Reactivation of demembranated, cytosol-free ram spermatozoa

A procedure for preparing cytosol-free ram sperm models was developed. Sperm are introduced to a Triton X-100-containing demembranation medium layered on top of a discontinuous Percoll gradient. After brief exposure to the demembranating solution, the sperm are separated from the detergent-soluble components by centrifugation through a 55% Percoll layer, finally collecting on top of a 90% Percoll cushion from where they are recovered. Optimum conditions consisted of Triton X-100 at 0.20% and a demembranation time of 35 sec. Cross-sections of midpieces and principal pieces of the demembranated sperm were examined by electron microscopy. With 0.20% Triton X-100 in the demembranation medium, 86% of the cross-sections showed no plasma membranes and the rest had broken plasma membranes. The remaining tail structures appeared to be morphologically intact. Assay of phosphoglucose isomerase as a marker enzyme confirmed that at least 98% of the cytosolic protein was removed. Ram sperm models obtained by this procedure could be reactivated, and the percent motility and beat parameters were similar to those of the intact sperm. Reconstitution with the detergent-soluble components was neither required for, nor enhanced, reactivation. Therefore, demembranated ram sperm do not require a detergent-soluble protein factor for reactivation.     

Degradation of bradykinin in semen of ram and boar

The pattern of bradykinin (BK; Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9)-inact iva ting peptidases in semen of boar and ram was investigated. The degradation of BK in semen was completely abolished by the metalloprotease inhibitors EDTA and o-phenanthroline. Inhibitors of angiotensin-converting enzyme (ACE; EC 3.4.15.1) and phosphoramidon, an inhibitor of neutral metalloendopeptidase (NEP; EC 3.4.24.11), were only partially effective in preventing BK degradation in semen. An additive effect was seen with simultaneous inhibition of both enzymes, resulting in complete abolition of BK degradation. HPLC analysis demonstrated that exogenous BK in semen is cleaved at Gly4-Phe5, Phe5-Ser6 and Pro7-Phe8. These results indicate that NEP and ACE are the main peptidases responsible for rapid BK inactivation in semen. The involvement of other peptidases known to be responsible for BK cleavage in other tissues and body fluids, namely carboxypeptidase N (EC 3.4.12.7), post proline cleaving enzyme (EC 3.4.21.26) and aminopeptidase P (EC 3.4.11.9) was excluded. NEP and ACE were shown to be localized mainly in seminal plasma and to a lesser extent on sperm cells.     

Assay of progressive motion of boar spermatozoa

The motility of ejaculated boar spermatozoa was assayed by measuring the number of organisms which have entered a capillary tube in an experimental setup with the spermatozoal suspension as well as the capillary content containing the same chemically defined motility medium. This medium, used both for washing and suspending spermatozoa, contained only an isotonic tris HCl (isotris) buffer at pH 7.0. The recommended assay conditions are the following: (1) wash the spermatozoa three times with isotris buffer, (2) suspend the spermatozoa in a concentration of 10(6) spermatozoa/ml, and (3) assay at 25 degrees C with an incubation period of 60 min.     

Role of cAMP in the reactivation of demembranated ram spermatozoa

Ejaculated ram sperm were demembranated with Triton X-100, separated from the detergent-soluble matrix, and reactivated [San Agustin and Witman (1993): Cell Motil. Cytoskeleton 24:264-273]. The percent motility of models prepared from freshly washed sperm was comparable to that of the washed sample before demembranation, regardless of whether cAMP was included in the reactivation medium. However, demembranated models derived from aging or metabolically inhibited sperm exhibited a lower percent reactivation and required cAMP to attain the level of motility of freshly washed sperm. Cyclic AMP was approximately 100 times more effective than cGMP. The requirement for cAMP could be bypassed by addition of porcine heart cAMP-dependent protein kinase (PKA) catalytic subunit to the reactivation medium, demonstrating that cAMP was acting via PKA. The cAMP stimulation of reactivation was not affected by inclusion of the PKA inhibitor PKI(5-24) in the reactivation medium, but was decreased when the models were preincubated with PKI(5-24) prior to reactivation. The cytosol-free models retained > 90% of the sperm PKA activity; therefore, the PKA appears to be anchored to internal sperm structures. This PKA could not be extracted by cAMP or Triton X-100 alone, but only by cAMP and Triton X-100 in combination. We conclude that cAMP-dependent protein phosphorylation is critical for sperm motility, but that the essential protein phosphate sites turn over slowly under our reactivation conditions, so that the cAMP requirement is apparent only in models prepared from sperm having a low internal ATP or cAMP content. Interestingly, reactivation was rapidly blocked by the peptide arg-lys-arg-ala-arg-lys-glu, which has been reported to be a selective inhibitor of cGMP-dependent protein kinase.     

In vitro capacitating effect of gamma-aminobutyric acid in ram spermatozoa

We have evaluated the capacitating effect of gamma-aminobutyric acid (GABA) in ram spermatozoa in vitro, in a chemically defined medium, by means of the chlortetracycline (CTC) binding assay. Semen from adult Australian Merino rams was collected in an artificial vagina; spermatozoa were washed once in modified Biggers, Whitten, and Wittingham medium (m-BWW), without BSA or serum, and incubated in m-BWW alone or in m-BWW containing GABA, GABA agonists, or antagonists for 2 h at 38.5 degrees C under 5% CO2 in air. Samples were taken for assessment of CTC binding pattern or were further incubated for 15 min in the presence of 5 microM calcium ionophore A23187. Acrosomal exocytosis was evaluated by Pisum sativum agglutinin binding. Addition of GABA to the incubation medium resulted in a concentration-dependent increase in the percentage of CTC forms II and III, corresponding to mid-capacitated and capacitated spermatozoa, respectively. The effect was marginally significant at 1 microM and maximal at 20 microM. The action of 20 microM GABA was mimicked by the GABAB-receptor agonist, muscimol, but not by the GABAA-receptor agonist, baclofen, and completely blocked by the GABAA-receptor antagonists, bicuculline and picrotoxin, which lacked effect per se. In a separate set of experiments, incubation of spermatozoa with GABA at a concentration of 1 microM, which was insufficient to stimulate sperm capacitation, together with the neuroactive steroid allopregnanolone (1 microM) provoked a capacitating effect similar to that achieved by 20 microM GABA alone. These results show that GABA has a capacitating action on ram spermatozoa through a GABAA receptor-mediated mechanism.     

Fructose stimulates shedding of cytoplasmic droplets from epididymal boar spermatozoa

The aim of the present study was to establish the presence of an inducer(s) for the shedding of cytoplasmic droplets from boar spermatozoa after ejaculation. Cauda epididymal spermatozoa were incubated with seminal plasma, seminal vesicular fluid (SVF) or chemical agents at 39 degrees C for 30 min. After fixation and staining, percentages of spermatozoa without a droplet were determined. In the samples incubated with seminal plasma, SVF and a filtrate of SVF obtained after passage through an ultrafilter (molecular weight cut-off, 10,000), 43%, 60-69% and 43% of the spermatozoa were without a droplet respectively. The percentage of spermatozoa without a droplet after incubation with D-fructose (1.0 mM), which was one of the energy substrates included in SVF, was 76%. Furthermore, percentages increased to 93% and 90% with the addition of caffeine (2.0 mM) and N6, 2'-O-dibutyryl cyclic adenosine 3',5'-monophosphate (1.0 mM), respectively, but decreased to 48% with the addition of imidazole (2.0 mM). Based on these results, it is suggested that the shedding of cytoplasmic droplets from boar spermatozoa is induced by fructose originating from SVF. It also appears that this event is mediated by increasing the concentration of intracellular cyclic adenosine 3',5'-monophosphate.     

Epididymal compounds and antioxidants in diluents for the frozen storage of ram spermatozoa

The epididymal compounds taurine, hypotaurine and inositol, and the antioxidants carnosine and ascorbic acid, were added to Tris-based diluents containing varying concentrations of glycerol, and their effect on the post-thaw motility characteristics and fertility of ram spermatozoa was examined. Overall, the post-thaw motility characteristics of spermatozoa were better when semen was frozen in the presence rather than in the absence of glycerol. Only taurine protected spermatozoa during cryopreservation; the presence of 25 mM or 50 mM taurine significantly improved the post-thaw percentage of motile spermatozoa but this had no effect on fertility after cervical or laparoscopic insemination of ewes. Increasing the concentration of taurine to more than 100 mM significantly reduced the percentage of motile spermatozoa, compared with the lower concentrations of the amino acid. The presence of more than 50 mM carnosine or ascorbic acid significantly reduced all motility characteristics compared with the control diluent. Given that hypotaurine, carnosine, or ascorbic acid did not improve post-thaw motility, the cryoprotective effect of taurine may be attributable to its osmoregulation rather than to its antioxidant properties.     

Viability of ram spermatozoa in relation to the abstinence period and successive ejaculations

For successful fertilization, a functionally constituted sperm plasma membrane is necessary, and this is clearly dependent on the sperm maturation process. The latter involves a series of complex changes which result from a sequence of events occurring at different points within the epididymis. The transit time through the epididymis can be influenced by external factors such as sexual stimulus and ejaculatory frequency. The present work was undertaken to determine changes in ram sperm viability and other sperm quality characteristics in relation to ejaculatory frequency. Three successive ejaculates were collected from rams during three different abstinence periods (collected every day, every 2 days and every 3 days). Cell viability (membrane integrity determined by fluorescence staining), progressive individual motility, and other in vitro parameters of sperm quality were evaluated. Second ejaculates showed the highest cell viability of the three periods studied, and increased as the abstinence period lengthened. The maximum proportion of viable cells (average 60%) was obtained in the second ejaculate after an abstinence period of 3 days. Likewise, overall and progressive individual motilities were higher in second ejaculates, the maximum value being 70% after 3 days of abstinence. The percentage of damaged or acrosome-reacted spermatozoa was greater after 1 day of abstinence than after the other periods analysed, whereas the first ejaculate showed the highest value in all periods. Differences in ejaculate volume were correlated strongly with both variables considered (abstinence period and ejaculate number). In the third ejaculate, about 27% more volume was obtained after 3 days of abstinence than after abstinence for 1 or 2 days. Sperm concentration increased significantly as the abstinence period lengthened, and also decreased significantly with ejaculate number in all cases. Therefore, the total number of spermatozoa in the ejaculate was clearly dependent on the abstinence period and the ejaculate number. In conclusion, the results obtained suggest that using the second and/or a mixture of second and third ejaculates would improve the results in artificial insemination and in fertility studies. In addition, the use of better quality semen would facilitate progress in semen cryopreservation studies.     

Motility of ram spermatozoa during storage in a chemically-defined diluent containing antioxidants

The effects of five antioxidants--Vitamin E (VE), butylated hydroxyanisole (BHA), n-propyl gallate (n-PG), deferoxamine mesylate (Desferal) and catalase (EC 1 . 11 . 1 . 6)--on the maintenance of motility of ram spermatozoa in a chemically-defined ram semen diluent (RSD-1) have been evaluated. VE, n-PG and Desferal inhibited spermatozoal motility. The relative inhibition (i.e., ratio of change in % motility over 24 h between the treatment group and the corresponding control) at equimolar concentrations (100 microM) of Desferal, VE and n-PG were 1.6, 1.8 and 3.6 respectively. BHA had no effect at 10 microM but at lower concentrations, gave a slight improvement in motility in freshly diluted spermatozoal samples and in those stored for 1 day at 15 degrees C. The addition of catalase to RSD-1 was also ineffective in improving the motility of spermatozoa. The lack of beneficial effects of the tested antioxidants suggests that RSD-1 itself may destroy reactive oxygen species and the antioxidant activity of RSD-1 components requires further study.     

Intramuscular collagen characteristics of ram, wether, and zeranol-implanted ram lambs

Eighteen spring-born Columbia ram, wether, and zeranol-implanted ram lambs were studied to determine the influence of castration or zeranol implants on intramuscular collagen (IMC) properties and muscle shear force values. Warner-Bratzler shear force values for longissimus muscle were greatest for ram lambs, intermediate for implanted rams, and least for wethers (P < .05). Nonreducible collagen crosslink concentration was greater in IMC of rams and implanted rams (P < .05). The IMC from rams compared with that from wethers contained proportionately more Type III than Type I collagen (P < .05); values for implanted rams were intermediate. Heat-soluble muscle collagen concentration was greater for rams and implanted rams than for wethers (P < .05); however, insoluble collagen concentration did not differ by treatment. Muscle collagen concentrations were not different for rams, wethers, or implanted rams. Increased shear force values in rams were associated with elevated collagen crosslink concentration and increased proportion of Type III collagen. Greater concentration of soluble collagen in ram IMC neither diminished nor diluted IMC crosslinking. The proportion of heat-labile collagen in the fractions did not reflect the IMC crosslinking profile for ram and wether lambs. Zeranol implantation modified IMC characteristics of rams such that shear force values and some collagen properties were similar to those of wethers.     

Correlation between motility of testicular spermatozoa, testicular histology and the outcome of intracytoplasmic sperm injection

Many studies have demonstrated the postoperative analgesic efficacy of fentanyl delivered i.v. by patient-controlled analgesia (PCA) devices at demand doses ranging from 10 to 50 microg, but none has sought to define the optimal fentanyl PCA dose. In this randomized, double-blind, multicenter study, we compared the safety and efficacy of three administered demand-dose sizes of fentanyl (20, 40, and 60 microg) in 150 patients after major surgery. Efficacy was dose-dependent; positive response rates (i.e., a global assessment score of "very good" or "excellent" and the absence of severe opioid adverse effects) were 42%, 52%, and 68% for the 20, 40, and 60 microg demand-dose groups, respectively, and were significantly higher in the 60 microg demand-dose group. The number of doses administered and missed attempts were significantly smaller in the 40 and 60 microg demand-dose groups compared with the 20 microg demand-dose group. This suggests that the 20 microg demand dose provided inadequate pain relief. Adverse respiratory events were more frequent and mean respiratory rates were significantly slower with the 60 microg demand dose, compared with the 20 or 40 microg demand doses. These results indicate that, of these three doses, the 40 microg demand dose was optimal for fentanyl PCA management of moderate to severe pain after major surgery. IMPLICATIONS: The postoperative analgesic efficacy of fentanyl delivered i.v. by patient-controlled analgesia devices has been demonstrated for demand doses ranging from 10 to 50 microg, but the optimal fentanyl dose remains unknown. In this randomized, double-blind study, we compared three demand dose sizes of fentanyl (20, 40, and 60 microg) and found that the 40 microg demand dose was the most appropriate for fentanyl patient-controlled analgesia management of postoperative pain.     

In-vitro effect of estrogen-antagonist on motility and penetration ability of human spermatozoa

We report a case of inflammatory tuberculosis of the breast in a 83-year-old French Caucasian woman. She presented with a diffuse inflammatory tuberculosis of the breast and an axillary enlarged lymph node but no tumor was palpable in the breast. A carcinoma was firstly suspected. The diagnosis was accessed after repeat punction of a lymph node, as a Mycobacterium tuberculosis was identified after culture. A medical treatment was sufficient in our case.     

Effect of culture, incubation and acrosome reaction of fresh and frozen-thawed ram spermatozoa for in vitro fertilization and intracytoplasmic sperm injection

This study evaluated different sperm treatments for fertilization of sheep oocytes by intracytoplasmic sperm injection (ICSI) and in vitro fertilization (IVF). In Experiment 1, fresh and frozen semen was separated by Percoll centrifugation and incubated at 30 degrees C or 39 degrees C in HSOF or BSOF medium for 1 h before use for IVF or ICSI. For IVF, oocytes were inseminated and incubated with sperm for 30 min, 4 h and 19 h. Sperm were assessed for acrosome integrity after Percoll centrifugation and 1 h incubation, and those used for IVF were assessed after each period of exposure to the oocytes. Fertilization rates after ICSI were higher for fresh than for frozen-thawed sperm and were highest 19 h after IVF with fresh or frozen-thawed sperm in the presence of HSOF at 30 degrees C. In Experiment 2, fresh semen was separated by Percoll centrifugation and incubated for 5 h in HSOF, and the acrosome reaction was induced with lysophosphatidylcholine. Acrosome integrity was then assessed. Fertilization rates after ICSI were similar for acrosome-reacted and control spermatozoa. These results suggest that induction of the acrosome reaction in spermatozoa before ICSI is unnecessary, whereas a capacitating treatment of spermatozoa is required before IVF.     

Internal pH of human spermatozoa: effect of ions, human follicular fluid and progesterone

The internal pH (pHi) of human spermatozoa was measured by the fluorescent indicator 2,7-bicarboxyethyl-5,6-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and the distribution of the radioactive [14C]-methylamine under different external ionic conditions. The effect of the addition of progesterone and human follicular fluid (HFF) on the spermatozoa pHi was also analysed. The pHi values obtained were almost identical with the two probes used. In sodium (NaM) and potassium (KM) media, a linear relationship between the internal and external pH was observed. In NaM, the pHi values were approximately 0.4 pH unit less than the external pH. In KM, the pHi measured was higher than in NaM and only slightly inferior to the external pH (0.1-0.2 pH unit). Addition of 10 microM progesterone, oestradiol 17 beta or 20% HFF to spermatozoa incubated at pH 7.2 in NaM did not induce any rapid variation of the BCECF fluorescence or change in the accumulation of methylamine. A slight change in pH (approximately 0.5 units) occurred with progesterone after 15 min. As a control, addition of 10 mM of NH4Cl induced a rapid alkalinization (0.4 pH unit) of the cell interior while 10 mM lactate produced only a slight acidification (approximately 0.2 pH unit). Under the same conditions (NaM, pH 7.2), the pHi of the spermatozoa prepared by Percoll gradient was found more acidic by 0.2 pH unit than washed unfractionated spermatozoa. Progesterone, oestradiol 17 beta and HFF had no effect on the pHi of these spermatozoa. The results obtained in this study show that it is possible to measure accurately the internal pH of human spermatozoa. Internal pH was found to be dependent upon the pH of the external medium and a quasi-linear relationship exists between the internal and external pH, suggesting no specific pH regulatory mechanisms. Our data suggest instead that the protons, under our experimental conditions, are passively distributed. Progesterone, oestradiol 17 beta and HFF, known to promote both capacitation and the acrosome reaction, do not act through a rapid pHi change.     

Stroboscopic illumination for the assessment of hyperactivated motility of mouse spermatozoa

Spermatozoa showing hyperactivated motility can be recognized in multiple-exposure micrographs. The cells were confined in 10-micrometer-deep chambers, illuminated in darkfield conditions, and photographed using a stroboscope (10 Hz). Hyperactivation, as judged from the pattern of ten superimposed images, could be promoted by raising the ionic strength of the medium but did not occur in the absence of calcium.     

Toxicity of ornidazole and its analogues to rat spermatozoa as reflected in motility parameters

Ornidazole, a 5-nitro-imidazole derivative, has contraceptive properties in rats. As some ornidazole passes through the body unmetabolized after administration, the aim of this study was to investigate if ornidazole itself has a direct effect on sperm motility and whether these effects are limited or potentiated by the epididymal epithelium or structural changes to the molecule. Cauda epididymal spermatozoa or cauda epididymal tubules were incubated with ornidazole or ornidazole analogues, and motility parameters were subsequently measured by means of a computer-assisted sperm analysis (CASA) system. Incubation of spermatozoa in 2.5 mmol/L ornidazole for 4 h reduced their motility significantly, whereas incubation of epididymal tubules for 8 h in 10 mmol/L ornidazole was required to alter the velocity parameters of the enclosed spermatozoa upon release, suggesting that extratubular non-metabolized ornidazole can participate in inhibiting the motility in vivo. The in vitro toxicity of ornidazole derivatives depends on the halogen present and on the position of the nitro-group. The putatively inactive (R)- and the active (S)-ornidazole exhibited equivalent depression of sperm motility by direct incubation. This observation, and the differences between the in vitro and the in vivo efficacies of various ornidazole analogues, indicates distinct mechanisms of motility inhibition in the two experimental systems.     

Movements of sodium and potassium into ejaculated boar spermatozoa suspended in seminal plasma and a biological salt solution

Uptake of 22Na and 42K into ejaculated boar spermatozoa was measured in vitro. Cells were suspended either in seminal plasma or in a biological salt solution of essentially the same composition as boar seminal plasma. Samples were incubated at 30 degrees C. Correction was made for extracellular space in the centrifuged sperm pellet. This was determined as 22Na-space, which was less (P less than 0.001) than [14C] carboxyinulin space. Large differences were observed among individual ejaculates. The half-time for potassium uptake into the spermatozoa averaged 11.5 min, which is much faster than that for leukocytes or erythrocytes. When the spermatozoa were suspended in the biological salt solution, the initial rate of 42K uptake was significantly decreased. This may be due to disturbances of the protein components of the sperm membrane. The uptake of 22Na into the spermatozoa was slow. Sodium and potassium transport appeared not to be coupled in the 3/2 ratio which has been reported for erythrocyte membranes. The average concentration of sodium was 108 mM in seminal plasma and 26 mM in the spermatozoa (112 mmol/kg water and 38 mmol/kg water, respectively). The corresponding figures for potassium were 26 mM and 51 mM (27 mmol/kg water and 74 mmol/kg water). The random error for a single determination for the various methods used varied between 2.4 and 13.3% of the mean.     

Improved motility recovery of human spermatozoa after freeze preservation via a new approach

A new sperm isolation technique was studied in conjunction with the short-term freeze preservation of human spermatozoa. The isolation procedure yielded subpopulations of spermatozoa with very high percentage motility and progressive motility score, and which were virtually free of seminal debris. For 24 semen samples from 13 donors, the mean prefreeze values of percentage motility and motility score were 53% (2.8), 73% (3.1), and 88% (3.5) for the parent semen, 7.5% BSA (middle) fractions, and 17.5% BSA (bottom) fractions, respectively. These samples were used without a priori constraint on semen quality. Eleven samples were preselected on the basis of good motility in the semen. These yielded prefreeze motility values of 66% (3.2), 70% (3.4), and 87% (3.9) for the semen and two fractions, respectively. Sperm motility in the middle fractions was thus intermediate to that in the semen and in the bottom fractions, although closer to the former in these 11 cases. The sperm freeze preservation procedure involved dilution of the semen samples and separated sperm fractions with a cryoprotective semen extender, and freezing and thawing in a conventional manner. Post-thaw percentage motility, motility score, and percentage survival were substantially higher for the separated fractions than for the parent semen. For the 24 cases, the bottom fractions yielded mean values of 57% motility, 3.0 motility score, and 70% survival, in comparison with the respective values of 20%, 2.3, and 34% for the semen. In the 11 preselected cases, the bottom fractions yielded post-thaw mean values of 60% motility, 3.3 motility score, and 72% survival; the middle fractions yielded respective values of 45%, 3.1, and 64%; and the parent semen yielded respective values of 27%, 2.3, and 41%. It was concluded that the major factor in improving post-thaw motility recovery was the separation process as a whole, rather than the degree of separation.