Identification of functional SNP associated with sperm quality in porcine ANXA5 that contributes to the growth of immature Sertoli cell

Abstract

AnnexinA5 (ANXA5) has been identified as a positional candidate gene for reproduction and fertility traits in boars, but its role in testicular tissue development, as well as genetic variations remain unclear. The aim of this study was to explore the effect of ANXA5 in the growth of swine Sertoli cells and identify its functional variations. Firstly, the expression of porcine ANXA5 in different tissues was detected by semi-quantitative RT-PCR and its effect on the proliferation of Sertoli cells was evaluated by CCK8, EdU, flow cytometry analyses and qRT-PCR. Then, putative causative variants were screened by integrating in silico analysis and DNA sequencing, and the subsequent association analysis was performed in Largewhite boars. Lastly, dual luciferase reporter assay was used to clarify the effect of specific SNP or ESR1 on ANXA5 transcription. The results showed that ANXA5 expressed in all the detected tissues, promoted proliferation of Sertoli cells by advancing cell cycle progression from the G1 to S phase and encouraging expression of PCNA. Putative causative variants, including two ns-SNPs within the coding region, and three closely linked SNPs in the promoter region were identified. Statistical analysis showed that the frequency of the T allele at g.-676 T > C, A allele at g.-674C > A, and T allele at g.-105G > T were each 0.75, the heterozygotes of Yorkshire boars had greater sperm motility as compared to TT, AA, and TT animals (p < 0.05). Luciferase reporter analysis suggested g.-105G > T and ESR1 modulated ANXA5 transcription. Taken together, this study demonstrated ANXA5 affected swine immature Sertoli cells growth and g.-105G > T was a candidate genetic marker for reproductive trait of boar.

Keywords: Anxa5; ESR1; Sertoli cell; boar; semen traits.

Figure 1

Characterization of porcine ANXA5. (A) Tissue expression profile of porcine ANXA5. (B) Multiple sequence alignment of ANXA5 protein in eight species. (C) Phylogenetic tree of ANXA5. (D) The predictive functional partner of human ANXA5. (E) The predictive functional partner of porcine ANXA5.

Figure 2

ANXA5 affects the proliferation of immature Sertoli cells. (A) The efficiency of porcine ANXA5 overexpression or interference. (B) Cell viability was detected by CCK8. (C) Cell proliferation was detected by EdU. Scale bar: 2 mm. (D) The effect of ANXA5 overexpression on the mRNA expression of PCNA. (E) The effect of ANXA5 interference on the mRNA expression of PCNA. (F) The effect of ANXA5 overexpression on the cell cycle. (G) The effect of ANXA5 interference on cell cycle. Values were shown as the mean ± SEM (n = 3). *p < 0.05; **p < 0.01.

Figure 3

Characterization of ANXA5 promoter. (A) Amplification of porcine ANXA5 promoter. (B) Verification of pGL3-ANXA5 and 5′-deletion plasmids. (C) 5′-deletion analysis of the pGL3-ANXA5. Values were shown as the mean ± SEM (n = 3). *p < 0.05; **p < 0.01.

Figure 4

In silico analysis of ns-SNPs in porcine ANXA5. (A) The sequencing results of rs711261805 locus as G. (B) The sequencing results of rs711261805 locus as C. (C) The sequencing results of rs708358082 locus with G were analyzed. (D) The sequencing results of rs343104241 locus as T. (E) The sequencing results of rs343104241 locus as C. (F) The sequencing results of rs328765480 locus as C. (G) The sequencing results of rs328765480 locus as A. (H) The sequencing results of rs346330091 locus as T. (I) The sequencing results of rs346330091 locus as G (J) Linkage disequilibrium analysis of g.-676 T > C, g.-674C > A and g.-105G > T.

Figure 5

Effect of ESR1 and g.-105G > T on ANXA5 transcription. (A) Predictive ESR1 binding site within ANXA5 promoter in ST cells. (B) Verification of pCMV-HA-ESR1 plasmids. (C) Overexpression of ESR1 up-regulate ANXA5 mRNA expression. (D) Overexpression of ESR1 up-regulate ANXA5 promoter activity. (E) Schematic presentation of putative ESR1-binding sites in ANXA5 promoter. (F) Overexpression of ESR1 up-regulate the promoter activity of ANXA5 with T allele at g.-105G > T. Values were shown as the mean ± SEM (n = 3). *p < 0.05; **p < 0.01.