Inside vitro follicle incubation that have radiolabeled steroid precursors

Inside vitro follicle incubation that have radiolabeled steroid precursors
Seafood and you may sampling

In the spawning season (later booleaf wrasse was basically caught by the hook up and range in coastal waters nearby the Fisheries Look Research, Kyushu School and you will gone to live in the new research. Seafood was indeed stored in five-hundred-litre fiberglass tanks having filtered seawater, lower than sheer date-duration and h2o heat, and fed krill and you will live hermit crab once a day. Immediately following verifying daily spawning, cuatro–6 female seafood (body weight – grams, complete duration 11step three–159 mm) was indeed sampled within , , , and hours. Fish was anesthetized having 2-phenoxyethanol (three hundred ppm), and bloodstream trials was amassed in the caudal vessel using syringes installing which have 25-g to possess 20 min. This new split serum is actually held from the ?30°C up to assayed to possess steroid peak. Just after bloodstream testing, fish were killed by the decapitation, in addition to ovaries was dissected away. Having ovarian histology, small ovarian fragments have been fixed in the Bouin’s service, dehydrated, and you can stuck during the Technovit resin (Kulzer, Wehrheim). The brand new developmental degrees regarding oocytes was indeed in past times claimed (Matsuyama et al., 1998b).

The latest developmental degrees of your own prominent oocytes throughout the seafood gathered during the , , and you may hour was basically tertiary yolk (TY), early migratory nucleus (EMN), and you may late migratory nucleus (LMN) amounts, respectively. The most significant hair follicles in the fish tested during the hour, where germinal vesicle breakdown (GVBD) got already happened as well as the cytoplasm was clear because of yolk proteolysis and you can hydration, have been named adult (M) stage.

Getting white microscopy, 4-?m-thick sections was clipped and you can stained that have step 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 indonesian cupid 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).