Development of non-competitive enzyme-linked immunosorbent assays for mummichog Fundulus heteroclitus gonadotropins - examining seasonal variations in plasma FSH and LH levels in both sexes

Abstract

The mummichog Fundulus heteroclitus is an excellent experimental fish for reproductive physiology because of its adequate size, easiness for rearing, and controllable reproduction under laboratory conditions. Furthermore, it is the only species that the native GtHs and their subunits have been purified among small experimental fishes. In this study, homologous non-competitive enzyme-linked immunosorbent assays (ELISAs) for the mummichog FSH and LH were developed by raising monoclonal and polyclonal antibodies against the purified GtHs or their subunits, and the plasma hormone levels in various seasons were examined. The cross-reactivity of LH in the FSH ELISA and the cross-reactivity of FSH in the LH ELISA were low, 2.3% and 0.2% respectively, indicating high specificities of both GtH assays. The practical detection limits were 10 pg/well (0.125 ng/ml plasma) for the FSH ELISA and 8 pg/well (0.1 ng/ml plasma) for the LH ELISA. Plasma FSH levels in females indicated distinct correlations with ovarian stages: they were almost undetectable (<0.125 ng/ml) during the post-spawning immature phase (September), low values (0.3 ng/ml) during the cortical alveoli accumulation phase (December), considerably high (1.8 ng/ml) in the vitellogenic phase (February), and very high values (12 ng/ml) during the spawning season (June). The male FSH levels showed similar pattern of changes to that of females, also indicating distinct correlations with testicular activities. Plasma LH levels were considerably high during the spawning period in both sexes (3.3 ng/ml in females and 4.5 ng/ml in males). They were low or undetectable values in non-spawning seasons, and clear correlation with the gonadal stages was not observed. These results indicate the importance of FSH for various reproductive events in multiple spawning fishes, and are consistent with the general understanding that the LH is responsible for final gametes maturation in both sexes. Nonetheless, they further suggest that the role of LH for various reproductive events other than the final maturation may be limited.