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2 CORA ET AL. TOXICOLOGIC PATHOLOGY
HISTORICAL PERSPECTIVE
The term estrus was first used (as oestrus2) in Heape’s
1
1.
Note that the word estrus is a noun, while the word estrous with an ‘‘o’’ is an
adjective, as in ‘‘the estrous cycle’’; the two words are not interchangeable.
2.
As is widely known, the British English, Greek-derived spelling of estrus FIGURE 1.—Historical representation of select stages of the rat estrous
with an ‘‘o’’ as in oestrus and the oestrous cycle and the use of an ‘‘o’’ in cycle as presented in Long and Evans (1922). These eloquent pencil
the spelling of the stages (pro-oestrus, metoestrus, dioestrus) is still used drawings illustrate proestrus (A), metestrus (B), and diestrus (C).
today in British-based publications. Additionally, older literature, regardless
of country of origin, generally used British English. This is important to note
and Evans (1922) eloquently expressed the importance of
when undertaking a comprehensive literature search on the subject of the Stockard and Papanicolaou’s (1917) findings with the
estrous cycle and its stages. following:
Vol. XX, No. X, 2015 VAGINAL CYTOLOGY IN RATS AND MICE 3
FIGURE 8.—A large nucleated epithelial cell containing many small FIGURE 10.—A keratin bar (arrow) is observed in a vaginal smear of
cytoplasmic granules (arrow) from a Sprague-Dawley rat. Modified estrus from a Sprague-Dawley rat. Note that many bacteria are either
Wright–Giemsa stain. Original oil objective magnification of 100. adhered to the cells or free in the background. Modified Wright–
Giemsa stain. Original objective magnification of 40.
FIGURE 11.—Estrous cycle wheels. Visual representation of the cell types and relative proportion of each cell type present during the 4 stages of the
estrous cycle in the mouse and rat. The size of each quadrant does not directly correlate to the length of each stage. Adapted from Byers et al.
(2012). The mouse wheel was modified from its original form to create the rat wheel. Adaptations and modifications by David Sabio.
TABLE 1.—Basic classification of the stages of estrous cycle based on cell types and relative numbers of these cell types in vaginal smears.
Stage Neutrophils Small nucleated epithelial cells Large nucleated epithelial cells Anucleated keratinized epithelial cells Relative cell density
slide. Conventionally, the cycle is divided into the four stages either proestrus or estrus. Figure 11 and Table 1 are graphical and
of proestrus, estrus, metestrus, and diestrus; however, some tabular representations of the estrous cycle adapted from various
researchers use a simple proestrus–estrus–diestrus classifica- sources and serve as good adjuncts to the subsequent narrative.
tion, while others subdivide the four stages, record transitional
stages, or roughly quantify the individual cell populations Proestrus
(Goldman, Murr, and Cooper 2007). The following will define Proestrus is a short stage, lasting an average of 14 hr in rats
and characterize the range in appearance of the four classic and less than 24 hr in mice (Astwood 1939; Long and Evans
stages, including species differences and a brief discussion of 1922; Mandl 1951; Allen 1922; Grasso, Rozhavskaya, and Rie-
transitional stages, from which criteria for stage subdivisions chert 1998). The predominant feature of this stage is the pres-
or transitional stages can be based. ence of small, round, nucleated epithelial cells of relatively
One general approach to staging vaginal smears is to first uniform appearance and size (Figures 6 and 12); these cells
assess for the presence of neutrophils—are there a good number may stain deeply basophilic. Often they will be seen in cohe-
of neutrophils observed or just an occasional one? If neutrophils sive clusters (grape clusters), sheets, or strands. However,
are a dominant feature, or consistently observed, the stage is either cohesive clusters, sheets, and strands are not always observed,
metestrus or diestrus; if neutrophils are rare to absent, the stage is especially in low cellularity samples, and should not be thought
Vol. XX, No. X, 2015 VAGINAL CYTOLOGY IN RATS AND MICE 9
FIGURE 12.—Vaginal smears of proestrus from Sprague-Dawley rats characterized by high numbers of small nucleated epithelial cells found
individually and in cohesive clusters (arrows). Proestrus appears the same in mice. Plate D shows the appearance of a so-called epithelial
‘‘strand’’ that is typical of proestrus. Modified Wright–Giemsa stain (A, D) and Toluidine blue O stain (B, C). Original objective magni-
fication of 10 (A, B), 20 (C), or 40 (D). Plate C is in Elmore et al. Proceedings of the 2014 National Toxicology Program satellite
symposium. Toxicol Pathol, 43:10-40, 2015.
of as a prerequisite in the determination of proestrus; cells may Riechert 1998). It is characterized by the presence of predomi-
only be seen individually throughout the smear. Typically, no nately anucleated keratinized epithelial cells (Figures 13 and
neutrophils will be seen. That said, rare to occasional neutro- 14). Numerous bacteria may be observed adhered to the cells
phils can be found in early proestrus as the rodent would have or free in the background. Occasional nucleated epithelial cells
recently transitioned from diestrus into proestrus. Relatively can be observed throughout the stage of estrus, and neutrophils
low numbers of large epithelial cells and keratinized anu- are absent or occasionally observed in late estrus. The first and
cleated cells may also be observed. As the cycle approaches second half of estrus, while consisting mostly of anucleated
estrus, keratinized cells will become more abundant. The pres- keratinized cells, have distinct differences in their appearances
ence of low numbers of neutrophils, or large and anucleated and also differ between rats and mice. These differences were
epithelial cells, does not preclude the diagnosis of proestrus distinct enough that Young, Boling, and Blandau (1941) and
when the predominating feature of the smear are the small, round Allen (1922) had different terminology or subdivisions for the
epithelial cell population. first and second ‘‘phases’’ of estrus in rats and mice, respec-
tively. Long and Evans (1922) described these different phases
Estrus in rats but did not distinguish them as subdivisions of estrus.
Estrus duration ranges between 24 and 48 hr in rats and In a mouse when 2 consecutive days of estrus are observed,
between 12 and 48 hr in mice (Astwood 1939; Long and Evans these different phases are generally appreciated; however, the
1922; Mandl 1951; Allen 1922; Grasso, Rozhavskaya, and second phase in rats occurs late in estrus and will not always
10 CORA ET AL. TOXICOLOGIC PATHOLOGY
FIGURE 13.—Representative photomicrographs of the first (A, C) and second (B, D) ‘‘phases’’ of estrus in mice. The first phase typically has
smaller anuclear epithelial cells that are arranged in loose clusters reminiscent of proestrus. The anuclear epithelial cells of the second phase are
generally larger, more evenly dispersed, and higher in numbers. Plates C and D are 2 consecutive days of estrus from the same B6C3F1/N mouse.
Modified Wright–Giemsa stain (A, B) and Toluidine blue O stain (C, D). Original objective magnification of 10.
be seen (i.e., it will be missed) even if 2 days of estrus are (Figures 7 and 14C–E). These cells are small to large; round,
recorded. In mice, at the start of estrus, the anucleated cells are oval, or spindle shaped with smooth to irregular borders; and
smaller and are typically arranged in loose clusters or sheets sometimes stain deeply basophilic. Long and Evans (1922)
reminiscent of proestrus (Figure 13A and C). As estrus pro- describe this phase as such:
gresses, the cell numbers generally increase, and the cells
appear larger and more evenly dispersed and can be seen in It is interesting that in a certain proportion of animals the
stacks or layers (Figures 9 and 13B, D). Allen (1922) notes this epithelial desquamation of the mucosa may proceed
distinction in mice as follows: somewhat further than to the cornified cell layer before
leucocytes come in, and hence we may have . . . Stage
By standardizing the technique of smear preparation, . . . Three [estrus] immediately succeeded by a few hours of a
[the different phases of estrus] are easily differentiated, stage in which rather large, spindle-like, nucleated
although the basis for this division is the number and epithelial cells are shed. In such cases, however, vestiges
clumping rather than any change in the [anucleated, of the cornified cell are always present and when leuco-
keratinized] character of the cells themselves. (p. 306) cytes appear three cell types area actually found. (p. 21)
In late estrus of rats, before the emergence of neutrophils and Young, Boling, and Blandau (1941) referred to the large oval
metestrus, higher numbers of nucleated epithelial cells appear nucleated epithelial cells of late estrus in the rat as ‘‘pavement
Vol. XX, No. X, 2015 VAGINAL CYTOLOGY IN RATS AND MICE 11
FIGURE 14.—Vaginal smears of estrus (A, B) and late estrus (C–F) in several Sprague-Dawley rats. Plates A and B represent a typical estrus stage
with high numbers of anucleated epithelial cells. Vaginal smears of the late estrus phase (C–F) are characterized by the presence of round-, oval-,
and spindle-shaped nucleated epithelial cells interspersed among anucleated epithelial cells. Modified Wright–Giemsa stain (A–C, E) and Tolui-
dine blue O stain (D, F). Original objective magnification of 10 (A, C, D), 20 (F), or 40 (B, E). Plate A is in Elmore et al. Proceedings of the
2014 National Toxicology Program satellite symposium. Toxicol Pathol, 43:10-40, 2015.
12 CORA ET AL. TOXICOLOGIC PATHOLOGY
cells’’ and their presence became an indication that metestrus diestrus usually count metestrus as a ‘‘diestrus’’ day (Goldman,
was fast approaching. This phase of late estrus should not be Murr, and Cooper 2007). While distinguishing and recording
mistaken for proestrus. In general, the nucleated cells of proes- metestrus is recommended, if it cannot be certain if a smear
trus have a higher N:C ratio, are round to slightly oval, and is (late) metestrus or diestrus, then it is best to be as consistent
have smooth cell borders. as possible and pragmatically ‘‘error’’ on the side of diestrus;
In conclusion, estrus is the stage predominated by kerati- there is minimal value in overly scrutinizing a late metestrus/
nized anucleated epithelial cells. Neutrophils may be occasion- early diestrus smear.
ally seen in late estrus, as the rodent is transitioning to
metestrus; but once they are observed with regularity through- Diestrus
out all fields, the stage should be interpreted as metestrus.
Diestrus is the longest stage of the estrus cycle with an aver-
Relatively high numbers of nucleated epithelial cells are
age duration of 48–72 hr in both mice and rats (Astwood 1939;
interspersed with the keratinized cells in late estrus of rats.
Long and Evans 1922; Mandl 1951; Allen 1922; Grasso, Roz-
havskaya, and Riechert 1998). This stage is characterized by a
Metestrus substantial decrease in the number of anucleated keratinized
It is well documented that metestrus is a short stage of 6–8 epithelial cells (but not necessarily an absence of) as the animal
hr in rats (Astwood 1939; Long and Evans 1922; Mandl 1951). transitions out of metestrus. Overall cellularity is moderate to
Based on the limited reports in the literature and personal expe- low with a combination of neutrophils, small and large
rience, the duration of metestrus in mice seems to be longer nucleated epithelial cells, and low numbers of anucleated ker-
than rats and can last up to 24 hr (Allen 1922; Grasso, Rozhavs- atinized cells (Figures 4 and 17). Neutrophil numbers can vary
kaya, and Riechert 1998) and, although infrequent, metestrus but are usually higher in number relative to the epithelial cells
(as described herein) has been identified for 2 consecutive days with smears sometimes being exclusively neutrophilic. Occa-
in control mice on reproductive studies (personal experience; sionally, in early diestrus, neutrophils may still appear in
Figure 15A and D). clumps. It is not unusual for diestrus smears to have a very low
Metestrus is characterized by a combination of anucleated cellularity, especially on day 2 or 3 of diestrus, with only a
keratinized epithelial cells and neutrophils. In mice, nucleated sparse scattering of neutrophils and epithelial cells (Figure
cells may appear occasionally throughout metestrus (Figure 17E). In late diestrus, the epithelial cells may become more
15). In rats, the small and large nucleated cells of late estrus are round or be organized in small clumps, indicating proestrus the
present in low to moderate numbers throughout the stage (Fig- next day; however, neutrophils will still be consistently
ure 16). observed (Figure 17F).
In early metestrus, neutrophils are interspersed with the During diestrus, macroscopic vaginal excretions are low.
epithelial cells and are sometimes tightly packed together or When extrections are present, however, they can be viscous
clumped around the cells; the epithelial cells usually predomi- and stringy (i.e., mucous) and this is especially true during
nate but may be in equal proportion to the neutrophils. As times of persistent diestrus. This excretion is sometimes aspi-
metestrus progresses, neutrophils become very high in num- rated during vaginal cytology sample collection. It will stain
ber—outnumbering epithelial cells by as much as 10-fold and appear as a thick pink to blue-violet material with
(Hubscher, Brooks, and Johnson 2005)—and the smear will entrapped neutrophils and epithelial cells. The cells may be dis-
be highly cellular and dense. Neutrophil and epithelial cell torted or elongated in the strings of mucous (Figure 18).
numbers decrease by late metestrus, with a decrease in smear
cellularity, before the transition to diestrus. Allen (1922) gives Transitional stages
the following simple description of metestrus in mice: For any given study, transitional stages will be encoun-
tered (Figure 19). There are several strategies or paradigms
. . . the smear shows polymorphonuclear leukocytes that can be used for the recording of a transitional stage. The
among the red, horny elements [anucleated epithelial strategy used would depend on the reason or purpose for doc-
cells]. A few polymorphs and many cornified cells is an umenting the stages of the estrous cycle; for example, a study
early [metestrus] stage, and a heavy leucocytic infiltra- designed to document potential changes in the frequency or
tion and decrease in number of the cornified cells is a late duration of a particular estrous stage may require a different
one. (p. 306) transitional stage paradigm than a study evaluating the
estrous cycle in a group of young rodents to determine sexual
The separation between where metestrus ends and diestrus maturity or their first ovulation. Following are 2 of the more
begins is not always obvious, as they (i.e., the end of metestrus common approaches for dealing with or recording transitional
and start of diestrus) are very similar in appearance and are stages.
defined by the same cell types. Early and mid metestrus, how- In studies where only the 4 classic stages are to be used to
ever, are easily identified if and when sample collection hap- identify the stages of the estrous cycle, then it is recommended
pens to occur at these times. Some investigators who are that with a transitional smear the predominating feature(s) of
only interested in cycle length and number of days in estrus and the slide be used to make the final call. For instance, when
Vol. XX, No. X, 2015 VAGINAL CYTOLOGY IN RATS AND MICE 13
FIGURE 15.—Metestrous smears from several B6C3F1/N mice. Metestrus begins with the emergence of neutrophils interspersed or clumped
(arrow) among the anucleated epithelial cells (A–D). Occasional nucleated epithelial cells may also be present. As metestrus progresses, neutro-
phil numbers increase resulting in smears of very high cellularity (E, F). Neutrophil and epithelial cell numbers decrease as the mouse transitions
to diestrus (G, H). Plates A and D illustrate 2 consecutive days from a control B6C3F1/N mouse, representing the uncommon occurrence of a
metestrus stage being recorded for 2 days in a row in a mouse. Modified Wright–Giemsa stain (A–D, G, H) and Toluidine blue O stain (E,
F). Original objective magnification of 10 (A, C–E, G), 20 (F, H), or 40 (B). Plate F is in Elmore et al. Proceedings of the 2014 National
Toxicology Program satellite symposium. Toxicol Pathol, 43:10-40, 2015.
14 CORA ET AL. TOXICOLOGIC PATHOLOGY
FIGURE 15.—Continued
confronted with a proestrus–estrus transition smear, if the predicting in advance a given animal’s cyclic pattern
impression is that more than 50% of the cells are small and (Hubscher, Brooks, and Johnson 2005).
nucleated, then the smear would be classified as proestrus. Further, there are preanalytical sources of variation that can
Another strategy for the classification of transition stages is affect cell density on a vaginal cytology preparation, including,
to use uppercase and lowercase letters. For example, the previ- collection technique, compliance of the rat or mouse during
ously described smear would be designated as P(e) indicating collection and preparing more than one smear for each collec-
that while the day was called proestrus (denoted by ‘‘P’’), anu- tion day. More specifically, preparation of more than one smear
cleated keratinized cells were also present in significant num- for each day can alter the cellularity enough as to cause a
bers, indicating that a transitioning toward estrus was metestrus smear to be classified as diestrus, for example.
occurring (denoted by ‘‘e’’). Similarly, if clumps of small Although this is not a common issue, one should be aware that
nucleated cells are observed in late diestrus, the smear can be this phenomenon could occur when making more than 1 smear
recorded as D(p), indicating a transition from diestrus into for each day.
proestrus. Regardless of what classification paradigm is used,
to stay consistent and thus minimize variability, it is important
that a concerted effort be made to ensure agreement on the cri- CONCLUSION
teria for stage definitions.
Evaluation of sequential vaginal cytology smears is a time-
honored tool for the assessment of the estrous cycle in labora-
Cellularity
tory rats and mice. Collection and preparation of vaginal cytol-
The cell density or cellularity of a cytology preparation may ogy samples is an essentially noninvasive procedure that, with
aid in defining a stage. Cellularity of a vaginal cytology, how- proper training, is easily mastered. For reliable and consistent
ever, should not be used in isolation because it can vary for data generated from vaginal cytology studies, evaluations of
numerous reasons including those independent of the actual the cytology smears must be based on well-established standar-
stage of the estrous cycle. The overall cellularity and ratio of dized criteria. Smear review and, ultimately, the data obtained,
cells during the estrous cycle can differ between individual however, can only be as ‘‘good’’ as the technical expertise and
rodents and this is particularly true for diestrus. Long and experience of the evaluator performing the microscopic exam-
Evans (1922) reference this with the statement that ‘‘both types ination. Thus, it is recommended that individual laboratories
of cell [neutrophils, nucleated epithelial cells] may occur [dur- define and codify their respective processes via laboratory pro-
ing diestrus] in considerable numbers in some animals or may cedures, and the proficiency of the personnel reviewing the
be both of them very sparse in others.’’ (p. 18). For a given ani- cytology materials must be established and is achieved through
mal, cellularity and cellular composition is usually consistent appropriate training, the use of vaginal cytology study sets of
from cycle to cycle. For example, one animal may consistently known diagnoses, and experience. As highlighted in this
have almost exclusively neutrophilic diestrus smears, while review, the cytological appearance of the rat and mouse estrous
another will have a good mixture of neutrophils and epithelial cycle differs in several ways. Thus, regardless of general simi-
cells. Thus, comparison of one animal to another should be larities, when it comes to the estrous cycle, a ‘‘mouse is not a
avoided, and comparison of day-to-day changes for each ani- rat’’ and proper training is required for each species to garner
mal individually is the best approach for identifying and full understanding of the differences.
Vol. XX, No. X, 2015 VAGINAL CYTOLOGY IN RATS AND MICE 15
FIGURE 16.—Metestrous vaginal smears from several Sprague-Dawley rats. Metestrus begins with the emergence of neutrophils interspersed or
clumped among the nucleated and anucleated epithelial cells (A, B). Note that the appearance of metestrus differs in rats and mice, as rats have
much higher numbers of nucleated epithelial cells. As metestrus progresses, neutrophil numbers increase resulting in smears of high cellularity
(C–F). Toluidine blue O stain. Original objective magnification of 10 (A, C, E) or 40 (B, D, F).
16 CORA ET AL. TOXICOLOGIC PATHOLOGY
FIGURE 17.—Diestrous vaginal smears from B6C3F1/N mice (A–E) and a Sprague-Dawley rat (F) characterized by a moderate to low cellularity with
predominately neutrophils that are mixed with lower numbers of epithelial cells. A small clump of round epithelial cells (arrow) is present on a diestrous
smear taken the day before the emergence of proestrus. Modified Wright–Giemsa stain (C–E) and Toluidine blue O stain (A, B, F). Original objective
magnification of 10 (A, C, E, F) or 40 (B, D). Plate E is in Elmore et al. Proceedings of the 2014 National Toxicology Program satellite symposium.
Toxicol Pathol, 43:10-40, 2015.
Vol. XX, No. X, 2015 VAGINAL CYTOLOGY IN RATS AND MICE 17
ACKNOWLEDGMENTS
The authors would like to thank Drs. Mark Cesta, NTP,
NIEHS, and Barry McIntyre, NTP, for their helpful suggestions
concerning the article, and Beth Mahler, EPL, for her help with
all the photomicrographs.
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