Synchronizing the estrous cycle increases the efficiency of the production of pseudopregnant mice. Herein, we ascertained whether LHRHa could synchronize the estrous cycle in mice.
We successfully established a scheduled production method for pregnant and pseudopregnant female mice using LHRHa. This method could increase the efficiency in producing pseudopregnant mice compared to the visual method, which is being widely used. While the visual method is the most non-invasive method for selecting the female in producing pregnant and pseudopregnant mice, this method requires long-time training and vast space for keeping numerous mice, both females’ and VAS male stock. Moreover, this method poses a challenge for beginner researchers and small-scale animal facilities [14, 17, 19].
We attempted the vaginal cytology method, which is relatively easy for experimental beginners, to assess the estrous cycles before mating. Approximately 60% of female mice, identified as on the receptive stage (proestrus or estrus) by the vaginal cytology method, successfully mated with the male mice (Supplementary Table S1). Usually, at least 50% of the selected mouse in the estrus stage will mate . The presence of a vaginal plug indicates that mating has occurred and must be checked carefully in the early morning, as they could fall out or no longer be detectable 12 hours past mating.
The vaginal cytology method is accepted as the most accurate method to evaluate all stages of the estrous cycle in mice . However, the process is laborious and requires a long training period to be a skillful observer, and often disparities among the observers might occur. A report on the estrus stage judgment following vaginal cytology has been developed by Sano et al. (2020) to avoid differences in the accuracy depending on the examiners , but it fails to mention the correlation between the estrus stage and the copulation frequency. Even though this method helps select the mice in their receptive stage, a large number of female stocks still needs to be maintained as only 12–15% of female mice would be on the estrous cycle normally. Therefore, this method alone is not efficient in producing pregnant or pseudopregnant female mice.
Progesterone treatment in mice was shown to synchronize the estrous cycle in mice and was established as a new effective method than the visual method . Since 63% of female mice had a vaginal plug after mating with male mice, the method reduced the number of required female mice and succeeded in the embryo-transfer experiment by preparing planned pseudopregnant female mice. However, this method needed the female to pair with a male three days before mating for the efficiency and was less than 50% copulation rate if there was no continuous pairing. Eventually, this method needed to increasing the number of male mice in stock.
Administration of LHRHa at a dose of 20 µg/mouse synchronized the estrous cycle at the estrus stage in most females (73.3%) on Day 4; however, most females were not mating with males (Fig. 1, Fig. 2, Table 1, and Table 2). Subsequent administration of 20 µg/mouse (group_3) LHRHa for two days confirmed 78% of copulation frequency with one day pairing only. Moreover, the frequency of copulation in LHRHa-treated group_3 was higher than the one using the vaginal cytology method (64%) (control in Table 2). Interestingly, the mice in group_3 showed higher copulation frequencies than those in group_1, wherein 73.3% of the mice synchronized at the estrus stage. This percentage represented the highest synchronization rate corresponding to the estrus stage (20 µg/mouse in Table 1).
Reportedly, a mouse is sexually receptive when it is either in the proestrus and estrus stage . Our results are consistent with this previous study as shown in Supplemental Table 1. However, the copulation frequency of the female mice was approximately 60% at both the proestrus and estrus stages. This observation suggested that female mice, whose estrous cycle may be the late proestrus to the early estrus stage, have a higher chance of copulation with male mice. Further, given that the number of female mice at the proestrus stage in group_1 was lower than that of the female mice at the metestrus stage, which is the stage that passed the estrus stage (20 µg/mouse in Table 1), it is probable that some of the female mice in group_1 at estrus stage were in the late estrus stage.
Conversely, in group_3 (15%, 3/20), the number of female mice at the proestrus stage was greater than the number of mice at the metestrus stage (5%, 1/20). Therefore, most of the female mice at the estrus stage (65%, 13/20) in group_3 may have been at the early estrus stage. Thus, the frequency of copulation may have increased to a greater extent relative to group_1.
Based on these observations, it is evident that estrus synchronization via LHRHa administration made mating between female and male mice within one day possible. Thus, via this strategy, the total number of mice required for mating decreases and scheduled pregnancy as well as pseudopregnancy in female mice becomes possible.
Reportedly, the induction of pseudopregnancy and pregnancy is related to the number and rate of intromission . Additionally, neural stimuli from the vagina, which integrate with other sensory inputs on mating, are essential for initiating the neural and endocrine mechanisms that support pseudopregnancy and/or pregnancy . Therefore, it is necessary to control the estrous cycle as well as the induction of copulation to bring about pseudopregnancy and pregnancy.
Reports have shown that gonadotropin-releasing hormone (GnRH), whose function is similar to LHRH, regulates ovulation and promotes female sex behavior, such as lordosis [30, 31]. However, progesterone, released from the corpus luteum in the ovary, affects the hypothalamus, including GnRH neurons, and inhibits GnRH secretion. Even though GnRH is indirectly involved in progesterone secretion, the direct function of GnRH is to secrete luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from the pituitary. FSH induces the secretion of progesterone. Therefore, the administration of LHRHa may have effectively synchronized the estrous cycle and the induction of female sex behavior by adjusting the progesterone secretion.
PMSG belongs to the class of gonadotropin hormones and has a similar function to GnRH. Even though PMSG treatment shows a high frequency of copulation, our study showed that PMSG-treated mice delivered newborns with low viability. Thus, PMSG is not an excellent option for producing pseudopregnant female mice. Previous studies have also reported harmful effects of PMSG on the fetus development [20, 25, 26]. In contrast, more than 90% LHRHa treated plug-positive female mice became pregnant, and more than 85% of pups survived with normal body weight (Fig. 3 and Table 2).
While the function of PMSG and LHRHa are the same, our study showed them to have different effects on fetal development. A comparative investigation of these components, such as chemical states, physical properties, and physiological functions, might lead to the development of new strategies for treating miscarriages and newborn mortality in the future.
Overall, we succeeded in establishing an effective production method for pregnancy and pseudopregnancy female mice using LHRHa administration. This method is easy to apply, even for beginner experimenters, and could be performed in small-scale animal facilities. We believe that this study could make the generation of GEMMs more feasible and help progress the research in reproduction and animal biology while supporting the principle of 3Rs- reduction, refinement, and replacement.