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The mammary gland of mammals possesses the specific function of synthesizing, secreting, and delivering milk. Notably, mammary epithelial cells are considered to be central to control the expansion and remodeling of mammary gland into a milk-secretory organ. And the biological function of mammary gland is mainly regulated by the endocrine system, especially for estrogen. G protein-coupled receptor 30 (GPR30), an estrogen membrane receptor, mediates estrogen-induced functions of physiology and pathophysiology. However, the relationship between estrogen/GPR30 signaling and proliferation of goat mammary epithelial cells (gMECs) is still unclear. Herein, estrogen promoted cell proliferation than control, as evidence by upregulation of cell numbers, BrdU-positive cell counts, and cell viability. Of note, these activities were all obviously reduced by treatment with GPR30 antagonist G15, yet GPR30 agonist G1 increased cell proliferation than control. Further, GPR30 silencing inhibited cell proliferation than negative control. This inhibition was accompanied by a G2/M phase arrest and downregulation of cell cycle regulators. Meanwhile, estrogen increased the phosphorylation of ERK1/2 and AKT. Further, the protein level of p-ERK1/2 and p-AKT was enhanced by GPR30 agonist G1 but inhibited by GPR30 antagonist G15 and GPR30 silencing. Importantly, MEK inhibitor and PI3K inhibitor decreased the expression of cell cycle regulators, and repressed estrogen-induced and G1-driven promotion of cell proliferation, suggesting that estrogen regulated cell proliferation of gMECs through mechanisms involving cell cycle, dependent of GPR30 and MEK/ERK and PI3K/AKT signaling pathway. This may provide a strong theoretical basis for researching estrogen sustained-release drugs promoting breast development and improving lactation performance. Abbreviations: gMECs, goat mammary epithelial cells; E2, 17β-estradiol; GPR30, G protein-coupled receptor 30; shRNA, small hairpin RNA; CDK, cyclin-dependent kinase; PI3K, phosphatidylinositol 3-kinase; AKT, proteinkinase B; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase kinase; ERK1/2, extracellular signal-regulated kinase 1/2.

Goat milk contains a rich source of nutrients, especially unsaturated fatty acids. However, the regulatory mechanism of milk fat and fatty acid synthesis remains unclear. Stearoyl-CoA desaturase 1 (SCD1) is the key enzyme catalyzing monounsaturated fatty acid synthesis and is essential for milk lipid metabolism. To explore milk lipid synthesis mechanism

G protein-coupled estrogen receptor (GPER), which is different from traditional estrogen nuclear receptors (ERs), mediates the rapid transduction of nongenomic signals in cells, and works by regulating transcription and intracellular second messengers. Studies have shown that GPER may regulate oocyte maturation, but the relevant mechanism is not entirely clear. Here, goat cumulus-oocyte complexes (COCs) were used as a model to explore the regulation and mechanism of GPER on oocyte maturation. Our study showed that 17β-estradiol (E2) significantly reduced cyclic guanosine monophosphate (cGMP) synthesis in COCs and accelerated the meiotic resumption of goat oocytes via GPER. Further investigation found that GPER mediated the downregulation of natriuretic peptide receptor 2 (NPR2) protein expression in goat cumulus cells by E2. In addition, we found that E2 significantly upregulated the mRNA levels of epidermal growth (EGF)-like factors in goat cumulus cells through GPER, and activated the downstream EGF receptor (EGFR) and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathways. Both AG1478 (EGFR inhibitor) and U0126 (ERK1/2 inhibitor) abolished the inhibitory effect of E2 on the protein expression of NPR2. These results indicate that, through GPER, E2 upregulates the mRNA levels of EGF-like factors in goat cumulus cells and activates the downstream EGF signaling network to suppress the expression of NPR2 protein, which results in a decrease in cGMP synthesis and acceleration of meiotic resumption in goat oocytes.

Decreased oocyte quality and compromised embryo development are particularly prevalent in older females, but the aging-related cellular processes and effective ameliorative approaches have not been fully characterized. Intermittent fasting (IF) can help improve health and extend lifespan; nevertheless, how it regulates reproductive aging and its mechanisms remain unclear. We used naturally aged mice to investigate the role of IF in reproduction and found that just one month of every-other-day fasting was sufficient to improve oocyte quality. IF not only increased antral follicle numbers and ovulation but also enhanced oocyte meiotic competence and embryonic development by improving both nuclear and cytoplasmic maturation in maternally aged oocytes. The beneficial effects of IF manifested as alleviation of spindle structure abnormalities and chromosome segregation errors and maintenance of the correct cytoplasmic organelle reorganization. Moreover, single-cell transcriptome analysis showed that the positive impact of IF on aged oocytes was mediated by restoration of the nicotinamide adenine dinucleotide (NAD+)/Sirt1-mediated antioxidant defense system, which eliminated excessive accumulated ROS to suppress DNA damage and apoptosis. Collectively, these findings suggest that IF is a feasible approach to protect oocytes against advanced maternal age-related oxidation damage and to improve the reproductive outcomes of aged females.

Mastitis is one of the most frequent clinical diseases in dairy animals. Epithelial cells undergoing epithelial-mesenchymal transition (EMT) promote the process of mastitis. Oestrogen deficiency is disadvantaged of many tissue inflammation and regeneration, while exogenous oestrogen treatment can reverse these effects. G protein-coupled estrogen receptor 1 (GPER1) is a membrane estrogen receptor. However, the potential effects of oestrogen via GPER1 on EMT in goat mammary epithelial cells (GMECs) are still unclear. Here, this study discovered that the activation of GPER1 by oestrogen could inhibit the EMT in GMECs via NF-κB signalling pathway. The activation of GPER1 by oestrogen inhibited the EMT accompanied by upregulation of E-cadherin and downregulation of N-cadherin and vimentin. Meanwhile, mRNA expression of transcription factors including Snail1 and ZEB1 was decreased. Further, like to oestrogen, GPER1 agonist G1 repressed the EMT progression. Conversely, GPER1 antagonist G15 reversed all these features induced by oestrogen. What's more, GPER1 silencing with shRNA promoted GMECs undergoing EMT. Additionally, oestrogen increased the phosphorylation of Erk1/2, which then decreased the phosphorylation and nuclear translocation of NF-κB, inhibiting the NF-κB signalling pathway activity. Taken, GPER1 may act as a suppressor through the regulation of EMT to prevent the development of mastitis.

Adipose tissue formation and moderate fat deposition are important for the production performance and eating quality of livestock meats. The self-renewal and adipogenic differentiation of adipose-derived stem cells are responsible for the formation and development of adipose tissue. In addition, estrogen targeting G protein-coupled estrogen receptor 1 (GPER1) has been reported to modulate cell proliferation and differentiation during tissue and organ development. However, the potential correlation among estrogen, GPER1, proliferation, and adipogenic differentiation in goat adipose-derived stem cells (gADSCs) is still unclear. Herein, we demonstrated that 17β-estradiol enhances the proliferative ability of gADSCs, indicated by the increased cell number and cell viability, accompanied by up-regulated expressions of cyclin D1 and PCNA. Meanwhile, the adipogenic differentiation is promoted by 17β-estradiol, supported by higher ccumulation of intracellular lipids and increased expressions of PPARγ, ACC, and FABP4. Notably, these activities are all obviously reduced by administration with GPER1 antagonist G15, but GPER1 agonist G1 enhances cell proliferation and adipogenic differentiation. Moreover, GPER1 silencing diminishes cell proliferation and adipogenic differentiation. In parallel, 17β-estradiol elevates the protein level of nuclear p-p65. Furthermore, the phosphorylation of p65 is enhanced by G1 but inhibited by G15 and GPER1 silencing. In addition, the phosphorylation of p65 is mediated by ERK1/2, suggesting that estrogen targeting GPER1 regulates cell proliferation and adipogenic differentiation of gADSCs through the ERK1/2-NF-κB signaling pathway. This study may provide a strong theoretical basis for improving meat quality, flavor, and cold resistance of livestock.

The estrogen membrane receptor GPR30 (also known as G-protein coupled receptor 30) has recently been shown to be involved in the regulation of oocyte maturation and cumulus expansion. However, whether GPR30 expression is regulated by gonadotropin stimulation and how it participates in the regulation of the maturation process is still not clear. In this study, we explored the mechanism underlying the synergy between luteinizing hormone and 17β-estradiol (17β-E2) to improve the epidermal growth factor (EGF) response in cumulus oocyte complexes (COCs) during oocyte maturation in mice. The expression and distribution of GPR30, EGFR, and EGF-like growth factors were examined by real-time quantitative PCR, western blot, and immunofluorescence staining. Lyso-Tracker Red labeling was performed to detect the lysosomal activity in follicle granular cells (FGCs). Cumulus expansion of COCs was evaluated after in vitro maturation for 16 h. We found that EGF-like growth factors transmit LH signals to increase GRP30 levels by inhibiting protein degradation in lysosomes. Meanwhile, 17β-E2 stimulates the GPR30 signaling pathway to increase EGF receptor levels, enhancing the response ability of EGF signaling in COCs and thus promoting cumulus expansion. In conclusion, our study reveals the synergistic mechanism between LH and estrogen in the regulation of cumulus expansion during oocyte maturation process.

Background: Concentrated growth factor (CGF) is a promising scaffold in regenerative endodontic procedures (REPs) for its abundant growth factors. However, no clinical study has evaluated the effectiveness of CGF compared with the conventional scaffold in REPs. The purpose of this study was to investigate and compare the success rate of CGF and blood clot (BC) as scaffolds in REPs.Methods: Immature permanent teeth diagnosed with pulp necrosis treated with REPs between 2012 and 2020 with a minimum of 6-month follow-up were included. These teeth were divided into the CGF and BC groups. Treatment outcomes were assessed using a combined clinical and radiographic scoring system. Preoperative and intraoperative data included tooth type, aetiology, capping materials, and restorative materials. Statistical analysis was performed using the chi-square test and Kaplan-Meier survival analysis at a significance level of 0.05.Results: A total of 121 teeth from 107 children were included. The CGF and BC groups included 53 and 68 teeth, respectively. The total success rate was 91.74% over a mean follow-up period of 23.15 months. There was no significant difference between the CGF group (86.79%) and BC group (95.59%). In both groups, a high proportion of teeth were scored 2 (increased root wall thickness and/or increased root length), and there was no significant difference in the scores between groups. The success rate of traumatic teeth (84.31%) was significantly lower than that of teeth with developmental dental anomalies (98.39%). Conclusions: CGF may be a suitable alternative scaffold in REPs when adequate bleeding cannot be achieved. Moreover, compared to developmental dental anomalies, traumatic teeth treated by REPs may be more vulnerable to failure.Trial registration: Retrospectively registered.

4-Vinylcyclohexene diepoxide (VCD) is a potentially hazardous industrial chemical that may enter a goat's body in various ways during industrial breeding. Ovarian granulosa cells (GCs) play a critical role in supporting follicle development and hormone synthesis. However, there are few studies on the effect of VCD on goat ovarian GCs. In this study, goat ovarian GCs were isolated and treated with VCD. The results showed that treatment with VCD increased the proportion of S phase and G2/M cells, but decreased the proportion of G1 phase. VCD treatment significantly inhibited the expression of cyclin A and cyclin-dependent kinase 2 (CDK2). But the expression levels of p21 and p27 were increased. VCD could induce an apparent increase in the proportion of apoptosis and the level of cleaved caspase 3. Treatment with VCD significantly reduced the progesterone and estrogen concentration in the medium in which goat ovarian GCs were cultured. Correspondingly, the expression level of steroidogenic acute regulatory protein (STAR) was significantly downregulated. Treatment with 0.25 and 0.5 mM VCD, the protein expression level of insulin-like growth factor 1 receptor (IGF1R) and Akt were significantly decreased. Moreover, treatment with 0.25 mM VCD significantly inhibited the phosphorylation of Akt. In conclusion, VCD exposure had cytotoxic effects such as decreased cell viability, disordered cell cycle, increased apoptosis, and interference with steroid hormone synthesis on goat GCs. These cytotoxic effects of VCD on goat GCs may be due to the downregulation of IGF1R and the inhibition of IGF1R/Akt signaling pathway.

Simple Summary Mastitis is a common and important clinical disease in ruminants, resulting in decreased milk production, infertility and delayed conception. If not treated promptly, mastitis may result in fibrotic mastitis. Although epithelial–mesenchymal transition (EMT) is a typical characteristic of fibrotic diseases, the relationship between EMT and mastitis remains largely unknown. NF-κB and Snail are key regulators of the EMT. In the present study, we found that lipopolysaccharide (LPS) induced EMT in primary goat mammary epithelial cells (GMECs). Additionally, the expression of Snail was induced by LPS and was inhibited by the suppression of the TLR4/NF-κB signaling pathway. The knockdown of Snail alleviated LPS-induced EMT and altered the expression of inflammatory cytokines. Finally, we found that the expression of key molecules of the TLR4/NF-κB/Snail signaling pathway was increased in mastitic tissues. This study provides evidence that LPS induces EMT in GMECs through the TLR4/NF-κB/Snail signaling pathway and lays a theoretical foundation for further exploration of the pathological mechanism and treatment of mastitis. Abstract Mastitis is a common and important clinical disease in ruminants. This may be associated with inflammatory fibrosis if not treated promptly. Inflammation-derived fibrosis is usually accompanied by epithelial–mesenchymal transition (EMT) in epithelial cells. However, the precise molecular mechanism underlying mastitis-induced fibrosis remains unclear. Nuclear factor kappa-B (NF-κB) and Snail are key regulators of EMT. In this study, primary goat mammary epithelial cells (GMECs) were treated with 10 μg/mL lipopolysaccharide (LPS) for 14 d to mimic the in vivo mastitis environment. After LPS treatment, the GMECs underwent mesenchymal morphological transformation and expressed mesenchymal cell markers. Snail expression was induced by LPS and was inhibited by suppression of the TLR4/NF-κB signaling pathway. Snail knockdown alleviated LPS-induced EMT and altered the expression of inflammatory cytokines. Finally, we found that the expression of key molecules of the TLR4/NF-κB/Snail signaling pathway was increased in mastitis tissues. These results suggest that Snail plays a vital role in LPS-induced EMT in GMECs and that the mechanism is dependent on the activation of the TLR4/NF-κB signaling pathway.

Mitochondria play a key role in embryo development by providing energy. However, vitrification often causes mitochondrion damage of embryo, which further impairs embryo development. Therefore, the efficiency of embryo development after vitrification could be improved by protecting mitochondrial function from vitrification injury. The purpose of this study was to investigate the effects of resveratrol on mitochondrial damage after vitrification. The results showed that vitrification induced the abnormal mitochondrial distribution and damage mitochondrial function of mouse 2-cell embryos. However, co-culturing with resveratrol for 2 h could repair the abnormal mitochondrial distribution and mitochondrial dysfunction of embryos after vitrification. More than anything, the subsequent development ability of vitrified-thawed 2-cell embryos was significantly higher than that with no resveratrol treatment. In conclusion, resveratrol could protect the mitochondrial from injury caused by vitrification.

Base editing has the potential to improve important economic traits in agriculture and can precisely convert single nucleotides in DNA or RNA sequences into minimal double-strand DNA breaks (DSB). Adenine base editors (ABE) have recently emerged as a base editing tool for the conversion of targeted A:T to G:C, but have not yet been used in sheep. ABEmax is one of the latest versions of ABE, which consists of a catalytically-impaired nuclease and a laboratory-evolved DNA-adenosine deaminase. The Booroola fecundity (FecBB) mutation (g.A746G, p.Q249R) in the bone morphogenetic protein receptor 1B (BMPR1B) gene influences fecundity in many sheep breeds. In this study, by using ABEmax we successfully obtained lambs with defined point mutations that result in an amino acid substitution (p.Gln249Arg). The efficiency of the defined point mutations was 75% in newborn lambs, since six lambs were heterozygous at the FecBB mutation site (g.A746G, p.Q249R), and two lambs were wild-type. We did not detect off-target mutations in the eight edited lambs. Here, we report the validation of the first gene-edited sheep generated by ABE and highlight its potential to improve economically important traits in livestock.

The CRISPR/Cas9 system has been extensively used to engineer genetic loci for the generation of knockouts, insertions, and point mutations in animal models. However, many mutations that have been reported in animals are small insertions or deletions. This study used the CRISPR/Cas9 system to induce large DNA fragment deletions in MSTN via three guide RNAs in sheep. This successfully achieved the precise gene editing of the ovine MSTN gene by injecting both Cas9 mRNA and sgRNAs into embryos at the one-cell stage. Of 10 edited animals, 3 animals (30%) exhibited large genomic fragment deletions (~5 kb). Furthermore, the body weights of these 3 animals were significantly different (P0>0.0001, P15=0.001, P30=0.005, P60=0.027) between lambs with large deletions and wildtype lambs. In addition, the edited lambs were also significantly different (P0>0.0001, P15>0.0001, P30=0.002, P60=0.011) compared with wildtype. These results suggest that the generated MSTN knockout sheep is a reliable and effective animal model for further study. Furthermore, this method is time- and labor-saving, and efficient for the creation of animal models for agriculture, biology, and medicine.

Background C- type natriuretic peptide (CNP) and its receptor natriuretic peptide receptor B (NPR-B) were members of natriuretic peptides system which were well known produced in cardiovascular and central nervous system. However, whether CNP/NPR-B is expressed in mammary gland, and what its role in mammary gland has not been reported. The antimicrobial peptide S100A7 is a critical component of the animal innate immune system and plays important roles to resist infection of pathogens in the mastitis. It is a hypothesis that CNP/NPR-B signaling pathway maybe induces the expression and secretion of S100A7 in mammary epithelial cells to take part in local mammary gland innate immune. In order to verify this hypothesis, goat mammary gland and isolated mammary epithelial cells (MECs) were used to explore the expression of CNP/NPR-B and their physiological roles in goat mammary gland. Results The results showed that goat mammary gland expressed NPR-B but not CNP. Activating NPR-B by treatment with CNP had no effect on synthesis of β-casein and lipid in goat MECs. But the expression and secretion of S100A7 in goat MECs were obviously induced by CNP/NPR-B signaling pathway. After treatment with CNP, the cGMP level in goat MECs were significantly up-regulated. Along with the up-regulation of cGMP level, the phosphorylation levels of JNK and its target c-Jun also increased gradually. KT-5823 is a specific inhibitor for PKG. KT5823 remarkably inhibited the phosphorylation of JNK and c-Jun induced by CNP. Correspondingly, KT5823 evidently inhibited the expression and secretion of S100A7 induced by CNP. On the other hand, the expression of NPR-B and S100A7 were up-regulated in mastitis goat mammary gland. But, there were no significant difference in expression of CNP between health and mastitis goat mammary gland tissues. Goat mastitis model was established in vitro using goat MECs treated by lipopolysaccharide (LPS). LPS treatment also could increase the expression of NPR-B and NPR-B. Conclusions Goat mammary gland expressed NPR-B, indicating mammary gland was target organ for natriuretic peptide system. Moreover, CNP, through NPR-B/JNK/c-Jun signaling pathway to regulate the expression and secretion of S100A7 in MECs, played an important role in mammary gland innate immune.

Background: The antimicrobial peptide (AMP) S100A7, with antimicrobial activities for a broad spectrum of bacteria, have attracted more and more attention for the prevention and treatment of mastitis. However, in goat mastitis, there is little information about the expression and regulation mechanism of S100A7. In present study, the immunolocalization of S100A7 in healthy and mastitis goat udder were compared. In order to further explore the regulatory mechanism of S100A7 expression in mammary epithelial cells (MECs), goat MECs were isolated and treated by 2.5, 5, 10 and 20 μg/mL lipopolysaccharide (LPS) respectively for different time.Results: Both in healthy and mastitis goat teat, S100A7 was mainly expressed in stratified squamous epithelium of teat skin and streak canal. In healthy goat mammary gland, weakly S100A7 immunoreactivity was present in the alveolus. But in the collapsed alveolus of mastitis goat mammary gland, densely S100A7 immunoreactivity could be observed.The goat MECs were treated by 2.5, 5, 10 and 20 μg/mL LPS respectively for different time. For all of these four groups, after treatment for 3 h, increase in S100A7 mRNA expression and protein secretion were detected compared to control (pConclusions: S100A7 was mainly expressed in stratified squamous epithelium of teat skin and streak canal. In mastitis goat mammary gland alveolus, the expression level of S100A7 was up-regulated compared to that in healthy goat. LPS induced the expression and secretion of S100A7 in goat MECs depended on concentration and treatment duration. Moreover, LPS induced the expression and secretion of S100A7 in goat MECs via TLR4/NF-κB signaling pathway.

Mammary epithelial cells have been suggested to be central to control the expansion and remodeling of mammary gland. Wnt/β-catenin signaling modulates cell fate in animals throughout their life span, and represents indispensable roles in tissue homeostasis, cell renewal, and regeneration in organs. Here, we utilized the small molecule 6-bromoindirubin-3'-oxime (BIO), an activator of Wnt/β-catenin signaling, and investigated whether Wnt/β-catenin signaling regulated the proliferation and acinus-like structure formation of goat mammary epithelial cells (GMECs). We showed that isolated GMECs displayed the typical epithelial cobblestone morphology and expressed specific markers of mammary epithelial cells. BIO inhibited the proliferation of GMECs and decreased the expression of proliferation marker c-myc and cell cycle protein cyclin D1. However, the ability of GMECs to form spheroids was accelerated, and the level of E-cadherin mRNA was upregulated with BIO treatment. E-cadherin showed a bright cytomembrane with DMSO treatment, yet E-cadherin was present in cytomembrane and cytoplasm in GMECs with BIO treatment. Meanwhile, BIO increased the protein level of β-catenin and enhanced the translocation of β-catenin into the nucleus in GMECs. Furthermore, the mRNA level of Axin2 was also upregulated. This study suggested that Wnt/β-catenin signaling may play an important role in the proliferation and the acinus-like formation of GMECs.

BackgroundTwo types of adipose tissue, white adipose and brown adipose, have been identified in mammals. For goat, adipose tissue also plays an important role in improving meat and milk quality. C-type natriuretic peptide (CNP) is a member of natriuretic peptide family. Once CNP binds to natriuretic peptide receptor B (NPR-B), NPR-B induces the production of cGMP, thereby activating PKG and downstream targets. The expression of CNP and NPR-B in adipose tissue led to a hypothesis that CNP could have roles involving in regulation of adipogenesis in goat. However, there are few studies on the relationship between CNP and adipogenesis in goat. In the present study, goat adipose derived stem cells (ADSCs) were isolated and employed to investigate the effect of CNP on adipogenesis in goat.ResultsDuring adipogenic differentiation in goat ADSCs, the expression of NPR-B increased at 4 d and 8 d and reduced at 12 d after differentiation. When goat ADSCs were treated with 100 nM and 1000 nM CNP during adipogenic differentiation, there are more accumulation of lipid droplets in the cytoplasm and the up-regulation in the expression of adipogenesis relative genes including PPAR-γ, FASN and LPL. Interestingly, the expression level of brown adipose genes UCP-1 and PGC-1α were also up-regulated. Furthermore, the cGMP level was increased after treatment with CNP. Adding cGMP analogues 8-pCPT-cGMP (PKG activator) could increase adipogenesis efficiency, and adding both PKG inhibitor Rp-8-CPT-cGMP and CNP inhibited adipogenic differentiation. After treatment with CNP or PKG activator, the phosphorylation of p38 MAPK, MK2 and ATF2 were up-regulated, and their phosphorylation were significantly inhibited when CNP and PKG inhibitor were added simultaneously. SB203580 is the specific inhibitor for p38 MAPK. After treatment with CNP and SB203580 simultaneously, the adipogenic differentiation efficiency of goat ADSCs was significantly inhibited, and the expression of UCP-1 and PGC-1α were also reduced. ConclusionsCNP could promote adipogenic differentiation of goat ADSCs. The stimulative effect of CNP on adipose differentiation depends on the cGMP/PKG/ p38 MAPK signal pathway. Our study will be helpful to understand the regulation mechanism of goat adipose differentiation, especially brown adipose differentiation.

Estrogen is an important modulator of reproductive activity through nuclear receptors and G protein–coupled estrogen receptor (GPER). Here, we observed that both estradiol and the GPER-specific agonist G1 rapidly induced cyclic adenosine monophosphate (cAMP) production in cumulus cells, leading to transient stimulation of phosphorylated cAMP response element binding protein (CREB), which was conducive to the transcription of epidermal growth factor (EGF)-like factors, amphiregulin, epiregulin, and betacellulin. Inhibition of GPER by G15 significantly reduced estradiol-induced CREB phosphorylation and EGF-like factor gene expression. Consistently, the silencing of GPER expression in cultured cumulus cells abrogated the estradiol-induced CREB phosphorylation and EGF-like factor transcription. In addition, the increase in EGF-like factor expression in the cumulus cells is associated with EGF receptor (EFGR) tyrosine kinase phosphorylation and extracellular signal–regulated kinase 1/2 (ERK1/2) activation. Furthermore, we demonstrated that GPER-mediated phosphorylation of EGFR and ERK1/2 was involved in reduced gap junction communication, cumulus expansion, increased oocyte mitochondrial activity and first polar body extrusion. Overall, our study identified a novel function for estrogen in regulating EGFR activation via GPER in cumulus cells during oocyte maturation.

BackgroundGoat is an important dairy animal. During lactation, maintaining a high proliferative activity in goat mammary epithelial cells (GMECs) is significant to improve the yield and composition of goat milk. Estrogen is an essential hormone in epithelial cell proliferation and ductal morphogenesis of mammary gland. G protein-coupled receptor 30 (GPR30) is a novel membrane receptor of estrogen. However, the relationship between estrogen/GPR30 signaling and proliferation of goat mammary epithelial cells has not been reported. And the molecular mechanisms underlying the proliferative effect of estrogen via GPR30 on GMECs remain unclear.ResultsTo investigate the effect of estrogen/GPR30 signaling on GMECs proliferation, goat mammary epithelial cells, which expressed cytokeratin 18 and β-casein, were isolated and identified, defining their mammary alveolar epithelium origination. Estrogen and GPR30 agonist G1 obviously promoted the proliferation of GEMCs, in contrast, GPR30 antagonist G15 partly abolished estrogen-induced cell proliferation. Remarkably, the stimulatory effect of estrogen and G1 on GMECs growth was suppressed by GPR30 knockdown detected by cell counting assay, CCK-8 assay, and BrdU assay, suggesting that estrogen/GPR30 signaling was involved in GMECs proliferation. Additionally, G15 decreased cyclin D1, cyclin B1, CDK1, and p-CDK1 expression, resulting in cell cycle arrest in the G2/M phase via a down-regulated phosphorylation of Erk1/2 and Akt compared with estrogen alone. What’s more, knock-down GPR30 led to an accumulation in the G2/M phase and inhibition of cyclin D1, cyclin B1, CDK1, and p-CDK1 expression via a down-regulation of phosphorylated Erk1/2 and Akt despite the presence of estrogen and G1. Furthermore, MEK inhibitor and PI3K inhibitor decreased the expression of cyclin D1, cyclin B1, CDK1, and p-CDK1, and repressed estrogen-induced and G1-driven promotion of cell growth. It indicated that estrogen/GPR30 signaling played an important role in GMECs proliferation by affecting cell cycle progression via MEK/Erk&PI3K/Akt signaling pathway.ConclusionThis study may provide a new insight into the effect of estrogen/GPR30 signaling on the regulatory action of goat mammary gland development.

Additional file 1. Table S1. sgRNA of target sites. Table S2. Oligonucleotides for generating transcription of sgRNA expression vectors. Table S3. Primers for genotyping and amplifying Cas9/sgRNA targeted BMPR1B fragment. Table S4. Detailed summary of the lambs generated with ABEmax-mediated base editing. Table S5. Primers for genotyping and amplifying predicted off-target site fragments. Table S6. List of predicted off-target sites.

Additional file 5: Figure S4. Detection of potential off-targeted sites by Sanger sequencing in sheep fibroblasts. Five potential off-targeted sites (OT1–OT5) were predicted by Cas-OFFinder. Sanger sequencing was used to determine substitutions at predicted target sites in sheep fibroblasts.

Additional file 3: Figure S2. Sanger sequencing maps of the DNA from the eight founder animals.

Additional file 4: Figure S3. Detection of potential off-targeted sites by Sanger sequencing in founder animals. Five potential off-targeted sites (OT1–OT5) were predicted by Cas-OFFinder. Sanger sequencing was used to determine substitution at predicted target sites for the eight founder animals.

Additional file 2: Figure S1. Overlapping peaks in sequencing maps of the DNA from transfected cells.

Mammalian oocyte restores meiosis can be stimulated by follicle-stimulating hormone (FSH) under normal physiological conditions. G-protein coupled receptor 30 (GPR30), an non-classical estrogen membrane receptor, has been widely reported in teleost oocyte maturation. However, it remains unknown whether GPR30 involves the role of FSH in mammalian cumulus expansion and oocyte maturation. Here, we used mouse cumulus-oocyte complexes (COCs) as a model to investigate how FSH affects the in vitro maturation of mouse oocytes mediated by 17β-estradiol (E

Bone marrow mesenchymal stem cells (BMSCs) can transdifferentiate into different types of cells and may serve as a cell source for tissue engineering. Resveratrol has been shown to possess many benefits, including activation of osteogenesis. Furthermore, Wnt/β-catenin signaling has been known to promote osteogenic differentiation in many cells. In this study, we investigated the role of resveratrol in osteoblastic differentiation of canine BMSCs. Resveratrol treatment of canine BMSCs remarkably increased alkaline phosphatase activity and calcium nodules, inhibited the function of glycogen synthase kinase 3β, led to an increase in stabilization and nuclear accumulation of β-catenin, and upregulated expression of osteoblast-related marker gene expression. In addition, resveratrol caused rapid activation of ERK1/2. Collectively, our results indicate that resveratrol promotes osteoblastic differentiation of canine BMSCs by activating the Wnt/β-catenin; ERK/MAPK signaling pathways are also involved in osteogenic differentiation of canine BMSCs.

Since its emergence, CRISPR/Cas9-mediated base editors (BEs) with cytosine deaminase activity have been used to precisely and efficiently introduce single-base mutations in genomes, including those of human cells, mice, and crop species. Most production traits in livestock are induced by point mutations, and genome editing using BEs without homology-directed repair of double-strand breaks can directly alter single nucleotides. The p.96R>C variant of Suppressor cytokine signaling 2 (SOCS2) has profound effects on body weight, body size, and milk production in sheep. In the present study, we successfully obtained lambs with defined point mutations resulting in a p.96R>C substitution in SOCS2 by the co-injection of Base Editor 3 (BE3) mRNA and a single guide RNA (sgRNA) into sheep zygotes. The observed efficiency of the single nucleotide exchange in newborn animals was as high as 25%. Observations of body size and body weight in the edited group showed that gene modification contributes to enhanced growth traits in sheep. Moreover, targeted deep sequencing and unbiased family trio-based whole genome sequencing revealed undetectable off-target mutations in the edited animals. This study demonstrates the potential for the application of BE-mediated point mutations in large animals for the improvement of production traits in livestock species.

Estrogen plays a critical role in the regulation of gap junctions between oocytes and granulosa cells in mammalian ovaries. G protein-coupled receptor 30 (GPR30) was identified as a membrane estrogen receptor, mediating rapid, nongenomic signaling events that might be responsible for the regulation of oocyte meiosis resumption and gap junction intercellular communications (GJICs). The present study aimed to determine the expression and localization of GPR30 and its role in oocyte meiotic progression and GJICs in goat cumulus-oocyte complexes (COCs). Immunofluorescence experiments revealed that GPR30 was primarily located in the plasma membrane of cumulus cells and oocytes in goats. 17β-estradiol could promote oocyte meiotic progression, which was blocked by G15 (a selective GPR30 antagonist) but not ICI182780 (a nuclear estrogen receptor inhibitor) in the early stage of in vitro culture. The effect of 17β-estradiol on the GJICs was quantified by lucifer yellow (LY) microinjection and calcein-AM fluorescent dye diffusion. 17β-estradiol treatment of goat COCs resulted in rapid downregulation of GJICs. The transfer of calcein from cumulus cells to oocytes could be significantly inhibited by carbenoxolone (a known gap junction blocker), 17β-estradiol or G1 (a GPR30 agonist), and this inhibition could be reversed by G15 but not ICI182780, indicating that GPR30 mediates the effect of 17β-estradiol on the rapid downregulation of GJICs. 17β-estradiol also stimulated the serine 368 phosphorylation of connexin 43 (Cx43) when COCs were in vitro cultured for 4 h, 6 h, and 8 h. More importantly, 17β-estradiol or G1 could separately promote the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK 1/2) and Cx43 significantly when COCs were cultured for 4 h. Furthermore, both ERK1/2 and Cx43 phosphorylation could be inhibited by G15 and the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor AG1478 or by the ERK1/2 inhibitor PD98059, indicating that EGFR-ERK1/2 signaling was involved in these events. These results supported the hypothesis that GPR30 mediated 17β-estradiol-stimulated meiotic resumption and GJIC reduction in goat COCs. Thus, the present study provides novel insights into elucidating the mechanisms for steroid hormone action in the regulation of oocyte maturation.

Cryopreservation of cumulus oocyte complexes (COCs) is important for reproductive medicine. However, the vitrified-warmed COCs have lower maturation rate and subsequent developmental competence compared with fresh COCs. The present study was aimed to evaluate the effects of supplementation of the maturation medium with C-type natriuretic peptide (CNP) on the developmental competence of vitrified-warmed mouse COCs. Addition of CNP to the maturation medium improved the maturation rate and enhanced the developmental competence of vitrified-warmed mouse COCs. The reason may be that vitrified COCs led to a decline in cyclic guanosine monophosphate (cGMP) levels. Furthermore, addition of CNP to the maturation medium elevated cGMP levels of the vitrified-warmed COCs. In conclusion, cryopreservation-associated lower maturation rate and developmental competence of COCs may be ameliorated by CNP during maturation culture after warming.

C-type natriuretic peptide (CNP) plays a role as an oocyte maturation inhibitor (OMI) in many species, including the bovine. However, the effects of luteinizing hormone (LH) on CNP expression and its potential mechanisms have not reported in the bovine. In the present study, we aimed to study the effects of LH on CNP expression and to illuminate the potential molecular mechanism in this process. Our results showed that LH induced epidermal growth factor receptor (EGFR) phosphorylation, mitogen-activated protein kinase3/1 (MAPK3/1) activation and CNP mRNA decrease in cultured bovine granulosa cells. Further study revealed that LH suppressed CNP expression via the MAPK3/1 signaling pathway, which was activated by the EGFR pathway. In conclusion, our research suggested that MAPK3/1 is involved in LH-mediated decrease of CNP and that this process is related to the EGFR and MAPK3/1 signal pathways.