Sestrin2 and Sestrin3 protect spermatogenesis against heat-induced meiotic defects†.

Heat stress induces testicular oxidative stress, impairs spermatogenesis, and increases the risk of male infertility. Recent studies have highlighted the antioxidative properties of the Sestrins family in reducing cellular oxidative damage. However, the role of Sestrins (Sestrin1, 2, and 3) in the testicular response to heat stress remains unclear. Here, we found that Sestrin2 and 3 were highly expressed in the testis relative to Sestrin1. Then, the Sestrin2-/- and Sestrin3-/- mice were generated by CRISPR/Cas9 to investigate the role of them on spermatogenesis after heat stress. Our data showed that Sestrin2-/- and Sestrin3-/- mice testes exhibited more severe damage manifested by exacerbated loss of germ cells and higher levels of oxidative stress as compared to wild-type counterparts after heat stress. Notably, Sestrin2-/- and Sestrin3-/- mice underwent a remarkable increase in heat-induced spermatocyte apoptosis than that of controls. Furthermore, the transcriptome landscape of spermatocytes and chromosome spreading showed that loss of Sestrin2 and Sestrin3 exacerbated meiotic failure by compromising DNA double-strand breaks repair after heat stress. Taken together, our work demonstrated a critical protective function of Sestrin2 and Sestrin3 in mitigating the impairments of spermatogenesis against heat stress.

Meiotic defects in human oocytes: Potential causes and clinical implications.

Meiotic defects cause abnormal chromosome segregation leading to aneuploidy in mammalian oocytes. Chromosome segregation is particularly error-prone in human oocytes, but the mechanisms behind such errors remain unclear. To explain the frequent chromosome segregation errors, recent investigations have identified multiple meiotic defects and explained how these defects occur in female meiosis. In particular, we review the causes of cohesin exhaustion, leaky spindle assembly checkpoint (SAC), inherently unstable meiotic spindle, fragmented kinetochores or centromeres, abnormal aurora kinases (AURK), and clinical genetic variants in human oocytes. We mainly focus on meiotic defects in human oocytes, but also refer to the potential defects of female meiosis in mouse models.

Decreased HAT1 expression in granulosa cells disturbs oocyte meiosis during mouse ovarian aging.

BACKGROUND: With advanced maternal age, abnormalities during oocyte meiosis increase significantly. Aneuploidy is an important reason for the reduction in the quality of aged oocytes. However, the molecular mechanism of aneuploidy in aged oocytes is far from understood. Histone acetyltransferase 1 (HAT1) has been reported to be essential for mammalian development and genome stability, and involved in multiple organ aging. Whether HAT1 is involved in ovarian aging and the detailed mechanisms remain to be elucidated. METHODS: The level of HAT1 in aged mice ovaries was detected by immunohistochemical and immunoblotting. To explore the function of HAT1 in the process of mouse oocyte maturation, we used Anacardic Acid (AA) and small interfering RNAs (siRNA) to culture cumulus-oocyte complexes (COCs) from ICR female mice in vitro and gathered statistics of germinal vesicle breakdown (GVBD), the first polar body extrusion (PBE), meiotic defects, aneuploidy, 2-cell embryos formation, and blastocyst formation rate. Moreover, the human granulosa cell (GC)-like line KGN cells were used to investigate the mechanisms of HAT1 in this progress. RESULTS: HAT1 was highly expressed in ovarian granulosa cells (GCs) from young mice and the expression of HAT1 was significantly decreased in aged GCs. AA and siRNAs mediated inhibition of HAT1 in GCs decreased the PBE rate, and increased meiotic defects and aneuploidy in oocytes. Further studies showed that HAT1 could acetylate Forkhead box transcription factor O1 (FoxO1), leading to the translocation of FoxO1 into the nucleus. Resultantly, the translocation of acetylated FoxO1 increased the expression of amphiregulin (AREG) in GCs, which plays a significant role in oocyte meiosis. CONCLUSION: The present study suggests that decreased expression of HAT1 in GCs is a potential reason corresponding to oocyte age-related meiotic defects and provides a potential therapeutic target for clinical intervention to reduce aneuploid oocytes.

Glutathione ameliorates the meiotic defects of copper exposed ovine oocytes via inhibiting the mitochondrial dysfunctions.

Regardless of the essential role of copper (Cu) in the physiological regulation process of mammalian reproduction, excessive exposure to Cu triggers the meiotic defects of porcine oocytes via compromising the mitochondrial functions. However, the connections between the excessive Cu exposure and meiotic defects of ovine oocytes have not been reported. In this study, the effect of copper sulfate (CuSO4) exposure on the meiotic potentials of ovine oocytes was analyzed. Subsequently, the ameliorative effect of glutathione (GSH) supplementation on the meiotic defects of CuSO4 exposed ovine oocytes was investigated. For these purposes, the in vitro maturation (IVM) of ovine cumulus oocyte complexes (COCs) was conducted in the presence of 5, 10, 20 and 40 µg/mL of CuSO4 supplementation. Subsequently, different concentrations of GSH (2, 4 and 8 mM) were added to the IVM medium containing CuSO4 solution. After IVM, the assay, including nuclear maturation, spindle organization, chromosome alignment, cytoskeleton assembly, cortical granule (CGs) dynamics, mitochondrial function, reactive oxygen species (ROS) generation, apoptosis, epigenetic modification and fertilization capacity of ovine oocytes were performed. The results showed that excessive Cu exposure triggered the meiotic defects of ovine oocytes via promoting the mitochondrial dysfunction related oxidative stress damage. Moreover, the GSH supplementation, not only ameliorated the decreased maturation potential and fertilization defect of CuSO4 exposed oocytes, but inhibited the mitochondrial dysfunction related oxidative stress damage, ROS generation, apoptosis and altered H3K27me3 expression in the CuSO4 exposed oocytes. Combined with the gene expression pattern, the finding in the present study provided fundamental bases for the ameliorative effect of GSH supplementation on the meiotic defects of CuSO4 exposed oocytes via inhibiting the mitochondrial dysfunctions, further benefiting these potential applications of GSH supplementation in the mammalian IVM system and livestock breeding suffering from the excessive Cu exposure.

Butylparaben weakens female fertility via causing oocyte meiotic arrest and fertilization failure in mice.

Butylparaben is an ubiquitous environmental endocrine disruptor, that is commonly used in cosmetics and personal care product due to its anti-microbial properties. Butylparaben has been shown to cause developmental toxicity, endocrine and metabolic disorders and immune diseases. However, little is known about the impact on female fertility, especially oocyte quality. In the present study, we reported that butylparaben influenced female fertility by showing the disturbed oocyte meiotic capacity and fertilization potential. Specifically, butylparaben results in the oocyte maturation arrest by impairing spindle/chromosome structure and microtubule stability. Besides, butylparaben results in fertilization failure by impairing the dynamics of Juno and ovastacin and the sperm binding ability. Last, single-cell transcriptome analysis showed that butylparaben-induced oocyte deterioration was caused by mitochondrial dysfunction, which led to the accumulation of ROS and occurrence of apoptosis. Collectively, our study indicates that mitochondrial dysfunction and redox perturbation is the major cause of the weakened female fertility expoesd to butylparaben.

IGF2 reduces meiotic defects in oocytes from obese mice and improves embryonic developmental competency.

BACKGROUND: Maternal obesity is a global issue that has devastating effects across the reproductive spectrum such as meiotic defects in oocytes, consequently worsening pregnancy outcomes. Different studies have shown that such types of meiotic defects originated from the oocytes of obese mothers. Thus, there is an urgent need to develop strategies to reduce the incidence of obesity-related oocyte defects that adversely affect pregnancy outcomes. Multiple growth factors have been identified as directly associated with female reproduction; however, the impact of various growth factors on female fertility in response to obesity remains poorly understood. METHODS: The immature GV-stage oocytes from HFD female mice were collected and cultured in vitro in two different groups (HFD oocytes with and without 50 nM IGF2), however; the oocytes from ND mice were used as a positive control. HFD oocytes treated with or without IGF2 were further used to observe the meiotic structure using different analysis including, the spindle and chromosomal analysis, reactive oxygen species levels, mitochondrial functional activities, and early apoptotic index using immunofluorescence. Additionally, the embryonic developmental competency and embryos quality of IGF2-treated zygotes were also determined. RESULTS: In our findings, we observed significantly reduced contents of insulin-like growth factor 2 (IGF2) in the serum and oocytes of obese mice. Our data indicated supplementation of IGF2 in a culture medium improves the blastocyst formation: from 46% in the HFD group to 61% in the HFD + IGF2-treatment group (50 nM IGF2). Moreover, adding IGF2 to the culture medium reduces the reactive oxygen species index and alleviates the frequency of spindle/chromosome defects. We found increased mitochondrial functional activity in oocytes from obese mice after treating the oocytes with IGF2: observed elevated level of adenosine triphosphate, increased mitochondrial distribution, higher mitochondrial membrane potentials, and reduced mitochondrial ultrastructure defects. Furthermore, IGF2 administration also increases the overall protein synthesis and decreases the apoptotic index in oocytes from obese mice. CONCLUSIONS: Collectively, our findings are strongly in favor of adding IGF2 in culture medium to overcome obesity-related meiotic structural-developmental defects by helping ameliorate the known sub-optimal culturing conditions that are currently standard with assisted reproduction technologies.

Altered expression of Aurora kinases in Arabidopsis results in aneu- and polyploidization.

Aurora is an evolutionary conserved protein kinase family involved in monitoring of chromosome segregation via phosphorylation of different substrates. In plants, however, the involvement of Aurora proteins in meiosis and in sensing microtubule attachment remains to be proven, although the downstream components leading to the targeting of spindle assembly checkpoint signals to anaphase-promoting complex have been described. To analyze the three members of Aurora family (AtAurora1, -2, and -3) of Arabidopsis we employed different combinations of T-DNA insertion mutants and/or RNAi transformants. Meiotic defects and the formation of unreduced pollen were revealed including plants with an increased ploidy level. The effect of reduced expression of Aurora was mimicked by application of the ATP-competitive Aurora inhibitor II. In addition, strong overexpression of any member of the AtAurora family is not possible. Only tagged or truncated forms of Aurora kinases can be overexpressed. Expression of truncated AtAurora1 resulted in a high number of aneuploids in Arabidopsis, while expression of AtAurora1-TAPi construct in tobacco resulted in 4C (possible tetraploid) progeny. In conclusion, our data demonstrate an essential role of Aurora kinases in the monitoring of meiosis in plants.

Nicotinamide mononucleotide supplementation improves the quality of porcine oocytes under heat stress.

BACKGROUND: Elevated ambient temperature-caused heat stress is a major concern for livestock production due to its negative impact on animal feed intake, growth, reproduction, and health. Particularly, the germ cells are extremely sensitive to the heat stress. However, the effective approach and strategy regarding how to protect mammalian oocytes from heat stress-induced defects have not been determined. METHODS: Germinal vesicle (GV) porcine oocytes were cultured at 41.5 °C for 24 h to induce heat stress, and then cultured at 38.5 °C to the specific developmental stage for subsequent analysis. Nicotinamide mononucleotide (NMN) was dissolved in water to 1 mol/L for a stock solution and further diluted with the maturation medium to the final concentrations of 10 µmol/L, 20 µmol/L, 50 µmol/L or 100 µmol/L, respectively, during heat stress. Immunostaining and fluorescence intensity quantification were applied to assess the effects of heat stress and NMN supplementation on the key processes during the oocyte meiotic maturation. RESULTS: Here, we report that NMN supplementation improves the quality of porcine oocytes under heat stress. Specifically, we found that heat stress resulted in oocyte maturation failure by disturbing the dynamics of meiotic organelles, including the cytoskeleton assembly, cortical granule distribution and mitochondrial function. In addition, heat stress induced the production of excessive reactive oxygen species (ROS) and DNA damage, leading to the occurrence of apoptosis in oocytes and subsequent embryonic development arrest. More importantly, we validated that supplementation of NMN during heat stress restored the meiotic defects during porcine oocyte maturation. CONCLUSIONS: Taken together, our study documents that NMN supplementation is an effective approach to improve the quality of oocytes under heat stress by promoting both nuclear and cytoplasmic maturation.