Within the vitro hair follicle incubation that have radiolabeled steroid precursors
For the spawning season (later booleaf wrasse was indeed stuck from the hook up and you may line when you look at the seaside oceans around the Fisheries Look Lab, Kyushu School and you may moved to the latest research. Fish was indeed kept in 500-litre fiberglass tanks having filtered seawater, lower than sheer date-duration and you can liquid temperatures, and you can fed krill and you can alive hermit crab daily. Once verifying every day spawning, cuatro–six girls fish (lbs – grams, total length 11step three–159 mm) was basically sampled within , , , and you can time. Seafood was indeed anesthetized that have dos-phenoxyethanol (300 ppm), and you can bloodstream products was amassed from the caudal vessel using syringes installing having twenty five-grams to possess 20 minute. This new split solution is actually stored at the ?30°C up to assayed to have steroid level. Once blood sampling, seafood have been killed from the decapitation, and also the ovaries had been dissected aside. Getting ovarian histology, quick ovarian fragments was fixed for the Bouin’s provider, dehydrated, and you will stuck into the Technovit resin (Kulzer, Wehrheim). The newest developmental degree out-of oocytes was basically in the past said (Matsuyama et al., 1998b).
This new developmental values of largest oocytes about fish built-up on , , and you will hours have been tertiary yolk (TY), very early migratory nucleus (EMN), and you may later migratory nucleus (LMN) values, respectively. The most significant hair follicles in the fish tested from the hours, where germinal vesicle dysfunction (GVBD) got already took place plus the cytoplasm is transparent because of yolk proteolysis and you can moisture, was indeed called https://datingranking.net/pl/crossdresser-heaven-recenzja/ mature (M) stage.
Getting white microscopy, 4-?m-heavy parts were cut and you will discolored that have 1% toluidine bluish 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).