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

Abstract

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.

Follicle selection in cattle and horses: role of intrafollicular factors

“…In this respect, FSH and IGF-I increased the activin tone in bovine granulosa cell culture media . Further, a recent study (Glister et al 2006) reported a sharp increase in intrafollicular activin tone in 3-6 mm follicles in cattle. Increased activin tone was reflected by a 30-fold increase in activin-A with a concomitant sixfold increase in inhibin-A.…”

Molecular determinants of a competent bovine corpus luteum: first- vs final-wave dominant follicles

“…These features are consistent with a number of previous observations. For example, increasing levels of LH (Byers et al 1997) and ovarian oestrogens (Sharma et al 1999) are each known to down-regulate ESR2 expression in granulosa cells and, in cattle, levels of inhibin-A and activin-A in follicular fluid are reduced in large (13-20 mm) follicles with high (>5) compared with low (<5) E2:P4 ratios (Glister et al 2006). Peripheral (Armstrong et al 2001) and follicular (Echternkamp et al 1994) concentrations of IGF1 also increase under these oestrogen-dominated conditions (Fig.…”

TGF-β superfamily members and ovarian follicle development

“…Whilst granulosa cells have the capacity to synthesise inhibins and activins from an early stage of follicle development (McNatty et al 2000, Montgomery et al 2001, there is evidence that smaller follicles preferentially produce more activin relative to inhibin, whilst larger selected follicles secrete proportionally more inhibin (Schwall et al 1990, Yamoto et al 1992, Glister et al 2006). However, a sharp increase in activin A:inhibin A and activin A:follistatin ratios occurs in bovine antral follicles as they reach the size at which the FSH-dependent follicle selection mechanism operates (Glister et al 2006).…”

“…However, a sharp increase in activin A:inhibin A and activin A:follistatin ratios occurs in bovine antral follicles as they reach the size at which the FSH-dependent follicle selection mechanism operates (Glister et al 2006). So far, there is no corresponding information available on the intrafollicular concentrations of other ligands (BMPs, TGF-b isoforms) although, as will be discussed later, these proteins have also been clearly implicated in the autocrine/paracrine regulation of granulosa and theca cell function in antral follicles.…”