Wook-Bin Im

Experimental Inhibition of Corneal Neovascularization by Photodynamic Therapy with Verteporfin

Purpose: To investigate the anti-angiogenic effects of photodynamic therapy with verteporfin in a rabbit model of corneal neovascularization. Methods: One week after suturing, the localization of verteporfin in the neovascularized cornea was examined through fluorescent microscopy 1 hr after administration. Rabbits were treated with one or two times of photodynamic therapy with verteporfin at 1-week intervals. Analysis of corneal neovascularization was performed by biomicroscopic and histological examinations. Results: Fluorescent microscopy showed green fluorescence in the vascular walls and interstitial tissue of the corneal stroma. The mean percentages of neovascularized corneal area at 3 days, 1 week, and 2 weeks after one time of photodynamic therapy were 90.3% ± 3.5%, 71.6% ± 6.2%, and 43.6% ± 15.1% in treated eyes and 96.4% ± 1.9% (p = 0.10), 88.6% ± 4.6% (p = 0.01), and 76.8% ± 4.4% (p < 0.01) in control eyes, respectively. The mean percentages 3 days, 1 week, and 2 weeks after two times of photodynamic therapy were also significantly lower in treated eyes compared with control eyes. In quantitative histological examination at 1 and 2 weeks after therapy, treated eyes showed significantly less neovascular area and number of vessels than control eyes. Conclusions: Photodynamic therapy with verteporfin is a safe and useful procedure to reduce experimental corneal neovascularization and can be used to inhibit angiogenesis in the cornea.

DIFFERENTIAL EFFECTS OF GONADOTROPIN AND ORTHOVANADATE ON OOCYTE MATURATION, OVULATION, AND PROSTAGLANDIN SYNTHESIS BY RANA OVARIAN FOLLICLES IN VITRO

Effects of gonadotropin and sodium orthovanadate on oocyte maturation, ovulation, prostaglandin, and progesterone synthesis were examined during in vitro culture of Rana ovarian follicles obtained from hibernating animals. Frog pituitary homogenates (FPH, 0.05 gland/ml) effectively induced oocyte maturation (germinal vesicle breakdown, GVBD) and ovulation (> 90%) in ovarian follicles obtained in mid-hibernation (mid-December through late January). In contrast, orthovanadate induced a limited amount (< 45%) of ovulation of some oocytes without concomitant induction of maturation during mid-hibernation. In late hibernation (February to early March), considerable spontaneous maturation and ovulation occurred in cultured ovarian follicles. During this period both FPH (0.0005-0.05 gland/ml) or orthovanadate (0.01-1 mM) treatment markedly increased the incidence of ovulation and accelerated the onset of ovulation in a dose dependent manner. When examined after 12 h of culture, few (< 5%) oocytes ovulated in response to orthovanadate had undergone GVBD whereas most (> 90%) of those ovulated in response to FPH had matured. However, the majority of oocytes ovulated (72%) within 6 h of exposure to FPH had intact GVs, indicating that GVBD is also not a prerequisite for ovulation induction in response to FPH. Orthovanadate stimulated PGF2 alpha in a dose dependent manner but failed to stimulate progesterone production whereas FPH stimulated secretion of both PGF2 alpha and progesterone. The amount and time course of PGF2 alpha secretion in response to orthovanadate were similar to results produced with FPH stimulation. Treatment of PGF2 alpha to follicles obtained in late hibernation also accelerated the ovulation of the oocytes. Taken together, the data suggest that orthovanadate enhanced ovulation of immature oocytes was mediated via enhanced PGF2 alpha production and that oocyte maturation is not essential or prerequisite for in vitro oocyte ovulation in Rana.

Visualization of progesterone binding to plasma membrane of xenopus oocytes

We have previously shown that oocyte maturation is induced by an immobilized progesterone, progesterone‐3‐carboxymethyloxime ‐ bovine serum albumin conjugate (P‐BSA) in Rana dybowskii. In this study, we confirmed the maturation inducing activity of P‐BSA on Xenopus oocyte and examined the binding character of the immobilized progesterone on the surface of Xenopus oocytes after removal of the vitelline layer. P‐BSA induced maturation of Xenopus oocytes but E‐BSA failed to do so as observed in Rana. Binding of the immobilized progesterone, fluorescein isothiocyanate‐labeled progesterone‐3‐O‐carboxymethyloxime‐BSA (P‐BSA‐FITC) on the devitellined oocytes surface was examined by fluorescence confocal microscopy. The binding affinity of P‐BSA‐FITC to the devitellined oocyte was higher than that of estrogen‐BSA‐FITC (E‐BSA‐FITQ or testo‐sterone‐BSA‐FITC (T‐BSA‐FITC). The binding disappeared in the presence of excess free progesterone but not in the presence of free estrogen. Maximum binding occurred after two‐hours of incubation with P‐BSA‐FITC at pH 7.5. Stronger binding occurred in oocytes at stage VI than stage IV, and in vitro treatment of hCG enhanced the binding. Taken together, these results suggest that a specific receptor for progesterone exists on the plasma membrane of Xenopus oocytes and that progesterone acts initially on this putative receptors and triggers generation of membrane‐mediated second messengers during the early stage of oocyte maturation in amphibians.

Ultrastructure of the Follicular Oocyte Surface in Rana dybowskii

Rana ovarian follicles consist of oocyte, vitelline envelope, granulosa cells, and theca/epithelial layer. Using scanning electron microscopy, the surface structure of each follicular component was investigated. Changes in oocyte surface during oocyte maturation were also examined. Theca/epithelial layer was almost transparent and some blood vessels and granulosa cells were observed underneath in intact follicle. The number of granulosa cells was estimated to be 6700–7200 per oocyte. The granulosa cells partially overlapped each other and their microvilli penetrated the vitelline membrane via holes present in the vitelline envelope and seemed to be linked to oocyte microvilli. After removal of the vitelline envelope by microforcep, oocyte microvilli were observed on the surface of the devitellined oocyte. The oocyte microvilli formed partial clusters on the surface of white spot area which appears just before germinal vesicle breakdown (GVBD), whereas they were evenly distributed in other areas. The microvilli became shorter and less dense with oocyte maturation. The lengths of oocyte microvilli in the immature and mature oocyte were 1.5 μm and 0.6 μm, respectively. The present study suggests a fundamental structural change occurring on the oocyte surface during maturation.