Background MiRNAs act as pivotal post-transcriptional gene mediators in the regulation of diverse biological processes, including proliferation, development and apoptosis. Our previous study has showed that miR-101-3p is differentially expressed in dairy goat ovaries compared single with multiple litters. The objective of this research was to explore the potential function and molecular mechanism of miR-101-3p via its target STC1 in goat ovarian growth and development. Results cDNA libraries were constructed using goat granulosa cells transfected with miR-101-3p mimics and negative control by RNA-sequencing. In total, 142 differentially expressed unigenes (DEGs) were detected between two libraries, including 78 down-regulated and 64 up-regulated genes. GO and KEGG enrichment analysis showed the potential impacts of DEGs on ovarian development. STC1 was singled out from DEGs for further research owing to it regulates reproductive-related processes. In vitro, bioinformatics analysis and 3′-UTR assays confirmed that STC1 was a target of miR-101-3p. ELISA was performed to detect the estrogen (E2) and progesterone (P4) levels. CCK8, EdU and flow cytometry assays were performed to detect the proliferation and apoptosis of granulosa cells. Results showed that miR-101-3p regulated STAR, CYP19A1, CYP11A1 and 3β-HSD steroid hormone synthesis-associated genes by STC1 depletion, thus promoted E2 and P4 secretions. MiR-101-3p also affected the key protein PI3K, PTEN, AKT and mTOR in PI3K-AKT pathway by STC1, thereby suppressing proliferation and promoting apoptosis of granulosa cells. In vivo, the distribution and expression levels of miR-101-3p in mouse ovaries were determined through fluorescence in situ hybridisation (FISH). Immunohistochemistry results showed that STC1 expression was suppressed in mouse ovaries in miR-101-3p-agonist and siRNA-STC1 groups. Small and stunted ovarian fragments, decreased numbers of follicles at diverse stages were observed using Hematoxylin-eosin (HE) staining, thereby showing unusual ovarian development after miR-101-3p overexpression or STC1 depletion. Inhibition of miR-101-3p manifested opposite results. Conclusions Taken together, our results demonstrated a regulatory mechanism of miR-101-3p via STC1 in goat granulosa cells, and offered the first in vivo example of miR-101-3p and STC1 functions required for ovarian development.

BackgroundDNA methylation plays a vital role in reproduction. Entire genome DNA methylation changes during the oestrous phase (ES) and dioestrous phase (DS) in the ovaries of Guanzhong dairy goats were investigated using bisulphite sequencing to understand the molecular biological mechanisms of these goats’ oestrous cycle.ResultsWe discovered distinct genome-wide DNA methylation patterns in ES and DS ovaries. A total of 26,910 differentially methylated regions were upregulated and 21,453 differentially methylated regions were downregulated in the ES samples compared with the DS samples (P-values ≤0.05 and fold change of methylation ratios ≥2). Differentially methylated region analysis showed hypomethylation in the gene body regions and hypermethylation in the joining region between upstream regions and gene bodies. The methylation ratios of the STAR, FGF2, FGF12, BMP5 and SMAD6 genes in the ES samples were lower than those of the DS samples (P-values ≤0.05 and fold change of methylation ratios ≥2). Conversely, the methylation ratios of the EGFR, TGFBR2, IGF2BP1 and MMD2 genes increased in the ES samples compared with the DS samples. In addition, 223 differentially methylated genes were found in the GnRH signalling pathway (KO04912), ovarian steroidogenesis pathway (KO04913), oestrogen signalling pathway (KO04915), oxytocin signalling pathway (KO04921), insulin secretion pathway (KO04911) and MAPK signalling pathway (KO04010).ConclusionsThis study is the first large-scale comparison of the high-resolution DNA methylation landscapes of oestrous and dioestrous ovaries from dairy goats. Previous studies and our investigations have shown that the NR5A2, STAR, FGF2 and BMP5 genes might have potential application value in regulating caprine oestrus.Electronic supplementary materialThe online version of this article (10.1186/s40104-018-0301-x) contains supplementary material, which is available to authorized users.

In this study, an RT-qPCR analysis showed that the expression levels of miR-449a in the pre-receptive endometrium were lower compared to the receptive endometrium, which is consistent with previous sequencing data (previous investigations). To detect the role of miR-449a in endometrial receptivity, we transfected caprine endometrial stromal cells (ESCs) cultured in vitro with miR-449a mimics. The results revealed that miR-449a decreased the mRNA and protein levels of LGR4 by targeting its 3′-untranslated region. The miR-449a mimics significantly reduced the G1 cell population from 52.56% (mimic NC) to 42.19% with a concordant increase in the G2 and S cell populations from 47.44% (mimic NC) to 57.81%, suggesting that miR-449a caused ESC cell cycle arrest. In addition, the number of apoptotic cells was significantly higher in ESCs transfected with miR-449a mimics (P < 0.05) than in ESCs transfected with mimic NC. In vivo, rich pinopodes were observed on the endometrial surface in the miR-449a agomir group compared with the miR-449a antagomir group. The results of hematoxylin-eosin staining showed that endometrial thickness was significantly increased in the miR-449a agomir group compared with the miR-449a antagomir group. These results suggest that miR-449a could enhance endometrial receptivity.

This study identified three novel single nucleotide polymorphisms (SNPs) (c.1389C > T, c.1457A > C and c.1520G > A) in the caprine KITLG 3′-UTR through DNA sequencing. The three SNP loci were closely linked in Guanzhong dairy (GD) goats. Two alleles of the c.1457A > C SNP introduced two miRNA sites (chi-miR-204-5p and chi-miR-211). Individuals with combined genotype TT-CC-AA had a higher litter size compared with those with combined genotypes CC-AA-GG, TC-CC-GA and CC-AC-GG (P < 0.05). Luciferase assays showed that chi-miR-204-5p and chi-miR-211 suppressed luciferase expression in the presence of allele 1457A compared with negative control (NC) and allele 1457C (P < 0.05). Western blot revealed that KITLG significantly decreased in the granulosa cells (GCs) of genotype AA compared with that in the GCs of genotype CC and NC (P < 0.05). The KITLG mRNA levels of the CC-AA-GG carriers significantly decreased compared with those of the TT-CC-AA, TC-CC-GA and CC-AC-GG carriers. In addition, cell proliferation was reduced in haplotype C-A-G GCs compared with that in haplotype T-C-A GCs. These results suggest that SNPs c.1389C > T, c.1457A > C and c.1520G > A account for differences in the litter size of GD goats because chi-miR-204-5p and chi-miR-211 could change the expression levels of the KITLG gene and reduce GC proliferation.