Into the vitro follicle incubation which have radiolabeled steroid precursors

Into the vitro follicle incubation which have radiolabeled steroid precursors
Seafood and you will testing

In spawning season (later booleaf wrasse was in fact stuck from the hook and you can line inside the coastal oceans close to the Fisheries Browse Lab, Kyushu University and you may transferred to the fresh laboratory. Seafood had been stored in 500-litre fiberglass tanks which have filtered seawater, around natural time-duration and you will h2o temperatures, and you can fed krill and alive hermit crab daily. Shortly after confirming every day spawning, 4–6 women fish (lbs – g, complete size 11step three–159 mm) was in fact tested from the , , , and you may hour. Seafood have been anesthetized which have dos-phenoxyethanol (3 hundred ppm), and you may blood examples was basically amassed throughout the caudal motorboat having fun with syringes suitable that have 25-grams having 20 min. The latest separated solution was held during the ?30°C up until assayed getting steroid top. Immediately after bloodstream testing, seafood was in fact slain from the decapitation, and ovaries was indeed dissected out. To have ovarian histology, brief ovarian fragments have been repaired in Bouin’s services, dehydrated, and you will inserted in the Technovit resin (Kulzer, Wehrheim). New developmental levels out-of oocytes were in the past claimed (Matsuyama et al., 1998b).

The developmental grade of the premier oocytes regarding the seafood amassed from the , , and hr was in fact tertiary yolk (TY), very early migratory nucleus (EMN), and you will late migratory nucleus (LMN) amounts, correspondingly. The biggest hair follicles about seafood tested at the hours, where germinal vesicle breakdown (GVBD) got currently taken place and also the cytoplasm was transparent because of yolk proteolysis and you may moisture, was basically also known as mature (M) stage.

To own light microscopy, 4-?m-thicker sections was in fact cut and discolored that have 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 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).