Cathepsin A is expressed in a cell- and region-specific manner in the testis and epididymis and is not regulated by testicular or pituitary factors.

The epithelial cells of the testis are involved in the production, differentiation, and sustenance of sperm, and those of the epididymis play a major role in sperm maturation, protection, and storage. These tissues express various proteins that respond differently to androgens. Cathepsin A is a multifunctional lysosomal carboxypeptidase that also functions as a protective and an activator protein for neuraminidase and beta-galactosidase. In this study, cathepsin A was immunolocalized by light and electron microscopy using a polyclonal affinity-purified antibody on the testis and epididymis of normal, orchidectomized with or without testosterone supplementation, efferent duct-ligated, and hypophysectomized adult rats. In normal rats, cathepsin A expression was noted in lysosomes of Sertoli and Leydig cells but not in germ cells of the testis, as well as nonciliated cells of the efferent ducts. In the epididymis, a cell- and region-specific distribution of cathepsin A was noted. In experimentally treated animals, no changes were noted in the expression of cathepsin A. Immunolabeling of tissues examined at the electron microscopic level revealed that lysosomes were reactive. These data indicate cell- and region-specific expression of cathepsin A in cells of the testis and epididymis and also indicate that cathepsin A expression is not regulated by testicular or pituitary factors.     

Abnormal zinc metabolism in unilateral maldescended testes of a mutant rat strain

The status of zinc in a mutant rat strain with heritable maldescended testes was examined. In rats with unilateral maldescended testis, the ectopic testis consistently had decreased zinc content (121.0 +/- 23.0 micrograms zinc/g dry wt), while the eutopic testis had zinc content similar to that of normal rats (182.0 +/- 5.0 micrograms zinc/g dry wt). Uptake of zinc by the ectopic testis was comparable to normal. Sephadex gel chromatography showed greatly reduced zinc content of one of the endogenous zinc binding fractions with a mol wt of 30,000 of cytosol of the ectopic testis in spite of a near normal protein content. Incorporation of zinc-65 into this fraction was also shown to be greatly reduced in ectopic testis. Sodium dodecylsulphate-polyacrylamide gel electrophoresis demonstrated that a protein of 23,000 Da was greatly reduced in quantity. This 23-kDa protein in ectopic testis may play a role in reduced testicular function of the ectopic testis.     

Expression of claudin-1 and -11 in immature and mature pheasant (Phasianus colchicus) testes

The expression of claudin-1 and -11, tight junctions (TJs) proteins was examined in immature and adult pheasant (Phasianus colchicus) testes. Claudin-1 and -11 cDNA were highly similar to those of human, mice, and chicken. Claudin-1 mRNA and protein (21 kDa) levels in immature testes were higher than those of adult testis. In immature testes until 6 weeks of age, Claudin-1 was found at contacts between adjacent Sertoli cells and between Sertoli cells and germ cells. In adult testis, Claudin-1 was found in early spermatocytes migrating the blood testis barrier (BTB). Blood vessels were positive for claudin-1. Claudin-11 mRNA and protein (21 kDa) increased during adulthood development of testis. In immature testis, Claudin-11 was found in apicolateral contacts between adjacent Sertoli cells, indicating its involvement in cell adhesion in immature testis. In adult testis, strong wavy Claudin-11 immunoreactivity was parallel to basal lamina at the basal part of seminiferous epithelium, indicating that Claudin-11 at the inter-Sertoli TJs may act as a structural element of the BTB. Weak Claudin-1 and -11 immunoreactivity at contacts between Sertoli cells to elongating/elongated spermatids, meiotic germ cells, and basal lamina suggests that they also participate in the cell-cell and cell-extracellular matrix adhesion in pheasant testis. Testosterone increased claudin-11 mRNA in testis organ culture and Sertoli cell primary culture, suggesting positive regulation of claudin-11 gene by androgen in Sertoli cells of pheasant testis. This is the first report on the claudins expression at BTB in avian testis.     

Feeding of a low-zinc diet lowers the tissue concentrations of alpha-tocopherol in adult rats

Previous work has shown that a low dietary intake of zinc for a short duration significantly lowers the lymphatic absorption of α-tocopherol (αTP) in adult male rats. The present study investigated whether the nutritional status of zinc is critical in maintaining the tissue levels of the vitamin. One group of rats was fed an AIN-93G diet containing 3 mg zinc/kg (low zinc, LZ) and the other was fed the same diet but containing 30 mg zinc/kg (adequate zinc, AZ). Food intakes between groups were matched by feeding two meals per day. At 6 wk, the body weights (356±8 g) of LZ rats reached 98% those (362±10 g) of AZ rats. Feeding of the LZ diet for 6 wk significantly lowered the concentrations of both αTP and zinc in the liver, kidney, heart, testis, and brain. No consistent relationships between αTP and zinc concentrations were observed in other tissues such as spleen, lung, gastrocnemius muscle, and retroperitoneal fat tissues. The concentrations of αTP in the liver, testis, brain, spleen, heart, and kidney were significantly correlated with the tissue concentrations of zinc. The LZ diet slightly but significantly increased the total lipid contents (mg/g) of liver, kidney, heart, and spleen. However, the tissue levels of phospholipid (μmol/100 mg lipid) in the heart, lung, testis, and spleen were decreased significantly in LZ rats. These findings indicate that low zinc intake results in a pronounced decrease in the animal’s αTP status under the conditions of matched food intakes, body weights, and feeding patterns. The lower tissue levels of αTP may explain in part the compromised antioxidant defense system and increased susceptibility to oxidative damage observed in zinc deficiency.     

Regional differences in the testosterone to dihydrotestosterone ratio in the epididymis and vas deferens of adult mice

The concentrations of testosterone and dihydrotestosterone (DHT) were measured in the testis and in different segments of the epididymis and vas deferens of adult mice. There were marked regional variations in the concentrations of testosterone and DHT from the testis to the caudal part of the vas deferens. In the testis, testosterone was the predominant androgen (364 +/- 90 ng/g) while DHT was weakly represented (8 +/- 2 ng/g). Qualitative and quantitative changes occurred in epididymis: DHT was the main steroid in the caput (29.3 +/- 2.7 ng/g) and corpus (33.1 +/- 4.4 ng/g) while testosterone and DHT were in similar quantities in the cauda (18.6 +/- 2.6 and 19.0 +/- 2.7 ng/g, respectively). The proximal region of the vas deferens contained higher amounts (71.4 +/- 8.0 ng/g) of androgens (testosterone + DHT) than did the caput epididymidis (39.1 +/- 3.3 ng/g). Testosterone was the predominant androgen in each part of the vas deferens and its concentrations decreased from the proximal (64.5 +/- 7.5 ng/g) to the caudal (26.9 +/- 4.3 ng/g) region. Castration and section of the efferent ducts of the testis showed that the epididymis received testosterone essentially via the blood supply and that epididymal DHT was produced locally from circulating testosterone.     

Expression of constitutively active Notch1 in male genital tracts results in ectopic growth and blockage of efferent ducts, epididymal hyperplasia and sterility

The Notch signaling pathway is involved in a variety of developmental processes. Here, we characterize the phenotypes developing in the reproductive organs of male transgenic (Tg) mice constitutively expressing the activated mouse Notch1 intracellular domain (Notch1(intra)) under the regulatory control of the mouse mammary tumor virus (MMTV) long terminal repeat (LTR). Tg expression was detected in testis, vas deferens and epididymis by Northern blot analysis. In situ hybridization with a Notch1-specific probe lacked sensitivity to detect expression in normal-appearing cells, but demonstrated expression in hyperplastic epithelial cells of the vas deferens, epididymis and efferent ducts. Tg males from three independent founder lines were sterile. Histological analysis of reproductive organs of young Tg males (postnatal ages 8 and 21) showed no difference compared to those of non-Tg males. In contrast, in adult Tg mice from day 38 onwards, the efferent ducts, the vas deferens and most epididymal segments revealed bilateral epithelial cell hyperplasia with absence of fully differentiated epithelial cells. Electron microscopy confirmed the uniformly undifferentiated state of these cells. Immunohistochemistry with anti-PCNA antibody also revealed enhanced proliferation of Tg epididymis. In adult Tg testis, the different generations of germ cells of seminiferous tubules appeared normal, although some tubules were highly dilated and revealed an absence of early and/or late spermatids. The epithelial cells of the Tg tubuli recti and rete testis were not abnormal, but the rete testis was highly dilated and contained numerous spermatozoa, suggesting a downstream blockage. Consistent with a blockage of efferent ducts often seen at the rete testis/efferent duct interface, spermatozoa were absent in epididymis of all adult Tg mice and in all highly hyperplastic efferent duct tubules of these Tg mice. Such a blockage was visualized by injection of Evans blue dye into the rete testis lumen. Finally, the presence of ectopic hyperplastic efferent duct tubules was observed within the testicular parenchyma itself, outside their normal territory, suggesting that Notch1 signaling is involved in the establishment of these borders. This phenotype seems to represent a novel developmental defect in mammals. Together, these results show that constitutive Notch1 signaling significantly affects the development of male reproductive organs.     

Morphology and immunolocalization of aquaporins 1 and 9 in the agouti (Dasyprocta azarae) testis excurrent ducts

This study investigated the morphology and immunoexpression of aquaporins (AQPs) 1 and 9 in the rete testis, efferent ducts, epididymis, and vas deferens in the Azara’s agouti (Dasyprocta azarae). For this purpose, ten adult sexually mature animals were used in histologic and immunohistochemical analyses. The Azara’s agouti rete testis was labyrinthine and lined with simple cubic epithelium. Ciliated and non-ciliated cells were observed in the epithelium of the efferent ducts. The epididymal cellular population was composed of principal, basal, apical, clear, narrow, and halo cells. The epithelium lining of vas deferens was composed of the principal and basal cells. AQPs 1 and 9 were not expressed in the rete testis. Positive reaction to AQP1 was observed at the luminal border of non-ciliated cells of the efferent ducts, and in the peritubular stroma and blood vessels in the epididymis, and vas deferens. AQP9 was immunolocalized in the epithelial cells in the efferent ducts, epididymis and vas deferens. The morphology of Azara’s agouti testis excurrent ducts is similar to that reported for other rodents such as Cuniculus paca. The immunolocalization results of the AQPs suggest that the expression of AQPs is species-specific due to differences in localization and expression when compared to studies in other mammals species. The knowledge about the expression of AQPs in Azara’s agouti testis excurrent ducts is essential to support future reproductive studies on this animal, since previous studies show that AQPs may be biomarkers of male fertility and infertility.     

Zinc metabolism in testicular feminization and surgical cryptorchid testes in rats

Rats with testicular feminization (Tfm) had been reported to have a testis specific zinc deficiency. In this report it is demonstrated that this organ specific zinc deficiency was not corrected by intraperitoneal zinc administration. Normal littermates on the other hand showed a positive testicular response to zinc administration. The increased testicular zinc level in control animals returned to normal 1 week after the zinc treatment probably due to the fast turnover of this element in the testis. Not only surgically induced cryptorchidism but also surgical cryptorchidism and epididymodeferentectomy (to simulate Tfm conditions in normal adult rats) caused a drastic reduction in testicular zinc level. Unlike in Tfm rats, however, the decrease in zinc content in operated animals was not accompanied by a corresponding decrease in alkaline phosphatase activity. Zinc concentration and alkaline phosphatase activity in plasma or other organs were not affected by the surgical procedure. The testicular copper content in the operated animals was higher than that of the unoperated controls.     

Effects of cryptorchidism and orchidopexy on deoxyribonucleoside-activated nucleotidase (DAN) in the rat testis

1. Unilateral cryptorchidism was associated with an increase in specific DAN activity in the abdominal testis of adult rats. 2. In the scrotal, immature testis, there was an age-dependent decrease in specific DAN activity similar to that normally seen in rats. This decrease was less pronounced in the abdominal testis of unilaterally cryptorchid rats. 3. Total DAN activity in the abdominal testis showed a decrease in both immature and adult rats, when compared to that of the scrotal testis. 4. Orchidopexy following cryptorchidism in adult rats, resulted in complete restoration of DAN activity in spite of poor recovery of spermatogenesis.     

Expression of nerve growth factor (NGF), and its receptors TrkA and p75 in the reproductive organs of the adult male rats

Immunolocalization of nerve growth factor (NGF) and its receptors, TrkA and p75 in the reproductive organs of adult male rats was investigated. Sections of the testis, efferent duct, epididymis, deferent duct, seminal vesicle, coagulating gland and prostate of adult male rats were immunostained by the avidin-biotin-peroxidase complex methods (ABC). NGF was expressed in Leydig cells, primary spermatocytes and pachytene spermatocytes in the testis. TrkA only immunoreacted to elongate spermatids and p75 showed positive immunostaining in the Sertoli cells, Leydig cells, the pachytene spermatocytes and elongate spermatids. Immunoreactions for NGF and its two receptors were detected in epithelial cells of efferent duct, deferent duct and epididymis. In addition, immunoreactions for NGF and its two receptors were also observed in columnar secretory epithelium lines of the seminal vesicles, prostate and coagulating gland. These results suggest that NGF is an important growth factor in gonadal function of adult male rats.     

Appearance of the rat testicular receptor for calcitriol (1,25-dihydroxyvitamin D3) during development

In the present study we have examined the developmental changes in the concentration of receptors for calcitriol in high-salt cytosol from the rat testis. Receptors for calcitriol were undetectable (less than 0.4 fmol/mg protein) until day 24, after which there was a rapid increase to reach adult levels (6-8 fmol/mg protein) between day 50-60. The lack of receptors in high-salt cytosol from the immature rat testis is not due to degrading enzymes, since cytosols prepared from the combination of equal volumes of testis homogenates from immature and adult rats had binding levels exactly half of that found in "adult controls". Furthermore, the increase in specific binding of [3H]calcitriol during development is due to an increase in the number of receptor sites, and is not due to a change in the apparent affinity of the receptors (Kd approximately equal to 1 X 10(-11) M at 0 degrees C). These results may explain why we previously were unable to demonstrate calcitriol receptors in cultured Sertoli cells and peritubular cells isolated from 19-day old rats. Furthermore, they indicate that calcitriol may be of minor importance for testicular function in the immature rat. The role of calcitriol in the pubertal and adult testis remains to be established.     

Effect of seasonal changes in Leydig cell number on the volume of smooth endoplasmic reticulum in Leydig cells and intratesticular testosterone content in stallions

Testes from 47 adult (4-20 years) stallions obtained in November-January (non-breeding season) and 41 adult stallions obtained in May-July (breeding season) were perfused with glutaraldehyde, placed in osmium and embedded in Epon 812. Percentage Leydig cell cytoplasm or nuclei in the testis was determined by point counting of 0.5 micron sections under bright-field microscopy. Testes from 6 randomly selected horses per season were processed for electron microscopy. The volume (ml) of SER/testis was calculated from the % SER in the cytoplasm % Leydig cell cytoplasm, and parenchymal volume. Number of Leydig cells was calculated from the % nuclei, parenchymal volume, histological correction factor, and volume of single nucleus. Intratesticular testosterone content was determined from the contralateral testis by radioimmunoassay. The volume of SER/g and testosterone/g tended to be higher in the breeding than non-breeding season. Leydig cell number/g, volume of SER/testis, testosterone/testis, and Leydig cell number/testis were significantly greater in the breeding than in the non-breeding season. Volume of SER/testis and testosterone/testis were related significantly to the cell number/testis, and SER/testis was related (P less than 0.05) to testosterone/testis. These results emphasize the importance of seasonal changes in the number of Leydig cells on the amount of SER available to produce testosterone and on testosterone content/testis in the stallion.     

Effects of FSH on cyclic nucleotide accumulation in testes of rats of various ages.

The accumulation of cyclic AMP in rat testis in response to FSH is much greater in immature rat compared to adult. Addition of 3-isobutyl-1-methylxanthine (MIX), a potent in vitro inhibitor of cyclic nucleotide phosphodiesterase activity, potentiates the effects of FSH but does not restore the cyclic AMP response of adult rat to that of immature rat testis. The apparent substrate affinities for cyclic AMP and cyclic GMP hydrolysis are similar in immature and mature testis. Developmental changes in cyclic AMP and cyclic GMP phosphodiesterase specific activities fail to account for the reduced responsiveness of adult testis to FSH. These results suggest a defect in synthetic rather than hydrolytic mechanisms in FSH-responsive cells.     

Vascularization of ovaries from golden hamsters following implantation into the chick chorioallantoic membrane

In order to study the angiogenic activity, entire ovaries of immature or adult golden hamsters in different functional stages were implanted into the chick chorioallantoic membrane (CAM). Ovaries or other organs (heart, lung, liver, spleen, kidney, adrenal gland, testis) were applied during days 9-14 of incubation. Histological serial sections and ultrastructural studies displayed vascularization from the host to the ovary. Vascularization was marked for unstimulated immature ovaries compared to superstimulated immature ovaries or adult ovaries obtained during the period of proestrus-estrus. When no vascularization developed, grafted ovaries of immature or adult animals induced a mild erosive reaction of the CAM. In contrast, few other implanted organs obtained from immature animals caused a response with vascularization which was absent in grafted organs from adult hamsters. The erosive reaction of the CAM was rare, but the ulcerative response accompanied by infiltrating eosinophils was frequently seen both in immature and adult animals. These results may indicate that angiogenetic activity of the ovary is not the sole cause for vascularization of grafts. Other factors such as tissue death or a low histocompatibility barrier are likely to be involved.     

Follitropin receptors in rat testis. Characterization with enzymatically 125I-labeled human follitropin.

The interaction between enzymatically radioiodinated human follitropin and the follitropin receptors in testis homogenate was investigated in immature and adult rats. The 125I-labeled human follitropin exhibited high binding activity with specific binding of up to 17% in the presence of an excess of testis homogenate. Approx. 50% of the bound hormone could be eluted at pH 5, and the receptor purified tracer exhibited a 3.6-fold increase in binding activity when compared with the original tracer preparation. Quantitative analysis of equilibrium binding data was performed with corrections for the measured specific activity and maximum binding activity of the tracer hormone. The equilibrium association constants (Ka) determined 24 degrees C were not significantly different in immature and adult rat testis, and the mean value for Ka was 3.9 . 10(9) M-1. At 37 degrees C, the Ka value obtained using immature rat testis was 1.3 . 10(10) M-1. The association of 125I-labeled human follitropin with immature rat testis homogenate was time and temperature dependent. In the presence of an excess of unlabeled hormone, 30--60% of the preformed hormone . receptor complex was dissociated after 24 h incubation. A specific and sensitive radioligand-receptor assay for follitropin was developed using immature rat testis homogenate. The minimum detectable dose of purified human follitropin was 0.6 ng, and human urinary and pituitary follitropin, ovine follitropin and pregnant mare serum gonadotropin reacted in the assay with equivalent slopes. The potencies of highly purified pregnent mare serum gonadotropin and highly purified human follitropin were similar in the radioligand-receptor assay, consistent with the follitropin bioactivity of the equine gonadotropin.     

Expression of p57 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.     

Immunohistochemistry of the cytoskeleton in the excurrent ducts of the testis in birds of the Galloanserae monophyly

The presence, location and degree of immunoexpression of various microfilament (MF) and intermediate filament (IF) systems (actin, cytokeratins, desmin, vimentin) were studied in the excurrent ducts of the testis in sexually mature and active galliform (Japanese quail, domestic fowl, turkey) and anseriform (duck) birds. These proteins were variably expressed between the epithelia and periductal tissue (periductal smooth muscle cell layer and interductal connective tissue) types and between species. Variable heterogeneous co-expression of filament systems was also found in the various duct epithelia and periductal tissue types: co-expression of filament systems was the rule rather than the exception. In the duck, neither vimentin nor cytokeratin was present in any of the tissues, whereas actin and desmin (absent in the rete testis) were co-expressed in the efferent ducts and epididymal duct unit (comprising the ductus conjugens, ductus epididymidis and ductus deferens). Actin, desmin and vimentin were generally co-expressed in the rete testis, efferent ducts and epididymal duct unit of the quail, domestic fowl and turkey, with vimentin being more strongly immunoreactive than actin and desmin in the epididymal duct unit, but more weakly immunoexpressed in the efferent ducts. Cytokeratin was present and co-expressed with actin, desmin and vimentin in the rete testis, efferent ducts and epididymal duct unit of the domestic fowl and turkey, but not in the quail and duck. The periductal smooth muscle cell layer and interductal tissue co-expressed actin, desmin and vimentin variably in all birds. Luminal spermatozoa of both the turkey and duck were immunonegative for all protein systems, whereas those of the quail and domestic fowl co-expressed actin, desmin and vimentin moderately or strongly. The tissues of the reproductive tract of male birds thus contain cytoskeletal protein systems that are variably but mostly co-expressed and whose contractile ability appears necessary and sufficient for transportation through the various excurrent ducts of the voluminous testicular fluid and its high sperm content, characteristic features of male avian reproduction.