Neuronal Nitric Oxide Synthase as a Substrate for the Evolution of Pseudosexual Behaviour in a Parthenogenetic Whiptail Lizard

Abstract

The evolution of neuroendocrine mechanisms governing sex-typical behaviour is poorly understood. An outstanding animal model is the whiptail lizard (Cnemidophorus) as both the ancestral and descendent species still exist. The ancestral little striped whiptail, C. inornatus, consists of males and females, which exhibit sex-specific mating behaviours. The descendent desert grassland whiptail, C. uniparens, consists only of females that alternately exhibit both female-like and male-like pseudosexual behaviour. Castrated male C. inornatus will mount a conspecific in response to exogenous androgen, but some are also sensitive to progesterone. This polymorphism in progesterone sensitivity in the ancestral species may have been involved in evolution of progesterone-mediated male-typical behaviour in the descendant unisexual lizards. We tested if progesterone activates the typically androgenic signalling pathway by investigating hormonal regulation of neuronal nitric oxide synthase (nNOS) using in situ hybridization and NADPH diaphorase histochemistry, a stain for nNOS protein. NADPH diaphorase is widely distributed throughout the brain of both species, but only in the periventricular nucleus of the preoptic area (pvPOA) are there differences between mounting and non-mounting individuals. The number of cells expressing nNOS mRNA and NADPH diaphorase is higher in the pvPOA of individuals that mount in response to progesterone or androgen. Further, the nNOS promoter has both androgen and progesterone response elements, and NADPH diaphorase colocalizes with the progesterone receptor in the pvPOA. These data suggest that a polymorphism in progesterone sensitivity in the sexual ancestor reflects a differential regulation of nNOS and may account for the male-typical behaviour in unisexual whiptail lizards.

Molecular characterization and brain distribution of the progesterone receptor in whiptail lizards

“…Given the extensive amount of post-translation regulation of proteins, as well as higher stability of proteins compared to mRNA, it is possible that the effects of reproductive state on transcription of PR mRNAs do not lead to (detectable) changes in the levels of PR proteins. Additionally, PR seems to have different transcriptional activity in C. uniparens and C. inornatus females, as PR upregulates neuronal nitric oxide synthase to facilitate male-typical mounting behavior in C. uniparens but not C. inornatus females [50]. Future studies will focus on the gene regulatory network of PR, as several gene networks may be differentially regulated by PR in C. uniparens in response to progesterone that facilitate male-like behavior compared to C. inornatus females.…”

“…For control sections, all procedures were the same except that primary antibody is omitted. This antibody has been confirmed to bind the appropriate lizard antigens by western blot in O’Connell et al [50]. …”

“…Brains were sectioned at 30µm onto SuperFrost Plus slides (Fisher Scientific, Itasca, IL) and stored at −80°C until processing for immunohistochemistry as previously described [50]. Briefly, slides were dried and then rinsed in PBS, and incubated in 3% hydrogen peroxide in PBS for 20 min.…”

“…The template used to make the PR probe was the same as in Young et al [76]. Sections were fixed and processed for in situ hybridization staining for mRNA as previously described [50]. Briefly, Slides were pre-equilibrated in hybridization buffer (50% formamide, 5× SSC, 5× Denhardt’s solution, 125 mg/mL Baker’s yeast tRNA, 250 mg/mL denatured herring sperm DNA) for 2 h at 65 °C.…”

The Vertebrate mesolimbic reward system and social behavior network: A comparative synthesis

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