Seminal ribonuclease synthesis in bull reproductive organs during ontogenesis

Seminal ribonuclease (AS RNase) is synthesized in the epididymis, ampullary glands and seminal vesicles of sexually mature bulls. During sexual maturation of Czech red-spotted bulls it first begins to be synthesized in the seminal vesicles and ampullary glands, after the age of 20 weeks. At this age practically none is synthesized in the epididymis. As soon as the epithelial cells of the seminal vesicles start to synthesize the enzyme and secretion of the fluids of this organ begins, synthesis per ml fluid is almost the same as in sexually mature bulls. AS RNase synthesis in the cauda epididymidis begins after the age of 27 weeks and is individually variable. AS RNase synthesis in the reproductive organs depends on the testosterone concentration in the blood plasma of the bull.     

Seminal ribonuclease of bulls - biosynthesis in normal and orchitic bulls and correlation with blood plasma hormone levels

In sexually mature and healthy bulls, seminal ribonuclease (AS RNase) is synthesized in the distal part of the corpus epididymidis, the cauda epididymidis, the ampullary glands and the seminal vesicles. Indirect immunofluorescence demonstrated AS RNase binding to the cytoplasmic droplets of bull spermatozoa.In bulls with orchitis, AS RNase synthesis decreases in accordance with the degree of damage to the Leydig cells and the drop in the blood plasma testosterone level. The organ most sensitive to decreased testosterone levels, from the aspect of AS RNase synthesis, is the epididymis and the least sensitive are the seminal vesicles. Hypertrophy of the adrenal cortex (in particular of the zona fasciculata and the zona reticularis) and elevated adrenocortical secretion - demonstrated by a raised cortisol concentration in the blood plasma of severely orchitic bulls - failed to inhibit AS RNase synthesis. Injections of Gn RH-LH and HCG raised the blood plasma cortisol, but not testosterone, concentrations in bulls with very severe orchitis. This also indicates serious damage to the Leydig cells in these bulls.     

Aspermatogenic effect of the bull seminal ribonuclease (BS RNase) in the presence of anti-BS RNase antibodies in mice

Injection of mouse scrotum with the bull seminal ribonuclease (BS RNase) isolated from bull seminal vesicle fluid inhibited spermatogenesis and caused a decrease in the weight of the testes. Long-term injection of BS RNase evoked the production of antibodies which reached the titre 524448. These antibodies did not prevent the aspermatogenic action of BS RNase in vivo when a twofold higher amount of this enzyme was injected into mouse scrotum. Aspermatogenesis was reversible in both the first and second part of the experiment. During the period of aspermatogenesis the males were sterile. Increasing the amount of BS RNase injections in the second part of experiments caused aspermatogenesis around 3 months. No malformations were observed among offspring of males recovered from the first stage of aspermatogenesis. The antigen—antibody complex prepared in vitro and injected into testes of mice evoked the same degree of aspermatogenesis as the enzyme itself.     

Prostaglandin F2α concentration in genital tract secretions of dairy bulls

Prostaglandin F_2α (PGF_2α) concentrations in genital tract secretions of conscious dairy bulls were determined by radioimmunoassay procedures and compared with peripheral blood plasma levels. The mean (± SD) PGF_2α concentration of coccygeal venous blood plasma from four bulls was 0.14 ± 0.05 ng/ml. Values for rete testis fluid and seminal plasma were the same, namely 0.17 ± 0.01 ng/ml (n = 5) and 0.17 ± 0.02 ng/ml (n = 4), respectively. However, the PGF_2α level in cauda epididymal plasma was 1.61 ± 0.41 ng/ml, or over 8 to 10 times (P < 0.01) the concentration of any other fluid studied.Added PGF_2α had no effect on the endogenous oxygen consumption of washed cauda epididymal spermatozoa or on the oxidative and glycolytic activities of washed ejaculated spermatozoa in vitro. No evidence was obtained suggesting that the prostaglandin may interact with the stimulatory effect of added testosterone or phosphatidylinositol (PI) on the motility, respiration or glucose uptake of ejaculated spermatozoa.     

Partial purification and properties of a protein kinase C type enzyme from plants

Partial purification of a protein kinase with a dependence on micromolar concentrations of free calcium has been achieved from seedlings of Amaranthus tricolor. The enzyme has a M_r of 77 600 as determined by gel filtration and 84 500 by SDS-PAGE analysis. Interaction of the enzyme with membranes (inside-out erythrocyte vesicles) is regulated by calcium, a characteristic of animal protein kinase C. Phospholipid and diolein activation of the enzyme is markedly dependent on the phospholipid used and on both calcium and phospholipid concentration. K_m values for Ca²⁺ in the absence of phospholipid was 20–40 μM and in the presence of phosphatidylserine 5–10 μM. Diolein plus phosphatidylserine lowered the K_m to < 1.5 μM. The best activation was achieved at 1OOμM calcium with 40μg/ml phosphatidylserine and 8μg/ml diolein. These properties indicate a protein kinase C type enzyme. The plant enzyme reacted with antiserum directed against the regulatory domain of bovine brain protein kinase C in an immunoblot experiment.     

Regulation of plasma membrane Ca2 +-ATPase activity by acetylated tubulin: Influence of the lipid environment

We demonstrated previously that acetylated tubulin inhibits plasma membrane Ca^2 +-ATPase (PMCA) activity in plasma membrane vesicles (PMVs) of rat brain through a reversible interaction. Dissociation of the PMCA/tubulin complex leads to restoration of ATPase activity. We now report that, when the enzyme is reconstituted in phosphatidylcholine vesicles containing acidic or neutral lipids, tubulin not only loses its inhibitory effect but is also capable of activating PMCA. This alteration of the PMCA-inhibitory effect of tubulin was dependent on concentrations of both lipids and tubulin. Tubulin (300 μg/ml) in combination with acidic lipids at concentrations > 10%, increased PMCA activity up to 27-fold. The neutral lipid diacylglycerol (DAG), in combination with 50 μg/ml tubulin, increased PMCA activity > 12-fold, whereas tubulin alone at high concentration (≥ 300 μg/ml) produced only 80% increase. When DAG was generated in situ by phospholipase C incubation of PMVs pre-treated with exogenous tubulin, the inhibitory effect of tubulin on PMCA activity (ATP hydrolysis, and Ca^2 + transport within vesicles) was reversed. These findings indicate that PMCA is activated independently of surrounding lipid composition at low tubulin concentrations (< 50 μg/ml), whereas PMCA is activated mainly by reconstitution in acidic lipids at high tubulin concentrations. Regulation of PMCA activity by tubulin is thus dependent on both membrane lipid composition and tubulin concentration.     

Effects of cold shock treatment on angiotensin-converting enzyme activity and on semen characteristics in roosters and bulls

Angiotensin-converting enzyme (ACE) activity has been determined in the semen of certain avian and mammalian species as well as its release during cold shock. The maximum and minimum levels of this enzyme were found in mammalian spermatozoa and in seminal plasma, respectively. It was found that ACE activity in mammalian spermatozoa was more pronounced than in the seminal plasma, whereas in the avian species a revers pattern was observed. However, there were no significant differences in ACE activity in spermatozoa and seminal plasma between layer and broiler strains of avian species. By contrast, ACE activity in the spermatozoa and seminal plasma of buffalo bulls was significantly higher (P/ 0.01) than in cattle bulls. Cold shock did not significantly alter semen characteristics in avian species, while a significant (P/ 0.01) decrease in sperm live counts and motility as well as a corresponding increase in morphological abnormalities were observed in the spermatozoa of cattle and buffalo bulls due to cold shock.     

Selenoprotein P in seminal fluid is a novel biomarker of sperm quality

Hepatically-derived selenoprotein P (SePP) transports selenium (Se) via blood to other tissues including the testes. Male Sepp-knockout mice are infertile. SePP-mediated Se transport to Sertoli cells is needed for supporting biosynthesis of the selenoenzyme glutathione peroxidase-4 (GPX4) in spermatozoa. GPX4 becomes a structural component of sperm midpiece during sperm maturation, and its expression correlates to semen quality. We tested whether SePP is also present in seminal plasma, potentially correlating to fertility parameters. Semen quality was assessed by sperm density, morphology and motility. SePP was measured by an immunoluminometric assay, and trace elements were determined by X-ray fluorescence spectroscopy. SePP levels were considerably lower in seminal plasma as compared to serum (0.4 ± 0.1 mg/l vs. 3.5 ± 1.0 mg/l); Se concentrations showed a similar but less pronounced difference (48.9 ± 20.7 μg/l vs. 106.7 ± 17.3 μg/l). Se and Zn correlated positively in seminal fluid but not in serum. Seminal plasma SePP concentrations were independent of serum SePP concentrations, but correlated positively to sperm density and fraction of vital sperm. SePP concentrations in seminal plasma of vasectomized men were similar to controls indicating that accessory sex glands are a testes-independent source of SePP. This notion was corroborated by histochemical analyses localizing SePP in epithelial cells of seminal vesicles. We conclude that SePP is not only involved in Se transport to testes supporting GPX4 biosynthesis but it also becomes secreted into seminal plasma, likely important to protect sperm during storage, genital tract passage and final journey.     

Effect of hCG injection on prostaglandin E concentrations in ram seminal plasma

Ram and bull seminal plasma, respectively, contain 0.5-20 microg PGE/ml and 5-10 ng PGE/ml. To demonstrate that PGE concentrations in the seminal plasma are related to sperm quality and could be affected by hormonal stimulation in vivo, four rams were injected with 500 IU hCG, in and out of season. The rams responded 1 week after hCG with a 1.5- to 4-fold increase in seminal plasma PGE. The PGE peak was temporally separate from the hCG-induced rise in seminal plasma testosterone which was observed after 1 day. Using a simulated cryptochid ram, peaks in seminal fluid PGE were found to be associated with increased sperm velocity and sperm counts. In bulls, PGE concentrations in the seminal plasma of good bulls were significantly higher than that found in poor and cryptorchid bulls.     

Prostaglandin endoperoxide and thromboxane generating systems and their selective inhibition

Two enzyme systems and their selective inhibition are described. Microsomes from ram seminal vesicles (RSV) incubated with arachidonic acid at 22° C generated a rabbit aorta contracting substance which, after rapid ether extraction, had characteristics similar to purified standard endoperoxides. Incubation of either purified endoperoxide or the product from RSV and arachidonic acid with horse platelet microsomes (HPM) yielded a more potent rabbit aorta contracting substance characterized as thromboxane A₂, with a half life of 35.9 ± 2.2 s at 37° C after ether extraction. Two inhibitors, indomethacin and benzydamine exhibited selectivity for the two enzyme systems. The IC₅₀ for benzydamine against thromboxane synthetase was 100 μg/ml and 250 μg/ml against RSV. Indomethacin showed a greater degree of selectivity with an IC₅₀ of 5 μg/ml for the ram seminal vesicle cyclo-oxygenase compared to 100 μg/ml for thromboxane synthetase.     

Age-related differences of semen quality,seminal plasma,and spermatozoa antioxidative and oxidative stress variables in bulls during cold and warm periods of the year

The aims of this study were to determine the presence and quantities of antioxidative status and oxidative stress (OS) variables in the seminal plasma and spermatozoa of bulls of varying age during cold and warm periods of the year, and to establish the correlation of these variables with semen quality parameters. The study was conducted on two groups each comprising nine Simmental bulls: one group contained younger animals (aged 2 to 4 years) and the second older animals (aged 5 to 10 years). Semen samples were collected using an artificial vagina for biochemical analysis. Seminal plasma and spermatozoa activities of total superoxide dismutase (TSOD), manganese superoxide dismutase (MnSOD), copper–zinc superoxide dismutase (CuZnSOD), catalase (CAT), selenium-dependent glutathione peroxidase, reduced glutathione and concentrations of total protein (TP), thiobarbituric acid reactive substances (TBARS) and protein carbonyl content (PCC) were determined. Several antioxidants in seminal plasma were also determined: total glutathione peroxidase (TGSH-Px), selenium-independent glutathione peroxidase (Non-SeGSH-Px), uric acid, albumins (ALB) and alkaline phosphatase (ALP). Significantly higher spermatozoa motility was observed during the cold v. warm period, and a significantly higher volume and total number of spermatozoa per ejaculate was observed in older than in younger bulls. Significantly higher values of ALP, TP and ALB were found in seminal plasma of older bulls than in younger bulls during the warm period. The seminal plasma of younger bulls showed significantly higher activities of TSOD, MnSOD, CuZnSOD, TGSH-Px and Non-SeGSH-Px. Younger bulls had significantly higher PCC concentration and activity of CAT in seminal plasma than older bulls during the cold period. Significantly higher concentrations of PCC and TBARS, and activities of TSOD, MnSOD and CuZnSOD were established in spermatozoa of the younger than in older bulls during the warm period. It could be concluded that antioxidative and OS variables differ significantly depending on bull age and time of year. Younger bulls were more sensitive to elevated ambient temperatures during the warm period, when the higher enzymatic antioxidative protection in seminal plasma and spermatozoa were insufficient to counteract the intensive oxidative processes in spermatozoa, which eventually resulted in decreased spermatozoa motility. The estimation of antioxidative and OS variables in seminal plasma and spermatozoa may have practical value for the assessment of bull semen quality.     

Regulation of plasma membrane Ca(2+)-ATPase activity by acetylated tubulin: influence of the lipid environment

We demonstrated previously that acetylated tubulin inhibits plasma membrane Ca(2+)-ATPase (PMCA) activity in plasma membrane vesicles (PMVs) of rat brain through a reversible interaction. Dissociation of the PMCA/tubulin complex leads to restoration of ATPase activity. We now report that, when the enzyme is reconstituted in phosphatidylcholine vesicles containing acidic or neutral lipids, tubulin not only loses its inhibitory effect but is also capable of activating PMCA. This alteration of the PMCA-inhibitory effect of tubulin was dependent on concentrations of both lipids and tubulin. Tubulin (300μg/ml) in combination with acidic lipids at concentrations >10%, increased PMCA activity up to 27-fold. The neutral lipid diacylglycerol (DAG), in combination with 50μg/ml tubulin, increased PMCA activity >12-fold, whereas tubulin alone at high concentration (≥300μg/ml) produced only 80% increase. When DAG was generated in situ by phospholipase C incubation of PMVs pre-treated with exogenous tubulin, the inhibitory effect of tubulin on PMCA activity (ATP hydrolysis, and Ca(2+) transport within vesicles) was reversed. These findings indicate that PMCA is activated independently of surrounding lipid composition at low tubulin concentrations (<50μg/ml), whereas PMCA is activated mainly by reconstitution in acidic lipids at high tubulin concentrations. Regulation of PMCA activity by tubulin is thus dependent on both membrane lipid composition and tubulin concentration.     

On the interaction of bovine seminal RNase with actin in vitro

Ribonuclease from bovine seminal plasma (RNase BS) interacts with skeletal muscle actin in the following way: it binds to actin with an apparent binding constant of 9.2 X 10(4) M-1 in 0.1 M KCl, induces the polymerization of actin below the critical concentration in depolymerization buffer, accelerates the salt-induced polymerization of actin even at a molar ratio of RNase to actin lower than 1/100, and bundles F-actin filaments. In the bundles the molar ratio of RNase to actin is about 0.66. Actin inhibits the enzymatic activity of RNase BS. RNase A from bovine pancreas, which is structurally almost identical to the subunits of RNase BS as well as a monomeric form of RNase BS, do not cross-link actin filaments and have a much smaller effect on the polymerization of actin. We conclude that the dimeric structure of the RNase BS, which consists of two identical subunits cross-linked by interchain disulfide bridges, is probably responsible for the bundling activity and the accelerating effect on the polymerization of actin.     

Endogenous antioxidant defenses in neonates

Deficiencies of antioxidant defenses have been postulated as possible mechanisms in the development of bronchopulmonary dysplasia (BPD). Neonates, especially prematures with respiratory distress syndrome (RDS), are exposed to high oxygen tensions for prolonged periods.To evaluate the neonates' ability to respond to an oxygen challenge with increased superoxide dismutase (SOD), 9 prematures were studied immediately at birth and on days I, 3, 5, and 7. An increase in plasma levels was noted during the first week of life in the patients who were exposed to oxygen. The mean endogenous SOD level at birth was 1.28 μg/ml. On day 1, plasma SOD rose to 1.53 μg/ml and to 2.25 μg/ml on day 3 (P =.003). This trend continued into the fifth and seventh days.Whether this increase in SOD has clinical significance in the prevention of BPD requires further investigation.     

Inhibition of thrombin,an unexplored function of retinoic acid

Retinoic acid, a derivative of vitamin A, is known to possess in vivo anti-inflammatory, anti-platelet and fibrinolytic activities. We have investigated the in vitro thrombin and platelet aggregation inhibitory activities of vitamin A (retinol) and its derivatives, retinoic acid and retinaldehyde. The thrombin enzymatic assay was performed fluorimetrically to assess the inhibition of thrombin (Sigma and plasma). Retinoic acid, retinaldehyde and retinol exhibited potent inhibition of thrombin, with IC₅₀ values of 67μg/ml, 74μg/ml and 152μg/ml, respectively for the inhibition of thrombin (Sigma); and 49μg/ml, 74μg/ml and 178μg/ml, respectively for the inhibition of thrombin (plasma). Amongst vitamin A and its derivatives, retinoic acid showed the highest inhibition of both the forms of thrombin. Vitamin A and its derivatives also displayed remarkable inhibition of platelet aggregation. This is the first report of vitamin A and its derivatives showing inhibition of thrombin and platelet aggregation in vitro.     

Bioactivity of ovulation inducing factor (or nerve growth factor) in bovine seminal plasma and its effects on ovarian function in cattle

To understand the role of ovulation-inducing factor (or nerve growth factor) (OIF [NGF]) in bovine seminal plasma, we (1) used an in vivo llama bioassay to test the hypothesis that bovine seminal plasma induces ovulation and CL development in llamas similar to that of llama seminal plasma when the dose of seminal plasma is adjusted to ovulation-inducing factor content (experiment 1) and (2) determined the effect of bovine seminal plasma on the interval to ovulation and luteal development in heifers (experiment 2). Within species, seminal plasma was pooled (n = 160 bulls, n = 4 llamas), and the volume of seminal plasma used for treatment was adjusted to a total dose of 250 μg of ovulation-inducing factor. In experiment 1, mature female llamas were assigned randomly to four groups and treated intramuscularly with either 10 mL of PBS (negative control, n = 5), 50-μg GnRH (positive control, n = 5), 6-mL of llama seminal plasma (n = 6), or 12 mL of bull seminal plasma (n = 6). Ovulation and CL development were monitored by transrectal ultrasonography. In experiment 2, beef heifers were given a luteolytic dose of prostaglandin followed by 25-mg porcine LH (pLH) 12 hours later to induce ovulation. Heifers were assigned randomly to three groups and given 12 mL bovine seminal plasma intramuscularly 12 hours after pLH treatment (n = 10), within 4 hours after ovulation (n = 9), or no treatment (control, n = 10). Ovulation was monitored by ultrasonography every 4 hours, and the CL development was monitored daily until the next ovulation. In experiment 1, ovulation was detected in 0/5, 4/5, 4/6, 4/6 llamas in the PBS, GnRH, llama seminal plasma, and bovine seminal plasma groups, respectively (P < 0.05). Luteal development was not different among groups. In experiment 2, the interval to ovulation was more synchronous (range: 4 vs. 22 hours; P < 0.0001) in heifers treated with seminal plasma before ovulation compared with the other groups. Luteal development was not different among groups; however, plasma progesterone concentrations tended to be greater in the postovulation treatment group compared with other groups. In summary, results confirmed the presence of bioactive ovulation-inducing factor in bull seminal plasma and supported the hypothesis that bovine and llama seminal plasma have similar ovulatory effects, using a llama bioassay. Treatment with bovine seminal plasma resulted in greater synchrony of ovulation in heifers pretreated with pLH. Plasma progesterone concentration tended to be higher in heifers given bovine seminal plasma within 4 hours after ovulation, suggesting that bovine ovulation-inducing factor is luteotrophic.     

Contraction of seminal vesicle and prostaglandin E1

It was studied how PGE₁ would affect the responses of isolated human seminal vesicles to adrenalin. PGE₁ in the final concentration of 1.3 μg/ml suppressed the contraction of human seminal vesicle that would have occurred in reaction to adrenalin added one minute later. When the concentration of PGE₁ was increased to 6.7 μg/ml, the inhibitory action was further enhanced. The meanings of this phenomenon were discussed.     

Novel mechanisms for maturation of chloroplast transfer RNA precursors

Despite the prokaryotic origins of chloroplasts, a plant chloroplast tRNA precursor is processed in a homologous in vitro system by a pathway distinct from that observed in Escherichia coli, but identical to that utilized for maturation of nuclear pre-tRNAs. The mature tRNA 5' terminus is generated by the site-specific endonucleolytic cleavage of an RNase P (or P-type) activity. The 3' end is likewise produced by a single precise endonucleolytic cut at the 3' terminus of the encoded tRNA domain. This is the first complete structural characterization of an organellar tRNA processing system using a homologous substrate. In contrast to eubacterial RNase P, chloroplast RNase P does not appear to contain an RNA subunit. The chloroplast activity bands with bulk protein at 1.28 g/ml in CsCI density gradients, whereas E.coli RNase P bands as ribonucleoprotein at 1.73 g/ml. Chloroplast RNase P activity survives treatment with micrococcal nuclease (MN) at levels 10- to 100-fold higher than those required to totally inactivate the E.coli enzyme. The chloroplast system is sensitive to a suppression of tRNA processing, caused by binding of inactive MN to pre-tRNA substrate, which is readily overcome by addition of carrier RNA to the assay.     

Effect of ionic strength on chain elongation in ADP-ribosylation of various nucleases

With the use of a reconstituted poly(ADP-ribosyl)ating enzyme system and three purified nucleases, micrococcal nuclease (MN), bull seminal RNase (BS RNase) and Ca2+, Mg2+-dependent endonuclease (BS DNase), as model acceptor proteins for ADP-ribose, the effect of ionic strength on the modification reaction was examined in detail. When these three nucleases were extensively poly(ADP-ribosyl)ated in this system at a low ionic strength (5 mM Tris), they were all inhibited by about 80% and the chain length of the polymer covalently bound to the nucleases was 13 to 23 ADP-ribose units. The observed inhibition was markedly prevented by increasing the ionic strength in the reaction mixture with a concomitant decrease in the polymer size bound to the nucleases. The NaCl concentrations required for decreasing the extent of the inhibition to half of the maximum were calculated to be 20, 50, and 100 mM for MN, BS RNase, and BS DNase, respectively. These values are similar to the NaCl concentrations required for decreasing the average chain lengths of the polymer to half, suggesting that the length of polymer is closely correlated to the extent of inhibition of these nucleases. DNA-binding affinities of these nucleases, expressed in terms of the NaCl concentrations required for eluting the enzymes from DNA-cellulose, were 140, 280, and 340 mM for MN, BS RNase, and BS DNase, respectively. Considering that maintainance of a ternary complex of poly(ADP-ribose) synthetase, acceptor and DNA may be essential for the modification reaction, the relatively strong salt effect observed in the modification of MN may be explained by its low DNA-binding affinity.     

Immunohistochemical localization of phospholipase A2 in the bovine seminal vesicle and on the surface of the ejaculated spermatozoa.

1. Approximately 150-fold purified phospholipase A2 (PLA2) from bovine seminal vesicle fluid was injected into rabbit to prepare antibodies. 2. Produced antisera blocked PLA2 activity in bovine seminal plasma, seminal vesicles and its fluid and it gave single precipitation lines with the same samples. No cross-reactivity was detected with other reproductive tissues of bull as well as human seminal plasma. 3. Using indirect peroxidase technique PLA2 was localized in the apical part of epithelia cells of the bull seminal vesicle and also some minor immunohistochemical reactions were observed in the tubular lumen. Indirect peroxidase staining gave weak or no reaction at all to seminal vesicles of immature bulls. This suggests that the enzyme may be under hormonal control. 4. By indirect immunofluorescence method ejaculated spermatozoa of bull revealed immunoreaction which was not uniform and it was restricted to the middle piece, acrosome as well as postacrosomal region, but no specific immunostaining could be found on the surface of the epididymal spermatozoa. 5. Enzyme visualization by immunoelectron microscopic labelling showed a predominant localization in membrane particles inside the lumen of bovine seminal vesicle but some gold particles were also seen in granules, larger vacuoles and in cytoplasm of epithelia cells.