Xiaopeng An

Identification and Profiling of microRNAs in Goat Endometrium during Embryo Implantation

Background MicroRNAs (miRNAs) are short, highly conserved small noncoding RNAs that had fundamental roles in post-transcriptional gene expression, and they are crucial for proper control of biological processes and known to participate in embryo implantation. However, miRNA expression profiles in the pre-receptive and receptive phases of the goat endometrium during embryo implantation are unknown. Results A total of 1,069 and 847 miRNAs were expressed in receptive (R) and pre-receptive (P) goat endometrium, and 632 miRNAs were co-expressed in both phases. We identified 545 (50.98%) known miRNAs in the R library and 522 (61.63%) in the P library. There were 110 up-expressed miRNAs and 33 down-expressed miRNAs in receptive endometrium compared with the pre-receptive endometrium meeting the criteria of P-values< 0.05. Moreover, GO and KEGG analysis of the target genes of the differentially expressed miRNAs revealed some candidate miRNAs, genes and pathways that may involve in the formation of the receptive endometrium. Based on stem-loop RT-qPCR, 15 miRNAs were detected and the results suggested that the majority of the miRNA expression data measured by Solexa deep sequencing could represent actual miRNA expression levels. Conclusions Our data revealed the first miRNA profile related to the biology of the goat receptive endometrium during embryo implantation, and the results suggested that a subset of miRNAs might play important roles in the formation of endometrial receptivity. Thus, elucidating the physiological roles of endometrial miRNAs will help us better understand the genetic control of embryo implantation in goats.

Novel polymorphisms of the growth hormone gene and their effect on growth traits in Chinese goats

The polymorphisms of the growth hormone (GH) gene were analyzed in 686 individuals from four goat populations, Three haplotypes (A, B and C) and three observed genotypes (AA, AB and AC) were detected at the P2 locus, and three haplotypes (E, F and G) and three observed genotypes (EE, EF and EG) were also detected at the P4 locus. In addition, five single nucleotide polymorphisms (SNPs)-A112G, C142T (Gly>Ser), C214T (P2 locus), C266A (Pro>His) and C214T (P4 locus, Arg>Trp), were identified by GH gene sequencing and PCR-SSCP analysis. The SNPs loci were in Hardy-Weinberg disequilibrium in three goat populations (P<0.05). Association of polymorphisms with growth traits was done in BG, F1 and F1 populations, which were shown to be associated with growth traits in three goat populations. The SNPs in the goat GH gene had significant effects on growth traits (P<0.05). suggesting that the GH gene is a strong candidate gene that affects growth traits in goat.

Identification and gene expression analyses of natriuretic peptide system in the ovary of goat (Capra hircus).

Natriuretic peptides (NPs) are involved in maintaining cardiovascular and fluid homeostasis, regulating reproductive processes and bone growth, and other numerous functions. To better understand the role of NPs in goat (Capra hircus), in the present study, full-length cDNAs of goat Nppa (natriuretic peptide precursor A), Nppb (natriuretic peptide precursor B) and Nppc (natriuretic peptide precursor C), respectively encoding ANP, BNP and CNP, were cloned from adult goat heart and ovary. The putative prepropeptide ANP (prepro-ANP) and prepro-CNP share a high amino acid sequence identity with other species. Real-time PCR showed that Nppa, Nppb and Nppc were widely expressed in adult goat tissues. The mRNA expression of Nppa and Nppb in the heart was extremely higher compared with other tissues. Nppc mRNA expression in the lung and uterus was also higher than in other tissues. The expression of Nppa, Nppb and Nppc genes was examined at different ovarian follicle stages using RT-PCR. The mRNAs of Nppa and Nppb were detected in secondary follicles as well as in COCs (cumulus-oocyte-complexes) and granulosa cells of antral follicles. However, the mRNA expression of Nppc was observed throughout ovarian follicle development, and it was especially higher in granulosa cells of antral follicles. In vitro, stimulating goat granulosa cells with FSH led to an increase in the expression of Nppc by dose- and time-dependent manners and a rapid decline was induced by LH stimulation, but the expression of Nppa and Nppb did not change after FSH or LH treatment. These results suggest that Nppc is a gonadotropin-induced gene in granulosa cells of goat ovary and CNP may be involved in the regulation of ovarian follicle development and oocyte maturation.

Association analysis between variants in KISS1 gene and litter size in goats

BackgroundKisspeptins are the peptide products of KISS1 gene, which operate via the G - protein-coupled receptor GPR54. These peptides have emerged as essential upstream regulators of neurons secreting gonadotropin-releasing hormone (GnRH), the major hypothalamic node for the stimulatory control of the hypothalamic–pituitary– gonadal (HPG) axis. The present study detected the polymorphisms of caprine KISS1 gene in three goat breeds and investigated the associations between these genetic markers and litter size.ResultsThree goat breeds (n = 680) were used to detect single nucleotide polymorphisms (SNPs) in the coding regions with their intron–exon boundaries and the proximal flanking regions of KISS1 gene by DNA sequencing and PCR–RFLP. Eleven novel SNPs (g.384G>A, g.1147T>C, g.1417G>A, g.1428_1429delG, g.2124C>T, g.2270C>T, g.2489T>C, g.2510G>A, g.2540C>T, g.3864_3865delCA and g.3885_3886insACCCC) were identified. It was shown that Xinong Saanen and Guanzhong goat breeds were in Hardy-Weinberg disequilibrium at g.384G>A locus (P < 0.05). Both g.2510G>A and g.2540C>T loci were closely linked in Xinong Saanen (SN), Guanzhong (GZ) and Boer (BG) goat breeds (r2 > 0.33). The g.384G>A, g.2489T>C, g.2510G>A and g.2540C>T SNPs were associated with litter size (P<0.05). Individuals with AATTAATT combinative genotype of SN breed (SC) and TTAATT combinative genotype of BG breed (BC) had higher litter size than those with other combinative genotypes in average parity. The results extend the spectrum of genetic variation of the caprine KISS1 gene, which might contribute to goat genetic resources and breeding.ConclusionsThis study explored the genetic polymorphism of KISS1 gene, and indicated that four SNPs may play an important role in litter size. Their genetic mechanism of reproduction in goat breeds should be further investigated. The female goats with SC1 (AATTAATT) and BC7 (TTAATT) had higher litter size than those with other combinative genotypes in average parity and could be used for the development of new breeds of prolific goats. Further research on a large number of animals is required to confirm the link with increased prolificacy in goats.

Characterization of the Transcriptional Complexity of the Receptive and Pre-receptive Endometria of Dairy Goats.

Endometrium receptivity is essential for successful embryo implantation in mammals. However, the lack of genetic information remains an obstacle to understanding the mechanisms underlying the development of a receptive endometrium from the pre-receptive phase in dairy goats. In this study, more than 4 billion high-quality reads were generated and de novo assembled into 102,441 unigenes; these unigenes were annotated using published databases. A total of 3,255 unigenes that were differentially expressed (DEGs) between the PE and RE were discovered in this study (P-values < 0.05). In addition, 76,729–77,102 putative SNPs and 12,837 SSRs were discovered in this study. Bioinformatics analysis of the DEGs revealed a number of biological processes and pathways that are potentially involved in the establishment of the RE, notably including the GO terms proteolysis, apoptosis, and cell adhesion and the KEGG pathways Cell cycle and extracellular matrix (ECM)-receptor interaction. We speculated that ADCY8, VCAN, SPOCK1, THBS1, and THBS2 may play important roles in the development of endometrial receptivity. The de novo assembly provided a good starting point and will serve as a valuable resource for further investigations into endometrium receptivity in dairy goats and future studies on the genomes of goats and other related mammals.

Expression and regulative function of tissue inhibitor of metalloproteinase 3 in the goat ovary and its role in cultured granulosa cells.

Tissue inhibitor of metalloproteinase 3 (TIMP3) played a key role in female reproduction. However, its expression and function in goat are still unclear. In the present study, the full-length cDNA of goat TIMP3 was cloned from adult goat ovary; meanwhile, we demonstrated that putative TIMP3 protein shared a highly conserved amino acid sequence with known mammalian homologs. Real-time PCR results showed that TIMP3 was widely expressed in the tissues of adult goat. In the ovary, increasing expression of TIMP3 mRNA was discovered during the growth process of follicle and corpus luteum. Immunohistochemistry results suggested that TIMP3 protein existed in oocytes of all types of follicles, corpus luteum and granulosa and theca cells of primary, secondary, and antral but not primordial follicles. In vitro, human chorionic gonadotropin (hCG) stimulated the expression of TIMP3 in goat granulosa cells. hCG-induced TIMP3 mRNA expression was reduced by the inhibitors of protein kinase A, protein kinase C, MAPK kinase, or p38 kinase. Functionally, over-expression of TIMP3 significantly increased apoptosis and decreased the viability of cultured granulosa cells. Knockdown of TIMP3 could decrease hCG-induced progesterone secretion and the mRNA abundance of key steroidogenic enzymes (StAR, p450scc and HSD3B) as well as ECM proteins (DCN and FN). These findings provided evidence that the hCG induced expression of TIMP3 may play an important role in regulating goat granulosa cell survival and steroidogenesis.

Combined effects of four SNPs within goat PRLR gene on milk production traits

Xinong Saanen (SN, n=323) and Guanzhong (GZ, n=197) goat breeds were used to detect single nucleotide polymorphisms (SNPs) in the coding regions with their intron-exon boundaries of prolactin receptor (PRLR) gene by DNA sequencing, primer-introduced restriction analysis-polymerase chain reaction (PIRA-PCR) and PCR-restriction fragment length polymorphism (PCR-RFLP). Four novel SNPs (g.40452T>C, g.40471G>A, g.61677G>A and g.61865G>A) were identified. The g.61677G>A and g.61865G>A SNPs caused amino acid variations p.Ser485Asn and p.Val548Met, respectively. Both g.40452T>C and g.40471G>A loci were closely linked in SN and GZ goat breeds (r(2)>0.33). In addition, there was also a close linkage between g.61677G>A and g.61865G>A loci in both goat breeds. Statistical results indicated that the g.40452T>C, g.61677G>A and g.61865G>A SNPs were significantly associated with milk production traits in SN and GZ breeds. Further analysis revealed that combinative genotype C1 (TTAAGGGG) was better than the others for milk yield in SN and GZ goat breeds. These results are consistent with the regulatory function of PRLR in mammary gland development, milk secretion, and expression of milk protein genes, and extend the spectrum of genetic variation of the caprine PRLR gene, which might contribute to goat genetic resources and breeding.

Polymorphism identification in the goat KITLG gene and association analysis with litter size.

This study reported the analysis of KIT ligand (KITLG) gene polymorphisms in 681 goats of three breeds: Xinong Saanen (SN), Guanzhong (GZ), and Boer (BG). In addition, the study identified three allelic variants: g.769T>C and g.817G>T in SN and GZ breeds, and g.9760G>C in the three goat breeds. The g.769T>C and g.817G>T loci were closely linked (r(2)  > 0.33). All the single nucleotide polymorphism loci were in Hardy-Weinberg disequilibrium (P < 0.05). Significant associations were found for litter size with all three loci. Therefore, these results suggest that the KITLG gene is a strong candidate gene affecting litter size in goats.

Analysis of Differentially Expressed Genes in Ovaries of Polytocous versus Monotocous Dairy Goats Using Suppressive Subtractive Hybridization

In this study, suppressive subtractive hybridization (SSH) was performed to screen differentially expressed genes in ovarian tissues between polytocous and monotocous goats. From the SSH cDNA library, we obtained 29 differentially expressed sequence tags (ESTs) that have high similarity with known genes in the public database, which were involved in signal transduction, transcriptional regulation, cellular molecular dynamics, cytoskeleton, metabolism and oxidation-reduction. In addition, one novel EST that has no similarity with known genes in the public database was obtained. Eight ESTs, TIMP1, NUCB1, OAZ1, CXXC5, CAPNS2, ATP5A1, Z and RPL5, were further examined for the reproducibility of the SSH data by the real-time quantitative PCR. The results confirmed an increased expression of respective mRNA in ovarian tissues of polytocous goats compared with those of monotocous goats. The study has identified several genes (known or unknown) that may have effect on follicular development, ovulation and egg activation in goats.

Identification and profiling of microRNAs in the ovaries of polytocous and monotocous goats during estrus

MicroRNAs (miRNAs) play critical roles in almost all ovarian biological processes, including folliculogenesis, ovulation, luteal development, and regression. The study identified known and novel miRNAs in the ovaries of polytocous and monotocous goats by combining Solexa sequencing with bioinformatics. In total, 862 known and 53 novel miRNAs were identified in the ovaries of polytocous and monotocous goats. A total of 771 miRNAs were co-expressed in both libraries. One hundred twenty miRNAs in the ovaries of polytocous goats and 24 miRNAs in the ovaries of monotocous goats were specifically expressed. In addition, 445 miRNAs were differentially expressed in the ovaries of polytocous and monotocous goats, of which 348 were upregulated, and 97 were downregulated in the ovaries of polytocous goats compared with the ovaries of monotocous goats (P values < 0.05 and |log2 (fold change)|> 1). The expression levels of 12 randomly selected miRNAs were analyzed by stem-loop real-time quantitative polymerase chain reaction, and the results demonstrated that the expression patterns were consistent with Solexa sequencing results. KEGG analysis showed that GnRH, transforming growth factor-beta, vascular endothelial growth factor, and mammalian target of rapamycin signaling pathways, oocyte meiosis and ovarian steroidogenesis participated in follicular development and ovulation. On the basis of miRNA-mRNA network analysis and luciferase reporter assays, the ggo-miR-4488-p3_1ss10CG, bta-miR-2892-p5_1ss8CG and hsa-miR-4532_L+1R-3 were closely related with prolific traits. The results will help to further understand the role of miRNAs in kidding rate regulation.