Debate
The functional life span of the ovaries is dictated in large part by the number of
oocytes present, a number that is known to decline precipitously during both fetal
development and postnatal life. Furthermore, studies from numerous laboratories have
shown that exposure of the ovaries to a variety of pathological insults, such as anti-cancer
therapies and environmental toxicants, accelerates oocyte and follicle depletion,
consequently hastening the time at which ovarian failure is observed. Accordingly,
over the past several years a tremendous amount of research effort has been expended
to uncover the genetic and molecular mechanisms responsible for determining the size
of the follicle reserve endowed at birth as well as the rate at which this stockpile
of follicles is subsequently depleted [reviewed in [1,2]]. Since, however, there are
no known serum markers that can be used to accurately estimate the number of follicles
in the ovaries – in particular follicles at the earliest stages of development (primordial,
primary, small preantral) – some type of histomorphometric evaluation of the ovary
is generally employed.
Although conceptually this sounds like a rather straightforward, albeit tedious, procedure,
the total number of follicles reportedly present in the ovaries of a particular species
at any given time in life can vary by 10-fold or more, depending on the study. In
fact, Pepling and Spradling [3] recently commented that "reports of germ cell number
in fetal and neonatal mouse female gonads have varied from 3,500 to 30,000 but these
experiments utilized different strains, different developmental times, and different
methods". These authors went on to state that "further studies will be required to
confirm the reality of such a large variation in the number of germ cells in different
strains" [3]. Such studies, using one outbred and five inbred mouse strains, have
recently been completed [4]. It was demonstrated that the total number of follicles
present in neonatal life, as well as the size of the primordial follicle reserve,
does vary depending on strain. In fact, up to 2-fold differences in follicle numbers
between different strains was observed [4]. However, as striking as this finding is,
it does not account for even one-quarter of the 10 (or more)-fold variation in absolute
follicle numbers reported by different laboratories studying the mouse ovary.
Probably the most widely used approach for estimating follicle numbers is one based
on a histological sampling of the total ovarian mass for the number of primordial,
primary and preantral follicles, often followed by the application of a 'correction
factor'. This approach essentially entails that an ovary is fixed, paraffin-embedded
and serially sectioned, usually at 6–8 μm widths. The serial sections are placed in
order on glass microscope slides and stained with a vital dye. Depending on the study,
every fifth to every tenth section is then analyzed by light microscopy for the presence
of primordial, primary and preantral follicles. The starting section is usually selected
randomly (for example, if one is counting follicles in every fifth section, any one
of the first five sections could be used to start the process), and only those follicles
in which the nucleus of the oocyte is clearly visible are scored. Assuming a random
distribution of follicles at the various stages of development throughout the ovary,
a close estimate of total follicle numbers per ovary can theoretically be obtained
if the ovarian sections not included in the analysis are accounted for (see below).
How reliable is this technique? A paper by Hirshfield and Midgley [5] published 25
years ago directly tested the accuracy of estimating the number of follicles of various
size categories in the adult rat ovary by sampling every fifth section versus the
actual number derived from counting follicles in every section. For those values obtained
by evaluating every fifth section, the cumulative follicle counts were multiplied
by a factor of 5 to account for the fact that four-fifths of the ovary was not analyzed.
After studying the results, the authors concluded, "the discrepancy between the two
estimates was so great that this method [sampling every fifth section] was discarded
because it was so inaccurate" [5].
This conclusion is somewhat puzzling, however, based on the fact that the estimated
number of total follicles, derived from sampling every fifth section, varied from
the actual number of total follicles by only 3–11% [5]. Furthermore, subsequent studies
published by Hirshfield and colleagues, which describe results from the assessment
of follicle numbers in mice, utilized the procedure of sampling every fifth [6] or
every tenth [7] section. Accordingly, the approach of sampling a fraction of the ovary
appears sound and accepted, a conclusion reinforced by a series of experiments published
in 1997 from a collaborative effort between the National Center for Toxicological
Research and the National Institute of Environmental Health Sciences [8,9]. Assuming,
then, that the sampling procedure provides a reliable estimate of the number of follicles
per ovary, there remains an issue that is probably the principal cause of the large
discrepancy in follicle numbers reported by various laboratories. This issue concerns
the correction factor that is used to account for that proportion of the ovary not
included in the sampling analysis.
For example, in 1978 Hirshfield and Midgley used a correction factor of 5 in their
studies that examined every fifth section of the ovary, based on the fact that only
one-fifth of the total ovarian mass was analyzed [5]. This correction factor was used
again by Hirshfield and co-workers in studies published 19 years later, which evaluated
the effects of chronically elevated luteinizing hormone on the primordial follicle
pool in mice after sampling every fifth ovarian section [6]. However, in 2001, results
from a study co-authored by Hirshfield of the effects of ectopic Bcl-2 expression
on the primordial follicle endowment in the rodent, ovary used a correction factor
of 80 after sampling every tenth ovarian section [7]. The rationale for this was that
"the number of primordial, primary, or preantral/antral follicles present in the marked
sections was multiplied by 10 to account for the fact that every tenth section was
used in the analysis and by 8 to account for section thickness" [7]. This type of
correction factor has also been used routinely by others [e.g., [10-12]] and us [e.g.,
[13-21]] in studies of oocyte and follicle development in the rodent ovary. Thus,
which correction factor should be applied?
Intuitively, section thickness would seem to be an important variable. For example,
if one serially sections a day 4 postpartum mouse ovary, which averages 400 μm in
length (n = 25 mice), in 6- versus 8-μm widths, there will be approximately 67 6-μm
sections and 50 8-μm sections. Irrespective of section thickness, however, only one-fifth
of the ovary will be sampled in both cases, and thus multiplying all values by 5 to
account for the remainder of the ovary not sampled should provide a reasonable estimate
of absolute follicle numbers. However, if one counts every fifth section, ovaries
sectioned at 6-μm widths would have approximately 3 more sections included in the
counting process than ovaries sectioned at 8-μm widths. This suggests that an ovary
sectioned at 6-μm widths will probably have more follicles tallied than the same ovary
sectioned at 8-μm widths. Therefore, accounting only for that proportion of the ovary
not included in the sampling analysis, while ignoring the effect of section thickness,
does not completely address the issue of obtaining a realistic estimate of absolute
follicle numbers. On the other hand, multiplying the values by section thickness probably
gives spuriously high numbers, leaving open the question of how to accurately account
for the effect of section thickness.
Whatever the case, a much more fundamental question remains. Does the application
of a correction factor, regardless of whether it is 5 or 80, change the final conclusions
of any study published to date? Absolutely not, as long as the correction factor was
applied uniformly to all of the values used to produce the final results for analysis.
Indeed, in a study of the relationship between ovarian innervation and folliculogenesis
in the rat ovary, Malamed and colleagues estimated follicle numbers by determining
the mean total number of follicles per section after sampling every fifth or sixth
section [22]. This mean value was then multiplied by the total number of sections
of the ovary to obtain an estimate of the total number of follicles per ovary. The
authors state that "values obtained by this method tend to be spuriously high because
the thickness of each section (5 μm) is about one third the mean diameter of a primordial
follicle" [22]. Importantly, however, the authors point out that "regardless of the
validity of the estimated absolute values for numbers of follicles per ovary, the
uniform application of the method of calculation to the data from ovaries of all ages
examined should produce valid relative values" [22]. This is a key point since in
all studies cited herein that used a correction factor, the application of the correction
factor was uniform. Therefore, the purpose of this commentary is not to call into
question the validity of conclusions drawn from past studies of follicle numbers in
the rodent ovary by any investigators, but rather to explore the basis of the variance
in follicle numbers per ovary reported by different laboratories.
It should be noted that there are also many studies that have used the sampling procedure
to estimate follicle numbers in mouse or rat ovaries without the application of any
correction factor prior to data presentation [e.g., [23-26]]. The conclusions drawn
from these studies are no more or less valid than conclusions drawn from studies that
have utilized a correction factor, despite the fact that the absolute values for follicle
numbers are quite different among the various reports. Moreover, other types of follicle
counting procedures have been reported for studies of the mouse ovary. These include
counting every primordial follicle, as well as every growing follicle with the oocyte
nucleus clearly visible, in every second section [27], to more intensive procedures
involving the use of germ cell-specific markers with correction factors for oocyte
and ovarian volume [3]. For example, in work published by Pepling and Spradling [3],
the volume of each ovary analyzed was measured. A representative section from the
ovary was then immunostained for the germ cell-specific protein, Vasa, and the number
of Vasa-labeled cells in this section was counted. The authors then stated that "using
the average diameter of a germ cell, the fraction of the ovarian volume represented
by the counted section was then calculated. This allowed the number of germ cells
in the whole ovary to be computed" [3].
With this information in mind, what assistance will this commentary be to the field
of ovarian biology? If one thinks only in terms of the final conclusions drawn from
the various studies of follicle endowment and depletion, the answer to this question
is 'none' since the validity of these conclusions are not in question. However, if
one thinks in terms of the absolute values for follicle numbers per ovary reported
by different laboratories, a discussion of the advantages and drawbacks of the various
approaches used to assess follicle numbers may begin to settle an emerging controversy.
At least for our research group, this commentary has forced us to critically evaluate
the correction factor we have employed in past studies of the postnatal mouse ovary
[e.g., [13-21]], which attempted to account for both the fraction of the ovary sampled
(×5, for every fifth section) and section thickness (×8, for 8 μm). Indeed, as indicated
in the papers by Canning et al. [4] and Takai et al. [28], we now believe that when
sampling every fifth section a correction factor of 5, rather than 40, provides a
more reasonable estimate of absolute follicle numbers per ovary. Will this change
anything? We believe it will, if one considers the following example. Using a germ
cell counting procedure discussed earlier, Pepling and Spradling recently estimated
that CD-1 female mice possess approximately 2,400 oocytes in their ovaries 4 days
after birth [3]. We recently estimated that CD-1 females possess approximately 5,000
oocytes (follicles) at day 4 postpartum [4], using the every fifth 8-μm section sampling
procedure with a correction factor of 5. Although there still remains a discrepancy
in the absolute number of follicles per ovary between the two studies, a 2-fold difference
is a vast improvement over the 16-fold difference that would have occurred if a correction
factor of 40 rather than 5 were used.