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To investigate the anti-angiogenic effects of photodynamic therapy with verteporfin in a rabbit model of corneal neovascularization.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.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% (p0.01) 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.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.

Plasma melatonin, thyroid‐stimulating hormone (TSH) and body temperature were measured simultaneously and continuously before and after the sleep‐wake cycle was shifted in 4 healthy males and changes in the circadian rhythm itself and in the phase relationship among these circadian rhythms were determined. Normal sleep‐wake cycle (sleep hours: 2300–0700) was delayed by 10 h (sleep hours: 0900–1700) during the experiment. Even after this shift the typical melatonin rhythm was maintained: low during daytime and high during night. The melatonin rhythm was gradually delayed day by day. The TSH rhythm was also maintained fundamentally during 3 consecutive days of altered sleep‐wake cycle. The phase was also delayed gradually but remarkably. The daily rhythm of body temperature was changed by the alteration of sleep‐wake cycle. The body temperature began to decrease at the similar clock time as in the control but the decline during night awake period was less steep and the lowered body temperature persisted dur...

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‐h...

We have isolated the cDNAs encoding the GnRH1 and GnRH2 precursors, respectively, from bullfrog (Rana catesbeiana) brain. The first cDNA consists of 648 bp and contains an open-reading frame of 270 nucleotides, encoding the bullfrog GnRH1 precursor. The second cDNA consists of 1053 bp and contains an open-reading frame of 255 nucleotides, encoding the bullfrog GnRH2 precursor. Both types of bullfrog GnRH precursor have a similar molecular architecture as observed in other GnRH precursors, consisting of a signal peptide, followed by the GnRH decapeptide, a conserved carboxy-terminal amidation and proteolytical processing site, and a GnRH-associated peptide (GAP). In addition, we have identified a third cDNA, containing 24 additional nucleotides in its GAP-coding region. Genomic PCR and sequence analysis confirmed that this cDNA represents an alternative splice variant of the bullfrog GnRH2-precursor pre-mRNA. The bullfrog GnRH1 precursor exhibits 60% and less than 40% amino acid identity to its Xenopus and mammalian counterparts, respectively, whereas the bullfrog GnRH2 precursor displays 50% to 60% amino acid identity to that of its nonmammalian counterparts, but shares only 25% amino acid identity with its mammalian counterparts. Northern blot analysis revealed a single GnRH1-precursor mRNA species of ∼0.75 kilobases, expressed in bullfrog forebrain, and a single GnRH2-precursor mRNA species of ∼1.1 kilobases, expressed in bullfrog midbrain/hindbrain. Furthermore, both bullfrog GnRH-precursor mRNAs exhibited a differential spatiotemporal expression pattern. Genomic Southern blot analysis indicated that both bullfrog GnRH genes are present as single copy genes. This is the first report on the molecular cloning of a GnRH2-precursor cDNA from an amphibian species. In addition, we present data showing that alternative splicing is utilized to generate different GnRH2-precursor mRNAs. J. Exp. Zool. 289:190–201, 2001. © 2001 Wiley-Liss, Inc.

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 iust 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 m and 0.6 m, respectively. The present study suggests a fundamental structural change occurring on the oocyte surface during maturation.

Changes in the levels of prostaglandin F2α (PGF2α) and E2 (PGE2) in culture medium during in vitro ovulation of Rana dybowskii follicles were examined. The ovulation was induced by frog pituitary homogenate (FPH) or TPA (12‐O‐tetradecanoylphorbol‐13‐acetate, a protein kinase activator) and the levels of PGs were measured by radioimmunoassay. When the ovarian follicles were cultured, only a few oocytes were ovulated by 12 h, but half of them were ovulated by 24 h in response to FPH, whereas around 30% of oocytes were ovulated by 12 h and maximum ovulation (around 50%) occurred by 24 h in response to TPA. Without any stimulation (control), no ovulation occurred. TPA elevated the level of PGF2α to high levels when compared to control (basal levels), but the increase by FPH was less evident. Likewise, the levels of PGE2 increased markedly in response to TPA, but rather decreased by FPH treatment. Interestingly, PGF2α induced ovulation but PGE2 suppressed FPH‐ or PGF2α‐induced oocyte ovulation. Basal levels of...

Changes in plasma and pituitary levels of two gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), in male and female Korean frogs (Rana dybowskii and Rana nigromaculata) were examined. Plasma levels of LH and FSH were measured by radioimmunoassay using antibodies raised against bullfrog LH and FSH. In male and female R. dybowskii, plasma concentrations of LH were low in early hibernation (October-January) and increased to high levels by late hibernation, when breeding occurs (late February-early March). Plasma FSH levels were also higher in the breeding period than during hibernation in male and female animals, but absolute levels of FSH were much lower than those of LH. In females, pituitary LH levels were higher in early hibernation, whereas pituitary FSH in females and pituitary LH and FSH in males changed little during hibernation. Plasma LH levels of frogs having uterine eggs or in amplexus were much higher (25 ng/ml) than those of frogs with unovulated follicles (December) (8 ng/ml) or frogs that had already spawned (11 ng/ml). In R. nigromaculata, plasma LH and FSH levels of females collected during hibernation (October-May) were relatively low; however, following hibernation, plasma LH and FSH levels were markedly elevated for a short period. Thus, these animals exhibit a clear gonadotropin surge prior to ovulation and spawning. Soon after spawning, however, gonadotropin levels dropped to basal levels. Following spawning in females, levels of FSH increased steadily and rapid follicle growth occurred until August. By September, FSH had dropped to basal levels. In males, a sharp elevation of LH and FSH levels occurred during the short breeding period followed by a second increase in August, when early stages of spermatogenesis were evident.

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 ( 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.

Earlier studies showed that the predominant steroid (estradiol [E 2 ], testosterone [T], progesterone [P 4 ]) secreted by in vitro cultured amphibian ( Rana nigromaculata ) ovarian follicles varied with their size. E 2 was mainly produced by medium-sized follicles, T by intermediate-sized follicles, and P 4 by the largest follicles. Experiments were carried out to ascertain whether the activities of steroidogenic enzymes changed during follicle development and were responsive to gonadotropic stimulation. Enzyme activities were measured indirectly by monitoring conversion of exogenously added substrates to products during in vitro culture of isolated follicles. Different stage follicles were cultured in the presence or absence of frog pituitary homogenate (FPH, 0.1 pituitary/2 ml) and/or various steroid precursors (25-200 ng/2 ml). Amounts of E 2 , T, androstenedione (AD), 17α-hydroxyprogesterone (17α-OHP 4 ), or P 4 secreted into the medium were measured by RIA. Exogenous pregnenolone (P 5 ) was converted to P 4 by all types of follicles in a dose-dependent manner in the absence of FPH. Addition of FPH markedly enhanced medium levels of P 4 in all sized follicles. Highest levels of P 4 were presented in cultures containing the largest follicles. Such follicles were much less efficient than intermediate follicles in metabolizing P 4 to AD or T. EPH suppressed conversion of exogenous 17α-OHP 4 but not androstenedione to testosterone by the largest follicles. Exogenous T was converted to E 2 only by medium-sized follicles and FPH had little or no stimulating or inhibiting effect on this process in either medium- or intermediate-sized follicles. Taken together, the data suggest that (1) the transition from E 2 to T secretion (Class III to IV) is associated with a loss of aromatase activity, (2) the transition from T to P 4 production (Class IV to V) is correlated with a decrease in the activity of enzymes which metabolize P 4 , and (3) activities of steroidogenic enzymes varied in their responsiveness to EPH stimulation.

Cadmium (Cd) is one of the environmental pollutants affecting various tissues and organs including testis. Harmful effect of Cd in testis is known to be germ cell degeneration and impairment of testicular steroidogenesis. Animals treated with high doses of Cd (0.2 and 0.3 mg/100 g BW) showed a significant decrease in serum testosterone (T) level, but a significant induction of testicular lipid peroxidation levels. TUNEL assay showed that low doses of Cd (0.13 and 0.15 mg/100 g BW) exhibited typical characteristics of apoptosis while high doses of Cd caused more necrosis than apoptosis. In contrast, supplementation with ascorbic acid reduced testicular lipid peroxidation levels. Ascorbic acid supplementation restored testicular 3β-hydroxysteroiddehydrogenase (HSD) and 17β-HSD enzyme activities, 3β-HSD and cytochrome P450 side chain cleavage (P450scc) mRNA levels and serum T concentration to normal in Cd-administered rats. Moreover, administration of ascorbic acid prevented germ cell apoptosis as demonstrated by the reduced number of TUNEL-positive cells in germinal epithelium and inhibited Cd-induced necrosis. These results indicate that ascorbic acid have protective roles in vivo on the Cd-induced overall testicular damage including impaired steroidogenesis and germ cell death possibly through scavenging the reactive oxygen species generated by Cd administration.

Recently, we identified three types of non-mammalian gonadotropin-releasing hormone receptors (GnRHR) in the bullfrog (designated bfGnRHR-1-3), and a mammalian type-II GnRHR in green monkey cell lines (denoted gmGnRHR-2). All these receptors responded better to GnRH-II than GnRH-I, while mammalian type-I GnRHR showed greater sensitivity to GnRH-I than GnRH-II. In the present study, we designed new GnRH-II analogs and examined whether they activated or inhibited non-mammalian and mammalian type-II GnRHRs. [D-Ala6]GnRH-II, with D-Ala substituted for Gly6 in GnRH-II, increased inositol phosphate (IP) production in cells stably expressing non-mammalian GnRHRs more effectively than native GnRH-II. However, it exhibited lower activity for mammalian type-I GnRHR than GnRH-I itself. Trptorelix-1, a GnRH-II antagonist, inhibited GnRH-induced IP production in cells expressing non-mammalian GnRHRs more effectively than Cetrorelix, a GnRH-I antagonist. Trptorelix-1, however, had lower potency for mammalian type-I GnRHR than Cetrorelix. Ligand-receptor binding assays revealed that [D-Ala6]GnRH-II and Trptorelix-1 have higher affinities for non-mammalian GnRHRs but lower affinities for mammalian type-I GnRHR than GnRH-II and Cetrorelix, respectively. Moreover, [D-Ala6]GnRH-II and Trptorelix-1 had a higher affinity for gmGnRHR-2 than GnRH-II and Cetrorelix, respectively. These results indicate that [D-Ala6]GnRH-II and Trptorelix-1 are highly effective agonist and antagonist, respectively, for non-mammalian and type-II mammalian GnRHRs.

We previously demonstrated the presence of three distinct types of the gonadotropin-releasing hormone receptor (GnRHR) in a bullfrog (denoted bfGnRHR-1, bfGnRHR-2, and bfGnRHR-3). The bfGnRHRs exhibited differential tissue distribution and ligand selectivity. In the present study, we demonstrated the desensitization and internalization kinetics of these receptors in both transiently-transfected HEK293 cells and retrovirus-mediated stable cells. The time-course accumulation of the inositol phosphate in response to GnRH revealed that bfGnRHR-1 and -2 were rapidly desensitized, whereas bfGnRHR-3 was slowly desensitized. A comparison of the internalization kinetics revealed the most rapid rate and highest extent of internalization of bfGnRHR-2 among the three receptors. Interestingly, the mechanisms that underlie the receptor internalization appear to differ from each other. Internalization of bfGnRHR-1 was dependent on both dynamin and beta-arrestin, whereas those of bfGnRHR-2 and -3 were dependent on dynamin, but not on arrestin. These results, therefore, suggest that differential regulatory mechanisms for desensitization and internalization of the GnRHR are involved in diverse cellular and physiological responses to GnRH stimulation.

Previously, we observed that 70-kDa ribosomal protein S6 kinase (p70(s6k)) plays an essential role during the early phase of oocyte maturation in Rana dybowskii. To investigate further the early signal transduction components involved in this process, the possible role of phosphatidylinositol-3 kinase (PI3 kinase) during oocyte maturation was examined. Progesterone-induced oocyte maturation was significantly inhibited by wortmannin and LY294002, specific inhibitors of PI3 kinase. In contrast, protein kinase C activator 12-0-tetradecanoylphorbol-13-acetate-induced oocyte maturation was not inhibited by wortmannin. Protein synthesis was also significantly suppressed by wortmannin treatment during oocyte maturation. Moreover, PI3 kinase inhibitor suppressed progesterone-induced phosphorylation of S6 kinase in a dose-dependent manner. Likewise, PI3 kinase inhibitors significantly inhibited the phosphorylation of mitogen-activated protein (MAP) kinase which was increased during oocyte maturation. Finally, progesterone-induced H1 kinase activity was also inhibited by PI3 kinase inhibitors in a dose-dependent manner. Taken together, these results suggest that PI3 kinase is an initial component of the signal transduction pathway which precedes p70(s6k), MAP kinase, and MPF production during progesterone-induced maturation of amphibian oocyte.

Seasonal changes in testicular structure and function in three species of Korean frogs ( Rana nigromaculata, R. rugosa, and R. dybowskii ) having different breeding seasons and habitats were examined throughout the year. R. nigromaculata live in rice fields and breed in early May. Their gonadosomatic index (GSI) and testis weight decreased slightly from May until July, but increased markedly from August to high levels through December. The diameter (cross-sectional area) of seminiferous tubules changed little from January until August, increased sharply from September through October, and decreased thereafter. In seminiferous tubules, the number of primary spermatogonia (I SPG) was low from January–March, increased from April to maximum levels by May–June, and decreased subsequently. The number of spermatids (SPT) was highest from November to March, decreased to nondetectable levels in May, and increased markedly from September to November. Spermiation was most active during March and April. R. rugosa, which live in streams and breed during May–June, exhibited no changes in GSI, testis weight, or seminiferous tubule size throughout the year. The number of I SPG was high during May–August and that of secondary spermatogonia (II SPG) was highest in August. The number of SPT increased to high levels in November–December. Active spermiation occurred from January to April in this frog. In R. dybowskii, which live in the mountains and breed from late February to early March, the number of I SPG gradually increased from April through August; however, essentially no other spermatogenic cells were observed from January to July. A marked increase in early spermatogenic cells appeared during August–September and was followed by an increase in SPT from November to December. From December to March the number of spermatozoa increased and spermiation occurred. In general, testicular testosterone levels were high in the winter and low in summer in all three species, and positively correlated with the number of interstitial cells and the size of their nucleus.

Experiments were carried out at different times of hibernation to ascertain whether prostaglandin is produced by Rana ovarian follicles during gonadotropin- or 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced in vitro ovulation. Ovarian fragments were cultured in amphibian Ringer in the presence or absence of frog pituitary homogenate (FPH, 0.05 gland/ml) or TPA (1 or 10 microM). After various periods of culture, incidence of ovulation was determined and prostaglandin F2 alpha (PGF2 alpha) accumulated in culture medium was measured by radioimmunoassay. FPH and TPA increased PGF2 alpha levels in medium in a dose-dependent manner. The time course of PGF2 alpha secretion and ovulation by FPH or TPA treatment varied during the hibernation period. In early-hibernation, FPH stimulated neither PGF2 alpha secretion nor ovulation while TPA stimulated PGF2 alpha secretion, although it failed to induce ovulation. In mid-hibernation, FPH and TPA effectively stimulated PGF2 alpha secretion and ovulation, but both events took place several hours later than those observed in late-hibernation. Some fragments obtained in mid-hibernation and most obtained in late-hibernation spontaneously produced PGF2 alpha in vitro without FPH or TPA treatment and in some instances spontaneously ovulated. Furthermore, FPH or TPA increased PGF2 alpha levels further or accelerated the time course of secretion by such fragments. In late-hibernation, PGF2 alpha secretion induced with FPH or TPA increased simultaneously with or later than onset of ovulation. Exogenous cAMP (2.5 mM) or 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7, 100 microM), a PKC inactivator, markedly suppressed FPH- or TPA-stimulated PGF2 alpha secretion and ovulation in mid-hibernation. Indomethacin (IM, 5 micrograms/ml) strongly suppressed TPA- or FPH-stimulated PGF2 alpha production by fragments but its effect on ovulation varied among animals and at different times. IM suppressed ovulation of some ovarian fragments obtained in mid-hibernation, but failed to suppress hormone-induced ovulation in late-hibernation. Cycloheximide (5 micrograms/ml) and actinomycin D (1 microgram/ml) effectively suppressed FPH-stimulated PGF2 alpha production and ovulation, whereas actinomycin D reduced but failed to significantly suppress TPA-induced PGF2 alpha production in mid-hibernation. In general, effects of ovulation inhibitors exhibited strong seasonal variations and were less efficient as the breeding season approached. Taken together, the data suggest that elevated levels of PGF2 alpha are associated with spontaneous and hormone-induced ovulation, and PKC mediates gonadotropin induction of PGF2 alpha but not steroid synthesis in Rana ovaries.

Previous reports suggested that Na,K-ATPase activity and Na+-pump capacity decrease with senescence in left atrial myocardium of F344 rats. Current experiments were designed to determine if this reduction in the Na+-pump affects free intracellular Na+ levels. Mean intracellular Na+ ion activity (aiNa) was measured with Na-selective microelectrodes in left atrial muscle isolated from hearts of 4-, 14- and 25-month-old F344 rats. Preparations were stimulated randomly at frequencies between 0 and 12 h. There were no age-associated differences in a iNa measured at any frequency or in the decay of Na+ activity following discontinuation of electrical stimulation. These data indicate that the aging-related decline in Na,K-ATPase does not result in elevated aiNa even at extremely high stimulation frequencies, thus suggesting that other routes of Na+ influx and efflux are also altered in atrial muscle.