Single-cell transcriptomic characterization of sheep conceptus elongation and implantation.

Bidirectional communication between the developing conceptus and endometrium is essential for pregnancy recognition and establishment in ruminants. We dissect the transcriptomic dynamics of sheep conceptus and corresponding endometrium at pre- and peri-implantation stages using single-cell RNA sequencing. Spherical blastocysts contain five cell types, with 68.62% trophectoderm cells. Strikingly, elongated conceptuses differentiate into 17 cell types, indicating dramatic cell fate specifications. Cell-type-specific gene expression delineates the features of distinctive trophectoderm lineages and indicates that the transition from polar trophectoderm to trophoblast increases interferon-tau expression and likely drives elongation initiation. We identify 13 endometrium-derived cell types and elucidate their molecular responses to conceptus development. Integrated analyses uncover multiple paired transcripts mediating the dialogues between extraembryonic membrane and endometrium, including IGF2-IGF1R, FGF19-FGFR1, NPY-NPY1R, PROS1-AXL, and ADGRE5-CD55. These data provide insight into the molecular regulation of conceptus elongation and represent a valuable resource for functional investigations of pre- and peri-implantation ruminant development.

The expression of histone methyltransferases and distribution of selected histone methylations in testes of yak and cattle-yak hybrid.

Interspecies hybridization exists widely in nature and plays an important role in animal evolution and adaptation. It is commonly recognized that male offspring of interspecies hybrid are often sterile, which presents a crucial way of reproductive isolation. Currently, the mechanisms underlying interspecies hybrid male sterility are not well understood. Cattle-yak, progeny of yak (Bos grunniens) and cattle (Bos taurus) cross, is a unique animal model for investigating hybrid male sterility. Because histone modifications are vital for spermatogenesis, herein, we examined expressions of histone methyltransferases (HMTs) and distributions of histone methylations in the yak and cattle-yak testis. Histological examination of seminiferous tubules revealed that gonocytes and spermatocytes were established normally, however, spermatogenesis was arrested at the meiosis phase began at 10 months after birth in the hybrids. SUV420H1 was the only HMT examined showing a significant enrichment in cattle-yak testes at 3 months. Relative expressions of MLL5, SETDB1 and SUV420H1 were increased while SETDB2 and EZH2 were decreased in cattle-yak testes at 10 months. Relative concentrations of MLL5 and SUV420H1 were again increased while EHMT2 and PRDM9 expressions were decreased at 24 months. Immunofluorescent detection of selected histone methylations in cross-sections of testicular tissues or meiotic chromosomes demonstrated that depletion of H3K4me3 and significant enrichment of H3K27me3 and H4K20me3 were observed in Sertoli cells of cattle-yak. Levels and localizations of H3K4me3, H3K9me1, H3K9me3 and H4K20me3 were strikingly different in meiotic chromosomes of cattle-yak spermatocytes. These results highlighted the potential roles of histone methylations in spermatogenic failure and hybrid male sterility.

Transcriptome analysis revealed key signaling networks regulating ovarian activities in the domestic yak.

Domestic yaks are the most important livestock species on the Qinghai-Tibetan Plateau. Adult female yaks normally breed in warm season and enter anestrous in cold season. Currently, how the ovarian activity is regulated at the molecular level remains to be determined. This study was conducted to investigate follicular development and gene expression patterns of yak ovarian tissues in the warm and cold seasons. Dynamics of follicular development was evaluated based on histological analyses and global gene expression was examined by using RNA-sequencing (RNA-seq) technology. Firstly, we found that follicle development of yak cows in cold season was different from that in warm season. Interestingly, ovaries collected from yaks in cold season contained a significant higher number of antral follicles and some of these follicles showed signs of polycystic structure, indicating abnormal granulosa cell function. RNA-seq analyses of ovarian tissues from non-pregnant adult yaks in cold and warm season revealed that a list of 320 transcripts were differentially expressed, specifically, 79 were up-regulated and 241 were down-regulated in the ovaries from yaks during the cold season. Further analysis demonstrated that transcripts associated with estrogen secretion and metabolism signaling pathway were altered, including FST, CYP1A1, PIK3R1 and PIK3R2. This study showed histological features of follicle development and revealed candidate genes that may have important roles in regulating ovarian activities in the yak seasonal reproduction.

The CXCL12-CXCR4 signaling promotes oocyte maturation by regulating cumulus expansion in sheep.

Gonadotropins and growth factors synergistically regulate folliculogenesis and oocyte development. C-X-C motif chemokine 12 (CXCL12) and its receptor CXCR4 are expressed in ovaries of sheep, cattle and other species, however, roles of this multifunctional signal axis in oocyte maturation are not defined. Using sheep as a model, we examined the expression patterns and functions of the CXCL12-CXCR4 axis during oocyte maturation. CXCL12 and CXCR4 mRNA and protein were present in oocytes and granulosa cells. Relative abundance of CXCR4 transcript was controlled by epidermal growth factor (EGF). Transient inhibition of CXCR4 suppressed oocyte nuclear maturation while supplementing recombination CXCL12 significantly increased percent of oocyte undergone metaphase I phase. Inhibition of CXCR4 function decreased cumulus expansion growth rate. Furthermore, granulosa cell migration was impaired and expression of hyaluronan synthase 2 (HAS2) and hyaluronan binding protein tumor necrosis factor-alpha-induced protein 6 (TNFAIP6) were downregulated by CXCR4 inhibition. These findings revealed a novel role of the CXCL12-CXCR4 signaling in oocyte development in sheep.