Scheduled simple production method of pseudopregnant female mice for embryo transfer using the luteinizing hormone-releasing hormone agonist

doi:10.21203/rs.3.rs-1637853/v1

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Scheduled simple production method of pseudopregnant female mice for embryo transfer using the luteinizing hormone-releasing hormone agonist

https://doi.org/10.21203/rs.3.rs-1637853/v1

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The use of mice as experimental animal models has been a practice since the development of genetically engineered mouse models (GEMMs) in the early 1980s. New technologies, including genome editing, have helped in the time- and cost-efficient generation of GEMMs. However, methods for preparing pseudopregnant females, essential for the generation of GEMMs, remain less advanced. This study proposes a new method to achieve simple production of pseudopregnant female mice using a luteinizing hormone-releasing hormone agonist (LHRHa). A 20 µg LHRHa/mouse was identified as the best dose for inducing estrus synchronization. However, the frequency of copulation was 40% on a single injection. With sequential injections of 20 µg LHRHa/mouse on Days-1 and − 2, followed by pairing on Day-5, 78% of LHRHa-treated females copulated with male mice. Moreover, LHRHa treatment did not affect fetal development and offspring viability. Eventually, successful generation of offspring via embryo transfer was attained using LHRHa-treated pseudopregnant females. LHRHa administration method is efficient in producing pseudopregnant female mice for the generation of GEMMs, and we expect that it will contribute towards advancing the clinical research.

Genetically engineered mouse models (GEMMs), such as knock-in, knock-out, and transgenic mice, have contributed significantly to clinical and biological research, serving as effective disease models. New technologies, including assisted reproductive technology (ART) [1–3] and gene targeting knock-out technology [4], have increased the number of GEMMs since the 1980s. Moreover, the advancements in gene-editing technologies, such as CRISPR/Cas9, have helped in the faster, accurate, and efficient development of GEMMs [5, 6].

Since the 1980s, genome engineering and ART have improved in a time- and cost-efficient manner. The ART in mice is on the rise with many recent advancements, including an increasing number of oocyte collection [7, 8], increased efficiency of in vitro fertilization [9, 10], and improved cryopreservation and resuscitation methods [11–13]. However, the process of embryo transfer has changed little since the 1980s. It remains a challenge to produce pseudopregnant female mice as recipients of the embryos in the embryo transfer process. Female mice in proestrus and estrus stages need to be selected by visual observation of the vagina before pairing with vasectomized (VAS) males. The accuracy and precision of the female mice selection depend solely on the operator's training and experience [14–15]. Moreover, since the length of the estrous cycle in mice is four to five days (proestrus: < 24 hours, estrus: 12–48 hours, metestrus; 8–24 hours, and diestrus: 48–72 hours) [15, 16], operators need to keep more than four times the number of female mice required for the experiment [14, 17, 18]. Even though methods for producing pseudopregnant female mice have been reported before, the efficiency of the process depends on the operator's method, training, and experience, and thus, these methods are relatively tricky for beginner operators.

Several researchers have attempted to overcome these issues by assessing estrus stages by combining visual observation of the vagina and vaginal cytology [17] and the synchronization of estrus stages [19, 20].

The protocol for producing pseudopregnant so far, the method of synchronization of estrus stages using progesterone was 63% of female mice had a vaginal plug after mating with male mice and the most effective method for beginner operators [19]. However, since this protocol needed three days of continuous paring with male mice before mating, there was a demerit that the number of keeping male mice increased.

In contrast, several efficient methods for producing pseudopregnant female rats using exogenous hormone treatment have been described previously [21–24].

This study focused on administering the luteinizing hormone-releasing hormone agonist (LHRHa) [22] for producing a pseudopregnant female mice, as LHRHa has the estrus synchronization potency. Moreover, LHRHa can be dissolved in water and administrated through intraperitoneal injection allowing for a simple administration technique.

We first determined an effective dose of LHRHa in synchronizing the estrus stage in mice. Following LHRHa-mediated estrous cycle synchronization, production of pseudopregnant mice was scheduled, and we defined the protocol that 78% of female mice were mated with male mice for one day only pairing. Finally, we performed embryo transfer with LHRHa-treated pseudopregnant mice as recipients to determine the effects of LHRHa treatment on the reproductive parameters. At last we could establish the new simple method for producing pseudopregnant female mice, which reduce the space and expense in maintaining female stocks and VAS male stocks.

Dose optimization of LHRHa for estrus synchronization

The optimal LHRHa dose required for estrous cycle synchronization in mice was identified by administrating different doses of LHRHa in ICR female mice and analyzing their estrus stage using the vaginal cytology method. Borjeson et al. (2014) reported that by administrating 40 µg LHRHa/rat and analyzing the vaginal cytology 96 hours later, 55% of female rats were in the late proestrus to estrus stage [22].

By referring to Borjeson et al. (2014) report, we designed four different doses (0 µg, 5 µg, 10 µg, 20 µg, or 40 µg) of LHRHa. Single dose-LHRHa was intraperitoneally administered to a female mouse, and the vaginal cytology examination was performed 96 hours later. The mice who received a dose of 20 µg showed 73.3% (11/15) synchronization in their estrous cycle (Fig. 1), suggesting the estrus synchronization potency of LHRHa.

Table 1

Estrus stages at Day 4 after LHRHa administration in ICR females.
dosage	n	proestrus	(	%	)	estrus	(	%	)	metestrus	(	%	)	diestrus	(	%	)
0 µg ^(#1)	1040	186	(	18	)	270	(	26	)	124	(	12	)	460	(	44	)
5 µg	15	1	(	6.7	)	9	(	60	)	0	(	0	)	5	(	33	)
10 µg	15	1	(	6.7	)	8	(	53	)	1	(	6.7	)	5	(	33	)
20 µg	15	1	(	6.7	)	11	(	73	)	2	(	13	)	1	(	6.7	)
40 µg	15	2	(	13	)	7	(	47	)	0	(	0	)	6	(	40	)
(#1) Vaginal smear samples were collected from randomly chosen female mice.

Frequency Of Copulation After Lhrha Treatment

We confirmed that 63% (26/41) of female mice at the proestrus stage and 64% (114/178) at the estrus stage copulated with the male mice (Supplementary Table S1). We examined the frequency of copulation after LHRHa treatment based on the presence of vaginal plug on female mice, compared with females in proestrus and estrus stage without any treatment (64%, 140/219) as the control.

We investigated the copulation frequency of mice with the highest estrus synchronization cycle (i.e., the mice who received 20 µg of LHRHa) (Fig. 1b). Since most of the female mice after LHRH administration were in estrus stage 92–96 hours (Day four) later, mating with male mice started at Days four (group_1) or three (group_2) after LHRHa administration (Fig. 2a). Both conditions (group_1: 40%, 4/10, and group_2: 30%, 3/10) showed no improvement in the copulation efficiency compared with the control (Fig. 2b and Table 2).

Table 2

Effects of LHRHa on copulation and fetal development.
exp. group	No. (%) of females		Litter size ^(#3)			Viability of offspring (%)	Body weight of offspring (g) ^(#4)
exp. group	With plug ^(#2)	Pregnant	Litter size ^(#3)			Viability of offspring (%)	Body weight of offspring (g) ^(#4)
control ^(#1)	140/219 (64)	6/6 (100)	15	±	2	91/91 (100)	1.8	±	0.2
group_1	4/10 (40)	4/4 (100)	15	±	1	59/59 (100)	1.8	±	0.1
group_2	3/10 (30)	3/3 (100)	15	±	4	46/46 (100)	1.8	±	0.2
group_3	21/27 (78)	10/11 (91)	12	±	3	92/106 (87)*	1.8	±	0.2
(#1) As a control, the data for calculating the frequency of copulation (data of “with plug”) in Fig. 2 were collected from the proestrus and estrus female mice using vaginal cytology and that in Fig. 3 were collected from LHRHa non-treated plug-positive female mice.
(#2) Plug were checked one day after pairing with VAS male or WT mice.
(#3) n: All pregnant females in each experimental group.
(#4) n: All viable offspring in each experimental group.
Fisher’s exact probability test was performed to calculate the probability of “with plug” and “pregnant” mice and viability of the offspring. Kruskal-Wallis test with Bonferroni correction was performed for calculating the litter size and body weight of the offspring. * P < 0.01 vs. the corresponding value of the control.

In a report on the estrus synchronization using progesterone, Hasegawa et al. administered progesterone once a day for two days [8]. We attempted a similar condition in group_3 (Fig. 2a). The copulation efficiency in group_3 was improved (78%, 21/27) and was higher than the control (Fig. 2b and Table 2), with no significant result (P = 0.064). Furthermore, the copulation efficiency was increased in group_3 combined with the visual method (91.6%, 11/12, n = 20), but four plug-positive female mice were excluded (Supplementary Fig. S2). Next, we investigated whether the critical point to improve the copulation efficiency was the administration schedule, which is once a day for two days or the total dose. Therefore, we attempted five additional conditions described in Supplementary Fig. S1 and Supplementary Table S2. Our results showed that none of these five conditions increased the frequency of copulation compared with the control or the group_3.

Embryo Development After Lhrha Treatment

The pregnant mare serum gonadotropin (PMSG), whose efficacy is similar to LHRHa in synchronizing the estrus, has been widely used to induce superovulation in mice. However, PMSG administration can disrupt the maintenance of pregnancy, affecting the development of the fetus [20, 25, 26].

We mated the LHRHa-treated female mice with wild-type ICR male mice, and the plug-positive female mice were observed and analyzed for their fetal developmental abilities such as pregnancy rate, litter size, the bodyweight of offspring and offspring viability. The offspring viability in group_3 showed a significant difference compared to the control, with a more than 85% survival rate. Therefore, we concluded that none of the categories of LHRHa treatments affected embryo development (Fig. 3 and Table 2).

Generation Of Pseudopregnant Female Mice Using Lhrha Treatment And Embryo Transfer

After confirming that LHRHa treatment did not affect embryo development, we investigated whether LHRHa treated female mice could be used as pseudopregnant female mice for recipients in embryo transfer. We examined birth rates following embryo transfer in LHRHa-treated group_3 mice. After obtaining pseudopregnant female mice from group_3, we transferred tetraploid complemented embryos into their uterus. Offspring were obtained by natural delivery or Caesarian section on embryonic day 19.5. Even though few offspring died at birth or until the next day, more than 60% of offspring viability was confirmed (Table 3).

Table 3

4n complementation of ESC lines using the LHRHa treated pseudopregnant female.
Exp. condition	ESC lines	No. of
Exp. condition	ESC lines	pseudopregnant	transferred	offspring (%)	wean (%)
control ^(#1)	R01-09	2	40	7 (18)	6 (86)
group_3	R01-09	3 ^(#2)	30	7 (23)	5 (71)
group_3	Ets2 ^{null (em3/em3)}	3 ^(#2)	30	9 (30)	6 (67)
(#1) Pseudopregnant female mice of control were prepared by the visual method.
(#2) In all three female mice, ten R01-09 embryos were transferred into the left uteri, and ten Ets2 null (em3/em3) embryos were transferred into the right uteri. R01-09 offspring were recognized by RFP fluorescence signal, and Ets2 null (em3/em3) offspring were recognized by wavy hairs, which is known as Ets2-null mutant phenotype, after 10-day old.

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 [18]. 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 [15]. 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 [27], 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 [19]. 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 [28]. 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 [29]. 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 [29]. 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.

Animals

All animal experiments were conducted in accordance with the guidelines of “Regulations and By-Laws of Animal Experimentation at the Nara Institute for Science and Technology,” and were approved by the Animal experimental Committee at the Nara Institute of Science and Technology (the approval no.1639 and 2103). Study of the animal experiments were carried out in compliance with the ARRIVE guidelines [32]. ICR mice and 129X1 mice were purchased from SLC (Japan).

The mouse is maintained under 12 hours light/ 12 hours dark cycle (light on 08:00 AM). Female ages are 8–24 weeks. Male vasectomized mice are prepared but cutting their vas deferens. Food and water are available ad libitum.

Treatment of the luteinizing hormone-releasing hormone agonist.

LHRHa ([des-Gly10, D-Ala6]-LH-RH ethylamide acetate salt hydrate Sigma-Aldrich, L4513) were solved in saline. Four conditions of LHRHa (5 µg, 10 µg, 20 µg, 40 µg/mouse) were administrated to 8–24 weeks ICR female mice between 11:00 am − 12:00 am, intraperitoneally. For the vaginal cytology method, LHRHa treated female mice were analyzed four days later. For the assessment of the frequency of copulation, and the production of pregnant or pseudopregnant female mice, LHRHa treated female mice were mated with wild type ICR male mice or vasectomized ICR male mice in various conditions, described as Fig. 2a. As the control, non-treated female mice were used each experiment.

Cytological assessment of the estrus stage

Cytological examination of the estrus stage performed as described by Byerset al. (2012) [15] with modification on cell transfer process to the glass slide. Vaginal cells are collected by swab method using a small size cotton-tipped wetted with room temperature phosphate buffer saline (PBS) and inserted into the vagina of the restrained mouse. The cotton tip then turned and rolled carefully against the wall of vagina. The cotton tip was then dipped into 25 ul PBS. The vaginal cells then transferred into glass slide (Matsunami micro slide glass S2112), and then dried inside 56°C oven for 20 minutes and then stained with crystal violet (Merck 109218) for 90 seconds. The slides washed carefully with tap water to rinse the excess staining. After dry, microscopic observation was done under light microscope with 20 x magnification to observe. All cell types samples were collected at 08:00 am − 11:00 am.

Inducing pregnancy and pseudopregnancy

8- to 24- week-old ICR female mice were paired with wild type ICR male mice for producing pregnant female mice or vasectomized ICR males for producing pseudopregnant female mice. ICR males are housed one per cage with a female introduced at 15:00–16:00 h and vaginal plug was checked at 09:00 pm – 11:00 pm the following morning. Females are independently examined for the presence of vaginal plug. The day of plug detection is defined as 0.5-day post coitum (E0.5).

ES cell culture

R01-09 ESC line was established from embryos by crossing 129X1 female mouse and EGR-R01 ESC (EGR-R01(CAG-mtDsRed2) BDF1x129Sv) derived male mouse. EGR-R01 ESC line was kindly gifted by Dr. Ikawa. The Ets2 ^em3/em3 ESC line was reported previously [33]. Establishment of ESC line and maintaining of ESCs were performed as same as previous report [33]. Both ESC lines were provided for tetraploid complementation.

Tetraploid complementation

Tetraploid embryos were prepared as described previously [33, 34]. In brief, ICR two-cell stage embryos were placed in the fusion buffer, and electrofusion was performed by applying 140 V for 50 ms after aligning embryos between the electrodes. CFB16-HB and LF501PT1-10 electrode (BEXCo.Ltd., Tokyo, Japan) were used for cell fusion. Fused embryos were incubated until use. Six to twelve ESCs were injected into a tetraploid four-cell embryo, and then cultured until the blastocysts stage and transferred into the uterus of E2.5 pseudopregnant ICR mice. Offspring were recovered by natural delivery or Caesarean section on E19.5.

Statistical analysis

The statistical difference was determined using the Fisher’s exact probability test for the frequency of copulation and pregnancy rate and offspring viability comparing with the control. Bonferroni correction on Kruskal-Wallis test was used for the litter size and the offspring body weight comparing with the control. A probability of P < 0.01 was considered statistically significant.

Acknowledgements

This work was supported by JSPS KAKENHI Grant Number 20H0046921 to A.H., 21K19267 to A.I, and KAC 40th Anniversary Research Grant to A.I.

We appreciate to the animal experimental facility stuff for supporting to perform this study. We would like to thank to Dr. Ikawa to provide EGR-R01 ESC line.

The authors would like to thank Enago (www.enago.jp) for the English language review.

Author contributions

G.PS and A.I. performed most experiments, assisted by A.H., who provided LHRHa information and discussed this experimental plan, and S.Y., performed to help accumulation of the data. G.PS and A.I. wrote the manuscript and all authors discussed the results and commented on the manuscript.

Competing interests

The authors declare no competing financial interests.

Data availability statement

All data generated or analyzed during this study are included in this published article (and its Supplementary Information files).

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No competing interests reported.

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Editorial decision: Major revision
12 Jul, 2022
Reviews received at journal
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Reviewers agreed at journal
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Reviewers invited by journal
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Editor invited by journal
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Scheduled simple production method of pseudopregnant female mice for embryo transfer using the luteinizing hormone-releasing hormone agonist

Status:

Version 1

Abstract

Figures

Introduction

Results

Dose optimization of LHRHa for estrus synchronization

Frequency Of Copulation After Lhrha Treatment

Embryo Development After Lhrha Treatment

Generation Of Pseudopregnant Female Mice Using Lhrha Treatment And Embryo Transfer

Discussion

Methods

Animals

Cytological assessment of the estrus stage

Inducing pregnancy and pseudopregnancy

ES cell culture

Tetraploid complementation

Statistical analysis

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1