Overripeness and the Mammalian Ova II. Delayed Ovulation and Chromosome Anomalies ROY L. BUTCHER, PH.D., and N. W. FUGO, PH.D., M.D. THE CAUSES of abortion and birth defects are undoubtedly multiple and for the most part not understood. It has been shown however that some anomalies are associated with abnormal chromosome counts. This is true of Klinefelter's syndrome, ovarian aplasia, and mongolism. Furthermore, the high incidence of chromosomal anomalies in human abortuses 7 suggests that this may be one of the primary causes of miscarriage. We have recently reported that a delay of ovulation for 48 hr. by phenobarbital sodium blockage in rats produces a marked increase in anomalies of early embryonic development. 2 These abnormalities may be the result of either cytoplasmic or chromosomal changes in the ova after delayed ovulation. A high incidence of gross and chromosomal abnormalities have been found in amphibian embryos after a delayed release of the ova. 8 This concept is substantiated further by the report by Iffy that of 19 cases of abortion in which the conception date was known, 14 had conceived after Day 17 of the menstrual cycle. 4 The purpose of the present study is to ascertain whether a delay of ovulation in rats produces alteration in chromosome number, such as could come about by nondisjunction during meiosis. MATERIALS AND METHODS Ovulation was delayed for 24 or 48 hr. in sexually mature female (Holtzman) rats 3-6 months of age with phenobarbital sodium, by the method of Everett and Sawyer. (For details of the procedure used see Fugo and Butcher.) These animals as weh as control rats were mated to young fertile males. The day that sperm were found in the vaginal smear was From the Department of Obstetrics and Gynecology, West Virginia University Medical Center, Morgantown, W. Va. Supported by Grant HD-00983-01 from the National Institute of Child Health and Human Development, U. S. Public Health Service, and by the Association for the Aid of Crippled Children. 297
298 BuTCHER & Fuco FERTILITY & STERILITY designated as Day 0 of pregnancy. Chromosome preparations were made using a modification of the method of Moorhead et al. Both experimental and control animals were killed on Day 11 of gestation. Uteri were removed immediately and placed in 0.9% NaCl solution. The opening of implantation sites was antimesometrial. Each embryo was transferred to a 15-ml. conical centrifuge tube containing 2 ml. of magnesium- and calcium-free saline with 1.5 p,g.jml. colchicine. The tubes were placed in a water bath at 37 C. and allowed to remain undisturbed for 50 min. Then 0.25 ml. of 0.25% trypsin was added to each tube and the contents of each tube were gently agitated with a Pasteur capillary pipette at intervals over a 20-min. period to disperse the cells. One drop of IN HCl was added and the tubes were centrifuged at 800 rpm for 8 min. Mter decantation, the tubes were returned to the water bath, 5 ml. of 1% sodium citrate was added to each tube, and the cells were resuspended. After 20 min. in hypotonic solution three or four drops of a methanol: acetic acid ( 3: 1) fixative was added and the tubes were recentrifuged at 800 rpm for 8 min. The HCl and fixative was included in the above procedure to prevent the formation of a gelatin-like clump of cells which would not spin down. Two milliliters of chilled methanol: acetic acid ( 3: 1) fixative were added to the cells and the tubes were placed in the refrigerator for a minimum of 30 min. The cells were then washed in three changes of the fixative and resuspended in about 0.25 ml. of a fixative. The cell suspension was then pipetted onto a tilted microscope slide and dried rapidly by gentle warming. Chromosomes were stained for 20 min. in a freshly prepared Giemsa stain, consisting of 150 ml. distilled water, 8 ml. of Giemsa stain, and 4 ml. of 0.1N NH 4 0H. Karyograms were prepared from 100 treated and 100 control embryos at the initiation of the experiment. Karyograms were then discontinued for embryos with the normal chromosome number of 42, since sex ratios were normal and no anomalies other than the chromosome number were found in the initial 200 embryos examined. Embryos with abnormal chromosome counts were subjected to a sufficient number of karyograms to positively identify the extra or missing chromosomes. Karyograms were prepared as described by Hungerford and Nowell except that the chromosome classified as 13 by them was numbered 18 by us, since it appeared to belong at this position on a basis of decreasing size (Fig. 1). RESULTS Chromosome counts were obtained for a total of 390 treated and 410 control embryos. Abnormal chromosome counts were found in 18 embryos
VoL.l8, No.3, 1967 DELAYED OVULATION: II. 299 from the treated animals and in 6 from the control group (Table 1). This difference was determined to be significant (p<0.01) by a chi-square analysis. In the experimental group, 3 monosomy, 2 trisomy, 2 triploids, 1 tetraploid, and 10 mosaics were discovered, while in the control animals, only 3 triploids and 3 mosaics were found. Figures 2-4 are karyograms obtained from 3 of the embryos with abnormal chromosome numbers. As. in cases of chromosomal aberrations in human material, the smaller chromosomes are involved most often in rats. Mosaics of the 41j42 and 42j43 types were found in both experimental and control animals while none of the expected 41j43 type was observed. Seven anomalies (3 monosomy and 4 mosaics) were in the 4-10+X group and may have been XO in 5 embryos or XXY in the other 2. It was not possible to determine whether the extra or missing chromosome in these 7 embryos was an X, since unlike the finding in other strains of rats3 6 the X chromosomes had terminal centromeres as did all other chromosomes of the 4-10 group. Fugo and Butcher found that ovulation rates are normal while fertilization rates decreased after a 2-day delay in ovulation. In the present study ovulation rates based on number of corpora lutea were 14.6 in the treated rats and 14.4 in the controls. The average number of implantation sites vs. number of embryos for the treated and control groups, respectively, were: 8.8 vs. 7.7 and 12.7 vs. 11.8. The percentage of degenerating implantation Fig. 1. Karyogram of normal male rat embryo.
300 BuTCHER & Fuco FERTILITY & STERIUTY TABLE 1. Chromosome Anomalies in Treated and Control Embryos Abnormality Sex Affected chromosome No. of embryos TREATED Triploid XXX 2 Tetraploid xxxx 1 Monosomy XO* 4-10+X 3 Trisomy XY 13-17 1 Trisomy XX 12 1 42-43 Mosaic XX 19-20 1 XY 13-17 2 XX 13-17 1 XYor XXY* 4-10+X 2 41-42 Mosaic XX 13-17 1 XX 19-20 1 XY 19-20 1 XYor XO* 4-lO+X 1 CONTROLS Triploid XXX 1 XXY 2 42-43 ~losaic XX 13-17 1 XY 13-17 1 41-42 Mosaic XX or XO* 4-10+X 1 *Sex not certain since X chromosome could not be distinguished from the 4--10 group. sites was 12.5 in the treated group and 7.1 in the controls. Although the embryos were not studied for gross anomalies, records were made of unusually small embryos found during transfer to the centrifuge tubes. Of 17 small embryos on which countable preparations were made, 4 had abnormal counts: 2 were triploids, 1 a trisomy, and 1 a monosomy. The lower implantation rate and higher incidence of degeneration embryos in the treated group may also have resulted from chromosomal changes. The most seriously affected zygotes are probably eliminated prior to Day 11 of gestation, others may die before birth, and some of the less serious defects may permit the birth of defective young. These problems are under study in our laboratory. DISCUSSION The results of these experiments as well as that of Fugo and Butcher clearly demonstrate the detrimental effect of delayed ovulation on embryonic development in the rat. Decreased fertilization rate, decreased implantation rate, increased embryonic death rate, and the increased inci-
1111,. It c.~).~. II AA aa..,..-,-' \lit -.-.-,,,.,. Fig. 2-4. Karyograms from rat embryos. Fig. 2 (top). Triploid condition with XXY sex chromosomes. Fig. 3 (center). Trisomy in 13-17 group in female. Fig. 4 (bottom). Trisomy of chromosome 12 in female.
302 BuTCHER & FuGo FERTILITY & STERILITY dence of chromosomal anomalies found after delayed ovulation in the rat are also the type of phenomena found in women. Although there is no proof that delayed ovulation is the cause of increased congenital anomalies, the report by Iffy mentioned above would suggest that conception late in the menstrual cycle is detrimental to normal embryonic development. Since alterations in the secretion of hypophyseal gonadotrophins accompany the climacteric it would seem possible that the time of ovulation might account for the gradual increase in congenital anomalies occurring with increased maternal age. Variations in the secretions of gonadotrophin could also affect the uterine environment through their action on synthesis of ovarian steroids. The possible effects of changes in uterine environment by blockage of ovulation is to be studied in later experiments. However, abnormalities found in the ova prior to their entering the uterus, 2 as well as the increased incidence of chromosomal anomalies, demonstrate changes in the ova that are independent of the uterine environment. SUMMARY Delayed ovulation resulted in chromosomal anomalies in 18 of 390 rat embryos compared to only 6 anomalies in 410 control embryos. A higher incidence of degenerating embryos and failure of implantation was found in the treated group. This study proves that overripeness of mammalian ova is an important factor in the occurrence of chromosomal abnormalities as well as being responsible for early embryonic death. Department of Obstetrics and Gynecology West Virginia University Medical Center Morgantown, W.Va. 26506 REFERENCES 1. EvERETT, J. W., and SAwYER, C. H. A 24-hour periodicity in the "LH-release apparatus" of female rats, disclosed by barbiturate sedation. Endocrinology 47:198, 1950. 2. FuGo, N. W., and BUTCHER, R. L. Overripeness and the mammalian ova. I. Overripeness and early embryonic development. Fertil Sterill7:804, 1966. 3. HUNGERFORD, D. A., and NowELL, P. C. Sex chromosome polymorphism and the normal karyotype in three strains of the laboratory rat. ] Morph 113:215, 1963. 4. IFFY, L. Time of conception in pathological gestation. Proc Roy Soc Med 56: 1098, 1963. 5. MooRHEAD, P. S., NowELL, P. C., MELLMAN, W. J., BATTIPS, D. M., and HUNGER FORD, D. A. Chromosome preparations of leukocytes cultured from human peripheral blood. Exp Cell Res 20:613, 1960. 6. RIEKE, W. 0., and ScHWARZ, M. R. The culture and karyotype of rat lymphocytes stimulated with phytohemagglutinin. Anat Rec 150:383, 1964. 7. SzULMAN, A. E. Chromosome aberrations in early human abortions. Fed Proc 23: 499, 1964. 8. WITscm, E., and LAGUENS, R. Chromosomal aberrations in embryos from overripe eggs. Develop Biol 7:605, 1963.