PRESTON, R.L.; CLIFFORD, R.J.; THOMPSON, J.A.; SLAGER, D.L.; PETERSEN, C.W.; KIDDER, G.W.; Illinois State Univ; Illinois State Univ; Illinois State Univ; Calvin College; College of the Atlantic; Mt Desert Island Biol Lab; ; : Changes in apparent CFTR mRNA expression in developing killifish oocytes.

Fundulus heteroclitus, spawn in estuaries during spring tides in salinities between 5 � 25 o/oo. The fertilized oocytes develop in moist air or shallow water. At the next spring tide (~2 weeks), the oocytes hatch synchronously. While these oocytes have fairly low water permeability, we suggest that they might have some osmoregulatory capacity. Furthermore, the hatchlings must deal with the variable salinities in the estuaries. We tested the hypothesis that oocytes show increasing expression of mRNA for critical transport systems by using RT-PCR and real-time PCR with specific primers for CFTR, a chloride channel. Oocytes were manually expressed from females and fertilized in vitro. The oocytes were held at 20 C in moist air. At various times, total RNA from 10 oocytes (in triplicate) was extracted, with an added DNAase treatment to degrade genomic DNA. Total RNA was measured spectrophometrically and with an Agilent RNA Analyzer. cDNA was prepared by using constant total RNA with oligo dT primer and reverse transcriptase and was screened using PCR and real-time PCR with specific primers for Fundulus CFTR and actin. By day 3, CFTR bands are detectable using PCR. Real-time PCR detected CFTR in both unfertilized and fertilized oocytes, in amounts approx. proportional to total RNA. The ratio of actin to CFTR mRNA was roughly constant. We also tested the water permeability of unfertilized oocytes. Apparent water loss or gain was measured by sedimentation pycnometry (Preston et al., Bull. MDIBL 31:138-140, 1992). Unfertilized oocytes show water loss or gain with salinity changes. These data support the notion that CFTR and perhaps other osmoregulatory proteins are expressed early in fish development to meet the needs of oocyte and hatchling osmoregulation.(NSF C-RUI 0111860)