International Symposium on Human Sperm Acrosome Reaction , 1995
The meeting was held on September 7-9, 1995 in Collioure, France and attended by 127 participants from 22 different countries. Following lectures by leading researchers, a large part of the meeting was devoted to discussion. The lectures as well as the poster abstracts are published by INSERM. This book entitled "Human sperm acrosome reaction" edited by P. Fénichel and J. Parinaud, is available at John Libbey Eurotext, 127 rue de la République, 92120 Montrouge France. The following is a brief report of the various sessions.
The capacitation process, established more than 50 years ago as the acquisition of fertilizing ability, remained unexplained for a long time. It now can be classed as molecular modifications of the sperm plasma membrane, which enable sperm binding to the zona pellucida and induction of the acrosome reaction (AR).
William Holt (UK) described, using recent molecular analysis, the lipids/specific proteins interactions, the complex architecture of the human sperm plasma membrane, constantly changing throughout time and space. The plasma membrane is organized into domains and is subject to standing changes from spermiogenesis up to fertilization. During capacitation, the plasma membrane is prepared for signal transduction and fusion.
Robin Harrison (UK) studied with a lipophilic fluorescent probe and labeled phospholipids, the changes of the lipid architecture of the plasma membrane induced by bicarbonates exposure. These modifications, related to enzymatic mechanisms, help the loss of cholesterol molecules, a major step in the capacitation process, by purifying factors in both genital fluids and appropriate culture media.
Charles Müller (USA) has isolated from follicular fluid and characterized a lipid transfer protein (LTP-1). This HDL-like protein is found as contaminant in commercial albumin preparation and might stimulate capacitation by lowering the cholesterol/phospholipids ration. Membrane fluidity induced by these lipid changes leads to protein migration, various membrane and cytoplasmic events (ionic fluxes, tyrosine phosphorylation of proteins) necessary to acrosome reaction.
Lynn Fraser (UK) elucidated ion fluxes mainly in head membranes. During capacitation in the mouse they involve: 1) a slow calcium influx modulated by inactivation of a Ca2+ ATPase, different from the rapid and important Ca2+ flux via calcium channels, required for acrosomal loss; 2) a Na+ influx from genital fluid; 3) bicarbonate ions which could both play an important role in controlling pH and stimulating adenylate cyclase activity; and finally 4) CI transport.
Harry Moore (UK) explained that achieving the capacitation process necessitates the priming of protein tyrosine kinase transduction pathways, and particularly of a 95 kD protein (probably ZP3 receptor), that, partially phosphorylated, leads to acrosome reaction either spontaneously or by ZP3 binding.
Induction of Acrosome Reaction
The second session of the meeting dealt with physiological induction of acrosome reaction. Patricia Saling (USA) demonstrated that this 95 kD protein, termed ZRK (zona receptor kinase), has been identified, cloned and sequenced in human sperm. Its characteristics are close to the tyrosine kinase family and it is involved in ZP3 binding as well as induction of acrosomal loss.
Christopher Barratt (UK) succeeded in producing a recombinant human ZP3 (rhuZP3) in transfected Chinese hamster ovary cells. RhuZP3 can induce a high rate of AR even in spermatozoa that had only been capacitated for a minimal period; it will, therefore, be very helpful in further detailed biochemical studies of ZP3/rZP3 binding and, after purification may be a good candidate for clinical exploration of AR dysfunctions. Jan Tesarik (France) talked about the possible role of proteases (acrosin and acrosin like) during the acrosome reaction process which is both a cell activation via receptors and a cell fusion. Patricio Morales (Chile) reviewed the protein involved in sperm and egg vestments interactions, including the oocyte membrane and reported the possible role of a trypsin/trypsin like activity associated with the sperm membrane to induce AR by ZP.
Stanley Meizel (USA) stated that progesterone has a nongenomic effect on human sperm that can induce a calcium influx, a chloride efflux and AR. He described a sperm steroid receptor/Cl-channel complex resembling but not completely identical to a GABAA receptor/Cl-channel complex and questioned the hypothesis of a second receptor controlling the calcium influx. The physiological role of progesterone in the induction of AR by potentiation of ZP3 was also discussed. Peter Blackmore (USA) examined the characteristics of the cell surface progesterone receptor responsible for Ca 2+ influx in human sperm, using 160 steroid analogs. The results evoked a receptor different from the GABAA receptor/Cl-channel complex, an important role of the C-21 methyl and a sperm receptor binding site located on the b face of the steroid C and D rings.
Transduction Pathways During Acrosome Reaction
This session was dedicated to the mechanisms introduced after binding of acrosome reaction inducers (zona pellucida, progesterone) to their membranes receptors. Harvey Florman (USA) described a model in which ZP3 activates a Ca2+ influx. As ZP3 binds to its receptor, on the one hand a cationic nonselective channel opens thus inducing a membrane depolarization while on the other hand, there is activation of a G protein dependent pHi regulator leading to an elevation of the pH. These two responses allow the opening of calcium channels thus triggering the acrosome reaction. Gregory Kopf (USA) recalled that human sperm, contains only Gi but not Gs class proteins. Gi proteins are stimulated by ZP3 (only from nonfertilized oocytes) and regulate the opening of calcium channels. ZP3 also activates adenylyl cyclase, increases cAMP and stimulates a protein kinase A. Christopher De Jonge (USA) reported that cAMP analogs and PKA stimulators induce acrosome reaction.
Eduardo Roldan (Spain) revealed that zona pellucida, progesterone, or calcium ionophore-induced acrosome reaction leads to hydrolysis of phosphatidylinositol. This PI breakdown is essential to generate DAG formation, which has a crucial role in both phospholipase A2 (increase DAG messenger) and PKC activation. Zvi Naor (Israel) reported that PKC is mainly located in the equatorial segment and in the flagellum. PKC activation by phorbol esters leads to a calcium-dependent increase in motility and acrosome reaction. C. De Jonge described cross talk mechanism between PKA and PKC signal transduction pathways. Combinations of stimulatory compounds of the PKA and PKC pathways do not cause an additive acrosome reaction response, nevertheless kinase inhibitors of each pathway prevented induction of the AR by a stimulator of the alternative pathway. Rajesh Naz (USA) showed that capacitation and acrosome reaction increase the phosphorylation of sperm membrane proteins. Two proteins of 95 kD and 51 kD (FA-1 antigen) are tyrosine phosphorylated and play a key role in sperm-zona binding. Finally as reported by G. Kopf, it seems that sperm zona binding involves numerous receptors, implicating different but related signal transduction pathways.
This session was devoted to the different methods for evaluating acrosome reaction, the origin and consequences of acrosomal dysfunctions and in vivo and in vitro modulation of acrosome reaction. The study of acrosomal function encounters two main problems: 1) choosing the technique and 2) determining the inducer used for dynamic studies of AR. Nicholas Cross (USA) introduced the various techniques and emphasized the difficulty of selection, as some of them visualize the early stages of the AR, while others display the complete acrosome reaction. In his opinion, the ideal assay should clearly identify the early stages of acrosome reaction, be readable in brightfield microscopy or flow cytometry and should not alter sperm function in order to be able to combine motility evaluation with acrosomal status assessment.
David Mortimer (Australia) reported that the most appropriate inducer of the AR would be ZP3, but the scant supply of human zonae precludes this approach, as recombinant human ZP3 is not yet commercially available. Among other inducers, only progesterone and calcium ionophore are the only viable alternatives since follicular fluid cannot be standardized. D. Mortimer recommends using a small amount of A23187 as acrosome reaction inducer. Patrick Fénichel (France) demonstrated that spontaneous and calcium ionophore-induced acrosome reactions were respectively higher and lower in IVF failures than in successes. This clinical approach has identified unknown sperm defects, as well as explaining some IVF failures with a normal spermocytogram.
Frank Kohn (Germany) reported that varicoceles or environmental factors, such as tobacco or platinum chloride, induce acrosomal dysfunctions prior any other sperm abnormalities. John Aitken (Scotland) explained that an excess in reactive oxygen species production by sperm leukocytes can cause peroxidase damages to the lipid components of the membranes, thus producing acrosomal dysfunctions. However, small concentrations of reactive oxygen species are necessary for sperm capacitation and acrosome reaction.
Acrosomal function can be modulated in vitro to increase the IVF success rate. Thus, Yves Menezo (France) reported that the ionic components of a medium can influence capacitation process and that bicarbonates increase cAMP and hence capacitation. The use of antioxidative agents (hypotaurine, N-acetylcysteine, glutathione) helps to prevent damage from reactive oxygen species and to maintain sperm function. Finally, D. Mortimer and P. Fénichel showed that it was possible to increase in vitro sperm capability to undergo acrosome reaction using pharmacological agents such as pentoxifylline or NECA.
During the past five years the number of microinjection techniques has greatly increased. André Van Steirteghem (Belgium) demonstrated that acrosome function is necessary for SUZI technique but not for ICSI. This is the reason why he obtained pregnancies in cases of globozoospermia. Jean Parinaud (France) reported results from an acrosome reacted sperm selection technique using immunobeads coated with an anti-inner acrosomal membrane antibody. This method allows recovery of a 90% acrosome reacted sperm population and is a new tool to study characteristics of the subpopulation capable of fertilizing eggs.
Acrosome plays an essential role in the fertilizing process and thus is a good candidate for contraception purposes. John Herr (USA) has isolated and cloned SP10 protein, localized in the matrix and membranes of the acrosome. When injected into baboons, SP10 induces antibodies production that inhibit in vitro fertilization in bovine species. This work is the first approach to vaccine contraception using acrosomal antigens.
In conclusion, Harrison contrasted the great progress, in understanding capacitation and acrosome reaction processes in vitro, with the difficulty in specifying which mechanisms are used in vivo.
Jean Parinaud, France