An acrosin inhibitor in ram spermatozoa that does not originate from the seminal plasma.

Ram seminal plasma, and ejaculated ram spermatozoa that have been washed with 0.25M sucrose, both contain acrosin inhibitor. The aim of this work was to determine whether the intracellular inhibitor originates from the seminal plasma. The amounts of inhibitor in ejaculated and epididymal spermatozoa were measured and compared with the amounts present in the seminal plasma of normal and vasectomized rams. One ejaculated ram spermatozoon contained 2.1 amol (2.1 X 10(-18) mol) of inhibitor and one epididymal spermatozoon contained 3.3 amol of inhibitor. (All molarities are mean values based on pooled ram semen or on single ejaculates from three vasectomized rams.) Calculations from results in earlier publications indicated that one ejaculated ram spermatozoon contains about 3 amol of acrosin; thus the inhibitor: acrosin ratio in washed ram spermatozoa is approximately 1. One ml of ram semen contains, on average, 3 X 10(9) spermatozoa and not more than 0.8 ml of seminal plasma. This number of ejaculated spermatozoa would contain 6.3 nmol of inhibitor, while the same number of epididymal spermatozoa would contain 9.9 nmol of inhibitor. These values exceed the quantities of inhibitor present in 0.8 ml of normal seminal plasma (approximately 1.6 nmol) or in 0.8 ml of seminal plasma from vasectomized rams (approximately 2.3 nmol). We conclude that seminal plasma is not a major source of the acrosin inhibitor that can be recovered from washed ejaculated ram spermatozoa.     

Major contribution of epididymis to alpha-glucosidase content of ram seminal plasma

Acid alpha-glucosidase and L-carnitine (a well-known epididymal marker) were measured in rete testis and epididymal fluids of adult male rams. These fluids were collected by selective catheterization or by a micropuncture technique, respectively. Both parameters remained at a low and constant level in rete testis and all along caput and corpus epididymidis. Then they increased at equivalent rates in cauda epididymidis to much higher levels than those in seminal plasma (5 mU/mg protein and 10 mM, respectively). An optimum pH study of alpha-glucosidase activity in these fluids showed two well-separated peaks in rete testis and caput epididymal fluids around pH 4 and 7, respectively, but only a single peak at pH 4 in cauda epididymidis that was comparable to the one in seminal plasma. Sucrose density gradient fractions analyzed for their enzyme content in the absence or presence of sodium dodecyl sulfate (1% w/v), a selective inhibitor of acid alpha-glucosidase activity, allowed the demonstration of two enzyme forms at pH 6.8 in rete testis fluid sedimenting in the 7S and 4S regions of the gradient, while a unique 4S form was encountered in cauda epididymidis and in seminal plasma. Although the fate of the minor 7S component of the rete testis fluid in its epididymal transit is presently unknown, similarities between the enzyme in cauda epididymidis and seminal plasma are strong enough to support the hypothesis that epididymis contributes primarily to the acid alpha-glucosidase content of ram seminal plasma.     

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.     

Spermiogenic,epididymal, and spermatozoal damage induced by a peritesticular injection of ethylene dibromide to rams

The effects of a peritesticular injection of ethylene dibromide (EDB) on the germ cells, epididymis, and spermatozoa of rams was examined by removing each injected testis and epididymis at different times after treatment and by monitoring the seminal characteristics of ejaculates.A high incidence of abnormal elongating and elongated spermatids was observed in the testes of treated rams 48 h after injection. At this time the epithelium of the corpus and cauda epididymidis was damaged in the majority of observed rams, and the spermatozoa in these segments were abnormal, mainly with acrosomal defects, denuded forms and denuded tailless forms. Apparent phagocytosis of spermatozoa was also observed in these portions of the epididymal duct. Ejaculates collected as early as 48 h after injection had a very low sperm density and large percentages of spermatozoa with acrosomal and tail abnormalities. A dose-reponse effect was obtained, and the reversibility of the effect of a low dose of EDB on sperm morphology was demonstrated in the rams by semen examination.     

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.     

A 105- to 94-kilodalton protein in the epididymal fluids of domestic mammals is angiotensin I-converting enzyme (ACE); evidence that sperm are the source of this ACE

SDS-PAGE analysis of luminal fluid from the ram testis and epididymis revealed a protein of about 105 kDa in the fluid in the caput epididymal region. The molecular mass of this fluid protein shifted from 105 kDa to 94 kDa in the distal caput epididymidis and remained at 94 kDa in the lower regions of the epididymis. The possible sperm origin of this protein was suggested by the decrease in intensity of a 105-kDa compound on the sperm plasma membrane extract and by its total disappearance from the fluid of animals with impaired sperm production caused by scrotal heating. The 94-kDa protein was purified from ram cauda epididymal fluid, and a rabbit polyclonal antiserum was obtained. This antiserum showed that membranes of testicular sperm and sperm from the initial caput were positive for the presence of an immunologically related antigen. The protein was immunolocalized mainly on the flagellar intermediate piece, whereas in some corpus and caudal sperm, only the apical ridge of the acrosomal vesicle was labeled. The purified protein was microsequenced: its N-terminal was not found in the sequence database, but its tryptic fragments matched the sequence of the angiotensin I-converting enzyme (ACE). Indeed, the purified 94-kDa protein exhibited a carboxypeptidase activity inhibited by specific blockers of ACE. All the soluble seminal plasma ACE activity in the ram was attributable to the 94-kDa epididymal fluid ACE. The polyclonal antiserum also showed that a soluble form of ACE appeared specifically in the caput epididymal fluid of the boar, stallion, and bull. This soluble form was responsible for all the ACE activity observed in the fluid from the distal caput to the cauda epididymidis in these species. Our results strongly suggest that the epididymal fluid ACE derives from the germinal form of ACE that is liberated from the testicular sperm in a specific epididymal area.     

Localization of androgen receptors in ram epididymal principal cells

Androgen receptor was immunolocalized in the epididymal epithelium of rams and in isolated cells using an antibody against a synthetic polypeptide representing a portion of the androgen receptor. Immunostaining was predominant in the epithelium in tissue sections. Concentrations of androgen receptor were determined in cells from the central caput, distal caput, and central corpus epididymidis enzymically dissociated and elutriated to provide two fractions. On the average (n = 18), Fraction I contained 8% principal cells while Fraction II contained 71% principal cells; the stromal cells in each fraction were primarily smooth muscle and fibroblasts. For each sample, the number of DHT receptors (fmol) per 10(6) total cells was greater in Fraction II than in Fraction I. Few cells in Fraction I were immunostained for androgen receptor, whereas most cells in Fraction II were intensely stained. The numbers of DHT receptors per cell, or per principal cell, were similar for the central caput and distal caput, but lower in the central corpus epididymidis. The results support our hypothesis that most epididymal DHT receptors are localized in principal cells and confirm that the region between the central caput and proximal corpus of the ram epididymis is most dependent on androgen stimulation.     

Glycoproteins: a variable factor in surface transformation of ram spermatozoa during epididymal transit

To determine sequential surface glycoprotein changes in ram spermatozoa during epididymal maturation, labeling procedures were used that were specific for galactosyl, galactosaminyl, and sialyl residues. Spermatozoa and fluids were collected from the rete testis through surgically inserted catheters or flushed from the lumen of selected regions of the epididymis: i.e., caput, proximal and distal corpus, and cauda epididymidis. Ejaculated spermatozoa were collected by electrical stimulation. Electrophorectic analysis of galactose (GAO)-sodium boro[3H]hydride (NaB3H4)-treated spermatozoa revealed a sharp overall decrease in carbohydrate residue labeling during sperm transport through the efferent ducts and caput epididymidis, whereas several high molecular weight components in the 600K to 250K zone persisted throughout epididymal transit. Preincubation of spermatozoa with neuraminidase (NEUA) exposed galactose residues that had not been labeled with GAO alone (i.e., 97K, 43K, 24K) in both cauda epididymal and ejaculated spermatozoa. Treatment with sodium metaperiodate-NaB3H4 labeled many of the surface components displayed by NEUA-GAO-treated spermatozoa and revealed an overall shift in sialyl residue labeling from high molecular weight components in immature testicular spermatozoa to low molecular weight components in mature cells. The labeling procedures applied allowed only a qualitative interpretation of the results and they presumably represent the minimum possible changes. Nonetheless, our results demonstrate that glycoproteins are a major factor in surface transformations of ram spermatozoa in the epididymis, especially during the initial stages of maturation.     

Clinical, ultrasonographic and pathological features following unilateral vasectomy in rams

The effects of vasectomy on testes and related structures of animal species and men are largely disputable. These possible effects were studied in the ram, an established experimental animal model used to investigate genitalia pathophysiology. In each of five rams, vasectomy in the left spermatic cord was carried out; subsequently, the clinical and ultrasonographic features were monitored up to 12 months post-operatively. The rams were sequentially euthanatized 1, 3, 6, 9 and 12 months post-operatively; gross- and histo-pathological examination of their testes and related structures were carried out. Four of the five rams developed sperm granulomas at the proximal to the testis end of vas deferens or/and at the tail of the epididymis; these were palpable from the first and the third month after vasectomy, respectively. Ultrasonographic findings on the vasectomy side were increased size and echogenicity of the epididymal tail, as well as anechoic areas, representing sperm granulomas, visible in the epididymal tail 1 week after vasectomy and in the proximal to the testis end of vas deferens 4 weeks after vasectomy. Gross pathological findings were limited on the vasectomy side and included adhesions between the parietal and the visceral vaginal tunic, enlarged and firm epididymal tail and presence of sperm granulomas at the epididymal tail or/and at the proximal to the testis end of vas deferens; the granulomas contained creamy material. Histopathological changes were observed mainly in the epididymal tails, consisting of a central mass of spermatozoa, surrounded by a layer of macrophages, surrounded in turn by loose vascular connective tissue rich in lymphocytes and plasma cells. With the exception of signs of mild hypospermatogenesis observed in one ram euthanatized 9 months after surgery, and of a slight increase in seminiferous tubule diameter and in seminiferous epithelium height in the rams euthanatized 6 and 9 months after surgery, which are both findings of no clinical importance, no clinical, ultrasonographic, gross- or other histo-pathological changes were observed in the testicular parenchyma during a 12-month post-operative period. These results demonstrate that vasectomy has little if any detrimental effect on the morphologic characteristics of the spermatogenesis in rams.     

Prior exposure of maiden ewes to rams enhances their behavioural interactions with rams but is not a pre-requisite to their endocrine response to the ram effect

In this study, we tested whether prior experience with rams would modify the behavioural and endocrine responses of maiden ewes to rams. During mid-anoestrus, sexually na?ve, maiden ewes were exposed to rams for 7 days (ram experienced, RE; n=61) or isolated from rams (ram na?ve, RN; n=63). All ewes were subsequently isolated from rams. In Experiment 1, RE (n=55) and RN (n=57) ewes were introduced to rams during late anoestrus. RE ewes had more total and positive interactions with rams than RN ewes (P<0.001). RE ewes showed more ram seeking behaviour and spent more time in proximity of rams than RN ewes (at least; P<0.05). In Experiment 2, RE (n=6) and RN (n=6) ewes were introduced to rams midway through a frequent blood sampling regime in late anoestrus. Ram introduction stimulated an increase in LH pulse frequency and basal LH in both RE and RN ewes (at least P<0.05). RE ewes had an increase in mean LH concentrations (P<0.01) that failed to reach significance in RN ewes (P<0.1). There was no significant effect of prior experience with rams on LH pulse frequency, amplitude or whether ewes had an LH surge. In conclusion, prior experience with rams is important in developing appropriate ewe-ram interactions but is not a pre-requisite to the endocrine response to the ram effect.     

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.     

Pasteurellosis as a cause of genital lesions in rams. A descriptive study

Pasteurellosis is one of the most prevalent diseases of sheep, but the involvement of Pasteurellae in genital pathology of rams has been described rarely. One hundred and eighty-four rams showing palpable lesions in testes, epididymides or scrotum were submitted to bacteriological studies, and seven mature rams found infected with bacterial species belonging to the Pasteurella cluster (i.e., Mannheimia, Pasteurella and Bibersteinia (M/P/B)). The M/P/B cultures obtained were pure and/or heavy, and were confirmed after necropsy in the five M/P/B infected rams that could be slaughtered for further pathological examinations. Pasteurella multocida infected rams exhibited fibrinous exudate and generalized adhesions between the vaginal and the external scrotal layers. Testicular atrophy and epididymal sperm granulomas were also evident in these rams. Microscopically, epithelial hyperplasia with intraepithelial cysts, fibrosis and spermatic granulomas were present in the epididymis, while testis showed sperm stasis foci, microcalcifications and fibrosis. Mannheimia haemolytica infected rams showed severe unilateral epididymitis and testicular atrophy, being microscopically similar to the lesions found in P. multocida infected rams. The ram found infected with B. threalosi had severe unilateral lesions in testis, epididymis and scrotum. Microscopically, abscesses in epididymis and testis, and severe fibrosis and interstitial round cells infiltrates in testis were observed. Further studies should be conducted to determine properly the role played by the Pasteurella cluster in the pathogenesis of genital lesions in rams.     

Computer-assisted visualization of the rat epididymis: a methodological study based on paraffin sections autometallographically stained for zinc ions

A concept for the computer-assisted visualization of tubular organs is presented. Unmarked histological zinc-stained serial sections from the epididymis of the Wistar rat were aligned to demonstrate the concept. Virtual images were made through the aligned sections and served as controls for the alignment process. Animation of the serial sections and the virtual images revealed new information about the structure of the organ under investigation. The analysis was used to upgrade the anatomical knowledge of rat epididymis by describing how the epididymal duct runs through the structure. The proximal parts of the epididymis contain large communicating septa of connective tissue dividing the caput and the upper part of the corpus epididymidis into segments. The tortuousness was high in the caput with many turns within a small area of the epididymis, whereas longer loops were found in the lower part of the corpus and cauda epididymidis. The tube of the vas deferens was found to become an integrated part of the ductal system in the cauda epididymidis, although it was histologically easy to distinguish from the epididymal duct. The total number of cross-sections of the ductus epididymidis in the 2254, 15-mu m-thick, tissue sections analysed was 104 700, giving a minimum length of the ductal system of 1.5 m. © 1998 Chapman & Hall     

Effect of physiological (in lamb) or drug-induced (in adult) immaturity of ram spermatozoa on seminal plasma alpha-glucosidase activity

We have been able to collect ejaculates from four pre-pubertal Finnish Landrace and Suffolk lambs. Respective seminal plasma alpha-glucosidase specific activity was low (less than 0.3 mU/mg) whatever the season of observation. At puberty, it reached a level higher than 1 mU/mg as observed in adult rams. Administration of alpha-chlorohydrin to 14 adult rams (25 mg/kg/day during 25 days) led to the appearance of immature sperm. Seminal plasma alpha-glucosidase activity dropped from 1.5 to 0.5 mU/mg in both breeds, while fructose was raised from 2 to 6 mg/ml. L-carnitine and blood plasma testosterone remained unchanged during treatment. Semen characteristics appeared normal one month after the end of treatment when fructose concentration decreased simultaneously and enzymatic activity increased during two months to normal levels. The present findings suggest that seminal plasma alpha-glucosidase may be considered as a useful epididymal marker in ram.     

Effect of epididymis handling conditions on the quality of ram spermatozoa recovered post-mortem

Post-mortem spermatozoa recovery is an important technique for obtaining germplasm reserves from genetically valuable animals or endangered species. However, there are many factors that influence the outcome of this technique. We have studied the effect of the interval between animal's death and sperm recovery (0, 24 or 48 h) on the quality and freezability of ram spermatozoa from cauda epididymidis. Storage temperature of epididymis (room temperature or 5 degrees C) was also analysed. Spermatozoa were diluted with Tes-Tris-Fructose solution supplemented with egg yolk (10%) and glycerol (4%), and frozen using a programmable biofreezer (-20 degrees C/min). Pre-freeze and post-thaw sperm samples showed viable spermatozoa up to 48 h after the animal's death, although their quality declined significantly as post-mortem storage time increased. Epididymis sperm stored at 5 degrees C showed better motility and a lower percentage of abnormal forms than epididymis stored at room temperature after 24 and 48 h. The fertilizing ability of cauda epididymis ram spermatozoa obtained at 0 and 24h after the animal's death is similar to that of ejaculated spermatozoa. Therefore, a good protocol for post-mortem semen collection in rams when epididymal spermatozoa cannot be collected immediately, is to preserve the epididymis at 5 degrees C and process the samples in the first 24h after the animal's death.     

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.     

5alpha-Reductase and 3alpha-hydroxysteroid oxidoreductase enzyme activities in epididymis and their control by androgen and the rete testis fluid

The 5alpha-reductase and 3alpha-hydroxysteroid oxidoreductase enzyme activities have been measured in epididymal tissues and the control of these activities by androgens and the rete testis fluid appreciated. The highest 5alpha-reductase enzyme activity was found in the caput, the lowest in the corpus epididymidis. Androgens have a positive control on the 5alpha-reductase but no effect on the 3alpha-hydroxysteroid oxidoreductase activity. Ligation of the efferent ducts decreased significantly both enzyme activities in the caput but not in the corpus or in the cauda epididymidis.     

Microvasculature of the epididymis in the boar

Summary Microvasculature of the epididymis was investigated by scanning electron microscopy of vascular corrosion casts. The basic structure of blood supply to the boar epididymis consists of two superimposed vascular networks. Capillaries surrounding the epididymal duct constitute the inner level. They form polygonal meshes around the efferent ductules whereas circular capillaries strongly predominate in the subsequent region of the caput epididymidis. This annulate feature is progressively lost from corpus to cauda, where the capillary network once again has a polygonal appearance. The outer network is composed of feeding and draining vessels. Intertubular arteries pass between the loops of the epididymal duct and give rise to longitudinally oriented vessels attributable to only one adjacent duct segment. They feed the capillary network via circular ramifications debouching in different sectors of its circumference. The sparse veins draining the capillaries encircling the efferent ductules give way to a gradually increasing number of confluent veins up to the cauda.     

The contribution of the male to ovine embryogenesis in an in vitro embryo production system

The differences in the embryo production potential of four rams used in a commercial embryo transfer program were examined in both in vivo and in vitro embryo production systems. Processing frozen-thawed spermatozoa through Percoll density gradients prior to in vitro insemination eliminated differences in the estimates of sperm viability between the four rams, and yet, differences in embryo production persisted throughout the in vitro culture period. However, there was no effect of ejaculate within ram on embryo production rates. In addition, the timing of the onset of the differences between the rams at each stage of in vitro embryo development were revealed. Ram 2 differed from ram 4 in the proportion of fertilized oocytes at 17 h post-insemination (pi) and by 52 h, ram 3 differed from ram 4 in the proportion of cleaved embryos, and the observed differences between ram 1 and ram 2 in their blastocyst production were initiated prior to activation of the embryonic genome. Once differences in embryo development rates were detected among the four rams, they persisted throughout the in vitro culture period. The reduced in vitro fertilization (IVF) rates from ram 2 compared with the other rams was paralleled in vivo by the significantly lower proportion of embryos recovered from ewes mated to ram 2, and this was further exacerbated by a significantly lower embryo survival rate after transfer. However, the subtle differences observed in the timing of the contribution of each sire to embryo development during in vitro culture were not able to be detected in vivo. However, the higher proportions of transferable quality blastocysts obtained from ewes mated to ram 4 did not result in increased embryo survival throughout the remainder of gestation. Therefore, in this study, the blastocyst production potential for a particular sire, either in vitro or in vivo, does not necessarily reflect the potential for the production of live offspring.