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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]

The p110δ isoform of the PI3K catalytic subunit (encoded by the PIK3CD gene) is a key component of the PI3K pathway for follicle growth in mammalian ovarian granulosa cells. Nevertheless, little is known about the association of its polymorphisms with ovine litter size. In this study, the distribution of different genotypes of two SNPs in the PIK3CD gene was calculated in more than forty sheep breeds, and the associations between SNPs and litter size in Small Tail Han (STH) sheep were also analyzed. Besides, the mRNA expression of the PIK3CD gene was also detected in some reproduction-related tissues. The results showed that the "A" allele frequency was higher in rs412889931 (g.41926327G > A) in a typical polytocous sheep breed (p < 0.01). The association's analysis showed rs412889931 was correlated with ovine fecundity as assessed by three parity litter sizes (p < 0.05). Finally, we found the expression of PIK3CD in the ovary had significant differences in different fecundity sheep breeds, indicating that SNP may regulate the litter size by influencing the PIK3CD gene expression. The present results demonstrated that rs412889931 could be used in the marker-assisted selection of the litter size in sheep breeding. [ABSTRACT FROM AUTHOR]

GNAQ, a member of the alpha subunit encoding the q-like G protein, is a critical gene in cell signaling, and multiple studies have shown that upregulation of GNAQ gene expression ultimately inhibits the proliferation of gonadotropin-releasing hormone (GnRH) neurons and GnRH secretion, and ultimately affects mammalian reproduction. Photoperiod is a key inducer which plays an important role in gene expression regulation by affecting epigenetic modification. However, fewer studies have confirmed how photoperiod induces epigenetic modifications of the GNAQ gene. In this study, we examined the expression and epigenetic changes of GNAQ in the hypothalamus in ovariectomized and estradiol-treated (OVX+E2) sheep under three photoperiod treatments (short photoperiod treatment for 42 days, SP42; long photoperiod treatment for 42 days, LP42; 42 days of short photoperiod followed by 42 days of long photoperiod, SP-LP42). The results showed that the expression of GNAQ was significantly higher in SP-LP42 than in SP42 and LP42 (p < 0.05). Whole genome methylation sequencing (WGBS) results showed that there are multiple differentially methylated regions (DMRs) and loci between different groups of GNAQ. Among them, the DNA methylation level of DMRs at the CpG1 locus in SP42 was significantly higher than that of SP-LP42 (p < 0.01). Subsequently, we confirmed that the core promoter region of the GNAQ gene was located with 1100 to 1500 bp upstream, and the DNA methylation level of all eight CpG sites in SP42 was significantly higher than those in LP42 (p < 0.01), and significantly higher than those in SP-LP42 (p < 0.01), except site 2 and site 4 in the first sequencing fragment (p < 0.05) in the core promoter region. The expression of acetylated GNAQ histone H3 was significantly higher than that of the control group under three different photoperiods (p < 0.01); the acetylation level of sheep hypothalamic GNAQ genomic protein H3 was significantly lower under SP42 than under SP-LP42 (p < 0.05). This suggests that acetylated histone H3 binds to the core promoter region of the GNAQ gene, implying that GNAQ is epigenetically regulated by photoperiod through histone acetylation. In summary, the results suggest that photoperiod can induce DNA methylation in the core promoter region and histone acetylation in the promoter region of the GNAQ gene, and hypothesize that the two may be key factors in regulating the differential expression of GNAQ under different photoperiods, thus regulating the hypothalamus–pituitary–gonadal axis (HPGA) through the seasonal estrus in sheep. The results of this study will provide some new information to understand the function of epigenetic modifications in reproduction in sheep. [ABSTRACT FROM AUTHOR]