Single-cell analysis identifies critical regulators of spermatogonial development and differentiation in cattle-yak bulls.

Spermatogenesis is a continuous process in which functional sperm are produced through a series of mitotic and meiotic divisions and morphological changes in germ cells. The aberrant development and fate transitions of spermatogenic cells cause hybrid sterility in mammals. Cattle-yak, a hybrid animal between taurine cattle (Bos taurus) and yak (Bos grunniens), exhibits male-specific sterility due to spermatogenic failure. In the present study, we performed single-cell RNA sequencing analysis to identify differences in testicular cell composition and the developmental trajectory of spermatogenic cells between yak and cattle-yak. The composition and molecular signatures of spermatogonial subtypes were dramatically different between these 2 animals, and the expression of genes associated with stem cell maintenance, cell differentiation and meiotic entry was altered in cattle-yak, indicating the impairment of undifferentiated spermatogonial fate decisions. Cell communication analysis revealed that signaling within different spermatogenic cell subpopulations was weakened, and progenitor spermatogonia were unable or delayed receiving and sending signals for transformation to the next stage in cattle-yak. Simultaneously, the communication between niche cells and germ cells was also abnormal. Collectively, we obtained the expression profiles of transcriptome signatures of different germ cells and testicular somatic cell populations at the single-cell level and identified critical regulators of spermatogonial differentiation and meiosis in yak and sterile cattle-yak. The findings of this study shed light on the genetic mechanisms that lead to hybrid sterility and speciation in bovid species.

Comparative proteomic analysis identifies differentially expressed proteins associated with meiotic arrest in cattle-yak hybrids.

Cattle-yak, the interspecific hybrid between yak and taurine cattle, exhibits male-specific sterility. Massive loss of spermatogenic cells, especially spermatocytes, results in azoospermia in these animals. Currently, the mechanisms underlying meiosis block and defects in spermatocyte development remain elusive. The present study was designed to investigate the differences in the protein composition of spermatocytes isolated from 12-month-old yak and cattle-yak testes. Histological analysis confirmed that spermatocytes were the most advanced germ cells in the testes of yak and cattle-yak at this developmental stage. Comparative proteomic analysis identified a total of 452 differentially abundant proteins (DAPs) in the fluorescence-activated cell sorting (FACS) isolated spermatocytes from cattle-yak and yak. A total of 291 proteins were only present in yak spermatocytes. Gene Ontology analysis revealed that the downregulated DAPs were mostly enriched in the cellular response to DNA damage stimulus and double-strand breaks (DSBs) repair via break-induced replication, while the proteins specific for yak were related to cell division and cycle, spermatogenesis, and negative regulation of the extrinsic apoptotic signaling pathway. Ultimately, these DAPs were related to the critical process for spermatocyte meiotic events, including DSBs, homologous recombination, synapsis, crossover formation, and germ cell apoptosis. The database composed of proteins associated with spermatogenesis, including KPNA2, HTATSF1, TRIP12, STIP1, LZTFL1, LARP7, MTCH2, STK31, ROMO1, CDK5AP2, DNMT1, RBM44, and CHRAC1, is the focus of further research on male hybrid sterility. In total, these results provide insight into the molecular mechanisms underlying failed meiotic processes and male infertility in cattle-yak.

Single-cell transcriptomic survey of cell diversity and functional changes in yak hearts at different altitude.

The yak (Bos grunniens) is a species adapted to the hypoxic environment in the plateau area. The heart is a hypoxia-sensitive organ involved in this adaptation. Herein, we used single-cell RNA-seq technology and clustering to determine the presence of 11 cell populations in the yak heart. We analyzed gene expression differences and expression patterns in each cell subpopulation at different altitudes. The cells related to altitude changes are mainly smooth muscle cells and vascular endothelial cells. Of the four transcription factors (TFs, MEF2B, FOXP4, ARID5A, and HES4) found in smooth muscle cells, only MEF2B was specifically expressed in vascular smooth muscle cells. Three key TFs (HNF1B, DMRTA1, and ARNTL2) were also found in the cardiomyocyte module. Compared with data extracted from low-altitude yak, we observed that the high altitude yak has enhanced contraction and relaxation of vascular smooth muscle cells and an increased metabolic level of cardiomyocytes. These may be strategies for the yak to adapt to high-altitude hypoxia environments.

Long read genome assemblies complemented by single cell RNA-sequencing reveal genetic and cellular mechanisms underlying the adaptive evolution of yak.

Wild yak (Bos mutus) and domestic yak (Bos grunniens) are adapted to high altitude environment and have ecological, economic, and cultural significances on the Qinghai-Tibetan Plateau (QTP). Currently, the genetic and cellular bases underlying adaptations of yak to extreme conditions remains elusive. In the present study, we assembled two chromosome-level genomes, one each for wild yak and domestic yak, and screened structural variants (SVs) through the long-read data of yak and taurine cattle. The results revealed that 6733 genes contained high-FST SVs. 127 genes carrying special type of SVs were differentially expressed in lungs of the taurine cattle and yak. We then constructed the first single-cell gene expression atlas of yak and taurine cattle lung tissues and identified a yak-specific endothelial cell subtype. By integrating SVs and single-cell transcriptome data, we revealed that the endothelial cells expressed the highest proportion of marker genes carrying high-FST SVs in taurine cattle lungs. Furthermore, we identified pathways which were related to the medial thickness and formation of elastic fibers in yak lungs. These findings provide new insights into the high-altitude adaptation of yak and have important implications for understanding the physiological and pathological responses of large mammals and humans to hypoxia.

Characteristics of pulmonary microvascular structure in postnatal yaks.

Yaks are typical plateau-adapted animals, however the microvascular changes and characteristics in their lungs after birth are still unclear. Pulmonary microvasculature characteristics and changes across age groups were analysed using morphological observation and molecular biology detection in yaks aged 1, 30 and 180 days old in addition to adults. Results: Our experiments demonstrated that yaks have fully developed pulmonary alveolar at birth but that interalveolar thickness increased with age. Immunofluorescence observations showed that microvessel density within the interalveolar septum in the yak gradually increased with age. In addition, transmission electron microscopy (TEM) results showed that the blood-air barrier of 1-day old and 30-days old yaks was significantly thicker than that observed at 180-days old and in adults (P < 0.05), which was caused by the thinning of the membrane of alveolar epithelial cells. Furthermore, Vegfa and Epas1 expression levels in 30-day old yaks were the highest in comparison to the other age groups (P < 0.05), whilst levels in adult yaks were the lowest (P < 0.05). The gradual increase in lung microvessel density can effectively satisfy the oxygen requirements of ageing yaks. In addition, these results suggest that the key period of yak lung development is from 30 to 180 days.