Scorpion toxins that block T-type Ca2+ channels in spermatogenic cells inhibit the sperm acrosome reaction

The acrosome reaction (AR) is a Ca(2+)-dependent event required for sperm to fertilize the egg. The activation of T-type voltage-gated Ca(2+) channels plays a key role in the induction of this process. This report describes the actions of two toxins from the scorpion Parabuthus granulatus named kurtoxin-like I and II (KLI and KLII, respectively) on sperm Ca(2+) channels. Both toxins decrease T-type Ca(2+) channel activity in mouse spermatogenic cells and inhibit the AR in mature sperm. Saturating concentrations of the toxins inhibited at most approximately 70% of the whole-cell Ca(2+) current, suggesting the presence of a toxin-resistant component. In addition, both toxins inhibited approximately 60% of the AR, which is consistent with the participation of T-type Ca(2+) channels in the sperm AR.     

Regulation of the sperm EGF receptor by ouabain leads to initiation of the acrosome reaction

The sperm acrosome reaction occurs after the binding of the capacitated sperm to the egg zona pellucida. This study describes a novel mode of regulation of the sperm epidermal growth factor receptor (EGFR) under physiological conditions and its relevance to the acrosome reaction. Ouabain, a known Na/K ATPase blocker is present in the blood and in the female reproductive tract. We show here that physiological concentrations (nM) of ouabain enhance phosphorylation of EGFR on tyr-845, stimulate Ca²⁺ influx and induce the acrosome reaction in sperm. These effects could be seen only in the presence of very low concentrations of EGF (0.1 ng/ml or 0.016 nM) added together with nano-molar ouabain. Phosphorylation, Ca²⁺ influx, and the acrosome reaction are inhibited by an EGFR blocker, suggesting that trans-activation of the EGFR is involved. Moreover, our data revealed that protein kinase A and the family of tyrosine kinase, SRC, shown before to be involved in EGFR activation in sperm, mediate the acrosome reaction induced by ouabain. Ouabain alone (without EGF) at relatively high concentration (10 µM) could enhance EGFR phosphorylation, Ca²⁺ influx and acrosome reaction, and these processes were inhibited by EGFR blockers. Moreover, we show here that PKA and SRC family are involved in the activation of EGFR by 10 µM ouabain, further demonstrating that ouabain induces the acrosome reaction by a mechanism mediated by the trans-activation of EGFR. In conclusion, this study describes an interesting regulatory path of EGFR by physiological concentrations of ouabain and EGF found in the female reproductive tract. Neither of these compounds can activate the EGFR alone at such low physiological levels; however, when both are present, the interaction of ouabain with the Na/K ATPase leads to the priming of the EGFR, which undergoes its full activation by EGF.     

Bovine sperm acrosome reaction induced by G protein-coupled receptor agonists is mediated by epidermal growth factor receptor transactivation

We have previously demonstrated the presence of active epidermal growth factor receptor (EGFR) and its involvement in sperm capacitation and the acrosome reaction; however, the mechanism of EGFR activation was not clear. We show here that the sperm EGFR can be transactivated by angiotensin II or by lysophosphatydic acid, two ligands which activate specific G-protein-coupled receptors (GPCR), or by directly activating protein kinase A using 8Br-cAMP. This transactivation occurs in noncapacitated sperm and is mediated by PKA, SRC and a metalloproteinase. We also show that the EGFR is activated in sperm incubated under in vitro capacitation conditions, without any added ligand, but not in bicarbonate-deficient medium or when PKA is blocked. Despite the fact that EGFR is activated in capacitated sperm, this state is not sufficient to induce the acrosome reaction. We conclude that the EGFR is stimulated during capacitation via PKA activation, while further activation of the EGFR in capacitated sperm is required in order to induce the acrosome reaction. The acrosome reaction can be induced by GPCR via the transactivation of the EGFR by a signaling pathway involving PKA, SRC and metalloproteinase and the EGFR down-stream effectors PI3K, PLC and PKC.     

Iberiotoxin and clofilium regulate hyperactivation,acrosome reaction,and ion homeostasis synergistically during human sperm capacitation

Potassium channels play essential roles in the regulation of male fertility. However, potassium channels mediating K⁺ currents in human sperm (I_KSper) remain controversial. Besides SLO3, the SLO1 potassium channel is a potential candidate for human sperm KSper. This study intends to elucidate the function of SLO1 potassium channel during human sperm capacitation. Human sperm were treated with iberiotoxin (IbTX, a SLO1 specific inhibitor) and clofilium (SLO3 inhibitor) separately or simultaneously during in vitro capacitation. A computer-assisted sperm analyzer was used to assess sperm motility. The sperm acrosome reaction (AR) was analyzed using fluorescein isothiocyanate-conjugated Pisum sativum agglutinin staining. Sperm protein tyrosine phosphorylation was studied using western blotting. Intracellular Ca²⁺, K⁺, Cl⁻, and pH were analyzed using ion fluorescence probes. Independent inhibition with IbTX or clofilium decreased the sperm hyperactivation, AR, and protein tyrosine phosphorylation, and was accompanied by an increase in [K⁺]_i, [Cl⁻]_i, and pH_i, but a decrease in [Ca²⁺]_i. Simultaneously inhibition with IbTX and clofilium lower sperm hyperactivation and AR more than independent inhibition. The increase in [K⁺]_i, [Cl⁻]_i, and pH_i, and the decrease in [Ca²⁺]_i were more pronounced. This study suggested that the SLO1 potassium channel may have synergic roles with SLO3 during human sperm capacitation.     

Xenoestrogenic compounds promote capacitation and an acrosome reaction in porcine sperm

There is growing evidence that endocrine disruptors bind to hormone receptors; since these receptors are present on the sperm membrane, sperm are potentially a useful model for examining estrogenic activities of endocrine disruptors. The objective of the present study was to compare the effects of two xenoestrogenic compounds (genistein and 4-tert-octylphenol) to those of two steroids (estrogen and progesterone) and heparin on in vitro capacitation and the acrosome reaction in a porcine sperm model. Porcine sperm were incubated with various concentrations (0.001-100 μM) of each chemical for 15 or 30 min, and then capacitation and the acrosome reaction were assessed using chlortetracycline. Estrogen and progesterone were considerably more potent than the other chemicals in stimulating capacitation. Estrogen stimulated sperm capacitation at all tested concentrations after 15 min of incubation (P < 0.05), whereas progesterone stimulated sperm capacitation at all tested concentrations after 15 and 30 min (P < 0.05). The effect of genistein on sperm capacitation was comparable with that of estrogen, and it was the most potent in stimulating the acrosome reaction. Genistein stimulated the acrosome reaction at all tested concentrations after 30 min (P < 0.05). However, 4-tert-octylphenol had the least effect on capacitation and the acrosome reaction. In summary, since all chemicals studied effectively altered capacitation and the acrosome reaction, it was concluded that porcine sperm could be a useful model for in vitro screening of potential endocrine disruptors. It was noteworthy that concurrent comparisons to steroids increased the ability to determine estrogenic characteristics of the tested chemicals.     

Relationship between plasma membrane Ca2+-ATPase activity and acrosome reaction in guinea pig sperm

The results obtained by biochemical measurement demonstrated for the first time that significant decrease of the plasma membrane Ca²⁺-ATPase activity occurred during capacitation and acrosome reaction of guinea pig sperm. Ethaorynic acid, one kind of Ca²⁺-ATPase antagonists, inhibited the plasma membrane Ca²⁺-ATPase activity, but calmodulin (50μg/mL) and trifluoperazine (200- 500μmol/L) did not, suggesting that calmodulin is not involved in ATP-driven Ca²⁺ efflux from sperm. However, calmodulin is involved in the control of Ca²⁺ influx. TFP, one kind of calmodulin antagonists, accelerated the acrosome reaction and Ca²⁺ uptake into sperm cells significantly. Ca²⁺-ATPase antagonists, quercetin, sodium orthovandate, furosemide and ethacrynic acid promoted the acrosome reaction, but inhibited Ca2+ uptake, which cannot be explained by their inhibitory effects on the plasma membrane Ca²⁺-ATPase activity. It is speculated that this phenomenon might be caused by simultaneous inhibitions of the activities of Ca²⁺-ATPase present in the plasma membrane, the outer acrosome membrane and the outer mitochondrion membrane resulting in Ca²⁺ accumulation in the cytoplasm, which in turn blocks further Ca2+ entry through some negative feedback mechanism(s). The inhibitory effect of Ca²⁺-ATPase antagonist on glycolytic activity may also be the reason for Ca²⁺ accumulation in cytoplasm and inhibition of Ca²⁺ uptake.     

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.     

Ram spermatozoa cocultured with epithelial cell monolayers: An in vitro model for the study of capacitation and the acrosome reaction

The effects of different epithelial cells, namely, hamster oviduct, sheep oviduct, and pig kidney epithelial cells (IBRS-2), on the viability, percentage of progressive motility (PPM), and acrosome reactions of ejaculated ram spermatozoa were investigated. Sperm aliquots were cultured on cells, cell-conditioned medium 199, or control medium 199. The PPM of unattached spermatozoa was estimated after 0, 3, 6, 9, 12, and 24 hr of incubation at 37°C under 5% CO₂ in air. Viability and the occurrence of true acrosome reactions were assessed using a triple-stain technique. Spermatozoa started to attach within 1 hr of coculture with the hamster or sheep oviductal epithelial cell (OEC) monolayers, and these spermatozoa showed vigorous tail motion. No spermatozoa were found to attach to the IBRS-2 monolayer. The PPM of unattached spermatozoa cocultured with the various types of epithelial cell monolayers for 12 hr was significantly higher than that of spermatozoa incubated in conditioned media or medium 199 alone (54% in hamster OEC vs. 40% in conditioned; 68% in sheep OEC vs. 38% in conditioned; 36% in control medium). On the other hand, after 24 hr of incubation, there were no differences in the PPM of spermatozoa cocultured with epithelial cells or incubated in conditioned media. The percentages of cells undergoing a true acrosome reaction reached maximum values (P < 0.05) in spermatozoa incubated for 9 hr in the presence of hamster OEC (22.5%) or for 12 hr on sheep OEC (20.5%) monolayers. IBRS-2, a commercial nonreproductive cell type, had a positive influence on both PPM and sperm viability but no effect on the occurrence of the acrosome reaction. Interactions leading to the acrosome reaction were thus observed only when spermatozoa were cocultured with OEC monolayers. The values of PPM in unattached sperm cells seen after 12 hr of coculture with OEC or IBRS-2 were still at a high level (52–67%) for in vitro fertilization. The coculture with OECs provides an “in vitro” model to study the capacitation processes in a situation that may resemble that occurring in vivo. Moreover, the coculture with hamster OECs may provide a convenient and standardized in vitro system to study mechanisms underlying capacitation and the acrosome reaction. © 1993 Wiley-Liss, Inc.     

Structural requirements for species-specific induction of the sperm acrosome reaction by sea urchin egg sulfated fucan

The sulfated fucan (SF) of egg jelly induces the acrosome reaction (AR) of sea urchin sperm. Strongylocentrotus franciscanus (Sf) SF is sulfated only at the 2-position. Strongylocentrotus purpuratus (Sp) has two SF isotypes, each one being female specific. One is rich in sulfate at both the 2- and 4-positionS (SF-1), and the other is rich in sulfate at the 4-position, but not the 2-position (SF-2). Sf SF is poor at inducing the AR of Sp sperm, presumably due to lack of 4-sulfation. Sp SF-1 is better at inducing the AR of Sf sperm than Sp SF-2, hypothetically due to increased 2-sulfation. Chemical oversulfation of Sf SF increases the percentage of AR of Sp sperm, showing that 4-sulfation is important for recognition of SF by Sp sperm. Chemically oversulfated Sp SF-2 is better at inducing the Sf sperm AR, presumably because of increased 2-sulfation. The species, Strongylocentrotus drobachiensis (Sd), has an SF-2 that is exclusively 2-sulfated (like Sf), except the glycosidic linkage in Sd is alpha(1-->4), whereas in Sf it is alpha(1-->3). Sd SF-2 does not induce the AR of Sf sperm, showing the strict requirement for the alpha(1-->3) linkage in recognition between Sf sperm and SF. Egg jelly from Echinometra lucunter (El) contains sulfated galactan (SG) which differs from Sf SF only in that the monosaccharide is L-galactose, not L-fucose. This SG and Sf SF are equally potent in inducing the AR of Sf sperm, showing that modification at C6 of L-fucose is not important for proper recognition between SF and Sf sperm receptors. This system permits study of the structural basis for recognition between sulfated polysaccharide and receptors controlling signal transduction pathways in animal cells.     

Egg fucose sulfate polymer,sialoglycan, and speract all trigger the sea urchin sperm acrosome reaction

Macromolecules surrounding eggs induce the acrosome reaction (AR) of spermatozoa. In sea urchins, three egg jelly (EJ) molecules: a fucose sulfate polymer (FSP), a sialoglycan (SG), and speract mediate ionic fluxes triggering the AR. SG and speract are noninductive without FSP. Speract's role in AR induction is controversial. Here we show that speract potentiates the FSP-induced AR at pH 7.0, approximately 1 pH unit lower than natural seawater. At pH 7.0, a mixture of FSP, SG, and speract produces the intracellular pH increase necessary for maximum AR induction. Each EJ component may mediate a distinct intracellular pH control mechanism, and all three may function synergistically to increase the intracellular pH permitting AR induction. Speract peptides are an ancient family. Although important for activating cyclic nucleotide-mediated pathways in today's seawater of pH approximately 8, speract may have been more important in AR induction in the paleo-ocean of pH approximately 7.     

Ion channel activity of membrane vesicles released from sea urchin sperm during the acrosome reaction

The sperm acrosome reaction (AR) involves ion channel activation. In sea urchin sperm, the AR requires Ca2+ and Na+ influx and K+ and H+ efflux. During the AR, the plasma membrane fuses with the acrosomal vesicle membrane forming hybrid membrane vesicles that are released from sperm into the medium. This paper reports the isolation and preliminary characterization of these acrosome reaction vesicles (ARVs), using synaptosome-associated protein of 25 kDa (SNAP-25) as a marker. Isolated ARVs have a unique protein composition. The exocytosis regulatory proteins vesicle-associated membrane protein and SNAP-25 are inside ARVs, as judged by protease protection experiments, and membrane associated based on Triton X-114 partitioning. ARVs fused with planar bilayers display three main types of single channel activity. The most frequently recorded channel is cationic, weakly voltage dependent and has a low open probability that increases with negative potentials. This channel is activated by cAMP, blocked by Ba2+, and has a PK+/PNa+ selectivity of 4.5. ARVs represent a novel membrane preparation suitable to deepen our understanding of ion channel activity in the AR and during fertilization.     

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.     

Tuning sperm chemotaxis by calcium burst timing

Marine invertebrate oocytes establish chemoattractant gradients that guide spermatozoa towards their source. In sea urchin spermatozoa, this relocation requires coordinated motility changes initiated by Ca²⁺-driven alterations in sperm flagellar curvature. We discovered that Lytechinus pictus spermatozoa undergo chemotaxis in response to speract, an egg-derived decapeptide previously noted to stimulate non-chemotactic motility alterations in Strongylocentrotus purpuratus spermatozoa. Sperm of both species responded to speract gradients with a sequence of turning episodes that correlate with transient flagellar Ca²⁺ increases, yet only L. pictus spermatozoa accumulated at the gradient source. Detailed analysis of sperm behavior revealed that L. pictus spermatozoa selectively undergo Ca²⁺ fluctuations while swimming along negative speract gradients while S. purpuratus sperm generate Ca²⁺ fluctuations in a spatially non-selective manner. This difference is attributed to the selective suppression of Ca²⁺ fluctuations of L. pictus spermatozoa as they swim towards the source of the chemoattractant gradient. This is the first study to compare and characterize the motility components that differ in chemotactic and non-chemotactic spermatozoa. Tuning of Ca²⁺ fluctuations and associated turning episodes to the chemoattractant gradient polarity is a central feature of sea urchin sperm chemotaxis and may be a feature of sperm chemotaxis in general.     

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.     

Calcium and copper transport ATPases: analogies and diversities in transduction and signaling mechanisms

The calcium transport ATPase and the copper transport ATPase are members of the P-ATPase family and retain an analogous catalytic mechanism for ATP utilization, including intermediate phosphoryl transfer to a conserved aspartyl residue, vectorial displacement of bound cation, and final hydrolytic cleavage of Pi. Both ATPases undergo protein conformational changes concomitant with catalytic events. Yet, the two ATPases are prototypes of different features with regard to transduction and signaling mechanisms. The calcium ATPase resides stably on membranes delimiting cellular compartments, acquires free Ca²⁺ with high affinity on one side of the membrane, and releases the bound Ca²⁺ on the other side of the membrane to yield a high free Ca²⁺ gradient. These features are a basic requirement for cellular Ca²⁺ signaling mechanisms. On the other hand, the copper ATPase acquires copper through exchange with donor proteins, and undergoes intracellular trafficking to deliver copper to acceptor proteins. In addition to the cation transport site and the conserved aspartate undergoing catalytic phosphorylation, the copper ATPase has copper binding regulatory sites on a unique N-terminal protein extension, and has also serine residues undergoing kinase assisted phosphorylation. These additional features are involved in the mechanism of copper ATPase intracellular trafficking which is required to deliver copper to plasma membranes for extrusion, and to the trans-Golgi network for incorporation into metalloproteins. Isoform specific glyocosylation contributes to stabilization of ATP7A copper ATPase in plasma membranes.     

ZD7288 inhibits low-threshold Ca(2+) channel activity and regulates sperm function

In this study, ZD7288, a blocker of hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels, has been found to inhibit the mouse sperm acrosome reaction (AR). HCN channels have not yet been either recorded or implicated in mouse sperm AR, but low-threshold (T-type) Ca(2+) channels have. Interestingly, ZD7288 blocked native T-type Ca(2+) currents in mouse spermatogenic cells with an IC(50) of about 100 microM. This blockade was more effective at voltages producing low levels of inactivation, suggesting a differential affinity of ZD7288 for different channel conformations. Furthermore, ZD7288 inhibited all cloned T-type but not high-threshold N-type channels heterologously expressed in HEK-293 cells. Our results further support the role of T-type Ca(2+) channels in the mouse sperm AR.     

Importance of mammalian sperm metalloendoprotease activity during the acrosome reaction to subsequent sperm-egg fusion: inhibitor studies with human sperm and zona-free hamster eggs.

We have previously shown that each of the metalloendoprotease (MEP) inhibitors phosphoramidon, diethylenetriaminepentaacetic acid, and carbobenzoxy-L-phenylalanine, when present only during the human sperm acrosome reaction (AR), will not inhibit the AR or sperm motility but will decrease the number of sperm that penetrate zona-free hamster eggs. The present study was designed to investigate whether this inhibition of penetration is due to an effect on sperm binding to the egg plasma membrane and/or to an effect on the actual membrane fusion event. In these studies we used ionomycin to initiate the AR and assayed binding in a Ca(2+)-free medium and fusion in Ca(2+)-containing medium in the same experiment. Eggs were loaded with the fluorescent dye Hoechst 33342, and the appearance of fluorescence in a sperm head indicated that fusion had occurred. The three MEP inhibitors reduced binding only slightly but inhibited the actual fusion step by 50-60% (determined with an equation that corrected for any inhibition of fusion due to inhibition of binding). MEP inhibitors present only during gamete interactions had little or no effect on fusion. We also found that phosphoramidon-inhibitable MEP activity was released during the ionomycin-initiated AR. Incubation of AR supernatant containing MEP activity with previously acrosome-reacted, phosphoramidon-treated sperm resulted in a large reversal of the phosphoramidon-inhibitory effect on sperm-egg fusion. These results support the hypothesis that the acrosomal phosphoramidon-inhibitable MEP released during the AR acts directly or indirectly during that event to increase the fusibility of the sperm plasma membrane region required for subsequent sperm-egg fusion.     

In vitro induction of the acrosome reaction in bull sperm and the relationship to field fertility using low-dose inseminations

The acrosome reaction (AR) is a prerequisite for normal sperm fertilizing capability and can be studied in vitro after induction by various agents. The efficacy of a sperm population to undergo the AR in vivo is expected to influence male fertilizing potential. During the past two decades, a number of attempts have been made to relate the in vitro-induced AR to field fertility in several species. However, to our knowledge, no studies have combined in vitro induction of the AR with the simultaneous detection of sperm viability and acrosomal status using a high-precision flow cytometric technique. Furthermore, large-scale fertility trials using low-dose inseminations are pending. In the current study, the relationship between field fertility and the in vitro-induced AR was investigated using three ejaculates from each of 195 bulls, 156 Holstein and 39 Jersey bulls (Bos taurus), participating in a progeny test program including low-dose inseminations. A range of insemination doses, varying from 2.0 × 10⁶ to 15 × 10⁶ sperm/dose, was obtained by a controlled dilution process applied to each ejaculate. Different insemination doses were distributed at random among 75,610 experimental first inseminations in 4721 herds and 208 artificial insemination (AI) technicians. Simultaneous detection of sperm viability and acrosomal status was achieved using a triple color flow cytometric technique. Sperm samples from the bulls displayed a wide range of ability to acrosome react in response to calcium ionophore A23187. Both reproducibility of the AR response after induction and relationship between ability to acrosome react and field fertility was highly dependent on the definition of AR inducibility. Six basic and six combined AR indices were assessed. The AR index expressing the fraction of acrosome reacted sperm in the live sperm population after induction by ionophore had the highest repeatability, best described the biological variation in the studied population, and yielded the best significant predictive values on field fertility among the 12 indices considered. Moreover, the ability of sperm to acrosome react appeared to be a noncompensable trait that affects fertility regardless of the number of sperm per insemination dose. The current results therefore indicate that this sperm parameter is important in the field and also may play a role in the IVF laboratory.     

Sperm membrane physiology and relevance for fertilization

This paper aims to overview recent insights in sperm surface remodelling pertinent to fertilization. A basic understanding of this remodelling is required to interpret the high amount of data appearing from high-throughput identification techniques for proteins presently applied in reproductive biology. From the extensive lists of protein candidates identified by proteomics, only a few are recognized to be directly involved in fertilization. Others are indirectly involved, but many are not yet considered to be involved in fertilization. Some of these newly identified and unexpected proteins may shed new light in the current molecular models for fertilization. However, the gathered lists of sperm proteins possibly involved in fertilization do only tell a part of the story regarding how fertilization is accomplished. When considering the identification of proteins involved in fertilization, one also needs to take into account the fundamental mechanisms involved in the redistribution of sperm surface proteins in membrane protein complexes and the involvement of cell signalling events that regulate their post-translational modification status. Both processes are likely requisite for protein configuration and grouping into functional membrane protein complexes necessary to elicit their delicate roles in fertilization. This paper emphasizes biochemical models for membrane surface modelling and their potential involvement for remodelling the sperm surface in the above described processes.