Walker, David Joshua, authorSeidel, George E., Jr., advisorClay, Colin M., committee memberBruemmer, Jason E., committee memberAnthony, Russell V., committee member2022-04-072022-04-072010https://hdl.handle.net/10217/234631Covers not scanned.Print version deaccessioned 2022.Oocyte maturation is a complex process consisting of signal transduction, ultrastructural changes, and mRNA transcription, translation, storage, and degradation. In vitro-matured oocytes initiate maturation in response to removal from an inhibitory follicular environment while in vivo-matured oocytes mature in response to the LH surge. Oocytes matured in vivo lead to more successful embryo production than those matured in vitro. This research concerned study of expression levels and action of selected transcripts involved in RNA processing that occur in in vivo oocyte maturation. The first experiment focused on the inability of GnRH to induce oocyte maturation in superstimulated cows during the luteal stage of the estrous cycle. Superstimulated cows were treated with PGF2a and GnRH to induce in vivo maturation or were treated with GnRH without PGF2a to induce an LH surge at 0, 3, 12, or 24 h before aspiration. While treatment with GnRH caused an increase in LH over no GnRH treatment, it was a smaller increase than that observed in cows treated with PGF2a before GnRH treatment (P<0.001) (No GnRH; 0.84 ng/mL, GnRH: 9.45 ng/mL, PGF2a: 93.86 ng/mL). Thus, increases in LH were sufficient to initiate epiregulin mRNA transcription in granulosa cells (P<0.06) with the greatest expression levels after 6h. However, germinal vesicle breakdown did not occur as reliably in cows with an intact corpus luteum 23 h after GnRH (treated with PGF2a 36 h prior to GnRH injection) (GV stage oocytes; Oh; 79%, 6h: 58%, 12h: 83%, 24h: 60%, vs. controls 6%)(P<0.001). In addition, cAMP levels remained stable in oocytes from cows treated with GnRH in the presence of progesterone regardless of post injection time, while control oocytes had a slightly elevated level of cAMP (Oh: 4.95, 6h: 3.98, 24h: 4.12, control: 7.41 fmol) (P>0.1). Phosphodiesterase 3A mRNA levels were unaffected by any treatment (P>0.10). These data suggest that although the stimulatory signaling of LH and epiregulin occur, cAMP levels are unaffected by GnRH treatment in the presence of progesterone. The second experiment evaluated mRNA concentrations in bovine oocytes of four transcripts involved in RNA regulation in mammalian cells; CUG-BP, PARK, eIF-4E, and PAP-1. In vivo- and in vitro-matured oocytes were collected 0, 3, or 6 hours after initiation of maturation via GnRH injection for in vivo-maturation and aspiration from follicles for in vitro-maturation. eIF-4E and PARN mRNA concentrations increased over time in both in vitro- and in vivo-matured bovine oocytes (P<0.05). In vivo-matured oocytes contained more eIF-4E mRNA molecules than in vitro-matured bovine oocytes (P<0.10). CUG-BP and PAP-1 concentrations remained stable over the first 6 h of maturation and were similar in the in vivo- and in vitro-matured oocytes. The final experiment concerned deadenylation patterns for the cyclin B1 3’ untranslated region (UTR) and GDF-9 3’UTR with a poly(A) tail of 60 adenosines. Bovine oocytes were injected with radiolabeled constructs after 0, 5, or 19 h of maturation and then cultured for 0, 1, or 3 h. Radiolabeled RNA was recovered from oocytes after culture and analyzed for changes in construct size, reflective of deadenylation or general degradation. Cyclin B1 underwent deadenylation before being degraded regardless of the stage of oocyte maturation. Analysis of gels showed an intermediate with 0 adenosines (AO) in the Cyclin B1 injected oocytes, while those injected with GDF-9 displayed no such intermediate. These findings indicate that injected GDF-9 transcript remains stable with a poly(A) tail of 60 adenosines or is simply degraded randomly in bovine oocytes matured for 0, 5, or 19 h. Deadenylation of Cyclin B1 mRNA begins immediately in bovine oocytes resulting in degradation of the Cyclin Bl.doctoral dissertationsengCopyright and other restrictions may apply. User is responsible for compliance with all applicable laws. For information about copyright law, please see https://libguides.colostate.edu/copyright.OvumCattle -- ReproductionLuteinizing hormone releasing hormoneMessenger RNAText