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. 2016 Sep;22(9):622-33.
doi: 10.1093/molehr/gaw044. Epub 2016 Jul 6.

BMP Signalling in Human Fetal Ovary Somatic Cells Is Modulated in a Gene-Specific Fashion by GREM1 and GREM2

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Free PMC article

BMP Signalling in Human Fetal Ovary Somatic Cells Is Modulated in a Gene-Specific Fashion by GREM1 and GREM2

Rosemary A Bayne et al. Mol Hum Reprod. .
Free PMC article

Abstract

Study question: Do 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 finding: BMPs 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 already: Extensive 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, methods: Expression 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 chance: We 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 caution: While 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 findings: This 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 data: Not applicable.

Study funding/competing interests: This 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.

Keywords: BMP; BMP antagonist; human fetal ovary; ovarian development; ovarian somatic cell; pre-granulosa cell.

Figures

Figure 1
Figure 1
QRT-PCR Analysis of bone morphogenetic protein (BMP) antagonist expression across gestation in the human fetal ovary. Expression levels of each gene are given, normalised to the level of RPL32 in each sample. Ovary gestations are grouped to represent different stages in development as described in the main text. Results for individual samples (n = 5–6 for each stage) are presented along with median values. Changes between stages were analysed by the Kruskal–Wallis Test with Dunn's multiple comparisons and different letters above groups represent statistically significant differences (p ≤ 0.05, or less as indicated in the main text).
Figure 2
Figure 2
Expression of germ cell and various somatic cell markers with passage of adherent cells following disaggregation of human fetal ovaries. Expression of the germ cell marker DAZL, markers of differentiating pre-granulosa cells (FOXL2), steroidogenic cell precursors (NR2F2), immature dividing pre-granulosa cell precursors (LGR5) and components of the BMP pathway (ID2, BMP4, GREM1 and GREM2) were examined by qRT-PCR across passages for a number of human fetal ovarian somatic cell cultures derived from 14 to 17 week disaggregated ovaries (n = 5 for DAZL, FOXL2, NR2F2 and ID2; n = 4 for BMP4; n = 3 for LGR5, GREM1 and GREM2). Results are expressed as mean expression relative to RPL32 ± sem. The 0 time point represents disaggregated ovaries prior to plating.
Figure 3
Figure 3
Components of the BMP signalling pathway are expressed in human fetal ovary and cultured ovarian somatic cells and regulate SMAD1/5/8 phosphorylation. A: Agarose gel analysis of RT-PCR products for the 3 BMP receptors (BMPR1A, BMPR1B, BMPR2). M is the 100 bp Hyperladder (Bioline). (+) and (−) represent reactions with RT(+) and RT(−) cDNAs respectively. Gestations of fetal ovaries and derived somatic cell cultures used are given in weeks (wk). T0 is cells from ovary disaggregation prior to plating (i.e. contains both germ and somatic cells), P0 is adherent somatic cells taken just prior to the first passage and P2-4 represents the number of passages undergone for each cell culture. B: Western blot analysis of GREM1 expression. M is the PageRuler Plus (10–250 kda) Prestained Protein Ladder (Fisher Scientific) and numbers beside individual marker bands give their MW as a guide. GREM1 (lower bands) with β-actin (upper bands) acting as a positive control. C: Western blot of protein extracts from cells (cultures FT3096 and FT2692) treated with vehicle (V), recombinant GREM1 (G), recombinant BMP4 (B) or BMP4 + GREM1 (BG) for 1 hour after overnight serum-starvation. M is the PageRuler Plus (10–250 kda) Prestained Protein Ladder (Fisher Scientific) and white numbers over individual bands indicate their MW. pSMAD1/5/8 signal (expected MW = 60) is red while the β-actin normalisation control (expected MW = 42) signal is green. D: Comparison of pSMAD1/5/8 levels normalised to β-actin signal for each treatment and cell culture (single replicates only performed).
Figure 4
Figure 4
Titration of BMP4 and BMP2 effects on downstream gene expression in human fetal ovarian somatic cells. FT2692 ovarian somatic cells were serum-starved overnight and then treated (2 replicate wells per treatment) with vehicle or 1, 10, 50 or 100 ng/ml of recombinant human BMP4 or BMP2 for 24 h and RNA extracted. QRT-PCR for the genes indicated was performed and normalised to levels of RPL32 before calculating fold changes relative to the vehicle control. Data points are mean ± sem. BMP2 treatment graphs are blue while BMP treatment graphs are red. Data were analysed by ANOVA with Tukey's multiple comparisons post-hoc tests and significant differences (P ≤ 0.05) between treatment concentrations are indicated by different letters (with colour matching key) above the relevant concentration.
Figure 5
Figure 5
GREM1 sensitivity of BMP4 target genes in human fetal ovarian somatic cells. FT3010 cells were serum-starved overnight and treated with ± 1 µg/ml GREM1, ± 50ng/ml BMP4 (n = 4 replicate wells for each treatment) for 24 hours before RNA extraction and qRT-PCR analysis of key BMP4 targets, with normalisation to RPL32 transcript levels. Columns represent mean ± sem for each treatment (white = vehicle, red = GREM1, blue = BMP4, purple = BMP4 + GREM1). Differences in expression levels between treatments were analysed by ANOVA with Tukey's multiple comparisons post-hoc tests and significant differences (P ≤ 0.05 or less, as indicated in the main text) between treatments are indicated by different letters above the columns, or * in the case of SMAD1 (P = 0.01).
Figure 6
Figure 6
GREM2 Sensitivity of BMP4 Target Genes in human fetal ovarian somatic cells. FT3330 cells were serum-starved overnight and treated with ± 100 ng/ml GREM2, ± 50 ng/ml BMP4 (n = 4 replicate wells for each treatment) for 24 h before RNA extraction and qRT-PCR analysis of key BMP4 targets, with normalisation to RPL32 transcript levels. Columns represent mean ± sem for each treatment (white = vehicle, red = GREM2, blue = BMP4, purple = BMP4 + GREM2). Differences in expression levels between treatments were analysed by ANOVA with Tukey's multiple comparisons post-hoc tests and significant differences (P ≤ 0.05 or less, as indicated in the main text) between treatments are indicated by different letters above the columns.

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