Lentiviral Mediated Transgenesis by In Vivo Manipulation of Spermatogonial Stem Cells

The blood–testis barrier (BTB) is thought to be indispensable for spermatogenesis because it creates a special environment for meiosis and protects haploid cells from the immune system. The BTB divides the seminiferous tubules into the adluminal and basal compartments. Spermatogonial stem cells (SSCs) have a unique ability to transmigrate from the adluminal compartment to the basal compartment through the BTB upon transplantation into the seminiferous tubule. Here, we analyzed the role ofCldn11, a major component of the BTB, in spermatogenesis using spermatogonial transplantation.Cldn11-deficient mice are infertile due to the cessation of spermatogenesis at the spermatocyte stage.Cldn11-deficient SSCs failed to colonize wild-type testes efficiently, andCldn11-deficient SSCs that underwent double depletion ofCldn3andCldn5showed minimal colonization, suggesting that claudins on SSCs are necessary for transmigration. However,Cldn11-deficient Sertoli cells increased SSC homing efficiency by >3-fold, suggesting that CLDN11 in Sertoli cells inhibits transmigration of SSCs through the BTB. In contrast to endogenous SSCs in intactCldn11-deficient testes, those from WT orCldn11-deficient testes regenerated sperm inCldn11-deficient testes. The success of this autologous transplantation appears to depend on removal of endogenous germ cells for recipient preparation, which reprogrammed claudin expression patterns in Sertoli cells. Consistent with this idea, in vivo depletion ofCldn3/5regenerated endogenous spermatogenesis inCldn11-deficient mice. Thus, coordinated claudin expression in both SSCs and Sertoli cells expression is necessary for SSC homing and regeneration of spermatogenesis, and autologous stem cell transplantation can rescue congenital defects of a self-renewing tissue.

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Production of fertile sperm from in vitro propagating enriched spermatogonial stem cells of farmed catfish, Clarias batrachus

Zygote ◽

10.1017/s0967199416000149 ◽

2016 ◽

Vol 24 (6) ◽

pp. 814-824 ◽

Cited By ~ 6

Author(s):

Swapnarani Nayak ◽

Shajahan Ferosekhan ◽

Sangram Ketan Sahoo ◽

Jitendra Kumar Sundaray ◽

Pallipuram Jayasankar ◽

...

Keyword(s):

Stem Cells ◽

Gradient Centrifugation ◽

Spermatogonial Stem Cells ◽

Clarias Batrachus ◽

Proliferating Cells ◽

Complex Process ◽

Ficoll Gradient ◽

Mrna Expression Profiling

SummarySpermatogenesis is a highly co-ordinated and complex process. In vitro propagation of spermatogonial stem cells (SSCs) could provide an avenue in which to undertake in vivo studies of spermatogenesis. Very little information is known about the SSC biology of teleosts. In this study, collagenase-treated testicular cells of farmed catfish (Clarias batrachus, popularly known as magur) were purified by Ficoll gradient centrifugation followed by magnetic activated cell sorting using Thy1.2 (CD90.2) antibody to enrich for the spermatogonial cell population. The sorted spermatogonial cells were counted and gave ~3 × 106 cells from 6 × 106 pre-sorted cells. The purified cells were cultured in vitro for >2 months in L-15 medium containing fetal bovine serum (10%), carp serum (1%) and other supplements. Microscopic observations depicted typical morphological SSC features, bearing a larger nuclear compartment (with visible perinuclear bodies) within a thin rim of cytoplasm. Cells proliferated in vitro forming clumps/colonies. mRNA expression profiling by qPCR documented that proliferating cells were Plzf + and Pou2+, indicative of stem cells. From 60 days onwards of cultivation, the self-renewing population differentiated to produce spermatids (~6 × 107 on day 75). In vitro-produced sperm (2260 sperm/SSC) were free swimming in medium and hence motile (non-progressive) in nature. Of those, 2% were capable of fertilizing and generated healthy diploid fingerlings. Our documented evidence provides the basis for producing fertile magur sperm in vitro from cultured magur SSCs. Our established techniques of SSC propagation and in vitro sperm production together should trigger future in vivo experiments towards basic and applied biology research.

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In Vivo Genetic Manipulation of Spermatogonial Stem Cells and Their Microenvironment by Adeno-Associated Viruses

Stem Cell Reports ◽

10.1016/j.stemcr.2018.03.005 ◽

2018 ◽

Vol 10 (5) ◽

pp. 1551-1564 ◽

Cited By ~ 9

Author(s):

Satoshi Watanabe ◽

Mito Kanatsu-Shinohara ◽

Narumi Ogonuki ◽

Shogo Matoba ◽

Atsuo Ogura ◽

...

Keyword(s):

Stem Cells ◽

Genetic Manipulation ◽

Spermatogonial Stem Cells

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Loss of Ubiquitin Carboxy-Terminal Hydrolase L1 Impairs Long-Term Differentiation Competence and Metabolic Regulation in Murine Spermatogonial Stem Cells

Cells ◽

10.3390/cells10092265 ◽

2021 ◽

Vol 10 (9) ◽

pp. 2265

Author(s):

Whitney F. Alpaugh ◽

Anna L. Voigt ◽

Rkia Dardari ◽

Lin Su ◽

Iman Al Khatib ◽

...

Keyword(s):

Stem Cells ◽

Metabolic Regulation ◽

Spermatogonial Stem Cells ◽

Seminiferous Tubules ◽

Testis Weight ◽

Stem And Progenitor Cells ◽

Cell Transcriptome ◽

Carboxy Terminal

Spermatogonia are stem and progenitor cells responsible for maintaining mammalian spermatogenesis. Preserving the balance between self-renewal of spermatogonial stem cells (SSCs) and differentiation is critical for spermatogenesis and fertility. Ubiquitin carboxy-terminal hydrolase-L1 (UCH-L1) is highly expressed in spermatogonia of many species; however, its functional role has not been identified. Here, we aimed to understand the role of UCH-L1 in murine spermatogonia using a Uch-l1−/− mouse model. We confirmed that UCH-L1 is expressed in undifferentiated and early-differentiating spermatogonia in the post-natal mammalian testis. The Uch-l1−/− mice showed reduced testis weight and progressive degeneration of seminiferous tubules. Single-cell transcriptome analysis detected a dysregulated metabolic profile in spermatogonia of Uch-l1−/− compared to wild-type mice. Furthermore, cultured Uch-l1−/− SSCs had decreased capacity in regenerating full spermatogenesis after transplantation in vivo and accelerated oxidative phosphorylation (OXPHOS) during maintenance in vitro. Together, these results indicate that the absence of UCH-L1 impacts the maintenance of SSC homeostasis and metabolism and impacts the differentiation competence. Metabolic perturbations associated with loss of UCH-L1 appear to underlie a reduced capacity for supporting spermatogenesis and fertility with age. This work is one step further in understanding the complex regulatory circuits underlying SSC function.

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Successful transplantation of spermatogonial stem cells into the seminiferous tubules of busulfan-treated mice

Reproductive Health ◽

10.1186/s12978-021-01242-4 ◽

2021 ◽

Vol 18 (1) ◽

Author(s):

Hossein Azizi ◽

Amirreza Niazi Tabar ◽

Thomas Skutella

Keyword(s):

Stem Cells ◽

Flow Cytometry ◽

Spermatogonial Stem Cells ◽

Pcr Analysis ◽

Seminiferous Tubules ◽

Flow Cytometry Analysis ◽

Rt Pcr ◽

Expression Levels ◽

Successful Transplantation

Abstract Background Spermatogonial stem cells (SSCs) in the testis are crucial for transferring genetic information to the next generation. Successful transplantation of SSCs to infertile men is an advanced therapeutic application in reproductive biology research. Methods In this experimental research, both in vitro and in vivo characterization of undifferentiated and differentiated SSCs were performed by morphology—immunocytochemistry (ICC), immunohistochemistry (IMH), Fluidigm Real-Time polymerase chain reaction (RT-PCR) and flow cytometry analysis. The isolated SSCs were finally microinjected into the rete testis of busulfan-treated mice. The compact undifferentiated and more loosely connected round differentiated SSCs were isolated during testicular cell expansion from their specific feeder layer. Results ICC analysis indicated high and low expression levels of Zbtb16 in undifferentiated and differentiated germ cells. Also, IMH analysis showed different expression levels of Zbtb16 in the two different germ stem cell populations of the testicular tissue. While Fluidigm RT-PCR analysis indicated overexpression of the TAF4B germ cell gene, the expression of DAZL, VASA, and Zbtb16 were down-regulated during the differentiation of SSCs (P < 0.05). Also, flow cytometry analysis confirmed the significant downregulation of Itgb1 and Itga4 during differentiation. By transplantation of SSCs into busulfan-treated NOD/SCID mice, GFP-labeled sperm cells developed. Conclusions In the current study, we performed a transplantation technique that could be useful for the future microinjection of SSCs during infertility treatment and for studying in vivo differentiation of SSCs into sperm.

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GDNF Maintains Mouse Spermatogonial Stem Cells In Vivo and In Vitro

Methods in Molecular Biology™ - Germline Stem Cells ◽

10.1007/978-1-60327-214-8_9 ◽

2008 ◽

pp. 127-135 ◽

Cited By ~ 13

Author(s):

Hannu Sariola ◽

Tiina Immonen

Keyword(s):

Stem Cells ◽

Spermatogonial Stem Cells

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Successful Transplantation of Spermatogonial Stem Cells Into the Seminiferous Tubules of Busulfan-treated Mice

10.21203/rs.3.rs-135556/v1 ◽

2020 ◽

Author(s):

Hossein Azizi ◽

Amirreza Niazi Tabar ◽

Thomas Skutella

Keyword(s):

Stem Cells ◽

Flow Cytometry ◽

Spermatogonial Stem Cells ◽

Pcr Analysis ◽

Seminiferous Tubules ◽

Flow Cytometry Analysis ◽

Rt Pcr ◽

Expression Levels ◽

Successful Transplantation

Abstract Background: Spermatogonial stem cells (SSCs) in the testis are crucial for transferring genetic information to the next generation. Successful transplantation of SSCs to infertile men is an advanced therapeutic application in reproductive biology research. Methods: In this experimental research, both in vitro and in vivo characterization of undifferentiated and differentiated SSCs were performed by morphology - immunocytochemistry (ICC), immunohistochemistry (IMH), Fluidigm Real-Time polymerase chain reaction (RT-PCR) and flow cytometry analysis. The isolated SSCs were finally microinjected into the rete testis of busulfan-treated mice. The compact undifferentiated and more loosely connected round differentiated SSCs were isolated during testicular cell expansion from their specific feeder layer.Results: ICC analysis indicated high and low expression levels of Zbtb16 in undifferentiated and differentiated germ cells. Also, IMH analysis showed different expression levels of Zbtb16 in the two different germ stem cell populations of the testicular tissue. While Fluidigm RT-PCR analysis indicated overexpression of the TAF4B germ cell gene, the expression of DAZL, VASA, and Zbtb16 were down-regulated during the differentiation of SSCs (P< 0.05). Also, flow cytometry analysis confirmed the significant downregulation of Itgb1 and Itga4 during differentiation. By transplantation of SSCs into busulfan-treated NOD/SCID mice, GFP-labeled sperm cells developed. Conclusions: In the current study, we performed a transplantation technique that could be useful for the future microinjection of SSCs during infertility treatment and for studying in vivo differentiation of SSCs into sperm.

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