Adjacent Sertoli Cells Research Articles

The functional development of Leydig cells in a marsupial

Leydig cells are the major source of androgen in the male mammal. We describe here for the first time the development of the Leydig cell in a macropodid marsupial, the tammar wallaby, Macropus eugenii. Leydig cells are first recognized morphologically 2 days after birth with the appearance of lipid droplets in the cytoplasm of certain interstitial cells. Lipid content closely matches the steroid content of the developing testis and marks the maturation of the steroid synthesis pathway in the tammar testis. Morphologically mature Leydig cells, marked by distinct mitochondria with tubular cristae and an extensive anastomosing network of smooth endoplasmic reticulum, are developed by day 10 after birth - the time of peak testosterone content in perinatal tammar testes. The volume percentage of each cell type in the testis does not change over time so the growth of each cellular component keeps pace with growth of the whole testis. There was no morphological or quantitative evidence of a change from one population of Leydig cells to another in the tammar testis as has been reported in several other species including the rat, mouse and human. Maturation of the testis is also marked by the development of tight junctions between the cell membranes of adjacent Sertoli cells. These appear around day 30 after birth and coincide with the onset of mitotic arrest in male germ cells. Overall, the development of the Leydig cell in the tammar wallaby follows a similar pattern to that seen in other mammals, although the start of Leydig cell differentiation is, like many other organ systems in marsupials, post natal, not fetal and there appears to be only a single population of Leydig cells.

Role of Connexin 43 in Sertoli Cells of Testis

Connexin 43 (CX43, also known as GJA1) is the predominant testicular gap-junction protein. In the seminiferous epithelium, gap junctions occur between adjacent Sertoli cells and between Sertoli and germ cells. The specific role of CX43 in Sertoli cell has been difficult to study because of the neonatal lethality of the Cx43 global knockout. Recently two laboratories have independently developed a Sertoli cell conditional Cx43 knockout using Cre-loxP methodology. These have allowed the role of CX43 in Sertoli cells to be determined while circumventing lethal abnormalities in organs such as the heart seen in Cx43 global knockouts. In this review the focus is on the insights into the role of CX43 in Sertoli cells derived from these new model systems. Results indicate that CX43 is essential for cessation of proliferation and normal maturation in Sertoli cells. In addition, CX43 in Sertoli cells is required for spermatogenesis, but not early germ cell proliferation. These model systems show that CX43 is important in Sertoli cell development, and will be useful in further studies of its role in Sertoli cell and testis biology.

Varicocele-Induced Testicular Dysfunction May Be Associated with Disruption of Blood-Testis Barrier

The objective of this study was to examine E-cadherin and alpha-catenin expression at the junctions between adjacent Sertoli cells in testicular specimens from patients with varicocele in order to determine the presence of a possible link between blood-testis barrier and pathophysiology of varicocele. A total of 51 testicular biopsies were obtained from 28 infertile men with unilateral or bilateral varicocele. Twenty-three patients had bilateral and 5 had unilateral varicocele, Grade I varicocele was detected in 30 (59%), grade II in 15 (29%) and grade III in 6 (12%) patients. Abnormal expression of E-cadherin and alpha-catenin at the junctions between adjacent Sertoli cells was demonstrated in 100% and 90% of the patients with varicocele, respectively. In those with grade I-III varicocele, the mean E-cadherin and alpha-catenin expression were 7.6 +/- 11.4 and 39 +/- 36; 7.6 +/- 0.0 and 49 +/- 30; 8.3 +/- 9.3 and 58 +/- 33, respectively, but the difference was not significant. Reduced E-cadherin and alpha-catenin expression at the junctions between adjacent Sertoli cells may be associated with disruption of blood-testis barrier in varicocele.

Altered Expression of ZO-1 and ZO-2 in Sertoli Cells and Loss of Blood-Testis Barrier Integrity in Testicular Carcinoma In Situ

Carcinoma in situ (CIS) is the noninvasive precursor of most human testicular germ cell tumors. In normal seminiferous epithelium, specialized tight junctions between Sertoli cells constitute the major component of the blood-testis barrier. Sertoli cells associated with CIS exhibit impaired maturation status, but their functional significance remains unknown. The aim was to determine whether the blood-testis barrier is morphologically and/or functionally altered. We investigated the expression and distribution pattern of the tight junction proteins zonula occludens (ZO) 1 and 2 in normal seminiferous tubules compared to tubules showing CIS. In normal tubules, ZO-1 and ZO-2 immunostaining was observed at the blood-testis barrier region of adjacent Sertoli cells. Within CIS tubules, ZO-1 and ZO-2 immunoreactivity was reduced at the blood-testis barrier region, but spread to stain the Sertoli cell cytoplasm. Western blot analysis confirmed ZO-1 and ZO-2, and their respective mRNA were shown by RT-PCR. Additionally, we assessed the functional integrity of the blood-testis barrier by lanthanum tracer study. Lanthanum permeated tight junctions in CIS tubules, indicating disruption of the blood-testis barrier. In conclusion, Sertoli cells associated with CIS show an altered distribution of ZO-1 and ZO-2 and lose their blood-testis barrier function.

FSH regulates the formation of adherens junctions and ectoplasmic specialisations between rat Sertoli cells in vitro and in vivo

Spermatogenesis is dependent on the ability of Sertoli cells to form mature junctions that maintain a unique environment within the seminiferous epithelium. Adjacent Sertoli cells form a junctional complex that includes classical adherens junctions and testis-specific ectoplasmic specialisations (ES). The regulation of inter-Sertoli cell junctions by the two main endocrine regulators of spermatogenesis, FSH and testosterone, is unclear. This study aimed to investigate the effects of FSH and testosterone on inter-Sertoli cell adherens junctions (as determined by immunolocalisation of cadherin, catenin and actin) and ES junctions (as determined by immunolocalisation of espin, actin and vinculin) in cultured immature Sertoli cells and GnRH-immunised adult rat testes given FSH or testosterone replacement in vivo. When hormones were absent in vitro, adherens junctions formed as discrete puncta between interdigitating, finger-like projections of Sertoli cells, but ES junctions were not present. The adherens junction puncta included actin filaments that were oriented perpendicularly to the Sertoli cell plasma membrane, but were not associated with the intermediate filament protein vimentin. When FSH was added in vitro, ES junctions formed, and adjacent adherens junction puncta fused into extensive adherens junction belts. After hormone suppression in vivo, ES junctions were absent, while FSH replacement restored ES junctions, as confirmed by electron microscopy and confocal analysis of ES-associated proteins. Testosterone alone did not affect adherens junctions or ES in vitro or in vivo. We conclude that FSH can regulate the formation of ES junctions and stimulate the organisation and orientation of extensive adherens junctions in Sertoli cells.

Transfection of Sertoli cells ex vivo; Opening the door to the in vitro study of junction turnover in the seminiferous epithelium

Sertoli cells interact with adjacent Sertoli cells and with spermatids through elaborate intercellular adhesion junctions termed ectoplasmic specializations (ESs). Among adhesion molecules present at the sites is Nectin-2. Tubulobulbar complexes have been proposed to play a role in junction internalization. Here we establish a culture model for exploring the role of tubulobulbar complexes during junction turnover. Cell clumps enriched for Sertoli cells were isolated from ten day-old rat pups and cultured on Matrigel coated Transwells. These cells established a columnar morphology and formed identifiable basal ESs. Significantly, structures resembling tubulobulbar complexes developed at the junction sites. We generated eGFP-tagged nectin-2 constructs and transfected them first into Hela cells (cells that do not normally form adherens junctions) and then into our primary cultures of morphologically differentiated Sertoli cells. In Hela cells, eGFP-Nectin-2 localized to sites of intercellular contact between adjacent cells indicating that the protein was expressed and targeted to the plasma membrane where it appeared to establish adherens junctions. In Sertoli cells, the protein was localized to areas consistent with the position of ESs and to tubular structures associated with them. Primary Sertoli cell cultures may be a model for studying the role of tubulobulbar complexes in junction turnover. CIHR MOP 62728

Evidence that a kinesin is associated with ectoplasmic specializations in the testis

During spermatogenesis, spermatids undergo a “down and up” translocation event in the seminiferous epithelium. This event has been proposed to be microtubule-based and to result from the movement of apical “ectoplasmic specializations” (adhesion junctions) along adjacent Sertoli cell microtubule tracts. Dynein is present at the junctions and is likely responsible for the upward movement of spermatids. A kinesin is likely responsible for the downward movement of spermatids. To test the hypothesis that a kinesin is associated with the junctions, we generated an antibody to the “LAGSE” sequence conserved amongst kinesins, and reacted the antibody with fixed frozen sections of epithelium or fixed epithelial fragments. In tissue processed for immunofluorescensce, the antibody reacted at sites known to contain apical ectoplasmic specializations, in addition to reacting with other structures in the epithelium known to contain kinesins. In material processed for immunogold localization, the antibody reacted with the cytoplasmic face of the endoplasmic reticulum component of ectoplasmic specializations. Kinesin mRNA transcript screens using mouse GeneChip arrays of testis and Sertoli cells indicated that a possible candidate kinesin for spermatid translocation is Rab6KIFL. Antibodies generated against a peptide sequence that is unique to this kinesin reacted with regions associated with spermatid heads, and with a band on immunoblots that is not present in other tissues and migrates at a higher molecular weight than that known for Rab6KIFL. We are currently identifying the protein. Our results are consistent with the prediction that a kinesin is associated with ectoplasmic specializations. CIHR MOP 62768

Role of Glial Cell Line‐Derived Neurotrophic Factor in Germ‐Line Stem Cell Fate

The overall goal of this study is to unravel the role(s) played by glial cell line-derived neurotrophic factor (GDNF) in the fate of spermatogonial stem cells. There is great interest in the biology of spermatogonial stem cells, or A(single) spermatogonia, because of their importance in the treatment of infertility, the development of contraceptives, and the understanding of the etiology of testicular cancer, particularly seminoma. In the mouse, spermatogonial stem cells express GFRalpha-1, the receptor for GDNF, and respond to this growth factor in vivo and in vitro. GDNF is produced by the adjacent Sertoli cells, which are part of the germ-line stem cell niche in vertebrates. We specifically isolated GFRalpha-1-positive spermatogonia using an immunomagnetic bead technique. We then stimulated the cells with 100 ng/mL of rGDNF for 10 hours; unstimulated cells served as negative controls. Microarray analysis, immunocytochemistry, and Western blotting revealed that Numb, a regulator of the Notch pathway, is upregulated by GDNF in spermatogonial stem cells. There are indications that in rats, mice, and humans, the Notch pathway promotes spermatogonial differentiation. We observed that an increase in Numb expression is concomitant with Notch degradation in these cells. Thus, through Numb, GDNF might inhibit differentiation and allows the maintenance of the stem cell pool in the mouse seminiferous epithelium.

Granular changes in Sertoli cells in children and pubertal patients

To characterize lysosomes and histochemical function of granular Sertoli cells in developmental alterations. Prospective and retrospective study. University hospital and research centers. Nineteen infantile and pubertal patients undergoing testicular biopsy; four rat testes for lysosomal study. CD-68, alpha-1-antitrypsin, vimentin, inhibin alpha subunit, and anti-mullerian hormone antibodies were evaluated. Morphometric measures in seminiferous tubules with and without granular Sertoli cells were obtained. Ultrastructural data of lysosomes in human and rat Sertoli cells were compared. Quantification of mean diameter of seminiferous tubules, tubular fertility index, and germ and Sertoli cell indexes were obtained in human testis. Granular changes in Sertoli cells are due to the accumulation of large amounts of lysosomes. Vimentin immunoexpression in infantile and pubertal granular Sertoli cells was lower than in adjacent nongranular Sertoli cells. Inhibin was negative in granular cells. Anti-mullerian hormone-positive and -negative granular Sertoli cells were present within the same tubules. The presence of early granular changes in Sertoli cells in childhood and pubertal cryptorchidic patients, associated with other developmental alterations, suggests an intense and irreversible dysfunction of phagocytosis in the granular Sertoli cells. These alterations might be considered primary and irreversible anomalies of Sertoli cells, which might be contributing factors in the infertility seen in these patients.

Effects of melatonin treatment on Leydig cell activity in the testis of the frog Rana esculenta

This study was conducted to verify the effect(s) of melatonin treatment on frog Leydig cells. Morphological observation after melatonin treatment indicates that many frog Leydig cells show degenerative changes (i.e. heterochromatic nuclei, loss of cellular adhesion) while in adjacent germinal tubules several Sertoli cells show heterochromatic nuclei, confirming the presence of a paracrine effect between interstitial and germinal compartments. The effect of melatonin on frog Leydig cell steroidogenesis was investigated in in vitro experiments; after 6 h of incubation melatonin severely inhibits both control and GnRH-induced testosterone secretion. In addition, in order to verify the effect of indolamine on frog Leydig cell activity, we investigated, by in situ hybridization, the presence of frog relaxin (fRLX, a transcript specifically expressed by these cells) in the testes of melatonin-injected animals after 48 h. fRLX signal completely disappeared from the testis of melatonin- injected frogs. The results of the present study indicate that melatonin treatment provokes Leydig cell morphological changes, blocks GnRH-antagonist-induced testosterone secretion and decreases fRLX expression. Taken together these results strongly indicate that melatonin acts on Leydig cells in the testis of the frog Rana esculenta.

The acrosome-acroplaxome-manchette complex and the shaping of the spermatid head

A combination of exogenous contractile forces generated by a stack of F-actin-containing hoops embracing the apical region of the elongating spermatid nucleus and an endogenous modulating mechanism dependent on the spermatid-containing acrosome-acroplaxome-manchette complex may play a cooperative role in the shaping of the spermatid head. In addition, the manchette is a key element in the transport of vesicles and macromolecules to the centrosome and developing spermatid tails as well as in nucleocytoplasmic transport. The proposed model of spermatid head shaping is based on: 1) currently known structural and molecular components of the F-actin hoops, the main cytoskeletal element of the Sertoli cell ectoplasmic specializations; 2) the molecular features of acrosome biogenesis; 3) the assembly of a subacrosomal cytoskeletal plate called the acroplaxome; and 4) the spatial relationship of the acrosome-acroplaxome complex with the manchette, a transient microtubular/actin-containing structure. During acrosome biogenesis, the acroplaxome becomes the nucleation site to which Golgi-derived proacrosomal vescicles tether and fuse. The acroplaxome has at least two functions: it anchors the developing acrosome to the elongating spermatid head. It may also provide a mechanical scaffolding plate during the shaping of the spermatid nucleus. The plate is stabilized by a marginal ring with junctional complex characteristics, adjusting to exogenous clutching forces generated by the stack of Sertoli cell F-actin-containing hoops applied to the elongating spermatid head. A tubulobulbar complex, formed by cytoplasmic processes protruding from the elongating spermatid head extending into the adjacent Sertoli cell, is located at the concave side of the spermatid head. The tubulobulbar complex might provide stabilizing conditions, together with the actin-afadin-nectin-2/nectin-3 adhesion unit, to enable sustained and balanced clutching exogenous forces applied during the elongation of the spermatid head.

Functional characterization of Cx43 based gap junctions during spermatogenesis.

In the testis, spermatogenesis is a highly regulated process that includes germ cell multiplication and differentiation supported by Sertoli cells. Gap junction intercellular communication (GJIC), that is known to play an important role in the control of cell proliferation and differentiation, allows communication between adjacent cells. Gap junctions are present within the seminiferous epithelium but the precise nature of coupled cells is not yet identified. By applying a dye-transfer assay to testis, we demonstrated that coupling was basally located in the tubular compartment between adjacent Sertoli cells, between Sertoli cells and spermatogonia and early and late spermatocytes, but not between Sertoli cells and spermatids. Furthermore, no dye transfer occurred from germ cells to Sertoli cells. Specificity of the gap junction coupling was verified with known gap junction inhibitors such as oleamide, heptanol, and glycyrrhetinic acid. We developed a sophisticated assay that allows correlating the in vivo dye transfer with cell morphological identification and Cx43 expression. This approach demonstrated the Cx43 participation in the coupling. Interestingly Cx43 expression and dye-coupling varied with the stages of spermatogenesis. Our results suggest that Cx43 based gap junctions form a transversal and longitudinal intercellular communication network within seminiferous tubules, and that specific communication territories are formed within the seminiferous tubules to ensure the synchronization of germ cell proliferation and differentiation.

EXPRESSION OF CLAUDIN-1 IN MOUSE TESTIS

In testis, tight junctions (TJs) between adjacent Sertoli cells are important for the formation of the blood-testis barrier (BTB) and crucial for spermatogenesis. The present study aimed to find postnatal changes in the expression of claudin-1, one of the TJ genes in mouse testis. By semiquantitative RT-PCR, it was found that claudin-1 expression in testis increased up to a peak at 10 days after birth and decreased thereafter. Western blot analysis showed abundant expression of 21-kDa protein in testis, lung, and brain from the adult mouse. The developmental change in the expression of claudin-1 protein in testis coincided with that from the RT-PCR. Testosterone treatment significantly increased claudin-1 expression in immature Sertoli cells, suggesting the possible regulation of claudin-1 expression by androgen in mouse Sertoli cells. Claudin-1 expression appears to be developmentally regulated in the mouse testis.

Chronological gene expression of ADAMs during testicular development: prespermatogonia (gonocytes) express fertilin beta (ADAM2).

Immediately after birth, primordial germinal cell-derived prespermatogonia (PSG), located in the center of the testicular cords, migrate between adjacent Sertoli cells to establish contact with the cord basal lamina. PSG migration suggests continued assembly and disassembly of cell-cell contacts by a molecular mechanism that may involve integrins and their ligands, the disintegrin domain of spermatogenic cell-specific plasma membrane proteins called ADAMs. We have analyzed the temporal gene expression of selected ADAMs in intact fetal, early postnatal, and pubertal rat testis and Sertoli-spermatogenic cell cocultures by reverse transcriptase-polymerase chain reaction, in situ hybridization, and immunocytochemistry. We report that several ADAM transcripts are expressed in fetal, neonatal, and prepubertal testes. Cyritestin (ADAM3), ADAM5, ADAM6, and ADAM15 are expressed in day 17 fetal testes. In contrast, no expression of fertilin alpha (ADAM1) and fertilin beta (ADAM 2) was detected in fetal testes. Fertilin beta gene expression starts after postnatal day 2, subsequent to the expression of fertilin alpha, which occurs on postnatal day 1. After postnatal day 2, all the indicated ADAMs, including the fertilin alpha and fertilin beta, continue to be expressed. Transcripts of spermatogenic cell-specific fertilin alpha, fertilin beta, ADAM3, and ADAM5 were detected during the coculture of PSG with Sertoli cells for up to 72 hr after plating. The presence of fertilin beta mRNA and protein in cocultured PSG was visualized by in situ hybridization and immunocytochemistry, respectively. These observations indicate that PSG in coculture with Sertoli cells provide a suitable approach for analyzing cell-cell adhesive responses involving spermatogenic cell-specific ADAMs.

Impaired Gap Junction Connexin43 in Sertoli Cells of Patients with Secretory Azoospermia: A Marker of Undifferentiated Sertoli Cells

Gap junctions are intercellular channels formed of connexins (Cx) at appositional plasma membranes between adjacent cells that have been involved in the control of cell proliferation and differentiation. Altered Cx expression is implicated consistently in several human diseases and in tumorigenesis. Although Cx43 plays a critical role in Sertoli cell control of spermatogenesis, there is no evidence of its altered expression in human testicular pathologies. We show here that Cx43 mRNA expression was significantly reduced in testes of infertile patients with secretory azoospermia (p < 0.05) compared with testes displaying normal spermatogenesis (excretory azoospermic patients). In Sertoli cell-only syndrome, in situ hybridization and immunohistochemistry analyses indicated that Cx43 mRNA and protein were undetectable in Sertoli cells but were still present in the interstitial compartment. In a rat model of Sertoli cell-only syndrome, the lack of Cx43 in Sertoli cells was associated with an impairment of gap junction intercellular communication between adjacent Sertoli cells. These results reveal that Cx43 mRNA and protein expression are markedly impaired in Sertoli cells of infertile patients. This defect could be a new functional marker of undifferentiated Sertoli cells and could be related to the increased risk of testicular cancer recently described in the population of infertile men.

Expression of claudin-1 in mouse testis.

In testis, tight junctions (TJs) between adjacent Sertoli cells are important for the formation of the blood-testis barrier (BTB) and crucial for spermatogenesis. The present study aimed to find postnatal changes in the expression of claudin-1, one of the TJ genes in mouse testis. By semiquantitative RT-PCR, it was found that claudin-1 expression in testis increased up to a peak at 10 days after birth and decreased thereafter. Western blot analysis showed abundant expression of 21-kDa protein in testis, lung, and brain from the adult mouse. The developmental change in the expression of claudin-1 protein in testis coincided with that from the RT-PCR. Testosterone treatment significantly increased claudin-1 expression in immature Sertoli cells, suggesting the possible regulation of claudin-1 expression by androgen in mouse Sertoli cells. Claudin-1 expression appears to be developmentally regulated in the mouse testis.

Actin-related intercellular adhesion junctions in the germinal compartment of the testis in the hagfish ( Eptatretus stouti) and lamprey ( Lampetra tridentatus)

The germinal epithelium of male vertebrates consists of Sertoli cells and spermatogenic cells. Intercellular junctions formed by Sertoli cells assume critical roles in the normal functions of this epithelium. While Sertoli cell junctions have been well characterized in mammals, similar junctions in nonmammalian vertebrates have received little attention. We examined the intercellular junctions found within the germinal epithelium of the hagfish ( Eptatretus stouti) and lamprey ( Lampetra tridentatus). Ultrastructurally, Sertoli cells were seen to form filament-associated junctions in both species. Adjacent Sertoli cells formed microfilament-related junctions near their apices. Filaments of these junctions were arranged in loose networks and were not associated with cisterns of endoplasmic reticulum. In fixed, frozen sections of hagfish testis, similar areas labeled with rhodamine phalloidin, indicating the filament type is actin. In the lamprey, desmosomes were observed immediately below the microfilament-related junctions. In appearance and location, the Sertoli cell junctions observed in these species resembled those of the typical junctional complex of other epithelial cell types. No junctions were observed between Sertoli cells and elongating spermatids. In the hagfish, but not the lamprey, an additional zone of microfilaments occurred near the base of Sertoli cells in areas of association with the basal lamina. Our observations are consistent with the proposal that the unique forms of intercellular attachment found in the testes of higher vertebrates evolved from a typical epithelial form of intercellular junction.

Hereditary defects in both germ cells and the blood-testis barrier system in as-mutant rats: evidence from spermatogonial transplantation and tracer-permeability analysis.

The rat mutant allele as is located on chromosome 12. Homozygous (as/as) males show arrested spermatogenesis, mainly at the pachytene spermatocyte stage. It is not clear whether this defective spermatogenesis is caused by a failure in a somatic cell component that supports spermatogenesis or in the germ cell itself. Spermatogonial transplantation was performed to identify the genetically defective site in the as/as testis. In experiment 1, germ cells collected from as/as testes were transplanted into the testes of immunodeficient mice and normal rats. In experiment 2, normal rat germ cells were transplanted into as/as testes. The results of experiment 1 showed arrest of spermatogenesis at the pachytene spermatocyte stage, accompanied by a characteristic morphological feature, i.e., the formation of inclusion-like bodies in the cytoplasm, in both rat and mouse recipients. These results revealed the intrinsic effect of the mutant gene(s) on germ cells. In experiment 2, no restoration of spermatogenesis was detected in the recipient testes despite thorough histological examination. These results suggest that defects in a somatic cell component in as/as testes prevent the donor germ cells from colonizing and regaining their spermatogenetic ability. When the seminiferous epithelium of the as/as testis was examined by electron microscopy, no morphological abnormalities, including the formation of ectoplasmic specializations between adjacent Sertoli cells, were observed in the somatic cell components. However, when cytochrome c was applied as a tracer material, it penetrated the tight junctions between the Sertoli cells, indicating dysfunction of the blood-testis barrier in the as/as testis. The lack of restoration of spermatogenesis in the as/as testis after transplantation of normal germ cells may have been caused by the unfavorable environment in the seminiferous epithelium resulting from the incomplete barrier system between adjoining Sertoli cells. The gene(s) at the as locus may have a role in both germ cell differentiation and the establishment of the blood-testis barrier.

Spermatogenesis in vitro: a model for studying gap junctions in mammal seminiferous epithelium

Objective: Sertoli cells in vivo form compartments to allow germ cells proliferation and differentiation in a specific microenvironment. These compartments are created between adjacent Sertoli cells by the blood testis barrier (BTB), with specialized intercellular tight junctions, complexly intermingled with Gap junctions. The Gap junction protein Connexin 43 is one of the predominant connexins (Cx) in the testis (Cell Tissue Res., 1999; 298:113–121). Cx43 KO present a rapid arrest of germ cells development (Biol. Reprod., 1999; 60:1263–1270), while KI replacement of C-terminal region in Cx 43 produces Sertoli cells only syndromes (Curr. Biol., 2000; 10:1083–1091). Thus, Cx 43 seems to play a major role in the control of spermatogenesis. The objectives of this study were to study the distribution pattern of the testis gap junctions protein Cx 43 in the model devised in our laboratory to culture seminiferous tubules segments in vitro.

Osteopontin expression and regulation in the testis, efferent ducts, and epididymis of rats during postnatal development through to adulthood.

Osteopontin (OPN), a multifunctional phosphoprotein found in both hard and soft tissues, was examined in the male reproductive tract. The expression and regulation of OPN in the rat testis, efferent ducts, and epididymis was examined during postnatal development through to adulthood using immunocytochemistry at the light- and electron-microscopic level. Immunoblot analysis revealed a major 30-kDa band for epididymal tissue and a major 60-kDa band for the testis. In the testis, immunostaining of OPN was noted in early germ cells from spermatogonia to early pachytene spermatocytes, suggesting a role for OPN as an adhesive protein binding these cells to the basement membrane and adjacent Sertoli cells. Nonciliated cells of the efferent ducts expressed OPN, whereas a cell- and region-specific distribution of OPN was observed in the epididymis. Reactivity of OPN in the apical region of the cell corresponded to labeling of microvilli, small endocytic vesicles, and endosomes, where OPN may serve to remove calcium from the epididymal lumen and, thus, prevent mineral accumulation and subsequent decrease in sperm fertility. Regulation and postnatal studies revealed that circulating androgens regulate OPN expression in principal cells of the epididymis only. Taken together, the data reveal cell- and region-specific expression and regulation of OPN in the epididymis.