Melatonin protects oogenesis from hypobaric hypoxia-induced fertility damage in mice.

Environmental hypoxia adversely affects reproductive health in humans and animals at high altitudes. Therefore, how to alleviate the follicle development disorder caused by hypoxia exposure and to improve the competence of fertility in plateau non-habituated female animals are important problems to be solved urgently. In this study, a hypobaric hypoxic chamber was used for 4 weeks to simulate hypoxic conditions in female mice, and the effects of hypoxia on follicle development, proliferation and apoptosis of granulosa cells, reactive oxygen species (ROS) levels in MII oocyte and 2-cell rate were evaluated. At the same time, the alleviating effect of melatonin on hypoxic exposure-induced oogenesis damage was evaluated by feeding appropriate amounts of melatonin daily under hypoxia for 4 weeks. The results showed that hypoxia exposure significantly increased the proportion of antral follicles in the ovary, the number of proliferation and apoptosis granulosa cells in the follicle, and the level of ROS in MII oocytes, eventually led to the decline of oocyte quality. However, these defects were alleviated when melatonin was fed under hypoxia conditions. Together, these findings suggest that hypoxia exposure impaired follicular development and reduced oocyte quality, and that melatonin supplementation alleviated the fertility reduction induced by hypoxia exposure.

Three Novel Players: PTK2B, SYK, and TNFRSF21 Were Identified to Be Involved in the Regulation of Bovine Mastitis Susceptibility via GWAS and Post-transcriptional Analysis.

Bovine mastitis is a common inflammatory disease caused by multiple factors in early lactation or dry period. Genome wide association studies (GWAS) can provide a convenient and effective strategy for understanding the biological basis of mastitis and better prevention. 2b-RADseq is a high-throughput sequencing technique that offers a powerful method for genome-wide genetic marker development and genotyping. In this study, single nucleotide polymorphisms (SNPs) of the immune-regulated gene correlative with mastitis were screened and identified by two stage association analysis via GWAS-2b-RADseq in Chinese Holstein cows. We have screened 10,058 high quality SNPs from 7,957,920 tags and calculated their allele frequencies. Twenty-seven significant SNPs were co-labeled in two GWAS analysis models [Bayesian (P < 0.001) and Logistic regression (P < 0.01)], and only three SNPs (rs75762330, C > T, PIC = 0.2999; rs88640083, A > G, PIC = 0.1676; rs20438858, G > A, PIC = 0.3366) were annotated to immune-regulated genes (PTK2B, SYK, and TNFRSF21). Identified three SNPs are located in non-coding regions with low or moderate genetic polymorphisms. However, independent sample population validation (Case-control study) data showed that three important SNPs (rs75762330, P < 0.025, OR > 1; rs88640083, P < 0.005, OR > 1; rs20438858, P < 0.001, OR < 1) were significantly associated with clinical mastitis trait. Importantly, PTK2B and SYK expression was down-regulated in both peripheral blood leukocytes (PBLs) of clinical mastitis cows and in vitro LPS (E. coli)-stimulated bovine mammary epithelial cells, while TNFRSF21 was up-regulated. Under the same conditions, expression of Toll-like receptor 4 (TLR4), AKT1, and pro-inflammatory factors (IL-1ß and IL-8) were also up-regulated. Interestingly, network analysis indicated that PTK2B and SYK are co-expressed in innate immune signaling pathway of Chinese Holstein. Taken together, these results provided strong evidence for the study of SNPs in bovine mastitis, and revealed the role of SYK, PTK2B, and TNFRSF21 in bovine mastitis susceptibility/tolerance.

Transcriptome profiling reveals the role of ZBTB38 knock-down in human neuroblastoma.

ZBTB38 belongs to the zinc finger protein family and contains the typical BTB domains. As a transcription factor, ZBTB38 is involved in cell regulation, proliferation and apoptosis, whereas, functional deficiency of ZBTB38 induces the human neuroblastoma (NB) cell death potentially. To have some insight into the role of ZBTB38 in NB development, high throughput RNA sequencing was performed using the human NB cell line SH-SY5Y with the deletion of ZBTB38. In the present study, 2,438 differentially expressed genes (DEGs) in ZBTB38-/- SH-SY5Y cells were obtained, 83.5% of which was down-regulated. Functional annotation of the DEGs in the Kyoto Encyclopedia of Genes and Genomes database revealed that most of the identified genes were enriched in the neurotrophin TRK receptor signaling pathway, including PI3K/Akt and MAPK signaling pathway. we also observed that ZBTB38 affects expression of CDK4/6, Cyclin E, MDM2, ATM, ATR, PTEN, Gadd45, and PIGs in the p53 signaling pathway. In addition, ZBTB38 knockdown significantly suppresses the expression of autophagy-related key genes including PIK3C2A and RB1CC1. The present meeting provides evidence to molecular mechanism of ZBTB38 modulating NB development and targeted anti-tumor therapies.

A potential role for SMAD9 in goose follicular selection through regulation of mRNA levels of luteinizing hormone receptor.

The egg production of poultry depends on follicular development and selection. Nonetheless, the mechanism underlying the priority of selecting of hierarchical follicles is completely unknown. SMAD9 is one of the important transcription factors in the BMP/SMAD pathway and is involved in goose follicular initiation. To identify its potential role in determination of the goose follicle hierarchy, we used BMP type I receptor inhibitor LDN-193189 both in vivo and in vitro and found that SMAD9 mRNA expression decreased in the presence of LDN-193189. While the level of SMAD9 mRNA decreased after treatment with LDN-193189, we found that the egg production (7.08 eggs per bird per year) of the animals increased, estradiol (E2) levels significantly increased, but the levels of progesterone (P4) remained unchanged. We also detected a significant increase in luteinizing hormone receptor (LHR) mRNA expression, but no change in follicle-stimulating hormone receptor (FSHR) mRNA amounts. The in vitro experimental results indicated that SMAD9 knockdown by RNA interference noticeably reduced E2 and P4 biosynthesis and FSHR and LHR mRNA expression in goose granulosa cells. Chromatin immunoprecipitation assay of goose granulosa cells revealed that phospho-SMAD9 bound to the LHR promoter and possibly regulated its transcriptional activity. These findings revealed that SMAD9 is differentially expressed in goose follicles, and acts as a key player in the control over goose follicular selection.

The effects of mouse ovarian granulosa cell function and related gene expression by suppressing BMP/Smad signaling pathway.

BMP I type receptor inhibitor can selectively inhibit BMP/Smad signaling pathways, mainly by inhibiting the BMP I type receptor activity to prevent phosphorylation of Smad1, Smad5 and Smad9. The aim of the present study was to explore the effects of mouse ovarian granulosa cell function and related gene expression by suppressing BMP/Smad signaling pathway with LDN-193189(A type of BMP I type receptor inhibitor). In this study, we cultivate the original generation of mouse ovarian granular cells then collect cells and cell culture medium after treatment. Cellular localization and expression of Smad9 and P-smad9 proteins was studied by immunofluorescence (IF) in the ovarian granulosa cells of mouse; Related genes mRNA and proteins expression was checked by QRT-PCR and Western blot; Detected the concentration of related hormones by using ELISA kit; finally, the growth of the cells was analyzed by plotting cell growth curve with CCK-8 assay. The results indicate that, suppression of BMP/Smad signaling pathway can inhibit the expression of LHR and FSHR, inhibit cell proliferation and decrease E2 secretion, the mechanism of action maybe reduce the expression of smad9, at the same time, we found that the feedback regulation of smad9 may affect the expression of FSHR and cell proliferation.

ZBTB38, a novel regulator of autophagy initiation targeted by RB1CC1/FIP200 in spinal cord injury.

Apoptosis is an important contributing factor in spinal cord injury (SCI). ZBTB38 is involved in the transcriptional regulation of multiple signaling pathways, is differentially expressed at different SCI stages, and may provide a therapeutic strategy for the treatment of patients with SCI. In this study, we found that autophagy is blocked in ZBTB38 knockdown SH-SY5Y cells and that the expression levels of LC3B II/I decreased and P62 increased. We used transcriptome high-throughput sequencing to identify the target in ZBTB38 knockdown cells. From the transcriptome profile, RB1CC1 (i.e., FIP200), a key component of the initiation machinery of autophagy (FIP200-ATG13-ULK1-ATG101), was found to decrease 4.2-fold following ZBTB38 knockdown. When RB1CC1-overexpressed plasmids were transfected into ZBTB38 knockdown cells, they rescued the phenotype of ZBTB38 knockdown cells. Cell proliferation and viability were significantly enhanced by RB1CC1 overexpression, and LC3B and P62 expression returned to their original levels. We also injected ZBTB38-overexpressed lentivirus into the injured center of the spinal cord and detected significant upregulation of RB1CC1 in the spinal cord. ZBTB38 overexpression can promote autophagy and partly rescue the secondary damage of SCI. Therefore, our findings provide a new strategy for the treatment of SCI.

Tauroursodeoxycholic acid alleviates secondary injury in the spinal cord via up-regulation of CIBZ gene.

Spinal cord injury (SCI) is generally divided into primary and secondary injuries, and apoptosis is an important event of the secondary injury. As an endogenous bile acid and recognized endoplasmic reticulum (ER) stress inhibitor, tauroursodeoxycholic acid (TUDCA) administration has been reported to have a potentially therapeutic effect on neurodegenerative diseases, but its real mechanism is still unclear. In this study, we evaluated whether TUDCA could alleviate traumatic damage of the spinal cord and improve locomotion function in a mouse model of SCI. Traumatic SCI mice were intraperitoneally injected with TUDCA, and the effects were evaluated based on motor function assessment, histopathology, apoptosis detection, qRT-PCR, and western blot at different time periods. TUDCA administration can improve motor function and reduce secondary injury and lesion area after SCI. Furthermore, the apoptotic ratios were significantly reduced; Grp78, Erdj4, and CHOP were attenuated by the treatment. Unexpectedly, the levels of CIBZ, a novel therapeutic target for SCI, were specifically up-regulated. Taken together, it is suggested that TUDCA effectively suppressed ER stress through targeted up-regulation of CIBZ. This study also provides a new strategy for relieving secondary damage by inhibiting apoptosis in the early treatment of spinal cord injury.