The Effect of RBP4 on microRNA Expression Profiles in Porcine Granulosa Cells.

Retinol binding protein 4 (RBP4) is a transporter of vitamin A that is secreted mainly by hepatocytes and adipocytes. It affects diverse pathophysiological processes, such as obesity, insulin resistance, and cardiovascular diseases. MicroRNAs (miRNAs) have been reported to play indispensable roles in regulating various developmental processes via the post-transcriptional repression of target genes in mammals. However, the functional link between RBP4 and changes in miRNA expression in porcine granulosa cells (GCs) remains to be investigated. To examine how increased expression of RBP4 affects miRNA expression, porcine GCs were infected with RBP4-targeted lentivirus for 72 h, and whole-genome miRNA profiling (miRNA sequencing) was performed. The sequencing data were validated using real-time quantitative polymerase chain reaction (RT-qPCR) analysis. As a result, we obtained 2783 known and 776 novel miRNAs. In the experimental group, 10 and seven miRNAs were significantly downregulated and upregulated, respectively, compared with the control group. Ontology analysis of the biological processes of these miRNAs indicated their involvement in a variety of biological functions. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses indicated that these miRNAs were involved mainly in the chemokine signaling pathway, peroxisome proliferators-activated receptors (PPAR) signaling pathway, insulin resistance pathway, nuclear factor-kappa B(NF-kappa B) signaling pathway, and steroid hormone biosynthesis. Our results indicate that RBP4 can regulate the expression of miRNAs in porcine GCs, with consequent physiological effects. In summary, this study profiling miRNA expression in RBP4-overexpressing porcine GCs provides an important reference point for future studies on the regulatory roles of miRNAs in the porcine reproductive system.

Altered expression of miRNAs in the uterus from a letrozole-induced rat PCOS model.

Polycystic ovary syndrome (PCOS) causes female subfertility with ovarian disorders and may be associated with increased rate of early-pregnancy failure. Rat PCOS models were established using letrozole to understand the uterine pathogenesis of PCOS. The differential expression of microRNAs (miRNAs) was observed in rat uterus with PCOS. After estrous cycles were disrupted, significantly abnormal ovarian morphology and hormone level were observed in rats with PCOS. A total of 148 miRNAs differentially expressed were identified in the uterus from the letrozole-induced rat model compared with the control. These miRNAs included 111 upregulated miRNAs and 37 downregulated miRNAs. The differential expression of miR-484, miR-375-3p, miR-324-5p, and miR-223-3p was further confirmed by quantitative reverse transcription polymerase chain reaction. Bioinformatic analysis showed that these four miRNAs were predicted to regulate a large number of genes with different functions. Pathway analysis supported that target genes of miRNAs were involved in insulin secretion and signaling pathways, such as wnt, AMPK, PI3K-Akt, and Ras. These data indicated that miRNAs differentially expressed in rat uterus with PCOS may be associated with PCOS pathogenesis in the uterus. Our findings can help clarify the mechanism of uterine defects in PCOS.

MicroRNA-16 Modulates Melatonin-Induced Cell Growth in the Mouse-Derived Spermatogonia Cell Line GC-1 spg Cells by Targeting Ccnd1.

Melatonin exerts a range of physiological effects. However, the functional significance of melatonin in spermatogenesis and the underlying mechanisms remain unclear. MicroRNAs (miRNAs) are essential in the initiation and progression of testicular development, including spermatogenesis. Thus far, limited information is known about the role of miRNAs in melatonin-mediated spermatogenesis. In this study, the expression levels of testicular miRNA machinery genes, namely, Dgcr8 and Xpo5, were significantly increased by melatonin. The miRNA expression profile was identified in the testes of melatonin-treated mice. Ten miRNAs were significantly up-regulated, and 15 miRNAs were down-regulated. Melatonin (25 µM) enhanced cell growth and reduced apoptosis in GC-1 spg cells. Among the down-regulated miRNAs, miR-16 expression was influenced by melatonin in GC-1 spg cells. The miR-16 mimics in GC-1 spg cells significantly suppressed cell growth and promoted cell apoptosis. Conversely, transfection of the miR-16 inhibitor significantly increased cell growth and decreased cell apoptosis. The protein expression level of CCND1 (Cyclin D1) in GC-1 spg cells was decreased by the miR-16 mimics and increased by knockdown of miR-16. Moreover, bioinformatics and reporter activity analyses showed that Ccnd1 was a potential target of miR-16. These results suggested that miR-16 may function as a novel regulator of testicular functions during melatonin stimulation by targeting Ccnd1.

BDNF-induced expansion of cumulus-oocyte complexes in pigs was mediated by microRNA-205.

The neurotrophin family of proteins is required for the survival and differentiation of the nervous system and is important to the development of reproductive tissues. The objectives of the present study were to detect the presence of the brain-derived neurotrophic factor (BDNF) and its tyrosine kinase receptor protein in cumulus-oocyte complexes in pigs and to explore the role of microRNAs in the BDNF-induced in vitro maturation of oocytes. We demonstrate that both BDNF and tyrosine kinase receptor protein are expressed in porcine cumulus oocyte complexes. BDNF supplementation promotes the in vitro maturation of porcine oocytes. MiRNA-205 is downregulated during the BDNF-induced maturation of oocytes. The overexpression of miRNA-205 in granulosa cells and reporter gene assay shows that the marker gene ptx3 for cumulus expansion is the putative target gene of miR-205. Our data provide evidence that the BDNF-induced maturation of oocytes in pigs may be mediated by miR-205 through the regulation of potential target gene, ptx3.

Downregulation of the expression of inhibin α subunit and betaglycan in porcine cystic follicles.

Inhibins, as members of the transforming growth factor beta (TGF-ß) superfamily, downregulate the synthesis and secretion of follicle-stimulating hormone (FSH) in an endocrine manner. The role of inhibin/betaglycan in the ovary regulation recently gained attention. To date, no data exist on the function of inhibin α subunit and betaglycan in cystic follicles. In this study, the expressions of inhibin α subunit and betaglycan in cystic follicles were investigated using immunohistochemistry, real-time PCR and Western blot analysis. Both inhibin α subunit and betaglycan immunoreactivities were mainly localized in the granulosa cells of follicles. Expression of inhibin α subunit and betaglycan was inferior in cystic follicles compared with that in normal large follicles. However, the result of enzyme-linked immunosorbent assay showed no significant difference in the decreasing in concentration of inhibin α subunit in cystic follicular fluid compared with the control (P>0.05). In this study, we explored the effects of FSH on betaglycan expression in granulosa cells in vitro. As expected, a significant increase in the expressions of betaglycan mRNA and protein in granulosa cells was observed in response to exogenous FSH (30 ng/ml) (P<0.05) compared with the control. Consequently, this study provides evidence that the expressions of inhibin α subunit and betaglycan are inferior in cystic follicles, and this may be caused by the decrease in FSH in the presence of a cystic follicle.

4-Methylcatechol-induced cell damage in TM4 Sertoli cells.

4-Methylcatechol (4-MC) is one of the metabolites of quercetin, which is a potential drug for neuroprotection and tumorigenesis inhibition. This study was performed to investigate the cytotoxic effect of 4-MC in mouse TM4 Sertoli cells. TM4 Sertoli cell viability was significantly inhibited by 4-MC in a time- and dose-dependent manner. The number of apoptotic and dead cells was significantly increased after 4-MC treatment. Caspase 3 activity increased by prolonged exposure of TM4 Sertoli cells to 200 µM 4-MC. The 4-MC significantly upregulated the mRNA level of Bax gene and considerably downregulated the Bcl-2 gene expression in a concentration-dependent manner. Results showed that 4-MC could induce TM4 Sertoli cell apoptosis, and the cytotoxic effect of 4-MC on TM4 Sertoli cells may be associated with upregulated Bax gene expression, which induced caspase cascade activation.

Analysis of the vp2 gene sequence of a new mutated mink enteritis parvovirus strain in PR China.

BACKGROUND: Mink enteritis virus (MEV) causes a highly contagious viral disease of mink with a worldwide distribution. MEV has a linear, single-stranded, negative-sense DNA with a genome length of approximately 5,000 bp. The VP2 protein is the major structural protein of the parvovirus encoded by the vp2 gene. VP2 is highly antigenic and plays important roles in determining viral host ranges and tissue tropisms. This study describes the bionomics and vp2 gene analysis of a mutated strain, MEV-DL, which was isolated recently in China and outlines its homologous relationships with other selected strains registered in Genbank. RESULTS: The MEV-DL strain can infect F81 cells with cytopathic effects. Pig erythrocytes were agglutinated by the MEV-DL strain. The generation of MEV-DL in F81 cells could infect mink within three months and cause a disease that was similar to that caused by wild-type MEV. A comparative analysis of the vp2 gene nucleotide (nt) sequence of MEV-DL showed that this was more than 99% homologous with other mink enteritis parvoviruses in Genbank. However, the nucleotide residues at positions 1,065 and 1,238 in the MEV-DL strain of the vp2 gene differed from those of all the other MEV strains described previously. It is noteworthy that the mutation at the nucleotide residues position 1,238 led to Asp/Gly replacement. This may lead to structural changes. A phylogenetic tree and sequence distance table were obtained, which showed that the MEV-DL and ZYL-1 strains had the closest inheritance distance. CONCLUSIONS: A new variation of the vp2 gene exists in the MEV-DL strain, which may lead to structural changes of the VP2 protein. Phylogenetic analysis showed that MEV-DL may originate from the ZYL-1 strain in DaLian.