Postnatal Testis Development Research Articles

Post-natal sexual development of testis and epididymis in the rabbit: Variability and relationships among macroscopic and microscopic markers

The present work was performed to examine the existence of some relationships between macroscopic and microscopic traits of testis and epididymis in rabbit. The variables studied were live weight (LW), testis length (TL), testis width (TWh), testis weight (TW), testis volume (TV), epididymis length (EL), epididymis width (EWh), epididymis weight (EW), epididymis volume (EV), percentage of seminiferous tubules with presence of lumen (STL), percentage of seminiferous tubules with presence of elongated spermatids (STES), percentage of seminiferous tubules with presence of spermatozoa (STS) and diameter of seminiferous tubules (STD). Measurements began after weaning and continued until males reached 33 weeks of age. Phenotypic correlations between testis and epidydimis traits and the principal component analysis were estimated as the residual correlation from an analysis of variance, including the effects of line, birth-season, age, and the double interactions line × age and birth-season × age. Four principal components (PCs) explained 79% of the total variation. The predominant variables defining the first PC were TL, TW and TV. Epididymis width and STS were located in the second PC. Epididymis weight and EV were important in the definition of the first and third PC. Tubular diameter seems important in the definition of the fourth PC. It has been not found correlation between traits related to either testis or epididymis size and variables related to active spermatogenesis. Therefore, TW and/or TV seemed not to be good markers of maturity.

Postnatal Sertoli and Leydig cell proliferation and the establishment of puberty and sexual maturity in Chinchilla lanigera (Rodentia, Chinchillidae)

The Chilean chinchilla (Chinchilla lanigera) is threatened in its natural habitat and there is very little information concerning the reproductive biology of this species. Our main objectives were to investigate the postnatal testis development in this rodent, with emphasis on Sertoli and Leydig cell proliferation and the establishment of puberty and sexual maturity. Forty-four animals from one day to 30 months of age had their testis and epididymis prepared (time of collection for animals from 5 to 30 months of age, May-November in the southern hemisphere) for histological and stereological analyses. Both Sertoli and Leydig cell proliferation occurred up to two months after birth and their total number per testis were stable thereafter. Based on spermatid release from the seminiferous epithelium and the presence of sperm in the epididymis, puberty in chinchilla took place at around three months of age. However, testis weight and tubular diameter and epithelium height appeared to stabilise only after the animals reached 17 months of age, indicating that the establishment of full sexual maturity in this species takes a relatively long period of time. This particular finding indicates that chinchilla might represent an interesting experimental model to investigate the mechanisms that regulate the establishment of this important event of reproductive physiology in mammals.

A Sertoli Cell-Specific Knockout of Connexin43 Prevents Initiation of Spermatogenesis

The predominant testicular gap junctional protein connexin43 (cx43) is located between neighboring Sertoli cells (SCs) and between SCs and germ cells. It is assumed to be involved in testicular development, cell differentiation, initiation, and maintenance of spermatogenesis with alterations of its expression being correlated with various testicular disorders. Because total disruption of the cx43 gene leads to perinatal death, we generated a conditional cx43 knockout (KO) mouse using the Cre/loxP recombination system, which lacks the cx43 gene solely in SCs (SCCx43KO), to evaluate the SC-specific functions of cx43 on spermatogenesis in vivo. Adult SCCx43KO(-/-) mice showed normal testis descent and development of the urogenital tract, but testis size and weight were drastically lower compared with heterozygous and wild-type littermates. Histological analysis and quantitation of mRNA expression of germ cell-specific marker genes revealed a significant reduction in the number of spermatogonia but increased SC numbers/tubule with only a few tubules left showing normal spermatogenesis. Thus, SC-specific deletion of cx43 mostly resulted in an arrest of spermatogenesis at the level of spermatogonia or SC-only syndrome and in intratubular SC clusters. Our data demonstrate for the first time that cx43 expression in SCs is an absolute requirement for normal testicular development and spermatogenesis.

Correlation of appearance of metastasis-associated protein1 (Mta1) with spermatogenesis in developing mouse testis

Mta1, a representative of the MTA gene family, is believed to be involved in the metastasis of malignant tumors. However, a systematic study of its physiological function has not been performed. It has been found in normal mouse organs at relatively low levels, except for in testis, suggesting a potential function in the male reproductive system. In order to explore the role of Mta1 protein during spermatogenesis, its expression in adult mouse testis was compared with that in developing mouse testis and in testis from adult mice treated with methoxyacetic acid, which selectively depletes primary spermatocytes. Quantitative analysis revealed that Mta1 protein gradually increased in the testis from 14 days postnatally. Immunolocalization analysis demonstrated strong signals in the seminiferous tubules, and Mta1 was predominantly present in the nucleus of primary spermatocytes and spermatogonia from 14 days postnatally. The most intensive staining was located in the nucleus of pachytene spermatocytes in mature testes. The expression pattern of Mta1 during spermatogenesis was also shown to be stage-specific by immunohistochemistry analysis. Finally, dramatic loss of Mta1 expression from pachytene spermatocytes was observed in the spermatogenic-arrested adult mouse testis. These results collectively demonstrate that Mta1 appears during postnatal testis development and suggest that this expression may be crucial for spermatogenesis.

Expression of CatSper family transcripts in the mouse testis during post-natal development and human ejaculated spermatozoa: relationship to sperm motility

CatSper is a unique sperm cation channel-like protein family exclusively expressed in the testis and plays important roles in sperm functions. The temporal expression profiles of CatSper1-4 mRNAs in the mouse testis during post-natal development through adulthood were investigated using real-time RT-PCR. The CatSper2 transcript was present in the testis of the 8-day-old mice, and was repressed in the adult testis after two sharp up-regulations at day 18 and 35. CatSper1 and CatSper3, 4 mRNAs were detectable in the testis of 18-day and 15-day-old mice, respectively. After sharp up-regulation at day 25 and 35, respectively, they were maximal at the adult testis stage. The differences between the temporal expression profiles of the CatSper transcripts in post-natal mouse testis development suggest different regulation to their transcription, and potentially contribute to the possibility of forming heteromeric channels among these four CatSper family members. CatSper1-3 transcripts were identified to be present in the human ejaculated spermatozoa by RT-PCR. Significantly higher levels of CatSper2 and CatSper3 mRNAs revealed by real-time RT-PCR were observed in the high-motile spermatozoa than in the low-motile fraction and suggests that CatSper2 and CatSper3 transcripts in the human ejaculated spermatozoa could be the potential targets for further study and male infertility screening.

The expression and cellular localization of phospholipase D isozymes in the developing mouse testis

To examine the involvement of phospholipase D (PLD) isozymes in postnatal testis development, the expression of PLD1 and PLD2 was examined in the mouse testis at postnatal weeks 1, 2, 4, and 8 using Western blot analysis and immunohistochemistry. The expression of both PLD1 and PLD2 increased gradually with development from postnatal week 1 to 8. Immunohistochemically, PLD immunoreactivity was detected in some germ cells in the testis and interstitial Leydig cells at postnatal week 1. PLD was mainly detected in the spermatocytes and residual bodies of spermatids in the testis after 8 weeks after birth. The intense immunostaining of PLD in Leydig cells remained unchanged by postnatal week 8. These findings suggest that PLD isozymes are involved in the spermatogenesis of the mouse testis.

Expression of the adaptor protein m-Numb in mouse male germ cells

Numb is an adaptor protein that is asymmetrically inherited at mitosis and controls the fate of sibling cells in different species. The role of m-Numb (mammalian Numb) as an important cell fate-determining factor has extensively been described mostly in neural tissues, particularly in progenitor cells, in the mouse. Biochemical and genetic analyses have shown that Numb acts as an inhibitor of the Notch signaling pathway, an evolutionarily conserved pathway involved in the control of cell proliferation, differentiation, and apoptosis. In the present study, we sought to determine m-Numb distribution in germ cells in the postnatal mouse testis. We show that all four m-Numb isoforms are widely expressed during postnatal testis development. By reverse transcriptase-PCR and western blot analyses, we further identify p71 as the predominantly expressed isoform in germ cells. Moreover, we demonstrate through co-immunoprecipitation studies that m-Numb physically associates with Ap2a1, a component of the endocytotic clathrin-coated vesicles. Finally, we employed confocal immunofluorescence microscopy of whole mount seminiferous tubules and isolated germ cells to gain more insight into the subcellular localization of m-Numb. These morphological analyses confirmed m-Numb and Ap2a1 co-localization. However, we did not observe asymmetric localization of m-Numb neither in mitotic spermatogonial stem cells nor in more differentiated spermatogonial cells, suggesting that spermatogonial stem cell fate in the mouse does not rely on asymmetric partitioning of m-Numb.

Expression and localization of androgen receptor-interacting protein-4 in the testis

Androgen receptor-interacting protein 4 (ARIP4) belongs to the SNF2 family of proteins involved in chromatin remodeling, DNA excision repair, and homologous recombination. It is a DNA-dependent ATPase, binds to DNA and mononucleosomes, and interacts with androgen receptor (AR) and modulates AR-dependent transactivation. We have examined in this study the expression and cellular localization of ARIP4 during postnatal development of mouse testis. ARIP4 was detected by immunohistochemistry in Sertoli cell nuclei at all ages studied, starting on day 5, and exhibited the highest expression level in adult mice. At the onset of spermatogenesis, ARIP4 expression became evident in spermatogonia, pachytene, and diplotene spermatocytes. Immunoreactive ARIP4 antigen was present in Leydig cell nuclei. In Sertoli cells ARIP4 was expressed in a stage-dependent manner, with high expression levels at stages II-VI and VII-VIII. ARIP4 expression patterns did not differ significantly in testes of wild-type, follicle-stimulating hormone receptor knockout, and luteinizing hormone receptor knockout mice. In testes of hypogonadal mice, ARIP4 was found mainly in interstitial cells and exhibited lower expression in Sertoli and germ cells. In vitro stimulation of rat seminiferous tubule segments with testosterone, FSH, or forskolin did not significantly change stage-specific levels of ARIP4 mRNA. Heterozygous ARIP4(+/-) mice were haploinsufficient and had reduced levels of Sertoli-cell specific androgen-regulated Rhox5 (also called Pem) mRNA. Collectively, ARIP4 is an AR coregulator in Sertoli cells in vivo, but the expression in the germ cells implies that it has also AR-independent functions in spermatogenesis.

Appearance of Hyaluronan Binding Protein 1 Proprotein in Pachytene Spermatocytes and Round Spermatids Correlates With Spermatogenesis

The proprotein form of hyaluronan binding protein 1 (HABP1) has been reported to be present in the pachytene spermatocytes and the round spermatids of the adult testis. To explore the role of HABP1 proprotein in spermatogenesis, its expression in the testes of adult rats was compared with that in the testes of developing rats and that in the testes of adult rats that received estriadiol to halt spermatogenesis. Immunoblotting revealed that the mature form of HABP1 was consistently present in the testis, but its precursor form was not found in the testis of animals aged 7, 14, 21, and 28 days. However, immunohistochemical analysis revealed the presence of the proprotein form in the pachytene spermatocytes and the round spermatids of testes from rats aged 21 and the 28 days, the appearance of which correlated well with the appearance of these cells during spermatogenesis. Reverse-transcriptase polymerase chain reaction revealed transcriptional upregulation of HABP1 in the testes of adult rats, compared with the testes of developing rats. Finally, loss of HABP1 proprotein expression from the pachytene spermatocytes and round spermatids was observed in the testes from rats in which spermatogenesis was arrested. Collectively, these findings demonstrate the appearance of HABP1 proprotein in the pachytene spermatocytes and the round spermatids during the initial stages of postnatal testis development and suggest that this expression may be crucial for spermatogenesis.

Expression of the spermatogenic cell‐specific glyceraldehyde 3‐phosphate dehydrogenase (GAPDS) in rat testis

The spermatogenic cell-specific variant of glyceraldehyde 3-phosphate dehydrogenase (GAPDS) has been cloned from a rat testis cDNA library and its pattern of expression determined. A 1,417 nucleotide cDNA has been found to encode an enzyme with substantial homology to mouse GAPDS (94% identity) and human GAPD2 (83% identity) isozymes. Northern blotting of rat tissue RNAs detected the 1.5 kb Gapds transcript in the testis and not in RNA from liver, spleen, epididymis, heart, skeletal muscle, brain, seminal vesicle, and kidney. The rat Gapds mRNA was first detected at day 29 of postnatal testis development, an age which coincides with the initial post-meiotic differentiation of round spermatids. When isolated rat spermatogenic cell RNA was probed for Gapds expression, transcripts were detected only in round spermatids and condensing spermatids, but not in pachytene spermatocytes, demonstrating haploid expression of the Gapds gene. However, immunohistochemical staining of rat testis sections with anti-GAPDS antisera did not detect GAPDS in round spermatids, but localized the protein only to stage XIII and later condensing spermatids as well as testicular spermatozoa, indicating that Gapds expression is translationally regulated. The current results are similar to those previously obtained for mouse GAPDS and human GAPD2, suggesting that reliable comparisons can be made between these species in toxicant screening and contraceptive development.

Hepatocyte growth factor modulates in vitro survival and proliferation of germ cells during postnatal testis development

The hepatocyte growth factor (HGF) is a pleiotropic cytokine that influences mitogenesis, motility and differentiation of many different cell types by its tyrosine kinase receptor c-Met. We previously demonstrated that the c-Met/HGF system is present and functionally active during postnatal testis development. We found also that spermatozoa express c-Met and that HGF has a positive effect on the maintenance of sperm motility. In the present paper, we extend our study on the germ cells at different stages of differentiation during the postnatal development of the testis. We demonstrate that c-met is present in rat spermatogonia, pachytene spermatocytes and round spermatids and that HGF significantly increases spermatogonial proliferation in 8- to 10-day-old pre-pubertal rats. At this age HGF does not affect Sertoli cells and peritubular myoid cells proliferation. In addition, we studied the effect of the factor on germ cell apoptosis and we show that HGF prevents the germ cell apoptotic process. We also studied the effect of HGF on 18- to 20-day-old and 28- to 30-day-old rat testes. At these ages also the factor significantly increases germ cell duplication and decreases the number of apoptotic cells. However, the effect on programmed cell death is higher in the 8- to 10-day-old rats and declines in the older animals. In conclusion, we report that rat germ cells (spermatogonia, pachytene spermatocytes and round spermatids) express c-met and that HGF modulates germ cell proliferating activity and apoptosis in vitro. These data indicate that the c-Met/HGF system is involved in male germ cell homeostasis and, consequently, has a role in male fertility.

Remarkable expansions of an X-linked reproductive homeobox gene cluster in rodent evolution

Rhox is a recently identified cluster of 12 X-linked homeobox genes in mice. The expression pattern of Rhox genes during postnatal testis development corresponds to their chromosomal position, much like the colinear gene regulation of the Hox gene clusters during animal embryonic development. We here report the identification of 18 additional Rhox genes and 3 pseudogenes in mice. Comparative analyses of the mouse, rat, human, dog, cow, opossum, and chicken genomes suggest that the Rhox cluster originated in the common ancestor of primates and rodents. It subsequently underwent two remarkable expansions, first in the common ancestor of mice and rats and then in mice. Positive selection promoting amino acid substitutions was detected in some young Rhox genes, suggesting adaptive functional diversification. The recent expansions of the Rhox cluster provide an opportunity to study the mechanism and origin of colinear gene regulation, but they may also undermine the utility of mouse models for understanding the development and physiology of the human reproductive system.

HGF and postnatal testis development

Hepatocyte growth factor regulates many cellular functions acting through c-met, its specific receptor with tyrosine kinase activity. We have previously reported that in prepubertal rats HGF is secreted in the seminiferous tubules by purified peritubular myoid cells whereas Sertoli cells do not express HGF mRNA. In the present paper we report that HGF is expressed by the myoid cells during the entire postnatal testicular development studied and secreted in the culture medium. On the contrary, in Sertoli cells HGF starts to be clearly detectable by northern blot at 25 days of age. HGF is expressed and secreted by Sertoli cells isolated from 35-day-old rats and is able to increase the levels of c-met expression of the Sertoli cells. Although the role of HGF during the development of the postnatal testis need further research to be clarified, the data here presented indicate that HGF is one of the growth factors regulating mammalian testicular function.

Postnatal changes in the expression of p60c-Src in mouse testes

Src family non-receptor tyrosine kinases are involved in signaling pathways which mediate cell growth, differentiation, transformation and tissue remodeling in various organs. In an effort to elucidate functional involvement of p60c-Src (c-Src) in spermatogenesis, the postnatal changes in c-src mRNA and c-Src protein together with kinase activity and subcellular localization were examined in mouse testes. c-src mRNA levels in testes increased during the first 2 weeks of postnatal development (PND). Following a decrease at puberty (PND 28), the c-src mRNA levels re-increased at adulthood (PND 50). Src kinase activity of testes was low at PND 7 but sharply increased prepubertally (PND 15) and highest at adulthood. Upon Western blotting, the level of c-Src protein was the highest in prepubertal testes but rather decreased in adult testes at PND 50. In adult testes, ubiquitination of c-Src proteins was apparent compared with immature one at PND 7, suggesting active turnover of c-Src by ubiquitination. In immature testes, c-Src immunoreactivity was largely found in the cytoplasm of the Sertoli cells. By contrast, in pubertal and adult testes intense immunoreactivity was localized at the adluminal and basal cytoplasm of Sertoli cells bearing elongated spermatids and early germ cells, respectively. The immunoreactivity of c-Src in the Leydig cells was increased during pubertal development, suggesting the functional involvement of c-Src in differentiated adult Leydig cells. Throughout postnatal development, some spermatogonia and spermatocytes showed intensive c-Src immunoreactivity compared with other germ cells, suggesting a possible role of c-Src in germ cell death. Taken together, it is suggested that c-Src may participate in the remodeling of the seminiferous epithelia and functional differentiation of Leydig cells during the postnatal development of mouse testes.

Rhox: A New Homeobox Gene Cluster

Homeobox genes encode transcription factors notable for their ability to regulate embryogenesis. Here, we report the discovery of a cluster of 12 related homeobox genes on the X chromosome expressed in male and female reproductive tissues in adult mice. These reproductive homeobox on the X chromosome (Rhox) genes are expressed in a cell type-specific manner; several are hormonally regulated, and their expression pattern during postnatal testis development corresponds to their chromosomal position. Most of the Rhox genes are expressed in Sertoli cells, the nurse cells in direct contact with developing male germ cells, suggesting that they regulate the expression of somatic-cell gene products critical for germ cell development. In support of this, targeted disruption of Rhox5 increased male germ cell apoptosis and reduced sperm production, sperm motility, and fertility. Identification of this family of homeobox genes provides an opportunity to study colinear gene regulation and the transcriptional control of reproduction.

Novel testis-expressed profilin IV associated with acrosome biogenesis and spermatid elongation

A novel profilin, named profilin IV, was cloned and characterized as a testicular isoform, distinct from the previously described testis-specific profilin III. Profilin IV showed only 30% amino acid identity with the other mammalian profilins; nevertheless, database searches produced significant alignments with the conserved profilin domain. Northern blot analysis and in situ transcript hybridization suggested that profilin IV, like profilin III, is transcribed in the germ cells. However, the timing of their expression during post-natal development of rat testis and in the rat spermatogenetic cycle was distinct. In the human testis, profilin IV mRNA expression correlates with the presence of germ cells suggesting that it may be a suitable molecular diagnostic parameter to supplement conventional histopathological diagnostics in the assessment of testicular biopsies. The predicted profilin IV protein was verified employing an anti-oligopeptide antibody. Western blot analysis detected an immunorelated testicular protein of approximately 14 kDa. Immunohistochemistry revealed an intracellular protein of the rat, the mouse and the human testis accumulating asymmetrically in the cytoplasm of round and elongating spermatids with its perinuclear location coinciding with the position of the developing acrosome-acroplaxome and the manchette. Profilin IV thus may regulate testicular actin cytoskeleton dynamics and play a role in acrosome generation and spermatid nuclear shaping.

Molecular markers for the assessment of postnatal male germ cell development in the mouse

A proliferation marker, proliferating cell nuclear antigen (PCNA), a Sertoli cell specific transcription factor, GATA-1 and the male germ cell specific, RNA binding motif (RBM), were used to identify different cellular populations during postnatal development of the mouse testis. Immunohistochemistry, RT-PCR and real-time quantitative RT-PCR (QRT-PCR) were used. PCNA was expressed in pre-Sertoli and germ cells on the day of birth. Both pre-meiotic germ cells and spermatocytes expressed RBM throughout postnatal development. RBM-positive cell counts and QRT-PCR of RBM showed that average level of RBM per cell is highest in juvenile males between 14 and 21 days. From 42 days onward, there was a dramatic decrease in RBM expression in individual pre-meiotic and meiotic germ cells. These markers were used to correlate cell proliferative capability, gene expression profile and anatomic location within the developing mouse testis. The majority of germ cells start active proliferation once they have migrated to the basement membrane or immediately before. RBM is more highly expressed during the first wave of spermatogenesis versus subsequent waves, suggesting that there may be a change in the activity of RBM.

EXPRESSION OF CANNABINOID RECEPTOR 1 IN MOUSE TESTES

Marijuana smoke and cannabinoids adversely affect male reproductive function in human and rodent through the cannabinoid receptors. To understand the possible function of cannabinoid receptor 1 (CB1) in spermatogenesis, expression of CB1 in testis during the postnatal development was examined in mice. Semiquantitative RT-PCR analysis revealed that testicular CB1 mRNA level was relatively high at 1 week post partum (p.p.). Following decrease during prepubertal development (2 weeks p.p.) and CB1 mRNA level re-increased during puberty (4 weeks p.p.) and reached the peak in adult testis. At 1 week p.p., some spermatogonia and Leydig cells showed strong immunoreactivity of CB1. At 2 weeks p.p., CB1 immunoreactivity was largely found in the primary spermatocytes as well as spermatogonia, and Leydig cells showed a weak signal. In adult testis, strong immunoreactivity was found in Leydig cells and luminal epithelia of seminiferous tubule. Germ cells including spermatozoa were positive for CB1 immunoreactivity. On Western blot, multiple forms of CB1 proteins were detected in testes, suggesting oligomerization of CB1. Ubiquitous, but spatiotemporal difference in expression of CB1 in soma and germ line during postnatal development of testis suggests functional involvement of CB1 signaling in steroidogenesis, spermatogenesis and fertilization.

Androgens and Postmeiotic Germ Cells Regulate Claudin-11 Expression in Rat Sertoli Cells

In the present study we investigated whether fetal exposure to flutamide affected messenger and protein levels of claudin-11, a key Sertoli cell factor in the establishment of the hemotesticular barrier, at the time of two key events of postnatal testis development: 1) before puberty (postnatal d 14) during the establishment of the hemotesticular barrier, and 2) at the adult age (postnatal d 90) at the time of full spermatogenesis. The data obtained show that claudin-11 expression was inhibited in prepubertal rat testes exposed in utero to 2 and 10 mg/kg x d flutamide. However, in adult testes, the inhibition was observed only with 2, and not with 10, mg/kg x d of the antiandrogen. It is shown here that these differences between prepubertal and adult testes could be related to dual and opposed regulation of claudin-11 expression resulting from positive control by androgens and an inhibitory effect of postmeiotic germ cells. Indeed, testosterone is shown to stimulate claudin-11 expression in cultured Sertoli cells in a dose- and time-dependent manner (maximum effect with 0.06 microm after 72 h of treatment). In contrast, postmeiotic germ cells potentially exert a negative effect on claudin-11 expression, because adult rat testes depleted in spermatids (after local irradiation) displayed increased claudin-11 expression, whereas in a model of cocultured Sertoli and germ cells, spermatids, but not spermatocytes, inhibited claudin-11 expression. The apparent absence of claudin-11 expression changes in adult rat testes exposed to 10 mg/kg x d flutamide therefore could result from the antagonistic effects of 1) the inhibitory action of the antiandrogen and 2) the stimulatory effect of the apoptotic germ cells on claudin-11 expression. Together, due to the key role of claudin-11 in the hemotesticular barrier, the present findings suggest that such regulatory mechanisms may potentially affect this barrier (re)modeling during spermatogenesis.

Expression of p57kip2 in mouse and human testes

The expression of cyclin‐dependent kinases inhibitors, p57kip2, was investigated during the postnatal development of mouse testis, and in adult human testis. Expression of p57kip2 mRNA was higher in immature than pubertal or adult mouse testes. In postnatal day 7 (PND7) testes, moderate p57kip2 immunoreactivity was found in spermatogonia, but signal was heterogeneous among the spermatogonia. In PND14 testes onward, strong immunoreactivity of p57kip2 was found in the nuclei of early spermatocytes but not in the late pachytene stage onward. In PND28 and PND50 testes, p57kip2 immunoreactivity was varying among the seminiferous tubules. There was no visible signal in late pachytene stage onward. In Leydig cells, heterogeneous immunoreactivity of p57kip2 was found in immature testis and the signal intensity was higher in adult testis than immature ones. In Sertoli cells, weak or negligible immunoreactivity of p57kip2 was found. In human seminiferous tubule, strong immunoreactivity of p57kip2 was found in the nucleus of early spermatocytes, but not in the late pachytene spermatocytes onward and Sertoli cells. These results suggest the possible role of p57kip2 in the regulation of early spermatogonial proliferation, meiotic progression of early spermatocytes and differentiation of Leydig cells in testis.