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Polycystic ovary syndrome (PCOS) appears to have a genetic predisposition and a fetal origin. We compared the expression levels of 25 PCOS candidate genes from adult control and PCOS human ovaries (n = 16) using microarrays. Only one gene was potentially statistically different. Using qRT-PCR, expression of PCOS candidate genes was examined in bovine fetal ovaries from early stages when they first developed stroma through to completion of development (n = 27; 60–270 days of gestation). The levels of ERBB3 mRNA negatively correlated with gestational age but positively with HMGA2, FBN3, TOX3, GATA4, and DENND1A.X1,2,3,4, previously identified as correlated with each other and expressed early. PLGRKT and ZBTB16, and less so IRF1, were also correlated with AMH, FSHR, AR, INSR, and TGFB1I1, previously identified as correlated with each other and expressed late. ARL14EP, FDFT1, NEIL2, and MAPRE1 were expressed across gestation and not correlated with gestational age as shown previously for THADA, ERBB4, RAD50, C8H9orf3, YAP1, RAB5B, SUOX, and KRR1. LHCGR, because of its unusual bimodal expression pattern, had some unusual correlations with other genes. In human ovaries (n = 15

Objective Mechanisms of insulin resistance in polycystic ovary syndrome (PCOS) remain ill defined, contributing to sub-optimal therapies. Recognising skeletal muscle plays a key role in glucose homeostasis we investigated early insulin signalling, its association with aberrant transforming growth factor β (TGFβ)-regulated tissue fibrosis. We also explored the impact of aerobic exercise on these molecular pathways. Methods A secondary analysis from a cross-sectional study was undertaken in women with (n = 30) or without (n = 29) PCOS across lean and overweight BMIs. A subset of participants with (n = 8) or without (n = 8) PCOS who were overweight completed 12 weeks of aerobic exercise training. Muscle was sampled before and 30 min into a euglycaemic-hyperinsulinaemic clamp pre and post training. Results We found reduced signalling in PCOS of mechanistic target of rapamycin (mTOR). Exercise training augmented but did not completely rescue this signalling defect in women with PCOS. Genes in the TGFβ signalling network were upregulated in skeletal muscle in the overweight women with PCOS but were unresponsive to exercise training except for genes encoding LOX, collagen 1 and 3. Conclusions We provide new insights into defects in early insulin signalling, tissue fibrosis, and hyperandrogenism in PCOS-specific insulin resistance in lean and overweight women. PCOS-specific insulin signalling defects were isolated to mTOR, while gene expression implicated TGFβ ligand regulating a fibrosis in the PCOS-obesity synergy in insulin resistance and altered responses to exercise. Interestingly, there was little evidence for hyperandrogenism as a mechanism for insulin resistance.

Bromine is one of the most abundant and ubiquitous trace elements in the biosphere and until recently had not been shown to perform any essential biological function in animals. A recent study demonstrated that bromine is required as a cofactor for peroxidasin-catalysed formation of sulfilimine crosslinks in Drosophila. In addition, bromine dietary deficiency is lethal in Drosophila, whereas bromine replenishment restores viability. The aim of this study was to examine the distribution and speciation of bromine in mammalian tissues and fluids to provide further insights into the role and function of this element in biological systems. In this study we used X-ray fluorescence (XRF) imaging and inductively coupled plasma-mass spectrometry (ICP-MS) to examine the distribution of bromine in bovine ovarian tissue samples, follicular fluid and aortic serum, as well as human whole blood and serum and X-ray absorption spectroscopy (XAS) to identify the chemical species of bromine in a range of mammalian tissue (bovine, ovine, porcine and murine), whole blood and serum samples (bovine, ovine, porcine, murine and human), and marine samples (salmon (Salmo salar), kingfish (Seriola lalandi) and Scleractinian coral). Bromine was found to be widely distributed across all tissues and fluids examined. In the bovine ovary in particular it was more concentrated in the sub-endothelial regions of arterioles. Statistical comparison of the near-edge region of the X-ray absorption spectra with a library of bromine standards led to the conclusion that the major form of bromine in all samples analysed was bromide.

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.

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.FBN3is highly expressed only in early ovarian development and then it declines. In contrast,FBN1and2are upregulated in later ovarian development. We examined the expression ofFBN1–3in 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 ofFBN1–3. When bovine fetal ovarian tissue was cultured,FBN3expression declined significantly. Treatment with TGFβ-1 increasedFBN1andFBN2expression in bovine fibroblasts, but did not affectFBN3expression. Additionally, in cultures of human fetal ovarian fibroblasts (9–17weeks gestational age), the expression ofFBN1andFBN2increased with passage, whereasFBN3dramatically 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 inFBN1–3expression that occurin vitroare those that occur with increasing gestational agein vivo, we suggest that the fetal ovarian fibroblasts maturein vitro.

In the ovarian follicular membrana granulosa there are morphological and functional differences between cells adjacent to the follicular fluid lumen, or aligning the basal lamina. Amongst the observed functional differences are steroidogenic capacity and expression levels of a novel basal lamina, focimatrix; both of which increase in the later stages of antral follicle growth. A number of different studies have produced apparently inconsistent results as to which cell layers are more steroidogenic. To examine this systematically, individual bovine follicles, confirmed as healthy by post hoc histological examination, were used to isolate populations of apical and basal granulosa cells. Cell counts revealed that the respective groups did not differ in the numbers of cells, thus confirming the separation of these populations. We measured gene expression (quantitative RT-PCR, n=8-10, follicle diameter 14.0±0.5 mm) and protein levels (Western immunoblotting, n=14, follicle diameter 11.9±0.5 mm) and hormone production from granulosa cells (2.5×10(5) viable cells/well in serum-free conditions for 24 h, n=15, diameter 12±0.5 mm). Levels of mRNA of HSD3B1 and CYP19A1 and three focimatrix genes COL4A1, HSPG2 and LAMB2 and LHCGR were significantly lower in apical granulosa cells (P0.05), whereas, expression of CYP11A1 and HSD17B1 were not different (P0.05). The protein levels of steroidogenic enzymes P450scc and P450arom were significantly higher in apical cells (P0.05), whereas those of 3β-hydroxysteroid dehydrogenase and 17β-hydroxysteroid dehydrogenase type 1 were not different (P0.05). Progesterone production was significantly lower and oestradiol production was significantly higher in apical granulosa cells (P0.05). These results confirm that apical and basal cells are functionally different, and the differences might be explained by the location of cells of different ages and maturity within the membrana granulosa. Discrepancies in the literature on their steroidogenic capacity may reflect differences in the steroidogenic parameters measured.

Several studies have demonstrated an association between polycystic ovary syndrome (PCOS) and the dinucleotide repeat microsatellite marker D19S884, which is located in intron 55 of the fibrillin-3 (FBN3) gene. Fibrillins, including FBN1 and 2, interact with latent transforming growth factor (TGF)-β-binding proteins (LTBP) and thereby control the bioactivity of TGFβs. TGFβs stimulate fibroblast replication and collagen production. The PCOS ovarian phenotype includes increased stromal collagen and expansion of the ovarian cortex, features feasibly influenced by abnormal fibrillin expression. To examine a possible role of fibrillins in PCOS, particularly FBN3, we undertook tagging and functional single nucleotide polymorphism (SNP) analysis (32 SNPs including 10 that generate non-synonymous amino acid changes) using DNA from 173 PCOS patients and 194 controls. No SNP showed a significant association with PCOS and alleles of most SNPs showed almost identical population frequencies between PCOS and control subjects. No significant differences were observed for microsatellite D19S884. In human PCO stroma/cortex (n = 4) and non-PCO ovarian stroma (n = 9), follicles (n = 3) and corpora lutea (n = 3) and in human ovarian cancer cell lines (KGN, SKOV-3, OVCAR-3, OVCAR-5), FBN1 mRNA levels were approximately 100 times greater than FBN2 and 200–1000-fold greater than FBN3. Expression of LTBP-1 mRNA was 3-fold greater than LTBP-2. We conclude that FBN3 appears to have little involvement in PCOS but cannot rule out that other markers in the region of chromosome 19p13.2 are associated with PCOS or that FBN3 expression occurs in other organs and that this may be influencing the PCOS phenotype. Refereed/Peer-reviewed

The X-ray Fluorescence Micro-spectroscopy (XFM) beamline at the Australian Synchrotron was used to image 97 follicle histological sections from 45 different bovine ovaries focusing on healthy antral follicles ranging from small (4 mm) up to preovulatory sizes (16 mm) and on antral follicles undergoing atresia. This analysis identified five elements (Cu, Fe, Zn, Se and Br) consistently present within the ovarian tissue with Fe, Zn and Se localised to specific structures. GeoPIXE v6.4g was subsequently used to extract quantitative information pertaining to the elemental concentrations surrounding each of these follicles. Statistical analysis suggested that significant elemental differences were evident between follicle groups sorted according to their health status (Fe and Br), and their size (Se). Se appeared to be the element which most greatly distinguished large antral follicles from smaller counterparts. The ability to use synchrotron radiation to measure trace element distributions in bovine follicles at such high resolutions could have a significant impact on understanding the mechanisms of follicular development. This research is intended to form a baseline study of healthy cycling ovaries which could later be extended to disease states, thereby improving our current understanding of infertility and endocrine diseases involving the ovary.

In studies using isolated ovarian granulosa and thecal cells it is important to assess the degree of cross contamination. Marker genes commonly used for granulosa cells include FSHR, CYP19A1 and AMH while CYP17A1 and INSL3 are used for thecal cells. To increase the number of marker genes available we compared expression microarray data from isolated theca interna with that from granulosa cells of bovine small (n = 10 for both theca and granulosa cells; 3-5 mm) and large (n = 4 for both theca and granulosa cells, > 9 mm) antral follicles. Validation was conducted by qRT-PCR analyses. Known markers such as CYP19A1, FSHR and NR5A2 and another 11 genes (LOC404103, MGARP, GLDC, CHST8, CSN2, GPX3, SLC35G1, CA8, CLGN, FAM78A, SLC16A3) were common to the lists of the 50 most up regulated genes in granulosa cells from both follicle sizes. The expression in theca interna was more consistent than in granulosa cells between the two follicle sizes. Many genes up regulated in theca interna were common to both sizes of follicles (MGP, DCN, ASPN, ALDH1A1, COL1A2, FN1, COL3A1, OGN, APOD, COL5A2, IGF2, NID1, LHFP, ACTA2, DUSP12, ACTG2, SPARCL1, FILIP1L, EGFLAM, ADAMDEC1, HPGD, COL12A1, FBLN5, RAMP2, COL15A1, PLK2, COL6A3, LOXL1, RARRES1, FLI1, LAMA2). Many of these were stromal extracellular matrix genes. MGARP, GLDC, CHST8, GPX3 were identified as new potential markers for granulosa cells, while FBLN5, OGN, RAMP2 were significantly elevated in the theca interna.

High levels of tumor necrosis factor-alpha (TNF-alpha), a pro-inflammatory cytokine, are present in the wound fluid of chronic nonhealing wounds. This leads to increased inflammation, cytokine expression, and ultimately results in impaired wound healing and tissue destruction. Etanercept is a recombinant fusion protein that consists of the soluble TNF receptor (p75) linked to the Fc portion of human IgG1. It is an effective inhibitor of TNF-alpha and has been shown to provide rapid and sustained improvement in rheumatoid arthritis by acting as a soluble receptor binding TNF-alpha and preventing its proinflammatory activities. Therefore, the aim of this study was to determine whether Etanercept could inhibit TNF-alpha activity in chronic wound fluid. Wound fluid was collected from the venous leg ulcers of 16 different patients. The effect of Etanercept on TNF-alpha activity was evaluated using both a TNF-alpha bioassay and an enzyme-linked immunoassay. Etanercept was found to reduce the cytotoxic effect of chronic wound fluid on L929 fibroblasts by approximately 30% and neutralized TNF-alpha binding in the enzyme-linked immunoassay by up to 80%. Direct application of Etanercept to chronic wounds may therefore reduce the inflammatory activity of TNF-alpha, which could reduce the chronicity of venous leg ulcers and thus aid in the healing of these wounds.

Skin from the embryonic day 17 rat retains the ability to epithelialize an excisional wound when isolated in serum-supplemented suspension culture. This ability is lost by embryonic day 19. We have investigated this effect of gestational age on fetal epithelial wound closure by correlating the involvement of filamentous actin (F-actin) and its associated proteins, paxillin and gelsolin, in the wound margins of embryonic day 17 and 19 rat skins, with the ability to close a full thickness excisional wound. Using fluorescent-phalloidin histochemistry and scanning confocal microscopy, actin polymerization was observed some five to six cells back from the margin of wounds in the embryonic day 17 skin as early as 3 h postwounding. As the wounds closed over the following 48–72 h, the actin further condensed around the epithelial margin before dispersing after wound closure. In contrast, no organization of actin was seen in the epithelial margin of wounds in skin from the embryonic day 19 embryos. Instead, actin filaments were observed surrounding the dermal wound margins. Chemical or mechanical disruption of the actin in wounded embryonic day 17 skins prevented epithelial closure, although wound repair was independent of cell division. In particular, incising the wound margin 24 h after wounding resulted in the “springing-open” of the embryonic day 17 wound but not the embryonic day 19 wound, reflecting the development of tension in the embryonic day 17 wound margin. Expression of paxillin mRNA was upregulated following wounding at embryonic day 17 but not at embryonic day 19. Paxillin was also observed to colocalize with actin in embryonic day 17 wounds, but not embryonic day 19 wounds, indicating a potential role for paxillin in epithelial repair of the fetal wound. In contrast, gelsolin mRNA was upregulated in embryonic day 19 fetal skin but not at embryonic day 17 and gelsolin protein was observed surrounding actin filaments at embryonic day 19 but not embryonic day 17. These results demonstrate a change in the mechanism of wound epithelialization at the same gestational age that fetal wounds change from scar-free to scar-forming wound repair.

The theca interna is a specialized stromal layer that envelops each growing ovarian follicle. It contains capillaries, fibroblasts, immune cells and the steroidogenic cells that synthesize androgens for conversion to estradiol by the neighboring granulosa cells. During reproductive life only a small number of follicles will grow to a sufficient size to ovulate, whereas the majority of follicles will undergo regression/atresia and phagocytosis by macrophages. To identify genes which are differentially regulated in the theca interna during follicular atresia, we undertook transcriptome profiling of the theca interna from healthy (n = 10) and antral atretic (n = 5) bovine follicles at early antral stages (

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.

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.

Background Oocytes mature in ovarian follicles surrounded by granulosa cells. During follicle growth, granulosa cells replicate and secrete hormones, particularly steroids close to ovulation. However, most follicles cease growing and undergo atresia or regression instead of ovulating. To investigate the effects of stimulatory (follicle-stimulating hormone; FSH) and inhibitory (tumour necrosis factor alpha; TNFα) factors on the granulosa cell transcriptome, bovine ovaries were obtained from a local abattoir and pools of granulosa cells were cultured in vitro for six days under defined serum-free conditions with treatments present on days 3–6. Initially dose–response experiments (n = 4) were performed to determine the optimal concentrations of FSH (0.33 ng/ml) and TNFα (10 ng/ml) to be used for the microarray experiments. For array experiments cells were cultured under control conditions, with FSH, with TNFα, or with FSH plus TNFα (n = 4 per group) and RNA was harvested for microarray analyses. Results Statistical analysis showed primary clustering of the arrays into two groups, control/FSH and TNFα/TNFα plus FSH. The effect of TNFα on gene expression dominated that of FSH, with substantially more genes differentially regulated, and the pathways and genes regulated by TNFα being similar to those of FSH plus TNFα treatment. TNFα treatment reduced the endocrine activity of granulosa cells with reductions in expression of FST, INHA, INBA and AMH. The top-ranked canonical pathways and GO biological terms for the TNFα treatments included antigen presentation, inflammatory response and other pathways indicative of innate immune function and fibrosis. The two most significant networks also reflect this, containing molecules which are present in the canonical pathways of hepatic fibrosis/hepatic stellate cell activation and transforming growth factor β signalling, and these were up regulated. Upstream regulator analyses also predicted TNF, interferons γ and β1 and interleukin 1β. Conclusions In vitro, the transcriptome of granulosa cells responded minimally to FSH compared with the response to TNFα. The response to TNFα indicated an active process akin to tissue remodelling as would occur upon atresia. Additionally there was reduction in endocrine function and induction of an inflammatory response to TNFα that displays features similar to immune cells.

Background The 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). Results Principal 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. Conclusions Small 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.

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.

To examine the detailed composition of glycosaminoglycans during bovine ovarian follicular development and atresia, the specialized stromal theca layers were separated from the stratified epithelial granulosa cells of healthy (n = 6) and atretic (n = 6) follicles in each of three size ranges: small (3–5 mm), medium (6-9 mm) and large (10 mm or more) (n = 29 animals). Fluorophore-assisted carbohydrate electrophoresis analyses (on a per cell basis) and immunohistochemistry (n = 14) were undertaken. We identified the major disaccharides in thecal layers and the membrana granulosa as chondroitin sulfate-derived ∆uronic acid with 4-sulfated N-acetylgalactosamine and ∆uronic acid with 6-sulfated N-acetylgalactosamine and the heparan sulfate-derived Δuronic acid with N-acetlyglucosamine, with elevated levels in the thecal layers. Increasing follicle size and atresia was associated with increased levels of some disaccharides. We concluded that versican contains 4-sulfated N-acetylgalactosamine and it is the predominant 4-sulfated N-acetylgalactosamine proteoglycan in antral follicles. At least one other non- or 6-sulfated N-acetylgalactosamine proteoglycan(s), which is not decorin or an inter-α-trypsin inhibitor family member, is present in bovine antral follicles and associated with hitherto unknown groups of cells around some larger blood vessels. These areas stained positively for chondroitin/dermatan sulfate epitopes [antibodies 7D4, 3C5, and 4C3], similar to stem cell niches observed in other tissues. The sulfation pattern of heparan sulfate glycosaminoglycans appears uniform across follicles of different sizes and in healthy and atretic follicles. The heparan sulfate products detected in the follicles are likely to be associated with perlecan, collagen XVIII or betaglycan., Highlights ► In ovarian follicles the major chondroitin sulfates are ∆uronic acid with 4- or 6-sulfated N-acetylgalactosamine. ► Versican is the predominant 4-sulfated N-acetylgalactosamine proteoglycan in follicles. ► An unidentified non- or 6-sulfated N-acetylgalactosamine proteoglycan is also present in follicles. ► 6-sulfated N-acetylgalactosamine epitopes [antibodies 7D4, 3C5, 4C3 and 3B3(+)] identify a potential stem cell niche. ► The major heparan sulfate are Δuronic acid with N-acetlyglucosamine, and the sulfation is uniform in the follicles examined.

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.—Hatzirodos, N., Bayne, R. A., Irving-Rodgers, H. F., Hummitzsch, K., Sabatier, L., Lee, S., Bonner, W., Gibson, M. A., Rainey, W. E., Carr, B. R., Mason, H. D., Reinhardt, D. P., Anderson, R. A., Rodgers, R. J. Linkage of regulators of TGF-β activity in the fetal ovary to polycystic ovary syndrome.

During the growth of bovine follicles, one emerges from a wave as the largest and dominant follicle. What regulates dominance is not known but candidates include oestradiol, transforming growth factor beta beta1 (TGFB1), and recently CYP11AI (cholesterol side-chain cleavage) and focal intra-epithelial matrix (focimatrix). To examine this, pairs of bovine ovaries with 2 or more follicles of equal size (>5mm) and hence in a wave before deviation, were collected at an abattoir (6.7+/-SEM 0.1mm diameter; n=14 animals, 35 follicles in total). These follicles were dissected and follicular fluid collected to measure progesterone and oestradiol concentrations. A portion of the follicle wall was processed for histological classification of health or atresia and granulosa cells were harvested for quantitative RT-PCR of focimatrix components [COL4A1 (collagen type IV alpha1), LAMB2 (laminin beta2) and HSPG2 (perlecan)], steroidogenic enzymes [CYP11A1 and CYP19A1] and TGFB1. For statistical analyses follicles within each animal were grouped into either the highest (oestradiol, CYP11A1) or lowest (TGFB1) expression (n=14) for comparison with the remaining follicles (n=21). When grouped on oestradiol no other parameters differed significantly, and when grouped on TGFB1 some parameters were different however the levels were also lower, and not higher as expected. When grouped on CYP11A1 other parameters were significantly elevated in the high CYP11A1 group (COL4A1P

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/ROBOandPI3Ksignalling, and (B)ITGB5and extracellular matrix signalling through extracellular signal related kinases (ERKs). Upstream regulator genes which were predicted to be active in large follicles includedSTATandXBP1.By comparison, developmental processes such as those stimulated byKIT,IHHandMESTwere most active in small follicles.MGEA5was 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.

Granulosa cells constitute the ovarian follicular epithelium which at the beginning of folliculogenesis forms a single layer of flattened cells. As the follicle matures the cells acquire a cuboidal morphology, proliferate and differentiate into the cumulus cells surrounding the oocyte, and the mural granulosa cells forming the inner layer of the follicle (the membrana granulosa). Mural granulosa cells may further differ in their functionality depending on whether they are situated apically or basally within the stratified membrana granulosa. Late in folliculogenesis granulosa cells develop the ability to produce oestradiol, and also a specialised extracellular matrix (focimatrix) which is more abundant between apical cells. In order to investigate possible differences between granulosa cells, the expression of genes for oestradiol synthesis (CYP11A1, CYP19A1), focimatrix components (LAMB2, COL4A1, HSPG2), FSH and LH receptors, and cell cycle genes (CCND2, CCNE1, CCNE2, CDKN1B, CDKN2D) were examined in apical and basal granulosa cells from large healthy bovine follicles [n = 18, 14.3 ± 0.3 mm (mean + SEM)] using quantitative RT-PCR. Apical granulosa cells were collected by flushing the follicle with balanced salt solution. The remaining cells were detached from the follicular basal lamina by gently scraping; these are the basal granulosa cells. This collection method resulted in equivalent cell yields of apical and basal cells. Expression for all genes was significantly higher in basal cells in comparison to apical cells (P < 0.05), except for the cycle genes CCND2 and CDKN2D, which did not differ between cell populations. These results suggest that functional heterogeneity exists within the membrana granulosa. How differences between apical and basal cells are established is unknown but may be due to the proximity of the basal cells to the follicular basal lamina. The relevance of this aspect of follicle maturation to the endocrine function of granulosa cells has yet to be determined.

Small antral follicles 5 mm there is upregulation in expression of focimatrix genes and later upregulation of the LH receptor and steroidogenic enzymes, especially aromatase, in the granulosa cells. For follicles at sizes >5 mm entering atresia the granulosa cells are the first in the follicle to die. Thus expression of genes in granulosa cells is critical to the fate of the follicle. To examine granulosa cells of small follicles we collected bovine ovaries and dissected follicles, removed part of the follicle wall for subsequent classification of health or atresia, and harvested the remaining granulosa cells for RNA isolation. Follicles examined included small follicles (10 mm, n = 4). RNA was hybridized to Affymetrix GeneChip Bovine Genome Arrays and the results were analysed using Partek Genomics Suite software. The number of genes which were 2 fold differentially regulated between large and small follicles by Benjamini Hochberg post hoc test (False Discovery Rate, P < 0.05) was 2408 and between healthy and atretic small follicles was 4931. The coefficient of variation (CV; SD/mean × 100) for the expression level of each gene for each group was calculated. A gene frequency distribution indicated greater heterogeneity in expression levels in small follicles in comparison to large follicles. Furthermore, the greatest variability in genes in small follicles includes those that are either up or down regulated due to atresia or growth. We therefore conclude that variability in small follicles is a consequence of alternative fates that small follicle can undergo.

Development of ovarian follicles involves changes in cell function and remodelling of the follicular wall. Remodelling necessitates changes in the extracellular matrix, including proteoglycans (PGs). PGs contain glycosaminoglycans (GAGs) covalently bound to a protein core. The length of GAG chains in PGs and the degree and pattern of sulphation differs between cell types and change as cells alter their phenotype. PGs that have been identified in follicles include the chondroitin sulphate (CS) PG, versican and inter-a trypsin inhibitor, and the heparan sulphate (HS) PGs, perlecan and type XVIII collagen. The latter two are found in focimatrix, the follicular and the thecal subendothelial basal laminas. To examine GAGs composition in follicles, bovine antral follicles of various sizes were collected. Follicles were dissected and a biopsy taken for histological classification of health. Theca layers and granulosa cells were collected separately and analysed by fluorophore-assisted carbohydrate (FACE) analysis of GAGs following digestion to disaccharides with chondroitinase ABC, hyaluronidase, heparinase, and heparitinases I and II. Four non GAG sugars and 12 different GAG derived disaccharides were identified and quantitated on a per DNA basis. Healthy versus atretic follicles for each cell type were compared and correlation analyses were also undertaken. Immunohistochemistry using CS specific antibodies was also conducted. There was no effect of size on the GAG content for either granulosa or theca cells. The 4- and 6- sulphated CS sugars were the most abundant following digestion in all tissues. Theca had higher levels than granulosa cells of HS derived disaccharides and also of un- or 4- or 6- N-sulphated CS derived disaccharides. Some sulphated CS moieties localised uniquely to the stroma surrounding blood vessels in the theca externa. Atretic follicles had lower amounts of disaccharides derived from HS in both granulosa and thecal cells, suggesting that heparanase may be activated upon atresia.

During growth of bovine follicles, one emerges as the largest and dominant follicle. What regulates dominance remains unknown, but candidates include oestradiol, TGFB1, and recently CYP11A1 and focal intra-epithelial matrix (focimatrix). The two to four largest follicles were dissected from pairs of bovine ovaries and follicular fluid collected. A portion of the follicle wall was histologically classified for follicle health or atretia, and granulosa cells harvested for quantitative RT-PCR. Messenger RNA levels of focimatrix (COL4A1, LAMB2, HSPG2), steroidogenic enzymes (CYP11A1, CYP19A1) and TGFB1 genes were measured. Follicular fluid progesterone and oestradiol concentrations were measured by RIA. Follicles were identified as pre-deviated (before size-deviation) if the largest two or more healthy follicles were of equal size (6.7±0.1 mm, n = 14 animals, 35 follicles), and as post-deviated (after size-deviation) if they differed in size by 0.5–1.0 mm (7.2±0.2 mm; n = 11 animals, 26 follicles). For analyses, pre-deviated follicles were grouped into either the highest (oestradiol, CYP11A1) or lowest (TGFB1) expression (n = 14) and compared to the remaining follicles (n = 21). Deviated follicles were classified into dominant (n = 12) and subordinate (n = 14) based on diameter. Dominant follicles did not differ from subordinate follicles in any parameters measured, but were significantly larger than subordinate or pre-deviated follicles (P