Molecular Mechanism Of HSD17B14 Gene Regulating Estrus Behavior Of Gilts

The estrus behavior plays an important role on identification of estrus and timely insemination of gilts and sows in intensive pig farms.Our previous studies have found that Chinese Mi gilts showed more obvious estrus behavior than large white gilts did.At the molecular genetic level,some candicate genes of estrus behavior had been screened through RNA-seq.Therefore,based on the previous studies of our lab,the HSD17B14 gene was selected in the present study.The aim of this study is to identify the molecular mechanism of HSD17B14 gene regulating the estrus behavior in pigs.As a member of the SDR family,HSD17B14 gene plays an important role in the estrogen metabolism pathway.It converts high-efficiency estradiol(E2)into low-efficiency estrone(E1).Therefore,we hypothesized that HSD17B14 gene has an important impact on estrus behavior of pigs by influencing the level of E2.The results are as follows:1.Bioinformatics analyses of porcine HSD17B14 geneThe promoter region,CpG islands as well as binding site of transcription factors of porcine HSD17B14 genewere predicted on Bioinformatics websites.The predicted results showed that five core regions of promoter were in the promoter region of porcine HSD17B14 gene and three transcription initiation sites were found.The SNP sites in these regions would cause a large number of changes in transcription factor binding sites.These results provide useful information for the experimental study on the molecular mechanism of HSD17B14 gene regulating the estrus behavior in pigs.2.Identification of functional SNPs in porcine HSD17B14 gene associated with estrus behavior in giltsTo identify the functional SNPs in porcine HSD17B14 gene,ear tissue samples from Large White and Mi gilts were collected to extract genomic DNA.Variable lengths of truncated promoter of HSD17B14 gene were used to determine the promoter activity by a dual luciferase reporter assay.Vector HSD17B14Phe or HSD17B14Val was transfected into GCs.The core promoter region was identified between-972bp and-218bp.Six of seven SNPs had significant differences of allele frequency between Large White and Mi gilts.The plasmids with wild genotype AA of rs329427898 had a less fraction of promoter activity compared with the plasmids with mutant genotype GG,while the plasmids with wild genotype TT of rs319864566 had a greater promoter activity than the plasmids with mutant genotype CC.A missense mutation(Phe73Val)caused changes in structural dynamics and function of HSD17B14 protein.The highly expressed HSD17B14Val degraded less estradiol into estrone,while the relatively lowly expressed HSD17B14Phe degraded more estradiol into estrone,suggesting the protein activity of HSD17B14Phe was greater than that of HSD17B14Val.Moreover,the HSD17B14Phe group has greater apoptosis rate of porcine GCs.These results suggest that these SNPs in HSD17B14 gene could been used as candidate molecular markers for estrus behavior in pigs.3.MiR-20b and miR-31 target the HSD17B14 to influence the steroid hormone metabolism and granulosa cell apoptosisTo explore the epigenetic regulatory mechanisms of HSD17B14 gene in porcine GCs,in this study,two miRNAs(miR-31 and miR-20b)were screened out which target the 3’-UTR of HSD17B14 mRNA through bioinformatics analyses and dual luciferase reporter assays.Since previous studies have found that overexpression of this gene catalyzed the conversion of E2 to E1,as well as the apoptosis of porcine GCs.Overexpression of miR-20b inhibited the expression of HSD17B14 gene,which decreased the rate of apoptosis and the level of progesterone(P4)in porcine GCs.In contrast,although miR-31 targets the HSD17B14 mRNA,it did not show the same experimental results with miR-20b.On the contrary,it increased the apoptosis rate of porcine GCs.Through further bioinformatics analysis,we found that miR-31 also targets the 3’-UTR of porcine FSHR mRNA.A dual luciferase activity reporter assay further validated our prediction.Therefore,both miR-31 and miR-20b targets porcine HSD17B14 mRNA,but they have different regulation modes.These results provided epigenetic regulatory of the development of porcine GCs and steroid hormone metabolism,and expanded our understanding of the molecular mechanism of HSD17B14 gene regulating the estrus behavior in pigs.In conclusion,the molecular regulatory mechanisms,including the promoter activity,missense mutation,and miRNAs,of HSD17B14 gene regulating the estrus behavior in gilts were investigated in this study.