Differential expression of the mouse mitochondrial genes and the mitochondrial RNA-processing endoribonuclease RNA by androgens.

Using subtractive hybridization to identify genes that are androgen regulated in the mouse epididymis, a number of cDNAs were identified that represented mitochondrial genes including cytochrome oxidase c subunits I, II, and III, cytochrome b, NADH dehydrogenase subunit 5, a region of the displacement loop, and the 16S rRNA. Northern blot analysis of RNA from intact, castrate, or testosterone-replaced epididymides confirmed that these mitochondrial mRNAs as well as the rRNA were androgen regulated with a 2- to 5-fold reduction in expression observed after 4 weeks castration with partial to full recovery to precastrate levels upon 4 weeks of testosterone replacement. In contrast to the mitochondrial genes, the expression of the RNA component of the mitochondrial RNA-processing endoribonuclease (RNAase MRP), a nuclear factor which is thought to be involved in the regulation of mitochondrial DNA synthesis, increased in the epididymis upon castration and then returned to precastrate levels after testosterone replacement. An examination of other androgen-responsive tissues showed that mitochondrial gene expression was also regulated by androgens in the kidney. The RNAase MRP RNA levels, however, showed an increase after castration only in the reproductive tissues (epididymis, vas deferens, and seminal vesicle) and not in the kidney. No correlative increase in mitochondrial DNA levels was observed for any of the tissues. Finally, an analysis of various mouse tissues as well as the different regions of the epididymis revealed large differences in mitochondrial mRNA levels. While for most tissues the mRNA levels correlated with the mitochondrial DNA content, the levels of the RNAase MRP RNA did not. Taken together, these findings not only show the large variations in mitochondrial gene expression between tissues but also demonstrate that the expression of mitochondrial genes and ultimately mitochondrial function are androgen regulated in the epididymis and kidney.     

Immunochemical distribution and immunohistochemical localization of 20β-hydroxysteroid dehydrogenase in neonatal pig tissues

Immunochemical distribution of 20β-hydroxysteroid dehydrogenase (HSD) in neonatal pig tissues was investigated by Western blot analysis of the proteins reacting with anti-20β-HSD antibody. 20β-HSD was present in all organs investigated: brain, lung, thymus, submandibular gland, heart, liver, kidney, spleen, adrenal gland, testis, epididymis, prostate, vas deferens and seminal vesicle. In particular, high concentrations of 20β-HSD were detected in the testis, followed by the kidney and liver, by the [¹²⁵I]-protein A binding method. Immunohistochemical localization of the enzyme was achieved in paraffin sections of the testis, kidney, liver, epididymis, and vas deferens by the streptoavidin-biotin complex method. In the testis, very strong immunostaining was found only in interstitial Leydig cells, whereas the cells in seminiferous tubules, such as Sertoli cells and spermatogenic cells, were entirely negative. In the kidney, strong immunostaining was detected in epithelial cells of Henle's loop. The immunoreactive proteins were also localized in the hepatic lobules of the liver, tall columnar cells of the ductus epididymidis of the epididymis, and mucosal epithelium cells and muscularis of the vas deferens. These observations indicate that tissue distribution of 20β-HSD is similar to that of carbonyl reductase in the human and rat. However, the specific and abundant expression of 20β-HSD in testicular Leydig cells of the neonatal pig, which are concerned with the synthesis of androgens, suggests that 20β-HSD has a very important physiological role in testicular function during the neonatal stage.     

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.     

C-Fos在db/db自发性糖尿病小鼠颌下腺的表达

目的观察转基因糖尿病小鼠下颌下腺形态学改变与原癌基因C-fos蛋白表达的关系,为糖尿病的临床及基础研究提供依据。方法引进日本C57BL/ksj-db/ m表型正常隐性基因小鼠,其近亲交配所得纯合子后代,即为db/db(单基因遗传自然发病型)糖尿病小鼠。取3、4、6、8、10月龄db/db糖尿病小鼠及相应月龄的db/ m正常小鼠下颌下腺,行HE染色及SP免疫组化染色后进行图象分析,统计各组下颌下腺C-fos阳性表达的细胞数,观察其形态学改变。结果糖尿病小鼠下颌下腺腺泡萎缩,细胞缩小,形态不规则,排列不整齐。各月龄糖尿病小鼠颌下腺C-Fos阳性细胞明显低于相应对照组(P<0.01),且逐渐减少,呈下降趋势。结论db/db糖尿病状态下颌下腺细胞表达C-Fos蛋白明显降低,c-fos低表达可能与下颌下腺实质细胞的增殖减弱性形态学变化密切相关。     

Preliminary characterization, androgen-dependence and ontogeny of an abundant protein from mouse vas deferens

Polyacrylamide gel electrophoresis analysis revealed that the vas deferens of adult mouse contains a major protein. Mouse vas deferens protein is a basic glycoprotein with a molecular weight of 34,800 +/- 300. The protein represents 17 +/- 0.7% and 42 +/- 2.4% of soluble proteins from homogenate and luminal fluid respectively, an estimate based on densitometric scanning of polyacrylamide gels. The protein originated from the vas deferens since it was not detected in blood plasma or in sexual organs and it was still present after ligation of the epididymis. Changes in androgen status of the animal markedly affected the vas deferens protein. After castration a progressive decrease in the protein was observed and its relative percentage dropped to 2 +/- 0.4% after 45 days. The concentration of the protein returned to precastration levels after 2 weeks of testosterone treatment but oestradiol, progesterone and corticosterone were ineffective in this respect. The vas deferens protein was not synthesized in significant amounts until animals were 20 days old and its concentration increased rapidly from 20 to 30 days in concert with the pubertal increase of androgens in the vas deferens.     

Defective epidermal growth factor gene expression in mice with polycystic kidney disease

The C57BL/6J-cpk mouse has an inheritable form of polycystic kidney disease similar to the autosomal recessive disorder seen in humans. Between approximately 1 and 3 weeks of age, affected cpk mice develop numerous large cysts in the collecting tubule segment of kidney nephrons. The present study examined the ontogeny of renal and submandibular gland prepro-epidermal growth factor (preproEGF) gene expression in the cpk mouse using Northern blot hybridization and immunohistochemistry. There was a virtual absence of renal preproEGF gene expression in cystic kidneys over the 3-week postnatal period, during which time renal preproEGF mRNA and proEGF/EGF protein normally reach significant levels. PreproEGF mRNA was expressed in salivary glands of cystic mice; however, this mRNA could not be further elevated with testosterone suggesting that there are abnormalities in the regulation of the preproEGF gene in the submandibular gland, as well as in the kidney. Since renal preproEGF expression during the early postnatal period occurs when collecting duct cysts form, it is possible that a deficiency in renal proEGF or EGF contributes to the rapid development of collecting duct cysts and the concomitant renal failure in the C57BL/6J-cpk cystic mouse.     

Distribution of the vacuolar H+ atpase along the rat and human male reproductive tract

Luminal acidification in parts of the male reproductive tract generates an appropriate pH environment in which spermatozoa mature and are stored. The cellular mechanisms of proton (H+) secretion in the epididymis and the proximal vas deferens involve the activity of an apical vacuolar H+ ATPase in specialized cell types, as well as an apical Na+/H+ exchanger in some tubule segments. In this study we used Western blotting and immunocytochemistry to localize the H+ ATPase in various segments of the male reproductive tract in rat and man as a first step toward a more complete understanding of luminal acidification processes in this complex system of tissues. Immunoblotting of isolated total cell membranes indicated a variable amount of H+ ATPase in various segments of the rat reproductive tract. In addition to its known expression in distinct cell types in the epididymis and vas deferens, the H+ ATPase was also localized at the apical pole and in the cytoplasm of epithelial cells in the efferent duct (nonciliated cells), the ampulla of the vas deferens and the ventral prostate (scattered individual cells), the dorsal and lateral prostate, the ampullary gland, the coagulating gland, and all epithelial cells of the prostatic and penile urethra. Both apical and basolateral localization of the protein were found in epithelial cells of the prostatic ducts in the lateral prostate and in periurethral tissue. Only cytoplasmic, mostly perinuclear localization of the H+ ATPase was found in all epithelial cells of the seminal vesicles and in most cells of the ventral prostate and coagulating gland. No staining was detected in the seminiferous tubules, rete testis, and bulbourethral gland. In human tissue, H+ ATPase-rich cells were detected in the epididymis, prostate, and prostatic urethra. We conclude that the vacuolar H+ ATPase is highly expressed in epithelial cells of most segments of the male reproductive tract in rat and man, where it may be involved in H+ secretion and/or intracellular processing of the material endocytosed from the luminal fluid or destined to be secreted by exocytosis.     

Species-specific expression of microsomal prostaglandin E synthase-1 and cyclooxygenases in male monkey reproductive organs

We investigated the tissue distribution and cellular localization of microsomal PGE synthase-1 (mPGES-1) and cyclooxygenase (COX)-1 and -2 in male monkey reproductive organs. Western blotting revealed that monkey mPGES-1 was expressed most intensely in the seminal vesicles, moderately in the testis, and weakly in the epididymis and vas deferens. The tissue distribution profile was quite different from those profiles for rats, rabbits, and pigs, e.g., rat mPGES-1 was the most abundant in the vas deferens, and the rabbit and pig enzymes, in the testis. Immunohistochemical staining with mouse monoclonal anti-human mPGES-1 antibody revealed that monkey mPGES-1 was localized in spermatogonia, Sertoli cells, and primary spermatocytes of testis and in epithelial cells of the epididymis, vas deferens, and seminal vesicles. In monkeys, COX-1 was localized in epithelial cells of the epididymis and vas deferens, whereas COX-2 was dominantly found in epithelial cells of the seminal vesicles.     

Specificity of androgen resistance inMus caroli kidney

Androgen controls the expression of beta-glucuronidase and several other proteins in the kidney of the standard laboratory mouse, Mus musculus. Other species within the genus Mus exhibit a variety of response patterns for kidney beta-glucuronidase and other markers of androgen action. We have investigated the mechanism of androgen action in M. caroli, a Mus species that does not produce beta-glucuronidase in response to testosterone. The failure of testosterone to induce beta-glucuronidase in M. caroli females cannot be overcome by treatment with dihydrotestosterone, with pharmacological doses of testosterone propionate or dihydrotestosterone propionate, or with a variety of potent androgen analogues. All of these compounds induce kidney beta-glucuronidase in M. musculus females and kidney ornithine decarboxylase, submandibular gland renin, and submandibular gland epidermal growth factor in both M. caroli and M. musculus females. Furthermore, kidney androgen receptor proteins from M. caroli and M. musculus animals have the same sedimentation characteristics on sucrose density gradients. These data indicate that androgen resistance in M. caroli is not due to deficient 5 alpha-reductase or aberrant hormone metabolism producing suboptimal levels of functional androgen and is not caused by a defective androgen receptor. They suggest that the resistance of beta-glucuronidase in M. caroli kidney to induction by androgen occurs at the level of the beta-glucuronidase gene.     

Metabolism of testosterone by tes epididymis and ventral prostate of rat and its inhibition by cyproterone acetate

The metabolism of ³H-testosterone by the epididymis and accessory organs of adult male rats exposed continuously to microdoses of cyproterone acetate from subcutaneous capsules were studied. The major metabolite of ³M-testosterone in the epididymis, vas deferens and ventral prostate of control rat was dihydrotestosterone while the formation of androstanediol by these tissues was low. The highest percentage of DHT was formed by the ventral prostate and cauda epididymis. In rats exposed to cyproterone acetate for four months, the conversion of testosterone to DHT was inhibited in all the tissues but maximally in the ventral prostate and cauda epididymis. In these rats, the secretory function of the ventral prostate was normal while that of the epididymis was markedly decreased. These data are discussed based on the differential thresholds of androgens required to regulate the functions of the accessory organs.     

Expression of the A-raf proto-oncogene in the normal adult and embryonic mouse.

We have determined the expression pattern of the A-raf proto-oncogene in the embryonic and adult mouse. Western blot analysis of protein lysates from tissues of adult mice show that p69A-raf is ubiquitously expressed, but that levels of expression vary among different tissues. To determine the cell-specific expression pattern of A-raf, we generated transgenic mice expressing the beta-galactosidase reporter gene from the A-raf promoter. We show that A-raf expression is highly specific within a given tissue, and we identify cell types expressing this gene in the adult testis, epididymis, vas deferens, seminal vesicle, ovary, oviduct, bladder, kidney, intestine, heart, spleen, thymus, and cerebellum. In the embryo, ubiquitous expression of the reporter gene is observed, but the highest levels of expression are specifically detected in the embryonic heart at stages 9.5-11.5 days post-coitum.     

Co-expression and interaction of cubilin and megalin in the adult male rat reproductive system

Cubilin is a peripheral membrane protein that cooperates with the endocytic receptor megalin to mediate endocytosis of ligands in various polarized epithelia. Megalin is expressed in the male reproductive tract where it has been implicated in the process of sperm membrane remodeling. A potential role for cubilin in the male reproductive tract has not been explored. Using RT-PCR, we found that cubilin and megalin mRNAs are expressed in the efferent ducts, corpus and cauda epididymis, and proximal and distal vas deferens. Immunohistological analysis revealed that cubilin was expressed in nonciliated cells of the efferent ducts, principal cells of the corpus and cauda epididymis and vas deferens. Immunogold EM showed cubilin in endocytic pits, endocytic vesicles, and endosomes of these cells. The expression profile of cubilin in the male reproductive tract was coincident with that of megalin except in principal cells of the caput epididymis. Double immunogold labeling showed that cubilin and megalin co-localized within the endocytic apparatus and recycling vesicles of efferent duct cells. Neither protein was found in lysosomes. Injection of RAP, an antagonist of megalin interaction with cubilin, reduced the level of intracellular cubilin in cells of the efferent ducts and vas deferens. In conclusion, cubilin and megalin are co-expressed in cells of the epididymis and vas deferens and the endocytosis of cubilin in these tissues is dependent on megalin. Together, these findings highlight the potential for a joint endocytic role for cubilin and megalin in the male reproductive tract.     

Zinc and copper content in rat epididymis and vas deferens

The zinc and copper content in the different epididymal segments and vas deferens of castrated rats were investigated with the help of atomic absorption spectrophotometer. The vas deferens showed maximum zinc content as compared to that of different parts of epididymis in all groups whether castrated unilaterally, bilaterally or in the intact control. Zinc content was reduced in the epididymis and vas deferens ipsilateral to the castrated side as compared to that of contralateral control and intake animals. Lowest zinc content was observed in the epididymis and vas deferens of bilaterally castrated animals from that of other groups. Absence of sperms was observed in all segments of epididymis and vas in bilaterally castrated animals and from the unilaterally castrated side. Copper content was unaltered in all epididymal segments and vas deferens. There appears to be a correlation between the absence of sperms in the male genital tract and the decrease in zinc content.     

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.     

Characterization of epidermal growth factor receptor in testis, epididymis and vas deferens of non-human primates.

The presence of the epidermal growth factor receptor (EGFR) in testis, epididymis and vas deferens of monkeys was demonstrated using a polyclonal antibody (RK2) raised against a peptide-specific sequence of the intracellular domain of the human EGFR. Immunoblotting of membrane preparations revealed a specific band at approximately 170 kDa corresponding to those of controls, A431 and monkey liver cells. Cryostat sections were stained by biotin-streptavidin peroxidase immunocytochemistry. The liver showed positive staining along the basolateral membranes of the hepatocytes lining the sinusoids. The testis showed positive staining indicating the presence of EGFR in Leydig cells, Sertoli cells and peritubular cells. In the epididymis, immunostaining of the EGFR was observed on both the basolateral and the luminal borders of the epididymal epithelium. Immunofluorescence studies revealed a similar pattern of EGFR distribution in the epididymis and indicated that the luminal immunostaining was vesicular. In the vas deferens, positive immunostaining was detected in a pattern very similar to that observed in the epididymis. There was no positive staining in the interstitium of the epididymis or in the smooth muscle cell layers of the vas deferens. The sections of all tissues treated with pre-immune serum were negative. These results suggest that EGF in the primate testis may act at the level of somatic cells. In addition, the basolateral and luminal EGFR staining in the epididymis and vas deferens suggest that these cells respond to an EGF, or EGF-like, source both at the basal, luminal or at both sides of the cells, or that these tissues serve as sites of EGF transcytosis across the epithelium.     

Immunolocalization of AE2 anion exchanger in rat and mouse epididymis.

A low-bicarbonate concentration and an acidic pH in the luminal fluid of the epididymis and vas deferens are important for sperm maturation. These factors help maintain mature sperm in an immotile but viable state during storage in the cauda epididymidis and vas deferens. Two proton extrusion mechanisms, an Na(+)/H(+) exchanger and an H(+)ATPase, have been proposed to be involved in this luminal acidification process. The Na(+)/H(+) exchanger has not yet been localized in situ, but we have reported that H(+)ATPase is expressed on the apical membrane of apical (or narrow) and clear cells of the epididymis. These cells are enriched in carbonic anhydrase II, indicating the involvement of bicarbonate in the acidification process and suggesting that the epididymis is a site of bicarbonate reabsorption. Previous unsuccessful attempts to localize the Cl/HCO(3) anion exchanger AE1 in rat epididymis did not investigate other anion exchanger (AE) isoforms. In this report, we used a recently described SDS antigen unmasking treatment to localize the Cl/HCO(3) exchanger AE2 in rat and mouse epididymis. AE2 is highly expressed in the initial segment, intermediate zone, and caput epididymidis, where it is located on the basolateral membrane of epithelial cells. The cauda epididymidis and vas deferens also contain basolateral AE2, but in lower amounts. The identity of the AE2 protein was further confirmed by the observation that basolateral AE2 expression was unaltered in the epididymis of AE1-knockout mice. Basolateral AE2 may participate in bicarbonate reabsorption and luminal acidification, and/or may be involved in intracellular pH homeostasis of epithelial cells of the male reproductive tract.     

Mus Spretus Line-1 Sequences Detected in the Mus Musculus Inbred Strain C57bl/6j Using Line-1 DNA Probes

The inbred mouse strain, C57BL/6J, was derived from mice of the Mus musculus complex. C57BL/6J can be crossed in the laboratory with a closely related mouse species, M. spretus to produce fertile offspring; however there has been no previous evidence of gene flow between M. spretus and M. musculus in nature. Analysis of the repetitive sequence LINE-1, using both direct sequence analysis and genomic Southern blot hybridization to species-specific LINE-1 hybridization probes, demonstrates the presence of LINE-1 elements in C57BL/6J that were derived from the species M. spretus. These spretus-like LINE-1 elements in C57BL/6J reveal a cross to M. spretus somewhere in the history of C57BL/6J. It is unclear if the spretus-like LINE-1 elements are still embedded in flanking DNA derived from M. spretus or if they have transposed to new sites. The number of spretus-like elements detected suggests a maximum of 6.5% of the C57BL/6J genome may be derived from M. spretus.