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.

BackgroundThe major function of the ovary is to produce oocytes for fertilisation. Oocytes mature in follicles surrounded by nurturing granulosa cells and all are enclosed by a basal lamina. During growth, granulosa cells replicate and a large fluid-filled cavity (the antrum) develops in the centre. Only follicles that have enlarged to over 10 mm can ovulate in cows. In mammals, the number of primordial follicles far exceeds the numbers that ever ovulate and atresia or regression of follicles is a mechanism to regulate the number of oocytes ovulated and to contribute to the timing of ovulation. To better understand the molecular basis of follicular atresia, we undertook transcriptome profiling of granulosa cells from healthy (n = 10) and atretic (n = 5) bovine follicles at early antral stages (< 5 mm).ResultsPrincipal Component Analysis (PCA) and hierarchical classification of the signal intensity plots for the arrays showed primary clustering into two groups, healthy and atretic. These analyses and size-frequency plots of coefficients of variation of signal intensities revealed that the healthy follicles were more heterogeneous. Examining the differentially-expressed genes the most significantly affected functions in atretic follicles were cell death, organ development, tissue development and embryonic development. The overall processes influenced by transcription factor gene TP53 were predicted to be activated, whereas those of MYC were inhibited on the basis of known interactions with the genes in our dataset. The top ranked canonical pathway contained signalling molecules common to various inflammatory/fibrotic pathways such as the transforming growth factor-β and tumour necrosis factor-α pathways. The two most significant networks also reflect this pattern of tissue remodelling/fibrosis gene expression. These networks also contain molecules which are present in the canonical pathways of hepatic fibrosis/hepatic stellate cell activation and transforming growth factor-β signalling and were up regulated.ConclusionsSmall healthy antral follicles, which have a number of growth outcomes, exhibit greater variability in gene expression, particularly in genes associated with cell division and other growth-related functions. Atresia, on the other hand, not only involves cell death but clearly is an active process similar to wound healing.

Although not often discussed, the ovaries of women with polycystic ovary syndrome (PCOS) show all the hallmarks of increased TGF-β activity, with increased amounts of fibrous tissue and collagen in the ovarian capsule or tunica albuginea and ovarian stroma. Recent studies suggest that PCOS could have fetal origins. Genetic studies of PCOS have also found linkage with a microsatellite located in intron 55 of the extracellular matrix protein fibrillin 3. Fibrillins regulate TGF-β bioactivity in tissues by binding latent TGF-β binding proteins. We therefore examined expression of fibrillins 1-3, latent TGF-β binding proteins 1-4, and TGF-β 1-3 in bovine and human fetal ovaries at different stages of gestation and in adult ovaries. We also immunolocalized fibrillins 1 and 3. The results indicate that TGF-β pathways operate during ovarian fetal development, but most important, we show fibrillin 3 is present in the stromal compartments of fetal ovaries and is highly expressed at a critical stage early in developing human and bovine fetal ovaries when stroma is expanding and follicles are forming. These changes in expression of fibrillin 3 in the fetal ovary could lead to a predisposition to develop PCOS in later life.

BackgroundAt later stages of folliculogenesis, the mammalian ovarian follicle contains layers of epithelial granulosa cells surrounding an antral cavity. During follicle development granulosa cells replicate, secrete hormones and support the growth of the oocyte. In cattle, the follicle needs to grow > 10 mm in diameter to allow an oocyte to ovulate, following which the granulosa cells cease dividing and differentiate into the specialised cells of the corpus luteum. To better understand the molecular basis of follicular growth and granulosa cell maturation, we undertook transcriptome profiling of granulosa cells from small (< 5 mm; n = 10) and large (> 10 mm, n = 4) healthy bovine follicles using Affymetrix microarrays (24,128 probe sets).ResultsPrincipal component analysis for the first two components and hierarchical clustering showed clustering into two groups, small and large, with the former being more heterogeneous. Size-frequency distributions of the coefficient of variation of the signal intensities of each probe set also revealed that small follicles were more heterogeneous than the large. IPA and GO enrichment analyses revealed that processes of axonal guidance, immune signalling and cell rearrangement were most affected in large follicles. The most important networks were associated with: (A) Notch, SLIT/ROBO and PI3K signalling, and (B) ITGB5 and extracellular matrix signalling through extracellular signal related kinases (ERKs). Upstream regulator genes which were predicted to be active in large follicles included STAT and XBP1. By comparison, developmental processes such as those stimulated by KIT, IHH and MEST were most active in small follicles. MGEA5 was identified as an upstream regulator in small follicles. It encodes an enzyme that modifies the activity of many target proteins, including those involved in energy sensing, by removal of N-acetylglucosamine from serine and threonine residues.ConclusionsOur data suggest that as follicles enlarge more genes and/or pathways are activated than are inactivated, and gene expression becomes more uniform. These findings could be interpreted that either the cells in large follicles are more uniform in their gene expression, or that follicles are more uniform or a combination of both and that additional factors, such as LH, are additionally controlling the granulosa cells.

Studies of selenium (Se) status indicate that Se is necessary for fertility but how precisely is not known. We aimed to show that Se was important in bovine female reproductive function. The elemental distribution in the bovine ovary (n = 45 sections) was identified by X-ray fluorescence (XRF) imaging. Se was consistently localized to the granulosa cell layer of large (>10 mm) healthy follicles. Inductively Coupled Plasma - Mass Spectrometry revealed tenfold higher Se in the bovine follicle wall compared to corpora lutea. Gene expression analysis of selenoprotein genes GPX1, GPX3, VIMP and SELM in bovine granulosa cells revealed that only GPX1 was significantly up-regulated in large healthy follicles compared to the small healthy or atretic follicles (P < 0.05). Western immunoblotting identified GPX1 protein in bovine granulosa cells of large healthy follicles, but not of small healthy follicles. To assess if GPX1 was important in human follicles, cumulus cells from women undergoing IVF/ICSI with single embryo transfer were collected. Oocytes and embryos were cultured and transferred independently in 30 patients undergoing elective single embryo transfer. Gene expression of GPX1 was significantly higher in human cumulus cells from cumulus-oocyte complexes yielding a pregnancy (P < 0.05). We present the first XRF imaging of mammalian ovaries showing that Se is consistently localized to the granulosa cells of large healthy follicles. We conclude that Se and selenoproteins are elevated in large healthy follicles and may play a critical role as an antioxidant during late follicular development.

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.

The transforming growth factor betas are of major importance in the wound repair process; however, no studies to date have investigated the role of the transforming growth factor beta receptors in chronic venous leg ulcers or what effect healing has on these proteins. To determine whether the transforming growth factor beta peptides and their receptors are expressed in chronic venous wounds, we used immunofluorescent analysis and quantitative competitive reverse transcription polymerase chain reaction to identify the protein and mRNA expression, respectively. Biopsy samples from wounds and normal skin were collected from 12 patients with chronic venous leg ulcers and three patients undergoing reconstructive surgery, respectively. Additionally four of the chronic venous leg ulcer patients were re-biopsied between 2 and 8 wk after the first biopsy when the wounds had entered the healing phase. The tissue excised from the ulcers included the surrounding intact skin, the ulcer edge, and the ulcer base. Immunofluorescent staining for transforming growth factors beta1, beta2, and beta3 was observed within the epidermis of the skin surrounding the chronic venous ulcers and in fibroblasts and inflammatory cells of the dermis, although this staining was not as strong as that seen in normal unwounded skin. Very little staining could be seen within the ulcers for any of the ligands, however. In contrast the transforming growth factor beta type I receptor was observed throughout the ulcers and the normal unwounded skin biopsies, particularly in the basal epidermal cells. No immunofluorescence for the type II transforming growth factor beta receptor was observed in any of the ulcer biopsies investigated, although it was observed throughout the epidermis and in fibroblasts and inflammatory cells in the surrounding skin. Quantitative, competitive reverse transcription polymerase chain reaction was used to analyze mRNA expression for transforming growth factor beta1 and the type II receptor in the nonhealing ulcers and normal unwounded skin biopsies. These studies revealed that transforming growth factor beta1 and transforming growth factor beta receptor II mRNA was expressed in all the chronic nonhealing ulcers albeit at very low levels for the type II receptor. In marked contrast to the staining observed in nonhealing chronic ulcers, positive immunostaining was observed for the transforming growth factor betas and both the type I and type II receptors in healing ulcers. These results suggest that the absence of a viable receptor complex for the transforming growth factor betas in nonhealing chronic venous ulcers may contribute to wound chronicity.

Hepatocyte growth factor (HGF) and macrophage-stimulating protein (MSP) are structurally related molecules that stimulate epithelial cell proliferation and migration. MSP also acts directly as a chemoattractant for resident macrophages. These activities are integral to the wound repair processes of inflammation, epithelialization and tissue remodelling. To begin to examine the involvement of HGF and MSP in healing of cutaneous wounds we have mapped the temporal expression of these two molecules and their receptors, MET and RON respectively, in adult rat excisional wounds. Four 2x2-cm full-thickness excisional wounds were created on the dorsum of 18 rats, and biopsies were taken through the wounds at 3, 5, 7, 14, 21, and 28 days postwounding. These biopsies were analyzed using immunofluorescent staining and in situ hybridization (ISH). The number of cells staining positively for HGF and MET significantly increased in response to wounding. HGF staining and mRNA peaked at 7 days postwounding whereas MET was upregulated earlier, peaking after 3 days. Both HGF and MET protein were observed in fibroblasts of the dermis and in the newly forming granulation tissue. ISH studies also revealed that fibroblasts at the wound edges and within the newly forming granulation tissue also expressed HGF and c-met mRNA. Immunofluorescent staining revealed both MSP and RON within the wound, with maximum staining occurring between 7 and 21 days for both the ligand and receptor. In addition, MSP co-localized with a small subset of ED1-positive cells (monocytes). In contrast, ED2-positive cells (macrophages) did not co-localize with MSP. Thus, increased expression of HGF, MSP and their receptors MET and RON respectively was observed in response to wounding. Furthermore, MSP co-localization with a subset of monocytes may confirm a role for MSP in the activation of mature macrophages, which may be important in tissue remodelling.