A quantitative method for assessing stages of the

rat estrous cycle

CH Hubscher, DL Brooks, JR Johnson
Department of Anatomical Sciences & Neurobiology, University of Louisville, Louisville, Kentucky 40292

Submitted June 17, 2004; revised February 2, 2005; accepted March 8, 2005

Abstract
The impact of gender and/or hormone variations on a wide variety of neural functions makes the
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choice between studying males or females (or both) of a given species difficult. Although female
rats are widely used experimentally, few studies control for the stage of estrus. More detailed
information about how to distinguish the various stages of the estrous cycle is needed. For
the present study, vaginal smears were obtained once a day and stained using an adaptation of
the Papanicolaou (PAP) procedure. Images are provided of unstained ‘‘wet’’ samples and the
corresponding PAP stained smears illustrating the cellular profile for each stage of the cycle as
well as post-ovariectomy. The different cell populations across the cycle were quantified and
ratios determined to show trends between the predominant and other cell types in each stage of
the estrous cycle. Both stained and unstained images and cell quantification data provide valuable
guidelines for distinguishing the stages of the estrous cycle.
For personal use only.

Key words: estrous cycle, estrus, ovariectomy, PAP stain, proestrus, reproduction, vagina

Rats are often used experimentally as a model to
study the neural mechanisms underlying a wide
variety of normal functions and pathophysiological
conditions that occur in humans. Gender differ-
ences have been identified for many conditions,
making the choice between studying males or
females of a given species (or using both) unclear.
The potential impact of hormonal variations be-
tween the sexes on the outcome of research as well
as normal cyclic hormonal fluctuations in females
requires the availability of more detailed informa-
tion about the rat’s reproductive cycle.
The ‘‘estrous cycle’’ in female rats has four
stages, proestrus, estrus, metestrus and diestrus,
and is under photoperiodic control. For the major-
ity of animals, the estrous cycle lasts 4
/5 days
(Long and Evans 1922, Mandl 1951, Feder 1981).
Some rats with 5 day cycles (Long and Evans 1922,
Nequin et al. 1979, Feder 1981) exhibit an addi-
Correspondence to: Dr. Charles H. Hubscher, Dept. of
Anatomical Sciences & Neurobiology, University of Louisville,
Louisville, KY 40292, USA. Tel: 502-852-3058; Fax: 502-852-
6228; E-mail: chhubs01@louisville.edu
– Biological Stain Commission
Biotechnic & Histochemistry 2005, 80(2): 79
/87.
tional day of estrus or diestrus (Freeman 1994),
possibly due to prolonged progesterone secretion
(Nequin et al. 1979). The estrous cycle is considered
irregular when the stages are not in sequence
or when a single stage lasts 4
/5 days (Marcondes
et al. 2002).
The stage of the cycle can be determined by
viewing at low microscopic magnification a sample
of cells obtained from the surface of the vaginal
epithelium (Feder 1981, Freeman 1994). The layers
of the vaginal mucosa include the stratum cor-
neum, stratum granulosum, rete mucosum and
stratum germinativum (Long and Evans 1922).
The cyclic differences in vaginal cytology occur in
response to the morphological changes of the
vaginal epithelium as cells desquamate. The 12
/
14 h proestrus stage is characterized by round
nucleated cells of uniform size (Long and Evans
1922, Freeman 1994). During this stage, the vaginal
epithelium is composed of 9
/12 layers of cells with
the mature cells at the surface. By the end of
proestrus, the surface layer of mature epithelial
cells has shed and the stratum corneum is exposed
(Long and Evans 1922). The next stage, estrus, lasts
25
/27 h, and is distinguished by the appearance
of irregularly shaped, un-nucleated cornified cells

DOI:10.1080/10520290500138422 79
 (Long and Evans 1922, Freeman 1994). During the
next stage, metestrus, lasting 6
/8 h, leukocytes
infiltrate the thinned vaginal epithelium owing to a
decline in estrogen secretion and pass into the
vaginal canal (Montes and Luque 1988). Vaginal
secretions make the smear appear white and
opaque (Long and Evans 1922). During diestrus,
which lasts 55
/57 h, more than half of the cycle
(Freeman 1994), the epithelium reaches its thinnest
point (4
/7 layers). It is during this stage that the
degeneration of the epithelium stops and the
height of the epithelium increases again because
of mitosis (Long and Evans 1922). Both leukocytes
and nucleated cells are present during this phase.
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In the study reported here, we obtained vaginal
smears from cycling and ovariectomized female
rats. The ‘‘wet’’ smear samples obtained from the
vaginal lumen were viewed immediately, docu-
mented, then viewed again after drying and stain-
ing using an adaptation of the Papanicolaou (PAP)
stain, which is used clinically as a screening tool for
cervical cancer and lesions associated with that
disease (Ducatman and Wang 2002). The cell types
present in all four stages, as well as those present
following ovariectomy, were identified qualitatively
For personal use only.
in wet versus stained samples, then quantified
using the stained samples. We present a flowchart
for identifying stages of the estrous cycle.
Materials and methods
Animal groups
Thirty-two 60
/70-day-old female Wistar rats (in-
itial weight 220
/270 g) were used in our study.
Each animal was handled daily for approximately
one week before beginning vaginal smears. Animals
were housed individually and exposed to a 12 h
light/dark cycle: 6:00 am-lights on, 6:00 pm-lights
off. Food and water were available at all times. The
stage of estrous for each animal was evaluated once
daily. Each animal’s cycle was followed for a
minimum of 15 days, allowing for at least three
cycles (4
/5 days) to be monitored and evaluated.
After the initial 15 day period, nine of the 32
animals were ovariectomized, and vaginal smears
were taken following ovariectomy. Both pre-ovar-
iectomy cycle data and the data obtained from the
normal cycling animals were used for evaluating
the normal progression of the estrous cycle.
Vaginal smear protocol
To assess the progression of the estrous cycle,
vaginal smears were taken once each day, between
80 Biotechnic & Histochemistry 2005, 80(2): 79
/87
11:00 am and 12:00 pm. Each animal was held
behind the shoulder blades in a supine position to
obtain the smears (see Fig. 1a in Marcondes et al.
2002). To obtain a sample, the tip of a 3 inch
borosilicate glass medicine dropper was filled with
approximately 0.2 ml of sterile water and inserted
approximately 3
/5 mm into the rat’s vagina
(Eckel et al. 2001). Care was exercised not to insert
the dropper too deeply, because inadvertent
cervix stimulation can initiate pseudopregnancy
(Freeman 1994). Sterile water was quickly released
from the dropper, then immediately drawn back
into it.
Initial evaluation of ‘‘wet’’ vaginal smear
The sample containing cells was placed on an
untreated glass microscope slide and viewed while
still wet under a light microscope at 100  magni-
fication. After determining the stage of estrous,
images were taken using a Spot Insight color
camera mounted on a Nikon E400 microscope.
After images were taken, the slides were air dried
prior to staining.
Papanicolaou (PAP) stain
Vaginal smears were stained using an adaptation
of the Papanicolaou (PAP) stain used for humans
as developed by Dr. George N. Papanicolaou
(Sheehan and Hrapchak 1980). The original PAP
staining used a regressive method, where tissue
was overstained followed by removal of excess
stain (Presnell and Schreibman 1997). We used a
progressive staining method where the sample was
stained until the desired color intensity was
reached. The procedure followed for the air dried
smears is presented in Table 1. Note that fresh
solutions were used each time a step was repeated.
Following the final change of xylene, each slide
was coverslipped with a 50:50 mixture of Gurr
mounting adhesive:xylene.
Ovariectomies
Ovariectomies were performed under aseptic con-
ditions. Animals were anesthetized with a mixture
of ketamine (80 mg/kg body weight) and xylazine
(10 mg/kg body weight) injected intraperitoneally
(Hubscher et al. 2001). Supplements of the mixture
were given as needed in 0.2 ml doses. After
induction, animals received a 0.5 ml subcutaneous
injection of Ambi-Pen (Penicillin G and Penicillin G
Procrain, Butler, Columbus, OH). Bilateral lumbar
incisions were made approximately 1/2 inch below
Biotech Histochem Downloaded from informahealthcare.com by University of Queensland on 05/07/13
For personal use only.

Fig. 1. Representative wet vaginal samples. Images were taken immediately after obtaining smears from 4 day cycling
rats. 200 /.

the ends of the ribs. The ovary was gently extracted The ovary, oviduct, and a small section of the uterus
by grasping the periovarian fat. The cranial portion were then removed (Olson and Bruce 1986). After
of the uterus and associated uterine vessels were suturing the muscle layer, the skin was closed with
clamped and the remaining tissue was tied off with one or two Michel clips. Once the ovariectomies
Ethicon 4
/0 monocryl sterile absorbable sutures. were completed, animals were given a 0.1 ml
injection of the analgesic Ketoprofen (2.5 mg/kg)
and monitored until fully recovered from the
Table 1. Adaptation of PAP stain for rat vaginal smears
anesthesia. Beginning the day after surgery,
Time per Repeat animals were given subcutaneous injections of
Step Solution change step Ketoprofen (2.5 mg/kg body weight) twice daily
for two days.
1 95% ethanol 5 min. 1
2 tap water 10 quick dips 1
3 Gill’s Hematoxylin 2 min. 0 Quantification of cell populations in vaginal
4 tap water 10 quick dips 1 smears
5 Scott’s tap water 1 min. 0
substitute PAP stained vaginal smears from ten of the normal
6 tap water 10 quick dips 1 cycling animals that were obtained daily for an
7 95% ethanol 10 quick dips 1 average of eight cycles, were used to quantify the
8 Orange G6 1 min. 0
different cell populations in each stage of the
9 95% ethanol 10 quick dips 1
estrous cycle. The area containing the densest
10 eosin-azure 50 10 min. 0
11 95% ethanol 20 quick dips 2 portion of cells in a given sample was used
12 100% ethanol 10 quick dips 2 for quantification. All cell counts were made at
13 xylene 10 quick dips 2 200  magnification, which comprised a 0.25 mm2
area. Micrographs were taken and each cell type

Rat estrous cycle 81

 (i.e., nucleated, leukocytes, and cornified cells) was
counted within the entire area. After counting cells
for designated animals, the average population of
each cell type was determined and the mean was
calculated for all of the animals during each stage
of the estrous cycle. Cells also were counted in
ovariectomized animals, beginning at 3 weeks
postovariectomy, for a five day period.
Statistical analysis
A standard t-test was used to compare individual
cell populations and Chi-square was used to
determine frequency differences between condi-
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tions. Differences with a probability value 5 0.05
were considered significant. Our study was per-
formed in accordance with guidelines of the
Animal Care and Use Committee (IACUC) of the
University of Louisville School of Medicine and
with the Guide for the Care and Use of Laboratory
Animals (National Academy of Sciences, publica-
tion No. 0-309-05377-3).
Results
For personal use only.
Estrous cycle in normal female rats
Consistent with Long and Evans (1922), we found
the estrous cycle to be 4 days long for most (88%) of
the animals studied. The remaining animals were
either 5 day cyclers (extra day of diestrus) or they
cycled irregularly (progression not in the usual
sequence).
Identification of cell populations in ‘‘wet’’
smears
In proestrus, nucleated cells were round to hex-
agonal in shape depending on how the cells were
packed together. The nucleated cells often were
so densely packed that spaces were not visible
between cells. Proestrus progressed to estrus,
which was characterized by the predominance of
Table 2. Comparison of cell populations across the estrous cycle
Stage Leukocytes
Proestrus (n/47) 18.89/6.1
Estrus (n /46) 5.99/2.1
Metestrus (n /25) 949.29/234.6*
Diestrus (n /101) 234.59/59.7*
Ovariectomy (n /45) 285.69/108*
n/number of samples observed.
*Data significantly different from proestrus (pB/0.05).
82 Biotechnic & Histochemistry 2005, 80(2): 79
/87
numerous cornified squamous cells. The cells in the
sample often were aggregated and arranged in
layers. Only a few leukocytes were present in the
vaginal smear. A greater number of nucleated cells
were observed when a smear in late estrus was
encountered (Feder 1981), although cornified cells
still predominated. By contrast, metestrus con-
tained many leukocytes, which were densely
packed around the nucleated cells. The final stage,
diestrus, had scattered leukocytes. Both nucleated
and cornified cells were present during diestrus.
Typical examples of all stages are shown in Fig. 1.
Identification of cell populations in PAP stained
smears
Vaginal smears stained using the PAP method are
shown in all four stages in Fig. 2. In proestrus,
nucleated cells with pink cytoplasms predomi-
nated. All of the densely packed nucleated cells
showed dark blue to purple staining granulated
nuclei. In estrus, cornified cells were arranged in
sheets and clumps, and were stained pale orange
and pink. The superficial cornified cells were pink
and the nucleated cells in estrus had pink, blue, or
orange cytoplasms and dark blue stained nuclei. In
metestrus, the leukocytes were stained blue. The
nucleated cells in metestrus had a distinct pale
blue border surrounding the cell membranes and
dark blue stained nuclei. The cytoplasms of the
nucleated cells were both pink (inner) and blue
(outer) (Fig. 2). Leukocytes were densely packed
in clusters around nucleated cells, and their
abundance gave the smear an overall dark blue
tint. In diestrus, the leukocytes were scattered
throughout the sample. Leukocytes, as in the
metestrus stage, stained dark blue to blue-purple.
The nucleated cells in the sample had large opaque
nuclei with pink or blue cytoplasms. The cyto-
plasms of the cornified cells were stained pink
(Fig. 2).
Cornified cells Nucleated Cells
43.49/6.3 207.99/97.7
68.99/12.4* 57.19/15.0*
6.89/1.9* 93.39/19.7
13.79/1.2* 78.59/11.0*
7.29/1.6* 69.09/16.3*
Biotech Histochem Downloaded from informahealthcare.com by University of Queensland on 05/07/13
For personal use only.

Fig. 2. Representative PAP stained vaginal samples from 4 day cycling rats. The image for each stage was taken from the
same smear as depicted in Fig. 1. 400 /.

Quantification of the cell populations present in
the vaginal smears
In general, the cell populations observed were
consistent with qualitative descriptions of the
stages described by Long and Evans (1922). Table 2
provides quantitative data for normal 4 day cycling
animals. The data illustrates the prevalence of
leukocytes, cornified cells, and nucleated epithelial
cells in the metestrus, estrus, and proestrus stages of

Fig. 3. Representative vaginal smear taken 3 weeks postovariectomy. The wet vaginal sample on the left was
photographed shortly after obtaining the smear. The image on the right was taken from the same sample, but after PAP
staining. 200/.

Rat estrous cycle 83

 the estrous cycle, respectively. In metestrus, leuko-
cytes outnumbered the second most dominant cell
type, nucleated cells, by a 10:1 ratio. In diestrus,
leukocytes outnumbered the nucleated cells by a 3:1
ratio (Table 2). Although some leukocytes were
present in both proestrus and estrus, there were
significantly fewer than the average number present
in either metestrus or diestrus (p B 0.05). Similarly,
some cornified cells were present in both metestrus
and diestrus, but they were significantly fewer than
in either proestrus or estrus (pB 0.05).

Ovariectomy
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Nine animals were ovariectomized. All of the

animals stopped cycling and remained in a phase
resembling diestrus (Fig. 3) for the duration of
the experiment. The smear samples were heavily
populated with dark blue leukocytes and nucleated
cells with pink or blue cytoplasms. Some cornified
cells also were present in the sample. The ratio of
leukocytes to nucleated cells was 4:1, which was
not significantly different from the ratio in diestrus
for normal cycling animals (p 0.05). Fig. 4. A protocol that can be used to identify the stage of
the estrous cycle by qualitative observation of ‘‘wet’’
For personal use only.

Discussion or stained samples and/or quantitative analysis of the

The purpose of this study was to use a combination
of currently available techniques to improve our
ability to identify the stages of the estrous cycle and
to quantify cell types using a multicomponent
histological stain. Monitoring the daily progression
of the estrous cycle in combination with the
staining profiles and cell quantification provides a
consistent way to classify the stages of the estrous
cycle. The flowchart presented in Fig. 4 provides a
protocol for identifying the stages of estrous. What
one should see, based on classical descriptions of a
‘‘perfect’’ sample, and what one actually sees, given
the many experimental and nonexperimental vari-
ables that exist, needs further investigation.
Variations in the estrous cycle of normal female
rats
In the present study, the predominant cell type
found in each stage of the cycle was in general
agreement with the classical qualitative description
of the estrous cycle presented by Long and Evans
(1922). Quantification of the different cells, how-
ever, revealed some subtle differences in cell
populations across the stages of the estrous cycle.
Contrary to earlier observations (Long and Evans
1922, Montes and Luque 1988), leukocytes were
cell populations. Cn, cornified cells; Lk, leukocytes; Nu,
nucleated cells.
present in all stages of the estrous cycle. There was
a significant increase in leukocyte population in
metestrus (113-fold) and diestrus (16-fold) com-
pared to proestrus. According to Long and Evans
(1922), leukocytes do not appear in the vaginal
smear until metestrus and diestrus. Long and
Evans (1922) also stated that cornified cells first
appear in vaginal smears at estrus. We found that
cornified cells appeared in proestrus and increased
significantly (2-fold) in number during estrus.
Some cornified cells, however, were observed in
all stages of the cycle. If the vaginal mucosa sheds
evenly throughout the vagina, as suggested by
Long and Evans (1922), only one type of epithelial
cell would be present in each vaginal smear. The
appearance of all three cell types throughout the
cycle, therefore, is an indication of uneven exfolia-
tion of the vaginal epithelium.
It is important to note that vaginal smears were
taken only once a day, between 11 am and 12 pm.
Because estrus is 25
/27 h long (Long and Evans
1922, Freeman 1994) and vaginal smears were taken
once every 24 h, this phase was always observed
in a four day cycling animal. Because metestrus is

84 Biotechnic & Histochemistry 2005, 80(2): 79
/87

 only 6
/8 h (Long and Evans 1922, Freeman 1994), it
is possible that this stage could have been missed.
In fact, two days of diestrus often were observed,
which is why the number of metestrus stages
sampled was lower and the number of diestrus
stages sampled in normal rats was higher (Table 2).
Diestrus (55
/57 h) was always observed at least
once, occasionally twice.
The timing of the estrous cycle may account for
some of the variations in cell populations in
proestrus (12
/14 h). The standard error of the
mean was very high for nucleated cells, the
predominant cell type. According to Feder (1981),
animals enter proestrus in the early to late after-
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noon and ovulation normally occurs 4
/6 h after the
dark phase begins (Becker et al. 2002); variations
can occur from animal to animal, which would
explain the high standard error for nucleated cells.
The small number of leukocytes observed in
proestrus also is likely due to the timing, as the
cycle emerges from diestrus into proestrus. Simi-
larly, the small number of leukocytes observed
during estrus likely indicates the approach of
metestrus. Thus, the best approach is to view each
sample as an indicator of a process that is con-
For personal use only.
stantly undergoing changes. A comparison of the
day to day changes in the cell populations for each
animal individually should be the most reliable
means for identifying and predicting in advance a
given animal’s cyclic pattern. It has been shown
previously that the variance in the duration of the
estrous cycle is significantly greater between litter-
mates than for individual rats (Mandl 1951).
The Papanicolaou (PAP) stain
Wet smears normally are viewed under a light
microscope, but once the sample dries, cells are
flattened and difficult to identify. The PAP stain
aids in preserving the smears, and cells are easily
described due to their consistent staining pattern.
Clinically, the main use of the PAP stain is to screen
for cervical cancer lesions. In addition, it has been
used as a tool to assess vaginal cornification after
injection with retinoids (Chateau et al. 1996) and to
predict ovulation in exotic cases such as the giant
panda (Durrant et al. 2002).
In the proestrus stage, when mature epithelial
cells are shed and predominate in the vaginal
smear, an overall pink cast was found due to eosin,
which stains the cytoplasms of mature squamous
cells pink (Keebler and Somrak 1993). The size of
the nuclei also indicated that the cells were shed
from mature epithelium. The nuclei, which were
either granulated or stippled, were stained blue by
hematoxylin nuclear staining followed by the blue-
ing of the nucleus by Scott’s tap water substitute.
The stippling of the nucleus is an indication of
maturity as measured by an increase in nuclear
DNA (Keebler and Somrak 1993). When cells begin
to degenerate, the nuclei become opaque indicating
pyknosis (Keebler and Somrak 1993).
In estrus, the large cornified cells appear orange
due to keratin, which is synthesized during cell
differentiation (Gimenez-Conti et al. 1994). As cells
migrate toward the free surface of the vaginal
epithelium, the nuclei and other organelles break
down and the plasma membrane thickens (Ross
et al. 1995). When the keratinized cells become the
superficial layer, they are sloughed off, possibly
owing to the accumulation of acid phosphatase in
the cells (Ross et al. 1995).
In metestrus, the overall appearance of the
vaginal smear had a blue
/violet cast due to the
dark blue staining of densely packed leukocytes by
hematoxylin and Scott’s tap water substitute.
Clumping of leukocytes around the nucleated cells
likely is caused by the action of chemokines,
specialized molecules that bind leukocytes in
tissues (Townson and Liptak 2003). Chemokines
are expressed as a result of luteal regression and
are associated with accumulation of leukocytes
(Townson and Liptak 2003).
In diestrus, the overall appearance of the smear is
blue
/violet, and the leukocytes are scattered and
there are significantly fewer of them (Table 2).
Cell quantification
Analysis of the populations of leukocytes, cornified
cells and epithelial cells confirmed that nucleated
cells predominated in proestrus, cornified cells
predominated in estrus, and leukocytes predomi-
nated in metestrus and diestrus. We also noted that
the smears from some animals had more cells
overall than others. For example, some animals
consistently had fewer cells during diestrus, and
this was consistent for a given animal from cycle to
cycle. These observations prompted us to calculate
ratios of the different cell types present to provide
more consistency and to depend less on the
number of cells obtained in a given smear, because
getting a good sample can sometimes be proble-
matic. Obtaining multiple samples at a time in-
creases the risk of inducing pseudopregnancy.
Although the ratios (Fig. 4) represent one time
point, they provide an expectation that can be used
to differentiate better among stages at a quick
glance when the stage is not obvious. For example,
the ratio of leukocytes to nucleated cells in metes-
Rat estrous cycle 85
 trus (10:1) is far greater than in diestrus (3:1).
Furthermore, although Marcondes et al. (2002)
characterized metestrus as having an equal propor-
tion of all cell types in the vaginal smear, our
quantitative analysis revealed that the cells were
present in different proportions. In fact, the 10:1
ratio of leukocytes to nucleated cells in metestrus
could be an underestimate, because some of the
smears may have been taken either late in metes-
trus or early in diestrus. The cell populations are in
constant flux, so the numbers of any given cell type
vary with time.
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Ovariectomy
All overiectomized animals stopped cycling after
completing their diestrus phase. Without input
from estrogen during the estrous cycle, the epithe-
lium does not proliferate and increase in size (Koss
1998). According to Montes and Luque (1988),
vaginal smears of ovariectomized rats have an
abundance of leukocytes, because they pass from
the diminished epithelium into the vagina. In our
study, although the presence of leukocytes in the
For personal use only.
ovariectomized vaginal smear confirmed that the
vaginal epithelium stopped proliferating without
hormonal input, neither the number of leukocytes
nor the ratio of leukocytes to nucleated cells
postovariectomy differed significantly from a nor-
mal diestrus (4:1 vs. 3:1).
Our qualitative presentation of cell populations
was in agreement with the classical description of
the rat estrous cycle (Long and Evans 1922);
however, quantitative analysis revealed differences
in the cycle that have not been reported previously.
The PAP staining method is a valuable tool for
distinguishing stages of the estrous cycle due to its
distinct staining patterns. We recommend that
experimental studies involving circumstances that
might alter the normal progression of the rat’s
estrous cycle (e.g., spinal cord injury; Hubscher
et al. 2004) include both qualitative and quantitative
analyses. Quantitative analysis can reveal subtle
differences in the vaginal cell population that might
otherwise be overlooked.
Acknowledgments
We thank James Armstrong, Deya Banerjee and
Amanda Fodrey for excellent technical assistance.
This study was supported by a grant from the
Christopher Reeve Paralysis Foundation.
86 Biotechnic & Histochemistry 2005, 80(2): 79
/87
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