Abstract
Phospholipase A2 (PLA2) activity has been shown to be involved in the sperm acrosome reaction (AR), but the molecular identity of PLA2 isoforms has remained elusive. Here, we have tested the role of two intracellular (iPLA2β and cytosolic PLA2α) and one secreted (group X) PLA2s in spontaneous and progesterone (P4)-induced AR by using a set of specific inhibitors and knock-out mice. iPLA2β is critical for spontaneous AR, whereas both iPLA2β and group X secreted PLA2 are involved in P4-induced AR. Cytosolic PLA2α is dispensable in both types of AR. P4-induced AR spreads over 30 min in the mouse, and kinetic analyses suggest the presence of different sperm subpopulations, using distinct PLA2 pathways to achieve AR. At low P4 concentration (2 μm), sperm undergoing early AR (0-5 min post-P4) rely on iPLA2β, whereas sperm undergoing late AR (20-30 min post-P4) rely on group X secreted PLA2. Moreover, the role of PLA2s in AR depends on P4 concentration, with the PLA2s being key actors at low physiological P4 concentrations (≤2 μm) but not at higher P4 concentrations (~10 μm).
Highlights
France, the **Centre Hospitalier Universitaire de Grenoble, Unité Fonctionnelle de Biochimie et Génétique Moléculaire, Grenoble F-38000, France, the ¶Division of Endocrinology, Metabolism and Lipid Research, Washington University School of Medicine, St
CPLA2␣ is a constitutive enzyme with a ubiquitous expression and an important role in lipid mediator release and vesicle trafficking [38, 39], and more importantly its enzymatic activity and distribution are modified in patients presenting asthenozoospermia [26], suggesting that it may be important for male fertility
Discussion independent PLA2s (iPLA2) and mGX secreted PLA2s (sPLA2) but Not cytosolic PLA2s (cPLA2)␣ Are Involved in the Mouse acrosome reaction (AR)—The importance of phospholipase activity in the sperm AR was demonstrated several decades ago [1]
Summary
Biological Preparation—All animal procedures were performed according to the French guidelines on the use of living animals in scientific investigations with the approval of the local Ethical Review Committee. mGX-KO mice (null for Pla2g10 gene) on a C57BL/6J background were obtained from Lexicon Inc. as described [36]. cPLA2␣ KO mice (null for the Pla2g4a gene) were obtained as described [37]. iPLA2 KO mice (null for the Pla2g6a gene) were obtained as described previously [23]. Sperm were capacitated for 35–55 min in M16 2% BSA (37 °C, 5% CO2) and introduced into droplets containing oocytes. For progesterone (P4) treatment, capacitated sperm (35 min) were incubated with P4 in M16 medium at 37 °C for the FEBRUARY 5, 2016 VOLUME 291 NUMBER 6. Preparation of the Cumulus-Oocyte Complex-conditioned Medium for Enzyme Immunoassay—Eggs were collected from mature OF1 females (6 weeks old) synchronized with 5 units of pregnant mare serum gonadotrophin and 5 units of human chorionic gonadotrophin. Cumulus-oocyte complexes harvested from 10 mice were placed in culture Petri dishes containing 250 l of M16 medium and incubated for 10 min, 1 and 2 h at 37 °C in a 5% CO2 atmosphere. For the calculation of intra-cumulus P4 concentrations, the total volume of the cumuli was estimated based on the number of oocytes per sample and on their average volume. Statistical tests with 2-tailed p values of Յ0.05 were considered significant
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