Transcriptome analysis reveals miRNA expression profiles in hypothalamus tissues during the sexual development of Jining grey goats.

BACKGROUND: Exploring the physiological and molecular mechanisms underlying goat sexual maturation can enhance breeding practices and optimize reproductive efficiency and is therefore substantially important for practical breeding purposes. As an essential neuroendocrine organ in animals, the hypothalamus is involved in sexual development and other reproductive processes in female animals. Although microRNAs (miRNAs) have been identified as significant regulators of goat reproduction, there is a lack of research on the molecular regulatory mechanisms of hypothalamic miRNAs that are involved in the sexual development of goats. Therefore, we examined the dynamic changes in serum hormone profiles and hypothalamic miRNA expression profiles at four developmental stages (1 day (neonatal, D1, n = 5), 2 months (prepubertal, M2, n = 5), 4 months (sexual maturity, M4, n = 5), and 6 months (breeding period, M6, n = 5)) during sexual development in Jining grey goats. RESULTS: Transcriptome analysis revealed 95 differentially expressed miRNAs (DEMs) in the hypothalamus of goats across the four developmental stages. The target genes of these miRNAs were significantly enriched in the GnRH signalling pathway, the PI3K-Akt signalling pathway, and the Ras signalling pathway (P < 0.05). Additionally, 16 DEMs are common among the M2 vs. D1, M4 vs. D1, and M6 vs. D1 comparisons, indicating that the transition from D1 to M2 represents a potentially critical period for sexual development in Jining grey goats. The bioinformatics analysis results indicate that miR-193a/miR-193b-3p-Annexin A7 (ANXA7), miR-324-5p-Adhesion G protein-coupled receptor A1 (ADGRA1), miR-324-3p-Erbb2 receptor tyrosine kinase 2 (ERBB2), and miR-324-3p-Rap guanine nucleotide exchange factor 3 (RAPGEF3) are potentially involved in biological processes such as hormone secretion, energy metabolism, and signal transduction. In addition, we further confirmed that miR-324-3p targets the regulatory gene RAPGEF3. CONCLUSION: These results further enrich the expression profile of hypothalamic miRNAs in goats and provide important insights for studying the regulatory effects of hypothalamic miRNAs on the sexual development of goats after birth.

Characterization of MicroRNA expression profiles in the ovarian tissue of goats during the sexual maturity period.

BACKGROUND: The ovary is an important reproductive organ in mammals, and its development directly affects the sexual maturity and reproductive capacity of individuals. MicroRNAs (miRNAs) are recognized as regulators of reproductive physiological processes in various animals and have been shown to regulate ovarian development through typical targeting and translational repression. However, little is known about the regulatory role of miRNAs in ovarian tissue development during sexual maturity in goats. To comprehensively profile the different physiological stages of sexual maturation in goats, we performed small-RNA sequencing of ovarian tissue samples collected at four specific time points (1 day after birth (D1), 2 months old (M2), 4 months old (M4), and 6 months old (M6)). In addition, we used ELISAs to measure serum levels of reproductive hormones to study their temporal changes. RESULTS: The results showed that serum levels of gonadotropin-releasing hormone, follicle-stimulating hormone, luteinizing hormone, oestradiol, progesterone, oxytocin, and prolactin were lower in goats at the D1 stage than at the other three developmental stages (P < 0.05). The secretion patterns of these seven hormones show a similar trend, with hormone levels reaching their peaks at 4 months of age. A total of 667 miRNAs were detected in 20 libraries, and 254 differentially expressed miRNAs and 3 groups of miRNA clusters that had unique expression patterns were identified (|log2-fold change|> 1, FDR < 0.05) in the 6 comparison groups. RT‒qPCR was employed to confirm that the expression pattern of the 15 selected miRNAs was consistent with the Illumina sequencing results. Gene ontology analyses revealed significant enrichment of GO terms such as cell proliferation regulation, epithelial cell development, and amino acid transport, as well as important signaling pathways including the MAPK signaling pathway, the PI3K-Akt signaling pathway, and the oestrogen signaling pathway. Further miRNA‒mRNA regulation network analysis revealed that 8 differentially expressed miRNAs (chi-miR-1343, chi-miR-328-3p, chi-miR-877-3p, chi-miR-296-3p, chi-miR-128-5p, chi-miR-331-3p, chi-miR-342-5p and chi-miR-34a) have important regulatory roles in ovarian cell proliferation, hormone secretion and metabolism-related biological processes. CONCLUSIONS: Overall, our study investigated the changes in serum hormone and miRNA levels in the ovaries. These data provide a valuable resource for understanding the molecular regulatory mechanisms of miRNAs in ovarian tissue during the sexual maturity period in goats.

Differentially Expressed MiRNAs of Goat Submandibular Glands Among Three Developmental Stages Are Involved in Immune Functions.

Submandibular glands (SMGs) are one of the primary components of salivary glands in goats. The proteins and biologically active substances secreted by the SMGs change with growth and development. Our previous studies showed that most of the differentially expressed genes in the SMGs of goats at different developmental stages are involved in immune-related signaling pathways, but the miRNA expression patterns in the same tissues are unknown. The aim of this study was to reveal the expression profile of miRNAs at three different developmental stages, detect differentially expressed miRNAs (DE miRNAs) and predict disease-related DE miRNAs. SMG tissue samples were collected from groups of 1-month-old kids, 12-month-old maiden goats and 24-month-old adult goats (three samples from each group), and high-throughout transcriptome sequencing was conducted. A total of 178, 241 and 7 DE miRNAs were discovered between 1-month-old kids and 12-month-old maiden goats, between 1-month-old kids and 24-month-old adult goats, and between 12-month-old maiden goats and 24-month-old adult goats, respectively. Among these DE miRNAs, 88 DE miRNAs with medium or high expression levels (TPM ≥50) were classified into five expression pattern clusters. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses indicated that some of the predicted target genes of the DE miRNAs in the five clusters were enriched in disease-related GO terms and pathways. MiRNA target genes in significant pathways were significantly enriched in Hepatitis B (FDR = 9.03E-10) and Pathways in cancer (FDR = 4.2E-10). Further analysis was performed with a PPI network, and 10 miRNAs were predicted to play an important role in the occurrence and prevention of diseases during the growth and development of goats.

Characterization of microRNA profiles in the mammary gland tissue of dairy goats at the late lactation, dry period and late gestation stages.

MicroRNAs (miRNAs) play an important role in regulating mammary gland development and lactation. We previously analyzed miRNA expression profiles in Laoshan dairy goat mammary glands at the early (20 d postpartum), peak (90 d postpartum) and late lactation (210 d postpartum) stages. To further enrich and clarify the miRNA expression profiles during the lactation physiological cycle, we sequenced miRNAs in the mammary gland tissues of Laoshan dairy goats at three newly selected stages: the late lactation (240 d postpartum), dry period (300 d postpartum) and late gestation (140 d after mating) stages. We obtained 4038 miRNAs and 385 important miRNA families, including mir-10, let-7 and mir-9. We also identified 754 differentially expressed miRNAs in the mammary gland tissue at the 3 different stages and 6 groups of miRNA clusters that had unique expression patterns. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed that GO terms such as mammary gland development (GO:0030879) and mammary gland morphogenesis (GO:0060443) and important signaling pathways, including the insulin signaling pathway (chx04910), hippo signaling pathway (chx04390) and estrogen signaling pathway (chx04915), were enriched. We screened miRNAs and potential target genes that may be involved in the regulation of lactation, mammary gland growth and differentiation, cell apoptosis, and substance transport and synthesis and detected the expression patterns of important genes at the three stages. These miRNAs and critical target genes may be important factors for mammary gland development and lactation regulation and potentially valuable molecular markers, which may provide a theoretical reference for further investigation of mammary gland physiology.

Genome­wide integrated analysis demonstrates widespread functions of lncRNAs in mammary gland development and lactation in dairy goats.

BACKGROUND: The mammary gland is a unique organ for milk synthesis, secretion and storage, and it undergoes cyclical processes of development, differentiation, lactation and degeneration. At different developmental periods, the biological processes governing mammary gland physiology and internal environmental homeostasis depend on a complex network of genes and regulatory factors. Emerging evidence indicates that lncRNAs have arbitrarily critical functions in regulating gene expression in many organisms; however, the systematic characteristics, expression, and regulatory roles of lncRNAs in the mammary gland tissues of dairy goats have not been determined. RESULT: In the present study, we profiled long noncoding RNA (lncRNA) expression in the mammary gland tissues of Laoshan dairy goats (Capra hircus) from different lactation periods at the whole-genome level, to identify, characterize and explore the regulatory functions of lncRNAs. A total of 37,249 transcripts were obtained, of which 2381 lncRNAs and 37,249 mRNAs were identified, 22,488 transcripts, including 800 noncoding transcripts and 21,688 coding transcripts, differed significantly (p ≤ 0.01) among the different lactation stages. The results of lncRNA-RNA interaction analysis showed that six known lncRNAs belonging to four families were identified as the precursors of 67 known microRNAs; 1478 and 573 mRNAs were predicted as hypothetical cis-regulation elements and antisense mRNAs, respectively. GO annotation and KEGG analysis indicated that the coexpressed mRNAs were largely enriched in biological processes related to such activities as metabolism, immune activation, and stress,., and most genes were involved in pathways related to such phenomena as inflammation, cancer, signal transduction, and metabolism. CONCLUSIONS: Our results clearly indicated that lncRNAs involved in responses to stimuli, multiorganism processes, development, reproductive processes and growth, are closely related to mammary gland development and lactation.

chi-miR-143-3p Promotes Apoptosis of Mammary Gland Epithelial Cells from Dairy Goats by Targeting Ndfip1.

The mammary gland is an important organ for lactation in dairy goats. Mammary gland development and lactation functions are primarily regulated by natural hormones and certain crucial regulatory factors. Nedd4 family-interacting protein 1 (Ndfip1) can specifically bind to neural precursor cell-expressed, developmentally downregulated protein 4 (Nedd4) family members to participate in ubiquitination, which in turn regulates a range of biological processes in the body. However, the effects of Ndfip1 expression regulation at the post-transcriptional level on the development of mammary gland cells have not been previously reported. To study the regulation of Ndfip1 at post-transcriptional level, the overexpression and interference vectors of Ndfip1 were constructed, and co-transfected into the primary mammary gland epithelial cells cultured in vitro with miR-143 mimics and inhibitor. Dual luciferase reporter gene system, real-time quantitative polymerase chain reaction, western blotting, cholecystokinin octapeptide assays, and flow cytometry were used to identify their regulation and function. As a result, Ndfip1 was targeted and regulated by miR-143, which influences the development of mammary gland epithelial cells in dairy goats cultured in vitro. This study will lay an experimental foundation for further understanding the functions of Ndfip1 and miR-143.

Transcriptome Analysis of Three Sheep Intestinal Regions reveals Key Pathways and Hub Regulatory Genes of Large Intestinal Lipid Metabolism.

The large intestine, also known as the hindgut, is an important part of the animal digestive system. Recent studies on digestive system development in ruminants have focused on the rumen and the small intestine, but the molecular mechanisms underlying sheep large intestine metabolism remain poorly understood. To identify genes related to intestinal metabolism and to reveal molecular regulation mechanisms, we sequenced and compared the transcriptomes of mucosal epithelial tissues among the cecum, proximal colon and duodenum. A total of 4,221 transcripts from 3,254 genes were identified as differentially expressed transcripts. Between the large intestine and duodenum, differentially expressed transcripts were found to be significantly enriched in 6 metabolism-related pathways, among which PPAR signaling was identified as a key pathway. Three genes, CPT1A, LPL and PCK1, were identified as higher expression hub genes in the large intestine. Between the cecum and colon, differentially expressed transcripts were significantly enriched in 5 lipid metabolism related pathways, and CEPT1 and MBOAT1 were identified as hub genes. This study provides important information regarding the molecular mechanisms of intestinal metabolism in sheep and may provide a basis for further study.

Molecular cloning, characterization and tissue specificity of the expression of the ovine CSRP2 and CSRP3 genes from Small-tail Han sheep (Ovis aries).

Two genes, cysteine- and glycine-rich protein 2 (CSRP2) and cysteine- and glycine-rich protein 3 (CSRP3), play important roles in tissue-specific cell growth and development. However, few CSRP2 and CSRP3 genes have been functionally characterized in sheep. In this study, the full-length cDNAs of the CSRP2 and CSRP3 genes were cloned from Small-tail Han sheep by rapid amplification of cDNA ends-PCR. The GenBank accession numbers of the full-length CSRP2 and CSRP3 cDNA sequences are KJ743957 and KJ743958, respectively. The full-length cDNA of ovine CSRP2 was 917bp, with a 582-bp open reading frame encoding 193 amino acids. The complete ovine CSRP3 cDNA was 917bp, with a 585-bp open reading frame encoding 194 amino acids. Alignment and phylogenetic analyses revealed that their amino acid sequences are highly similar to those of other vertebrates, all of which contain two conserved LIM-only domains and a relatively conserved nuclear targeting sequence. To further validate the functions of the two genes, their mRNA expression patterns were evaluated in various Small-tail Han and Dorper sheep tissues using qRT-PCR analyses. CSRP2 was mainly detected in the aorta, whereas CSRP3 was highly concentrated in the heart and the muscle. CSRP3 was expressed to a higher level in the hearts of Small-tail Han sheep than in Dorper sheep (P<0.05). However, the opposite was found in the muscle (the longissimus dorsi and biceps femoris); CSRP3 was expressed to a higher level in Dorper sheep than in Small-tail Han sheep (P<0.05). We quantified the CRP3 protein (coded by the CSRP3 gene) levels in different tissues in Small-tail Han and Dorper sheep. We also detected a putative isoform of the CRP3 protein in sheep, which was significantly different in the heart tissue of the two breeds (P<0.05). The expression patterns of the two genes' mRNAs and CRP3 protein showed clear tissue specificity in both sheep breeds.

miR-135a Targets and Regulates Prolactin Receptor Gene in Goat Mammary Epithelial Cells.

Mammary gland development and lactation are typical traits controlled by multiple genes, hormones, and regulatory factors. Prolactin receptor (PRLR), a specific receptor of prolactin, has been reported to have important physiological functions in regulating mammogenesis and lactogenesis. However, the post-transcriptional regulation mechanisms of PRLR expression have not yet been shown in detail. In this study, the expression of miR-135a and PRLR at different development stages of Laoshan dairy goat mammary gland tissues was investigated. After overexpression and silencing expression of miR-135a in cultured primary mammary epithelial cells, the regulatory relationship between miR-135a and PRLR was examined through dual-luciferase reporter assay, and the expression of PRLR at both mRNA and protein levels was examined by real-time quantitative polymerase chain reaction (RT-qPCR) and western blot. Collectively, our results suggested that PRLR is a direct target gene of miR-135a, miR-135a is a novel regulator of PRLR, and it might play an essential role in the regulation of animal mammary gland development and lactation.