The specific aims of the proposed research are to test the hypotheses that the rate of phospholipid turnover in immature mammalian oocytes is altered during the resumption of meiosis, and that phospholipid turnover is prerequisite for maturation. Attention will be focused particularly on the role of phosphoinositides in this process. Phospholipid turnover will therefore be studied (i) in the immature oocyte; (ii) just prior to the period at which the oocytes become irreversibly committed to mature; (iii) at various stages during maturation; (iv) and subsequent to this process. Bovine oocytes will be mass harvested and labeled with (32P)orthophosphate, (14C)arachidonate or (3H)myo-inositol. Prelabeled cells will subsequently be incubated either in culture medium conducive to spontaneous maturation or in medium containing testosterone and follicle stimulating hormone to maintain transient meiotic arrest. Oocytes will be removed at intervals representing different meiotic stages, previously determined by cytogenetic analysis. After cellular extraction, individual phospholipids and diacylglycerol will be separated and identified by thin layer chromatography, and inositol phosphates will be separated by anion exchange chromatography. The distribution of radioactivity in the individual phospholipids, inositol phosphates and diacylglycerol will be compared among oocytes at different stages of meiosis. The direct effects of diacylglycerol and inositol 1,4,5-trisphosphate on meiotic maturation will be examined. Immature oocytes will be incubated with a diacylglycerol analogue. Inositol 1,4,5- trisphosphate will be introduced either into permeabilized oocytes or by intracellular iontophoretic injection into intact cells. The long-term objectives of the proposed research are to elucidate the mechanisms by which phosphoinositide metabolism can override adenosine 3',5'-cyclic monophosphate maintained meiotic arrest, thereby regulating the maturation process. This research area relates directly to the health of mankind. It is crucial to understand the biochemical mechanisms which regulate oocyte maturation in order to prevent abnormal oocyte development which may result in potential embryonic defects, and in order to treat human infertility that may occur as a direct result of alterations in oocyte development.
