Molecular and expression characterization of three gonadotropin subunits common α, FSHβ and LHβ 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 α, FSHβ and LHβ were cloned and sequenced from the library. The nucleotide sequences of common α, FSHβ and LHβ 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 α, FSHβ and LHβ 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 α, FSHβ and LHβ 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 α was almost not detected in pituitaries, and FSHβ and LHβ 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 FSHβ cells mainly distributed in the middle area of PPD, while the LHβ cells distributed more widely, including in the area similar to the FSHβ cells and at the external periphery of pituitary near to the PI side. The common α might be expressed in both FSHβ and LHβ cells. Double immunofluoresence localization further demonstrated FSHβ and LHβ expression in distinct cells in the PPD area, although the FSHβ and LHβ cells were detected in the identical area of PPD.