Date Published: March 30, 2017
Publisher: Public Library of Science
Author(s): Damien B. Wilburn, Kari A. Doty, Adam J. Chouinard, Sarah L. Eddy, Sarah K. Woodley, Lynne D. Houck, Richard C. Feldhoff, Jorge Mpodozis.
Chemical communication via chemosensory signaling is an essential process for promoting and modifying reproductive behavior in many species. During courtship in plethodontid salamanders, males deliver a mixture of non-volatile proteinaceous pheromones that activate chemosensory neurons in the vomeronasal epithelium (VNE) and increase female receptivity. One component of this mixture, Plethodontid Modulating Factor (PMF), is a hypervariable pheromone expressed as more than 30 unique isoforms that differ between individual males—likely driven by co-evolution with female receptors to promote gene duplication and positive selection of the PMF gene complex. Courtship trials with females receiving different PMF isoform mixtures had variable effects on female mating receptivity, with only the most complex mixtures increasing receptivity, such that we believe that sufficient isoform diversity allows males to improve their reproductive success with any female in the mating population. The aim of this study was to test the effects of isoform variability on VNE neuron activation using the agmatine uptake assay. All isoform mixtures activated a similar number of neurons (>200% over background) except for a single purified PMF isoform (+17%). These data further support the hypothesis that PMF isoforms act synergistically in order to regulate female receptivity, and different putative mechanisms are discussed.
Information exchange in animals is mediated through a range of sensory systems—visual, auditory, chemical, vibrational, etc.—yet the broad steps can be generally described through a basic model of communication : an information source produces a message, the message is encoded in a signal, the signal is broadcast, a receiver acquires the signal, and the signal is decoded. Arguably, chemical signaling—the most ancient form of cellular communication—relies on a “simple” system of direct biochemical interactions between ligands from a sender binding to target receptors in a receiver . Olfaction is one form of chemical communication in vertebrates, and signal transduction is mediated through G-protein coupled receptors (GPCR) from three divergent but highly duplicated gene families [3, 4]. Of the various types of semiochemicals that may stimulate these receptors, pheromones are of particular interest for their ability to elicit preprogrammed behavioral or neuroendocrine responses [5, 6]. Despite >50 years of research , only a limited number of specific pheromone-receptor pairs have been identified [7–10]. A different but related challenge in pheromone research remains multicomponent signals: pheromones are generally released from glands as complex chemical mixtures [9, 11, 12], and pheromone activity is often dependent on the combination of several components in specific ratios [13, 14]. While multicomponent signals have been most well characterized in invertebrate systems, there has been limited investigation into how complex pheromone mixtures may influence vertebrate behaviors, and how these pathways are neurophysiologically mediated.
Pheromone mixtures were prepared containing different degrees of PMF isoform diversity, with pheromone whole extract (WE) and 0.5X PBS used as a positive and negative controls, respectively (Fig 1). Prior to full immunohistochemical (IHC) processing, we optimized methods from the original Wirsig-Wiechmann et al.  protocol to reduce background staining and enhance resolution (Fig 2). Pheromone treatment significantly affected the number of AGB reactive neurons, determined by likelihood ratio test (χ2(6) = 38.5, p = 8.8 x 10−7). Comparison of effects are summarized in Table 1. As expected, WE activated the most neurons on average of any treatment (x¯=106.5), and 0.5X PBS the fewest (x¯=7.0). Three of the five PMF treatments (PMF-EFG, PMF-GHI, PMF-EFGHI) significantly activated more neurons relative to saline (Table 1, Fig 3), and all to a similar degree (x¯=38.3, 33.4, 33.8 neurons, respectively). While PMF-EF was not significant at p < 0.05 (x¯=22.7, p=0.057), the observed effect relative to 0.5X PBS (+225%) was nearly twice the effect observed in Wirsig-Wiechmann et al.  (+124%). Hence, this marginal p-value is likely a result of reduced power from the large number of treatment groups and correction for multiple comparisons. Except for PMF-G, there was no statistical difference between any of the PMF isoform mixtures. In the current study, we evaluated the response of female vomeronasal neurons to different isoform combinations of PMF, a hypervariable salamander courtship pheromone. Behavioral studies of courtship indicate that PMF can either increase or decrease female receptivity depending on the isoform composition [33, 34]. The principal goal of this study was to determine if these different behaviors map proportionally onto VNE neuron activation. When the different mixtures of PMF isoforms were tested in the current study, all but the single isoform (PMF-G) elicited a similar response >200% over background. The non-significant +17% increase in neuronal activation from PMF-G was consistent with courtship trials where there was no detectable response over vehicle . However, the mixture of the three highly abundant isoforms, PMF-GHI, elicited a robust response compared to G alone, suggesting that (1) PMF-H and PMF-I are independently producing large effects, or (2) PMF isoforms act synergistically to stimulate females. While we cannot definitively exclude the hypothesis that PMF-HI has a large independent effect, this seems unlikely for several reasons. First, PMF-G is consistently more abundant than PMF-H or PMF-I in the pheromone extract . Second, the three-dimensional structure of PMF-G was recently solved by NMR, with homology modeling suggesting that PMF-H and PMF-I are structurally very similar to PMF-G such that they may bind similar receptors . Third, the only solution missing PMF-G (PMF-EF) activated the fewer neurons compared to other PMF mixtures, and while not statistically significant, it is interesting that the same mixture plus PMF-G (PMF-EFG) activated approximately twice as many neurons. Consequently, these data further support the hypothesis that synergistic interactions between different PMF isoforms are required in order to enhance female receptivity, but not in a linear relationship between VNE neuron activation and reduction in courtship time.