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BackgroundEstrogen receptor 2 (ESR2) plays significant biological roles in the reproductive system and ovarian follicle development. This study, therefore, aimed to reveal the expression pattern and cell-specific localization of ESR2 in the ovarian follicles of Leizhou black ducks. MethodFour laying Leizhou black ducks at 43 weeks old were annihilated and different grade-sized follicles were collected for immunohistochemistry and expression profile study. The follicles were grouped into seven (7) as small white follicles (SWF), large white follicles (LWF), small yellow follicles (SYF), large yellow follicles (LYF), follicle 5 (F5), follicle 2 (F2), and follicle 1 (F1). ResultsThe qRT/PCR results displayed that ESR2 mRNA was expressed in all follicles with the highest (P < 0.05) level of expression found in F1 compared to other follicles. Immunohistochemistry analysis of the cell-specific localization of ESR2 protein revealed that ESR2 was distributed in both granulosa and theca cells region in all the follicles examined. There was a significantly higher localization of ESR2 protein in the granulosa cells than the theca cells of SWF, SYF, LYF, F2, and F1. Comparatively, ESR2 was highly expressed in the granulosa cells of LYF than in all the other follicles. ConclusionThese results provide theoretical knowledge for the in-depth study of the related biological functions of the ESR2 gene and its application at the cellular level.

Our previous study on the ovarian transcriptomic analysis in Leizhou black duck revealed that the ESR2 gene was involved in hormone regulation in reproduction and the estrogen signaling pathway related to reproductive performance was enriched. This suggested that ESR2 may have a functional role in the reproductive performance of the Leizhou black duck. Thus, this study aimed at evaluating the polymorphism of the ESR2 gene and its association with egg-laying traits and the distribution pattern of ESR2 mRNA in laying and non-laying Leizhou black ducks. In this study, genomic DNA was extracted from blood samples of 101 Leizhou black ducks to identify single nucleotide polymorphisms (SNPs) of the ESR2 gene to elucidate molecular markers highly associated with egg-laying traits. Four each of laying and non-laying Leizhou black ducks were selected to collect different tissues to analyze the ESR2 gene expression. A total of 23 SNPs were identified and association analysis of the single SNP sites showed that SNPs g.56805646 T>C and exon 3-20G>A were significantly (P < 0.05) associated with egg weight. Ducks with CT and AG genotypes had significantly higher (P < 0.05) egg weights than their respective other genotypes. Haplotype association analysis of g.56805646 T>C and exon 3-20G>A showed that the haplotypes were significantly associated with egg weight. Higher egg weight was seen in individuals with H3H4 haplotypes. In the hypothalamus-pituitary-gonadal (HPG) axis, the results of qRT/PCR showed that ESR2 mRNA was significantly (P < 0.05) expressed in the ovaries of both duck groups than in the hypothalamus and pituitary. In the oviduct, ESR2 was significantly (P < 0.05) higher in the infundibulum and magnum of laying and non-laying ducks respectively. This study provides a molecular marker for selecting Leizhou black ducks for egg production. In addition, it offers theoretical knowledge for studying the related biological functions of the ESR2 gene at the cellular level.

Skeletal muscle is one of the most important economic traits in the poultry industry whose development goes through several processes influenced by several candidate genes. This study explored the regulatory role of DCN on MSTN and the influence of these genes on the proliferation and differentiation of embryonic myoblasts in Leizhou black ducks. Embryonic myoblasts were transfected with over-expressing DCN, Si-DCN, and empty vector and cultured for 24 h, 48 h, and 72 h of proliferation and the comparative expression of DCN and MSTN were measured. The results showed that cells transfected with the over-expression DCN had a significantly (P 0.05) higher expression of DCN mRNA than the normal group and the expression of MSTN mRNA showed a downward trend during the proliferation of myoblasts. DCN mRNA expression was lower in cells transfected with Si-DCN than the normal group in all stages of proliferation. While the expression of MSTN in the Si-DCN transfected group was higher than the normal group with a significant (P 0.05) difference at the 72 h stage. DCN mRNA increased at the early stage of differentiation but decreased (P 0.05) from the 6th day to the 8th day of differentiation. The level of MSTN increased gradually during the differentiation process of myoblasts until it decreased significantly on the 8th day. These results show that DCN enhances the proliferation and differentiation of Leizhou black duck myoblasts and suppresses MSTN activity.