When you look at the spawning 12 months (later booleaf wrasse were trapped of the connect and you may line within the seaside seas nearby the Fisheries Research Lab, Kyushu College or university and transferred to new lab. Fish was indeed kept in five hundred-litre fiberglass tanks having blocked seawater, significantly less than natural go out-size and you may h2o temperatures, and provided krill and you can alive hermit crab once a day. Just after verifying each and every day spawning, 4–6 women fish (weight – grams, total size 11step three–159 mm) was in fact tested during the , , , and you will hours. Fish have been anesthetized which have dos-phenoxyethanol (three hundred ppm), and bloodstream samples was basically obtained on caudal boat using syringes fitting that have twenty-five-grams for 20 minute. The newest split up solution are held within ?30°C until assayed getting steroid peak. Immediately following bloodstream sampling, seafood was indeed killed by decapitation, while the ovaries were dissected aside. To have ovarian histology, small ovarian fragments were fixed inside Bouin’s provider, dehydrated, and you can embedded in the Technovit resin (Kulzer, Wehrheim). New developmental values out-of oocytes was in past times said (Matsuyama mais aussi al., 1998b).
The newest developmental level of the prominent oocytes regarding the fish accumulated at the , , and you may hr had been tertiary yolk (TY), early migratory nucleus (EMN), and you may late migratory nucleus (LMN) amounts, correspondingly. The biggest follicles on the fish sampled from the hour, where germinal vesicle description (GVBD) got currently took place and also the cytoplasm is clear on account of yolk proteolysis and you may moisture, was named adult (M) phase.
Having light microscopy, 4-?m-thicker parts was basically cut and you may tarnished which have 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 https://datingranking.net/tr/maiotaku-inceleme/ 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).