It takes several hours for mammalian sperm to migrate from the ejaculation or insemination site to the fertilization site in the female reproductive tract in which glucose, amino acids, and fatty acids are regarded as the primary substrates for ATP generation. The present study was designed to investigate whether oleic acid and palmitic acid were beneficial to boar sperm in vitro; and if yes, to elucidate the mechanism that regulates sperm motility. Therefore, the levels of oleic acid and palmitic acid, motility, membrane integrity, acrosome integrity, and apoptosis of sperm were evaluated. Moreover, the enzymes involved in mitochondrial β-oxidation (CPT1: carnitine palmitoyltransferase 1; ACADVL: long-chain acyl-coenzyme A dehydrogenase) were detected with immunofluorescence and Western blotting. Consequently, the ATP content and the activities of CPT1, ACADVL, malate dehydrogenase (MDH), and succinate dehydrogenase (SDH) were also measured. We observed that CPT1 and ACADVL were expressed in boar sperm and localized in the midpiece. The levels of oleic acid and palmitic acid were decreased during storage at 17 °C. The addition of oleic acid and palmitic acid significantly increased sperm motility, progressive motility, straight-line velocity (VSL), membrane integrity, and acrosome integrity with a simultaneous decrease in sperm apoptosis after seven days during storage. When sperm were incubated with oleic acid and palmitic acid at 37 °C for 3 h, the activities of CPT1 and ACADVL, the ATP level, the mitochondrial membrane potential, the activities of MDH and SDH, as well as sperm motility patterns were significantly increased compared to the control (p < 0.05). Moreover, the addition of etomoxir to the diluted medium in the presence of either oleic acid or palmitic acid and the positive effects of oleic acid and palmitic acid were counteracted. Together, these data suggest that boar sperm might utilize oleic acid and palmitic acid as energy substrates for ATP production via β-oxidation. The addition of these acids could improve sperm quality.

Infertility caused by male factors is routinely diagnosed by assessing traditional semen parameters. Growing evidence has indicated that the tsRNAs carried in sperm act as epigenetic factors and potential biomarkers for the assessment of sperm quality. We recently demonstrated that tRNAGln-TTG derived small RNAs played notable roles in the first cleavage of a porcine embryo. However, the function of human sperm tRNAGln-TTG derived small RNAs as a diagnostic biomarker and its role in early embryo development remains unclear. In this study, we found that human sperm tRNAGln-TTG derived small RNAs were highly associated with sperm quality. By microinjecting the antisense sequence into human tripronuclear (3PN) zygotes followed by single-cell RNA-sequencing, we found that human sperm tRNAGln-TTG derived small RNAs participated in the development of a human embryo. Furthermore, Gln-TTGs might influence embryonic genome activation by modulating noncoding RNA processing. These findings demonstrated that human sperm tRNAGln-TTG derived small RNAs could be potential diagnostic biomarkers and could be used as a clinical target for male infertility.

Background: Spermatogenesis is the process by which male gametes are formed from spermatogonial stem cells and it is essential for the reliable transmission of genetic information between generations. To date, the dynamic transcriptional changes of defined populations of male germ cells in pigs have not been reported.Results: To characterize the atlas of porcine spermatogenesis, we profiled the transcriptomes of ~ 16,966 testicular cells from a 150-day-old pig testis through single-cell RNA-sequencing (scRNA-seq). The scRNA-seq analysis identified spermatogonia, spermatocytes, spermatids and three somatic cell types in porcine testes. The functional enrichment analysis demonstrated that these cell types played diverse roles in porcine spermatogenesis. The accuracy of the defined porcine germ cell types was further validated by comparing the data from scRNA-seq with those from bulk RNA-seq. Since we delineated four distinct spermatogonial subsets, we further identified CD99 and PODXL2 as novel cell surface markers for undifferentiated and differentiating spermatogonia, respectively.Conclusions: The present study has for the first time analyzed the transcriptome of male germ cells and somatic cells in porcine testes through scRNA-seq. Four subsets of spermatogonia were identified and two novel cell surface markers were discovered, which would be helpful for studies on spermatogonial differentiation in pigs. The datasets offer valuable information on porcine spermatogenesis, and pave the way for identification of key molecular markers involved in development of male germ cells.

Proline was reported to improve sperm quality in rams, stallions, cynomolgus monkeys, donkeys, and canines during cryopreservation. However, the underlying mechanism remains unclear. The aim of this study was to investigate the effect of proline on boar semen during liquid storage at 17 °C and explore the underlying mechanism. Freshly ejaculated boar semen was supplemented with different concentrations of proline (0, 25, 50, 75, 100, 125 mM) and stored at 17 °C for nine days. Sperm motility patterns, membrane integrity, ATP (adenosine triphosphate), reactive oxygen species (ROS), and GSH (glutathione) levels, and the activities of catalase (CAT) and superoxide dismutase (SOD) were evaluated after storage for up to five days. It was observed that boar sperm quality gradually decreased with the extension of storage time, while the ROS levels increased. Addition of 75 mM proline not only significantly improved sperm membrane integrity, motility, and ATP levels but also maintained the redox homeostasis via increasing the GSH levels and activities of CAT and SOD. When hydrogen peroxide (H₂O₂) was used to induce oxidative stress, addition of proline significantly improved sperm quality and reduced ROS levels. Moreover, addition of proline also improved sperm quality during the rapid cooling process. Notably, addition of DL-PCA (DL-pipecolinic acid) rescued the reduction of progressive motility and total motility caused by H₂O₂, and THFA (tetrahydro-2-furoic acid) failed to provide protection. Furthermore, addition of proline at 75 mM increased the activity of proline dehydrogenase (PRODH) and attenuated the H₂O₂-induced reduction in progressive motility. These data demonstrate that proline protects sperm against oxidative stress through the secondary amine structure and proline dehydrogenase-mediated metabolism.

FGF13, a member of the FGF subfamily, has been found to be highly expressed in cancer cells such as prostate cancer, melanoma, glioma and multiple myeloma. However, the mechanism of FGF13 function ...