Induced Ovulation in the Mouse and the Measurement of Its Inhibition

Transcription

1 Induced Ovulation in the Mouse and the Measurement of Its Inhibition t' NATESAIER PURSHOTTAM, ~ MARCUS M. MASON, AND GREGORY PINCUS RUNNER has shown that the immature mouse ovary responds by superovulation to appropriate injections of pregnant mare serum gonadotrophin (PMS ) t followed by human chorionic gonadotrophin (HCG) t or pituitary luteinizing hormone (LH) and has suggested the use of this response as a basis for gonadotrophin assay.4 A similar ovarian response in the immature rat was developed as a quantitative assay for ovulating hormone, 6 but its use for the determination of human urinary LH and HCG was found impractical because of the presence of interfering substances in urinary gonadotrophin preparations. 7 Other attempts to utilize the response of the immature mouse ovary to urinary gonadotrophin in an assay procedure disclosed variable inhibitory effects in urinary extracts. 5 It occurred to us that this apparently quantifiable ovarian response might be employed as a basis for the assessment of the inhibiting effects of a variety of compounds on ovulation. In this paper we present the results of our efforts to develop methods for quantifying the ovarian response in mice and for the measurement of its inhibition. METHODS & MATERIAL To study the superovulatory response, female Swiss mice, weighing 7-10 From the Worcester Foundation for Experimental Biology, Shrewsbury, Mass., and the Biologics Testing Laboratory, Worcester, Mass. These investigations were carried out under a contract (No. SA-43pH-1713) with the National Cancer Chemotherapy Service Center of the National Institutes of Health and a grant-inaid from the Population Council. " Now at the Department of Obstetrics and Gynecology, Stanford University School of Medicine, Palo Alto, Calif. f The PMS used was Equinex and the HCG was A.P.L., both kindly supplied by Dr. J. Jewell of Ayerst and McKenna, Ltd., New York City, N. Y. 346

2 VOL. 12, No.4, 1961 INDUCED OVULATION 847 gm., were obtained at exactly 20 days of age. On the afternoon of the twentyfirst day of age, each test animal received an intraperitoneal injection of PMS and hours later (on the twenty-third day) a similar injection of ReG. Autopsy of the animals was carried out 20 hours after the administration of the ReG. Examination of the oviducts at low magnification under transmitted light revealed in the cumulus mass the presence of ova, which were dissected out onto a microscope slide and counted according to the technic of Rowlands. 3 Quantifying the Ovulation RESULTS In a preliminary study we confirmed the lack of an ovulating effect of PMS but obtained definite ovarian and uterine stimulation. The induction of superovulation in a large proportion of the test animals was possible only when ReG was injected 42 hours later (Table 1). TABLE 1. Induction of Superovulation with PMS and HCG Mean egg No. of Uterine weight Ovarian weight count Injection animals (mg.) (mg.) per mouse 0.1 ml. saline PMS-4I.U PMS-8I.U PMS & ReG U. of each Remarks 1 animal dead 65% had eggs in tubes Using a minimum of 10 animals to each test group, we determined the effects of various dosage combinations of BMS and ReG on the percentage of ovulating females and the mean number of ova ovulated (Fig. 1). It is clear that a priming dose of 2 LU. of PMS followed by 1 LU. of ReG gives a maximal yield of ova (mean of 27 per animal) and a maximum percentage of animals ovulating (90 per cent). Forty-nine groups of 10 animals each were studied for their ovulatory response to the two successive injections of non-pituitary gonadotrophins in optimal dosage and a certain time sequence. In the large majority of these groups, every animal ovulated with an ovulation rate of 100 per cent, while in a few groups the ovulation rate was 90 per cent, giving a mean of 97.75

3 348 PURSHOTTAM ET AL. FERTILITY & STERILITY per cent for all the animals induced to ovulate on this schedule. The frequency distribution of the mean number of eggs per animal was as follows: Average no. of eggs/animal No. of groups The mean number of eggs per animal in all groups was 27.89, with standard deviation plus and minus.76. The standard error for the 5-animal group was 7.3, and for the 10-animal group, 5.2. The variance analysis indicates that the ovulation of an average of less than eggs per animal in groups of 10 animals indicates a significant inhibition of the standard response; in groups of 5 animals a P value of 0.01 or less is obtained when an average of less than 8.41 eggs per animal is found. The distribution of percentages of 32.0 Ovulation [7 rate Meanno..LI aggs per animal 16.0 ~?; 8.0 on,; (; w ~ '" a o o Fig DOSAGE OF H.C.G. IN I.U. Effect on ovulation of various combinations of PMS and ReG. 16.0

4 VOL. 12, No.4, 1961 INDUCED OVULATION 349 animals ovulating is markedly skewed, with no occurrence of less than 90 per cent ovulating. Nonetheless, it may be calculated that for a group of 10 animals, ovulation in 40 per cent would be a highly significant departure (P equals 0.01) from the mean of per cent given by these 49 groups, and for a group of 5 animals, ovulation in 20 per cent represents significant inhibition. Effects of Reserpine In preliminary studies of a series of potential inhibitors of superovulation it was found that reserpine is highly active. When injected in a dose of 0.1 mg. per mouse on any of the 4 experimental days (Day 20-Day 23) it inhibits ovulation in 100 per cent of the animals. With a submaximal dose of 0.01 mg. per mouse the percentage of animals ovulating is not significantly reduced in any single group, but a significant reduction in the mean number of ova ovulated is obtained. In the hopes of establishing a scale of inhibition, a study of the effects of varying dosages of reserpine on 5-animal groups was undertaken with administration of the test dose on the day of ReG injection (Day 23). The data obtained are presented in Table 2. They demonstrate: (1) a reduction TABLE 2. The Effect of Varying Doses of Reserpine on PMS- and HCG-induced Ovulation Mean gain in Reserpine bodywt. Uterine wt. Ovarianwt. No. Ovulating (mg.) (mg.) (mg.) (mg.) eggs/mouse (%) O. 2.8 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ( ± ± ± ± ± ± ± ± ± in the gain of body weight in animals receiving more than mg. of reserpine but no proportionality between dose and degree of weight gain; (2) no significant reduction in the uterine weight response to the gonadotrophins, but a significant inhibition of ovarian weight increase at dosages of 0.08 mg. or greater, and a fairly regular relationship between the degree of inhibition of ovarian growth and the dosage of reserpine; and (3) a significant reduction in percentage of animals ovulating and in mean numbers of ova shed at doses of 0.01 mg. and higher, with complete inhibition with mg.

5 350 PURSHOTTAM ET AL. FERTILITY & STERILITY Over the dosage range used in this study neither the mean number of eggs ovulated nor the percentage of animals ovulating is linearly proportional to either the dose or log dose of reserpine. It has, therefore, been difficult to set up a standard for the assessment of relative potency of agents inhibiting ovulation. Nonetheless, we have examined the effects of a number of compounds on the response to the standard regime of PMS-RCG administration. Differential Effects of Various Drugs A wide range of drugs were tried to see whether any had inhibiting effects in this system of superovulation. The trials were conducted on tranquilizers, sedatives, narcotics, antispasmodics, steroids and antihistamines. During these trials it was customary to use only two dose levels, and only in the case of few compounds was a multiple dose response titration carried out. The dose was administered orally or by injection. From the perusal of Table 3 it can be seen that reserpine, chlorpromazine, Trilafon and Sparine gave fairly consistent inhibition of ovulation if the doses were high enough. The other tranquilizers tested all showed some degree of ovulatory inhibition but reacted in a rather irregular pattern. A number of salicylates and other analgesics were tested but they yielded no significant inhibition of ovulation. When other drugs affecting the central nervous system were tried it was found that there was very slight inhibition and then only if the dose of the drug approached the range of acute toxicity. Of the eight steroids tested in these trials only progesterone and 17 a-ethynylestratl5 lo-enolone gave a clear cut inhibition. DISCUSSION Gonadotrophic activity is usually estimated by weight increases of the ovary and uterus in either immature rats or mice or in suitable parabiotic rats. Ovulation results from the complex interaction of at least two of the gonadotrophic hormones. We feel that ovulation is a far more sensitive endpoint than organ weight and is physiologically more reactive to abnormal conditions such as excessive dosage of drugs. Lamond and Emmens assert the similarity of PMS to FSR and also that of RCG to LR. We feel that for the purposes of assaying ovulatory or antiovulatory influences these similarities are great enough to allow substitution, respectively. They also feel that the injection of PMS and/or RCG do not stimulate endogenous gonadotrophins in the immature mouse and therefore would not interfere in an assay procedure. 2 Ladman and Runner have introduced a slightly disturbing note by their

6 TABLE 3. The Effects of Various Compounds on Quantified Ovulation No. animals Dosage/animal animals Mean no. Inhibition of Compound in test (mg.) ovuluting of eggs ovulation* Equanil so O.S S.O ') Sparine Chlorpromazine SO Atarax Trilafon Paxitol Compazine Stelazine Atropine Metropine S.6 + Demerol ls.8 Phenobarbital Dibenamine Benadryl Estrone a-ethinyl estradiol SME Cortisone 10 O.S S.O Progesterone a-ethyl 19nor testosterone a-ethynyl estra enolone a-methallyl nor testosterone a-chloro 11 acetoxy progesterone * + indicates significant reduction in number of eggs; ++, significant reduction in both ovulation rate and number of eggs; and +++, complete suppression of ovulation. %of

7 352 PURSHOTT AM ET AL. FERTILITY & STERILITY observations that FSH when given to intact or hypophysectomized mice previously primed with PMS, would induce ovulation. 1 Their hypothesis is that a component other than LH is present that facilitates ovulation. A careful study of the ovulatory response may lead to an understanding of such factors involved. It is beyond the scope of this early study to delve into the questions of pituitary activation or repression and the site or mechanism of action of ovulatory hormones. The assay procedure outlined does offer a tool for its investigation and for the screening of compounds to enhance or inhibit ovulation. f SUMMARY The developmen! of an assay is described dealing with ovulation in the prepubertal mouse. The mice are superovulated by intraperitoneal injections of 2 1. U. units of pregnant mare serum followed in 42 hours by intraperitoneal injection of 1 1.U. of human chorionic gonadotrophin. Autopsy in 20 hours reveals the ova lying in the first loops of the oviduct. These may easily be removed and counted. To demonstrate inhibition of this process, the test substances were administered orally or by injection, at or before the time of the HCG injection. Many substances interfere with the ovarian response up to 50 per cent, but only a few groups of compounds are effective in totally abolishing ovulation. Compounds yielding the greatest inhibition were in the class of tranquilizers. Of these, reserpine, Sparine, chlorpromazine, and Trilafon have been very effective. Some steroids are very active while others are entirely without inhibiting properties. REFERENCES 1. LADMAN, A. J., and RUNNER, M. N. Induction of ovulation in normal and hypophysectomized immature mice with purified pituitary extracts of FSH and LH. 1st Internat. Congress of Endocrinology, July 1960, Abstract LAMOND, D. R., and EMMENS, C. W. The effect of hypophysectomy on the mouse uterine response to gonadotrophins. J. Endocrinol. 18: , ROWLANDS, 1. W. Collection of eggs from the fallopian tube of the rat. Nature 150:267, RUNNER, M. N. Ovulation in the prepuberal mouse; a delicate bioassay. Anat. Rec. 128:514, WILSON, E. D., and ZARROW, M. X. Induction of superovulation with HCG in immature mice primed with PMS. Proc. Am. Soc. Zool. 1958, Paper ZARROW, M. X., HAFEZ, E. S. E., and PINCUS, G. New bioassay for urinary LH. Fed. Proc. 15:205, ZARROW, M. X., CALDWELL, A. L. JR., HAFEZ, E. S. E., and PINCUS, G. Superovulation in the immature rat as a possible assay for LH and HCG. Endocrinology 63:748, 1958.