Testicular Germ Cell Differentiation Research Articles

Investigation of VASA Gene and Protein Expression in Neonate and Adult Testicular Germ Cells in Mice In Vivo and In Vitro.

Objective We aimed to examine the expression levels of the VASA gene and protein in testis sections of neonate and adult mice as well as testicular cell cultures. Materials and Methods In this experimental study, in order to investigate the expression of this germ cell marker gene in more detail, we analyzed the expression of VASA by immunocytochemistry, immunohistochemistry and fluidigm reverse transcription-polymerase chain reaction (RT-PCR). Results The immunohistochemical assays showed that the VASA protein was exclusively expressed in germ cells in the seminiferous tubules of the neonate and adult testis and not in somatic cells. VASA was not detectable in PLZF positive spermatogonial stem cells (SSCs), was weakly expressed in proliferating spermatogonia, and became abundant in spermatocytes and round spermatozoa. Counting VASA-positive cells in the seminiferous tubules of the neonate and adult testis depicted significant higher expression (P<0.05) of VASA in the adult testis in comparison to its neonate counterpart. SSC colonies were established in vitro after digestion of the testis and characterized by immunocytochemistry for CD90 and stage-specific embryonic antigens 3 (SSEA3). Immunocytochemistry confirmed that in contrast to the not detectable signal in vivo, VASA protein was strongly localized in the cytoplasm of both neonate and adult mouse SSCs under in vitro conditions. The results of Fluidigm RT-PCR revealed a significant higher expression of the germ cell gene VASA in adult SSCs in comparison to neonate SSCs in cell culture (P<0.05). ConclusionThe VASA protein is, therefore, an extremely specific marker of testicular germ cell differentiation in vivo and mostly expressed in the adult testis in spermatocytes and round spermatids. The immunohistochemical signal in spermatogonia is very low. So, PLZF positive SSCs are negative for VASA in vivo, while in contrast, once isolated from the testicular niche VASA is also strongly expressed in SSCs under in vitro conditions.

Effect of resveratrol on restoring spermatogenesis in experimental cryptorchid mice and analysis of related differentially expressed proteins

The present study aimed to evaluate the effect of trans-Resveratrol on spermatogenesis. Male Kunming suckling mice (10 days old) were surgically rendered cryptorchid and subcutaneously injected with trans-Resveratrol at doses of 5, 10, 20, and 40 µg/g/day as groups I, II, III, and IV, respectively, for 35 days. Animals in the control group received 10 µL/mouse/day of olive oil. Serum estradiol, testosterone, FSH, and LH levels were measured on day 45. Tissue analysis and sperm morphological abnormalities analysis were done. Results showed that in the control group and group I only spermatogonia and primary spermatocytes were present, whereas spermatogenesis was totally restored in groups II, III, and IV. Sperm counts in groups III and IV were remarkably higher than the control group (P<0.05). The morphological abnormalities in resveratrol-treated groups were higher than the mature mice. Serum estradiol levels in the resveratrol-treated groups were not significantly different from the control group, but were lower than the mature mice (P<0.05). There was no significant difference in serum testosterone levels between the resveratrol-treated groups and mature mice, but the levels in the resveratrol-treated groups was significantly lower than the control group (P<0.05). No significant influence of trans-Resveratrol was observed on serum FSH levels in all cryptorchid mice. Serum LH levels in groups I, II, and III were higher than the control group. These results indicate that trans-Resveratrol restores spermatogenesis in cryptorchid mice. In addition, proteomic analysis between the 20 μg/g/day resveratrol-treated group and the control group was carried out, and five kinds of proteins (BAF250, ZFP261, CHD1L, RBBP9, and SOHLH2) were identified. The expression of SOHLH2 increased, while that of BAF250, ZFP261, CHD1L, and RBBP9 decreased in the 20 µg/g/day resveratrol-treated group, indicating that SOHLH2 may contribute to testicular germ cell differentiation.

Application of three-dimensional culture systems to study mammalian spermatogenesis, with an emphasis on the rhesus monkey (Macaca mulatta).

In vitro culture of spermatogonial stem cells (SSCs) has generally been performed using two-dimensional (2D) culture systems; however, such cultures have not led to the development of complete spermatogenesis. It seems that 2D systems do not replicate optimal conditions of the seminiferous tubules (including those generated by the SSC niche) and necessary for spermatogenesis. Recently, one of our laboratories has been able to induce proliferation and differentiation of mouse testicular germ cells to meiotic and postmeiotic stages including generation of sperm in a 3D soft agar culture system (SACS) and a 3D methylcellulose culture system (MCS). It was suggested that SACS and MCS form a special 3D microenvironment that mimics germ cell niche formation in the seminiferous tubules, and thus permits mouse spermatogenesis in vitro. In this review, we (1) provide a brief overview of the differences in spermatogenesis in rodents and primates, (2) summarize data related to attempts to generate sperm in vitro, (3) report for the first time formation of colonies/clusters of cells and differentiation of meiotic (expression of CREM-1) and postmeiotic (expression of acrosin) germ cells from undifferentiated spermatogonia isolated from the testis of prepubertal rhesus monkeys and cultured in SACS and MCS, and (4) indicate research needed to optimize 3D systems for in vitro primate spermatogenesis and for possible future application to man.

Reconstruction of a seminiferous tubule-like structure in a 3 dimensional culture system of re-aggregated mouse neonatal testicular cells within a collagen matrix

Male gonad development is initiated by the aggregation of pre-Sertoli cells (SCs), which surround germ cells to form cords. Several attempts to reconstruct testes from dissociated testicular cells have been made; however, only very limited morphogenesis beyond seminiferous cord formation has been achieved. Therefore, we aimed to reconstruct seminiferous tubules using a 3-dimensional (D) re-aggregate culture of testicular cells, which were dissociated from 6-dpp neonatal mice, inside a collagen matrix. We performed a short-term culture (for 3days) and a long-term culture (up to 3 wks). The addition of KnockOut Serum Replacement (KSR) promoted (1) the enlargement of SC re-aggregates; (2) the attachment of peritubular myoid (PTM) cells around the SC re-aggregates; (3) the sorting of germ cells inside, and Leydig cells outside, seminiferous cord-like structures; (4) the alignment of SC polarity inside a seminiferous cord-like structure relative to the basement membrane; (5) the differentiation of SCs (the expression of the androgen receptor); (6) the formation of a blood–testis-barrier between the SCs; (7) SC elongation and lumen formation; and (8) the proliferation of SCs and spermatogonia, as well as the differentiation of spermatogonia into primary spermatocytes. Eventually, KSR promoted the formation of seminiferous tubule-like structures, which accompanied germ cell differentiation. However, these morphogenetic events did not occur in the absence of KSR. This in vitro system presents an excellent model with which to identify the possible factors that induce these events and to analyze the mechanisms that underlie cellular interactions during testicular morphogenesis and germ cell differentiation.

Critical function of RA-GEF-2/Rapgef6, a guanine nucleotide exchange factor for Rap1, in mouse spermatogenesis

Small GTPase Rap1 has been implicated in the proper differentiation of testicular germ cells. In the present study, we investigated the functional significance of RA-GEF-2/Rapgef6, a guanine nucleotide exchange factor for Rap1, in testicular differentiation using mice lacking RA-GEF-2. RA-GEF-2 was expressed predominantly on the luminal side of the seminiferous tubules in wild-type mice. No significant differences were observed in the body weights or hormonal parameters of RA-GEF-2−/− and wild-type mice. However, the testes of RA-GEF-2−/− male mice were significantly smaller than those of wild-type mice and were markedly atrophied as well as hypospermatogenic. The concentration and motility of epididymal sperm were also markedly reduced and frequently had an abnormal shape. The pregnancy rate and number of fetuses were markedly lower in wild-type females after they mated with RA-GEF-2−/− males than with wild-type males, which demonstrated the male infertility phenotype of RA-GEF-2−/− mice. Furthermore, a significant reduction and alteration were observed in the expression level and cell junctional localization of N-cadherin, respectively, in RA-GEF-2−/− testes, which may, at least in part, account for the defects in testicular differentiation and spermatogenesis in these mice.

Slide Preparation Method to Preserve Three-dimensional Chromatin Architecture of Testicular Germ Cells

During testicular germ cell differentiation, the structure of nuclear chromatin dynamically changes. The following describes a method designed to preserve the three-dimensional chromatin arrangement of testicular germ cells found in mice; this method has been termed as the three-dimensional (3D) slide method. In this method, testicular tubules are directly treated with a permeabilization step that removes cytoplasmic material, followed by a fixation step that fixes nuclear materials. Tubules are then dissociated, the cell suspension is cytospun, and cells adhere to slides. This method improves sensitivity towards detection of subnuclear structures and is applicable for immunofluorescence, DNA, and RNA fluorescence in situ hybridization (FISH) and the combination of these detection methods. As an example of a possible application of the 3D slide method, a Cot-1 RNA FISH is shown to detect nascent RNAs. The 3D slide method will facilitate the detailed examination of spatial relationships between chromatin structure, DNA, and RNA during testicular germ cell differentiation.

Fertility preservation of pre-pubertal cancer patient boys before aggressive chemotherapy. peliminary results from in vitro cultue of fresh testicular tissue from three pre-pubertal patients

To evaluate the possibility of inducing growth, proliferation and differentiation of testicular germ cells form prepubertal cancer patients (PBCP) in a novel three-dimensional Methylcellulose- Culture System (MCS).

Perinatal germ cell development and differentiation in the male marmoset (Callithrix jacchus): similarities with the human and differences from the rat

STUDY QUESTIONIs perinatal germ cell (GC) differentiation in the marmoset similar to that in the human?SUMMARY ANSWERIn a process comparable with the human, marmoset GC differentiate rapidly after birth, losing OCT4 expression after 5–7 weeks of age during mini-puberty.WHAT IS KNOWN ALREADYMost of our understanding about perinatal GC development derives from rodents, in which all gonocytes (undifferentiated GC) co-ordinately lose expression of the pluripotency factor OCT4 and stop proliferating in late gestation. Then after birth these differentiated GC migrate to the basal lamina and resume proliferation prior to the onset of spermatogenesis. In humans, fetal GC differentiation occurs gradually and asynchronously and OCT4+ GC persist into perinatal life. Failure to switch off OCT4 in GC perinatally can lead to development of carcinoma in situ (CIS), the precursor of testicular germ cell cancer (TGCC), for which there is no animal model. Marmosets show similarities to the human, but systematic evaluation of perinatal GC development in this species is lacking. Similarity, especially for loss of OCT4 expression, would support use of the marmoset as a model for the human and for studying CIS origins.STUDY DESIGN, SIZE AND DURATIONTestis tissues were obtained from marmosets (n = 4–10 per age) at 12–17 weeks' gestation and post-natal weeks 0.5, 2.5, 5–7, 14 and 22 weeks, humans at 15–18 weeks' gestation (n = 5) and 4–5 weeks of age (n = 4) and rats at embryonic day 21.5 (e21.5) (n = 3) and post-natal days 4, 6 and 8 (n = 4 each).PARTICIPANTS/MATERIALS, SETTING AND METHODSTestis sections from fetal and post-natal marmosets, humans and rats were collected and immunostained for OCT4 and VASA to identify undifferentiated and differentiated GC, respectively, and for Ki67, to identify proliferating GC. Stereological quantification of GC numbers, differentiation (% OCT4+ GC) and proliferation were performed in perinatal marmosets and humans. Quantification of GC position within seminiferous cords was performed in marmosets, humans and rats.MAIN RESULTS AND ROLE OF CHANCEThe total GC number increased 17-fold from birth to 22 post-natal weeks in marmosets; OCT4+ and VASA+ GC proliferated equally in late gestation and early post-natal life. The percentage of OCT4+ GC fell from 54% in late fetal life to <0.5% at 2.5 weeks of age and none were detected after 5–7 weeks in marmosets. In humans, the percentage of OCT4+ GC also declined markedly during the equivalent period. In marmosets, GC had begun migrating to the base of seminiferous cords at ∼22 weeks of age, after the loss of GC OCT4 expression.LIMITATIONS, REASONS FOR CAUTIONThere is considerable individual variation between marmosets. Although GC development in marmosets and humans was similar, there are differences with respect to proliferation during fetal life. The number of human samples was limited.WIDER IMPLICATIONS OF THE FINDINGSThe similarities in testicular GC differentiation between marmosets and humans during the perinatal period, and their differences from rodents, suggest that the marmoset may be a useful model for studying the origins of CIS, with relevance for the study of TGCC.STUDY FUNDING/COMPETING INTERESTSThis work was supported by Grant G33253 from the Medical Research Council, UK. No external funding was sought and there are no competing interests.

Attenuation of Spermatogonial Stem Cell Activity in Cryptorchid Testes

To elucidate the mechanism of infertility caused by cryptorchidism we focused on early stage spermatogenesis and spermatogonial stem cell activity in undifferentiated spermatogonia in cryptorchid testes. Histological findings and expression patterns of the stem cell marker undifferentiated embryonic cell transcription factor 1 were examined in a unilateral cryptorchid rat model. We removed unilateral descended testis and contralateral descended testis from cryptorchid and normal rats (control), respectively, 18 days postcoitum to 144 days postpartum. In descended testes gonocyte differentiation into early A spermatogonia occurred at 9 days postpartum. However, this transformation was altered in undescended testes. Furthermore, the undifferentiated embryonic cell transcription factor 1 negative early A spermatogonia-to-positive early A spermatogonia ratio was significantly higher in the undescended testis group (mean ± SD 0.69 ± 0.04) than in the control (0.46 ± 0.10, p = 0.037) and descended testis (0.44 ± 0.05, p = 0.022) groups, indicating decreased early A spermatogonia with spermatogonial stem cell activity in cryptorchid testes. In cryptorchid testes the differentiation from gonocytes into early A spermatogonia and the stem cell activity of early A spermatogonia were altered during the early stage of spermatogenesis, suggesting that the loss of spermatogonial stem cell activity in cryptorchid rats resulted in altered spermatogenesis, thus interfering with fertility.

New horizons for in vitro spermatogenesis? An update on novel three-dimensional culture systems as tools for meiotic and post-meiotic differentiation of testicular germ cells

Culture and differentiation of male germ cells has been performed for various purposes in the past. To date, none of the studies aimed at in vitro spermatogenesis has resulted in a sufficient number of mature gametes. Numerous studies have revealed worthy pieces of information, building up a body of information on conditions that are required to maintain and mature male germ cells in vitro. In this review, we report on previously published and unpublished experiments addressing murine germ cell differentiation in three-dimensional (3D) in vitro culture systems. In a systematic set of experiments, we examined the influence of two different matrices (soft agar and methylcellulose) as well as the need for gonadotrophin support. For the first time, we demonstrate that pre-meiotic male germ cells [revealed by the absence of meiotic marker expression (e.g. Boule)] obtained from immature mice pass through meiosis in vitro. After several weeks of culture, we obtained morphologically normal spermatozoa embedded in the matrix substance. Complete maturation relied on support from somatic testicular cells and the presence of gonadotrophins but appeared independent from the matrix in a 3D culture environment. Further research efforts are required to reveal the applicability of this culture technique for human germ cells and the functionality of the spermatozoa for generating offspring.

Maturation of Testicular Tissue from Infant Monkeys after Xenografting into Mice

In juvenile monkeys, precocious puberty can be induced by administration of gonadotropins resulting in testicular somatic cell maturation and germ cell differentiation. It is, however, unknown whether testicular maturation can also be induced in younger monkeys. Here we used testis tissue xenografting to investigate whether infant monkey testis tissue will undergo somatic cell maturation and/or spermatogenesis in response to endogenous adult mouse gonadotropins or exogenous gonadotropins. Testicular tissue pieces from 3- and 6-month-old rhesus monkeys were grafted to immunodeficient, castrated mice. Recipient mice were either left untreated or treated with pregnant mare serum gonadotropin and/or human chorionic gonadotropin twice weekly and were killed 28 weeks after grafting. Testicular maturation in grafted tissue was assessed based on morphology and the most advanced germ cell type present and by immunohistochemistry for expression of proliferating cell nuclear antigen, Mullerian-inhibiting substance, and androgen receptor. Testis grafts, irrespective of donor age or treatment, contained fewer germ cells than donor tissue. Grafts from 6-month-old donors showed tubular expansion with increased seminiferous tubule diameter and lumen formation, whereas those harvested from gonadotropin-treated mice contained elongated spermatids. Grafts from 3-month-old donors recovered from gonadotropin-treated mice contained pachytene spermatocytes, whereas those recovered from untreated mice showed only slight tubular expansion. Immunohistochemistry revealed that exposure to exogenous gonadotropins supported Sertoli cell maturation, irrespective of donor age. These results indicate that sustained gonadotropin stimulation of immature (<12 months old) monkey testis supports Sertoli cell maturation, thereby terminating the unresponsive phase of the germinal epithelium and allowing complete spermatogenesis in testis tissue from infant rhesus monkeys.

Over-expression of IL-18, ICE and IL-18 R in testicular tissue from sexually immature as compared to mature mice.

In this study we examined the cellular origin and the expression levels of interleukin-18 (IL-18), IL-18 receptor (IL-18R) and IL-1beta-converting enzyme (ICE), which activates pro-IL-18, during normal maturation of murine testis. The levels of IL-18, IL-18R and ICE were significantly higher in testicular tissues and homogenates (but not in the spleen or liver) from sexually immature than mature mice. Immunohistochemical staining of testicular tissues from sexually immature and mature mice shows that testicular germ cells and Leydig cells/interstitial cells express higher levels of IL-18, as compared to other testicular cells. Peritubular cells of sexually immature and mature mice also expressed IL-18. Our results demonstrate, for the first time, over-expression of the IL-18 family in testicular tissues of sexually immature mice, as compared to mature mice, as well as the expression of IL-18 in the different stages of differentiation of testicular germ cells. Thus, our results may indicate involvement of the endocrine system (gonadotropins and testosterone) in the regulation of the testicular IL-18 family, which could be involved in the regulation of testicular functions, development and spermatogenesis under physiological conditions.

Immunohistochemical profiling of germ cells within the human fetal testis: identification of three subpopulations.

In the human fetal testis, germ cells that have migrated to the genital ridges become enclosed within testicular cords by 8 wk of gestation. Most papers refer to all types of germ cells as being "gonocytes" or "prespermatogonia," giving the impression that they are identical. Detailed morphological studies, however, have suggested a heterogeneous population. We have used single, double, and triple immunohistochemistry to evaluate the differentiation of cells within fetal testes recovered during the first (7-9 wk) and second (14-19 wk) trimesters. In the first trimester, differentiation of Sertoli cells preceded the formation of testicular cords and the differentiation of interstitial (Leydig, peritubular myoid) cells. Immunostaining for CHK2, C-KIT, placental alkaline phosphatase, PCTAIRE-1, and MAGE-A4 revealed that the proportion of germ cells expressing each of these proteins was correlated with gestational age. Expression of the pluripotency marker OCT4 was restricted to a population of small, round germ cells. Three types of germ cell were identified, and we propose that these should be known as gonocytes (OCT4pos/C-KITpos/MAGE-A4neg), intermediate germ cells (OCT4low/neg/C-KITneg/MAGE-A4neg), and prespermatogonia (OCT4neg/C-KITneg/MAGE-A4pos). In the first trimester, most germ cells had a gonocyte phenotype; however, from 18 wk of gestation, prespermatogonia were the most abundant cell type. These data provide evidence for the functional differentiation of human testicular germ cells during the second trimester of pregnancy, and they argue against these germ cells being considered as a homogeneous population, as in rodents.

Mutational analysis of the human FATE gene in 144 infertile men.

The FATE gene maps to Xq28 where one case of a translocation breakpoint has been found in an infertile man. Moreover, the FATE promoter contains a putative SF-1-binding site, and FATE has been proposed as representing a target gene of SF-1 in testicular development or germ cell differentiation. This study presents a complete mutational screening of the FATE gene in a random group of 144 infertile males. Four polymorphisms and two mutations were found. Three of the polymorphisms, viz., 741C-->T, 905A-->C, and 3985C-->T, occurred in exon 5 and intron 2 and did not alter the deduced polypeptide. One polymorphism resulted in the conservative amino acid exchange, A10 V, in 16.0% of the patients. This substitution occurred with similar frequencies in the control groups, indicating that the mutation does not affect fertility in men or women. The two mutations caused the non-conservative amino acid substitutions S125R (patient 1) and I34T (patient 2). A family study (patient 1) revealed, however, that S125R was inherited and that a fertile male family member carried the mutation. Patient 2 did not have relevant family members who could be examined. Thus, this study has shown that only 1.4% of infertile men have mutations in the FATE gene, and that some of these mutations do not singly cause infertility. Hence, FATE may not play an important role in the disease-state of infertile men attending fertility clinics. However, FATE mutations cannot be excluded as being a contributing factor in some cases of male infertility.

Real-time observation of transplanted 'green germ cells': proliferation and differentiation of stem cells.

To elucidate the mechanism of proliferation and differentiation of testicular germ cells, donor testicular germ cells labeled with enhanced green fluorescent protein (eGFP) were transplanted to recipient seminiferous tubules. The kinetics of colonization as well as of differentiation of the donor cells was followed in the same transplanted tubules (alive) under ultraviolet light. One week after transplantation, clusters of fluorescent cells were randomly spread as dots in the recipient seminiferous tubule, whereas non-homed cells flowed out from the testis to the epididymis. By 4 weeks after transplantation, green germ cells were observed with weak and moderate fluorescence along the recipient seminiferous tubule. By 8 weeks, proliferation and differentiation of the germ cells occurred, resulting in strong fluorescence in the middle part of the seminiferous tubule but in weak and moderate fluorescence at both terminals. The length of the fluorescent positive seminiferous tubule became longer. Detailed histological analyses of the recipient tubules indicated that the portions of the seminiferous tubule in weak, moderate, and strong fluorescence contained the spermatogonia, spermatogonia with spermatocytes, and all types of germ cells including spermatids, respectively. Thus, testicular stem cells colonized first as dots within 1 week, and then proliferated along the basement membrane of the seminiferous tubules followed by differentiation.

Molecular cloning and characterization of a novel dual-specificity protein phosphatase possibly involved in spermatogenesis

Dual-specificity protein phosphatases (DSPs) play roles in the regulation of mitogenic signal transduction for extracellular stimulation and the cell cycle. In the present study, we identified a novel DSP, termed TMDP (testis- and skeletal-muscle-specific DSP). Nucleotide sequence analysis of TMDP cDNA indicated that the open reading frame of 597 bp encodes a protein of 198 amino acid residues with a predicted molecular mass of 22.5 kDa. The deduced amino acid sequence contains a motif for a conserved catalytic domain of DSPs and shows highest similarity to human Vaccinia HI-related phosphatase (45.5% identity) but low homology to the mitogen-activated protein kinase phosphatase and CDC25 subfamilies of DSPs. Recombinant TMDP protein exhibited intrinsic phosphatase activity towards both phospho-seryl/threonyl and -tyrosyl residues of myelin basic protein, with similar specific activities in vitro. Northern-blot analysis revealed that TMDP is most abundantly expressed in the testis. The expression in the testis is characterized as follows: (i) TMDP mRNA first appeared 3 weeks after birth, corresponding to the time that meiosis begins; (ii) TMDP mRNA was abundant in fractionated spermatocytes and round spermatids; and (iii) hybridization in situ showed that the TMDP mRNA is localized in spermatocytes and/or spermatids in seminiferous tubules. These data demonstrate that TMDP is a novel DSP abundantly expressed in the testis and suggest that TMDP may be involved in the regulation of meiosis and/or differentiation of testicular germ cells during spermatogenesis.

Molecular cloning and characterization of a novel dual-specificity protein phosphatase possibly involved in spermatogenesis

Dual-specificity protein phosphatases (DSPs) play roles in the regulation of mitogenic signal transduction for extracellular stimulation and the cell cycle. In the present study, we identified a novel DSP, termed TMDP (testis- and skeletal-muscle-specific DSP). Nucleotide sequence analysis of TMDP cDNA indicated that the open reading frame of 597 bp encodes a protein of 198 amino acid residues with a predicted molecular mass of 22.5 kDa. The deduced amino acid sequence contains a motif for a conserved catalytic domain of DSPs and shows highest similarity to human Vaccinia HI-related phosphatase (45.5% identity) but low homology to the mitogen-activated protein kinase phosphatase and CDC25 subfamilies of DSPs. Recombinant TMDP protein exhibited intrinsic phosphatase activity towards both phospho-seryl/threonyl and -tyrosyl residues of myelin basic protein, with similar specific activities in vitro. Northern-blot analysis revealed that TMDP is most abundantly expressed in the testis. The expression in the testis is characterized as follows: (i) TMDP mRNA first appeared 3 weeks after birth, corresponding to the time that meiosis begins; (ii) TMDP mRNA was abundant in fractionated spermatocytes and round spermatids; and (iii) hybridization in situ showed that the TMDP mRNA is localized in spermatocytes and/or spermatids in seminiferous tubules. These data demonstrate that TMDP is a novel DSP abundantly expressed in the testis and suggest that TMDP may be involved in the regulation of meiosis and/or differentiation of testicular germ cells during spermatogenesis.

Pituitary Tumor-transforming Gene Protein Associates with Ribosomal Protein S10 and a Novel Human Homologue of DnaJ in Testicular Cells

Pituitary tumor-transforming gene (PTTG) is a recently characterized proto-oncogene that is expressed specifically in adult testis. In this study, we have used in situ hybridization and developmental Northern blot assays to demonstrate that PTTG mRNA is expressed stage-specifically in spermatocytes and spermatids during rat spermatogenic cycle. We have used the yeast two-hybrid system to identify proteins that interact with PTTG in testicular cells. Two positive clones were characterized. One of the clones is the ribosomal protein S10, the other encodes a novel human DnaJ homologue designated HSJ2. Northern blot analysis showed that testis contains higher levels of HSJ2 mRNA than other tissues examined, and the expression pattern of HSJ2 mRNA in postnatal rat testis is similar to PTTG. S10 mRNA levels do not vary remarkably among different tissues and remains unchanged during testicular germ cell differentiation. In vitro binding assays demonstrated that both S10 and HSJ2 bind to PTTG specifically and that PTTG can be co-immunoprecipitated with S10 and HSJ2 from transfected cells. Moreover, the binding sites for both proteins were located within the C-terminal 75 amino acids of the PTTG protein. These results suggest that PTTG may play a role in spermatogenesis.

Mapping of Eight Testis-Specific Genes to Mouse Chromosomes

We previously identified eight testis-specific genes using antibodies raised against testicular germ cells. They are expressed during spermatogenesis and are presumed to be involved in testicular germ cell differentiation and sperm formation. We have mapped the genomic loci for these testis-specific genes using restriction fragment length variants in interspecific backcross mice. The calmegin gene (Clgn) was mapped to Chr 8. The synaptonemal complex protein gene 1 (Sycp1) probe hybridized with two sequences on different chromosomes;Sycp1-rs2was mapped to Chr 3, whereasSycp1-rs3was mapped to Chr 7. The relaxin-like factor gene (Rlnl) was mapped to Chr 8, and collapsin response mediator protein 1 (Crmp1) was mapped to Chr 5. Three novel genes encoding testis-specific proteins A2 (Tsga2), A8 (Tsga8), and A12 (Tsga12) were mapped to chromosomes 3, X, and 10, respectively.

Inhibition of testicular germ cell apoptosis and differentiation in mice misexpressing Bcl-2 in spermatogonia

During normal spermatogenesis, more than half of the germ cells undergo apoptosis, but the physiological significance and molecular mechanisms of this programmed cell death are largely unknown. Because Bcl-2 functions as a death repressor, we have investigated the effect of misexpressing Bcl-2 in spermatogonia in transgenic mice using the human bcl-2 cDNA under the control of the human polypeptide chain elongation factor 1alpha (EF-1alpha) promoter. In the 2-week-old transgenic testes, exogenous Bcl-2 was expressed in spermatogonia and massive accumulation of spermatogonia was observed in seminiferous tubules by 4 weeks. At this time, only a few spermatocytes were apparent, and the accumulated cells degenerated, leading to vacuolization in some seminiferous tubules by 7 weeks. In older transgenic mice, abnormal accumulation of spermatogonia and degeneration of these germ cells was still observed, but some seminiferous tubules in which the level of Bcl-2 expression was reduced recovered normal spermatogenesis. These observations indicate that spermatogonial apoptosis is part of the normal program of mammalian spermatogenesis and is regulated by a pathway affected by Bcl-2.