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Gonadal sex determination and reversal in the mouse
The Sry gene on the Y-chromosome is transiently expressed in the XY gonadal primordium to upregulate the Sox9 gene, which in turn upregulates a set of genes such as Fgf9, Ptgds, and Amh to initiate and stabilize testicular differentiation. In the absence of Sry expression in the XX gonadal primordium, genes such as Rspo1, Wnt4, and Foxl2 are expressed at high levels to support ovarian differentiation. These two pathways antagonize each other to ensure the development of only one gonadal sex in normal development. Genetic manipulations in the mouse served as powerful tools to identify and delineate the molecular mechanisms of gonadal sex determination. Nonetheless, more studies are needed to elucidate how the balance between testicular and ovarian differentiation is adjusted during gonadal differentiation in vivo. In the consomic mouse strains, named B6.YTIR and B6.YPOS, the Y-chromosome from variants of Mus musculus domesticus caught in Tirano, Italy (YTIR) or Poschiavinus Valley, Switzerland (YPOS) has been placed on the C57BL/6 (B6) genetic background by repeating backcross (Nagamine et al., 1987). Both mouse strains fail to develop normal testes and instead develop XY ovaries or ovotestes. We showed that Sry is expressed normally, but its target Sox9 is inefficiently upregulated while Rspo1 failed to be repressed in the B6.XYTIR gonad at Embryonic Day (ED) 11.5-12.5, preceding morphological sex differentiation (Taketo et al., 2005; Cao et al., 2024). Furthermore, the low Sox9 transcript levels result in fewer SOX9-positive cells and allow for the appearance of FOXL2-positive cells in a mosaic pattern in the entire B6.YTIR gonad. Regulation of the genes downstream of Sox9 or Rspo1 in the B6.YTIR gonad was as efficient as in the B6.XY gonad at these stages. However, these molecular and morphological features were consistently found in all B6.YTIR gonads and do not explain their subsequent development into either ovaries or ovotestes. We hypothesize that there is a developmental window of opportunity for amplifying the Sox9-Fgf9 feedback loop to secure testis cord formation in the B6.YTIR gonad between ED 12.5 and 13.5. Finding developmental cues that affect the frequency or area of testis cord formation in the B6.YTIR gonad would help understand the mechanism of ultimate morphological gonadal sex determination.

Surveillance mechanism of oocytes in mouse ovaries during fetal and neonatal development
The germ cells enter meiosis to become oocytes and undergo homologous chromosome synapsis and recombination in the fetal mouse ovary. During this period, 70-80% of the initial oocyte population is eliminated to limit the oocytes in the ovarian reserve for later reproduction. The cause or mechanism of this major oocyte loss is not fully understood. We have established cytogenetic methods to analyze the oocyte number and chromosomal alignment in mouse ovaries (McClellan et al., 2003). Using this method, we showed that the total number of oocytes continuously declines while they go through different stages of Meiotic Prophase I (MPI), suggesting that multiple causes provoke distinct oocyte surveillance mechanisms. We found that the oogonia in mitosis are eliminated by a Bax-dependent apoptotic pathway whereas the oocytes are eliminated by a Bax-independent, Caspase9-dependent mitochondrial apoptotic pathway (Alton & Taketo, 2007; Ene et al., 2013). However, Caspase9 is constitutively activated in all oocytes, some of which should survive, in the fetal ovary. We found that the oocytes are protected from apoptotic demise by the balancing act of endogenous X-linked inhibitor of apoptosis XIAP (Liu et al., 2019). What determines the balance between Caspase9 and XIAP towards oocyte survival or death remains to be determined. We also defined how homologous chromosomes find each other and initiate synapsis during the MPI progression (Kazemi & Taketo, 2021). Furthermore, we showed that the mixture of acrocentric and metacentric chromosomes, like in most mammalian species, gives a disadvantage in efficient homologous chromosome recombination, compared to all acrocentric chromosomes in the mouse (Kazemi & Taketo, 2022). This may explain the exceptionally low rate of aneuploidy (chromosome segregation errors in the first meiotic division) in the mouse. It is well established that in the postnatal ovary, errors in chromosome synapsis are recognized for oocyte elimination by CHK2-dependent apoptosis. XO oocytes, in which the single X chromosome remains unsynapsed, are subject to elimination during this phase. Consequently, the XO female mouse on the B6 genetic background results in early fertility loss (Vaz et al., 2020). These findings provide the foundation for studying how chromosomal structures affect the success in meiotic recombination and lead to early oocyte loss or meiotic division errors.
Influence of the sex chromosome complement, XX, XO, and XY, on the competence of oocytes for the second meiotic division and embryonic development
When the X monosomy or XY sex-reversal occurs, XO and XY females exhibit subfertility and infertility in the mouse on the B6 genetic background, suggesting that functional germ cell differentiation requires the proper sex chromosome complement (Taketo-Hosotani et al. 1989; Amleh et al., 1998; Alton & Taketo, 2008; Taketo & Naumova, 2013; Vaz et al., 2020). The oocyte populations in neonatal XO and XY females are comparable and smaller than in XX females, suggesting that the monosomy X, not the presence of the Y-chromosome, plays the dominant role in exacerbating the oocyte loss during neonatal development. Thereafter, the XY female becomes infertile while the XO female remains fertile during postnatal development. We compared XX, XO, and XY oocytes during follicular growth, during which the oocyte accumulates mRNAs and proteins that play essential roles for subsequent meiotic divisions and embryonic development (Xu et al., 2014; Yamazaki et al., 2021). We found that the transcript levels of most genes increase with oocyte growth while largely maintaining the X-chromosome-dosage-dependence. Near the end of the growth phase, however, the XY oocyte exhibits abnormal chromatin condensation and mitochondria distribution, and stops prematurely de novo transcription. Furthermore, transcript levels of some X-linked genes did not increase in XY oocyte as much as the XX or XO oocyte. Despite these abnormalities, the XY oocyte goes through the first meiotic division and reaches the second meiotic metaphase (Amleh et al., 1996, Villemure et al., 2007). However, the oocyte of XY females fails in extruding chromatids into the polar body upon the second meiotic division. Such meiotic defect can be attributed to the cytoplasm of the XY oocyte since the nucleus of XY oocyte placed into an enucleated XX oocyte can produce healthy offspring (Obata et al., 2008). We are currently studying the mechanisms leading to cytoplasmic defects and ultimate failure of the second meiotic division in the XY oocyte.
Development of mouse oocytes with gene mutations linked to hydatidiform mole pregnancy in humans (collaboration with Dr. Rima Slim)
Hydatidiform moles (HMs) are abnormal human pregnancies with impaired embryonic development and excessive trophoblastic proliferation. Using exome sequencing, Dr. Slim's team, has identified biallelic pathogenic mutations in nine genes in the patients with androgenetic recurrent HM (RHM), all chromosomes originating from the father (Nguyen e t al., 2018; Raezei et al., 2024). All but one of these genes are known to play essential roles in homologous chromosome synapsis in the oocytes during fetal development and result in early oocyte loss or, otherwise, a failure in embryonic development in the mouse, suggesting the existence of a common cause for androgenetic RHM. We aim at delineating the defects of oocytes in Mei1, Hfm1 and Majin homozygous and heterozygous mutant female mice, which may explain development of androgenetic RHM in humans.