Dynamics of extracellular matrix in ovarian follicles and corpora lutea of mice

Despite the mouse being an important laboratory species, little is known about changes in its extracellular matrix (ECM) during follicle and corpora lutea formation and regression. Follicle development was induced in mice (29 days of age/experimental day 0) by injections of pregnant mare’s serum gonadotrophin on days 0 and 1 and ovulation was induced by injection of human chorionic gonadotrophin on day 2. Ovaries were collected for immunohistochemistry (n=10 per group) on days 0, 2 and 5. Another group was mated and ovaries were examined on day 11 (n=7). Collagen type IV α1 and α2, laminin α1, β1 and γ1 chains, nidogens 1 and 2 and perlecan were present in the follicular basal lamina of all developmental stages. Collagen type XVIII was only found in basal lamina of primordial, primary and some preantral follicles, whereas laminin α2 was only detected in some preantral and antral follicles. The focimatrix, a specialised matrix of the membrana granulosa, contained collagen type IV α1 and α2, laminin α1, β1 and γ1 chains, nidogens 1 and 2, perlecan and collagen type XVIII. In the corpora lutea, staining was restricted to capillary sub-endothelial basal laminas containing collagen type IV α1 and α2, laminin α1, β1 and γ1 chains, nidogens 1 and 2, perlecan and collagen type XVIII. Laminins α4 and α5 were not immunolocalised to any structure in the mouse ovary. The ECM composition of the mouse ovary has similarities to, but also major differences from, other species with respect to nidogens 1 and 2 and perlecan

Could perturbed fetal development of the ovary contribute to the development of polycystic ovary syndrome in later life?

Polycystic ovary syndrome (PCOS) affects around 10% of young women, with adverse consequences on fertility and cardiometabolic outcomes. PCOS appears to result from a genetic predisposition interacting with developmental events during fetal or perinatal life. We hypothesised that PCOS candidate genes might be expressed in the fetal ovary when the stroma develops; mechanistically linking the genetics, fetal origins and adult ovarian phenotype of PCOS. In bovine fetal ovaries (n = 37) of 18 PCOS candidate genes only SUMO1P1 was not expressed. Three patterns of expression were observed: early gestation (FBN3, GATA4, HMGA2, TOX3, DENND1A, LHCGR and FSHB), late gestation (INSR, FSHR, and LHCGR) and throughout gestation (THADA, ERBB4, RAD50, C8H9orf3, YAP1, RAB5B, SUOX and KRR1). A splice variant of FSHB exon 3 was also detected early in the bovine ovaries, but exon 2 was not detected. Three other genes, likely to be related to the PCOS aetiology (AMH, AR and TGFB1I1), were also expressed late in gestation. Significantly within each of the three gene groups, the mRNA levels of many genes were highly correlated with each other, despite, in some instances, being expressed in different cell types. TGFβ is a well-known stimulator of stromal cell replication and collagen synthesis and TGFβ treatment of cultured fetal ovarian stromal cells inhibited the expression of INSR, AR, C8H9orf3 and RAD50 and stimulated the expression of TGFB1I1. In human ovaries (n = 15, < 150 days gestation) many of the same genes as in bovine (FBN3, GATA4, HMGA2, FSHR, DENND1A and LHCGR but not TOX3 or FSHB) were expressed and correlated with each other. With so many relationships between PCOS candidate genes during development of the fetal ovary, including TGFβ and androgen signalling, we suggest that future studies should determine if perturbations of these genes in the fetal ovary can lead to PCOS in later life

Regulation of fibrillins and modulators of TGFβ in fetal bovine and human ovaries

Fibrillins 1–3 are stromal extracellular matrix proteins that play important roles in regulating TGFβ activity, which stimulates fibroblasts to proliferate and synthesize collagen. In the developing ovary, the action of stroma is initially necessary for the formation of ovigerous cords and subsequently for the formation of follicles and the surface epithelium of the ovary. FBN3 is highly expressed only in early ovarian development and then it declines. In contrast, FBN1 and 2 are upregulated in later ovarian development. We examined the expression of FBN1–3 in bovine and human fetal ovaries. We used cell dispersion and monolayer culture, cell passaging and tissue culture. Cells were treated with growth factors, hormones or inhibitors to assess the regulation of expression of FBN1–3. When bovine fetal ovarian tissue was cultured, FBN3 expression declined significantly. Treatment with TGFβ-1 increased FBN1 and FBN2 expression in bovine fibroblasts, but did not affect FBN3 expression. Additionally, in cultures of human fetal ovarian fibroblasts (9–17 weeks gestational age), the expression of FBN1 and FBN2 increased with passage, whereas FBN3 dramatically decreased. Treatment with activin A and a TGFβ family signaling inhibitor, SB431542, differentially regulated the expression of a range of modulators of TGFβ signaling and of other growth factors in cultured human fetal ovarian fibroblasts suggesting that TGFβ signaling is differentially involved in the regulation of ovarian fibroblasts. Additionally, since the changes in FBN1–3 expression that occur in vitro are those that occur with increasing gestational age in vivo, we suggest that the fetal ovarian fibroblasts mature in vitro.Nicole A Bastian, Rosemary A Bayne, Katja Hummitzsch, Nicholas Hatzirodos, Wendy M Bonner, Monica D Hartanti, Helen F Irving-Rodgers, Richard A Anderson and Raymond J Rodger

Transcript abundance of stromal and thecal cell related genes during bovine ovarian development

<div><p>Movement and expansion of mesonephric-derived stroma appears to be very important in the development of the ovary. Here, we examined the expression of 24 genes associated with stroma in fetal ovaries during gestation (n = 17; days 58–274) from <i>Bos taurus</i> cattle. RNA was isolated from ovaries for quantitative RT-PCR. Expression of the majority of genes in TGFβ signalling, stromal transcription factors (<i>NR2F2</i>, <i>AR)</i>, and some stromal matrix genes (<i>COL1A1</i>, <i>COL3A1</i> and <i>FBN1</i>, but not <i>FBN3</i>) showed a positive linear increase with gestational age. Expression of genes associated with follicles (<i>INSL3</i>, <i>CYP17A1</i>, <i>CYP11A1</i> and <i>HSD3B1</i>), was low until mid-gestation and then increased with gestational age. <i>LHCGR</i> showed an unusual bimodal pattern; high levels in the first and last trimesters. <i>RARRES1</i> and <i>IGFBP3</i> also increased with gestational age. To relate changes in gene expression in stromal cells with that in non stromal cells during development of the ovary we combined the data on the stromal genes with another 20 genes from non stromal cells published previously and then performed hierarchical clustering analysis. Three major clusters were identified. Cluster 1 genes (<i>GATA4</i>, <i>FBN3</i>, <i>LHCGR</i>, <i>CYP19A1</i>, <i>ESR2</i>, <i>OCT4</i>, <i>DSG2</i>, <i>TGFB1</i>, <i>CCND2</i>, <i>LGR5</i>, <i>NR5A1</i>) were characterised by high expression only in the first trimester. Cluster 2 genes (<i>FSHR</i>, <i>INSL3</i>, <i>HSD3B1</i>, <i>CYP11A1</i>, <i>CYP17A1</i>, <i>AMH</i>, <i>IGFBP3</i>, <i>INHBA</i>) were highly expressed in the third trimester and largely associated with follicle function. Cluster 3 (<i>COL1A1</i>, <i>COL3A1</i>, <i>FBN1</i>, <i>TGFB2 TGFB3</i>, <i>TGFBR2</i>, <i>TGFBR3</i>, <i>LTBP2</i>, <i>LTBP3</i>, <i>LTBP4</i>, <i>TGFB1I1</i>, <i>ALDH1A1</i>, <i>AR</i>, <i>ESR1</i>, <i>NR2F2</i>) had much low expression in the first trimester rising in the second trimester and remaining at that level during the third trimester. Cluster 3 contained members of two pathways, androgen and TGFβ signalling, including a common member of both pathways namely the androgen receptor cofactor TGFβ1 induced transcript 1 protein (<i>TGFB1I1</i>; hic5). <i>GATA4</i>, <i>FBN3</i> and <i>LHCGR</i>, were highly correlated with each other and were expressed highly in the first trimester during stromal expansion before follicle formation, suggesting that this could be a critical phase in the development of the ovarian stroma.</p></div

A new model of development of the mammalian ovary and follicles

Ovarian follicular granulosa cells surround and nurture oocytes, and produce sex steroid hormones. It is believed that during development the ovarian surface epithelial cells penetrate into the ovary and develop into granulosa cells when associating with oogonia to form follicles. Using bovine fetal ovaries (n = 80) we identified a novel cell type, termed GREL for Gonadal Ridge Epithelial-Like. Using 26 markers for GREL and other cells and extracellular matrix we conducted immunohistochemistry and electron microscopy and chronologically tracked all somatic cell types during development. Before 70 days of gestation the gonadal ridge/ovarian primordium is formed by proliferation of GREL cells at the surface epithelium of the mesonephros. Primordial germ cells (PGCs) migrate into the ovarian primordium. After 70 days, stroma from the underlying mesonephros begins to penetrate the primordium, partitioning the developing ovary into irregularly-shaped ovigerous cords composed of GREL cells and PGCs/oogonia. Importantly we identified that the cords are always separated from the stroma by a basal lamina. Around 130 days of gestation the stroma expands laterally below the outermost layers of GREL cells forming a sub-epithelial basal lamina and establishing an epithelial-stromal interface. It is at this stage that a mature surface epithelium develops from the GREL cells on the surface of the ovary primordium. Expansion of the stroma continues to partition the ovigerous cords into smaller groups of cells eventually forming follicles containing an oogonium/oocyte surrounded by GREL cells, which become granulosa cells, all enclosed by a basal lamina. Thus in contrast to the prevailing theory, the ovarian surface epithelial cells do not penetrate into the ovary to form the granulosa cells of follicles, instead ovarian surface epithelial cells and granulosa cells have a common precursor, the GREL cell.Katja Hummitzsch, Helen F. Irving-Rodgers, Nicholas Hatzirodos, Wendy Bonner, Laetitia Sabatier, Dieter P. Reinhardt, Yoshikazu Sado, Yoshifumi Ninomiya, Dagmar Wilhelm and Raymond J. Rodger

Transcriptome profiling of the theca interna in transition from small to large antral ovarian follicles.

The theca interna layer of the ovarian follicle forms during the antral stage of follicle development and lies adjacent to and directly outside the follicular basal lamina. It supplies androgens and communicates with the granulosa cells and the oocyte by extracellular signaling. To better understand developmental changes in the theca interna, we undertook transcriptome profiling of the theca interna from small (3-5 mm, n = 10) and large (9-12 mm, n = 5) healthy antral bovine follicles, representing a calculated >7-fold increase in the amount of thecal tissue. Principal Component Analysis and hierarchical classification of the signal intensity plots for the arrays showed no clustering of the theca interna samples into groups depending on follicle size or subcategories of small follicles. From the over 23,000 probe sets analysed, only 76 were differentially expressed between large and small healthy follicles. Some of the differentially expressed genes were associated with processes such as myoblast differentiation, protein ubiquitination, nitric oxide and transforming growth factor β signaling. The most significant pathway affected from our analyses was found to be Wnt signaling, which was suppressed in large follicles via down-regulation of WNT2B and up-regulation of the inhibitor FRZB. These changes in the transcriptional profile could have been due to changes in cellular function or alternatively since the theca interna is composed of a number of different cell types it could have been due to any systematic change in the volume density of any particular cell type. However, our study suggests that the transcriptional profile of the theca interna is relatively stable during antral follicle development unlike that of granulosa cells observed previously. Thus both the cellular composition and cellular behavior of the theca interna and its contribution to follicular development appear to be relatively constant throughout the follicle growth phase examined

Measurement of gene expression by qRT-PCR.

<p>The data are shown as the mean ± SEM (n = 7 for small follicle group, n = 4 for large follicle group). qRT-PCR values were determined from the mean of the ratio of 2^−ΔCt of the target genes to cyclophilin A (<i>PPIA</i>) and glyceraldehyde phosphate dehydrogenase (GAPDH), and the microarray values are signal intensities (normalized but not log transformed). Significantly different results for qRT-PCR were determined by Student's <i>t</i>-test. The <i>P</i> values for the microarray results are corrected for multiple testing using the FDR (*<i>P</i><0.05, **<i>P</i><0.01 and ***<i>P</i><0.001).</p

Top canonical pathways mapped in IPA (A) and GO terms (B) classified under biological process.

<p>Data set analysed were genes differentially regulated (2 fold with FDR <i>P</i><0.05) between atretic and healthy samples. In (A) the bar chart on the left represents the percentage of genes from the data set that map to each canonical pathway, showing those which are up-regulated (in red) and down-regulated (in blue) in atretic compared with healthy follicles. The line chart on the right ranks these pathways, from the highest to lowest degree of association based on the value of Benjamini-Hochberg test for multiple corrections (bottom to top in graph on right). In (B) the bar chart on the left represents the proportion of genes which map to a GO term associated with a biological process. The line chart on the right ranks these pathways from the highest to lowest degree of association (bottom to top) using the Benjamini-Yuketeli test for multiple corrections.</p

Expression data for up regulated genes in granulosa cells from small and large follicles.

<p>The data are shown as the mean ± SEM (n = 10 for small follicle group, n = 4 for large follicle group, GC = granulosa cells, TI = theca interna). qRT-PCR values were determined from the geometric mean of 2^-ΔΔCt of the target genes to the cyclophilin A (<i>PPIA</i>) and glyceraldehyde phosphate dehydrogenase (<i>GAPDH</i>), and the microarray values are signal intensities (normalized but not log transformed). Significantly different results for qRT-PCR were determined by one-way ANOVA with Tukey’s post hoc test. The <i>P</i> values for the microarray results were corrected for multiple testing using the FDR. All values which were statistically different from each other are indicated by the different alphabetical symbols in the graphs.</p