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After ovulation, the mitochondrial enzyme CYP11A1 cleavage the cholesterol into pregnenolone for progesterone synthesis, suggesting that mitochondrial dynamics play a vital role in the female reproductive system. The changes in the mitochondria dynamics throughout the ovarian cycle have been reported in literature, but the correlation to its role in the ovarian cycle remains unclear. In this study, mitochondrial fusion promotor, M1, was used to study the impact of mitochondria dynamics in the female reproductive system. Our results showed that M1 treatment in mice can lead to the disruptions of estrous cycles in vagina smears. The decrease in serum LH was recorded in the animal. And the inhibitions of progesterone secretion and ovulations were observed in ovarian culture. Although no significant changes in mitochondrial networks were observed in the ovaries, significant up-regulation of mitochondrial respiratory complexes was revealed in M1 treatments through transcriptomic analysis. In contrast to the estrogen and steroid biosynthesis up-regulated in M1, the molecules of extracellular matrix, remodeling enzymes, and adhesion signalings were decreased. Collectively, our study provides novel targets to regulate the ovarian cycles through the mitochondria. However, more studies are still necessary to provide the functional connections between mitochondria and the female reproductive systems.

BACKGROUND Kisspeptin (KISS1) and kisspeptin receptor (KISS1R) are essential gatekeepers of the reproductive system. The functions of KISS1 and KISS1R in corpus luteal cells remain ambiguous. The objective was to observe normal physiologic functions of corpus luteal cells in vivo and clarify the functions of KISS1 in vitro. METHODS We conducted an in vivo observation of cellular patterns as well as the levels of steroidogenic enzymes and KISS1/KISS1R in corpus luteal cells obtained from female crossbred Taiwan native goats in the estrous cycle; the observation was performed using hematoxylin and eosin and immunohistochemistry staining. Subsequently, we used kisspeptin-10 (Kp-10) to stimulate temperature sensitive-caprine luteal cell line (ts-CLC-D) cells to investigate the progesterone (P4) levels, steroidogenic messenger RNA (mRNA)/protein levels, cell survival rate, intracellular Ca2+ concentration, and cell proliferation-related mRNA/protein levels in the mitogen-activated protein kinase pathway in vitro by applying immunofluorescence staining, Western blotting, 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide assay, and real-time polymerase chain reaction. RESULTS We observed the presence of proteins and mRNAs for STAR, CYP11A1, HSD3B, KISS1, and KISS1R in the corpus luteal cells from goats in vivo. In vitro, the addition of Kp-10 reduced the P4 levels (p < 0.01) and increased cell proliferation (p < 0.05) of the ts-CLC-D cells. Furthermore, we found that the levels of proteins and mRNA for STAR, CYP11A1, and HSD3B decreased significantly when Kp-10 was added (p < 0.05). However, adding Kp-10 did not affect the mRNA levels for PLCG2, DAG1, PRKCA, KRAS, RAF1, MAP2K1, MAP2K2, MAPK3, MAPK1, and MAPK14. CONCLUSION We determined that KISS1 could affect the P4 levels, steroidogenesis, and cell proliferation in luteal cells. However, further research is required to clarify how KISS1 regulates proliferation and steroid production in luteal cells.

Kisspeptin and its receptor KISS1R have been proven as pivotal regulators on controlling the hypothalamus–pituitary–gonad axis. Inactivating mutations in one of them cause idiopathic hypogonadotropic hypogonadism in human as well as rodent models. Notably, gonadotropin insensitivity, failure in hCG response, was presented in the male patients with loss-function-mutations in KISS1R gene; this reveals the essential role of KISS1R signaling in regulating testosterone production beyond the hypothalamic functions of kisspeptin. In this study, we hypothesized that the autocrine action of kisspeptin on Leydig cells may modulate steroidogenesis. Based on the mouse cell model, we first demonstrated that the cAMP/protein kinase A (PKA)/cAMP response element-binding protein (CREB) signaling pathway mediated gonadotropin-induced kisspeptin expression. By using siRNA interfering technique, knockdown of Kiss1r in MA-10 cells, a mouse Leydig tumor cell line, significantly reduced progesterone productions in both basal and hCG-treated conditions. Integrating the results from both quantitative real-time PCR and steroidogenic enzyme-activity assay, we found that this steroidogenic defect was associated with decreased luteinizing hormone/choriogonadotropin receptor (Lhcgr) and StAR protein (Star) expressions. Furthermore, exogenous expression of human LHCGR completely rescued hCG-stimulated progesterone production in the KISS1R-deficient cells. In conclusion, we proposed that the reproductive functions of KISS1R signaling in Leydig cell include modulating Lhcgr and steroidogenic gene expressions, which may shed the light on the pathophysiology of gonadotropin insensitivity.

In mammalian ovaries, mitochondria are integral sites of energy production and steroidogenesis. While shifts in cellular activities and steroidogenesis are well characterized during the differentiation of large luteal cells in folliculogenesis and luteal formation, mitochondrial dynamics during this process have not been previously evaluated. In this study, we collected ovaries containing primordial follicles, mature follicles, corpus hemorrhagicum, or corpus luteum from goats at specific times in the estrous cycle. Enzyme histochemistry, ultrastructural observations, and 3D structural analysis of serial sections of mitochondria revealed that branched mitochondrial networks were predominant in follicles, while spherical and tubular mitochondria were typical in large luteal cells. Furthermore, the average mitochondrial diameter and volume increased from folliculogenesis to luteal formation. In primordial follicles, the signals of cytochrome c oxidase and ATP synthase were undetectable in most cells, and the large luteal cells from the corpus hemorrhagicum also showed low enzyme signals and content when compared with granulosa cells in mature follicles or large luteal cells from the corpus luteum. Our findings suggest that the mitochondrial enlargement could be an event during folliculogenesis and luteal formation, while the modulation of mitochondrial morphology and respiratory enzyme expressions may be related to tissue remodeling during luteal formation.

Obese men have lower circulating testosterone than men with an optimal body mass index. Elevated fatty acids (FAs) caused by obesity have been reported to suppress the steroidogenesis of Leydig cells. Recent studies have demonstrated that autophagy regulates steroidogenesis in endocrine cells; however, few studies have investigated the molecular mechanisms of FA-impaired steroidogenesis. To study FA regulation in the steroidogenesis of Leydig cells, MA-10 cells were treated with an FA mixture and co-treated with 8-Br-cAMP to stimulate the steroidogenesis capacity. We showed that FAs led to cellular lipid accumulation and decreased steroidogenesis of MA-10 cells, and FA-suppressed steroidogenesis was largely recovered by P5 treatment but not by 22R-OHC treatment, suggesting the primary defect was the deficiency of CYP11A1. To examine the involvement of autophagy in the steroidogenesis of Leydig cells, we treated MA-10 cells with autophagy regulators, including rapamycin, bafilomycin, and chloroquine. Inhibition of late-stage autophagy including FA-upregulated Rubicon suppressed the steroidogenesis of MA-10 cells. More interestingly, Rubicon played a novel regulatory role in the steroidogenesis of MA-10 cells, independent of inhibitors of late-stage autophagy. Collectively, this study provides novel targets to investigate the interaction between FAs and steroidogenesis in steroidogenic cells.

BACKGROUND Kisspeptin and its receptor KISS1R have been found to be essential regulators of reproductive function. Previous data have revealed the presence of Kiss1 and Kiss1r mRNAs in the hypothalamus and the testis of humans and rodents. However, the precise location and possible physiological role of the kisspeptin/KISS1R system in the testis remain ambiguous. METHODS We first produced an anti-KISS1R immunoglobulin Y antibody for KISS1R identification. To detect the exact sites of KISS1R and kisspeptin expression in the testis, we conducted immunohistochemistry assays on sections of testes. We used real-time polymerase chain reactions to identify Kiss1r in mice and to determine the expression levels of testicular genes. Finally, to verify the upstream regulation on the Kisspeptin/KISS1 receptor system, we treated primary mouse Leydig cells and MA-10 cells with luteinizing hormone (LH) and Br-cAMP, respectively, and examined Kiss1 and Kiss1r mRNA expression. RESULTS Immunohistochemistry assays revealed that kisspeptin was expressed in Leydig cells and KISS1R was localized in the seminiferous tubules. With real-time polymerase chain reactions, we found Kiss1r mRNA was constitutively expressed in the mouse testis from birth until the postnatal fourth week. Furthermore, mRNA expression of Kiss1 was synchronized with that of Insl3 and Cyp19a. However, the expression of the LH receptor-encoding gene increased 1 week earlier than did Kiss1 expression. This indicated that the kisspeptin/KISS1R system in the testis may be controlled by LH and cAMP signaling pathways. Finally, we confirmed that Kiss1 mRNA expression was increased in both LH-treated primary Leydig cells and Br-cAMP-treated MA-10 cells (p

Thioacetamide (TAA), usually used as a fungicide to control the decay of citrus products, itself is not toxic to the liver, but its intermediates are able to increase oxidative stress in livers and further cause fibrosis. Ophiocordyceps sinensis mycelium (OSM) which contains 10% polysaccharides and 0.25% adenosine decreased (P < 0.05) the lipid accumulation and increased (P

Previous studies have demonstrated the important role of kisspeptin in impaired glucose-stimulated insulin secretion (GSIS). In addition, it was reported that the activation of autophagy in pancreatic β-cells decreases insulin secretion by selectively degrading insulin granules. However, it is currently unknown whether kisspeptin suppresses GSIS in β-cells by activating autophagy. To investigate the involvement of autophagy in kisspeptin–regulated insulin secretion, we overexpressed Kiss1 in NIT-1 cells to mimic the long-term exposure of pancreatic β-cells to kisspeptin during type 2 diabetes (T2D). Interestingly, our data showed that although kisspeptin potently decreases the intracellular proinsulin and insulin ((pro)insulin) content and insulin secretion of NIT-1 cells, autophagy inhibition using bafilomycin A1 and Atg5 siRNAs only rescues basal insulin secretion, not kisspeptin-impaired GSIS. We also generated a novel in vivo model to investigate the long-term exposure of kisspeptin by osmotic pump. The in vivo data demonstrated that kisspeptin lowers GSIS and (pro)insulin levels and also activated pancreatic autophagy in mice. Collectively, our data demonstrated that kisspeptin suppresses both GSIS and non-glucose-stimulated insulin secretion of pancreatic β-cells, but only non-glucose-stimulated insulin secretion depends on activated autophagic degradation of (pro)insulin. Our study provides novel insights for the development of impaired insulin secretion during T2D progression.

BACKGROUND Cardio-renal syndrome (CRS) is an integrative problem related to chronic malnutrition, obesity, etc. Amino acids and peptides are regarded as protective and essential for tissues. Pepsin-digested chicken liver hydrolysates (CLHs), which are made from the byproducts of the poultry industry, are amino-acid based and of animal origin, and may be protective against the myocardial and renal damage induced by a high-fat diet (HFD). RESULTS Our results showed that CLHs contain large quantities of anserine, taurine, and branched-chain amino acids (BCAAs), and supplementing the diet with CLHs reduced (P < 0.05) weight gain, liver weight, peri-renal fat mass / adipocyte-area sizes, serum total cholesterol (TC), aspartate aminotransferase (AST), and low-density lipoprotein cholesterol (LDLC) levels in HFD-fed mice but increased (P < 0.05) serum high-density lipoprotein cholesterol (HDLC) levels. By histological analyses, CLHs alleviated (P < 0.05) renal lipid deposition and fibrosis, as well as cardiac fibrosis and inflammation of HFD-fed mice. Meanwhile, increased (P < 0.05) inflammatory and fibrotic cytokines levels in the myocardia of the HFD-fed mice were downregulated (P < 0.05) by CLH supplementation. Regarding autophagy-related protein levels, protective effects of CLHs on the myocardia against HFD feeding may result from the early blockade of the autophagy pathway to prevent autophagosome accumulation. CONCLUSION Functional CLHs could be a novel food ingredient as a cardio-renal protective agent against a high-fat dietary habit in a niche market. © 2020 Society of Chemical Industry.

Patients with lung cancer harboring activating epidermal growth factor (EGFR) mutations and pre-existing diabetes have been demonstrated to exhibit poor responses to first-line EGFR-tyrosine kinase inhibitor (TKI) therapy. Strategies for the management of acquired resistance to EGFR-TKIs in patients with advanced non-small cell lung cancer (NSCLC) are urgently required. Only a limited number of studies have been published to date on the effects of insulin on EGFR-TKI resistance in NSCLC. Hence, the aim of the present study was to investigate the roles of hyperinsulinemia and hyperglycemia in mediating gefitinib resistance in NSCLC cells with activating EGFR mutations. In the present study, the HCC4006 cell line, which harbors EGFR mutations, was co-treated with gefitinib and long-acting insulin glargine. Whether hyperinsulinemia is able to mediate EGFR-TKI resistance in the NSCLC cell line harboring activating EGFR mutations was also investigated, and the possible underlying mechanisms responsible for these actions were explored. The inhibition of cell proliferation, and the potential mechanism of gefitinib resistance, were examined using an MTS proliferation assay and western blot analysis, and through the transfection of siRNAs. Whether the inhibition of AKT is able to overcome EGFR-TKI resistance induced by long-acting insulin was also investigated. The results obtained suggested that hyperinsulinemia induced by glargine upregulated NSCLC cell proliferation and survival, and induced gefitinib resistance. By contrast, the morphology and proliferation of the cells in a medium containing a 2-fold concentration of glucose were not significantly affected. Gefitinib resistance induced by hyperinsulinemia may have been mediated via the phosphoinositide 3-kinase (PI3K)/AKT pathway rather than the mitogen-activated protein kinase extracellular signal regulated kinase (MAPK/ERK) pathway. AKT serine/threonine kinase 1 knockdown by siRNA rescued the gefitinib resistance that was induced by hyperinsulinemia. In conclusion, hyperinsulinemia, but not hyperglycemia, was identified to cause the development of gefitinib resistance in NSCLC cells with activating EGFR mutations. However, additional studies are required to investigate strategies, such as co targeting hyperinsulinemia and the PI3K/AKT pathway, for overcoming EGFR-TKI resistance in patients with NSCLC.

The present study was designed to study the ultrastructure of goat corpora lutea (CL, n=10) and structural changes as related to steroidogenic functions during the estrous cycle. The reproduction status of goats was estimated by analyzing serum progesterone concentrations. The CL at various stages was surgically collected. To characterize ultrastructural features associated with steroidogenesis, tissue and cellular structures were studied. Blood supplies were examined based on features of the endothelial cells and capillary structures in the CL. Activated endothelial cells and developing vessels were observed in the early stage, whereas mature endothelial cells, accumulating extracellular matrix fibers, and stabilized vessels were observed in the middle and late stages of assessment. In the late stage of assessment, shrunken goat luteal cells scattered around the capillaries were detected and formed circular regression areas. Features of autophagy and luteal cell apoptosis were noted. In large luteal cells, steroidogenic organelles were present, including microvillar channels, endoplasmic reticulum, and mitochondria. Conformational changes in the endoplasmic reticulum and increased mitochondria with tubular cristae were observed in the early-middle CL transitions. In contrast, mitochondria swelled and the cristae transformed to the lamellar type in the late stage, suggesting that organelle plasticity could contribute to steroidogenesis in goat CL. In conclusion, results suggest angiogenesis occurs in early developing CL and programmed cell death occurred in the late stage of CL assessment in the present study. Structures and quantiles of steroidogenic organelles are correlated with the steroidogenic functions in goats.

Patients with lung cancer harboring activating epidermal growth factor (EGFR) mutations and pre-existing diabetes have been demonstrated to exhibit poor responses to first-line EGFR-tyrosine kinase inhibitor (TKI) therapy. Strategies for the management of acquired resistance to EGFR-TKIs in patients with advanced non-small cell lung cancer (NSCLC) are urgently required. Only a limited number of studies have been published to date on the effects of insulin on EGFR-TKI resistance in NSCLC. Hence, the aim of the present study was to investigate the roles of hyperinsulinemia and hyperglycemia in mediating gefitinib resistance in NSCLC cells with activating EGFR mutations. In the present study, the HCC4006 cell line, which harbors EGFR mutations, was co-treated with gefitinib and long-acting insulin glargine. Whether hyperinsulinemia is able to mediate EGFR-TKI resistance in the NSCLC cell line harboring activating EGFR mutations was also investigated, and the possible underlying mechanisms responsible for these actions were explored. The inhibition of cell proliferation, and the potential mechanism of gefitinib resistance, were examined using an MTS proliferation assay and western blot analysis, and through the transfection of siRNAs. Whether the inhibition of AKT is able to overcome EGFR-TKI resistance induced by long-acting insulin was also investigated. The results obtained suggested that hyperinsulinemia induced by glargine upregulated NSCLC cell proliferation and survival, and induced gefitinib resistance. By contrast, the morphology and proliferation of the cells in a medium containing a 2-fold concentration of glucose were not significantly affected. Gefitinib resistance induced by hyperinsulinemia may have been mediated via the phosphoinositide 3-kinase (PI3K)/AKT pathway rather than the mitogen-activated protein kinase extracellular signal regulated kinase (MAPK/ERK) pathway. AKT serine/threonine kinase 1 knockdown by siRNA rescued the gefitinib resistance that was induced by hyperinsulinemia. In conclusion, hyperinsulinemia, but not hyperglycemia, was identified to cause the development of gefitinib resistance in NSCLC cells with activating EGFR mutations. However, additional studies are required to investigate strategies, such as co targeting hyperinsulinemia and the PI3K/AKT pathway, for overcoming EGFR-TKI resistance in patients with NSCLC.

Although curcumin was widely applied as a functional food for different diseases, it was found to reduce serum testosterone level and fertility in male animals by unknown molecular mechanisms. Here in our study, we investigated the possible mechanisms of curcumin-suppressed testosterone production in Leydig cells. Our enzyme immunoassay results showed that curcumin cell-autonomously suppressed ovine luteinizing hormone-stimulated testosterone production in primary Leydig cells and 8-bromo-cyclic adenosine monophosphate (8-br-cAMP)-induced progesterone production in MA-10 cells. Furthermore, our real-time PCR, Western blot, and 22R-OHC/pregnenolone supplementing experiment data demonstrated that curcumin suppressed 8-br-cAMP-induced steroidogenesis in Leydig cells by inhibiting the expression of StAR and Cyp11a1. Interestingly, our Western blot data showed that although curcumin suppressed PKA activity, it did not alter the 8-br-cAMP-induced phosphorylation of CREB. On the contrary, the real-time PCR results showed that curcumin suppressed 8-br-cAMP-induced expression of Nr5a1 and Fos, which are crucial for cAMP-stimulated StAR and Cyp11a1 expression in Leydig cells. Collectively, our data demonstrated that curcumin may suppress cAMP-induced steroidogenesis in mouse Leydig cells by down-regulating Nr5a1/Fos-controlled StAR and Cyp11a1 expression independently of the PKA-CREB signaling pathway.

Valproic acid (VPA) is one of the most widely used antiepilepsy drugs. However, several side effects, including weight gain and fatty liver, have been reported in patients following VPA treatment. In this study, we explored the molecular mechanisms of VPA-induced hepatic steatosis using FL83B cell line-basedin vitromodel. Using fluorescent lipid staining technique, we found that VPA enhanced oleic acid- (OLA-) induced lipid accumulation in a dose-dependent manner in hepatocytes; this may be due to upregulated lipid uptake, triacylglycerol (TAG) synthesis, and lipid droplet formation. Real-time PCR results showed that, following VPA treatment, the expression levels of genes encoding cluster of differentiation 36 (Cd36), low-density lipoprotein receptor-related protein 1 (Lrp1), diacylglycerol acyltransferase 2 (Dgat2), and perilipin 2 (Plin2) were increased, that of carnitine palmitoyltransferase Ia(Cpt1a) was not affected, and those of acetyl-Co A carboxylaseα(Acca) and fatty acid synthase (Fasn) were decreased. Furthermore, using immunofluorescence staining and flow cytometry analyses, we found that VPA also induced peroxisome proliferator-activated receptorγ(PPARγ) nuclear translocation and increased levels of cell-surface CD36. Based on these results, we propose that VPA may enhance OLA-induced hepatocyte steatosis through the upregulation of PPARγ- and CD36-dependent lipid uptake, TAG synthesis, and lipid droplet formation.

Background Kisspeptin, encoded by the Kiss1 gene, has many forms including kisspeptin54, kisspeptin14, kisspeptin13, and kisspeptin10, and all these peptides have the same affinity to their receptor KISS1R encoded by the Kiss1r gene. The KISS1–KISS1R system was discovered in neurons, and many reports stress on their function in the brain. However, recent studies have shown that Kiss1 and Kiss1r are expressed in the testes. The goal of this study was to demonstrate the roles of Kiss1 and Kiss1r in testicular function, especially their steroidogenic activity. Methods Kisspeptin10 and the kisspeptin10 antagonist peptide234 were used to determine their effect on testosterone production. Moreover, expression of steroidogenic genes in mouse testes and their gonadosomatic index (weight of the testes divided by the total body weight) and also serum testosterone level were studied between the ages of 2 weeks and 15 weeks. Results Kisspeptin10 and peptide234 did not affect testosterone production in primary Leydig cells from adult mice. Kiss1 and Esr1 expression also increased during puberty. The peak gonadosomatic index occurred at 4 weeks of age, and serum testosterone levels plateaued after the age of 4 weeks. Conclusion Our results suggest that kisspeptin10 does not affect steroidogenesis in adult Leydig cells, but its pattern of expression follows the stages of testicular development. Future studies should determine if kisspeptin regulates testicular development during puberty.

D-Glucuronolactone (C6H8O6, lactone), naturally found in plant gums, is commercially acclaimed for its hepatoprotective effects. This study was to investigate whether lactone can attenuate thioacetamide (TAA)-induced liver fibrosis in a rat model. Results showed that lactone supplementation (75 mg kg−1 bw glucuronolactone) alleviated AST values in TAA-intraperitoneally-injected rats (100 mg kg−1 bw TAA) and increased antioxidant capacity of liver via elevations of antioxidant enzymes activities [superoxide dismutase (SOD) and glutathione peroxidase (GPx)], glutathione (GSH) and trolox equivalent antioxidative capacity (TEAC) levels (p

Androgens, especially testosterone produced in Leydig cells, play an essential role in development of the male reproductive phenotype and fertility. However, testicular oxidative stress may cause a decline in testosterone production. Many antioxidants have been used as reactive oxygen species (ROS) scavengers to eliminate oxidative stress to protect steroidogenesis. Astaxanthin (AST), a natural extract from algae and plants ubiquitous in the marine environment, has been shown to have antioxidant activity in many previous studies. In this study, we treated primary mouse Leydig cells or MA-10 cells with hydrogen peroxide (H2O2) to cause oxidative stress. Testosterone and progesterone production was suppressed and the expression of the mature (30 kDa) form of StAR protein was down-regulated in MA-10 cells by H2O2 and cAMP co-treatment. However, progesterone production and expression of mature StAR protein were restored in MA-10 cells by a one-hour pretreatment with AST. AST also reduced ROS levels in cells so that they were lower than the levels in untreated controls. These results provide additional evidence of the potential health benefits of AST as a potential food additive to ease oxidative stress.

Background During the prediabetic development, the changes in β-cell function and tissue-specific insulin resistance have been described. However, there are conflicting views in insulin secretory capacity between early clinical observation and recent proposed mathematical model. On the basis of digestive and metabolic similarities with humans, swine have great potential as an animal model to investigate the progressive mechanisms of prediabetes. The aim of this study was to investigate the insulin secretory response and tissue-specific insulin resistance in a dietary-induced prediabetic porcine model. Methods Adult male Taiwan Lee-Sung miniature pigs were randomized into two groups: (1) low-fat diet and (2) high-fat plus high-fructose diet (HFHF; 20.9% crude fat and 17.8% fructose). During the 12-month dietary intervention, body weights and blood glucose levels were measured monthly. Intravenous glucose tolerance test was used for measuring glucose tolerance and insulin secretory capacity. At the end of the experiment, liver and soleus muscle specimens were collected for ex vivo insulin sensitivity testing. Results The results showed that the HFHF group had obesity, hyperinsulinemia, and dyslipidemia, but normal fasting glucose levels. The HFHF pigs exhibited enhanced first- and second-phase insulin secretion and high 2-h postload glucose levels in intravenous glucose tolerance test. Furthermore, the skeletal muscle specimens from the HFHF group were desensitized to insulin stimulation as shown by the lack of AKT Ser473 phosphorylation; however, the liver specimens remained a normal response. Conclusions In conclusion, the HFHF diet-fed pigs developed isolated impaired glucose tolerance corresponding to prediabetes with an intense insulin secretory response and skeletal muscle insulin resistance. Electronic supplementary material The online version of this article (doi:10.1186/s13098-017-0281-6) contains supplementary material, which is available to authorized users.

Kisspeptin acts as an upstream regulator of the hypothalamus–pituitary–gonad axis, which is one of the main regulatory systems for mammalian reproduction.Kiss1and its receptorKiss1r(also known as G protein-coupled receptor 54 (Gpr54)) are expressed in various organs, but their functions are not well understood. The purpose of this study was to investigate the expression profiles and functions of kisspeptin and KISS1R in the reproductive tissues of imprinting control region mice. To identify the expression pattern and location of kisspeptin and KISS1R in gonads, testes and ovarian tissues were examined by immunohistochemical or immunofluorescent staining. Kisspeptin and KISS1R were expressed primarily in Leydig cells and seminiferous tubules respectively. KISS1R was specifically localized in the acrosomal region of spermatids and mature spermatozoa. Kisspeptin, but not KISS1R, was expressed in the cumulus–oocyte complex and oviductal epithelium of ovarian and oviductal tissues. The sperm intracellular calcium concentrations significantly increased in response to treatment with kisspeptin 10 in Fluo-4-loaded sperm. The IVF rates decreased after treatment of sperm with the kisspeptin antagonist peptide 234. These results suggest that kisspeptin and KISS1R might be involved in the fertilization process in the female reproductive tract. In summary, this study indicates that kisspeptin and KISS1R are expressed in female and male gametes, respectively, and in mouse reproductive tissues. These data strongly suggest that the kisspeptin system could regulate mammalian fertilization and reproduction.

The mating system of the Taiwan field vole (Microtus kikuchii) has been proposed to be monogamous. In monogamous animals, individuals should exhibit monogamy syndromes, such as little sexual dimorphism and strong pair bonding (a strong social preference for a familiar partner versus a strange one). In this study, we examined the effect of cohabitation on the partner preference. In a reciprocal experiment, all test individuals were cohabited with a heterosexual vole for 24 hr prior to the partner preference trials. We collected the feces of voles before and after the trials, and analyzed the concentration of fecal steroid hormones, including testosterone of males, progesterone and estradiol of females, and corticosterone of all voles. The results showed that the behaviors of focal voles were not influenced by the status (partner or stranger) of stimulus vole. There was no significant relationship between steroid hormones and partner preference. Furthermore, the degree of sexual dimorphism in the Taiwan field vole was low, and similar to that of the monogamous prairie vole (M. ochrogaster). In light of this study and other recent findings, we propose that the mating system of the Taiwan field vole is not strictly monogamy, but flexible depending on environmental conditions.

Nonalcoholic fatty liver disease (NAFLD) commonly results from excessive dietary fat intake which characterized by obesity and insulin resistance. Wild fruiting body of Antrodia camphorata (AC) was assayed for alleviative effects on NAFLD. An NAFLD animal model was successfully established in male 7-week-old C57BL/6 mice fed with high-fat diet (HFD) for 36 weeks. The HFD mice exhibited obese and impaired glucose metabolism. After an induction of NAFLD syndrome, AC was given for one week via gavage. Mice with AC treatment showed lowered (p

Additional file 1: Table S1. Compositions of experimental diets. Figure S1. The effect of HFHF diet on the triglyceride accumulations in the skeletal muscle and hepatic tissues. Figure S2. Liver histology of Lee-Sung pigs at the end of the dietary intervention.

Gentisic acid and epicatechin are two major compounds in phenolic acids and flavonoids of litchi-flower-water extracts (LFWEs), respectively. Increased (p

Thioacetamide (TAA), usually used as a fungicide to control the decay of citrus products, itself is not toxic to the liver, but its intermediates are able to increase oxidative stress in livers and further cause fibrosis. Ophiocordyceps sinensis mycelium (OSM) which contains 10% polysaccharides and 0.25% adenosine decreased (P0.05) the lipid accumulation and increased (P 0.05) antioxidative capacity in livers of thioacetamide (TAA) injected rats. Meanwhile, the increased (P0.05) liver sizes, serum alanine transaminase (AST) and aspartate transaminase (ALT) values in thioacetamide (TAA)-injected rats were ameliorated (P 0.05) by OSM supplementation. Moreover, the levels of proinflammatory cytokines, such as the tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), were also reduced (P 0.05). The fibrosis phenomena in pathological (Masson's trichrome and HE stainings) and immunohistochemical [α-smooth actin (αSMA) and CD86/ED1] observations in TAA-treated rats were reduced (P0.05) by OSM cotreatment. The protective effect of OSM against TAA-induced liver inflammation/fibrosis may be via downregulations (P 0.05) of TGF-β pathways and NFκB which further influenced (P 0.05) the expressions of fibrotic and inflammatory genes (i. e., αSMA, Col1α, COX2). Therefore, OSM shows preventive effects on the development of TAA-induced hepatic fibrosis.

Inflammation and hepatic stellate cell (HSC) activation are the most crucial steps in the formation of hepatic fibrosis. Hepatocytes damaged by viral or bacterial infection, alcohol or toxic chemicals initiate an inflammatory response that activates collagen production by HSCs. Recent studies indicate curcumin has liver-protective effects due to its anti-inflammatory, antioxidant and anticancer activities; however, the mechanisms are not well understood. In this study, we show that curcumin protected against hepatic fibrosis in BALB/c mice in vivo by inhibiting HSC activation, inflammatory responses and inducing apoptosis of damaged hepatocytes. Using the thioacetamide (TAA)-induced hepatic fibrosis animal model, we found that curcumin treatment up-regulated P53 protein expression and Bax messenger RNA (mRNA) expression and down-regulated Bcl-2 mRNA expression. Together, these responses increased hepatocyte sensitivity to TAA-induced cytotoxicity and forced the damaged cells to undergo apoptosis. Enhancing the tendency of damaged hepatocytes to undergo apoptosis may be the protective mechanism whereby curcumin suppresses inflammatory responses and hepatic fibrogenesis. These results provide a novel insight into the cause of hepatic fibrosis and the cytoprotective effects curcumin has on hepatic fibrosis suppression.

An alcoholic fatty liver disease was induced by feeding an ethanol diet. Eight mice per group were randomly assigned to each following group: (1) control liquid diet + 0.5 mL ddH2O; (2) control liquid diet + 150 mg silymarin/kg body weight (BW) in 0.5 mL ddH2O; (3) ethanol liquid diet + 0.5 mL ddH2O; and (4) ethanol liquid diet + 150 mg silymarin/kg BW in 0.5 mL ddH2O. The ethanol diet increased (P

BACKGROUND: Liver fibrosis is chronic liver damage usually caused by alcohol, viruses or other toxins and is characterised by an excessive accumulation of extracellular matrix proteins such as collagen. The aim of this study was to establish an animal model of chronic liver damage and investigate molecular mechanisms of silymarin hepatoprotective effects. RESULTS: Thioacetamide (TAA; 100 mg kg−1 intraperitoneal (i.p.) injection three times weekly) effectively induced chronic liver fibrosis in male ICR mice. Then 24 ICR mice were randomly divided into four groups: (1) saline (i.p.) + water (gavage); (2) saline (i.p.) + 150 mg kg−1 silymarin (gavage); (3) 100 mg kg−1 TAA (i.p.) + water (gavage); (4) 100 mg kg−1 TAA (i.p.) + 150 mg kg−1 silymarin (gavage). Eight weeks of TAA treatment resulted in lower body weight, serum cholesterol and triglycerides as well as increased liver size, ALT, AST and LDH values (P < 0.05). These TAA-induced effects were attenuated by silymarin (P < 0.05); therefore silymarin also ameliorated TAA-induced liver lesions. Effects of silymarin on TAA-induced chronic liver damage may be attributed to down-regulation of hepatic MMP-2, MMP-13, TIMP-1, TIMP-2, AP-1, KLF6, TGF-β1, α-SMA and COL-α1. CONCLUSION: A mouse model of chronic liver fibrosis was successfully established by injecting 100 mg kg−1 TAA three times weekly in male ICR mice. Meanwhile, silymarin showed hepatoprotection against TAA-induced damage. Copyright © 2011 Society of Chemical Industry

Chronic alcohol consumption or alcohol abuse is the main cause of alcoholic steatohepatitis or further cirrhosis. This study was to exam if the antioxidant capacity and alcohol metabolism in livers of chronic alcohol-fed rats were improved by supplementing taurine (Tau). Rats were randomly divided into four groups with five times per week of treatment: 1) isocaloric solution; 2) 3 g alcohol/kg BW/day; 3) 3 g alcohol/kg BW/day + 1 g taurine (Tau)/kg BW/day; and 4) 3 g alcohol/kg BW/day + 2 g Tau/kg BW/day. A 6-week alcohol consumption resulted in lower ( p p p p

Hypoxia-inducible factor 1 (HIF-1) is a transcription factor that senses and adapts cells to hypoxic environmental conditions. HIF-1 is composed of an oxygen-regulated α subunit (HIF-1α) and a constitutively expressed β subunit (HIF-1β). Taiwan voles (Microtus kikuchii) are an endemic species in Taiwan, found only in mountainous areas greater than 2000m above sea level. In this study, the full-length HIF-1α cDNA was cloned and sequenced from liver tissues of Taiwan voles. We found that HIF-1α of Taiwan voles had high sequence similarity to HIF-1α of other species. Sequence alignment of HIF-1α functional domains indicated basic helix-loop-helix (bHLH), PER-ARNT-SIM (PAS) and C-terminal transactivation (TAD-C) domains were conserved among species, but sequence variations were found between the oxygen-dependent degradation domains (ODDD). To measure Taiwan vole HIF-1α responses to hypoxia, animals were challenged with cobalt chloride, and HIF-1α mRNA and protein expression in brain, lung, heart, liver, kidney, and muscle was assessed by quantitative RT-PCR and Western blot analysis. Upon induction of hypoxic stress with cobalt chloride, an increase in HIF-1α mRNA levels was detected in lung, heart, kidney, and muscle tissue. In contrast, protein expression levels showed greater variation between individual animals. These results suggest that the regulation of HIF-1α may be important to the Taiwan vole under cobalt chloride treatments. But more details regarding the evolutionary effect of environmental pressure on HIF-1α primary sequence, HIF-1α function and regulation in Taiwan voles remain to be identified.

Nonalcoholic fatty liver (NAFL) is also called hepatic steatosis and has become an emergent liver disease in developed and developing nations. This study was to exam the preventive effects of taurine (Tau) on the development of hepatic steatosis via a hamster model. Although hepatic steatosis of hamsters was induced by feeding a high-fat/cholesterol diet, drinking water containing 0.35 and 0.7% Tau improved (p < 0.05) the serum lipid profile. Meanwhile, the smaller (p < 0.05) liver sizes and lower (p < 0.05) hepatic lipids in high-fat/cholesterol dietary hamsters drinking Tau may be partially due to higher (p < 0.05) fecal cholesterol, triacylglycerol, and bile acid outputs. In the regulation of lipid homeostasis, drinking a Tau solution upregulated (p < 0.05) low-density lipoprotein receptor and CYP7A1 gene expressions in high-fat/cholesterol dietary hamsters, which result in increased fecal cholesterol and bile acid outputs. Drinking a Tau solution also upregulated (p < 0.05) peroxisome proliferator-activated receptor-α (PPAR-α) and uncoupling protein 2 (UPC2) gene expressions in high-fat/cholesterol dietary hamsters, thus increasing energy expenditure. Besides, Tau also enhanced (p < 0.05) liver antioxidant capacities (GSH, TEAC, SOD, and CAT) and decreased (p < 0.05) lipid peroxidation (MDA), which alleviated liver damage in the high-fat/cholesterol dietary hamsters. Therefore, Tau shows preventive effects on the development of hepatic steatosis induced by a high-fat/cholesterol dietary habit.

Consumption of eggs contaminated with Salmonella Enteritidis (SE) has been recognized as one of the important causes of human foodborne salmonellosis. Chicken granulosa cells (cGCs) comprise the last tissue layer surrounding the yolk in preovulatory follicles and are a preferred site for SE invasion. To understand the cGC response to SE infection, we conducted an in vitro time-course study to identify cGC transcriptional changes using chicken whole genome microarrays. The expression of 135 (4 h postinfection) and 120 cGC genes (48 h postinfection) were altered (P < .01) compared to uninfected cells. Many of the altered genes were related to immune response, physiological processes, signal transduction, and transcription. Furthermore, we also found that the Jak-STAT pathway, which is essential in the regulation of cellular cytokines and growth factors, was highly active in this study. Among the genes identified by microarray, the mRNA levels of TLR15, IL-6, CXCLi1, CXCLi2, and K203 were shown to be upregulated by real-time RT-PCR (qRT-PCR). In contrast, the mRNA levels of RASD1 and HB-EGF decreased according to both microarray and qRT-PCR analyses. These results suggest that during the SE infection, cGCs recruit cells of the innate immune responses; the infection may also induce suppression of cGC cell proliferation, which alters follicular development and ovulation.

The corpus luteum (CL) is a transient endocrine gland that produces progesterone, a product required for the establishment and maintenance of pregnancy. In the absence of pregnancy, the production of progesterone in the CL decreases and the structure itself regresses in size. The life span and function of the CL are regulated by complex interactions between stimulatory (luteotrophic) and inhibitory (luteolytic) mediators. When an ovum is released from a mature follicle, angiogenesis and rapid growth of follicular cells form the CL. The purpose of the present study was to determine whether steroidogenesis, proliferation, and hypoxia-related proteins are expressed in caprine CL. The expression of proliferating cell nuclear antigen (PCNA), vascular endothelial growth factor (VEGF), and hypoxia-inducible factor la (HIF-la) were determined in caprine CL during the estrous cycle. Cytochrome P450 side chain cleavage protein did not vary significantly during the estrous cycle; however, there was an increased expression of 3β-hydroxysteroid dehydrogenase in the early and middle stages, which rapidly decreased in the late stage. The same observations were made with respect to steroidogenic acute regulatory protein. Variations in progesterone content and expression of PCNA. HIF-la, and VEGF were consistent with this result. Thus. the steroidogenic proteins, PCNA, HIF-la, and VEGF in caprine CL are dependent on the stage of the estrous cycle.

The biosynthesis of steroids from steroidogenic cells are catalyzed by the two major enzymes, P450 side-chain cleavage enzyme (P450scc) and 3beta-hydroxysteroid dehydrogenase (3beta-HSD). This article describes the construction of two novel polyclonal antibodies against conserved recombinant protein and the validation of these antibodies on fixed tissue sections of bovine corpus luteum. The polyclonal antibodies were used successfully in Western blots and specifically reacted with P450scc and 3beta-HSD protein in bovine luteal cell extracts. Thus, P450scc and 3beta-HSD are two specific polyclonal antibodies that are integral products in the investigation of the biological function and regulatory mechanism involved in steroidogenesis.