Germ cell tumours (GCTs) are clinically and pathologically heterogeneous neoplasms that arise at gonadal (testicular/ovarian) and extra-gonadal sites. The chemotherapy burden for patients with malignant germ cell tumours (mGCTs) that require treatment results in substantial longterm side-effects, and, furthermore, poor-risk patients have < 50% survival. Consequently, identifying common molecular changes and novel therapeutic targets in mGCTs is of major clinical importance. MicroRNAs are short, non-protein coding RNAs that regulate gene expression. We previously showed that miR-99a-5p/-100-5p and miR-125b-5p are among the most frequently underexpressed microRNAs in mGCTs, regardless of anatomical site, histological type or patient age. The present study investigates the upstream causes and downstream consequences of such under-expression. The mature form of miR-125b-5p is the product of two genomic loci, which form a cluster with either miR-99a-5p (on chromosome 21q) or miR-100-5p (on chromosome 11q). MiR-99a-5p/- 100-5p share identical 'seed' regions (at nucleotide positions 2-7), which determine their mRNA targets. Cross-reactivity experiment revealed that both miR-99a-5p and miR-100-5p probes were highly cross-reactive to each other's target (from 91% to 95%), indicating functional overlap. Linear regression analysis of qRT-PCR data reveals a strong positive correlation between miR-99a-5p/-100-5p and miR-125b-5p levels (R2 =0.989) in mGCTs, strongly suggesting co-regulation. Primary microRNA transcripts (pri-miR-99a/-100 and pri-miR-125b), and other genes that colocalise to these miRNA clusters (e.g. BLID on chromosome 11), were quantified by RT-qPCR in four representative cell lines - TCam2, 1411H, 2102Ep, and GCT44 - which were derived from a range of common histological types of mGCTs. A significant down-regulation (p < 0.0001) of all primary transcripts was observed, suggesting transcriptional repression of the entire cluster regions. Treatment of the cell lines with 5'-azacytidine resulted in significant upregulation of all three miRNAs (p < 0.002), as well as BLID (p < 0.02). The methylation status of potential CpG islands at the region of interest on chromosome 11 and chromosome 21 was therefore investigated by Pyrosequencing. Significant hyper methylation was found in 2102Ep, 1411H and GCT44 cell lines, suggesting that the miR-99a-5p/-100-5p and miR-125b-5p clusters are likely transcriptionally silenced by DNA methylation. To assess the functional relevance of these microRNAs in GCT progression, co-transfection of microRNA mimics (8.3 nM miR-99a-5p/-100-5p + 8.3 nM miR-125b-5p) was performed. A significant decrease in cell growth was seen in 1411H (p < 0.01) and TCam2 (p < 0.03) cells. To identify the mimics' downstream mRNA targets, HumanHT-12 v4 Expression Bead Chip (Illumina) mRNA arrays were used and data analysed using Sylamer. This analysis showed that mimic-treated cells were enriched in downregulated genes involved in pro-proliferative mechanisms. Among those, further functional characterisation focussed in particular on TRIM71, FGFR3, E2F7 and LIN28A. Moreover, restoring miR-99a-5p/-100-5p and miR-125b-5p in TCam2 cells also resulted in G0-G1 accumulation, consistent with a cell cycle effect. These data support a functionally important role for miR99a-5p/-100-5p and miR-125b-5p in GCT progression. They also raise the possibility of a therapeutic replenishment approach for treating these, and potentially other, tumours.

Yes / In each generation, the germline is tasked with producing somatic lineages that form the body, and segregating a population of cells for gametogenesis. During animal development, when do cells of the germline irreversibly commit to producing gametes? Integrating findings from diverse species, we conclude that the final commitment of the germline to gametogenesis - the process of germ cell determination - occurs after primordial germ cells (PGCs) colonize the gonads. Combining this understanding with medical findings, we present a model whereby germ cell tumors arise from cells that failed to undertake germ cell determination, regardless of their having colonized the gonads. We propose that the diversity of cell types present in these tumors reflects the broad developmental potential of migratory PGCs. / This work was supported by the Howard Hughes Medical Institute where D.C.P. is an Investigator, and the Frontier Research Program from the Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology. P.K.N. is a recipient of the Hope Funds for Cancer Research Fellowship (HFCR-15-06-06) and an Early Career Fellowship from the National Health and Medical Research Council, Australia (GNT1053776).

Normal germ cell development in the human testis is crucial for subsequent fertility and reproductive health. Disruption of testis development in fetal life can result in deleterious health consequences such as testicular dysgenesis syndrome (TDS), which includes disorders, such as cryptorchidism, hypospadias, infertility and testicular germ cell tumours (TGCT). A rat model of TDS in which rats are exposed to phthalates in utero has been validated, but does result in the development of TGCT. In humans, TGCTs result from transformation of pre-neoplastic carcinoma in-situ (CIS) cells and these CIS cells are believed to arise from human fetal germ cells during their transition from gonocyte to spermatogonia, based on their morphology and protein expression profile. It has been proposed asynchronous differentiation of germ cells in the human fetal testis may predispose fetal germ cells to become CIS cells. Studying the development of these tumours in humans is difficult because of their fetal origins and prolonged duration from initiation of impaired development to invasive disease. For this reason the use of relevant animal models that can mimic normal and abnormal germ cell development may provide new insight into how TGCT develop. The Common Marmoset monkey, a New World primate exhibits many similarities to the human in terms of reproductive biology and could represent such a model. This thesis aimed to further characterise the origins of CIS cells in the human testis by investigating the protein expression profile of CIS cells in patients with TGCT and comparing them to established markers of human fetal germ cell types using immunohistochemistry and immunofluorescence. Quantification of the various subpopulations of CIS and proliferation within these populations was performed. The thesis also investigated the Common Marmoset monkey as a potential model of normal testis and germ cell development by comparing the differentiation and proliferation profile of germ cells with those of the human during fetal and early postnatal life. During the present studies methods were successfully developed that enabled us to use testicular xenografts to recapitulate normal development of immature testes from marmoset and human. This involved grafting pieces of testis tissue subcutaneously under the dorsal skin of immunodeficient mice and retrieving them several weeks later to investigate their development during the grafting period. Xenografts using tissue from fetal, neonatal and juvenile marmosets were performed in addition to testes from first and second trimester human fetuses. Finally the present studies aimed to use the marmoset and the xenografting approach as systems in which to examine the effects of gonadotrophin suppression and phthalate treatment on germ cell differentiation and proliferation, with particular attention to the potential for development of CIS and TGCT. Heterogeneous phenotypes of CIS cells were identified, mostly consistent with those seen in the normal human fetal testis, however some of these CIS cells did not exhibit the same phenotype as germ cells identified in normal fetal testes. In addition it was shown that some of the proteins considered to be ‘classical’ markers of CIS cells, such as the pluripotent transcription factor OCT4, were not expressed in a proportion of the CIS cells. The proliferation index of CIS cells is also significantly higher in those subpopulations with the most ‘undifferentiated’ phenotype (i.e. OCT4+/VASA-). The present studies have generated novel data showing that the marmoset is a good model of fetal and neonatal germ cell development, with similarities to the human in terms of an asynchronous and prolonged period of differentiation and proliferation of germ cells from gonocyte to spermatogonia. This feature is also common to the human, but not a characteristic of the rodent. Fetal, neonatal and pre-pubertal germ cell development can be re-capitulated by xenografting tissue from marmoset and human testes into nude mouse hosts. Human fetal testis grafts produced testosterone and were responsive to hCG stimulation. First trimester human testis xenografts that have not developed fully formed seminiferous cords prior to grafting can complete the process of cord formation whilst grafted in host mice. In addition, germ cells in fetal human and marmoset xenografts can differentiate and proliferate in a similar manner to that seen in the intact non-grafted testis. In the intact neonatal marmoset, suppression of gonadotrophins resulted in a 30% decrease in proliferation, however differentiation of gonocytes is not affected. In-utero treatment of neonatal marmosets with mono-n-butyl phthalate was associated with unusual ‘gonocyte’ clusters, however, di-n-butyl phthalate treatment of mice carrying fetal marmoset xenografts resulted in no visible effects on germ cell differentiation or proliferation and did not result in the development of CIS or TGCT. In conclusion, this thesis has shown that there are many subpopulations of CIS cells of which many have not been previously described. These subpopulations have different characteristics, such as variable proliferation rates and this may indicate the potential for progression or invasiveness. These subpopulations have similar protein expression phenotypes to normal human fetal germ cells although the present studies have identified some CIS cells with phenotypes that are not found in the normal human testis. This thesis has demonstrated that the marmoset is a comparable model to the human in terms of asynchronous fetal germ cell development, which may predispose this species to the development of CIS/TGCT. In addition to the use of intact marmosets, these studies have also demonstrated for the first time that testis xenografting provides a comparable system for testis cord formation, germ cell differentiation and proliferation in fetal/postnatal marmosets and fetal human testis. In addition the marmoset and xenografting models have indicated that phthalates may have minor effects on testis development in the human and marmoset but do not result in CIS or TGCT. These model systems are suitable for further investigation of normal and disrupted testis development.