Diaph1 knockout inhibits mouse primordial germ cell proliferation and affects gonadal development.

BACKGROUND: Exploring the molecular mechanisms of primordial germ cell (PGC) migration and the involvement of gonadal somatic cells in gonad development is valuable for comprehending the origins and potential treatments of reproductive-related diseases. METHODS: Diaphanous related formin 1 (Diaph1, also known as mDia1) was screened by analyzing publicly available datasets (ATAC-seq, DNase-seq, and RNA-seq). Subsequently, the CRISPR-Cas9 technology was used to construct Diaph1 knockout mice to investigate the role of Diaph1 in gonad development. RESULTS: Based on data from public databases, a differentially expressed gene Diaph1, was identified in the migration of mouse PGC. Additionally, the number of PGCs was significantly reduced in Diaph1 knockout mice compared to wild type mice, and the expression levels of genes related to proliferation (Dicer1, Mcm9), adhesion (E-cadherin, Cdh1), and migration (Cxcr4, Hmgcr, Dazl) were significantly decreased. Diaph1 knockout also inhibited Leydig cell proliferation and induced apoptosis in the testis, as well as granulosa cell apoptosis in the ovary. Moreover, the sperm count in the epididymal region and the count of ovarian follicles were significantly reduced in Diaph1 knockout mice, resulting in decreased fertility, concomitant with lowered levels of serum testosterone and estradiol. Further research found that in Diaph1 knockout mice, the key enzymes involved in testosterone synthesis (CYP11A1, 3ß-HSD) were decreased in Leydig cells, and the estradiol-associated factor (FSH receptor, AMH) in granulosa cells were also downregulated. CONCLUSIONS: Overall, our findings indicate that the knockout of Diaph1 can disrupt the expression of factors that regulate sex hormone production, leading to impaired secretion of sex hormones, ultimately resulting in damage to reproductive function. These results provide a new perspective on the molecular mechanisms underlying PGC migration and gonadal development, and offer valuable insights for further research on the causes, diagnosis, and treatment of related diseases.

Methyltransferase like-14 suppresses growth and metastasis of non-small-cell lung cancer by decreasing LINC02747.

Multiple epigenetic regulatory mechanisms exert critical roles in tumor development, and understanding the interactions and impact of diverse epigenetic modifications on gene expression in cancer is crucial for the development of precision medicine. We found that methyltransferase-like 14 (METTL14) was significantly downregulated in non-small-cell lung cancer (NSCLC) tissues. Functional experiments demonstrated that overexpression of METTL14 inhibited the proliferation and migration of NSCLC cells both in vivo and in vitro, and the colorimetric m6A quantification assay also showed that knockdown of METTL14 notably reduced global m6A modification levels in NSCLC cells. By using the methylated-RNA immunoprecipitation-qPCR and dual-luciferase reporter assays, we verified that long noncoding RNA LINC02747 was a target of METTL14 and was regulated by METTL14-mediated m6A modification, and silencing LINC02747 inhibited the malignant progression of NSCLC by modulating the PI3K/Akt and CDK4/Cyclin D1 signaling pathway. Further studies revealed that overexpression of METTL14 promoted m6A methylation and accelerated the decay of LINC02747 mRNA via increased recognition of the "GAACU" binding site by YTHDC2. Additionally, histone demethylase lysine-specific histone demethylase 5B (KDM5B) mediated the demethylation of histone H3 lysine 4 tri-methylation (H3K4me3) in the METTL14 promoter region and repressed its transcription. In summary, KDM5B downregulated METTL14 expression at the transcriptional level in a H3K4me3-dependent manner, while METTL14 modulated LINC02747 expression via m6A modification. Our results demonstrate a synergy of multiple mechanisms in regulating the malignant phenotype of NSCLC, revealing the complex regulation involved in the occurrence and development of cancer.

NFIC mediates m6A mRNA methylation to orchestrate transcriptional and post-transcriptional regulation to represses malignant phenotype of non-small cell lung cancer cells.

BACKGROUND: Multiple genetic and epigenetic regulatory mechanisms are crucial in the development and tumorigenesis process. Transcriptional regulation often involves intricate relationships and networks with post-transcriptional regulatory molecules, impacting the spatial and temporal expression of genes. However, the synergistic relationship between transcription factors and N6-methyladenosine (m6A) modification in regulating gene expression, as well as their influence on the mechanisms underlying the occurrence and progression of non-small cell lung cancer (NSCLC), requires further investigation. The present study aimed to investigate the synergistic relationship between transcription factors and m6A modification on NSCLC. METHODS: The transcription factor NFIC and its potential genes was screened by analyzing publicly available datasets (ATAC-seq, DNase-seq, and RNA-seq). The association of NFIC and its potential target genes were validated through ChIP-qPCR and dual-luciferase reporter assays. Additionally, the roles of NFIC and its potential genes in NSCLC were detected in vitro and in vivo through silencing and overexpression assays. RESULTS: Based on multi-omics data, the transcription factor NFIC was identified as a potential tumor suppressor of NSCLC. NFIC was significantly downregulated in both NSCLC tissues and cells, and when NFIC was overexpressed, the malignant phenotype and total m6A content of NSCLC cells was suppressed, while the PI3K/AKT pathway was inactivated. Additionally, we discovered that NFIC inhibits the expression of METTL3 by directly binding to its promoter region, and METTL3 regulates the expression of KAT2A, a histone acetyltransferase, by methylating the m6A site in the 3'UTR of KAT2A mRNA in NSCLC cells. Intriguingly, NFIC was also found to negatively regulate the expression of KAT2A by directly binding to its promoter region. CONCLUSIONS: Our findings demonstrated that NFIC suppresses the malignant phenotype of NSCLC cells by regulating gene expression at both the transcriptional and post-transcriptional levels. A deeper comprehension of the genetic and epigenetic regulatory mechanisms in tumorigenesis would be beneficial for the development of personalized treatment strategies.

Unveiling cancer dormancy: Intrinsic mechanisms and extrinsic forces.

Tumor cells disseminate in various distant organs at early stages of cancer progression. These disseminated tumor cells (DTCs) can stay dormant/quiescent without causing patient symptoms for years or decades. These dormant tumor cells survive despite curative treatments by entering growth arrest, escaping immune surveillance, and/or developing drug resistance. However, these dormant cells can reactivate to proliferate, causing metastatic progression and/or relapse, posing a threat to patients' survival. It's unclear how cancer cells maintain dormancy and what triggers their reactivation. What are better approaches to prevent metastatic progression and relapse through harnessing cancer dormancy? To answer these remaining questions, we reviewed the studies of tumor dormancy and reactivation in various types of cancer using different model systems, including the brief history of dormancy studies, the intrinsic characteristics of dormant cells, and the external cues at the cellular and molecular levels. Furthermore, we discussed future directions in the field and the strategies for manipulating dormancy to prevent metastatic progression and recurrence.

YY1-induced lncRNA00511 promotes melanoma progression via the miR-150-5p/ADAM19 axis.

Increasing evidence indicates that long noncoding RNAs (lncRNAs) are therapeutic targets and key regulators of tumors development and progression, including melanoma. Long intergenic non-protein-coding RNA 511 (LINC00511) has been demonstrated as an oncogenic molecule in breast, stomach, colorectal, and lung cancers. However, the precise role and functional mechanisms of LINC00511 in melanoma remain unknown. This study confirmed that LINC00511 was highly expressed in melanoma cells (A375 and SK-Mel-28 cells) and tissues, knockdown of LINC00511 could inhibit melanoma cell migration and invasion, as well as the growth of subcutaneous tumor xenografts in vivo. By using Chromatin immunoprecipitation (ChIP) assay, it was demonstrated that the transcription factor Yin Yang 1 (YY1) is capable of binding to the LINC00511 promoter and enhancing its expression in cis. Further mechanistic investigation showed that LINC00511 was mainly enriched in the cytoplasm of melanoma cells and interacted directly with microRNA-150-5p (miR-150-5p). Consistently, the knockdown of miR-150-5p could recover the effects of LINC00511 knockdown on melanoma cells. Furthermore, ADAM metallopeptidase domain expression 19 (ADAM19) was identified as a downstream target of miR-150-5p, and overexpression of ADAM19 could promote melanoma cell proliferation. Rescue assays indicated that LINC00511 acted as a competing endogenous RNA (ceRNA) to sponge miR-150-5p and increase the expression of ADAM19, thereby activating the PI3K/AKT pathway. In summary, we identified LINC00511 as an oncogenic lncRNA in melanoma and defined the LINC00511/miR-150-5p/ADAM19 axis, which might be considered a potential therapeutic target and novel molecular mechanism the treatment of patients with melanoma.

Diagnostic and prognostic potential of exosome non-coding RNAs in bladder cancer: a systematic review and meta-analysis.

Background: Bladder cancer stands as the predominant malignant tumor in the urological system, presenting a significant challenge to public health and garnering extensive attention. Recently, with the deepening research into tumor molecular mechanisms, non-coding RNAs (ncRNAs) have emerged as potential biomarkers offering guidance for the diagnosis and prognosis of bladder cancer. However, the definitive role of ncRNAs in bladder cancer remains unclear. Hence, this study aims to elucidate the relevance and significance of ncRNAs through a Meta-analysis. Methods: A systematic meta-analysis was executed, including studies evaluating the diagnostic performance of ncRNAs and their associations with overall survival (OS) and disease-free survival (DFS). Key metrics such as hazard ratios, sensitivity, specificity, and diagnostic odds ratios were extracted and pooled from these studies. Potential publication bias was assessed using Deeks' funnel plot, and the robustness of the results was ascertained through a sensitivity analysis. Results: Elevated ncRNA expression showed a positive correlation with improved OS, evidenced by a hazard ratio (HR) of 0.82 (95% CI: 0.66-0.96, P<0.001). Similarly, a significant association was observed between heightened ncRNA expression and DFS, with an HR of 0.86 (95% CI: 0.73-0.99, P<0.001). Diagnostic performance analysis across 17 articles yielded a pooled sensitivity of 0.76 and a specificity of 0.83. The diagnostic odds ratio was recorded at 2.71, with the area under the ROC curve (AUC) standing at 0.85. Conclusion: Exosome ncRNAs appear to possess potential significance in the diagnostic and prognostic discussions of bladder cancer. Their relationship with survival outcomes and diagnostic measures suggests a possible clinical utility. Comprehensive investigations are needed to fully determine their role in the ever-evolving landscape of bladder cancer management, especially within the framework of personalized medicine.

MiR-290 Family Maintains Pluripotency and Self-Renewal by Regulating MAPK Signaling Pathway in Intermediate Pluripotent Stem Cells.

Mouse embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs) are derived from pre- and post-implantation embryos, representing the initial "naïve" and final "primed" states of pluripotency, respectively. In this study, novel reprogrammed pluripotent stem cells (rPSCs) were induced from mouse EpiSCs using a chemically defined medium containing mouse LIF, BMP4, CHIR99021, XAV939, and SB203580. The rPSCs exhibited domed clones and expressed key pluripotency genes, with both X chromosomes active in female cells. Furthermore, rPSCs differentiated into cells of all three germ layers in vivo through teratoma formation. Regarding epigenetic modifications, the DNA methylation of Oct4, Sox2, and Nanog promoter regions and the mRNA levels of Dnmt3a, Dnmt3b, and Dnmt1 were reduced in rPSCs compared with EpiSCs. However, the miR-290 family was significantly upregulated in rPSCs. After removing SB203580, an inhibitor of the p38 MAPK pathway, the cell colonies changed from domed to flat, with a significant decrease in the expression of pluripotency genes and the miR-290 family. Conversely, overexpression of pri-miR-290 reversed these changes. In addition, Map2k6 was identified as a direct target gene of miR-291b-3p, indicating that the miR-290 family maintains pluripotency and self-renewal in rPSCs by regulating the MAPK signaling pathway.

A meta-analysis of the risk factors for surgical site infection in patients with colorectal cancer.

The purpose of the meta-analysis was to evaluate and compare the surgical site infection (SSI) risk factors in patients with colorectal cancer (CC). The results of this meta-analysis were analysed, and the odds ratio (OR) and mean difference (MD) with 95% confidence intervals (CIs) were calculated using dichotomous or contentious random or fixed-effect models. For the current meta-analysis, 23 examinations spanning from 2001 to 2023 were included, encompassing 89 859 cases of CC. Clean-contaminated surgical site wounds had significantly lower infections (OR, 0.36; 95% CI, 0.20-0.64, p < 0.001) compared to contaminated surgical site wounds in patients with CCs. Males had significantly higher SSIs (OR, 1.18; 95% CI, 1.12-1.24, p < 0.001) compared to females in patients with CC. American Society of Anesthesiology score ≥3 h had a significantly higher SSI (OR, 1.42; 95% CI, 1.18-1.71, p < 0.001) compared to <3 score in patients with CCs. Body mass index ≥25 had significantly higher SSIs (OR, 1.54; 95% CI, 1.11-2.14, p = 0.01) compared to <25 in patients with CCs. The presence of stoma creation had a significantly higher SSI rate (OR, 2.28; 95% CI, 1.37-3.79, p = 0.001) compared to its absence in patients with CC. Laparoscopic surgery had significantly lower SSIs (OR, 0.68; 95% CI, 0.59-0.78, p < 0.001) compared to open surgery in patients with CC. The presence of diabetes mellitus had a significantly higher SSI rate (OR, 1.24; 95% CI, 1.15-1.33, p < 0.001) compared to its absence in patients with CCs. No significant difference was found in SSI rate in patients with CCs between <3 and ≥3 h of operative time (OR, 1.07; 95% CI, 0.75-1.51, p = 0.72), between the presence and absence of blood transfusion (OR, 1.60; 95% CI, 0.69-3.66, p = 0.27) and between the presence and absence of previous laparotomies (OR, 1.47; 95% CI, 0.93-2.32, p = 0.10). The examined data revealed that contaminated wounds, male sex, an American Society of Anesthesiology score ≥3 h, a body mass index ≥25, stoma creation, open surgery and diabetes mellitus are all risk factors for SSIs in patients with CC. However, operative time, blood transfusion and previous laparotomies were not found to be risk factors for SSIs in patients with CC. However, given that several comparisons had a small number of chosen research, consideration should be given to their values.

METTL3 exerts synergistic effects on m6A methylation and histone modification to regulate the function of VGF in lung adenocarcinoma.

BACKGROUND: Multiple genetic and epigenetic regulatory mechanisms play a vital role in tumorigenesis and development. Understanding the interplay between different epigenetic modifications and its contribution to transcriptional regulation in cancer is essential for precision medicine. Here, we aimed to investigate the interplay between N6-methyladenosine (m6A) modifications and histone modifications in lung adenocarcinoma (LUAD). RESULTS: Based on the data from public databases, including chromatin property data (ATAC-seq, DNase-seq), methylated RNA immunoprecipitation sequencing (MeRIP-seq), and gene expression data (RNA-seq), a m6A-related differentially expressed gene nerve growth factor inducible (VGF) was identified between LUAD tissues and normal lung tissues. VGF was significantly highly expressed in LUAD tissues and cells, and was associated with a worse prognosis for LUAD, silencing of VGF inhibited the malignant phenotype of LUAD cells by inactivating the PI3K/AKT/mTOR pathway. Through the weighted correlation network analysis (WGCNA) and integration of TCGA-LUAD RNA-seq and m6A methyltransferase METTL3-knockdown RNA-seq data, a significant positive correlation between METTL3 and VGF was observed. By using the MeRIP-qPCR and dual-luciferase reporter assays, we demonstrated that METTL3 knockdown decreased m6A modification level of VGF coding sequences in LUAD cells, the colorimetric m6A quantification assay also showed that METTL3 knockdown significantly decreased global m6A modification level in LUAD cells. Interestingly, we found that METTL3 knockdown also reduced VGF expression by increasing H3K36me3 modification at the VGF promoter. Further research revealed that METTL3 knockdown upregulated the expression of histone methylase SETD2, the major H3K36me3 methyltransferase, by methylating the m6A site in the 3'UTR of SETD2 mRNA in LUAD cells. CONCLUSIONS: Overall, our results reveal that the expression of VGF in LUAD cells is regulated spatio-temporally by METTL3 through both transcriptional (via histone modifications) and post-transcriptional (via m6A modifications) mechanisms. The synergistic effect of these multiple epigenetic mechanisms provides new opportunities for the diagnosis and precision treatment of tumors.

A homozygous EVC mutation in a prenatal fetus with Ellis-van Creveld syndrome.

BACKGROUND: Ellis-van Creveld (EvC) syndrome, caused by variants in EVC, is a rare genetic skeletal dysplasia. Its clinical phenotype is highly diverse. EvC syndrome is rarely reported in prenatal stages because its presentation overlaps with other diseases. METHODS: A Chinese pedigree diagnosed with EvC syndrome was enrolled in this study. Whole-exome sequencing (WES) was applied in the proband to screen potential genetic variant(s), and then Sanger sequencing was used to identify the variant in family members. Minigene experiments were applied. RESULTS: WES identified a homozygous variant (NM_153717.3:c.153_174 + 42del) in EVC which was inherited from the heterozygous parents and confirmed by Sanger sequencing. Further experiments demonstrated that this variant disrupts the canonical splicing site and produces a new splicing site at NM_153717.3: c.-164_174del, which ultimately leads to a 337 bp deletion at the 3' end of exon 1 and loss of the start codon. CONCLUSION: This is the first reported case of EvC syndrome based on a splicing variant and detailed delineation of the aberrant splicing effect in the fetus. Our study demonstrates the pathogenesis of this new variant, expands the spectrum of EVC mutations, and demonstrates that WES is a powerful tool in the clinical diagnosis of diseases with genetic heterogeneity.

Effects of New Mutations in BMPRIB, GDF9, BMP15, LEPR, and B4GALNT2 Genes on Litter Size in Sheep.

Prolificacy is a crucial characteristic of livestock, particularly for species such as sheep that have many births. The objectives of this study were as follows: (1) to investigate the genetic diversity of the 13 new and 7 known variants in the BMPRIB, GDF9, BMP15, LEPR, and B4GALNT2 genes in Ujimqin (UM), the F1 population of Dorper × Ujimqin crossbred (DPU), the F1 population of Suffolk × Ujimqin crossbred (SFKU), Sonid sheep (SN), Tan sheep (Tan), Hu sheep (Hu), and Small-tailed Han sheep (STH) sheep breeds/populations; (2) to perform an association analysis of the above 20 variants with litter size in 325 UM, 304 DPU, and 66 SFKU sheep populations; (3) to compare the frequencies of the litter-size-related alleles of these 20 variants among 8 sheep breeds/populations (the above seven sheep breeds + Mongolia sheep breed). With the use of the Sequenom MassARRAY®SNP assay technology, these 20 mutations were genotyped. The association analysis results showed that the c.746A>G (FecB) mutation in BMPR1B was significantly associated with the litter size of UM and DPU, the c.994A>G (FecGA) in GDF9 was significantly associated with the litter size of SFKU, and the c.31_33CTTinsdel (B1) in BMP15 was significantly associated with the litter size of UM. Our findings might provide valuable genetic markers for expanding sheep litter sizes.

Effects of novel variants in BMP15 gene on litter size in Mongolia and Ujimqin sheep breeds.

Reproductive traits, such as ovulation rate and litter size, are important factors influencing the sheep industry. The bone morphogenetic protein 15 (BMP15) is a major gene affecting the reproductive traits in sheep, and multiple mutations in BMP15 gene could affect the ovulation rate and litter size in many sheep breeds, showing high breed specificity. However, identification of novel variations and seeking breed-specific markers associated with litter size in other sheep breeds are still important. In this study, we sequenced the BMP15 gene of Mongolia sheep, and 12 novel variants were detected by direct sequencing and whole-genome resequencing. Among them, the g.50985975 G > A polymorphism in intron and synonymous c.755 T > C (Leu252Pro) in exon 2 of BMP15 were significantly associated with the litter sizes of Mongolia ewes (P < 0.01 and P < 0.01, respectively), as well as the g.50988478C > A and g.50987863G > A in the promoter region of BMP15 were significantly associated with the litter sizes of Ujimqin ewes (P < 0.05 and P < 0.01, respectively). The c.755 T > C mutation is predicted to change the tertiary structure of BMP15. Our findings may provide potentially useful genetic markers for increasing litter size in sheep.

Integrated bioinformatics analysis of the transcription factor-mediated gene regulatory networks in the formation of spermatogonial stem cells.

Background: In vitro induction of spermatogonial stem cells (SSCs) from embryonic stem cells (ESCs) provides a promising tool for the treatment of male infertility. A variety of molecules are involved in this complex process, which needs to be further clarified. Undoubtedly, the increased knowledge of SSC formation will be beneficial to facilitate the currently complex induction process. Methods: Based on ATAC-seq, DNase-seq, RNA-seq, and microarray data from GEO datasets, chromatin property data (ATAC-seq, DNase-seq) and gene expression data (RNA-seq, microarray data) were combined to search for SSC-specific transcription factors (TFs) and hub SSC-specific genes by using the WGCNA method. Then, we applied RNA-seq and microarray data screening for key SSC-specific TFs and constructed key SSC-specific TF-mediated gene regulatory networks (GRNs) using ChIP-seq data. Results: First, after analysis of the ATAC-seq and DNase-seq data of mouse ESCs, primordial germ cells (PGCs), and SSCs, 33 SSC-specific TFs and 958 targeting genes were obtained. RNA-seq and WGCNA revealed that the key modules (turquoise and red) were the most significantly related to 958 SSC-specific genes, and a total of 10 hub SSC-specific genes were identified. Next, when compared with the cell-specific TFs in human ESCs, PGCs, and SSCs, we obtained five overlapping SSC-specific TF motifs, including the NF1 family TF motifs (NFIA, NFIB, NFIC, and NFIX), GRE, Fox:Ebox, PGR, and ARE. Among these, Nfib and Nfix exhibited abnormally high expression levels relative to mouse ESCs and PGCs. Moreover, Nfib and Nfix were upregulated in the testis sample with impaired spermatogenesis when compared with the normal group. Finally, the ChIP-seq data results showed that NFIB most likely targeted the hub SSC-specific genes of the turquoise module (Rpl36al, Rps27, Rps21, Nedd8, and Sec61b) and the red module (Vcam1 and Ccl2). Conclusion: Our findings preliminarily revealed cell-specific TFs and cell-specific TF-mediated GRNs in the process of SSC formation. The hub SSC-specific genes and the key SSC-specific TFs were identified and suggested complex network regulation, which may play key roles in optimizing the induction efficiency of the differentiation of ESCs into SSCs in vitro.

Implementation of Photosensitive, Injectable, Interpenetrating, and Kartogenin-Modified GELMA/PEDGA Biomimetic Scaffolds to Restore Cartilage Integrity in a Full-Thickness Osteochondral Defect Model.

Cartilage defects caused by mechanical tear and wear are challenging clinical problems. Articular cartilage has unique load-bearing properties and limited self-repair ability. The current treatment methods, such as microfractures and autogenous cartilage transplantation to repair full-thickness cartilage defects, have apparent limitations. Tissue engineering technology has the potential to repair cartilage defects and directs current research development. To enhance the regenerative capacities of cartilage in weight-bearing areas, we attempted to develop a biomimetic scaffold loaded with a chondroprotective factor that can recreate structure, restore mechanical properties, and facilitate anabolic metabolism in larger joint defects. For enhanced spatial control over both bone and cartilage layers, it is envisioned that biomaterials that meet the needs of both tissue components are required for successful osteochondral repair. We used gelatin methacrylate (GELMA) and polyethylene glycol diacrylate (PEGDA) light-cured dual-network cross-linking modes that can significantly increase the mechanical properties of scaffolds and are capable of restoring function and prolonging the degradation time. Once the hydrogel complex was injected into the osteochondral defect, in situ UV light curing was applied to seamlessly connect the defect repair tissue with the surrounding normal cartilage tissue. The small molecule active substance kartogenin (KGN) can promote cartilage repair. We encapsulated KGN in biomimetic scaffolds so that, as the scaffold degrades, scaffold-loaded KGN was slowly released to induce endogenous mesenchymal stem cells to home and differentiate into chondrocytes to repair defective cartilage tissue. Our experiments have proven that, compared with the control group, GELMA/PEGDA + KGN repaired cartilage defects and restored cartilage to hyaline cartilage. Our study suggests that implementing photosensitive, injectable, interpenetrating, and kartogenin-modified GELMA/PEDGA biomimetic scaffolds may be a novel approach to restore cartilage integrity in full-thickness osteochondral defects.

Association analysis between novel variants in LEPR gene and litter size in Mongolia and ujimqin sheep breeds.

Prolificacy is an important trait of livestock, especially for species with multiple births, such as sheep. The leptin receptor (LEPR) is considered as a major candidate gene that affects the reproductive traits of sheep. The known R62H mutation in the LEPR gene is associated with the hyperprolific phenotype of Davisdale sheep. However, studies identifying novel variations and seeking breed-specific markers associated with litter size in other sheep breeds are rare. In this study, we sequenced all coding regions of the LEPR gene of Mongolia sheep, and nine novel variants of the LEPR gene were detected by direct sequencing. Among them, the synonymous c.240C > T and c.279C > T mutations were significantly associated with the litter sizes of Mongolia ewes (P < 0.05 and P < 0.01, respectively) and Ujimqin ewes (P < 0.05 and P < 0.01, respectively). The silent c.240C > T and c.279C > T mutations are predicted to change the stability of the mRNA secondary structure by reducing the minimum free energy and are predicted to change the mRNA secondary structure of LEPR. Our findings may provide potentially useful genetic markers for increasing litter size in sheep.

MiR-290 family maintains developmental potential by targeting p21 in mouse preimplantation embryos‡.

The miR-290 family is a mouse-specific microRNA cluster, which maintains mouse embryonic stem cells (ESCs) pluripotency by increasing OCT3/4 and C-MYC expression. However, its functions in mouse preimplantation embryos remain unclear, especially during zygotic genome activation (ZGA). In this study, miR-290 family expression increased from the two-cell embryo stage through the blastocyst stage. Inhibition of miR-294-3p/5p did not affect ZGA initiation or embryo development, whereas pri-miR-290 knockdown decreased ZGA gene expression and slowed embryonic development. In addition, pluripotency decreased in ESCs derived from pri-miR-290 knockdown blastocysts. To clarify the mechanism of action, 33 candidate miR-294-3p target genes were screened from three databases, and miR-294-3p directly targeted the 3'-untranslated region of Cdkn1a (p21) mRNA. Similar to pri-miR-290 knockdown, P21 overexpression impeded embryonic development, whereas simultaneous overexpression of P21 and pri-miR-290 partially rescued embryonic development. The results indicate that the miR-290 family participates in promoting ZGA process and maintaining developmental potency in embryos by targeting p21.

Enzalutamide-induced and PTH1R-mediated TGFBR2 degradation in osteoblasts confers resistance in prostate cancer bone metastases.

Enzalutamide resistance has been observed in approximately 50% of patients with prostate cancer (PCa) bone metastases. Therefore, there is an urgent need to investigate the mechanisms and develop strategies to overcome resistance. We observed enzalutamide resistance in bone lesion development induced by PCa cells in mouse models. We found that the bone microenvironment was indispensable for enzalutamide resistance because enzalutamide significantly inhibited the growth of subcutaneous C4-2B tumors and the proliferation of C4-2B cells isolated from the bone lesions, and the resistance was recapitulated only when C4-2B cells were co-cultured with osteoblasts. In revealing how osteoblasts contribute to enzalutamide resistance, we found that enzalutamide decreased TGFBR2 protein expression in osteoblasts, which was supported by clinical data. This decrease was possibly through PTH1R-mediated endocytosis. We showed that PTH1R blockade rescued enzalutamide-mediated decrease in TGFBR2 levels and enzalutamide responses in C4-2B cells that were co-cultured with osteoblasts. This is the first study to reveal the contribution of the bone microenvironment to enzalutamide resistance and identify PTH1R as a feasible target to overcome the resistance in PCa bone metastases.

Association between novel variants in BMPR1B gene and litter size in Mongolia and Ujimqin sheep breeds.

Prolificacy is an important trait of animals, specifically for sheep. The Bone morphogenetic protein receptor 1B (BMPR1B) is a major gene affecting the litter size of many sheep breeds. The well-known FecB mutation (Q249R) was associated fully with the hyper prolific phenotype of Booroola Merino. However, the identification of variation in all exonic regions of BMPR1B was rare. In this study, we sequenced all exonic regions of BMPR1B gene of Mongolia sheep breed, and ten novel variants were detected by direct sequencing. Among them, the litter size of the Mongolia ewes with the CC genotype was significantly higher (0.34 additional lambs, p < .05) than those with the TT genotype of the g.29346567C>T single nucleotide polymorphism (SNP). The litter size of the Mongolia ewes with the TT genotype was significantly higher (0.19 additional lambs, p < .05 and .31 additional lambs, p < .01, respectively) than those with the GT and GG genotypes of the c.1470G>T SNP. The silent c.1470G>T mutation is predicted to increase the stability of the mRNA secondary structure through reducing minimum free energy and is predicted to change the mRNA secondary structure of BMPR1B. Our findings may give potentially useful genetic markers for increasing litter size in sheep.

The effects of cryopreservation on the acrosome structure, enzyme activity, motility, and fertility of bovine, ovine, and goat sperm.

The study was designed to investigate the effects of cryopreservation on bovine, ovine, and goat sperm motility, acrosome structure, enzyme activity, and fertilization ability. Percentage of sperm with hyaluronidase enzyme (HYD) activity was detected by a modified sodium hyaluronate-gelatin membrane. The N-α-benzoyl-DL-arginine-p-nitroanilide (BNPNA) method was used to assess the sperm acrosome enzyme (ACE). The mean percentage of sperm acrosome integrity dropped significantly (P < 0.01) after cryopreservation. The ACE activity of bovine sperm (100.48) was higher (P < 0.01) than that of ovine (57.88) or goat sperm (50.30), while the percentage of sperm with HYD activity of bovine (71.10%) and ovine (67.60%) sperm was higher than that of goat sperm (58.52%) after cryopreservation (P < 0.01). Sperm motility was positively correlated with the activity of the two acrosome enzymes before and after cryopreservation (P < 0.01). Cryopreservation had a negative effect on acrosomal morphology, motility, and acrosomal enzyme activity in their sperm. The fertilization ability of ovine and goat sperm decreased significantly after cryopreservation, but that of frozen bovine sperm did not differ significantly when compared with fresh sperm. There was no significant difference between ovine and goat sperm indices, except for percentage of sperm with HYD activity.