The histochemical localization of prostaglandin synthetase activity in reproductive tract of the male rat.

PG synthetase activity was assessed histochemically in the reproductive tract of male rats. Moderate activity was observed in tails of spermatozoa within the corpus and cauda epididymidis but there was no activity in the caput epididymidis or the seminiferous tubules. The sperm tail activity was maximal for cells within the vas deferens. PG synthetase activity was also observed in individual adipose cells adhering to the testicular capsule, epididymis and vas deferens, and in isolated interstitial cells of the testis and the caput, corpus and cauda epididymidis. Specific cells in the capsules of the testes, epididymis and vas deferens also produced PGs. The activity observed in the interstitial cells of the testis and the caput epididymidis was less than that for the other tissues in terms of the proportion of possible cells. The demonstration of PG synthetase activity paralleled to known loss of arachidonic acid from the phospholipids of the spermatozoa as they pass through the male tract. Endogenous substrate was not limiting in the assay system, even in the testis and caput epididymidis where PG synthesis was not normally observed, indicating that a PG synthesis inhibitor may be present in these two tissues. PG synthetase activity within teased seminiferous tubules was markedly increased by physical trauma. Indomethacin diminished but did not eliminate synthesis.     

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.     

Effect of mating on sperm distribution in the reproductive tract of the male rat

Extragonadal sperm reserves in male rats were measured in different regions of the genital tract before and subsequent to normal ejaculation. In sexually rested rats, the sperm count (million spermatozoa for the paired organs) in different regions was: distal vas, 18; proximal vas, 9.8; cauda epididymidis, 229; caput + corpus epididymidis, 154. Following mating, the sperm count was reduced in the proximal and distal vas deferens and in the cauda epididymidis. The reproductive tract of mated females was found to contain 29% (no copulatory plug) or 59% (with copulatory plug) of the estimated mean ejaculate, which was estimated from the difference between the sperm counts in the sexually rested rat and following ejaculation. It is concluded that in the rat the immediate source of spermatozoa for ejaculation is the cauda epididymidis, with a smaller contribution arising from the vas deferens.     

Effects of age and frequency of ejaculation on sperm production and extragonadal sperm reserves in stallions.

Extragonadal reserves totalled 89 X 10(9) spermatozoa for 5--16-year-old sexually rested stallions and 60 X 10(9) for 2--4-year-olds. Regardless of age, the cauda epididymidis contained 62% of the total reserves and the vas deferens, including the ampulla, contained 7% of the total reserves of spermatozoa. The caput plus corpus epididymidis from 5--16-year-old stallions (N = 41) contained 14.9 X 10(9) spermatozoa per side as compared (P less than 0.01) to 8.5 X 10(9) for 2--4-year olds (N = 30). Frequency of ejaculation did not influence the number of spermatozoa found in caput plus corpus epididymidis. Across all ages, the number of spermatozoa potentially available for ejaculation from the cauda epididymidis and vas deferens on each side totalled 54 X 10(9). Collection of 5 successive ejaculates from sexually rested stallions removed 40--60% of the available spermatozoa while ejaculation every 2nd day reduced (P less than 0.05) the number of spermatozoa potentially available for ejaculation by 27%. Nevertheless, sufficient spermatozoa are produced daily (6.4 and 4.2 X 10(9) for 5--16-and 2--4-year-olds) to permit use of an average stallion once or twice daily, during spring and summer, providing sexual behaviour is adequate.     

Interaction of the human epididymal protein CD52 (HE5) with epididymal spermatozoa from men and cynomolgus monkeys

A monoclonal antibody (CAMPATH-1G) against the human lymphocyte surface protein CD52, which is similar to the epididymal secretion HE5, was used to ascertain the presence of this protein on maturing primate spermatozoa by flow cytometry. The percentage of human viable spermatozoa stained specifically with this antibody increased from sperm in spermatocoeles (0.5%), to the efferent ducts (3.8%), corpus (47.2%), and cauda (85.7%) epididymidis. Positive cells revealed staining mainly over the whole tail and postacrosomal region of the sperm head. Spermatozoa (∼10%) from both the efferent ducts and corpus epididymidis took up additional antigen when incubated with human distal cauda epididymidal plasma as a source of CD52, and 12–22% of human testicular sperm (from spermatocoeles) took up CD52 from human seminal plasma. In the cynomolgus monkey, nonspecific binding of control IgG was greater than that in human males and net CD52 staining was measurable only on ∼30% of corpus sperm where it was mainly on the principal piece. Neither caput nor cauda sperm took up human CD52 upon incubation with human seminal plasma, but an additional 27% of corpus sperm expressed CD52. Such uptake of CD52 was drastically reduced, or did not occur, when seminal plasma had been fractionated by filtration through 0.1 μm filters (filtrate II) or 300,000 Da cutoff filters (filtrate III), respectively. Western blots revealed that CD52 contents were much reduced in filtrate II and nondetectable in filtrate III of seminal plasma. Similar reduction of CD52 in the filtrate of cauda epididymidal plasma indicates the association of this epididymal secretion with large molecular factors and suggests their involvement as carriers in the in vivo transfer of the secretion onto the epididymal sperm surface. The in vitro uptake of CD52 by some but not all immature sperm and the detection by Western blotting of much less CD52 in the corpus than the cauda luminal plasma suggest that the acquisition of this epididymal secretion by spermatozoa depends on their maturation status as well as the availability of the protein in the epididymal lumen. Mol. Reprod. Dev. 48:267–275, 1997. © 1997 Wiley-Liss, Inc.     

Spontaneous electrical activity of the rat epididymis in vitro

Smooth muscle electrical activity was recorded with suction electrodes from the partly or completely uncoiled epididymal duct of the rat in vitro. The electrical activity of the cauda epididymidis consisted of one or few spikes followed by a plateau of 1-2 sec. The frequency of electrical activity declined from the thicker-walled initial segment of the thin-walled initial segment, was increased to the level seen in the initial segment in the thicker, major portion of the caput epididymidis, declined in the corpus and fell steeply in the cauda epididymidis towards the vas deferens. Electrical activity spread over long distances in the distal cauda and epididymal vas. Elsewhere in the epididymis activity remained synchronous only for a short period in short segments.     

Electron Microscopic (Energy Dispersive X-ray Analysis) Study of Human Male Reproductive Organs and Semen

Recently, technological advancement helped to improve our knowledge on trace elements in human male reproductive organs and its secretion, semen. In this study, employing energy dispersive x-ray analysis facilities on electron microscope, presence of different elements in human male reproductive organs-??testis, epididymis, caput, corpus and cauda, prostate gland, seminal vesicle, Cowper??s gland and vas deferens??seminal plasma and spermatozoa pellet was studied. Several elements were observed. Gold was one among them that was present in seminal plasma and spermatozoa. It was also present in epididymis caput. Authors consider epididymis caput as the source of gold in semen.     

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.     

beta-N-acetylglucosaminidase in the reproductive organs and seminal plasma of the bull

The highest specific activity of beta-N-acetylglucosaminidase (beta-NAG) was found in the different parts of the epididymis, where the activity seemed to be partly in secretory and partly in non-secretory, tissue-bound form. Epididymal spermatozoa also contained moderate beta-NAG activity. The beta-NAG was separated by chromatofocussing and anion exchange chromatography with HPLC into multiple forms with distinct pI values (8.0-4.0). The cauda epididymidis, ampulla and the seminal vesicles formed the major secretory sources of the high beta-NAG activity in bull seminal plasma. The major secretory forms of beta-NAG in caput and cauda epididymidis showed distinct elution profiles. In the fractionation with gel filtration on Sepharose 6B, the beta-NAG activities derived from bull testis and caput epididymidis had smaller molecular weights than did the secretory enzymes in seminal plasma, seminal vesicle secretion and cauda epididymidis. Maximum activity of all beta-NAG isoenzymes was observed at pH 5.0. They were almost totally inactivated at 60 degrees C and about 75-80% of the activity was lost at 55 degrees C. All the isoenzymes were strongly inhibited by thiol reagents but not with other metal ions and chelating agents. Histochemical studies showed a strong granular (lysosomal) reaction for beta-NAG in basal cells and basal parts of the principal cells in all but the initial segment of the epididymis. An apical (secretory) reaction was prominent in the distal caput and corpus as well as in distal cauda. After the distal caput the luminal sperm mass became increasingly mixed with a beta-NAG-positive material. The epithelial cells of the ampulla and seminal vesicle displayed a moderate apical (secretory) reaction.     

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.     

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

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

Production and characterization of monoclonal antibodies to the sperm acrosome stabilizing factor (ASF): utilization for purification and molecular analysis of ASF

Utilizing hybridoma technology and highly purified acrosome stabilizing factor (ASF), six monoclonal antibodies (mAbs) specific for ASF were produced and characterized. Specificity and binding properties of each clone were examined by immunoperoxidase labeling of electrophoretic blots of rabbit serum, seminal plasma, cauda epididymal fluid and vasectomized seminal plasma separated on native and sodium dodecyl sulfate (SDS)-polyacrylamide gels. All mAbs recognize ASF in seminal plasma and cauda epididymal fluid but do not bind components in serum or vasectomized seminal plasma. Purification of ASF by affinity chromatography utilizing the mAbs, has shortened the 6-day isolation procedure for ASF used previously to less than 2 h and has increased the yield from 2 micrograms to 300 micrograms of ASF obtained per ml of seminal plasma. Three mAbs were used in conjunction with Cleveland digest with Staphylococcus aureus V8 protease, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoperoxidase labeling of Western Blots, to identify peptides containing specific determinants recognized by each mAb. At least five separate determinants were recognized by the six mAbs. The sensitivity of the Western blotting technique in conjunction with the specificity of the mAbs was exploited to detect polymeric forms of ASF in seminal plasma and cauda epididymal fluid. ASF is shown to be a 360-kd dimer consisting of two identical 180-kd monomers. Tools are now available to develop sensitive qualitative and quantitative assays for ASF, thus providing rapid, extremely sensitive methods for evaluating experiments designed to probe the molecular mechanism of capacitation.     

The lipid content of epididymal spermatozoa of Rattus norvegicus.

1. Seven major lipids of rat spermatozoa from the caput, corpus and cauda epididymidis were separated and quantitated by TLC. 2. Spermatozoa from the caput epididymidis had a significantly greater (P less than 0.05) content of phospholipid, cholesterol, cholesterol ester and free fatty acid than those from the cauda epididymidis. 3. Spermatozoa from the corpus epididymidis had a significantly greater (P less than 0.05) content of monoglyceride than those from the caput epididymidis and a greater content of phospholipid, cholesterol, free fatty acids and monoglyceride than those from the cauda epididymidis.     

Different mechanisms are responsible for 3H-androgen movement across the rat seminiferous and epididymal epithelia in vivo.

Mechanisms involved in the maintenance of the microenvironment of the seminiferous and epididymal tubules were examined in a series of experiments utilizing in vivo microperifusion, microperfusion, and micropuncture. Intraluminal 3H-androgen concentrations in seminiferous tubules increased linearly as interstitial 3H-androgen concentrations increased from 10 nM to 2,000 nM, but in caput epididymidal tubules, intraluminal 3H-androgen concentrations increased hyperbolically across the same range of peritubular 3H-androgen concentrations. Intraluminal 3H-androgen concentrations in the caput epididymidis did not rise above approximately 340 nM even if peritibular 3H-androgen concentrations exceeded 2,000 nM. Perifusion of caput tubules with 0.1 mM dinitrophenol or potassium cyanide or 100 micrograms/ml cyclohexamide significantly reduced proluminal 3H-androgen movement, but tubules perifused with control medium would not support antigrade 3H-androgen movement in the absence of native lumen fluids which contain androgen-binding protein. Antigrade proluminal 3H-androgen movement was not inhibited by competition with estradiol at ten-times 3H-androgen concentrations. Thus, energy-requiring protein synthesis is necessary for antigrade 3H-androgen movement in the caput epididymidis, but the mechanism for the interaction of intracellular protein(s) and 3H-androgen movement remains undetermined.     

Resorption versus secretion in the rat epididymis

Micropuncture samples were taken from the rete testis, caput epididymidis and cauda epididymidis of anaesthetized adult rats and assayed for total protein, sodium and potassium concentrations. Intraluminal sperm concentrations were determined and used to calculate the amount of fluid resorbed from the efferent duct and epididymal lumen. It was demonstrated that large amounts of protein (30.2 mg/ml cauda volume) and sodium (241.8 mequiv./l) and smaller amounts of potassium (19.4 mequiv./l) are resorbed from the rat epididymal lumen between the caput and corpus epididymidis. This occurs despite increases in intraluminal concentrations of protein (from 22 to 28 mg/ml) and potassium (from 16 to 50 mequiv./l). Resorption is an important aspect of epididymal control of the intraluminal environment.     

Toluidine blue staining reveals changes in chromatin stabilization of mouse spermatozoa during epididymal maturation and penetration of ova

Sperm samples, recovered from various regions of the reproductive tract of male and female mice, were air dried, fixed for 2 min in acetic alcohol and stained with toluidine blue. Testicular sperm heads were deeply stained, while in the samples from the successive regions of caput epididymidis the proportion of stained heads gradually decreased and many of them were stained in their distal part only. Spermatozoa from the corpus and cauda epididymidis, vas deferens, uterus, oviduct and from the perivitelline space were colourless (except for one type of misshapen head). Sperm heads that had penetrated the vitellus became stained distally. Vas deferens sperm heads were fully stained after treatment with dithiothreitol for 30 min. We suggest that the inability to stain with toluidine blue is characteristic of sperm chromatin stabilized by disulphide bonds.     

Regional differences in the testosterone to dihydrotestosterone ratio in the epididymis and vas deferens of adult mice

The concentrations of testosterone and dihydrotestosterone (DHT) were measured in the testis and in different segments of the epididymis and vas deferens of adult mice. There were marked regional variations in the concentrations of testosterone and DHT from the testis to the caudal part of the vas deferens. In the testis, testosterone was the predominant androgen (364 +/- 90 ng/g) while DHT was weakly represented (8 +/- 2 ng/g). Qualitative and quantitative changes occurred in epididymis: DHT was the main steroid in the caput (29.3 +/- 2.7 ng/g) and corpus (33.1 +/- 4.4 ng/g) while testosterone and DHT were in similar quantities in the cauda (18.6 +/- 2.6 and 19.0 +/- 2.7 ng/g, respectively). The proximal region of the vas deferens contained higher amounts (71.4 +/- 8.0 ng/g) of androgens (testosterone + DHT) than did the caput epididymidis (39.1 +/- 3.3 ng/g). Testosterone was the predominant androgen in each part of the vas deferens and its concentrations decreased from the proximal (64.5 +/- 7.5 ng/g) to the caudal (26.9 +/- 4.3 ng/g) region. Castration and section of the efferent ducts of the testis showed that the epididymis received testosterone essentially via the blood supply and that epididymal DHT was produced locally from circulating testosterone.     

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     

Maturation of spermatozoa in the epididymis of the Chinese hamster

Chinese hamster spermatozoa gain their ability to move when they descend from the testis to the distal part of the caput epididymis, but it is not until they enter the corpus epididymis that they become capable of fertilizing eggs. The maturation of the spermatozoa proceeds as they further descend the tract and perhaps continues even in the vas deferens. During transit between the distal caput and proximal cauda epididymides, small membrane-limited vesicles (and tubules) appear on the plasma membrane over the acrosomes of the spermatozoa. The number of vesicles appearing on the sperm brane reaches a maximum when the spermatozoa are in the proximal cauda epididymis. It declines sharply in the distal cauda epididymis. Spermatozoa in the vas deferens are free of the vesicles. The origin, chemical nature, and functional role of the vesicles that appear on the sperm surface during epididymal transit must be the subject of further investigation.