This study investigates the role of p38 MAPK, inducible nitric oxide synthase (iNOS), and the intrinsic pathway signaling in male germ cell death in rats after hormonal deprivation by a potent GnRH antagonist treatment. Germ cell apoptosis, involving exclusively middle (VII-VIII) stages, was activated by d 5 after GnRH antagonist treatment. Initiation of germ cell apoptosis was preceded by p38 MAPK activation and induction of iNOS. p38 MAPK activation and iNOS induction were further accompanied by a marked perturbation of the BAX/BCL-2 rheostat, cytochrome c, and DIABLO release from mitochondria, caspase activation, and poly(ADP-ribose) polymerase cleavage. Concomitant administration of aminoguanidine, a selective iNOS inhibitor, significantly prevented hormone deprivation-induced germ cell apoptosis. Inhibitors of iNOS or p38 MAPK were also effective in preventing human male germ cell apoptosis induced by hormone-free culture conditions. Together, these results establish a new signal transduction pathway involving p38 MAPK and iNOS that, through activation of the intrinsic pathway signaling, promotes male germ cell death in response to a lack of hormonal stimulation across species.
The understanding of testicular physiology, pathology, and male fertility issues requires knowledge of male germ cell death and energy production. Here, we induced human male germ cell apoptosis (detected by Southern blot analysis of DNA fragmentation, TUNEL, activation of caspases-3 and -9, and electron microscopy) by incubating seminiferous tubule segments under hormone-and serum-free conditions. Inhibitors of complexes I to IV of mitochondrial respiration, exposure to anoxia, and inhibition of F0F1-ATPase (with oligomycin) decreased the ATP levels (analyzed by HPLC) and suppressed apoptosis at 4 h. Uncoupler 2,4-dinitrophenol (DNP) and oligomycin combination also suppressed death at 4 h, as did the DNP alone. Inhibition of glycolysis by 2-deoxyglucose neither suppressed nor further induced apoptosis nor altered the antiapoptotic effects of the mitochondrial inhibitors. Furthermore, Fas system activation did not modify the effects of mitochondrial modulators. After 24 h, delayed male germ cell apoptosis was observed despite the presence of the mitochondrial inhibitors. We conclude that the mitochondrial ATP production machinery plays an important role in regulating in vitroinduced primary pathways of human male germ apoptosis. The ATP synthesized by the F0F1-ATPase seems to be the crucial death regulator, rather than any of the complexes (I-IV) alone, the functional electron transport chain, or the membrane potential. We also conclude that there seem to be secondary pathways of human testicular cell apoptosis that do not require mitochondrial ATP production. The present study emphasizes the role of the main catabolic pathways in the complex network of regulating events of male germ cell life and death.testis; spermatogenesis; apoptosis; mitochondria; oxidative phosphorylation THE ENERGY METABOLISM and catabolism in the testis involve a unique network of reactions and include several testis-specific enzymes, hormonal regulation, and essential cell-to-cell interactions (1,8,27,29,41,43,55,56,58). The proper functioning of this network is critical for testicular physiology. Another crucial regulator of normal testicular function is appropriate germ cell death, and the disruption of this orderly process is associated with several male reproductive disorders (15,16,30,38,47,54,60). How these two entities, i.e., energy metabolism/catabolism and male germ cell death, are linked is presently unclear.The cell types of the seminiferous epithelium differ from each other in their sensitivity to death-inducing signals and with their preferred substrates for energy metabolism. Spermatogonia, mature spermatozoa, and the somatic Sertoli cells exhibit high glycolytic activity, whereas spermatocytes and spermatids produce ATP mainly by mitochondrial oxidative phosphorylation (OXPHOS; see Refs. 3,28,31,37,45,53,64). Interestingly, the cell types that use OXPHOS for energy production (i.e., spermatids and spermatocytes) are sensitive to death-inducing signals such as hormonal deprivation, Fas activation, and elevated temperature ...
IntroductionAppropriate apoptosis is important for testicular cell homeostasis and disruption of this orderly process may be associated with several male reproductive disorders. The purpose of our study was to evaluate the previously undefined role of glutathione-to-glutathione disulfide (GSH-to-GSSG) redox balance in human male germ cell death. We also investigated the antiapoptotic mechanism(s) of N-acetyl-l-cysteine (NAC), a clinically used compound with multiple mechanisms of action in many nontesticular tissues that include antioxidation, modulation of AP-1 and NF-κB activation, and regulation of death pathways through phosphorylation of stress kinase p38 MAPK.Results and MethodsIncubation of human seminiferous tubule segments under serum- and hormone-free conditions induced germ cell apoptosis as demonstrated by Southern blot analysis of DNA fragmentation, in situ TUNEL, electron microscopy, and Western blot detection of caspase 3 and 9 activation. Apoptosis was associated with increased NF-κB- and AP-1-DNA-binding activities (EMSA) and lowered glutathione levels as well as decreased ratio of reduced (GSH) to oxidized (GSSG) glutathione (HPLC). NAC, while not affecting the NF-κB- or AP-1 binding activities, effectively prevented the germ cell death and fully restored the GSH-to-GSSG ratios. Two inhibitors of p38 MAPK suppressed human male germ cell apoptosis, suggesting a role of the stress kinases in regulating male germ cell death.ConclusionWe conclude that the hormone- and serum-free culture condition disturbs the GSH-to-GSSG balance, indicating oxidative stress, and induces human male germ cell death. NAC prevents these events, which were associated with p38 MAPK activation. The present study may lead to the clinical use of NAC to prevent inappropriate male germ cell death.
Addison's disease (AD) is a rare autoimmune disorder affecting approximately 1/10,000 in the general population. It typically occurs sporadically with a single member of a family affected (simplex families). Rarely, multiple members of a family can be affected with AD (multiplex families). The purpose of this study is to characterize the HLA haplotypes and genotypes of a group of individuals with AD and evaluate the differences between simplex and multiplex cases. Individuals with AD were recruited for participation in this study through the National Adrenal Diseases Foundation and through screening individuals with type 1 diabetes (T1D) for AD. A total of 62 families and 239 individuals with at least one member with AD were identified. They were divided into four groups: multiplex AD (9 families, 82 members), simplex AD (34 families, 91 members), multiplex T1D (5 families, 32 members) and T1D with AD (14 families, 34 members). HLA haplotypes were remarkably restricted in this group of families with AD. Seventy-seven percent of unrelated probands had at least one DR3 and 47% had at least one DR4. Five of the seven unrelated probands who had neither DR3 nor DR4 were either DR2/2, DR2/5 or DR 5/5. The DR2 haplotype was concentrated in the simplex AD probands in which there was a 16% haplotype frequency of DR2 compared with 0% in the other groups (p=0.0015). Unrelated probands from multiplex AD families had an increase of the DRB1*0404, DQ8/ DR3 genotype (56%) compared with 10% of simplex AD probands (p≤0.01). Similarly, unrelated probands of multiplex T1D families had a higher rate of this genotype compared with simplex T1D probands (80% vs. 23%, p=0.047). T1D tended to occur at an increased frequency in the patients of multiplex AD families compared with the patients of the simplex AD families (24% vs. 3%, p=0.07). In conclusion, there is a restricted pattern of HLA haplotypes in these individuals with AD. DRB1*0404,DQ8/DR3 was highly associated with multiplex AD and T1D families. This may suggest that refined HLA analysis can identify groups of people with risk for AD approaching that found in the population with T1D, a group in which some centers are screening for the adrenal autoimmunity associated with AD.
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