The in vitro perfused rabbit ovary preparation has been effective in studying local factors involved in the ovulatory process and for evaluating effects of specific agents on phenomena associated with ovulation. Using this in vitro model: (1) observations will be made of follicular development and rupture, ovarian smooth muscle contractions, oocyte maturation, and fertilizability of in vitro ovulated ova and of follicular oocytes; (2) progesterone, estradiol, and prostaglandin assays will be performed on serial aliquots of perfusion effluent; (3) morphology of the wall of preovulatory follicles will be examined by TEM and SEM and perifollicular microvasculature of corrosion casts by SEM. Observations will be made under control conditions to compare characteristics of ovulation in vitro with those in vivo. The influence on follicular morphology of conditions known to stimulate (PGF2Alpha, histamine, norepinephrine, perfusion in Ca++/Mg++ free medium) or inhibit (indomethacin, EGTA, phenoxybenzamine, phentolamine) the ovulatory process in vitro will be further investigated using SEM and TEM. Additional studies will be performed to examine the effects of these inhibitory and stimulatory agents as well as the effects of clomiphene citrate and calcium deprivation on ovum fertilizability. A hypothetical enzymatic pathway leading to follicle wall disruption will be tested by determining the effects on ovulation of inclusion in the perfusate of specific enzyme inhibitors and mediators of enzyme activity. The relationship of ovarian enzymatic activity to ovarian prostaglandins in preovulatory phenomena will also be evaluated. The effect of oocyte maturation inhibitor (OMI) on intrafollicular oocyte development will be studied in the in vitro model. Ultimately in vitro ovarian perfusion may serve as a basis for assaying OMI activity. The major objective of this proposal is to delineate the sequence of events leading to disruption of the follicle wall and release of an oocyte capable of fertilization and to determine the specific points(s) at which individual factors influence these processes.
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