Characterization Of Serum Reproductive Hormones And Ovarian MicroRNAs Of Bamei Mutton Sheep During Estrus

Reproductive trait is one of most important economic traits in the sheep industry.It’s an effective measure to increase litter size per fetus to reduce production costs andincrease productivity, with important theoretical and practical significance. Sheep littersize is affected by ovulation rate. Follicular development is an extremely complex andhighly coordinated process, which is precisely regulated by the endocrine,paracrine/autocrine factors from hypothalamus, pituitary and ovary as well as manyother growth factors. So far however, the molecular mechanism of folliculardevelopment, maturation, ovulation and the reasons caused ovulation rate differences isnot fully understood. In recent years, microRNAs have been discovered to play a role inmany biological processes, such as body growth, differentiation, metabolism anddisease, and its role in mammalian reproduction is also gradually taken seriously. Inthis study, two aspects were researched about reproductive endocrine hormone andovarian microRNAs. The study performed ECLIA and miRNA microarray assay toresearch reproductive hormones change and ovarian microRNAs expression profileduring estrus of Bamei mutton sheep, and compare their differences betweensingle-bearing and biparous ewes, in order to discuss the factors and molecularregulation mechanism causing differences in sheep litter size.1. Research of serum reproductive hormone changes and their relationship withlitter size in Bamei mutton sheep during estrusRules and characterization of4kinds of reproductive hormone FSH, LH, E2and P4were analyzed during estrus of single-bearing and biparous Bamei mutton sheep usingECLIA technology. The results showed that:(1) Serum FSH and LH were secreted in a pulsatile way. Concentration of serumFSH peaked at1h after estrus (P<0.001), and concentration of serum LH peak during12~24h after estrus; Serum E2and P4appeaed different changing rules betweensingle-bearing and biparous groups: in single-bearing group, E2showed a "V" typechanging trend, while in biparous group, E2showed a downward changing trend; P4showed an upward changing trend in single-bearing group, but showed an inverted "V"type changing trend in biparous group.(2) Variance analysis showed that, serum FSH level in biparous ewes was extreme significantly higher than that of single-bearing ewes at1h after estrus(P<0.001), andwas significantly higher than that of single-bearing ewes at12h after estrus (P<0.05);serum LH level in biparous ewes was extreme significantly higher than that ofsingle-bearing ewes at12h after estrus (P<0.001), and was significantly higher thanthat of single-bearing ewes at24h after estrus (P<0.05). This indicated that, increase inBamei mutton sheep litter size may be associated with raised serum FSH and LHconcentration.(3) Serum E2and P4levels at different time points showed no significant differencebetween single-bearing and biparous group (P>0.05), but the two hormones showeddifferent changing rules between the two groups. Their relationship with Bamei muttonsheep fecundity remains to be further studied.2. MicroRNAs expression profiling and differentially expressed microRNAsscreening, identification and characterization in ovarian tissue of Bamei mutton sheep(1) Multiple species miRNA microarrays were applied to3cases of single-bearingewes and3cases of biparous ewes to analysis ovarian miRNAs expression. The resultsshowed that, in all the detected miRNAs, there were5448miRNAs expressed in6ovarian samples. Among them,22miRNAs were specifically expressed insingle-bearing samples, and15miRNAs were specifically expressed in biparoussamples. In103detected miRNAs from sheep, there were47miRNAs expressed in6ovarian samples.(2) Differential expression analysis was performed on the103miRNAs from sheep.Compared with single-bearing ewes, biparous ewes contained11differentiallyexpressed miRNAs in the ovarian tissue, of which4miRNAs were up-regulated (FoldChange≥2),7miRNAs were down-regulated (Fold Change≤0.5). These miRNAs maybe related to sheep follicular development and litter size. Up-regulated geneoar-miR-381-5P and down-regulated gene oar-miR-1185-5p were randomly selected tovalidate the accuracy of the microarray using miRNA Real Time RT-PCR technology.The results showed that, miRNA microarray results were accurate and credible.(3) Bioinformatics analysis indicated that, the11differentially expressed miRNAs,except for oar-miR-544-5p which had no similar sequence with sheep reference genome,were all intron miRNAs located in sheep chromosome18. Target genes of thedifferentially expressed miRNAs were predicted using two different methods (microTand miRDB), then took the intersection, and finally obtained a total of target genes of7miRNAs: oar-miR-370-5p, oar-miR-376b-5p, oar-miR-381-5p, oar-miR-412-5p, oar-miR-541-3p, oar-miR-544-5p and oar-miR-1185-5p, these miRNAs obtained115,71,1,5,8,135and23target genes, respectively.(4) GO annotation results of partial target genes showed that: the target genes ofmiR-376b-5p and miR-1185-5p were distributed in most functional classifications ofthe cell component, molecular function and biological process. Referring to the existingliteratures, the target genes of miR-376b-5p, such as IGF-1, DAZL, MTOR et al, andtarget gene of miR-376b-5p such as AHR were closely related to mammalianreproductive process such as follicular growth and development, reproductive hormonesecretion. At the same time, this also indicated that miR-376b-5p and miR-1185-5p mayplay important roles in the regulation of mammalian reproduction through these targetgenes.