Long noncoding RNAs (lncRNAs) are increasingly being recognized as key regulators in many cellular processes. However, few reports of them in livestock have been published. Here, we describe the identification and characterization of lncRNAs in ovine skeletal muscle. Eight libraries were constructed from the gastrocnemius muscle of fetal (days 85 and 120), newborn and adult Texel and Ujumqin sheep. The 2002 identified transcripts shared some characteristics, such as their number of exons, length and distribution. We also identified some coding genes near these lncRNA transcripts, which are particularly associated with transcriptional regulation- and development-related processes, suggesting that the lncRNAs are associated with muscle development. In addition, in pairwise comparisons between the libraries of the same stage in different breeds, a total of 967 transcripts were differentially expressed but just 15 differentially expressed lncRNAs were common to all stages. Among them, we found that TCONS_00013201 exhibited higher expression in Ujumqin samples, while TCONS_00006187 and TCONS_00083104 were higher in Texel samples. Moreover, TCONS_00044801, TCONS_00008482 and TCONS_00102859 were almost completely absent from Ujumqin samples. Our results suggest that differences in the expression of these lncRNAs may be associated with the muscular differences observed between Texel and Ujumqin sheep breeds.

The expression characteristics of the prolific candidate genes, BMPR1B, BMP15, and GDF9, in the major visceral organs and hypothalamic⁻pituitary⁻gonadal (HPG) axis tissues of three FecB genotypes (FecB BB, FecB B+, and FecB ++) were explored in STH ewes using RT-PCR and qPCR. The results were as follows, BMPR1B was expressed in all FecB BB genotype (Han BB) tissues, and GDF9 was expressed in all selected tissues, but BMP15 was specifically expressed in the ovaries. Further study of ovarian expression indicated that there was no difference in BMPR1B expression between genotypes, but the FecB B+ genotype (Han B+) had greater expression of GDF9 and BMP15 than Han BB and FecB ++ genotype (Han ++) (p < 0.05, p < 0.01). BMP15 expression was lower in the ovaries of Han BB than in Han ++ sheep, but the reverse was shown for GDF9. The gene expression in non-ovarian tissues was also different between genotypes. Therefore, we consider that the three genes have an important function in ovine follicular development and maturation. This is the first systematic analysis of the tissue expression pattern of BMPR1B, BMP15, and GDF9 genes in STH sheep of the three FecB genotypes. These results contribute to the understanding of the molecular regulatory mechanism for ovine reproduction.

The diverse functions of long noncoding RNAs (lncRNAs), which execute their functions mainly through modulating the activities of their target genes, have been have been widely studied for many years (including a number of studies involving lncRNAs in the ovary and uterus). Herein, for the first time, we detect lncRNAs in sheep hypothalami with FecB++ through RNA Sequencing (RNA-Seq) and identify a number of known and novel lncRNAs, with 622 and 809 found to be differentially expressed in polytocous sheep in the follicular phase (PF) vs. monotocous sheep in the follicular phase (MF) and polytocous sheep in the luteal phase (PL) vs. monotocous sheep in the luteal phase (ML), respectively. Then, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed based on the predicted target genes. The most highly enriched GO terms (at the molecular function level) included carbonyl reductase (NADPH), 15-hydroxyprostaglandin dehydrogenase (NADP+), and prostaglandin-E2 9-reductase activity in PF vs. MF, and phosphatidylinositol-3,5-bisphosphate binding in PL vs. ML was associated with sheep fecundity. Interestingly, the phenomena of valine, leucine, and isoleucine degradation in PL vs. ML, and valine, leucine, and isoleucine biosynthesis in PF vs. MF, were present. In addition, the interactome of lncRNA and its targets showed that MSTRG.26777 and its cis-targets ENSOARG00000013744, ENSOARG00000013700, and ENSOARG00000013777, and MSTRG.105228 and its target WNT7A may participate in the sheep reproductive process at the hypothalamus level. Significantly, MSTRG.95128 and its cis-target Forkhead box L1 (FOXG1) were shown to be upregulated in PF vs. MF but downregulated in PL vs. ML. All of these results may be attributed to discoveries of new candidate genes and pathways related to sheep reproduction, and they may provide new views for understanding sheep reproduction without the effects of the FecB mutation.

Bamei mutton sheep is a Chinese domestic sheep breed developed by crossing German Mutton Merino sheep and indigenous Mongolian sheep for meat production. Here, we focused on detecting candidate genes associated with the increasing of the litter size in this breeds under recent artificial selection to improve the efficiency of mutton production. We selected five high- and five low-fecundity Bamei mutton sheep for whole-genome resequencing to identify candidate genes for sheep prolificacy. We used the FST and XP-EHH statistical approach to detect the selective sweeps between these two groups. Combining the two selective sweep methods, the reproduction-related genes JUN, ITPR3, PLCB2, HERC5, and KDM4B were detected. JUN, ITPR3, and PLCB2 play vital roles in GnRH (gonadotropin-releasing hormone), oxytocin, and estrogen signaling pathway. Moreover, KDM4B, which had the highest FST value, exhibits demethylase activity. It can affect reproduction by binding the promoters of estrogen-regulated genes, such as FOXA1 (forkhead box A1) and ESR1 (estrogen receptor 1). Notably, one nonsynonymous mutation (p.S936A) specific to the high-prolificacy group was identified at the TUDOR domain of KDM4B. These observations provide a new opportunity to research the genetic variation influencing fecundity traits within a population evolving under artificial selection. The identified genomic regions that are responsible for litter size can in turn be used for further selection.

Photoperiod is one of the important factors leading to seasonal reproduction of sheep. However, the molecular mechanisms underlying the photoperiod regulation of seasonal reproduction remain poorly understood. In this study, we compared the expression profiles of mRNAs, lncRNAs, and circRNAs in uterine tissues from Sunite sheep during three different photoperiods, namely, the short photoperiod (SP), short transfer to long photoperiod (SLP), and long photoperiod (LP). The results showed that 298, 403, and 378 differentially expressed (DE) mRNAs, 171, 491, and 499 DE lncRNAs, and 124, 270, and 400 DE circRNAs were identified between SP and LP, between SP and SLP, and between LP and SLP, respectively. Furthermore, functional enrichment analysis showed that the differentially expressed RNAs were mainly involved in the GnRH signaling pathway, thyroid hormone synthesis, and thyroid hormone signaling pathway. In addition, co-expression networks of lncRNA-mRNA were constructed based on the correlation analysis between the differentially expressed RNAs. Our study provides new insights into the expression changes of RNAs in different photoperiods, which might contribute to understanding the molecular mechanisms of seasonal reproduction in sheep.

Prolificacy of most local goat breeds in China is low. Jining Grey goat is one of the most prolific goat breeds in China, it is an important goat breed for the rural economy. ASMT (acetylserotonin O-methyltransferase) and ADAMTS1 (ADAM metallopeptidase with thrombospondin type 1 motif) are essential for animal reproduction. Single nucleotide polymorphisms (SNPs) of ASMT and ADAMTS1 genes in the highly prolific breed (Jining Grey goats), medium prolific breed (Boer goats and Guizhou White goats) and low prolific breeds (Angora goats, Liaoning Cashmere goats and Inner Mongolia Cashmere goats) were detected by polymerase chain reaction-restriction fragment length polymorphism and sequencing. Two SNPs (g.158122T>C, g.158700G>A) of ASMT gene and two SNPs (g.7979798A>G, g.7979477C>T) of ADAMTS1 gene were identified. For g.158122T>C of ASMT gene, further analysis revealed that genotype TC or CC had 0.66 (p < 0.05) or 0.75 (p < 0.05) kids more than those with genotype TT in Jining Grey goats. No significant difference (p > 0.05) was found in litter size between TC and CC genotypes. The SNP (g.158122T>C) caused a p.Tyr298His change and this SNP mutation resulted in changes in protein binding sites and macromolecule-binding sites. The improvement in reproductive performance may be due to changes in the structure of ASMT protein. For g.7979477C>T of ADAMTS1 gene, Jining Grey does with genotype CT or TT had 0.82 (p < 0.05) or 0.86 (p < 0.05) more kids than those with genotype CC. No significant difference (p > 0.05) was found in litter size between CT or TT genotypes. These results preliminarily indicated that C allele (g.158122T>C) of ASMT gene and T allele (g.7979477C>T) of ADAMTS1 gene are potential molecular markers which could improve litter size of Jining Grey goats and be used in goat breeding.

MicroRNA (miRNA) is a kind of noncoding RNA whose function involved in various biological processes in neuronal maturation and adipocyte cells, such as differentiation, proliferation, development, apoptosis, and metabolism. Herein, miRNA-Seq was used to identify miRNAs in the tail fat tissue of Hu sheep (short-fat-tailed) and Tibetan sheep (short-thin-tailed). In this study, 155 differentially expression miRNAs (DE miRNAs) were identified, including 78 up-regulated and 77 down-regulated. Among these DE miRNAs, 17 miRNAs were reported and related with lipid metabolism. MiRanda and RNAhybrid software were used to predict the target genes of DE miRNAs, obtaining the number of targeting relationships is 38553. Target genes were enriched by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). 742 terms and 302 single pathways are enriched, including lipid metabolic process, response to lipid, cellular lipid catabolic process, lipid catabolic process, cellular lipid metabolic process, inositol lipid-mediated signaling, calcium channel activity, PI3K-Akt signaling pathway, MAPK signaling pathway, ECM-receptor interaction, AMPK signaling pathway, Wnt signaling pathway and TGF-beta signaling pathway. Notably, miR-379-5p was associated with tail fat deposition of sheep. Dual-Luciferase reporter assays showed miR-379-5p and HOXC9 had targeted relationship. The result of RT-qPCR showed that the expression trend of miR-379-5p and HOXC9 was opposite. miR-379-5p was down-regulated and highly expressed in tail adipose tissue of Tibetan sheep. HOXC9 was highly expressed in adipose tissue of Hu sheep. These results could provide a meaningful theoretical basis for studying the molecular mechanisms of sheep tail adipogenesis.

Small Tail Han (STH) sheep, a unique Chinese breed, is recognized for its early maturity, year-round estrus, and prolificacy. However, the molecular mechanism of its high prolificacy has not been fully elucidated. The Proteomics approach is feasible and effective to reveal the proteins involved in the complex physiological processes of any organism. Given this, we performed the protein expression profiling of ovarian tissues during the luteal phase using polytocous STH sheep (litter size ≥2, three consecutive lambings) and monotocous STH sheep (litter size =1, three consecutive lambings) (PL vs. ML), and the follicular phase using polytocous STH sheep (litter size ≥2, three consecutive lambings) and monotocous STH sheep (litter size =1, three consecutive lambings) (PF vs. MF), respectively. Parallel Reaction Monitoring (PRM) was conducted to validate the differentially abundant proteins (DAPs). The tandem mass tag (TMT) quantitative proteomic results showed that a total of 5,237 proteins were identified, of which 49 and 44 showed differential abundance in the PL vs. ML and PF vs. MF groups, respectively. Enrichments analyses indicated that the DAPs including TIA1 cytotoxic granule-associated RNA-binding protein-like 1 (TIAL1), nicotinamide phosphoribosyltransferase (NAMPT), and cellular retinoic acid-binding protein 1 (CRABP1) were enriched at the luteal phase, while TIAL1, inhibin beta-a-subunit (A2ICA4), and W5PG55 were enriched at the follicular phase, potentially mediating reproductive processes in polytocous ewes. Furthermore, six DAPs were verified using PRM, confirming the accuracy of the TMT data acquired in this study. Together, our work expanded the database of indigenous sheep breeds and provided new ovarian candidate molecular targets, which will help in the study of the genetic mechanisms of ovine prolificacy.

Circular RNAs (circRNAs) are a specific type of noncoding RNA, and some have defined roles in cellular and biological processes. However, little is known about the role of circRNAs in follicular development in sheep with FecB (fecundity Booroola) mutations. Here, the expression profiles of circRNAs were investigated using RNA sequencing (RNA-seq) in the follicular phase (F) and the luteal phase (L) of FecB mutant homozygous (BB) and wild-type (WW) Small Tail Han sheep. A total of 38,979 circRNAs were identified, and 314, 343, 336, and 296 of them were differentially expressed (DE) between BB_F and BB_L, WW_F and WW_L, BB_F and WW_F, and BB_L and WW_L, respectively. The length, type, and chromosome distribution of the circRNAs and the expression characteristic between the circRNAs and their host genes in the sheep hypothalamus were ascertained. Enrichment analysis showed that the host genes of DE circRNAs in the follicular and luteal phases were annotated to MAPK, gap junctions, progesterone-mediated oocyte maturation, oocyte meiosis, and other hormone-related signaling pathways, and the different FecB genotypes were annotated to the gap junctions, circadian entrainment, MAPK, and other hormone-related signaling pathways. The competing endogenous RNA network prediction revealed that the 129 target miRNAs might be bound to 336 DE circRNAs. oar_circ_0000523 and oar_circ_0028984, which were specifically expressed during the follicular phase in the BB genotype sheep, probably acted as miRNA sponges involved in the regulation of LH synthesis and secretion. This study reveals the expression profiles and characterization of circRNAs at two phases of follicular development considering different FecB genotypes, thereby providing an improved understanding of the roles of circRNAs in the sheep hypothalamus and their involvement in follicular development and ovulation.

Long non-coding RNAs (lncRNAs) regulate endometrial secretion and uterine volume. However, there is little research on the role of lncRNAs in the uterus of Small Tail Han sheep (FecB++). Herein, RNA-seq was used to comparatively analyze gene expression profiles of uterine tissue between polytocous and monotocous sheep (FecB++) in follicular and luteal phases.

Although it has been investigated for many years, the physiological processes regulating prolificacy in sheep remains unclear because of regulation by many genes. To better understand the effects of three single nucleotide polymorphisms (SNPs) comprising g.48462350C>T in BMP2, g.58171856C>G and g.58171886A>C in BMP7, a population genetic analysis was conducted using data obtained from genotyping in 768 sheep from six breeds (three polytocous and three monotocous). The results indicate that all the sheep breeds were considered to conform to the Hardy-Weinberg equilibrium (P > 0.05). The associations of these three SNPs with litter size in 384 Small Tail Han sheep were analyzed, therefore, and found to be correlated with fecundity as assessed by mean litter size (P < 0.05). Bioinformatic analysis indicated there was a transmembrane domain change that occurred after a mutation in BMP2 at g.48462350C>T, and changes involving transcription factors such as USF1, USF2 and INMS1 in the BMP7 promoter region might be involved in greater sheep prolificacy.

TGF-β induced factor homeobox 1 (TGIF1) and splicing factor 1 (SF1) are important for mammalian reproduction; however, the effects of these genes on litter size in sheep remain unexplored. In this study, we genotyped 768 ewes from seven sheep breeds at two loci: g.37871539C>T, a synonymous mutation of TGIF1; and g.42314637T>C, a 3'UTR variant of SF1. Our analysis of polymorphism revealed only two genotypes at locus g.37871539C>T in TGIF1, with most sheep populations being moderately polymorphic (0.25 < PIC < 0.5) at this site. In contrast, most breeds exhibited low polymorphism (PIC ≤0.25) at the SF1 locus g.42314637T>C. The association analysis revealed that a synonymous mutation at g.37871539C>T in TGIF1 was highly associated with litter size in Small Tail Han sheep, in which it causes a significant decrease in litter size. Conversely, while the SF1 3'UTR variant g.42314637T>C was also highly associated with litter size in sheep, it causes a significant increase in the number of litter size. Combined, these data provide valuable information regarding candidate genetic markers for sheep breeding programs.

Previous studies revealed that alternative splicing (AS) events and gene variants played key roles in reproduction; however, their location and distribution in hypothalamic fecundity-related genes in sheep without the FecB mutation remain largely unknown. Therefore, in this study, we described the hypothalamic AS events and variants in differentially expressed genes (DEGs) in Small-tailed Han sheep without the FecB mutation at polytocous sheep in the follicular phase vs. monotocous sheep in the follicular phase (PF vs. MF) and polytocous sheep in the luteal phase vs. monotocous sheep in the luteal phase (PL vs. ML) via an RNA-seq study for the first time. We found 39 DEGs with AS events (AS DEGs) in PF vs. MF, while 42 AS DEGs were identified in PL vs. ML. No DEGs with single nucleotide polymorphisms (SNPs) were observed in PF vs. MF, but five were identified in PL vs. ML. We also performed a correlation analysis of transcriptomics and proteomics, and the results suggested several key DEGs/differentially expressed proteins (DEPs), such as LGALS3 in PF vs. MF and aspartoacylase (ASPA) and transthyretin (TTR) in PL vs. ML, could be candidate genes influencing ovine litter size. In addition, further analyses suggested that AS events, SNPs and miRNA-binding sites existed in key DEGs/DEPs, such as ASPA and TTR. All in all, this study provides a new insight into ovine and even other mammalian reproduction.

The expression characteristics of the hypothalamic-pituitary-gonadal (HPG) axis-related candidate genes, DIO2, EYA3, KISS1 and GPR54, were analyzed in year-round estrous rams (small-tail Han sheep, STH) and seasonally estrous rams (Sunite sheep, SNT) using qPCR. The results were as follows: DIO2 was mainly expressed in pituitary, and KISS1 was specifically expressed in hypothalamus in the two groups. However, EYA3 and GPR54 were widely expressed in the cerebrum, cerebellum, hypothalamus, pituitary, testis, epididymis, vas deferens and adrenal gland tissues in both breeds, with significant differences in the cerebellum, hypothalamus, pituitary, testis and vas deferens tissues. We speculated that DIO2 and KISS1 may have positive roles in different regions in ram year-round estrus. Moreover, the expression patterns of EYA3 and GPR54 suggested that they may regulate the estrous mode of ram via testis and vas deferens. This is the first study to systematically analyze the expression patterns of HPG axis-related genes in rams.

The fat tail is a unique characteristic of sheep that represents energy reserves and is a complex adaptative mechanism of fat-tailed sheep to environmental stress. MicroRNA plays a significant role as regulators at the posttranscriptional level, but no studies have explained the molecular mechanisms of miRNA which regulate fat deposition in sheep tails. In this study, mRNA and miRNA analysis examined tail fat tissue from three Hu fat-tailed and three Tibetan thin-tailed sheep. After aligning to the reference sequences, 2,108 differentially expressed genes and 105 differential expression miRNAs were identified, including 1,247 up- and 861 downregulated genes and 43 up- and 62 downregulated miRNAs. Among these differentially expressed miRNAs, oar-miR-432 was one of the most downregulated miRNAs between Hu sheep and Tibetan sheep, and 712 genes were predicted to be targeted by oar-miR-432, 80 of which overlapped with DEGs. The Gene Ontology analysis on these genes showed that BMP2, LEP, GRK5, BMP7, and RORC were enriched in fat cell differentiation terms. The genes for BMP2 targeted by oar-miR-432 were examined using dual-luciferase assay. The oar-miR-432 mimic transfected into preadipocytes resulted in increased expression of BMP2. The marker gene PPAR-γ of fat differentiation had a lower expression than the negative control on days 0, 2, and 4 after induced differentiation. The decrease in the number of lipids in the oar-miR-432 mimic group detected by oil red O stain was also less than that in the negative control. This is the first study to reveal the fat mechanisms by which oar-miR-432 inhibits fat differentiation and promotes the expression of BMP2 in sheep tails.

Many recent studies have shown that miRNAs play important roles in the regulation of animal reproduction, including seasonal reproduction. The pineal gland is a crucial hub in the regulation of seasonal reproduction. However, little is known about the expression characteristics of pineal miRNAs in different reproductive seasons (anestrus and breeding season). Therefore, the expression profiles and regulatory roles of ovine pineal miRNAs were investigated during different reproductive stages using Solexa sequencing technology and dual luciferase reporter assays.

A new fecundity gene named the FecL (mutation), which regulates the ovulation rate, was discovered in French Lacaune sheep. The B4GALNT2 (beta-1, 4-N-acetyl-galactosaminyl transferase 2) gene was considered as the potential FecL mutation gene. This study explores whether the effect of the FecL mutation exists in other sheep breeds, and the features of the B4GALNT2 gene in terms of the molecular structure and its expression profile. Using Sanger sequencing, we found that high and low fecundity breeds from among 11 measured sheep breeds all had no variation in the three specific mutation sites, which were linked with the FecL mutation. However, two mutations of g.36946470C > T and g.36933082C > T in the exon of B4GALNT2 had a significant effect on litter size in the first parity for Small Tail Han (STH) Sheep (p < 0.05). Two transcription start sites (TSS) of B4GALNT2 in its 5'-flanking region were discovered in ovine granule cells in vitro, through the RACE (Rapid amplification of cDNA ends) method. Except for in the kidney and oviduct, no significant difference in expression levels had been found between STH sheep and Tan sheep breeds. The B4GALNT2 gene, as a candidate for FecL, may have a relationship with the differences in litter size in STH sheep. B4GALNT2 is mainly expressed in the ovine ovary, which also suggests that B4GALNT2 plays an important role in sheep reproduction.

Animal domestication has been extensively studied, but the process of feralization remains poorly understood.

Litter size is an economically important trait in sheep, and it is a complex trait controlled by multiple genes in multiple organs. Among them, the regulation of lamb number trait by the thyroid gland is a very important part. However, the molecular mechanisms of the thyroid gland in sheep reproduction remain unclear. Here, RNA-seq was used to detect transcriptome expression patterns in the thyroid gland between follicular phase (FP) and luteal phase (LP) in FecB BB (MM) and FecB ++ (ww) STH sheep, respectively, and to identify differentially expressed circRNAs (DECs) associated with reproduction. Bioinformatic analysis of the source genes of these DECs revealed that they can be enriched in multiple signaling pathways involved in the reproductive process of animals. We found that the source genes of these DECs, such as GNAQ, VEGFC, MAPK1, STAT1, and HSD17B7, may play important roles in the reproductive process of animals. To better understand the function of these DECs, we constructed circRNA-miRNA co-expression networks. Dual luciferase reporter assays suggested that a ceRNA regulatory mechanism between circ_0003259-oar-miR-133-TXLNA and circ_0012128-oar-miR-370-3p-FGFR1 may hold. All of these DEC expression profiles in the thyroid gland provide a novel resource for elucidating the regulatory mechanisms underlying STH sheep prolificacy.

Understanding the molecular mechanism of mammalian reproduction (puberty and prolificacy) will play a part in improving animal reproductive performance. GLUD1 (glutamate dehydrogenase 1) is important for mammalian reproduction, as shown in previous studies; however, its roles in puberty and prolificacy have rarely been reported. In this study, we designed seven pairs of primers (P1 to P7) for cloning and sequencing genomic DNA of Jining Grey goats and Liaoning Cashmere goats. Primer 8 (P8) was designed to detect single nucleotide polymorphism (SNP) of the GLUD1 in both sexually precocious and high-fecundity breeds (Jining Grey, Nanjiang Brown and Matou goats) and sexually late-maturing and low-fecundity breeds (Liaoning Cashmere, Inner Mongolia Cashmere and Taihang goats) by PCR-RFLP (restriction fragment length polymorphism). The real-time quantitative polymerase chain reaction (RT-qPCR) technique was used to detect the expression of GLUD1 in a variety of tissues. The results showed that the A197C mutation was only found in the amplification product of P6. For this SNP locus, only two genotypes (AA and AC) were detected in Nanjiang Brown goats, while three genotypes (AA, AC and CC) were detected in the other five breeds. In Jining Grey goats, the frequency of genotypes AA, AC and CC was 0.69, 0.26 and 0.05, respectively. In Jining Grey goats, AA genotype had 0.54 (P<0.05) and 0.3 (P<0.05) more kids than the CC and AC genotype, respectively, and no significant difference (P>0.05) was found in kidding number between the AC and CC genotype. GLUD1 was expressed in five tissues of different developmental stages. The expression level of GLUD1 in the hypothalamus was higher than that in the other four tissues except during puberty of Liaoning Cashmere goats. In puberty in goats, GLUD1 expression was significantly higher in ovaries than that in the juvenile period (P<0.01). RT-qPCR results showed that the expression of GLUD1 in ovaries may relate to the puberty of goats. The present study preliminarily indicated that there might be an association between the 197 locus of GLUD1 and sexual precocity in goats, and allele A of GLUD1 was a potential DNA marker for improving kidding number in Jining Grey goats.