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Invited Reviews

Toxicologic Pathology, 36: 375-384, 2008


Copyright © 2008 by Society of Toxicologic Pathology
ISSN: 0192-6233 print / 1533-1601 online
DOI: 10.1177/0192623308315665

The Female Rat Reproductive Cycle: A Practical


Histological Guide to Staging
F. RUSSELL WESTWOOD
Global Safety Assessment, AstraZeneca, Macclesfield, Cheshire SK10 4TG, United Kingdom

ABSTRACT

During preclinical investigations into the safety of drugs and chemicals, many are found to interfere with reproductive function in the female rat.
This interference is commonly expressed as a change in normal morphology of the reproductive tract or a disturbance in the duration of particular
phases of the estrous cycle. Such alterations can be recognized only if the pathologist has knowledge of the continuously changing histological
appearance of the various components of the reproductive tract during the cycle and can accurately and consistently ascribe an individual tract to a
particular phase of the cycle. Unfortunately, although comprehensive reports illustrating the normal appearance of the tract during the rat estrous cycle
have been available over many years, they are generally somewhat ambiguous about distinct criteria for defining the end of one stage and the begin-
ning of another. This detail is absolutely essential to achieve a consistent approach to staging the cycle. For the toxicologic pathologist, this report
illustrates a pragmatic and practical approach to staging the estrous cycle in the rat based on personal experience and a review of the literature from
the last century.

Keywords: rat; reproductive; cycle; estrous; proestrus; estrus; metestrus; diestrus; histological; staging.

INTRODUCTION beginning of another. This detail is absolutely essential to


achieve a consistent approach to staging the cycle. Indeed, some
During preclinical investigations into the safety of drugs and
authors provide only a single description of regions of the tract
chemicals, many are found to interfere with the reproductive
from rats with a four-day estrous cycle, collected at a specific
function of the female rat (Yuan and Foley 2002). This interfer-
time on each day, as typical of a particular phase (Yuan and
ence is commonly expressed as a change in normal morphology
Foley 2002), which can lead to inconsistency when assessing
of the reproductive tract or a disturbance in the duration of
progressive and cyclical changes. My aim here is to illustrate,
particular phases of the estrous cycle. These alterations can be
for the toxicologic pathologist, a pragmatic and practical
recognized only if the pathologist has knowledge of the contin-
approach to staging the estrous cycle in the rat based on per-
uously changing histological appearance of the various com-
sonal experience and literature references from the last century.
ponents of the reproductive tract during the cycle and can
The images presented (hematoxylin-and-eosin–stained sections
accurately and consistently ascribe an individual tract to a par-
processed by standard histological techniques) are examples
ticular phase of the cycle. Indeed, functional alterations may be
from young adult undosed Wistar rats (Alderley Park strain) but
apparent only following such an assessment and may manifest
are entirely consistent with, and illustrative of, other strains.
themselves by a change in the number of animals in a particu-
I have also included descriptions of the appearance of the tract
lar phase of the cycle, a disturbance in the coordinated mor-
during aging. Some details of the associated hormonal changes
phology of the components of the tract, or abnormal appearance
are included in relation to the morphological changes, particu-
of a particular region. Unfortunately, although comprehensive
larly for the aging rat, but they are necessarily cursory, as this is
reports illustrating the normal appearance of the tract during
not the main purpose of the report. For a complete review of the
the rat estrous cycle have been available over many years
hormonal correlates, see The Physiology of Reproduction, edited
(Hubscher et al. 2005; Long and Evans 1922; Marshall 1960;
by Knobil and Neil (1994).
Yuan and Foley 2002), they are generally somewhat ambiguous
about distinct criteria for defining the end of one stage and the THE ESTROUS CYCLE

Address correspondence to: Dr. F. Russell Westwood, AstraZeneca,


Estrus was used first by Heape (1900) as a Latin adaptation
Mereside, Alderley Park, Macclesfield, Cheshire SK10 4TG, United Kingdom; of the Greek word oistros, meaning gadfly, sting, or frenzy to
e-mail: russell.westwood@astrazeneca.com. describe the “special period of sexual desire of the female”
375
376 WESTWOOD TOXICOLOGIC PATHOLOGY

(Freeman 1994). He also used: anestrus, nonbreeding season The stage of the reproductive cycle cannot easily be determined
when reproductive organs are quiescent; proestrus, animal com- from the appearance of the ovarian follicles in a species like the
ing on heat; metestrus, in the absence of conception, when estrus rat, which has a short estrous cycle. Follicles in virtually all
changes in the reproductive tract subside; and diestrus, repro- stages of development are generally present within the ovaries
ductive tract prepares for receipt of the ovum. Clearly, these at all phases of the cycle. However, the formation, progression,
“periods,” although they are accompanied by morphological and regression of the corpora lutea are somewhat synchronized,
changes, they are not described by them, although the morpho- and they can be used as an aid (Yuan and Foley 2002). It is of
logical appearances of the reproductive organs of the rat have note that three or more generations of corpora lutea may be
been well characterized under this general behavioral scheme, as present in an individual ovary from the preceding ovulatory
noted in the introduction. Vaginal smears are also widely used for cycles, as each corpora lutea persists morphologically for
this purpose; their characteristics have been described in detail twelve to fourteen days (Long and Evans 1922; Yuan and Foley
elsewhere (Hubscher et al. 2005; Long and Evans 1922; Maeda 2002). Thus, the use of corpora luteal morphology as an accurate
et al. 2000). However, they will also be discussed alongside the tool for estrous staging can be confusing. But the presence of
morphological features of the estrous stages under “Staging the particular morphologies of corpora lutea can be used to support
Cycle” below in support of the proposed method. staging, even though this is not definitive.
The onset of puberty in the female rat results from a cascade Table 1 summarizes the characteristics of the vaginal, uterine,
of events following establishment of a pulsatile luteinizing and ovarian morphology during the four estrous stages seen in
hormone (LH) release after the fourth postnatal week (approxi- the normally cycling adult rat. These cycles are described below.
mately thirty days of age) that leads to ovarian maturation
(Andrews and Ojeda 1981). Before this time, the reproductive
tract is inactive. This change in LH release is apparent eight to Diestrus
nine days before the first proestrus, and this period of change in Figure 1 illustrates the diestrus stage.
mode of LH release is considered anestrus (Urbanski and Ojeda
1985). The first proestrus, estrus, and diestrus periods then follow Vagina: At the start of diestrus, the vaginal epithelium is at its low-
(Advis et al. 1979; Ojeda et al. 1976). est level of approximately three to seven cells thick consisting
Ovulation occurs in the young adult laboratory rat every four simply of the stratum germinativum. The stratum germinativum
to five days throughout the year (Ojeda and Urbanski 1994). consists of stratum basale as a single layer of columnar epithe-
The normal length of the estrous cycle has been investigated over lial cells and an outer stratum spinosum as multiple layers of
many decades. Long and Evans (1922) used approximately 2000 polyhedral cells. There is a variable infiltration by leukocytes.
young adult rats derived from descendents of a cross between A reduction in the infiltration of leukocytes and a notable
several white females and a wild grey caught in Berkeley, epithelial cell proliferation occurs toward the end of the phase
California, and fed on table scraps and other supplements, with thickening of the epithelium, but with no clear stratum
including raw liver. They found marked individual variations, granulosum. The formation of a stratum granulosum is the defin-
with cycle lengths from three to thirty-eight days, but, excluding ing characteristic for the practical staging of the end of diestrus
those of more than eight days, the average was 4.8 days, and this and the beginning of proestrus. The vaginal smear is consistent
duration is reasonably consistent with findings from subsequent with this histological appearance, which is characterized by
investigators (Astwood 1939; Blandau et al. 1941). Based on little mucus with some leukocytes, nucleated basophilic cells,
vaginal smears, the duration of the individual components of and occasional vacuolated cells.
the estrous cycle for rats with a four- or five-day cycle are
proestrus, twelve to fourteen hours; estrus, twenty-five to Uterus: At the start of diestrus, the uterus is small and inactive,
twenty-seven hours; metestrus, six to eight hours; and diestrus, and the horns lack a prominent vasculature and generally show
fifty-five to fifty-seven hours (Astwood, 1939; Hartman 1944; a slit-like lumen. They are lined by a low cuboidal or columnar
Long and Evans 1922; Mandl 1951). However, as noted above, epithelium showing occasional degenerate cells. There are few
many authors refer to the day of the cycle, with each period mitoses at the start of the stage and the glands are particularly
having its own day, and those in a five-day cycle generally inactive, but there is some increase in activity during the pro-
showing either an extra day of vaginal cornification (extra day gression of the phase. Also, toward the end of the phase, a slight
of estrus) or an extra day of leukocyte infiltration (extra day of edema of the stroma adjacent to the endometrial epithelium can
diestrus) (vom Sall et al. 1994). be seen.
STAGING THE CYCLE
Ovaries: By diestrus, in the ovary, the newly formed corpora
Complete longitudinal sections of the vagina and cervix, lutea from the previous ovulation have attained their maximal
transverse sections of the mid portion of both uterine horns, and size, and this is the best ovarian marker for diestrus, although
medial sections of both ovaries are the minimal requirement for degenerate corpora lutea will also be present. Vacuoles are
an adequate evaluation. commonly present, particularly in the cells in the center of these
Observation of the coordinated morphology of the vagina large corpora lutea, indicative of active steroidogenesis, and early
and uterus is key to consistent staging of the estrous cycle, and fibrous tissue formation may be seen in what was previously
this will be the main focus of the descriptions provided here. the central, fluid-filled cavity.
Vol. 36, No. 3, 2008 STAGING THE FEMALE RAT REPRODUCTIVE CYCLE 377

TABLE 1.—Summary of the defining histological features of the rat female reproductive tract
during diestrus, proestrus, estrus, and metestrus.

Vagina Uterus Ovaries

Diestrus Start defined by epithelium at lowest level with Small, avascular, slit-like lumen. Lined by low Large corpora lutea. May be finely
variable leukocyte infiltration. Subsequent columnar epithelium. Initially few mitoses, but vacuolated. Fibrous tissue formation
epithelial proliferation and thickening (no some increase during phase. Only occasional in central cavity.
clear stratum granulosum) with reduction in degenerate cells. Stromal edema at end of stage.
leukocyte infiltration.
Proestrus Mitotic figures present. Occasional polymorphs. Epithelium cuboidal to columnar. Mitoses present Corpora lutea often degenerate.
Little if any degeneration or desquamation. in epithelial cells with little or no degeneration Cytoplasmic vacuoles generally
Formation of stratum granulosum (defines and little inflammatory cell infiltration. present. Fibrous tissue proliferation
start), superficial mucoid layer and stratum Dilatation, particularly toward end of stage. in central cavity.
corneum progressively. At end of stage, fully
cornified and generally showing superficial
mucoid layer with some desquamation of
mucoid cells.
Estrus Progressive shedding of superficial mucoid and Start of estrus defined by appearance of Degenerate corpora lutea often present.
cornified layers. Reduction in height of notable degeneration/necrosis of epithelial Some small corpora lutea with
epithelium. Cell debris present. Loss of mitotic cells, glands generally first. Loss of mitotic basophilic cell cytoplasm, central
figures. Progressive leukocyte infiltration. activity. Leukocyte infiltration. Dilatation may fluid-filled cavity, and no fibrous
persist to late estrus. tissue.
Metestrus Start defined by virtually complete detachment Continued degeneration of endometrial epithelial Corpora lutea may still contain fluid
of cornified layer (generally residual squames cells. Return of mitotic activity; both (mitotic cavity. Smaller than at diestrus.
in lumen). Continued desquamation with loss of activity and degeneration) seen together. Slightly basophilic cells. Generally
stratum granulosum and upper germinativum. devoid of fibrous tissue.
Leukocyte infiltration.

Proestrus (the appearance of notable endometrial epithelial cell degen-


eration marks the end of proestrus/start of estrus) and little
Proestrus is illustrated in Figure 2.
inflammatory cell infiltration, although mitotic figures disappear
at the end of the stage. The endometrial vasculature becomes
Vagina: Formation of the stratum granulosum over the stratum
more prominent, and the stroma can show some edema, with
germinativum of the vaginal epithelium, consisting of flattened
the lumen generally becoming markedly dilated toward the
epithelial cells containing many keratohyalin granules, marks
end of the stage.
the start of proestrus. The vaginal epithelium shows mitotic
figures throughout the stage, although they are less numerous
at the end. Following the early formation of the stratum granu- Ovaries: Ovarian corpora lutea are often degenerate, with central
losum, there is a progressive development of the superficial fibrous tissue formation, and the cells commonly contain cyto-
mucoid layer (stratum mucification according to Yuan and Foley plasmic vacuoles.
[2002], or rete mucosum according to Hubscher et al. [2005]),
characterized by layers of cuboidal to ovoid cells with mucin- Estrus
containing cytoplasmic vacuoles, and the formation of a stratum
Figure 3 illustrates estrus.
corneum of dense, cornified cells. There is little if any degen-
eration or desquamation during the early- or mid-proestrus Vagina: In the vagina, there is a loss of mitotic figures and a
period, and occasional infiltrates of leukocytes are seen. At the progressive shedding of the superficial mucoid and cornified
end of the stage, the epithelium is fully cornified and generally layers during estrus, with a reduction in the height of the epithe-
shows a superficial mucoid layer exhibiting some desquama- lium, and cell debris is present in the lumen. There is also a vari-
tion of mucoid cells. The vaginal smear is again consistent with able and progressive leukocyte infiltration. The end of the stage
this histological appearance, showing a disappearance of leuko- is characterized by detachment of the cornified epithelium,
cytes and the presence of sheets of, or isolated, nucleated epithe- although some may persist, particularly adjacent to the vaginal
lial cells, which become progressively acidophilic with the opening. So, if there is a predominance of attached cornified
appearance of cornified cells. epithelium, then the stage is still “estrus.” Virtually complete
detachment of the cornified epithelium of the vagina marks the
Uterus: During this stage, the uterine endometrial lining end of estrus and the start of metestrus. The vaginal smear shows
progresses to large cells, forming a tall cuboidal to columnar non-nucleated cornified cells, which by late estrus have dimin-
epithelium. There are frequent mitoses with only limited or no ished when leukocytes appear. Also, large basophilic epithelial
epithelial cell degeneration of the glands and lining epithelium cells are present in the smear.
378 WESTWOOD TOXICOLOGIC PATHOLOGY

FIGURE 1.—Diestrus. Black bars = 100 μm; red bars = 1000 μm.

Uterus: Changes in the endometrial epithelium define the start activity and leukocyte infiltration. Luminal dilatation may per-
of estrus, with the appearance of cellular degeneration/necrosis sist into late estrus, although this is generally not the case.
in the glands first, followed by the lining epithelium, which Some endometrial epithelial mitotic activity returns by the end
becomes quite marked. It is accompanied by a loss of mitotic of estrus.
Vol. 36, No. 3, 2008 STAGING THE FEMALE RAT REPRODUCTIVE CYCLE 379

FIGURE 2.—Proestrus. Black bars = 100 μm; red bars = 1000 μm.

Ovaries: Degenerate ovarian corpora lutea are often present at Metestrus


estrus, but others (the newly formed corpora lutea) are small,
Metestrus is illustrated in Figure 4.
with cells showing a basophilic cytoplasm and occasionally a
central fluid-filled cavity retained from the follicular stage. Vagina: The start of metestrus is marked by the mid region of
Central fibrous tissue is generally not present. the vagina showing a complete detachment of the cornified
380 WESTWOOD TOXICOLOGIC PATHOLOGY

FIGURE 3.—Estrus. Black bars = 100 μm; red bars = 1000 μm.

epithelium, generally with residual squames present in the lumen. spinosum). There is an accompanying variable leukocyte infil-
As noted above, some cornified epithelium may persist, partic- tration. As noted under diestrus, the end of metestrus/start of
ularly adjacent to the vaginal opening. There is a continued desqua- diestrus is marked by the epithelium reaching its lowest level.
mation of epithelial cells throughout the stage, with a progressive The smear during this stage shows leukocytes, a few cornified
loss of the stratum granulosum and upper germinativum (stratum cells, and basophilic cells.
Vol. 36, No. 3, 2008 STAGING THE FEMALE RAT REPRODUCTIVE CYCLE 381

FIGURE 4.—Metestrus. Black bars = 100 μm; red bars = 1000 μm.

Uterus: During metestrus, the uterine endometrial epithelium THE CYCLE IN AGING RATS
shows continued vacuolar degeneration, but also a marked return
of mitotic activity, so both are seen together. There is also a During the midlife period in rats, the estrous cycle length
variable leukocyte infiltration increases, and some histological inconsistency is occasionally
seen between the various components of the tract. Progesterone
Ovaries: Ovarian corpora lutea may still contain a fluid-filled plays an important role in influencing cycle length in rodents
central cavity generally devoid of fibrous tissue, and the cyto- (Nequin et al. 1979), and progesterone and estrogen act syner-
plasm of the new corpora lutea are somewhat less basophilic gistically (Morali and Beyer 1979). The estrogen/progesterone
than at estrus but smaller than that seen at diestrus. ratio is elevated for prolonged periods before ovulation in most
382 WESTWOOD TOXICOLOGIC PATHOLOGY

FIGURE 5.—Persistent estrus and repetitive pseudopregnancy.

middle-aged rats, and this age-related deficit in progesterone Persistent Estrus


has a significant responsibility for this increase in cycle length
Persistent estrus is illustrated in Figure 5.
(vom Saal et al. 1994). At any time from six to eighteen
months, but seldom later, following lengthening of the cycle or Subsequent to the lengthening of the estrous cycle, most
interspersed long cycles, a constant state of vaginal cornifica- aging females between six and eighteen months of age exhibit
tion or “persistent estrus” can be observed (vom Saal et al. periods of persistent estrus, with constant sexual receptivity
1994). This may be followed by states in which cycles are ten (Felicio et al. 1984). This state results from estradiol levels
to fourteen days long, suggestive of a prolonged luteal phase, being maintained at a tonic level (Lu et al. 1979; Nelson et al.
which is termed “repetitive pseudopregnancy” (Nelson and 1981), reflecting the presence of numbers of ovarian follicular
Felicio 1985). The final stage of reproductive senescence is cysts (Gosden, Laing, Flurkey et al. 1983) and a tall columnar
“persistent anestrus” (vom Saal et al. 1994). endometrial epithelium. The tonic estrogen secretion stimulates
Vol. 36, No. 3, 2008 STAGING THE FEMALE RAT REPRODUCTIVE CYCLE 383

FIGURE 6.—Persistent anestrus. Black bars = 100 μm; red bars = 1000 μm.

cornification of the vaginal epithelium, so this state is also referred Persistent Anestrus
to as persistent vaginal cornification (vom Saal et al. 1994). Low
Figure 6 illustrates persistent anestrus.
levels of progesterone during this period (Miller and Riegle 1980;
Nelson et al. 1981) are considered responsible for the maintenance Old rats eventually reach a final stage of reproductive senes-
of this state (vom Saal et al. 1994) and are associated with an cence. This is termed persistent anestrus, or persistent diestrus.
absence of corpora lutea (Gosden, Laing, Flurkey et al. 1983). After the cessation of ovulation and entry into persistent estrus,
there is still some maturation of follicles, followed by atresia
without ovulation, culminating in complete follicular depletion
Repetitive Pseudopregnancy (Gosden, Laing, Felicio at al. 1983; Gosden, Laing, Flurkey et
Repetitive pseudopregnancy is illustrated in Figure 5. al. 1983). Morphological features of the reproductive tract are
characterized by a low, somewhat mucified vaginal epithelium
In young adult female rats, whether sterile or fertile, for the and a low inactive endometrial epithelium. The loss of follicles
first nine or ten days after mating, prolactin secreted by the results in estradiol levels similar to those seen in ovariec-
pituitary maintains the corpora lutea. The placenta of a suc- tomized rats (vom Saal et al. 1994). There are low progesterone
cessfully mated female then takes over (Smith 1980; Smith et al. levels, reflecting the absence of corpora lutea (Lu et al. 1979),
1975). If mating is unsuccessful, the corpora lutea degenerate and high prolactin levels, generally from pituitary tumors,
by Day 12 or 13. which have an inhibitory effect on LH (vom Saal et al. 1994).
Therefore, the repetitive states of pseudopregnancy that can
occur following persistent estrus in aging rats are typically ten
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