Submitted to: Biology of Reproduction
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 16, 2000
Publication Date: N/A
Interpretive Summary: Meiotic competence of oocytes dependent on the level of follicular/oocyte maturation, is likely expressed though factors produced by oocytes and somatic cells in the follicular environment. Because of our lack of knowledge of the factors controlling porcine oocyte maturation, effective methods control meiotic competence and induce superovulation to increase numbers of quality embryos have been elusive in swine. Protease inhibitors were used to test the hypothesis that members of the interleukin-1beta- converting enzyme family, caspases, and other proteases were active during apoptosis in cultured porcine granulosa cells. We found that caspases and possibly other proteases play a role in initiating apoptosis in cultured porcine granulosa cells. In contrast, a serine protease inhibitor, TPCK, was lethal to porcine granulosa cells through a non-apoptotic death mechanism. The results of this research will be useful to other scientists showing that endogenous proteases can be very disruptive causing death in cultured granulosa cells and hence a potential detriment to maturation of the oocyte.
Technical Abstract: Granulosa cells from gilts were cultured with seven different protease inhibitors to determine proteases play a role in apoptosis. Cells were incubated for 24 hr in Dulbecco's modified Eagles medium: Hams F12 (1:1) containing 1% FBS. Dead, membrane compromised cells were identified by uptake of ethidium homodimer (EH). The percent apoptotic (%Ao) cells was determined by DNA fluorescence flow cytometry. The percent EH permeable (%EHP) cells was 43 at 24 hr (no inhibitor). Serine protease inhibitors N- tosyl-L-phenylalanine chloromethyl ketone (TPCK) and N-p-tosyl-L-lysine chloromethyl ketone (TLCK) at 125 uM increased (p<.05) %EHP cells to 92 and 63, respectively, and were designated lethal. The inhibitors of ICE: VAD- FK; serine proteases: phenylmethylsulfonyl fluoride (PMSF); cysteine proteases: leupeptin and trans-epoxysuccininyl-L-leucylamido-(4-guadidino) butane (E64); and calpain enzyme: N-acetyl-leucine-leucine-normethional (AllnM) had no significant effect on %EHP and were designated nonlethal. %Ao cells increased (p<.05) from 1.7 to 29 between 0 and 24 hr (no inhibitor). Culture with > 1 uM of VAD-FK, TLCK, leupeptin, E64, and AllnM decreased (p<.05) %Ao cells by 50%. VAD-FK at 25 uM further decreased (p<.05) %Ao cells to 3.1. TPCK at 5 uM had no significant effect on %Ao cells; whereas at doses of 25 and 125 uM, %Ao cells was < 2. Compared to no inhibitor, 125 uM TPCK and TLCK, decreased (p<.05) estradiol production by 98 and 39%. Progesterone production decreased (p<.05) by 94% with 125 uM TPCK, whereas TLCK had no significant effect at the same dose. None of the nonlethal inhibitors had an effect on steroidogenesis. We conclude that a protease cascade and, specifically, ICE-like proteases play a role in initiating apoptosis in cultured porcine granulosa cells.