Vaginal smear cytology

Determination of the estrous stage in females was performed by assessing vaginal smear cytology. To assess the phase of the estrous cycle that female mice presented during FA, all female mice were monitored for 3 to 4 consecutive cycles (approximately 10 to 14 days) before learning to test for the regularity of the cycle. We performed a vaginal lavage with a 20-μl pipette that was loaded with 10 μl of standard NaCl 0.9% (w/v) solution, and later, the tip was softly placed on the vaginal aperture. In case of urination when grabbing the animal, urine was cleaned using a regular tissue. Male mice received the same amount of handling as female mice. The 10 μl of saline was unloaded and collected five consecutive times to collect enough cells for assessment and later placed on an adhesion slide (Superfrost Plus, Thermo Fisher Scientific, Barcelona, Spain). All vaginal smear samples were collected between 9:30 and 11:30 a.m. Slides were dried using a hot plate (HI1220, Leica, Madrid, Spain) at 37°C for 30 min and later stained in Cresyl Violet acetate (C5042, Sigma-Aldrich, Spain) 0.1% (v/v), washed twice for 1 min in distilled water, and read in bright-field microscopy with a 10× or 20× objective in an Eclipse 80i microscope (Zeiss, Spain).

Three different cell types may appear in the preparation: cornified epithelial cells, round nucleated epithelial cells, or leukocytes. The different stages of the estrous cycle were assessed depending on the proportion of the abovementioned cells. Proestrus is characterized by a high proportion (>80%) of nucleated epithelial cells that might present very small amounts of cornified epithelial cells or leukocytes. Estrus is typically presented with cornified epithelial cells with a lower grade of staining than leukocytes and nucleated epithelial cells. Metestrus presents a mixture of cornified epithelial cells and a considerable proportion of leukocytes. Diestrus is characterized by >90% of leukocytes that might present a very small proportion of round nucleated epithelial cells. After the assessment of regular cycling, females were distributed in groups according to the stage of the estrous cycle they presented before FA, which is also the day they received the CNO injection after FA (6).

For the electrophysiology experiment, vaginal swabs were collected for at least 1 week before sacrifice at proestrus, consistent with previous work (6, 35). For identification of the estrous cycle stage, vaginal smears were stained using a Shorr stain (Sigma-Aldrich, catalog no. 109275). The staging was identified using the Track-by-Day method, predicting proestrus as 3 days after estrus. Estrus was identified by a fully cornified cell histology smear, appearing in sheets and clumps with little to no nucleated epithelial and leukocyte presence (36).