The Role of TcCYP6K1 and TcCYP9F2 Influences Trehalose Metabolism under High-CO2 Stress in Tribolium castaneum (Coleoptera).

Cytochrome P450 monooxygenases (CYP), crucial detoxification enzymes in insects, are involved in the metabolism of endogenous substances as well as the activation and degradation of exogenous compounds. In this study, T. castaneum was utilized to investigate the roles of TcCYP6K1 and TcCYP9F2 genes influencing in the trehalose metabolism pathway under high-CO2 stress. By predicting the functional sequences of TcCYP6K1 and TcCYP9F2 genes and analyzing their spatiotemporal expression patterns, it was discovered that both genes belong to the CYP3 group and exhibit high expression levels during the larval stage, decreasing during the pupal stage, while showing high expression in the fatty body, intestine, and malpighian tubules. Furthermore, following the knockdown of TcCYP6K1 and TcCYP9F2 genes in combination with treating larvae with 75% CO2, it was observed that larval mortality increased, and glycogen content significantly decreased, while trehalose content increased significantly. Additionally, membrane-bound trehalase enzyme activity declined, TPS gene expression was significantly upregulated, GS gene expression was significantly downregulated, and ATP content showed a marked decrease. In conclusion, CYP genes are critical responsive genes of T. castaneum to high CO2 levels, potentially impacting the insect's resistance to carbon dioxide through their involvement in the synthesis or breakdown of the carbohydrate metabolism pathway. These findings could serve as a theoretical basis for the utilization of novel pesticides in low-oxygen grain storage techniques and offer new insights for environmentally friendly pest control strategies in grain storage.

Identification and functional prediction of miRNAs that regulate ROS levels in dielectric barrier discharge plasma-treated boar spermatozoa.

Dielectric barrier discharge (DBD) plasma regulates the levels of reactive oxygen species (ROS), which are critical for sperm quality. MicroRNAs (miRNAs) are non-coding single-stranded RNA molecules encoded by endogenous genes, which regulate post-transcriptional gene expression in animals. At present, it is unknown whether DBD plasma can regulate sperm ROS levels through miRNAs. To further understand the regulatory mechanism of DBD plasma on sperm ROS levels, miRNAs in fresh boar spermatozoa were detected using Illumina deep sequencing technology. We found that 25 known miRNAs and 50 novel miRNAs were significantly upregulated, and 14 known miRNAs and 74 novel miRNAs were significantly downregulated in DBD plasma-treated spermatozoa. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that target genes of differentially expressed miRNAs were involved in many activities and pathways associated with antioxidants. We verified that DBD plasma significantly increased boar sperm quality and reduced ROS levels. These results suggest that DBD plasma can improve sperm quality by regulating ROS levels via miRNAs. Our findings provide a potential strategy to improve sperm quality through miRNA-targeted regulation of ROS, which helps to increase male reproduction and protect cryopreserved semen in clinical practice.

Glutamate rescues heat stress-induced apoptosis of Sertoli cells by enhancing the activity of antioxidant enzymes and activating the Trx1-Akt pathway in vitro.

Heat stress reduces the number of Sertoli cells, which is closely related to an imbalanced redox status. Glutamate functions to maintain the equilibrium of redox homeostasis. However, the role of glutamate in heat treated Sertoli cells remains unclear. Herein, Sertoli cells from 3-week-old piglets were treated at 44 °C for 30 min (heat stress). Glutamate levels increased significantly following heat stress treatment, followed by a gradual decrease during recovery, while glutathione (GSH) showed a gradual increase. The addition of exogenous glutamate (700 µM) to Sertoli cells before heat stress significantly reduced the heat stress-induced apoptosis rate, mediated by enhanced levels of antioxidant substances (superoxide dismutase (SOD), total antioxidant capacity (TAC), and GSH) and reduced levels of oxidative substances (reactive oxygen species (ROS) and malondialdehyde (MDA)). Glutamate addition to Sertoli cells before heat stress upregulated the levels of glutamate-cysteine ligase, modifier subunit (Gclm), glutathione synthetase (Gss), thioredoxin (Trx1) and B-cell leukemia/lymphoma 2 (Bcl-2), and the ratio of phosphorylated Akt (protein kinase B)/total Akt. However, it decreased the levels of Bcl2-associated X protein (Bax) and cleaved-caspase 3. Addition of the inhibitor of glutaminase (Gls1), Bptes (Bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide, 30 µM)to Sertoli cells before heat stress reversed these effects. These results inferred that glutamate rescued heat stress-induced apoptosis in Sertoli cells by enhancing activity of antioxidant enzymes and activating the Trx1-Akt pathway. Thus, glutamate supplementation might represent a novel strategy to alleviate the negative effect of heat stress.

Non-thermal plasma promotes boar sperm quality through increasing AMPK methylation.

Boar sperm quality, as an important indicator of reproductive efficiency, directly affects the efficiency of livestock production. Here, this study was conducted to improve the boar sperm quality by using a non-thermal dielectric barrier discharge (DBD) plasma. Our results showed that DBD plasma exposure at 2.1 W for 15 s could improve boar sperm quality by increasing exon methylation level of adenosine monophosphate-activated protein kinase (AMPK) and thus improving the glycolytic flux, mitochondrial function, and antioxidant capacity without damaging the integrity of sperm DNA and acrosome. In addition, DBD plasma could rescue DNA methyltransferase inhibitor decitabine-caused low sperm quality through reducing the oxidative stress and mitochondrial damage. Therefore, the application of non-thermal plasma provides a new strategy for reducing sperm oxidative damage and improving sperm quality, which shows a great potential in assisted reproduction to solve the problem of male infertility.

Effects and Mechanisms of Non-Thermal Plasma-Mediated ROS and Its Applications in Animal Husbandry and Biomedicine.

Non-thermal plasma (NTP) is an ionized gas composed of neutral and charged reactive species, electric fields, and ultraviolet radiation. NTP presents a relatively low discharge temperature because it is characterized by the fact that the temperature values of ions and neutral particles are much lower than that of electrons. Reactive species (atoms, radicals, ions, electrons) are produced in NTP and delivered to biological objects induce a set of biochemical processes in cells or tissues. NTP can mediate reactive oxygen species (ROS) levels in an intensity- and time-dependent manner. ROS homeostasis plays an important role in animal health. Relatively low or physiological levels of ROS mediated by NTP promote cell proliferation and differentiation, while high or excessive levels of ROS mediated by NTP cause oxidative stress damage and even cell death. NTP treatment under appropriate conditions not only produces moderate levels of exogenous ROS directly and stimulates intracellular ROS generation, but also can regulate intracellular ROS levels indirectly, which affect the redox state in different cells and tissues of animals. However, the treatment condition of NTP need to be optimized and the potential mechanism of NTP-mediated ROS in different biological targets is still unclear. Over the past ten decades, interest in the application of NTP technology in biology and medical sciences has been rapidly growing. There is significant optimism that NTP can be developed for a wide range of applications such as wound healing, oral treatment, cancer therapy, and biomedical materials because of its safety, non-toxicity, and high efficiency. Moreover, the combined application of NTP with other methods is currently a hot research topic because of more effective effects on sterilization and anti-cancer abilities. Interestingly, NTP technology has presented great application potential in the animal husbandry field in recent years. However, the wide applications of NTP are related to different and complicated mechanisms, and whether NTP-mediated ROS play a critical role in its application need to be clarified. Therefore, this review mainly summarizes the effects of ROS on animal health, the mechanisms of NTP-mediated ROS levels through antioxidant clearance and ROS generation, and the potential applications of NTP-mediated ROS in animal growth and breeding, animal health, animal-derived food safety, and biomedical fields including would healing, oral treatment, cancer therapy, and biomaterials. This will provide a theoretical basis for promoting the healthy development of animal husbandry and the prevention and treatment of diseases in both animals and human beings.

Melatonin or vitamin C attenuates lead acetate-induced testicular oxidative and inflammatory damage in mice by inhibiting oxidative stress mediated NF-κB signaling.

Lead (Pb) acts as an environmental endocrine disruptor and has negative effects in animals; excessive accumulation of lead causes reproductive dysfunction in male animals. Oxidative stress plays a vital role in Pb-induced injury. However, the mechanisms underlying chronic testicular toxicity of Pb remain unclear. In this study, we aimed to determine the effects of lead acetate on reproductive function in male mice, identify the underlying mechanisms, and test counter measures to alleviate the toxic effects. Male mice were dosed with lead acetate (500 mg/L) in free drinking water for 12 weeks, and administered melatonin (5 mg/kg) or vitamin C (500 mg/kg) by intraperitoneal injection. Blood from the eyeball, testicles, and sperm from the caudal epididymis were collected after 12 weeks and analyzed. Pb exposure reduced sperm count and motility, increased sperm malformation (P < 0.01), disrupted testicular morphology and structure, and decreased the expression of steroid hormone synthesis-related enzymes and serum testosterone concentration (P < 0.01). Pb also increased the number of inflammatory cells and the levels of the pro-inflammatory cytokines TNF-α and IL-6 (P < 0.01), and activated NF-κB signaling. Furthermore, the ROS yield and oxidation indicators LPO and MDA were significantly increased (P < 0.01), and the antioxidant indicators T-AOC, SOD, and GSH were significantly reduced (P < 0.01). Treatment with melatonin or vitamin C reversed the effects of lead acetate; vitamin C was more effective in restoring SOD activity (P < 0.01) and enhancing ZO-1 protein levels (P < 0.01). Thus, long-term exposure to lead acetate at low concentrations could adversely affect sperm quality and induce inflammatory damage by oxidative stress mediated NF-κB signaling. Vitamin C could act as a protective agent and improve reproductive dysfunction in male animals after lead accumulation.

Regulation of Vicia faba L. Response and Its Effect on Megoura crassicauda Reproduction under Zinc Stress.

The heavy metal zinc (Zn) is known to be transmitted in the food chain; however, the effect of Zn stress on beans and herbivorous insects is largely unclear. This study aimed to investigate the resistance of broad bean plants to Zn stress and the consequent changes in their physiological and biochemical metabolism by simulating heavy metal pollution in soil. Simultaneously, the effects of aphid progeny treated with different Zn concentrations on the expression of carbohydrate and related genes were analyzed. The results showed that Zn had no effect on the germination rate of broad beans, but other effects mainly manifested as follows. (1) Chlorophyll content decreased. (2) The total soluble sugar and Zn content in stems and leaves increased with increasing Zn content. (3) The proline content first increased and then decreased with increasing Zn content. (4) The height of the seedlings indicates that low concentrations promote growth and high concentrations inhibit growth. In addition, only the first-generation fecundity decreased significantly when aphids fed on heavy metal broad beans. Continuous high Zn levels increase the trehalose content of aphid F1 and F2, while F3 decreases. These results can not only provide a theoretical basis for exploring the impact of soil heavy metal pollution on ecosystems but also preliminarily evaluate the possibility of broad beans as a means of pollution remediation.

Novel Functional Soft Magnetic CoFe2O4/Fe Composites: Preparation, Characterization, and Low Core Loss.

In this study, composites CoFe2O4/Fe were successfully synthesized by in situ oxidation, and their composition, structure, and magnetic properties have been investigated. According to the analysis of X-ray photoelectron spectrometry measure results, the cobalt ferrite insulating layer was completely coated on the surface of Fe powder particles. The evolution of the insulating layer during the annealing process has been discussed, which is correlated to effects on the magnetic properties of the composites CoFe2O4/Fe. The amplitude permeability of the composites reached a maximum of 110, and their frequency stability reached 170 kHz with a relatively low core loss of 253.6 W/kg. Therefore, the composites CoFe2O4/Fe has potential application in the field of integrated inductance and high-frequency motor, which is conducive to energy conservation and carbon reduction.

The Regulatory Mechanism of Cold Plasma in Relation to Cell Activity and Its Application in Biomedical and Animal Husbandry Practices.

As an innovative technology in biological applications, cold plasma is widely used in oral treatment, tissue regeneration, wound healing, and cancer therapy, etc., because of the adjustable composition and temperature which allow the plasma to react with bio-objects safely. Reactive oxygen species (ROS) produced by cold plasma regulate cell activity in an intensity- and time-dependent manner. A low level of ROS produced by cold plasma treatment within the appropriate intensities and times promotes proliferation of skin-related cells and increases angiogenesis, which aid in the acceleration of the wound healing process, while a high level of ROS produced by cold plasma treatment performed at a high intensity or over a long period of time inhibits the proliferation of endothelial cells, keratinocytes, fibroblasts, and cancer cells. Moreover, cold plasma can regulate stem cell proliferation by changing niche interface and producing nitric oxide directly. However, the molecular mechanism of cold plasma regulating cell activity and its potential application in the field of animal husbandry remain unclear in the literature. Therefore, this paper reviews the effects and possible regulatory mechanisms of cold plasma on the activities of endothelial cells, keratinocytes, fibroblasts, stem cells, and cancer cells to provide a theoretical basis for the application of cold plasma to skin-wound healing and cancer therapy. In addition, cold plasma exposure at a high intensity or an extended time shows excellent performances in killing various microorganisms existing in the environment or on the surface of animal food, and preparing inactivated vaccines, while cold plasma treatment within the appropriate conditions improves chicken growth and reproductive capacity. This paper introduces the potential applications of cold plasma treatment in relation to animal-breeding environments, animal health, their growth and reproduction, and animal food processing and preservation, which are all beneficial to the practice of animal husbandry and guarantee good animal food safety results.

Melatonin alleviates the heat stress-induced impairment of Sertoli cells by reprogramming glucose metabolism.

Sertoli cells (SCs) provide structural and nutritional support for developing germ cells. Normal glucose metabolism of SCs is necessary for spermatogenesis. Melatonin could alleviate the effects of heat stress on spermatogenesis. However, the influences of heat stress on glucose metabolism in SCs remain unclear, and the potential protective mechanisms of melatonin on SCs need more exploration. In this study, boar SCs were treated at 43°C for 30 min, and different concentrations of melatonin were added to protect SCs from heat stress-induced impairment. These results showed that heat stress-induced oxidative stress caused cell apoptosis, inhibited the pentose phosphate pathway, and decreased the ATP content. Furthermore, heat stress increased the expressions of glucose intake- and glycolytic-related enzymes, which enhanced the glycolysis activity to compensate for the energy deficit. Melatonin relieved heat stress-induced oxidative stress and apoptosis by activating the Kelch-like ECH-associated protein 1 (KEAP1)/NF-E2-related factor 2 signaling pathway to increase the capacity of antioxidants. In addition, melatonin enhanced heat-shock protein 90 (HSP90) expression through melatonin receptor 1B (MTNR1B), thereby stabilizing hypoxia-inducible factor-1α (HIF-1α). Activation of the HIF-1α signaling pathway enhanced glycolysis, promoted the pentose phosphate pathway, and increased cell viability. Our results suggest that melatonin reprograms glucose metabolism in SCs through the MTNR1B-HSP90-HIF-1α axis and provides a theoretical basis for preventing heat stress injury.

The role of ALOX15B in heat stress-induced apoptosis of porcine sertoli cells.

Heat stress reduces the number of Sertoli cells and impairs spermatogenesis. Mounting evidence indicates that lipid homeostasis is fundamental to cell survival. However, little is known about the role of lipid peroxides in the heat stress-induced apoptosis of Sertoli cells. In the present study, we used metabolomics to explore the changes of lipid peroxides in porcine Sertoli cells under heat stress using liquid chromatograph-mass spectrometry. These results showed a notable increase in the content of 8-hydroxyeicosatetraenoic acid (8-HETE), and 15-hydroxyeicosatetraenoic acid (15-HETE). Furthermore, we found that among arachidonate lipoxygenases, heat stress significantly increased the expression of arachidonate 15-lipoxygenase type B (ALOX15B). Moreover, baicalein, a specific inhibitor of ALOX15B, reduced the content of 8-HETE and 15-HETE, and decreased the apoptosis rate in heat stress-treated porcine Sertoli cells. In addition, baicalein and small interfering RNAs targeting ALOX15B increased the content of 8-HETE and 15-HETE, and activated the p38-p53 pathway, causing apoptosis in heat stress treated porcine Sertoli cells. Interestingly, a p38 inhibitor decreased the expression of ALOX15B, reduced the content of 8-HETE and 15-HETE, and decreased the expression of p53 and the apoptosis rate in heat stress treated porcine Sertoli cells. A p53 inhibitor had similar effect on Sertoli cells. These results indicated that heat stress enhanced the expression of ALOX15B, increased the content of 8-HETE and 15-HETE, and activated the p38-p53 pathway to cause apoptosis. ALOX15B and lipid peroxides obtained feedback from the p38-p53 pathway. Our findings will help to reveal the mechanism of lipid metabolism in Sertoli cells, and could provide a new targeted substrate for anti-heat stress strategies.

Heat treatment reduced the expression of miR-7-5p to facilitate insulin-stimulated lactate secretion by targeting IRS2 in boar Sertoli cells.

Insulin dysfunction of diabetes mellitus (DM) disorders the glucose metabolism in Sertoli cells (SCs), resulting in the impairment of spermatogenesis.Insulin signaling system in Sertoli cells (SCs) plays an important role in regulating lactate secretion. Heat treatment could increase the lactate secretion of boar SCs, but whether heat treatment participates in lactate secretion by improving the sensitivity of insulin is unknown. In the current study, the primary SCs from 21-day-old boar were employed to treat with 100 nM insulin for 24 h or heat treatment (43 °C, 30 min). Heat treatment strengthened the effect of insulin on the effect of lactate secretion. In addition, heat treatment increased the expression of insulin-induced insulin receptor substrate 2 (IRS2), but reduced the expression of miR-7-5p. Using dual luciferase reporter assay and Western blot, the study found that IRS2 is a potential target gene of miR-7-5p. Heat treatment also enhanced the Phosphorylation of insulin-stimulated PI3K/Akt, and increased lactate secretion by promoting the expression of Glucose Transporter 3 (GLUT3), Lactate Dehydrogenase-A (LDHA) and monocarboxylate transporter 1 (MCT1). Furthermore, miR-7-5p inhibitor could partly mimic the effects of heat treatment on lactate production of SCs, indicating that heat treatment improves insulin sensitivity by regulating the expression of miR-7-5p/IRS2/PI3K/Akt. These results reveal a novel miRNA-mediated mechanism of heat treatment on the regulation of lactate metabolism production, and suggest that targeting miR-7-5p is a probably therapeutic method to insulin dysfunction-induced metabolic diseases.

(Li,Na)SbS2 as a promising solar absorber material: A theoretical investigation.

NaSbS2 has been proposed as a novel photovoltaic material, but its band gap is not suitable for single-junction solar cells. In the present study, the systematic first-principles calculations were carried out to investigate the structural, mechanical, electronic and optical properties of ASbS2 (A = Li, Na, K) and Na1-xLixSbS2 solid solutions. These structures show good structural stability compared to CH3NH3PbI3. The results indicate that all the structures are indirect band gap semiconductors. The band gap of ASbS2 increases gradually when the alkali metal changes from Li to K. The band gap of NaSbS2 can be tuned by manipulating the amount of Li doping. The Na1-xLixSbS2 solid solutions have suitable band gaps for light-absorber semiconductors in solar cells. Moreover, the suitable band gap of NaSbS2 can be also obtained under moderate pressure. The mechanical properties of these materials are also analyzed, and the results indicate that they are brittle materials except for KSbS2. The optical absorption coefficients of these compounds are large over 10-5 cm-1 in the visible light region. We find that alloying can provide a feasible and effective approach for improving the photovoltaic performance of NaSbS2-based solar cells.

Oxidative stress mediates heat-induced changes of tight junction proteins in porcine sertoli cells via inhibiting CaMKKß-AMPK pathway.

Heat stress causes reversible changes in tight junction proteins in immature Sertoli cells via inhibition of the AMPK signaling pathway; these effects are accompanied by an increase in the early apoptotic rate and decrease in the cell viability of Sertoli cells. Since heat stress is known to also cause oxidative damage, in the present study, we investigated whether the earlier mentioned effects of heat stress were brought about via the induction of oxidative stress in boar Sertoli cells. Immature Sertoli cells obtained from 3-week-old piglets were subjected to heat treatment (43 °C, 30 min), and the percentage of ROS-positive cells, the malonaldehyde (MDA) concentration, and the activity of the antioxidases, including superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT) were measured. Next, the Sertoli cells were treated with N-acetyl-l-cysteine (NAC) (1 mmol/L, 2 h), an antioxidant agent, before they were exposed to heat stress. The effects of NAC on ROS accumulation, MDA levels, antioxidase activity, the CaMKKß-AMPK signaling pathway and expression of tight junction proteins were assessed. The results showed that heat stress reversibly increased the percentage of ROS-positive cells and MDA levels, and decreased the activity of SOD, GSH-Px, and CAT. Pretreatment with NAC abrogated these effects of heat stress. Additionally, NAC reversed the heat stress-induced decrease in the expression of CaMKKß and dephosphorylation of AMPK. NAC also obviously rescued the heat stress-induced downregulation of tight junction proteins (claudin-11, JAM-A, occludin, and ZO-1) both at the mRNA and protein level. In conclusion, the findings indicate that oxidative damage participates in heat stress-induced downregulation of tight junction proteins in Sertoli cells by inhibiting the CaMKKß-AMPK axis. Further, NAC reversed the effects of heat stress on tight junction proteins; this means that it has potential as a protective agent that can prevent reproductive dysfunction in boars under conditions of heat stress.

MiR-8-3p regulates hyperthermia-induced lactate secretion by targeting PPP2R5B in boar Sertoli cells.

Lactate produced by glycolysis in Sertoli cells (SCs) is the main energy substrate for developing germ cells and plays a vital role in spermatogenesis. MicroRNAs (miRNAs) function as posttranscriptional regulators of gene expression in biological processes. We have previously shown that hyperthermia (43°C, 30 min) promotes lactate secretion by inhibiting phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) in cultured immature boar SCs. However, it is unclear whether miRNAs are involved in AMPK-modulated glycolysis in SCs. In the present study, we identified 349 miRNAs (227 upregulated and 122 downregulated) in hyperthermia-treated boar SCs by next-generation high-throughput RNA sequencing. MiR-8-3p, which was found to be a novel upregulated miRNA in hyperthermia-treated SCs, suppressed the expression of AMPK upstream genes (protein phosphatase 2 subunit B, PPP2R5B), and further downregulated the expression of p-AMPK. The miR-8-3p mimic upregulated expression of glucose transporter 3, lactate dehydrogenase A and monocarboxylate transporter 1, and increased lactic acid dehydrogenase activity, lactate secretion, and ATP depletion in SCs; the miR-8-3p inhibitor had the opposite effects on these parameters. Our findings indicate that miR-8-3p acts as a novel regulator of AMPK-modulated lactate secretion by targeting PPP2R5B in hyperthermic boar SCs.

Ssc-novel-miR-106-5p reduces lipopolysaccharide-induced inflammatory response in porcine endometrial epithelial cells by inhibiting the expression of the target gene mitogen-activated protein kinase kinase kinase 14 (MAP3K14).

As an important gram-negative bacterial outer membrane component, lipopolysaccharide (LPS) plays an important role in bacterial-induced endometritis in sows. However, how LPS induces endometritis is unclear. We stimulated sow endometrial epithelial cells (EECs) with LPS and detected cell viability and tumour necrosis factor-α (TNF-α) and interleukin-1 (IL-1) secretion. LPS affected EEC viability and TNF-α and IL-1 secretion in a dose-dependent manner. LPS induced differential expression in 10 of 393 miRNAs in the EECs (downregulated, nine; upregulated, one). MicroRNA (miRNA) high-throughput sequencing of the LPS-induced EECs plus bioinformatics analysis and the dual-luciferase reporter system revealed a novel miRNA target gene: mitogen-activated protein kinase kinase kinase 14 (MAP3K14). Ssc-novel-miR-106-5p mimic, inhibitor and the nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα) phosphorylation inhibitor Bay11-7085 were used to detect EEC nuclear factor-κB phosphorylation levels (p-NF-κB) and TNF-α and IL-1 secretion. MiR-106-5p mimic downregulated MAP3K14 mRNA and protein expression levels, inhibited p-NF-κB levels and decreased IL-1 and TNF-α secretion, whereas miR-106-5p inhibitor had the opposite effect. Bay11-7085 inhibited p-NF-κB expression and TNF-α and IL-1 secretion. These results suggest that LPS downregulates ssc-novel-miR-106-5p expression in sow EECs to increase MAP3K14 expression, which increases p-NF-κB to promote IL-1 and TNF-α secretion.

AMP-activated protein kinase negatively regulates heat treatment-induced lactate secretion in cultured boar sertoli cells.

Lactate secreted by Sertoli cells plays an important role in spermatogenesis. Heat stress changes AMP-activated protein kinase (AMPK) activity in many tissues and cells, and enhances lactate secretion in Sertoli cells. However, it is unclear whether heat stress affects lactate secretion by regulating the phosphorylation level of AMPK in boar immature Sertoli cells. In this study, immature boar Sertoli cells were treated at 43 °C for 30 min in an incubator. From 0 to 48 h post-heat stress, lactate secretion was enhanced and reached the maximum level (175% of the control) at 12 h. However, with increased recovery time, the phosphorylation level of AMPK decreased gradually, and reached the minimum level (58% of the control) at 12 h. Compared with heat treatment alone, pretreatment with the AMPK agonist AICAR (2 mmol/L, 2 h) reduced lactate secretion by 42.6%. Additionally, AICAR significantly decreased the lactate dehydrogenase (LDH) activity, and the mRNA and protein expression levels of GLUT3, LDHA, and MCT1. In addition, AMPK overexpression reduced lactate secretion by 22.5%, significantly decreased the LDH activity, and mRNA and protein expression levels of GLUT3, LDHA, and MCT1. These results showed that AMPK reduces heat-induced lactate secretion by decreasing the expression levels of GLUT3, LDHA and MCT1. The results also suggested that AMPK is a negative regulator of heat treatment-induced lactate secretion in cultured boar Sertoli cells.

Role of AMPK in the expression of tight junction proteins in heat-treated porcine Sertoli cells.

Hyperthermia can cause dysfunction of the tight junctions (TJs) in testes. Adenosine 5'-monophosphate-activated protein kinase (AMPK) participates in the regulation of TJs in testis. However, whether AMPK regulates the expression of TJ proteins in the response of Sertoli cells to heat treatment remains unknown. We subjected Sertoli cells from 3-week-old piglets to heat treatment (43 °C, 30 min), which decreased cell viability, and increased the early apoptosis rate. These effects were reversible and the cells gradually recovered to normal viability at 48 h post-heat treatment. Expression of TJ proteins (claudin 11, JAMA, occludin, and ZO1) was detected in immature porcine Sertoli cells. The mRNA and protein levels of TJ proteins significantly decreased at 1 h after heat exposure, but recovered with increasing recovery time. Additionally, the expression of claudin 11 in the cytoplasm was also markedly decreased by heat treatment. AMPK phosphorylation, the cellular ATP level, and Ca2+/calmodulin-dependent protein kinase kinase B (CaMKKB) level, but not the liver kinase B1 (LKB1) level, were downregulated by heat treatment. More importantly, activation or overexpression of AMPK, which is a regulator of the assembly of TJs, partially rescued the heat treatment-induced downregulation of TJ proteins. By contrast, AMPK knockdown using small interfering RNA (siRNA) further decreased the expression levels of TJ proteins. In addition, claudin 11 was almost undetectable post heat treatment. Collectively, this study demonstrated that heat treatment could reversibly perturb the expression of TJ proteins in immature porcine Sertoli cells by inhibiting the AMPK signaling pathway.

ERK1/2 regulates heat stress-induced lactate production via enhancing the expression of HSP70 in immature boar Sertoli cells.

Lactate produced by Sertoli cells plays an important role in spermatogenesis, and heat stress induces lactate production in immature boar Sertoli cells. Extracellular signaling regulated kinase 1 and 2 (ERK1/2) participates in heat stress response. However, the effect of ERK1/2 on heat stress-induced lactate production is unclear. In the present study, Sertoli cells were isolated from immature boar testis and cultured at 32 °C. Heat stress was induced in a 43 °C incubator for 30 min. Proteins and RNAs were detected by western blotting and RT-PCR, respectively. Lactate production and lactate dehydrogenase (LDH) activity were detected using commercial kits. Heat stress promoted ERK1/2 phosphorylation, showing a reducing trend with increasing recovery time. In addition, heat stress increased heat shock protein 70 (HSP70), glucose transporter 3 (GLUT3), and lactate dehydrogenase A (LDHA) expressions, enhanced LDH activity and lactate production at 2-h post-heat stress. Pretreatment with U0126 (1 × 10-6 mol/L), a highly selective inhibitor of ERK1/2 phosphorylation, reduced HSP70, GLUT3, and LDHA expressions and decreased LDH activity and lactate production. Meanwhile, ERK2 siRNA1 reduced the mRNA level of ERK2 and weakened ERK1/2 phosphorylation. Additionally, ERK2 siRNA1 reduced HSP70, GLUT3, and LHDA expressions decreased LDH activity and lactate production. Furthermore, HSP70 siRNA3 downregulated GLUT3 and LDHA expressions and decreased LDH activity and lactate production. These results show that activated ERK1/2 increases heat stress-induced lactate production by enhancing HSP70 expression to promote the expressions of molecules related to lactate production (GLUT3 and LDHA). Our study reveals a new insight in reducing the negative effect of heat stress in boars.

Heat stress-induced autophagy promotes lactate secretion in cultured immature boar Sertoli cells by inhibiting apoptosis and driving SLC2A3, LDHA, and SLC16A1 expression.

This study aimed to determine whether heat stress (HS) could induce autophagy in immature boar Sertoli cells (SCs) and test whether HS-induced autophagy could regulate lactate secretion by SCs. Cultured immature boar SCs were incubated at 43 °C for 30 minutes. The ratio of LC3B-II to LC3B-I and the mRNA transcript levels of LC3B showed time-dependent changes 0 to 48 hours after HS treatment, which peaked at 24 hours and increased by 30.25% or 260%, respectively, compared with control SCs. The density of autolysosomes, which were labeled with a red dye, was higher at 24 hours than at any other time point. However, the apoptosis rate, cleavage of caspase-3, and mRNA transcript levels of CASP3 (caspase-3) at 24 hours after HS were lower than at 12 hours. Furthermore, lactate secretion, and mRNA transcript levels of SLC2A3 (GLUT3), LDHA (LDHA), and SLC16A1 (MCT1) also showed time-dependent changes with a peak at 24 hours. In addition, LY294002 (20 µM) significantly inhibited changes in ratio of LC3B-II to LC3B-I, LC3B mRNA transcript levels, and autolysosome formation. It also resulted in significantly less lactate secretion and increased apoptosis but showed no effect on B-cell lymphoma-2 expression in heat-treated immature SCs. These findings indicated that HS-induced autophagy regulates lactate secretion by inhibiting apoptosis and increasing mRNA transcript and protein levels of SLC2A3, LDHA, and SLC16A1, which suggests that HS-induced autophagy may enhance lactate secretion by SCs.