Fish and you may sampling
When you look at the spawning seasons (late booleaf wrasse have been trapped of the hook up and you may range inside the seaside seas around the Fisheries Research Lab, Kyushu School and you will transferred to the brand new lab. Seafood have been stored in five hundred-litre fiberglass tanks having blocked seawater, around absolute go out-length and you will h2o temperature, and you can given krill and you can live hermit crab once a day. Immediately following confirming everyday spawning, cuatro–6 female fish (weight – grams, overall duration 11step 3–159 mm) have been tested at the , , , and you will hours. Seafood was anesthetized that have 2-phenoxyethanol (3 hundred ppm), and you can bloodstream samples have been collected on caudal vessel using syringes fitting having twenty-five-g to own 20 minute. The broke up gel are held on ?30°C up until assayed to possess steroid height. Just after bloodstream sampling, fish was indeed murdered of the decapitation, in addition to ovaries was indeed dissected aside. Getting ovarian histology, short ovarian fragments have been repaired into the Bouin’s provider, dehydrated, and embedded inside the Technovit resin (Kulzer, Wehrheim). This new developmental grade away from oocytes were in the past stated (Matsuyama mais aussi al., 1998b).
The fresh developmental amounts of one’s prominent oocytes about seafood amassed within , , and you may hours have been tertiary yolk (TY), early migratory nucleus (EMN), and you will late migratory nucleus (LMN) amount, correspondingly. The largest hair follicles on fish tested during the time, where germinal vesicle breakdown (GVBD) got already occurred in addition to cytoplasm try transparent on account of yolk proteolysis and you will moisture, was basically described as adult (M) phase.
To have light microscopy, 4-?m-dense areas had been slashed and you can discolored having step 1% toluidine bluish soluton
Ovarian follicles collected at hr were used for in vitro incubation with https://datingranking.net/pl/blued-recenzja/ 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, 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).