Germ Cell Apoptosis Research Articles

The Impact of N-Acetylcysteine on Hypoxia-Induced Testicular Apoptosis in Male Rats: TUNEL and IHC Findings

The present study aimed to evaluate the impact of N-acetylcysteine (NAC) on testicular hypoxia caused by varicocele, focusing specifically on the regulation of genes related to apoptosis and oxidative stress in the testes of mature Wistar rats.Thirty-two rats were divided into four groups: Control (Sham), hypoxia, testicular hypoxia treated with NAC (Hypoxia + NAC), and healthy animals treated with NAC. After the 8-week treatment period, testicular histopathology and the levels of oxidative stress markers—superoxide dismutase (SOD), glutathione peroxidase (GPx), and malondialdehyde (MDA)—in serum were examined. The expression of Bax and Bcl-2 mRNA was analyzed using immunocytochemistry and RT-qPCR assays, while the apoptosis rate was determined using the TUNEL method.Histopathological evaluations showed that parameters such as Johnsen’s score, epithelium width, and seminiferous tubule diameter indicated significant improvement in the Hypoxia + NAC group compared to the Hypoxia group. NAC administration resulted in elevated serum levels of GPx and SOD, accompanied by a reduction in MDA levels (p < 0.003). Furthermore, the study revealed that NAC decreased Bax expression and enhanced Bcl-2 gene and protein expression compared to the varicocele group (p < 0.05). Additionally, NAC administration significantly decreased the rate of apoptosis in germ cells (p < 0.05).These findings suggest that NAC administration can mitigate testicular damage induced by hypoxia from varicocele in rats, primarily due to its antioxidant properties.

Beneficial effects of pentoxifylline on spermatogenesis and germ cell apoptosis in stallions subjected to scrotal heat stress

Beneficial effects of pentoxifylline on spermatogenesis and germ cell apoptosis in stallions subjected to scrotal heat stress

Reproductive Toxicity and Multi/Transgenerational Effects of Emerging Pollutants on C. elegans

Emerging pollutants (EPs) are receiving increasing attention due to the threats they pose to the environment and human health. As EPs continue to emerge, risk assessment requires many model animals. Caenorhabditis elegans (C. elegans) has been an outstanding toxicological model organism due to its growth and development characteristics. Particularly, in studying the transgenerational influences of EPs, C. elegans has advantages in saving time and cost due to its short generation cycle. As infertility has become a major problem in human reproductive health, reproductive toxicities of EPs on contemporary nematodes and across generations of C. elegans were introduced in this review. Moreover, the underlying mechanisms involved in germ cell apoptosis, spermatogenesis, and epigenetic alteration were discussed. Future research opportunities and challenges are also discussed to expand our understanding of the reproductive influences of EPs.

RNA-binding protein IGF2BP1 is required for spermatogenesis in an age-dependent manner.

Post-transcriptional regulation mediated by RNA binding proteins is crucial for male germline development. Insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1), an RNA binding protein, is specifically expressed in human and mouse male gonads and is involved in manifold biological processes and tumorigenesis. However, the function of IGF2BP1 in mammalian spermatogenesis remains poorly understood. Herein, we generated an Igf2bp1 conditional knockout mouse model using Nanos3-Cre. Germ cell deficiency of Igf2bp1 in mice caused spermatogenic defects in an age-dependent manner, resulting in decreased numbers of undifferentiated spermatogonia and increased germ cell apoptosis. Immunoprecipitation-mass spectrometry analysis revealed that ELAV-like RNA binding protein 1, a well-recognized mRNA stabilizer, interacted with IGF2BP1. Single cell RNA-sequencing showed distinct mRNA profiles in spermatogonia from conditional knockout versus wide type mice. Further research showed that IGF2BP1 plays a vital role in the modulation of spermatogenesis by regulating Lin28a mRNA, which is essential for clonal expansion of undifferentiated spermatogonia. Thus, our results highlight the crucial effects of IGF2BP1 on spermatogonia for the long-term maintenance of spermatogenesis.

BCL-2 and BAX expression and germ cell apoptosis following the intervention of 1-isothiocyanato-4-methylsulfinylbutane in cisplatin-induced testicular toxicity and sperm DNA fragmentation in Sprague-Dawley rat

Cisplatin (CP) has been used in clinical oncology but causes spermatogenesis damage. Isothiocyanato-4-methylsulfonylbutane (SFN) is a potent dietary bioactive agent that has been extensively studied for its effects on disease prevention. This study focused on the intervention of SFN on Germ cell apoptosis in CP-induced testicular toxicity and sperm DNA fragmentation (SDF). A total of ninety (90) male and ninety (90) female rats (weighing, 150–200 g, 12–14 weeks old) were assigned randomly into nine groups of ten (n = 10) rats each. Group A received normal saline, group B received a single dose of 10 mg/kg CP (i.p.), group C received 50 mg/kg bwt of SFN, group D received 100 mg/kg bwt of SFN, group E received 10 mg/kg bwt CP and 50 mg/kg bwt of SFN, group F received 10 mg/kg bwt CP and 100 mg/kg bwt of SFN, group G received 10 mg/kg bwt CP and 50 mg/kg bwt vitamin C, group H received 50 mg/kg bwt of SFN and 10 mg/kg bwt CP, Group I received 100 mg/kg bwt of SFN and 10 mg/kg bwt CP. The procedure lasted for 56 days. At the end of each treatment, the 90 male rats were introduced to the 90 female rats on the proestrus at a ratio of 1:1 for fertility tests. Testicular histopathological, apoptotic marker, immunoreactivity, sperm parameters, and SDF were investigated.Cisplatin significantly decreases chromatin condensation/de-condensation levels, haploid germ cells, the number of fetuses, and BCL-2 expression. Also, CP increases SDF and BAX expression relative to control. Treatment with SFN increased BCL-2 expression, haploid germ cells, protected sperm chromatin condensation, improved microarchitecture of testes, and decreased SDF and BAX expression.Therefore, SFN protects against CP-induced apoptosis by controlling BCL-2 and BAX expression and ameliorates SDF.

Aspirin enhances radio/chemo-therapy sensitivity in C. elegans by inducing germ cell apoptosis and suppresses RAS overactivated tumorigenesis via mtROS-mediated DNA damage and MAPK pathway

Previous studies have demonstrated that combination therapy involving radiotherapy and aspirin decreases the survival rate of cancer cells. However, the mechanism by which aspirin exerts its radiation sensitization effect at the in vivo level remains largely unclear. In this study, we employed Caenorhabditis elegans (C. elegans) as a model organism to investigate the effect of aspirin combined with radio/chemo-therapy on tumors at the individual level. Here, we illustrate that high-dose aspirin increases the expression of genes involved in core apoptosis pathways (egl-1, ced-9, ced-4 and ced-3) and induces germ cell apoptosis in C. elegans through mitochondrial outer membrane permeabilization (MOMP) and elevation of reactive oxygen species (ROS) levels. Crucially, aspirin-induces ROS upregulates the expression of genes critical for DNA damage response (hus-1, clk-2 and cep-1) and genes involved in MAPK pathways (lin-45, mek-2, mpk-1, sek-1 and pmk-1), thereby mediating the enhanced sensitivity of radio/chemo-therapy by aspirin. Notably, aspirin fails to induce germ cell apoptosis and enhance radio/chemo-therapy in C. elegans lacking the expression of each of those genes. Furthermore, in a C. elegans tumor-like symptom model, aspirin enhances radio/chemo-therapy sensitivity through ROS induction. However, low-dose aspirin can diminish the apoptotic signal of reproductive cells in C. elegans and exert anti-inflammatory effects. Our research results suggest that the tumor-suppressive and radio/chemo-therapy sensitizing effects of aspirin provide robust experimental evidence for improving the clinical efficacy of tumor radio/chemo-therapy and deepening our understanding of aspirin's mechanism of action in cancer.

Molecular mechanisms underlying the effects of hypo- and hyper-prolactinemia on spermatogenesis and fertility in male rats.

Hypo- and hyper-prolactinemia have deleterious effects on male reproduction, yet there is a dearth of information regarding the underlying mechanisms. The aim of this study was to delineate the molecular mechanisms by which hypo- and hyper-prolactinemia affects spermatogenesis and fertility in male rats. In vivo male rat models for hypo- and hyper-prolactinemia were established using dopamine receptor agonist, Bromocriptine (Brm), and antagonist, Fluphenazine (Flu), respectively. Effects on fertility and spermatogenesis were assessed by studying pre- and post-implantation loss, litter size, sperm parameters, hormonal profile, testicular histology, testicular cell population, and testicular transcriptome in rats. Treatment with Brm and Flu for 60 days led to subfertility, which was indicated by an increase in pre- and post-implantation loss and decrease in litter size, when mated with control female rats. Decreased sperm count was observed after both treatments, whereas reduced sperm motility was noted in Flu group. Serum FSH was unaffected, and LH was decreased by Flu treatment. Testosterone was decreased in both the groups, whereas estradiol was decreased in the Flu group. An arrest in spermatogenic cycle beyond round spermatids was observed in the Flu group. Additionally, testicular apoptosis in germ cells, mostly spermatocytes of Stage IX-XIV was noted in both the groups. Further, testicular RNA-Seq analysis revealed a total of 1539 and 824 differentially expressed genes/DEGs in Brm and Flu, respectively (Sequence Read Archive/SRA Database accession number: PRJNA1150513). Gene ontology and pathway analysis of DEGs highlighted enrichment of steroid metabolic pathway and ribosomal biogenesis pathway. Hub genes identified from the DEGs were validated by qPCR and the results showed that Uba52, Rps27a, Rpl23, Rps5, Rps16 were significantly down-regulated by Brm, whereas Rps27a, Rps29, Rps15, Rps27, Faul1 were significantly down-regulated by Flu. Hypo- and hyper-prolactinemia leads to subfertility and decreased sperm parameters possibly through an effect on steroid metabolism and ribosomal biogenesis pathway. Therefore, maintaining prolactin levels in physiological range is crucial.

Deficiency in the Rab25 gene leads to a decline in male fertility and testicular injury: Impact on the regulation of germ cell proliferation and apoptosis

BackgroundRab25 is a member of the Rab family, functioning as a regulatory molecule in intracellular transport. Although its involvement in cellular functions and disease development is well-established, its precise roles in male reproductive physiology remain elusive. MethodsTo explore the specific roles of Rab25 in testicular development and spermatogenesis, we established the Rab25−/− mouse model and Rab25 knockdown germ cell line (GC-2). We compared the fertility, sperm analysis, and testicular tissues between Rab25−/− and wild-type male mice. To delve deeper into potential mechanisms, we employed immunohistochemistry, TUNEL assay, Western Blotting, CCK-8 assay, etc. to evaluate cell proliferation and apoptosis in testicular tissues and GC-2 cells. ResultsOur findings indicated that Rab25 was expressed in germ cells and Leydig cells in the testes. Although the weight of Rab25−/− mice testes exhibited no significant changes, fertility was compromised, with a decrease in sperm quantity and reduced motility. HE staining revealed a disorganized arrangement of germ cells and vacuolization. Additionally, chromatin marginalization and nuclear pyknosis were observed in the Rab25−/− mice. In both Rab25−/− mice testes and Rab25 knockdown GC-2 cells, we found that germ cell proliferation was reduced, while apoptosis was increased. ConclusionsIn conclusion, our study proposes that Rab25 plays a vital role in spermatogenesis by regulating the proliferation and apoptosis of germ cells.

Exposure to benzyl butyl phthalate (BBP) leads to increased double-strand break formation and germline dysfunction in Caenorhabditis elegans.

Benzyl butyl phthalate (BBP), a plasticizer found in a wide range of consumer products including vinyl flooring, carpet backing, food packaging, personal care products, and children's toys, is an endocrine-disrupting chemical linked to impaired reproduction and development in humans. Despite evidence that BBP exposure perturbs the integrity of male and female gametes, its direct effect on early meiotic events is understudied. Here, using the nematode Caenorhabditis elegans, we show that BBP exposure elicits a non-monotonic dose response on the rate of X-chromosome nondisjunction measured using a high-throughput screening platform. From among the range of doses tested (1, 10, 100 and 500 μM BBP), we found that 10 μM BBP elicited the strongest effect on the germline, resulting in increased germ cell apoptosis and chromosome organization defects. Mass spectrometry analysis shows that C. elegans efficiently metabolizes BBP into its primary metabolites, monobutyl phthalate (MBP) and monobenzyl phthalate (MBzP), and that the levels of BBP, MBP, and MBzP detected in the worm are within the range detected in human biological samples. Exposure to 10 μM BBP leads to germlines with enlarged mitotic nuclei, altered meiotic progression, activation of a p53/CEP-1-dependent DNA damage checkpoint, increased double-strand break levels throughout the germline, chromosome morphology defects in oocytes at diakinesis, and increased oxidative stress. RNA sequencing analysis indicates that BBP exposure results in the altered expression of genes involved in xenobiotic metabolic processes, extracellular matrix organization, oocyte morphogenesis, meiotic cell cycle, and oxidoreduction. Taken together, we propose that C. elegans exposure to BBP leads to increased oxidative stress and double-strand break formation, thereby compromising germline genomic integrity and chromosome segregation.

Arecoline Hydrobromide Promotes the Apoptosis of Male Germ Cells

Arecoline Hydrobromide Promotes the Apoptosis of Male Germ Cells

Nutritional vitamin B12 regulates RAS/MAPK-mediated cell fate decisions through one-carbon metabolism

Vitamin B12 is an essential nutritional co-factor for the folate and methionine cycles, which together constitute one-carbon metabolism. Here, we show that dietary uptake of vitamin B12 modulates cell fate decisions controlled by the conserved RAS/MAPK signaling pathway in C. elegans. A bacterial diet rich in vitamin B12 increases vulval induction, germ cell apoptosis and oocyte differentiation. These effects are mediated by different one-carbon metabolites in a tissue-specific manner. Vitamin B12 enhances via the choline/phosphatidylcholine metabolism vulval induction by down-regulating fat biosynthesis genes and increasing H3K4 tri-methylation, which results in increased expression of RAS/MAPK target genes. Furthermore, the nucleoside metabolism and H3K4 tri-methylation positively regulate germ cell apoptosis and oocyte production. Using mammalian cells carrying different activated KRAS and BRAF alleles, we show that the effects of methionine on RAS/MAPK-regulated phenotype are conserved in mammals. Our findings suggest that the vitamin B12-dependent one-carbon metabolism is a limiting factor for diverse RAS/MAPK-induced cellular responses.

Benzo(a)pyrene exposure during pregnancy leads to germ cell apoptosis in male mice offspring via affecting histone modifications and oxidative stress levels

Infertility has gradually become a global health concern, and evidence suggests that exposure to environmental endocrine-disrupting chemicals (EDCs) represent one of the key causes of infertility. Benzo(a)pyrene (BaP) is a typical EDC that is widespread in the environment. Previous studies have detected BaP in human urine, semen, cervical mucus, oocytes and follicular fluid, resulting in reduced fertility and irreversible reproductive damage. However, the mechanisms underlying the effects of gestational BaP exposure on offspring fertility in male mice have not been fully explored. In this study, pregnant mice were administered BaP at doses of 0, 5, 10 and 20 mg/kg/day via gavage from Days 7.5 to 12.5 of gestation. The results revealed that BaP exposure during pregnancy disrupted the structural integrity of testicular tissue, causing a disorganized arrangement of spermatogenic cells, compromised sperm quality, elevated levels of histone modifications and increased apoptosis in the testicular tissue of F1 male mice. Furthermore, oxidative stress was also increased in the testicular tissue of F1 male mice. BaP activated the AhR/ERα signaling pathway, affected H3K4me3 expression and induced apoptosis in testicular tissue. AhR and Cyp1a1 were overexpressed, and the expression of key molecules in the antioxidant pathway, including Keap1 and Nrf2, was reduced. The combined effects of these molecules led to apoptosis in testicular tissues, damaging and compromising sperm quality. This impairment in testicular cells further contributed to compromised testicular tissues, ultimately impacting the reproductive health of F1 male mice.

Trehalose attenuates testicular aging by activating autophagy and improving mitochondrial quality.

Reproductive aging can adversely affect male fertility and the health of offspring. The aging process is accompanied by impaired autophagy. Recent studies have shown that Trehalose plays an important role in the prevention of various diseases by regulating autophagy. However, the roles of Trehalose in testicular aging and reproductive decline remain to be clarified. The present study aimed to evaluate the protective effects of Trehalose on testes in an aging mouse model. In this study, an in vivo aging model in mice by administering D-galactose was established to explore the protective effect of Trehalose on testicular aging. We examined histological changes and related indicators of apoptosis, autophagy, mitochondrial biogenesis, and sperm quality. D-galactose treatment induced oxidative stress, apoptosis, and impairment of autophagy of testicular cells in mouse testes. Trehalose administration significantly reduced germ cell apoptosis and DNA damage caused by D-galactose-induced oxidative stress. Notably, Trehalose activated autophagy activity and improved mitochondrial function in testicular cells. Furthermore, Trehalose treatment increased the expression level of the tight junction protein ZO-1, and accelerated clearance of damaged mitochondria in Sertoli cells, indicating that Trehalose ameliorated Sertoli cell function in D-galactose-induced aging testes. These findings suggest that Trehalose administration activated the autophagy activity in testicular cells and improved mitochondrial function, thereby effectively preventing testicular aging. Trehalose and its activated autophagy are crucial for preventing testicular aging, thus restoring autophagy activity by administering Trehalose could be a promising means to delay aging.

Actomyosin-mediated apical constriction promotes physiological germ cell death in C. elegans.

Germ cell apoptosis in Caenorhabditis elegans hermaphrodites is a physiological process eliminating around 60% of all cells in meiotic prophase to maintain tissue homeostasis. In contrast to programmed cell death in the C. elegans soma, the selection of germ cells undergoing apoptosis is stochastic. By live-tracking individual germ cells at the pachytene stage, we found that germ cells smaller than their neighbors are selectively eliminated through apoptosis before differentiating into oocytes. Thus, cell size is a strong predictor of physiological germ cell death. The RAS/MAPK and ECT/RHO/ROCK pathways together regulate germ cell size by controlling actomyosin constriction at the apical rachis bridges, which are cellular openings connecting the syncytial germ cells to a shared cytoplasmic core. Enhancing apical constriction reduces germ cell size and increases the rate of cell death while inhibiting the actomyosin network in the germ cells prevents their death. We propose that actomyosin contractility at the rachis bridges of the syncytial germ cells amplifies intrinsic disparities in cell size. Through this mechanism, the animals can adjust the balance between physiological germ cell death and oocyte differentiation.

Cadmium exposure induces multigenerational inheritance of germ cell apoptosis and fertility suppression in Caenorhabditis elegans

Cadmium is a significant environmental pollutant that poses a substantial health hazard to humans due to its propensity to accumulate in the body and resist excretion. We have a comprehensive understanding of the damage caused by Cd exposure and the mechanisms of tolerance, however, the intricate mechanisms underlying multigenerational effects resulting from Cd exposure remain poorly understood. In this study, Caenorhabditis elegans were used as a model organism to investigate Cd-induced multigenerational effects and its association with epigenetic modifications. The results showed that Cd exposure leads to an increase in germ cell apoptosis and a decrease in fertility, which can be passed down to subsequent generations. Further analysis revealed that transcription factors DAF-16/FOXO and SKN-1/Nrf2 play essential roles in responding to Cd exposure and in the transgenerational induction of germ cell apoptosis. Additionally, histone H3K4 trimethylation (H3K4me3) marks stress-responsive genes and enhances their transcription, ultimately triggering multigenerational germ cell apoptosis. This study provides compelling evidence that the detrimental effects of Cd on the reproductive system can be inherited across generations. These findings enhance our understanding of the multigenerational effects of environmental pollutants and may inform strategies for the prevention and control of such pollutants.

EHDPP induces proliferation inhibition and apoptosis to spermatocyte: Insights from transcriptomic and metabolomic profiles

Background2-ethylhexyldiphenyl phosphate (EHDPP) was used widespread in recent years and it was reported to impair reproductive behaviors and decrease fertility in male Japanese medaka. However, whether EHDPP causes spermatogenesis disturbance remains uncertain. ObjectivesWe aimed to study the male reproductive toxicity of EHDPP and its related mechanism. MethodsHuman spermatocyte cell line GC-2 was treated with 10 µM, 50 µM or 100 µM EHDPP for 24 h. Male CD-1 mice aged 6 weeks were given 1, 10, or 100 mg/kg/d EHDPP daily for 42 days and then euthanized to detect sperm count and motility. Proliferation, apoptosis, oxidative stress was detected in mice and cell lines. Metabolome and transcriptome were used to detect the related mechanism. Finally, anti-oxidative reagent N-Acetylcysteine was used to detect whether it could reverse the side-effect of EHDPP both in vivo and in vitro. ResultsOur results showed that EHDPP inhibited proliferation and induced apoptosis in mice testes and spermatocyte cell line GC-2. Metabolome and transcriptome showed that nucleotide metabolism disturbance and DNA damage was potentially involved in EHDPP-induced reproductive toxicity. Finally, we found that excessive ROS production caused DNA damage and mitochondrial dysfunction; NAC supplement reversed the side effects of EHDPP such as DNA damage, proliferation inhibition, apoptosis and decline in sperm motility. ConclusionROS-evoked DNA damage and nucleotide metabolism disturbance mediates EHDPP-induced germ cell proliferation inhibition and apoptosis, which finally induced decline of sperm motility.

A testis-specific lncRNA functions as a post-transcriptional regulator of MDM2 and stimulates apoptosis of testicular germ cell tumor cells

Germ cells preferentially induce apoptosis in response to DNA damage to avoid genomic mutations. Apoptosis of germ cells is closely related to cancer development and chemotherapy resistance; however, its regulatory mechanism is unclear. Here, we suggest that testis-specific lncRNA LINC03074 is involved in male germ cell apoptosis by regulating the expression of the proto-oncogene MDM2. LINC03074 is highly expressed in the sperm of healthy adult testes and cancer cells of testes with testicular germ cell tumors (TGCTs). LINC03074 binds to MDM2 mRNA via an Alu element, thereby reducing MDM2 protein levels. LINC03074 stimulates STAU1-mediated nuclear export of MDM2 mRNA by increasing STAU1 binding to MDM2 mRNA in the cell nucleus, thereby promoting PKR-mediated translational repression in the cytoplasm. The induction of apoptosis in TGCT cells and their responsiveness to the anticancer drug cisplatin is enhanced by LINC03074. Notably, LINC03074 increased E2F1 expression without increasing p53, the primary target of MDM2, and upregulated the apoptotic gene p73, the target gene of E2F1. LINC03074-mediated regulation of apoptosis contributes to the responsiveness of TGCTs to anticancer drug-induced DNA damage.

The effects of olibanum on male reproductive system damage in a lipopolysaccharide induced systemic inflammation model in rat

BackgroundLipopolysaccharide (LPS) as a particle of Gram-negative bacteria is a main contributer in the pathogenesis of the male reproductive system infectious. Male infertility due to LPS is reported to be related to overproduction reactive oxygen species. This study aimed to investigate the effects of olibanum on oxidative stress and apoptosis in testes and sperm dysfunction induced by LPS. MethodsThe male (n = 28) rats were allocated in four groups: control, LPS (1 mg/kg, i.p., 14 days), LPS + Olibanum 100 (100 mg/kg, i.p., 14 days), and LPS + Olibanum 200 (200 mg/kg, i.p., 14 days). Germ cell apoptosis was determined by TUNEL assays and computed using the stereological method. Additionally, semen samples of the animals were analyzed for sperm count and morphology. Oxidative stress indicators were also determined. ResultsThe count of TUNEL-positive germ cells in LPS-treated rats was more than that in the controls. Treatment of the animals with olibanum significantly attenuated the number of apoptotic cells compared to the LPS group. The sperm count and those with a normal morphology in LPS-treated rats was lower than that in the controls. Administration of olibanum significantly improved the sperms with normal morphology and sperm count. Olibanum treatment also improved superoxide dismutase, catalase, and total thiol in testicular tissue and decreased malondialdehyde. ConclusionAdministering both doses of olibanum in LPS-treated rats had potentially a therapeutic value in reducing germ cell apoptosis, as well as improving sperm parameters.

Heat Shock Related Protein Expression in Abdominal Testes of Asian Elephant (Elephas maximus).

The abdominal testes of Asian elephants show normal spermatogenesis. Heat shock in cryptorchid testes elevates heat shock factor (HSF) expression, leading to germ cell apoptosis, while increased heat shock proteins (HSPs) levels provide protection. To investigate how heat shock affects elephant spermatogenic cells, focusing on heat shock-related molecules and the cell death mechanism, immunohistochemistry and TUNEL staining were employed to assess the immunoexpression of several heat shock-related molecules and the status of apoptosis in elephant fibroblasts (EF) induced by heat shock stimulus. Additionally, the immunoexpression of heat shock-related molecules and cell proliferation status in the elephant spermatogenic cells. Our finding indicated that heat shock-induced HSF1 immunoexpression in EF leads to apoptosis mediated by T-cell death-associated gene 51 (TDAG51) while also upregulating HSP70 to protect damaged cells. In elephant spermatogenic cells, immunostaining revealed a predominance of proliferating cell nuclear antigen (PCNA)-positive cells with minimal TDAG51- and TUNEL-positive cells, suggesting active proliferation and apoptosis suppression during normal spermatogenesis in the abdominal testis. Interestingly, spermatogonia co-immunoexpressed HSF1 and HSP90, potentially reducing apoptosis through protective mechanisms different from those observed in other mammals. Spermatogenic cells did not show immunolocalisation of HSP70, and hence, it may not contribute to protecting the spermatogonia from heat shock because the transcriptional activity of HSF1 is suppressed by HSP90A binding. This study provides insight into the specific heat shock response and defence mechanisms in elephant spermatogenic cells and may contribute to our understanding of species-specific adaptation to environmental stresses of the testis.

Nur77 improves ovarian function in reproductive aging mice by activating mitophagy and inhibiting apoptosis

Reproductive aging not only affects the fertility and physical and mental health of women but also accelerates the aging process of other organs. There is an urgent need newfor novel mechanisms, targets, and drugs to break the vicious cycle of mitochondrial dysfunction, redox imbalance, and germ cell apoptosis associated with ovarian aging. Autophagy, recognized as a longevity mechanism, has recently become a focal point in anti-aging research. Although mitophagy is a type of autophagy, its role and regulatory mechanisms in ovarian aging, particularly in age-related ovarian function decline, remain unclear. Nerve growth factor inducible gene B (Nur77) is an early response gene that can be stimulated by oxidative stress, DNA damage, metabolism, and inflammation. Recent evidence recommends that decreased expression of Nur77 is associated with age-related myocardial fibrosis, renal dysfunction, and Parkinson's disease; however, its association with ovarian aging has not been studied yet. We herein identified Nur77 as a regulator of germ cell senescence, apoptosis, and mitophagy and found that overexpression of Nur77 can activate mitophagy, improve oxidative stress, reduce apoptosis, and ultimately enhance ovarian reserve in aged mice ovaries. Furthermore, we discovered an association between Nur77 and the AKT pathway through String and molecular docking analyses. Experimental confirmation revealed that the AKT/mTOR signaling pathway is involved in the regulation of Nur77 in ovarian function. In conclusion, our results suggest Nur77 as a promising target for preventing and treating ovarian function decline related to reproductive aging.