:1591-1602. [PMID: 34494084 PMCID: PMC8689118 DOI: 10.1093/biolre/ioab160] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/05/2021] [Revised: 07/30/2021] [Accepted: 08/13/2021] [Indexed: 01/15/2023] Open
Abstract
Sertoli cells are a critical component of the testis environment for their role in maintaining seminiferous tubule structure, establishing the blood-testis barrier, and nourishing maturing germ cells in a specialized niche. This study sought to uncover how Sertoli cells are regulated in the testis environment via germ cell crosstalk in the mouse. We found two major clusters of Sertoli cells as defined by their transcriptomes in Stages VII-VIII of the seminiferous epithelium and a cluster for all other stages. Additionally, we examined transcriptomes of germ cell-deficient testes and found that these existed in a state independent of either of the germ cell-sufficient clusters. Together, we highlight two main transcriptional states of Sertoli cells in an unperturbed testis environment, and a germ cell-deficient environment does not allow normal Sertoli cell transcriptome cycling and results in a state unique from either of those seen in Sertoli cells from a germ cell-sufficient environment.
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Affiliation(s)
- Rachel L Gewiss
- School of Molecular Biosciences, Washington State University, Pullman, Washington, USA.,Center for Reproductive Biology, Washington State University, Pullman, Washington, USA
| | - Nathan C Law
- Center for Reproductive Biology, Washington State University, Pullman, Washington, USA.,Department of Animal Sciences, Washington State University, Pullman, Washington, USA
| | - Aileen R Helsel
- School of Molecular Biosciences, Washington State University, Pullman, Washington, USA.,Center for Reproductive Biology, Washington State University, Pullman, Washington, USA
| | - Eric A Shelden
- School of Molecular Biosciences, Washington State University, Pullman, Washington, USA.,Center for Reproductive Biology, Washington State University, Pullman, Washington, USA
| | - Michael D Griswold
- School of Molecular Biosciences, Washington State University, Pullman, Washington, USA.,Center for Reproductive Biology, Washington State University, Pullman, Washington, USA
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17
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Zhang Y, Liu T, Hu X, Wang M, Wang J, Zou B, Tan P, Cui T, Dou Y, Ning L, huang Y, Rao S, Wang D, Zhao X. CellCall: integrating paired ligand-receptor and transcription factor activities for cell-cell communication. Nucleic Acids Res 2021; 49:8520-8534. [PMID: 34331449 PMCID: PMC8421219 DOI: 10.1093/nar/gkab638] [Citation(s) in RCA: 156] [Impact Index Per Article: 39.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/06/2021] [Revised: 06/24/2021] [Accepted: 07/16/2021] [Indexed: 12/14/2022] Open
Abstract
With the dramatic development of single-cell RNA sequencing (scRNA-seq) technologies, the systematic decoding of cell-cell communication has received great research interest. To date, several in-silico methods have been developed, but most of them lack the ability to predict the communication pathways connecting the insides and outsides of cells. Here, we developed CellCall, a toolkit to infer inter- and intracellular communication pathways by integrating paired ligand-receptor and transcription factor (TF) activity. Moreover, CellCall uses an embedded pathway activity analysis method to identify the significantly activated pathways involved in intercellular crosstalk between certain cell types. Additionally, CellCall offers a rich suite of visualization options (Circos plot, Sankey plot, bubble plot, ridge plot, etc.) to present the analysis results. Case studies on scRNA-seq datasets of human testicular cells and the tumor immune microenvironment demonstrated the reliable and unique functionality of CellCall in intercellular communication analysis and internal TF activity exploration, which were further validated experimentally. Comparative analysis of CellCall and other tools indicated that CellCall was more accurate and offered more functions. In summary, CellCall provides a sophisticated and practical tool allowing researchers to decipher intercellular communication and related internal regulatory signals based on scRNA-seq data. CellCall is freely available at https://github.com/ShellyCoder/cellcall.
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Affiliation(s)
- Yang Zhang
- Department of Bioinformatics, School of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, China
| | - Tianyuan Liu
- Department of Bioinformatics, School of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, China
| | - Xuesong Hu
- State Key Laboratory of Organ Failure Research, Department of Developmental Biology, School of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, China
| | - Mei Wang
- State Key Laboratory of Organ Failure Research, Department of Developmental Biology, School of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, China
| | - Jing Wang
- Department of Bioinformatics, School of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, China
| | - Bohao Zou
- Department of Statistics, University of California Davis, Davis, CA, USA
| | - Puwen Tan
- Department of Bioinformatics, School of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, China
| | - Tianyu Cui
- Department of Bioinformatics, School of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, China
| | - Yiying Dou
- Department of Bioinformatics, School of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, China
| | - Lin Ning
- Department of Bioinformatics, School of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, China
| | - Yan huang
- Department of Bioinformatics, School of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, China
| | - Shuan Rao
- Department of Thoracic Surgery, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China
| | - Dong Wang
- Department of Bioinformatics, School of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, China
| | - Xiaoyang Zhao
- State Key Laboratory of Organ Failure Research, Department of Developmental Biology, School of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, China
- Guangdong Key Laboratory of Construction and Detection in Tissue Engineering, Southern Medical University, Guangzhou 510515, China
- Department of Gynecology, Zhujiang Hospital, Southern Medical University, Guangzhou 510280, China
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18
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Figueiredo AFA, Hess RA, Batlouni SR, Wnuk NT, Tavares AO, Abarikwu SO, Costa GMJ, França LR. Insights into differentiation and function of the transition region between the seminiferous tubule and rete testis. Differentiation 2021; 120:36-47. [PMID: 34229995 DOI: 10.1016/j.diff.2021.06.002] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/23/2021] [Revised: 06/18/2021] [Accepted: 06/27/2021] [Indexed: 01/15/2023]
Abstract
Seminiferous tubules physically connect to the rete testis through short segments called the transition region (TR). During fetal development, this specialized junction is considered the initial site where testis cords begin to form and to grow in length well beyond birth and into adulthood and form convoluted tubular cores. Mitotic activity of the Sertoli cell, the somatic cell of the epithelium, ceases before puberty, but modified Sertoli cells in the TR remain immature and capable of proliferation. This review presents what is known about this specialized region of the testis, with an emphasis on the morphological, molecular and physiological features, which support the hypothesis that this short region of epithelial transition serves as a specialized niche for undifferentiated Sertoli cells and spermatogonial stem cells. Also, the region is populated by an elevated number of immune cells, suggesting an important activity in monitoring and responding to any leakage of autoantigens, as sperm enter the rete testis. Several structure/function characteristics of the transition region are discussed and compared across species.
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Affiliation(s)
- A F A Figueiredo
- Laboratory of Cellular Biology, Department of Morphology, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil
| | - Rex A Hess
- Department of Comparative Biosciences, University of Illinois, Urbana-Champaign, IL, USA
| | - S R Batlouni
- Aquaculture Center (CAUNESP), São Paulo State University, São Paulo, SP, Brazil
| | - N T Wnuk
- Laboratory of Cellular Biology, Department of Morphology, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil
| | - A O Tavares
- Laboratory of Cellular Biology, Department of Morphology, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil
| | - S O Abarikwu
- Department of Biochemistry, University of Port Harcourt, Choba, Nigeria
| | - G M J Costa
- Laboratory of Cellular Biology, Department of Morphology, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil.
| | - L R França
- Laboratory of Cellular Biology, Department of Morphology, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil.
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19
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Higuch K, Matsumura T, Akiyama H, Kanai Y, Ogawa T, Sato T. Sertoli cell replacement in explanted mouse testis tissue supporting host spermatogenesis. Biol Reprod 2021; 105:934-943. [PMID: 34057178 DOI: 10.1093/biolre/ioab104] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/03/2020] [Revised: 03/25/2021] [Accepted: 05/27/2021] [Indexed: 11/13/2022] Open
Abstract
Spermatogenesis takes place in the seminiferous tubules, starting from the spermatogonial stem cell and maturing into sperm through multiple stages of cell differentiation. Sertoli cells, the main somatic cell constituting the seminiferous tubule, are in close contact with every germ cell and play pivotal roles in the progression of spermatogenesis. In this study, we developed an in vitro Sertoli cell replacement method by combining an organ culture technique and a toxin receptor-mediated cell knockout (Treck) system. We used Amh- diphtheria toxin receptor (DTR) transgenic mice, whose Sertoli cells specifically express human DTR, which renders them sensitive to diphtheria toxin (DT). An immature Amh-DTR testis was transplanted with donor testis cells followed by culturing in a medium containing DT. This procedure successfully replaced the original Sertoli cells with the transplanted Sertoli cells, and spermatogenesis originating from resident germ cells was confirmed. In addition, Sertoli cells in the mouse testis tissues were replaced by transplanted rat Sertoli cells within culture conditions, without requiring immunosuppressive treatments. This method works as a functional assay system, making it possible to evaluate any cells that might function as Sertoli cells. It would also be possible to investigate interactions between Sertoli and germ cells more closely, providing a new platform for the study of spermatogenesis and its impairments.
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Affiliation(s)
- Kazusa Higuch
- Laboratory of Biopharmaceutical and Regenerative Sciences, Institute of Molecular Medicine and Life Science, Yokohama City University Association of Medical Science, Yokohama 236-0004, Japan
| | - Takafumi Matsumura
- Laboratory of Biopharmaceutical and Regenerative Sciences, Institute of Molecular Medicine and Life Science, Yokohama City University Association of Medical Science, Yokohama 236-0004, Japan
| | - Haruhiko Akiyama
- Department of Orthopedics, Gifu University School of Medicine, Gifu, Japan
| | - Yoshiakira Kanai
- Department of Veterinary Anatomy, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan
| | - Takehiko Ogawa
- Laboratory of Biopharmaceutical and Regenerative Sciences, Institute of Molecular Medicine and Life Science, Yokohama City University Association of Medical Science, Yokohama 236-0004, Japan.,Department of Urology, Yokohama City University Graduate School of Medicine, Yokohama 236-0004, Japan
| | - Takuya Sato
- Laboratory of Biopharmaceutical and Regenerative Sciences, Institute of Molecular Medicine and Life Science, Yokohama City University Association of Medical Science, Yokohama 236-0004, Japan
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20
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Liu W, Wang Z, Hu X. Identification of Competing Endogenous RNA and Micro-RNA Profiles and Regulatory Networks in 4-Nonylphenol-induced Impairment of Sertoli Cells. Front Pharmacol 2021; 12:644204. [PMID: 34084133 PMCID: PMC8167654 DOI: 10.3389/fphar.2021.644204] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/20/2020] [Accepted: 05/04/2021] [Indexed: 12/02/2022] Open
Abstract
The xenoestrogens nonylphenols (NPs), which are materials used in the plastic polymer industry, are considered endocrine disruptors in a wide range of organisms. Studies have shown that human health problems, such as infertility and reproductive toxicology, are linked with NPs. However, the mechanism by which NPs interfere with male reproduction is not fully elucidated. Here, we found that 4-NP can result in male reproductive impairment and reduce androgen receptor (AR) protein levels in rat sertoli cells in vitro and in vivo. Moreover, we performed RNA sequencing to assess the differential expression of ceRNAs in rat primary sertoli cells treated with 4-NP. Bioinformatics methods, such as Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) database and ceRNA functional network analyses, were used to investigate the sequencing data and gain further understanding of the biological processes. Our analysis revealed a core set of mRNAs (Ar, Atf6 and Cbp), and circRNAs (circ673, circ1377, circ1789, and circPTEN) that were selected and validated by RT-qPCR. In addition, the head-to-tail splicing of circ673, circ1377, circ1789, and circPTEN was identified by Sanger sequencing. These findings provide the first insight into the ceRNA expression profiles of rat sertoli cells and reveal that ceRNAs participate in 4-NP-induced impairment of sertoli cell function, thereby indicating potential therapies for both reproductive toxicology and male infertility.
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Affiliation(s)
- Wenjie Liu
- School of Pharmaceutical Sciences, Fujian Provincial Key Laboratory of Innovative Drug Target Research, Xiamen University, Xiamen, China
| | - Zhaokai Wang
- Technical Innovation Center for Utilization of Marine Biological Resources, Third Institute of Oceanography, Ministry of Natural Resources, Xiamen, China
| | - Xiaopeng Hu
- Bio-X Institutes, Shanghai Jiao Tong University, Shanghai, China
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21
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Washburn RL, Hibler T, Thompson LA, Kaur G, Dufour JM. Therapeutic application of Sertoli cells for treatment of various diseases. Semin Cell Dev Biol 2021; 121:10-23. [PMID: 33910764 DOI: 10.1016/j.semcdb.2021.04.007] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/15/2021] [Accepted: 04/07/2021] [Indexed: 12/11/2022]
Abstract
Sertoli cells (SCs) are immune privileged cells found in the testis that function to immunologically protect maturing germ cells from immune destruction. This immune protection is due to the blood-testis-barrier, which prevents infiltration of cytotoxic immune cells and antibodies, and SC production of immunomodulatory factors, that favor a tolerogenic environment. The ability of SCs to create an immune privileged environment has led to the exploration of their potential use in the treatment of various diseases. SCs have been utilized to create a tolerogenic ectopic microenvironment, to protect co-grafted cells, and to deliver therapeutic proteins through gene therapy. To date, numerous studies have reported the potential use of SCs for the treatment of diabetes, neurodegenerative disorders, and restoration of spermatogenesis. Additionally, SCs have been investigated as a delivery vehicle for therapeutic products to treat other diseases like Laron syndrome, muscular dystrophy, and infections. This review will provide an overview of these therapeutic applications.
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Affiliation(s)
- Rachel L Washburn
- Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA
| | - Taylor Hibler
- Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA
| | - Lea Ann Thompson
- Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA
| | - Gurvinder Kaur
- Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA; Department of Medical Education, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA.
| | - Jannette M Dufour
- Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA; Department of Medical Education, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA.
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22
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Zhang XY, Li TT, Liu YR, Geng SS, Luo AL, Jiang MS, Liang XW, Shang JH, Lu KH, Yang XG. Transcriptome analysis revealed differences in the microenvironment of spermatogonial stem cells in seminiferous tubules between pre-pubertal and adult buffaloes. Reprod Domest Anim 2021; 56:629-641. [PMID: 33492695 DOI: 10.1111/rda.13900] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/09/2020] [Accepted: 01/19/2021] [Indexed: 12/21/2022]
Abstract
The microenvironment in the seminiferous tubules of buffalo changes with age, which affects the self-renewal and growth of spermatogonial stem cells (SSCs) and the process of spermatogenesis, but the mechanism remains to be elucidated. RNA-seq was performed to compare the transcript profiles of pre-pubertal buffalo (PUB) and adult buffalo (ADU) seminiferous tubules. In total, 17,299 genes from PUB and ADU seminiferous tubules identified through RNA-seq, among which 12,271 were expressed in PUB and ADU seminiferous tubules, 4,027 were expressed in only ADU seminiferous tubules, and 956 were expressed in only PUB seminiferous tubules. Of the 17,299 genes, we identified 13,714 genes that had significant differences in expression levels between PUB and ADU through GO enrichment analysis. Among these genes, 5,342 were significantly upregulated and possibly related to the formation or identity of the surface antigen on SSCs during self-renewal; 7,832 genes were significantly downregulated, indicating that genes in PUB seminiferous tubules do not participate in the biological processes of sperm differentiation or formation in this phase compared with those in ADU seminiferous tubules. Subsequently, through the combination with KEGG analysis, we detected enrichment in a number of genes related to the development of spermatogonial stem cells, providing a reference for study of the development mechanism of buffalo spermatogonial stem cells in the future. In conclusion, our data provide detailed information on the mRNA transcriptomes in PUB and ADU seminiferous tubules, revealing the crucial factors involved in maintaining the microenvironment and providing a reference for further in vitro cultivation of SSCs.
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Affiliation(s)
- Xiao-Yuan Zhang
- State Key Laboratory for Conservation and Utilisation of Subtropical Agro-bioresources, Guangxi University, Nanning, China.,College of Animal Science & Technology, Guangxi University, Nanning, China
| | - Ting-Ting Li
- State Key Laboratory for Conservation and Utilisation of Subtropical Agro-bioresources, Guangxi University, Nanning, China.,College of Animal Science & Technology, Guangxi University, Nanning, China.,HeNan Provincial People's Hospital, China
| | - Ya-Ru Liu
- State Key Laboratory for Conservation and Utilisation of Subtropical Agro-bioresources, Guangxi University, Nanning, China.,College of Animal Science & Technology, Guangxi University, Nanning, China
| | - Shuang-Shuang Geng
- State Key Laboratory for Conservation and Utilisation of Subtropical Agro-bioresources, Guangxi University, Nanning, China.,College of Animal Science & Technology, Guangxi University, Nanning, China
| | - Ao-Lin Luo
- State Key Laboratory for Conservation and Utilisation of Subtropical Agro-bioresources, Guangxi University, Nanning, China.,College of Animal Science & Technology, Guangxi University, Nanning, China
| | - Ming-Sheng Jiang
- College of Animal Science & Technology, Guangxi University, Nanning, China
| | - Xing-Wei Liang
- State Key Laboratory for Conservation and Utilisation of Subtropical Agro-bioresources, Guangxi University, Nanning, China.,College of Animal Science & Technology, Guangxi University, Nanning, China
| | - Jiang-Hua Shang
- Guangxi Key Laboratory of Buffalo Genetics, Reproduction and Breeding, Guangxi Buffalo Research Institute, Nanning, China
| | - Ke-Huan Lu
- State Key Laboratory for Conservation and Utilisation of Subtropical Agro-bioresources, Guangxi University, Nanning, China.,College of Animal Science & Technology, Guangxi University, Nanning, China
| | - Xiao-Gan Yang
- State Key Laboratory for Conservation and Utilisation of Subtropical Agro-bioresources, Guangxi University, Nanning, China.,College of Animal Science & Technology, Guangxi University, Nanning, China
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23
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Liu HC, Xie Y, Deng CH, Liu GH. Stem cell-based therapies for fertility preservation in males: Current status and future prospects. World J Stem Cells 2020; 12:1097-1112. [PMID: 33178394 PMCID: PMC7596443 DOI: 10.4252/wjsc.v12.i10.1097] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/26/2020] [Revised: 05/13/2020] [Accepted: 08/26/2020] [Indexed: 02/06/2023] Open
Abstract
With the decline in male fertility in recent years, strategies for male fertility preservation have received increasing attention. In this study, by reviewing current treatments and recent publications, we describe research progress in and the future directions of stem cell-based therapies for male fertility preservation, focusing on the use of spermatogonial stem cells (SSCs), SSC niches, SSC-based testicular organoids, other stem cell types such as mesenchymal stem cells, and stem cell-derived extracellular vesicles. In conclusion, a more comprehensive understanding of the germ cell microenvironment, stem cell-derived extracellular vesicles, and testicular organoids will play an important role in achieving male fertility preservation.
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Affiliation(s)
- Han-Chao Liu
- Department of Andrology, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou 510080, Guangdong Province, China
| | - Yun Xie
- Department of Andrology, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou 510080, Guangdong Province, China
| | - Chun-Hua Deng
- Department of Andrology, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou 510080, Guangdong Province, China
| | - Gui-Hua Liu
- Reproductive Medicine Research Center, The Sixth Affiliated Hospital of Sun Yat-sen University, Guangzhou 510655, Guangdong Province, China
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24
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Xu D, Yoshino T, Konishi J, Yoshikawa H, Ino Y, Yazawa R, Dos Santos Nassif Lacerda SM, de França LR, Takeuchi Y. Germ cell-less hybrid fish: ideal recipient for spermatogonial transplantation for the rapid production of donor-derived sperm†. Biol Reprod 2020; 101:492-500. [PMID: 31132090 DOI: 10.1093/biolre/ioz045] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 05/24/2019] [Indexed: 12/14/2022] Open
Abstract
An interspecific hybrid marine fish that developed a testis-like gonad without any germ cells, i.e., a germ cell-less gonad, was produced by hybridizing a female blue drum Nibea mitsukurii with a male white croaker Pennahia argentata. In this study, we evaluated the suitability of the germ cell-less fish as a recipient by transplanting donor testicular cells directly into the gonads through the urogenital papilla. The donor testicular cells were collected from hemizygous transgenic, green fluorescent protein (gfp) (+/-) blue drum, and transplanted into the germ cell-less gonads of the 6-month-old adult hybrid croakers. Fluorescent and histological observations showed the colonization, proliferation, and differentiation of transplanted spermatogonial cells in the gonads of hybrid croakers. The earliest production of spermatozoa in a hybrid recipient was observed at 7 weeks post-transplantation (pt), and 10% of the transplanted recipients produced donor-derived gfp-positive spermatozoa by 25 weeks pt. Sperm from the hybrid recipients were used to fertilize eggs from wild-type blue drums, and approximately 50% of the resulting offspring were gfp-positive, suggesting that all offspring originated from donor-derived sperm that were produced in the transplanted gfp (+/-) germ cells. To the best of our knowledge, this is the first report of successful spermatogonial transplantation using a germ cell-less adult fish as a recipient. This transplantation system has considerable advantages, such as the use of comparatively simple equipment and procedures, and rapid generation of donor-derived spermatogenesis and offspring, and presents numerous applications in commercial aquaculture.
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Affiliation(s)
- Dongdong Xu
- Marine Fishery Institute of Zhejiang Province, Key Lab of Mariculture and Enhancement of Zhejiang Province, Zhoushan, Zhejiang Province, PR China.,Division of Fisheries Resource Sciences, Faculty of Fisheries, Kagoshima University, Shimoarata 4-50-20, Kagoshima City, Japan
| | - Tasuku Yoshino
- Department of Marine Bioscience, Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato, Tokyo, Japan
| | - Junpei Konishi
- Department of Marine Bioscience, Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato, Tokyo, Japan
| | - Hiroyuki Yoshikawa
- Department of Applied Aquabiology, National Fisheries University, Japan Fisheries Research and Education Agency, 2-7-1 Nagata-Honmachi, Shimonoseki, Japan
| | - Yasuko Ino
- Department of Applied Aquabiology, National Fisheries University, Japan Fisheries Research and Education Agency, 2-7-1 Nagata-Honmachi, Shimonoseki, Japan
| | - Ryosuke Yazawa
- Department of Marine Bioscience, Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato, Tokyo, Japan
| | | | - Luiz Renato de França
- Laboratory of Cellular Biology, Department of Morphology, Federal University of Minas Gerais, Belo Horizonte, Brazil
| | - Yutaka Takeuchi
- Division of Fisheries Resource Sciences, Faculty of Fisheries, Kagoshima University, Shimoarata 4-50-20, Kagoshima City, Japan.,The United Graduate School of Agricultural Sciences, Kagoshima University, Kagoshima, Japan
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25
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Javadi A, Mokhtari S, Moraveji SF, Sayahpour FA, Farzaneh M, Gourabi H, Esfandiari F. Short time exposure to low concentration of zinc oxide nanoparticles up-regulates self-renewal and spermatogenesis-related gene expression. Int J Biochem Cell Biol 2020; 127:105822. [PMID: 32771442 DOI: 10.1016/j.biocel.2020.105822] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/13/2020] [Revised: 07/21/2020] [Accepted: 07/31/2020] [Indexed: 12/16/2022]
Abstract
Extensive application of zinc oxide (ZnO) nanoparticles (NPs) in everyday life results in increased exposure to these NPs. Spermatogonial stem cells (SSCs) guarantee sperm production throughout the male reproductive life by providing a balance between self-renewal and differentiation. We used an in vitro platform to investigate the ZnO NPs effects on SSCs. We successfully synthesized ZnO NPs. In order to investigate these NPs, we isolated SSCs from mouse testes and cultured them in vitro. Our results confirmed the uptake of ZnO NPs by the cultured SSCs. We observed a dose- and time-dependent decrease in SSC viability. Both spherical and nanosheet ZnO NPs had the same cytotoxic effects on the SSCs, irrespective of their shapes. Moreover, we have shown that short time (one day) exposure of SSCs to a low concentration of ZnO NPs (10 μg/mL) promoted expressions of specific genes (Plzf, Gfr α1 and Bcl6b) for SSC self-renewal and differentiation genes (Vasa, Dazl, C-kit and Sycp3) expressed by spermatogonia during spermatogenesis. Our study provides the first insight into ZnO NPs function in SSCs and suggests a new function for ZnO NPs in the male reproductive system. We demonstrated that ZnO NPs might promote spermatogenesis via upregulation of gene expression related to SSC self-renewal and differentiation at low concentrations. Additional research should clarify the possible effect of ZnO NPs on the SSC genome and its effects on human SSCs.
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Affiliation(s)
- Azam Javadi
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran; Department of Genetics, Faculty of Basic Science and Advanced Technologies, University of Science and Culture, Tehran, Iran
| | - Saadat Mokhtari
- Department of Physics, Shahid Beheshti University, Tehran, Iran
| | - Seyedeh-Faezeh Moraveji
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
| | - Forough-Azam Sayahpour
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
| | - Maryam Farzaneh
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran; Department of Molecular and Cellular Biology, Faculty of Basic Science and Advanced Technologies, University of Science and Culture, Tehran, Iran
| | - Hamid Gourabi
- Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
| | - Fereshteh Esfandiari
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.
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26
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Karimipour M, Ahmadi A, Zirak Javanmard M, Jafari A, Mohebi M, Hosseinalipour E. The effects of exposure to fluoxetine during lactation on testicular tissue and sperm parameters in mice offspring. VETERINARY RESEARCH FORUM : AN INTERNATIONAL QUARTERLY JOURNAL 2020; 11:35-42. [PMID: 32537105 PMCID: PMC7282220 DOI: 10.30466/vrf.2018.82090.2082] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 03/06/2018] [Accepted: 09/25/2018] [Indexed: 11/02/2022]
Abstract
Fluoxetine is a selective serotonin reuptake inhibitor is commonly prescribed to treat maternal depression in pregnancy and lactation. This study aimed to investigate the effects of maternal exposure to fluoxetine via lactation on testicular tissue, sperm parameters including count, motility, viability, and normal morphology and testicular oxidative stress status in male mice offspring. Ten mice dams were divided into control and experimental groups. The control group received water and the experimental group received fluoxetine (20.00 mg kg-1) by gavage daily from postnatal days of 0-21. Histology of testis, sperm parameters and oxidative stress in the testicular tissue were analyzed at 80 days after birth in their male offspring (n = 8). Significant reductions in the body and testes weights were observed in animals exposed to fluoxetine. Additionally, fluoxetine exposure significantly reduced all sperm parameters, tubular diameter and epithelial height of the seminiferous tubules as well as Leydig cells number. Significant increases in the testicular malondialdehyde levels and percentage of sperm with chromatin/DNA damage were observed in mice exposed to fluoxetine compared to control. These findings suggest that maternal exposure to fluoxetine during lactation in mice has a negative effect on the testicular tissue of their offspring and impairs the spermatogenesis process which in turn can induce infertility.
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Affiliation(s)
- Mojtaba Karimipour
- Department of Anatomy and Histology, Faculty of Medicine, Urmia University of Medical Sciences, Urmia, Iran
| | - Abbas Ahmadi
- Department of Basic Sciences, Faculty of Veterinary Medicine, Urmia University , Urmia, Iran
| | - Masoumeh Zirak Javanmard
- Department of Anatomy and Histology, Faculty of Medicine, Urmia University of Medical Sciences, Urmia, Iran
| | - Abbas Jafari
- Department of Occupational Health, School of Health, Urmia University of Medical Sciences, Urmia, Iran
| | - Maryam Mohebi
- Department of Anatomy and Histology, Faculty of Medicine, Urmia University of Medical Sciences, Urmia, Iran
| | - Elnaz Hosseinalipour
- Department of Anatomy and Histology, Faculty of Medicine, Urmia University of Medical Sciences, Urmia, Iran
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27
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Cui L, Gu Y, Liu S, Li M, Ye J, Zhang F, Luo X, Chang WL, Gui Y. TBC1D20 Is Essential for Mouse Blood-Testis Barrier Integrity Through Maintaining the Epithelial Phenotype and Modulating the Maturation of Sertoli Cells. Reprod Sci 2020; 27:1443-1454. [PMID: 31994000 DOI: 10.1007/s43032-020-00156-z] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/19/2019] [Accepted: 12/10/2019] [Indexed: 11/30/2022]
Abstract
Sertoli cells are important for spermatogenesis not only by directly interacting with germ line cells in the seminiferous epithelium but also by constituting the blood-testis barrier (BTB) structure to create a favorable environment for spermatogenesis. Blind sterile (bs) male mice are infertile, with excessive germ cell apoptosis and spermatogenesis arrest. TBC1D20 (TBC1 domain family member 20) deficiency has been identified as the causative mutation in bs mice. However, whether TBC1D20 loss of function also impairs BTB integrity, which further contributes to the failed spermatogenesis of bs male mice, remains unclear. In the present study, biotin tracer assay and transmission electron microscopy showed severely disrupted BTB integrity in bs testes. Compared to the wild-type Sertoli cells, BTB components of cultured bs Sertoli cells in vitro was perturbed with downregulation of E-cadherin, ZO-1, β-catenin, and Claudin 11. The obvious rearrangement of F-actin indicated disrupted epithelial-mesenchymal balance in TBC1D20-deficient Sertoli cells. The ability of bs Sertoli cells to maintain the clone formation of spermatogonia stem cells was also obviously limited. Furthermore, the decreasing of SOX9 (sex-determining region Y box 9) and WT1 (Wilms' tumor 1) and increasing of vimentin in bs Sertoli cells indicated that TBC1D20 loss of function attenuated the differentiation progression of bs Sertoli cells. In summary, TBC1D20 loss of function impedes the maturation of adult Sertoli cells and resulted in impaired BTB integrity, which is further implicated in the infertile phenotype of bs male mice.
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Affiliation(s)
- Lina Cui
- Guangdong and Shenzhen Key Laboratory of Male Reproductive Medicine and Genetics, Institute of Urology, Peking University Shenzhen Hospital, Shenzhen Peking University-The Hong Kong University of Science and Technology Medical Center, Shenzhen, 518036, China
| | - Yanli Gu
- Department of Obstetrics, the People's Hospital of Longhua, Shenzhen, 518109, China
| | - Shuo Liu
- College of Biological Sciences and Technology, Beijing Forestry University, Beijing, 10083, China
| | - Minghua Li
- Guangdong and Shenzhen Key Laboratory of Male Reproductive Medicine and Genetics, Institute of Urology, Peking University Shenzhen Hospital, Shenzhen Peking University-The Hong Kong University of Science and Technology Medical Center, Shenzhen, 518036, China
| | - Jing Ye
- Guangdong and Shenzhen Key Laboratory of Male Reproductive Medicine and Genetics, Institute of Urology, Peking University Shenzhen Hospital, Shenzhen Peking University-The Hong Kong University of Science and Technology Medical Center, Shenzhen, 518036, China
| | - Fanting Zhang
- Guangdong and Shenzhen Key Laboratory of Male Reproductive Medicine and Genetics, Institute of Urology, Peking University Shenzhen Hospital, Shenzhen Peking University-The Hong Kong University of Science and Technology Medical Center, Shenzhen, 518036, China
| | - Xiaomin Luo
- Guangdong and Shenzhen Key Laboratory of Male Reproductive Medicine and Genetics, Institute of Urology, Peking University Shenzhen Hospital, Shenzhen Peking University-The Hong Kong University of Science and Technology Medical Center, Shenzhen, 518036, China
| | - Wen-Lin Chang
- Department of Obstetrics, the People's Hospital of Longhua, Shenzhen, 518109, China.
| | - Yaoting Gui
- Guangdong and Shenzhen Key Laboratory of Male Reproductive Medicine and Genetics, Institute of Urology, Peking University Shenzhen Hospital, Shenzhen Peking University-The Hong Kong University of Science and Technology Medical Center, Shenzhen, 518036, China.
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28
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Yokonishi T, McKey J, Ide S, Capel B. Sertoli cell ablation and replacement of the spermatogonial niche in mouse. Nat Commun 2020; 11:40. [PMID: 31896751 PMCID: PMC6940386 DOI: 10.1038/s41467-019-13879-8] [Citation(s) in RCA: 47] [Impact Index Per Article: 9.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/25/2019] [Accepted: 11/14/2019] [Indexed: 01/15/2023] Open
Abstract
Spermatogonia, which produce sperm throughout the male lifetime, are regulated inside a niche composed of Sertoli cells, and other testis cell types. Defects in Sertoli cells often lead to infertility, but replacement of defective cells has been limited by the inability to deplete the existing population. Here, we use an FDA-approved non-toxic drug, benzalkonium chloride (BC), to deplete testis cell types in vivo. Four days after BC administration, Sertoli cells are preferentially depleted, and can be replaced to promote spermatogenesis from surviving (host) spermatogonia. Seven days after BC treatment, multiple cell types can be engrafted from fresh or cryopreserved testicular cells, leading to complete spermatogenesis from donor cells. These methods will be valuable for investigation of niche-supporting cell interactions, have the potential to lead to a therapy for idiopathic male infertility in the clinic, and could open the door to production of sperm from other species in the mouse. Sertoli cells and other somatic cells of the testis comprise the germ cell niche and are critical to regulate spermatogenesis. Here the authors present a method in which Sertoli cells are selectively targeted for ablation by the compound benzalkonium chloride (BC) in mice, and the spermatogenic niche is subsequently repopulated in regions that have been affected by BC treatment.
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Affiliation(s)
- Tetsuhiro Yokonishi
- Department of Cell Biology, Duke University Medical Center, Durham, NC, 27710, USA. .,Department of Urology, Yokohama City University, Yokohama, Japan.
| | - Jennifer McKey
- Department of Cell Biology, Duke University Medical Center, Durham, NC, 27710, USA
| | - Shintaro Ide
- Division of Nephrology, Department of Medicine, Duke University School of Medicine, Durham, NC, 27710, USA
| | - Blanche Capel
- Department of Cell Biology, Duke University Medical Center, Durham, NC, 27710, USA.
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29
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Vermeulen M, Giudice MG, Del Vento F, Wyns C. Role of stem cells in fertility preservation: current insights. STEM CELLS AND CLONING-ADVANCES AND APPLICATIONS 2019; 12:27-48. [PMID: 31496751 PMCID: PMC6689135 DOI: 10.2147/sccaa.s178490] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 01/16/2019] [Accepted: 05/24/2019] [Indexed: 12/11/2022]
Abstract
While improvements made in the field of cancer therapy allow high survival rates, gonadotoxicity of chemo- and radiotherapy can lead to infertility in male and female pre- and postpubertal patients. Clinical options to preserve fertility before starting gonadotoxic therapies by cryopreserving sperm or oocytes for future use with assisted reproductive technology (ART) are now applied worldwide. Cryopreservation of pre- and postpubertal ovarian tissue containing primordial follicles, though still considered experimental, has already led to the birth of healthy babies after autotransplantation and is performed in an increasing number of centers. For prepubertal boys who do not produce gametes ready for fertilization, cryopreservation of immature testicular tissue (ITT) containing spermatogonial stem cells may be proposed as an experimental strategy with the aim of restoring fertility. Based on achievements in nonhuman primates, autotransplantation of ITT or testicular cell suspensions appears promising to restore fertility of young cancer survivors. So far, whether in two- or three-dimensional culture systems, in vitro maturation of immature male and female gonadal cells or tissue has not demonstrated a capacity to produce safe gametes for ART. Recently, primordial germ cells have been generated from embryonic and induced pluripotent stem cells, but further investigations regarding efficiency and safety are needed. Transplantation of mesenchymal stem cells to improve the vascularization of gonadal tissue grafts, increase the colonization of transplanted cells, and restore the damaged somatic compartment could overcome the current limitations encountered with transplantation.
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Affiliation(s)
- Maxime Vermeulen
- Gynecology-Andrology Research Unit, Institut de Recherche Expérimentale et Clinique (IREC), Université Catholique de Louvain, Brussels, 1200, Belgium
| | - Maria-Grazia Giudice
- Gynecology-Andrology Research Unit, Institut de Recherche Expérimentale et Clinique (IREC), Université Catholique de Louvain, Brussels, 1200, Belgium.,Department of Gynecology-Andrology, Cliniques Universitaires Saint-Luc, Brussels 1200, Belgium
| | - Federico Del Vento
- Gynecology-Andrology Research Unit, Institut de Recherche Expérimentale et Clinique (IREC), Université Catholique de Louvain, Brussels, 1200, Belgium
| | - Christine Wyns
- Gynecology-Andrology Research Unit, Institut de Recherche Expérimentale et Clinique (IREC), Université Catholique de Louvain, Brussels, 1200, Belgium.,Department of Gynecology-Andrology, Cliniques Universitaires Saint-Luc, Brussels 1200, Belgium
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30
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Kubota H, Brinster RL. Spermatogonial stem cells. Biol Reprod 2019; 99:52-74. [PMID: 29617903 DOI: 10.1093/biolre/ioy077] [Citation(s) in RCA: 156] [Impact Index Per Article: 26.0] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/14/2017] [Accepted: 03/29/2018] [Indexed: 12/19/2022] Open
Abstract
Spermatogonial stem cells (SSCs) are the most primitive spermatogonia in the testis and have an essential role to maintain highly productive spermatogenesis by self-renewal and continuous generation of daughter spermatogonia that differentiate into spermatozoa, transmitting genetic information to the next generation. Since the 1950s, many experimental methods, including histology, immunostaining, whole-mount analyses, and pulse-chase labeling, had been used in attempts to identify SSCs, but without success. In 1994, a spermatogonial transplantation method was reported that established a quantitative functional assay to identify SSCs by evaluating their ability to both self-renew and differentiate to spermatozoa. The system was originally developed using mice and subsequently extended to nonrodents, including domestic animals and humans. Availability of the functional assay for SSCs has made it possible to develop culture systems for their ex vivo expansion, which dramatically advanced germ cell biology and allowed medical and agricultural applications. In coming years, SSCs will be increasingly used to understand their regulation, as well as in germline modification, including gene correction, enhancement of male fertility, and conversion of somatic cells to biologically competent male germline cells.
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Affiliation(s)
- Hiroshi Kubota
- Laboratory of Cell and Molecular Biology, Department of Animal Science, School of Veterinary Medicine, Kitasato University, Towada, Aomori, Japan
| | - Ralph L Brinster
- Department of Biomedical Sciences, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA
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31
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Savvulidi F, Ptacek M, Savvulidi Vargova K, Stadnik L. Manipulation of spermatogonial stem cells in livestock species. J Anim Sci Biotechnol 2019; 10:46. [PMID: 31205688 PMCID: PMC6560896 DOI: 10.1186/s40104-019-0355-4] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/05/2018] [Accepted: 04/17/2019] [Indexed: 12/12/2022] Open
Abstract
We are entering an exciting epoch in livestock biotechnology during which the fundamental approaches (such as transgenesis, spermatozoa cryopreservation and artificial insemination) will be enhanced based on the modern understanding of the biology of spermatogonial stem cells (SSCs) combined with the outstanding recent advances in genomic editing technologies and in vitro cell culture systems. The general aim of this review is to outline comprehensively the promising applications of SSC manipulation that could in the nearest future find practical application in livestock breeding. Here, we will focus on 1) the basics of mammalian SSC biology; 2) the approaches for SSC isolation and purification; 3) the available in vitro systems for the stable expansion of isolated SSCs; 4) a discussion of how the manipulation of SSCs can accelerate livestock transgenesis; 5) a thorough overview of the techniques of SSC transplantation in livestock species (including the preparation of recipients for SSC transplantation, the ultrasonographic-guided SSC transplantation technique in large farm animals, and the perspectives to improve further the SSC transplantation efficiency), and finally, 6) why SSC transplantation is valuable to extend the techniques of spermatozoa cryopreservation and/or artificial insemination. For situations where no reliable data have yet been obtained for a particular livestock species, we will rely on the data obtained from studies conducted in rodents because the knowledge gained from rodent research is translatable to livestock species to a great extent. On the other hand, we will draw special attention to situations where such translation is not possible.
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Affiliation(s)
- Filipp Savvulidi
- Department of Animal Science, Faculty of Agrobiology, Food and Natural Resources, Czech University of Life Sciences Prague, Kamýcká 129, 165 00 Prague, Suchdol Czech Republic
- Institute of Pathological Physiology, First Faculty of Medicine, Charles University in Prague, U Nemocnice 5, 128 53 Prague, Czech Republic
| | - Martin Ptacek
- Department of Animal Science, Faculty of Agrobiology, Food and Natural Resources, Czech University of Life Sciences Prague, Kamýcká 129, 165 00 Prague, Suchdol Czech Republic
| | - Karina Savvulidi Vargova
- Institute of Pathological Physiology, First Faculty of Medicine, Charles University in Prague, U Nemocnice 5, 128 53 Prague, Czech Republic
| | - Ludek Stadnik
- Department of Animal Science, Faculty of Agrobiology, Food and Natural Resources, Czech University of Life Sciences Prague, Kamýcká 129, 165 00 Prague, Suchdol Czech Republic
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32
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Baert Y, Dvorakova-Hortova K, Margaryan H, Goossens E. Mouse in vitro spermatogenesis on alginate-based 3D bioprinted scaffolds. Biofabrication 2019; 11:035011. [PMID: 30921781 DOI: 10.1088/1758-5090/ab1452] [Citation(s) in RCA: 48] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
In vitro spermatogenesis (IVS) has already been successfully achieved in rodents by organotypic and soft matrix culture systems. However, the former does not allow single cell input, and the latter presents as a simple thick layer in which all cells are embedded. We explored a new culture system using a mouse model by employing an alginate-based hydrogel and 3D bioprinting, to control scaffold design and cell deposition. We produced testicular constructs consisting of printed cell-free scaffolds (CFS) with prepubertal testicular cells (TC) in their easy-to-access macropores. Here, the pores represented the only cell compartment (TC/CFS). Double-cell compartment testicular constructs were achieved by culturing magnetic-activated cell sorting-enriched epithelial cells in the pores of interstitial cell-laden scaffolds (CD49f+/CLS). Cell spheres formed in the pores in the weeks following cell seeding on both CFS and CLS. Although restoration of the tubular architecture was not observed, patches of post-meiotic cells including elongated spermatids were found in 66% of TC/CFS. Differentiation up to the level of round spermatids and elongated spermatids was observed in all and 33% of CD49f+/CLS constructs, respectively. Organ culture served as the reference method for IVS, with complete spermatogenesis identified in 80% of cultivated prepubertal tissue fragments. So far, this is the first report applying a 3D bioprinting approach for IVS. Further optimization of the scaffold design and seeding parameters might be permissive for tubular architecture recreation and thereby increase the efficiency of IVS in printed testicular constructs. While it remains to be tested whether the gametes generated on the alginate-based scaffolds can support embryogenesis following IVF, this IVS approach might be useful for (patho)physiological studies and drug-screening applications.
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Affiliation(s)
- Yoni Baert
- Biology of the Testis, Research Laboratory for Reproduction, Genetics and Regenerative Medicine, Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, B-1090 Brussels, Belgium. Polymer Chemistry & Biomaterials Research Group, Department of Organic Chemistry, Ghent University, Krijgslaan 281 S4 Bis, B-9000 Ghent, Belgium
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Co-transplantation of mesenchymal stem cells improves spermatogonial stem cell transplantation efficiency in mice. Stem Cell Res Ther 2018; 9:317. [PMID: 30463610 PMCID: PMC6249754 DOI: 10.1186/s13287-018-1065-0] [Citation(s) in RCA: 28] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/11/2018] [Revised: 10/19/2018] [Accepted: 10/31/2018] [Indexed: 12/27/2022] Open
Abstract
Background Spermatogonial stem cell transplantation (SSCT) could become a fertility restoration tool for childhood cancer survivors. However, since in mice, the colonization efficiency of transplanted spermatogonial stem cells (SSCs) is only 12%, the efficiency of the procedure needs to be improved before clinical implementation is possible. Co-transplantation of mesenchymal stem cells (MSCs) might increase colonization efficiency of SSCs by restoring the SSC niche after gonadotoxic treatment. Methods A mouse model for long-term infertility was developed and used to transplant SSCs (SSCT, n = 10), MSCs (MSCT, n = 10), a combination of SSCs and MSCs (MS-SSCT, n = 10), or a combination of SSCs and TGFß1-treated MSCs (MSi-SSCT, n = 10). Results The best model for transplantation was obtained after intraperitoneal injection of busulfan (40 mg/kg body weight) at 4 weeks followed by CdCl2 (2 mg/kg body weight) at 8 weeks of age and transplantation at 11 weeks of age. Three months after transplantation, spermatogenesis resumed with a significantly better tubular fertility index (TFI) in all transplanted groups compared to non-transplanted controls (P < 0.001). TFI after MSi-SSCT (83.3 ± 19.5%) was significantly higher compared to MS-SSCT (71.5 ± 21.7%, P = 0.036) but did not differ statistically compared to SSCT (78.2 ± 12.5%). In contrast, TFI after MSCT (50.2 ± 22.5%) was significantly lower compared to SSCT (P < 0.001). Interestingly, donor-derived TFI was found to be significantly improved after MSi-SSCT (18.8 ± 8.0%) compared to SSCT (1.9 ± 1.1%; P < 0.001), MSCT (0.0 ± 0.0%; P < 0.001), and MS-SSCT (3.4 ± 1.9%; P < 0.001). While analyses showed that both native and TGFß1-treated MSCs maintained characteristics of MSCs, the latter showed less migratory characteristics and was not detected in other organs. Conclusion Co-transplanting SSCs and TGFß1-treated MSCs significantly improves the recovery of endogenous SSCs and increases the homing efficiency of transplanted SSCs. This procedure could become an efficient method to treat infertility in a clinical setup, once the safety of the technique has been proven. Electronic supplementary material The online version of this article (10.1186/s13287-018-1065-0) contains supplementary material, which is available to authorized users.
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Seol DW, Park S, Shin EY, Chang JH, Lee DR. In Vitro Derivation of Functional Sertoli-Like Cells from Mouse Embryonic Stem Cells. Cell Transplant 2018; 27:1523-1534. [PMID: 30215278 PMCID: PMC6180718 DOI: 10.1177/0963689718797053] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022] Open
Abstract
Sertoli cells (SCs) in the mammalian testes are well known as supporting cells of spermatogenesis, but have recently become an attractive source of cell therapy because of their capacity for immune modulation and trophic effects. In order to increase their applicable efficacy, we demonstrate a novel differentiation method for mouse embryonic stem cell (ESC)-derived Sertoli-like cells (SLCs) via the intermediate mesoderm (IM). We show that IM derived from an induction of 6 days expressed markers such as Wt1, Lhx1, Pax2 and Osr1, and that a sequential induction of 6 days resulted in ESC-SLCs. The SLCs expressed their marker genes ( Sf1, Sox9, Gata4, Wt1, Fshr and Scf), but the pluripotency-marker gene Oct4 was decreased. After sorting by FSHR expression, high-purity (> 90%) SLCs were collected that showed distinct characteristics of SCs such as high phagocytic and immune modulation activities as well as the expression of immune-related genes. In addition, when transplanted into the seminiferous tubule of busulfan-treated mice, SLCs re-located and were maintained in the basal region of the tubule. These results demonstrated that our robust sequential differentiation system produced functional SLCs from mouse ESCs in vitro.
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Affiliation(s)
- Dong-Won Seol
- 1 Department of Biomedical Science, College of Life Science, CHA University, Gyeonggi-do, Korea
| | - Seah Park
- 1 Department of Biomedical Science, College of Life Science, CHA University, Gyeonggi-do, Korea
| | - Eun Young Shin
- 1 Department of Biomedical Science, College of Life Science, CHA University, Gyeonggi-do, Korea
| | - Jae Ho Chang
- 2 Department of Bio-Convergence, Underwood International College, Yonsei University, Seoul, Korea
| | - Dong Ryul Lee
- 1 Department of Biomedical Science, College of Life Science, CHA University, Gyeonggi-do, Korea
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Comparison of cryosurvival and spermatogenesis efficiency of cryopreserved neonatal mouse testicular tissue between three vitrification protocols and controlled-rate freezing. Cryobiology 2018; 84:4-9. [PMID: 30195700 DOI: 10.1016/j.cryobiol.2018.09.001] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/18/2018] [Revised: 09/03/2018] [Accepted: 09/05/2018] [Indexed: 01/15/2023]
Abstract
Grafting of cryopreserved testicular tissue is a promising tool for fertility and testicular function preservation in endangered species, mutant animals, or cancer patients for future use. In this study, we aimed to improve the whole neonatal mouse testicular tissue cryopreservation protocols by comparing cryosurvival, spermatogenesis, and androgen production of grafted testicular tissue after cryopreservation with three different vitrification protocols and an automated computed controlled-rate freezing. Whole neonatal mouse testes were vitrified with various vitrification solutions (V1) 40% EG + 18% Ficoll + 0.35 M Sucrose, (V2) DAP 213 (2 M DMSO + 1 M Acetamid + 3 M PG), or (V3) 15% EG + 15% PG + 0.5 M Sucrose (total solute concentration V1:74.34%, V2:44.0%, and V3:49.22% wt/vol). Alternatively, neonatal testicular tissue was also frozen in 0.7 M DMSO +5% fetal bovine serum using controlled-rate freezing and compared to fresh grafted testicular tissue, sham grafted controls, and the vitrification protocol groups. Fresh (n = 4) and frozen-thawed (n = 4) testes tissues were grafted onto the flank of castrated male NCr Nude recipient mouse. The grafts were harvested after three months. Fresh or frozen-thawed grafts with controlled-rate freezing had the highest rate of tissue survival compared to other vitrified protocols after harvesting (p < 0.05). Both controlled-rate freezing and V1 protocol groups displayed the most advanced stages of spermatogenesis with elongated spermatids and spermatozoa in 17.6 ± 1.3% and 16.3 ± 1.9% of seminiferous tubules based on histopathological evaluation, respectively. Hosts of the testicular graft from controlled-rate freezing had higher levels of serum testosterone compared to all other vitrified-thawed graft groups (p < 0.05). This study shows that completed spermatogenesis from whole neonatal mouse testes were obtained when frozen with controlled-rate freezing and V1 vitrification solution and that testicular cryopreservation efficacy vary with the protocol and vitrification technique.
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Griswold MD. 50 years of spermatogenesis: Sertoli cells and their interactions with germ cells. Biol Reprod 2018; 99:87-100. [PMID: 29462262 PMCID: PMC7328471 DOI: 10.1093/biolre/ioy027] [Citation(s) in RCA: 167] [Impact Index Per Article: 23.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/13/2017] [Revised: 01/22/2018] [Accepted: 02/02/2018] [Indexed: 01/15/2023] Open
Abstract
The complex morphology of the Sertoli cells and their interactions with germ cells has been a focus of investigators since they were first described by Enrico Sertoli. In the past 50 years, information on Sertoli cells has transcended morphology alone to become increasingly more focused on molecular questions. The goal of investigators has been to understand the role of the Sertoli cells in spermatogenesis and to apply that information to problems relating to male fertility. Sertoli cells are unique in that they are a nondividing cell population that is active for the reproductive lifetime of the animal and cyclically change morphology and gene expression. The numerous and distinctive junctional complexes and membrane specializations made by Sertoli cells provide a scaffold and environment for germ cell development. The increased focus of investigators on the molecular components and putative functions of testicular cells has resulted primarily from procedures that isolate specific cell types from the testicular milieu. Products of Sertoli cells that influence germ cell development and vice versa have been characterized from cultured cells and from the application of transgenic technologies. Germ cell transplantation has shown that the Sertoli cells respond to cues from germ cells with regard to developmental timing and has furthered a focus on spermatogenic stem cells and the stem cell niche. Very basic and universal features of spermatogenesis such as the cycle of the seminiferous epithelium and the spermatogenic wave are initiated by Sertoli cells and maintained by Sertoli-germ cell cooperation.
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Affiliation(s)
- Michael D Griswold
- Center for Reproductive Biology, Washington State University, Pullman, Washington, USA
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Underlying Mechanisms that Restore Spermatogenesis on Transplanting Healthy Niche Cells in Busulphan Treated Mouse Testis. Stem Cell Rev Rep 2017; 12:682-697. [PMID: 27663915 DOI: 10.1007/s12015-016-9685-1] [Citation(s) in RCA: 77] [Impact Index Per Article: 9.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/02/2023]
Abstract
Very small embryonic-like stem cells (VSELs) exist among spermatogonial stem cells and survive chemotherapy in both mice and human testes because of their relatively quiescent nature. Our earlier study revealed that inter-tubular transplantation of niche (Sertoli or bone marrow derived mesenchymal) cells can restore spermatogenesis from endogenous surviving VSELs. Present study was undertaken to delineate the effect of busulphan on testicular stem/germ/Sertoli cells and to comprehend the underlying mechanisms of how transplanted niche cells restore spermatogenesis. Ploidy analysis showed an increase in diploid cells on D7 and VSELs (2-6 μm; LIN-/CD45-/SCA-1+) were detected at all time-points studied and were maximum on D15 after busulphan treatment. They were visualized in cell smears, expressed nuclear NANOG and SOX2 and BrdU uptake on D15 suggested they were proliferating in response to stress induced by busulphan. Verapamil-sensitive side population detected comprised SCA-1 positive stem cells (5 ± 0.02 % in normal and 8.6 ± 2.02 % in chemoablated testis). Adverse effects of busulphan on Sertoli cells by transcriptome analysis included altered expression of Gdnf, Scf, Fgf, Bmp4, androgen binding protein, components of blood-testis-barrier and also stem cells related signaling pathways including Wnt. GFP positive transplanted cells aligned themselves as 'neo-tubules' and were visualized adjacent to 'native' germ cells depleted tubules. 'Neo-tubules' provide paracrine support to endogenous VSELs to undergo spermatogenesis. Quantitative analysis was done to track proliferation (PCNA) and differentiation (MVH) of stem cells by immuno-localization studies at different time intervals. Results provide an alternative strategy to restore spermatogenesis in cancer survivors from endogenous stem cells which needs to be further researched.
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Malolina EA, Kulibin AY, Kushch AA. Neonatal testicular cell transplantation restores murine spermatogenesis damaged in the course of herpes simplex virus-induced orchitis. Reprod Fertil Dev 2017; 28:757-64. [PMID: 25399480 DOI: 10.1071/rd14255] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/18/2014] [Accepted: 09/19/2014] [Indexed: 01/15/2023] Open
Abstract
Genital tract infection and inflammation may affect male fertility, causing germ and Sertoli cell loss. We determined if testicular cell transplantation is effective at repairing testicular injury induced by herpes simplex virus (HSV) orchitis. ROSA26 mice were used as donors and the recipients were C57BL/6 mice after HSV testicular inoculation; some of the recipients were treated with the antiviral drug acyclovir (ACV). ACV reduced the amount of HSV antigen in testes on Day 3 after transplantation and enhanced the efficacy of transplantation at Day 30. In recipient testes, donor Sertoli cells formed new seminiferous tubules; significantly more new tubules were observed in the testes of ACV-treated mice compared with mice not treated with ACV (17.8% vs 3.6%). Over half (50.4%) of new tubules in ACV-treated testes contained germ cells and round spermatids were detected in 14.2% of new tubules compared with 15.9% and 5.3% in testes not treated with ACV, respectively. At Day 150 the seminiferous epithelium was completely recovered in some donor tubules and elongated spermatids were observed inside it. Thus, our findings reveal the effectiveness of the combination of antiviral therapy with neonatal testis-cell transplantation for the restoration of spermatogenesis damaged by viral infection.
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Affiliation(s)
- Ekaterina A Malolina
- Ivanovsky Institute of Virology, Ministry of Health of the Russian Federation, Gamaleya str. 16, 123098, Moscow, The Russian Federation
| | - Andrey Yu Kulibin
- Kol'tsov Institute of Developmental Biology, Russian Academy of Sciences, Vavilova str. 26, 119071, Moscow, The Russian Federation
| | - Alla A Kushch
- Ivanovsky Institute of Virology, Ministry of Health of the Russian Federation, Gamaleya str. 16, 123098, Moscow, The Russian Federation
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Xu ML, Hu J, Guo BP, Niu YR, Xiao C, Xu YX. Exploration of intrinsic and extrinsic apoptotic pathways in zearalenone-treated rat sertoli cells. ENVIRONMENTAL TOXICOLOGY 2016; 31:1731-1739. [PMID: 26460601 DOI: 10.1002/tox.22175] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/16/2015] [Revised: 07/10/2015] [Accepted: 07/12/2015] [Indexed: 06/05/2023]
Abstract
Zearalenone (ZEA) is a nonsteroidal estrogenic mycotoxin produced mainly by Fusarium. ZEA causes reproductive disorders and is both cytotoxic and genotoxic in animals; however, little is known regarding the molecular mechanism(s) leading to ZEA toxicity. Sertoli cells are somatic cells that support the development of spermatogenic cells. The objective of this study was to explore the effects of ZEA on the proliferation, apoptosis, and necrosis of rat Sertoli cells to uncover signaling pathways underlying ZEA cytotoxicity. ZEA reduced the proliferation of rat Sertoli cells in a dose-dependent manner, as indicated by a CCK8 assay, while flow cytometry revealed that ZEA caused both apoptosis and necrosis. Immunoblotting revealed that ZEA treatment increased the ratio of Bax/Bcl-2, as well as the expression of FasL and caspases-3, -8, and -9, in a dose-dependent manner. Collectively, these data suggest that ZEA induced apoptosis and necrosis in rat Sertoli cells via extrinsic and intrinsic apoptotic pathways. This study provides new insights into the molecular mechanisms by which ZEA exhibits cytotoxicity. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1731-1739, 2016.
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Affiliation(s)
- Ming-Long Xu
- College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, 210095, People's Republic of China
| | - Jin Hu
- College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, 210095, People's Republic of China
| | - Bao-Ping Guo
- College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, 210095, People's Republic of China
| | - Ya-Ru Niu
- College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, 210095, People's Republic of China
| | - Cheng Xiao
- College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, 210095, People's Republic of China
| | - Yin-Xue Xu
- College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, 210095, People's Republic of China
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Kulibin AY, Malolina EA. Only a small population of adult Sertoli cells actively proliferates in culture. Reproduction 2016; 152:271-81. [DOI: 10.1530/rep-16-0013] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/10/2016] [Accepted: 07/04/2016] [Indexed: 11/08/2022]
Abstract
Adult mammalian Sertoli cells (SCs) have been considered to be quiescent terminal differentiated cells for many years, but recently, proliferation of adult SCs was demonstrated in vitro and in vivo. We further examined mouse SC behavior in culture and found that there are two populations of adult SCs. The first population is SCs from seminiferous tubules that hardly proliferate in vitro. The second population is small and consists of SCs with atypical nuclear morphology from the terminal segments of seminiferous tubules, a transitional zone (TZ). TZ SCs multiply in culture and form colonies, display mixture of mature and immature SC characteristics, and generate cord-like structures in a collagen matrix. The specific features of TZ SCs are ACTA2 expression in vitro and DMRT1 low levels in vivo and in vitro. Although the in vivo function of TZ SCs still remains unclear, this finding has significant implications for our understanding of SC differentiation and functioning in adult mammals.
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Deng B, Bondarenko T, Pakhomov O. Changes in Sexual Behavior of Orchidectomized Rats Under Influence of Allotransplantation of Testicular Interstitial Cell Suspension. Cell Transplant 2016; 26:795-803. [PMID: 27697096 DOI: 10.3727/096368916x693301] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2023] Open
Abstract
Transplantation of hormone-producing cells is an experimental endocrine dysfunction treatment. The present study investigated the effects of orchidectomy (OE) and transplantation of interstitial cell suspension (ICS) on rat sexual behavior. Adult experimental animals were divided into two populations. One of these populations had sexual experience before the experiment and the other did not. Each population was divided into three groups: control group and two orchidectomized groups. One of the orchidectomized groups was treated with ICS, and the other was treated with the vehicle. The changes in the sexual behavior were investigated on the following parameters: mount latency (ML), intromission latency (IL), ejaculation latency (EL), mount frequency (MF), intromission frequency (IF), copulatory efficacy (CE), and IF/EL ratio. The investigation of these changes lasted 4 weeks after ICS transplantation. The parameters of sexual behavior reflected a decrease in sexual function after OE at the beginning of the observation, especially for the animals that did not have a sexual experience. However, it was shown that sexual activity increased in the following 4 weeks. We have indicated that the loss of gonads attenuated the capacity to acquire sexual experience; nonetheless, it did not mean that the animals completely lost this capacity. Transplantation of ICS facilitated the maintenance of male sexual behavior after OE, fractionally enlarged the size of regressed seminal vesicles of the animals, and increased the free testosterone (T) level. These findings suggest the ICS can be considered as a temporal source of androgens, which can facilitate a restoration of sexual activity.
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Bhartiya D, Shaikh A, Anand S, Patel H, Kapoor S, Sriraman K, Parte S, Unni S. Endogenous, very small embryonic-like stem cells: critical review, therapeutic potential and a look ahead. Hum Reprod Update 2016; 23:41-76. [PMID: 27614362 DOI: 10.1093/humupd/dmw030] [Citation(s) in RCA: 87] [Impact Index Per Article: 9.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/16/2016] [Revised: 07/27/2016] [Accepted: 08/04/2016] [Indexed: 12/15/2022] Open
Abstract
BACKGROUND Both pluripotent very small embryonic-like stem cells (VSELs) and induced pluripotent stem (iPS) cells were reported in 2006. In 2012, a Nobel Prize was awarded for iPS technology whereas even today the very existence of VSELs is not well accepted. The underlying reason is that VSELs exist in low numbers, remain dormant under homeostatic conditions, are very small in size and do not pellet down at 250-280g. The VSELs maintain life-long tissue homeostasis, serve as a backup pool for adult stem cells and are mobilized under stress conditions. An imbalance in VSELs function (uncontrolled proliferation) may result in cancer. SEARCH METHODS The electronic database 'Medline/Pubmed' was systematically searched with the subject heading term 'very small embryonic-like stem cells'. OBJECTIVE AND RATIONALE The most primitive stem cells that undergo asymmetric cell divisions to self-renew and give rise to progenitors still remain elusive in the hematopoietic system and testes, while the presence of stem cells in ovary is still being debated. We propose to review the available literature on VSELs, the methods of their isolation and characterization, their ontogeny, how they compare with embryonic stem (ES) cells, primordial germ cells (PGCs) and iPS cells, and their role in maintaining tissue homeostasis. The review includes a look ahead on how VSELs will result in paradigm shifts in basic reproductive biology. OUTCOMES Adult tissue-specific stem cells including hematopoietic, spermatogonial, ovarian and mesenchymal stem cells have good proliferation potential and are indeed committed progenitors (with cytoplasmic OCT-4), which arise by asymmetric cell divisions of pluripotent VSELs (with nuclear OCT-4). VSELs are the most primitive stem cells and postulated to be an overlapping population with the PGCs. Rather than migrating only to the gonads, PGCs migrate and survive in various adult body organs throughout life as VSELs. VSELs express both pluripotent and PGC-specific markers and are epigenetically and developmentally more mature compared with ES cells obtained from the inner cell mass of a blastocyst-stage embryo. As a result, VSELs readily differentiate into three embryonic germ layers and spontaneously give rise to both sperm and oocytes in vitro. Like PGCs, VSELs do not divide readily in culture, nor produce teratoma or integrate in the developing embryo. But this property of being relatively quiescent allows endogenous VSELs to survive various kinds of toxic insults. VSELs that survive oncotherapy can be targeted to induce endogenous regeneration of non-functional gonads. Transplanting healthy niche (mesenchymal) cells have resulted in improved gonadal function and live births. WIDER IMPLICATIONS Being quiescent, VSELs possibly do not accumulate genomic (nuclear or mitochondrial) mutations and thus may be ideal endogenous, pluripotent stem cell candidates for regenerative and reproductive medicine. The presence of VSELs in adult gonads and the fact that they survive oncotherapy may obviate the need to bank gonadal tissue for fertility preservation prior to oncotherapy. VSELs and their ability to undergo spermatogenesis/neo-oogenesis in the presence of a healthy niche will help identify newer strategies toward fertility restoration in cancer survivors, delaying menopause and also enabling aged mothers to have better quality eggs.
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Affiliation(s)
- Deepa Bhartiya
- Stem Cell Biology Department, National Institute for Research in Reproductive Health (Indian Council of Medical Research), Jehangir Merwanji Street, Parel, Mumbai 400 012, India
| | - Ambreen Shaikh
- Stem Cell Biology Department, National Institute for Research in Reproductive Health (Indian Council of Medical Research), Jehangir Merwanji Street, Parel, Mumbai 400 012, India
| | - Sandhya Anand
- Stem Cell Biology Department, National Institute for Research in Reproductive Health (Indian Council of Medical Research), Jehangir Merwanji Street, Parel, Mumbai 400 012, India
| | - Hiren Patel
- Stem Cell Biology Department, National Institute for Research in Reproductive Health (Indian Council of Medical Research), Jehangir Merwanji Street, Parel, Mumbai 400 012, India
| | - Sona Kapoor
- Stem Cell Biology Department, National Institute for Research in Reproductive Health (Indian Council of Medical Research), Jehangir Merwanji Street, Parel, Mumbai 400 012, India
| | - Kalpana Sriraman
- Stem Cell Biology Department, National Institute for Research in Reproductive Health (Indian Council of Medical Research), Jehangir Merwanji Street, Parel, Mumbai 400 012, India.,The Foundation for Medical Research, 84-A, RG Thadani Marg, Worli, Mumbai 400018, India
| | - Seema Parte
- Stem Cell Biology Department, National Institute for Research in Reproductive Health (Indian Council of Medical Research), Jehangir Merwanji Street, Parel, Mumbai 400 012, India.,Department of Physiology, James Graham Brown Cancer Centre, University of Louisville School of Medicine, 2301 S 3rd St, Louisville, KY 40202, USA
| | - Sreepoorna Unni
- Stem Cell Biology Department, National Institute for Research in Reproductive Health (Indian Council of Medical Research), Jehangir Merwanji Street, Parel, Mumbai 400 012, India.,Inter Disciplinary Studies Department, University College, Zayed University, Academic City, PO Box 19282, Dubai, United Arab Emirates
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González R, Dobrinski I. Beyond the mouse monopoly: studying the male germ line in domestic animal models. ILAR J 2016; 56:83-98. [PMID: 25991701 DOI: 10.1093/ilar/ilv004] [Citation(s) in RCA: 28] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
Spermatogonial stem cells (SSCs) are the foundation of spermatogenesis and essential to maintain the continuous production of spermatozoa after the onset of puberty in the male. The study of the male germ line is important for understanding the process of spermatogenesis, unravelling mechanisms of stemness maintenance, cell differentiation, and cell-to-cell interactions. The transplantation of SSCs can contribute to the preservation of the genome of valuable individuals in assisted reproduction programs. In addition to the importance of SSCs for male fertility, their study has recently stimulated interest in the generation of genetically modified animals because manipulations of the male germ line at the SSC stage will be maintained in the long term and transmitted to the offspring. Studies performed mainly in the mouse model have laid the groundwork for facilitating advancements in the field of male germ line biology, but more progress is needed in nonrodent species in order to translate the technology to the agricultural and biomedical fields. The lack of reliable markers for isolating germ cells from testicular somatic cells and the lack of knowledge of the requirements for germ cell maintenance have precluded their long-term maintenance in domestic animals. Nevertheless, some progress has been made. In this review, we will focus on the state of the art in the isolation, characterization, culture, and manipulation of SSCs and the use of germ cell transplantation in domestic animals.
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Affiliation(s)
- Raquel González
- Raquel González, DVM, PhD, is a postdoctoral research fellow at the Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Canada. Ina Dobrinski, DVM, MVSc, PhD, Dipl ACT, is a professor and the head of the Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Canada
| | - Ina Dobrinski
- Raquel González, DVM, PhD, is a postdoctoral research fellow at the Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Canada. Ina Dobrinski, DVM, MVSc, PhD, Dipl ACT, is a professor and the head of the Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Canada
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Smith LB, O'Shaughnessy PJ, Rebourcet D. Cell-specific ablation in the testis: what have we learned? Andrology 2015; 3:1035-49. [PMID: 26446427 PMCID: PMC4950036 DOI: 10.1111/andr.12107] [Citation(s) in RCA: 47] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/19/2015] [Revised: 08/19/2015] [Accepted: 08/19/2015] [Indexed: 01/15/2023]
Abstract
Testicular development and function is the culmination of a complex process of autocrine, paracrine and endocrine interactions between multiple cell types. Dissecting this has classically involved the use of systemic treatments to perturb endocrine function, or more recently, transgenic models to knockout individual genes. However, targeting genes one at a time does not capture the more wide‐ranging role of each cell type in its entirety. An often overlooked, but extremely powerful approach to elucidate cellular function is the use of cell ablation strategies, specifically removing one cellular population and examining the resultant impacts on development and function. Cell ablation studies reveal a more holistic overview of cell–cell interactions. This not only identifies important roles for the ablated cell type, which warrant further downstream study, but also, and importantly, reveals functions within the tissue that occur completely independently of the ablated cell type. To date, cell ablation studies in the testis have specifically removed germ cells, Leydig cells, macrophages and recently Sertoli cells. These studies have provided great leaps in understanding not possible via other approaches; as such, cell ablation represents an essential component in the researchers’ tool‐kit, and should be viewed as a complement to the more mainstream approaches to advancing our understanding of testis biology. In this review, we summarise the cell ablation models used in the testis, and discuss what each of these have taught us about testis development and function.
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Affiliation(s)
- L B Smith
- MRC Centre for Reproductive Health, University of Edinburgh, The Queen's Medical Research Institute, Edinburgh, UK
| | - P J O'Shaughnessy
- College of Medical, Veterinary and Life Sciences, Institute of Biodiversity, Animal Health and Comparative Medicine, University of Glasgow, Garscube Campus, Glasgow, UK
| | - D Rebourcet
- MRC Centre for Reproductive Health, University of Edinburgh, The Queen's Medical Research Institute, Edinburgh, UK
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Mehrabani D, Hassanshahi MA, Tamadon A, Zare S, Keshavarz S, Rahmanifar F, Dianatpour M, Khodabandeh Z, Jahromi I, Tanideh N, Ramzi M, Aqababa H, Kuhi-Hoseinabadi O. Adipose tissue-derived mesenchymal stem cells repair germinal cells of seminiferous tubules of busulfan-induced azoospermic rats. J Hum Reprod Sci 2015; 8:103-10. [PMID: 26157302 PMCID: PMC4477447 DOI: 10.4103/0974-1208.158618] [Citation(s) in RCA: 64] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2014] [Revised: 01/15/2015] [Accepted: 05/19/2015] [Indexed: 12/24/2022] Open
Abstract
CONTEXT: Adipose tissue-derived mesenchymal stem cells (AT-MSCs) are less invasive than bone marrow mesenchymal stem cells to obtain for cell therapy. AIMS: The aims of this study were to evaluate the germinal cells characteristics and repairs in seminiferous tubules of busulfan-induced azoospermic rats after AT-MSCs transplantation. SETTINGS AND DESIGN: Experimental case-control study. MATERIALS AND METHODS: In the present experimental study, donors AT-MSCs were isolated from subcutaneous adipose tissue of two Sprague-Dawley rats. The recipients (n = 5) were received two doses of 10 mg/kg of busulfan with 21 days interval to stop endogenous spermatogenesis. After induction of azoospermia by busulfan, rats were injected with the AT-MSCs into the efferent duct of right testes. After 60 days, the right testes were injected AT-MSCs were compared to left azoospermic testes. Five untreated male rats served as negative control. STATISTICAL ANALYSIS USED: Stereological indices were analyzed by one-way ANOVA and LSD post-hoc test. The spermatogenesis index was compared using Mann–Whitney U test. RESULTS: After stereological analyses, the seminiferous tubules treated with AT-MSCs had normal morphology. The untreated seminiferous tubules were empty. Spermatogenesis was observed in most cell-treated seminiferous tubules. CONCLUSIONS: The testis of busulfan-induced azoospermic rats accepted transplanted AT-MSCs. The transplanted AT-MSCs could induce spermatogenesis in seminiferous tubules of the rat.
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Affiliation(s)
- Davood Mehrabani
- Stem Cell and Transgenic Technology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Mohammad Amin Hassanshahi
- Stem Cell and Transgenic Technology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Amin Tamadon
- Stem Cell and Transgenic Technology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Shahrokh Zare
- Stem Cell and Transgenic Technology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Saeideh Keshavarz
- Stem Cell and Transgenic Technology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Farhad Rahmanifar
- Department of Basic Sciences, School of Veterinary Medicine, Shiraz University, Shiraz, Iran
| | - Mehdi Dianatpour
- Stem Cell and Transgenic Technology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran ; Department of Medical Genetics, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Zahra Khodabandeh
- Stem Cell and Transgenic Technology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
| | - ImanRazeghian Jahromi
- Stem Cell and Transgenic Technology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Nader Tanideh
- Stem Cell and Transgenic Technology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Mani Ramzi
- Hematology and Bone Marrow Transplant Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Heydar Aqababa
- Department of Biology, Islamic Azad University, Arsanjan Branch, Arsanjan, Iran
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Luca G, Mancuso F, Calvitti M, Arato I, Falabella G, Bufalari A, De Monte V, Tresoldi E, Nastruzzi C, Basta G, Fallarino F, Lilli C, Bellucci C, Baroni T, Aglietti MC, Giovagnoli S, Cameron DF, Bodo M, Calafiore R. Long-term stability, functional competence, and safety of microencapsulated specific pathogen-free neonatal porcine Sertoli cells: a potential product for cell transplant therapy. Xenotransplantation 2015; 22:273-83. [PMID: 26134468 DOI: 10.1111/xen.12175] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/09/2015] [Accepted: 06/04/2015] [Indexed: 01/13/2023]
Abstract
BACKGROUND Porcine Sertoli cells (pSCs) have been employed for cell therapy in pre-clinical studies for several chronic/immune diseases as they deliver molecules associated with trophic and anti-inflammatory effects. To be employed for human xenografts, pSCs products need to comply with safety and stability. To fulfill such requirements, we employed a microencapsulation technology to increase pre-transplant storage stability of specific pathogen-free pSCs (SPF-pSCs) and evaluated the in vivo long-term viability and safety of grafts. METHODS Specific pathogen free neonatal pigs underwent testis excision under sterility. pSCs were isolated, characterized by immunofluorescence (IF) and cytofluorimetric analysis (CA) and examined in terms of viability and function [namely, production of anti-müllerian hormone (AMH), inhibin B, and transforming growth factor beta-1 (TFGβ-1)]. After microencapsulation in barium alginate microcapsules (Ba-MC), long-term SPF-pSCs (Ba-MCpSCs) viability and barium concentrations were evaluated at 1, 24 throughout 40 h to establish pre-transplant storage conditions. RESULTS The purity of isolated pSCs was about 95% with negligible contaminating cells. Cultured pSCs monolayers, both prior to and after microencapsulation, maintained high function and full viability up to 24 h of storage. At 40 h post-encapsulation, pSCs viability decreased to 80%. Barium concentration in Ba-MCpSCs lagged below the normal maximum daily allowance and was stable for 4 months in mice with no evident side effects. CONCLUSIONS Such results suggest that this protocol for the isolation and microencapsulation of pSCs is compatible with long-haul transportation and that Ba-MCpSCs could be potentially employable for xenotransplantation.
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Affiliation(s)
- Giovanni Luca
- Department of Experimental Medicine, University of Perugia, Perugia, Italy.,Division of Medical Andrology and Endocrinology of Reproduction, Saint Mary Hospital, Terni, Italy
| | - Francesca Mancuso
- Department of Experimental Medicine, University of Perugia, Perugia, Italy
| | - Mario Calvitti
- Department of Experimental Medicine, University of Perugia, Perugia, Italy
| | - Iva Arato
- Department of Experimental Medicine, University of Perugia, Perugia, Italy
| | - Giulia Falabella
- Department of Experimental Medicine, University of Perugia, Perugia, Italy
| | - Antonello Bufalari
- Department of Pathology, Diagnostic and Clinical Veterinary Medicine, University of Perugia, Perugia, Italy
| | - Valentina De Monte
- Department of Pathology, Diagnostic and Clinical Veterinary Medicine, University of Perugia, Perugia, Italy
| | - Enrico Tresoldi
- Experimental Zooprophylactic Institute of Lombardia and Emilia Romagna, Brescia, Italy
| | - Claudio Nastruzzi
- Department of Life Sciences and Biotechnology, University of Ferrara, Ferrara, Italy
| | - Giuseppe Basta
- Department of Medicine, University of Perugia, Perugia, Italy
| | | | - Cinzia Lilli
- Department of Experimental Medicine, University of Perugia, Perugia, Italy
| | - Catia Bellucci
- Department of Experimental Medicine, University of Perugia, Perugia, Italy
| | - Tiziano Baroni
- Department of Experimental Medicine, University of Perugia, Perugia, Italy
| | | | - Stefano Giovagnoli
- Department of Pharmaceutical Sciences, University of Perugia, Perugia, Italy
| | - Don F Cameron
- Department of Internal Medicine, University of South Florida, Tampa, FL, USA
| | - Maria Bodo
- Department of Experimental Medicine, University of Perugia, Perugia, Italy
| | - Riccardo Calafiore
- Division of Medical Andrology and Endocrinology of Reproduction, Saint Mary Hospital, Terni, Italy.,Department of Medicine, University of Perugia, Perugia, Italy
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47
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Shinomura M, Kishi K, Tomita A, Kawasumi M, Kanezashi H, Kuroda Y, Tsunekawa N, Ozawa A, Aiyama Y, Yoneda A, Suzuki H, Saito M, Picard JY, Kohno K, Kurohmaru M, Kanai-Azuma M, Kanai Y. A novel Amh-Treck transgenic mouse line allows toxin-dependent loss of supporting cells in gonads. Reproduction 2014; 148:H1-9. [PMID: 25212783 DOI: 10.1530/rep-14-0171] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
Cell ablation technology is useful for studying specific cell lineages in a developing organ in vivo. Herein, we established a novel anti-Müllerian hormone (AMH)-toxin receptor-mediated cell knockout (Treck) mouse line, in which the diphtheria toxin (DT) receptor was specifically activated in Sertoli and granulosa cells in postnatal testes and ovaries respectively. In the postnatal testes of Amh-Treck transgenic (Tg) male mice, DT injection induced a specific loss of the Sertoli cells in a dose-dependent manner, as well as the specific degeneration of granulosa cells in the primary and secondary follicles caused by DT injection in Tg females. In the testes with depletion of Sertoli cell, germ cells appeared to survive for only several days after DT treatment and rapidly underwent cell degeneration, which led to the accumulation of a large amount of cell debris within the seminiferous tubules by day 10 after DT treatment. Transplantation of exogenous healthy Sertoli cells following DT treatment rescued the germ cell loss in the transplantation sites of the seminiferous epithelia, leading to a partial recovery of the spermatogenesis. These results provide not only in vivo evidence of the crucial role of Sertoli cells in the maintenance of germ cells, but also show that the Amh-Treck Tg line is a useful in vivo model of the function of the supporting cell lineage in developing mammalian gonads.
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Affiliation(s)
- Mai Shinomura
- Department of Veterinary AnatomyThe University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, JapanDepartment of Experimental Animal Model for Human DiseaseCenter for Experimental Animals, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo 113-8510, JapanGraduate School of Biological SciencesNara Institute of Science and Technology, 8916-5, Takayama, Ikoma, Nara 630-0192, JapanINSERM U1133BFA, University Paris VII, 75205 Paris Cedex 13, France
| | - Kasane Kishi
- Department of Veterinary AnatomyThe University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, JapanDepartment of Experimental Animal Model for Human DiseaseCenter for Experimental Animals, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo 113-8510, JapanGraduate School of Biological SciencesNara Institute of Science and Technology, 8916-5, Takayama, Ikoma, Nara 630-0192, JapanINSERM U1133BFA, University Paris VII, 75205 Paris Cedex 13, France
| | - Ayako Tomita
- Department of Veterinary AnatomyThe University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, JapanDepartment of Experimental Animal Model for Human DiseaseCenter for Experimental Animals, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo 113-8510, JapanGraduate School of Biological SciencesNara Institute of Science and Technology, 8916-5, Takayama, Ikoma, Nara 630-0192, JapanINSERM U1133BFA, University Paris VII, 75205 Paris Cedex 13, France
| | - Miyuri Kawasumi
- Department of Veterinary AnatomyThe University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, JapanDepartment of Experimental Animal Model for Human DiseaseCenter for Experimental Animals, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo 113-8510, JapanGraduate School of Biological SciencesNara Institute of Science and Technology, 8916-5, Takayama, Ikoma, Nara 630-0192, JapanINSERM U1133BFA, University Paris VII, 75205 Paris Cedex 13, France
| | - Hiromi Kanezashi
- Department of Veterinary AnatomyThe University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, JapanDepartment of Experimental Animal Model for Human DiseaseCenter for Experimental Animals, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo 113-8510, JapanGraduate School of Biological SciencesNara Institute of Science and Technology, 8916-5, Takayama, Ikoma, Nara 630-0192, JapanINSERM U1133BFA, University Paris VII, 75205 Paris Cedex 13, France
| | - Yoshiko Kuroda
- Department of Veterinary AnatomyThe University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, JapanDepartment of Experimental Animal Model for Human DiseaseCenter for Experimental Animals, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo 113-8510, JapanGraduate School of Biological SciencesNara Institute of Science and Technology, 8916-5, Takayama, Ikoma, Nara 630-0192, JapanINSERM U1133BFA, University Paris VII, 75205 Paris Cedex 13, France
| | - Naoki Tsunekawa
- Department of Veterinary AnatomyThe University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, JapanDepartment of Experimental Animal Model for Human DiseaseCenter for Experimental Animals, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo 113-8510, JapanGraduate School of Biological SciencesNara Institute of Science and Technology, 8916-5, Takayama, Ikoma, Nara 630-0192, JapanINSERM U1133BFA, University Paris VII, 75205 Paris Cedex 13, France
| | - Aisa Ozawa
- Department of Veterinary AnatomyThe University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, JapanDepartment of Experimental Animal Model for Human DiseaseCenter for Experimental Animals, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo 113-8510, JapanGraduate School of Biological SciencesNara Institute of Science and Technology, 8916-5, Takayama, Ikoma, Nara 630-0192, JapanINSERM U1133BFA, University Paris VII, 75205 Paris Cedex 13, France
| | - Yoshimi Aiyama
- Department of Veterinary AnatomyThe University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, JapanDepartment of Experimental Animal Model for Human DiseaseCenter for Experimental Animals, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo 113-8510, JapanGraduate School of Biological SciencesNara Institute of Science and Technology, 8916-5, Takayama, Ikoma, Nara 630-0192, JapanINSERM U1133BFA, University Paris VII, 75205 Paris Cedex 13, France
| | - Asuka Yoneda
- Department of Veterinary AnatomyThe University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, JapanDepartment of Experimental Animal Model for Human DiseaseCenter for Experimental Animals, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo 113-8510, JapanGraduate School of Biological SciencesNara Institute of Science and Technology, 8916-5, Takayama, Ikoma, Nara 630-0192, JapanINSERM U1133BFA, University Paris VII, 75205 Paris Cedex 13, France
| | - Hitomi Suzuki
- Department of Veterinary AnatomyThe University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, JapanDepartment of Experimental Animal Model for Human DiseaseCenter for Experimental Animals, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo 113-8510, JapanGraduate School of Biological SciencesNara Institute of Science and Technology, 8916-5, Takayama, Ikoma, Nara 630-0192, JapanINSERM U1133BFA, University Paris VII, 75205 Paris Cedex 13, France
| | - Michiko Saito
- Department of Veterinary AnatomyThe University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, JapanDepartment of Experimental Animal Model for Human DiseaseCenter for Experimental Animals, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo 113-8510, JapanGraduate School of Biological SciencesNara Institute of Science and Technology, 8916-5, Takayama, Ikoma, Nara 630-0192, JapanINSERM U1133BFA, University Paris VII, 75205 Paris Cedex 13, France
| | - Jean-Yves Picard
- Department of Veterinary AnatomyThe University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, JapanDepartment of Experimental Animal Model for Human DiseaseCenter for Experimental Animals, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo 113-8510, JapanGraduate School of Biological SciencesNara Institute of Science and Technology, 8916-5, Takayama, Ikoma, Nara 630-0192, JapanINSERM U1133BFA, University Paris VII, 75205 Paris Cedex 13, France
| | - Kenji Kohno
- Department of Veterinary AnatomyThe University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, JapanDepartment of Experimental Animal Model for Human DiseaseCenter for Experimental Animals, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo 113-8510, JapanGraduate School of Biological SciencesNara Institute of Science and Technology, 8916-5, Takayama, Ikoma, Nara 630-0192, JapanINSERM U1133BFA, University Paris VII, 75205 Paris Cedex 13, France
| | - Masamichi Kurohmaru
- Department of Veterinary AnatomyThe University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, JapanDepartment of Experimental Animal Model for Human DiseaseCenter for Experimental Animals, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo 113-8510, JapanGraduate School of Biological SciencesNara Institute of Science and Technology, 8916-5, Takayama, Ikoma, Nara 630-0192, JapanINSERM U1133BFA, University Paris VII, 75205 Paris Cedex 13, France
| | - Masami Kanai-Azuma
- Department of Veterinary AnatomyThe University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, JapanDepartment of Experimental Animal Model for Human DiseaseCenter for Experimental Animals, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo 113-8510, JapanGraduate School of Biological SciencesNara Institute of Science and Technology, 8916-5, Takayama, Ikoma, Nara 630-0192, JapanINSERM U1133BFA, University Paris VII, 75205 Paris Cedex 13, France
| | - Yoshiakira Kanai
- Department of Veterinary AnatomyThe University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, JapanDepartment of Experimental Animal Model for Human DiseaseCenter for Experimental Animals, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo 113-8510, JapanGraduate School of Biological SciencesNara Institute of Science and Technology, 8916-5, Takayama, Ikoma, Nara 630-0192, JapanINSERM U1133BFA, University Paris VII, 75205 Paris Cedex 13, France
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Oliveira PF, Martins AD, Moreira AC, Cheng CY, Alves MG. The Warburg effect revisited--lesson from the Sertoli cell. Med Res Rev 2014; 35:126-51. [PMID: 25043918 DOI: 10.1002/med.21325] [Citation(s) in RCA: 129] [Impact Index Per Article: 11.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Otto Warburg observed that cancerous cells prefer fermentative instead of oxidative metabolism of glucose, although the former is in theory less efficient. Since Warburg's pioneering works, special attention has been given to this difference in cell metabolism. The Warburg effect has been implicated in cell transformation, immortalization, and proliferation during tumorigenesis. Cancer cells display enhanced glycolytic activity, which is correlated with high proliferation, and thus, glycolysis appears to be an excellent candidate to target cancer cells. Nevertheless, little attention has been given to noncancerous cells that exhibit a "Warburg-like" metabolism with slight, but perhaps crucial, alterations that may provide new directions to develop new and effective anticancer therapies. Within the testis, the somatic Sertoli cell (SC) presents several common metabolic features analogous to cancer cells, and a clear "Warburg-like" metabolism. Nevertheless, SCs actively proliferate only during a specific time period, ceasing to divide in most species after puberty, when they become terminally differentiated. The special metabolic features of SC, as well as progression from the immature but proliferative state, to the mature nonproliferative state, where a high glycolytic activity is maintained, make these cells unique and a good model to discuss new perspectives on the Warburg effect. Herein we provide new insight on how the somatic SC may be a source of new and exciting information concerning the Warburg effect and cell proliferation.
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Affiliation(s)
- Pedro F Oliveira
- CICS-UBI, Health Sciences Research Centre, University of Beira Interior, Covilhã, Portugal
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50
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Starvation is more efficient than the washing technique for purification of rat Sertoli cells. In Vitro Cell Dev Biol Anim 2014; 50:723-30. [PMID: 24789729 DOI: 10.1007/s11626-014-9762-1] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/23/2014] [Accepted: 04/09/2014] [Indexed: 12/16/2022]
Abstract
Sertoli cells (SCs), one of the most important components of seminiferous tubules, are vital for normal spermatogenesis and male fertility. In recent years, numerous in vitro studies have shown the potential and actual activities of SCs. However, pure SCs are necessary for various in vitro studies. In this study, we have evaluated the efficiency of the starvation method for SC purification as compared with the washing method. Seminiferous tubule-derived cells (STDCs) of rats' testes underwent two different techniques for SC purification. In the first group (washing group), the medium was changed every 3-4 d, and cells were washed twice with phosphate-buffered saline that lacked CaC12 and MgSO4 (PBS(-)) before the addition of fresh medium. In the second group (starvation), the medium was changed every 7-8 d. Primary culture (P0), passage 1 (P1), and passage 2 (P2) cells were analyzed for the expression of SC-specific genes, vimentin, Wilm's tumor 1 (WT1), germ cell gene (vasa), Leydig cell marker, 17beta-hydroxysteroid dehydrogenase type 3 (Hsd17b3), and a marker of peritubular myoid cells, alpha smooth muscle actin (αSma), by reverse transcriptase polymerase chain reaction (RT-PCR) and real-time RT-PCR. Gene expression analysis showed that P0 cells expressed all tested genes except Hsd17b3. The starvation method caused significant downregulation of vasa and αSma expression in P0, P1, and P2 cells, whereas vimentin and WT1 were upregulated. In contrast, the washing method was less effective than the starvation method for the removal of germ and pretubular myoid cells (p < 0.001). Totally, the results have revealed that although washing is the only common technique for elimination of contaminant cells in SC cultures, starvation has a stronger effect and is a suitable, affordable technique for SC purification. We propose that starvation is an efficient, inexpensive method that can be used for purification of SCs in animal species.
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