ALTERATIONS IN AMOUNTS OF DIFFERENT FORMS OF INHIBIN DURING FOLLICULAR ATRESIA

An inhibin immunoblot procedure was validated for measurement of inhib ins (alpha beta) and inhibin cu subunits in bovine follicular fluid (b FF) from individual follicles. Bovine FF was removed from nonovulatory follicles (greater than or equal to 6 mm in diameter) of cows during early diestrus. Ratio of progesterone (P) to estradiol (E) concentrati ons was used to classify follicles into three different stages of diff erentiation and atresia: estrogen-active (EA; PE ratio < 1), atretic ( PE = 1-100), and highly atretic (PE = > 100). Bovine inhibin (bINH) wa s analyzed by an immunoblot procedure, an RIA that cross-reacts with i nhibin alpha beta dimers and alpha subunits, and a two-site alpha beta dimer-specific immunoradiometric assay (IRMA). Immunoblots consistent ly detected 9 different bINH forms in bFF of each follicle. The averag e molecular mass for the bINH forms ranged from 29 to > 160 kDa, which was similar to those for purified bINH and bINH alpha subunits. The p redominant bINH forms were greater than or equal to 122 kDa. Amounts o f most bINH forms decreased several fold during atresia of EA follicle s. However, the 34-kDa form increased 1.9-fold. To examine whether amo unts of each bINH form varied independently during atresia, proportion of total bINH immunoblot activity represented by each bINH form was s tatistically analyzed. The results indicated that the proportion of to tal immunoblot activity for the 34-, 68-, and > 160-kDa bINH forms inc reased during atresia, whereas proportions for the 48-, 49-, and 122-k Da forms decreased. Alterations in amounts of most bINH forms were pos itively correlated with intrafollicular concentrations of E and negati vely correlated with P. In contrast, the 34-kDa bINH form was negative ly correlated with E, but positively correlated with P. Total bINH con centrations determined by RIA decreased 3-fold during atresia. However , amounts of most forms of bINH, except the 29-kDa form (r = 0.72), we re relatively weakly correlated (r < 0.41) or not correlated with the RIA results. In contrast to RIA results, concentration of dimeric form s of bINH measured by IRMA increased 2.5-fold during atresia, but only the 34-kDa form of bINH was correlated (r = 0.77) with IRMA results. We concluded that during differentiation and atresia of estrogen-activ e follicles, 1)production of a 34-kDa form of bINH was selectively enh anced, 2) amounts of most forms of bINH were differentially regulated, 3) production of 34-kDa bINH and E was not enhanced simultaneously, a nd 4) neither RIA nor IRMA accurately measured alterations in all form s of inhibin in bFF.