|Existence of Female Germline Progenitors in Doubt - “Experimental evidence showing that no mitotically active female germline progenitors exist in postnatal mouse ovaries”|
Results of a recent study in Nature Medicine (White et al), backed up by other research of the last ten years, indicated that the understanding that oocytes in postnatal mammals cannot renew and their number is fixed (Telfer et al and Zuckerman) may be wrong (Johnson et al, Pacchiarotti et al, White YA et al, Zou et al 2009, Zou et al 2011). Most recently, using antibody-based separation techniques with the germ cell marker Ddx4, two groups have postulated the existence of functional female germline stem cells (FGSCs) and oogonial stem cells (OSCs) in adult mouse and human ovaries (White et al, Zou et al 2009, Zou et al 2011). However for many researchers, questions still remained unanswered about these potentially exciting findings. This led researchers from the laboratory of Kui Liua at the University of Gothenburg, Sweden to use an endogenous genetic approach in mouse to trace the proliferation and differentiation of Ddx4 cells in vitro and in vivo, finding that Ddx4-expressing cells in postnatal mouse ovaries do not proliferate and nor do they contribute to oocytes during de novo folliculogenesis (Zhang and Zheng et al).
The genetic approach utilised a cross between mice bearing Cre driven by Ddx4 regulatory regions (Ddx4-Cre) and mice bearing a Rainbow fluorescence cassette (Rosa26rbw/+). When Cre is activated by Ddx4 promoter activity, enhanced green-fluorescent protein (EGFP) randomly switches to red- (RFP), orange- (OFP) or cyan-(CFP) fluorescent protein allowing Ddx4+ cells (Red, Orange or Cyan) to be distinguished from Ddx4- cells (Green). Initial studies used the injection of EGFP+ ovarian cells containing primordial germ cells (PGCs) from Rosa26rbw/+ to ascertain the existence of female germline progenitors in postnatal mouse ovaries. At both 4 weeks and 8 weeks, EGFP-positive follicles (oocytes and supporting granulosa cells) at different stages of development were observed in the ovaries of recipient females indicating that adult mouse ovaries supported de novo follicular formation. However, no cells from the adult mouse ovaries were seen to contribute to oocytes or granulosa cells during de novo folliculogenesis, (all follicles were GFP+, with no apparent non-GFP cells) suggesting that previously reported germline progenitors were not in fact active; if they existed at all. The chemotherapeutic reagents busulfan and cyclophosphamide are often used in the study of postnatal follicular renewal as they deplete endogenous oocytes and follicles in mouse ovaries. Mice treated with both agents were injected with EGFP+ ovarian cells from Rosa26rbw/+ mice at two weeks with new EGFP+ follicles found at 4 weeks, suggesting the capability of the chemotherapeutically-treated ovary to support de novo folliculogenesis when exogenous support from cell progenitors and germline progenitors is provided. However, again, no proof of cells from the adult mouse ovaries contributing to oocytes or granulosa cells during de novo folliculogenesis was observed, overall suggesting that these drugs do not stimulate adult ovarian cells to contribute oocytes to follicular regeneration.
Next the Rosa26rbw/+;Ddx4-Cre mice were studied and, as expected, germline cells in the mouse ovary (Ddx4+) were found to express RFP, OFP, or CFP, whereas gonadal somatic cells still expressed EGFP, allowing the tracing of possibly proliferative Ddx4+ germline progenitors by following the mitotic divisions of RFP+, OFP+ or CFP+ cells using live cell imaging. Analysis of RFP+ cells from the ovary found that no positive cell underwent any cell divisions and all cells died during the course of prolonged culture, suggesting that RFP+ and therefore Ddx4+ cells did not enter mitosis. Additionally, RFP+ cells could not form colonies further demonstrating the lack of mitotically active cells within this fraction. However, during long term culture EGFP+ (Ddx4-) colonies were discovered which were morphologically similar to those reported for OSCs and could be stably passaged, but did not express pluripotency (Sox2) or germ cell (Oct4, Stella, Ddx4) markers suggesting that these EGFP+ clonal cells were not germline cells. To confirm this, EGFP+ clonal cells from cultured Rosa26rbw/+; Ddx4-Cre ovarian cells were transplanted into wild type mouse ovaries. At 4 weeks, EGFP+ cells were observed in some non-fluorescent follicles, although no RFP+, OFP+ or CFP+ oocytes or granulosa cells could be identified. Further evidence came from a follicular reconstitution system, where EGFP+ ovarian cells from 12.5 dpc Rosa26rbw/+ female mice or EGFP+ clonal cells from cultured Rosa26rbw/+;Ddx4-Cre ovarian cells were mixed with ovarian cells from 14.5 dpc wild type foetuses and transplanted under the kidney capsules of ovariectomized adult wild type females. At 4 weeks, the 12.5-dpc EGFP+ Rosa26rbw/+ ovarian cells were found to contribute to EGFP+ oocytes in the reconstituted follicles, although the EGFP+ clonal cells did not, overall suggesting that these cells, while having a stem cell-like morphology, were not functional female germline progenitors. When EGFP+ clonal cells were maintained for three weeks without passaging, large spherical cells with an oocyte-like morphology appeared. However, these cells still expressed EGFP, and were therefore Ddx4-, and not oocytes or any cells of the germline.
In conclusion, this new study provides strong evidence that no postnatal follicular renewal occurs in mammals, and no mitotically active Ddx4-expressing germline progenitors exist in postnatal mouse ovaries. Additional work is required to fully understand why these studies have produced differing data and further, to fully understand mammalian development and the impact this may have on clinical approaches to fertility treatment.
Johnson J, et al. (2004)
Pacchiarotti J, et al. (2010)
Telfer EE, et al. (2005)
White YA, et al. (2012)
Zhang H, Zheng W, et al. (2012)
Zou K, et al. (2009)
Zou K, et al. (2011)
Zuckerman S (1951)
STEM CELLS correspondent Stuart P. Atkinson reports on those studies appearing in current journals that are destined to make an impact on stem cell research and clinical studies.
Original study from PNAS.