Spermatogenesis is an important biological process in male reproduction. The interaction between male germ cells and somatic cells during spermatogenesis, is necessary for male reproductive activities. This cellular heterogeneity has made it difficult to profile distinct cell types at different stages of development. Here, we present the first comprehensive, unbiased single‐cell transcriptomic study of sheep spermatogenesis using 10× genomics single cell sequencing (scRNA‐seq). We collected scRNA‐seq data from 11 772 cells from the adult sheep testis and identified all known germ cells (including early primary spermatocytes, late primary spermatocytes, round spermatids, elongated spermatids, and sperm), and somatic cells (Sertoli cells and Leydig cells), as well as one somatic cell that unexpectedly contained leukocytes. The functional enrichment analysis indicated that several pathways of cell cycle, gamete generation, protein processing, and mRNA surveillance pathways were significantly enriched in testicular germ cell types, and ribosome pathway was significantly enriched in testicular somatic cell types. Further analysis identified several stage‐specific marker genes of sheep germ cells, such as EZH2 , SOX18 , SCP2 , PCNA, and PRKCD . Our research explored for the first time of the changes in the transcription level of various cell types during the process of sheep spermatogenesis, providing new insights for sheep spermatogenesis and spermatogenic cell development.

The pituitary gland directly regulates the reproduction of domestic animals. Research has increasingly focused on the potential regulatory mechanism of non-coding RNA in pituitary development. Little is known about the differential expression pattern of lncRNAs in Hu sheep, a famous sheep breed with high fecundity, and its role in the pituitary gland between the follicular phase and luteal phase. Herein, to identify the transcriptomic differences of the sheep pituitary gland during the estrus cycle, RNA sequencing (RNA-Seq) was performed. The results showed that 3529 lncRNAs and 16,651 mRNAs were identified in the pituitary gland. Among of them, 144 differentially expressed (DE) lncRNA transcripts and 557 DE mRNA transcripts were screened in the follicular and luteal phases. Moreover, GO and KEGG analyses demonstrated that 39 downregulated and 22 upregulated genes interacted with pituitary functions and reproduction. Lastly, the interaction of the candidate lncRNA XR_001039544.4 and its targeted gene LHB were validated in sheep pituitary cells in vitro. LncRNA XR_001039544.4 and LHB showed high expression levels in the luteal phase in Hu sheep. LncRNA XR_001039544.4 is mainly located in the cytoplasm, as determined by FISH analysis, indicating that XR_001039544.4 might act as competing endogenous RNAs for miRNAs to regulate LHB. LncRNA XR_001039544.4 knockdown significantly inhibited LH secretion and cell proliferation. LncRNA XR_001039544.4 may regulate the secretion of LH in the luteal-phase pituitary gland via affecting cell proliferation. Taken together, these findings provided genome-wide lncRNA- and mRNA-expression profiles for the sheep pituitary gland between the follicular and luteal phases, thereby contributing to the elucidation of the molecular mechanisms of pituitary function.

Pituitary gonadotropins play a pivotal role in reproduction. Long noncoding RNAs (lncRNAs) have been identified as important regulators in the hypothalamic–pituitary–ovarian (HPO) axis associated with reproduction. However, the contributions of lncRNAs to pituitary gonadotropin secretion remain largely unknown. Therefore, this work was performed to uncover the functional mechanisms of the novel lncRNA TCONS_00083279 (lncRNA SM2) and its potential targeting pathway oar-miR-16b/TGF-beta/SMAD2, which is associated with gonadotropin secretion in sheep pituitary cells. In the present study, the lncRNA SM2 showed high expression levels in the sheep pituitary gland, and it was located in both the nucleus and the cytoplasm of pituitary cells. lncRNA SM2 knockdown inhibited pituitary cell proliferation and FSH and LH secretion. The function of the lncRNA SM2 was sponged by oar-miR-16b, and this regulated the growth and gonadotropin secretion of pituitary cells by modulating SMAD2, as shown by the dual-luciferase reporter assay. FSH and LH levels were both upregulated by SMAD2 overexpression. Moreover, the levels of the lncRNA SM2, SMAD2 and TGFR1, as well as FSH and LH, in sheep pituitary cells increased significantly under gonadotropin-releasing hormone (GnRH) stimulation (p < 0.05). This work illustrates that the lncRNA SM2 regulates gonadotropin secretion in the Hu sheep anterior pituitary by targeting the oar-miR-16b/TGF-β/SMAD2 signaling pathway, providing a valuable resource for understanding the molecular mechanisms underlying sheep reproduction.