Molecular and expression characterization of three gonadotropin subunits common alpha, FSHbeta and LHbeta in groupers

A SMART cDNA plasmid library was constructed from protogyous greasy grouper (Epinephelus coioides) pituitary, and the full-length cDNAs of three gonadotropin (GTH) subunits common alpha, FSHbeta and LHbeta were cloned and sequenced from the library. The nucleotide sequences of common alpha, FSHbeta and LHbeta subunit cDNAs are 647, 594 and 574 bp in length, and encode for mature peptides of 94, 99 and 115 aa, respectively. High homology was observed by amino acid sequence alignment and identity comparison of the grouper mature peptides of common alpha, FSHbeta and LHbeta with that of other fishes. Phylogenetic tree analyses of the three GTH mature subunits revealed similar phylogeny relationships among the studied fish species. Three polyclonal antibodies were prepared from the in vitro expressed common alpha, FSHbeta and LHbeta mature proteins, respectively. Western blot analysis and immunofluoresence localization were performed on two typical stages of ovarian development stages in red-spotted grouper. Significant differences in protein expression levels of three gonadotropin subunits were revealed between the two ovarian development stages. In the individuals with resting ovary, common alpha was almost not detected in pituitaries, and FSHbeta and LHbeta expression levels were very low. While in the individuals with developing ovary, the expression of all three gonadotropin subunits reached to a high level. Immunofluoresence localization indicated that the grouper FSHbeta cells mainly distributed in the middle area of PPD, while the LHbeta cells distributed more widely, including in the area similar to the FSHbeta cells and at the external periphery of pituitary near to the PI side. The common alpha might be expressed in both FSHbeta and LHbeta cells. Double immunofluoresence localization further demonstrated FSHbeta and LHbeta expression in distinct cells in the PPD area, although the FSHbeta and LHbeta cells were detected in the identical area of PPD.