ALTERATIONS IN AMOUNTS OF DIFFERENT FORMS OF INHIBIN DURING FOLLICULAR ATRESIA

An inhibin immunoblot procedure was validated for measurement of inhibins (alpha beta) and inhibin cu subunits in bovine follicular fluid (bFF) 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) concentrations was used to classify follicles into three different stages of differentiation and atresia: estrogen-active (EA; PE ratio < 1), atretic (PE = 1-100), and highly atretic (PE = > 100). Bovine inhibin (bINH) was analyzed by an immunoblot procedure, an RIA that cross-reacts with inhibin alpha beta dimers and alpha subunits, and a two-site alpha beta dimer-specific immunoradiometric assay (IRMA). Immunoblots consistently detected 9 different bINH forms in bFF of each follicle. The average 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 predominant bINH forms were greater than or equal to 122 kDa. Amounts of most bINH forms decreased several fold during atresia of EA follicles. However, the 34-kDa form increased 1.9-fold. To examine whether amounts of each bINH form varied independently during atresia, proportion of total bINH immunoblot activity represented by each bINH form was statistically analyzed. The results indicated that the proportion of total immunoblot activity for the 34-, 68-, and > 160-kDa bINH forms increased during atresia, whereas proportions for the 48-, 49-, and 122-kDa forms decreased. Alterations in amounts of most bINH forms were positively correlated with intrafollicular concentrations of E and negatively correlated with P. In contrast, the 34-kDa bINH form was negatively correlated with E, but positively correlated with P. Total bINH concentrations determined by RIA decreased 3-fold during atresia. However, amounts of most forms of bINH, except the 29-kDa form (r = 0.72), were relatively weakly correlated (r < 0.41) or not correlated with the RIA results. In contrast to RIA results, concentration of dimeric forms 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-active follicles, 1)production of a 34-kDa form of bINH was selectively enhanced, 2) amounts of most forms of bINH were differentially regulated, 3) production of 34-kDa bINH and E was not enhanced simultaneously, and 4) neither RIA nor IRMA accurately measured alterations in all forms of inhibin in bFF.