Sci. Aging Knowl. Environ., 10 March 2004
Vol. 2004, Issue 10, p. pe11
[DOI: 10.1126/sageke.2004.10.pe11]

PERSPECTIVES

Can the Clock Be Turned Back on Ovarian Aging?

Patricia B. Hoyer

Patricia B. Hoyer is in the Department of Physiology at the University of Arizona, Tucson, AZ 85721, USA. E-mail: hoyer{at}u.arizona.edu

http://sageke.sciencemag.org/cgi/content/full/2004/10/pe11

Key Words: germ cells • oocyte • ovary • follicle • menopause • fertility

Introduction

The ovary, the basic unit of female reproduction, is complex and dynamic in structure. The oocyte is the germ cell within the ovary, and it can be ovulated and fertilized for the generation of progeny. During fetal ovarian development, the germ cell pool develops from mitotic proliferation of oogonia, which are transformed into oocytes when they stop dividing and begin to undergo meiosis. Primordial follicles are formed once the oocytes have become surrounded by single layers of somatic pre-granulosa cells [reviewed in (1); see also Walter Perspective for a description of oocyte development]. It has long been assumed that all mammalian females are born with a preset number of ovarian follicles, because the oocyte in a primordial follicle is arrested in the prophase of the first meiotic division. This means that during the reproductive life span, once the cohort of primordial follicles formed during fetal development is depleted by ovulation or cell death by atresia (apoptosis), ovarian failure results. This is the basic process by which menopause is assumed to result in women living into the fifth decade of life.

New Findings Challenge Old Beliefs About Postnatal Oocyte Production

However, a recent article by Johnson et al. (2) challenges these long-held beliefs about ovarian function. This study presents findings that collectively suggest that the postnatal murine ovary contains oocytes with the capacity for mitotic division. The authors first considered this possibility using a comparison of the number of follicles lost by atresia versus the number of healthy follicles present in the ovaries of juvenile and young adult mice. Johnson et al. observed that although the number of healthy follicles decreases over time, the extent of loss is much less than would be predicted by the high degree of follicle loss by atresia, suggesting the possibility of follicle renewal.

This hypothesis was supported by the identification and immunohistochemical evaluation of presumptive germ cells in ovaries from young postnatal mice. These cells displayed staining for both the mouse Vasa homolog (MVH), a protein expressed exclusively in germ cells (3), and 5-bromodeoxyuridine (BrdU, a thymidine analog that is incorporated into DNA during S phase, indicating an actively dividing cell), which had been injected into the mice 1 hour before ovaries were collected (Fig. 1). The double staining was seen in cells associated with the surface epithelium that were not encircled by granulosa cells. Thus, the proliferating cells had not become incorporated into follicular structures. It is known that the functional competence of an ovarian follicle requires the oocyte to be surrounded by a complete layer of viable granulosa cells (4). The double-labeled oocytes, then, would not be capable of follicular development or eventual ovulation. However, in another experiment, the authors also demonstrated migration of oocytes with incorporation into a follicular structure. Transgenic mice constitutively expressing green fluorescent protein (GFP) were used as a host for an ovarian graft from sibling wild-type (WT) mice. WT ovarian fragments were grafted onto one-half of the host's ovary. When collected and visualized by confocal microscopy, primary follicles in the grafted WT ovarian region contained GFP-expressing oocytes surrounded by non-GFP-expressing granulosa cells. This result suggests that oocytes from the GFP region of the host ovary have migrated into the grafted region and become incorporated into follicles. Thus, these results indicate that the capacity for follicular formation is retained in the adult ovary.



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Fig. 1. Germ cell proliferation in juvenile and young adult mouse ovaries. (A) Presumptive germline stem cells (GSCs, arrowheads) in the surface epithelium. (B) Immunohistochemical detection of MVH (brown reaction product) in presumptive GSCs (arrowheads). (C) Presumptive GSC undergoing mitosis across the surface epithelium. (D and E) Dual immunostaining for BrdU (red) and MVH (green) in juvenile and young adult mouse ovaries. (F) Hematoxylin and eosin staining of the section shown in (E), highlighting the dividing germ cell. (G and H) Chromatin morphology (propidium iodide counterstaining, violet) within MVH-positive cells (green) shows a germ cell in prometaphase (G) and a germ cell in metaphase [(H); sister chromatids are highlighted by arrowheads]. [Figure and legend are reproduced from (2) with permission from Nature]

 
Another experiment proposed to support the hypothesis was one in which mice were injected with the germ cell toxicant busulfan. After two injections with busulfan, young adult mice exhibited a dramatic loss of healthy primordial follicles, although all other aspects of ovarian morphology were unaffected. The number of atretic primordial follicles was minimally and transiently increased in these animals, which the authors explain as being caused by the cytotoxic effects of busulfan rather than by induction of atresia (apoptosis). The interpretation of these results is not clearly discussed; however, the implications are that (i) the great degree of follicle loss resulted from the targeting of mitotically active germ cells by busulfan, and (ii) the observed 20-day delay in the loss of primordial follicles after the injection of busulfan could not result from an immediate cytotoxicity of this drug but rather is due to the maintenance of the primordial follicle pool by replacement through oocyte replication and follicle formation. In other words, busulfan did not kill follicles that were already present on the day of injection, but affected dividing cells only.

Although the authors' interpretation might be a correct explanation for those observations, there are other factors that must be considered. For example, previous studies using busulfan have demonstrated that it is toxic only to mitotically dividing germ cells in rats exposed in utero on gestational day 12 [to a dose of 10 mg per kg of body mass (mg/kg)] (5). Furthermore, a similar effect has been shown to be time- and dose-dependent over a range of 1 to 20 mg/kg (6). However, when given in larger doses (40 mg/kg) to postnatal mice, busulfan was also observed to cause complete oocyte loss 2 to 17 weeks after exposure (7). The delay in follicle depletion probably occurred as a result of selective targeting of small pre-antral follicles--those in the immature stages of development. These findings demonstrate that the results of exposure to busulfan can be affected by the doses given. A relation is known to exist between the dose of a xenobiotic chemical, the duration of exposure, and the type of cell death that follows (8). As a specific example, there was a similar degree of toxicity to primordial follicles within 2 days in mice given a single high dose of the polycyclic aromatic hydrocarbon 7,8-dimethyl benzanthracene (9), as occurred in mice given much lower doses in the form of 15 daily injections (10). Thus, without consideration of the dosing regimen followed for a given xenobiotic agent, conclusions about the timing and mechanisms of ovotoxicity should be tempered with caution.

Implications of Follicle Renewal in the Postnatal Ovary

At any rate, the findings reported in the paper by Johnson et al. (2) are provocative and lend themselves to more in-depth investigations into the potential for primordial follicle proliferation in postnatal mammalian ovaries. If proven to be the case, this finding could have profound implications, not only because it would alter the historical perspective with which ovarian physiology has been viewed, but also for the potential of developing methods to delay the onset of menopause in women. Such an ability would serve two potentially beneficial purposes. First, it could prolong the childbearing years. In recent years in Western societies, there has been an increase in the number of working women and a tendency for women to postpone the start of a family. These trends have heightened an awareness of reproductive life span. Thus, the ability to bear children at a later age might be attractive to career-minded women. However, in simply prolonging ovarian life span, the effects of aging on oocyte quality must also be taken into account. A recent study examining ultrastructural changes in human ovaries reported that resting follicles in older women showed evidence of greater oxidative damage than those in younger women (11). The authors concluded that this in part explains the reduced quality of oocytes in women in the fourth decade of life.

The other potential benefit of prolonging reproductive life span in women would be to delay the onset of menopause. In the United States, with the average age of menopause at 51 years, and the average life span of women at 82, a woman can expect to spend 30% of her life in the postmenopausal period (see Holmes Perspective). Many health risks are known to be associated with menopause. These include, among others, osteoporosis, cardiovascular disease, and Alzheimer's disease (12-15) (see "Detangling Alzheimer's Disease" and "More Than a Hot Flash"). As a result of findings of the Women's Health Initiative study announced in 2002, the advisability of using hormone replacement therapy (HRT) to reduce the incidence of those diseases in postmenopausal women is in question (16) (see "Weathering the HRT Storm"). A recently developed follicle-depleted, ovary-intact mouse model for designing menopause-related studies (17) promises to help resolve the HRT issue; however, a better solution would be to acquire the ability to delay the onset of natural menopause. Thus, the potential of being able to manipulate a woman's ovaries to replenish the supply of primordial follicles lost throughout her lifetime would be a particularly attractive prospect looming on the horizon. The findings reported by Johnson et al. (2) are suggestive of such a potential.


March 10, 2004
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Citation: P. B. Hoyer, Can the Clock Be Turned Back on Ovarian Aging? Sci. Aging Knowl. Environ. 2004 (10), pe11 (2004).








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