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Transcription factors (TFs) are known to be involved in regulating the expression of several classes of genes during folliculogenesis. However, the regulatory role of TFs during oxidative stress (OS) is not fully understood. The current study was aimed to investigate the regulation of the TFs in bovine granulosa cells (bGCs) during exposure to OS induced by H2O2 in vitro. For this, bGCs derived from ovarian follicles were cultured in vitro till their confluency and then treated with H2O2 for 40 min. Twenty-four hours later, cells were subjected to various phenotypic and gene expression analyses for genes related to TFs, endoplasmic reticulum stress, apoptosis, cell proliferation, and differentiation markers. The bGCs exhibited higher reactive oxygen species accumulation, DNA fragmentation, and endoplasmic reticulum stress accompanied by reduction of mitochondrial activity after exposure to OS. In addition, higher lipid accumulation and lower cell proliferation were noticed in H2O2-challenged cells. The mRNA level of TFs including NRF2, E2F1, KLF6, KLF9, FOS, SREBF1, SREBF2, and NOTCH1 was increased in H2O2-treated cells compared with non-treated controls. However, the expression level of KLF4 and its downstream gene, CCNB1, were downregulated in the H2O2-challenged group. Moreover, targeted inhibition of NRF2 using small interference RNA resulted in reduced expression of KLF9, FOS, SREBF2, and NOTCH1 genes, while the expression of KLF4 was upregulated. Taken together, bovine granulosa cells exposed to OS exhibited differential expression of various transcription factors, which are mediated by the NRF2 signaling pathway.

So far the intimate link between serum microRNA (miRNA) and uterine inflammation in mares is unknown. We aimed (I) to investigate expression profile of eca-miR-155, eca-miR-223, eca-miR-17, eca-miR-200a, and eca-miR-205 (II) and to measure concentrations of interleukin 6 (IL-6), and prostaglandins (PGF2α and PGE2) in serum of mares with healthy and abnormal uterine status (endometritis). This study was conducted on 80 Arabian mares: young (4–7 years), and old (8–14 years). Mares were divided into 48 sub-fertile (endometritis) and 32 fertile (control) at stud farms. Serum was collected for measuring IL-6, PGF2α, and PGE2, as well as miRNA isolation and qRT-PCR. Concentrations of IL-6, PGE2, and PGF2α were higher in mares with endometritis compared to control. Age of mares had a remarkable effect on IL-6, PGE2, and PGF2α concentrations. Relative abundance of eca-miR-155, eca-miR-223, eca-miR-17, eca-miR-200a, and eca-miR-205 was higher in both young and old mares with endometritis. We noticed that eca-miR-155, eca-miR-223, eca-miR-200a, and eca-miR-205 revealed higher expression level in old than young mares with endometritis. This is the first study that has revealed the changes in cell free miRNA and serum inflammatory mediators during endometritis, and these findings could be used for a better understanding the pathophysiology mechanisms of endometritis in equine.

Background: So far the intimate link between serum microRNA (miRNA) and uterine inflammation in mares is unknown. We aimed (I) to investigate the expression profile of eca-miR-155, eca-miR-223, eca-miR-17, eca-miR-200a, and eca-miR-205 (II) and to measure the concentrations of interleukin 6 (IL-6), and prostaglandins (PGF2α& PGE2) in serum of Arabian mares with healthy and abnormal uterine status (endometritis).Methods and Results: This study was conducted on 80 Arabian mares; young (4-7 years), and old (8-14 years). These animals were divided into 48 sub-fertile including 16 young and 32 old mares suspected of endometritis and 32 fertile as control (24 young and 8 old) at stud farms. Serum samples were collected for measuring IL-6, PGF2α, and PGE2 concentrations, as well as serum miRNA isolation and qRT-PCR. Serum concentrations of IL-6, PGE2, and PGF2α were higher (P≤0.001) in mares with endometritis (young and old) compared to the control ones. Age of mares had a remarkable effect(0.001≤P≤0.01) onIL-6, PGE2, and PGF2αconcentrations. The relative abundance of eca-miR-155, eca-miR-223, eca-miR-17, eca-miR-200a, and eca-miR-205 was higher (P≤0.001) in both young and old mares with endometritis. We noticed that eca-miR-155, eca-miR-223, eca-miR-200a, and eca-miR-205 revealed higher (0.001≤P≤0.01) expression level in old than young mares with endometritis. Conclusions: To the best of our knowledge, this is the first study revealed that serum miRNA and serum inflammatory mediators (IL-6, PGE2, and PGF2α) could be used as non-invasive gold standard biomarkers, and therefore might be served as an important additional diagnostic tool for endometritis in Arabian mares.

The present study aimed to determine the effects of zinc oxide nanoparticles (ZnO-NPs), thyme oil (THO), or their combination on the nutrient digestibility coefficients, reproductive parameters, and some blood metabolites of male Californian rabbits. One hundred rabbits, 29-weeks of age (initial body weight 3.48 &plusmn, 0.08 kg) were randomly distributed into four groups, 25 rabbits each. Treatment groups were fed a control diet, a control diet supplemented with ZnO-NPs (100 mg/kg), THO (500 mg/kg), or combination of ZnO-NPs (100 mg/kg) and THO (500 mg/kg). The feeding trial lasted for 35 days. Results showed improvements in dry matter, crude protein, ether extract, and crude fiber in ZnO-NPs, THO, and their combination treated groups compared to those of control. Furthermore, semen volume, sperm motility, vitality, and morphology were significantly improved (p &lt, 0.01) in ZnO-NPs and THO groups rather than the control. Both ZnO-NPs and THO, as either individual or combined treatments significantly improved the serum alanine amino-transferase (ALT), aspartate amino-transferase (AST), urea, and creatinine compared to the control. Moreover, serum concentrations of testosterone were significantly increased in rabbits supplemented with ZnO-NPs, THO, or their combination compared to those of control (p &lt, 0.05). In conclusion, ZnO-NPs, THO, or their combination improved the digestibility of nutrients, liver/ kidney functions, semen characteristics, and testosterone concentration in male rabbits.

Sexually dimorphic differences in genome activity, which is orchestrated by transcription factors (TFs), could explain the differential response of male and female embryos to environmental stressors. To proof this hypothesis, the expression of cellular and extracellular TFs was investigated in male and female bovine embryos in vitro cultured either under low (5%) or high (20%) oxygen levels. The intracellular reactive oxygen species (ROS), total cell number, expression of nuclear factor (erythroid-derived 2) factor 2 (NFE2L2), Kruppel-like factor 4 (KLF4), notch receptor 1 (NOTCH1), E2F transcription factor 1 (E2F1), and SREBF2 along with extracellular vesicles (EVs) biogenesis genes were assessed at the blastocyst stage and their released EVs. Low blastocyst rate in both sexes due to oxidative stress (OS) was accompanied by increased ROS accumulation and reduced cell number in female embryos. The messenger RNA and protein levels of NFE2L2, as well as KLF4 expression, were higher in male embryos exposed to OS compared with female embryos. However, the expression of NOTCH1 and E2F1 was higher in female embryos cultured in high oxygen level. Male embryos exposed to OS released more EVs enriched with NFE2L2, superoxide dismutase 1, and NOTCH1 accompanied by elevated expression of EVs biogenesis genes. Accordingly, differential expression of TFs and their release into spent media could partially explain the sexual dimorphic response of bovine embryos to environmental stresses.

Nrf2 is a redox sensitive transcription factor regulating the expression of antioxidant genes as defense mechanism against various stressors. The aim of this study is to investigate the potential role of noncoding miRNAs as endogenous and quercetin as exogenous regulators of Nrf2 pathway in bovine granulosa cells. For this cultured granulosa cells were used for modulation of miRNAs (miR-28, 153 and miR-708) targeting the bovine Nrf2 and supplementation of quercentin to investigate the regulatory mechanisms of the Nrf2 antioxidant system. Moreover, cultured cells were treated with hydrogen peroxide to induce oxidative stress in those cells. Our results showed that, oxidative stress activated the expression of Nrf2 as a defense mechanism, while suppressing the expression of those miRNAs. Overexpression of those miRNAs resulted in downregulation of Nrf2 expression resulted in higher ROS accumulation, reduced mitochondrial activity and cellular proliferation. Quercetin supplementation showed its protective role against oxidative stress induced by H2O2 by inducing the expression of antioxidant enzymes. In conclusion, this study highlighted the involvement of miR-153, miR-28 and miR-708 in regulatory network of Nrf2 mediated antioxidant system in bovine granulosa cells function. Furthermore, quercetin at a low dose played a protective role in bovine granulosa cells against oxidative stress damage.

Pre-implantation embryo development is a critical stage, in which several development and stress-response related transcription factors (TF) are involved. Exposing embryos to environmental insults alter some of these stress response-related TF. However, their expression pattern in male and female embryos and their release via exosomes is still unclear. Here, we aimed to investigate the effect of culture-induced oxidative stress on development and expression pattern of stress-related TF in male and female embryos and in respective spent media coupled with exosomes. For this, bovine male and female zygotes were in vitro produced using sexed semen and cultured under 5% and 20% oxygen in exosome-depleted SOFaa medium (SOF with amino acids). Blastocysts were subjected to total RNA isolation followed by quantitative RT-PCR analysis of the selected TF (Nrf2, KLF4, NOTCH1, SREBF2, E2F1, CAT1, SOD1, and OCT4), as well as protein abundance analysis using immunofluorescence and related phenotypes analysis, including reactive oxygen species (ROS) level and total cell count. Furthermore, the spent embryo culture media were collected for exosomes isolation and expression analysis of candidate TF. The data were statistically analysed using one-way ANOVA followed by multiple pair-wise comparisons using the Tukey post hoc test. Results showed that the blastocyst rates of both male (29.9% v. 34.9%) and female (16.7% v. 26.5%) bovine embryos were significantly lower in 20% than in 5% oxygen level. Female blastocysts subjected to the higher oxygen level showed increased ROS level (37.66 ± 1.70 v. 45.32 ± 2.05 in male and 29.42 ± 1.44 v. 45.51 ± 2.06 in female) and significantly reduced total cell count compared with the male embryo counterpart (136.55 ± 7.8 v. 112.75 ± 2.9 in male and 138.75 ± 2.0 v. 88.25 ± 4.3 in female cultured in 5% and 20% oxygen levels, respectively). Consequently, the expression levels of Nrf2, KLF4, SREBF2, CAT1, SOD1, and OCT4 were significantly increased in male embryos exposed to oxidative stress compared with those cultured under the lower oxygen level. However, NOTCH1 and E2F1 were significantly increased in female embryos exposed to oxidative stress compared with the male counterparts. The mRNA level of SREBF2 was significantly increased in male embryos cultured under both 5% and 20% O2 compared with female embryos. The protein expression level of Nrf2 and KLF4 was higher in embryos cultured at 20% v. 5% O2 with greater Nrf2 abundance in male embryos. Consequently, the male embryos produced at 20% O2 released a higher number of exosomes enriched with Nrf2, SOD1, and NOTCH1 mRNA than the other groups. Interestingly, the exosomal mRNA expression level of E2F1 tended to be higher in female embryos exposed to oxidative stress than their male counterparts. Taken together, the male embryos were more tolerant to oxidative stress than female embryos via the activation Nrf2-mediated oxidative stress response and development related TF. The release of these TF via exosomes could enhance cellular homeostasis maintenance under oxidative stress.