Acrosome Reaction In Bovine Spermatozoa Research Articles

The Modulation of Functional Status of Bovine Spermatozoa by Progesterone.

Simple SummaryProgesterone is an endogenous steroid hormone, which can induce capacitation and/or acrosome reactions in semen of certain mammalian species. Our study aimed to investigate the effect of progesterone on the functional status of fresh bovine spermatozoa using a chlortetracycline fluorescent probe. Results showed that heparin induced capacitation in spermatozoa incubated with or without progesterone. The destruction of microfilaments by an inhibitor of cytochalasin D blocked the stimulating effect of heparin. Steroid hormone in mixture with prolactin stimulated the acrosome reaction in spermatozoa, which was blocked by an inhibitor of microtubule polymerization (nocodazole). At the acrosome stage, prolactin provided the undergoing of acrosome reaction in male gametes. This effect was noted both in the presence and absence of progesterone and inhibited by nocodazole. The supplementation of dibutyryl cyclic adenosine monophosphate during the acrosome reaction to progesterone-untreated spermatozoa did not cause changes in proportion of acrosome-reacted cells. However, when progesterone was added during capacitation, a significant increase in the proportion of capacitated cells was noted, which was inhibited by nocodazole. Thus, progesterone under the action of prolactin and dibutyryl cyclic adenosine monophosphate determines the functional status of fresh spermatozoa, which indicates progesterone-modulating effect on the indicators of post-ejaculatory maturation of male gametes.The aim of this study is to identify the effects of progesterone (PRG) on the capacitation and the acrosome reaction in bovine spermatozoa. The fresh sperm samples were incubated with and without capacitation inductors (heparin, dibutyryl cyclic adenosine monophosphate (dbcAMP)), hormones (prolactin (PRL), PRG), inhibitors of microfilaments (cytochalasin D) and microtubules (nocodazole) during capacitation and acrosome reactions. The functional status of spermatozoa was examined using the chlortetracycline assay. Supplementation of heparin stimulated capacitation in the presence and absence of PRG. Cytochalasin D blocked the stimulating effect of heparin on capacitation. The addition of PRL during capacitation (without PRG) did not affect the functional status of spermatozoa, while in PRG-treated cells PRL stimulated the acrosome reaction. PRL (with and without PRG) increased the acrosome reaction in capacitated cells. These PRL-dependent effects were inhibited by nocodazole. During the acrosome reaction, in presence of dbcAMP, PRG decreased the proportion of acrosome-reacted cells compared to PRG-untreated cells. This effect in PRG-treated cells was canceled in the presence of nocodazole. In conclusion, PRG under the action of PRL and dbcAMP determines the changes in the functional status of native sperm cells, which indicates PRG modulating effect on the indicators of post-ejaculatory maturation of spermatozoa.

112 The effect of biological extenders on invitro induction of the acrosome reaction in bovine spermatozoa

Biological extenders in semen are used to protect the integrity of the sperm cells during cryopreservation. The two most common extenders are egg yolk and milk, and initial experiments describing heparin capacitation for IVF were conducted with semen frozen in egg yolk extender. However, semen of many bulls used for IVF is frozen in milk-based extender, and it is not clear whether this affects the response to heparin capacitation. This study aims to assess whether the use of milk- or egg-based extenders in frozen-thawed bovine semen affects sperm's ability to undergo the acrosome reaction, utilising either the endogenous progesterone pathway or calcium ionophore A23187 to induce the acrosome reaction following heparin capacitation. Frozen semen from 12 bulls (6 using egg yolk citrate-based extender containing 20% egg yolk, and 6 using milk-based extender) was used for no less than 3 replicates each. All semen was commercially processed and cryopreserved using the same methodology. Semen was thawed and separated using a Percoll gradient and centrifugation at 400×g for 10min, then incubated for 4h in a capacitating medium containing heparin (10μgmL−1) at a concentration of 1×106 spermmL−1. Capacitated sperm were then introduced to progesterone (10μM) or A23187 (10 μM) for acrosome induction and stained with fluorescein isothiocyanate-conjugated peanut agglutinin (5μgmL−1). After treatment and staining, the sperm were analysed using flow cytometry to determine the percentage of populations with fluorescent acrosomes, indicating an exposed acrosome and an ongoing acrosome reaction. Fluorescent microscopy was also used to confirm fluorescence in acrosome-reacting samples. Results were analysed as a 2×3 factorial design by ANOVA with extender and acrosome induction treatment as factors. For egg yolk-extended semen, the mean percent of acrosome-reacted sperm was 83.7 (±6.8), 81.8 (±7), and 15.0 (±7.5) for sperm treated with progesterone, A23187, and heparin only, respectively. Compared to milk-extended semen, for which the mean percent acrosome-reacted was 15.0 (±8.2), 14.8 (±7.4), and 12.0 (±6.8) for progesterone, A23187, and heparin only, respectively. Significant differences were observed between extenders, with egg yolk-extended semen having a significantly greater response to acrosome reaction-inducing agents than semen extended with milk-based extenders (P<0.05). Also, it was observed that progesterone induced a similar percentage of acrosome reactions as A23187. Variation between bulls in the response to heparin capacitation has been observed and cannot be eliminated in this study. The difference observed between extenders warrants further study with a split ejaculate design. The possibility that the semen extender used during cryopreservation affects the response to heparin capacitation suggests that this should be accounted for in IVF protocols to increase the efficiency of this technology.

Expression of Syntaxin‐2 in Bovine Sperm

Syntaxins are membrane integrated Q‐SNARE proteins known to participate in exocytosis. Vesicle docking involves the binding of two plasma membrane proteins, syntaxin and SNAP‐25, to the vesicle membrane protein VAMP to form a stable trimeric core complex; synaptophysin is thought to regulate the formation of this complex. Although the members of Q‐SNARE proteins are characterized in somatic cells, it is not known whether related proteins function in the sperm acrosome reaction. The objective of the present study is to identify and localize syntaxin and synaptophysin in bovine epididymal spermatozoa. Western blots of caput and cauda sperm lysates and plasma membrane fractions stained with anti‐syntaxin and anti‐synaptophysin showed the presence of a 31kDa and 38kDa band in both sperm lysates and plasma membrane fractions, respectively. Indirect immunofluorescence localized syntaxin to the anterior but not equatorial regions of the acrosomal segment. Syntaxin was recovered in the anti‐syntaxin immunoprecipitation pellet. Coimmunoprecipitation analysis will be utilized to identify the syntaxin‐associated protein(s) complex during capacitation and acrosome reaction of bovine spermatozoa. It is assumed that syntaxin‐2 and synaptophysin may be involved in triggering the acrosome reaction through a ligand‐receptor mediated signal transduction pathway. NIH 1SC3GM096875 and FSU‐RISE ‐ STEM Grants.

Relocation of myosin and actin, kinesin and tubulin in the acrosome reaction of bovine spermatozoa

The mammalian acrosome reaction is a specialised exocytotic event. Although molecular motors are known to be involved in exocytosis in many cell types, their potential involvement in the acrosome reaction has remained unknown. Here, it has been shown that actin is localised within the equatorial segment and in the marginal acrosomal ridge of the heads of unreacted bull spermatozoa. Myosins IIA and IIB are found within the anterior acrosomal margins of virtually all sperm cells and, less prominently, within the equatorial segment. Tubulin was detected in the equatorial segment and around the periphery of the acrosome while kinesin was prominent in the equatorial segment. After induction of the acrosome reaction by means of the calcium ionophore A23187, the number of cells exhibiting actin fluorescence intensity in the anterior acrosomal margin decreased four-fold and those displaying equatorial segment fluorescence decreased 3.5-fold; myosin IIA immunofluorescence decreased in intensity with most spermatozoa losing equatorial staining, whereas there was little change in the distribution or intensity of myosin IIB immunofluorescence, except for approximately 20% decrease in the number of cells exhibiting acrosomal staining. Tubulin became largely undetectable within the head and kinesin staining spread rostrally over the main acrosome region. A possible sequence of events that ties in these observations of molecular motor involvement with the known participation of SNARE proteins is provided.

Effect of Gonadotropin Releasing Hormone Agonist-Buserelin on In Vitro Induction of Acrosome Reaction in Bovine Spermatozoa.

Acrosome reaction (AR) is an important step in the process of fertilization in mammals which takes place in the oviduct. Recent studies suggest that Gonadotropin Releasing Hormone (GnRH) can act in an autocrine or paracrine manner in reproductive tissues and cells to modulate their physiological functions. It is also evident that functional forms of GnRH- GnRH receptor system could be present in the oviductal microenvironment. Thus this study was performed to assess the direct effect of GnRH on induction of AR in bovine spermatozoa. Frozen thawed bovine spermatozoa after swim up separation in modified Tyrodes medium were treated with a GnRH agonist, buserelin at the dose rates of 0 (control), 0.2, 0.4, 0.8, 1.2μgmL-1 and 1 μgmL-1 progesterone (positive control) and incubated at 38.50C in a humidified atmosphere of 5% CO2 for 3 hours. Acrosomal status at 0 hour and after incubation in each treatment group was determined with fluorescent microscopy, using fluorescein isothiocyanate labeled Pisum sativum agglutinin (FITC-PSA) staining. Increase in percent acrosome reacted after 3 hours were significantly higher (p<0.001) in the presence of 0.4 and 0.8 μgmL-1 when compared with the control group that was incubated in the absence of buserelin. While 0.2 and 1.2 μgmL-1 were also able to increase the percent acrosome reacted insignificantly (p >0.05).Increase in the percentage of acrosome reacted spermatozoa in different treatment groups were 24.115 ± 1.201, 29.65 ± 0.8685, 36.39 ± 2.165, 48.32 ±1.254, 27.05 ± 0.6074 and 34.836 ± 1.143 for 0, 0.2, 0.4, 0.8, 1.2μgmL-1 buserelin and 1 μgmL-1 progesterone respectively. In addition, it was also revealed that this stimulatory effect of GnRH agonist could be blocked by introduction of 1000ng mL-1 Antide (GnRH antagonist) in to the incubation medium, suggesting a receptor mediated stimulatory effect of GnRH agonist on bovine spermatozoa. The results of this study further strengthens our knowledge on the presence of GnRH-R system and the role of GnRH in bovine reproductive biology which would contribute to the more meaningful application of GnRH and its potent synthetic analogues, in both domestic animal and human reproductive management and in vitro fertilization programs.

Effect of Progesterone and Oestradiol on Sperm–zona Binding and Acrosome Reaction in Bovine Spermatozoa after Thawing

This study was conducted assess spermatozoa binding capacity to the oocyte in the presence of 0.1 or 0.5 microg/ml progesterone (P4) or a combination of 0.5 microg/ml P4 with 0.1 microg/ml oestradiol (OE). The number of oocyte-bound spermatozoa in the presence of progesterone was significantly higher (p < 0.05 to p < 0.001) when compared with the control samples, that were incubated in the absence of P4. Spermatozoa binding to the zona pellucida (ZP) of the oocyte were concentration-dependent - significantly higher numbers of spermatozoa were bound in the presence of 0.5 microg/ml P4, when compared with that of 0.1 microg/ml P4. Oestradiol at 0.1 microg/ml concentration used impaired the effect of progesterone-mediated sperm-oocyte binding. The incidences of acrosome-reacted (AR) spermatozoa bound to the ZP - following 0, 60, 120 and 180 min. incubation in the presence and absence of 1 microg/ml progesterone was also assessed. Only at 180 min of incubation a higher (p < 0.001) incidence of the AR-spermatozoa was found in sperm-ZP complexes incubated in the presence or absence of progesterone, being 56.5 +/- 11.1 and 43.2 +/- 8.8 % respectively. In conclusion, progesterone enhances the sperm-ZP binding capacity. Progesterone affects the incidences of AR on zona-bound spermatozoa only after prolonged co-culture.

Acrosome reaction in bovine spermatozoa: Role of reactive oxygen species and lactate dehydrogenase C4

After capacitation, mammalian spermatozoa accomplish the acrosome reaction (AR), a well-controlled exocytosis process crucial to fertilize mature oocytes that involves several protein kinases such as protein kinase A (PKA), C (PKC), and tyrosine kinase (PTK). Reactive oxygen species (ROS) are involved in both bovine sperm capacitation and AR. Lactate dehydrogenase C4 (LDH-C4) was associated with bovine and mouse sperm capacitation. Our aims were to study the participation of LDH-C4 to contribute with the status redox required for AR and the role of ROS in the regulation of PKA, PKC, and PTK involved in the exocytotic event. Sodium oxamate, an inhibitor of LDH-C4, prevented the AR induced by lysophosphatidylcholine (LPC) or NADH. Hydrogen peroxide promoted and superoxide dismutase (scavenger of superoxide), catalase (scavenger of hydrogen peroxide), diphenyleneiodinum, diphenyliodonium, cibacron blue, and lapachol (inhibitors of NADPH oxidase) prevented the AR, suggesting that ROS and a sperm oxidase are involved in the AR induced by these compounds. Inhibitors of PKA, PKC, and PTK also prevented the AR induced by LPC or NADH, suggesting the involvement of these kinases in the process. These results suggest that LDH-C4 may participate in the regulation of the redox status required to achieve the AR in bovine spermatozoa and that ROS are key elements in the regulation of protein kinases associated with the AR process.

A synthetic decapeptide from a conserved ZP3 protein domain induces the G protein-regulated acrosome reaction in bovine spermatozoa

In some animal species, the zona pellucida protein 3 (ZP3) plays a central role during fertilization, functioning as a specific receptor for sperm and as an inducer of the acrosome reaction. On the other hand, the zona pellucida protein 2 (ZP2) acts as a secondary receptor, binding to acrosome-reacted sperm. The objective of these studies was to identify ZP2 and ZP3 domains that may be of importance for the induction of the acrosome reaction. For this purpose, we synthesized a number of ZP2 and ZP3 peptides that were either conserved among species or that were species-specific according to their respective primary structures. We identified a defined, conserved ZP3 decapeptide (ZP3-6 peptide) that bound to the surface of the acrosomal region and induced the acrosome reaction in a concentration-dependent manner in capacitated bovine sperm; this effect was significant in the nanomolar range. Pertussis toxin inhibited the ZP3-6 peptide-induced acrosome reaction but had no effect on the progesterone-induced exocytotic event. Our data are in accordance with previous studies showing that progesterone induces acrosomal exocytosis via a different pathway than ZP3 and strengthen the hypothesis that the effect of ZP3-6 peptide upon acrosomal exocytosis is G protein regulated. Despite the commonly accepted idea that glycosylation of ZP proteins is required for successful sperm–oocyte interaction, we found that acrosomal exocytosis can be induced by a synthetic ZP3 peptide that is not glycosylated. The results presented in this study may be useful for the investigation of the molecular mechanisms of sperm–egg interaction in bovine and other species.

Effect of exogenous progesterone on post-thaw capacitation and acrosome reaction of bovine spermatozoa.

The aim of this work was to study the effect of progesterone (P4) on capacitation and acrosome reaction (AR) of post-thaw bovine spermatozoa in vitro. Spermatozoa were incubated (0-180 min) in capacitation medium supplemented with 0, 0.1, 1.0 and 10.0 microg/ml of P4. At different time intervals aliquots were taken to determine sperm plasma membrane lipid destabilization, or capacitation (AR induced by lysophosphatidylcholine) in spermatozoa. The second experiment aimed to study the effects of P4, as potential inducer of AR in heparin-capacitated spermatozoa. The acrosomal status and viability of spermatozoa were evaluated under an epifluorescence microscope using Ethidium homodimer/peanut agglutinin fluorescein isothiocyanate staining method. Plasma membrane scrambling in spermatozoa was assessed by a flow cytometer, using merocyanine staining. The results show that P4 at the concentrations used had no negative effects on sperm viability. Progesterone significantly enhanced sperm capacitation (p < 0.001), but had no effect on plasma membrane lipid stability (p > 0.05) and did not significantly increase the AR of heparin-capacitated spermatozoa (p > 0.05). Progesterone displayed its effects in a dose-dependent manner with a maximum effect of 10 microg/ml P4 at 180 min of incubation. The results demonstrate that in cryopreserved bovine semen, P4 acts as capacitating, but not as an AR-inducing agent.

Evaluation of acrosomal status of bovine spermatozoa using concanavalin a lectin

We report here that fluorescein isothiocyanate-conjugated concanavalin A (FITC-ConA) specifically labels the acrosomal region of acrosome-reacted bovine spermatozoa. This labeling is found to be useful in evaluating the acrosome status of bovine spermatozoa. When fresh bovine spermatozoa that had been fixed with 4% formaldehyde, smeared on glass slides and then air-dried were stained by FITC-ConA, weak fluorescence was observed on the acrosomal region, although almost all the spermatozoa appeared to be acrosome-intact. However, when fresh sperm suspensions were incubated with FITC-ConA and then mounted on glass slides, no fluorescence was observed on the acrosomal region. Therefore, in the ensuing experiments, both the fixation and the FITC-ConA staining of spermatozoa were done in suspension. When ethanol-treated spermatozoa, whose outer membrane may be permeabilized, were stained with FITC-ConA, the fluorescence was extensively observed on the inner acrosomal region. This fluorescence was inhibited in the presence of 0.2 M D-mannose, a competitive sugar, suggesting that FITC-ConA binds specifically to glycocomponents on the inner acrosomal membrane. We next tried to stain fresh or frozen-thawed spermatozoa from 3 different bulls that had been treated with the calcium ionophore A23187, which is known to induce acrosome reaction of bovine spermatozoa, with FITC-ConA. A significant correlation between the percentage of ConA-labeled spermatozoa and that of rose bengal stained negative ones at various time points during A23187 incubation was achieved. Furthermore, suitability of dual staining to distinguish between physiological acrosome reaction (acrosome-lost and live) and degenerative acrosomal loss (acrosome-lost and dead) using FITC-ConA and Hoechst bis-benzimide 33258 (H258) supravital stain was also confirmed. From these results, it was concluded that the FITC-ConA labeling procedure is a feasible and reliable method for the assessment of physiological acrosome reaction of bovine spermatozoa.

Bovine oviductal epithelial cells: their cell culture and applications in studies for reproductive biology.

Epithelial cells of the mammalian oviduct play an important role in reproductive and developmental events that occur there. Oviductal epithelial cells from several mammalian species can be isolated and cultured in serum or serum-free medium in vitro and cell culture of bovine oviductal epithelial cells (BOEC) has been described by many investigators. Cultured BOEC show a wide variety of secretory activities and these secretory factors may influence early embryonic development or sperm function. Monolayer cultures of BOEC have been widely used for in vitro co-culture of bovine preimplantation embryos. The use of BOEC co-culture systems has improved embryonic development in nearly all the studies conducted. In addition, interaction of bovine spermatozoa with BOEC, in a similar manner to that observed for spermatozoa in vivo, induced specific changes in sperm capacitation and consequently improved the fertilizing capacity of bovine spermatozoa in vitro. Thus co-culture systems with BOEC may not only offer an excellent model for studying the mechanisms of capacitation and acrosome reaction of bovine spermatozoa but also provide a useful tool for the improvement of embryo development in vitro.

Evaluating Acrosome Interference Contrast Reaction Steps with Brightfield and Differential Microscopy Techniques

Acrosomal integrity of bovine spermatozoa was evaluated using naphthol yellow S-erythrosine B stain with bright field microscopy, .2% glutaraldehyde fixation with Nomarski optics, and live wet smears with Nomarski optics to compare the evaluation techniques. Four ejaculates per two Holstein bulls were enriched through a Percoll gradient, capacitated with heparin, and acrosome-reacted with lysophosphatidylcholine to prepare spermatozoa for in vitro fertilization. Spermatozoa samples were taken after each preparatory step, and percentage of intact acrosomes was evaluated with the stain, .2% glutaraldehyde, and live wet smear. Acrosomal integrity decreased with each preparatory step across all techniques. The decrease in integrity from heparin to lysophosphatidylcholine indicated that all methods detected the acrosome reaction. Technique means across preparation steps showed no differences. Correlations between microscopy techniques were high. The live wet smear used no fixation methods. Both the naphthol yellow S-erythrosine B stain and the .2% glutaraldehyde techniques employed fixation steps that may have introduced artifacts that could influence the data. The live wet smear evaluated by Nomarski optics allowed a comparable evaluation of the acrosome-reacted bovine spermatozoa. Comparing brightfield with differential interference contrast microscopy for detecting the acrosome reaction in bovine spermatozoa showed that both methods were equally accurate.

Effects of platelet activating factor on the motility and acrosome reaction of bovine spermatozoa

The effects of platelet activating factor (PAF) on motility and the acrosome reaction of ejaculated bull spermatozoa were evaluated. Washed spermatozoa (30 × 10 6/ml) were incubated (39°C) for up to 2 h with 10 to 200 μM PAF in a modified Tyrode's solution (pH 7.4) containing 3 mg/ml bovine serum albumin. Sperm motility was evaluated subjectively and by computer-assisted semen analysis. Percent acrosome-reacted spermatozoa was quantified microscopically from fixed smears following Giemsa staining. Percent fertilization by PAF-treated spermatozoa was determined using in vitro-matured bovine ova. Percent sperm motility decreased with ≥ 50 μM PAF, while the rate of motility loss increased with PAF concentration (P<0.001). Percent acrosome reactions increased with PAF concentration during incubation (P<0.001). Acrosomal loss was rapid and complete with 200 μM PAF. At concentrations between 80 to 120 μM PAF, bull spermatozoa underwent acrosome reactions without a rapid loss of motility and penetrated in vitro-matured bovine ova at a rate comparable to that of heparin-capacitated spermatozoa (68 versus 54%, respectively). Incubation of bull spermatozoa with 10 to 50 μM PAF for 45 min had no effect on percent progressive motility, sperm velocity or other motility parameters. These results indicate that PAF can be used to induce acrosome reactions in bull spermatozoa and to promote in vitro fertilization of bovine ova. Under the conditions used in this study, PAF did not stimulate bovine sperm motility.

Penetration of bovine follicular oocytes by frozen-thawed spermatozoa in the presence of caffeine and heparin.

Bovine follicular oocytes matured in culture were inseminated with frozen-thawed spermatozoa which were either preincubated for 5-5.5 h or not preincubated in a medium with caffeine (5 mM) and heparin (10 micrograms/ml). When the oocytes with cumulus and corona cells were inseminated, spermatozoa started to penetrate oocytes 3 h later regardless of whether spermatozoa were preincubated or not. However, a significantly higher proportion of oocytes was penetrated by preincubated than non-preincubated spermatozoa. When the oocytes were freed from cumulus and corona cells, penetration was observed to start 1 h after insemination and there were no differences in penetration rates 1-5 h after insemination between preincubated and non-preincubated spermatozoa. This study demonstrates that capacitation and the acrosome reaction of bovine spermatozoa can be induced within 1 h in a medium containing both caffeine and heparin when denuded oocytes are inseminated.

Regulation of acrosomal exocytosis: II. The zona pellucida-induced acrosome reaction of bovine spermatozoa is controlled by extrinsic positive regulatory elements

The effects of accessory sex gland secretions on the zona pellucida-induced acrosome reaction of bovine spermatozoa were investigated. Soluble extracts of zonae pellucidae initiated exocytosis in ejaculated spermatozoa. This process had an ED 50 of 20 ng/μl zona pellucida protein and saturated at 50 ng/μl ( Florman and First, 1988, Dev. Biol. 128, 453–463). In epididymal sperm this dose-response relationship was shifted toward greater agonist concentrations by at least a factor of 10 3. Reconstitution of high potency agonist response was achieved in vitro by incubation of epididymal sperm with bovine seminal plasma. Reconstitution was dependent on the seminal plasma protein concentration. The ED 50 of this process was 62 μg protein/10 8 sperm and saturation was observed with 124 μg protein/10 8 sperm. Agonist responses in reconstituted epididymal sperm and in ejaculated sperm were indistinguishable with regard to dependence on the zona pellucida protein concentration and the kinetics of induced acrosome reactions. Kinetic studies suggest that reconstitution is due to adsorption of regulatory factors from seminal plasma. In addition to the positive regulatory elements responsible for reconstituting activity, seminal plasma also contains negative regulatory elements which inhibit agonist response. These negative factors are inactivated during sperm capacitation, permitting the expression of positive regulators. Acting together, these regulatory elements could coordinate high affinity agonist response with the availability of eggs in vivo.

Acrosome reaction of bovine spermatozoa in vivo: sites and effects of stages of the estrous cycle.

Thirty-two cows were inseminated near the uterotubal junction at various stages of the estrous cycle and slaughtered 16 h later to determine the effects of stage of the estrous cycle and tubal site of sperm recovery on the frequency of acrosome-reacted bull spermatozoa. Slaughter times were 46, 70, 144, or 168 h after each cow was injected with prostaglandin (PG) F 2 alpha or during the luteal phase of the estrous cycle. Sperm were recovered from the upper uterus and the isthmus and ampulla of the oviducts and stained for both viability and acrosome reaction. The highest frequency of acrosome-reacted sperm was found in the ampulla ipsilateral to a dominant follicle (largest follicle present) or recent ovulation and primarily at 70 h after PGF2 alpha (P less than 0.05). Also, fewer sperm were acrosome reacted prior to (46 h post-PGF2 alpha) and well after (168 h post-PGF2 alpha) estrus than during or immediately postestrus (70, 90, and 144 h post-PGF2 alpha; P less than 0.05). Except for two cows, one at 46 h and one at 70 h, all cows with more than 50% acrosome-reacted sperm in the ampulla had ovulated before slaughter. These data suggest that capacitated sperm become localized in the ampulla of the oviduct of the ovulatory side around the time of ovulation.

In vitro maturation and fertilization of bovine oocytes are temperature-dependent processes.

Effects of temperature on bovine sperm acrosome reaction, oocyte maturation, hyaluronic acid production by cumulus cells and in vitro fertilization were studied. Viability and a true acrosome reaction of bovine spermatozoa were impaired at 40 degrees C. Temperatures lower than 35 degrees C did not enhance the acrosome reaction. However, viability between 30 degrees C-38 degrees C was not altered after 22 h of incubation. The optimal temperature for the acrosome reaction was 38 degrees C. Labeled glucosamine incorporation into glycosaminoglycans was not different among temperatures of 35 degrees C, 37 degrees C or 39 degrees C, whereas 41 degrees C caused a significant reduction (P less than 0.02). Temperatures ranging between 35 degrees C-39 degrees C had no deleterious effects on resumption and completion of meiosis, but at 41 degrees C the frequency of oocytes that progressed to Metaphase II was significantly reduced (P less than 0.0001). Ova matured at 39 degrees C had significantly higher rates of fertilization than at 35 degrees C, 37 degrees C, or 41 degrees C. Killed spermatozoa (control) had no effect on ovum activation at 39 degrees C. From these results it was concluded that events occurring prior to and during fertilization are temperature sensitive.

Chondroitin sulfate facilitates an acrosome reaction in bovine spermatozoa as evidenced by light microscopy, electron microscopy and in vitro fertilization.

Bovine epididymal spermatozoa were incubated for 22 h in a modified Tyrode's medium. The percentages of sperm exhibiting an acrosome reaction were determined morphologically after fixing and staining specimens. The addition of chondroitin sulfate A (CS-A) significantly increased the incidence of acrosome reaction. When observed by electron microscopy, acrosome-reacted sperm had undergone vesiculation of the plasma and outer acrosomal membranes. Sperm incubated in the presence of CS-A demonstrated a significantly higher incidence of vesiculation when compared to the controls. Additionally, rates of in vitro fertilization of bovine oocytes were significantly elevated when sperm and ova were exposed to CS-A. These results suggest that glycosaminoglycans in the female reproductive tract may be responsible for some of the biochemical changes associated with fertilization, and a light microscope procedure can be used to assess occurrence of the acrosome reaction.

Structural comparisons among glycosaminoglycans to promote an acrosome reaction in bovine spermatozoa

Bovine epididymal spermatozoa were cultured for 22 hr. in a chemically defined medium containing glycosaminoglycans. Sperm were stained and examined for degree of acrosome reactions and viability after treatment. Viability did not differ significantly among treatments or doses. Highly significant treatment and dose effects resulted for induction of the acrosome reaction. Heparin was the most potent glycosaminoglycan and hyaluronic acid was least effective. Chondroitin sulfate isomers yielded a response intermediate to heparin or hyaluronic acid. These results suggested that the degree of sulfation of the glycosaminoglycans was partly responsible for promoting the acrosome reaction. Treatment of chondroitin sulfate isomers with chondro-4-sulfatase or chondro-6-sulfatase showed that the respective stimulatory effects of chondroitin-4-sulfate and chondroitin-6-sulfate were negated. It was concluded that the ability of glycosaminoglycans to induce the acrosome reaction in bovine spermatozoa in vitro resides in the amino-sugar complex and is enhanced by the presence of a sulfate moiety on the amino-sugar disaccharide.

Proteoglycan from bovine follicular fluid enhances an acrosome reaction in bovine spermatozoa

Bovine epididymal and ejaculated spermatozoa were incubated for 22 and 9.5 h respectively, in a chemically defined medium. The percentages of sperm exhibiting an acrosome reaction were determined morphologically after fixing and staining specimens. Addition of bovine follicular proteoglycan or chondroitin sulfates ABC significantly increased the incidence of acrosome reaction. The stimulatory effects of the proteoglycan or chondroitin sulfates were negated by exposure to the enzyme chondroitinase ABC. Viability of spermatozoa was not affected by the various experimental treatments. Transmission electron microscopy of spermatozoa showed that vesiculation had occurred between the plasma and outer acrosomal membrane. These results suggest that proteoglycan present in follicular fluid at the time of ovulation may promote the acrosome reaction which precedes the ability of sperm to fertilize an ovum.