Search

Abstract Background Myoblasts serve as the fundamental building blocks of muscle fibers, and there is a positive correlation between the diameter of myofibers during the juvenile phase and the rate of muscle growth, which does not change in adulthood. However, the molecular mechanisms governing myofiber diameter across various developmental stages in goats remain largely unclear. Results In this study, we examined miRNA expression in the longissimus dorsi muscle tissue of goats at two distinct ages: one month, a period characterized by robust muscle growth, and nine months, when muscle development plateaus in adulthood. A total of 408 known miRNAs and 86 novel miRNAs were identified, with 32 miRNAs exhibiting differential expression between the two groups. A functional enrichment analysis of these targeted genes revealed significant enrichment in pathways closely correlated with skeletal muscle growth, development, and senescence. Notably, chi-miR-103-3p was identified among the DE miRNAs and appeared to play an important role in skeletal myoblast proliferation. Bioinformatics analysis, complemented by dual luciferase activity assays revealed that chi-miR-103-3p specifically targets the 3’UTR of FGF18. Subsequent cell transfection experiments demonstrated that chi-miR-103-3p suppresses the expression of FGF18 in goat myoblasts, thereby inhibiting cell proliferation. Moreover, FGF18 was observed to enhance the proliferation of goat myoblasts. Conclusions Collectively, our data indicated that the elevated expression of chi-miR-103-3p in adult goat myoblasts significantly repressed FGF18 expression, thereby limiting rapid muscle growth. Proliferation and differentiation of myoblasts can affect myofiber number and cell volume expansion. These findings lay the foundation for further elucidation of the molecular mechanisms underlying muscle growth and development across different life stages of goats. Additionally, it could be a potential molecular marker for improving muscle production in goats.

Background The thyroid gland is an important endocrine gland in animals that secretes thyroid hormones and acts on various organs throughout the body. lncRNAs are long non-coding RNAs that play an important role in animal reproduction; however, there is a lack of understanding of their expression patterns and potential roles in the thyroid gland of the Small Tail Han (STH) sheep. In this study, we used RNA-Seq technology to examine the transcriptome expression pattern of the thyroid from the luteal phase (LP) and follicular phase (FP) of FecB BB (MM) STH sheep.Results We identified a total of 122 and 1287 differential expression lncRNAs (DELs) and differential expression mRNAs (DEGs), respectively, which were significantly differentially expressed. These DELs target genes and DEGs can be enriched in several signalling pathways related to the animal reproduction process.Conclusions The expression profiles of DELs and DEGs in thyroid glands provide a more comprehensive resource for elucidating the reproductive regulatory mechanisms of STH sheep.

Litter size in sheep is a complex trait controlled by micro-effective polygenes. APAF1, CLSTN2, CTH, PLCB1, PLCB4, and CHST11 are all involved in mammalian reproduction. However, the effects of these genes on litter size in sheep are still unclear. Therefore, in this study, we used Sequenom MassARRAY® SNP assay technology to type the single nucleotide polymorphisms (SNPs) loci of six genes in five sheep breeds. The results showed that most sheep breeds contain three genotypes at each locus. Then, we conducted population genetic analysis on the SNPs of six genes and found that the polymorphic information content in all sheep breeds ranged from 0 to 0.37, and most sheep breeds were in Hardy-Weinberg equilibrium (p > 0.05). In addition, association analysis in Small Tail Han sheep indicated that the rs399534524 locus in CLSTN2 was highly associated with first parity litter size, and litter size in ewes with CT genotype was higher than that in ewes with CC genotype or TT genotype. Furthermore, the rs407142552 locus in CTH was highly associated with second parity litter size in Small Tail Han sheep, and litter size in ewes with CT genotype was higher than that in ewes with TT genotype. Finally, we predicted the CTH and CLSTN2 protein interaction network and found that HTR1E, NOM1, CCDC174 and ALPK3 interact with CLSTN2 and have been reported as candidate genes related to litter size in sheep. These results suggest that they may be useful genetic markers for increasing litter size in sheep.

The thyroid gland is an important endocrine gland in animals, which mainly secretes thyroid hormones and acts on various organs of the body. Long-chain non-coding RNA (lncRNA) plays an important role in animal reproduction. However, there is still a lack of understanding of their expression patterns and potential roles in the thyroid of Small Tail Han (STH) sheep. In this study, RNA-seq was used to examine the transcriptome expression patterns of lncRNAs and mRNAs in the follicular phase (ww_FT) and luteal phase (ww_LT) in FecB++ genotype STH Sheep. A total of 17,217 lncRNAs and 39,112 mRNAs were identified including 96 differentially expressed lncRNAs (DELs) and 1054 differentially expressed mRNAs (DEGs). Functional analysis of genes with significant differences in expression level showed that these genes could be enriched in Ras signalling pathway, hedgehog (HH) signalling pathway, ATP-binding cassette (ABC) transporters and other signalling pathways related to animal reproduction. In addition, through correlation analysis for lncRNA–mRNA co-expression and network construction, we found that LNC_009115 and LNC_005796 trans target NIK-related kinase (NRK) and poly(A)-specific ribonuclease (PARN). LNC_007189 and LNC_002045 trans target progesterone-induced blocking factor 1 (PIBF1), LNC_009013 trans targets small mothers against decapentaplegic (SMAD1) are related to animal reproduction. These genes add new resources for elucidating the regulatory mechanisms of reproduction in sheep with different reproductive cycles of the FecB++ genotype STH sheep.

Abstract Genetic variations in the ovine ovulation rate, which are associated with the FecB mutation, provide useful models by which to explore the mechanisms regulating the development of mammalian antral follicles. In order to study the effects of the FecB mutation on cumulus cell differentiation, preovulatory follicles were aspirated and cumulus cells were isolated from three FecB genotypes (homozygous, heterozygous and wild type) of Small Tail Han (STH) sheep superstimulated with FSH. Transcriptome information from tens of thousands of cumulus cells was determined with the 10 × Genomics single-cell RNA-seq technology. Under the superovulation treatment, the observed number of preovulatory follicles in the ovaries of FecB carriers was still significantly higher than that in the wild-type (P

Abstract Background The pituitary directly regulates the reproductive process through follicle-stimulating hormone (FSH) and luteinizing hormone (LH). Transcriptomic research on the pituitaries of ewes with different FecB (fecundity Booroola) genotypes has shown that some key genes and lncRNAs play an important role in pituitary function and sheep fecundity. Our previous study found that ewes with FecB + + genotypes (without FecB mutation) still had individuals with more than one offspring per birth. It is hoped to analyze this phenomenon from the perspective of the pituitary transcriptome. Results The 12 Small Tail Han Sheep were equally divided into polytocous sheep in the follicular phase (PF), polytocous sheep in the luteal phase (PL), monotocous sheep in the follicular phase (MF), and monotocous sheep in the luteal phase (ML). Pituitary tissues were collected after estrus synchronous treatment for transcriptomic analysis. A total of 384 differentially expressed genes (DEGs) (182 in PF vs. MF and 202 in PL vs. ML) and 844 differentially expressed lncRNAs (DELs) (427 in PF vs. MF and 417 in PL vs. ML) were obtained from the polytocous-monotocous comparison groups in the two phases. Functional enrichment analysis showed that the DEGs in the two phases were enriched in signaling pathways known to play an important role in sheep fecundity, such as calcium ion binding and cAMP signaling pathways. A total of 1322 target relationship pairs (551 pairs in PF vs. MF and 771 pairs in PL vs. ML) were obtained for the target genes prediction of DELs, of which 29 DEL-DEG target relationship pairs (nine pairs in PF vs. MF and twenty pairs in PL vs. ML). In addition, the competing endogenous RNA (ceRNA) networks were constructed to explore the regulatory relationships of DEGs, and some important regulatory relationship pairs were obtained. Conclusion According to the analysis results, we hypothesized that the pituitary first receives steroid hormone signals from the ovary and uterus and that VAV3 (Vav Guanine Nucleotide Exchange Factor 3), GABRG1 (Gamma-Aminobutyric Acid A Receptor, Gamma 1), and FNDC1 (Fibronectin Type III Domain Containing 1) played an important role in this process. Subsequently, the reproductive process was regulated by gonadotropins, and IGFBP1 (Insulin-like Growth Factor Binding Protein 1) was directly involved in this process, ultimately affecting litter size. In addition, TGIF1 (Transforming Growth Factor-Beta-Induced Factor 1) and TMEFF2 (Transmembrane Protein With EGF Like And Two Follistatin Like Domains 2) compensated for the effect of the FecB mutation and function by acting on TGF-β/SMAD signaling pathway, an important pathway for sheep reproduction. These results provided a reference for understanding the mechanism of multiple births in Small Tail Han Sheep without FecB mutation.

The photoperiod has been considered to be a key environmental factor in sheep reproduction, and some studies have shown that the thyroid gland plays an important role in mammalian reproduction, but the molecular mechanism is still unclear. In this study, we used the artificial-light-controlled, ovariectomized, and estradiol-treated model (OVX + E2 model); healthy and consistent 2–3-year-old Sunite multiparous ewes were collected; and thyroids were collected for testing, combined with RNA-seq technology and bioinformatics analysis, to analyze the effects of different photoperiods (long photoperiod treatment for 42 days, LP42; short photoperiod treatment for 42 days, SP42; SP42 transferred to LP42, SPLP42) on the variations in the miRNA profiles of the thyroid gland. A total of 105 miRNAs were differentially expressed in the thyroid gland, most of which were new miRNAs. Through GO and KEGG enrichment analysis, the results showed that the photoperiod response characteristics of Sunite ewes were affected by Olfactory transduction, Wnt signaling pathways, and Apelin signaling pathways. A different illumination time may have a certain influence on the downstream of these pathways, which leads to the change in animal estrus state. In addition, lncRNA-mRNA-miRNA network analysis revealed the target binding sites of identified miRNAs in DE-circRNA and DE-mRNA, such as Novel_369, Novel_370, Novel_461, and so on. The results of this study will provide some new insights into the function of miRNA and the changes in sheep thyroid glands under different photoperiods.

The Booroola fecundity mutation (FecB) in Small Tail Han sheep has been shown to enhance ovulation rates and litter sizes by affecting the hypothalamic–pituitary–gonadal (HPG) axis. Despite the pituitary’s role in reproductive regulation, its involvement in FecB-induced ovulation remains understudied. Our study aimed to fill this gap by analyzing pituitary tissues from FecB homozygous (BB) and wild-type (WW) ewes during luteal and follicular phases using tandem mass tag–based protein quantification and the DIABLO framework for proteomic and transcriptomic data integration. Significant differences in 277 proteins were observed across estrus periods, with network analysis highlighting the voltage-dependent calcium channel L-type alpha-1C as a key convergence point in oxytocin signaling and GnRH secretion pathways. The DIABLO method revealed a strong correlation (0.98) between proteomic and transcriptomic datasets, indicating a coordinated response in FecB ewes. Notably, higher expression levels of Follicle Stimulating Hormone Subunit Beta (FSHB) and Luteinizing Hormone Subunit Beta (LHB) were found in BB ewes during the follicular phase, potentially due to elevated E2 concentrations. Furthermore, our analysis identified genes related to the Gamma–aminobutyric acid type A receptor family (GABRA2, GABRG1, GABRB1) in the pituitary, with GABRB1 showing higher expression in BB ewes. This suggests a role for GABA in modulating GnRH and gonadotropin feedback loops, potentially contributing to the FecB mutation’s effect on ovulation. This study provides novel insights into the pituitary’s role in fertility among FecB sheep, identifying GABA as a potential regulatory factor within the HPG axis. The findings also open avenues for discovering new biomarkers in pituitary endocrinology for sheep breeding purposes.

Neurotrophin receptor B (NTRK2), also named TRKB, belongs to the neurotrophic factor family. Previous studies have shown that NTRK2 is associated with high fertility in mammals. However, the molecular mechanism and regulatory pathway of this neurotrophic factor remain unclear. In this study, NTRK2 overexpression and NTRK2-siRNA were constructed to detect the effects of NTRK2 on the proliferation and hormone secretion of the ovarian granulosa cells (GCs) of sheep. We successfully isolated follicular phase granulosa cells in vitro from the ovaries of sheep in simultaneous estrus, and the immunofluorescence results confirmed that NTRK2 was expressed in the collected cells. Subsequently, the effect of NTRK2 on the proliferation of sheep granulosa cells was examined via cell transfection experiments. The results showed that the expression of CDK4 and CyclinD2 was significantly increased after NTRK2 overexpression, while the opposite trend was observed after the inhibition of NTRK2 expression (p < 0.05). The EdU and CCK-8 assays showed that the proliferation rate of sheep GCs was significantly increased after NTRK2 overexpression, while the opposite trend was observed after the inhibition of NTRK2 expression (p < 0.05). Moreover, NTRK2 significantly increased the expression of steroidogenesis-related genes, including steroidogenic acute regulatory protein (STAR) and hydroxy-δ-5-steroid dehydrogenase (HSD3B1), and cytochrome P450 family 19 subfamily A member 1 (CYP19A1). The ELISA results showed that the secretion levels of E2 and P4 significantly increased after NTRK2 overexpression, while the opposite trend was observed after the inhibition of NTRK2 expression (p < 0.05). Previous studies had confirmed that NTRK2 gene belongs to the PI3K-AKT signaling pathway and participates in the signaling of this pathway. This was demonstrated by protein–protein interaction analysis and NTRK2 belongs to the PI3K-AKT pathway. The modification of PI3K and AKT, markers of the PI3K-AKT pathway, via phosphorylation was increased after NTRK2 overexpression in the sheep GCs, while the opposite trend was observed after the inhibition of NTRK2 expression (p < 0.05). Overall, these results suggest that the NTRK2 gene regulates the proliferation of GCs and the secretion of steroid hormones in sheep, and that it influences the phosphorylation level of the PI3K/AKT signaling pathway. These findings provided a theoretical basis and new perspectives for exploring the regulation of NTRK2 gene in the development of ovine follicles.

IntroductionSuper-enhancers (SEs) are clusters of enhancers that act synergistically to drive the high-level expression of genes involved in cell identity and function. Although SEs have been extensively investigated in humans and mice, they have not been well characterized in pigs.MethodsHere, we identified 42,380 SEs in 14 pig tissues using chromatin immunoprecipitation sequencing, and statistics of its overall situation, studied the composition and characteristics of SE, and explored the influence of SEs characteristics on gene expression.ResultsWe observed that approximately 40% of normal enhancers (NEs) form SEs. Compared to NEs, we found that SEs were more likely to be enriched with an activated enhancer and show activated functions. Interestingly, SEs showed X chromosome depletion and short interspersed nuclear element enrichment, implying that SEs play an important role in sex traits and repeat evolution. Additionally, SE-associated genes exhibited higher expression levels and stronger conservation than NE-associated genes. However, genes with the largest SEs had higher expression levels than those with the smallest SEs, indicating that SE size may influence gene expression. Moreover, we observed a negative correlation between SE gene distance and gene expression, indicating that the proximity of SEs can affect gene activity. Gene ontology enrichment and motif analysis revealed that SEs have strong tissue-specific activity. For example, the CORO2B gene with a brain-specific SE shows strong brain-specific expression, and the phenylalanine hydroxylase gene with liver-specific SEs shows strong liver-specific expression.DiscussionIn this study, we illustrated a body map of SEs and explored their functions in pigs, providing information on the composition and tissue-specific patterns of SEs. This study can serve as a valuable resource of gene regulatory and comparative analyses to the scientific community and provides a theoretical reference for genetic control mechanisms of important traits in pigs.

In humans, variation of the ATP7A gene may cause cranial exostosis, which is similar to “human horn,” but the function of the ATP7A gene in sheep is still unknown. Tissue expression patterns and potential functional loci analysis of the ATP7A gene could help understand its function in sheep horn. In this study, we first identified tissue, sex, breed, and species-specific expression of the ATP7A gene in sheep based on the RNA-sequencing (RNA-seq) data. Second, the potential functional sites of the ATP7A gene were analyzed by using the whole genome sequencing (WGS) data of 99 sheep from 10 breeds. Last, the allele-specific expression of the ATP7A gene was explored. Our result showed the ATP7A gene has significantly higher expression in the big horn than in the small horn, and the ATP7A gene has high expression in the horn and skin, suggesting that this gene may be related to the horn. The PCA results show that the region around the ATP7A can distinguish horned and hornless groups to some extent, further indicating that the ATP7A may be related to horns. When compared with other species, we find seven ruminate specific amino acid sites of the ATP7A protein, which can be important to the ruminate horn. By analyzing WGS, we found 6 SNP sites with significant differences in frequency in horned and hornless populations, and most of these variants are present in the intron. But we still find some potential functional sites, including three missenses, three synonymous mutations, and four Indels. Finally, by combining the RNA-seq and WGS functional loci results, we find three mutations that showed allele-specific expression between big and small horns. This study shows that the ATP7A gene in sheep may be related to horn size, and several potential functional sites we identified here can be useful molecular markers for sheep horn breeding.

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.

In previous studies, NOX4, PDE11A and GHR genes have been screened as important candidate genes for litter size in sheep by using the GWAS method; however, neither their effects on litter size nor the loci associated with litter size have been identified. In this study, three candidate loci (c.1057-4C > T in NOX4, c.1983C > T in PDE11A and c.1618C > T in GHR) were first screened based on our previous resequencing data of 10 sheep breeds. After the three loci were genotyped using Sequenom MassARRAY technology, we carried out population genetics analysis on the three loci and performed association analysis between the polymorphism of the three loci and the litter size of sheep. The results of population genetics analysis suggested that c.1057-4C > T in NOX4 and c.1983C > T in PDE11A may be subject to natural or artificial selection. The results of association analysis indicated that litter size was significantly associated with c.1057-4C > T in NOX4 and c.1983C > T in PDE11A (p < 0.05) in Small Tail Han sheep, and there was no significant interaction effect between the two loci on the litter size. In summary, c.1057-4C > T in NOX4 and c.1983 C > T in PDE11A can be considered candidate molecular markers for improving litter size in sheep.

Pregnancy loss that occurs in the uterus is an important and widespread problem in humans and farm animals and is also a key factor affecting the fecundity of livestock. Understanding the differences in the fecundity of goats may be helpful in guiding the breeding of goats with high fecundity. In this study, we performed RNA sequencing and bioinformatics analysis to study the uterus of Yunshang black goats with high and low fecundity in the proliferative period. We identified mRNAs, long non-coding RNAs (lncRNAs), and microRNAs (miRNAs) by analyzing the uterine transcriptomes. The target genes of the identified miRNAs and lncRNAs were predicted, and miRNA–mRNA interaction and competitive endogenous RNA (ceRNA) networks were constructed. By comparisons between low- and high-fecundity groups, we identified 1,674 differentially expressed mRNAs (914 were upregulated, and 760 were downregulated), 288 differentially expressed lncRNAs (149 were upregulated, and 139 were downregulated), and 17 differentially expressed miRNAs (4 were upregulated, and 13 were downregulated). In addition, 49 miRNA–mRNA pairs and 45 miRNA–lncRNA pairs were predicted in the interaction networks. We successfully constructed a ceRNA interaction network with 108 edges that contained 19 miRNAs, 11 mRNAs, and 73 lncRNAs. Five candidate genes (PLEKHA7, FAT2, FN1, SYK, and ITPR2) that were annotated as cell adhesion or calcium membrane channel protein were identified. Our results provide the overall expression profiles of mRNAs, lncRNAs, and miRNAs in the goat uterus during the proliferative period and are a valuable reference for studies into the mechanisms associated with the high fecundity, which may be helpful to guide goat to reduce pregnancy loss.

Abstract Background The adrenal gland participates in the process of sheep reproduction. MicroRNAs (miRNAs), endogenous small noncoding RNAs, regulate gene expression at the posttranscriptional level. However, the miRNA-mRNA network profile of adrenal glands relating to reproduction in sheep is still not well-studied. As sheep with FecBBB genotype show higher lambing number compare with the sheep with FecB++ genotype. This research aims to compare gene expression by small RNA-seq in adrenal tissues at follicular (F) and luteal (L) phases in FecB BB (MM) and FecB ++ (ww) sheep. After analysis of gene expression, significant differentially expressed microRNAs (DEMs) and corresponding target genes were identified. Results A total of 180 miRNAs were found in this study, of which 19 DEMs were expressed in the four comparison groups (MM_F_A vs. MM_L_A, MM_F_A vs. ww_F_A, MM_L_A vs. ww_L_A, ww_F_A vs. ww_L_A). Subsequently, 354 target genes of 19 DEMs were predicted by integrated analysis. Cluster analysis was performed by K_means_cluster, and the expression patterns of these DEMs were separated into four subclusters. Functional analysis of target genes was performed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). The results indicated that the target genes were involved mainly in the Notch signaling pathway, signal transduction, cell communication, innate immune response and amino acid metabolism. Specifically, the Notch signaling pathway, biosynthetic process and metabolic process of pyrimidine nucleotide and amino acid metabolism appear to play key regulatory roles in the sheep fertility trait. Furthermore, miRNA-mRNA interaction networks were constructed by differentially expressed genes combined with our previous study of transcriptome data. The results showed that several key genes, including TDRD3, ANAPC7, CCNL2, BRD2 and MUT, were related to the transformation from the follicular phase to the luteal phase. PLAC8L1, NFAT5, DDX24 and MBD1 were related to the high fecundity of small tail Han sheep. Conclusions In this study, the miRNA transcriptome profile was identified, and miRNA-mRNA interaction networks were constructed in adrenal gland tissue of small tail Han sheep, the interaction between miR-370-3p and its targets were considered to play a major role in the reproduction regulation process. The results enriched the number of known miRNAs in adrenal glands and provided novel ideas and further information to demonstrate posttranscriptional regulation mechanisms at follicular and luteal phases in different genotypes of small tail Han sheep.

Abstract Background Hu sheep and Tibetan sheep in China are characterized by fat tails and thin tails, respectively. Several transcriptomes have been conducted in different sheep breeds to identify the differentially expressed genes (DEGs) underlying this trait. However, these studies identified different DEGs in different sheep breeds. Results Hence, RNA sequencing was performed on Hu sheep and Tibetan sheep. We obtained a total of 45.57 and 43.82 million sequencing reads, respectively. Two libraries mapped reads from 36.93 and 38.55 million reads after alignment to the reference sequences. 2108 DEGs were identified, including 1247 downregulated and 861 upregulated DEGs. GO and KEGG analyses of all DEGs demonstrated that pathways were enriched in the regulation of lipolysis in adipocytes and terms related to the chemokine signalling pathway, lysosomes, and glycosaminoglycan degradation. Eight genes were selected for validation by RT–qPCR. In addition, the transfection of BMP2 overexpression into preadipocytes resulted in increased PPAR-γ expression and expression. BMP2 potentially induces adipogenesis through LOX in preadipocytes. The number of lipid drops in BMP2 overexpression detected by oil red O staining was also greater than that in the negative control. Conclusion In summary, these results showed that significant genes (BMP2, HOXA11, PPP1CC and LPIN1) are involved in the regulation of adipogenesis metabolism and suggested novel insights into metabolic molecules in sheep fat tails.

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.

The Booroola fecundity (FecB) mutation in the bone morphogenetic protein receptor type 1B (BMPR1B) gene increases ovulation in sheep. However, its effect on follicular maturation is not fully understood. Therefore, we collected granulosa cells (GCs) at a critical stage of follicle maturation from nine wild-type (WW), nine heterozygous FecB mutant (WB), and nine homozygous FecB mutant (BB) Small Tail Han sheep. The GCs of three ewes were selected at random from each genotype and consolidated into a single group, yielding a total of nine groups (three groups per genotype) for proteomic analysis. The tandem mass tag technique was utilized to ascertain the specific proteins linked to multiple ovulation in the various FecB genotypes. Using a general linear model, we identified 199 proteins significantly affected by the FecB mutation with the LIMMA package (p < 0.05). The differential abundance of proteins was enriched in pathways related to cholesterol metabolism, carbohydrate metabolism, amino acid biosynthesis, and glutathione metabolism. These pathways are involved in important processes for GC-regulated ‘conservation’ of oocyte maturation. Further, the sparse partial least-squares discriminant analysis and the Fuzzy-C-mean clustering method were combined to estimate weights and cluster differential abundance proteins according to ovulation to screen important ovulation-related proteins. Among them, ZP2 and ZP3 were found to be enriched in the cellular component catalog term “egg coat”, as well as some apolipoproteins, such as APOA1, APOA2, and APOA4, enriched in several Gene Ontology terms related to cholesterol metabolism and lipoprotein transport. A higher abundance of these essential proteins for oocyte maturation was observed in BB and WB genotypes compared with WW ewes. These proteins had a high weight in the model for discriminating sheep with different FecB genotypes. These findings provide new insight that the FecB mutant in GCs improves nutrient metabolism, leading to better oocyte maturation by altering the abundance of important proteins (ZP2, ZP3, and APOA1) in favor of increased ovulation or better oocyte quality.

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.

Previous studies have screened key candidate genes for litter size in sheep, including fibrillin-1 (FBN1), family with sequence similarity 184 member B (FAM184B) and zinc finger and AT-hook domain containing (ZFAT). Therefore, it is necessary to verify these genes in the Xinggao mutton sheep population and determine the associated loci for litter size. In this study, three loci (FBN1 g.160338382 T > C, FAM184B g.398531673 C > T and ZFAT g.20150315 C > T) were firstly screened based on the population differentiation coefficient between the polytocous and monotocous sheep groups. Then, population genetic analysis and association analysis were performed on these loci. The results revealed that the g.160338382 T > C in FBN1 was significantly associated with the litter size of sheep. Moreover, there was no significant interaction effect between the g.160338382 T > C locus and FecB on litter size. Notably, g.160338382 T > C is adjacent to the anterior border of exon 58 and belongs to a splice polypyrimidine tract variant, which may lead to alternative splicing and ultimately cause changes in the structure and function of the protein. In summary, our results provided a potentially effective genetic marker for improving the litter size of sheep.

Objective Runs of homozygosity (ROH) are contiguous lengths of homozygous genotypes that can reveal inbreeding levels, selection pressure, and mating schemes. In this study, ROHs were evaluated in Wannan Black pigs to assess the inbreeding levels and the genome regions with high ROH frequency. Methods In a previous study, we obtained 501.52 GB of raw data from resequencing (10×) of the genome and identified 21,316,754 single-nucleotide variants in 20 Wannan Black pig samples. We investigated the number, length, and frequency of ROH using resequencing data to characterize the homozygosity in Wannan Black pigs and identified genomic regions with high ROH frequencies. Results In this work, 1,813 ROHs (837 ROHs in 100 to 500 kb, 449 ROHs in 500 to 1,000 kb, 527 ROHs in >1,000 kb) were identified in all samples, and the average genomic inbreeding coefficient (FROH) in Wannan Black pigs was 0.5234. Sixty-one regions on chromosomes 2, 3, 7, 8, 13, 15, and 16 harbored ROH islands. In total, 105 genes were identified in 42 ROH islands, among which some genes were related to production traits. Conclusion This is the first study to identify ROH across the genome of Wannan Black pigs, the Chinese native breed of the Anhui province. Overall, Wannan Black pigs have high levels of inbreeding due to the influence of ancient and recent inbreeding due to the genome. These findings are a reliable resource for future studies and contribute to save and use the germplasm resources of Wannan Black pigs.

Abstract Background Litter size is an important index of mammalian prolificacy and is determined by the ovulation rate. The ovary is a crucial organ for mammalian reproduction and is associated with follicular development, maturation and ovulation. However, prolificacy is influenced by multiple factors, and its molecular regulation in the follicular phase remains unclear. Methods Ten female goats with no significant differences in age and weight were randomly selected and divided into either the high-yielding group (n = 5, HF) or the low-yielding group (n = 5, LF). Ovarian tissues were collected from goats in the follicular phase and used to construct mRNA and miRNA sequencing libraries to analyze transcriptomic variation between high- and low-yield Yunshang black goats. Furthermore, integrated analysis of the differentially expressed (DE) miRNA-mRNA pairs was performed based on their correlation. The STRING database was used to construct a PPI network of the DEGs. RT–qPCR was used to validate the results of the predicted miRNA-mRNA pairs. Luciferase analysis and CCK-8 assay were used to detect the function of the miRNA-mRNA pairs and the proliferation of goat granulosa cells (GCs). Results A total of 43,779 known transcripts, 23,067 novel transcripts, 424 known miRNAs and 656 novel miRNAs were identified by RNA-seq in the ovaries from both groups. Through correlation analysis of the miRNA and mRNA expression profiles, 263 negatively correlated miRNA-mRNA pairs were identified in the LF vs. HF comparison. Annotation analysis of the DE miRNA-mRNA pairs identified targets related to biological processes such as “estrogen receptor binding (GO:0030331)”, “oogenesis (GO:0048477)”, “ovulation cycle process (GO:0022602)” and “ovarian follicle development (GO:0001541)”. Subsequently, five KEGG pathways (oocyte meiosis, progesterone-mediated oocyte maturation, GnRH signaling pathway, Notch signaling pathway and TGF-β signaling pathway) were identified in the interaction network related to follicular development, and a PPI network was also constructed. In the network, we found that CDK12, FAM91A1, PGS1, SERTM1, SPAG5, SYNE1, TMEM14A, WNT4, and CAMK2G were the key nodes, all of which were targets of the DE miRNAs. The PPI analysis showed that there was a clear interaction among the CAMK2G, SERTM1, TMEM14A, CDK12, SYNE1 and WNT4 genes. In addition, dual luciferase reporter and CCK-8 assays confirmed that miR-1271-3p suppressed the proliferation of GCs by inhibiting the expression of TXLNA. Conclusions These results increase the understanding of the molecular mechanisms underlying goat prolificacy. These results also provide a basis for studying interactions between genes and miRNAs, as well as the functions of the pathways in ovarian tissues involved in goat prolificacy in the follicular phase.

As a novel class of small RNAs, piRNAs are highly expressed in the animal gonads and their main known role is to inhibit transposon activity for ensuring the correctness and integrity of genome. In order to explore the characteristics of piRNAs in sheep testis and their possible regulatory roles on male reproduction, deep sequencing technology was used to sequence small RNAs and identify piRNAs in testes of sheep. The length of piRNAs in sheep testes showed a unimodal distribution between 26 and 31 nt, with a peak at 29 nt. These piRNAs exhibited obvious ping-pong signature and strand specificity. In the genome, they were mainly aligned to CDS, intron, repetitive sequence regions and unannotated regions. Furthermore, in transposon analysis, piRNAs were aligned predominantly to LINE, SINE, and LTR types of retrotransposon in sheep testes, and the piRNAs derived from each type showed obvious ping-pong signature. The piRNA clusters identified in sheep testes were mainly distributed on chromosomes 3, 7, 15, 17, 18 and 20. The results combining semen determination with pathway enrichment analysis implied that differentially expressed piRNAs between the testes of rams with different fertility might participate in spermatogenesis by regulating multiple pathways closely related to stabilization of blood-testis barrier and renewal and differentiation of spermatogonial stem cell. Taken together, the study provided new insights into the characteristics, origin and expression patterns of piRNAs in sheep testes tissue, which would help us better understand the role of piRNAs in sheep reproduction.

CircRNAs have been found to play key roles in many biological processes and have diverse biological functions. There have been studies on circRNAs in sheep pituitary, and some important circRNAs have been found. But there are still few studies on circRNAs in sheep pituitary with different fecundity. In this study, we obtained the circRNAs expression profiles in the pituitary of FecB ++ genotype Small Tail Han sheep with different fecundity and estrous phases. A total of 34,878 circRNAs were identified in 12 pituitary samples, 300 differentially expressed circRNAs (DE circRNAs) (down: 104; up: 196) were identified in polytocous sheep in the follicular phase (PF) and monotocous sheep in the follicular phase (MF) (PF vs. MF), and 347 DE circRNAs (down: 162; up: 185) were identified in polytocous sheep in the luteal phase (PL) and monotocous sheep in the luteal phase (ML) (PL vs. ML). Cortisol synthesis and secretion pathway (follicular phase) and estrogen signaling pathway (luteal phase) were obtained by functional enrichment analysis of circRNAs source genes. Competing endogenous RNA (ceRNA) network analysis of key DE circRNAs revealed that oar-circ-0022776 (source gene ITPR2, follicular phase) targeted oar-miR-432, oar-circ-0009003 (source gene ITPR1, luteal phase) and oar-circ-0003113 (source gene PLCB1, luteal phase) targeted oar-miR-370-3p. We also explored the coding ability of DE circRNAs. In conclusion, our study shows that changes in the pituitary circRNAs may be related to the response of the pituitary to steroid hormones and regulate the reproductive process of sheep by affecting the pituitary function. Results of this study provide some new information for understanding the functions of circRNAs and the fecundity of FecB ++ genotype sheep.

PIWI-interacting RNAs (piRNAs), as a novel class of small non-coding RNAs that have been shown to be indispensable in germline integrity and stem cell development. However, the expressed characteristics and regulatory roles of piRNAs during different reproductive phases of animals remain unknown. In this study, we investigated the piRNAs expression profiles in ovaries of sheep during the luteal phase (LP) and follicular phase (FP) using the Solexa sequencing technique. A total of 85,219 and 1,27,156 piRNAs tags were identified in ovine ovaries across the two phases. Most expressed piRNAs start with uracil. piRNAs with a length of 24 nt or 27–29 nts accounted for the largest proportion. The obvious ping-pong signature appeared in the FP ovary. The piRNA clusters in the sheep ovary were unevenly distributed on the chromosomes, with high density on Chr 3 and 1. For genome distribution, piRNAs in sheep ovary were mainly derived from intron, CDS, and repeat sequence regions. Compared to the LP ovary, a greater number of expressed piRNA clusters were detected in the FP ovary. Simultaneously, we identified 271 differentially expressed (DE) piRNAs between LP and FP ovaries, with 96 piRNAs upregulated and 175 piRNAs downregulated, respectively. Functional enrichment analysis (GO and KEGG) indicated that their target genes were enriched in reproduction-related pathways including oocyte meiosis, PI3K-Akt, Wnt, and TGF-β signaling pathways. Together, our results highlighted the sequence and expression characteristics of the piRNAs in the sheep ovary, which will help us understand the roles of piRNAs in the ovine estrus cycle.

The mammalian oviduct is functionally highly diverse during the estrus cycle. It provides a suitable milieu for oocyte maturation, sperm capacitation, fertilization, early embryo development and transportation. While there have been many studies of molecular mechanisms on the kidding number of goats, a systematic analysis by which the underlying circular RNAs (circRNAs) changes in the oviduct related to prolificacy traits is lacking. Herein, we present a comprehensive circRNA atlas of the oviduct among high- and low-fecundity goats in the follicular phase (FH vs. FL), luteal phase (LH vs. LL), and estrus cycle (FH vs. LH; FL vs. LL) to unravel their potential regulatory mechanisms in improving kidding number. We generated RNA sequencing data, and identified 4,078 circRNAs from twenty sampled Yunshang black goats. Many of these circRNAs are exon-derived and differentially expressed between each comparison group. Subsequently, eight differentially expressed (DE) circRNAs were validated by RT‒qPCR, which was consistent with the RNA-seq data. GO and KEGG enrichment analyses suggested that numerous host genes of DE circRNAs were involved in the hormone secretion, gamete production, fertilization, and embryo development processes. The competing endogenous RNA (ceRNA) interaction network analysis revealed that 2,673 circRNA–miRNA–mRNA axes (including 15 DE circRNAs, 14 miRNAs, and 1,699 mRNAs) were formed, and several target genes derived from the ceRNA network were associated with oviduct functions and reproduction, including SMAD1, BMPR1B, IGF1, REV1, and BMP2K. Furthermore, miR-15a-5p, miR-181b-5p, miR-23b-5p, miR-204-3p, and miR-145-5p might play important roles in reproduction. Finally, a novel circRNA, circIQCG, was identified as potentially involved in embryo development. Overall, our study provides a resource of circRNAs to understand the oviductal function and its connection to prolificacy trait of goats in the differentiation estrus cycle.

Circular RNAs (circRNAs) are non-coding RNAs newly identified and play important roles in RNA regulation. However, little is known regarding photoperiods induced circRNAs in the thyroid gland. In this study, we performed a comprehensive analysis of circRNA profiles in the thyroid gland of OVX+E2 ewes at different photoperiods by whole transcriptome sequencing. A total of 37,470 novel circRNAs were detected in different photoperiods (42 days of short photoperiod treatment, SP42; 42 days of long photoperiod treatment, LP42; SP42 transfer to LP42, SPLP42), with a total of 817 circRNAs for SP42-LP42 (down: 132; up: 114), LP42-SPLP42 (down: 136; up: 112) and SP42-SPLP42 (down: 182; up: 141) having differentially expressed. Functional enrichment annotation analysis of DE-circRNAs for GO and KEGG by R package, features that influence photoperiod response in Sunite ewes through the Inositol phosphate metabolism, cGMP-PKG signaling pathway, Calcium signaling pathway, MAPK signaling pathway, and Oocyte meiosis. In addition, competitive endogenous RNA (ceRNA) network analysis revealed target binding sites for identified miRNAs in DE-cirRNAs such as oar-miR-10b, oar-miR-200c, oar-miR-21, oar-miR-370-3p, oar-miR-377-3p, oar-miR-181a, oar-miR-432, and oar-miR-495-3p. These results of this study will provide some new information for understanding circRNA function as well as the changes in the sheep thyroid gland under different photoperiods.

Research has shown that MTNR1A plays an essential role in the estrus cycle and seasonal reproduction changes in sheep. However, few people have focused on the DNA methylation of MTNR1A by season or photoperiod. In this study, using qPCR and Western blotting, we measured the MTNR1A expression in the hypothalamus of ovariectomized and estradiol-treated (OVX + E2) sheep under different photoperiod treatment conditions. Subsequently, the core promoter of the MTNR1A gene was identified, and its methylation level in sheep exposed to different photoperiod treatments was measured by pyrosequencing. The results showed that MTNR1A gene expression significantly differed between the short 42-day photoperiod (SP42) and the LP42 or combined SP-LP42 treatment groups (p < 0.05). In addition, we determined that the core MTNR1A promoter region was within 540 bp upstream of the transcriptional start site (TSS) and that the DNA methylation levels at CpG sites in the SP42 vs. LP42 and SP42 vs. SP-LP42 groups significantly differed. Finally, a significant negative correlation (p < 0.001) between gene expression and DNA methylation levels was revealed, suggesting that photoperiod may induce DNA methylation of the MTNR1A gene and thus change its expression. The findings provide valuable bases for the further study of seasonal reproduction in sheep.

Abstract Background JUNO and IZUMO1 are the first receptor-ligand protein pairs discovered to be essential for sperm-oocyte fusion; their interaction is indispensable for fertilization. Methods PCR was used to clone the full-length DNA sequence of the Juno gene in sheep. The single nucleotide polymorphism (SNP) loci of Juno were genotyped by Sequenom MassARRAY®. PCR combined with rapid amplification of cDNA Ends were used to clone the full-length cDNA sequence of Juno and Izumo1. Reverse transcriptase-PCR (RT-PCR) and real time-quantitative-PCR (RT-qPCR) were used to analyze the genes’ expression in tissues of sheep, and single cell RNA-seq was used to analyze the genes’ expression in oocytes, granulosa cells and follicular theca of polytocous and monotocous Small Tail Han ewes. Bioinformatics was used to analyze advanced structure and phylogeny of JUNO and IZUMO1 proteins. Results The full-length DNA sequence of the Juno gene in sheep was cloned and nine SNPs were screened. We found a significant association between the g.848253 C > A locus of Juno and litter size of Small Tail Han sheep (P A locus of Juno results in a nonconservative missense point mutation leading to a change from Phe to Leu at position 219 in the amino acid sequence. Conclusions For the first time, this study systematically analyzed the expression, structure and function of Juno and Izumo1 genes and their encoded proteins in Small Tail Han sheep, providing the basis for future studies of the regulatory mechanisms of Juno and Izumo1 genes.

In seasonal estrus, it is well known that melatonin-regulated biorhythm plays a key role. Some studies indicate that the adrenal gland plays an important role in reproduction in mammals, but the molecular mechanism is not clear. This study used an artificially controlled light photoperiod model, combined with RNA-seq technology and bioinformatics analysis, to analyze the messenger RNA (mRNA) and microRNA (miRNA) of ewe (Sunite) adrenal glands under different photoperiod treatments. After identification, the key candidate genes GRHL2, CENPF, FGF16 and SLC25A30 that photoperiod affects reproduction were confirmed. The miRNAs (oar-miR-544-3p, oar-miR-411b-5p, oar-miR-376e-3p, oar-miR-376d, oar-miR-376b-3p, oar-miR-376a-3p) were specifically expressed in the adrenal gland. The candidate mRNA-miRNA pairs (e.g., SLC25A30 coagulated by novel miRNA554, novel miRNA555 and novel miRNA559) may affect seasonal estrus. In summary, we constructed relation network of the mRNAs and miRNAs of sheep adrenal glands using RNA sequencing and bioinformatics analysis, thereby, providing a valuable genetic variation resource for sheep genome research, which will contribute to the study of complex traits in sheep.

IGF1, a member of the insulin-like growth factor (IGF) superfamily, is also known as the growth-promoting factor (somatomedin C). IGF1 is involved in vertebrate growth and development, immunity, cell metabolism, reproduction, and breeding. However, there are relatively few studies on the relationship between IGF1 and goat reproduction. In this study, a new transcription factor SP1 bound to the IGF1 g. 64943050T>C promoted granulosa cell (GC) proliferation. A mutation g.64943050T>C located in the promoter region of IGF1 was identified. Association analysis revealed that the kidding number in the first and second litters and the average number of first three litters of the CC genotype (2.206 ± 0.044, 2.254 ± 0.056, and 2.251 ± 0.031) were significantly higher than those in the TC genotype (1.832 ± 0.049, 1.982 ± 0.06, and 1.921 ± 0.034) and TT genotype (1.860 ± 0.090, 1.968 ± 0.117, and 1.924 ± 0.062) (p < 0.05). The kidding number in the third litter of the CC genotype (2.355 ± 0.057) was significantly higher than that in the TT genotype (2.000 ± 0.107) (p < 0.05). Then, the function of this mutation was validated by the dual-luciferase reporter assay and EMSA. The results showed that the luciferase activity of IGF1-mutant-C was significantly higher than that of IGF1-Wild-T (p < 0.05). The EMSA also showed that the binding ability of IGF1-mutant-C was higher than that of IGF1-Wild-T (p < 0.05). Subsequently, the transcription factor SP1 was predicted to bind to the mutation of IGF1 (g.64943050T>C). Overexpression of SP1 promotes the expression of IGF1 in the primary granulosa cells (GCs). The results of the CCK-8 assay and the expression of GC proliferation factors (CDK4, cyclin D1, and cyclin D2) demonstrated that SP1 promoted GC proliferation by regulating IGF1 expression. Our results suggested that the IGF1 g.64943050T>C was significantly associated with the kidding number of Yunshang black goats, and SP1 as a transcription factor of IGF1 binding to the mutation T>C regulated the expression of IGF1. Furthermore, SP1 promoted goat GC proliferation by regulating the expression of IGF1, which provides a new insight for the goat fertility trait.

The granulosa cell (GC) is the basic functional unit of follicles, and it is important for promoting follicle growth and sex hormones, as well as growth factor secretion in the process of reproduction. A variety of factors influence granulocyte proliferation, yet there are still many gaps to be filled in target and non-coding RNA regulation. In our study, the differentially expressed (DE) mRNAs and miRNAs were detected by using RNA-seq, and we constructed a mRNA–miRNA network related to goat prolificacy. Then, the goat primary GCs were isolated from the follicle for the function validation of candidate genes and their regulator miRNAs. A total of 2,968 DE mRNAs and 99 DE miRNAs were identified in the high- and low-prolificacy goat by RNA-seq, of which there were 1,553 upregulated and 1,415 downregulated mRNAs, and 80 upregulated and 19 downregulated miRNAs, respectively. JAK3 was identified as highly expressed in the low-prolificacy goats (3 times higher than high-prolificacy goats), and the integrated analysis showed that chi-miR-493-3p was a potential regulator of JAK3. The analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) showed that JAK3 was involved in the PI3K-Akt signaling pathway, the Jak-STAT signaling pathway, and signaling pathways regulating pluripotency of stem cells. In particular, the PI3K-Akt signaling pathway was a typical pathway for cell proliferation, differentiation, apoptosis, and migration. We found that the chi-miR-493-3p targets JAK3 directly via RT-qPCR, dual fluorescence assays, and Western blot. Furthermore, the expression of JAK3 was significantly decreased by the chi-miR-493-3p mimic and increased by the chi-miR-493-3p inhibitor. The CCK-8 assay showed that overexpression of JAK3 promoted cell proliferation, while inhibiting JAK3 had the opposite effect. The expression of cell proliferation markers CDK4 and cyclin D2 also showed the same results. Moreover, the enzyme-linked immunosorbent assay showed that steroid hormones E2 and PROG were increased by overexpressing JAK3 and decreased by inhibiting JAK3. Therefore, our results identified a chi-miR-439-3p-JAK3 regulatory pathway, which provided a new insight into the GC proliferation and prolificacy of goat.

Intense artificial selection has been imposed to Luzhong mutton sheep population in the past years. Improvements on growth and reproductive performance are two breeding goals in the present herd. Although some progresses were phenotypically observed possibly due to inbreeding induced by strong selection in terms of these traits, the genomic evaluation was poorly understood. Therefore, a high-density SNP array was used to characterize the pattern of runs of homozygosity (ROH), estimate inbreeding and inbreeding depressions on early growth performance and litter size based upon ROH, and scan positive selection signatures of recent population. Consequently, a low inbreeding level was observed which had negative effects on litter size, but not on early growth performance. And 160 genes were under selection, of which some were reported to be linked to several traits of sheep including body weight, litter size, carcass and meat quality, milk yield and composition, fiber quality and health, and the top genes were associated with growth (growth hormone [GH]- growth hormone receptor [GHR]- Insulin-like growth factor 1 [IGF1] axis) and litter size (bone morphogenic proteins [BMPs]-associated). The effectiveness of previous breeding measures was highlighted, but purging selection was proposed to alleviate the inbreeding depression on litter size, providing some genomic insights to breeding management of Luzhong mutton sheep.

Abstract Background Long non-coding RNAs (lncRNAs) can play important roles in uterine and ovarian functions. However, little researches have been done on the role of lncRNAs in the adrenal gland of sheep. Herein, RNA sequencing was used to compare and analyze gene expressions in adrenal tissues between follicular phases and luteal phases in FecB BB (MM) and FecB ++ (WW) sheep, respectively, and differentially expressed lncRNAs and genes associated with reproduction were identified. Results In MM sheep, 38 lncRNAs and 545 mRNAs were differentially expressed in the adrenal gland between the luteal and follicular phases; In WW sheep, 513 differentially expressed lncRNAs and 2481 mRNAs were identified. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses indicated that differentially expressed lncRNAs and their target genes are mainly involved in the circadian rhythm, the mitogen activated protein kinase, thyroid, ovarian steroidogenesis and transforming growth factor beta signaling pathways. Differentially expressed lncRNAs can regulate reproduction by modulating genes involved in these signaling pathways and biological processes. Specifically, XLOC_254761, XLOC_357966, 105,614,839 and XLOC_212877 targeting CREB1, PER3, SMAD1 and TGFBR2, respectively, appear to play key regulatory roles. Conclusion These results broaden our understanding of lncRNAs in adrenal gland of sheep and provide new insights into the molecular mechanisms underlying sheep reproduction.

Abstract Background Circular RNA (circRNA) is produced during the splicing of mRNA (in addition to linear splicing) and is part of the gene regulatory network. The temporal expression patterns the different developmental stages were inseparable from these molecules’ function. Results Skeletal muscles of Anhui white goat (AWG) across seven fetal to postnatal development stages were sequenced and 21 RNA sequencing libraries were constructed. We thereby identified 9090 circRNAs and analyzed their molecular properties, temporal expression patterns, and potential functions at the different stages. CircRNAs showed complexities and diversity of formation as the same host gene produces multiple isoforms of these nucleic acids with different expression profiles. The differential expression of 2881 circRNAs (DECs, P 2 and P-adj value

The piRNA-PIWI protein complex plays crucial roles in safeguarding the genome against inordinate transposon mobilization and regulation of embryonic development. A previous study indicated the presence of piRNA in sheep reproductive organs. However, the tissue distribution and cellular localization of PIWI proteins in sheep remains unclear. Therefore the present study aimed to explore the expression profiles of mRNAs of mammalian PIWI proteins (PIWIL1, PIWIL2, PIWIL4 and AGO3) in 9 tissues derived from adult male and female sheep. Results showed the expression of PIWIL1, PIWIL2, and PIWIL4 was significantly higher in the testis and ovary than in the other tissues. Immunohistochemistry analysis of testes indicated that each of the 4 proteins had specific cellular localizations, and some of the localizations were different from those of other species. All the proteins were mainly localized in the primary spermatocytes, suggesting that they are crucial for silencing of transposon to guarantee the integrity of the gamete genome during early stage of spermatogenesis. In the ovaries, the PIWI proteins were mainly localized in oocytes from antral follicles and leukocytes in ovarian blood. Our results provide insights to better understand the functions of PIWI proteins during spermatogenesis, oogenesis and immune defence in sheep.

Accurate timing of seasonal changes is an essential ability for an animal’s survival, and the change in the photoperiod is the key factor affecting reproductive seasonality in mammals. Emerging evidence has suggested that multiple hypothalamic genes participate in the photoperiod-induced regulation of reproductive activities in sheep, but the mechanism is still unclear. In this study, we initially examined the plasma level of two major reproductive hormones, namely, follicle-stimulating hormone (FSH) and prolactin (PRL), under different photoperiods in ovariectomized and estradiol-treated (OVX + E2) sheep using radioimmunoassay (RIA). Of the two hormones, the concentration of PRL significantly increased with the extension of the photoperiod, while FSH showed the opposite trend. Subsequently, an examination of the transcriptomic variation between the short photoperiod (SP) and long photoperiod (LP) was conducted. Differential expression analyses and functional annotation showed that several key genes in the insulin secretion (VAMP2, PRKACB, PRKCG, and PLCB1), GnRH (MAPK13, CGA, CDC42, ATF4, and LHB) pathways, and circadian entrainment (KCNJ5, PER1, GNB2, MTNR1A, and RASD1), as well as numerous lncRNAs, including XR_173257.3, XR_173415.3, XR_001435315.1, XR_001024596.2, and XR_001023464.2, were shown potentially vital for the hypothalamic photoperiodic response. Four of the differentially expressed mRNAs and lncRNAs were validated by qPCR. The constructed mRNA–mRNA interaction networks further revealed that transcripts potentially participated in hypothalamic thyroid hormone synthesis, endocrine resistance, and neuroactive ligand–receptor interactions. The interactome analysis of lncRNAs and their targets implied that XR_173257.3 and its target arylalkylamine N-acetyltransferase (AANAT) and XR_173415.3 and its target TH might participate in the regulation of seasonal reproduction. Together, the changes in reproductive hormones and transcriptome will help to determine the important photoperiod-induced lncRNAs and mRNAs and provide a valuable resource for further research on reproductive seasonality in sheep.

The hypothalamic–pituitary–adrenal (HPA) axis plays an important role in the growth and development of mammals. Recently, lncRNA transcripts have emerged as an area of importance in sheep photoperiod and seasonal estrus studies. This research aims to identify lncRNA and mRNA that are differentially expressed in the sheep adrenal gland in long (LP) or short (SP) photoperiods using transcriptome sequencing and bioinformatics analysis based on the OVX + E2 (Bilateral ovariectomy and estradiol-implanted) model. We found significant differences in the expression of lncRNAs in LP42 (where LP is for 42 days) vs. SP-LP42 (where SP is for 42 days followed by LP for 42 days) (n = 304), SP42 (where SP is for 42 days) vs. SP-LP42 (n = 1,110) and SP42 vs. LP42 (n = 928). Cluster analysis and enrichment analysis identified SP42 vs. LP42 as a comparable group of interest and found the following candidate genes related to reproductive phenotype: FGF16, PLGF, CDKN1A, SEMA7A, EDG1, CACNA1C and ADCY5. FGF16 (Up-regulated lncRNA MSTRG.242136 and MSTRG.236582) is the only up-regulated gene that is closely related to oocyte maturation. However, EDG1 (Down-regulated lncRNA MSTRG.43609) and CACNA1C may be related to precocious puberty in sheep. PLGF (Down-regulated lncRNA MSTRG.146618 and MSTRG.247208) and CDKN1A (Up-regulated lncRNA MSTRG.203610 and MSTRG.129663) are involved in the growth and differentiation of placental and retinal vessels, and SEMA7A (Up-regulated lncRNA MSTRG.250579) is essential for the development of gonadotropin-releasing hormone (GnRH) neurons. These results identify novel candidate genes that may regulate sheep seasonality and may lead to new methods for the management of sheep reproduction. This study provides a basis for further explanation of the basic molecular mechanism of the adrenal gland, but also provides a new idea for a comprehensive understanding of seasonal estrus characteristics in Sunite sheep.

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.

CircRNAs acting as miRNA sponges play important roles in the growth process of animal individuals. The prolificacy trait of goats is involved in many pathways, however, the variation of circRNA expression profiles in the different phases of the estrus cycle at high and low fecundity groups is still unknown. Here, we analyzed the circRNA profiles of ovarian tissues among high and low fecundity groups in the follicular phase (HF vs LF), high and low fecundity groups in the luteal phase (HL vs LL), and high and low fecundity in the whole estrus cycle (HF vs HL and LF vs LL) using RNA-seq. A total of 283 (114 upregulated and 169 downregulated), 559 (299 upregulated and 260 downregulated), 449 (254 upregulated and 195 downregulated), and 314 (210 upregulated and 104 downregulated) differentially expressed (DE) circRNAs were screened in HF vs LF, HF vs HL, HL vs LL, and LF vs LL groups, respectively. Enrichment analysis suggested that the targeting of DE circRNAs was mainly enriched in oocyte meiosis, the GnRH signaling pathway, and estrogen signaling pathway. After integrating our previous study of miRNA-seq, there were 56 miRNAs that could target to 192 DE circRNAs, including the miR-133 family (including miR-133a-3p and miR-133b), miR-129-3p, and miR-21, which also had important influence on the prolificacy trait of goats. Then, 18 circRNAs with coding potential were obtained by four software predictions, and 9 circRNAs were validated by RT-qPCR. Together, circRNAs play a key role in the prolificacy trait and the transformation of the follicular phase to the luteal phase in the estrus cycle of goats.

The pituitary is a remarkably dynamic organ with roles in hormone (FSH and LH) synthesis and secretion. In animals with the FecB (fecundity Booroola) mutation, the pituitary experiences hormone fluctuations during the follicular–luteal transition, which is implicated in the expression and regulation of many genes and regulators. Long non-coding RNAs (lncRNAs) are a novel type of regulatory factors for the reproductive process. Nevertheless, the expression patterns of lncRNAs and their roles in FecB-mediated follicular development and ovulation remain obscure. Thus, we profiled the pituitary transcriptome during the follicular (F, 45 h after evacuation vaginal sponges) and luteal (L, 216 h after evacuation vaginal sponges) phases in FecB-mutant homozygous (BB) and wild-type (WW) Small Tail Han sheep. We identified 78 differentially expressed genes (DEGs) and 41 differentially expressed lncRNAs (DELs) between BB_F and BB_L, 32 DEGs and 26 DELs between BB_F and WW_F, 16 DEGs and 29 DELs between BB_L and WW_L, and 50 DEGs and 18 DELs between WW_F and WW_L. The results of real-time quantitative PCR (RT-qPCR) correlated well with the transcriptome data. In both the follicular and luteal phases, DEGs (GRID2, glutamate ionotropic receptor delta type subunit 2; ST14, ST14 transmembrane serine protease matriptase) were enriched in hormone synthesis, secretion, and action. MSTRG.47470 and MSTRG.101530 were the trans-regulated elements of ID1 (inhibitor of DNA binding 3, HLH protein) and the DEG ID3 (inhibitor of DNA binding 3, HLH protein), and EEF2 (eukaryotic translation elongation factor 2), respectively; these factors might be involved in melatonin and peptide hormone secretion. In the FecB-mediated follicular phase, MSTRG.125392 targeted seizure-related 6 homolog like (SEZ6L), and MSTRG.125394 and MSTRG.83276 targeted the DEG KCNQ3 (potassium voltage-gated channel subfamily Q member 3) in cis, while MSTRG.55861 targeted FKBP4 (FKBP prolyl isomerase 4) in trans. In the FecB-mediated luteal phase, LOC105613905, MSTRG.81536, and MSTRG.150434 modulated TGFB1, SMAD3, OXT, respectively, in trans. We postulated that the FecB mutation in pituitary tissue elevated the expression of certain genes associated with pituitary development and hormone secretion. Furthermore, this study provides new insights into how the pituitary regulates follicular development and ovulation, illustrated by the effect of the FecB mutation.

Pituitary pars tuberalis (PT) plays an important role as the transmission center in the seasonal reproduction of animals. It helps convert external photoperiod signals into intrinsic seasonal reproduction signals. In sheep PT, specific expression patterns of several genes (including short photoperiod-induced gene CHGA and long photoperiod genes EYA3 and TSHβ) under different photoperiods are crucial characteristics during this signal transduction. Recent studies have revealed the role of epigenetics in regulating the expression of seasonal reproductive key genes. Therefore, we explored whether microRNAs and LncRNAs regulated the expressions of the above key genes. Firstly, the expression of miR-25 and CHGA showed a significant negative correlation in sheep PT. Results of the dual luciferase reporter assay and miR-25 overexpression indicated that miR-25 could inhibit the expression of CHGA by specifically binding to its 3′UTR region in pituitary cells. Then, expression negative correlation and dual luciferase reporter analyses were used to screen and identify the candidate LncRNA (Lnc107153) targeted by miR-25. Finally, the results of fluorescence in situ hybridization and Lnc107153 overexpression suggested that Lnc107153 and miR-25 were involved in the epigenetic regulation of CHGA expression. However, the expressions of EYA3 and TSHβ were not regulated by miRNAs. These results will provide new insights into the epigenetic regulatory network of key genes in sheep seasonal reproduction.

The pituitary gland is an important organ. It is a complex area of the brain involved in endocrine function and reproductive regulation. However, the function of the pituitary in goat reproduction is still unclear. Herein, RNA sequencing was used to explore the expression patterns of circle RNAs (circRNAs) in the pituitary of Yunshang black goats during the various estrus phases. Then the host genes of the circRNAs were predicted, and a competing endogenous RNA (ceRNA) network was constructed. The results showed a total of 6,705 circRNAs in the pituitary of Yunshang black goats, among which 388 differentially expressed (DE) circRNAs (214 were upregulated, while 174 were downregulated) were identified between high- and low-yield Yunshang black goats in the follicular phase (HF vs. LF); moreover, 361 DE circRNAs (136 were upregulated, while 225 were downregulated) were identified between high- and low-yield Yunshang black goats in the luteal phase (HL vs. LL). There were 65 DE circRNAs targeting 40 miRNAs in the HF vs. LF comparison and 46 DE circRNAs targeting 31 miRNAs in the HL vs. LL comparison. We identified chi_circ_0030920, chi_circ_0043017, chi_circ_0008353, chi_circ_0041580, and chi_circ_0016478 as the key circRNAs through functional enrichment analysis. The ceRNA network analysis showed that chi_circ_0031209 and chi_circ_0019448 might play an important role in reproduction by influencing the expression of prolactin receptor (PRLR) in high- and low-yielding goats during the luteal phase, whereas chi_circ_0014542 regulates the expression of WNT5A during the follicular phase. Our study provided the overall expression profiles of circRNAs in the goat pituitary during the estrus phase, which provides new insight into the mechanism of high-yield goats, which can be helpful to guide goat breeding.

The Booroola fecundity gene (FecB) has a mutation that was found to increase the ovulation rate and litter size in Booroola Merino sheep. This mutation is also associated with the fecundity of small-tail han (STH) sheep, an important maternal breed used to produce hybrid offspring for mutton production in China. Previous research showed that the FecB gene affects reproduction in STH sheep, based on litter size records. However, the effects of this gene on estrus, ovulation, and endocrine characteristics in these sheep remain unclear. Here, we analyzed the traits mentioned earlier and compared them among the three FecB genotypes of STH ewes using estrus synchronization. Overall, 53 pluriparous ewes were selected from among 890 STH ewes and subjected to FecB genotyping for experiments to characterize estrous and ovulation rates. FecB heterozygous (+B) ewes presented an earlier onset of estrus (42.9 ± 2.2 h) and a shorter estrous cycle (17.2 ± 0.2 days) (P ≤ 0.05). The ovulation rates increased with the increasing copy number of the B allele (P ≤ 0.01). Ovulation time showed no significant differences among the three FecB genotypes. The serum concentrations of follicle-stimulating hormone (FSH), luteinizing hormone, estrogen (E2), and progesterone (P4) were measured in 19 of the ewes. Serum concentrations of E2 and FSH dramatically varied around the time of behavioral estrus. In FecB mutant homozygous (BB) ewes, E2 concentration had two peaks, which were higher (P ≤ 0.05) than those of ++ genotypes. FSH concentration of BB ewes was higher (P ≤ 0.05) than that of the ++ ewes just after estrus. The expression of the estrogen receptor 1 (ESR1) gene in the +B genotype was higher than in the other genotypes. Based on the data for the reproductive performance of STH ewes with the three FecB genotypes, our study suggests that the development of follicles in ewes with the B allele is dependent on the response to FSH regulated by E2 in the early stage. +B ewes, exhibiting moderate ovulation and litter size and a shorter estrous cycle, can be highly recommended in sheep crossbreeding systems for commercial mutton production. Moreover, this study provides useful information to conserve better and use the genetic resources of STH sheep in China.

The hypothalamus was the coordination center of the endocrine system, which played an important role in goat reproduction. However, the molecular mechanism of hypothalamus regulating litter size in goats was still poorly understood. This study aims to investigate the key functional genes associated with prolificacy by hypothalamus transcriptome analysis of goats. In this research, an integrated analysis of microRNAs (miRNAs)-mRNA was conducted using the hypothalamic tissue of Yunshang black goats in the follicular stage. A total of 72,220 transcripts were detected in RNA-seq. Besides, 1,836 differentially expressed genes (DEGs) were identified between high fecundity goats at the follicular phase (FP-HY) and low fecundity goats at the follicular phase (FP-LY). DEGs were significantly enriched in 71 Gene Ontology (GO) terms and 8 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The transcriptome data suggested that DEGs such as BMPR1B, FGFR1, IGF1 and CREB1 are directly or indirectly involved in many processes like hypothalamic gonadal hormone secretion. The miRNA-seq identified 1,837 miRNAs, of which 28 differentially expressed miRNAs (DEMs). These DEMs may affect the nerve cells survival of goat hypothalamic regulating the function of target genes and further affect the hormone secretion activities related to reproduction. They were enriched in prolactin signaling pathway, Jak-STAT signaling pathway and GnRH signaling pathway, as well as various metabolic pathways. Integrated analysis of DEMs and DEGs showed that 87 DEGs were potential target genes of 28 DEMs. After constructing a miRNA-mRNA pathway network, we identified several mRNA-miRNAs pairs by functional enrichment analysis, which was involved in hypothalamic nerve apoptosis. For example, NTRK3 was co-regulated by Novel-1187 and Novel-566, as well as another target PPP1R13L regulated by Novel-566. These results indicated that these key genes and miRNAs may play an important role in the development of goat hypothalamus and represent candidate targets for further research. This study provides a basis for further explanation of the basic molecular mechanism of hypothalamus, but also provides a new idea for a comprehensive understanding of prolificacy characteristics in Yunshang black goats.

Granulosa cell (GC) proliferation provides essential conditions for ovulation in animals. A previous study showed that DENND1A plays a significant role in polycystic ovary syndrome. However, the modulation of DENND1A in GCs remains unclear. Our previous integrated analysis of miRNA–mRNA revealed that the 3'-untranslated region of DENND1A could be a target of chi-miR-324-3p. In this study, we used quantitative reverse transcription polymerase chain reaction (RT-qPCR) to investigate DENND1A expression in ovarian tissues of high- and low-yielding goats. Furthermore, dual-fluorescent reporter vector experiments, Cell Counting Kit-8 (CCK-8) assay, and RT-qPCR were used to elucidate the regulatory pathway of chi-miR-324-3p-DENND1A in GCs. The results revealed an opposite tendency between the expressions of chi-miR-324-3p and DENND1A in the ovaries of high- and low-yielding goats. The CCK-8 assay indicated that chi-miR-324-3p overexpression significantly suppressed GC proliferation, whereas chi-miR-324-3p inhibition promoted GC proliferation. In addition, the expressions of GC proliferation markers LHR, Cylin D2, and CDK4 showed the same tendency. The dual-fluorescent reporter assay revealed that chi-miR-324-3p directly targeted DENND1A, and the RT-qPCR results revealed that DENND1A expression was inhibited by chi-miR-324-3p. In summary, chi-miR-324-3p inhibited the proliferation of GCs by targeting DENND1A.

A normal estrus cycle is essential for the breeding of goats, and the luteal phase accounts for most of the estrus cycle. The corpus luteum (CL) formed during the luteal phase is a transient endocrine gland that is crucial for the reproductive cycle and pregnancy maintenance, and is controlled by many regulatory factors. However, the molecular mechanism of the hypothalamus effect on the reproductive performance of different litter sizes during the luteal phase of goats has not been elucidated. In this study, RNA-sequencing was used to analyze the mRNA and miRNA expression profiles of the hypothalamic tissues with the high-fecundity goats during the luteal phase (LP-HF) and low-fecundity goats during the luteal phase (LP-LF). The RNA-seq results found that there were 1963 differentially expressed genes (DEGs) (890 up-regulated and 1073 down-regulated). The miRNA-seq identified 57 differentially expressed miRNAs (DEMs), including 11 up-regulated and 46 down-regulated, of which 199 DEGs were predicted to be potential target genes of DEMs. Meanwhile, the functional enrichment analysis identified several mRNA-miRNA pairs involved in the regulation of the hypothalamic activity, such as the common target gene MEA1 of novel-miR-972, novel-miR-125 and novel-miR-403, which can play a certain role as a related gene of the reproductive development in the hypothalamic–pituitary–gonadal (HPG) axis and its regulated network, by regulating the androgen secretion. While another target gene ADIPOR2 of the novel-miR-403, is distributed in the hypothalamus and affects the reproductive system through a central role on the HPG axis and a peripheral role in the gonadal tissue. An annotation analysis of the DE miRNA-mRNA pairs identified targets related to biological processes, such as anion binding (GO:0043168) and small molecule binding (GO: 0036094). Subsequently, the KEGG(Kyoto Encyclopedia of Genes and Genomes) pathways were performed to analyze the miRNA-mRNA pairs with negatively correlated miRNAs. We found that the GnRH signaling pathway (ko04912), the estrogen signaling pathway (ko04915), the Fc gamma R-mediated phagocytosis (ko04666), and the IL-17 signaling pathway (ko04657), etc., were directly and indirectly associated with the reproductive process. These targeting interactions may be closely related to the reproductive performance of goats. The results of this study provide a reference for further research on the molecular regulation mechanism for the high fertility in goats.

The litter size and ovulation rate are different among ewes of different FecB genotypes in Small Tail Han sheep. These variants in reproductive phenotypes may be regulated by hormones released by the hypothalamic–pituitary–ovarian axis. However, there have been few reports on the hypothalamus regarding regulating an increase in ovulation in sheep with FecB mutation at different estrous stages. Thus, we examined the abundance of hypothalamus tissue protein profiles of six FecB mutant homozygous (BB) and six wild-type (WW) ewes at the luteal and follicular phases. We determined this abundance by tandem mass tag-based quantitative analysis and parallel reaction monitoring methods. Furthermore, an integrated proteotranscriptomic analysis was performed by the Data Integration Analysis for Biomarker discovery using the latent variable approaches for Omics studies (DIABLO) framework to examine biological processes and pathway alterations by the FecB mutant. The abundance of 154 proteins was different between the two estrous stages. Growth hormone and prolactin were particularly enriched in the neuroactive ligand–receptor interactions, the prolactin signaling pathway, and the PI3K-Akt signaling pathway which are related to hypothalamic function and reproduction. We combined proteome and transcriptome data from different estrous stages and genotypes. There is a high correlation (Pearson correlation coefficient = 0.99) between the two datasets in the first two components. We applied the traditional single-omic multivariate approach to obtain differentially abundant proteins and differentially expressed genes. The major fertility related biomarkers were selected using the two approaches mentioned above. Several key pathways (GABAergic synapse, neuroactive ligand–receptor interaction, estrogen and MAPK signaling pathways) were enriched, which are central to gonadotrophin-releasing hormone (GnRH) secretion and reproduction. A higher level of gamma-aminobutyric acid type A receptor subunit alpha1 (GABRA1) and gamma-aminobutyric acid type A receptor subunit beta2 (GABRB2) expression was observed in BB ewes as compared to WW ewes. This finding suggested that a greater production of GnRH during follicular development in BB ewes may explain the higher mature follicle number in mutant ewes. FKBP prolyl isomerase 1A (FKBP1A), which was a major feature factor in the proteome selected by DIABLO, was an important switch for activating the transforming growth factor beta (TGFβ) pathway, and its expression was higher in the WW ewes than in the BB ewes. We suggest that BB sheep maintain TGFβ pathway activity by reducing FKBP1A protein abundance. This innovative data integration in the hypothalamus may provide fresh insight into the mechanisms by which the FecB mutation affects sheep fertility, while providing novel biomarkers related to reproductive endocrinology in sheep breeding.

The pituitary pars tuberalis (PT) is the regulating center of seasonal reproduction, which can sense the melatonin signal and eventually cause downstream changes of GnRH secretion through TSHβ. Recently, lncRNAs have been identified in animal reproductive-related tissues, and they play important roles in reproductive regulation. Therefore, in this study, we expect to identify photoperiod-induced lncRNAs and genes in pituitary PT of sheep by comparison of expression profiles between short photoperiod (SP) and long photoperiod (LP). Through RNA-Seq, a total of 55,472 lncRNAs were identified in pituitary PT of Sunite ewes. The number of differentially expressed (DE) genes and lncRNAs between SP and LP increased gradually with the extension of LP (from LP7 to LP42). The notable LP-induced candidate genes included EYA3, TSHB, SIX1, DCT, VMO1, AREG, SUV39H2, and EZH2, and SP-induced genes involved ENSOARG00000012585, CHGA, FOS, SOCS3, and TH. In enriched pathways for DE genes and lncRNA target genes between SP and LP, the reproduction- and circadian-related pathways were highlighted. In addition, the interactome analysis of lncRNAs and their targets implied that MSTRG.209166 and its trans-target TSHB, MSTRG.288068 and its cis-target SIX1, and ENSOARG00000026131 and its cis-target TH might participate in regulation of seasonal reproduction. Together, these results will help to determine important photoperiod-induced lncRNAs and genes and give us some new insights into the epigenetic regulation of seasonal reproduction in sheep.