# Gonadal sex determination in vertebrates: rethinking established mechanisms

Dagmar Wilhelm, Aitana Perea-Gomez, Axel Newton, Marie-Christine

Chaboissier

► To cite this version:

Dagmar Wilhelm, Aitana Perea-Gomez, Axel Newton, Marie-Christine Chaboissier. Gonadal sex determination in vertebrates: rethinking established mechanisms. Development (Cambridge, England), 2025, 152 (6), pp.dev204592. 10.1242/dev.204592. hal-05166957

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25 Abstract

26 Sex determination and differentiation are fundamental processes that are not only essential for fertility but
27 also influence the development of many other organs and hence are important for species diversity and
28 survival. In mammals, sex is determined by the inheritance of an X or a Y chromosome from the father. The
29 Y chromosome harbours the testis-determining gene SRY, and it has long been thought that its absence is
30 sufficient for ovarian development. Consequently, the ovarian pathway has been treated as a default
31 pathway, in the sense that ovaries do not have or need a female determining factor. Recently, a female-
32 determining factor has been identified in mouse as the master regulator of ovarian development.

33 Interestingly, this scenario was predicted as early as 1983. In this Review, we discuss the model predicted in
34 1983, how the mechanisms and genes currently known to be important for sex determination and
differentiation in mammals have changed or supported this model and finally reflect on what these findings
35 might mean for sex determination in other vertebrates.
36
37 **Introduction**

38 One of the first questions a family asks when a baby is born is “is it a boy or a girl?”. In many cases this is an
39 easy question to answer. However, between 1:2,500 to 1:5,500 births (Bashamboo and McElreavey, 2016)
40 the baby is born with ambiguous genitalia and this poses challenges for the medical team to decide the sex
41 of rearing for the child. These conditions are part of a larger group known as differences of sex development
42 (DSDs, also known as variants of sex development, VSDs), which are defined as congenital conditions in
43 which the development of chromosomal, gonadal or anatomical sex is “atypical” (Hughes et al., 2006).
44 While mechanisms of sex determination have fascinated scientists for hundreds of years (Stevant et al.,
45 2018b), in recent times, more and more people are becoming aware of them as topics such as DSD are
46 more openly discussed. To avoid causing harm through misinformation and to help improve DSD diagnosis
47 we need to understand how these processes are normally regulated during embryogenesis and what
48 happens when things do not go to plan.

49

50 In anisogamous sexual reproduction, two types of gametes are produced: the smaller and more mobile are
51 referred to male gametes and define the male, while the female gametes are larger and with a cytoplasm
52 that can provide sufficient nutrients for the zygote. The two sexes can then acquire different morphological
53 characteristics during development, depending on the species. The differentiation of males and females
54 start to diverge when the embryonic primordium of the gonads, which is initially similar in both sexes, start
55 to form either a testis or an ovary, often referred to as gonadal sex determination. The trigger for this
56 decision varies widely between different species but can be broadly divided into environmental sex
determination (ESD) where, for example, in many reptiles the incubation temperature of the egg
57 determines the sex of the offspring, genetic sex determination (GSD), such as in mammals and birds, or
58 even a combination of these (Schartl et al., 2023, Weber and Capel, 2021). In vertebrates, GSD can be
59 further divided into either a XX/XY or a ZZ/ZW system (Schartl et al., 2023). In the XX/XY system, the males
60 are heterogametic (i.e. XY) and the females are homogametic (XX), while in the ZZ/ZW system, the females
61 are heterogametic (i.e. ZW) and the males are homogametic (ZZ). Interestingly, beyond vertebrates, there is
62 also a U/V GSD system, in which sex is expressed in the haploid phase of the life cycle with females carrying
63
the U chromosome and males the V chromosome (Luthringer et al., 2024). Finally, the sex chromosome-linked master regulator that initiates the distinct differentiation process of testes or ovaries, varies widely independent of the sex determination system (Nagahama et al., 2021). In contrast, the molecular pathways that are downstream of the initial switch and the cellular composition of testes and ovaries show a much higher conservation between different species.

In this Review, we discuss the mechanisms, factors and genes known to be important for vertebrate sex determination and differentiation with a focus on somatic cells in the mouse and insights we gained through *in vivo* transgenic and knockout mouse models without going into details regarding complementary studies using *in vitro* reconstitution of reproductive organoids (see Box 1). We integrate the most recent data with previously identified mechanisms, highlighting species variability and, finally, we reflect on what it might mean for gonadal sex determination in general.

## Historic models of gonadal sex determination

Over 40 years ago, in 1983, Eva Eicher and Linda L. Washburn (Eicher and Washburn, 1983) proposed a genetic model of gonadal sex determination in mouse based on the data available at that time. Thanks to early reports examining human patients with Turner syndrome (XO females) and Klinefelter syndrome (XXY males) (Ford et al., 1959, Jacobs and Strong, 1959), it was known that in mammals the Y chromosome is necessary for the differentiation of the genital ridges into testes. This gene was referred to as *Tdy* (testis-determining on the Y) in mice and TDF (testis determining factor) in human, but its identity was unknown at the time. In addition, Eicher and colleagues made the serendipitous discovery that offspring from XY mice (*Mus musculus domesticus*, subsequently named *Mus poschiavinus*) derived from the valley Val Poschiavio in Switzerland, as well as other XY *Mus musculus domesticus* trapped in three different regions of Italy, showed a skewing of the sex ratio towards females when bred with XX C57BL/6J (Eicher et al., 1982).

Chromosomal analysis revealed that, indeed, some of these female offspring were XY. Further investigations showed that they were either completely XY sex reversed (i.e. had ovaries) or were partially XY sex reversed with both ovarian and testicular tissue (Eicher et al., 1982). Interestingly, XY females were only produced
91 when the Y chromosome was inherited from the original *Mus poschiavinus* male but not from a C57BL/6

92 male, indicating that the *Mus poschiavinus* Y chromosome carries a variant of *Tdy*, called *Tdy*<sup>POS</sup> or *Y*<sup>POS</sup>, that

93 is not fully functional on a C57BL/6J background (Eicher et al., 1982).

94

95 From these data, Eicher and Washburn (Eicher and Washburn, 1983) proposed the following model of

96 gonadal sex determination in mammals (Fig. 1A): the undifferentiated gonad is bipotential and has the

97 capacity to develop into either a testis or an ovary depending on coordinated networks of gene expression

98 to drive the testicular and the ovarian differentiation program, respectively. Both testicular and ovarian

99 pathways are triggered by a master sex determining gene, *Tdy*, from the Y chromosome in testes and ovary-

100 determining (*Od*) from the X chromosome or an autosome in ovaries. *Tdy* and *Od* then activate downstream

101 genes, such as testis-determining (*Td*)-1, *Td*-2, etc. and (*Od*)-1, *Od*-2, etc., respectively, to drive

102 differentiation (Fig. 1A). The authors predicted that the *Tdy* locus functions before *Od* to ensure testis

103 development and that *Tdy* or its downstream gene *Td*-1 inhibits *Od* or the *Od*-1 locus to ensure testis

104 differentiation (Fig. 1A). Hence, in the *Y*<sup>POS</sup> model of XY sex reversal, *Tdy* from the *Y*<sup>POS</sup> chromosome and the

105 testis pathway are not activated in a coordinated or timely manner and/or *Od* and the ovarian pathway in

106 C57BL/6 mice is initiated earlier or stronger compared to *Od* in *Mus poschavinus*.

107

108 A second model was proposed a few years later by McElreavey and colleagues (McElreavey et al., 1993),

109 after the discovery of *Sry* as *Tdy/TDF* (Berta et al., 1990, Gubbay et al., 1990, Sinclair et al., 1990). The

110 authors based their model on the analysis of human XX DSD cases who exhibited male characteristics with

111 or without the testis-determining gene *SRY* (McElreavey et al., 1993). They concluded that the most

112 straightforward interpretation of their results would be the presence of a recessive allele in an autosomal

113 locus, called *Z*, which allows testis development in the absence of *SRY*. In this model, *Z* inhibits the

114 expression of testicular genes in XX females, while in XY males *SRY* inhibits the expression or the function of

115 *Z* (Fig. 1B)(McElreavey et al., 1993).

116
117 These models formed the basis of our understanding of the molecular mechanisms driving gonadal sex determination in mammals. In the following sections, we describe what is known today and how the insight we gained confirmed, disproved or enhanced these models.

120

121 Established mechanism of gonadal sex determination

122 *Formation of the genital ridge*

123 The gonads develop as part of the urogenital system and, in mice, both gonads and adrenal glands are believed to develop from a common precursor called the adrenogonadal primordium (AGP), which is visible as a thickening of the coelomic epithelium (CE) overlying the mesonephros and functions as an embryonic kidney in some species (Hatano et al., 1996, Ikeda et al., 1994). The AGP had been described to originate from the intermediate mesoderm (Fraser, 1950) but single-cell transcriptomic signatures have challenged this observation, suggesting that the AGP arises from the lateral plate rather than the intermediate mesoderm (Neirijnck et al., 2023, Qiu et al., 2024). This may also be the case in humans because *in vitro* differentiation of human embryonic cells into gonadal cells is more efficient when they first undergo lateral mesodermal differentiation (Knarston et al., 2020).

132

133 In mice, it had been described that the AGP starts to develop at around 9.5 days *post coitum* (dpc) through proliferation of coelomic epithelial cells and fragmentation of the underlying basement membrane, allowing epithelial cells to ingress to form a multilayered structure (Brambell, 1927, Kusaka et al., 2010).
134
135
136 Approximately one day later, at 10.5 dpc, the AGP splits into the more cranial adrenal primordium (AP) and the more caudal gonadal primordium (GP). Interestingly, recent single-cell RNA sequencing (scRNAseq) data suggested that, at the molecular level, CE cells are already specified (most likely driven by *Hox* gene expression) into AP and GP before ingression of cells and the formation of a multilayered ridge (Fig. 2) (Neirijnck et al., 2023).

141

142 The AGP is marked by the expression of several transcription factors, such as GATA-binding protein 4 (GATA4) (Hu et al., 2013) and Wilms' tumour suppressor 1 (WT1) (Wilhelm and Englert, 2002), followed by
144 Lim homeobox protein 9 (LHX9) (Birk et al., 2000) and homeobox protein EMX2 (Miyamoto et al., 1997),
145 and subsequently the nuclear receptor subfamily 5, group A, member 1 (NR5A1, also known as
146 steroidogenic factor 1, SF1) (Neirijnck et al., 2023). For quite some time, NR5A1 has been used as an early
147 marker for the AGP (Ikeda et al., 1996), but the transcripts of this factor become abundant only after the
148 AGP splits into the AP and GP. Consequently, NR5A1 appears to be (a) independently upregulated in these
149 two cell lineages; and (b), not a suitable marker for early AGP cells. In addition, scRNAseq analyses in mouse
150 have revealed that AP and GP identity are associated with an upregulation of RHO and WNT/β-catenin
151 signalling, respectively (Neirijnck et al., 2023).

152
153 Curiously, when comparing scRNAseq analyses in different species, greater variability between species in
154 these early processes has been discovered than what has been previously assumed (Fig. 2). While in mouse,
155 AP and GP appear to be specified at around the same time [right next to each other in the CE (Fig. 2)] in
156 chicken, two CE cell layers have been described with the one lining the coelomic cavity giving rise to the
157 gonads, and the one below likely being the AP (Saito et al., 2017). However, this is in contradiction to other
158 studies that analysed Nr5a1 expression and showed that, at early stages, there is a single Nr5a1-positive
159 AGP cell group within the mesonephric mesenchyme, which splits into two subgroups shortly after: one
160 ventral, next to the CE and assumed to be the GP, and one dorsal, next to the aorta and assumed to be the
161 AP (Fig. 2) (Yoshioka et al., 2005). These data suggested that the GP in chicken is not derived from the CE,
162 but actually from the mesonephric mesenchyme. This was confirmed in more recent publications, which
163 have shown that the gonadal anlage in chicken consists of cells not only from the CE but also from the
164 nephrogenic mesenchyme, with interstitial/stromal cells derived from the CE, but supporting and
165 steroidogenic cells derived from the nephrogenic mesenchyme (Estermann et al., 2020, Sekido and Lovell-
166 Badge, 2007). A third variation is found in humans and cynomolgus monkeys in which the AP and GP are
167 spatially, temporally and phenotypically distinct with the AP developing earlier and clearly separated from
168 the GP, although also driven by HOX gene expression as in mice (Fig. 2) (Cheng et al., 2022). Similarly in
169 zebrafish, scRNAseq data has suggested that there is no AGP, and that the AP and GP develop
170 independently, but further lineage tracing experiments are required to rule out a common origin (Yan et al., 2021).

172

173 Further growth of the GP is achieved by continuous proliferation of CE cells, possibly by asymmetric division, with some daughter cells remaining in the epithelium, while others undergo epithelial-to-mesenchymal transition, leading to the expansion of the GP in both XY and XX (Karl and Capel, 1998, Kusaka et al., 2010, Lin et al., 2017, Schmahl et al., 2000). Many factors are involved in these early processes including Insulin/Insulin-like growth factor 1 receptor (IGF1R) and R-SPONDIN-1 (RSPO1)/WNT signalling pathways (reviewed in (Chassot et al., 2012, Nef et al., 2019). The CE-derived somatic cells are multipotent progenitors that give rise to most somatic cell lineages in the gonads (Capel, 2000, Garcia-Alonso et al., 2022, Karl and Capel, 1998, Mayere et al., 2022, Stevant et al., 2018a). Unexpectedly, in both mouse and human, recent scRNAseq has shown that the first cell lineage to be specified is a novel cell lineage named 'supporting-like cell lineage', which is marked by the expression of *Pax8* (Fig. 3) and contributes to the formation of the rete testis and rete ovarii (Garcia-Alonso et al., 2022, Kulibin and Malolina, 2020, Mayere et al., 2022). Subsequently, the next cell lineage specified is the pre-supporting cells, which differentiate into Sertoli cells in XY and pre-granulosa cells in the XX gonads, respectively (Mayere et al., 2022, Stevant et al., 2019, Stevant et al., 2018a).

187

188 The expansion of the GP at 10-10.5 dpc is accompanied by the arrival and incorporation of primordial germ cells (PGCs) into the GP (Molyneaux et al., 2001). PGCs are the precursor cells of sperm and eggs, which are specified extra-gonadally and migrate towards the developing gonads to coalesce with the somatic cells. In mice, PGCs are specified in the epiblast during gastrulation through inductive bone morphogenetic protein (BMP) signalling at 6.5 dpc (Lawson et al., 1999, Roelen and Chuva de Sousa Lopes, 2022, Saitou and Hayashi, 2021, Saitou and Yamaji, 2012). From there they migrate to the endoderm by 7.5 dpc, and along the developing hindgut and dorsal mesentery to reach the developing genital ridge at around 10.5 dpc (Tam and Snow, 1981). Remarkably, the migration path along the hindgut appears to be conserved in many species ranging from *Drosophila* to mammals (Richardson and Lehmann, 2010). One of the exceptions are
birds, where migration towards the gonads occurs using the bloodstream (Swift, 1913). While germ cells are an integral part of gonad development, in the remainder of this Review, we focus on somatic cells and refer to comprehensive reviews covering germ cell specification, migration and differentiation (Cooke and Moris, 2021, Hancock et al., 2021, McLaren, 1983, Roelen and Chuva de Sousa Lopes, 2022, Saitou and Yamaji, 2012).

202

### Tdy, Td-1, etc. and testis differentiation

In the original model, Eicher and Washburn (Eicher and Washburn, 1983) had predicted that the testis-determining gene *Tdy* on the Y chromosome is the first to be activated (Fig. 1). It took several years before the mammalian testis determining gene *Tdy* was identified and named *Sry* for sex determining region of chromosome Y (Berta et al., 1990, Gubbay et al., 1990, Sinclair et al., 1990). Subsequent experiments in mice proved that *Sry* is not only necessary but also sufficient for testis development (Koopman et al., 1991).
*Sry* has been described as a single-exon gene in eutherian mammals, which is believed to have evolved from *Sox3* (Katsura et al., 2018). Interestingly, recent long-read transcriptomic analysis has revealed a second *Sry* transcript in mouse arising from a cryptic second exon, named *Sry-T* as opposed to the single exon transcript *Sry-S* (Miyawaki et al., 2020). The two different transcripts encode proteins that differ in their last 15 and 18 amino acids, respectively (Miyawaki et al., 2020). The protein encoded by the originally identified SRY-S contains a degron sequence in its C-terminus, which is not present in SRY-T, resulting in destabilisation of the SRY-S protein. Hence, while both proteins are capable of driving testis development when expressed at sufficiently high levels, in wildtype mice, it is predominantly the SRY-T that is present and responsible for the switch to the testis program (Miyawaki et al., 2020). Interestingly, while the degron sequence is present in the mRNA of other rodents, such as rats (*Rattus norvegicus*), Okinawa spiny rats (*Tokudaia muenninki*) and target rats (*Stochomys longicaudatus*), it is not part of the translated protein in these species due to upstream stop codons (Miyawaki et al., 2020) and, therefore, does not result in destabilisation of the protein. Given that there are known differences with respect to sex determination between different mouse species, as shown by the Y<sup>POS</sup> mouse model, it would be interesting to see if the second transcript is present in all mouse strains.
224
225 In mice, *Sry* is expressed in the developing ridge from approximately 10.5 to 12.5dpc. The expression of *Sry* is highly regulated by signalling cascades and epigenetic factors (reviewed in (Kashimada and Koopman, 2010, Svingen and Koopman, 2013) and must occur in a tight time window (Hiramatsu et al., 2009). *Sry* encodes a transcription factor with an HMG domain as DNA-binding domain that binds the minor groove and thereby bends the DNA (Pontiggia et al., 1994, Pontiggia et al., 1995, Ferrari et al., 1992). The HMG domain of SRY protein is highly conserved between different species, in contrast to regions N- and C-terminal to this domain. Specifically, the C-terminal domain in mouse and rat contains a unique Q-rich domain connected to the HMG domain via a bridge domain (Dubin and Ostrer, 1994), both of which are not present in any other species. Nevertheless, transgenic mouse models and analysis of human DSD patients with mutations in *SRY* suggest that even though these domains outside the HMG are poorly conserved, they still contribute to the function of SRY protein (for review see: (Zhao and Koopman, 2012)).
236
237 Although progress was made in characterising the SRY protein, the target gene *Td-1* that had been predicted by Eicher and Washburn (Fig. 1) remained elusive for a long time, although many speculated that it would be the related gene *Sox9* (Sekido et al., 2004, Wilhelm et al., 2005). *Sox9* is expressed and plays a crucial role in several tissues (Ming et al., 2022), and hence its regulatory region includes long-range elements and enhancers (Bagheri-Fam et al., 2001), which made it difficult to identify and define the elements that are important for its expression in the developing testis and to prove that it is regulated by SRY. Eventually, in 2008, Sekido and Lovell-Badge showed that SRY, together with NR5A1, directly regulates *Sox9* expression through an enhancer called *TES* (testis-specific enhancer of *Sox9*) and its core element *TESCO* (*TES* core) (Sekido and Lovell-Badge, 2008). However, subsequent loss-of-function analysis of this enhancer demonstrated that, firstly, although *Sox9* expression is reduced, it does not lead to XY sex reversal (Gonen et al., 2017). Secondly, to date, no human DSD patient has been identified with pathogenic variants in this enhancer (Georg et al., 2010). These data suggest that there might be additional elements that are important for the expression of *Sox9* in mouse and human testes. To identify other *Sox9* regulatory sequences, Gonen and colleagues (Gonen et al., 2018) used DNasel-seq (deoxyribonuclease I
hypersensitivity site sequencing) and ATAC-seq (assay for transposase-accessible chromatin using
sequencing), followed by interrogation of the identified elements for their ability to drive expression in
testes using *LacZ* transgenic mice. The ultimate test the authors used was genome editing to delete putative
enhancers in mice, which proved that either deletion of enhancer 13 (*Enh13*) or deletion of two SRY/SOX
binding sites in this enhancer resulted in a loss of *Sox9* expression and complete male-to-female sex
reversal (Gonen et al., 2018, Ridnik et al., 2024). Excitingly, analysis of human 46,XX and 46,XY DSD patients
with copy number variants within the upstream regulatory region of *SOX9* revealed the human *Enh13*
ortholog, named *eSR-A* (enhancer sex reversal-A) (Croft et al., 2018). However, the authors showed that
there are, at least *in vitro*, species differences with the mouse *Enh13* being activated by SRY and SOX9, while
human *eSR-A* is only activated by SOX9 but not SRY (Croft et al., 2018). Instead, two additional enhancers
were identified in human, *eSR-B* and *eALDI* (enhancer alternate long-distance initiator), of which only the
latter is activated *in vitro* by SRY, but all three (*eSR-A*, *eSR-B* and *eALDI*) function synergistically to ensure
*SOX9* expression in the testis (Croft et al., 2018).

264

To identify other SRY target genes in addition to *Sox9*, *in vivo* chromatin immunoprecipitation (ChIP) experiments were performed (Bradford et al., 2009, Li et al., 2014). These identified a number of putative target genes, including genes that, in cell culture experiments, are upregulated by SRY such as cerebellin 4 precursor gen (*Cbln4*) and monocyte to macrophage differentiation-associated 2 (*Mmd2*), or repressed, for example, follistatin (*Fst*) and Iroquois related homeobox 3 (*Irx3*), which have been shown to be important for ovarian development (Fu et al., 2018, Jorgez et al., 2004, Matzuk et al., 1995). However, the evidence that SRY indeed influences the expression of these genes *in vivo* is weak.

272

Similar to SRY, SOX9 is an HMG domain transcription factor; however, unlike SRY, for which only one target (*Sox9*) has been confirmed today, SOX9 upregulates a number of downstream targets including fibroblast growth factor 9 (*Fgf9*), anti-Müllerian hormone (*Amh*), and prostaglandin D2 synthase (*Ptgds*), which could be considered Td-2, Td-3, etc. (Arango et al., 2008, Kim et al., 2006, Rahmoun et al., 2017, Wilhelm et al., 2007)(Fig. 1). FGF9 and PTGDS contribute to the maintenance of *Sox9* expression in the testis (Kim et al.,
278 2006, Wilhelm et al., 2007, Wilhelm et al., 2005, Moniot et al., 2009), which is crucial for continued development and testicular integrity (Gao et al., 2006, Georg et al., 2012).

280
281 Both *Sry* and *Sox9* are expressed in pre-supporting cells and drive their differentiation into Sertoli cells (Fig. 3) (Hiramatsu et al., 2009, Sekido et al., 2004, Chaboissier et al., 2004, Barrionuevo et al., 2006), which is accompanied by downregulation of the WNT/β-catenin signalling pathway (Fig. 4) (Chang et al., 2008, Harris et al., 2018, Kim et al., 2006). This downregulation occurs in wildtype XY gonads at around 11.5 dpc (Chassot et al., 2012, Munger et al., 2013). *Wnt4* expression is detected in *Fgf9*-null XY gonads at 12.5 dpc (Kim et al., 2006), indicating that FGF9 signalling and hence stabilisation of *Sox9* expression is required to repress WNT signalling in the XY gonad, however, a direct link between the two has not been confirmed so far. The differentiation of Sertoli cells is also marked by the polarization and mesenchymal-to-epithelial transition of Sertoli cells to assemble around clusters of germ cells (Fig. 3) (Fleming et al., 2012, Nel-Themaat et al., 2009). These clusters are partitioned into testis cords, the precursors of the seminiferous tubules, by wedges of proliferating interstitial cells and endothelial cells that migrate into the developing testis from the underlying mesonephros to form the testis-specific vasculature (Combes et al., 2009, Cool et al., 2012, Cool et al., 2011). Sertoli cells also secrete factors, such as desert hedgehog (DHH) and PDGFA platelet growth factor A (PDGFA), which induce the differentiation of steroidogenic fetal Leydig cells (FLCs) by 12.5dpc and peritubular myoid cells (PM cells) by about 13.5 dpc from interstitial progenitor cells (Brennan et al., 2003, Clark et al., 2000, Pierucci-Alves et al., 2001, Schmahl et al., 2008, Yao et al., 2002).

297
298 FLCs produce INSL3 hormone, which is involved in testicular descent (Nef and Parada, 1999), and contribute to the production of androgens that virilise primary sexual characteristics such as external genitalia. Their number increases in the mouse fetal testis until around the time of birth, before declining to be replaced by adult Leydig cells (ALCs) that have distinct morphological and molecular characteristics (Shima, 2019, Shima and Morohashi, 2017). The increase in Leydig cell number is not due to proliferation but depends on differentiation from interstitial steroidogenic progenitor cells that are marked by the expression of *Pdgfra*, *Arx*, *Nr2f2* and *Tcf21* (Brennan et al., 2003, Cui et al., 2004, Eliveld et al., 2019, Ge et al., 2006, Kilcoyne et
al., 2014, Miyabayashi et al., 2013, Perea-Gomez, 2025, Shen et al., 2021), as well as the newly identified marker Wnt5a (Ademi et al., 2022). Several origins of steroidogenic progenitor cells have been hypothesised over the years (Barsoum and Yao, 2006, Defalco et al., 2011, Griswold and Behringer, 2009, Merchant-Larios and Moreno-Mendoza, 1998), and only recent in vivo lineage tracing and scRNAseq experiments have demonstrated that there are two sources of steroidogenic progenitor cells, CE- and mesonephros-derived cells (Ademi et al., 2022, Kumar and DeFalco, 2018). The first FLCs specified from 12.5 dpc are CE-derived, but by the end of gestation approximately 33% of FLCs have been recruited from mesonephros-derived progenitor cells (Kumar and DeFalco, 2018). Interstitial progenitors are multipotent, and their fate varies depending on the developmental stage, with early progenitors giving rise to FLCs while late progenitors contribute to PM cells and later to ALCs (Ademi et al., 2022). The differentiation of FLCs is positively regulated by Sertoli cell-derived factors including DHH and PDGFRA (Barsoum and Yao, 2011, Brennan et al., 2003, Schmahl et al., 2008, Yao et al., 2002) and negatively controlled by vascular and peri-vascular derived NOTCH ligands (Defalco et al., 2013, Kumar and DeFalco, 2018, Liu et al., 2016, Tang et al., 2008).
Interestingly, Sertoli derived signals are no longer required after 14.5 dpc for the differentiation or maintenance of FLCs (Souali-Crespo et al., 2023). While DHH is also required for PM cell differentiation (Pierucci-Alves et al., 2001, Yao et al., 2002), the factors controlling the commitment to FLC versus PM cell fate of interstitial progenitors are currently unknown.

Overall, the prediction from Eicher and Washburn (Eicher and Washburn, 1983) regarding male sex determination and the differentiation of the testis have been confirmed and great progress has been made regarding details of factors and mechanisms that are required to form a functional testis. In the next sections, we look at ovarian development and if the predictions made also can be confirmed for females.

## Od, Od-1, etc. and ovary differentiation

The identification of SRY as the testis-determining factor provided an anchor point to elucidate the male pathway (Fig. 4). Together with the obvious morphological changes, relatively fast progress was made to identify additional genes involved in testis differentiation. In contrast, ovarian differentiation had been
considered the "default" pathway and molecular markers for specific somatic cells were not known, hence
resulting in a delay in the identification of cellular and molecular pathways driving ovarian development.
Things started to turn around with the discovery of partial XX female-to-male sex reversal in a mouse model
lacking the signalling molecule, WNT4 (Vainio et al., 1999). Similar to mice, variants in WNT4 lead to
virilisation in 46,XX SRY-negative humans affected by the SERKAL syndrome (46,XY sex reversal with
dysgenesis of kidneys, adrenals, and lungs; OMIM #611812) (Biason-Lauber et al., 2004, Mandel et al.,
2008), showing that WNT4 plays an important role in ovarian differentiation. WNTs function by binding to
Frizzled receptors, of which there are 10 different (FZD1 to FZD10) encoded in the mouse and human
genome. These often act redundantly (Wang et al., 2016), hence we do not know yet which FZDs are
involved in ovarian development. WNT4 has been shown to act context dependent either through the
canonical WNT/β-catenin or non-canonical pathways (Zhang et al., 2021). However, with respect to ovarian
differentiation, WNT4 has been shown to activate the bona fide Wnt/β-catenin target genes Axin2 and
lymphoid enhancer binding factor 1 (Lef1) (Chassot et al., 2008), supporting the notion that it functions at
least partly through the canonical pathway to promote ovarian differentiation. Nevertheless, the absence of
early sex reversal in Wnt4-deficient XX gonads, highlighted by the lack of stable expression of specific
markers for Sertoli cells, suggesting that the absence of Wnt4 does not affect primary gonadal sex
determination. Thus, Wnt4 might not be the ovarian determining gene.

349

The next important regulator of ovarian development identified was the forkhead transcription factor
FOXL2 (Crisponi et al., 2001, Ottolenghi et al., 2005). Foxl2 is ovarian-enriched expressed in many species
(Cocquet et al., 2002) and loss-of-function of FOXL2 in goats is involved in polled intersex syndrome, which
is associated with XX female-to-male sex reversal (Boulanger et al., 2014, Pailhoux et al., 2001, Pannetier et
al., 2012) and implying Foxl2 could be the female determining gene Od in mammals. Supporting this idea
were reports that FOXL2 is an important ovarian determining gene in various fish, amphibian and reptile
species (Alam et al., 2008, Baron et al., 2004, Govoroun et al., 2004, Hu et al., 2014, Liu et al., 2012,
Nakamoto et al., 2006, Okada et al., 2009, Oshima et al., 2008, Shoemaker-Daly et al., 2010, Yamaguchi et
al., 2007). However, analysis of human patients with heterozygous, and rare instances of homozygous FOXL2
359 variants suffering from type 1 BPES (blepharophimosis, ptosis, and epicanthus inversus syndrome;

360 OMIM#110100) (Crisponi et al., 2001, Kaur et al., 2011, Nallathambi et al., 2007) and knockout experiments

361 in mice (Ottolenghi et al., 2005) have demonstrated that, in these species loss-of-function of FOXL2 results
362 in premature ovarian insufficiency, but not sex reversal. Another transcription factor, runt related

363 transcription factor 1 (RUNX1), was recently identified that has conserved ovary-enriched expression and
364 plays complementary and redundant functions with FOXL2 in the developing ovary (Nicol et al., 2019).

365 While RUNX1 loss-of-function in mice do not result in severe defects and females are fertile, the combined
366 knockout with *Foxl2* leads to masculinisation with visible morphological changes of the fetal ovary (Nicol et
367 al., 2019).

368

369 The next candidate for *Od*, the ovarian determining gene in mammals, that was discovered was RSPO1 a
370 signalling molecule that functions to enhance canonical WNT signalling through binding to LGR (leucine-rich
371 repeat-containing G-protein coupled) receptors. Variants in *RSPO1* are causative for a human disorder
372 named palmoplantar hyperkeratosis with squamous cell carcinoma of skin, which is associated with 46,XX
373 sex reversal (OMIM #610644, (Parma et al., 2006)). Deletion of *Rspo1* in mouse results in partial XX sex
374 reversal, and this phenotype can be rescued by overexpression of constitutively activated β-catenin in the
375 developing XX *Rspo1* mutant gonads (Chassot et al., 2008, Tomizuka et al., 2008). These data suggested that
376 RSPO1, similar to WNT4, functions in ovarian development through the stabilisation of β-catenin (Fig. 4).

377 Indeed, ectopic expression of constitutively activated β-catenin in XY mouse gonads results in XY male-to-
378 female sex reversal (Maatouk et al., 2008), indicating that canonical WNT signalling can promote ovarian
379 development. However, the dominant mutant β-catenin used in these experiments lacks exon 3 of the
380 *Ctnnb1* allele that encodes β-catenin. Exon 3 contains all residues that, when phosphorylated, lead to
381 degradation of β-catenin, and hence the lack of exon 3 results in non-physiological high levels of the
382 protein. In contrast, loss-of-function of ZNRF3, a ubiquitin ligase that inhibits canonical WNT/β-catenin by
383 promoting the degradation of the WNT receptor complex (Hao et al., 2012), also results in the activation of
384 β-catenin but to a more physiological level. In these mice XY gonads display a variable phenotype ranging
385 from almost normal testis development to complete XY sex reversal (Harris et al., 2018). Interestingly, more
detailed analysis of these mutant gonads by scRNAseq has shown that there is an increase in pre-supporting cells that fail to differentiate into pre-Sertoli cells and instead a fraction of these commit to the pre-granulosa or supporting-like cell fate (Kay et al., 2024). Thus, mutation of *Znrf3* and therefore the increase in WNT/β-catenin signalling tips the balance from Sertoli to granulosa cell differentiation. The molecular mechanism of how WNT signalling leads to this could be by promoting the granulosa cell fate through the upregulation of *Foxl2* (Li et al., 2017, Nicol et al., 2018) and/or by directly interfering with *Sox9* expression (Bernard et al., 2012). Finally, the conditional deletion of β-catenin in the gonadal somatic cells, similar to the deletion of *Wnt4* or *Rspo1* in XX gonads, allows pre-granulosa cell differentiation but results in the trans-differentiation of a proportion of them into Sertoli-like cells perinatally (Le Rolle et al., 2021, Liu et al., 2009, Maatouk et al., 2013, Nicol and Yao, 2015). Taken together, these data indicate that the RSPO1/WNT4/β-catenin signalling pathway is involved in the stabilization of ovarian development but is not *Od*.

With the identification of these important ovarian regulators (RSPO1, WNT4 and FOXL2), the question became: if canonical WNT signalling (initiated by RSPO1 and WNT4) and FOXL2 function in the same or in parallel pathways in the fetal ovary? On the one hand, *Rspo1* expression is not affected in *Foxl2*-null mice, but *Foxl2* expression is reduced in *Rspo1* knockout ovaries (Auguste et al., 2011), suggesting that *Foxl2* expression is, directly or indirectly, downstream of *Rspo1*. However, on the other hand, deletion of *Foxl2* on a *Wnt4*-null or *Rspo1*-null exacerbates the sex reversal phenotype of *Wnt4* and *Rspo1* knockout, respectively (Auguste et al., 2011, Ottolenghi et al., 2007), indicating that they function – at least to some extent – in parallel pathways. Of note, neither double knockout *Wnt4/Foxl2* nor *Rspo1/Foxl2* leads to lack of ovarian differentiation (Auguste et al., 2011, Ottolenghi et al., 2007).

The identification of these molecular players has allowed the elucidation of cellular events during early ovarian development (Fig. 3). Lineage tracing in mice has demonstrated that supporting precursor cells, which in the developing testis express *Sry* and *Sox9*, upregulate *Foxl2* in the developing ovary (Albrecht and Eicher, 2001, Mork et al., 2011). However, surprisingly, even though in the adult ovary the supporting
413 granulosa cells all express *Foxl2*, the *Foxl2*-positive pre-supporting cells in the fetal ovary mainly give rise to
414 granulosa cells of the medullary follicles, which are activated shortly after birth and contribute to puberty
415 and early fertility but not the granulosa cells of cortical follicles (Mork et al., 2011, Niu and Spradling, 2020,
416 Zheng et al., 2014), which encompass the ovarian reserve. Instead, these granulosa cells are derived from
417 cells in the CE of fetal ovaries that express LGR5 (Fukuda et al., 2021, Gustin et al., 2016, Niu and Spradling,
418 2020, Rastetter et al., 2014), one of the receptors of RSPO1 (Carmon et al., 2011, Chen et al., 2013). In
419 addition, around 7% of FOXL2 positive granulosa cells in the newborn ovary, mainly located close to the rete
420 ovary, are derived from *Pax8*-positive supporting-like cells (Mayere et al., 2022).

421
422 Hence, most – if not all – supporting precursor cells in the developing ovary are derived from the CE, similar
423 to the supporting cells in a testis. However, the timing of supporting cell differentiation is crucially different
424 in testes and ovaries. Single-cell transcriptomic analyses have shown in detail that the establishment of
425 sexual dimorphism in the supporting lineage occurs at different times in XY and XX mice (Stevant et al.,
426 2019). In the testes, pre-supporting cells adopt a Sertoli cell identity at 11.5 dpc and rapidly lose the
427 transcriptomic signature characteristic of the bipotential supporting cell state. In contrast, XX cells adopt a
428 pre-granulosa cell transcriptional program one day later, from 12.5 dpc, and maintain some degree of
429 bipotential supporting cell signature throughout fetal life (Stevant et al., 2019, Stevant et al., 2018a).
430 Moreover, Sertoli cell recruitment from the CE ceases around 11.7 dpc in the testis (Karl and Capel, 1998),
431 while the recruitment phase in the ovary from the CE is much prolonged until about day 7 after birth (Mork
432 et al., 2011).

433
434 A difference between testis and ovary in timing of somatic cell differentiation also exists for the
435 steroidogenic cell lineage. While in testes, steroidogenic Leydig cells differentiate shortly after Sertoli cells,
436 in an ovary, steroidogenic theca cells cannot be observed in the fetal, only in the postnatal, ovary in mouse
437 (Magoffin, 2005, Mannan and O'Shaughnessy, 1991). Theca cells differentiate from *Gli1*-positive progenitors
438 that origin from the CE as well as the mesonephros in response to Hedgehog signalling, specifically DHH and
439 Indian hedgehog (IHH) secreted by granulosa cells (Liu et al., 2015). In the human ovary, multi-omics
analysis has identified putative steroidogenic progenitor cells, as well as three theca cell lineages including
structural, androgenic and perifollicular theca cells, with each lineage marked by specific gene expression
(Guahmich et al., 2023). In fetal mouse ovaries, interstitial or stromal cells are likely the source of the
steroidogenic progenitors giving rise to theca cells in the postnatal ovary (Mayere et al., 2022, Stevant et al.,
2019).

445

Antagonism between testicular and ovarian pathways

The two pathways, testicular and ovarian pathway do not function in isolation. Eicher and Washburn (Eicher
and Washburn, 1983) hypothesised that *Tdy* or downstream effectors inactivate or inhibit the ovarian
pathway (Fig.1A). This idea of antagonising the other pathway was further expanded on by McElreavey and
colleagues (McElreavey et al., 1993), who predicted that there is an autosomal gene Z, which is not only
inhibited by SRY in XY males but also inhibits the expression of testicular genes in XX females (Fig. 1B,
McElreavey et al., 1993). Indeed, although years later, Kim and colleagues (Kim et al., 2006) showed that
antagonistic pathways are involved in the sexual differentiation of the gonad. The authors showed through
gain- and loss-of-function analysis in mice that FGF9, which in a developing testis is upregulated by SOX9 and
forms a positive feedback loop (Kim et al., 2006), inhibits WNT4. However, we still do not know the molecular
mechanism of how FGF signalling represses WNT4. On the other hand, WNT4 represses FGF9 and thereby
the testicular pathway. Since then, additional antagonistic actions of the testicular and ovarian pathways have
been proposed at different levels. These include the inhibition of β-catenin activity and the inhibition of FOXL2
expression or activity by SRY and SOX9 (Bernard et al., 2008, Nicol et al., 2018, Rahmoun et al., 2017) and the
inhibition of SOX9 expression or activity by β-catenin and FOXL2 (Fig. 4) (Bernard et al., 2008, Migale et al.,
2024, Nicol et al., 2018, Uhlenhaut et al., 2009).

462

The discovery of Od

Overall, data to date have confirmed and expanded almost all predictions made in the early days (Eicher
and Washburn, 1983, McElreavey et al., 1993) – except for the ovarian determining gene *Od* predicted by
Eicher and Washburn (Eicher and Washburn, 1983). WNT signalling and FOXL2 have been identified as
important regulators of ovarian development; however, they do not fit the criteria of being *Od*, but rather downstream regulators *Od-1* and *Od-2*. Nevertheless, all molecular and cellular processes driving gonadal sex determination and differentiation can be explained with *Sry* as the testis determining gene and canonical WNT signalling, together with *FOXL2*, as drivers of ovarian differentiation. In other words, no ovarian *Sry* equivalent is needed, suggesting that the predictions were wrong with respect to *Od* in mice.

There is, however, another explanation of how gonadal sex is determined that requires the existence of OD. In this scenario, *Od* must be expressed not only in XX but also in XY gonads in order for ovaries to develop in cases where *Sry* and/or *Sox9* are absent or their function is impaired, which can be seen in mouse models in which these genes are deleted (Barrionuevo et al., 2006, Lavery et al., 2012). This scenario also requires that the testicular pathway downstream of *Sry* is activated before the ovarian pathway driven by *Od* to ensure testis differentiation, as has been predicted (Eicher and Washburn, 1983). Therefore, if *Sry* expression is inhibited or delayed, or if *Od* expression is activated before *Sry* expression, the bipotential gonad can become an XY ovary. scRNAseq data demonstrating that Sertoli cell differentiation occurs around 11.5 dpc in XY, about a day earlier than pre-granulosa cell differentiation in XX mouse gonads (Stevant et al., 2019), has confirmed the prediction of Eicher and Washburn (Fig. 1)(Eicher and Washburn, 1983).

Furthermore, weak or delayed expression of *Sry*, such as in *B6-XYPOS* mice, allows the ovarian programme to kick in, resulting in a variable phenotype ranging from ovotestis to ovarian development (e.g. (Eicher et al., 1982, Hiramatsu et al., 2009, Pitetti et al., 2013, Wilhelm et al., 2009, Windley et al., 2022).

In 1997, the identification of the molecular basis of Frasier syndrome (OMIM #136680), characterised by focal segmental glomerulosclerosis and the presence of female genitalia in a large number of 46,XY patients (Barbaux et al., 1997, Ezaki et al., 2015), was the first step towards the identification of OD. This syndrome is associated with a variant in intron 9 of the Wilms' tumour suppressor gene *WT1*, which leads to an imbalance in the ratio of the two major splice isoforms termed +KTS and -KTS that differ by the presence or absence of the three amino acids KTS (Barbaux et al., 1997, Klamt et al., 1998). *In vitro*, the -KTS and +KTS isoforms have different biochemical properties (Hastie, 2017). While *WT1-KTS* variants are potent DNA binding proteins that regulate a large number of genes (Hartwig et al., 2010), the *WT1+KTS* variants have a
higher affinity to RNA, co-localise with nuclear speckles and associate with splicing complexes (Englert et al., 1995, Larsson et al., 1995). Andreas Schedl's team recapitulated the renal failure and genital feminisation phenotype of Frasier syndrome in mice by introducing a mutation in the *Wt1* gene that prevents alternative splicing so that only the -KTS isoform is produced (Hammes et al., 2001). However, while these mice do not produce any WT1+KTS, it is important to note that there is an increase in the WT-KTS isoform compared to the wildtype mice. As this mouse model reproduces the XY sex reversal observed in human patients with Frasier syndrome, it suggested that either the absence of the +KTS isoform and/or the increase in the -KTS isoform may be involved in gonadal sex determination. Further investigations using a WT1-KTS knockout mouse model have revealed that gonadal differentiation is blocked in the absence of WT1-KTS and that XX gonads do not develop into ovaries but remain in an undifferentiated state instead, suggesting that the -KTS isoform is important for ovarian development (Gregoire et al., 2023). This is further supported by the fact that ovarian differentiation is accelerated in the Frasier syndrome mouse model, which produces only the -KTS isoform but at a higher level than wildtype, thereby preventing the establishment of high levels of *Sry* and testicular differentiation in XY individuals (Bullejos and Koopman, 2005, Gregoire et al., 2023). These data suggest that the WT1-KTS isoform has all the characteristics of OD: it is necessary for ovarian differentiation and its early expression prevents testis development. Finally, transgenic XY embryos containing additional copies of WT1-KTS have been generated by additive transgenesis of *Wt1-KTS* using the *Wt1* regulatory region, resulting in XY male-to-female sex reversal with differentiation of pre-granulosa cells (Gregoire et al., 2023), which rules out the involvement of +KTS isoform and proves that the -KTS isoform is responsible for the initiation of ovarian development, thereby demonstrating that WT1-KTS is OD, and proving that also these predictions (Eicher and Washburn, 1983) were correct.

Recently, a conditional *Wt1-KTS* gain-of-function model, in which transgenic WT1-KTS expression is activated in XY gonads using the *Nr5a1-Cre* line, has been reported (Cen et al., 2024). The *Nr5a1-Cre* (Bingham et al., 2006) exhibits limited activity in the CE, as evidenced by sparse reporter expression in CE cells at 11.5 dpc (Kim et al., 2007), after the peak of *Sry* expression and the commitment to testis differentiation (Hiramatsu et al., 2009, Wilhelm et al., 2005). In *Wt1+/Ctg;Nr5a1-Cre* XY gonads, expression
521 of the -KTS isoform is increased at 12.5 dpc, which does not result in ovarian development. However,
522 interestingly, FOXL2-positive cells are observed between the testis cords, suggesting abnormal
523 differentiation of XY interstitial cells into theca cells (Cen et al., 2024). Taken together, the WT1-KTS isoform
524 not only plays a role in the sexual differentiation of pre-supporting cells but may also contribute to the
525 sexual differentiation of the steroidogenic cell lineage.

526

527 **Conclusions: rethinking gonadal sex determination in vertebrates**

528 There is a great variability in how sex is determined in different species. With the discovery of SRY as the
529 male determining factor in humans and mice, it became quickly clear that while SRY is necessary and
530 sufficient for testis development in mammals, it is not present in other vertebrates and even some
531 mammals appear to have lost *Sry* such as the mole voles and spiny rats (Fredga, 1983, Graves, 2001). This
532 led to extensive research in finding the master regulator of gonadal sex determination in other vertebrates.
533 Surprisingly, over the years a wide variety of factors that function as master regulators were discovered in
534 various species (Nagahama et al., 2021). Interestingly, all these regulators are testis-determining factors
535 except for *DM-W* in *Xenopus laevis* that fulfils the criteria of being a female determining gene, i.e. when
536 mutated results in ZW female-to-male sex reversal (Yoshimoto et al., 2010) and gain-of-function in ZZ leads
537 to ovarian cavity formation and primary oocyte development (Yoshimoto et al., 2008). For all the other
538 species, as in mammals with *Sry*, all aspects of gonadal sex determination can be explained with “just” a
539 male-determining factor. However, the discovery of WT1-KTS as the ovarian determining factor, in addition
540 to a testis-determining factor, in mice raises the question: are mice an exception or are we missing
541 especially ovarian-determining genes in other species? There are some indications that WT1-KTS could also
542 be important for ovary determination in human. The experiments in mice showed that increased levels of
543 WT1-KTS resulted in precocious differentiation of ovaries, thus preventing testis differentiation (Gregoire et
544 al., 2023). This phenocopies the human syndrome known as “Frasier syndrome” in which XY individuals are
545 feminised due to variants in the WT1 gene that skew the alternative splicing event towards an increased
546 generation of the -KTS isoform (Barbaux et al., 1997, Klamt et al., 1998), suggesting that also in humans
547 WT1-KTS is the ovary determining gene. Interestingly, there are data that implicate WT1-KTS in promoting
548 ovarian development beyond mammals. In reptiles, sex is determined by the temperature at which the eggs
549 are incubated. In the snapping turtle *Chelydra serpentina* it appears that a change to the female-
550 determining temperature rapidly increased not only the expression levels of *Wt1* mRNA but also alternative
551 splicing of *Wt1* with an increase in the relative abundance of the WT-KTS isoform (Rhen et al., 2015). In
552 conclusion, there are still many unknowns when it comes to how sex is determined in different species. And
553 even if a testis (or ovary) determining genes has been identified for any given species, it does not mean that
554 there is not also an ovary (or testis) determining gene still to be discovered.

555

556

557

558 Acknowledgments

559 We are grateful to Yasmine Neirijnck for helpful discussions. This work was supported by the Agence

560 Nationale de la Recherche (ANR-23-CE14-0012 HeteroSex; ANR-24-CE14-2476 Anaform).

561

562

563 References

564 ADEMI, H., DJARI, C., MAYERE, C., NEIRIJNCK, Y., SARAROLS, P., RANDS, C. M., STEVANT, I.,
565 CONNE, B. & NEF, S. 2022. Deciphering the origins and fates of steroidogenic lineages
566 in the mouse testis. *Cell Rep*, 39, 110935.

567 ALAM, M. A., KOBAYASHI, Y., HORIGUCHI, R., HIRAI, T. & NAKAMURA, M. 2008. Molecular
568 cloning and quantitative expression of sexually dimorphic markers Dmrt1 and Foxl2
569 during female-to-male sex change in Epinephelus merra. *Gen Comp Endocrinol*, 157,
570 75-85.

571 ALBRECHT, K. & EICHER, E. 2001. Evidence that *Sry* is expressed in pre-Sertoli cells and
572 Sertoli and granulosa cells have a common precursor. *Developmental Biology*, 240, 92-
573 107.

574 ARANGO, N. A., KOBAYASHI, A., WANG, Y., JAMIN, S. P., LEE, H. H., ORVIS, G. D. &
575 BEHRINGER, R. R. 2008. A mesenchymal perspective of Mullerian duct differentiation
576 and regression in Amhr2-lacZ mice. *Mol Reprod Dev*, 75, 1154-62.

577 AUGUSTE, A., CHASSOT, A. A., GREGOIRE, E. P., RENAULT, L., PANNETIER, M., TREIER, M.,
578 PAILHOUX, E. & CHABOISSIER, M. C. 2011. Loss of R-spondin1 and Foxl2 amplifies
579 female-to-male sex reversal in XX mice. *Sex Dev*, 5, 304-17.

580 BAGHERI-FAM, S., FERRAZ, C., DEMAILLE, J., SCHERER, G. & PFEIFER, D. 2001. Comparative
581 genomics of the SOX9 region in human and *Fugu rubripes*: Conservation of short
582 regulatory sequence elements within large intergenic regions. *Genomics*, 78, 73-82.
583 BARBAUX, S., NIAUDET, P., GUBLER, M. C., GRUNFELD, J. P., JAUBERT, F., KUTTENN, F., FEKETE, C. N., SOULEYREAU-THERVILLE, N., THIBAUD, E., FELLOUS, M. & MCELREAVEY, K. 1997. Donor splice-site mutations in WT1 are responsible for Frasier syndrome. *Nat Genet*, 17, 467-70.

587 BARON, D., COCQUET, J., XIA, X., FELLOUS, M., GUIGUEN, Y. & VEITIA, R. A. 2004. An evolutionary and functional analysis of FoxL2 in rainbow trout gonad differentiation. *J Mol Endocrinol*, 33, 705-15.

590 BARRIONUEVO, F., BAGHERI-FAM, S., KLATTIG, J., KIST, R., TAKETO, M. M., ENGLERT, C. & SCHERER, G. 2006. Homozygous Inactivation of Sox9 Causes Complete XY Sex Reversal in Mice. *Biol Reprod*, 74, 195-201.

593 BARSOUM, I. & YAO, H. H. 2006. The road to maleness: from testis to Wolffian duct. *Trends Endocrinol Metab*, 17, 223-8.

595 BARSOUM, I. & YAO, H. H. 2011. Redundant and differential roles of transcription factors Gli1 and Gli2 in the development of mouse fetal Leydig cells. *Biol Reprod*, 84, 894-9.

597 BASHAMBOO, A. & MCELREAVEY, K. 2016. Mechanism of Sex Determination in Humans: Insights from Disorders of Sex Development. *Sex Dev*, 10, 313-325.

599 BERNARD, P., RYAN, J., SIM, H., CZECH, D. P., SINCLAIR, A. H., KOOPMAN, P. & HARLEY, V. R. 2012. Wnt signaling in ovarian development inhibits Sf1 activation of Sox9 via the Tesco enhancer. *Endocrinology*, 153, 901-12.

602 BERNARD, P., SIM, H., KNOWER, K., VILAIN, E. & HARLEY, V. 2008. Human SRY inhibits betacatenin-mediated transcription. *Int J Biochem Cell Biol*, 40, 2889-900.

604 BERTA, P., HAWKINS, J. R., SINCLAIR, A. H., TAYLOR, A., GRIFFITHS, B. L., GOODFELLOW, P. N. & FELLOUS, M. 1990. Genetic evidence equating SRY and the male sex determining gene. *Nature*, 348, 448-450.

607 BIASON-LAUBER, A., KONRAD, D., NAVRATIL, F. & SCHOENLE, E. J. 2004. A WNT4 mutation associated with Mullerian-duct regression and virilization in a 46,XX woman. *N Engl J Med*, 351, 792-8.

610 BINGHAM, N. C., VERMA-KURVARI, S., PARADA, L. F. & PARKER, K. L. 2006. Development of a steroidogenic factor 1/Cre transgenic mouse line. *Genesis*, 44, 419-24.

612 BIRK, O., CASIANO, D., WASSIF, C., COGLIATI, T., ZHAO, L., ZHAO, Y., GRINBERG, A., HUANG, S., KREIDBERG, J., PARKER, K., PORTER, F. & WESTPHAL, H. 2000. The LIM homeobox gene *Lhx9* is essential for mouse gonad formation. *Nature*, 403, 909-913.

615 BOULANGER, L., PANNETIER, M., GALL, L., ALLAIS-BONNET, A., ELZAIAT, M., LE BOURHIS, D., DANIEL, N., RICHARD, C., COTINOT, C., GHYSELINCK, N. B. & PAILHOUX, E. 2014. *FOXL2* is a female sex-determining gene in the goat. *Curr Biol*, 24, 404-8.

618 BRADFORD, S. T., HIRAMATSU, R., MADDUGODA, M. P., BERNARD, P., CHABOISSIER, M. C., SINCLAIR, A., SCHEDL, A., HARLEY, V., KANAI, Y., KOOPMAN, P. & WILHELM, D. 2009. The cerebellin 4 precursor gene is a direct target of SRY and SOX9 in mice. *Biol Reprod*, 80, 1178-88.

622 BRAMBELL, F. W. R. 1927. The Development and Morphology of the Gonads of the Mouse.- Part I. The Morphogenesis of the Indifferent Gonad and of the Ovary. *Proceedings of the Royal Society of London*, 101, 391-409.

625 BRENNAN, J., TILMANN, C. & CAPEL, B. 2003. Pdgfr-alpha mediates testis cord organization and fetal Leydig cell development in the XY gonad. *Genes Dev*, 17, 800-10.

627 BULLEJOS, M. & KOOPMAN, P. 2005. Delayed Sry and Sox9 expression in developing mouse gonads underlies B6-Y(DOM) sex reversal. *Dev Biol*, 278, 473-81.

629 CAPEL, B. 2000. The battle of the sexes. *Mechanisms of Development*, 92, 89-103.
630 CARMON, K. S., GONG, X., LIN, Q., THOMAS, A. & LIU, Q. 2011. R-spondins function as
631 ligands of the orphan receptors LGR4 and LGR5 to regulate Wnt/beta-catenin
632 signaling. *Proc Natl Acad Sci U S A*, 108, 11452-7.

633 CEN, C., LIU, B., LIN, L., SHEN, Z., WANG, N., ZHANG, L., MENG, K., CHEN, M. & GAO, F. 2024.
634 The -KTS isoform of Wt1 induces the transformation of Leydig cells into granulosa-like
635 cells. *Cell Discov*, 10, 113.

636 CHABOISSIER, M. C., KOBAYASHI, A., VIDAL, V. I., LUTZKENDORF, S., VAN DE KANT, H. J.,
637 WEGNER, M., DE ROOIJ, D. G., BEHRINGER, R. R. & SCHEDL, A. 2004. Functional
638 analysis of Sox8 and Sox9 during sex determination in the mouse. *Development*, 131,
639 1891-901.

640 CHANG, H., GAO, F., GUILLOU, F., TAKETO, M. M., HUFF, V. & BEHRINGER, R. R. 2008.
641 negatively regulates β-catenin signaling during testis development. *Development*, 135,
642 1875-1885.

643 CHASSOT, A. A., BRADFORD, S. T., AUGUSTE, A., GREGOIRE, E. P., PAILHOUX, E., DE ROOIJ,
644 D. G., SCHEDL, A. & CHABOISSIER, M. C. 2012. WNT4 and RSPO1 together are
645 required for cell proliferation in the early mouse gonad. *Development*, 139, 4461-72.

646 CHASSOT, A. A., RANC, F., GREGOIRE, E. P., ROEPERS-GAJADIEN, H. L., TAKETO, M. M.,
647 CAMERINO, G., DE ROOIJ, D. G., SCHEDL, A. & CHABOISSIER, M. C. 2008. Activation
648 of beta-catenin signaling by Rspo1 controls differentiation of the mammalian ovary.
649 *Hum Mol Genet*, 17, 1264-77.

650 CHEN, P. H., CHEN, X., LIN, Z., FANG, D. & HE, X. 2013. The structural basis of R-spondin
651 recognition by LGR5 and RNF43. *Genes Dev*, 27, 1345-50.

652 CHENG, K., SEITA, Y., MORIWAKI, T., NOSHIRO, K., SAKATA, Y., HWANG, Y. S., TORIGOE, T.,
653 SAITOU, M., TSUCHIYA, H., IWATANI, C., HOSAKA, M., OHKOUCHI, T., WATARI, H.,
654 UMAZUME, T. & SASAKI, K. 2022. The developmental origin and the specification of the
655 adrenal cortex in humans and cynomolgus monkeys. *Sci Adv*, 8, eabn8485.

656 CLARK, A. M., GARLAND, K. K. & RUSSELL, L. D. 2000. Desert hedgehog (Dhh) gene is required
657 in the mouse testis for formation of adult-type Leydig cells and normal development of
658 peritubular cells and seminiferous tubules. *Biol Reprod*, 63, 1825-38.

659 COCQUET, J., PAILHOUX, E., JAUBERT, F., SERVEL, N., XIA, X., PANNETIER, M., DE BAERE, E.,
660 MESSIAEN, L., COTINOT, C., FELLOUS, M. & VEITIA, R. A. 2002. Evolution and
661 expression of FOXL2. *J Med Genet*, 39, 916-21.

662 COMBES, A. N., WILHELM, D., DAVIDSON, T., DEJANA, E., HARLEY, V., SINCLAIR, A. &
663 KOOPMAN, P. 2009. Endothelial cell migration directs testis cord formation. *Dev Biol*,
664 326, 112-20.

665 COOKE, C. B. & MORIS, N. 2021. Tissue and cell interactions in mammalian PGC
666 development. *Development*, 148.

667 COOL, J., DEFALCO, T. & CAPEL, B. 2012. Testis formation in the fetal mouse: dynamic and
668 complex de novo tubulogenesis. *Wiley Interdiscip Rev Dev Biol*, 1, 847-59.

669 COOL, J., DEFALCO, T. J. & CAPEL, B. 2011. Vascular-mesenchymal cross-talk through Vegf
670 and Pdgf drives organ patterning. *Proc Natl Acad Sci U S A*, 108, 167-72.

671 CRISPONI, L., DEIANA, M., LOI, A., CHIAPPE, F., UDA, M., AMATI, P., BISCEGLIA, L., ZELANTE,
672 L., NAGARAJA, R., PORCU, S., RISTALDI, M. S., MARZELLA, R., ROCCHI, M., NICOLINO,
673 M., LIENHARDT-ROUSSIE, A., NIVELON, A., VERLOES, A., SCHLESSINGER, D.,
674 GASPARINI, P., BONNEAU, D., CAO, A. & PILIA, G. 2001. The putative forkhead
675 transcription factor FOXL2 is mutated in blepharophimosis/ptosis/epicanthus inversus
676 syndrome. *Nat Genet*, 27, 159-66.

677 CROFT, B., OHNESORG, T., HEWITT, J., BOWLES, J., QUINN, A., TAN, J., CORBIN, V., PELOSI,
678 E., VAN DEN BERGEN, J., SREENIVASAN, R., KNARSTON, I., ROBEVSKA, G., VU, D. C.,
679 HUTSON, J., HARLEY, V., AYERS, K., KOOPMAN, P. & SINCLAIR, A. 2018. Human sex reversal is caused by duplication or deletion of core enhancers upstream of SOX9. *Nat Commun*, 9, 5319.

680
681
682 CUI, S., ROSS, A., STALLINGS, N., PARKER, K. L., CAPEL, B. & QUAGGIN, S. E. 2004. Disrupted gonadogenesis and male-to-female sex reversal in Pod1 knockout mice. *Development*, 131, 4095-105.

685 DEFALCO, T., SARASWATHULA, A., BRIOT, A., IRUELA-ARISPE, M. L. & CAPEL, B. 2013. Testosterone levels influence mouse fetal Leydig cell progenitors through notch signaling. *Biol Reprod*, 88, 91.

688 DEFALCO, T., TAKAHASHI, S. & CAPEL, B. 2011. Two distinct origins for Leydig cell progenitors in the fetal testis. *Dev Biol*, 352, 14-26.

690 DUBIN, R. A. & OSTRER, H. 1994. Sry is a transcriptional activator. *Mol Endocrinol*, 8, 1182-92.

691 EICHER, E. M. & WASHBURN, L. L. 1983. Inherited sex reversal in mice: identification of a new primary sex-determining gene. *J Exp Zool*, 228, 297-304.

693 EICHER, E. M. & WASHBURN, L. L. 1986. Genetic control of primary sex determination in mice. *Annu Rev Genet*, 20, 327-60.

695 EICHER, E. M., WASHBURN, L. L., WHITNEY, J. B., 3RD & MORROW, K. E. 1982. Mus poschiavinus Y chromosome in the C57BL/6J murine genome causes sex reversal. *Science*, 217, 535-7.

698 ELIVELD, J., VAN DEN BERG, E. A., CHIKHOVSKAYA, J. V., VAN DAALEN, S. K. M., DE WINTER-KORVER, C. M., VAN DER VEEN, F., REPPING, S., TEERDS, K. & VAN PELT, A. M. M. 2019. Primary human testicular PDGFRalpha+ cells are multipotent and can be differentiated into cells with Leydig cell characteristics in vitro. *Hum Reprod*, 34, 1621-1631.

703 ENGLERT, C., VIDAL, M., MAHESWARAN, S., GE, Y., EZZELL, R. M., ISSELBACHER, K. J. & HABER, D. A. 1995. Truncated WT1 mutants alter the subnuclear localization of the wild-type protein. *Proc Natl Acad Sci U S A*, 92, 11960-4.

706 ESTERMANN, M. A., WILLIAMS, S., HIRST, C. E., ROLY, Z. Y., SERRALBO, O., ADHIKARI, D., POWELL, D., MAJOR, A. T. & SMITH, C. A. 2020. Insights into Gonadal Sex Differentiation Provided by Single-Cell Transcriptomics in the Chicken Embryo. *Cell Rep*, 31, 107491.

710 EZAKI, J., HASHIMOTO, K., ASANO, T., KANDA, S., AKIOKA, Y., HATTORI, M., YAMAMOTO, T. & SHIBATA, N. 2015. Gonadal tumor in Frasier syndrome: a review and classification. *Cancer Prev Res (Phila)*, 8, 271-6.

713 FERRARI, S., HARLEY, V. R., PONTIGGIA, A., GOODFELLOW, P. N., LOVELL-BADGE, R. & BIANCHI, M. E. 1992. SRY, like HMG1, recognizes sharp angles in DNA. *Embo J*, 11, 4497-506.

716 FLEMING, A., GHAHRAMANI, N., ZHU, M. X., DELOT, E. C. & VILAIN, E. 2012. Membrane beta-catenin and adherens junctions in early gonadal patterning. *Dev Dyn*, 241, 1782-98.

718 FORD, C. E., JONES, K. W., POLANI, P. E., DE ALMEIDA, J. C. & BRIGGS, J. H. 1959. A sex-chromosome anomaly in a case of gonadal dysgenesis (Turner's syndrome). *Lancet*, 1, 711-3.

721 FRASER, E. A. 1950. The development of the vertebrate excretory system. *Biol Rev Camb Philos Soc*, 25, 159-87.

723 FREDGA, K. 1983. Aberrant sex chromosome mechanisms in mammals. Evolutionary aspects. *Differentiation*, 23 Suppl, S23-30.

725 FU, A., OBERHOLTZER, S. M., BAGHERI-FAM, S., RASTETTER, R. H., HOLDREITH, C., CACERES, V. L., JOHN, S. V., SHAW, S. A., KRENTZ, K. J., ZHANG, X., HUI, C. C., WILHELM, D. & JORGENSEN, J. S. 2018. Dynamic expression patterns of Irx3 and Irx5
728 during germline nest breakdown and primordial follicle formation promote follicle
729 survival in mouse ovaries. *PLoS Genet*, 14, e1007488.

730 FUKUDA, K., MURAOKA, M., KATO, Y. & SAGA, Y. 2021. Decoding the transcriptome of pre-
731 granulosa cells during the formation of primordial follicles in the mousedagger. *Biol
732 Reprod*, 105, 179-191.

733 GAO, F., MAITI, S., ALAM, N., ZHANG, Z., DENG, J. M., BEHRINGER, R. R., LECUREUIL, C.,
734 GUILLOU, F. & HUFF, V. 2006. The Wilms tumor gene, Wt1, is required for Sox9
735 expression and maintenance of tubular architecture in the developing testis. *Proc Natl
736 Acad Sci U S A*, 103, 11987-92.

737 GARCIA-ALONSO, L., LORENZI, V., MAZZEO, C. I., ALVES-LOPES, J. P., ROBERTS, K., SANCHO-
738 SERRA, C., ENGELBERT, J., MARECKOVA, M., GRUHN, W. H., BOTTING, R. A., LI, T.,
739 CRESPO, B., VAN DONGEN, S., KISELEV, V. Y., PRIGMORE, E., HERBERT, M., MOFFETT,
740 A., CHEDOTAL, A., BAYRAKTAR, O. A., SURANI, A., HANIFFA, M. & VENTO-TORMO, R.
741 2022. Single-cell roadmap of human gonadal development. *Nature*, 607, 540-547.

742 GE, R. S., DONG, Q., SOTTAS, C. M., PAPADOPOULOS, V., ZIRKIN, B. R. & HARDY, M. P. 2006. In
743 search of rat stem Leydig cells: identification, isolation, and lineage-specific
744 development. *Proc Natl Acad Sci U S A*, 103, 2719-24.

745 GEORG, I., BAGHERI-FAM, S., KNOWER, K. C., WIEACKER, P., SCHERER, G. & HARLEY, V. R.
746 2010. Mutations of the SRY-responsive enhancer of SOX9 are uncommon in XY
747 gonadal dysgenesis. *Sex Dev*, 4, 321-5.

748 GEORG, I., BARRIONUEVO, F., WIECH, T. & SCHERER, G. 2012. Sox9 and Sox8 are required for
749 basal lamina integrity of testis cords and for suppression of FOXL2 during embryonic
750 testis development in mice. *Biol Reprod*, 87, 99.

751 GONEN, N., EOZENOU, C., MITTER, R., ELZAIAT, M., STEVANT, I., AVIRAM, R., BERNARDO, A.
752 S., CHERVOVA, A., WANKANIT, S., FRACHON, E., COMMERE, P. H., BRAILLY-TABARD,
753 S., VALON, L., BARRIO CANO, L., LEVAYER, R., MAZEN, I., GOBAA, S., SMITH, J. C.,
754 MCELREAVEY, K., LOVELL-BADGE, R. & BASHAMBOO, A. 2023. In vitro cellular
755 reprogramming to model gonad development and its disorders. *Sci Adv*, 9, eabn9793.

756 GONEN, N., FUTTNER, C. R., WOOD, S., GARCIA-MORENO, S. A., SALAMONE, I. M., SAMSON,
757 S. C., SEKIDO, R., POULAT, F., MAATOUK, D. M. & LOVELL-BADGE, R. 2018. Sex reversal
758 following deletion of a single distal enhancer of Sox9. *Science*, 360, 1469-1473.

759 GONEN, N., QUINN, A., O'NEILL, H. C., KOOPMAN, P. & LOVELL-BADGE, R. 2017. Normal
760 Levels of Sox9 Expression in the Developing Mouse Testis Depend on the TES/TESCO
761 Enhancer, but This Does Not Act Alone. *PLoS Genet*, 13, e1006520.

762 GOVOROUN, M. S., PANNETIER, M., PAILHOUX, E., COCQUET, J., BRILLARD, J. P., COUTY, I.,
763 BATELLIER, F. & COTINOT, C. 2004. Isolation of chicken homolog of the FOXL2 gene
764 and comparison of its expression patterns with those of aromatase during ovarian
765 development. *Dev Dyn*, 231, 859-70.

766 GRAVES, J. A. 2001. From brain determination to testis determination: evolution of the
767 mammalian sex-determining gene. *Reprod Fertil Dev*, 13, 665-72.

768 GREGOIRE, E. P., DE CIAN, M. C., MIGALE, R., PEREA-GOMEZ, A., SCHAUB, S., BELLIDO-
769 CARRERAS, N., STEVANT, I., MAYERE, C., NEIRIJNCK, Y., LOUBAT, A., RIVAUD, P.,
770 SOPENA, M. L., LACHAMBRE, S., LINSSEN, M. M., HOHENSTEIN, P., LOVELL-BADGE,
771 R., NEF, S., CHALMEL, F., SCHEDL, A. & CHABOISSIER, M. C. 2023. The -KTS splice
772 variant of WT1 is essential for ovarian determination in mice. *Science*, 382, 600-606.

773 GRISWOLD, S. L. & BEHRINGER, R. R. 2009. Fetal Leydig cell origin and development. *Sex
774 Dev*, 3, 1-15.

775 GUAHMICH, N. L., MAN, L., WANG, J., ARAZI, L., KALLINOS, E., TOPPER-KROOG, A.,
776 GRULLON, G., ZHANG, K., STEWART, J., SCHATZ-SIEMERS, N., JONES, S. H., BODINE,
777 R., ZANINOVIC, N., SCHATTMAN, G., ROSENWAKS, Z. & JAMES, D. 2023. Human theca arises from ovarian stroma and is comprised of three discrete subtypes. *Commun Biol*, 778 6, 7.

780 GUBBAY, J., COLLIGNON, J., KOOPMAN, P., CAPEL, B., ECONOMOU, A., MÜNSTERBERG, A., 781 VIVIAN, N., GOODFELLOW, P. & LOVELL-BADGE, R. 1990. A gene mapping to the sex- 782 determining region of the mouse Y chromosome is a member of a novel family of 783 embryonically expressed genes. *Nature*, 346, 245-250.

784 GUSTIN, S. E., HOGG, K., STRINGER, J. M., RASTETTER, R. H., PELOSI, E., MILES, D. C., 785 SINCLAIR, A. H., WILHELM, D. & WESTERN, P. S. 2016. WNT/beta-catenin and 786 p27/FOXL2 differentially regulate supporting cell proliferation in the developing ovary. 787 *Dev Biol*, 412, 250-60.

788 HAMMES, A., GUO, J. K., LUTSCH, G., LEHESTE, J. R., LANDROCK, D., ZIEGLER, U., GUBLER, 789 M. C. & SCHEDL, A. 2001. Two splice variants of the Wilms' tumor 1 gene have distinct 790 functions during sex determination and nephron formation. *Cell*, 106, 319-29.

791 HANCOCK, G. V., WAMAITHA, S. E., PERETZ, L. & CLARK, A. T. 2021. Mammalian primordial 792 germ cell specification. *Development*, 148.

793 HAO, H. X., XIE, Y., ZHANG, Y., CHARLAT, O., OSTER, E., AVELLO, M., LEI, H., MICKANIN, C., 794 LIU, D., RUFFNER, H., MAO, X., MA, Q., ZAMPONI, R., BOUWMEESTER, T., FINAN, P. M., 795 KIRSCHNER, M. W., PORTER, J. A., SERLUCA, F. C. & CONG, F. 2012. ZNRF3 promotes 796 Wnt receptor turnover in an R-spondin-sensitive manner. *Nature*, 485, 195-200.

797 HARRIS, A., SIGGERS, P., CORROCHANO, S., WARR, N., SAGAR, D., GRIMES, D. T., SUZUKI, 798 M., BURDINE, R. D., CONG, F., KOO, B. K., CLEVERS, H., STEVANT, I., NEF, S., WELLS, 799 S., BRAUNER, R., BEN RHOUMA, B., BELGUITH, N., EOZENOU, C., BIGNON- 800 TOPALOVIC, J., BASHAMBOO, A., MCELREAVEY, K. & GREENFIELD, A. 2018. ZNRF3 801 functions in mammalian sex determination by inhibiting canonical WNT signaling. *Proc 802 Natl Acad Sci U S A*, 115, 5474-5479.

803 HARTWIG, S., HO, J., PANDEY, P., MACISAAC, K., TAGLIENTI, M., XIANG, M., ALTEROVITZ, G., 804 RAMONI, M., FRAENKEL, E. & KREIDBERG, J. A. 2010. Genomic characterization of 805 Wilms' tumor suppressor 1 targets in nephron progenitor cells during kidney 806 development. *Development*, 137, 1189-203.

807 HASTIE, N. D. 2017. Wilms' tumour 1 (WT1) in development, homeostasis and disease. 808 *Development*, 144, 2862-2872.

809 HATANO, O., TAKAKUSU, A., NOMURA, M. & MOROHASHI, K. 1996. Identical origin of adrenal 810 cortex and gonad revealed by expression profiles of Ad4BP/SF-1. *Genes Cells*, 1, 663- 811 71.

812 HAYASHI, K., OHTA, H., KURIMOTO, K., ARAMAKI, S. & SAITOU, M. 2011. Reconstitution of the 813 mouse germ cell specification pathway in culture by pluripotent stem cells. *Cell*, 146, 814 519-32.

815 HAYASHI, K. & YOSHINO, T. 2022. Reconstitution of reproductive organ system that produces 816 functional oocytes. *Curr Opin Genet Dev*, 77, 101982.

817 HIRAMATSU, R., MATOBA, S., KANAI-AZUMA, M., TSUNEKAWA, N., KATOH-FUKUI, Y., 818 KUROHMARU, M., MOROHASHI, K., WILHELM, D., KOOPMAN, P. & KANAI, Y. 2009. A 819 critical time window of Sry action in gonadal sex determination in mice. *Development*, 820 136, 129-38.

821 HU, Q., GUO, W., GAO, Y., TANG, R. & LI, D. 2014. Molecular cloning and analysis of gonadal 822 expression of Foxl2 in the rice-field eel Monopterus albus. *Sci Rep*, 4, 6884.

823 HU, Y. C., OKUMURA, L. M. & PAGE, D. C. 2013. Gata4 is required for formation of the genital 824 ridge in mice. *PLoS Genet*, 9, e1003629.
825 HUGHES, I. A., HOUK, C., AHMED, S. F., LEE, P. A. & LAWSON WILKINS PEDIATRIC

826 ENDOCRINE SOCIETY/EUROPEAN SOCIETY FOR PAEDIATRIC ENDOCRINOLOGY

827 CONSENSUS, G. 2006. Consensus statement on management of intersex disorders. *J Pediatr Urol*, 2, 148-62.

829 IKEDA, Y., SHEN, W. H., INGRAHAM, H. A. & PARKER, K. L. 1994. Developmental expression of mouse steroidogenic factor-1, an essential regulator of the steroid hydroxylases. *Mol Endocrinol*, 8, 654-62.

832 IKEDA, Y., SWAIN, A., WEBER, T. J., HENTGES, K. E., ZANARIA, E., LALLI, E., TAMAI, K.,
833 SASSONE-CORSI, P., LOVELL-BADGE, R., CAMERINO, G. & PARKER, K. L. 1996.

834 Steroidogenic factor 1 and Dax-1 colocalize in multiple cell lineages: Potential links in
835 endocrine development. *Molecular Endocrinology*, 10, 1261-1272.

836 JACOBS, P. A. & STRONG, J. A. 1959. A case of human intersexuality having a possible XXY sex-determining mechanism. *Nature*, 183, 302-3.

838 JORGEZ, C. J., KLYSIK, M., JAMIN, S. P., BEHRINGER, R. R. & MATZUK, M. M. 2004. Granulosa cell-specific inactivation of follistatin causes female fertility defects. *Mol Endocrinol*, 18, 953-67.

841 KARL, J. & CAPEL, B. 1998. Sertoli cells of the mouse testis originate from the coelomic
842 epithelium. *Developmental Biology*, 203, 323-333.

843 KASHIMADA, K. & KOOPMAN, P. 2010. Sry: the master switch in mammalian sex determination. *Development*, 137, 3921-30.

845 KATSURA, Y., KONDO, H. X., RYAN, J., HARLEY, V. & SATTA, Y. 2018. The evolutionary process of mammalian sex determination genes focusing on marsupial SRYs. *BMC Evol Biol*, 18, 3.

848 KAUR, I., HUSSAIN, A., NAIK, M. N., MURTHY, R. & HONAVAR, S. G. 2011. Mutation spectrum of fork-head transcriptional factor gene (FOXL2) in Indian Blepharophimosis Ptosis Epicanthus Inversus Syndrome (BPES) patients. *Br J Ophthalmol*, 95, 881-6.

851 KAY, R. G. G., REEVES, R., SIGGERS, P., GREENAWAY, S., MALLON, A. M., WELLS, S., KOO, B. K., MAYERE, C., NEF, S., GREENFIELD, A. & SIMON, M. M. 2024. Gonadal sex reversal at single-cell resolution in Znrf3-deficient mice. *Development*, 151.

854 KILCOYNE, K. R., SMITH, L. B., ATANASSOVA, N., MACPHERSON, S., MCKINNELL, C., VAN DEN DRIESCHE, S., JOBLING, M. S., CHAMBERS, T. J., DE GENDT, K., VERHOEVEN, G., O'HARA, L., PLATTS, S., RENATO DE FRANCA, L., LARA, N. L., ANDERSON, R. A. & SHARPE, R. M. 2014. Fetal programming of adult Leydig cell function by androgenic effects on stem/progenitor cells. *Proc Natl Acad Sci U S A*, 111, E1924-32.

859 KIM, Y., BINGHAM, N., SEKIDO, R., PARKER, K. L., LOVELL-BADGE, R. & CAPEL, B. 2007. Fibroblast growth factor receptor 2 regulates proliferation and Sertoli differentiation during male sex determination. *Proc Natl Acad Sci U S A*, 104, 16558-63.

862 KIM, Y., KOBAYASHI, A., SEKIDO, R., DINAPOLI, L., BRENNAN, J., CHABOISSIER, M. C., POULAT, F., BEHRINGER, R. R., LOVELL-BADGE, R. & CAPEL, B. 2006. Fgf9 and Wnt4 act as antagonistic signals to regulate mammalian sex determination. *PLoS Biol*, 4, e187.

866 KLAMT, B., KOZIELL, A., POULAT, F., WIEACKER, P., SCAMBLER, P., BERTA, P. & GESSLER, M. 1998. Frasier syndrome is caused by defective alternative splicing of WT1 leading to an altered ratio of WT1 +/-KTS splice isoforms. *Hum Mol Genet*, 7, 709-14.

869 KNARSTON, I. M., PACHERNEGG, S., ROBEVSKA, G., GHOBRIAL, I., ER, P. X., GEORGES, E., TAKASATO, M., COMBES, A. N., JORGENSEN, A., LITTLE, M. H., SINCLAIR, A. H. & AYERS, K. L. 2020. An In Vitro Differentiation Protocol for Human Embryonic Bipotential Gonad and Testis Cell Development. *Stem Cell Reports*, 15, 1377-1391.
873 KOOPMAN, P., GUBBAY, J., VIVIAN, N., GOODFELLOW, P. & LOVELL-BADGE, R. 1991. Male
874 development of chromosomally female mice transgenic for *Sry*. *Nature*, **351**, 117-121.

875 KULIBIN, A. Y. & MALOLINA, E. A. 2020. Formation of the rete testis during mouse embryonic
development. *Dev Dyn*, **249**, 1486-1499.

877 KUMAR, D. L. & DEFALCO, T. 2018. A perivascular niche for multipotent progenitors in the fetal
testis. *Nat Commun*, **9**, 4519.

879 KUSAKA, M., KATOH-FUKUI, Y., OGAWA, H., MIYABAYASHI, K., BABA, T., SHIMA, Y., SUGIYAMA,
880 N., SUGIMOTO, Y., OKUNO, Y., KODAMA, R., IIZUKA-KOGO, A., SENDA, T., SASAOKA, T.,
881 KITAMURA, K., AIZAWA, S. & MOROHASHI, K. 2010. Abnormal epithelial cell polarity
882 and ectopic epidermal growth factor receptor (EGFR) expression induced in Emx2 KO
883 embryonic gonads. *Endocrinology*, **151**, 5893-904.

884 LARSSON, S. H., CHARLIEU, J. P., MIYAGAWA, K., ENGELKAMP, D., RASSOULZADEGAN, M.,
885 ROSS, A., CUZIN, F., VAN HEYNINGEN, V. & HASTIE, N. D. 1995. Subnuclear
886 localization of WT1 in splicing or transcription factor domains is regulated by
887 alternative splicing. *Cell*, **81**, 391-401.

888 LAVERY, R., CHASSOT, A. A., PAUPER, E., GREGOIRE, E. P., KLOPFENSTEIN, M., DE ROOIJ, D.
889 G., MARK, M., SCHEDL, A., GHYSELINCK, N. B. & CHABOISSIER, M. C. 2012. Testicular
890 differentiation occurs in absence of R-spondin1 and Sox9 in mouse sex reversals. *PLoS
891 Genet*, **8**, e1003170.

892 LAWSON, K. A., DUNN, N. R., ROELEN, B. A., ZEINSTRA, L. M., DAVIS, A. M., WRIGHT, C. V.,
893 KORVING, J. P. & HOGAN, B. L. 1999. Bmp4 is required for the generation of primordial
germ cells in the mouse embryo. *Genes Dev*, **13**, 424-36.

895 LE ROLLE, M., MASSA, F., SIGGERS, P., TURCHI, L., LOUBAT, A., KOO, B. K., CLEVERS, H.,
896 GREENFIELD, A., SCHEDL, A., CHABOISSIER, M. C. & CHASSOT, A. A. 2021. Arrest of
897 WNT/beta-catenin signaling enables the transition from pluripotent to differentiated
germ cells in mouse ovaries. *Proc Natl Acad Sci USA*, **118**.

899 LI, Y., ZHANG, L., HU, Y., CHEN, M., HAN, F., QIN, Y., CHEN, M., CUI, X., DUO, S., TANG, F. &
900 GAO, F. 2017. beta-Catenin directs the transformation of testis Sertoli cells to ovarian
901 granulosa-like cells by inducing Foxl2 expression. *J Biol Chem*, **292**, 17577-17586.

902 LI, Y., ZHENG, M. & LAU, Y. F. 2014. The sex-determining factors SRY and SOX9 regulate similar
target genes and promote testis cord formation during testicular differentiation. *Cell
903 Rep*, **8**, 723-33.

905 LIN, Y. T., BARSKE, L., DEFALCO, T. & CAPEL, B. 2017. Numb regulates somatic cell lineage
commitment during early gonadogenesis in mice. *Development*, **144**, 1607-1618.

907 LIU, C., PENG, J., MATZUK, M. M. & YAO, H. H. 2015. Lineage specification of ovarian theca
cells requires multicellular interactions via oocyte and granulosa cells. *Nat Commun*,
909 6, 6934.

910 LIU, C., RODRIGUEZ, K. & YAO, H. H. 2016. Mapping lineage progression of somatic progenitor
cells in the mouse fetal testis. *Development*, **143**, 3700-3710.

912 LIU, C. F., BINGHAM, N., PARKER, K. & YAO, H. H. 2009. Sex-specific roles of beta-catenin in
mouse gonadal development. *Hum Mol Genet*, **18**, 405-17.

914 LIU, X. L., ZHANG, Z. F., SHAO, M. Y., LIU, J. G. & MUHAMMAD, F. 2012. Sexually dimorphic
expression of foxl2 during gametogenesis in scallop Chlamys farreri, conserved with
vertebrates. *Dev Genes Evol*, **222**, 279-86.

917 LUTHRINGER, R., RAPHALEN, M., GUERRA, C., COLIN, S., MARTINHO, C., ZHENG, M.,
918 HOSHINO, M., BADIS, Y., LIPINSKA, A. P., HAAS, F. B., BARRERA-REDONDO, J., ALVA, V.
919 & COELHO, S. M. 2024. Repeated co-option of HMG-box genes for sex determination
920 in brown algae and animals. *Science*, **383**, eadk5466.
921 MAATOUK, D. M., DINAPOLI, L., ALVERS, A., PARKER, K. L., TAKETO, M. M. & CAPEL, B. 2008.

922 Stabilization of beta-catenin in XY gonads causes male-to-female sex-reversal. *Hum Mol Genet*, 17, 2949-55.

923

924 MAATOUK, D. M., MORK, L., CHASSOT, A. A., CHABOISSIER, M. C. & CAPEL, B. 2013.

925 Disruption of mitotic arrest precedes precocious differentiation and

926 transdifferentiation of pregranulosa cells in the perinatal Wnt4 mutant ovary. *Dev Biol*,

927 383, 295-306.

928 MAGOFFIN, D. A. 2005. Ovarian theca cell. *Int J Biochem Cell Biol*, 37, 1344-9.

929 MANDEL, H., SHEMER, R., BOROCHOWITZ, Z. U., OKOPNIK, M., KNOPF, C., INDELMAN, M.,

930 DRUGAN, A., TIOSANO, D., GERSHONI-BARUCH, R., CHODER, M. & SPRECHER, E.

931 2008. SERKAL syndrome: an autosomal-recessive disorder caused by a loss-of-

932 function mutation in WNT4. *Am J Hum Genet*, 82, 39-47.

933 MANNAN, M. A. & O'SHAUGHNESSY, P. J. 1991. Steroidogenesis during postnatal

934 development in the mouse ovary. *J Endocrinol*, 130, 101-6.

935 MATZUK, M. M., LU, N., VOGEL, H., SELLHEYER, K., ROOP, D. R. & BRADLEY, A. 1995. Multiple

936 defects and perinatal death in mice deficient in follistatin. *Nature*, 374, 360-3.

937 MAYERE, C., REGARD, V., PEREA-GOMEZ, A., BUNCE, C., NEIRIJNCK, Y., DJARI, C., BELLIDO-

938 CARRERAS, N., SARAROLS, P., REEVES, R., GREENAWAY, S., SIMON, M., SIGGERS, P.,

939 CONDREA, D., KUHNE, F., GANTAR, I., TANG, F., STEVANT, I., BATTI, L., GHYSELINCK,

940 N. B., WILHELM, D., GREENFIELD, A., CAPEL, B., CHABOISSIER, M. C. & NEF, S. 2022.

941 Origin, specification and differentiation of a rare supporting-like lineage in the

942 developing mouse gonad. *Sci Adv*, 8, eabm0972.

943 MCELREAVEY, K., VILAIN, E., HERSKOWITZ, I. & FELLOUS, M. 1993. A regulatory cascade

944 hypothesis for mammalian sex determination: SRY represses a negative regulator of

945 male development. *Proceedings of the National Academy of Sciences USA*, 90, 3368-

946 3372.

947 MCLAREN, A. 1983. Primordial germ cells in mice. *Bibl Anat*, 24, 59-66.

948 MERCHANT-LARIOS, H. & MORENO-MENDOZA, N. 1998. Mesonephric stromal cells

949 differentiate into Leydig cells in the mouse fetal testis. *Exp Cell Res*, 244, 230-8.

950 MIGALE, R., NEUMANN, M., MITTER, R., RAFIEE, M. R., WOOD, S., OLSEN, J. & LOVELL-

951 BADGE, R. 2024. FOXL2 interaction with different binding partners regulates the

952 dynamics of ovarian development. *Sci Adv*, 10, eadl0788.

953 MING, Z., VINING, B., BAGHERI-FAM, S. & HARLEY, V. 2022. SOX9 in organogenesis: shared

954 and unique transcriptional functions. *Cell Mol Life Sci*, 79, 522.

955 MIYABAYASHI, K., KATOH-FUKUI, Y., OGAWA, H., BABA, T., SHIMA, Y., SUGIYAMA, N.,

956 KITAMURA, K. & MOROHASHI, K. 2013. Aristaless related homeobox gene, Arx, is

957 implicated in mouse fetal Leydig cell differentiation possibly through expressing in the

958 progenitor cells. *PLoS One*, 8, e68050.

959 MIYAMOTO, N., YOSHIDA, M., KURATANI, S., MATSUO, I. & AIZAWA, S. 1997. Defects of

960 urogenital development in mice lacking *Emx2*. *Development*, 124, 1653-1664.

961 MIYAWAKI, S., KUROKI, S., MAEDA, R., OKASHITA, N., KOOPMAN, P. & TACHIBANA, M. 2020.

962 The mouse Sry locus harbors a cryptic exon that is essential for male sex

963 determination. *Science*, 370, 121-124.

964 MOLYNEAUX, K. A., STALLOCK, J., SCHAIBLE, K. & WYLIE, C. 2001. Time-lapse analysis of

965 living mouse germ cell migration. *Dev Biol*, 240, 488-98.

966 MONIOT, B., DECLOSMENIL, F., BARRIONUEVO, F., SCHERER, G., ARITAKE, K., MALKI, S.,

967 MARZI, L., COHEN-SOLAL, A., GEORG, I., KLATTIG, J., ENGLERT, C., KIM, Y., CAPEL, B.,

968 EGUCHI, N., URADE, Y., BOIZET-BONHoure, B. & POULAT, F. 2009. The PGD2
969 pathway, independently of FGF9, amplifies SOX9 activity in Sertoli cells during male
970 sexual differentiation. *Development*, 136, 1813-21.

971 MORK, L., MAATOUK, D. M., MCMAHON, J. A., GUO, J. J., ZHANG, P., MCMAHON, A. P. &
972 CAPEL, B. 2011. Temporal Differences in Granulosa Cell Specification in the Ovary
973 Reflect Distinct Follicle Fates in Mice. *Biol Reprod*.

974 MUNGER, S. C., NATARAJAN, A., LOOGER, L. L., OHLER, U. & CAPEL, B. 2013. Fine time
975 course expression analysis identifies cascades of activation and repression and maps
976 a putative regulator of mammalian sex determination. *PLoS Genet*, 9, e1003630.

977 NAGAHAMA, Y., CHAKRABORTY, T., PAUL-PRASANTH, B., OHTA, K. & NAKAMURA, M. 2021.
978 Sex determination, gonadal sex differentiation, and plasticity in vertebrate species.
979 *Physiol Rev*, 101, 1237-1308.

980 NAKAMOTO, M., MATSUDA, M., WANG, D. S., NAGAHAMA, Y. & SHIBATA, N. 2006. Molecular
981 cloning and analysis of gonadal expression of Foxl2 in the medaka, Oryzias latipes.
982 *Biochem Biophys Res Commun*, 344, 353-61.

983 NALLATHAMBI, J., MOUMNE, L., DE BAERE, E., BEYSEN, D., USHA, K., SUNDARESAN, P. &
984 VEITIA, R. A. 2007. A novel polyalanine expansion in FOXL2: the first evidence for a
985 recessive form of the blepharophimosis syndrome (BPES) associated with ovarian
986 dysfunction. *Hum Genet*, 121, 107-12.

987 NEF, S. & PARADA, L. F. 1999. Cryptorchidism in mice mutant for Insl3. *Nat Genet*, 22, 295-9.

988 NEF, S., STEVANT, I. & GREENFIELD, A. 2019. Characterizing the bipotential mammalian
989 gonad. *Curr Top Dev Biol*, 134, 167-194.

990 NEIRIJNCK, Y., SARAROLS, P., KUHNE, F., MAYERE, C., WEERASINGHE ARACHCHIGE, L. C.,
991 REGARD, V., NEF, S. & SCHEDL, A. 2023. Single-cell transcriptomic profiling redefines
992 the origin and specification of early adrenogonadal progenitors. *Cell Rep*, 42, 112191.

993 NEL-THEMAAT, L., VADAKKAN, T. J., WANG, Y., DICKINSON, M. E., AKIYAMA, H. & BEHRINGER,
994 R. R. 2009. Morphometric analysis of testis cord formation in Sox9-EGFP mice. *Dev
995 Dyn*, 238, 1100-10.

996 NICOL, B., GRIMM, S. A., CHALMEL, F., LECLUZE, E., PANNETIER, M., PAILHOUX, E., DUPIN-
997 DE-BEYSSAT, E., GUIGUEN, Y., CAPEL, B. & YAO, H. H. 2019. RUNX1 maintains the
998 identity of the fetal ovary through an interplay with FOXL2. *Nat Commun*, 10, 5116.

999 NICOL, B., GRIMM, S. A., GRUZDEV, A., SCOTT, G. J., RAY, M. K. & YAO, H. H. 2018. Genome-
1000 wide identification of FOXL2 binding and characterization of FOXL2 feminizing action in
1001 the fetal gonads. *Hum Mol Genet*, 27, 4273-4287.

1002 NICOL, B. & YAO, H. H. 2015. Gonadal Identity in the Absence of Pro-Testis Factor SOX9 and
1003 Pro-Ovary Factor Beta-Catenin in Mice. *Biol Reprod*, 93, 35.

1004 NIU, W. & SPRADLING, A. C. 2020. Two distinct pathways of pregranulosa cell differentiation
1005 support follicle formation in the mouse ovary. *Proc Natl Acad Sci U S A*, 117, 20015-
1006 20026.

1007 OKADA, E., YOSHIMOTO, S., IKEDA, N., KANDA, H., TAMURA, K., SHIBA, T., TAKAMATSU, N. &
1008 ITO, M. 2009. Xenopus W-linked DM-W induces Foxl2 and Cyp19 expression during
1009 ovary formation. *Sex Dev*, 3, 38-42.

1010 OSHIMA, Y., UNO, Y., MATSUDA, Y., KOBAYASHI, T. & NAKAMURA, M. 2008. Molecular cloning
1011 and gene expression of Foxl2 in the frog Rana rugosa. *Gen Comp Endocrinol*, 159, 170-
1012 7.

1013 OTTOLENGHI, C., OMARI, S., GARCIA-ORTIZ, J. E., UDA, M., CRISPONI, L., FORABOSCO, A.,
1014 PILIA, G. & SCHLESSINGER, D. 2005. Foxl2 is required for commitment to ovary
differentiation. *Hum Mol Genet*, 14, 2053-62.
1016 OTTOLENGHI, C., PELOSI, E., TRAN, J., COLOMBINO, M., DOUGLASS, E., NEDOREZOV, T.,
1017 CAO, A., FORABOSCO, A. & SCHLESSINGER, D. 2007. Loss of Wnt4 and Foxl2 leads to
1018 female-to-male sex reversal extending to germ cells. *Hum Mol Genet*, **16**, 2795-804.

1019 PAILHOUX, E., VIGIER, B., CHAFFAUX, S., SERVEL, N., TAOURIT, S., FURET, J. P., FELLOUS, M.,
1020 GROSCLAUDE, F., CRIBIU, E. P., COTINOT, C. & VAIMAN, D. 2001. A 11.7-kb deletion
1021 triggers intersexuality and polledness in goats. *Nat Genet*, **29**, 453-8.

1022 PANNETIER, M., ELZAIAT, M., THEPOT, D. & PAILHOUX, E. 2012. Telling the story of XX sex
1023 reversal in the goat: highlighting the sex-crossroad in domestic mammals. *Sex Dev*, **6**,
1024 33-45.

1025 PARMA, P., RADI, O., VIDAL, V., CHABOISSIER, M. C., DELLAMBRA, E., VALENTINI, S.,
1026 GUERRA, L., SCHEDL, A. & CAMERINO, G. 2006. R-spondin1 is essential in sex
determination, skin differentiation and malignancy. *Nat Genet*, **38**, 1304-9.

1028 PEREA-GOMEZ, A. B.-C., N.; DHELLEMMES, M.; TANG, F., LE GALLO, C.; CHABOISSIER, M.C.
1029 2025. NR2F2 is required in the embryonic testis for Fetal Leydig Cell development.
1030 *eLife*, Reviewed Preprint.

1031 PIERUCCI-ALVES, F., CLARK, A. M. & RUSSELL, L. D. 2001. A developmental study of the
1032 Desert hedgehog-null mouse testis. *Biol Reprod*, **65**, 1392-402.

1033 PITETTI, J. L., CALVEL, P., ROMERO, Y., CONNE, B., TRUONG, V., PAPAIOANNOU, M. D.,
1034 SCHAAD, O., DOCQUIER, M., HERRERA, P. L., WILHELM, D. & NEF, S. 2013. Insulin and
1035 IGF1 receptors are essential for XX and XY gonadal differentiation and adrenal
1036 development in mice. *PLoS Genet*, **9**, e1003160.

1037 PONTIGGIA, A., RIMINI, R., HARLEY, V. R., GOODFELLOW, P. N., LOVELL-BADGE, R. &
1038 BIANCHI, M. E. 1994. Sex-reversing mutations affect the architecture of SRY-DNA
1039 complexes. *EMBO Journal*, **13**, 6115-6124.

1040 PONTIGGIA, A., WHITFIELD, S., GOODFELLOW, P. N., LOVELL-BADGE, R. & BIANCHI, M. E.
1041 1995. Evolutionary conservation in the DNA-binding and -bending properties of HMG-
1042 boxes from SRY proteins of primates. *Gene*, **154**, 277-80.

1043 QIU, C., MARTIN, B. K., WELSH, I. C., DAZA, R. M., LE, T. M., HUANG, X., NICHOLS, E. K.,
1044 TAYLOR, M. L., FULTON, O., O'DAY, D. R., GOMES, A. R., ILCISIN, S., SRIVATSAN, S.,
1045 DENG, X., DISTECHE, C. M., NOBLE, W. S., HAMAZAKI, N., MOENS, C. B., KIMELMAN,
1046 D., CAO, J., SCHIER, A. F., SPIELMANN, M., MURRAY, S. A., TRAPNELL, C. &
1047 SHENDURE, J. 2024. A single-cell time-lapse of mouse prenatal development from
1048 gastrula to birth. *Nature*, **626**, 1084-1093.

1049 RAHMOUN, M., LAVERY, R., LAURENT-CHABALLIER, S., BELLORA, N., PHILIP, G. K.,
1050 ROSSITTO, M., SYMON, A., PAILHOUX, E., CAMMAS, F., CHUNG, J., BAGHERI-FAM, S.,
1051 MURPHY, M., BARDWELL, V., ZARKOWER, D., BOIZET-BONHOURE, B., CLAIR, P.,
1052 HARLEY, V. R. & POULAT, F. 2017. In mammalian foetal testes, SOX9 regulates
1053 expression of its target genes by binding to genomic regions with conserved signatures.
1054 *Nucleic Acids Res*, **45**, 7191-7211.

1055 RASTETTER, R. H., BERNARD, P., PALMER, J. S., CHASSOT, A. A., CHEN, H., WESTERN, P. S.,
1056 RAMSAY, R. G., CHABOISSIER, M. C. & WILHELM, D. 2014. Marker genes identify three
1057 somatic cell types in the fetal mouse ovary. *Dev Biol*, **394**, 242-52.

1058 RHEN, T., FAGERLIE, R., SCHROEDER, A., CROSSLEY, D. A., 2ND & LANG, J. W. 2015.
1059 Molecular and morphological differentiation of testes and ovaries in relation to the
1060 thermosensitive period of gonad development in the snapping turtle, Chelydra
1061 serpentina. *Differentiation*, **89**, 31-41.

1062 RICHARDSON, B. E. & LEHMANN, R. 2010. Mechanisms guiding primordial germ cell
1063 migration: strategies from different organisms. *Nat Rev Mol Cell Biol*, **11**, 37-49.
1064 RIDNIK, M., ABBERBOCK, E., ALIPOV, V., LHERMANN, S. Z., KAUFMAN, S., LUBMAN, M.,
1065 POULAT, F. & GONEN, N. 2024. Two redundant transcription factor binding sites in a
1066 single enhancer are essential for mammalian sex determination. *Nucleic Acids Res,
1067 52, 5514-5528.

1068 RODRIGUEZ GUTIERREZ, D., EID, W. & BIASON-LAUBER, A. 2018. A Human Gonadal Cell
1069 Model From Induced Pluripotent Stem Cells. *Front Genet*, 9, 498.

1070 ROELEN, B. A. J. & CHUVA DE SOUSA LOPES, S. M. 2022. Stay on the road: from germ cell
1071 specification to gonadal colonization in mammals. *Philos Trans R Soc Lond B Biol Sci,
1072 377, 20210259.

1073 SAITO, D., TAMURA, K. & TAKAHASHI, Y. 2017. Early segregation of the adrenal cortex and
gonad in chicken embryos. *Dev Growth Differ*, 59, 593-602.

1075 SAITOU, M. & HAYASHI, K. 2021. Mammalian in vitro gametogenesis. *Science*, 374, eaaz6830.

1076 SAITOU, M. & YAMAJI, M. 2012. Primordial germ cells in mice. *Cold Spring Harb Perspect Biol*,
1077 4.

1078 SASAKI, K., YOKOBAYASHI, S., NAKAMURA, T., OKAMOTO, I., YABUTA, Y., KURIMOTO, K.,
1079 OHTA, H., MORITOKI, Y., IWATANI, C., TSUCHIYA, H., NAKAMURA, S., SEKIGUCHI, K.,
1080 SAKUMA, T., YAMAMOTO, T., MORI, T., WOLTJEN, K., NAKAGAWA, M., YAMAMOTO, T.,
1081 TAKAHASHI, K., YAMANAKA, S. & SAITOU, M. 2015. Robust In Vitro Induction of Human
1082 Germ Cell Fate from Pluripotent Stem Cells. *Cell Stem Cell*, 17, 178-94.

1083 SCHARTL, M., GEORGES, A. & MARSHALL GRAVES, J. A. 2023. Polygenic sex determination in
vertebrates - is there any such thing? *Trends Genet*, 39, 242-250.

1085 SCHMAHL, J., EICHER, E., WASHBURN, L. & CAPEL, B. 2000. *Sry* induces cell proliferation in
the mouse gonad. *Development*, 127, 65-73.

1087 SCHMAHL, J., RIZZOLO, K. & SORIANO, P. 2008. The PDGF signaling pathway controls
multiple steroid-producing lineages. *Genes Dev*, 22, 3255-67.

1089 SEKIDO, R., BAR, I., NARVAEZ, V., PENNY, G. & LOVELL-BADGE, R. 2004. SOX9 is up-regulated
1090 by the transient expression of SRY specifically in Sertoli cell precursors. *Dev Biol*, 274,
1091 271-9.

1092 SEKIDO, R. & LOVELL-BADGE, R. 2007. Mechanisms of gonadal morphogenesis are not
conserved between chick and mouse. *Dev Biol*, 302, 132-42.

1094 SEKIDO, R. & LOVELL-BADGE, R. 2008. Sex determination involves synergistic action of SRY
1095 and SF1 on a specific Sox9 enhancer. *Nature*, 453, 930-4.

1096 SHEN, Y. C., SHAMI, A. N., MORITZ, L., LAROSE, H., MANSKE, G. L., MA, Q., ZHENG, X.,
1097 SUKHWANI, M., CZERWINSKI, M., SULTAN, C., CHEN, H., GURCZYNSKI, S. J., SPENCE,
1098 J. R., ORWIG, K. E., TALLQUIST, M., LI, J. Z. & HAMMOUD, S. S. 2021. TCF21(+)
1099 mesenchymal cells contribute to testis somatic cell development, homeostasis, and
1100 regeneration in mice. *Nat Commun*, 12, 3876.

1101 SHIMA, Y. 2019. Development of fetal and adult Leydig cells. *Reprod Med Biol*, 18, 323-330.

1102 SHIMA, Y. & MOROHASHI, K. I. 2017. Leydig progenitor cells in fetal testis. *Mol Cell
Endocrinol*, 445, 55-64.

1104 SHOEMAKER-DALY, C. M., JACKSON, K., YATSU, R., MATSUMOTO, Y. & CREWS, D. 2010.
1105 Genetic network underlying temperature-dependent sex determination is
1106 endogenously regulated by temperature in isolated cultured Trachemys scripta
1107 gonads. *Dev Dyn*, 239, 1061-75.

1108 SINCLAIR, A. H., BERTA, P., PALMER, M. S., HAWKINS, J. R., GRIFFITHS, B. L., SMITH, M. J.,
1109 FOSTER, J. W., FRISCHAUF, A.-M., LOVELL-BADGE, R. & GOODFELLOW, P. N. 1990. A
gene from the human sex-determining region encodes a protein with homology to a
1110 conserved DNA-binding motif. *Nature*, 346, 240-244.
1112 SOUALI-CRESPO, S., CONDREA, D., VERNET, N., FERET, B., KLOPFENSTEIN, M.,
1113 GRANDGIRARD, E., ALUNNI, V., CERCIAT, M., JUNG, M., MAYERE, C., NEF, S., MARK,
1114 M., CHALMEL, F. & GHYSELINCK, N. B. 2023. Loss of NR5A1 in mouse Sertoli cells
1115 after sex determination changes cellular identity and induces cell death by anoikis.
1116 *Development*, 150.

1117 STEVANT, I., KUHNE, F., GREENFIELD, A., CHABOISSIER, M. C., DERMITZAKIS, E. T. & NEF, S.
1118 2019. Dissecting Cell Lineage Specification and Sex Fate Determination in Gonadal
1119 Somatic Cells Using Single-Cell Transcriptomics. *Cell Rep*, 26, 3272-3283 e3.

1120 STEVANT, I., NEIRIJNCK, Y., BOREL, C., ESCOFFIER, J., SMITH, L. B., ANTONARAKIS, S. E.,
1121 DERMITZAKIS, E. T. & NEF, S. 2018a. Deciphering Cell Lineage Specification during
1122 Male Sex Determination with Single-Cell RNA Sequencing. *Cell Rep*, 22, 1589-1599.

1123 STEVANT, I., PAPAIOANNOU, M. D. & NEF, S. 2018b. A brief history of sex determination. *Mol
1124 Cell Endocrinol*, 468, 3-10.

1125 SVINGEN, T. & KOOPMAN, P. 2013. Building the mammalian testis: origins, differentiation, and
1126 assembly of the component cell populations. *Genes Dev*, 27, 2409-26.

1127 SWIFT, C. 1913. Origin and early history of the primordial germ-cells in the chick. *American
1128 Journal of Anatomy*, 15, 483-514.

1129 TAM, P. P. & SNOW, M. H. 1981. Proliferation and migration of primordial germ cells during
1130 compensatory growth in mouse embryos. *J Embryol Exp Morphol*, 64, 133-47.

1131 TANG, H., BRENNAN, J., KARL, J., HAMADA, Y., RAETZMAN, L. & CAPEL, B. 2008. Notch
1132 signaling maintains Leydig progenitor cells in the mouse testis. *Development*, 135,
1133 3745-53.

1134 TOMIZUKA, K., HORIKOSHI, K., KITADA, R., SUGAWARA, Y., IBA, Y., KOJIMA, A., YOSHITOME,
1135 A., YAMAWAKI, K., AMAGAI, M., INOUE, A., OSHIMA, T. & KAKITANI, M. 2008. R-
1136 spondin1 plays an essential role in ovarian development through positively regulating
1137 Wnt-4 signaling. *Hum Mol Genet*, 17, 1278-91.

1138 UHLENHAUT, N. H., JAKOB, S., ANLAG, K., EISENBERGER, T., SEKIDO, R., KRESS, J., TREIER, A.
1139 C., KLUGMANN, C., KLASEN, C., HOLTER, N. I., RIETHMACHER, D., SCHUTZ, G.,
1140 COONEY, A. J., LOVELL-BADGE, R. & TREIER, M. 2009. Somatic sex reprogramming of
1141 adult ovaries to testes by FOXL2 ablation. *Cell*, 139, 1130-42.

1142 VAINIO, S., HEIKKILA, M., KISPERT, A., CHIN, N. & MCMAHON, A. P. 1999. Female
1143 development in mammals is regulated by Wnt-4 signalling. *Nature*, 397, 405-409.

1144 WANG, Y., CHANG, H., RATTNER, A. & NATHANS, J. 2016. Frizzled Receptors in Development
1145 and Disease. *Curr Top Dev Biol*, 117, 113-39.

1146 WEBER, C. & CAPEL, B. 2021. Sex determination without sex chromosomes. *Philos Trans R
1147 Soc Lond B Biol Sci*, 376, 20200109.

1148 WILHELM, D. & ENGLERT, C. 2002. The Wilms tumor suppressor WT1 regulates early gonad
1149 development by activation of Sf1. *Genes Dev*, 16, 1839-51.

1150 WILHELM, D., HIRAMATSU, R., MIZUSAKI, H., WIDJAJA, L., COMBES, A. N., KANAI, Y. &
1151 KOOPMAN, P. 2007. SOX9 regulates prostaglandin D synthase gene transcription in
1152 vivo to ensure testis development. *J Biol Chem*, 282, 10553-60.

1153 WILHELM, D., MARTINSON, F., BRADFORD, S., WILSON, M. J., COMBES, A. N., BEVERDAM, A.,
1154 BOWLES, J., MIZUSAKI, H. & KOOPMAN, P. 2005. Sertoli cell differentiation is induced
1155 both cell-autonomously and through prostaglandin signaling during mammalian sex
determination. *Dev Biol*, 287, 111-24.

1157 WILHELM, D., WASHBURN, L. L., TRUONG, V., FELLOUS, M., EICHER, E. M. & KOOPMAN, P.
1158 2009. Antagonism of the testis- and ovary-determining pathways during ovotestis
d Development in mice. *Mech Dev*, 126, 324-36.
1160 WINDLEY, S. P., MAYERE, C., MCGOVERN, A. E., HARVEY, N. L., NEF, S., SCHWARZ, Q.,
1161 KUMAR, S. & WILHELM, D. 2022. Loss of NEDD4 causes complete XY gonadal sex
1162 reversal in mice. *Cell Death Dis*, 13, 75.

1163 YAMAGUCHI, T., YAMAGUCHI, S., HIRAI, T. & KITANO, T. 2007. Follicle-stimulating hormone
1164 signaling and Foxl2 are involved in transcriptional regulation of aromatase gene during
1165 gonadal sex differentiation in Japanese flounder, *Paralichthys olivaceus*. *Biochem
1166 Biophys Res Commun*, 359, 935-40.

1167 YAN, Y. L., TITUS, T., DESVIGNES, T., BREMILLER, R., BATZEL, P., SYDES, J., FARNSWORTH, D.,
1168 DILLON, D., WEGNER, J., PHILLIPS, J. B., PEIRCE, J., DOWD, J., UNDIAGNOSED
1169 DISEASES, N., BUCK, C. L., MILLER, A., WESTERFIELD, M. & POSTLETHWAIT, J. H.
1170 2021. A fish with no sex: gonadal and adrenal functions partition between zebrafish
1171 NR5A1 co-orthologs. *Genetics*, 217.

1172 YAO, H. H., WHORISKEY, W. & CAPEL, B. 2002. Desert Hedgehog/Patched 1 signaling
1173 specifies fetal Leydig cell fate in testis organogenesis. *Genes Dev*, 16, 1433-40.

1174 YOSHIMOTO, S., IKEDA, N., IZUTSU, Y., SHIBA, T., TAKAMATSU, N. & ITO, M. 2010. Opposite
1175 roles of DMRT1 and its W-linked paralogue, DM-W, in sexual dimorphism of Xenopus
1176 laevis: implications of a ZZ/ZW-type sex-determining system. *Development*, 137, 2519-
1177 26.

1178 YOSHIMOTO, S., OKADA, E., UMEMOTO, H., TAMURA, K., UNO, Y., NISHIDA-UMEHARA, C.,
1179 MATSUDA, Y., TAKAMATSU, N., SHIBA, T. & ITO, M. 2008. A W-linked DM-domain gene,
1180 DM-W, participates in primary ovary development in Xenopus laevis. *Proc Natl Acad
1181 Sci U S A*, 105, 2469-74.

1182 YOSHINO, T., SUZUKI, T., NAGAMATSU, G., YABUKAMI, H., IKEGAYA, M., KISHIMA, M., KITA, H.,
1183 IMAMURA, T., NAKASHIMA, K., NISHINAKAMURA, R., TACHIBANA, M., INOUE, M.,
1184 SHIMA, Y., MOROHASHI, K. I. & HAYASHI, K. 2021. Generation of ovarian follicles from
1185 mouse pluripotent stem cells. *Science*, 373.

1186 YOSHIOKA, H., ISHIMARU, Y., SUGIYAMA, N., TSUNEKAWA, N., NOCE, T., KASAHARA, M. &
1187 MOROHASHI, K. 2005. Mesonephric FGF signaling is associated with the development
1188 of sexually indifferent gonadal primordium in chick embryos. *Dev Biol*, 280, 150-61.

1189 ZHANG, Q., PAN, Y., JI, J., XU, Y., ZHANG, Q. & QIN, L. 2021. Roles and action mechanisms of
1190 WNT4 in cell differentiation and human diseases: a review. *Cell Death Discov*, 7, 287.

1191 ZHAO, L. & KOOPMAN, P. 2012. SRY protein function in sex determination: thinking outside
1192 the box. *Chromosome Res*, 20, 153-62.

1193 ZHENG, W., ZHANG, H., GORRE, N., RISAL, S., SHEN, Y. & LIU, K. 2014. Two classes of ovarian
1194 primordial follicles exhibit distinct developmental dynamics and physiological
1195 functions. *Hum Mol Genet*, 23, 920-8.

1196

1197
1198 **Figure legends**

1199 **Fig. 1. Models for gonadal sex determination in mammals. (A)** Gonadal sex determination model in mammals as proposed by Eicher and Washburn (Eicher and Washburn, 1986). *Tdy* (testis-determining on the Y) from the Y chromosome in testes and *Od* (ovary-determining gene) from the X chromosome or an autosome in ovaries activate downstream genes, testis-determining (*Td*)-1 and ovary-determining (*Od*)-1, to drive testicular and ovarian differentiation respectively. **(B)** Gonadal sex determination model in mammals as proposed by McElreavey and colleagues (McElreavey et al., 1993). A recessive autosomal gene called *Z*, inhibits the expression of testicular genes in XX females, while in XY males *SRY* inhibits the expression or the function of *Z*.

1207

1208 **Fig. 2. Formation of the adrenal and gonadal primordia in vertebrates. (A)** The adrenogonadal primordium (AGP, indicated in colour with the future AP in pink and the future GP in yellow) arises from lateral plate mesoderm at 9.5 dpc in mouse embryos, at HH13 (Hamburger Hamilton stage) in chicken embryos and at Carnegie stage (CS)14 in human embryos. **(B)** The spatial and temporal appearance of the adrenal primordium (AP) and gonadal primordium vary between species with the AP and GP are spatially, temporally and phenotypically distinct with the AP developing earlier and clearly separated from the GP in human (left). In mouse (centre), the AGP splits into the more cranial adrenal primordium (AP), and the more caudal gonadal primordium (GP) at 10.5 dpc, and in chicken (right), the AP and GP primordia are derived from two adjacent layers of coelomic epithelium and/or the mesonephric mesenchyme. MT, mesonephric tubules; MD, mesonephric ducts.

1218

1219 **Fig. 3. Somatic cell differentiation during gonadal development in the mouse embryo.** In XY embryos, the bipotential gonad develops as a testis with the differentiation of Sertoli cells (around 11.5 dpc), the formation of testis cords (starting at 12.5 dpc) and the differentiation of fetal Leydig cells from steroidogenic progenitors present in the interstitial space between the testis cords. In XX embryos, the bipotential gonad develops as an ovary with the differentiation of pre-granulosa cells from 12.5 dpc. The first wave of pre-granulosa cells generated in the embryonic ovary contribute to the medullary follicles activated shortly after
1225 birth, while the second wave of pre-granulosa cells will contribute to cortical follicles which encompass the
1226 ovarian reserve. Lineage tracing and single cell RNA sequencing experiments have revealed the lineage
1227 relationships of gonadal somatic cells in XY and XX mouse gonads. The colors and shapes of the cells in the
1228 top panel correspond to the ones shown in the bottom panel.

1229

1230 **Fig. 4. Updated model of the genetic control of sex determination in mouse.** *Sry* initiates testicular
1231 development in XY gonads through the activation of *Sox9* expression that, in turn, controls Sertoli cell
1232 differentiation and the activation of downstream target genes in Sertoli cells. In XX gonads, *Wt1-KTS*
1233 initiates ovarian development and pre-granulosa cell differentiation, which is then maintained by the
1234 combined action of *Rspo1/Wnt4/Ctnnb1* pathway, *Foxl2* and *Runx1* controlling the expression of
1235 downstream target genes in pre-granulosa cells.

1236

1237

1238 **Box 1. Reproductive organoids.**

1239 More recent advances in generating mouse and human reproductive organoids provide a novel tool, which
1240 is complementary to *in vivo* analysis of genetically modified mouse lines, to better understand gonadal sex
1241 determination and differentiation. To faithfully reconstitute testes and ovaries, both germ cells and somatic
1242 cells need to be derived from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). Both
1243 sources have been used to successfully differentiate mouse and human primordial germ cell-like cells
1244 (PGCLSs) that have similar genetic and epigenetic properties as PGCs in vivo (Hayashi et al., 2011, Sasaki et
1245 al., 2015). The derivation of gonadal cells proved to be more difficult (Gonen et al., 2023, Knarston et al.,
1246 2020, Rodriguez Gutierrez et al., 2018). Recently, fetal ovarian somatic cell-like cells, which were able to
1247 support all steps of oogenesis, were generated from ESCs (Hayashi and Yoshino, 2022, Yoshino et al., 2021).
1248 Despite these advances some characteristics of *in vivo* follicles were missing such as their ability to arrest at
1249 the primordial follicle stage. Going forward, gaining new understanding of gonadal somatic cell
1250 differentiation *in vivo* will inform approaches to optimise these processes *in vitro*, and *in vitro* generation of
1251 reproductive organoids offers a feasible way to further investigate possible roles of factors and signalling

1252 pathways through genetic manipulation of pluripotent stem cells.
<figure><img src="image_2.png" /><figcaption>Figure 1</figcaption></figure>

<figure><img src="image_3.png" /></figure>
<figure><img src="image_4.png" /><figcaption>Figure 2</figcaption></figure>

<figure><img src="image_5.png" /></figure>
<figure><img src="image_6.png" /><figcaption>Figure 3</figcaption></figure>
<figure><img src="image_7.png" /><figcaption>Figure 4</figcaption></figure>