A novel regulatory pathway in granulosa cells, the LH/human chorionic gonadotropin-microRNA-125a-3p-Fyn pathway, is required for ovulation.

Granulosa cells support the developing oocytes and serve as transducers of the ovulatory stimulus induced by LH surge. Fyn kinase is expressed in granulosa cells, though its role in these cells has not been studied. In human embryonic kidney 293T cells, microRNA (miR)-125a-3p down-regulates Fyn expression, causing a decrease in cells' migratory ability. Our aim was to explore the role of miR-125a-3p and Fyn in granulosa cells toward ovulation, focusing on migration as a possible mechanism. We demonstrate expression of miR-125a-3p and Fyn in mouse mural granulosa cells of preovulatory follicles and miR-125a-3p-induced down-regulation of Fyn expression in a granulosa cell line (rat). Administration of human chorionic gonadotropin (hCG; LH analog) caused a 75% decrease in the in vivo miR-125a-3p:Fyn mRNA ratio, followed by a 2-fold increased migratory ability of mural granulosa cells. In the hCG-treated granulosa cell line, miR-125a-3p expression was decreased, followed by Fyn up-regulation and phosphorylation of focal adhesion kinase and paxillin, enabling cell migration. An in vivo interference with miR-125a-3p:Fyn mRNA ratio in granulosa cells by intrabursal injections of Fyn small interfering RNA or miR-125a-3p mimic caused a 33 or 55% decrease in the number of ovulated oocytes, respectively. These observations reveal a new regulatory pathway in mural granulosa cells under the regulation of LH/hCG. Modulation of cell migration may account for the significance of the LH/hCG-miR-125a-3p-Fyn pathway to ovulation.

Hormonal regulation of pigment epithelium-derived factor (PEDF) in granulosa cells.

Angiogenesis is critical for the development of ovarian follicles. Blood vessels are abrogated from the follicle until ovulation, when they invade it to support the developing corpus luteum. Granulosa cells are known to secrete anti-angiogenic factors that shield against premature vascularization; however, their molecular identity is yet to be defined. In this study we address the physiological role of pigment epithelium-derived factor (PEDF), a well-known angiogenic inhibitor, in granulosa cells. We have shown that human and mouse primary granulosa cells express and secrete PEDF, and characterized its hormonal regulation. Stimulation of granulosa cells with increasing doses of estrogen caused a gradual decrease in the PEDF secretion, while stimulation with progesterone caused an abrupt decrease in its secretion. Moreover, We have shown, by time- and dose-response experiments, that the secreted PEDF and vascular endothelial growth factor (VEGF) were inversely regulated by hCG; namely, PEDF level was nearly undetectable under high doses of hCG, while VEGF level was significantly elevated. The anti-angiogenic nature of the PEDF secreted from granulosa cells was examined by migration, proliferation and tube formation assays in cultures of human umbilical vein endothelial cells. Depleting PEDF from primary granulosa cells conditioned media accelerated endothelial cells proliferation, migration and tube formation. Collectively, the dynamic expression of PEDF that inversely portrays VEGF expression may imply its putative role as a physiological negative regulator of follicular angiogenesis.

A physiological approach for treating endometriosis by recombinant pigment epithelium-derived factor (PEDF).

STUDY QUESTION: Is pigment epithelium-derived factor (PEDF) expressed in the rodent endometrium and can it be utilized to treat endometriosis without negatively affecting reproductive parameters? SUMMARY ANSWER: PEDF is dynamically expressed in rat endometrium throughout the estrous cycle in a reciprocal manner to vascular endothelial growth factor (VEGF); it possesses potent therapeutic properties for endometriosis that do not compromise the reproductive parameters. WHAT IS KNOWN ALREADY: Endometriosis pathogenesis depends mainly on neovascularization, with a high local level of VEGF. PEDF, a 50 kDa secreted glycoprotein with a potent anti-angiogenic activity, negates several strong pro-angiogenic factors, such as VEGF. STUDY DESIGN, SIZE, DURATION: Rat endometrial samples were collected at various days of the estrous cycle (n = 5 rats/day) and mRNA of VEGF and PEDF was determined. Endometriosis was induced by transplanting uterine pieces onto the inner surface of the abdominal wall of recipient rats, resulting in proliferation of the endometrial transplants. Recipient rats were randomly injected intravenously (IV), every third day for the next 3 weeks, with either Tris ('control'; n = 7) or recombinant PEDF (rPEDF; 2 mg/kg/day; 'PEDF prevention'; n = 7), while others were IV injected every third day starting from Day 9 after grafting until the end of 3 weeks, with rPEDF (2 mg/kg/day; 'PEDF treatment'; n = 6). The effect of rPEDF on the duration of the estrous cycle and on the number of ovulated oocytes was evaluated in rats that were randomly divided into four groups and were injected with either Tris or rPEDF every third day for 3 weeks: naive rats (n = 6); rPEDF-treated rats (n = 5); endometriosis-induced rats (n = 5); or endometriosis + rPEDF rats (n = 6). MATERIALS, SETTING, METHODS: Reproductive parameters: the estrous cycle was evaluated by daily vaginal smears, and the number of ovulated oocytes in the oviductal ampullae of estrus rats was counted. The efficiency of endometriosis induction and treatment was evaluated on the third week after endometrial transplantation, on the day of pro-estrus. Endometrial transplants were isolated and weighted. PEDF and VEGF were monitored by quantitative PCR and immunohistochemistry using confocal microscopy. MAIN RESULTS AND THE ROLE OF CHANCE: PEDF mRNA and protein were dynamically expressed in the endometrium all throughout the estrous cycle, reciprocally to VEGF; VEGF was highly expressed during estrus while PEDF expression was low, and vice versa at metestrus II. The weight of the endometrial transplants was significantly reduced after PEDF administration (13% of control for 'PEDF treatment' rats; 7% of control for 'PEDF prevention' rats; P < 0.001). Histology of the transplants' remnants showed a complete loss of their endometrial characteristics. Furthermore, the level of VEGF mRNA in the transplants of PEDF-administered rats was significantly lower (P < 0.05) than in transplants of control rats. Administration of rPEDF had no effect on the estrous cycle or ovulation rate of naive rats, while it had a significantly beneficial effect on the low ovulation rate of endometriosis-induced rats (P < 0.05). LIMITATIONS, REASONS FOR CAUTION: The experiments were performed in a rat model. WIDER IMPLICATIONS OF THE FINDINGS: The endometriosis therapeutic potency of PEDF that is exerted reciprocally to VEGF and does not compromise reproductive parameters offers a rational for using PEDF as a treatment for endometriosis with a potential of treating other reproductive angiogenic-related pathologies.

De novo synthesis of protein phosphatase 1A, magnesium dependent, alpha isoform (PPM1A) during oocyte maturation.

Oocyte maturation in mammals is a multiple-stage process that generates fertilizable oocytes. Ovarian oocytes are arrested at prophase of the first meiotic division characterized by the presence of a germinal vesicle. Towards ovulation, the oocytes resume meiosis and proceed to the second metaphase in a process known as maturation; they undergo nuclear and cytoplasmic changes that are accompanied by translation and degradation of mRNA. Protein phosphatase 1A, magnesium dependent, alpha isoform (PPM1A), which belongs to the metal-dependent serine/threonine protein phosphatase family, is highly conserved during evolution. PPM1A plays a significant role in many cellular functions such as cell cycle progression, apoptosis and cellular differentiation. It works through diverse signaling pathways, including p38 MAP kinase JNK and transforming growth factor beta (TGF-ß). Herein we report that PPM1A is expressed in mouse oocytes and that its mRNA level rises during oocyte maturation. Using quantitative real-time polymerase chain reaction (qPCR) and western blot analysis, we found that PPM1A mRNA is synthesized at the beginning of the maturation process and remains elevated in the mature oocytes, promoting the accumulation of PPM1A protein. Since PPM1A function is mainly affected by its level, we propose that it might have an important role in oocyte maturation.

Regulation of division in mammalian oocytes: implications for polar body formation.

Meiosis in mammalian oocytes includes two asymmetric meiotic divisions that result in extrusion of the first and second polar bodies (PBI and PBII, respectively). Fyn, an Src family kinase (SFK), colocalizes with filamentous actin (F-actin) at the meiotic cleavage furrow area of mouse oocytes. In this paper, these studies are extended to rat oocytes. Furthermore, inhibition of Fyn decreased the rate of PBs extrusion and led to formation of larger PBs (PBI and PBII). This effect differs from the effect of Fyn inhibition on the first mitotic symmetric cell division where only the rate of cleavage was affected but the two daughter cells were of regular size. Inhibition of Fyn resulted in a significant decrease in cortical F-actin in the oocytes. We suggest a meiotic model for mammalian oocytes in which Fyn is recruited to the meiotic area of cleavage furrow formation and induces polymerization and stabilization of F-actin, possibly by regulating F-actin effectors, such as RhoA, Arp2/3 and formins, thus allowing ingression of the cleavage furrow. In the context of PB formation, we suggest that SFKs are involved in maintaining the precise temporal restrains of the asymmetric divisions and in regulation of PBs size by inducing polymerization and stabilization of F-actin during the formation and ingression of the cleavage furrow.

Myristoylated alanine-rich C kinase substrate, but not Ca2+/calmodulin-dependent protein kinase II, is the mediator in cortical granules exocytosis.

Sperm-egg fusion induces cortical granules exocytosis (CGE), a process that ensures the block to polyspermy. CGE can be induced independently by either a rise in intracellular calcium concentration or protein kinase C (PKC) activation. We have previously shown that myristoylated alanine-rich C kinase substrate (MARCKS) cross-links filamentous actin (F-actin) and regulates its reorganization. This activity is reduced either by PKC-induced MARCKS phosphorylation (PKC pathway) or by its direct binding to calmodulin (CaM; CaM pathway), both inducing MARCKS translocation, F-actin reorganization, and CGE. Currently, we examine the involvement of Ca(2)(+)/CaM-dependent protein kinase II (CaMKII) and MARCKS in promoting CGE and show that PKC pathway can compensate for lack of Ca(2)(+)/CaM pathway. Microinjecting eggs with either overexpressed protein or complementary RNA of constitutively active alphaCaMKII triggered resumption of second meiotic division, but induced CGE of an insignificant magnitude compared with CGE induced by wt alphaCaMKII. Microinjecting eggs with mutant-unphosphorylatable MARCKS reduced the intensity of 12-O-tetradecanoylphorbol 13-acetate or ionomycin-induced CGE by 50%, indicating that phosphorylation of MARCKS by novel and/or conventional PKCs (n/cPKCs) is a pivotal event associated with CGE. Moreover, we were able to demonstrate cPKCs involvement in ionomycin-induced MARCKS translocation and CGE. These results led us to propose that MARCKS, rather than CaMKII, as a key mediator of CGE.

The role of pigment epithelium-derived factor in the pathophysiology and treatment of ovarian hyperstimulation syndrome in mice.

CONTEXT: Ovarian hyperstimulation syndrome (OHSS) is a potentially life-threatening complication of assisted reproduction. OHSS is induced by an ovarian release of vasoactive, angiogenic substances that results in vascular hyperpermeability, leakage, and shift of fluids from blood vessels into the extravascular space with consequent ascites and edema that are attributed to vascular endothelial growth factor (VEGF). OBJECTIVE: Our objective was to examine a physiological approach for preventing and treating OHSS, based on negating the VEGF network. DESIGN: We used a mouse OHSS model and cultured granulosa cells. MAIN OUTCOME: Changes in pigment epithelium-derived factor (PEDF) and VEGF were measured by quantitative PCR and Western blot analysis. OHSS was recorded by changes in body weight and in peritoneal vascular leakage, quantified by the modified Miles vascular permeability assay. RESULTS: Granulosa cells produced and secreted the anti-angiogenic factor, PEDF, in an inverse fashion to VEGF. The physiological PEDF-VEGF counterbalance was found to be impaired in the mouse OHSS model. Treatment of OHSS-induced mice with low doses of recombinant PEDF (rPEDF) alleviated OHSS signs including edema (P < .001) and vascular leakage (P < .001) and reduced the level of ovarian VEGF mRNA. Low doses of rPEDF also reduced VEGF mRNA levels in granulosa cells in vitro. However, these effects were not seen at higher doses of rPEDF, suggesting a hormetic mechanism of rPEDF action. CONCLUSION: These observations provide a new perspective into the pathophysiology of OHSS, namely, high expression level of VEGF together with a nearly undetectable level of PEDF. A replacement therapy with rPEDF is suggested as an innovative physiological treatment for OHSS. Finally, control of the PEDF-VEGF reciprocal relationship could open new therapeutic avenues for other angiogenic-related fertility pathologies.