Assessment of protein synthesis and secretion by rat seminiferous and epididymal tubules in vivo

In vivo microperifbsion and micropuncture were used to study tubule protein synthesis and proluminal secretion by the male reproductive tract in vivo . Somniferous and caput and cauda epididymal tubules were perihsed for 3 h. with [³⁵S]-methionine. Perifused interstitial fluid (IF), lumen fluid (LF), and tubule extract (TE) were collected. Proteins were separated by SDS-PAGE, and autoradiograms were developed.
Trichloroacetic acid precipitable proteins in each fluid were determined and a protein synthesis index (PSI) was calculated. PSI values demonstrated that the cauda epididymis synthesized less protein in vivo than did either seminiferous or caput tubules.
Seminiferous tubules synthesized and secreted into the tubule lumen a relatively constant panel of proteins. Epididymal tubules synthesized and secreted proteins in a region-specific manner. In the caput epididymis the most prominent secreted bands were consistent with the heavy and light chains of epididymal clusterin. In the cauda epididymis, the most prominent synthesized and secreted protein was a 25 kDa protein consistent with the protein D. The above approach to studying protein synthesis and secretion will allow direct study of the physiological and path physiological effects on this important epithelial hnction in vim     

Effects of antifertility drugs on epididymal protein secretion, acquisition of sperm surface proteins and fertility in male rats

The possibility that α-chlorohydrin, 6-chloro-6-deoxyglucose (6CDG) and cyproterone acetate (CPA) might affect epididymal protein secretion or acquisition of sperm surface proteins as the cause of their antifertility action in male rats was investigated. Daily administration of 9 mg/kg α-chlorohydrin for 7–14 days and 24 mg/kg 6CDG for 14–21 days induced sterility in male rats and impaired the capacity of the cauda epididymal spermatozoa to initiate motility. Treatment with CPA (30 mg/kg/day) for 21–28 days, however, was found to have no effect on fertility and initiation of sperm motility, although the epididymis of the treated animals underwent a loss in weight. The antifertility effects of α-chlorohydrin or 6CDG did not seem to be attributed to an interference with epididymal protein secretion. The cauda epididymal fluids of the α-chlorohydrin, 6CDG and CPA treated animals have similar protein patterns compared to those of the control animals. However, when the surface proteins of the spermatozoa were labelled with radioactive iodine, the sperm surface proteins of the α-chlorohydrin and 6CDG treated animals were found to differ from those of the control animals. Two peaks (MW 32 000 and 70 000) and one peak (70 000) were significantly reduced in the α-chlorohydrin treated and 6CDG treated animals, respectively. Additional bands appeared on the surface of the treated (infertile) animals. In contrast, CPA treatment did not affect the surface protein pattern of the epididymal spermatozoa. It was concluded that the antifertility affects of α-chlorolydrin and 6CDG are not due to an interference with epididymal secretion of specific proteins but to an intervention of the subsequent acquisition of these proteins by epididymal spermatozoa. This results in a decrease in the capacity of the epididymal sperm to initiate motility and hence a loss of fertilizing capacity.     

Restricted entry of an anti-rat epididymal protein IgG into the rat epididymis

A rat epididymal protein (MW 32 000) was isolated and purified from the rat caudal epididymal fluid. Mono-specific antiserum against this protein was raised in rabbits, purified and labelled with ¹²⁵I. The labelled IgG was infused intravenously into anaesthetized male rats, and the transfer of the labelled IgG across the luminally perfused cauda epididymidis was studied. It was found that during the 2 h infusion period, radioactivity in blood rose, but no radioactivity could be detected in the perfusates, irrespective of whether the epididymis was perfused with Krebs bicarbonate solution or a solution which resembled the rat caudal fluid in ionic composition. In some experiments, rats were given a single intravenous injection of labelled IgG and radioactivity in the epididymal content was measured 10 days later. It was found that despite a high IgG level in blood and liver, no radioactivity could be detected in epididymal fluid and sperm. It is concluded that the blood-epididymis barrier restricts the passage from blood to lumen, an immunologlobulin directed against an epididymal protein.     

降钙素合成分泌及生理作用

降钙素是一种由甲状腺滤泡旁细胞产生和分泌、含有32个氨基酸、参与钙磷代谢调节的多肽类激素。其主要生理作用是降钙素通过与靶细胞膜上的降钙素受体(calcitonin receptor,CTR)特异性结合,发挥降低破骨细胞活性、减少破骨细胞数量、抑制破骨细胞吸收的作用,并具有稳定血钙的作用。笔者综述了降钙素的化学结构、降钙素的合成与分泌、降钙素对破骨细胞、成骨细胞、血钙的调节等生理作用、降钙素实验研究的临床意义,以及降钙素的实验研究进展、降钙素的临床应用和研究成果分享。降钙素在临床骨质疏松、骨质疏松性骨折、变形性骨炎等的治疗中具有重要的应用价值。     

Origin of the luminal fluid proteins of the rat epididymis

Using a combined microperfusion and high resolution gel electrophoresis technique, the origin of the epididymal fluid proteins of the rat has been investigated. Some proteins originate from the testis, others are secreted by the epididymis or are released by spermatozoa. Of particular interest is a 32 000 dalton protein found to be actively secreted by the caput epithelium in situ and concenrated in the lumen. The cauda epididymidis contained the highest concentration of this protein. Radioactive labelling of the sperm surface proteins revealed that this protein was present on the surface of the mature cauda but not on the immature caput or corpus sperm, suggesting its acquisition by the sperm surface during epididymal transit. Another sperm surface protein of interest (MW 40 000) is present only on the plasma membrane of the cauda but not on that of the caput or corpus sperm. Since this protein was not identified in the epididymal perfusates or luminal fluids, its presence may result from some modification events taking place in the sperm membrane during maturation.     

Localization of antigenic determinants recognized by a monoclonal antibody in rat epididymal spermatozoa, with particular reference to sperm maturation

Summary.  This study localized antigenic determinants recognized by a mouse anti-human sperm monoclonal antibody TüS10 immunocytochemically and immunoelectron microscopically in the rat sperm recovered from the caput and cauda epididymidis. Immunocytochemistry showed that the antibody bound specifically to the plasma membrane overlying the principal piece of membrane-intact sperm from the caput and cauda epididymidis. Demembranation by Triton X-100 significantly decreased the affinity of the monoclonal antibody TüS10 to the caput sperm but did not obviously change that to the cauda sperm. Immunoelectron microscopy with biotinstreptavidin peroxidase complex pre-embedding method confirmed the localization of the antigenic determinants over the cell surface of the principal piece of the membrane-intact spermatozoa from the caput and cauda epididymidis. The demembranated sperm from the caput epididymidis showed no intracellular labelling, while those from the cauda displayed labelling on their external surface of the fibrous sheath. Using monoclonal antibody TüS10 as a probe, we detected different distribution patterns of the antigenic determinants between the spermatozoa in the caput and cauda epididymidis. These results suggest that spermatozoa mature with immunologically detectable changes in the fibrous sheath during their epididymal transit.     

In vitro synthesis and glycosylation of androgen-dependent secretory proteins of rat dorsal prostate and coagulating gland

The two major androgen-dependent secretory proteins of rat dorsal prostate and coagulating gland, DP I and DP II, were compared by in vitro translation of total poly(A)RNA and by pulse-chase techniques by means of [35S]methionine and tissue minces of coagulating gland. DP I is a major in vitro translation product of isolated poly(A)RNA, whereas DP II is undetectable in a mouse embryo fibroblast cell-free system where glycosylation does not occur. DP I is synthesized within 20 min in minces of coagulating gland incubated in the presence of [35S]methionine and is secreted in 40 min. DP II is detectable in the medium only after 8 hr of labeling. Inhibition of asparagine-linked protein glycosylation with tunicamycin (10 micrograms/ml) blocked the synthesis and secretion of DP II with an apparent increase in DP I secretion. Inhibition of DP II synthesis by monensin implicates the Golgi in the processing of DP II oligosaccharides. The data are consistent with the proposal that DP I enters a pathway of rapid secretion that is enhanced by the absence of core glycosylation, whereas DP II follows a slow pathway through the Golgi that involves extensive glycosylation.     

Effects of ethanol ingestion on epididymal glycosidases and fertility in the rat

Epididymal glycosidases play a role in sperm maturation by modifying sperm surface glycoproteins. To study the effects of ethanol on epididymal sperm maturation, ethanol (3 g/kg body weight as 25%, v/v) was administered to a group of rats by gastric-intubation twice daily for 30 days. In another group, rats were also treated with alcohol for 30 days but were then withdrawn from treatment for 30 days to assess the reversibility of ethanol-induced effects. Ethanol-induced changes in epididymal tissue and sperm glycosidases, cauda epididymal sperm motility and the fertility of rats were assessed. Ethanol treatment caused a marked decrease in the specific activities of glycosidases in both tissues and spermatozoa from epididymal segments. Cauda epididymal sperm motility and the fertility of ethanol-treated rats were significantly impaired compared to control rats fed an isocaloric diet. These changes are likely to be the consequence of direct and indirect effects of ethanol mediated through subnormal testosterone and dihydrotestosterone. Most of these changes were found to be reversible. The present study suggests that impaired activity of sperm glycosidases may be one of the factors responsible for defective sperm motility and fertilizing potential in ethanol-treated rats.     

Protein profiles in various epididymal segments of normal and castrated rats

Aim: Epididymal proteins are known to play an important role in the maturation of spermatozoa. We ought to determine if there are regional differences in androgen-dependent epididymal proteins. Methods: A group of adult rats was castrated and epididymides were removed three days following castration. The epididymides were dissected into caput,corpus and cauda segments, homogenized, and proteins were fractionated by anion exchange HPLC. Proteins in selected fractions were resolved by SDS-PAGE and visualized by silver staining. Results: It was observed that the levels of multiple proteins drastically reduced in the various regions of epididymis of the orchiectomized rats. Conclusion: The epididymal proteins appear to be useful markers to study androgenic action in the epididymis. ( Asian J Androl 2000 ;2: 57-64)     

Increase in fluid viscosity during epididymal transit and the immobilization of rat epididymal spermatozoa

A microcapillary method was developed to measure the viscosity of small volumes of undiluted epididymal fluid. Fluid from the cauda epididymis registered 82 +/- 17 centipoise which was much more viscous than fluid from the caput region (8 +/- 2 centipoise). Initiation of sperm motility was strongly suppressed in the viscosity range of 7 to 150 centipoise. A significant increase in the viscosity of fluid from the caput region was observed when immobilin in the fluid binds with a lectin from Jack fruit (Artocarpus heterophyllus). Thus, it is postulated that aggregation of the immobilin induced by a lectin-like material produced by the cauda epididymis may be a mechanism by which fluid viscosity is increased during epididymal transit.     

Participation of epididymal cysteine-rich secretory proteins in sperm-egg fusion and their potential use for male fertility regulation

Rat protein DE is an androgen-dependent cysteine-rich secretory protein （CRISP） synthesized by proximal epididymal regions. DE, also known as CRISP-1, is localized on the equatorial segment of acrosome-reacted spermatozoa and participates in gamete fusion through binding to egg complementary sites. Immunization of rats with DE inhibits fertility and sperm fusion ability, suggesting that DE represents a good epididymal contraceptive target. Recombinant DE fragments and synthetic peptides revealed that DE binds to the egg via a 12-amino acid region of an evolutionarily conserved motif, Signature 2 （S2）. The ability of other CRISP to bind to the rat egg was correlated with their S2 amino acid sequences. Although testicular protein Tpx- 1 （CRISP-2） was capable of binding to rodent eggs, human epididymal AEG-related protein （ARP） and helothermine （from lizard saliva） were not. The S2 region presented only two substitutions in Tpx-1 and four in ARP and helothermine, compared with the DE S2, suggesting that this amino acid sequence was relevant for egg interaction. Studies with Tpx- 1 and anti-Tpx- 1 revealed the participation of this protein in gamete fusion through binding to complementary sites in the egg. In competition studies, DE reduced binding of Tpx- 1 dose-dependently, indicating that both CRISP share the egg complementary sites. That anti-DE and anti-Tpx-1 inhibit sperm-egg fusion while recognizing only the corresponding proteins, suggests functional cooperation between these homologous CRISP to ensure fertilization success. These results increase our understanding of the molecular mechanisms of gamete fusion and contribute to the development of new and safer fertility regulating methods. （Asian J Androl 2007 July; 9： 528-532）     

Purification and Localization of Acidic Epididymal Glycoprotein (AEG): A Sperm Coating Protein Secreted by the Rat Epididymis

A protein designated acidic epididymal glycoprotein (AEG) was purified from rat epididymis using ion exchange chromatography on DEAE-Sephadex, gel filtration in Sephadex G-75 and affinity chromatography on Concanavalin-A Sepharose. AEG is a major secretory product of the epididymis making up 2–3 per cent of total soluble protein. Antibody to AEG was raised in rabbits and purified by affinity chromatography on AEG-Sepharose. Quantitation of AEG in cytosol using "rocket" immunoelectrophoresis showed AEG to increase in epididymal segments from caput to cauda. Ligation of the midcorpus decreased AEG in the cauda. Localization of AEG using an immunoperoxidase method revealed that it is secreted largely by the epithelium of caput and corpus beginning with the region distal to the initial segment. It appears to be secreted by a specific cell type, probably the so-called "principal" cell. Specific staining of AEG was also noted in "clear" cells in the cauda. Spermatozoa become coated with AEG as they leave the initial segment and remain so during passage through the cauda.     

Molecular cloning and identification of mouse epididymis-specific gene mHong1, the homologue of rat HongrES1

Previous studies have shown that rat epididymis-specific gene HongrES1 plays important roles in sperm capacitation and fertility. In this study, we cloned the mouse homologue gene by sequence alignment and RT-PCR methods and designated it as mHong1. The mHong1 gene is located on chromosome 12p14, spanning five exons. The cDNA sequence consists of 1257 nucleotides and encodes a 419 amino-acid protein with a predicted N-terminal signal peptide of 20 amino acids. The mHong1 mRNA shows similarity with HongrES1 in the expression patterns: (i) specific expression in epididymal tissue, especially in the cauda region; and (ii) androgen-dependence but testicular fluid factor independence. Its protein product shows 71% similarity with HongrES1 and contains a classical serpin domain as does HongrES1. A polyclonal antibody against mHong1 with high specificity and sensitivity was raised. Like HongrES1, the mHong1 protein shows a checker-board expression pattern in the epididymal epithelium and is secreted into the epididymal lumen. The mHong1 protein shows higher glycosylation than HongrES1. Although both of them are deposited onto the sperm head surface, mHong1 is localized to the equatorial segment, which is different from that of HongrES1. The mHong1 protein can be removed from the sperm membrane by high ionic strength and therefore can be classed as an extrinsic membrane protein. Collectively, we conclude that mHong1 is the homologue of HongrES1 and the present work paves the way for establishing animal models to elucidate the precise functions of HongrES1 and mHong1.     

Decreased expression of AKAP4 and TyrPho proteins in testis,epididymis, and spermatozoa with low sexual performance of mice induced by modified CUMS

The molecular mechanism of chronic stress especially reduced motility, a major cause of male infertility, has not been proved. It is known that A-kinase anchor protein 4 (AKAP4) and tyrosine-phosphorylated (TyrPho) proteins are involved in progressive motility. This study aimed to investigate the effect of chronic unpredictable mild stress (CUMS) on sexual behaviours, sperm quality, and expressions of AKAP4 and TyrPho proteins in testis, epididymis, and spermatozoa. Sixteen male mice were divided into control and CUMS groups (n = 8/group). Animals were induced by a stressor from twelve stressors for 36 days. Sexual behaviours, corticosterone and testosterone, sperm parameters, and histopathology were observed. The expressions of AKAP4 and TyrPho proteins in testis, epididymis, and spermatozoa were examined. Results showed that CUMS significantly increased corticosterone while serum testosterone level was decreased. Sexual behaviours and sperm parameter quality were significantly decreased. CUMS mice showed vacuolisation and pyknotic cells in seminiferous epithelium and less sperm mass was observed within epididymal lumen. CUMS decreased expressions of AKAP4 and TyrPho proteins in testis, epididymis, and spermatozoa. In conclusion, the decreased expression of AKAP4 and TyrPho proteins may be a mechanism associated with low semen qualities particularly decrease of sperm motility in CUMS.     

Purification and characterization of β-galactosidase from rat epididymal fluid

Summary. β-Galactosidase from rat epididymal fluid was purified by a combination of chromatographic techniques and precipitation with ammonium sulphate. Specific activity of the enzyme in the final precipitate was 18 times greater than in the original fluid, and it was practically free of N-acetyl-β-D-glucosaminidase. A single major band was seen when the precipitate was analysed by sodium dodecylsulphate polyacrylamide gelectrophoresis (SDS-PAGE).
The activity of the purified enzyme has an optimum at pH 4.5, and the temperature optimum is around 45 °C. The activity was inhibited by p-chloromercuribenzoic acid and ions such as Cd(II), Co(II), Cu(II) and Ag(I). Lactose does not appear to be a substrate for this enzyme.     

Chronic stress affects tyrosine phosphorylated protein expression and secretion of male rat epididymis

Chronic stress (CS) is shown to decrease the semen quality with changed expression of tyrosine phosphorylated (TyrPho) proteins in testicular and seminal tissues. However, the alterations of such proteins and fluid contents in the epididymis, producing sperm maturation factors, have never been reported. Sixteen adult rats were randomly divided into 2 groups (n = 8). The control animals were not subjected to stressors whereas CS rats were immobilised within restraint cage (4 hr/day) before cold forced-water swimming (15 min/day) for 60 days. Corticosterone, testosterone, blood glucose level (BGL), malondialdehyde (MDA) and biochemical components in epididymal fluid were assayed. Expressions of heat shock protein 70 (HSP-70), androgen receptor (AR) and TyrPho protein were investigated in epididymal tissue and fluid. Significantly, CS increased the corticosterone and BGL but decreased testosterone and epididymal substance levels. MDA level in tail epididymal fluid and HSP-70 expression in both regions of epididymal tissues and fluids, except in head epididymal fluid of CS were increased. Epididymal tissues showed the decrease of AR expression. Presence and changes of many TyrPho proteins were observed in CS. In conclusion, CS could affect functional proteins particularly TyrPho in epididymis, resulted in low semen quality.     

Influence of liver transplantation and cyclosporin on bile secretion —an experimental study in the rat

Bile secretion is reduced after liver transplantation. It has been suggested that this is due either to the effect of cyclosporin or to the damage to the liver graft during preservation and reperfusion. The aim of this study was to explore the influence of cyclosporin as well as of liver transplantation on bile secretion. Bile flow was studied in an experimental model in the rat. In syngeneic liver-transplanted animals, the bile flow was increased compared to the bile flow in the control group (1.29±0.09 ml/h vs 0.66±0.03 ml/h; P<0.01), mainly due to an increased bile acid-independent flow (0.76 ml/h vs 0.50 ml/h; P<0.01). The findings in the livertransplanted rats contrasted with those in a group of nontransplanted animals treated with cyclosporin. Cyclosporin treatment resulted in a reduced bile acid-independent fraction (0.37 ml/h vs 0.50 ml/h, P<0.05) of the bile flow, although no biochemical signs of hepatotoxicity were present. This reduction in the bile acid-independent fraction could, however, not be demonstrated when cyclosporin was given to a group of liver-transplanted rats, although a reduced total bile flow was recorded in the 1st hour measurements. In contrast to previous studies, we found that the cyclosporin vehicle (Cremophor EL), when administered chronically, induced a higher bile flow than that in the control rats. This effect was not seen in the transplanted rats. Our findings in this experimental rat model indicate that cyclosporin will influence and reduce bile secretion and bile acid secretion even if no other signs of liver dysfunction are present. On the other hand, the preservation and reperfusion in this model resulted in an increased bile flow, while bile acid secretion remained constant.     

Synthesis and secretion of proteins in vitro by isolated epithelial strips from the proximal and distal vas deferens of the rat

Epithelial strips of rat vas deferens were isolated by a new technique and used to study differences in protein synthesis and secretion between morphologically defined segments of the vas deferens. The isolated strips were viable as judged by linear oxygen uptake over the incubation period and by preservation of structure. Epithelium from the proximal vas deferens incorporated more labelled amino acids into cytosolic (P less than 0.02) and incubation medium (P less than 0.01) proteins than did epithelium from distal vas deferens; this incorporation was inhibited by cycloheximide. Although some of the incubation medium proteins arose by leakage from damaged cells, specific protein secretion was indicated by differences in SDS-PAGE autoradiogram banding patterns and by differences in glycosylation between proteins in the incubation medium and those in the cytosol. Thus, the former contained more label from [14C]galactose incorporation than did cytosolic proteins (proximal: P less than 0.05; distal: P less than 0.005).     

Binding of seminal vesicle proteins to the plasma membrane of rat spermatozoa in vivo and in vitro

The association of seminal vesicle (SV) proteins with rat spermatozoa has been studied in vivo and in vitro. SV proteins bind to the sperm plasma membrane after ejaculation but are removed progressively from the sperm plasma membrane in the female genital tract. Although some of these remain bound to spermatozoa when they reach the oviducts, they do not seem to be present at the time of fertilization. This could indicate a putative role for these SV proteins in pre-fertilization events. In addition, the binding of SV antigens was studied in vitro. It was observed that the ability to bind SV proteins is gained by the spermatozoa during epididymal maturation, and is first detectable in spermatozoa collected from the cauda epididymis. On the other hand, the binding is regulated by other proteins present in the ejaculate which are secreted by the coagulating glands. Experiments also showed that mouse spermatozoa are able to bind rat SV proteins, indicating that the binding is not a highly species-specific phenomenon.