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Figure 1: schematic representation of experimental design Figure 2: Histological manifestation of treated uteri at the short-term setting Figure 3: uterine characteristics following "rPEDF prevention" regimen

Fertilization triggers physiological degradation of maternal-mRNAs, which are then replaced by embryonic transcripts. Ample evidence suggests that Argonaut 2 (AGO2) is a possible post-fertilization regulator of maternal-mRNAs degradation; but its role in degradation of maternal-mRNAs during oocyte maturation remains obscure. Fyn, a member of the Src family kinases (SFKs), and an essential factor in oocyte maturation, was reported to inhibit AGO2 activity in oligodendrocytes. Our aim was to examine the role of Fyn and AGO2 in degradation of maternal-mRNAs during oocyte maturation by either suppressing their activity with SU6656 – an SFKs inhibitor; or by microinjecting DN-Fyn RNA for suppression of Fyn and BCl-137 for suppression of AGO2. Batches of fifteen mouse oocytes or embryos were analyzed by qPCR to measure the expression level of nine maternal-mRNAs that were selected for their known role in oocyte growth, maturation and early embryogenesis. We found that Fyn/SFKs are involved in maintaining the stability of at least four pre-transcribed mRNAs in oocytes at the germinal vesicle (GV) stage, whereas AGO2 had no role at this stage. During in-vivo oocyte maturation, eight maternal-mRNAs were significantly degraded. Inhibition of AGO2 prevented the degreadation of at least five maternal-mRNAs, whereas inhibition of Fyn/SFK prevented degradation of at least five Fyn maternal-mRNAs and two SFKs maternal-mRNAs; pointing at their role in promoting the physiological degradation which occurs during in-vivo oocyte maturation. Our findings imply the involvement of Fyn/SFKs in stabilization of maternal-mRNA at the GV stage and the involvement of Fyn, SFKs and AGO2 in degradation of maternal mRNAs during oocyte maturation.

Background: Testicular toxicity following chemotherapy is of increasing importance with the continuous improvement of survival rates. Gonadotropin-releasing hormone (GnRH) was suggested to protect testis against such toxicity; however, its suppressive quality and mechanism of action are still unclear. We examined whether and how pretreatment with GnRH antagonist protects against the testicular damage caused by chemotherapy. Methods: Mature male mice were injected subcutaneously eight times in 2-day intervals with either saline or GnRH antagonist (Cetrotide; 1 g/mg), followed by an intraperitoneal injection with either saline or cyclophosphamide (CTX;100 mg/kg BW) and sacrificed 2 weeks or 3 months later. Testicular weight, epididymis weight, epididymal sperm count and sperm motility were measured. Serum anti-Müllerian hormone (AMH) was measured by enzyme-linked immunosorbent assay. Immunohistochemistry (Ki-67), immunofluorescence (PCNA, CD34), terminal transferase-mediated deoxyuridine 5-triphosphate nick-end labeling (TUNEL) and computerized analysis were performed to examine testicular proliferation, apoptosis and vascularization. Quantitative real-time PCR was used to assess the amount of spermatogonial reserve (Id4 and Gfra1 mRNAs). Results: Pretreatment with GnRH antagonist transiently reduced testicular weight, epididymal weight, germinal proliferation and sperm count; it also abolished the permanent long-term effect of CTX on these parameters and prevented cyclophosphamide-induced testicular toxicity characterized by apoptosis and serum AMH increase and irreversible loss of spermatogonial reserve. Conclusions: Our findings imply that pretreatment with GnRH antagonist temporarily reduces spermatogenesis and may be used as pretreatment for reducing chemotherapeutic testicular toxicity.

Summary Ultraviolet (UV) light affects endocrinological and behavioral aspects of sexuality via an unknown mechanism. Here we discover that ultraviolet B (UVB) exposure enhances the levels of sex-steroid hormones and sexual behavior, which are mediated by the skin. In female mice, UVB exposure increases hypothalamus-pituitary-gonadal axis hormone levels, resulting in larger ovaries; extends estrus days; and increases anti-Mullerian hormone (AMH) expression. UVB exposure also enhances the sexual responsiveness and attractiveness of females and male-female interactions. Conditional knockout of p53 specifically in skin keratinocytes abolishes the effects of UVB. Thus, UVB triggers a skin-brain-gonadal axis through skin p53 activation. In humans, solar exposure enhances romantic passion in both genders and aggressiveness in men, as seen in analysis of individual questionaries, and positively correlates with testosterone level. Our findings suggest opportunities for treatment of sex-steroid-related dysfunctions., Graphical abstract, Highlights • UVB exposure increases circulating sex-steroid levels in mice and humans • UVB exposure enhances female attractiveness and receptiveness toward males • UVB exposure increases females’ estrus phase, HPG axis hormones, and follicle growth • Skin p53 regulates UVB-induced sexual behavior and ovarian physiological changes, Parikh et al. find that UVB exposure triggers a skin-brain-gonadal axis through skin p53 activation. UVB exposure increases in female mice sexual responsiveness and attractiveness, hypothalamus-pituitary-gonadal axis hormone levels, ovary size, and estrus duration, as well as male-female interactions. Solar exposure in humans enhances romantic passion and positively correlates with male testosterone levels.

Molecular changes, caused by various environmental factors, affect the quality and developmental potential of oocytes. Oxidative stress (OS) is a major factor involved in various gynecologic disorders and/or in aging. Recent studies suggest that elevated reactive oxygen species (ROS) hamper oocyte quality and future embryonic development. Pigment epithelium-derived factor (PEDF) is a pleiotropic protein, known for its antiangiogenic, anti-inflammatory, and antioxidative properties. Our previous findings demonstrate the antioxidative role of rPEDF in maintaining granulosa cell viability. In the current study, we examined the ability of PEDF to negate the adverse impact of OS on oocytes. Maturation rate of oocytes exposed to OS was significantly lower than that of control oocytes. The number of mtDNA copies in OS-exposed oocytes was significantly higher than in control oocytes (>3 times), whereas ATP concentration was significantly lower. Oocytes exposed to OS demonstrated impaired chromosome arrangement at the metaphase plate. PEDF significantly improved maturation rate of untreated OS-exposed oocytes. Moreover, mtDNA copy number, ATP concentration, and chromosome arrangement at the metaphase plate in rPEDF-treated OS-exposed oocytes were restored to the level of control oocytes. Our findings demonstrate that OS hampers the ability of oocytes to undergo proper in vitro maturation. The energetic balance of OS-exposed oocyte is characterized by excessive mtDNA replication and reduced ATP concentration; it hampers the ability of oocytes to perform high fidelity chromosome segregation. PEDF alleviates this damage, improves the rate of oocyte maturation, and preserves mtDNA level and ATP content, thus enabling oocytes to form proper metaphase plate and improve oocyte competence.

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-M&uuml, llerian 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 (p &lt, 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.

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&rsquo, 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&ndash, 35). The immunohistochemistry of the EPI-treated human placentae showed enhanced proliferation and apoptosis as compared with matched chemo-na&iuml, ve 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.

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.

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.

Ultraviolet (UV) light affects endocrinological and behavioral aspects of human sexuality via an unknown mechanism. Using a unique male-female comparative approach, we discovered that the sexual behavioral and hormonal features enhanced by UVB are mediated by the skin. In mice, UV exposure increases hypothalamus-pituitary-gonadal axis hormone levels, resulting in enhanced ovary size, extended estrus days, and anti-Mullerian hormone (AMH) expression. It likewise enhances sexual responsiveness and attractiveness of females and heterosexual interactions of both males and females. Conditional knockout of p53 specifically in skin keratinocytes abolished UV’s effects. In humans, UV exposure enhanced romantic passion in both genders, increased testosterone levels in men and correlated with heightened plasma AMH levels in women. Our data, revealing that UVB triggers a skin-brain-gonadal axis through skin p53 activation, offers therapeutic opportunities for sex-steroid-related dysfunctions. We speculate that during human furless skin evolution, the skin became the front-line regulator of the response to UVB.

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.

// Mattan Levi 1 , Salomon M. Stemmer 2, 3 , Jerry Stein 2, 3 , Ruth Shalgi 1 and Irit Ben-Aharon 2, 3 1 Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel 2 Institute of Oncology, Davidoff Center, Rabin Medical Center, Beilinson Campus, Petah-Tiqva, Israel 3 Sackler Faculty of Medicine, Tel-Aviv University, Tel Aviv, Israel Correspondence to: Irit Ben-Aharon, email: iritbenaharon@gmail.com Keywords: busulfan; treosulfan; gonadal toxicity Received: December 01, 2017 Accepted: March 19, 2018 Published: April 10, 2018 ABSTRACT 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.

// Irit Ben-Aharon 1, 5, * , Mattan Levi 2, * , David Margel 1, 5 , Rinat Yerushalmi 1, 5 , Shulamith Rizel 1 , Shlomit Perry 1, 5 , Eran Sharon 3, 5 , Noa Hasky 2 , Ronit Abir 4, 5 , Benny Fisch 4, 5 , Ana Tobar 6 , Ruth Shalgi 2 and Salomon Marcello Stemmer 1, 5 1 Institute of Oncology, Davidoff Center, Rabin Medical Center, Beilinson Campus, Petach Tikva, Israel 2 Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel-Aviv University, Ramat Aviv, Tel-Aviv, Israel 3 Department of Surgery, Rabin Medical Center, Beilinson Campus, Petach Tikva, Israel 4 IVF and Infertility Unit, Schneider Women Hospital, Rabin Medical Center, Beilinson Campus, Petach Tikva, Israel 5 Sackler Faculty of Medicine, Tel-Aviv University, Ramat Aviv, Tel-Aviv, Israel 6 Department of Pathology, Rabin Medical Center, Petach Tikva, Israel * These authors have contributed equally to this work Correspondence to: Irit Ben-Aharon, email: iritbenaharon@gmail.com Keywords: BRCA; ovarian aging; systemic precocious aging Received: December 01, 2017 Accepted: February 27, 2018 Published: March 23, 2018 ABSTRACT Purpose: Though former evidence implies a correlation of breast cancer susceptibility gene ( BRCA ) 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 BRCA mutation. We hypothesized that the role played by BRCA genes in aging pathways is not exclusive to the ovary. Experimental Design: Healthy female BRCA carriers, 40 years or younger and healthy male BRCA 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 BRCA 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) BRCA carriers were enrolled into the study and matched to control non-carriers (34y and 43y, respectively). Serum AMH level was significantly lower in BRCA 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 BRCA mutation, accelerated ovarian aging and systemic aging-related pathophysiology.

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.

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.

miR-125a-3p, a post-transcription regulator of Fyn kinase, is expressed in mouse pre-ovulatory follicles; its expression within the follicle decreases toward 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 overnight 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 toward ovulation.

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.

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.

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.The objective of the study was to explore whether GnRH-a triggering could directly modulate PEDF/VEGF balance in granulosa cells.The design of the study was a mouse model and cultured granulosa cells.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.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.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.

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.

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.

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.

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.

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.

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).Our objective was to examine a physiological approach for preventing and treating OHSS, based on negating the VEGF network.We used a mouse OHSS model and cultured granulosa cells.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.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.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.

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.

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.

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.

Autoproteolytic cleavage of the inactive acid ceramidase (AC) precursor into the active heterodimer exposes a free cysteine residue, leading us to study whether AC could be regulated by one or more members of the cystatin family. Co-expression of the full-length AC and cystatin SA (cysSA) cDNAs led to significant reduction of AC activity in the transfected cells. Expression of cysSA also inhibited endogenous AC activity in cells and increased ceramide. Conversely, cysSA siRNA expression led to elevated AC activity and reduction in ceramide. The effects of cysSA siRNA expression could be reversed by the addition of recombinant cysSA into the culture media. These results were consistent with detection of a physical interaction between AC and cysSA, assessed by co-immunoprecipitation and nickel-nitrilotriacetic acid affinity chromatography, and further supported by co-localization of the endogenous proteins using confocal microscopy. In vitro kinetic analysis of purified, recombinant AC and cysSA confirmed the transfection results and suggested a non-competitive type of inhibition with a K(i) in the low micromolar range. Processing of the AC precursor into the active form was not affected by cysSA expression, suggesting that it likely inhibits AC by allosteric interference. Computer modeling and expression studies identified several potential inhibitory domains in cysSA, including a small "AC-like" domain (identical to the AC cleavage site, TICT). Small peptides, synthesized with combinations of this and a "cystatin-like" domain (QXVXG), exhibited significant AC inhibition as well. Such peptide-based AC inhibitors could potentially be used to regulate AC activity in cancer cells that are known to overexpress this enzyme alone and in combination with conventional anti-cancer drugs.

Ovarian failure is a-known side-effect observed in women treated for cancer. Doxorubicin (DXR) was found to be detrimental to MII mouse oocytes. We aimed at characterizing the effect of DXR on germinal vesicle (GV) oocytes that comprise the majority of oocytes within ovaries encountering chemotherapy. Mouse follicles and oocytes were exposed to DXR in vitro. DXR localization and its possible cellular targets were examined by fluorescence confocal microscopy. We demonstrated that DXR crosses the blood-follicle barrier and accumulates in oocytes and granulosa cells. The mechanism of DXR-induced apoptosis involves chromosomal disintegration, activation of the mitochondria followed by activation of PERK and caspase-12 and inactivation of PARP. The follicular GV oocytes were more vulnerable to the toxic effect of DXR than ovulated MII oocytes. We suggest that DXR elicits apoptotic signal within GV oocytes that involves activation of the mitochondria, induction of ER-stress and a possible increase in intracellular calcium.

e13097Background: Several studies have addressed gestational and neonatal outcomes of chemotherapy during pregnancy (CDP) and demonstrated increased complications as pre-eclampsia and intrauterine ...

Meiosis in mammalian oocytes starts during embryonic life and arrests for the first time before birth, at prophase of the first meiotic division. The second meiotic arrest occurs after spindle formation at metaphase of the second meiotic division (MII) in selected oocytes designated for ovulation. The fertilizing spermatozoon induces the release from MII arrest only after the oocyte's spindle assembly checkpoint (SAC) was deactivated. Src family kinases (SFKs) are nine non-receptor protein tyrosine kinases that regulate many key cellular functions. Fyn is an SFK expressed in many cell types, including oocytes. Recent studies, including ours, imply a role for Fyn in exit from meiotic and mitotic metaphases. Other studies demonstrate that SFKs, particularly Fyn, are required for regulation of microtubules polymerization and spindle stabilization. Altogether, Fyn is suggested to play an essential role in signaling events that implicate SAC pathway and hence in regulating the exit from metaphase in oocytes and zygote.

We report that full-length and truncated transcripts of Fyn tyrosine protein kinase are expressed during testicular development. Truncated Fyn (tr-Fyn) transcripts encode a 24 kDa protein with a N-terminal (NT) domain, a complete Src homology (SH) 3 domain and an incomplete SH2 domain. The kinase domain is missing in tr-Fyn. In contrast, full-length Fyn transcripts encode a 59-55 kDa protein. Fractionated spermatids by centrifugal elutriation express tr-Fyn transcripts and protein, but not full-length Fyn transcripts and protein. Neither full-length Fyn nor tr-Fyn transcripts and encoded proteins are detected in elutriated pachytene spermatocytes. Sertoli cells express full-length and truncated Fyn throughout testicular development. In contrast, sperm contain full-length Fyn transcripts and protein but not the truncated form. tr-Fyn protein is visualized at the cytosolic side of Golgi membranes, derived proacrosomal vesicles, along the outer acrosome membrane and the inner acrosome membrane-acroplaxome complex anchoring the acrosome to the spermatid nuclear envelope. Fyn and phosphotyrosine immunoreactivity coexist in the tail of capacitated sperm. During fertilization, the Fyn-containing acroplaxome seen in the egg-bound and egg-fused sperm is no longer detected upon decondensation of the sperm nucleus. tr-Fyn expands the catalog of truncated tyrosine protein kinases expressed during spermiogenesis. We suggest that the NT and SH3 domains of tr-Fyn may recruit adaptor and effector proteins, in particular GTPase activating proteins, required for acrosome-acroplaxome biogenesis, acroplaxome F-actin dynamics and Sertoli cell function. During fertilization, full-length Fyn in the acroplaxome may contribute to a transient local signaling burst during the early events of sperm-egg interaction.

Herein we report the mechanism of human acid ceramidase (AC; N-acylsphingosine deacylase) cleavage and activation. A highly purified, recombinant human AC precursor underwent self-cleavage into α and β subunits, similar to other members of the N-terminal nucleophile hydrolase superfamily. This reaction proceeded with first order kinetics, characteristic of self-cleavage. AC self-cleavage occurred most rapidly at acidic pH, but also at neutral pH. Site-directed mutagenesis and expression studies demonstrated that Cys-143 was an essential nucleophile that was required at the cleavage site. Other amino acids participating in AC cleavage included Arg-159 and Asp-162. Mutations at these three amino acids prevented AC cleavage and activity, the latter assessed using BODIPY-conjugated ceramide. We propose the following mechanism for AC self-cleavage and activation. Asp-162 likely forms a hydrogen bond with Cys-143, initiating a conformational change that allows Arg-159 to act as a proton acceptor. This, in turn, facilitates an intermediate thioether bond between Cys-143 and Ile-142, the site of AC cleavage. Hydrolysis of this bond is catalyzed by water. Treatment of recombinant AC with the cysteine protease inhibitor, methyl methanethiosulfonate, inhibited both cleavage and enzymatic activity, further indicating that cysteine-mediated self-cleavage is required for ceramide hydrolysis.

Ovulated mammalian eggs remain arrested at the second meiotic metaphase (MII) until fertilization. The fertilizing spermatozoon initiates a sequence of biochemical events, collectively referred to as 'egg activation', which overcome this arrest. The initial observable change within the activated egg is a transient rise in intracellular Ca2+ concentration ([Ca2+]i) followed by cortical granule exocytosis (CGE) and resumption of the second meiotic division (RMII). To date, the mechanism by which the fertilizing spermatozoon activates the signaling pathways upstream to the Ca2+ release and the manner by which the signals downstream to Ca2+ release evoke RMII are not well documented. Protein tyrosine kinases (PTKs) were suggested as possible inducers of some aspects of egg activation. Src family kinases (SFKs) constitute a large family of evolutionarily conserved PTKs that mediate crucial biological functions. At present, the theory that one or more SFKs are necessary and sufficient for Ca2+ regulation at fertilization is documented in eggs of marine invertebrates. The mechanism leading to Ca2+ release during fertilization is less established in mammalian eggs. A controversy still exists as to whether SFKs within the mammalian egg are sufficient and/or necessary for Ca2+ release, or whether they play a role during egg activation via other signaling pathways. This article summarizes the possible signaling pathways involved upstream to Ca2+ release but focuses mainly on the involvement of SFKs downstream to Ca2+ release toward RMII, in invertebrate and vertebrate eggs.

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.

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 (