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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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Chen, Guo, He, Di, Zhang, Zhang, Wang and Chu.)

Follistatin-like 3 (FSTL3) is a regulator of cellular apoptosis and was previously identified via RNA-Seq to be associated with follicular development in mammalian ovaries. However, the mechanism underlying the FSTL3 regulation of oestrus in sheep remained poorly understood. In this study, the oestrogen (E2) and progesterone (P4) concentrations in blood were detected, and the expression level and functional analysis of FSTL3 in the ovary were studied during the different reproductive stage in Aohan fine wool sheep (seasonal breeding breed in China). The concentrations of E2 and P4 at the anestrus were significantly lower compared to dioestrus, proestrus and oestrus stages. Higher expression levels of FSTL3 were observed in the sheep ovary, hypothalamus, and thyroid. During different reproductive stages, higher expression levels were found during the stages of dioestrus and proestrus, while lower levels were found during the oestrus and anestrus stages. Functional analysis of FSTL3 was performed in primary granulosa cells (GCs) of sheep. The concentration of E2 increased significantly after RNAi interference of FSTL3, while the P4 level decreased. FSTL3 can decrease P4 levels, which might be involved in mediating oestrous cycle in sheep., (© 2020 The Authors. Reproduction in Domestic Animals published by Wiley-VCH GmbH.)

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.

To investigate the genetic mechanism of sheep prolificacy, protein profiling of ovaries in the follicular and luteal phases was conducted. The tandem mass tag technique was used to analyze the proteomes of ovaries from STH sheep that did not have the FecB mutation in the bone morphogenetic protein receptor 1B gene. Parallel Reaction Monitoring (PRM) was operated to validate the target differentially abundant proteins (DAPs). The result showed, a total of 34,037 peptides were found, and 5074 proteins were identified. The screened DAPs strictly related to energy metabolism, hormone synthesis, ovarian function were significantly enriched in oxidative phosphorylation(COX7A, ND5, and UQCR10), ovarian steroidogenesis(StAR and HSD3B), taurine and hypotaurine metabolism(CSAD), glycosaminoglycan biosynthesis-heparin sulfate/heparin(GLCE), necroptosis(H2AX, AIFM1, and FTH1), protein digestion and absorption(COL4A1 and COL4A5) and glycosaminoglycan degradation(HYAL2 and HEXB) pathways. These analyses indicated that the reproductive performance of sheep is regulated through different pathways. In consequence, these findings are an important resource that can be used in future studies of the genetic mechanism of high fecundity traits in sheep, and these DAPs can be further investigated as candidate markers to predict prolificacy of sheep. SIGNIFICANCE: Litter size is an important quantitative trait, but the genetic mechanism of high-prolificacy is still unclear in sheep. Our study identified potential signaling pathways and differentially abundant proteins related to reproductive performance. These findings will facilitate a better revealing the mechanism and provide possible targets for molecular design breeding for the formation of polytocous traits in sheep., (Copyright © 2019. Published by Elsevier B.V.)

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., (Copyright © 2019. Published by Elsevier B.V.)

Background: Sheep have developed the ability to store fat in their tails, which is a unique way of reserving energy to survive a harsh environment. However, the mechanism underlying this adaptive trait remains largely unsolved., Results: In the present study, we provide evidence for the genetic determinants of fat tails, based on whole genome sequences of 89 individual sheep. A genome-wide scan of selective sweep identified several candidate loci including a region at chromosome 13, a haplotype of which underwent rapid evolution and spread through fat-tailed populations in China and the Middle East. Sequence analysis revealed an inter-genic origin of this locus, which later became a hotspot of ruminant-specific retro-transposon named BovB. Additionally, the candidate locus was validated based on a fat- and thin-tailed cross population. The expression of an upstream gene BMP2 was differentially regulated between fat-tailed and thin-tailed individuals in tail adipose and several other tissue types., Conclusions: Our findings suggest the fixation of fat tails in domestic sheep is caused by a selective sweep near a retro-transposable hotspot at chromosome 13, the diversity of which specifically affects the expression of BMP2. The present study has shed light onto the understanding of fat metabolism.

Seasonal breeding is an important factor limiting sheep production efficiency. Detailed analysis on the molecular mechanisms of seasonal breeding is the premise for improving estrus and mating rate of sheep during anestrus. Recent research showed that under long-photoperiod and short-photoperiod conditions, a series of changes in signaling molecules and cell morphology could be observed in ovine seasonal reproduction pathway. Based on the molecular mechanisms of seasonal reproduction, several technologies or methods for inducing estrus and mating of ewes in anestrus have been developed. In this review, photoperiod-induced changes in signaling molecules and cell morphology in pituitary and hypothalamic tissue are first summarized in terms of the molecular mechanisms and characteristics of seasonal reproduction. The application effect, advantages and disadvantages for applying these technologies for inducing estrus and mating of ewes in anestrus are then discussed, thereby providing the critical insights in identifying a new technology, which is environmentally friendly and efficient, to improve breeding rate in anestrus.

Background: Animal domestication has been extensively studied, but the process of feralization remains poorly understood., Results: Here, we performed whole-genome sequencing of 99 sheep and identified a primary genetic divergence between 2 heterogeneous populations in the Tibetan Plateau, including 1 semi-feral lineage. Selective sweep and candidate gene analysis revealed local adaptations of these sheep associated with sensory perception, muscle strength, eating habit, mating process, and aggressive behavior. In particular, a horn-related gene, RXFP2, showed signs of rapid evolution specifically in the semi-feral breeds. A unique haplotype and repressed horn-related tissue expression of RXFP2 were correlated with higher horn length, as well as spiral and horizontally extended horn shape., Conclusions: Semi-feralization has an extensive impact on diverse phenotypic traits of sheep. By acquiring features like those of their wild ancestors, semi-feral sheep were able to regain fitness while in frequent contact with wild surroundings and rare human interventions. This study provides a new insight into the evolution of domestic animals when human interventions are no longer dominant.

The KiSS-1 and GPR54 genes were studied as candidate genes for the prolificacy in sheep. Four pairs of primers were designed to detect single nucleotide polymorphisms of exon 1 of KiSS-1 gene and exon 1, exon 2 and partial exon 5 of GPR54 gene in high fecundity breeds (Small Tail Han and Hu sheep) and low fecundity breeds (Dorset, Texel and Corriedale sheep) by PCR-SSCP. Polymorphisms in exon 1 of KiSS-1 gene were detected in prolific Small Tail Han sheep (AA, AB and BB genotypes) and Hu sheep (AA and CC genotypes), no polymorphism was found in low fecundity sheep breeds (only AA genotype). Polymorphisms in exon 2 of GPR54 gene were detected in prolific Hu sheep (DD and EE genotypes) and no polymorphism was found in prolific Small Tail Han sheep and low fecundity sheep breeds (only DD genotype). No polymorphism was detected in exon 1 and partial exon 5 of GPR54 gene in five sheep breeds. The polymorphic genotypes were sequenced. While compared the BB genotype with the AA genotype, one nucleotide mutation (G1035A) was detected, which resulted in amino acid change, Val25Met. Five nucleotide mutations were detected from AA to CC genotype (C981T, C996T, T997C, C1034G, C1039T), and among them four caused amino acid changes, that is, Arg7Trp, Phe12Leu, Asn24Lys, Ala26Val. While compared the EE genotype with the DD genotype, two nucleotide mutations (T2360C, A2411C) were detected, which gave rise to amino acid changes, Met90Thr and Asp107Ala, respectively. Genotype frequencies of AA, BB and AB were 0.62, 0.05 and 0.33 in Small Tail Han sheep, respectively. The Small Tail Han sheep ewes with genotype BB or AB had 0.88 (P < 0.05) or 0.51 (P < 0.05) lambs more than those with genotype AA; the Small Tail Han sheep ewes with genotype BB had 0.37 (P > 0.05) lambs more than those with genotype AB. These results preliminarily indicated that the KiSS-1 gene may have some association with prolificacy in sheep.

The bone morphogenetic protein receptor IB (BMPR-IB) was studied as a candidate gene for the prolificacy of sheep. Nine pairs of primers (P1-P9) were designed to detect single nucleotide polymorphisms (SNPs) of exons 1-4 and 6-10 of the BMPR-IB gene in both high (Small Tail Han and Hu sheep) and low prolificacy breeds (Texel and Chinese Merino sheep) by polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP). Only the products amplified by primers P2, P5, P6, P7, P8 and P9 displayed polymorphisms. The present study identified 22 SNPs in partial coding regions of ovine BMPR-IB, in which 20 SNPs were reported for the first time. In total of the 22 mutations, 18 DNA variations were originated from the Hu breed, three were found in the Small Tail Han breed (two of them were found in other sheep breeds), three in the Chinese Merino breed, and none in the Texel breed. These results preliminarily demonstrated that BMPR-IB is a major gene affecting the hyperprolificacy in Small Tail Han and Hu sheep, and could be used as a molecular genetic marker for early auxiliary selection for hyperprolificacy in sheep.

The inhibin β(B) (INHBB) gene was studied as a candidate gene for the prolificacy of Small Tail Han and Hu sheep. According to the sequence of exon 1 and 2 of bovine INHBB gene, six pairs of primers were designed to detect single nucleotide polymorphisms of exon 1 and 2 of INHBB gene in both high (Small Tail Han and Hu sheep) and low prolificacy breeds (Dorset, Texel and German Mutton Merino sheep) by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). Three pairs of primers (primers 1-1, 1-2 and 1-3) were used to amplify the exon 1, and others (primers 2-1, 2-2 and 2-3) to the exon 2. Only the products amplified by primer 2-3 displayed polymorphism. For primer 2-3, three genotypes (AA, AB and BB) were detected in Hu sheep and only AA genotype in other breeds. In Hu sheep, frequency of AA, AB and BB genotypes was 0.636, 0.046 and 0.318, respectively. Sequencing revealed 276A > G mutation (based on the amplification region of primer 2-3) which did not cause any amino acid change because it lay in the 3' untranslated region. The ewes with genotype BB had 0.58 (P < 0.01) lambs more than those with AA in Hu sheep., (© 2010 The Authors. Journal compilation © 2010 Japanese Society of Animal Science.)

Genetic polymorphisms of microsatellite locus BMS2508, which was closely linked to the ovine fecundity gene FecB, were detected in prolific (Small Tail Han sheep) and non-prolific breeds of sheep (Texel, Dorset and Chinese Merino). The linkage disequilibrium between microsatellite locus BMS2508 and FecB gene of Small Tail Han sheep was also analyzed. There was the same mutation (A746G) of BMPR-IB gene in Small Tail Han sheep as that of FecB in Booroola Merino ewes, but the FecB mutation was absent in Texel, Dorset and Chinese Merino sheep. The genotype frequencies of BB, B+ and ++ were 0.485, 0.398 and 0.117 in Small Tail Han sheep, respectively. There were eight alleles varied from 94 bp to 116 bp and 15 genotypes detected at BMS2508 locus in four sheep breeds totally 438 individual. The preponderant allele was 100 bp, 94 bp, 94 bp, 112 bp, 100 bp, 100 bp, 112 bp, and the frequency was 0.453, 0.544, 0.802, 0.475, 0.483, 0.439, 0.389 in Small Tail Han (n=307), Texel (n=45), Dorset (n=46), Chinese Merino (n=40), and BB group (n=149), B+ group (n=122), ++ group (n=36) from Small Tail Han, respectively. In Small Tail Han sheep, linkage analysis indicated that there was certain linkage disequilibrium between 100 bp allele of microsatellite BMS2508 and B allele of FecB gene (D' =0.408), and certain linkage disequilibrium between 110 bp and 114 bp alleles of microsatellite BMS2508 and + allele of FecB gene (D'=0.513).

PCR-RFLP was applied to analyze the polymorphism of MSTN gene UTR in 345 sheep that comprised of eleven sheep breeds, namely Texel sheep, Charolais sheep, Small-tailed Han sheep, Monggolian sheep, Ujumqin sheep, Altay Fat-rumped sheep, Hulunbeir sheep, Tashikurgan sheep, Duolang sheep, Hu sheep, and Gangba sheep. A 271 bp and a 1 003 bp long PCR products were digested with Mboand Bsato demonstrate polymorphism in the eleven sheep breeds, which were all at Hardy-Weinberg equilibrium (P>0.05). The distribution of 3 genotypes in 11 sheep breeds was significantly different (P<0.01). Digestion of the PCR products with HpyCH4 proved that 9 domestic local sheep breeds were different from Texel sheep in the SNP site that was associated with muscularity. The individual mutation base could generate the motifs for miRNA in the 3'UTR, and sequencing analysis demonstrated high frequency of mutation in the 3'UTR region.

Simple Summary: NOX4, PDE11A and GHR have previously been screened as candidate genes for litter size in sheep. Therefore, it is necessary to validate these genes in the sheep population and identify loci that are associated with litter size. In this study, the results of association analysis showed that c.1057-4C > T in NOX4 and c.1983C > T in PDE11A were significantly associated with litter size in Small Tail Han sheep, and their effects were independent of each other. In summary, this study provided two new genetic markers for improving litter size in sheep. 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. [ABSTRACT FROM AUTHOR]

Simple Summary: This study investigates the expression characteristics of circRNAs during follicular development in sheep with the FecB gene mutation through the use of RNA sequencing. A total of 38,979 circRNAs were identified, and differential expression was observed between different genotypes and developmental stages. Functional enrichment analysis revealed that these differentially expressed circRNAs are associated with signaling pathways such as MAPK, gap junctions, and progesterone-mediated oocyte maturation. This study also predicted a competitive endogenous RNA network, suggesting that specific circRNAs may be involved in regulating LH synthesis and secretion, thereby influencing the process of ovulation. These findings provide important insights into the role of circRNAs in follicular development and ovulation in the sheep hypothalamus and have practical implications for the improvement of sheep breeding practices. 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. [ABSTRACT FROM AUTHOR]

Simple Summary: The lambing trait is an important production trait in sheep, and the FecB gene is one of the major genes affecting sheep reproduction. In order to explore the molecular regulatory mechanisms affecting lambing number in sheep, we used RNA-seq technology to detect the thyroid transcircRNA recombinome expression patterns during the luteal and luteal phases in different FecB genotypes of small-tail Han sheep (STH), identified reproduction-related circRNAs in the thyroid gland of STH sheep, and performed bioinformatic analysis, RT-qPCR validation of these genes, and dual luciferase reporter gene experimental validation to predict their potential functions in repopulation and better reveal the molecular mechanisms of thyroid organization during reproduction in sheep. 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. [ABSTRACT FROM AUTHOR]

Simple Summary: Our study aimed to investigate the impact of the FecB mutation on ovulation in sheep. We analyzed the proteomic profiles of granulosa cells from wild-type, heterozygous, and mutant homozygous sheep. Through our analysis, we identified 199 proteins that showed differential abundance and were involved in important metabolic pathways related to oocyte development. These findings provide insights into the processes of oocyte conservation during follicular development and highlight the role of nutrient metabolism in oocyte maturation. Additionally, we introduced a heterozygous sheep to study the additive effect of the FecB mutation on ovulation. By using advanced clustering and machine learning algorithms, we identified potential biomarkers associated with multi-ovulation. These biomarkers, such as ZP2, ZP3, and APOA1 proteins, have the potential to assess oocyte quality and regulate hormone synthesis, thereby controlling ovulation number. Overall, our study contributes to the understanding of how FecB mutations affect ovulation in sheep and has implications for sheep breeding. 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. [ABSTRACT FROM AUTHOR]

Simple Summary: FBN1, ZFAT and FAM184B have been screened as candidate genes for the reproduction of sheep. Therefore, it is necessary to verify these genes in the population and determine the associated loci for litter size. The association of litter size with the genotypes of three candidate genes was analyzed using the fixed effects model in Xinggao mutton sheep. The results showed that the g.160338382 T > C in FBN1 was significantly associated with litter size in Xinggao mutton sheep and that this effect was independent of the FecB mutation. Overall, this study provides a useful genetic marker for improving sheep fecundity. 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. [ABSTRACT FROM AUTHOR]

MicroRNA (miRNA) is a type of endogenous short−stranded ncRNA that influences many biological processes such as animal growth, development and metabolism. The thyroid gland is an important endocrine gland in sheep, and an increasing number of studies have shown that the thyroid gland plays an important role in animal reproduction, but the molecular mechanisms of the thyroid gland in sheep reproduction are poorly understood. In this study, RNA-seq was used to detect transcriptome expression patterns in the thyroid gland between the follicular phase (FP) and luteal phase (LP) in FecB BB (MM) and FecB ++ (ww) small-tail Han (STH) sheep, respectively, and to identify differentially expressed miRNAs (DEMs) associated with reproduction. Bioinformatic analysis of the target genes of these DEMs revealed that they can be enriched in multiple GO terms associated with the reproductive process in animals and in the KEGG signaling pathway. The miRNA–mRNA coexpression network revealed that oar-miR-133 and oar-miR-370-3p may play an important role in sheep reproduction. The results of the dual-luciferase reporter assay suggest a possible targeting relationship between novel-51 and TARBP2. These results provided a novel resource for elucidating regulatory mechanisms underlying STH sheep prolificacy. [ABSTRACT FROM AUTHOR]

Simple Summary: Booroola fecundity gene (FecB) is known as the main gene for multiple lambs in sheep, which has been shown to affect the reproduction of Small Tail Han sheep. The three genotypes of FecB are BB (two copies of FecB mutation), +B (one copy of FecB mutation), and ++ (without FecB mutation). FecB ++ genotype Small Tail Han sheep usually have one offspring. Interestingly, we found that FecB ++ Small Tail Han sheep also had high-reproductive-capacity individuals (multiple lambs per litter). The pituitary affects reproductive processes of animals through a variety of hormones. How pituitary circular RNAs (circRNAs) affect fecundity in FecB ++ genotype sheep remains unknown. Through RNA sequencing, we speculated that changes in circRNAs may be related to the response of the pituitary to steroid hormones, and directly or indirectly affect the pituitary function and the fecundity of sheep. These results provide new insights into pituitary function and high fecundity in sheep. 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. [ABSTRACT FROM AUTHOR]

Melatonin (MLT) protects cells by reducing reactive oxygen species (ROS) levels, which are key for inducing cellular autophagy. The aim of this study was to investigate the molecular mechanisms underlying MLT regulation of autophagy in granulosa cells (GCs) with BMPR‐1B homozygous (FecB BB) and wild type (FecB ++) mutations. GCs collected from small‐tailed Han sheep with different FecB genotypes were typed using a TaqMan probe assay, and autophagy levels were found to be significantly higher in GCs with FecB BB than the levels in those with FecB ++. Autophagy‐related 2 homolog B (ATG2B) was associated with cell autophagy and was highly expressed in GCs with the FecB BB genotype in small‐tailed Han sheep. Overexpression of ATG2B in the GCs of sheep with both FecB genotypes promoted GC autophagy, and the contrary was observed after the inhibition of ATG2B expression. Subsequently, treatment of GCs with different genotypes of FecB and MLT revealed a significant decrease in cellular autophagy and an increase in ATG2B expression. Addition of MLT to GCs with inhibited ATG2B expression revealed that MLT could protect GCs by decreasing ROS levels, especially in GCs with FecB ++ genotype. In conclusion, this study determined that autophagy levels were significantly higher in sheep GCs with FecB BB genotype than the levels in those with FecB ++ genotype, which may have contributed to the difference in lambing numbers between the two FecB genotypes. Autophagy was regulated by ATG2B and was able to protect GCs by reducing the high levels of ROS produced following inhibition of ATG2B through the addition of MLT in vitro. [ABSTRACT FROM AUTHOR]

Simple Summary: In sheep pituitary pars tuberalis tissue, EYA3, TSHβ and CHGA are key genes regulating seasonal reproduction. Their high expression is the characteristic signal under long photoperiods (EYA3, TSHβ) and short photoperiods (CHGA). The epigenetic regulation of microRNAs and LncRNAs on their expression remains unknown. In this study, our results indicated that miR-25 could inhibit the expression of CHGA by specifically binding to its 3′UTR region in pituitary cells. Lnc107153 played the role of CeRNA, which can adsorb miR-25 and weaken the inhibitory effect of miR-25 on CHGA expression. Together, these results suggested the epigenetic regulative network of Lnc107153-miR-25-CHGA was involved in sheep seasonal reproduction. 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. [ABSTRACT FROM AUTHOR]

Simple Summary: Increasing sheep litter size is essential to improve lamb production efficiency. In Small Tail Han sheep, the large variation in litter size is determined by segregation of the bone morphogenetic receptor type 1B (BMPR1B) gene. BMPR1B is the major gene for fertility that affects the ovulation rate, and a prolific allele is FecBB, also called the FecB mutation. The mechanism of how the hypothalamus regulates ovulation number increase in sheep with the FecB mutation is not clear. Using a systems biology workflow in DIABLO, we integrated hypothalamic transcriptomics and proteomics data to predict the main fertility-related biomarkers (gamma-aminobutyric acid type A receptor subunit alpha 1, gamma-aminobutyric acid type A receptor subunit beta 2 and FKBP prolyl isomerase 1A) in the hypothalamus that may regulate changes in the ovulation rate of FecB mutant sheep by participating in gonadotrophin-releasing hormone synthesis and secretion. These results may provide potential genetic markers for breeding multi-lambing meat sheep These results also provide new insight into the reproductive endocrine regulation of fecundity and potential genetic markers of breeding for prolificacy in meat sheep. 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The reproduction of sheep is affected by many factors such as light, nutrition and genetics. The Hypothalamic-pituitary-gonadal (HPG) axis is an important pathway for sheep reproduction, and changes in HPG axis-related gene expression can affect sheep reproduction. In this study, a model of bilateral ovarian removal and estrogen supplementation (OVX + E2) was applied to screen differentially expressed genes and miRNAs under different photoperiods using whole transcriptome sequencing and reveal the regulatory effects of the photoperiod on the upstream tissues of the HPG axis in sheep. Whole transcriptome sequencing was performed in ewe hypothalamus (HYP) and distal pituitary (PD) tissues under short photoperiod 21st day (SP21) and long photoperiod 21st day (LP21). Compared to the short photoperiod, a total of 1813 differential genes (up-regulation 966 and down-regulation 847) and 145 differential miRNAs (up-regulation 73 and down-regulation 72) were identified in the hypothalamus of long photoperiod group. Similarly, 2492 differential genes (up-regulation 1829 and down-regulation 663) and 59 differential miRNAs (up-regulation 49 and down-regulation 10) were identified in the pituitary of long photoperiod group. Subsequently, GO and KEGG enrichment analysis revealed that the differential genes and target genes of differential miRNA were enriched in GnRH, Wnt, ErbB and circadian rhythm pathways associated with reproduction. Combined with sequence complementation and gene expression correlation analysis, several miRNA-mRNA target combinations (e.g., LHB regulated by novel-414) were obtained. Taken together, these results will help to understand the regulatory effect of the photoperiod on the upstream tissues of HPG in sheep. [ABSTRACT FROM AUTHOR]

Long non-coding RNA (lncRNA) accounts for a large proportion of RNA in animals. The thyroid gland has been established as an important gland involved in animal reproduction, however, little is known of its gene expression patterns and potential roles in the sheep. Herein, RNA-Seq was used to detect reproduction-related differentially expressed lncRNAs (DELs) and mRNAs (DEGs) in the follicular phase (FT) FecBBB (MM) and FecB++ (ww) genotypes of Small Tail Han (STH) sheep thyroids. Overall, 29 DELs and 448 DEGs in thyroid between MM and ww sheep were screened. Moreover, GO and KEGG enrichment analysis showed that targets of DELs and DEGs were annotated in biological transitions, such as cell cycle, oocyte meiosis and methylation, which in turn affect reproductive performance in sheep. In addition, we constructed co-expression and networks of lncRNAs-mRNAs. Specifically, XLOC_075176 targeted MYB, XLOC_014695 targeted VCAN, 106991527 targeted CASR, XLOC_075176 targeted KIFC1, XLOC_360232 targeted BRCA2. All these differential lncRNAs and mRNAs expression profiles in the thyroid provide a new resource for elucidating the regulatory mechanism underlying STH sheep prolificacy. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The thyroid gland is a vital endocrine organ involved in the reproduction of animals via the regulation of hormone synthesis and secretion. LncRNAs have been proven to play important roles in reproductive regulation; however, the associated mechanism in the thyroid gland has not been clarified. In this study, we investigated to identify photoperiod-induced lncRNAs and mRNAs in the thyroid gland in Sunite ewes by comparing the expression profiles of short photoperiod (SP) and long photoperiods (LP). A total of 41,088 lncRNAs were identified in the thyroid gland through RNA-Seq. Functional analysis of differentially expressed lncRNAs using the R package revealed that reproductive hormone- and photoperiod response-related pathways, including the prolactin signaling, cAMP signaling, and circadian rhythm pathways, were significantly enriched. An mRNA-lncRNA interaction analysis suggested that the lncRNA LOC1056153S88 trans targets ARG2 and CCNB3, and the lncRNA LOC105607004 trans targets DMXL2, both of these might be involved in seasonal sheep breeding reproduction. Together, these results will provide resources for further studies on seasonal reproduction in sheep. [ABSTRACT FROM AUTHOR]

Summary: Improvement of ewe reproduction is considerable by appropriately increasing litter size and sustaining non‐seasonal breeding. However, their genetic makeups have not been entirely elucidated. Genome‐wide analyses of 821 individuals were performed by combining three genomic approaches (genome‐wide association study, XP‐nSL, and runs of homozygosity). Consequently, 35 candidate genes including three domestication genes (TSHR, GTF2A1, and KITLG) were identified. Other than the FecB mutation at BMPR1B, we described a significant association of a missense mutation rs406686139 at seasonal lambing‐associated TSHR gene with litter size. Some promising novel genes may be relevant for sheep reproduction by multitude biological processes, such as FETUB functioning in fertilization, HNRNPA1 in oogenesis, DCUN1D1 in spermatogenesis, and HRG in fertility outcome. The present study suggests that improvement of ewe reproduction is attributed to selective breeding, and casts light on the genetic basis and improvement of sheep reproduction. [ABSTRACT FROM AUTHOR]

Previous studies have indicated that FLT3 , NLRP5 , and TGIF1 play a pivotal role in sheep fecundity. Nevertheless, little is known about the association of the polymorphisms of these genes with litter size (LS). In this study, the selected single-nucleotide polymorphisms (SNPs) were genotyped using a Sequenom MassARRAY® platform, and the distribution of different genotypes of the SNPs in the seven sheep breeds (Small-tailed Han, Hu, Cele Black, Suffolk, Tan, Prairie Tibetan, and Sunite sheep) were analyzed. The reliability of the estimated allele frequency for all seven SNPs was at least 0.9545. Given the association of the TGIF1 g.37866222C > T polymorphism with LS in Small-tailed Han sheep (p<0.05), fecundity differences might be caused by the change in amino acid from proline (Pro) to serine (Ser), which has an impact on secondary, tertiary protein structures with concomitant TGIF1 functionality changes. The FLT3 rs421947730 locus has a great effect on the LS (p<0.05), indicating that the locus of FLT3 in synergy with KILTG is likely to facilitate ovarian follicle maturation and ovulation. Moreover, NLRP5 rs426897754 is associated with the LS of the second and third parities (p<0.05). We speculate that a synonymous variant of NLRP5 may be involved in folliculogenesis accompanied by BMP15, FSHR, BMPR1B, AMH, and GDF9, resulting in the different fecundity of Small-tailed Han sheep. Our studies provide valuable genetic markers for sheep breeding. [ABSTRACT FROM AUTHOR]

The circadian rhythm is a biological rhythm that is closely related to the rhythmic expression of a series of clock genes. Results from several studies have indicated that clock genes are associated with the estrous cycle in female animals. Until now, the relationship between estrus cycle transition and clock gene expression in reproductive-axis-related tissues has remained unknown in Small-tailed Han (STH) sheep. This study was conducted to analyze the expression patterns of six canonical clock genes (Clock , BMAL1 , Per1 , Per2 , Cry1 , and Cry2) in the follicle phase and luteal phase of STH sheep. We found that all six genes were expressed in the brain, cerebellum, hypothalamus, pituitary, ovary, uterus, and oviduct in follicle and luteal phases. The results indicated that Clock expression was significantly higher in the cerebellum, hypothalamus, and uterus of the luteal phase than that of the follicle phase, whereas BMAL1 expression was significantly higher in the hypothalamus of the luteal phase than that of the follicle phase. Per1 expression was significantly higher in the brain, cerebellum, hypothalamus, and pituitary of the luteal phase than that of the follicle phase, and Per2 expression was significantly higher in the hypothalamus, pituitary, and uterus of the luteal phase than that of the follicle phase. Cry1 expression was significantly higher in the brain, cerebellum, and hypothalamus of the luteal phase than that of the follicle phase, whereas Cry2 expression was significantly higher in the pituitary of the luteal phase than that of the follicle phase. The clock gene expression in all tissues was different between follicle and luteal phases, but all clock gene mRNA levels were found to exhibit higher expression among seven tissues in the luteal phase. Our results suggest that estrous cycles may be associated with clock gene expression in the STH sheep. This is the first study to systematically analyze the expression patterns of clock genes of different estrous cycle in ewes, which could form a basis for further studies to develop the relationship between clock genes and the estrous cycle. [ABSTRACT FROM AUTHOR]

The objective of this study was to assess the genetic diversity and phylogenetic relationship of nine sheep populations, including two famous high prolific populations and seven popular mutton populations raised in China. Overall, these sheep populations in this study exhibited a rich genetic diversity. Both the expected heterozygosity and Nei's unbiased gene diversity ranged from 0.64 to 0.75, with the lowest value found in Dorset sheep (DST) and the highest in Hu sheep (HUS) and Ba Han sheep (BAS). The polymorphic information content (PIC) varied between 0.59 in DST and 0.71 in HUS and BAS. Specifically, for individual breeds, the small-tail Han sheep (STH) and the four introduced populations did not display the expected diversity; therefore more attention should be paid to the maintenance of diversity during management of these populations. The results of un-weighted pair-group method (UPGMA) phylogenetic tree and structure analysis indicated that the nine investigated populations can be divided into two groups. Suffolk (SUF) and DST were clustered in one group, and the other group can be further divided into three clusters: German Mutton Merino (GMM)–BAS–Bamei Mutton sheep (BAM), HUS–STH and Du Han (DOS)–Dorper (DOP). This clustering result is consistent with sheep breeding history. TreeMix analysis also hinted at the possible gene flow from GMM to SUF. Together, an in-depth view of genetic diversity and genetic relationship will have important implications for breed-specific management. [ABSTRACT FROM AUTHOR]

The objective of the current study was to analyze expression levels of synapse differentiation inducing 1-like (SYNDIG1L) and unc-13 homolog C (UNC13C) genes in different tissues, while single-nucleotide polymorphisms (SNPs) of two genes were associated with multiple thoracic vertebrae traits in both Small-tailed Han sheep (STH) and Sunite sheep (SNT). The expression levels of SYNDIG1L and UNC13C were analyzed in the brain, cerebellum, heart, liver, spleen, lung, kidney, adrenal gland, uterine horn, longissimus muscle, and abdominal adipose tissues of two sheep breeds with different thoracic vertebral number (TVN) sheep (T13 groups and T14 groups) by real-time quantitative polymerase chain reaction (RT-qPCR). Meanwhile, the polymorphisms of UNC13C gene g.52919279C > T and SYNDIG1L gene g.82573325C > A in T14 and T13 were genotyped by the Sequenom MassARRAY® SNP assay, and association analysis was performed with the TVN. The results demonstrated that UNC13C gene was extensively expressed in 11 tissues. The expression of UNC13C gene in longissimus muscle of T14 groups of STH was significantly higher than that of T13 groups (P<0.05). SYNDIG1L gene was overexpressed in brain and cerebellum tissues, and the expression level of UNC13C gene in the brain and cerebellum of T13 groups in SNT was significantly higher than that of T14 groups (P<0.01). Association analysis showed that SNPs found in the UNC13C gene had no significant effects on TVN for both two genes. The polymorphism of SYNDIG1L g.82573325C > A was significantly correlated with the TVN in both STH (P<0.05) and SNT (P<0.01). Taken together, the SYNDIG1L gene was related to thoracic vertebral development, and this variation may be potentially used as a molecular marker to select the multiple thoracic vertebrae in sheep. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Early studies have provided a wealth of information on the functions of microRNAs (miRNAs). However, less is known regarding their functions in the hypothalamus involved in sheep reproduction. To explore the potential roles of hypothalamic messenger RNAs (mRNAs) and miRNAs in sheep without FecB mutation, in total, 172 and 235 differentially expressed genes (DEGs) and 42 and 79 differentially expressed miRNAs (DE miRNAs) were identified in polytocous sheep in the follicular phase versus monotocous sheep in the follicular phase (PF vs. MF) and polytocous sheep in the luteal phase versus monotocous sheep in the luteal phase (PL vs. ML), respectively, using RNA sequencing. We also identified several key mRNAs (e.g., POMC , GNRH1 , PRL , GH , TRH , and TTR) and mRNA–miRNAs pairs (e.g., TRH co-regulated by oar-miR-379-5p, oar-miR-30b, oar-miR-152, oar-miR-495-3p, oar-miR-143, oar-miR-106b, oar-miR-218a, oar-miR-148a, and PRL regulated by oar-miR-432) through functional enrichment analysis, and the identified mRNAs and miRNAs may function, conceivably, by influencing gonadotropin-releasing hormone (GnRH) activities and nerve cell survival associated with reproductive hormone release via direct and indirect ways. This study represents an integral analysis between mRNAs and miRNAs in sheep hypothalamus and provides a valuable resource for elucidating sheep prolificacy. [ABSTRACT FROM AUTHOR]

Background. 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. Methods. To identify lncRNA and mRNA expressed in the uterus, the expression of lncRNA and mRNA in the uterus of Small Tail Han sheep (FecB++) from the polytocous group (n = 6) and the monotocous group (n = 6) using RNA-sequencing and real-time polymerase chain reaction (RT-PCR). Identification of differentially expressed lncRNAs and mRNAs were performed between the two groups and two phases . Gene ontology (GO) and pathway enrichment analyses were performed to analyze the biological functions and pathways for the differentially expressed mRNAs. LncRNA-mRNA co-expression network was constructed to further analyses the function of related genes. Results. In the follicular phase, 473 lncRNAs and 166 mRNAs were differentially expressed in polytocous and monotocous sheep; in the luteal phase, 967 lncRNAs and 505 mRNAs were differentially expressed in polytocous and monotocous sheep. GO and KEGG enrichment analysis showed that the differentially expressed lncRNAs and their target genes are mainly involved in ovarian steroidogenesis, retinol metabolism, the oxytocin signaling pathway, steroid hormone biosynthesis, and the Foxo signaling pathway. Key lncRNAs may regulate reproduction by regulating genes involved in these signaling pathways and biological processes. Specifically, UGT1A1, LHB, TGFB1, TAB1, and RHOA, which are targeted by MSTRG.134747, MSTRG.82376, MSTRG.134749, MSTRG.134751, and MSTRG.134746, may play key regulatory roles. These results offer insight into molecular mechanisms underlying sheep prolificacy. [ABSTRACT FROM AUTHOR]

Most ewes in China are seasonally polyestrous with normal ovulatory cycles, which is controlled by photoperiod (length of the daily light phase). These ewes are estrous in the short-day season and anestrus in the long-day season and cannot mate during anestrus. Thus seasonal breeding limits both diversification and intensification of production. If sheep can estrus all round year, it can be mated twice per year, which can greatly improve the economic benefits. To change seasonal estrus at the genetic level and cultivating new sheep breeds, it is important to understand the molecular mechanisms of seasonal breeding trait in sheep. The BCL2-associated agonist of cell death (BAD) gene being a regulator of cellular apoptosis was identified by our previous RNA-Seq, which is associated with follicular development in mammalian ovaries. However, the mechanism how BAD can regulate estrus in sheep was poorly understood. In this study, we characterized ovine BAD , including full-length mRNA cloning and protein sequence prediction, as well as BAD expression profile in Small-tailed Han (STH) sheep. The highest expression levels of BAD were observed in sheep hypothalamus, lung, and pituitary, while the lowest expression was in liver. Functional analysis of BAD was performed in primary granulosa cells of sheep. The concentration of P4 was significantly increased after RNAi interference of BAD , while P4 level was shown to be opposite after BAD overexpression in vitro. It has been found that BAD can reduce progesterone levels by promoting ovarian GC apoptosis, which might be involved in regulating the estrus cycle in sheep. [ABSTRACT FROM AUTHOR]

The FecB gene has been discovered as an important gene in sheep for its high relationship with the ovulation rate, but its regulatory mechanism remains unknown. In the present study, liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS) techniques were adopted to detect the metabolic effects of FecB gene in follicular fluid (FF) and ovarian vein serum (OVS) in Small Tail Han (STH) sheep. ANOVA and random forest statistical methods were employed for the identification of important metabolic pathways and biomarkers. Changes in amino acid metabolism, redox environment, and energy metabolism were observed in FF from the three FecB genotype STH ewes. Principal component analysis (PCA) and hierarchical clustering analysis (HCA) showed that metabolic effects of FecB gene are more pronounced in FF than in OVS. Therefore, the difference of the metabolic profile in FF is also affected by the FecB genotypes. In Spearman correlation analysis, key metabolites (e.g., glucose 6-phosphate, glucose 1-phosphate, aspartate, asparagine, glutathione oxidized (GSSG), cysteine-glutathione disulfide, γ-glutamylglutamine, and 2-hydrosybutyrate) in ovine FF samples showed a significant correlation with the ovulation rate. Our findings will help to explain the metabolic mechanism of high prolificacy ewes and benefit fertility identification. [ABSTRACT FROM AUTHOR]

Abstract 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 FROM AUTHOR]

Simple Summary: BMP15 is a critical gene in sheep reproduction. Most of its variations have been reported in European sheep. In this study, the entire open reading frame (ORF) region of BMP15 was sequenced in 154 Luzhong mutton sheep. Among 13 identified variations, six were novel. Four SNPs (ENSOART00000010201.1:c.352+342C>A, c.352+1232T>C, c.352+1165A>G and c.353-2036T>A) were significantly associated with litter size, and could be used as candidate genetic markers for improving litter size. The results also suggested possible interaction between BMP15 and FecB/GDF9. Litter size is an important economic trait in the mutton sheep industry. BMP15 is one of the key candidate genes for litter size in sheep. In this study, the entire ORF region of BMP15 was sequenced in 154 Luzhong mutton ewes, and the novel variations were determined. The association between polymorphism in BMP15 and litter size was analyzed using a general linear model. Six out of a total of thirteen variations were identified to be novel. Association analysis indicated that four (SNPs ENSOART00000010201.1:c.352+342C>A, c.352+1232T>C, c.352+1165A>G and c.353-2036T>A) were significantly associated with litter size. The joint analysis among three major genes (BMP15, BMPR1B and GDF9) exhibited significant interaction effects in three combinations (FecB and c.352+1232T>C of BMP15; FecB and c.352+1165A>G of BMP15; c.352+342C>A of BMP15 and ENSOART00000014382.1:c.994G>A of GDF9). For the SNPs c.352+1232T>C and c.352+342C>A, the global distribution of allele frequencies showed that the highest variation frequency occurs in Western Europe. In conclusion, the results demonstrated that BMP15 is a major gene for litter size in Luzhong mutton sheep and candidate SNPs associated with litter size were identified. [ABSTRACT FROM AUTHOR]

Most sheep are seasonal estrus, and they breed in autumn when the days get shorter. Seasonal estrus is an important factor that affects the productivity and fertility of sheep. The key point to solve this problem is to explore the regulation mechanism of estrus in sheep. Therefore, in this study, transcriptomic sequencing technology was used to identify differentially expressed mRNAs in the hypothalamus, pituitary and ovary of Small Tail Han sheep (year-round estrus) and tan sheep (seasonal estrus) among luteal, proestrus and estrus stages. There were 256,923,304,156 mRNAs being identified in the hypothalamus, pituitary and ovary, respectively. Functional analysis showed that the photosensor, leucine and isoleucine biosynthesis pathways were enriched significantly. It is speculated that photoperiod may initiate estrus by stimulating the corresponding pathways in hypothalamus. ODC1, PRLH, CRYBB2, SMAD5, OPN1SW, TPH1 are believed to be key genes involved in the estrogen process. In conclusion, this study expanded the database of indigenous sheep breeds, and also provided new candidate genes for future genetic and molecular studies on the seasonal estrus trait in sheep. [ABSTRACT FROM AUTHOR]

Simple Summary: GDF9 and BMPR1B are two important reproduction genes. In this study, the whole coding region of GDF9 was sequenced, of which the mutations were detected in Luzhong mutton sheep. The results suggested that two single nucleotide polymorphisms (SNPs), g.41768501A > G and g.41768485 G > A in GDF9 gene were associated with litter size. The g.41768485 G > A is a missense mutation which is predicted to affect the tertiary structure of the protein. Thus, these two mutations may be potential effective genetic markers to improve the litter size in sheep. Litter size is one of the most important economic traits in sheep. GDF9 and BMPR1B are major genes affecting the litter size of sheep. In this study, the whole coding region of GDF9 was sequenced and all the SNPs (single nucleotide polymorphisms) were determined in Luzhong mutton ewes. The FecB mutation was genotyped using the Sequenom MassARRAY®SNP assay technology. Then, the association analyses between polymorphic loci of GDF9 gene, FecB, and litter size were performed using a general linear model procedure. The results showed that eight SNPs were detected in GDF9 of Luzhong mutton sheep, including one novel mutation (g.41769606 T > G). The g.41768501A > G, g.41768485 G > A in GDF9 and FecB were significantly associated with litter size in Luzhong mutton ewes. The g.41768485 G > A is a missense mutation in the mature GDF9 protein region and is predicted to affect the tertiary structure of the protein. The results preliminarily demonstrated that GDF9 was a major gene affecting the fecundity of Luzhong mutton sheep and the two loci g.41768501A > G and g.41768485 G > A may be potential genetic markers for improving litter size. [ABSTRACT FROM AUTHOR]

Ovine litter size (LS) is an important trait showing variability within breeds. It remains largely unknown whether inbreeding depression on LS exists based on genomic homozygous regions, and whether the homozygous regions resulted from inbreeding are significantly associated with LS in sheep. We here reanalyze a set of single nucleotide polymorphism (SNP) chip of six breeds to characterize the patterns of runs of homozygosity (ROH), to evaluate inbreeding levels and inbreeding depressions on LS, and to identify candidate homozygous regions responsible for LS. Consequently, unique ROH patterns were observed among six sheep populations. Inbreeding depression on LS was only found in Hu sheep, where a significant reduction of 0.016, 0.02, and 0.02 per 1% elevated inbreeding FROH4–8, FROH>8 and the total inbreeding measure was observed, respectively. Nine significantly homozygous regions were found for LS in Hu sheep, where some promising genes for LS possibly via regulation of the development of oocytes (NGF, AKT1, and SYCP1), fertilization (SPAG17, MORC1, TDRD9, ZFYVE21, ADGRB3, and CKB), embryo implantation (PPP1R13B, INF2, and VANGL1) and development (DPPA2, DPPA4, CDCA4, CSDE1, and ADSSL1), and reproductive health (NRG3, BAG5, CKB, and XRCC3) were identified. These results from the present study would provide insights into the genetic management and complementary understandings of LS in sheep. [ABSTRACT FROM AUTHOR]

Simple Summary: Bamei mutton sheep is a Chinese domestic sheep breed developed by crossing German Mutton Merino sheep and indigenous Mongolian sheep for meat production. There is large variation in the reproductive abilities of Bamei mutton sheep. After recent artificial selection, the average lambing rate of the Bamei mutton nucleus group was over 150%. We used the FST (Fixation Index) and XP-EHH (The Cross-Population Extended Haplotype Homozygosity) statistical approach to detect the selective sweeps between high- and low-fecundity Bamei mutton sheep groups. JUN (JUN proto-oncogene, AP-1 transcription factor subunit), ITPR3 (inositol 1,4,5-trisphosphate receptor type 3, PLCB2 (phospholipase C beta 2), HERC5 (HECT and RLD domain containing E3 ubiquitin protein ligase 5), and KDM4B (lysine demethylase 4B) were detected that are potential responsible for litter size. These observations provide a new opportunity to research the genetic variation influencing fecundity traits within a population evolving under artificial selection. 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. [ABSTRACT FROM AUTHOR]

Simple Summary: 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. In this study, we performed a correlation analysis of transcriptomics and proteomics, and the results suggested several differentially expressed genes (DEGs)/differentially expressed proteins (DEPs), including galectin 3 (LGALS3), aspartoacylase (ASPA) and transthyretin (TTR), could be candidate genes influencing ovine litter size. Further analysis suggested that AS events, single nucleotide polymorphisms (SNPs) and microRNA (miRNA)-binding sites existed in key DEGs/DEPs, such as ASPA and TTR. This study provides a new insight into ovine and even other mammalian reproduction. 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 Tail 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 galectin 3 (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. [ABSTRACT FROM AUTHOR]

Simple Summary: The NLR family pyrin domain-containing 5 (NLRP5) and NLRP9 genes are two important reproductive genes; however, their effects on litter size in sheep are unknown. In this study, we conducted population genetic and association analyses on five NLRP5 and NLRP9 loci of sheep. Our results suggested that a mutation in g.60495363G > A may decrease interactions of NLRP5 with proteins, such as the growth differentiation factor 9 (GDF9), whereas a mutation in g. g.59030623T > C may enhance the NLRP9-combining capacity with these proteins. Consequently, these mutations may lead to differences in ovulation rate and even litter size. Overall, this study provided useful genetic markers that can be used to improve sheep breeding. Previous studies showed that the NLR family pyrin domain-containing 5 (NLRP5) and NLRP9 genes are two important reproductive genes; however, their effects on sheep litter size are unknown. Therefore, in this study, we first genotyped seven sheep breeds via the MassARRAY® SNP system at the loci g.60495375A > G, g.60495363G > A, and g.60499690C > A in NLRP5, and g.59030623T > C and g.59043397A > C in NLRP9. Our results revealed that each locus in most sheep breeds contained three genotypes. Then, we conducted population genetic analysis of single nucleotide polymorphisms in NLRP5 and NLRP9, and we found that the polymorphism information content value in all sheep breeds ranged from 0 to 0.36, and most sheep breeds were under Hardy–Weinberg equilibrium (p > 0.05). Furthermore, association analysis in Small Tail Han sheep indicated that two loci, g.60495363G > A in NLRP5 and g.59030623T > C in NLRP9, were highly associated with litter size. The mutation in g.60495363G > A may decrease interactions of NLRP5 with proteins, such as GDF9, whereas the mutation in g.59030623T > C may enhance the combining capacity of NLRP9 with these proteins; consequently, these mutations may influence the ovulation rate and even litter size. The findings of our study provide valuable genetic markers that can be used to improve the breeding of sheep and even other mammals. [ABSTRACT FROM AUTHOR]