Fish and you will testing
In the spawning year (later booleaf wrasse have been caught of the link and you may line in the coastal oceans nearby the Fisheries Search Research, Kyushu College or university and you will moved to the fresh new laboratory. Seafood had been stored in five-hundred-litre fiberglass tanks that have blocked seawater, significantly less than natural date-size and liquids temperature, and you can fed krill and alive hermit crab daily. Once verifying daily spawning, 4–6 female seafood (body weight – grams, full duration 11step three–159 mm) was tested at the , , , and you can hours. Seafood was indeed anesthetized having dos-phenoxyethanol (300 ppm), and you can bloodstream examples was accumulated from the caudal motorboat having fun with syringes suitable that have twenty-five-grams to possess 20 minute. The brand new split up solution try kept at ?30°C up until assayed for steroid level. Once bloodstream sampling, seafood had been murdered because of the decapitation, together with ovaries have been dissected out. To have ovarian histology, small ovarian fragments have been repaired when you look at the Bouin’s provider, dried, and you can embedded into the Technovit resin (Kulzer, Wehrheim). The fresh new developmental level off oocytes had been previously reported (Matsuyama et al., 1998b).
The brand new developmental grade of your own prominent oocytes in the seafood amassed within , , and hour was tertiary yolk (TY), early migratory nucleus (EMN), and later migratory nucleus (LMN) degrees, respectively. The biggest follicles throughout the seafood sampled on time, in which germinal vesicle dysfunction (GVBD) had already took place additionally the cytoplasm was transparent due to yolk proteolysis and you can hydration, were called adult (M) stage.
Getting light microscopy, 4-?m-dense areas were reduce and you can discolored which 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 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 https://datingranking.net/tr/okcupid-inceleme/ 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).