All of the procedures performed in this study were carried out in accordance with the guidelines and regulations set forth by the University of Ottawa Animal Care Committee and the Canadian Council on Animal Care. A rapid means of assessing reproductive status and rodents is useful not only in the study of reproductive dysfunction, but is also required for production of new mouse models of disease and investigations into the hormonal regulation of tissue degeneration or regeneration following pathological challenge. Here we describe how to identify the stage of the reproductive cycle of female mouses in on any given day by simple, non-invasive cytological assessment of the predominant cell type present in vaginal smears.
The mirroring reproductive or estro cycle is divided into four stages. PROEs es, metes, and drisk define fluctuations in the circulating levels of the ovarian steroids, 17 beta estradiol and progesterone, the gonadotropins, luteinizing, and follicle stimulating hormones, and the lute atropic hormone prolactin signal transition through these reproductive stages. The proestrus phase of the esra cycle corresponds to the human follicular phase of the menstrual cycle and is defined by a pre ovulatory increase in circulating 17 beta estradiol levels, as well as a small surge in prolactin.
The increase in 17 beta estradiol is followed by a rise in circulating luteinizing hormone and follicle stimulating hormone levels. The peak in follicle stimulating hormone levels signals ovulation and entry into es. During ES stress, 17 beta estradiol levels decline in prolactin levels.
Peak entry into met estro coincides with a small surge in circulating follicle stimulating hormone levels and corresponds to the beginning of the human luteal phase. Progesterone levels start to rise and there is a second small surge in 17 beta estradiol levels. In response to the corpus lium activation entry into diastrous and mice occurs when the circulating progesterone levels peak and corresponds to the human.
Late luteal phase. Regression of the corpus luteum leads to a subsequent sharp decline in progesterone levels and entry into prote. Changes in cell typology reflect these underlying endocrine events in the relative ratio of epithelial cells.
Quantified cells and leukocytes detected in vaginal smears can be used to identify each of the esra stages. Hello, my name is Ashley McLean. I'm a researcher in the neuro regeneration laboratory under the direction of Dr.Stephanie Bennett at the University of Ottawa.
And I'm Nicholas Valenzuela. I work with Dr.Steven Phi at the Carlton Immersive Media Studio at Carlton University in Ottawa. In this video, we describe a simple protocol that can be used for EST extra cycle stage determination.
This protocol will detail how you can differentiate between the four mouse reproductive stages of proestrus, met Es and d Este by assessing predominant cell typology and vaginal smears. So let's get started. For this protocol, you'll need to prepare two items, autoclave, double distilled water, and a 0.1%crystal violet solution.
The staining solution can be stored in a tightly sealed container at room temperature until needed. Start by placing a latex bulb on the end of a sterile 200 microliter tip and draw up approximately a hundred microliters of the sterile double distilled water. Use the gradations on the tip as a volume guideline.
Place your mouse on the hopper such that her hind end is facing towards you. Firmly grasp her tail and gently elevate her rear end. The mouse will use her front paws to grasp the hopper.
At this point, the mouse may urinate. If so, rinse off the region with double distilled water from a separate tip. Place the end of your double distilled water filled tip at the opening of the vaginal canal.
Gently depress the bulb and expel a quarter to half the volume of water into the vaginal canal. Then slowly release the pressure exerted on the bulb, withdrawing the fluid back into the tip. Repeat this previous step four to five times using the same tip bulb and fluid.
Avoid releasing pressure too quickly to avoid aspirating fluid into the bulb. A filter tip may be useful for this purpose. Return the mouse to her cage and expel the fluid onto the glass slide.
Allow the slide to completely dry at room temperature. Once dry, these Este extra smears can be stained immediately or at a later date. Place the air dried slide in the crystal violet stain for one minute.
Wash the slide with double distilled water for one minute. Repeat, remove the excess double distilled water off the slide with a light duty tissue wiper. Avoiding the stained smear region pipe had approximately 20 microliters of glycerol on top of the smear and cover slip.
You should examine the stained smears as soon as possible under light microscopy, as the stain will diffuse from cells in the glycerol mounting over time. Start by examining the entire smear at a lower magnification. Select a representative area and move to a higher magnification.
During the Pro Astra stage, cells are almost exclusively nucleated epithelial cells. During the extra stage, the cells you'll see are predominantly squamous cornified epithelial cells, usually forming densely packed clusters. These cells are irregularly shaped and have no visible nucleus.
During met estro stage, rare cornified squamous epithelial cells are still observed, but now there's a predominance in smaller, darker stained leukocytes. During the diastrous stage, there are few, if any, squamous cornified epithelial cells. Remaining leukocytes are present in large numbers.
Nucleated epithelial cells are observed, found both as single cells and in clusters. One issue you may encounter is urine contaminating your smear. Should this happen, you should discard the smear and not use it for staging.
We've just shown you how to perform vaginal lavages. Collect samples, stain these samples and assess cell typology to determine the stage of the est extra cycle of female mouses in at time of analysis. To summarize, during the pre ovulatory phase in PROEs, a peak in circulating 17 beta estradiol levels is reflected by a predominance of well-formed nucleated epithelial cells in the vaginal smear samples.
A subsequent peak in follicle stimulating hormone levels signals ovulation and entry into estro cornified squamous epithelial cells predominate in the smears. During metes, progesterone levels rise. The cell type present in vaginal smears during this time point are fragmented cornified epithelial cells, and smaller, darker stained leukocytes.
During dste, with a peak in progesterone, nucleated, epithelial cells begin to appear. Leukocytes are still present. To ensure a female mouse is cycling regularly and to establish the length of estro cycle, these assessments should be performed daily and assessed over two complete cycles is normal to see extended diastrous and esra stages in different mice.
We hope this helps you in your gender and hormonal focused studies, and good luck with your experiments.
