Distal Cauda Epididymidis Research Articles

Lectin Staining of Rat Testis and Epididymis: Effect of Cyproterone Acetate and Testosterone

Control and castrated (C) adult rats were treated with cyproterone acetate (CA), testosterone (T) or their combination (CA + T) for 15 days and the affinity of the testicular and epididymal tissue for seven rhodamine-conjugated lectins was analysed in fluorescence microscopy. In the testis CA caused the appearance of degenerating cells in the basal part of the seminiferous epithelium. These cells (spermatogonia, early spermatocytes) appear to be the most vulnerable germinal cells to anti-androgen treatment and this effect could be prevented by simultaneous administration of T. Castration caused a marked reduction in lectin staining of principal and light cells of distal caput (Dcp) and distal cauda (Dcd) epididymidis accompanied by the disappearance of the intratubular sperm mass. In castrated animals CA caused a moderate and T as well as CA + T a marked increase in the stainability of these cells and the appearance of homogeneous secretory material in the tubules. This material gave moderate or strong affinity for most of the lectins. Some minor differences were found in the staining pattern of the cells and the secretory material in Dcp and Dcd as well as after different treatments. This is consistent with local and qualitative differences in the epididymal secretory and absorptive activity, which can be further modified by the hormonal milieu.

Evidence for blood myo-inositol as a source of the epididymal secretion in the perfused cauda epididymidis of the rat.

The movement of radioactive inositol and glucose across the epididymal epithelium have been studied by perfusion of fluid, which has the major electrolyte compositions resemble those in the native epididymal fluid, through the lumen of a sperm free tubule of the distal cauda epididymidis of intact and nephrectomized rats. During intravenous infusion of (3H)-myo-inositol into the intact rats, labelled myo-inositol and its metabolites entered the lumen. However, only labelled inositol was found in the luminal perfusate when radioactive inositol was infused into the nephrectomized rat. On the other hand, after infusion of (14C)-glucose no trace of labelled inositol appeared in the lumen. Instead, the luminal radioactivity could be accounted for by labelled glucose. The results indicate that the myo-inositol present in the luminal fluid of the rat cauda epididymidis originates, in part, from blood inositol and, in part, from blood glucose.

Na+/H+-exchange activity and immunolocalization of NHE3 in rat epididymis.

An acidic luminal pH in the epididymis and vas deferens (VD) helps maintain mature sperm in an immotile state during storage. We have previously shown that the majority of proton secretion in the VD is due to the activity of the vacuolar H+-ATPase. Acidification is dependent on luminal sodium in more proximal regions of the epididymis, and we examined the distribution of the Na+/H+ exchanger, NHE3, by immunofluorescence and measured Na+/H+ exchange (NHE) activity in isolated epididymal tubules. NHE3 was detected in the apical pole of nonciliated cells of the efferent ducts and principal cells (PC) of the epididymis. No staining was seen in the distal cauda epididymidis and the VD. Isolated tubules from the distal initial segment (DIS) and proximal cauda epididymidis were perfused in vitro and loaded with the pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6')-carboxyfluorescein. Ethylisopropyl amiloride (EIPA) (50 microM) reduced the initial rate of intracellular pH recovery (dpH(i)/dt), in response to an acute acid load, by 51% and 45% in the DIS and cauda epididymidis, respectively. In the DIS, removal of luminal sodium reduced dpH(i)/dt by 52%. HOE694 (50 microM) inhibited all EIPA-sensitive dpH(i)/dt in the DIS, despite the previously reported absence of NHE2 in this region (Cheng Chew SB, Leung GPH, Leung PY, Tse CM, and Wong PYD, Biol Reprod 62: 755-758, 2000). These data indicate that HOE694- and EIPA-sensitive Na+/H+ exchange may participate, together with the H+-ATPase, in luminal acidification in the male excurrent duct.

Fertility of rat epididymal sperm after chemically and surgically induced sympathectomy.

Guanethidine, a chemical that selectively blocks sympathetic noradrenergic neurons, was used to investigate the role of sympathetic innervation in the fertility of rat epididymal sperm, using both natural mating and in utero insemination protocols. This animal model correlates, at least in part, with spinal cord injury (SCI) in men. Adult male rats were treated daily by i.p. injections, for 21 or 42 days, with 0 or 6.25 mg/kg guanethidine. To compare the effects of guanethidine-induced sympathectomy with those following surgically induced sympathectomy, the inferior mesenteric ganglion and the proximal hypogastric nerves were removed in another group of rats. Both chemically and surgically induced sympathectomy increased the weight of the epididymis and seminal vesicles/coagulating glands as well as the number and the transit time of cauda epididymal sperm. Neither serum testosterone levels nor LH was affected by treatment with guanethidine. Using natural mating, no litters were produced by guanethidine-treated rats. Chemically denervated rats failed to produce copulatory plugs or ejaculate into the uterus. However, distal cauda epididymal sperm from chemically or surgically denervated rats displayed normal fertilization ability (80%) using in utero inseminations. In addition, the sperm of denervated rats did not show abnormal sperm chromatin structure using an assay that detects DNA damage. We conclude that sympathectomy delays the transit of sperm through the cauda epididymidis and produces ejaculatory dysfunction but does not compromise sperm quality in the distal cauda epididymidis. Moreover, these data provide compelling evidence that there is no association between the prolonged transit time of sperm within the epididymis, i.e., pre-ejaculatory sperm aging, and the fertility of those sperm, which has important implications for artificial insemination using sperm from men with SCI.

Epididymal maturation of chromatin in spermatozoa from control monkeys (Macaca fascicularis) and those treated with Cetrorelix, a gonadotropin-releasing hormone antagonist.

In order to examine changes in sperm chromatin upon epididymal maturation in the macaque epididymis (Macaca fascicularis), spermatozoa were obtained from six regions of the duct and examined for the state of their chromatin condensation by flow cytometry after staining with acridine orange. To see whether changes were affected by androgens, tissue was obtained from five monkeys treated with the gonadotropin releasing hormone (GnRH) antagonist Cetrorelix. Spermatozoa were recovered from treated and control animals after 16 days (at hemicastration) and another 9 days of treatment. Chromatin condensation of epididymal spermatozoa from controls displayed an increase upon maturation. After 16 days of GnRH-antagonist treatment, spermatozoa in the caput epididymidis displayed greater fluorescence than those from controls, but this was reduced during epididymal transit to values found in the distal epididymal regions of the controls. It is concluded that epididymal chromatin condensation 1) is normal in GnRH-antagonist-treated monkeys as long as sperm are being produced and 2) can compensate for poor testis function so that spermatozoa with normal states of chromatin condensation are found in the distal cauda epididymidis and probably the ejaculate.

Effects of pivalic acid and sodium pivalate on L-carnitine concentrations in the cauda epididymidis and on male fertility in the hamster.

In this study, administration of pivalic acid or its sodium salt was found to decrease the L-carnitine concentration in the epididymal lumen of the hamster; it also tested whether this decrease affected sperm cell motility, chromatin structure, or fertilizing capacity. Provision of pivalic acid or its sodium salt (20 mM or 40 mM) in the drinking water of mature male golden hamsters for 30 days reduced (by 72%, 75%, and 83% in three experiments) the L-carnitine concentration of the cauda epididymidis but did not inhibit sperm chromatin condensation, as assessed by flow cytometry. The treatments did not alter the location of motile sperm in the epididymidis nor did they appreciably affect the motility of sperm obtained from the distal cauda epididymidis. The numbers and percentage of ova that reached the 2-cell stage 36-40 h after uterine insemination with spermatozoa from control and treated hamsters served as a measure of sperm fertility. Treatment with pivalic acid or sodium pivalate did not render male hamsters infertile although it appeared to reduce the fertilizing ability of their spermatozoa. These results suggest that the high concentration of L-carnitine present in the lumen of the cauda epididymidis is not required for maturation of sperm chromatin or development of sperm motility.

Fertilizing capacity of rat spermatozoa is correlated with decline in straight-line velocity measured by continuous computer-aided sperm analysis: epididymal rat spermatozoa from the proximal cauda have a greater fertilizing capacity in vitro than those from the distal cauda or vas deferens.

Rat spermatozoa recovered from different regions of the excurrent ducts of 10 adult males (proximal cauda epididymidis [PC], distal cauda epididymidis [DC], and vas deferens [VD]) were assessed by in vitro fertilization (LVF) using limited sperm numbers, and by continuous evaluation of motility parameters during 5 hours of incubation in vitro with automated computer-aided sperm analysis (CASA). Spermatozoa from the PC region fertilized (68 +/- 6%) a significantly greater (P < or = 0.005) number of oocytes than those from the DC (44 + 5%) or VD (47 +/- 7%). For pooled samples from all three regions, the mean fertilization rate (51 +/- 14%) was less tan for spermatozoa from the PC (P < 0.05) but was not significantly different from spermatozoa from the DC or VD. For each time point and sample, 1,592 +/- 428 sperm tracks were analyzed. CASA was verified by comparison with manual still-frame analysis of video recordings, by repeated analysis of the same or different samples of spermatozoa, and by examination of computer tracks. The coefficients of variation for various motion parameters suggested that the CASA obtained a high degree of precision. There were no significant differences in motility parameters for spermatozoa recovered from equivalent regions of the left or right tract or in motility parameters for spermatozoa from different regions of the tract immediately after recovery. However, during incubation in vitro, spermatozoa from the DC or VD regions exhibited a marked decline in straight-line velocity (VSL) compared with spermatozoa from the PC region. The reduction in VSL (combined values from right and left tract) for DC or VD spermatozoa compared with PC spermatozoa was significant at 2.5 hours of incubation (P < or = 0.05) and highly significant (P < or = 0.005) by the end of the incubation period. Differences in average path velocity (VAP) were also apparent after 4 hours (p < or = 0.05), but no significant differences were observed for measurements of curvilinear velocity (VCL) or lateral bead displacement (ALH). Overall, the decline in VSL over 5 hours was highly correlated (P < or = 0.001) with the outcome of fertilization in vitro. In contrast, initial VSL and changes in VCL of spermatozoa were not correlated with fertilization rate. These results indicate that the in vitro fertilizing capacity of rat spermatozoa is correlated with 1) the decline in straight-line velocity (VSL) as measured by repeated CASA during incubation in vitro and 2) with the site of recovery of mature rat spermatozoa from the distal excurrent duct. It is suggested that the deterioration of the quality of rat spermatozoa in the distal epididymidis and vas deferens during storage may occur sooner than previously realized, and therefore care must be taken when recovering samples for fertility assessment. In keeping with findings in other species, immediate "snapshot" analysis of rat motility was a poor predictor of sperm fertility. In contrast, continuous CASA provided significant information for determining sperm fertilizing capacity and will be a useful technique for reproductive toxicology.

Morphological characteristics of boar efferent ductules and epididymal duct

The aim of the present study was to provide a comprehensive morphological analysis of the porcine epididymis in view of the specific functions being performed in different regions of this organ. Blood supply and microvasculature of efferent ductules and epididymal duct were investigated by means of corrosion casts which were analysed macroscopically and by scanning electron microscopy. This revealed blood supply to the testis and epididymis to be closely related. The capillary pattern was typical for the efferent ductules, the caput, corpus, and distal cauda epididymidis, respectively. Corrosion casts were also used to visualize the course of the efferent ductules themselves. Tissue samples from different regions of the efferent ductules and epididymal duct were examined by light microscopy and both scanning and transmission electron microscopy, with special attention being payed to transitional areas. Morphological criteria allowed the distinction of three segments within the efferent ductules and of the initial segment, proximal caput, distal caput, corpus, proximal cauda, and distal cauda regions of the epididymal duct. Components of the endocytic apparatus of efferent ductule principal cells were identified by ferritin uptake. Ultrastructural evidence of absorption in the epididymal duct was particularly prominent in proximal and distal caput. Extensive cisternae of rough endoplasmic reticulum and a well-developed Golgi apparatus were indicative of active protein synthesis and secretion especially in the distal caput and corpus regions. However, assignment of various organelles in principal cells of the epididymal duct to either absorptive or secretory pathways still remains tentative.

Developmental expression of sulfated glycoprotein-2 in the epididymis of the rat.

Sulfated glycoprotein-2 (SGP-2), also designated as clusterin, is a protein secreted by the epididymis and which binds to spermatozoa. In adult rats it is secreted at high levels by principal cells of the distal initial segment, intermediate zone and caput epididymidis, and at relatively lower levels by principal cells of the corpus and cauda epididymidis. The objective of this study was to correlate the developmental events in the maturation of the epididymis with the timing of SGP-2 expression in order to evaluate the testicular or epididymal factors which may regulate it. Our approach was to follow and compare the developmental expression of SGP-2 by immunocytochemistry in normal untreated control rats and rats whose efferent ducts were ligated on day 15 and examined at different postnatal ages thereafter. In control animals, SGP-2 expression in principal cells of the distal initial segment, intermediate zone, and caput and distal cauda epididymidis, as characterized in normal 90-day-old adult animals, was attained between postnatal days 39 and 49. However, only by postnatal day 56 did SGP-2 display in the corpus and proximal cauda the characteristic secretory pattern found in adult rats. In contrast, in efferent duct ligated rats examined at postnatal day 64, SGP-2 was absent in principal cells of the corpus and proximal cauda epididymidis but continued to be secreted by the distal initial segment, intermediate zone, and caput and distal cauda epididymidis. Furthermore, unlike the case in control rats, SGP-2 was secreted at high levels by the principal cells of the proximal initial segment. Thus during normal postnatal development, in the proximal initial segment, the production of SGP-2 is suppressed by luminal factors originating from the testis, while in the distal initial segment, intermediate zone, and caput epididymidis, it is unaffected by these factors. On the other hand, the production of SGP-2 in the corpus and proximal region of the cauda epididymidis is normally stimulated by luminal factors originating from the testis, while in the distal cauda, it is unaffected by these factors. Our results thus show a differential regulation of SGP-2 expression in principal cells of the proximal versus distal regions of the epididymis and even within subdivisions of each region. In some regions of the epididymis, SGP-2 production appears to be unaffected by luminal factors originating from the testis, while in other regions it is either inhibited or stimulated by these factors.

Developmental expression of immobilin in the rat epididymis

Immobilin is a protein secreted by principal cells of the distal initial segment, intermediate zone and caput epididymidis of adult rats, which serves to immobilize spermatozoa. In the distal cauda, epithelial clear cells are involved in its endocytosis. The objective of this study was to correlate the developmental events in the maturation of the epididymis with the timing of immobilin secretion and endocytosis in order to evaluate the testicular or epididymal factors which may influence or regulate immobilin expression. Our approach was to follow and compare the developmental expression of immobilin by light microscope immunocytochemistry in control and efferent duct ligated rats of different postnatal ages. Coincident with the morphological maturation of the principal cells by postnatal day 39, immobilin displayed the characteristic secretory immunostaining pattern found in adults. This adult-like expression occurred despite the absence of spermatozoa in the lumen but was coincident with high levels of circulating and luminal androgens. In contrast, immobilin secretion in rats whose efferent ducts were ligated at day 15 was weak to non-existent in the principal cells of the caput epididymidis at day 28 and remained so into adulthood, indicating that principal cells of this region of the epididymis are dependent either directly or indirectly upon testicular factors present in the lumen for immobilin expression. However, secretion of immobilin in the principal cells of the distal initial segment was unaffected by ligation and unlike the case in control rats high levels of immobilin also continued to be secreted into adulthood by the principal cells of the proximal initial segment. Thus in the distal initial segment immobilin secretion is not regulated by luminal factors originating from the testis, while in the proximal initial segment the normal suppression of immobilin that occurs by postnatal day 39 is. Despite ligation, endocytosis of immobilin by clear cells of the distal cauda epididymidis occurred by day 49, indicating that luminal testicular factors are not essential for stimulating the uptake of immobilin by these cells. The results taken together suggest that there are stimulatory and inhibitory luminal testicular factors involved in the regional development of immobilin secretion in the epididymis. There are also immobilin secreting regions in the epididymis, whose secretory development is independent of luminal testicular factors.

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 &amp; 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.

β-D-Galactosidase of Rat Spermatozoa: Subcellular Distribution, Substrate Specificity, and Molecular Changes during Epididymal Maturation1

In previous studies, we reported that rat epididymal fluid acid beta-D-galactosidase, which optimally cleaves a synthetic substrate (PNP beta-D-galactoside) at pH 3.5, shows maximum activity at pH 6.8 when a glycoprotein is used as a substrate [Skudlarek MD, Tulsiani DRP, Orgebin-Crist M-C. Biochem J 1992; 286: 907-914]. We now describe a similar pH-dependent substrate preference for rat sperm beta-D-galactosidase. We found that only 10-14% of total beta-D-galactosidase (and other glycosidase) activity was associated with spermatozoa. The remaining enzyme activities were present in soluble form in the luminal fluid. When the glycosidase levels were expressed per 10(6) sperm, all enzymes showed a progressive increase in spermatozoa from the caput to the corpus or proximal cauda followed by a sharp decline in spermatozoa from the distal cauda epididymidis. The observed decrease in beta-D-galactosidase activity could not be explained by the loss of cytoplasmic droplets (which have a low enzyme activity relative to spermatozoa) or the presence of inhibitors/activators of the enzyme activity in spermatozoa from the proximal or distal epididymis. However, we found that the changes in beta-D-galactosidase activity during sperm maturation in the epididymis were accompanied by changes in the molecular form(s) of the enzyme. Western blot analysis using an antibody to beta-D-galactosidase showed a progressive processing of the 82-kDa immunoreactive band in caput spermatozoa to an 80-kDa diffuse band in cauda spermatozoa. The sperm-associated beta-D-galactosidase form(s) does not appear to be due to adsorption and/or binding of the luminal fluid beta-D-galactosidase, which contained a 97-kDa form in fluid from the caput and two forms, of 97 kDa and 84 kDa, in corpus and cauda fluids. The observed difference in the molecular forms of the sperm and luminal fluid was found to be due to differential glycosylation, since de-N-glycosylation of various forms of beta-D-galactosidase generated a single immunoreactive form of 70 kDa. Subcellular localization studies and assay for the beta-D-galactosidase activity in the enriched plasma membrane and acrosomal membrane fractions suggested the likelihood that the activity of beta-D-galactosidase and other glycosidases is present in the acrosome and is readily released during sperm disruption. The evidence suggests that sperm beta-D-galactosidase may be functional within the acidic environment of the acrosome during sperm maturation as well as in the neutral environment of the oviduct after the zona-induced acrosome reaction.

Microassay of sperm concentration in the rat epididymis by micropuncture technique.

Micropuncture samples taken from the rete testis, caput, corpus and cauda epididymidis of adult rats were assayed for intraluminal sperm concentrations. The amount of fluid resorbed from the efferent duct and epididymal lumen was calculated based on the sperm concentration. Epididymal sperm concentrations increased from the rete fluid to the cauda fluid through the caput fluid. Eighty-nine percent of the fluid leaving the rete testis is resorbed by the efferent ducts and proximal epididymal tubule, and 96% of the fluid leaving the rete testis is resorbed in the distal cauda epididymidis. Resorption is important for the control of intraluminal fluid by the epididymis.

Acute effects of ethane dimethane sulfonate (EDS) on the structure of the cauda epididymidis in the rat: selective destruction of clear cells in the proximal cauda region.

Of eight groups of five adult male Sprague-Dawley rats, three groups received an intraperitoneal dose of EDS (75 mg kg-1) in DMSO (dimethylsulfoxide in water; 1:3) and three control groups received DMSO only. One EDS and one control group were killed 1, 2 and 3 days after treatment. One EDS+T group, given a 5-cm implant of T sufficient to maintain normal serum T concentrations, and one untreated control group were killed on Day 3. Epididymides were fixed by vascular perfusion and embedded in Epon/Araldite for light and electron microscopic studies. Epididymal duct diameters and epithelial heights were measured from 1-micron sections and structural changes were assessed from thin sections using a Jeol 100B electron microscope. Morphological studies showed a reduction in duct diameter and an associated increase in epithelial height in the proximal cauda epididymidis of the experimental groups. In the EDS+T implant group, epithelial heights were significantly greater than in controls but duct diameters remained unchanged. The most obvious structural change in the proximal cauda epididymidis was the selective destruction of clear cells in the epithelium. Initially, vacuoles were observed in the lateral intercellular spaces of the epithelium; large autophagic vacuoles then appeared in the clear cells, which had degenerated and disappeared from the epithelium by Day 3. Progressive infiltration of leucocytes into the intertubular interstitium, the epithelium and lumen of the proximal cauda was also observed. Loss of clear cells in the proximal cauda epididymidis was also seen in the EDS+T group, suggesting that clear-cell degeneration was not associated with reduced concentrations of circulating androgen. In all EDS-treated groups, however, clear cells and duct profiles in the distal cauda epididymidis remained unaffected. The reason for their protection from the effects of EDS has not yet been determined. These results suggest that, in addition to other specific lesions described in previous studies, EDS also has a direct effect on the rat epididymis that appears to be specifically targeted to the clear cells in the proximal caudal region.

Characterization of the motility of maturing rat spermatozoa by computer-aided objective measurement.

A computer-aided sperm analysis system was optimized for objective assessment of the movement characteristics of mature and immature rat spermatozoa by testing different settings. Measurements of straight line velocity of individual motile cells were validated by manual tracking with a digitizer. Better agreement between the two methods and better performance in distinguishing between mature and immature spermatozoa was obtained by reducing the tracking rate to increase the time of analysis. However, numbers of motile and immotile cells could not be determined accurately. Manual counting of videotaped images revealed no significant differences in percentage motility of spermatozoa from five epididymal regions. Caput spermatozoa were characterized by low straight-line (VSL) and averaged-path (VAP) velocities and low path straightness (STR), whereas mature cells displayed high VSL, VAP and STR. An increase in curvilinear velocity on maturation was less obvious. Spermatozoa in the proximal corpus epididymidis were heterogeneous in their acquisition of motility maturation and the uniformity of movement pattern achieved in the distal corpus and proximal cauda regions tended to decrease again in the distal cauda epididymidis. Such objective measurements of motility patterns will facilitate studies on the regulation of motility development upon sperm maturation.

Rat sperm motility analysis: Methodologic considerations

The objective of these studies was to optimize conditions for computer-assisted sperm analysis (CASA) of rat epididymal spermatozoa. Methodologic issues addressed include sample collection technique, sampling region within the epididymis, type of diluent medium used, and sample chamber depth. In addition, sources of variation were identified and accuracy of the analysis was examined. All samples in this report were analyzed using a Hamilton Thorn Motility Analyzer (HTM-2000; Hamilton Thorn Research, Danvers, MA). We found that allowing the sperm to swim out from cuts made in the distal cauda epididymidis yielded samples with percentages of motile sperm 60% higher than samples collected using an aspiration method. Furthermore, sperm isolated from the distal cauda epididymidis exhibited slightly but significantly greater percentages of motile sperm and swimming speeds than sperm isolated from the proximal cauda epididymidis. Of the four motility media examined, all maintained a high percentage of motile sperm over an hour-long incubation period, but Medium 199 and modified Hank's Balanced Salt supported substantially greater sperm velocity than Dulbecco's Phosphate Buffered Saline (with Ca ++ and Mg ++), with or without glucose. Motility and velocity endpoints were comparable in 200-, 100-, or 40-μm deep chambers, but significantly lower in 20-μm-deep chambers. Since these and presumably other variables in the preparation and analysis of rat sperm do influence the assessed motility endpoints, it is important to standardize these methods and to consider these issues when interpreting CASA data.

Inflammatory Changes in the Epididymis After Vasectomy in the Lewis Rat1

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.

Histochemical Localization and Quantification of α-Glucosidase in the Epididymis of Men and Laboratory Animals1

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.

Effects of breeding season and mating on total number and distribution of spermatozoa in the epididymis of the brown marsupial mouse, Antechinus stuartii

Changes in the number and distribution of spermatozoa in the epididymis of the adult brown marsupial mouse were examined during July/August in mated and unmated males. The effects of mating on epididymal sperm populations were studied in 2 groups of males each mated 3 times and compared with the number and distribution of spermatozoa in the epididymides of 4 unmated control groups. One testis and epididymis were removed from each animal (hemicastration) either before or early in the mating season to provide information on initial sperm content and distribution. The contralateral side was removed later in the mating season to examine the effects of mating or sexual abstinence on epididymal sperm distribution. Epididymal sperm number peaked in both the distal caput and distal corpus/proximal cauda epididymidis in late July. The total number of spermatozoa, including those remaining in the testis, available to each male at the beginning of the mating season in early August was approximately 4.4 x 10(6)/side. Although recruitment of spermatozoa into the epididymis from the testis continued until mid-August, sperm content of the epididymis reached a peak of about 3.5 x 10(6)/epididymis in early August. At this time approximately 0.9 x 10(6) spermatozoa remained in the testis which had ceased spermatogenic activity. Throughout the mating season, epididymal spermatozoa were concentrated in the distal corpus/proximal cauda regions of the epididymis and were replenished by spermatozoa from upper regions of the duct. Relatively few spermatozoa were found in the distal cauda epididymidis, confirming a low sperm storage capacity in this region. A constant loss of spermatozoa from the epididymis, probably via spermatorrhoea, occurred throughout the mating season and very few spermatozoa remained in unmated males in late August before the annual male die-off. Mating studies showed that an average of 0.23 x 10(6) spermatozoa/epididymis were delivered per mating in this species, but the number of spermatozoa released at each ejaculation may be as few as 0.04 x 10(6)/epididymis when sperm loss via spermatorrhoea is taken into account. We suggest that the unusual structure of the cauda epididymidis, which has a very restricted sperm storage capacity, may function to limit the numbers of spermatozoa available at each ejaculation and thus conserve the dwindling epididymal sperm reserves in order to maximize the number of successful matings which are possible during the mating season.

Transport of inositol into the distal cauda epididymidis of the rat.

The transport of [3H]myo-inositol into the lumen of the rat distal cauda epididymidis was studied by luminal perfusion in vitro. Entry was time and tubule length-dependent and saturable transport could be demonstrated with Vmax of 237 pmol/(30 min. cm) and K+ of 1 mM. Secretion of unlabeled inositol into the epididymal lumen was maintained for 5 hours at a concentration 6 times greater than that of the bathing solution. The turnover time of the epithelial pool of inositol was 4.5 hours, from which the intracellular concentration was estimated to be 26.6 mM. Transport was not reduced by metabolic inhibitors, and it was demonstrated that exchange diffusion across the basolateral membranes could drive the uptake in this region.