Germ cell neoplasia in situ: the precursor cell for invasive germ cell tumors of the testis

Germ cell neoplasia in situ is the non-invasive precursor cell of origin for type II testicular germ cell tumors. It has long been postulated that germ cell neoplasia in situ is derived from defective germ cell development during embryonic life, and although it is impossible to trace in vivo the progression from fetal germ cell to germ cell neoplasia in situ to tumor, there is a large volume of evidence supporting this theory. Current studies focus on understanding how germ cell neoplasia in situ forms, how these cells are activated at puberty and how they transform to invasive tumors of various subtypes. Such information is informing novel diagnostic and therapeutic options

Cripto: Expression, epigenetic regulation and potential diagnostic use in testicular germ cell tumors

Type II germ cell tumors arise after puberty from a germ cell that was incorrectly programmed during fetal life. Failure of testicular germ cells to properly differentiate can lead to the formation of germ cell neoplasia in situ of the testis; this precursor cell invariably gives rise to germ cell cancer after puberty. The Nodal co-receptor Cripto is expressed transiently during normal germ cell development and is ectopically expressed in non-seminomas that arise from germ cell neoplasia in situ, suggesting that its aberrant expression may underlie germ cell dysregulation and hence germ cell cancer. Here we investigated methylation of the Cripto promoter in mouse germ cells and human germ cell cancer and correlated this with the level of CRIPTO protein expression. We found hypomethylation of the CRIPTO promoter in undifferentiated fetal germ cells, embryonal carcinoma and seminomas, but hypermethylation in differentiated fetal germ cells and the differentiated types of non-seminomas. CRIPTO protein was strongly expressed in germ cell neoplasia in situ along with embryonal carcinoma, yolk sac tumor and seminomas. Further, cleaved CRIPTO was detected in media from seminoma and embryonal carcinoma cell lines, suggesting that cleaved CRIPTO may provide diagnostic indication of germ cell cancer. Accordingly, CRIPTO was detectable in serum from 6/15 patients with embryonal carcinoma, 5/15 patients with seminoma, 4/5 patients with germ cell neoplasia in situ cells only and in 1/15 control patients. These findings suggest that CRIPTO expression may be a useful serological marker for diagnostic and/or prognostic purposes during germ cell cancer management

Transcriptomic analysis of mRNA expression and alternative splicing during mouse sex determination

Mammalian sex determination hinges on sexually dimorphic transcriptional programs in developing fetal gonads. A comprehensive view of these programs is crucial for understanding the normal development of fetal testes and ovaries and the etiology of human disorders of sex development (DSDs), many of which remain unexplained. Using strand-specific RNA-sequencing, we characterized the mouse fetal gonadal transcriptome from 10.5 to 13.5 days post coitum, a key time window in sex determination and gonad development. Our dataset benefits from a greater sensitivity, accuracy and dynamic range compared to microarray studies, allows global dynamics and sex-specificity of gene expression to be assessed, and provides a window to non-transcriptional events such as alternative splicing. Spliceomic analysis uncovered female-specific regulation of Lef1 splicing, which may contribute to the enhanced WNT signaling activity in XX gonads. We provide a user-friendly visualization tool for the complete transcriptomic and spliceomic dataset as a resource for the field

Cell cycle analysis of fetal germ cells during sex differentiation in mice

Background information. Primordial germ cells in developing male and female gonads are responsive to somatic cell cues that direct their sex-specific differentiation into functional gametes. The first divergence of the male and female pathways is a change in cell cycle state observed from 12.5 dpc (days post coitum) in mice. At this time XY and XX germ cells cease mitotic division and enter G1/G0 arrest and meiosis prophase I respectively. Aberrant cell cycle regulation at this time can lead to disrupted ovarian development, germ cell apoptosis, reduced fertility and/or the formation of germ cell tumours

Investigating Sex Specific Cell Cycle Regulation in Fetal Germ Cells

During development, somatic cell cues direct sex-specific differentiation of germ cells that is characterised by two distinct cell cycle states. At 12.5 days post coitum (dpc) in a testis, XY germ cells stop proliferating and enter G1/G0 arrest. In the ovary, XX germ cells bypass G1/G0 arrest and instead enter the first phase of meiosis I from 13.5 dpc. Whilst it is hypothesised that errors in cell cycle control during development precede the formation of testicular germ cell tumours, the mechanism of cell cycle control at this time has not been thoroughly investigated. This project therefore sought to explore the mechanism of XY germ cell G1/G0 arrest using several approaches. Although cell cycle regulation for somatic cells is well established, we know very little regarding germ cell control of this process. Therefore my first aim was to profile this machinery at the transcript level using a cell cycle cDNA array. Purified populations of germ cells were isolated both before and after sex differentiation and expression of 112 cell cycle related genes was assessed. From this study a comprehensive network governing apoptosis and calcium signalling that was common to both XX and XY germ cells was observed. Importantly, the retinoblastoma family and cyclin dependent kinase inhibitor p21 was implicated in the regulation of G1/G0 arrest in XY germ cells. Lastly, XX germ cells displayed a down-regulation of genes involved in both G1 and G2 phases of the cell cycle consistent with their progression past G1 phase. This study has provided a detailed analysis of cell cycle gene expression during fetal germ cell development and identified candidate factors for future investigation in order to understand cases of aberrant cell cycle control in these specialised cells. In order to investigate several candidate genes identified within the cell cycle array, I next sought to generate a germ cell-specific Cre recombinase mouse model for use in conditional knockout studies. As current Cre lines lack specificity or appropriate temporal expression, we used the germ cell-specific regions of the fragilis promoter to drive Cre expression during germ cell specification. Eleven founder lines were generated using this construct and four were analysed using a reporter line. Although we have not achieved germ cell expression from these lines to date, analysis continues in order to identify an invaluable new tool for germ cell research. Following the implication of the retinoblastoma family in XY germ cell G1/G0 arrest, I next investigated the role of RB in these cells using the Rb null mutant. RB is a known cell cycle suppressor that controls this process in many cell types and, subsequently, mice homozygous for the Rb deletion die in utero at 14.5 dpc. Using this model we analysed developing gonads from 14.5 – 16.5 dpc using ex vivo culture techniques. At 14.5 dpc when wild type germ cells have arrested, proliferating germ cells were detected in the absence of Rb using proliferation marker Ki67. This proliferation was accompanied by a slight increase in germ cell number at 14.5 dpc, however, two days later at 16.5 dpc germ cell numbers were slightly decreased in the Rb-/- testes. During this time we could also detect increased expression of other RB family members p107 and p130, suggesting that these factors may compensate for the loss of Rb in the germ line. This investigation has implicated RB in the regulation of XY germ cell G1/G0 arrest and will form the basis for future work aimed at understanding the initiation of this cell cycle state. In addition to RB, a lesser-known transcription factor was also investigated in the initiation and maintenance of XY germ cell G1/G0 arrest. The high mobility group box transcription factor 1 (HBP1) suppresses proliferation and promotes differentiation in various cell types and was recently identified within the XY germ cells at the appropriate time of sex differentiation. In my analysis two Hbp1 transcripts were identified within the XY germ cells that display different sub-cellular localisations in vitro. Next, Hbp1-LacZ reporter lines were generated to aid in understanding the germ cell-specific regulation of these transcripts and lastly, I analysed the genetrap mutation for Hbp1. Surprisingly, this model revealed no aberrations to germ cell-cell cycle control during development. In summary, I have performed the first comprehensive study of the cell cycle machinery utilised by germ cells as they undergo the first stages of sex differentiation. Using loss-of-function models I was able to implicate the cell cycle regulator RB specifically in XY germ cell G1/G0 arrest and, conversely, demonstrate that the transcription factor HBP1 is not required for this process

Cell cycle control of germ cell differentiation

The germ cell lineage is our lifelong reservoir of reproductive stem cells and our mechanism for transmitting genes to future generations. These highly specialised cells are specified early during development and then migrate to the embryonic gonads where sex differentiation occurs. Germ cell sex differentiation is directed by the somatic gonadal environment and is characterised by two distinct cell cycle states that are maintained until after birth. In the mouse, XY germ cells in a testis cease mitotic proliferation and enter G1/G0 arrest from 12.5 dpc, while XX germ cells in an ovary enter prophase I of meiosis from 13.5 dpc. This chapter discusses the factors known to control proliferation and survival of germ cells during their journey of specification to sex differentiation during development

Sex determination in mammalian germ cells

Germ cells are the precursors of the sperm and oocytes and hence are critical for survival of the species. In mammals, they are specified during fetal life, migrate to the developing gonads and then undergo a critical period during which they are instructed, by the soma, to adopt the appropriate sexual fate. In a fetal ovary, germ cells enter meiosis and commit to oogenesis, whereas in a fetal testis, they avoid entry into meiosis and instead undergo mitotic arrest and mature toward spermatogenesis. Here, we discuss what we know so far about the regulation of sex-specific differentiation of germ cells, considering extrinsic molecular cues produced by somatic cells, as well as critical intrinsic changes within the germ cells. This review focuses almost exclusively on our understanding of these events in the mouse model

Sex Determination and Gonadal Development

In mammals, sex determination is a result of chromosomal constitution determined at fertilization, with females harboring XX and males XY sex chromosomes. The apparent simplicity of this system belies the intricate genetic and cellular interactions that direct differentiation of the bipotential gonadal primordium into either a testis or an ovary. These organs house, nurture, and direct the differentiation of the germ cells, which in turn are required for reproduction and for transmission of genetic information to future generations. Specification, migration, and differentiation of germ cells are controlled by the somatic cell environment. Once sex differentiation has occurred, the germ cells are directed to develop into oocytes (female) or spermatozoa (male), both highly specialized cell types. This chapter traces the key events in the genesis of the mammalian germline, and describes how genetic and cellular control mechanisms in the gonadal somatic cell lineages influence the development of germ cells

Regulation of fetal male germ cell development by members of the TGFβ superfamily

There is now substantial evidence that members of the transforming growth factor-β (TGFβ family) regulate germ cell development in the mouse fetal testis. Correct development of germ cells during fetal life is critical for establishment of effective spermatogenesis and for avoiding the formation of testicular germ cell cancer in later life. Here we consider the evidence for involvement of various TGFβ family members, attempt to reconcile discrepancies and clarify what we believe to be the likely in vivo roles of these factors