Abstract
STUDY QUESTIONDo changes in the expression of bone morphogenetic proteins (BMPs) 2 and 4, and their antagonists Gremlin1 (GREM1) and Gremlin2 (GREM2) during human fetal ovarian development impact on BMP pathway activity and lead to changes in gene expression that may influence the fate and/or function of ovarian somatic cells?STUDY FINDINGBMPs 2 and 4 differentially regulate gene expression in cultured human fetal ovarian somatic cells. Expression of some, but not all BMP target genes is antagonised by GREM1 and GREM2, indicating the existence of a mechanism to fine-tune BMP signal intensity in the ovary. Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5), a marker of immature ovarian somatic cells, is identified as a novel transcriptional target of BMP4.WHAT IS KNOWN ALREADYExtensive re-organisation of the germ and somatic cell populations in the feto-neonatal ovary culminates in the formation of primordial follicles, which provide the basis for a female's future fertility. BMP growth factors play important roles at many stages of ovarian development and function. GREM1, an extracellular antagonist of BMP signalling, regulates the timing of primordial follicle formation in the mouse ovary, and mRNA levels of BMP4 decrease while those of BMP2 increase prior to follicle formation in the human fetal ovary.STUDY DESIGN, SAMPLES/MATERIALS, METHODSExpression of genes encoding BMP pathway components, BMP antagonists and markers of ovarian somatic cells were determined by quantitative (q)RT-PCR in human fetal ovaries (from 8 to 21 weeks gestation) and fetal ovary-derived somatic cell cultures. Ovarian expression of GREM1 protein was confirmed by immunoblotting. Primary human fetal ovarian somatic cell cultures were derived from disaggregated ovaries by differential adhesion and cultured in the presence of recombinant human BMP2 or BMP4, with or without the addition of GREM1 or GREM2.MAIN RESULTS AND THE ROLE OF CHANCEWe demonstrate that the expression of BMP antagonists GREM1, GREM2 and CHRD increases in the lead-up to primordial follicle formation in the human fetal ovary, and that the BMP pathway is active in cultured ovarian somatic cells. This leads to differential changes in the expression of a number of genes, some of which are further modulated by GREM1 and/or GREM2. The positive transcriptional regulation of LGR5 (a marker of less differentiated somatic cells) by BMP4 in vitro suggests that increasing levels of GREM1 and reduced levels of BMP4 as the ovary develops in vivo may act to reduce LGR5 levels and allow pre-granulosa cell differentiation.LIMITATIONS, REASONS FOR CAUTIONWhile we have demonstrated that markers of different somatic cell types are expressed in the cultured ovarian somatic cells, their proportions may not represent the same cells in the intact ovary which also contains germ cells.WIDER IMPLICATIONS OF THE FINDINGSThis study extends previous work identifying germ cells as targets of ovarian BMP signalling, and suggests BMPs may regulate the development of both germ and somatic cells in the developing ovary around the time of follicle formation.LARGE SCALE DATANot applicable.STUDY FUNDING/COMPETING INTERESTSThis work was supported by The UK Medical Research Council (Grant No.: G1100357 to RAA), and Medical Research Scotland (Grant No. 345FRG to AJC). The authors have no competing interests to declare.
Highlights
Female reproductive lifespan is determined before birth, with the formation of a pool of primordial follicles within the fetal ovaries, which constitute the supply of eggs for her entire life
To determine whether Bone Morphogenetic Proteins (BMPs) may play a role in the specification or development of ovarian somatic cells, we examined whether expression of somatic cell markers Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) and FOXL2 were modulated by BMPs or GREM1/2 in the cultured somatic cells
Expression of BMP antagonists NOG, GREM1, GREM2, CHRD, SMAD6 and SMAD7 was examined by Quantitative reverse transcriptase-PCR (qRT-PCR) using cDNA samples corresponding to three stages of human fetal ovarian development, namely: 8–11 weeks, 14–16 weeks and 17–21 weeks (Fig. 1)
Summary
Female reproductive lifespan is determined before birth, with the formation of a pool of primordial follicles within the fetal ovaries, which constitute the supply of eggs for her entire life. Primordial follicle formation results from a highly dynamic period of germ and somatic cell reorganisation within the ovary. This occurs during fetal life in humans, and around the time of birth in mice. Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5; a known marker of adult stem cells (Hirsch et al, 2014, Ng et al, 2014)) is expressed by somatic cells in the cortical region of fetal and postnatal mouse ovary, and LGR5-positive cells appear to be the progenitors of the pregranulosa cells of follicles which form in the ovarian cortex (Rastetter et al, 2014). The intrinsic and extrinsic mechanisms governing the specification and maintenance of these different populations of somatic cells remains to be established, their distinct spatial distributions may support a role for localised growth factor signalling in regulating their ontogeny
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