Follicular fluid HD-sevs-mir-128-3p is a key molecule in regulating bovine granulosa cells autophagy.

Follicular fluid (FF) is rich in extracellular vesicles (EVs). EVs carries a variety of miRNA involved in regulating follicular development, the function of cells in follicles, primordial follicular formation, follicular recruitment and selection, follicular atresia, oocyte communication, granulosa cells (GCs) function and luteinization and other biological processes of follicular development. Previous studies in our laboratory have shown that bovine follicular fluid (bFF) high density-small extracellular vesicles (HD-sEVs)-miRNA was enriched in autophagy-related pathways. However, the mechanism of bFF EVs carrying miRNA regulating GCs autophagy is not clear. Thus, this study carried out a series of studies on the previous HD-sEVs sequencing data and miR-128-3p contained in bFF HD-sEVs. A total of 38 differentially expressed genes were detected by RNA-Seq after overexpression of miR-128-3p in bovine GCs (bGCs). Through cell transfection, Western blot (WB) and Immunofluorescence (IF), it was proved that overexpression of miR-128-3p could promote the expression of LC3 (microtubule-associated protein I light chain 3), inhibit p62, promote the number of autophagosome, promote the formation of autophagy lysosome and autophagy flow, and activate bGCs autophagy. MiR-128-3p inhibitor significantly inhibited the expression of LC3 and monodansylcadaverine (MDC) in bGCs, and promoted the expression of autophagy substrate p62, indicating that HD-sEVs-miR-128-3p could activate bGCs autophagy. In addition, through double luciferase assay, bioinformatics analysis, WB and RT-qPCR, it was concluded that bFF HD-sEVs-miR-128-3p could target TFEB (transcription factor EB) and FoxO4 (Forkhead box O4) and activate GCs autophagy.

Large extracellular vesicles in bovine follicular fluid inhibit the apoptosis of granulosa cell and stimulate the production of steroid hormones.

The cargo carried by extracellular vesicles (EVs) plays an important physiological role in their corresponding target organs or target tissue cells. Extracellular vesicles are classified into large extracellular vesicles (LEVs) and small extracellular vesicles (SEVs) according to their diameters. Since different subtypes contain different contents, their roles are also different. In this study, the morphology and size of LEVs were analyzed by transmission electron microscopy and nanoparticle size, and the marker proteins of LEVs (CD63, GP96, TSG101, ALB) were identified by western blot, and high-purity LEVs were obtained. Through the uptake of extracellular vesicles by purified ovarian granulosa cells and the determination of granulosa cell viability, cell apoptosis, and steroid hormone production, the result indicated that LEVs significantly enhanced cell viability (P < 0.05), reduced the rate of granulosa cell apoptosis (P < 0.05). Meanwhile, LEVs promoted the secretion of estradiol in granulosa cells (P < 0.05). This study provides a reference for the in-depth study of the function of follicular fluid extracellular vesicle subtypes and the research on the regulation of extracellular vesicles on follicle and oocyte development.

Extracellular vesicles of bovine small follicular fluid promote ovarian cortical stromal cell proliferation and steroidogenesis.

The aim of this study was to investigate the effects of extracellular vesicles (EVs) on the proliferation and steroid hormone synthesis of bovine ovarian cortical stromal cells in vitro. The release and uptake of EVs are the new mechanisms of cell-to-cell communication. Using reverse transcriptase polymerase chain reaction, enzyme-linked immunosorbent assay, TUNEL and other experiments, we found that EVs in bovine follicular fluid can promote the proliferation and synthesis of androstenedione and progesterone in ovarian cortical stromal cells. Moreover, 100 µg/ml EVs caused the most significant effect. We conclude that EVs at 100 µg/ml can significantly promote the proliferation and synthesis of androstenedione and progesterone in ovarian cortical stromal cells. This research is of great significance for further elucidating the regulatory role of follicular fluid EVs in follicular development and atresia and for research on the interaction of ovarian stromal cells, granulosa cells and oocytes.

Effect of extracellular vesicles and microRNAs in follicular fluid on follicular development / 生物工程学报

Extracellular vesicles (EVs) refer to bilayer membrane transport vesicles secreted by cells. EVs can take macromolecules from cells and transfer them to receptor cells. Among these macromolecular substances, the most studied are microRNAs (miRNAs). miRNA is non-coding RNA involved in the regulation of gene expression. It has been confirmed that there are different non-coding RNAs in mammalian follicular fluid EVs. EVs carrying miRNA can act as an alternative mechanism for autocrine and paracrine, affecting follicular development. This paper systematically introduced the kinds, characteristics and methods of isolation and identification of EVs, focusing on the effects of EVs and miRNAs on follicular development, including early follicular development, oocyte maturation, follicular dominance and effects on granulosa cell function. At the same time, the authors prospected the future research of EVs and microRNAs in follicular fluid, and provided ideas and directions for the research and application of EVs and miRNA functions in follicular fluid.

Site-directed mutagenesis of MSTN gene by gene targeting in Qinchuan cattle / 生物工程学报

Myostatin, a member of the transforming growth factor beta (TGF-beta) family, is a negative regulator for muscle growth. Loss of the function of this gene is associated with the phenotype described as "double muscling", an extreme form of muscle development characterized by a large increase in muscle mass. Two replacement vectors, pA2T-Mstn4.0 and pA2T-Mstn3.2, were constructed, linearized, and transfected into the bovine fetal fibroblasts through electroporation. 170 drug-resistant cell colonies were obtained in cell culture medium containing 600 microg/mL G418 and 50 nmol/L GCV. Targeted homologous integration occurred in colony No. 58 as identified by PCR, and the targeted colony was further confirmed by sequencing and Southern blotting. This suggested that one allele of myostatin was successfully mutagenized in bovine fetal fibroblasts.

Nuclear localization of oligonucleotides decoy effect on nuclear factor-kappaB activity / 生物工程学报

To investigate the effect of the localization of oligonucleotides decoy (ODNs decoy) on the activation of nuclear factor-kappaB (NF-kappaB) in TNF-alpha induced HeLa cells. The mercapto group-modified nuclear localization signal (NLS) peptide was covalently conjugated to amino group-modified NF-kappaB ODNs decoy by Sulfo-SMCC cross-linker. The NLS-ODNs decoy was transfected into HeLa cells by TransME transfection reagent. The intracellular distribution of fluorescent labeled NLS-ODNs decoy was detected with a microscope. The cell viability was detected by MTT assay, and then the activity of NF-kappaB in cell nuclear extract was assayed by electrophoretic mobility shift assay (EMSA). The results showed that NLS peptide was successfully conjugated to ODNs decoy by Sulfo-SMCC cross-linker. The NLS-ODNs decoy effectively entered into nucleus with high rate of 17.9%. It was observed that the cell viability of HeLa cell was not significantly affected by the transfection of NLS-ODNs decoy, while NLS-ODNs decoy significantly inhibited the activation of NF-kappaB in TNF-alpha induced HeLa cells nuclear extracts. This experiment can provide a new covalent conjugation of NLS peptide to ODNs can effectively drive decoy into nucleus, and thus improve its inhibitory effects on the activation a transcription factor.

Co-culture of mouse blastocysts and their epigenetic modification / 生物工程学报

To discuss the effect of co-culture on the quality of mouse blastocysts and their epigenetic modification. We divided mouse zygotes into three co-culture experiment groups : with granular cells (group I ), oviduct epithelium cells (group II) and oviduct tissue (group III). Meanwhile, we set up control A (cultured in vitro, only KSOM (KCl+ simplex optimized medium)) and control B (cultured in vivo). Then we compared cleavage rate and blastocyst rate among different groups. After that we evaluated the quality of blastocysts by using ICM/TE (Inner cell mass/Trophectoderm cells) ratio via staining with propidium iodide and Hoechest333258, and analyzed the level of genome methylation and histone acetylation by immunofluorescence. Compared with the control group A, the co-culture groups had increased cleavage rate and blastocyst rate (P < 0.05), blastocyst cells and the ICM/TE ratio of co-culture groups were higher (P < 0.05), the level of genome methylation and histone acetylation had no significant difference between groups in vitro (P > 0.05), but the level of genome methylation in vivo was significantly higher than that of in vitro (P < 0.05). The co-culture methods can successfully promote the development rate of embryos in vitro, and improve the quality of the blastocyst. However, the methods have drawbacks in changing the abnormal genome methylation with in vitro culture.