Spontaneous Mammary Cancer Research Articles

Permanent cure of some spontaneous mammary cancers of mice with analogs of 1,2-dimethyl-4,5-diaminobenzene

A mixture of 1,2-dimethyl-4-( p-carboxyphenylazo)-5-hydroxybenzene (CPA) and 1,2-dichloro-4-benzenesulfonamido-5-nitrobenzene (DCBN), when fed to white mice of the SPFS strain bearing spontaneous mammary cancers, cured permanently about one-third of the animals. In others, transient cures were observed, but the cancers eventually returned. The maximal number of cures was obtained with 6 g CPA and 0·5 g DCBN per kg of ration. Additional amounts did not increase the percentage of cures. A related compound, 1,2-dimethyl-4,5- bis(benzenesulfonamido)benzene (DMDB), also cured some cancers but seemed less potent than the combination of CPA and DCBN. Some other relatives were apparently inactive. In C3H mice, bearing spontaneous mammary cancers, CPA plus DCBN caused transient regressions of the neoplasms but did not result in permanent cures. Several substances such as aminopterin, 5-fluororacil and 5-fluorodeoxycytidine, which inhibit the growth of certain transplanted cancers of mice, did not exert any detectable effect on these spontaneous cancers. The effective compounds were conceived on the basis of the demonstration that the spontaneous cancers differed from normal host tissues in that they synthesized vitamin B 12. The new compounds are members of a series of antimetabolites which have been shown to inhibit the biosynthesis of this vitamin in microorganims. However, there was no direct proof that the effect on the cancers was the result of the relationship to vitamin B 12 synthesis. The compounds exerted no detectable harmful effects on the host mice at the doses used to bring about cure of the cancers. This selective toxicity was predicted from knowledge of the relationship of the analogs to the biosynthesis of vitamin B 12 and riboflavin.

Spontaneous mammary cancer developing in rats born and nursed by mothers treated with 3-methylcholanthrene.

SummarySpontaneous mammary cancers were observed in 4 female rats among a total of 28 (7♂ and 21♀) offspring of mothers treated with 3-methylcholanthrene before pregnancy. These tumors developed at the age of 49. 85. 90. and 90 days. Histologically, 3 were mammary adenocarcinoma and 1 an undifferentiated breast cancer. One female rat among a total of 12 (3♂ and 9♀) offspring of mothers treated with 3-methylcholanthrene during pregnancy developed spontaneous mammary cancer at the age of 200 days. No mammary cancer was observed in rats whose mothers were fed 3-methylcholanthrene during lactation. Also, injection of cell-free filtrate prepared from the mammary cancer induced with 3-methylcholanthrene to the normal newborn rats of less than 24 hours old failed to induce mammary tumors in these animals. These observations are of great interest for further investigation.

Mammary cancer in fostered CBA mice and CBA hybrids.

In the absence of the mammary-tumor agent (MTA), breeding females of the CBA stock, observed since 1951, have remained free of spontaneous mammary cancer. After CBA young had been nursed by females of the cancerous Z (C3H) stock, to obtain the MTA, CBA breeders of the fostered and succeeding 3 generations had an incidence of 99 percent, with an average cancer age of 252 days. Because gonadectomized mice of the CBA stock developed adrenal cortical lesions, CBA males were mated with females of the A strain, which have a low incidence of mammary cancer in nonbreeders. The F1 females, maintained as virgins, showed a high incidence. These observations demonstrate that mice of the inbred CBA stock are susceptible to spontaneous mammary cancer and, in addition, transmit the mammary-cancer-inducing inherited hormonal pattern associated with postcastrational adrenal alterations.

Incorporation of P32 into mouse mammary cancer; influence of estrogen and testosterone.

Introduction The effect of estrogens and androgens on some neoplasms of the human reproductive system, though unpredictable, has been sufficiently dramatic to warrant continued investigation. The growth rate of mammary carcinoma in particular appears to be subject to modification by these hormones in a substantial number of cases. The mechanism of action, however, is unknown. Strains of mice developing spontaneous mammary cancer in a high proportion of the females offer an appropriate laboratory preparation for the study of basic growth mechanisms involved. These tumors can be readily grafted into males of the same strain. Endogenous estrogen or androgen production can be reduced by castration in both sexes, and the influence of exogenous hormone on basic biochemical alterations is then more clearly defined. We have chosen to study the effect of 17β-estradiol and testosterone propionate on incorporation of P 32 into the total nucleic acids of these tumors. In a previous

The production of mammary cancer in rats using oestrogens.

An attempt to confirm previous observations that mammary cancer can be induced in rats by prolonged stimulation with estrogenic substances and to determine any variation in the response of different strains of rats to the carcinogenic action of estrogens on their mammary glands is presented. The results while confirming the previous reports varied with the strain of rats employed. A strain of albino rats used had been randomly bred over many years without a single case of malignant breast tumor developing. Another strain of albino rats used (WAG rats) had been maintained by brother-to-sister mating for about 50 generations. Spontaneous mammary cancers had been very rare. Black and white rats formed the 3rd group used. Although many of the older females had developed benign mammary tumors only occasionally had a mammary carcinoma been found. The estrogens used - estradiol estradiol dipropionate and stilbestrol - were implanted as pellets of 5-6 mg each. Each rat was implanted with 2 pellets of the same substance 1 at the age of 4 weeks and the other 1-3 months later. All animals were observed until they died. At death postmortem examinations were made of all organs. There was no evidence of tumor production at implantation sites. The total number of female animals used was 140. Estradiol or its dipropionate resulted in marked retardation of growth loss of activity partial loss of hair and ataxic symptoms. Stilbestrol showed less difference in weight compared with controls but no other symptoms except slight ataxia. Estradiol and the dipropionate produced similar findings. The main effect on the breast tissue was 1 of atrophy. There was also cyst formation with minimal stromal reaction. Occasionally the cellular appearance was that of a premalignant change. There were 18 rats with malignant breast tumors. In 8 of them there were multiple tumors. These were adenocarcinoma papillary carcinoma and anaplastic carcinoma. Squamous metaplasia was not seen. No tumors were found in control rats of the WAG and other albino group. Among the black and white strain there were 5 benign breast tumors. Enlargement of the pituitary gland was a consistent finding in all experimental animals. Cystic ovaries were frequent. In those implanted with stilbestrol there were 2 malignant ovarian tumors and 2 of uterine origin. Of a total of 140 female animals of 3 strains treated with estrogens 21 had developed malignant mammary tumors and 4 had malignant tumors of the ovaries or uterus.

Genetic aspect of cancer research

Many inbred strains of mice have been selected which differ in their inherent ability to develop various types of spontaneous neoplasms. This material is to the biologist what pure chemicals are to the chemist. However, as animals are subject to mutational changes, each experimental series should have adequate controls. Many studies have been reported on the influence of environmental factors upon the genesis of cancer and consequently data obtained in one laboratory should never be used as controls for those secured elsewhere. Only the experimental variable being tested should exist between the animals of the two groups. Reciprocal crosses between inbred strains with either high or low incidences of tumors have demonstrated that the genetic constitution of the host plays an important part in the genesis of leukemia, pulmonary and mammary cancer in mice. In the case of leukemia, while most of the workers agree that susceptibility may be transmited as a dominant complex, the number of genes could not be ascertained because of the action of undetermined environmental factors or, according to MacDowell, a complex interrelationship of modifying influences. Certain unknown factors probably operate also in the development of lung carcinoma and the development of the disease in very old animals makes it difficult to obtain ratios for genetic interpretation. Three primary factors have been demonstrated to interact in the development of spontaneous mammary tumors— genetic susceptibility, hormonal stimuli and a virus, the mammary tumor milk agent. In several studies low incidences have been found in mice that possessed the milk agent and had an adequate hormonal stimulation. Such groups may be considered to be relatively resistant; whether they may be called “non-susceptible” or not is problematic. The interaction of these primary causative factors is complex as shown by the observations that reciprocal hybrids between various cancerous strains may or may not have the same incidences and average cancer ages. The difference observed between two groups of susceptible mice with different agents may be of statistical significance whereas reciprocal groups with the same genetic constitution and milk agent may or may not show comparable data. If two groups of virgin females show a significant variation in incidence and average cancer age, there may be no difference in the incidences in the breeders and the group with the later age as virgins may have an earlier cancer age when maintained as breeders (unpublished). Although not characteristic for all series, in one study the incidence found among the progeny was influenced by the age at which the mothers developed cancer and the litter in which the young were born. Also, the young fostered by mothers that die either cancerous or non-cancerous may show different incidences, regardless of the genetic constitution of the mothers. Analogous results may be noted for the progeny of females of the same litter of an inbred stock. The genetic constitution of the animals also plays an important role in controlling various hormonal mechanisms, one of which, called the inherited hormonal influence, may influence the development of mammary cancer in virgin mice. Various studies were cited to determine the physiologic effects of these hormonal factors and their possible relationship to the genesis of mammary cancer in inbred stocks and their hybrids. Recently it was observed that mice of inbred strains and their hybrids with the mammary tumor milk agent excreted less 17-ketosteroids than did mice of the same stock, or hybrid generation, without the agent. The injection of extracts with the agent altered the excretion rate to that found for animals which obtained the milk virus by nursing. Considerable difference was noted between various susceptible strains. In most of the groups, but not all, the presence of the milk agent was associated with an increased number of positive smears observed during a definite period. The long interval between the introduction of the mammary tumor milk agent, a virus, and the appearance of spontaneous mammary cancer would become apparent if the virus had some role in controlling the metabolism of the hormones. Through such a process the mice with the milk agent might possess an excess of what might be called “carcinogenic” hormones as compared with mice without the agent. These hormones, acting upon susceptible cells, could be the “primary” cause of mammary cancer in mice although the interaction of all the causative factors would be required before a high incidence would be obtained. Thus genetic factors have been demonstrated in the etiology of several types of tumors in mice. The exact genic make-up of the susceptibilities has not been definitely determined because of the possible interaction of other genetic and perhaps unknown “environmental” influences. In the cancerous A strain, linkage between one of the susceptibility genes for spontaneous mammary cancer and the brown gene has been demonstrated.

Antireticular cytotoxic serum (ACS). III. Reduced incidence of spontaneous mammary cancer in R3 mice treated with ACS

Antireticular cytotoxic serum (ACS). III. Reduced incidence of spontaneous mammary cancer in R3 mice treated with ACS

The growth-inhibitory and carcinogenic properties of 4-aminostilbene and derivatives

Marked inhibition of the growth of the Walker rat carcinoma 256 is produced by administration of 4-aminostilbene and of 4-dimethylaminostilbene. From similar experiments with the Crocker sarcoma 180, the carcinoma C63, and spontaneous mammary cancer, it appears that the growth inhibitory action of these compounds is much less pronounced in mice. In a dosage of 200 to 250 mg. /kg. in the rat, both 4-amino- and 4-dimethylaminostilbene produce ( a ) gastro-intestinal submucous haemorrhage most evident in the pyloric portion of the stomach, ( b ) haematuria, and ( c ) haemolymph changes, while the former compound induces methaemoglobinaemia in addition. Using the Walker rat carcinoma 256 as the biological test object, a series of derivatives of 4-aminostilbene has been examined to determine the relationship between the growth-inhibitory effect and chemical constitution. The great majority of the active compounds can be defined as stilbenes with a basic substituent, the position of which is of paramount importance; thus o -dimethylaminostilbene is very much less active than the p -isomeride, and the m -compound is completely inactive. A further essential feature is the ethylene bridge, since activity disappears when either of its hydrogen atoms is substituted, when the bridge is reduced, when it is extended to contain three or four carbon atoms, or when either methine group is replaced by a nitrogen atom. Compounds in which the ethylene bridge is absent, or is replaced by oxygen or sulphur, are also inactive, and activity is further dependent upon the trans configuration of the molecule about the ethylenic bond, and to a large extent upon a free p' -position. These and other facts have suggested the working hypothesis that one of the features required for activity is an unbroken conjugation of the amino group with both nuclei, enabling the compound to assume some dipolar quinonoid character, which depends, among other things, on the co-planar arrangement of the two benzene nuclei which characterizes the trans form of the stilbenes. When evidence concerning steric conditions in the molecules (obtained mainly from the ultraviolet spectroscopy of 4-dimethylaminostilbene and thirteen of its alkyl derivatives) is compared with the biological activities of these compounds, a close parallel is suggested between lack of growth-inhibitory power and buckling of the molecule. Thus in the 4-dimethylaminostilbene derivatives with substituents on the x- and ?-carbon atoms of the ethylenic double bond, or with methyl groups at two ortho positions in a phenyl group, steric factors reduce the planarity of the molecule, thus affecting the conjugation resonance characteristic of the whole molecule. These compounds had previously been found to be non-inhibitory. All the evidence from diagrams, models and spectra suggests that steric interference with the planar configuration of molecules in this series varies continuously from the planar 4-dimethylaminostilbene to the highly buckled a/?-diethyl derivative. Inhibitory activity within this series appears to depend upon a conjunction of such factors as molecular size and shape, and the apposition of a planar molecule to a hypothetical adsorbing surface. In view of the previously suggested connexion between growth-inhibitory activity and tumour production, a number of selected aminostilbenes has been tested for carcinogenicity. Of seventy-two rats exposed to the action of 4-amino-, 4-acetamido-, 4-dimethylamino-, or 4-diethyIaminostilbene, twenty-three developed a total of eight sarcomata and thirty distant tumours mostly comprising squamous keratinizing carcinomata of the external acoustic duct, mammary adenomata, and cholangiomata. In a second series, the compounds 4-amino-, 4-dimethylamino- and 2,-methyl- 4-dimethylaminostilbene, and 1-(4'-dimethylaminophenyl)-2-(T-naphthyl)ethylene, were tested in male and female mice and rats. Of 120 rats exposed, forty-eight developed a total of twenty sarcomata at the site of injection and fifty-one tumours in organs distant from the site of injection, while of 120 mice similarly treated only four sarcomata and two distant tumours were recorded in five. The nature and distribution of the distant tumours induced in the rat strongly suggest some common feature in the carcinogenic action of aminostilbenes and 2-acetamidofluorene. Other points discussed include ( a ) the association between haemolymphatic changes and carcinogenesis, ( b ) the possible significance of the nitrogenous analogue 2-(4'-dimethylaminostyryl) quinoline in relation to the carcinogenic action of ‘styryl 430’, ( c ) a comparison of the biological properties of 4-dimethylaminostilbene and 4-dimethylaminoazobenzene, and ( d ) the dependence for manifestation of the growth-inhibiting action of the aminostilbenes upon a sufficiently low intake of dietary protein.

Propagation of the Mammary Tumor Milk Agent in Tumors from C57 Black Mice.

SummaryMice of some sublines of the C57 black stock may propagate and transmit the mammary tumor agent. The agent may be recovered from spontaneous mammary cancers which developed in mice of the strain and may survive for at least 10 passages of these tumors in mice without the agent.

Characteristics of the Mammary Tumor Milk Agent in Serial Dilution and Blood Studies.

The mammary tumor milk agent has been shown to be essential for the regular development of mammary cancer in mice. It is usually transferred by nursing and is present in various tissues3-6 as well as the milk. The infectious agent has the characteristics of a virus. This report gives data on the activity of the milk agent for mammary cancer in blood plasma, blood-cell suspensions, and serial dilution of extracts made from mammary tissue. Blood was obtained from the hearts of mice known to harbor the agent. Either females of the A stock, 75 to 105 days of age, or females (A and C3H stocks and their hybrids) that had developed spontaneous mammary cancer were used. The blood was citrated to prevent coagulation but moderate hemolysis occurred. Separation of the cells and the plasma was by centrifugation (approximately 4,000 r.p.m. for 10 minutes) and the cells were washed with saline several times before they were used. The cells were suspended in saline and the plasma was injected either undiluted or diluted in saline (see table for details). As test animals mice were used that were susceptible for mammary cancer and lacking the milk agent. They were injected intra-peritoneally when from 3 to 5 weeks of age. The incidence in control mice maintained as breeding females was approximately 1%. The results for the blood studies are presented in Table I. Many of the mice in one series are living at 15 months of age. The donors of the lactating mammary tissue were AZF1 females that had had from 5 to 7 litters. The glands were macerated by means of a press, and serial dilutions were made with triple distilled water. Each animal received 1 cc of the suspension intraperitoneally and the surviving animals are 15 months of age. The preliminary results are tabulated in Table II.

The Prevention of Spontaneous Mammary Cancer in Mice by an Anterior Pituitary Hormone

In 1938 E. S. Horning and I (1) recorded the results of an experiment in which the development of spontaneous mammary cancer in an inbred high-cancer strain—the Paris strain RIII—was completely prevented by the continued injection of an anterior pituitary hormone preparation containing the so-called thyrotropic hormone. This experiment has been repeated by Haagensen, Randall and Auchincloss (2) with the same tumor strain and the same hormone preparation, using the same dosage. Their results, published under the title: “Failure of Thyrotropic Hormone to Prevent Spontaneous Mammary Cancer in Mice,” lead them to conclude “that the treatment prevented, to some extent, pregnancy in our mice and therefore [our italics] lowered slightly the incidence of mammary carcinoma.” In their summary they state that the treatment with the anterior pituitary hormone preparation “did not definitely affect the incidence of mammary carcinoma in female mice of the RIII strain.” If this conclusion is correct, the difference between their results and ours is inexplicable. An analysis of their own figures shows, however, that their conclusion is not correct.

Failure of Thyrotropic Pituitary Hormone to Prevent Spontaneous Mammary Cancer in Mice

SummaryLong continued subcutaneous injection of an anterior pituitary preparation which contained thyrotropic hormone and some gonadotropic hormone did not definitely affect the incidence of mammary carcinoma in female mice of the RIII strain.

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Evidence suggests that maternal/extrachromosomal influence may be a factor in mammary carcinogenesis in mice. There are 3 possible methods of transmission to the progeny: 1) by means of the cytoplasm or cytoplasmic inheritance 2) during intrauterine development and 3) by way of the mothers milk while nursing. Various experiments in breast cancer etiology in mice have assumed a correlation between milk/hormonal/susceptibility factors and cancer development. For stock A high-breast tumor breeding females this would suggest development of spontaneous mammary cancer in every female. However this had not been the case. If noncancerous animals of this stock are mated and their progeny continued the incidence of breast tumors among the offspring is very similar to that of the control stock suggesting that these mice are genetically cancerous and would probably develop the condition if they live longer. Other experiments show that breast tumors can occur in a small percentage of animals where none were to be expected suggesting that there may be other contributing factors of unknown origin and/or there may be physiological upsets in the equilibrium or relationship of the known factors and changes characteristic of somatic mutation. Experimental evidence also suggests that an excessive dose of estrogenic hormone may produce carcinoma more easily in males of a strain not susceptible to breast cancer but having the milk factor active than in males of a susceptible strain having the milk factor that is not active.

The Influence of Foster Nursing upon the Incidence of Spontaneous Mammary Cancer in Resistant and Susceptible Mice

The Influence of Foster Nursing upon the Incidence of Spontaneous Mammary Cancer in Resistant and Susceptible Mice

Tumor Incidence in Line a Albino Mice following Injections of Progynon-B

The appearance of mammary tumors in male mice following the injection of estrogenic hormones has been reported by a number of investigators. Comprehensive reviews of the literature have appeared in papers by Loeb et al. (1), Cook et al. (2), and Gardner (3). It is now well established that the incidence of these mammary adenocarcinomas varies with the strain of mice under investigation and with the type and amount of estrogen used. Thus, male mice from stocks in which there is a high rate of spontaneous mammary cancer in the females have a higher percentage of tumors following treatment than males from low-cancer strains. Treated females in some instances have not shown as high tumor incidence as treated males of the same strain. The present investigation was begun in 1935 to test the effect of long-continued injections of estradiol benzoate on the production of tumors in the A strain of albino mice, in which 80 per cent of the bred females were reported to develop spontaneous mammary cancers. The results for the most part confirm the findings of other investigators which have been published since this experiment was started, but as the behavior of the A strain in this laboratory is at variance with that reported elsewhere, it seems of value to record the results.

Simple Experimental Cancer Research

We have nearly every year summaries of the current status of cancer research. These orient the worker and are valuable. One learns from them that steps of progress are visible, though often small. Without attempting much summation, I shall describe briefly the course of a single simple but long continued experiment with animal cancer, hoping that it may be one of these short steps, and shall then suggest a much longer step. This experiment concerns the helminth parasites which infest the intestinal tract of mice fated to develop spontaneous mammary cancer. The mice in question are laboratory albinos of the common house mouse, and the parasites are cestodes of occasional and nematodes of almost universal presence. These orders have already been linked with cancer. It is pertinent to recall that some cancers have a parasitic origin and that cestodes and nematodes furnish striking and authentic examples. It is true that in such cases the cancer has arisen at the site of infestation or in tissues closely adjacent, and not at a distance, though we now know that the action of cancerogenic agents is not necessarily confined to the area of application.

Carcinoma mammae occurring in a male mouse under continued treatment with oestrin.

Many years ago Lathrop and Leo Loeb showed by experimental work that some connection exists in mice between ovarian activity and carcinoma of the mamma. This general conclusion was confirmed and amplified by further experiments carried out by Cori and by W. S. Murray. The discovery by Doisy, Veler, and Thayer, and by Butenandt, in 1929, of methods for preparing the ovarian hormone, oestrin, in a pure form opened the way to further and more exact inquiry into the problem of this association between ovarian activity and cancer of the breast. Lacassagne (1932, 1933) gave weekly injections of oestrin (folliculin) to 5 male mice belonging to a strain in which 72 per cent of the females are subject to spontaneous mammary cancer, the males not being subject to this development. Mammary cancer appeared in every one of the 5 males and in 5 of 7 females treated in this manner. Using mice belonging to strains in which mammary cancer occurred spontaneously in only 2 per cent of the females, and treating them in the same way as those of the preceding experiment, he found that mammary tumours developed in 2 of 8 males and in 1 of 7 females. These tumours appeared at a more advanced age than those produced in the strain which had a high natural incidence of cancer. In yet another strain of mice no neoplasia took place among 7 males treated by weekly injections of oestrin in the same manner as in the preceding experiments.

A comparison of the normal œstrous cycle and of the response to the administration of œstrin in two strains of mice differing greatly in incidence of spontaneous mammary cancer

A comparison of the normal œstrous cycle and of the response to the administration of œstrin in two strains of mice differing greatly in incidence of spontaneous mammary cancer

The Development of Multiple Tumors in Tarred and Radiated Mice: Part II

In an earlier paper (1) the preliminary results of a study of the effect of radiation on the subsequent development of tar tumors were given. The present paper deals with the final results of this experiment. As outlined previously, the experiment consisted of painting with a sample of coal tar of unknown carcinogenic power, the interscapular region of 139 albino mice, chosen from an inbred strain. This strain, called strain 3, developed by the biologist of the State Institute, Mr. Marsh, is a high-cancer strain, in which upwards of 80 per cent of the bred females develop spontaneous mammary cancer, while the unbred females have a cancer incidence which is slightly lower and somewhat retarded. The mice used in this experiment were unbred females and therefore belong in the latter group. The tar was applied twice weekly, one group receiving 8 applications, another 16 applications, and a third group 24 applications. Following the tarring, each large group was divided into two subgroups, one of which was radiated with heavy doses of gamma rays, while the other was held as a control group. Approximately six months after cessation of tarring, when the mice were ten to eleven months old, all of the animals still alive (103 in number), irrespective of the grouping, showed widely distributed multiple tumors on the skin (Figs. 1–4). The location of these tumors bore no relation to the site painted; in fact, this site rarely showed any tumor formation. The widespread occurrence of the tumors is indicated by the fact that 26 separate and distinct nodules were counted on one animal.

Ovarian Hormone and Traumatic Stimulation of Monkey's Cervix to a Condition Resembling Early Cancer

The ovarian follicular hormone has been shown by Allen1, 2 to be a powerful stimulator of epithelial growth in the female genital tract and breast. Large amounts of this hormone have been reported in the blood of cancer patients, both male and female. Also cancer tissue from men has been found to have a high content of theelin. Lacassagne gave theelin injections to 3 male mice from a strain in which 72% of the females developed spontaneous mammary cancer. The males of this strain normally never developed mammary cancer but the theelin injections produced mammary cancer in 5 to 6 months in each of the 3 males. Removal of the ovaries in mammary cancer strains of mice and rats markedly decreases or entirely prevents the development of spontaneous mammary cancer.6, 7, 8 Hofbauer reported that anterior hypophysis transplants and extracts in guinea pigs produced a precancerous condition in the cervix. His anterior pituitary treatment should. have increased the production of the follicular hormone, although he considers that the pituitary hormone caused a direct stimulation of the cervical epithelium. These facts led us to attempt to produce cancer by injections of genital growth hormones combined with chronic trauma. Nine female Macacus rhesus monkeys were used. All except one were ovariectomized several months to 20 days before the beginning of the experiment. The cervices were traumatized by scissor cuts through a vaginal speculum. This procedure was repeated at 7 to 10 day intervals throughout the experiment. In addition to the cuts a small metal clip was clamped on the cervix in some of the animals. Subcutaneous injections of hormone were made twice daily, morning and evening as a rule.