Immunocytochemical localization of the subunits of glycoprotein hormones (LH, FSH, and TSH) in the bullfrog pituitary gland using monoclonal antibodies and polyclonal antiserum.

Using beta and alpha subunits of bullfrog follitropin (FSH) III (pI 6.2), which were highly purified by HPLC, we generated three monoclonal antibodies (MCAs) to FSH beta subunit (FSH beta) and six to FSH alpha subunit (FSH alpha). They were produced by hybridomas derived from the myeloma X63.Ag8.653 and spleen lymphocytes from mice immunized with each subunit. Non-competitive binding tests revealed that one of the MCAs against FSH beta (BF3B25) bound strongly to intact FSH and its beta subunit, but not FSH alpha, lutropin (LH), LH alpha, and LH beta. The immunoblotting results also showed a similar immunological specificity for BF3B25. Cross-reactivity of bullfrog FSH against BF3B25 was 19.4%, when compared with FSH beta in the competitive inhibition assay system. On the other hand, noncompetitive binding tests and immunoblotting results showed that one of the MCAs against FSH alpha (BF3A20) bound strongly to intact LH and FSH and their alpha subunits, but not their beta subunits. The inhibition curves obtained using the alpha subunits of LH and FSH were similar. In the sexually mature bullfrog pituitary, immunoreactive FSH cells stained with MCA BF3B25 were distributed throughout the pars distalis, except for the rostral region, and were polygonal in shape, with well-developed cytoplasm. With respect to distribution and histological characteristics, the immunoreactive LH cells were very similar to the immunoreactive FSH cells when consecutive sections were stained with LH beta-specific MCA (BL4B11). However, immunoreactive TSH cells, revealed by anti-human TSH beta serum, formed clusters in the ventrocentral region of the pars distalis. In young adult pituitary, almost all of the gonadotrophs showed the coexistence of FSH and LH, but some gonadotrophs contained only FSH. The number of immunoreactive alpha-subunit cells stained by BF3A20 was always higher than the sum of the numbers of cells stained by the three beta-subunit-specific antibodies.