Incidence Of Acrosome Reaction Research Articles

Variability in in vitro fertilization outcomes of prepubertal goat oocytes explained by basic semen analyses.

This work analyses the changes that caprine spermatozoa undergo during in vitro fertilization (IVF) of in vitro matured prepubertal goat oocytes and their relationship with IVF outcome, in order to obtain an effective model that allows prediction of in vitro fertility on the basis of semen assessment. The evolution of several sperm parameters (motility, viability and acrosomal integrity) during IVF and their relationship with three IVF outcome criteria (total penetration, normal penetration and cleavage rates) were studied in a total of 56 IVF replicates. Moderate correlation coefficients between some sperm parameters and IVF outcome were observed. In addition, stepwise multiple regression analyses were conducted that considered three grouping of sperm parameters as potential explanatory variables of the three IVF outcome criteria. The proportion of IVF outcome variation that can be explained by the fitted models ranged from 0.62 to 0.86, depending upon the trait analysed and the variables considered. Seven out of 32 sperm parameters were selected as partial covariates in at least one of the nine multiple regression models. Among these, progressive sperm motility assessed immediately after swim-up, the percentage of dead sperm with intact acrosome and the incidence of acrosome reaction both determined just before the gamete co-culture, and finally the proportion of viable spermatozoa at 17 h post-insemination were the most frequently selected sperm parameters. Nevertheless, the predictive ability of these models must be confirmed in a larger sample size experiment.

Use of cholesterol-loaded cyclodextrin: An alternative for bad cooler stallions

During the cooling process, sperm may suffer irreversible damage that compromises the fertility rate. Incorporating cholesterol-loaded cyclodextrin (CLC) represents a strategy to increase sperm resistance at low temperatures; however, high levels of cholesterol in the cell membrane can interfere with sperm capacitation. The goals of this study were to determine the CLC concentration and cooling temperature that produce optimal kinetic parameters and viability of sperm from stallions identified as bad coolers (BCs) and good coolers (GCs), as well as the effect of adding CLC on the occurrence of the acrosome reaction (ACR) and on the fertility rate of cooled sperm. In experiment 1, each ejaculate was divided into four groups: Control and treated with 1 (CLC-1), 1.5 (CLC-1.5), or 2 mg (CLC-2) of CLC/120 × 106 sperm and cooled for 48 hours at 5 °C. In experiment 2, each ejaculate was divided into four groups: Control and CLC-1.5 cooled at 15 °C or 5 °C for 24 hours. For experiment 3, GC and BC stallions were used, and the ejaculates were divided into control and CLC-1.5 cooled at 5 °C for 48 hours. According to experiment, the sperm kinetics (SK) and plasma membrane integrity (PMI) were analyzed before and after 24 and 48 hours of cooling. In experiment 4, the ejaculates were divided into four groups: Control and CLC-1.5 maintained at room temperature or cooled at 5 °C for 24 hours. Each group was incubated with ionophore calcium at 37 °C for 3 hours. The incidence of ACR was analyzed before and after 1, 2, and 3 hours of incubation. For experiment 5, two cycles of 10 mares for a GC stallion and two cycles of 25 for a BC stallion were used. The inseminations were performed with control and CLC-1.5 groups cooled at 5 °C for 24 hours. According to results, all groups treated with CLC exhibited higher PMI compared with controls, and CLC-1.5 and CLC-2 exhibited the best SK results. The cooling temperature of 5 °C was superior to 15 °C when the sperm was treated with CLC. The GC and BC stallions benefited from the CLC-1.5 treatment, but the BCs were more evident, which presented greatly increased PMI and SK. There was a delay in capacitation of at least 3 hours for the fresh sperm and at least 1 hour for cooled sperm supplemented with CLC-1.5. After adding CLC-1.5, the fertility of BC stallion significantly increased, but that of the GC was not altered. Thus, incorporating CLC is an effective technique to cool equine semen, although it is indicated mainly for BC stallions.

163 HYPERACTIVATION OF STALLION SPERM IN FOLLICULAR FLUID FOR IN VITRO FERTILIZATION OF EQUINE OOCYTES

In vitro fertilization has remained elusive in the horse, as evidenced by low sperm penetration rates when IVF has been attempted with in vivo- or in vitro-matured oocytes. It is likely that the low sperm penetration rates observed in IVF studies are due to the inability to appropriately capacitate or hyperactivate, or both, stallion sperm in the laboratory. The acquisition of hyperactivated sperm motility has been observed within the oviducts of mammals at the time of fertilization and is required for zona pellucida penetration in conjunction with the acrosome reaction (AR). Although the zona pellucida is considered the prime physiological inducer of AR, previous studies have shown a low incidence of AR in zona pellucida-bound stallion spermatozoa after 1 h of in vitro binding. This low incidence suggests that, besides the zona pellucida glycoproteins, another major factor might be responsible for AR. Protein-bound progesterone, present in equine follicular fluid (FF), has been demonstrated to induce AR in stallion spermatozoa. In this context, the aims of this study were (1) to hyperactivate stallion sperm in FF and (2) to verify whether this hyperactivation supports equine IVF. Pooled FF, aspirated from the preovulatory follicles of oestrous mares, was used and its progesterone concentration was determined by immune enzymatic assay. Spermatozoa from fertile stallions selected by a swim-up procedure were pre-incubated for 6 h in capacitating medium (modifed Whittens's medium (WM) supplemented with 25 mM NaHCO3 and 7 mg mL–1 of BSA) and then incubated for 6 h at 37°C in either FF or capacitating WM. Sperm motility was assayed by computer-assisted semen analysis, rates of AR were assessed by fluorescein isothiocyanate-PNA staining and rate of apoptosis was assessed by an annexin V test. For IVF, spermatozoa were incubated at 10 × 106 sperm mL–1 in capacitating WM for 6 h and then diluted to 1 × 106 sperm mL–1 in capacitating WM with or without 10% of FF. Five mature mare oocytes were transferred into droplets (100 μL) of the sperm suspensions covered with mineral oil and then incubated for 18 h at 38.5°C in 5% CO2 in humidified air. After that, oocytes were transferred to an embryo culture medium (DMEM/F-12) for an additional 3 days. Data were analysed by ANOVA. Treatment of sperm with FF resulted in a significant (P ≤ 0.05) decrease of 3 motility variables indicative of hyperactivation: straight line velocity, straightness and linearity. The highest rate of AR (29.44%) and a lower rate of apoptosis (16.93%) were obtained after 4 h of incubation in follicular fluid. By coupling capacitating conditions with the induction of hyperactivation using follicular fluid, we have obtained reproducible percentages of 8-cell-stage embryos (18.56%) in our IVF experiments. Conversely, sperm incubated in capacitating conditions but not treated with FF did not fertilize (0%). It is concluded that mare FF does not impair sperm viability, stimulates equine sperm hyperactivation in vitro, induces the AR and supports equine IVF.

311 MELATONIN PROMOTES IN VITRO SPERM CAPACITATION IN BUFFALO (BUBALUS BUBALIS)

Melatonin, the main hormone secreted by the pineal gland, plays many roles in reproduction. In ram spermatozoa, melatonin administration increases plasminogen activator activity (Tsantarliotou MP et al. 2007 Theriogenology 69, 458-465), known to be involved in sperm capacitation and acrosome reaction (Taitzoglou IA et al. 1996 Mol. Androl. 8, 187-197). The aim of this study was to evaluate the efficiency of melatonin to induce buffalo in vitro sperm capacitation, indirectly assessed by estimating the capability of spermatozoa to acrosome-react. Frozen-thawed semen from 4 different bulls was pooled and treated by swim-up in order to select only the motile population. Spermatozoa (n = 829) were assessed immediately after swim- up separation, to evaluate the incidence of acrosomal loss in non-treated cells (time 0). The remaining spermatozoa were incubated in the absence of capacitating agents (negative control; n = 513), in the presence of 0.01 mM heparin (positive control; n = 775), 10 μM melatonin (n = 684), 100 μM melatonin (n = 751), and 1 mM melatonin (n = 650), for 2 h. Sperm were then exposed for 15 min to 60 μg mL-1 of lysophosphatidylcholine, an agent known to induce acrosome reaction (AR) only on capacitated spermatozoa. Trypan blue was used first to differentiate live from dead spermatozoa and the dried smears were then fixed in 37% formaldehyde and stained with Giemsa for acrosome evaluation by microscopic examination. The percentage of acrosome-reacted spermatozoa in each group was used to assess the efficiency of capacitation under different incubation conditions. The experiment was repeated 4 times. Differences among groups were analyzed by chi-square test. Acrosomal loss was observed only in 2.1% of the sperm population at time 0. Interestingly, sperm treatment with both heparin and the different concentrations of melatonin resulted in a significantly higher incidence of AR compared to the negative control (24.4, 20.5, 20.0, 23.6 v. 8.0% for the positive control, 10 μM melatonin, 100 μM melatonin, 1 mM melatonin, and the negative control, respectively; P < 0.01). These results demonstrated that melatonin determines capacitation of buffalo spermatozoa in vitro. Furthermore, the effect of melatonin was comparable to that of heparin, that is, the capacitating agent currently used in the IVF system. The capacitating effect was observed at all the tested concentrations, and viability was not affected. This suggests to extend the range of concentrations to test in future studies, in order to identify the optimal dose. Moreover, considering the seasonality of the species and the great differences in fertility attitude of buffalo bulls, it would be interesting to investigate the capacitation effect of melatonin in relation to both the season and the bull.

Antihyaluronidase Action of Ellagic Acid Effectively Prevents Polyspermy As a Result of Suppression of the Acrosome Reaction Induced by Sperm-Zona Interaction During In Vitro Fertilization of Porcine Oocytes

The present study was conducted to examine the effects of three tannin relatives (tannic acid, TA; gallic acid, GA; and ellagic acid, EA) on antihyaluronidase and reactive oxygen species (ROS) scavenging activity, in vitro fertilization (IVF) parameters, and the acrosome reaction (AR) induced by sperm-zona interaction. Among the three tannin relatives, TA and EA showed the strongest potency for blocking the hyaluronidase activity of boar sperm, with concentration-dependent inhibition over the range of 2-10 microg/ml. In contrast, ROSs were effectively scavenged by TA and GA, but not EA. When cumulus-free oocytes were inseminated in IVF medium containing 5 microg/ml of the tannin relatives, polyspermy was significantly reduced by TA and EA (32 and 29%, respectively) compared with oocytes treated with or without GA (51 and 69%, respectively) under conditions that maintained a high sperm penetration rate (P<0.05). Interestingly, induction of the AR by treatment of preincubated sperm with progesterone was blocked by TA and GA as a result of their higher levels of ROS scavenging activity, while EA, which possessed weak ROS scavenging activity, did not disturb induction of the AR with progesterone. However, the incidence of AR induced by sperm-zona interaction was significantly decreased by the strong antihyaluronidase actions of TA and EA compared with that in the absence of these compounds. Treatment with the compounds caused neither a protective proteolytic modification of the zona pellucida matrix before fertilization nor a reduction in acrosomal proteolytic activity or the number of zona-bound sperm. These findings suggest that the antihyaluronidase action of EA effectively prevents polyspermy by suppression of AR functionality induced by sperm-zona interaction and that hyaluronidase intervention is therefore required during porcine IVF.

322 EFFECT OF SODIUM NITROPRUSSIDE ON BUFFALO SPERM CAPACITATION IN VITRO

The overall in vitro embryo production efficiency in buffalo is hampered by the poor fertilization rate. It is known that the quality of the frozen semen may affect fertilization efficiency. However, it is not possible to rule out that the process of capacitation, required by spermatozoa to acquire the fertilizing ability, is impaired in the in vitro fertilization (IVF) system. Although several agents have been proven to induce sperm capacitation in vitro, heparin treatment is still the most efficient method in most of the domestic species. There is evidence that capacitation is part of an oxidative process and that nitric oxide (NO) acts as a capacitation inducer in human (Herrero et al. 1999 Biol. Reprod. 61, 575–581) and bovine (Rodriguez et al. 2005 Anim. Reprod. Sci. 85, 231–242) spermatozoa. The aim of the present study was to evaluate whether sodium nitroprusside (SNP), a well-known generator of NO in vitro, improves buffalo sperm capacitation in vitro. Frozen–thawed sperm from a bull previously tested for IVF were treated by swim-up in order to select only the motile population. Spermatozoa were incubated in the presence of 0.01 mM heparin (control group) for 1 h (n = 266), 2 h (n = 270), and 3 h (n = 306), and in the presence of 10 �M SNP for 1 h (n = 302), 2 h (n = 286), and 3 h (n = 260). The concentration of SNP was chosen on the basis of a preliminary dose-response trial (0.1 �M, 1 �M, and 10 �M). Following incubation with these agents, sperm were exposed for 15 min to 60 �g mL-1 of lysophosphatidylcholine, an agent known to induce acrosome reaction only on capacitated spermatozoa. Trypan blue was used first to differentiate live from dead spermatozoa and the dried smears were then fixed in 37% formaldehyde and stained with Giemsa for acrosome evaluation by microscopic examination. The proportion of acrosome-reacted spermatozoa in each group was used to assess the efficiency of capacitation under different incubation conditions. Differences between groups were analyzed by chi-squared test. No dead spermatozoa were found in all groups. Following 1-h sperm treatment with either heparin or SNP, the proportion of acrosome-reacted spermatozoa was similar (35.3% vs. 28.5%, respectively). However, extending the incubation time to 2 h, SNP significantly (P &amp;lt; 0.01) increased the incidence of acrosome reaction compared to heparin (60.1% vs. 44.1%, respectively). Analogously, when the sperm treatment was prolonged to 3 h, SNP gave a significantly (P &amp;lt; 0.01) higher percentage of acrosome reaction compared to the control (68.8% vs. 36.6%, respectively). In conclusion, sperm treatment with SNP for either 2 or 3 h significant improved the efficiency of buffalo sperm capacitation in vitro compared with heparin, that is, the capacitating agent currently used in the IVF system. The promoting effect of SNP indirectly indicates that NO acts as a capacitation inducer in buffalo spermatozoa. Finally, these results suggest the need to evaluate the effect of SNP on the fertilizing capability of buffalo spermatozoa in vitro.

Characterization of the endocannabinoid system in boar spermatozoa and implications for sperm capacitation and acrosome reaction

Anandamide (AEA) is the endogenous ligand of cannabinoid (CB) receptors, and as such it plays several central and peripheral activities. Regulation of female fertility by AEA has attracted growing interest, yet a role for this endocannabinoid in controlling sperm function and male fertility in mammals has been scarcely investigated. In this study we report unprecedented evidence that boar sperm cells have the biochemical machinery to bind and degrade AEA, i.e. type-1 cannabinoid receptors (CB1R), vanilloid receptors (TRPV1), AEA-synthesizing phospholipase D (NAPE-PLD), AEA transporter (AMT) and AEA hydrolase (FAAH). We also show that the non-hydrolyzable AEA analogue methanandamide reduces sperm capacitation and, as a consequence, inhibits the process of acrosome reaction (AR) triggered by the zona pellucida, according to a cyclic AMP-dependent pathway triggered by CB1R activation. Furthermore, activation of TRPV1 receptors seems to play a role of stabilization of the plasma membranes in capacitated sperm, as demonstrated by the high incidence of spontaneous AR occurring during the cultural period when TRPV1 activity was antagonized by capsazepine. We show that sperm cells have a complete and efficient endocannabinoid system, and that activation of cannabinoid or vanilloid receptors controls, at different time-points, sperm functions required for fertilization. These observations open new perspectives on the understanding and treatment of male fertility problems.

Optimizing incubation conditions for the preservation of sperm motility in processed semen samples

To determine the optimal conditions to maintain sperm motility and viability for prolonged periods, semen specimens were processed and cultured in different temperature and conditions. We conclude that processing of semen and incubation of sperm in protein-supplemented N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) human tubal fluid medium at room temperature best preserved viability and motility of sperm.

Effects of human follicular fluid on spermatozoa that have been cocultured with human oviductal cells

Objective: To investigate the sequential effects of human oviductal cells and human follicular fluid (hFF) on various sperm functions. Design: Laboratory experimental study. Setting: University gynecology unit. Patient(s): Fallopian tubes were from patients undergoing tubal ligation or hysterectomy. Semen was from men attending the subfertility clinics. Intervention(s): Spermatozoa were treated with [1] 6 hours in Earle’s balanced salt solution (EBSS-BSA; control); [2] 5 hours in EBSS-BSA and 1 hour with hFF (hFF); [3] 5 hours with oviductal cells and 1 hour in EBSS-BSA (coculture); and [4] 5 hours with oviductal cells and 1 hour with hFF (sequential). Main Outcome Measure(s): Motility, acrosome reaction, zona binding, and oocyte fusion. Result(s): Groups II and III spermatozoa had similar motility and were better than that of group I. Group IV displayed higher motility parameters than the other groups. Human follicular fluid induced acrosome reaction. The incidence of acrosome reaction in group IV was significantly lower than that in group II. Group III did not affect the acrosome reaction. Spermatozoa in groups II–IV had lower zona binding capacity than those in group I. Human follicular fluid stimulated oocyte penetration, whereas oviductal cells suppressed this effect of hFF. Conclusion(s): Oviductal cells maintained the fertilizing capacity of spermatozoa, whereas hFF facilitated the fertilization process of oviductal spermatozoa.

O-163. Observation of the incidence of acrosome reaction in human spermatozoa treated with mibefradil as a T-type Ca2+ ionophore channel inhibitor

O-163. Observation of the incidence of acrosome reaction in human spermatozoa treated with mibefradil as a T-type Ca2+ ionophore channel inhibitor

Peritoneal fluid modulates the sperm acrosomal exocytosis induced by N-acetylglucosaminyl neoglycoprotein.

The effect of peritoneal fluid (PF) on the human sperm acrosome reaction (AR) was tested. Sperm was pre-incubated with PF and the AR was induced by calcium ionophore A23187 and a neoglycoprotein bearing N-acetylglycosamine residues (NGP). The AR induced by calcium ionophore was inhibited 40% by PF from controls (PFc) and 50% by PF from the endometriosis (PFe) group, but not by PF from infertile patients without endometriosis (PFi). No significant differences were found in the spontaneous AR. When the AR was induced by NGP, pre-incubation with PFc reduced (60%) the percentage of AR, while PFe and PFi caused no significant differences. The average rates of acrosome reactions obtained in control. NGP- and ionophore-treated sperm showed that NGP-induced exocytosis differed significantly between the PFc (11%) and PFe/PFi groups (17%), and the ionophore-induced AR was higher for PFi (33%) than PFc/PFe (25%). The incidence of the NGP-induced AR was reduced in the first hour of pre-incubation with PFc and remained nearly constant throughout 4 h of incubation. The present data indicate that PF possesses a protective factor which prevents premature AR.

Induction of acrosome reaction in human spermatozoa accelerates the time of pronucleus formation of hamster oocytes after intracytoplasmic sperm injection

To assess the relationship between the incidence of acrosome reaction (AR) and the timing of pronucleus (PN) formation after intracytoplasmic sperm injection (ICSI). Prospective study. Infertility Research Center, Jeil Women's Hospital. Human semen obtained from fertile donors was prepared by one of the following methods: washing only (washed control); Percoll gradient; pentoxifylline; human follicular fluid (FF); pentoxifylline + FF; or platelet-activating factor (PAF) treatment. The AR of each group was assessed by fluorescein isothiocyanate-conjugated Pisum sativum agglutinin or Arachis hypogea agglutinin. Spermatozoa of washed control, pentoxifylline + FF, and PAF treated groups, with significantly higher AR rate than others, were injected into mature hamster oocytes. Spermatozoon-injected oocytes were cultured for 6, 9, 12, or 15 hours. Then they were stained with Toluidine blue for PN formation examination under a light microscope. Acrosome reaction rates of washed control, Percoll gradient, pentoxifylline, FF, pentoxifylline + FF, and PAF treated groups were 10.5% +/- 2.6%, 10.3% +/- 1.7%, 16.4% +/- 1.8%, 24.8% +/- 5.6%, 28.4% +/- 3.8%, and 33.3% +/- 5.2%, respectively. Pronuclear formation rate in washed control, pentoxifylline + FF, and PAF treated groups were 5.6% (3/54), 19.0% (11/58), and 18.9% (10/53) at 6 hours; 32.7% (18/55), 51.8% (29/56), and 57.4% (31/54) at 9 hours; 36.1% (22/61), 53.6% (30/56), and 50.0% (27/54) at 12 hours; and 47.2% (25/53), 64.8% (35/54), 53.6% (30/56) at 15 hours after ICSI. Pronuclear formation rate was significantly higher in pentoxifylline + FF, and PAF treated groups than that in the washed control group at 6 and 9 hours after ICSI. Pronuclear formation of oocytes takes place faster on those that were injected with acrosome-reacted spermatozoon than those injected with acrosome-intact spermatozoon. It could be concluded that induction of the AR of spermatozoa accelerates the time of PN formation and early development of the embryo in ICSI.

Influence of progesterone on boar sperm capacitation

This research investigates the effect of progesterone (P4) on boar sperm capacitation. Ejaculated spermatozoa were washed and incubated under capacitating conditions with or without P4. At different times of incubation samples of sperm were exposed to solubilized zonae pellucidae (ZP) and the degree of capacitation was evaluated by the incidence of zona-induced acrosome reaction (AR). The status of the acrosome was studied by using an FITC-conjugated lectin (Pisum sativum agglutinin; FITC-PSA). The effect of P4 on the fertilizing ability of semen was then evaluated in an in vitro fertilization system by exposing in vitro matured oocytes to sperm preincubated for 2 or 4 h with or without P4, under capacitating conditions. PSA staining showed that P4 does not affect the incidence of spontaneous AR. By contrast, spermatozoa incubated with P4 showed a higher percentage of AR than controls after the exposure to solubilized ZP. This enhanced reactivity to ZP suggests a direct effect of P4 on sperm capacitation. The in vitro fertilization assay was consistent with these results demonstrating a higher fertilizing ability in sperm preincubated with P4 than in controls while the steroid was without effect when added only during the fertilization step. These results demonstrate that P4 improves the fertilizing ability of boar semen essentially by facilitating the process of capacitation.

Acrosome reaction of human spermatozoa in zona-free hamster egg penetration test

The acrosomal status of human sperm during preparation for the process of zona-free hamster egg penetration test (ZFHEPT) was determined. The incidence of acrosome reaction (AR), as assessed by triple-stain technique, was significantly increased after 24 hours of incubation at 4 degrees C in TES-Tris (TEST)-yolk buffer, but the absolute values were relatively low (20% or less). Sperm from fertile donors and infertile patients with normal or abnormal semen analysis displayed similar capacity to undergo the AR in vitro. Although a positive correlation was found between the incidence of AR and the score of ZFHEPT, a remarkable individual variation was noted. The incidence of AR in freely swimming human sperm does not accurately reflect the fertilizing ability of the sperm.

Epididymal maturation and the acrosome reaction in mouse sperm: response to zona pellucida develops coincident with modification of M42 antigen.

Development of the sperm's capacity to interact with the zona pellucida was investigated at the stage when the acrosome reaction (AR) is induced. The response of epididymal sperm to agents that affect the occurrence of the AR was used to monitor maturational changes. Despite the finding that sperm from the three main epididymal regions were competent to undergo ARs induced by the divalent cation ionophore A23187 (56% AR, 74% AR, and 83% AR in caput, corpus, and cauda, respectively), the cells' responses to solubilized zonae pellucidae were different. When challenged with 5 zonae equivalents/microliter, both corpus and cauda sperm shed their acrosomes in high numbers (75% AR and 86% AR, respectively), whereas caput sperm did not (23% AR). Previous work has shown that the presence of M42 monoclonal antibody (mAb) during in vitro and in vivo fertilization inhibits sperm penetration through the zona pellucida by specific interference with zonae pellucidae-induced ARs. In this study, presence of the M42 mAb did not affect the incidence of A23187-induced ARs, whereas the zona-induced ARs that occurred in both corpus and cauda sperm were inhibited fully with M42 immunoglobulin (Ig) G. In addition, the antigen recognized by M42 mAb on sperm, termed M42 Ag, was examined during epididymal maturation. Although antigen localization appeared indistinguishable by immunofluorescence on sperm taken from the caput, corpus, and cauda regions of the epididymis, modification of this antigen during epididymal transit was detected. Equilibrium-binding studies using 125I-M42 IgG demonstrated a progressive increase during epididymal transit in the amount of M42 mAb that bound to fixed cells. Corpus and cauda sperm bound 185% and 240%, respectively, of the 125I-M42 IgG detected on caput sperm. These changes in expression of M42 Ag paralleled a structural change: the Mr of the antigen decreased from a 195,000/210,000 doublet in caput sperm to a 185,000/200,000 doublet in corpus and cauda sperm, as determined by immunoblot analysis of sodium dodecyl sulfate (SDS)-extracted sperm. Results presented here demonstrate that mouse sperm develop the capacity to undergo a zona-induced AR during epididymal maturation. The M42 antigen, which is involved in the zona-induced AR, is modified during epididymal transit coincident with development of the sperm's responsiveness to zonae. Our working hypothesis, based on these results, is that development of the sperm's capacity to undergo a physiological AR is related to modification of M42 Ag.

Glycosaminoglycans in ewe reproductive tracts and their influence on acrosome reactions in bovine spermatozoa in vitro.

Glycosaminoglycans (GAG) promote acrosome reactions (AR) in bovine and rabbit spermatozoa in vitro. Female bovine reproductive tract secretions contained GAG and the concentrations and composition of those GAG varied with different regions and stages of the estrous cycle. This study was designed to evaluate the types of GAG found in reproductive tract secretions of ewes at different stages of the estrous cycle and the ability of the secretions to promote AR. Ewes (n = 48) were slaughtered at 0, 12, 24, 36, 72 and 144 h following observations of standing estrus. Reproductive tracts were flushed with a modified Tyrode's medium (TALP: 7 ml) not containing calcium. Concentrations and composition of GAG in the tract were determined by high performance liquid chromatography. Concentrations of GAG decreased anterior to the cervix: 2.28 to .74 mg/100 mg protein (P less than .05). Increases in heparin-like GAG during the estrual phase and chondroitin sulfate GAG during the luteal phase were noted. Bovine sperm were incubated in tract flushings standardized to contain 4 micrograms/ml of GAG and supplemented with calcium. Sperm incubated in estrual flushings for 9 h exhibited a higher incidence of AR than those incubated in luteal flushings or control, 50% vs 36% and 29%, respectively (P less than .0001). It is concluded that during the estrous cycle there were changes in concentrations and composition of GAG in ewe reproductive tracts and the potencies of those female secretions causing AR varied.

The effects of washing and protein supplementation on the acrosome reaction of ram spermatozoa in vitro

Ram spermatozoa were incubated for 12 h at a concentration of 1–5 × 10 6 ml −1 in a modified BWW medium with TRIS (pH 8.0 in air, 308 mOsm) and a variety of pre-treatments were examined. These included washing in hypertonic (360 mOsm) medium by centrifugation and also supplementing with purified bovine serum albumin (BSA), with fatty acid-depleted BSA and with 5% v/ v heat-inactivated sheep serum. Motility was assessed by phase contrast microscopy, and the incidence of acrosome reactions among live spermatozoa was estimated from nigrosin-eosin live-dead smears. All treatments showed a steady decline in sperm motility with time, and progressive increases in the proportion of live, acrosome-reacted spermatozoa (% ARS). However, there were no significant differences between washing regimes except for the treatment with hypertonic medium., where % ARS was elevated significantly after 9 and 12 h incubation. No differences were seen in % ARS between the various protein supplementations, although serum promoted significantly better survival.

Effects of epididymal occlusion on sperm maturation in the hamster.

Hamster epididymal spermatozoa were isolated in the caput, corpus and cauda regions for one, two and three days. Sperm suspensions from these regions were assessed for morphology, motility, viability; and for fertility by A.I. The ability to capacitate was tested by checking for acrosome reactions when the sperm were cultured under defined capacitating conditions at 37 degrees C in vitro. Normal, mature cauda sperm were highly fertile (overall 77% of ova fertilized) and showed 50-80% incidence of acrosome reactions after six hours in culture. Isolation for up to three days had no effect. Immature sperm from the caput and corpus were poorly motile, infertile, and did not manifest an acrosome reaction. Isolation for one and two days produced improvements in motility, and distal migration of the cytoplasmic droplet skin to normal maturation, however the sperm remained infertile and did not capacitate in vitro. Survival after three days isolation was poor. Thus the development of fertilizing ability in hamster epididymal spermatozoa is closely related to the ability to manifest an acrosome reaction in vitro; however it is only poorly correlated with motility and morphology. Completion of sperm maturation in this species appears to require the environment of the cauda epididymidis.