11.1. Repowering the ovary
In a recent article in Science–Business eXchange (SciBX) of the Nature Publishing Group , the Senior Editor Tracey Baas indicated:
“Up until the 1990s, the central dogma of reproductive biology was that female mammals have a restricted capacity for generating oocytes before birth, and once born the ovaries cannot renew egg cells that die because of aging or disease. Consequently, infertility resulting from oocyte loss had been considered irreversible.
However, multiple papers now cast doubt on that belief through the identification of a population of stem cells that give rise to functional oocytes.
First, Antonin Bukovsky and colleagues at The University of Tennessee Knoxville published in the American Journal of Reproductive Immunology in 1995 that a subpopulation of human germline stem cells, now known as oogonial stem cells (OSCs), could be collected from the ovaries of women undergoing surgery and used to generate what were perceived as oocytes in cell culture, based on detection of oocyte markers [7, 11].
Almost a decade later, Jonathan Tilly and colleagues at Massachusetts General Hospital (MGH) and Harvard Medical School (HMS) produced multiple datasets that ran counter to the belief that germline stem cells disappear from ovaries at birth .
In 2009, Ji Wu and colleagues at Shanghai Jiao Tong University used a unique biomarker of murine OSCs—dubbed DEAD box polypeptide 4 (Ddx4)—to isolate OSCs from adult mouse ovaries. The team used the marker to purify OSCs, which were transplanted into ovaries of infertile mice to generate functional oocytes capable of producing offspring .
Those data were published in Nature Cell Biology. The open question was whether OSCs purified from humans had a similar ability to generate functional ovarian follicles.
To answer that question, researchers at MGH and HMS started by modifying Wu’s Ddx4-based purification protocol to improve its selectivity for OSCs. The team, which was once again led by Tilly, used fluorescence-activated cell sorting (FACS), which is less likely to be contaminated by oocytes than Wu’s technique of magnetic bead sorting.
His group isolated DDX4-positive cells from murine ovarian tissue as well as from human ovarian tissue derived from whole ovaries of reproductive-age women undergoing elective surgery. In vitro, both human and murine DDX4-positive cells expressed primitive germline markers but not oocyte markers, suggesting the procedure had indeed purified OSCs. To determine whether the OSCs could differentiate into viable oocytes, the team cultured the cells on mouse embryo fibroblasts used as feeder cells, which supplied a cellular matrix upon which the stem cells grew. The human and mouse OSCs spontaneously differentiated into oocytes, as assessed by morphology, gene expression patterns and progression through meiosis.
The next step was to look at whether the OSCs could generate oocytes in vivo. To do so, the team engineered the mouse and human OSCs to express GFP, which made it possible to visualize and track the cells. When GFP-expressing mouse OSCs were injected into the ovaries of mice, primary ovarian follicles developed that contained GFP-positive oocytes. Moreover, those oocytes led to the development of GFP expressing mouse embryos following fertilization.
Finally, GFP-expressing human OSCs were injected into human ovarian tissue and transplanted into immunodeficient mice. The result was primary ovarian follicles that contained GFP-expressing oocytes.
That suggested OSCs might indeed give rise to functional oocytes if transplanted into humans.
Results were published in Nature Medicine . The team also included researchers from Saitama Medical University. Tilly and colleagues are now optimizing the conditions for production of human oocytes in vitro and have started studies to assess whether OSCs in nonhuman primates can generate functional oocytes for IVF.
Bukovsky, professor of reproductive biology associated with the Institute of Biotechnology of the Academy of Sciences of the Czech Republic, said he is most interested in seeing the work translated from ovarian tissue obtained from reproductive-aged women to women with ovarian failure who are unable to produce follicle cells or oocytes. “Tilly’s work supports the existing idea that purified germ cells transplanted into mouse ovaries will utilize existing immature follicle cells to produce new follicles and that the survival and function of oocytes in vivo requires interaction with these immature follicle cells,” he said. “It will be very important that the team reproduce the experiments using ovarian tissue biopsies obtained from reproductive-aged women and from women with premature ovarian failure. I would very much like to see the mechanistic details”.
Questions aside, Oktay said the new study is “a game changer because it increases the options available for obtaining viable eggs. When it comes to fertility restoration, it is all about options”.
Wu, professor of molecular reproduction and stem cell biology at the Bio-X Center of Shanghai Jiao Tong University, thinks there are still unaddressed technical challenges. “It is very satisfying to see our protocol optimized and fine-tuned to obtain and differentiate human female germline stem cells into oocytes, but the team’s culturing conditions will have to be optimized to avoid use of animal components, and mouse feeder cells will need to be replaced with either human feeder cells or a nonfeeder culturing system,” Wu told SciBX ”.
For complete SciBX article see Additional file 2
, Supplemental material.
The article published in Nature Medicine  indicates that primitive germ cells purified from the cortex of functional adult human ovaries form new ovarian follicles when injected into human ovarian cortical biopsies and xeno-transplanted into immunodeficient NOD-SCID mice. The article is important, since it is the first confirmation of former observations on neo-oogenesis and follicular renewal in adult human ovaries published in 1995  and expanded thereafter [7, 11, 68, 94, 134, 135, 139, 192]. In addition, neo-oogenesis from secondary germ cells is already present in human fetal ovaries, during the second trimester of pregnancy [9, 100].
It has to be noted, that the earlier rarely used term “oogonial stem cells” [193–195], now considered as a new term , is doubtful, since such cells are in reality well known germ cells capable to proliferate, originating in fetal and adult human ovaries by asymmetric division of OSC progenitor cells (see Chapters 4-6), and not persisting in adult ovaries from the fetal period.
In a recent commentary , Telfer and Albertini indicated:
“Work of White et al.  represents an advance that has the potential to change the nature of future infertility treatments, although many practical and conceptual obstacles remain before the clinical utility of their methods can be realized. Much effort will be required to improve the efficiency of isolation and transformation of OSCs into oocytes, as the number of OSCs that went on to form follicle-enclosed oocytes in the study by White et al. was small.”
Even in the case of the mouse studies, very few GFP-positive oocytes were shown to fertilize and undergo even minimal embryogenesis, with many of these oocytes clearly arresting at the preblastocyst stage. Further, a more detailed characterization of genetic integrity (euploidy and the appropriate retention of epigenetic marks) and other hallmarks of oocyte quality (such as meiotic and developmental competence) are required before any clinical application of these techniques can be considered.
Given the response to earlier work from this laboratory, questions will undoubtedly linger as to whether these cells are only activated in vitro or whether they indeed contribute to de novo neo-oogenesis in vivo. Further research will be required before these issues can be fully resolved. Nonetheless, the findings of this study will change the tone of future discourse on the subject toward measured enthusiasm and, most importantly, will prompt speculation and tempered progress into what remains a major obstacle in the treatment of various forms of human infertility ”.
In an additional commentary  Oatley and Hunt indicated:
“For OSCs, doubt will persist until clear evidence is provided that they give rise to genetically normal, developmentally competent eggs. In the meantime, skeptics are plagued by several nagging questions: What do these cells do in the ovary? Where do they come from? And, most importantly, if they can and do give rise to viable eggs in the adult ovary, why is female reproduction of such limited duration? .”