In vitro follicle incubation that have radiolabeled steroid precursors
Fish and sampling

Into the spawning seasons (late booleaf wrasse were trapped by hook up and range within the coastal seas around the Fisheries Lookup Research, Kyushu College and relocated to this new lab. Seafood was basically kept in 500-litre fiberglass tanks that have filtered seawater, significantly less than pure time-duration and you will liquid temperature, and you may fed krill and you will alive hermit crab daily. Once verifying every day spawning, cuatro–6 female seafood (body weight – grams, overall size 11step 3–159 mm) was tested within , , , and hours. Fish was anesthetized with dos-phenoxyethanol (3 hundred ppm), and you may bloodstream examples were amassed on caudal vessel playing with syringes fitting that have twenty-five-g for 20 minute. Brand new separated gel was kept at ?30°C up until assayed to possess steroid height. Just after blood testing, seafood were killed of the decapitation, as well as the ovaries was in fact dissected out. Having ovarian histology, short ovarian fragments have been repaired within the Bouin’s solution, dehydrated, and you will stuck inside Technovit resin (Kulzer, Wehrheim). This new developmental grade out of oocytes have been in earlier times reported (Matsuyama ainsi que al., 1998b).

The developmental grade of the biggest oocytes on fish gathered on , , and you can hr was in fact tertiary yolk (TY), early migratory nucleus (EMN), and you can late migratory nucleus (LMN) amount, respectively. The most significant follicles on fish tested at the time, where germinal vesicle malfunction (GVBD) had already occurred together with cytoplasm was transparent because of yolk proteolysis and you will hydration, had been named mature (M) phase.

To have light microscopy, 4-?m-thick areas was indeed slash and stained with 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 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, https://www.datingranking.net/tr/millionairematch-inceleme 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).

In vitro follicle incubation that have radiolabeled steroid precursors

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