Renewed debate overpostnatal oogenesis in themammalian ovaryChuck Greenfeld1 and Jodi A. Flaws2*

SummaryThe central dogma of female reproductive biology haslong held that oogenesis ceases prior to birth in mam-mals. During the first half of the last century, there wasmuch debate about whether this was the case or whetheroogenesis continued in the postnatal ovary. A report in1951 effectively put an end to this debate and laid thefoundation for the dogma. A new paper by Johnson et al.(2004)(1) resurrects the debate over whether postnataloogenesis occurs in the mammalian ovary. If confirmed,this would have tremendous impact on issues related tofemale fertility and reproductive longevity. BioEssays26:829–832, 2004. � 2004 Wiley Periodicals, Inc.

Introduction

The idea that oogenesis, the production of ova, occurs in the

adult mammalian ovary was proposed as early as themid 19th

century, andwas a source of debate throughout the first half of

the 20th century. It was a commonly held belief throughout the

first half of the 1900s that the ova initially present in theovary at

birth, the primitive ova, degenerated prior to sexual maturity

and were replaced by new ova, the definitive ova.(2–4) The

most commonly held view was that definitive ova arise from

proliferation of the ovarian surface epithelium,(4–7) though

some investigators proposed that they arose from differentia-

tion of somatic cells.(3) It was alleged that postnatal oogenesis

occurs in a variety of mammalian species, including mice,(2,6)

rats,(4,5,7) cats(4) and humans.(3)

In a comprehensive review, however, Zuckerman (1951)(8)

effectively refuted all of the supportive evidence that postnatal

oogenesis occurred in the mammalian ovary. Zuckerman(8)

argued that the majority of studies supporting the idea of

postnatal oogenesis were ‘‘inadequate and inexact’’ because

they relied on histological evidence for the demonstration of a

dynamic process for which there were no distinct markers. In

addition, Zuckerman(8) argued that quantitative studies of

changes in follicle numbers were inaccurate because they

often ignored primordial follicles, the quiescent, most abun-

dant follicle type. Zuckerman(8) highlighted, among other

things, the fact that removal of the surface epithelium by

chemical or physical means did not affect follicle numbers.

This is contrary to what would be expected if new ova arose

from the surface epithelium. Additionally, Zuckerman(8)

pointed out that no study had observed meiotic prophase, or

more specifically synapsis, occurring in ova within the

postnatal ovary. Most supporters of the postnatal oogenesis

hypothesis overlooked this fact, believing that meiosis was

either unnecessary for the production of the definitive ova, or

that the process had been modified from that observed in the

primitive ova. Zuckerman(8) concluded that the body of

evidence supporting the concept of postnatal oogenesis was

unsound, and that it is more likely that oogenesis ceases prior

to or just after birth. This notion grew to be widely accepted,

and became the central dogma of mammalian female

reproductive biology.

The dogma is challenged

The idea that oogenesis continues in the mammalian adult

ovary has been resurrected in a new report by Johnson et al.

(2004).(1) The authors of this manuscript propose that germ-

line stem cells (GSCs) exist in the mouse ovary and that they

serve as a source of new oocytes in the adult. The evidence

presented in support of this hypothesis is: (1) quantitation of

healthy and atretic (dying) immature follicles (i.e. primordial,

primary, and small preantral follicles), (2) evidence of

proliferating cells of apparent germ lineage in adult ovaries,

(3) detection of proteins restricted to early meiotic events and

that are not expressed within follicle-enclosed oocytes, (4)

delayed onset of sterility following treatment of juvenile

females with the germ cell toxicant busulphan, and (5) the

apparent demonstration of new follicle production in adult

ovaries. The first piece of evidence that the authors present in

support of their hypothesis was obtained throughmorphologic

examination of the numbers of healthy and atretic immature

follicles. The authors observed large numbers of misshapen

immature follicles, and concluded that these follicles were

BioEssays 26:829–832, � 2004 Wiley Periodicals, Inc. BioEssays 26.8 829

1Department of Physiology, University of Maryland School of

Medicine, Baltimore.2Department of Epidemiology and Preventive Medicine, University of

Maryland School of Medicine, Baltimore

Funding agency: Chuck Greenfeld was supported by NIH 38955,

T32HD07170.

*Correspondence to: Jodi A. Flaws, Department of Epidemiology and

Preventive Medicine, 660 W. Redwood Street, Howard Hall 133B,

Baltimore, MD, 21201. E-mail: jflaws@epi.umaryland.edu

DOI 10.1002/bies.20094

Published online in Wiley InterScience (www.interscience.wiley.com).

What the papers say

atretic, and that such large numbers of atretic follicles were

incompatible with the observed relatively small changes in

healthy follicle numbers. They state that this suggests that new

follicles must be produced or the follicular reserve would be

exhausted at a greater rate than observed in their experi-

ments. While provocative, care should be taken in the

interpretation of these morphological results because it is

unclear whether morphological determination of the numbers

of atretic primordial follicles is completely accurate.(9) Further,

formalin fixation is very harsh on morphology and the atretic

appearance could be due to fixation damage. In addition,

atresia of primary and small preantral follicles is very rare.(9,10)

Thus, it is possible that primordial follicles might have been

misclassified as healthy or atretic in the study by Johnson

et al.(1) The atresia data would be stronger if they were

confirmed with some molecular marker for atresia, such as

caspase activation or TUNEL staining. That said, the results of

Johnson et al.(1) may be consistent with previous estimates of

follicle exit from the primordial pool. For example, Faddy

et al.(9) modeled the numbers of follicles exiting the primordial

pool per day in mice. They calculated that a diminishing

number of follicles exit the primordial follicle pool per day and

that, by 2months of age, an estimated 19 follicles exit the pool

daily. If no new oocytes were produced, the pool should be

exhausted by 6 months of age. This, however, does not occur

because the pool is not exhausted until approximately 1–

2 years of age. While such models support Johnson et al.,(1)

they assume that the rate of exit does not changewith age and

it has been shown that the number of follicles that exit the

primordial pool is inversely correlated with age.(11)

Johnson et al.(1) next present evidence that proliferating

cells of germ lineage exist in or near the ovarian surface

epithelium. The functional significance of this finding is not

clear as it is unknown whether these cells become incorpo-

rated into follicles. In addition, the authors report that proteins

restricted to early events of prophase I, events that cease

before the oocyte enters into meiotic arrest, were detected in

the adult ovary. While these data offer some support for the

hypothesis that there is production of new oocytes in the adult

ovary, it is important to note that other investigators have

reported the absence of these proteins in adult ovaries.

Hodges et al. (2001)(12) reported that Spc3 became undetect-

able by western blot analysis by two weeks of age in mouse

ovaries. Keeney et al. (1999),(13) using an in situ hybridization

approach,were unable to detectSpo11 in the postnatalmouse

ovary and, using RT–PCR, they were unable to detect Spo11

or Dmc1. All three of these genes (Spc3, Spo11 and Dmc1)

were detected by Johnson et al.(1) Reasons for the differences

between these reports may be due to subtle differences in the

age or strain of mice used in each study.

Johnson et al.(1) next report that evidence for the existence

of ovarian GSCs comes from experiments in which they at-

tempted to kill GSCs by treating juvenile mice with busulphan.

In these experiments, mice received two doses of busulphan,

one at postnatal day (PN) 25 and the other at PN35. The

authors used busulphan because it had been shown to target

spermatogonial stem cells in testes.(14) The primordial follicle

pool was observed to be nearly depleted at PN45. Johnson

et al.(1) interpreted this to be the result of failed follicular

replacement due to toxicity of busulphan to presumptive

GSCs.While these data are interesting, the authors base their

interpretation upon the assumption that busulphan targets

only the presumptive GSCs and that it is not toxic to primordial

follicles. Johnson et al.(1) indicate that they demonstrate a lack

of toxicity of busulphan to primordial follicles because there

was only a small peak in the number of atretic primordial

follicles two days after the first dose of busulphan. In an earlier

experiment utilizing the follicle toxicant 9,10-dimethylbenz-

[a]anthracene (DMBA), however, Johnson et al.(1) demon-

strated that atretic primordial follicles are very rapidly cleared

from the ovary, and thus it is possible that busulphan was

acutely toxic to primordial follicles and that their death was not

observed due to their very rapid clearance. Additionally, the

assumption that busulphan doesnot kill primordial follicles, but

rather only targetsmitotic germ cells, was based uponwork by

Pelloux et al. (1988),(15) in which rats exposed to busulphan in

utero at embryonic day (E) 12, but not at E18, had a reduced

number of oocytes compared to vehicle-treated controls at

PN5andPN10, suggestinganeffect only onmitotic germcells.

As primordial follicles are not yet formed at E18, it is not

possible to conclude from these data that primordial follicles

are refractory tobusulphan toxicity. SinceJohnsonet al.(1) only

reported the number of healthy primordial follicles at the end of

the busulphan experiment rather than at the same multiple

timepoints at which they reported numbers of atretic follicles, it

is impossible to determine the rate of loss of primordial follicles

from the follicle pool. The rate of primordial follicle loss is

crucial to the interpretation of data obtained by Johnson

et al.,(1) andwould havehad to bedemonstrated to beslowand

steady if busulphan was toxic only to GSCs, and had no effect

on primordial follicles.

In a final experiment, Johnson et al.(1) report that they

demonstrated the formation of new follicles by producing

chimeric ovaries, composed of half wild-type (WT) tissue and

half transgenic tissue that constitutively expressed green

fluorescent protein (GFP). The authors report that, in WT

tissue, there were follicles composed of transgenic GFP-

expressing oocytes surrounded by WT non-GFP-expressing

granulosa cells. It is possible that this situation would arise if

new oocytes were being produced in the ovary. A limitation of

this experiment, however, is that no quantification of these

follicles was presented, nor were there any low magnification

photographs of the ovaries to demonstrate that the few

presented follicles were not aberrations. Additionally, no

follicles were presented that were composed of a WT oocyte

surrounded by transgenic GFP-expressing granulosa cells,

What the papers say

830 BioEssays 26.8

the presence of which would be expected given the experi-

mental design.

A new era in ovarian research?

While the results of Johnson et al.(1) are suggestive of the

presence ofGSCs in the adultmouse ovary, some of their data

are difficult to reconcilewith data frompast studies. The results

of their busulphan experiments suggest that the primordial

pool almost completely turns over in a 20-day period. This is in

contrast to the results of Meredith et al. (2000),(16) in which

adult female rats were exposed to bromodeoxyuridine (BrdU),

amarker that is incorporated into proliferative cells, for 7 days,

after which the presence of labeled granulosa cells in

primordial follicles containing at least one cuboidal granulosa

cell was examined at various times following cessation of

exposure. The results of Meredith et al.(16) indicated that 57%

of thoseprimordial follicles that contained at least one cuboidal

granulosa cell labeledwithBrdU remained throughout the150-

day study. In addition, Hirshfield (1992)(17) exposed rat

embryos to tritiated thymidine late in gestation and observed

a subset of oocytes that were labeled and that remained

throughout the length of the study until PN40. Further,

Hirshfield (1994)(18) treated rats in utero with busulphan and

found the continued presence of primordial and growing

follicles at PN59. These results would not be expected if the

primordial follicle pool were actively turning over and if

busulphan killed GSCs. Perhaps, these studies highlight

species differences in the rate of turnover of the primordial

follicle pool, or that the presence of GSCs is unique to the

mouse. Future studies will need to determine whether GSCs

exist in the ovaries of other species, including humans. If they

exist in human ovaries, the implications of this would be

unclear because the onset of menopause is a stereotypical,

tightly regulated process.

If the presence of GSCs in adult ovaries were confirmed by

future studies, such a finding would raise a number of

questions pertaining to follicular endowment. One question

to be addressed would center around the origin of GSCs.

Primordial germ cells migrate to and colonize the primitive

genital ridge, during and after which their proliferation

produces a large population of germ cells.(19) Thus, it would

be important to know if GSCs are members of the initial

migratory population or if they arise from differentiation of

migratory primordial germ cells. As these migratory and

proliferative processes are critical for determining future

reproductive function in the adult,(20) it would seem likely that

residency or functionality of GSCs in the ovary would be

established at this stage.

Further questions are related to the function of germ cell

nests. These structures, which arise due to incomplete

cytokinesis in proliferating oogonia in the embryonic ovary,

have been hypothesized to be involved in the synchronization

of germ cell development or organelle biogenesis.(21) Germ

cell nests are not present in the adult ovary, thus raising

questions about their functional importance during prenatal

oogenesis. The potential existence ofGSCswould also raise a

question as to the role of a resting pool of primordial follicles.

The reports discussed above(16–18) suggest that if turnover of

the follicle pool does occur in the rat, it does so slowly. What

then is the purpose of a resting follicle pool andwhywould it be

more advantageous to maintain the presence of a resting pool

rather than simply producing new follicles as needed?

Confirmation of GSCs would also potentially require re-

evaluation of various gene-deletion studies in which follicular

endowment is altered by the deletion. For example, the Dazla

deficient female hasnormal numbers of pachytene stagegerm

cells at E15.5. By E19.5, many of these germ cells are

undergoing apoptosis, and the adult ovary is devoid of

oocytes.(22) Deletion of the anti-apoptotic Bcl-Xl results in a

greatly reduced follicular endowment and premature ovarian

failure.(23) Deletion of the zinc-finger transcription factor Zfx

results in a small primordial germ cell population, likely due to

reduced proliferation of primordial germ cells, and this

translates into reduced follicle populations in the postnatal

animal as well as premature ovarian failure.(24) In these

models, it would be interesting to know if GSCs are similarly

affected such that they have loss of function or decreased

survival, or whether they remain viable and functional. These

types of models may be useful in the determination of factors

that regulate GSC survival and function.

Perhaps the most important question to arise from the

results of Johnson et al.(1) relates to why reproductive sen-

escence (menopause) occurs if GSCs are present in the ovary.

Johnson et al.(1) suggest that the potential for GSCs and

follicular renewal exists, but this seems contrary to data

indicating that mammalian ovaries gradually lose follicles with

age, and that aged animals become infertile.(25) Schlessinger

andVanZant (2001)(26) argue that stemcells acquire functional

deficits that arise with age and that this may be responsible for

the aging of the organism in general. Using hematopoietic stem

cells as a model, they argue that stem cells become quiescent

as they age and that this acquired quiescence may be involved

in theagingprocess.Asimilar situationmayoperate in theovary

such thatGSCsbecomequiescent with increasing age, leading

to depletion of the follicle pool.

Conclusion

The history of science has abundant examples of the hazards

of slavish adherence to long-standing paradigms. Central

premises should be re-examined and challenged on a periodic

basis to ensure that they continue to remain robust. Johnson

et al.(1) have performed a service to the field of ovarian biology

by prompting re-examination of the ‘‘central dogma’’ of the

primordial follicle pool that has shaped our understanding of

ovarian function since 1951. However, the central dogma has

withstood the test of time in that it has remainedconsonantwith

What the papers say

BioEssays 26.8 831

nearly every empirical observation concerning ovarian folli-

cular dynamics in the past 50 years. Johnson et al.’s(1)

observations need to be confirmed by others and extended by

additional supporting evidence before they could be consid-

ered to constitute a serious challenge to the central dogma

crystallized by Zuckerman(8) in his landmark work.

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