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LeBlanc DPM, Zhou G, Williams A, Meier MJ, Valentine CC, Salk JJ, Yauk CL, Marchetti F. Duplex sequencing identifies unique characteristics of ENU-induced mutations in male mouse germ cells†. Biol Reprod 2025; 112:1015-1027. [PMID: 39936590 PMCID: PMC12078080 DOI: 10.1093/biolre/ioaf029] [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] [Subscribe] [Scholar Register] [Received: 10/15/2024] [Revised: 01/21/2025] [Accepted: 02/11/2025] [Indexed: 02/13/2025] Open
Abstract
Germ cell mutagenicity testing is increasingly required for chemical risk assessment. Duplex sequencing is rapidly gaining acceptance as a method to assess in vivo mutagenesis, and as a valid alternative to transgenic rodent mutation models such as the MutaMouse. We used a duplex sequencing panel of 20 genomic targets and the transgenic rodent assay to measure mutations in the germ cells of MutaMouse males exposed to 0, 1, 2, or 5 mg/kg N-ethyl-N-nitrosourea for 28 days. Germ cells from the seminiferous tubules were collected 28 days post-exposure. The transgenic rodent assay showed a significant increase in mutant frequencies at the high (P < 0.001) and medium (P = 0.01) N-ethyl-N-nitrosourea doses relative to controls, while duplex sequencing revealed a significant increase (P < 0.001) in N-ethyl-N-nitrosourea-induced mutations only at the high dose. Duplex sequencing mutation frequencies were lower in genic than in intergenic targets, suggesting a protective role for transcription-coupled repair. Interestingly, we observed several unique germ cell characteristics with respect to duplex sequencing data from rodent somatic tissues: 1) larger inter-animal variability in clonally expanded mutations that affects the ability to detect significant increases in mutation frequency; 2) a target on chromosome 2 showing much higher susceptibility to spontaneous and chemical-induced mutagenesis than other targets; and 3) a mutation spectrum consistent with that observed in the offspring of N-ethyl-N-nitrosourea-treated males but not with the spectrum in bone marrow of directly-exposed males. These results suggest that duplex sequencing is a promising approach for characterizing germ cell mutagenesis and that mutagenic mechanisms operating in germ cells differ from those in somatic tissues.
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Affiliation(s)
- Danielle P M LeBlanc
- Environmental Health Science and Research Bureau, Health Canada, Ottawa, Ontario, Canada
| | - Gu Zhou
- Environmental Health Science and Research Bureau, Health Canada, Ottawa, Ontario, Canada
| | - Andrew Williams
- Environmental Health Science and Research Bureau, Health Canada, Ottawa, Ontario, Canada
| | - Matthew J Meier
- Environmental Health Science and Research Bureau, Health Canada, Ottawa, Ontario, Canada
| | | | - Jesse J Salk
- Division of Hematology and Oncology, University of Washington School of Medicine, Seattle, Washington, USA
| | - Carole L Yauk
- Department of Biology, University of Ottawa, Ottawa, Ontario, Canada
| | - Francesco Marchetti
- Environmental Health Science and Research Bureau, Health Canada, Ottawa, Ontario, Canada
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Bashghareh A, Rastegar T, Modarresi P, Kazemzadeh S, Salem M, Hedayatpour A. Recovering Spermatogenesis By Protected Cryopreservation Using Metformin and Transplanting Spermatogonial Stem Cells Into Testis in an Azoospermia Mouse Model. Biopreserv Biobank 2024; 22:68-81. [PMID: 37582284 DOI: 10.1089/bio.2022.0178] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 08/17/2023] Open
Abstract
Cryopreservation and transplantation of spermatogonial stem cells (SSCs) may serve as a new method to restore male fertility in patients undergoing chemotherapy or radiotherapy. However, SSCs may be damaged during cryopreservation due to the production of reactive oxygen species (ROS). Therefore, different antioxidants have been used as protective agents. Studies have shown that metformin (MET) has antioxidant activity. The aim of this study was to assess the antioxidant and antiapoptotic effects of MET in frozen-thawed SSCs. In addition, the effect of MET on the proliferation and differentiation of SSCs was evaluated. To this end, SSCs were isolated from mouse pups aged 3-6 days old, cultured, identified by flow cytometry (ID4, INTEGRIN β1+), and finally evaluated for survival and ROS rate. SSCs were transplanted after busulfan and cadmium treatment. Cryopreserved SSCs with and without MET were transplanted after 1 month of cryopreservation. Eight weeks after transplantation, the recipient testes were evaluated for the expression of apoptosis (BAX, BCL2), proliferation (PLZF), and differentiation (SCP3, TP1, TP2, PRM1) markers using immunohistochemistry, Western blot, and quantitative real-time polymerase chain reaction. The findings revealed that the survival rate of SSCs was higher in the 500 μm/mL MET group compared to the other groups (50 and 5000 μm/mL). MET significantly decreased the intracellular ROS production. Transplantation of SSCs increased the expression level of proliferation (PLZF) and differentiation (SCP3, TP1, TP2, PRM1) markers compared to azoospermia group, and their levels were significantly higher in the MET group compared to the cryopreservation group containing basic freezing medium (p < 0.05). MET increased the survival rate of SSCs, proliferation, and differentiation and decreased the ROS production and the apoptosis rate. Cryopreservation by MET seems to be effective in treating infertility.
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Affiliation(s)
- Alieh Bashghareh
- Department of Anatomy, School of Medicine, Tehran University of Medical Science, Tehran, Iran
| | - Tayebeh Rastegar
- Department of Anatomy, School of Medicine, Tehran University of Medical Science, Tehran, Iran
| | - Peyman Modarresi
- Department of Veterinary Medicine, Faculty of Veterinary Medicine, Islamic Azad University, Shabestar, Iran
| | - Shokoofeh Kazemzadeh
- Department of Anatomy, Faculty of Medicine, Shoushtar University of Medical Sciences, Shoushtar, Iran
| | - Maryam Salem
- Department of Anatomy, School of Medicine, Tehran University of Medical Science, Tehran, Iran
| | - Azim Hedayatpour
- Department of Anatomy, School of Medicine, Tehran University of Medical Science, Tehran, Iran
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Aponte PM, Gutierrez-Reinoso MA, Garcia-Herreros M. Bridging the Gap: Animal Models in Next-Generation Reproductive Technologies for Male Fertility Preservation. Life (Basel) 2023; 14:17. [PMID: 38276265 PMCID: PMC10820126 DOI: 10.3390/life14010017] [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: 09/05/2023] [Revised: 12/12/2023] [Accepted: 12/19/2023] [Indexed: 01/27/2024] Open
Abstract
This review aims to explore advanced reproductive technologies for male fertility preservation, underscoring the essential role that animal models have played in shaping these techniques through historical contexts and into modern applications. Rising infertility concerns have become more prevalent in human populations recently. The surge in male fertility issues has prompted advanced reproductive technologies, with animal models playing a pivotal role in their evolution. Historically, animal models have aided our understanding in the field, from early reproductive basic research to developing techniques like artificial insemination, multiple ovulation, and in vitro fertilization. The contemporary landscape of male fertility preservation encompasses techniques such as sperm cryopreservation, testicular sperm extraction, and intracytoplasmic sperm injection, among others. The relevance of animal models will undoubtedly bridge the gap between traditional methods and revolutionary next-generation reproductive techniques, fortifying our collective efforts in enhancing male fertility preservation strategies. While we possess extensive knowledge about spermatogenesis and its regulation, largely thanks to insights from animal models that paved the way for human infertility treatments, a pressing need remains to further understand specific infertility issues unique to humans. The primary aim of this review is to provide a comprehensive analysis of how animal models have influenced the development and refinement of advanced reproductive technologies for male fertility preservation, and to assess their future potential in bridging the gap between current practices and cutting-edge fertility techniques, particularly in addressing unique human male factor infertility.
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Affiliation(s)
- Pedro M. Aponte
- Colegio de Ciencias Biológicas y Ambientales (COCIBA), Universidad San Francisco de Quito (USFQ), Quito 170901, Ecuador
- Instituto de Investigaciones en Biomedicina “One-Health”, Universidad San Francisco de Quito (USFQ), Campus Cumbayá, Quito 170901, Ecuador
| | - Miguel A. Gutierrez-Reinoso
- Facultad de Ciencias Agropecuarias y Recursos Naturales, Carrera de Medicina Veterinaria, Universidad Técnica de Cotopaxi (UTC), Latacunga 050150, Ecuador;
- Laboratorio de Biotecnología Animal, Departamento de Ciencia Animal, Facultad de Ciencias Veterinarias, Universidad de Concepción (UdeC), Chillán 3780000, Chile
| | - Manuel Garcia-Herreros
- Instituto Nacional de Investigação Agrária e Veterinária (INIAV), 2005-048 Santarém, Portugal
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Shakeel M, Yoon M. Effects of insulin-like growth factor-1 on the proliferation and apoptosis of stallion testicular cells under normal and heat stress culture conditions. Anim Reprod Sci 2023; 256:107319. [PMID: 37633109 DOI: 10.1016/j.anireprosci.2023.107319] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/23/2023] [Revised: 08/13/2023] [Accepted: 08/14/2023] [Indexed: 08/28/2023]
Abstract
This study investigated the effect of heat stress on stallion testicular cells (TCs) and the effect of insulin-like growth factor (IGF)-1 on TC viability, proliferation, and apoptosis, including different stages of germ cells. TCs were divided into control or treatment groups with 0.01, 0.1, 1, 10, and 100 ng/mL of recombinant human IGF-1 (rhIGF-1) for 24 h at 34 °C and 37 °C. The population and viability were measured before and after treatment. The effects of rhIGF-1 on TC viability, proliferation, and apoptosis were determined using RT-qPCR. Proliferating cell nuclear antigen (PCNA) and marker of proliferation Ki-67 (MKI-67) were used as proliferation markers. Myeloid leukemia-1 (MCL-1) was used as an antiapoptotic marker. BCL2 antagonist/killer-1 (BAK-1) was used as a proapoptotic marker. The relative abundance of mRNA transcript of undifferentiated cell transcription factor 1 (UTF-1), protein gene product 9.5 (PGP9.5), and deleted in azoospermia-like (DAZL), was measured for spermatogenesis progression. TCs treated with 1 ng/mL rhIGF-1 at 34 °C exhibited the highest viability. Significant upregulation of the relative abundance of mRNA transcript of PCNA, MKI-67, and MCL-1 was observed in treated TCs compared with untreated TCs; however, BAK-1 was significantly downregulated in treated TCs. Germ cells treated with 1 ng/mL rhIGF-1 exhibited the highest relative abundance of mRNA transcript of UTF-1 and DAZL, whereas TCs exposed to 0.1 ng/mL showed the highest PGP9.5 level. These data confirm that heat stress in stallions decreases TC viability. These findings may help identify a basal IGF-1 level for TC proliferation and apoptosis during heat stress-induced testicular degeneration in stallions.
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Affiliation(s)
- Muhammad Shakeel
- Department of Animal Science and Biotechnology, Kyungpook National University, Sangju 37224, Republic of Korea; Department of Clinical Studies, Faculty of Veterinary and Animal Sciences, Pir Mehr Ali Shah, Arid Agriculture University, Rawalpindi 44000, Pakistan
| | - Minjung Yoon
- Department of Animal Science and Biotechnology, Kyungpook National University, Sangju 37224, Republic of Korea; Department of Horse, Companion, and Wild Animal Science, Kyungpook National University, Sangju 37224, Republic of Korea; Research Centre for Horse Industry, Kyungpook National University, Sangju 37224, Republic of Korea.
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Jorge AS, Recchia K, Glória MH, de Souza AF, Pessôa LVDF, Fantinato Neto P, Martins DDS, de Andrade AFC, Martins SMMK, Bressan FF, Pieri NCG. Porcine Germ Cells Phenotype during Embryonic and Adult Development. Animals (Basel) 2023; 13:2520. [PMID: 37570330 PMCID: PMC10417053 DOI: 10.3390/ani13152520] [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: 06/12/2023] [Revised: 07/31/2023] [Accepted: 08/01/2023] [Indexed: 08/13/2023] Open
Abstract
Primordial germ cells (PGCs) are the precursors of gametes. Due to their importance for the formation and reproduction of an organism, understanding the mechanisms and pathways of PGCs and the differences between males and females is essential. However, there is little research in domestic animals, e.g., swine, regarding the epigenetic and pluripotency profiles of PGCs during development. This study analyzed the expression of epigenetic and various pluripotent and germline markers associated with the development and differentiation of PGCs in porcine (pPGCs), aiming to understand the different gene expression profiles between the genders. The analysis of gonads at different gestational periods (from 24 to 35 days post fertilization (dpf) and in adults) was evaluated by immunofluorescence and RT-qPCR and showed phenotypic differences between the gonads of male and female embryos. In addition, the pPGCs were positive for OCT4 and VASA; some cells were H3k27me3 positive in male embryos and adult testes. In adults, the cells of the testes were positive for germline markers, as confirmed by gene expression analysis. The results may contribute to understanding the pPGC pathways during reproductive development, while also contributing to the knowledge needed to generate mature gametes in vitro.
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Affiliation(s)
- Amanda Soares Jorge
- Department of Veterinary Medicine, School of Animal Sciences and Food Engineering, University of Sao Paulo, Pirassununga 13635-900, SP, Brazil
| | - Kaiana Recchia
- Department of Surgery, Faculty of Veterinary Medicine and Animal Sciences, University of Sao Paulo, São Paulo 01001-010, SP, Brazil
| | - Mayra Hirakawa Glória
- Department of Veterinary Medicine, School of Animal Sciences and Food Engineering, University of Sao Paulo, Pirassununga 13635-900, SP, Brazil
| | - Aline Fernanda de Souza
- Department Biomedical Science, Ontario Veterinary College (OVC), University of Guelph, Guelph, ON N1G 2W1, Canada
| | - Laís Vicari de Figueirêdo Pessôa
- Department of Veterinary Medicine, School of Animal Sciences and Food Engineering, University of Sao Paulo, Pirassununga 13635-900, SP, Brazil
| | - Paulo Fantinato Neto
- Department of Veterinary Medicine, School of Animal Sciences and Food Engineering, University of Sao Paulo, Pirassununga 13635-900, SP, Brazil
| | - Daniele Dos Santos Martins
- Department of Veterinary Medicine, School of Animal Sciences and Food Engineering, University of Sao Paulo, Pirassununga 13635-900, SP, Brazil
| | - André Furugen Cesar de Andrade
- Department of Animal Reproduction, Faculty of Veterinary Medicine and Animal Sciences, University of Sao Paulo, Pirassununga 13635-900, SP, Brazil
| | | | - Fabiana Fernandes Bressan
- Department of Veterinary Medicine, School of Animal Sciences and Food Engineering, University of Sao Paulo, Pirassununga 13635-900, SP, Brazil
- Department of Surgery, Faculty of Veterinary Medicine and Animal Sciences, University of Sao Paulo, São Paulo 01001-010, SP, Brazil
| | - Naira Caroline Godoy Pieri
- Department of Veterinary Medicine, School of Animal Sciences and Food Engineering, University of Sao Paulo, Pirassununga 13635-900, SP, Brazil
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Rahbar M, Asadpour R, Azami M, Mazaheri Z, Hamali H. Improving the process of spermatogenesis in azoospermic mice using spermatogonial stem cells co-cultured with epididymosomes in three-dimensional culture system. Life Sci 2022; 310:121057. [DOI: 10.1016/j.lfs.2022.121057] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/04/2022] [Revised: 09/28/2022] [Accepted: 10/06/2022] [Indexed: 11/09/2022]
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A Comparative Cross-Platform Analysis to Identify Potential Biomarker Genes for Evaluation of Teratozoospermia and Azoospermia. Genes (Basel) 2022; 13:genes13101721. [PMID: 36292606 PMCID: PMC9602071 DOI: 10.3390/genes13101721] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/19/2022] [Revised: 09/12/2022] [Accepted: 09/19/2022] [Indexed: 11/24/2022] Open
Abstract
Male infertility is a global public health concern. Teratozoospermia is a qualitative anomaly of spermatozoa morphology, contributing significantly to male infertility, whereas azoospermia is the complete absence of spermatozoa in the ejaculate. Thus, there is a serious need for unveiling the common origin and/or connection between both of these diseases, if any. This study aims to identify common potential biomarker genes of these two diseases via an in silico approach using a meta-analysis of microarray data. In this study, a differential expression analysis of genes was performed on four publicly available RNA microarray datasets, two each from teratozoospermia (GSE6872 and GSE6967) and azoospermia (GSE145467 and GSE25518). From the analysis, 118 DEGs were found to be common to teratozoospermia and azoospermia, and, interestingly, sperm autoantigenic protein 17 (SPA17) was found to possess the highest fold change value among all the DEGs (9.471), while coiled-coil domain-containing 90B (CCDC90B) and coiled-coil domain-containing 91 (CCDC91) genes were found to be common among three of analyses, i.e., Network Analyst, ExAtlas, and GEO2R. This observation indicates that SPA17, CCDC90B, and CCDC91 genes might have significant roles to play as potential biomarkers for teratozoospermia and azoospermia. Thus, our study opens a new window of research in this area and can provide an important theoretical basis for the diagnosis and treatment of both these diseases.
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Hasani Fard AH, Kamalipour F, Mazaheri Z, Hosseini SJ. The Effect of miR-106b-5p Expression in The Production of iPS-Like Cells from Mice SSCs during The Formation of Teratoma and The Three Embryonic Layers. CELL JOURNAL 2022; 24:442-448. [PMID: 36093803 PMCID: PMC9468720 DOI: 10.22074/cellj.2022.8147] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 07/24/2021] [Indexed: 11/24/2022]
Abstract
<strong>Objective:</strong> According to the mounting data, microRNAs (miRNAs) may play a key role in reprogramming. miR-106b<br />is considered as an enhancer in reprogramming efficiency. Based on induced pluripotent stem cells (iPSCs), cell treatments have a huge amount of potential. One of the main concerns about using iPSCs in therapeutic settings is the possibility of tumor formation. It is hypothesized that a procedure that can reprogram cells with less genetic manipulation reduces the possibility of tumorigenicity.<br /><strong>Materials and Methods:</strong> In this experimental study, miR-106b-5p transduced by pLV-miRNA vector into mice isolated spermatogonial stem cells (SSCs) to achieve iPS-like cells. Then the transduced cells were cultured in specific conditions to study the formation of three germ layers. The tumorigenicity of these iPS-like cells was investigated by transplantation into male BALB/C mice.<br /><strong>Results:</strong> We show that SSCs can be successfully reprogrammed into induced iPS-like cells by pLV-miRNA vector to transduce the hsa-mir-106b-5p into SSCs and generating osteogenic, neural and hepatoblast lineage cells in vitro as a result of pluripotency. Although these iPS-like cells are pluripotent, they cannot form palpable tumors in vivo.<br /><strong>Conclusion:</strong> These results demonstrate that infection of hsa-mir-106b-5p into SSCs can reprogram them into iPSCs<br />and advanced germ cell lineages without tumorigenicity. Also, a novel approach for studying the generation of iPSCs<br />and the application of iPS or iPS-like cells in regenerative medicine is presented.
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Affiliation(s)
- Amir Hossein Hasani Fard
- Men’s Health and Reproductive Health Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran
| | - Fatemeh Kamalipour
- Men’s Health and Reproductive Health Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran
| | - Zohreh Mazaheri
- Department of Anatomical Sciences, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
| | - Seyed Jalil Hosseini
- Men’s Health and Reproductive Health Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran,P.O.Box: 19899/34148Men’s Health and Reproductive Health Research CenterShahid Beheshti University of
Medical SciencesTehranIran
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Hasani Fard AH, Valizadeh M, Mazaheri Z, Hosseini SJ. MiR-106b-5p Regulates the Reprogramming of Spermatogonial Stem Cells into iPSC (Induced Pluripotent Stem Cell)-Like Cells. IRANIAN BIOMEDICAL JOURNAL 2022; 26:291-300. [PMID: 35791490 PMCID: PMC9432470 DOI: 10.52547/ibj.3594] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 10/03/2021] [Accepted: 01/05/2022] [Indexed: 11/18/2022]
Abstract
Background Recent years have brought notable progress in raising the efficiency of the reprogramming technique so that approaches have evolved from known transgenic factors to only a few miRNAs. Nevertheless, there is a poor understanding of both the key factors and biological networks underlying this reprogramming. The present study aimed to investigate the potential of miR-106b-5p in regulating spermatogonial stem cells (SSCs) to induced pluripotent stem cell (iPSC)-like cells. Methods We used SSCs because pluripotency is inducible in SSCs under defined culture conditions, and they have a few issues compared to other adult stem cells. As both signaling and post-transcriptional gene controls are critical for pluripotency regulation, we traced the expression of Oct-4, Sox-2, Klf-4, c-Myc, and Nanog (OSKMN). Besides, we considered miR-106b-5p targets using bioinformatic methods. Results Our results showed that transfected SSCs with miR-106b-5p increased the expression of the OSKMN factors, which was significantly more than negative control groups. Moreover, using the functional miRNA enrichment analysis, online tools, and databases, we predicted that miR-106b-5p targeted a signaling pathway gene named MAPK1/ERK2, related to regulating stem cell pluripotency. Conclusion Together, our data suggest that miR-106b-5p regulates the reprogramming of SSCs into iPSC-like cells. Furthermore, noteworthy progress in the in vitro development of SSCs indicates promise reservoirs and opportunities for future clinical trials.
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Affiliation(s)
- Amir Hossein Hasani Fard
- Men’s Health and Reproductive Health Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran
| | - Mahmoud Valizadeh
- Men’s Health and Reproductive Health Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran
| | - Zohreh Mazaheri
- Department of Anatomical Sciences, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
| | - Seyed Jalil Hosseini
- Men’s Health and Reproductive Health Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran
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Thiageswaran S, Steele H, Voigt AL, Dobrinski I. A Role for Exchange of Extracellular Vesicles in Porcine Spermatogonial Co-Culture. Int J Mol Sci 2022; 23:ijms23094535. [PMID: 35562927 PMCID: PMC9103065 DOI: 10.3390/ijms23094535] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/24/2022] [Revised: 04/14/2022] [Accepted: 04/16/2022] [Indexed: 12/22/2022] Open
Abstract
Spermatogonial stem cells (SSCs) provide the basis for lifelong male fertility through self-renewal and differentiation. Prepubertal male cancer patients may be rendered infertile by gonadotoxic chemotherapy and, unlike sexually mature men, cannot store sperm. Alternatively, testicular biopsies taken prior to treatment may be used to restore fertility in adulthood. Testicular SSC populations are limited, and in vitro culture systems are required to increase numbers of SSCs for treatment, demanding culture systems for SSC propagation. Using the pig as a non-rodent model, we developed culture systems to expand spermatogonia from immature testis tissue, comparing different feeders (Sertoli cells, peritubular myoid cells (PMCs) and pig fetal fibroblasts (PFFs)). Spermatogonia co-cultured with Sertoli cells, PMCs and PFFs had comparable rates of proliferation and apoptosis. To elucidate the mechanism behind the beneficial nature of feeder layers, we investigated the role of extracellular vesicles in crosstalk between spermatogonia and feeder cells. Sertoli cell-released exosomes are incorporated by spermatogonia, and inhibition of exosomal release reduces spermatogonial proliferation. Together, these results show that PMCs, PFFs and Sertoli cells promote spermatogonial proliferation in co-culture, with exosomal exchange representing one possible mechanism. Further characterization of exosomal cargo may ultimately allow the development of feeder-free culture systems for clinical use.
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Affiliation(s)
- Shiama Thiageswaran
- Department of Biochemistry and Molecular Biology, Cumming School of Medicine, University of Calgary, Calgary, AB T2N 4N1, Canada;
| | - Heather Steele
- Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Calgary, AB T2N 1N4, Canada; (H.S.); (A.L.V.)
| | - Anna Laura Voigt
- Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Calgary, AB T2N 1N4, Canada; (H.S.); (A.L.V.)
| | - Ina Dobrinski
- Department of Biochemistry and Molecular Biology, Cumming School of Medicine, University of Calgary, Calgary, AB T2N 4N1, Canada;
- Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Calgary, AB T2N 1N4, Canada; (H.S.); (A.L.V.)
- Correspondence: ; Tel.: +1-403-210-6532
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11
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Diaz EA, Donoso G, Saenz C, Aponte PM. Spermatogenesis in a vulnerable South American cervid, dwarf red brocket (Mazama rufina). Anat Histol Embryol 2021; 51:91-102. [PMID: 34820886 DOI: 10.1111/ahe.12766] [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: 08/23/2021] [Revised: 10/12/2021] [Accepted: 11/15/2021] [Indexed: 11/29/2022]
Abstract
The brocket deer (Genus Mazama) is a highly diverse cervid group distributed from Mexico to Argentina, with a downward population trend. However, literature on the basic reproductive biology of the genus is scarce. This work aimed to study biometric, histological and stereological aspects of the testes of Dwarf Red Brocket (Mazama rufina). Testes from free-ranging adult brockets (n = 3) were retrieved from necropsies. Testes were histologically processed. From histological images, several stereological parameters were estimated, and seminiferous epithelium cycle morphology was described. Testes volumes were between 8.2 and 18.4 ml and weights from 8.3 to 19.4 g. Gonadosomatic index (% paired-testes weight to body weight) went from 0.17 to 0.64. The tubular cross-sectional diameter was 179.8 ± 2.8 µm. Estimated volume densities for parenchyma and interstitium were 78.8% and 21.2% respectively. There were (in millions/ml) 96.0 ± 13.1 germ cells and 37.7 ± 6.0 somatic cells. Specific cell densities were (all expressed in millions/ml) as follows: spermatogonia 13.1 ± 4.2; primary spermatocytes 43.1 ± 5.0; round spermatids 36.8 ± 8.0 (lower density near the caudal pole, p < 0.01); sustentacular (Sertoli) cells 16.8 ± 4.1 and interstitial endocrine (Leydig) cells 17.4 ± 3.4. Sertoli cell index (germ cells per Sertoli cell) was 6.72. Eight stages of the cycle were described, and frequencies estimated, resembling those of goats. M. rufina adult testis anatomy is similar to that of other cervids and domestic ruminants, with an apparently lower spermatogenic efficiency. This work is a first approximation to the physiology of the testis of M. rufina. Basic knowledge of the reproductive physiology of vulnerable species may allow biotechnological approaches for the restitution of animal populations.
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Affiliation(s)
- Eduardo A Diaz
- Escuela de Medicina Veterinaria, Colegio de Ciencias de la Salud, Universidad San Francisco de Quito (USFQ), Quito, Ecuador.,Hospital de Fauna Silvestre Tueri, Instituto iBIOTROP, Universidad San Francisco de Quito (USFQ), Quito, Ecuador
| | - Gustavo Donoso
- Hospital de Fauna Silvestre Tueri, Instituto iBIOTROP, Universidad San Francisco de Quito (USFQ), Quito, Ecuador
| | - Carolina Saenz
- Hospital de Fauna Silvestre Tueri, Instituto iBIOTROP, Universidad San Francisco de Quito (USFQ), Quito, Ecuador
| | - Pedro M Aponte
- Escuela de Medicina Veterinaria, Colegio de Ciencias de la Salud, Universidad San Francisco de Quito (USFQ), Quito, Ecuador.,Colegio de Ciencias Biológicas y Ambientales, Universidad San Francisco de Quito (USFQ), Quito, Ecuador.,Instituto de Investigaciones en Biomedicina iBIOMED, Universidad San Francisco de Quito (USFQ), Quito, Ecuador
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Binsila B, Selvaraju S, Ranjithkumaran R, Archana SS, Krishnappa B, Ghosh SK, Kumar H, Subbarao RB, Arangasamy A, Bhatta R. Current scenario and challenges ahead in application of spermatogonial stem cell technology in livestock. J Assist Reprod Genet 2021; 38:3155-3173. [PMID: 34661801 DOI: 10.1007/s10815-021-02334-7] [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: 01/06/2021] [Accepted: 09/27/2021] [Indexed: 11/28/2022] Open
Abstract
PURPOSE Spermatogonial stem cells (SSCs) are the source for the mature male gamete. SSC technology in humans is mainly focusing on preserving fertility in cancer patients. Whereas in livestock, it is used for mining the factors associated with male fertility. The review discusses the present status of SSC biology, methodologies developed for in vitro culture, and challenges ahead in establishing SSC technology for the propagation of superior germplasm with special reference to livestock. METHOD Published literatures from PubMed and Google Scholar on topics of SSCs isolation, purification, characterization, short and long-term culture of SSCs, stemness maintenance, epigenetic modifications of SSCs, growth factors, and SSC cryopreservation and transplantation were used for the study. RESULT The fine-tuning of SSC isolation and culture conditions with special reference to feeder cells, growth factors, and additives need to be refined for livestock. An insight into the molecular mechanisms involved in maintaining stemness and proliferation of SSCs could facilitate the dissemination of superior germplasm through transplantation and transgenesis. The epigenetic influence on the composition and expression of the biomolecules during in vitro differentiation of cultured cells is essential for sustaining fertility. The development of surrogate males through gene-editing will be historic achievement for the foothold of the SSCs technology. CONCLUSION Detailed studies on the species-specific factors regulating the stemness and differentiation of the SSCs are required for the development of a long-term culture system and in vitro spermatogenesis in livestock. Epigenetic changes in the SSCs during in vitro culture have to be elucidated for the successful application of SSCs for improving the productivity of the animals.
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Affiliation(s)
- Balakrishnan Binsila
- Reproductive Physiology Laboratory, Animal Physiology Division, Indian Council of Agricultural Research-National Institute of Animal Nutrition and Physiology, Bengaluru, 560 030, India.
| | - Sellappan Selvaraju
- Reproductive Physiology Laboratory, Animal Physiology Division, Indian Council of Agricultural Research-National Institute of Animal Nutrition and Physiology, Bengaluru, 560 030, India
| | - Rajan Ranjithkumaran
- Reproductive Physiology Laboratory, Animal Physiology Division, Indian Council of Agricultural Research-National Institute of Animal Nutrition and Physiology, Bengaluru, 560 030, India
| | - Santhanahalli Siddalingappa Archana
- Reproductive Physiology Laboratory, Animal Physiology Division, Indian Council of Agricultural Research-National Institute of Animal Nutrition and Physiology, Bengaluru, 560 030, India
| | - Balaganur Krishnappa
- Reproductive Physiology Laboratory, Animal Physiology Division, Indian Council of Agricultural Research-National Institute of Animal Nutrition and Physiology, Bengaluru, 560 030, India
| | - Subrata Kumar Ghosh
- Animal Reproduction Division, Indian Council of Agricultural Research-Indian Veterinary Research Institute, Izatnagar, 243 122, India
| | - Harendra Kumar
- Animal Reproduction Division, Indian Council of Agricultural Research-Indian Veterinary Research Institute, Izatnagar, 243 122, India
| | - Raghavendra B Subbarao
- Reproductive Physiology Laboratory, Animal Physiology Division, Indian Council of Agricultural Research-National Institute of Animal Nutrition and Physiology, Bengaluru, 560 030, India
| | - Arunachalam Arangasamy
- Reproductive Physiology Laboratory, Animal Physiology Division, Indian Council of Agricultural Research-National Institute of Animal Nutrition and Physiology, Bengaluru, 560 030, India
| | - Raghavendra Bhatta
- Indian council of Agricultural Research-National Institute of Animal Nutrition and Physiology, Bengaluru, 560 030, India
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de Souza AF, Pieri NCG, Martins DDS. Step by Step about Germ Cells Development in Canine. Animals (Basel) 2021; 11:ani11030598. [PMID: 33668687 PMCID: PMC7996183 DOI: 10.3390/ani11030598] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/20/2020] [Revised: 01/13/2021] [Accepted: 01/19/2021] [Indexed: 12/26/2022] Open
Abstract
Simple Summary The progression of germ cells is a remarkable event that allows biological discovery in the differ-entiation process during in vivo and in vitro development. This is crucial for understanding one toward making oogenesis and spermatogenesis. Companion animals, such as canine, could offer new animal models for experimental and clinical testing for translation to human models. In this review, we describe the latest and more relevant findings on germ cell development. In addition, we showed the methods available for obtaining germ cells in vitro and the characterization of pri-mordial germ cells and spermatogonial stem cells. However, it is necessary to further conduct basic research in canine to clarify the beginning of germ cell development. Abstract Primordial germ cells (PGCs) have been described as precursors of gametes and provide a connection within generations, passing on the genome to the next generation. Failures in the formation of gametes/germ cells can compromise the maintenance and conservation of species. Most of the studies with PGCs have been carried out in mice, but this species is not always the best study model when transposing this knowledge to humans. Domestic animals, such as canines (canine), have become a valuable translational research model for stem cells and therapy. Furthermore, the study of canine germ cells opens new avenues for veterinary reproduction. In this review, the objective is to provide a comprehensive overview of the current knowledge on canine germ cells. The aspects of canine development and germ cells have been discussed since the origin, specifications, and development of spermatogonial canine were first discussed. Additionally, we discussed and explored some in vitro aspects of canine reproduction with germ cells, such as embryonic germ cells and spermatogonial stem cells.
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Kokkorakis N, Gaitanou M. Minibrain-related kinase/dual-specificity tyrosine-regulated kinase 1B implication in stem/cancer stem cells biology. World J Stem Cells 2020; 12:1553-1575. [PMID: 33505600 PMCID: PMC7789127 DOI: 10.4252/wjsc.v12.i12.1553] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/10/2020] [Revised: 09/29/2020] [Accepted: 10/15/2020] [Indexed: 02/06/2023] Open
Abstract
Dual-specificity tyrosine phosphorylation-regulated kinase 1B (DYRK1B), also known as minibrain-related kinase (MIRK) is one of the best functionally studied members of the DYRK kinase family. DYRKs comprise a family of protein kinases that are emerging modulators of signal transduction pathways, cell proliferation and differentiation, survival, and cell motility. DYRKs were found to participate in several signaling pathways critical for development and cell homeostasis. In this review, we focus on the DYRK1B protein kinase from a functional point of view concerning the signaling pathways through which DYRK1B exerts its cell type-dependent function in a positive or negative manner, in development and human diseases. In particular, we focus on the physiological role of DYRK1B in behavior of stem cells in myogenesis, adipogenesis, spermatogenesis and neurogenesis, as well as in its pathological implication in cancer and metabolic syndrome. Thus, understanding of the molecular mechanisms that regulate signaling pathways is of high importance. Recent studies have identified a close regulatory connection between DYRK1B and the hedgehog (HH) signaling pathway. Here, we aim to bring together what is known about the functional integration and cross-talk between DYRK1B and several signaling pathways, such as HH, RAS and PI3K/mTOR/AKT, as well as how this might affect cellular and molecular processes in development, physiology, and pathology. Thus, this review summarizes the major known functions of DYRK1B kinase, as well as the mechanisms by which DYRK1B exerts its functions in development and human diseases focusing on the homeostasis of stem and cancer stem cells.
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Affiliation(s)
- Nikolaos Kokkorakis
- Laboratory of Cellular and Molecular Neurobiology-Stem Cells, Hellenic Pasteur Institute, Athens 11521, Greece
| | - Maria Gaitanou
- Laboratory of Cellular and Molecular Neurobiology-Stem Cells, Hellenic Pasteur Institute, Athens 11521, Greece.
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15
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Binsila BK, Selvaraju S, Ghosh SK, Ramya L, Arangasamy A, Ranjithkumaran R, Bhatta R. EGF, GDNF, and IGF-1 influence the proliferation and stemness of ovine spermatogonial stem cells in vitro. J Assist Reprod Genet 2020; 37:2615-2630. [PMID: 32821972 DOI: 10.1007/s10815-020-01912-5] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/29/2019] [Accepted: 08/03/2020] [Indexed: 11/25/2022] Open
Abstract
PURPOSE The objective of the present study was to purify sheep spermatogonial stem cells (SSCs) from testicular isolate using combined enrichment methods and to study the effect of growth factors on SSC stemness during culture. METHODS The testicular cells from prepubertal male sheep were isolated, and SSCs were purified using Ficoll gradients (10 and 12%) followed by differential plating (laminin with BSA). SSCs were cultured with StemPro®-34 SFM, additives, and FBS for 7 days. The various doses (ng/ml) of growth factors, EGF at 10, 15, and 20, GDNF at 40, 70, and 100 and IGF-1 at 50, 100, and 150 were tested for the proliferation and stemness of SSCs in vitro. The stemness in cultured cells was assessed using SSC markers PLZF, ITGA6, and GFRα1. RESULTS Ficoll density gradient separation significantly (p < 0.05) increased the percentage of SSCs in 12% fraction (35.1 ± 3.8 vs 11.2 ± 3.7). Subsequently, purification using laminin with BSA plating further enriched SSCs to 61.7 ± 4.7%. GDNF at 40 ng/ml, EGF at 15 and 20 ng/ml and IGF1 at 100 and 150 ng/ml significantly (p < 0.05) improved proliferation and stemness of SSCs up to 7 days in culture. GDNF at 40 ng/ml outperformed other growth factors tested and could maintain the ovine SSCs proliferation and stemness for 36 days. CONCLUSIONS The combined enrichment method employing density gradient centrifugation and laminin with BSA plating improves the purification efficiency of ovine SSCs. GDNF at 40 ng/ml is essential for optimal proliferation and sustenance of stemness of ovine SSCs in vitro.
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Affiliation(s)
- B K Binsila
- Reproductive Physiology Laboratory, Animal Physiology Division, Indian Council of Agricultural Research-National Institute of Animal Nutrition and Physiology, Bengaluru, 560 030, India.
| | - S Selvaraju
- Reproductive Physiology Laboratory, Animal Physiology Division, Indian Council of Agricultural Research-National Institute of Animal Nutrition and Physiology, Bengaluru, 560 030, India
| | - S K Ghosh
- Animal Reproduction Division, Indian Council of Agricultural Research-Indian Veterinary Research Institute, Izatnagar, 243 122, India
| | - L Ramya
- Reproductive Physiology Laboratory, Animal Physiology Division, Indian Council of Agricultural Research-National Institute of Animal Nutrition and Physiology, Bengaluru, 560 030, India
| | - A Arangasamy
- Reproductive Physiology Laboratory, Animal Physiology Division, Indian Council of Agricultural Research-National Institute of Animal Nutrition and Physiology, Bengaluru, 560 030, India
| | - R Ranjithkumaran
- Reproductive Physiology Laboratory, Animal Physiology Division, Indian Council of Agricultural Research-National Institute of Animal Nutrition and Physiology, Bengaluru, 560 030, India
| | - R Bhatta
- Indian Council of Agricultural Research-National Institute of Animal Nutrition and Physiology, Bengaluru, 560 030, India
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Ghanbari E, Khazaei M, Ghahremani-Nasab M, Mehdizadeh A, Yousefi M. Novel therapeutic approaches of tissue engineering in male infertility. Cell Tissue Res 2020; 380:31-42. [PMID: 32043209 DOI: 10.1007/s00441-020-03178-w] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/17/2019] [Accepted: 01/23/2020] [Indexed: 12/25/2022]
Abstract
Male reproductive organ plays an important role in sperm production, maintenance and entry to the female reproductive tract, as well as generation and secretion of male sex hormones responsible for the health of male reproductive system. The purpose of this paper is to discuss the experimental and clinical evidence on the utilization of tissue engineering techniques in treating male infertility. Tissue engineering (TE) and regenerative medicine have developed new approaches to treat patients with reproductive disorders such as iatrogenic injuries, congenital abnormalities, and trauma. In some cases, including congenital defects and undescended testis or hypogonadism, the sperm samples are not retrieved. This makes TE a possible future strategy for restoration of male fertility. Here, we have summarized the recent advances in experimental and clinical application of cell-, tissue-, and organ-based regenerative medicine in male reproductive disorders.
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Affiliation(s)
- Elham Ghanbari
- Fertility and Infertility Research Center, Health Technology Institute, Kermanshah University of Medical Sciences, Kermanshah, Iran
| | - Mozafar Khazaei
- Fertility and Infertility Research Center, Health Technology Institute, Kermanshah University of Medical Sciences, Kermanshah, Iran
| | | | - Amir Mehdizadeh
- Endocrine Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.,Comprehensive Health Laboratory, Tabriz University of Medical Sciences, Tabriz, Iran
| | - Mehdi Yousefi
- Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. .,Aging Research Institute, Tabriz University of Medical Sciences, Tabriz, Iran. .,Department of Immunology, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran.
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Aponte PM. Isolation, Culture, Cryopreservation, and Identification of Bovine, Murine, and Human Spermatogonial Stem Cells. Methods Mol Biol 2020; 2155:151-164. [PMID: 32474875 DOI: 10.1007/978-1-0716-0655-1_13] [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] [Indexed: 12/13/2022]
Abstract
Spermatogonial stem cells (SSCs) are the germ cells at the basis of spermatogenesis in adult mammals. SSCs offer many biotechnological possibilities and are fundamental cells in the study of spermatogenesis (Aponte, World J Stem Cells 7:669-680, 2015). This chapter describes detailed procedures for SSC isolation, culture, cryopreservation, and characterization in bovine, murine, and human models.
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Affiliation(s)
- Pedro M Aponte
- Colegio de Ciencias Biológicas y Ambientales, Universidad San Francisco de Quito (USFQ), Quito, Ecuador. .,Colegio de Ciencias de la Salud, Escuela de Medicina Veterinaria, Universidad San Francisco de Quito (USFQ), Quito, Ecuador. .,Instituto de Investigaciones en Biomedicina "One-health", Universidad San Francisco de Quito (USFQ), Quito, Ecuador.
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Tian J, Ma K, Pei CB, Zhang SH, Li X, Zhou Y, Yan B, Wang HY, Ma LH. Relative safety of various spermatogenic stem cell purification methods for application in spermatogenic stem cell transplantation. Stem Cell Res Ther 2019; 10:382. [PMID: 31842987 PMCID: PMC6916234 DOI: 10.1186/s13287-019-1481-9] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/30/2019] [Revised: 10/18/2019] [Accepted: 10/31/2019] [Indexed: 11/29/2022] Open
Abstract
BACKGROUND Spermatogonial stem cell (SSC) transplantation technology as a promising option for male fertility preservation has received increasing attention, along with efficient SSC purification technology as a necessary technical support; however, the safety of such application in patients with tumors remains controversial. METHODS In this study, we used a green fluorescent protein mouse xenograft model of B cell acute lymphocytic leukemia. We isolated and purified SSCs from the testicular tissue of model mice using density gradient centrifugation, immune cell magnetic bead separation, and flow cytometry. The purified SSCs were transplanted into convoluted seminiferous tubules of the nude mice and C57BL/6 male mice subjected to busulfan. The development and proliferation of SSCs in the recipient testis were periodically tested, along with whether B cell acute lymphocytic leukemia was induced following SSC implantation. The genetic characteristics of the offspring obtained from natural mating were also observed. RESULTS In testicular leukemia model mice, a large number of BALL cells infiltrated into the seminiferous tubule, spermatogenic cells, and sperm cells in the testis tissue decreased. After spermatogonial stem cell transplantation, the transplanted SSCs purified by immunomagnetic beads and flow cytometry methods colonized and proliferated extensively in the basement of the seminiferous tubules of mice; a large number of spermatogenic cells and sperm were found in recipient testicular tissue after 12 weeks of SSC transplantation. In leukemia detection in nude mice after transplantation in the three SSC purification groups, a large number of BALL cells could be detected in the blood of recipient mice 2-3 weeks after transplantation in the density gradient centrifugation group, but not in the blood of the flow cytometry sorting group and the immunomagnetic bead group after 16 weeks of observation. CONCLUSIONS In this study, we confirmed that immunomagnetic beads and flow cytometry methods of purifying SSCs from the testicular tissue of the testicular leukemia mouse model could be safely applied to the SSC transplantation technology without concomitant tumor implantation. The results thus provide a theoretical basis for the application of tumor SSC cryopreservation for fertility preservation in patients with tumors.
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Affiliation(s)
- Jia Tian
- General Hospital of Ningxia Medical University/Human Sperm Bank of Ningxia, Key Laboratory of Fertility Preservation and Maintenance of Ministry of Education, Ningxia Medical University, Yinchuan, 750001, China
| | - Ke Ma
- Clinical College, Ningxia Medical University, Yinchuan, 750001, China
| | - Cheng-Bin Pei
- General Hospital of Ningxia Medical University/Human Sperm Bank of Ningxia, Key Laboratory of Fertility Preservation and Maintenance of Ministry of Education, Ningxia Medical University, Yinchuan, 750001, China
| | - Shao-Hua Zhang
- Clinical College, Ningxia Medical University, Yinchuan, 750001, China
| | - Xue Li
- Key Laboratory of Fertility Preservation and Maintenance of Ministry of Education, Ningxia Medical University, Yinchuan, 750001, China
| | - Yue Zhou
- General Hospital of Ningxia Medical University/Human Sperm Bank of Ningxia, Key Laboratory of Fertility Preservation and Maintenance of Ministry of Education, Ningxia Medical University, Yinchuan, 750001, China
| | - Bei Yan
- General Hospital of Ningxia Medical University/Human Sperm Bank of Ningxia, Key Laboratory of Fertility Preservation and Maintenance of Ministry of Education, Ningxia Medical University, Yinchuan, 750001, China
| | - Hong-Yan Wang
- General Hospital of Ningxia Medical University/Human Sperm Bank of Ningxia, Key Laboratory of Fertility Preservation and Maintenance of Ministry of Education, Ningxia Medical University, Yinchuan, 750001, China
| | - Liang-Hong Ma
- General Hospital of Ningxia Medical University/Human Sperm Bank of Ningxia, Key Laboratory of Fertility Preservation and Maintenance of Ministry of Education, Ningxia Medical University, Yinchuan, 750001, China.
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Zinovieva NA, Volkova NA, Bagirov VA. Genome Editing: Current State of Research and Application to Animal Husbandry. APPL BIOCHEM MICRO+ 2019. [DOI: 10.1134/s000368381907007x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/23/2022]
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Lee J, Lee Y, Lee GS, Lee ST, Lee E. Comparative study of the developmental competence of cloned pig embryos derived from spermatogonial stem cells and fetal fibroblasts. Reprod Domest Anim 2019; 54:1258-1264. [PMID: 31283039 DOI: 10.1111/rda.13507] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/22/2019] [Accepted: 07/04/2019] [Indexed: 01/27/2023]
Abstract
Spermatogonial stem cells (SSC) are promising resources for genetic preservation and restoration of male germ cells in humans and animals. However, no studies have used SSC as donor nuclei in pig somatic cell nuclear transfer (SCNT). This study investigated the potential for use of porcine SSC as a nuclei donor for SCNT and developmental competence of SSC-derived cloned embryos. In addition, demecolcine was investigated to determine whether it could prevent rupture of SSC during SCNT. When the potential of SSC to support embryonic development after SCNT was compared with that of foetal fibroblasts (FF), SSC-derived SCNT embryos showed a higher (p < .05) developmental competence to the blastocyst stage (47.8%) than FF-derived embryos (25.6%). However, when SSC were used as donor nuclei in the SCNT process, cell fusion rates were lower (p < .05) than when FF were used (61.9% vs. 75.8%). Treatment of SSC with demecolcine significantly (p < .05) decreased rupture of SSC during the SCNT procedure (7.5% vs. 18.8%) and increased fusion of cell-oocyte couplets compared with no treatment (74.6% vs. 61.6%). In addition, SSC-derived SCNT embryos showed higher blastocyst formation (48.4%) than FF-derived embryos without (28.4%) and with demecolcine treatment (17.4%), even after demecolcine treatment. Our results demonstrate that porcine SSC are a desirable donor cell type for production of SCNT pig embryos and that demecolcine increases production efficiency of cloned embryos by inhibiting rupture of nuclei donor SSC during SCNT.
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Affiliation(s)
- Joohyeong Lee
- Institute of Veterinary Science, Kangwon National University, Chuncheon, Korea
| | - Yongjin Lee
- College of Veterinary Medicine, Kangwon National University, Chuncheon, Korea
| | - Geun-Shik Lee
- Institute of Veterinary Science, Kangwon National University, Chuncheon, Korea.,College of Veterinary Medicine, Kangwon National University, Chuncheon, Korea
| | - Seung Tae Lee
- College of Animal Life Science, Kangwon National University, Chuncheon, Korea
| | - Eunsong Lee
- Institute of Veterinary Science, Kangwon National University, Chuncheon, Korea.,College of Veterinary Medicine, Kangwon National University, Chuncheon, Korea
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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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Pieri NCG, de Souza AF, Botigelli RC, Machado LS, Ambrosio CE, Dos Santos Martins D, de Andrade AFC, Meirelles FV, Hyttel P, Bressan FF. Stem cells on regenerative and reproductive science in domestic animals. Vet Res Commun 2019; 43:7-16. [PMID: 30656543 DOI: 10.1007/s11259-019-9744-6] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/30/2018] [Accepted: 01/09/2019] [Indexed: 12/12/2022]
Abstract
Stem cells are undifferentiated and self-renewable cells that present new possibilities for both regenerative medicine and the understanding of early mammalian development. Adult multipotent stem cells are already widely used worldwide in human and veterinary medicine, and their therapeutic signalling, particularly with respect to immunomodulation, and their trophic properties have been intensively studied. The derivation of embryonic stem cells (ESCs) from domestic species, however, has been challenging, and the poor results do not reflect the successes obtained in mouse and human experiments. More recently, the generation of induced pluripotent stem cells (iPSCs) via the forced expression of specific transcription factors has been demonstrated in domestic species and has introduced new potentials in regenerative medicine and reproductive science based upon the ability of these cells to differentiate into a variety of cells types in vitro. For example, iPSCs have been differentiated into primordial germ-like cells (PGC-like cells, PGCLs) and functional gametes in mice. The possibility of using iPSCs from domestic species for this purpose would contribute significantly to reproductive technologies, offering unprecedented opportunities to restore fertility, to preserve endangered species and to generate transgenic animals for biomedical applications. Therefore, this review aims to provide an updated overview of adult multipotent stem cells and to discuss new possibilities introduced by the generation of iPSCs in domestic animals, highlighting the possibility of generating gametes in vitro via PGCL induction.
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Affiliation(s)
- Naira Caroline Godoy Pieri
- Department of Animal Reproduction, Faculty of Veterinary Medicine and Animal Sciences, University of São Paulo, São Paulo, Brazil
| | - Aline Fernanda de Souza
- Department of Veterinary Medicine, Faculty of Animal Sciences and Food Engineering, University of São Paulo, Pirassununga, Brazil
| | - Ramon Cesar Botigelli
- Department of Pharmacology, Institute of Biosciences, São Paulo State University, Botucatu, Brazil
| | - Lucas Simões Machado
- Department of Surgery, Faculty of Veterinary Medicine and Animal Sciences, University of São Paulo, São Paulo, Brazil
| | - Carlos Eduardo Ambrosio
- Department of Veterinary Medicine, Faculty of Animal Sciences and Food Engineering, University of São Paulo, Pirassununga, Brazil
| | - Daniele Dos Santos Martins
- Department of Veterinary Medicine, Faculty of Animal Sciences and Food Engineering, University of São Paulo, Pirassununga, Brazil
| | - André Furugen Cesar de Andrade
- Department of Animal Reproduction, Faculty of Veterinary Medicine and Animal Sciences, University of São Paulo, São Paulo, Brazil
| | - Flavio Vieira Meirelles
- Department of Veterinary Medicine, Faculty of Animal Sciences and Food Engineering, University of São Paulo, Pirassununga, Brazil
| | - Poul Hyttel
- Department of Veterinary and Animal Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Fabiana Fernandes Bressan
- Department of Veterinary Medicine, Faculty of Animal Sciences and Food Engineering, University of São Paulo, Pirassununga, Brazil.
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Identification of a Technique Optimized for the Isolation of Spermatogonial Stem Cells from Mouse Testes. JOURNAL OF ANIMAL REPRODUCTION AND BIOTECHNOLOGY 2018. [DOI: 10.12750/jet.2018.33.4.327] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022] Open
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Sisakhtnezhad S. In silico analysis of single‐cell RNA sequencing data from 3 and 7 days old mouse spermatogonial stem cells to identify their differentially expressed genes and transcriptional regulators. J Cell Biochem 2018; 119:7556-7569. [DOI: 10.1002/jcb.27066] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/29/2018] [Accepted: 04/23/2018] [Indexed: 02/06/2023]
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Sisakhtnezhad S, Heshmati P. Comparative analysis of single-cell RNA sequencing data from mouse spermatogonial and mesenchymal stem cells to identify differentially expressed genes and transcriptional regulators of germline cells. J Cell Physiol 2018; 233:5231-5242. [PMID: 29194616 DOI: 10.1002/jcp.26303] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/06/2017] [Accepted: 11/28/2017] [Indexed: 12/17/2022]
Abstract
Identifying effective internal factors for regulating germline commitment during development and for maintaining spermatogonial stem cells (SSCs) self-renewal is important to understand the molecular basis of spermatogenesis process, and to develop new protocols for the production of the germline cells from other cell sources. Therefore, this study was designed to investigate single-cell RNA-sequencing data for identification of differentially expressed genes (DEGs) in 12 mouse-derived single SSCs (mSSCs) in compare with 16 mouse-derived single mesenchymal stem cells. We also aimed to find transcriptional regulators of DEGs. Collectively, 1,584 up-regulated DEGs were identified that are associated with 32 biological processes. Moreover, investigation of the expression profiles of genes including in spermatogenesis process revealed that Dazl, Ddx4, Sall4, Fkbp6, Tex15, Tex19.1, Rnf17, Piwil2, Taf7l, Zbtb16, and Cadm1 are presented in the first 30 up-regulated DEGs. We also found 12 basal transcription factors (TFs) and three sequence-specific TFs that control the expression of DEGs. Our findings also indicated that MEIS1, SMC3, TAF1, KAT2A, STAT3, GTF3C2, SIN3A, BDP1, PHC1, and EGR1 are the main central regulators of DEGs in mSSCs. In addition, we collectively detected two significant protein complexes in the protein-protein interactions network for DEGs regulators. Finally, this study introduces the major upstream kinases for the main central regulators of DEGs and the components of core protein complexes. In conclusion, this study provides a molecular blueprint to uncover the molecular mechanisms behind the biology of SSCs and offers a list of candidate factors for cell type conversion approaches and production of germ cells.
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Affiliation(s)
| | - Parvin Heshmati
- Faculty of Dentistry, Department of Endodontics, Kermanshah University of Medical Sciences, Kermanshah, Iran
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Active immunization against GnRH in pre-pubertal domestic mammals: testicular morphometry, histopathology and endocrine responses in rabbits, guinea pigs and ram lambs. Animal 2018; 12:784-793. [DOI: 10.1017/s1751731117002129] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/06/2022] Open
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Jung SE, Kim YH, Cho S, Kim BJ, Lee HS, Hwang S, Kim GB, Kim YH, Pang MG, Lee S, Ryu BY. A Phytochemical Approach to Promotion of Self-renewal in Murine Spermatogonial Stem Cell by Using Sedum Sarmentosum Extract. Sci Rep 2017; 7:11441. [PMID: 28900261 PMCID: PMC5595968 DOI: 10.1038/s41598-017-11790-0] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/16/2017] [Accepted: 08/25/2017] [Indexed: 01/15/2023] Open
Abstract
Spermatogonial stem cells (SSCs) are the basis of spermatogenesis, which is dependent on the ability to self-renew and differentiation. Controlling self-renewal and differentiation of SSCs could apply to treatment of disease such as male infertility. Recently, in the field of stem cell research, it was demonstrated that effective increase in stem cell activity can be achieved by using growth factors derived from plant extracts. In this study, our aim is to investigate components from natural plant to improve the self-renewal of SSCs. To find the components, germ cells were cultured with comprehensive natural plant extracts, and then the more pure fraction, and finally single compound at different concentrations. As a result, we found 5H-purin-6-amine at 1 µg/mL, originated from Sedum sarmentosum, was a very effective compound induced SSCs proliferation. Our data showed that germ cells cultured with 5H-purin-6-amine could maintain their stable characteristics. Furthermore, transplantation results demonstrated that 5H-purin-6-amine at 1 µg/mL increased the activity of SSCs, indicating the compound could increase true SSC concentration within germ cells to 1.96-fold. These findings would be contributed to improve further reproductive research and treat male infertility by using natural plant extracts.
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Affiliation(s)
- Sang-Eun Jung
- Department of Animal Science & Technology, Chung-Ang University, Anseong, Republic of Korea
| | - Yong-Hee Kim
- Department of Animal Science & Technology, Chung-Ang University, Anseong, Republic of Korea
| | - Sunghun Cho
- Department of Integrative Plant Science, Chung-Ang University, Anseong, Republic of Korea
| | - Bang-Jin Kim
- Department of Cancer Biology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, 19104, USA
| | - Hee-Seok Lee
- Food Safety Risk Assessment Division, National Institute of Food and Drug Safety Evaluation, MFDS Korea, Chungcheongbuk-do, Republic of Korea
| | - Seongsoo Hwang
- Animal Biotechnology Division, National Institute of Animal Science, Rural Development Administration, Jeollabuk-do, Republic of Korea
| | - Geun-Bae Kim
- Department of Animal Science & Technology, Chung-Ang University, Anseong, Republic of Korea
| | - Young-Hyun Kim
- National Primate Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 30 Yeongudangi-ro, Ochang-eup, Cheongwon-gu, Cheongju-si, Chungcheongbuk-do, Republic of Korea.,Department of Functional Genomics, KRIBB School of Bioscience, Korea University of Science and Technology (UST), 217 Gajeong-ro, Yuseong-gu, Deajeon, Republic of Korea
| | - Myung-Geol Pang
- Department of Animal Science & Technology, Chung-Ang University, Anseong, Republic of Korea
| | - Sanghyun Lee
- Department of Integrative Plant Science, Chung-Ang University, Anseong, Republic of Korea.
| | - Buom-Yong Ryu
- Department of Animal Science & Technology, Chung-Ang University, Anseong, Republic of Korea.
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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: 4] [Impact Index Per Article: 0.4] [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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Galuppo AG. Spermatogonial stem cells as a therapeutic alternative for fertility preservation of prepubertal boys. EINSTEIN-SAO PAULO 2016; 13:637-9. [PMID: 26761559 PMCID: PMC4878644 DOI: 10.1590/s1679-45082015rb3456] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/15/2015] [Accepted: 11/04/2015] [Indexed: 01/15/2023] Open
Abstract
Spermatogonial stem cells, which exist in the testicles since birth, are progenitors cells of male gametes. These cells are critical for the process of spermatogenesis, and not able to produce mature sperm cells before puberty due to their dependency of hormonal stimuli. This characteristic of the reproductive system limits the preservation of fertility only to males who are able to produce an ejaculate. This fact puts some light on the increase in survival rates of childhood cancer over the past decades because of improvements in the diagnosis and effective treatment in pediatric cancer patients. Therefore, we highlight one of the most important challenges concerning male fertility preservation that is the toxic effect of cancer therapy on reproductive function, especially the spermatogenesis. Currently, the experimental alternative for fertility preservation of prepubertal boys is the testicular tissue cryopreservationfor, for future isolation and spermatogonial stem cells transplantation, in order to restore the spermatogenesis. We present a brief review on isolation, characterization and culture conditions for the in vitro proliferation of spermatogonial stem cells, as well as the future perspectives as an alternative for fertility preservation in prepubertal boys. The possibility of restoring male fertility constitutes a research tool with an huge potential in basic and applied science. The development of these techniques may be a hope for the future of fertility preservation in cases that no other options exist, e.g, pediatric cancer patients.
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Affiliation(s)
- Andrea Giannotti Galuppo
- Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil
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Abstract
Spermatogenesis is a stem cell based system. Both therapeutic and biomedical research applications of spermatogonial stem cells require a large number of cells. However, there are only few germ line stem cells in the testis, contained in the fraction of undifferentiated spermatogonia. The lack of specific markers makes it difficult to isolate these cells. The long term maintenance and proliferation of nonrodent germ cells in culture has so far been met with limited success, partially due to the lack of highly enriched starting populations. Differential plating, which depends on the differential adhesion properties of testicular somatic and germ cells to tissue culture dishes, has been the method of choice for germ cell enrichment, especially for nonrodent germ cells. However, for large animals, this process becomes labor intensive and increases variability due to the need for extensive handling. Here, we describe the use of stirred suspension bioreactors, as a novel system for enriching undifferentiated germ cells from 1-week-old pigs. This method capitalizes on the adherent properties of somatic cells within a controlled environment, thus promoting the enrichment of progenitor cells with minimal handling and variability.
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