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

Localization of cannabinoid receptor type 1 (CB1) immunoreactivity on mitochondrial membranes, at least their outer membranes distinctly, was detected in progesterone-producing cells characterized by mitochondria having tubular cristae and aggregations of lipid droplets in ovarian interstitial glands in situ of adult mice. Both immunoreactive and immunonegative mitochondria were contained in one and the same cell. Considering that the synthesis of progesterone is processed in mitochondria, the mitochondrial localization of CB1 in the interstitial gland cells suggests the possibility that endocannabinoids modulate the synthetic process of progesterone in the cells through CB1.

Localization of phosphatidylinositol 4-phosphate 5-kinase (PIP5K) α confined to the surface of lipid droplets and adjacent narrow cytoplasm in progesterone-producing cells of in situ ovaries of adult mice.

Phosphatidylinositol(4,5)bisphosphate (PI(4,5)P2) produced by phosphatidylinositol phosphate 5 kinase (PIP5K) plays not only as a precursor of second messengers in the phosphoinositide signal transduction, but also multiple roles influencing a variety of cellular activities. From this viewpoint, the present study attempted to localize PIP5Kα in the ovaries in situ of adult mice. PIP5Kα-immunoreactivity was confined to the surfaces of lipid droplets (LDs) and their adjacent cytoplasm in progesterone-producing cells of the interstitial glands, corpora lutea and theca interna. The LDs often contained membranous tubules/lamellae along their surfaces and within their interior whose membranes were continuous with those delineating LDs composed of a monolayer of phospholipids and were partially PIP5Kα-immunoreactive. Although granulosa cells of healthy-looking follicles were immunonegative, as the atresia progressed, PIP5Kα-immunoreactivity first appeared in sparsely dispersed dot forms in mural cells of the follicular epithelia, and then were dominant in almost all mural cells that remained after desquamation of the antral cells. The present study provides evidence suggesting that PI(4,5)P2 locally synthesized by PIP5K in LDs is involved in the lipid transfer between lipid droplets (LDs) and the endoplasmic reticulum, which eventually regulates ovarian progesterone production through control of multiple dynamic activities of LDs. It is also suggested that PIP5Kα and PI(4,5)P2 are implicated in the modulation of programmed cell death and/or acquiring the ability of progesterone production in some follicular cells surviving atresia.

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

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 ß are involved in the synthesis of an eCB 2-arachidonoylglycerol (2-AG). DGLα/ß 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 ß were weakly detected throughout the ooplasm in light microscopy for which the intracellular membranes of vesicles forming tiny scattered aggregates were responsible. Secondly, DGLß-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.

Different expression and subcellular localization of vesicular inhibitory amino acid transporter in ducts of major salivary glands: An in situ study in mice.

OBJECTIVE: The aim of this study was to clarify the mechanism of GABA (□-amino butyric acid)-signaling in the salivary glands by localization of vesicular inhibitory amino acid transporter, a key molecule in GABA-synthesis. DESIGN: Parotid, sublingual and submandibular glands of mice at various postnatal stages were examined in immuno-light and electron microscopy as well as immuno-blotting. RESULTS: Expression for vesicular inhibitory amino acid transporter was detected in parotid and sublingual glands of both sexes and female submandibular gland throughout postnatal development, while it was negligible in male submandibular glands at and after puberty. The expression in female submandibular glands attenuated after testosterone injection. The immunoreactivity was localized in striated ductal cells, but not acinar cells, in the salivary glands, and it occurred in association with intracellular and plasma membranes of the cells. It also occurred in myoepithelial and vascular smooth muscle cells. CONCLUSIONS: GABA-signaling was suggested to be a significant signaling pathway in salivary ductal cells, which was suppressed in male submandibular glands at and after puberty. The suppression in the submandibular duct was by testosterone. In addition, the participation of vesicular inhibitory amino acid transporter in GABA signaling through plasma membranes of the ductal cells was suggested. The significance of occurrence of the immunoreactivity in myoepithelial and smooth muscle cells remains to be further elucidated in terms of implication in GABA signaling.