Persistent organic pollutants dysregulate energy homeostasis in human ovaries in vitro.

Exposure to persistent organic pollutants (POPs), such as dichlorodiphenyltrichloroethane (DDT) and polychlorinated biphenyls (PCBs), has historically been linked to population collapses in wildlife. Despite international regulations, these legacy chemicals are still currently detected in women of reproductive age, and their levels correlate with reduced ovarian reserve, longer time-to-pregnancy, and higher risk of infertility. However, the specific modes of action underlying these associations remain unclear. Here, we examined the effects of five commonly occurring POPs - hexachlorobenzene (HCB), p,p'-dichlorodiphenyldichloroethylene (DDE), 2,3,3',4,4',5-hexachlorobiphenyl (PCB156), 2,2',3,4,4',5,5'-heptachlorobiphenyl (PCB180), perfluorooctane sulfonate (PFOS) - and their mixture on human ovaries in vitro. We exposed human ovarian cancer cell lines COV434, KGN, and PA1 as well as primary ovarian cells for 24 h, and ovarian tissue containing unilaminar follicles for 6 days. RNA-sequencing of samples exposed to concentrations covering epidemiologically relevant levels revealed significant gene expression changes related to central energy metabolism in the exposed cells, indicating glycolysis, oxidative phosphorylation, fatty acid metabolism, and reactive oxygen species as potential shared targets of POP exposures in ovarian cells. Alpha-enolase (ENO1), lactate dehydrogenase A (LDHA), cytochrome C oxidase subunit 4I1 (COX4I1), ATP synthase F1 subunit alpha (ATP5A), and glutathione peroxidase 4 (GPX4) were validated as targets through qPCR in additional cell culture experiments in KGN. In ovarian tissue cultures, we observed significant effects of exposure on follicle growth and atresia as well as protein expression. All POP exposures, except PCB180, decreased unilaminar follicle proportion and increased follicle atresia. Immunostaining confirmed altered expression of LDHA, ATP5A, and GPX4 in the exposed tissues. Moreover, POP exposures modified ATP production in KGN and tissue culture. In conclusion, our results demonstrate the disruption of cellular energy metabolism as a novel mode of action underlying POP-mediated interference of follicle growth in human ovaries.

Inhibition of epigenetic and cell cycle-related targets in glioblastoma cell lines reveals that onametostat reduces proliferation and viability in both normoxic and hypoxic conditions.

The choice of targeted therapies for treatment of glioblastoma patients is currently limited, and most glioblastoma patients die from the disease recurrence. Thus, systematic studies in simplified model systems are required to pinpoint the choice of targets for further exploration in clinical settings. Here, we report screening of 5 compounds targeting epigenetic writers or erasers and 6 compounds targeting cell cycle-regulating protein kinases against 3 glioblastoma cell lines following incubation under normoxic or hypoxic conditions. The viability/proliferation assay indicated that PRMT5 inhibitor onametostat was endowed with high potency under both normoxic and hypoxic conditions in cell lines that are strongly MGMT-positive (T98-G), weakly MGMT-positive (U-251 MG), or MGMT-negative (U-87 MG). In U-251 MG and U-87 MG cells, onametostat also affected the spheroid formation at concentrations lower than the currently used chemotherapeutic drug lomustine. In T98-G cell line, treatment with onametostat led to dramatic changes in the transcriptome profile by inducing the cell cycle arrest, suppressing RNA splicing, and down-regulating several major glioblastoma cell survival pathways. Further validation by immunostaining in three cell lines confirmed that onametostat affects cell cycle and causes reduction in nucleolar protein levels. In this way, inhibition of epigenetic targets might represent a viable strategy for glioblastoma treatment even in the case of decreased chemo- and radiation sensitivity, although further studies in clinically more relevant models are required.

Sperm plasma membrane ion transporters and male fertility potential: A perspective under the prism of cryopreservation.

Intracellular calcium homeostasis plays a crucial role in spermatozoa by regulating physiological functions associated with sperm quality and male fertility potential. Intracellular calcium fine balance in the sperm cytoplasm is strictly dependent on sperm surface channels including the CatSper channel. CatSpers' role is to ensure the influx of extracellular calcium, while intracellular pH alkalinization serves as a stimulus for the activation of several channels, including CatSper. Overall, the generation of intracellular calcium spikes through CatSper is essential for fertilization-related processes, such as sperm hyperactivation, acrosome reaction, egg chemotaxis, and zona pellucida penetration. Multiple lines of evidence suggest that disruption in the close interaction among ions, pH, and CatSper could impair male fertility potential. In contemporary times, the growing reliance on Medically Assisted Reproduction procedures underscores the impact of cryopreservation on gametes. In fact, a large body of literature raises concerns about the cryo-damages provoked by the freeze-thawing processes, that can affect the plasma membrane integrity, thus the structure of pivotal ion channels, and the fine regulation of both intracellular calcium and pH. This review aims to provide an overview of the importance of the CatSper channel in sperm quality and further fertilization potential. Additionally, it addresses the emerging issue of cryopreservation's impact on the functionality of this sperm channel.

Repeated Rounds of Gonadotropin Stimulation Induce Imbalance in the Antioxidant Machinery and Activation of Pro-Survival Proteins in Mouse Oviducts.

Controlled ovarian stimulation (COS) through gonadotropin administration has become a common procedure in assisted reproductive technologies. COS's drawback is the formation of an unbalanced hormonal and molecular environment that could alter several cellular mechanisms. On this basis, we detected the presence of mitochondrial DNA (mtDNA) fragmentation, antioxidant enzymes (catalase; superoxide dismutases 1 and 2, SOD-1 and -2; glutathione peroxidase 1, GPx1) and apoptotic (Bcl-2-associated X protein, Bax; cleaved caspases 3 and 7; phosphorylated (p)-heat shock protein 27, p-HSP27) and cell-cycle-related proteins (p-p38 mitogen-activated protein kinase, p-p38 MAPK; p-MAPK activated protein kinase 2, p-MAPKAPK2; p-stress-activated protein kinase/Jun amino-terminal kinase, p-SAPK/JNK; p-c-Jun) in the oviducts of unstimulated (Ctr) and repeatedly hyperstimulated (eight rounds, 8R) mice. While all the antioxidant enzymes were overexpressed after 8R of stimulation, mtDNA fragmentation decreased in the 8R group, denoting a present yet controlled imbalance in the antioxidant machinery. Apoptotic proteins were not overexpressed, except for a sharp increase in the inflammatory-related cleaved caspase 7, accompanied by a significant decrease in p-HSP27 content. On the other hand, the number of proteins involved in pro-survival mechanisms, such as p-p38 MAPK, p-SAPK/JNK and p-c-Jun, increased almost 50% in the 8R group. Altogether, the present results demonstrate that repeated stimulations cause the activation of the antioxidant machinery in mouse oviducts; however, this is not sufficient to induce apoptosis, and is efficiently counterbalanced by activation of pro-survival proteins.

Quantitative analysis of ovarian surface photographs as a tool for assessment of chemical effects on folliculogenesis and ovulation in rats.

Female reproductive toxicity assessments rely on histological evaluation of ovaries by hematoxylin & eosin (H&E)-stained cross-sections. This is time-consuming, labor-intensive and costly, thus alternative methods for ovarian toxicity assessment could be valuable. Here, we report on an improved method based on quantification of antral follicles (AF) and corpora lutea (CL) using ovarian surface photographs, called 'surface photo counting' (SPC). To validate a potential utility for the method to detect effects on folliculogenesis in toxicity studies, we investigated ovaries from rats exposed to two well-known endocrine disrupting chemicals (EDCs), diethylstilbestrol (DES) and ketoconazole (KTZ). Animals were exposed to DES (0.003, 0.012, 0.048 mg/kg body weight (bw)/day) or KTZ (3, 12, 48 mg/kg bw/day) during puberty or adulthood. At the end of the exposure, ovaries were photographed under stereomicroscope and subsequently processed for histological assessments to allow for direct comparison between the two methods by quantifying AF and CL. There was a significant correlation between the SPC and histology methods, albeit CL counts correlated better than AF counts, potentially due to their larger size. Effects of DES and KTZ were found by both methods, suggesting applicability of the SPC method to chemical hazard and risk assessment. Based on our study, we propose that SPC can be employed as a fast and cheap tool for assessment of ovarian toxicity in in vivo studies to prioritize chemical exposure groups for further histological assessment.

A comprehensive review and update on human fertility cryopreservation methods and tools.

The broad conceptualization of fertility preservation and restoration has become already a major concern in the modern western world since a large number of individuals often face it in the everyday life. Driven by different health conditions and/or social reasons, a variety of patients currently rely on routinely and non-routinely applied assisted reproductive technologies, and mostly on the possibility to cryopreserve gametes and/or gonadal tissues for expanding their reproductive lifespan. This review embraces the data present in human-focused literature regarding the up-to-date methodologies and tools contemporarily applied in IVF laboratories' clinical setting of the oocyte, sperm, and embryo cryopreservation and explores the latest news and issues related to the optimization of methods used in ovarian and testicular tissue cryopreservation.

Endocannabinoid System Components of the Female Mouse Reproductive Tract Are Modulated during Reproductive Aging.

The endocannabinoid (eCB) system has gained ground as a key modulator of several female fertility-related processes, under physiological/pathological conditions. Nevertheless, its modulation during reproductive aging remains unclear. This study aimed to investigate the expression levels of the main receptors (cannabinoid receptor 1,CB1; cannabinoid receptor 2, CB2; G-protein coupled receptor, GPR55; and transient receptor potential vanilloid type 1 channel, TRPV1) and metabolic enzymes (N-acylphosphatidylethanolamine phospholipase D, NAPE-PLD; fatty acid amide hydrolase, FAAH; monoacylglycerol lipase, MAGL; and diacylglycerol lipase, DAGL-α and -ß) of this system in the ovaries, oviducts, and uteri of mice at prepubertal, adult, late reproductive, and post-reproductive stages through quantitative ELISA and immunohistochemistry. The ELISA showed that among the receptors, TRPV1 had the highest expression and significantly increased during aging. Among the enzymes, NAPE-PLD, FAAH, and DAGL-ß were the most expressed in these organs at all ages, and increased age-dependently. Immunohistochemistry revealed that, regardless of age, NAPE-PLD and FAAH were mainly found in the epithelial cells facing the lumen of the oviduct and uteri. Moreover, in ovaries, NAPE-PLD was predominant in the granulosa cells, while FAAH was sparse in the stromal compartment. Of note, the age-dependent increase in TRPV1 and DAGL-ß could be indicative of increased inflammation, while that of NAPE-PLD and FAAH could suggest the need to tightly control the levels of the eCB anandamide at late reproductive age. These findings offer new insights into the role of the eCB system in female reproduction, with potential for therapeutic exploitation.

Do Aging and Parity Affect VEGF-A/VEGFR Content and Signaling in the Ovary?-A Mouse Model Study.

In this study, the effects of aging and parity on VEGF-A/VEGFR protein content and signaling in the mice ovaries were determined. The research group consisted of nulliparous (virgins, V) and multiparous (M) mice during late-reproductive (L, 9-12 months) and post-reproductive (P, 15-18 months) stages. Whilst ovarian VEGFR1 and VEGFR2 remained unchanged in all the experimental groups (LM, LV, PM, PV), protein content of VEGF-A and phosphorylated VEGFR2 significantly decreased only in PM ovaries. VEGF-A/VEGFR2-dependent activation of ERK1/2, p38, as well as protein content of cyclin D1, cyclin E1, and Cdc25A were then assessed. In ovaries of LV and LM, all of these downstream effectors were maintained at a comparable low/undetectable level. Conversely, the decrease recorded in PM ovaries did not occur in the PV group, in which the significant increase of kinases and cyclins, as well phosphorylation levels mirrored the trend of the pro-angiogenic markers. Altogether, the present results demonstrated that, in mice, ovarian VEGF-A/VEGFR2 protein content and downstream signaling can be modulated in an age- and parity-dependent manner. Moreover, the lowest levels of pro-angiogenic and cell cycle progression markers detected in PM mouse ovaries sustains the hypothesis that parity could exert a protective role by downregulating the protein content of key mediators of pathological angiogenesis.

The Upper Reproductive System Microbiome: Evidence beyond the Uterus.

The microbiome of the female upper reproductive system has garnered increasing recognition and has become an area of interest in the study of women's health. This intricate ecosystem encompasses a diverse consortium of microorganisms (i.e., microbiota) and their genomes (i.e., microbiome) residing in the female upper reproductive system, including the uterus, the fallopian tubes, and ovaries. In recent years, remarkable advancements have been witnessed in sequencing technologies and microbiome research, indicating the potential importance of the microbial composition within these anatomical sites and its impact in women's reproductive health and overall well-being. Understanding the composition, dynamics, and functions of the microbiome of the female upper reproductive system opens up exciting avenues for improving fertility, treating gynecological conditions, and advancing our comprehension of the intricate interplay between the microbiome and the female reproductive system. The aim of this study is to compile currently available information on the microbial composition of the female upper reproductive system in humans, with a focus beyond the uterus, which has received more attention in recent microbiome studies compared with the fallopian tubes and ovaries. In conclusion, this review underscores the potential role of this microbiome in women's physiology, both in health and disease.

In vivo and in vitro postovulatory aging: when time works against oocyte quality?

In mammalian species an optimal fertilization window during which successful fertilization occurs. In the majority of mammals estrus marks ovulation time and coincident with mating, thereby allowing the synchronized meeting in the fallopian tubes, between freshly ejaculated sperm and freshly ovulated oocytes. Conversely, women do not show natural visual signs of ovulation such that fertilization can occur hours later involving an aged oocyte and freshly ejaculated spermatozoa. During this time, the oocyte undergoes a rapid degradation known as "postovulatory aging" (POA). POA may become particularly important in the human-assisted reproductive technologies, as the fertilization of retrieved mature oocytes can be delayed due to increased laboratory workload or because of unforeseeable circumstances, like the delayed availability of semen samples. This paper is an updated review of the consequences of POA, either in vivo or in vitro, on oocyte quality with particular attention to modifications caused by POA on oocyte nuclear, cytoplasmic, genomic, and epigenetic maturation, and embryo development.

Modulating the dose-rate differently affects the responsiveness of human epithelial prostate- and mesenchymal rhabdomyosarcoma-cancer cell line to radiation.

Purpose: Radiation therapy (RT), by using ionizing radiation (IR), destroys cancer cells inducing DNA damage. Despite several studies are continuously performed to identify the best curative dose of IR, the role of dose-rate, IR delivered per unit of time, on tumor control is still largely unknown.Materials and methods: Rhabdomyosarcoma (RMS) and prostate cancer (PCa) cell lines were irradiated with 2 or 10 Gy delivered at dose-rates of 1.5, 2.5, 5.5 and 10.1 Gy/min. Cell-survival rate and cell cycle distribution were evaluated by clonogenic assays and flow cytometry, respectively. The production of reactive oxygen species (ROS) was detected by cytometry. Quantitative polymerase chain reaction assessed the expression of anti-oxidant-related factors including NRF2, SODs, CAT and GPx4 and miRNAs (miR-22, -126, -210, -375, -146a, -34a). Annexin V and caspase-8, -9 and -3 activity were assessed to characterize cell death. Senescence was determined by assessing ß-galactosidase (SA-ß-gal) activity. Immunoblotting was performed to assess the expression/activation of: i) phosphorylated H2AX (γ-H2AX), markers of DNA double strand breaks (DSBs); ii) p19Kip1/Cip1, p21Waf1/Cip1 and p27Kip1/Cip1, senescence-related-markers; iii) p62, LC3-I and LC3-II, regulators of autophagy; iv) ATM, RAD51, DNA-PKcs, Ku70 and Ku80, mediators of DSBs repair.Results: Low dose-rate (LDR) more efficiently induced apoptosis and senescence in RMS while high dose-rate (HDR) necrosis in PCa. This paralleled with a lower ability of LDR-RMS and HDR-PCa irradiated cells to activate DSBs repair. Modulating the dose rate did not differently affect the anti-oxidant ability of cancer cells.Conclusion: The present results indicate that a stronger cytotoxic effect was induced by modulating the dose-rate in a cancer cell-dependent manner, this suggesting that choose the dose-rate based on the individual patient's tumor characteristics could be strategic for effective RT exposures.

The (endo)cannabinoid signaling in female reproduction: What are the latest advances?

Cannabis extracts like marijuana have the highest consumption rate worldwide. Yet, their societal acceptance as recreational and therapeutic drugs could represent a serious hazard to female human reproduction, because cannabis ingredients [termed (phyto)cannabinoids] can perturb an endogenous system of lipid signals known as endocannabinoids. Accumulated evidence on animal models and humans has demonstrated a crucial role of these endogenous signals on different aspects of female reproduction, where they act through an ensamble of proteins that synthesize, transport, degrade and traffic them. Several reports have recently evidenced the potential role of endocannabinoids as biomarkers of female infertility for disease treatment and prevention, as well as their possible epigenetic effects on pregnancy. The purpose of this review is to provide an update of data collected in the last decade on the effects of cannabinoids and endocannabinoids on female reproductive events, from development and maturation of follicles and oocytes, to fertilization, oviductal transport, implantation and labor. In this context, a particular attention has been devoted to the ovary and the production of fertilizable oocytes, because recent studies have addressed this hot topic with conflicting results among species.

Increased levels of proapoptotic markers in normal ovarian cortex surrounding small endometriotic cysts.

Endometriosis can impair fertility by reducing ovarian reserve and the production of good-quality oocytes. The surgical removal of endometriotic lesions is generally recommended for women who wish to conceive. In this paper we studied whether ovarian cortex adjacent to excised small (diameter ≤ 4 cm) endometriotic cyst (here referred as Cortex Surrounding Endometriotic Cyst, CSEC) showed signs of tissue damages by evaluating the expression of proteins involved in DNA repair and apoptosis. To this end, phosphorylated H2A.X, Chk1 and 2, ATM and ATR, Bcl-2, Bid, phosphorylated and total p53, caspases (9, 8 and 3), XIAP, phosphorylated and total NFκB were analyzed by western blot. Results showed that caspase 8, XIAP, p53/p-p53 and NFκB were more abundantly expressed in all samples of CSEC group in comparison with ovarian cortex of controls. Conversely, the levels of the other proteins were comparable between the two groups. In conclusion, these results suggest that NFκB, caspase 8 and p53/p-p53 elevated expressions in samples of CSEC can be considered as an early sign of tissue injury, indicating that ovarian cortex is already sensitized to apoptosis and inflammation. Therefore, excision of EC should occur very early, to avoid further ovarian damages.

Pro-differentiating and radiosensitizing effects of inhibiting HDACs by PXD-101 (Belinostat) in in vitro and in vivo models of human rhabdomyosarcoma cell lines.

This study describes the in vitro and in vivo activity of PXD-101 (Belinostat), a novel hydroxamic acid-type pan-HDACs inhibitor characterized by a larger safety and efficacy, on myogenic-derived PAX3/FOXO1 fusion protein positive (RH30) or negative (RD) expressing rhabdomyosarcoma (RMS) cell lines. PXD-101 at low doses efficiently inhibited HDACs activity and counteracted the transformed phenotype of RMS by inducing growth arrest and apoptosis, affecting cancer stem cells population and inducing differentiation in RD. Notably, PXD-101 induced oxidative stress promoting DNA damages and affected the ability of RMS to assemble mitotic spindle. PXD-101 radiosensitized by inducing G2 cell cycle growth arrest, enhancing the radiation's ability to induce ROS accumulation and compromising both the ability of RMS to detoxify from ROS and to repair DNA damage. PXD-101 transcriptionally and post-transcriptionally affected c-Myc expression, key master regulator of rhabdomyosarcomagenesis and RMS radioresistance. All in vitro data were corroborated by in vivo experiments showing the cytostatic effects of PXD-101 when used alone and at low dose and its ability to promote the RT-induced killing of RMS. Taken together, our data confirm that altered HDACs activity plays a key role in RMS genesis and suggest PXD-101 as a valid therapeutic strategy particularly in combination with RT.

Role of Major Endocannabinoid-Binding Receptors during Mouse Oocyte Maturation.

Endocannabinoids are key-players of female fertility and potential biomarkers of reproductive dysfunctions. Here, we investigated localization and expression of cannabinoid receptor type-1 and -2 (CB1R and CB2R), G-protein coupled receptor 55 (GPR55), and transient receptor potential vanilloid type 1 channel (TRPV1) in mouse oocytes collected at different stages of in vivo meiotic maturation (germinal vesicle, GV; metaphase I, MI; metaphase II, MII) through qPCR, confocal imaging, and western blot. Despite the significant decrease in CB1R, CB2R, and GPR55 mRNAs occurring from GV to MII, CB2R and GPR55 protein contents increased during the same period. At GV, only CB1R was localized in oolemma, but it completely disappeared at MI. TRPV1 was always undetectable. When oocytes were in vitro matured with CB1R and CB2R but not GPR55 antagonists, a significant delay of GV breakdown occurred, sustained by elevated intraoocyte cAMP concentration. Although CBRs antagonists did not affect polar body I emission or chromosome alignment, GPR55 antagonist impaired in ~75% of oocytes the formation of normal-sized MI and MII spindles. These findings open a new avenue to interrogate oocyte pathophysiology and offer potentially new targets for the therapy of reproductive alterations.

VEGFR2 Expression Is Differently Modulated by Parity and Nulliparity in Mouse Ovary.

Parity and nulliparity exert opposite effects on women's health, as parity is considered a protective factor for several reproductive diseases. This study is aimed at determining if ovarian VEGF and VEGFR2 expression are differently modulated in the ovaries of parous and nulliparous mice. To this end primiparous and nulliparous fertile mice were sacrificed at postovulatory stage. Whole ovaries, corpus luteum, and residual stromal tissues were analyzed to assess VEGF/VEGFR2 expression levels. Ovarian mRNA amounts of Vegfa (120 and 164) and Vegfr2 were comparable between primiparous and nulliparous mice; both isoforms and receptor were accumulated mainly in corpus luteum tissues. VEGF 120 and 164 protein accumulation and distribution mirrored that of mRNA. Conversely, VEGFR2 protein content was significantly higher in ovaries of nulliparous mice and was more efficiently phosphorylated in ovaries of primiparous mice. In both groups, VEGFR2 was preferentially expressed in corpus luteum, while its phosphorylated form was equally distributed in two somatic compartments. We suggest that parity influences VEGFR2/phospho-VEGFR2 expression and tissue distribution. This difference could be part of a more complex mechanism that at least in mice is activated after the first pregnancy and likely aims to preserve female health.

Increased rounds of gonadotropin stimulation have side effects on mouse fallopian tubes and oocytes.

In this study, it was evaluated if increased rounds of gonadotropin stimulation could affect in mice: (i) expression levels of proteins regulating cell cycle and DNA repair in fallopian tubes and (ii) meiotic spindle morphology of ovulated oocytes. To this end, adult female mice were subjected or not (Control) to 6 or 8 rounds of gonadotropin stimulation. Ovulated oocytes were incubated with anti A/B tubulin to evaluate spindle morphology. Fallopian tubes were analyzed to detect Cyclin D1, phospho-p53/p53, phospho-AKT/AKT, phospho-GSK3B/GSK3B, SOX2, OCT3/4, phospho-B-catenin/B-catenin, phospho-CHK1 and phospho-H2A.X protein levels. After 6 rounds, Cyclin D1, p53 and phospho-p53 contents were higher than Control. After 8 rounds, the contents of phosphorylated AKT, GSK3B and p53 as well as of total p53, Cyclin D1 and OCT3/4 significantly increased in comparison with Control. Conversely, SOX2 and B-catenin were similarly expressed among all experimental groups. The finding that phospho-CHK1 and phospho-H2A.X protein levels were undetectable supported the absence of extensive DNA damage. Oocytes number and percentage of normal meiotic spindles drastically decreased from 6 rounds onward. Altogether, our results demonstrated that 6 and 8 cycles of gonadotropin stimulation reduce mouse reproductive performances by inducing over-expression and over-activation of proteins controlling cell cycle progression in fallopian tubes and by impairing oocyte spindle.

The pesticide Lindane induces dose-dependent damage to granulosa cells in an in vitro culture.

Lindane, which is one of the most persistent organochlorine pesticide contaminating the Aral Sea region, is associated with numerous pathologies of the female reproductive system, including infertility, due to its gap junction blocker activity. By using an in vitro model of reproductive toxicity consisting of mouse parietal granulosa cells (GCs) exposed to increasing concentrations of Lindane ranging from 1 to 100µM (L1; L10; L100), we aimed to ascertain the Lindane toxicity by evaluating the ultrastructure and expression of the cell death protein p53. GCs exposed to L1 showed an early sign of degeneration as chromatin marginalization and initial reduction of cell-to-cell contacts. Such effects increased at L10 with nuclear membrane invagination, cytoplasmic blebbing, reduction of microvilli and intercellular connections. L100 induced evident cellular damages with an extensive presence of vacuoles, cytoplasmic fragments, nuclear membrane vesiculation and abundant cellular debris. A dose-dependent increase of p53 expression was evident in the L1 and L10 groups but not in L100. These data provide evidence for a dose-dependent reproductive toxicity of the gap junction blocker Lindane, as seen in mouse GCs cultured in vitro by ultrastructural damage compatible with apoptosis. Since gap junctions may play a critical role in FSH-stimulated progesterone production, the ultrastructural damage here evidenced could explain the increase in the prevalence of reproductive pathologies and infertility in exposed women. Finally, this study provided a useful and repeatable model of reproductive toxicity in vitro, which is applicable to evaluate the detrimental effects of toxicants or the reversing effect of protective substances.