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.     

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.     

Electrolyte and Water Transport in Rat Epididymis; Its Possible Role in Sperm Maturation

Electrolyte and water transport in different regions of the rat epididymis has been studied using a microperfusion technique. The caput and proximal corpus epididymides were found to absorb NaCl and water and secrete K⁺ at a lower rate than the cauda epididymidis. The secretion rate of protein was the same in both regions. In the caput and proximal corpus, reabsorption of chloride was hypertonic. Reabsorption of sodium could not account for water reabsorption. In contrast, water reabsorption in the cauda epididymidis was dependent upon the intraluminal sodium ions. Amiloride inhibited both the Na⁺ and water reabsorption in this region. It was concluded that in the proximal regions of the rat epididymidis, water reabsorption may be secondary to an active transport of chloride, whereas in the cauda, a net transepithelial transport of sodium ions is the driving force for water reabsorption.
Transport of electrolytes and water across the perfused rat cauda epididymidis has also been studied under various experimental conditions. Treatment of rats with alpha-chlorohydrin (9 mg/kg/day) for 7 days inhibited the rate of sodium and water reabsorption without affecting the secretion of proteins. Ligation of the testicular efferent duct or the corpus epididymidis had no significant effect on the transport functions of the cauda epididymidis. When cyproterone acetate (10 mg/rat/day) was injected into male rats, the rate of sodium and water reabsorption was reduced. This effect was accompanied by a loss of sperm motility. It is concluded that the transport functions of the cauda do not require the normal flow of testicular fluid, but may depend on the supply of circulating androgen in the blood. Alpha-chlorohydrin and cyproterone acetate may affect sperm maturation by disrupting the normal milieu of the epididymal duct.     

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.     

The presence of α-glucosidase, glycerophosphocholine and carnitine in the epididymis and ejaculate of the vervet monkey (Cercopithecus aethiops) and the Chacma baboon (Papio ursinus)

Summary.  The epididymis is the site of post-testicular sperm maturation in the male genital tract. Studies on human epididymides are hampered by the practical inaccessibility of epididymides of healthy men in their reproductive years. The limited use of laboratory animals therefore seems unavoidable. The objective was to establish baseline values of the epididymal markers α-glucosidase, glycerophosphocholine (GPC) and carnitine in the lumen of the caput, corpus and cauda epididymidis and in the ejaculate of adult male Chacma baboons and vervet monkeys. In both primates, α-glucosidase was found throughout the epididymis and in the ejaculate; values did not vary significantly. In monkeys, the highest concentration of GPC was found in the cauda epididymidis, but smaller amounts were found in the other regions and the ejaculate. In baboons, GPC was absent from the caput, but present in the other regions, including the ejaculate. Carnitine concentrations increased significantly from the caput to the cauda in monkeys and from the caput to the corpus in baboons. With this study, the relative concentration ranges in which these markers are present in the epididymides of these primates have been established. In future studies, changes in concentrations of these substances would probably indicate changes in epididymal function.     

Zinc content in the epididymis, vas deferens, prostate, and seminal vesicles of juvenile rhesus monkey (Macaca mulatta): effect of androgen and estrogen

The zinc content in the three segments of the epididymis (caput, corpus, and cauda), vas deferens, seminal vesicles, and prostate of juvenile monkeys was determined by atomic absorption spectrophotometry. Zinc content (micrograms/gm wet weight) was found to be maximum (328) in the vas deferens; in the other organs it measured in the following order: caput 191, corpus 238, cauda 193, prostate 133 and seminal vesicles 85. In order to investigate the endocrine control of the zinc in these organs, two groups of animals were treated with testosterone propionate (2 mg) or estradiol dipropionate (10 micrograms) once daily for 30 days. In response to androgen, a rise in both concentration and content of zinc was evident only in the prostate. The results further suggested that the prostatic zinc may be under dual hormonal control, but in the epididymis and vas deferens it may be under the influence of estrogen. It is concluded that the hormonal effects on zinc content and growth stimulation in accessory sex organs are quite separate and may be under different hormonal control.     

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.     

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.     

Epididymal carbohydrate metabolism in experimental hypercorticosteronism: studies on mature male rats

Corticosterone induced changes in serum hormonal profiles and the key enzymes involved in glycolytic and pentose phosphate pathways were studied in caput, corpus and cauda epididymides of mature male rats (200–250 g body weight). Corticosterone (3.5 mg/100 g body weight sc. for 20 days) treatment was found to depress serum testosterone and prolactin while the gonadotrophins were unaltered. Enzymes of both the glycolytic and pentose phosphate pathways were significantly decreased in caput epididymidis. But in corpus and cauda epididymides only the glycolytic enzymes were reduced. Withdrawal of treatment (for 20 days), resulted in restoration of the glycolytic enzymes to normalcy. The serum hormonal profiles were also found to be within the normal range. The pentose phosphate pathway in caput epididymidis showed a significant increase in enzyme activities following withdrawal of treatment. From the present investigation it is clear that hypercorticosteronism had a definite influence on epididymal enzymes involved in carbohydrate metabolism.     

Loss of sperm surface sialic acid induces phagocytosis: an assay with a monoclonal antibody T21, which recognizes a 54K sialoglycoprotein

Using a monoclonal antibody T21, we reported that a mouse sperm maturation-associated antigen sialoglycoprotein of 54000 daltons (54K sialoglycoprotein) was secreted at the distal caput to proximal corpus epididymidis and that the 54K sialoglycoprotein had a hidden determinant (cryptodeterminant), which could be eliminated by sialidase treatment (Toshimori et al. (1988): Histochemistry 90:195-200; (1990a): Biol Reprod 42:151-160; (1990b): Arch Histol Cytol 53:339-349). This study evaluated the mouse sperm susceptibility to phagocytosis by macrophage in vitro. Comparisons were made between sperm from the caput epididymidis (caput sperm) incubated in modified Krebs Ringer's solution (MKR) and caput sperm incubated in MKR containing cauda fluid, and between sialylated (sialidase-untreated) sperm from the corpus and cauda epididymidis (corpus/cauda sperm) and desialylated (sialidase-treated) corpus/cauda sperm. The results showed that macrophages were least actively engaged in phagocytosis for caput sperm incubated in MKR containing cauda fluid, and most active for desialylated corpus/cauda sperm. Incubation of caput sperm in MKR containing cauda fluid revealed that the 54K sialoglycoprotein in cauda fluid could be bound to the flagellar surface of caput sperm. These results together with previous findings strongly suggest that the 54K sialoglycoprotein bound to immature sperm during maturation in the epididymis is implicated in the protection of sperm from phagocytosis with the aid of sialic acid residues.     

Further characterization of a secreted epididymal glycoprotein in mice that binds to sperm tails

Sperm maturation antigen 4 (SMA-4) is a surface component of the mouse sperm tail. Previously, immunofluorescence studies indicated that SMA-4 may be secreted by principal cells of the distal caput epididymidis and bound to spermatozoa as they pass through that region of the duct. In the present study, detergent extracts of spermatozoa from the cauda epididymidis were subjected to polyacrylamide gel electrophoresis under reducing and denaturing conditions, transferred to nitrocellulose, and immunostained with a monoclonal antibody against SMA-4. A band of approximately 54,000 molecular weight was revealed. The band was also stained by the periodic acid-Schiff (PAS) procedure. This glycoprotein was not detected in extracts of spermatozoa from the proximal caput epididymidis or of spermatozoa from the cauda epididymidis that were preincubated for 4 hours in an in vitro fertilization environment. Blots of sperm-free fluid from the corpus and cauda epididymidis displayed an immunoreactive and PAS-positive band of about 85,000 molecular weight that was not observed in fluid from the caput epididymidis. The difference in the molecular weights of the antigen in the fluid and that in extracts of cauda spermatozoa suggests that SMA-4 may be modified chemically upon association with the sperm surface.     

Experimental chlamydial epididymitis

Male Wistar rats were infected with the chlamydial agent of guinea pig inclusion conjunctivitis by inoculation of chlamydiae into the vas deferens. Epididymitis was observed in all infected animals clinically and histologically. Chlamydiae were detected in the epithelium of epididymal tubules by immunohistochemical staining (alkaline phosphatase anti-alkaline phosphatase technique). Inflammation progressed from the cauda to the corpus and caput epididymidis leading to fibrosis of the cauda epididymidis 28 days after infection. Animals responded to the infection with a rise of both serum IgM and IgG antibodies.     

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.     

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.     

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.     

Regional Differences in Steroidogenesis and Hormone Levels in the Epididymis and Vas Deferens of Adult Rats

In vivo and in vitro studies with different parts of the epididymis and vas deferens were carried out to determine their inherent capacity to synthesize steroids and to correlate with the endogenous levels with or without the administration of hCG.
Incubation with ¹⁴C-labelled pregnenolone and testosterone demonstrated that caput epididymidis was more active than other parts in synthesizing testosterone from ¹⁴C-pregnenolone and in converting labelled testosterone to 5α-dihydrotestosterone (DHT). The cauda epididymidis and vas deferens accumulated more radioactivity in progesterone and dehydroepiandrosterone (DHEA) than the caput epididymidis.
The levels of DHT, testosterone and 4-androstene-3,17-dione in the caput epididymidis were reduced after ligation of ipselateral efferent ductules indicating the testicular origin of these steroids. The cauda epididymidis and vas deferens had higher levels of progesterone as compared to the other regions of the epididymis, which were decreased after the ligation. Intravenous injection of hCG increased the levels of oestradiol-17β in all tissues and markedly in the cauda epididymidis and vas deferens. The high levels of progesterone and oestradiol-17β present in these organs may be of importance in maintaining fertilizing ability of spermatozoa stored in the cauda epididymidis and vas deferens and their transport.     

Immunohistochemical Localization of Neuropeptide Y in Nerves of the Male Genital Tract of the Photoperiodically Responsive Djungarian Hamster, Phodopus Sungoras/Immunhistochemische Lokalisation von Neuropeptid Y in Nerven der männlichen Genitalorgane des photoperiodisch sensiblen Djungarischen Zwerghamsters, Phodopus sungorus

Neuropeptide Y (NPY) was demonstrated by immunohistochemistry in nerves of the male genital tract of Phodopus sungorus at long (LD 16:8) und short (LD 8:16) photoperiods. No immunoreactive nerve fibres could be demonstrated in the testis, caput and corpus epididymidis and the ventral prostate gland. Dense networks of NPY-containing nerve fibers were demonstrated in the smooth muscle layer of the sperm-transporting duct, beginning in the cauda epididymidis with increasing density towards the distal part of the ductus deferens, and in the smooth muscle layer of the seminal vesicles. At short photoperiods, the density of the NPY-containing nerve plexus decreased only in the smooth muscle layer of the ductus deferens. A "trophic" influence of the large smooth muscle cells of the ductus deferens on their nerves not only in regard to their noradrenaline, but also on their NPY content is discussed.     

Cell- and region-specific localization of lysosomal and secretory proteins and endocytic receptors in epithelial cells of the cauda epididymidis and vas deferens of the adult rat.

The epithelial cells lining the cauda epididymidis and vas deferens are active in endocytosis and have an abundance of lysosomes and a well-characterized secretory apparatus. However, little is known about the nature of lysosomal proteins contained within lysosomes, the types of receptors on the cell surface, and the types of proteins secreted by these cells. In the present study, cathepsins A, D, B, and sulfated glycoprotein (SGP)-1, well-characterized lysosomal proteins, as well as SGP-2, a secretory protein and low-density lipoprotein receptor-related protein-2 (LRP-2), an endocytic receptor, were immunolocalized at the light-microscopic level within epithelial cells of the cauda epididymidis and vas deferens. Principal cells showed numerous intensely reactive lysosomes for cathepsins A, D, and SGP-1 in all regions of the cauda and vas deferens and for cathepsin B only in the cauda epididymidis. Basal cells were intensely reactive for cathepsin A, unreactive for cathepsins D and B, and weakly reactive for SGP-1 in the cauda region. In the vas deferens, these cells were intensely reactive for cathepsin A and SGP-1 and unreactive for cathepsin B; in the case of cathepsin D, basal cells were weakly reactive in the proximal vas deferens but intensely reactive in the middle and distal vas deferens. Clear cells, present in the cauda region and proximal vas deferens, were intensely reactive for cathepsin A, weakly reactive for SGP-1, and unreactive for cathepsins D and B, while narrow cells found mainly in the proximal vas deferens were intensely reactive for cathepsins A, D, and SGP-1 and unreactive for cathepsin B. Thus, the expression of different lysosomal enzymes in the cauda epididymidis and vas deferens is not only cell- but also region-specific, suggesting differences in the type of substrates internalized by these cells. SGP-2, a secretory protein, showed a checkerboardlike staining pattern in the cytoplasm of principal cells of the cauda epididymidis, while the cytoplasm of all principal cells were intensely reactive in the vas deferens. This type of reaction, as well as staining of sperm, suggests that SGP-2 is secreted into the lumen, where it functions in relation to sperm. The endocytic receptor LRP-2 was noted only on the apical surface of principal cells of the cauda and vas deferens and in spherical structures indicative of endosomes suggestive of their role in the uptake of various ligands, including SGP-2, for which it has a high binding affinity. Thus SGP-2 in the cauda and vas deferens is not only secreted but endocytosed by principal cells, suggestive of an active turnover in the lumen. In summary, the epithelial cells of the cauda and vas deferens show marked differences in expression of lysosomal proteins, SGP-2, and LRP-2 suggestive of differences in their functional activity while sperm are stored and protected in these regions.     

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.