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Peg3) promotes sexual dimorphism in metabolism and behavior

['Karo Tanaka', 'Stem Cells', 'Regenerative Medicine', 'Institute Of Cardiometabolism', 'Nutrition', 'Ican', 'Inserm', 'University Of Pierre', 'Marie Curie Paris Vi', 'Paris']

Date: 2022-03

The paternally expressed gene 3 (Pw1/Peg3) is a mammalian-specific parentally imprinted gene expressed in stem/progenitor cells of the brain and endocrine tissues. Here, we compared phenotypic characteristics in Pw1/Peg3 deficient male and female mice. Our findings indicate that Pw1/Peg3 is a key player for the determination of sexual dimorphism in metabolism and behavior. Mice carrying a paternally inherited Pw1/Peg3 mutant allele manifested postnatal deficits in GH/IGF dependent growth before weaning, sex steroid dependent masculinization during puberty, and insulin dependent fat accumulation in adulthood. As a result, Pw1/Peg3 deficient mice develop a sex-dependent global shift of body metabolism towards accelerated adiposity, diabetic-like insulin resistance, and fatty liver. Furthermore, Pw1/Peg3 deficient males displayed reduced social dominance and competitiveness concomitant with alterations in the vasopressinergic architecture in the brain. This study demonstrates that Pw1/Peg3 provides an epigenetic context that promotes male-specific characteristics through sex steroid pathways during postnatal development.

Pw1/Peg3 is under parental specific epigenetic regulation. We propose that Pw1/Peg3 confers a selective advantage in mammals by regulating sexual dimorphism. To address this question, we examined the consequences of Pw1/Peg3 loss of function in mice in an age- and sex-dependent context and found that Pw1/Peg3 mutants display reduced sexual dimorphism in growth, metabolism and behaviors. Our findings support the intralocus sexual conflict model of genomic imprinting where it contributes in sexual differentiation. Furthermore, our observations provide a unifying role of sex steroid signaling as a common property of Pw1/Peg3 expressing stem/progenitor cells and differentiated endocrine cells, both of which remain proliferative in response to gonadal hormones in adult life.

Funding: This work was supported by the French Ministry of Research Chaire d'Excellence and the European Community Seventh Framework Program projects ENDOSTEM (Activation of vasculature associated stem cells and muscle stem cells for the repair and maintenance of muscle tissue-agreement number 241440) and support from the Agence Nationale de la Recherche (Laboratoire d'Excellence Revive, Investissement d'Avenir; ANR-10-LABX-73) and Carmaa (RHU-ANR). We also thank Inserm and the University of Paris (VI) Sorbonne for institutional support. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

In this study, we characterized paternally inherited Pw1 deficient phenotypes in male and female mice at different stages of postnatal development. We identified specific growth factor and hormonal axes that are deregulated at critical stages of postnatal development. At the cellular level, we demonstrate co-localization of Pw1 in sex-hormone dependent cell types in various organs. Our results point to a central role for Pw1 in establishing sexual dimorphism in mammals that regulates overall sex-specific physical traits, metabolism and behavior.

The Pw1/paternally expressed gene 3 (hereafter referred to as Pw1) is a mammalian-specific, parentally imprinted gene that is widely expressed during early embryonic development and becomes restricted to subset of tissues in adulthood [ 15 , 16 ]. Using a Pw1 reporter transgenic mouse line (Pw1 IRESnLacZ ), we showed that Pw1 is expressed in a wide array of adult stem/progenitor cells [ 17 ]. Studies of different types of progenitor cells, all of which express high levels of Pw1, demonstrated that Pw1 dysfunction alters stem cell competence, self-renewal capacity, and cell cycle behaviors [ 18 – 21 ]. At a molecular level, Pw1 modulates cell stress pathways including TNFα-NFκB signaling in cell growth and survival [ 22 ], p53 signaling in apoptosis [ 23 , 24 ], and decolin-induced autophagy [ 25 ]. The PW1 protein also acts as a transcription factor that is shown to regulate expression of mitochondrial genes in the brain [ 26 ] as well as oxytocin receptor [ 27 ]. To date, several lines of Pw1 mutant mice have been generated by different gene targeting strategies [ 28 – 30 ]. Mice carrying a paternally inherited mutant allele for Pw1 consistently displayed reduced pre- and postnatal growth in all models. Pw1 +/p- adult males were also shown to have altered energy homeostasis such as increased body fat and reduced thermogenesis, whereas metabolic phenotypes of female counterparts were not fully characterized in detail [ 31 ]. By contrast, a delayed onset of oestrus cycle and alterations in the reproductive physiology, such as smaller litter size and mature oocytes, were demonstrated [ 30 , 31 ]. It has been also reported that Pw1 deficient mice display deficits in adaptive traits, such as maternal care in females [ 28 ], and sexual experience-dependent olfactory learning in males [ 32 ]. All these findings indicate a significant involvement of Pw1 in sex-hormone dependent physiology, but the underlying mechanism by which this paternally expressed gene exerts such diverse biological functions remained unresolved.

Human diseases associated with deregulated genomic imprinting and gene knockout studies in mice have established pivotal roles of genomic imprinting in growth, metabolism, reproduction, and behavior [ 9 – 11 ]. In human and mouse, many imprinted genes are clustered in distinct chromosomal regions and are typically co-expressed in organs and tissues that regulate homeostasis of the whole-body energy metabolism, such as the brain hypothalamic region, liver, pancreas, muscle, fat, gonads, and placenta [ 9 , 12 , 13 ]. The generation of mutants corresponding to several imprinted genes in mice demonstrated global metabolic changes and their imprinting status (i.e. maternal or paternal) often correlates with inverse metabolic outcomes. Specifically, paternally expressed genes such as Magel2, Dlk1 and Zac1 promote growth and energy expenditure and restrict adiposity whereas the maternally expressed genes, H19 and Grb10, suppress growth and increase adiposity ([ 10 ] and references therein). Genome-wide transcriptome analyses have further demonstrated that inactivation or overexpression of a single imprinted gene alters the expression profile of multiple imprinted genes, suggesting that imprinted genes act in networks to coordinate cellular and organ development and functions [ 14 ].

Parental genomic imprinting is a form of epigenetic regulation by which one allele of a gene is expressed according to its parent-of-origin. In vertebrates, this form of imprinting is unique to placental mammals and its evolutionary advantage is still under active debate [ 1 – 3 ]. The parental conflict (or kinship) [ 4 ] and maternal-offspring coadaptation theories [ 5 ] are two widely recognized concepts to explain why parental genomic imprinting arose in mammals. Independently, Day and Bonduriansky proposed an ‘intralocus sexual conflict’ model [ 6 ] that predicts a physiological role for genomic imprinting in the genetic architecture of sexually dimorphic traits. This hypothesis is applicable to any species and traits under sex-specific selection pressure. However, empirical exploration of the role of imprinted genes in sexual differentiation is relatively limited [ 7 , 8 ].

Results

Reduced masculinization of growth and metabolism in Pw1 deficient males Mice carrying a paternally inherited mutant allele (Pw1+/pat) were distinguishable from their wildtype (WT) littermates (Pw1+/+) by a smaller size at birth and a reduced postnatal weight gain, as previously reported by our group and others [19,28,29]. Comparison of male and female littermates in the postnatal growth phase revealed that body weight was identical between males and females up to 3.5 weeks of age in both genotypes and Pw1+/p- mice were significantly smaller (Fig 1A). Sex differences emerge at 4 weeks of age in both genotypes, with slight delay in the Pw1+/pat littermates. Body weight at 7 weeks of age revealed a significant interaction between sex and genotype (p<0.05), and Pw1+/pat- are significantly smaller in both sexes (p<0.0001) while females are significantly smaller than males (p<0.0001). Multiple comparisons revealed all four groups are different (p<0.0001), however notably, there were no differences detected between Pw1+/+ males and Pw1+/pat- females. We observed a positive correlation between random-fed blood glucose and body weight during the postnatal growth phase regardless of sex and genotype (Fig 1B, left), and the Pw1+/pat- mice displayed reduced glucose levels up to 2 months of age (Fig 1B, right, p<0.001). Concomitantly, food consumption between 2 months and 2.5 months of age was reduced in a sex dependent manner (Fig 1C). We noted that for all sexually dimorphic parameters examined, Pw1+/pat- males were similar to Pw1+/+ female littermates, which indicated a role for Pw1 in the control of male-specific sexual differentiation during postnatal development. PPT PowerPoint slide

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TIFF original image Download: Fig 1. Reduced masculinization of metabolisms in Pw1+/pat- males. (A) Postnatal growth of Pw1+/pat- compared with Pw1+/+ littermates showing Pw1+/pat- animals are smaller in both sexes throughout the growing phase (p<0.001), and the onset of sexual dimorphism in body weight is delayed in Pw1+/pat- at four weeks of age. Two-way ANOVA test revealed significant interaction between genotype and sex after 6 weeks of age. (B) Random-fed glucose levels at 2 month-old and its positive correlation with body weight (r = 0.690, p<0.0001). Each symbol represents independent measurement. (C) Sex dimorphic food consumption at 2 month-old. Two way ANOVA test with multiple comparisons demonstrated significant interaction between sex and genotype. (D) Sexual dimorphisms in body composition in young adults (n = 8–14 each group). Male-specific increase in lean mass and decrease in fat mass in Pw1+/+ males at 2–3 month of age was less prominent in Pw1+/pat- males, while the females Pw1+/pat- are proportionally smaller in lean and fat mass. * in black represents comparison in males and * in red represents comparison in females. (E) An inverse correlation between lean mass and fat mass at 10 month of age was only found in the Pw1+/+ males (r = 0.757, p<0.001) and not in the Pw1+/p- males (r = 0.076, p = 0.771) nor in the females (r = 0.006, p = 0.981). Comparison between genotypes and sexes was performed using two-way ANOVA with Tukey’s multiple comparisons. Correlation was determined with simple linear regression analysis. *P<0.05, **P<0.01, and ***P<0.001. NS: non-significant. Values are mean ± SEM. Each symbol represents individual animals in (B) and (E). https://doi.org/10.1371/journal.pgen.1010003.g001 In adult mammals, including humans and mice, males are typically larger with an increased skeletal mass, whereas females are smaller with higher adiposity. To further monitor the sex-dependent postnatal development and maturation, we performed a longitudinal analysis of body composition (lean/fat mass) of Pw1+/pat- males and females in comparison to Pw1+/+ littermates using non-invasive NMR imaging. During secondary sexual maturation, both male and female Pw1+/pat- animals showed reduced lean mass at all time points analyzed, but the difference became more marked in males (by 20%, p<0.001) than in females (by 15%, p<0.001) (Fig 1D, top left). In contrast, the fat mass development was highly sex-dependent. Pw1+/+ males manifested transient reduction of fat mass at 3 months of age whereas Pw1+/pat- males did not undergo this transition resulting in an accelerated fat accumulation in later adulthood (Fig 1D, bottom left). When expressed in percentage, the composition of lean mass shows a steady increase up to three months of age in Pw1+/+ males (Fig 1D, top right), while fat mass decrease comparatively (Fig 1D, bottom right). These male-specific changes in body composition were less prominent in Pw1+/pat- males. Furthermore, the phenotype is highly specific to male, as lean and fat mass were proportionally reduced in the Pw1+/pat- females as compared to their Pw1+/+ littermates. Therefore, there was no difference in % of body composition in females (Fig 1D, right). Two-way ANOVA test revealed an interaction between genotype and sex with age, indicating that loss of Pw1 has significantly different impacts on body composition in males and females. There was an inverse correlation between lean mass and fat mass in mature age specifically in Pw1+/+ adult males (r = 0.757, p<0.001) (Fig 1E). Taken together, Pw1+/pat- mice displayed a significant reduction in male-specific body growth.

Pw1 deficient mice have altered GH/IGF signaling that reduces body size and sexual dimorphism during postnatal development The growth hormone (GH)/insulin-like growth factor-1 (IGF-1) axis plays a pivotal role in directing postnatal growth and regulates fat metabolism [33], whereas gonadal androgens stimulate the male-specific pulsatile secretion of GH in early puberty [34,35] thereby promoting sexually dimorphic patterns of somatic growth and body composition. The anabolic effect of GH is exerted by the stimulation of endocrine IGF-1 production primarily in the liver, and the circulating IGF-1 levels are considered as an indicator of GH activity in the postnatal growth phase [33]. Therefore, we examined IGF-1 activity in the Pw1 mutant mice during postnatal development. Plasma IGF-1 levels correlated with body weight at 3 weeks old, as commonly expected (Fig 2A), and IGF-1 levels were reduced in the Pw1+/pat- mice as compared to Pw1+/+ littermates (Fig 2B). We further monitored the circulating levels of IGF-1 in the same animals weekly up to 6 weeks of age corresponding to the period when the circulating IGF-1 levels dynamically change in a sex-dependent manner [36]. At five weeks of age, IGF-1 levels were significantly different between sexes (p<0.001) when IGF-1 levels decline in females corresponding to an earlier cessation of growth and increase in males to further promote their growth. Therefore, the levels of IGF-1 in Pw1+/+ males were significantly higher as compared to Pw1+/+ and Pw1+/pat- females (p<0.05 and p<0.01, respectively). Remarkably, no statistical differences were found between Pw1+/pat- males and females of both genotypes. We conclude that the Pw1+/pat- animals display reduced sex-specific regulation in IGF-1 secretion compared to the wild-type littermates. PPT PowerPoint slide

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TIFF original image Download: Fig 2. Deregulated GH/IGF axis in Pw1+/pat- youngs and insulin homeostasis in Pw1+/pat- adult males. (A) Circulating IGF-1 levels at 3 weeks of age and its positive correlation with body weight. Correlation was determined with simple linear regression analysis. (B) Blunted sexual dimorphism of circulating IGF-1 dynamics in Pw1+/pat- mice as compared to that of Pw1+/+. N = 4–8 each group from 5 litters. Two-way ANOVA showed that the IGF-1 levels at 3 weeks old are significantly lower in Pw1+/pat- mice, with no difference between sexes. **P<0.01 in black represent comparison between genotypes, whereas ***P<0.001 in red represent comparison between sex. (C) mRNA expression of growth hormone (Gh) in the pituitary gland, and GH receptor (Ghr) and insulin-like growth factor (Igf1) in the liver at 3 weeks of age. Values were normalized with Tbp and presented relative to the Pw1+/+ male littermates (n = 6–12 each group). (D) Random-fed blood insulin levels at 3 months and 6 months of age in Pw1+/pat- and Pw1+/+ littermates (n = 4–6). (E) Representative insulin tolerance test (ITT) on Pw1+/pat- and Pw1+/+ males from a single litter (n = 3 each genotype) at 3 months and 6 months of age. Similar results were obtained from two other litters. (F) Representative oral glucose tolerance test (OGTT) with insulin secretion measurement on the same set of mice as in ITT. (G) Liver size and mRNA expression of lipogenic genes in the liver at 3 months and 6 months of age showing an age-dependent development of hepatic steatosis in Pw1+/pat- mice as compared to Pw1+/+ littermates. Srebp1, sterol regulatory element binding protein 1; Acaca, acetyl-CoA carboxylase alpha; Fasn, fatty acid synthese; Scd1, Stearoyl-CoA desaturase; Pparg1 & 2, peroxisome proliferator activated receptor gamma 1 & 2. (H) Fat deposition revealed by Oil Red-O staining in 8-month-old livers (n = 4–6 for each group). Lipid droplets were quantified in number and in size using particle analysis tool in Image-J software. Values are mean ± SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.001 by two-way ANOVA with Tukey’s multiple comparisons. https://doi.org/10.1371/journal.pgen.1010003.g002 Based on the observation that circulating IGF-1 levels are reduced in Pw1+/pat- mice at 3 weeks of age, we performed gene expression analysis on pituitary gland and liver of different sets of littermates. Consistently, the expression of Gh in the pituitary gland was significantly reduced in Pw1+/pat- males as compared to Pw1+/+ littermates, whereas growth hormone receptor (Ghr) and Igf1 expression levels in the liver also showed a trend of down-regulation (Fig 2C). Taken together, these results show a global suppression of GH/IGF-1 activity during postnatal growth in Pw1+/pat- mice in a sex-dependent manner.

Pw1 deficiency deregulates insulin sensitivity and increases adiposity in adult males Insulin is a key regulator of energy and fat metabolism throughout life. Its anabolic action promotes postnatal growth after weaning [37], however, chronically elevated insulin levels are associated with obesity and abnormal fat metabolism [38]. To evaluate the steady state insulin levels, we measured blood insulin levels in fed animals. At 3 months of age, circulating insulin levels were lower in females than males (p<0.001) corresponding to their lower levels of glycemia (Fig 2D). The insulin levels of Pw1+/pat- were also reduced at this age although this trend is only confirmed with Fisher’s LSD test. By contrast, the Pw1+/pat- genotype exhibited a male specific increase in insulin levels at 6 months of age. Linear regression between insulin levels and body composition revealed a positive correlation between plasma insulin levels and lean mass at 3 months of age in Pw1+/+ and Pw1+/pat- males (r = 0.714, p<0.01, and r = 0.699, p<0.01), respectively (S1A Fig). At 6 months of age, on the other hand, the insulin levels correlated better with fat mass in Pw1+/+ and Pw1+/pat- males (r = 0.875, **p<0,01 and r = 0.644, p = 0.118, respectively). An insulin tolerance test (ITT) was performed in a set of Pw1+/+ and Pw1+/pat- male littermates, which revealed no differences between genotypes at 3 months of age, whereas the Pw1+/pat- males developed a modest insulin resistance at 6 months of age as compared to Pw1+/+ males (Fig 2E). Oral glucose tolerance test (OGTT) on the animals of the same litter showed that insulin secretion and glucose clearance were slightly lower in Pw1+/pat- at 3 months of age (Fig 2F). Notably, these patterns were inverted at 6 months of age and Pw1+/pat- males displayed a higher insulin secretion and clearance. Pw1 reporter gene expression was high in pancreatic β-cells and in hepatocytes (S2A and S2B Fig), both of which were characterized by the presence of sex steroid hormone receptors [39,40]. Co-localization of Pw1 with ERα in various endocrine cells indicates a pivotal role of Pw1 in these cell types via sex steroid signaling. Paternal loss of Pw1 has been shown to lead to increased β-cells cycling in Pw1+/pat- males at 3 months of age [41]. The increase of proliferation at a younger age may result in increased insulin production in later adulthood. We analyzed the insulin content of pancreas in mature adult males and observed that the pancreatic insulin is slightly elevated in the Pw1+/pat- males (S1B Fig, top). In addition, random-fed glycemia was significantly elevated in Pw1+/pat- males, in agreement with their insulin resistance in adulthood (S1B Fig, bottom). Pw1+/pat- animals also exhibited age- and sex-dependent hepatic phenotypes: liver size was significantly higher in Pw1+/pat- males at 6 months of age as compared to Pw1+/+ males and females (Fig 2G, left). Gene expression of major adipogenic genes in these animals demonstrated significant changes in the 6-month-old Pw1+/pat- livers in a sex-dependent manner (Fig 2G, right). Notably, the two major isoforms of Pparg1 and Pparg2, differentially expressed between males and females [42], were differently affected by Pw1 loss of function. While the Pparg1 is similarly expressed in Pw1+/+ and Pw1+/pat- livers, Pparg2 expression levels were significantly increased in 6-month-old male livers. In contrast, Pparg2 levels were significantly lower in female livers as compared to male livers at 3 months of age and no increase was observed in Pw1+/pat- female livers at 6 months of age. PPARG2 is selectively increased in human obesity [43] and is specifically elevated in the steatotic livers of ob/ob mice [44]. We therefore performed hepatic histology using Oil Red-O staining on the 8-month-old livers of both sexes (Figs 2H and S1C). Pw1+/+ livers revealed multiple small lipid droplets in both sexes. In contrast, Pw1+/pat- mice showed abundant, large lipid droplets that were more marked in males. Digital quantification revealed that the total number of lipid droplets and Oil Red-O positive area size were significantly increased in Pw1+/pat- livers (p<0.001) in mature adulthood (Fig 2H). We note that smaller droplets are more abundant in Pw1+/+, whereas larger droplets increased by age in Pw1+/pat- livers, and that this trend was more pronounced in males (S1C Fig). Taken together, our findings demonstrated that paternal Pw1 deficiency affected multiple stages of early life that proceeded to an age- and sex-dependent global shift of body metabolism towards accelerated adiposity, diabetic-like insulin resistance, and fatty liver in later adulthood, and the impact is more profound in males.

Paternal Pw1 deficiency reduces aggressive behavior and social dominance in males During routine handling of the Pw1 mutant colony, we observed that adult Pw1+/pat- males seldom display typical aggressive behavior as compared to their Pw1+/+ littermates. We scored incidents of spontaneous fights among Pw1+/+ (n = 75) and Pw1+/pat- (n = 57) male offspring that were group-caged with littermates, and found that Pw1+/pat- males were significantly less aggressive (S3A Fig). When male offspring were separated according to genotype at the time of weaning (Pw1+/+ or Pw1+/pat-), we observed little incidents of fight in the Pw1+/pat- cages, suggesting that the reduced aggressive behavior is, at least in part, intrinsic to the paternal Pw1 loss of function. To quantitatively assess the competitive ability of Pw1+/pat- males, we used a social-confrontation tube test [45] on adult offspring derived from Pw1+/pat- breeder males. The first test was to examine whether Pw1 is involved in establishing social hierarchy among littermates by using litters consisting of two genotypes. We observed a typical social dominance pattern in which Pw1+/+ males dominate the siblings in a given cage at 10 months of age (S3B Fig, squared in red). Each animal was ranked within each litter by the number of wins and the score was compared between genotypes. This ranking revealed that the Pw1+/+ males rank higher and there is a significant difference between genotype (***p<0.001) (Fig 3A). Notably, the same analysis on younger litters at 3 months of age revealed no significant difference in the inter-litter rank between genotypes, suggesting that younger males have not yet established social rank at this age. A second test was performed in the context of stranger encounter as described by Garfield et. al. [46] in which animals were tested against unfamiliar opponents from different cages with mixed genotypes. The winning rate was determined by the percentage of win in all matches against unknown opponents (S3B Fig). This test demonstrated that the Pw1+/+ males have a greater likelihood of winning in a forced encounter (*P<0.05) (Fig 3B). PPT PowerPoint slide

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TIFF original image Download: Fig 3. Altered social behavior and brain architecture in Pw1+/pat- males. (A) Interlitter social rank by tube test in Pw1+/+ and Pw1+/pat- males from mixed genotypes at 10 months of age (n = 11 vs n = 10, from 5 litters) and at 3 months of age (n = 7 vs n = 10, from 4 litters). (B) Assessment of social dominance in the stranger encounter tube test. Animals used were listed in S3B Fig. The winning rate was calculated from 17–18 matches against unfamiliar opponents. (C) Pw1 reporter expression (β-gal+) is observed in the vasopressinergic neurons of PVN (top), whereas β-gal signals are strongly co-localizing with ERα receptor in SON (bottom) in the hypothalamus (x400). (D) Representative images of AVP expressing neurons in the PVN of hypothamamus in the Pw1+/+ and Pw1+/pat- male brains (x40). Coronal sections at 120μm intervals through PVN from anterior to posterior axis were immunostained with an anti-AVP antibody. AVP positive area size (dotted line) and cell count were quantified from five sequential sections. E. Digital quantification of AVP+ area size and the total cell count in PVN and their positive correlation in the Pw1+/+ male brain. Columns, mean; bars, SEM; *, P < 0.05, Mann-Whitney U test. https://doi.org/10.1371/journal.pgen.1010003.g003 In the female Pw1+/p- brains, the oxytocinergic architecture appeared under-developed concomitant with alteration in maternal care [28], a female specific behavior that is acquired at pregnancy. On the other hand, the aggressive behavior commonly observed in laboratory mice is male specific and develop during postnatal growth period. Oxytocin and arginine-vasopressin (AVP) are the two major neuropeptide that regulates sex-specific mammalian behaviors (reviewed in [47,48]). In particular, the AVP system is androgen-dependent [49] and central AVP plays a pivotal role in inter-male aggressive behavior [50,51]. Pw1 is shown to be expressed in both oxytocinergic and vasopressinergic neurons [52]. Therefore, we hypothesized that Pw1 plays a pivotal role in regulating the function of these cell types through sex hormone signaling. We first examined Pw1 expression in the brain using the Pw1 reporter transgenic mouse line Pw1IRESnLacZ [17]. As predicted, we found high levels of reporter gene expression in brain nuclei known to be sexually dimorphic and express sex steroid hormone receptors [53,54], including paraventricular nucleus (PVN) of hypothalamus, the bed nucleus of stria terminalis (BnST), the medial preoptic area (mPOA), and the medial amygdala (MeA) (S3C Fig). These regions are primary sites of AVP production and vasopressinergic neuronal projections [50,55]. We therefore examined the brains from Pw1 reporter mice by immunofluorescence using anti-β-gal and anti-AVP antibodies and found that the vasopressinergic cells in the PVN and SON are the sites of high Pw1 reporter gene expression (Fig 3C, top) which co-express ERα (Fig 3C, bottom), suggesting a role of Pw1 in this cell type. We next examined the architecture of AVP+ cells in the Pw1+/+ and Pw1+/pat- males whose competitive ability had been already established by the tube test (Litter 1–6 in S3B Fig). Using anti-AVP antibody, we immunostained the coronal sections of entire brain and the total AVP+ cell number in the PVN and its area size were determined (Fig 3D). Concordant with the reduced social competitiveness, the AVP+ PVN area size was significantly reduced in the Pw1+/+ brains at 10 months of age (Fig 3E). In the Pw1+/+ brain, we found a strong positive correlation between the area size and the cell number (Fig 3E). Remarkably, this correlation is abolished in the Pw1+/pat- brain, implying that the proliferation and/or expansion of the AVP+ cells are deregulated. Finally, we examined the correlation between the AVP+ cell structure and social behavior in the litter 1 which consists of four Pw1+/+ males. Both area size and cell count in the PVN showed positive correlation with the winning rate in this set of animals (S3D Fig). These data suggests that Pw1 promotes acquired social dominance and aggressive behavior by modulating the AVP+ neuroendocrine architecture in male mice.

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