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Lina Tsaadon, Ruth Kaplan-Kraicer, and Ruth Shalgi

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 Ca2 +/CaM-dependent protein kinase II (CaMKII) and MARCKS in promoting CGE and show that PKC pathway can compensate for lack of Ca2 +/CaM pathway. Microinjecting eggs with either overexpressed protein or complementary RNA of constitutively active αCaMKII triggered resumption of second meiotic division, but induced CGE of an insignificant magnitude compared with CGE induced by wt αCaMKII. 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.

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Irit Ben-Aharon and Ruth Shalgi

Seminal advances in anticancer therapy as well as supportive care strategies have led to improved survival rates, posing an emphasis on preserving an optimum quality of life after cancer treatment. This recognition has paved the way to an increasing research of long-term side effects, both clinical and preclinical and to an ongoing design of a supportive care system to evaluate and treat long-term adverse effects of anticancer treatments, including the impact on fertility. As with many adverse effects induced by anticancer treatments, the literature comprised mostly clinical data with regard to chemotherapy-induced gonadotoxicity, while understanding of the biological mechanism is lagging. The impact of anticancer treatments on female fertility depends on the women's age at the time of treatment, the chemotherapy protocol, the duration, and total cumulative dose administered. Several suggested mechanisms that underlie chemotherapy-induced gonadotoxicity have been described. This review illustrates the clinical evidence, as well as its supportive preclinical studies, while proceeding from the ‘bedside to the bench work’ and provides an insight to what lies behind chemotherapy-induced gonadotoxicity.

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Samples were obtained from ovarian tissue excised during the second quarter of the menstrual cycle. Sodium, potassium and chloride concentrations resembled those of blood, while pO2 was highly variable, and not correlated with follicular histology. The pCO2 was correlated with pH, but follicular fluid had a more acid pH than blood plasma. It is concluded that culture media based on 'physiological' saline solutions are appropriate for culture of oocytes.

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Ruth Shalgi, Nava Dekel, and P. F. Kraicer

Summary. The effect of adding LH (10 μg NIH-LH-B8/ml) to the medium in which oocytes were undergoing maturation in vitro was studied. The fertilizability of the oocytes was evaluated in the sterile oviduct of a unilaterally ovariectomized, mated recipient. Freshly ovulated oocytes, used as a control of the method, were fertilized at a rate of 72%. Only 14% of oocytes matured in culture (without LH) were penetrated by spermatozoa, and 11% were fertilized normally. Addition of LH to the medium increased these proportions to 43 and 33% respectively. Oocytes matured in the presence of LH were able to develop into apparently normal rats. It is concluded that, although oocytes can mature in vitro spontaneously, and that these matured oocytes can be fertilized, addition of LH increases the numbers 3-fold. LH therefore has a direct maturation-promoting action on the rat oocyte–cumulus complex in vitro.

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Mattan Levi, Bernard Maro, and Ruth Shalgi

Fertilization of mammalian oocytes triggers their exit from the second meiotic division metaphase arrest. The extrusion of the second polar body (PBII) that marks the completion of meiosis is followed by the first mitotic cleavage of the zygote. Several lines of evidence in somatic cells imply the involvement of Fyn, an Src family kinase (SFK), in cell cycle control and actin functions. In this study, we demonstrate, using live cell confocal imaging and microinjection of Fyn cRNAs, the recruitment of Fyn to the oocyte's cortical area overlying the chromosomes and its colocalization with filamentous actin (F-actin) during exit from the meiotic metaphase. Fyn concentrated asymmetrically at the cortical site designated for ingression of the PBII cleavage furrow, where F-actin had already been accumulated, and then redispersed throughout the entire cortex only to be recruited again to the cleavage furrow during the first mitotic division. Although microinjection of dominant negative Fyn did not affect initiation of the cleavage furrow, it prolonged the average duration of ingression, decreased the rates of PB extrusion and of the first cleavage, and led to the formation of bigger PBs and longer spindles. Extrusion of the PBII was blocked in oocytes exposed to SU6656, an SFK inhibitor. Our results demonstrate, for the first time, a continuous colocalization of Fyn and F-actin during meiosis and imply a role for the SFKs, in general, and for Fyn, in particular, in regulating pathways that involve actin cytoskeleton, during ingression of the meiotic and mitotic cleavage furrows.

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Efrat Eliyahu, Nataly Shtraizent, Alina Tsaadon, and Ruth Shalgi

Cortical granule exocytosis (CGE), following egg activation, is a secretory process that blocks polyspermy and enables successful embryonic development. CGE can be triggered independently by either a rise in intracellular calcium concentration ([Ca2+]i) or activation of protein kinase C (PKC). The present study investigates the signal transduction pathways leading to CGE through activation of PKC or stimulation of a rise in [Ca2+]i. Using Western blot analysis, co-immunoprecipitation and immunohistochemistry, combined with various inhibitors or activators, we investigated the link between myristoylated alanin-rich C kinase substrate (MARCKS) translocation and CGE. We were able to demonstrate translocation of MARCKS from the plasma membrane to the cortex, in fertilized as well as in parthenogenetically activated eggs. MARCKS phosphorylation was demonstrated upon PKC activation, whereas a PKC inhibitor (myrPKCψ) prevented both MARCKS translocation and CGE in 12-O-tetradecanoyl phorbol-13-acetate (TPA)-activated eggs. We have further shown that upon egg activation the amount of phosphorylated MARCKS (p-MARCKS) and the amount of calmodulin bound to MARCKS were increased. MARCKS translocation in ionomycin activated eggs was also inhibited by the calmodulin inhibitor N-(6-aminohexyl)-5-chloro-1-napthalenesulfonamide hydrochloride (W7). These results complement other studies showing MARCKS requirement for exocytosis and imply that upon fertilization, MARCKS translocation is followed by CGE. These findings present a significant contribution to our understanding of CGE in mammalian eggs in particular, as well as cellular exocytosis in general.

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Dana Chuderland, Ido Ben-Ami, Hadas Bar-Joseph, and Ruth Shalgi

The physiological function of the female reproductive organs is hormonally controlled. In each cycle, the reproductive organs undergo tissue modifications that are accompanied by formation and destruction of blood vessels. Proper angiogenesis requires an accurate balance between stimulatory and inhibitory signals, provided by pro- and anti-angiogenic factors. As with many other tissues, vascular endothelial growth factor (VEGF) appears to be one of the major pro-angiogenic factors in the female reproductive organs. Pigment epithelium-derived factor (PEDF) is a non-inhibitory member of the serine protease inhibitors (serpin) superfamily, possessing potent physiologic anti-angiogenic activity that negates VEGF activity. The role of PEDF in decreasing abnormal neovascularization by exerting its anti-angiogenic effect that inhibits pro-angiogenic factors, including VEGF, has been investigated mainly in the eye and in cancer. This review summarizes the function of PEDF in the reproductive system, showing its hormonal regulation and its anti-angiogenic activity. Furthermore, some pathologies of the female reproductive organs, including endometriosis, ovarian hyperstimulation syndrome, polycystic ovary syndrome, and others, are associated with a faulty angiogenic process. This review illuminates the role of PEDF in their pathogenesis and treatment. Collectively, we can conclude that although PEDF seems to play an essential role in the physiology and pathophysiology of the reproductive system, its full role and mechanism of action still need to be elucidated.

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Hadas Bar-Joseph, Ido Ben-Ami, Raphael Ron-El, Ruth Shalgi, and Dana Chuderland

Human chorionic gonadotropin (hCG) is a known trigger of ovarian hyperstimulation syndrome (OHSS), a potentially life-threatening complication of assisted reproduction. Administration of hCG results in the release of vascular endothelial growth factor (VEGF) from the ovary. We have previously shown that expression of pigment epithelium-derived factor (PEDF) in granulosa cell line is regulated by hCG, reciprocally to VEGF, and that the PEDF–VEGF balance is impaired in OHSS. Our aim was to explore the signaling network by which hCG downregulates the expression of PEDF mRNA and protein in granulosa cells. We applied specific chemical inhibitors and stimuli to human primary granulosa cells and rat granulosa cell line. We found that PKA and protein kinase C, as well as EGFR, ERK1/2 and PI3K, participate in the signaling network. The finding that hCG-induced PEDF downregulation and VEGF upregulation are mediated by similar signaling cascades emphasizes the delicate regulation of ovarian angiogenesis.

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Irit Ben-Aharon, Karin Haim, Ruth Shalgi, and Dalit Ben-Yosef

At fertilization in mammals, the spermatozoon triggers a unique signal transduction mechanism within the egg, leading to its activation. It is well accepted that the earliest event observed in all activated eggs is an abrupt rise in intracellular calcium concentrations. However, little is known regarding the downstream proteins that are activated by this rise in calcium. Calpains constitute a family of intracellular calcium-dependent cysteine proteases whose members are expressed widely in a variety of cells. We investigated the expression and possible role of the calpain isoforms μ and m throughout egg activation. Both calpains were expressed in the rat egg and localized at the egg cortex as well as in the meiotic spindle. m Calpain translocated to the membrane and to the spindle area during parthenogenetic egg activation and during in vivo fertilization, upon sperm binding to the egg. The cytoskeletal protein α-spectrin (fodrin) was proteolysed by calpain during the egg-activation process, as demonstrated by specific calpain-breakdown products. Following parthenogenetic activation by ionomycin or puromycin, the calpain-selective permeable inhibitor, calpeptin, inhibited the resumption of meiosis and cortical reaction in a dosedependent manner. Calpeptin was also effective in inhibiting in vitro fertilization. These results may imply a correlation between calpain activation and mammalian egg activation at fertilization and a possible role for calpain in the cascade of cellular events leading to resumption of meiosis.

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Mattan Levi, Moran Tzabari, Naphtali Savion, Salomon M Stemmer, Ruth Shalgi, and Irit Ben-Aharon

Infertility induced by anti-cancer treatments pose a major concern for cancer survivors. Doxorubicin (DXR) has been previously shown to exert toxic effects on the testicular germinal epithelium. Based upon the cardioprotective traits of dexrazoxane (DEX), we studied its potential effect in reducing DXR-induced testicular toxicity. Male mice were injected with 5 mg/kg DXR, 100 mg/kg DEX, combination of both or saline (control) and sacrificed either 1, 3 or 6 months later. Testes were excised and further processed. Glutathione and apoptosis assays were performed to determine oxidative stress. Immunohistochemistry and confocal microscopy were used to study the effects of the drugs on testicular histology and on spermatogonial reserve. DXR and the combined treatment induced a striking decline in testicular weight. DEX prevented DXR-induced oxidative stress, but enhanced DXR-induced apoptosis within the testes. Furthermore, the combined treatment depleted the spermatogonial reserve after 1 month, with impaired recovery at 3 and 6 months post-treatment. This resulted in compromised sperm parameters, testicular and epididymal weights as well as significantly reduced sperm motility, all of which were more severe than those observed in DXR-treated mice. The activity of DEX in the testis may differ from its activity in cardiomyocytes. Adding DEX to DXR exacerbates DXR-induced testicular toxicity.