Effect of follicular or oviductal fluids on movement characteristics of bovine sperm during capacitation in vitro

Mammalian sperm exhibit characteristic motility changes associated with capacitation. Movement characteristics of bovine sperm incubated in noncapacitating (control, medium alone), capacitating (oviduct fluid, nonluteal, and luteal), or capacitating, acrosome reaction inducing (follicular fluid) conditions were investigated using a computer-assisted automated semen analysis system. Sperm were incubated up to 4 hours in a modified Tyrode's medium (control), 20 and 60% nonluteal (NL) or luteal (L) oviduct fluid (ODF), or 20 and 60% follicular fluid (FF). Relative to sperm incubated in control medium, motility of sperm treated with ODF or FF had increased linearity and vigorous motility. Sperm incubated in 60% ODF or FF showed a small decrease in mean trajectory/path straightness and velocity over time compared to 20% fluid treatments and control. Frequency distribution graphs were symmetric for 20% NL- and L-ODF treated sperm. However, 20% FF and 60% ODF and FF treatments had distributions skewed to the left, indicating smaller values for lateral head displacement (ALH) and curvilinear velocity (VCL). Median values for ALH and VCL were determined for control-treated sperm, and subtracted from individual sperm values for all treatments to estimate deviation from control, designated ALHc and VCLc. Three-dimensional plots of ALHc, VCLc and corresponding frequency indicated shifts in peak patterns for fluid-treated sperm compared to control sperm. Incubation in 20% ODF and FF resulted in peak shift for ALH and VCL values; yet, little change in peak position was observed in sperm incubated in 60% ODF and FF.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of natural antioxidants, superoxide dismutase and hydrogen peroxide on capacitation of frozen-thawed bull spermatozoa

This study comprised 100 persons with antibodies to hepatitis C virus (HCV), including 77 intravenous drug users (IVDUs). They were tested with serological HCV typing assays (Murex HCV serotyping 1-6 assay; Chiron RIBA HCV Serotyping SIA). Patients with a positive polymerase chain reaction (PCR) for HCV (n = 66) were tested with genotyping molecular assays (Inno-Lipa HCV II test; Sorin GEN-ETI-K HCV typing assay). Comparison of the results of these tests showed that (a) 92% of samples could be typed by one test at least; 44% could be typed by all four tests; 88% could be typed by one serological test at least and 66% by one molecular test at least; (b) 81% of the samples successfully tested with both serological tests gave comparable results; 95% of the samples successfully tested with both molecular tests gave comparable results; (c) when serological and molecular tests yielded different results, sequences in the 5'-non-coding (5' NC) or E1 regions always confirmed the results of the molecular tests; (d) in case of discrepancy between the results of the molecular tests the E1 region sequences confirmed the Sorin test results. It is concluded that the molecular tests compared gave similar results. The fact that the Murex serological test gave comparable results in more than 80% of cases indicates that it is an alternative to the molecular tests for routine diagnosis. However, comparison of the results of this test with those obtained in patients consulting a hepatology department showed that it gave the best results in a population of patients not exposed repeatedly to HCV.     

Role of zinc during hamster sperm capacitation

Zinc stabilizes somatic cell membranes and DNA, inhibits respiration, is present in high concentrations in the male reproductive tract, and may stabilize sperm during storage and ejaculation. Zinc removal from sperm may be necessary to prepare sperm for fertilization (capacitation). Incubation with Zn2+ chelators, e.g., D-penicillamine, can capacitate hamster sperm (Andrews and Bavister, Gamete Res 1989; 23:159-70). In the present study, the Zn(2+)-specific fluorochrome N-(6-methoxy-8-quinolyl)-p-toluenesulfonamide (TSQ) and the vital stain propidium iodide were used to assess the zinc content of live hamster sperm with flow cytometry before and after capacitation. Capacitation was monitored with a salt-stored zona pellucida penetration assay or the occurrence of spontaneous or induced (with lysophosphatidylcholine) acrosome reactions. The effect of added zinc on sperm capacitation was also evaluated. Image Analysis was used to determine the subcellular location of zinc (TSQ fluorescence) and atomic absorption to determine whether the total zinc content of sperm changes during capacitation. Sperm incubated under non-capacitating conditions had high TSQ fluorescence and could not penetrate zonae pellucidae. Sperm incubated under capacitating conditions (plus BSA or D-penicillamine) were zinc-depleted (low fluorescence) and penetrated 90% or 78% of zonae, respectively. Image analysis showed a significant reduction in zinc in the acrosomal region during capacitation with BSA, but this did not correlate with the occurrence of spontaneous acrosome reactions. The atomic absorption data showed that the total zinc content of sperm was reduced by 44% or 40% when sperm were incubated under capacitating conditions (BSA or D-penicillamine, respectively). Zona pellucida penetration was completely inhibited when zinc was present throughout the capacitation period but not when it was added at the end of incubation. These data indicate that removal of zinc from hamster sperm is correlated with capacitation and may play a key regulatory role in this process.     

Characterization of the oviductal sperm reservoir in cattle

A reservoir for sperm has been found in the oviductal isthmus in several species. Sperm are apparently trapped in the reservoir by binding to the oviductal epithelium, although other factors may be involved. We hypothesized that binding sites for bovine sperm are limited to the isthmus and are regulated by the hormonal state of the cow. Ipsilateral oviducts were obtained from heifers that were preovulatory (in estrus), had ovulated recently (within 12 h), or were in diestrus (Day 10). The isthmic and the ampullar epithelium were milked out and incubated separately in serum-free (SFRE-199-2) medium, at 39 degrees C in 5% CO2. Frozen-thawed sperm from bulls were added to the epithelium and coincubated for 15 min. The number of spermatozoa that bound to explants was not affected by stage of cycle or by anatomic origin of the explants (p > 0.05). In an additional experiment, oviducts were infused with sperm in vivo and then prepared for scanning electron microscopy, which revealed that sperm were associated with ciliated epithelium in both the isthmus and ampulla. Thus, bovine sperm may form a reservoir in the isthmic end of the oviduct because it is the first oviductal region that they encounter.     

Fertility-associated antigen on bull sperm indicates fertility potential

A 30-kDa heparin-binding protein named fertility-associated antigen (FAA) was identified in sperm membranes of beef bulls with greater fertility potential. In a survey of 2,191 beef bulls, 88% had FAA present in sperm membranes (FAA-positive), and 12% were FAA-negative. In the first study, 54 Santa Gertrudis and 51 Santa Cruz bulls were grouped (1 to 14 bulls per group) according to FAA profiles and were bred to 2,403 cows at ratios of 1 bull: 25 cows. Fertility for 14 groups of FAA-positive bulls averaged 88%, whereas three groups of FAA-negative bulls impregnated 79% of the cows. Thus, FAA-positive bulls were nine percentage points more (P < .01) fertile than FAA-negative bulls. In the second study, 2-yr-old Santa Cruz bulls (n = 26) were grouped according to FAA profiles and serving capacity. The fertility of the group of 12 high-serving-capacity, FAA-positive bulls was 87% of 270 cows. The group of six FAA-negative bulls with high serving capacity impregnated 78% of 143 cows. Among the groups of bulls with high serving capacity, FAA-positive bulls were nine percentage points more (P < .05) fertile than FAA-negative bulls. The group of eight FAA-positive bulls with low serving capacity impregnated the least (P < .01) percentage (69%) of 238 cows. Serving capacity of bulls should be considered when optimizing fertility potential. Among bulls with acceptable physical characteristics and serving capacity, determination of FAA profiles in sperm can be used as a tool to identify subfertile bulls.     

Atypical cilia in the oviductal epithelium of healthy dogs

Specimens of the uterine tube (ampulla) were obtained from seven healthy, ovario-hysterectomized dogs. Ultrastructurally, a total of 35,000 cilia were examined. Compound cilia ranged from 0.0 to 0.4%; both intracytoplasmic and swollen cilia ranged from 0.1% to 0.4%. The microtubular pattern was studied in 3,500 cross-sectioned cilia and an abnormal pattern was found in 2-5%. Similarly to the other animal species, abnormalities involving the peripheral microtubules were the prevailing defect. An electron-dense plug into the lumen was seen in 1-3% of the basal bodies; occasionally an abnormal spatial configuration of them was also observed. The incidence of abnormal cilia hence is lower than found in the tracheae.     

Effects of modulators of protein kinase C on human sperm capacitation

OBJECTIVE: To examine the effects of stimulators or inhibitors of protein kinase C on capacitation and protein phosphorylation in human sperm. DESIGN: Capacitated sperm treated with or without modulators of protein kinase C were monitored by the chlortetracycline fluorescence assay. Capacitation was confirmed by the ability of sperm to undergo the acrosomal reaction in response to mouse zonae pellucidae. 32P-labeled sperm phosphoproteins were analyzed by one-dimensional gel electrophoresis to detect the effect of protein kinase C stimulator, 12-O-tetradecanoyl-phorbol-13-acetate, on protein phosphorylation. RESULTS: The treatment of sperm with protein kinase C stimulators resulted in the following: [1] the rapid appearance of the clear perimeter pattern, featuring distribution of fluorescence over the entire head exhibiting a bright perimeter and bright midpiece; [2] an accelerated ability to undergo the acrosomal reaction; and [3] an enhanced phosphorylation of 57.5-kd sperm phosphoprotein. Furthermore, these stimulatory effects were inhibited by protein kinase C inhibitors. CONCLUSION: Protein phosphorylation mediated by protein kinase C may be involved in the regulation of human sperm capacitation.     

Association of glycerylphosphorylcholine with human sperm and effect of capacitation on their metabolism

The presence and localization of glycerylphosphorylcholine (GPC) on the surface of human sperm, as well as the metabolism of its breakdown product L-glycerol 3 phosphate (G3P), were investigated. GPC was found to be associated with sperm after penetrating cervical mucus and was present after repeated washing of the sperm. GPC was partially released by treatment with 0.4 M NaCl in 0.01 M sodium phosphate buffer (pH 7.4) and localized to the head region after sperm fractionation. G3P did not increase O2 uptake of uncapacitated human sperm. However, under aerobic conditions, lactate accumulated when exogenous G3P or uterine GPC diesterase was added to sperm in suspension. The uptake of O2 by washed capacitated sperm pre-incubated with 1 unit of rat uterine GPC diesterase for 30 min was significant. This effect was inhibited by 2 microM oligomycin indicating that oxidative phosphorylation had occurred. The present study indicates that GPC may play a role in the metabolism of human sperm after capacitation.     

Major proteins of bovine seminal plasma modulate sperm capacitation by high-density lipoprotein

Bovine seminal plasma (BSP) contains four similar proteins secreted by the seminal vesicles, designated BSP-A1, -A2, -A3, and -30 kDa. These proteins bind to choline phospholipids on the surface of the sperm after ejaculation. These BSP proteins also interact with heparin, apolipoprotein A-I (apoA-I) and apoA-I associated with high-density lipoprotein (HDL). The HDL and heparin present in the female reproductive tract have been implicated in sperm capacitation and the acrosome reaction (AR). This study was undertaken to determine whether or not these BSP proteins and HDL could modulate the capacitation of sperm, and to determine the combined effect of HDL and heparin on capacitation. Washed bovine epididymal sperm were preincubated in buffer containing BSP proteins, washed, and incubated with lipoproteins (HDL, and low- and very low-density lipoproteins) or liposomes with or without apoA-I in the presence or absence of heparin. The percentage of capacitated sperm was evaluated after the AR was induced with lysophosphatidylcholine. HDL alone (160 microg/ml) after an 8-h incubation stimulated the AR of epididymal sperm. The percentage of HDL-enhanced AR further increased when sperm were preincubated with BSP proteins. ApoA-I-liposomes stimulated the AR more rapidly (5 h, 160 microg/ml) than HDL. When sperm were preincubated with BSP proteins, the percentage of apoA-I-enhanced AR further increased. In contrast, when liposomes without apoA-I or when low- or very low-density lipoproteins or lipoprotein-depleted serum was used, no significant increase in the AR was detected with or without BSP proteins. When heparin and HDL or apoA-I-liposomes were used together, their combined effects on the AR were not additive. These results indicate that BSP proteins modulate the process of capacitation induced by heparin, HDL, and apoA-I-liposomes.     

Regulation of protein-tyrosine phosphorylation and human sperm capacitation by reactive oxygen derivatives

Spermatozoa undergoing capacitation, a necessary prerequisite event to successful fertilization that can be induced in vitro by reactive oxygen species (ROS), generate superoxide anion (O2.-). Because, in neutrophils, the generation of O2.- is associated with tyrosine phosphorylation of several proteins, the aim of the present study was to investigate the association between protein-tyrosine phosphorylation and ROS-induced human sperm capacitation. Human spermatozoa express two major phosphotyrosine-containing proteins of 105 and 81 kDa, the phosphotyrosine content of which is increased when spermatozoa are incubated under capacitating conditions. Superoxide dismutase and catalase abolish both sperm capacitation and tyrosine phosphorylation of p105 and p81, suggesting the involvement of O2.- and hydrogen peroxide in these two processes. Inhibitors of NADPH oxidase, the enzyme responsible for the neutrophil's respiratory burst, decrease both p105 and p81 tyrosine phosphorylation and sperm capacitation while hydrogen peroxide stimulates these two processes. Tyrosine phosphorylation of p105 and p81 occurs through a herbimycin A-sensitive tyrosine kinase, and sperm incubation with phosphotyrosine-protein phosphatase inhibitors results in an increase in phosphotyrosine content of these two proteins. Indirect immunocytochemical studies reveal phosphotyrosine-containing proteins mostly in the principal piece of the flagellum, in agreement with the localization of p105 and p81 in the human sperm fibrous sheath. Although tyrosine phosphorylation of p105 and p81 and sperm capacitation are related in a time-dependent fashion, some discrepancies are observed in the regulation of these two processes according to the redox status of the spermatozoa.     

Paradoxical effect of reagents for sulfhydryl and disulfide groups on human sperm capacitation and superoxide production

Spermatozoa must undergo capacitation prior to fertilization. In humans, this process appears regulated by oxidoreduction reactions. We investigated the possibility that these reactions involved the sulfhydryl-disulfide pair, which offers a reversible regulation of cellular processes. The effects of reagents targeted for sulfhydryl and disulfide groups on human sperm capacitation, superoxide (O2-.) generation and protein tyrosine phosphorylation were evaluated. The sulfhydryl targeted agents, phenylarsine oxide (PAO), diamide, dithiopyridine (DTP), N-ethylmaleimide (NEM), maleimidylpropionyl biocytin (MPB), p-chloromercuribenzoic acid (PCMB), and bromobimane analogs (mBBr and qBBr) triggered sperm capacitation to levels comparable to those observed with a biological inducer, fetal cord serum ultrafiltrate (FCSu). Capacitation induced by NEM, MPB, PCMB, and PAO was prevented by superoxide dismutase (SOD) and associated with an increased sperm production of O2-.. However, SOD did not affect the increase in protein tyrosine phosphorylation of spermatozoa treated with NEM, PAO, or MPB. Disulfide reductants, dithiothreitol (DTT), thioredoxin (TRX), glutathione (GSH), tris-(2-carboxyethyl) phosphine (TCEP), and tris-(2-cyanoethyl) phosphine (TCP) partially to totally inhibited FCSu-induced sperm capacitation and O2-. production. TCEP, DTT, and TRX decreased the capacitation-associated tyrosine phosphorylation of sperm proteins. The strong time-dependent increase of sperm membrane sulfhydryl groups exposed to the extracellular space occurring during the first hour of capacitation could indicate an important rearrangement of sulfhydryl carrying proteins during the initiation of capacitation. Therefore, protein sulfhydryl-disulfide status may be important for the regulation of human sperm capacitation and the mechanisms involved may be complex and multifactorial.     

Factors affecting preservation and fertility of bull sperm: a brief review

This paper is a brief review of the factors that determine the number of sperm required for insemination to obtain high fertility and ways that sperm viability might be prolonged. Damage to sperm during freezing results in a requirement, after thawing, of about 6 x 10(6) motile sperm (> 10 x 10(6) total) per insemination to achieve near-maximal fertility, whereas 2.5 x 10(6) motile fresh sperm result in high nonreturn rates. Multiple inseminations to bracket the time of ovulation are usually not economical except in superovulated cows. Earlier unpublished work on sperm packaging for slow release in the cow and methods for stabilizing membranes to increase sperm survival time in the cow are discussed. Current studies are directed towards reducing catabolic metabolism of sperm and studying membrane changes during freezing and thawing and during incubation with bovine oviduct epithelial cells. Studies with bull sperm indicate that the choline and ethanolamine phosphoglyceride components of their membranes represent an unstable configuration. Exposure of sperm to liposomes with the sterol cholesterol can alter the phospholipid bilayer and increase capacitation time. Similar approaches may produce sperm with a longer fertilizing life following insemination. New procedures in vitro permit low cost modelling of fertilization, which will facilitate research by reducing the cost of studies in vivo.     

Survival of bull sperm frozen at different rates in media varying in osmolarity

The microscopic method of age at death determination was introduced by Kerley in 1965. The method, which relies on the quantification of selected elements in cortical bone tissue, has been widely used, and several other researchers have modified or added to the method. Yet, very few studies have been carried out dealing with the intra- and inter-observer error. Furthermore, when such studies have been completed, the statistical tools for assessing variability have not been adequate. This study presents the results of applying simple quantitative statistics on several counts of microscopic elements as observed on photographic images of cortical bone, in order to assess intra- and inter-observer error. Overall, substantial error was present at the level of identifying and counting secondary osteons, osteon fragments and Haversian canals. Only secondary osteons can be reliably identified, precluding the use of osteon fragments and Haversian canals. The observers in this study included experienced and inexperienced users of the microscopic method, yet the variability was uniformly large for all observers, suggesting fundamental problems in definition and identification of the structural elements. Until more rigorous definitions of such elements have been agreed upon, the use of microscopical methods must be discouraged as a sole or uncontrolled method of evaluating age at death.     

Effect of seminal plasma on capacitation and hyperactivation in human spermatozoa

While hyperactivated motility is known to be a concomitant of capacitation, and a prerequisite for fertilization, the specific interdependence of capacitation and hyperactivation in human spermatozoa has not been investigated. This study was designed to determine the effect of seminal plasma contamination on the expression of hyperactivated motility and the relationship between hyperactivation and capacitation, since seminal plasma contains decapacitation factor(s). Seminal plasma was obtained by centrifugation of aliquots of liquefied semen layered over 1.5 ml 40.5% Percoll and mixed with human tubal fluid (HTF) medium containing 30 mg/ml human serum albumin (HSA) (HTF) to a final concentration of 5% (v/v) seminal plasma (SP). Motile spermatozoa were isolated from the remainder of the semen by swim-up into either HTF or SP medium. Samples were taken from each treatment immediately post-harvest (0 h) and after 60 min at 37 degrees C (1 h) for hyperactivation and capacitation assessment. The treatments were then divided into two portions, centrifuged and resuspended in either HTF or SP, giving HTF control and SP control treatments and two crossover treatments, 1 h HTF then 1 h SP (H/SP) and 1 h SP then 1 h HTF (SP/H). All tubes were incubated for a further 60 min at 37 degrees C before aliquots were taken for hyperactivation and capacitation assessments. Hyperactivation was estimated using an IVOS v10.6t (Hamilton Thorne Research, Beverly, MA, USA) 60 Hz CASA instrument, and capacitation was estimated using the chlortetracycline (CTC) method. The presence of seminal plasma in the capacitation medium for 60-120 min post-swim-up inhibited the development of hyperactivated motility. This inhibition was reversible, and was not prevented by preincubation for 1 h in HTF medium. There was no difference in the CTC binding patterns between treatments at 2 h, indicating that the capacitation-associated membrane changes were not affected by the presence of a low concentration of seminal plasma. There was no correlation between percentage capacitated and percentage hyperactivated spermatozoa for any treatment. Since the proportions of hyperactivated spermatozoa and capacitated spermatozoa were not related, we conclude that the processes leading to hyperactivation and to the membrane changes associated with capacitation are not tightly interlinked and consider this finding to be due to hyperactivated motility being associated with flagellar movement, while the CTC assay assesses changes in the Ca2+ levels of the sperm head plasma membrane.     

Anti-bull sperm monoclonal antibodies: I. Identification of major antigenic domains of bull sperm and manifestation of interspecies cross-reactivity

The overall objective of this series of experiments is to generate immunological markers that may elucidate bull sperm surface changes in vitro. Here we report the initial experiments of the study, involving the production and characterization of monoclonal antibodies (mAbs) again bull sperm. BALB/c mice were immunized with phosphate-buffered saline (PBS)-washed whole bull sperm, and their spleen cells were fused with NS-1 myeloma cells in two separate cell fusion experiments, resulting in the generation of 15 mAbs. The mAbs were specific to antigens of either the posterior tail or the head regions of bull sperm and detected five major domains of antigen localization in the bull sperm (apical crescent, equatorial band, principal acrosomal, whole head, and posterior tail). Eleven of the 13 head-specific mAbs recognized intra-acrosomal antigens, whereas 2 mAbs recognized antigens that were localized in the plasma membrane. One mAb specific to the tail region was of the IgM class; the remaining 14 mAbs were of the IgG class. They were all sperm specific, with no cross-reactivity to bovine oocytes or to any of the 12 bovine somatic tissues tested. The mAbs were not species specific, however, because 11, 10, 2, and 1 of the 15 mAbs cross-reacted with sheep, pig, mouse, and human sperm, respectively. None of the mAbs cross-reacted with rooster sperm. The cognate antigens of the 11 tested mAbs were of testicular origin, but several of them showed enhanced binding to epididymal sperm. In western blot analysis, 3 of the 13 mAbs tested identified more than one protein band (40-200 kDa). Seven others recognized proteins of > or = 200 kDa, whereas three mAbs recognized no proteins.     

Increased capacitation of buffalo sperm by heparin as confirmed by electron microscopy and in vitro fertilization

Capacitation of buffalo sperm was evaluated by induced acrosome reaction (AR) upon the exposure of 10 mM Ca2+. Culture of sperm for 8 hr in BO medium supplemented with 10 micrograms/ml heparin significantly (P < 0.01) increased the percentage of AR and confirmed by transmission electron microscopy. Vesiculization of outer acrosomal membrane and plasma membrane was observed significantly higher (P < 0.01) following 8 hr of sperm culture with heparin. Culture of sperm with heparin also increased rate of fertilization of in vitro matured oocytes and their subsequent development up to morula/blastocyst stage (P < 0.01). The study demonstrates that capacitation of buffalo sperm by heparin required at least 8 hr exposure of sperm to heparin for maximum acrosome reaction.     

Extraction of sp18 family membrane proteins on posterior head of bull sperm and the effect of anti-sp18 IgG on sperm motility and murine in vitro fertilization

BACKGROUND AND PURPOSE: We evaluated sucralfate, well-known in the treatment of gastric ulcers, in relation to its possible reduction of radiation-induced acute complications in the treatment of head and neck cancers. MATERIALS AND METHODS: One hundred two patients were randomized in a double-blind placebo-controlled prospective setting. All patients were treated to a minimum dose of 55 Gy in 5 weeks. Oral intake of sucralfate was started at the beginning of radiotherapy and continued during the whole treatment at a dose of 1 g six times a day. All patients were scored according to a scoring system developed in our department. Weight was checked once a week. RESULTS: Comparing the time course of the mean scores for subjective intolerance, mucositis, dysphagia, dermatitis and nausea, no statistically significant differences between the two treatment arms (sucralfate, n = 38; placebo, n = 45) were observed. The mean weight loss in the sucralfate arm was 1.6 +/- 3.4 kg while it was 1.3 +/- 2.0 kg in the placebo arm. Apart from gastrointestinal upset, the administration of sucralfate did not cause any side-effects. CONCLUSION: This trial produced no clinical evidence indicating that the oral intake of sucralfate reduces the acute radiation-induced side-effects. Therefore, we do not recommend the prophylactic use of sucralfate in patients with head and neck cancer treated by radiotherapy.     

Stimulating effect of pyroglutamylglutamylprolineamide, a prostatic, TRH-related tripeptide, on mouse sperm capacitation and fertilizing ability in vitro

The morbidity associated with total glossectomy for treatment of base of tongue carcinomas provides the impetus to investigate techniques to salvage the uninvolved normal anterior tongue. This report describes a method of reinnervation of the anterior tongue using a hypoglossal-lingual transfer. In Cynomolgus monkeys, unilateral transfers with and without a subsequent muscle fillet resulted in reinnervation from the base to the tip of the tongue. It is proposed that hypoglossal-lingual nerve transfers be considered to allow sparing and return of function to the anterior tongue in conjunction with a resection of the tongue base. Additional experiments confirmed that the base of the tongue, like other midline muscles, has bilateral and separate innervation. The presence of a physiological lingual-hypoglossal reflex in the normal animal was documented.