ABSTRACT
Increasing evidences showed that ovulatory dysfunction, possibly caused by luteinized unruptured follicular follicle syndrome (LUFS), is one of the reasons for endometriosis-related infertility. The present study was conducted to explore the potential effect of elevated progesterone in follicular fluid (FF) on ovulation in endometriosis. A prospective study including 50 ovarian endometriosis patients and 50 control patients with matched pairs design was conducted with alterations in FF and peritoneal fluid (PF) components identified by metabolomics analyses and differentially expressed genes in granulosa cells (GCs) identified by transcriptome analysis. Patients with endometriosis exhibited a significantly higher progesterone level in serum, FF, and PF. Granulosa cells from endometriosis patients revealed decreased expression of HPGD, COX-2, and suppressed NF-ĸB signaling. Similarly, progesterone treatment in vitro downregulated HPGD and COX2 expression and suppressed NF-ĸB signaling in granulosa tumor-like cell line KGN (Bena Culture Collection, China) and primarily cultured GCs, as manifested by decreased expressions of IL1R1, IRAK3, reduced pIĸBα/IĸBα ratio, and nucleus translocation of p65. On the contrary, TNF-α treatment increased expression of IL1R1, IRAK3, pIĸBα, p65, and HPGD in GCs. One potential p65 binding site was identified in the promoter region of HPGD by chromatin immunoprecipitation. In conclusion, we found that intrafollicular progesterone might downregulate HPGD and COX-2 in GCs via suppressing the NF-ĸB signaling pathway, shedding light on the mechanism underlying the endometriosis-related ovulatory dysfunction.
Subject(s)
Endometriosis , Infertility, Female , Female , Humans , Progesterone/pharmacology , Progesterone/metabolism , Follicular Fluid/metabolism , Endometriosis/genetics , Endometriosis/metabolism , Cyclooxygenase 2/genetics , Cyclooxygenase 2/metabolism , NF-kappa B/genetics , NF-kappa B/metabolism , Prospective Studies , Granulosa Cells/metabolism , Infertility, Female/metabolismABSTRACT
Phosphodiesterase (PDE)-mediated reduction of cyclic adenosine monophosphate (cAMP) activity can initiate germinal vesicle (GV) breakdown in mammalian oocytes. It is crucial to maintain oocytes at the GV stage for a long period to analyze meiotic resumption in vitro. Meiotic resumption can be reversibly inhibited in isolated oocytes by cAMP modulator forskolin, cAMP analog dibutyryl cAMP (dbcAMP), or PDE inhibitors, milrinone (Mil), Cilostazol (CLZ), and 3-isobutyl-1-methylxanthine (IBMX). However, these chemicals negatively affect oocyte development and maturation when used independently. Here, we used ICR mice to develop a model that could maintain GV-stage arrest with minimal toxic effects on subsequent oocyte and embryonic development. We identified optimal concentrations of forskolin, dbcAMP, Mil, CLZ, IBMX, and their combinations for inhibiting oocyte meiotic resumption. Adverse effects were assessed according to subsequent development potential, including meiotic resumption after washout, first polar body extrusion, early apoptosis, double-strand DNA breaks, mitochondrial distribution, adenosine triphosphate levels, and embryonic development. Incubation with a combination of 50.0 µM dbcAMP and 10.0 µM IBMX efficiently inhibited meiotic resumption in GV-stage oocytes, with low toxicity on subsequent oocyte maturation and embryonic development. This work proposes a novel method with reduced toxicity to effectively arrest and maintain mouse oocytes at the GV stage.
Subject(s)
1-Methyl-3-isobutylxanthine/pharmacology , Bucladesine/pharmacology , Cell Nucleus/metabolism , Meiosis/drug effects , Oocytes/metabolism , Phosphodiesterase Inhibitors/pharmacology , Animals , Apoptosis/drug effects , Cell Nucleus/drug effects , Cells, Cultured , Colforsin/pharmacology , Cyclic AMP/metabolism , DNA Breaks/drug effects , Embryonic Development/drug effects , Female , Mice , Mice, Inbred ICR , Oocytes/drug effectsABSTRACT
In our present work, seven conotoxins and conopeptides were cloned from four cone snail species based on the M-superfamily signal peptides. Among them, two conopeptides, Vt3.1 and Vt3.2, showed unusual sequence characteristics. Both of them contained two cysteines that are separated by just one non-cysteine residue. In vitro, the chemically synthesized Vt3.1 formed dimers with different intermolecular disulfide linkages. Only the dimer with crossed disulfides showed bioactivity when injected into the intraventricular region of mice brains. Therefore, Vt3.1 and Vt3.2 represent a new group of conopeptides that form disulfide-crosslinked dimers in vitro and probably in vivo.
