Positive In Vitro Effect of ROCK Pathway Inhibitor Y-27632 on Qualitative Characteristics of Goat Sperm Stored at Low Temperatures.

Y-27632, as a cytoskeleton protector, is commonly used for low-temperature preservation of cells. Goat sperm are prone to damage to the cytoskeleton under low-temperature conditions, leading to a loss of sperm vitality. However, the Y-27632 small molecule has not yet been used in research on low-temperature preservation of goat semen. This study aims to address the issue of low temperature-induced loss of sperm motility in goats by using Y-27632, and explore the regulation of Y-27632 on goat sperm metabolism. At a low temperature of 4 °C, different concentrations of Y-27632 were added to the sperm diluent. The regulation of Y-27632 on the quality of low temperature-preserved goat semen was evaluated by detecting goat sperm motility, antioxidant capacity, mitochondrial activity, cholesterol levels, and metabolomics analysis. The results indicated that 20 µM Y-27632 significantly increased plasma membrane integrity (p < 0.05), and acrosome integrity (p < 0.05) and sperm motility (p < 0.05), increased levels of superoxide dismutase (SOD) and catalase (CAT) (p < 0.01), increased total antioxidant capacity (T-AOC) (p < 0.05), decreased levels of malondialdehyde (MDA) and reactive oxygen species (ROS) (p < 0.01), and significantly increased mitochondrial membrane potential (MMP). The levels of ATP, Ca2+, and TC in sperm increased (p < 0.01). Twenty metabolites with significant differences were identified, with six metabolic pathways having a significant impact, among which the D-glutamic acid and D-glutamine metabolic pathways had the most significant impact. The artificial insemination effect of goat semen treated with 20 µM Y-27632 was not significantly different from that of fresh semen. This study indicates that Y-27632 improves the quality of low-temperature preservation of sperm by protecting the sperm plasma membrane, enhancing sperm antioxidant capacity, regulating D-glutamine and D-glutamate metabolism, and promoting the application of low-temperature preservation of semen in artificial insemination technology.

Multi-Omics Analysis Reveals the Regulatory Mechanism of Probiotics on the Growth Performance of Fattening Sheep.

Probiotics have been proven to improve the growth performance of livestock and poultry. The aim of this experiment was to investigate the effects of probiotic supplementation on the growth performance; rumen and intestinal microbiota; rumen fluid, serum, and urine metabolism; and rumen epithelial cell transcriptomics of fattening meat sheep. Twelve Hu sheep were selected and randomly divided into two groups. They were fed a basal diet (CON) or a basal diet supplemented with 1.5 × 108 CFU/g probiotics (PRB). The results show that the average daily weight gain, and volatile fatty acid and serum antioxidant capacity concentrations of the PRB group were significantly higher than those of the CON group (p < 0.05). Compared to the CON group, the thickness of the rumen muscle layer in the PRB group was significantly decreased (p < 0.01); the thickness of the duodenal muscle layer in the fattening sheep was significantly reduced; and the length of the duodenal villi, the thickness of the cecal and rectal mucosal muscle layers, and the thickness of the cecal, colon, and rectal mucosal layers (p < 0.05) were significantly increased. At the genus level, the addition of probiotics altered the composition of the rumen and intestinal microbiota, significantly upregulating the relative abundance of Subdivision5_genera_incertae_sedis and Acinetobacter in the rumen microbiota, and significantly downregulating the relative abundance of Butyrivibrio, Saccharofermentans, and Fibrobacter. The relative abundance of faecalicoccus was significantly upregulated in the intestinal microbiota, while the relative abundance of Coprococcus, Porphyromonas, and Anaerobacterium were significantly downregulated (p < 0.05). There were significant differences in the rumen, serum, and urine metabolites between the PRB group and the CON group, with 188, 138, and 104 metabolites (p < 0.05), mainly affecting pathways such as vitamin B2, vitamin B3, vitamin B6, and a series of amino acid metabolisms. The differential genes in the transcriptome sequencing were mainly enriched in protein modification regulation (especially histone modification), immune function regulation, and energy metabolism. Therefore, adding probiotics improved the growth performance of fattening sheep by altering the rumen and intestinal microbiota; the rumen, serum, and urine metabolome; and the transcriptome.

Multi-omics analysis on the mechanism of the effect of Isatis leaf on the growth performance of fattening sheep.

Introduction: This study evaluated the effects of Isatis Leaf (ISL) on the growth performance, gastrointestinal tissue morphology, rumen and intestinal microbiota, rumen, serum and urine metabolites, and rumen epithelial tissue transcriptome of fattening sheep. Methods: Twelve 3.5-month-old healthy fattening sheep were randomly divided into two groups, each with 6 replicates, and fed with basal diet (CON) and basal diet supplemented with 80 g/kg ISL for 2.5 months. Gastrointestinal tract was collected for histological analysis, rumen fluid and feces were subjected to metagenomic analysis, rumen fluid, serum, and urine for metabolomics analysis, and rumen epithelial tissue for transcriptomics analysis. Results: The results showed that in the ISL group, the average daily gain and average daily feed intake of fattening sheep were significantly lower than those of the CON group (P < 0.05), and the rumen ammonia nitrogen level was significantly higher than that of the CON group (P < 0.01). The thickness of the reticulum and abomasum muscle layer was significantly increased (P < 0.05). At the genus level, the addition of ISL modified the composition of rumen and fecal microorganisms, and the relative abundance of Methanobrevibacter and Centipeda was significantly upregulated in rumen microorganisms, The relative abundance of Butyrivibrio, Saccharofermentans, Mogibacterium, and Pirellula was significantly downregulated (P < 0.05). In fecal microorganisms, the relative abundance of Papillibacter, Pseudoflavonifractor, Butyricicoccus, Anaerovorax, and Methanocorpusculum was significantly upregulated, while the relative abundance of Roseburia, Coprococcus, Clostridium XVIII, Butyrivibrio, Parasutterella, Macellibacteroides, and Porphyromonas was significantly downregulated (P < 0.05). There were 164, 107, and 77 different metabolites in the rumen, serum, and urine between the ISL and CON groups (P < 0.05). The differential metabolic pathways mainly included thiamine metabolism, niacin and nicotinamide metabolism, vitamin B6 metabolism, taurine and taurine metabolism, beta-Alanine metabolism and riboflavin metabolism. These metabolic pathways were mainly involved in the regulation of energy metabolism and immune function in fattening sheep. Transcriptome sequencing showed that differentially expressed genes were mainly enriched in cellular physiological processes, development, and immune regulation. Conclusion: In summary, the addition of ISL to the diet had the effect of increasing rumen ammonia nitrogen levels, regulating gastrointestinal microbiota, promoting body fat metabolism, and enhancing immunity in fattening sheep.

Antioxidant activity and metabolic regulation of sodium salicylate on goat sperm at low temperature.

OBJECTIVE: The purpose of this study was to explore the effect of sodium salicylate (SS) on semen preservation and metabolic regulation in goats. METHODS: Under the condition of low temperature, SS was added to goat semen diluent to detect goat sperm motility, plasma membrane, acrosome, antioxidant capacity, mitochondrial membrane potential (MMP) and metabonomics. RESULTS: The results show that at the 8th day of low-temperature storage, the sperm motility of the 20 µM SS group was 66.64%, and the integrity rates of the plasma membrane and acrosome were both above 60%, significantly higher than those of the other groups. The activities of catalase and superoxide dismutase in the sperm of the 20 µM SS group were significantly higher than those of the control group, the contents of reactive oxygen species and malondialdehyde were significantly lower than those in the control group, the MMP was significantly higher than that in the control group, and the contents of Ca2+ and total cholesterol were significantly higher than those in the control group. Through metabonomics analysis, there were significant metabolic differences between the control group and the 20 µM SS group. Twenty of the most significant metabolic markers were screened, mainly involving five metabolic pathways, of which nicotinic acid and nicotinamide metabolic pathways were the most significant. CONCLUSION: The results indicate that SS can effectively improve the low-temperature preservation quality of goat sperm.

Roles of Y-27632 on sheep sperm metabolism.

To investigate the effect of Y-27632 on low-temperature metabolism of sheep sperm, different concentrations of Y-27632 were added to sheep semen at 4 °C in this experiment to detect indicators such as sperm motility, plasma membrane, acrosome, antioxidant performance, mitochondrial membrane potential (MMP), and metabolomics. The results showed that the addition of 20 µM Y-27632 significantly increased sperm motility, plasma membrane integrity rate, acrosome integrity rate, antioxidant capacity, MMP level, significantly increased sperm adenosine triphosphate (ATP) and total cholesterol content, and significantly reduced sperm Ca2+ content. In metabolomics analysis, compared with the control group, the 20 µM Y-27632 group screened 20 differential metabolites, mainly involved in five metabolic pathways, with the most significant difference in Histidine metabolism (P = 0.001). The results confirmed that Y-27632 significantly improved the quality of sheep sperm preservation under low-temperature conditions.

Sheep semen preservation and artificial insemination is an important reproductive technology that supports the large-scale and intensive development of the sheep farming industry. Under low-temperature condition, sperm metabolic activity slows down or pauses, energy consumption decreases, thereby prolonging sperm preservation time and motility. During the process of sperm preservation, sperm are susceptible to cold shock damage, which affects the quality of sperm preservation. Y-27632 is a rho-associated cooled-coil kinase (ROCK) inhibitor that competes with ATP to inhibit the kinase activity of ROCK-I and ROCK-II. However, the study of Y-27632 used in sheep semen preservation and its protective mechanism is less. In this study, we used the ROCK inhibitor Y-27632 and the ROCK activator arachidonic acid (AA) for low-temperature preservation of sheep semen and related metabolic regulation mechanisms. This experiment confirmed that Y-27632 played a significant protective role by regulating sperm metabolism and protecting sperm plasma membrane in sheep.

Multi-Omics Analysis of the Mechanism of Mentha Haplocalyx Briq on the Growth and Metabolic Regulation of Fattening Sheep.

Mentha haplocalyx Briq (MHB) and its components have been proven to improve the growth performance of livestock and poultry. The aim of this experiment was to investigate the effects of MHB addition on growth performance, rumen and fecal microbiota, rumen fluid, serum and urine metabolism, and transcriptomics of rumen epithelial cells in meat sheep. Twelve Hu sheep were selected for the experiment and fed with basic diet (CON) and a basal diet supplemented with 80 g/kg DM of Mentha haplocalyx Briq (MHB). The experimental period was 10 weeks with the first 2 weeks as the pre-trial period. The results showed that compared with the CON group, the average daily weight gain of meat sheep in the MHB group increased by 20.1%; the total volatile fatty acid (VFA) concentration significantly increased (p < 0.05); The thickness of the cecal mucosal layer was significantly reduced (p < 0.01), while the thickness of the colonic mucosal layer was significantly increased (p < 0.05), the length of ileal villi significantly increased (p < 0.01), the thickness of colonic mucosal layer and rectal mucosal muscle layer significantly increased (p < 0.05), and the thickness of cecal mucosal layer significantly decreased (p < 0.05); The serum antioxidant capacity has increased. At the genus level, the addition of MHB changed the composition of rumen and fecal microbiota, increased the relative abundance of Paraprevotella, Alloprevotella, Marinilabilia, Saccharibacteria_genera_incertae_sedis, Subdivision5_genera_incertae_sedis and Ornatilinea in rumen microbiota, and decreased the relative abundance of Blautia (p < 0.05). The relative abundance of Prevotella, Clostridium XlVb and Parasutterella increased in fecal microbiota, while the relative abundance of Blautia and Coprococcus decreased (p < 0.05). There were significant differences in the concentrations of 105, 163, and 54 metabolites in the rumen, serum, and urine between the MHB group and the CON group (p < 0.05). The main metabolic pathways of the differences were pyrimidine metabolism, taurine and taurine metabolism, glyceride metabolism, and pentose phosphate pathway (p < 0.05), which had a significant impact on protein synthesis and energy metabolism. The transcriptome sequencing results showed that differentially expressed genes were mainly enriched in immune regulation, energy metabolism, and protein modification. Therefore, adding MHB improved the growth performance of lambs by altering rumen and intestinal microbiota, rumen, serum and urine metabolomics, and transcriptome.

CRISPR-based m6A modification and its potential applications in telomerase regulation.

Telomerase determines cell lifespan by controlling chromosome stability and cell viability, m6A epigenetic modification plays an important role in the regulation of telomerase activity. Using CRISPR epigenome editing to analyze specific m6A modification sites in telomerase will provide an important tool for analyzing the molecular mechanism of m6A modification regulating telomerase activity. In this review, we clarified the relevant applications of CRISPR system, paid special attention to the regulation of m6A modification in stem cells and cancer cells based on CRISPR system, emphasized the regulation of m6A modification on telomerase activity, pointed out that m6A modification sites regulate telomerase activity, and discussed strategies based on telomerase activity and disease treatment, which are helpful to promote the research of anti-aging and tumor related diseases.

MiR-188-5p regulates the proliferation and differentiation of goat skeletal muscle satellite cells by targeting calcium/calmodulin dependent protein kinase II beta.

OBJECTIVE: The aim of this study was to reveal the role and regulatory mechanism of miR-188-5p in the proliferation and differentiation of goat muscle satellite cells. METHODS: Goat skeletal muscle satellite cells isolated in the pre-laboratory were used as the test material. First, the expression of miR-188-5p in goat muscle tissues at different developmental stages was detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR). In addition, miR-188-5p was transfected into goat skeletal muscle satellite cells by constructing mimics and inhibitors of miR-188-5p, respectively. The changes of differentiation marker gene expression were detected by qPCR method. RESULTS: It was highly expressed in adult goat latissimus dorsi and leg muscles, goat fetal skeletal muscle, and at the differentiation stage of muscle satellite cells. Overexpression and interference of miR-188-5p showed that miR-188-5p inhibited the proliferation and promoted the differentiation of goat muscle satellite cells. Target gene prediction and dual luciferase assays showed that miR-188-5p could target the 3'untranslated region of the calcium/calmodulin dependent protein kinase II beta (CAMK2B) gene and inhibit luciferase activity. Further functional studies revealed that CAMK2B promoted the proliferation and inhibited the differentiation of goat muscle satellite cells, whereas si-CAMK2B restored the function of miR-188-5p inhibitor. CONCLUSION: These results suggest that miR-188-5p inhibits the proliferation and promotes the differentiation of goat muscle satellite cells by targeting CAMK2B. This study will provide a theoretical reference for future studies on the molecular mechanisms of skeletal muscle development in goats.

Analysis of serum reproductive hormones and ovarian genes in pubertal female goats.

BACKGROUND: Age at puberty is an important factor affecting goat fertility, with endocrine and genetic factors playing a crucial role in the onset of puberty. To better understand the relationship between endocrine and genetic factors and mechanisms underlying puberty onset in goats, reproductive hormone levels were analyzed by ELISA and ultraperformance liquid chromatography-multiple reaction monitoring-multistage/mass spectrometry and RNA sequencing was performed to analyze ovarian genes. RESULTS: Serum follicle stimulating hormone, luteinizing hormone, estradiol, 11-deoxycortisol, 11-deoxycorticosterone, corticosterone, cortisone, and cortisol levels were found to be higher but progesterone were lower in pubertal goats as compared to those in prepubertal goats (P < 0.05). A total of 18,139 genes were identified in cDNA libraries, and 75 differentially expressed genes (DEGs) were identified (|log2 fold change|≥ 1, P ≤ 0.05), of which 32 were significantly up- and 43 were down-regulated in pubertal goats. Gene ontology enrichment analyses indicated that DEGs were mainly involved in "metabolic process," "signaling," "reproduction," and "growth." Further, DEGs were significantly enriched in 91 Kyoto Encyclopedia of Genes and Genomes pathways, including estrogen signaling pathway, steroid hormone biosynthesis, and cAMP signaling pathway. Bioinformatics analysis showed that PRLR and THBS1 were highly expressed in pubertal ovaries, and ZP3, ZP4, and ASTL showed low expression, suggesting their involvement in follicular development and lutealization. CONCLUSIONS: To summarize, serum hormone changes and ovarian DEGs expression were investigated in our study. Further studies are warranted to comprehensively explore the functions of DEGs in goat puberty.

Metabolomics Analysis of Sodium Salicylate Improving the Preservation Quality of Ram Sperm.

The aim of this study was to investigate the effects of sodium salicylate (SS) on the preservation and metabolic regulation of sheep sperm. Under 4 °C low-temperature conditions, SS (at 10 µM, 20 µM, 30 µM, and 50 µM) was added to the semen diluent to detect sperm motility, plasma membrane, and acrosome integrity. Based on the selected optimal concentration of SS (20 µM), the effects of 20 µM of SS on sperms' antioxidant capacity and mitochondrial membrane potential (MMP) were evaluated, and metabolomics analysis was conducted. The results showed that on the 20th day of low-temperature storage, the sperm motility of the 20 µM SS group was 62.80%, and the activities of catalase (CAT) and superoxide dismutase (SOD) were significantly higher than those of the control group (p < 0.01). The content of Ca2+, reactive oxygen species (ROS), and malondialdehyde (MDA) were significantly lower than those of the control group (p < 0.01), and the total antioxidant capacity (T-AOC) was significantly higher than that of the control group (p < 0.05); mitochondrial activity and the total cholesterol (TC) content were significantly higher than those in the control group (p < 0.01). An ultrastructural examination showed that in the SS group, the sperm plasma membrane and acrosome were intact, the fibrous sheath and axoneme morphology of the outer dense fibers were normal, and the mitochondria were arranged neatly. In the control group, there was significant swelling of the sperm plasma membrane, rupture of the acrosome, and vacuolization of mitochondria. Using metabolomics analysis, 20 of the most significant differential metabolic markers were screened, mainly involving 6 metabolic pathways, with the amino acid biosynthesis pathway being the most abundant. In summary, 20 µM of SS significantly improved the preservation quality of sheep sperm under low-temperature conditions of 4 °C.

Genome-Wide Association Study of Egg-Laying Traits and Egg Quality in LingKun Chickens.

Egg production is the most important trait of laying hens. To identify molecular markers and candidate genes associated with egg production and quality, such as body weight at first oviposition (BWF), the number of eggs produced in 500 days (EN500), egg weight (EW), egg shell thickness (EST), egg shell strength (ESS), and Haugh unit (HU), a genome-wide analysis was performed in 266 LingKun Chickens. The results showed that thirty-seven single nucleotide polymorphisms (SNPs) were associated with all traits (p < 9.47 × 10-8, Bonferroni correction). These SNPs were located in close proximity to or within the sequence of the thirteen candidate genes, such as Galanin And GMAP Prepropeptide (GAL), Centromere Protein (CENPF), Glypican 2 (GPC2), Phosphatidylethanolamine N-Methyltransferase (PEMT), Transcription Factor AP-2 Delta (TFAP2D), and Carboxypeptidase Q (CPQ) gene related to egg-laying and Solute Carrier Family 5 Member 7 (SLC5A7), Neurocalcin Delta (NCALD), Proteasome 20S Subunit Beta 2 (PSMB2), Slit Guidance Ligand 3 (SLIT3), and Tubulin Tyrosine Ligase Like 7 (TTLL7) genes related to egg quality. Interestingly, one of the genes involved in bone formation (SLIT3) was identified as a candidate gene for ESS. Our candidate genes and SNPs associated with egg-laying traits were significant for molecular breeding of egg-laying traits and egg quality in LingKun chickens.

Genetic Regulation of N6-Methyladenosine-RNA in Mammalian Gametogenesis and Embryonic Development.

Emerging evidence shows that m6A is the most abundant modification in eukaryotic RNA molecules. It has only recently been found that this epigenetic modification plays an important role in many physiological and pathological processes, such as cell fate commitment, immune response, obesity, tumorigenesis, and relevant for the present review, gametogenesis. Notably the RNA metabolism process mediated by m6A is controlled and regulated by a series of proteins termed writers, readers and erasers that are highly expressed in germ cells and somatic cells of gonads. Here, we review and discuss the expression and the functional emerging roles of m6A in gametogenesis and early embryogenesis of mammals. Besides updated references about such new topics, readers might find in the present work inspiration and clues to elucidate epigenetic molecular mechanisms of reproductive dysfunction and perspectives for future research.

Analysis of protein phosphorylation sites in the hypothalamus tissues of pubescent goats.

Protein phosphorylation plays an important role in animal reproduction. However, its role in the onset of puberty in goats remains largely unexplored. Accordingly, in the present study, the molecular changes controlling the onset of puberty in goats were investigated by identifying the differentially phosphorylated proteins (DPPs) and phosphorylation sites (DPSs) in the hypothalami of prepubertal and pubertal female goats using LC-MS/MS and tandem mass tag labelling. A total of 3265 phosphopeptides corresponding to 1628 phosphoproteins were identified, including 234 upregulated and 342 downregulated phosphopeptides. The DPSs HTT, MAP1B, CAMKK1, MAP2, DNAJC5, and GAP43 were identified. These DPPs are enriched in the endocytosis, cAMP signaling, Rap1 signaling, melanogenesis, and insulin secretion pathways. These pathways are related to gonadotropin-releasing hormone and puberty. In particular, glucose-6-phosphate isomerase, fructose-bisphosphate aldolase C, and fructose-bisphosphate aldolase A occupy important locations in the protein-protein interaction network. These data provide evidence for a complex interaction network in goat hypothalamus proteins that affects puberty. Furthermore, they may help identify new puberty-regulating candidates and/or serve as an important resource for exploring the physiological mechanism of puberty onset in mammals. SIGNIFICANCE: This study provides evidence for a complex interaction network in goat hypothalamus proteins that affects puberty. Furthermore, our data may help identify new puberty-regulating candidates and/or serve as an important resource for exploring the physiological mechanism of puberty onset in mammals.

Effects of Coated Sodium Butyrate and Polysaccharides From Cordyceps cicadae on Intestinal Tissue Morphology and Ileal Microbiome of Squabs.

This experiment was conducted to investigate the effects of dietary supplementation with different levels of coated sodium butyrate (CSB) and polysaccharides extracted from Cordyceps cicadae (CCP) on growth performance, intestinal tissue morphology and ileum microbiome in squabs. A total of 420 1-day-old squabs were randomly divided into seven groups with 5 replicates each and 12 squabs per replicate. The squabs were fed basal diet (control group) and basal diet supplemented with different levels of CSB (275, 550, and 1,100 mg/kg, groups CSB-275, CSB-550, CSB-1100) and CCP (27.5, 55, and 110 mg/kg, groups CCP-27.5, CCP-55, and CCP-110), respectively. The experiment was conducted for 28 days. The results revealed that the final BW and average daily gain concentration were higher (P < 0.05) in squabs of CSB-275 and CCP-110 groups than those in the CON group. Comparing with control group, the squabs in the groups CSB-275, CSB-550, and CCP-55 obtained higher villus height/crypt depth (VH/CD) of the duodenum and higher VH of the jejunum (P < 0.05). Operational taxonomic units in the groups CSB-550 and CCP-27.5 were also increased (P < 0.05). Regarding the relative abundance of flora, the Actinobacteria abundance in the groups CSB-550, CSB-1100, and CCP-55 were higher than in control group (P < 0.05), and the Aeriscardovia abundance of CSB-275, CSB-550, CSB-1100, and CCP-110 were elevated (P < 0.05). However, the Enterococcus abundance in CSB-275, CSB-550, CSB-1100, and CCP-27.5 decreased (P < 0.05). In summary, results obtained in the present study indicate that CSB and CCP can improve growth performance, intestinal microbial balance and gut health of squabs.

Proteomic analysis of hypothalamus in prepubertal and pubertal female goat.

The functions of proteins at the onset of puberty in goats remain largely unexplored. To identify the proteins regulating puberty in goats, we analysed protein abundance and pathways in the hypothalamus of female goats. We applied tandem mass tag (TMT) labelling, liquid chromatography-tandem mass spectrometry (LC-MS/MS), and parallel reaction monitoring (PRM) to examine hypothalamus of pubertal (cases; n = 3) and prepubertal (controls; n = 3) goats. We identified 5119 proteins, including 69 differentially abundant proteins (DAPs), of which 35 were upregulated and 34 were downregulated. Fourteen DAPs were randomly selected to verify these results using PRM, and the results were consistent with the TMT quantitative results. DAPs were enriched in MAPK signalling pathway, Ras signalling pathway, Autophagy-animal, Endocytosis, and PI3K/Akt/mTOR signalling pathway categories. These pathways are related to embryogenesis, cell proliferation, cell differentiation, and promoting the release of gonadotropin-releasing hormone (GnRH) in the hypothalamus. In particular, PDGFRß and MAP3K7 occupied important locations in the protein-protein interaction network. The results demonstrate that DAPs and their related signalling pathways are crucial in regulating puberty in goats. However, further research is needed to explore the functions of DAPs and their pathways to provide new insights into the mechanism of puberty onset. SIGNIFICANCE: In domestic animals, reaching the age of puberty is an event that contributes significantly to lifetime reproductive potential. And the hypothalamus functions directly in the complex systemic changes that control puberty. Our study was the first TMT proteomics analysis on hypothalamus tissues of pubertal goats, which revealed the changes of protein and pathways that are related to the onset of puberty. We identified 69 DAPs, which were enriched in the MAPK signaling pathway, the Ras signaling pathway, and the IGF-1/PI3K/Akt/mTOR pathway, suggesting that these processes were probably involved in the onset of puberty.

Knockdown of Ptprn-2 delays the onset of puberty in female rats.

In the present study, we evaluated how Ptprn-2 (encoding tyrosine phosphatase, receptor type, N2 polypeptide protein) affects the onset of puberty in female rats. We evaluated the expression of Ptprn-2 mRNA and protein in the hypothalamus-pituitary-ovary axis of female rats using real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) and immunofluorescence at infancy, prepuberty, puberty, peripuberty, and adulthood. We evaluated the effects of Ptprn-2 gene knockdown on different aspects of reproduction-related biology in female rats, including the expression levels of puberty-related genes in vivo and in vitro, the time to onset of puberty, the concentration of serum reproductive hormones, the morphology of ovaries, and the ultrastructure of pituitary gonadotropin cells. Our results demonstrated that PTPRN-2 was primarily distributed in the arcuate nucleus (ARC), periventricular nucleus (PeN), adenohypophysis, and the ovarian follicular theca, stroma, and granulosa cells of female rats at various stages. Ptprn-2 mRNA levels significantly varied between peripuberty and puberty (P < 0.05) in the hypothalamus and pituitary gland. In hypothalamic cells, Ptprn-2 knockdown decreased the expression of Ptprn-2 and Rfrp-3 mRNA (P < 0.05) and increased the levels of Gnrh and Kiss-1 mRNA (P < 0.05). Ptprn-2 knockdown in the hypothalamus resulted in delayed vaginal opening compared to the control group (n = 12, P < 0.01), and Ptprn-2, Gnrh, and Kiss-1 mRNA levels (P < 0.05) all decreased, while the expression of Igf-1 (P < 0.05) and Rfrp-3 mRNA (P < 0.01) increased. The concentrations of FSH and P4 in the serum of Ptprn-2 knockdown rats were lower than in control animals (P < 0.05). Large transverse perimeters and longitudinal perimeters (P < 0.05) were found in the ovaries of Ptprn-2 knockdown rats. There were fewer large secretory particles from gonadotropin cells in adenohypophysis tissue of the Ptprn-2 knockdown group compared to the control group. This indicates that Ptprn-2 knockdown can regulate levels of Gnrh, Kiss-1, and Rfrp-3 mRNA in the hypothalamus, regulate the concentration of serum FSH and P4, and alter the morphology of ovarian and gonadotropin cells, delaying the onset of puberty in female rats.

Establishment of lncRNA-mRNA network in bovine oocyte between germinal vesicle and metaphase II stage.

Long non-coding RNA (lncRNA), a type of non-protein coding transcripts with lengths exceeding 200 nucleotides, is reported to be widely involved in many cellular and developmental processes. However, few roles of lncRNA in oocyte development have been defined. In this study, to uncover the effect of lncRNA during oocyte maturation, bovine germinal vesicle (GV) and in vitro matured metaphase II (MII) oocytes underwent RNA sequencing. Results revealed a wealth of candidate lncRNAs, which might participate in the biological processes of stage-specific oocytes. Furthermore, their trans- and cis-regulatory effects were investigated in-depth by using bioinformatic software. Functional enrichment analysis of target genes showed that these lncRNAs were likely involved in the regulation of many key signaling pathways during bovine oocyte maturation from GV to MII stage, as well as multiple lncRNA-mRNA networks. One novel lncRNA (MSTRG.19140) was particularly interesting, as it appeared to mediate the regulation of oocyte meiotic resumption, progesterone-mediated oocyte maturation, and cell cycle. Therefore, this study enhanced insights into the regulation of molecular mechanisms of bovine oocyte maturation from a lncRNA-mRNA network perspective.

Genome-Wide Network of lncRNA-mRNA During Ovine Oocyte Development From Germinal Vesicle to Metaphase II in vitro.

Long non-coding RNA (lncRNA) is involved in many biological processes, and it has been closely investigated. However, research into the role of lncRNA in ovine ovarian development is scant and poorly understood, particularly in relation to the molecular mechanisms of ovine oocyte maturation. In the current study, RNA sequencing was performed with germinal vesicle (GV) and in vitro matured metaphase II (MII) stage oocytes, isolated from ewes. Through the use of bioinformatic analysis, abundant candidate lncRNAs in stage-specific ovine oocytes were identified, and their trans- and cis-regulatory effects were deeply dissected using computational prediction software. Functional enrichment analysis of these lncRNAs revealed that they were involved in the regulation of many key signaling pathways during ovine oocyte development, which was reflected by their targeted genes. From this study, multiple lncRNA-mRNA networks were presumed to be involved in key signaling pathways regarding ovine oocyte maturation and meiotic resumption. In particular, one novel lncRNA (MSTRG.17927) appeared to mediate the regulation of phosphatidylinositol 3-kinase signaling (PI3K) signaling during ovine oocyte maturation. Therefore, this research offers novel insights into the molecular mechanisms underlying ovine oocyte meiotic maturation regulated by lncRNA-mRNA networks from a genome-wide perspective.

Effect of GABA-T on Reproductive Function in Female Rats.

This study explored the role of γ-aminobutyric acid transaminase (GABA-T) in the puberty and reproductive performance of female rats. Immunofluorescence technique, quantitative real-time PCR (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA) were used to detect the distribution of GABA-T and the expression of genes and hormones in female rats, respectively. The results showed that GABA-T was mainly distributed in the arcuate nucleus (ARC), paraventricular nucleus (PVN) and periventricular nucleus (PeN) of the hypothalamus, and in the adenohypophysis, ovarian granulosa cells and oocytes. Abat mRNA level at 28 d was lowest in the hypothalamus and the pituitary; at puberty, it was lowest in the ovary. Abat mRNA level was highest in adults in the hypothalamus; at infancy and puberty, it was highest in the pituitary; and at 21 d it was highest in the ovary. After vigabatrin (GABA-T irreversible inhibitor) was added to hypothalamus cells, the levels of Abat mRNA and Rfrp-3 mRNA were significantly reduced, but Gnrh mRNA increased at the dose of 25 and 50 µg/mL; Kiss1 mRNA was significantly increased but Gabbr1 mRNA was reduced at the 50 µg/mL dose. In prepubertal rats injected with vigabatrin, puberty onset was delayed. Abat mRNA, Kiss1 mRNA and Gnrh mRNA levels were significantly reduced, but Rfrp-3 mRNA level increased in the hypothalamus. Vigabatrin reduced the concentrations of GABA-T, luteinizing hormone (LH) and progesterone (P4), and the ovarian index. Lactation performance was reduced in adult rats with vigabatrin treatment. Four hours after vigabatrin injection, the concentrations of GABA-T and LH were significantly reduced in adult and 25 d rats, but follicle-stimulating hormone (FSH) increased in 25 d rats. In conclusion, GABA-T affects the reproductive function of female rats by regulating the levels of Gnrh, Kiss1 and Rfrp-3 in the hypothalamus as well as the concentrations of LH and P4.