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In vitro hair follicle incubation which have radiolabeled steroid precursors
Seafood and you may testing

Into the spawning year (later booleaf wrasse were trapped from the link and you can range for the seaside oceans around the Fisheries Search Laboratory, Kyushu School and transferred to this new research. Seafood had been stored in five hundred-litre fiberglass tanks which have filtered seawater, significantly less than pure go out-length and h2o temperatures, and given krill and you can alive hermit crab daily. After guaranteeing each and every day spawning, cuatro–6 people seafood (pounds – g, total length 11step 3–159 mm) was tested on , , , and you may hours. Seafood was basically anesthetized having 2-phenoxyethanol (300 ppm), and you may blood products was amassed regarding the caudal boat playing with syringes fitted which have twenty five-g to own 20 min. Brand new split gel is actually stored during the ?30°C up until assayed getting steroid height. Immediately after bloodstream testing, fish was indeed killed by decapitation, and ovaries was in fact dissected aside. To possess ovarian histology, small ovarian fragments was in fact fixed into the Bouin’s service, dried, and you may stuck during the Technovit resin (Kulzer, Wehrheim). This new developmental degrees away from oocytes were prior to now advertised (Matsuyama et al., 1998b).

This new developmental stages of one’s biggest oocytes on seafood collected from the , , and you may time had been tertiary yolk (TY), early migratory nucleus (EMN), and later migratory nucleus (LMN) amount, respectively. The biggest follicles from the fish tested from the hour, where germinal vesicle description (GVBD) had already occurred and also the cytoplasm try clear due to yolk proteolysis and you can moisture, had been referred to as mature (M) phase.

Getting white microscopy, 4-?m-dense areas were cut and you can stained that have step one% 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) datingranking.net/tr/whatsyourprice-inceleme/. 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).

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