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Title: CHANGES IN AMOUNTS OF DIFFERENT MOLECULAR WEIGHT FORMS OF INHIBIN IN ATRETIC AND NONATRETIC FOLLICLES DURING THE EARLY LUTEAL PHASE AND ALTRENOGEST-SYNCHRONIZED FOLLICULAR PHASE IN PIGS

Author
item Guthrie, Howard
item IRELAND, J.L.H. - EAST LANSING, MI
item GOOD, T.E.M. - EAST LANSING, MI
item IRELAND, J - EAST LANSING, MI

Submitted to: Biology of Reproduction
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/11/1996
Publication Date: N/A
Citation: N/A

Interpretive Summary: We characterized changes in amounts and proportions of different molecular forms of inhibin in porcine follicular fluid in 37 follicles recovered during the early luteal phase of the estrous cycle (days 5-7) and 34 recovered during a follicular phase synchronized by altrenogest. We compared atretic (if incidence of apoptotic granulosa cells was >10% and nonatretic follilces. 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 c1-26 gly.tyr antiserum and quantified. Ten different inhibin forms ranging in size from 27- to greater than >227-kDa were detected. Eighty-three % of the forms, those > 58 kDa may be dimeric, biologically active inhibin that could exert negative feedback on pituitary secretion of follicle-stimulating hormone. The 32 kDa dimeric form was barely detectable in pigs. The amounts of all the forms were greater in nonatretic and atretic follicles. In nonatretic follicles, the 121 kDa form decreased during the early luteal and follicular phases. During the follicular phase, the 44-kDa form increased 10-fold. These results are important information to other scientists because they indicate that production of inhibin dimer and/or alpha subunits are altered differently during follicular growth and atresia thus supporting a possible physiological role for these peptides.

Technical Abstract: We characterized changes in amounts and proportions of different molecular forms of inhibin in porcine follicular fluid of 37 follicles recovered during the early luteal phase of the estrous cycle (days 5-7) and 34 recovered during a follicular phase synchronized by altrenogest. Follicles were designated atretic if incidence of apoptotic granulosa cells was >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 c1-26 gly.tyr antiserum and quantified. Immunoblots detected seven bands corresponding to inhibin forms 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 >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. 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 to 80% lower in atretic than in nonatretic follicles. In nonatretic follicles, the 121 kDa form decreased during the early luteal and follicular phases. During the follicular phase, the 44-kDa form increased 10-fold. In atretic follicles, neither amount nor proportion of inhibin forms differed among days. We conclude that production of inhibin dimer and/or alpha subunits are altered differently during follicular growth and atresia thus supporting a possible physiological role for these peptides.