A new tool to generate transgenic rats using female germline stem cells from post-natal ovaries.

Primordial germ cells (PGCs) are the precursors of reproductive gametes. They were first identified in the posterior region of embryos at the angle between the allantois and the wall of the yolk sac, and were shown to subsequently migrate into the forming gonadal ridges in early mouse and human embryos of both sexes (Eddy et al., 1981; Bendel-Stenzel et al., 1998). PGCs will eventually differentiate into oogonia/oocytes in the female mammals. At birth, ovaries are filled with primordial follicles, which are differentiated from PGCs/oogonia (De Felici, 2010; De Felici and Barrios, 2013). The field of reproductive biology has maintained a long-standing dogma for decades, stating that a non-renewable pool of oocytecontaining follicles is established in female mammals at birth. This is perceived tobedifferent fromthe renewable spermpool in the testis of adult males, where spermatogonial stem cells can proliferate and produce sperm throughout the lifetime of the male (de Rooij and Kramer, 1968). It is generally regarded that the germcell generation inmammalian females ceases prior to birth, and that female mammals lose the capability to replenish or regenerate their oocyte reserve, resulting in a progressive and irreversible decrease in follicle numbers until the pool is exhausted at middle age (Faddy et al., 1992; Tilly et al., 2009; Tilly and Telfer, 2009). In 2004, this long-held concept was challenged by a study from Jonathan Tilly’s group reporting the presence of ovarian germline cells, which had germ cell morphology, mitotic activity and the expression of MVH (mouse vasa homolog), a conserved germlinespecificmarker. These cells were designated as putative female germline stemcells (FGSCs) and identified in or proximal to the surface epithelium of juvenile and adultmouse ovaries ( Johnson et al., 2004). Since then, the existence of FGSCs in the post-natal ovary has been much debated. In 2004, Roger Gosden wrote a commentary ‘Germline stem cells in the post-natal ovary: is the ovary more like a testis?’ (Gosden, 2004) and in the following year, Telfer et al. responded with an article ‘On regenerating the ovary and generating controversy’ (Telfer et al., 2005). The subsequent experiments from the Tilly group indicated a possible extra-ovarian origin of FGSCs in the adult mouse from circulating bone marrow or peripheral blood cells ( Johnson et al., 2005). Gradually there have been more studies in different species from different laboratories to add to the above findings. In 2006, Eggan et al. demonstrated that the circulating germ cells from bone marrow or blood cells failed to generate oocytes in adult mice after induction (Eggan et al., 2006). However, in 2009, Ji Wu and colleagues published a study showing that mouse offspring could be produced using an FGSC stem cell line derived from neonatal or adult mouse ovaries (Zou et al., 2009). This was the first publication reporting that the proliferative FGSCs can be purified from the post-natal ovary by using immunomagnetic sorting with antibodies against MVH. FGSC culture can be maintained in vitro formonths; furthermore, these FGSCs can restore oogenesis after transplantation into the ovaries of chemotherapy-sterilized recipients and produce fertile offspring. Although these findings do not confirm that oogenesis can occur in adult humans under physiological conditions, they provide strong evidence for the existence of FGSCs in post-natal mouse ovaries, and have attracted a substantial amount of interest from scientists in the field of reproductive biology (Normile, 2009; Tilly andTelfer, 2009).Recently, other groupshaveexploredhowtoproliferate and differentiate FGSCs from the post-natal mouse ovaries (Pacchiarotti et al., 2010; Hu et al., 2012). All these findings from murine models have stimulated interest in using FGSCs for future clinical application, and several studies of FSGCs in human ovaries have emerged (Liu et al., 2007; Virant-Klun et al., 2008, 2009; Byskov et al., 2011). Some of them claimed no presence of FGSCs in post-natal human ovaries. However, in 2012, White et al. from the Tilly group revived interest for clinical FGSC application by their observation that mitotically active germ cells purified from ovaries of reproductive-age women were able to form oocytes (White et al., 2012). The morphology of the FGSCs from this study is different from that shown by Virant-Klun et al. (2008, 2009). All these experimental results from post-natal ovaries in human have raised the prospect of using FGSCs to combat aging and female-related reproductive diseases. However, in 2012, a study from Kui Liu’s group provided contrary evidence that no mitotically active female germline progenitors exist in post-natal mouse ovaries (Zhang et al., 2012). In this study, unlike most of the above studies, which used immunomagnetic or FACS sorting with Vasa or other germ cell markers to isolate putative FGSCs (Zou et al., 2009; White et al., 2012), Zhang et al. took an endogenous genetic approach by generating a multiple fluorescent Rosa26rbw/+; Ddx4-Cre germline reporter mouse model for in vivo and in vitro tracing of the development of FGSC lineage. Through live cell imaging and de novo folliculogenesis