Urocortin (UCN1) is a member of corticotrophin-releasing factor (CRF) family, which has been proven to participate in inflammation. Previous work showed that dihydrotestosterone (DHT) could promote the inflammatory process. Little is known about the effect of DHT on UCN1 expression. The aim of our study is to investigate the effects and underlying mechanisms of DHT on endothelial UCN1 expression in the absence and presence of induced inflammation. Therefore, we tested the alterations of endothelial UCN1 expression treated with DHT in the presence or absence of lipopolysaccharide (LPS). Our data showed that DHT alone decreased UCN1 levels, which were attenuated in the presence of the androgen receptor (AR) antagonist flutamide. Conversely, in the presence of LPS, DHT augmented the LPS-induced increase in UCN1 expression, which was, interestingly, not affected by flutamide. When cells were treated with DHT alone, AR was upregulated and translocated into the nuclei, which might repress UCN1 expression via a potential androgen-responsive element found in human CRF family promoter. In the presence of LPS, DHT did not influence AR expression and location while it increased toll-like receptor 4 expression and activation, which was not altered by flutamide. DHT enhanced LPS-induced p38MAPK, ERK1/2, and nuclear factor κB pathway activation, which may contribute to the elevated expression of UCN1. These data suggest that DHT differentially influences UCN1 levels under normal and inflammatory conditions in human umbilical vein endothelial cells, which involves AR-dependent and -independent mechanisms respectively.

Locally synthesized atrial natriuretic peptide (ANP) and its receptors have been found in reproductive tissues of various mammals, and play an important role in the acrosome reaction of human sperm. The objective of the present study was to examine the expression of ANP and its receptors in pig spermatozoa and oviduct, and the effect of ANP on pig spermatozoa function. The expression of ANP and its receptors was analyzed by RT-PCR. Only natriuretic peptide receptors-A (NPRA) mRNA was detected in fresh sperm. While the levels of natriuretic peptide receptors-C (NPRC) mRNA were low with no obvious change among different oviductal phases, the levels of ANP mRNA were high in oviduct(OT)1 , OT3 and OT5, but were very low in OT2. On the other hand, the levels of NPRA mRNA were low in OT1 and OT2, increased in OT3 and reached a maximum in OT4 and OT5. Western blot analysis revealed that the level of ANP was high in OT1, decreased in OT2 and OT3, and arrived at the nadir in OT4 and OT5. The effect of ANP on spermatozoa function was studied by the acrosome reaction and IVF. Incubation with ANP for 1 h significantly induced acrosome reaction of preincubated spermatozoa, and maximal response of acrosome reaction (34.1 ± 2.3%) was achieved at 1 nM ANP treatment. Both C-ANP-(4–23), a selective ligand of NPRC, and caffeine had no effect on the acrosome reaction. The stimulatory effect of ANP on acrosome reaction could be mimicked by the permeable cGMP analog, 8-Br-cGMP. ANP and caffeine had a similar effect on improving the oocytes penetration rate, polyspermy rate and the average number of sperm per penetrated oocyte. Also, ANP treatment had a similar effect on cleavage rate, blastocyst formation rate and the number of cells per blastocyst as that of caffeine treatment. The effects of ANP on the acrosome reaction and the parameters of oocyte penetration could be blocked by cGMP-dependent protein kinase (PKG) inhibitors KT5823 and/or Rp-8-pCPT-cGMPS. These results suggest that the expression of ANP in the oviduct may be involved in the regulation of the acrosome reaction and the fertilising ability of pig spermatozoa, and the PKG pathway possibly participates in the process.

Hormonal signals help to maintain glucose and lipid homeostasis in the liver during the periods of fasting. Glucagon, a pancreas-derived hormone induced by fasting, promotes gluconeogenesis through induction of intracellular cAMP production. Glucagon also stimulates hepatic fatty acid oxidation but the underlying mechanism is poorly characterized. Here we report that following the acute induction of gluconeogenic genes Glucose 6 phosphatase (G6Pase) and Phosphoenolpyruvate carboxykinase (Pepck) expression through cAMP-response element-binding protein (CREB), glucagon triggers a second delayed phase of fatty acid oxidation genes Acyl-coenzyme A oxidase (Aox) and Carnitine palmitoyltransferase 1a (Cpt1a) expression via extracellular cAMP. Increase in extracellular cAMP promotes PPARα activity through direct phosphorylation by AMP-activated protein kinase (AMPK), while inhibition of cAMP efflux greatly attenuates Aox and Cpt1a expression. Importantly, cAMP injection improves lipid homeostasis in fasted mice and obese mice, while inhibition of cAMP efflux deteriorates hepatic steatosis in fasted mice. Collectively, our results demonstrate the vital role of glucagon-stimulated extracellular cAMP in the regulation of hepatic lipid metabolism through AMPK-mediated PPARα activation. Therefore, strategies to improve cAMP efflux could serve as potential new tools to prevent obesity-associated hepatic steatosis.

Obesity is a worldwide health problem. Semaphorins are involved in axonal guidance; however, the role of secretory semaphorin 3G (SEMA3G) in regulating adipocyte differentiation remains unclear. Microarray analysis showed that the SEMA3G gene was upregulated in an in vitro model of adipogenesis. In this study, SEMA3G was highly expressed in the white adipose tissue and liver. Analysis of 3T3-L1 cell and primary mouse preadipocyte differentiation showed that SEMA3G mRNA and protein levels were increased during the middle stage of cell development. In vitro experiments also showed that adipocyte differentiation was promoted by SEMA3G; however, SEMA3G inhibition using a recombinant lentiviral vector expressing a specific shRNA showed the opposite results. Mice were fed a chow or high-fat diet (HFD); knockdown of SEMA3G was found to inhibit weight gain, reduce fat mass in the tissues, prevent lipogenesis in the liver tissue, reduce insulin resistance and ameliorate glucose tolerance in HFD mice. Additionally, the effect of SEMA3G on HFD-induced obesity was activated through PI3K/Akt/GSK3β signaling in the adipose tissue and the AMPK/SREBP-1c pathway in the liver. Moreover, the plasma concentrations of SEMA3G and leptin were measured in 20 obese and 20 non-obese human subjects. Both proteins were increased in obese subjects, who also exhibited a lower level of adiponectin and presented with insulin resistance. In summary, we demonstrated that SEMA3G is an adipokine essential for adipogenesis, lipogenesis, and insulin resistance and is associated with obesity. SEMA3G inhibition may, therefore, be useful for treating diet-induced obesity and its complications.

Tryptophan 2,3-dioxygenase (T do 2) is a rate-limiting enzyme which directs the conversion of tryptophan to kynurenine. The aim of this study was to examine the expression and regulation of T do 2 in mouse uterus during decidualization. T do 2 mRNA was mainly expressed in the decidua on days 6–8 of pregnancy. By real-time PCR, a high level of T do 2 expression was observed in the uteri from days 6 to 8 of pregnancy, although T do 2 expression was observed on days 1–8. Simultaneously, T do 2 mRNA was also detected under in vivo and in vitro artificial decidualization. Estrogen, progesterone, and 8-bromoadenosine-cAMP could induce the expression of T do 2 in the ovariectomized mouse uterus and uterine stromal cells. T do 2 could regulate cell proliferation and stimulate the expression of decidual marker Dtprp in the uterine stromal cells and decidual cells. Overexpression of T do 2 could upregulate the expression of Ahr, Cox2, and Vegf genes in uterine stromal cells, while T do 2 inhibitor 680C91 could downregulate the expression of Cox2 and Vegf genes in uterine decidual cells. These data indicate that T do 2 may play an important role during mouse decidualization and be regulated by estrogen, progesterone, and cAMP.

Polycystic ovary syndrome (PCOS) is a condition resulting from the interaction between environmental factors and hereditary components, profoundly affecting offspring development. Although the etiology of this disease remains unclear, aberrant in utero androgen exposure is considered one of the pivotal pathogenic factors. Herein, we demonstrate the intergenerational inheritance of PCOS-like phenotypes in F2 female offspring through F1 males caused by maternal testosterone exposure in F0 mice. We found impaired serum hormone expression and reproductive system development in prenatal testosterone-treated F1 male and F2 female mice (PTF1 and PTF2). In addition, downregulated N6-methyladenosine (m6A) methyltransferase and binding proteins induced mRNA hypomethylation in the PTF1 testis, including frizzled-6 (Fzd6). In the PTF2 ovary, decreased FZD6 protein expression inhibited the mammalian target of rapamycin (mTOR) signaling pathway and activated Forkhead box O3 (FoxO3) phosphorylation, which led to impaired follicular development. These data indicate that epigenetic modification of the mTOR signaling pathway could be involved in the intergenerational inheritance of maternal testosterone exposure-induced impairments in the PTF2 ovary through male PTF1 mice.

Prenatal caffeine exposure (PCE) can induce testicular developmental toxicity. Here, we aimed to explore the underlying mechanism of this process in reference to its intrauterine origin. Pregnant rats were intragastrically administrated caffeine (30 and 120 mg/kg/day) from gestational days 9 to 20. The results showed that the male fetuses exposed to high dose of caffeine (120 mg/kg/day) had a decreased bodyweight and inhibited testosterone synthetic function. Meanwhile, their serum corticosterone concentration was elevated and their testicular insulin-like growth factor 1 (Igf1) expression was decreased. Moreover, the histone 3 lysine 14 acetylation (H3K14ac) level in the Igf1 promoter region was reduced. Low-dose (30 mg/kg/day) caffeine exposure, however, increased steroidogenic enzymes expression in male fetuses. After birth, the serum corticosterone concentration gradually decreased in the PCE (120 mg/kg/day) offspring rats, whereas the expression and H3K14ac level of Igf1 gradually increased, with obvious catch-up growth and testicular development compensation. Intriguingly, when we subjected the offspring to 2 weeks of chronic stress to elevate the serum corticosterone concentration, the expression of Igf1 and testosterone synthesis were inhibited again in the PCE (120 mg/kg/day) group, accompanied by a decrease in the H3K14ac level in the Igf1 promoter region. In vitro, corticosterone (rather than caffeine) was proved to inhibit testosterone production in Leydig cells by altering the H3K14ac level and the expression of Igf1. These observations suggested that PCE-induced testicular developmental toxicity is related to the negative regulation of corticosterone on H3K14ac levels and the expression of Igf1.

Oxidative stress is a major cause of islet injury and dysfunction during isolation and transplantation procedures. Cyanidin-3-O-glucoside (C3G), which is present in various fruits and vegetables especially in Chinese bayberry, shows a potent antioxidant property. In this study, we determined whether C3G could protect neonatal porcine islets (NPI) from reactive oxygen species (H₂O₂)-induced injury in vitro and promote the function of NPI in diabetic mice. We found that C3G had no deleterious effect on NPI and that C3G protected NPI from damage induced by H₂O₂. Significantly higher hemeoxygenase-1 (HO1) gene expression was detected in C3G-treated NPI compared to untreated islets before and after transplantation (P < 0.05). Western blot analysis showed a significant increase in the levels of phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) and phosphatidylinositol 3-kinase (PI3K/Akt) proteins in C3G-treated NPI compared to untreated islets. C3G induced the nuclear translocation of nuclear erythroid 2-related factor 2 (NRF2) and the significant elevation of HO1 protein. Recipients of C3G-treated NPI with or without C3G-supplemented drinking water achieved normoglycemia earlier compared to recipients of untreated islets. Mice that received C3G-treated islets with or without C3G-supplemented water displayed significantly lower blood glucose levels at 5–10 weeks post-transplantation compared to mice that received untreated islets. Mice that received C3G-treated NPI and C3G-supplemented drinking water had significantly (P < 0.05) lower blood glucose levels at 7 and 8 weeks post-transplantation compared to mice that received C3G-treated islets. These findings suggest that C3G has a beneficial effect on NPI through the activation of ERK1/2- and PI3K/AKT-induced NRF2-mediated HO1 signaling pathway.

Previous research on the role of insulin has focused on metabolism. This study investigated the effect of insulin on angiogenesis in endometrial decidualization. High insulin-treated mouse model was constructed by subcutaneous injection of insulin. Venous blood glucose, serum insulin, P4, E2, FSH and LH levels in the pregnant mice were detected by ELISA. Decidual markers, angiogenesis factors and decidual vascular network were detected during decidualization in the pregnant mouse model and an artificially induced decidualization mouse model. Tube formation ability and angiogenesis factors expression were also detected in high insulin-treated HUVECS cells. To confirm whether autophagy participates in hyperinsulinemia-impaired decidual angiogenesis, autophagy was detected in vivo and in vitro. During decidualization, in the condition of high insulin, serum insulin and blood glucose were significantly higher, while ovarian steroid hormones were also disordered (P < 0.05), decidual markers BMP2 and PRL were significantly lower (P < 0.05). Uterine CD34 staining showed that the size of the vascular sinus was significantly smaller than that in control. Endometrial VEGFA was significantly decreased after treatment with high insulin in vivo and in vitro (P < 0.05), whereas ANG-1 and TIE2 expression was significantly increased (P < 0.05). In addition, aberrant expression of autophagy markers revealed that autophagy participates in endometrial angiogenesis during decidualization (P < 0.05). After treatment with the autophagy inhibitor 3-MA in HUVEC, the originally damaged cell tube formation ability and VEGFA expression were repaired. This study suggests that endometrial angiogenesis during decidualization was impaired by hyperinsulinemia in early pregnant mice.