Fish and you will testing
In spawning season (later booleaf wrasse was stuck of the connect and you may range from inside the seaside seas near the Fisheries Lookup Laboratory, Kyushu University and transferred to the brand new laboratory. Fish were stored in five-hundred-litre fiberglass tanks having blocked seawater, less than sheer go out-length and you will water temperature, and you may fed krill and live hermit crab daily. Once confirming everyday spawning, cuatro–six girls seafood (pounds – grams, full size 11step 3–159 mm) was basically sampled in the , , , and hours. Fish was anesthetized with dos-phenoxyethanol (300 ppm), and blood samples was obtained throughout the caudal vessel having fun with syringes fitting with 25-grams to have 20 minute. The newest split serum are kept in the ?30°C until assayed to possess steroid level. Just after bloodstream sampling, seafood was slain of the decapitation, and ovaries have been dissected away. Getting ovarian histology, short ovarian fragments had been repaired for the Bouin’s service https://datingranking.net/tr/korean-cupid-inceleme/, dehydrated, and you will embedded into the Technovit resin (Kulzer, Wehrheim). The newest developmental stages off oocytes was indeed before advertised (Matsuyama et al., 1998b).
Brand new developmental amounts of the largest oocytes on seafood gathered within , , and you may hour had been tertiary yolk (TY), early migratory nucleus (EMN), and you will late migratory nucleus (LMN) degrees, respectively. The greatest follicles on fish sampled on hours, where germinal vesicle description (GVBD) got currently took place together with cytoplasm was transparent due to yolk proteolysis and you may moisture, was indeed also known as mature (M) phase.
For light microscopy, 4-?m-dense areas was in fact slash and discolored having step one% 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, 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).