Vaginal Cycles in the Rat after X-ray or Other Damage to One or Both Ovaries' JAMES C. PLAGGE AND ROBERT H. KREHBIEL DepaTtment of Anatomy, University of Illinois, College of Medicine, Chicago, Illinois ABSTRACT Damage to the rat ovary at 60 days of age causes irregularities in the estrous cycle and leads to continuous comification of the vagina. These vaginal changes occurred within two to three months after bilateral irradiation (800 or 2,000 r ) or after bilateral castration and autotransplantation of a portion of one ovary. Unilateral irradiation or ligation of the ovarian arteries at their junction with the uterine artery produces similar changes within five to seven months. Comparable vaginal changes did not occur in control animals until they reached the age of ten months. The estrous cycle of rats exhibits periods of irregularity during the last two thirds of life in which extended periods of continuous estrus are characteristic (Asdell and Crowell, '35; Bloch, '57; Everett, '39; Ingram, '59 and others). Bloch and Flury ('59) found continuous estrus in the rat to last for varying periods up to the age of 30 months. Thereafter, their animals showed an anestrus. Comparable irregularities have been noted after various insults to the ovary. In both the mouse (Parkes, '27) and the rat (Drips and Ford, '32) x-ray irradiation of the ovaries may be followed by regular cycles or, after a latent interval, by periods of continuous estrus (Mandl and Zuckerman, '56; Ingram, '58; and Slate and Bradbury, '62). The continuous estrus lasts for varying periods and is followed by diestrus. Continuous corniiication is also found after transplantation of the ovary or interference with its normal innervation and blood supply (Fels, '44; Hernandez, '43; Kullander, '59, and '60; Lipschutz, Mena and Vinals, '34 and others). The time at which constant estrus appears, and its extent, may be dependent upon the degree of ovarian damage (Fels, '35 and Slate and Bradbury, '62) and may also be an indication of an acceleration of the processes which are seen in the untreated animals. In most observations all the ovarian tissue of the animal was subjected to the injury. Ingram ('58) and others suggest that the alteration may be due in part to the loss of ova in the damaged ovary. Pencharz and Long ('32) found that when only one ovary was irradiated the injured ovary was inactive in the presence of an untreated ovary. There is some question of whether the control of the estrous cycle by the normal ovary is affected by the presence of a damaged ovary.. The age of the animal at the time of ovarian damage may also be a significant factor. This investigation compares the effects of various doses of x-ray or other damaging agents administered to one or both ovaries of the rat at 60 days of age. MATERIAL AND METHODS Vaginal cycles were followed in 169 rats. Smears were started at 45 to 50 days of age and irradiations, transplantations and other procedures were done on day 60. Irradiations with 800,2,000or 3,000 roentgens were applied to the exteriorized ovary or ovaries with the animal under Nembutal anesthesia. The female was placed in a lead compartment and the gonad or gonads brought through both the dorsal body wall and an opening in the lead shield. Lead plates were placed around the mesovanum with care to avoid trauma to the ovary or its blood vessels. The entire unit was then placed under the x-ray tube to irradiate (140 KV and a current of 5 mA at a dose of 520 r/min.) the gonad or gonads. A dosimeter in the lead container during irradiation had an average reading of 0.6 r of scatter rays in an 800 r exposure. Sham 1Aided by grants from National Institutes of Health United States Public Health Service and the Univerhty of Illinois Research Board, The Graduate College. 287 288 JAMES C . PLAGGE AND ROBERT H. KREHBIEL irradiation, irradiation of the left ear or only anesthesia with Nembutal, as well as unilateral and bilateral castrates and untreated animals served as controls. Ovarian transplantations were made to the subcutaneous tissue of the dorsal body wall. Both ovaries were removed and onehalf of the left ovary was transplanted. In another series the blood supply to both ovaries was disturbed to the extent of placing a ligature on each ovarian artery at its junction with the uterine artery. OBSERVATIONS The most evident effects of irradiation on the vaginal cycle as reported in the literature and in our observations was the appearance of a continuous cornification (CC will be used to designate this type of smear). The first change was usually an irregularity in the cycle in which extended periods of cornification occurred. The time at which the irregularity of the cycle appeared varied and will be considered with specific groups. As the interval following irradiation became longer, the periods of cornification increased in length to replace the cycle with CC. The length of a given period of CC varied. At times a typical diestrous smear would appear for one to several days with a return to the CC state. Unless the break was longer than a few days the smear was recorded as CC. There were, however, periods of diestrus, periods of irregularity or even periods of regular cycling after an extended period of CC. As described by others, the cell type in the smear was not always typically that of estrus. There were periods of large nucleated epithelial cells, with or without white blood cells. Another occasional variation was the presence of dark fluid in the vagina resembling that found at the time of the placental sign. This type of smear often lasted for a considerable number of days and the blood was thought to be uterine in origin. In some of the females which had had extended periods of CC the uterus was greatly distended. Volumes of 15 to 30 ml of fluid were obtained from the uterus of a single rat. It was either clear estrous fluid or contained blood. Not all uteri contained fluid but all showed stimulation. The mammary glands in many of the fe- males in CC were stimulated and contained secretion. The vaginal cycles in ten of the 36 females in which both ovaries received 800 r were normal prior to irradiation and for approximately the first two months after irradiation (fig. 1 ) . In the other 26 females of this series smearing was not started until one and one-half, two and onehalf, three and one-half or four and onehalf months after the irradiation date. It is seen that after two months post irradiation the cycles became either irregular or continuously cornified. Once the CC had been established, it generally continued during the period of observation. Normal cycling did not occur during interruptions of the CC. After a long period of diestrus female 868 returned to CC, as did several animals in the 2,000 r series (fig. 3). The mechanical stimulus of smearing (noted by Wade and Doisey, '35) was not the cause of cornification since CC was present in the animals when smearing was started three and one-half or four and onehalf months after irradiation. One ovary was removed from 13 of these animals. In seven (662, 733, 748, 725, 775, 776 and 676 as well as one animal in the 2,000 r series, 811) ovariectomy was done during a period of CC. With the exception of 776 there was no effect, or only a short break in the CC due to the removal of one ovary. Removal of both ovaries (807, 808, 809) resulted in an immediate interruption of cornification. The effects of mating are to be reported elsewhere but it may be mentioned that in the early post irradiation period fertilization occurred (867). On the other hand, seven females placed with males two and one-half months after irradiation did not become pregnant even though sperm were found occasionally in the smear. Irradiation of the left ovary by 800 r and removal of the right ovary did not cause a marked change in the vaginal state from that seen in the animals with 800 r to both ovaries. The smear records of five of 12 animals so treated are shown in figure 2. After 2,000 r to both ovaries ( 8 femdes) disturbances in the cycle appeared somewhat earlier than in the 800 r animals (fig. 3). Irregularities and CC were preva- RAT CYCLES AFTER OVARIAN X-RAY Figs. 1 to 9. Graphic representation of vaginal cycles of female rats which received various treatment at 60 days of age. AGE IN MONTHS Fig. 1 Fig. 2 Fig. 3 800 r to both ovaries. 800 r to left ovary; right ovary removed. 2,000 r to both ovaries. 289 290 JAMES C. PLAGGE AND ROBERT H. KREHBIEL lent during the second month. Thereafter extended periods of diestrus were seen in all animals but one (811). Prolonged CC appeared in three of these animals after months of diestrus (896, 897,898). Three females which received 3,000 r to both ovaries had irregular cycles during the first 50 days after irradiation and then had diestrous smears throughout the period of observation ( 6 months). Vaginal records of 18 of the 30 females in which only the left ovary was exposed to 800 r are shown in figure 4. In general the cycles were normal during the first three to five months. The 12 animals which were followed for only five months are not included in the figure. It is evident that at six to seven months post irradiation (8-9 months of age) irregularities and CC became prevalent. The onset of CC was later than in the bilaterally irradiated females and there were extended periods of irregular cycles which appeared at about the time CC appeared in the bilateral group. Periods of regular cycles followed CC and were observed up to ten months after irradiation. When the normal (right) ovary was removed the animal showed a period of diestrus. Two of these (547, 797) returned to normal cycling. Continuous comification occured in the others about a month after the ovariectomy. Removal of the irradiated ovary (798,799) caused no change. Removal of both ovaries (799,547) produced anestrus. Unilateral irradiation with 2,000 r had much the same effect as with 800 r (fig. 5). The greater damage produced by the 2,000 r to the single ovary of these seven animals did not seem to change the time at which the irregular and CC vaginal smears appeared. Irregular cycles or CC were observed seven to ten months after irradiation in 36 females which were unilaterally irradiated and the irradiated ovary removed six to 90 days later. Four typical cases are shown in figure 6. In four females the left ear was irradiated as one of the control procedures. Rats 873 and 874 received 800 r and numbers 875 and 876 received 2,000 r (fig. 7). Continuous cornification appeared in two of these animals at 8 months post irradiation. The cycles of five untreated control animals (889, 891, 892, 899 and 924) were essentially normal (fig. 8). Exceptions were found in that 899 had some CC at 380 days of age and 924 showed an extended period of irregularity between 9 and 11 months of age. Eight females were unilaterally castrated. Their cycles were essentially normal and three are shown in figure 8 (836, 918, 920). The one exception (920) showed cornification between 10 and 11 months of age. The one sham irradiated animal (834) cycled normally whereas the animal with Nembutal anesthesia (840) showed irregularity and CC (fig. 8). Five females were castrated bilaterally at 60 days of age and their smears were recorded at intervals for periods of 14 days. These females showed no vaginal cornification through the ten months of observation. Transplantation of half an ovary to the dorsal body wall and the removal of the remainder of the ovarian tissue produced marked changes in the vaginal picture. Four of seven animals are shown in figure 9 (942, 943, 945, 947). Vaginal changes in these animals began at a time comparable to, and were similar to, those in the bilaterally irradiated series (800 r). Continuous cornification also was found in six animals after ligatures had been placed on the ovarian arteries at their junction with the uterine arteries. Two of these (948, 951) are shown in figure 9. Cornification appeared during the sixth month after surgery, and their uteri showed distention beyond the limits of normal estrus. Fig. 4 800 r to left ovary only. Fig. 5 2,000 r to left ovary only. Fig. 6 800 r to left ovary; left ovary removed 6 to 24 days post irradiation. Fig. 7 800 r (873,874) or 2,000 r (875,876) to left ear. Fig. 8 Untreated (889, 891, 892, 899, 924); unilaterally castrated (836, 918, 920); sham irradiated (834); Nembutal anesthesia (840). Fig. 9 Subcutaneous autotransplantation of an ovarian fragment after bilateral castration (942, 943,945, 947); bilateral ligation at junction of ovarian and uterine vessels (948, 951). RAT CYCLES AFTER OVARIAN X-RAY 547 662 795 796 797 798 799 800 851 852 853 854 856 859 861 864 893 895 877 879 880 881 882 883 884 814 816 821 823 873 874 875 876 889 891 892 899 924 836 918 920 834 840 942 943 945 947 948 951 Figures 4-9. 291 292 JAMES C. PLAGGE AND ROBERT H. KREHBIEL DISCUSSION The continuous cornification found in the vaginal smears of animals after ovarian irradiation was not due to the effects of continuous smearing noted by Wade and Doisey ('35). The changes in the vaginal cycle produced by damage to the ovary appeared at an earlier age than comparable changes which occur in the aging female rat. Normal cycles continued for a period which is, in general, inversely proportional to the degree of damage to the ovary (Fels, '35; Ingram, '59 and Slate and Bradbury, '62). Unilateral ovariectomy did not hasten the appearance of the vaginal changes. Vaginal changes appeared at the same time as in untreated animals and as in those of Bloch and Flury ('59). The effects of aging may be the determining factor in animals which received irradiation to the ear or nembutal anesthesia. There is no apparent explanation of why irradiation of one ovary, followed by its removal a short time later, resulted in a higher percentage of cornification than was seen in the untreated or unilaterally ovariectomized female. Again, this may have been an aging process. From our observations there is no evidence why alterations occurred in the vaginal cycle after unilateral irradiation. Damage to one ovary may introduce some new factor. The same hypothesis may also be applied to bilateral irradiation and to the other forms of ovarian damage. It is clear that the damaged ovaries are responsible for the observed changes, because in every case anestrus followed bilateral ovariectomy. This would support the observations of Mandl and Zuckerman ('56) that the suprarenals do not maintain the cornification. Whatever the changes may be, the endocrine balance is disturbed. This leads to a masking of the cyclic activity, resulting in a continuous cornification of the vagina and an excessive stimulation of the uterus and mammary gland. There may be an altered estrogen effect, as suggested by Fels ('55) or Ingram and Mandl ('58), which may activate the accessory reproductive organs but have no effect on the pituitary. Or there may be a difference in the threshold at which the various end organs will respond to estrogen stimula- tion. Ingram and Mandl ('58) have shown that gonadotrophins are necessary for the damaged ovary to continue its activity. Their evidence suggests that the damaged ovary may not be able to respond fully to the gonadotrophins. It is not possible to determine if the effects are due to interference of the nerve (Hill, '48) or blood supply (Fels, '44) or to a direct effect on the ovarian tissue. Since the ovary is capable of maintaining an estrogenic influence on the vagina, uterus and mammary gland, it would seem that the damage leads to an endocrine imbalance which begins in, and is mediated through, the ovary. LITERATURE CITED Asdell, S. A., and Mary F. Crowell 1935 The effect of retarded growth upon the sexual development of rats. J. Nutrition, 10: 13-24. Bloch, Suzanne 1957 Untersuchungen iiber das funktionelle Altern tierischer Genitalorgane. Gynaecologia, 144: 313-316. Bloch, Suzanne, and E. Flury 1959 Untersuchungen iiber Klimakterium und Menopause an Albino Ratten. Ibid., 147: 414-438. Drips, Della G., and Frances A. Ford 1932 The study of the effects of roentgen rays on the estrual cycle and the ovaries of the white rat. Surg. Gynec. and Obst., 55: 596-606. Everett, J. W. 1939 Spontaneous persistent estrus in a strain of albino rats. Endocrinology, 25: 123-127. Fels, E. 1935 Ergebnisse expenmenteller Eierstocks und Nierenbestrahlung bei der weissen Ratte. Strahlentherapie, 54: 279-293. 1944 El efecto de la inyecci6n de hormona estrdgeno sobre la hipdfisis de rata con ovarios ligados. Rev. Assoc. Med. Argentina, 58: 778. 1955 Transformations tumorales des greffes ovariennes dans le rein de la Ratte castree. Compt. Rend. SOC.Biol., 149: 16661667. Hernandez, T. 1943 Hormonal ambisexuality of ovarian grafts in female rats. Amer. J. Anat., 73: 127-151. Hill, R. T. 1948 Effect of cutting the ovarian artery and nerves. Anat. Rec., 100: 42. Ingram, D. L. 1958 Fertility and oocyte numbers after x-irradiation of the ovary. J. Endocrin., 17: 81-90. 1959 The vaginal smear of senile laboratory rats. Ibid., 19: 182-188. Ingram, D. L., and Anita M. Mandl 1958 The hypophyseal control of x-ray sterilized ovary. Ibid., 17: 1-12. Kullander, S. 1959 Studies on the histogenesis of experimental granulosa cell tumours in rats. 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Long 1932 The effect on a subsequent pregnancy after x-raying one ovary of a rat. Am. J. Anat., 50: 1-8. Slate, W.G.,and J. T. Bradbury 1962 Ovarian function and histology after x-ray irradiation in albino rats. Endocrinology, 70: 1-6. Wade, N. J., and E. A. Doisey 1935 Cornification of vaginal epithelium of ovariectomized rat produced by smearing. Proc. SOC.Exp. Biol. and Med., 32: 707-709.