BackgroundThis study was conducted to clarify the effect of the inhibiting action of inhibin on porcine granulosa cell proliferation and function, and to investigate the underlying intracellular regulatory molecular mechanisms.MethodsPorcine granulosa cells were cultured in vitro, and were treated with an anti-inhibin alpha subunit antibody, with or without co-treatment of follicle-stimulating hormone (FSH) in the culture medium.ResultsTreatment with anti-inhibin alpha subunit antibody led to a significant increase in estradiol (E2) secretion and cell proliferation. Anti-inhibin alpha subunit antibody worked synergistically with FSH at low concentrations (25 microg/mL) to stimulate E2 secretion, but attenuated FSH action at high concentrations (50 microg/mL). Immunoneutralization of inhibin bioactivity increased FOXL2, Smad3, and PKA phosphorylation, and mRNA expression of the transcription factors CEBP and c-FOS. The expression of genes encoding gonadotropin receptors, FSHR and LHR, and of those involved in steroidogenesis, as well as IGFs and IGFBPs, the cell cycle progression factors cyclinD1 and cyclinD2, and the anti-apoptosis and anti-atresia factors Bcl2, TIMP, and ADAMTS were upregulated following anti-inhibin alpha-subunit treatment. Treatment with anti-inhibin alpha subunit down regulated expression of the pro-apoptotic gene encoding caspase3. Although expression of the pro-angiogenesis genes FN1, FGF2, and VEGF was upregulated, expression of the angiogenesis-inhibiting factor THBS1 was downregulated following anti-inhibin alpha subunit treatment.ConclusionsThese results suggest that immunoneutralization of inhibin bioactivity, through augmentation of the activin and gonadotropin receptor signaling pathways and regulation of gene expression, permits the development of healthy and viable granulosa cells. These molecular mechanisms help to explain the enhanced ovarian follicular development observed following inhibin immunization in animal models.

N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL), a quorum-sensing (QS) molecule produced by Gram-negative bacteria in the gastrointestinal tract, adversly impacts host cells. Our previous study demonstrated that 3-oxo-C12-HSL induced a decrease in cell viability via cell apoptosis and eventually disrupted mucin synthesis from LS174T goblet cells. However, the molecular mechanism underlying cell apoptosis and whether pyroptosis was involved in this process are still unknown. In this study, we emphasized on the caspases signal pathway and sterile inflammation to reveal the harmful effects of 3-oxo-C12-HSL on LS174T goblet cells. Our data showed that 3-oxo-C12-HSL is a major inducer of oxidative stress indicated by a high level of intracellular reactive oxygen species (ROS). However, TQ416, an inhibitor of paraoxonase 2, can effectively block oxidative stress. A higher ROS level is the trigger for activating the caspase-1 and 3 cascade signal pathways. Blockade of ROS synthesis and caspase-1 and 3 cascades can obviously rescue the viability of LS174T cells after 3-oxo-C12-HSL treatment. We also found that paralleled with a higher level of ROS and caspases activation, an abnormal expression of proinflammatory cytokines was induced by 3-oxo-C12-HSL treatment; however, the blockage of TLRs-NF-κB pathway cannot restore cell viability and secretary function. These data collectively indicate that 3-oxo-C12-HSL exposure induces damages to cell viability and secretary function of LS174T goblet cells, which is mediated by oxidative stress, cell apoptosis, and sterile inflammation. Overall, the data in this study will provide a better understanding of the harmful impacts of some QS molecules on host cells and their underlying mechanism.

LPS and heat stress have been shown to exert various toxic effects in animals, as they induce estradiol biosynthesis dysfunction in granulosa cells (GCs) and result in low reproductive performance. However, there is limited information regarding their detailed mechanisms. In the present study, primary cultured porcine GCs were treated with LPS (1000ng/mL for 48h), or heat stress (41°C for 3h), in vitro, with or without the HSP70 inhibitor VER155008 (10μM), to investigate their potential mechanisms. To mimic the spike in HSP70 from LPS and heat stress, treatments with only the HSP70 activator STA-4783 (10μM for 3h or 48h) were also performed. We found that LPS and heat stress treatments could significantly reduce the expressions of FSHR and CYP19A1; associated with a reduction in estradiol concentrations; and increased in HSP70 expression both at mRNA and protein levels. While, VER155008 attenuation of LPS and heat stress induced HSP70 upregulation can restore the expressions of FSHR and CYP19A1. Furthermore, STA-4783 treatment alone can mimic the effects of LPS and heat stress treatments. Following immunofluorescence staining and western blot analysis showed that Smad3 phosphorylation and nuclear translocation were also inhibited by LPS, heat stress and STA-4783 treatments. We also examined the interactions between HSP70 and Smad3 by yeast two-hybrid screening, the results revealed that HSP70 indirectly interacted with Smad3. Thus, our results suggested that LPS and heat stress could impair estradiol biosynthesis in GCs via increased HSP70 and indirect inhibition of Smad3 phosphorylation and nuclear translocation.

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Subacute ruminal acidosis (SARA) is a common metabolic disease in the dairy farming industry which is usually caused by an excessive amount of high concentrate diet. SARA not only threatens animal welfare but also leads to economic losses in the farming industry. The liver plays an important role in the distribution of nutritional substances and metabolism; however, a high concentrate diet can cause hepatic metabolic disorders and liver injury. Recently, noncoding RNA has been considered as a critical regulator of hepatic disease, however, its role in the bovine liver is limited. In this study, 12 mid-lactating dairy cows were randomly assigned to a control (CON) group (40% concentrate of dry matter, n = 6) and a SARA group (60% concentrate of dry matter, n = 6). After 21 d of treatment, all cows were sacrificed, and liver tissue samples were collected. Three dairy cows were randomly selected from the CON and SARA groups respectively to perform whole transcriptome analysis. More than 20,000 messenger RNA (mRNA), 10,000 long noncoding RNA (lncRNA), 3,500 circular RNA (circRNA) and 1,000 micro RNA (miRNA) were identified. Furthermore, 43 mRNA, 121 lncRNA and 3 miRNA were differentially expressed, whereas no obvious differentially expressed circRNA were detected between the 2 groups. Gene Ontology (GO) annotation revealed that the differentially expressed genes were mainly enriched in oxidoreductase activity, stress, metabolism, the immune response, cell apoptosis, and cell proliferation. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that the deferentially expressed genes were highly enriched in the phosphatidylinositol 3 kinase ( PI3K ) - serine/threonine kinase ( AKT ) signaling pathway ( P < 0.05). According to KEGG pathway analysis, the differentially expressed lncRNA (DElncRNA) target genes were mainly related to proteasomes, peroxisomes, and the hypoxia-inducible factor-1 signaling pathway ( P < 0.005). Further bioinformatics and integrative analyses revealed that the lncRNA were strongly correlated with mRNA; therefore, it is reasonable to speculate that lncRNA potentially play important roles in the liver dysfunction induced by SARA. Our study provides a valuable resource for future investigations on the mechanisms of SARA to facilitate an understanding of the importance of lncRNA, and offer functional RNA information.