During the vitro hair follicle incubation which have radiolabeled steroid precursors

During the vitro hair follicle incubation which have radiolabeled steroid precursors
Fish and you may sampling

During the spawning season (later booleaf wrasse have been trapped by hook and you may range inside the seaside seas nearby the Fisheries Look Lab, Kyushu College and you can gone to live in this new research. Seafood were kept in 500-litre fiberglass tanks that have blocked seawater, below absolute go out-size and you will liquid heat, and you can given krill and you will alive hermit crab once a day. After guaranteeing each day spawning, cuatro–6 people seafood (fat – grams, complete length 11step three–159 mm) was sampled at , , , and time. Fish was in fact anesthetized that have dos-phenoxyethanol (three hundred ppm), and you can blood samples was in fact amassed about caudal motorboat having fun with syringes suitable having 25-grams for 20 minute. The fresh separated gel was stored within ?30°C up until assayed having steroid peak. Immediately following bloodstream sampling, fish were killed because of the decapitation, and ovaries have been dissected away. To have ovarian histology, brief ovarian fragments was fixed inside the Bouin’s service, dehydrated, and embedded from inside the Technovit resin (Kulzer, Wehrheim). The fresh developmental levels regarding oocytes was in fact prior to now stated (Matsuyama et al., 1998b).

The new developmental levels of the prominent oocytes on fish compiled from the , , and you can hr was indeed tertiary yolk (TY), early migratory nucleus (EMN), and you can later migratory nucleus (LMN) stages, respectively. The greatest follicles regarding seafood sampled on hours, in which germinal vesicle breakdown (GVBD) had currently happened in addition to cytoplasm are clear due to yolk proteolysis and you may hydration, were called adult (M) phase.

To possess light microscopy, 4-?m-dense areas had been slash and you can discolored that have step 1% 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 gleeden ücretsiz deneme 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).

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