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Litter size is one of the important economic traits of livestock. Seasonal oestrus, ovulation and lambing of sheep have severely restricted the development of sheep farming in Xinjiang, China. The purpose of this study was to investigate the polymorphisms and genetic correlation between GRM1, GNAQ and HCRTR1 genes and the seasonal reproduction and litter size in three sheep breeds. The DNA mixed pool sequencing and PCR‐SSCP methods were used to detect single nucleotide polymorphisms (SNPs) of GRM1, GNAQ and HCRTR1 genes in seasonal oestrous (Kazakh and Chinese Merino [Xinjiang Junken type]) and perennial oestrous (Hu) sheep breeds. The association between genetic polymorphism and litter size was also analysed. The results showed that T945C in exon 2 of GRM1 gene, C589T in exon 2 of HCRTR1 gene and A191G in exon 2 of GNAQ gene were identified by Sanger sequencing, and three genotypes were existed in each SNP site, which all belonged to the synonymous mutation. GRM1 (CC), GNAQ (GA) and HCRTR1 (TC) were the dominant genotypes of seasonal reproduction and litter size in Kazakh sheep and Chinese Merino sheep, respectively, while, in perennial oestrous Hu sheep populations, the dominant genotypes were GRM1 (TC), GNAQ (GA) and HCRTR1 (TC), respectively, and association analysis also confirmed the results. The above results implied that GRM1, GNAQ and HCRTR1 genes are significantly associated with lambing traits in Kazakh, Chinese Merino and Hu sheep. Among them, the locus of GRM1 (T945C), GNAQ (A191G) and HCRTR1 (C589T) might be considered as a potential molecular marker, which controls seasonal reproduction and litter size in sheep. [ABSTRACT FROM AUTHOR]

Kazakh sheep are typical seasonal estrus animals. Their reproductive system regulation mainly involves the complex regulation of the hypothalamic–pituitary–gonadal axis (HPGA), which is also closely related to reproductive hormone secretion. Gonadotropin-releasing hormone (GnRH), synthesized and secreted by the hypothalamus, is the key to controlling sheep reproductive activity. We studied how GNAQ (G protein subunit alpha q) regulates estrus in sheep by controlling GnRH expression and secretion. We used hypothalamic nerve cells as the research model. GNAQ overexpression and RNA interference vectors were constructed and transfected into the hypothalamic nerve cells of fetal Kazakh sheep. qPCR, western blotting, and enzyme-linked immunosorbent assay were used to detect GNAQ gene expression in Kazakh ewe tissues and analyze its regulatory effect on GnRH expression in the hypothalamic nerve cells. The fetal sheep hypothalamic nerve cells were successfully isolated and cultured. qPCR and cell immunofluorescence showed that the purity of positive cells was >95%. The tissue expression profile showed that there were different degrees of GNAQ gene expression in the Kazakh ewe tissue. Expression levels were relatively higher in the hypothalamus, pituitary, brain, and uterine tissues. When GNAQ expression was downregulated in the hypothalamic nerve cells, the upstream genes KISS1 (kisspeptin), GPR54 (KISS1 receptor), and ER (estrogen receptor) were all upregulated, as were the downstream genes PLCB1 (phospholipase C beta 1), PRKCB (protein kinase C beta), and GNRH. At the same time, GnRH secretion levels were also upregulated. GNAQ regulated its downstream gene PLCB1 in the hypothalamic nerve cells, and directly regulated GnRH expression and secretion through the calcium and PRKC signaling pathways. GNAQ also regulated kisspeptin expression, subsequently regulating GnRH expression and secretion indirectly through the kisspeptin–GPR54 signaling pathway. Our results are of great importance for improving the reproductive performance of seasonal-estrus sheep. • The highest expressions of the GNAQ gene in Kazakh ewes were in the hypothalamus, pituitary, brain, and uterus. • The GNAQ gene can target the calcium signaling pathway and the PRKC signaling pathway in the hypothalamic nerve cells. • Downregulation of the GANQ gene contributes to upregulation of the PLCB1 , PRKCB , KISS1 , GPR54 , ER and GnRH genes in sheep. [ABSTRACT FROM AUTHOR]