Proteomic analyses of limbic regions in neonatal male, female and androgen receptor knockout mice

BMC Neuroscience, Jan 2017

Background It is well-established that organizational effects of sex steroids during early development are fundamental for sex-typical displays of, for example, mating and aggressive behaviors in rodents and other species. Male and female brains are known to differ with respect to neuronal morphology in particular regions of the brain, including the number and size of neurons, and the density and length of dendrites in nuclei of hypothalamus and amygdala. The aim of the present study was to use global proteomics to identify proteins differentially expressed in hypothalamus/amygdala during early development (postnatal day 8) of male, female and conditional androgen receptor knockout (AR NesDel ) male mice, lacking androgen receptors specifically in the brain. Furthermore, verification of selected sexually dimorphic proteins was performed using targeted proteomics. Results Our proteomic approach, iTRAQ, allowed us to investigate expression differences in the 2998 most abundantly expressed proteins in our dissected tissues. Approximately 170 proteins differed between the sexes, and 38 proteins between AR NesDel and control males (p < 0.05). In line with previous explorative studies of sexually dimorphic gene expression we mainly detected subtle protein expression differences (fold changes <1.3). The protein MARCKS (myristoylated alanine rich C kinase substrate), having the largest fold change of the proteins selected from the iTRAQ analyses and of known importance for synaptic transmission and dendritic branching, was confirmed by targeted proteomics as differentially expressed between the sexes. Conclusions Overall, our results provide solid evidence that a large number of proteins show sex differences in their brain expression and could potentially be involved in brain sexual differentiation. Furthermore, our finding of a sexually dimorphic expression of MARCKS in the brain during development warrants further investigation on the involvement in sexual differentiation of this protein.

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Proteomic analyses of limbic regions in neonatal male, female and androgen receptor knockout mice

Zettergren et al. BMC Neurosci Proteomic analyses of limbic regions in neonatal male, female and androgen receptor knockout mice Anna Zettergren 0 2 3 Sara Karlsson 0 3 Erik Studer 0 3 Anna Sarvimäki 0 3 Petronella Kettunen 1 2 Annika Thorsell 4 Carina Sihlbom 4 Lars Westberg 0 3 0 Department of Pharmacology, Institute of Neuroscience and Physiology, Sahlgrenska Academy, University of Gothenburg , POB 431, 405 30 Göteborg , Sweden 1 Department of Neuropathology, Nuffield Department of Clinical Neurosciences, University of Oxford, John Radcliffe Hospital , Oxford , UK 2 Department of Psychiatry and Neurochemistry, Institute of Neu- roscience and Physiology, Sahlgrenska Academy, University of Gothenburg , Göteborg , Sweden 3 Department of Pharmacology, Institute of Neuroscience and Physiology, Sahlgrenska Academy, University of Gothenburg , POB 431, 405 30 Göteborg , Sweden 4 The Proteomics Core Facility, Sahlgrenska Academy, University of Gothen- burg , Göteborg , Sweden Background: It is well-established that organizational effects of sex steroids during early development are fundamental for sex-typical displays of, for example, mating and aggressive behaviors in rodents and other species. Male and female brains are known to differ with respect to neuronal morphology in particular regions of the brain, including the number and size of neurons, and the density and length of dendrites in nuclei of hypothalamus and amygdala. The aim of the present study was to use global proteomics to identify proteins differentially expressed in hypothalamus/amygdala during early development (postnatal day 8) of male, female and conditional androgen receptor knockout (ARNesDel) male mice, lacking androgen receptors specifically in the brain. Furthermore, verification of selected sexually dimorphic proteins was performed using targeted proteomics. Results: Our proteomic approach, iTRAQ, allowed us to investigate expression differences in the 2998 most abundantly expressed proteins in our dissected tissues. Approximately 170 proteins differed between the sexes, and 38 proteins between ARNesDel and control males (p < 0.05). In line with previous explorative studies of sexually dimorphic gene expression we mainly detected subtle protein expression differences (fold changes <1.3). The protein MARCKS (myristoylated alanine rich C kinase substrate), having the largest fold change of the proteins selected from the iTRAQ analyses and of known importance for synaptic transmission and dendritic branching, was confirmed by targeted proteomics as differentially expressed between the sexes. Conclusions: Overall, our results provide solid evidence that a large number of proteins show sex differences in their brain expression and could potentially be involved in brain sexual differentiation. Furthermore, our finding of a sexually dimorphic expression of MARCKS in the brain during development warrants further investigation on the involvement in sexual differentiation of this protein. Proteomics; Sex differences; Androgen receptor; Neonatal; Amygdala; Hypothalamus - Background There are striking sex differences in many aspects of behaviors, not least in social behaviors such as mating and displaying of aggression [1]. Most psychiatric disorders also display sex differences in prevalence and/ or symptomatology [2]. Although it is known that both sex steroids and sex chromosomes are essential for these behavioral differences as well as for sexual dimorphisms in brain anatomy and function [3], the knowledge about the proteins mediating these effects is sparse. The underlying neural circuits of social behaviors are controlled by sensory cues as well as by physiological signals, including the sex steroids. In rodents, these behaviors differ quantitatively as well as qualitatively between males and females, and rely on activity in several sexually dimorphic regions, e.g. in the amygdala and hypothalamus. In short, the medial amygdala neurons receive pheromonal input © The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. and subsequently provide afferents to specific hypothalamic nuclei [1, 4, 5], which execute social behaviors, e.g., aggression, parental behaviors and mating behaviors. One of the most important factors responsible for sexual differentiation of the brain is testosterone. By acting during critical periods of neural development, testosterone and its metabolites cause male and female brains to (...truncated)


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Anna Zettergren, Sara Karlsson, Erik Studer, Anna Sarvimäki, Petronella Kettunen, Annika Thorsell, Carina Sihlbom, Lars Westberg. Proteomic analyses of limbic regions in neonatal male, female and androgen receptor knockout mice, BMC Neuroscience, 2017, pp. 9, 18, DOI: 10.1186/s12868-016-0332-1