H2O2 induces apoptosis in variety of cells; however, the sensitivities of testicular germ cells to H2O2 are not known. In the present study, H2O2, at concentrations in the range 1–10 μm, was found to induce apoptosis in testicular germ cells in vitro. Following 1 h of treatment with 10 μm H2O2, a 10‐fold rise in the percentage of apoptotic cells was observed. Induction of germ cell apoptosis was directly associated with a significant (P < 0.01) increase in lipid peroxidation and a concomitant decrease in superoxide dismutase and catalase activity. Examination of apoptotic signalling pathways revealed an increased expression of extrinsic (Fas, FasL and caspase‐8) and intrinsic (Bid, Bak, Bad, Bax and caspase‐9) markers, as well as p53, along with a simultaneous decrease in the Bcl‐2 protein at the highest concentration of H2O2 exposure. Both, c‐jun N‐terminal kinase and p38 phosphorylated forms were found to be up‐regulated. Interestingly, up‐regulation of the nuclear transcription factor kappa B was also observed. The respective transcripts for many of the above proteins followed an identical trend. Caspase‐3 activity was also estimated to be 30‐fold higher. Taken together, the above data indicate that testicular germ cells are prone to apoptosis at very low concentrations of H2O2, the mechanism of which involves extrinsic and intrinsic as well other regulatory pathways.
Insecticides, a heterogeneous group of chemicals, are widely used in agriculture and household practices to avoid insect‐inflicted damage. Extensive use of insecticides has contributed substantially to agricultural production and the prevention of deadly diseases by destroying their vectors. On the contrary, many of the insecticides are associated with several adverse health effects like neurological and psychological diseases, metabolic disorders, hormonal imbalance, and even cancer in non‐target species, including humans. Reproduction, a very selective process that ensures the continuity of species, is affected to a greater extent by the rampant use of insecticides. In females, exposure to insecticides leads to reproductive incapacitation primarily through disturbances in ovarian physiology. Disturbed ovarian activities encompass the alterations in hormone synthesis, follicular maturation, ovulation process, and ovarian cycle, which eventually lead to decline in fertility, prolonged time‐to‐conceive, spontaneous abortion, stillbirths, and developmental defects. Insecticide‐induced ovarian toxicity is effectuated by endocrine disruption and oxidative stress. Oxidative stress, which occurs due to suppression of antioxidant defense system, and upsurge of reactive oxygen and nitrogen species, potentiates DNA damage and expression of apoptotic and inflammatory markers. Insecticide exposure, in part, is responsible for ovarian malfunctioning through disruption of hypothalamic–pituitary–gonadal axis. The current article is focused on the adverse effects of insecticides on ovarian functioning, and consequently, on the reproductive efficacy of females. The possible strategies to combat insecticide‐induced toxicity are also discussed in the latter part of this review. Environ. Mol. Mutagen. 61:369–392, 2020. © 2020 Wiley Periodicals, Inc.
The mechanism of H(2)O(2) induced oxidative stress leading to male germ cell apoptosis was earlier reported from our laboratory. In the present study, we investigated the mechanisms by which N-acetyl-L-cysteine (NAC, which is highly cell specific with strong antioxidant and anti-genotoxic properties), stimulated cell survival under such conditions. Co-incubation with 5 mM NAC significantly (P<0.001) reduced the germ cell apoptosis induced by 10 µM H(2)O(2). Lipid peroxidation was brought down with significant restoration of activities of antioxidant enzymes, SOD, GST, and catalase. Expression of pro-apoptotic marker, Bax up-regulated following H(2)O(2) exposure, was reversed back to control levels. In contrast, expression of anti-apoptotic Bcl-2 and phospho-Akt revealed a completely opposite trend. While caspase-8 activity remained unaffected, NAC successfully attenuated the increased activities of caspase-3 and -9 in the H(2) O(2) treated cells. Simultaneously, the increased expression of caspase-9, phospho-JNK, and phospho-c-Jun after H(2)O(2) treatment was down-regulated by NAC. The above findings indicate that the mechanism of inhibition of H(2)O(2) induced male germ cell apoptosis by NAC is mediated through regulation of caspase-9 and JNK.
Summary The unique phenomenon of cell proliferation and apoptosis is encountered in the ovarian follicles undergoing early stages of atresia. The aim of this study was to verify the morphological variations in these two physiologically distinct processes operating in antral follicles of caprine ovaries using histological and ultrastructural techniques. Histologically the degenerating granulosa cells were characterized by condensed cytoplasm, and nucleus fragmentation in hazy cytosol. The pyknotic nuclei of degenerating cells stained darkly with haematoxylin and giemsa while the cytoplasm was eosinophilic. Under electron microscopy, apoptosis was marked by asymmetrical shrinkage, vacuolization of cytoplasm, swollen and vacuolated mitochondria, increased irregularity and/or fragmentation of nucleus, chromatin condensation and finally, production of membrane enclosed nuclear fragments containing intracellular material, the apoptotic bodies. The parallel use of these two methods on caprine ovaries has enabled us to analyse the decline in the frequency of granulosa cells during follicular atresia due to apoptosis.
The present study was aimed to investigate the beneficial effects of N-acetyl-L: -cysteine (NAC, 150 mg/kg bw twice/week) against testicular germ cell apoptosis in rats induced by chronic hCG administration (100 IU/rat/day for 30 days). NAC co-treatment improved serum testosterone, prevented rise in lipid peroxidation, intracellular H(2)O(2) and the activities of antioxidant enzymes in germ cells. Replenishment of intracellular GSH and total antioxidant capacity was seen. There was a marked reduction in TUNEL positive germ cells and expression of caspase-3 (p < 0.01) and PARP cleavage. Pro-apoptotic markers Fas, FasL, caspase-8 were also significantly downregulated. While Bcl-2 was fully restored, rise in Bax, caspase-9, phospho-JNK/JNK and phospho-c-Jun/c-Jun expression was significantly arrested. Anti-apoptotic phospho-Akt/Akt and NF-κB were otherwise found upregulated. Taken together, the above findings demonstrate that NAC intervention rescued the testicular germ cells from demise following chronic hCG treatment through regulation of multiple signaling mechanisms of metazoan apoptosis.
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