Male germ cells
Germ cell homing to the Stem Cell Niche
Chemokines and spermatogenesis: roles of SDF-1 and CXCR-4
In the mammalian testes, numerous spermatozoa are continuously produced throughout adult reproductive life. This process, known as spermatogenesis, is dependent on the establishment early in neonatal development, of a population of self renewing germ line stem cells known as GSC's, from which the highly differentiated haploid spermatozoa are produced. Little is known about the molecular and cellular mechanisms underlying the creation of GSC's from their immediate progenitors, the gonocytes, and how the stem cell niche in which they reside in the testes is defined. Recent evidence from our laboratory suggests that a chemokine SDF-1 and its receptor CXCR-4 are intimately involved in the processes of gonocyte survival and differentiation in the fetal testes and the eventual migration of the GSC population to the stem cell "niche" located in the basement membrane in the post natal testes. The aims of this proposal are: to elucidate the mechanism of maintenance and migration of male germ cells in the developing testis and the role of SDF-1/CXCR-4 interaction in the establishment of stem cell populations in the germ cell niche, to characterize the signalling pathways activated by CXCR-4 and their role in germ cell differentiation and to investigate the use of knockdown and overexpression technologies in vivo and in vitro to manipulate germ cell survival and differentiation.
Retention of Spermatogonial Stem cell phenotype
Crucial to spermatogenesis are a number of RNA binding proteins, which are expressed in germ cells. These proteins are responsible for the control of post-transcriptional regulation of the multitude of mRNAs coding for proteins essential for latter stages of germ cell and spermatozoal development. Musashi (Msi) is an RNA binding protein family and we recently investigated the role of Musashi family in the Drosophila and mouse testes and showed that Drosophila Msi is both expressed and required in spermatogonial stem cells for maintenance of stem cell fate. We also found that cell-autonomous loss of Msi in the Drosophila testis results in the premature differentiation of spermatogonial stem cells, indicating an intrinsic requirement for Msi for regulation of stem cell maintenance – we have produced a transgenic msi1 mouse for overexpression studies and this project aims to characterise this mouse.
Molecular Pathways of Germ Cell Differentiation
We are seeking to understand the transition from spermatogonia to spermatocyte at the molecular level. We have identified 2 signalling pathways that interact during spermatogonial differentiation: the BMP4 and retinoid pathways. Previously, we have found that the metabolites of vitamin A, known as retinoids, regulate BMP4 gene expression. The major metabolite of vitamin A is not the active molecule in this case. This challenges a dogma. Testis and germ cells will be examined for vitamin A metabolites. Retinoids, both extracted and commercially available, will be tested by assessing their affects on BMP4 gene expression in isolated cells.
Using array technology we have previously identified a number of other genes also regulated by retinoids in the testis. We now intend to determine which of these regulated genes are expressed in spermatogonia. A separate array approach has suggested some candidates. One project involves confirming the expression and regulation of candidates in spermatogonia treated with a number of retinoids with a focus on the regulation of BMP4 gene expression. Another project involves further analysis of the two sets of array data in combination with published data sets relating to stem cells from other populations. We are also developing a number of models of BMP4 signalling in the germline. These will allow us to determine the role of BMP4 in spermatogonial differentiation. A project exists in characterising the molecular response of the germ line to BMP4.
Supervisor: Dr Shaun Roman
DNA damage in the Germline
DNA damage in the male germline is associated with poor fertilization rates following IVF, defective preimplantation embryonic development, and high rates of miscarriage and morbidity in the offspring, including childhood cancer. This damage is poorly characterized, but is known to involve hypomethylation of key genes, oxidative base damage, endonuclease mediated cleavage and the formation of adducts with xenobiotics and the products of lipid peroxidation. There are many possible causes of such DNA damage, including abortive apoptosis, the oxidative stress associated with male genital tract infection, exposure to redox cycling chemicals, and defects of spermiogenesis associated with the retention of excess residual cytoplasm. Physical factors such as exposure to radiofrequency electromagnetic radiation or mild scrotal heating can also induce DNA damage in mammalian spermatozoa, although the underlying mechanisms are unclear. Ultimately, resolving the precise nature of the DNA lesions present in the spermatozoa of infertile men will be an important step towards uncovering the aetiology of this damage and developing strategies for its clinical management.
We are interested in the response of the male germ line to exposure with foreign compounds (xenobiotics). Such exposure can lead to defects in the offspring indicating damage at the level of DNA. We are trying to elucidate the mechanism of damage generation. Using microarray technology we established the cyp gene profile in the male germ line. Cyp genes encode for the phase I detoxifying enzymes; the cytochrome P450s. Further studies on gene expression and toxicology in germ cells are required. In particular we are interested in assessing changes in cyp expression in response to toxicant treatment. We are also looking to measure the formation toxicant-DNA adducts. Currently we are establishing a DNA damage assay based on real time PCR. This work has been carried out on human DNA. We are interested in establishing this assay in mouse germ cells with a view to high throughput testing of the germ cell response to xenobiotic compounds.
Supervisor: Dr Shaun Roman