Male pheromone protein components activate female vomeronasal neurons in the salamander <it>Plethodon shermani</it>

<p>Abstract</p> <p>Background</p> <p>The mental gland pheromone of male Plethodon salamanders contains two main protein components: a 22 kDa protein named Plethodon Receptivity Factor (PRF) and a 7 kDa protein named Plethodon Modulating Factor (PMF), respectively. Each protein component individually has opposing effects on female courtship behavior, with PRF shortening and PMF lengthening courtship. In this study, we test the hypothesis that PRF or PMF individually activate vomeronasal neurons. The agmatine-uptake technique was used to visualize chemosensory neurons that were activated by each protein component individually.</p> <p>Results</p> <p>Vomeronasal neurons exposed to agmatine in saline did not demonstrate significant labeling. However, a population of vomeronasal neurons was labeled following exposure to either PRF or PMF. When expressed as a percent of control level labeled cells, PRF labeled more neurons than did PMF. These percentages for PRF and PMF, added together, parallel the percentage of labeled vomeronasal neurons when females are exposed to the whole pheromone.</p> <p>Conclusion</p> <p>This study suggests that two specific populations of female vomeronasal neurons are responsible for responding to each of the two components of the male pheromone mixture. These two neural populations, therefore, could express different receptors which, in turn, transmit different information to the brain, thus accounting for the different female behavior elicited by each pheromone component.</p

Gonadotropin-Releasing Hormone Modulates Vomeronasal Neuron Response to Male Salamander Pheromone

Electrophysiological studies have shown that gonadotropin-releasing hormone (GnRH) modifies chemosensory neurons responses to odors. We have previously demonstrated that male Plethodon shermani pheromone stimulates vomeronasal neurons in the female conspecific. In the present study we used agmatine uptake as a relative measure of the effects of GnRH on this pheromone-induced neural activation of vomeronasal neurons. Whole male pheromone extract containing 3 millimolar agmatine with or without 10 micromolar GnRH was applied to the nasolabial groove of female salamanders for 45 minutes. Immunocytochemical procedures were conducted to visualize and quantify relative agmatine uptake as measured by labeling density of activated vomeronasal neurons. The relative number of labeled neurons did not differ between the two groups: pheromone alone or pheromone-GnRH. However, vomeronasal neurons exposed to pheromone-GnRH collectively demonstrated higher labeling intensity, as a percentage above background (75%) as compared with neurons exposed to pheromone alone (63%, P < 0.018). Since the labeling intensity of agmatine within neurons signifies the relative activity levels of the neurons, these results suggest that GnRH increases the response of female vomeronasal neurons to male pheromone

Measurements of structural and sequence variability in PMF.

<p>(a) Backbone amide (H_N) exchange H/D exchange rates measured by half life (in hours), with proline residues omitted; (b) Root mean squared fluctation (RMSF) per residue in the PMF structural ensemble (blue) and predicted from the random coil index (red); (c) spectral density functions at 0, ω_N, and ω_H; J(0) is sensitive to fast (ns) and slow (µs-ms) motions, J(ω_N) to motiions on time scales faster than (1/ω_N = 2 ns), and J(ω_H) to motions faster than ¹H (1/ω_H = 0.2 ns); (d) Sequence variability (Shannon entropy index) at each residue measured for all Class I PMFs, shaded according to likelihood of positive selection at each position (red p<0.01, orange p<0.05; yellow = neutral selection). Seven out of the nine non-conserved amino acids in finger 3 display signatures of positive selection, suggesting combined structural flexibility and rapid evolution in this region.</p

NMR-derived structural ensemble of PMF-G.

<p>(a) Backbone model of PMF-G with the twenty lowest-energy conformers, color coded from N- to C-terminus (blue to red), and peptide finger numbers denoted (1–3); (b) disulfide bonds in PMF-G from underside view (same color scheme as a) and a representative TFP (1IQ9, carbons in magenta, sulfurs in green).</p

Summary of mass spectral analysis in PMF-G disulfide bonding pattern determination.

<p>Mass spectral analyses was performed on the three-disulfide species of PMF-G purified by RP-HPLC. Differential treatment included proteolytic enzyme (Enz; chymotrypsin [C] or AspN [A]), reduction with dithiothreitol (DTT), and alkylation with iodoacetamide (addition of a carboxyamidomethyl (CAM) group). Observed monoisotopic masses were compared to theoretical masses with no free sulfhydryls, and mass shifts used to determine peptide modification. All assignments were confirmed by analysis of the fragmented ion series.</p