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Effect of Blastocyst Transfer on Gestation Period in Rats
Effect of Blastocyst Transfer on Gestation Period in Rats SERGE J. MANTALENAKIS, M.D., and JOHN M. DANEZIS, M.D.
FERTILIZED RAT ovA enter the uterus on Day 4. The ova remain free and unattached in the uterine lumen for about 24 hr.; during this period they undergo evolution from morulae to blastocysts. Normally, if no lactation occurs up to this point, the blastocysts become attached to the endometrium in the afternoon of Day 5. Dickmann and Noyes, and Noyes and Dickmann, utilizing ovum transfer technics, established that 5-day ova transplanted into pseudopregnant recipients either on Day 4 or 5 became implanted. During the course of experiments designed for another purpose, we observed that the synchronous transfer of 5-day blastocysts to the endometrium of pseudopregnant rats on Day 5 resulted in extension of the gestation period. 4 The present study was undertaken to examine the validity of the preliminary observation. METHODS AND MATERIALS
Nulliparous female Sprague-Dawley rats weighing 190-240 gm. were housed at a constant 23.3-24.4 o C., with an automatic lighting cycle of 12-hr. periods of light and dark. They were placed in mating cages in the evening with normal males to obtain pregnant females, or with vasectomized males to obtain pseudopregnant females. Pregnancy or pseudopregnancy was inferred from the presence of a vaginal plug early the following day, which was then designated Day 1. Blastocysts were obtained from normal pregnant donors sacrificed between 9A.M. and 11 A.M. on Day 5 of pregnancy. The uteri were flushed with Ringer's phosphate buffer (pH 7.1 to 7.2). Seven to 9 blastocysts were transferred by means of a micropipet into the left horn of the recipient either on Day 4, 5, or 6 of pseudopregnancy. Near the time of parturition the rats were observed at 2-hr. intervals to record the time of parturition and the number of young. From the University of Athens, Medical School, Maternity Hospital "Alexandra," Department of Fertility & Sterility, Athens, Greece.
910
-
VoL. 19, No. 6, 1968
BLASTOCYST TRANSFER
911
Pregnant animals in which the right uterine tube was ligated on Day 3 were used as control. The newborn litters were weighed. RESULTS
The results of the experiments are shown in Table 1. Blastocysts transferred to pseudopregnant recipients either on Day 4 (Group I) or on Day 5 (Group II) showed a high rate of development, resulting in fetuses at term. The percentage of viable young obtained from Group I and Group II were 67% and 60% respectively. Only 2 young were born from 110 fiveday blastocysts transferred into the uterus of pseudopregnant rats on Day 6. Although the mean weights of young at birth in Group I did not diller significantly from those of Group II, the mean gestation periods within the same groups were significantly different ( P = 0.01). Gestation was about 1 day longer for animals carrying fetuses derived from blastocysts transplanted on Day 5 of pseudopregnancy (Group II) as compared to those with blastocysts transplanted on Day 4 (Group I). The mean length of gestation in the Group I was found not significantly diHerent from that of the control animals. In both groups the mean litter size was similar. DISCUSSION
I
The results indicate that 5-day blastocysts transferred on Day 4 begin attachment to the uterine epithelium on Day 5, as in normal pregnancy. The extension of the gestation period for about a day in Group II suggests that an inhibition in the initiation of the implantation occurred rather than a delay of the onset of parturition. The longer gestation in the synchronous transfer of 5-day blastocysts to endometrium on Day 5 could not be explained by the surgical operation on the uterus, or by the reduction of the litter size; 1 the last factor was excluded by ligation of the one tube on Day 3 in the control pregnant animals. McLaren and Michie transplanted 3~-day fertilized ova into 2~-day mouse endometrium, and they observed that the gestation period was one-half day shorter. Noyes and coworkers7 • 8 showed that precociously transferred fertilized rat ova were heavier than synchronously transferred ova at the same stage of late pregnancy. It may be speculated then, that during ovum transfer experiments, the early or late contact of the fertilized ovum with the endometrium during the sensitive period-from noon of Day 4 to Day 6-affects the length of gestation. 2 SUMMARY
A total of 158 of 249 five-day blastocysts transferred into the uterus either on Day 4, or on Day 5 of pseudopregnancy developed to viable
TABLE 1. Results of Asynchronous Transplantation of Blastocysts into Pseudopregnant Recipients
Group
I II III
Day of transfer
4 5 6 Control (pregnant, one tube tied)
* P = NS from control. t P = 0.01 from control.
No. of rats
16 18 15 24
No. of blastocysts transf.
117 132 110
No. of young
No. of young per rat (mean)
Wt. at birth (mg.; mean± S.E.)
Gestation (days; mean± S.E.)
78 80 2 121
4.9 4.4
4893 ± 145* 4957 ± 123* 5106 5002 ± 97
22.7 ± 0.26* 23.8 ±0.24t 24.1 22.6 ± 0.18
5.0
I
VoL. 19, No.6, 1968
BLASTOCYST TRANSFER
913
young. Only 2 young were born of 110 five-day blastocysts transferred on Day 6. Gestation was about 1 day longer for animals carrying fetuses derived from synchronous blastocyst transplantation on Day 5. 10 Kerasountos St. Athefl8 611, Greece
REFERENCES 1. BIGGERRS, J. D., CURNow, R. N., FINN, C. A., and McLAREN, A. Regulation of the gestation period in mice. ] Reprod Fertil6:125, 1963. 2. DE FEo, V. J. Determination of the sensitive period for the induction of deciduomata in the rat by different inducing procedures. Endocrinology 73:488, 1963. 3. DICKMANN, Z., and NoYES, R. W. The fate of ova transferred into the uterus of the rat. ] Reprod Fertill:191, 1960. 4. MANTALENAKIS, S. J., and KETCHEL, M. M. Blastocyst implantation during postpartum pseudopregnancy in the rat. ] Endocr 37:385, 1967. 5. McLAREN, A., and MicmE, D. Nature of the systemic effect of litter size on gestation period in mice. ] Reprod Fertil 6:139, 1963. 6. NoYES, R. W., and DICKMANN, Z. Relationship of ovular age to endometrial development. ] Reprod Fertil1:186, 1960. 7. NoYEs, R. W., DoYLE, L. L., and BENTLEY, D. L. Effect of preimplantation development on fetal weight in the rat. ] Reprod Fertil2:238, 1961. 8. NoYES, R. W., DoYLE, L. L., GATES, A. H., and BENTLEY, D. L. Ovular maturation and fetal development. Fertil Steril12:405, 1961.
FERTILIZED RAT ovA enter the uterus on Day 4. The ova remain free and unattached in the uterine lumen for about 24 hr.; during this period they undergo evolution from morulae to blastocysts. Normally, if no lactation occurs up to this point, the blastocysts become attached to the endometrium in the afternoon of Day 5. Dickmann and Noyes, and Noyes and Dickmann, utilizing ovum transfer technics, established that 5-day ova transplanted into pseudopregnant recipients either on Day 4 or 5 became implanted. During the course of experiments designed for another purpose, we observed that the synchronous transfer of 5-day blastocysts to the endometrium of pseudopregnant rats on Day 5 resulted in extension of the gestation period. 4 The present study was undertaken to examine the validity of the preliminary observation. METHODS AND MATERIALS
Nulliparous female Sprague-Dawley rats weighing 190-240 gm. were housed at a constant 23.3-24.4 o C., with an automatic lighting cycle of 12-hr. periods of light and dark. They were placed in mating cages in the evening with normal males to obtain pregnant females, or with vasectomized males to obtain pseudopregnant females. Pregnancy or pseudopregnancy was inferred from the presence of a vaginal plug early the following day, which was then designated Day 1. Blastocysts were obtained from normal pregnant donors sacrificed between 9A.M. and 11 A.M. on Day 5 of pregnancy. The uteri were flushed with Ringer's phosphate buffer (pH 7.1 to 7.2). Seven to 9 blastocysts were transferred by means of a micropipet into the left horn of the recipient either on Day 4, 5, or 6 of pseudopregnancy. Near the time of parturition the rats were observed at 2-hr. intervals to record the time of parturition and the number of young. From the University of Athens, Medical School, Maternity Hospital "Alexandra," Department of Fertility & Sterility, Athens, Greece.
910
-
VoL. 19, No. 6, 1968
BLASTOCYST TRANSFER
911
Pregnant animals in which the right uterine tube was ligated on Day 3 were used as control. The newborn litters were weighed. RESULTS
The results of the experiments are shown in Table 1. Blastocysts transferred to pseudopregnant recipients either on Day 4 (Group I) or on Day 5 (Group II) showed a high rate of development, resulting in fetuses at term. The percentage of viable young obtained from Group I and Group II were 67% and 60% respectively. Only 2 young were born from 110 fiveday blastocysts transferred into the uterus of pseudopregnant rats on Day 6. Although the mean weights of young at birth in Group I did not diller significantly from those of Group II, the mean gestation periods within the same groups were significantly different ( P = 0.01). Gestation was about 1 day longer for animals carrying fetuses derived from blastocysts transplanted on Day 5 of pseudopregnancy (Group II) as compared to those with blastocysts transplanted on Day 4 (Group I). The mean length of gestation in the Group I was found not significantly diHerent from that of the control animals. In both groups the mean litter size was similar. DISCUSSION
I
The results indicate that 5-day blastocysts transferred on Day 4 begin attachment to the uterine epithelium on Day 5, as in normal pregnancy. The extension of the gestation period for about a day in Group II suggests that an inhibition in the initiation of the implantation occurred rather than a delay of the onset of parturition. The longer gestation in the synchronous transfer of 5-day blastocysts to endometrium on Day 5 could not be explained by the surgical operation on the uterus, or by the reduction of the litter size; 1 the last factor was excluded by ligation of the one tube on Day 3 in the control pregnant animals. McLaren and Michie transplanted 3~-day fertilized ova into 2~-day mouse endometrium, and they observed that the gestation period was one-half day shorter. Noyes and coworkers7 • 8 showed that precociously transferred fertilized rat ova were heavier than synchronously transferred ova at the same stage of late pregnancy. It may be speculated then, that during ovum transfer experiments, the early or late contact of the fertilized ovum with the endometrium during the sensitive period-from noon of Day 4 to Day 6-affects the length of gestation. 2 SUMMARY
A total of 158 of 249 five-day blastocysts transferred into the uterus either on Day 4, or on Day 5 of pseudopregnancy developed to viable
TABLE 1. Results of Asynchronous Transplantation of Blastocysts into Pseudopregnant Recipients
Group
I II III
Day of transfer
4 5 6 Control (pregnant, one tube tied)
* P = NS from control. t P = 0.01 from control.
No. of rats
16 18 15 24
No. of blastocysts transf.
117 132 110
No. of young
No. of young per rat (mean)
Wt. at birth (mg.; mean± S.E.)
Gestation (days; mean± S.E.)
78 80 2 121
4.9 4.4
4893 ± 145* 4957 ± 123* 5106 5002 ± 97
22.7 ± 0.26* 23.8 ±0.24t 24.1 22.6 ± 0.18
5.0
I
VoL. 19, No.6, 1968
BLASTOCYST TRANSFER
913
young. Only 2 young were born of 110 five-day blastocysts transferred on Day 6. Gestation was about 1 day longer for animals carrying fetuses derived from synchronous blastocyst transplantation on Day 5. 10 Kerasountos St. Athefl8 611, Greece
REFERENCES 1. BIGGERRS, J. D., CURNow, R. N., FINN, C. A., and McLAREN, A. Regulation of the gestation period in mice. ] Reprod Fertil6:125, 1963. 2. DE FEo, V. J. Determination of the sensitive period for the induction of deciduomata in the rat by different inducing procedures. Endocrinology 73:488, 1963. 3. DICKMANN, Z., and NoYES, R. W. The fate of ova transferred into the uterus of the rat. ] Reprod Fertill:191, 1960. 4. MANTALENAKIS, S. J., and KETCHEL, M. M. Blastocyst implantation during postpartum pseudopregnancy in the rat. ] Endocr 37:385, 1967. 5. McLAREN, A., and MicmE, D. Nature of the systemic effect of litter size on gestation period in mice. ] Reprod Fertil 6:139, 1963. 6. NoYES, R. W., and DICKMANN, Z. Relationship of ovular age to endometrial development. ] Reprod Fertil1:186, 1960. 7. NoYEs, R. W., DoYLE, L. L., and BENTLEY, D. L. Effect of preimplantation development on fetal weight in the rat. ] Reprod Fertil2:238, 1961. 8. NoYES, R. W., DoYLE, L. L., GATES, A. H., and BENTLEY, D. L. Ovular maturation and fetal development. Fertil Steril12:405, 1961.