Jonathan A. Schmidt

Prepubertal human spermatogonia and mouse gonocytes share conserved gene expression of germline stem cell regulatory molecules

In the human testis, beginning at ≈2 months of age, gonocytes are replaced by adult dark (Ad) and pale (Ap) spermatogonia that make up the spermatogonial stem cell (SSC) pool. In mice, the SSC pool arises from gonocytes ≈6 days after birth. During puberty in both species, complete spermatogenesis is established by cells that differentiate from SSCs. Essentially pure populations of prepubertal human spermatogonia and mouse gonocytes were selected from testis biopsies and validated by confirming the presence of specific marker proteins in cells. Stem cell potential of germ cells was demonstrated by transplantation to mouse testes, following which the cells migrated to the basement membrane of the seminiferous tubule and were maintained similar to SSCs. Differential gene expression profiles generated between germ cells and testis somatic cells demonstrated that expression of genes previously identified as SSC and spermatogonial-specific markers (e.g., zinc-finger and BTB-domain containing 16, ZBTB16) was greatly elevated in both human spermatogonia and mouse gonocytes compared to somatic cells. Several genes were expressed at significantly higher levels in germ cells of both species. Most importantly, genes known to be essential for mouse SSC self-renewal (e.g., Ret proto-oncogene, Ret; GDNF-family receptor α1, Gfrα1; and B-cell CLL/lymphoma 6, member B, Bcl6b) were more highly expressed in both prepubertal human spermatogonia and mouse gonocytes than in somatic cells. The results indicate remarkable conservation of gene expression, notably for self-renewal genes, in these prepubertal germline cells between two species that diverged phylogenetically ≈75 million years ago.

Effect of Vascular Endothelial Growth Factor and Testis Tissue Culture on Spermatogenesis in Bovine Ectopic Testis Tissue Xenografts

Abstract Bovine ectopic testis tissue grafting is a technique that can be used to study bovine spermatogenesis and for the production of germ cells for a variety of applications. Approximately 10% of seminiferous tubule cross sections in testis grafts contain spermatids, providing a unique tool to investigate what regulates germ cell differentiation. We hypothesized that manipulation of testis tissue grafts would increase the percentage of seminiferous tubule cross sections undergoing complete germ cell differentiation. To test this hypothesis, bovine testis tissue was treated with vascular endothelial growth factor (VEGF) at the time of grafting or explant cultured for 1 wk prior to grafting. For the VEGF experiment, 8-wk donor tissue and graft sites were treated with 1 μg of VEGF in order to increase angiogenesis at the graft site. For the testis tissue culture experiment, 4-wk-old donor testis was cultured for 1 wk prior to grafting to stimulate spermatogonial stem cell proliferation. Testis tissue grafts were removed from the mice 24 wk after grafting. VEGF treatment increased graft weight and the percentage of seminiferous tubule cross sections with elongating spermatids at the time of graft removal. Cultured testis tissue grafts were smaller and had fewer seminiferous tubules per graft. However, there was no difference in the percentage of seminiferous tubule cross sections that contained any germ cell type between groups. These data indicate for the first time that bovine testis tissue can be manipulated to better support germ cell differentiation in grafted tissue.

Spermatogonial stem cells derived from infertile Wv/Wv mice self-renew in vitro and generate progeny following transplantation.

Abstract Loss-of-function mutation of the Kit gene causes a severe defect in spermatogenesis that results in infertility due to the inability of its cognate ligand, KIT ligand (KITL), to stimulate spermatogonial proliferation and differentiation. Although self-renewal of mouse spermatogonial stem cells (SSCs) depends on glial cell line-derived neurotrophic factor (GDNF), there is no unequivocal evidence that SSCs with a KIT deficiency can self-renew in vivo or in vitro. In the testis of Wv/Wv mice, in which the KIT tyrosine kinase activity is impaired, spermatogonia with SSC phenotype were identified. When Wv/Wv spermatogonia were cultured in an SSC culture system supplemented with GDNF in a 10% O2 atmosphere, they formed clumps and proliferated continuously. An atmosphere of 10% O2 was better than 21% O2 to support SSC self-renewal. When Wv/Wv clump-forming germ cells were transplanted into testes of infertile wild-type busulfan-treated mice, they colonized the seminiferous tubules but did not differentiate. However, when transplanted into the testes of infertile W/Wv pups, they restored spermatogenesis and produced spermatozoa, and progeny were generated using microinsemination. These results clearly show that SSCs exist in Wv/Wv testes and that they proliferate in vitro similar to wild-type SSCs, indicating that a functional KIT protein is not required for SSC self-renewal. Furthermore, the results indicate that a defect of KIT/KITL signaling of Wv/Wv SSCs does not prevent spermatogonial differentiation and spermatogenesis in some recipient strains.

Cryopreservation in trehalose preserves functional capacity of murine spermatogonial stem cells.

Development of techniques to isolate, culture, and transplant human spermatogonial stem cells (SSCs) has the future potential to treat male infertility. To maximize the efficiency of these techniques, methods for SSC cryopreservation need to be developed to bank SSCs for extended periods of time. Although, it has been demonstrated that SSCs can reinitiate spermatogenesis after freezing, optimal cryopreservation protocols that maximize SSC proliferative capacity post-thaw have not been identified. The objective of this study was to develop an efficient cryopreservation technique for preservation of SSCs. To identify efficient cryopreservation methods for long-term preservation of SSCs, isolated testis cells enriched for SSCs were placed in medium containing dimethyl sulfoxide (DMSO) or DMSO and trehalose (50 mM, 100 mM, or 200 mM), and frozen in liquid nitrogen for 1 week, 1 month, or 3 months. Freezing in 50 mM trehalose resulted in significantly higher cell viability compared to DMSO at all thawing times and a higher proliferation rate compared to DMSO for the 1 week freezing period. Freezing in 200 mM trehalose did not result in increased cell viability; however, proliferation activity was significantly higher and percentage of apoptotic cells was significantly lower compared to DMSO after freezing for 1 and 3 months. To confirm the functionality of SSCs frozen in 200 mM trehalose, SSC transplantation was performed. Donor SSCs formed spermatogenic colonies and sperm capable of generating normal progeny. Collectively, these results indicate that freezing in DMSO with 200 mM trehalose serves as an efficient method for the cryopreservation of SSCs.

Identification of Glial Cell Line-Derived Neurotrophic Factor-Regulated Genes Important for Spermatogonial Stem Cell Self-Renewal in the Rat

Abstract Spermatogonial stem cells (SSCs) provide the foundation for spermatogenesis throughout the life of a male. Because SSCs of many species can colonize the mouse testis, and glial cell line-derived neurotrophic factor (GDNF) is responsible for stimulating SSC self-renewal in rodents, we reasoned that molecular mechanisms of SSC self-renewal are similar across species. GDNF-regulated genes have been identified in mouse SSCs; however, downstream targets of GDNF are unknown in other species. The objective of this work was to identify GDNF-regulated genes in rat SSCs and to define the biological significance of these genes for rat SSC self-renewal. We conducted microarray analysis on cultured rat germ cells enriched for SSCs in the presence and absence of GDNF. Many GDNF-regulated genes were identified, most notably, Bcl6b and Etv5, which are important for mouse SSC self-renewal. Bcl6b was the most highly regulated gene in both the rat and mouse. Additionally, we identified three novel GDNF-regulated genes in rat SSCs: Bhlhe40, Hoxc4, and Tec. Small interfering RNA treatment for Bcl6b, Etv5, Bhlhe40, Hoxc4, and Tec resulted in a decrease in SSC number, as determined by transplantation, without a change in total cell number within the culture. These data indicate that, like in the mouse SSC, Bcl6b and Etv5 are important for rat SSC self-renewal, suggesting that these genes may be important for SSCs in all mammals. Furthermore, identification of three novel GDNF-regulated genes in the rat SSC extends our knowledge of SSC activity and broadens the foundation for understanding this process in higher species, including humans.

Analysis of Gene Expression in Bovine Testis Tissue Prior to Ectopic Testis Tissue Xenografting and During the Grafting Period

Abstract The purpose of this study was to identify factors that contribute to bovine testis development and donor age-dependent differences in the abilities of bovine ectopic testis tissue grafts to produce elongated spermatids. We used real-time RT-PCR and microarrays to evaluate and to identify the expression of genes that are involved in Sertoli and germ cell development in bovine testis tissues. Testis tissues were obtained from 2-, 4-, and 8-wk-old bull calves and were grafted immediately. Grafted bovine testis tissue was removed from mice, RNA was isolated from the grafts, and real-time RT-PCR was used to evaluate gene expression during the grafting period. In addition, the gene expression in the donor tissue was analyzed using Affymetrix Bovine GeneChips, to identify differentially expressed genes. Examination of the testis tissue grafts indicated that Sertoli cell-specific gene expression was lower in 8-wk donor tissue grafts compared to the donors of other ages. Furthermore, the expression of KIT, which is a germ cell-specific gene, was low in testis tissue grafts. Microarray analysis of the donor tissue showed that several genes that are involved in angiogenesis or tissue growth were differentially expressed in 2-, 4-, and 8-wk-old bovine testes. The levels of expression of the genes for angiogenin, transgelin, thrombomodulin, early growth response 1, insulin-like growth factor 2, and insulin-like growth factor-binding protein 3 were lower in testis tissues from older animals. Using these data, it will be possible in the future to manipulate the testis xenograft microenvironment so as to improve the efficiency of sperm production within the graft.

Grafting Period and Donor Age Affect the Potential for Spermatogenesis in Bovine Ectopic Testis Xenografts

Abstract Bovine testis tissue xenografts contain elongating spermatids 6 mo after grafting. The percentage of seminiferous tubule cross sections with elongating spermatids at the time of graft removal varies depending on donor age and rarely exceeds 10%. These data indicate significant changes are occurring to bovine testicular cells during the first weeks of life. The objective of this research was to xenograft testis tissue from multiple ages of bull calves for 24 or 36 wk in order to gain a better understanding of early bovine testis development. Testis tissue from 1-, 2-, 4-, and 8-wk-old calves was grafted onto the backs of castrated immunodeficient mice. Testis tissue from all donor ages grew, differentiated, and produced testosterone and elongating spermatids. Testis tissue grafts from 1- and 8-wk-old calves had elongating spermatids in greater than 5.5% of seminiferous tubule cross sections at the time of graft removal regardless of grafting period. Four-week-old donor tissue never had more than 5.2% of seminiferous tubule cross sections with elongating spermatids. Extending the grafting period from 24 to 36 wk resulted in an increase in the percentage of seminiferous tubule cross sections with elongating spermatids from 2% to 10% in 2-wk donor tissue. These data demonstrate that both donor age and grafting period may be important factors regulating the maturation of bovine testis xenografts, indicating that intrinsic differences exist within testis tissue at these donor ages. These data provide the framework for further study of bovine spermatogenesis using ectopic testis xenografting.

In Vivo and In Vitro Aging Is Detrimental to Mouse Spermatogonial Stem Cell Function

The development of techniques to maintain the spermatogonial stem cell (SSC) in vivo and in vitro for extended periods essentially allows for the indefinite continuation of an individual germline. Recent evidence indicates that the aging of male reproductive function is due to failure of the SSC niche. SSCs are routinely cultured for 6 mo, and no apparent effect of culture over this period has been observed. To determine the effects of SSC aging, we utilized an in vitro culture system, followed by quantitative transplantation experiments. After culture for 6 mo, SSCs that had been aged in vivo for 1500 days had a slower proliferation rate than SSCs that were aged in vivo to 8 or 300 days. Examination of methylation patterns revealed no apparent difference in DNA methylation between SSCs that were aged 8, 300, or 1500 days before culture. Long-term culture periods resulted in a loss of stem cell potential without an obvious change in the visual appearance of the culture. DNA microarray analysis of in vivo- and in vitro-aged SSCs identified the differential expression of several genes important for SSC function, including B-cell CLL/lymphoma 6, member B (Bcl6b), Lim homeobox protein 1 (Lhx1), and thymus cell antigen 1, theta (Thy1). Collectively, these data indicate that, although both in vitro and in vivo aging are detrimental to SSC function, in vitro aging results in greater loss of function, potentially due to a decrease in core SSC self-renewal gene expression and an increase in germ cell differentiation gene expression.

Endocrine Regulation of the Establishment of Spermatogenesis in Pigs

Somatic and germ cell maturation precedes the start of spermatogenesis and is coordinated, so efficient spermatogenesis will occur in the adults. The present study was conducted to evaluate endocrine regulation of germ and somatic cell homeostasis in the neonatal boar testis associated with the establishment of spermatogenesis. Testis tissue obtained from 3-, 5-, 7- and 14-day-old piglets were ectopically xenografted onto castrated, immunodeficient nude mice. Grafts were removed 22 weeks later and evaluated for growth and the establishment of spermatogenesis. Recipient mouse testosterone biosynthesis and follicle-stimulating hormone (FSH) concentrations were also assayed. Testis tissue graft growth was significantly greater in testis grafts from 3-day donor tissue when compared to all other ages; 5-, 7- and 14-day-old donor tissue weights were not significantly different at removal. Follicle-stimulating hormone concentrations in recipient mice supporting testis grafts from 5-, 7- and 14-day-old donor tissues did not differ and were similar to normal physiological levels in age-matched, intact nude mice. Serum FSH levels were significantly lower in recipient mice supporting testis grafts from 3-day-old donor tissue. Radioimmunoassay and biological assay indicated no differences in testosterone production by testis tissue grafts of varying donor age. Porcine testis tissue obtained from 3-, 5-, 7- and 14-day-old neonatal boars were all capable of producing round and elongate spermatids after 22 weeks of grafting, but testis grafts from 14-day-old donors had a significantly greater (eightfold) percentage of seminiferous tubules with spermatids compared to all other donor ages (p < 0.05). Cryopreservation did not affect the ability of testis tissue grafts to grow, produce testosterone or establish spermatogenesis when compared to controls (p < 0.05). Collectively, these data demonstrate intrinsic differences in the biological activity of germ and somatic cell populations during neonatal boar testis development associated with the establishment of spermatogenesis.

Cryopreservation of porcine spermatogonial stem cells by slow-freezing testis tissue in trehalose.

Spermatogonial stem cells provide the foundation for continued adult spermatogenesis and their manipulation can facilitate assisted reproductive technologies or the development of transgenic animals. Because the pig is an important agricultural and biomedical research animal, the development of practical application techniques to manipulate the pig Spermatogonial stem cell is needed. The ability to preserve porcine Spermatogonial stem cell or testis tissue long term is one of these fundamental techniques. The objective of this study was to optimize methods to cryopreserve porcine Spermatogonial stem cell when freezing testis cells or testis tissue. To identify the most efficient cryopreservation technique, porcine testis cells (cell freezing) or testis tissue (tissue freezing) were frozen in medium containing dimethyl sulfoxide (DMSO) and fetal bovine serum (FBS) or DMSO, FBS, and various concentrations of trehalose (50, 100, or 200 mM). After thawing, undifferentiated germ cells were enriched and treatments were evaluated for cryopreservation efficiency. The tissue freezing method resulted in significantly greater germ cell recovery (P = 0.041) and proliferation capacity (P < 0.001) compared to the cell freezing treatment. Regardless of freezing method (cell vs. tissue), addition of 200 mM trehalose to freezing medium increased germ cell recovery and proliferation capacity compared to cells frozen using the same freezing method without trehalose. Interestingly, addition of trehalose to the tissue freezing medium significantly increased germ cell recovery (P = 0.012) and proliferation capacity (P = 0.004) compared to the cell freezing treatment supplemented with trehalose. To confirm that cryopreservation in trehalose improves the survival of Spermatogonial stem cell, testis cells enriched for undifferentiated germ cells were xenotransplanted into recipient mouse testes. Germ cells recovered from tissue frozen with 200 mM trehalose generated significantly more (P < 0.001) donor derived colonies than tissue frozen without trehalose. Regardless of cryopreservation medium or freezing method, testis cell recovery, viability, and proliferation capacity of germ cells after thawing were significantly lower compared to those of untreated fresh control. Nevertheless, these data demonstrate that undifferentiated porcine germ cells can be efficiently cryopreserved in the presence of 200 mM trehalose.