Elucidating metformin action on the ovary and fertility in healthy mice.

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.

Platelet-derived growth factor B restores vascular barrier integrity and diminishes permeability in ovarian hyperstimulation syndrome.

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-ß 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 ß-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.

PDGFB as a vascular normalization agent in an ovarian cancer model treated with a gamma-secretase inhibitor.

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.

Ceramide-1-phosphate has protective properties against cyclophosphamide-induced ovarian damage in a mice model of premature ovarian failure.

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.

Ovarian angiogenesis in polycystic ovary syndrome.

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.

In vivo intrabursal administration of bioactive lipid sphingosine-1-phosphate enhances vascular integrity in a rat model of ovarian hyperstimulation syndrome.

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 3ß-hydroxysteroid dehydrogenase (3ßHSD) 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.

Tankyrase inhibition regulates corpus luteum development and luteal function in gonadotropin-treated rats.

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/ß-catenin signaling appears to participate in the regulation of corpus luteum development and luteal cell function.

Inhibition of angiopoietin-1 (ANGPT1) affects vascular integrity in ovarian hyperstimulation syndrome (OHSS).

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 αvß3 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 ß-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.

Platelet-derived growth factor BB and DD and angiopoietin1 are altered in follicular fluid from polycystic ovary syndrome patients.

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, ß-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.

Effects of an inhibitor of the γ-secretase complex on proliferation and apoptotic parameters in a FOXL2-mutated granulosa tumor cell line (KGN).

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.

Resveratrol alleviates doxorubicin-induced damage in mice ovary.

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.

Beneficial effects of metformin on mice female fertility after a high-fat diet intake.

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.

SARS-CoV-2 infection negatively affects ovarian function in ART patients.

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-1ß 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 ß (Erß), and vascular endothelial growth factor (VEGF) expression were significantly decreased, whereas estrogen-receptor α (ERα) and 3ß-hydroxysteroid dehydrogenase (3ß-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.

Altered expression of Bcl-2 and Bax in follicles within dehydroepiandrosterone-induced polycystic ovaries in rats.

PCOS (polycystic ovary syndrome) is a heterogeneous disease characterized by hyperandrogenaemia, hirsutism, oligo- or amenorrhea, insulin resistance and anovulation. The aim of the present study was to evaluate if the balance between the ovarian expression of Bax (proapoptotic protein) and Bcl-2 (antiapoptotic protein) is altered in a PCOS model developed in rats by DHEA (dehydroepiandrosterone) administration. In addition, the ovarian morphology and the circulating progesterone levels were evaluated. Histological studies confirmed the presence of follicular cysts, atretic follicles and the absence of corpora lutea in the ovaries from the PCOS group and a significant decrease in circulating progesterone levels. Immunohistochemical studies showed that the expression of Bcl-2 and Bax were mainly localized in granulosa cells of AFs (antral follicles) in both groups. Bax expression was greater in preantral and AFs from PCOS ovarian sections than in the controls. In contrast, intense Bcl-2 immunostaining was observed in the control AFs, while Bcl-2 protein was either absent in PFs (preantral follicles) or weakly expressed in AFs from PCOS rats. These results were partially confirmed by Western studies. Data revealed that the ovarian level of Bcl-2 protein was lower in PCOS than in the control and that there were no differences in Bax ovarian levels between groups. However, Bax/Bcl-2 ratio was significantly higher in PCOS group than in the control group. In conclusion, an increase in ovarian apoptosis through an imbalance among the Bcl-2 family members may be involved in the transformation of growing follicles in cystic follicles in the ovaries from DHEA-induced PCOS rats.

Local application of low level laser therapy in mice ameliorates ovarian damage induced by cyclophosphamide.

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/cm2) 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.

Modulation of the Notch System in Response to Wnt Inhibition Induces Restoration of the Rat Luteal Function.

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 has a direct effect on ovarian cells that is dependent on organic cation transporters.

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.

Spatiotemporal analysis of the protein expression of angiogenic factors and their related receptors during folliculogenesis in rats with and without hormonal treatment.

This study investigated the protein expression and cellular localization of ANGPT1, ANGPT2, and their receptor TEK, as well as vascular endothelial growth factor A (VEGFA) and its receptor KDR (VEGFR2) during folliculogenesis. To obtain follicles at different stages for immunochemistry and western analyses, we used prepubertal untreated, diethylstilbestrol- and equine chorionic gonadotropin-treated rats. To confirm that these hormonal treatments reflect physiological change, we used non-treated adult rats. No expression of ANGPT1 was observed in granulosa cells (Gc) from immature hormone-treated and non-treated rats at any follicular stage. By contrast, ANGPT1 expression in theca cells (Tc) increased with follicular maturation. ANGPT2 protein was either absent or weakly expressed in Gc at all follicular stages. In Tc, minimal expression of ANGPT2 protein was detected in the preantral follicle (PF), whereas it was stronger in the early antral follicle (EAF) and preovulatory follicle (POF). TEK staining was absent in Gc but was intense in Tc at every follicular stage. Staining for VEGFA was either absent or weakly present in Gc and Tc in PF and EAF, although in POF it was stronger in Gc and Tc. Staining for KDR was absent in Gc and very low in Tc from PF. Gc and Tc of EAF showed positive staining for KDR and in POF the staining was stronger. These results were confirmed by western immunoblot. A similar pattern of expression of these proteins was observed in cycling rats. In conclusion, we observed that the protein expression of ANGPT1, ANGPT2, VEGFA and their receptors increased during follicular development in rats.

Low level laser therapy (LLLT) modulates ovarian function in mature female mice.

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/cm2) 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 P4 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.

Local administration of platelet-derived growth factor B (PDGFB) improves follicular development and ovarian angiogenesis in a rat model of Polycystic Ovary Syndrome.

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.