Secretion of glycosidases in human epididymal cell cultures

The dynamics of glycosidase secretion was evaluated in human epididymal cell culture. Epithelial cells from caput, corpus, and cauda epididymis were isolated from tissue obtained from patients undergoing therapeutic orchidectomy due to prostatic carcinoma. The activities of alpha-glucosidase, N-acetylglucosaminidase, beta-glucuronidase, and alpha-mannosidase were analyzed in conditioned culture media. Glycosidase activity was significantly higher in corpus and/or cauda than in caput epididymis. There was a time-dependent increase in enzyme activities that was maximal between 10 and 14 days of culture in all epididymal regions. Epididymal glycosidases are secreted by cultured epithelial cell from human epididymis with an increase toward the distal regions of this organ, which may be related to the dynamics of sperm maturation. Cultures from different epididymal regions may represent a valuable tool to study of human epididymal function.     

Sperm maturation in dogs: sperm profile and enzymatic antioxidant status in ejaculated and epididymal spermatozoa

Spermatozoa become more susceptible to the attack of reactive oxygen species during maturation. To avoid oxidative damage, the epididymis must provide the necessary antioxidant protection. The aim of this study was to compare the canine sperm profile and the enzymatic antioxidant status of the ejaculated fractions and samples collected from the different segments of the epididymis (caput, corpus and cauda). Five adult dogs were used, and after 1–3 weeks, subsequently to bilateral orchiectomy and epididymal storage, sperm samples were collected from the different segments of the epididymis. Samples were evaluated for conventional microscopy and computer‐assisted motility analysis: sperm plasma membrane permeability and the activity of the antioxidant enzymes catalase, glutathione peroxidase (GPx) and superoxide dismutase (SOD). Samples collected from the caput and corpus showed lower values for most of the motility variables evaluated, indicating different levels of immaturity. Catalase activity was observed only in ejaculated samples. Conversely, GPx activity was higher in the cauda epididymidis. Correlations were found between SOD and GPx and SOD and sperm motility in the epididymal cauda and corpus, highlighting the importance of the enzymes for the protection of spermatozoa during the transit along the epididymis.     

Tissue Specificity of the Epididymal Androgen Dependent Phospholipase A

The effect of androgens on phospholipase A in the rat epididymis, liver, heart, kidney, prostate and epididymal fat pad has been investigated both in the cytosol and in a postmitochondrial partiulate cell fraction.
In both cell fractions, phospholipase A activity was 10–15 times higher in the cauda epididymidis than in the other organs, including the caput epididymidis, while the corpus showed an intermediate activity. In the cauda and corpus, castration reduced phospholipase A activity in both subcellular fractions to the same level as found in the other organs, and testosterone supplementation restored the enzyme activity. No evidence of a similar androgen controlled phospholipase A was found in any other tissue examined, including the prostate.     

Glutathione-related enzymes in cell cultures from different regions of human epididymis

Protection of maturing sperm from potential endogenous or exogenous harmful substances during their transit throughout the epididymis is a critical event. The authors studied the activity of gamma-glutamyl transpeptidase (GGT) and glutathione S-transferase (GST), and glutathione (GSH) levels in epithelial cell cultures from human caput, corpus, and cauda epididymides. Tissue was obtained from patients undergoing therapeutic orchidectomy for prostatic cancer. Enzymatic activity was measured in conditioned media and cellular fractions. Androgen influence was also evaluated. Both enzymatic activities were found in cellular homogenates and conditioned media from cultures of all epididymal regions. GGT activity was highest in cultures from cauda epididymis, both in conditioned media and cell fractions, while GST activity did not show regional differences in conditioned media, but exhibited higher activity in cell homogenates from cauda cultures than those obtained from corpus and caput epididymis. GSH level showed no regional difference in cell homogenates and it could not be detected in conditioned media by the method used. Presence of different concentrations of dihydrotestosterone (DHT) had no influence neither on the enzymatic activities nor GSH concentration. The results indicate that GGT and GST are present along the human epididymis and a fraction or isoform of these enzymes might be secreted to the luminal fluid to play a detoxificative role in sperm maturation.     

Effect of a nonsteroidal antiandrogen, flutamide on intraluminal acidification in rat testis and epididymis

Summary. Flutamide, a pure antiandrogen was administered to intact adult male rats to study the effect of altered availability of hormones on in situ pH, PCO₂ and bicarbonate concentration ([HCO⁻₃]) of seminiferous tubules, proximal caput, middle caput, middle corpus, and proximal cauda epididymidis. The weights of the epididymis and ventral prostate as well as the plasma testosterone level showed antiandrogenic effects of flutamide. Relative to controls, flutamide elevated significantly in situ pH in proximal caput, middle caput, middle corpus and proximal cauda epididymidis but not in seminiferous tubules. In situ PCO₂ values in the above segments, after flutamide, were indistinguishable from controls and from each other but all values remained significantly higher than systemic arterial blood PCO₂. Flutamide treatment did not change the [HCO⁻₃] in systemic arterial blood or seminiferous tubules but increased markedly the values in proximal caput and middle caput. The results of the present studies support the view that luminal acidification in the rat epididymis is under androgen control and may be important for sperm maturation and storage.     

Erratum

During passage of hamster spermatozoa through the epididymis their maturation is shown to involve changes in the sperm head, midpiece (mitochondria) and tail. The sum of these changes results in a dramatic increase in the fertilizing potential of the spermatozoa. When comparable numbers of spermatozoa from the caput or corpus epididymis were injected into one uterine horn of mature females, following ovulation induction, and spermatozoa from the cauda epididymis were injected into the contralateral horn, no fertilization was observed with caput epididymal spermatozoa, 1.7% of oocytes were fertilized by corpus epididymal spermatozoa, whereas 79.5% fertilization was obtained with cauda epididymal spermatozoa. Total sperm numbers increased from caput to corpus to cauda [28.3 ± 12.2, 40.6 ± 20.8, 144 ± 62 million, respectively]. The percentage of progressively motile spermatozoa increased from 27.9 ± 6.4 to 33.8 ± 4.8 to 70 ± 10.7 during this passage. Viability, measured by exclusion of the dye, propidium iodide, was significantly less in spermatozoa from the cauda than from the proximal or mid-caput epididymis. The percentage of the live cells that were stained intensely by rhodamine-123 (a measure of mitochondria1 membrane potential) increased during epididymal passage from 22.8 ± 7.8% in the proximal caput epididymis to 57.2 ± 16.5% in the cauda epididymis. Staining with acridine orange (a measure of DNA packaging in the sperm head) indicated an increase in chromatin condensation in cauda epididymal spermatozoa, when compared to those obtained from the caput or corpus.     

Investigation of epididymal sperm maturation in the golden hamster

During passage of hamster spermatozoa through the epididymis their maturation is shown to involve changes in the sperm head, midpiece (mitochondria) and tail. The sum of these changes results in a dramatic increase in the fertilizing potential of the spermatozoa. When comparable numbers of spermatozoa from the caput or corpus epididymis were injected into one uterine horn of mature females, following ovulation induction, and spermatozoa from the cauda epididymis were injected into the contralateral horn, no fertilization was observed with caput epididymal spermatozoa, 1.7% of oocytes were fertilized by corpus epiddymal spermatozoa, whereas 79.5% fertilization was obtained with cauda epididymal spermatozoa. Total sperm numbers increased from caput to corpus to cauda [28.3 ± 12.2, 40.6 ±20.8, 1434 ±62 mihon, respectively]. The percentage of progressively motile spermatozoa increased from 27.9 ±6.4 to 33.8 ± 4.8 to 70 ± 10.7 during this passage. Viability, measured by exclusion of the dye, propidium iodide, was significantly less in spermatozoa from the cauda than from the proximal or mid-caput epididymis. The percentage of the live cells that were stained intensely by rhodamine-123 (a measure of mitochondrial membrane potential) increased during epididymal passage from 22.8 ±7.8% in the proximal caput epididymis to 57.2 ± 16.5% in the cauda epididymis. Staining with acridine orange (a measure of DNA packaging in the sperm head) indicated an increase in chromatin condensation in cauda epididymal spermatozoa, when compared to those obtained from the caput or corpus.     

Two-dimensional gel electrophoretic analysis of rat sperm membrane interaction with cauda epididymal fluid

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) was used to analyze the polypeptide composition of rat cauda epididymal fluid, blood serum and membrane-enriched fractions of caput, corpus, and cauda epididymal spermatozoa. Several polypeptides were found in both cauda fluid and blood serum, and in both cauda fluid and epididymal spermatozoa. Prominent cauda epididymal fluid polypeptides that were associated with caput, corpus, and cauda sperm membranes were 32 and 33 kDa. Passage of spermatozoa from the caput to the cauda epididymidis was characterized by the loss of three glycopolypeptides of 32, 30 and 29 kDa, and by the addition of a 37-kDa glycopolypeptide. Incubation of intact caput, corpus and cauda spermatozoa with cauda epididymal fluid revealed major changes in the polypeptide maps of the incubation fluid and the membrane-enriched fractions of caput and corpus, but not cauda spermatozoa. The incubation of cauda fluid with caput and corpus sperm cells was characterized by a loss of several polypeptides and the addition of a 24-kDa glycopolypeptide. The most striking change in spermatozoa incubated with cauda epididymal fluid was the addition of two glycopolypeptides of 32 and 33 kDa to the polypeptide maps of caput sperm cells. These data demonstrate that rat spermatozoa undergo surface modifications during epididymal maturation and that these modifications can be influenced by epididymal fluid.     

Hormonal regulation of bovine secretory proteins derived from caput and cauda epididymal epithelial cell cultures

The goal of this study was to investigate the effect of hormones (testosterone, dihydrotestosterone [DHT], and hydrocortisone) on the protein secretion of caput and cauda epididymal epithelial cells cultured in principal cell medium (PCM). A confluent monolayer of caput and cauda epididymal epithelial cells was obtained from serum-containing PCM in the presence or absence of hormones after 7 days of culture at 38.5 degrees C (5% CO(2) in air). The protein secretion of epididymal epithelial monolayers incubated in serum-free PCM for 3 days was examined. The secreted proteins were separated by 2-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (2D SDS-PAGE). A comparison of the different protein patterns showed 61 spots, of which 11 were secreted only in the presence of hormones, 3 appeared to show hormone-related changes, and 25 were region-specific. Most of these secreted proteins were low-molecular-weight acidic proteins. To obtain evidence of the epididymal origin of the secreted proteins, proteins present in caput and cauda epididymal plasma were analyzed. In conclusion, our data indicate that hormones influence the synthesis of a number of caput and cauda epididymal proteins. Some of these proteins could be important for improving our understanding of spermatozoa maturation and storage and their acquisition of fertilizing ability.     

大鼠精子在附睾成熟过程中肉毒碱量变化的研究

肉毒碱被认为是与精子在附睾中成熟有关的成熟因子。本文报告测定大鼠附睾头部,体部及尾部精子中的肉毒碱。结果表明精子在循附睾头、体、尾运行过程中,精子中肉毒碱含量逐步增加,在附睾体一头部,附睾尾一头部的精子内的肉毒碱量呈现十分显著性差异。结果提示附睾体部或体一头部交界部位可能是精子在附睾中成熟的关键部位。     

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.     

Androgen Dependence of Testicular and Epididymal Angiotensin Converting Enzyme

Treatment with cadmium chloride (CdCl2) and cyproterone acetate (CA) depressed angiotensin converting enzyme (ACE) activity significantly in testes and epididymal regions of the adult rats compared to the corresponding untreated controls. Exogenous testosterone to CA-treated rats significantly increased the enzyme activity both in the testes and epididymis, the effect in the latter being very significant comparable to CA-treated and untreated controls. Testosterone failed to induce ACE activity in the testes and caput epididymis of 30 day-old immature rats, but the enzyme activity was detected in corpus and cauda epididymis. Our findings indicate that ACE activity in the testicular complex is possibly linked with androgen and is concerned with spermatogenesis and sperm maturation.     

The Presence of an Androgen Controlled Phospholipase A in Rat Epididymis

The presence of a phospholipase A (EC 3.1.1.4) and a lysophospholipase (EC 3.1.1.5) has been demonstrated in a postmitochondrial preparation from rat epididymis. Three different enzyme assays using both endogenous and exogenous substrates were employed. Phospholipase A was the rate limiting enzyme in the hydrolysis of lecithin to sn -3-glycerophosphorylcholine which also was the main product of the hydrolysis. The enzyme preparation did not show any phospholipase D (EC 3.1.4.4) nor glycerophosphinicocholine glycerophosphohydrolase (EC 3.1.4.2) activity. A small amount of phosphorylcholine was liberated during the hydrolysis indicating the presence of a glycerophosphinicocholine cholinephosphohydrolase (EC 3.1.4.38).
The phospholipase A activity per mg of protein was 10 times higher in the cauda than in the caput epididymidis, while the corpus epididymidis possessed an intermediate specific activity. Castration reduced the specific activity of the enzyme with about 50% in the caput and with about 95% in the cauda epididymidis. Testosterone supplementation restored the enzyme activity nearly to normal in the castrated animals.
This work suggests that the androgen control of the epididymal concen tration of sn-3-glycerophosphorylcholine is exerted via a control of the phospholipase A activity in the epididymal epithelial cells. It is suggested that this enzyme controls the concentration of epididymal glycerophosphorylcholine and the supply of long chain fatty acids to epididymal spermatozoa.     

有机阳离子转运子2在人类附睾中的表达及其意义

目的:研究人类附睾有机阳离子转运子2(OCTN2)mRNA的表达,探讨附睾肉碱转运机制,为探索男性避孕节育新技术提供理论依据。方法:应用RT-PCR方法检测人类附睾头、体、尾组织中OCTN2 mRNA的表达。结果:人类附睾头、体、尾组织中都存在OCTN2 mRNA表达。结论:人类附睾可能依赖OCTN2转运肉碱进入附睾管,为精子提供能量,促进精子成熟。对人类附睾OCTN2的进一步研究,将成为男性节育研究中新的分子靶标。     

Gamma-glutamyl transpeptidase activity in the epididymis during fetal and postnatal development in rats.

The histochemical and biochemical distributions of gamma-glutamyl transpeptidase (gamma-GT) were investigated in the epididymis of rats during fetal and postnatal development. In the epididymal homogenates, gamma-GT activity was detected on the fifth day after birth. A sharp increase was observed after 30 days of life in the caput homogenates. Moderate levels of the enzyme were found in the cauda epididymis. Gamma-GT is histochemically detected from the 15th day of gestation in Wolffian ducts and in 17- to 18-day-old fetuses in newly differentiated epididymal tubules. Enzyme activity, was associated with the plasma membranes (apical, lateral, and basal), was preponderant on the apical part of the epithelial cells. During the first 15 days of the postnatal life, the histochemical reaction intensities were identical from the caput to the cauda epididymidis. From the 18th day onwards, enzyme activity decreased in the corpus and in the cauda, while gamma-GT increased in the caput epididymidis, and a strong activity was found on the apical surface of epithelial cells. Weak or moderate gamma-GT activity of spermatozoa in the caput tubules, increasing steadily from caput to cauda epididymidis, suggests that gamma-GT may be related to the functional maturation of spermatozoa.     

Differential effect of hyperthyroidism on rat epididymal glycosidases

The impact of hyperthyroidism on epididymal glycosidases was studied in albino rats. Hyperthyroidism was induced in Wistar rats aged 30 days by daily injection of T4 (25 microg/100 g body weight/day intramuscularly) for 30 or 60 days; control rats were injected with vehicle (alkaline saline, pH 7.8). One set of hyperthyroid rats was reverted to euthyroid status by withdrawing T4 treatment after 30 days of hyperthyroidism. To asses the direct effect of thyroid hormone on epididymal hexosaminidases, caput, corpus and cauda tissues were stimulated with 25, 50 or 100 ng/mL T3 for 24 h, after an initial culture of 24 h. The activity of beta-glucosidase decreased in caput, corpus and cauda epididymis of hyperthyroid rats. beta-Galactosidase activity increased in the caput epididymis irrespective of the duration of hyperthyroidism. While a similar decrease occurred in the corpus and cauda epididymis in the 30 day hyperthyroid group, an opposite trend was observed in 60 day hyperthyroid rats. Caput beta-N-acetylglucosaminidase activities increased at both time points, whereas activity decreased in the corpus and cauda in 30 day, but increased in 60 day hyperthyroid rats. Hyperthyroidism consistently increased caput and corpus beta-N-acetylgalactosaminidase activity irrespective of the duration. Cauda epididymal beta-N-acetylgalactosaminidase activity was decreased in 30 day and increased in 60 day hyperthyroid rats. Hyperthyroidism induced changes in caput beta-galactosidase, beta-N-acetylgalactosaminidases, corpus beta-N-acetylglucosaminidase and cauda beta-N-acetylgalactosaminidase which were irreversible while the remaining actvities were brought back to normal when T4 treatment was withdrawn. In vitro studies showed that T3 stimulates epididymal hexosaminidases (beta-N-acetylglucosaminidase and beta-N-acetylgalactosaminidase) irrespective of the dose. These data suggest that thyroid hormones have a specific and direct influence on glycosidases in specific regions of the epididymis.     

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.     

Zinc Content in Epididymal Spermatozoa of Metoclopramide-Treated Rats

Zinc content was determined separately in spermatozoa taken from epididymal caput and cauda in rats. It was revealed that spermatozoa transported from the epididymal caput to the cauda reduce about 54% of zinc. This reduction is significantly inhibited in spermatozoa of rats receiving metoclopramide. That is also accompanied by a fall of testosterone level in blood serum and of delta 5, 3 beta-HSD activity in Leydig cells. It was found out that the reduction of zinc in spermatozoa at the time of their passage through the epididymis is the process that depends on androgens.     

The testicular and epididymal luminal amino acid microenvironment in the rat

Concentrations of amino acids were measured in arterial and testicular venous blood, and in fluids from the seminiferous tubule, rete testis, and the caput, corpus, and cauda epididymidis. There were no significant differences in the concentrations of any amino acids between arterial and testicular venous blood, whereas there were significant differences between arterial/venous blood and testicular interstitial fluid. The predominant amino acids measured within seminiferous tubule fluid (STF) and rete testis fluid (RTF) were glycine, alanine, glutamate, and glutamine. RTF contained approximately equal concentrations of basic and total amino acids, but 17 times higher acidic amino acids and 1.2 and 1.3 times lower uncharged polar and nonpolar amino acids, respectively, compared to STF. The concentration of total amino acids within caput fluid reached over 50 nmol/L, but then declined to approximately 50% and 0.1% of caput for corpus and cauda, respectively. The predominant amino acids measured within epididymal luminal fluids were glutamate and taurine; glutamate contributed to approximately 90% of the total amino acids measured in caput fluid. The presence of glutamate and taurine within the epididymal lumen is due primarily to a direct contribution from the epididymal epithelium, as measured using the split-drop stopped-flow microperfusion technique. Several other amino acids within the lumen also originate from the epididymal epithelium. Amino acids contribute approximately 20%, 9%, and 2% of the total osmolality of caput, corpus, and cauda fluid, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)