Follicle oocyte interactions and embryo development in the bovine: an in vitro study

Abstract

In current systems for production of bovine embryos in vitro, the frequency of development to the blastocyst stage and subsequently production of live offspring is lower than that observed in vivo. Oocytes are aspirated from a wide range of follicle sizes (2-8 mm in diameter) for in vitro maturation and fertilisation. During development oocytes increase in size with enlargement of follicles, this may reflect different cytoplasmic maturation levels of the oocytes. Oocytes from larger follicles have a higher developmental competence. In other word, oocytes from small antral follicles which are more abundant on the ovaries, have an intrinsic deficiency in some vital factors, which are important for embryonic development. In vivo, follicle enclosed oocytes are arrested at germinal vesicle (GV) stage. When aspirated from the follicle they are released from the inhibitory function of the follicle wall and develop to metaphase of the second (Mil) meiotic division. It was hypothesised that if oocytes could be cultured in vitro under conditions which maintain GV arrest, prior to in vitro maturation, then the oocyte may have the opportunity to synthesise or modify the factors which are required for subsequent embryonic development. To this end a system was established for culture of intact large antral follicles of 3-8mm in diameter for different periods of time up to 7 days. The majority of oocytes (96.8%) recovered following 24 hours of follicle culture remained at the GV stage. After recovery, 80% of these oocytes resumed meiosis and developed to the Mil stage. However, with increasing periods of follicle culture, both the number of oocytes remaining arrested at GV stage and also the number able to resume meiotic maturation were significantly reduced. On the basis of these results, follicle culture for a period of 24 hours was used for the following experiments. In a comparative study, follicle culture derived oocytes resulted in a significantly higher rate of blastocyst production than directly aspirated oocytes used as the control group. However, there was no difference in embryo quality based on total cell number. It was concluded that oocytes gain a higher developmental competence during follicle culture.Histological and ultrastructural aspects of both the follicles and oocytes were analysed following different periods of follicle culture. With increasing periods of follicle culture, the number of pycnotic cells in the granulosa and cumulus compartments increased and some oocytes resumed meiosis within the follicle. The cumulus cells became expanded and abnormal chromatin condensation was observed in the GV. The ultrastructure of oocytes was analysed using transmission electron microscopy. Immature oocytes recovered following 24 hours of follicle culture and exhibited characteristics typical of immature oocytes. However, oocytes recovered following longer periods of follicle culture, showed some abnormal ultrastructure. It was concluded that follicle culture up to 24 hours has no detrimental effect on oocyte quality and structure.The patterns of total and de novo proteins of oocytes were studied. During oocyte maturation, some new protein bands were observed as evidenced by both 1D and 2DSDS PAGE followed by silver staining. There were differences in both the protein profile and concentrations of some individual proteins between immature oocytes from aspirated and follicle cultured groups. Any differences in de novo protein synthesis during follicle culture could not be demonstrated by J5S methionine labelling due to technical difficulties. Following maturation, oocytes from both groups showed identical protein profiles. It was concluded that follicle culture has no detrimental effect on protein synthesis in the oocyte.Insulin is a known regulator of ovarian function and is also a follicular survival factor. The effects of addition of insulin (5pg/ml) during bovine antral follicle culture on oocyte quality and development, protein profile and Insulin-like Growth Factor Binding Proteins (IGFBPs) expression were examined. Insulin adversely affected the cleavage rate, however, there were no differences in the proportion of cleaved embryos which subsequently developed to blastocyst stage. The ultrastructure and total protein profile of oocytes from the insulin positive group indicated that these oocytes gain a cytoplasmic structure similar to Mil oocytes. IGFBPs profiles in follicular fluid and culture media in small (<4mm) and large (>4mm) follicles were demonstrated. Two strong bands of 25 (IGFBP-4) and 34 (IGFBP-2) kDa disappeared from both the follicular fluid and culture media in large follicles. Since the concentrations of these proteins have been reported to increase during follicular atresia, it was concluded that insulin prevents follicular atresia in larger follicles and promotes cytoplasmic features of oocyte maturation. However, insulin does not improve oocyte quality or pre implantation embryo development in vitro.Apoptosis is known to be the mechanism by which large follicles become atretic. Many hormones and growth factors have been reported as follicular survival factors in vivo. The effects of various factors on prevention of apoptosis in the granulosa cells from large antral follicle were assessed both individually and in combinations by adding to the culture media during 2 days of follicle culture. Growth hormone (GH) and luteinising hormone (LH), did not prevent apoptosis. In contrast, IGF-I and FSH supported follicle survival and addition of a combination of oestradiol, FSH, LH, IGFI, EGF and GH completely prevented apoptosis. When the above combination was added to the culture media in the presence or absence of 10% FCS there were no differences between the two groups, whilst follicles cultured in the absence of both showed a severe pattern of apoptotic cell death.In conclusion, culture of bovine large antral follicles in vitro for up to 24 hours maintain GV arrest and has no subsequent detrimental effect on oocyte quality. In fact during this culture period it appears that the oocytes acquire a greater developmental competence perhaps by de novo synthesis of proteins, mRNAs or other factors which are necessary for subsequent embryonic development or by post translational or post transcriptional modifications. The addition of factors which support follicular survival to the culture media did not improve the rate of subsequent embryo production following follicle culture. Therefore to improve oocyte quality other known or unknown factor (s) may be required to supplement the culture media. Alternatively improvements may be achieved through changes in the composition of culture media or the follicle culture system.