Stability of human follicle-stimulating hormone receptor mRNA in stably transfected cells

Abstract

In order to assess the impact of mRNA degradation on steady state levels of follicle-stimulating hormone receptor (FSHR) mRNA and on regulation of FSHR gene expression, the stability and half-life of FSHR mRNA were determined in transfected cells expressing recombinant FSHR. Time-dependent changes in FSHR mRNA content were determined by nuclease protection-solution hybridization assay (NPA) or by qualitative reverse transcription-competitive polymerase chain reaction (RT-PCR) in cultured hFSHR-YI cells, cell lines stably transfected with a human FSHR cD-NA. FSHR mRNA content remained constant during 8 h control incubations of hFSHR-YI cells (NPA, 2. 9±0. 3 μg/mg RNA; RT-PCR, 2. 7±0. 3 μg/mg RNA). Actinomycin D (ActD, 5 μg/ ml) inhibited mRNA synthesis, as assessed by incorporation of [³H]uridine into total RNA, by 90 % within 1 h in hFSHR-Y1 cells. No effect of ActD on cellular morphology or viability was observed. ActD caused a time-dependent decrease in FSHR mRNA content in hFSHR-Y1 cell lines with a lag time of 1 h. There were no significant differences in the rate of FSHR mRNA degradation between the two methods of mRNA quantification. The half-life of hFSHR mRNA was 3. 6±0. 2 h by NPA and 3. l±0. 1 h by RT-PCR. The results indicated that degradation of mRNA was an important process in maintenance of steady state expression of the FSHR gene in cells stably expressing recombinant receptor.

Keywords

gonadotropin / granulosa cell / folliculogenesis / follicle-stimulating hormone mRNA

References

[1] NimrodA, EricksonG F, RyanK J. A specific FSH receptor in rat cells: Properties of bindingin vitro. Endocrinology, 1976, 98: 56-56

[2] GromollJ, DankbarB, SharmaR S, et al.. Molecular cloning of the testicular follicle stimulating hormone receptor of the non human primate macaca fascicularis and identification multiple transcripts in the testis. Biochem Biophys Res Commun, 1993, 196: 1066-1066

[3] RossJ. mRNA stability in mammalian cells. Microbiol Rev, 1995, 59: 423-423

[4] KindyM S, SonensheinG E. Regulation of oncogene expression in cultured aortic smooth cells. Post-transcriptional control of c-myc mENA. J Biol Chem, 1986, 261: 12865-12865

[5] SachsA B. Messenger RNA degradation in eukaryotes. Cell, 1993, 74: 413-413

[6] DiviaccoS, NorioP, ZentilinL, et al.. A novel procedure for quantitative polymerase chain reaction by coamplification of competitive templates. Gene, 1992, 15: 313-313

[7] DurnamD M, PalmiterR D. A Practical approach for quantitating specific mRNAs by solution hybridization. Anal Biochem, 1983, 131: 385-385

[8] GromollJ, PekelE, NieschlagE. The structure and organization of the human follicle-stimulating hormone receptor (FSHR) gene. Genomics, 1996, 35: 308-308

[9] AttardiB, WintersSJ. Decay of follicle-stimulating hormone-(messenger RNA in the presence of transcriptional inhibitors and/or inhibin, activin, or follistatin. Mol Endocrinol, 1993, 7: 668-668

[10] DankbarB, SohnM, NieschlagE, et al.. Quantification of androgen receptor and follicle-stimulating hormone receptor mRNA levels in human and monkey testis by a ribonuclease protection assay. Int J Androl, 1995, 18: 88-88

[11] ChuzelF, SchteingartH, VigierM, et al.. Transcriptional and post-transcriptional regulation of luteotropin/ chorionic gonadotropin receptor by the agonist in Leydig cells. Eur J Biochem, 1995, 220: 316-316

[12] ShiH, SegaloffD L. A role for increased lutropin/choriogonadotropin receptor (LHR) gene transcription in the follitropin-stimulated induction of the LHR in granulosa cells. Mol Endocrinol, 1995, 9: 734-734

[13] CollinsS, BouvierM, BolanowskiM A, et al.. cAMP stimulates transcription of the (2-adrenergic receptor gene in response to short-term agonist exposure. Proc Natl Acad Sci USA, 1989, 86: 4853-4853

[14] TanoM, MinegishiT, NakmuraK, et al.. Transcriptional and post-transcriptional regulation of FSH receptor in rat granulosa cells by cAMP and activin. J Endocrinol, 1997, 153: 465-465