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

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.

Genotypic sex shapes maternal care in the African pygmy mouse, Mus minutoides.

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.

Multiple sex chromosome drivers in a mammal with three sex chromosomes.

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 are 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 modeling, 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.

Does high mitochondrial efficiency carry an oxidative cost? The case of the African pygmy mouse (Mus mattheyi).

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.

Unusual Mammalian Sex Determination Systems: A Cabinet of Curiosities.

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.

Meiotic Behavior of Achiasmate Sex Chromosomes in the African Pygmy Mouse Mus mattheyi Offers New Insights into the Evolution of Sex Chromosome Pairing and Segregation in Mammals.

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.

A supernumerary "B-sex" chromosome drives male sex determination in the Pachón cavefish, Astyanax mexicanus.

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.

A brief review of vertebrate sex evolution with a pledge for integrative research: towards 'sexomics'.

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)'.

Meiosis reveals the early steps in the evolution of a neo-XY sex chromosome pair in the African pygmy mouse Mus minutoides.

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.

Improved mitochondrial coupling as a response to high mass-specific metabolic rate in extremely small mammals.

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 for energetic losses dissipated as the form of heat (proton leak) during oxidative phosphorylation. Consequently, ATP synthesis and proton leak compete for the same electrochemical gradient. Because proton leak co-varies negatively with body mass, it is unknown whether extremely small mammals further decouple their mitochondria to maintain their body temperature or whether they implement metabolic innovations to ensure cellular homeostasis. The present study investigated the impact of body mass variation on cellular and mitochondrial functioning in small mammals, comparing two extremely small African pygmy mice (Mus mattheyi, ∼5 g, and Mus minutoides, ∼7 g) with the larger house mouse (Mus musculus, ∼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 the cellular and mitochondrial levels. Mus mattheyi had generally the lowest cellular and mitochondrial fluxes, depending on the tissue considered (liver or skeletal muscle), as well as having more-efficient muscle mitochondria than the other two species. Mus mattheyi presents metabolic innovations to ensure its homeostasis, by generating more ATP per oxygen consumed.

ZP4 Is Present in Murine Zona Pellucida and Is Not Responsible for the Specific Gamete Interaction.

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.

Contrasting patterns of ERK activation in the tail of the striatum in response to aversive and rewarding signals.

The caudal part of the striatum, also named the tail of the striatum (TS), defines a fourth striatal domain. Determining whether rewarding, aversive and salient stimuli regulate the activity of striatal spiny projections neurons (SPNs) of the TS is therefore of paramount importance to understand its functions, which remain largely elusive. Taking advantage of genetically encoded biosensors (A-kinase activity reporter 3) to record protein kinase A signals and by analyzing the distribution of dopamine D1R- and D2R-SPNs in the TS, we characterized three subterritories: a D2R/A2aR-lacking, a D1R/D2R-intermingled and a D1R/D2R-SPNs-enriched area (corresponding to the amygdalostriatal transition). In addition, we provide evidence that the distribution of D1R- and D2R-SPNs in the TS is evolutionarily conserved (mouse, rat, gerbil). The in vivo analysis of extracellular signal-regulated kinase (ERK) phosphorylation in these TS subterritories in response to distinct appetitive, aversive and pharmacological stimuli revealed that SPNs of the TS are not recruited by stimuli triggering innate aversive responses, fasting, satiety, or palatable signals whereas a reduction in ERK phosphorylation occurred following learned avoidance. In contrast, D1R-SPNs of the intermingled and D2R/A2aR-lacking areas were strongly activated by both D1R agonists and psychostimulant drugs (d-amphetamine, cocaine, 3,4-methyl enedioxy methamphetamine, or methylphenidate), but not by hallucinogens. Finally, a similar pattern of ERK activation was observed by blocking selectively dopamine reuptake. Together, our results reveal that the caudal TS might participate in the processing of specific reward signals and discrete aversive stimuli. Cover Image for this issue: doi: 10.1111/jnc.14526. Open Science: This manuscript was awarded with the Open Materials Badge For more information see: https://cos.io/our-services/open-science-badges/.

Sex chromosome quadrivalents in oocytes of the African pygmy mouse Mus minutoides that harbors non-conventional sex chromosomes.

Eutherian mammals have an extremely conserved sex-determining system controlled by highly differentiated sex chromosomes. Females are XX and males XY, and any deviation generally leads to infertility, mainly due to meiosis disruption. The African pygmy mouse (Mus minutoides) presents an atypical sex determination system with three sex chromosomes: the classical X and Y chromosomes and a feminizing X chromosome variant, called X*. Thus, three types of females coexist (XX, XX*, and X*Y) that all show normal fertility. Moreover, the three chromosomes (X and Y on one side and X* on the other side) are fused to different autosomes, which results in the inclusion of the sex chromosomes in a quadrivalent in XX* and X*Y females at meiotic prophase. Here, we characterized the configurations adopted by these sex chromosome quadrivalents during meiotic prophase. The XX* quadrivalent displayed a closed structure in which all homologous chromosome arms were fully synapsed and with sufficient crossovers to ensure the reductional segregation of all chromosomes at the first meiotic division. Conversely, the X*Y quadrivalents adopted either a closed configuration with non-homologous synapsis of the X* and Y chromosomes or an open chain configuration in which X* and Y remained asynapsed and possibly transcriptionally silenced. Moreover, the number of crossovers was insufficient to ensure chromosome segregation in a significant fraction of nuclei. Together, these findings raise questions about the mechanisms allowing X*Y females to have a level of fertility as good as that of XX and XX* females, if not higher.

Repeat associated mechanisms of genome evolution and function revealed by the Mus caroli and Mus pahari genomes.

Understanding the mechanisms driving lineage-specific evolution in both primates and rodents has been hindered by the lack of sister clades with a similar phylogenetic structure having high-quality genome assemblies. Here, we have created chromosome-level assemblies of the Mus caroli and Mus pahari genomes. Together with the Mus musculus and Rattus norvegicus genomes, this set of rodent genomes is similar in divergence times to the Hominidae (human-chimpanzee-gorilla-orangutan). By comparing the evolutionary dynamics between the Muridae and Hominidae, we identified punctate events of chromosome reshuffling that shaped the ancestral karyotype of Mus musculus and Mus caroli between 3 and 6 million yr ago, but that are absent in the Hominidae. Hominidae show between four- and sevenfold lower rates of nucleotide change and feature turnover in both neutral and functional sequences, suggesting an underlying coherence to the Muridae acceleration. Our system of matched, high-quality genome assemblies revealed how specific classes of repeats can play lineage-specific roles in related species. Recent LINE activity has remodeled protein-coding loci to a greater extent across the Muridae than the Hominidae, with functional consequences at the species level such as reproductive isolation. Furthermore, we charted a Muridae-specific retrotransposon expansion at unprecedented resolution, revealing how a single nucleotide mutation transformed a specific SINE element into an active CTCF binding site carrier specifically in Mus caroli, which resulted in thousands of novel, species-specific CTCF binding sites. Our results show that the comparison of matched phylogenetic sets of genomes will be an increasingly powerful strategy for understanding mammalian biology.

X inactivation in a mammal species with three sex chromosomes.

X inactivation is a fundamental mechanism in eutherian mammals to restore a balance of X-linked gene products between XY males and XX females. However, it has never been extensively studied in a eutherian species with a sex determination system that deviates from the ubiquitous XX/XY. In this study, we explore the X inactivation process in the African pygmy mouse Mus minutoides, that harbours a polygenic sex determination with three sex chromosomes: Y, X, and a feminizing mutant X, named X*; females can thus be XX, XX*, or X*Y, and all males are XY. Using immunofluorescence, we investigated histone modification patterns between the two X chromosome types. We found that the X and X* chromosomes are randomly inactivated in XX* females, while no histone modifications were detected in X*Y females. Furthermore, in M. minutoides, X and X* chromosomes are fused to different autosomes, and we were able to show that the X inactivation never spreads into the autosomal segments. Evaluation of X inactivation by immunofluorescence is an excellent quantitative procedure, but it is only applicable when there is a structural difference between the two chromosomes that allows them to be distinguished.

Extensive Amplification of Telomeric Repeats in the Karyotypically Highly Diverse African Pygmy Mice.

Telomeres are ribonucleoprotein structures protecting the physical ends of eukaryotic chromosomes. However, telomeric sequences can also occur at non-terminal regions of chromosomes, forming the so-called interstitial telomeric sequences (ITSs). Some ITSs are considered as relics of past chromosomal rearrangements and as such provide important insights into karyotype evolution. By FISH, we explored the distribution of telomeric motifs in the genome of a complex of mammalian species that has long been recognized for its extraordinary karyotypic diversity: the African pygmy mice. This survey involved 5 species, representing 10 highly diverse karyotypes with or without autosomal and sex-autosome robertsonian (Rb) fusions. The study revealed that in species with an ancestral-like karyotype (i.e., no fusions; Mus mattheyi and M. indutus), only terminal telomeres were observed, whereas in species experiencing intense chromosomal evolution (e.g., M. minutoides, M. musculoides), a large amplification of telomeric repeats was also identified in the pericentromeric region of acrocentrics and most metacentrics. We concluded that (i) the mechanism of Rb fusion in the African pygmy mice is different than the one highlighted in the house mouse; (ii) the intensity of the ITS hybridization signal could be a signature of the age of formation of the Rb fusion; (iii) the large amplification of pericentromeric telomeric sequences in acrocentrics may mediate the formation of Rb fusions, and (iv) the ITSs on the sex-autosome fusion Rb(X.1) may participate to the insulation buffer between the sexual and autosomal arms to prevent X inactivation from spreading and silencing autosomal genes and allow the independent regulation of replication timing of both segments.

Sex reversal induces size and performance differences among females of the African pygmy mouse, Mus minutoides.

Differences in biological performance, at both intra- and inter-specific levels, have often been linked to morphology but seldom to behavioural or genotypic effects. We tested performance at the intraspecific level by measuring bite force in the African pygmy mouse, Mus minutoides. This species displays an unusual sex determination system, with sex-reversed, X*Y females carrying a feminizing X* chromosome. X*Y females cannot be differentiated from XX females based on external or gonadal morphology; however, they are known to be more aggressive. We found that bite force was higher in X*Y females than in other females and males. We then performed geometric morphometric analyses on their skulls and mandibles and found that the higher performance of X*Y females was mainly explained by a greater overall skull size. The effects of the X* chromosome thus go beyond feminization, and extend to whole-organism performance and morphology. Our results also suggest limited effects of behaviour on bite force.

Reduced Activity of SRY and its Target Enhancer Sox9-TESCO in a Mouse Species with X*Y Sex Reversal.

In most eutherian mammals, sex determination is governed by the Y-linked gene Sry, but in African pygmy mice Mus minutoides, Sry action is overridden by a variant X chromosome (X*), yielding X*Y females. We hypothesized that X*Y sex reversal may be underpinned not only by neomorphic X chromosome functionality, but also by a compromised Sry pathway. Here, we show that neither M. minutoides SRY nor its target, the Sox9-TESCO enhancer, had appreciable transcriptional activity in in vitro assays, correlating with sequence degradation compared to Mus musculus counterparts. However, M. minutoides SRY activated its cognate TESCO to a moderate degree, and can clearly engage the male pathway in M. minutoides in the wild, indicating that SRY and TESCO may have co-evolved in M. minutoides to retain function above a threshold level. We suggest that weakening of the SRY/TESCO nexus may have facilitated the rise and spread of a variant X* chromosome carrying female-inducing modifier gene(s).