Publications by authors named "Ruth Shalgi"

67 Publications

Chemotherapy-induced acute vascular injury involves intracellular generation of ROS via activation of the acid sphingomyelinase pathway.

Cell Signal 2021 Jun 26;82:109969. Epub 2021 Feb 26.

Department of Radiation Oncology, Memorial Sloan-Kettering Cancer Center, New York, NY, USA. Electronic address:

Several categories of chemotherapy confer substantial risk for late-term vascular morbidity and mortality. In the present study, we aimed to investigate the mechanism of acute chemotherapy-induced vascular injury in normal tissues. Specifically, we looked at activation of the acid sphingomyelinase (ASMase)/ceramide pathway, which leads to generation of reactive oxygen species (ROS) and induction of oxidative stress that may result in vascular injury. In particular, we focused on two distinct drugs, doxorubicin (DOX) and cisplatin (CIS) and their effects on normal endothelial cells. In vitro, DOX resulted in increased ASMase activity, intra-cellular ROS production and induction of apoptosis. CIS treatment generated significantly reduced effects in endothelial cells. In-vivo, murine femoral arterial blood flow was measured in real-time, during and after DOX or CIS administration, using fluorescence optical imaging system. While DOX caused constriction of small vessels and disintegration of large vessels' wall, CIS induced minor vascular changes in arterial blood flow, correlating with the in vitro findings. These results demonstrate that DOX induces acute vascular injury by increased ROS production, via activation of ASMase/ceramide pathway, while CIS increases ROS production and its immediate extracellular translocation, without causing detectable acute vascular injury. Our findings may potentially lead to the development of new strategies to prevent long-term cardiovascular morbidity in cancer survivors.
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http://dx.doi.org/10.1016/j.cellsig.2021.109969DOI Listing
June 2021

Anti-HER2/neu Antibody Reduces Chemotherapy-Induced Ovarian Toxicity-From Bench to Bedside.

Biomedicines 2020 Dec 7;8(12). Epub 2020 Dec 7.

Division of Oncology, Rambam Health Care Campus, Haifa 3109601, Israel.

Background: Trastuzumab, a humanized anti-human epidermal growth factor receptor 2 (HER2/neu) antibody, is considered a standard treatment in addition to chemotherapy in the adjuvant setting for HER2/neu-positive breast cancer, yet its impact on fertility and ovarian reserve remains obscure. We aimed to study the effect of anti-HER2/neu on chemotherapy-induced ovarian toxicity in both clinical and preclinical settings.

Methods: We prospectively enrolled breast cancer patients below the age of 42 years who were treated with chemotherapy with or without trastuzumab into the study. Anti-Müllerian hormone (AMH) was measured 6 and 12 months post-chemotherapy as an ovarian reserve indicator. In the animal model, pubertal mice were injected with cyclophosphamide or paclitaxel with or without anti-HER2/neu, or saline, and sacrificed 1 week or 3 months later. Ovarian apoptosis, proliferation and vascularity were measured by immunohistochemistry and ovarian reserve was measured by morphometric analysis and serum-AMH.

Results: Thirty-three patients with early breast cancer were enrolled into the study. Nineteen patients had HER2/neu negative cancer and were treated with chemotherapy and 14 had HER2/neu positive cancer and were treated with chemotherapy and trastuzumab. In all patients, AMH levels declined to undetectable values immediately post-treatment, but regained for 57.1% of the HER2/neu positive cohort and 36.8% of the negative cohort ( < 0.05). In the preclinical setting, anti-HER2/neu antibody, in combination with chemotherapy, displayed lessened ovarian and vascular damage.

Conclusions: Our results indicate that trastuzumab may alleviate chemotherapy-induced ovarian toxicity that may be mediated via its effect on ovarian vasculature.
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http://dx.doi.org/10.3390/biomedicines8120577DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7762209PMC
December 2020

Pigment epithelium derived factor as a novel multi-target treatment for uterine fibroids.

Reprod Biomed Online 2020 Aug 29;41(2):335-342. Epub 2020 Apr 29.

Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv 69978, Ramat-Aviv, Israel.

Research Question: Does recombinant pigment epithelium derived factor (PEDF) have potential in treating uterine fibroids?

Design: In-vitro models that used human leiomyoma and Eker rat uterine leiomyoma (ELT-3) cell lines. The ELT-3 cell line was used to examine cellular targets after adding recombinant PEDF to the culture media. Athymic nude female mice were used as an in-vivo model. They were injected with ELT-3 cells to induce ectopic fibroid lesions, then treated with recombinant PEDF.

Results: RNA expression of PEDF and its receptors was found in both leiomyoma cell lines, as well as the expression of PEDF receptors. Addition of recombinant PEDF to the culture medium of leiomyoma cell lines activated ERK in a time-dependent manner, induced down-regulation of vascular endothelial growth factor mRNA and protein, as well as the mRNAs of oestrogen receptors alpha and beta and inhibited cellular proliferation. Treatment of mice-bearing fibroids with recombinant PEDF reduced fibroid growth rate and resulted in smaller tumours.

Conclusions: This study suggests that recombinant PEDF is a putative novel potent physiological treatment for uterine fibroids. It targets several cornerstones of fibroid pathobiology in parallel, including vascular endothelial growth factor and oestrogen receptors, which are needed for vascularization, and restricts fibroid growth and final size in an animal model.
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http://dx.doi.org/10.1016/j.rbmo.2020.03.024DOI Listing
August 2020

Male sterility and reduced female fertility in SCAPER-deficient mice.

Hum Mol Genet 2020 Aug;29(13):2240-2249

Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa 31096, Israel.

Mutations in S-phase cyclin A-associated protein in the endoplasmic reticulum (SCAPER) cause a recessively inherited multisystemic disorder whose main features are retinal degeneration and intellectual disability. SCAPER, originally identified as a cell cycle regulator, was also suggested to be a ciliary protein. Because Scaper mutant males are sterile, we set up to characterize their phenotype. The testes of Scaper mutant mice are significantly smaller than those of WT mice. Histology revealed no signs of spermatogenesis, and seminiferous tubules contained mainly Sertoli cells with a few spermatogonia/spermatogonial stem cells (SSCs). In WT testes, SCAPER is expressed by SSCs and in the various stages of spermatogenesis, as well as in Sertoli cells. In WT spermatozoa SCAPER is not expressed in the flagellum but rather in the head compartment, where it is found both in the nucleus and in the perinuclear region. Scaper mutant females present reduced fertility, manifested by a significantly smaller litter size compared to WT females. Mutant ovaries are similar in size but comprised of significantly less primordial and antral follicles, compared to WT ovaries, while the number of atretic follicles is significantly higher. In WT ovarian follicles SCAPER is expressed in the somatic granulosa cells as well as in the oocyte. In conclusion, our data demonstrate that SCAPER is a crucial component in both male and female reproductive systems. We hypothesize that the reproductive phenotype observed in Scaper mutant mice is rooted in SCAPER's interaction with cyclin A/Cdk2, which play an important role, however different, in male and female gonads.
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http://dx.doi.org/10.1093/hmg/ddaa113DOI Listing
August 2020

Cancer During Pregnancy: The Role of Vascular Toxicity in Chemotherapy-Induced Placental Toxicity.

Cancers (Basel) 2020 May 18;12(5). Epub 2020 May 18.

Oncology, Rambam Health Care Center, Haifa 3109601, Israel.

Breast cancer is diagnosed in ~0.3% of pregnant women. Studies that have addressed gestational and neonatal outcomes of chemotherapy during pregnancy have demonstrated increased gestational complications including preeclampsia and intrauterine growth retardation. We hypothesized that anthracycline-induced gestational complications could be derived from direct toxicity on the placenta vasculature. Pregnant ICR mice (day E12.5) were treated with doxorubicin (DXR; 8 mg/kg) or saline, while their umbilical cord blood flow was imaged by pulse-wave (PW) Doppler. Mice were euthanized on day E18.5, and their embryos and placentae were collected for further analysis. Unlike control mice, the DXR-treated mice presented an acute change in the umbilical cord's blood flow parameters (velocity time integral and heart rate interval), reduced embryos' weight, reduced placenta efficiency, and modulation in vascular-related pathways of treated placenta proteomics. Apoptosis and proliferation were also enhanced, as demonstrated by TUNEL and proliferating cell nuclear antigen (PCNA) analysis. We further examined the placentae of patients treated with epirubicin (EPI), who had been diagnosed with breast cancer during pregnancy (weeks 27-35). The immunohistochemistry of the EPI-treated human placentae showed enhanced proliferation and apoptosis as compared with matched chemo-naïve placentae, as well as reduced neovascularization (CD34). Our findings suggest that anthracycline-induced vascular insult promotes placental toxicity, and could point to potential agents designated to offset the damage and to reduce gestational complications in pregnant cancer patients.
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http://dx.doi.org/10.3390/cancers12051277DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7281110PMC
May 2020

miR-125a Induces HER2 Expression and Sensitivity to Trastuzumab in Triple-Negative Breast Cancer Lines.

Front Oncol 2020 28;10:191. Epub 2020 Feb 28.

Division of Oncology, Rambam Health Care, Haifa, Israel.

The EGFR/HER2 signaling network is an effective therapeutic target for HER2-positive cancers, which are known for their aggressive biological course. Evidence indicates that the EGFR/HER2 network plays a role in the aggressive basal-like subtype as well. Here, we studied the potential role of miR-125a-3p as a modulator of the EGFR/HER2 pathway in basal-like breast cancer. Over-expression of miR-125a-3p reduced the migratory capability of MDA-MB-231 cells and led to an increase in the expression of ErbB2 transcript and protein. The induced ErbB2 responded to trastuzumab and underwent internalization and subsequent intra-lysosomal degradation. Trastuzumab treatment further reduced the migratory capability and induced the apoptosis of the cells. An mouse model, which supported the findings, showed a synergistic effect for miR-125a-3p and trastuzumab. Trastuzumab-treated miR-125a-3p-induced tumors were significantly smaller than control induced tumors. Our findings indicate that, in the basal-like subtype of breast cancer, miR-125a-3p may act as a tumor suppressor. miR-125a-3p induces an increase in the expression of ErbB2 that may render the cells suitable for treatment with anti-HER2 therapies.
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http://dx.doi.org/10.3389/fonc.2020.00191DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7058585PMC
February 2020

Pigment epithelium-derived factor (PEDF) negates hyperandrogenic PCOS features.

J Endocrinol 2020 05;245(2):291-300

Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel-Aviv University, Ramat-Aviv, Tel-Aviv, Israel.

Polycystic ovary syndrome (PCOS), one of the most common female endocrine disorder, is a prevalent cause of infertility. Hyperandrogenism is a key feature in PCOS and is correlated with increased expression of VEGF and cytokines in the ovaries. We have previously shown that pigment epithelium-derived factor (PEDF), an endogenous protein, presents potent anti-angiogenic and anti-inflammatory activities in the ovary and negates the effects of cytokines and VEGF. Additionally, PEDF plays a role in both pathophysiology and treatment of ovarian-hyperstimulation syndrome (OHSS), frequently seen in PCOS patients. We established hyperandrogenic-PCOS models, both in vivo, using mice exposed prenatally to dihydrotestosterone (DHT) and, in vitro, using human primary granulosa cells (hpGCs) and human granulosa cell line (KGN). In PCOS-induced mice, the mRNA levels of I l-6, V egf and Amh were higher than those of control; yet, treatment with rPEDF decreased these levels. Moreover, treating OHSS-induced PCOS-mice with rPEDF alleviated all OHSS symptoms. Stimulation of hpGCs with DHT resulted in downregulation of PEDF mRNA expression, concomitantly with a significant increase in IL-6 and IL-8 mRNAs expression. However, co-stimulation of DHT with rPEDF attenuated the increase in cytokines expression. The anti-inflammatory effect of PEDF was found to be mediated via PPARγ pathway. Our findings suggest that rPEDF treatment may normalize the ovarian angiogenic-inflammatory imbalance, induced by PCOS-associated hyperandrogenism. Moreover, the therapeutic potency of PEDF in preventing OHSS symptomes offers a rationale for using PEDF as novel physiological treatment for PCOS sequels.
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http://dx.doi.org/10.1530/JOE-19-0603DOI Listing
May 2020

Pre-ovulatory intercellular regulation of miR-125a-3p within mouse ovarian follicles.

Reproduction 2019 Dec 1. Epub 2019 Dec 1.

R Shalgi, Cell and Developmental Biology, Medical School, Tel Aviv University, Tel Aviv, Israel.

miR-125a-3p, a post-transcription regulator of Fyn kinase, is expressed in mouse pre-ovulatory follicles; its expression within the follicle decreases towards ovulation. Our aim was to follow the synthesis of miR-125a-3p and regulation of its expression in all follicular compartments, focusing on intercellular communication. Mural granulosa cells (GCs) or cumulus cells (CCs) were transfected with either scrambled-miR (negative control) or miR-125a-3p-mimic. Freshly isolated GCs or CCs were incubated over-night in culture media conditioned by transfected cells. To examine a possible role of gap-junctions in the regulation of miR-125a-3p, we incubated large antral-follicles in the presence of Carbenoxolone, a gap-junction inhibitor, and triggered them to mature with hGC. Levels of miR-125a family members in GCs, CCs, oocytes and culture media were measured by qPCR. We showed that miR-125a-3p is synthesized by all follicular components, but is regulated within the follicle as a whole. It is secreted by mural-GCs and taken up by CCs, where it remains functional, and vice versa, mural-GCs can take up miR-125a-3p secreted by CCs. miR-125a-3p is transcribed and accumulated in oocytes throughout oogenesis. Transcriptionally quiescent GV oocytes utilize their accompanying follicular cells to monitor the level of miR-125a-3p within them, as indicated in an ex-vivo follicle culture. Our study reveals that miR-125a-3p expression is modulated by a network of intercellular communications within pre-ovulatory follicles; thus, enabling a coordinated decrease of miR-125a-3p towards ovulation.
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http://dx.doi.org/10.1530/REP-19-0419DOI Listing
December 2019

Elucidating the role of pigment epithelium-derived factor (PEDF) in metabolic PCOS models.

J Endocrinol 2020 02;244(2):297-308

Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel.

PCOS is the most common endocrinopathy in women; associated with obesity and insulin resistance (IR). IR leads to accumulation of advanced-glycation-end-products (AGEs) and their receptor, RAGE. PCOS patients have increased levels of vascular endothelial growth factor (VEGF), interleukin 6/8 (IL-6/8) and anti-Mϋllerian-hormone (AMH). PEDF is a secreted-glycoprotein known for its anti-angiogenic and anti-inflammatory properties. We aimed to elucidate the role of PEDF in the pathogenesis and treatment of PCOS. We used a prenatal PCOS mouse model and fed the female offspring a high-fat diet, inducing metabolic PCOS (met.PCOS) characteristics. Female offspring were divided into three groups: control; met.PCOS; met.PCOS + recombinant PEDF (rPEDF). Met.PCOS mice gained more weight, had elevated serum IL-6 and higher mRNA levels of AMH, PEDF and RAGE in their granulosa cells (GCs) than met.PCOS + rPEDF mice. An in vitro Met.PCOS model in human GCs (KGN) line was induced by prolonged incubation with insulin/AGEs, causing development of IR. Under the same conditions, we observed an elevation of VEGF, IL-6/8 mRNAs, concomitantly with an increase in PEDF mRNA, intracellular protein levels, and an elevation of PEDF receptors (PEDF-Rs) mRNA and protein. Simultaneously, a reduction in the secretion of PEDF from GCs, was measured in the medium. The addition of rPEDF (5 nM) activated P38 signaling, implying that PEDF-Rs maintained functionality, and negated AGE-induced elevation of IL-6/8 and VEGF mRNAs. Decreased PEDF secretion may be a major contributor to hyperangiogenesis and chronic inflammation, which lie at the core of PCOS pathogenesis. rPEDF treatment may restore physiological angiogenesis inflammatory balance, thus suggesting a potential therapeutic role in PCOS.
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http://dx.doi.org/10.1530/JOE-19-0297DOI Listing
February 2020

Treosulfan induces distinctive gonadal toxicity compared with busulfan.

Oncotarget 2018 Apr 10;9(27):19317-19327. Epub 2018 Apr 10.

Institute of Oncology, Davidoff Center, Rabin Medical Center, Beilinson Campus, Petah-Tiqva, Israel.

Treosulfan (L-treitol-1,4-bis-methanesulfonate) has been increasingly incorporated as a main conditioning protocol for hematopoietic stem cell transplantation in pediatric malignant and non-malignant diseases. Treosulfan presents lower toxicity profile than other conventional alkylating agents containing myeloablative and immunosuppressive traits such as busulfan. Yet, whereas busulfan is considered highly gonadotoxic, the gonadal toxicity profile of treosulfan remains to be elucidated. To study the gonadotoxicity of treosulfan, pubertal and prepubertal male and female mice were injected with treosulfan or busulfan and sacrificed one week, one month or six months later. Testicular function was assessed by measurements of sperm properties, testes and epididymides weights as well as markers for testicular reserve, proliferation and apoptosis. Ovarian function was assessed by measurements of ovary weight and markers for ovarian reserve, proliferation and apoptosis. Treosulfan testicular toxicity was milder than that of busulfan toxicity; possibly by sparing the stem spermatogonia in the testicular sanctuary. By contrast, ovarian toxicity of both treosulfan and busulfan was severe and permanent and displayed irreversible reduction of reserve primordial follicles in the ovaries. Our data indicate that treosulfan exerts a different gonadal toxicity profile from busulfan, manifested by mild testicular toxicity and severe ovarian toxicity.
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http://dx.doi.org/10.18632/oncotarget.25029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5922399PMC
April 2018

Premature ovarian aging in carriers: a prototype of systemic precocious aging?

Oncotarget 2018 Mar 23;9(22):15931-15941. Epub 2018 Mar 23.

Institute of Oncology, Davidoff Center, Rabin Medical Center, Beilinson Campus, Petach Tikva, Israel.

Purpose: Though former evidence implies a correlation of breast cancer susceptibility gene () mutation with reduced ovarian reserve, the data is yet inconsistent. Our aim was to investigate biomarkers of ovarian aging in a cohort of young healthy carriers of the mutation. We hypothesized that the role played by genes in aging pathways is not exclusive to the ovary.

Experimental Design: Healthy female carriers, 40 years or younger and healthy male carriers, 50 years or younger, were enrolled in the study. Serum anti-mullerian Hormone (AMH), fibroblast growth factor-23 (FGF-23), Klotho and IL-1 were measured by enzyme-linked immunosorbent assay (ELISA). Ovarian AMH and protein kinase B (AKT) mRNA from carriers who underwent prophylactic oophorectomy and from age-matched, healthy, non-carriers who underwent partial oophorectomy due to benign conditions were analyzed by qPCR.

Results: Thirty-three female (median age 35y) and 20 male (44y) carriers were enrolled into the study and matched to control non-carriers (34y and 43y, respectively). Serum AMH level was significantly lower in female carriers than in both non-carrier controls and age-matched nomograms. The levels of ovarian AMH and AKT mRNA were significantly lower in carriers than in controls. The systemic aging cytokines FGF-23, klotho and IL-1 displayed a differential expression in carriers of both genders. FGF-23 level was higher in carriers (P=0.06).

Conclusions: Our results suggest a link between mutation, accelerated ovarian aging and systemic aging-related pathophysiology.
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http://dx.doi.org/10.18632/oncotarget.24638DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5882308PMC
March 2018

Regulation of GVBD in mouse oocytes by miR-125a-3p and Fyn kinase through modulation of actin filaments.

Sci Rep 2017 05 22;7(1):2238. Epub 2017 May 22.

Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel-Aviv University, Ramat-Aviv, Tel-Aviv, 69978, Israel.

Meiotically arrested oocytes are characterized by the presence of the nuclear structure known as germinal-vesicle (GV), the breakdown of which (GVBD) is associated with resumption of meiosis. Fyn is a pivotal factor in resumption of the first meiotic division; its inhibition markedly decreases the fraction of oocytes undergoing GVBD. Here, we reveal that in mouse oocytes Fyn is post-transcriptionally regulated by miR-125a-3p. We demonstrate that in oocytes resuming meiosis miR-125a-3p and Fyn exhibit a reciprocal expression pattern; miR-125a-3p decreases alongside with an increase in Fyn expression. Microinjection of miR-125a-3p inhibits GVBD, an effect that is markedly reduced by Fyn over-expression, and impairs the organization of the actin rim surrounding the nucleus. Lower rate of GVBD is also observed in oocytes exposed to cytochalasin-D or blebbistatin, which interfere with actin polymerization and contractility of actin bundles, respectively. By down-regulating Fyn in HEK-293T cells, miR-125a-3p reduces the interaction between actin and A-type lamins, which constitute the nuclear-lamina. Our findings suggest a mechanism, by which a decrease in miR-125a-3p during oocyte maturation facilitates GVBD by allowing Fyn up-regulation and the resulting stabilization of the interaction between actin and A-type lamins.
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http://dx.doi.org/10.1038/s41598-017-02071-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5440411PMC
May 2017

Interleukin 1-alpha deficiency increases the expression of Follicle-stimulating hormone receptors in granulosa cells.

Mol Reprod Dev 2017 Jun 17;84(6):460-467. Epub 2017 Apr 17.

Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel.

Follicle-stimulating hormone receptor (FSHR) is a pivotal regulator of ovarian response to hormonal stimulation. Inflammatory conditions have been linked to lower FSHR expression in granulosa cells (GCs) as well as an attenuated response to hormonal stimulation. The current study aimed to reveal if deficiency and/or blockage of the pro-inflammatory cytokine interleukin 1-alpha (IL1A) increased Fshr expression in rodent GCs. We found elevated Fshr transcript abundance, as assessed by quantitative PCR, in primary GCs isolated from Il1a-knockout compared to wild-type mice, and that the expression of FSHR is significantly higher in Il1a-knockout compared to wild-type ovaries. Supplementing GC cultures with recombinant IL1A significantly lowered Fshr expression in these cells. In accordance with the Fshr expression pattern, proliferation of GCs was higher in follicles from Il1a-knockout mice compared to wild-type mice, as indicated by the MKI67 immunohistochemical staining. Furthermore, treating wild-type mice with anakinra, an IL1 receptor 1 antagonist, significantly increased the expression of Fshr in primary GCs from treated compared to control mice. These data highlight an important interdependency between the potent pro-inflammatory cytokine IL1A and Fshr expression.
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http://dx.doi.org/10.1002/mrd.22799DOI Listing
June 2017

The Dual Role of PEDF in the Pathogenesis of OHSS: Negating Both Angiogenic and Inflammatory Pathways.

J Clin Endocrinol Metab 2016 12 28;101(12):4699-4709. Epub 2016 Sep 28.

Department of Cell and Developmental Biology (I.M., D.C., H.G., R.S.), Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv 69978, Israel; and IVF and Infertility Unit (R.R.-E., I.B.-A.), Department of Obstetrics and Gynecology, Assaf Harofeh Medical Center, Zerifin 70300, Israel.

Context: Ovarian hyperstimulation syndrome (OHSS) is a potentially life-threatening complication of assisted reproductive technologies. This complex syndrome is known to involve massive angiogenesis and inflammation. We have previously established the anti-angiogenic involvement of pigment epithelium-derived factor (PEDF) in the pathophysiology and treatment of OHSS.

Objective: Evaluate the anti-inflammatory role of PEDF in OHSS.

Design: In vivo mouse OHSS model and in vitro cultures of granulosa cells.

Main Outcome: Changes in the expression of PEDF, IL-6, IL-8, and vascular endothelial growth factor (VEGF) were measured by quantitative PCR and ELISA; OHSS symptoms were recorded (body and ovarian weight gain and peritoneal vascular leakage quantified by the modified Miles's assay).

Results: Rat granulosa cell-line stimulated with lysophosphatidic acid (LPA), exhibited a significant increase in IL-6 expression, concomitantly with a decrease in PEDF level (P < .01). Co-stimulation with recombinant PEDF (rPEDF) decreased the expression of IL-6 significantly (P < .05). Furthermore, the expression of IL-6 and IL-8 increased in LPA-stimulated human primary granulosa cells (P < .01). Co-stimulation with rPEDF decreased the expression of LPA-induced IL-6 and IL-8 mRNA and protein by 4- and 2- to 5-fold, respectively. IL-8-stimulated human primary granulosa cells exhibited increased expression of VEGF mRNA; co-stimulation with hCG induced a significantly higher increase in the expression of VEGF mRNA (P < .001), which was counteracted by rPEDF. Subcutaneous injection of 0.5 mg/kg rPEDF to OHSS-induced mice reduced the increased expression of IL-6 in the ovary (P < .01) and alleviated the severity of all OHSS parameters.

Conclusions: Our findings provide a framework that correlates down-regulation of OHSS symptoms caused by PEDF with both angiogenic and inflammatory pathways.
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http://dx.doi.org/10.1210/jc.2016-1744DOI Listing
December 2016

A Role of MicroRNAs in Cell Differentiation During Gonad Development.

Results Probl Cell Differ 2016 ;58:309-36

Department of Cell Biology and Development, Tel Aviv University, Ramat Aviv, Israel.

MicroRNAs (miRNAs) are a group of small noncoding RNA molecules that play a major role in posttranscriptional regulation of gene expression and are expressed in an organ-specific manner. One miRNA can potentially regulate the expression of several genes, depending on cell type and differentiation stage. miRNAs are differentially expressed in the male and female gonads and have an organ-specific reproductive function. Exerting their affect through germ cells and gonadal somatic cells, miRNAs regulate key proteins necessary for gonad development. The role of miRNAs in the testes is only starting to emerge though they have been shown to be required for adequate spermatogenesis. Widely explored in the ovary, miRNAs were suggested to play a fundamental role in follicles' assembly, growth, differentiation, and ovulation. In this chapter, we focus on data obtained from mice in which distinct proteins that participate in the biosynthesis of miRNAs were conditionally knocked out from germ cells (spermatogonial cells or oocytes) or gonadal somatic cells (Sertoli or granulosa cells). We detail recent advances in identification of particular miRNAs and their significance in the development and function of male and female gonads. miRNAs can serve as biomarkers and therapeutic agents of pathological conditions; thus, elucidating the branched and complex network of reproduction-related miRNAs will aid understanding of gonads' physiology and managing reproduction disorders.
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http://dx.doi.org/10.1007/978-3-319-31973-5_12DOI Listing
January 2017

Cetuximab intensifies cisplatin-induced testicular toxicity.

Reprod Biomed Online 2016 Jul 20;33(1):102-10. Epub 2016 Apr 20.

Institute of Oncology, Davidoff Center, Rabin Medical Center, Beilinson Campus, Petah-Tiqva, and Sackler Faculty of Medicine, Tel-Aviv University, Israel. Electronic address:

Epidermal growth factor receptor (EGFR) has proliferative properties in the testis. Cetuximab, an anti-EGFR, is administered together with chemotherapy to patients with various types of cancer. This studies aim was to investigate the effect of cetuximab on testicular function. Adult male mice were injected with cetuximab (10 mg/kg), cisplatin (8 mg/kg) or a combination of both, and killed one week or one month later. The doses were chosen by human equivalent dose calculation. Testicular function was evaluated by epididymal-spermatozoa total motile count and sperm motility, weights of testes and epididymides, and the level of anti-Müllerian hormone (AMH) in the serum. Immunohistochemistry was performed to examine germ cell proliferation (Ki-67), apoptosis (Terminal transferase-mediated deoxyuridine 5-triphosphate nick-end labelling), reserve (DAZL-Deleted in azoospermia-like, Promyelocytic leukaemia zinc-finger), blood vessels (CD34) and Sertoli cells (GATA-4). Administration of cetuximab alone increased testicular apoptosis and decreased epididymal-spermatozoa total motile count over time. When added to cisplatin, cetuximab exacerbated most of the recorded testicular parameters, compared with the effect of cisplatin alone, including testis and epididymis weights, epididymal-spermatozoa total motile count, AMH concentration, meiosis and apoptosis. In conclusion, cetuximab has only a mild effect on testicular reserve, but when added to cisplatin, it exacerbates cisplatin-induced testicular toxicity.
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http://dx.doi.org/10.1016/j.rbmo.2016.04.004DOI Listing
July 2016

Pigment epithelium-derived factor regulation by human chorionic gonadotropin in granulosa cells.

Reproduction 2016 Feb 26;151(2):179-85. Epub 2015 Nov 26.

Department of Cell and Developmental BiologySackler Faculty of Medicine, Tel-Aviv University, Ramat-Aviv, Tel-Aviv 69978, IsraelIVF and Infertility UnitDepartment of Obstetrics and Gynecology, Assaf Harofeh Medical Center, Zerifin 70300, IsraelSackler Faculty of MedicineTel-Aviv University, Ramat-Aviv, Tel-Aviv 69978, Israel

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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http://dx.doi.org/10.1530/REP-15-0478DOI Listing
February 2016

Pigment Epithelium-Derived Factor Alleviates Tamoxifen-Induced Endometrial Hyperplasia.

Mol Cancer Ther 2015 Dec 8;14(12):2840-9. Epub 2015 Oct 8.

Institute of Oncology, Davidoff Center and Rabin Medical Center, Petah-Tiqva and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel.

Tamoxifen is a cornerstone component of adjuvant endocrine therapy for patients with hormone-receptor-positive breast cancer. Its significant adverse effects include uterine hyperplasia, polyps, and increased risk of endometrial cancer. However, the underlying molecular mechanism remains unclear. Excessive angiogenesis, a hallmark of tumorigenesis, is a result of disrupted balance between pro- and anti-angiogenic factors. VEGF is a pro-angiogenic factor shown to be elevated by tamoxifen in the uterus. Pigment epithelium-derived factor (PEDF) is a potent anti-angiogenic factor that suppresses strong pro-angiogenic factors, such as VEGF. Our aim was to investigate whether angiogenic balance plays a role in tamoxifen-induced uterine pathologies, elucidate the molecular impairment in that network, and explore potential intervention to offset the proposed imbalance elicited by tamoxifen. Using in vivo mouse models, we demonstrated that tamoxifen induced a dose-dependent shift in endogenous uterine angiogenic balance favoring VEGF over PEDF. Treatment with recombinant PEDF (rPEDF) abrogated tamoxifen-induced uterine hyperplasia and VEGF elevation, resulting in reduction of blood vessels density. Exploring the molecular mechanism revealed that tamoxifen promoted survival and malignant transformation pathways, whereas rPEDF treatment prevents these changes. Activation of survival pathways was decreased, demonstrated by reduction in AKT phosphorylation concomitant with elevation in JNK phosphorylation. Estrogen receptor-α and c-Myc oncoprotein levels were reduced. Our findings provide novel insight into the molecular mechanisms tamoxifen induces in the uterus, which may become the precursor events of subsequent endometrial hyperplasia and cancer. We demonstrate that rPEDF may serve as a useful intervention to alleviate the risk of tamoxifen-induced endometrial pathologies.
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http://dx.doi.org/10.1158/1535-7163.MCT-15-0523DOI Listing
December 2015

The impact of oxaliplatin on the gonads: from bedside to the bench.

Mol Hum Reprod 2015 Dec 6;21(12):885-93. Epub 2015 Oct 6.

Institute of Oncology, Davidoff Center, Rabin Medical Center, Beilinson Campus, Petah-Tiqva, Israel Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel

Study Hypothesis: What is the impact of oxaliplatin on gonadal function?

Study Finding: Our results in both the clinical and pre-clinical settings indicate that oxaliplatin exerts moderate transient gonadal toxicity.

What Is Known Already: Recent studies have indicated a significant increase in survivorship of colorectal cancer patients of reproductive age, who may then face fertility concerns. The impact of oxaliplatin on gonadal function is yet to be discovered.

Study Design, Samples/materials, Methods: Eleven female (<43 years) and eight male (<45 years) patients recently diagnosed with colorectal cancer, who were candidates for oxaliplatin-based protocol, were enrolled into the study. FSH, estradiol, anti-Müllerian hormone (AMH) and menstrual pattern were measured in female patients, whereas FSH, inhibin-B, testosterone, and steroid-hormone binding globulin were measured in male patients. Hormones were measured at baseline and 6 months post-treatment (last chemotherapy administration) in men and women. In the animal model, pubertal mice were injected with oxaliplatin and sacrificed 1 week, 1 month and 3 months later. Ovarian reserve was estimated by serum AMH measurements. Testicular function was evaluated by serum inhibin-B and sperm evaluation. Gonadal apoptosis (TUNEL), proliferation (Ki-67), repair (PCNA), ovarian reserve (AMH) and testicular reserve (DAZL) were measured by immunohistochemistry.

Main Results And The Role Of Chance: In all women, AMH decreased post-treatment, but remained above the detection limit in 9/11 patients (P < 0.05). FSH was elevated, but did not exceed the premenopausal range in 9/11 patients. All patients remain menstruating or resumed menstruation post-treatment. In female mice oxaliplatin induced transient apoptosis at 1-month post-treatment. In men Inhibin-B was slightly reduced post-treatment. In male mice oxaliplatin did not affect spermatozoa concentration, but was associated with transient, moderate reductions of spermatocytes-spermatogonia numbers and spermatozoa motility.

Limitations, Reasons For Caution: Future prospective large-scale studies are warranted in order to affirm these outcomes.

Wider Implications Of The Findings: Due to high survival rates of colorectal cancer patients of reproductive age that were diagnosed at early stages of the disease, the issue of treatment-induced gonadotoxicity gains significance. Since at the individual level there might be a risk of infertility, a detailed discussion and referral to fertility preservation prior to initiation of treatment is recommended. Nevertheless, oxaliplatin-based protocols appear to be less gonadotoxic than other chemotherapeutic protocols.

Large Scale Data: None.

Study Funding And Competing Interests: This study was supported by the Israeli Science Foundation (ISF) grant 13-1816 (I.B.-A.). There is no conflict of interest.
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http://dx.doi.org/10.1093/molehr/gav055DOI Listing
December 2015

Dexrazoxane exacerbates doxorubicin-induced testicular toxicity.

Reproduction 2015 Oct;150(4):357-66

Department of Cell and Developmental BiologySackler Faculty of Medicine, Tel Aviv University, Ramat Aviv, Tel Aviv 69978, IsraelThe Goldschleger Eye Research InstituteSheba Medical Center, Tel Hashomer 52621, IsraelInstitute of OncologyDavidoff Center, Rabin Medical Center, Beilinson Campus, 39 Jabotinski Street, Petah-Tiqva 49100, IsraelSackler Faculty of MedicineTel Aviv University, Ramat Aviv, Tel Aviv 69978, Israel Department of Cell and Developmental BiologySackler Faculty of Medicine, Tel Aviv University, Ramat Aviv, Tel Aviv 69978, IsraelThe Goldschleger Eye Research InstituteSheba Medical Center, Tel Hashomer 52621, IsraelInstitute of OncologyDavidoff Center, Rabin Medical Center, Beilinson Campus, 39 Jabotinski Street, Petah-Tiqva 49100, IsraelSackler Faculty of MedicineTel Aviv University, Ramat Aviv, Tel Aviv 69978, Israel

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.
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http://dx.doi.org/10.1530/REP-15-0129DOI Listing
October 2015

GnRH Agonist Triggering Modulates PEDF to VEGF Ratio Inversely to hCG in Granulosa Cells.

J Clin Endocrinol Metab 2015 Nov 26;100(11):E1428-36. Epub 2015 Aug 26.

Department of Cell and Developmental Biology (I.M., D.C., R.S.), Sackler Faculty of Medicine, and IVF and Infertility Unit (R.R.-E., I.B.-A.), Department of Obstetrics and Gynecology, Assaf Harofeh Medical Center, Zerifin 70300, Israel.

Context: GnRH agonist (GnRH-a) triggering is associated with a reduced risk of ovarian hyperstimulation syndrome (OHSS) compared with human chorionic gonadotropin (hCG) in assisted reproduction technology cycles. We have shown that ovarian pigment epithelium derived factor (PEDF), a potent antiangiogenic factor, counteracts vascular endothelial growth factor (VEGF) expression and that OHSS is correlated with hCG-induced impaired PEDF to VEGF ratio.

Objective: The objective of the study was to explore whether GnRH-a triggering could directly modulate PEDF/VEGF balance in granulosa cells.

Design: The design of the study was a mouse model and cultured granulosa cells.

Main Outcome: Changes in PEDF and VEGF were measured by quantitative PCR and Western blot analysis. OHSS symptoms were recorded by changes in body weight and in peritoneal vascular leakage, quantified by the modified Miles vascular permeability assay.

Results: GnRH-a stimulation significantly increased PEDF and decreased VEGF mRNA and protein levels both in rat granulosa cell line and human primary granulosa cells in vitro. GnRH-a and hCG triggering inversely modulated PEDF mRNA and protein level in human granulosa cells in vivo. In the GnRH-a triggering mouse model, we showed similar increase in PEDF to VEGF ratio as in the in vitro results. OHSS-predisposed mice did not develop OHSS parameters after GnRH-a triggering, opposed to hCG-triggered mice. Finally, GnRH-a triggering of OHSS-predisposed mice significantly increased ovarian PEDF to VEGF ratio compared with hCG-triggered mice and control mice.

Conclusions: GnRH-a triggering induces a direct effect on PEDF/VEGF balance in granulosa cells inversely to hCG. Our results suggest a novel elucidation to the GnRH-a triggering-mediated risk reduction of OHSS and may clarify the pros and cons of this triggering method.
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http://dx.doi.org/10.1210/jc.2015-2312DOI Listing
November 2015

Anti-Müllerian Hormone Is a Marker for Chemotherapy-Induced Testicular Toxicity.

Endocrinology 2015 Oct 7;156(10):3818-27. Epub 2015 Aug 7.

Department of Cell and Developmental Biology (M.L., N.H., R.S.), Sackler Faculty of Medicine, Tel Aviv University, Israel and Institute of Oncology (S.M.S., I.B-A.), Davidoff Center, Rabin Medical Center, Beilinson Campus, Petah-Tiqva 49100, and Sackler Faculty of Medicine, Tel-Aviv University, Israel.

Due to increased numbers of young cancer patients and improved survival, the impact of anticancer treatments on fertility has become a major health concern. Despite mounting research on ovarian toxicity, there is paucity of data regarding reliable biomarkers of testicular toxicity. Our aim was to evaluate anti-Müllerian hormone (AMH) as a marker for chemotherapy-induced testicular toxicity. Serum AMH and a panel of gonadal hormones were measured in male cancer patients at baseline and after chemotherapy. In the preclinical setting, mice were injected with diverse chemotherapies and were killed 1 week or 1, 3, or 6 months later. We evaluated spermatogenesis by AMH as well as qualitative and quantitative sperm parameters. Nineteen patients were enrolled, the median age was 38 years (21-44 y). Serum AMH was correlated with increased FSH and T and decreased inhibin-B in gonadotoxic protocols (cisplatin or busulfan) and remained unchanged in nongonadotoxic protocols (capecitabine). AMH expression had the same pattern in mice serum and testes; it was negatively correlated with testicular/epididymal weight and sperm motility. The increase in testicular AMH expression was also correlated with elevated apoptosis (terminal transferase-mediated deoxyuridine 5-triphosphate nick-end labeling) and reduced proliferation (Ki67, proliferating cell nuclear antigen; all seminiferous tubules cells were analyzed). Severely damaged mice testes demonstrated a marked costaining of AMH and GATA-4, a Sertoli cell marker; staining that resembled the pattern of the Sertoli cell-only condition. Our study indicates that the pattern of serum AMH expression, in combination with other hormones, can delineate testicular damage, as determined in both experimental settings. Future large-scale clinical studies are warranted to further define the role of AMH as a biomarker for testicular toxicity.
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http://dx.doi.org/10.1210/en.2015-1310DOI Listing
October 2015

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

FASEB J 2015 Aug 28;29(8):3206-16. Epub 2015 Apr 28.

Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel Aviv University, Ramat-Aviv, Tel Aviv, Israel

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.
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http://dx.doi.org/10.1096/fj.14-269449DOI Listing
August 2015

Gonadotrophin-releasing hormone agonists for fertility preservation: unraveling the enigma?

Hum Reprod 2015 May 21;30(5):1089-101. Epub 2015 Mar 21.

Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel.

Study Question: Can gonadotrophin-releasing hormone agonists (GnRH-a) preserve long-term fertility when administered prior to and concomitantly with chemotherapy?

Summary Answer: GnRH-a display a differential protective effect on fertility, depending upon the specific chemotherapy-induced mechanism of ovarian injury.

What Is Known Already: The role of GnRH-a in fertility preservation has been constantly debated and their use is considered experimental due to conflicting clinical evidence and paucity of data regarding their mechanism for ovarian protection.

Study Design, Size, Duration: In vivo model: 7-8 weeks old imprinting control region (ICR) mice were injected with GnRH-a (Leuprolide-acetate) or saline prior to and concomitantly with cyclophosphamide, doxorubicin or saline and sacrificed at various time-points on a longitudinal follow-up; 24 h (n = 36), 1 week (n = 40), 1 month (n = 36) and 9 months (n = 66) post chemotherapy treatment. Blood samples were drawn on Day 0 and on a monthly basis after chemotherapy treatment. On the day of sacrifice, blood samples were drawn and ovaries excised and processed for either immunohistochemistry (IHC), protein or RNA extraction. In vitro model: 21-23 days old Wistar-derived rats were sacrificed, their ovaries excised and primary granulosa cells (PGC) were either isolated for in vitro culture, or processed for immunofluorescence (IF) as well as for protein or RNA extraction.

Materials, Setting, Methods: Ovarian reserve was estimated by serial measurements of serum anti-mullerian hormone (AMH), quantified by the AMH Gen II ELISA assay. Ovarian AMH and phosphorylated Akt (pAkt) were detected by immunoblotting. Vascular endothelial growth factor (VEGF) was measured by quantitative PCR. Ovarian GnRH receptor (GnRHR), AMH and CD34 were visualized by IHC, and apoptosis was evaluated using TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labeling (TUNEL).

Main Results And The Role Of Chance: Cyclophosphamide-induced ovarian injury caused a prompt decrease in AMH level (P < 0.01) and a further long-term decline in serum AMH (P = 0.017), indicating damage to the ovarian reserve. Pretreatment with GnRH-a diminished AMH-decrease (P < 0.05) and maintained serum AMH level in the long run (P < 0.05). Doxorubicin-exerted ovarian-vascular-injury is also displayed by an acute increase in ovarian VEGF level (P < 0.05) and a sustained decrease in serum AMH level (P < 0.001). This was followed by ovarian recovery manifested by increased neovascularization. GnRH-a delayed the recovery in AMH level and decreased the level of VEGF (P < 0.001), thus interfering with the vascular recovery subsequent to doxorubicin-induced vascular damage.

Limitations, Reasons For Caution: To portray the differential mechanism of each chemotherapy, cyclophosphamide and doxorubicin were given separately, whereas most of the clinical protocols include several types of chemotherapies. Thus, future study should explore a prospective evaluation of various chemotherapies, as well as combined chemotherapeutic protocols.

Wider Implications Of The Findings: Our study demonstrates that different chemotherapy agents affect the ovary via diverse mechanisms and thus the administration of GnRH-a concomitantly, could be beneficial to a subpopulation of patients treated with cyclophosphamide-based protocols.

Study Funding/competing Interests: This work was partially supported by a grant from the Israel Science Foundation (ISF) to I.B.-A. The authors have no conflict of interest to disclose.
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http://dx.doi.org/10.1093/humrep/dev037DOI Listing
May 2015

miR-125a--does the difference lie in the isoform?

Cell Cycle 2015 ;14(6):785-6

a Department of Cell and Developmental Biology ; Sackler Faculty of Medicine ; Tel-Aviv University ; Tel-Aviv , Israel.

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http://dx.doi.org/10.1080/15384101.2015.1010961DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4614516PMC
May 2015

MicroRNA miR-125a-3p modulates molecular pathway of motility and migration in prostate cancer cells.

Oncoscience 2014 30;1(4):250-261. Epub 2014 Apr 30.

Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel-Aviv University, Israel.

Fyn kinase is implicated in prostate cancer. We illustrate the role of miR-125a-3p in cellular pathways accounted for motility and migration of prostate cancer cells, probably through its regulation on Fyn expression and Fyn-downstream proteins. Prostate cancer PC3 cells were transiently transfected with empty miR-Vec (control) or with miR-125a-3p. Overexpression of miR-125a-3p reduced migration of PC3 cells and increased apoptosis. Live cell confocal imaging indicated that overexpression of miR-125a-3p reduced the cells' track speed and length and impaired phenotype. Fyn, FAK and paxillin, displayed reduced activity following miR-125a-3p overexpression. Accordingly, actin rearrangement and cells' protrusion formation were impaired. An inverse correlation between miR-125a-3p and Gleason score was observed in human prostate cancer tissues. Our study demonstrated that miR-125a-3p may regulate migration of prostate cancer cells.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4278297PMC
http://dx.doi.org/10.18632/oncoscience.30DOI Listing
January 2015

Chemotherapy-induced vascular toxicity--real-time in vivo imaging of vessel impairment.

J Vis Exp 2015 Jan 7(95):e51650. Epub 2015 Jan 7.

Sackler Faculty of Medicine, Tel Aviv University; Institute of Oncology, Davidoff Center and Rabin Medical Center;

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an increased risk of vascular morbidity. Yet, albeit the mounting clinical evidence, there is a paucity of clear studies of vascular toxicity and therefore the etiology of a heterogeneous group of vascular/cardiovascular disorders remains to be elucidated. Moreover, the mechanism that may underlie vascular toxicity can completely differ from the principles of chemotherapy-induced cardiotoxicity, which is related to direct myocyte injury. We have established a real-time, in vivo molecular imaging platform to evaluate the potential acute vascular toxicity of anti-cancer therapies. We have set up a platform of in vivo, high-resolution molecular imaging in mice, suitable for visualizing vasculature within confined organs and reference blood vessels within the same individuals whereas each individual serve as its own control. Blood vessel walls were impaired after doxorubicin administration, representing a unique mechanism of vascular toxicity that may be the early event in end-organ injury. Herein, the method of fibered confocal fluorescent microscopy (FCFM) based imaging is described, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.
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http://dx.doi.org/10.3791/51650DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4354501PMC
January 2015

Excess cholesterol induces mouse egg activation and may cause female infertility.

Proc Natl Acad Sci U S A 2014 Nov 3;111(46):E4972-80. Epub 2014 Nov 3.

Departments of Biology and

The HDL receptor scavenger receptor, class B type I (SR-BI) controls the structure and fate of plasma HDL. Female SR-BI KO mice are infertile, apparently because of their abnormal cholesterol-enriched HDL particles. We examined the growth and meiotic progression of SR-BI KO oocytes and found that they underwent normal germinal vesicle breakdown; however, SR-BI KO eggs, which had accumulated excess cholesterol in vivo, spontaneously activated, and they escaped metaphase II (MII) arrest and progressed to pronuclear, MIII, and anaphase/telophase III stages. Eggs from fertile WT mice were activated when loaded in vitro with excess cholesterol by a cholesterol/methyl-β-cyclodextrin complex, phenocopying SR-BI KO oocytes. In vitro cholesterol loading of eggs induced reduction in maturation promoting factor and MAPK activities, elevation of intracellular calcium, extrusion of a second polar body, and progression to meiotic stages beyond MII. These results suggest that the infertility of SR-BI KO females is caused, at least in part, by excess cholesterol in eggs inducing premature activation and that cholesterol can activate WT mouse eggs to escape from MII arrest. Analysis of SR-BI KO female infertility raises the possibility that abnormalities in cholesterol metabolism might underlie some cases of human female infertility of unknown etiology.
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http://dx.doi.org/10.1073/pnas.1418954111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4246315PMC
November 2014

Interleukin-1 deficiency prolongs ovarian lifespan in mice.

Proc Natl Acad Sci U S A 2014 Aug 11;111(34):12492-7. Epub 2014 Aug 11.

The Bert W. Strassburger Lipid Center, Sheba Medical Center, Tel-Hashomer 52621, Israel;

Oocyte endowment dwindles away during prepubertal and adult life until menopause occurs, and apoptosis has been identified as a central mechanism responsible for oocyte elimination. A few recent reports suggest that uncontrolled inflammation may adversely affect ovarian reserve. We tested the possible role of the proinflammatory cytokine IL-1 in the age-related exhaustion of ovarian reserve using IL-1α and IL-1β-KO mice. IL-1α-KO mice showed a substantially higher pregnancy rate and litter size compared with WT mice at advanced age. The number of secondary and antral follicles was significantly higher in 2.5-mo-old IL-1α-KO ovaries compared with WT ovaries. Serum anti-Müllerian hormone, a putative marker of ovarian reserve, was markedly higher in IL-1α-KO mice from 2.5 mo onward, along with a greater ovarian response to gonadotropins. IL-1β-KO mice displayed a comparable but more subtle prolongation of ovarian lifespan compared with IL-1α-KO mice. The protein and mRNA of both IL-1α and IL-1β mice were localized within the developing follicles (oocytes and granulosa cells), and their ovarian mRNA levels increased with age. Molecular analysis revealed decreased apoptotic signaling [higher B-cell lymphoma 2 (BCL-2) and lower BCL-2-associated X protein levels], along with a marked attenuation in the expression of genes coding for the proinflammatory cytokines IL-1β, IL-6, and TNF-α in ovaries of IL-1α-KO mice compared with WT mice. Taken together, IL-1 emerges as an important participant in the age-related exhaustion of ovarian reserve in mice, possibly by enhancing the expression of inflammatory genes and promoting apoptotic pathways.
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http://dx.doi.org/10.1073/pnas.1323955111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4151745PMC
August 2014

Pigment epithelium-derived factor exerts antioxidative effects in granulosa cells.

Fertil Steril 2014 Sep 23;102(3):891-898.e3. Epub 2014 Jul 23.

Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel-Aviv University, Ramat-Aviv, Tel Aviv, Israel. Electronic address:

Objective: To determine whether supplementing granulosa cells cultures with pigment epithelium-derived factor (PEDF) can protect them from oxidative stress.

Design: We used either granulosa cell line or human primary granulosa cell culture from women undergoing in vitro fertilization (IVF) treatments.

Setting: University research facilities.

Animal(s): Imprinting control region female mice.

Intervention(s): Recombinant PEDF (rPEDF) was added to cultures of either primary granulosa cell culture or granulosa cell line in the present or absence of H2O2 triggering.

Main Outcome Measure(s): We followed cell viability with the use of methylthiazolyl tetrazolium assay and tracked PEDF mechanism of action with the use of Western blot analysis, measuring the level of SOD-1 and GPX-1 mRNA, protein level of BAX, and phosphorylation of AKT.

Result(s): We found that granulosa cell viability and the level of PEDF mRNA were both significantly reduced, in a dose-dependent manner, after exposure to H2O2. The rate of H2O2-induced apoptosis was significantly attenuated in granulosa cells treated with rPEDF. We showed that granulosa cells, of both humans and rodents, express the PEDF receptor, PNPLA2; once stimulated by rPEDF, the cells exhibited phosphorylation of AKT. Finally, we showed that PEDF exerts its antioxidative activity through the AKT signaling pathway.

Conclusion(s): This study demonstrates that PEDF represents a novel intrinsic antioxidant of granulosa cells.
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http://dx.doi.org/10.1016/j.fertnstert.2014.06.012DOI Listing
September 2014