Cloning and sequence analysis of cDNA species coding for the two subunits of inhibin from bovine follicular fluid.

Abstract

The primary amino acid structures of the 43-kDa (A) and 15-kDa (B) subunits of the 58-kDa form of the hormone inhibin have been elucidated by cloning and analysis of cDNA species derived from bovine granulosa cell mRNA.The A subunit (Mr = 32,298) is a protein of 300 amino acids with two potential N-glycosylation sites and two potential proteolytic processing sites and'has a pre-pro region of 60 amino acids. The mature B sublinit (Mr = 12,977) is a protein of 116 amino acids synthesized from a separate mRNA. These data establish that a 31-kDa forn of inhibin also isolated from bovine follicular fluid, with subunits qf 20 kJa (Ac) and 15 kDa (B), is derived from the 58-kDa form by proteolytic processing of the A subunit.Considerable evidence has now accumulated to support the concept that the gonads produce a protein termed inhibin (1) that selectively suppresses the pituitary secretion of folliclestimulating hormone (2), a key hormone in controlling folliculogenesis and spermatogenesis. Controversy still exists concerning the nature of inhibin (3) partly because of the fact that some of the bioassays used in its characterization do not monitor follicle-stimulating hormone directly (4). For instance, inhibin-like activity has been detected in seminal plasma; the proteins associated with this activity have been purified and characterized but subsequently they were shown not to be of gonadal origin (5)(6)(7)(8)(9).We have isolated inhibin from a gonadal source, bovine follicular fluid, with an apparent molecular mass of 58 kDa, composed of two subunits now designated A and B, of 43 kDa and 15 kDa, respectively, linked together by disulfide bonds (10). Subsequently, 32-kDa inhibin was isolated by others from porcine follicular fluid, with two subunits of 18/20 kDa and 13/14 kDa (11,12). We have also shown that a 31-kDa form of bFF inhibin, composed of 20-kDa and 15-kDa subunits, is generated during a pH precipitation step in the purification procedure (13) but the precise relationship between the different forms has been unknown. However, the biological activity of the 31-kDa form is neutralized in vitro by an antiserum raised against the 58-kDa form (13), suggesting that the 31-kDa inhibin is a processed form of the 58-kDa inhibin.This article describes the amino acid sequences of the two subunits of the 58-kDa bovine inhibin as determined fromn cDNA sequencing and reports that the 20-kDa subunit of the 31-kDa inhibin is derived from the 43-kDa subunit of the 58-kDa inhibin, thus clarifying the relationship between these different forms.MATERIALS AND MIETIODS NH2-Terminal Amino Acid Sequencing of the 58-kDa Inhibin. The 58-kDa inhibin was isolated from bFF (10).Briefly, this involved a four-step' purjfication procedure: (i) gel permeation chromatography on Sephacryl 3200 in 0.05 M ammonium acetate, (it) gel permeation chromatography on Sephadex G100 in 4 M acetic acid, (iii) reversed-phase HPLC on an Ultrapore RPSC column-(Beckman) using a 0.1% trifluoroacetic acid-acetonitrile-H2O gradient, and (iv) prepara...

Genomic Cloning and Sequence Analyses of the Bovine α‐, βA‐ and βB‐Inhibin/activin Genes

“…The sequence of part of this fragment was similar to that described by Forage et al (1986) for a bovine…”

“…The nucleotide sequence determined for a 907-bp PstI bovine genomic a-inhibin fragment contained within the XhoI fragment, that was identified by comparison of data from restriction mapping and sequence information with the complete bovine cDNA sequence reported by Forage et al (1986), is shown in Fig. 1.…”

“…Nick-translated cDNAs of the bovine a-inhibin gene (902 bp partial cDNA provided by Drs S. Sunderland and D. R. Headon; bases 280-1182 of the sequence reported by Forage et al, 1986), the bovine P,-inhibin/activin gene (718 bp partial cDNA provided by Drs S. Sunderland and D. R. Headon; bases 1202-1919 of the sequence reported in Fig. 2) and the human P,-inhibirdactivin gene (1.25 kb sequence provided by Dr A. J.…”

New targets for old hormones: inhibins clinical role revisited [Review]

“…Later, isolation and characterization of inhibins was made possible with the development of an improved bioassay based on measurement of FSH production by rat pituitary cells in vitro [13][14][15][16]. Subsequent breakthroughs in the molecular biology and its applications, cloning of the gene encoding inhibin was made possible following characterization and purification from the bovine follicular fluid [17][18][19]. Following, large scale productions of recombinant DNA-derived inhibin, great strides were made in elucidating its role and other functions [20].…”

The Role of Inhibin on Pituitary FSH Secretion and Maturation of Pituitary-Gonadal Axis in Immature Female Rats.

“…The sequence of inhibin in several species have been elucidated by many investigators (Mason et al 1985(Mason et al , 1986Forage et al 1986) and there are 3 subunits (alpha, beta-A, beta-B) (Mason et al 1985). Inhibin is a combination of alpha either with beta-A (inhibin A) or beta-B (inhibin B) which acts as a FSH suppressor.…”