REPRODUCTIONRESEARCH Granulosal and thecal expression of bone morphogenetic protein- and activin-binding protein mRNA transcripts during bovine follicle development and factors modulating their expression in vitro Claire Glister, Leanne Satchell and Phil G Knight School of Biological Sciences, University of Reading, Hopkins Building, Whiteknights, Reading RG6 6UB, UK Correspondence should be addressed to P G Knight; Email: [email protected] Abstract Evidence supports local roles for transforming growth factor b superfamily members including activins and bone morphogenetic proteins (BMP) in follicle development. Access of these ligands to signalling receptors is likely modulated by extracellular binding proteins (BP). In this study, we compared ex vivo expression of four BPs (chordin, gremlin, noggin and follistatin) in granulosal (GC) and theca interna (TC) compartments of developing bovine antral follicles (1–18 mm). Effects of FSH and IGF on BMP and BP expression by cultured GC, and effects of LH and BMPs on BPexpression by cultured TC were also examined. Follicular expression of all four BP transcripts was higher in GC than TC compartments (P!0.001) a finding confirmed by immunohistochemistry. Follicle category affected (P!0.01) gremlin and follistatin mRNA abundance, with a significant cell-type!follicle category interaction for chordin, follistatin and noggin. Noggin transcript abundance was lower (P!0.05) in GC of large ‘E-active’ than ‘E-inactive’ follicles while follistatin mRNA level was higher (P!0.01). FSH enhanced CYP19, FSHR, INHBA and follistatin by GC without affecting BMPor BMP–BPexpression. IGF increased CYP19 and follistatin, reduced BMP4, noggin and gremlin but did not affect chordin or FSHR mRNA levels. LH increased TC androgen secretion but had no effect on BMP or BP expression. BMPs uniformly suppressed TC androgen production whilst increasing chordin, noggin and gremlin mRNA levels up to 20-fold (P!0.01). These findings support the hypothesis that extracellular BP, mostly from GC, contribute to the regulation of intrafollicular BMP/activin signalling. Enhancement of thecal BP expression by BMP implies an autoregulatory feedback role to prevent excessive signalling. Reproduction (2011) 142 581–591 Introduction and related this to the pattern of expression of type 1 and type 2 signalling receptors. Significant cell-type and During recent years a wealth of investigation has been follicle stage-dependent changes in expression of b directed at transforming growth factor (TGFB) super- individual ligands and receptors were revealed but, in family members, in particular the largest subset within general, changes in receptor transcript abundance were the family, the bone morphogenetic proteins (BMP). relatively modest, supporting the notion that additional More than 20 BMPs have been identified in vertebrates levels of control may be operational to modulate BMP and non-vertebrate species and these multifunctional signalling at the local level. proteins contribute to the regulation of cellular prolifer- Despite the likely functional importance of BMP ation, differentiation and apoptosis in a variety of tissues, signalling within the ovary, few studies have addressed including the ovary (Miyazono et al. 2001, Chen et al. the potential role of extracellular BMP-binding proteins 2004, Shimasaki et al. 2004, Knight & Glister 2006). (BP) in modulating BMP access to their signalling A number of BMPs are expressed in theca cells (TC), receptors on different ovarian cell-types. Much of the granulosa cells (GC) and oocytes in a cell- and follicle research on BMP-BPs has been in the developmental stage-specific manner and evidence supports their biology field where their crucial roles have been involvement in the regulation of folliculogenesis, identified in establishing BMP morphogen gradients ovulation and corpus luteum function (reviews: Shimasaki responsible for embryonic patterning (Thomsen 1997, et al. 2004, Juengel & McNatty 2005, Knight & Glister Balemans & Van Hul 2002, Chen et al. 2004, Kishigami 2006). Recently, Glister et al. (2010) reported a survey of & Mishina 2005). To our knowledge no comprehensive BMP2, BMP4, BMP6 and BMP7 expression in TC and surveys of BMP-BP expression throughout ovarian GC throughout bovine antral follicular development follicle growth have yet been carried out in any species. q 2011 Society for Reproduction and Fertility DOI: 10.1530/REP-11-0150 ISSN 1470–1626 (paper) 1741–7899 (online) Online version via www.reproduction-online.org Downloaded from Bioscientifica.com at 09/29/2021 12:39:17AM via free access 582 C Glister and others Similarly, information is lacking on the factors (systemic and GC is modulated by gonadotrophins or insulin-like or intraovarian) that potentially modulate expression of growth factor 1 (IGF1) and iii) examine the effect of BMP-BPs by follicle cells. As such, with the exception of exogenous BMP2, BMP4, BMP6 and BMP7 on follistatin (predominantly an activin-BP), the significance expression of BP transcripts by cultured TC. of secreted BMP-BPs as intrafollicular modulators of BMP action has received little attention. Follistatin was first isolated by Robertson et al. (1987) Results from bovine follicular fluid based on its ability to More detailed characteristics of the follicle sample set suppress FSH secretion from pituitary cells and was analysed in this study, including follicular fluid steroid subsequently shown to function by binding activin with concentrations and relative expression of GC and TC high affinity, thus neutralising its bioactivity (Nakamura ‘marker’ transcripts including FSHR, CYP19, LHCGR et al. 1990). Follistatin has also been shown to bind and CYP17, are documented in our recent companion directly to several other TGFB superfamily members, paper focusing on follicular expression of BMP ligands including inhibin (Shimonaka et al. 1991) and BMP2, and receptors (Glister et al. 2010). BMP4, BMP6, BMP7 and BMP15 (Otsuka et al. 2001, Amthor et al. 2002, Glister et al. 2004) albeit with lower affinity. Noggin, a homodimeric glycoprotein, mRNA expression profiles for gremlin, chordin, was first isolated from Xenopus embryonic tissues where follistatin and noggin in developing follicles it blocks BMP action in the dorsal organising centre mRNA transcripts for each of the four BPs were readily (Lamb et al. 1993, Smith et al. 1993). Noggin displays a detectable in all GC and TC samples with average qPCR Ct high-affinity association with BMP2 and BMP4, but less values of 26, 28, 26 and 21 for noggin, chordin, gremlin so with BMP7 and BMP6; binding directly prevents and follistatin respectively. In Fig. 1, the relative interaction of ligand with type 1 receptor (Zimmerman abundance of mRNA transcripts for all four BPs examined et al. 1996). Chordin, a cysteine-rich secreted protein, was greater in GC than TC (two-way ANOVA: effect of was also first identified in Xenopus; located in the cell-type: P!0.0001). Gremlin expression was highest in dorsal organising centre (Sasai et al. 1994, 1995). It GC of 1–2 mm follicles and declined progressively as also binds directly to BMP2, BMP4 and BMP7 to prevent follicles developed to 11–18 mm (approximately fivefold BMP–receptor interaction. However, the affinity of reduction; P!0.002); no difference in GC gremlin chordin for BMP4 is some tenfold less than its counterpart expression was evident between large oestrogen-active noggin (Piccolo et al. 1996). Gremlin belongs to a (LEA) and large oestrogen-inactive (LEI) follicles. subfamily of BMP antagonists all of whom contain a Although there was no overall effect of follicle characteristic cysteine-rich domain (CAN domain) conta- category on the relative abundance of chordin mRNA, ining a particular consensus sequence (Avsian-Kretchmer & a highly significant follicle category!cell-type Hsueh 2004). As with chordin and noggin, gremlin was first interaction (P!0.0001) justified a series of post hoc identified in a Xenopus screen for genes with dorsalising pairwise comparisons within cell-type. In GC, chordin activity (Hsu et al. 1998) and has since been shown to mRNA abundance fell (approximately sixfold; P!0.05) regulate murine limb, kidney and lung development as follicles grew from 1–2 to 7–8 mm but then increased (Zuniga et al. 1999, Khokha et al. 2003, Michos et al. again (approximately sixfold; P!0.01) between the 2004). Gremlin can bind to and inhibit BMP2 and BMP4, 9–10 and 11–18 mm (LEA) follicle categories; GC chordin but does not interact with activin (Hsu et al. 1998). expression did not differ significantly between LEA and LEI Interestingly, expression of noggin and gremlin is follicles although the mean value was 50% lower in LEI enhanced by the very BMPs they act to inhibit, indicative category. In contrast to GC, chordin expression in the TC of negative feedback loops in operation (Kameda et al. layer increased from 1–2 to 9–10 mm follicles (approxi- 1999, Pereira et al. 2000). In addition, certain isoforms of mately twofold; P!0.05), before falling (approximately follistatin, noggin and chordin bind to cell-surface threefold; P!0.01) to its lowest level in LEA follicles; TC associated heparin-sulphate proteoglycans. This associ- chordin expression was approximately fivefold higher ation may serve to limit diffusion and ‘action-range’ of (P!0.01) in LEI than in LEA follicles. either antagonist and/or ligand. It may also promote Follistatin expression patterns in GC and TC were endocytosis and lysosomal degradation of the target similar to those for chordin: in