1Department of Biochemistry, Microbiology and Immunology, Neural Regeneration Laboratory and Ottawa Institute of Systems Biology, 2Department of Cellular and Molecular Medicine, University of Ottawa, 3CIHR Program in Neurodegenerative Lipidomics, University of Ottawa, 4Carleton Immersive Media Studio, Azrieli School of Architecture and Urbanism
McLean, A. C., Valenzuela, N., Fai, S., Bennett, S. A. L. Performing Vaginal Lavage, Crystal Violet Staining, and Vaginal Cytological Evaluation for Mouse Estrous Cycle Staging Identification. J. Vis. Exp. (67), e4389, doi:10.3791/4389 (2012).
A rapid means of assessing reproductive status in rodents is useful not only in the study of reproductive dysfunction but is also required for the production of new mouse models of disease and investigations into the hormonal regulation of tissue degeneration (or regeneration) following pathological challenge. The murine reproductive (or estrous) cycle is divided into 4 stages: proestrus, estrus, metestrus, and diestrus. Defined fluctuations in circulating levels of the ovarian steroids 17-β-estradiol and progesterone, the gonadotropins luteinizing and follicle stimulating hormones, and the luteotropic hormone prolactin signal transition through these reproductive stages. Changes in cell typology within the murine vaginal canal reflect these underlying endocrine events. Daily assessment of the relative ratio of nucleated epithelial cells, cornified squamous epithelial cells, and leukocytes present in vaginal smears can be used to identify murine estrous stages. The degree of invasiveness, however, employed in collecting these samples can alter reproductive status and elicit an inflammatory response that can confound cytological assessment of smears. Here, we describe a simple, non-invasive protocol that can be used to determine the stage of the estrous cycle of a female mouse without altering her reproductive cycle. We detail how to differentiate between the four stages of the estrous cycle by collection and analysis of predominant cell typology in vaginal smears and we show how these changes can be interpreted with respect to endocrine status.
1. Preparing Reagents
2. Collecting Vaginal Cells (Vaginal Lavage)
3. Cytological Staining using Crystal Violet*4
* The staining method described here is the simplest procedure that can be performed in any laboratory. Other methods can provide additional details. For example, using Papanicolaou staining, the maturity of nucleated epithelial cells can be distinguished with less mature cells stained turquoise and more mature cells pink- or orange-stained. These differences can be used to stage early or late proestrus.4
4. Vaginal Cytology
5. Representative Results
Cytology: Three primary cell types can be detected in vaginal smear samples: (1) nucleated epithelial cells (Figure 1A), (2) cornified squamous epithelial cells (Figure 1B), and (3) leukocytes (Figure 1C). Nucleated epithelial cells have a lightly stained cytoplasm, darker stained plasma membrane, and an oval nucleus (Figure 1A). Cornified squamous epithelial cells are uniformly stained, more polygonal in shape than their nucleated epithelial predecessors, and lack a nucleus (Figure 1B). Polymorphonuclear leukocytes can be distinguished from epithelial cells by their irregular shape, darkly stained polymorphic nuclei, and small size (Figure 1C, black arrows). Should urine contamination be present in the smear, uric acid crystals are readily detected by their crystalline structures dissimilar to any expected cell types (Figure 3). Should this occur, and obscure detection of predominant cell type, the smear should be discarded and not used for staging purposes.
Staging: The relative ratio of cell types observed in smears can be used to identify the stage of the estrous cycle of your mouse on the day of sample collection (Figure 1D-G). During proestrus, cells are almost exclusively clusters of round, well-formed nucleated epithelial cells (Figure 1D, representative cell indicated by white arrow). During estrus, cells are predominantly cornified squamous epithelial cells, present in densely packed clusters (Figure 1E, representative cell indicated by arrowhead). During metestrus, small darkly stained leukocytes predominate (Figure 1F, representative cell indicated by black arrow). Cornified squamous epithelial cells may be observed, often in fragments, (Figure 1F, representative cell indicated by black arrowhead). During diestrus, rare cornified squamous epithelial cells may still be present (Figure 1G, representative cell indicated by black arrowhead), however leukocytes still predominate (Figure 1G, representative cell indicated by black arrow). Metestrus can be distinguished from diestrus by the appearance of nucleated epithelial cells in diestrus (Figure 1G, representative cell indicated by white arrow).
Figure 1. Cytological assessment of vaginal smears can be used to identify estrous stage. Three main cell types are detected in vaginal smear samples: (A) nucleated epithelial cells, (B) cornified squamous epithelial cells, and (C) leukocytes. The ratio of these cell types present in the smear can be used to identify mice in (D) proestrus, (E) estrus, (F) metestrus, or (G) diestrus as described in representative results. Black arrowheads in E, F and G point to representative cornified squamous epithelial cells. Black arrows in C, F and G point to representative leykocytes. White arrows in D and G highlight representative nucleated epithelial cells.
Figure 2. Vaginal smear cytology reflects underlying endocrine events. Details are also provided in Discussion. Click here to view larger figure.
Figure 3. Uric acid crystals may be present following crystal violet staining of urine-contaminated samples. (A) Crystals are transparent and can be of various sizes (arrows and boxed region magnified in (B)). No cells are present in this field. Should uric acid crystal contamination within fields used for cytological staining be present, it may be difficult to accurately identify cell types present and the smear should be discarded. Scale bars = 50 μm.
These changes in cell typology are indicative of underlying endocrine events. The proestrus phase of the estrous cycle corresponds to the human follicular phase of the menstrual cycle5 and is defined by a pre-ovulatory increase in circulating 17-β-estradiol levels6, as well as a small surge in prolactin7 (Figure 2, Proestrus, left panel). The increase in 17-β-estradiol indirectly stimulates gonadotropin-releasing hormone neurons in the hypothalamus and septum that, in turn, activate responsive cells in the anterior pituitary to release luteinizing hormone and follicle-stimulating hormone into the circulation8,9 (Figure 2, Proestrus, left panel). In vaginal smears taken from animals in proestrus, cells are almost exclusively oval nucleated epithelial cells (Figure 1D, Figure 2, Proestrus, right panel). The peak in follicle-stimulating hormone levels signals ovulation and entry into estrus10,11. During estrus, 17-β-estradiol levels decline and prolactin levels peak6,7 (Figure 2, Estrus, left panel). Vaginal smears are characterized by almost exclusive detection of irregular-shaped cornified squamous epithelial cells often in clumps (Figure 1E, Figure 2, Estrus, right panel). Entry into metestrus coincides with a continuous rise in progesterone hormone levels6 and corresponds to the beginning of human luteal phase12 (Figure 2, Metestrus, left panel). As progesterone levels start to rise and there is a small surge in 17-β-estradiol levels in response to corpus luteum activation6,13,14 (Figure 2, Metestrus, left panel). The cell types present in vaginal smears during this stage are fragmented, cornified epithelial cells and smaller darker stained leukocytes (Figure 1F, Figure 2, Metestrus, right panel). Finally, entry into diestrus in mice occurs and circulating progesterone levels peak6, corresponding to the human late luteal phase12. Regression of the corpus luteum leads to a subsequent sharp decline in progesterone levels15,16 (Figure 2, Diestrus, left panel). Leukocytes predominate in smears during diestrus. The frequency of cornified epithelial cells is reduced and nucleated epithelial cells begin to be detected just prior to transition to proestrus (Figure 1G, Figure 2, Diestrus, right panel).
In summary, this simple, routine protocol can be used to estimate daily hormonal fluctuations and establish estrous stage in experimental mice without altering reproductive status if the following precautions are taken. Sampling should be performed no more than once daily using the non-invasive protocol described here as compared to repeated penetration of the vaginal canal, aspiration, and agitation. This can cause vaginal irritation resulting in an inflammatory response17 resulting in leukocytes and other cell types to be present in smears that may confound cytological assessment. Moreover, even in colony-housed females, it is normal to see extended diestrus and estrus stages in different mice as well induction of anestrous18 and this identification is useful in the interpretation of hormonal impact in reproductive, gender, and disease studies. Variability in cycle length is also introduced with age and by housing differences within colonies (individual or group-housing) of females7,19-21. Females housed in female-only colonies can cease cycling and enter a state of prolonged diestrus18,22,23 although cycling can be re-instated by exposure to cages pretreated with male urine to elicit cycling24,25. Thus, to establish individual cycle lengths for a given mouse, it is recommended that the non-invasive assessments described here be performed daily, with care, until two complete cycles are observed.
The authors declare no conflict of interest. All experiments on animals were performed in strict accordance with the guidelines and regulations set forth by the University of Ottawa Animal Care Committee and the Canadian Council on Animal Care.
We would like to thank Marc Leonard from Carleton Immersive Media Studio for expert technical assistance in video production and editing and Dr. Martin Bertrand from Carleton Immersive Media Studio / Neural Regeneration Laboratory for visual model assistance. The authors gratefully acknowledge the expert advice of Dr. Marilyn Keaney and all of her dedicated staff at the University of Ottawa Animal Care and Veterinary Services. This work was funded by the Canadian Institute of Health Research (CIHR, MOP 62826) to SALB, the CIHR Institute of Aging and Strategic Training Initiative in Health Research/CIHR Training Program in Neurodegenerative Lipidomics (TGF 96121) to SALB and SF, Canadian Foundation for Innovation to SF, Ontario Innovation Trust to SF, and Autodesk Research to SF. ACM receives a CIHR Banting and Best doctoral award. NV receives a post-professional fellowship from Institute of Aging and CIHR Training Program in Neurodegenerative Lipidomics.
|Sterile 200 μl pipette tips||Diamed||E340901|
|Latex bulb (1 ml)||Fisher||03-488-21|
|Glass microscope slides||Fisher||12-550-15|
|Crystal Violet stain (25 g)||Fisher||C581-25|
|Light-duty Tissue Wipers||VWR||82003-820|
|Microscope Cover Glass (22x30)||Fisher||12-544A|