Forebrain epileptiform activity is not required for seizure-induced apnea in a mouse model of Scn8a epilepsy.

Sudden unexpected death in epilepsy (SUDEP) accounts for the deaths of 8-17% of patients with epilepsy. Although the mechanisms of SUDEP are essentially unknown, one proposed mechanism is respiratory arrest initiated by a convulsive seizure. In mice, we have previously observed that extended apnea occurs during the tonic phase of seizures. Although often survived, tonic seizures became fatal when breathing did not immediately recover postictally. We also found that respiratory muscles were tonically contracted during the apnea, suggesting that muscle contraction could be the cause of apnea. In the present study, we tested the hypothesis that pyramidal neurons of the motor cortex drive motor units during the tonic phase, which produces apnea. Mice harboring the patient-derived N1768D point mutation of an Scn8a allele were crossed with transgenic mice such that inhibitory Designer Receptors Exclusively Activated by Designer Drugs (DREADD) receptors were selectively expressed in excitatory forebrain neurons. We then triggered audiogenic and hippocampal (HC) stimulated seizures under control conditions and when excitatory forebrain neurons were inhibited with the synthetic ligand Clozapine-N-Oxide (CNO). We found that inhibition with CNO was sufficient to increase seizure threshold of HC stimulated, but not audiogenic, seizures. In addition, regardless of seizure type, CNO nearly eliminated epileptiform activity that occurred proximal to the tonic phase; however, the seizure behaviors, notably the tonic phase and concomitant apnea, were unchanged. We interpret these results to indicate that while cortical neurons are likely critical for epileptogenesis and seizure initiation, the behavioral manifestations of tonic seizures are generated by neural circuitry in the mid- and/or hindbrain.

Ameliorative potential of vitamin C and E against Roundup-glyphosate induced genotoxicity triggering apoptosis in caprine granulosa cells.

The present study was aimed to investigate the genotoxic and apoptotic effects of glyphosate (GLP) in Roundup formulation along with mitigation of two potent antioxidants that is, vitamin C and E in caprine granulosa cells in vitro. The entire work was done in a dose and time dependent manner where different concentrations of GLP (0.1, 2.0, and 4.0 mg/ml) in Roundup and antioxidants (0.5 and 1.0 mM) were employed to culture of granulosa cells for exposure durations of 24, 48, and 72 h. Analysis of GLP-induced geno-toxicity was accomplished by using single cell gel electrophoresis (comet assay) assay. Results have shown increased incidences of DNA fragmentation, evidenced by presence of different types of comets (Type 1-Type 4) in Roundup-GLP- exposed groups in contrast to the control group (Type 0 comet). However, mitigation by both vitamin C and E was significant (p < .05) in combating the GLP-induced genotoxicity in granulosa cells in a concentration- and time-dependent manner. The results of our study provide a clear indication of the ameliorative actions of vitamin C and E against Roundup-GLP-induced genotoxicity that instigate apoptosis in ovarian granulosa cells of caprine.

Role of autophagy in follicular development and maintenance of primordial follicular pool in the ovary.

The reproductive life span of the organism mainly depends on follicular development that maintains the primordial follicle pool in the cohort of follicles within the ovary. The total count of primordial follicles decreases with age due to ovulation and follicular atresia. Follicular atresia, a process of ovarian follicles degradation, mainly occurs via apoptosis, but recent studies also favor autophagy existence. Autophagy is a cellular and energy homeostatic response that helps to maintain the number of healthy primordial follicles, germ cell survival, and removal of corpus luteum remnants. But the excessive autophagic cell death changes both the quality and quantity of oocytes that ultimately affect female reproductive health. Autophagy regulation occurs by various autophagy-regulated genes like BECN1 and LC3-II (autophagy marker genes). Their abnormal regulation or mutation highly influences follicular development by alteration of primordial follicles formation, the decline in oocytes count, and germ cell loss. Various classical signaling pathways such as PI3K/AKT/mTOR, MAPK/ERK1/2, AMPK, and IRE1 are involved in granulosa and oocytes autophagy, while mTOR signaling is the primary mechanism. Along with basal level autophagy, chemical/hormone/stress-mediated autophagy also affects follicular development and female reproduction. In this review, we have primarily focused on granulosa cell and oocytes' autophagy, mechanism, and the role of autophagy determining marker genes in follicular development.

Efficacy of N-acetyl- l-cysteine against glyphosate induced oxidative stress and apoptosis in testicular germ cells preventing infertility.

The present era's demand for continuous pesticides' use to increase the agriculture outcome, has caused numerous health effects among which mammalian infertility, owing to reproductive toxicity, is serious. Thus, the present study emphasizes upon glyphosate (GLY) induced toxicity and mitigating effects of N-acetyl cysteine (NAC) in testicular cells of caprine by using various cytotoxic and biochemical parameters. GLY was found to induce several apoptotic attributes such as pyknotic nuclei, tubular degeneration, increased vacuolization, and so on, in testicular cells. GLY also decreased the cell viability and increased the incidence of apoptosis in testicular cells in a dose- and time-dependent manner as revealed by MTT assay and Fluorescence (ethidium bromide/acridine orange) assay, respectively. It also increased the level of oxidative stress as evident with an increase in lipid peroxidation and decline in antioxidant power along with the decreased enzymatic activity of different antioxidant enzymes (SOD, CAT, and GST). However, NAC supplementation showed antagonistic results in GLY-treated testicular tissues with maximum amelioration at the highest dose, thereby decreasing GLY-mediated apoptosis rate and oxidative stress. Maximum amelioration was reported at 10 mM NAC concentration. Reduced GLY toxicity due to NAC will prove NAC to be an excellent approach for dealing with male reproductive toxicity at the cellular level, benefiting the mammalian reproductive status.

Effects of heavy metals on reproduction owing to infertility.

The reproductive performance of most of the species is adversely affected by hazardous heavy metals like lead, cadmium, mercury, arsenic, zinc, and copper. Heavy metals are liberated in the environment by natural sources like rock weathering, volcanic eruption, and other human activities like industrial discharge, mineral mining, automobile exhaust, and so forth. Heavy metals alter several reproductive functions in both males and females like a decrease in sperm count, motility, viability, spermatogenesis, hormonal imbalance, follicular atresia, and delay in oocyte maturation, and so forth, and thus, forms an important aspect of reproductive toxicology. The present review compiles toxicity aspects of various heavy metals and their efficacy and mechanism of action in mammals.

Postictal Death Is Associated with Tonic Phase Apnea in a Mouse Model of Sudden Unexpected Death in Epilepsy.

OBJECTIVE: Sudden unexpected death in epilepsy (SUDEP) is an unpredictable and devastating comorbidity of epilepsy that is believed to be due to cardiorespiratory failure immediately after generalized convulsive seizures. METHODS: We performed cardiorespiratory monitoring of seizure-induced death in mice carrying either a p.Arg1872Trp or p.Asn1768Asp mutation in a single Scn8a allele-mutations identified from patients who died from SUDEP-and of seizure-induced death in pentylenetetrazole-treated wild-type mice. RESULTS: The primary cause of seizure-induced death for all mice was apnea, as (1) apnea began during a seizure and continued for tens of minutes until terminal asystole, and (2) death was prevented by mechanical ventilation. Fatal seizures always included a tonic phase that was coincident with apnea. This tonic phase apnea was not sufficient to produce death, as it also occurred during many nonfatal seizures; however, all seizures that were fatal had tonic phase apnea. We also made the novel observation that continuous tonic diaphragm contraction occurred during tonic phase apnea, which likely contributes to apnea by preventing exhalation, and this was only fatal when breathing did not resume after the tonic phase ended. Finally, recorded seizures from a patient with developmental epileptic encephalopathy with a previously undocumented SCN8A likely pathogenic variant (p.Leu257Val) revealed similarities to those of the mice, namely, an extended tonic phase that was accompanied by apnea. INTERPRETATION: We conclude that apnea coincident with the tonic phase of a seizure, and subsequent failure to resume breathing, are the determining events that cause seizure-induced death in Scn8a mutant mice. ANN NEUROL 2021;89:1023-1035.

Diacetoxy iodobenzene mediated regioselective synthesis and characterization of novel [1,2,4]triazolo[4,3-a]pyrimidines: apoptosis inducer, antiproliferative activities and molecular docking studies.

Prompt and regioselective synthesis of eleven novel [1,2,4]triazolo[4,3-a]pyrimidines 2a-2k, via intramolecular oxidative-cyclization of 2-(2-arylidenehydrazinyl)-4-methyl-6-phenylpyrimidine derivatives 1a-1k has been demonstrated here using diacetoxy iodobenzene (DIB) as inexpensive and ecofriendly hypervalent iodine(III) reagent in CH2Cl2 at room temperature. Regiochemistry of final product has been established by developing single crystal and studied X-ray crystallographic data for two derivatives 2c and 2h without any ambiguity. These prominent [1,2,4]triazolo[4,3-a]pyrimidines were evaluated for human osteosarcoma bone cancer (MG-63) and breast cancer (MCF-7) cell lines using MTT assay to find potent antiproliferative agent and also on testicular germ cells to find potent apoptotic inducing activities. All compounds show significant cytotoxicity, particularly 3-(2,4-dichlorophenyl)-5-methyl-7-phenyl-[1,2,4]triazolo[4,3-a]pyrimidine (2g) was found significant apoptotic inducing molecule, as well as the most potent cytotoxic agent against bone cancer (MG-63) and breast cancer (MCF-7) cell lines with GI50 value 148.96 µM and 114.3 µM respectively. Molecular docking studies has been carried out to see the molecular interactions of synthesized compounds with the protein thymidylate synthase (PBD ID: 2G8D).Communicated by Ramaswamy H. Sarma.

Cadmium as a testicular toxicant: A Review.

Cadmium (Cd) is a toxic heavy metal with no known biological functions in the human body. Due to a considerably long biological half-life and very low rate of excretion, accumulation of Cd in different body organs (eg, liver, kidney, and testes) over time is associated with perturbed functioning of these organs. Recent studies have shown the extreme sensitivity of the testes to Cd toxicity. In testes, Cd has been reported to induce oxidative stress, apoptosis of spermatogenic cells, reduction in androgen production and sperm functions. Moreover, Cd in combination with other environmental toxicants may be responsible for the declining fertility of males in both animals and humans. Pinpointing how Cd toxicity affects various testicular processes will be imperative for the development of preventative measures to promote fertility among males. Therefore, in the present review, we summarize the recent findings related to the Cd-induced oxidative toxicity, apoptotic toxicity, steroidogenic toxicity, and spermatotoxicity, along with their possible mechanisms in testicular tissue of different animal species. In addition, the utilization of various antioxidant compounds, medicinal plants and other compounds for the management of Cd toxicity in testes is discussed.

Ameliorating Effects of Natural Antioxidant Compounds on Female Infertility: a Review.

The prevalence of female infertility cases has been increasing at a frightening rate, affecting approximately 48 million women across the world. However, oxidative stress has been recognized as one of the main mediators of female infertility by causing various reproductive pathologies in females such as endometriosis, PCOS, preeclampsia, spontaneous abortion, and unexplained infertility. Nowadays, concerned women prefer dietary supplements with antioxidant properties over synthetic drugs as a natural way to lessen the oxidative stress and enhance their fertility. Therefore, the current review is an attempt to explore the efficacy of various natural antioxidant compounds including vitamins, carotenoids, and plant polyphenols and also of some medicinal plants in improving the fertility status of females. Our summarization of recent findings in the current article would pave the way toward the development of new possible antioxidant therapy to treat infertility in females. Natural antioxidant compounds found in fruits, vegetables, and other dietary sources, alone or in combination with other antioxidants, were found to be effective in ameliorating the oxidative stress-mediated infertility problems in both natural and assisted reproductive settings. Numerous medicinal plants showed promising results in averting the various reproductive disorders associated with female infertility, suggesting a plant-based herbal medicine to treat infertility. Although optimum levels of natural antioxidants have shown favorable results, however, their excessive intake may have adverse health impacts. Therefore, larger well-designed, dose-response studies in humans are further warranted to incorporate natural antioxidant compounds into the clinical management of female infertility.

N-acetyl-l-cysteine mediated regulation of DNA fragmentation, an apoptotic event, against methoxychlor toxicity in the granulosa cells of ovarian antral follicles.

Methoxychlor (MXC), an organo-chlorine insecticide, is a reproductive toxicant in females, causing apoptosis-mediated follicular atresia. To elucidate the potentials of Methoxychlor as a geno-toxicant, granulosa cells of healthy antral follicles, exposed to MXC and antioxidant, N-acetyl-l-cysteine, were studied by the terminal deoxynucleotidyltransferase-dUTP nick end-labelling and single-cell gel electrophoresis (comet) assays. MXC caused DNA fragmentation, as revealed by the increased incidence of dark brown condensed TUNEL positive cells in contrast with lightly brown TUNEL negative cells with maximum TUNEL positive cells were observed in 100 µg/mL MXC treated groups. Quantitatively, maximum geno-toxicity was exhibited at highest MXC treatment with percent tail DNA as 17.87 ± 0.85, 41.16 ± 3.94, and 47.73 ± 3.71 in comparison with control (0.65 ± 0.03, 2.91 ± 0.27, and 7.16 ± 1.39) after 24, 48 and 72 h exposure duration, respectively. MXC treated groups exhibited Type 1-Type 3 comets as compared to Type 0 comets in control groups. Supplementation of NAC led to significant (p < 0.05) decline in geno-toxicity in MXC treated groups with maximum amelioration observed at 5 and 10 mM. Consequently, increased DNA damage attributed to the granulosa cells apoptosis in response to Methoxychlor exposure was significantly combated by NAC supplementation, preventing the geno-toxicity induced cyto-toxicity in GCs.

Effective attenuation of glyphosate-induced oxidative stress and granulosa cell apoptosis by vitamins C and E in caprines.

Pesticides are known to cause a wide range of reproductive problems that possess degenerative effects on mammalian fertility. Glyphosate (GLP), a broad-spectrum organophosphate herbicide, is known to be a potent mammalian toxicant. The present study aims at assessing the GLP-induced (0.1, 2.0, and 4.0 mg/ml) granulosa cells toxicity and evaluating the mitigating effects of vitamins C and E (0.5 mM and 1.0 mM) in healthy caprine antral follicles, cultured in vitro in a dose- and time-dependent manner (24, 48, and 72 hr) and subjected to various cytotoxic and geno-toxic analysis, namely, classic histology, EB/AO differential staining, oxidative stress parameters, and antioxidant enzymatic activity. The histomorphological analysis and EB/AO staining elucidated increase in the incidence of apoptotic attributes within granulosa cells with increasing dose and duration of the GLP treatment. The highest apoptotic frequency was observed at 4.0 mg/ml GLP after 72-hr exposure duration in comparison with the control. GLP exposure also led to a significant decline in the antioxidant enzymes' activity, namely, SOD, catalase, and GST along with enhanced lipid peroxidation and reduced FRAP activity in a dose- and time-dependent manner. Vitamins C and E supplementation decreased oxidative stress-mediated granulosa cells apoptosis, suggesting its efficiency to diminish GLP-mediated GCs cytotoxicity and thereby, preventing associated fertility disorders.

Antiapoptotic effects of vitamins C and E against cypermethrin-induced oxidative stress and spermatogonial germ cell apoptosis.

Toxicological studies have demonstrated the relation between use of agrochemicals and fertility issues within males. Thus, the present study aimed to elucidate the propensity of cypermethrin (CYP) in bringing testicular germ cell apoptosis and effective attenuation by vitamins C and E in caprines. Reproductive toxicity of CYP was evaluated using histomorphological, cytological, and biochemical changes in the testicular germ cells in dose-dependent (1, 5, 10 µg/mL) and time-dependent (4, 6, 8 h) manner. Histological and ethidium bromide/acridine orange fluorescence staining exhibited that vitamins C and E (0.5 and 1.0 mM) successfully diminished the CYP-induced testicular germ cells apoptosis. CYP exposure along with vitamins C and E supplementation also resulted in significantly increased ferric reducing antioxidant power activity along with the antioxidant enzymes, namely catalase, superoxide dismutase, and glutathione-s-transferase, and decreased lipid peroxidation in testicular germ cells. Thus, vitamins C and E ameliorated CYP-induced testicular germ cell apoptosis, thereby preventing spermatogonial cells degeneration and male infertility.

N-Acetyl-cysteine mediated inhibition of spermatogonial cells apoptosis against malathion exposure in testicular tissue.

Toxicological studies so far suggest that excessive use of malathion, an organophosphate insecticide, causes serious ill-effects in mammalian reproductive physiology. The present study aims at assessing malathion-induced toxicity in a dose- and time-dependent manner with mitigating effects of N-acetyl-l-cysteine. The testicular germ cell viability was monitored using MTT assay, where NAC, being an antioxidant significantly reduced malathion-induced toxicity by enhancing the frequency of cell viability. The histomorphological analysis showed that NAC successfully diminished several apoptotic features in testicular cells, induced by malathion. The differential EB/AO staining revealed a significant decline in the percentage of apoptosis after NAC supplementation. NAC also diminished the malathion-induced DNA fragmentation along with significantly reduction in oxidative stress parameters causing decrease in lipid peroxidation and enhancement of ferric reducing antioxidant power within testicular germ cells. Thus, NAC mitigated the malathion-induced toxicity, proving its potential in infertility treatment.

Multicomponent Synthesis of Some Molecular Hybrid Containing Thiazole Pyrazole as Apoptosis Inducer.

The present study describes a multicomponent synthesis of molecular hybrid containing pyrazole, thiazole moiety using hydrazone as a linker, which have been synthesized by condensation of 1-phenyl-3-(aryl)-1H-pyrazole-4-carbaldehydes 1A-B: , thiosemicarbazide and α-bromoketones 2A-C: .The target hybrid compounds, 1-((1-phenyl-3-aryl-1H-pyrazole-4-yl)methylene)-2-(4-arylthiazole-2-yl)hydrazine 3A-F: are characterized by 1H-NMR, 13C NMR, FT-IR and mass. Apoptosis inducing ability and cytotoxic nature of all the hybrid compounds having thiazole, pyrazole and hydrazone were assessed by using biological assays viz morphological, fluorescence and tunel assays on granulosa cells of ovarian antral follicles of goat (Capra hircus) in vitro. Apoptosis was recognized and quantified using differential staining of ethidium bromide and acridine orange where apoptotic cells exhibited red fluorescence and live normal cells with intact cell membrane and normal nucleus displayed bright green fluorescence. Among the tested compounds, compound 3E: and 3B: showed the maximum potency to induce apoptosis with percentage of apoptosis 25.61±2.95and 23.45±1.46 respectively followed by 3F: (20.95±0.40) and 3D: (20.44±1.60) in comparison with control (5.14±0.44).

Design, Synthesis, and Screening of Triazolopyrimidine-Pyrazole Hybrids as Potent Apoptotic Inducers.

An efficient synthesis of novel 3-(3-aryl-1-phenyl-1H-pyrazol-4-yl)-5,7-dimethyl-[1,2,4]triazolo[4,3-a]-pyrimidines was accomplished by the oxidation of pyrimidinylhydrazones by using organoiodine(III) reagent. All new triazolopyrimidine derivatives bearing the pyrazole scaffold were screened to evaluate them as a reproductive toxicant in the testicular germ cells of goat (Capra hircus). This study aimed at assessing the cytological and biochemical changes in testicular germ cells after the exposure to triazolopyrimidines in a dose- and time-dependent manner. Histomorphological analysis, fluorescence assays, apoptosis quantification, and terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling (TUNEL) assays were performed to determine cytological changes, whereas thiobarbituric acid-reactive substance (TBARS) and ferric reducing antioxidant power (FRAP) assays were carried out to measure the oxidative stress in triazolopyrimidines treated germ cells. The parallel use of these methods enabled us to determine the role of triazolopyrimidines in inducing apoptosis as a consequence of cytogenetic damage and oxidative stress generated in testicular germ cells of goat.

N-acetyl cysteine-mediated effective attenuation of methoxychlor-induced granulosa cell apoptosis by counteracting reactive oxygen species generation in caprine ovary.

Methoxychlor (MXC), an organochloride insecticide, is a potent toxicant-targeting female reproductive system and known to cause follicular atresia by inducing apoptosis within granulosa cells. Oxidative stress plays a pivotal role in apoptosis; thus, this study focuses on the ameliorative action of N-acetyl cysteine (NAC) on MXC-induced oxidative stress and apoptosis within granulosa cell of caprine ovary. Classic histology, fluorescence assay, and biochemical parameters were employed to evaluate the effect of varied concentration of NAC (1, 5, and 10 mM) on granulosa cell apoptosis after 24, 48, and 72 h exposure duration. Histomorphological studies revealed that NAC diminished the incidence of apoptotic attributes like condensed or marginated chromatin, pyknosis, crescent-shaped nucleus, empty cell spaces, and degenerated cellular structure along with the presence of cytoplasmic processes within granulosa cells in dose- and time-dependent manner. NAC significantly downregulated the percentage of MXC-induced granulosa cell apoptosis within healthy ovarian follicle with its increasing dose, maximum at 10 mM concentration. It also significantly (p < 0.05) upregulated the activity of antioxidant enzymes, namely catalase, superoxide dismutase, and glutathione-s-transferase, along with ferric reducing antioxidant power further declining lipid peroxidation in the MXC-treated caprine ovary. The results revealed a negative correlation between apoptosis frequency and antioxidant enzymes' activity (rCAT = -0.67, rSOD = -0.56, rGST = -0.31; p < 0.05) while a positive correlation was observed with lipid peroxidation (r = 0.63; p < 0.05) after NAC supplementation. Thus, NAC supplementation reduces the MXC-generated oxidative stress that perhaps declines the ROS generating signal transduction pathway of apoptosis, thereby preventing MXC-induced granulosa cell apoptosis and follicular atresia. © 2015 Wiley Periodicals, Inc. Environ Toxicol 32: 156-166, 2017.

Granulosa cell apoptosis by impairing antioxidant defense system and cellular integrity in caprine antral follicles post malathion exposure.

Toxicological studies have demonstrated the exposure-risk relationship of several pesticides on reproduction of living organisms. To evaluate the role of malathion as a reproductive toxicant, this study aims at assessing the cytological and biochemical changes in the granulosa cells after malathion exposure in dose (1 nM, 10 nM, 100 nM) and time (4 h, 6 h, 8 h) dependent manner. Histomorphological analysis, fluorescence assay, apoptosis quantification, and terminal deoxynucleotidyl transferase d-UTP mediated nick end labeling (TUNEL) assay were done to determine cytological changes, whereas antioxidant enzyme assays were done to measure the oxidative stress in malathion treated ovarian antral follicles. Histological studies exhibited the occurrence of highly condensed or marginated chromatin with fragmented nucleus, pyknosis, loss of membrane integrity, increased empty spaces, and vacuolization in malathion treated granulosa cells. Ethidium bromide/acridine orange (EB/AO) fluorescence staining demonstrated a significant increase in incidence and percentage of apoptosis after malathion exposure (p < 0.001), both between and within the groups. Malathion exposure also resulted in increased DNA fragmentation and decline in both antioxidant enzymes activity namely catalase (CAT) and superoxide dismutase (SOD) in granulosa cells of antral follicles. Moreover, there was found a significant negative correlation between the apoptosis incidence and the level of antioxidant enzymes activity, SOD (r = -0.73 p < 0.01) and CAT (r = -0.80 p < 0.01), in malathion treated ovarian antral follicles. Thus, highlighting the role of DNA fragmentation and declining antioxidant level as a possible mechanism underlying malathion induced reproductive toxicity. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1944-1954, 2016.

Transmission electron microscopic analysis of malathion-induced cytotoxicity in granulosa cells of caprine antral follicles.

Malathion, one of the most abundantly used organophosphate pesticides, has immoderate potency as a cytotoxic and genotoxic compound that induces toxicity in granulosa cells, resulting in its apoptosis. Thus, the present study aims to employ ultrastructural analysis for assessing the cytotoxicity of malathion at nanomolar concentrations (1 nM and 10 nM) in granulosa cells of caprine antral follicles at different exposure durations. Transmission electron microscopy revealed diminished cell-cell contact and cellular integrity, presence of crescent-shaped nucleus, chromatin condensation, and pyknosis with nuclear membrane folding, accumulation of lipid droplets with occurrence of cytoplasmic protrusions in granulosa cells treated with 1 nM malathion, whereas at 10 nM concentration, along with apoptotic attributes, prominent association of nucleus, endoplasmic reticulum, mitochondria and lipid droplets, nucleus invagination into lipid droplets, apical localization of lipid bodies, and occurrence of autophagic body were observed as compared to healthy granulosa cells in control with normal intact cellular integrity, well-developed cellular association, and doubled membrane nuclear lamina with homogenously dispersed chromatin surrounded by intact mitochondria with well-developed cristae. Thus, the results of ultrastructural analysis clearly suggest that nanomolar concentration of malathion induces apoptotic hallmarks within the granulosa cells of antral follicles that play a consequential role in increasing the incidence of follicular atresia, thereby affecting the overall fertility.

Malathion-induced granulosa cell apoptosis in caprine antral follicles: an ultrastructural and flow cytometric analysis.

Organophosphate pesticides (OPs) like malathion interfere with normal ovarian function resulting in an increased incidence of atresia and granulosa cell apoptosis that plays a consequential role in the loss of ovarian follicles or follicular atresia. The aim of present study was to assess malathion-induced (100 nM) reproductive stress, ultrastructural damage and changes in apoptosis frequency in ovarian granulosa cells of antral follicles. Transmission electron microscopy (TEM) was employed for ultrastructural characterization, oxidative stress was evaluated using thiobarbituric acid reactive substances (TBARS) assay to measure lipid peroxidation, and apoptosis was quantified via flow cytometry. By TEM, apoptosis was identified by the presence of an indented nuclear membrane with blebbing, pyknotic crescent-shaped fragmented nuclei, increased vacuolization, degenerating mitochondria, and lipid droplets. The results indicate a significant increase in malondialdehyde (MDA) level (nmols/g wet tissue) at a 100 nM dose of malathion i.e. 7.57±0.033*, 8.53±0.12*, and 12.87±0.78** at 4, 6, or 8 h, respectively, as compared with controls (6.07±0.033, p<0.01*, p<0.05**) showing a positive correlation between malathion-induced lipid peroxidation and percentage of granulosa cell apoptosis (r=1; p<0.01). The parallel use of these three methods enabled us to determine the role of malathion in inducing apoptosis as a consequence of cytogenetic damage and oxidative stress generated in granulosa cells of antral follicles.