Changes in bacterial community composition in the uterus of Holstein cow with endometritis before and after treatment with oxytetracycline.

It is important to study the bacteria that cause endometritis to identify effective therapeutic drugs for dairy cows. In this study, 20% oxytetracycline was used to treat Holstein cows (n = 6) with severe endometritis. Additional 10 Holstein cows (5 for healthy cows, 5 for cows with mild endometritis) were also selected. At the same time, changes in bacterial communities were monitored by high-throughput sequencing. The results show that Escherichia coli, Staphylococcus aureus and other common pathogenic bacteria could be detected by traditional methods in cows both with and without endometritis. However, 16S sequencing results show that changes in the abundance of these bacteria were not significant. Endometritis is often caused by mixed infections in the uterus. Oxytetracycline did not completely remove existing bacteria. However, oxytetracycline could effectively inhibit endometritis and had a significant inhibitory effect on the genera Bacteroides, Trueperella, Peptoniphilus, Parvimonas, Porphyromonas, and Fusobacterium but had no significant inhibitory effect on the bacterial genera Marinospirillum, Erysipelothrix, and Enteractinococcus. During oxytetracycline treatment, the cell motility, endocrine system, exogenous system, glycan biosynthesis and metabolism, lipid metabolism, metabolism of terpenoids, polyketides, cofactors and vitamins, signal transduction, and transport and catabolism pathways were affected.

TBX2 affects proliferation, apoptosis and cholesterol generation by regulating mitochondrial function and autophagy in bovine cumulus cell.

BACKGROUND: T-box transcription factor 2 (TBX2) is a member of T-box gene family whose members are highly conserved in evolution and encoding genes and are involved in the regulation of developmental processes. The encoding genes play an important role in growth and development. Although TBX2 has been widely studied in cancer cell growth and development, its biological functions in bovine cumulus cells remain unclear. OBJECTIVES: This study aimed to investigate the regulatory effects of TBX2 in bovine cumulus cells. METHODS: TBX2 gene was knockdown with siRNA to clarify the function in cellular physiological processes. Cell proliferation and cycle changes were determined by xCELLigence cell function analyzer and flow cytometry. Mitochondrial membrane potential and autophagy were detected by fluorescent dye staining and immunofluorescence techniques. Western blot and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) were used to detect the expression changes of proliferation and autophagy-related proteins. Aadenosine triphosphate (ATP) production, glucose metabolism, and cholesterol synthesis of cumulus cells were measured by optical density and chemiluminescence analysis. RESULTS: After inhibition of TBX2, the cell cycle was disrupted. The levels of apoptosis, ratio of light chain 3 beta II/I, and reactive oxygen species were increased. The proliferation, expansion ability, ATP production, and the amount of cholesterol secreted by cumulus cells were significantly decreased. CONCLUSIONS: TBX2 plays important roles in regulating the cells' proliferation, expansion, apoptosis, and autophagy; maintaining the mitochondrial function and cholesterol generation of bovine cumulus cells.

KRAS Affects Adipogenic Differentiation by Regulating Autophagy and MAPK Activation in 3T3-L1 and C2C12 Cells.

Kirsten rat sarcoma 2 viral oncogene homolog (Kras) is a proto-oncogene that encodes the small GTPase transductor protein KRAS, which has previously been found to promote cytokine secretion, cell survival, and chemotaxis. However, its effects on preadipocyte differentiation and lipid accumulation are unclear. In this study, the effects of KRAS inhibition on proliferation, autophagy, and adipogenic differentiation as well as its potential mechanisms were analyzed in the 3T3-L1 and C2C12 cell lines. The results showed that KRAS was localized mainly in the nuclei of 3T3-L1 and C2C12 cells. Inhibition of KRAS altered mammalian target of rapamycin (Mtor), proliferating cell nuclear antigen (Pcna), Myc, peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer binding protein beta (C/ebp-ß), diacylglycerol O-acyltransferase 1 (Dgat1), and stearoyl-coenzyme A desaturase 1 (Scd1) expression, thereby reducing cell proliferation capacity while inducing autophagy, enhancing differentiation of 3T3-L1 and C2C12 cells into mature adipocytes, and increasing adipogenesis and the capacity to store lipids. Moreover, during differentiation, KRAS inhibition reduced the levels of extracellular regulated protein kinases (ERK), c-Jun N-terminal kinase (JNK), p38, and phosphatidylinositol 3 kinase (PI3K) activation. These results show that KRAS has unique regulatory effects on cell proliferation, autophagy, adipogenic differentiation, and lipid accumulation.

Regulation of FSH Synthesis by Differentially Expressed miR-488 in Anterior Adenohypophyseal Cells.

Gonadotropin-releasing hormone (GnRH), which is synthesized and released by the hypothalamus, promotes the synthesis and secretion of follicle-stimulating hormone (FSH), thereby regulating the growth and reproduction of animals. GnRH analogues have been widely used in livestock production. MiRNAs, which are endogenous non-coding RNAs, have been found to play important roles in hormone regulation and other physiological processes in recent years. However, the roles of miRNAs in GnRH-mediated regulation of FSH secretion have rarely been studied. Herein, we treated bovine anterior adenohypophyseal cells with an exogenous GnRH analogue and found that miR-488 was differentially expressed. Through a combination of TargetScan prediction and dual luciferase reporter analysis, miR-488 was confirmed to be able to target the FSHB gene. Based on this finding, we verified the expression of Fshß and Lhß mRNA in the rat adenohypophysis before and after exogenous GnRH treatment in vivo and in vitro. Experiments on rat anterior adenohypophyseal cells showed that overexpression of miR-488 significantly inhibited Fshß expression and FSH synthesis, while knockdown of miR-488 had the opposite effects. Our results demonstrate that GnRH relies on miR-488 to regulate FSH synthesis, providing additional useful evidence for the significance of miRNAs in the regulation of animal reproduction.

Schisanhenol improves early porcine embryo development by regulating the phosphorylation level of MAPK.

Schisanhenol (SAL), a biphenyl cyclooctene-type lignin compound which can be extracted and isolated from many plants of the Schisandra family, exhibits a variety of biological activities including anti chronic cough, night sweating, thirst, diabetes, and obesity. However, its effects on the female reproductive system are unclear. Previous studies showed that SAL had potential antioxidant activity in heart, liver, and brain. Therefore, we hypothesized that SAL could improve porcine early development by reducing oxidative stress. The purpose of this study was to investigate the effects of SAL on preimplantation porcine embryos and the potential mechanisms. In this study, we analyzed the effects of SAL on embryo quality, reactive oxygen species (ROS) accumulation, mitochondrial function, cell proliferation and apoptosis, and the activation of MAPK pathway. The results showed that 10 µM SAL significantly increased the blastocyst formation rate, proliferation ability, and mitochondrial activity while reducing ROS accumulation and apoptosis level. During this process, the phosphorylation levels of ERK1/2, JNK1/2/3, and p38 were decreased. In summary, 10 µM SAL improves porcine preimplantation embryo development by reducing ROS accumulation.

Carnosic acid improves porcine early embryonic development by inhibiting the accumulation of reactive oxygen species.

Carnosic acid (CA), a natural catechol rosin diterpene, is used as an additive in animal feeds and human foods. However, the effects of CA on mammalian reproductive processes, especially early embryonic development, are unclear. In this study, we added CA to parthenogenetically activated porcine embryos in an in vitro culture medium to explore the influence of CA on apoptosis, proliferation, blastocyst formation, reactive oxygen species (ROS) levels, glutathione (GSH) levels, mitochondrial membrane potential, and embryonic development-related gene expression. The results showed that supplementation with 10 µM CA during in vitro culture significantly improved the cleavage rates, blastocyst formation rates, hatching rates, and total numbers of cells of parthenogenetically activated porcine embryos compared with no supplementation. More importantly, supplementation with CA also improved GSH levels and mitochondrial membrane potential, reduced natural ROS levels in blastomeres, upregulated Nanog, Sox2, Gata4, Cox2, Itga5, and Rictor expression, and downregulated Birc5 and Caspase3 expression. These results suggest that CA can improve early porcine embryonic development by regulating oxidative stress. This study elucidates the effects of CA on early embryonic development and their potential mechanisms, and provides new applications for improving the quality of in vitro-developed embryos.

CPEB3 regulates the proliferation and apoptosis of bovine cumulus cells.

Cytoplasmic polyadenylation element-binding protein 3 (CPEB3) is a member of the Cytoplasmic polyadenylation element-binding family, which has been found to regulate the translation of dormant and masked mRNA in Xenopus oocytes and plays potential roles in regulating biological functions in cells and tissues. However, its role in cumulus cells is not clear. In this study, the mRNA expression of CPEB3 in bovine cumulus cells was inhibited with small interfering RNA. Cell cycle progression, proliferation, and apoptosis were measured after inhibition of CPEB3. Subsequently, changes in intracellular Reactive oxygen species content, mitochondrial membrane potential and expansion-related gene expression were examined. The results showed that after CPEB3 inhibition, cumulus cells had an abnormal cell cycle, the numbers of cells in the S and G2/M phases were significantly increased, cell proliferation was increased and apoptosis rates were decreased. These effects were likely due CPEB3 inhibition-induced decreases in intracellular Reactive oxygen species levels; increases in mitochondrial membrane potential; decreases in apoptosis; downregulation of CCNA, CCND, CCNE, CDK2, CDK4, CDK6, p21, and p27 mRNA expression; and upregulation of CCNB, CDK1, HAS2, PTGS2, PTX3, and CEBPB mRNA expression. Therefore, CPEB3 plays potential roles in regulating the biological and physiological functions of bovine cumulus cell.

Imperatorin improves in vitro porcine embryo development by reducing oxidative stress and autophagy.

Imperatorin (IMP), a furanocoumarin derivative with many biological properties and pharmacological activities, is widely used as an antibacterial, anti-inflammatory, antiviral, anticancer, cardiovascular and neuroprotective agent. The purpose of this study was to explore the effects of IMP on early embryo development in pigs as well as the potential mechanisms. Our results showed that IMP can enhance the developmental competence of porcine early embryos. Supplementation of in vitro culture medium with 40 µM IMP significantly increased the blastocyst rate and total cell number. At the same time, apoptosis of blastocysts was also significantly decreased in the supplemented group compared with the control group, in accordance with the subsequent results of FAS and CASP3 gene expression analysis. Furthermore, IMP attenuated intracellular reactive oxygen species (ROS) generation, increased fluorescein diacetate (FDA) and glutathione (GSH) levels. Importantly, IMP not only improved the activity of mitochondria but also inhibited the occurrence of autophagy. In addition, pluripotency-related genes (OCT4, NANOG, and SOX2) and a growth and metabolism regulatory gene (mTOR) were upregulated after IMP supplementation on Day 7. These results demonstrate that IMP exerts a beneficial effect on preimplantation embryo development by reducing oxidative stress and autophagy.

Dual-specificity phosphatase 1 regulates cell cycle progression and apoptosis in cumulus cells by affecting mitochondrial function, oxidative stress, and autophagy.

Dual-specificity phosphatase 1 (DUSP1) is differentially expressed in cumulus cells of different physiological states, but its specific function and mechanism of action remain unclear. In this study, we explored the effects of DUSP1 expression inhibition on cell cycle progression, proliferation, apoptosis, and lactate and cholesterol levels in cumulus cells and examined reactive oxygen species levels, mitochondrial function, autophagy, and the expression of key cytokine genes. The results showed that inhibition of DUSP1 in cumulus cells caused abnormal cell cycle progression, increased cell proliferation, decreased apoptosis rates, increased cholesterol synthesis and lactic acid content, and increased cell expansion. The main reason for these effects was that inhibition of DUSP1 reduced ROS accumulation, increased glutathione level and mitochondrial membrane potential, and reduced autophagy levels in cells. These results indicate that DUSP1 limits the biological function of bovine cumulus cells under normal physiological conditions and will greatly contribute to further explorations of the physiological functions of cumulus cells and the interactions of the cumulus-oocyte complex.

COL1A1 affects apoptosis by regulating oxidative stress and autophagy in bovine cumulus cells.

The collagen type I alpha 1 chain (COL1A1), as a major component of extracellular matrix, plays a potential role in the growth and development of bovine follicles. However, its specific role in bovine cumulus cells remains unclear. In this study, we examined apoptosis, the cell cycle and reactive oxygen species after inhibition of COL1A1 expression by siRNA in bovine cumulus cells. Cell proliferation was measured by CCK-8, and mitochondrial membrane potential was detected by fluorescence intensities of JC-1 staining. Moreover, cell autophagy was detected by immunofluorescence, and cell migration was detected by a cell scratch assay. Lactic acid and cholesterol concentration were measured to evaluate the glucose utilization and cholesterol synthesis activity in cumulus cell by optical density detection method. RT-qPCR and Western blot analysis were used to measure changes in key gene expression. The results showed that cumulus cells were found to have an abnormal cell cycle, and the numbers of cells in S phase were significantly reduced, accompanied by decreases in cholesterol synthesis, and cell proliferation ability and an increase in apoptosis rate with siRNA-COL1A1 treatment. These findings were likely due to inhibition of COL1A1 resulting in high levels of ROS in the cells, a decrease in mitochondrial membrane potential, an increase in intracellular autophagy, activation of the apoptotic pathway, and a decrease in lactic acid conversion ability. COL1A1 plays an important role in regulating the physiological and biological functions of bovine cumulus cells.

Identification of circular RNAs in the immature and mature rat anterior pituitary.

Circular RNAs (circRNAs) are a new class of RNA that have a stable structure characterized by covalently closed circular molecules and are involved in invasive pituitary adenomas and recurrent clinically nonfunctioning pituitary adenomas. However, information on circRNAs in the normal pituitary, especially in rats, is limited. In this study, we identified 4123 circRNAs in the immature (D15) and mature (D120) rat anterior pituitary using the Illumina platform, and 32 differentially expressed circRNAs were found. A total of 150 Gene Ontology terms were significantly enriched, and 16 KEGG pathways were found to contain differentially expressed genes. Moreover, we randomly selected eight highly expressed circRNAs and detected their relative expression levels in the mature and immature rat pituitary by qPCR. In addition, we predicted 90 interactions between 53 circRNAs and 57 miRNAs using miRanda. Notably, circ_0000964 and circ_0001303 are potential miRNA sponges that may regulate the Fshb gene. The expression profile of circRNAs in the immature and mature rat anterior pituitary may provide more information about the roles of circRNAs in the development and reproduction in mammals.

MiR-31 and miR-143 affect steroid hormone synthesis and inhibit cell apoptosis in bovine granulosa cells through FSHR.

The regulatory role of microRNAs (miRNAs) has been explored in ovarian cells, and the effects of miRNAs on gonadal development, apoptosis, ovulation, and steroid production have been reported. In this study, we analyzed the effects of follicle stimulating hormone (FSH) on miR-31 and miR-143 expression levels in bovine granulosa cells (GCs). Our results demonstrated that the FSH receptor (FSHR) is a common target gene of miR-31 and miR-143 in bovine GCs. We further analyzed the roles of miR-31 and miR-143 in bovine GCs by transfecting miR-31 and miR-143 mimics and inhibitors. The Western blot and RT-PCR results showed that miR-31 and miR-143 reduced the mRNA and protein expression levels of FSHR. Moreover, miR-31 overexpression decreased the secretion of progesterone (P4), and miR-143 overexpression decreased both the synthesis of P4 and the secretion of estrogen (E2). In contrast, miR-31 inhibition increased the secretion of progesterone (P4), and miR-143 inhibition increased both the synthesis of P4 and the secretion of E2. Finally, we analyzed the possible effects of miR-31 and miR-143 on bovine GC apoptosis. The results showed that transfection with miR-31 and miR-143 mimics promoted GC apoptosis and that miR-143 and miR-31 inhibition reduced the rate of apoptosis in bovine GCs. Taken together, our results indicate that miR-31 and miR-143 decrease steroid hormone synthesis and inhibit bovine GC apoptosis by targeting FSHR.

Identification of long non-coding RNAs in the immature and mature rat anterior pituitary.

Many long non-coding RNAs (lncRNAs) have been identified in several types of human pituitary adenomas and normal anterior pituitary, some of which are involved in the pathogenesis of pituitary adenomas. However, a systematic analysis of lncRNAs expressed at different developmental stages of normal pituitary, particularly in rats, has not been performed. Therefore, we contrasted two cDNA libraries of immature (D15) and mature (D120) anterior pituitary in rat that were sequenced on an Illumina HiSeq Xten platform, and a total of 29,568,806,352 clean reads were identified. Notably, 7039 lncRNA transcripts corresponded to 4442 lncRNA genes, and 1181 lncRNA transcripts were significantly differentially expressed in D15 and D120. In addition, 6839 protein-coding genes (<100 kb upstream and downstream) were the nearest neighbors of 4074 lncRNA genes. An interaction network of lncRNAs and the follicle-stimulating hormone beta-subunit (FSHb) gene was constructed using the lncRNATargets platform, and three novel lncRNAs were obtained. Furthermore, we detected the expression of the novel lncRNAs and ten highly expressed lncRNAs that were randomly selected through quantitative PCR (qPCR). The rat anterior pituitary lncRNA content identified in this study provides a more in-depth understanding of the roles of these lncRNAs in hormone and reproduction development and regulation in mammals.

Roles of differential expression of microRNA-21-3p and microRNA-433 in FSH regulation in rat anterior pituitary cells.

Follicle-stimulating hormone (FSH) secreted by adenohypophyseal cells plays an important role in the regulation of reproduction, but whether microRNAs (miRNAs) regulate the secretion of FSH remains unclear. In the present study, we predicted and screened miRNAs that might act on the follicle-stimulating hormone beta-subunit (FSHb) gene of rats using the TargetScan program and luciferase reporter assays, and the results identified two miRNAs, miR-21-3p and miR-433. We then transfected these miRNAs into rat anterior adenohypophyseal cells and assessed the FSHb expression levels in and FSH secretion by the transfected cells through quantitative PCR and ELISA. The results showed that both miR-21-3p and miR-433 down-regulated the expression levels of FSHb and resulted in the decrease of the secretion of FSH compared with the control group, and treatment with miR-21-3p and miR-433 inhibitors up-regulated the expression levels of FSHb and resulted in the increase of the secretion of FSH. Taken together, our results indicate that miR-21-3p and miR-433 can down-regulate the expression of FSHb by directly targeting the FSHb 3'UTR in rat primary pituitary cells. Our findings provide evidence that miRNAs can regulate FSHb expression and further affect the secretion of FSH and might contribute to the use of miRNAs for the regulation of animal reproduction.

A comprehensive expression profile of micrornas in rat's pituitary.

MicroRNAs (miRNAs) are a class of small, non-coding RNA molecules that act as a negative regulator of most mRNAs. miRNAs influence the gene expression as transcriptional regulators and play an important role in many fundamental biological processes. It is generally acknowledged that miRNAs have a very important affection on mammalian pituitary. However, the answers of which role miRNAs play in the development of sexual function or how much they contribute to the pituitary function are not exactly. In our study, we used three female 21-day-old rats and three female 12-month-old rats to analysis the function of miRNAs. By the analyses of microarray data, we finished the stem-loop real-time RT-PCR for the differentially expressed miRNAs. We detected a total of 93 differentially expressed miRNAs between 21-day-old rats' pituitary and 12-month-old rats'. Stem-loop real-time RT-PCR suggests that the obtained data is of high credibility. Among these miRNAs, 7 miRNAs' expression (rno-miR-880, rno-miR-503, rno-miR-125a-3p, rno-miR-3596b, rno-miR-30e, rno-miR-214 and rno-miR-22) are significant different (P≤0.05). In a word, this study identified a number of specific changes in the expression of miRNAs, in rats by detecting the expression profile of miRNAs in rat's pituitary, and all of that lay the foundation for elucidating the regulatory mechanisms of miRNAs in rat's reproduction process. These differentially expressed miRNAs may play a very important role in rat's reproduction process.