The sex of an individual is determined by the fate of the gonad. While the expression of Sry and Sox9 is sufficient to induce male development, we here show that female differentiation requires activation of the canonical beta-catenin signaling pathway. beta-catenin activation is controlled by Rspo1 in XX gonads and Rspo1 knockout mice show masculinized gonads. Molecular analyses demonstrate an absence of female-specific activation of Wnt4 and as a consequence XY-like vascularization and steroidogenesis. Moreover, germ cells of XX knockout embryos show changes in cellular adhesions and a failure to enter XX specific meiosis. Sex cords develop around birth, when Sox9 becomes strongly activated. Thus, a balance between Sox9 and beta-catenin activation determines the fate of the gonad, with Rspo1 acting as a crucial regulator of canonical beta-catenin signaling required for female development.
Differentiation of male germ cells requires a continuous cross-talk with their somatic support, the Sertoli cell. An in vitro model of Sertoli cells was recently provided by established cell lines which maintain Sertoli-specific characteristics, among which is a regulated phagocytic capacity. In vivo, Sertoli cells take up the residual cytoplasm expelled from the maturing sperm, a process restricted to a limited period of germinal maturation, and they also eliminate abnormally differentiated germ cells in case of hormonal deficiency. Cells of the Sertoli line efficiently take up latex beads, as well as dead cells in the cultures. A semiquantitative assay of phagocytosis was developed, based on the uptake of fluorescent latex beads. 15P-1 cultures were found to contain a minor fraction of active phagocytes. After addition of a defined fraction of germ cells, however, all the cells internalized beads as efficiently as macrophages. The inducing cell was identified as the pachytene spermatocyte, a cell type which, in vivo, is associated with Sertoli cells when they express their phagocytic potential. These inducing meiotic cells were not internalized themselves. Rather, they interacted with Sertoli cells via a surface signal that was resistant to formaldehyde fixation. The whole induction process does not involve changes in Sertoli gene expression, since it occurs even in the presence of high doses of cycloheximide. After the required initial contact, further maintenance of the activity was dependent on factor(s) secreted in the medium of the activated culture. Phagocytosis was, on the other hand, abrogated in the presence of factor(s) secreted by a distinct fraction of germ cells, enriched in the late stages (second division) of meiosis.
Journal of Cell Science was alerted to duplicated HPRT blots in Fig. 4A and Fig. 7C of this paper. The authors state that the conclusions of the paper are not affected by the duplicated control blots, but were unable to locate the original data from almost 20 years ago. Without the original full blots, the journal is unable to determine whether the results and conclusions reported in the paper are compromised. The journal is publishing this Expression of Concern to make readers aware of these issues. The authors offer cell lines used in the paper for replication by any interested investigators and apologise to readers for any inconvenience caused.
Analysis of complex signalisation networks involving distinct cell types is required to understand most developmental processes. Differentiation of male germ cells in adult mammals involves such a cross-talk between Sertoli cells, the somatic component which supports and controls germinal differentiation, and germ cells at their successive maturation stages. We developed a gene trapping strategy to identify genes, which, in Sertoli cells, are either up- or down-regulated by signals emitted by the germinal component. A library of approximately 2,000 clones was constituted from colonies independently selected from the Sertoli line 15P-1 by growth in drug-containing medium after random integration of a promoter-less (beta)geo transgene (neo(r)-lacZ fusion), which will be expressed as a fusion transcript from a ‘trapped’ cellular promoter, different in each clone. A first screen conducted on 700 events identified six clones in which beta-galactosidase activity was increased and one in which it was repressed upon addition of germ cells. The targeted loci were identified by cloning and sequencing the genomic region 5′ of the insert. One of them was identified as the gene encoding Fra1, a component of the AP1 transcription regulatory complex. Accumulation of Fra1 mRNA was induced, both in 15P-1 and in freshly explanted Sertoli cells, by addition of either round spermatids or nerve growth factor (NGF). The effect of NGF was mediated by the TrkA receptor and the ERK1-ERK2 kinase kinase pathway. Fos and Fra1 transcription were induced within the first hour after addition of the neurotrophin, but, unlike what is observed after serum induction in the same cells, a second wave of transcription of Fra1, but not of Fos, started 16 hours later and peaked at higher levels at about 20 hours. These results suggest that AP1 activation may be an important relay in the Sertoli-germ cell cross-talk, and validate the gene trapping approach as a tool for the identification of target genes in cell culture systems.
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