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      Sequential exposure of porcine cumulus cells to FSH and/or LH is critical for appropriate expression of steroidogenic and ovulation-related genes that impact oocyte maturation in vivo and in vitro.

      Reproduction (Cambridge, England)
      ADAM Proteins, genetics, Animals, Blotting, Western, methods, Cell Culture Techniques, Chorionic Gonadotropin, pharmacology, Cumulus Cells, drug effects, metabolism, DNA Primers, Dose-Response Relationship, Drug, Estradiol, analysis, Female, Follicle Stimulating Hormone, Follicular Fluid, chemistry, Gene Expression, Gene Expression Regulation, Developmental, Gonadotropins, Equine, Granulosa Cells, Luteinizing Hormone, Oogenesis, Progesterone, RNA, Messenger, Receptors, FSH, Receptors, LH, Receptors, Progesterone, Reverse Transcriptase Polymerase Chain Reaction, Superovulation, Swine, Testosterone

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          In this study, we collected follicular fluid, granulosa cells, and cumulus cells from antral follicles at specific time intervals following equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG) treatment of gilts. The treatment with eCG increased the production of estrogen coordinately with up-regulated proliferation of granulosa and cumulus cells. eCG also induced the expression of LHCGR and PGR in cumulus cells and progesterone accumulation was detected in follicular fluid prior to the LH/hCG surge. Moreover, progesterone and progesterone receptor (PGR) were critical for FSH-induced LHCGR expression in cumulus cells in culture. The expression of LHCGR mRNA in cumulus cells was associated with the ability of LH to induce prostaglandin production, release of epidermal growth factor (EGF)-like factors, and a disintegrin and metalloprotease with thrombospondin-like repeats 1 expression, promoting cumulus cell oocyte complexes (COCs) expansion and oocyte maturation. Based on the unique expression and regulation of PGR and LHCGR in cumulus cells, we designed a novel porcine COCs culture system in which hormones were added sequentially to mimic changes observed in vivo. Specifically, COCs from small antral follicles were pre-cultured with FSH and estradiol for 10 h at which time progesterone was added for another 10 h. After 20 h, COCs were moved to fresh medium containing LH, EGF, and progesterone. The oocytes matured in this revised COC culture system exhibited greater developmental competence to blastocyst stage. From these results, we conclude that to achieve optimal COC expansion and oocyte maturation in culture the unique gene expression patterns in cumulus cells of each species need to be characterized and used to increase the effectiveness of hormone stimulation.

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