The comprehensive detection of miRNA, lncRNA, and circRNA in regulation of mouse melanocyte and skin development

BACKGROUND: Pigmentation development, is a complex process regulated by many transcription factors during development. With the development of the RNA sequencing (RNA-seq), non-coding RNAs, such as miRNAs, lncRNAs, and circRNAs, are found to play an important role in the function detection of related regulation factors. In this study, we provided the expression profiles and development of ncRNAs related to melanocyte and skin development in mice with black coat color skin and mice with white coat color skin during embryonic day 15 (E15) and postnatal day 7 (P7). The expression profiles of different ncRNAs were detected via RNA-seq and also confirmed by the quantitative real-time PCR (qRT-PCR) method. GO and KEGG used to analyze the function the related target genes. RESULTS: We identified an extensive catalogue of 206 and 183 differently expressed miRNAs, 600 and 800 differently expressed lncRNAs, and 50 and 54 differently expressed circRNAs, respectively. GO terms and pathway analysis showed the target genes of differentially expressed miRNA and lncRNA. The host genes of circRNA were mainly enriched in cellular process, single organism process. The target genes of miRNAs were mainly enriched in chromatin binding and calcium ion binding in the nucleus. The function of genes related to lncRNAs are post translation modification. The competing endogenous RNA (ceRNA) network of lncRNAs and circRNAs displays a complex interaction between ncRNA and mRNA related to skin development, such as Tcf4 , Gnas , and Gpnms related to melanocyte development. CONCLUSIONS: The ceRNA network of lncRNA and circRNA displays a complex interaction between ncRNA and mRNA related to skin development and melanocyte development. The embryonic and postnatal development of skin provide a reference for further studies on the development mechanisms of ncRNA during pigmentation.

Study on the relationship between expression patterns of cocaine-and amphetamine regulated transcript and hormones secretion in porcine ovarian follicles

Abstract Background Cocaine-and amphetamine regulated transcript (CART) is an endogenous neuropeptide, which is widespread in animals, plays a key role in regulation of follicular atresia in cattle and sheep. Among animal ovaries, CART mRNA was firstly found in the cattle ovaries. CART was localized in the antral follicles oocytes, granulosa and cumulus cells by immunohistochemistry and in situ hybridization. Further research found that secretion of E2 was inhibited in granulosa cells with a certain dose of CART, the effect depends on the stage of cell differentiation, suggesting that CART could play a crucial role in regulating follicle atresia. The objective of this study was to characterize the CART expression model and hormones secretion in vivo and vitro in pig follicle granulosa cells, preliminarily studied whether CART have an effect on granulosa cells proliferation and hormones secretion in multiparous animals such as pigs. Methods The expression levels of CART mRNA in granulosa cells of different follicles were analyzed using qRT-PCR technology. Immunohistochemistry technology was used to localize CART peptide. Granulosa cells were cultured in medium supplemented with different concentrations of CART and FSH for 168 h using Long-term culture system, and observed using a microscope. The concentration of Estradiol (E2) and progesterone (P) in follicular fluids of different test groups were detected by enzyme linked immunosorbent assay (ELISA). Results Results showed that expression level of CART mRNA was highest in medium follicles, and significantly higher than that in large and small follicles (P < 0.05). Immunohistochemical results showed that CART were expressed both in granulosa cells and theca cells of large follicles, while CART were detected only in theca cells of medium and small follicles. After the granulosa cells were cultured for 168 h, and found that concentrations of E2 increase with concentrations of follicle-stimulating hormone (FSH) increase when the CART concentration was 0 μM. And the concentration of FSH reached 25 ng/mL, the concentration of E2 is greatest. It shows that the production of E2 needs induction of FSH in granulosa cells of pig ovarian follicles. With the increasing of CART concentrations (0.01, 0.1, 1 μM), E2 concentration has a declining trend, when the FSH concentrations were 25 and 50 ng/mL in the medium, respectively. Conclusions These results suggested that CART plays a role to inhibit granulosa cells proliferation and E2 production, which induced by FSH in porcine ovarian follicular granulosa cells in vitro, but the inhibition effect is not significant. So we hypothesis CART maybe not a main local negative regulatory factor during porcine follicular development, which is different from the single fetal animals.

Expression of cocaine- and amphetamine-regulated transcript (CART) in hen ovary

BACKGROUND: Cocaine- and amphetamine-regulated transcript (CART), discovered initially by via differential display RT-PCR analysis of brains of rats administered cocaine, is expressed mainly in central nervous system or neuronal origin cells, and is involved in a wide range of behaviors, such as regulation of food intake, energy homeostasis, and reproduction. The hens egg-laying rate mainly depends on the developmental status of follicles, expression of CART have not been identified from hen follicles, the regulatory mechanisms of CART biological activities are still unknown. The objective of this study was to characterize the mRNA expression of CART in hen follicular granulosa cells and determine CART peptide localization and regulatory role during follicular development. METHODS: Small white follicles (1-2 mm in diameter) were treated for RNA isolation; Small white follicles (1-2 mm in diameter) and large white follicles (4-6 mm in diameter) were treated for immunohistochemical localization and large white follicles (4-6 mm in diameter), small yellow follicles (6-8 mm in diameter), large yellow follicles (9-12 mm in diameter), mature follicles (F5, F4, F3, F2, F1, >12 mm in diameter) were treated for RNA isolation and Real time PCR. RESULTS: The results showed that full length of the CDS of hen CART was 336 bp encoding a 111 amino acid polypeptide. In the hen ovary, CART peptide was primarily localized to the theca layer, but not all, the oocyte and granulosa layer, with diffused, weaker staining than relative to the theca cell layer. Further, amount of CART mRNA was more (P < 0.05) in granulosa cells of 6-8 mm follicles compared with that in granulosa cells of other follicles. However, CART mRNA amount was greater in theca cells of 4-6 mm follicles relative to follicles of other sizes (P < 0.05). CONCLUSIONS: Results suggest that CART could play a potential role in developmental regulation of chicken follicles.

Depressed nNOS expression during spine transition in the developing hippocampus of FMR1 KO mice

Nitric oxide (NO), synthesized as needed by NO synthase (NOS), is involved in spinogenesis and synaptogenesis. Immature spine morphology is characteristic of fragile X syndrome (FXS). The objective of this research was to investigate and compare changes of postnatal neuronal NOS (nNOS) expression in the hippocampus of male fragile X mental retardation 1 gene knockout mice (FMR1 KO mice, the animal model of FXS) and male wild-type mice (WT) at postnatal day 7 (P7), P14, P21, and P28. nNOS mRNA levels were analyzed by real-time quantitative PCR (N = 4-7) and nNOS protein was estimated by Western blot (N = 3) and immunohistochemistry (N = 1). In the PCR assessment, primers 5’-GTGGCCATCGTGTCCTACCATAC-3’ and 5’-GTTTCGAGGCAGGTGGAAGCTA-3’ were used for the detection of nNOS and primers 5’-CCGTTTCTCCTGGCTCAGTTTA-3’ and 5’-CCCCAATACCACATCATCCAT-3’ were used for the detection of β-actin. Compared to the WT group, nNOS mRNA expression was significantly decreased in FMR1 KO mice at P21 (KO: 0.2857 ± 0.0150, WT: 0.5646 ± 0.0657; P < 0.05). Consistently, nNOS immunoreactivity also revealed reduced staining intensity at P21 in the FMR1 KO group. Western blot analysis validated the immunostaining results by demonstrating a significant reduction in nNOS protein levels in the FMR1 KO group compared to the WT group at P21 (KO: 0.3015 ± 0.0897, WT: 1.7542 ± 0.5455; P < 0.05). These results suggest that nNOS was involved in the postnatal development of the hippocampus in FXS and impaired NO production may retard spine maturation in FXS.