Genetic and epigenetic changes in sperm caused by male aging may be essential factors affecting semen parameters, but the effects and specific molecular mechanisms of aging on male reproduction have not been fully clarified. In this study, to explore the effect of aging on male fertility and seek the potential molecular etiology, we performed high-throughput RNA-sequencing in isolated spermatogenic cells, including pachytene spermatocytes (marked by the completion of chromosome synapsis) and round spermatids (produced by the separation of sister chromatids) from the elderly and the young men. Functional enrichment analysis of differentially expressed genes (DEGs) in round spermatids between the elderly and young showed that they were significantly enriched in gamete generation, spindle assembly, and cilium movement involved in cell motility. In addition, the expression levels of DEGs in round spermatids (post-meiotic cells) were found to be more susceptible to age. Furthermore, ten genes (AURKA, CCNB1, CDC20, CCNB2, KIF2C, KIAA0101, NR5A1, PLK1, PTTG1, RAD51AP1) were identified to be the hub genes involved in the regulation of sperm quality in the elderly through Protein-Protein Interaction (PPI) network construction and measuring semantic among GO terms and gene products. Our data provide aging-related molecular alterations in meiotic and post-meiotic spermatogenic cells, and the information gained from this study may explain the abnormal aging-related male fertility decline.

Some X-linked genes necessary for spermiogenesis are specifically activated in the postmeiotic germ cells. However, the regulatory mechanism about this activation is not clearly understood. Here, we examined the potential mechanism controlling the transcriptional activation of the mouse testis specific gene A8 (Tsga8) gene in round spermatids. We observed that the Tsga8 expression was negatively correlated with the methylation level of the CpG sites in its core promoter. During spermatogenesis, the Tsga8 promoter was methylated in spermatogonia, and then demethylated in spermatocytes. The demethylation status of Tsga8 promoter was maintained through the postmeiotic germ cells, providing a potentially active chromatin for Tsga8 transcription. In vitro investigation showed that the E12 and Spz1 transcription factors can enhance the Tsga8 promoter activity by binding to the unmethylated E-box motif within the Tsga8 promoter. Additionally, the core Tsga8 promoter drove green fluorescent protein (GFP) expression in the germ cells of Tsga8-GFP transgenic mice, and the GFP expression pattern was similar to that of endogenous Tsga8. Moreover, the DNA methylation profile of the Tsga8-promoter-driven transgene was consistent with that of the endogenous Tsga8 promoter, indicating the existence of a similar epigenetic modification for the Tsga8 promoter to ensure its spatiotemporal expression in vivo. Taken together, this study reports the details of a regulatory mechanism that includes DNA methylation and transcription factors to mediate the postmeiotic expression of an X-linked gene.

UNASSIGNED: To investigate the application value of the frozen-thawed round spermatids of the mouse in in vitro fertilization (IVF).
METHODS: Haploid spermatids of the mouse obtained in vitro were divided into a frozen-thawed and a fresh group and oocytes were collected from 6－8 weeks old female mice. After diamidino-phenyl-indole (DAPI) staining, the oocytes were subjected to intracytoplasmic round spermatid injection (ROSI), 259 in the frozen-thawed and 238 in the fresh group. Comparisons were made between the two groups in the capacities of fertilization and embryonic development.
RESULTS: The survival rate of the frozen-thawed haploid spermatids was (75.9 ± 2.3) %. No statistically significant differences were observed between the frozen-thawed and fresh groups after ROSI in the rates of fertilization (51.9 vs 55.7%, P >0.05), 2-cell embryos (51.0 vs 62.2%, P >0.05), 4－8-cell embryos (41.8 vs 42.9%, P >0.05), or morula-blastocysts (12.2 vs 21.4%, P >0.05).
CONCLUSIONS: Frozen-thawed round spermatids of the mouse are similar to fresh ones in their capacities of fertilization and embryonic development.
目的： 探讨冻融小鼠圆形精子细胞在体外受精中的应用价值。方法： 体外培养获得的小鼠单倍体精子细胞冻融复苏后，经 4\'，6-二眯基-2-苯基吲哚(DAPI)荧光染料标记后，行小鼠卵胞质内圆形精子细胞显微注射(ROSI) (冻融组，n=259)，同时以体外培养获得的新鲜单倍体精子细胞行ROSI (新鲜组，n=238)作对照，比较两组精子细胞受精能力和后续胚胎发育能力。结果： 体外培养获得的小鼠单倍体精子细胞冻融后存活率为(75.9±2.3)%。冻融组与新鲜组受精率(51.9% vs 55.7%)、2细胞率(51.0% vs 62.2%)、4~8细胞率(41.8% vs 42.9%)及桑囊胚形成率(12.2% vs 21.4%)均无显著性差异(P>0.05)。结论： 冻融复苏后的小鼠圆形精子细胞与冷冻前相似，具有一定的受精和胚胎发育能力。.