TGFβ signaling in male germ cells regulates gonocyte quiescence and fertility in mice

During testis development, proliferation and death of gonocytes are highly regulated to establish a standard population of adult stem spermatogonia that maintain normal spermatogenesis. As Transforming Growth Factor beta (TGFbeta) can regulate proliferation and apoptosis, we investigated its expression and functions during testis development. We show that TGFbeta2 is only expressed in quiescent gonocytes and decreases gonocyte proliferation in vitro. To study the functions of TGFbeta2, we developed conditional mice that invalidate the TGFbeta receptor type II in germ cells. Most of the knock-out animals die during fetal life, but the surviving adults show a reduced pool of spermatogonial stem/progenitor cells and become sterile with time. Using an organ culture system mimicking in vivo development, we show higher proportions of proliferating and apoptotic gonocytes from 13.5 dpc until 1 dpp, suggesting a reduction of germinal quiescence in these animals. Conversely, a 24-hour TGFbeta2-treatment of explanted wild-type testes, isolated every day from 13.5 dpc until 1 dpp, increased the duration of quiescence.These data show that the TGFbeta signaling pathway plays a physiological role during testis development by acting directly as a negative regulator of the fetal and neonatal germ cell proliferation, and indicate that the TGFbeta signaling pathway might regulate the duration of germ cell quiescence and is necessary to maintain adult spermatogenesis.     

Regulation of the proliferation of cocultured gonocytes and Sertoli cells by retinoids,triiodothyronine, and intracellular signaling factors: differences between fetal and neonatal cells

The regulation of early fetal germ cell growth has not been studied in cell culture, probably due to the poor survival of these cells. However, cell culture is the only system in which the control of cell growth can be studied independently of the influence of secreted testicular factors, which are diluted in the medium. We successfully cultured dispersed testicular cells from 16.5-day-old rat fetuses in defined medium and compared the growth of these cells with that of cells from 3-day-old neonates. In this system, fetal gonocytes displayed low levels of mitotic activity and their numbers remained stable. In contrast, neonatal gonocytes displayed high levels of mitotic activity and increased in number, these characteristics resembling those observed in vivo. We found that retinoic acid had deleterious effects on the number of gonocytes but did not affect Sertoli cell proliferation in fetal and neonatal cell cultures. Moreover, in fetal cell cultures, the decrease in the number of gonocytes resulted from a decrease in mitotic activity, probably due to a direct effect of retinoids on fetal gonocytes. Among the selective agonists for the retinoic acid receptor (RARalpha agonist, RARbeta agonist, and RARgamma agonist) and the retinoic X receptor (pan-RXR agonist) tested, only the RARalpha agonist reproduced the effects of retinoic acid at concentrations lower than its Kd value in both fetal and neonatal cell cultures. As both RARalpha and RXRalpha are present in fetal and neonatal gonocytes, we suggest that retinoic acid exerts its effects on gonocytes via a RARalpha-RXRalpha heterodimer, with RARalpha functioning as an active partner and RXRalpha as a passive partner. In this culture system, we show for the first time that triiodothyronine (T3) inhibits testicular fetal Sertoli cell and germ cell growth. We also tested intracellular signaling factors and found that a cAMP analog increased Sertoli cell proliferation and germ cell survival in both fetal and neonatal cells whereas phorbol esters (PMA) strongly inhibited the proliferation of fetal but not of neonatal gonocytes. None of the tested factors (T3, dbcAMP, and PMA) seemed to interact with the all-trans retinoic acid pathway. Thus, fetal gonocytes and neonatal gonocytes differ in intrinsic properties, and their growth is not regulated in the same manner. Despite their low level of mitotic activity, fetal gonocytes were more sensitive to various factors than neonatal gonocytes.     

Development of Fetal Testicular Cells in Androgen Receptor Deficient Mice

During mammalian development, androgens produced by the fetal testis are the most important hormones controlling the masculinization of the reproductive tract and the genitalia. New findings show that the male germ line is the most sensitive to anti-androgenic endocrine disruptors during the embryonic period. In a recent study, we reported that endogenous androgens physiologically control germ cell growth in the male mouse fetus during early fetal life. In the present study, we extended this result by showing the presence of a functional androgen receptor in the gonocytes in the latter part of the fetal life. We also studied the effect of androgens on the development of the somatic testicular cells using the Tfm mice which carry a naturally inactivating mutation of the androgen receptor. Fetal Leydig cell are largely independent of endogenous androgens during fetal development whereas fetal Sertoli cell number is decreased following a default of peritubular myoid cells differenciation. They also point to the gonocyte as a special target for androgens during the embryonic period and indicate a novel mechanism of androgen action on gonocytes. Elucidation of this new pathway in the fetal testis will clarify not only fetal testis physiology but also the effects of environmental anti-androgens that act during fetal life and open new perspectives for future investigations into the sensitivity of fetal germ cell to androgens.     

Retinoic acid receptors and retinoid X receptors in the rat testis during fetal and postnatal development: immunolocalization and implication in the control of the number of gonocytes

Retinoids have pleiotropic effects on embryonic development and are essential for spermatogenesis in the adult, where they act via nuclear retinoid receptors: retinoic acid receptors (RARs) and retinoid X receptors (RXRs). We used immunohistochemistry to examine the cellular localization of RARs and RXRs in the rat testis from Day 13.5 postconception (13.5 dpc) until Day 8 postpartum (8 dpp), and these findings were compared with those for immature and adult testes. RARalpha and RARbeta were detected in the interstitial tissue from 14.5 dpc, with intense staining in the gonocytes from 20. 5 dpc to 8 dpp. The nuclei of all cell types stained faintly for RARgamma from 8 dpp. Immunoreactivity for RXRalpha was intense in the gonocytes from 13.5 dpc and in the Leydig cells from 16.5 dpc, and persisted throughout the period studied. RXRbeta was always detected in the Leydig cells and during a short neonatal period in the gonocytes. RXRgamma gave a faint reaction in the nuclei of all cell types from 20.5 dpc. Unexpectedly, immunostaining for all the receptors tested, except RARgamma and RXRgamma, was detected in the cytoplasmic compartment of the cells of fetal and neonatal testes, while it was found in the nuclei in immature and adult testes. In cultures of dispersed testicular cells from 3 dpp pups, retinoic acid had a dose-dependent deleterious effect on the survival of the gonocytes and, to a lesser extent, of the somatic cells. These results suggest that retinoids act on the testicular development, especially on germ cells, via RARs and/or RXRs.     

Expression of the scaffolding subunit A of protein phosphatase 2A during rat testicular development

Previously, we found that the poly(A)+ RNA of the scaffolding subunit A (alpha isoform) of protein phosphatase 2A (PP2A-Aalpha) was clearly expressed by fetal gonocytes but weakly expressed by adult single (As), paired (Apr), and aligned (Aal) A spermatogonia. The scaffolding subunit A of PP2A (PP2A-A) is the major subunit in the formation of a functional PP2A holoenzyme. In this study, we investigated the expression of PP2A-A during testicular development in more detail using in situ hybridization, immunohistochemistry, and Western blot with testes of rats of various ages from 16 days postcoitum (pc) to adulthood. The expression of PP2A-A was detected in fetal proliferative gonocytes at 16 days pc, declining thereafter during the quiescent period of the gonocytes. From the day of birth to the start of spermatogenesis (Day 4 postpartum [pp]), the number of PP2A-A-immunopositive gonocytes increased. At Day 4 pp, the first A1 spermatogonia appeared along the basement membrane; all were PP2A-A positive. In the adult, PP2A-A was upregulated during the differentiation of the As, Apr, and Aal spermatogonia to the A1 spermatogonia and expressed thereafter by all other spermatogonia. Spermatocytes from the pachytene stage onward and all spermatids in the adult testis also showed clear expression of PP2A-A. In Sertoli cells, PP2A-A was detected during their proliferative period at 19 days pc to 15 days pp. The presence of a functional enzyme was confirmed by the additional detection of the catalytic subunit C of PP2A using Western blot analyses at various ages during testicular development. This apparent pattern of expression of PP2A-A during testicular development suggests that PP2A may play an important role in the proliferation of distinct populations of testicular cells and during meiosis and sperm maturation.     

DeltaNp63 plays an anti-apoptotic role in ventral bladder development

The bladder, the largest smooth-muscle organ in the human body, is responsible for urine storage and micturition. P63, a homolog of the p53 tumor-suppressor gene, is essential for the development of all stratified epithelia, including the bladder urothelium. The N-terminal truncated isoform of p63, DeltaNp63, is known to have anti-apoptotic characteristics. We have established that DeltaNp63 is not only the predominant isoform expressed throughout the bladder, but is also preferentially expressed in the ventral bladder urothelium during early development. We observed a host of ventral defects in p63-/- embryos, including the absence of the abdominal and ventral bladder walls. This number of ventral defects is identical to bladder exstrophy, a congenital anomaly exhibited in human neonates. In the absence of p63, the ventral urothelium was neither committed nor differentiated, whereas the dorsal urothelium was both committed and differentiated. Furthermore, in p63-/- bladders, apoptosis in the ventral urothelium was significantly increased. This was accompanied by the upregulation of mitochondrial apoptotic mediators Bax and Apaf1, and concurrent upregulation of p53. Overexpression of DeltaNp63gamma and DeltaNp63beta in p63-/- bladder primary cell cultures resulted in a rescue, evidenced by significantly reduced expressions of Bax and Apaf1. We conclude that DeltaNp63 plays a crucial anti-apoptotic role in normal bladder development.     

Developmental expression of the translocator protein 18 kDa (TSPO) in testicular germ cells

Translocator protein (TSPO) is a high affinity 18 kDa drug- and cholesterol-binding protein strongly expressed in steroidogenic tissues where it mediates cholesterol transport into mitochondria and steroid formation. Testosterone formation by Leydig cells in the testis is critical for the regulation of spermatogenesis and male fertility. Male germ cell development comprises two main phases, the pre-spermatogenesis phase occurring from fetal life to infancy and leading to spermatogonial stem cell (SSC) formation, and spermatogenesis, which consists of repetitive cycles of germ cell mitosis, meiosis and differentiation, starting with SSC differentiation and ending with spermiogenesis and spermatozoa formation. Little is known about the molecular mechanisms controlling the progression from one germ cell phenotype to the next. Here, we report that testicular germ cells express TSPO from neonatal to adult phases, although at lower levels than Leydig cells. TSPO mRNA and protein were found at specific steps of germ cell development. In fetal and neonatal gonocytes, the precursors of SSCs, TSPO appears to be mainly nuclear. In the prepubertal testis, TSPO is present in pachytene spermatocytes and dividing spermatogonia. In adult testes, it is found in a stage-dependent manner in pachytene spermatocyte and round spermatid nuclei, and in mitotic spermatogonia. In search of TSPO function, the TSPO drug ligand PK 11195 was added to isolated gonocytes with or without the proliferative factors PDGF and 17β-estradiol, and was found to have no effect on gonocyte proliferation. However, TSPO strong expression in dividing spermatogonia suggests that it might play a role in spermatogonial mitosis. Taken together, these results suggest that TSPO plays a role in specific phases of germ cell development.     

Multiple effects of retinoids on the development of Sertoli, germ, and Leydig cells of fetal and neonatal rat testis in culture

We investigated the effect of retinoids on the development of Sertoli, germ, and Leydig cells using 3-day culture of testes from fetuses 14.5 and 18.5 days post-conception (dpc) and from neonates 3 days postpartum (dpp). Addition of 10(-6) M and 3.10(-8) M retinoic acid (RA) caused a dose-dependent disruption of the seminiferous cords in 14.5-day-old fetal testes, without any change in the 5-bromo-2'-deoxyuridine (BrdU) labeling index of the Sertoli cells. RA caused no disorganization of older testes, but it did cause hyperplasia of the Sertoli cells in 3-dpp testes. Fragmentation of the Sertoli cell DNA was not detected in control or RA-treated testes at any age studied. The cAMP produced in response to FSH was significantly decreased in RA-treated testes for all studied ages. Both 10(-6) M and 3.10(-8) M RA dramatically reduced the number of gonocytes per 14.5-dpc testis. This resulted from a high increase in apoptosis, which greatly exceeded the slight increase of mitosis. RA caused no change in the number of gonocytes in testes explanted on 18.5 dpc (the quiescent period), whereas it increased this number in testes explanted on 3 dpp (i.e., when gonocyte mitosis and apoptosis resume). Lastly, RA and retinol (RE) reduced both basal and acute LH-stimulated testosterone secretion by 14.5-dpc testis explants, without change in the number of 3beta-hydroxysteroid dehydrogenase-positive cells per testis. Retinoids had no effect on basal or LH-stimulated testosterone production by older testes. In conclusion, RE and RA are potential regulators of the development of the testis and act mainly negatively during fetal life and positively during the neonatal period on the parameters we have studied.     

Expression of Fas,p53 and AFP in development of human fetal germ cells in vitro

In the present study we employed a two-step culture system to study the expression of Fas, p53 and alpha-fetoprotein (AFP) in the development in vitro of human fetal germ cells. p53 mRNA was determined by Northern blotting, and Fas content was assessed by western blotting. RT-nested polymerase chain reaction (RT-nPCR) analysis was performed to determine the expression of AFP mRNA in different stages of fetal follicular development. Follicular cell apoptosis was evaluated by DNA fragmentation analyses (DNA ladder). The results showed that by day 7 of culture approximately one-sixth of fetal germ cells grew to class C oocytes (primary oocytes) from class B oocytes (primordial oocytes) or class A oocytes. On day 45 of culture, one-third of these primary follicles doubled in size. In the meantime, there was a high proportion apoptosis of follicular cells on days 35 or 45 of culture, as evident by a clear ladder pattern of DNA fragmentation upon electrophoretic analysis. Expression of Fas antigen and p53 mRNA increased in a time-dependent manner, while AFP mRNA was expressed on days 10 to 35, and disappeared on day 45. These results indicate that human fetal germ cells can develop in a two-step culture system and AFP may play an active role in the proliferation of these germ cells. At the late stage of follicular development in vitro, a number of follicular cells became apoptotic. Moreover, apoptosis may be the mechanism responsible for fetal germ cell regression and the Fas antigen and/or p53-mediated death pathway may be central in the induction of germ cell regression.     

Effects of mono-(2-ethylhexyl) phthalate on fetal and neonatal rat testis organ cultures

Di-(2-ethylhexyl) phthalate (DEHP) and its active metabolite, mono-(2-ethylhexyl) phthalate (MEHP), have been shown to cause reproductive toxicity in both developing and adult animals. In this study, we used organ cultures of fetal and neonatal rat testes to assess the in vitro effect of MEHP on seminiferous cord formation in Embryonic Day 13 (E13) testes and on the development of E18 and Postnatal Day 3 (P3) testes. Interestingly, MEHP had no effect on cord formation in the organ cultures of E13 testes, indicating that it has no effect on sexual differentiation of the indifferent gonad to testis. Consistently, the expression of a Sertoli cell-specific protein, mullerian inhibiting substance (MIS), or the number of gonocytes did not change in E13 testes after MEHP treatment. In contrast, MEHP decreased the levels of MIS and GATA-4 proteins in Sertoli cells and impaired Sertoli cell proliferation in the organ cultures of E18 and P3 testes. These results suggest that MEHP negatively influences proliferation and differentiation of Sertoli cells in both fetal and neonatal testes. In addition, MEHP treatment did not alter the number of gonocytes in E18 testes, whereas the number of gonocytes in P3 testes decreased in a dose-dependent manner, apparently due to enhanced apoptosis. These results suggest that MEHP adversely affects the gonocytes, which are mitotically active and undergoing migration and differentiation in neonatal testes, but it has no effect on fetal gonocytes that are mitotically quiescent.     

Développementin vitro de la lignée germinale foetale mâle chez le rat, la souris et l’homme

The key role of the foetal germ cell line in the reproductive capacity of the adult has been known for a long time. More recently, the observed increase in male reproductive disorders such as the decline of sperm count and quality and the increased incidence of testicular cancer has been postulated to be due to alterations of foetal and neonatal testicular development in response to increasing environmental pollution. However, few tools are available to study foetal and neonatal germ cell line development and the effects of physiological or toxic substances on this process. The authors have developed an organ culture system in which foetal or neonatal testis is grown on a filter floating on a synthetic medium free of serum, hormones or biological factors. This study, using rats and mice, first compared the long-term morphological and functional development of Sertoli and germ cells in thisin vivo system. In rats, these cells developed normally over a period of two weeksin vitro. Fewer cells were produced thanin vivo, but a similar level of differentiated function was observed. Germ cells, which are difficult to maintainin vitro, resumed mitosis after a quiescent period, at the same time asin vivo. Similar results were obtained with mouse fetuses, but this model was less efficient. This culture model can be used to study post-natal development of the germ cell lineage in testes derived from foetuses on the last day of foetal life and invalidated for P63, that do not survive after birth. This gene was found to be involved in the regulation of germ apoptosis which resumes after birth in the mouse. Lastly, this model applied to the human species (from 6 to 12 weeks of gestation) showed that testicular architecture and germ cells can be maintained for 4 days with better efficiency at younger stages than at older stages. p]In conclusion, testicular architecture and intercellular communications are sufficiently preserved to sustain gametogenesisin vitro with no added factors. This method is potentially useful to study the effects of various factors, particularly xenobiotics.     

Short-type PB-cadherin promotes survival of gonocytes and activates JAK-STAT signalling

Neonatal development of the rat testis involves a number of critical events including re-entry of gonocytes into the cell cycle and eventual loss of many of these cells and their progeny via apoptosis. Since surviving gonocytes give rise to subsequent generations of germ cells, regulation of their fate is critical for adult testicular function. Here, we have identified a role for short-type PB-cadherin (STPB-C) in promoting survival of gonocytes in neonatal rats and we have linked its expression to the JAK-STAT signaling pathway. These findings were obtained with varied approaches including use of transgenic rats overexpressing STPB-C which were studied with protein microarrays and other techniques, direct examination of germ cell apoptosis and survival in gonocyte-Sertoli cell co-cultures, and direct study of the JAK-STAT pathway in these models and in L cells transfected with STPB-C. These data provide new information on the regulation of gonocyte fate and exciting new evidence supporting a link between the JAK-STAT pathway and cadherin-based cell-cell interactions.     

Apoptosis of fetal testicular cells is regulated by both p53-dependent and independent mechanisms

A portion of fetal germ cells undergoes apoptosis in the physiological context, but the molecular mechanisms of their apoptosis are largely unknown. Because p53 tumor suppressor gene product promotes apoptosis in various types of cells, we have investigated the expression of p53 in fetal gonads and examined the influence of loss of p53 function in fetal gonad cells using mice deficient in the p53 gene. We found that the expression of p53 protein in fetal testis was induced after 15.5 dpc (days post coitum), while the expression was not detected in fetal ovary. The number of apoptotic cells found in the seminiferous tubules of fetal testes was not significantly different between p53-deficient and wild-type mice until 16.5 dpc. At 17.5 dpc, however, more apoptotic cells were observed in wild-type testes than in the p53-deficient mice. In contrast, a similar number of apoptotic cells was found in fetal ovaries throughout these developmental stages. These observations indicate that p53 promotes apoptosis of fetal testicular cells after 16.5 dpc.     

Is fetal testis in danger?

Estrogens are classically known to play a major role in female reproduction, but there is now compelling evidence that they may also be involved in the regulation of male reproductive function. In humans, a decrease in sperm count and an increase in the incidences of testicular cancer, cryptorchidism and hypospadia have been observed in many countries over the last 50 years. Male reproductive alterations were also observed in wildlife. Such male reproductive disorders have been attributed to the increase in concentration of xenobiotics, and of xenoestrogens in particular, in the environment and in food. Epidemiological, clinical and experimental studies have suggested that excessive exposure to estrogens during fetal/neonatal life can lead to reproductive disorders in adulthood. Using an in vitro model, we showed that estrogens directly affected the development of the fetal testis. Lastly, we clearly demonstrated that the fetal and neonatal testis is very sensitive to estrogens since the invalidation of estrogen receptor alpha leads to an increase of steroidogenesis and the invalidation of estrogen receptor beta enhances the development of the germ cell lineage in the male.     

Status of p53, p21, mdm2, pRb proteins, and DNA methylation in gonocytes of control and gamma-irradiated rats during testicular development

In fetal and newborn rat testes, gonocytes, which stop cycling for about 8 days, become highly radiosensitive. The presence of p53, p21, mdm2, and pRb, which are involved in cell cycle, apoptosis control, or both, were studied by immunohistochemistry to determine if their expression is related to this radiosensitivity. A strong cytoplasmic expression of p53 and p21 was detected. Cytoplasmic expression of p53 occurred only in arrested gonocytes, whereas that of p21 was observed before and after the block. P21 was found to colocalize with mitochondria. No expression of mdm2 was detected and pRb was present only when the gonocytes started cycling again. In animals exposed to 1.5 Gy of gamma-irradiation at Day 19 postcoitum, p53 expression was prolonged in time, whereas no change was observed in p21 amounts and localization, compared with controls. Using antibodies against 5-methyl cytosine, it was shown that gonocyte DNA passed from a hypomethylated to a methylated status 1 day after gonocytes stopped cycling. A prolonged survival of gonocytes after exposure to radiation was followed by their progressive apoptosis, which finally involved the entire gonocyte population between Days 6 and 12 postpartum. The elevated but delayed sensitivity of gonocytes to genotoxic stress may be related to the unusual expression of p53 and p21, which may itself be related to the large DNA methylation changes.     

A unique combination of male germ cell miRNAs coordinates gonocyte differentiation

The last 100 years have seen a concerning decline in male reproductive health associated with decreased sperm production, sperm function and male fertility. Concomitantly, the incidence of defects in reproductive development, such as undescended testes, hypospadias and testicular cancer has increased. Indeed testicular cancer is now recognised as the most common malignancy in young men. Such cancers develop from the pre-invasive lesion Carcinoma in Situ (CIS), a dysfunctional precursor germ cell or gonocyte which has failed to successfully differentiate into a spermatogonium. It is therefore essential to understand the cellular transition from gonocytes to spermatogonia, in order to gain a better understanding of the aetiology of testicular germ cell tumours. MicroRNA (miRNA) are important regulators of gene expression in differentiation and development and thus highly likely to play a role in the differentiation of gonocytes. In this study we have examined the miRNA profiles of highly enriched populations of gonocytes and spermatogonia, using microarray technology. We identified seven differentially expressed miRNAs between gonocytes and spermatogonia (down-regulated: miR-293, 291a-5p, 290-5p and 294*, up-regulated: miR-136, 743a and 463*). Target prediction software identified many potential targets of several differentially expressed miRNA implicated in germ cell development, including members of the PTEN, and Wnt signalling pathways. These targets converge on the key downstream cell cycle regulator Cyclin D1, indicating that a unique combination of male germ cell miRNAs coordinate the differentiation and maintenance of pluripotency in germ cells.     

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.     

Metastasis tumor antigen 1 (MTA1) might contribute to maintain the cellular integrity of germ cells by coordination with p53 through EGF-pathway stimulation during spermatogenesis

DNA quality control is expected to be highly regulated in tetraploid spermatocytes, since chromosome crossover, which involves DNA strand break, transfer, and renaturation, is an active event at this stage of germ cell development. p53-mediated apoptosis is an effector of cellular proofreading that acts to maintain the cellular integrity of germ cells during spermatogenesis and the highest levels of testicular p53 are found in tetraploid spermatocytes. MTA1 has been previously implicated in cellular transformation through its ability to regulate the epithelial-to-mesenchymal transition and to enhance metastasis. MTA1 is a component of the Mi-2/nucleosome remodeling and deacetylating (NURD) complex that contains both histone deacetylase and nucleosome remodeling activity, and its expression and deacetylase activity could be remarkably upregulated by the stimulation of epidermal growth factor (EGF). Recent studies revealed that MTA1 inhibits p53-induced apoptosis in both cancer cells and testicular function. All these observations lent support to the hypotheses that up-regulation of MTA1 by EGF might repress testicular expression of p53 and lead to low abundance of p53 expression during adult spermatogenesis. This repression as well as p53-mediated apoptosis may play important roles in the regulation of homeostasis of germ cell proliferation and death. Further investigation in this field is urgently requested.     

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.