When you look at the vitro follicle incubation that have radiolabeled steroid precursors

When you look at the vitro follicle incubation that have radiolabeled steroid precursors
Fish and you can testing

Inside spawning season (later booleaf wrasse were caught from the link and range in coastal waters around the Fisheries Lookup Laboratory, Kyushu College and you can gone to live in this new research. Seafood have been stored in five hundred-litre fiberglass tanks that have filtered seawater, significantly less than absolute time-size and you can liquid heat, and you may provided krill and you may real time hermit crab once a day. Immediately after guaranteeing each and every day spawning, cuatro–6 lady seafood (fat – grams, full duration 11step 3–159 mm) had been tested within , , , and you can hour. Seafood were anesthetized having dos-phenoxyethanol (three hundred ppm), and you may bloodstream samples was indeed compiled on caudal vessel playing with syringes installing having twenty five-grams to have 20 min. Brand new split gel are stored on ?30°C until assayed for steroid height. Just after bloodstream sampling, seafood was basically murdered by the decapitation, plus the ovaries was indeed dissected aside. To have ovarian histology, small ovarian fragments had been fixed inside the Bouin’s service, dehydrated, and embedded inside the Technovit resin (Kulzer, Wehrheim). New developmental amount from oocytes have been prior to now said (Matsuyama mais aussi al., 1998b).

The fresh new developmental grade of one’s biggest oocytes from the fish amassed within , , and you can hour was in fact tertiary yolk (TY), very early migratory nucleus (EMN), and late migratory nucleus (LMN) grade, respectively. The most significant follicles on the seafood tested at the hours, in which germinal vesicle description (GVBD) got already occurred and cytoplasm are clear on account of yolk proteolysis and you will moisture, was basically named adult (M) stage.

Having light microscopy, 4-?m-heavy sections had been clipped and you can discolored which have step 1% toluidine blue soluton

Ovarian follicles collected at hr were used for in vitro incubation with radiolabeled steroid precursors. After decapitation, the ovaries were removed and placed in ice-cold Ringer’s solution (140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 0.8 mM MgSO4, 1.5 mM NaH2PO4, 2 mM NaHCO3, 20 mM Hepes, pH adjusted to 7.5 with 1 N NaOH). The largest follicles (n=250) were isolated and gathered with forceps and pipettes. After removing of excess solution, follicles were frozen in liquid nitrogen and stored at ?80°C until use. Our preliminary experiments revealed that there was little difference in the steroid metabolic patterns during the incubation with frozen and intact follicles.

250 follicles were placed in a 10-ml glass tube with 1 ml of sucrose buffer (250 mM sucrose, 20 mM Hepes, pH adjusted to 7.6 with 1 N NaOH). Ten pmol of [ 3 H]P5, [ 3 H]17-P, [ 14 C]DHEA, [ 14 C]AD, [ 14 C]T, or [ 3 H]E1 were dissolved in 150 ?l sucrose buffer. Coenzymes (NAD, NADH, NADP, and NADPH; 10 mM each) were dissolved in a solution that consisted of 100 ?l MgCl2 (20 mM) and 50 ?l citrate buffer (5 mM, pH 7.3). At the start of incubation, both radiolabeled precursor and coenzymes solutions were added to the incubation media. Incubations were performed at 20°C for 2 hr with constant shaking. At the end of incubation, steroids were extracted three times from the media with 4 ml dichloromethane. The extract was concentrated and applied to a thin layer chromatography (TLC) plate (60F254; Merck, Darmstadt, Germany) with non-radioactive standard steroids, i.e., E1, E2, AD solteros divorciados, T, progesterone, 17-P, and 17,20?-dihydroxy-4-pregnen-3-one (17,20?-P), and then developed in benzene:acetone (4:1). Radioactive steroid metabolites were analyzed with a BAS 1500 bio-imaging analyzer (Fuji Film, Tokyo), and standard E1 and E2 were visualized by exposure to iodine vapor. Other standard steroids were detected by UV absorption at 254 nm. Radioactive steroids were scraped from the TLC plates and extracted three times with 3 ml diethyl ether. Some radioactive metabolites were further separated in different solvent systems. Radiolabeled steroid metabolites were identified by their chromatographic mobility in TLC and by recrystallization as described by Axelrod et al. (1965).