γ-Glutamyl transpeptidase in rat epididymis: effects of castration, hemicastration and efferent duct ligation

Gamma-Glutamyl transpeptidase (gamma-GT) was studied histochemically and biochemically in the rat epididymis after castration with or without testosterone treatment, or after hemicastration and ligation of the efferent ducts. There was a strong reaction to gamma-GT in the apical part of the epithelium in the caput epididymis, while in the corpus and cauda the reaction was confined mainly to the luminal contents. Castration caused a marked decline in epithelial gamma-GT activity within 10 days. Subsequent testosterone treatment (1 mg/day for 10 days) restored gamma-GT activity in the apical surface and lumen. After hemicastration of adult rats, and after hemicastration or ligation of the efferent ducts in immature 28-day-old rats, a small but significant (P less than 0.001) decrease was observed in gamma-GT activity in the epididymal caput compared to controls. The quantities of six other enzymes (beta-N-acetylglucosaminidase, beta-galactosidase, angiotensin-converting enzyme, alanyl amino-peptidase, dipeptidyl peptidase IV, acid phosphatase) also displayed significant changes after castration and restoration of activities by testosterone treatment. However, their distribution in the caput and cauda epididymis was more even than that of gamma-GT, and the changes after castration were less drastic. It is concluded that gamma-GT is a highly sensitive androgen-dependent secretory marker in the caput epididymis and may have an important function in 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.     

Glutathione, L-glutamic acid and γ-glutamyl transpeptidase in the bull reproductive tissues

The distribution of glutathione (GSH), L-glutamic acid (Glu) and gamma-glutamyl transpeptidase (gamma-GT) was studied in bull reproductive organs and fluids. Glutathione, the physiological substrate of gamma-GT, was localized specifically by a fluorescence method in the testis, epididymis and spermatozoa. Of the reproductive tissues, the testis, caput epididymis and ampulla had the highest levels of GSH, but it was also present in seminal fluid. Washed caput epididymal sperm had three times the GSH content of cauda epididymal or ejaculated sperm. In spermatozoa, GSH displayed maximal staining in the midpiece and tail regions. The highest levels of gamma-GT were encountered in the epididymis. The concentration of Glu was also high in the epididymis. Its formation may be due to the hydrolytic activity of gamma-GT, which, in addition, may have an important role in the transfer of Glu residues to reactive groups on the sperm surface.     

Nonprotein thiols and disulfides in rat epididymal spermatozoa and epididymal fluid: role of gamma-glutamyl-transpeptidase in sperm maturation

Sperm thiol oxidation during sperm maturation is important for sperm component stabilization, the acquisition of sperm motility, and fertilizing ability. A correct degree of oxidation is required, since spermatozoa are very susceptible to oxidative damage. The pathways involved in physiologic sperm thiol oxidation in the epididymis are not completely understood. The nonprotein thiol glutathione (GSH), in addition to playing a major role as an antioxidant and in eliminating toxic compounds, has been implicated in prooxidation processes in various cells, via gamma-glutamyl-transpeptidase (gamma-GT)-dependent catabolism. Little information is available on the dynamics of nonprotein thiols (NPSHs) and disulfides (NPSSNPs) in spermatozoa and epididymal fluid (EF) during sperm passage in the epididymis. It is not clear whether NPSHs and NPSSNPs are involved in sperm protein thiol (PSH) oxidation or whether GSH catabolism in the epididymis can serve as a pathway for sperm PSH oxidation. In the present study, we used the thiol fluorescence labeling agent monobromobimane to analyze NPSHs and nonprotein disulfides (NPSSRs) (R, nonprotein or protein) in spermatozoa and EF in the rat caput and cauda epididymis. NPSH levels are shown to be significantly higher in the caput than in the cauda (spermatozoa and fluid). GSH in the caput lumen is subject to high gamma-GT activity. A marked loss of sperm GSH and a shift to an oxidized state (resulting in a significantly higher concentration of glutathione disulfides [GSSRs] than GSH) occur during the passage of spermatozoa from the caput to the cauda epididymis. Caput EF and extracellular NPSSNPs induce sperm thiol oxidation. The results suggest that epididymal NPSH/NPSSNP participates in sperm PSH oxidation and that some reactions of GSH in the gamma-GT pathway (in the epididymis) provide oxidizing power, leading to physiologic sperm thiol oxidation.     

The ultrastructure of dog epididymis

Summary The ultrastructure of the epididymal duct and ductuli efferentes in the dog has been studied by electron microscopy. The epididymidis can be separated into the classical divisions of caput, corpus and cauda epididymidis on the basis of general morphology and ultrastructure. The ductuli efferentes have a low epithelium with pronounced cilia at the apices of cells and appear to provide primarily a transport role for spermatozoa. In the epididymis proper the caput region is characterized by an extremely large Golgi apparatus with large numbers of lysosomes and nuclear inclusions. Secretory activity appears to be most common in the corpus region. Absorption and secretion are most active in the first two segments while in the cauda epideidymidis the long-term storage of spermatozoa in the lumen is associated with many dense crystalline bodies formed in the epithelial cells within the Golgi apparatus and possibly deriving from absorbed macromolecular material from the lumen. The theory of whole sperm cell resorption by the epididymal duct is not supported by this study.     

Modification of the rat sperm flagellar plasma membrane during maturation in the epididymis

Previously, we demonstrated that surface radiolabeling of rat epididymal spermatozoa by lactoperoxidase-catalyzed iodination reveals a major component with an apparent molecular weight of 26,000 to 28,000 daltons (26 kDa) on spermatozoa from the cauda but not the caput epididymidis. To characterize this surface component further, sperm surface constituents radiolabeled by lactoperoxidase-catalyzed iodination were separated by 2-D PAGE. The 26 kDa component was localized by autoradiography and appeared as the major labeled acidic spot on cauda spermatozoa, but neither a radiolabeled spot nor a corresponding stained spot was present on caput spermatozoa. The 26 kDa spot was excised from 2-D gels of plasma membranes from cauda spermatozoa and utilized for immunization. The monospecific antiserum stained a single band of 26 kDa on Western blots of SDS-PAGE-separated plasma membranes from cauda spermatozoa and in a 100,000 X g supernatant fluid of the luminal contents of the cauda epididymidis. Immunohistochemical staining of cauda spermatozoa revealed antigen exclusively on the flagellar domain; the antigen was not seen on caput spermatozoa but first appeared in spermatozoa from the proximal corpus epididymidis. Immunoelectron microscopy confirmed the 26 kDa component was localized to the external face of the flagellar plasma membrane. Immunohistochemical staining of caput spermatozoa incubated in vitro with cauda epididymal luminal fluid revealed the 26 kDa component specifically bound the flagellar domain of immature spermatozoa.     

Glutathione peroxidase activity in cell cultures from different regions of human epididymis

AIM: To study the secretory activity and androgen regulation of glutathione peroxidase (GPx) in epithelial cell cultures from human epididymis. METHODS: Tissue was obtained from patients undergoing therapeutic orchidectomy for prostatic cancer. Epithelial cell cultures were obtained from the caput, corpus and cauda epididymides. Enzymatic activity was measured in conditioned media by colorimetric methods in absence or presence of 1, 10 or 100 nmol/L testosterone. The effect of 1 micromol/L flutamide was also evaluated. RESULTS: GPx activity was higher in cultures from corpus and cauda than caput epididymidis. The presence of different concentrations of testosterone increase enzyme activity in cell cultures from all epididymal regions. Addition of flutamide reverses the androgen dependent increase of GPx activity. CONCLUSION: GPx activity is secreted from human epididymal cells in a region dependent manner and is regulated by androgens.     

Changes in surface protein structure of bull spermatozoa during epididymal maturation

The surface proteins of bull spermatozoa from caput and cauda epididymidis were labelled by lactoperoxidase-catalyzed radioiodination and solubilized and analysed by SDS-PAG-electrophoresis. The surface protein patterns of caput and cauda epididymal spermatozoa resembled each other but some distinct differences could be found. Caput epididymal spermatozoa revealed a protein peak with molecular weight of 15 000 - 18 000 daltons but this peak was not found on cauda epididymal spermatozoa. On caput epididymal epermatozoa the most intensely labelled protein peak was located between 90 000 and 100 000 daltons but on cauda epididymal spermatozoa the corresponding peak was only weakly labelled and had a molecular weight of 80 000–90 000 daltons. Surface protein with molecular weight of 42 000–47 000 daltons was dominating on cauda epididymal spermatozoa. The surface protein structure of cytoplasmic droplets did not drastically differ from that of epididymal spermatozoa.     

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.     

Alterations in protein profile of rat spermatozoa during maturation

Alterations in the polypeptide pattern of rat spermatozoa during epididymal transit were studied by SDS-PAGE and compared with that of epididymal cytosol and luminal fluid. The total number of cytosol and luminal fluid polypeptides increase from caput to cauda epididymidis but sperm associated polypeptides decrease during epididymal transit. Changes in polypeptide pattern of spermatozoa are due to their acquisition, loss or modification. Spermatozoa acquire seven polypeptides, of which six are acquired in corpus (MW 16.5, 38, 41, 72, 75 and 100 Kdal) and one (MW 28.5 Kdal) in cauda epididymidis. Spermatozoa lose one polypeptide of MW 72.5 Kdal in caput and two polypeptides of MW 70 and 115 Kdal in cauda epididymidis. Four polypeptides of MW 18.5, 19.5, 64 and 67.5 Kdal disappear from cauda spermatozoa without appearing in the luminal fluid. Polypeptide of MW 62.5 Kdal is observed only in spermatozoa and luminal fluid from cauda epididymidis.     

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.     

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.     

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.     

The Effect of Epididymal and Testicular Fluids on the Fertilising Capacity of Testicular and Epididymal Spermatozoa

It is possible that the fertilising capacity of spermatozoa in the epididymis is influenced by the epididymal secretion. We have studied this problem by obtaining spermatozoa before entry into the epididymis and after passage through it, incubating both types of spermatozoa in fluids from the rete testis and cauda epididymidis and then checking their fertilising capacity. While spermatozoa from the rete testis were infertile, rete testis fluid did not decrease the fertilising capacity of epididymal sperm from the cauda epididymidis. Fluid from the cauda epididymidis did not promote the fertilising capacity of testicular spermatozoa. These results are discussed in the light of the current understanding of epididymal physiology.     

Immunolocalization of NBC3 and NHE3 in the rat epididymis: colocalization of NBC3 and the vacuolar H+-ATPase

In the male reproductive tract, the epididymis plays an important role in mediating transepithelial bicarbonate transport and luminal acidification. In the proximal vas deferens, a significant component of luminal acidification is Na+-independent, and mediated by specific cells that possess apical vacuolar proton pumps. In contrast, luminal acidification in the cauda epididymidis is an Na+-dependent process. The specific apical Na+-dependent H+/base transport process(es) responsible for luminal acidification have not been identified. A potential clue as to the identity of these apical Na+-dependent H+/base transporter(s) is provided by similarities between the transport properties of the epididymis and the mammalian nephron. Specifically, the H+/base transport properties of caput epididymidis resemble the mammalian renal proximal tubule, whereas the distal epididymis and vas deferens have characteristics in common with renal collecting duct intercalated cells. Given the known expression of the Na+/H+ antiporter, NHE3, in the proximal tubule, and of the electroneutral sodium bicarbonate cotransporter, NBC3, in renal intercalated cells, we determined the localization of NHE3 and NBC3 in various regions of rat epididymis. NBC3 was highly expressed on the apical membrane of apical (narrow) cells in caput epididymidis, and light (clear) cells in corpus and cauda epididymidis. The number of cells expressing apical NBC3 was highest in cauda epididymidis. The localization of NBC3 in the epididymis was identical to the vacuolar H+-ATPase. The results indicate that colocalization of NBC3 and the vacuolar H+-ATPase is not restricted to kidney intercalated cells. Moreover, the close association of the two transporters appears to be a more generalized phenomenon in cells that express high levels of vacuolar H+-ATPase. Unlike NBC3, NHE3 was most highly expressed on the apical membrane of all epithelial cells in caput epididymidis, with less expression in the corpus, and no expression in the cauda. These results suggest that apical NBC3 and NHE3 potentially play an important role in mediating luminal H+/base transport in epididymis.     

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.     

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.     

Transepithelial movement of non-polar and polar compounds in male rat reproductive tubule examined by in vivo microperifusion and in vivo micropuncture

Proluminal movement of 3H-testosterone and 3H-sucrose from peritubular to intratubular fluids of the adult rat testis and epididymis was investigated by using in vivo microperifusion and subsequent micropuncture of seminiferous tubules and caput, corpus, and cauda epididymal tubules. Tubules were perifused with Minimum Essential Medium containing 3H-testosterone or 3H-sucrose. 14C-polyethyleneglycol was included in the perifusion fluid as a marker for contamination of intraluminal fluid by interstitial fluid. Radioactivity of isotopes in perifusion and intraluminal fluids was determined at one and two hours after perifusion and the percentage of peritubular isotopes appearing in intraluminal fluid was determined. Net entry of 3H-sucrose into the seminiferous and epididymal tubules was significantly reduced. Proluminal movement of 3H-androgen across the seminiferous epithelium was also restricted. In contrast, intraluminal 3H-androgen concentrations in caput epididymal fluid were 200 to 300% of those in peritubular fluid at both one and two hours after perifusion. Similar results were found in the corpus epididymidis. 3H-androgen concentrations in cauda epididymal fluid were approximately 125% of peritubular isotope concentrations. The exact mechanism underlying this uphill proluminal movement of 3H-androgen into the rat epididymal lumen remains to be elucidated.     

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.     

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。