Sperm survival versus degradation in the Mammalian epididymis: a hypothesis

A long-standing problem in epididymal physiology is the fate of unejaculated spermatozoa in the cauda epididymidis under conditions such as congenital absence of the vas deferens, long-term vasectomy, or castration. There is no convincing evidence for significant absorption of spermatozoa, defective or otherwise, by spermiophagy or dissolution in the epididymis of normal animals. Spermiophagy by epithelial cells or intraluminal macrophages may take place if the duct ruptures and granulomas form (e.g., after experimental ligation), although there is no quantitative information on the rate of sperm removal by this means. In one animal model (the rabbit), the epididymis is unusually resistant to granuloma formation and has provided unique insights into a phenomenon that is suggested to be present in all species. Spermatozoa retained in the rabbit cauda epididymidis by placing ligatures on the vas deferens and corpus epididymidis degenerate after several weeks but do not decrease significantly in numbers. After castration, however, they die very rapidly and >90% disappear. It is hypothesized that, in the normal androgen-maintained epididymis, degradative pathways are present in the luminal fluid that are constitutively inhibited by survival signals emanating from the epithelium. In the absence of androgen, the intraluminal mileau changes and death signals predominate that activate degradative pathways via the ubiquitin-proteasome system, DNAses, etc., to mediate dissolution of sperm organelles and nucleoprotein. It is suggested that the latter condition is the default situation and is only prevented by the stimulatory action of androgens on the epididymal epithelium.     

Maturation and regulation of the motility of spermatozoa in the epididymis of the tammar wallaby (Macropus eugenii).

Demembranated spermatozoa from the rete testis developed vigorous flagellation when reactivated with ATP, but showed no forward progression such as that seen in samples from the cauda epididymidis. The proportion of spermatozoa that were reactivated was smaller for samples from the rete testis than from the cauda epididymidis. Studies in vitro of undiluted micropuncture samples from the epididymis indicated that the activity of spermatozoa is suppressed as they develop the capacity for motility. However, as spermatozoa spontaneously became activated during the collection or subsequent incubation of undiluted samples, it was concluded that the suppressive action is labile. The activity of spermatozoa in vitro was examined in diluted samples from the cauda epididymidis. A concentration of 2.5 mmol extracellular calcium/l was better than lower concentrations. Diluents at pH 5.5 completely inhibited sperm motility when they contained 20 mmol lactate/l (but not glutamate) and the effect was reversed by readjusting the diluent to pH 7.4. However, lactate was not considered to suppress sperm motility in situ, as the plasma from the cauda epididymidis contained only 2.7 +/- 0.5 mmol lactate/l. There was no effect of sodium concentration (1 and 115 mmol/l), pH (5.5 and 7.4) or amiloride (0 and 1 mmol/l) on sperm motility, indicating that motility is not dependent on the concentration of sodium above 1 mmol/l or on a sodium-proton exchange system. The relative viscosity of plasma from the cauda epididymidis did not affect the motility of spermatozoa.     

Changes in protein composition of the luminal fluids along the epididymis of the tammar, Macropus eugenii

Micropuncture samples of luminal fluid were collected from the rete testis and along the epididymis. Quantitative analyses showed that the ductuli efferentes reabsorb about half the protein leaving the testis. Considerable protein is secreted by the caput epididymidis (initial segment) and there is a net loss of protein from the corpus and cauda epididymidis. Denatured, polyacrylamide gel electrophoresis showed that there are 5 proteins in rete testis fluid which are not present in blood (Mr of 14,700, 22,800, 24,100, 43,000 and 44,800). One of these proteins (Mr 14,700) is lost from plasma in the ductuli efferentes and 2 (Mr 43,200 and 44,800) are lost in the corpus epididymidis. Twelve proteins appear in the epididymal plasma and are not present in rete testis fluid or blood: 6 appear in the caput epididymidis (Mr 30,000, 31,000, 32,300, 17,400, 18,700 and 21,400), 3 in the corpus epididymidis (Mr 12,800, 39,800 and 90,600) and 3 in the cauda epididymidis (Mr 10,900, 56,300 and 63,000). A protein with the same molecular weight as a blood protein (149,500) accumulates in the corpus and cauda epididymidis. None of the samples of luminal fluid contained particulate matter other than spermatozoa, indicating that the tammar is a useful animal for micropuncture studies.     

Distribution of 5 alpha-reductase in the epididymis of the tammar wallaby (Macropus eugenii) and dependence of the epididymis on systemic testosterone and luminal fluids from the testis

The activity of 5 alpha-reductase was much higher in the caput and corpus epididymidis than in the cauda epididymidis. Orchidectomy caused a reduction in 5 alpha-reductase activity in the caput and corpus epididymidis, and regression of the epithelium and reduction in mass of all regions of the epididymis. Subsequent testosterone therapy caused a substantial increase in amount of epithelium and overall mass of the cauda epididymidis but showed little or no increase in any of the responses measured in the caput and corpus epididymidis. We concluded that the caput and corpus epididymidis of the tammar respond to factors other than testosterone, probably some constituent in the luminal fluid, and therefore are homologous with the initial segments of the epididymis in eutherians.     

Biochemical characterization of two ram cauda epididymal maturation-dependent sperm glycoproteins

Rabbit polyclonal antibodies were raised against ram cauda epididymal sperm proteins solubilized by N-octyl-beta-D-glucopy-ranoside (anti-CESP) and against proteins of the fluid obtained from the cauda epididymidis (anti-CEF). The anti-CESP polyclonal antibody reacted with several bands from 17 to 111 kDa with different regionalization throughout the epididymis. The strongest epitopes at 17 kDa and 23 kDa were restricted to the cauda epididymidis. The anti-CEF polyclonal antibody reacted mainly with a 17-kDa and a 23-kDa compound in the cauda sperm extract. These cauda epididymal 17- and 23-kDa proteins disappeared after orchidectomy, but they reappeared in the same regions after testosterone supplementation, indicating that they were secreted by the epithelium. The fluid and membrane 17- and 23-kDa antigens had a low isoelectric point and were glycosylated. The fluid 17- and 23-kDa proteins had hydrophobic properties: they were highly enriched in the Triton X-114 detergent phase and could be extracted from the cauda epididymal fluid by a chloroform-methanol mixture. These proteins were further purified, and their N-terminal sequences did not match any protein in current databases. A polyclonal antibody against the fluid 17-kDa protein recognized the protein in the cauda epididymal sperm extract and immunolocalized it on the sperm flagellum membrane and at the luminal border of all cells in the cauda epididymal epithelium. These results indicated that secreted glycoproteins with hydrophobic properties could be directly integrated in a specific domain of the sperm plasma membrane.     

Cauda epididymal sperm motility: a comparison among five species

Rat spermatozoa are immotile in the cauda epididymidis and are kept quiescent by a protein which increases viscoelasticity of cauda luminal fluid. How species-specific this phenomenon is, is unknown. In the present study, the motility of cauda epididymal spermatozoa of rats, hamsters, guinea pigs, rabbits and humans have been investigated. Sperm motility was observed in undiluted cauda sperm samples and in samples diluted with physiological diluents with or without Ca++, among others. Hamster sperm were studied in further detail to determine if the motility inhibiting factor in hamster cauda lumen fluid had characteristics similar to those previously described in the rat. Cauda fluid protein concentrations and apparent viscoelasticity were also determined and related to cauda sperm motility in all species. The results demonstrated that all species studied except rabbits have immotile sperm in their native cauda fluid and that additional Ca++ is not a factor in the initiation of motility. Cauda sperm immotility is not always related to fluid viscosity, however, so other as yet unknown mechanisms must be called upon in some species. The vigorous motility of rabbit spermatozoa in their native fluid implies that a fundamental difference exists in the relationship between epididymis and spermatozoa in rabbits from that observed in other species.     

Regulation of the elemental composition of the epididymal fluids in the tammar, Macropus eugenii

Micropuncture, microanalytical and microelectrode techniques were used to study electrochemical aspects of 7 elements and fluid in the ductuli efferents and ductus epididymidis of the tammar. Rete testis fluid was isosmotic with blood and had a lower pH. It also contained lower concentrations of bicarbonate, sodium, calcium, magnesium, phosphorus and sulphur and higher concentrations of potassium and chloride than blood. The luminal fluid was acidified further during passage through the sperm ducts and all of the elements which were studied moved in or out of the lumen, usually against an electrochemical gradient. The ductuli efferents reabsorbed 87% of the fluid leaving the testis without changing the intraluminal concentrations of sodium, potassium and calcium, but the concentrations of magnesium, phosphorus and sulphur increased. The caput epididymidis reabsorbed about half the fluid entering it: sodium concentrations decreased and those of potassium and phosphorus increased. There was also some fluid reabsorption and an increase in the values of potassium and phosphorus in the corpus epididymidis. There was little net transport of fluid in the cauda epididymidis; sodium, chloride, magnesium and phosphorus concentrations decreased and potassium values increased. Studies involving filtration through a dialysis membrane of blood and fluid from the rete testis and cauda epididymidis showed that, whilst some of the calcium, magnesium, phosphorus and sulphur was associated with high molecular weight compounds in blood, the association was not significant in the reproductive fluids.     

Evidence for the presence of oxytocin in the ovine epididymis

The testes of several species contain oxytocin and/or neurophysin, but the content or localization of oxytocin in epididymal tissue has not been studied. The present study was undertaken to localize oxytocin and neurophysin in epididymal tissue of the ram, and to quantify oxytocin in the ductus epididymidis and fluids entering and leaving the ductus epididymidis. Neurophysin was not detected in the epididymis; thus, synthesis of oxytocin by the epididymis is unlikely. Immunohistochemical localization of oxytocin was confined to the epithelium and capillaries. Oxytocin immunostaining was most intense for epithelium of the caput and declined in corpus and cauda regions. However, based on radioimmunoassay, no difference in oxytocin concentration was detected among regions of the epididymis. Since rete testis fluid entering and cauda epididymal fluid leaving the epididymis contained at least fourfold more oxytocin than testicular venous plasma, it was concluded that regional differences in epithelial concentration of oxytocin may have been masked by oxytocin contained in the luminal fluid. It was concluded further that the epididymis of the ram does not synthesize oxytocin, but about 22 ng/day enters the epididymis in rete testis fluid. Most of this luminal oxytocin apparently is absorbed by the epithelium of the caput epididymidis, with additional adsorption in the corpus and cauda. Although a role for oxytocin in ductal contractility cannot be excluded, it is more likely that the luminal oxytocin influences epithelial or sperm function.     

Sperm maturation, fluid transport, and secretion and absorption of protein in the epididymis of the echidna, Tachyglossus aculeatus

Extratesticular sperm maturation in the echidna mainly occurs in the initial segment of the ductus epididymidis. The process involves the development of motility, migration and loss of the cytoplasmic droplet, a decrease in permeability to Congo red and the formation of sperm bundles. The spermatozoa are supported in the bundles by a matrix of electron-dense material; the bundles are very motile is undiluted samples of luminal fluids. Micropuncture studies of anaesthetized echidnas showed that the ductuli efferentes absorb 74% of the fluid and 46% of the soluble protein that enters them. The initial segment of the ductus epididymidis absorbs 83% of the fluid which enters it, and its secretions increase the concentration of protein in luminal fluid by 107%. Denatured, linear-gradient polyacrylamide gel electrophoresis of micropuncture samples showed that 1 protein (apparent M4 = 100 500) which is not present in blood plasma is present in rete testis fluid, and a glycoprotein which is present in rete testis fluid (apparent Mr = 78500) is absorbed by the ductuli efferentes. Six proteins which are not present in blood plasma are secreted into the initial segment of the ductus epididymidis; 5 are glycoproteins (apparent Mr = 48500, 39000, 32000, 20500 and 19000) and one (apparent Mr = 82500) is not. The most prominent electrophoresis bands corresponded to the glycoproteins with apparent molecular weights of 48500, 20500 and 19000.     

Effects of caput ligation on rat sperm and epididymis: protein thiols and fertilizing ability.

Mammalian spermatozoa mature while passing through the epididymis. Maturation is accompanied by thiol oxidation to disulfides. In rats, sperm become motile and fertile in the cauda. We have previously demonstrated that rat caput sperm contain mostly thiols and that upon passage from the corpus to the cauda epididymidis, sperm protein thiols are oxidized. The present work was undertaken to study the role of the regions of the epididymis in sperm maturation as reflected in the thiol status, fertility, and motility of the spermatozoa. The distal caput epididymidis of mature albino rats was ligated on one side. After 5 days, sperm were isolated from the ligated caput and from caput and cauda of the control side. Thiol groups in sperm, epididymal luminal fluid (EF), and epididymal tissue were labeled using the fluorescent thiol-labeling agent monobromobimane. After ligation, changes were observed in a) sperm proteins, sperm nuclear proteins, and epididymal fluid by electrophoresis; b) epididymal tissues by histochemistry; c) progressive motility by phase microscopy; and d) fertilizing ability after insemination into uteri of immature females. We found that after ligation, caput sperm thiols, especially protamine thiols, are oxidized, rendering them similar to mature sperm isolated from the cauda epididymidis. Spermatozoa from ligated caput epididymidis gain progressive motility and partial fertilizing ability. Morphology of epithelial cells of ligated caput is similar to that of cauda cells. However, other changes in caput EF and epithelium induced by ligation render the ligated caput epididymidis different from either control caput or cauda. Hence, sperm thiol oxidation, along with the development of fertilizing ability, can occur in sperm without necessity for sperm transit through the corpus and cauda epididymidis.     

Structural features of the epididymis in a dasyurid marsupial (Antechinus stuartii)

Summary The ductus epididymidis of the marsupial mouse Antechinus stuartii was divided into caput, corpus, and caudal regions using several constant morphological landmarks. Tubule diameter and epithelial height increased gradually from caput to cauda. In contrast, the surface area of the lumen of the ductus epididymidis increased to a maximum in the distal caput region, but decreased markedly in the distal cauda in association with characteristic changes in lumen shape (from circular to slit-shaped) and epithelial height. Epithelial cells of the ductus epididymidis were generally similar in structure to those described in other mammalian species. Principal and basal cells were common throughout the epithelium. Clear and mitochondria-rich cells were also identified, but occurred less frequently. Regional variations in cell ultrastructure were observed only in principal cells. Numerous vesicular inclusions occurred in the apical cytoplasm of cells in caput segments, membrane-bounded, electron-dense bodies were common in distal corpus regions, and a brush border of microvilli characterized the luminal surface of principal cells in caudal segments. Sperm index increased in the proximal caput, declined to basal levels in the distal caput and proximal corpus, and then increased to a maximum in segment 9 of the distal corpus and remained at about this level throughout the cauda epididymidis. Nuclear rotation, loss of cytoplasmic droplets, and other sperm maturational changes were observed along the epididymis. Discarded cytoplasmic droplets collected in large masses interspersed between aggregates of spermatozoa throughout the distal regions of the duct. There was no evidence of phagocytosis by principal cells of cytoplasmic droplets. The epididymis of A. stuartii differs from that of other mammals. The unusual caudal region, which has little storage capacity for sperm, is an unusual adaptation in a species in which the male is known to be polygamous.     

Clusterin (SGP-2) in epididymal luminal fluid and its association with epididymal spermatozoa in androgen-deprived rats.

Clusterin is a heterodimeric glycoprotein synthesized and secreted by rat Sertoli cells and epididymal epithelium. The goal of this study was to determine the presence of clusterin in the luminal fluid of the cauda epididymides and its association with the membranes of developing spermatozoa in the presence and absence of androgen. We have previously demonstrated by two-dimensional (2-D) Western blot probing for clusterin that in epididymal fluid the amounts of clusterin were: caput greater than corpus greater than cauda. Luminal fluid from cauda epididymides was collected from control and orchiectomized rats (6 and 12 days) and orchiectomized animals that received testosterone implants. Equal volumes of fluid were analyzed by 2-D Western blot probing for clusterin. Following orchiectomy, there was an increase in clusterin in the luminal fluid after 6 days and maximal amount after 12 days compared with control cauda fluid. Orchiectomized animals which received testosterone treatment showed levels of clusterin comparable to that of controls. Serum clusterin was detected in fluid of orchiectomized animals with and without testosterone. Western blots of cauda sperm membrane extracts of control animals and orchiectomized animals treated with testosterone had a very low level of epididymal clusterin, whereas extracts collected from orchiectomized animals revealed high levels of clusterin. We suggest that, in the normal animal, clusterin is secreted into the lumen of the proximal epididymis where it binds to the sperm membrane. In the distal epididymis, clusterin dissociates from sperm and is processed (proteolysis/endocytosis). We hypothesize that, in the absence of androgen, the processing and regulation of clusterin is disrupted.     

Fine structure distribution of non-specific acid phosphatase in the head region of mouse spermatozoa from various regions of the male reproductive tract.

The fine structure distribution of non-specific acid phosphatase was determined in the head region of mouse spermatozoa from the testes, the caput, corpus and cauda epididymidis and the ductus deferens. Enzymatic localization was achieved by the Gomori technique. The postacrosomal dense lamina, the nuclear side of the inner acrosomal membrane and the space between the plasmalemma and the outer acrosomal membrane showed reaction product in spermatozoa from the testis and caput epididymidis. Spermatozoa from the cauda epididymidis exhibited reaction product only between the plasmalemma and the outer acrosomal membrane. Spermatozoa from the corpus epididymidis and from the ductus deferens showed no reaction product in the head region. The changes observed in the distribution of acid phosphatase in the sperm head during epididymal transport may reflect maturational events.     

Enhancement of sperm transport through the rat epididymis after castration

Transport of spermatozoa through different regions of the epididymis has been followed by labelling testicular spermatozoa with [3H]thymidine in intact rats and in rats in which the efferent ducts were ligated or the testes were removed. In intact rats, the transit times of epididymal spermatozoa from the initial segment to the caput, from the caput to the corpus, and from the corpus to the cauda were 2, 4 and 2 days, respectively, giving a total transit time of 8 days. After bilateral castration, labelled spermatozoa were transferred from the initial segment into the proximal cauda by 2 days and appeared in the ductus deferens by 4 days. This effect was prevented by a daily subcutaneous injection of testosterone propionate (0.2 mg/kg). Bilateral efferent duct ligation had only a slight effect on the passage of epididymal spermatozoa. The results indicate that epididymal sperm transport is enhanced after androgen withdrawal.     

Distribution of carnitine and acylcarnitine in the hamster epididymis and in epididymal spermatozoa during maturation

The highest levels of carnitine and acylcarnitine were found in the cauda epididymidis, and spermatozoa from the cauda contained greater amounts of total carnitine (free carnitine plus acylcarnitine) than those removed from the corpus or caput epididymidis. Spermatozoa from the distal cauda contained significantly greater amounts of both free and total carnitine than those removed from the proximal cauda epididymidis. The acylcarnitine:carnitine ratio was 1.7 and 0.37 in caput and cauda spermatozoa, respectively and 1.7 and 1.3 in caput and cauda fluid, respectively. It is suggested that the accumulation of carnitine is involved in sperm maturation and that acylcarnitine serves as an energy substrate for epididymal spermatozoa.     

On the proluminal movement of 3H-androgens across the rat epididymal epithelium

3H-Androgens in rat epididymal interstitium have previously been shown to move into the epididymal lumen against a concentration gradient. This is true especially in the caput epididymidis. The present investigation used the technique of in vivo epididymal perifusion and tubule micropuncture to demonstrate that the proluminal movement of 3H-androgens is subject to competitive inhibition (unlabeled testosterone in the perifusion fluid at 10 times and 100 times the concentration of 3H-testosterone significantly reduced proluminal movement of isotope) and is not energy-dependent (1 mM 2,4-dinitrophenol in perifusion fluid did not reduce the proluminal movement of isotope). Additionally, dry-mount autoradiography demonstrated high intraluminal concentrations of isotope relative to interstitial concentrations after caput tubule incubation in 3H-dihydrotestosterone (3H-DHT), and showed that the high intraluminal concentrations of isotope were not dependent on the presence of spermatozoa, i.e. proluminal movement of 3H-androgens was not due to binding to intraluminal spermatozoa. Isolation of caput epididymidal sperm on filters followed by 3H-DHT binding experiments also failed to demonstrate the presence of specific binding of this androgen to spermatozoa. Finally, it was confirmed that electrophoresed epididymal lumen fluid contains a single 3H-DHT binding peak that is at its highest concentration in the caput epididymal fluid. These data are consistent with the conclusion that intraluminal androgen-binding protein is an important factor in transepithelial androgen movement.     

Temperature and androgens regulate the biosynthesis of secretory proteins from rabbit cauda epididymidis

We have compared the biosynthesis of secretory proteins in rabbit cauda epididymidis maintained for 15 days at abdominal temperature with that of the scrotal cauda. Explants from both situations were incubated in vitro in the presence of [³⁵S] methionine, and the labelled proteins released into the incubation medium were analyzed by polyacrylamide gel electrophoresis. Body temperature specifically inhibited the synthesis of at least two polypeptides of 43 kDa and 21 kDa (designated EP21), whereas the synthesis of polypeptides of 80, 39, 31, and 24 kDa was increased. These changes resembled those produced by castration, but androgen treatment was not able to reverse the effect of body temperature. To confirm these observations, poly (A)⁺ RNA from the scrotal and the abdominal cauda respectively, was translated in vitro and the synthesized products were immunoprecipi-tated with an antibody against EP21 polypeptide. Both castration and body temperature strongly decreased the concentration of EP21 mRNA. In vivo testosterone administration restored the content of EP21 mRNA in cauda from castrated animals, but not in cauda maintained at body temperature. The changes observed might be related to the adverse effect of body temperature on sperm storage in the cauda epididymidis. © 1993 Wiley-Liss, Inc.     

Comparative studies of epididymal morphology and sperm distribution in dasyurid marsupials during the breeding season

The structural features of the epididymis and the number and distribution of spermatozoa along the duct, during the breeding season, were examined in two semelparous and three iteroparous dasyurid marsupials. Total numbers of epididymal spermatozoa were extremely low in all of these species when compared with epididymal sperm numbers in most other marsupials and eutherian mammals. Although semelparous dasyurids had significantly more epididymal spermatozoa than itcroparous species, very few spermatozoa were seen in the distal cauda epididymidis of any of the species examined. This coincided with distinct changes in duct shape and the surface area of the lumen in caudal regions which resulted in a reduced sperm storage capacity in the cauda epididymidis of these species. The data suggest that, like Antechinus stuartii (Taggart & Temple-Smith, 1990a), sperm content of the ejaculates in these species will be extremely low, and that sperm motility and/or transport in the female tract is highly efficient. The functional and evolutionary significance of the reproductive strategies of semelparous and iteroparous dasyurid marsupials is still obscure and further study is needed to determine if the length of sperm storage in the female and sperm competition for storage sites is related to sperm distribution in the male and mating activities. This study does, however, clearly indicate that large numbers of spermatozoa are not required to ensure successful fertilization in either semelparous or iteroparous members of the family Dasyuridae.     

Glutathione-independent prostaglandin D2 synthase in ram and stallion epididymal fluids: origin and regulation

Microsequencing after two-dimensional electrophoresis revealed a major protein, glutathione-independent prostaglandin D2 synthase (PGDS) in the anterior epididymal region fluid of the ram and stallion. In this epididymal region, PGDS was a polymorphic compound with a molecular mass around 30 kDa and a range of pI from 4 to 7. PGDS represented 15% and 8% of the total luminal proteins present in this region in the ram and stallion, respectively. The secretion of the protein as judged by in vitro biosynthesis, and the presence of its mRNA as studied by Northern blot analysis, were limited to the proximal caput epididymidis. Using a specific polyclonal antibody raised against a synthetic peptide, PGDS was found throughout the epididymis, decreasing in concentration toward the cauda region. PGDS was also detected in the testicular fluid and seminal plasma by Western blotting. Castration and efferent duct ligation in the ram led to a decrease in PGDS mRNA and secretion. PGDS mRNA was not detected in the stallion 1 mo after castration, and it was restored by testosterone supplementation. This study showed that PGDS is present in the environment of spermatozoa throughout the male genital tract. Its function in the maturation and/or protection of spermatozoa is unknown.     

The concentration of carnitine in the luminal fluid of the testis and epididymis of the rat and some other mammals.

Luminal fluid was collected by micropuncture techniques from the testis and epididymis of the rat, hamster, rabbit, boar and ram and the concentration of free L-carnitine in the fluid was estimated using enzymic methods. Carnitine was present in the testicular fluid of the rat in concentrations less than 1 mM but increased down the epididymis to reach 53 mM in luminal fluid from the cauda epididymidis, approximately 2000 times higher than in blood plasma. A high concentration was first found in the luminal fluid from the distal caput epididymidis, at about the point where the spermatozoa become motile. Carnitine was also present in the epididymal luminal fluid of the other species studied; the amounts were not as high as those in the rat but were still higher than those in blood plasma.