Identification of FASN Gene Polymorphisms, Expression and Their Relationship with Body Size Traits in Guizhou White Goat (Capra hircus) with Different Genders.

To investigate the nucleotide variation sites (SNPs) and expression differences of the fatty acid synthase gene (FASN) in Guizhou white goats, the relationship between the variation and body size traits was investigated. In this study, DNA was extracted from the blood of 100 samples of white goats from different regions in Guizhou province, China, and the variation sites were screened using pooled sequencing by mixing DNA samples, and 242 blood samples with body size traits were used for association analysis. The allele frequency, genotype frequency, homozygosity, heterozygosity and effective gene number were calculated by using PopGene 32.0 software, the population polymorphism information content was calculated by using PIC software (Version 0.6), and the state of genetic balance of the genes was analyzed by using the chi-square test. The mRNA of FASN gene expression levels in male and female goats were investigated by using real-time fluorescence quantitative PCR (RT-qPCR). The general linear mixed model of MINTAB software (Version 16.0) was used to analyze the association between FASN gene nucleotide mutation sites and body size traits. The results showed that there was one nucleotide mutation site g.141 C/T in the target fragment of FASN gene amplification, and revealed two alleles, C and T, and three genotypes CC, CT and TT. The genotype frequencies for CC, CT and TT were 0.4308, 0.4205 and 0.1487, respectively. The allele frequencies for C and T were 0.6410 and 0.3590, respectively. The genetic homozygosity (Ho) was higher than the heterozygosity (He). The χ2 test showed that the mutation site was in the Hardy-Weinberg equilibrium state (p > 0.05). The RT-qPCR results showed that the FASN gene had different expression levels in the longissimus dorsi muscle of male and female goats, and its expression was significantly higher in male goats than in female goats. The association analysis results showed that the mutation of the FASN gene had different effects on body size traits of male and female goats, and the presence of the populations of the T allele and the TT genotype recorded higher body size traits (body weight, heart girth and wither height) in female populations. Therefore, the site of the FASN gene can be used as a candidate marker for the early selection of growth traits in Guizhou white goats.

Deciphering the miRNA transcriptome of granulosa cells from dominant and subordinate follicles at first follicular wave in goat.

In goats, most follicles in the ovaries will be atresia and only a few dominant follicles (DFs) may eventually mature and ovulate at a follicular wave. To investigate the potential microRNAs (miRNAs) that regulate the expression of genes associated with follicular dominance or atresia, small RNA sequencing was performed on granulosa cells of DF and subordinate follicle at the first follicular wave in goats. A total of 108 differentially expressed miRNAs were detected in the two types of follicle granulosa cells: 16 upregulated miRNAs and 92 downregulated miRNAs. Kyoto Encyclopedia of Genes and Genomes analysis of the target genes showed that TKTL1, LOC102187810, LOC102184409 and ALDOA are closely associated with follicle dominance and are involved in the pentose phosphate pathway. Furthermore, a coexpression network of miRNAs and follicular dominance-related genes was constructed. The qPCR results well correlated with the small RNA sequencing data. Our findings provide new insight for exploring the molecular mechanism of miRNAs in regulating follicular development in goats.

m6A mRNA Methylation Analysis Provides Novel Insights into Pigmentation in Sheep Skin.

N6-methyladenosine (m6A) is the most universal post-transcriptional modification of mRNA which may play important roles in verious species. However, the potential roles of m6A in the pigmentation of skin are not completely understood. To explore the role of m6A modification in pigmentation of sheep skin, we used MeRIP-seq and RNA-seq to profile the skin transcriptome in black and white coat color (n=3). Our results showed that an average of 7701 m6A peaks were obtained for all samples and the average length was 305.89 bp. The GGACUU sequence was the most enrichment motif and shared in black skin and white skin. The m6A peaks were mainly enriched in the CDS, 3'UTR and 5'UTR, especially in CDS region near the stop codon of the transcript. 235 significantly differential peaks were found in black skin vs. white skin. The KEGG signaling pathways of downregulated and upregulated m6A peaks were mainly enriched in AGE-RAGE signaling pathway in diabetic complications, Viral carcinogenesis, Transcriptional misregulation in cancer, ABC transporters, Basal transcription factors and Thyroid hormone synthesis (P value <0.05). For RNA-seq, 71 differently expressed genes (DEGs) were scanned in black skin vs. white skin. DEGs were significantly enriched in tyrosine metabolism, melanogenesis, neuroactive ligand-receptor interaction pathway (P value <0.05). Combined m6A-seq and RNA-seq analysis showed that the hyper-up genes and hypo-up genes were both enriched in ErbB signaling pathway (P value <0.05). In conclusion, it provide a basis for further research into the functions of m6A methylation modifications in pigmentation.

The First High-quality Reference Genome of Sika Deer Provides Insights into High-tannin Adaptation.

Sika deer are known to prefer oak leaves, which are rich in tannins and toxic to most mammals; however, the genetic mechanisms underlying their unique ability to adapt to living in the jungle are still unclear. In identifying the mechanism responsible for the tolerance of a highly toxic diet, we have made a major advancement by explaining the genome of sika deer. We generated the first high-quality, chromosome-level genome assembly of sika deer and measured the correlation between tannin intake and RNA expression in 15 tissues through 180 experiments. Comparative genome analyses showed that the UGT and CYP gene families are functionally involved in the adaptation of sika deer to high-tannin food, especially the expansion of the UGT family 2 subfamily B of UGT genes. The first chromosome-level assembly and genetic characterization of the tolerance to a highly toxic diet suggest that the sika deer genome may serve as an essential resource for understanding evolutionary events and tannin adaptation. Our study provides a paradigm of comparative expressive genomics that can be applied to the study of unique biological features in non-model animals.

USP9X promotes the proliferation, invasion and metastasis of liver cancer cells through regulating the JAK2/STAT3 signaling.

X-linked ubiquitin-specific peptidase 9 (USP9X) serves important roles in the development and progression of various human cancers. However, its role and molecular mechanism in liver cancer require further elucidation. In the present study, USP9X was found to be upregulated in liver cancer tissues. At the same time, overexpression of USP9X promoted the proliferation, invasiveness and migration of liver cancer cells, which were subsequently suppressed by USP9X silencing. On a molecular level, the results revealed that USP9X knockdown suppressed elements of the Janus kinase 2 (JAK2)/STAT3 signaling pathway, including JAK2, STAT3, matrix metalloproteinase-2 and c-Myc. By contrast, overexpression of USP9X had the opposite effect. In conclusion, the results of the present study suggest that USP9X is upregulated in patients with liver cancer, which may accelerate the proliferation, invasiveness and migration of liver cancer cells by regulating the JAK2/STAT3 signaling pathway.

Identification of key genes associated with spermatogenesis arrest in fox hybrids using weighted gene co-expression network analysis.

The silver fox and the blue fox represent different genera, but produce viable offspring. Although these hybrids show obvious heterosis, they are completely sterile due to spermatogenic arrest at the early stages of spermatogenesis, especially mitosis and meiosis I; the hybrids produce few spermatogonia and primary spermatocytes, and no secondary spermatocytes. Although the mechanisms of spermatogenic arrest have been well investigated, transcriptomic differences between hybrid and the pure-species testes have not clarified. In the present study, we used RNA sequencing (RNA-Seq) to generate testicular transcriptomic profiles for silver foxes, blue foxes, and reciprocal hybrids during the pre-breeding period and the breeding season. In total, 1,344,022 transcripts (≥200 bp) were generated; 1,057,724 genes were obtained; and 33,423 genes were shown to have fragments per kilobase of transcript per million mapped reads (FPKM) > 0.3. To identify the hub genes associated with spermatogenesis arrest, weighted gene co-expression network analysis (WGCNA) was used. Nine modules were explored. Genes in only a single module were consistently downregulated in the hybrids as compared to the pure species; these genes were significantly associated with fox hybrid male infertility. Six of the genes in this module (CATSPERD, DMRTC2, RNF17, NME5, SPEF2, SPINK2) also play key roles in mitosis and meiosis during spermatogenesis. Therefore, these six genes might be associated with fox hybrid male infertility.

Epigallocatechin-3-Gallate Promotes the Growth of Mink Hair Follicles Through Sonic Hedgehog and Protein Kinase B Signaling Pathways.

Background: Hair follicles play an essential role in the growth of hair. Epigallocatechin-3-gallate (EGCG), a catechin polyphenol in green tea, has various bioactivities. The present study aims to evaluate the effect of EGCG on the growth of mink hair follicles and investigate the possible molecular mechanisms. Methods: The length of hair follicles was recorded up to 6 days in presence of 0.1-5 µM EGCG. Primary dermal papilla cells (DPCs) and outer root sheath cells (ORSCs) were treated with 0.25-4 µM EGCG, and their growth was evaluated by MTT assay and cell cycle detection. The levels of key molecules in sonic hedgehog (Shh) and protein kinase B (AKT) signaling pathways were further assessed by quantitative real-time PCR, western blot and immunofluorescence. To determine the involvement of Shh and AKT pathways in EGCG-mediated growth-promotion of ORSCs and DPCs, Shh pathway inhibitors cyclopamine and GANT61 or AKT pathway inhibitor LY294002 were employed, and then cell proliferation and cell cycle were analyzed. Results: Data from ex vivo culture showed that, in presence of 0.5-2.5 µM EGCG, the growth of mink hair follicles was promoted. In vitro, the proliferation of DPCs and ORSCs was enhanced by 0.5-4 µM EGCG treatment. More cells entered S phase upon treatment of EGCG, accompanied with upregulation of cyclin D1 and cyclin E1. Furthermore, when exposed to EGCG, the Shh and AKT signaling pathways were activated in both hair follicles and primary DPCs and ORSCs. Inhibiting either of these two pathways partly reversed the effect of EGCG on proliferation and cell cycle of DPCs and ORSCs. Conclusion: EGCG promotes the growth of mink hair follicles at concentrations of 0.5-2.5 µM. This growth-promoting effect of EGCG may be associated with the increased proliferation of DPCs and ORSCs through activating Shh and AKT signaling pathways.

Knockdown of HIF-1α inhibits the proliferation and migration of outer root sheath cells exposed to hypoxia in vitro: An involvement of Shh pathway.

AIMS: Outer root sheath (ORS) is a highly proliferative component of a hair follicle. This study is performed to investigate whether hypoxia-induced elevation of hypoxia-inducible factor (HIF)-1α, a transcriptional activator, contributes to the outgrowth of ORS cells in vitro. MAIN METHODS: Hair follicles with intact ORS collected from 4-month old male American minks were cultured in normoxic or hypoxic condition (3% oxygen) for 7days. Primary ORS cells isolated from the mink hair follicles were exposed to hypoxia for 12, 24 or 48h, and their proliferation was analyzed with immunofluorescence assay using anti-proliferating cell nuclear antigen (PCNA) antibody. The migratory ability of ORS cells was detected via the transwell chamber. The endogenous HIF-1α was knocked down with its specific siRNA in ORS cells. KEY FINDINGS: Hypoxic exposure induced an elevation of HIF-1α in ex vivo cultured hair follicles. The mRNA and protein levels of sonic hedgehog (Shh), Shh receptor Patched 1, Smoothened and glioma-associated oncogene homologue 1 were upregulated. In vitro, hypoxia induced an increase in HIF-1α in ORS cells. Further, under hypoxic condition, the number of PCNA-positive cells was increased, and more cells migrated towards high serum media. Hypoxia-enhanced proliferation and migration of ORS cells were suppressed either by HIF-1α siRNA or by pharmacological inhibitors of Shh pathway, cyclopamine and GANT61. The activation of Shh pathway was attenuated in HIF-1α-silenced ORS cells under hypoxic condition. SIGNIFICANCE: Our work demonstrates a direct role of activated HIF-1/Shh biological axis in sustaining the development of ORS in vitro.

Comparative Transcriptome Analysis of Mink (Neovison vison) Skin Reveals the Key Genes Involved in the Melanogenesis of Black and White Coat Colour.

Farmed mink (Neovison vison) is one of the most important fur-bearing species worldwide, and coat colour is a crucial qualitative characteristic that contributes to the economic value of the fur. To identify additional genes that may play important roles in coat colour regulation, Illumina/Solexa high-throughput sequencing technology was used to catalogue the global gene expression profiles in mink skin with two different coat colours (black and white). RNA-seq analysis indicated that a total of 12,557 genes were differentially expressed in black versus white minks, with 3,530 genes up-regulated and 9,027 genes down-regulated in black minks. Significant differences were not observed in the expression of MC1R and TYR between the two different coat colours, and the expression of ASIP was not detected in the mink skin of either coat colour. The expression levels of KITLG, LEF1, DCT, TYRP1, PMEL, Myo5a, Rab27a and SLC7A11 were validated by qRT-PCR, and the results were consistent with RNA-seq analysis. This study provides several candidate genes that may be associated with the development of two coat colours in mink skin. These results will expand our understanding of the complex molecular mechanisms underlying skin physiology and melanogenesis in mink and will provide a foundation for future studies.

Comparative Transcriptome Analysis of Raccoon Dog Skin to Determine Melanin Content in Hair and Melanin Distribution in Skin.

The raccoon dog (Nyctereutes procyonoides) is an important canid fur-bearing animal species worldwide. Chinese raccoon dogs that present a white mutation, especially those with a white coat. Exploring melanin biosynthesis in the hair and skin of raccoon dogs is important for understanding the survival and evolutionary mechanisms of them. In this study, we measured the content of melanin in the hair of two types of raccoon dog and generated stained slices of skin tissue. The results indicated that melanin biosynthesis occurs in the wild-type (W) and white-type (B) raccoon dog skin, although less melanin is produced in B skin. We then sequenced the skin transcriptomes of W and B, compared the similarities and differences in expressed genes. A comparison of the gene expression showed 60 up-regulated genes and 127 down-regulated genes in B skin. We analyzed the unigenes and pathways related to the melanogenesis pathway and found that TYR, TYRP1, MC1R, SLC24a5, SLC45a2 and OCA2 were significantly down-regulated in B skin and these results were verified via qRT-PCR. We surmised that the phenotypic characteristics of the white mutation might be caused by the reduced expression of these genes and this finding provides new insights for future experiments in raccoon dogs.