Differential gene expression in ovaries of pregnant pigs with high and low prolificacy levels and identification of candidate genes for litter size

Previous results from a genome scan in an F(2) Iberian × Meishan pig intercross showed several chromosome regions associated with litter size traits in this species. In order to identify candidate genes underlying these quantitative trait loci (QTL), we performed an ovary gene expression analysis during the sow's pregnancy. F(2) sows were ranked by their estimated breeding values for prolificacy: six sows with the highest estimated breeding value (EBV) (i.e., high prolificacy) and six sows with the lowest EBV (low prolificacy) were selected. Samples were hybridized using an Affymetrix GeneChip porcine genome array. Statistical analysis with a mixed model approach identified 221 differentially expressed probes, representing 189 genes. These genes were functionally annotated in order to identify genetic pathways overrepresented in this list. Among the functional groups most represented was, in first position, immune system response activation against external stimulus. The second group consisted of integrated genes that regulate maternal homeostasis by complement and coagulation cascades. A third group was involved in lipid and fatty acid enzymes of metabolic processes, which participate in the steroidogenesis pathway. In order to identify powerful candidate genes for prolificacy, the second approach of this study was to merge microarray data with the QTL positional information affecting litter size, previously detected in the same experimental cross. As a result, we have identified 27 differentially expressed genes colocalizing with QTL for litter size traits, which fulfill the biological, positional, and functional criteria.     

Identification and differential expression of microRNAs in the ovaries of pigs (Sus scrofa) with high and low litter sizes

Litter size affects profitability in the swine industry. Mammalian ovaries play important roles during reproduction, including ovulation and hormone secretion, which are tightly regulated by specific microRNAs (miRNAs). In this study, we investigated the effects of specific miRNAs on porcine litter size. We compared the ovarian miRNAs of Yorkshire pigs with high (YH) and low (YL) litter sizes using Solexa sequencing technology. We identified 327 and 320 miRNAs in the ovaries of YH and YL pigs respectively. A total of 297 miRNAs were co‐expressed; 30 and 23 miRNAs respectively were specifically expressed in the two libraries. A total of 83 novel miRNAs were predicted; 37 specific miRNAs were obtained, of which 21 miRNAs were upregulated and 16 miRNAs were downregulated in YH compared with YL. Additionally, 19 628 and 19 250 target genes were predicted in the two libraries respectively. The results revealed that specific miRNAs (i.e., miR‐224, miR‐99a, let‐7c, miR‐181c, miR‐214 and miR‐21) may affect porcine litter size. The results of this study will help in gaining understanding of the role of miRNAs in porcine litter size regulation.     

Proteomic Analysis of Fetal Ovary Reveals That Ovarian Developmental Potential Is Greater in Meishan Pigs than in Yorkshire Pigs

Time-dependent expression of functional proteins in fetal ovaries is important to understand the developmental process of the ovary. This study was carried out to enhance our understanding of the developmental process of porcine fetal ovaries and to better address the differences in fetal ovary development of local and foreign pigs. The objective of the present study is to test the expression of key proteins that regulate the growth and development of fetal ovaries in Meishan and Yorkshire porcine breeds by using proteomics technology. Six Meishan and 6 Yorkshire pregnant gilts were used in this experiment. Fetal ovaries were obtained from Yorkshire and Meishan gilts on days 55 and 90 of the gestation period. Using 2D-DIGE (two dimensional-difference in gel electrophoresis) analysis, the results showed that there are about 1551 and 1400 proteins in gilt fetal ovaries on days 55 and 90, respectively of the gestation. Using MALDI TOF-TOF MS analysis, 27 differentially expressed proteins were identified in the fetal ovaries of the 2 breeds on day 55 of gestation, and a total of 18 proteins were identified on day 90 of gestation. These differentially expressed proteins were involved in the regulation of biological processes (cell death, stress response, cytoskeletal proteins) and molecular functions (enzyme regulator activity). We also found that alpha-1-antitrypsin, actin, vimentin, and PP2A proteins promote the formation of primordial follicles in the ovaries of Yorkshire pigs on day 55 of gestation while low expression heat shock proteins and high expression alpha-fetoproteins (AFP) may promote Meishan fetal ovarian follicular development on day 90 of gestation. These findings provide a deeper understanding of how reduced expression of heat shock proteins and increased expression of AFP can significantly reduce the risk of reproductive disease in obese Meishan sows. Our study also shows how these proteins can increase the ovulation rate and may be responsible for the low reproductive efficiency reported in other obese breeds. The ovarian developmental potential was found to be greater in Meishan pigs than in Yorkshire pigs.     

Exploring differentially expressed genes in the ovaries of uniparous and multiparous goats using the RNA-Seq (Quantification) method

Fecundity improvement is one of the most important objectives for goat breeders as it greatly increases production efficiency. To investigate the genes associated with litter sizes in the Anhui White goat (AWG), gene expression differences in the ovaries of uniparous and multiparous AWG were assessed using the RNA-Seq (Quantification) method. This analysis generated 6,027,714 and 5,884,062 clean reads in uniparous and multiparous libraries, respectively. A total of 2201 differentially expressed genes (DEGs) were thereby identified (FDR ≤ 0.001, |log₂Ratio| ≥ 1). There were 1583 up-regulated and 618 down-regulated genes in the multiparous samples compared with the uniparous samples. A large number of these DEGs were related to the terms cellular process, cell & cell part and binding. Twelve genes which may be associated with the high prolificacy of AWG were identified using a bioinformatic screen. In addition, pathway analysis revealed that these DEGs were significantly enriched in 11 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, including ECM–receptor interactions, focal adhesion, and regulation of the actin cytoskeleton among others. This suggested a role for these pathways in the prolificacy of AWG. These results provide a list of new candidate genes for goat prolificacy.     

Comparison of stomach microRNA transcriptomes of Tibetan and Yorkshire pigs by deep sequencing

MiRNAs regulate the expression of target genes in diverse cellular processes and hence play important roles in different physiological processes, yet little is known about the stomach microRNAome (miRNAome) of the Tibetan pig. The objective of this experiment was to investigate differentially expressed stomach miRNAs participating in digestion. Firstly, we isolated total RNA by Trizol reagent from three Tibetan and three Yorkshire purebred pigs stomach samples at 90-day-old. Secondly, a comprehensive analysis of Tibetan and Yorkshire pig stomach miRNAomes was performed by small RNA sequencing in the Illumina HiSeq 2000 system. Finally, SYBR Green Real-time RT-PCR was performed to validate the differentially expressed miRNAs. We identified 318 unique miRNAs, 260 were co-expressed in both libraries, 17 and 31 miRNAs were specifically expressed in Tibetan and Yorkshire pigs respectively. Fifty six differentially expressed miRNAs were identified by the identifying differentially expressed genes 6 (IDEG6). Kyoto encyclopedia of genes and genomes analysis revealed that some of the differentially expressed miRNAs were associated with protein and fat digestion. Two differentially expressed miRNAs (miR-214-3p and ssc-un39) participating in the digestion of lipid were identified. Additionally, qRT-PCR results suggested that a higher expression of miR-214-3p in the Tibetan pig stomach could lead to relatively lower expression of calcium-dependent phospholipase A2, which is an enzyme important for the digestion of glycerol phospholipid. This study has delineated the different stomach miRNAs expression patterns of Tibetan and Yorkshire pigs, which would help explain the regulatory mechanisms of miRNAs in digestion of Tibetan pigs, and contribute to utilize a the unique digestion merits of Tibetan pig in future porcine hybridization breeding.     

Microarray profiling for differential gene expression in ovaries and ovarian follicles of pigs selected for increased ovulation rate

A unique index line of pigs created by long-term selection ovulates on average 6.7 more ova than its randomly selected control line. Expression profiling experiments were conducted to identify differentially expressed genes in ovarian tissues of the index and control lines during days 2-6 of the follicular phase of the estrous cycle. Fluorescently labeled cDNAs derived from ovary and follicle RNA were cohybridized on microarray slides (n = 90) containing 4608 follicle-derived probes printed in duplicate. Statistical analysis of the resulting approximately 1.6 million data points with a mixed-model approach identified 88 and 74 unique probes, representing 71 and 59 unique genes, which are differentially expressed between lines in the ovary and ovarian follicles of different size classes, respectively. These findings indicate that long-term selection for components of litter size has caused significant changes in physiological control of the dynamics of follicular maturation. Genes involved with steroid synthesis, tissue remodeling, and apoptosis, in addition to several genes not previously associated with ovarian physiology or with unknown function, were found to be differentially expressed between lines. This study reveals many potential avenues of investigation for seeking new insights into ovarian physiology and the quantitative genetic control of reproduction.     

Transcriptome analysis of differentially expressed genes in rabbits’ ovaries by digital gene-expression profiling

Reproduction is a complex physiological process that is regulated by multiple genes and pathways. Compared with studies of common livestock, fewer studies of genes related to the fertility of rabbits (Oryctolagus cuniculus) have been reported, and the molecular mechanism of their high productivity is still poorly understood. To identify candidate genes associated with development and prolificacy in rabbits, we analyzed gene expression differences among the ovaries of mature Californian rabbit (LC), and mature (HH) and immature Harbin white rabbit (IH) using digital gene expression technology. We detected 885 and 321 genes that were significantly differentially expressed in comparisons between HH/IH and HH/LC, respectively. The functions of the differentially expressed genes (DEGs) were determined by GO classification and KEGG pathway analysis. The results suggest that most of the DEGs between the mature and immature developmental stages were predominantly associated with DNA replication, cell cycle, and progesterone-mediated oocyte maturation, and most were up-regulated in the IH group compared with the HH group. The DEGs involved in disparate fecundities between HH and LC were associated with reproduction, fructose and mannose metabolism, steroid hormone biosynthesis, and pyruvate metabolism. Our results will contribute to a better understanding of changes in the regulatory network in ovary at different developmental stages and in different fertility of rabbit.     

Comparison of microRNAs in adipose and muscle tissue from seven indigenous Chinese breeds and Yorkshire pigs

Elucidation of the pig microRNAome is essential for interpreting functional elements of the genome and understanding the genetic architecture of complex traits. Here, we extracted small RNAs from skeletal muscle and adipose tissue, and we compared their expression levels between one Western breed (Yorkshire) and seven indigenous Chinese breeds. We detected the expression of 172 known porcine microRNAs (miRNAs) and 181 novel miRNAs. Differential expression analysis found 92 and 12 differentially expressed miRNAs in adipose and muscle tissue respectively. We found that different Chinese breeds shared common directional miRNA expression changes compared to Yorkshire pigs. Some miRNAs differentially expressed across multiple Chinese breeds, including ssc‐miR‐129‐5p, ssc‐miR‐30 and ssc‐miR‐150, are involved in adipose tissue function. Functional enrichment analysis revealed that the target genes of the differentially expressed miRNAs are associated mainly with signaling pathways rather than metabolic and biosynthetic processes. The miRNA–target gene and miRNA–phenotypic traits networks identified many hub miRNAs that regulate a large number of target genes or phenotypic traits. Specifically, we found that intramuscular fat content is regulated by the greatest number of miRNAs in muscle tissue. This study provides valuable new candidate miRNAs that will aid in the improvement of meat quality and production.     

Detection of SNPs in Porcine Haptoglobin and Apolipoprotein Genes

To find differentially expressed protein spots using two-dimensional electrophoresis proteomic analysis, we took blood serum samples from 40 purebred Yorkshire pigs at 12, 18, 24, and 30 weeks. Each growth stage contained 10 male pigs having half-sib pedigrees. With the pooled serum samples, two interesting spots, differentially expressed in the growth stages, were identified using MALDI-TOF-TOF MS/MS analysis as haptoglobin alpha 1S (Hp) and apolipoprotein A-IV (APOA4) gene products. The Hp was down-regulated from 12 to 30 weeks, and APOA4 was not expressed much before 18 weeks but was highly expressed in the late growth stages. There may be an inverse relationship between the Hp and APOA4 genes. Four segments for the Hp and APOA4 genes were successfully amplified with sizes around 500 bp. The porcine Hp and APOA4 genes were screened in the 40 purebred Yorkshire pigs and a random cross population (90 pigs), resulting in the location of 6 single nucleotide polymorphisms (SNPs) in the coding regions. The mutations resulted in amino acid changes in segments of Hp627, Hp742, and APOA41203. Further investigation of the function of the Hp and APOA4 genes with SNPs will be necessary to understand fully the different expression profiles and association studies.     

Mapping of the porcine oestrogen receptor 2 gene and association study with litter size in Iberian pigs

Genes encoding the oestrogen receptors (ESR) are considered candidate genes for prolificacy traits due to the key role these molecules play in the regulation of reproductive physiology. In this paper, we report the assignment of the pig ESR2 gene to porcine chromosome 1 by radiation hybrid mapping. Most of the ESR2 cDNA was sequenced from Iberian pig ovarian RNA samples and one A/G single nucleotide polymorphism (SNP) was found at exon 5, being associated with a Met/Val substitution at position 949. This SNP was genotyped using a PCR-RFLP (Hsp92II) protocol and its potential effect on litter size was evaluated in two Iberian pig populations. However, no statistically significant association between the ESR2 polymorphism and litter size was found.     

影响二花脸猪高产仔性能的生理及遗传机制研究进展

二花脸猪是我国太湖流域以产仔数高而闻名于世界的优质猪种。已有的研究发现,二花脸猪排卵数高、胚胎死亡率低和子宫容积大是其高产性能的重要生理特征,通过候选基因法鉴别到其高产的重要候选基因——促卵泡素β亚基(follicular-stimulating hormone beta, FSH?),采用QTL定位方法在猪第2、6、7、8、12、13和15号染色体上定位到影响其产仔性状的QTL。但是,目前影响二花脸猪产仔数的主效基因尚不清楚。本文主要介绍了二花脸猪的高排卵数、低胚胎死亡率和高子宫容积特性对高产仔性能形成的生理作用;概括了基于传统方法鉴别出的影响二花脸猪高产仔性能的候选基因和QTL,以及基于组学方法鉴别出的二花脸猪高产候选基因;探讨了如何利用基因组、转录组、蛋白质组和功能组等多组学手段深入研究二花脸猪高产性能的成因,以期为深入解析二花脸猪高产性能的分子遗传机制提供参考。     

Genome‐wide association study to identify genomic regions affecting prolificacy in Lori‐Bakhtiari sheep

Several causative mutations in candidate genes affecting prolificacy have been detected in various sheep breeds. A genome‐wide association study was performed on estimated breeding values for litter size in Lori‐Bakhtiari sheep. Prolific ewes with twinning records and others with only singleton records were genotyped using the medium‐density Illumina Ovine SNP50 array. Four single nucleotide polymorphisms (SNPs) associated with litter size were identified on chromosomes 3, 6 and 22. The region on sheep chromosome 3 between 75 739 167 and 75 745 152 bp included two significant SNPs (s52383.1 and OAR3_80038014_X.1) in high linkage disequilibrium with each other. The region that surrounds these SNPs contains a novel putative candidate gene: luteinizing hormone/choriogonadotropin receptor (LHCGR), known to be involved in ovarian steroidogenesis and organism‐specific biosystem pathways in sheep. Known prolificacy genes BMPR1B, BMP15 and GDF9 were not associated with litter size in Lori‐Bakhtiari sheep, suggesting that other biological mechanisms could be responsible for the trait's variation in this breed.     

Mapping quantitative trait loci affecting female reproductive traits on porcine chromosome 8

An understanding of the genetic control of porcine female reproductive performance would offer the opportunity to utilize natural variation and improve selective breeding programs through marker-assisted selection. The Chinese Meishan is one of the most prolific pig breeds known, farrowing three to five more viable piglets per litter than the European Large White breed. This difference in prolificacy is attributed to the Meishan's superior prenatal survival levels. The present study utilized a three-generation cross in which the founder grandparental animals were purebred Meishan and Large White pigs in a scan for quantitative trait loci (QTL) on porcine chromosome 8 (SSC8) associated with reproductive performance. Reproductive traits, including number of corpora lutea (ovulation rate), teat number, litter size, and prenatal survival, were recorded for as many as 220 F2 females. Putative QTL for the related traits of litter size and prenatal survival were identified at the distal end of the long arm of SSC8. A physiological candidate gene, SPP1, was found to lie within the 95% confidence interval of these QTL. A suggestive QTL for teat number was revealed on the short arm of SSC8. The present study demonstrates, to our knowledge, the first independent confirmation of QTL for fecundity on SSC8, and these QTL regions provide a crucial starting point in the search for the causal genetic variants.     

Sequence and Expression Characteristics of Long Noncoding RNAs in Honey Bee Caste Development – Potential Novel Regulators for Transgressive Ovary Size

Division of labor in social insect colonies relies on a strong reproductive bias that favors queens. Although the ecological and evolutionary success attained through caste systems is well sketched out in terms of ultimate causes, the molecular and cellular underpinnings driving the development of caste phenotypes are still far from understood. Recent genomics approaches on honey bee developmental biology revealed a set of genes that are differentially expressed genes in larval ovaries and associated with transgressive ovary size in queens and massive cell death in workers. Amongst these, two contigs called special attention, both being over 200 bp in size and lacking apparent coding potential. Herein, we obtained their full cDNA sequences. These and their secondary structure characteristics placed in evidence that they are bona fide long noncoding RNAs (lncRNA) differentially expressed in larval ovaries, thus named lncov1 and lncov2. Genomically, both map within a previously identified QTL on chromosome 11, associated with transgressive ovary size in honey bee workers. As lncov1 was over-expressed in worker ovaries we focused on this gene. Real-time qPCR analysis on larval worker ovaries evidenced an expression peak coinciding with the onset of autophagic cell death. Cellular localization analysis through fluorescence in situ hybridization revealed perinuclear spots resembling omega speckles known to regulate trafficking of RNA-binding proteins. With only four lncRNAs known so far in honey bees, two expressed in the ovaries, these findings open a novel perspective on regulatory factors acting in the fine tuning of developmental processes underlying phenotypic plasticity related to social life histories.