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Transcriptomic analyses of germ cells at different stages of differentiation have shed light on the transcriptional and post-transcriptional mechanisms regulating gene expression that ensure the correct progression of spermatogenesis and male fertility. In this chapter, we describe a method to isolate meiotic and post-meiotic germ cells, based on gravimetric sedimentation, starting from a testicular germ cell suspension isolated from a single adult mouse. We also describe how to assess the purity and quality of the collected fractions of germ cells and how to optimize the extraction from these samples of RNA for subsequent RNA-sequencing experiment. In our experience, this protocol is suitable for germ cell isolation and transcriptomic analysis for mouse models with spermatogenic defects, overcoming the limits that reduced fertility poses to the obtaining of experimental animals., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The male infertility of cattleyak resulted from spermatogenic arrest has greatly restricted the effective utilization of the heterosis from crossbreeding of cattle and yak. Based on our previous studies, the significant divergences of the transcriptomic and proteomic sequencing between yak and cattleyak prompt us to investigate the critical roles of microRNAs in post-transcriptional regulation of gene expression during spermatogenesis. TUNEL-POD analysis presented sharply decreased spermatogenic cell types and the increased apoptotic spermatogonia in cattleyak. The STA-PUT velocity sedimentation was employed to obtain spermatogonia and spermatocytes from cattle, yak and cattleyak and these spermatogenic cells were verified by the morphological and phenotypic identification. MicroRNA microarray showed that 27 differentially expressed miRNAs were simultaneously identified both in cattleyak vs cattle and in cattleyak vs yak comparisons. Further analysis revealed that the down-regulation of bta-let-7 families, bta-miR-125 and bta-miR-23a might impair the RA-induced differentiation of spermatogonia. Target gene analysis for differentially expressed miRNAs revealed that miRNAs targeted major players involved in vesicle-mediated transport, regulation of protein kinase activity and Pathways in cancer. In addition, spermatogonia transfection analysis revealed that the down-regulation of bta-miR-449a in the cattleyak might block the transition of male germ cells from the mitotic cycle to the meiotic program. The present study provided valuable information for future elucidating the regulatory roles of miRNAs involved in spermatogenic arrest of cattleyak., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)