Discovery and evaluation of novel spiroheterocyclic protective agents via a SIRT1 upregulation mechanism in cisplatin-induced premature ovarian failure.

Currently, no effective treatment exists for premature ovarian failure (POF). To obtain compounds with protective effects against POF, we aimed to design and synthesize a series of spiroheterocyclic protective agents with a focus on minimizing toxicity while enhancing their protective effect against cisplatin-induced POF. This was achieved through systematic modifications of Michael receptors and linkers within the molecular structure of 1,5-diphenylpenta-1,4-dien-3-one analogs. To assess the cytotoxicity and activity of these compounds, we constructed quantitative conformational relationship models using an artificial intelligence random forest algorithm, resulting in R2 values exceeding 0.87. Among these compounds, j2 exhibited optimal protective activity. It significantly increased the survival of cisplatin-injured ovarian granulosa KGN cells, improved post-injury cell morphology, reduced apoptosis, and enhanced cellular estradiol (E2) levels. Subsequent investigations revealed that j2 may exert its protective effect via a novel mechanism involving the activation of the SIRT1/AKT signal pathway. Furthermore, in cisplatin-injured POF in rats, j2 was effective in increasing body, ovarian, and uterine weights, elevating the number of follicles at all levels in the ovary, improving ovarian and uterine structures, and increasing serum E2 levels in rats with cisplatin-injured POF. In conclusion, this study introduces a promising compound j2 and a novel target SIRT1 with substantial protective activity against cisplatin-induced POF.

Roscovitine and Trichostatin A promote DNA damage repair during porcine oocyte maturation.

Faithful repair of DNA double-strand breaks in mammalian oocytes is essential for meiotic maturation and embryonic development. In the present study we investigated the roles of Roscovitine and Trichostatin A (TSA) in DNA damage recovery during invitro maturation of porcine oocytes. Etoposide was used to trigger DNA damage in oocytes. When these DNA-damaged oocytes were treated with 2µM Roscovitine, 50nM TSA or both for 22h, first polar body extrusion and blastocyst formation in all treated groups were significantly improved compared with the etoposide-only group. The most significant improvement was observed when Roscovitine was present. Further immunofluorescent analysis of γH2A.X, an indicator of DNA damage, indicated that DNA damage was significantly decreased in all treated groups. This observation was further supported by analysing the relative mRNA abundance of DNA repair-related genes, including meiotic recombination 11 homolog A (MRE11A), breast cancer type 1 susceptibility protein (BRCA1), Recombinant DNA Repair Protein 51 (RAD51), DNA-dependent protein kinase catalytic subunit (PRKDC) and X-ray cross complementing gene 4 (XRCC4). Compared with the etoposide-only group, the experimental group with combined treatment of Roscovitine and TSA showed a significant decrease of all genes at germinal vesicle and MII stages. The Roscovitine-only treatment group revealed a similar tendency. Together, these results suggest that Roscovitine and TSA treatments could increase the capacity of oocytes to recover from DNA damage by enlisting DNA repair processes.

Effect of EZH2 knockdown on preimplantation development of porcine parthenogenetic embryos.

The EZH2 protein endows the polycomb repressive complex 2 (PRC2) with histone lysine methyltransferase activity that is associated with transcriptional repression. Recent investigations have documented crucial roles for EZH2 in mediating X-inactivation, stem cell pluripotency and cancer metastasis. However, there is little evidence demonstrating the maternal effect of EZH2 on porcine preimplantation development. Here, we took parthenogenetic activation embryos to eliminate the confounding paternal influence. We showed that the dynamic expression of EZH2 during early development was accompanied by changes in H3K27me3 levels. Depletion of EZH2 in MII oocytes by small interfering RNA not only impaired embryonic development at the blastocyst stage (P < 0.05), but also disrupted the equilibrium of H3K4me3 and H3K27me3 in the embryo. Interestingly, the expression of TET1, a member of Ten-Eleven Translocation gene family for converting 5-methylcytosine (5 mC) to 5-hydroxymethylcytosine (5hmC), was decreased after EZH2 knockdown, in contrast to the increase of the other two members, TET2 and TET3 (P < 0.05). These results indicate a correlation between histone methylation and DNA methylation, and between EZH2 and TET1. Along with the downregulation of TET1, the expression of the pluripotency gene NANOG was decreased (P < 0.05), which is consistent with a previous finding in mouse ES cells. Meanwhile, the abundance of OCT4 and SOX2 were also down-regulated. Moreover, EZH2 knockdown reduced the capacity of cells in the blastocysts to resist apoptosis. Taken together, our data suggest that EZH2 is integral to the developmental program of porcine parthenogenetic embryos and exerts its function by regulating pluripotency, differentiation and apoptosis.