Isolation of bovine ovarian inhibin, its immunoneutralization in vitro and immunolocalization in bovine ovary.
A purification scheme involving gel permeation chromatography, anion exchange chromatography and reversed-phase high performance liquid chromatography (RP-HPLC) was used to isolate from bovine follicular fluid (FF) biologically-active inhibin of molecular weight 32 kDa. Chromatographic fractions were monitored for inhibin-like biological activity (ILA) using a simplified bioassay procedure in which a suppression of total basal FSH production by rat pituitary cells in monolayer culture indicates the presence of ILA. Approximately 3 mg protein having an ILA potency (ED50 value in in vitro bioassay) of 1.7 ng/ml was obtained from 4 1 crude bovine FF (260 g protein; ILA potency 3750 ng/ml) reflecting an approximate 2200-fold purification factor with an overall recovery of about 3%. The isolated material appeared as a single major UV absorbance peak on RP-HPLC and as a single band (32 kDa) when subjected to SDS-PAGE (15% gel) under non-reducing conditions. Under reducing conditions the molecule dissociated into 2 subunits of apparent molecular weight 22 and 14 kDa confirming that it is probably identical to the 31/32 kDa form of bovine ovarian inhibin previously reported by two other independent research groups. An antiserum raised in a chicken against the isolated material completely neutralized the suppressive effects of both 32 kDa inhibin and bovine FF on basal production of FSH by rat pituitary cells in vitro but only partially reversed the suppressive effects of both porcine and human FF. Immunohistochemical staining of sections of bovine ovary and of isolated preparations of bovine granulosa cells using this antiserum confirmed that granulosa cells are a major source of inhibin. The observation that specific immunostaining was not confined to these cells, however, suggests that they may not be the exclusive source of immunoreactive inhibin in the bovine ovary.
Knight
P G
PG
Department of Biochemistry & Physiology, The University, Whiteknights, Reading, U.K.
Castillo
R J
RJ
Glencross
R G
RG
Beard
A J
AJ
Wrathall
J H
JH
D000818:Animals; D001681:Biological Assay; D002417:Cattle / Q000378:metabolism*; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D007106:Immune Sera / Q000276:immunology; D007150:Immunohistochemistry; D007265:Inhibins / Q000032:analysis / Q000276:immunology / Q000302:isolation & purification*; D009500:Neutralization Tests; D010053:Ovary / Q000032:analysis*
D007106:Immune Sera; D007265:Inhibins