Progesterone regulates inflammation and receptivity of cells via the NF-κB and LIF/STAT3 pathways.

A high concentration of blood ammonia can lead to failure of early embryo implantation in dairy cows. Progesterone (P4) is an important hormone that regulates the changes of uterine receptivity and participates in pregnancy during early implantation. However, whether P4 alleviates the harmful effects of blood ammonia is unknown. Therefore, this experiment treated bovine endometrial epithelial cells (BEEC) with ammonia chloride and P4. The results showed that ammonia decreased the viability of BEEC, upregulated the ROS level and apoptosis rate, and upregulated the expressions of Bax, Bcl-2, P53, Cyt-c, caspase-3, caspase-8 and caspase-9. It also upregulated the expressions of TLR4, NF-κB, and in turn, produced a large amount of IL-6 and downregulated the expressions of EGF and VEGF. After co-treating BEEC with ammonia and P4, P4 had no alleviating effect on cell viability, ROS and apoptosis, but downregulated the expressions of TLR4, NF-κB, IL-6, and upregulated the expressions of LIF, EGF and VEGF. Inhibition of STAT3 signaling pathway, LIF expression was not affected, and VEGF expression was down-regulated. Using LIF treated BEEC, VEGF expression was upregulated, but after the addition of inhibitors, the expression of VEGF was not upregulated by LIF. Therefore, progesterone could not alleviate the oxidative stress and apoptosis induced by ammonia in BEEC. However, progesterone ameliorated the inflammation and receptivity genes of BEEC induced by ammonia via the NF-κB and LIF/STAT3 signaling pathways.

Cadmium induces testosterone synthesis disorder by testicular cell damage via TLR4/MAPK/NF-κB signaling pathway leading to reduced sexual behavior in piglets.

Cadmium (Cd) is a highly toxic metal pollutant that can endanger the life and health of animals. Toll-like receptor 4 (TLR4) can result in testicular cell damage by positively regulating mitogen-activated protein kinase (MAPK)/nuclear factor-kappaB (NF-κB) signaling pathway. Meanwhile, Testosterone (T) synthesis disorder can affect sexual behavior. However, the harmful influence of Cd on animal sexual behavior during its growth and development and the role of TLR4/MAPK/NF-κB signaling pathway in testicular cell damage and testosterone production remained poorly understood. Forty-two-day-old male piglets were fed with diets that contained CdCl2 (20 mg Cd/kg) for 40 days to explore the toxic effects of Cd on sexual behavior. The results showed that Cd activated TLR4, promoted MAPK (p-ERK, p-JNK, and p-p38)/NF-κB expression, induced apoptosis (Caspase-3, Cleaved Caspase3, Bax, Cyt-c, and Caspase-9 expression increased, but Bcl-2 expression decreased) and necroptosis (MLKL, RIPK1, and RIPK3 expression increased) in piglet testis. In addition, Cd exposure decreased mRNA expression of STAR, CYP11A1, 3ß-HSD, CYP17A1, and 17ß-HSD of testis and the concentrations of T and thyroid-stimulating hormone (TSH). Both the mRNA and protein expression levels of the major genes in TLR4/MAPK/NF-κB signaling pathway, apoptosis signaling pathway, and necroptosis signaling pathway increased significantly and the expression levels of testosterone decreased gradually in pig Leydig cells cultured in vitro after being treated with different concentrations of Cd. Moreover, Cd reduced sexual behavior (the parameters of sniffing, chin resting, and mounting decreased) in piglets. In conclusion, Cd induced testicular cell damage via TLR4/MAPK/NF-κB signaling pathway leading to testosterone synthesis disorder and sexual behavior reduction in piglets.

Alleviative effect of melatonin on the decrease of uterine receptivity caused by blood ammonia through ROS/NF-κB pathway in dairy cow.

High concentration of blood ammonia can affect the uterus receptivity and decrease fecundity in dairy cow. Melatonin can reduce reactive oxygen species (ROS) level and has antioxidant and anti-inflammatory effects. However, it is not clear whether melatonin can alleviate ammonia-induced apoptosis of endometrial epithelial cell (EEC) and reduced uterus receptivity. The bovine EEC were treated with ammonium chloride and/or melatonin. Cell viability, apoptosis, oxidative stress and mitochondrial membrane potential were measured and the expression of apoptosis-related genes (p53, Cyt-c, Bax, Bcl-2, caspase-8, caspase-9 and caspase-3), uterus receptivity related genes (VEGF, LIF and EGF) and inflammatory factors (TLR-4, IL-6 and NF-κB) were detected. In addition, the expression of VEGF was detected after adding NF-κB inhibitor (40 µM) and IL-6 (1 ng/mL and 50 ng/mL). The results showed that ammonia significantly increased intracellular ROS level, mRNA and protein expression of Bax, p53, Cyt-c, caspase-9, caspase-8, caspase-3, TLR-4, NF-κB and IL-6, promoted cell apoptosis, while decreased mitochondrial membrane potential, the mRNA and protein expression of VEGF and EGF. Interestingly, melatonin significantly mitigated ammonia-induced changes. However, melatonin could not alleviate ammonia-induced changes of IL-6 and VEGF when NF-κB signal pathway was inhibited. The addition of IL-6 significantly reduced mRNA and protein expression of VEGF. In conclusion, ammonia induced EEC apoptosis through ROS production and activation of mitochondrial apoptosis pathway, and induced inflammatory response through TLR4/NF-κB/IL-6 pathway. Melatonin alleviated EEC apoptosis by inhibiting ROS pathway, and reduced IL-6 expression by inhibiting TLR-4/NF-κB signal pathway, which eventually improved VEGF expression and uterus receptivity in dairy cows.

The Antioxidant Role of Selenium via GPx1 and GPx4 in LPS-Induced Oxidative Stress in Bovine Endometrial Cells.

This study investigated the antioxidant role of selenium (Se) in the form of selenomethionine (SLM) in LPS-induced oxidative stress via the glutathione peroxidase (GPx) enzymes and the Nrf2/HO-1 transcription factor. The impact of serum supplementation in culture media on GPxs was also studied. The bovine uterus is constantly exposed to exogenous pathogens postpartum, and the endometrium is the first contact against bacteria invasion. Endometritis is an inflammation of the endometrium and is brought about by bacterial lipopolysaccharide capable of inducing oxidative stress. The BEND cells were supplemented at the point of seeding with the following SLM concentrations 0, 100, 500, and 1000 nM for 48 h. BEND cells, cultured with or without SLM (100 nM), were initially incubated for 48 h, and then, we serum starved the SLM group for 24, 48, and 72 h. Similarly, an assay involving serum volume (0, 2, 5, and 10%) supplementation in culture media (v/v) with or without SLM (100 nM) was performed for 48 h. The BEND cells were also seeded into four experimental groups and cultured for an initial 48 h as follows: control, LPS (20 µg/mL), SLM (100 nM), and SLM + LPS groups followed by 6-h LPS treatment. The role of SLM in modulating the expressions of GPx1 and GPx4 and the Nrf2 transcription factor-related genes was assessed using qRT-PCR and Western blot techniques. The results showed serum starvation in the presence of SLM supplementation decreased the expression of GPx1 enzyme but increased GPx4 compared to the control. The addition of SLM to cell culture media in an FBS limiting condition improved the expressions of both GPx1 and GPx4. SLM supplementation promoted GPx enzymes' expressions in a serum-free media (0%) and at 2% FBS in media. However, it did not improve their expressions at 10% FBS in media than the untreated groups. Together, our data show the protective role of Se by regulating the expressions of GPx1 and GPx4 enzymes in BEND cells. It also shows that SLM promoted the expression of Nrf2 transcription factor-related genes at both the mRNA and protein levels in BEND cells during LPS stimulation.

The ameliorative effect of melatonin on LPS-induced Sertoli cells inflammatory and tight junctions damage via suppression of the TLR4/MyD88/NF-κB signaling pathway in newborn calf.

The blood-testicular barrier (BTB) is involved in spermatogenesis, protects sperm development, and plays a crucial role in the reproductive process. Tight junctions (TJs) between Sertoli cells (SCs) are the key structure of (BTB), and if its structure is damaged, BTB function is affected. The cellular inflammation caused by Gram-negative bacteria affects the structural integrity of TJs. Melatonin (MT) has anti-inflammatory effects; however, the effect of MT in newborn calf SCs is unknown. Therefore, this experiment studied the protective effect of MT. The results showed that LPS upregulated TLR4, MyD88, and NF-κB expressions, in turn, activated the TLR4/MyD88/NF-κB signaling pathway, produced a large amount of IL-6 and IL-1ß, downregulated the expression of ZO-1 and Occludin, and reduced the viability of SCs, which resulted in the inflammatory response of SCs and damage of TJs. The addition of MT decreased TLR4, MyD88, and NF-κB expressions, it then inhibited the activation of TLR4/MyD88/NF-κB signaling pathway, downregulated the expression of IL-6 and IL-1ß, upregulated the expression of ZO-1 and Occludin, and increased the cell viability, thereby alleviating the inflammatory response of SCs, and restored the TJs structure. Overall, our results reveal that MT can alleviate LPS-induced in newborn calf SCs Inflammation and TJs injury through TLR4/MyD88/NF-κB signaling pathway.

The alleviative effect of thyroid hormone on cold stress-induced apotosis via HSP70 and mitochondrial apoptosis signal pathway in bovine Sertoli cells.

Thyroid hormone was involved in gene expression and functional regulation in various signal pathways. Cold stress can increase triiodothyronine (T3) level in the blood. The aim of this study was to investigate the effect of T3 on HSP70 expression and apoptosis in Sertoli cells (SCs) under cold stress in vitro culture at 26 °C, and provide a theoretical and practical basis for improving the reproductive efficiency of bulls in cold areas. SCs were treated with different cold stress duration and different T3 concentrations for pre-screening. HSP70 inhibitor was added later, and the apoptotic rate was measured using flow cytometry. The expression of HSP70 and the main genes of mitochondrial apoptosis pathway were determined by means of real-time PCR and western-blot, respectively. The localization of HSP70 was assessed by immunofluorescence. The results showed that cold stress (26 °C, 6 h) played an inductive role in SCs apoptotic rate (P < 0.01) and the transfer of HSP70 into the nucleus. 100 nM T3 further promoted HSP70 expression and its transfer into the nucleus, which significantly inhibited the expression of vital genes (cyt-c, Caspase-9 and Caspase-3) in mitochondrial pathway (P < 0.05). Subsequently, higher survival and lower apoptotic rates of SCs (P < 0.01) were observed. When T3 and HSP70 inhibitor were added together, the expression of cyt-c, Caspase-9 and Caspase-3 were inhibited (P < 0.05), and then the declining apoptotic rate increased again (P < 0.01). In conclusion, T3 can regulate HSP70 expression and translocation to mediate mitochondrial apoptosis pathway to inhibit SCs apoptosis induced by cold stress.

LRH-A3 and HCG increase pregnancy rate during timed artificial insemination in dairy cows.

The objective of this study was to use luteinizing hormone-releasing hormone A3 (LRH-A3) and human chorionic gonadotrophin (HCG) to improve pregnancy rate of dairy cows during timed artificial insemination (TAI). In experiment 1, the TAI process (0 d, GnRH, 100 µg; 7 d, PGF2α, 0.4 mg; 56 hr, GnRH, 100 µg; 16 hr, AI) was applied to 160 dairy cows on 50th and 60th days after parturition respectively. In experiment 2, 320 postpartum dairy cows were treated with TAI (Group A), TAI + 25 µg LRH-A3 (Group B), TAI + 1,500 IU HCG 5 days after AI (Group C), and TAI + 25 µg LRH-A3 + 1,500 IU HCG 5 days after AI (Group D). In experiment 3, endometrial cells were treated with HCG. The results showed that TAI did not affect the pregnancy rate, while LRH-A3 and HCG increased the pregnancy rate of the cow. HCG of 5 IU/ml and 10 IU/ml increased the expressions of leukemia inhibitory factor but decreased those of interleukin-6, epidermal growth factor and vascular endothelial growth factor in endometrial cells. This study provided a plan for the use of LRH-A3 and HCG to increase pregnancy rate during TAI in dairy cows.

3, 3', 5-Triiodo-L-thyronine affects polarity proteins of bovine Sertoli cells via WT1/non-canonical Wnt signaling pathway.

To determine the role of 3, 3', 5-triiodo-L thyroxine (T3) in the differentiation of Sertoli cells (SCs) and the factors influencing maturity via the Wilms' tumor 1 (WT1)/non-canonical Wnt signaling pathway, high purity SCs were isolated from newborn calves' testes and cultured in vitro. The SCs were stimulated with T3, and co-treated with short interference (si) RNA to knockdown endogenous WT1 and non-canonical Wnt signalling inhibitor Wnt-c59. Our results suggested that the addition of different concentrations (0, 25, 50, and 100 nM) of T3 in the culture medium changed the expression of KRT-18 (SCs immature marker) and accelerated the differentiation of SCs. T3 (100 nM) treatment induced up-regulated expression of WT1 over time (p < 0.05), while the expression of polarity proteins (Par3, Par6b, and E-cadherin) and Wnt4 were affected to varying degrees (p < 0.05). SCs were treated simultaneously with T3 + Wnt-c59 and T3 + WT1 siRNA, and the results showed that T3 could affect the expression of polarity proteins via WT1/non-canonical Wnt signaling pathway. These data put together indicate that T3 plays a dependent role in the induction of bovine SCs differentiation via WT1/non-canonical Wnt signaling pathway in vitro. This study proposes for the first time that WT1 is a major target for T3.

Wilms' tumour 1 (WT1) negatively regulates the expression of connexin 43 via a non-canonical Wnt signalling pathway in cultured bovine Sertoli cells.

The gap junction protein connexin (Cx) 43 between adjacent Sertoli cells (SCs) is the main testicular factor regulating the growth and development of SCs, and plays a vital role in controlling cell differentiation and maturation. However, the endogenous testicular factors that regulate Cx43 and the downstream signalling pathways that mediate Cx43-dependent SC differentiation are unclear. In this study, high-purity SCs were isolated from newborn calves' testes by differential adherence. The SCs were then cultured invitro and treated with short interference RNA to knockdown endogenous Wilms' tumour 1 (WT1). In WT1-knockdown SCs, Cx43 expression was upregulated. To elucidate the intracellular signalling mechanism of Cx43 in the differentiation and maturation of immature SCs, SCs were treated simultaneously with non-canonical Wnt signalling inhibitors CCG-1423 and GO-6983; in these SCs, Cx43 expression was upregulated. Together, these data indicate that WT1 negatively regulates the expression of Cx43 produced from SCs via a non-canonical Wnt signalling pathway in cultured bovine SCs.

Influence of Wilms' tumor suppressor gene WT1 on bovine Sertoli cells polarity and tight junctions via non-canonical WNT signaling pathway.

Sertoli cells (SCs) are polarized epithelial cells and provide a microenvironment for the development of germ cells (GCs). The Wilms' tumor suppressor gene WT1 which support spermatogenesis is expressed explicitly in SCs. This study investigated the effect of WT1 on the polarity and blood-testis barrier (BTB) formation of bovine SCs in order to provide theoretical and practical bases for the spermatogenic process in mammals. In this study, newborn calf SCs were used as research material, and the RNAi technique was used to knockdown the endogenous WT1. The results show that WT1 knockdown did not affect the proliferation ability of SCs, but down-regulated the expression of polarity-associated proteins (Par3, Par6b, and E-cadherin), junction-associated protein (occludin) and inhibits transcription of downstream genes (WNT4, JNK, αPKC, and CDC42) in non-canonical WNT signaling pathway. WT1 also altered ZO-1 and occludin protein distribution. Overexpression of WNT1 did not affect the expression of Par6b, E-cadherin, and occludin, whereas the non-canonical WNT signaling pathway inhibitors wnt-c59, CCG-1423, and GO-6983 down-regulated the expression of Par6b, E-cadherin, and occludin respectively. This study indicates that WT1 mediates the regulation of several proteins involved in bovine SCs polarity maintenance and intercellular tight junctions (TJ) by non-canonical WNT signaling pathway.

Thyroid hormone (T3) is involved in inhibiting the proliferation of newborn calf Sertoli cells via the PI3K/Akt signaling pathway in vitro.

The experiment was designed to study the effects of Thyroid hormone (T3) on the proliferation and differentiation of newborn calf Sertoli cells (SCs) to provide a theoretical and practical basis for increased testicular semen production. In this experiment, the cck8 method was used to detect the effects of different concentrations of T3 on the proliferation rate of newborn calf SCs. qPCR and Western Blot methods were used to explore the effects of T3 on the proliferation and differentiation of calves SCs and whether T3 through Wnt/ß-catenin and PI3K/Akt pathways can regulate the proliferation and differentiation of SCs. We found that dosage (T3) and time correlated with proliferation inhibition of SC. T3 inhibited the proliferation of SC by down-regulating cyclinD1, upregulating p21Cip, p27Kip1, and other cell-cycle factors. By up-regulating AR and down-regulating KRT-18, T3 promoted the maturated differentiation of SC. T3 could not affect the expression of ß-catenin in SC of newborn calf, indicating that T3 may not regulate SCs proliferation through the Wnt pathway. T3 also negatively regulated the gene expression and protein levels of some genes in the PI3K/Akt signaling pathway. We concluded that T3 inhibited newborn calf SCs proliferation through the PI3K/Akt signaling pathway and possibly promoted their differentiation.