Spermatogenesis and germ cell transgene expression in xenografted bovine testicular tissue

The present study was conducted to evaluate the development of spermatogenesis and utility of using electroporation to stably transfect germ cells with the beta-galactosidase gene in neonatal bovine testicular tissue ectopically xenografted onto the backs of recipient nude mice. Bull testicular tissue from 4-wk donor calves, which contains a germ cell population consisting solely of gonocytes or undifferentiated spermatogonia, was grafted onto the backs of castrated adult recipient nude mice. Testicular grafts significantly increased in weight throughout the grafting period and the timing of germ cell differentiation in grafted tissue was consistent with postnatal testis development in vivo relative to the bull. Seminiferous tubule diameter also significantly increased with advancing time after grafting. At 1 wk after grafting, gonocytes in the seminiferous cords completed migration to the basement membrane and differentiated germ cell types could be observed 24 wk after grafting. The presence of elongating spermatids at 24 wk confirmed that germ cell differentiation occurred in the bovine tissue. Leydig cells in the grafted bovine tissue were also capable of producing testosterone in the castrated recipient mice from 4 wk to 24 wk after grafting at concentrations that were similar to levels in intact, nongrafted control mice. The testicular tissue that had been electroporated with a beta-galactosidase expression vector showed tubule-specific transgene expression 24 wk after grafting. Histological analysis showed that transgene expression was present in both Sertoli and differentiated germ cells but not in interstitial cells. The system reported here has the potential to be used for generation of transgenic bovine spermatozoa.     

Characterization of spermatogonial stem cell maturation and differentiation in neonatal mice

Initiation of the first wave of spermatogenesis in the neonatal mouse testis is characterized by the differentiation of a transient population of germ cells called gonocytes found in the center of the seminiferous tubule. The fate of gonocytes depends upon these cells resuming mitosis and developing the capacity to migrate from the center of the seminiferous tubule to the basement membrane. This process begins approximately Day 3 postpartum in the mouse, and by Day 6 postpartum differentiated type A spermatogonia first appear. It is essential for continual spermatogenesis in adults that some gonocytes differentiate into spermatogonial stem cells, which give rise to all differentiating germ cells in the testis, during this neonatal period. The presence of spermatogonial stem cells in a population of cells can be assessed with the use of the spermatogonial stem cell transplantation technique. Using this assay, we found that germ cells from the testis of Day 0-3 mouse pups can colonize recipient testes but do not proliferate and establish donor-derived spermatogenesis. However, germ cells from testes of Day 4-5 postpartum mice colonize recipient testes and generate large areas of donor-derived spermatogenesis. Likewise, germ cells from Day 10, 12, and 28 postpartum animals and adult animals colonize and establish donor-derived spermatogenesis, but a dramatic reduction in the number of colonies and the extent of colonization occurs from germ cell donors Days 12-28 postpartum that continues in adult donors. These results suggest spermatogonial stem cells are not present or not capable of initiating donor-derived spermatogenesis until Days 3-4 postpartum. The analysis of germ cell development during this time frame of development and spermatogonial stem cell transplantation provides a unique system to investigate the establishment of the stem cell niche within the mouse testis.     

White-spotting mutations affect the regenerative differentiation of testicular germ cells: demonstration by experimental cryptorchidism and its surgical reversal.

The effect of white-spotting (W) mutations on differentiation of testicular germ cells was investigated by using experimental cryptorchidism and its surgical reversal. All mutant mice used in this study (Wv/+, Wsh/+, Wf/+ and Wf/Wf) showed normal fertility and well-ordered spermatogenesis, as in congenic +/+ mice. In the cryptorchid testis, which contains only type A spermatogonia as germ cells, the number and the proliferative activity of type A spermatogonia in mutant mice were comparable to +/+ mice. On the other hand, surgical reversal of the cryptorchid testis in mutants resulted in impaired regenerative differentiation of germ cells. Although complete recovery of spermatogenesis was observed in +/+ mice, testicular weight in Wsh/+, Wf/+ and Wf/Wf mice recovered to approximately 60-70% of intact levels, and some portions of seminiferous epithelium showed incomplete spermatogenesis. In Wv/+ mice, however, ability to recover the weight was completely lost, and only type A spermatogonia existed as germ cells in seminiferous tubules 3 mo after surgical reversal. These results suggest that W mutation affects the differentiation through type A spermatogonia to type B spermatogonia, indicating the functional significance of W (c-kit) in early spermatogenesis.     

Hormonal induction of all stages of spermatogenesis in germ-somatic cell coculture from immature Japanese eel testis

In cultivated male eel, spermatogonia are the only germ cells present in testis. Our previous studies using an organ culture system have shown that gonadotropin and 11-ketotestosterone (11-KT, a potent androgen in teleost fishes) can induce all stages of spermatogenesis in vitro. for detailed investigation of the control mechanisms of spermatogenesis, especially of the interaction between germ cells and testicular somatic cells during 11-KT-induced spermatogenesis in vitro, we have established a new culture system in which germ cells and somatic cells are cocultured after they are aggregated into pellets by centrifugation. Germ cells (spermatogonia) and somatic cells (mainly Sertoli cells) were isolated from immature eel testis. Coculture of the isolated germ cells and somatic cells without forming aggregation did not induce spermatogenesis, even in the presence of 11-KT. In contrast, when isolated germ cells and somatic cells were formed into pellets by centrifugation and were then cultured with 11-KT for 30 days, the entire process of spermatogenesis from premitotic spermatogonia to spermatozoa was induced. However, in the absence of 11-KT in the culture medium spermatogenesis was not induced, even when germ cell and somatic cells were aggregated. These results demonstrate that physical contact of germ cells to Sertoli cells is required for inducing spermatogenesis in response to 11-KT.     

Functional analysis of the p53 gene in apoptosis induced by heat stress or loss of stem cell factor signaling in mouse male germ cells

Apoptosis plays an important role in controlling germ cell numbers and restricting abnormal cell proliferation during spermatogenesis. The tumor suppressor protein, p53, is highly expressed in the testis, and is known to be involved in apoptosis, which suggests that it is one of the major causes of germ cell loss in the testis. Mice that are c-kit/SCF mutant (Sl/Sld) and cryptorchid show similar testicular phenotypes; they carry undifferentiated spermatogonia and Sertoli cells in their seminiferous tubules. To investigate the role of p53-dependent apoptosis in infertile testes, we transplanted p53-deficient spermatogonia that were labeled with enhanced green fluorescence protein into cryptorchid and Sl/Sld testes. In cryptorchid testes, transplanted p53-deficient spermatogonia differentiated into spermatocytes, but not into haploid spermatids. In contrast, no differentiated germ cells were observed in Sl/Sld mutant testes. These results indicate that the mechanism of germ cell loss in the c-kit/SCF mutant is not dependent on p53, whereas the apoptotic mechanism in the cryptorchid testis is quite different (i.e., although the early stage of differentiation of spermatogonia and the meiotic prophase is dependent on p53-mediated apoptosis, the later stage of spermatids is not).     

Lymphoid-specific helicase (HELLS) is essential for meiotic progression in mouse spermatocytes

Lymphoid-specific helicase (HELLS; also known as LSH) is a member of the SNF2 family of chromatin remodeling proteins. Because Hells-null mice die at birth, a phenotype in male meiosis cannot be studied in these animals. Allografting of testis tissue from Hells(-/-) to wild-type mice was employed to study postnatal germ cell differentiation. Testes harvested at Day 18.5 of gestation from Hells(-/-), Hells(+/-), and Hells(+/+) mice were grafted ectopically to immunodeficient mice. Bromodeoxyuridine incorporation at 1 wk postgrafting revealed fewer dividing germ cells in grafts from Hells(-/-) than from Hells(+/+) mice. Whereas spermatogenesis proceeded through meiosis with round spermatids in grafts from Hells heterozygote and wild-type donor testes, spermatogenesis arrested at stage IV, and midpachytene spermatocytes were the most advanced germ cell type in grafts from Hells(-/-) mice at 4, 6, and 8 wk after grafting. Analysis of meiotic configurations at 22 days posttransplantation revealed an increase in Hells(-/-) spermatocytes with abnormal chromosome synapsis. These results indicate that in the absence of HELLS, proliferation of spermatogonia is reduced and germ cell differentiation arrested at the midpachytene stage, implicating an essential role for HELLS during male meiosis. This study highlights the utility of testis tissue grafting to study spermatogenesis in animal models that cannot reach sexual maturity.     

The testicular cycle of Gambusia affinis holbrooki (Teleostei: Poeciliidae)

Fifteen male mosquito fish ( Gambusia affinis holbrooki ) were collected in 1989 on the 15th of each month to perform a quantitative histologic study of the annual testicular cycle including a calculation of the gonadosomatic index, testicular volume, and the total volume per testis occupied by each germ cell type. The cycle comprises two periods: spermatogenesis and quiescence. The spermatogenic period begins in April with the development of primary spermatogonia into secondary spermatogonia, spermatocytes and round spermatids. In May, the first spermatogenic wave is completed and the testicular volume begins to increase up to June when the maximum testicular volume and gonadosomatic index are reached. Germ cell proliferation with successive spermatogenetic waves continues until August. In September germ cell proliferation ceases and neither secondary spermatogonia nor spermatocytes are observed. However, spermiogenesis continues until October. In November, spermiogenesis has stopped and the testis enters the quiescent period up to April. During this period only primary spermatogonia and spermatozoa are present in the testis. In addition, a few spermatids whose spermiogenesis was arrested in November are observed. Testicular release of spermatozoa is continuous during the entire spermatogenesis period. The spermatozoa formed at the end of this period (September-October) remain in the testis during the quiescent period and are released at the beginning of the next spermatogenesis period in April. Developed Leydig cells appear all year long in the testicular interstitium, mainly around both efferent ducts and the testicular tubule sections showing S₄ spermatids.     

Failure of spermatogenesis in mice lacking connexin43

Connexin43 (Cx43), a gap junction protein encoded by the Gja1 gene, is expressed in several cell types of the testis. Cx43 gap junctions couple Sertoli cells with each other, Leydig cells with each other, and spermatogonia/spermatocytes with Sertoli cells. To investigate the role of this communication pathway in spermatogenesis, we studied postnatal testis development in mice lacking Cx43. Because such mice die shortly after birth, it was necessary to graft testes from null mutant fetuses under the kidney capsules of adult males for up to 3 wk. Grafted wild-type testes were used as controls. In our initial experiments with wild-type testes, histological examination indicated that the development of grafted testes kept pace with that of nongrafted testes in terms of the onset of meiosis, but this development required the presence of the host gonads. When excised grafts were stimulated in vitro with cAMP or LH, there was no significant difference in androgen production between null mutant and wild-type testes, indicating that the absence of Cx43 had not compromised steroidogenesis. Previous research has shown that Cx43 null mutant neonates have a germ cell deficiency that arises during fetal life, and our analysis of grafted testes demonstrated that this deficiency persists postnatally, giving rise to a "Sertoli cell only" phenotype. These results indicate that intercellular communication via Cx43 channels is required for postnatal expansion of the male germ line.     

The inflammatory mediators TNFα and nitric oxide arrest spermatogonia GC-1 cell cycle

During an inflammatory process of the testis, the network of somatic, immune, and germ cell interactions is altered leading to organ dysfunction. In testicular biopsies of infertile men, spermatogenesis impairment is associated with reduced spermatogonia proliferation, increased number of immune cells, and content of pro-inflammatory cytokines. TNFα-TNFR and nitric oxide (NO)-NO synthase systems are up-regulated in models of testicular damage and in human testis with maturation arrest. The purpose of this study was to test the hypothesis that TNFα-TNFR system and NO alter the function of spermatogonia in the inflamed testis. We studied the effect of TNFα and NO on GC-1 spermatogonia cell cycle progression and death by flow cytometry. GC-1 cells expressed TNFR1 and TNFR2 (immunofluorescence). TNFα (10 and 50 ng/ml) and DETA-Nonoate (0.5 and 2 mM), a NO releaser, increased the percentage of cells in S-phase of the cell cycle and reduced the percentage in G1, inducing also cell apoptosis. TNFα effect was not mediated by oxidative stress unlike NO, since the presence of N-acetyl-l-cysteine (2.5 and 5.0 mM) prevented NO induced cell cycle arrest and death. GC-1 spermatogonia overpass NO induced cell cycle arrest but no TNFα, since after removal of NO, spermatogonia progressed through the cell cycle. We propose TNFα and NO might contribute to impairment of spermatogenesis by preventing adequate functioning of the spermatogonia population. Our results showed that TNFα and NO impaired spermatogonia cell cycle, inducing GC-1 arrest in the S phase.     

Localization of the epidermal growth factor (EGF) and epidermal growth factor receptor (EGFR) in the bovine testis

In the last few decades, several growth factors were identified in the testis of various mammalian species. Growth factors are shown to promote cell proliferation, regulate tissue differentiation, and modulate organogenesis. In the present investigation we have studied the localization of EGF and EGFR in the adult bovine testis by means of immunohistochemical method. Our results demonstrated that EGF and EGFR were localized solely to the bovine testicular germ cells (spermatogonia, spermatocytes, and round spermatids). In contrast, the somatic testicular cells (i.e., Sertoli, Leydig, and myofibroblast cells) exhibited no staining affinity. EGF and EGFR were additionally detected in the epithelial lining of straight tubules and rete testis. Interestingly, the distribution of EGF and EGFR in the germ cells was mainly dependent upon the cycle of the seminiferous epithelium since their localization appeared to be preponderant during the spermatogonia proliferation and during the meiotic and spermiogenic processes. In conclusion, such findings may suggest that EGF and EGFR are important paracrine and/or autocrine regulators of spermatogenesis in bovine.     

Recombinant human insulin-like growth factor I stimulates all stages of 11-ketotestosterone-induced spermatogenesis in the Japanese eel, Anguilla japonica, in vitro.

In this study, we examined the in vitro effects of insulin-like growth factor I (IGF-I) in the presence or absence of 11-ketotestosterone (11-KT: the spermatogenesis-inducing hormone) on the proliferation of Japanese eel (Anguilla japonica) testicular germ cells. Initially, a short-term culture (15 days) of testicular tissue with only type A and early type B spermatogonia (preproliferated spermatogonia) was carried out in Leibovitz-15 growth medium supplemented with different concentrations of recombinant human IGF (rhIGF)-I or -II in the presence or absence of 10 ng/ml of 11-KT. Late type B spermatogonia (proliferated spermatogonia) were observed in treatments of 100 ng/ml of both rhIGF-I and -II in combination with 11-KT, indicating the onset and progression of spermatogenesis. In all tested rhIGF-I concentrations (except 0.1 ng/ml) supplemented with 11-KT, late type B spermatogonia were detected in at least one individual. Then, we proceeded with an in vitro 45-day culture of testicular tissue with 100 ng/ml of rhIGF-I in the presence or absence of 10 ng/ml of 11-KT to test the long-term effects of rhIGF-I on the spermatogenetic cycle. The presence of all types of germ cells, including spermatozoa, in the testis cultured with the admixture of the two hormones indicated that the germ cells underwent complete spermatogenesis whereas no germ cell proliferation was observed when the rhIGF-I was applied alone. These results suggest that IGF-I in the presence of 11-KT plays an essential role in the onset, progress, and regulation of spermatogenesis in the testis of the Japanese eel.     

Impaired spermatogenesis in the Japanese eel, Anguilla japonica: Possibility of the existence of factors that regulate entry of germ cells into meiosis

In the cultivated male Japanese eel, spermatogonia are the only germ cells present in the testis. Weekly injections of human chorionic gonadotropin (HCG) can induce complete spermatogenesis from proliferation of spermatogonia to spermiogenesis. In some cases, however, HCG injection fails to induce complete spermatogenesis. Testicular morphological observations revealed that HCG-injected eels could be classified into three types based on their testicular conditions. Type 1 eels had a well-developed testis and the milt could be acquired by hand-stripping. In type 2 eels, spermatogenesis was also induced by HCG injection, but testicular size was remarkably smaller than that of type 1 eels, and the milt could not be hand-stripped. At the end of the experiment, type 2 fish had only spermatogonia and a small amount of spermatozoa, but no spermatocytes or spermatids, in their testis. Type 3 eels had thready testis, which did not develop any germ cells during the experimental period. These results suggest that, despite elevations of plasma 11–ketotestosterone levels, HCG injections were not successful in inducing the completion of spermatogenesis in type 2 and type 3 eels. In most spermatogonia of type 2 eels, meiosis was not induced by HCG injections. Furthermore, only few mitotic divisions had occurred as evidenced by the presence of 2³ to 2⁶ late type B spermatogonia in most cysts. This suggests that spermatogonial stem cells undergo four or five, and occasionally six, mitotic divisions before the interruption of spermatogenesis in type 2 eels. It is proposed that those numbers of mitotic divisions are related to a mediator that regulates entry of spermatogonia of the Japanese eel into meiosis.     

Establishment of spermatogenesis in neonatal bovine testicular tissue following ectopic xenografting varies with donor age

Ectopic testicular xenografting can be used to investigate spermatogenesis and as an alternative means for generating transgenic spermatozoa in many species. Improving the efficiency of spermatogenesis in xenografted testicular tissue will aid in the application of using this approach. The present study was conducted to evaluate age-related differences in the establishment of spermatogenesis in grafted testicular tissue from bulls between 2 and 16 wk of life. Testicular tissue was ectopically xenografted under the skin on the backs of castrated nude mice and subsequently evaluated for growth, testosterone production, and establishment of spermatogenesis 24 wk after grafting. The greatest weight increases occurred in donor tissue from calves of the ages 2, 4, and 8 wk compared with the ages of 12 and 16 wk. Recipient mouse serum testosterone concentration was at normal physiological levels 24 wk after grafting and no significant differences were detected between recipients grafted with testicular tissue from bull calves of different ages. The development of germ cells to elongated spermatids were observed in seminiferous tubules of grafts from donor calves of the ages 4, 8, 12, and 16 wk but not observed in grafts from 2-wk donors, which contained round spermatids as the most advanced germ cell stage. Grafts from 8-wk donors contained a significantly higher (10-fold) average percentage of seminiferous tubules with elongated spermatids than all other donor ages. These data demonstrate differences in the ability of testicular tissue from donor animals of different ages to establish spermatogenesis following ectopic testicular xenografting.     

Germ Cell Transplantation and Testis Tissue Xenografting in Mice

Germ cell transplantation was developed by Dr. Ralph Brinster and colleagues at the University of Pennsylvania in 1994^1,2. These ground-breaking studies showed that microinjection of germ cells from fertile donor mice into the seminiferous tubules of infertile recipient mice results in donor-derived spermatogenesis and sperm production by the recipient animal². The use of donor males carrying the bacterial β-galactosidase gene allowed identification of donor-derived spermatogenesis and transmission of the donor haplotype to the offspring by recipient animals¹. Surprisingly, after transplantation into the lumen of the seminiferous tubules, transplanted germ cells were able to move from the luminal compartment to the basement membrane where spermatogonia are located³. It is generally accepted that only SSCs are able to colonize the niche and re-establish spermatogenesis in the recipient testis. Therefore, germ cell transplantation provides a functional approach to study the stem cell niche in the testis and to characterize putative spermatogonial stem cells. To date, germ cell transplantation is used to elucidate basic stem cell biology, to produce transgenic animals through genetic manipulation of germ cells prior to transplantation^4,5, to study Sertoli cell-germ cell interaction^6,7, SSC homing and colonization^3,8, as well as SSC self-renewal and differentiation^9,10.Germ cell transplantation is also feasible in large species¹¹. In these, the main applications are preservation of fertility, dissemination of elite genetics in animal populations, and generation of transgenic animals as the study of spermatogenesis and SSC biology with this technique is logistically more difficult and expensive than in rodents. Transplantation of germ cells from large species into the seminiferous tubules of mice results in colonization of donor cells and spermatogonial expansion, but not in their full differentiation presumably due to incompatibility of the recipient somatic cell compartment with the germ cells from phylogenetically distant species¹². An alternative approach is transplantation of germ cells from large species together with their surrounding somatic compartment. We first reported in 2002, that small fragments of testis tissue from immature males transplanted under the dorsal skin of immunodeficient mice are able to survive and undergo full development with the production of fertilization competent sperm¹³. Since then testis tissue xenografting has been shown to be successful in many species and emerged as a valuable alternative to study testis development and spermatogenesis of large animals in mice¹⁴.     

An overview of cell renewal in the testis throughout the reproductive cycle of a seasonal breeding teleost, the gilthead seabream (Sparus aurata L)

The gilthead seabream is a protandrous hermaphrodite seasonal breeding teleost with a bisexual gonad that offers an interesting model for studying the testicular regression process that occurs in both seasonal testicular involution and sex change. Insofar as fish reproduction is concerned, little is known about cell renewal and elimination during the reproductive cycle of seasonal breeding teleosts with asynchronous spermatogenesis. We have previously described how acidophilic granulocytes infiltrate the testis during postspawning where, surprisingly, they produce interleukin-1beta, a known growth factor for mammalian spermatogonia, rather than being directly involved in the elimination of degenerative germ cells. In this study, we are able to discriminate between spermatogonia stem cells and primary spermatogonia according to their nuclear and cytoplasmic diameters and location in the germinal epithelium, finding that these two cell types, together with Sertoli cells, proliferate throughout the reproductive cycle with a rate that depends on the reproductive stage. Thus, during spermatogenesis the spermatogonia stem cells, the Sertoli cells, and the developing germ cells (primary spermatogonia, A and B spermatogonia, and spermatocytes) in the germinal compartment, and cells with fibroblast-shaped nuclei in the interstitial tissue proliferate. However, during spawning, the testis shows few proliferating cells. During postspawning, the resumption of proliferation, the occurrence of apoptotic spermatogonia, and the phagocytosis of nonshed spermatozoa by Sertoli cells lead to a reorganization of both the germinal compartment and the interstitial tissue. Finally, the proliferation of spermatogonia increases during resting when, unexpectedly, both oogonia and oocytes also proliferate. This proliferative pattern was correlated with the gonadosomatic index, testicular morphology, and testicular and gonad areas, suggesting that complex mechanisms operate in the regulation of gonocyte proliferation in hermaphrodite fish.     

Bax-dependent spermatogonia apoptosis is required for testicular development and spermatogenesis

Bax is a multidomain, proapoptotic member of the Bcl-2 family that is required for normal spermatogenesis in mice. Despite its proapoptotic function, previous results found that Bax-deficient mature male mice demonstrate increased cell death and dramatic testicular atrophy. The present study examined the role of Bax during the normal development of the testis to determine whether the increased cell death in mature mice could be explained by decreased apoptosis earlier in development. Consistent with this hypothesis, testicular atrophy is preceded by increased testicular weight and hypercellular tubules in immature Bax-deficient mice. TUNEL staining at Postnatal Day (P) 7 and morphological quantitation between P5 and P15 demonstrates decreased germ cell apoptosis in Bax-deficient mice. By P15, increased numbers of type A spermatogonia, and at P12 and P15, an increase in intermediate type spermatogonia were noted in Bax-deficient animals. By P25, the number of basal compartment cells was greatly increased in Bax-deficient animals compared with controls such that four or five layers of preleptotene spermatocytes were routinely present within the basal compartment of the testis. Although the Sertoli cell barrier was significantly removed from the basement membrane, it appeared intact as judged by the hypertonic fixation test. During late pubertal development, massive degeneration of germ cells took place, including many of those cell types that previously survived in the first wave of spermatogenesis. The data indicate that Bax is required for normal developmental germ cell death in the type A spermatogonia, specifically dividing (A(2), A(3), and A(4)) spermatogonia, at a time at which the number of spermatogonia is regulated in a density-dependent manner. The massive hyperplasia that occurs in Bax-deficient mice subsequently results in Bax independent cell death that may be triggered by overcrowding of the seminiferous epithelium.     

Primate spermatogonial stem cells colonize mouse testes

In mice, transplantation of spermatogonial stem cells from a fertile male to the seminiferous tubules of an infertile recipient male results in progeny with donor-derived haplotype. Attempts to extend this approach by transplanting human testis cells to mice have led to conflicting claims that no donor germ cells persisted or that human spermatozoa were produced in the recipient. To examine this issue we used the baboon, a primate in which testis cell populations of several ages could be obtained for transplantation, and demonstrate that donor spermatogonial stem cells readily establish germ cell colonies in recipient mice, which exist for periods of at least 6 mo. However, differentiation of germ cells toward the lumen of the tubule and production of spermatozoa did not occur. The presence of baboon spermatogonial stem cells and undifferentiated spermatogonia in mouse seminiferous tubules for long periods after transplantation indicates that antigens, growth factors, and signaling molecules that are necessary for interaction of these cells and the testis environment have been preserved for 100 million years of evolutionary separation. Because germ cell differentiation and spermatogenesis did not occur, the molecules necessary for this process appear to have undergone greater divergence between baboon and mouse.     

Testis-specific sulfoglycolipid, seminolipid, is essential for germ cell function in spermatogenesis

More than 90% of the glycolipid in mammalian testis consists of a unique sulfated glyceroglycolipid, seminolipid. The sulfation of the molecule is catalyzed by a Golgi membrane-associated sulfotransferase, cerebroside sulfotransferase (CST). Disruption of the Cst gene in mice results in male infertility due to the arrest of spermatogenesis prior to the metaphase of the first meiosis. However, the issue of which side of the cell function-germ cells or Sertoli cells-is deteriorated in this mutant mouse remains unknown. Our findings show that the defect is in the germ cell side, as evidenced by a transplantation analysis, in which wild-type spermatogonia expressing the green fluorescent protein were injected into the seminiferous tubules of CST-null testis. The transplanted GFP-positive cells generated colonies and spermatogenesis proceeded over meiosis in the mutant testis. The findings also clearly show that the seminolipid is expressed on the plasma membranes of spermatogonia, spermatocytes, spermatids, and spermatozoa, as evidenced by the immunostaining of wild-type testes using an anti-sulfogalactolipid antibody, Sulph-1 in comparison with CST-null testes as a negative control, and that seminolipid appears as early as day 8 of age, when Type B spermatogonia emerge.     

Spermatogonial depletion in adult Pin1-deficient mice

Spermatogonia in the mouse testis arise from early postnatal gonocytes that are derived from primordial germ cells (PGCs) during embryonic development. The proliferation, self-renewal, and differentiation of spermatogonial stem cells provide the basis for the continuing integrity of spermatogenesis. We previously reported that Pin1-deficient embryos had a profoundly reduced number of PGCs and that Pin1 was critical to ensure appropriate proliferation of PGCs. The current investigation aimed to elucidate the function of Pin1 in postnatal germ cell development by analyzing spermatogenesis in adult Pin1-/- mice. Although Pin1 was ubiquitously expressed in the adult testis, we found it to be most highly expressed in spermatogonia and Sertoli cells. Correspondingly, we show here that Pin1 plays an essential role in maintaining spermatogonia in the adult testis. Germ cells in postnatal Pin1-/- testis were able to initiate and complete spermatogenesis, culminated by production of mature spermatozoa. However, there was a progressive and age-dependent degeneration of the spermatogenic cells in Pin1-/- testis that led to complete germ cell loss by 14 mo of age. This depletion of germ cells was not due to increased cell apoptosis. Rather, detailed analysis of the seminiferous tubules using a germ cell-specific marker revealed that depletion of spermatogonia was the first step in the degenerative process and led to disruption of spermatogenesis, which resulted in eventual tubule degeneration. These results reveal that the presence of Pin1 is required to regulate proliferation and/or cell fate of undifferentiated spermatogonia in the adult mouse testis.