Teruko Taketo
Associate Professor, Department of Surgey, McGill University
Associate Member, Department of Biology, McGill University
Associate Member, Department of Obstetrics and Gynecology, McGill University
   

Contact Information:
Urology Research Laboratory
Room H6-19
Royal Victoria Hospital
687 Pine Avenue, West
Montreal, Quebec
Canada H3A 1A1

Tel.: (514)-934-1934 ext. -34197
Fax: (514)-843-1457
e-mail: teruko.taketo@mcgill.ca

RESEARCH

The sex of an individual is determined by the combination of sex chromosomes, XX or XY, in mammals. Once the gonadal sex is established, however, most of sex characteristics are determined by the hormones produced by the gonad regardless of the chromosomal sex. Exception is the germ line; proper set of sex chromosomes maximizes the fertility of each sex. We are particularly interested in the role of sex chromosomes in female germ cell differentiation, in addition to the mechanism of gonadal sex determination.

1. Mechanism of sex determination and sex reversal in the mouse gonad

Sry, a single copy gene on the Y-chromosome, acts dominantly to trigger differentiation of a testis from a gonadal primordium that otherwise differentiates into an ovary in mammals. Sry encodes a protein with many features typical of a transcription factor and activates the cascade of molecular events leading to testis-specific Sertoli cell differentiation. However, the mode of SRY action in testis determination is not yet fully understood. Despite the conservative function of Sry, its DNA sequence is diverse among mammalian species or even among mouse strains. When the Y-chromosome (YTIR) from local variants of Mus musculus domesticus caught in Tirano, Italy is transferred onto the genetic background of B6 inbred strain, the normal testis determining mechanism is interrupted and results in complete or partial sex reversal of XY individuals. While the B6 mouse carrying its own Y-chromosome develops normal testes, YTIR acts properly on any genetic background other than B6. These results indicate the importance of coordination between SRY and autosomal genes in testis determination. The objective of our study is to clarify the mechanism of sex reversal in the B6. YTIR gonad. Our results support the hypothesis that at least two mechanisms in combination are responsible for this sex reversal. First, Sry transcription from YTIR is inefficient on the B6 genetic background. Second, the SRY protein encoded on YTIR is inefficient in up-regulating its downstream gene Sox9. Consequently, the delay and low levels of Sox9 expression allows ovarian differentiation, which, in turn, destabilizes Sox9 expression and predisposes testicular differentiation.

 

Fig. 1. (a) XY gonad of the CD-1 mouse strain at 12.5 days postcoitum (dpc). Intense SRY staining (green) is seen in the nuclei of abundant cells in the posterior pole (P), where testis cords have not yet formed, while much reduced staining is seen in the Sertoli cells within the testis cords near the medial region (M). On the other hand, intense MIS staining (red) is seen in the cytoplasm of Sertoli cells within the testis cords near the medial region while it is less frequently seen near the posterior pole. Both types of staining are seen in the same cells in an area indicated by *** (b) and also in a few cells within the testis cords indicated by * (c). The bar indicates 80 mm. (Taketo et al., 2005). These results indicate that Sry is expressed in the Sertoli cell lineage but downregulated upon Sertoli cell differentiation.

 

Publications (Members of our laboratory are indicated in bold-face)

  • Nagamine, C.M., Taketo, T., Koo, G.C. (1987) Studies on the genetics of tda-1 XY sex reversal in the mouse. Differentiation 33: 223-231.
  • Taketo, T., Saeed, J., Nishioka, Y., Donahoe, P.K. (1991) Delay of testicular differentiation in the B6.YDOM ovotestis demonstrated by immunocytochemical staining for Mullerian inhibiting substance. Developmental Biology 146: 386-395.
  • Lee, C.-H., Taketo, T. (1994) Normal onset, but prolonged expression of Sry gene in the B6.YDOM sex-reversed mouse gonad. Developmental Biology 165: 442-452 .
  • Lee, C.-H., Taketo, T. (2001) Low levels of Sry transcripts cannot be the sole cause of B6.YTIR sex reversal. Genesis 30: 7-11.
  • Taketo, T., Lee, C.-H., Zhang, J., Li, Y.-M., Lee, C.-Y.G., Lau, Y.-F.C. (2005) Expression of SRY proteins in both normal and sex-reversed XY fetal mouse gonads. Developmental Dynamics 233: 612-622.
  • Park, S., Zeidan, K.T., Shin, J.S., Taketo, T. (2011) SRY upregulation of SOX9 is inefficient and delayed, allowing ovarian differentiation, in the B6.YTIR gonad. Differentiation 82: 18-27.


2. Regulation of oocyte population in fetal and neonatal mouse ovaries

The primodial germ cells originate in an extra-embryonic site and migrate into the gonadal primordium. Subsequently, germ cells undergo sexual differentiation dependent on the gonadal environment. In the fetal ovary, most germ cells cease proliferation, enter meiosis, and become primary oocytes. Thus, oocytes reserve becomes finite and eventually limits female reproductive life. In addition, more than half of the initial oocyte population is eliminated in fetal and neonatal life, further restricting the oocyte reserve. The role or cause of this oocyte loss in the normal ovary remains poorly understood. We have been testing a hypothesis that oocytes with meiotic errors are eliminated by a checkpoint for raising the quality of the surviving oocytes. In order to delineate the association between a failure in homologous chromosome synapsis and death in oocytes, we have been studying the mode of oocyte death. Our previous studies have suggested that while a mitochondrial apoptotic pathway mediated by caspase 9 is constitutively activated, endogenous inhibitors of apoptosis such as XIAP protect oocytes from apoptosis execution. To provide evidence to support this hypothesis, we plan to either inhibit caspase 9 activity or overexpress XIAP specifically in oocytes by constructing conditional mutant mice. In the mouse models with enlarged oocyte reserves, thus produced, we will address whether or not the prevention of physiological oocyte loss renders the survival of poor quality oocytes.
   
Most previous studies of female meiosis used either histological sections or the squashed cells of ovaries; the former method provides a limited resolution, whereas the latter provides a limited representation. We have developed a new method to overcome these limitations (Taketo, 2012). In this method, termed “microspread cell preparation”, we dissociate ovarian cells and spin them down onto histology slides by centrifugation. By treating the cells in a hypotonic solution prior to centrifugation, we eliminate the cell membrane and the cytoplasmic components for better exposure of the nucleus. These preparations are excellent for immuncytochemical staining of chromosomal components and for visualizing homologous chromosome pairing (Fig. 2).

Fig. 2. Pairing between homologous chromosomes at the pachytene stage of meiotic prophase is shown in chromosome spreading preparations from neonatal ovaries. (a) XX ovary. Synaptonemal complex (red) is formed between 20 sets of homologous chromosomes including the X-X pair (not identified). (b) XY ovary. The single Y-chromosome (arrow) is left alone while the single X-chromosome with fainter SC staining (arrowhead) is tangled with autosomal pairs. (c) XO ovary. The single X-chromosome is left alone while 19 sets of autosomes are completely paired. The partial SC staining on the X-chromosome may indicate self-pairing. (Alton et al., 2008)


Publications (Members of our laboratory are indicated in bold-face)

  • Mertineit, C., Yoder, J.A., Taketo, T., Laird, D.W., Trasler, J.M., Bestor, T.H. (1998) Sex-specific exons control DNA methyltransferase in germ cells. Development 125: 889-897.
  • McClellan, K.A., Gosden, R., Taketo, T. (2003) Continuous loss of oocytes throughout meiotic prophase in the normal mouse ovary. Developmental Biology 258: 334-348.
  • Park, E.-H., Taketo, T. (2003) Onset and progress of meiotic prophase in the oocytes in the B6.YTIR sex-reversed mouse ovary. Biology of Reproduction 69: 1879-1889.
  • La Salle, S., Mertineit, C., Taketo, T., Moens, P.B., Bestor, T.H., Trasler, J.M. (2004) Windows for sex-specific methylation marked by DNA methyltransferase expression profiles in mouse germ cells. Developmental Biology, 268: 403-415.
  • Alton, M., Taketo. T. (2007) Switch from BAX-dependent to BAX-independent germ cell loss during the development of fetal mouse ovaries. Journal of Cell Science 120: 417-424.
  • Alton, M., Lau, M.P., Villemure, M., and Taketo, T. (2008) The behavior of the X- and Y-chromosomes in the oocyte during meiotic prophase in the B6.YTIR sex-reversed mouse ovary. Reproduction 135: 241-252.
  • Taketo, T. (2012) Microspread oocyte preparations for the analysis of meiotic prophase progression with improved recovery by cytospin centrifugation. Methods in Molecular Biology 825: 173-181.
  • Ene, A.C., Park, S., Edelmann, W., Taketo, T. Caspase 9 is constitutively activated in the mouse oocyte during meiotic prophase progression. Reproduction (under revision).


3. Regulation of meiotic divisions in the mouse oocyte

Female fertility depends on availability and quality of oocytes, both of which decline with maternal age. A major cause of the age-related infertility is the aneuploidy; chromosomes do not segregate properly in the oocyte upon ovulation or fertilization. The aneuploidy could be caused by a defect in the oocyte nucleus, cytoplasm, or both. Little is known about the specific nature of such defects. We have previously demonstrated that the oocytes of B6.YTIR emales reach the second metaphase but the second meiotic spindle is not adequately assembled and sister chromatids fail to segregate upon fertilization. these meiotic errors can be prevented by transferring the XY nucleus into an enucleated XX oocyte, thereby enabling the production of healthy offspring from the XY oocyte nucleus (Obata et al, 2008). We thus find that the XY female mouse is suitable for studying the mechanism of chromosome segregation failure inflicted by the cytoplasmic defects of oocytes. We have further found that the XY oocyte influences gene expression involved in glycolysis in its neighboring follicular cells, and consequently produces low ATPs, which may be responsible for its ooplasmic defects (Xu et al, under revision). We are currently addressing the role of communication between the oocyte and its surrounding follicular cells for the development of oocyte competence for the second meiotic division and embryonic development. .



Fig. 3. Cell cycle progression in the oocytes from XX (a-e) and XY (f-k) females after in vitro maturation for 19 h, SrCl2 activation for 2 h, and further culture. Immunolabeling of microtubules (green) and DAPI staining of chromosomes (blue). a. At MII before activation. Meiotic spindle is assembled in a barrel shape on both sides of condensed chromosomes (arrow). b. At AII (2.5 h post-activation). Condensed chromosomes (arrows) are migrated into the two poles of meiotic spindle. c. At TII (4 hpa). A set of condensed chromosomes (arrow) are extruding from the oocyte. d. At PN-stage (4 hpa). One prominent PN (arrow) has been formed. e. At the 2-cell-stage (24 hpa). Two symmetrical blastomeres have been formed after the first cell cleavage. f. At MII (2.5 hpa). Chromosomes are condensed at the second metaphase plate (arrow) encompassed by bulky microspindles. g. At AII (2.5 hpa). Chromosomes are segregating towards the both poles of meitoic spindle but some are bridging between the two groups (arrowhead in inset). h. At TII (2.5 hpa). A group of chromosomes are extruding from the oocyte, but many are bridging between the two groups of chromosomes (in inset). i. At TII- to PN-stage (4 hpa). A pole of meiotic spindle is extruding from the oocyte as if at TII. However, all nuclear materials (arrows) remain within the oocyte. j. At PN-stage (10 hpa). Three PN have been formed (arrows). k. At the 2-cell-stage (24 hpa). Asymmetrical two blastomeres contain multinuclei (arrows). (Villemure et al., 2007).



Publications (Members of our laboratory are indicated in bold-face)

  • Taketo-Hosotani, T., Nishioka, Y., Nagamine, C.M., Villalpando, I., Merchant-Larios, H. (1989) Development and fertility of ovaries in the B6.YDOM sex-reversed female mouse. Development 107: 95-105.
  • Merchant-Larios, H., Clarke, H. J., Taketo, T. (1994) Developmental arrest of fertilized eggs from the B6.YDOM sex-reversed female mouse. Developmental Genetics 15:435-442.
  • Amleh, A., Ledee, N., Saeed, J., Taketo, T. (1996) Competence of XY oocytes from the B6.YDOM sex-reversed female mouse for maturation, fertilization, and embryonic development in vitro. Developmental Biology 178:263-275.
  • Vanderhyden, B.C., Macdonald, E.A., Merchant-Larios, H., Fernandez, A., Amleh, A., Nasseri, R., Taketo, T. (1997) Interactions between the oocyte and cumulus cells in the ovary of the B6.YTIR sex-reversed female mouse. Biology of Reproduction 57: 641-646.
  • Amleh, A., Taketo, T. (1998) Live-borns from XX but not XY oocytes in the chimeric ovary composed of B6.YTIR and XX cells. Biology of Reproduction 58: 574-582.
  • Amleh, A., Smith, L., Chen, H.-Y., Taketo, T. (2000) Both nuclear and cytoplasmic components are defective in oocytes of the B6.YTIR sex-reversed female mouse. Developmental Biology 219: 277-286.
  • Wong, J., Luckers, L., Okawara, Y., Pelletier, R.-M., Taketo, T. (2000) Follicular development and atresia in the B6.YTIR sex-reversed mouse ovary. Biology of Reproduction 63: 756-762.
  • Villemure, M., Chen, H.-Y., Kurokawa, M., Fissore, R.M., and Taketo, T. (2007) The presence of X-and Y-chromosomes in oocytes leads to impairment in the progression of the second meiotic division. Developmental Biology 301: 1-13.
  • Obata, Y., Villemure, M., Kono, T., and Taketo, T. (2008) Transmission of Y-chromosomes from XY female mice was made possible by the replacement of cytoplasm during oocyte maturation. Proceedings of the National Academy of Sciences of the United States of America 105: 13918-13923.
  • Xu, B.-Z., Obata, Y., Cao, F., Shin, J.S., Taketo T. The expression of Y-encoded genes makes the cytoplasm defective in the XY oocyte by influencing its neighboring cumulus cells during the growth phase. Developmental Biology (under revision).

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[Updated: Jan. 9, 2012]