11Beta-hydroxysteroid dehydrogenase enzymes in the testis and male reproductive tract of the boar (Sus scrofa domestica) indicate local roles for glucocorticoids in male reproductive physiology.

11Beta-hydroxysteroid dehydrogenase (11betaHSD) enzymes modulate the target cell actions of corticosteroids by catalysing metabolism of the physiological glucocorticoid (GC), cortisol, to inert cortisone. Recent studies have implicated GCs in boar sperm apoptosis. Hence, the objective of this study was to characterise 11betaHSD enzyme expression and activities in the boar testis and reproductive tract. Although 11betaHSD1 and 11betaHSD2 mRNA transcripts and proteins were co-expressed in all tissues, cortisol-cortisone interconversion was undetectable in the corpus and cauda epididymides, vas deferens, vesicular and prostate glands, irrespective of nucleotide cofactors. In contrast, homogenates of boar testis, caput epididymidis and bulbourethral gland all displayed pronounced 11betaHSD activities in the presence of NADPH/NADP(+) and NAD(+), and the penile urethra exhibited NAD(+)-dependent 11beta-dehydrogenase activity. In kinetic studies, homogenates of boar testis, caput epididymidis and bulbourethral gland oxidised cortisol with K(m) values of 237-443 and 154-226 nmol/l in the presence of NADP(+) and NAD(+) respectively. Maximal rates of NADP(+)-dependent cortisol oxidation were 7.4- to 28.5-fold greater than the V(max) for NADPH- dependent reduction of cortisone, but were comparable with the rates of NAD(+)-dependent cortisol metabolism. The relatively low K(m) estimates for NADP(+) -dependent cortisol oxidation suggest that either the affinity of 11betaHSD1 has been increased or the cortisol inactivation is catalysed by a novel NADP(+)-dependent 11betaHSD enzyme in these tissues. We conclude that in the boar testis, caput epididymidis and bulbourethral gland, NADP(+)- and NAD(+)-dependent 11betaHSD enzymes catalyse net inactivation of cortisol, consistent with a physiological role in limiting any local actions of GCs within these reproductive tissues.

Determination of glutathione peroxidase and superoxide dismutase-like activities in equine spermatozoa, seminal plasma, and reproductive tissues.

OBJECTIVE: To determine glutathione peroxidase (GPX) and superoxide dismutase (SOD)-like activities in spermatozoa, seminal plasma, and reproductive tissues (ie, testis, epididymis, bulbourethral gland, prostate, vesicular gland, and ampulla) in horses. SAMPLE POPULATION: Seminal plasma from 17 stallions, spermatozoa from 5 stallions, and reproductive tissues from 3 stallions. PROCEDURE: Activity of GPX was determined by use of assays measuring oxidation of NADPH in the presence of exogenous glutathione, cumene hydroperoxide, and glutathione reductase. Activity of SOD-like enzymes was determined by use of the nitroblue tetrazolium assay. RESULTS: Mean GPX and SOD-like activities in seminal plasma were 1.3 +/- 0.1 nmol of NADPH oxidized/min/mg of protein and 29.2 +/- 6.6 U/mg of protein, respectively. Mean GPX activities in spermatozoa separated from seminal plasma by centrifugation and via Percoll gradient were 2.2 +/- 0.3 nmol and 6.1 +/- 1.3 nmol of NADPH oxidized/min/mg of protein, respectively. Mean SOD-like activity of spermatozoa separated by centrifugation was 58.6 +/- 22.3 U/mg of protein; SOD-like activity was not detected in Percoll-separated spermatozoa. Among reproductive tissues, the ampulla and prostate had the highest SOD-like activity, although this was not significantly different from activity in other tissues. Testes and spermatozoa from the cauda epididymis contained significantly more GPX activity than other tissues. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that although equine seminal plasma contains high SOD-like enzyme activity, spermatozoa have limited GPX and SOD-like activity. Enzymatic antioxidant activity in equine spermatozoa appears to be predominantly derived from seminal plasma adsorbed onto the plasma membrane. Removal of seminal plasma during semen processing may increase oxidative stress in equine-spermatozoa.

Cloning and seasonal secretion of the pancreatic lipase-related protein 2 present in goat seminal plasma.

The storage of frozen semen for artificial insemination is usually performed in the presence of egg yolk or skimmed milk as protective agents. In goats, the use of skimmed milk extenders requires, however, that most of the seminal plasma is removed before dilution of spermatozoa because it is deleterious for their survival. It has been previously demonstrated that a lipase (BUSgp60) secreted by the accessory bulbourethral gland was responsible for the cellular death of goat spermatozoa, through the lipolysis of residual milk lipids and the release of toxic free fatty acids. This lipase was purified from the whole seminal plasma of goat and was found to display both lipase and phospholipase A activities, this latter activity representing the main phospholipase activity detected in goat seminal plasma. Based on its N-terminal amino acid sequence, identical to that of BUSgP60 purified from bulbourethral gland secretion, and the design of degenerated oligonucleotides, the lipase was cloned from total mRNA isolated from bulbourethral gland. DNA sequencing confirmed it was the goat pancreatic-lipase-related protein 2 (GoPLRP2). The physiological role of GoPLRP2 is still unknown but this enzyme might be associated with the reproductive activity of goats. A significant increase in lipase secretion was observed every year in August and the level of lipase activity in the semen remained high till December, i.e., during the breeding season. A parallel increase in the plasmatic levels of testosterone suggested that GoPLRP2 expression might be regulated by sexual hormones. The lipase activity level measured in goat seminal plasma, which could reach 1000 U/ml during the breeding season, was one of the highest lipase activity measured in natural sources, including gastric and pancreatic juices.

Expression of the lipocalin-type prostaglandin D synthase gene in the reproductive tracts of Holstein bulls.

The aim of this study was to localize expression of the prostaglandin D synthase gene in the reproductive tracts of Holstein bulls using northern blotting and in situ hybridization. For northern blotting, a digoxigenin-labelled prostaglandin D synthase cDNA probe was used to probe blots containing RNA isolated from the testes, epididymides, vas deferens, ampullae, seminal vesicles, prostate and bulbourethral glands of bulls. The digoxigenin-labelled cDNA for the bovine homologue of prostaglandin D synthase hybridized to a single band (approximately 0.9 kb) to RNA samples from the caput, corpus and cauda epididymides, as well as RNA samples from the vas deferens and the ampulla. The probe also detected a single band in testis samples, although the transcript size was slightly larger (approximately 1.0 kb) than the transcript found in the other tissues. The highest expression of prostaglandin D synthase was observed in the testes and caput epididymides. Prostaglandin D synthase transcripts were not found in the seminal vesicles or the prostate or bulbourethral glands using northern blotting. For in situ hybridization, antisense and sense riboprobes were synthesized and used to hybridize to cryosections obtained from the reproductive tissues of bulls. In situ hybridization of bull testes showed that prostaglandin D synthase transcripts were present within the germ cells in the adluminal compartment of the seminiferous tubules containing round and elongated spermatids, indicating that expression varied with stage of development of the seminiferous tubules. Prostaglandin D synthase expression was observed in the epithelial cells of the epididymides with greatest expression occurring in the caput epididymidis. Some expression was also observed in the epithelial cells of the vas deferens and a few cells of some lobules in the prostate and bulbourethral glands. Expression of the prostaglandin D synthase gene was not detected in ampullae or seminal vesicles by in situ hybridization.

Deterioration of goat sperm viability in milk extenders is due to a bulbourethral 60-kilodalton glycoprotein with triglyceride lipase activity.

The aim of this work was to purify, identify, and characterize the component of goat bulbourethral gland secretion (BUS) responsible for goat sperm deterioration in skim milk extender. BUS extracts promote a decrease in the percentage of motile spermatozoa, deterioration in the quality of movement, breakage of acrosomes, and cellular death of goat spermatozoa diluted in skim milk. A 55- to 60-kDa monomeric glycoprotein (BUSgp60) was purified by cation-exchange, concanavalin A, and heparin-affinity chromatography and was identified as the only BUS component responsible for the deterioration of spermatozoa in milk. The BUSgp60 was shown to display triacylglycerol hydrolase activity, and partial sequences (37 amino acids in all) exhibited 50-70% homology with sequences of various types of pancreatic lipases (PLs), especially PL-related protein 2 (PL-RP2). In addition, porcine PL produced deterioration of goat sperm properties in milk as effectively as BUS and BUSgp60. These results support the preliminary identification of the goat BUSgp60 as a bulbourethral lipase belonging to the PL-RP2 family. Since seminal plasma also contains factors favorable for sperm survival, it is envisaged that, for the best preservation of goat semen, specific inhibitors of this family of lipase could be added into milk-based extenders without eliminating seminal plasma.

Immunohistolocalization of the carbonic anhydrase isoenzymes (CA-I, CA-II, and CA-III) in the reproductive tract of male horses.

OBJECTIVE: To elucidate locations of cytosolic carbonic anhydrase isoenzyme (CA-I, CA-II, and CA-III)-positive epithelial cells in equine male reproductive organs. DESIGN: Descriptive and immunohistochemical study. ANIMALS: 4 clinically normal male horses. PROCEDURE: The testis (seminiferous tubules, rete tubules), epididymis (initial, middle, and terminal segments), proximal and distal portions of the ductus deferens, ampulla ductus deferentis, seminal vesicle, prostate, and bulbourethral gland were excised from euthanatized horses after administration of an overdose of pentobarbital. The tissue specimens were quickly placed in fixative solution, dehydrated in ethanol, and embedded; then thin sections were cut. For immunohistochemical staining, antibodies against purified equine CA-I, CA-II, and CA-III were raised in rabbits. After examination of the specificity of each antiserum, the monospecific antisera against carbonic anhydrase isoenzymes were used to localize the isoenzymes. RESULTS: Specific staining for CA-III was found in the Sertoli and basal cells of the ductus deferens. Most of the testicular and epididymal tissue, as well as ductus deferens, were virtually negative for the enzymes when stained with the antibody to CA-I and CA-II. In the initial segment of the epididymis, a few principal cells had intense cytoplasmic staining with anti-CA-II. In the male accessory glands, CA-I, CA-II, and CA-III were detected in the epithelial cells of the seminal vesicle, prostate, and bulbourethral gland. CONCLUSIONS: In the equine male reproductive tract, the bicarbonate in semen originates mainly from accessory reproductive glands. All 3 isoenzymes may have central roles in the regulation of bicarbonate concentration in seminal plasm and, accordingly, regulate seminal plasma pH. Distribution of CA-III in Sertoli and basal cells of the ductus deferens suggests other specialized physiologic roles.

Immunohistochemical localization of phospholipase A2 in human and bovine male reproductive organs.

Phospholipase A2 (PLA2) taken from human seminal plasma and purified about 1450-fold was injected into rabbits to prepare polyclonal antibodies. The antiserum produced and the IgG purified from the antiserum were used to compare the immunohistochemical localization of PLA2 in the human male reproductive system with that in the bull. In Western blot analyses, the polyclonal antibodies cross-reacted with purified PLA2s from human seminal plasma, bovine Cowper's glands and with partly purified PLA2 from bovine seminal vesicle fluid. On the other hand, purified PLA2s from Crotalus adamanteus venom, bovine pancreas or from bovine prostate were not recognized by the polyclonal antibodies. With an indirect peroxidase technique, PLA2 was localized in the epithelial cells of the human prostate and bovine seminal vesicles as well as in the fibrous connective tissue of bovine Cowper's gland. Indirect peroxidase staining also gave an immunoreaction in the enlarged acrosomes of round spermatids in the human testis. Using an indirect immunofluorescence method, ejaculated human spermatozoa revealed an immunoreaction which was not uniform, and the reaction was restricted to the middle piece and the acrosomal and postacrosomal regions. In conclusion, human seminal plasma, spermatozoa and prostate PLA2s were immunohistochemically related to those from bovine Cowper's gland, seminal vesicles and seminal fluid.

Immunohistochemical study of phospholipase A2 in bovine prostate.

Approximately 1,100-fold purified phospholipase A2 (PLA2) from bovine prostate was injected into rabbit to prepare polyclonal antibodies. Antibodies produced showed specific immunoprecipitation only with the purified enzyme, as well as with homogenate of bovine prostatic tissue. By Western blot analysis or by immunodiffusion test, no cross-reactivity with PLA2 purified from human seminal plasma, bovine pancreas, Crotalus adamanteus venom, or with partially purified PLA2 from bovine seminal vesicle fluid or Cowper's glands was observed. Using the indirect peroxidase technique, PLA2 was localized in the cytoplasm of bovine prostatic epithelial cells. By immunogold microscopy, this enzyme was directly visualized inside the lysosomes, as well as in the endoplasmic reticulum of the glandular epithelial cells. Enzyme activity was localized in two principal subcellular sites: the mitochondria and lysosome-enriched fraction, and in the microsomal fraction.

Phospholipases A2 in the reproductive system of the bull.

1. Phospholipase A2 activities were studied in the reproductive organs, seminal plasma and spermatozoa of adult bulls. 2. Phosphatidylethanolamine and phosphatidylcholine with 14C-labelled linoleic (lino-PE, lino-PC) or arachidonic acid (ara-PE, ara-PC) at sn-2 position as well as a fluorescent derivative (4-pyrenylbutyric acid) of phosphatidylcholine (PPC) were used as substrates. 3. The radioactive substrates were hydrolysed most strongly by homogenates of the prostate and Cowper's gland, but also seminal vesicle and its secretory fluid, seminal plasma and ejaculated spermatozoa contained hydrolytic activity. The fluorescence substrate was most strongly hydrolysed by homogenates of ampulla and seminal vesicle as well as its secretory fluid, seminal plasma and ejaculated spermatozoa. 4. Seminal plasma and seminal vesicle fluid contained a Ca2(+)-independent enzyme (enzyme I), which hydrolysed only PPC, while another Ca2(+)-dependent enzyme (enzyme II) hydrolysed only the radioactive substrates. 5. Both enzymes were purified from the seminal vesicle fluid and their biochemical properties were analysed. In SDS-PAGE enzyme I preparation resulted in two major bands with molecular weights of 16,000 and 60,000 in equal quantities and minor band at 15,000. The binding of the enzyme I to Con A-Sepharose indicated that it is a glycoprotein and it had multiple pI-values from 3.75 to 5.0. Enzyme II gave in SDS-PAGE two closely located bands with molecular weights of about 15,000 and 16,000 (major band). Isoelectric focusing showed one band at pI 4.7. Both enzymes appear to bind to spermatozoa at ejaculation but their function remains to be shown.

Hydrolases from bovine seminal vesicle, prostate and Cowper's gland.

The bovine seminal plasma is formed mainly by secretions of epididymis and the glandular epithelia in ampulla, seminal vesicles, prostate and Cowper's glands. The contribution of each organ to the hydrolytic enzyme activities (glycosidases, exopeptidases, phospholipases) of the bull seminal plasma has been analyzed and is reviewed in this paper with special emphasis on the role of the accessory glands. Seminal vesicles seem to have a major role in the secretion of seminal plasma acid alpha-glucosidase, acid alpha-mannosidase and beta-N-acetylhexosaminidase, aminopeptidase A, dipeptidyl peptidase II and IV and gamma-glutamyl transpeptidase as well as Ca(2+)-dependent and Ca(2+)-independent phospholipases A2 with distinct substrate specificities, a choline-specific phospholipase C and a Co2+ (Mn2+)-activated sphingomyelinase. The enzyme pattern in the ampulla closely resembled that of the seminal vesicles and obviously contributes to the seminal plasma level of these hydrolases. The bull prostate and Cowper's glands contained a strong Ca(2+)-dependent phospholipase A2 activity. However, these glands may not contribute to the seminal plasma PLA2 activity. At ejaculation the epididymal spermatozoa are exposed to these enzymes. They may have a specific affinity to sugar, peptide or phospholipid residues at distinct sites of the sperm surface. These enzymes may also participate in the digestion of various other semen components to create a suitable milieu for the emitted spermatozoa.

Dipeptidyl peptidases in bovine reproductive organs and secretions.

Dipeptidyl peptidases (DPP) I-IV were analysed in homogenates of bovine reproductive organs as well as in seminal vesicle secretions and seminal plasma. The presence of various molecular forms of these enzymes was studied by fractionation using gel filtration, anion exchange chromatography and chromatofocusing. The eluting enzymes were pooled, and their biochemical properties were briefly characterized. The histochemical localization of DPP II and IV was carried out with the most active tissues. DPP I and III were absent from seminal plasma, but their highest activity was found in the epididymis and increased during sexual maturation. DPP II was found mainly in a single molecular form and displayed a wide distribution in the reproductive organs. Its activity in seminal plasma may be derived from various organs, although the major sources are probably the apical activity in the epididymis, ampulla and seminal vesicle. DPP IV activity was high in the cauda epididymis, and ampulla, and in the seminal vesicles and their secretions. The high activity of DPP IV in seminal plasma appeared to derive from these organs, which showed a strong apical reaction of the epithelial lining. In seminal vesicles the enzyme was mainly secreted attached to membrane particles called vesiculosomes.

Observations under the electron microscope on the localization of phosphatases in epithelial cells of Cowper's glands in rats.

The positive results of the reactions for alkaline and acid phosphatases were obtained in epithelial cells of Cowper's glands in rats. Observations under the electron microscope allowed us to state that alkaline phosphatase was localized in the cell's areola, in membranes of smooth intraplasmic reticulum and in the basement membrane of the epithelium. The acid phosphatase was seen in primary lysosomes as well as in the secondary ones, which are seldom seen on unincubated specimens.

[Histotopography of glycosidases in the accessory glands of the boar before and after castration]. / Histotopik von Glykosidasen in den akzessorischen Geschlechtsdrüsen des Ebers vor und nach Kastration.

The histochemical distribution of six glycosidases (N-acetyl-beta-glucosaminidase, beta-galactosidase, beta-glucuronidase, alpha-galactosidase, alpha-mannosidase and alpha-fucosidase) was investigated in the prostate, glandula vesicularis and glandula bulbourethralis of castrated and non-castrated adult boars. The functions of the glycosidases in the male accessory sex glands of the boar and their androgen dependence are discussed briefly.

Localization of certain phosphatases in the male sex accessory glands of Suncus murinus sindensis, Anderson, the common shrew.

Histochemical localization of various phosphatases, alkaline phosphatase, acid phosphatase, adenosine-tri-phosphatase and glucose-6-phosphatase, have been carried out in the male sex accessory glands of Suncus murinus sindensis, ANDERSON. The seminal vesicle and the COWPER'S gland in Suncus display strong phosphatases activities in the epithelium, except the alkaline phosphatase in the in the COWPER'S gland which is more pronounced in the stroma. The possible role of these phosphatases in the secretory activities of the organ where they are localized have been discussed. In the prostate gland, no phosphatase activity could be revealed in the epithelium and the secretions.