Monoclonal antibodies reveal cell-type-specific antigens in the sexually dimorphic olfactory system of Manduca sexta. I. Generation of monoclonal antibodies and patial characterization of the antigens

The olfactory system of the moth Manduca sexta is sexually dimorphic. Male moths possess a male-specific olfactory 'subsystem,' comprising olfactory receptor cells (ORCs) and CNS neurons and synaptic areas associated with the detection of female sex pheromones, in addition to elements common to males and females. In order to explore the molecular differences between cells that subserve the sexual dimorphism and odor-specificity of components of the olfactory system, we generated monoclonal antibodies (Mabs) against tissue of the olfactory system of the moth. In 2 fusions, we screened 1105 hybridoma lines and obtained 272 lines that secreted antibodies against Manduca nervous tissue, as assayed immunocytochemically on sections of the primary olfactory center (the antennal lobe) in the brain of Manduca. We describe here 3 classes of Mabs exemplifying the several cell-type-specific antibodies obtained through the screening procedure. Seven hybridoma lines secrete antibodies that specifically recognize cell bodies, axons, and initial segments of dendrites of many or all ORCs of both males and females (classified as olfactory-specific antibodies, OSAs). Electron-microscopic studies of 2 of the Mabs in this class showed that they recognize antigens associated with the cell membrane and that the immunoreactive ORC axons are bundled together in fascicles in the antennal nerve. On immunoblots, one of the OSA Mabs recognizes 3 distinct protein bands of apparant M,s 42,000, 59,000, and 66,000 Da. When tissue samples enriched in either receptor cell bodies, dendrites, and initial segments of axons or in distal segments of axons and their terminals and synapses were extracted separately, different patterns of bands were detected - 42,000 and 59,000 Da bands from cell bodies and initial segments of axons and dendrites, and 42,000 and 66,000 Da bands from distal segments of axons and their terminals - suggesting that the 59,000 Da protein is modified to the 66,000 Da protein during axonal transport. The second Mab we describe here, the male olfactory-specific antibody (MOSA), selectively recognizes the sexually dimorphic ORCs that are present only in males. The antigen recognized by this antibody is found in cell bodies, dendrites, axons, and axon terminals. By electron-microscopic immunocytochemistry, the MOSA immunoreactivity is found in the cytoplasm and appears not to be associated with particular subcellular organelles. This antibody demonstrates that male-specific ORCs are molecularly distinct from other types of ORCs. The third antibody, the mechanosensory-preferring antibody (MPA), recognizes the mechanosensory receptor cells of the antenna but not the ORCs; it also recognizes some other cells in the CNS. In electron micrographs, MPA appears to bind to intermediate filaments in mechanoreceptor cells.