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OVARIAN STEM CELLS
A SCIENTIFIC REVIEW
Introduction

In the field of developmental and reproductive biology there has been a longstanding dogma that mammalian females produce a finite number of oocytes in the prenatal phase of life, and this production ceases after birth (Bukovsky et al; 2005; Bristol-Gould et al, 2006; Hutt and Albertini, 2006). Over the past several years, however, emergent data has refuted the dogma and supported post-natal development of oocytes and follicles in female mouse ovaries (Johnson et al, 2004; Johnson et al, 2005b). Before continuing we will briefly introduce reproductive biology and relevant terms of the mammalian female and discuss the development of the oocytes.


Development of the female reproductive system starts with the migration of primordial germ cells (PGCs) along the hindgut of the fetus to the gonadal ridge; the cells along the gonadal ridge are mostly mesenchymal cells. When arriving at the gonadal ridge, the PGCs are now considered oogonia (McLaren, 2003). The oogonia continue to divide mitotically until meiosis is initiated. The oogonia undergo the meiosis I and are arrested in this phase when complete. They are now referred to as primary oocytes. It has been traditionally believed that there are a finite number of primary oocytes, all of which are produced at this stage in development that will last the individual organism throughout its reproductive life until the menopause stage.


The continuation of the oocyte’s life is initiated in the puberty stage in human females, which is under control of sex hormones. During each menstrual cycle, a primary oocyte is engulfed by a surrounding Graffian follicle through a process called folliculogenesis. The follicle matures as the primary oocyte becomes a secondary oocyte and then is eventually released from the follicle into the fallopian tube where it is viable for fertilization by sperm (Figure 1).


The female process is contrasting to the male process of gametogenesis. Male gonads have unipotent germline stem cells called spermatogonium that can either duplicate by mitosis or undergo meiosis to ultimately give rise to four sperm that have the potential to fertilize an egg. Due to the presence of these germ cells the male may be reproductively viable as long as the germ cells persist in duplication and differentiation into sperm (Kubota and Brinster, 2006). The female mammalian reproductive time-frame, on the other hand, is limited by the finite number of primary oocytes produced prenatally.


The convention that most mammals do not continue producing oocytes during adulthood has been upheld for over half a century (Bukovsky et al, 2005; Bristol-Gould et al, 2006; Hutt and Albertini, 2006; Telfer et al, 2005). Reports now are beginning to support the production of follicles and oocytes in animal models during adulthood. Among the first labs to produce data against the dogma was the Tilly lab at Harvard University, starting in 2002 (Johnson 2005a).



FIGURE 1
(A. fallopian tube, B. ovary, C. follicle, D. oocyte)
The Johnson Study (Conducted in the Tilly lab):

The Tilly lab began by characterizing the kinetics of decreasing follicle numbers throughout the life of a female mouse. Mice were euthanized at various stages in life. The ovaries were frozen, cross-sectioned and stained to count atretic follicles, which are follicles containing oocytes that have undergone apoptosis without being released during a menstrual cycle, and healthy follicles. The estimated rate of follicle loss plus the loss of follicles due to atresia was compared to the number of healthy follicles per ovary at various time periods (ranging from 8 to 120 days). Atresia of follicles was markedly increased at two time points: once by day 30 and again by day 40. Thus, without postnatal production of follicles from germline stem cells the mouse could not account for the number of follicles required for its expected reproductive lifespan (Johnson et al., 2004).

To confirm that the atretic follicles were not taking longer than expected to be cleared from the ovary and therefore being double-counted in the original experiment, the mice were exposed to 5-bromodeoxyuridine (BrdU). BrdU is a known agent that effectively initiates follicle atresia. The BrdU experiment was successful in widespread atresia between hours 24 and 48 and eventually cleared all follicles by hour 96. This showed that the atretic follicles were cleared from the ovary within three days. The original kinetic experiment was repeated in other strains of mice to confirm that it was not a strain-specific phenomenon. The data was consistent and, interestingly, a specific mouse strain showed a marked increase in the number of healthy follicles on day 42 compared to day 4 despite an increase in follicle atresia (Johnson et al., 2004).


The following year, the Tilly lab continued with experimentation to further explore this phenomenon and determine the validity of their previous results. They hypothesized that there was a germline stem cell that was providing the post-natal production of follicles to support the reproductive lifespan in a mouse. A search for the germline stem cells within the ovary were unsuccessful and led Tilly to search extraembryonically. They searched by targeting specific genes that are known to be involved in folliculogenesis and restricted to germ cells such as Oct4, Mvh, Dazl, Stella and Fragilis. Positive signals for these genes were found on adult mouse bone marrow (BM) cells. Subsequent experiments showed a correlation between increased levels of Mvh, which regulate Oct4 levels, in mouse bone marrow and the peak of the mouse menstrual cycle. At this peak there were also increased numbers of oocytes and follicles. In vivo studies were conducted to determine if bone marrow transplantation (BMT) or peripheral blood cell transfusion could generate oocytes in a sterilized mouse and both resulted in recovery of oocytes in the sterilized ovary (Johnson et al., 2005).

Recently, the Tilly lab has furthered this work by demonstrating the ability of bone marrow transplant (BMT) to rescue ovarian function in recipients who have undergone chemotherapy. (Lee et al, 2007). An additional function of this study was to dispel the claim made by critics that claimed ovulated eggs found in ovaries (report discussed above), were not BM-derived oocytes, but were instead a yet unidentified immune cell.  The methods of this study proceeded as follows. Female mice underwent chemotherapy, which prematurely terminated primary oocytes, eliminating the ability to become pregnant, followed by BMT from recipients of a different genotype.  The mice were housed with male mice and the number of pregnancies as well as the genotypes of offsprings was recorded. Donor-derived oocytes were showed to be generating in the recipients ovary after transplant and were clearly shown to be oocytes and not immune cells. However, all offspring of post-treatment pregnancies were of the recipient’s germline, not the donors as might be expected. This interesting data presents a somewhat ambiguous view of oocyte recovery, but provide clear evidence that the addition of BM-derived stem cells allows for recovery of primary oocytes using a post-natal process (Lee et al, 2007). In conjunction with other data obtained by the Tilly lab, it is suggested that the development of primary oocytes can occur post-nataly, with help for some component of the bone marrow.


In Support

In response to the Tilly lab hypothesis and experimental data, other labs have attempted to recreate the results and tried new approaches. In one study, immunolabeling of germ cell nuclear antigen (GCNA) and proliferating cell nuclear antigen (PCNA) were performed to count oocyte meiosis and ovarian cell proliferation (Kerr et al, 2006). Using the same strain of mouse as the Johnson experiments, the mean number of healthy follicles was not largely contrasting between day 100 and day 7. These experiments concluded that there was postnatal renew of follicles in adult mouse ovaries, but did not show evidence for germline stem cells (Kerr et al, 2006).


In Disagreement


The current literature on ovarian stem cells shows more evidence in disagreement with the Johnson study than evidence in support of it. The BrdU experiment was repeated and showed no significant DNA synthesis in a range of ages in the mouse model (Byskov and Faddy, 2005; Hutt and Albertini, 2006).


The kinetics of follicle-unloading by the ovary, by atresia or ovulation, were reconsidered. A complex mathematical formula was derived and applied to two models: an initial pool of primordial follicles and the stem cell model. The resulting data showed support for the dogma of a finite number of oocytes determined during the prenatal stage of life and did not confirm with the results from the Tilly kinetic experiments. However, it is important to note that this experiment used a different mouse strain than the Johnson lab. (Bristol-Gould et al, 2006).


 Another experiment conducted in the lab of Amy Wagner et al, further examined the bone marrow transplantation experiments where the authors looked for chimerism in the blood. Distinguishing between the chimeric sets of circulation blood there was evidence of engraftment of donor BM cells, labeled with green fluorescence protein (GFP), into the host’s BM, but there was no cross-engraftment of GFP-labeled BM cells into the host’s ovary. Recovery of ovaries sterilized by chemotherapy was also looked at and in this experiment it was found that the chemotherapy did not completely ablate all oocytes in the ovary. Both of these findings undermine the Johnson experiments. (Eggan  et al, 2006; Hutt and Albertini, 2006). However, since the assay utilized in this experiment was different than that used in the Tilly experiments, this comparison difficult to make. The Tilly group data looked primarily at the formation of immature oocytes, whereas Wagners looked solely at ovulation as a measure of oocyte formation, which could potentially lead to differences in the interpretation of the results. Additionally, the two studies utilized different mouse strains, thereby making genetic differences between the strains a potential factor contributing to differences in the data. However, the results of this study contribute to the continuation of this scientific debate by contrasting with those obtained by the Tilly group.


Direction of Ovarian Stem Cell Research


The subject of oogenesis, over the last two years, has turned into a heated scientific debate where some of the debate has been fought with words, but most has been fought with scientific data (Johnson 2005b; Telfer et al, 2005). Whether the fifty-year old dogma of no post-natal oogenesis will remain upheld or the theory of an ovarian germline stem cell confirmed can only be answered by scientific research (Kayisli 2006; Hutt 2005). In the research that has already been done, there exist many gaps that must be filled in. Although data have confirmed for and against the existence of ovarian germline stem cells, the experiments have not been consistent with each other, as outlined in the differences discussed above. Additionally, regarding the chemotherapy experiment to sterilize the mouse ovary conducted by the Tilly lab, very few oocytes were found after chemotherapy (Johnson et al, 2005a). The BMT resulted in, what the Tilly lab considered a marked increase in oocytes in the ovary (Johnson et al, 2005a). Without a source of more oocytes and follicles, how is the increase in number of oocytes post-BMT in the Johnson study explained? These discrepancies and others need to be resolved and explained before any conclusion.


Beyond the Scientific Debate


Examples of recovering viable oocytes following chemotherapy have been documented in human cases. In 2002, a retrospective study followed four women, two with Hodgkin’s disease and two with advanced breast carcinoma. After BMT and estrogen treatment, chemotherapy-induced menopause was reversed and all four women were viable and became pregnant. (Herschlag and Schuster, 2002). In another case of Hodgkins disease, a woman had experienced two years of menopause as a result of sterilizing chemotherapy. After BMT, the patient experienced recovery of her existing ovary and conceived twice. (Oktay, 2006). More recently, an similar report further confirmed the benefits of stem cell transplant and hormone therapy following chemotherapy, describing recovery of menstruation and subsequent pregnancy in several women that have undergone premature ovarian failure brought on by common chemotherapeutic methods (Liu and Malhotra, 2007). The ovarian recovery found in these reports is inexplicable and the mechanisms by which they occurred are completely unknown. It is predicted that germline stem cells that potentially came from the BMT (Hutt and Albertini, 2006; Oktay et al, 2005).  Taken together, these studies may eventually lead to work that will further support the data obtained in the Tilly lab, and more importantly, extend the implications of the research to humans.


Another possible source of the stem cells is mesenchymal cells in ovarian tunica albuginea (TA), which have been found to differentiate into surface epithelium (Bukovsky et al, 2005).  It has been found that extracting ovarian surface epithelium (OSE) from human ovaries can serve as a source of oocytes and granulosa cells. When the OSE cells are placed in a medium of phenol red (PhR), the cells differentiate into cells of a oocyte phenotype. In the absence of PhR, there is differentiation into granulosa phenotypes as well as epithelial, neural and mesenchymal type cells. (Bukovsky et al, 2005).


In the United States, there are approximately 180,000 patients a year that develop breast cancer and 15% of the women are of reproductive age. Research that helps explain the phenomenon in the above cases could pave the way to new therapies that can help maintain a woman’s reproductive capacity and quality of life despite having undergone chemotherapy to treat cancer. As an example of therapy, research has shown that the wasted follicular aspirate at fertility clinics can be saved for it is a good source of immature ovarian follicles (Heng et al, 2005). These follicles can then be cultivated with mature oocytes to enhance fertility treatment, and the follicles have also been found to be a potential stem cell niche (Heng et al, 2005).With successful collaboration and sharing of findings, the field of reproductive biology and fertility treatment can develop exciting new strategies to help treat thousands of infertile women that underwent chemotherapy or suffered from a disease affecting their reproductive capacity.


REFERENCES

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Eggan K, Jurga S, et al. (2006) Ovulated oocytes in adult mice derive from non-circulating germ cells. Nature 441: 1109-14.
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Acknowledgments

This review was prepared by the following graduate students in the Stem Cell Biology Class, Graduate School of Biomedical Sciences, University of Medicine and Dentistry of New Jersey:
Fall 2006: Steven Nguyen and Rami Rafeh (in alphabetical order)
Teaching Assistant: Katherine Liu
Fall 2007: Modified by Joseph Capecci and Jun Zhao (in alphabetical order)
Teaching Assistant: Katherine Liu