Molecular cloning of two gonadotropin receptors in mummichog Fundulus heteroclitus and their gene expression during follicular development and maturation.

Two cDNAs encoding gonadotropin receptors, follicle-stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHR) were cloned from mummichog (Fundulus heteroclitus) ovary. Deduced amino acid sequences of the mummichog FSHR (fhFSHR) and LHR (fhLHR) showed high homologies to teleost FSHRs (77-53%) and teleost LHRs (76-62%), respectively. Both the fhFSHR and fhLHR are composed of a typical structural architecture of glycoprotein hormone receptors consisting of the large N-terminal extracellular domain, the transmembrane domain containing seven cell surface membrane-spanning regions, and the intracellular domain. Functional analysis using HEK293 cells stably expressing the fhFSHR or fhLHR demonstrated that both the receptors are specifically activated by mummichog FSH or LH, respectively. Reverse transcription-polymerase chain reaction revealed that both the fhFSHR and fhLHR were expressed in the ovary, testis, and pituitary, and the fhLHR was also expressed in several extra-gonadal tissues. Real-time quantitative-PCR analysis revealed that the fhFSHR gene was abundantly expressed in developing follicles whereas expression of the fhLHR gene markedly increased in follicles of the final maturational stage. These results indicate that gonadotropin stimulation on follicles is regulated by the two distinct pathways via their cognate receptors.