EFFECT OF FOLLICLE CELLS AND STEROIDOGENESIS ON MATURATION AND FERTILIZATION INVITRO OF PIG OOCYTES

Follicle cell numbers, steroidogenesis, and culture conditions were manipulated in a series of experiments designed to identify their effect during in vitro maturation of pig oocytes upon meiotic cycle progression, sperm penetration and male pronuclear formation. In Experiment 1 a static culture system was used to determine the effect of follicle cell numbers on maturation. When the follicle cell number was 3.5 x 10(3) cells per complex or less, cell cycle progression to metaphase II (M-II) and male pronuclear formation were both low (<30%). Increasing follicle cell numbers to 15 x 10(3) per complex significantly enhanced both meiotic maturation (93%) and male pronuclear formation (83%). A further 10-fold increase in follicle cell numbers (150 x 10(3)), however, reduced subsequent male pronuclear formation (33%). The purpose of Experiment 2 was to determine whether the observed follicle cell effects were influenced by the type of culture system used for oocyte maturation. Comparisons were made between both static and non-static forms of culture. Meiotic progression was significantly depressed when non-static culture systems were used for the maturation of oocyte-cumulus complexes containing low numbers of somatic cells (<15 x 10(3) cells). However, the addition of somatic cells (follicle shells) to non-static, but not static, culture systems significantly enhanced male pronuclear formation in all groups irrespective of the number of cumulus cells in the oocyte complex. Experiment 3 investigated the effects of the steroid environment on maturation and male pronuclear formation. The addition of high levels of progesterone (1 mg/ml) from the beginning of maturation was detrimental for maturation. More interestingly, inhibition of steroidogenesis using an inhibitor of 20alpha-cholesterol oxidase was without effect on meiotic progression or sperm penetration but reduced male pronuclear formation. The combined results demonstrate the importance of appropriate somatic support of oocytes as measured by their subsequent capacity to induce normal pronuclear formation after fertilization. Furthermore, studies on the required hormonal environment support published data showing that progesterone is required during maturation for subsequent male pronuclear formation.