The definition of biological sex has become a renewed focus of societal debate, fuelled by the conflation of biological principles with discussions of human gender diversity. Here, we argue that conceptual clarity critically depends on separating these domains. Drawing on evolutionary theory and empirical evidence, we maintain that biological sex is best defined as a binary classification of male and female reproductive strategies rooted in anisogamy, characterised by the production of two discrete gamete types of different sizes. We stress that gamete size constitutes the ultimate criterion for biological sex and that this definition applies consistently across sexual systems, from separate-sexed species to hermaphrodites, irrespective of variation in karyotype, hormonal profile, somatic phenotype, or behaviour. Further, we emphasise that evolutionary insights offer a coherent explanation for recurring, though not universal, associations between biological sex and patterns of sex-specific selection, sexual dimorphism and parental care. We conclude that the definition of biological sex as a binary classification based on gamete size is a powerful scientific framework compatible with the diversity of sexual phenotypes found in anisogamous organisms and distinct from the concept of human gender.

Somatic cell reprogramming into induced pluripotent stem cells (iPSCs) has been achieved in various mammals; however, assessing iPSC contribution to full-term chimeras beyond laboratory rodents remains challenging. Here, we demonstrate induction of pluripotency in male and female fibroblasts from the African pygmy mouse (APM), one of the smallest mammals. Using transcription factors and small molecules, we derived expandable APM-iPSCs that express pluripotency markers, differentiate into various cell types in vitro, and form gastruloids. Injection of APM-iPSCs into house mouse blastocysts generated full-term interspecies chimeras demonstrating extensive contribution to various tissues, including testicular germ cells. Notably, high APM contribution in organs such as heart and testes correlated with reduced organ size compared with mouse organs. Lastly, injection of APM-iPSCs into mouse blastocysts carrying a Pax7 ablation system enabled substantial production of APM muscle stem cells in chimeras. Collectively, this study establishes APM-iPSCs as a developmental model for pluripotency, differentiation, and interspecies chimerism.

Sex differences in social aggression are widespread across the animal kingdom, with males typically displaying greater territoriality. While this dimorphism has traditionally been attributed to sex hormones, sex chromosomes can also contribute to it independently of hormonal influence. In the African pygmy mouse Mus minutoides, naturally occurring sex-reversed XY females (named X*Y due to a mutation on the X chromosome) are highly territorial in comparison to the other female genotypes present in the population (XX and XX*). However, the molecular basis of this phenotype remains unknown. Here, we evaluate molecular factors, known to correlate with aggressiveness, following a standardized behavioural assessment of aggression. We focus on i) the androgen pathway by quantifying testosterone serum levels and expression of its receptor in the brain; ii) the brain dopaminergic system through expression of dopamine-regulating genes, including the sex-determining Sry gene; and iii) neuroendocrine circuits, via vasopressin and oxytocin expression. These systems, although distinct, converge on hypothalamic-limbic circuits that regulate social behaviour and threat responses, providing an integrated framework for the modulation of aggression. Contrary to earlier reports, we found reduced level of aggression in X*Y females, likely to reflect a breeding facility effect. We also observe no correlations between aggressiveness and androgen levels or gene expression of the tested factors. However, our results support a stimulation of the dopaminergic system and of the oxytocin pathway following the agonistic assay suggesting their potential involvement in aggression-related responses. This further supports the idea that aggression is multifactorial. It is shaped by the interaction of several neuroendocrine and neurotransmitter pathways rather than a single determinant. ### Competing Interest Statement The authors have declared no competing interest. Agence Nationale de la Recherche, https://ror.org/00rbzpz17, SEXREV no. 18‐CE02‐0018‐01

Sex chromosomes are expected to play a role in shaping the transcriptional architecture of sexual dimorphism, through the direct expression of sex-linked genes, by regulating autosomal genes, or in interactions with hormones. Yet, their degree of involvement remains elusive partly because chromosomal sex (e.g. XX/XY) and gonadal sex (ovaries or testes) are usually inextricably intertwined. They are, however, dissociated in the African pygmy mouse, Mus minutoides, in which a feminizing X (X*) has evolved, resulting in three female genotypes (XX, XX*, and X*Y) and one male genotype (XY). Furthermore, all sex chromosomes are fused to autosomes (neo-sex chromosomes: neo-X, neo-X* and neo-Y). Despite complete sex reversal, X*Y females show distinctive phenotypes with greater fertility, divergent maternal care strategies, and the masculinization of some traits (e.g. enhanced aggressiveness). By comparing the brain transcriptome of the four sexual genotypes, we show that differential gene expression is mainly linked to gonadal sex but also, and significantly, to chromosomal sex. Genes influenced by chromosomal sex are overrepresented on sex-linked genomic regions, and some are strong candidates to explain X*Y-specific behavioral and reproductive traits. Our results also suggest the preferential inactivation of the X* chromosome in XX* females, only in the brain, which could explain their trait similarities with XX females. Overall, we show that sex and neo-sex chromosomes have profoundly impacted the brain transcriptome in ways that reflect their new transmission modes, evolutionary trajectories, and resulting genomic conflicts.

A wave of studies has recently emphasized the influence of sex chromosomes on both lifespan and actuarial senescence patterns across vertebrates and invertebrates. Basically, the heterogametic sex (XY males in XX/XY systems or ZW females in ZW/ZZ systems) typically displays a lower lifespan and a steeper rate of actuarial senescence than the homogametic sex. However, whether these effects extend to the senescence patterns of other phenotypic traits or physiological functions is yet to be determined. Here, we investigated whether sex chromosomes modulate reproductive senescence using females from the African pygmy mouse (Mus minutoides). This biological model exhibits an odd sex determining system with a third, feminizing sex chromosome, X*, resulting in three distinct female genotypes (XX, X*X, or X*Y) that coexist in natural populations. We found that the rate of senescence in litter size at birth is much more pronounced in heterogametic X*Y females than in homogametic XX or X*X females that may support the unguarded X or toxic Y hypotheses and can be directly linked to the complex and unique X*Y phenotype. A decrease in neonatal survival with mother's age has also been found, but this decline is not different between the three female genotypes.

Thermoregulation is a major challenge for extremely small mammals such as the African pygmy mice Mus mattheyi and Mus minutoides, weighing less than 12g. A previous study showed that these tiny mice exhibit different mitochondrial energy efficiency for ATP synthesis, with a higher efficiency in M. mattheyi (∼6 g) than in M. minutoides (∼10 g). This result suggests a lower mitochondrial heat production at rest in M. mattheyi, despite its lower body weight and inevitably greater heat loss from its body surface, than in M. minutoides. Consequently, a compensatory thermoregulatory strategy should exist in M. mattheyi to maintain homeothermy. The present study aimed to assess whether M. mattheyi uses non-shivering thermogenesis in brown adipose tissue (BAT) and/or activity-thermoregulatory heat substitution. For this purpose, multidisciplinary approaches involving behavioral, physiological, biochemical and molecular analyses were used. Mus mattheyi showed higher daily mass-specific energy expenditure and food intake per unit weight and allocated more daily energy to vital function, spent less time moving in their cage during daytime but exhibited higher non-locomotor activity and higher movement-related energy cost compared to M. minutoides. BAT of M. mattheyi, was metabolically more active, exhibiting higher mitochondrial respiration rates and citrate synthase activity than in M. minutoides, but lower uncoupling protein 1 content. Altogether, these results suggest that the tiny M. mattheyi mainly uses non-exercise activity thermogenesis, increased cost of movement, and to a lesser extent BAT non-shivering thermogenesis for remaining warm.

Le système de déterminisme du sexe des mammifères (XX/XY) est extrêmement ancien et conservé : toute déviation mène généralement à la stérilité. Cependant, quelques espèces dérogent à la règle. C’est le cas de la souris naine africaine Mus minutoides, qui possède un déterminisme du sexe unique avec un troisième chromosome sexuel : un variant féminisant du X, nommé X* et présent en forte fréquence dans les populations naturelles, qui produit des femelles fertiles X*Y. Ainsi, les femelles sont soit XX, XX* ou X*Y, et tous les mâles XY. Nous avons montré que la combinaison d’un chromosome X* féminisant et d’un chromosome Y a donné naissance à un troisième phénotype sexuel, tant les femelles X*Y diffèrent considérablement des autres femelles sur de nombreux traits. Par exemple, elles ont une plus grande fécondité et procurent de meilleurs soins maternels, en revanche elles présentent aussi des caractères masculinisés comme une plus grande agressivité, malgré des taux d’hormones stéroïdes circulantes similaires. Nous avons étendu nos recherches à l’expression des gènes du cerveau. En comparant le transcriptome des 4 génotypes sexuels, nous montrons qu’une part importante des gènes du cerveau des femelles X*Y s’expriment soit de manière unique (expression différente de tous les autres génotypes) soit identique à celle des mâles. Ces gènes sont surreprésentés sur les chromosomes sexuels, et certains sont de bons candidats pour expliquer les traits comportementaux et reproductifs spécifiques aux femelles X*Y. Au-delà du rôle majeur des hormones, nos résultats mettent en évidence l’importance des chromosomes sexuels dans la construction des différences entre sexes ; ils contribuent également à repenser la vision binaire des sexes.

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.

Sexually dimorphic behaviours, such as parental care, have long been thought to be mainly driven by gonadal hormones. In the past two decades, a few studies have challenged this view, highlighting the direct influence of the sex chromosome complement (XX versus XY or ZZ versus ZW). The African pygmy mouse, Mus minutoides , is a wild mouse species with naturally occurring XY sex reversal induced by a third, feminizing X* chromosome, leading to three female genotypes: XX, XX* and X*Y. Here, we show that sex reversal in X*Y females shapes a divergent maternal care strategy (maternal aggression, pup retrieval and nesting behaviours) from both XX and XX* females. Although neuroanatomical investigations were inconclusive, we show that the dopaminergic system in the anteroventral periventricular nucleus of the hypothalamus is worth investigating further as it may support differences in pup retrieval behaviour between females. Combining behaviours and neurobiology in a rodent subject to natural selection, we evaluate potential candidates for the neural basis of maternal behaviours and strengthen the underestimated role of the sex chromosomes in shaping sex differences in brain and behaviours. All things considered, we further highlight the emergence of a third sexual phenotype, challenging the binary view of phenotypic sexes.

Eukaryotes with separate males and females display a great diversity in the way they determine sex, but it is still unclear what evolutionary forces cause transitions between sex-determining systems. Rather that the lack of hypotheses, the problem is the scarcity of adequate biological systems to test them. Here, we take advantage of the recent evolution of a feminizing X chromosome (called X*) in the African pygmy mouse Mus minutoides, to investigate one of the evolutionary forces hypothesized to cause such transitions, namely sex chromosome drive (i.e., biased transmission of sex chromosomes to the next generation). Through extensive molecular sexing of pups at weaning, we reveal the existence of a remarkable male sex chromosome drive system in this species, whereby direction and strength of drive is conditional upon the genotype of males’ partners: males transmit their Y at a rate close to 80% when mating with XX or XX* females, and only 36% when mating with X*Y females. Using mathematical modelling, we explore the joint evolution of these unusual sex-determining and drive systems, revealing that different sequences of events could have led to the evolution of this bizarre system, and that the “conditional” nature of sex chromosome drive plays a crucial role in the short- and long-term maintenance of the three sex chromosomes.

Skeletal muscle mitochondria of the African pygmy mouse Mus mattheyi exhibit markedly reduced oxygen consumption and ATP synthesis rates but a higher mitochondrial efficiency than what would be expected from allometric trends. In the present study, we assessed whether such reduction of mitochondrial activity in M. mattheyi can limit the oxidative stress associated with an increased generation of mitochondrial reactive oxygen species. We conducted a comparative study of mitochondrial oxygen consumption, H2O2 release, and electron leak (%H2O2/O) in skeletal muscle mitochondria isolated from the extremely small African pygmy mouse (M. mattheyi, ~5 g) and Mus musculus, which is a larger Mus species (~25 g). Mitochondria were energized with pyruvate, malate, and succinate, after which fluxes were measured at different steady-state rates of oxidative phosphorylation. Overall, M. mattheyi exhibited lower oxidative activity and higher electron leak than M. musculus, while the H2O2 release did not differ significantly between these two Mus species. We further found that the high coupling efficiency of skeletal muscle mitochondria from M. mattheyi was associated with high electron leak. Nevertheless, data also show that, despite the higher electron leak, the lower mitochondrial respiratory capacity of M. mattheyi limits the cost of a net increase in H2O2 release, which is lower than that expected for a mammals of this size.

Sex chromosomes are generally derived from a pair of classical type-A chromosomes, and relatively few alternative models have been proposed up to now.1,2 B chromosomes (Bs) are supernumerary and dispensable chromosomes with non-Mendelian inheritance found in many plant and animal species3,4 that have often been considered as selfish genetic elements that behave as genome parasites.5,6 The observation that in some species Bs can be either restricted or predominant in one sex7-14 raised the interesting hypothesis that Bs could play a role in sex determination.15 The characterization of putative B master sex-determining (MSD) genes, however, has not yet been provided to support this hypothesis. Here, in Astyanax mexicanus cavefish originating from Pachón cave, we show that Bs are strongly male predominant. Based on a high-quality genome assembly of a B-carrying male, we characterized the Pachón cavefish B sequence and found that it contains two duplicated loci of the putative MSD gene growth differentiation factor 6b (gdf6b). Supporting its role as an MSD gene, we found that the Pachón cavefish gdf6b gene is expressed specifically in differentiating male gonads, and that its knockout induces male-to-female sex reversal in B-carrying males. This demonstrates that gdf6b is necessary for triggering male sex determination in Pachón cavefish. Altogether these results bring multiple and independent lines of evidence supporting the conclusion that the Pachón cavefish B is a "B-sex" chromosome that contains duplicated copies of the gdf6b gene, which can promote male sex determination in this species.

Mammalian eggs are surrounded by an extracellular matrix called the zona pellucida (ZP). This envelope participates in processes such as acrosome reaction induction, sperm binding, protection of the oviductal embryo, and may be involved in speciation. In eutherian mammals, this coat is formed of three or four glycoproteins (ZP1–ZP4). While Mus musculus has been used as a model to study the ZP for more than 35 years, surprisingly, it is the only eutherian species in which the ZP is formed of three glycoproteins Zp1, Zp2, and Zp3, Zp4 being a pseudogene. Zp4 was lost in the Mus lineage after it diverged from Rattus , although it is not known when precisely this loss occurred. In this work, the status of Zp4 in several murine rodents was tested by phylogenetic, molecular, and proteomic analyses. Additionally, assays of cross in vitro fertilization between three and four ZP rodents were performed to test the effect of the presence of Zp4 in murine ZP and its possible involvement in reproductive isolation. Our results showed that Zp4 pseudogenization is restricted to the subgenus Mus , which diverged around 6 MYA. Heterologous in vitro fertilization assays demonstrate that a ZP formed of four glycoproteins is not a barrier for the spermatozoa of species with a ZP formed of three glycoproteins. This study identifies the existence of several mouse species with four ZPs that can be considered suitable for use as an experimental animal model to understand the structural and functional roles of the four ZP proteins in other species, including human.

Therian mammals have among the oldest and most conserved sex-determining systems known to date. Any deviation from the standard XX/XY mammalian sex chromosome constitution usually leads to sterility or poor fertility, due to the high differentiation and specialization of the X and Y chromosomes. Nevertheless, a handful of rodents harbor so-called unusual sex-determining systems. While in some species, fertile XY females are found, some others have completely lost their Y chromosome. These atypical species have fascinated researchers for over 60 years, and constitute unique natural models for the study of fundamental processes involved in sex determination in mammals and vertebrates. In this article, we review current knowledge of these species, discuss their similarities and differences, and attempt to expose how the study of their exceptional sex-determining systems can further our understanding of general processes involved in sex chromosome and sex determination evolution.

Triggers and biological processes controlling male or female gonadal differentiation vary in vertebrates, with sex determination (SD) governed by environmental factors or simple to complex genetic mechanisms that evolved repeatedly and independently in various groups. Here, we review sex evolution across major clades of vertebrates with information on SD, sexual development and reproductive modes. We offer an up-to-date review of divergence times, species diversity, genomic resources, genome size, occurrence and nature of polyploids, SD systems, sex chromosomes, SD genes, dosage compensation and sex-biased gene expression. Advances in sequencing technologies now enable us to study the evolution of SD at broader evolutionary scales, and we now hope to pursue a sexomics integrative research initiative across vertebrates. The vertebrate sexome comprises interdisciplinary and integrated information on sexual differentiation, development and reproduction at all biological levels, from genomes, transcriptomes and proteomes, to the organs involved in sexual and sex-specific processes, including gonads, secondary sex organs and those with transcriptional sex-bias. The sexome also includes ontogenetic and behavioural aspects of sexual differentiation, including malfunction and impairment of SD, sexual differentiation and fertility. Starting from data generated by high-throughput approaches, we encourage others to contribute expertise to building understanding of the sexomes of many key vertebrate species. This article is part of the theme issue 'Challenging the paradigm in sex chromosome evolution: empirical and theoretical insights with a focus on vertebrates (Part I)'.

Stressful events can alter organism physiology at several levels triggering allostatic responses. Telomeres are well-conserved repetitive DNA sequences mainly localised at chromosome's ends, playing a crucial role in DNA stability. Analyses of telomere dynamics are new tools to assess consequences of environmental stress in non-model organisms like fish. In this study, the relationship between freshwater tolerance and telomere dynamics was investigated in the gills of the European sea bass Dicentrarchus labrax. Fluorescent in situ hybridisation of telomeric sequences revealed distal telomeres as well as intrachromosomal telomeres known as interstitial telomere sequences. In order to better understand telomere dynamics in the gills of D. labrax, we used quantitative PCR to measure telomere length and mRNA expression of the catalytic subunit of telomerase reverse transcriptase tert. For the calculation of the relative telomere length, two reference genes were tested: the single copy gene mc2r, encoding melanocortin 2 receptor and the multicopy gene 18S, encoding the 18S ribosomal RNA. We proposed a novel normalisation method to calculate the relative telomere length using both, single and multiple copy genes as references. Cell dynamics was also investigated by measuring mRNA expression of genes involved in apoptosis (caspase 8 and 9), cell proliferation (proliferation cell nuclear antigen), aerobic mitochondrial metabolism (ATP citrate-synthase), anaerobic metabolism (lactate dehydrogenase a) and antioxidant enzymatic defences (superoxide dismutase 1 and 2, catalase). Following a 15-days fresh water exposure, telomere dynamics was not significantly modified in the gills of freshwater tolerant fish. But freshwater intolerant fish exhibited telomere attrition relative to saltwater controls, and lower expression of tert in gills relative to freshwater tolerant fish. This modification of telomere dynamics in intolerant individuals was found to be correlated with lower antioxidant enzymatic defences, a higher aerobic metabolic marker and a lower cellular turnover. These data bring new perspectives for the use of telomere dynamics as an integrative marker to study environmental stress in fish, while considering individual phenotypic plasticity in response to freshwater exposure.

X and Y chromosomes in mammals are different in size and gene content due to an evolutionary process of differentiation and degeneration of the Y chromosome. Nevertheless, these chromosomes usually share a small region of homology, the pseudoautosomal region (PAR), which allows them to perform a partial synapsis and undergo reciprocal recombination during meiosis, which ensures their segregation. However, in some mammalian species the PAR has been lost, which challenges the pairing and segregation of sex chromosomes in meiosis. The African pygmy mouse Mus mattheyi shows completely differentiated sex chromosomes, representing an uncommon evolutionary situation among mouse species. We have performed a detailed analysis of the location of proteins involved in synaptonemal complex assembly (SYCP3), recombination (RPA, RAD51 and MLH1) and sex chromosome inactivation (γH2AX) in this species. We found that neither synapsis nor chiasmata are found between sex chromosomes and their pairing is notably delayed compared to autosomes. Interestingly, the Y chromosome only incorporates RPA and RAD51 in a reduced fraction of spermatocytes, indicating a particular DNA repair dynamic on this chromosome. The analysis of segregation revealed that sex chromosomes are associated until metaphase-I just by a chromatin contact. Unexpectedly, both sex chromosomes remain labelled with γH2AX during first meiotic division. This chromatin contact is probably enough to maintain sex chromosome association up to anaphase-I and, therefore, could be relevant to ensure their reductional segregation. The results presented suggest that the regulation of both DNA repair and epigenetic modifications in the sex chromosomes can have a great impact on the divergence of sex chromosomes and their proper transmission, widening our understanding on the relationship between meiosis and the evolution of sex chromosomes in mammals.

Sex chromosomes of eutherian mammals are highly different in size and gene content, and share only a small region of homology (pseudoautosomal region, PAR). They are thought to have evolved through an addition-attrition cycle involving the addition of autosomal segments to sex chromosomes and their subsequent differentiation. The events that drive this process are difficult to investigate because sex chromosomes in almost all mammals are at a very advanced stage of differentiation. Here, we have taken advantage of a recent translocation of an autosome to both sex chromosomes in the African pygmy mouse Mus minutoides , which has restored a large segment of homology (neo-PAR). By studying meiotic sex chromosome behavior and identifying fully sex-linked genetic markers in the neo-PAR, we demonstrate that this region shows unequivocal signs of early sex-differentiation. First, synapsis and resolution of DNA damage intermediates are delayed in the neo-PAR during meiosis. Second, recombination is suppressed or largely reduced in a large portion of the neo-PAR. However, the inactivation process that characterizes sex chromosomes during meiosis does not extend to this region. Finally, the sex chromosomes show a dual mechanism of association at metaphase-I that involves the formation of a chiasma in the neo-PAR and the preservation of an ancestral achiasmate mode of association in the non-homologous segments. We show that the study of meiosis is crucial to apprehend the onset of sex chromosome differentiation, as it introduces structural and functional constrains to sex chromosome evolution. Synapsis and DNA repair dynamics are the first processes affected in the incipient differentiation of X and Y chromosomes, and they may be involved in accelerating their evolution. This provides one of the very first reports of early steps in neo-sex chromosome differentiation in mammals, and for the first time a cellular framework for the addition-attrition model of sex chromosome evolution.

Mass-specific metabolic rate negatively co-varies with body mass from the whole-animal to the mitochondrial levels. Mitochondria are the mainly consumers of oxygen inspired by mammals to generate ATP or compensate energetic losses dissipated as the form of heat (proton leak) during oxidative phosphorylation. Consequently, ATP synthesis and proton leak thus compete for the same electrochemical gradient. Because proton leak co-varies negatively with body mass, it is unknown if extremely small mammals further decouple their mitochondria to maintain their body temperature or if they implement metabolic innovations to ensure cellular homeostasis. The present study investigates the impact of body mass variation on cellular and mitochondrial functioning in small mammals, comparing the two extremely small African pygmy mice (Mus mattheyi, approx. 5 g and Mus minutoides, approx. 7 g) with the larger house mouse (Mus musculus, approx. 22 g). Oxygen consumption rates were measured from the animal to the mitochondrial levels. We also measured mitochondrial ATP synthesis in order to appreciate the mitochondrial efficiency (ATP/O). At the whole-animal scale, mass- and surface-specific metabolic rates co-varied negatively with body mass, whereas this was not necessarily the case at cellular and mitochondrial levels. M. mattheyi had generally the lowest cellular and mitochondrial fluxes, depending on the tissue considered (liver or skeletal muscle), as well as having higher efficient muscle mitochondria than the other two species. M. mattheyi presents metabolic innovations to ensure its homeostasis, by generating more ATP per oxygen consumed.