In the ovary, greater than 99% of the follicles present at birth are destined to degenerate during life. In humans, less than 400 of the more than 400,000 follicles found at puberty will eventually ovulate whereas the overwhelming majority of follicles undergo atresia. Although follicular atresia plays a critical role during the recruitment of follicles for ovulation, the exact mechanism of this process is unknown. In chicken and porcine ovaries, atretic follicles can be morphologically distinguished from their healthy counterparts of the same size. Adapting a sensitive 3'-end labeling method for DNA analysis, we identified internucleosomal cleavage of cellular DNA in atretic (but not normal) follicles of both animal species, resembling that found during programmed cell death in embryogenesis, autoimmune T-cell removal and prostate regression. The present findings provide a basis for elucidating the hormonal signals involved in the initiation of follicular atresia during follicle recruitment, reproductive aging and premature ovarian failure.

The reproductive strategy for avian species that produce a sequence (or clutch) of eggs is dependent upon the maintenance of a small cohort of viable, undifferentiated (prehierarchal) follicles. It is from this cohort that a single follicle is selected on an approximate daily basis to initiate rapid growth and final differentiation before ovulation. This review describes a working model in which follicles within this prehierarchal cohort are maintained in an undifferentiated state by inhibitory cell signaling until the time of selection. Ultimately, follicle selection represents a process in which a single undifferentiated follicle per day is predicted to escape such inhibitory mechanisms to begin rapid growth and final maturation before ovulation. Several processes initiated within the granulosa cell layer at selection are dependent upon G protein-coupled receptors signaling via cyclic adenosine monophosphate (cAMP), and several critical processes are described herein. Finally, reference is made to several practical outcomes that can result from understanding the process of selection, including applications within the poultry industry. Proximal factors and processes that mediate follicle selection can either extend or decrease the length of the laying sequence, and thus directly influence overall egg production. In particular, any aberration that results in the selection of more than one follicle per day will result in decreased egg production. More generally, in wild birds these processes are modified by prevailing environmental conditions and by social interactions to influence clutch size. The elucidation of cellular processes that regulate follicle selection can assist in the development of assisted reproductive technologies for application in threatened and endangered avian species.

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The present studies were conducted to evaluate whether apoptosis occurs during spontaneous and prostaglandin F2 alpha (PGF2 alpha)-induced luteolysis and, if so, to determine the relationship between the onset of luteolysis and oligonucleosome formation (a characteristic of apoptosis). In the first study, nine normally cycling heifers were ovariectomized (ovx) during the midluteal phase (day 10 or 15; day 0 = estrus) or after luteal regression (day 19; n = 3/time point). While there was no evidence of oligonucleosome formation in DNA from corpora lutea (CL) collected on days 10 and 15, each CL collected on day 19 exhibited DNA fragmentation, represented by distinct bands of DNA in approximately 185-basepair multiples. In the second study, heifers were ovx (n = 5; controls) or given 25 mg PGF2 alpha 15-16 days after estrus. Heifers receiving PGF2 alpha were subsequently ovx 4, 8, 12, 24, or 48 h (n = 5/time point) after the injection of PGF2 alpha. The concentration of progesterone in venous sera collected at ovx was not different (P > 0.20) in control and 4 h groups, but was decreased (P < 0.01) in the 8, 12, 24, and 48 h groups. Total CL weight (mean +/- SEM; grams) did not change (P > 0.10) from 0 h (controls) to 24 h after injection (range, 3.2 +/- 0.5 to 4.1 +/- 0.6), but decreased (P < 0.06) to 2.0 +/- 0.3 at 48 h. With ethidium bromide (EtBr) staining, no oligonucleosome formation was detected in CL collected from 0-12 h after PGF2 alpha injection. However, pronounced oligonucleosome formation was observed in all 10 CL collected 24 and 48 h after the injection of PGF2 alpha. The absence of oligonucleosomes in 0 and 4 h samples was confirmed by the more sensitive technique of 3'-end labeling of DNA fragments. Some samples in both the 8 and 12 h groups had slight oligonucleosome formation, while all samples in the 24 and 48 h groups showed evidence of intense oligonucleosome formation. Histological analysis of tissue sections indicated an increase (P < 0.001) in the percentage of degenerated luteal cells in the 24 and 48 h groups compared to that in the 0-12 h groups. These data indicate that apoptosis occurs during both spontaneous and PGF2 alpha-induced luteal regression in cattle; however, apoptosis, as indicated by oligonucleosome formation, is not apparent until after serum progesterone concentrations have begun to decrease.(ABSTRACT TRUNCATED AT 400 WORDS)

Previously described models for avian ovarian steroidogenesis, using mature, 25-40-mm preovulatory follicles as the source of tissues, were based on the assumption that interaction of the granulosa layer, as the predominant source of progesterone, with adjacent theca cells is required for maximal production of C21, C19, and C18 steroids. In the present study, we evaluated the steroidogenic capacity of ovarian cells isolated from less mature, 6-8-mm and 9-12-mm follicles in the chicken ovary (representative of a stage of development 2-3 wk prior to ovulation) to determine at which stage of follicular development granulosa and/or theca cells become steroidogenically competent. Granulosa cells collected from 6-8-mm follicles were found to be virtually incompetent to produce steroids, containing extremely low basal levels of progesterone (12 pg/5 x 10(5) cells) and failing to respond with increased steroid output following a 3-h exposure to ovine LH (oLH; 0.1 and 100 ng/0.5 ml), ovine FSH (oFSH; 100, 500, and 1,000 ng/0.5 ml), 8-bromo-cyclic adenosine monophosphate (8-bromo-cAMP; 0.33 and 3.33 mM) or 25-hydroxycholesterol (250 and 2,500 ng/0.5 ml). However, addition of pregnenolone (20 and 200 ng/0.5 ml) to granulosa incubations resulted in significantly increased progesterone levels. Granulosa cells of 6-8-mm follicles also failed to increase cAMP formation in the presence of oLH (10, 100, and 1,000 ng/0.5 ml) and 3-isobutyl-1-methylxanthine (IBMX; 10 microM), but responded to stimulation with 1,000 ng oFSH (4.4-fold increase over basal) or 10 microM forskolin (32-fold increase over basal) in the presence of IBMX. In contrast, granulosa cells isolated from 9-12-mm follicles and incubated for 3 h in vitro were found to contain basal progesterone levels 200-fold higher than those found in granulosa cells of 6-8-mm follicles. Furthermore, granulosa cells of 9-12-mm follicles markedly increased progesterone production following incubation in the presence of oFSH (100-1,000 ng/0.5 ml), 8-bromo-cAMP (0.33 and 3.33 mM), or 25-hydroxycholesterol (250 and 2,500 ng/0.5 ml). However, these granulosa cells remained unresponsive to oLH (0.1, 10, and 100 ng/0.5 ml), failing to increase cAMP accumulation (in the presence of IBMX) and progesterone output. Theca cells of small yellow follicles were found to produce measurable basal levels of progesterone, androstenedione, and estradiol, and levels of each steroid were significantly increased following a 3-h challenge with oLH, 8-bromo-cAMP, 25-hydroxycholesterol, and pregnenolone.(ABSTRACT TRUNCATED AT 400 WORDS)

Both the viability of hen prehierarchal follicles and subsequent differentiation associated with the selection of a single follicle per day into the preovulatory hierarchy depend on circulating FSH and the expression of FSH receptor (FSH-R) in granulosa cells. The present study addresses mechanisms that mediate both basal expression plus selective up-regulation of FSH-R mRNA in granulosa cells from prehierarchal follicles. Results demonstrate that FSH-R mRNA is both expressed and functional in granulosa cells collected from growing prehierarchal follicles as small as those of 1-2 mm in diameter, as indicated by rapid induction of steroidogenic acute regulatory (StAR) protein expression by FSH in vitro. Real-time polymerase chain reaction determined that relative FSH-R expression within the granulosa layer from individual prehierarchal follicles of 6-8 mm in diameter was similar among the 8-13 follicles within this cohort, with the notable exception that the granulosa layer from a single follicle (presumably the selected follicle) showed elevated expression. Levels of FSH-R mRNA expression were enhanced by both recombinant human (rh) transforming growth factor (TGF) beta1 and, to a lesser extent, rh-activin A after 20 h of culture. This stimulatory effect was effectively blocked by mitogen-activated protein (MAP) kinase signaling induced by TGF alpha treatment. Finally, inhibition of MAP kinase signaling, using the selective inhibitor U0126, promoted FSH-R expression and further enhanced TGF beta1-induced FSH-R expression in vitro. Collectively, results suggest that premature granulosa cell differentiation normally is suppressed by tonic MAP kinase signaling. At the time of follicle selection, a release from inhibitory MAP kinase signaling is proposed to occur, which enables the full potentiation of FSH-R expression mediated by intrafollicular factors.

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