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The aim of this study was to identify genes related to precocious puberty expressed in the pituitary of goats at different growth stages by suppression subtractive hybridization (SSH). The pituitary glands from Jining Gray (JG) goats (early puberty) and Liaoning Cashmere (LC) goats (late puberty) at 30, 90, and 180 days were used in this study. To identify differentially expressed genes (DEGs) in the pituitary glands, mRNA was extracted from these tissues, and SSH libraries were constructed and divided into the following groups: juvenile group (30-JG vs. 30-LC, API), puberty group (90-JG vs. 180-LC, BPI), and control group (90-JG vs. 90-LC, EPI). A total of 60, 49, and 58 DEGs were annotated by 222 Gene Ontology (GO) terms and 75 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Most of the DEGs were significantly enriched in GO terms related to 'structural constituent of ribosome', 'translation' and 'GTP binding', and numerous DEGs were also significantly enriched in KEGG terms related to the Jak-STAT signaling and oocyte meiosis pathways. Candidate genes associated with precocious puberty and sexual development were screened from the SSH libraries. These genes were analyzed to determine if they were expressed in the pituitary tissues of the goats at different growth stages and to identify genes that may influence the hypothalamic-pituitary-gonadal (HPG) axis. In this study, we found precocious puberty-related genes (such as PRLP0, EIF5A, and YWHAH) that may be interesting from an evolutionary perspective and that could be investigated for use in future goat breeding programs. Our results provide a valuable dataset that will facilitate further research into the reproductive biology of goats. [ABSTRACT FROM AUTHOR]

The oviduct is a dynamic reproductive organ for mammalian reproduction and is required for gamete storage, maturation, fertilization, and early embryonic development, and it directly affects fecundity. However, the molecular regulation of prolificacy occurring in estrous periods remain poorly understood. This study aims to gain a better understanding of the genes involved in regulating goat fecundity in the proteome and transcriptome levels of the oviducts. Twenty female Yunshang black goats (between 2 and 3 years old, weight 52.22 ± 0.43 kg) were divided into high- and low-fecundity groups in the follicular (FH and FL, five individuals per group) and luteal (LH and LL, five individuals per group) phases, respectively. The DIA-based high-resolution mass spectrometry (MS) method was used to quantify proteins in twenty oviducts. A total of 5409 proteins were quantified, and Weighted gene co-expression network analysis (WGCNA) determined that the tan module was highly associated with the high-fecundity trait in the luteal phase, and identified NUP107, ANXA11, COX2, AKP13, and ITF140 as hub proteins. Subsequently, 98 and 167 differentially abundant proteins (DAPs) were identified in the FH vs. FL and LH vs. LL comparison groups, respectively. Parallel reaction monitoring (PRM) was used to validate the results of the proteomics data, and the hub proteins were analyzed with Western blot (WB). In addition, biological adhesion and transporter activity processes were associated with oviductal function, and several proteins that play roles in oviductal communication with gametes or embryos were identified, including CAMSAP3, ITGAM, SYVN1, EMG1, ND5, RING1, CBS, PES1, ELP3, SEC24C, SPP1, and HSPA8. Correlation analysis of proteomics and transcriptomic revealed that the DAPs and differentially expressed genes (DEGs) are commonly involved in the metabolic processes at the follicular phase; they may prepare the oviductal microenvironment for gamete reception; and the MAP kinase activity, estrogen receptor binding, and angiotensin receptor binding terms were enriched in the luteal phase, which may be actively involved in reproductive processes. By generating the proteome data of the oviduct at two critical phases and integrating transcriptome analysis, we uncovered novel aspects of oviductal gene regulation of fecundity and provided a reference for other mammals. [ABSTRACT FROM AUTHOR]

The production of goat meat is determined by the growth speed of muscle fibers, and the autophagy and apoptosis of myoblast cells is a crucial process in the growth of muscle fibers. The rapid growth of muscle fibers occurs from one month old to nine months old in goats; however, the mechanisms of myoblast cells' autophagy and apoptosis in this process are still unknown. To identify candidate genes and signaling pathway mechanisms involved in myoblast apoptosis and autophagy, we compared the expression characteristics of longissimus dorsi tissues from Wu'an goats—a native goat breed of China—at 1 month old (mon1 group) and 9 months old (mon9 group). Herein, a total of 182 differentially expressed mRNAs (DEGs) in the mon1 vs. mon9 comparison, along with the KEGG enrichments, showed that the PI3K-Akt pathway associated with autophagy and apoptosis was significantly enriched. Among these DEGs, expression of vacuole membrane protein 1 (VMP1)—a key gene for the PI3K-Akt pathway—was significantly upregulated in the older goats relative to the 1-month-old goats. We demonstrated that VMP1 promotes the proliferation and autophagy of myoblasts, and inhibits their apoptosis. The integration analysis of miRNA–mRNA showed that miR-124a was a regulator of VMP1 in muscle tissue, and overexpression and inhibition of miR-124a suppressed the proliferation and autophagy of myoblasts. The PI3K/Akt/mTOR pathway was an important pathway for cell autophagy. Additionally, the activator of the PI3K/Akt/mTOR pathway, the expression of VMP1, and ULK1 were higher than the negative control, and the expression of mTOR was depressed. The expression of VMP1, ULK1, and mTOR was the opposite when the inhibitor was added to the myoblasts. These results show that the PI3K/Akt/mTOR pathway promoted the expression of VMP1 and ULK1. By using adenovirus-mediated apoptosis and proliferation assays, we found that that miR-124a inhibits myoblast proliferation and autophagy, and promotes their apoptosis by targeting VMP1. In conclusion, our results indicated that VMP1 was highly expressed in the LD muscle tissues of nine-month-old goats, and that it was regulated by miR-124a to inhibit myoblast cells' apoptosis through the PI3K/Akt/mTOR pathway, and to promote proliferation and autophagy. These findings contribute to the understanding of the molecular mechanisms involved in myoblast proliferation, autophagy, and apoptosis. [ABSTRACT FROM AUTHOR]

Retinol-binding protein 4 (RBP4), a member of the lipocalin family, is a specific carrier of retinol (vitamin A) in the blood. Numerous studies have shown that RBP4 plays an important role in mammalian embryonic development and that mutations in RBP4 can be used for the marker-assisted selection of animal reproductive traits. However, there are few studies on the regulation of reproduction and high-prolificacy traits by RBP4 in goats. In this study, the 5′ flanking sequence of RBP4 was amplified, and a G>C polymorphism in the promoter region -211 bp (g.36491960) was detected. An association analysis revealed that the respective first, second and third kidding number and mean kidding number of nanny goats with CC and GC genotypes (2.167 ± 0.085, 2.341 ± 0.104, 2.529 ± 0.107 and 2.189 ± 0.070 for CC and 2.052 ± 0.047, 2.206 ± 0.057, 2.341 ± 0.056 and 2.160 ± 0.039 for GC) were significantly higher (p < 0.05) than those with the GG genotype (1.893 ± 0.051, 2.027 ± 0.064, 2.107 ± 0.061 and 1.74 ± 0.05). The luciferase assay showed that luciferase activity was increased in C allele individuals compared with that in G allele individuals. A competitive electrophoretic mobility shift assay (EMSA) showed that individuals with the CC genotype had a stronger promoter region binding capacity than those with the GG genotype. In addition, transcription factor prediction software showed that the RBP4 g.36491960G>C mutation added a novel binding site for transcription factor DP-1 (TFDP1). RT–qPCR results showed that the expression of TFDP1 was significantly higher in the high-prolificacy group than in the low-prolificacy group, and the expression of RBP4 was higher in both the CC and GC genotypes than that in the GG genotype. TFDP1 overexpression significantly increased the expression of RBP4 mRNA (p < 0.05) and the expression of the cell proliferation factors cyclin-D1, cyclin-D2 and CDK4 (p < 0.05). The opposite trend was observed after interference with TFDP1. Both the EdU and CCK-8 results showed that TFDP1 expression could regulate the proliferation of goat ovarian granulosa cells. In summary, our results showed that RBP4 g.36491960G>C was significantly associated with fecundity traits in goats. The g.36491960G>C mutation enhanced the transcriptional activity of RBP4 and increased the expression of RBP4, thus improving the fertility of Yunshang black goats. [ABSTRACT FROM AUTHOR]

Simple Summary: The kidding number is an important reproductive trait in domestic goats. The oviduct, as one of the most major organs, is directly involved in the reproductive process, providing nutrition and a location for early embryonic development. The current study provides genome-wide expression profiles of mRNA and long noncoding RNAs (lncRNAs) expression in Yunshang black goat, a new breed of meat goat bred in China with a high kidding number. During the luteal phases, oviduct mRNAs and lncRNAs associated with high- and low-fecundity Yunshang black goats were identified, and their potential biological functions were predicted using GO, KEGG, and GSEA enrichment analysis. These findings shed light on the oviduct-based prolificacy mechanism in goats. The oviduct is associated with embryo development and transportation and regulates the pregnancy success of mammals. Previous studies have indicated a molecular mechanism of lncRNAs in gene regulation and reproduction. However, little is known about the function of lncRNAs in the oviduct in modulating goat kidding numbers. Therefore, we combined RNA sequencing (RNA-seq) to map the expression profiles of the oviduct at the luteal phase from high- and low-fecundity goats. The results showed that 2023 differentially expressed mRNAs (DEGs) and 377 differentially expressed lncRNAs (DELs) transcripts were screened, and 2109 regulated lncRNA-mRNA pairs were identified. Subsequently, the genes related to reproduction (IGF1, FGFRL1, and CREB1) and those associated with embryonic development and maturation (DHX34, LHX6) were identified. KEGG analysis of the DEGs revealed that the GnRH- and prolactin-signaling pathways, progesterone-mediated oocyte maturation, and oocyte meiosis were related to reproduction. GSEA and KEGG analyses of the target genes of DELs demonstrated that several biological processes and pathways might interact with oviduct functions and the prolificacy of goats. Furthermore, the co-expression network analysis showed that XLOC_029185, XLOC_040647, and XLOC_090025 were the cis-regulatory elements of the DEGs MUC1, PPP1R9A, and ALDOB, respectively; these factors might be associated with the success of pregnancy and glucolipid metabolism. In addition, the GATA4, LAMA2, SLC39A5, and S100G were trans-regulated by lncRNAs, predominantly mediating oviductal transport to the embryo and energy metabolism. Our findings could pave the way for a better understanding of the roles of mRNAs and lncRNAs in fecundity-related oviduct function in goats. [ABSTRACT FROM AUTHOR]

Artificial directional selection has replaced natural selection and resulted in trait differences across breeds in domestic animal breeding. However, the molecular mechanism by which the oviduct regulates litter size remains largely elusive in goats during the follicular phase. Accumulating data have linked lncRNAs to reproductive activities; however, little is known about the modulation mechanism in the oviduct. Herein, RNA-seq was used to measure mRNA and lncRNA expression levels in low- and high-fecundity goats. We observed distinctive differences in mRNA and lncRNA in terms of different kidding numbers and detected the differential expression of 1640 mRNA transcripts and 271 lncRNA transcripts. Enrichment analysis of differentially expressed mRNAs (DEGs) suggested that multiple pathways, such as the AMPK, PI3K–Akt, calcium signaling pathway, oocyte meiosis, ABC transporter, and ECM–receptor interaction pathways, directly or indirectly affected goat reproduction. Additionally, coexpression of differentially expressed lncRNAs (DEL)-genes analysis showed that XLOC_021615, XLOC_119780, and XLOC_076450 were trans-acting as the DEGs ATAD2, DEPDC5, and TRPM6, respectively, and could regulate embryo development. Moreover, XLOC_020079, XLOC_107361, XLOC_169844, XLOC_252348 were the trans-regulated elements of the DEGs ARHGEF2 and RAPGEF6, and the target DEGs CPEB3 of XLOC_089239, XLOC_090063, XLOC_107409, XLOC_153574, XLOC_211271, XLOC_251687 were associated with prolificacy. Collectively, our study has offered a thorough dissection of the oviduct lncRNA and mRNA landscapes in goats. These results could serve as potential targets of the oviduct affecting fertility in goats. [ABSTRACT FROM AUTHOR]

ALAS1 is a member of the α‐oxoamine synthase family, which is the first rate‐limiting enzyme for heme synthesis and is important for maintaining intracellular heme levels. In the ovary, ALAS1 is associated with the regulation of ovulation‐related mitochondrial P450 cytochromes, steroid metabolism, and steroid hormone production. However, there are few studies on the relationship between ALAS1 and reproductive traits in goats. In this study, a mutation located in the promoter region of ALAS1 (g.48791372C>A) was found to be significantly (p < 0.05) associated with the kidding number of Yunshang black goats. Specifically, the mean kidding number in the first three litters and the kidding numbers of all three litters were significantly (p < 0.05) higher in individuals with the CA genotype or AA genotype than in those with the CC genotype. To further investigate the regulatory mechanism of ALAS1, the expression of ALAS1 in goat ovarian tissues with different genotypes was verified by real‐time quantitative PCR. The results showed that the expression of ALAS1 was significantly higher in the ovaries of individuals with AA genotype than those with AC and CC genotypes (p < 0.01), and the expression trend of transcription factor ASCL2 was consistent with ALAS1. Additionally, the ALAS1 g.48791372C>A mutation created a new binding site for the transcription factor ASCL2. The luciferase activity assay indicated that the mutation increased the promoter activity of ALAS1. Overexpression of the transcription factor ASCL2 induced increased expression of ALAS1 in goat granulosa cells (p < 0.05). The opposite trend was shown for the inhibition of ASCL2 expression. The results of real‐time quantitative PCR, EdU and Cell Counting Kit‐8 assays indicated that the transcription factor ASCL2 increased the proliferation of goat granulosa cells by mediating the expression of ALAS1. In conclusion, the transcription factor ASCL2 positively regulated the transcriptional activity and expression levels of ALAS1, altering granulosa cell proliferation and the kidding number in goats. [ABSTRACT FROM AUTHOR]

Simple Summary: The protein phosphatase 2 regulatory subunit B'gamma (PPP2R5C) and solute carrier family 39 member 5 (SLC39A5) genes are essential in mammalian growth and development. Here, KASP genotyping was used to analyze the association between polymorphisms of PPP2R5C and SLC39A5 and litter size in Yunshang black goats, a cultivated goat breed with high prolificacy in China. The results show that PPP2R5C g.65977743C>T and SLC39A5 g.50676693T>C were significantly associated with litter size of the third parity in Yunshang black goats. Luciferase assays and RT-qPCR showed that individuals with the CC genotype of PPP2R5C and TT genotype of SLC39A5 showed higher expression of these genes in goat ovarian tissues than those with other genotypes. Transcription factor predictions showed that SOX18, ZNF418, and ZNF667 and NKX2-4 and TBX6 bind to the polymorphisms of two respective genes. These two polymorphisms provide new insights into the study of fertility in goats. Screening for candidate genes and genetic variants associated with litter size is important for goat breeding. The aim of this study was to analyze the relationship between single nucleotide polymorphisms (SNPs) in PPP2R5C and SLC39A5 and litter size in Yunshang black goats. KASP genotyping was used to detect the SNP genetic markers in the PPP2R5C and SLC39A5 in a population of 569 Yunshang black goats. The results show that there were two SNPs in the PPP2R5C and SLC39A5 promoter regions. Association analysis revealed that the polymorphisms PPP2R5C g.65977743C>T and SLC39A5 g.50676693T>C were significantly associated with the litter size of the third parity of Yunshang black goats (p < 0.05). To further explore the regulatory mechanism of the two genes, the expression of different genotypes of PPP2R5C and SLC39A5 was validated by RT-qPCR and Western blotting. The expression of PPP2R5C was significantly higher in individuals with the TT genotype than in those with the TC and CC genotypes (p < 0.05). The expression of SLC39A5 was also significantly higher in individuals with the TT genotype than in TC and CC genotypes (p < 0.05). Dual luciferase reporter analysis showed that the luciferase activity of PPP2R5C-C variant was significantly higher than that of PPP2R5C-T variant (p < 0.05). The luciferase activity of SLC39A5-T variant was significantly higher than that of SLC39A5-C variant (p < 0.05). Software was used to predict the binding of transcription factors to the polymorphic sites, and the results show that SOX18, ZNF418, and ZNF667 and NKX2-4 and TBX6 might bind to PPP2R5C g.65977743C>T and SLC39A5 g.50676693T>C, respectively. These results provide new insights into the identification of candidate genes for marker-assisted selection (MAS) in goats. [ABSTRACT FROM AUTHOR]

Simple Summary: The skeletal muscle growth and development affects the production of goat meat. Here, we performed transcriptome sequencing of longissimus dorsi muscle of goats at two development stages (namely, 1-month-old and 9-month-old) by RNA sequencing, assessing the lncRNA expression profile during skeletal muscle development. This study identified regulatory lncRNAs and pathways related to the development of skeletal muscle, providing a reference for future study on the molecular mechanism that regulates the skeletal growth and development. LncRNAs are essential for regulating skeletal muscle. However, the expression profile and function of lncRNAs in goat muscle remains unclear. Here, an average of ~14.58 Gb high-quality reads were obtained from longissimus dorsi tissues of 1-month-old (n = 3) and 9-month-old (n = 3) Wu'an black goats using RNA sequencing. Of a total of 3441 lncRNAs, 1281 were lincRNAs, 805 were antisense lncRNAs, and 1355 were sense_overlapping lncRNAs. These lncRNAs shared some properties with goats, such as fewer exons, shorter transcript, and open reading frames (ORFs) length. Among them, 36 differentially expressed lncRNAs (DE lncRNA) were identified, and then 10 random lncRNAs were validated by RT-qPCR. Furthermore, 30 DE lncRNAs were neighboring 71 mRNAs and several genes were functionally enriched in muscle development-related pathways, such as APC, IFRD1, NKX2-5, and others. Additionally, 36 DE lncRNAs and 2684 mRNAs were included in co-expression interactions. A lncRNA-miRNA-mRNA network containing 4 lncRNAs, 3 miRNAs, and 8 mRNAs was finally constructed, of which XR_001296113.2 might regulate PDLIM7 expression by interaction with chi-miR-1296 to affect skeletal muscle development. This study revealed the expression profile of goat lncRNAs for further investigative studies and provides a fuller understanding of skeletal muscle development. [ABSTRACT FROM AUTHOR]

Long noncoding RNAs (lncRNAs), as competitive endogenous RNAs (ceRNAs), can directly or indirectly affect the proliferation and apoptosis of granulosa cells by regulating microRNA (miRNA) pathways. A ceRNA network of the SLC19A1-AS-miR-1343-WNT11 axis was constructed via comprehensive transcriptome sequencing of ovaries from goats with various fertility levels to further elucidate the function and regulatory mechanism of SLC19A1-AS in modulating miR-1343 and WNT11 during granulosa cell proliferation and apoptosis. Subsequent validation experiments were conducted in vitro using granulosa cells. In these experiments, we performed RNA immunoprecipitation (RIP) and identified SLC19A1-AS as a ceRNA in goat granulosa cells that promoted proliferation. Through bioinformatics prediction, luciferase reporter gene assays, and RNA pulldown assays, we confirmed that SLC19A1-AS acts as a sponge for miR-1343, preventing its binding to WNT11 mRNA and thereby increasing the expression of WNT11. This interaction also influenced the proliferation and apoptosis of granulosa cells. Our study systematically validated the biological function of the lncRNA–miRNA-mRNA ceRNA network in goat ovaries and revealed the potential regulatory mechanism by which SLC19A1-AS functions as a ceRNA in granulosa cells. These findings are expected to provide an important experimental foundation for further elucidating the physiological regulatory network of the ovary and contributing to reproductive health in goats. [Display omitted] [ABSTRACT FROM AUTHOR]