# Hormone profiles of the African pygmy mouse Mus minutoides, a species with XY female sex reversal

Frédéric Veyrunes, Julie Perez, Louise D Heitzmann, Paul Saunders, Laurent Givalois

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Frédéric Veyrunes, Julie Perez, Louise D Heitzmann, Paul Saunders, Laurent Givalois. Hormone profiles of the African pygmy mouse Mus minutoides, a species with XY female sex reversal. Journal of Experimental Zoology Part A: Ecological and Integrative Physiology, 2023, 341, pp.130 - 137. 10.1002/jez.2767. hal-04782336

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# Hormone profiles of the African pygmy mouse *Mus minutoides*, a species with XY female sex reversal

Frederic Veyrunes<fnref n="1" /> D
Laurent Givalois<fnref n="2" />,<fnref n="3" />,<fnref n="4" />

Julie Perez<fnref n="1" />

Louise D. Heitzmann<fnref n="1" />

Paul A. Saunders<fnref n="1" />

**Correspondence**

Frederic Veyrunes, ISEM, Institut des Sciences de l'Evolution de Montpellier UMR 5554, CNRS, Université Montpellier, IRD, Montpellier, France.

Email: frederic.veyrunes@umontpellier.fr

**Funding information**

ANR grant SEXREV

**Abstract**

In mammals, most sex differences in phenotype are controlled by gonadal hormones, but recent work on transgenic mice has shown that sex chromosomes can have a direct influence on sex-specific behaviors. In this study, we take advantage of the naturally occurring sex reversal in a mouse species, *Mus minutoides*, to investigate for the first time the relationship between sex chromosomes, hormones, and behaviors in a wild species. In this model, a feminizing variant of the X chromosome, named X*, produces three types of females with different sex chromosome complements (XX, XX*, and X*Y), associated with alternative behavioral phenotypes, while all males are XY. We thus compared the levels of three major circulating steroid hormones (testosterone, corticosterone, and estradiol) in the four sex genotypes to disentangle the influence of sex chromosomes and sex hormones on behavior. First, we did not find any difference in testosterone levels in the three female genotypes, although X*Y females are notoriously more aggressive. Second, in agreement with their lower anxiety-related behaviors, X*Y females and XY males display lower baseline corticosterone concentration than XX and XX* females. Instead of a direct hormonal influence, this result rather suggests that sex chromosomes may have an impact on the baseline corticosterone level, which in turn may influence behaviors. Third, estradiol concentrations do not explain the enhanced reproductive performance and maternal care behavior of the X*Y females compared to the XX and XX* females. Overall, this study highlights that most of the behaviors varying along with sex chromosome complement of this species are more likely driven by genetic factors rather than steroid hormone concentrations.

**KEYWORDS**

African pygmy mouse, behavior, sex chromosomes, sex-reversed females, steroid hormones

<figure><img src="image_2.png" /></figure>

<footnote n="1">¹ISEM, Institut des Sciences de l'Evolution de Montpellier UMR 5554, CNRS, Université Montpellier, IRD, Montpellier, France</footnote>

<footnote n="2">²MMDN, Molecular Mechanisms in Neurodegenerative Dementia Laboratory, Université Montpellier, EPHE-PSL, INSERM U1198, Montpellier, France</footnote>

<footnote n="3">³Department of Psychiatry and Neurosciences, CR-CHUQ, Faculty of Medicine, Laval University, Québec City, Canada</footnote>

<footnote n="4">⁴CNRS, Paris, France</footnote>
# 1 | INTRODUCTION

The developmental processes leading from a single genome (at the exception of the small sex-specific region when sex chromosomes are present) to two sexually dimorphic phenotypes are remarkably complex and remain poorly understood (Mank, 2017; Naqvi et al., 2019). Sex development can be divided into two main components: sex determination and sex differentiation. In mammals, sex determination is the process by which the bipotential embryonic gonads develop into either testes or ovaries, which depends exclusively on the XY sex chromosomes; sex differentiation involves the subsequent events that ultimately produce either the male or female phenotype, driven mostly by gonadal steroid hormones (review in Arnold, 2012). However, in the last two decades, several studies on genetically manipulated mice have challenged this view, highlighting the direct influence of the sex chromosomes on sex differences in behavior and brain, such as some aspects of aggressiveness, parental behavior, food consumption, social interactions, anxiety, or brain structures and functions (e.g., Arnold, 2012, 2019; Corre et al., 2016; Gatewood et al., 2006; Lentini et al., 2013; De Vries et al., 2002).

Although mammalian XX/XY sex determination is extremely conserved, a few species escape this convention and present unusual sex determination systems (Parma et al., 2016; Saunders & Veyrunes, 2021). The African pygmy mouse, *Mus minutoides* is the latest addition to this short list of odd mammals, owing to the presence of a third sex chromosome named X\* (Veyrunes et al., 2010). This is a mutant copy of the X chromosome that differs genetically from the ancestral X and is feminizing. Therefore, three female genotypes are found: XX, XX\*, and X\*Y, while all males are XY. The X\*-linked mutation at the origin of the sex reversal is still unknown. Previous works showed that X\*Y females may represent up to 75% of the females in some wild populations (Veyrunes et al., 2010). They are fully fertile with typical ovaries without apparent signs of testicular tissue (Rahmoun et al., 2014), and they have a higher lifetime reproductive success than the XX and XX\* females, including larger litter size despite the cost of losing lethal YY embryos (Saunders et al., 2014), and they also show some enhanced maternal care behaviors (e.g., pup retrieval) compared to other females (Heitzmann et al., 2023). On the other hand, they display some masculinized behaviors, such as greater aggressiveness, lower anxiogenic response (Saunders et al., 2016), higher bite-force performance (Ginot et al., 2017), and poor nest-building skills (Heitzmann et al., 2023). Thus, the X\* effect goes well beyond sex reversal and is at the origin of a complex X\*Y phenotype. In the African pygmy mouse, chromosomal sex (XX vs. XY), gonadal sex (ovaries vs. testes), and phenotypic sex (here mostly sexually dimorphic behaviors) are partially uncoupled: some individuals carrying a Y chromosome bear fully functional ovaries, but display partial masculinization of behaviors. This unique system for a wild mammal species offers a promising model to investigate the influence of sex chromosome complements versus steroid hormones on sexual dimorphism in brain and behavioral traits. To date, the behavioral differences observed in the three *M. minutoides* female genotypes have been mostly attributed to genetic effects (Ginot et al., 2017;

Saunders et al., 2016) on the basis that X\*Y females are characterized by complete sex reversal with typical ovarian anatomy (no ovotestes) and normal anogenital distance. This suggests a low level of circulating androgens (Rahmoun et al., 2014), but the question remains open. In this study, we thus compared the levels of three circulating steroid hormones (testosterone, corticosterone and estradiol) in the four sex genotypes (XY males, and XX, XX\*, and X\*Y females) to assess hormonal profiles and disentangle the influence of sex-linked genes and steroid hormones on the masculinized behaviors observed in X\*Y females (Ginot et al., 2017; Heitzmann et al., 2023; Saunders et al., 2016).

# 2 | MATERIALS AND METHODS

## 2.1 | Animals

Mice were bred in our own breeding colony (CECEMA, University of Montpellier), established from wild-derived animals (for further details see Saunders et al., 2014). Females were housed in same-sex groups of three to four individuals per cage, and males were housed with brothers or in individual cages (to prevent agonistic behaviors). They were provided with ad libitum food and water, and the light regime was set to 15:9 h (light:dark). The genotype of females was determined by polymerase chain reaction amplification of the Y-specific Sry gene from tail biopsies or culture of fibroblasts from skin biopsies as previously described (Veyrunes et al., 2010). The animal age varied between 8 and 10 months, and all blood/urine samples were collected at the same time of the day (±2 h). Experimental procedures were in accordance with the European guidelines and were approved by the French Ethical Committee on Animal Care and Use (No. CEEA-LR-12170).

## 2.2 | Hormone assays

As the African pygmy mouse is one of the smallest mammal species in the world (Boel et al., 2020), the amount of blood that could be collected from each individual was limited. This prevented testing all samples in duplicate and measuring the three steroid hormones in the same animal (one mouse for each hormone). Moreover, for a limited number of animals, reduced volumes of plasma, serum, or urine were collected and had to be used diluted with the kit's sample diluents or SAMPLEDIL (ref: KLZZ731). Nevertheless, all samples were within the range of robust minimal assay sensitivity. The numbers of animals used for the different measurements are listed in Table 1.

### 2.2.1 | Testosterone

Animals were euthanized by decapitation and trunk blood was collected in Eppendorf tubes. After 30 min at room temperature, blood samples were centrifuged and serum was collected and stored
<table><caption>TABLE 1 Number of mice involved in each hormone assay.</caption><thead><tr><th rowspan="2">Sex<br>Genotype</th><th colspan="3">Female</th><th>Male</th></tr><tr><th>XX</th><th>XX*</th><th>X*Y</th><th>XY</th></tr></thead><tbody><tr><td>Testosterone</td><td>10</td><td>10</td><td>10</td><td>10</td></tr><tr><td>Corticosterone (control)</td><td>8</td><td>10</td><td>10</td><td>13</td></tr><tr><td>Corticosterone (stress)</td><td>8</td><td>12</td><td>13</td><td>11</td></tr><tr><td>Estradiol (diestrus)</td><td>12</td><td>11</td><td>13</td><td>NA</td></tr><tr><td>Estradiol (estrus)</td><td>13</td><td>13</td><td>12</td><td>NA</td></tr></tbody></table>

Abbreviation: NA, not applicable.

at -20°C. Testosterone concentrations were measured using an enzyme-linked immunosorbent assay (ELISA) kit (IBL International ref: RE52151) following the manufacturer's instructions. To fall within the sensitivity range of the assay, serum from males were tested at two dilutions (1:5 and 1:10), and the hormone concentration was calculated by the mean of values obtained at each dilution. This also allowed to obtain duplicates that confirmed a good assay precision with an intra-assay coefficient of variation at 8.4%.

### 2.2.2 | Corticosterone

Plasma corticosterone concentrations were measured at baseline (no stress) and after stress induction. For baseline corticosterone, mice were taken from the cage and rapidly decapitated to collect trunk blood. For the poststress corticosterone measurement, mice were placed in a glass beaker (diameter 12.5 cm), filled with 1.4 L of water (22 ± 1°C), and forced to swim for 10 min, then animals were rapidly decapitated. In both cases, blood samples were collected in ethylenediaminetetraacetic acid-coated Eppendorf tubes, centrifuged at 4°C, and plasma was collected and stored at -20°C. Corticosterone concentrations were measured with a conventional ELISA kit (ENZO Life Sciences; ref: ADI-900-097) following the manufacturer's instructions, as previously reported (Canet et al., 2020).

### 2.2.3 | Estradiol

the inconclusive results obtained with serum (most concentrations below the standard curve) led to its quantification in urine samples that have shown to give interpretable results in mice (e.g., deCantazaro et al., 2004). Urine was collected upon handling mice and pipetted on a clean surface, and stored at -20°C. As estradiol concentration varies along the estrous cycle (e.g., Wood et al., 2007), this study focused arbitrarily on the diestrus and estrus stages. To determine the estrous cycle phase, just after urine collection, females were held by the tail, and the vagina was flushed twice with 10 µL of sterile water using a pipette tip. The vaginal smear was placed on a glass slide, stained with Giemsa, and viewed under a light microscope (×10 magnification). The diestrus phase was defined by the exclusive presence of leukocytes, and the estrus phase by the presence of large

and squamous-type epithelial cells without nucleus. Urinary estradiol concentrations were measured with an ELISA kit (IBL International ref: 30121045) following the manufacturer's instructions and protocol in deCatanzaro et al. (2004).

## 2.3 | Statistical analysis

Final steroid concentrations were calculated from hormone-specific standard curves corrected for an individual's sample volume. All statistical analyses were performed on R v3.6.3. We used a nonparametric test, Kruskall-Wallis, to test differences in testosterone levels between the four genotypes. Post-hoc pairwise comparisons were made using *Dunn test* with a Benjamini-Hochberg *p* value adjustment to account for multiple testing. Baseline and stress-induced corticosterone concentration were analyzed separately fitting a linear model with individuals' genotype as the only predictor. We used a log-transformation of corticosterone baseline level to account for heteroscedasticity. Post-hoc pairwise comparisons were made using *emmeans* package with Tukey adjustments of *p* values to account for multiple testing. We also fitted a linear model to investigate estradiol level using "genotype" and "estrous cycle phase" (estrus vs. diestrus) as predictors. We included an interaction between both predictors with the assumption that female estradiol level may vary with the estrous cycle and independently between genotypes (i.e., estradiol variation between estrus and diestrus states may differ between genotypes). Post-hoc comparisons were made using *emmeans* package with Tukey adjustments of *p* values to account for multiple testing.

# 3 | RESULTS AND DISCUSSION

In *M. minutoides*, sex-reversed females show a combination of phenotypic traits not seen in any other individuals. For some phenotypic traits, X*Y females can be viewed as "super females" as called by Knight (2017), with a greater reproductive output (Saunders et al., 2014) and enhanced maternal care (for instance, they retrieve pups faster and with a higher probability; Heitzmann et al., 2023) compared to XX and XX* females. Conversely, for other aspects, they are more similar to males: lower anxiety-related behavior, enhanced territorial aggressiveness (Saunders et al., 2016), and impaired nest-building behavior (Heitzmann et al., 2023). That similarity between X*Y females and males extends to whole-organism performance with higher bite force than other females, and even a tendency (though not statistically significant) to bite harder than males (Ginot et al., 2017). In summary, sex reversal changes not only the gonadal sex, but also some behavioral and life history traits, and is at the origin of a new female sexual phenotype (Heitzmann et al., 2023). Therefore, to untangle the role of genetic versus hormonal effects on these behavioral differences observed among females, we compared the levels of three major circulating sex steroid hormones in the different genotypes.
Testosterone is ubiquitous in vertebrates and is the key mediator in the expression of many male morphological and behavioral traits (Giammanco et al., 2005; McEvoy et al., 2015; Preston et al., 2012; Trainor & Marler, 2002; and references therein). More particularly, it is thought to be the major sex steroid regulating male aggressive behavior and dominance, in a wide variety of fish, reptile, bird, and mammal species (including humans). For example, high rates of aggression generally correlate with elevated testosterone levels (Cantarero et al., 2015; Denson et al., 2018; Giammanco et al., 2005; St Clair Yewers et al., 2017; Yu & Shi, 2009), and male aggressiveness can be reduced by castration and restored by administration of androgens (Giammanco et al., 2005). In vertebrates, testosterone is mostly produced by Leydig cells in testes, while smaller quantities are also produced by ovaries, and adrenal glands in both sexes (Nelson, 2010). Although aggressiveness and testosterone may be lower in females, the more limited literature data shows a similar positive relationship between testosterone and aggression, albeit less so (Denson et al., 2018; Giammanco et al., 2005). In the African pygmy mice, we found a high sexual dimorphism of serum testosterone levels as expected (p < 0.001 between males and XX and XX\* females; p = 0.002 between males and X\*Y females); however, we did not detect any difference among the three female genotypes (Figure 1). Therefore, the aggressive behavior of X\*Y females (in a context of territorial aggression, they show an attack latency at least twice shorter and a number of aggressions at least twice higher than in the other female genotypes, and concerning maternal aggression, they have three-time higher probabilities than other females to severely aggress mates; Heitzmann et al., 2023; Saunders et al., 2016) and the stronger bite force (another indicator of aggressive performance; Ginot et al., 2017) are unlikely to be directly mediated by the circulating testosterone level. This result adds to the increasing evidence that the relationship between aggression and testosterone is not straightforward (e.g., Cantarero

<figure><img src="image_3.png" /><figcaption>**FIGURE 1** Serum testosterone concentrations of the four sex genotypes, XX, XX\*, and X\*Y females (F), and XY males (M). Values are means ± 95% IC. \*\*\*p < 0.001, \*\*p < 0.01.</figcaption></figure>

et al., 2015; Huyghe et al., 2009; McEvoy et al., 2015; St Clair Yewers et al., 2017), which might be explained by the fact that aggressiveness is highly context-dependent (e.g., social experience or motivation), includes complex behavioral patterns with different underlying physiological mechanisms and metabolic pathways, and is influenced by other sexual hormones as well. This is especially relevant in females: in females more than males, it would be simplistic to reduce hormone-mediated aggressive behavior to circulating testosterone concentration only (reviews in Denson et al., 2018; Trainor et al., 2009). More importantly, exposure to androgen levels during prenatal development can also determine the propensity to show adult aggressive behaviors, this is called the organizational effect (Phoenix et al., 1959; review in Arnold, 2009). The level of perinatal androgens is correlated to the number of male womb-mates and to the intrauterine position, that is, female fetuses developing between two males tend to have higher fetal testosterone levels and to be more aggressive at adulthood than female fetuses developing without adjacent males (review in Ryan & Vandenbergh, 2002). Interestingly, in *M. minutoides*, due to a complex sex chromosome drive the more aggressive X\*Y female fetuses tend to be surrounded by less male fetuses in-utero than XX female fetuses (Saunders et al., 2022), which does not support an organizational (perinatal) effect of androgen on aggressive behaviors in X\*Y females. The clear-cut contrast of aggressiveness in controlled experiments between X\*Y females and XX and XX\* females may actually suggest a direct effect of the sex chromosome complement on the behavior of X\*Y females. Specifically, the Y chromosome includes at least two genes, *Sts* and *Sry*, that have been suggested to influence aggressiveness in mice (Lee & Harley, 2012; Mortaud et al., 2010). Two other important candidates are carried by the X chromosome, the androgen receptor which mediates the actions of testosterone (Davey & Grossmann, 2016), and the monoamine oxidase A (*MaoA*) which is well known to influence aggressiveness (Cases et al., 1995; Dorfman, 2014; Freudenberg et al., 2015; McDermott et al., 2009). Following the rise of the X\* chromosome, differences of expression could have evolved between the X and X\* copies of these genes.

Although glucocorticoids (i.e., mainly corticosterone in rodents) are involved in aggression as well, they are best known as stress hormones (Bonier et al., 2009; Romero, 2004). Corticosterone secreted by adrenal glands is increasingly employed as a physiological indices of the relative health conditions and as a stress index of individuals or populations (review in Bonier et al., 2009). The interpretation of corticosterone concentrations is complicated by the fact that its secretion can be classified into two types based on the secretion dynamics, the receptors bound, and the physiological and behavioral effects (Rensel & Schoech, 2011; Romero, 2004). The baseline corticosterone is the amount of hormone secreted on a continuous low-level basis and is involved in the maintenance of energetic balances and homeostasis. Elevated baseline corticosterone levels are assumed to predict a low relative fitness of individuals, associated with multiple deleterious characteristics, such as chronic stress, poor body conditions, reallocation of resources away from reproductive behaviors, and infertility (e.g., Romero & Wikelski, 2001;
Strasser & Heath, 2013; review in Bonier et al., 2009). The second type of secretion takes place when a stressful stimulus is perceived, stress responses are thus initiated which trigger activation of the hypothalamic-pituitary-adrenal axis with a rapid and intense release of corticosterone. This abrupt increase of circulating corticosterone modulates specific physiological and behavioral traits to help the organism to cope with the stressor, thus promoting short-term survival, and will remain high for a few hours (Romero, 2004; Wingfield & Sapolsky, 2003). Acute elevation of corticosterone has been associated with escape behavior, suppression of territorial behavior and reproductive activity, reduction of parental care, and energy reallocation (Miller et al., 2009; Sapolsky et al., 2000; Wingfield et al., 1998). Therefore, differences in baseline or stress-induced corticosterone concentrations among individuals may have completely different physiological and behavioral consequences, and it is essential to consider both. In *M. minutoides*, X*Y females and XY males display lower baseline corticosterone levels than XX and XX* females; concentrations were significantly lower in sex-reversed females than in XX* (*p* = 0.003) and XX females (*p* = 0.01), tended to be lower though not significant in males than in XX* (*p* = 0.054) and XX females (*p* = 0.14), and were similar between X*Y females and males (*p* = 0.54), as well as between XX and XX* females (*p* = 0.99); whereas the stress-induced concentrations were comparable in the four groups (Figure 2a,b). The lower baseline corticosterone concentrations in X*Y females and males suggest lower anxiety, and is congruent with behavioral traits measured in captivity, that is, X*Y females and males have a lower anxiogenic response to novelty (latency before movement in a new environment) compared with XX and XX* females (Saunders et al., 2016). According to the Glucocorticoid-Fitness hypothesis (Bonier et al., 2009), the reduced baseline corticosterone level may also explain the higher breeding success of the X*Y females; they are less stressed and, therefore, they reproduce better. Indeed, X*Y females display a greater probability of breeding and produce significantly more offspring during their lifetime than other females (Saunders et al., 2014). On the whole, this might suggest that anxiety-related behaviors are influenced by sex hormones only; however, the finding that animals harboring a Y chromosome have lower baseline corticosterone than XX and XX* females, rather suggests a direct effect of the sex

chromosome complements. We show that irrespective of the gonadal sex, the chromosomal sex also may have an impact on steroid concentrations, which in turn may influence behaviors.

Finally, we investigated estradiol levels in females since the reproductive performances (Saunders et al., 2014) and the maternal care behavior (Heitzmann et al., 2023) of the three female genotypes differ. Estrogens, particularly estradiol, exert powerful influences on the mammalian female reproductive physiology and behavior. For example, estradiol plays critical roles in regulating estrous cycling, preparing the uterus for embryo implantation, inducing sexual receptivity and facilitating lordosis, and on the onset and maintenance of maternal behaviors (reviews in Bridges, 2015; Numan, 2020). In *M. minutoides*, urinary estradiol levels were comparable among female genotypes at diestrus, and marginally lower in XX females during estrus (*p* = 0.053 and *p* = 0.11 in comparison to X*Y and XX*, respectively) (Figure 3). Our data also show a trend for higher estradiol levels at estrus than diestrus in X*Y (*p* = 0.048) and XX* females (*p* = 0.08), as reported in the house mouse (e.g., Wood et al., 2007), but not in XX females (*p* = 0.92) (Figure 3). In our laboratory colony, XX females show lower breeding performance and lower pup retrieval behavior than X*Y females (Heitzmann et al., 2023; Saunders et al., 2014), which fits with the trend in estradiol level differences observed. However, the finding that XX* have comparable reproductive traits and maternal care behaviors to XX females, but the same estradiol concentration as X*Y females rules out any causal link. Conversely, these results strongly suggest an advantageous feature of the X*Y complement, and it is tempting to attribute it to the X* chromosome that evolved toward a female uniparental transmission and may have accumulated female-beneficial genes. But this hypothesis does not explain why XX* females, which also harbor the X* chromosome and retain random inactivation of the X and X* chromosomes (Veyrunes & Perez, 2018), do not present a more intermediate phenotype. Other hypotheses are discussed by Saunders et al. (2014), particularly concerning the X chromosome copy number (one in X*Y vs. two in XX and XX*) that has been shown to influence the expression of hundreds of autosomal genes in the house mouse (Wijchers et al., 2010). Finally, the low level of estradiol concentration and the absence of detectable variations between estrus and diestrus in XX females are noteworthy, and might be

<figure><img src="image_4.png" /><figcaption>**FIGURE 2** Plasma corticosterone concentrations of the four sex genotypes, XX, XX*, and X*Y females (F), and XY males (M). (a) Baseline and (b) stress-induced concentrations. Values are means ± IC 95%. **$p < 0.01$, *$p < 0.05$.</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>**FIGURE 3** Urinary estradiol concentrations of the three female genotypes, XX, XX*, and X*Y, at diestrus and estrus stages. Values are means ± IC 95%. \* *p* < 0.05.</figcaption></figure>

related to their apparent smaller ovaries and fewer follicles compared to XX* and X*Y females (Rahmoun et al., 2014); this result requires further investigations.

Overall, there is a substantial body of evidence about sex differences in many aspects of brain development and function and also behavior (reviews in Arnold, 2012; Arnold & Chen, 2009; Ngun et al., 2011; De Vries & Forger, 2015). Although such differences are mostly due to steroid hormone levels, recent data indicate that the sex-specific genetic architecture and expression of sex-linked genes also play a direct role on sexual dimorphism (e.g., Arnold, 2019; Corre et al., 2016; Gatewood et al., 2006; Lentini et al., 2013; McPhie-Lalmansingh et al., 2008; De Vries et al., 2002). The direct effect of the sex chromosome complement on behavior and brain has been mostly assessed using transgenic mouse strains, called the “Four core genotypes” model (see Cox et al., 2014; De Vries et al., 2002 for a description). But these strains show some limitations, including the fact that these mice are genetically manipulated and that the Sry gene is absent in XY females (Ngun et al., 2011). The sex determination system of *M. minutoides*, shaped by one million years of natural selection (Veyrunes et al., 2013), in which gonadal sex and chromosomal sex are partially dissociated, offers a unique framework to investigate the relationship between sex chromosomes, hormones, and behaviors in a wild species. The hormonal profiles of each female genotype obtained in this study suggest that most of the sexually dimorphic behaviors of this species are more likely driven by genetic factors rather than steroid hormone concentrations. However, the results have to be put into perspective as there are limitations. First, it would be too simplistic to reduce one behavior to one hormone; second, hormone concentrations are not fixed and vary with environment and social context, so the measurements of hormone levels at a single point in time may not be representative of the context in which the behavioral experiments have been performed; third, behaviors and hormone concentrations have not been

investigated on the same specimens. Further analyses are thus required, for instance by using the same animals and the same point in time of sampling for behavioral experiments and hormonal concentrations. Accordingly, this study does not permit to completely rule out the effects of sex hormones on these behaviors, but at least highlights the absence of main differences on hormonal profiles between sex-reversed females and XX and XX* females, and opens avenues for future studies on the link between behaviors, hormones, sex chromosomes, and genes. As such, the Y-linked Sry gene provides a prime candidate to investigate the genetic basis of behavioral uniqueness of X*Y females. We previously demonstrated that it is expressed in X*Y female brain (Rahmoun et al., 2014), and outside its maleness function in testes, emerging evidence indicates that Sry has also multiple brain functions related to behavior. In mice for instance, it has been shown that Sry can stimulate the expression of tyrosine hydroxylase, the rate-limiting enzyme for dopamine synthesis, a neurotransmitter that is involved in many social behaviors, including aggression, behavioral response to stress, and maternal care (Dewing et al., 2006; Lee & Harley, 2012; Mahadevia et al., 2021; Rosenfeld, 2017; Sekido, 2014; Scott et al., 2015). In addition, Sry has been identified to bind directly to the promoter of *MaoA* and mediate the expression of this gene involved in aggressive behaviors (Wu et al., 2009). These results also highlight the need to consider naturally occurring sex reversal with alternative phenotypes, as observed in the pygmy mouse, in the bearded dragon (Castelli et al., 2021; Li et al., 2016) or in the Nile tilapia (Dussenne et al., 2020; Gennotte et al., 2017), to better understand the large spectrum of behaviors under the direct influence of genetic factors and as such, to challenge the classical view of steroid hormone predominance in the induction of sex differences in nongonadal organs and behavior.

## ACKNOWLEDGMENTS

We are grateful to RAM-CECEMA animal facility of University Montpellier, Marie Challe for her help in maintaining the breeding colony, Laurence Meslin for the illustration, and anonymous reviewers for insightful comments to the manuscript. This study was supported by the ANR grant SEXREV (no. 18-CE02-0018-01).

## CONFLICT OF INTEREST STATEMENT

The authors declare no conflict of interest.

## DATA AVAILABILITY STATEMENT

The data that support the findings of this study are openly available in Hormone profiles of the African pygmy mouse *Mus minutoides*, at https://doi.org/10.5281/zenodo.10089782.

## ORCID

Frederic Veyrunes http://orcid.org/0000-0002-1706-9915

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**How to cite this article:** Veyrunes, F., Perez, J., Heitzmann, L. D., Saunders, P. A., & Givalois, L. (2024). Hormone profiles of the African pygmy mouse *Mus minutoides*, a species with XY female sex reversal. *Journal of Experimental Zoology Part A: Ecological and Integrative Physiology, 341*, 130-137.
https://doi.org/10.1002/jez.2767