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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 tional day of estrus or diestrus (Freeman 1994),
study the neural mechanisms underlying a wide possibly due to prolonged progesterone secretion
variety of normal functions and pathophysiological (Nequin et al. 1979). The estrous cycle is considered
conditions that occur in humans. Gender differ- irregular when the stages are not in sequence
ences have been identified for many conditions, or when a single stage lasts 4 /5 days (Marcondes
making the choice between studying males or et al. 2002).
females of a given species (or using both) unclear. The stage of the cycle can be determined by
The potential impact of hormonal variations be- viewing at low microscopic magnification a sample
tween the sexes on the outcome of research as well of cells obtained from the surface of the vaginal
as normal cyclic hormonal fluctuations in females epithelium (Feder 1981, Freeman 1994). The layers
requires the availability of more detailed informa- of the vaginal mucosa include the stratum cor-
tion about the rat’s reproductive cycle. neum, stratum granulosum, rete mucosum and
The ‘‘estrous cycle’’ in female rats has four stratum germinativum (Long and Evans 1922).
stages, proestrus, estrus, metestrus and diestrus, The cyclic differences in vaginal cytology occur in
and is under photoperiodic control. For the major- response to the morphological changes of the
ity of animals, the estrous cycle lasts 4/5 days vaginal epithelium as cells desquamate. The 12 /
(Long and Evans 1922, Mandl 1951, Feder 1981). 14 h proestrus stage is characterized by round
Some rats with 5 day cycles (Long and Evans 1922, nucleated cells of uniform size (Long and Evans
Nequin et al. 1979, Feder 1981) exhibit an addi- 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
Correspondence to: Dr. Charles H. Hubscher, Dept. of proestrus, the surface layer of mature epithelial
Anatomical Sciences & Neurobiology, University of Louisville, cells has shed and the stratum corneum is exposed
Louisville, KY 40292, USA. Tel: 502-852-3058; Fax: 502-852-
6228; E-mail: chhubs01@louisville.edu
(Long and Evans 1922). The next stage, estrus, lasts
– Biological Stain Commission 25/27 h, and is distinguished by the appearance
Biotechnic & Histochemistry 2005, 80(2): 79 /87. of irregularly shaped, un-nucleated cornified cells
DOI:10.1080/10520290500138422 79
(Long and Evans 1922, Freeman 1994). During the 11:00 am and 12:00 pm. Each animal was held
next stage, metestrus, lasting 6/8 h, leukocytes behind the shoulder blades in a supine position to
infiltrate the thinned vaginal epithelium owing to a obtain the smears (see Fig. 1a in Marcondes et al.
decline in estrogen secretion and pass into the 2002). To obtain a sample, the tip of a 3 inch
vaginal canal (Montes and Luque 1988). Vaginal borosilicate glass medicine dropper was filled with
secretions make the smear appear white and approximately 0.2 ml of sterile water and inserted
opaque (Long and Evans 1922). During diestrus, approximately 3 /5 mm into the rat’s vagina
which lasts 55 /57 h, more than half of the cycle (Eckel et al. 2001). Care was exercised not to insert
(Freeman 1994), the epithelium reaches its thinnest the dropper too deeply, because inadvertent
point (4 /7 layers). It is during this stage that the cervix stimulation can initiate pseudopregnancy
degeneration of the epithelium stops and the (Freeman 1994). Sterile water was quickly released
height of the epithelium increases again because from the dropper, then immediately drawn back
of mitosis (Long and Evans 1922). Both leukocytes into it.
and nucleated cells are present during this phase.
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In the study reported here, we obtained vaginal Initial evaluation of ‘‘wet’’ vaginal smear
smears from cycling and ovariectomized female
rats. The ‘‘wet’’ smear samples obtained from the The sample containing cells was placed on an
vaginal lumen were viewed immediately, docu- untreated glass microscope slide and viewed while
mented, then viewed again after drying and stain- still wet under a light microscope at 100 magni-
ing using an adaptation of the Papanicolaou (PAP) fication. After determining the stage of estrous,
stain, which is used clinically as a screening tool for images were taken using a Spot Insight color
cervical cancer and lesions associated with that camera mounted on a Nikon E400 microscope.
disease (Ducatman and Wang 2002). The cell types After images were taken, the slides were air dried
present in all four stages, as well as those present prior to staining.
following ovariectomy, were identified qualitatively
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
tions. Differences with a probability value 5 0.05 Identification of cell populations in PAP stained
were considered significant. Our study was per- smears
formed in accordance with guidelines of the
Animal Care and Use Committee (IACUC) of the Vaginal smears stained using the PAP method are
University of Louisville School of Medicine and shown in all four stages in Fig. 2. In proestrus,
with the Guide for the Care and Use of Laboratory
nucleated cells with pink cytoplasms predomi-
Animals (National Academy of Sciences, publica-
nated. All of the densely packed nucleated cells
tion No. 0-309-05377-3).
showed dark blue to purple staining granulated
nuclei. In estrus, cornified cells were arranged in
Results sheets and clumps, and were stained pale orange
and pink. The superficial cornified cells were pink
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 stages described by Long and Evans (1922). Table 2
the vaginal smears provides quantitative data for normal 4 day cycling
animals. The data illustrates the prevalence of
In general, the cell populations observed were leukocytes, cornified cells, and nucleated epithelial
consistent with qualitative descriptions of the 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/.
Ovariectomy
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noon and ovulation normally occurs 4 /6 h after the superficial layer, they are sloughed off, possibly
dark phase begins (Becker et al. 2002); variations owing to the accumulation of acid phosphatase in
can occur from animal to animal, which would the cells (Ross et al. 1995).
explain the high standard error for nucleated cells. In metestrus, the overall appearance of the
The small number of leukocytes observed in vaginal smear had a blue /violet cast due to the
proestrus also is likely due to the timing, as the dark blue staining of densely packed leukocytes by
cycle emerges from diestrus into proestrus. Simi- hematoxylin and Scott’s tap water substitute.
larly, the small number of leukocytes observed Clumping of leukocytes around the nucleated cells
during estrus likely indicates the approach of likely is caused by the action of chemokines,
metestrus. Thus, the best approach is to view each specialized molecules that bind leukocytes in
sample as an indicator of a process that is con- tissues (Townson and Liptak 2003). Chemokines
For personal use only.
stantly undergoing changes. A comparison of the are expressed as a result of luteal regression and
day to day changes in the cell populations for each are associated with accumulation of leukocytes
animal individually should be the most reliable (Townson and Liptak 2003).
means for identifying and predicting in advance a In diestrus, the overall appearance of the smear is
given animal’s cyclic pattern. It has been shown blue /violet, and the leukocytes are scattered and
previously that the variance in the duration of the there are significantly fewer of them (Table 2).
estrous cycle is significantly greater between litter-
mates than for individual rats (Mandl 1951). Cell quantification
ovariectomized vaginal smear confirmed that the Gimenez-Conti IB, Lynch M, Roop D, Bhowmik S,
Majeski P, Conti CJ (1994) Expression of keratins in
vaginal epithelium stopped proliferating without
mouse vaginal epithelium. Differentiation 56: 143 /151.
hormonal input, neither the number of leukocytes Hubscher CH, Brooks DL, Johnson JR (2004) Effects of
nor the ratio of leukocytes to nucleated cells spinal cord injury on the rat estrous cycle. No. 458.15.
postovariectomy differed significantly from a nor- 2004 Abstract. Viewer/Itinerary Planner, Society for
mal diestrus (4:1 vs. 3:1). Neuroscience, Washington DC.
Our qualitative presentation of cell populations Hubscher CH, Petruska JC, Rau KK, Johnson RD (2001)
was in agreement with the classical description of Co-expression of P2X receptor subunits on rat nodose
neurons that bind the isolectin GS-I-B4. Neuroreport 12:
the rat estrous cycle (Long and Evans 1922);
2995 /2997.
however, quantitative analysis revealed differences Keebler CM, Somrak TM (1993) The Manual of Cytotech-
in the cycle that have not been reported previously. nology. 7th ed. American Society of Clinical Pathologists,
The PAP staining method is a valuable tool for Chicago.
distinguishing stages of the estrous cycle due to its Koss LG (1998) Gynecologic Cytopathology. Williams &
distinct staining patterns. We recommend that Wilkins, Baltimore.
experimental studies involving circumstances that Long JA, Evans HM (1922) The oestrous cycle in the rat
and its associated phenomena. Mem. Univ. California 6:
might alter the normal progression of the rat’s 1 /148.
estrous cycle (e.g., spinal cord injury; Hubscher Mandl AM (1951) The phases of the oestrous cycle in the
et al. 2004) include both qualitative and quantitative adult white rat. J. Exp. Biol. 28: 576 /584.
analyses. Quantitative analysis can reveal subtle Marcondes FK, Bianchi FJ, Tanno AP (2002) Determina-
differences in the vaginal cell population that might tion of the estrous cycle phases of rats: some helpful
otherwise be overlooked. considerations. Braz. J. Biol. 62: 609 /614.
Montes GS, Luque EH (1988) Effects of ovarian steroids
on vaginal smears in the rat. Acta Anat. (Basel) 133: 192 /
199.
Acknowledgments Nequin LG, Alvarez J, Schwartz NB (1979) Measure-
ment of serum steroid and gonadotropin levels and
uterine and ovarian variables throughout 4 day and 5
We thank James Armstrong, Deya Banerjee and day estrous cycles in the rat. Biol. Reprod. 20: 659 /670.
Amanda Fodrey for excellent technical assistance. Olson ME, Bruce J (1986) Ovariectomy, ovariohysterect-
This study was supported by a grant from the omy and orchidectomy in rodents and rabbits. Can. Vet. J.
Christopher Reeve Paralysis Foundation. 27: 523 /527.