Heat stress induces ferroptosis of porcine Sertoli cells by enhancing CYP2C9-Ras- JNK axis.

Heat stress leads to the accumulation of lipid peroxides in Sertoli cells. Unrestricted lipid peroxidation of catalyzed polyunsaturated fatty acids by Cytochrome P450 (CYP) drive the ferroptosis. However, little is known about the role of CYP cyclooxygenase in heat stress-induced ferroptosis in Sertoli cells. In this study, we investigated the relationship between CYP cyclooxygenase and heat stress-induced ferroptosis in porcine Sertoli cells, as well as whether Ras-JNK signaling is involved in the process. The results showed that heat stress significantly increased the expression of cytochrome P450 cyclooxygenase 2C9 (CYP2C9) and the content of epoxyeicosatrienoic acids (EETs), although there are no significant effect on the expression of cytochrome P450 cyclooxygenase 2J2 (CYP2J2) and cytochrome P450 cyclooxygenase 2C8 (CYP2C8). In addition, heat stress reduced the cell viability, the protein expression level of glutathione peroxidase 4 (GPX4) and Ferritin (all P < 0.01) while increased the level of intracellular reactive oxygen species (ROS) and the protein level of Transferrin receptor 1(TFR1) (both P < 0.01), as well as activating the Ras-JNK signaling pathway. Ferrostatin-1, a ferroptosis-specific inhibitor, reduced ROS levels and the protein level of TFR1 (both P < 0.01), but elevated the cell viability, the protein level of GPX4, and Ferritin (all P < 0.01). Sulfaphenazole, a specific inhibitor of CYP2C9 or two small interfering RNAs targaring CYP2C9 enhanced the cell viability (all P < 0.01), while reduced the content of EETs (all P < 0.01) and inhibited the Ras-JNK signaling and ferroptosis under heat stress. Salirasib, a specific inhibitor of Ras, significantly elevated the cell viability, whereas reduced the level of intracellular ROS and inhibited the phosphorylation of JNK, and alleviated heat stress-induced ferroptosis in porcine Sertoli cells. Notably, there is no effect on the expression of CYP2C9 and the content of EETs. These results indicate that heat stress can induce ferroptosis in Sertoli cells by increasing the expression of CYP2C9 and the content of EETs, which in true activates the Ras-JNK signaling pathway, but there is no feedback from Ras-JNK signaling to the expression of CYP2C9. Our study finds a novel heat stress-induced cell death model of Sertoli cells as well as providing the therapeutic potential for anti-ferroptosis.

Colon-Targeted Release of Turmeric Nonextractable Polyphenols and Their Anticolitis Potential via Gut Microbiota-Dependent Alleviation on Intestinal Barrier Dysfunction in Mice.

Solid evidence has emerged supporting the role of nonextractable polyphenols (NEPs) and dietary fibers (DFs) as gut microbiota modulators. This study aims to elucidate gut microbiota-dependent release of turmeric NEPs and examine the possible anti-inflammatory mechanism in the dextran sulfate sodium-induced ulcerative colitis (UC) model. 1.5% DSS drinking water-induced C57BL/6J mice were fed a standard rodent chow supplemented with or without 8% extractable polyphenols (EPs), NEPs, or DFs for 37 days. The bound curcumin, demethoxycurcumin, and bisdemethoxycurcumin in NEPs were released up to 181.5 ± 10.6, 65.2 ± 6.0, and 69.5 ± 7.6 µg/mL by in vitro gut microbiota-simulated fermentation and released into the colon of NEP-supplemented mice by 5.7-, 11.0-, and 7.8-fold higher than pseudo germ-free mice, respectively (p < 0.05). NEPs also enhanced the colonic microbiota-dependent production of short-chain fatty acids in vitro and in vivo (p < 0.05). Interestingly, NEP feeding significantly improved the DSS-caused gut microbiota disorder, epithelial barrier damage, and inflammation of UC mice better than EPs or DFs (p < 0.05). Meanwhile, the pseudo germ-free mice supplemented with NEPs failed to ameliorate UC symptoms. These findings manifest that turmeric NEPs as macromolecular carriers exert the target delivery of polyphenols into the colon for regulating gut microbiota to restore the impaired gut barrier function for alleviation of inflammation.

Optimization of Piezoresistive Strain Sensors Based on Gold Nanoparticle Deposits on PDMS Substrates for Highly Sensitive Human Pulse Sensing.

In this study, highly-sensitive piezoresistive strain sensors based on gold nanoparticle thin films deposited on a stretchable PDMS substrate by centrifugation were developed to measure arterial pulse waveform. By controlling carbon chain length of surfactants, pH value and particle density of the colloidal solutions, the gauge factors of nanoparticle thin film sensors can be optimized up to 677 in tensile mode and 338 in compressive mode, and the pressure sensitivity up to 350. Low pH and thin nanoparticle films produce positive influences to superior gauge factors. It has been demonstrated that nanoparticle thin film sensors on PDMS substrates were successfully applied to sense arterial pulses in different body positions, including wrist, elbow crease, neck, and chest.

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.

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.

Acetamiprid inhibits testosterone synthesis by affecting the mitochondrial function and cytoplasmic adenosine triphosphate production in rat Leydig cells.

The insecticide acetamiprid is used to control noxious agricultural pests. However, it can cause mammalian toxicity. We evaluated the reproductive toxicity of acetamiprid in adult male Sprague Dawley rats. Rats were given oral acetamiprid alone or with vitamin E for 35 days. Rat plasma testosterone concentration and sperm quality decreased significantly as the levels of luteinizing hormone (LH) increased after exposure. At the same time, acetamiprid increased malondialdehyde and nitric oxide (NO) levels of Leydig cells. Further analysis showed that acetamiprid reduced the adenosine triphosphate (ATP) and cyclic adenosine monophosphate (cAMP) production of Leydig cells, but the expression of luteinizing hormone/choriogonadotropin receptor (LHCGR) and the activity of adenylyl cyclase were not changed. Acetamiprid exposure also significantly diminished protein levels of steroidogenic acute regulatory protein (STAR), hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase cluster (HSD3B), and cytochrome P450, family 11, subfamily a, polypeptide 1 (CYP11A1), and testicular mRNA levels, which are cAMP-dependent proteins that are essential for steroidogenesis. Electron microscopy indicated mitochondrial membrane damage in the Leydig cells of the testes of exposed rats. Vitamin E ameliorated the impairment of acetamiprid on Leydig cells. Our results indicate that acetamiprid causes oxidative stress and mitochondrial damage in Leydig cells and inhibits the synthesis of testicular ATP and cAMP. Acetamiprid disrupts subsequent testosterone biosynthesis by decreasing the rate of conversion of cholesterol to testosterone and by preventing cholesterol from entering the mitochondria within the Leydig cells. These effects caused reproductive damage to the rats.

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.

PI3K/Akt Activated by GPR30 and Src Regulates 17ß-Estradiol-Induced Cultured Immature Boar Sertoli Cells Proliferation.

Sertoli cell (SC) is a key element in the process of spermatogenesis. Accumulating research show that estrogen plays an important role in regulating boar SC proliferation. However, it is unclear whether phosphatidylinositol-4,5-bisphosphate 3-kinase/protein kinase B (PI3K/Akt) is involved in this process. In the present study, the role of PI3K/Akt on the 17ß-estradiol-induced piglet SC proliferation was explored. In addition, we also explained the roles of G-protein-coupled estrogen receptor (GPR30) and Sarcoma protein (Src) in this process. Our study demonstrated that, 17ß-estradiol induced activation of PI3K in a time-dependent manner. Both G-15 (an antagonist of GPR30, 0.1 µmol/L) and PP2 (an inhibitor of Src, 2.0 µmol/L) inhibited 17ß-estradiol-induced activation of PI3K, reduced SC proliferation, and decreased messenger RNA (mRNA) and protein expression of S-phase kinase-associated protein 2 (Skp2). We also found that 17ß-estradiol induced activation of Akt in a time-dependent manner. Both LY294002 (an inhibitor of PI3K) and 10-DEBC (an inhibitor of Akt) significantly reduced 17ß-estradiol-induced SC proliferation and reduced mRNA and protein expression of Skp2. In addition, LY294002 inhibited 17ß-estradiol-induced activation of Akt. The results indicated that 17ß-estradiol regulates SC proliferation by activating PI3K/Akt. Both GPR30 and Src are involved in 17ß-estradiol-induced phosphorylation of PI3K/Akt. Activation of PI3K/Akt enhances the expression of Skp2, which promotes SC proliferation.

Lipopolysaccharide-induced expression of FAS ligand in cultured immature boar sertoli cells through the regulation of pro-inflammatory cytokines and miR-187.

Lipopolysaccharide (LPS) induces germ cell apoptosis, but its mechanism of action is not clear. One possibility is that LPS regulates the expression of FAS ligand (FASLG) in Sertoli cells, which will then influence germ cell apoptosis. In this study, LPS reduced the viability of cultured, immature boar Sertoli cells in a time- and dose-dependent manner; enhanced the production of pro-inflammatory cytokines including tumor necrosis factor α (TNFA), interleukin-1ß (IL1B), nitric oxide (NO), and transforming growth factor-ß (TGFB); and increased the expression of FASLG in a dose-dependent manner. While 10 µg/ml LPS enhanced the expression of FASLG, reduced cell cycle progression, and impaired the ultrastructure of Sertoli cells, this dose did not induce apoptosis. LPS also had no effect on the activity or expression of matrix metalloproteinases 2 or 9 (MMP2 or MMP9). In contrast, the expression of ssc-miR-187 increased following LPS challenge, and inhibition of ssc-miR-187 blocked LPS-induced expression of FASLG. Our results therefore suggest that LPS reduces the viability of and enhances FASLG expression in cultured, immature boar Sertoli cells through elevated secretion of TNFA, IL1B, NO, and TGFB as well as through the regulation of ssc-miR-187 potency.

mTOR is involved in 17ß-estradiol-induced, cultured immature boar Sertoli cell proliferation via regulating the expression of SKP2, CCND1, and CCNE1.

Mammalian target of rapamycin (mTOR) is known to be involved in mammalian cell proliferation, while S-phase kinase-associated protein 2 (SKP2) plays a vital role in the cell cycle. Within the testis, estrogen also plays an important role in Sertoli cell proliferation, although it is not clear how. The present study asked if mTOR is involved in 17ß-estradiol-dependent Sertoli cell proliferation. We specifically assessed if extracellular signal-regulated kinase 1/2 (ERK1/2) and/or phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) exert convergent effects toward the activation of mTOR signaling, and if this signaling regulates the expression of SKP2 through retinoblastoma (RB) and early mitotic inhibitor 1 (EMI1) protein and on CCNE1 and CCND1 mRNA levels. Treatment with 17ß-estradiol for 15-90 min activated mTOR, with mTOR phosphorylation peaking after 30 min. U0126 (5 µM), a specific inhibitor of (MEK1/2), and 10-DEBC (2 µM), a selective inhibitor of AKT, both significantly reduced 17ß-estradiol-induced phosphorylation of mTOR. Rapamycin suppressed 17ß-estradiol-induced Sertoli cell proliferation, appearing to act by reducing the abundance of SKP2, CCND1, and CCNE1 mRNA as well as RB and EMI1 protein. These data indicated that 17ß-estradiol enhances Sertoli cell proliferation via mTOR activation, which involves both ERK1/2 and PI3K/AKT signaling. Activated mTOR subsequently increases SKP2 mRNA and protein expression by enhancing the expression of CCND1 and CCNE1, and inhibits SKP2 protein degradation by increasing EMI1 abundance.

Thyroid hormone inhibits the proliferation of piglet Sertoli cell via PI3K signaling pathway.

Accumulating researches show that thyroid hormone (TH) inhibits Sertoli cells (SCs) proliferation and stimulates their functional maturation in prepubertal rat testis, confirming that TH plays a key role in testicular development. However, the mechanism under the T3 regulation of piglet SC proliferation remains unclear. In the present study, in order to investigate the possible mechanism of T3 on the suppression of SC proliferation, the expression pattern of TRα1 and cell cycle-related molecules, effect of T3 on SC proliferation, and the role of phosphoinositide 3-kinase (PI3K)/Akt signaling pathway on the T3-mediated SC proliferation in piglet testis were explored. Our results demonstrated that TRα1 was expressed in all tested stages of SCs and decreased along with the ages. T3 inhibited the proliferation of SCs in a time- and dose-dependent manner, and T3 treatment downregulated the expressions of cell cycling molecules, such as cyclinA2, cyclinD1, cyclinE1, PCNA, and Skp2, but upregulated the p27 expression in SCs. Most importantly, the suppressive effects of T3 on SC proliferation seemed dependent on the inhibition of PI3K/Akt signaling pathway, and pre-stimulation of PI3K could enhance such suppressive effects. Together, our findings demonstrate that TH inhibits the proliferation of piglet SCs via the suppression of PI3K/Akt signaling pathway.