Forskolin Research Articles

Endocrine-Disruptive Effects of Adenylate Cyclase Activator Forskolin: In Vitro and In Vivo Evidence.

Forskolin (FSK) is a potent adenylate cyclase activator and may display endocrine-disruptive effects via the disruption of steroidogenesis. Here, we tested this hypothesis by use of the in vitro H295R steroidogenesis assay and the in vivo long-term medaka (Oryzias latipes) exposure assay. The results from the H295R assay demonstrated that the transcriptional levels of a series of genes involved in steroidogenesis, including HSD3B2, CYP11A, CYP11B2, CYP17, CYP19, and CYP21, were remarkably up-regulated. Meanwhile, the productions of estrogens (17β-estradiol (17β-E2) and estrone (E1)) and progestins (progesterone (PGT) and 17-hydroxyprogesterone (17-HPT)) were significantly increased, and those of androgens (androstenedione (ADD) and testosterone (TTR)) were significantly inhibited. After waterborne exposure of medaka to FSK for 100 days, the gene expressions of HMGR, HSD17B1, CYP17B, CYP19A, and CYP21A were significantly enhanced in the gonads of male medaka. 17β-E2 was remarkably induced, although without statistical significance. In addition, the biomarker genes for estrogenicity, including VTG-I, VTG-II, CHG-H, and CHG-L, were significantly induced in male medaka livers. Pathological damage to their gonads was further identified. Therefore, the results demonstrated that FSK modulates the transcriptions of steroidogenesis genes and alters hormone levels in vitro and in vivo, which is a mark of endocrine disruption in organisms.

Role of phosphorylation and vanilloid ligand structure in ligand-dependent differential activations of transient receptor potential vanilloid 1.

Vanilloid analogs, which can activate transient receptor potential vanilloid 1 (TRPV1), have been classified into two types based on susceptibility to forskolin (FSK). Treatment of cells expressing TRPV1 with FSK enhances TRPV1 responses to capsaicin-type ligands while diminishing the responses to eugenol-type ligands. In this study, we determined the effect of FSK on the activation of TRPV1 stimulated with vanilloid ligands, through the influx of Ca2+ in HEK293T cells expressing TRPV1. Our findings suggest that the effects of FSK can be attributed to the phosphorylation of TRPV1, as evidenced by using a protein kinase A inhibitor and TRPV1 mutants at potential phosphorylation sites. Furthermore, we examined the structure-activity relationship of 13 vanilloid analogs. Our results indicated that vanilloid compounds could be classified into three types, that is the previously reported two types and a novel type of 10-shogaol, by which TRPV1 activation was insusceptible to the FSK treatment.

Forskolin improves experimental autoimmune encephalomyelitis in mice probably by inhibiting the calcium and the IL-17-STEAP4 signaling pathway

Multiple sclerosis (MS) is a chronic autoimmune disease in the central nervous system. Forskolin (FSK) is a plant-derived diterpene with excellent immunomodulatory properties and has not been systematically reported for treating MS. This study investigated the therapeutic effects of FSK on cellular and animal MS models and preliminarily explored related mechanisms. The results showed that FSK suppressed the inflammatory response, reduced the expression of STEAP4, and relieved iron deposition in BV-2 cells pretreated by LPS at the cellular level. Meanwhile, at the animal level, FSK treatment halted the progression of experimental autoimmune encephalomyelitis (EAE), alleviated the damage at the lesion sites, reduced the concentration of proinflammatory factors in peripheral blood, and inhibited the immune response of peripheral immune organs in EAE mice. Besides, FSK treatment decreased the expression of STEAP4 in the spinal cord and effectively restored the iron balance in the brain, spinal cord, and serum of EAE mice. Further investigation showed that FSK can reduce IL-17 expression, prevent the differentiation of TH17 cells, and inhibit the calcium signaling pathway. Thus, these results demonstrate that FSK may have the potential to treat MS clinically.

Forskolin is an effective therapeutic small molecule for the treatment of hypertrophic cardiomyopathy through ADCY6/cAMP/PKA pathway

Hypertrophic cardiomyopathy (HCM) arises from a pathogenic variant in the gene responsible for encoding the myocardium-associated protein. Forskolin (FSK), a labdane diterpene isolated from Sphingomonas capillaris, exhibits diverse pharmacological effects, including bronchospasm relief, intraocular pressure reduction, and glaucoma treatment. However, whether FSK could regulate HCM and its associated mechanism remains unclear. Here, we discovered that FSK could mitigate cardiac hypertrophy in two HCM mouse models (Myh6R404Q and Tnnt2R109Q) in vivo. Additionally, FSK could prevent norepinephrine (NE)-induced cardiomyocyte hypertrophy in vitro. It reversed cardiac dysfunction, reduced enlarged cell size, and downregulated the expression of hypertrophy-related genes. We further demonstrated that FSK's mechanism in alleviating HCM relied on the activation of ADCY6. In conclusion, our findings demonstrate that FSK alleviates hypertrophic cardiomyopathy by modulating the ADCY6/cAMP/PKA pathway, suggesting that FSK holds promise as a therapeutic agent for HCM.

Divergent effects of olfactory receptors on transient receptor potential vanilloid 1 activation by capsaicin and eugenol.

We analyzed the effects of olfactory receptors (ORs) on transient receptor potential vanilloid 1 (TRPV1) activation using HEK293T cells co-expressing TRPV1 and OR51E1. We demonstrate here that the effect of OR51E1 on TRPV1 activation varies depending on the two TRPV1 ligands: capsaicin and eugenol. Notably, both of these ligands are vanilloid analogs. OR51E1 enhanced the response of TRPV1 to capsaicin but diminished that to eugenol. OR51E2 also showed similar effects. Based on the susceptibility to the OR's modulatory effects, various TRPV1 ligands could be classified into capsaicin and eugenol types. Activation of OR51E1 enhanced cAMP production. In addition, forskolin exhibited almost identical effects as ORs on TRPV1 responses to both types of ligands. These results suggest that OR51E1-induced cAMP elevation leads to a modification of TRPV1, presumably phosphorylation of TRPV1, which amplifies the susceptibility of TRPV1 to the two types of ligands differently.

Plasma adiponectin/leptin ratio associates with subcutaneous abdominal and omental adipose tissue characteristics in women

BackgroundA better understanding of adipose tissue (AT) dysfunction, which includes morphological and functional changes such as adipocyte hypertrophy as well as impaired adipogenesis, lipid storage/mobilization, endocrine and inflammatory responses, is needed in the context of obesity. One dimension of AT dysfunction, secretory adiposopathy, often assessed as a low plasma adiponectin (A)/leptin (L) ratio, is commonly observed in obesity. The aim of this study was to examine markers of AT development and metabolism in 67 women of varying age and adiposity (age: 40-62 years; body mass index, BMI: 17-41 kg/m2) according to levels of adiponectinemia, leptinemia or the plasma A/L ratio.MethodsBody composition, regional AT distribution and circulating adipokines were determined. Lipolysis was measured from glycerol release in subcutaneous abdominal (SCABD) and omental (OME) adipocytes under basal, isoproterenol-, forskolin (FSK)- and dibutyryl-cyclic AMP (DcAMP)-stimulated conditions. Adipogenesis (C/EBP-α/β/δ, PPAR-γ2 and SREBP-1c) and lipid metabolism (β2-ARs, HSL, FABP4, LPL and GLUT4) gene expression (RT-qPCR) was assessed in both fat depots. Participants in the upper versus lower tertile of adiponectin, leptin or the A/L ratio were compared.ResultsBasal lipolysis was similar between groups. Women with a low plasma A/L ratio were characterized by higher adiposity and larger SCABD and OME adipocytes (p<0.01) compared to those with a high ratio. In OME adipocytes, women in the low adiponectinemia tertile showed higher isoproterenol-stimulated lipolysis (0.01<p<0.05), while those in the high leptinemia tertile displayed increased lipolytic response to this agent (p<0.05). However, lipolysis stimulated by isoproterenol was enhanced in both compartments (0.01<p<0.05) in women with a low plasma A/L ratio. AT abundance of selected transcripts related to adipogenesis or lipid metabolism did not differ between women with or without secretory adiposopathy, except for lower GLUT4 mRNA levels in OME fat.ConclusionsSecretory adiposopathy assessed as the plasma A/L ratio, more so than adiponectin or leptin levels alone, discriminates low and elevated lipolysis in OME and SCABD adipocytes despite similar AT expression of selected genes involved in lipid metabolism.

Patchouli Alcohol: A Potent Tyrosinase Inhibitor Derived from Patchouli Essential Oil with Potential in the Development of a Skin-Lightening Agent

The inhibitory effects of Pogostemon cablin essential oil (patchouli essential oil, PEO) and its primary bioactive compound, patchouli alcohol (PA), on tyrosinase and melanin were investigated in vitro and ex vivo. Treatment with PEO and PA significantly, as well as dose-dependently, reduced forskolin (FRK)-induced melanin biosynthesis, cellular tyrosinase activity, and tyrosinase (TYR) protein expression. However, the transcriptional levels of TYR and tyrosinase-related proteins (TRP-1 and TRP-2) remained unaffected. These results suggest that PEO and PA may directly interrupt tyrosinase enzyme activity, leading to a reduction in melanin biosynthesis. Further experiments supported this notion, revealing that both PEO and PA significantly and dose-dependently inhibited mushroom tyrosinase activity in both the monophenolase and diphenolase phases. Additionally, an in silico molecular docking analysis was performed, utilizing a homology model of human tyrosinase. In conclusion, these findings strongly suggest that patchouli essential oil and its primary bioactive component, patchouli alcohol, hold promise as potential treatments for hyperpigmentary skin conditions and in the development of cosmetic products designed to lighten the skin.

Lipolysis inhibition improves the survival of fat grafts through ameliorating lipotoxicity and inflammation.

Fat grafting is a promising technique for correcting soft tissue abnormalities, but oil cyst formation and graft fibrosis frequently impede the therapeutic benefit of fat grafting. The lipolysis of released oil droplets after grafting may make the inflammation and fibrosis in the grafts worse; therefore, by regulating adipose triglyceride lipase (ATGL) via Atglistatin (ATG) and Forskolin (FSK), we investigated the impact of lipolysis on fat grafts in this study. After being removed from the mice and chopped into small pieces, the subcutaneous fat from wild-type C57BL/6J mice was placed in three different solutions for two hours: serum-free cell culture medium, culture medium+FSK (50 μM), and culture medium+ATG (100 μM). Following centrifugation to remove water and free oil droplets, 0.3 mL of the fat particles per mouse was subcutaneously injected into the back of mice. Additionally, the subcutaneous fat grafting area was immediately injected with PBS (control group), ATG (30 mg/kg), and FSK (15 mg/kg) following fat transplantation. Detailed cellular events after grafting were investigated by histological staining, real-time polymerase chain reaction, immunohistochemistry/immunofluorescent staining, and quantification. Two weeks after grafting, grafts treated with ATG showed lower expression of ATGL and decreased mRNA levels of TNFα and IL-6. In contrast, grafts treated with ATG showed elevated expression levels of IL-4 and IL-13 compared to the control grafts. In addition, fewer apoptotic cells and oil cysts were observed in ATG grafts. Meanwhile, a higher CD206+/CD68+ ratio of macrophages and more CD31+ vascular endothelial cells existed in the 2-month ATG grafts. In comparison to the control, ATG treatment improved the volume retention of grafts, and decreased graft fibrosis and oil cyst formation. By preventing oil droplet lipolysis, pharmacological suppression of ATGL shielded adipocytes from lipotoxicity following grafting. Additionally, ATG ameliorated the apoptosis and inflammation brought on by adipocyte death and oil droplet lipolysis in grafted fat. These all indicate that lipolysis inhibition improved transplanted fat survival and decreased the development of oil cysts and graft fibrosis, offering a potential postoperative pharmacological intervention for bettering fat grafting.

Epigenetic drug screening for trophoblast syncytialization reveals a novel role for MLL1 in regulating fetoplacental growth.

Abnormal placental development is a significant factor contributing to perinatal morbidity and mortality, affecting approximately 5-7% of pregnant women. Trophoblast syncytialization plays a pivotal role in the establishment and maturation of the placenta, and its dysregulation is closely associated with several pregnancy-related disorders, including preeclampsia and intrauterine growth restriction. However, the underlying mechanisms and genetic determinants of syncytialization are largely unknown. We conducted a systematic drug screen using an epigenetic compound library to systematically investigate the epigenetic mechanism essential for syncytialization, and identified mixed lineage leukemia 1 (MLL1), a histone 3 lysine 4 methyltransferase, as a crucial regulator of trophoblast syncytialization. BeWo cells were utilized to investigate the role of MLL1 during trophoblast syncytialization. RNA sequencing and CUT&Tag were further performed to search for potential target genes and the molecular pathways involved. Human placenta tissue was used to investigate the role of MLL1 in TEA domain transcription factor 4 (TEAD4) expression and the upstream signaling during syncytialization. A mouse model was used to examine whether inhibition of MLL1-mediated H3K4me3 regulated placental TEAD4 expression and fetoplacental growth. Genetic knockdown of MLL1 or pharmacological inhibition of the MLL1 methyltransferase complex (by MI-3454) markedly enhanced syncytialization, while overexpression of MLL1 inhibited forskolin (FSK)-induced syncytiotrophoblast formation. In human placental villous tissue, MLL1 was predominantly localized in the nuclei of cytotrophoblasts. Moreover, a notable upregulation in MLL1 expression was observed in the villus tissue of patients with preeclampsia compared with that in the control group. Based on RNA sequencing and CUT&Tag analyses, depletion of MLL1 inhibited the Hippo signaling pathway by suppressing TEAD4 expression by modulating H3K4me3 levels on the TEAD4 promoter region. TEAD4 overexpression significantly reversed the FSK-induced or MLL1 silencing-mediated trophoblast syncytialization. Additionally, decreased hypoxia-inducible factor 1A (HIF1A) enrichment at the MLL1 promoter was observed during syncytialization. Under hypoxic conditions, HIF1A could bind to and upregulate MLL1, leading to the activation of the MLL1/TEAD4 axis. In vivo studies demonstrated that the administration of MI-3454 significantly enhanced fetal vessel development and increased the thickness of the syncytial layer, thereby supporting fetoplacental growth. These results revealed a novel epigenetic mechanism underlying the progression of syncytialization with MLL1, and suggest potential avenues for identifying new therapeutic targets for pregnancy-related disorders.

Forskolin rescues hypoxia-induced cognitive dysfunction in zebrafish with potential involvement of O-GlcNAc cycling regulation

Repeated sublethal hypoxia exposure induces brain inflammation and affects the initiation and progression of cognitive dysfunction. Experiments from the current study showed that hypoxic exposure downregulates PKA/CREB signaling, which is restored by forskolin (FSK), an adenylate cyclase activator, in both Neuro2a (N2a) cells and zebrafish brain. FSK significantly protected N2a cells from hypoxia-induced cell death and neurite shrinkage. Intraperitoneal administration of FSK for 5 days on zebrafish additionally led to significant recovery from hypoxia-induced social interaction impairment and learning and memory (L/M) deficit. FSK suppressed hypoxia-induced neuroinflammation, as indicated by the observed decrease in NF-κB activation and GFAP expression. We further investigated the potential effect of FSK on O-GlcNAcylation changes induced by hypoxia. Intriguingly FSK induced marked upregulation of the protein level of O-GlcNAc transferase catalyzing addition of the GlcNAc group to target proteins, accompanied by elevated O-GlcNAcylation of nucleocytoplasmic proteins. The hypoxia-induced O-GlcNAcylation decrease in the brain of zebrafish was considerably restored following FSK treatment. Based on the collective results, we propose that FSK rescues hypoxia-induced cognitive dysfunction, potentially through regulation of HBP/O-GlcNAc cycling.

27-Hydroxycholesterol inhibits trophoblast fusion during placenta development by activating PI3K/AKT/mTOR signaling pathway.

Trophoblast cell syncytialization is essential for placental and fetal development. Abnormal trophoblast cell fusion leads to pregnancy pathologies, such as preeclampsia (PE), intrauterine growth restriction (IUGR), and miscarriage. 27-hydroxycholesterol (27-OHC) is the most abundant oxysterol in human peripheral blood synthesized by sterol 27-hydroxylase (CYP27A1) and is considered a critical mediator between hypercholesterolemia and a variety of related disorders. Gestational hypercholesterolemia was associated with spontaneous preterm delivery and low birth weight (LBW) in term infants, yet the mechanism is unclear. In this study, two trophoblast cell models and CD-1 mice were used to evaluate the effects of 27-OHC on trophoblast fusion during placenta development. Two different kinds of trophoblast cells received a dosage of 2.5, 5, or 10 uM 27-OHC. Three groups of pregnant mice were randomly assigned: control, full treatment (E0.5-E17.5), or late treatment (E13.5-E17.5). All mice received daily intraperitoneal injections of saline (control group) and 27-OHC (treatment group; 5.5mg/kg). In vitro experiments, we found that 27-OHC inhibited trophoblast cell fusion in primary human trophoblasts (PHT) and forskolin (FSK)-induced BeWo cells. 27-OHC up-regulated the expression of the PI3K/AKT/mTOR signaling pathway-related proteins. Moreover, the PI3K inhibitor LY294002 rescued the inhibitory effect of 27-OHC. Inhibition of trophoblast cell fusion by 27-OHC was also observed in CD-1 mice. Furthermore, fetal weight and placental efficiency decreased and fetal blood vessel development was inhibited in pregnant mice treated with 27-OHC. This study was the first to prove that 27-OHC inhibits trophoblast cell fusion by Activating PI3K/AKT/mTOR signaling pathway. This study reveals a novel mechanism by which dyslipidemia during pregnancy results in adverse pregnancy outcomes.

Zika Virus Infection Alters the Circadian Clock Expression in Human Neuronal Monolayer and Neurosphere Cultures.

Rhythmic regulations are virtually described in all physiological processes, including central nervous system development and immunologic responses. Zika virus (ZIKV), a neurotropic arbovirus, has been recently linked to a series of birth defects and neurodevelopmental disorders. Given the well-characterized role of the intrinsic cellular circadian clock within neurogenesis, cellular metabolism, migration, and differentiation among other processes, this study aimed to characterize the influence of ZIKV infection in the circadian clock expression in human neuronal cells. For this, in vitro models of human-induced neuroprogenitor cells (hiNPCs) and neuroblastoma cell line SH-SY5Y, cultured as monolayer and neurospheres, were infected by ZIKV, followed by RNA-Seq and RT-qPCR investigation, respectively. Targeted circadian clock components presented mRNA oscillations only after exogenous synchronizing stimuli (Forskolin) in SH-SY5Y monolayer culture. Interestingly, when these cells were grown as 3D-arranged neurospheres, an intrinsic oscillatory expression pattern was observed for some core clock components without any exogenous stimulation. The ZIKV infection significantly disturbed the mRNA expression pattern of core clock components in both neuroblastoma cell culture models, which was also observed in hiNPCs infected with different strains of ZIKV. The ZIKV-mediated desynchronization of the circadian clock expression in human cells might further contribute to the virus impairment of neuronal metabolism and function observed in adults and ZIKV-induced congenital syndrome. In vitro models of Zika virus (ZIKV) neuronal infection. Human neuroprogenitor cells were cultured as monolayer and neurospheres and infected by ZIKV. Monolayer-cultured cells received forskolin (FSK) as a coupling factor for the circadian clock rhythmicity, while 3D-arranged neurospheres showed an intrinsic oscillatory pattern in the circadian clock expression. The ZIKV infection affected the mRNA expression pattern of core clock components in both cell culture models. The ZIKV-mediated desynchronization of the circadian clock machinery might contribute to the impairment of neuronal metabolism and function observed in both adults (e.g., Guillain-Barré syndrome) and ZIKV-induced congenital syndrome (microcephaly). The graphical abstract has beencreated with Canva at the canva.com website.

The secreted peptide BATSP1 promotes thermogenesis in adipocytes

Although brown adipose tissue (BAT) has historically been viewed as a major site for energy dissipation through thermogenesis, its endocrine function has been increasingly recognized. However, the circulating factors in BAT that play a key role in controlling systemic energy homeostasis remain largely unexplored. Here, we performed a peptidomic analysis to profile the extracellular peptides released from human brown adipocytes upon exposure to thermogenic stimuli. Specifically, we identified a secreted peptide that modulates adipocyte thermogenesis in a cell-autonomous manner, and we named it BATSP1. BATSP1 promoted BAT thermogenesis and induced browning of white adipose tissue in vivo, leading to increased energy expenditure under cold stress. BATSP1 treatment in mice prevented high-fat diet-induced obesity and improved glucose tolerance and insulin resistance. Mechanistically, BATSP1 facilitated the nucleocytoplasmic shuttling of forkhead transcription factor 1 (FOXO1) and released its transcriptional inhibition of uncoupling protein 1 (UCP1). Overall, we provide a comprehensive analysis of the human brown adipocyte extracellular peptidome following acute forskolin (FSK) stimulation and identify BATSP1 as a novel regulator of thermogenesis that may offer a potential approach for obesity treatment.

A forskolin-mediated increase in cAMP promotes T helper cell differentiation into the Th1 and Th2 subsets rather than into the Th17 subset

The adenylyl cyclase (AC) signaling pathway is suggested to be a key regulator of immune system functions. However, specific effects of cyclic adenosine monophosphate (cAMP) on T helper (Th) cell differentiation and functions are unclear. The involvement of cAMP in the Th cell differentiation program, in particular the development of Th1, Th2, and Th17 subsets, was evaluated employing forskolin (FSK), a labdane diterpene well known as an AC activator. FSK mediated an elevation in Th1-specific markers reinforcing the Th1 cell phenotype. The Th2 differentiation was supported by FSK, though cell metabolism was negatively affected. In contrast, the Th17 immunophenotype was severely suppressed leading to the highly specific upregulation of CXCL13. The causality between FSK-elicited cAMP production and the observed reinforcement of Th2 differentiation was established by using AC inhibitor 2′,5′-dideoxyadenosine, which reverted the FSK effects. Overall, an FSK-mediated cAMP increase affects Th1, Th2 and Th17 differentiation and can contribute to the identification of novel therapeutic targets for the treatment of Th cell-related pathological processes.

Effect and mechanism of Zexie Decoction in promoting white adipose tissue browning/brown adipose tissue activation based on GLP-1R/cAMP/PKA/CREB pathway

This study investigated the mechanism of Zexie Decoction(ZXD) in promoting white adipose tissue browning/brown adipose tissue activation based on the GLP-1R/cAMP/PKA/CREB pathway. A hyperlipidemia model was induced by a western diet(WD) in mice, and the mice were divided into a control group, a model group(WD), and low-, medium-, and high-dose ZXD groups. An adipogenesis model was induced in 3T3-L1 cells in vitro, and with forskolin(FSK) used as a positive control, low-, medium-, and high-dose ZXD groups were set up. Immunohistochemistry and immunofluorescence results showed that compared with the WD group, ZXD promoted the expression of UCP1 in white and brown adipose tissues, and also upregulated UCP1, CPT1β, PPARα, and other genes in the cells. Western blot analysis showed a dose-dependent increase in the protein expression of PGC-1α, UCP1, and PPARα with ZXD treatment, indicating that ZXD could promote the white adipose tissue browning/brown adipose tissue activation. Hematoxylin-eosin(HE) staining results showed that after ZXD treatment, white and brown adipocytes were significantly reduced in size, and the mRNA expression of ATGL, HSL, MGL, and PLIN1 was significantly upregulated as compared with the results in the WD group. Oil red O staining and biochemical assays indicated that ZXD improved lipid accumulation and promoted lipolysis. Immunohistochemistry and immunofluorescence staining for p-CREB revealed that ZXD reversed the decreased expression of p-CREB caused by WD. In vitro intervention with ZXD increased the protein expression of CREB, p-CREB, and p-PKA substrate, and increased the mRNA level of CREB. ELISA detected an increase in intracellular cAMP concentration with ZXD treatment. Molecular docking analysis showed that multiple active components in Alismatis Rhizoma and Atractylodis Macrocephalae Rhizoma could form stable hydrogen bond interactions with GLP-1R. In conclusion, ZXD promotes white adipose tissue browning/brown adipose tissue activation both in vivo and in vitro, and its mechanism of action may be related to the GLP-1R/cAMP/PKA/CREB pathway.

FRI392 Functional Interrelationship Between Oxytocin And Bone Morphogenetic Proteins In Ovarian Steroidogenesis By Granulosa Cells

Abstract Disclosure: K. Yamamoto: None. Y. Nakano: None. N. Iwata: None. Y. Soejima: None. A. Suyama: None. T. Hasegawa: None. F. Otsuka: None. Oxytocin (OXT) is a neuropeptide that is produced in the hypothalamus and is secreted from the posterior pituitary. OXT has diverse physiological roles mediated by the OXT receptor (OXTR), including not only stimulation of uterine contractions during labor and peripheral functions of milk ejection during lactation but also functions in pair bonding, maternal nurturing, and social behavior related to the action of OXT on the brain. It has been shown that OXT and OXTR are expressed in ovarian granulosa cells (GCs) in several species including humans. Secretion of OXT by bovine GCs is reported to be increased after the preovulatory luteinizing hormone surge, which is coincident with an increase in progesterone secretion and a decrease in secretion of estradiol and androgen, suggesting a functional interrelationship between OXT and ovarian steroidogenesis. It has been demonstrated that application of OXT to cultures of GCs from preovulatory follicles in bovines, marmoset monkeys, and humans led to increases in progesterone production. However, the precise molecular mechanism by which OXT modulates ovarian steroidogenesis in GCs remains unknown. Bone morphogenetic proteins (BMPs), which belong to the transforming growth factor beta (TGF-β) superfamily, play important roles in steroidogenesis and cellular proliferation in ovarian follicles via autocrine/paracrine fashion. BMP ligands and receptors are expressed in a cell-dependent manner in the ovary and have a pivotal role in female fertility. Throughout folliculogenesis, BMPs commonly inhibit follicle-stimulating hormone (FSH)-induced progesterone production, and oocyte-derived factors BMP-6 and BMP-15 are known to act on GCs through BMP receptors and suppress FSH-induced progesterone synthesis. To clarify the reproductive role of OXT in ovarian steroidogenesis and its functional interaction with BMPs, the effects of OXT on ovarian steroidogenesis were investigated by using primary culture of rat GCs and human granulosa KGN cells. Here we found that the OXT receptor was expressed in both rat GCs and human KGN cells and that OXT treatment significantly increased FSH- and forskolin (FSK)-induced progesterone production, but not estradiol production, by rat GCs and KGN cells, respectively. In accordance with the effects of OXT on progesterone production, OXT enhanced mRNA levels of CYP11A1 and HSD3B2 induced by FSK in KGN cells. Of note, OXT enhanced the phosphorylation of SMAD1/5/9 and the mRNA level of ID1 induced by BMP-15, but not those induced by BMP-6, in KGN cells. It was also revealed that OXT application upregulated the expression of BMPR2, a crucial type-II receptor of BMP-15, and enhanced the BMP-15-induced expression of inhibitory SMAD6 by KGN cells. Taken together, it was shown that OXT enhances ovarian progesterone production with upregulation of BMP-15 activity, leading to a fine-tuning of ovarian steroidogenesis. Presentation: Friday, June 16, 2023

Lipid modulation during IVM increases the metabolism and improves the cryosurvival of cat oocytes

This study investigated the time course of lipid accumulation during IVM and assessed the role of lipid modulators added during IVM on lipid content, nuclear maturation, oxidative stress, mitochondrial activity, gene expression, and cryosurvival of cat oocytes. First, the lipid content of immature COCs was compared to those subjected to different IVM duration times (24, 28, and 32 h). Then, the lipid content was investigated after the use of different lipid modulators [conjugated linoleic acid (CLA), forskolin (FSK), l-carnitine (LC)]. Subsequently, both the CONTROL group and MIX 18 (CLA+FSK+LC) were compared regarding nuclear maturation, mitochondrial activity, reactive oxygen 19 species (ROS), and glutathione (GSH) levels, to the expression of SDHA, GDF9, BMP15, ZAR-1, 20 PRDX1, SIRT1, and SIRT3 genes (normalized by ACTB and YWHAZ genes); and to vitrification and 21 post-warming viability assessment. When not using any lipid modulator, an increase (P < 0.05) in lipid content could be observed after 28 h of IVM. The MIX group showed the greatest (P < 0.05) reduction in oocyte lipid content after 28 h of IVM. No difference (P > 0.05) was observed in the MII rate in the CONTROL (45%) and MIX (41%) groups and in mitochondrial activity ((1.00 ± 0.35 A U vs 1.19 ± 0.14 A U). Although ROS and GSH levels were higher (P < 0.05) in MIX than in CONTROL, the redox balance (ROS/GSH) was greater (P < 0.05) in the latter (C:1.00 ± 0.20b vs M:0.26 ± 0.06 a A.U). The GDF9, HSP70, PRDX1, and SIRT1 transcripts were downregulated (P < 0.05) in MIX-oocytes, compared to the CONTROL. After vitrification, MIX (74%) presented a higher (P < 0.05) viability compared to control (53%). In conclusion, MIX can reduce the total lipid content and improve viability after cryopreservation, however, it seems to affect the oocyte metabolism in a way that still needs to be better understood in the cat biological model.

SAT269 Orexin Activates Adrenocortical Steroidogenesis With Upregulation Of BMP Signaling By Human H295R Cells

Abstract Disclosure: Y. Soejima: None. N. Iwata: None. R. Nishioka: None. M. Honda: None. Y. Nakano: None. K. Yamamoto: None. A. Suyama: None. F. Otsuka: None. Orexins are neuropeptides that express primarily in the hypothalamus and are composed of two isoforms, orexin A and orexin B. Orexin A binds to orexin receptor type 1 (OX1R) and type 2 (OX2R), while orexin B binds selectively to OX2R. Orexin has been reported to have important roles in various systems including sleep-wake regulation and feeding behavior in the central nervous system, whereas its receptors are also expressed in peripheral tissues including the endocrine system. The expression of orexin receptors and the effects of orexins on adrenocortical steroidogenesis have been shown in experiments using adrenocortical cells. Bone morphogenetic proteins (BMPs), which are members of the transforming growth factor (TGF)-β superfamily, also play functional roles in various endocrine tissues. In our previous experiments, we have revealed that BMP-6 enhances aldosterone production which is induced by angiotensin II through activation of mitogen-activated protein kinase (MAPK) signaling. We have also shown that peripheral clock genes have functional roles in modulating adrenocortical steroidogenesis by interacting with adrenocortical BMP system. However, the molecular mechanism of physiological effects of orexin and its interaction with BMP signaling remains unclear. In the present study, we investigated the effects of orexin on adrenal steroidogenesis using human adrenocortical H295R cells, by focusing on the interaction with adrenocortical BMP signaling. First, it was confirmed that both OX1R and OX2R were expressed in H295R cells. It was revealed that orexin A (10-300 nM) stimulation for 24 h directly increased mRNA levels of steroidogenic acute regulatory protein (StAR), cytochrome P450 family 11 subfamily B member 2 (CYP11B2) and cytochrome P450 family 17 subfamily A member 1 (CYP17A1) in a concentration-dependent manner. Orexin A further upregulated the expression of these steroidogenic enzymes in H295R cells co-treated with forskolin (FSK). Next, the interrelationship between orexin and BMP signaling was examined. Stimulation with BMP-6 readily activated Smad1/5/9 phosphorylation in H295R cells and the pretreatment with orexin A for 24 h enhanced the Smad1/5/9 phosphorylation induced by BMP-6. On the other hand, BMP-6 significantly suppressed the expression of OX1R but not that of OX2R. In our previous study, we have reported that BMP-6 upregulates the adrenocortical steroidogenesis via the IP3 and MAPK signaling in H295R cells, suggesting that orexin may have a pivotal role as a functional modulator of BMP-6. Collectively, it was revealed that orexin enhances adrenocortical steroidogenesis by regulating BMP signaling with a feedback system in human adrenocortical cells. Here, we will also discuss the relationship with BMP receptors and the effects on endogenous BMP expression. Presentation: Saturday, June 17, 2023

Reproductive toxicity of bisphenol A, at environmentally relevant concentrations, on ovarian redox balance, maturational response, and intra-oocyte signalling events in Labeo bata

Bisphenol A (BPA) is a widely used plastic monomer that potentially interferes with ovarian neuroendocrine, endocrine, and autocrine/paracrine factors, causing reproductive dysfunction. However, the influence of BPA on redox balance, estrogen receptor (ER) expression vis-à-vis meiotic cell cycle progression, and intra-oocyte signalling events has not been extensively investigated. The present study examines the impact of BPA on reproductive toxicity in female Labeo bata (Order Cypriniformes, Family Cyprinidae), a freshwater teleost preferred as a food fish in the Indian subcontinent. Our results show that while ovarian weight (gonadosomatic index, GSI) and dynamics of follicular growth undergo pronounced changes during the annual reproductive cycle, chronic BPA exposure at environmentally relevant concentrations promotes follicular atresia concomitant with reduced GSI during the spawning phase, the highest response being observed due to low-dose (0.1 μg/L, 0.438 nM) BPA exposure in vivo. Furthermore, BPA perturbation of ovarian StAR expression and ERα/ERβ homeostasis corroborates with elevated oxidative stress in BPA-treated ovary, FG follicles, and follicular cells. A sharp increase in ROS accumulation and nitric oxide (NO) levels in BPA-treated full-grown (FG) follicles coupled with loss of redox balance, elevated follicular cell death, and activation of apoptotic markers (caspase -8, -9, -3, Bax) indicate poor oocyte health and reproductive toxicity. Importantly, maturational steroid (MIS, 17,20β-P)-induced cyclin B-p34cdc2 activation and elevated GVBD (germinal vesicle breakdown) response require protein kinase A (PKA) inhibition and participation of Mos/MAPK- and cdc25-mediated signalling events. While the adenylate cyclase activator forskolin (FK) abrogates, priming with a PKA inhibitor (H89) promotes the meiotic G2-M1 transition, confirming the role of PKA in meiotic cell cycle progression in this species. Furthermore, the negative influence of BPA priming on 17,20β-P-induced oocyte maturation involves elevated PKAc phosphorylation (activation) and significant alteration in Mos/MAPK signalling, indicating derailed meiotic maturational competence and disrupted oocyte quality.

The Adenylyl Cyclase Activator Forskolin Increases Influenza Virus Propagation in MDCK Cells by Regulating ERK1/2 Activity.

Vaccination is the most effective method for preventing the spread of the influenza virus. Cell-based influenza vaccines have been developed to overcome the disadvantages of egg-based vaccines and their production efficiency has been previously discussed. In this study, we investigated whether treatment with forskolin (FSK), an adenylyl cyclase activator, affected the output of a cell-based influenza vaccine. We found that FSK increased the propagation of three influenza virus subtypes (A/H1N1/California/4/09, A/H3N2/Mississippi/1/85, and B/Shandong/7/97) in Madin-Darby canine kidney (MDCK) cells. Interestingly, FSK suppressed the growth of MDCK cells. This effect could be a result of protein kinase A (PKA)-Src axis activation, which downregulates extracellular signal-regulated kinase (ERK)1/2 activity and delays cell cycle progression from G1 to S. This delay in cell growth might benefit the binding and entry of the influenza virus in the early stages of viral replication. In contrast, FSK dramatically upregulated ERK1/2 activity via the cAMP-PKA-Raf-1 axis at a late stage of viral replication. Thus, increased ERK1/2 activity might contribute to increased viral ribonucleoprotein export and influenza virus propagation. The increase in viral titer induced by FSK could be explained by the action of cAMP in assisting the entry and binding of the influenza virus. Therefore, FSK addition to cell culture systems could help increase the production efficiency of cell-based vaccines against the influenza virus.