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Two hundred and five ram lambs originating from several sources were assembled for a ram performance trial. All rams were immunized with a commercial Brucella ovis bacterin. Four rams developed clinical evidence of epididymitis during the 150 day trial. Actinobacillus seminis was identified as the causative agent.
Fifteen Red Sokoto goats (bucks) aged 2 to 2 1 2 years with a mean body weight of 17.80+/-1.24 kg were used for studying daily spermatozea output, testicular and epididymal sperm reserves. Semen ejaculates were obtained from each buck and evaluated daily for six weeks. Semen characteristics were as follows: volume, 0.72+/-0.91ml; colour, milky/creamy; sperm concentration/ml, 0.61+/-0.05 x 10(9); mass motility, 3.80+/-0.33; individual metility, 77.50+/-5.60; and normal sperm morphology, 80.00+/-10.50. Mean scretal circumference was 21.80+/-0.29. Mean testicular and spididymal (capita, corpora and caudas) weights (gm) were 83.74+/-5.33, 7.27+/-0.63, 2.22+/-0.23 and 6.13+/-0.73 respectively. Corresponding sperm reserves were 44.32+/-5.79, 8.82+/-1.95, 4.99+/-0.86 and 45.64+/-7.87 x 10(9). Scrotal circumference was positively correlated to testicular weight (r = 0.86), testicular spermatids (r = 0.77), and caudas spermatozoa (r = 0.83).
Most of the epididymal spermatozoa collected in all the seasons examined maintained an ability to move progressively, had a cytoplasmic droplet in the distal site of the middle piece, and were morphologically normal. Reduced desire to mount a dummy was not observed during the experimental period. Characteristics of ejaculated semen were not significantly altered throughout the year. However, progressive motility and acrosomal integrity of spermatozoa ejaculated between July and September were more susceptible to storage at 4 degrees C than spermatozoa ejaculated during the other months and acrosomal integrity of spermatozoa ejaculated during the 3 months was to freezing-thawing. These results indicate that the reproductive activity of Meishan boars in Japan is only slightly influenced by season, but semen ejaculated during the summer is less suitable for storage than that ejaculated during the other seasons of the year.
This study was designed to determine the effects of Sephadex filtration (SpermprepI method) on the separation of motile, morphologically normal, mouse epididymal spermatozoa and to study the viability of the recovered spermatozoa over a 3-h incubation period. Spermatozoa were harvested from the caudae epididymie (5 animals per run or replication; n=10) following bilateral testicular excision, after which they were incubated in 2-ml of Test-Yolk buffer (TYB) at 37 degrees C for 15-min. The specimens were then split into 2 1-ml aliquots, with Aliquot 1 as the control and Aliquot 2 as the filtered sample. The SpermprepI column was employed according to the manufacturer's specifications using TYB. During filtration (10-min), 2 different fractions were obtained: first 5-min (Sample 1) and second 5-min (Sample 2). The 2 fractions were evaluated and incubated at 37 degrees C and assessed for percentage of motility and grade of motility (0 to 4) every 30-min for 3-h. Filtration resulted in a significant improvement in the percentage and grade of motility (91.5% and 3.0 vs 76.5% and 2.5, respectively). The results indicate that filtration with the SpermprepI method improved the percentage and grade of motility (P<0.05) but not the percentage of normal morphology of the spermatozoa. In addition, the SpermprepI method enabled the recovery of 45% (8.3x10(6) spermatozoa recovered) of the total number of spermatozoa processed in the control aliquot (18.4x10(6) spermatozoa), which is consistent with previous observations. Most importantly, filtered spermatozoa incubated for 3-h showed a greater percentage and grade of motility than the control spermatozoa (63% and 1.66 vs 39% and 0.82, respectively. The SpermprepI filtration method selected a higher proportion of quality spermatozoa, which also displayed significant long-term motility (longevity) during in vitro incubation.
A solution hybridization assay is systematically characterized and used to quantitate glucose-6-phosphate dehydrogenase (G6PD) mRNA from epididymal fat pads in fasted and glucose-induced rats. G6PD mRNA and specific activity increase 9-fold and 2-fold, respectively. The 9-fold increase in G6PD synthesis reported previously (Wolfe et al. (1979) Biochem. Biophys. Res. Commun. 89, 108-115) can, therefore, be accounted for by the increase in G6PD mRNA. This solution hybridization assay is sensitive enough to quantitative levels of G6PD mRNA in total liver RNA from a fasted rat, one of the least abundant sources of this mRNA. It can, therefore, be used to answer several questions about the regulation of G6PD synthesis in rat tissues. Preliminary results suggest that the dietary regulation of G6PD mRNA in rat liver is much larger than previously reported.
Human epididymal tubules from 9 patients undergoing orchidectomy were cultured for periods of up to 8 days with preservation of the histological structure of the tissue. Addition of androgens (testosterone, 5 alpha-dihydrotestosterone and 5 alpha-androstane-3 alpha, 17 beta-diol) significantly increased the epithelial height, the incorporation of [3H]amino acids and [3H]thymidine. The effect on protein synthesis was significant 48 h after the onset of treatment and was maximal after 3 days. The effect on DNA replication was maximal during the 3rd day of treatment. In both instances maximal activity was achieved at a concentration of 10(-7) M of testosterone. Cyproterone acetate (10(-5) M) was able to negate all effects of androgens.
The effects of insulin on the turnover of glucose-6-phosphate dehydrogenase in rat epididymal adipose tissue were studied by immunochemical technique in in vitro incubations. Insulin increased the relative rate of synthesis of glucose-6-phosphate dehydrogenase by two-fold in tissue obtained from normal rats. Insulin also had an effect on the rate of degradation of this enzyme. In the absence of insulin in the incubation medium the rate constant of degradation was 0.11 h-1 (half-life, 6.3 h). When insulin was added to the medium degradation of this enzyme was slowed. The new rate constant of degradation was 0.04 h-1 (half-life, 17 h). In the presence of insulin, the rate constant of degradation of total protein in adipose tissue was unchanged; therefore the effects of insulin on the degradation of glucose-6-phosphate dehydrogenase are specific to that protein and perhaps to a few other specific proteins.
Spermadhesins are multifunctional proteins involved in boar sperm capacitation and gamete recognition. Using anti-AWN antibodies, we have followed the fate of spermadhesin AWN along the maturation and capacitation stages of boar spermatozoa. In addition, the amount of spermadhesins AQN-1, AQN-2, and AQN-3 relative to that of AWN was determined by amino acid analysis after reverse-phase HPLC isolation. Our data show that AWN-1 is the only spermadhesin on the surface of epididymal sperm and that a large amount of AQN-1, AQN-2, AQN-3, AWN-1 and AWN-2 become coated on ejaculated spermatozoa. The number of spermadhesin molecules on ejaculated sperm (12-60 x 10(6)/spermatozoa) is sufficient to form a many-molecules-thick coat over the sperm head. However, 50-75% of the AQN-1, AQN-2, and AQN-3 population, and around 90% of coated AWN (1 + 2) are released from ejaculated sperm during capacitation. This indicates that a large subpopulation of each boar spermadhesin is loosely associated to the sperm surface and may function as decapacitation factors. The remaining spermadhesin molecules, which are tightly bound to the sperm head's surface may play a role as either positive capacitation factors and/or in gamete recognition and binding.
Lactate dehydrogenase isozyme X (LDH X), malate dehydrogenase (MDH) and total soluble protein have been determined in lysates of spermatozoa isolated from caput, corpus and cauda of rat epididymis. Transit of spermatozoa through epididymis is accompanied by a reduction of LDH X, MDH and total protein per cell in sexually rested animals. The profiles of reduction along epididymal segments are different for the three variables studied. Mating with receptive females during the 5 days prior to determinations increases significantly the levels of MDH in spermatozoa from all sections of epididymis and produces increase of total soluble protein in the cells contained in cauda.
The inhibitory effects of atropine and pirenzepine (nonselective and M1-selective antagonists, respectively) on the contractile responses of the epididymal half of the rat vas deferens to methacholine included a depression of the maximal response. In the presence of pirenzepine, atropine was a competitive antagonist of the responses to methacholine. However in the presence of atropine, pirenzepine remained a non-competitive inhibitor of the response. It is suggested that low concentrations of methacholine predominantly stimulate M2-receptors and that at high concentrations, methacholine also stimulates M1-receptors.
Quantitative morphometrical parameters were compared in epididymal adipose tissue of adult (6 months old) and old (24 months old) Wistar rats, using light and electron microscopy of embedded material and freeze-etch replicas, and taking into account the functional unit of adipose tissue: the capillary-adipocyte. Despite an insignificant reduction of the adipocyte number and size in old rats when the whole population was sampled, the confounding factor of size of adipocytes could be excluded from morphometric computations in adult and old rats. Morphometric measures were performed on the whole transit from capillaries to adipocytes. They revealed that the plasma membranes, as seen in freeze etching, and the thicknesses of endothelial and adipocyte cytoplasms, as seen in ultra-thin sections, remained unaltered with aging. By contrast, the basement membranes were changed but differently around capillaries and adipocytes. The capillary-adipocyte distances were shorter and the vascularization density was higher in old rats.
Quantitative histologic evaluation was performed on the testes of albino rats exposed to scrotal gamma irradiation of 2.5 Gy and 10.0 Gy. The animals were sacrificed at 1, 2, 4, and 16 weeks posttreatment. As a result of irradiation, there was a gradual decrease in testicular weight. The seminiferous tubules of irradiated animals showed arrest of spermatogenesis, desquamation, vacuolization of germinal cells, and multinucleated giant cells. The extent of damage increased with posttreatment interval and with increasing dose. Counting of germinal cells at stage VII of the seminiferous epithelium revealed that the preleptotene spermatocyte is the most sensitive to radiation as compared to other cells. The number of epididymal sperm decreased, whereas the proportion of nonmotile and morphologically abnormal sperm increased following irradiation.
Random and nonrandom factors associated with sample preparation and the automated analysis (CellSoft) of rat cauda epididymal sperm motion were studied. Random factors included inherent system variation at both the individual cell level and at the multiple cell level. Repeated analyses of identical tracks across grey level revealed a statistical interaction between grey settings and curvilinear velocity. However, in multiple track analyses, grey level was seen to be a factor only at higher settings. Nonrandom factors included time after sample preparation, dilution medium, and sample preparation procedures. Using a nicked preparation of the entire cauda epididymis from Long-Evans rats, the effects of time were studied on sperm suspended in 1) phosphate-buffered saline + 10 mg BSA/mL, 2) TEST yolk buffer, and 3) Medium 199. In PBS/BSA, the percent motile sperm estimate decreased (50% to 30%) over an hour, while the curvilinear velocity increased (127 to 142 microns/sec). Both sperm motion parameters were maintained in the TEST yolk buffer and in the Medium 199, although at lower values for the latter. Evaluation of the relative contribution of several factors, nested within sample, to the overall variance of three separate motion endpoints revealed that there was a large variation from field to field, negligible variation between overall CellSoft analyses of 200 cells or more, low variation at the preparation aliquot level, and moderate variation at the animal level. In planning experiments to test for effects on sperm motion endpoints, consideration of the relative contribution of the individual study factors to the overall variance of the parameter estimates will result in more sensitive experimental designs.
Both NaCl and NaF promoted PGE2 binding to epididymal adipocyte membranes by apparent increase in the binding affinity. In order to distinguish between the effect of fluoride and the 'salt effect' of sodium on PGE2 binding, the effects of Mg2+ and guanyl nucleotides on PGE2 binding in the presence of NaCl or NaF were compared. Mg2+ decreased PGE2 binding; high NaF concentration abolished this inhibition, while increased NaCl concentrations did not affect the Mg2+ inhibition. In the presence of Mg2+ the effects of NaCl and NaF were additive. The enhancement of PGE2 binding by fluoride, unlike sodium, was dependent on the presence of Mg2+. Incubation of the membranes with GDP beta S, Gpp(NH)p, GTP or GTP gamma S increased PGE2 binding. Gradual increase in NaF concentrations in the presence of guanyl nucleotides resulted in stimulation of PGE2 binding at low NaF concentrations and inhibition of PGE2 binding at high NaF concentrations. No changes in the stimulatory action of NaCl on PGE2 binding were observed in the simultaneous presence of NaCl and guanyl nucleotides. A biphasic effect on PGE2 binding was observed with a wide concentration range of guanyl nucleotides. Treatment of the isolated membranes with cholera or pertussis toxins stimulated the adenylyl cyclase activity of the membranes, but failed to influence PGE2 binding. The implications of these findings are discussed.
Soluble and particulate fractions from extracts of rat epididymal fat cells were shown to exhibit a number of different phosphodiesterase activities, as determined by substrate specificity and sensitivity to activators and inhibitors. These activities were then further characterized following separation by MonoQ fast protein liquid chromatography (FPLC). A cyclic AMP-specific activity, unaffected by the presence of calcium and calmodulin and inhibited by rolipram, was the major soluble phosphodiesterase. This fraction also contained distinct calcium and calmodulin- and cyclic-GMP-stimulated activities. Over 80% of the phosphodiesterase activity in the particulate fraction could be accounted for by an insulin-activated cyclic AMP and cyclic GMP-hydrolysing enzyme, which was sensitive to inhibition by cyclic GMP, SKF 94120, SKF 95654 and cilostamide, and eluted as a single peak during MonoQ chromatography. At 1 microM cyclic AMP, the phosphodiesterase activity in the soluble fraction was about eight times greater than in the particulate fraction. Specific inhibitors to the particulate phosphodiesterase (cilostamide and SKF 95654) were added to incubations of isolated fat cells, and were able to potentiate sub-maximal concentrations of isoproterenol in the stimulation of lipolysis. These inhibitors were also able to reverse the antilipolytic effect of insulin, demonstrating the importance of the particulate phosphodiesterase in insulin action, despite the fact that its activity represents only a small proportion of the total phosphodiesterase activity in fat cells. Inhibitors of the major soluble phosphodiesterase had no effect on lipolysis.
Measurements of prostaglandin E2 (PGE2)-induced adenylyl cyclase activity in membranes isolated from epididymal rat adipocytes revealed inhibition of cAMP production at low concentrations of PGE2 (less than 10 mM) and stimulation at higher concentrations. This biphasic effect of PGE2 was obtained when adenylyl cyclase was stimulated with GTP or NaF. In the presence of forskolin only the inhibitory phase by PGE2 was observed. Sulprostone, a PGE2 analogue, did not affect cAMP synthesis in the presence of either GTP or NaF; however, in the presence of forskolin, it inhibited cAMP production similarly to PGE2. Treatment of the membranes with cholera or pertussis toxin did not alter the biphasic effect of PGE2 on cAMP production. These findings raise the possibility that PGE2 acts through several receptor subtypes which are coupled to GTP binding proteins different from the classical Gi or Gs proteins.
The 2-hydroxy and 4-hydroxyestradiols (2-/4-OHE2) caused marked cytotoxic effects, including vacuolation and nuclear changes, in rat epididymal epithelia, after exposure to very low levels (40 ng/rat/week) for 20 weeks. The effects of the 2-/4-OHE2 metabolites were more pronounced than that of estradiol-17 beta (E2).
Seventy semen ejaculates were obtained from 14 Murrah buffalo bulls and were subjected to plasma separation immediately after collection by centrifugation at 2000 rpm for 20 min and stored in liquid nitrogen until analysis. In the seminal plasma the total protein concentration were estimated and the heparin and gelatin binding (HB and GB) proteins were isolated using heparin and gelatin affinity column chromatography. The molecular weight of individual isolated HB and GB protein was determined by SDS-PAGE analysis. Buffalo bull spermatozoa was collected from cauda epididymis under aseptic conditions and was used for the in vitro fertility tests (i.e. bovine cervical mucus penetration test (BCMPT) and hypo-osmotic swelling test (HOST)). The heparin and gelatin binding buffalo seminal plasma proteins were used in six concentrations i.e. 10, 20, 30, 40, 50 and 60 microg/ml to test their effect on in vitro fertility assessment of cauda epididymal spermatozoa. The overall mean values of total protein, HB and GB proteins were recorded as 29+/-2.7, 2.61 and 0.2mg/ml, respectively. Eighteen total protein bands were observed in the range of 12-127 kDa. Eight major HB proteins were isolated in the range of 13-71 kDa. Seven major GB proteins were isolated in the range of 13-61 kDa in the buffalo seminal plasma. The mean penetration distance (mm) travelled by the buffalo cauda spermatozoa was maximum in HB proteins (26.9+/-0.6) followed by GB proteins (25.4+/-0.6) and control (21.2+/-1.4). The difference in BCMPT values between protein treated and control group was significant (P<0.05). Almost similar trend in the effect of protein on values of HOST percentage in both HB and GB proteins treated semen samples were recorded (66.4+/-0.65 and 66.1+/-0.6, respectively). The difference in HOST values between proteins treated and control group (50.4+/-2.0) was significant (P<0.05). The present results indicate that among the isolated proteins, 4 proteins were commonly seen in both the heparin and gelatin-sepharose affinity column chromatography, and the addition of buffalo seminal plasma proteins improved the in vitro sperm functions (40 microg/ml gave best results) of buffalo cauda spermatozoa.