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Hypoxia-inducible factors (HIFs) are transcription factors responsible for sensing low oxygen levels and, in response, inducing the transcription of numerous genes. One of the main processes stimulated by HIFs is the formation of new vessels to increase oxygen supply to the tissue. Formation of the corpus luteum strongly depends on the vasculature and an active angiogenesis occurs during luteinization. In this study, we aimed to analyze the role of HIF in corpora lutea early formation and function, and in female fertility. To this aim, we superovulated mice by eCG and hCG, and administered the HIF inhibitor acriflavine (ACF) to the mice 3 h before hCG. We found a decrease in ovarian HIF1A and VEGFA, and in the vascular area in the animals treated with ACF. Moreover, we observed an increase in aberrant structures in the ovaries and in luteal cell apoptosis. Serum progesterone levels were decreased together with ovarian STAR expression. However, the animals treated with ACF during the early formation of the corpus luteum were completely fertile and no alterations were observed when the treated females were mated with fertile males. These results collectively suggest that HIF regulates gonadotropin-induced corpus luteum formation acting on luteal blood vessel formation, luteal cell survival, and progesterone synthesis. However, adequate HIF activity may not be essential to achieve and maintain pregnancy. These findings are significant to better understand the complex mechanisms of corpus luteum formation and identify potential abnormalities to allow better knowledge of ovarian physiology and pathologies in which this factor could be involved.

In Brief: The hypoglycemic drug metformin has shown reproductive effects in women, although its mechanism of action is not fully understood. In this study, we demonstrate the direct effects of metformin on the ovary of healthy mice, with no alterations in fertility., Abstract: Metformin is a hypoglycemic drug widely used in type-2 diabetes (T2D) patients. In recent years, this drug has been suggested as a treatment for gestational diabetes and recommended to women with ovarian hyperstimulation syndrome (PCOS) to increase the chances of pregnancy or avoid early miscarriages. However, the exact effects of metformin on the female reproductive tract in general, and on the ovary in particular, are still not completely understood. In this study, we analyzed the effect of metformin on fertility and ovarian physiology in healthy female mice. We found that this drug altered the estrous cycle, early follicular development, serum estradiol and progesterone levels, and ovarian steroidogenic enzyme expression. Moreover, ovarian angiogenesis was lower in metformin-treated animals compared with untreated ones, whereas natural or gonadotropin-induced fertilization rates remained unchanged. However, offspring of metformin-treated animals displayed decreased body weight at birth. In this work, we unraveled the main effects of metformin on the ovary, isolated from other conditions such as hyperglycemia and hyperandrogenism, which is essential for a better understanding of metformin's mechanisms of action on reproduction and fertility.

Allopregnanolone (ALLO) is a known neurosteroid and a progesterone metabolite synthesized in the ovary, CNS, PNS, adrenals and placenta. Its role in the neuroendocrine control of ovarian physiology has been studied, but its in situ ovarian effects are still largely unknown. The aims of this work were to characterize the effects of intrabursal ALLO administration on different ovarian parameters, and the probable mechanism of action. ALLO administration increased serum progesterone concentration and ovarian 3β-HSD2 while decreasing 20α-HSD mRNA expression. ALLO increased the number of atretic follicles and the number of positive TUNEL granulosa and theca cells, while decreasing positive PCNA immunostaining. On the other hand, there was an increase in corpora lutea diameter and PCNA immunostaining, whereas the count of TUNEL-positive luteal cells decreased. Ovarian angiogenesis and the immunohistochemical expression of GABA A receptor increased after ALLO treatment. To evaluate if the ovarian GABA A receptor was involved in these effects, we conducted a functional experiment with a specific antagonist, bicuculline. The administration of bicuculline restored the number of atretic follicles and the diameter of corpora lutea to normal values. These results show the actions of ALLO on the ovarian physiology of the female rat during the follicular phase, some of them through the GABA A receptor. Intrabursal ALLO administration alters several processes of the ovarian morpho-physiology of the female rat, related to fertility and oocyte quality., (© 2024. The Author(s).)

Female fertility is highly dependent on energy balance. High fat diet (HFD) intake entails a risk of infertility and ovulatory disorders. Considering the increase in the prevalence of overweight and obesity over the last decades, it is crucial to understand the mechanisms involved in overweight-associated infertility. In this study, we evaluated the reproductive performance of female mice fed with a HFD and the effects of metformin administration on ovarian function in these mice. We hypothesized that one of the mechanisms involved in subfertility due to a HFD intake is the alteration of ovarian blood vessel formation. We found that mice fed with HFD had altered estrous cycles and steroidogenesis, increased ovarian fibrosis, fewer pups per litter and require more time to achieve pregnancy. HFD-fed mice also presented dysregulated ovarian angiogenesis and an increase in nuclear DNA damage in ovarian cells. Ovulation rates were lower in these animals, as evidenced both in natural mating and after ovulation induction with gonadotropins. Metformin ameliorated ovarian angiogenesis, improved steroidogenesis, fibrosis, and ovulation, decreased the time to pregnancy and increased litter sizes in HFD-fed mice. We conclude that ovarian angiogenesis is one of the mechanisms detrimentally affected by HFD intake. Since metformin could improve ovarian microvasculature, it may be an interesting strategy to study in women to shed light on new targets for patients with metabolic disturbances., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Dalhia Abramovich reports financial support was provided by National Scientific and Technical Research Council., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Introduction: The mechanisms that govern fibroblast behavior during the vascular adaptations of the uterus at early pregnancy remain unknown. Anandamide, an endocannabinoid, binds to cannabinoid receptors (CBs), and regulates gestation and angiogenesis. Its tone is regulated by fatty acid amide hydrolase (FAAH) within the uterus. We investigated the role of anandamide in endometrial fibroblasts migration and whether anandamide modulates fibroblasts-endothelial crosstalk., Methods: T-hESC and EA.hy926 cell lines were used as models of endometrial stromal and endothelial cells, respectively. T-hESC were incubated with anandamide plus different agents. Migration was tested (wound healing assay and phalloidin staining). Protein expression and localization were studied by Western blot and immunofluorescence. To test fibroblast-endothelial crosstalk, EA.hy926 cells were incubated with fibroblast conditioned media obtained after T-hESC migration., Results: Anandamide 1 nM increased T-hESC migration via CB1 and CB2. Cyclooxygenase-2 participated in anandamide-stimulated fibroblast migration. Prostaglandin F2alpha, and not prostaglandin E2, increased fibroblast wound closure. CB1, CB2, cyclooxygenase-2 and FAAH were expressed in T-hESC. Anandamide did not alter cyclooxygenase-2 localization but induced its cytoplasmic and nuclear expression through CB1 and CB2. URB-597, a FAAH selective inhibitor, also increased T-hESC migration via both CBs, and augmented cyclooxygenase-2 expression. Conditioned media from anandamide-induced T-hESC wound healing closure stimulated endothelial migration and did not alter their proliferation. Soluble factors from cyclooxygenase-2 were secreted by T-hESC and participated in T-hESC-induced EA.hy926 migration. Although anandamide-conditioned media augmented in EA.hy926 the expression of γH2AX, a marker of DNA damage, cyclooxygenase-2 was not involved in this effect., Discussion: Our results provide novel evidence about an active role of anandamide on endometrial fibroblast behavior as a mechanism regulating uterine vascular adaptations in early gestation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

While oocytes and embryos cryopreservation can favor some patients with cancer-induced infertility to achieve pregnancy, the development of effective therapeutic strategies to preserve ovarian function during chemotherapy would be a significant advantage. The aim of the present study is to analyze whether Resveratrol treatment (Res) can preserve ovarian function from doxorubicin (Doxo)-induced gonadotoxicity using a mice model of premature ovarian failure. Res (7 and 15 mg/kg) increased the percentage of primary and antral follicles whilst decreasing the percentage of atretic follicles compared to Doxo alone. Res preserved the number of primordial follicles compared with those in the Doxo group but they did not change from those in the control group. Res treatment increased the number of AMH positive follicles compared to Doxo alone. Res increased proliferation index in follicular cells and reduced the DNA damage and apoptosis in preantral and early antral follicles compared to Doxo alone. Additionally, Doxo administration caused a severe endothelial damage and affected microvasculature stability in the ovary. However, Res was able to increase the recruitment of pericytes and smooth muscle cells in the Doxo-treated group. We also found that Res increased the expression of VEGF compared to Doxo alone. By H&E staining, Doxo-treated mice demonstrated endometrial alterations compared to controls, affecting both epithelial and stromal compartments. Nonetheless, Res restored the architecture of uterine tissue. Moreover, we also showed that Res administration is able to maintain antioxidant defenses through the increase of SOD expression in the Doxo-induced POF model. In conclusion, Res administration prior to and during Doxo treatment might serve as a noninvasive and low-cost protocol to preserve ovarian function in female cancer survivors., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Complex immune regulation during pregnancy is required to ensure a successful pregnancy outcome. Vasoactive intestinal peptide (VIP) has local immunoregulatory effects on the ovary, uterus and maternal-fetal interface that favor a tolerogenic maternal microenvironment. Since the VIP Knockout (KO) mice are subfertile, we investigated the mechanisms underlying the effects of VIP deficiency on ovarian physiology and immune homeostasis. Therefore, we studied VIP KO, deficient (HT) and wild type (WT) female mice in estrus at 3 or 8 months of age. Young KO mice showed abnormal cycle timing and regularity associated with dysfunctional ovaries. Ovaries presented higher number of atretic follicles and reduced number of corpora lutea leading to a lower ovulation rates. Part of the VIP KO mice (25 %) failed to ovulate or ovulated oocytes incompetent to be fertilized (50 %). In particular, ovaries of young KO mice exhibited features of premature aging accompanied by a pro-inflammatory milieu with increased levels of IL-1β. A unique macrophage subpopulation identified as "foamy macrophages" was found. On the other hand, aged VIP KO females did not gain body weight probably due to the sustained production of E2. Finally, the adoptive transfer of FOXP3+ cells to infertile VIP KO females resulted in their selective recruitment to the ovary. It increased FOXP3/RORγt and TGFβ/IL-6 ratio improving ovarian microenvironment and pregnancy rate. The present results suggest that VIP contributes to ovarian homeostatic mechanisms required for a successful pregnancy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier B.V.)

Context: The FMR1 gene consists of 17 exons and codes for the FMRP protein. FMR1 is involved in four genetic disorders depending on the CGG repeats length in its 5'UTR: the full mutation is responsible for the Fragile X syndrome while the premutation is associated with the Fragile X-associated Tremor/Ataxia Syndrome, Fragile X-associated Primary Ovarian Insufficiency (FXPOI) and Fragile X-associated neuropsychiatric disorders. FMR1 presents multiple isoforms resulting from skipping of exons 12 and 14 and the use of alternative splice sites in exons 15 and 17., Aims: To investigate the expression of Fmr1 splicing variants during folliculogenesis in the rat., Methods: We used preantral, early antral and preovulatory follicles to isolate RNA and characterise, by fluorescent PCR followed by sequencing, all the isoforms present in the different follicular stages., Key Results: We identified two isoforms resulting from splicing of exon 12, six isoforms resulting from splicing of exon 14 and 15 and one isoform for exon 17., Conclusions: The expression levels of the isoforms vary within each follicular stage but not between different stages of folliculogenesis. Importantly, we identify for the first time in rat, an isoform that contains exon 12 and two isoforms, one that includes and one that excludes exon 14 and use the third acceptor site in exon 15., Implications: Characterisation of the different FMR1 variants expressed during folliculogenesis will help to understand the potential distinct cellular roles of each of them and the possible implication in the development of FXPOI.

Several organs, such as the heart, breasts, intestine, testes, and ovaries, have been reported to be target tissues of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. To date, no studies have demonstrated SARS-CoV-2 infection in the female reproductive system. In the present study, we investigated the effects of SARS-CoV-2 infection on ovarian function by comparing follicular fluid (FF) from control and recovered coronavirus disease 2019 (COVID-19) patients and by evaluating the influence of these FF on human endothelial and non-luteinized granulosa cell cultures. Our results showed that most FFs (91.3%) from screened post COVID-19 patients were positive for IgG antibodies against SARS-CoV-2. Additionally, patients with higher levels of IgG against SARS-CoV-2 had lower numbers of retrieved oocytes. While VEGF and IL-1β were significantly lower in post COVID-19 FF, IL-10 did not differ from that in control FF. Moreover, in COV434 cells stimulated with FF from post COVID-19 patients, steroidogenic acute regulatory protein (StAR), estrogen-receptor β (Erβ), and vascular endothelial growth factor (VEGF) expression were significantly decreased, whereas estrogen-receptor α (ERα) and 3β-hydroxysteroid dehydrogenase (3β-HSD) did not change. In endothelial cells stimulated with post COVID-19 FF, we observed a decrease in cell migration without changes in protein expression of certain angiogenic factors. Both cell types showed a significantly higher γH2AX expression when exposed to post COVID-19 FF. In conclusion, our results describe for the first time that the SARS-CoV-2 infection adversely affects the follicular microenvironment, thus dysregulating ovarian function., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The aim of the present study is to assess whether low level laser therapy (LLLT) can protect ovaries from chemotherapy-induced gonadotoxicity using a mice model of premature ovarian failure induced by cyclophosphamide (CTX). LLLT (64 J/cm 2 ) increased the number of antral follicles whilst decreasing the number of atretic follicles compared to CTX alone. LLLT increased the number of primordial follicles compared with those in the CTX group but they did not differ from those in the control group. LLLT treatment increased the number of AMH-positive follicles compared to CTX alone. LLLT application increased ovarian weight, serum progesterone concentration and P450scc protein levels compared to CTX alone. LLLT reduced the apoptosis in antral follicles and the BAX/BCL-2 ratio compared to CTX alone. Vascular morphology, analysed by CD31 and α-SMA immunostaining, was restored in LLLT-treated ovaries compared to CTX alone. In conclusion, application of LLLT prior to CTX might serve as a promising and novel protocol to preserve female fertility in cancer survivors., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Although advances in the prediction and management of ovarian hyperstimulation syndrome (OHSS) have been introduced, complete prevention is not yet possible. Previously, we and other authors have shown that vascular endothelial growth factor, angiopoietins (ANGPTs) and sphingosine-1-phosphate are involved in OHSS etiology. In addition, we have demonstrated that ovarian protein levels of platelet-derived growth factor (PDGF) ligands -B and -D decrease in an OHSS rat model, whilst PDGFR-β and ANGPT2 remain unchanged. In the present work, we investigated the role of PDGF-B in OHSS by evaluating ligand protein levels in follicular fluid (FF) from women at risk of developing OHSS and by using an immature rat model of OHSS. We demonstrated that PDGF-B and PDGF-D are lower in FF from women at risk of developing OHSS compared to control patients (P < 0.05). In the OHSS rat model, PDGF-B (0.5 µg/ovary) administration decreased ovarian weight (P < 0.05), reduced serum progesterone (P < 0.05) and lowered the percentage of cysts (P < 0.05), compared to untreated OHSS rats, but had no effect on the proportion of follicles or corpora lutea (CL). PDGF-B treatment also restored the expression of steroidogenic acute regulatory protein (P < 0.05) and P450 cholesterol side-chain cleavage enzyme (P < 0.01) to control levels. In addition, PDGF-B increased the peri-endothelial cell area in CL and cystic structures, and reduced vascular permeability compared to untreated OHSS ovaries. Lastly, PDGF-B increased the levels of junction proteins claudin-5 (P < 0.05), occludin (P < 0.05) and β-catenin (P < 0.05), while boosting the extracellular deposition of collagen IV surrounding the ovarian vasculature (PP < 0.01), compared to OHSS alone. In conclusion, our findings indicate that PDGF-B could be another crucial mediator in the onset and development of OHSS, which may lead to the development of novel prediction markers and therapeutic strategies., (© The Author(s) 2020. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Normal placentation entails highly regulated interactions of maternal leukocytes with vascular and trophoblast cells to favor vascular transformation. Neutrophil activation and neutrophil extracellular trap (NET) formation associate with poor placentation and severe pregnancy complications. To deepen into the mechanisms of trophoblast-neutrophil interaction, we explored the effects of NETs on trophoblast cell function and, conversely, whether trophoblast cell-derived factors condition neutrophils to favor angiogenesis and anti-inflammatory signals required for fetal growth. NETs isolated from activated neutrophils hindered trophoblast cell migration. Trophoblast conditioned media prevented the effect as well as the vasoactive intestinal peptide (VIP) known to regulate trophoblast and neutrophil function. On the other hand, factors released by trophoblast cells and VIP shaped neutrophils to a proangiogenic profile with increased vascular endothelial growth factor synthesis and increased capacity to promote vascular transformation. Results presented here provide novel clues to reconstruct the interaction of trophoblast cells and neutrophils in vivo during placentation in humans., (© 2019 Wiley Periodicals, Inc.)

The aim of this study was to investigate whether the Notch pathway is modulated in response to the downregulation of the Wnt/Β-catenin system in corpora lutea (CLs) from superovulated rats. To this end, we analyzed the effect of in vitro CL Wnt/Β-catenin inhibition on the expression of Notch members and on luteal function. Mechanically isolated rat CLs were cultured with ICG-001, a Wnt/B-catenin inhibitor. In this system, Wnt/B-catenin inhibition reduced progesterone production and decreased StAR protein levels. Besides, Wnt/B-catenin inhibition stimulated the Notch system, evidenced by an increase in Hes1 expression, and promoted the expression of selected Notch family members. At long incubation times, StAR levels and progesterone concentration reached the control values, effects probably mediated by the Notch pathway. These results provide the first evidence of a compensatory mechanism between Wnt/B-catenin signaling and the Notch system, which contributes to the homeostasis of luteal cells.

Metformin (MET) is the most widely prescribed hypoglycemic drug in type 2 diabetes and Polycystic Ovary Syndrome. Besides its effects on glucose metabolism, MET exerts beneficial effects on these patients' fertility. However, the exact mechanisms of action of MET on female fertility are still unclear. In this work, we analyzed a possible direct effect of MET on ovarian cells. We found expression of the organic cation transporters OCT1, OCT2 and OCT3, responsible for MET uptake into the cells, in rat granulosa cells and human cumulus cells. Furthermore, MET increased pAMPK and decreased VEGF levels both in vivo and in rat granulosa cells in culture. These last effects were reversed when OCTs were inhibited. Our results suggest that MET acts directly on ovarian cells regulating cell metabolism and VEGF expression. Our findings are relevant to optimize PCOS fertility treatment and to explore ovarian MET actions in other female pathologies., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Objective: Ceramide 1-phosphate (C1P) is a bioactive sphingolipid highly augmented in damaged tissues. Because of its abilities to stimulate migration of murine bone marrow-derived progenitor cells, it has been suggested that C1P might be involved in tissue regeneration. In the present study, we aimed to investigate whether C1P regulates survival and angiogenic activity of human progenitor cells with great therapeutic potential in regenerative medicine such as endothelial colony-orming cells (ECFCs). Approach and Results: C1P protected ECFC from TNFα (tumor necrosis factor-α)-induced and monosodium urate crystal-induced death and acted as a potent chemoattractant factor through the activation of ERK1/2 (extracellular signal-regulated kinases 1 and 2) and AKT pathways. C1P treatment enhanced ECFC adhesion to collagen type I, an effect that was prevented by β1 integrin blockade, and to mature endothelial cells, which was mediated by the E-selectin/CD44 axis. ECFC proliferation and cord-like structure formation were also increased by C1P, as well as vascularization of gel plug implants loaded or not with ECFC. In a murine model of hindlimb ischemia, local administration of C1P alone promoted blood perfusion and reduced necrosis in the ischemic muscle. Additionally, the beneficial effects of ECFC infusion after ischemia were amplified by C1P pretreatment, resulting in a further and significant enhancement of leg reperfusion and muscle repair., Conclusions: Our findings suggest that C1P may have therapeutic relevance in ischemic disorders, improving tissue repair by itself, or priming ECFC angiogenic responses such as chemotaxis, adhesion, proliferation, and tubule formation, which result in a better outcome of ECFC-based therapy.

It is known that LLLT has beneficial effects on several pathological conditions including wound healing, pain and inflammation. LLLT modulates biological processes, including cell proliferation, apoptosis and angiogenesis. In the present study, we examined the effect of local application of LLLT on follicular dynamics, ovarian reserve, AMH expression, progesterone levels, apoptosis, angiogenesis, and reproductive outcome in adult mice. LLLT (200 J/cm 2 ) increased the percentage of primary and preantral follicles, whilst decreasing the percentage of corpora lutea compared to control ovaries. LLLT-treated ovaries did not exhibit any changes regarding the number of primordial follicles. We observed a higher percentage of AMH-positive follicles (in early stages of development) in LLLT-treated ovaries compared to control ovaries. LLLT reduced the P 4 concentration and the apoptosis in early antral follicles compared to control ones. LLLT caused a reduction in the endothelial cell area and an increase in the periendothelial cell area in the ovary. Additionally, LLLT was able to improve oocyte quality. Our findings suggest that local application of LLLT modulates follicular dynamics by regulating apoptosis and the vascular stability in mouse ovary. In conclusion, these data indicate that LLLT might become a novel and useful tool in the treatment of several pathologies, including female reproductive disorders., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Spiral artery remodeling at the maternal-fetal interface is crucial for successful pregnancy and requires the interaction between the first trimester trophoblast and the endothelial cells of the maternal vessels. However, the precise mechanism of this dialog has yet to be determined. The current study investigated whether lysophosphatidic acid (LPA) modulates trophoblast-endothelial crosstalk in vitro. HTR-8/SVneo trophoblast cell line (H8) was seeded on top of Geltrex, incubated with LPA or LPA + NS-398 (selective cyclooxygenase-2 inhibitor), LPA + 1400W (selective inducible nitric oxide synthase inhibitor) or LPA + IL-6 neutralizing antibody and assayed for tube formation to model the acquisition of trophoblast endovascular phenotype. The supernatants were collected and used as conditioned media (CM). To test trophoblast-endothelial crosstalk, the endothelial cell line EA.hy926 was incubated with trophoblast CM. The CM from LPA-induced tubulogenesis stimulated endothelial cells migration and did not modify the apoptosis. Soluble factors derived from cyclooxygenase-2 and IL-6 pathways were involved in H8-EA.hy926 interaction under the LPA effect. Moreover, LPA increased the levels of IL-6 mRNA by cyclooxygenase-2 pathway in H8 cells. Collectively, LPA promotes trophoblast-endothelial crosstalk in vitro and induces the release of trophoblast soluble factors that stimulate endothelial cells migration without changes in apoptosis. The evidence presented here provides new insights about an active role of LPA as a lipid mediator regulating vascular remodeling at the maternal-fetal interface., (© 2018 Wiley Periodicals, Inc.)

Ovarian cancer is the fifth leading cause of cancer-related deaths in women. In the past 20 years, the canonical types of drugs used to treat ovarian cancer have not been replaced and the survival rates have not changed. These facts show the clear need to find new therapeutic strategies for this illness. Thus, the aim of the present study was to investigate the effect of a gamma-secretase inhibitor (DAPT) in combination with the Platelet-derived growth factor B (PDGFB) on an ovarian cancer xenograft model. To achieve this goal, we analyzed the effect of the administration of DAPT alone and the co-administration of DAPT and recombinant PDGFB on parameters associated with tumor growth and angiogenesis in an orthotopic experimental model of ovarian cancer. We observed that the dose of DAPT used was ineffective to reduce ovarian tumor growth, but showed anticancer activity when co-administered with recombinant PDGFB. The administration of PDGFB alone normalized tumor vasculature by increasing periendothelial coverage and vascular functionality. Interestingly, this effect exerted by PDGFB was also observed in the presence of DAPT. Our findings suggest that PDGFB is able to improve tumor vascularity and allows the anticancer action of DAPT in the tumor. We propose that this therapeutic strategy could be a new tool for ovarian cancer treatment and deserves further studies., (© 2017 Wiley Periodicals, Inc.)

Polycystic ovary syndrome (PCOS) is the most prevalent endocrine pathology among women in reproductive age. Its main symptoms are oligo or amenorrhea, hyperandrogenism and the presence of ovarian cysts. It is also associated with infertility, obesity and insulin resistance. Mainly due to its heterogeneity, PCOS treatments are directed to manage its symptoms and to prevent associated diseases. The correct formation and regression of blood vessels during each ovarian cycle is indispensable for proper follicular development, ovulation and corpus luteum formation. The importance of these processes opened a new and promising field: ovarian angiogenesis. Vascular alterations characterize numerous pathologies, either with increased, decreased or abnormal angiogenesis. In the last years, several anomalies of ovarian angiogenesis have been described in women with PCOS. Therefore, it has been suggested that these alterations may be associated with the decreased - or lack of - ovulation rates and for the formation of cysts in the PCOS ovaries. Restoration of a proper vessel formation in the ovaries may lead to improved follicular development and ovulation in these patients. In the present review, we attempt to summarize the alterations in ovarian angiogenesis that have been described in women with PCOS. We also discuss the therapeutic approaches aimed to correct these alterations and their beneficial effects on the treatment of infertility in PCOS., (© 2018 Society for Reproduction and Fertility.)

Study Question: Is ceramide-1-phosphate (C1P) an ovarian protective agent during alkylating chemotherapy?, Summary Answer: Local administration of C1P drastically reduces ovarian damage induced by cyclophosphamide (Cy) via protection of follicular reserve, restoration of hormone levels, inhibition of apoptosis and improvement of stromal vasculature, while protecting fertility, oocyte quality and uterine morphology., What Is Known Already: Cancer-directed therapies cause accelerated loss of ovarian reserve and lead to premature ovarian failure (POF). Previous studies have demonstrated that C1P regulates different cellular processes including cell proliferation, cell migration, angiogenesis and apoptosis. This sphingolipid may be capable of modulating vascular development and apoptosis in ovaries affected by chemotherapy., Study Design, Size, Duration: The 6-8-week-old mice were weighed and administered either a single intraperitoneal injection of Cy (75 mg/kg) or an equal volume of saline solution only for control mice. Control and Cy mice underwent sham surgery and received an intrabursal injection of saline solution, while Cy + C1P animal groups received 5 μl C1P, either 0.5 or 1 mM, under the bursa of both ovaries 1 h prior to Cy administration., Participants/materials, Setting, Methods: Animals were euthanized by cervical dislocation or cardiac puncture 2 weeks after surgery for collection of blood orovary and uterus samples, which were cleaned of adhering tissue in culture medium and used for subsequent assays. Ovaries were used for Western blotting or immunohistochemical and/or histological analyses or steroid extraction, as required (n = 5-8 per group). A set of mice (n = 3/group) was destined for oocyte recovery and IVF. Finally, another set (n = 5-6/group) was separated to study fertility parameters., Main Results and the Role of Chance: The number of primordial (P < 0.01), primary (P < 0.05) and preantral follicles (P < 0.05) were decreased in Cy-treated mice compared to control animals, while atretic follicles were increased (P < 0.001). In Cy + C1P mice, the ovaries recovered control numbers of these follicular structures, in both C1P doses studied. Cy affected AMH expression, while it was at least partially recovered when C1P is administered as well. Cy caused an increase in serum FSH concentration (P < 0.01), which was prevented by C1P coadministration (P < 0.01). E2 levels in Cy-treated ovaries decreased significantly compared to control ovaries (P < 0.01), whilst C1P restored E2 levels to those of control ovaries (P < 0.01). Cy increased the expression of BAX (P < 0.01) and decreased the expression of BCLX-L compared to control ovaries (P < 0.01). The ovarian BCLX-L:BAX ratio was also lower in Cy-treated mice (P < 0.05). In the Cy + C1P group, the expression levels of BAX, BCLX-L and BCLX-L:BAX ratio were no different than those in control ovaries. In addition, acid sphingomyelinase (A-SMase) expression was higher in Cy-treated ovaries, whilst remaining similar to the control in the Cy + C1P group. Cy increased the apoptotic index (TUNEL-positive follicles/total follicles) in preantral and early antral stages, compared to control ovaries (P < 0.001 and P < 0.01, respectively). C1P protected follicles from this increase. No primordial or primary follicular cells stained for either cleaved caspase-3 or TUNEL when exposed to Cy, therefore, we have found no evidence for follicular reserve depletion in response to Cy being due to apoptosis. Cy caused evident vascular injury, especially in large cortical stromal vessels, and some neovascularization. In the Cy + C1P group, the disruptions in vascular wall continuity were less evident and the number of healthy stromal blood vessels seemed to be restored. In Cy-treated ovaries α-SMA-positive cells showed a less uniform distribution around blood vessels. C1P coadministration partially prevented this Cy-induced effect, with a higher presence of α-SMA-positive cells surrounding vessels. By H&E staining, Cy-treated mice showed endometrial alterations compared to controls, affecting both epithelial and stromal compartments. However, C1P allowed that the stromal tissue to maintain its loose quality and its glandular branches. Cy-treated animals had significantly lower pregnancy rates and smaller litter sizes compared with control mice (P = 0.013 and P < 0.05, respectively), whereas cotreatment with C1P preserved normal fertility. Furthermore, a higher (P < 0.05) proportion of abnormal oocytes was recovered from Cy-treated mice compared to the control, which was prevented by C1P administration., Large Scale Data: N/A., Limitations Reasons for Caution: The results of this study were generated from an in-vivo animal experimental model, already used by several authors. Further studies on C1P functions in female reproduction in pathological conditions such as chemotherapy-induced ovarian failure and on the safety of use of this sphingolipid are required., Wider Implications of the Findings: The present findings showed that C1P administration prior to Cy might be a promising fertility preservation strategy in female patients who undergo chemotherapy., Study Funding/competing Interest(s): This work was supported by grants from ANPCyT (PICT 2015-1117), CONICET (PIP 380), Cancer National Institute (INC) and Roemmers Foundation, Argentina. The authors declare no conflicts of interest.

Background: Allopregnanolone is a neurosteroid synthesized in the central nervous system independently of steroidogenic glands; it influences sexual behavior and anxiety. The aim of this work is to evaluate the indirect effect of a single pharmacological dose of allopregnanolone on important processes related to normal ovarian function, such as folliculogenesis, angiogenesis and luteolysis, and to study the corresponding changes in endocrine profile and enzymatic activity over 4 days of the rat estrous cycle. We test the hypothesis that allopregnanolone may trigger hypothalamus - hypophysis - ovarian axis dysregulation and cause ovarian failure which affects the next estrous cycle stages., Methods: Allopregnanolone was injected during the proestrous morning and then, the animals were sacrificed at each stage of the estrous cycle. Ovarian sections were processed to determine the number and diameter of different ovarian structures. Cleaved caspase 3, proliferating cell nuclear antigen, α-actin and Von Willebrand factor expressions were evaluated by immunohistochemistry. Luteinizing hormone, prolactin, estrogen and progesterone serum levels were measured by radioimmunoassay. The enzymatic activities of 3β-hydroxysteroid dehydrogenase, 3α-hydroxysteroid oxidoreductase and 20α-hydroxysteroid dehydrogenase were determined by spectrophotometric assays. Two-way ANOVA followed by Bonferroni was performed to determine statistical differences between control and treated groups along the four stages of the cycle., Results: The results indicate that allopregnanolone allopregnanolone decreased the number of developing follicles, while atretic follicles and cysts increased with no effects on normal cyclicity. Some cysts in treated ovaries showed morphological characteristics similar to luteinized unruptured follicles. The apoptosis/proliferation balance increased in follicles from treated rats. The endocrine profile was altered at different stages of the estrous cycle of treated rats. The angiogenic markers expression increased in treated ovaries. As regards corpora lutea, the apoptosis/proliferation balance and 20α-hydroxysteroid dehydrogenase enzymatic activity decreased significantly. Progesterone levels and 3β-hydroxysteroid dehydrogenase enzymatic activity increased in treated rats. These data suggest that allopregnanolone interferes with steroidogenesis and folliculogenesis at different stages of the cycle., Conclusion: Allopregnanolone interferes with corpora lutea regression, which might indicate that this neurosteroid exerts a protective role over the luteal cells and prevents them from luteolysis. Allopregnanolone plays an important role in the ovarian pathophysiology.

Tankyrases are physiological regulators of Axin, a protein involved in several cellular processes, including Wnt signaling. Here, we investigated the effect of a specific Tankyrase inhibitor (XAV939) in follicular-luteal dynamics, and its possible relationship with ovarian vascular development. Studies were designed to analyze the effect of intrabursa administration of XAV939 in gonadotropin-treated prepubertal rats. In particular, we examined follicle and corpus luteum development, steroidogenesis, angiogenic markers, and apoptotic parameters. We found that in vivo inhibition of Wnt signaling impaired corpus luteum development, with a decrease in the number of corpora lutea balanced by a high number of cysts; decreased circulating progesterone levels, likely due to a decrease in Steroidogenic acute regulatory protein content in the corpus luteum; and increased pro-apoptotic parameters. In addition, Extracellular signal-regulated kinase phosphorylation, Vascular endothelium growth factor 120 content, and endothelial cell area were diminished in corpora lutea of inhibitor-treated ovaries. Thus, Wnt/β-catenin signaling appears to participate in the regulation of corpus luteum development and luteal cell function., (© 2017 Wiley Periodicals, Inc.)

Study Question: Can the bioactive lipid sphingosine-1 phosphate (S1P) act as an endothelial barrier-enhancing molecule and, in turn, restore the vascular integrity and homoeostasis in a rat model of ovarian hyperstimulation syndrome (OHSS)., Study Answer: In vivo administration of S1P may prevent the early onset of OHSS and decrease its severity., What Is Known Already: Although advances in the prediction and treatment of OHSS have been made, complete prevention has not been possible yet. S1P in follicular fluid from women at risk of developing OHSS are lower in comparison from women who are not at such risk and administration of S1P in an OHSS rat model decreases ovarian capillary permeability., Study Design, Size, Duration: We used an animal model that develops OHSS in immature Sprague-Dawley rats. The rats were randomly divided into three groups: the control group, which was injected with 10 IU of pregnant mare's serum gonadotropin (PMSG), and 10 IU of hCG 48 h later; the OHSS group, which was injected with excessive doses of PMSG (50 IU/day) for four consecutive days, followed by hCG; and the OHSS + S1P group, which was injected with the same doses of PMSG and hCG as the OHSS group and then treated with 5 μl S1P (1 mM) under the bursa of both ovaries, whereas the other groups of animals received the S1P vehicle., Participants /materials, Setting, Methods: Rats were killed by decapitation 48 h after the hCG injection for ovary, endometrium and blood collection. The ovaries were weighed and then used for subsequent assays, while the serum was used for hormone assays. One of the ovaries from each rat (n = 6) was used for Western immunoblot and the other for immunohistochemical analysis. Statistical comparisons between groups were carried out., Main Results and the Role of Chance: S1P administration reduced the ovarian weight (P < 0.05), and decreased the concentration of serum progesterone in the OHSS group compared to the OHSS group without treatment (P < 0.001). The percentage of antral follicles in the OHSS group was lower than that in the control group. S1P increased the percentage of antral follicles (P < 0.05) and decreased the percentage of corpora lutea (P < 0.01) and cystic structures in the OHSS group (P < 0.05). S1P had no effect on the expression levels of the enzymes 3β-hydroxysteroid dehydrogenase (3βHSD) or cholesterol side-chain cleavage enzyme (P450scc), but reduced the levels of steroidogenic acute regulatory protein (StAR) in OHSS rat ovaries (P < 0.05). S1P decreased the endothelial (P < 0.05) and periendothelial (P < 0.01) cell area in OHSS rat ovaries. S1P restored the levels of N-cadherin and VE-cadherin proteins to control values. Furthermore, S1P enhanced the levels of claudin-5, occludin (P < 0.05) and sphingosine-1-phosphate receptor 1 (S1PR1) in OHSS (P < 0.01). In addition, no histological differences were found in endometrium between OHSS and S1P-treated OHSS animals., Limitations Reasons for Caution: The results of this study were generated from an in vivo OHSS experimental model, which has been used by several authors and our group due to the similarity between the rat and human angiogenic systems. Further studies in patients will be needed to evaluate the effects of S1P in the pathogenesis of OHSS., Wider Implications of the Findings: These findings concern the pathophysiological importance of S1P in OHSS. More studies on the regulation of endothelial cell barrier function by S1P in reproductive pathological processes and its therapeutic application are required., Large Scale Data: N/A., Study Funding and Competing Interest(s): This work was supported by grants from ANPCyT (PICT 2012-897), CONICET (PIP 5471), Roemmers and Baron Foundations, Argentina. The authors declare no conflicts of interest., (© The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Allopregnanolone, a progesterone metabolite, is one of the best characterized neurosteroids. In a dose that mimics serum levels during stress, allopregnanolone inhibits sexual receptivity and ovulation and induces a decrease in luteinizing hormone levels. The aim of this work was to examine the effect of an intracerebroventricular administration of allopregnanolone on ovarian morphophysiology, serum and tissue levels of progesterone and estrogen, and enzymatic activity of 3β-hydroxysteroid dehydrogenase, 20α-hydroxysteroid dehydrogenase and 3α-hydroxysteroid oxido-reductase in the ovary and in the medial basal hypothalamus on the morning of estrus. Ovarian morphology was analyzed under light microscopy. The hormone assays were performed by radioimmunoassay. The enzymatic activities were measured by spectrophotometric analysis. The morphometric analysis revealed that, in allopregnanolone-treated animals, the number of secondary and Graafian follicles was decreased while that of atretic follicles and cysts was significantly increased. Some cysts showed luteinized unruptured follicles. There were no differences in the number of tertiary follicles or corpora lutea in comparison with the corresponding control groups. In allopregnanolone-treated animals, progesterone serum levels were increased, while ovarian progesterone levels were decreased. Moreover, 3β-HSD and 3α-HSOR enzymatic activities were increased in the medial basal hypothalamus while ovarian levels were decreased. The enzyme 20α-hydroxysteroid dehydrogenase showed the opposite profile. The results of this study showed that allopregnanolone interferes on ovarian steroidogenesis and ovarian morphophysiology in rats, providing a clear evidence for the role of this neurosteroid in the control of reproductive function under stress situations.

Alterations in ovarian angiogenesis are common features in Polycystic Ovary Syndrome (PCOS) patients; the most studied of these alterations is the increase in vascular endothelial growth factor (VEGF) production by ovarian cells. Platelet-derived growth factor B (PDGFB) and D (PDGFD) are decreased in follicular fluid of PCOS patients and in the ovaries of a rat model of PCOS. In the present study, we aimed to analyze the effects of local administration of PDGFB on ovarian angiogenesis, follicular development and ovulation in a DHEA-induced PCOS rat model. Ovarian PDGFB administration to PCOS rats partially restored follicular development, decreased the percentage of cysts, increased the percentage of corpora lutea, and decreased the production of anti-Müllerian hormone. In addition, PDGFB administration improved ovarian angiogenesis by reversing the increase in periendothelial cell area and restoring VEGF levels. Our results shed light into the mechanisms that lead to altered ovarian function in PCOS and provide new data for potential therapeutic strategies., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Ovarian hyperstimulation syndrome (OHSS) is a complication of ovarian stimulation with gonadotrophins following human chorionic gonadotrophin (hCG) administration. The relationship between hCG and OHSS is partly mediated via the production of angiogenic factors, such as vascular endothelial growth factor A (VEGFA) and angiopoietins (ANGPTs). Here, we investigated the effect of ANGPT1 inhibition on ovarian angiogenesis in follicular fluid (FF) from women at risk of OHSS, using the chorioallantoic membrane (CAM) of quail embryos as an experimental model. We also analysed cytoskeletal changes and endothelial junction protein expression induced by this FF in the presence or absence of an ANGPT1-neutralising antibody in endothelial cell cultures. The presence of this antibody restored the number of vascular branch points and integrin αvβ3 levels in the CAMs to control values. ANGPT1 inhibition in FF from OHSS patients also restored the levels of claudin-5, vascular endothelial cadherin and phosphorylated β-catenin and partially reversed actin redistribution in endothelial cells. Our findings suggest that ANGPT1 increases pathophysiological angiogenesis in patients at risk of OHSS by acting on tight and adherens junction proteins. Elucidating the mechanisms by which ANGPT1 regulates vascular development and cell-cell junctions in OHSS will contribute to identifying new therapeutic targets for the treatment of human diseases with aberrant vascular leakage.

The platelet-derived growth factor (PDGF) system is crucial for blood vessel stability. In the present study, we evaluated whether PDGFs play a critical intraovarian survival role in gonadotropin-dependent folliculogenesis. We examined the effect of intrabursal administration of a selective platelet-derived growth factor receptor (PDGFR) inhibitor (AG1295) on follicular development, proliferation, apoptosis and blood vessel formation and stability in ovaries from rats treated with equine chorionic gonadotropin (eCG). The percentages of preantral follicles (PAFs) and early antral follicles (EAFs) were lower in AG1295-treated ovaries than in control ovaries (p < 0.01-0.05). The percentage of atretic follicles (AtrFs) increased in AG1295-treated ovaries compared to control (p < 0.05). The ovarian weight and estradiol concentrations were lower in AG1295-treated ovaries than in the control group (p < 0.01 and p < 0.05, respectively), whereas progesterone concentrations did not change. AG1295 decreased the proliferation index in EAFs (p < 0.05) and increased the percentage of nuclei positive for cleaved caspase-3 and apoptotic DNA fragmentation (p < 0.01-0.05). AG1295 increased the expression of Bax (p < 0.05) without changes in the expression of Bcl-2 protein. AG1295-treated ovaries increased the cleavage of caspase-8 (p < 0.05) and decreased AKT and BAD phosphorylation compared with control ovaries (p < 0.05). AG1295 caused a decrease not only in the endothelial cell area but also in the area of pericytes and vascular smooth muscle cells (VSMCs) in the ovary (p < 0.05). Our findings suggest that the local inhibition of PDGFs causes an increase in ovarian apoptosis through an imbalance in the ratio of antiapoptotic to proapoptotic proteins, thus leading a larger number of follicles to atresia. PDGFs could exert their mechanism of action through an autocrine/paracrine effect on granulosa and theca cells mediated by PDGFRs. In conclusion, these data clearly indicate that the PDGF system is necessary for follicular development induced by gonadotropins., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Polycystic ovary syndrome (PCOS) is a frequent pathology that affects more than 5% of women of reproductive age. Among other heterogeneous symptoms, PCOS is characterized by abnormalities in angiogenesis. Metformin has been introduced in the treatment of PCOS to manage insulin resistance and hyperglycemia. Besides its metabolic effects, metformin has been shown to improve ovulation, pregnancy and live birth rates in PCOS patients. In the present study, we used a dehydroepiandrosterone-induced PCOS rat model to analyze the effect of metformin administration on ovarian angiogenesis. We found that metformin was able to restore the increased levels of vascular endothelial growth factor, angiopoietin (ANGPT)1, and ANGPT1/ANGPT2 ratio and the decreased levels of platelet-derived growth factor B and platelet-derived growth factor D observed in the dehydroepiandrosterone-treated rats. These effects could take place, at least in part, through a decrease in the levels of serum insulin. We also found an improvement in follicular development, with a lower percentage of small follicles and cysts and a higher percentage of antral follicles and corpora lutea after metformin administration. The improvement in ovarian angiogenesis is likely to restore the accumulation of small follicles observed in PCOS rats and to reduce cyst formation, thus improving follicular development and the percentage of corpora lutea. These results open new insights into the study of metformin action not only in glucose metabolism but also in ovarian dysfunction in PCOS women.

Knockout models have shown that the coagulation system has a role in vascular development and angiogenesis. Herein, we report for the first time that zymogen FX and its active form (FXa) possess anti-angiogenic properties. Both the recombinant FX and FXa inhibit angiogenesis in vitro using endothelial EA.hy926 and human umbilical cord vascular endothelial cells (HUVEC). This effect is dependent on the Gla domain of FX. We demonstrate that FX and FXa use different mechanisms: the use of Rivaroxaban (RX) a specific inhibitor of FXa attenuated its anti-angiogenic properties but did not modify the anti-angiogenic effect of FX. Furthermore, only the anti-angiogenic activity of FXa is PAR-1dependent. Using in vivo models, we show that FX and FXa are anti-angiogenic in the zebrafish intersegmental vasculature (ISV) formation and in the chick embryo chorioallantoic membrane (CAM) assays. Our results provide further evidence for the non-hemostatic functions of FX and FXa and demonstrate for the first time a biological role for the zymogen FX., (© 2014 Wiley Periodicals, Inc.)

The relationship between human chorionic gonadotropin and ovarian hyperstimulation syndrome (OHSS) is partially mediated by vascular endothelial growth factor A (VEGF). The aim of this study was to investigate the effects of VEGF inhibition on the development of corpora lutea (CL) and cystic structures, steroidogenesis, apoptosis, cell proliferation, endothelial cell area, VEGF receptors (KDR and Flt-1), claudin-5 and occludin levels in ovaries from an OHSS rat model. The VEGF inhibitor used (VEGF receptor-1 (FLT-1)/Fc chimera, TRAP) decreased the concentrations of progesterone and estradiol as well as the percentage of CL and cystic structures in OHSS rats, and increased apoptosis in CL. Endothelial cell area in CL and KDR expression and its phosphorylation were increased, whereas claudin-5 and occludin levels were decreased in the OHSS compared to the control TRAP reversed these parameters. Our findings indicate that VEGF inhibition prevents the early onset of OHSS and decreases its severity in rats., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Polycystic ovary syndrome (PCOS) is the most common endocrinological pathology among women of reproductive age, and is characterized by abnormalities in ovarian angiogenesis, among other features. Consistent with this association, follicular fluid (FF) concentration and ovarian expression of vascular endothelial growth factor (VEGF) are increased in PCOS patients. In this study, we examined the protein levels of platelet-derived growth factor (PDGF) BB and DD (PDGFBB and PDGFDD), angiopoietin 1 and 2 (ANGPT1 and ANGPT2), and their soluble receptor sTIE2 in FF from PCOS and control patients undergoing assisted reproductive techniques. We also analyzed the effect of FF from PCOS and control patients on tight and adherens junction protein expression in an endothelial cell line. PDGFBB and PDGFDD were significantly lower whereas ANGPT1 concentration was significantly higher in FF from PCOS patients than from control patients. No changes were found in the concentration of ANGPT2 or sTIE2. Expression of claudin-5 was significantly increased in endothelial cells incubated for 24 hr in the presence of FF from PCOS versus from control patients, while vascular-endothelial cadherin, β-catenin, and zonula occludens 1 expression were unchanged. The changes observed in the levels of PDGF isoforms and ANGPT1 may prevent VEGF-induced vascular permeability in the PCOS ovary by regulating endothelial-cell-junction protein levels. Restoring the levels of angiogenic factors may provide new insights into PCOS treatment and the prevention of ovarian hyperstimulation syndrome in affected women., (© 2014 Wiley Periodicals, Inc.)

Ovarian granulosa cell tumors (GCTs) represent 3%-5% of all ovarian malignancies. Treatments have limited proven efficacy and biologically targeted treatment is lacking. The aim of this study was to investigate the role of Notch signaling in the proliferation, steroidogenesis, apoptosis, and phosphatidylinositol 3-kinase (PI3K)/AKT pathway in a FOXL2-mutated granulosa tumor cell line (KGN) representative of the adult form of GCTs. When Notch signaling is initiated, the receptors expose a cleavage site in the extracellular domain to the metalloproteinase TACE and, following this cleavage, Notch undergoes another cleavage mediated by the presenilin-gamma-secretase complex. To achieve our goal, DAPT, an inhibitor of the gamma-secretase complex, was used to investigate the role of the Notch system in parameters associated with cell growth and death, using a human granulosa cell tumor line (KGN) as an experimental model. We observed that JAGGED1, DLL4, NOTCH1, and NOTCH4 were highly expressed in KGN cells as compared to granulosa-lutein cells obtained from assisted reproductive techniques patients. The proliferation and viability of KGN cells, as well as progesterone and estradiol production, decreased in the presence of 20 μM DAPT. Apoptotic parameters like PARP and caspase 8 cleavages, BAX, and BCLXs increased in KGN cells cultured with DAPT, whereas others such as BCL2, BCLXl, FAS, and FAS ligand did not change. AKT phosphorylation decreased and PTEN protein increased when Notch signaling was inhibited in KGN cells. We conclude that the Notch system acts as a survival pathway in KGN cells, and might be interacting with the PI3K/AKT pathway.

Reproductive success stems from a finely regulated balance between follicular maturation and atresia, in which the role of carbohydrate structure is poorly understood. Here, we describe for the first time a fraction of purified recombinant human FSH that is capable of bringing about the cell death of granulosa cells and preventing follicular maturation in a rat model. Further analysis by mass spectrometry revealed the presence of the lectin Concanavalin-A (Con-A) within this fraction of recombinant FSH. Using both the fractionated FSH and Con-A, the observed cell death was predominantly located to the granulosa cells. Ex vivo culture of rat follicles demonstrated that follicle degeneration occurred and resulted in the release of a denuded and deteriorated oocyte. Moreover, in vivo experiments confirmed an increase in atresia and a corresponding reduction confined to follicle in early antral stage. As a mechanism of action, Con-A reduces ovarian proliferation, Von Willebrand staining, and angiogenesis. Based on the observation that Con-A may induce granulosa cell death followed by follicle death, our results further demonstrate that follicular carbohydrate moiety is changing under the influence of FSH, which may allow a carbohydrate-binding lectin to increase granulosa cell death. The physiological consequences of circulating lectin-like molecules remain to be determined. However, our results suggest a potential exploitation of carbohydrate binding in fertility and ovarian cancer treatment. This work may shed light on a key role of carbohydrates in the still obscure physiological process of follicular selection and atresia.

Fragile X mental retardation protein (FMRP) belongs to a small family of RNA-binding proteins. Its absence or inactivity is responsible for fragile X syndrome, the most common cause of inherited mental retardation. Despite its ubiquitous expression, FMRP function and expression remain almost understudied in non-neuronal tissues, though previous studies on germline development during oogenesis may suggest a special function of this protein also in ovarian tissue. In addition, the well-documented association of FMR1 premutation state with fragile X-related premature ovarian insufficiency adds interest to the role of FMRP in ovarian physiology. The aim of the present work was to investigate the expression of Fmr1 mRNA and its protein, FMRP, at different stages of rat follicular development. By immunohistochemical studies we demonstrated FMRP expression in granulosa, theca and germ cells in all stages of follicular development. In addition, changes in Fmr1 expression, both at the protein and mRNA levels, were observed. FMRP levels increased upon follicular development while preantral and early antral follicles presented similar levels of Fmr1 transcripts with decreased expression in preovulatory follicles. These observations suggest that Fmr1 expression in the ovary is regulated at different and perhaps independent levels. In addition, our results show expression of at least four different isoforms of FMRP during all stages of follicular growth with expression patterns that differ from those observed in brain and testis. Our study shows a regulated expression of Fmr1, both at mRNA and protein levels, during rat follicular development.

Ovarian hyperstimulation syndrome (OHSS) is a disorder associated with ovarian stimulation. OHSS features are ovarian enlargement with fluid shifting to the third space. Disturbances in the vasculature are considered the main changes that lead to OHSS. Our aim was to analyze the levels of angiopoietins 1 and 2 (ANGPT1 and 2) and their soluble and membrane receptors (s/mTie-2) in follicular fluid (FF) and in granulosa-lutein cells culture (GLCs) from women at risk of developing OHSS. We also evaluated the effect of ANGPT1 on endothelial cell migration. In ovaries from an OHSS rat model, we analyzed the protein concentration of ANGPTs, their mTie-2 receptor, and platelet-derived growth factor PDGF-B, -D and PDGFR-β. ANGPT1 levels were increased in both FF and GLCs from women at risk of OHSS. Incubation of these FF with an ANGPT1 neutralizing antibody decreased endothelial cell migration. In the ovaries of OHSS rat model, mTie-2 protein levels increased and PDGF-B and -D decreased. In summary, these results suggest that ANGPT1 could be another mediator in the development of OHSS., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Polycystic ovary syndrome (PCOS) is the most common endocrinological pathology among women of reproductive age. It is characterized by anovulation, oligo- or amenorrhea, hyperandrogenism, obesity, and insulin resistance. PCOS patients present with elevated levels of vascular endothelial growth factor (VEGF) in serum and follicular fluid. In this study, we examined the ovarian expression of angiopoietins (ANGPT) and their receptor tyrosine kinase receptor (TIE2), involved in the stabilization of blood vessels, in a rat model of dehydroepiandrosterone-induced PCOS. We also analyzed the effect of ovarian VEGF inhibition on ANGPT/TIE2, follicular development, and vascular stability. VEGF levels were increased in the PCOS ovaries, whereas the levels of its receptor fetal liver kinase-1 were decreased. In addition, the periendothelial cell area and the ANGPT1 to ANGPT2 ratio in the ovary were increased in the PCOS group. Percentage of primary follicles was increased and the percentage of preantral follicles and corpora lutea was decreased in the PCOS group. VEGF inhibition decreased the percentage of primary follicles close to control values. Interestingly, despite the presence of cysts in the ovaries from VEGF inhibitor-treated PCOS rats, its percentage was lower than the PCOS group without treatment. In summary, this study describes an alteration not only in the VEGF/fetal liver kinase-1 system but also in the ANGPT/TIE2 system in a dehydroepiandrosterone-induced PCOS rat model. This leads to an increase in periendothelial cell recruitment. We also demonstrated that ovarian VEGF inhibition can partially restore the accumulation of small follicles in PCOS rats and reduces cyst formation, improving ovulation and follicular development. Therefore, the inhibition of VEGF could be considered, in addition to other currently applied treatments, as a new strategy to be studied in PCOS patients to restore ovarian function.

The aim of this study was to determine the effect of the local inhibition of ANGPT1 on steroid production, proliferation and apoptosis of ovarian follicular cells and on the PI3K/AKT pathway. We also examined the effect of ANGPTs on follicular cell apoptosis and proliferation in early antral follicles (EAFs) in culture. Follicular cells expressing PCNA decreased after ANGPT1 Ab treatment. Moreover, ANGPT1 inhibition increased the levels of active caspase 3 and androsterone, but decreased estradiol, AKT phosphorylation and the area of smooth muscle cell actin. In cultured EAFs from prepubertal rats treated with diethylstilbestrol (DES), ANGPT1 increased PCNA and decreased apoptosis while ANGPT2 reversed these effects. These results show that ANGPT1 alters steroidogenesis, reduces ovarian apoptosis, and stimulates cell proliferation in antral follicles. ANGPT1 may exert these roles by regulating ovarian vascular stability and/or by a direct effect on follicular cells, possibly involving the PI3K/AKT pathway., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Ovarian hyperstimulation syndrome (OHSS) is a complication of ovarian stimulation with gonadotropins followed by the administration of human chorionic gonadotropin (hCG) to trigger the final steps of oocyte maturation. Gonadotropin-releasing hormone (GnRH) analogs are thought to be effective in preventing this complication and a clinical trial has found a lower incidence of OHSS in patients treated with these molecules. Our aim was to analyze the in vivo effect of a GnRH-I agonist on corpus luteum development and regression, ANGPT-1, ANGPT-2 and Tie-2 protein expression and luteal blood vessel stabilization, the expression of the steroidogenic acute regulatory protein (StAR) and the cytochrome P450 side-chain cleavage enzyme (P450scc) and cell proliferation, in ovaries from an OHSS rat model. To this end immature female Sprague-Dawley rats were hyperstimulated and treated with a GnRH-I agonist from the start of pregnant mare serum gonadotropin (PMSG) administration until the day of hCG injection for 5 consecutive days. Blood and tissue samples were collected 48h after hCG injection. Vascular endothelial growth factor VEGF levels were evaluated in the peritoneal fluid by ELISA. Serum progesterone and estradiol were measured by RIA. Histological features of sectioned ovaries were assessed in hematoxylin and eosin (H&E) stained slides. Luteal blood vessel stability, cell proliferation and apoptosis were assessed by immunohistochemistry for SMCA, PCNA, and TUNEL, respectively. P450scc, StAR, FLK-1, ANGPT-1, ANGPT-2, Tie-2 and PCNA protein levels were evaluated by Western blot from dissected corpora lutea (CL). The treatment with the GnRH-I agonist significantly decreased serum progesterone and estradiol levels as well as P450scc and StAR protein expression in the untreated OHSS group. In addition, the agonist significantly decreased the number of CL in the OHSS group, as compared with the untreated OHSS group. In the OHSS group, the area of periendothelial cells in the CL was larger than that of the control group. However, the treatment with the GnRH-I agonist significantly reduced the area of periendothelial cells in the CL in the OHSS group. The luteal levels of ANGPT-1 and its receptor Tie-2 significantly increased in the OHSS group when compared with the control group. Conversely, the administration of the GnRH-I agonist significantly decreased the levels of these factors in the CL from the OHSS group, as compared with the untreated OHSS group. In addition, the treatment with the GnRH-I agonist reduced the diameter of CL and decreased CL cell proliferation as compared with that observed in the untreated OHSS group. Finally, the GnRH-I agonist increased apoptosis in the CL from the OHSS group. In conclusion, these results show that GnRH-I agonist exerts diverse actions on the CL from a rat OHSS model. The decrease in P450scc, StAR, ANGPT-1 and Tie-2 expression, blood vessel stability and luteal proliferation leads to CL regression in the ovaries from OHSS rats. Moreover, our results suggest that the downregulation of ANGPT-1 and its receptor is a possible mechanism whereby GnRH-I agonists could prevent early OHSS., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Acute hyperandrogenism decreases serum P levels and induces early apoptosis of antral follicles by a mechanism mediated by the peroxisome proliferator-activated receptor gamma system and independent of the steroidogenic acute regulator protein., (Copyright © 2011 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

The aim of the present work was to analyze the direct effect of VEGF in follicular cell proliferation, apoptosis and activation of the PI3K/AKT and ERK/MEK signaling pathways in early antral follicles or granulosa cells. Antral follicles or granulosa cells were isolated from prepubertal female Sprague Dawley rats treated with DES.VEGF directly stimulates follicular cell proliferation and it also decreases apoptosis by inhibiting caspase 3 activation. In addition, VEGF increases the proliferation and inhibits the apoptosis of isolated granulosa cells in culture. VEGF activates the PI3K/AKT pathway evidenced by an increase in AKT phosphorylation levels and induces the phosphorylation of ERK1/2 in cultured antral follicles. These results demonstrate for the first time that VEGF has a proliferative and cytoprotective role in early antral follicles and in granulosa cells isolated from DES treated prepubertal rats and suggest that PI3K/AKT and ERK/MEK signaling pathways are involved in these processes., (Copyright (c) 2010 Elsevier Ireland Ltd. All rights reserved.)

Objective: To investigate the effects of local inhibition of vascular endothelial growth factor A (VEGFA) on proliferation and apoptosis of follicular cells in rat ovaries. To analyze the role of the PI3K/AKT signaling pathway on VEGFA effects., Design: Experimental study., Setting: Research laboratory., Animal(s): Female Sprague Dawley rats, 21 days old, treated with equine chorionic gonadotropin (eCG)., Main Outcome Measure(s): Follicular cell proliferation, apoptosis, and activation of the PI3K/AKT signaling pathway after intrabursal injection of a VEGFA inhibitor., Result(s): Inhibition of VEGFA leads to a decrease in the expression of the proliferation marker proliferating cell nuclear antigen (PCNA) in theca and granulosa cells (GC) and an increase in the activation of caspase 3 in antral follicles. Furthermore, we observed a decrease in the phosphorylation of RAC-alpha serine/threonine-protein kinase (AKT) and its target Bcl2 antagonist of cell death (BAD). No differences were found in the levels of kinase insert domain receptor (KDR) protein or in endothelial cell density., Conclusion(s): The VEGFA prevents apoptosis and stimulates proliferation of follicular cells, regulating follicular growth and development. The PI3K/AKT signaling pathway is one of the pathways involved in this mechanism. Therefore, VEGFA has a role as an antiapoptotic and proliferative factor in follicular cells from the rat ovary., (Copyright 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

The aim of the present study was to determine the endocrine activity of cultured early antral follicles (EAF) isolated from prepubertal diethylstilbestrol-treated rats. The effect of steroidogenic substrates and FSH on steroid, inhibin A and B, Pro-alphaC and activin A production was evaluated. Androsterone was the predominant steroid produced by EAF. The addition of androstenedione, androstenedione+FSH and progesterone stimulated oestradiol production, whereas 25-hydroxycholesterol (25-OH-Chol) increased progesterone production. Inhibin A, B, Pro-alphaC, and activin A were produced under basal conditions. The predominance of inhibin B over inhibin A was not affected by the addition of androstenedione or progesterone. Inhibin A and activin A production was stimulated by FSH. 25-OH-Chol increased Inha, Inhba and Inhbb mRNA expression and the production of the three molecular forms of inhibins but decreased activin A production. These results show that FSH and the steroid follicular microenvironment differentially modulate the gene expression of inhibin/activin subunits, their assembly and secretion.