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).

male sex-determining function in XY males, leading us to hypothesize that molecular function of the Sry pathway might be more subtly compromised in this species.
In the current study, we test this hypothesis by examining in detail the structure and function of SRY in M. minutoides. We find that both SRY and its target enhancer, Sox9-TESCO, are strongly debilitated in M. minutoides, but they appear to have co-evolved such that they remain able to function together in XY males. We propose a model in which weakening of the SRY/TESCO nexus may have facilitated the rise and stabilization of an X*-based feminizing mechanism in M. minutoides.

Results
A degraded C-terminal polyQ tract in M. minutoides SRY. We began by investigating whether structural changes in SRY might contribute to the mechanism of X*Y sex reversal. We sequenced the entire Sry coding region in M. minutoides and M. mattheyi (Fig. 1a), a close relative with a typical XX/XY system 15 , and identified 5 and 7 Sry haplotypes from M. minutoides and M. mattheyi respectively (Supplementary Figs S1-4 and Table 1), consistent with previous reports of multiple non-identical Sry copies in these species 14,16 . In subsequent analyses, a reference clone representing the respective consensus of all identified Sry haplotypes in M. mattheyi or M. minutoides was used (Table 1).
Mouse SRY protein comprises an N-terminal HMG domain responsible for DNA binding, a short bridge domain of unknown function, and a large C-terminal polyQ domain composed of 8 (Mus musculus domesticus; hereafter referred to as M. domesticus) to 20 (Mus musculus molossinus; hereafter referred to as M. musculus) blocks of 2-13 glutamine residues interspersed by a short histidine-rich spacer sequence 17,18 . Compared with M. musculus SRY (musSRY), most sequence variations in M. mattheyi and M. minutoides SRY (mat-and minSRY) were found in the C-terminal polyQ tract, whereas the HMG box and bridge domains are relatively conserved (Fig. 1b,c, and Supplementary Figs S5,S6), consistent with previous analyses 12, 14 . The C-terminus of matSRY, despite the truncation caused by an internal stop codon ( Supplementary Fig. S5), retains 11 glutamine blocks and histidine-rich spacers, resembling a typical SRY polyQ tract (Fig. 1c). In contrast, the C-terminus of minSRY was further shortened and contains 4 blocks of only 2 glutamine residues and no histidine-rich spacers (Fig. 1b,c).
The fact that other murine species, like Rattus, Arvicanthini tribe, and other Mus species including pygmy mice share a long glutamine-rich C-terminal domain 18,19 (and our unpublished data) suggests the M. minutoides polyQ tract has evolved by degradation of a longer and more organized polyQ tract present in common ancestors to M. minutoides, M. mattheyi and M. musculus. Supporting this view, further degradation of the polyQ tract has occurred in some of the identified Sry haplotypes when compared with the reference ones (M. minutoides haplotype a and M. mattheyi haplotypes c, g; Table 1 and Supplementary Figs S1-4).

Loss of transactivation ability of M. minutoides SRY.
We have previously established that the polyQ tract is essential for protein stabilization and transactivation by SRY in M. musculus 11 . Because minSRY has a highly degraded polyQ tract, we examined whether the stability and transactivation potential of minSRY is compromised. We found that, unlike a EGFP-tagged M. musculus SRY mutant protein completely lacking the polyQ tract that is barely detectable when stably expressed in mouse Sertoli-like 15P-1 cell line 11 , EGFP-tagged min-and matSRY protein (Fig. 2a) were readily detected by Western blot and immunofluorescence (Fig. 2b,c) in stable 15P-1 cell lines, indicating that the stability of minSRY is not affected by its degraded polyQ tract.
In mouse sex determination, the 1.4-kb TESCO enhancer element plays a significant role in mediating the induction of Sox9 expression by SRY in the presence of SF1 5 , although it is likely that other, yet to be identified testis-specific Sox9-enhancer elements may also contribute to SRY's regulation of Sox9. We have previously shown that the ability of a series of SRY mutant proteins to transactivate a M. musculus TESCO-luciferase reporter construct (musTESCO-Luc) 5 in the heterologous cell line HEK293 correlates closely with their ability to induce Sox9 expression and direct male sex determination in transgenic mouse embryos 11 . We therefore examined the ability of min-or matSRY protein to activate the musTESCO-Luc reporter in the presence of M. musculus SF1 (musSF1) in HEK293 cells. We observed activation by matSRY, albeit weaker than musSRY (Fig. 3a), consistent with our previous observation that, compared with musSRY, M. domesticus SRY exhibits reduced TESCO activation due to its shortened polyQ tract 11 . Strikingly, minSRY failed to activate musTESCO-Luc at all in this system (Fig. 3a, compare green arrows).
The failure of minSRY to activate musTESCO-Luc could be caused by either its intrinsic structural changes or incompatibility between minSRY and musSF1. We therefore investigated whether potential sequence variations between M. musculus and M. minutoides Sf1 could account for the loss of musTESCO activation by minSRY. To this end, we sequenced coding exons 2-7 of Sf1 gene in M. minutoides and M. mattheyi ( Supplementary Fig. S7) and found four and one amino acid substitutions in M. minutoides and M. mattheyi SF1 (min-and matSF1) respectively, compared with musSF1 (Supplementary Figs. S8 and S9a). Nevertheless, these sequence changes had no measurable effect on SF1's ability to synergize with SRY to activate musTESCO ( Supplementary Fig. S9b,c). Importantly, minSRY failed to activate the musTESCO-Luc reporter in the presence of either mat-or minSF1. These results indicate that the sequence changes in mat/minSF1 do not significantly alter their ability to synergize with SRY to activate TESCO (at least in these in vitro reporter assays), and that the loss of transactivation ability of minSRY is most likely caused by its intrinsic structural changes.
We reasoned that either the degraded polyQ domain of minSRY had lost transactivation potential, or variations in the HMG + bridge domains ( Supplementary Fig. S6) 14  interaction with SF1. To distinguish these possibilities, we generated two mutant constructs and analyzed their ability to activate musTESCO-Luc in the presence of musSF1. The mutant musHBminQ-combining the musSRY HMG + bridge domains with the M. minutoides polyQ tract-failed to activate musTESCO-Luc, similar to minSRY (Fig. 3b, compare orange arrows). In contrast, the mutant minHBmusQ-combining the minSRY HMG + bridge domains with the M. musculus polyQ tract-showed fully restored ability to activate musTESCO-Luc (Fig. 3b, compare blue arrows). The use of mat/min SF1 appeared to have no effect on the activities of these SRY mutants ( Supplementary Fig. S9b 23 , and the Japanese spiny rats Tokudaia osimensis and T. tokunoshimensis 24 . We therefore analyzed TESCO sequences in M. minutoides and M. mattheyi (minTESCO and matTESCO) to look for structural irregularities that might similarly disrupt this nexus. matTESCO and minTESCO have sequence identities of approximately 93% with musTESCO ( Fig. 4a). Most sequence variations lie outside regulatory elements previously identified in M. musculus, including the SRY binding sites R4-R6 5 and the evolutionarily conserved regions ECRi-v 25 ( Supplementary Fig. S10). Notably, sequence variations were identified in both mat-and minTESCO at SF1 binding sites F1-F4 5 (Fig. 4b,c) which may impair SF1 binding 26,27 . Sequences at SF1 binding sites F5-F6 in mat/minTESCO remain identical to those in musTESCO ( Supplementary Fig. S10). Moreover, two haplotypes minTESCO.a and -.b differ at three sites (V1-3; Figs 4b and 5a).
We next examined the transcriptional activities conferred by mat/minTESCO enhancers using luciferase reporter assays in HEK293 cells. The transcriptional activities of mat/minTESCO in the presence of musSF1 decreased significantly compared to musTESCO (Fig. 4c), due to a severely diminished response to musSF1: fold induction by musSF1 (that is, the ratio of activity in the presence:absence of SF1) fell to approximately 8 or 24% in the case of matTESCO and minTESCO.b respectively ( Supplementary Fig. S11). As a result, minTESCO.a (a) Schematic of EGFP-tagged matSRY and minSRY proteins (g-matSRY and g-minSRY, respectively). EGFPtagged musSRY (g-musSRY) and musSRY lacking the polyQ domain (g-musSRYΔ Q) are also shown here for comparison. (b) Both g-minSRY and g-matSRY were detected in 15P-1 stable cell lines by Western blotting using an anti-EGFP antibody. g-musSRY and g-musSRYΔ Q were included as positive and negative controls, respectively. Predicted molecular weight: g-musSRY, 77.1 kDa; g-musSRY Δ Q, 47.9 kDa; g-minSRY, 50.7 kDa; g-matSRY, 59.9 kDa. A blot using anti-α -Tubulin served as loading control. Full-length blots are presented in Supplementary Fig. S15. (c) Both g-matSRY and g-minSRY were detected in the nuclei of 15P-1 cells by immunofluorescence using an anti-EGFP antibody. g-musSRY and g-musSRYΔ Q were included as positive and negative controls, respectively. retained ~10% of musTESCO activity, while the activities of matTESCO and minTESCO.b were 1-3% of that of musTESCO (Fig. 4c).
The severely decreased ability of mat/minTESCO to respond to SF1 may be a direct consequence of the sequence variations at known SF1 binding sites F1-4 (Fig. 4b,c). We therefore mutated these sites in musTESCO-Luc to the corresponding sequences in mat/minTESCO (musTESCO.mut1/2; Fig. 4c). Surprisingly, the two TESCO mutants showed only mildly reduced reporter activities (Fig. 4c) and response to musSF1 ( Supplementary Fig. S11), nowhere near those of mat/minTESCO, suggesting the likely presence of unidentified SF1 binding sites within TESCO. Supporting this view, mutation of the V3 site in minTESCO.b to the corresponding motif found in minTESCO.a (minTESCO.b.mut; Fig. 5a), fully restored its activity and response to musSF1 to the levels of minTESCO.a ( Fig. 5b and Supplementary Fig. S12). Similar results were obtained with mat-and minSF1 (Supplementary Fig. S13 and data not shown).
Together, these results demonstrate that TESCO enhancers in M. minutoides and M. mattheyi are barely functional, compared to M. musculus TESCO. Moreover, our data strongly indicate that sequences outside the previously identified SF1 (and SRY) binding sites may play hitherto unappreciated roles in modulating TESCO activity and Sox9 expression in Mus species.
M. minutoides SRY activates its cognate TESCO in the presence of SF1. The failure of minSRY to activate musTESCO (Fig. 3a) may indicate that, in M. minutoides, SRY is no longer functioning as the testis-determining gene and a newly evolved Y-linked testis-determining gene may be operative. Alternatively, TESCO and SRY may have co-evolved in this species, such that minSRY remains able to activate its cognate TESCO. To distinguish these possibilities, we performed luciferase reporter assays in HEK293 cells testing synergistic activation of min/matTESCO luciferase reporters by various combinations of SRY and SF1 constructs. We found that, in the presence of musSF1, minSRY robustly activated its cognate minTESCO.a/b (Fig. 6a,b, compare green arrows). However, the fold induction by minSRY was much weaker compared to musSRY which has a longer polyQ tract (Fig. 6a,b). minSRY also activated matTESCO ( Supplementary Fig. S14c), despite its lack of activity on musTESCO (Fig. 3a). Similar results were obtained with mat/minSF1 constructs ( Supplementary Fig. S14). Thus, Sry and TESCO appear to have co-evolved to retain functional compatibility in M. minutoides.
We reasoned that that mat/minTESCO may have acquired sequence changes making them more responsive to SRY, thus allowing a weaker SRY such as minSRY to function. Consistent with this hypothesis, minTESCO was activated > 7 fold by musSRY in the presence of SF1 (Fig. 6a,b), while musTESCO was only activated ~2 fold by musSRY (Fig. 3a).
Furthermore, minSRY may have also acquired adaptive changes, presumably within the HMG box and bridge domains, since its degraded polyQ domain represents a much weaker transactivation domain compared with its counterpart in musSRY (Fig. 3b), and is unlikely to offer any adaptive advantages. To test this possibility, we made various combinations of the HMG (H), bridge (B) and polyQ-domains (Q), and assayed their ability to activate both minTESCO.a/b luciferase reporters. Consistent with our hypothesis, minSRY outperformed the musHBminQ mutant, and the minHBmusQ mutant eclipsed musSRY (Fig. 6c,d, compare orange and blue arrows respectively).
These results support the notion that Sry and TESCO may have co-evolved in M. minutoides, and further indicate that the HMG box and bridge domains of SRY may have a mild impact on TESCO activation. This conclusion is in accordance with a previous report that M. musculus polyQ tract, as a strong transactivation domain, allows and compensates for otherwise deleterious amino acid substitutions in the HMG box 28 .
Taken together, our results indicate that the SRY/SF1/TESCO nexus has been severely weakened in M. minutoides through two different mechanisms. M. minutoides SRY's capacity to activate TESCO is severely compromised primarily due to its degraded polyQ tract, as SRY binding sites within TESCO remain intact. Secondly, SF1 protein in M. minutoides possesses comparable activity compared with M. musculus SF1, but DNA sequence changes in its target sites in TESCO cause a significantly reduced response.

Discussion
In contrast to the high plasticity seen in many vertebrate classes, sex determination in mammals is relatively static and almost invariably based on an XX female/XY male system. Exceptions are rare 29 , and afford the potential to gain insights into the evolution of sex-determining systems as well as into mechanistic issues such as the interplay between testis-and ovarian-determining pathways. Such is the case with the unusual X*Y sex reversal system found in M. minutoides, the focus of the present study. By studying critical elements of the testis-determining pathway, we have shown that the activities of M. minutoides SRY, SF1 and the Sox9 enhancer TESCO are Sequence changes at sites F1-4 may impair SF1 binding and are indicated with different colours. The F3 site predicted to have SF1 binding disrupted is removed from the matTESCO diagram. (c) Sequence comparison of F1-4 sites from the three species, and the mutants musTESCO.mut1/2. Sequence changes compared with musTESCO are in red and mutated sequences are underlined. Compared with musTESCO, mat/minTESCO showed markedly reduced activities in the presence of musSF1. Mutations of SF1 binding sites F1-4 in musTESCO to the corresponding sequence in matTESCO (musTESCO.mut1) or minTESCO (musTESCO. mut2) caused mildly reduced reporter activities in the presence of musSF1. Data are presented as TESCO luciferase activity normalized to co-transfected CMV-renilla luciferase activity. Error bars: s.e.m. (n = 3). Dashed lines indicate the levels of musTESCO activity. (**) P < 0.01 vs. musTESCO, one-way repeated measures ANOVA with Dunnett's multiple comparisons test.
compromised but still capable of functioning such that they are able to generate XY males. Assuming our in vitro data broadly reflect the activities of these elements in vivo, the phenomenon of X*Y sex reversal in this species is likely to be due to a combination of an as-yet unidentified variant gene(s) on the X* chromosome and a weakening of the male sex-determining pathway. Conversely, minHBmusQ mutant significantly outperformed musSRY (compare blue arrows). The luciferase activity of each luciferase reporter co-transfected with the empty vector in the absence of SF1 was set to 1. SRY constructs do not activate TESCO in the absence of SF1, and thus the − SF1 data essentially showed unchanged base level activities of TESCO-Luc reporter. Therefore, for simplicity, only the + SF1 data are presented here as mean ± s.e.m (n = 3). Dashed lines indicate the levels of baseline (empty vector + musSF1) or synergistic activation by musSRY + musSF1. (*) P < 0.05, (**) P < 0.01 vs. empty vector, or as indicated, one-way repeated measures ANOVA with Holm-Sidak multiple comparisons test. ns, not significant. Co-evolution of SRY and TESCO in M. minutoides. Our analyses reveal that M. minutoides SRY has completely lost its ability to activate TESCO from other species, and that M. minutoides TESCO also has degenerated so that it retains only very limited ability to respond to SF1, compared with its counterpart in M. musculus. However, M. minutoides SRY is clearly able to activate its cognate TESCO, albeit weakly, indicating that SRY and its target TESCO enhancer may have co-evolved in this species to secure male sex determination. TESCO in M. mattheyi and M. minutoides may have acquired sequence changes outside the previously identified SRY binding sites R4-R6 to enable itself to respond more strongly to SRY, thus compensating for the weaker transactivating capacity of SRY in these species. In addition, SRY itself has also evolved. It has been suggested that Sry is under positive selection 9,30,31 . Supporting this view, we found several amino acid changes in the sequences of the HMG and bridge domains of matSRY and minSRY. These adaptive sequence changes in TESCO and SRY may improve the binding affinity of SRY to TESCO and/or the interaction between SRY and SF1. In this regard, we note an arginine to cysteine change at amino acid 17 (R17C) in the HMG box ( Supplementary Fig. S6), a site involved in electrostatic and hydrophobic interactions between SRY and its target DNA 32 .
Despite the co-evolution of SRY and TESCO in M. minutoides, the absolute activity of the SRY/SF1/TESCO nexus in this species is reduced to less than 10% of that in M. musculus, due to the severely impaired transactivating capacity of SRY and reduced response of TESCO to SF1.
Interaction of the X* chromosome with the weakened male sex-determining pathway. Even though the Sry pathway is weakened in M. minutoides, it is able to function in the presence of a normal X but not in the presence of an X*. We hypothesize that the X* chromosome carries a stronger pro-ovarian allele, a stronger anti-testis allele, or a weaker allele essential for testis-determining signalling, compared to a normal X. We further hypothesize that this X*-linked modifier gene favours ovarian development but does not cause XY sex reversal in combination with a strong Sry allele such as those in M. musculus or M. mattheyi. Only when combined with a weakened male sex-determining pathway such as that found in M. minutoides does XY femaleness result.
A similar scenario has been described previously in laboratory mice M. musculus, where additional copies of the X-linked Dax1 gene in transgenic mice do not affect normal testis development in the presence of an M. musculus Y chromosome, but cause ovarian or ovotestis development 33 when combined with an M. domesticus poschiavinus Y chromosome carrying a weak Sry allele 34 . Nevertheless, Dax1 does not seem to be involved in X*Y sex reversal in M. minutoides, as the X*-linked Dax1 copy shows no sign of changes in either copy number or expression 13 . Therefore, a true test of the hypothesis that the X* harbours a pro-ovarian modifier gene rests on identification of suitable candidates for such a role and testing their activity in different mouse strains.
Origin and fixation of X* chromosome. The finding that the Sry-driven male pathway is compromised to some extent in M. minutoides logically suggests that it is the combination of a neomorphic gene on the X* chromosome and the weakened Sry pathway that underpin the X*Y sex reversal in M. minutoides. How might this unusual sex-determination system have arisen? Our findings lead us to propose the model illustrated in Fig. 7. In this model, in a common ancestor to M. mattheyi and M. minutoides, TESCO acquired sequence variations that caused both decreased basal activity and reduced response to SF1. Subsequently, in an ancestor to M. minutoides, the SRY polyQ tract degenerated further, likely due to the rapid sequence evolution and degradation of the Y chromosome-linked genes and/or the CAG microsatellite instability 30,35 , causing diminished capacity to activate TESCO. With the activity of both SRY and its key target TESCO being compromised, the male pathway in M. minutoides became vulnerable to the influence of a modifier gene. It is conceivable that a subsequent chromosomal rearrangement event of the ancestral X created a tight linkage between this modifier and genes conferring enhanced reproductive outcome, thus generating the X*. In the presence of a weakened M. minutoides type Sry allele, the X*-borne modifier caused ovarian development, resulting in X*Y females with high fecundity and a skewed sex ratio.
In contrast to laboratory mouse models where sex reversed XY females are almost always sterile 36 , X*Y females in M. minutoides show enhanced reproductive performance compared with the XX and XX* 37 . Increased fecundity of X*Y over XX females has also been reported in several other rodent species with sex determination systems similar to that in M. minutoides, including the lemming Myopus schisticolor and Dicrostonyx torquatus 38,39 , and the South American grass mouse Akodon azarae 40,41 .
Another common feature of species with X*Y females is an expected female-biased sex ratio 38,41 , which has been suggested to contribute to the invasion and maintenance of the X* in certain circumstances [42][43][44] . Hence it is possible that the X* subsequently was stabilized in the population due to the reproductive advantage of X*Y females over XX and XX* and/or a selection for favouring a female-biased sex ratio or for restoring a 1:1 sex ratio after the invasion of a Y chromosome distorter 43-46 . Implications for evolution of mammalian sex-determining systems independent of Sry. From the time when Sry first arose as a variant of the X-chromosomal gene Sox3 [47][48][49] and became the male sex determinant in a mammalian ancestor, most genes on the neo-Y chromosome began an inexorable process of loss or pseudogenization, thus resulting in the highly evolved and decayed Y chromosomes currently found in mammals [50][51][52][53] . Our data show that Sry function has deteriorated in M. minutoides, likely due to ongoing degradation of Sry in African pygmy mice. With the activities of both SRY and its target TESCO being compromised, the testis-determining pathway in M. minutoides is likely operating at a threshold level.
The current situation in M. minutoides may represent an intermediate state in which both Sry-dependent testis-determination and an X*-dependent dominant feminizing mechanism are in operation. With the further passage of evolutionary time, SRY's polyQ tract may undergo further degradation in M. minutoides, as has already occurred in some identified Sry haplotypes. With the translocation of Y-linked essential male fertility genes to other chromosomes 54,55 , Y chromosome would no longer be required and would eventually be lost, supplanted by a neo-Y chromosome (and a new male sex-determining gene that should overpower the weakened Scientific RepoRts | 7:41378 | DOI: 10.1038/srep41378 Sry) or replaced by a XO/X*O sex determination system with a masculinizing X and a feminizing X* chromosome, as has likely occurred in the Japanese spiny rats T. osimensis and T. tokunoshimensis 56,57 and the mole vole E. lutescens 58,59 .
In this regard, another species of African pygmy mice, M. triton, has lost the Y chromosome, with both males and females having an XO karyotype 60 and may represent this ultimate evolutionary step. Undoubtedly, more examples remain to be discovered. Further study of these exceptions to the rule, in particular the identification of the X*-borne modifier gene(s) that cause X*Y sex reversal and genes that trigger sex development in the absence of Sry, will illuminate the normal process of sex development and identify new candidate genes whose loss of function might cause human disorders of sex development.  Supplementary Table S1. Eight (for Sry), four (for TESCO), or three (for each Sf1 coding exon) independent clones from each PCR product were sequenced. Sequences were aligned using ClustalW 61 . Identity scores were calculated using GeneStream II 62 .

Methods
Phylogenetic reconstruction using TESCO sequence. TESCO sequences in M. musculus and Rattus norvegicus were retrieved from mm10 or rn6 reference genome, respectively. The maximum likelihood phylogeny was reconstructed using BEAST 2.0 63 and visualised using FigTree. R. norvegicus was included as an outgroup.

Expression analyses of SRY protein in stable mouse Sertoli-like 15P-1 cell lines. The M. mattheyi
or M. minutoides Sry coding region with a preceding EGFP coding sequence inserted in frame was subcloned into pMIH retroviral vector 64 . Stable 15P-1 cell lines were established by infection of individual retrovirus produced as described 11,65 . Western blot and immunofluorescence analyses were performed as described 11 with anti-EGFP (Abcam, Ab5450) or anti-α -Tubulin (Sigma, T5168) antibodies.
Luciferase reporter assays were conducted as described 11 . Briefly, HEK293 cells were co-transfected with a TESCO luciferase construct, a pMIH empty vector or a pMIH construct containing various Sry or Sry mutant sequences, and an empty pcDNA3 or a pcDNA3 construct containing various Sf1 sequence. A cytomegalovirus (CMV)-renilla luciferase plasmid 11 was included as a control for transfection efficiency. Multiplicity-adjusted P values were calculated using GraphPad Prism 6. Invasion and fixation of the X* Figure 7. A model for the evolution of the atypical sex determination system in M. minutoides. In a common ancestor to M. mattheyi and M. minutoides, multiple sequence variations within TESCO occurred. This first strike significantly reduced the basal transcriptional activity of TESCO and attenuated its response to SF1. Subsequently, a second strike hit Sry in an ancestor to M. minutoides, causing a severe degradation of its polyQ tract. Such a degenerated SRY retains only weak capacity to activate TESCO and Sox9 expression. Together, these genetic events have rendered the male sex-determining pathway vulnerable in M. minutoides, which may have facilitated the invasion and subsequent spread of the X* chromosome (and feminising modifier(s) thereon). The presence of X* chromosome in X*Y individuals overwrites the fragile male sexdetermining pathway and leads to female sex reversal.