In situ localization of diacylglycerol lipase α and β producing an endocannabinoid 2‐arachidonoylglycerol and of cannabinoid receptor 1 in the primary oocytes of postnatal mice

Abstract

In order to understand the mechanism of the endocannabinoid (eCB) signal, which has so far been shown to work in oocyte genesis and maturation, it is critical to clarify detailed localization of the eCB synthesizing enzyme molecules as well as receptors for eCBs in oocytes in the ovary in situ. For this purpose, diacylglycerol lipase (DGL) α and β are involved in the synthesis of an eCB 2-arachidonoylglycerol (2-AG). DGLα/β and the cannabinoid receptor 1 (CB1) for 2-AG were shown to be localized to the primary oocytes of postnatal mice using immuno-light and electron microscopy. It was found that two types of localization existed: first, immunoreactivities for DGLα and β were weakly detected throughout the ooplasm in light microscopy for which the intracellular membranes of vesicles forming tiny scattered aggregates were responsible.Secondly, DGLβ-immunoreactivity was distinctly confined to the nuage of Balbiani bodies and small nuage-derivative structures; both amorphous materials and membranes of vesicles were responsible for their localization. On the other hand, the weak immunoreactivity for CB1 was localized in a pattern similar to the first one for DGLs, but not found in a pattern for the Balbiani nuage. Two routes of functional exertion of 2-AG synthesized by DGLs were suggested from the two types of localization: one was that the eCB synthesized at all the sites of DGLs is released from the oocytes and exerts paracrine or autocrine effects on adjacent intra-ovarian cells as well as the oocytes themselves. The other was that the eCB synthesized within the nuage was involved in the modulation of the posttranscriptional processing of oocytes. Owing to the failure in the detection of CB1 in the Balbiani nuage, however, the validity of the latter possibility remains to be elucidated.

Mitochondrial Localization of CB1 in Progesterone-producing Cells of Ovarian Interstitial Glands of Adult Mice

“…In our recent study, 6 the expression of CB1 in the ovary of adult female mice was confirmed with a band of 51 kDa in Western blots, whose size is compatible with that reported in mouse brain with the same antibody. 7…”

“…In total, 10 female ICR mice at a stage of postnatal 8 weeks were purchased from the National Laboratory Animal Center, Bangkok, Thailand. As the expression bands for CB1 at its authentic molecular size and no differences in expression of the molecule among the four estrous stages were already confirmed in Western blots of adult ovaries of the same resource in our previous study, 6 mice at the diesterus stage were used in this study.…”

“…The specimen preparation for routine immuno-DAB light and electron microscopy, including the treatment of adult mice and the control with antigen absorption, was done in the same way as that in our previous study. 6…”

“…In view of this point, our recent study has clarified for the first time the detailed localization of CB1, DGLα, and DGLβ in oocytes in situ of mouse ovary. 6 This study was thus undertaken as the second to clarify in immuno-light and electron microscopy the expression and localization of these eCB signaling molecules in various ovarian cells in situ of adult mice. During this attempt, CB1 immunoreactivity was found on mitochondria containing tubular cristae in steroidogenic cells characterized by mitochondria having tubular cristae and aggregations of lipid droplets in the interstitial gland.…”

Expression and Subcellular Localization of Endocannabinoid System in Primary Interstitial Glandular Cells In Situ of Mice Ovary

“…There are many studies for effects of the eCB system on the ovarian events such as folliculogenesis, oogenesis and oocyte maturity until fertilization and preimplantation of embryo [9] and presents the eCB system immunoreactivities by several authors to assume the results of eCB, which localized on the oocyte maturity after restart of meiosis I [8,10,11]. In my recent study, the novel knowledge showed the expression and localization of CB1, DAGL-α and DAGL-β that it was first reported in oocytes of mouse ovary [12]. Additionally, the eCB signaling molecules including CB1, CB2 and enzymes for degradation as well as FAAH and MGLL were studied to locate in the follicle, corpus luteum and corpus albicans of human ovary [2,8].…”