Diestrus

A schematic of the experimental design is shown in Fig. 1A. Preinjection estrous cycle monitoring was performed for at least 2 weeks to confirm that mice had regular cycles before any experimentation. All mice tested for the present studies showed at least two consecutive regular cycles prior to injection. After KA/saline injection, mice were allowed to rest for a 2‐week recovery period, and then were monitored daily for estrous cycle stages for an average of 66 days (range from 60 to 75 days) postsurgery. Daily vaginal cytology was performed to determine the estrous cycle stages (proestrus, estrus, metestrus, and diestrus). The vaginal lavage method20 was used for vaginal cytology examination and was performed between 10 am and 12 pm (relative to lights off at 7 pm). Twenty microliters of sterile 1% phosphate‐buffered saline (PBS) was inserted into the vaginal cavity, withdrawn, and smeared on a microscope slide. Estrous cycle stages were determined according to the type, number, and morphology of cells in the smear. Proestrus is indicated by the presence of mostly nucleated epithelial cells, estrus by cornified epithelial cells, metestrus by cornified epithelial cells with leukocytes, and diestrus by leukocytes21 (Fig. 1B). Regularly cycling mice showed cycles with an average 5‐day periodicity (1 day each of proestrus, estrus, metestrus, diestrus day I, and diestrus day II; Fig. 1B).20Estrous cyclicity was evaluated from two aspects. The cycle length was calculated by counting the days between two successive estrus stages with both metestrus and diestrus stages occurring in between. The percentage of time spent in each stage was calculated by the number of days spent in each stage divided by the total number of days of monitoring. In order to determine whether each mouse displayed regular or irregular estrous cyclicity, the average cycle length and percentages of time in each stage were calculated from the data collected from 42 days postinjection until the last day of monitoring. If a mouse spent more than 50% of time in one stage, or the average cycle length was 7 days or longer, this mouse was considered to have an irregular estrous cycle. Note that here we use the term “regular” to refer to mice whose cycle parameters fall within a normal range, as assessed through these measurements. For rodents, prolonged diestrus may indicate an acyclic period or pseudopregnancy; these two situations are usually accompanied by heavy mucus secretion.22, 23 The mice in this study, however, did not show this type of secretion. The ages of saline‐treated and KA‐treated mice were not significantly different at the time of injection (saline 11.5 + 0.3 weeks, KA 11.9 + 0.7 weeks, p = 0.45 Mann–Whitney test). The estrous cycle of both groups was monitored for 2 months after injections, and then the mice were euthanized, at which time the ovaries were collected for histology (time postinjection: saline 9.4 ± 0.9 weeks, KA 9.5 ± 0.6 weeks, p = 0.67 two‐sample t test). The body weight of each mouse was measured on the day of surgery and the last day of monitoring, and these values were used to calculate mean weight gain per week and final body weight values.