Inflammatory changes in the epididymis after vasectomy in the Lewis rat

The epididymides of Lewis rats were studied at intervals up to 7 months after vasectomy, vasectomy followed 3 months later by vasovasostomy, or sham operations. Epididymal histology was related to testicular alterations and to serum antisperm antibodies as determined with an enzyme-linked immunosorbent assay. In vasectomy and vasovasostomy groups. 13 of 33 rats had testicular alterations, which consisted mainly of pronounced depletion of germ cells. Over half of the rats with testicular alterations also had severe epididymal lesions that included interstitial changes characteristic of an inflammatory response. These consisted of aggregates of mononuclear cells, including lymphocytes, plasma cells, and macrophages. The lumina of epididymides with interstitial changes contained polymorphonuclear leukocytes and/or macrophages. All animals with altered testes had greatly decreased numbers of epididymal sperm. In many instances, the lumen of the proximal cauda epididymidis was collapsed, and columnar cells of the epididymal epithelium contained many very large lysosomes. The distal cauda epididymidis was distended with sperm and debris. None of the rats that lacked testicular alterations showed epididymal changes. Mean serum antisperm antibody levels were significantly higher for rats with epididymal interstitial changes than for animals without such epididymal alterations. Infiltrations of inflammatory cells into the epididymal interstitium and lumen are part of the constellation of changes that occurs after immunization with testicular homogenates to produce experimental allergic orchitis. The observations reported here support the hypothesis that reproductive tract alterations after vasectomy in this model have an immune basis.     

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.     

Histochemical localization and quantification of alpha-glucosidase in the epididymis of men and laboratory animals

The seminal marker of epididymal function alpha-1, 4-glucosidase was localized histochemically in the cytoplasm of the efferent duct epithelium and the brush border of the entire length of the human epididymis. Quantification using the specific inhibitor castanospermine revealed strongest activity in the corpus and cauda regions. Selective inhibition of the brush border enzyme activities by maltotriose identified these as the neutral isoenzymes. Despite detection of alpha-glucosidase in the renal tubules of all the animals studied, the enzyme was not detectable in epididymides of hamsters or mice. In rabbits and monkeys, it was absent from the entire brush border but present weakly in the cytoplasm of the proximal epididymides. An enzyme distribution pattern similar to that in the human epididymis was found in rats, except for the absence of histochemical staining at pH 6.5 from the initial segment and distal cauda epididymidis. Experiments in which endogenous testosterone was depleted in rats demonstrated the dependence of epididymal alpha-glucosidase on androgen, albeit with a low sensitivity. This study suggests the rat to be a suitable model for the investigation of the role of epididymal alpha-glucosidase in fertility.     

Two-dimensional electrophoresis of proteins in principal cells, spermatozoa, and fluid associated with the rat epididymis

Spermatozoa, fluids, and principal cells from different regions of the epididymis were characterized by two-dimensional electrophoresis. Rete testis fluid was collected after 36-h efferent duct ligation, and cauda epididymal fluid was collected by retrograde perfusion through the vas deferens. Spermatozoa were collected after their exudation from minced caput and corpus epididymal tissue. Principal cells were recovered after enzymatic disaggregation and centrifugal elutriation of epididymides. Two-dimensional polyacrylamide gel electrophoresis was used to prepare protein profiles of all samples. Comparison of the proteins found in rete testis fluid versus those found in cauda epididymal fluid revealed a dramatic change in composition, including the loss, addition, or retention of specific proteins as well as changes in the relative concentrations of certain proteins. Prominent cauda epididymal fluid proteins, possibly contributed by the epididymal epithelium, were detected at 16, 23, and 34 kDa. After epididymal transit, a considerable decrease was observed in the number of aqueous-soluble sperm proteins. Differences in the protein composition of epididymal epithelial principal cells from the caput versus corpus epididymidis were also noted, suggesting that functional differences exist for these epididymal regions. Of particular interest was the occurrence of a prominent protein of approximately 20-23 kDa found in all sperm samples, in fluids, and in caput and corpus principal cells. However, this protein was absent in cauda epididymal sperm after 36-h efferent duct ligation. The rapid loss of this protein from sperm after efferent duct ligation suggests that this surgical intervention may affect spermatozoa residing within the epididymis.     

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.     

Immunocytochemical localization of alpha-lactalbumin in the male reproductive tract

The immunocytochemical localization of the milk protein alpha-lactalbumin in the male reproductive tract is described. Using a primary antiserum raised against highly purified rat milk alpha-lactalbumin, specific staining was consistently shown in the supranuclear Golgi region of the principal cells of the proximal caput epididymidis but only occasionally in epithelial cells from other regions of the duct. Staining was also found in the epididymal lumen and associated with spermatozoa. This luminal staining persisted throughout the distal caput, corpus and cauda epididymidis. Staining was rarely associated with spermatozoa in the efferent ducts and initial segment. Alpha-lactalbumin immunoreactivity was also detected in the seminiferous epithelium. Staining was confined to the Golgi-acrosome region of spermatids. These results indicate that an alpha-lactalbumin-like molecule, or molecules, is present in the male reproductive tract and that it is localized specifically in principal cells from the proximal caput epididymidis and germ cells from the seminiferous epithelium.     

Morphological changes in the testis and epididymis of rats treated with cyclophosphamide: a quantitative approach

Cyclophosphamide is a widely used anticancer and immunosuppressive drug that affects fertility in men. In a previous study, we found that chronic, daily treatment of male rats with low doses of cyclophosphamide had no apparent effect on the pituitary-gonadal axis, whereas it had time- and dose-dependent effects on male reproductive organ weights, the hematologic system, and on pregnancy outcome. To determine whether cyclophosphamide induces morphological changes within the male reproductive system, a detailed qualitative and quantitative evaluation of changes in the histology of the testis and epididymis was undertaken. Adult male Sprague-Dawley rats were gavage-fed for 1, 3, 6, and 9 wk with saline (control), 5.1 (low dose) or 6.8 (high dose) mg/kg/day of cyclophosphamide; the testes and epididymides were prepared for light and electron microscopy. At the light microscopic level, the orderly process of spermatogenesis in the seminiferous tubules was not affected at any time point with either dose of the drug. A number of time-dependent drug-induced changes in the histology of the epididymis, however, were apparent: 1) an increase in the relative number and a change in the distribution of halo cells in the caput epididymidis, 2) an increase in the number and size of clear cells in the caput and/or cauda epididymidis, and 3) an increase in the size of clear cells in both the caput and cauda epididymides; these changes were time dependent. At the electron microscopic level, there was a dose-dependent, two- to threefold increase in the number of spermatozoa with abnormal flagellar midpieces in the lumen of both the caput and cauda epididymides. Although the 9 plus 2 axonemal complex and the 9 outer dense fibers were present and appeared normal, the close approximation of these two structures was lost in these abnormal spermatozoa. Such abnormal flagellar midpieces were also found in the testes of control and treated rats. Electron microscopic examination of the testis revealed that both Sertoli and Leydig cells were normal in appearance. The type and timing of the effects of cyclophosphamide on the histology of the testis and epididymis suggest that the drug could be affecting germ cells by 1) inducing changes in the developing spermatozoa in the testis, some of which are seen microscopically in the epididymal lumen, and/or 2) affecting epididymal morphology and function.     

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.     

Immunocytochemical localization of the major glycoprotein of epididymal fluid from the cauda in the epithelium of the mouse epididymis

Summary The most abundant protein in fluid from the mouse cauda epididymidis, designated CP 27, is a glycoprotein that migrates at approximately 27000 daltons on SDS-polyacrylamide gels. Samples of CP 27 were isolated by preparative gel electrophoresis and were used to raise a guinea-pig polyclonal antiserum, which reacted with a single band on western blots of caudal epididymal fluid. This antiserum was used for immunocytochemical localization of CP 27 in histological sections of mouse epididymis using the peroxidase-antiperoxidase and protein A-gold methods. The most proximal staining with anti-CP 27 was in segment 6 of the distal caput epididymidis, where the lumen and a portion of the supranuclear cytoplasm of principal cells were stained. In contrast, in the distal corpus and cauda epididymidis (segments 8–11), there was pronounced staining of the luminal contents, stereocilia, and scattered cells identified as the light cells of the epididymal epithelium. Although CP 27 was found in the epididymal lumen of all segments distal to segment 6, the intensity of staining appeared to decline distally in the cauda epididymidis. Control sections exposed to pre-immune serum instead of anti-CP 27 showed no reaction. The results suggest that CP 27, the major glycoprotein of cauda epididymal fluid, is synthesized by principal cells of segment 6 of the distal caput epididymidis. CP 27 may be among the substances absorbed from the lumen by the light cells of the distal epididymis.     

过氧化物酶6在大鼠附睾中的表达及分布

通过双向电泳结合质谱技术分离鉴定正常成年大鼠附睾头段与尾段管腔液中的蛋白组成,从附睾头段及尾段管腔液的22个差异蛋白点中鉴定出12个蛋白质.其中11个蛋白质在不同种属哺乳动物的附睾组织中已有鉴定报道,而过氧化物酶6(peroxiredoxin 6,Prdx6)为新发现的存在于附睾头段及尾段管腔液中的体液蛋白.采用RT-PCR、Western印迹及免疫组化技术,对该蛋白在大鼠附睾中的表达及分布进行了分析.实验表明,Prdx6与精子的成熟、贮存及保护有一定关系,其具体机制值得进一步深入研究.     

Effect of diabetes mellitus on epididymal enzymes of adult rats.

Diabetes mellitus caused significant reduction in serum testosterone and accessory sex glands weight. The sperm content of epididymal regions also decreased. Among the epididymal regions, the cauda epididymidal tissue alone showed significant reduction in Na(+)-K+ ATPase activity. However, Mg2+ ATPase activity was lowered in caput epididymidis only. Specific activity of Ca2+ ATPase significantly decreased in caput and cauda epididymides. All three ATPases decreased significantly in caput epididymidal spermatozoa leaving cauda epididymidal spermatozoa unaffected. Specific activity of alkaline phosphatase was suppressed in caput epididymidis and in the spermatozoa collected from caput and cauda epididymides, while the acid phosphatase was unaffected. In general, the results are suggestive of definite influence of diabetes on epididymal phosphatases which is region specific. Diabetes induced decrease in phosphatases may have an impact on secretory and absorptive functions of epididymis and thus on sperm maturation.     

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.     

Effects of sulphapyridine on sperm transport through the rat epididymis and contractility of the epididymal duct

This study was undertaken to investigate the effects of sulphapyridine on the transport of spermatozoa through different regions of the epididymis and on the contractility of the epididymal duct in the rat. Sperm transport was investigated by labelling testicular spermatozoa with [3H]thymidine and measuring intraluminal pressures of the epididymis by micropuncture, using a servo-nulling pressure transducer system. In control rats, the transit times of epididymal spermatozoa from the initial segment to the caput, from the caput to the proximal cauda, and from the proximal cauda to the distal cauda were 2, 6 and 3 days, respectively, giving a total transit time of 11 days. The total transit time was shortened to 8 days after treatment with sulphapyridine at a dosage of 450 mg kg-1 for 38-52 days. The rate of sperm transport was most affected in the caput epididymidis. Measurements of intraluminal pressures showed that sulphapyridine had no effect on spontaneous contractions in any regions of the epididymis. However, the frequency of contraction of the corpus and cauda epididymides in response to administration of 10 micrograms noradrenaline kg-1 in the sulphapyridine-treated rats was significantly higher (P < 0.05) than it was in the controls. Methacholine, at a dose of 20 micrograms kg-1, produced a smaller increase in basal pressure in the caput epididymidis of sulphapyridine-treated rats (P < 0.05) compared with controls. The results led to the conclusion that sulphapyridine increases the rate of sperm transport from the caput through the cauda epididymidis, in part, by changes in the responsiveness of the epididymis to the autonomic nervous system.     

狗施行附睾尾化学注射绝育后寒九附睾内的免疫复合物观察

５只性成熟成年雄性比格犬被施行附睾尾逆向注射１０％精氨酸锌０．５毫升。一般饲料饲养两月后处死作病理检查,发现精子在睾丸曲细精管内的生成及在附睾管内的成熟均受到影响,附睾尾管腔内精子降到了极少程度且多已变性。免疫组织化学反应发现睾丸间质及附睾管上皮间隙内,附睾间质中均有大量免疫复合物的沉积,而其它主要生命器官均未发现异常。结果表明,精氨酸锌附睾尾注射的绝育作用是肯定的,其伴随出现的抗精子抗体及免疫复合物仅对睾丸、附睾及生育功能产生影响,对机体主要生命器官并无损害。     

Segmental expression of the bradykinin type 2 receptor in rat efferent ducts and epididymis and its role in the regulation of aquaporin 9

Water and solute transport in the efferent ducts and epididymis are important for the establishment of the appropriate luminal environment for sperm maturation and storage. Aquaporin 9 (AQP9) is the main water channel in the epididymis, but its regulation is still poorly understood. Components of the kinin-kallikrein system (KKS), leading to the production of bradykinin (BK), are highly expressed in the lumen of the male reproductive tract. We report here that the epididymal luminal fluid contains a significant amount of BK (2 nM). RT-PCR performed on epididymal epithelial cells isolated by laser capture microdissection (LCM) showed abundant BK type 2 receptor (Bdkrb2) mRNA expression but no type 1 receptor (Bdkrb1). Double-immunofluorescence staining for BDKRB2 and the anion exchanger AE2 (a marker of efferent duct ciliated cells) or the V-ATPase E subunit, official symbol ATP6V1E1 (a marker of epididymal clear cells), showed that BDKRB2 is expressed in the apical pole of nonciliated cells (efferent ducts) and principal cells (epididymis). Triple labeling for BDKRB2, AQP9, and ATP6V1E1 showed that BDKRB2 and AQP9 colocalize in the apical stereocilia of principal cells in the cauda epididymidis. While uniform Bdkrb2 mRNA expression was detected in the efferent ducts and along the epididymal tubule, marked variations were detected at the protein level. BDKRB2 was highest in the efferent ducts and cauda epididymidis, intermediate in the distal initial segment, moderate in the corpus, and undetectable in the proximal initial segment and the caput. Functional assays on tubules isolated from the distal initial segments showed that BK significantly increased AQP9-dependent glycerol apical membrane permeability. This effect was inhibited by BAPTA-AM, demonstrating the participation of calcium in this process. This study, therefore, identifies BK as an important regulator of AQP9.     

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.     

Ultrastructural study of the boar seminiferous epithelium: changes in cryptorchidism

The present study compares the ultrastructural features of Sertoli cells and germ cells between scrotal testes of healthy boars and abdominal testes of unilateral and bilateral cryptorchid boars. In healthy boars, spermatogonia are flat cells lying in close association with the basal lamina. As differentiation progresses, spermatogonia acquire an oval profile and lose their contact with the basal lamina. Spermatocytes are round cells moving from the basal compartment of the seminiferous epithelium to the luminal compartment. Spermatids exhibit complex morphological changes leading to the formation of spermatozoa. Sertoli cells extend from the basal lamina to the tubular lumen. The nucleus encloses fine euchromatin and one or two nucleoli; the nuclear envelope has a few deep infoldings. The lateral cell membranes form junctional specializations that constitute the blood-testis barrier. The cytoplasm encloses smooth endoplasmic reticulum, vesicles, aggregates, and scattered mitochondria. The seminiferous epithelium of abdominal testes from unilateral and bilateral cryptorchid boars contains few spermatogonia with an abnormal appearance; the alteration in germ cell number is more severe in the bilateral disease. In unilateral cryptorchid boars, spermatogonia appear as either large pyramidal cells or roundish cells; in bilateral cryptorchid boars, spermatogonia show roundish profiles and degenerative patterns. Abdominal testes of both unilateral and bilateral cryptorchid boars are constituted by immature Sertoli cells that show abnormal cytoplasmic content, defective development of the blood-testis barrier, and atypical nuclear appearance; in bilateral cryptorchid boars, immature Sertoli cells exhibit degenerative signs. At postpubertal age, unilateral and bilateral cryptorchidism induce total arrest of spermatogenesis at spermatogonial stage as a result of an abnormal differentiation of the Sertoli cells. Moreover, the degeneration of abdominal testes initiates earlier in bilateral cryptorchidism than in unilateral cryptorchidism.     

Galactosyltransferase, pyrophosphatase and phosphatase activities in luminal plasma of the cauda epididymidis and in the rete testis fluid of some mammals

Galactosyltransferase activity was measured in the luminal plasma of the cauda epididymidis of mice, rats, rabbits, rams and boars, and in the rete testis fluid of rams and boars. The activities of nucleotide pyrophosphatase and alkaline phosphatase, which compete with galactosyltransferase for substrate, were also determined. In these species, galactosyltransferase activity in the luminal plasma of the cauda epididymidis was similar when the inhibitory effect of pyrophosphatase and phosphatase was minimized by assay conditions. However, under assay conditions that did not minimize the effect of these enzymes, the galactosyltransferase activities of these species were very different and were inversely correlated with the activities of pyrophosphatase and phosphatase. The ratio of galactosyltransferase activity to pyrophosphatase and phosphatase activity was much higher in the rete testis fluid than in the luminal plasma of the cauda epididymidis in both rams and boars. In rams, galactosyltransferase in the luminal plasma of the cauda epididymidis was more heat resistant than that in serum. These results suggest that there is a species difference in the availability of galactosyltransferase activity in the luminal plasma of the cauda epididymidis and that in some species, galactosyltransferase in the luminal fluid is unlikely to have any function. The results are also discussed with respect to the possible function of galactosyltransferase, pyrophosphatase and phosphatase in epididymal luminal plasma and rete testis fluid.