GRANULOSA-CELL APOPTOSIS, AROMATASE-ACTIVITY, CYCLIC ADENOSINE-3',5'-MONOPHOSPHATE RESPONSE TO GONADOTROPINS, AND FOLLICULAR-FLUID STEROID-LEVELS DURING SPONTANEOUS AND INDUCED FOLLICULAR ATRESIA IN EWES

The aims of the present study in ewes were 1) to test the hypothesis t hat apoptosis in granulosa cells is one of the processes involved in t he structural demise of follicles;and 2) to define the temporal relati onships among the occurrence and degree of apoptosis in granulosa cell s, aromatase activity, production of cyclic AMP (cAMP) by granulosa ce lls in response to FSH or LH, concentrations of estradiol 17 beta (E(2 )) and progesterone in follicular fluid, and the characteristic morpho metric changes associated with the process of follicular atresia. To a ddress these aims, ewes were treated with either saline or steroid-fre e bovine follicular fluid (bFF) at 60 h after estrus, and ovarian foll icles greater than or equal to 3 mm diameter were recovered at 0, 12, 18, or 24 h later. Apoptotic granulosa cells were identified by the pr esence of oligonucleosomes after 3'-end labeling of extracted DNA with [P-32]alpha dideoxy ATP (ddATP). The degree of oligonucleosome format ion, based on the intensity of radiolabeling, was given an apoptosis s core (AP) of 0 (nondetectable), 1 (slight), 2 (moderate), or 3 (marked ). Moreover, a labeling index (Ll)was calculated from the amount of ra diolabeled ddATP incorporated into low-molecular weight (< 4.2 kb) DNA fragments. On the basis of morphometric criteria, 73% (141 of 194) of the follicles classified as healthy had apoptotic granulosa cells com pared to 86% (? a of 21) of the follicles classified as atretic. In th e bFF-but not saline-treated ewes, the concentrations of plasma FSH ha d declined to basal values at 12 h after treatment. At the beginning o f the treatment period, the degree of granulosa cell apoptosis was eit her undetectable (AP = 0, 47% of follicles) or slight (AP 1, 44% of fo llicles) in the majority of follicles. After 12 h from the bFF but not the saline injection, there was a significant increase in the proport ion of follicles (greater than or equal to 3 mm diameter) per ewe cont aining apoptotic granulosa cells (p < 0.001) and a significant decreas e in the number of follicles per ewe with aromatase activity (p < 0.05 ) and with follicular fluid E(2) > 20 ng/ml (p < 0.05). By 24 h after bFF treatment, apoptosis was evident in all follicles (greater than or equal to 3 mm diameter), fewer follicles contained FSH-responsive gra nulosa cells in terms of cAMP production (p < 0.05), and none were LH- responsive. A significant negative relationship was found between the degree of granulosa cell death as measured by LI and follicular fluid E(2) concentrations. In summary, the presence of apoptotic granulosa c ells in an appreciable number of follicles considered to be healthy by morphometric criteria and before their commitment to preovulatory enl argement and ovulation suggests that apoptosis may be a physiological process in developing follicles and/or a very early event in atresia. Collectively, these data provide strong evidence that granulosa cells may die by apoptosis before there is an appreciable decrease in the ca pacity of the granulosa cell layer as a whole to respond to gonadotrop ins or to produce E(2).