B. Mairet

Bio: B. Mairet is an academic researcher. The author has contributed to research in topics: Propidium iodide & Fluo-3. The author has an hindex of 1, co-authored 1 publications receiving 168 citations.

Flow cytometric studies of bicarbonate‐mediated Ca2+ influx in boar sperm populations

Abstract: Boar spermatozoa loaded with the Ca2+ probe fluo-3 were incubated in various Tyrode's-based media similar to those used for in vitro fertilization (IVF), and samples were then analysed by two-colour flow cytometry; propidium iodide was included in the media to detect membrane-damaged ("dead") cells. If media contained bicarbonate/CO2 (a component thought to promote capacitation), part of the live sperm population experienced a considerable influx of Ca2+ into both head and tail compartments. The percentage of responding cells reached a maximum after about 30 min, but both during and after this period there was also a steady increase in the number of dead cells. This bicarbonate-mediated increase in cell death took place in the absence of external Ca2+. Evidence was obtained that the entry of propidium iodide was preceded by a change in permeability of the plasma membrane, detectable by leakage of carboxydichlorofluorescein, and it was therefore deduced that the Ca2+ influx detected by fluo-3 was due to destabilization of the plasma membrane. A similar response could be produced by both caffeine and papaverine (best known as phosphodiesterase inhibitors), but neither cyclic AMP nor activators of adenylate cyclase had any effect. There was no influence of substrate on the process, but, in comparison to poly(vinyl alcohol), serum albumin enhanced it. The precise relevance of this destabilization to capacitation is not yet clear, but it seems significant that the process is mediated or enhanced by components often specifically included in IVF media, and that different individual cells respond after different times.

The capacitating agent bicarbonate induces protein kinase A-dependent changes in phospholipid transbilayer behavior in the sperm plasma membrane.

Abstract: A flow cytometric procedure was used to follow the effect of bicarbonate, a key inducer of sperm capacitation in vitro, on the transbilayer behavior of C6NBD-phospholipids in the plasma membrane of living acrosome-intact boar spermatozoa under physiological conditions. In the absence of bicarbonate, 97% of C6NBD-phosphatidylserine and 78% of C6NBD-phosphatidylethanolamine was rapidly translocated from the outer leaflet to the inner, whereas relatively little C6NBD-phosphatidylcholine and C6NBD-sphingomyelin was translocated (15% and 5%, respectively). Inclusion of 15 mM bicarbonate/5%CO(2) markedly slowed down the rates of translocation of the aminophospholipids without altering their final distribution, whereas it increased the proportions of C6NBD-phosphatidylcholine and C6NBD-sphingomyelin translocated (30% and 20%, respectively). Bicarbonate activated very markedly the outward translocation of all four phospholipid classes. The changes in C6NBD-phospholipid behavior were accompanied by increased membrane lipid disorder as detected by merocyanine 540, and also by increased potential for phospholipase catabolism of the C6NBD-phospholipid probes. All three changes were mediated via a cAMP-dependent protein phosphorylation pathway. We suspect that the changes result from an activation of the non- specific bidirectional translocase ('scramblase'). They have important implications with respect to sperm fertilizing function.

Fertilization and subsequent development in vitro of pig oocytes inseminated in a modified tris-buffered medium with frozen-thawed ejaculated spermatozoa.

Abstract: The present study examined the penetrability of pig oocytes by frozen-thawed ejaculated boar spermatozoa, prepared by the pellet method, coincubated in a modified Tris-buffered medium. Subsequent embryonic development of fertilized oocytes was also determined. Porcine oocyte-cumulus complexes were cultured in BSA-free North Carolina State University (NCSU) 23 medium containing porcine follicular fluid (10%), cysteine (0.1 mg/ml), and hormonal supplements (eCG and hCG: 10 IU/ml each) for 20-22 h. They were then cultured in the same medium but without hormonal supplements for an additional 20-22 h. After culture, cumulus cells were removed and oocytes were coincubated for 12 h with three different (1 x 10(5), 5 x 10(5) and 1 x 10(6)/ml) sperm concentrations (experiment 1). In experiment 2, oocytes were coincubated with sperm (5 x 10(5)/ml) for 3, 6, 9, and 12 h. In experiment 3, at 6 h after coincubation with sperm at 5 x 10(5)/ml concentration, oocytes were transferred into NCSU 23 + 0.4% BSA medium. At 48 and 144 h, cleavage and blastocyst formation rates, respectively, were evaluated. Insemination with 1 x 10(5)/ml resulted in a 40% sperm penetration rate of oocytes with 16% polyspermy. Mean number of sperm (MNS) per oocyte was 1.2 +/- 0.1. At 5 x 10(5) and 1 x 10(6)/ml, penetration rate (84-87%) and polyspermy (57-64%) increased (p < 0.001), with no difference between the two concentrations. However, MNS per oocyte increased (p < 0.05) with increasing sperm concentration. Penetration rate was 31% at 3 h and increased (p < 0.001) at 6-12 h (80-88%), with no difference between these time points. Polyspermy increased (p < 0.05) in a time-dependent manner up to 9 h, with no difference between 9 and 12 h. Compared to 3 h, MNS per oocyte increased (p < 0.05) at 9 and 12 h, with no mean difference at 6 h. At 48 after culture, the cleavage rate was 40%, and at 144 h, the blastocyst rate was 19%. This study describes the cryopreservation of ejaculated boar semen by the pellet method and the successful in vitro fertilization of pig oocytes by frozen-thawed spermatozoa with subsequent development to the blastocyst stage.

Bicarbonate/CO2, an effector of capacitation, induces a rapid and reversible change in the lipid architecture of boar sperm plasma membranes

Abstract: Bicarbonate/CO2 is believed to be the key in vitro effector of sperm capacitation, a process which induces major changes in the sperm plasma membrane in preparation for fertilization. In a flow cytometric study, we examined the effect of bicarbonate on boar spermatozoa using merocyanine, an impermeant lipophilic probe which binds to plasma membranes with increasing affinity as their lipid components become more disordered. We found that bicarbonate causes a rapid increase in the ability of live boar spermatozoa to bind merocyanine. First detected about 100 sec after exposure to bicarbonate and largely complete by 300 sec, this increase appears to result from individual cells within the sperm population switching from a low merocyanine-binding state to a high binding state. The majority of live spermatozoa are capable of responding in this way, and do so in proportion to bicarbonate concentration, half-maximal response being induced by about 3 mM bicarbonate; however, overall population response varies greatly between ejaculates. Increased merocyanine stainability is observed over the whole surface area of the cell, and is reversible both with respect to temperature (it is only manifested above 30 degrees C) and with respect to presence of bicarbonate. A similar effect can be induced by phosphodiesterase inhibitors such as isobutylmethylxanthine, and enhanced by a permeant cyclic nucleotide analogue. We conclude that bicarbonate causes a major alteration in sperm plasma membrane lipid architecture, apparently by perturbing enzymic control processes. This novel action of bicarbonate may represent an initial permissive event in the capacitation sequence.

Bicarbonate stimulated phospholipid scrambling induces cholesterol redistribution and enables cholesterol depletion in the sperm plasma membrane

Abstract: Mammalian sperm cells are activated prior to fertilization by high bicarbonate levels, which facilitate lipoprotein-mediated cholesterol efflux. The role of bicarbonate and cholesterol acceptors on the cholesterol organization in the sperm plasma membrane was tested. Bicarbonate induced an albumin-independent change in lipid architecture that was detectable by an increase in merocyanine staining (due to protein kinase A-mediated phospholipid scrambling). The response was limited to a subpopulation of viable sperm cells that were sorted from the non-responding subpopulation by flow cytometry. The responding cells had reduced cholesterol levels (30% reduction) compared with non-responding cells. The subpopulation differences were caused by variable efficiencies in epididymal maturation as judged by cell morphology. Membrane cholesterol organization was observed with filipin, which labeled the entire sperm surface of non-stimulated and non-responding cells, but labeled only the apical surface area of bicarbonate-responding cells. Addition of albumin caused cholesterol efflux, but only in bicarbonate-responding cells that exhibited virtually no filipin labeling in the sperm head area. Albumin had no effect on other lipid components, and no affinity for cholesterol in the absence of bicarbonate. Therefore, bicarbonate induces first a lateral redistribution in the low cholesterol containing spermatozoa, which in turn facilitates cholesterol extraction by albumin. A model is proposed in which phospholipid scrambling induces the formation of an apical membrane raft in the sperm head surface that enables albumin mediated efflux of cholesterol.