Measurement of dimeric inhibin using a modified two-site immunoradiometric assay specific for oxidized (Met O) inhibin

Knight, P. G.; Muttukrishna, Shanthi

doi:10.1677/joe.0.1410417

Cited by 54 publications

(24 citation statements)

References 15 publications

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“…Concentrations of inhibin-A were determined using the two-site IRMA described by Knight & Muttukrishna (1994), which has a detection limit of 250 pg/ml and within-and between-assay coefficients of variation (CV) of 5.2 and 7.4% respectively. Activin-A and FS levels were measured by ELISA (Knight et al 1996, Tannetta et al 1998, both with detection limits of 100 pg/ml.…”

Section: Immunoassaysmentioning

confidence: 99%

Bone morphogenetic protein (BMP) ligands and receptors in bovine ovarian follicle cells: actions of BMP-4, -6 and -7 on granulosa cells and differential modulation of Smad-1 phosphorylation by follistatin

Glister¹,

Kemp²,

Knight³

2004

Reproduction

246

223

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Given the paucity of information on the potential roles of bone morphogenetic proteins (BMPs) in the ruminant ovary we conducted immunolocalization and functional studies on cells isolated from bovine antral follicles. Immunocytochemistry revealed expression of BMP-4 and -7 in isolated theca cells whereas granulosa cells and oocytes selectively expressed BMP-6. All three cell types expressed a range of BMP-responsive type-I (BMPRIB, ActRI) and type-II (BMPRII, ActRII, ActRIIB) receptors supporting autocrine/paracrine roles within the follicle. This was reinforced by functional experiments on granulosa cells which showed that BMP-4, -6 and -7 promoted cellular accumulation of phosphorylated Smad-1 but not Smad-2 and enhanced 'basal' and IGF-stimulated secretion of oestradiol (E2), inhibin-A, activin-A and follistatin (FS). Concomitantly, each BMP suppressed 'basal' and IGF-stimulated progesterone secretion, consistent with an action to prevent or delay atresia and/or luteinization. BMPs also increased viable cell number under 'basal' (BMP-4 and -7) and IGF-stimulated (BMP-4, -6 and -7) conditions. Since FS, a product of bovine granulosa cells, has been shown to bind several BMPs, we used the Biacore technique to compare its binding affinities for activin-A (prototype FS ligand) and BMP-4, -6 and -7. Compared with activin-A (K d 0.28 6 0.02 nM; 100%), the relative affinities of FS for BMP-4, -6 and -7 were 10, 5 and 1% respectively. Moreover, studies on granulosa cells showed that preincubation of ligand with excess FS abolished activin-A-induced phosphorylation of Smad-2 and BMP-4-induced phosphorylation of Smad-1. However, FS only partially reversed BMP-6-induced Smad-1 phosphorylation and had no inhibitory effect on BMP-7-induced Smad-1 phosphorylation. These findings support functional roles for BMP-4, -6 and -7 as paracrine/autocrine modulators of granulosa cell steroidogenesis, peptide secretion and proliferation in bovine antral follicles. The finding that FS can differentially modulate BMP-induced receptor activation and that this correlates with the relative binding affinity of FS for each BMP type implicates FS as a potential modulator of BMP action in the ovary.

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Section: Immunoassaysmentioning

confidence: 99%

Bone morphogenetic protein (BMP) ligands and receptors in bovine ovarian follicle cells: actions of BMP-4, -6 and -7 on granulosa cells and differential modulation of Smad-1 phosphorylation by follistatin

Glister¹,

Kemp²,

Knight³

2004

Reproduction

246

223

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“…Concentrations of inh-A were determined using the two-site IRMA described by Knight and Muttukrishna (1994). Purified 32 kDa bovine inh-A (Knight et al 1990) was used as a standard.…”

Section: Hormone Immunoassaysmentioning

confidence: 99%

Bovine follicle development is associated with divergent changes in activin-A, inhibin-A and follistatin and the relative abundance of different follistatin isoforms in follicular fluid

Glister

Groome²,

Knight

2006

Journal of Endocrinology

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The aim was to determine whether follicle growth in cattle is accompanied by changes in levels of inhibin-A (inh-A), activin-A (act-A) and different Mr isoforms of follistatin (FS) in bovine follicular fluid (bFF), reflecting differential roles of these proteins during folliculogenesis. Follicles (n=146) from 2-20 mm diameter were dissected from ovaries of 40 cattle. Immunoassays were used to measure total FS, act-A, inh-A, oestradiol (E) and progesterone (P) levels; immunoblotting was used to quantify the relative abundance of different FS isoforms. Follicle growth from 2-6 mm was associated with a 6-fold increase in inh-A and 30-fold increase in act-A; FS remained uniformly high from 2-10 mm. From 6-2 mm, inh-A remained high while act-A and FS fell 3-fold and 2-fold, respectively. Act-A/FS ratio increased 20-fold from 2-6 mm before falling slightly through to 20 mm. Act-A/inh-A ratio increased 6-fold from 2-6 mm before falling 2-fold from 6 to 17-20 mm. These findings imply a marked increase in relative activin 'tone' around the stage at which dominant follicle selection occurs. When larger follicles (13-20 mm) were subdivided according to E/P ratio, those with high (>5) E/P ratio had lower (2-fold; P<0·001) levels of inh-A and act-A in comparison to follicles with low (<5) E/P ratio, but there were no significant differences in FS, act-A/inh-A ratio or act-A/FS ratio. Thus follicle size, but not oestrogenic status, has a major influence on the intrafollicular balance between act-A and its opposing factors, inh-A and FS. Six FS isoforms were detected in bFF (apparent Mr: 65, 41, 37, 35, 33 and 31 kDa) averaging 6, 13, 24, 26, 13 and 17% respectively of total FS. During growth from 2-20 mm the proportion of total FS represented by 65, 41 and 37 kDa isoforms increased 2-fold while the proportion represented by the 33 and 31 kDa isoforms decreased by 3-fold and 1·6-fold, respectively. Treatment of bovine granulosa cells in vitro with FSH and IGF alone or in combination increased total FS secretion up to 12-fold but did not affect the relative abundance of the five different FS isoforms detected. While the functional significance of the intriguing shift in FS isoform abundance in bFF during follicle development remains to be established, we have shown that a marked increase in intrafollicular activin 'tone' accompanies bovine follicle growth from 3-6 mm, corresponding to the stage at which the FSH-dependent follicle selection mechanism operates in this species.

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“…Samples and standard (32 kDa human recombinant inhibin; Genentech, South San Francisco, CA, USA) were diluted in assay diluent (0·1 M Tris HCl pH 7·5, containing 0·15 M sodium chloride, 5% Triton X-100, 10% BSA and 5% mouse serum) to a volume of 150 µl, incubated with 15 µl 10% hydrogen peroxide for 30 min at room temperature, then diluted to 300 µl with assay diluent. The oxidation step has been shown to improve antibody (E4) recognition of the -subunit (Knight & Muttukrishna 1994). Aliquots of 100 µl sample solutions were assayed in duplicate.…”

Section: Assaysmentioning

confidence: 99%

Effect of FSH and cell localization on dimeric inhibin-A secretion from bovine granulosa cells in culture

Boudjemaa

Rouillier²,

Bhatia³

et al. 2000

Journal of Endocrinology

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We tested the hypotheses that the secretion of dimeric inhibin-A from cultured bovine granulosa cells is stimulated by FSH, and that antral cells secrete more inhibin-A than do mural cells. Cells from the antral or mural compartment of follicles were cultured in defined medium in two culture systems, and dimeric inhibin-A was measured by two-site ELISA or by Western immunoblotting. In the first culture system, dimeric inhibin-A secretion declined with time in culture, but was significantly (P<0·05) higher from antral than from mural cells (as was total inhibin-measured by RIA). The secretion of dimeric inhibin-A and inhibin-from antral but not mural cells was responsive to FSH. In the second culture system, dimeric inhibin-A secretion increased with time in culture, and was significantly stimulated by FSH, but FSH responsiveness was dependent on the concentrations of insulin in the culture medium. The major forms of inhibin-A secreted had molecular masses of approximately 58, 62, 103-116 and >116 kDa; the 32 kDa form was barely detectable. These different forms were all stimulated by FSH, but the >116 and 62 kDa forms were most responsive to FSH. We conclude that (i) FSH stimulates dimeric inhibin-A secretion from bovine granulosa cells, (ii) the 62 kDa form of inhibin-A may be more responsive to FSH than the 58 kDa form, and (iii) the spatial differentiation of granulosa cell function within the follicle previously observed for oestradiol secretion was also observed for inhibin-and dimeric inhibin-A secretion.

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Measurement of dimeric inhibin using a modified two-site immunoradiometric assay specific for oxidized (Met O) inhibin

Cited by 54 publications

References 15 publications

Bone morphogenetic protein (BMP) ligands and receptors in bovine ovarian follicle cells: actions of BMP-4, -6 and -7 on granulosa cells and differential modulation of Smad-1 phosphorylation by follistatin

Bone morphogenetic protein (BMP) ligands and receptors in bovine ovarian follicle cells: actions of BMP-4, -6 and -7 on granulosa cells and differential modulation of Smad-1 phosphorylation by follistatin

Bovine follicle development is associated with divergent changes in activin-A, inhibin-A and follistatin and the relative abundance of different follistatin isoforms in follicular fluid

Effect of FSH and cell localization on dimeric inhibin-A secretion from bovine granulosa cells in culture

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