Katarina Nordqvist

Male-specific cell migration into the developing gonad

BACKGROUND The gene Sry acts as a developmental switch, initiating a pathway of gene activity that leads to the differentiation of testis rather than ovary from the indifferent gonad (genital ridge) in mammalian embryos. The early events following Sry expression include rapid changes in the topographical organization of cells in the XY gonad. To investigate the contribution of mesonephric cells to this process, gonads from wild-type mice (CD1), and mesonephroi from a transgenic strain ubiquitously expressing beta-galactosidase (ROSA26), were grafted together in vitro. After culture, organs were fixed and stained for beta-galactosidase activity to identify cells contributed from the mesonephros to the male or female gonad. RESULTS Migration of mesonephric cells occurred into XY but not XX gonads from 11.5-16.5 days post coitum (dpc). Somatic cells contributed from the mesonephros were distinguished by their histological location and by available cell-specific markers. Some of the migrating cells were endothelial; a second population occupied positions circumscribing areas of condensing Sertoli cells; and a third population lay in close apposition to endothelial cells. CONCLUSIONS OFFgration from the mesonephros to the gonad is male specific at this stage of development and depends on an active signal that requires the presence of a Y chromosome in the gonad. The signals that trigger migration operate over considerable distances and behave as chemoattractants. We suggest that migration of cells into the bipotential gonad may have a critical role in initiating the divergence of development towards the testis pathway.

Normal sexual development and fertility in testatin knockout mice

ABSTRACT The testatin gene was previously isolated in a screen focused on finding novel signaling molecules involved in sex determination and differentiation. testatin is specifically upregulated in pre-Sertoli cells in early fetal development, immediately after the onset of Sry expression, and was therefore considered a strong candidate for involvement in early testis development. testatin expression is maintained in the adult Sertoli cell, and it can also be found in a small population of germ cells. Testatin shows homology to family 2 cystatins, a group of broadly expressed small secretory proteins that are inhibitors of cysteine proteases in vitro but whose in vivo functions are unclear. testatin belongs to a novel subfamily among the cystatins, comprising genes that all show expression patterns that are strikingly restricted to reproductive tissue. To investigate a possible role of testatin in testis development and male reproduction, we have generated a mouse with targeted disruption of the testatin gene. We found no abnormalities in the testatin knockout mice with regard to fetal and adult testis morphology, cellular ultrastructure, body and testis weight, number of offspring, spermatogenesis, or hormonal parameters (testosterone, luteinizing hormone, and follicle-stimulating hormone).

Sry and the testis: Molecular pathways of organogenesis

The gene Sry acts as a switch, initiating pathways leading to the differentiation of a testis rather than an ovary from the indifferent gonad (genital ridge) in mammals. The early events following Sry expression include rapid changes in the topographical organization of cells in the XY gonad. Sry must therefore initiate signaling pathways that direct male-specific patterns of proliferation, migration, cell-cell organization, and vascularization. We have identified an increase in male-specific proliferation by 12.0 days post coitum, while proliferation in the female gonad declines. We have also observed male-specific cell migration from the mesonephros into the gonad in a composite organ culture system in which gonads from wild-type mice (CD1) and mesonephroi from a transgenic strain expressing beta-galactosidase in all its cells (ROSA26) were grafted together in vitro at the indifferent stage of gonadogenesis. Migration depends on an active signal that requires the presence of a Y chromosome in the gonadal portion of the graft. The signals that trigger migration operate over considerable distances, suggesting either a long-range diffusible factor or the involvement of a rapid and efficient relay mechanism. Identification of the somatic cells contributed from the mesonephros with cell-specific markers indicated that some of the migrating cells were endothelial, revealing differences in processes of vascularization between male and female gonads. A second distinct population of migrating cells lay in close apposition to endothelial cells, and a third population occupied positions circumscribing areas of condensing Sertoli cells.

Isolation of sex-specific cDNAs from fetal mouse brain using mRNA differential display and representational difference analysis

Comparing female and male brain structures reveals a variety of sex differences in many vertebrates. These differences are manifested throughout the brain, in regions such as the hypothalamus, the preoptic area and the amygdala. Some are thought to be induced during the fetal period by the effect of steroid hormones produced in the gonads. It is well-established that fetal androgens, probably through the conversion to estrogen by the enzyme aromatase, masculinize the nervous system and set adult mounting behavior in rodents. However, less is known about molecular mechanisms involved in gender-specific development of the brain. We have taken a broad approach to isolate sex-specific genes from fetal brain. mRNAs from 18.5 days post-coitum (dpc) female and male mouse brain were screened with the classical and the recently developed signal peptide differential display (SPDD) and with representational difference analysis of cDNA (cDNA-RDA). Two sex-specific cDNAs were isolated, F29 and M17, corresponding to the female-specific Xist gene and the male-specific Smcy gene, respectively.

Protocol for using signal peptide differential display and representational difference analysis to isolate differentially expressed cDNAs from fetal mouse brain.

Comparing female and male brain structures reveals a variety of sex differences in many vertebrates. Some of these differences are thought to be induced during the fetal period by the effect of steroid hormones produced in the gonads. Not much is known about molecular mechanisms involved in gender-specific development of the brain. We have taken a broad approach to isolate sex-specific genes from 18.5 days post coitum brain (A. Eriksson, C. Wahlestedt and K. Nordqvist. 1999. Isolation of sex-specific cDNAs from fetal mouse brain using mRNA differential display and representational difference analysis. Mol. Brain Res., 74, 91-97). Female and male mouse brains were screened with the signal peptide differential display, developed in our laboratory, and with a modified representational difference analysis of cDNA. The resulting sex-specific fragments were verified by semi-quantitative RT-PCR. Here we describe these methods in detail.