Capacitation of hamster spermatozoa with the divalent cation chelators D-penicillamine,L-histidine,and L-cysteine in a protein-free culture medium

By using a chemically defined (protein-free) culture medium that supports sperm viability but not capacitation or the acrosome reaction, we have determined that hamster spermatozoa can be chemically capacitated in vitro by the divalent cation chelators D-penicilla-mine, L-histidine, and L-cysteine in the absence of bovine serum albumin (BSA). Washed cauda epididymal spermatozoa were preincubated (1–2 × 10⁶ sperm/ml) for 3, 4, or 6 hr at 37°C in 5% CO₂ in air. The basic culture medium used for sperm preincubation and for sperm:egg coincubation was a modified Tyrode's solution (protein-free) containing 10 mM sodium lactate, 100 μM sodium pyruvate, and 1.0 mg/ml polyvinylalcohol (TLP-PVA). Sperm viability was maintained in all preincubation and coincubation media with PHE (20 μM D-penicillamine, 100 μM hypotaurine, and 1.0 μM epinephrine). The low control sperm preincubation medium consisted of TLP-PVA. In some cases the high control preincubation medium also contained 3 mg/ml BSA (TALP-PVA). The experimental preincubation medium was TLP-PVA with additional D-penicillamine (125 or 500 μM), or L-histidine (10, 100, or 1,000 μM) or L-cysteine (25, 75, or 125 μM). After preincubation, sperm were coincubated (2 × 10⁴ sperm/ml) with cumulus-free hamster eggs in TALP-PVA ± additional D-penicillamine, L-histidine, or L-cysteine for 1.5 hr, fixed, and evaluated for percent egg penetration as an index of sperm capacitation. The results demonstrate that hamster spermatozoa can be chemically capacitated in vitro with D-penicillamine (500 μM: range of mean penetration values, 53.6%–84.3%), L-histidine (100 μM: range of mean values, 24.8%–56.3%) or L-cysteine (75 μM: 51.3%) in the absence of exogenous protein.     

Effects of various lipids on the acrosome reaction and fertilizing capacity of guinea pig spermatozoa with special reference to the possible involvement of lysophospholipids in the acrosome reaction

The effects of lipids on the survival, acrosome reaction, and fertilizing capacity of guinea pig spermatozoa were studied by incubating the spermatozoa in media containing various concentrations of the lipids. Lipids tested were: phosphatidyl-choline (PC), -ethanolamine (PE), -inositol (PI), -serine (PS), sphingomyelin (S), cholesterol (C), lysophosphatidyl-choline (LC), -ethanolamine (LE), -inositol (LI), -serine (LS), and glyceryl monooleate (M). When spermatozoa were incubated in a regular medium (containing 2 mM Ca²⁺) with M, the majority underwent the acrosome reaction within 1 hour. None of the other lipids were as effective as M, and some were totally ineffective under the same conditions. However, when spermatozoa were preincubated in Ca²⁺-free medium containing LC, LE, or LI, they gained the ability to undergo the acrosome reaction. One hour of preincubation in Ca²⁺-free medium with LC, LE, or LI was enough to render the vast majority of spermatozoa capable of undergoing the acrosome reaction in response to Ca²⁺. The optimum concentrations for LC, LE, and LI were approximately 85 μg/ml, 210 μg/ml, and 140 μg/ml, respectively. Spermatozoa that had undergone the acrosome reaction by pretreatment with LC, LE, or LI remained actively motile and were capable of fertilizing eggs. LS was totally ineffective in rendering the spermatozoa capable of undergoing the acrosome reaction, and in fact it inhibited the acrosome reaction by itself and also inhibited the LC-, LE-, or LI-mediated acrosome reaction. LS did not prevent acrosome-reacted spermatozoa from penetrating the zona pellucida, but did prevent sperm-egg fusion. Based on these findings, it is suggested that lysophospholipids are intricately involved in the sperm acrosome reaction and perhaps in sperm-egg fusion.     

Induction of the acrosome reaction in guinea pig spermatozoa by calmodulin antagonist W-7

The effect of the calmodulin antagonist W-7 on the capacitation and the acrosome reaction of guinea pig spermatozoa was examined. The characteristic features of the acrosome reaction induced by W-7 were the dependence on the composition and pH of the medium and on the presence of sodium bicarbonate. The most effective concentration of W-7 for inducing the acrosome reaction was approximately 5 μM, which is far less than the Kd for calmodulin. Moreover, W-7 enhanced the ability of spermatozoa to acquire capacitation in a Ca²⁺-free medium. The spermatozoa induced to undergo the acrosome reaction by W-7 were capable of penetrating the zona-free hamster eggs. W-5, which has a lower affinity for calmodulin than W-7, also induced the acrosome reaction in the same manner as W-7. These results suggest that the naphthalenesulfonamide derivatives W-7 and W-5 can induce the acrosome reaction in guinea pig spermatozoa via capacitation in a pH-dependent, Ca²⁺-calmodulin-independent manner.     

Effect of hen's egg yolk on capacitation and acrosome reaction of diluted canine spermatozoa

The aim of this study was to investigate the influence of progesterone, cholesterol and calcium (Ca²⁺) in an egg-yolk-containing extender on capacitation and acrosome reactions (AR) of diluted canine spermatozoa during 4 days of cooled-storage. For this purpose, we first investigated the effect of supplementation of a Tris–citrate–fructose buffer (TCF) with progesterone in a final concentration of 0.1, 0.2 and 1.0 μg progesterone/ml TCF-diluted semen. We then compared the effects of TCF and the same buffer-containing 20% egg yolk (TCF–EY). In egg yolks and the TCF–EY, progesterone was measured by enzyme immunoassay, cholesterol by enzymatic colorimetry and Ca²⁺ by flame atomic absorption spectrophotometry. For both experiments, ejaculates from eight dogs were used. For the comparison of diluents, one ejaculate was divided and one half diluted with TCF, the other with TCF–EY. One half of each TCF- and TCF–EY-diluted sample was evaluated immediately (D1), the other after storage for 4 days at +4 °C (D4). In diluted semen, motility and viability were measured by a computer assisted sperm analyzer (CASA; Sperm Vision, Minitüb, Germany), capacitation and AR were evaluated with a modified chlortetracycline assay (CTC) and the AR additionally by flow cytometry. Results: Supplementation of progesterone revealed, that between D1 and D4, total and progressive motility decreased with all progesterone concentrations, while viability as well as percentage of capacitated and acrosome reacted spermatozoa stayed constant. Progesterone-, cholesterol- and Ca²⁺ concentrations in egg yolks were 524.8 ± 131.4 ng/g, 13.9 ± 2.03 mg/g and 1.27 ± 0.17 mg/g, respectively. In the TCF–EY-diluent, the respective values were 210.9 ng/g, 2.52 mg/g and 1.1 mg/g. In TCF–semen, at D1, motility and viability were significantly higher than in TCF–EY-samples (p < 0.05), however at D4, no significant differences were detectable. Further, in TCF–semen, percentages of spermatozoa with intact membranes decreased significantly (p < 0.05) and capacitated spermatozoa increased (p < 0.05), which was not seen in TCF–EY-samples. In all samples, low percentages of AR were detected and after 4 days, the highest value of AR in TCF–EY-samples was 5.3% on average, as detected by flow cytometry. We therefore conclude that progesterone from egg yolk in routine extenders does not substantially influence semen longevity or AR of canine semen during cold-storage for 4 days. In contrary, egg yolk seems to prevent a significant increase in capacitated spermatozoa.     

Development of ability to penetrate the cumulus oophorus by hamster spermatozoa capacitated in vitro,in relation to the timing of the acrosome reaction

Hamster spermatozoa were tested for their ability to penetrate the intact cumulus matrix at low sperm:egg ratios (approximately 3:1). Uncapacitated spermatozoa attached to the surface of the cumulus and could not penetrate. Spermatozoa capacitated in vitro began to be able to penetrate after about 2 hr of preincubation, coincidentally with the first appearance of hyperactivation and spontaneous acrosome reactions. In all, 628 in vitro incubated spermatozoa were evaluated on and in cumuli: 270 could penetrate, but only ten of these were judged to have intact, “unmodified” acrosomes. Almost all spermatozoa capable of penetrating showed optically “modified” and swelling acrosomal caps, and this confirms previous observations on cumulus penetration in vivo. Penetration appeared limited to a phase in capacitation prior to completion of the acrosome reaction, as spermatozoa that had lost the acrosomal cap penetrated poorly and showed reduced viability. Penetration of the cumulus was inhibited by the hyaluronidase inhibitor sodium aurothiomalate. Cumulus penetrating ability could result either from a change in surface properties of the sperm at capacitation, which renders them less “sticky” to the matrix, or from release or activation of a “cumulus lysin.” We conclude that the ability to enter the cumulus matrix coincides with physiological changes in spermatozoa that occur during a terminal phase of capacitation preceding complete loss of the acrosomal cap, and that initiation of this process in vivo must precede sperm-zona contact.     

Capacitation and the acrosome reaction in squirrel monkey (Saimiri sciureus) spermatozoa evaluated by the chlortetracycline fluorescence assay

Capacitation and the acrosome reaction in squirrel monkey seminal spermatozoa diluted in Tyrode's medium (TALP) and TC-199 were monitored by a chlortetracycline (CTC) fluorescence assay. Four CTC patterns, similar to those found in human sperm, were readily characterized by fluorescent staining on the heads of the spermatozoa. The appearance of the capacitated (CP) pattern was dependent on the concentration of the bovine serum albumin. Acrosomal loss was observed in a maximum of 15% of the sperm in the populations studied here. Calcium ionophore A23187 (5 μM to 20μM) induced acrosomal loss in 60–70% of capacitated spermatozoa. However in freshly ejaculated sperm incubated under capacitating conditions or in spermatozoa incubated in Ca^{+ +}-free medium, A23187 failed to induce acrosomal loss. Furthermore, spermatozoa incubated in the presence of seminal plasma or spermatozoa obtained following a 1-hour “swim-up” procedure showed an identical timecourse of appearance of the CP pattern, indicating the lack of effect of seminal plasma on capacitation in the squirrel monkey.     

Inhibition of domestic cat spermatozoa acrosome reaction and zona pellucida penetration by tyrosine kinase inhibitors

Spermatozoa from teratospermic domestic cats (>60% morphologically abnormal spermatozoa per ejaculate) consistently exhibit lower levels of oocyte penetration in vitro than their normospermic (<40% abnormal spermatozoa per ejaculate) counterparts. This could be caused by structural abnormalities or intracellular defects resulting in disruption of normal cellular functions. Spermatozoa from teratospermic cats also are compromised in the ability to capacitate and undergo the acrosome reaction (AR) in vitro. Further, we recently identified two tyrosine phosphorylated proteins (95- and 160-kDa) localized over the acrosome region in domestic cat spermatozoa. Phosphorylation of these proteins is reduced in teratospermic compared with normospermic ejaculates. To begin to understand the relationship between tyrosine phosphorylation and sperm function, we examined the effects of two protein tyrosine kinase inhibitors (tyrphostin RG-50864 and genistein) on (1) sperm motility; (2) protein tyrosine phosphorylation; (3) the ionophore A23187-induced AR; (4) the spontaneous and zona pellucida (ZP)–induced AR, and (5) the ability of spermatozoa from normospermic cats to penetrate conspecific ZP-intact oocytes. Over a wide range of concentrations, neither inhibitor affected sperm percentage motility during incubation (P > 0.05). Preincubation with either inhibitor reduced tyrosine phosphorylation of both (95- and 160-kDa) sperm proteins. Although both inhibitors blocked the ZP-induced AR, neither influenced the spontaneous AR nor the A23187-induced AR, suggesting that tyrosine phosphorylation may be involved in physiologic AR. No differences (P > 0.05) were observed in the ability of control or inhibitor-treated spermatozoa to bind to or penetrate the outer ZP layer. However, percentages of oocytes with treated spermatozoa in the inner ZP (tyrphostin, 8.7%; genistein, 20.4%) and perivitelline space (tyrphostin, 0%; genistein, 2.3%) were less (P < 0.001) than untreated controls (inner ZP, 62.7%; perivitelline space, 10.2%). These results (1) demonstrate that ZP-induced acrosomal exocytosis in domestic cat spermatozoa is regulated via a tyrosine kinase–dependent pathway and (2) suggest that defects in these signaling pathways may represent one of the causes for compromised sperm function in teratospermic males. Mol. Reprod. Dev. 49:48–57, 1998. © 1998 Wiley-Liss, Inc.     

A molecular membrane model of sperm capacitation and the acrosome reaction of mammalian spermatozoa

The abundance of data pertaining to the metabolism of lipids in relation to mammalian fertilization has warranted an effort to assemble a molecular membrane model for the comprehensive visualization of the biochemical events involved in sperm capacitation and the acrosome reaction. Derived both from earlier models as well as from current concepts, our membrane model depicts a lipid bilayer assembly of space-filling molecular models of sterols and phospholipids in dynamic equilibrium with peripheral and integral membrane proteins. A novel feature is the possibility of visualizing individual lipid molecules such as phosphatidylcholine, phosphatidylethanolamine, lysophospholipids, fatty acids, and free or esterified cholesterol. The model illustrates enzymatic reactions which are believed to regulate the permeability and integrity of the plasma membrane overlying the acrosome during interactions between the male gamete and capacitation factors present in fluids of the female genital tract. The use of radioactive lipids as molecular probes for monitoring the metabolism of cholesterol and phosphatidylcholine revealed the presence of (1) steroid sulfatase in hamster cumulus cells, (2) lecithin: cholesterol acyltransferase in human follicular fluid, (3) phospholipase A₂, and (4) lysophospholipase in human spermatozoa. These enzymatic reactions can be integrated into a pathway that provides a link between the concepts of lysophospholipid accumulation in the sperm membranes and alteration of the cholesterol/phospholipid ratio as factors involved in the preparation of the membranes for the acrosome reaction. Capacitation is viewed as a reversible phenomenon which, upon completion, results in a decrease in negative surface charge, an efflux of membrane cholesterol, and an influx of calcium between the plasma and outer acrosomal membranes. Triggered by the entry of calcium, the acrosome reaction involves phospholipase A₂ activation followed by a transient accumulation of unsaturated fatty acids and lysophospholipids implicated in membrane fusion which occurs during the formation of membrane vesicles in spermatozoa undergoing the acrosome reaction.     

Rac1 is necessary for capacitation and acrosome reaction in guinea pig spermatozoa

Actin cytoskeleton remodeling is a critical process for the acquisition of fertilizing capacity by spermatozoa during capacitation. However, the molecular mechanism that regulates this process has not been fully elucidated. In somatic cells, Ras-related C3 botulinum toxin substrate 1 protein (Rac1) promotes the polymerization of actin by participating in the modeling of two structures: lamellipodia and adhesion complexes linked with the plasma membrane. Rac1 is expressed in mammalian spermatozoa; however, the role of Rac1 in sperm physiology is unknown. This study aimed to elucidate the participation of Rac1 in capacitation and acrosome reaction (AR). Rac1 was found to be dispersed throughout the acrosome and without changes in the middle piece. After 60 minutes of capacitation, Rac1 was found in the apical region of the acrosome only, which concurred with an increase in Rac1-GTP. Rac1 inhibition prevented such changes. In the middle piece, Rac1 localization remained unchanged. Besides, Rac1 inhibition blocked capacitation and AR. The present study demonstrates that Rac1 participates only in the actin cytoskeleton remodeling that occurs in the acrosomal apical region during capacitation, a region where a large amount of actin is polymerized and shaped in a diadem-like structure. Our data also show that this actin cytoskeleton organized by Rac1 interacts with filamin-1, and such interaction was blocked by the inhibition of Rac1, which led to a different organization of the actin cytoskeleton. All these outcomes imply that the formation of an F-actin cytoskeleton in the acrosomal apical region is a necessary event for capacitation and AR, and which is Rac1 driven.     

Capacitation of mammalian spermatozoa in vivo, with a specific focus on events in the Fallopian tubes

This essay argues strongly that for those sperm cells involved in fertilisation, the process of capacitation represents an active and specific coordination within succeeding regions of the female tract and one whose completion is synchronised with the events of ovulation. Observations on the time-course of capacitation when spermatozoa are first exposed to the uterus and then progress to the Fallopian tubes indicate a synergistic influence of these adjoining portions of the female tract on the rate of capacitation. Three concepts on the control of capacitation are introduced to emphasise the importance of integration in vivo, namely that (1) completion of capacitation is a peri-ovulatory event, (2) suppression of completion of capacitation is an essential storage strategy during a long pre-ovulatory interval, and (3) the process of capacitation comes under the influence of local and systemic ovarian control mechanisms, especially the secretion of progesterone from Graafian follicles soon to ovulate. The last would act to coordinate the final maturation and meeting of male and female gametes. Despite the preceding points, the requirement for such integrated in vivo programming of sperm cell maturation can clearly be overridden in systems of culture. The most reasonable interpretation here would be that a microdrop of culture medium containing eggs, follicular cells and components of follicular fluid would to a considerable extent represent a post-ovulatory environment. Within such a preparation, there would be leaching of the sperm surface among the relatively vast and heterogeneous population of cells, and a proportion of spermatozoa could then respond to 'post-ovulatory signals', not least to molecular influences of the zona pellucida and vitelline products for completion of capacitation. Nonetheless, a physiologically meaningful interpretation of capacitation calls for a stepwise analysis of the dynamic interactions between sperm cell and female tract at successive stages between the uterus and ampullary-isthmic junction.     

Effects of surfactants on the fertilizing capacity and acrosome reaction of sea urchin spermatozoa

The effects of seven surfactants on spermatozoa of the sea urchin, Hemicentrotus pulcherrimus, were studied. All these surfactants induced the acrosome reaction and inhibited the fertilizing capacity of spermatozoa. There was a statistically significant correlation between the concentrations that induce the acrosome reaction and inhibit fertilization. The critical micelle concentrations (CMC) of surfactants in sea water were almost even and these values, which are inherent physical properties of surfactants, did not provide a direct measure of their inhibitory effect of fertilization. Among seven surfactants, p-menthanyl-phenol polyoxyethylene (8.8) ether (TS-88) with a characteristic hydrophobes was the most potent both in the induction of acrosome reaction and in the inhibition of fertilization. Various ethylene oxide adducts to p-menthanyl-phenol were also tested for the purpose of comparison. It is suggested that the effects of surfactants on sea urchin spermatozoa at low concentrations reflect their activity associated with the hydrophobic group inherent in each surfactant.     

Solubilized zona pellucida proteins and progesterone induce calcium influx and the acrosome reaction in capacitated dog spermatozoa.

Spermatozoa from the sperm-rich fractions of the semen of 6 beagle dogs were capacitated and the effect of both zona pellucida (ZP) proteins and progesterone on calcium flux and the acrosome reaction measured. Sperm calcium flux was determined using the dual wavelength calcium probe indo-1/AM (6 microM) in a flow cytometric assay (one ejaculate from each dog examined; n = 6). No calcium flux was observed in the negative control treatments (RPMI medium or DMSO). Both heat-solubilized bitch ZP proteins and progesterone caused a similar response characterized by a gradual but marked influx of calcium ions which was sustained over 2 min. Acrosomal status was assessed by indirect immunofluorescence using a specific monoclonal antibody following 1 hr incubation for each treatment (four ejaculates from each dog examined; n = 24). The level of acrosomal exocytosis was very high for samples treated with ZP proteins (70.3 +/- 2.1%) and progesterone (84.6 +/- 1.5%) and was significantly different from the respective controls (P < 0.001). Interestingly the patterns of calcium flux in response to both ZP proteins and progesterone were in contrast to the situation in other species studied to date raising the possibility that the mechanism for triggering the acrosome reaction may be different in dog spermatozoa. In addition the high degree of progesterone-induced acrosomal exocytosis compared to other species raises the probability that the majority of dog spermatozoa are already undergoing the acrosome reaction before they reach the egg ZP.     

Induction of the acrosome reaction in ejaculated goat spermatozoa by preincubation in chemically defined medium

The aim of the present study was to examine the effects of the elimination of energy substrate from the medium and the effects of the preincubation vessel, temperature, and time on the induction of the acrosome reaction in ejaculated goat spermatozoa in chemically defined medium. Washed spermatozoa were resuspended in Brackett-Oliphant (BO) medium or substrate-free BO medium to give a high concentration and preincubated in open test tubes or sealed glass tubes at 37.0 or 39.5 degrees C for 1, 2, or 3 h. Sperm acrosome reaction was evaluated using a simplified triple-stain technique and a hamster test. It was found that the goat sperm acrosome reaction occurred more readily in the sealed glass tubes than in open test tubes, in substrate-free BO medium than in BO medium, and at 39.5 than at 37.0 degrees C. During preincubation with substrate-free BO medium in sealed glass tubes at 39.5 degrees C, the optimum time necessary to induce the acrosome reaction in goat spermatozoa was 2-3 h.     

Capacitation in vitro of bovine spermatozoa by oviduct epithelial cell monolayer conditioned medium

The effect of the active capacitating factor secreted from oviduct epithelial cell monolayers (OECM) in different environments on in vitro fertilization was evaluated. Capacitation was determined as the ability of sperm to fertilize bovine oocytes in vitro. When the mTALP was supplemented with glucose during conditioning, the sperm penetration rate was significantly reduced (P ≤ 0.05) compared to the control (7% ± 1 vs. 30% ± 4). The percentages of sperm penetrated oocytes were higher following insemination in the OECM-conditioned medium derived from the early stage (48% ± 7) of the estrous cycle than in the OECM-conditioned medium derived from either mid (35% ± 2) or late stages (28% ± 3) of the estrous cycle. When the medium was supplemented with 0.1 or 0.5 μg/ml estradiol-17β during medium conditioning, sperm penetration rates increased (P ≤ 0.05) compared to the control group (55% ± 4 vs. 40% ± 3 and 54% ± 2 vs. 41% ± 3, respectively). In addition, the percentages of penetrated oocytes significantly decreased (P ≤ 0.05) following insemination when the OECM-conditioned medium was added to 0.01%, 0.05%, and 0.1% ethanol compared to the control (25% ± 4, 19% ± 2, 18% ± 3, and 45% ± 3, respectively). Sperm penetration rates significantly (P ≤ 0.01) decreased when the OECM-conditioned medium was heated to 100°C for 5 min (10% ± 1 vs. 40% ± 3). These results suggest that the active capacitating factor was sereted by the OECM and that this capacitating factor in the OECM-conditioned medium was inhibited by the presence of glucose. This factor was found to be heat-sensitive and its action was affected by ethanol. The OECM derived from the three phases of the estrous cycle as well as the presence of estradiol-17β influenced the capacity of the OECM to secrete this capacitating factor in Vitro. © 1995 wiley-Liss, Inc.     

Dithiothreitol,a disulfide-reducing agent,inhibits capacitation,acrosome reaction,and interaction with eggs by guinea pig spermatozoa

Capacitation of guinea pig spermatozoa in vitro was inhibited by the disulfide-reducing agent dithiothreitol (DTT). Even a brief treatment with DTT inhibited capacitation unless an oxidizing agent (glutathione disulfide) was present in the posttreatment medium. Precapacitated spermatozoa were unable to undergo the acrosome reaction in the presence of DTT, indicating that this reagent also blocks the acrosome reaction. Acrosome-reacted spermatozoa were incapable of attaching to and penetrating the zona pellucida in the presence of DTT. Even when acrosome-reacted spermatozoa were directly brought to the surface of zona-free eggs, they were unable to bind to and fuse with the egg plasma membrane so long as DTT was present in the medium. These observations suggest that the tertiary and quaternary structures of sperm surface proteins regulated by their thioldisulfide status are of critical importance in the physiology and function of spermatozoa preliminary to and in the process of fertilization.     

Induction of the acrosome reaction in human spermatozoa by a fraction of human follicular fluid

Human ejaculated spermatozoa were washed through a Percoll gradient, preincubated for 10 hr in a defined medium containing serum albumin, and then induced to undergo rapid acrosome reactions by addition of human follicular fluid or a Sephadex G-75 column fraction of the fluid. Induction by follicular fluid did not occur when the spermatozoa were preincubated for only 0 or 5 hr. The reactions were detected by indirect immunofluorescence using a monoclonal antibody directed against the human sperm acrosomal region. The percentage of acrosomal loss counted by transmission electron microscopy agreed with that counted by immunofluorescence. The apparent molecular weight of the Sephadex G-75 fraction containing the peak of acrosome reaction-inducing activity was 45,000 ± 4,200 (SD). The occurrence of physiological acrosome reactions was supported by: assessing motility (no significant loss of motility occurred during the treatment period when sperm were preincubated with bovine serum albumin), transmission electron microscopy (the ultrastructural criteria for the acrosome reaction were met), and zona-free hamster oocyte binding and penetration (spermatozoa pretreated with the active fraction of follicular fluid, then washed and incubated with oocytes, showed significantly greater binding to and penetration of oocytes). The stimulation of the acrosome reaction by follicular fluid is apparently not due to blood serum contamination; treatment of preincubated spermatozoa with sera from the follicular fluid donors had no effect on the spermatozoa. The nature of the active component(s) in that fraction is currently being investigated.     

Changes in calmodulin compartmentalization throughout capacitation and acrosome reaction in guinea pig spermatozoa

Calmodulin has been postulated as a mediator in the calcium-dependent processes that culminate in the acrosome reaction. Changes in calmodulin compartmentalization as a consequence of the increased permeability to extracellular calcium during capacitation and acrosome reaction have been suggested. In the present study the temporal localization of calmodulin in guinea pig spermatozoa was studied during in vitro capacitation and acrosome reaction by indirect immunofluorescence. Capacitation was achieved by incubation in Tyrode medium supplemented with pyruvate, lactate, and glucose in the presence and in the absence of calcium. Acrosome reaction was elicited in three different conditions: 1) by transfer to minimal culture medium containing pyruvate and lactate (MCM-PL) after in vitro capacitation 2) by 0.003% Triton-X 100 treatment, and 3) by A 23187 addition to sperm samples incubated in MCM-PL. During capacitation, calmodulin was observed both in the acrosome and in the flagellum; this localization seemed to be independent of the presence of extracellular calcium and of exogenous substrates. Throughout the acrosome reaction, different stages of calmodulin compartmentalization were observed. It became clustered around the equatorial region just before or a little after the acrosome reaction had occurred. Later, it was observed around the postacrosomal region in the acrosome-reacted sperm. The changes in calmodulin distribution were found to be dependent on the stage in the acrosome reaction.     

Observations on the acrosome reaction of human sperm in vitro

Human sperm were incubated in vitro in serum or the defined medium TMPA and were periodically assessed for acrosome reactions using two new methods of assay. The first method, FITC-RCA labeling, was previously shown to be valid for estimating the percentage of normal acrosome reactions of human sperm. The second method, a triple staining technique, is shown in this study to give results comparable to those obtained with FITC-RCA labeling. The percentage of acrosome-reacted sperm was determined at 0, 2.5, 5, and 7 hr of incubation. In both media, some sperm had reacted by 2.5 hr; a maximum percentage of reactions occurred between 5 and 7 hr. The maximum percentage never exceeded 20–25%, which represents only one-third of the live sperm, ie, those potentially able to undergo normal acrosome reactions. It will be important in future studies to determine if this low-peak percentage is due to the fact that: (1) Commonly used culture media are suboptimal or (2) only about 25% of the sperm in a human ejaculate are capable of undergoing normal acrosome reactions.     

Development of a simple chemically defined medium for Porphyromonas gingivalis: requirement for α-ketoglutarate

Abstract The aim of this study was the development of a simple, defined medium for the growth of laboratory and clinical isolates of Porphyromonas gingivalis . A medium was designed in which the carbon and nitrogen requirements were provided by a single protein source — bovine serum albumin. High cell yields were achieved in this medium but growth was accompanied by a heavy blackening of the cells due to the deposition of metal sulfide(s), most probably iron(II) sulfide, at the cell surface. Good growth in the absence of blackening was achieved when the iron salt in the medium was substituted with α-ketoglutarate. The resultant α-ketoglutarate/BSA medium was able to support the growth of all laboratory and clinical P. gingivalis strains examined and should prove useful in the investigation of the physiology and nutritional regulation of virulence of this organism.     

Induction of the acrosome reaction in the spermatozoa of the goat by secretions of the male accessory glands and milk

The balancing effects of bulbourethral gland secretion (BUS) and of seminal vesicle secretion (SVS) on goat semen quality were previously demonstrated. In the present study, electron microscope observations revealed a high frequency of spermatozoa with a reacted acrosome among spermatozoa from cauda epididymis exposed to BUS in the presence of milk. This frequency was significantly reduced when SVS had been added either before or after BUS. No reacted acrosome was observed in the absence of milk. All mount spermatozoa were incubated with milk or SVS or BUS or combinations of the three materials labeled with colloidal gold. SVS attached specifically on the plasma membrane covering the anterior part of the acrosome, whereas BUS spread all over the sperm head. Milk attached on the anterior half of the sperm head only when BUS was present in the sperm environment. It is concluded that BUS plays an active role in the induction of the acrosome reaction in the presence of milk and that SVS counteracts this role.