Adult Human Testis Research Articles

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.

High levels of the extracellular matrix proteoglycan decorin are associated with inhibition of testicular function

Decorin (DCN), a component of the extracellular matrix of the peritubular wall and the interstitial areas of the human testis, can interact with growth factor (GF) signalling, thereby blocking downstream actions of GFs. In the present study the expression and regulation of DCN using both human testes and two experimental animal models, namely the rhesus monkey and mouse, were examined. DCN protein was present in peritubular and interstitial areas of adult human and monkey testes, while it was almost undetectable in adult wild type mice. Interestingly, the levels and sites of testicular DCN expression in the monkeys were inversely correlated with testicular maturation markers. A strong DCN expression associated with the abundant connective tissue of the interstitial areas in the postnatal through pre-pubertal phases was observed. In adult and old monkeys the DCN pattern was similar to the one in normal human testes, presenting strong expression at the peritubular region. In the testes of both infertile men and in a mouse model of inflammation associated infertility (aromatase-overexpressing transgenic mice), the fibrotic changes and increased numbers of tumour necrosis factor (TNF)-α-producing immune cells were shown to be associated with increased production of DCN. Furthermore, studies with human testicular peritubular cells isolated from fibrotic testis indicated that TNF-α significantly increased DCN production. The data, thus, show that an increased DCN level is associated with impaired testicular function, supporting our hypothesis that DCN interferes with paracrine signalling of the testis in health and disease.

Chromosome X-encoded cancer/testis antigens show distinctive expression patterns in developing gonads and in testicular seminoma

Cancer/testis (CT) antigens are cancer antigens normally expressed in adult testicular germ cells. The expression of chromosome X-encoded CT antigens (CT-X antigens) in human fetal gonads and in testicular seminomas was examined. The expression of 10 CT-X antigens (MAGEA, NY-ESO-1, GAGE, CT7/MAGEC1, CT10/MAGEC2, CT45, SAGE1, SSX2, NXF2 and SPANX) was studied immunohistochemically. In adult human testis, SPANX is expressed in late spermatids and spermatozoa, whereas all other CT-X antigens are predominantly expressed in spermatogonia or primary spermatocytes. All CT-X antigens except SPANX are expressed in human fetal germ cells. CT-X-positive germ cells appear as early as 13 weeks after gestation, increase with age and reach a plateau at around 22 weeks. In the fetal ovary, CT-X-positive oogonia are most abundant at around 24 weeks and sharply decrease subsequently. CT-X antigens are almost exclusively expressed in OCT3/4-negative gonocytes and their expression appears to coincide with the loss of pluripotency. Spermatocytic seminoma, a neoplasm derived from adult pre-meiotic germ cells, showed uniform expression of all CT-X antigens except SPANX. In contrast, most seminomas (>80%) express CT7, CT45, GAGE and CT10 but express MAGEA, NXF2 and NY-ESO-1 at lower frequency, and very rarely express SSX2 and SAGE1. Most CT-X antigens are expressed in human fetal germ cells after they have lost stem cell characteristics, with predominant expression in pre-meiotic germ cells. Spermatocytic seminomas showed expression of all CT-X antigens except SPANX, whereas classical seminomas only express some CT-X antigens, reflecting their different origins from adult versus fetal germ cells.

In vitro differentiation of haploid cells after spermatogonial co-culture with sertoli cells from adult human testes

In vitro differentiation of haploid cells after spermatogonial co-culture with sertoli cells from adult human testes

Changing subcellular localization of nuclear transport factors during human spermatogenesis

Spermatogenesis requires progressive changes in gene expression mediated by hormonal and local factors. Regulated macromolecular movement between nuclear and cytoplasmic compartments enables these essential responses to changing extracellular cues, and dynamic production of the nucleocytoplasmic transporters and importin proteins, throughout gametogenesis in rodents implicates them as key mediators of germline differentiation. We examined normal adult human testis expression profiles of six importins plus five additional proteins involved in nucleocytoplasmic transport. Although most were detected in the nucleus during germline differentiation, importin α4 was exclusively observed in Sertoli and germ cell cytoplasm. Many proteins were present in round spermatid nuclei (importins α1, α3, β1, β3; exportin-1, Nup62, Ran, RanBP1, RCC1), and remarkable intense nuclear and/or nuclear-associated signals were detected for importin α1, importin α3 and Nup62 in spermatocytes. This study identifies conserved aspects of nucleocytoplasmic transport during spermatogenesis and extends our knowledge of the dynamic presence of these proteins, which indicates that they contribute to germ cell-specific cargo trafficking and potentially to other functions during human spermatogenesis. We also demonstrate for the first time that importin α3 is nuclear in spermatocytes, when exportin-1 is cytoplasmic, suggesting that nuclear transport is altered during meiosis.

Heterochromatin marks HP1γ, HP1α and H3K9me3, and DNA damage response activation in human testis development and germ cell tumours

Heterochromatinization has been implicated in fundamental biological and pathological processes including differentiation, senescence, ageing and tumourigenesis; however, little is known about its regulation and roles in human cells and tissues in vivo. Here, we show distinct cell-type- and cancer-stage-associated patterns of key heterochromatin marks: histone H3 trimethylated at lysine 9 (H3K9me3) and heterochromatic adaptor proteins HP1α and HP1γ, compared with the γH2AX marker of endogenously activated DNA damage response (DDR) and proliferation markers in normal human foetal (n=4) and adult (n=29) testes, pre-invasive carcinoma in situ (CIS; n=26) lesions and a series of overt germ cell tumours, including seminomas (n=26), embryonal carcinomas (n=18) and teratomas (n=11). Among striking findings were high levels of HP1γ in foetal gonocytes, CIS and seminomas; enhanced multimarker heterochromatinization without DDR activation in CIS; and enhanced HP1α in teratoma structures with epithelial and neuronal differentiation. Differential expression of the three heterochromatin markers suggests their partly non-overlapping roles, and separation of heterochromatinization from DDR activation highlights distinct responses of germ cells vs. somatic tissues in early tumourigenesis. Conceptually interesting findings were that subsets of human cells in vivo proliferate despite enhanced heterochromatinization, and that cells can strongly express even multiple heterochromatin features in the absence of functional retinoblastoma protein and without DDR activation. Overall, these results provide novel insights into cell-related and tumour-related diversity of heterochromatin in human tissues in vivo, relevant for andrology and intrinsic anti-tumour defence roles attributed to activated DDR and cellular senescence.

Distribution and expression of alternative splice isoforms of NOR1 in human tissues and cell lines.

To analyze the distribution and subcellular localization of NOR1 alternative splice isoforms in human tissues and cell lines. NOR1 open reading frame(ORF) was amplified from human fetal brain cDNA library and subcloned into pCMV/myc vector. The level of NOR1 mRNA in human tissues was determined by real-time RT-PCR. Region spanning exon 2 was amplified from cDNA or genomic DNA by specific primers and sequenced. The expression plasmids of NOR1 were transfected into cells and immunofluoresence assay was performed to determine the subcellular localization of NOR1 protein isoforms in human cells. The expression of NOR1 mRNA was high in human adult testis, moderate in human fetus nasopharynx, trachea, brain, and kidney tissues, and weak or undetectable in other tissues. Two splice variants of human NOR1 gene resulted from alternative splicing at exon 2 were identified. Both of isoform 1 and isoform 2 mRNA were detected in human fetus brain. Isoform 2 was the sole isoform in other tissues but brain. Only isoform 2 mRNA was detected in cell lines used in this study, though no genomic deletion of exon 2 could be found in all these cell lines. Immunofluoresence assay showed both isoform 1 and isoform 2 proteins were distributed in cytoplasm. Alternative splice isoforms of tumor suppressor gene NOR1 are identified. NOR1 isoform 1 and isoform 2 are both detected in fetus brain. NOR1 isoform 2 lacking of exon 2 is the sole isoform in multiple tissues except for brain. The exon 2 encoded peptide does not affect the subcellular location of NOR1 protein.

Stem cell research for male infertility.

Stem cells have the ability both to differentiate into numerous tissues and to self-renew. Because of these unique properties, stem cells are promising candidates for use in regenerative medicine. Among stem cell types, embryonic stem (ES) cells have been the most studied; however, alternatives such as induced pluripotent stem cells or other adult stem cells are now being established. In this review, we focus on stem cell research that may have applications in treating male infertility. Stem cells with ES-like properties have been generated from adult human testis tissue. We expect that breakthroughs in stem cell research will increase our understanding of male infertility and lead to treatments in the near future.

Identification and characterization of repopulating spermatogonial stem cells from the adult human testis

This study was conducted to identify and characterize repopulating spermatogonial stem cells (SSCs) in the adult human testes. Testes biopsies from obstructive azoospermic patients and normal segments of human testicular tissue were used. Flow cytometry, real-time PCR and immunohistochemical analysis were performed. Purified human spermatogonia were transplanted into busulfan-treated recipient mouse testes and integrated cells were detected by human nuclear protein antibody co-localized with stem cell and germ cell markers. Testicular biopsies collected from obstructive azoospermic men showed similar morphology and distribution of markers to the normal human testes. Flow cytometry showed distinct populations of stage-specific embryonic antigen-4 (SSEA-4), CD49f and CD90 positive cells in the adult human testes. SSEA-4 (+) cells showed high expression levels of SSC-specific genes and high levels of telomerase activity. Extensive colonization of human cells in the mouse testes indicates the presence of highly enriched populations of SSCs in the SSEA-4 (+) sorted cells. All the HNP (+) cells in the mouse testes were positive for germ cell marker dead box mRNA helicase and only half of them were dimly positive for c-kit. In addition, subpopulations of human spermatogonia that colonized mouse testes were positively stained for CD49f, GPR-125, Nanog and Oct-4 indicating the existence of population of cells among human spermatogonia with SSC and pluripotent characteristics. This study clearly demonstrates that repopulating human SSCs have phenotypic characteristics of SSEA-4(+), CD49f(+), GPR-125(+)and c-Kit (neg/low). The results have direct implications for enrichment of human spermatogonia for further culture and germ cell differentiation studies.

Newer Insights Into Premeiotic Development of Germ Cells in Adult Human Testis Using Oct-4 as a Stem Cell Marker

The transcription factor octamer-binding transforming factor 4 (Oct-4) is central to the gene regulatory network responsible for self-renewal, pluripotency, and lineage commitment in embryonic stem (ES) cells and induced pluripotent stem cells (PSCs). This study was undertaken to evaluate differential localization and expression of two major transcripts of Oct-4, viz. Oct-4A and Oct-4B, in adult human testis. A novel population of 5- to 10-μm PSCs with nuclear Oct-4A was identified by ISH and immunolocalization studies. Besides Oct-4, other pluripotent markers like Nanog and TERT were also detected by RT-PCR. A(dark) spermatogonial stem cells (SSCs) were visualized in pairs and chains undergoing clonal expansion and stained positive for cytoplasmic Oct-4B. Quantitative PCR and Western blotting revealed both the transcripts, with higher expression of Oct-4B. It is proposed that PSCs undergo asymmetric cell division and give rise to A(dark) SSCs, which proliferate and initiate lineage-specific differentiation. The darkly stained nuclei in A(dark) SSCs may represent extensive nuclear reprogramming by epigenetic changes when a PSC becomes committed. Oct-4B eventually disappeared in mature germ cells, viz. spermatocytes, spermatids, and sperm. Besides maintaining normal testicular homeostasis, PSCs may also be implicated in germ cell tumors and ES-like colonies that have recently been derived from adult human testicular tissue.

Human Spermatogonial Stem Cells and Pluripotent Stem Cells from Adult Human Testes

Abstract: Continuation of the spermatogenic process throughout life relies on proper regulation of selfrenewal and differentiation in spermatogonial stem cells. Mouse spermatogonial stem cells can be cultured with established methods; however, human spermatogonial stem cells are not well understood. Recent reports have shown that adult cells can be reprogrammed to pluripotency if specific genes are delivered and almost established assay. These cells are called iPS (induced pluripotent stem) cells. In addition, it has been reported that pluripotent stem cells can be generated from adult human testes. Stem cell science has progressed remarkably quickly over the last 5 years. Future research into stem cells may reveal the mechanisms underlying male infertility and allow for patient-specific treatments. In this review, we provide an overview of the current understanding of human spermatogonial stem cells and pluripotent stem cells from adult human testes.

Expression of a truncated form of KIT tyrosine kinase in human spermatozoa correlates with sperm DNA integrity

TR-KIT, a truncated form of KIT (the KITL receptor), corresponding to the c-terminal half of the intracellular split tyrosine kinase domain, is expressed during the haploid stages of mouse spermatogenesis, and is one of the candidate sperm factors possibly involved in egg activation at fertilization. Immunocytochemistry of adult human testis, and studies of human semen samples from volunteer donors through immunofluorescence, confocal microscopy, flow cytometry, western blot and RT-PCR analyses were performed. We show that the TR-KIT is expressed during spermiogenesis in the human testis, and that it is maintained in human ejaculated spermatozoa. TR-KIT is localized both in the equatorial segment and in the sub-acrosomal region of the human sperm head. The equatorial localization of the TR-KIT persists after the spontaneous acrosome reaction. Cytometric analysis of several sperm samples from volunteer donors, showed variable degrees of the TR-KIT-specific immunolabeling, and a significant inverse correlation (Pearson's coefficient, r = -0.76, P < 0.0001, n = 23) of the TR-KIT positivity with markers of sperm damage, i.e. DNA fragmentation, as revealed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-nick end labeling (TUNEL) analysis and the intense clusterin positivity. We also found less significant inverse correlation with altered head morphology (r = -0.47, P < 0.05, n = 23) and direct correlation with sperm forward motility parameters (r = 0.59, P < 0.01, n = 23). The TR-KIT is present in the equatorial region of human spermatozoa, which is the first sperm component entering into the oocyte cytoplasm after fusion with the egg. This localization is consistent with the function previously proposed for this protein in mice. In addition, the TR-KIT represents a potential predictive parameter of human sperm quality.

Human tissue distribution of paraoxonases 1 and 2 mRNA

We have studied the distribution of mRNA for paraoxonases (PON) 1 and 2 in 24 human tissues using Gene Expression Panels. PON1 mRNA was restricted to adult kidney, liver, and colon as well as fetal liver, whereas PON2 mRNA was more widely distributed in adult human brain, heart, kidney, spleen, liver, colon, lung, small intestine, muscle, stomach, testis, placenta, salivary, thyroid and adrenal glands, pancreas, skin, and bone marrow, as well as fetal brain and liver. PON2 mRNA was not found in ovary, uterus, or plasma leukocytes using the panels. However, using real time PCR, we found PON2 mRNA expression in human plasma leukocytes. There were differences between the tissue distribution of mRNAs found in this study and the immunohistochemical localization of the PON1 and PON2 proteins reported previously. In particular, PON1 protein is much more widely distributed than its mRNA, possibly indicating the delivery of PON1 to various tissues by HDL. In addition, differences between PON2 mRNA and protein distributions could be due to missence mutations in the PON2 gene, causing nontranslation of mRNA to protein in some tissues.

Isolation, Characterization, and Culture of Human Spermatogonia1

This study was designed to isolate, characterize, and culture human spermatogonia. Using immunohistochemistry on tubule sections, we localized GPR125 to the plasma membrane of a subset of the spermatogonia. Immunohistochemistry also showed that MAGEA4 was expressed in all spermatogonia (A(dark), A(pale), and type B) and possibly preleptotene spermatocytes. Notably, KIT was expressed in late spermatocytes and round spermatids, but apparently not in human spermatogonia. UCHL1 was found in the cytoplasm of spermatogonia, whereas POU5F1 was not detected in any of the human germ cells. GFRA1 and ITGA6 were localized to the plasma membrane of the spermatogonia. Next, we isolated GPR125-positive spermatogonia from adult human testes using a two-step enzymatic digestion followed by magnetic-activated cell sorting. The isolated GPR125-positive cells coexpressed GPR125, ITGA6, THY1, and GFRA1, and they could be cultured for short periods of time and exhibited a marked increase in cell numbers as shown by a proliferation assay. Immunocytochemistry of putative stem cell genes after 2 wk in culture revealed that the cells were maintained in an undifferentiated state. MAPK1/3 phosphorylation was increased after 2 wk of culture of the GPR125-positive spermatogonia compared to the freshly isolated cells. Taken together, these results indicate that human spermatogonia share some but not all phenotypes with spermatogonial stem cells (SSCs) and progenitors from other species. GPR125-positive spermatogonia are phenotypically putative human SSCs and retain an undifferentiated status in vitro. This study provides novel insights into the molecular characteristics, isolation, and culture of human SSCs and/or progenitors and suggests that the MAPK1/3 pathway is involved in their proliferation.

Embryonic stem cell-like cells derived from adult human testis

Given the significant drawbacks of using human embryonic stem (hES) cells for regenerative medicine, the search for alternative sources of multipotent cells is ongoing. Studies in mice have shown that multipotent ES-like cells can be derived from neonatal and adult testis. Here we report the derivation of ES-like cells from adult human testis. Testis material was donated for research by four men undergoing bilateral castration as part of prostate cancer treatment. Testicular cells were cultured using StemPro medium. Colonies that appeared sharp edged and compact were collected and subcultured under hES-specific conditions. Molecular characterization of these colonies was performed using RT-PCR and immunohistochemistry. (Epi)genetic stability was tested using bisulphite sequencing and karyotype analysis. Directed differentiation protocols in vitro were performed to investigate the potency of these cells and the cells were injected into immunocompromised mice to investigate their tumorigenicity. In testicular cell cultures from all four men, sharp-edged and compact colonies appeared between 3 and 8 weeks. Subcultured cells from these colonies showed alkaline phosphatase activity and expressed hES cell-specific genes (Pou5f1, Sox2, Cripto1, Dnmt3b), proteins and carbohydrate antigens (POU5F1, NANOG, SOX2 and TRA-1-60, TRA-1-81, SSEA4). These ES-like cells were able to differentiate in vitro into derivatives of all three germ layers including neural, epithelial, osteogenic, myogenic, adipocyte and pancreatic lineages. The pancreatic beta cells were able to produce insulin in response to glucose and osteogenic-differentiated cells showed deposition of phosphate and calcium, demonstrating their functional capacity. Although we observed small areas with differentiated cell types of human origin, we never observed extensive teratomas upon injection of testis-derived ES-like cells into immunocompromised mice. Multipotent cells can be established from adult human testis. Their easy accessibility and ethical acceptability as well as their non-tumorigenic and autogenic nature make these cells an attractive alternative to human ES cells for future stem cell therapies.

Pluripotent Stem Cells Derived From Adult Human Testes

Recent reports have demonstrated that adult tissue cells can be induced to pluripotency, the iPS cells, mostly with the addition of genes delivered using viruses. Also, several publications both in mouse and in human have demonstrated that spermatogonial stem cells (SSCs) from testes can convert back to embryonic stem (ES)-like cells without the addition of genes. Furthermore, these pluripotent ES-like cells can differentiate into all three germ layers and organ lineages. Thus, SSCs have great potential for cell-based, autologous organ regeneration therapy for various diseases. We obtained testes from organ donors and using 1 g pieces of tissue (biopsy size) we demonstrate that testis germ cells (putative SSCs and/or their progenitors) reprogram to pluripotency when removed from their stem cell niche and when appropriate growth factors and reagents in embryonic stem cell medium are added. In addition, our method of obtaining pluripotent ES-like cells from germ cells is simpler than the described methods and may be more suitable if this procedure is developed for the clinic to obtain pluripotent cells to cure disease.

Analysis of activin/TGFB-signaling modulators within the normal and dysfunctional adult human testis reveals evidence of altered signaling capacity in a subset of seminomas

Activin is a pleiotropic growth factor belonging to the transforming growth factor-beta (TGFB) superfamily of signaling molecules. Regulated activin signaling is known to influence several steps in rodent male gamete differentiation. TGFB ligand isoforms, TGFB1-B3, also influence germ cell survival in the rodent testis at the onset of spermatogenesis and around the time of puberty. Given the importance of regulated activin and TGFB signaling in testis development and function, we sought to investigate the cellular production sites of activin/TGFB-signaling modulators in normal and dysfunctional adult human testes samples. Signaling transducers phosphorylated SMAD2/3, and signaling modulators SMAD6, MAN-1, inhibin alpha (INHA), and beta-glycan were detected in Bouins fixed, paraffin-embedded adult human testis sections using immunohistochemistry. Additional samples examined were from testicular cancer patients and from normal men subjected to gonadotropin suppression with androgen-based contraceptives. Our findings identify distinct differences between normal and gonadotropin-deprived human testis in the expression and cellular localization of activin/TGFB-signaling modulators. The presence of a nuclear phosphorylated SMAD2/3 signal in all analyzed seminoma specimens indicated active activin/TGFB signaling. Moreover, a subset of seminoma specimens exhibited selective enhanced expression of beta-glycan (4 out of 28 seminoma tumors), INHA (6 out of 28), and MAN-1 (6 out of 28), highlighting potential functional differences between individual tumors in their capacity to regulate activin/TGFB signaling. Within the heterogenous nonseminomas, expression of signaling modulators was variable and reflected the degree of somatic differentiation. Thus, synthesis of activin and TGFB-signaling modulators may be affected by spermatogenic disruption and altered hormone levels in the testis.

A putative mesenchymal stem cells population isolated from adult human testes

Mesenchymal stem cells (MSCs) isolated from several adult human tissues are reported to be a promising tool for regenerative medicine. In order to broaden the array of tools for therapeutic application, we isolated a new population of cells from adult human testis termed gonadal stem cells (GSCs). GSCs express CD105, CD166, CD73, CD90, STRO-1 and lack hematopoietic markers CD34, CD45, and HLA-DR which are characteristic identifiers of MSCs. In addition, GSCs express pluripotent markers Oct4, Nanog, and SSEA-4. GSCs propagated for at least 64 population doublings and exhibited clonogenic capability. GSCs have a broad plasticity and the potential to differentiate into adipogenic, osteogenic, and chondrogenic cells. These studies demonstrate that GSCs are easily obtainable stem cells, have growth kinetics and marker expression similar to MSCs, and differentiate into mesodermal lineage cells. Therefore, GSCs may be a valuable tool for therapeutic applications.

Localization of the Chromatin Remodelling Protein, ATRX in the Adult Testis

Mutations in ATRX (alpha-thalassaemia and mental retardation on the X-chromosome) can give rise to ambiguous or female genitalia in XY males, implying a role for ATRX in testicular development. Studies on ATRX have mainly focused on its crucial role in brain development and α-globin regulation; however, little is known about its function in sexual differentiation and its expression in the adult testis. Here we show that the ATRX protein is present in adult human and rat testis and is expressed in the somatic cells; Sertoli, Leydig, and peritubular myoid cells, and also in germ cells; spermatogonia and early meiotic spermatocytes. The granular pattern of ATRX staining is consistent with that observed in other cell-types and suggests a role in chromatin regulation. The findings suggest that ATRX in humans may play a role in adult spermatogenesis as well as in testicular development.

Stage-specific Localization and Expression of c-kit in the Adult Human Testis

The c-kit receptor (KIT) and its ligand, stem cell factor (SCF), represent one of the key regulators of testicular formation, development, and function and have been extensively studied in various animal models. The present study was undertaken to characterize the pattern of localization and expression of c-kit in normal adult human testis. Immunohistochemical analysis showed that KIT is expressed in the cytoplasm of spermatogonia, acrosomal granules of spermatids, and Leydig cells. Interestingly, a rather heterogenous pattern of expression of the protein along the basement membrane was observed. Intense protein localization in spermatogonia was detected in stages I-III, whereas low expression was observed in stages IV-VI of the seminiferous epithelium, indicating that the expression of the molecule was stage specific. In situ hybridization studies revealed that the transcripts of the gene were also localized in a similar non-uniform pattern. To the best of our knowledge, such a stage-specific expression of KIT has not been reported previously in the human testis. The results of the present study may expand current knowledge about the c-kit/SCF system in human spermatogenesis.