Fish and you will testing
During the spawning year (later booleaf wrasse was basically caught by the hook up and range when you look at the coastal waters nearby the Fisheries Look Lab, Kyushu University and transferred to the fresh research. Seafood were stored in five hundred-litre fiberglass tanks that have filtered seawater, below absolute go out-duration and liquids temperature, and you can fed krill and you will live hermit crab once a day. Immediately after confirming every day spawning, cuatro–six people seafood (body weight – grams, complete duration 11step 3–159 mm) was indeed tested within , , , and you will hour. Fish was indeed anesthetized having dos-phenoxyethanol (three hundred ppm), and you may blood examples was accumulated regarding caudal boat having fun with syringes installing having twenty-five-grams to possess 20 min. Brand new separated serum is stored at ?30°C up to assayed to own steroid height. Immediately after bloodstream testing, seafood was basically killed by decapitation, plus the ovaries were dissected aside. Getting ovarian histology, quick ovarian fragments had been fixed inside Bouin’s provider, dried, and you will embedded inside Technovit resin (Kulzer, Wehrheim). This new developmental stages from oocytes was prior to now stated (Matsuyama et al., 1998b).
New developmental values of the premier oocytes throughout the seafood amassed in the , , and you may hr was basically tertiary yolk (TY), early migratory nucleus (EMN), and you will later migratory nucleus (LMN) grade, correspondingly. The biggest follicles regarding seafood sampled at the hr, where germinal vesicle malfunction (GVBD) had already took place additionally the cytoplasm try clear on account of yolk proteolysis and you can moisture, had been known as adult (M) stage.
For light microscopy, 4-?m-thick sections was basically reduce and you will tarnished having 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 https://datingranking.net/tr/hi5-inceleme 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, 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).