2.1. Primordial germ cell specification and candidate genes controlling germline establishment

The establishment of early germ cells and their successful maturation are complex processes, and require frequent changes in physiology, location and transcriptional profile of involved cells. Germline establishment in mammals occurs via inductive signaling, in contrast to lower organisms such as flies and worms where the germ cell identity is transmitted via the inheritance of germ plasm (McLaren, 1999, 2000; Saitou et al., 2002). In rodents and humans, the first glimpses of primordial germ cell (PGC) formation are observed in the embryo after implantation and gastrulation, when the epiblast, endoderm, mesoderm and ectoderm are first established (Matsui & Okamura, 2005). At this time, in response to molecular cues including Bone Morphogenetic Proteins (BMP4 and BMP8a) from the yolk sac, a population of pluripotent stem cells is segregated from the ICM and set physically apart from the extra-embryonic ectoderm or yolk sac of the embryo (Figure 1) (Lawson et al., 1999; Ying 2000, 2001). While BMPs provide the inductive signal to epiblast stem cells to supply PGCs, it is not clear what signals control the size of the PGC founder population or what molecules signal the termination of PGC specification. BMPs are crucial to specification of PGCs due to their activation of the ALK2 receptor and Smad-1/5 signaling pathways, as evidenced in mice (Hayashi et al., 2002). In the human embryo, the effects of BMPs on germline specification are unclear because there is very limited access to early embryonic samples. From work in our laboratory, it appears that similar BMP protein pathways are activated in the human embryo during gastrulation that direct PGC specification (Clark & Reijo Pera, 2006, Kee et al, 2006).

Additional mechanisms that may assist germline specification are the activation of pathways that either promote PGC survival and/or inhibit molecules that promote somatic differentiation of PGCs (Ewen & Koopman, 2010). As such, mouse and human PGCs retain expression of several biomarkers of pluripotency, including Oct4, Nanog and Sox2 which underlies their close resemblance to other pluripotent stem cells (Clark & Reijo Pera, 2006; Medrano et al., 2010; Nicholas et al., 2009). Interestingly, the transcription factor Sox2 is expressed on both mouse and human embryonic stem cells but unlike in the mouse, human Sox2 expression is diminished when PGCs migrate to the fetal gonad (Perrett et al., 2008). Coincident with the timing of germline specification in the mouse (E7.5), PGCs near the extraembryonic ectoderm begin to express germ-cell specific markers such as Blimp-1 (Prdm1 in human), Stella (Dppa3 in human), E-Cadherin, and Dazl and harbor alkaline phosphatase activity. Blimp-1/Prdm-1 is a transcriptional repressor whose activity is restricted to the germ lineage and appears to be critical for maintaining a PGC fate. There is strong evidence to suggest that in the mouse, Blimp-1 actively represses the somatic fate of PGCs by inhibiting expression of key somatic regulators during development (Ohinata et al., 2005; Hayashi et al., 2007). It is unclear whether Prdm-1 carries out a similar function in human germline formation. Once cell-cell communication has been established, the Fragilis (IFITIM-1 in human) and Stella/ Dppa3 genes promote further development of PGCs and may do so in a similar fashion as Blimp-1 in mice (Saitou et al., 2002).

Migration of germ cells to the gonad begins at E8.5 in the mouse and during weeks 4 to 6 of human gestation (first trimester). At this stage, PGCs accumulating at the base of the allantois exit the extraembryonic ectoderm and begin migration to the developing gonads, also known as the genital ridge. During migration, PGCs also proliferate by undergoing mitosis and express a new set of biomarkers, including the CXCR4 receptor in mice and the proto-oncogene c-KIT and its ligand, KIT in both mice and humans (Molyneaux & Wylie, 2004; Gomperts et al., 1994). The DAZ gene homologue, DAZL is also expressed on migrating PGCs. In the mouse, migratory PGCs display pseudopodia that may assist in movement through the hindgut and it is plausible that human germ cells behave similarly since they are also observed in the hindgut during migration. Various somatic tissues interact with PGCs during the migratory path to the gonad. It is likely therefore, that these tissues express molecules and factors that guide or cue the PGCs and help maintain their survival. In the mouse, several candidate molecules have been identified, including receptors such as -1 Integrin and extracellular matrix components such as Collagen I (Chuva de Sousa Lopes et al., 2005; De Felici et al., 2005). Although migration of PGCs is less well understood in human development, germ cells have been histologically observed during the late first trimester, when they undergo migration to the hindgut (Fujimoto et al., 1977; Gaskell et al., 2004; Goto et al., 2004). Male and female PGCs have been isolated from

Developmental Cycle of Mammalian Germ Cells. Life cycle of the mouse and human embryo following fertilization, progressing through gastrulation and producing the germline. The germline develops in the gonads and transmits genetic information to the next generation, thus completing the cycle. Fertilization of oocytes by sperm promotes the formation of a 1-cell zygote that undergoes cell division and cleavage to form a blastocyst. The outer layer of blastocyst gives rise to the trophectoderm while the inner cell mass (ICM) contains embryonic stem cells (ESCs). During gastrulation (E7.5 in mouse; Day 15+ in human), the blastocyst cavitates and develops the three germ layers and the epiblast. The primordial germ cells (PGCs) are specified and localize near the extra-embryonic ectoderm, at the base of the allantois. Once PGCs are specified, they migrate to the fetal gonads and undergo sex-specific developmental to male and female gonocytes. Subsequently, male gonocytes undergo spermatogenesis while female gonocytes enter meiotic prophase I and begin oogenesis. Adapted from Schuh-Huerta et al., 2011.

10-week old fetuses and were observed to express Alkaline Phosphatase (AP), a marker of PGCs (Goto et al., 2004). The morphology of human PGCs also resemble the rounded shape of mouse PGCs. Female-specific germ cells have also been visualized at the ultrastructural level during gonadal development in human fetuses (Motta et al., 1997). Finally, recent studies by Kerr et al. with human fetal gonads (testis and ovary) provide a detailed analysis of pluripotency and germ cell-specific markers. The germ cells of the fetal testis are Oct4 +/Nanog +/ c-Kit+ from week 7 to 15 after which these cells become localized to the testis periphery. Meanwhile, the presumptive gonocytes in the week 15 testis show strong expression of Pumilio2 (PUM2), VASA and DAZL and express low to no pluripotency markers (Oct4, Nanog, c-Kit, Tra1-60, Tra1-81) (Kerr et al., 2007). In the human fetal ovary, as in the testis, the expression of pluripotency markers peaks by week 8 and then declines after week 9, as oocytes enter meiosis (Kerr et al., 2008). Interestingly, the cell surface markers SSEA-1 and SSEA-4 are co-expressed on the female germ cells from week 5 onwards although only SSEA-1 is restricted to the germ cell lineage.

Upon arrival at the genital ridge, germ cells express another germ-cell specific marker, VASA, a cytoplasmic protein that is implicated in translational regulation. The gene encoding VASA expression, DDX4 (Mvh in mouse) is highly conserved among species and is expressed exclusively in both male and female pre-meiotic germ cells (Gustafson & Wessel, 2010). This finding underlines the importance of the VASA protein in germline function and makes it an attractive candidate for further study. Along with VASA, other factors produced are germ cell nuclear antigen-1 (GCNA-1) and E-cadherin. The sex-specific character of the developing gonad is controlled by the chromosomal constitution of gonadal somatic cells. In particular, the SRY gene expressed on the Y chromosome in mammals is thought to be an essential regulator of various downstream targets including the Sox9 gene that controls male gonadal development (McLaren, 1995, 2003). Once within the gonad, germ cells associate with Sertoli cells to form testis cords and this interaction induces the expression of VASA in post-migratory PGCs. VASA expression is induced in both male and female PGCs and persists until these cells enter meiosis and after which its levels diminish (Castrillon et al., 2000; Toyooka et al., 2000).

During germline development, an extensive remodeling of the epigenetic landscape occurs. This takes place during embryogenesis and during PGC migration to the gonad (Nicholas et al, 2009). The first wave of epigenetic remodeling occurs during implantation of the blastocyst and involves the erasure of all DNA methylation at CpG islands except those at imprinted gene loci. This transition is observed in all cells of the embryo, including the primitive germline. In female PGCs, there is another level of epigenetic change in the form of random X inactivation wherein one copy of the X chromosome pair is silenced. The second wave of epigenetic remodeling occurs when PGCs migrate to the primitive gonads and their paternal or maternal imprinted loci undergo a gradual process of erasure (Hajkova et al., 2002; Yamazaki et al., 2003). This phase occurs only in germ cells and may help to prime them for sex-specific DNA remethylation, when their developmental programs are established (Durcova-Hills et al., 2006). In the mouse, the re-establishment of imprints takes place prior to birth in the male prospermatogonia (E15) and only after birth in oocytes (Lucifero et al., 2002). In addition to DNA methylation changes, male and female germ cells also undergo post-translational histone modifications and RNA-mediated silencing (Reviewed in Tasler, 2009 & Nicolas et al., 2009).

The successful passage of germ cells through meiosis is a unique and highly rigorous process. However, between male and female embryos, the timing of meiosis is different. Male germ cells are restricted from entering meiosis while female germ cells enter meiosis within the embryo. Although the mechanisms for these contrasting behaviors are unclear, it appears that the gonadal cells provide the signal for (or against) meiotic entry (Brennan & Capel, 2004; Ewen & Koopman, 2010). One signal could be retinoic acid (RA) produced in the fetal ovary, which in turn induces Stra8, a key regulator of meiotic entry. In female germ cells destined to become oocytes, mitotic divisions cease and meiotic prophase begins with the correct stimuli (Borum, 1961); eventually oocytes arrest during Meiosis I prior to fetal birth and will only resume meiosis upon receiving hormonal signals during adulthood (Peters, 1970). Meanwhile, male germ cells transition from primordial status to the gonocyte stage, stop proliferating and remain quiescent in the fetal seminiferous tubules until after birth. The post-natal gonocytes then commit to a spermatogonial stem cell (SSC) fate and amplify through self-renewal or enter meiosis to initiate spermatogenesis. In both male and female germ cells, the synaptonemal complex proteins (SCPs) SCP-1, SCP-2 and SCP-3 are critical components of the meiotic machinery during chromosomal segregation (Chuma et al., 2001; Parra et al., 2004; Yuan et al., 2000). The completion of meiosis signals that germ cells have matured into haploid male and female gametes. At this stage, oocytes exclusively express GDF9 and spermatocytes express TEKT1. However, one feature that distinguishes human and mouse germline differentiation is the synchronization of meiotic entry, as in human fetal gonads, one can observe both pre-meiotic and meiotic germ cells in close proximity (Anderson, 2007).

Significant efforts have been made to culture mouse and human PGCs and gonocyes in vitro. In the case of mouse germ cells, the addition of endogenous factors known to affect germ cell development such as BMPs, RA, LIF and Forskolin have produced mixed results in maintaining PGCs in culture. For example, adding LIF enhanced PGC survival but the observations with the use of other factors not as clear. PGCs may also behave erratically in culture (showing low survival rates and non sex-specific behavior) because of the lack of a normal somatic environment (Childs et al., 2008). Studies of PGCs by Shamblott et al., Turnpenny et al. and Tu et al. with fetal human gonocytes resulted in a mixture of cellular phenotypes. Some gonocytes appeared rounded while others took on an elongated or spindly appearance. In addition, they appeared to have different proliferation rates (Shamblott et al., 1998; Turnpenny et al., 2003; Tu et al., 2007). Currently, it is not at all clear that these cells resemble their germ cell counterparts in vivo, but improvements in culture conditions and the cellular microenvironment will certainly help in this regard.

As delineated earlier, mammalian germ cells populate the testis and ovary during development in a incredibly dynamic manner. During early prenatal mice and human embryo development, PGCs migrate to the primitive gonad (gonadal ridge) and associate with Sertoli cells to form primitive testicular cords (Brennan et al., 2004). Within the testicular cords, the primitive germline stem cells (now termed gonocytes) remain in the testis as the gonad differentiates. Eventually, Sertoli cells, peritubular myoid cells and gonocytes form more compact structures known as the seminiferous tubules. When the gonocytes migrate to the periphery of the tubules, they transform into prospermatogonia and then into spermatogonia (Gondos & Hobel, 1971). In the fetal testis, prospermatogonia enter mitotic arrest, a feature observed at E12.5 to E14.5 in the mouse. At the molecular level, during entry into meiosis, both male and female human gonocytes express DAZL proteins and Vasa transcripts and downregulate OCT3/4 expression (Anderson et al., 2007). Interestingly, it is the early migrating germ cells that share similar properties with embryonic stem cells and testicular germ cell tumors (Ezeh et al., 2005). From what is known, the development of gonocytes in the fetal ovary follows a similar path in that OCT3/4 expression is reduced while VASA, Germ Cell Nuclear Antigen (GCNA) and DAZL are expressed (McLaren, 2003). In contrast to the male gonocytes, the female gonocytes receive sex-specific signals from the fetal gonad to enter meiotic prophase. After initiating meiosis, the female gonocytes will develop into primordial follicles and subsequently into primary follicles at puberty. A key difference between mouse and human systems is the timing of primary follicle formation: the mouse achieves this stage at birth while in the human ovary, this occurs at puberty (Bukovsky et al., 2005). There is some speculation whether this difference in follicle development is due to autocrine signals produced from the oocyte itself or from the ovarian environment (Hutt & Albertini, 2007). A plausible hypothesis is that the immediate environment of early germ cells determines whether they are commit to spermatogenesis or oogenesis. The most obvious source of signals are the mesonephros, primitive Sertoli cells in the testis, and primitive Granulosa cells in the ovary.

Timeline of Germline Specification and Germ Cell Marker Expression. A temporal representation of the stages of human and mouse germline differentiation in vivo. At each cellular stage, important molecular and somatic signals controlling that stage are indicated above the diagram. Specific germ cell molecular markers are indicated on left with arrows depicting the duration of development during which they expression has been observed. Genes that are italicized are present in both mouse and human germ cells. At the bottom, an approximate timing of each stage during mouse or human germline development is indicated. Adapted from Schuh-Huerta & Reijo Pera, 2011.

The seminiferous tubule serves as the sperm production center, where approximately 123 x 106 spermatozoa are produced from germ cells daily, or about 1000 sperm/second (Amann et al., 1980; de Rooij, 2009). Developing germ cells are arranged along the basement membrane in a highly ordered sequence and extend into the lumen of the tubule. At the most basal portion of the tubules lies the spermatogonial stem cell (SSC) population, closely associated with the adjacent Sertoli cells. Morphologic analysis of the various germ cells reveals at least 13 recognizable germ cell types in the human testis (Heller & Clermont, 1963, 1964). Each cell type is thought to represent a different step in spermatogenesis. From the least to the most differentiated, they have been named dark type A spermatogonia (Ad); pale type A spermatogonia (Ap); type B spermatogonia (B); preleptotene (R), leptotene (L), zygotene (z) and pachytene primary spermatocytes (p); secondary spermatocytes (II); and Sa, Sb, Sc, Sd1, and Sd2 spermatids (Figure 3). The early, type A spermatogonia are the most interesting germ cell type from a stem cell point of view (de Rooij, 2009). In fact, time-course studies using GFP-based reporters with early type A, type Ad and type Ap spermatogonia in the mouse revealed that the early type A cell has the ability to divide, self-renew, and give rise to the Ad and Ap sun-populations (Nakagawa T. et al., 2007). These observations provide evidence for the existence of SSCs in the testis and their clonal behavior is protypical of other adult stem cells.

It is currently thought that pale type A (Ap) spermatogonia in the basal, stem cell niche of the seminiferous tubule divide at 16-day intervals and differentiate to type B spermatogonia, which then become spermatocytes (Clermont, 1972). The ability of SSCs within the testis stem cell niche to undergo stem cell renewal is governed by several known factors. The growth factor-receptor kit ligand/c-kit receptor system and the niche factor glial cell line-derived neurotrophic factor (GDNF) are important in this process (Oatley & Brinster, 2008). In fact, spermatogenesis in the rat is dependent on c-Kit receptor activity, whereas spermatogonial stem cell renewal may be c-kit independent (Dym, 1994). GDNF appears to provide a significant stimulus to self-renewal of SSCs through receptors for GDNF on SSCs such as c-Ret and GFR-1(Meng et al., 2000). Despite this, our knowledge of other receptor-ligand systems that control human SSC renewal is limited at this point. During spermatogenesis, the cytoplasm between spermatogonial daughter cells remains conjoined after mitosis, forming cytoplasmic bridges between adjacent cells (Ewing et al., 1980). Cytoplasmic bridges are thought to be important for synchronized cellular proliferation, differentiation, and possibly regulation of gene expression. Thus, SSCs and early spermatogonia in the adult testis are critical for germ cell renewal and differentiation into sperm and raise important questions about the source of proliferative and differentiation signals for spermatogenesis. The majority of stages in mouse spermatogenesis delineated above are translatable to the human germ cell development pathway except for differences in the timing of each stage during development.

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