Fundamental aspects of bovine oocyte maturation : the role of estradiol, VIP and GHRH

Abstract

Chapter 1 presents an overview on the aspects of oocyte maturation. Growth hormone (GH), released from the pituitary by the stimulus of GHRH, increases cumulus expansion and improves cytoplasmic maturation in bovine oocytes. GHRH is also expressed in extraneural tissues suggesting that GHRH also plays a regulatory role in peripheral tissues. Therefore in Chapter 2 we studied the role of GHRH and its related peptide VIP on the nuclear maturation and cumulus expansion. We observed that exposure of bovine cumulus oocyte complexes (COCs) to GHRH or VIP did not affect nuclear maturation or cumulus expansion, but it retarded cytoplasmic maturation. At the time of the LH surge, follicular fluid of preovulatory follicles has a high concentration of estradiol (E2), but 6h after the LH peak a sharp decline of E2 occurs, coinciding with the germinal vesicle breakdown (GVBD). Although most in vivo effects of E2 are well identified, the role of E2 during in vitro maturation (IVM) remains contradictory. In Chapter 3 we investigated the effects of E2 on bovine oocyte IVM and subsequent embryo development. Both COCs and denuded oocytes (DO) were used to study whether effects of E2 are exerted via cumulus cells or directly on the oocyte. We observed that presence of E2 during IVM decreased the percentage of metaphase II (MII) stage oocytes and increased the percentage of nuclear aberrations. This effect of E2 was stronger in DO compared with COCs. Concomitant presence of FSH and E2 during IVM did not influence the proportion of MII oocytes, although nuclear aberrations were still observed. Presence of E2 during IVM also decreased the blastocyst rate. However, when FSH was present, E2 had no effect on the cleavage rate and blastocyst formation. We concluded that supplementation of E2 to maturation medium negatively affects bovine oocyte nuclear maturation and subsequent embryo development. Although these effects are attenuated in the presence of FSH, we suggest omission of E2 in maturation protocols of bovine oocytes. To investigate whether the effects of E2 were mediated via genomic or nongenomic pathways, we cultured oocytes in the presence of E2 either before or after GVBD (the period when transcription ceased) (Chapter 4). Additionally, an estradiol membrane-impermeable conjugate (E2-BSA) was used to investigate the presence of a putative membrane receptor for estradiol. By using RT-PCR we observed that oocytes expressed estrogen receptor (ER)β mRNA, while cumulus cells expressed both ERα and ERβ mRNA. Exposure to E2 during the first 8h of culture (before GVBD) induced a block at the metaphase I stage. However, the presence of E2 after GVBD induced an increase of oocytes exhibiting nuclear aberrations. Exposure of oocytes to E2-BSA did not affect nuclear maturation, suggesting that the effects of E2 on in vitro nuclear maturation are not exerted via a plasma membrane receptor. Although oocytes from small-sized antral follicles can resume meiosis, the blastocyst yield obtained with such oocytes is low. It was hypothesized that chemically preventing GVBD by roscovitine (ROS), until the initiation of IVM might allow the oocyte more time to undergo cytoplasmic maturation and consequently acquire a higher developmental competence. In Chapter 5 we demonstrated that exposure to E2, during temporary inhibition of the GVBD with ROS, affected neither nuclear nor cytoplasmic maturation of oocytes originating from small-sized follicles. Possibly, the increase of E2 during follicular growth in vivo is more important for selection of the dominant follicle than for the cytoplasmic maturation of the oocyte. Finally, Chapter 6 presents a summarizing discussion