Postnatal expression and androgen regulation of HOXBES2 homeoprotein in rat epididymis

The multifunctional and androgen-regulated epididymis is known to provide a conducive microenvironment for the maturation and storage of mature spermatozoa. HOXB2 homeodomain-containing epididymis-specific sperm protein (HOXBES2), a molecule first reported by our group, exhibits cell- and region-specific expression. It was found in the cytoplasm of the principal epithelial cells with maximum in the distal segments of the rat epididymis. The present study was undertaken to determine whether HOXBES2 expression is regulated by androgens and postnatal epididymal development. Toward this, the epididymis was disallowed access to circulating androgens either by chemical or biologic castration. In bilaterally orchidectomized animals, the levels of immunoreactive HOXBES2 declined to <5 % of those seen in sham-operated animals. Exogenous dihydrotestosterone (DHT) supplementation (250 microg/kg body weight) for 7 days restored the expression levels to >or= 90 % of that observed in intact animals. Ethylene dimethane sulfonate (EDS) administration completely abolished HOXBES2 expression in the epididymis, and supplementation with DHT or DHT + estradiol for 10 days re-established HOXBES2 expression to near normalcy. However, in the estradiol alone-supplemented EDS-treated group, HOXBES2 remained undetected. The unaltered HOXBES2 expression following efferent duct ligation suggested that HOXBES2 is not critically dependent on testicular factors. During postnatal development, protein expression in the epididymis begins to appear from day 40 and 50 and increased from day 60 onward, coinciding with the mature levels of circulating androgens and the well-differentiated epididymis. Thus, the data obtained from this study suggests that HOXBES2 expression could be regulated by androgens, and its expression level is closely associated with the postnatal development of the epididymis.     

Structural features of cultured epithelial cells from the adult rat epididymis

Epithelial cells isolated from the caput epididymidis of adult rats were placed in primary culture and examined daily for ten days for changes in external anatomy, reorganization of cytoskeletal components, maintenance of characteristic cytoplasmic features, and response to media formulated to minimize nonepithelial cell proliferation. Significant cell attachment to the substrate began after the first 24 hours of culture. After attachment, the cells underwent a progressive flattening and became closely applied to the substrate. This was accompanied by a redistribution of microvilli on the cell surface and a reorganization of cytoskeletal elements within the cell. After flattening, the cultured cells displayed an extensive array of 10-nm filaments which were associated with the desmosomes attaching adjacent cells. Immunofluorescence studies demonstrated that these were keratin-containing intermediate filaments and 2-D gel electrophoresis of intact cells and cell cytoskeletons revealed that a family of "keratin-like" polypeptides were major components of the cells. Epithelial cell attachment, morphology, and maintenance in the primary culture were unaffected by D-valine, cytosine arabinoside, or both; however, these agents, either individually or in combination, reduced significantly the number of cells incorporating 3H-thymidine. These data show that isolated epithelial cells retain some differentiated structural features that characterize the intact cell and that enriched cultures of epithelial cells can be maintained under conditions where fibroblast proliferation is inhibited.     

Hormonal regulation of specific proteins in the rat epididymis

In the adult caput epididymidis, three specific prealbumin proteins (B2, B3, and B4) could be visualized by polyacrylamide gel electrophoresis. Castration caused the disappearance of B2 and B4, and B3 became faint. Androgen administration to castrated rat restored the protein pattern to normal. In the cauda epididymidis of the control rat, four prealbumin proteins (B1-B4) were seen of which only B3 was evident after castration. The dose of androgen administered was insufficient to restore the protein pattern of the cauda epididymidis. The caput epididymidis thus appears to have a lower androgen requirement than the cauda in regard to its protein pattern.     

Maturation of sperm volume regulation in the rat epididymis

Sperm maturation in the epididymis may involve differences between mature and immature spermatozoa in their volume regulatory osmolyte response. Spermatozoa obtained from the rat caput and cauda epididymidis were examined for their ability to regulate volume after transfer from in situ epididymal osmolality （measured to be 343 ± 13 and 365 ± 19 mmol kg^-1, respectively） to that of the female tract in single- and multiple-step protocols. Cells withstood the single-step treatment better than the multistep protocol. Sperm volume estimates by flow cytometric measure- ments of forward scatter of cells with intact head membranes was more sensitive than those by assessing cell coiling microscopically. At osmolalites below 210 mmol kg l both caput and cauda cells ruptured, limiting the use of flow cytometry. Above this critical value, the use of quinine showed that both caput and cauda cells could regulate volume, but cauda cells were the more effective. Of several organic osmolytes studied, myo-inositol, glutamate and KCl caused only temporary and slight swelling of spermatozoa cells in hypotonic medium. Spermatozoa of both maturities seemed to use potassium as the preferred osmolyte for regulating volume.     

Immunolocalization of CA II and H+ V-ATPase in epithelial cells of the mouse and rat epididymis

Acidification of the epididymal lumen has been suggested to play an important role in sperm functions; however, the cell types, pumps, and mechanisms involved have not been fully addressed. In this study, carbonic anhydrase II (CA II) and a 67-kd subunit of Neurospora crassa vacuolar proton adenosinetriphosphatase (H+ V-ATPase) pump were immunolocalized using light microscopy and electron microscopy (EM) in the epididymis of rats and mice. In both animals, narrow cells, identified in the initial segment and intermediate zone of the epididymis, contained numerous small vesicles in their apical region, often cup-shaped in appearance. In the mouse but not rat, these cells also possessed numerous cisternae of smooth endoplasmic reticulum, suggesting steroid synthesis; and cytoplasmic blebs of their apical cell surface, which appeared to detach, suggesting apocrine secretion. Anti-CA II antibody was immunocytochemically localized in the light microscope within narrow cells but not over any other cell types of the entire epididymis. Anti-H+ V-ATPase antibody was also localized in narrow cells of the initial segment and intermediate zone; as well as clear cells of the caput, corpus, and cauda regions. Using EM, gold particles for anti-CA II and H+ V-ATPase antibodies were noted in the apical region of narrow cells in relation to the numerous, small, cup-shaped vesicles. Although CA II was mainly located in the cytosol near these vesicles, H+ V-ATPase appeared on their delimiting membrane and on the apical plasma membrane of these cells. A similar distribution was noted for H+ V-ATPase in clear cells. The nature of the small vesicles of the apical region of narrow cells was examined with electron-dense fluid phase tracers that were introduced into the epididymal lumen. The tracers appeared within these vesicles and a few endosomes 1 hour after injection, suggesting that they contact the apical plasma membrane. Since these vesicles are also related to CA II and H+ V-ATPase, the data suggests that, as the site of proton production, the vesicles recycle to and from the apical cell surface, and in this way, deliver protons to the epididymal lumen for acidification. Clear cells and their expression of H+ V-ATPase may also serve in this function. In summary, both narrow and clear cells appear to be involved in luminal acidification, an activity that may be essential for sperm as they traverse and are stored in the epididymis.     

II. Characterization and development of the regional- and cellular-specific abnormalities in the epididymis of mice with beta-hexosaminidase A deficiency

Beta-hexosaminidase (Hex) is a lysosomal enzyme that exists as two isoenzymes: Hex A (subunit structure alphabeta) and Hex B (betabeta). Its presence in the testis and epididymis suggests important roles for Hex and its substrates in male fertility and reproductive functions. Disruption of the Hexa gene encoding the alpha-subunit of Hex has led to the generation of a mildly affected mouse model of human Tay-Sachs disease, allowing us the opportunity to analyze the effects of isolated Hex A deficiency on epithelial cellular morphology of the male reproductive tract. At 5 weeks and at 3, 5, and 12 months, the testes, efferent ducts and epididymides of Hex A-deficient (Hexa -/-) and wild-type (Hexa +/+) mice were perfuse fixed and analyzed by routine light and electron microscopy as well as with immunocytochemistry employing antibodies to lysosomal enzymes. In the testis, the seminiferous epithelium of Hexa -/- mice appeared comparable to that of wild-type mice in appearance and topographical arrangement of its cell types at all ages examined. Also, no differences were noted for the efferent ducts. In contrast, there were striking abnormalities in the epididymides of the mutant mice; however, the abnormalities were mainly restricted to the initial segment and intermediate zone. Principal cells of these regions at 5 weeks showed a dramatic increase in the number of lysosomes as compared with those from wild-type animals, and this progressed with increasing age. Furthermore, unlike the few small lysosomes present in wild-type mice, those of Hexa -/- mice were at times enlarged and often filled the supranuclear and basal regions of these cells. In the light microscope, large, dense cellular aggregates were noted at the base of the epithelium in the proximal initial segment that corresponded in the electron microscope to two different cell types, both of which increased in size with age. One aggregate was considered to belong to narrow cells on the basis of the presence of numerous cup-shaped vesicles characteristic of these cells; they appeared to be dislocated from the upper half of the epithelium. In the distal initial segment and intermediate zone, narrow cells were readily identified, but rather than being slender as in the control animals, they were greatly enlarged and filled with pale lysosomes in mutant mice. The second type of cellular aggregate noted in the proximal initial segment corresponded to halo cells. They contained numerous small and large lysosomes and small, Golgi-related, dense, core granules characteristic of halo cells. On the basis of the large size of these cells, they appeared to be actively internalizing substances from the intercellular space. In contrast, principal and clear cells of the caput, corpus, and cauda regions did not appear to show a significant increase in number or size of lysosomes as compared with those of wild-type animals. All structures identified as lysosomes in the various cell types were immunoreactive for cathepsin D. The present data thus reveal that isolated Hex A deficiency results in region- and cell-specific abnormalities in the epididymis but in no apparent abnormalities in the testis or efferent ducts. Specific roles for Hex A that cannot be compensated for by other isozymes of Hex appear to exist within lysosomes of epithelial cells predominantly of the initial segment and intermediate zone. Taken together, the results also suggest that the inability to degrade endocytosed substrates normally acted upon by Hex A in lysosomes of principal and narrow cells leads to their accumulation, eventual fusion, and increased size.     

Changes in the number, proliferation rates, and bcl-2 protein immunoexpression of epithelial and periductal cells from rat epididymis during postnatal development

To investigate 1) the correlation between the proliferative activity of epididymal epithelium plus myoid cells and the increase in the number of these cells and 2) the role of the basal epithelial cells in the renewal of epididymal epithelium, a quantitative evaluation of the proliferation of both epithelial cells and periductal myoid cells in the different epididymal regions (caput, corpus, and cauda) has been carried out during postnatal development of the rat by immunohistochemical evaluation of BrdU-labeling indices. These data were correlated with cell numbers and counted by the optical dissector method. The presence of bcl-2 protein was immunohistochemically detected and evaluated. No significant differences in BrdU indices were observed among epididymal regions in any stage studied. Cell proliferation decreased from the prepubertal period to adulthood in both epithelial and myoid cells in the three regions of the epididymis, suggesting a close relationship between epithelial and mesenchymal components. The numbers of both cell types were significantly higher in the caput than in the corpus and cauda in all stages studied, suggesting functional differences between regions. A negative linear correlation between proliferative activity and cell numbers was noted that might be related to regulation of the cell population size. Basal cells showed a lower proliferation rate than principal cells, but most of the immunoreactive bcl-2 protein, in pubertal and adult epididymides, was observed in basal cells. Therefore, these cells might comprise a low-proliferating and apoptosis-resistant population.     

Effect of testosterone on epithelial cell proliferation in the regressed rat epididymis

It is well established that testosterone plays a crucial role in maintaining the integrity of epididymal structure and function. However, the role of testosterone in restoring the cellular architecture of the regressed epididymis is not well known. The present study was undertaken to test the hypothesis that testosterone triggers the regressed epididymis by re-expanding existing cells and inducing cell proliferation. Testosterone-dependent epididymal morphology was evaluated in orchidectomized, regressed rats after initiation of treatment with testosterone. Besides that, the proliferative activity of epithelial cells in all regions of the epididymis of the orchidectomized, regressed rats was assessed at 1, 3, 7, and 28 days after testosterone replacement. Epithelial cell proliferation decreased after testosterone withdrawal and increased following testosterone administration. We found that bromodeoxyuridine incorporation and proliferating nuclear antigen expression increased significantly 3 days after testosterone replacement in all regions of the regressed epididymis except in the initial segment. The highest mitotic activity was seen in the corpus epididymidis at 3 days postimplantation. Using specific markers for each cell type, we found no significant changes in the proportion of each cell type compared with the control. We observed labeled nuclei in all epithelial cell types in the control; however, principal cells were the major cell types that responded to testosterone after regression. These observations demonstrate that the mammalian epididymis is not a static tissue without any significant cell renewal, either under control conditions or when androgen exposure is altered, thus providing new insight in the role of androgen in restoration and maintenance of the architecture of the epididymis.     

Immunolocalization and regulation of cystic fibrosis transmembrane conductance regulator in the adult rat epididymis

Cystic fibrosis is the most common serious autosomal recessive condition in whites, and more than 95% of men with cystic fibrosis are infertile. The cystic fibrosis transmembrane conductance regulator (CFTR), a cyclic adenosine monophosphate (cAMP)-regulated chloride channel, has been localized in the efferent ducts; however, to our knowledge, its expression and regulation in the epididymis by testicular factors have not been examined. In the present study, these parameters were examined immunocytochemically by the light microscope with an anti-CFTR antibody in Bouin-fixed, paraffin-embedded control adult rat epididymides and both orchidectomized adult rats with or without testosterone supplementation and efferent duct-ligated rats sacrificed at different time points. In control animals, a thick dense band of immunoperoxidase reaction product was visualized over the apical plasma membrane of the principal cells but not their microvilli. The apical band was prominent only in the corpus and cauda regions. While there was no CFTR expression in basal cells, clear cells of the corpus and cauda regions showed a weak-to-moderate band of apical plasma membrane staining. An examination of orchidectomized, orchidectomized and testosterone, and efferent duct-ligated rats revealed that CFTR was no longer expressed as an intense band on the apical plasma membrane of the principal cells of the corpus and cauda regions. However, under these conditions, an intense apical/supranuclear reaction was noted in the form of small vesicular structures. Clear cells were unaffected by the different experimental treatments. Together, these data indicate that CFTR is expressed in a cell- and region-specific manner and that, while its synthesis in principal cells is not under the control of testicular factors, targeting to the apical plasma membrane is regulated by a testicular luminal factor.     

Postnatal differentiation of the enzymatic activity of the mouse epididymis

Dehydrogenase and hydrolase activities were assessed histochemically during postnatal development of the mouse epididymis. At birth the activities of various enzymes were demonstrable along the epithelium at the same intensity. Variations occurred in the intensity of enzyme activities in principal cells, leading to regional differentiation which progressed according to an ascending pattern from the distal part (2nd week) to the medial and proximal parts (3rd week). The proximal part reached its definitive differentiation at the 4th week when the 5 segments characteristic of the adult state were distinguishable. At the same time, 3 types of "apical cells" (narrow, prominent and mitochondria-goblet cells) in the proximal part and "clear cells" in the medial and distal parts showed higher activity of several enzymes (dehydrogenases, acid phosphatase, Ca2+-ATPase) than did adjacent principal cells. This histochemical data has led us to propose a model for epididymal cell differentiation in the mouse. The role of androgens in the development of those regional differences is discussed.     

Appearance of specific proteins in rat epididymis during postnatal development

In the immature 30-day-old caput and cauda epididymides of the rat, only a single prealbumin protein was visible. Additional epididymis-specific proteins appeared in the caput on day 45, coinciding with the entry of testicular fluid containing first wave of spermatozoa. In the cauda epididymides, additional proteins appeared only on day 50. These results indicate that the functional differentiation of caput precedes that of the cauda epididymidis.     

I. Abnormalities in cells of the testis, efferent ducts, and epididymis in juvenile and adult mice with beta-hexosaminidase A and B deficiency

Beta-hexosaminidase (Hex) is a lysosomal enzyme that exists as two major isoenzymes: Hex A (subunit structure, alphabeta) and Hex B (betabeta). The presence of Hex in the testis and epididymis suggests important roles for the enzyme and its substrates in male fertility and reproductive functions. Disruption of the Hexb gene encoding the beta-subunit of Hex has led to the generation of a mouse model of human Sandhoff disease that survives to adulthood, enabling us to analyze the effects of Hex A and Hex B deficiency on epithelial cellular morphology of the male reproductive tract. At 1 and 3 months of age, the testes, efferent ducts, and epididymides of Hex-deficient (Hexb -/-) and wild-type (Hexb +/+) mice were perfuse fixed and analyzed by routine light and electron microscopy (LM and EM, respectively) as well as with immunocytochemistry employing antibodies to lysosomal proteins. In the testis, the morphological appearance and topographical arrangement of the cell types of the seminiferous epithelium of Hexb -/- mice were similar to those of wild-type animals at both ages. Both Sertoli and germ cells appeared to be unaffected. However, at both ages, myoid cells and macrophages showed an increased number of lysosomes in their cytoplasm as compared with the number seen in controls. The epithelial cells of the efferent ducts also showed an accumulation of lysosomes that increased with age as compared with controls. Principal cells of the entire epididymis revealed an increase in the size and number of lysosomes at 1 month of age as compared with those of controls, and by 3 months, these lysosomes often filled the supranuclear and basal regions of the cells. Narrow cells of the distal initial segment and intermediate zone, normally slender cells showing several lysosomes, became greatly enlarged and entirely filled with lysosomes in Hexb -/- mice. Clear cells of the caput, corpus, and cauda regions also showed a progressive increase in the size and number of lysosomes with age as compared with controls; the clear cells of the mutant mice were often enlarged and at times bulged into the lumen. Some basal cells of each epididymal region in Hexb -/- mice were similar to controls at 1 and 3 months, showing few lysosomes, while others showed an accumulation of lysosomes. Lysosomes of all affected epithelial cells were of varying sizes, but many large ones were present, apparently resulting from lysosomal fusion. Although pale stained, their identification as lysosomes was confirmed by EM immunocytochemistry with anti-cathepsin D and anti-Hex A antibodies. Predominantly in the proximal initial segment, large, pale cellular aggregates were noted in the LM analysis at the base of the epithelium, which by EM analysis were identified as belonging to two different cell types, narrow cells and halo cells. Taken together, these data reveal an increase in the size and number of lysosomes in all epithelial cell types lining the efferent ducts and entire epididymis as well as in myoid cells and macrophages of the testis. In the light of data showing epididymal defects restricted predominantly to the initial segment in Hexa -/- (Hex A-deficient) mice, our data on the Hexb -/- mice demonstrate a major role for Hex that can be fulfilled by either Hex A or Hex B in the epididymis.     

Proliferation of epithelial cells of vas deferens and epididymis in young adult rabbits.

Epithelial proliferation of the vas deferens and epididymis was studied in rabbits after flash or continuous labeling with DNA precursor by autoradiography. A high proliferative capacity of the epithelium was found: after 2 days of labeling, 11% of labeled cells were found in the vas deferens and 20% in the ductus epididymidis.     

Androgenic regulation of novel genes in the epididymis

The epididymis is critically dependent on the presence of the testis. Although several hormones, such as retinoids and progestins, and factors secreted directly into the epididymal lumen, such as androgen binding protein and fibroblast growth factor, might play regulatory roles in epididymal function, testosterone （T） and its metabolites, dihydrotestosterone （DHT） and estradiol （E2）, are accepted as the primary regulators of epididymal structure and functions, with the former playing the greater role. To ascertain the molecular action of androgens on the epididymis, three complementary approaches were pursued to monitor changes in gene expression in response to different hormonal milieux. The first was to establish changes in gene expression along the epididymis as androgenic support is withdrawn. The second was to determine the sequence of responses that occur in an androgen deprived tissue upon re-administration of the two metabolites of T, DHT and E2. The third was to study the effects of androgen withdrawal and re-administration on gene expression in immortalized murine caput epididymidal principal cells. Specific responses were observed under each of these conditions, with an expected major difference in the panoply of genes expressed upon hormone withdrawal and re-administration; however, some key common features were the common roles of genes in insulin like growth factor/epidermal growth factor and the relatively minor and specific effects of E2 as compared to DHT. Together, these results provide novel insights into the mechanisms of androgen regulation in epididymal principal cells. （Asian J Androl 2007 July; 9： 545-553）     

Spatiotemporal expression patterns of natriuretic peptides in rat testis and epididymis during postnatal development

Natriuretic peptide (NP) family is composed of atrial, brain and C‐type NP (NPPA, NPPB and NPPC). Here, we aimed to investigate NP expression in testis and epididymis during postnatal development. NPPA expression was observed in gonocytes at prepubertal period but in only spermatocytes in pachytene and leptotene/zygotene stage at pubertal period. In prepubertal and pubertal periods, we detected NPPB expression in only Leydig cells. However, NPPC expression was detected in all of the gonocytes and Sertoli cells, spermatocytes and some interstitial cells in prepubertal and pubertal periods. In postpubertal and mature periods, NPPA and NPPB staining were detected in Leydig cells, elongated and round spermatids but not in spermatogonia and spermatocytes. However, we observed NPPC expression in all cells of the seminiferous tubules and Leydig cells in the postpubertal and mature periods. Epididymal epithelium showed intense NPPC expression during postnatal period but weak NPPA and NPPB expression in prepubertal and pubertal periods. The expression of three NPs in the testis significantly increased after puberty. In conclusion, puberty had a significant effect on NP expression in testis. Unlike NPPA and NPPB, expression of NPPC in all cells of the seminiferous tubule suggests that NPPC is effective in each step of spermatogenesis.     

Expression profile of Toll-like receptor 4 in rat testis and epididymis throughout postnatal development

Toll-like receptors (TLRs) belonging to pattern recognition receptors are involved in maintaining testicular and epididymal immune homeostasis. The purpose of the current study was to investigate TLR4 expression in rat testis and epididymis throughout postnatal development. Weak staining was detected in peritubular myoid cells and immature Sertoli cells while no staining was observed in gonocytes during prepubertal period. However, TLR4 expression began to appear in spermatocytes in pubertal period and gradually increased in spermatids. An intense staining was observed in steps 5–19 spermatids in post pubertal and mature periods. Similarly, TLR4 expression in the testes steadily increased from pubertal period to mature period. Puberty also caused a significant increase in TLR4 expression in epididymis. TLR4 expression in cauda epididymis was lower as compared to those of other epididymal segments. The majority of epididymal epithelial cells exhibited apical TLR4 expression, whereas basal cells showed intense intracytoplasmic immunoreaction. We detected an intense staining in epididymal smooth muscle cells. The expression levels of TLR4 showed dynamic changes in both spermatogenic cells, and entire testicular and epididymal tissues during postnatal development. These results suggest that TLR4 expression contributes not only to inflammation but also to the development of spermatogenic cells.