Activin, Inhibin, and Follistatin in Zebrafish Ovary: MATERIALS AND METHODS(1)

MATERIALS AND METHODS(1)

Animals

Adult zebrafish, Danio rerio, were obtained from a local aquarium and maintained in the laboratory according to the procedures of Westerfield. They were kept in filtered and aerated tanks at 28°C under a 14L:10D photoperiod. Experiments were performed according to the Guide to the Care and Use of Experimental Animals published by Canadian Council on Animal Care.

Total RNA Extraction, Reverse Transcription (RT), and Polymerase Chain Reaction (PCR)

Zebrafish were anesthetized with MS222 (Sigma-Aldrich Canada, Oakville, ON, Canada) and killed by decapitation. Ovaries were removed, and total RNA was extracted using Trizol reagent (Canadian Life Technologies, Burlington, ON, Canada). Five micrograms of total RNA were reverse-transcribed using Moloney murine leukemia virus (MMLV) reverse transcriptase (Canadian Life Technologies) and oli-go-dT12-18 primer (Amersham-Pharmacia Biotech, Oakville, ON, Canada) in a 50-^l reaction. Two microliters from the first-strand cDNA synthesis reaction were used in one PCR. Primers made to detect (3A subunit mRNA were a sense primer based on a conserved region of the known (3A cDNA sequences and an antisense primer specific for the zebrafish activin (3A (according to the partial sequence deposited in the Genbank, accession #AJ238980). Primers for FS were designed on the basis of the zebrafish FS cDNA sequence. The sequences of the primers are were as follows: 0A1, 5′ ACT TTG AGA TTT CCA AGG AAG 3′; (3a2, 5′ TGT ACC CTC GGA TAC GGT AGT 3′; FS1, 5′ GAT CCA TCG GCG TGG CAT ATG 3′; and FS2, 5′ GTA GCT TAA CTC TTA GCA GCA 3′. PCR was carried out in the presence of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2.5 mM MgCl2, 50 |xM dNTPs, 10 pmol primers, and 1 U Taq DNA polymerase (Canadian Life Technologies). Ten cycles of PCR (denaturing at 94°C for 20 sec; annealing for 30 sec at 57-52°C for FS or 55-50°C for activin (3A, with a decrease of 0.5°C for each cycle; and extension at 72°C for 50 sec) were performed, followed by 25 cycles with the same profile except that the annealing temperatures were 52°C for FS and 50°C for activin.

This entry was posted in Ovary and tagged Activin, Inhibin, Oocyte, Zebrafish Ovary.