The epithelial content of an estradiol-sensitive immunological marker (CVA) has been quantified by mixed haemagglutination on tissue sections from the vagina of neonatal mice exposed to different schedules of estradiol treatment.Daily administration of estradiol-17$ (5 ,ug/day) was especially efficient in elevating the CVA content when the hormone was administered during the first four days after birth.Following a single injection of estradiol-17,5 (5 ,ug in aqueous suspension) on one of the first five days of life, the vaginal epithelium reacted with a more vigourous CVA accumulation when challenged with estradiol at a later time.The possibility that this early effect of estradiol may involve other mechanisms than those operative in the estradiol action at later stages, is discussed.The effect was most pronounced for days 2 and 3.The cervicovaginal epithelium of adult mice has been shown to contain a specific cell product (CVA) with antigenic properties when injected into rabbits. By immunofluorescence and immunoferritin techniques the epithelial content of this substance was found to vary during the estrous cycle (24) and to increase after estradiol treatment of spayed mice (8). We have demonstrated CVA in fetuses and newborn mice (3). The highly sensitive indirect mixed haemagglutination method of TONDER et al. (21), was modified to allow a relative quantification of the epithelial CVA content in tissue sections. The present study was undertaken to get a more detailed information on the effect of estradiol on epithelial CVA-accumulation in neonatal stages in mouse. The duration of estradiol treatment and the age of the animals at the time of the first hormone injection were systematically varied. Another aspect was to try to find out if there was a critical period in neonatal life where estradiol might have effects influencing a later CVA-response to the hormone. MATERIALS AND METHODSAll mice used belonged to a randomly bred stock of the NMRI strain. They were fed a standard pellet diet and given water ad libitum. The day of birth was considered as day 1 of life. Animal treatmentIn the first series of experiments, animals were given subcutaneous injections of 5 pg estradiol-17,5 (Sigma) in 0.02 ml olive oil (experimental animals) or 0.02 ml olive oil only (control animals). The animals were divided into the following subgroups: A : Animals given one single injection of estradiol 24 hr before death, the animals were killed on day 2 to 8 after birth.B: Animals given injections of estradiol, 48 and 24 hr before death. The animals were killed on day 3-9 or day 14. 111

Regulation of intercellular junctional complexes is critical for the control of endothelial barrier function and dependent on cAMP‐mediated Rac1 activation. Recently, the exchange protein activated by cAMP (Epac1) has been shown to serve as a tonic stabilizer of the endothelial barrier. Here, we further elucidate the role of Epac1 in cAMP and Rac1‐mediated endothelial barrier regulation.Transendothelial‐electrical‐resistance (TER) measurements in newly generated immortalized myocardial endothelial cells derived from Epac1‐knockout (KO) mice revealed significant reduction in the baseline TER, when compared to wild‐type (WT) cells, indicating that Epac1 is required for maintenance of endothelial barrier function. This effect was associated with fragmented VE‐cadherin staining and reduced localization of tight junctional proteins ZO‐1 and occludin along cell borders. In WT‐cells, application of forskolin (F) and rolipram (R) resulted in increased TER associated with linearization and augmentation of VE‐cadherin immunostaining at adherens junctions. In contrast, Epac1‐KO‐cells did not respond to F/R treatment indicating that Epac1 is crucial for cAMP‐mediated endothelial barrier stabilization. In comparison to WT‐cells, Epac1‐KO‐cells revealed significantly increased protein levels of Rac1, whereas Rac1 basal activity was not enhanced, indicating a defect in Rac1 activation. However, in Epac1‐KOcells, cAMP‐mediated Rac1 activation was not abolished and the basal cAMP‐concentration was significantly increased. Additionally, in Epac1‐deficient cells F/R‐mediated increase in cAMP levels was less compared to WT cells. Taken together, our data show that Epac1 is crucial for cAMP‐mediated barrier stabilization by mechanisms which at least in part are independent of Rac1 regulation.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.