This study aimed to find the SNPs in the SORBS1 gene of cattleyak, analyze the relationship between its polymorphisms and the milk fat traits, and find potential molecular markers for the milk fat traits of cattleyak. The polymorphism of the SORBS1 gene in 350 cattleyak from Hongyuan County (Sichuan, China) were detected by PCR and DNA sequencing, and the correlation between these SNPs and the milk production traits of cattleyak was analyzed. The results showed that there were nine SNPs in the CDS and their adjacent non-coding regions of the SORBS1 gene, and all SNPs have three genotypes. The correlation analysis found that the genotypes with superior milk fat traits in the other eight alleles were homozygous genotypes with a high genotype frequency except the g.96284 G > A (c.3090 G > A) (p < 0.05). However, at locus g.96284 G > A, the milk fat percentage, monounsaturated fatty acids (MUFAs), polyunsaturated fatty acids (PUFAs) and saturated fatty acids (SFAs) of the GA genotype were significantly higher than that of GG and AA genotypes (p < 0.05). Among these SNPs, three SNPs (g.6256 C > T (c.298 C > T), g.24791 A > G (c.706 A > G) and g.29121 A > G (c.979 A > G)) caused the amino acids change. The genotypes of the three SNPs consist of three haplotypes and four diplotypes. The amino acid mutation degree of diplotype H1–H1 (CCAAAA) was the highest, and its milk fat percentage, MUFAs, PUFAs and SFAs were also the highest (p < 0.05). Taken together, we found nine SNPs in the SORBS1 gene that are closely related to the milk fat traits of cattleyak. Moreover, the mutation of amino acids caused by SNPs had positive effects on the milk fat traits of cattleyak. H1-H1 is the dominant diplotype which significantly related to the milk fat traits of cattleyak. This study provides a new molecular marker and theoretical basis for screening the milk fat traits of cattleyak.
The methylation status of histones plays a crucial role in many cellular processes, including follicular and oocyte development. Lysine-specific demethylase 2a (KDM2a) has been reported to be closely associated with gametogenesis and reproductive performance, but the specific function and regulatory mechanism have been poorly characterized in vivo. We found KDM2a to be highly expressed in growing follicles and oocytes of mice in this study. To elucidate the physiological role of Kdm2a, the zona pellucida 3-Cre (Zp3-Cre)/LoxP system was used to generate an oocyte Kdm2a conditional knockout (Zp3-Cre; Kdm2aflox/flox, termed Kdm2a cKO) model. Our results showed that the number of pups was reduced by approximately 50% in adult Kdm2a cKO female mice mating with wildtype males than that of the control (Kdm2aflox/flox) group. To analyze the potential causes, the ovaries of Kdm2a cKO mice were subjected to histological examination, and results indicated an obvious difference in follicular development between Kdm2a cKO and control female mice and partial arrest at the primary antral follicle stage. The GVBD and matured rates of oocytes were also compromised after conditional knockout Kdm2a, and the morphological abnormal oocytes increased. Furthermore, the level of 17β-estradiol of Kdm2a cKO mice was only 60% of that in the counterparts, and hormone sensitivity decreased as the total number of ovulated and matured oocytes decreased after superovulation. After deletion of Kdm2a, the patterns of H3K36me2/3 in GVBD-stage oocytes were remarkedly changed. Transcriptome sequencing showed that the mRNA expression profiles in Kdm2a cKO oocytes were significantly different, and numerous differentially expressed genes were involved in pathways regulating follicular and oocyte development. Taken together, these results indicated that the oocyte-specific knockout Kdm2a gene led to female subfertility, suggesting the crucial role of Kdm2a in epigenetic modification and follicular and oocyte development.
The aims of this study were to analyse the protein phosphatase 1 regulatory subunit 11 (PPP1R11) expression and cellular localization in yak follicles and investigate its effects on cell proliferation, apoptosis and oestrogen secretion in granulosa cells (GCs). Ten healthy and non‐pregnant female yaks (4‐year‐old) were used as experimental animals. The mRNA relative expression level of PPP1R11 in GCs from small (<3.0 mm), medium (3.0–5.9 mm) and large (6.0–9.0 mm) follicles was detected by RT‐qPCR, and the cellular localization of PPP1R11 protein was detected by immunohistochemistry staining (IHC). After isolation, culture and identification of yak GCs in vitro, si‐PPP1R11 and si‐NC (negative control) were transfected into GCs. RT‐qPCR and immunofluorescence staining were used to evaluate the interference efficiency, and ELISA was performed to detect oestrogen concentration. Then, EdU staining and TUNEL staining were conducted to analyse cell proliferation and apoptosis. In addition, the oestrogen synthesis, proliferation‐ and apoptosis‐related genes were detected by RT‐qPCR after knockdown PPP1R11. The results showed that PPP1R11 is mainly located in ovarian GCs, and the expression levels of PPP1R11 in GCs from large follicles were significantly higher than that from medium and small follicles. Transfection of si‐PPP1R11 into GCs could significantly inhibit the expression of PPP1R11. Interestingly, the oestrogen secretion ability and the expression level of oestrogen pathway‐related genes (STAR, CYP11A1, CYP19A1 and HSD17B1) were also significantly downregulated. Moreover, the proportion of positive cells was decreased, and cellular proliferation‐related genes (PCNA, CCNB1 and CDC25A) were significantly downregulated after knockdown PPP1R11. However, the proportion of apoptotic cells was increased, and apoptosis‐related genes (BAX, CASP3 and P53) were significantly upregulated. Taken together, this study was the first revealed the expression and cellular localization of PPP1R11 in yak follicles. Interference PPP1R11 could reduce oestrogen secretion, inhibit proliferation and promote apoptosis in GCs, which provided a basis for further studies on the regulatory mechanism of PPP1R11 in follicle development.
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