Studies were conducted to evaluate the susceptibility of avian ovarian granulosa cells to apoptosis when incubated in vitro and to relate this relative susceptibility to both the stage of follicle development from which granulosa cells were collected (atresia-prone vs. -resistant) and to the expression of a gene previously linked to the regulation of cell viability, bcl-xlong. Granulosa cells from slow growing, prehierarchal (4- to 8-mm diameter; atresia-prone) follicles were found to undergo rapid and progressively extensive apoptosis after incubation in defined medium for 6- 24 h (P < 0.05 vs. unincubated controls). By contrast, cells from the largest preovulatory (F1) follicle, as well as from follicles most recently recruited into the follicle hierarchy (9- to 12-mm diameter), showed significantly less low molecular wt labeling at 6 h of incubation (P < 0.05 vs. 4- to 8-mm follicles). Furthermore, the amount of low molecular wt labeling did not significantly increase in cells from either stage of follicle development at 12 or 24 b of incubation (P > 0.05 vs. 6 h incubation). This biochemical indication of ongoing apoptosis in prehierarchal follicle granulosa cells was confirmed by an increased incidence of pyknotic nuclei detected by morphological analysis. Thus, increased susceptibility to apoptosis in incubated prehierarchal follicle granulosa cells is correlated with the high rate of follicle atresia that is known to occur at this stage of development in vivo. Recombinant human FSH (100 mIU) and transforming growth factor-α (3.3 nM) partially suppressed apoptosis in prehierarchal follicle granulosa cells after 6 h of incubation (by 46 -57%; P < 0.05 vs. control), as did the cAMP analog, 8-Br-cAMP (1 mM; by 59%; P < 0.05). A single form of the bcl-2- related gene, bcl-x, was detected in hen ovarian tissues; this transcript corresponded to bcl-xlong, the death-suppressing form of bcl-x. The highest levels of bcl-xlong messenger RNA were found in granulosa tissue from preovulatory follicles, with significantly lower levels detected in prehierarchal follicle granulosa tissue (P < 0.05). Elevated expression of bcl-xlong in preovulatory follicles was correlated to increased resistance to the process of apoptosis, in vitro, and the virtual absence of follicle atresia at this stage of development, in vivo. We conclude that there is a direct relationship between the inherent susceptibility of avian granulosa cells to apoptosis and the high rate of follicle atresia in follicles not yet selected into the preovulatory hierarchy. Moreover, our results are consistent with the proposal that the expression of death-suppressing genes, including bcl-xlong, is capable of rendering cells resistant to the process of apoptosis. The findings reported herein provide the foundation for a novel model with which to further elucidate molecular mechanisms related not only to the initiation of follicle atresia, but also events associated with the process of follicle selection.
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