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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 2005, Bristol-Gould 2006, Hutt 2006). Over the past two years, though, emergent data from research labs have come to refute the dogma and support post-natal development of oocytes and follicles in female mouse ovaries (Johnson 2005, 2006). Before continuing we must first introduce the reproductive biology and relevant terms of the mammalian female and the development of the oocytes.
            The beginning of the 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. According to the dogma, it is believed that there are a finite number of primary oocytes, which are all produced at this stage in development and 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. (see 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 2006). The female 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 2005, Bristol-Gould 2006, Hutt 2006, Telfer 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 2006).

FIGURE 1
(A. fallopian tube, B. ovary, C. follicle, D. oocyte)

The Johnson Study
            The studies began by characterizing the kinetics of the decreasing amount of follicles through the life of a mouse. Mice were euthanized at various stages in life. The ovaries were frozen, cross-sectioned and stained to count atretic follicles, which are follicles with oocytes that undergo apoptosis without ever 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 amount of healthy follicles per ovary at different time periods (from 8 days to 120 days). It was observed that there was a marked increase in atresia of follicles by day 30 and another increase by day 40. The resulting data of this initial kinetics experiment showed that without postnatal production of follicles from germline stem cells the mouse would not be reach 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 hour 24 and 48 and eventually cleared all follicles by hour 96. It showed that the atretic follicles were cleared from the ovary by 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 Johnson 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. Their search for the germline stem cells within the ovary were unsuccessful and led them 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 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 see if bone marrow transplantation 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)

In Support
            In reaction to the Johnson lab hypothesis and experimental data, other labs have attempted to recreate the results or try new approaches. Immunolabeling germ cell nuclear antigen (GCNA) and proliferating cell nuclear antigen (PCNA) were performed to count oocyte meiosis and ovarian cell proliferation (Kerr 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 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 2005, Hutt 2006).
            The kinetics of follicle-unloading by the ovary, either 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 Johnson kinetic experiments. This experiment used a different mouse strain than the Johnson lab. (Bristol-Gould 2006)
            In another experiment focusing on the BMT experiments of the Johnson lab, chimerism in the blood was looked at. 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 2006, Hutt 2006)

Direction of Ovarian Stem Cell Research
            The subject, 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 2006, Telfer 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. For example, in the Eggan study, which focused on whether allogenic BM cells would home to the ovary, the strain of mouse was different than the one used in the Johnson study. Also, in regards to the chemotherapy experiment to sterilize the mouse ovary, the Johnson lab did mention very few oocytes post-chemotherapy (Johnson 2005). The BMT resulted in, what the Johnson lab considered a marked increase in oocytes in the ovary (Johnson 2005). 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 conclusions can be made.

Beyond the Scientific Debate
            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 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).
            It has also 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 2005)
            The ovarian recovery found in the Oktay and Herschlag trials are inexplicable and the mechanisms by which they occurred are completely unknown. It is predicted that there are possibly germline stem cells that came from the BMT (Hutt 2006, Oktay 2005). Another possible source of the stem cells are mesenchymal cells in ovarian tunica albuginea (TA), which have been found to differentiate into surface epithelium (Bukovsky 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 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 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

1) Antonin Bukovsky, M. S. a. M. R. C. (2005). "Oogenesis in cultures derived from adult human ovaries." Reproductive Biology and Endocrinology 3(17).

2) Bristol-Gould, S. K., PK.; Selkirk, CG.; Kilen, SM.; Mayo, KE.; Shea, LD.; Woodruff, TK. (2006). "Fate of the initial follicle pool: Empirical and mathematical evidence supporting its sufficiency for adult fertility." Developmental Biology 298(1): 149-154.

3) Byskov, A. G., M. J. Faddy, et al. (2005). "Eggs forever?" Differentiation 73(9-10): 438-46.

4) Eggan, K., S. Jurga, et al. (2006). "Ovulated oocytes in adult mice derive from non-circulating germ cells." Nature 441(7097): 1109-14.

5) Heng, B. C., T. Cao, et al. (2005). ""Waste" follicular aspirate from fertility treatment--a potential source of human germline stem cells?" Stem Cells Dev 14(1): 11-4.

6) Hershlag, A. and M. W. Schuster (2002). "Return of fertility after autologous stem cell transplantation." Fertil Steril 77(2): 419-21.

7) Hutt, K. J. and D. F. Albertini (2006). "Clinical applications and limitations of current ovarian stem cell research: a review." J Exp Clin Assist Reprod 3: 6.

8) Johnson, J., J. Bagley, et al. (2005). "Oocyte generation in adult mammalian ovaries by putative germ cells in bone marrow and peripheral blood." Cell 122(2): 303-15.

9) Johnson, J., J. Canning, et al. (2004). "Germline stem cells and follicular renewal in the postnatal mammalian ovary." Nature 428(6979): 145-50.

10) Johnson, J., M. Skaznik-Wikiel, et al. (2005). "Setting the record straight on data supporting postnatal oogenesis in female mammals." Cell Cycle 4(11): 1471-7.

11) Kayisli, U. A. and E. Seli (2006). "Stem cells and fertility: what does the future hold?" Curr Opin Obstet Gynecol 18(3): 338-43.

12)Kerr, J. B., R. Duckett, et al. (2006). "Quantification of healthy follicles in the neonatal and adult mouse ovary: evidence for maintenance of primordial follicle supply." Reproduction 132(1): 95-109.

13) Kubota, H. and R. L. Brinster (2006). "Technology insight: In vitro culture of spermatogonial stem cells and their potential therapeutic uses." Nat Clin Pract Endocrinol Metab 2(2): 99-108.

14) McLaren, A. (2003). "Primordial germ cells in the mouse." Dev Biol 262(1): 1-15.

15) Oktay, K. H. (2005). "Options for preservation of fertility in women." N Engl J Med 353(13): 1418-20; author reply 1418-20.

16) Oktay, K. (2006). "Spontaneous conceptions and live birth after heterotopic ovarian transplantation: is there a germline stem cell connection?" Hum Reprod 21(6): 1345-8.

17) Oktay, K., E. Buyuk, et al. (2005). "Fertility preservation in breast cancer patients: a prospective controlled comparison of ovarian stimulation with tamoxifen and letrozole for embryo cryopreservation." J Clin Oncol 23(19): 4347-53.

18) Telfer, E. E., R. G. Gosden, et al. (2005). “On regenerating the ovary and generating controversy.” Cell 122(6): 821-2.

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: Steven Nguyen and Rami Rafeh (in alphabetical order)

Teaching Assistant: Katherine Liu