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.

The in vivo comet assay is widely used to measure genotoxicity; however, the current OECD test guideline (TG 489) does not recommend using the assay to assess testicular germ cells, due to the presence of testicular somatic cells. An adapted approach to specifically assess testicular germ cells within the comet assay is certainly warranted, considering regulatory needs for germ cell-specific genotoxicity data in relation to the increasing global production of and exposure to potentially hazardous chemicals. Here, we provide a proof-of-concept to selectively analyze round spermatids and primary spermatocytes, distinguishing them from other cells of the testicle. Utilizing the comet assay recordings of DNA content (total fluorescence intensity) and DNA damage (% tail intensity) of individual comets, we developed a framework to distinguish testicular cell populations based on differences in DNA content/ploidy and appearance. Haploid round spermatid comets are identified through (1) visual inspection of DNA content distributions, (2) setting DNA content thresholds, and (3) modeling DNA content distributions using a normal mixture distribution function. We also describe an approach to distinguish primary spermatocytes during comet scoring, based on their high DNA content and large physical size. Our concept allows both somatic and germ cells to be analyzed in the same animal, adding a versatile, sensitive, rapid, and resource-efficient assay to the limited genotoxicity assessment toolbox for germ cells. An adaptation of TG 489 facilitates accumulation of valuable information regarding distribution of substances to germ cells and their potential for inducing germ cell gene mutations and structural chromosomal aberrations.

Spermatogenesis is tightly controlled at transcriptional, post-transcriptional, and epigenetic levels by various regulators, including miRNAs. This study deals with the identification of miRNAs critical to the three important stages of germ cell development (spermatocytes, round spermatids, and mature sperm) during spermatogenesis. We used high-throughput transcriptome sequencing to identify the differentially expressed miRNAs in the pachytene spermatocytes, round spermatids, and mature sperm of rat. We identified 1843 miRNAs that were differentially expressed across the three stages of germ cell development. These miRNAs were further categorized into three classes according to their pattern of expression during spermatogenesis: class 1 - miRNAs found exclusively in one stage and absent in the other two stages; class 2 - miRNAs found in any two stages but absent in the third stage; class 3 - miRNAs expressed in all the three stages. Six hundred forty-six miRNAs were found to be specific to one developmental stage, 443 miRNAs were found to be common across any two stages, and 754 miRNAs were common to all the three stages. Target prediction for ten most abundant miRNAs specific to each category identified miRNA regulators of mitosis, meiosis, and cell differentiation. The expression of each miRNA is specific to a particular developmental stage, which is required to maintain a significant repertoire of target mRNAs in the respective stage. Thus, this study provided valuable data that can be used in the future to identify the miRNAs involved in spermatogenic arrest at a particular stage of the germ cell development.

It has been documented that the inefficacy of round spermatid injection (ROSI) might be caused by abnormal epigenetic modifications. Therefore, this study aimed to evaluate the effect of trichostatin A (TSA) as an epigenetic modifier of preimplantation embryo development in activated ROSI oocytes. Matured oocytes were collected from superovulated female mice. Testes were placed in human tubal fluid medium and masses were then cut into small pieces to disperse spermatogenic cells. Round spermatids were treated with TSA and subsequently injected into oocytes. The expression level of the development-related genes including Oct4, Sox2, Nanog, Dnmt and Hdac transcripts were evaluated using qRT-PCR. Immunohistochemistry was performed to confirm the presence of Oct-4 protein at the blastocyst stage. There was no statistically significant difference in fertilization rate following ROSI/+TSA compared with the non-treated ROSI and intracytoplasmic sperm injection (ICSI) groups. Importantly, TSA treatment increased blastocyst formation from 38% in non-treated ROSI to 68%. The relative expression level of developmentally related genes increased and Dnmt transcripts decreased in ROSI/+TSA-derived embryos, similar to the expression levels observed in the ICSI-derived embryos. In conclusion, our results indicate that spermatid treatment with TSA prior to ROSI would increase the success rate of development to the blastocyst stage and proportion of pluripotent cells.

暂无摘要。

Cytosolic Ca2+ concentration ([Ca2+ ]) has an important role in spermatozoa and hence it regulates fertilization. In male germinal cells, there are indirect evidences that this ion could regulate physiological processes in spermatogenesis. Since little is known about Ca2+ homeostasis in spermatogenic cells, in this work we propose a mathematical model that accounts for experimental [Ca2+ ] dynamics triggered by blockade of the SERCA transport ATPase with thapsigargin in round rat spermatids, without external Ca2+ and with different extracellular lactate concentrations. The model included three homogeneous calcium compartments and Ca2+-ATPase activities sensitive and insensitive to thapsigargin, and it adjusted satisfactorily the experimental calcium dynamic data. Moreover, an extended version of the model satisfactorily adjusted the stationary states of calcium modulated by extracellular lactate, which is consistent with the participation of a low affinity lactate transporter and further lactate metabolism in these cells. Further studies and modeling would be necessary to shed some light into the relation between Ca2+-lactate-ATP homeostasis and cell-cell interactions in the seminiferous tubules that are expected to modulate Ca2+ dynamics by hormonal factors or energetic substrates in meiotic and postmeiotic spermatogenic cells.

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.

Sperm granuloma may develop in the epididymis following vasectomy or chemical insults. Inflammation due to sperm granuloma causes abdominal and scrotal pain. Prolonged and persistent inflammation in the epididymis due to sperm granuloma may lead to infertility. Extravasation of germ cells into the interstitium of epididymis following damage of the epididymal epithelium is one of the primary reasons for sperm granuloma-associated pathology. Since testosterone is vital for the maintenance of epididymal epithelium, we investigated the pathology of sperm granuloma and its relationship with testosterone. Adult rats were treated with a Leydig cell-specific toxicant ethylene dimethane sulfonate (EDS) to eliminate testosterone. At 7 days post-EDS, disrupted epididymal epithelium and sperm granuloma were observed in the caput epididymis. Sperm granuloma and caput were collagen-filled indicating fibrosis. Numerous round apoptotic cells were localized inside the caput lumen and dispersed through the sperm granuloma. Tnp1 (round spermatid marker) was significantly higher in the epididymis of the EDS-treated group compared to controls suggesting the apoptotic cells were round spermatids. Increases in CD68+ macrophages and T cells (CD4 and CD8) support an inflammatory immune infiltration in post-EDS epididymis. However, testosterone replacement following EDS prevented the sperm granuloma-associated pathology. We suggest that the immune response in the sperm granuloma may be due to the increased numbers of apoptotic round spermatids or other testicular tissue components that may be released, in addition to the regression of epididymal epithelium due to testosterone loss. Thus, testosterone replacement prevents EDS-induced sperm granuloma and ameliorates sperm granuloma-associated pathology.

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。.

Alternative pre-mRNA splicing is a tightly controlled process conducted by the spliceosome, with the assistance of several regulators, resulting in the expression of different transcript isoforms from the same gene and increasing both transcriptome and proteome complexity. The differences between alternative isoforms may be subtle but enough to change the function or localization of the translated proteins. A fine control of the isoform balance is, therefore, needed throughout developmental stages and adult tissues or physiological conditions and it does not come as a surprise that several diseases are caused by its deregulation. In this review, we aim to bring the splicing machinery on stage and raise the curtain on its mechanisms and regulation throughout several systems and tissues of the human body, from neurodevelopment to the interactions with the human microbiome. We discuss, on one hand, the essential role of alternative splicing in assuring tissue function, diversity, and swiftness of response in these systems or tissues, and on the other hand, what goes wrong when its regulatory mechanisms fail. We also focus on the possibilities that splicing modulation therapies open for the future of personalized medicine, along with the leading techniques in this field. The final act of the spliceosome, however, is yet to be fully revealed, as more knowledge is needed regarding the complex regulatory network that coordinates alternative splicing and how its dysfunction leads to disease.