Palmitoylated GLB1L4 transfers via exosomes to maintain sperm function in rat epididymis.

Epididymal specific proteins play a crucial role in sperm maturation. Some of the post-translational modified proteins are transported from the caput to the cauda of the epididymis through exosomes which regulate the function of sperm in cauda epididymis. Rat beta-galactosidase-1-like protein 4 (GLB1L4) expressed specifically in the caput epididymis, localizes on the sperm; however, the regulatory ways in which GLB1L4 protein interacts with sperm to maintain sperm function are unclear. In this study, knockdown of rat GLB1L4 could inhibit in vitro capacitation of sperm in cauda epididymis and reduce the fertility of the male rats by injection of special lentivirus-shRNA into caput epididymis. Moreover, a considerable proportion of GLB1L4 proteins from rat caput epididymis were loaded on exosomes. The exosomes loaded GLB1L4 from in vitro primary rat caput epididymal epithelial cells could bind with spermatozoa in cauda epididymis. Further, the palmitoylation status of cysteine residues at the 12th and 15th sites of the protein molecule could significantly affect cellular localization of GLB1L4 protein. It was identified that most of GLB1L4 was palmitoylated in the presence of exosomes from primary caput epididymal cells and the level of palmitoylated GLB1L4 in the exosomes could be inhibited by 2-bromopalmitate (2-BP). These results suggested that the palmitoylated GLB1L4 from rat caput epididymis could be transported to the cauda epididymis to regulate the sperm function by exosomes.

Melatonin regulates ATP content and fertilising capacity of Onychostoma macrolepis spermatozoa by inhibiting ROS accumulation during semen storage in vitro.

Melatonin (MLT) is an efficient antioxidant that protects spermatozoa against damages caused by oxidative stress. In this study, to maintain good function of Onychostoma macrolepis spermatozoa during semen preservation invitro at 4°C, different concentrations of MLT (0.5, 1 and 2µM) were added to the semen. After storage (0, 24, 48 and 72h), 1µM MLT in semen markedly improved sperm quality, as reflected by better plasma membrane integrity, the relative steady level of reactive oxygen species (ROS) and slower rate of decrease in mitochondrial membrane potential. Activated spermatozoa in semen with 1µM MLT had higher kinematic performance (i.e. percentage of motile and progressive spermatozoa and the beat cross frequency; P<0.05) and longer duration of sperm motility (P<0.05) compared with spermatozoa in semen withother MLT concentrations. Furthermore, 1µM MLT maintained higher ATP concentrations in spermatozoa during semen storage and significantly improved the fertilising capacity of spermatozoa after 72h semen storage compared with the other MLT concentrations. To expand wild resources of O. macrolepis, 1µM MLT can be used as a semen additive to maintain better sperm function and enhance sperm fertilising capacity in artificial insemination (AI).

Characteristics of steroidogenesis-related factors in the musk gland of Chinese forest musk deer (Moschus berezovskii).

Musk secreted by Chinese forest musk deer (FMD; Moschus berezovskii) is a highly valuable ingredient in the fields of perfumery and medicine, and the main factor affecting the production of musk is the androgen level of male FMD. To clarify whether the musk gland of FMD can synthesize androgen, we compared and analyzed the expression patterns of steroid hormone biosynthesis-related genes in the musk gland and testis of FMD by RNA-seq and RT-qPCR. We obtained 33,308 and 38,602 unigenes from the musk gland and testis, respectively, and 26,780 co-expressed unigenes. Analysis of co-expressed genes revealed that 12,647 genes were annotated to 11,484 Gene Ontology terms and 10,941 genes were annotated to 6120 pathways, including several pathways important in metabolic and synthetic activity. Next, 21 steroid hormone synthesis-related genes were screened from the transcriptome of the musk gland of 4-month-old FMD. The expression levels of three key genes of steroid hormone biosynthesis (CYP11A1, CYP17A1, and HSD3B) in the musk gland differed from their expression levels in the testis based on RT-qPCR. Furthermore, immunohistochemistry indicated that CYP11A1, CYP17A1, and HSD3B were localized in the glandular tubular columnar cells of the musk gland. These results suggested that the musk gland of male FMD has the potential to locally synthesize steroid hormone and thus plays a critically important role in musk secretion.

Overexpression of MicroRNA-10a in Germ Cells Causes Male Infertility by Targeting Rad51 in Mouse and Human.

Spermatogenesis is a complicated process including spermatogonial stem cells self-renewal and differentiates into mature spermatozoa. MicroRNAs (miRNAs) as a class of small non-coding RNAs play a crucial role during the process of spermatogenesis. However, the function of a plenty of miRNAs on spermatogenesis and the potential mechanisms remain largely unknown. Here, we show that genetically conditional overexpressed miR-10a in germ cells caused complete male sterility, characterized by meiotic arrested in germ cells. Analysis of miR-10a overexpression mouse testes reveals that failure of double strand break (DSB) repairs and aberrant spermatogonial differentiation. Furthermore, we identified Rad51 as a key target of miR-10a in germ cell by bioinformatics prediction and luciferase assay, which may be responsible for the infertility of the miR-10a overexpressed mice and germ cell arrested patients. Our data show that miR-10a dependent genetic regulation of meiotic process is crucial for male germ cell development and spermatogenesis in both mouse and human. These findings facilitate our understanding of the roles of miRNA-10a in spermatogenesis and male fertility.

Influence of outer membrane vesicles of Proteus mirabilis isolated from boar semen on sperm function.

This study focuses on the effect of outer membrane vesicles (OMVs) in gram-negative bacteria on boar sperm function during in vitro storage. In the 40 ejaculates collected from Guangzhong Black boar, six gram-negative bacterial species were detected by 16S rDNA sequencing, of which Proteus mirabilis was the main contaminating bacterium. The OMVs of P. mirabilis were isolated by gradient ultracentrifugation. To reveal the effect of OMVs on boar sperm, different OMV concentrations were added to the Modena medium during sperm storage at 17 °C. Even after 3 days of storage, it was noted that low OMV dose (<5 µg/mL) in the extender did not significantly reduce sperm quality as compared with that in the control semen samples; however, sperm motility and sperm morphology were significantly altered in the extender owing to a high OMV dose (>10 µg/mL). The relative ROS level successively increased with OMV dose in sperm samples and storage time. Meanwhile, OMVs dramatically elevated the mitochondrial potential of sperm. OMVs could bind with the sperm membrane to further influence the capacity of sperm-oocyte binding; they also increased the expression of LC3 and caspase 3 and decreased that of anti-apoptosis-related protein, Bcl2, in sperm. It was concluded that OMVs of P. mirabilis influenced the function of boar sperm by inducing sperm membrane reconstruction as well as autophagy and apoptosis of sperm.