Role of actin cytoskeleton in mammalian sperm capacitation and the acrosome reaction

In order to fertilize, the mammalian spermatozoa should reside in the female reproductive tract for several hours, during which they undergo a series of biochemical modifications collectively called capacitation. Only capacitated sperm can undergo the acrosome reaction after binding to the egg zona pellucida, a process which enables sperm to penetrate into the egg and fertilize it. Polymerization of globular (G)-actin to filamentous (F)-actin occurs during capacitation, depending on protein kinase A activation, protein tyrosine phosphorylation, and phospholipase D activation. F-actin formation is important for the translocation of phospholipase C from the cytosol to the sperm plasma membrane during capacitation. Prior to the occurrence of the acrosome reaction, the F-actin should undergo depolymerization, a necessary process which enables the outer acrosomal membrane and the overlying plasma membrane to come into close proximity and fuse. The binding of the capacitated sperm to the zona pellucida induces a fast increase in sperm intracellular calcium, activation of actin severing proteins which break down the actin fibers, and allows the acrosome reaction to take place.     

The association between CDC42 and caveolin-1 is involved in the regulation of capacitation and acrosome reaction of guinea pig and mouse sperm

In the mammalian sperm, the acrosome reaction (AR) is considered to be a regulated secretion that is an essential requirement for physiological fertilization. The AR is the all-or-nothing secretion system that allows for multiple membrane fusion events. It is a Ca(2)(+)-regulated exocytosis reaction that has also been shown to be regulated by several signaling pathways. CDC42 has a central role in the regulated exocytosis through the activation of SNARE proteins and actin polymerization. Furthermore, the lipid raft protein caveolin-1 (CAV1) functions as a scaffold and guanine nucleotide dissociation inhibitor protein for CDC42, which is inactivated when associated with CAV1. CDC42 and other RHO proteins have been shown to localize in the acrosome region of mammalian sperm; however, their relationship with the AR is unknown. Here, we present the first evidence that CDC42 and CAV1 could be involved in the regulation of capacitation and the AR. Our findings show that CDC42 is activated early during capacitation, reaching an activation maximum after 20 min of capacitation. Spontaneous and progesterone-induced ARs were inhibited when sperm were capacitated in presence of secramine A, a specific CDC42 inhibitor. CAV1 and CDC42 were co-immunoprecipitated from the membranes of noncapacitated sperm; this association was reduced in capacitated sperm, and our data suggest that the phosphorylation (Tyr14) of CAV1 by c-Src is involved in such reductions. We suggest that CDC42 activation is favored by the disruption of the CAV1-CDC42 interaction, allowing for its participation in the regulation of capacitation and the AR.     

Localization of phosphatidylserine in boar sperm cell membranes during capacitation and acrosome reaction

One of the essential properties of mammalian, including sperm, plasma membranes is a stable transversal lipid asymmetry with the aminophospholipids, phosphatidylserine (PS) and phosphatidylethanolamine (PE), typically in the inner, cytoplasmic leaflet. The maintenance of this nonrandom lipid distribution is important for the homeostasis of the cell. To clarify the relevance of lipid asymmetry to sperm function, we have studied the localization of PS in boar sperm cell membranes. By using labeled annexin V as a marker for PS and propidium iodide (PI) as a stain for nonviable cells in conjunction with different methods (flow cytometry, fluorescence and electron microscopy), we have assessed the surface exposure of PS in viable cells during sperm genesis, that is, before and during capacitation as well as after acrosome reaction. An approach was set up to address also the presence of PS in the outer acrosome membrane. The results show that PS is localized in the cytoplasmic leaflet of the plasma membrane as well as on the outer acrosome membrane. Our results further indicate the cytoplasmic localization of PS in the postacrosomal region. During capacitation and acrosome reaction of spermatozoa, PS does not become exposed on the outer surface of the viable cells. Only in a subpopulation of PI-positive sperm cells does PS became accessible upon capacitation. The stable cytoplasmic localization of PS in the plasma membrane, as well as in the outer acrosome membrane, is assumed to be essential for a proper genesis of sperm cells during capacitation and acrosome reaction.     

Fate of lactadherin P47 during post-testicular maturation and capacitation of boar spermatozoa

Polyclonal avian antibody was used partially to characterize the pig sperm lactadherin P47. P47 is a mosaic protein, composed of two epidermal growth factor (EGF)-like domains and two C1/C2 domains. P47 is homologous to the bovine mammary gland protein MGP 53/57 and mouse milk fat globule protein. Expression of P47 along the male genital tract and its localization on spermatozoa during post-testicular maturation and capacitation were studied. P47 was detected in the testis and in all parts of the epididymis by immunohistochemistry and by western blots of tissue extracts. By indirect immunocytochemistry, P47 was localized at the apical ridge of the sperm head in testicular, epididymal and ejaculated spermatozoa. The fluorescence intensity progressed during sperm transit from caput to cauda epididymis, probably caused by the ongoing expression and subsequent accumulation of P47 on the sperm surface. During the time course of capacitation, P47 appears to be unmasked by the release of coating proteins and appears to migrate from the apical ridge onto the entire acrosomal region, showing an intensive fluorescence pattern after 3 h capacitation in vitro. The kinetics of signal changes during in vitro capacitation were different in epididymal and ejaculated spermatozoa, indicating accelerated capacitational plasma membrane destabilization in epididymal spermatozoa.     

The slower the better: how sperm capacitation and acrosome reaction is modified in the presence of estrogens

In order for mammalian sperm to obtain a fertilizing ability, they must undergo a complex of molecular changes, called capacitation. During capacitation, steroidal compounds can exert a fast nongenomic response in sperm through their interaction with plasma membrane receptors, and activate crucial signaling pathways leading to time-dependent protein tyrosine phosphorylation (TyrP). Estrogen receptor beta was detected in epididymal mouse sperm; therefore, the effect of 17B-estradiol, estrone, estriol, and 17A-ethynylestradiol on mouse sperm capacitation in vitro was investigated. The effect was evaluated by positive TyrP in sperm heads and in the whole sperm lysates. Simultaneously, the state of the acrosome after the calcium ionophore-induced acrosome reaction was assessed. Generally, estrogens displayed a time and concentration-dependent stimulatory effect on sperm TyrP during capacitation. In contrast, the number of sperm that underwent the acrosome reaction was lower in the experimental groups. It has been demonstrated that both natural and synthetic estrogens can modify the physiological progress of mouse sperm capacitation. The potential risk in the procapacitation effect of estrogens can also be seen in the decreased ability of sperm to undergo the acrosome reaction. In conclusion, the capacitating ability of sperm can be significantly lowered by increasing the level of estrogens in the environment.     

Changes in oocyte plasma membrane binding sites on boar spermatozoa with capacitation and acrosome reactions

The objective of this study was to determine the localization and distribution of oocyte plasma membrane binding sites on capacitated and acrosome-reacting live boar spermatozoa. Localization of oocyte plasma membrane binding sites on boar spermatozoa was determined with fluorescence microscopy and population distribution was examined with flow cytometry. The number of spermatozoa with oocyte plasma membrane bound to the equatorial segment and postacrosomal region of the sperm head significantly increased with capacitation. Equatorial segment labelling further increased with induced acrosome reactions. When the population distribution of oocyte plasma membrane binding sites on live boar spermatozoa was analysed, the percentage of spermatozoa with bound oocyte plasma membrane significantly increased after capacitation compared with that of washed spermatozoa. Binding of oocyte plasma membrane did not increase in control spermatozoa incubated under non-capacitating conditions and was not correlated with the percentage of dead spermatozoa. A change in localization of oocyte plasma membrane binding sites on the sperm head was demonstrated using fluorescence microscopy and an increase in oocyte plasma membrane binding sites after capacitation was shown using flow cytometry.     

Acrosomal integrity and capacitation are not influenced by sperm cryopreservation in the giant panda

Sperm cryopreservation and artificial insemination are important management tools for giant panda breeding and the preservation of extant genetic diversity. This study examined the influence of freeze-thawing on sperm function, specifically capacitation. Sperm from nine giant pandas were assessed before and after rapid (- 40 and - 100 degrees C/min) cryopreservation by incubation in HEPES-buffered Ham's F10 medium with and without the capacitation accelerators, 3-isobutyl-1-methylxanthine (IBMX) and dibutyryl cyclic AMP (dbcAMP). At 0, 3 and 6 h of exposure, aliquots were assessed for sperm motility traits and capacitation, defined as the proportion of sperm with intact acrosomes following exposure to solubilised zonae pellucidae (ursid or felid) or calcium ionophore subtracted from the proportion of sperm with intact acrosomes before exposure. Although mean+/-S.E.M. sperm motility post-thaw (56.1 +/- 3.9% at 0 h) was less (P < 0.05) than pre-freeze (71.7 +/- 6.0%), there was no difference (P > 0.05) in the proportion of acrosome-intact sperm (fresh, 93.0 +/- 1.7% versus cryopreserved-thawed, 81.7 +/- 4.7% at 0 h). Incidence of capacitation was greater (P < 0.05) in fresh sperm incubated with capacitation accelerators IBMX and dbcAMP (9 h: 50.9 +/- 1.1) compared with fresh sperm incubated without accelerators (9 h: 41.2 +/- 1.1%). Frozen-thawed sperm preincubated without accelerators underwent capacitation (49.6 +/- 1.1%) to a greater extent (P < 0.05) compared with these fresh counterparts. Thawed samples with (9 h: 45.9 +/- 1.4%) and without accelerators (9 h: 41.2 +/- 1.1%) did not differ (P > 0.05) during the 9-h incubation. We conclude that giant panda spermatozoa (1) undergo capacitation in vitro with or without chemical accelerators and (2) withstand a rapid cryopreservation protocol, including retaining normal acrosomal integrity and functional capacitation ability.     

Evaluation of calcium-free Tyrode's sperm capacitation medium for use in bovine in vitro fertilization

Our objective was to determine if a bovine sperm capacitation technique, developed with zona-free hamster oocytes, could be used for the in vitro fertilization of in vitro matured bovine zona-intact oocytes. Bovine cumulus-enclosed primary oocytes from 2- to 5-mm follicles were matured in tissue culture Medium 199 containing Earle's salts and bicarbonate and supplemented with 10% fetal calf serum, FSH (10 micrograms/ml), and estradiol-17 beta (1.5 microgram/ml) for 24 h at 37 degrees C under paraffin oil. Ejaculated bovine sperm, washed thrice in bovine serum albumin-saline (pH 7.6) and capacitated for 4 h in Ca(++)-free Tyrode's medium (pH 7.6), were diluted to 2 x 10(6) sperm/ml in Medium 199 supplemented with 10% fetal calf serum. Oocytes were added (10/500 microliters droplet) to this medium containing the capacitated sperm, freeze-thawed killed sperm, or no sperm and incubated for 8 h before transfer to fresh medium and then incubated for 40 h. At the end of each incubation, a portion of the oocytes were stained and evaluated for development or fertilization. After 24 h of culture, 49% of the oocytes had matured (metaphase II). Fertilization rates were 55.6% after exposure of all oocytes to Ca(++)-free Tyrode's capacitated sperm and 82.5% if only metaphase II oocytes were selected. The parthenogenetic controls were negative (1.4% and 0%). Therefore, the Ca(++)-free Tyrode's sperm capacitation technique can be used for bovine in vitro fertilization studies.     

Evaluation of a TEST-yolk sperm capacitation system for use in bovine in vitro fertilization.

Bovine sperm acquire the ability to penetrate zona-free hamster oocytes (capacitation) after incubation in TEST-yolk buffer. Our objective was to determine whether such sperm could penetrate zona-intact bovine oocytes in vitro. Bovine cumulus enclosed oocytes from 2- to 5-mm follicles were incubated in maturation medium for 24 h at 37 degrees C. Ejaculated bovine semen was diluted 1: 10 in TEST-yolk buffer, cooled to 4 degrees C, and stored for 8 h to induce capacitation. Sperm were then washed thrice in pH 7.6, .15 M NaCl containing .1% bovine serum albumin V (37 degrees C) and diluted to 2 x 10(6) sperm/ml in fertilization medium. Droplets of fertilization medium containing capacitated sperm, killed sperm, or no sperm were made under paraffin oil. Oocytes (matured 24 h) were added and cocultured with sperm for 8 h and then transferred to fresh fertilization medium for 40 h. After 24 h, 53% of the oocytes had matured (metaphase II). The fertilization rate of the metaphase II oocytes (203) with TEST-yolk capacitated sperm was 87%, whereas the parthenogenetic controls were 2 and 0%, respectively. Therefore, TEST-yolk buffer can be used to capacitate bull sperm for in vitro fertilization.     

Hypotonic resistance of boar spermatozoa: sperm subpopulations and relationship with epididymal maturation and fertility

Hypotonic resistance of boar spermatozoa was investigated by measuring the ratio of live/dead spermatozoa (SYBR-14/propidium iodide) by flow cytometry after hypotonic stress. The survival rate of ejaculated spermatozoa incubated in hypotonic solutions ranging from 3 to 330 mmol/kg followed a sigmoid curve that fitted a simple logistic model. The critical osmolality value (Osm(crit)) at which 50% of spermatozoa died was determined with this model. Hypotonic resistance of spermatozoa increased with temperature between 15 and 39 degrees C and decreased after hydrogen superoxide treatment, but was not modified during 8 days of preservation in Beltsville thawing solution. Hypotonic resistance markedly decreased during epididymal maturation and after ejaculation as Osm(crit) at 15 degrees C was 54.7+/-3.2, 68.5+/-10.6, 116.7+/-2.1 and 194.3+/-3.7 mmol/kg for the caput, corpus, cauda and ejaculated spermatozoa respectively. Hypo-osmotic stress of 100 mmol/kg revealed a sperm subpopulation exhibiting increased hypotonic resistance compared with the whole ejaculate (Osm(crit)=67.8+/-2.1 mmol/kg). Consistent differences were observed between lean and standard breeds (Pietrain versus Large White) and between boars within the same breed. According to data collected by artificial insemination centers during a large-scale field trial, hypotonic resistance of ejaculates was found to be positively correlated with in vivo fertility.     

Induced hyperactivity in boar spermatozoa and its evaluation by computer-assisted sperm analysis

Hyperactivity, a form of sperm motility characterized by vigorous flagellar movements, has been proposed as essential for fertilization in mammals. The objective of the present study was to establish a method for inducing hyperactivity in vitro in boar spermatozoa and to define threshold values to differentiate between hyperactive and non-hyperactive spermatozoa by computer-assisted sperm analysis (CASA) as a prerequisite for analyzing the energy metabolism during hyperactivity. In TALP-HEPES medium, non-frozen boar spermatozoa were stimulated to hyperactivity by 50 micromol l(-1) Ca2+ within 15 min at 37 degrees C if 5 micromol l(-1) of the Ca2+ ionophore A23187 was present. If 25% seminal plasma was present, boar spermatozoa required higher Ca2+ concentrations (about 700 micromol l(-1)) for hyperactivity. Under both conditions, immobilization and head-to-head agglutination were low so that hyperactive spermatozoa could be analyzed for at least 40 min. The transition from normal to hyperactive movement was characterized by an increase in flagellar beat angle from 49 degrees +/- 12 degrees to 200 degrees +/- 36 degrees (n = 32) and a decrease in flagellar curvature ratio from 0.89 +/- 0.04 to 0.47 +/- 0.11 (n = 32). For quantification of hyperactive boar sperm, kinematic parameters of hyperactive and non-hyperactive spermatozoa were measured by CASA and statistically evaluated (receiver operating characteristic (ROC) curve analysis). The threshold values of the following four parameters were well suited for differentiating between hyperactive and non-hyperactive boar spermatozoa (ROC curve analysis: > 50% specificity at 100% sensitivity). Hyperactive boar spermatozoa showed mean lateral head displacement > 3.5 microm, curvilinear velocity > 97 microm s(-1), linearity < 32% and wobble < 71%. According to this multiparametric definition, induction of hyperactivity increased significantly (P < 0.0001) the fraction of hyperactive spermatozoa in semen samples from 5.1 +/- 4.3% (n = 13) to 48.3 +/- 6.6% (n = 7) in the absence and to 44.2 +/- 7.6% (n = 10) in the presence of 25% seminal plasma, while the overall percentage of motile spermatozoa did not change significantly.     

Effect of estrogen on leptin and expression of leptin receptor transcripts in prepubertal dairy heifers

Plasma leptin concentrations increase as growing dairy heifers approach puberty and have greater plasma estrogen. In intact and ovariectomized rodents, estrogen has been shown to modulate expression of leptin and its receptor (Ob-R). To determine if estrogen regulates the bovine leptin system, prepubertal dairy heifers were ovariectomized at 140 d of age or left intact. A month later, both groups received a subcutaneous injection of excipient or 17β-estradiol for 3 consecutive days. Neither ovarian status nor 17β-estradiol injection altered plasma leptin or leptin mRNA abundance in adipose tissue depots. To assess whether these factors affected Ob-R expression, we tested 20 bovine tissues for leptin receptor (Ob-R) by using quantitative real-time PCR assays for the short receptor isoform (Ob-Ra), the long receptor isoform (Ob-Rb), and all receptor isoforms (Ob-R_TOTAL). Ob-R_TOTAL was detected in all tissues, with copy numbers covering 3 orders of magnitude between the lowest and highest expressing tissues (kidney cortex vs. liver). The Ob-Rb isoform accounted for 40% of Ob-R_TOTAL in the hypothalamus, but averaged less than 3% of Ob-R_TOTAL in peripheral tissues. Reciprocally, Ob-Ra accounted for only 19% of Ob-R_TOTAL in the hypothalamus and for nearly all of Ob-R_TOTAL in most peripheral tissues. Finally, we evaluated the effects of ovarian status and 17β-estradiol on Ob-R expression in selected tissues. Treatment with 17β-estradiol reduced Ob-R_TOTAL, Ob-Rb, and Ob-Ra expression by 70% in the uterine endometrium and tended to do the same in mammary adipose tissue. There was no effect of 17β-estradiol on Ob-R in the hypothalamus, liver, soleus muscle, or subcutaneous adipose tissue. We conclude that greater estrogen secretion does not cause increased plasma leptin in prepubertal dairy heifers but estradiol can modulate Ob-R expression in some estrogen-responsive tissues.     

Regulation of acrosome reaction of fowl spermatozoa: evidence for the involvement of protein kinase C and protein phosphatase-type 1 and/or -type 2A

The signal transduction pathways involved in the regulation of the acrosome reaction and motility of fowl spermatozoa were investigated. The motility and acrosomal integrity of fowl spermatozoa in TES/NaCl buffer, with or without homogenised inner perivitelline layers (IPVL), prepared from laid fowl eggs, was almost negligible at 40 degrees C. In the presence of 2 mmol CaCl(2)/l at 40 degrees C, motility became vigorous and the acrosome reaction was stimulated when IPVL was added. In the absence of Ca(2+), motility was stimulated by the addition of calyculin A and okadaic acid, both specific inhibitors of protein phosphatase-type 1 (PP1) and -type 2A (PP2A), but Okadaic acid, which is a weaker inhibitor of PP1, did not completely restore motility at 40 degrees C. However, the acrosome reaction was significantly and equally stimulated in a dose-dependent manner by both inhibitors in the range of 10-1000 nmol/l, when spermatozoa were incubated with IPVL but without Ca(2+). These inhibitors did not stimulate the acrosome reaction in the absence of IPVL. The vigorous motility of spermatozoa, stimulated by the addition of Ca(2+), was reduced gradually as the concentrations of SC-9, a selective activator of protein kinase C (PKC), were increased and a similar SC-9-induced inhibition was observed in the acrosome reaction in the presence of Ca(2+) and IPVL. These results confirm that IPVL is necessary for the activation of the acrosome reaction in fowl spermatozoa and that Ca(2+) plays an important role in the stimulation of motility and acrosomal exocytosis. Furthermore, it appears that the intracellular molecular mechanisms for the regulation of acrosome reaction of fowl spermatozoa are different from those for the restoration of motility, i.e., protein dephosporylation involving PP1 and/or PP2A in the former, and PP1 alone in the latter case. In addition, the activation of PKC may contribute to a decrease in the flagellar movement and acrosome reaction of fowl spermatozoa.     

Segregation of spermatozoa within sperm storage tubules of fowl and turkey hens

In avian species, spermatozoa reside in the oviduct for prolonged periods in specialized structures known as sperm storage tubules, but little is known about the relative distribution of spermatozoa in these tubules after successive inseminations by different males. The staining efficacies of various fluorescent dyes for fowl and turkey spermatozoa were evaluated to investigate one proposed mechanism of sperm competition. Hens were then inseminated at different intervals with stained and unstained spermatozoa to observe the spatial distribution of spermatozoa within the storage tubules. Several novel fluorescent lipophilic tracers that successfully stain mammalian spermatozoa either did not stain fowl or turkey spermatozoa, or greatly impaired sperm motility. In contrast, Hoechst 33342 readily stained sperm nuclei (fowl: 25 nmol l-1; turkey: 77 nmol l-1) within 4 h without inhibiting sperm motility, or affecting fertility or the hatching ability of the eggs. Hens were tandemly inseminated with equal numbers of stained or unstained spermatozoa at 24 h intervals and were killed 24 h after the final insemination to study sperm entry and storage within the tubules. Oviductal mucosa containing sperm storage tubules was removed, and individual tubules were classified as containing stained spermatozoa, unstained spermatozoa, a mixture of stained and unstained spermatozoa, or as not containing spermatozoa. Results from the present study indicate that spermatozoa from two different inseminations generally segregate into different storage tubules in both fowl and turkey hens. Storage tubules containing mixed populations of spermatozoa were found in only 4% of fowl and 12% of turkey storage tubules examined. Thus, the mechanism of last-male precedence does not appear to be due to the stratification of spermatozoa within the tubules.     

Pre-treatment of sperm reduces success of ICSI in the pig

In pigs, although ICSI is a feasible fertilization technique, its efficiency is low. In general, injected pig sperm are insufficient to induce oocyte activation and embryonic development. Pretreatments for disrupting sperm membranes have been applied to improve the fertility of ICSI oocytes; however, we hypothesize that such pretreatment(s) may reduce the ability of the sperm to induce oocyte activation. We first evaluated the effects of sperm pretreatments (sonication (SO) to isolate the sperm heads from the tails, Triton X-100 (TX), and three cycles of repeated freezing/thawing (3×-FT) for disrupting sperm membranes) on the rate of pronucleus (PN) formation after ICSI. We found that oocytes injected with control (whole) sperm had higher rates of PN formation than those obtained after subjecting the sperm to SO, TX, and 3×-FT. The amounts of phospholipase Cζ (PLCζ), which is thought to be the oocyte-activating factor in mammalian sperm, in sperm treated by each method was significantly lower than that in whole untreated sperm. Furthermore, using immunofluorescence, it was found that in pig sperm, PLCζ was localized to both the post-acrosomal region and the tail area. Thus we demonstrated for the first time that sperm pretreatment leads to a reduction of oocyte-activating capacity. Our data also show that in addition to its expected localization to the sperm head, PLCζ is also localized in the tail of pig sperm, thus raising the possibility that injection of whole sperm may be required to attain successful activation in pigs.     

Effect of platelet-activating factor (PAF) on stallion sperm motility, capacitation and the acrosome reaction

Phospholipids are an essential component of all mammalian cells; platelet activating factor (PAF=1-O-alkyl-acetyl-sn-glycero-3-phosphocholine) is a signalling phospholipid that has many biological properties in addition to platelet activation. PAF receptors have been detected on stallion spermatozoa; therefore, the aim of this study was to evaluate the effect of synthetic PAF on the motility, capacitation and the acrosome reaction of stallion spermatozoa. Treatment of ten stallion semen samples with 10(-4)-10(-13) mol PAF l(-1) resulted in significant differences in motility and capacitation (r(2)=0.81 and 0.83, respectively). Statistical analysis indicated that PAF also has an effect on acrosome reaction (r(2)=0.20). PAF concentrations, incubation time and their interaction had a highly significant (P<0.01) effect on motility. After capacitation in vitro with PAF, and induction of the acrosome reaction by progesterone, transmission electron microscopy was conducted on the spermatozoa of three stallions to detect the true acrosome reaction. Differences in PAF concentrations were highly significant (r(2) for intact: 97.2; reacted: 89.8; and vesiculated: 98.1). The results indicate that a lower concentration of PAF enhances motility and induces capacitation of stallion spermatozoa, whereas a higher concentration of PAF induces the acrosome reaction.     

Effect of osmolality and glycosaminoglycans on motility, capacitation, acrosome reaction, and in vitro fertilizability of bovine ejaculated sperm

Bovine ejaculated semen was placed in a modified Tyrode's medium with albumin, lactate, and pyruvate. The sperm were washed three times and subjected to nine treatment in a 3 X 3 factorial arrangement. Treatments consisted of osmolality (exposure to 380 mOsmol/kg medium for 5 min, exposure to 340 or 295 mOsmol/kg medium for the entire incubation period), and the presence or absence of glycosaminoglycans (100 micrograms/ml chondroitin sulfate A or 10 micrograms/ml heparin). Sperm were examined at 4.5 h, 8 to 9 h, and 24 to 25 h of incubation (37 degrees C, 5% CO2, and 95% air). Heparin caused head-to-head agglutination of sperm, raised the percent sperm without seminal antigens over the acrosome (capacitated) by 20% at 4.5 h, and doubled the percent of acrosome-reacted sperm. However, this stimulation did not improve in vitro fertilizability. Chondroitin sulfate A tended to maintain motility, but did not affect capacitation or the acrosome reaction, possibly due to glucose inhibition. Both high osmolality treatments tended to reduce motility, especially after 24 h of incubation when the 340 osmolality treatment reduced motility by 14% over the 295 treatment. No consistent effect on capacitation was observed. The 340 and 380 osmolality treatments induced 8.6 and 6.1% more acrosome reactions by 24 h than the 295 treatment. The 340 mOsmol/kg treatment yielded insignificantly higher in vitro fertilization rates, as evidenced by development of zygotes to the two-cell stage. Lack of statistical significance was due to high variation with in vitro fertilization rates.