Pattern of secretion of immunoreactive inhibin/activin subunits by avian granulosa cells

The messenger RNA expression for the inhibin/activin subunits in the granulosa layer of avian follicles of different developmental stages has previously been reported. In the present study, we examined the pattern of secretion of these protein subunits from cultured granulosa cells (GC) of avian follicles of defined maturity. Laying hens were euthanized and the F1, F2, F3, F4, small yellow follicles (SYF; 6-10 mm) and large white follicles (LWF; 3-5 mm) were removed. GC were isolated from the follicles, plated by size at a density of 6.25 x 10(5)cells per well (3 wells per follicle size) and cultured for 48 h in medium 199 with 5% FBS, antibiotics, and 1.0 microg/ml bovine insulin. After 48 h, the cultures were terminated and the media were saved (n = 6 replications). Proteins were precipitated from media, reconstituted for electrophoresis (SDS-PAGE), and analyzed by Western blot. Progesterone was also measured in the medium. For detection of the inhibin alpha-subunit, a rabbit antibody against the chicken inhibin alpha-subunit (1-26 aa) was used. The betaA-subunit was detected with rabbit anti-betaA-subunit (81-113 aa) and the betaB-subunit was detected with rabbit anti-betaB-subunit (80-112 aa). Under reduced conditions, GC from the larger follicle sizes (F1-F4) secreted the most (p < 0.05) immunoreactive inhibin alpha-subunit compared to smaller follicle sizes. Under non-reduced conditions, a band at approximately 32 kDa was detected by both the alpha-subunit antibody and by the betaA-subunit antibody in media from GC of the F1-F4 follicles, suggesting secretion of intact inhibin A. Immunoreactive alpha-subunit and betaB-subunit were detected under reduced conditions in media from the GC of the SYF, suggesting that this follicle population may secrete intact inhibin B. In addition, under non-reduced conditions, cells from the SYF secreted the greatest amount of intact inhibin B (p < 0.05) at a size of approximately 32 kDa. Cells from the LWF expressed low levels of all inhibin subunits. Progesterone concentration in the media from the F1 follicle was greatest and was decreased in media from smaller follicles. It is suggested that the largest follicles in the hierarchy are the source of most circulating intact inhibin A while the small follicles are the source of intact inhibin B.