r 2005, Copyright the Authors

Differentiation (2005) 73:438–446 DOI:10.1111/j.1432-0436.2005.00045.x Journal compilation r 2005, International Society of Differentiation

COMMENTARY

Anne Grete Byskov . Malcolm J. Faddy . Josephine G.

Lemmen . Claus Yding Andersen

Eggs forever?

Received September 14, 2005; accepted in revised form September 23, 2005

Abstract A group of scientists from Harvard Medical Key words oogenesis
mammal
postnatal

School (Johnson et al., 2004) claims to have ‘‘estab- germline stem cells
lished the existence of proliferative germ cells that sus-
tain oocyte and follicle production in the postnatal
mammalian ovary,’’ expressing no doubts about their Introduction: Learning from the past
methods, results and conclusion. Johnson et al. based
their conclusions of oocyte and follicular renewal from The article by Johnson et al. (2004) marks a distinct
existing germline stem cells (GSC) in the postnatal departure from the old paradigm that oogonial stem cells
mouse ovary on three types of observations: (1) A become extinct early in life after transformation into
claimed discordance in follicle loss versus follicle atresia oocytes. It claims to show that functional germline stem
in the neonatal period and in the following pubertal cells (GSC) persist in adult mouse ovaries, which is par-
and adult period; (2) immunohistochemical detection adoxical in view of the decline in follicle number with age
of proliferating GSC with meiotic capacity using com- seen in the mouse and in all other mammalian species.
bined markers for meiosis, germline, and mitosis; and The classical definition of stem cells is that they perpet-
(3) neo-folliculogenesis in ovarian chimeric grafting ex- ually renew themselves and generate differentiated prog-
periments with adult mice. eny, which in the ovary will be the oocytes. Thus, if the
Oogenesis is the process that transforms the prolif- follicle population is being replenished continuously dur-
erative oogonium into an oocyte through meiosis, fol- ing adult life, as claimed, GSCs must progressively lose
lowed by folliculogenesis and follicular and oocyte their ability to multiply to account for declining fecun-
maturation. The most crucial part in producing a func- dity. Such an aging behavior has been considered excep-
tional oocyte is firstly, initiation and completion of the tional among stem cells, although stem cells may suffer
first meiotic prophase, and secondly, enclosure of the from age-related functional deletions which could pos-
resulting diplotene oocyte in a follicle. Neither of these sibly explain ageing (Schlessinger and Van Zant, 2001).
two events has been shown to take place in Johnson There can be no doubt that the human menopause is
et al.’s study of the postnatal mouse ovary. We hereby universal. It is caused by continuous disappearance of
address the observations underpinning their hypothesis follicles by atresia and, to a minor degree, ovulation. In
and conclude that it is premature to replace the para- many other mammalian species disappearance of folli-
digm that adult mammalian neo-oogenesis/folliculogen- cles and cessation of reproduction are also connected to
esis does not take place. aging (vom Saal et al., 1994). Today most of us believe
that all oogonia, i.e., the oocyte stem cells, become
Anne Grete Byskov (* . )
Josephine G. Lemmen
extinct early in life as they transform into oocytes, but
Claus Yding Andersen during the past 150 years neo-oogenesis in adult mam-
Laboratory of Reproductive Biology mals has been vigorously discussed. Waldeyer in
Juliane Marie Centre, Rigshospital his famous publication ‘‘Eierstock und Ei’’ from 1870
Copenhagen, Denmark maintained the idea that no new formation of oocytes
E-mail: [Link]@[Link]
took place in adult mammals and birds, but that the
oocytes arose in the ovarian surface epithelium, the so-
Malcolm J. Faddy
School of Mathematical Sciences called ‘‘germinal’’ epithelium, during a limited period
Queensland University of Technology early in life (Waldeyer, 1870). His hypothesis was
Brisbane, Qld, Australia temporarily overturned 50 years later by Kingery (1917)

U.S. Copyright Clearance Center Code Statement: 0301–4681/2005/7309–438 $ 15.00/0

 439

who claimed that all oocytes formed during fetal life meiotic prophase early in life, often before birth; the
degenerated and were replaced by oocytes arising in the second wave hits the oocytes during follicle enclosure
adult ovarian ‘‘germinal’’ epithelium. Allen (1923) sup- around the time when the first cohort of follicles begin
ported this theory and stated that mitotic divisions in to grow; the last wave is introduced at early puberty
the ‘‘germinal’’ epithelium resulted in new oocytes. when small antral follicles become atretic owing to go-
During the following decades numerous studies sup- nadotropin shortage. This last wave of atresia is recur-
ported the concept that neo-oogenesis in the adult rently seen throughout fertile life as it fluctuates with
mammalian ovary does not take place. Many experi- the reproductive cycle and continues until no more fol-
ments focussed on evaluating fertility after decreasing licles are present.
the oocyte pool, e.g., by irradiation (Everett, 1943; It appears that the large number of small atretic fol-
Mandl, 1959; Peters and Levy, 1963), or semiovariec- licles found during early neonatal and again in late ju-
tomy (Mandl et al., 1952; Jones and Krohn, 1960). Both venile and adult mouse ovaries has driven Johnson et al.
treatments resulted in a faster depletion of the remain- to propose that new formation of oocytes and follicles
ing oocyte pool and curtailed the reproductive lifespan. must take place to account for the number of healthy
None of these studies supported the hypothesis that follicles that is actually present in these ovaries. Diag-
neo-oogenesis took place in the adult ovaries. In com- nosis of atresia is difficult and often dependent on sub-
parison, in the adult frog, which notoriously has a pool jective judgments. Cell death of follicles may affect the
of GSC, reduction of the pool by UV irradiation results granulosa cells or oocyte or both, depending on the size
in even greater proliferation of the remaining GSC. In of the follicle and the physiological circumstances. At-
the mammalian male the situation is quite different and resia of primordial follicles seems to affect the oocyte
similar experiments will often result in replenishment before the granulosa cells (Depalo et al., 2003), but in
of functional germ cells from the male GSC, i.e., the growing and larger follicles atresia is first characterized
spermatogonia (Puscheck et al., 2004). by granulosa cell apoptosis (Hirshfield, 1988; Tilly,
The discussion of neo-oogenesis in the adult mam- 1996; Reynaud and Driancourt, 2000; de Bruin et al.,
malian ovary was almost ended by the studies of 2002). Surprisingly, Johnson et al. (2004) use a defini-
Zuckerman and his group (Zuckerman, 1951) and in tion of follicle atresia solely based on oocyte morphol-
particular those of the Peters group (Peters et al., 1962). ogy: ‘‘Follicles at the primordial, primary and preantral
Using delayed autoradiography after injection of triti- (immature) stages of development were scored as atretic
ated thymidine Peters et al., showed that the premeiotic if the oocyte was degenerating (convoluted, condensed)
S phase leading to the formation of oocytes in the mouse or fragmented’’. They found that atretic follicles only
only takes place during a limited prenatal period and accumulate from day 30 onwards, and they assume that
that these oocytes remain in the adult ovary. In the years the number of 1,200 atretic follicles present on day 42 is
to follow most scientists accepted that there are no so high that the oocyte pool would soon disappear if
oogonia in the adult mammalian ovary that can multi- new oocytes were not formed. This assumption was
ply, enter meiosis and form new follicles. It seems, how- based on the very fast disappearance rate of small
ever, that a few species prove to be the exceptions to the follicles calculated in three different situations: (1) The
rule. In prosimian monkeys, oocytes in transitory stages loss of oocytes during the first 4 days of life; (2) The
of the first meiotic prophase confined to ‘‘germ cell daily number of atretic follicles after 9,10-dimethyl-
nests’’ are present in juvenile and adult ovaries as first benz[a]anthracene (DMBA) injection on day 25; (3) The
described in 1920 (Gerard, 1920) and later confirmed by number of atretic primordial follicles after injections of
many others (Ioannou, 1967). Also the armadillo’s ovary busulfan on days 25 and 35, evaluated every 5th day
contains germ cells in meiosis but in this species they are until day 45.
confined to cord-like structures within the cortex (End- In order to get an idea of the nature of these small
ers, 1960). However, and most importantly, none of atretic follicles with ‘‘convoluted, condensed or frag-
these studies showed that new functional follicles were mented’’ oocytes as defined by Johnson et al., we in-
formed in the adult ovary of any of these species. jected four 30-day-old mice with the DNA marker
bromodeoxyuridine (BrdU) five times, once every hour
and removed the ovaries 1 hr (two mice) and 8 days
(two mice) after the last injection. In such a setup
Follicle atresia: How and how fast growing cells can be traced in time and place. The mice
were the same strain (C57Bl/6) and ovaries were proc-
One of the striking features of the mammalian ovary is essed as Johnson et al. (2004). Healthy and atretic fol-
the amazing disappearance of oocytes by atresia, a licles were counted in the 30-day-old mouse ovaries
process that begins early in life and proceeds until no according to their definitions and methods. We found
more follicles are present in old age. Oocyte atresia that between 0% and 0.5% of primordial follicles and
comes in three waves. The first wave is confined to none of the primary or early preantral follicles were
oocytes passing through the transitory stages of the first atretic according to these criteria and none were
440

fragmented. None of the oocytes—primordial, primary Based on the assumption that a large fraction of
growing or large—were labeled with BrdU, neither at small follicles die, Johnson et al. go on to show that
1 hr or 8 days after the last of the five BrdU injections. small follicles seem to be cleared very fast from the
Among the 100 follicles classified as early antral, ovary. After injection of the toxicant DMBA on day 25
around 60 had some pyknotic granulosa cells but the the number of small follicles was counted daily for the
oocytes looked healthy (stages I–II atresia, (Byskov, next 4 days. During that period 3,500 follicles disap-
1974) and they were therefore not counted as atretic in peared, but atretic follicles were only seen on the second
the present context. However, 185 and 370 very atretic and third day after injection, 800 each day, suggesting
follicles (atretic stages III–IV, Byskov, 1974) with ‘‘con- that they were removed within hours or a few days. The
voluted, condensed or fragmented oocytes’’ were pre- authors take these results as proof that the small atretic
sent in the two 30-day-old mouse ovaries, and were follicles (our interpretation: previously larger growing
situated almost exclusively in the center of the ovary ones) seen from day 30 and onwards disappear very fast
where no primordial follicles are found (Fig. 1A). This and that GSC is needed to maintain ovarian function.
number is comparable with Johnson et al.’s small atretic Johnson et al. based the next part of their evidence
follicles at day 30, namely 200–400. Stage III atresia for the existence of GSC on a decrease of the primordial
would have referred to healthy late preantral or early follicle pool after two injections of busulfan, a com-
antral follicles 4–6 days earlier as previously shown pound with cytotoxicity toward proliferating GSC,
(Byskov, 1974) and stage IV would have been growing oogonia and spermatogonia, on day 25 and 35. Ten
follicles even before that. At a first glance they may days after the last injection the pool was decreased to
resemble small growing follicles with ‘‘convoluted, con- around 200, but the authors were puzzled that ovaries
densed or fragmented oocytes’’ (Fig. 1B). The somatic still contained growing and large follicles as well as cor-
cells of such remnants are unlabeled with BrdU after pora lutea. Then they counted the number of atretic pri-
1 hr (Fig. 1C,1D) but heavily labeled 8 days after injec- mordial follicles every fifth day after onset of the
tions (Fig. 1E,1F). This supports previous results that busulfan treatment and found 45 to be atretic on days
follicles with ‘‘convoluted, condensed or fragmented 2, 5, and 10 after the first injection, but strangely they
oocytes’’ represent collapsed atretic follicles that were found no effect of the second injection. The authors’
growing follicles several days before. Early antral fol- dilemma, they state, was that the decrease of the pri-
licles begin to accumulate in the prepubertal mouse mordial follicle pool by 2,000 after busulfan treatment
ovary but become atretic as a result of gonadotropin did not relate to the number of atretic follicles that
shortage (Hillier, 1994). In the mouse more than half was actually counted. This dilemma, however, seems
the growing follicles are atretic by day 21 (Byskov, easily explained using the authors’ own calculations of
1978) and these plus those arising in the following days atretic follicles seen after DMBA treatment. After this
are the likely ones to be recognized as atretic stages III– treatment the authors observed a total of 1,600 atretic
IV on day 30 and later. Thus, the pool of atretic follicles follicles, equivalent to about 50% of the number of
observed on day 30 by Johnson et al. (2004), most likely lost follicles (3,500) (Johnson et al., 2004; Fig. 1C).
represents follicular remnants that have been degener- According to the authors, 45 follicles are expected to be
ating for many days. It is not known how long they seen as atretic per day. This would add up to a total
remain in the ovary before being completely removed of 900 atretic follicles in 20 days, equivalent to about
but the zona pellucida is likely to be the last remnant. 50% of the follicles actually lost (around 2,000).
At the age of 100 days estimates based on stereology Therefore, the observed follicle pool decrease after busul-
showed that there are 500 such zona-remnants in a fan is comparable with what the authors report after
C57BL/6 mouse ovary (Myers et al., 2004). We deduce DMBA, the process is just slower after busulfan treat-
that Johnson’s definition of atresia in pubertal and ment. Apparently busulfan not only exerts its toxic effect
young adult mice covers very atretic, collapsed or on proliferating GSC but also on the primordial follicles,
shrunken, previously growing follicles. Indeed, the pres- as previously suggested (Generoso et al., 1971).
ence of very atretic follicles is not per se a measure for Johnson et al. are also puzzled about the presence of
the clearance rate of the follicle pool. That the number growing and large follicles as well as corpora lutea 20
of atretic stages III–IV follicles remained at a steady days after the initial busulfan treatment. The explana-
level after day 42 only signifies that some follicles con- tion seems obvious. Busulfan, as DMBA, seems to de-
tribute continuously to the atretic pool and that an stroy the primordial follicles and not the growing ones,
equal number leave it. The 1,200 atretic follicles present so they keep growing (Krarup, 1970). Krarup found
on day 42 do not say anything about how many will that the mouse ovary contained small, growing and
become atretic per unit time. Johnson et al.’s estimate large follicles more than 4 weeks after DMBA injection.
that the follicle pool would be eliminated very fast ap- Moreover, the number of large antral follicles remains
parently relates to a misinterpretation of the nature of normal until the follicle pool is depleted (Hirshfield,
atretic follicles, and lack of a dynamic evaluation of the 1994). As it takes around 3 weeks to grow from the
follicular clearance rate. primary follicle stage to large antral follicles (Pedersen,
 441

Fig. 1 Histological sections of mouse ova-

ries, stained with hematoxylin and peri-
odic-Schiff, aged 30 days postpartum
(p.p.) (A, B, G) or immunostained for de-
tection of bromodeoxyuridine (BrdU).
BrdU was given on day 30 p.p. as five in-
jections 1 hr apart and animals sacrificed
1 hr (C, D) and 8 days after (E, F) the last
injection. (A) Thirty-day-old mouse ovary
with all types of follicles, including two
atretic follicles with ‘‘convoluted, con-
densed, or fragmented’’ oocytes (arrows)
at the inner border of cortex. (B) Higher
magnification of the two atretic follicles
with ‘‘convoluted, condensed, or fragment-
ed’’ oocytes. (C) Thirty-day-old mouse
ovary immunostained for BrdU 1 hr after
the last BrdU injection. All medium-sized
and large growing follicles are heavily
labeled. Notice that virtually no cells are
labeled in medulla. The arrow points at
an unlabeled atretic follicle with ‘‘convo-
luted, condensed, or fragmented’’ oocyte.
D: Larger magnification of the unlabeled
atretic follicle with ‘‘convoluted, con-
densed or fragmented’’ oocyte shown in
(C). (E) Thirty-eight-day-old mouse ovary
immunostained for BrdU 8 days after the
last BrdU injection. Only a few cells are
labeled in the medium-sized and large
growing follicles whereas atretic follicles
squized between them or situated at the
inner border of cortex (arrow) are heavily
labeled. (F) Larger magnification of the
heavily labeled atretic follicle with a ‘‘con-
voluted, condensed, or fragmented’’ oo-
cyte as shown in (E). (G) Primordial
follicles with normal looking oocytes sit-
uated within or at top of the surface ep-
ithelium.

1970) and perhaps another couple of weeks to accom- larger follicles (Pedersen and Krarup, 1969) and also
plish transition from the primordial to the primary fol- decrease atresia of growing ones (Guigon et al., 2003).
licle stage, it is not surprising that corpora lutea are We are, therefore, convinced that new formation of
found in the treated ovaries 20 days after treatment is follicles is not at all needed to explain the presence of
initiated or even several weeks after treatment (Gene- large follicles and corpora lutea after treatment with
roso et al., 1971). In addition, insults that will decrease busulfan in analogy to treatment with DMBA and X-
the pool of small follicles induce growth acceleration of irradiation, as mentioned above. Ironically, it seems
442

contradictory to propose the presence of GSC to pro- whether the putative GSC renew themselves and the fol-
duce new oocytes in the busulfan-treated mice because licle pool in a comparable temporal age related way.
this compound is known for its very focussed toxicity In addition, it has been known for a long time that
on exactly this cell type. We conclude that Johnson oocyte quality in mammals declines with age. Genetic
et al. lack evidence for the existence of new follicle for- disorders characterize old oocytes, as exemplified in
mation in their study. humans by the increasing number of Down’s syndrome
children born to women after the age of 35 years
(Eichenlaub-Ritter, 1996). If GSC renewed the oocyte
pool, should this not occur? On the other hand, the
Discordance in follicle loss versus atresia? children of elderly men also experience a higher rate of
genetically transmitted diseases, although this occurs
Johnson et al.’s conclusion that a replenishment of the later in life than in women (Stene et al., 1987). Perhaps
ovarian follicle pool in the mouse must take place arises women’s putative GSC grow older earlier than men’s?
from their estimates of follicle number, healthy and at-
retic, in particular between days 16 and 40 postpartum.
During these 24 days the actual number of primordial
follicles declined by 294 and total non-atretic follicles by Is the ovarian surface epithelium a ‘‘germinal’’
800. Then they estimated the rate of primordial follicle epithelium?
depletion using the mathematical model from Faddy
et al. (1987), which on day 21 predicts a daily loss of 89 Johnson et al. (2004) claim that functional GSC reside
primordial follicles. Thus they find that 2,136 follicles in the surface epithelium of the adult mouse ovary, and
should be lost during 24 days. In other words, the dif- that they can form follicles. They counted the ‘‘large
ference of 1,842 would represent the number of new ovoid germ cells’’ in the surface epithelium at day 30
follicles produced between days 16 and 40, correspond- postpartum and found 63 such cells per ovary. This
ing to 77 new follicles per day. A number of criticisms number is close to that expected for a small pool of
can be made of this reasoning. The estimated loss of asymmetrically dividing germ cells, referring to their
follicles (Faddy et al., 1987) is based on a different own calculation of ‘‘77 new primordial follicles per
strain of mouse (CBA/Ca) to theirs (C57BL/6) and ovary per day.’’ The only ‘‘large ovoid germ cells’’ in the
there is reported evidence that loss of follicles differs surface epithelium at day 30 postpartum we can find in
greatly between strains (Jones and Krohn, 1961; Faddy the two ovaries of the mice injected with BrdU and
et al., 1983; Canning et al., 2003). It is therefore quite otherwise prepared as Johnson et al. (see above) are
unjustifiable to take one strain as a benchmark and confined to primordial follicles. When corrected for
compare follicle loss with another strain, suggesting counting only every fifth section primordial follicles
that the difference is because of new follicles being pro- amount to a total number of 2,705 and 1,365, respec-
duced. An alternative conclusion is simply that the es- tively. It has previously been shown that primordial
timated rate of follicle loss for the one strain (C57BL/6) oocytes leave the ovary through the surface epithelium,
is 77 follicles a day lower than that for the other strain in particular during early stages of development, and in
(CBA/Ca), although this quantity is a very imprecise the prepubertal mouse they accumulate in the ovarian
estimate with a standard error of about 30. bursa cavity (Byskov and Rasmussen, 1973). In fact,
There is also a curious piece of circular arithmetical oocytes of primordial follicles situated within the ovar-
reasoning toward the end of the paper, where the au- ian surface epithelium of the immature mouse may be
thors seem to be arguing that the closeness of their es- released into the peritoneal cavity leaving their granu-
timated decline of ‘‘a total of 288 follicles’’ and ‘‘that losa cells within the epithelium (Wordinger et al., 1990).
derived from comparing the actual counts of non-atretic Small oocytes within or even on top of the surface ep-
primordial follicles on days 16 versus 40 (2,334 versus ithelium are also seen in the 30-day-old mouse ovaries
2,040, or a net loss of 294 primordial follicles)’’ in some that we prepared according to Johnson et al. (2004)
way supports their inferences. It does nothing of the (Fig. 1G). We did not recognize transitory stages of the
sort—the difference here is entirely because of their first meiotic prophase in any of such ‘‘large ovoid germ
earlier rounding of 1,842 / 24 5 76.75 follicles per day cells’’ or in any other ovarian cell wherever we looked.
to 77, with the rounding error of 0.25 when multiplied The role of the ovarian surface epithelium as the site of
by the number of days, 24, giving rise to the small dif- neo-oogenesis has previously been intensely studied. Allen
ference of 6. (1923) presented a series of figures from mouse ovarian
Another aspect concerning decrease of the follicular ‘‘germinal’’ epithelium containing mitotic figures and
pool is the relation to age. Numerous studies have shown small primordial oocytes, much like Johnson et al.’s
that the decrease of the oocyte pool is inversely correlated Fig. 2. Allen concluded that all postnatal oocytes arose in
with age: The older the mouse, the slower is the decrease this epithelium. In the meantime, experiments have
(Faddy et al., 1987). It would be interesting to study shown that growing follicles and corpora lutea were still
 443

developing and present in the ovaries of rat, guinea-pig, could sustain oocyte production in adulthood’’. (John-
cat, and opossum a year after destruction of the ovarian son et al., 2005).
surface epithelium (Moore and Wang, 1947; Mandl et al., Functional evidence for GSC in respect to formation
1952). Similar results were found in grafted monkey ova- of new oocytes must be the presence of oocytes in tran-
ries where the surface epithelium had been eliminated sitory stages of the first meiotic prophase, namely the
during the grafting period (Mandl and Zuckerman, 1949). leptotene, zygotene, and pachytene stages. This process
It therefore seems obvious that the ovarian surface ep- is slow and distinctive, taking 4 days in the fetal mouse
ithelium is not needed for maintenance of follicle growth. (Peters et al., 1962; Peters, 1970) and probably much
In order to discriminate GSC from somatic cells of longer in humans. If 77 follicles were being renewed
the postnatal mouse ovary, Johnson et al. (2004) used daily between days 16 and 40, approximately 300 or
expression of different genes involved in the early stages 10% of all oocytes would be in meiosis at the same time.
of meiosis: the synaptonemal complex protein, Scp3, In contradistinction, the abundant literature on oogen-
endonuclease Spo11, the recombinase Dmc1 and a esis provides no support for the existence of early me-
marker for the germ cell lineage, mouse vasa homo- iotic germ cells in ovaries of either mouse, rat, or human
logue, Mvh, i.e. Ddx4. Scp3 is expressed in germ cells after infancy. Only in a few mammalian species have
that pass through the first meiotic prophase and re- oocytes in the transitory stages of meiosis has been de-
mains in mouse spermatocytes in the meiotic metaphase scribed (see section ‘‘Germ cell nests with meiosis in the
I and up to anaphase II (Viera et al., 2003). Hodges adult ovaries’’). The new publication by Johnson et al.
et al. (2001) showed that 90% of the diplotene oocytes (2005) also claims that a spontaneous regeneration of
from ovaries of 3-day-old mice exhibited staining for primordial follicles after ablation with the oocyte-toxic
SCP3, but SCP3 became undetectable by immunolabe- chemical doxorubicin takes place after injection and
ling and western analysis during the second week after that the number of primordial follicles increases from
birth. Thus, expression of Scp3 is not restricted to early around 400 to almost 1,500 within 12 hr. Following the
transitory stages of meiosis as suggested by Johnson same arguments as above, this seems impossible. This
et al. As both studies used the same mouse strain, conclusion, among other criticisms of Johnson et al.’s
C57BL/6 as Johnson et al. the different results might be (2005) publication, was also reached by a newly pub-
related to the use of different antibodies raised against lished multi-author Correspondence in Cell (Telfer
SCP3. In parallel to the results of SCP3, Johnson et al. et al., 2005).
find an almost identical age-related expression of Spo11 Finally, it seems conceivable that GSC would enter
and Dmc1. This is partly in contradiction to a previous the premeiotic S phase before being transformed into
report (Keeney et al., 1999) who found no expression of oocytes. We therefore carefully examined all oocytes in
Spo11 in the postnatal mouse ovary whereas Dmc1 was the two ovaries from animals killed 8 days after the five
weakly expressed. Interestingly, these mice also had the consecutive BrdU injections for possible labeling. No
C57BL/6 background as those used by Johnson et al. labeled oocytes were found which is surprising if 77
Mvh is specifically expressed in the germ cell lineage oocytes are renewed daily.
(Toyooka et al., 2000; Shibata et al., 2004). The pictures We therefore cannot find convincing evidence for a
in Johnson et al. (2004; Fig. 2) derive from juvenile and functioning ‘‘germinal’’ epithelium.
young mouse ovaries with many primordial follicles. It
is therefore not surprising to see small oocytes close to
or within the surface epithelium as mentioned above.
Co-staining with BrdU and MVH seemingly, confined Ovarian plasticity
to the same cell, was taken to indicate a germ cell that
incorporated BrdU injected 1 hr earlier (Johnson et al., Johnson et al. (2004) created an interesting chimaeric
2004; Fig. 2D, E). Johnson et al. claim that this DNA ovary model by combining fractions of wild-type (wt)
labeling is too high to be the repair mechanism that and transgenic (ubiquitous green fluorescent protein
seems to exist in oocytes of young mice (Crone and (GFP) expressing) ovaries under the bursa of the GFP
Peters, 1968). A valid comparison between normal S- mouse. Within 3–4 weeks chimeric ovaries had formed.
phase incorporation of BrdU and repair is needed to Only results from the chimeric wt tissue were reported.
justify this claim. Interestingly, in a very recently pub- This tissue contained small and antral follicles com-
lished paper in Cell by Johnson et al. (2005) have re- prising GFP labeled oocytes and unlabeled granulosa
counted some of their findings and now find that ‘‘the cells embedded in tissue that was exclusively or partly
number of these cells drops precipitously as female mice GFP-labeled. The authors conjectured that new follicles
transit through puberty (63  8 versus 6  3 cells per had formed, containing labeled oocytes and unlabeled
ovary at postpartum days 30 versus 40, respectively), granulosa cells from the two different tissue sources.
suggesting that these cells could not represent adult However, at least in the case shown in Johnson et al.
GSCs.’’ Instead, this new paper suggests that cells of the (2004, Fig. 5A), this might rather have taken place in
bone marrow are a ‘‘potential source of germ cells that the GFP host tissue into which unlabeled wt granulosa
444

cells or granulosa cell precursors have invaded. In con- oogonia enter meiosis within a limited period early in
trast, Fig. 5B seems to show a wt graft with a small GFP life, whether this occurs before or after birth. Oogenesis
oocyte and a few GFP somatic cells. Previous studies seems always to begin in the innermost situated oogonia
have revealed that small follicles can be disaggregated in the medulla, gradually spreading toward the surface
and reformed spontaneously (Gosden, 1990), even com- of the ovary (Winiwarter, 1901). In some species it
bining cell types of separate species (Eppig and Wiggles- happens that during this process some oogonia and
worth, 2000). Thus, we suspect that the new observations oocytes in early meiotic stages become trapped and iso-
reflect primordial follicle plasticity and that chimaerism lated in clusters or nests in the medulla or hilus of the
was a product of tissue injury after transplantation rath- ovary, e.g. cat (Winiwarter, 1920), opossum (Hartman,
er than being because of the generation of follicles de 1926), armadillo (Hamlett, 1935; Enders, 1960), pro-
novo. Such mixture may also explain the GFP-labeled simian monkeys (for a review, see Ioannou, 1967), and
stroma cells surrounding the chimeric follicles. Impor- in the late fetal and newborn human ovary (Byskov and
tant information would have been obtained if the au- Nielsen, 2003; Hoyer et al., 2005). It was suggested that
thors had presented whole sections of the chimeric such nests of oogonia persisted, renewed themselves and
ovaries, including the areas where the wt and the GFP- resulted in delayed meiosis a long time after this process
expressing tissue were growing together. The time se- was finished in the remaining part of the ovary (Bram-
quence for a GSC to develop into a large antral follicle is bell, 1930). Occasionally incorporation of tritiated thy-
also an important parameter to consider for the authors’ midine into diplotene oocytes occurs shortly after birth
conclusion of neo-formation of oocytes and follicles in in the mouse (Crone and Peters, 1968), a phenomenon
this system. The transitory stages of the first meiotic likely to relate to DNA repair during degeneration.
prophase take around a week and the time required for However, autoradiographic studies performed on adult
follicle formation is unknown, growth from a primordial prosimian monkey ovaries showed that oogonia-like
follicle to a small primary follicle needs perhaps 1–2 cells in mitosis confined to cell nests, which also con-
weeks; growth of a small primary to a large antral follicle tained oocytes in early stages of meiosis, were labeled
takes 3 weeks. Luck would be needed to make a large 24 hr after injection of tritiated thymidine (Ioannou,
antral follicle with an oocyte that developed from a pre- 1967; David et al., 1974). David et al. concluded that
meiotic GSC within 4 weeks. It is unlikely that this the incorporated thymidine was unusual and character-
process can be accomplished within 3 weeks. ized it as unscheduled DNA synthesis. But another
Lastly, GFP transfer from transplanted stem cells to study on adult prosimian monkey ovaries showed that
host cells resulting from cell fusion (Medvinsky and 10 days after injection of tritiated thymidine some of the
Smith, 2003) may have been misinterpreted as stem cell zygotene stages were heavily labeled (Butler and Juma,
‘‘transdifferentiation,’’ forcing concerns about alterna- 1972). Some of these studies support the notion adult
tive explanations for the GFP signal in the somatic cells neo-oogenesis takes place in a few species. However,
and perhaps even oocytes. It would have been interest- there are still no experimental results that provide that
ing if Johnson et al. had published their evaluation of evidence for new formation of follicles in any mamma-
the chimeric model in more detail, e.g., by giving an lian species.
numerical evaluation of the number of chimeric follicles
in the host tissue and the grafted tissue.

Conclusions
Germ cell nests with meiosis in adult ovaries. ‘‘There have been instances in the history of biological
Archaic leftovers or functional neo-oogenesis? sciences where the acceptance of a theory has prevented
the acquisition of new facts’’ said Evans and Swezy,
Initiation of oogenesis denotes the transformation of (1931) in the Introduction to their monograph, Ovo-
mitotically dividing female germ cells, oogonia, to genesis and the Normal Follicular Cycle in Adult Mam-
oocytes as they enter the first meiotic prophase. This malia. And, according to Greenfield and Flaws ‘‘The
event occurs in a species-specific manner at different history of science has abundant examples of the hazards
times after gonadal sex determination has taken place of slavish adherence to long-standing paradigms’’ (But-
early in life. In species with immediate meiosis, such as ler and Juma, 1972; Greenfeld and Flaws, 2004).
the mouse and rat, meiosis begins shortly after sex de- After 50 years, acceptance of the dogma that adult
termination. In species with delayed meiosis, e.g. sheep, neo-oogenesis does not take place in the mammalian
rabbit, human, monkey, meiosis begins after an extend- ovary (Zuckerman, 1951; Butler and Juma, 1972) it is
ed time from sex determination, i.e. delayed meiosis therefore refreshing and provocative to learn that Jona-
(Mauleon, 1969; Byskov, 1986). The time of birth seems than Tilly’s group has challenged this important issue.
to have no influence on this event. Many experiments The approaches that Tilly and his group used to prove
from different species support the notion that all that new formation of oocytes and follicles takes place
 445

in the adult mouse ovary are multifaceted and include Brambell, F.W.R. (1930) The development and sex in vertebrates.
modern techniques as well as established methods. Sidgwich and Jackson, London.
Butler, H. and Juma, M.B. (1972) Oogenesis in the adult prosimian.
However, we find that their conclusions lack sufficient Mammalian oogenesis. MSS Information Corporation, New York,
proof of concept. pp. 38–41.
Our overall conclusion is that the results presented by Byskov, A.G. (1974) Cell kinetic studies of follicular atresia in the
Johnson et al. (2004) cannot support the concept of mouse ovary. J Reprod Fertil 37:277–285.
neo-oogenesis in the postnatal mouse ovary. Nor does Byskov, A.G. (1978) Follicular atresia. In: Jones, R.E. (ed.). The
vertebrate ovary. Plenum Press, New York, pp. 533–562.
there exist any evidence for neo-folliculogenesis in the Byskov, A.G. (1986) Differentiation of mammalian embryonic
adult mammalian ovary. gonad. Physiol Rev 66:71–117.
Byskov, A.G. and Nielsen, M. (2003) Ontogeny of the mammalian
ovary. In: Trounson, AO and Gosden, R (eds.). Biology and
pathology of the oocyte. Cambridge University Press, Cam-
Methods bridge, pp. 13–28.
Byskov, A.G. and Rasmussen, G. (1973) Ultrastructural studies of
Animals and exposure the developing follicle. In: Peters, H. (ed.). International Con-
gress Series No. 267. The development and maturation of the
Four mice, strain C57BL/6 (Taconic, Ry, Denmark), were injected ovary and its functions. Excerpta Medica, Amsterdam, pp. 55–62.
at day 30 postpartum with BrdU (Sigma-Vallensbæk Strand, Den- Canning, J., Takai, Y. and Tilly, J.L. (2003) Evidence for genetic
mark) dissolved in saline at a concentration of 0.1 mg/ml. 50 mg/kg modifiers of ovarian follicular endowment and development from
BrdU was injected intraperitoneally five times with a 1 hr interval studies of five inbred mouse strains. Endocrinology 144:9–12.
and the animals sacrificed by cervical dislocation 1 hr (two mice) Crone, M. and Peters, H. (1968) Unusual incorporation of tritiated
and 8 days (two mice) after the last injection. Animal experiments thymidine into early diplotene oocytes of mice. Exp Cell Res
were performed with approval of the Danish Animal Ethics Com- 50:664–668.
mittee of the Ministry of Justice (file 2003/561–713). David, G.F.X., Anand Kumar, T.C. and Baker, T.G. (1974) Up-
take of tritiated thymidine by primordial germinal cells in the
ovaries of the adult slender loris. J Reprod Fertil 41:447–451.
Histology and immunohistochemistry de Bruin, J.P., Dorland, M., Spek, E.R., Posthuma, G., van Ha-
aften, M., Looman, C.W. and te Velde, E.R. (2002) Ultrastruc-
The ovaries were fixed (0.34 N glacial acetic acid, 10% formalin, ture of the resting ovarian follicle pool in healthy young women.
and 28% ethanol) and processed for paraffin embedding and se- Biol Reprod 66:1151–1160.
rially sectioned at 8 mM according to Johnson et al. (2004). From Depalo, R., Nappi, L., Loverro, G., Bettocchi, S., Caruso, M.L.,
each mouse the sections from one ovary were stained with hem- Valentini, A.M. and Selvaggi, L. (2003) Evidence of apoptosis
atoxylin and periodic-Schiff and on sections from the other ovary in human primordial and primary follicles. Hum Reprod 18:
BrdU was visualized by immunohistochemistry. After de-paraffin- 2678–2682.
ation, the sections were incubated for 10 min in 0.3% H2O2 in H2O Eichenlaub-Ritter, U. (1996) Parental age-related aneuploidy in
and washed in PBS. DNA was denatured in 7.5 mM NaAc in human germ cells and offspring: a story of past and present.
formamide for 1 hr at 701C and the reaction stopped by washing Environ Mol Mutagen 28:211–236.
twice for 1 min in 0.1 M borate buffer (pH 8.1). After washing twice Enders, A.C. (1960) A histological study of the cortex of the ovary
for 4 min in PBS, the sections were treated with 5% goat serum in of the adult armadillo, with special reference to the question of
PBS and incubated with primary antibody (rat anti-BrdU antibody; neoformation of oocytes. Anat Rec 136:491–499.
Seralab, Trichem Aps, Copenhagen, Denmark) 1:50 in PBS-T (PBS Eppig, J.J. and Wigglesworth, K. (2000) Development of mouse
with 0.5% between 20) or IgG -control for 1 hr at RT, washed twice and rat oocytes in chimeric reaggregated ovaries after interspe-
for 4 min in PBS-T and incubated 30 min with the secondary per- cific exchange of somatic and germ cell components. Biol Reprod
oxidase-conjugated antibody (Envision1Dual link system, Da- 63:1014–1023.
kocytomation, Glostrup, Denmark). After another wash in PBS the Evans, H.M. and Swezy, O. (1931) Ovogenesis and the normal
sections were incubated with Liquid DAB1solution (Dakocyto- follicular cycles in adult mammalia. University of California
mation), counterstained with PAS and mounted. Press, Berkeley, pp. 119–224.
Everett, N.B. (1943) Observational and experimental evidences re-
lating to the origin and differentiation of the definitive germ cells
Counting of follicles in mice. J Exp Zool 92:49–91.
Faddy, M.J., Gosden, R.G. and Edwards, R.G. (1983) Ovarian
In the ovary stained with hematoxylin and PAS follicles were follicle dynamics in mice: a comparative study of three inbred
counted in every fifth section. Healthy primordial, primary, prean- strains and an F1 hybrid. J Endocrinol 96:23–33.
tral and antral follicles, and atretic follicles with ‘‘convoluted, con- Faddy, M.J., Telfer, E. and Gosden, R.G. (1987) The kinetics of
densed or fragmented’’ oocytes were counted according to Johnson pre-antral follicle development in ovaries of CBA/Ca mice during
et al. (2004). the first 14 weeks of life. Cell Tissue Kinet 20:551–560.
Generoso, W.M., Huff, S.W. and Stout, S.K. (1971) Chemically
Acknowledgments We thank Dr. Hannah Peters for valuable induced dominant-lethal mutations and cell killing mouse
discussions. The technical assistance of Inga Husum is appreciated. oocytes in the advanced stages of follicular development. Mu-
This work was supported by The Danish National Science tat Res 11:411–420.
Foundation #22-02-0233 (AGB), #22-03-0200 (AGB). Gerard, P. (1920) Contribution a l’etude de l’ovaire des Mammif-
eres. L’ovaire de Galago mossambicus (Young). Arch Biol
30:357–391.
Gosden, R.G. (1990) Restitution of fertility in sterilized mice by
References transferring primordial ovarian follicles. Hum Reprod 5:499–
504.
Allen, E. (1923) Ovogenesis during sexual maturity. Am J Anat Greenfeld, C. and Flaws, J.A. (2004) Renewed debate over post-
31:439–482. natal oogenesis in the mammalian ovary. Bioessays 26:829–832.
446

Guigon, C.J., Mazaud, S., Forest, M.G., Brailly-Tabard, S., Co- Myers, M., Britt, K.L., Wreford, N.G., Ebling, F.J. and Kerr, J.B.
udouel, N. and Magre, S. (2003) Unaltered development of the (2004) Methods for quantifying follicular numbers within the
initial follicular waves and normal pubertal onset in female rats mouse ovary. Reproduction 127:569–580.
after neonatal deletion of the follicular reserve. Endocrinology Pedersen, T. (1970) Determination of follicle growth rate in the
144:3651–3662. ovary of the immatura mouse. J Reprod Fertil 21:81–93.
Hamlett, G.W.D. (1935) Extra-ovarial sex cords on an armadillo Pedersen, T. and Krarup, T. (1969) Cell population kinetics in the
ovary. Anat Rec 62:195–199. mouse ovary after treatment with a chemical carcinogen
Hartman, C.G. (1926) Postpubertal oögenesis in the opossum. (DMBA). Int J Cancer 4:495–506.
Anat Rec 32:209. Peters, H. (1970) Migration of gonocytes into the mammalian
Hillier, S.G. (1994) Current concepts of the roles of follicle stim- gonad and their differentiation. Philos Trans R Soc Lond B Biol
ulating hormone and luteinizing hormone in folliculogenesis. Sci 259:91–101.
Hum Reprod 9:188–191. Peters, H. and Levy, E. (1963) Effect of irradiation in infancy on
Hirshfield, A.N. (1988) Size-frequency analysis of atresia in cycling the fertility of female mice. Radiat Res 18:421–428.
rats. Biol Reprod 38:1181–1188. Peters, H., Levy, E. and Crone, M. (1962) DNA synthesis in
Hirshfield, A.N. (1994) Relationship between the supply of pri- oocytes of mouse embryos. Nature 195:915–916.
mordial follicles and the onset of follicular growth in rats. Biol Puscheck, E., Philip, P.A. and Jeyendran, R.S. (2004) Male ferti-
Reprod 50:421–428. lity preservation and cancer treatment. Cancer Treat Rev 30:
Hodges, C.A., LeMaire-Adkins, R. and Hunt, P.A. (2001) Coor- 173–180.
dinating the segregation of sister chromatids during the first Reynaud, K. and Driancourt, M.A. (2000) Oocyte attrition. Mol
meiotic division: evidence for sexual dimorphism. J Cell Sci Cell Endocrinol 163:101–108.
114:2417–2426. Schlessinger, D. and Van Zant, G. (2001) Does functional
Hoyer, P.E., Byskov, A.G. and Mollgard, K. (2005) Stem cell fac- depletion of stem cells drive aging? Mech Ageing Dev 122:
tor and c-Kit in human primordial germ cells and fetal ovaries. 1537–1553.
Mol Cell Endocrinol 234:1–10. Shibata, N., Tsunekawa, N., Okamoto-Ito, S., Akasu, R., To-
Ioannou, J.M. (1967) Oogenesis in adult prosimians. J Embryol kumasu, A. and Noce, T. (2004) Mouse RanBPM is a partner
Exp Morphol 17:139–145. gene to a germline specific RNA helicase, mouse vasa homolog
Johnson, J., Bagley, J., Skaznik-Wikiel, M., Lee, H.-J., Adams, protein. Mol Reprod Dev 67:1–7.
G.B., Niikura, Y., Tschudy, K.S., Tilly, J.C., Cortes, M.L., Stene, E., Stene, J. and Stengel-Rutkowski, S. (1987) A reanalysis
Forkert, R., Spitzer, T., Iacomini, J., Scadden, D.T. and Tilly, of the New York state prenatal diagnosis data on Down’s syn-
J.L. (2005) Oocyte generation in adult mammalian ovaries by drome and paternal age effects. Hum Genet 77:299–302.
putative germ cells in bone marrow and peripheral blood. Cell Telfer, E.E., Gosden, R.G., Byskov, A.G., Spears, N., Albertini,
122:303–315. D., Yding Andersen, C., Anderson, R., Braw-Tal, R., Clarke, H.,
Johnson, J., Canning, J., Kaneko, T., Pru, J.K. and Tilly, J.L. Gougeon, A., McLaughlin, E., McLaren, A., McNatty, K.,
(2004) Germline stem cells and follicular renewal in the postnatal Schatten, G., Silber, S. and Tsafriri, A. (2005) On regenerating
mammalian ovary. Nature 428:145–150. the ovary and generating controversy. Cell 122:821–822.
Jones, E.C. and Krohn, P.L. (1960) The effect of unilateral ova- Tilly, J.L. (1996) Apoptosis and ovarian function. Rev Reprod
riectomy on the reproductive lifespan of mice. J Endocrinol 1:162–172.
20:129–134. Toyooka, Y., Tsunekawa, N., Takahashi, Y., Matsui, Y., Satoh,
Jones, E.C. and Krohn, P.L. (1961) The effect of hypophysectomy M. and Noce, T. (2000) Expression and intracellular localization
on age changes in the ovaries of mice. J Endocrinol 21:497–509. of mouse Vasa-homologue protein during germ cell development.
Keeney, S., Baudat, F., Angeles, M., Zhou, Z.H., Copeland, N.G., Mech Dev 93:139–149.
Jenkins, N.A., Manova, K. and Jasin, M. (1999) A mouse ho- Viera, A., Parra, M.T., Page, J., Santos, J.L., Rufas, J.S. and
molog of the Saccharomyces cerevisiae meiotic recombination Suja, J.A. (2003) Dynamic relocation of telomere complexes
DNA transesterase Spo11p. Genomics 61:170–182. in mouse meiotic chromosomes. Chromosome Res 11:797–
Kingery, H.M. (1917) Oogenesis in the white mouse. J Morphol 807.
30:261–316. vom Saal, F.S., Finch, C.E. and Nelson, J.F. (1994) Natural history
Krarup, T. (1970) Oocyte survival in the mouse ovary after treat- and mechanisms of reproductive aging in humans, laboratory
ment with 9,10-dimethyl-1,2-benzanthracene. J Endocrinol 46: rodents, and other selected vertebrates. In: Knobil, E. and Neill,
483–495. J.D. (eds.). The physiology of reproduction. 2nd ed. Raven Press,
Mandl, A.M. (1959) A quantitative study of the sensitivity of Ltd., New York, pp. 1213–1314.
oocytes to x-irradiation. Proc R Soc Lond B Biol Sci 150:53–71. Waldeyer, W. (1870) Eierstock und Ei. Engleman, Leipzig.
Mandl, A.M. and Zuckerman, S. (1949) Ovarian autografts in Winiwarter, H. (1901) Recherche sur l’ovogenèse de l’ovaire des
monkeys. J Anat 83:315–324. mammifères (lapin et homme). Arch de Biol 17:33–200.
Mandl, A.M., Zuckerman, S. and Patterson, H.D. (1952) The Winiwarter, H. (1920) Formation de la couche corticale definitive et
number of oocytes in ovarian fragments after compensatory origine des oeufs definitifs dans l’ovaire de chatte. C R Soc Biol
hypertrophy. J Endocrinol 8:347–356. (Paris) 83:1403–1405.
Mauleon, P. (1969) Oogenesis and folloculogenesis. In: Cole, H.H. Wordinger, R., Sutton, J. and Brun-Zinkernagel, A.M. (1990) Ul-
and Cupps, P.T. (eds.). Reproduction in domestic animals. Ac- trastructure of oocyte migration through the mouse ovarian sur-
ademic Press, Davis, CA, pp. 187–215. face epithelium during neonatal development. Anat Rec 227:
Medvinsky, A. and Smith, A. (2003) Stem cells: Fusion brings 187–198.
down barriers. Nature 422:823–825. Zuckerman, S. (1951) The number of oocytes in the mature ovary.
Moore, C.R. and Wang, H. (1947) Ovarian activity in mammals Recent Prog Horm Res 6:63–108.
subsequent to chemical injury of cortex. Physiol Zool 20:300–321.