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Sharbatoghli M, Hajiaghalou S, Deheshkar Gooneh Farahani NS, Aghajanpour S, Shahverdi A, Rezazadeh Valojerdi M, Ebrahimi B. Effect of vitrification on in vitro developmental competence of rat testicular tissue. Cryobiology 2025; 119:105213. [PMID: 39952345 DOI: 10.1016/j.cryobiol.2025.105213] [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] [Received: 08/12/2024] [Revised: 02/09/2025] [Accepted: 02/11/2025] [Indexed: 02/17/2025]
Abstract
Cryopreservation of testicular tissue has been proposed as a potential technique for preserving fertility in pre-pubertal boys with various malignancies. The present study aimed to compare the effects of two vitrification techniques-solid surface vitrification (SSV) and needle-immersed vitrification (NIV)-on the integrity, development, cell viability, and apoptosis of rat testicular tissue. Testes from 4-week-old Wistar rats underwent a two-step vitrification process. Tissue pieces were allocated to either the SSV or NIV group. Equilibration involved a solution containing 7.5 % dimethyl sulfoxide (DMSO) and 7.5 % ethylene glycol (EG), followed by a vitrification solution with 0.07 mol/L sucrose, 15 % DMSO, and 15 % EG. The optimal protocol was determined after vitrification using either the NIV or SSV technique. Samples from the control and selected vitrification (SSV) groups were cultured for 3 weeks. Tissue integrity, cell viability, apoptosis, and gene expression were evaluated using hematoxylin and eosin staining, trypan blue staining, annexin V-PI staining, and real-time PCR. Morphological changes were more pronounced in the NIV group compared to the SSV group (P < 0.05). Although the percentage of viable cells did not significantly differ between the NIV and SSV groups, it was slightly higher in the SSV group. Thus, SSV was identified as the optimum vitrification method. Real-time PCR analysis revealed altered gene expression: spermatogonial-related genes (Lrp4, Egr3, Nanos, Gfra1, C-kit, and Sohlh1) were significantly decreased in the SSV group, while somatic-cells-related genes (Gdnf, Csf1, and Fgf2) were higher. Overall, SSV appears suitable for rat testis tissue vitrification, although it induces some molecular changes. Optimization of the culture medium is essential to support successful spermatogenesis.
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Affiliation(s)
- Mina Sharbatoghli
- Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran
| | - Samira Hajiaghalou
- Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran
| | | | - Samaneh Aghajanpour
- Department of Endocrinology and Female Infertility, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran
| | - Abdolhossein Shahverdi
- Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran
| | - Mojtaba Rezazadeh Valojerdi
- Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran; Department of Anatomy, Faculty o Medical Sciences, Tarbiat Modares University, P. O. Box: 14115-111, Tehran, Iran
| | - Bita Ebrahimi
- Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
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Tan J, Li J, Lin C, Ye N, Zhang H, Liu C, Han S, Li Z, Zhou X. Generation of mouse testicular organoids with highly compartmentalized tubular lumen structure and their cryopreservation. Life Sci 2024; 355:122980. [PMID: 39147312 DOI: 10.1016/j.lfs.2024.122980] [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] [Received: 05/24/2024] [Revised: 07/29/2024] [Accepted: 08/12/2024] [Indexed: 08/17/2024]
Abstract
Testicular organoids have great potential for maintaining male fertility and even restoring male infertility. However, existing studies on generating organoids with testis-specific structure and function are scarce and come with many limitations. Research on cryopreservation of testicular organoids is even more limited, and inappropriate cryopreservation methods may result in the loss of properties in resuscitated or regenerated organoids, rendering them unsuitable for clinical or research needs. In this paper, we investigated the effects of mouse age and cell number on the self-aggregation of testicular cells into spheres in low-adsorption plates. Various media compositions, culture systems, and cell numbers were used to culture cell spheres for 14 days to form testicular organoids, and the self-organization of the organoids was assessed by histological and immunofluorescence staining. We determined the appropriate cryopreservation conditions for testicular cells, cell spheres, and tissues. Subsequently, organoids derived from cryopreserved testicular tissues, testicular cells, and testicular cell spheres were compared and evaluated by histological and immunofluorescence staining. The results indicate that testicular cell spheres consisting of 30 × 104 testicular cells from 2-week-old mice were able to form organoids highly similar to the luminal structure and cell distribution of natural mouse testicular tissues. This transformation occurred over 14 days of incubation in α-MEM medium containing 10 % knockout serum replacer (KSR) using an agarose hydrogel culture system. Additionally, the Sertoli cells were tightly connected to form a blood-testis barrier. The relative rates of tubular area, germ cells, Sertoli cells, and peritubular myoid cells were 36.985 % ± 0.695, 13.347 % ± 3.102, 47.570 % ± 0.379, and 27.406 % ± 1.832, respectively. The optimal cryopreservation protocol for primary testicular cells involved slow freezing with a cryoprotectant consisting of α-MEM with 10 % dimethyl sulfoxide (DMSO). Slow freezing with cryoprotectants containing 5 % DMSO and 5 % ethylene glycol (EG) was optimal for all different volumes of testicular cell spheres. Compared to testicular organoids generated from frozen testicular tissue and cell spheres, freezing testicular cells proved most effective in maintaining organoid differentiation characteristics and cell-cell interactions. The findings of this study contribute to a "universal" testicular organoid in vitro culture protocol with promising applications for fertility preservation and restoration in prepubertal cancer patients and adult infertile patients.
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Affiliation(s)
- Jia Tan
- Institute of Biomedical Technology, University of Shanghai for Science and Technology, Shanghai 200093, China
| | - Jiahui Li
- Institute of Biomedical Technology, University of Shanghai for Science and Technology, Shanghai 200093, China
| | - Chunyan Lin
- Institute of Biomedical Technology, University of Shanghai for Science and Technology, Shanghai 200093, China
| | - Na Ye
- Institute of Biomedical Technology, University of Shanghai for Science and Technology, Shanghai 200093, China
| | - Hui Zhang
- Institute of Biomedical Technology, University of Shanghai for Science and Technology, Shanghai 200093, China
| | - Chenxi Liu
- Institute of Biomedical Technology, University of Shanghai for Science and Technology, Shanghai 200093, China
| | - Sha Han
- Department of Andrology, The Center for Men's Health, Urologic Medical Center, Shanghai Key Laboratory of Reproductive Medicine, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200080, China
| | - Zheng Li
- Department of Andrology, The Center for Men's Health, Urologic Medical Center, Shanghai Key Laboratory of Reproductive Medicine, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200080, China
| | - Xinli Zhou
- Institute of Biomedical Technology, University of Shanghai for Science and Technology, Shanghai 200093, China.
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Damyanova KB, Nixon B, Johnston SD, Gambini A, Benitez PP, Lord T. Spermatogonial stem cell technologies: applications from human medicine to wildlife conservation†. Biol Reprod 2024; 111:757-779. [PMID: 38993049 PMCID: PMC11473898 DOI: 10.1093/biolre/ioae109] [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: 05/01/2024] [Revised: 07/05/2024] [Accepted: 07/10/2024] [Indexed: 07/13/2024] Open
Abstract
Spermatogonial stem cell (SSC) technologies that are currently under clinical development to reverse human infertility hold the potential to be adapted and applied for the conservation of endangered and vulnerable wildlife species. The biobanking of testis tissue containing SSCs from wildlife species, aligned with that occurring in pediatric human patients, could facilitate strategies to improve the genetic diversity and fitness of endangered populations. Approaches to utilize these SSCs could include spermatogonial transplantation or testis tissue grafting into a donor animal of the same or a closely related species, or in vitro spermatogenesis paired with assisted reproduction approaches. The primary roadblock to progress in this field is a lack of fundamental knowledge of SSC biology in non-model species. Herein, we review the current understanding of molecular mechanisms controlling SSC function in laboratory rodents and humans, and given our particular interest in the conservation of Australian marsupials, use a subset of these species as a case-study to demonstrate gaps-in-knowledge that are common to wildlife. Additionally, we review progress in the development and application of SSC technologies in fertility clinics and consider the translation potential of these techniques for species conservation pipelines.
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Affiliation(s)
- Katerina B Damyanova
- Discipline of Biological Sciences, The University of Newcastle, Callaghan, NSW 2308, Australia
- Infertility and Reproduction Program, Hunter Medical Research Institute, New Lambton Heights, NSW 2305, Australia
| | - Brett Nixon
- Discipline of Biological Sciences, The University of Newcastle, Callaghan, NSW 2308, Australia
- Infertility and Reproduction Program, Hunter Medical Research Institute, New Lambton Heights, NSW 2305, Australia
| | - Stephen D Johnston
- School of Environment, The University of Queensland, Gatton, QLD 4343, Australia
- School of Veterinary Science, The University of Queensland, Gatton, QLD 4343, Australia
| | - Andrés Gambini
- School of Veterinary Science, The University of Queensland, Gatton, QLD 4343, Australia
- School of Agriculture and Food Science, The University of Queensland, Gatton, QLD 4343, Australia
| | - Patricio P Benitez
- School of Agriculture and Food Science, The University of Queensland, Gatton, QLD 4343, Australia
| | - Tessa Lord
- Discipline of Biological Sciences, The University of Newcastle, Callaghan, NSW 2308, Australia
- Infertility and Reproduction Program, Hunter Medical Research Institute, New Lambton Heights, NSW 2305, Australia
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Bashiri Z, Hosseini SJ, Salem M, Koruji M. In vivo and in vitro sperm production: an overview of the challenges and advances in male fertility restoration. Clin Exp Reprod Med 2024; 51:171-180. [PMID: 38525520 PMCID: PMC11372308 DOI: 10.5653/cerm.2023.06569] [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: 10/03/2023] [Accepted: 12/14/2023] [Indexed: 03/26/2024] Open
Abstract
Male infertility can be caused by genetic anomalies, endocrine disorders, inflammation, and exposure to toxic chemicals or gonadotoxic treatments. Therefore, several recent studies have concentrated on the preservation and restoration of fertility to enhance the quality of life for affected individuals. It is currently recommended to biobank the tissue extracted from testicular biopsies to provide a later source of spermatogonial stem cells (SSCs). Another successful approach has been the in vitro production of haploid male germ cells. The capacity of SSCs to transform into sperm, as in testicular tissue transplantation, SSC therapy, and in vitro or ex vivo spermatogenesis, makes them ideal candidates for in vivo fertility restoration. The transplantation of SSCs or testicular tissue to regenerate spermatogenesis and create embryos has been achieved in nonhuman mammal species. Although the outcomes of human trials have yet to be released, this method may soon be approved for clinical use in humans. Furthermore, regenerative medicine techniques that develop tissue or cells on organic or synthetic scaffolds enriched with bioactive molecules have also gained traction. All of these methods are now in different stages of experimentation and clinical trials. However, thanks to rigorous studies on the safety and effectiveness of SSC-based reproductive treatments, some of these techniques may be clinically available in upcoming decades.
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Affiliation(s)
- Zahra Bashiri
- Endometrium and Endometriosis Research Center, Hamadan University of Medical Sciences, Hamadan, Iran
- Department of Anatomy, School of Medicine, Iran University of Medical Sciences, Tehran, Iran
- Omid Fertility and Infertility Clinic, Hamedan, Iran
| | - Seyed Jamal Hosseini
- Biomedical Engineering Department, Amirkabir University of Technology, Tehran, Iran
- Department of Pharmaceutical Biomaterials and Medical Biomaterials Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran
| | - Maryam Salem
- Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
| | - Morteza Koruji
- Department of Anatomy, School of Medicine, Iran University of Medical Sciences, Tehran, Iran
- Stem Cell and Regenerative Medicine Research Center, Iran University of Medical Sciences, Tehran, Iran
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Richer G, Goyvaerts C, Marchandise L, Vanhaecke T, Goossens E, Baert Y. Spermatogenesis in mouse testicular organoids with testis-specific architecture, improved germ cell survival and testosterone production. Biofabrication 2024; 16:045024. [PMID: 38986466 DOI: 10.1088/1758-5090/ad618f] [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] [Received: 12/29/2023] [Accepted: 07/10/2024] [Indexed: 07/12/2024]
Abstract
This study presents a biphasic approach to overcome the limitations of current testicular organoid (TO) cultures, including histological heterogeneity, germ cell loss and absence of spermatogenesis. Agarose microwells were utilized to create TOs from prepubertal C57BL/6 J testicular cells. First emphasis was on improving germ cell survival during the initial 2-week reorganization phase by comparingα-MEM + 10% knockout serum replacement (KSR) medium, known to support TO generation in mice, to three optimized media (1-3). Cell densities and culture dynamics were also tested to recreate histological resemblance to testes. After optimizing germ cell survival and cell organization, the effect of growth factors and immunomodulation through CD45+immune cell depletion or dexamethasone (DEX) supplementation were assessed for enhancing spermatogenesis during the subsequent differentiation phase. Testicular cells self-reorganized into organoids resembling the testicular anatomical unit, characterized by one tubule-like structure surrounded by interstitium. Media 1-3 proved superior for organoid growth during the reorganization phase, with TOs in medium 3 exhibiting germ cell numbers (7.4% ± 4.8%) comparable to controls (9.3% ± 5.3%). Additionally, 37% ± 30% demonstrated organized histology from 32 × 103cells under static conditions. Switching toα-MEM + 10% KSR during the differentiation phase increased formation efficiency to 85 ± 7%, along with elevated germ cell numbers, testosterone production (3.1 ± 0.9 ng ml-1) and generation ofγ-H2AX+spermatid-like cells (steps 8-11, 1.2% ± 2.2% of the total). Adding differentiation factors to theα-MEM increased spermatid-like cell numbers to 2.9% ± 5.9%, confirmed through positive staining for CREM, transition protein 1, and peanut agglutinin. Although, these remained diploid with irregular nuclear maturation. DEX supplementation had no additional effect, and immune cell depletion adversely impacted TO formation. The manipulability of TOs offers advantages in studying male infertility and exploring therapies, with scalability enabling high-throughput chemical screening and reducing animal usage in reproductive toxicity and drug discovery studies.
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Affiliation(s)
- Guillaume Richer
- Biology of the Testis (BITE) laboratory, Genetics Reproduction and Development (GRAD) research group, Vrije Universiteit Brussel (VUB), Brussels, Belgium
| | - Cleo Goyvaerts
- Laboratory for Molecular and Cellular Therapy (LMCT), Translational Oncology Research Center (TORC), VUB, Brussels, Belgium
- Department of Medical Imaging, Molecular Imaging and Therapy (MITH), VUB, Brussels, Belgium
| | - Lorna Marchandise
- Department of In Vitro Toxicology and Dermato-Cosmetology (IVTD), Center for Pharmaceutical Research, VUB, Brussels, Belgium
| | - Tamara Vanhaecke
- Department of In Vitro Toxicology and Dermato-Cosmetology (IVTD), Center for Pharmaceutical Research, VUB, Brussels, Belgium
| | - Ellen Goossens
- Biology of the Testis (BITE) laboratory, Genetics Reproduction and Development (GRAD) research group, Vrije Universiteit Brussel (VUB), Brussels, Belgium
| | - Yoni Baert
- Biology of the Testis (BITE) laboratory, Genetics Reproduction and Development (GRAD) research group, Vrije Universiteit Brussel (VUB), Brussels, Belgium
- Department of In Vitro Toxicology and Dermato-Cosmetology (IVTD), Center for Pharmaceutical Research, VUB, Brussels, Belgium
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Lopez I, Truskey GA. Multi-cellular engineered living systems to assess reproductive toxicology. Reprod Toxicol 2024; 127:108609. [PMID: 38759876 PMCID: PMC11179964 DOI: 10.1016/j.reprotox.2024.108609] [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: 12/21/2023] [Revised: 05/07/2024] [Accepted: 05/09/2024] [Indexed: 05/19/2024]
Abstract
Toxicants and some drugs can negatively impact reproductive health. Many toxicants haven't been tested due to lack of available models. The impact of many drugs taken during pregnancy to address maternal health may adversely affect fetal development with life-long effects and clinical trials do not examine toxicity effects on the maternal-fetal interface, requiring indirect assessment of safety and efficacy. Due to current gaps in reproductive toxicological knowledge and limitations of animal models, multi-cellular engineered living systems may provide solutions for modeling reproductive physiology and pathology for chemical and xenobiotic toxicity studies. Multi-cellular engineered living systems, such as microphysiological systems (MPS) and organoids, model of functional units of tissues. In this review, we highlight the key functions and structures of human reproductive organs and well-known representative toxicants afflicting these systems. We then discuss current approaches and specific studies where scientists have used MPS or organoids to recreate in vivo markers and cellular responses of the female and male reproductive system, as well as pregnancy-associated placenta formation and embryo development. We provide specific examples of organoids and organ-on-chip that have been used for toxicological purposes with varied success. Finally, we address current issues related to usage of MPS, emerging techniques for improving upon these complications, and improvements needed to make MPS more capable in assessing reproductive toxicology. Overall, multi-cellular engineered living systems have considerable promise to serve as a suitable, alternative reproductive biological model compared to animal studies and 2D culture.
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Affiliation(s)
- Isabella Lopez
- Department of Biomedical Engineering, Duke University, Durham, NC 27708, United States
| | - George A Truskey
- Department of Biomedical Engineering, Duke University, Durham, NC 27708, United States.
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Mecca R, Tang S, Jones C, Coward K. The limitations of testicular organoids: are they truly as promising as we believe? Reprod Fertil Dev 2024; 36:RD23216. [PMID: 38935835 DOI: 10.1071/rd23216] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/07/2023] [Accepted: 05/31/2024] [Indexed: 06/29/2024] Open
Abstract
Organoid systems have revolutionised various facets of biological research by offering a three-dimensional (3D), physiologically relevant in vitro model to study complex organ systems. Over recent years, testicular organoids have been publicised as promising platforms for reproductive studies, disease modelling, drug screening, and fertility preservation. However, the full potential of these systems has yet to be realised due to inherent limitations. This paper offers a comprehensive analysis of the current challenges associated with testicular organoid models. Firstly, we address the inability of current organoid systems to fully replicate the intricate spatial organisation and cellular diversity of the in vivo testis. Secondly, we scrutinise the fidelity of germ cell maturation within the organoids, highlighting incomplete spermatogenesis and epigenetic inconsistencies. Thirdly, we consider the technical challenges faced during organoid culture, including nutrient diffusion limits, lack of vasculature, and the need for specialised growth factors. Finally, we discuss the ethical considerations surrounding the use of organoids for human reproduction research. Addressing these limitations in combination with integrating complementary approaches, will be essential if we are to advance our understanding of testicular biology and develop novel strategies for addressing reproductive health issues in males.
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Affiliation(s)
- R Mecca
- Nuffield Department of Women's and Reproductive Health, University of Oxford, Level 3, Women's Centre, John Radcliffe Hospital, Oxford OX3 9DU, UK
| | - S Tang
- Radcliffe Department of Medicine, MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK
| | - C Jones
- Nuffield Department of Women's and Reproductive Health, University of Oxford, Level 3, Women's Centre, John Radcliffe Hospital, Oxford OX3 9DU, UK
| | - K Coward
- Nuffield Department of Women's and Reproductive Health, University of Oxford, Level 3, Women's Centre, John Radcliffe Hospital, Oxford OX3 9DU, UK
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Bashiri Z, Gholipourmalekabadi M, Khadivi F, Salem M, Afzali A, Cham TC, Koruji M. In vitro spermatogenesis in artificial testis: current knowledge and clinical implications for male infertility. Cell Tissue Res 2023; 394:393-421. [PMID: 37721632 DOI: 10.1007/s00441-023-03824-z] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/16/2022] [Accepted: 08/14/2023] [Indexed: 09/19/2023]
Abstract
Men's reproductive health exclusively depends on the appropriate maturation of certain germ cells known as sperm. Certain illnesses, such as Klinefelter syndrome, cryptorchidism, and syndrome of androgen insensitivity or absence of testis maturation in men, resulting in the loss of germ cells and the removal of essential genes on the Y chromosome, can cause non-obstructive azoospermia. According to laboratory research, preserving, proliferating, differentiating, and transplanting spermatogonial stem cells or testicular tissue could be future methods for preserving the fertility of children with cancer and men with azoospermia. Therefore, new advances in stem cell research may lead to promising therapies for treating male infertility. The rate of progression and breakthrough in the area of in vitro spermatogenesis is lower than that of SSC transplantation, but newer methods are also being developed. In this regard, tissue and cell culture, supplements, and 3D scaffolds have opened new horizons in the differentiation of stem cells in vitro, which could improve the outcomes of male infertility. Various 3D methods have been developed to produce cellular aggregates and mimic the organization and function of the testis. The production of an artificial reproductive organ that supports SSCs differentiation will certainly be a main step in male infertility treatment.
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Affiliation(s)
- Zahra Bashiri
- Stem Cell and Regenerative Medicine Research Center, Iran University of Medical Sciences, Shahid Hemmat Highway, Tehran, 1449614535, Iran.
- Department of Anatomy, School of Medicine, Iran University of Medical Sciences, Shahid Hemmat Highway, Tehran, 1449614535, Iran.
- Omid Fertility & Infertility Clinic, Hamedan, Iran.
| | - Mazaher Gholipourmalekabadi
- Cellular and Molecular Research Center, Iran University of Medical Sciences, Tehran, Iran
- Department of Tissue Engineering & Regenerative Medicine, Faculty of Advanced Technologies in Medicine, Iran University of Medical Sciences, Tehran, Iran
- Department of Medical Biotechnology, Faculty of Allied Medicine, Iran University of Medical Sciences, Tehran, Iran
| | - Farnaz Khadivi
- Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
- Department of Anatomy, School of Medicine, Shahrekord University of Medical Sciences, Shahrekord, Iran
| | - Maryam Salem
- Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
| | - Azita Afzali
- Hajar Hospital, Shahrekord University of Medical Sciences, Shahrekord, Iran
| | - Tat-Chuan Cham
- Department of Veterinary Biomedical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, SK, S7N 5B4, Canada
| | - Morteza Koruji
- Stem Cell and Regenerative Medicine Research Center, Iran University of Medical Sciences, Shahid Hemmat Highway, Tehran, 1449614535, Iran.
- Department of Anatomy, School of Medicine, Iran University of Medical Sciences, Shahid Hemmat Highway, Tehran, 1449614535, Iran.
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Jokar J, Abdulabbas HT, Alipanah H, Ghasemian A, Ai J, Rahimian N, Mohammadisoleimani E, Najafipour S. Tissue engineering studies in male infertility disorder. HUM FERTIL 2023; 26:1617-1635. [PMID: 37791451 DOI: 10.1080/14647273.2023.2251678] [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] [Received: 06/11/2022] [Accepted: 07/06/2023] [Indexed: 10/05/2023]
Abstract
Infertility is an important issue among couples worldwide which is caused by a variety of complex diseases. Male infertility is a problem in 7% of all men. In vitro spermatogenesis (IVS) is the experimental approach that has been developed for mimicking seminiferous tubules-like functional structures in vitro. Currently, various researchers are interested in finding and developing a microenvironmental condition or a bioartificial testis applied for fertility restoration via gamete production in vitro. The tissue engineering (TE) has developed new approaches to treat male fertility preservation through development of functional male germ cells. This makes TE a possible future strategy for restoration of male fertility. Although 3D culture systems supply the perception of the effect of cellular interactions in the process of spermatogenesis, formation of a native gradient of autocrine/paracrine factors in 3D culture systems have not been considered. These results collectively suggest that maintaining the microenvironment of testicular cells even in the form of a 3D-culture system is crucial in achieving spermatogenesis ex vivo. It is also possible to engineer the testicular structures using biomaterials to provide a supporting scaffold for somatic and stem cells. The insemination of these cells with GFs is possible for temporally and spatially adjusted release to mimic the microenvironment of the in situ seminiferous epithelium. This review focuses on recent studies and advances in the application of TE strategies to cell-tissue culture on synthetic or natural scaffolds supplemented with growth factors.
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Affiliation(s)
- Javad Jokar
- Department of Tissue Engineering, Faculty of Medicine, Fasa University of Medical Science, Fasa, Iran
| | | | - Hiva Alipanah
- Department of Physiology, School of Medicine, Fasa University of Medical Science, Fasa, Iran
| | - Abdolmajid Ghasemian
- Noncommunicable Diseases Research Center, Fasa University of Medical Sciences, Fasa, Iran
| | - Jafar Ai
- Tissue Engineering and Applied Cell Sciences Department, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran
| | - Niloofar Rahimian
- Department of Biotechnology, Faculty of Medicine, Fasa University of Medical Sciences, Fasa, Iran
| | - Elham Mohammadisoleimani
- Department of Biotechnology, Faculty of Medicine, Fasa University of Medical Sciences, Fasa, Iran
| | - Sohrab Najafipour
- Department of Microbiology, Faculty of Medicine, Fasa University of Medical Sciences, Fasa, Iran
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Xin B, Wang Q, Wang X, Li F, Bai M, Fu H, Yan Z, Zhu Y, Huang X. Reduction of excessive unfolded protein response by 4-phenylbutyric acid may mitigate procymidone-induced testicular damage in mice by changing the levels of circRNA Scar and circZc3h4. PESTICIDE BIOCHEMISTRY AND PHYSIOLOGY 2023; 197:105689. [PMID: 38072544 DOI: 10.1016/j.pestbp.2023.105689] [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: 05/12/2023] [Revised: 11/02/2023] [Accepted: 11/05/2023] [Indexed: 12/18/2023]
Abstract
Procymidone (PCM) exposure below the no-observed-effect level triggers changes in circRNA Scar and circZc3h4 and overactivation of the unfolded protein response (UPR) in mice, culminating in testicular injury. The 4-phenyl butyric acid (4-PBA) is known to stabilize proteins and reduce the UPR. This study employed an in vitro system in which mouse testes were cultured with 1 × 10-5 M PCM and varying concentrations (0, 20, 40, and 80 mM) of 4-PBA; 4-week-old male mice were subsequently treated with 100 mg/kg/d PCM (suspended in corn oil) and/or 100 mg/kg/d 4-PBA for 21 d, consecutively. The treatments were as follows: the negative control (NC) group was orally administered corn oil; the positive control (PC) group was orally administered PCM; the 4-PBA group was intraperitoneally injected with 4-PBA; the 4-PBA-I group was orally administered PCM and 4-PBA simultaneously; the 4-PBA-II group received daily administration of 4-PBA 24 h prior to PCM; and the 4-PBA-III group was intraperitoneally injected with 4-PBA for 7 d after 21 d of PCM administration. However, the 4-PBA intervention groups showed no considerable changes in the overall or testicular appearance of mice. In vitro, 4-PBA inhibited the PCM-induced testicular injury, with the most significant effect observed at 80 mM. In vivo, the 4-PBA-III group exhibited the best in vivo effects. Our findings indicate that 4-PBA conferred testicular protection by decreasing PCM-induced circRNA Scar, elevating circZc3h4, and suppressing UPR both in vitro and in vivo. It has been hypothesized that 4-PBA mitigates testicular damage by reducing excessive UPR levels.
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Affiliation(s)
- Bingyan Xin
- Key Laboratory of Study and Discovery of Small Targeted Molecules of Hunan Province, Medical School, Hunan Normal University, Changsha 410013, China; Department of Preventive Medicine, Medical School, Hunan Normal University, Changsha 410013, China
| | - Qing Wang
- Key Laboratory of Study and Discovery of Small Targeted Molecules of Hunan Province, Medical School, Hunan Normal University, Changsha 410013, China; Department of Preventive Medicine, Medical School, Hunan Normal University, Changsha 410013, China
| | - Xuning Wang
- Key Laboratory of Study and Discovery of Small Targeted Molecules of Hunan Province, Medical School, Hunan Normal University, Changsha 410013, China; Department of Preventive Medicine, Medical School, Hunan Normal University, Changsha 410013, China
| | - Fan Li
- Key Laboratory of Study and Discovery of Small Targeted Molecules of Hunan Province, Medical School, Hunan Normal University, Changsha 410013, China; Department of Preventive Medicine, Medical School, Hunan Normal University, Changsha 410013, China
| | - Mingxin Bai
- Key Laboratory of Study and Discovery of Small Targeted Molecules of Hunan Province, Medical School, Hunan Normal University, Changsha 410013, China; Department of Preventive Medicine, Medical School, Hunan Normal University, Changsha 410013, China
| | - Hu Fu
- Key Laboratory of Study and Discovery of Small Targeted Molecules of Hunan Province, Medical School, Hunan Normal University, Changsha 410013, China; Department of Preventive Medicine, Medical School, Hunan Normal University, Changsha 410013, China
| | - Zhengli Yan
- Key Laboratory of Study and Discovery of Small Targeted Molecules of Hunan Province, Medical School, Hunan Normal University, Changsha 410013, China; Department of Preventive Medicine, Medical School, Hunan Normal University, Changsha 410013, China
| | - Yongfei Zhu
- Key Laboratory of Study and Discovery of Small Targeted Molecules of Hunan Province, Medical School, Hunan Normal University, Changsha 410013, China; Department of Preventive Medicine, Medical School, Hunan Normal University, Changsha 410013, China.
| | - Xin Huang
- Key Laboratory of Study and Discovery of Small Targeted Molecules of Hunan Province, Medical School, Hunan Normal University, Changsha 410013, China; Department of Preventive Medicine, Medical School, Hunan Normal University, Changsha 410013, China.
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11
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Hau RK, Wright SH, Cherrington NJ. In Vitro and In Vivo Models for Drug Transport Across the Blood-Testis Barrier. Drug Metab Dispos 2023; 51:1157-1168. [PMID: 37258305 PMCID: PMC10449102 DOI: 10.1124/dmd.123.001288] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/03/2023] [Revised: 05/10/2023] [Accepted: 05/25/2023] [Indexed: 06/02/2023] Open
Abstract
The blood-testis barrier (BTB) is a selectively permeable membrane barrier formed by adjacent Sertoli cells (SCs) in the seminiferous tubules of the testes that develops intercellular junctional complexes to protect developing germ cells from external pressures. However, due to this inherent defense mechanism, the seminiferous tubule lumen can act as a pharmacological sanctuary site for latent viruses (e.g., Ebola, Zika) and cancers (e.g., leukemia). Therefore, it is critical to identify and evaluate BTB carrier-mediated drug delivery pathways to successfully treat these viruses and cancers. Many drugs are unable to effectively cross cell membranes without assistance from carrier proteins like transporters because they are large, polar, and often carry a charge at physiologic pH. SCs express transporters that selectively permit endogenous compounds, such as carnitine or nucleosides, across the BTB to support normal physiologic activity, although reproductive toxicants can also use these pathways, thereby circumventing the BTB. Certain xenobiotics, including select cancer therapeutics, antivirals, contraceptives, and environmental toxicants, are known to accumulate within the male genital tract and cause testicular toxicity; however, the transport pathways by which these compounds circumvent the BTB are largely unknown. Consequently, there is a need to identify the clinically relevant BTB transport pathways in in vitro and in vivo BTB models that recapitulate human pharmacokinetics and pharmacodynamics for these xenobiotics. This review summarizes the various in vitro and in vivo models of the BTB reported in the literature and highlights the strengths and weaknesses of certain models for drug disposition studies. SIGNIFICANCE STATEMENT: Drug disposition to the testes is influenced by the physical, physiological, and immunological components of the blood-testis barrier (BTB). But many compounds are known to cross the BTB by transporters, resulting in pharmacological and/or toxicological effects in the testes. Therefore, models that assess drug transport across the human BTB must adequately account for these confounding factors. This review identifies and discusses the benefits and limitations of various in vitro and in vivo BTB models for preclinical drug disposition studies.
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Affiliation(s)
- Raymond K Hau
- College of Pharmacy, Department of Pharmacology & Toxicology, (R.K.H., N.J.C.) and College of Medicine, Department of Physiology, The University of Arizona, Tucson, Arizona (S.H.W.)
| | - Stephen H Wright
- College of Pharmacy, Department of Pharmacology & Toxicology, (R.K.H., N.J.C.) and College of Medicine, Department of Physiology, The University of Arizona, Tucson, Arizona (S.H.W.)
| | - Nathan J Cherrington
- College of Pharmacy, Department of Pharmacology & Toxicology, (R.K.H., N.J.C.) and College of Medicine, Department of Physiology, The University of Arizona, Tucson, Arizona (S.H.W.)
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12
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Kulibin AY, Malolina EA. In vitro spermatogenesis: In search of fully defined conditions. Front Cell Dev Biol 2023; 11:1106111. [PMID: 36910153 PMCID: PMC9998899 DOI: 10.3389/fcell.2023.1106111] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/23/2022] [Accepted: 02/08/2023] [Indexed: 03/14/2023] Open
Abstract
A complete reconstruction of spermatogenesis in vitro under fully defined conditions still has not been achieved. However, many techniques have been proposed to get closer to that aim. Here we review the current progress in the field. At first, we describe the most successful technique, the organ culture method, which allows to produce functional haploid cells. However, this method is based on the culturing of intact testis tissue with unknown factors acting inside it. Then we discuss different types of 3D-cultures where specific testicular cell populations may be aggregated and the impact of each cell population may be examined. Unfortunately, germ cell development does not proceed further than the pachytene stage of meiosis there, with rare exceptions. Finally, we describe recent studies that focus on germ cells in a conventional adherent cell culture. Such studies thoroughly examine issues with in vitro meiosis and provide insight into the mechanisms of meiotic initiation.
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Affiliation(s)
- A Yu Kulibin
- Koltzov Institute of Developmental Biology of the Russian Academy of Sciences, Moscow, Russia
| | - E A Malolina
- Koltzov Institute of Developmental Biology of the Russian Academy of Sciences, Moscow, Russia
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13
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Inoue M, Baba T, Takahashi F, Terao M, Yanai S, Shima Y, Saito D, Sugihara K, Miura T, Takada S, Suyama M, Ohkawa Y, Morohashi KI. Tmsb10 triggers fetal Leydig differentiation by suppressing the RAS/ERK pathway. Commun Biol 2022; 5:974. [PMID: 36109592 PMCID: PMC9478096 DOI: 10.1038/s42003-022-03941-5] [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: 03/18/2022] [Accepted: 09/02/2022] [Indexed: 11/24/2022] Open
Abstract
Leydig cells in fetal testes play crucial roles in masculinizing fetuses through androgen production. Gene knockout studies have revealed that growth factors are implicated in fetal Leydig cell (FLC) differentiation, but little is known about the mechanisms regulating this process. We investigate this issue by characterizing FLC progenitor cells using single-cell RNA sequencing. The sequence datasets suggest that thymosin β10 (Tmsb10) is transiently upregulated in the progenitors. While studying the function of Tmsb10, we reveal that platelet-derived growth factor (PDGF) regulates ciliogenesis through the RAS/ERK and PI3K/AKT pathways, and thereby promotes desert hedgehog (DHH)-dependent FLC differentiation. Tmsb10 expressed in the progenitor cells induces their differentiation into FLCs by suppressing the RAS/ERK pathway. Through characterizing the transiently expressed Tmsb10 in the FLC progenitors, this study unveils the molecular process of FLC differentiation and shows that it is cooperatively induced by DHH and PDGF. Investigation of fetal Leydig progenitors shows that thymosin β10 (Tmsb10) suppresses the RAS/ERK pathway, inducing progenitor differentiation into fetal Leydig cells.
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14
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Liu X, Wu K, Gao L, Wang L, Shi X. Biomaterial strategies for the application of reproductive tissue engineering. Bioact Mater 2022; 14:86-96. [PMID: 35310354 PMCID: PMC8892081 DOI: 10.1016/j.bioactmat.2021.11.023] [Citation(s) in RCA: 17] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/08/2021] [Revised: 11/13/2021] [Accepted: 11/22/2021] [Indexed: 02/06/2023] Open
Abstract
Human reproductive organs are of vital importance to the life of an individual and the reproduction of human populations. So far, traditional methods have a limited effect in recovering the function and fertility of reproductive organs and tissues. Thus, aim to replace and facilitate the regrowth of damaged or diseased tissue, various biomaterials are developed to offer hope to overcome these difficulties and help gain further research progress in reproductive tissue engineering. In this review, we focus on the biomaterials and their four main applications in reproductive tissue engineering: in vitro generation and culture of reproductive cells; development of reproductive organoids and models; in vivo transplantation of reproductive cells or tissues; and regeneration of reproductive tissue. In reproductive tissue engineering, designing biomaterials for different applications with different mechanical properties, structure, function, and microenvironment is challenging and important, and deserves more attention.
Various biomaterials have been developed and used in reproductive tissue engineering. 3D culture systems can lead to better cell-cell interactions for in vitro production of reproductive cells. Reproductive organoids and models are formed by biomaterials to simulate the environment of natural reproductive organs. Biomaterials should promote vascular regeneration and resist inflammation for in-situ reproductive tissue regeneration.
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15
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Diao L, Turek PJ, John CM, Fang F, Reijo Pera RA. Roles of Spermatogonial Stem Cells in Spermatogenesis and Fertility Restoration. Front Endocrinol (Lausanne) 2022; 13:895528. [PMID: 35634498 PMCID: PMC9135128 DOI: 10.3389/fendo.2022.895528] [Citation(s) in RCA: 27] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/14/2022] [Accepted: 03/31/2022] [Indexed: 01/21/2023] Open
Abstract
Spermatogonial stem cells (SSCs) are a group of adult stem cells in the testis that serve as the foundation of continuous spermatogenesis and male fertility. SSCs are capable of self-renewal to maintain the stability of the stem cell pool and differentiation to produce mature spermatozoa. Dysfunction of SSCs leads to male infertility. Therefore, dissection of the regulatory network of SSCs is of great significance in understanding the fundamental molecular mechanisms of spermatogonial stem cell function in spermatogenesis and the pathogenesis of male infertility. Furthermore, a better understanding of SSC biology will allow us to culture and differentiate SSCs in vitro, which may provide novel stem cell-based therapy for assisted reproduction. This review summarizes the latest research progress on the regulation of SSCs, and the potential application of SSCs for fertility restoration through in vivo and in vitro spermatogenesis. We anticipate that the knowledge gained will advance the application of SSCs to improve male fertility. Furthermore, in vitro spermatogenesis from SSCs sets the stage for the production of SSCs from induced pluripotent stem cells (iPSCs) and subsequent spermatogenesis.
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Affiliation(s)
- Lei Diao
- The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, China
| | | | | | - Fang Fang
- The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, China
| | - Renee A. Reijo Pera
- McLaughlin Research Institute, Touro College of Osteopathic Medicine – Montana (TouroCOM-MT), Great Falls, MT, United States
- Research Division, Touro College of Osteopathic Medicine – Montana (TouroCOM-MT), Great Falls, MT, United States
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16
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Tran KTD, Valli-Pulaski H, Colvin A, Orwig KE. Male fertility preservation and restoration strategies for patients undergoing gonadotoxic therapies†. Biol Reprod 2022; 107:382-405. [PMID: 35403667 PMCID: PMC9382377 DOI: 10.1093/biolre/ioac072] [Citation(s) in RCA: 29] [Impact Index Per Article: 9.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2021] [Revised: 03/29/2022] [Accepted: 04/04/2022] [Indexed: 11/22/2022] Open
Abstract
Medical treatments for cancers or other conditions can lead to permanent infertility. Infertility is an insidious disease that impacts not only the ability to have a biological child but also the emotional well-being of the infertile individuals, relationships, finances, and overall health. Therefore, all patients should be educated about the effects of their medical treatments on future fertility and about fertility preservation options. The standard fertility preservation option for adolescent and adult men is sperm cryopreservation. Sperms can be frozen and stored for a long period, thawed at a later date, and used to achieve pregnancy with existing assisted reproductive technologies. However, sperm cryopreservation is not applicable for prepubertal patients who do not yet produce sperm. The only fertility preservation option available to prepubertal boys is testicular tissue cryopreservation. Next-generation technologies are being developed to mature those testicular cells or tissues to produce fertilization-competent sperms. When sperm and testicular tissues are not available for fertility preservation, inducing pluripotent stem cells derived from somatic cells, such as blood or skin, may provide an alternative path to produce sperms through a process call in vitro gametogenesis. This review describes standard and experimental options to preserve male fertility as well as the experimental options to produce functional spermatids or sperms from immature cryopreserved testicular tissues or somatic cells.
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Affiliation(s)
- Kien T D Tran
- Molecular Genetics and Developmental Biology Graduate Program, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA,Department of Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA,Magee-Womens Research Institute, Pittsburgh, PA, USA
| | - Hanna Valli-Pulaski
- Department of Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA,Magee-Womens Research Institute, Pittsburgh, PA, USA
| | - Amanda Colvin
- Department of Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA,Magee-Womens Research Institute, Pittsburgh, PA, USA
| | - Kyle E Orwig
- Correspondence: Department of Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh School of Medicine, Magee-Womens Research Institute, 204 Craft Avenue, Pittsburgh, PA 15213, USA. Tel: 412-641-2460; E-mail:
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17
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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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18
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Gholami K, Solhjoo S, Aghamir SMK. Application of Tissue-Specific Extracellular Matrix in Tissue Engineering: Focus on Male Fertility Preservation. Reprod Sci 2022; 29:3091-3099. [PMID: 35028926 DOI: 10.1007/s43032-021-00823-9] [Citation(s) in RCA: 12] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/01/2021] [Accepted: 12/03/2021] [Indexed: 11/28/2022]
Abstract
In vitro spermatogenesis and xenotransplantation of the immature testicular tissues (ITT) are the experimental approaches that have been developed for creating seminiferous tubules-like functional structures in vitro and keeping the integrity of the ITTs in vivo, respectively. These strategies are rapidly developing in response to the growing prevalence of infertility in adolescent boys undergoing cancer treatment, by the logic that there is no sperm cryopreservation option for them. Recently, with the advances made in the field of tissue engineering and biomaterials, these methods have achieved promising results for fertility preservation. Due to the importance of extracellular matrix for the formation of vascular bed around the grafted ITTs and also the creation of spatial arrangements between Sertoli cells and germ cells, today it is clear that the scaffold plays a very important role in the success of these methods. Decellularized extracellular matrix (dECM) as a biocompatible, functionally graded, and biodegradable scaffold with having tissue-specific components and growth factors can support reorganization and physiologic processes of originated cells. This review discusses the common protocols for the tissue decellularization, sterilization, and hydrogel formation of the decellularized and lyophilized tissues as well as in vitro and in vivo studies on the use of the testis-derived dECM for testicular organoids.
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Affiliation(s)
- Keykavos Gholami
- Urology Research Center, Tehran University of Medical Sciences, Tehran, Iran
| | - Somayeh Solhjoo
- Department of Anatomy, Kerman University of Medical Sciences, Kerman, Iran
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19
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Richer G, Hobbs RM, Loveland KL, Goossens E, Baert Y. Long-Term Maintenance and Meiotic Entry of Early Germ Cells in Murine Testicular Organoids Functionalized by 3D Printed Scaffolds and Air-Medium Interface Cultivation. Front Physiol 2022; 12:757565. [PMID: 35002756 PMCID: PMC8739976 DOI: 10.3389/fphys.2021.757565] [Citation(s) in RCA: 14] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/12/2021] [Accepted: 11/18/2021] [Indexed: 01/15/2023] Open
Abstract
Short-term germ cell survival and central tissue degeneration limit organoid cultures. Here, testicular organoids (TOs) were generated from two different mouse strains in 3D printed one-layer scaffolds (1LS) at the air-medium interface displaying tubule-like structures and Leydig cell functionality supporting long-term survival and differentiation of germ cells to the meiotic phase. Chimeric TOs, consisting of a mixture of primary testicular cells and EGFP+ germline stem (GS) cells, were cultured in two-layer scaffolds (2LSs) for better entrapment. They showed an improved spheroidal morphology consisting of one intact tubule-like structure and surrounding interstitium, representing the functional unit of a testis. However, GS cells did not survive long-term culture. Consequently, further optimization of the culture medium is required to enhance the maintenance and differentiation of germ cells. The opportunities TOs offer to manipulate somatic and germ cells are essential for the study of male infertility and the search for potential therapies.
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Affiliation(s)
- Guillaume Richer
- Biology of the Testis Lab, Vrije Universiteit Brussel (VUB), University Medical Campus, Brussels, Belgium
| | - Robin M Hobbs
- Centre for Reproductive Health, Hudson Institute of Medical Research, Clayton, VIC, Australia
| | - Katherine L Loveland
- Centre for Reproductive Health, Hudson Institute of Medical Research, Clayton, VIC, Australia.,Department of Molecular and Translational Sciences, School of Clinical Sciences, Monash Medical Centre, Monash University, Clayton, VIC, Australia
| | - Ellen Goossens
- Biology of the Testis Lab, Vrije Universiteit Brussel (VUB), University Medical Campus, Brussels, Belgium
| | - Yoni Baert
- Biology of the Testis Lab, Vrije Universiteit Brussel (VUB), University Medical Campus, Brussels, Belgium
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20
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Cham TC, Ibtisham F, Fayaz MA, Honaramooz A. Generation of a Highly Biomimetic Organoid, Including Vasculature, Resembling the Native Immature Testis Tissue. Cells 2021; 10:cells10071696. [PMID: 34359871 PMCID: PMC8305979 DOI: 10.3390/cells10071696] [Citation(s) in RCA: 23] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/29/2021] [Revised: 06/27/2021] [Accepted: 07/04/2021] [Indexed: 12/25/2022] Open
Abstract
The creation of a testis organoid (artificial testis tissue) with sufficient resemblance to the complex form and function of the innate testis remains challenging, especially using non-rodent donor cells. Here, we report the generation of an organoid culture system with striking biomimicry of the native immature testis tissue, including vasculature. Using piglet testis cells as starting material, we optimized conditions for the formation of cell spheroids, followed by long-term culture in an air–liquid interface system. Both fresh and frozen-thawed cells were fully capable of self-reassembly into stable testis organoids consisting of tubular and interstitial compartments, with all major cell types and structural details expected in normal testis tissue. Surprisingly, our organoids also developed vascular structures; a phenomenon that has not been reported in any other culture system. In addition, germ cells do not decline over time, and Leydig cells release testosterone, hence providing a robust, tunable system for diverse basic and applied applications.
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21
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Wyns C, Kanbar M, Giudice MG, Poels J. Fertility preservation for prepubertal boys: lessons learned from the past and update on remaining challenges towards clinical translation. Hum Reprod Update 2020; 27:433-459. [PMID: 33326572 DOI: 10.1093/humupd/dmaa050] [Citation(s) in RCA: 34] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/01/2020] [Revised: 09/25/2020] [Indexed: 01/01/2023] Open
Abstract
BACKGROUND Childhood cancer incidence and survivorship are both on the rise. However, many lifesaving treatments threaten the prepubertal testis. Cryopreservation of immature testicular tissue (ITT), containing spermatogonial stem cells (SSCs), as a fertility preservation (FP) option for this population is increasingly proposed worldwide. Recent achievements notably the birth of non-human primate (NHP) progeny using sperm developed in frozen-thawed ITT autografts has given proof of principle of the reproductive potential of banked ITT. Outlining the current state of the art on FP for prepubertal boys is crucial as some of the boys who have cryopreserved ITT since the early 2000s are now in their reproductive age and are already seeking answers with regards to their fertility. OBJECTIVE AND RATIONALE In the light of past decade achievements and observations, this review aims to provide insight into relevant questions for clinicians involved in FP programmes. Have the indications for FP for prepubertal boys changed over time? What is key for patient counselling and ITT sampling based on the latest achievements in animals and research performed with human ITT? How far are we from clinical application of methods to restore reproductive capacity with cryostored ITT? SEARCH METHODS An extensive search for articles published in English or French since January 2010 to June 2020 using keywords relevant to the topic of FP for prepubertal boys was made in the MEDLINE database through PubMed. Original articles on fertility preservation with emphasis on those involving prepubertal testicular tissue, as well as comprehensive and systematic reviews were included. Papers with redundancy of information or with an absence of a relevant link for future clinical application were excluded. Papers on alternative sources of stem cells besides SSCs were excluded. OUTCOMES Preliminary follow-up data indicate that around 27% of boys who have undergone testicular sampling as an FP measure have proved azoospermic and must therefore solely rely on their cryostored ITT to ensure biologic parenthood. Auto-transplantation of ITT appears to be the first technique that could enter pilot clinical trials but should be restricted to tissue free of malignant cells. While in vitro spermatogenesis circumvents the risk linked to cancer cell contamination and has led to offspring in mice, complete spermatogenesis has not been achieved with human ITT. However, generation of haploid germ cells paves the way to further studies aimed at completing the final maturation of germ cells and increasing the efficiency of the processes. WIDER IMPLICATIONS Despite all the research done to date, FP for prepubertal boys remains a relatively young field and is often challenging to healthcare providers, patients and parents. As cryopreservation of ITT is now likely to expand further, it is important not only to acknowledge some of the research questions raised on the topic, e.g. the epigenetic and genetic integrity of gametes derived from strategies to restore fertility with banked ITT but also to provide healthcare professionals worldwide with updated knowledge to launch proper multicollaborative care pathways in the field and address clinical issues that will come-up when aiming for the child's best interest.
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Affiliation(s)
- Christine Wyns
- Andrology lab, Institut de Recherche Expérimentale et Clinique (IREC), Université Catholique de Louvain, Brussels, Belgium.,Department of Gynecology-Andrology, Cliniques Universitaires Saint-Luc, Brussels, Belgium
| | - Marc Kanbar
- Andrology lab, Institut de Recherche Expérimentale et Clinique (IREC), Université Catholique de Louvain, Brussels, Belgium.,Department of Gynecology-Andrology, Cliniques Universitaires Saint-Luc, Brussels, Belgium
| | - Maria Grazia Giudice
- Andrology lab, Institut de Recherche Expérimentale et Clinique (IREC), Université Catholique de Louvain, Brussels, Belgium.,Department of Gynecology-Andrology, Cliniques Universitaires Saint-Luc, Brussels, Belgium
| | - Jonathan Poels
- Andrology lab, Institut de Recherche Expérimentale et Clinique (IREC), Université Catholique de Louvain, Brussels, Belgium.,Department of Gynecology-Andrology, Cliniques Universitaires Saint-Luc, Brussels, Belgium
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22
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Sabetkish S, Kajbafzadeh AM, Sabetkish N. Recellularization of testicular feminization testis in C57bl6 as a natural bioreactor for creation of cellularized seminiferous tubules: an experimental study. Cell Tissue Bank 2020; 22:287-295. [PMID: 33146808 DOI: 10.1007/s10561-020-09877-6] [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: 07/06/2019] [Accepted: 10/22/2020] [Indexed: 11/24/2022]
Abstract
We determined histological aspects of implanted human decellularized testicular matrix (DTM) in C57BL6 as a primitive step for further testis tissue engineering. A total of 4 immature human testicles were obtained after bilateral orchiectomy from patients with testicular feminization syndrome. The optimal decellularization protocol was determined and the efficacy of decellularization was evaluated in two of the testicles. The remaining scaffolds were cut in 3 × 3 mm3 pieces and implanted between the tight muscles in 32 C57BL6. Biopsies were taken at 2, 4, 8, and 24 weeks postoperatively and stained with PLZF, protamine, and tekt1 markers. Histological examination of DTMs confirmed complete absence of nuclear remnants and preservation of the extracellular matrix. Successful cell seeding was observed in all follow-ups confirmed by H&E and IHC staining that increased continuously during the whole study. Interestingly, spermatogonial stem-like cells were observed on decellularized implants that were well differentiated during the follow-ups. Natural bioreactors may provide a good cell source for testes tissue regeneration. This technique may provide testis bioscaffold as a three-dimensional platform and further successful cell seeding to produce a functional testis. This novel technique may be beneficial for patients who require testicular supplementation.
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Affiliation(s)
- Shabnam Sabetkish
- Pediatric Urology and Regenerative Medicine Research Center, Section of Tissue Engineering and Stem Cells Therapy, Children's Hospital Medical Center, Tehran University of Medical Sciences, No. 62, Dr. Gharib's Street, Keshavarz Boulevard, Tehran, 1419433151, Iran
| | - Abdol-Mohammad Kajbafzadeh
- Pediatric Urology and Regenerative Medicine Research Center, Section of Tissue Engineering and Stem Cells Therapy, Children's Hospital Medical Center, Tehran University of Medical Sciences, No. 62, Dr. Gharib's Street, Keshavarz Boulevard, Tehran, 1419433151, Iran.
| | - Nastaran Sabetkish
- Pediatric Urology and Regenerative Medicine Research Center, Section of Tissue Engineering and Stem Cells Therapy, Children's Hospital Medical Center, Tehran University of Medical Sciences, No. 62, Dr. Gharib's Street, Keshavarz Boulevard, Tehran, 1419433151, Iran
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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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Edmonds ME, Woodruff TK. Testicular organoid formation is a property of immature somatic cells, which self-assemble and exhibit long-term hormone-responsive endocrine function. Biofabrication 2020; 12:045002. [PMID: 32492667 DOI: 10.1088/1758-5090/ab9907] [Citation(s) in RCA: 33] [Impact Index Per Article: 6.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Testicular organoid models are tools to study testicular physiology, development, and spermatogenesis in vitro. However, few side-by-side comparisons of organoid generation method have been evaluated. Here, we directly tested whether the culture microenvironment is the prime determinant promoting testicular organoid self-assembly. Using Matrigel as a representative extracellular matrix (ECM), we compared multiple culture environments, 2D and 3D, ECM-free and ECM, for organoid self-assembly with immature murine testicular cells. De novo tissues were observed to self-assemble in all four culture environments tested within 72 h, however, these tissues only met requirements to be named organoids in 2D ECM and 3D ECM-free (3DF) culture methods. Based on these results, 3DF was selected for further study, and used to examine animal age as an independent variable. Organoid assembly was significantly delayed when using pubertal murine cells and entirely absent from adult murine and adult human cells. Organoid-conditioned medium and medium supplemented with 1% Matrigel did not improve organoid assembly in pubertal murine cells, but immature murine cells rescued the assembly of adult murine cells when cultured together as age-chimeric cell mixtures. In murine organoids cultured for 14 d, tubule-like structures exhibiting a highly biomimetic architecture were characterized, including some rare germ and spermatogonial stem cells. These structural organoids secreted high levels of testosterone and inhibin B over 12 weeks with preserved responsivity to gonadotropins. Collectively these studies, in which cellular self-assembly and organoid formation was achieved independent of the culture microenvironment, suggest that self-assembly is an innate property of immature testicular cells independent from, but capable of being promoted by, the culture environment. This study provides a template for studying testicular organoid self-assembly and endocrine function, and a platform for improving the engineering of functional testicular tissues.
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Affiliation(s)
- Maxwell E Edmonds
- Department of Obstetrics and Gynecology, Northwestern University Feinberg School of Medicine, Chicago, IL, United States of America
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25
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Gao H, Liu C, Wu B, Cui H, Zhao Y, Duan Y, Gao F, Gu Q, Wang H, Li W. Effects of Different Biomaterials and Cellular Status on Testicular Cell Self-Organization. ACTA ACUST UNITED AC 2020; 4:e1900292. [PMID: 32453509 DOI: 10.1002/adbi.201900292] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/10/2019] [Revised: 05/03/2020] [Indexed: 01/12/2023]
Abstract
A multicellular organism's development is coupled with cellular self-organization, which is regulated by cell-cell interactions and cell-extracellular matrix (ECM) crosstalk. Testicular cells from different species such as mouse, rat, and porcine can self-organize into seminiferous tubules both in vitro and in vivo, but the understanding of the functional role of the ECM during this process is limited. Here, it is shown that mouse testicular cells encapsulated with the biomaterial Matrigel can self-organize into seminiferous tubules with blood-testis barrier (BTB) formation and Leydig cell differentiation. By varying the encapsulation method, a combination of sodium alginate and collagen is used to promote reorganization of seminiferous tubules, which resemble those in vivo. In addition, the self-organization ability of testicular cells declines with advanced cell age, and those germ cells play a pivotal role in this process. These findings will be helpful to understand the self-organization process of testicular cells and provide insights for the reconstruction of testes.
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Affiliation(s)
- Hui Gao
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, P. R. China
| | - Chao Liu
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, P. R. China
| | - Bingbing Wu
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, P. R. China.,Colleague of Life Science, University of Chinese Academy of Sciences, Beijing, 100049, P. R. China
| | - Hang Cui
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, P. R. China
| | - Yan Zhao
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, P. R. China.,Colleague of Life Science, University of Chinese Academy of Sciences, Beijing, 100049, P. R. China
| | - Yongchao Duan
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, P. R. China
| | - Fei Gao
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, P. R. China.,Colleague of Life Science, University of Chinese Academy of Sciences, Beijing, 100049, P. R. China
| | - Qi Gu
- State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, P. R. China.,Stem cell and Regenerative Medicine Innovation Institute, Chinese Academy of Sciences, Beijing, 100101, P. R. China
| | - Hongmei Wang
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, P. R. China.,Colleague of Life Science, University of Chinese Academy of Sciences, Beijing, 100049, P. R. China
| | - Wei Li
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, P. R. China.,Colleague of Life Science, University of Chinese Academy of Sciences, Beijing, 100049, P. R. China
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26
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Ibtisham F, Honaramooz A. Spermatogonial Stem Cells for In Vitro Spermatogenesis and In Vivo Restoration of Fertility. Cells 2020; 9:E745. [PMID: 32197440 PMCID: PMC7140722 DOI: 10.3390/cells9030745] [Citation(s) in RCA: 62] [Impact Index Per Article: 12.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/01/2020] [Revised: 03/15/2020] [Accepted: 03/16/2020] [Indexed: 12/14/2022] Open
Abstract
Spermatogonial stem cells (SSCs) are the only adult stem cells capable of passing genes onto the next generation. SSCs also have the potential to provide important knowledge about stem cells in general and to offer critical in vitro and in vivo applications in assisted reproductive technologies. After century-long research, proof-of-principle culture systems have been introduced to support the in vitro differentiation of SSCs from rodent models into haploid male germ cells. Despite recent progress in organotypic testicular tissue culture and two-dimensional or three-dimensional cell culture systems, to achieve complete in vitro spermatogenesis (IVS) using non-rodent species remains challenging. Successful in vitro production of human haploid male germ cells will foster hopes of preserving the fertility potential of prepubertal cancer patients who frequently face infertility due to the gonadotoxic side-effects of cancer treatment. Moreover, the development of optimal systems for IVS would allow designing experiments that are otherwise difficult or impossible to be performed directly in vivo, such as genetic manipulation of germ cells or correction of genetic disorders. This review outlines the recent progress in the use of SSCs for IVS and potential in vivo applications for the restoration of fertility.
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Affiliation(s)
| | - Ali Honaramooz
- Department of Veterinary Biomedical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, SK S7N 5B4, Canada;
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27
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Richer G, Baert Y, Goossens E. In-vitro spermatogenesis through testis modelling: Toward the generation of testicular organoids. Andrology 2020; 8:879-891. [PMID: 31823507 PMCID: PMC7496450 DOI: 10.1111/andr.12741] [Citation(s) in RCA: 49] [Impact Index Per Article: 9.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/29/2019] [Revised: 10/17/2019] [Accepted: 12/04/2019] [Indexed: 01/09/2023]
Abstract
Background The testicular organoid concept has recently been introduced in tissue engineering to refer to testicular cell organizations modeling testicular architecture and function. The testicular organoid approach gives control over which and how cells reaggregate, which is not possible in organotypic cultures, thereby extending the applicability of in‐vitro spermatogenesis (IVS) systems. However, it remains unclear which culture method and medium allow reassociation of testicular cells into a functional testicular surrogate in‐vitro. Objective The aim of this paper is to review the different strategies that have been used in an attempt to create testicular organoids and generate spermatozoa. We want to provide an up‐to‐date list on culture methodologies and media compositions that have been used and determine their role in regulating tubulogenesis and differentiation of testicular cells. Search method A literature search was conducted in PubMed, Web of Science, and Scopus to select studies reporting the reorganization of testicular cell suspensions in‐vitro, using the keywords: three‐dimensional culture, in‐vitro spermatogenesis, testicular organoid, testicular scaffold, and tubulogenesis. Papers published before the August 1, 2019, were selected. Outcome Only a limited number of studies have concentrated on recreating the testicular architecture in‐vitro. While some advances have been made in the testicular organoid research in terms of cellular reorganization, none of the described culture systems is adequate for the reproduction of both the testicular architecture and IVS. Conclusion Further improvements in culture methodology and medium composition have to be made before being able to provide both testicular tubulogenesis and spermatogenesis in‐vitro.
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Affiliation(s)
- Guillaume Richer
- Biology of the Testis research Lab, Department of Reproduction, Genetics and Regenerative Medicine, Vrije Universiteit Brussel (VUB), Brussels, Belgium
| | - Yoni Baert
- Biology of the Testis research Lab, Department of Reproduction, Genetics and Regenerative Medicine, Vrije Universiteit Brussel (VUB), Brussels, Belgium
| | - Ellen Goossens
- Biology of the Testis research Lab, Department of Reproduction, Genetics and Regenerative Medicine, Vrije Universiteit Brussel (VUB), Brussels, Belgium
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28
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Vermeulen M, Del Vento F, Kanbar M, Pyr Dit Ruys S, Vertommen D, Poels J, Wyns C. Generation of Organized Porcine Testicular Organoids in Solubilized Hydrogels from Decellularized Extracellular Matrix. Int J Mol Sci 2019; 20:E5476. [PMID: 31684200 PMCID: PMC6862040 DOI: 10.3390/ijms20215476] [Citation(s) in RCA: 64] [Impact Index Per Article: 10.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/27/2019] [Revised: 10/25/2019] [Accepted: 10/30/2019] [Indexed: 01/15/2023] Open
Abstract
Cryopreservation of immature testicular tissue (ITT) prior to chemo/radiotherapy is now ethically accepted and is currently the only way to preserve fertility of prepubertal boys about to undergo cancer therapies. So far, three-dimensional culture of testicular cells isolated from prepubertal human testicular tissue was neither efficient nor reproducible to obtain mature spermatozoa, and ITT transplantation is not a safe option when there is a risk of cancer cell contamination of the testis. Hence, generation of testicular organoids (TOs) after cell selection is a novel strategy aimed at restoring fertility in these patients. Here, we created TOs using hydrogels developed from decellularized porcine ITT and compared cell numbers, organization and function to TOs generated in collagen only hydrogel. Organotypic culture of porcine ITT was used as a control. Rheological and mass spectrometry analyses of both hydrogels highlighted differences in terms of extracellular matrix stiffness and composition, respectively. Sertoli cells (SCs) and germ cells (GCs) assembled into seminiferous tubule-like structures delimited by a basement membrane while Leydig cells (LCs) and peritubular cells localized outside. TOs were maintained for 45 days in culture and secreted stem cell factor and testosterone demonstrating functionality of SCs and LCs, respectively. In both TOs GC numbers decreased and SC numbers increased. However, LC numbers decreased significantly in the collagen hydrogel TOs (p < 0.05) suggesting a better preservation of growth factors within TOs developed from decellularized ITT and thus a better potential to restore the reproductive capacity.
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Affiliation(s)
- Maxime Vermeulen
- Gynecology-Andrology Research Unit, Institut de Recherche Expérimentale et Clinique, Medical School, Université Catholique de Louvain, 1200 Brussels, Belgium.
| | - Federico Del Vento
- Gynecology-Andrology Research Unit, Institut de Recherche Expérimentale et Clinique, Medical School, Université Catholique de Louvain, 1200 Brussels, Belgium.
| | - Marc Kanbar
- Gynecology-Andrology Research Unit, Institut de Recherche Expérimentale et Clinique, Medical School, Université Catholique de Louvain, 1200 Brussels, Belgium.
| | - Sébastien Pyr Dit Ruys
- Phosphorylation - MassProt Unit, Institut de Duve, Université Catholique de Louvain, 1200 Brussels, Belgium.
| | - Didier Vertommen
- Phosphorylation - MassProt Unit, Institut de Duve, Université Catholique de Louvain, 1200 Brussels, Belgium.
| | - Jonathan Poels
- Department of Gynecology-Andrology, Cliniques Universitaires Saint-Luc, 1200 Brussels, Belgium.
| | - Christine Wyns
- Gynecology-Andrology Research Unit, Institut de Recherche Expérimentale et Clinique, Medical School, Université Catholique de Louvain, 1200 Brussels, Belgium.
- Department of Gynecology-Andrology, Cliniques Universitaires Saint-Luc, 1200 Brussels, Belgium.
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29
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Li N, Ma W, Shen Q, Zhang M, Du Z, Wu C, Niu B, Liu W, Hua J. Reconstitution of male germline cell specification from mouse embryonic stem cells using defined factors in vitro. Cell Death Differ 2019; 26:2115-2124. [PMID: 30683919 PMCID: PMC6748084 DOI: 10.1038/s41418-019-0280-2] [Citation(s) in RCA: 24] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/17/2018] [Revised: 11/24/2018] [Accepted: 01/02/2019] [Indexed: 01/15/2023] Open
Abstract
In vitro induction of functional haploid cells from embryonic stem cells (ESCs) has been reported by several groups. However, these reports either involve complex induction process with undefined induction factors or show low-induction efficiency. Here, we report complete meiosis in vitro from ESCs with defined induction factors. ESCs were first induced into primordial germ cell-like cells, which were further induced into male germline cells, including spermatogonial stem cell-like cells (SSCLCs) and spermatid-like cells. Importantly, the obtained SSCLCs were functional as infertile male mice sired healthy offspring via SSCLC transplantation. Further, we found that eukaryotic translation initiation factor 2 subunit 3 and structural gene Y-linked (Eif2s3y) was essential for spermatogenesis. Eif2s3y-overexpressing ESCs showed enhanced spermatogenesis in vitro, as demonstrated by higher expression levels of SSC-specific markers during SSCLC induction process, improved reproductive ability recovery of infertile male mice, and increased efficiency of haploid cell induction. Our work provides a convenient and efficient approach to obtain functional male germline cells.
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Affiliation(s)
- Na Li
- College of Veterinary Medicine, Shaanxi Center of Stem Cells Engineering & Technology, Northwest A & F University, Yangling, Shaanxi, China
| | - Wentao Ma
- College of Veterinary Medicine, Shaanxi Center of Stem Cells Engineering & Technology, Northwest A & F University, Yangling, Shaanxi, China
| | - Qiaoyan Shen
- College of Veterinary Medicine, Shaanxi Center of Stem Cells Engineering & Technology, Northwest A & F University, Yangling, Shaanxi, China
| | - Mengfei Zhang
- College of Veterinary Medicine, Shaanxi Center of Stem Cells Engineering & Technology, Northwest A & F University, Yangling, Shaanxi, China
| | - Zhaoyu Du
- College of Veterinary Medicine, Shaanxi Center of Stem Cells Engineering & Technology, Northwest A & F University, Yangling, Shaanxi, China
| | - Chongyang Wu
- College of Veterinary Medicine, Shaanxi Center of Stem Cells Engineering & Technology, Northwest A & F University, Yangling, Shaanxi, China
| | - Bowen Niu
- College of Veterinary Medicine, Shaanxi Center of Stem Cells Engineering & Technology, Northwest A & F University, Yangling, Shaanxi, China
| | - Wenqing Liu
- College of Veterinary Medicine, Shaanxi Center of Stem Cells Engineering & Technology, Northwest A & F University, Yangling, Shaanxi, China
| | - Jinlian Hua
- College of Veterinary Medicine, Shaanxi Center of Stem Cells Engineering & Technology, Northwest A & F University, Yangling, Shaanxi, China.
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30
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Sakib S, Goldsmith T, Voigt A, Dobrinski I. Testicular organoids to study cell-cell interactions in the mammalian testis. Andrology 2019; 8:835-841. [PMID: 31328437 DOI: 10.1111/andr.12680] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/16/2019] [Revised: 06/03/2019] [Accepted: 06/19/2019] [Indexed: 12/12/2022]
Abstract
BACKGROUND Over the last ten years, three-dimensional organoid culture has garnered renewed interest, as organoids generated from primary cells or stem cells with cell associations and functions similar to organs in vivo can be a powerful tool to study tissue-specific cell-cell interactions in vitro. Very recently, a few interesting approaches have been put forth for generating testicular organoids for studying the germ cell niche microenvironment. AIM To review different model systems that have been employed to study germ cell biology and testicular cell-cell interactions and discuss how the organoid approach can address some of the shortcomings of those systems. RESULTS AND CONCLUSION Testicular organoids that bear architectural and functional similarities to their in vivo counterparts are a powerful model system to study cell-cell interactions in the germ cell niche. Organoids enable studying samples in humans and other large animals where in vivo experiments are not possible, allow modeling of testicular disease and malignancies and may provide a platform to design more precise therapeutic interventions.
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Affiliation(s)
- S Sakib
- Biochemistry & Molecular Biology, Cumming School of Medicine, University of Calgary, Calgary, AL, Canada.,Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Calgary, AL, Canada
| | - T Goldsmith
- Biochemistry & Molecular Biology, Cumming School of Medicine, University of Calgary, Calgary, AL, Canada.,Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Calgary, AL, Canada
| | - A Voigt
- Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Calgary, AL, Canada
| | - I Dobrinski
- Biochemistry & Molecular Biology, Cumming School of Medicine, University of Calgary, Calgary, AL, Canada.,Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Calgary, AL, Canada
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31
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Sakib S, Voigt A, Goldsmith T, Dobrinski I. Three-dimensional testicular organoids as novel in vitro models of testicular biology and toxicology. ENVIRONMENTAL EPIGENETICS 2019; 5:dvz011. [PMID: 31463083 PMCID: PMC6705190 DOI: 10.1093/eep/dvz011] [Citation(s) in RCA: 23] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/26/2019] [Revised: 06/06/2019] [Accepted: 07/03/2019] [Indexed: 05/05/2023]
Abstract
Organoids are three dimensional structures consisting of multiple cell types that recapitulate the cellular architecture and functionality of native organs. Over the last decade, the advent of organoid research has opened up many avenues for basic and translational studies. Following suit of other disciplines, research groups working in the field of male reproductive biology have started establishing and characterizing testicular organoids. The three-dimensional architectural and functional similarities of organoids to their tissue of origin facilitate study of complex cell interactions, tissue development and establishment of representative, scalable models for drug and toxicity screening. In this review, we discuss the current state of testicular organoid research, their advantages over conventional monolayer culture and their potential applications in the field of reproductive biology and toxicology.
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Affiliation(s)
- Sadman Sakib
- Biochemistry & Molecular Biology, Cumming School of Medicine, University of Calgary, Canada
- Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Canada
| | - Anna Voigt
- Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Canada
| | - Taylor Goldsmith
- Biochemistry & Molecular Biology, Cumming School of Medicine, University of Calgary, Canada
- Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Canada
| | - Ina Dobrinski
- Biochemistry & Molecular Biology, Cumming School of Medicine, University of Calgary, Canada
- Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Canada
- Correspondence address. Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Room 404, Heritage Medical Research Building, 3300 Hospital Drive NW, Calgary, Alberta T2N 4N1, Canada. Tel: 4032106523; Fax: 4032108821; E-mail:
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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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33
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von Kopylow K, Schulze W, Salzbrunn A, Schaks M, Schäfer E, Roth B, Schlatt S, Spiess AN. Dynamics, ultrastructure and gene expression of human in vitro organized testis cells from testicular sperm extraction biopsies. Mol Hum Reprod 2019; 24:123-134. [PMID: 29304256 DOI: 10.1093/molehr/gax070] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/12/2017] [Accepted: 12/28/2017] [Indexed: 12/25/2022] Open
Abstract
STUDY QUESTION Is it possible to induce in vitro reorganization of primary human testis cells from testicular sperm extraction (TESE) biopsies, maintain their long-term cultivation in a 2D system and identify cellular compositions? SUMMARY ANSWER In vitro reorganization of primary human testis cells from TESE biopsies and their long-term cultivation on uncoated cell culture dishes is feasible and the cellular compositions can be uncovered through gene expression and microscopic analyses. WHAT IS KNOWN ALREADY It has been shown in the rodent model that mixtures of testicular cell types are able to reassemble into clusters when cultivated on different kinds of surfaces or three-dimensional matrices. Two recent publications demonstrated the ability of primary human testicular cells to assemble into testicular organoids and their cultivation for a period of 3-4 weeks. STUDY DESIGN SIZE, DURATION Primary human testis cells from TESE biopsies from 16 patients were reorganized in vitro and the clusters were cultivated long term on uncoated cell culture dishes, providing a solid ground for in vitro spermatogenesis. Gene expression analysis as well as fluorescence/transmission electron microscopy (TEM) were employed to uncover the cellular composition of the clusters. PARTICIPANTS/MATERIALS, SETTING, METHODS Testis biopsies from adult, normogonadotropic patients displaying full spermatogenesis (n = 11), hypospermatogenesis (n = 2), predominantly full spermatogenesis with some hypospermatogenic tubules (n = 1), meiotic arrest (n = 1) or mixed atrophy (n = 1) were enzymatically digested and dispersed cells were cultivated on 96-well plates or chamber dishes as aggregate-free cell suspensions. Time-lapse imaging of cluster formation was performed over a period of 48 h. For receptor tyrosine kinase inhibition of cluster formation, cells were treated twice with K252a within 2-3 days. Immunofluorescence staining and confocal microscopy was carried out on clusters after 1-3 weeks of cultivation to identify the presence of Sertoli cells (SC) (SOX9), peritubular myoid cells (SMA), Leydig cells (LC) (STAR), undifferentiated spermatogonia (FGFR3), differentiating spermatogonia/spermatocytes (DDX4) and postmeiotic germ cells (PRM1). Single clusters from four patients and a pool of eight larger clusters from another patient were manually picked and subjected to quantitative real-time PCR to evaluate the presence of SC (SOX9, AR), LC (INSL3, STAR, HSD3B1), peritubular myoid cells (ACTA2), fibroblasts (FSP1), endothelial cells (CD34), macrophages (CD68), undifferentiated spermatogonia (FGFR3), differentiating spermatogonia/spermatocytes (DDX4) and postmeiotic germ cells (PRM1). Finally, an ultrastructural investigation was conducted based on TEM of clusters from six different patients, among them 3-month cultivated large clusters from two patients. MAIN RESULTS AND THE ROLE OF CHANCE Quantitative PCR-based analysis of single-picked testicular cell clusters identified SC, peritubular myoid cells, endothelial cells, fibroblasts, macrophages, spermatids and LC after 1, 2 or 3 weeks or 3 months of cultivation. Immunofluorescence positivity for SC and peritubular myoid cells corroborated the presence of these two kinds of testis niche cells. In addition, round as well as elongated spermatids were frequently encountered in 1 and 2 weeks old clusters. Transmission electron microscopical classification confirmed all these cell types together with a few spermatogonia. Macrophages were found to be of the proinflammatory M1 subtype, as revealed by CD68+/CD163-/IL6+ expression. Time-lapse imaging uncovered the specific dynamics of cluster fusion and enlargement, which could be prevented by addition of protein kinase inhibitor K252a. LARGE SCALE DATA N/A. LIMITATIONS REASON FOR CAUTION Cell composition of the clusters varied based on the spermatogenic state of the TESE patient. Although spermatids could be observed with all applied methods, spermatogonia were only detected by TEM in single cases. Hence, a direct maintenance of these germ cell types by our system in its current state cannot be postulated. Moreover, putative dedifferentiation and malignant degeneration of cells in long-term cluster cultivation needs to be investigated in the future. WIDER IMPLICATIONS OF THE FINDINGS This work demonstrates that the reorganization of testicular cells can be achieved with TESE biopsies obtained from men enroled in a standard clinical assisted reproduction program. The formed clusters can be cultivated for at least 3 months and are composed, to a large extent, of the most important somatic cell types that are essential to support spermatogenesis. These findings may provide the cellular basis for advances in human in vitro spermatogenesis and/or the possibility for propagation of spermatogonia within a natural stem cell niche-like environment. STUDY FUNDING AND COMPETING INTERESTS The project was funded by a DFG grant to K.v.K. (KO 4769/2-1). The authors declare they have no conflicts of interest.
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Affiliation(s)
- Kathrein von Kopylow
- Department of Andrology, University Hospital Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, Germany
| | - Wolfgang Schulze
- MVZ Fertility Center Hamburg GmbH, Amedes Group, Speersort 4, 20095 Hamburg, Germany
| | - Andrea Salzbrunn
- Department of Andrology, University Hospital Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, Germany
| | - Matthias Schaks
- Department of Andrology, University Hospital Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, Germany
| | - Elke Schäfer
- Institute of Neuroanatomy, University Hospital Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, Germany
| | - Beate Roth
- Department of Andrology, University Hospital Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, Germany
| | - Stefan Schlatt
- Centre of Reproductive Medicine and Andrology, University Hospital of Münster, Albert-Schweitzer Campus 1, 48149 Münster, Germany
| | - Andrej-Nikolai Spiess
- Department of Andrology, University Hospital Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, Germany
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Fang F, Li Z, Zhao Q, Li H, Xiong C. Human induced pluripotent stem cells and male infertility: an overview of current progress and perspectives. Hum Reprod 2019; 33:188-195. [PMID: 29315416 PMCID: PMC5850345 DOI: 10.1093/humrep/dex369] [Citation(s) in RCA: 43] [Impact Index Per Article: 7.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/08/2017] [Indexed: 01/30/2023] Open
Abstract
Recently, significant progress has been made in ART for the treatment of male infertility. However, current ART has failed to help infertile patients with non-obstructive azoospermia, unless donor sperm is used. In fact, most couples wish to have their own genetically related child. Human induced pluripotent stem cells (hiPSCs) can be generated from patients’ somatic cells and in vitro derivation of functional germ cells from patient-specific iPSCs may provide new therapeutic strategies for infertile couples. The overall developmental dynamics of human primordial germ cells are similar to that in mice, but accumulating evidence suggests that there are crucial differences between human and mouse PGC specification. Unlike mouse iPSCs (miPSCs) in naive state, hiPSCs exhibit a primed pluripotency which possess less potential for the germ cell fate. Based on research in mice, male germ cells at different stages have been derived from hiPSCs with different protocols, including spontaneous differentiation, overexpression of germ cell regulators, addition of cytokines, co-culture with gonadal cells in vitro and xeno-transplantation. The aim of this review is to summarize the current advances in derivation of male germ cells from hiPSCs and raise the perspectives of hiPSCs in medical application for male infertility, as well as in basic research for male germ cell development.
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Affiliation(s)
- Fang Fang
- Family Planning Research Institute, Tongji Medical College, Huazhong University of Science and Technology, 13 Hangkong Road, Wuhan 430030, China
| | - Zili Li
- Family Planning Research Institute, Tongji Medical College, Huazhong University of Science and Technology, 13 Hangkong Road, Wuhan 430030, China.,Center for Reproductive Medicine, Wuhan Tongji Reproductive Medicine Hospital, 128 Sanyang Road, Wuhan 430013, China
| | - Qian Zhao
- Family Planning Research Institute, Tongji Medical College, Huazhong University of Science and Technology, 13 Hangkong Road, Wuhan 430030, China
| | - Honggang Li
- Family Planning Research Institute, Tongji Medical College, Huazhong University of Science and Technology, 13 Hangkong Road, Wuhan 430030, China
| | - Chengliang Xiong
- Family Planning Research Institute, Tongji Medical College, Huazhong University of Science and Technology, 13 Hangkong Road, Wuhan 430030, China.,Center for Reproductive Medicine, Wuhan Tongji Reproductive Medicine Hospital, 128 Sanyang Road, Wuhan 430013, China
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Baert Y, Rombaut C, Goossens E. Scaffold-Based and Scaffold-Free Testicular Organoids from Primary Human Testicular Cells. Methods Mol Biol 2019; 1576:283-290. [PMID: 28674937 DOI: 10.1007/7651_2017_48] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/07/2023]
Abstract
Organoid systems take advantage of the self-organizing capabilities of cells to create diverse multi-cellular tissue surrogates that constitute a powerful novel class of biological models. Clearly, the formation of a testicular organoid (TO) in which human spermatogenesis can proceed from a single-cell suspension would exert a tremendous impact on research and development, clinical treatment of infertility, and screening of potential drugs and toxic agents. Recently, we showed that primary adult and pubertal human testicular cells auto-assembled in TOs either with or without the support of a natural testis scaffold. These mini-tissues harboured both the spermatogonial stem cells and their important niche cells, which retained certain specific functions during long-term culture. As such, human TOs might advance the development of a system allowing human in vitro spermatogenesis. Here we describe the methodology to make scaffold-based and scaffold-free TOs.
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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, Brussels, 1090, Belgium.
| | - Charlotte Rombaut
- Biology of the Testis, Research Laboratory for Reproduction, Genetics and Regenerative Medicine, Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, Brussels, 1090, Belgium
| | - Ellen Goossens
- Biology of the Testis, Research Laboratory for Reproduction, Genetics and Regenerative Medicine, Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, Brussels, 1090, Belgium
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Yoshida S. Heterogeneous, dynamic, and stochastic nature of mammalian spermatogenic stem cells. Curr Top Dev Biol 2019; 135:245-285. [DOI: 10.1016/bs.ctdb.2019.04.008] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
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Efficiency of colony formation and differentiation of human spermatogenic cells in two different culture systems. Reprod Biol 2018; 18:397-403. [DOI: 10.1016/j.repbio.2018.09.006] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/27/2018] [Revised: 08/08/2018] [Accepted: 09/22/2018] [Indexed: 01/15/2023]
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Komeya M, Sato T, Ogawa T. In vitro spermatogenesis: A century-long research journey, still half way around. Reprod Med Biol 2018; 17:407-420. [PMID: 30377394 PMCID: PMC6194268 DOI: 10.1002/rmb2.12225] [Citation(s) in RCA: 43] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/04/2018] [Accepted: 07/19/2018] [Indexed: 01/15/2023] Open
Abstract
BACKGROUND Spermatogenesis is one of the most complicated cellular differentiation processes in a body. Researchers struggled to find and develop a micro-environmental condition that can support the process in vitro. Such endeavors can be traced back to a century ago and are yet continuing. METHODS Reports on in vitro spermatogenesis and related works were selected and classified into four categories based on the method used; organ culture, tubule culture, cell culture, and 3-dimensional cell culture methods. Each report was critically reviewed from the present point of view by authors who have been working on in vitro spermatogenesis with organ culture method over a decade. RESULTS The organ culture method has the longest history and is the most successful method, which produced fertile mouse sperm from spermatogonial stem cells. Formulation of the medium was a key factor, most importantly serum-derived substances. However, factors in the serum that induce and support spermatogenesis in the cultured tissue remain to be identified. In addition, the success of mouse spermatogenesis is yet to be applied to other animals. On looking into the history of cell culture method, it became clear that Sertoli cells as feeder cells play an important role. Even with Sertoli cells, however, spermatogenic development has been limited to small parts of spermatogenesis, a segmented period of meiotic prophase for instance. Recent developments of organoid or 3-dimensional culture techniques are promising but they still need further refinements. CONCLUSION The study of in vitro spermatogenesis progressed significantly over the last century. We need more work, however, to establish a culture system that can induce and maintain complete spermatogenesis of many if not all mammalian species.
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Affiliation(s)
- Mitsuru Komeya
- Department of UrologyYokohama City University Graduate School of MedicineYokohamaKanagawaJapan
- Laboratory of Biopharmaceutical and Regenerative SciencesInstitute of Molecular Medicine and Life ScienceYokohama City University Association of Medical ScienceYokohamaKanagawaJapan
| | - Takuya Sato
- Laboratory of Biopharmaceutical and Regenerative SciencesInstitute of Molecular Medicine and Life ScienceYokohama City University Association of Medical ScienceYokohamaKanagawaJapan
| | - Takehiko Ogawa
- Department of UrologyYokohama City University Graduate School of MedicineYokohamaKanagawaJapan
- Laboratory of Biopharmaceutical and Regenerative SciencesInstitute of Molecular Medicine and Life ScienceYokohama City University Association of Medical ScienceYokohamaKanagawaJapan
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Gholami K, Pourmand G, Koruji M, Ashouri S, Abbasi M. Organ culture of seminiferous tubules using a modified soft agar culture system. Stem Cell Res Ther 2018; 9:249. [PMID: 30257723 PMCID: PMC6158910 DOI: 10.1186/s13287-018-0997-8] [Citation(s) in RCA: 22] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/01/2018] [Revised: 08/24/2018] [Accepted: 08/28/2018] [Indexed: 11/10/2022] Open
Abstract
BACKGROUND In-vitro spermatogenesis in mammalian species is considered an important topic in reproductive biology. New strategies for achieving a complete version of spermatogenesis ex vivo have been conducted using an organ culture method or culture of testicular cells in a three-dimensional soft agar culture system (SACS). The aim of this study was to develop a new method that supports spermatogenesis to the meiotic phase and morphologically mature spermatozoa through the culture of testicular cells and seminiferous tubules (STs) in a modified SACS, respectively. METHODS First, enzymatically dissociated testicular cells and mechanically dissociated STs of neonatal mice were separately embedded in agarose and then placed on the flat surface of agarose gel half-soaked in the medium to continue culture with a gas-liquid interphase method. RESULTS Following 40 days of culture, the meiotic (Scp3) and post-meiotic (Acr) gene expression in aggregates and STs was confirmed by real-time polymerase chain reaction. These results were complemented by immunohistochemistry. The presence of morphologically mature spermatozoa in the frozen sections of STs was demonstrated with hematoxylin and eosin staining. We observed Plzf- or Integrin α6-positive spermatogonia in both cultures after 40 days, indicating the potency of the culture system for both self-renewal and differentiation. CONCLUSIONS This technique can be used as a valuable approach for performing research on spermatogenesis and translating it into the human clinical setting.
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Affiliation(s)
- Keykavos Gholami
- Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
| | - Gholamreza Pourmand
- Urology Research Center, Sina Hospital, Tehran University of Medical Sciences, Tehran, Iran
| | - Morteza Koruji
- Cellular and Molecular Research Center & Department of Anatomical Sciences, Iran University of Medical Sciences, Tehran, Iran
| | - Sepideh Ashouri
- Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
| | - Mehdi Abbasi
- Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
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Inoue M, Baba T, Morohashi KI. Recent progress in understanding the mechanisms of Leydig cell differentiation. Mol Cell Endocrinol 2018; 468:39-46. [PMID: 29309805 DOI: 10.1016/j.mce.2017.12.013] [Citation(s) in RCA: 12] [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: 10/04/2017] [Revised: 12/25/2017] [Accepted: 12/26/2017] [Indexed: 01/26/2023]
Abstract
Leydig cells in fetal and adult testes play pivotal roles in eliciting male characteristics by producing androgen. Although numerous studies of Leydig cells have been performed, the mechanisms for differentiation of the two cell types (fetal Leydig and adult Leydig cells), their developmental and functional relationship, and their differential characteristics remain largely unclear. Based on recent technical progress in genome-wide analysis and in vitro investigation, novel and fascinating observations concerning the issues above have been obtained. Focusing on fetal and adult Leydig cells, this review summarizes the recent progress that has advanced our understanding of the cells.
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Affiliation(s)
- Miki Inoue
- Division of Molecular Life Sciences, Graduate School of Systems Life Sciences, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka 812-8582, Japan
| | - Takashi Baba
- Division of Molecular Life Sciences, Graduate School of Systems Life Sciences, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka 812-8582, Japan; Department of Molecular Biology, Graduate School of Medical Sciences, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka 812-8582, Japan
| | - Ken-Ichirou Morohashi
- Division of Molecular Life Sciences, Graduate School of Systems Life Sciences, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka 812-8582, Japan; Department of Molecular Biology, Graduate School of Medical Sciences, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka 812-8582, Japan.
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Use of a three-layer gradient system of cells for rat testicular organoid generation. Nat Protoc 2018; 13:248-259. [DOI: 10.1038/nprot.2017.140] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
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Mincheva M, Sandhowe-Klaverkamp R, Wistuba J, Redmann K, Stukenborg JB, Kliesch S, Schlatt S. Reassembly of adult human testicular cells: can testis cord-like structures be created in vitro? Mol Hum Reprod 2017; 24:55-63. [DOI: 10.1093/molehr/gax063] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/08/2017] [Accepted: 12/11/2017] [Indexed: 02/06/2023] Open
Affiliation(s)
- M Mincheva
- Centre of Reproductive Medicine and Andrology, University Hospital of Münster, Albert-Schweitzer Campus 1, 48149 Münster, Germany
| | - R Sandhowe-Klaverkamp
- Centre of Reproductive Medicine and Andrology, University Hospital of Münster, Albert-Schweitzer Campus 1, 48149 Münster, Germany
| | - J Wistuba
- Centre of Reproductive Medicine and Andrology, University Hospital of Münster, Albert-Schweitzer Campus 1, 48149 Münster, Germany
| | - K Redmann
- Centre of Reproductive Medicine and Andrology, University Hospital of Münster, Albert-Schweitzer Campus 1, 48149 Münster, Germany
| | - J -B Stukenborg
- Department of Women’s and Children’s Health, NORDFERTIL research lab Stockholm, Pediatric Endocrinology Unit, Q2:08, Karolinska Institutet and University Hospital, SE-17176 Stockholm, Sweden
| | - S Kliesch
- Centre of Reproductive Medicine and Andrology, University Hospital of Münster, Albert-Schweitzer Campus 1, 48149 Münster, Germany
| | - S Schlatt
- Centre of Reproductive Medicine and Andrology, University Hospital of Münster, Albert-Schweitzer Campus 1, 48149 Münster, Germany
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Alves-Lopes JP, Stukenborg JB. Testicular organoids: a new model to study the testicular microenvironment in vitro? Hum Reprod Update 2017; 24:176-191. [PMID: 29281008 DOI: 10.1093/humupd/dmx036] [Citation(s) in RCA: 51] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/04/2017] [Revised: 11/20/2017] [Accepted: 11/27/2017] [Indexed: 12/11/2022] Open
Abstract
BACKGROUND In recent decades, a broad range of strategies have been applied to model the testicular microenvironment in vitro. These models have been utilized to study testicular physiology and development. However, a system that allows investigations into testicular organogenesis and its impact in the spermatogonial stem-cell (SSC) niche in vitro has not been developed yet. Recently, the creation of tissue-specific organ-like structures called organoids has resurged, helping researchers to answer scientific questions that previous in vitro models could not help to elucidate. So far, a small number of publications have concerned the generation of testicular organoids and their application in the field of reproductive medicine and biology. OBJECTIVE AND RATIONALE Here, we aim to elucidate whether testicular organoids might be useful in answering current scientific questions about the regulation and function of the SSC niche as well as germ cell proliferation and differentiation, and whether or not the existing in vitro models are already sufficient to address them. Moreover, we would like to discuss how an organoid system can be a better solution to address these prominent scientific problems in our field, by the creation of a rationale parallel to those in other areas where organoid systems have been successfully utilized. SEARCH METHODS We comprehensively reviewed publications regarding testicular organoids and the methods that most closely led to the formation of these organ-like structures in vitro by searching for the following terms in both PubMed and the Web of Science database: testicular organoid, seminiferous tubule 3D culture, Sertoli cell 3D culture, testicular cord formation in vitro, testicular morphogenesis in vitro, germ cell 3D culture, in vitro spermatogenesis, testicular de novo morphogenesis, seminiferous tubule de novo morphogenesis, seminiferous tubule-like structures, testicular in vitro model and male germ cell niche in vitro, with no restrictions to any publishing year. The inclusion criteria were based on the relation with the main topic (i.e. testicular organoids, testicular- and seminiferous-like structures as in vitro models), methodology applied (i.e. in vitro culture, culture dimensions (2D, 3D), testicular cell suspension or fragments) and outcome of interest (i.e. organization in vitro). Publications about grafting of testicular tissue, germ-cell transplantation and female germ-cell culture were excluded. OUTCOMES The application of organoid systems is making its first steps in the field of reproductive medicine and biology. A restricted number of publications have reported and characterized testicular organoids and even fewer have denominated such structures by this method. However, we detected that a clear improvement in testicular cell reorganization is recognized when 3D culture conditions are utilized instead of 2D conditions. Depending on the scientific question, testicular organoids might offer a more appropriate in vitro model to investigate testicular development and physiology because of the easy manipulation of cell suspensions (inclusion or exclusion of a specific cell population), the fast reorganization of these structures and the controlled in vitro conditions, to the same extent as with other organoid strategies reported in other fields. WIDER IMPLICATIONS By way of appropriate research questions, we might use testicular organoids to deepen our basic understanding of testicular development and the SSC niche, leading to new methodologies for male infertility treatment.
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Affiliation(s)
- João Pedro Alves-Lopes
- Department of Women's and Children's Health, NORDFERTIL Research Lab Stockholm, Paediatric Endocrinology Unit, Q2:08, Karolinska Institutet and Karolinska University Hospital, SE-17176 Stockholm, Sweden
| | - Jan-Bernd Stukenborg
- Department of Women's and Children's Health, NORDFERTIL Research Lab Stockholm, Paediatric Endocrinology Unit, Q2:08, Karolinska Institutet and Karolinska University Hospital, SE-17176 Stockholm, Sweden
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Abstract
PURPOSE OF REVIEW This review evaluates the state of the art in terms of challenges and strategies used to restore fertility with spermatogonial stem cells retrieved from prepubertal boys affected by cancer. Although these boys do not yet produce spermatozoa, the only option to preserve their fertility is cryopreservation of spermatogonial stem cells in the form of testicular cell suspensions or whole tissue pieces. Different techniques have been described to achieve completion of spermatogenesis from human, spermatogonial stem cells but none is yet ready for clinical application. A crucial point to address is gaining a full understanding of spermatogonial stem cell niche pathophysiology, where germ cells undergo proliferation and differentiation. Various fertility restoration approaches will be presented depending on the presence of an intact niche, dissociated niche, or reconstituted niche. RECENT FINDINGS Testicular organoids open the way to providing further insights into the niche. They can recreate the three-dimensional architecture of the testicular microenvironment in vitro, allowing a large number of applications, from physiology to drug toxicity investigations. SUMMARY In addition to the full elucidation of the niche microenvironment, achieving fertility restoration from cryopreserved human spermatogonial stem cells implies overcoming other important challenges. Testicular organoids might prove to be essential tools to progress in this field.
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Affiliation(s)
- Francesca de Michele
- aInstitut de Recherche Expérimentale et Clinique, Université Catholique de Louvain bDepartment of Gynecology-Andrology, Cliniques Universitaires Saint-Luc, Brussels, Belgium
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Zhang X, Wang L, Zhang X, Ren L, Shi W, Tian Y, Zhu J, Zhang T. The use of KnockOut serum replacement (KSR) in three dimensional rat testicular cells co-culture model: An improved male reproductive toxicity testing system. Food Chem Toxicol 2017; 106:487-495. [DOI: 10.1016/j.fct.2017.05.001] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/14/2016] [Revised: 03/30/2017] [Accepted: 05/02/2017] [Indexed: 10/19/2022]
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Baert Y, De Kock J, Alves-Lopes JP, Söder O, Stukenborg JB, Goossens E. Primary Human Testicular Cells Self-Organize into Organoids with Testicular Properties. Stem Cell Reports 2017; 8:30-38. [PMID: 28017656 PMCID: PMC5233407 DOI: 10.1016/j.stemcr.2016.11.012] [Citation(s) in RCA: 108] [Impact Index Per Article: 13.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/20/2016] [Revised: 11/25/2016] [Accepted: 11/25/2016] [Indexed: 01/08/2023] Open
Abstract
So far, successful de novo formation of testicular tissue followed by complete spermatogenesis in vitro has been achieved only in rodents. Our findings reveal that primary human testicular cells are able to self-organize into human testicular organoids (TOs), i.e., multi-cellular tissue surrogates, either with or without support of a biological scaffold. Despite lacking testis-specific topography, these mini-tissues harbored spermatogonia and their important niche cells, which retained specific functionalities during long-term culture. These observations indicate the posibility of in vitro re-engineering of a human testicular microenvironment from primary cells. Human TOs might help in the development of a biomimetic testicular model that would exert a tremendous impact on research and development, clinical treatment of infertility, and screening in connection with drug discovery and toxicology.
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Affiliation(s)
- Yoni Baert
- Biology of the Testis, Research Laboratory for Reproduction, Genetics and Regenerative Medicine, Vrije Universiteit Brussel (VUB), 1090 Brussels, Belgium.
| | - Joery De Kock
- Department of In Vitro Toxicology and Dermato-Cosmetology, Center for Pharmaceutical Research, Vrije Universiteit Brussel (VUB), 1090 Brussels, Belgium
| | - João P Alves-Lopes
- Pediatric Endocrinology Unit; Q2:08, Department of Women's and Children's Health, Karolinska Institutet and University Hospital, 17176 Stockholm, Sweden
| | - Olle Söder
- Pediatric Endocrinology Unit; Q2:08, Department of Women's and Children's Health, Karolinska Institutet and University Hospital, 17176 Stockholm, Sweden
| | - Jan-Bernd Stukenborg
- Pediatric Endocrinology Unit; Q2:08, Department of Women's and Children's Health, Karolinska Institutet and University Hospital, 17176 Stockholm, Sweden
| | - Ellen Goossens
- Biology of the Testis, Research Laboratory for Reproduction, Genetics and Regenerative Medicine, Vrije Universiteit Brussel (VUB), 1090 Brussels, Belgium
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Abstract
Twenty years ago, the transplantation of spermatogonial stem cells (SSCs) from a mouse to other recipient mice was shown to be feasible, which clearly demonstrated the functional identity of SSCs. Since then, several important new findings and other technical developments have followed, which included a new hypothesis on their cell kinetics and spermatogonial hierarchy in the testis, a culture method allowing their self-renewal and proliferation, a testis tissue organ culture method, which induced their complete differentiation up to sperm, and the in vitro induction of germ cells from embryonic stem cells and induced pluripotent stem cells. These advancements reinforced or advanced our understanding of this unique cell. Nonetheless, there are many unresolved questions in the study of spermatogonial stem cells and a long road remains until these cells can be used clinically in reproductive medicine.
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Affiliation(s)
| | - Takehiko Ogawa
- Department of Urology, Graduate School of Medicine, Yokohama City University; Laboratory of Proteomics, Institute of Molecular Medicine and Life Science, Yokohama City University Association of Medical Science, Yokohama 236 0004, Japan,
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Okada R, Hara T, Sato T, Kojima N, Nishina Y. The mechanism and control of Jagged1 expression in Sertoli cells. Regen Ther 2016; 3:75-81. [PMID: 31245476 PMCID: PMC6581826 DOI: 10.1016/j.reth.2016.02.005] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2015] [Revised: 02/08/2016] [Accepted: 02/15/2016] [Indexed: 01/25/2023] Open
Abstract
The regulation of Sertoli cells by some hormones and signaling factors is important for normal spermatogenesis. Notch signaling is considered to be necessary for normal spermatogenesis in mouse. In this study, we revealed two new facts about Sertoli cells by western blotting experiments on different types of primary cells and microdissected tubules. The first is that Sertoli cells express the Jagged1 ligand in mice testes. The second is that the expression level of Jagged1 oscillates in the seminiferous epithelial cycle. Therefore, we inferred that Jagged1 in Sertoli cells contributes to the Notch signaling involved in spermatogenesis. Furthermore, we examined the regulation of Jagged1 expression and found that Jagged1 expression was suppressed by cAMP signaling and was promoted by TNF-α signaling in Sertoli cells. When cAMP and TNF-α were simultaneously added to Sertoli cells, Jagged1 expression was suppressed. Therefore, cAMP signaling dominates Jagged1 expression over TNF-α signaling. These results suggest that cAMP signaling may cause the periodicity of Jagged1 expression in the seminiferous epithelial cycle, and controlling Jagged1 expression by adding TNF-α or cAMP may contribute to normal spermatogenesis in vitro.
Jagged1 was expressed in Sertoli cells in mouse testes. The expression of Jagged1 oscillated in the seminiferous epithelial cycle. The expression of Jagged1 in Sertoli cells was upregulated by TNF-α and downregulated by cAMP.
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Affiliation(s)
- Ryu Okada
- Graduate School of Nanobioscience, Yokohama City University, 22-2 Seto, Kanazawa-ku, Yokohama 236-0027, Japan
| | - Taro Hara
- Graduate School of Nanobioscience, Yokohama City University, 22-2 Seto, Kanazawa-ku, Yokohama 236-0027, Japan
| | - Tomomi Sato
- Graduate School of Nanobioscience, Yokohama City University, 22-2 Seto, Kanazawa-ku, Yokohama 236-0027, Japan
| | - Nobuhiko Kojima
- Graduate School of Nanobioscience, Yokohama City University, 22-2 Seto, Kanazawa-ku, Yokohama 236-0027, Japan
| | - Yukio Nishina
- Graduate School of Nanobioscience, Yokohama City University, 22-2 Seto, Kanazawa-ku, Yokohama 236-0027, Japan
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49
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Zhou Q, Wang M, Yuan Y, Wang X, Fu R, Wan H, Xie M, Liu M, Guo X, Zheng Y, Feng G, Shi Q, Zhao XY, Sha J, Zhou Q. Complete Meiosis from Embryonic Stem Cell-Derived Germ Cells In Vitro. Cell Stem Cell 2016; 18:330-340. [PMID: 26923202 DOI: 10.1016/j.stem.2016.01.017] [Citation(s) in RCA: 255] [Impact Index Per Article: 28.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/17/2015] [Revised: 12/23/2015] [Accepted: 01/21/2016] [Indexed: 02/08/2023]
Abstract
In vitro generation of functional gametes is a promising approach for treating infertility, although faithful replication of meiosis has proven to be a substantial obstacle to deriving haploid gamete cells in culture. Here we report complete in vitro meiosis from embryonic stem cell (ESC)-derived primordial germ cells (PGCLCs). Co-culture of PGCLCs with neonatal testicular somatic cells and sequential exposure to morphogens and sex hormones reproduced key hallmarks of meiosis, including erasure of genetic imprinting, chromosomal synapsis and recombination, and correct nuclear DNA and chromosomal content in the resulting haploid cells. Intracytoplasmic injection of the resulting spermatid-like cells into oocytes produced viable and fertile offspring, showing that this robust stepwise approach can functionally recapitulate male gametogenesis in vitro. These findings provide a platform for investigating meiotic mechanisms and the potential generation of human haploid spermatids in vitro.
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Affiliation(s)
- Quan Zhou
- State Key Laboratory of Reproductive Medicine, Department of Histology and Embryology, Nanjing Medical University, Nanjing 210029, China; State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
| | - Mei Wang
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; College of the Life Sciences, Hunan Normal University, Changsha, Hunan 410081, China
| | - Yan Yuan
- State Key Laboratory of Reproductive Medicine, Department of Histology and Embryology, Nanjing Medical University, Nanjing 210029, China; State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
| | - Xuepeng Wang
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; University of the Chinese Academy of Sciences, Beijing 100049, China
| | - Rui Fu
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
| | - Haifeng Wan
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
| | - Mingming Xie
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; College of Life Science, Anhui University of China, Hefei 230601, China
| | - Mingxi Liu
- State Key Laboratory of Reproductive Medicine, Department of Histology and Embryology, Nanjing Medical University, Nanjing 210029, China
| | - Xuejiang Guo
- State Key Laboratory of Reproductive Medicine, Department of Histology and Embryology, Nanjing Medical University, Nanjing 210029, China
| | - Ying Zheng
- Department of Histology and Embryology, Medical College of Yangzhou University, Yangzhou 225001, Jiangsu, China
| | - Guihai Feng
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
| | - Qinghua Shi
- Molecular and Cell Genetics Laboratory, Chinese Academy of Sciences Key Laboratory of Innate Immunity and Chronic Disease, Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Hefei 230027, China
| | - Xiao-Yang Zhao
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China.
| | - Jiahao Sha
- State Key Laboratory of Reproductive Medicine, Department of Histology and Embryology, Nanjing Medical University, Nanjing 210029, China.
| | - Qi Zhou
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China.
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50
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Inoue M, Shima Y, Miyabayashi K, Tokunaga K, Sato T, Baba T, Ohkawa Y, Akiyama H, Suyama M, Morohashi KI. Isolation and Characterization of Fetal Leydig Progenitor Cells of Male Mice. Endocrinology 2016; 157:1222-33. [PMID: 26697723 DOI: 10.1210/en.2015-1773] [Citation(s) in RCA: 38] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
Abstract
Fetal and adult Leydig cells develop in mammalian prenatal and postnatal testes, respectively. In mice, fetal Leydig cells (FLCs) emerge in the interstitial space of the testis at embryonic day 12.5 and thereafter increase in number, possibly through differentiation from progenitor cells. However, the progenitor cells have not yet been identified. Previously, we established transgenic mice in which FLCs are labeled strongly with enhanced green fluorescent protein (EGFP). Interestingly, fluorescence-activated cell sorting provided us with weakly EGFP-labeled cells as well as strongly EGFP-labeled FLCs. In vitro reconstruction of fetal testes demonstrated that weakly EGFP-labeled cells contain FLC progenitors. Transcriptome from the 2 cell populations revealed, as expected, marked differences in the expression of genes required for growth factor/receptor signaling and steroidogenesis. In addition, genes for energy metabolisms such as glycolytic pathways and the citrate cycle were activated in strongly EGFP-labeled cells, suggesting that metabolism is activated during FLC differentiation.
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Affiliation(s)
- Miki Inoue
- Division of Molecular Life Science (M.I., Y.S., T.B., K.-i.M.), Graduate School of Systems Life Science; Department of Molecular Biology (Y.S., K.M., K.T., T.B., K.-i.M.), Graduate School of Medical Sciences; Division of Bioinformatics (T.S., M.S.), Medical Institute of Bioregulation; and Department of Advanced Medical Initiatives (Y.O.), Japan Science and Technology Agency-Core Research for Evolutional Science and Technology, Faculty of Medicine, Kyushu University, Higashi-ku, Fukuoka 812-8582, Japan; and Department of Orthopaedics (H.A.), Gifu University Graduate School of Medicine, Gifu 501-1194, Japan
| | - Yuichi Shima
- Division of Molecular Life Science (M.I., Y.S., T.B., K.-i.M.), Graduate School of Systems Life Science; Department of Molecular Biology (Y.S., K.M., K.T., T.B., K.-i.M.), Graduate School of Medical Sciences; Division of Bioinformatics (T.S., M.S.), Medical Institute of Bioregulation; and Department of Advanced Medical Initiatives (Y.O.), Japan Science and Technology Agency-Core Research for Evolutional Science and Technology, Faculty of Medicine, Kyushu University, Higashi-ku, Fukuoka 812-8582, Japan; and Department of Orthopaedics (H.A.), Gifu University Graduate School of Medicine, Gifu 501-1194, Japan
| | - Kanako Miyabayashi
- Division of Molecular Life Science (M.I., Y.S., T.B., K.-i.M.), Graduate School of Systems Life Science; Department of Molecular Biology (Y.S., K.M., K.T., T.B., K.-i.M.), Graduate School of Medical Sciences; Division of Bioinformatics (T.S., M.S.), Medical Institute of Bioregulation; and Department of Advanced Medical Initiatives (Y.O.), Japan Science and Technology Agency-Core Research for Evolutional Science and Technology, Faculty of Medicine, Kyushu University, Higashi-ku, Fukuoka 812-8582, Japan; and Department of Orthopaedics (H.A.), Gifu University Graduate School of Medicine, Gifu 501-1194, Japan
| | - Kaori Tokunaga
- Division of Molecular Life Science (M.I., Y.S., T.B., K.-i.M.), Graduate School of Systems Life Science; Department of Molecular Biology (Y.S., K.M., K.T., T.B., K.-i.M.), Graduate School of Medical Sciences; Division of Bioinformatics (T.S., M.S.), Medical Institute of Bioregulation; and Department of Advanced Medical Initiatives (Y.O.), Japan Science and Technology Agency-Core Research for Evolutional Science and Technology, Faculty of Medicine, Kyushu University, Higashi-ku, Fukuoka 812-8582, Japan; and Department of Orthopaedics (H.A.), Gifu University Graduate School of Medicine, Gifu 501-1194, Japan
| | - Tetsuya Sato
- Division of Molecular Life Science (M.I., Y.S., T.B., K.-i.M.), Graduate School of Systems Life Science; Department of Molecular Biology (Y.S., K.M., K.T., T.B., K.-i.M.), Graduate School of Medical Sciences; Division of Bioinformatics (T.S., M.S.), Medical Institute of Bioregulation; and Department of Advanced Medical Initiatives (Y.O.), Japan Science and Technology Agency-Core Research for Evolutional Science and Technology, Faculty of Medicine, Kyushu University, Higashi-ku, Fukuoka 812-8582, Japan; and Department of Orthopaedics (H.A.), Gifu University Graduate School of Medicine, Gifu 501-1194, Japan
| | - Takashi Baba
- Division of Molecular Life Science (M.I., Y.S., T.B., K.-i.M.), Graduate School of Systems Life Science; Department of Molecular Biology (Y.S., K.M., K.T., T.B., K.-i.M.), Graduate School of Medical Sciences; Division of Bioinformatics (T.S., M.S.), Medical Institute of Bioregulation; and Department of Advanced Medical Initiatives (Y.O.), Japan Science and Technology Agency-Core Research for Evolutional Science and Technology, Faculty of Medicine, Kyushu University, Higashi-ku, Fukuoka 812-8582, Japan; and Department of Orthopaedics (H.A.), Gifu University Graduate School of Medicine, Gifu 501-1194, Japan
| | - Yasuyuki Ohkawa
- Division of Molecular Life Science (M.I., Y.S., T.B., K.-i.M.), Graduate School of Systems Life Science; Department of Molecular Biology (Y.S., K.M., K.T., T.B., K.-i.M.), Graduate School of Medical Sciences; Division of Bioinformatics (T.S., M.S.), Medical Institute of Bioregulation; and Department of Advanced Medical Initiatives (Y.O.), Japan Science and Technology Agency-Core Research for Evolutional Science and Technology, Faculty of Medicine, Kyushu University, Higashi-ku, Fukuoka 812-8582, Japan; and Department of Orthopaedics (H.A.), Gifu University Graduate School of Medicine, Gifu 501-1194, Japan
| | - Haruhiko Akiyama
- Division of Molecular Life Science (M.I., Y.S., T.B., K.-i.M.), Graduate School of Systems Life Science; Department of Molecular Biology (Y.S., K.M., K.T., T.B., K.-i.M.), Graduate School of Medical Sciences; Division of Bioinformatics (T.S., M.S.), Medical Institute of Bioregulation; and Department of Advanced Medical Initiatives (Y.O.), Japan Science and Technology Agency-Core Research for Evolutional Science and Technology, Faculty of Medicine, Kyushu University, Higashi-ku, Fukuoka 812-8582, Japan; and Department of Orthopaedics (H.A.), Gifu University Graduate School of Medicine, Gifu 501-1194, Japan
| | - Mikita Suyama
- Division of Molecular Life Science (M.I., Y.S., T.B., K.-i.M.), Graduate School of Systems Life Science; Department of Molecular Biology (Y.S., K.M., K.T., T.B., K.-i.M.), Graduate School of Medical Sciences; Division of Bioinformatics (T.S., M.S.), Medical Institute of Bioregulation; and Department of Advanced Medical Initiatives (Y.O.), Japan Science and Technology Agency-Core Research for Evolutional Science and Technology, Faculty of Medicine, Kyushu University, Higashi-ku, Fukuoka 812-8582, Japan; and Department of Orthopaedics (H.A.), Gifu University Graduate School of Medicine, Gifu 501-1194, Japan
| | - Ken-ichirou Morohashi
- Division of Molecular Life Science (M.I., Y.S., T.B., K.-i.M.), Graduate School of Systems Life Science; Department of Molecular Biology (Y.S., K.M., K.T., T.B., K.-i.M.), Graduate School of Medical Sciences; Division of Bioinformatics (T.S., M.S.), Medical Institute of Bioregulation; and Department of Advanced Medical Initiatives (Y.O.), Japan Science and Technology Agency-Core Research for Evolutional Science and Technology, Faculty of Medicine, Kyushu University, Higashi-ku, Fukuoka 812-8582, Japan; and Department of Orthopaedics (H.A.), Gifu University Graduate School of Medicine, Gifu 501-1194, Japan
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