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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

When first formed, the ovary only has an established epithelium at its base or hilum. Later, an epithelium is established around the rest of the ovary. To examine this further, we conducted scanning electron microscopy of the surface of bovine fetal ovaries and immunohistochemistry of ovarian cross-sections. From the earliest time point, the cells on the surface of the base or hilum of the ovary were cuboidal. On the remainder of the ovary, the surface was more irregular. By mid-development, the surface was covered completely with either a stratified or simple epithelium of cuboidal cells. Clefts were observed in the surface and appeared to form due to the expansion of stroma surrounding each open ovigerous cord, elevating the areas surrounding each cord, while leaving the opening of the cord to form the base of each cleft. The continued expansion of the surrounding stroma below the surface appeared not only to close the ovigerous cords from the surface but to compress the clefts into the shape of a groove. Later, most of the ovarian surface was covered with a simple cuboidal epithelium. The changes to the ovarian surface during fetal development coincide with the remodeling of the stroma and cords below.

During ovarian development stroma from the mesonephros penetrates and expands into the ovarian primordium and thus appears to be involved, at least physically, in the formation of ovigerous cords, follicles and surface epithelium. Cortical stromal development during gestation in bovine fetal ovaries (n=27) was characterised by immunohistochemistry and by mRNA analyses. Stroma was identified by immunostaining of stromal matrix collagen type I and proliferating cells were identified by Ki67 expression. The cortical and medullar volume expanded across gestation, with the rate of cortical expansion slowing over time. During gestation, the proportion of stroma in the cortex and total volume in the cortex significantly increased (P0.05). The expression levels of 12 genes out of 18 examined, including osteoglycin (OGN) and lumican (LUM), were significantly increased later in development (P

Background Annexin A2 is increased in serous ovarian cancer and plays an essential role in ovarian cancer invasion and metastasis. In combination with S100A10, annexin A2 plays an important role in the plasminogen activator system regulating plasmin production. The aim of this study was to investigate the potential utility of all-trans retinoid acid (ATRA), an inhibitor of the annexin A2-S100A10 signalling pathway, as a new therapeutic against serous ovarian cancer. Methods In this study we determined the effects of ATRA treatment (1-5 μM) on annexin A2 and S100A10 expression, plasmin activation, and the ability of ATRA to inhibit serous ovarian cancer cell survival, motility and invasion in vitro. We also employed an ex vivo tissue explant assay to assess response to ATRA treatment in serous ovarian cancers. Cryopreserved serous ovarian cancer tissues were cultured on gelatin sponges for 72 h with ATRA (1 μM). Effects on apoptosis and proliferation were assessed by immunohistochemistry using antibodies to cleaved caspase 3 or Ki67, respectively. Results Survival of serous ovarian cancer cells (OVCAR-3, OV-90, & OAW28) was significantly decreased by ATRA treatment (1-5 μM). ATRA (1 μM) also significantly decreased proliferation (Ki67 positivity, p = 0.0034), S100A10 protein levels (p = 0.0273), and increased cell apoptosis (cleaved caspase-3 positivity, p = 0.0024) in serous ovarian cancer tissues using the ex vivo tissue explant assay. In OAW28 cells, reduced cell survival following ATRA treatment was associated with a reduction of S100A10 mRNA and protein levels, S100A10 and annexin A2 membrane localization, plasmin generation, motility and invasion. In contrast, ATRA inhibited OV-90 cell survival and invasion but did not affect plasmin activation or S100A10 and annexin A2 expression or membrane localization. Conclusions These findings suggest that ATRA inhibits serous ovarian cancer proliferation and invasion via both S100A10 dependant and S100A10 independent mechanisms. Our results show that ATRA has promising potential as a novel therapy against serous ovarian cancer that warrants further evaluation. Electronic supplementary material The online version of this article (10.1186/s13046-018-1017-7) contains supplementary material, which is available to authorized users.

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.

The majority of ovarian cancer patients present with advanced disease and despite aggressive treatment, prognosis remains poor. Response to first-line carboplatin-containing chemotherapy is usually good, however, recurrence rates and subsequent chemoresistance are very high and ultimately responsible for the fatal outcome of the disease. To improve treatment outcomes pre-clinical models that can predict individual patient response to 1st line chemotherapy and novel therapeutics are urgently required. In this study, we employed an ex vivo ovarian cancer tissue explant assay to assess response to carboplatin and an inhibitor of the extracellular matrix molecule, hyaluronan (4-methylubelliferone, 4-MU), shown to inhibit cancer metastasis. Cryopreserved ovarian cancer tissues were cultured on gelatine sponges for 48-120 h with increasing concentrations of carboplatin (0-400 mu M) or 4-MU (1 mM) alone or the combination of both drugs. Effects on apoptosis and proliferation were assessed by immunohistochemistry using antibodies to cleaved caspase 3 or Ki67, respectively. The ex vivo tissue explant assay maintained viable tumor cells in an intact tumor microenvironment similar to the in vivo situation over the 120 h culture period. Carboplatin treatment promoted apoptosis in chemosensitive (P = 0.0047) but not chemoresistant cancer tissues. The combination of 4-MU (1 mM) and carboplatin (100 mu M) significantly increased apoptosis (P = 0.0111) and reduced proliferation (P = 0.0064) in chemoresistant tissues. Overall, our results show that the ex vivo explant assay is a robust and cost effective model to assess chemosensitivity and the effect of novel therapeutics in ovarian cancer. Refereed/Peer-reviewed

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.

Annexin A2 is increased in serous ovarian cancer and plays an essential role in ovarian cancer invasion and metastasis. In combination with S100A10, annexin A2 plays an important role in the plasminogen activator system regulating plasmin production. The aim of this study was to investigate the potential utility of all-trans retinoid acid (ATRA), an inhibitor of the annexin A2-S100A10 signalling pathway, as a new therapeutic against serous ovarian cancer. Survival of serous ovarian cancer cells (OVCAR-3, OV-90, & OAW28) was significantly decreased by ATRA treatment (1-5µM). ATRA (1µM) also significantly decreased proliferation (Ki67 positivity, p=0.0034), S100A10 protein levels (p=0.0273), and increased cell apoptosis (cleaved caspase-3 positivity, p=0.0024) in serous ovarian cancer tissues using an ex vivo explant assay. In OAW28 cells, reduced cell survival following ATRA treatment was associated with a reduction of S100A10 protein levels, S100A10 and annexin A2 membrane localization, plasmin generation, motility and invasion. In contrast, ATRA inhibited OV-90 cell survival and invasion but did not affect plasmin activation or S100A10 and annexin A2 membrane localization or protein levels. These findings suggest that ATRA inhibits serous ovarian cancer proliferation and invasion via both S100A10 dependant and S100A10 independent mechanisms. Our results show that ATRA has promising potential as a novel therapy against serous ovarian cancer that warrants further evaluation. Citation Format: Noor A Lokman,Rachel Ho, Kavyadharshini Gunasegaran, Wendy M Bonner , Martin K Oehler, Carmela Ricciardelli. ANTI-TUMOUR EFFECTS OF ALL-TRANS RETINOID ACID, AN ANNEXIN A2-S100A10 PATHWAY INHIBITOR ON SEROUS OVARIAN CANCER [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr NT-105.

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.

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 alpha1 and alpha2, laminin alpha1, beta1 and gamma1 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 alpha2 was only detected in some preantral and antral follicles. The focimatrix, a specialised matrix of the membrana granulosa, contained collagen type IV alpha1 and alpha2, laminin alpha1, beta1 and gamma1 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 alpha1 and alpha2, laminin alpha1, beta1 and gamma1 chains, nidogens 1 and 2, perlecan and collagen type XVIII. Laminins alpha4 and alpha5 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.

X-ray fluorescence (XRF) was used to image 40 histological cross-sections of bovine ovaries (n=19), focusing on structures including: antral follicles at different stages of growth or atresia, corpora lutea at three stages of development (II–IV), and capillaries, arterioles, and other blood vessels. This method identified three key trace elements [iron (Fe), zinc (Zn), and selenium (Se)] within the ovarian tissue which appeared to be localized to specific structures. Owing to minimal preprocessing of the ovaries, important high-resolution information regarding the spatial distribution of these elements was obtained with elemental trends and colocalizations of Fe and Zn apparent, as well as the infrequent appearance of Se surrounding the antrum of large follicles, as previously reported. The ability to use synchrotron radiation to measure trace element distributions in bovine ovaries at such high resolution and over such large areas could have a significant impact on understanding the mechanisms of ovarian development. This research is intended to form a baseline study of healthy ovaries which can later be extended to disease states, thereby improving our current understanding of infertility and endocrine diseases involving the ovary.

The microsatellite D19S884, located in intron 55 of fibrillin-3 (FBN3) gene, associates with polycystic ovary syndrome (PCOS) in familial studies. The family of fibrillin proteins (FBN1-3), which includes latent TGF-β binding proteins (LTBP-1 to -4), are extracellular matrix proteins. We localized and examined the expression of these proteins in the adult bovine ovaries (n = 7–10 per group, average age 681 days) born to mothers fed high (13% protein per total dry weight) or a low protein diet (5%) in each of the first and second trimesters of pregnancy (n = 4 groups). FBN1 and LTBP-1 and -2 were the major members expressed in the mature ovary. Each protein had a unique localization pattern but all were associated with stromal tissue including the tunica albuginea (FBN1 and LTBP-2 near surface, and FBN1 and LTBP-1 deeper in the tunica), cortical stroma (FBN1 and LTBP-1) and follicular thecal layers (FBN1 in theca interna, LTBP-1 in the inner regions of the theca externa, and LTBP-2 in the outer regions of the theca externa). No significant (P > 0.05) effects of maternal diet were observed on either the localization or the levels of mRNA of any of these proteins in the tunica. Expression levels of all three FBNs were positively correlated with each other, and FBN1 and 2 were positively correlated with LTBP-2, suggesting some level of co-ordinate regulation. This is the first study to investigate the expression and localization of these genes affecting TGFβ bioavailability in the ovary.

Extracellular matrix (ECM) has been shown to have distinct expression patterns during bovine follicle and corpus luteum (CL) formation and regression. To date little is known about ECM patterns during follicle and CL formation in the mouse ovary. Twenty nine day old mice were treated with PMSG on experimental day 0 and 1 to induce follicle development, and subsequently with hCG on day 2 to induce ovulation. Ovaries were collected for immunohistochemistry on days 0, 2 and 5 (n = 10 per group). Another group was similarly treated but was additionally mated, and ovaries examined in the pregnant mice on experimental day 11 (n = 7). The follicular basal lamina (BM) of all developmental stages contained collagen IV α1 and α2, laminin α1, β1 and γ1 chains, nidogen 1 and 2, and perlecan. Collagen XVIII was only found in BMs of primordial, primary and some preantral follicles, whereas laminin α2 was only present in some preantral and antral follicles. BMs of atretic follicles showed similar composition to healthy follicles. A specialized matrix of the membrana granulosa (focimatrix) was detected in both healthy and atretic follicles. The focimatrix contained collagen IV α1 and α2, laminin α1, β1 and γ1 chains, nidogen 1 and 2, perlecan and collagen XVIII, but not laminin α2. The immunostaining in CL was restricted to capillary sub-endothelial basal laminas and contained collagen IV α1 and α2, laminin α1, β1 and γ1 chains, nidogen 1 and 2, perlecan and collagen XVIII. No parenchymal matrix, as observed in cow and human, was detected. In addition, laminin α4 and α5 were not immunolocalised to any structure in the mouse ovary. The composition of the BM of follicles in the mouse ovary is similar to cow and rat, but the appearance and composition of the focimatrix differs from bovine ovaries.

Heparan sulphate proteoglycans such as perlecan and collagen XVIII, are present in the follicular basal lamina, focimatrix and sub-endothelial basal laminas in follicles, but following the LH surge the follicular basal lamina and focimatrix are degraded. Heparanase, which degrades the glycosaminoglycan heparan sulphate, is upregulated in granulosa cells during ovulation. We examined the effects of heparanase inhibition by maltohexaose sulphate treatment on ovulation, formation and vascularisation of corpora lutea, and the establishment of pregnancy. Female twenty-five days old mice were treated with 0.3 mg (>25 mg/kg) maltohexaose sulphate or saline daily for four days from time 0 h and subjected to induction of ovulation by treatment with PMSG (5 IU at 0 h) and hCG (5 IU at 48 h) and mated with a fertile male. Mice were sacrificed at time 0 h, 48 h, 120 h and day 11 (relative to the first injection of PMSG) (n = 20 per group), and ovaries were collected and pregnancy status examined. One ovary was stained with haematoxylin and eosin to measure numerical densities of antral follicles and corpora lutea in the ovary, as well as volume density of luteal tissue. Immunohistochemistry for CD34 (endothelial cell marker) and perlecan (localized to sub-endothelial basal lamina) was conducted to quantify the degree of vascularisation of the corpora lutea. In control mice there was a reduction in the number of antral follicles with increasing age, but no effect of treatment with sulphated maltohexaose was observed on antral follicle development. Nor was there any effect of sulphated maltohexaose on corpora lutea formation, or embryo implantation. In addition, this treatment did not have any effect on the vascularisation of the corpora lutea. These findings suggest that heparanase activity may not be critical during the later stages of follicle growth, ovulation and formation of corpora lutea.

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

Although numerous familial studies have indicated a genetic component to polycystic ovary syndrome (PCOS), a consistent and repeatable association with any particular gene has not been definitively established. However, one genetic marker, the microsatellite D19S884, has shown repeatable and consistent association with PCOS. D19S884 maps to intron 55 of the fibrillin-3 gene. The family of fibrillin extracellular matrix proteins includes the family of latent TGF-β binding proteins which are known to have roles in ovarian function. These provide structural support to tissues and regulate TGF-β bioavailability and are candidates for involvement in clinical manifestation of PCOS. Many of the risk factors associated with PCOS including type II diabetes, heart disease, and obesity are also known to be affected by in utero experience. In addition, although diet has not been examined in the PCOS outcome of offspring, some fetal experiences including androgen exposure have been linked to PCOS outcome. We therefore investigated the effect of protein content of maternal diet on expression of fibrillins-1, –2, and –3 and LTBPs-1 and –2 in the tunica albuginea of ovaries from female offspring by QRT–PCR. Diets were either high (13% protein per total dry weight) or a low protein (5%) during the first two trimesters. All three fibrillins were expressed in the tunica albuginea with the highest level of expression observed for fibrillin-1 followed by fibrillin-2, with low levels of expression of fibrillin-3. Both LTBP-1 and LTBP-2 were expressed, with the highest expression observed for LTBP-2. No significant (P > 0.05) effects of diet upon gene expression were observed. Expression levels of all three fibrillins were positively correlated with each other, and fibrillins-1 and 2 were positively correlated with LTBP-2. This is the first demonstration of the expression of fibrillins and LTBPs in the ovary, however these were unaffected by the maternal diets examined.