Expression of different molecular mass forms of inhibin in atretic and nonatretic follicles during the early luteal phase and altrenogest-synchronized follicular phase in pigs

This experiment characterized changes in amounts and proportions of different molecular forms of inhibin in porcine follicular fluid as related to stage of follicular development. Thirty-seven follicles (2-4 per pig) were dissected from 12 pigs during early luteal phase of the estrous cycle on Days 5, 6, and 7 of the estrous cycle, whereas 34 follicles (2-4 per pig) were dissected from 11 pigs on Days 1, 3, 5, and 7 of a follicular phase synchronized by altrenogest. Follicles were designated atretic if incidence of apoptotic granulosa cells was greater than or equal to 10% as determined by DNA fluorescence flow cytometry, Porcine follicular fluid was fractionated on 12% SDS-PAGE gels under non-reducing conditions and electroblotted to Immobilin P membranes. Inhibin forms were detected by immunoblot analysis using a mink anti-bovine inhibin alpha(C)(1-26)gly tyr antiserum and quantified. Immunoblots detected seven bands corresponding to inhibin forms of 44, 49, 58, 69, 121, 227, and > 227 kDa in > 91% of porcine follicular fluid samples. Three additional forms of 27, 29, and 32 kDa were detectable in only 52%, 64%, and 48% of samples,respectively. Forms greater than or equal to 69 kDa represented 83% of total inhibin immunoblot activity. The 121-kDa form was most abundant, with 39% of the total immunoblot activity in nonatretic follicles. The proportions of individual forms and total immunoblot activity pooled over days did not differ between early luteal and follicular phase follicles. Total inhibin immunoblot activity was 59% less in atretic than in nonatretic follicles. Amounts of the 44-, 49-, 69-, 121-, and 227-kDa forms were 50-80% lower (p less than or equal to 0.05) in atretic than in nonatretic follicles. Total inhibin immunoblot activity in nonatretic follicles decreased (p less than or equal to 0.05) by 60% during the early luteal phase but did not change significantly during the follicular phase. In nonatretic follicles, the 121-kDa form decreased (p < 0.05) during the early luteal and follicular phases. During the early luteal phase, amounts of the other forms did not change, whereas during the follicular phase the 44-kDa form increased (p < 0.05) 10-fold. In atretic follicles, neither amount nor proportion of inhibin forms differed among days. We conclude that follicular production and/or intracellular processing of inhibin dimer and/or inhibin alpha subunits changes during different phases of follicular development, supporting the notion of physiological roles Car these peptides.