Factors affecting the developmental competence of pig oocytes matured in vitro.

Abstract

Pre-pubertal pig oocytes possess lower developmental competence than those from adult pigs following in vitro maturation (IVM). Previous studies have demonstrated that exposure of pre-pubertal oocytes to 1 mM dibutyryl cAMP (dbcAMP), a membrane permeable cyclic adenosine monophosphate (cAMP) analogue, for the first 20 h of IVM improves the rate of blastocyst development. Developmental competence of in vitro matured pig oocytes has been reported to increase with increasing follicle size. In this thesis, experiments were carried out using pre-pubertal and adult pig oocytes to investigate the relationship between donor age, intra-oocyte cAMP level and follicle size in terms of oocyte maturation and developmental competence. These experiments demonstrated that, while ovarian, follicular and oocyte morphology are immediately altered with the onset of puberty, pre-pubertal oocytes must be exposed to more than the first oestrous cycle to achieve improved developmental competence in vitro. Later experiments demonstrated that pre-pubertal oocytes accumulate less cAMP during IVM, undergo more rapid meiotic progression and display reduced rates of blastocyst development compared to in vitro matured adult oocytes. Treatment with dbcAMP for 22 h IVM increased the cAMP content of pre-pubertal oocytes, slowed meiotic progression during IVM and improved the rate of blastocyst formation. While the cAMP concentration of pre-pubertal oocytes was increased to levels similar to that of adult oocytes, rates of blastocyst formation remained lower, suggesting that additional factor(s) are required for oocyte maturation. This thesis also examined the follicle size cohorts that make up the 3-8 mm aspiration range on pig ovaries. The surface of pre-pubertal ovaries contained around double the number of 3 mm follicles compared with adult ovaries. Blastocyst development of pre-pubertal oocytes increased with increasing follicle size and was highest using oocytes from 5-8 mm follicles, while adult oocytes from all follicle size cohorts displayed similar high rates of blastocyst formation. The interaction between follicle size and cAMP content in pre-pubertal oocytes was examined next. Cumulus-oocyte complexes (COCs) from 3 mm follicles accumulated less intra-oocyte and inter-COC cAMP and displayed reduced cumulus expansion compared with COCs from 5-8 mm follicles. While dbcAMP treatment increased the cAMP content of oocytes from 3 mm follicles, it had no effect on the cAMP content of the whole COC. These findings suggest that inadequate levels of intra-oocyte cAMP during IVM contribute to the low developmental competence of pre-pubertal oocytes from 3 mm follicles, suggesting that cAMP transfer, production or degradation processes are incomplete. Analysis of steroid content from different follicle size cohorts revealed that the progesterone content of prepubertal follicular fluid (FF) increased with increasing follicle size, yet overall was lower than that of adults. This suggests that differences may exist in the gonadotropinstimulated steroidogenic activity of granulosa cells of pre-pubertal COCs from different follicle sizes. Since progesterone secretion did not differ between pre-pubertal and adult COCs, it appears that the downstream pathway from the granulosa cell response rather than the actual quantity of progesterone is important for subsequent maturation processes. These studies then examined gap junction communication (GJC) within the pre-pubertal COC during IVM to examine whether the positive effects of increasing follicle size and dbcAMP on intra-oocyte cAMP levels relates to improved cAMP transfer between the cumulus cell layer and oocyte. Cumulus cell-oocyte GJC during IVM was maintained for a longer period in pre-pubertal COCs from 3 mm follicles than in those from 5-8 mm follicles. Treatment with dbcAMP had minimal effect on GJC in either COC type, thus the dbcAMP-induced increase in intra-oocyte cAMP levels appears independent of GJC. Differences in GJC during IVM together with the COCs ability to increase intraoocyte cAMP levels during IVM, suggests that differences may exist in the quantity of gonadotropin receptors, which are responsible for cAMP production, within the cumulus layer of COCs from 3 mm compared with 5-8 mm follicles. In conclusion, this thesis has demonstrated that an increase in intra-oocyte cAMP is necessary during maturation for completion and synchronisation of maturation and high developmental competence of the pig oocyte. Comparison of 3, 4 and 5-8 mm follicle sizes in the pre-pubertal pig, as described here, provides an excellent model for further investigation into the role of cAMP and the other factors required for co-ordination of oocyte nuclear and cytoplasmic maturation and subsequent embryo production.