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 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

Mouse fetal germ cell isolation and culture techniques

The fetal gonad contains a great variety of differentiating cell populations, of which germ cells make up a small percentage. In order to study germ cell-specific gene and protein expression, as well as determine direct effects of signaling molecules, it is necessary to prepare enriched populations of germ cells and maintain them in culture for several hours to multiple days. The protocols in this chapter are designed to provide a guide for the isolation or enrichment of mouse primordial germ cells (from 9.5 days postcoitum (dpc) to 18.5 dpc) by flow cytometry (Subheading 3.1) or magnetic sorting (Subheading 3.2), followed by primary germ cell culture (Subheading 3.3)

Nodal/Cripto signaling in fetal male germ cell development: implications for testicular germ cell tumors

Testicular cancer is the most frequent cancer in young men aged 15-40 years and ac-counts for 1% of all cancer diagnosed in males. Testicular germ cell tumors (TGCT) encompass a broad group of cancers, each displaying different levels of pluripotency and differentiation as well as malignancy potential. The TGCT cell of origin is thought to be a fetal germ cell that failed to cor-rectly differentiate during development: this is known as the 'fetal origins hypothesis'. This theory predicts that developmental pathways that control germ cell pluripotency or differentiation may be involved in the malignant transformation of these cells. Recently the Nodal/Cripto signaling pathway, known to control pluripotency and differentiation in embryonic stem (ES) cells, was implicated in regulating normal male fetal germ cell pluripotency. Although genes of this pathway are not normally expressed in germ cells during adult life, ectopic expression of this pathway was detected in several sub-groups of TGCTs. In this review, we consider the evidence for the fetal origins of TGCT and discuss the implications of Nodal/Cripto signaling in various aspects of germ cell development and cancer progression

Regulation of germ cell meiosis in the fetal ovary

Fertility depends on correct regulation of meiosis, the special form of cell division that gives rise to haploid gametes. In female mammals, germ cells enter meiosis during fetal ovarian development, while germ cells in males avoid entering meiosis until puberty. Decades of research have shown that meiotic entry, and germ cell sex determination, are not initiated intrinsically within the germ cells. Instead, meiosis is induced by signals produced by the surrounding somatic cells. More recently, retinoic acid (RA), the active derivative of vitamin A, has been implicated in meiotic induction during fetal XX and postnatal XY germ cell development. Evidence for an intricate system of RA synthesis and degradation in the fetal ovary and testis has emerged, explaining past observations of infertility in vitamin A-deficient rodents. Here we review how meiosis is triggered in fetal ovarian germ cells, paying special attention to the role of RA in this process

Retinoblastoma 1 protein modulates XY germ cell entry into G1/G0 arrest during fetal development in mice

During mouse germ cell development, the first sign of sex differentiation occurs when XY germ cells enter G1/G0 arrest from 12.5 days postcoitum (dpc). Retinoblastoma 1 (RB1), a potent cell cycle regulator, was investigated in XY germ cell arrest by studying germ cell proliferation in Rb1−/− mutant mouse embryos. Because mice homozygous for the Rb1 deletion die in utero around 14.5 dpc, we used ex vivo culture techniques to allow analysis of developing gonads to 16.5 dpc. In Rb1−/− gonads, we observed normal somatic cell development, assessed by immunofluorescence for markers HSD3B1 and anti-Müllerian hormone. However, at 14.5 dpc, when wild-type XY germ cells had arrested, we could detect actively proliferating germ cells using the proliferation markers MKI67, pHH3, and bromodeoxyuridine incorporation. The increased proliferation was reflected with a trend of increased germ cell number and expression of germ cell markers Ddx4 and Pou5f1 in the Rb1−/− testes. By 16.5 dpc, this phenotype was resolved such that the entire germ cell population had entered G1/G0 arrest, although the total germ cell number remained elevated. At each stage analyzed, we saw no increase in expression of RB1 family members Rbl1 and Rbl2 in the Rb1−/− testes, but we saw a significant increase of cyclin-dependent kinase (CDK) inhibitor Cdkn1b and Cdkn2b expression. We conclude that Rb1 is required for correct germ cell entry into G1/G0 arrest in the wild-type gonad at 14.5 dpc, but in its absence, upregulation of other cell cycle suppressors, including Cdkn1b and Cdkn2b, can induce delayed germ cell arrest

Developmental biology meets toxicology: contributing reproductive mechanisms to build adverse outcome pathways

An adverse outcome pathway (AOP) is a simplified description of the sequence of mechanistic events that lead to a particular toxicological effect, from initial trigger to adverse outcome. Although designed to inform regulatory risk assessors, the AOP framework also provides a platform for innovative collaborations between experts from relevant research fields and the regulatory community. The underpinning for any AOP is basic knowledge about molecular and developmental processes; such knowledge can only be attained by solid bioscientific research. Starting with this fundamental knowledge, the objective is to devise novel testing strategies that focus on key events in a causative pathway. It is anticipated that such a knowledge-based approach will ultimately alleviate many of the burdens associated with classical chemical testing strategies that typically involve large-scale animal toxicity regimens. This hails from the notion that a solid understanding of the underlying mechanisms will allow the development and use of alternative test methods, including both in vitro and in silico approaches. This review is specifically targeted at professionals working in bioscientific fields, such as developmental and reproductive biology, and aims to (i) inform on the existence of the AOP framework and (ii) encourage new cross-disciplinary collaborations. It is hoped that fundamental biological knowledge can thus be better exploited for applied purposes: firstly, an improved understanding of how our perpetual exposure to environmental chemicals is causing human reproductive disease and, secondly, new approaches to screen for harmful chemicals more efficiently. This is not an instructional manual on how to create AOPs; rather, we discuss how to harness fundamental knowledge from the biosciences to assist regulatory toxicologists in their efforts to protect humans against chemicals that harm human reproductive development and function