Paternally expressed gene 3 (Pw1/Peg3) promotes sexual dimorphism in metabolism and behavior.

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.

Introduction

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].

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].

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 (Pw1IRESnLacZ), 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.

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.

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.

Fig 1

Reduced masculinization of metabolisms in Pw1 males.

(A) Postnatal growth of Pw1 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 males, while the females Pw1 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 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).

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 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 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 males. Furthermore, the phenotype is highly specific to male, as lean and fat mass were proportionally reduced in the Pw1 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 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 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.

Fig 2

Deregulated GH/IGF axis in Pw1 youngs and insulin homeostasis in Pw1 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 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 and Pw1 littermates (n = 4–6). (E) Representative insulin tolerance test (ITT) on Pw1 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 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.

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 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 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 were also reduced at this age although this trend is only confirmed with Fisher’s LSD test. By contrast, the Pw1 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 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 at 3 months of age (Fig 2F). Notably, these patterns were inverted at 6 months of age and Pw1 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 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 males (S1B Fig, top). In addition, random-fed glycemia was significantly elevated in Pw1 males, in agreement with their insulin resistance in adulthood (S1B Fig, bottom).

Pw1 animals also exhibited age- and sex-dependent hepatic phenotypes: liver size was significantly higher in Pw1 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 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 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 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 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 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 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 males seldom display typical aggressive behavior as compared to their Pw1+/+ littermates. We scored incidents of spontaneous fights among Pw1+/+ (n = 75) and Pw1 (n = 57) male offspring that were group-caged with littermates, and found that Pw1 males were significantly less aggressive (S3A Fig). When male offspring were separated according to genotype at the time of weaning (Pw1 or Pw1), we observed little incidents of fight in the Pw1 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 males, we used a social-confrontation tube test [45] on adult offspring derived from Pw1 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).

Fig 3

Altered social behavior and brain architecture in Pw1 males.

(A) Interlitter social rank by tube test in Pw1 and Pw1 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 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.

In the female Pw1 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 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 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.

Pw1 promotes testosterone production in young male mice

Perinatal androgen secretion leads to changes in the CNS and underlie sexual dimorphism in the brain [56]. The testis produce androgens in adolescence that contribute to the development of adult male characteristics in metabolism and behavior that typically underlies reproductive success including male mating and aggression [57-59]. We therefore measured levels of testosterone in young males during peripubertal development when testosterone secretion peaks in postnatal growth (between 6–9 weeks)[60]. First, we measured testosterone levels of male Pw1 and Pw1 littermates derived from 3 litters and found that the Pw1 mice display a delayed pubertal surge (Fig 4A). Based on this timing, we further analyzed different young males from multiple cages containing mixed genotypes in the entire colony at the age between 2.5 and 3 month old. These analyses revealed that Pw1 males have significantly lower levels of testosterone as compared to Pw1 males (Fig 4B). In agreement with these observations, mRNA expression of steroidogenic genes encoding the rate limiting enzymes for testosterone biosynthesis (Cyp17a, Cyp11a, and 3β-HSD), as well as that of luteinizing hormone receptor (LHR) in the testes are significantly reduced in Pw1 males (Fig 4C). Notably, there was no significant difference in the expression of aromatase Cyp19 or 17β-HSD3.

Fig 4

Reduced testosterone production in the Pw1 males.

(A) Plasma testosterone measurements at peripubertal age in Pw1 and Pw1 males (n = 7 and 5, respectively, from 3 litters). Two-way repeated measures ANOVA with Sidak’s multiple comparisons test revealed a significant difference between genotypes at 9 weeks of age (*P<0.05). (B) Plasma testosterone levels in Pw1 and Pw1 non-breeder littermates (n = 47 and n = 42) at 2.5–3 months of age. Each symbol represents independent animals. Bars represent mean ± SEM; **, P<0.01, by Mann Whitney U test. (C) mRNA expression of steroidogenic genes in the 2.5–3 month-old testes of Pw1 and Pw1 non-breeder littermates (n = 15 versus n = 11). StAR, Steroidogenic acute regulatory protein; Cyp17a1, cytochrome P-450 17a; Cyp11a1, cholesterol side-chain cleavage enzyme; Hsd3b1, 3-b-hydroxysteroid dehydrogenase; Lhr, luteinizing hormone receptor; Cyp19a1, aromatase enzyme; Hsd17b3, 17-b-hydroxysteroid dehydrogenase. (D) Pw1 reporter expression in the 2.5-month-old testis of Pw1IRESnLacZ mice, showing X-gal staining (x100) and β-gal immunofluorescence (x400) in the interstitial compartment and in the epithelium of seminiferous tubules. The β-gal immunofluorescence overlapped with that of androgen receptor (AR) in the Leydig and Sertoli cells (arrows). L, interstitial Leydig cells; S, seminiferous tubule.

A previous study from our laboratory using the Pw1 reporter transgenic mouse line (Pw1IRESnLacZ) demonstrated that Pw1 is highly expressed in the peritubular cells near the basement membrane of seminiferous tubules, a part of which were identified as Bmi1+ spermatogonia [17]. Since the Pw1 mice displayed reduced steroidogenesis, we were interested in whether Pw1 is expressed in the cell types responsible for the testosterone production in the testis. We performed histological analyses on the testis of Pw1IRESnLacZ mice using an anti-β-gal antibody and antibodies against sex steroid hormone receptors and found that reporter gene expression colocalized with androgen receptor expression (Fig 4D). In the testis, Leidig cells and Sertoli cells are the two cell types predominantly express androgen receptor (AR) [61]. Therefore, we conclude that Pw1 reporter gene is abundantly expressed in the testosterone producing Leydig cells in the interstitial compartment and in the supporting Sertoli cells near the basement membrane of seminiferous tubules. We note that other imprinted genes are highly expressed in the Leidig cells and that a number of imprinted genes are simultaneously deregulated in Leidig cells of human patients with idiopathic germ cell aplasia [62].

Taken together, our data demonstrate that male sex-hormone signaling during secondary sexual maturation is suppressed in Pw1 male mice, concomitant with reduced gene expression for testosterone biosynthesis in the testis, which may account for the reduced masculinization in metabolism and social behavior during the postnatal development.

Discussion

We demonstrate that Pw1 plays a key role at specific stages of postnatal development for sex-specific growth, metabolism and behavior. Pw1 deficient male mice exhibit a smaller body size and a reduced masculinization of body composition followed by a global shift of metabolism leading to early onset obesity and related metabolic changes. A marked reduction of growth promoting GH/IGF-1 and insulin characterized early postnatal life and a decrease in testosterone activity at puberty coupled with insulin-resistance lead to a male-specific deficiency by adulthood (Fig 5). These observations are concomitant with a significantly reduced sexual dimorphism in body composition. In addition, our data suggests a model in which a paternally expressed gene promotes male-specific brain development and behavior through a sex steroid pathway that contribute to reproductive success in mammals. Our finding support the intralocus sexual conflict model of genomic imprinting [6] for the control of mammalian sexual dimorphism.

Fig 5

Hormonal cascades are deregulated in Pw1 mice.

Pw1 is abundantly expressed in the gonads and target organs and colocalizes with steroid hormone receptors in hormone secreting cells. GHRH, growth hormone-releasing hormone; GH, growth hormone; IGF-1, insulin-like growth factor 1; GnRH, gonadotropin-releasing hormone; LH, luteinizing hormone; FSH, follicle-stimulating hormone; AVP, arginine vasopressin; +, stimulation; -, inhibition by Pw1.

The physiopathological alterations in Pw1 deficient mice identified in this study are similar to the phenotypic spectrum of Prader-Willi syndrome (PWS), a disorder with hypothalamic dysfunction and hypopituitarism due to imprinting errors [63]. PWS is characterized by biphasic clinical manifestations, i.e., reduced growth velocity, hypoglycemia, and hypotonia at infancy, followed by hyperphagia, extreme obesity, and hypogonadism in childhood through adulthood. In addition, patients present low levels of GH and IGF-1, gonadotropins, and gonadal sex steroids, hence, the established treatment includes GH administration at infancy and sex hormone replacement in young adult that improve their growth and metabolism by enhancing muscle mass and reducing fat mass [64].

We found that the expression and function of Pw1 are particularly concentrated in the hypothalamus, pituitary gland, and gonads. Accordingly, our findings suggest that a primary Pw1 deficiency involves hypothalamic-pituitary-gonadal dysfunction at critical periods of postnatal development, leading to changes in sex steroids levels and GH/IGF signaling. Our observations are in line with several studies reporting that mouse mutants for paternally expressed genes typically display hypothalamo-pituitary phenotypes [65,66]. For instance, a loss of Grf1 or Dlk1 resulted in reduced GH content and secretion [67,68], while disruption of the imprinting domain encompassing Dlk1, Rtl1, and Dio3 resulted in transient perturbation in the GH/IGF-1 growth pathway with hypothyroidism [69]. Together, these observations pointed that imprinted genes play pivotal roles for the regulation of hypothalamo-pituitary-gonadal (HPG) axis governing growth, metabolism and reproduction.

Sexual dimorphism in mammalian growth is regulated by the sex steroid-dependent GH/IGF-1 pathway. During postnatal development, testosterone and 17-estradiol evoke pituitary production of GH in discrete manners [70,71]. GH in turn stimulates systemic IGF-1 through the JAK2/Stat5b pathways [72,73]. Estrogen-bound estrogen receptor (ER) also induces transcription of IGF-1 and IGF receptor through estrogen-responsive elements at their gene loci [74]. The activation of IGF-1/IGF1R signaling further activates transcription factors including ERs, forming a complex crosstalk between IGF-1 and sex steroid signaling that amplifies somatic growth in puberty [75]. Notably, sex differences during postnatal growth are diminished in the Stat5b deficient mice [76], the GH receptor (Ghr)- and Igf1-deficient mice [77]. Hence, reduced sexual dimorphism in Pw1 deficient young is likely due to an attenuated hypothalamic regulation of sex hormones and downstream GH/IGF-1 signaling.

Androgens and their cognate receptors regulate male secondary sexual differentiation [78], and testosterone deficiency is related to metabolic syndromes in men [79,80]. In androgen receptor deficient (ARKO) mice, late-onset obesity and fatty liver, as well as insulin- and leptin-resistance have been reported which is restricted to males [81-83]. Elevated levels of circulating leptin and leptin-resistance were also reported previously in the Pw1 adult males [31]. Leptin was shown to reciprocally regulate gonadal hormone production at central and peripheral levels [84] and the steroidogenesis in the Leydig cells was impaired in the leptin-deficient ob/ob males [85] concomitant with loss of skeletal masculinization [86]. Therefore, the defect in leptin signaling in Pw1 males may causally relate to further reduction in gonadal steroidogenesis. We note, however, that the Pw1 deficient phenotypes were not restricted to males, as both Pw1 males and females displayed delayed postnatal growth until weaning. Smaller body size of Pw1 mice before weaning may originate, at least in part, from changes arising during prenatal development, as observed by the lower body weight and myofiber numbers at birth [19]. Sex specificity of resulting phenotypes upon Pw1 loss is presumably due to sex dimorphic postnatal dependency to sex steroid signaling in males and females. Sex-dependent phenotypes in Pw1 deficiency have also been demonstrated in placental function, where female placenta compensates the loss of Pw1 better than male placenta [30,87]. This indicates that the sex-dependent Pw1 function starts much earlier than the offspring’s postnatal manifestation of sexually dimorphism in growth.

Testosterone exposure at specific phases of development, such as perinatal and pubertal periods, is crucial for the brain masculinization [56,58]. This study shows that Pw1 expression and sex steroid receptors highly overlap in key endocrine and neural tissues that regulate sex-specific behaviors. Specifically, we found that paternal loss of Pw1 function attenuated social dominance and correlates with the decreased expansion of the testosterone-sensitive vasopressinergic nuclei in the hypothalamus. In general, the sexually dimorphic nuclear volume/neuron number is ascribed to the sex steroid control of proliferation and cell death [88]. Given that Pw1 regulates cell survival and/or apoptosis at a cellular level, it is conceivable that Pw1 participates in sex-specific brain formation and neuronal proliferation/differentiation for various acquired behaviors. In contrast, aggression in males is ERα dependent [89] and the inter-male competitive ability reflects the level of ERα expression in BnST [45]. Pw1 may contribute to masculinization by regulating the expression of cognate receptors in sex-hormone dependent cells, thereby determining the dependency of organisms on sex steroid signaling.

Our finding suggests that Pw1 plays a role in regulating the proliferative and/or cell number in various tissues through sex steroid signaling during tissue remodeling in adult life. We demonstrate further that Pw1 expressing adult stem/progenitor cells are characterized by the presence of sex steroid receptors. Consistent with this observation, the number of Pw1 expressing cells in the heart were shown to increase during pregnancy [90], while the pregnancy-induced expansion of pancreatic β-cells inversely correlates with the levels of Pw1 expression [41]. Accumulating evidence also demonstrates the interaction of sex hormones in stem cell behaviors. Kim et. al. demonstrated that sex steroid hormones promote the establishment of the adult quiescent satellite cell pool through the HPG axis [91]. Likewise, ERα signaling has been shown to drive pancreatic β-cell replication in development as well as in post-injury regeneration [92]. How Pw1 loss of function alters adult stem/progenitor cell behaviors or steroid hormones per se affects pluripotency is beyond the scope of this study, however, we have shown previously that loss of Pw1 function leads to a loss in self-renewal capacity in adult muscle stem cells [19] as well as skin stem cell [17]. The link between self-renewal, stem cell competence and sex steroid hormones presents an emerging concept in stem cell biology.

This study uncovers a key role of the parentally imprinted gene Pw1 in establishing male-specific characteristics in metabolism, brain structure and behavior by modulating sex steroid pathways. We note that similar metabolic phenotypes observed in Pw1 deficient mice were reported in the progeny of human populations that have been subjected to overnutrition or prolonged nutritional deprivation [93], coinciding with a deregulation of several parentally imprinted genes [94]. Our findings show that Pw1 provides an epigenetic context that links energy metabolism to the generation of male-specific traits in mice. Analogous mechanisms may also underlie the sex differences in the metabolic syndrome in humans.

Materials and methods

Ethics statement

All experiments were in adherence to the institutional guideline for experiment and husbandry of laboratory animals. Approval for the animal (mouse) work performed in this study was obtained through review by the French Ministry of Education, Agreement #A751320.

Animals

The Pw1IRESnLacZ reporter mice [17] and the Pw1 deficient mice carrying targeted mutation Pw1Δ9 [29] were generated in our laboratory and maintained in C57BL/6J genetic background. Eight Pw1 males (two generation after the first deletion of Pw1 allele, designated F3) were used as breeders. In order to compare Pw1 offspring to their Pw1+/+ littermates, Pw1 breeder males under the age of 8 months old were crossed with young C57BL/6J females (purchased from Janvier Laboratory, Le Genest St Isle, France), and large litters with both genotypes were pooled for each analysis whenever available. All metabolic phenotypes presented in this study were obtained from offspring of F3-F4, and behavior phenotypes from F5 generations. Offspring were weaned at the age between 3–4 weeks, and housed in groups with littermates unless otherwise stated (maximum 6 per cage). Mice were kept on 12-hour light/dark cycle at 24°C with ad libitum access to water and standard chow diet.

Postnatal growth and body composition

Postnatal growth of Pw1 mice was measured along with Pw1+/+ littermates, which were nursed together by C57BL/6J females. Longitudinal body composition analysis was realized using Brucker minispec nuclear magnetic resonance (NMR) imaging (Brucker, USA), starting from 1 month of age. The lean and fat mass expressed in percentage (%) was converted to weight (g) in respect to the total body mass (g).

Blood glucose and food intake

Blood was sampled from the tail tip and glycemia was measured using an Accu-Check glucometer with disposable test strips (Roche Diagnostics, Basel, Switzerland). Food intake was assessed on N = 5–7 mice per genotype per sex, individually housed at 8 weeks of age and food consumption was measured twice a week for 3 weeks.

Plasma hormone measurement

Blood was collected either from the tail tip or from the facial vein of randomly fed animals at dormant period of circadian rhythms (11am-16pm), using Microvette CB300 capillary action blood collection tubes (Sarstedt, Nümbrecht, Germany). Plasma IGF, insulin, and testosterone levels were determined using ELISA kits for mouse (Chrystal Chem. Ltd, NJ, USA).

Glucose and insulin tolerance test

Oral glucose tolerance test (OGTT) and insulin tolerance test (ITT) were performed on 5–6 hours fasted mice. D-glucose (2 g/kg) was orally administered, and human insulin (0.25–0.55 units/kg) was intraperitoneally injected. For both tests, tail blood was sampled at 0, 15, 30, 60, and 90 minutes after the administration, and glycemia was immediately measured as described above. For monitoring insulin secretion upon OGTT, tail blood was collected at 0, 15, 30 minutes and subjected for ELISA protocol.

Quantification of fatty liver

To visualize lipid droplets, Oil red O-staining was performed in the 10um frozen sections of 8-month-old liver. Number of lipid droplets and the total oil Red positive area were quantified using ‘particle measurement’ in the Image-J software [95].

Gene expression analysis

Total RNA was isolated using TRIzol reagent (Ambion) and subjected to cDNA synthesis (Lifetechnology), followed by quantitative PCR analysis (LightCycler, Roche). The expression levels of target genes were normalized with the levels of a housekeeping gene coding TATA binding protein (Tbp). Primer sequences are available upon request.

Assessment of social dominance by confrontation tube test

We applied the social confrontation tube test previously described by Garfield et. al. on Grb10 deficient mice [46] to determine social dominance among Pw1 and Pw1 males. All mice used in the test were group-housed with littermates consist of both Pw1 and Pw1 genotypes (n = 4–6) at mature age between 10–12 months. Independent sets of mice were analyzed at 3 months old for comparison.

To determine the social hierarchy within a given litter, animals from a given litter were challenged with their littermate. On subsequent days, the same animals were also challenged with unfamiliar opponents from different litters. On the day of experiment, the animals were removed from home cages, and isolated for the duration of the test. All the tests were performed during daylight hours. The test apparatus was a 32-cm semi-transparent tube with adjustable internal diameter to prevent crossing over or turning of animals. Test animals were placed opposite ends of the tube and released simultaneously. Winners and losers were scored as one animal retreated from the tube completely. Tests in which the animal remained in the tube (five minutes maximum) were scored as draw. Inter-litter rank was determined by the number of winning within littermates (most win to least win = from 1, 2, 3, etc.).

Histology and Immunohistochemistry

X-Gal staining and immunohistochemistry were performed following standard protocols described previously [17]. All tissues were fixed in 4% paraformaldehyde and embedded in Optimal Temperature Compound (OTC) after cryoprotection in 20–30% sucrose. 12 μm cryosections were used for X-gal staining and co-immunofluorescences. The Pw1nLacZ reporter gene expression faithfully identified the Pw1 expressing cells [17], therefore, mouse anti-β-galactocidase antibody (1:500, Z3781: Promega) was used to represent Pw1 expression in combination with either rabbit anti-AR (1: 1000, Santa-cruz, sc-816), anti-ERα (1:1000, Santa-cruz, sc-542), or AVP (1:1000, AB1565: Millipore) in co-localization study. For brain structure analysis, mice were anesthetized with Ketamine/Xylazine and transcardially perfused with 4% paraformaldehyde. Brains were post-fixed overnight and 30μm serial sections were obtained from the entire brains using a vibratome. Comparable regions consist of 13–16 coronal cross-sections. The brain section images were collected from the same brain coordinates (Bregma: between -0.70 to -0.94 mm). Typically the AVP+ PVN structure spanning in four sections were subjected for analysis. Floating sections were incubated with rabbit anti-AVP (1, 1000, AB1565: Millipore) and detected using Vectastain ABC HRP kit (Vector Labs). Number of AVP+ cells and the total area were quantified using ImageJ software [95].

Statistical analysis

All figures and statistical analyses were generated using Prism 5 (GraphPad). Comparison between genotypes and sexes was performed using two-way ANOVA with Tukey’s multiple comparison test. For male specific variables, Mann-Whitney U-test was applied for independent measurement. Correlation between two variables was tested by a simple linear regression analysis. P value < 0.05 was considered statistically significant.

Insulin action in Pw1 adult males.

(A) Positive correlation of insulin levels with lean mass (top) and with body fat (bottom) was observed at at 3 months and 6 months of age, respectively. Correlation was determined by simple linear regression analysis. (B) Pancreatic insulin content and blood glucose levels at 10 months of age. Pancreatic insulin content was measured using acid-ethanol extraction protocol, followed by the insulin ELISA and normalized with total protein content. Columns, mean; bars, SEM; **, P< 0.01 by Mann-Whitney U test. (C) Typical images of Oil Red-O stained 8-month-old livers (top) and the quantification of lipid droplet number and size distribution (bottom), illustrating the differences in lipid content between the groups. P<0.001 by two-way ANOVA with Tukey’s multiple comparisons. Original magnifications: x200.

(TIF)

Click here for additional data file.

Pw1 colocalisation with sex hormone receptors.

Pw1 reporter expression predominantly co-localizing with the nuclear expression of sex hormone receptors in diverse cell types. (A) pancreatic islets, (B) mono- and dinucleated hepatocytes, (C) anterior pituitary cells, (D) adipocytes, and (E) skeletal muscle.

(TIF)

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Reduced aggressive behavior and social rank of Pw1 adult males.

(A) Spontaneous fights among group-housed littermates were monitored per cage and individuals involved in fights were identified. ***, P< 0.001 by Fisher’s exact test. (B) Result of social confrontation tube test in Pw1 (+/+) and Pw1 (pat-) littermates from six litters. Pw1 males (n = 10) and Pw1 littermates (n = 13) were subjected for confrontation against each other and their i) intra-litter rank and ii) winning rate against unfamiliar opponents was determined for each animal. Squared in red on the diagonal line show matches within littermates. Mice were derived from C57B6 x Pw1 breeding except for Litter 1, which was derived from C57B6 x Pw1 breeding). (C) Coronal sections of Pw1IRESnLacZ transgenic brain at 2.5 months of age revealing Pw1 reporter gene expression (X-gal staining) in sexually dimorphic brain regions. BnST, the bed nucleus of the stria terminalis; mPOA, medial preoptic area; PVN, paraventricular nucleus of hypothalamus; MeA, medial amygdala; PIR, piriform cortex (x40). (D) Size comparisons of AVP+ PVN area in four Pw1 male siblings from C57B6 x Pw1 breeding (Litter 1). Positive correlation was found between winning rate and AVP+ cells.

(TIF)

Click here for additional data file.

15 Jun 2021

Dear Dr SASSOON,

Thank you very much for submitting your Research Article entitled 'Paternally expressed gene 3 (Pw1/Peg3) promotes sexual dimorphism in metabolism and behavior' to PLOS Genetics.

The manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers appreciated the attention to an important problem, but raised some substantial concerns about the current manuscript. Based on the reviews, we will not be able to accept this version of the manuscript, but we would be willing to review a much-revised version. We cannot, of course, promise publication at that time.

All three reviewers feel that the statistical analysis of the data is not appropriate and would need to be addressed throughout the manuscript. The use of appropriate statistical tests are likely to change the results and therefore the conclusions that can be drawn from the data presented. There are also some data apparently missing from the submission. The conclusion that 'Pw1 deficient males develop a gender-specific global shift of body metabolism towards accelerated adiposity, diabetic-like insulin resistance, and fatty liver in adult life' has been contested as not a conclusion that can be drawn from the data that have been presented and the idea that there is sexual dimorphism in lean growth phenotype of Pat animals is not supported here. A re-analysis of the data would be necessary to provide a basis for a new manuscript to be considered as well as attention to the way the data are presented/organised and how the Figures are constructed to illustrate those data.

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John Greally

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PLOS Genetics

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: This submission reports on the characterisation of behavior and metabolism in a mouse model in which the imprinted Pw1/Peg3 gene has been ablated. Previous studies on this model and a previous independent targeting of this gene identified both metabolic and behavioral changes, albeit with some differences in findings in the different models/strain backgrounds as highlighted by the authors in their introduction. A key conclusion of this new study is that loss of Pw1/Peg3 results in gender-dependent metabolic and behavioural alterations whereby male mutant exhibit a “reduced masculinization of body composition, followed by a global shift of metabolism towards accelerated progression of obesity and related metabolic syndromes”.

Overall, this is an important finding which will be of interest to the wider genomic imprinting community and also those researching the sexually dimorphic consequence of early life adversities whereby, at least in some domains, males appear relatively more impacted than females.

A major comment on the manuscript is on the statistical approach. The authors have used paired comparisons but they should use ANOVA, with sex and genotype as factors. By using ANOVA, the authors will be able to make statistically supported conclusions regarding sex effects. For example, in Figure 1A this approach will likely show no effect of SEX and no interaction between SEX and GENOTYPE for body weight and loss of Peg3/Pw1 reduces body weight in both males and females. However, in terms of fat mass (Figure 1E & F), it is likely that an ANOVA would show an interaction between SEX and GENOTYPE, whereby loss of Peg3/Pw1 has little effect in females, but makes males more like WT females. Once this is done, the authors will need to update their abstract ect.

As an additional point, the authors keep switching comparisons between separating males and females, and Peg3/Pw1 and WT (e.g. Fig 1 D v E; Fig 2 C v G). They should be consistent in their comparisons which could also be resolved with the suggested statistical approach.

Tube test

There is some confusion regarding which animals are used in the tests to generate scores. The authors mention the use of “adult offspring derived from either Pw1+/+ or Pw1+/pat- males mated with C57/B6 WT females” and Pw1+/+ and Pw1+/pat littermates". Are the authors testing WT and mutant littermates against WT from a fully WT litter bred from Pw1+/+ males or, as indicated in S3, within litter bouts? It is also not clear what is meant by “found that the male progeny derived from Pw1+/pat- males were statistically less aggressive as compared to the ones derived from Pw1+/+ males?” The sentence implies that both mutant and WT males generated from Pw1+/pat- males are less aggressive than WT males from Pw1+/+ males? But there is no data on animals derived from Pw1+/+ males. A true test of social dominance takes into account rank within home cages which means animals must be tested against littermates, and rank order determined. Sentences should be modified in titles and elsewhere to reflect this.

Another point is that litter 1 is all WT and litter 4 has 2 mutant Peg3 males and no WT. If data from these two litters are excluded, are the findings still significant? Ideally for this test, litters should have mixed genotype in order for rank to be established in the context of genotype.

A third point is that the authors report that animals were initially housed together and then socially isolated before testing, as was done for the Garfield paper cited. There is a second paper on this model where the social group was maintained with no evidence of a social dominance phenotype (Rienecker et al., GBB 2020). This section and the M&M needs to be more clearly written and the conclusions reconsidered.

In the introduction, it would be helpful if the authors provided more explicit details on what behavioural and metabolic phenotypes have been reported in response to loss of Peg3 in their and other models. For example, has the timing of sexual maturity in males and females been reported? What precise behavioural phenotypes have been reported? Similarly, the authors should comment on previous reports of sexually dimorphic outcomes in response to loss of Peg3/Pw1 (Kim et al., Plos One 2013; Tunster et al., Front Dev Biol 2018). This would provide a more cohesive basis for the work undertaken and it’s interpretation.

Minor points

Introduction

Should “we characterized Pw1 deficient phenotypes in male and female mice at different postnatal development” read “we characterized Pw1 deficient phenotypes in male and female mice at different stages of postnatal development”

Results

“Consistent with changes in IGF-1 levels, we observed that gene expression levels of pituitary Gh, hepatic growth hormone receptor (Ghr) and Igf1 were reduced in Pw1+/pat- males as compared to Pw1+/+ littermates (Fig 2B).” change to “Consistent with changes in IGF-1 levels, we observed that gene expression levels of Gh in the pituitary gland and growth hormone receptor (Ghr) and Igf1 in the liver were reduced in Pw1+/pat- males as compared to Pw1+/+ littermates (Fig 2B).”

To avoid the use of “trends” suggest “Notably, these trends were inverted at 6-months of age that displayed higher insulin secretion and clearance in Pw1+/pat- males.” changes to “Notably, these patterns were inverted at 6-months of age that displayed higher insulin secretion and clearance in Pw1+/pat- males.”

“Prader-Willy syndrome (PWS)” change to “Prader-Willi syndrome (PWS)”

Reviewer #2: Review uploaded as an attachment.

Reviewer #3: Paternally expressed gene 3 (Pw1/Peg3) promotes sexual dimorphism in metabolism and behaviour.

Here the authors present interesting data regarding the function of the paternally-expressed imprinted gene Pw1/Peg3. While many experiments were performed to a good standard, I do not feel that the paper in its current form has sufficient novelty, explanatory power or statistical rigor to merit publication in PLoS genetics. Below I present my view of the weaknesses in the authors' argument, and some suggestions of how the work might be improved.

The authors state 3 key claims for this work.

Mice carrying paternally inherited Pw1/Peg3 allele manifest deficits in:

1) GH/IGF1 dependent growth before weaning.

2) Sex steroid-dependent masculinisation during puberty.

3) Insulin-dependent fat accumulation in adulthood.

Pw1/Peg3 paternal mutant mice (hereafter Pats) are born small and maintain their growth deficit into adult life, in both sexes.

• It is difficult to determine to what extent the growth retardation in Pat mice is due to early growth deficit, or the combination of this and reduced postnatal growth trajectory. The data in Fig1A should be also plotted as % wild-type (WT) weight, or and/growth rates should be calculated. In this way, deceleration of growth associated with Pw1/Peg3 deficit can be easily visualised and the timing pinpointed.

• In Fig1B body weight and glycemia are said to be correlated, yet no statistical value is stated. R or R-squared values as well as p-values should be reported.

• Food consumption is stated to be lower in mutant mice, yet this is expected if body mass is reduced. These data should be normalised to total body mass and lean body mass to determine if the mutant animals eat a disproportional amount, which would be indicative in a change in central metabolic homeostasis.

• The authors claim that because the Pat males resemble the WT females in size, there is therefore an absence of sexual dimorphism in the absence of Pw1/Peg3. This is faulty logic. In terms of the early life growth phenotype the males and females respond similarly to Pw1/Peg3 deletion (i.e., both are growth restricted to a similar extent, as is the body composition prior to 3 months). These data are obscured by the presentation in Figure 1D. These data should be shown as in E, with males and females on separate axes, allowing comparison between WT and Pat animals over time. Indeed, Fig1E clearly shows that both males and females have reduced lean mass to a similar degree.

• Further to this, the puberty-associated lean mass does not appear to be affected in the Pat males – compare the gradient of the lean mass increase in Fig1E between genotypes, it is identical. I would rather interpret this to indicate that the developmental deficit in Pw1/Peg3 has caused a reduction in muscle mass (as previously reported by this group and others), and that is maintained into adulthood.

• Statistics applied to repeated measures of body mass is inappropriate. Each animal at a different timepoint, nor fat and lean mass are independent variables, therefore pairwise statistics without multiple comparison correction are not appropriate. Consequently the authors should have performed a 2-Way ANOVA for each sex comparing the effects of time and genotype on lean and fat mass.

Link to IGF1/GH axis.

The authors report the interesting finding that IGF1 levels are reduced in 3-week old Pat mice. They attribute this to an alteration in the pituitary GH axis since in males pituitary Gh mRNA levels are reduced. This is an interesting finding, but there are some discrepancies.

• Growth retardation is evident from birth, yet in rodents GH does not drive hepatic IGF1 secretion until around the second postnatal week.

• The reduction in IGF1 is seen in both sexes (though the female data is underpowered) and is correlated with body size in both sexes, yet pituitary Gh and hepatic Igf1 mRNA levels are only different in males, what is the explanation for this?

• Regarding Figure 2C the authors state that there is sexually dimorphic behaviour regarding the male-specific rise in IGF1 post-puberty (which they say does not happen in Pat males). This way of showing the data distorts the picture. Clearly Pats of both sexes have an early IGF1 deficit (~200ng/mL vs ~350ng/mL) which is mostly normalised in the adults of both sexes. The data should be shown with sexes separated and WT and Pat on the same graph.

Metabolic phenotype

• Is the data in Fig2D from fasted or free-fed animals? The error bar appears to be missing for the 3mo pat females. What is the n for these data?

• Fig2G, the paper text says the animals are 6 months old, the figure legend say 10 mo, which is it?

• What is the n for Fig2H?

In conclusion, I do not feel that the data presented in this paper supports a conclusion that there is sexual dimorphism in lean growth phenotype of Pat animals. Therefore, the later association of this phenotype with masculinising hormones cannot be upheld. Moreover, while the observation of reduced IGF1 in Pats is interesting, it is present in both sexes and cannot be entirely explained by alteration of the pituitary GH axis. However, there is convincing evidence of male-specific adipose weight gain in the mutant animal in later life, and this is associated with the expected glucose intolerance, beta cell expansion and hyperglycaemia. While interesting, the authors are not able to provide an explanation for this at the molecular level. Moreover, such changes in body composition and metabolism have previously been reported by the Keverne/Curley group in an independent Pw1/Peg3 deletion model.

Behavioural phenotyping and localisation of PW1/PEG3 in the adult male brain.

• What statistics were performed on the behavioural data? This should be stated.

• Expression of PEG3 in the AVP neurons of the PVN has been previously reported: https://pubmed.ncbi.nlm.nih.gov/12399444/

• In Fig4A how many animals were assayed, this is not stated? Also, the stats is inappropriate, it should be analysed by One-Way ANOVA since the individuals at multiple timepoints are not independent. The error bar for the Pat at 9 weeks is missing.

• The ovelap between PW1/PEG3 expression and AR in the gonads is minimal – would this be sufficient to account for the reduction in testosterone observed in Fig4B?

• Would a transient reduction of testosterone observed be sufficient to account for the metabolic phenotype? The critical causal experiment would be to replace testosterone levels in mutant mice at this age, and see if the metabolic and behavioural phenotypes were reversed.

In conclusion, the observed transient reduction in testosterone levels, and association of PW1/PEG3 expression with sex hormone-responsive cell populations is a very interesting finding. However, at this stage the work appears preliminary with not much causal link to phenotype. A hormone replacement/conditional deletion experiment would be required to elevate this work.

**********

Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Genetics data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: Yes

Reviewer #2: No: Numerical data underlying graphs or summary statistics have not been provided in spreadsheet form in supporting information.

Reviewer #3: Yes

**********

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Reviewer #3: No

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28 Oct 2021

Submitted filename: Response to Reviewers v6.docx

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20 Nov 2021

Dear Dr SASSOON,

Thank you very much for submitting your Research Article entitled 'Paternally expressed gene 3 (Pw1/Peg3) promotes sexual dimorphism in metabolism and behavior' to PLOS Genetics.

The manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers appreciated the attention to an important topic but identified some concerns that we ask you address in a revised manuscript. These include a general attention to improve the language used and some specific issues around particular over-stretching of statements in the text from the data presented.

We therefore ask you to modify the manuscript according to the review recommendations. Your revisions should address the specific points made by each reviewer.

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Please be aware that our data availability policy requires that all numerical data underlying graphs or summary statistics are included with the submission, and you will need to provide this upon resubmission if not already present. In addition, we do not permit the inclusion of phrases such as "data not shown" or "unpublished results" in manuscripts. All points should be backed up by data provided with the submission.

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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To resubmit, you will need to go to the link below and 'Revise Submission' in the 'Submissions Needing Revision' folder.

[LINK]

Please let us know if you have any questions while making these revisions.

Yours sincerely,

Rebecca J Oakey

Guest Editor

PLOS Genetics

John Greally

Section Editor: Epigenetics

PLOS Genetics

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: The authors have undertaken considerable work to address comments made by reviewers which significantly improve the understanding of the manuscript. As a minor point, while tests for aggression have predominantly been developed on male rodents, non-pregnant females can exhibit aggression (Oliveira 2021). Moreover, there is evidence of increased aggression in lactating Peg3 mutant females (Champagne 2009). The authors have not tested aggression in females in this study. The title of the section related to tube test should be amended.

From

Paternal Pw1 deficiency reduces male-specific aggressive behaviors and social dominance.behavior.

To

Paternal Pw1 deficiency reduces aggressive behaviors and social dominance.behavior in males.

Reviewer #2: The authors have done a good job responding to my concerns and the manuscript is significantly improved. I suggest that the manuscript requires 'minor revisions' only because there are a few errors throughout the text. Some examples include "Body weight at 7-week-old revealed..." (this should read "Body weight at 7 weeks of age revealed...") and "Sex differences emerges..." (this should read "Sex differences emerge..."). I suggest the authors go through the manuscript one more time to correct these and other grammatical and sentence structure issues.

**********

Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Genetics data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: Yes

Reviewer #2: Yes

**********

PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

Reviewer #2: No

13 Dec 2021

Submitted filename: Responses to the reviewers 2nd.docx

Click here for additional data file.

20 Dec 2021

Dear Dr SASSOON,

We are pleased to inform you that your manuscript entitled "Paternally expressed gene 3 (Pw1/Peg3) promotes sexual dimorphism in metabolism and behavior" has been editorially accepted for publication in PLOS Genetics. Congratulations!

Before your submission can be formally accepted and sent to production you will need to complete our formatting changes, which you will receive in a follow up email. Please be aware that it may take several days for you to receive this email; during this time no action is required by you. Please note: the accept date on your published article will reflect the date of this provisional acceptance, but your manuscript will not be scheduled for publication until the required changes have been made.

Once your paper is formally accepted, an uncorrected proof of your manuscript will be published online ahead of the final version, unless you’ve already opted out via the online submission form. If, for any reason, you do not want an earlier version of your manuscript published online or are unsure if you have already indicated as such, please let the journal staff know immediately at plosgenetics@plos.org.

In the meantime, please log into Editorial Manager at https://www.editorialmanager.com/pgenetics/, click the "Update My Information" link at the top of the page, and update your user information to ensure an efficient production and billing process. Note that PLOS requires an ORCID iD for all corresponding authors. Therefore, please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field.  This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager.

If you have a press-related query, or would like to know about making your underlying data available (as you will be aware, this is required for publication), please see the end of this email. If your institution or institutions have a press office, please notify them about your upcoming article at this point, to enable them to help maximise its impact. Inform journal staff as soon as possible if you are preparing a press release for your article and need a publication date.

Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Genetics!

Yours sincerely,

Rebecca J Oakey

Guest Editor

PLOS Genetics

John Greally

Section Editor: Epigenetics

PLOS Genetics

www.plosgenetics.org

Twitter: @PLOSGenetics

----------------------------------------------------

Comments from the reviewers (if applicable):

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Data Deposition

If you have submitted a Research Article or Front Matter that has associated data that are not suitable for deposition in a subject-specific public repository (such as GenBank or ArrayExpress), one way to make that data available is to deposit it in the Dryad Digital Repository. As you may recall, we ask all authors to agree to make data available; this is one way to achieve that. A full list of recommended repositories can be found on our website.

The following link will take you to the Dryad record for your article, so you won't have to re‐enter its bibliographic information, and can upload your files directly:

http://datadryad.org/submit?journalID=pgenetics&manu=PGENETICS-D-21-00562R2

More information about depositing data in Dryad is available at http://www.datadryad.org/depositing. If you experience any difficulties in submitting your data, please contact help@datadryad.org for support.

Additionally, please be aware that our data availability policy requires that all numerical data underlying display items are included with the submission, and you will need to provide this before we can formally accept your manuscript, if not already present.

----------------------------------------------------

Press Queries

If you or your institution will be preparing press materials for this manuscript, or if you need to know your paper's publication date for media purposes, please inform the journal staff as soon as possible so that your submission can be scheduled accordingly. Your manuscript will remain under a strict press embargo until the publication date and time. This means an early version of your manuscript will not be published ahead of your final version. PLOS Genetics may also choose to issue a press release for your article. If there's anything the journal should know or you'd like more information, please get in touch via plosgenetics@plos.org.

10 Jan 2022

PGENETICS-D-21-00562R2

Paternally expressed gene 3 (Pw1/Peg3) promotes sexual dimorphism in metabolism and behavior

Dear Dr SASSOON,

We are pleased to inform you that your manuscript entitled "Paternally expressed gene 3 (Pw1/Peg3) promotes sexual dimorphism in metabolism and behavior" has been formally accepted for publication in PLOS Genetics! Your manuscript is now with our production department and you will be notified of the publication date in due course.

The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript.

Soon after your final files are uploaded, unless you have opted out or your manuscript is a front-matter piece, the early version of your manuscript will be published online. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.

Thank you again for supporting PLOS Genetics and open-access publishing. We are looking forward to publishing your work!

With kind regards,

Olena Szabo

PLOS Genetics

On behalf of:

The PLOS Genetics Team

Carlyle House, Carlyle Road, Cambridge CB4 3DN | United Kingdom

plosgenetics@plos.org | +44 (0) 1223-442823

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