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Research Article |
Centre de recherche en reproduction animale, Faculté de médecine vétérinaire, Université de Montréal, St-Hyacinthe, Québec, Canada J2S 7C6
ABSTRACT
Prostaglandins are required for the ovulatory process, and their biosynthesis depends on the initial release of arachidonic acid from membrane phospholipids. We hypothesized that phospholipase A2 group IVA (PLA2G4A) expression is upregulated in granulosa cells (GC) at ovulation. We have characterized bovine PLA2G4A cDNA, and investigated its spatiotemporal regulation at the mRNA and protein levels in hCG-induced ovulatory follicles and in vitro, using forskolin-stimulated GC. Regulation of PLA2G4A mRNA expression was studied in GC obtained from bovine follicles collected at different developmental stages: small follicles (2–4 mm), dominant follicles at Day 5 (D5) of the estrous cycle, ovulatory follicles 24 h following injection of hCG, and corpus luteum at D5. PLA2G4A mRNA increased by 14-fold in GC of hCG-stimulated versus dominant follicles (P < 0.0001). Follicular walls obtained from ovulatory follicles recovered at 0, 6, 12, 18, and 24 h post-hCG injection showed an initial 16-fold increase in PLA2G4A transcript at 12 h that reached a 45-fold increase at 24 h, as compared to 0 h (P < 0.0001). Immunoblots of GC extracts showed an initial induction of the PLA2G4A protein at 18 h post-hCG, reaching a maximum at 24 h. Immunohistochemistry observations showed that PLA2G4A signal was mainly observed in mural GC compared to antral GC in hCG-stimulated follicles. Stimulation of cultured bovine GC with 10 µM of forskolin caused an increase in PLA2G4A mRNA and protein. Ovulation is associated with an LH/hCG-dependent induction of PLA2G4A in GC via the adenylyl cyclase/cAMP pathway.
cPLA2
, follicle, follicular development, gene expression, granulosa cells, ovary, ovulation, phospholipase A2, PLA2A, PLA2G1B, PLA2G4A
1 Supported by a Discovery Grant (to J.G.L.) from the Natural Sciences and Engineering Research Council of Canada (NSERC), a FQRNT grant from le Fonds Québécois de la Recherche sur la Nature et les Technologies to J.G.L., D.W.S., and J.S., and an Islamic Development Bank fellowship to M.N.D. Presented in part at the 37th Annual Meeting of the Society for the Study of Reproduction, August 1–4, 2004, Vancouver, British Columbia, Canada. The nucleotide sequence reported in this paper has been submitted to GenBank with accession no. AY363688.
2 Correspondence: Jacques G. Lussier, Faculté de médecine vétérinaire, Université de Montréal, P.O. Box 5000, St-Hyacinthe, Québec, Canada J2S 7C6. FAX: 450 778 8103; jacques.lussier{at}umontreal.ca
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