Introduction Endocannabinoid system (ECS) includes lipid messengers termed endocannabinoids (eCBs), their receptor (CBRs) targets type-1 (CB1) and type-2 (CB2) cannabinoid receptors, G-protein coupled receptor 55 (GPR55), transient receptor potential vanilloid type 1 channel (TRPV1) and a number of metabolic enzymes. ECS has a key-role in virtually all steps of female reproduction. To date, among the 4 main receptors, only 2 receptors have been extensively studied in the mammalian oocytes, i.e. CB1 and CB2, both modulated during meiotic maturation. The aim of this thesis was (a) to determine expression levels of all these CBRs and (b) to investigate their role in the process of mouse oocyte meiotic maturation. Methods Adult CD1 female mice were primed with 5 IU PMSG and sacrificed: (a) 44 h later to obtain GV oocytes, (b) 8 or 12h after hCG (5 IU) injection to obtain MI or MII oocytes. CBRs mRNA and protein contents were assessed by qRT-PCR and Western blot; CBRs localization by confocal microscopy. CB1, CB2 and GPR55 roles during meiotic resumption (germinal vesicle breakdown, GVBD) or maturation up to MI and MII stages were assessed by incubating oocytes in the presence/absence of receptor antagonists (SR1, SR2, and ML193, respectively). cAMP concentration was assessed by EIA kit; MI/MII spindle morphology by appropriate immunofluorescent antibodies. Experiments were repeated at least 3 times. Results Despite a significant decrease of CB1, CB2 and GPR55 mRNAs occurring after GVBD, CB2 and GPR55 protein contents increased significantly from GV to MI and MII. At GV, only CB1 was localized in the oolemma, although it disappeared at MI. TRPV1 (mRNA/protein) was always undetectable. When oocytes were in vitro matured with CB1 and CB2 antagonists, a significant delay of GVBD was recorded, sustained by higher intraoocyte cAMP concentration. Although CBRs antagonists did not affect polar body I emission nor chromosome alignment at metaphase I or II plates, ML193 impaired the formation of normal MI or MII spindles in about 70% of oocytes. Indeed, ML193-incubated oocytes showed a significant reduction of spindle length as compared with control. Conclusion In mouse oocytes, all the major eCB-receptors are differentially expressed during meiotic maturation. CB1 and CB2 have a prominent role in the control of meiosis resumption, while GPR55 could be involved in the assembly of MI and MII spindles. These findings offer potentially novel biomarkers involved in the physiological maturation of mouse oocytes and could be a starting point for investigating female fertility issues also in women.
The endocannabinoid system in female reproduction: characterization of major endocannabinoid-binding receptors expression and function during mouse oocyte meiotic maturation / DI NISIO, VALENTINA. - (2020 Jun 30).
|Titolo:||The endocannabinoid system in female reproduction: characterization of major endocannabinoid-binding receptors expression and function during mouse oocyte meiotic maturation.|
|Data di pubblicazione:||30-giu-2020|
|Citazione:||The endocannabinoid system in female reproduction: characterization of major endocannabinoid-binding receptors expression and function during mouse oocyte meiotic maturation / DI NISIO, VALENTINA. - (2020 Jun 30).|
|Appare nelle tipologie:||8.1 Tesi di dottorato|