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Biology of Reproduction 61, 367-371 (1999)
©Copyright 1999 Society for the Study of Reproduction, Inc.

Articles

Participation of Embryonic Genotype in the Pregnancy Block Phenomenon in Mice1

Hak Jae Chunga, Wei Dong Zhaoa, and Noboru Wakasugi2,a

a Laboratory of Animal Reproduction, School of Agricultural Sciences, Nagoya University, Chikusa, Nagoya, 464–8601, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Pregnancy block by male pheromones in mice differs in incidence depending on the combination of strains. Female mice of BALB/cA strain mated with BALB/cA males show a 100% pregnancy block when exposed to males of inbred strain DDK shortly after copulation (Chung et al., Biol Reprod 1997; 57:312–319). In the present study, BALB/cA females mated with the males of other strains—CBA/J, C3H/HeN, C57BL/6Cr, and IXBL—showed higher pregnancy rates (66.6–87.5%) even when they were exposed to DDK males. In the pharmacological induction of pregnancy block with dopamine agonist (bromocriptine, 4 mg/kg BW), BALB/cA females mated with BALB/cA males showed a 100% pregnancy block. In contrast, BALB/cA females mated with CBA/J, C3H/HeN, and C57BL/6Cr males showed higher pregnancy rates (40–70%). These results suggest that the better pregnancy rate of BALB/cA females mated with alien males may be due to the stronger viability of F1 embryos. This interpretation was confirmed by an embryo transfer experiment in which a higher implantation rate was observed when BALB/cA embryos grown in BALB/cA females exposed to BALB/cA males were transferred into recipient BALB/cA females exposed to DDK males. These results suggest that the embryonic genotype or viability of the embryo is one factor contributing to the occurrence of pregnancy block by male pheromones in mice.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Pregnancy block caused by male pheromones in mice (Bruce effect) has been investigated from various points of view, including the physiologic [1], endocrinologic [2], neuroendocrinologic [3], genetic [4–7], and embryologic [8]. Neuroendocrinological studies have demonstrated that pheromonal effects are mediated through the vomeronasal organ and that they enhance the activity of tuberoinfundibular dopaminergic neurons that in turn block prolactin release from the pituitary. Administration of the dopamine agonist bromocriptine synchronized with the time of prolactin release can reproduce the pregnancy block by male pheromones in mice [2, 9]. It has been pointed out that the vomeronasal-accessory olfactory system plays important roles in pheromonal recognition and memory formation [10]. The sensitivity of females to male pheromones has been shown to be controlled by a single recessive gene [4]. Moreover, it has been found that the major histocompatibility complex plays an important role in this phenomenon [6, 7].

Recently, we found that BALB/cA females mated with BALB/cA males showed a 100% pregnancy block when exposed to DDK males within 3 days after copulation. However, a gradual increase in survival rates of embryos was seen when the start of exposure to DDK males was delayed, and it was suggested that the developmental state of embryos or embryonic viability at the critical period might be a factor [8]. In effect, embryonic development is influenced by both environmental [11] and genetic factors [12]. Therefore, we investigated the effect of embryonic genotype or the viability of embryos on pregnancy block induced by either male pheromonal effects or a dopamine agonist. In the present report, we describe evidence that an embryonic factor plays a significant role in pregnancy block in mice.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

Strains of mice used in the present study were BALB/cA, CBA/J, C3H/HeN, C57BL/6Cr, DDK, and IXBL. BALB/cA and C57BL/6Cr were purchased from Nippon Clea Ltd. (Tokyo, Japan) and Shizuoka Laboratory Animal Corporation (Hamamatsu, Japan), respectively. CBA/J and C3H/HeN were introduced from the Institute for Laboratory Animal Research, School of Medicine, Nagoya University. DDK and IXBL were obtained from the Laboratory of Animal Genetics, School of Agricultural Sciences, Nagoya University. Production colonies were developed and their descendants used for the experiments. Mice were kept under controlled conditions (23.0 ± 1°C, 14L:10D photoperiod) and given continuous access to a pelleted diet (CA-1; Nippon Clea, Tokyo, Japan) and water. All animal procedures were performed according to the Guidelines for Animal Experimentation of Nagoya University.

Mating and Exposure to Stimulus Males

BALB/cA females were caged with stud males and checked daily for a vaginal plug. In the morning when the vaginal plug was found, the female was removed from the stud male and exposed to stimulus males in the exposure cage partitioned by wire netting. The methods of exposure were the same as described previously [8]. The day the vaginal plug was found was considered Day 0 of pregnancy. The animals were killed by cervical dislocation. The females were dissected at Day 14 of pregnancy, and numbers of corpora lutea, implantation sites, and live fetuses were counted.

Administration of Dopamine Agonist

Mice were kept in the animal room maintained at 23.0 ± 1°C and 12L:12D (lights-on 1000–2200 h). On Day 1 of pregnancy, BALB/cA females mated with stud males were injected i.p. with the dopamine agonist (bromocriptine methanesulfonate; RBI Research Biochemical L.P., Natick, MA) at a dose of 2 mg or 4 mg/kg BW at 0600 h and 2100 h, the times of nocturnal and diurnal prolactin surges [2, 13]. Control groups were treated with saline in the same way. The females were killed as described above and dissected at Days 5 and 14 of pregnancy, and the absence of implantation sites was taken to indicate nonpregnancy. At Day 5, numbers of corpora lutea and implantation sites were counted, and at Day 14, numbers of corpora lutea, implantation sites, and live fetuses were counted for the pregnant females.

Embryo Transfer

Donor embryos were collected on Day 3 of pregnancy from BALB/cA females mated with BALB/cA males and exposed to DDK or BALB/cA males on Day 0 of pregnancy. Embryos were collected by flushing the uterine horns with M2 medium [14] supplemented with 60 µg/ml penicillin G, 50 µg/ml streptomycin, and 4 mg/ml BSA (Sigma Chemical Co., St. Louis, MO). Embryos were transferred to the uterine horns of pseudopregnant recipients (BALB/cA females mated with vasectomized BALB/cA males and exposed either to BALB/cA or DDK males at 2.5 days postcoitus under ether (Wako Chemical Co., Tokyo, Japan) anesthesia. Six to nine embryos were transferred into one uterine horn of the recipient. Recipients were killed 2 days after transfer, and the number of implantation sites was counted.

Statistical Analysis

Statistical significance was analyzed by Tukey's studentized range test, Duncan's new multiple range test, or chi-square test. Statistical significance was taken as p < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Incidence in Pregnancy Block in the Intra- and Interstrain Crosses

Table 1 shows the reproductive performance of BALB/cA females mated with BALB/cA males or males of other strains and exposed to DDK males or males from the same strain as stud males. BALB/cA females mated with BALB/cA males showed a 100% pregnancy block when exposed to DDK males. BALB/cA females mated with males of other strains and exposed to DDK males had pregnancy rates ranging from 66.6% to 87.5%. The BALB/cA females that had been mated with CBA/J, C3H/HeN, C57BL/6Cr, and IXBL males and exposed to males from the same strain as the stud males showed 75.0–88.8% pregnancy rates. There were no significant differences in the number of corpora lutea, implantation sites, and live fetuses on Day 14 of pregnancy between the experimental groups exposed to DDK males and the control groups exposed to males from the same strain as the stud males. These results may be interpreted in the following two ways: 1) pheromonal stimuli of the males other than BALB/cA may be similar to those of DDK males, and thus the physiological condition of BALB/cA females that have copulated may not be disturbed by exposure to DDK males; or 2) the viability of the F1 embryos may be such that they can survive the deteriorated condition in the reproductive tract and direct the physiological condition of the BALB/cA females to establishment of pregnancy.


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  		TABLE 1. Fertility of BALB/cA females mated with males of five strains and exposed to DDK males and males from the same strain as stud males for Days 0–14 of pregnancy.

Effect of Dopamine Agonist

Rosser and colleagues [2] published the method of inducing pregnancy block through administration of dopamine agonist instead of exposing newly copulated females to stimulus males. This method can be used to discriminate whether pregnancy block is due to the effects of stud males or stimulus males. Table 2 shows the results observed on Day 5 of pregnancy in the experiment using the dopamine agonist. BALB/cA females mated with BALB/cA males and treated with dopamine agonist (bromocriptine methanesulfonate, 2 mg/kg i.p.) synchronously with the times of the prolactin surges [2, 13] (0600 h and 2100 h) on Day 1 of pregnancy showed a less than 10% pregnancy rate. On the other hand, BALB/cA females mated with CBA/J, C3H/HeN, and C57BL/6Cr males and treated in the same way showed significantly higher pregnancy rates (45.4–75.0%). The difference was statistically significant (p < 0.05, Duncan's new multiple range test). However, the pregnant females showed significantly smaller numbers of corpora lutea and implantation sites than the control group (administration of saline). In the experiment using a higher dose of bromocriptine (4 mg/kg), BALB/cA females mated with BALB/cA males showed a 100% pregnancy block. In contrast, BALB/cA females mated with CBA/J, C3H/HeN, and C57BL/6Cr males showed 58.3–70.0% pregnancy rates, but numbers of corpora lutea and implantation sites were smaller than in the lower-dose groups (p < 0.05, Duncan's new multiple range test), indicating that the effect is slightly stronger at the higher dose. The results of dissection on Day 14 of pregnancy for the higher-dose group are shown in Figure 1. Numbers of live fetuses (2.0–2.4) were similar to those of implantation sites observed at Day 5 (2.0–2.1) and Day 14 (2.2–2.8) of pregnancy, indicating that embryonic losses rarely occurred after Day 5.


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  		TABLE 2. Effect of bromocriptine administered synchronously with the times of prolactin surges in Day 1 of pregnancy on pregnancy rate and numbers of corpora lutea and implantation sites at Day 5 of pregnancy.



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  		FIG. 1. Reproductive performance of BALB/cA females mated with males of three strains (CBA/J, C3H/HeN, C57BL/6Cr) and treated with bromocriptine (4 mg/kg) synchronously with the times of prolactin surges (0600 and 2100 h) on Day 1 of pregnancy (means ± SEM). Pregnancy rate (no. of pregnant females/no. of females that copulated and were treated) is given under each mating formula. No significant differences were seen among three mating types for each item (p > 0.05, Duncan's new multiple range test).

Embryo Transfer

Morulae and blastocysts from BALB/cA females mated with BALB/cA males and exposed to either BALB/cA or DDK males were transferred to BALB/cA recipients exposed to either BALB/cA or DDK males, and implantation sites were examined on Day 5 of pregnancy (2 days after transfer). The results of three kinds of experiments are shown in Table 3. Pregnancy rate and implantation rate were 75% (9 of 12) and 39.4% (54 of 137), respectively, in experiment 1, and 53.3% (8 of 15) and 20.8% (33 of 159), respectively, in experiment 2. The values were 10.0% (1 of 10) and 3.1% (3 of 96), respectively, in experiment 3, in which embryos from the BALB/cA donor females exposed to DDK males were transferred to BALB/cA pseudopregnant females exposed to BALB/cA males. Implantation rate in experiment 3 was significantly different (p > 0.05, chi-square test) from those in experiments 1 and 2. These results indicate that transfer of embryos grown in less optimal uterine conditions into the undisturbed reproductive tract of BALB/cA females resulted in a low pregnancy rate. In contrast, a higher pregnancy rate could be obtained when embryos grown in the undisturbed condition were transferred to recipient females with a uterine condition negatively altered by exposure to DDK males.


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  		TABLE 3. Transfer experiments of BALB/cA embryos with three combinations of donors and recipients


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
It was previously shown that BALB/cA females mated with BALB/cA males exhibit a 100% pregnancy block when exposed to DDK males [8]. In the present experiments, BALB/cA females mated with the males of other strains and exposed to DDK males showed normal pregnancy rates (66.6–87.5%), and the pregnant females had normal numbers of corpora lutea, implantation sites, and live fetuses at Day 14 of pregnancy. This result raised two possibilities: 1) pheromonal stimuli of the alien males and DDK males are similar, and no disturbance occurs in the physiological state of the BALB/cA females after exposure to DDK males; and 2) viability of F1 embryos is stronger than that of BALB/cA embryos, and the F1 embryos can survive deteriorated oviduct and uterine conditions, leading to the establishment of pregnancy.

To discriminate between these two possibilities, two experiments were performed. First, pharmacological induction of pregnancy block with a dopamine agonist (bromocriptine) was carried out. Almost all BALB/cA females mated with BALB/cA males showed pregnancy block when treated with the dopamine agonist. In contrast, BALB/cA females mated with alien males showed significantly higher pregnancy rates, although the rates were slightly lower than those for the groups exposed to DDK males (comparison between Tables 1 and 2). The number of corpora lutea, implantation sites, and live fetuses at Day 14 of pregnancy was also significantly smaller (comparison between Table 1 and Fig. 1, p < 0.01, Duncan's new multiple range test). Second, a higher pregnancy rate was obtained when embryos grown in the normal condition were transferred into recipients with a uterine condition deteriorated as a result of exposure to DDK males; and a low pregnancy rate was observed when embryos grown in the deteriorated condition were transferred into recipients with undisturbed uterine condition. Considering these two possibilities on the basis of the results, the second possibility may be supported, and it is suggested that the viability of the F1 embryos is stronger than that of BALB/cA embryos, indicating that genotypes of embryos must be taken into consideration in investigations of pregnancy block.

The mechanisms for pregnancy block in mice, including male pheromones and sensitivity of females, have been investigated [4, 5]. It is generally accepted that failure in the development of corpora lutea due to suppression of prolactin secretion is the cause of pregnancy block, and Rajendren and Dominic [15] have reported that luteal failure in newly mated females is prevented only when administration of progesterone is begun early during the male exposure period. However, the present study showed that the embryonic genotype or viability of the embryos is also an important factor involved in the occurrence of pregnancy block. It is inferred that the BALB/cA embryos are more sensitive than F1 embryos to a deteriorated condition in the reproductive tracts of BALB/cA females. Wang et al. [16] have reported a similar strain difference in the pregnancy block by passive immunization against progesterone; i.e., the BALB/cA strain is more sensitive to administration of the antibody to progesterone than the CBA/J strain. It is possible that the strain difference may be attributable to the sensitivity of embryos to the altered condition in the female reproductive tract; i.e., BALB/cA embryos may be more sensitive than CBA embryos.

Gene expression and metabolism in early embryos have been shown to be dependent on the genotype [17], and it is well known that implantation is established with the interaction of embryos and the uterine wall, i.e., the signals from embryos and the receptivity of the uterus. Estrogen is necessary to make the uterine environment receptive for embryos [18] and also seems to promote embryonic development [19]. Nitric oxide (NO) production at the morula and blastocyst stages is regulated by estrogen and appears to be required for normal embryonic development [20]. This embryonic NO may act as an implantation signal. Cell adhesion molecules also play important roles in the interaction of embryos and uterine epithelium or stroma cells at early stages of pregnancy [21]. In mice, embryo attachment induces local maternal epithelial retraction and apoptosis [22]. In humans, also, protrusive penetration of trophoblast occurs through the epithelium [23]. Strong signals from the embryo to the uterine epithelium and stromal cells may in turn be sent to the ovaries and stimulate development of corpora lutea.

The survival rate of F1 embryos was lower in the bromocriptine-treated BALB/cA females than in the BALB/cA females exposed to DDK males. Besides the blocking effect of prolactin and thyroid-stimulating hormone release [24], bromocriptine may have some other effects to alter the physiology of female mice that have just copulated, although it is known to have fewest side effects in humans [24]. It may be inferred that the condition in the reproductive tract of BALB/cA females is more severely disturbed by administration of bromocriptine than exposure to DDK males, and the embryos may incur more damage in bromocriptine-treated females. In view of the foregoing discussion, it would be interesting to investigate how development of F1 embryos differs from that of BALB/cA embryos in the deteriorated reproductive tract of BALB/cA females and to compare their interaction with uterine epithelium or stromal cells. In nature, pregnancy block has an adaptive significance for a newly arriving male to increase his reproductive success by terminating the pregnancy of females that have been impregnated by previous males. In contrast, a genetic factor conferring strong viability to embryos during implantation stages is considered to have a counteracting effect in favor of previous males by preventing pregnancy block.


   FOOTNOTES
 
1 This work was supported in part by a grant-in-aid for scientific research to N.W. from the Ministry of Education, Science, Sports and Culture of Japan (No. 05304022). Back

2 Correspondence: FAX: 81 52 789 4012; w16341a{at}nuagrl.agr.nagoya-u.ac.jp Back

Accepted: March 5, 1999.

Received: November 9, 1998.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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