Stage of Ovarian Follicular Development Associated with the Initiation of Steroidogenic Competence in Avian Granulosa Cells1

Abstract

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)

Bone morphogenetic protein 2 inhibits FSH responsiveness in hen granulosa cells

“…It has previously been demonstrated that significant FSH-induced cAMP production in granulosa cells occurs only subsequent to follicle selection (e.g. in 9-12 mm follicles; Tilly et al 1991). Nevertheless, granulosa cells from follicles at all stages Figure 5 Inhibition of ERBB receptor kinase activity by pretreatment (20 min) with the selective inhibitor, AG1478 (AG; 10 mM), prevents the inhibitory effects of BMP2 (10 ng/ml) on TGFB (10 ng/ml)-and FSH (100 ng/ml)-induced FSHR expression.…”

“…Accordingly, granulosa cell differentiation associated with preovulatory follicle growth is proposed to occur only following selective release from inhibitory MAP kinase/ERK signaling. Established characteristics of differentiated hen granulosa cells from follicles subsequent to selection include enhanced capacity for FSH-induced cAMP formation (Tilly et al 1991), significant up-regulation of LH receptor expression and LH responsiveness , STAR protein expression (Johnson & Bridgham 2001), and cytochrome P450 side chain cleavage enzyme activity (Li & Johnson 1993), together with a greatly enhanced capacity for progesterone production.…”

Toll-like receptor signaling in hen ovarian granulosa cells is dependent on stage of follicle maturation

“…For cell culture experiments, granulosa cells were collected and dispersed as described previously (Tilly et al 1991. Cells were incubated for up to 6 h at 40 8C in 12!75 mm polypropylene tubes at a density of 5!10 5 , or for 20 h in 6-or 96-well polystyrene culture dishes (Thermo Fisher Scientific, Rockford, IL, USA) at a density of 1!10 6 and 1.5!10 4 respectively in DMEM (Invitrogen), containing 2.5% FBS (Invitrogen), 0.1 mM non-essential amino acids, and 1% antibiotic-antimycotic mixture (Invitrogen).…”

Multiple Involvement of Clusterin in Chicken Ovarian Follicle Development

“…These cells are thought to support the oocyte during its entire growth and development (42,43). In the rapid oocyte growth phase, granulosa cells presumably undergo a crucial step in their differentiation program in preparation for ovulation.…”