Castrated Female Rats Research Articles

Receptor progesterone complex in the nuclear fraction of the rat uterus: Demonstration by 3H-progesterone exchange

We have previously shown that 3H-estradiol exchange can be used to measure the quantity of estrogen receptor complex in the nuclear fraction of target tissue cells. This method has been modified for the measurement of the progesterone receptor complex (R n·P) in the nuclear fraction of uterine cells. Nuclear fractions were incubated for 5 hrs. at 15°C in the presence of varying concentrations of 3H-progesterone ( 3H-P) with or without a 250-fold excess of non-labeled progesterone (P). R n·P was determined by subtracting the 3H-P bound in the presence of excess P (non-specific binding) from 3H-P bound in the absence of excess P (total binding). All R n·P studies were done in adult castrate female rats that had received estradiol benzoate (0.4 mg) one week before use. The quantity of R n·P increased in the uterine nuclear fractions by 280% 30 min. after injection of 5 mg of P. R n·P was not increased in muscle or fat pad by this treatment. Injections of corticosterone (B), cortisol (F), dexamethasone (Dx) or testosterone (T) failed to increase R n·P. The exchange reaction was specific for P; B, F, Dx or T did not compete. These results demonstrate the existence of a low capacity, high affinity, stereospecific progesterone binding site in the nuclear fraction of the uterus.

Testerone concentrations in the peripheral plasma of adrogenized female rats and in the estrous cycle of normal female rats.

Previous in vitro data have suggested that the ovaries of androgen sterilized female rats (ASFR) have the capacity to incorporate more labeled precursors into androgens than ovaries from normal rats. In order to test the biosynthetic capacity of the ASFR ovary in vivo, the following experiments were carried out. Blood was collected from 120-day-old intact, castrated or adrenalectomized ASFR. In addition, blood was collected from diestrous, estrous, proestrous, adrenalectomized proestrous and castrated female rats. The plasma was assayed for testosterone by a radioimmunologic system which did not require chromatography. The plasma testosterone concentration in ASFR was lower than that found in proestrous and diestrous females, similar to that found in estrous females, and higher than that found in castrated females. The proestrous females had significantly higher peripheral testosterone concentrations than either estrous or diestrous females. Furthermore, the testosterone concentration in the peripheral bl...

Irreversible neonatal differentiation of corticosterone metabolism in rats in vivo.

The excretion of corticosterone metabolites in bile from gonadectomized male and female rats was studied; the effects of testosterone propionate treatment on steroid excretion in intact and castrated female rats were also studied. Postpubertal ovariectomy did not change the female biliary corticosteroid pattern (mainly characterized by excretion of mono‐ and disulphurylated 3α,11β,15α,21‐tetrahydroxy‐5α‐pregnan‐20‐one); likewise postpubertal testectomy or even testectomy of rats 14 days of age did not change the male biliary corticosteroid pattern (mainly characterized by disulphurylated 3β,11β,21‐trihydroxy‐5α‐pregnan‐20‐one and 5α‐pregnane‐3β,11β,20β,21‐tetrol). On the other hand neonatal testectomy led to a feminized biliary corticosteroid pattern in the adult rats. The results indicate an irreversible suppression of the hepatic 15α‐hydroxylase activity by testicular secretion products, presumably androgen(s), in the neonatal period. When adult intact or gonadectomized female rats were treated with testosterone propionate the female biliary corticosteroid pattern changed to a male pattern but reverted to a female pattern some time after the therapy was stopped. These results indicate that the 15α‐hydroxylase activity in female rats may be reversibly suppressed by androgen treatment postpubertally.

Adrenal Progesterone: Factors Controlling Its Secretion1

Plasma levels of progesterone have been measured, using a competition binding technique, in the systemic circulation of normal and castrated female and male rats of the Sprague Dawley strain. Plasma progesterone exhibits a large fluctuation in normal females, with a peak during the day of proestrus. In females, progesterone does not disappear completely from the systemic circulation after castration, even if its levels are much lower than those of normally cycling animals. Progesterone is detectable in the systemic circulation of normal males, and its levels increase significantly after orchidectomy. The ip administration of 25 μg/100 g body wt of dexamethasone (a potent ACTH-blocking agent) to female animals castrated IS days previously induces, in 4 hr, a significant decrease of plasma levels of progesterone. Plasma levels of corticosterone (measured with a fluorometric procedure) are also significantly decreased after dexamethasone. Exogenous ACTH (administered iv 15 min before sacrifice) to dexamethas...

Regulation of Hydroxylation of 5α-Androstane-3α, 17β-diol in Liver Microsomes from Male and Female Rats

Abstract The metabolism of 5α-[4-14C]androstane-3α, 17β-diol was studied in the microsomal fraction of livers from adult male and female rats after castration and treatment with various steroid hormones and drugs. In intact male control rats 5α-[4-14C]androstane-3α, 17β-diol was hydroxylated in positions 2α, 2β, 7α, 7β, and 18, whereas female control rats only hydroxylated in positions 2α and 7α. Castration had no effect on the activities of the 2α- and 7α-hydroxylase systems in female rats but led to decreased activities of the 2α-, 2β-, 7β-, and 18-hydroxylase systems in male rats. The activities of these four enzyme systems were restored by treatment with testosterone propionate, 5α-dihydrotestosterone and 5α-androstane-3α, 17β-diol but similar treatment of castrated female rats had a very small effect on the hydroxylase activities and demonstrated a relative androgen unresponsiveness in postpubertal female rats. The 7α-hydroxylase system active on 5α-[4-14C]androstane-3α, 17β-diol was more active in female rats; this enzyme system was found to be primarily regulated by nongonadal factors and only slightly affected by androgens. Treatment of castrated male rats with estradiol benzoate led to a complete feminization of the activities of the liver microsomal hydroxylase systems. The activities of all hydroxylase systems active on 5α-[4-14C]androstane-3α, 17β-diol in both male and female rats decreased in incubations carried out in the presence of carbon monoxide indicating that carbon monoxide binding pigments were involved in all hydroxylations studied. Administration of phenobarbital and 16α-cyanopregnenolone to castrated male and female rats led to increased activities of all hydroxylase systems in both sexes even if quantitative sexual differences in responses were observed. Methylcholanthrene treatment specifically stimulated 7αhydroxylation, especially in female rats. It is speculated that 7α-hydroxylation of 5α-[4-14C]androstane-3α, 17β-diol is mainly carried out with cytochrome P-448 as the hemoprotein component and that the 2α-, 2β-, 7β-, and 18-hydroxylase systems mainly use cytochrome P-450 as hemoprotein. The hydroxylation of 5α-[4-14C]androstane-3α, 17β-diol was also studied in male and female rats 0, 7, 18, 28, 35, 42, and 56 days of age. No sexual differences were found in prepubertal rats and in these animals 2α-, 2β-, 7α-, and 18-hydroxylations were of the same order of magnitude as in adult male rats and regulated by nongonadal factors. At the time of puberty, sexual differences were found to develop and the control of the activities of the 2α-, 2β-, 7α-, and 18-hydroxylase systems changed from nongonadal to androgenic regulation.

Studies on the folate coenzyme metabolism in the castrated rat and treated with 17 -estradiol.

SummaryThe effect of 17β-estradiol on folate coenzyme metabolism was studied in normal and castrated female rats. The capacity of conversion in vivo of folic acid into activated forms was investigated by measuring the quantity of folate derivatives in the urine and the liver folate coenzyme contents after injection of folic acid. An increase in the urinary excretion of folate metabolites and a decrease in the liver content of reduced folate forms in castrated rats was seen compared with normal rats. Treatment of castrated animals with 17β-estradiol were corrective. The enzymic activities involved in the folate coenzyme metabolism were assayed in vitro both in the liver and in the uterus. Severe alterations were observed in the enzymes of castrated animals and here, too, 17β-estradiol administration restored the biochemical lesions almost completely. The results show a possible influence on folate coenzyme synthesis by 17β-estradiol.

Synergistic role of progestogens in estrogen induction of LH and FSH surge.

A synergistic effect of 20∞OH pregn-en-3-one (20∞P) on estrogen induced LHFSH peak was demonstrated in castrate female rats. When high doses of 40 μg and 100 μg/day ethinyl estradiol (EE) were administered over 5 days time to castrate rats, a biphasic response in serum LH was seen. During the first 4 days, LH concentrations progressively fell and then on day 5 a surge or peak occurred. FSH also fell progressively, but no peak occurred on day 5. Lower doses of EE resulted in suppression of both serum LH and FSH, but no surge of LH occurred. However, when 20∞P was added on the Sth day of treatment with a subthreshold (for stimulation of LH) dose of EE (0.4 μg/day), a surge in both serum LH and FSH occurred. If progesterone was given instead of 20∞P on day 5, a surge of serum LH but not FSH occurred. 17∞OH progesterone (17∞P) had no synergistic effect. P, 20∞P or 17∞P given alone to castrate rats had no effect on serum LH or FSH. We postulate that in rats 20∞P may have a synergistic effect with estradiol in ...

Evidence for a periodic release of LH in castrated male and female rats.

Radioimmunoassay systems were used to measure LH and FSH concentrations in sequential samples of whole blood obtained at frequent (2–10 minute) intervals from cannulated rats which had been castrated at least 45 days earlier. In castrated rats of both sexes, plasma LH patterns were characterized by brief periods of rapid increase, alternating with more prolonged periods during which LH levels decreased at a rate comparable to the disappearance rate (“halflife”) for LH following hypophysectomy. Plasma LH concentrations, which varied from 80 to 300 ng B160/ml, were consistently greater than levels measured in intact males or diestrous females ( @ 5 ng/ml) and consistently less than maximum levels associated with ovulation (400–800 ng/ml). These data suggest that LH is released at regular intervals ranging from 20 minutes to one hour and that the periodicity of this LH release is reasonably consistent within individual castrated rats during 2 to 3 hour intervals. It appears that there are periods of time dur...

The effect of growth hormone, insulin and alloxan-induced diabetes on carcinogenesis in the genital tract of intact and castrate female rats.

Castrate female rats given weekly applications of DMBA to the genital tract and treated additionally with growth hormone, insulin or alloxan (to induce diabetes) are heavier and have more sarcomatous and epithelial cervico-vaginal neoplasms than intact animals under the same experimental conditions. Promotion of carcinogenesis and gain in body weight are independent phenomena caused by castration in the medicated rats. Growth hormone is most effective in enhancing body weight in all animals, but least as regards tumour formation. It reduces the incidence of sarcomas in intacts, but raises that of epithelial neoplasms, and promotes both types of neoplasms in castrates. The highest incidence of cervico-vaginal epithelial and sarcomatous tumours occurs in spayed diabetics.Squamous celled epitheliomas of the vulva are not affected by castration or additional medication, while basal celled neoplasms tend to be more frequent in intacts than in castrates and particularly numerous in intact failed diabetics. Vulval sarcomas are usually rare but are increased in numbers in diabetic and in insulin treated intacts.Granular myoblastomas of the cervico-vaginal tract occur in intacts only and particularly in diabetics and those medicated with growth hormone or insulin.

Effect of oral contraceptive steroids on jejunal pyruvate kinase and adenyl cyclase activities.

Castrate female rats were intubated with physiologic sex steroids and oral contraceptive synthetic steroids to determine whether these substances produced adaptive changes in the activity of jejunal pyruvate kinase and the membrane-bound enzyme adenyl cyclase. Rats received 100 mcg test substances/100 gm body weight in 3 ml propylene glycol for 1 day and were sacrificed 16 hours later. Pyruvate kinase was assayed according to Bucher and Pfleiderer. Adenyl cyclase activity was measured by method of Krishna. Oral 17beta-estradiol and various estrogenlike oral contraceptives produce significant (p less than .01) adaptive increases in activity of jejunal pyruvate kinase and adenyl cyclase. Progesterone and progesteronelike oral contraceptives have no effect on pyruvate kinase activity while only Provera effected the adenyl cyclase causing a decrease in activity.

The influence of carcinogenic dosage and of sex on the induction of epitheliomas and sarcomas in the dorsal skin of rats.

The effect of varying the numbers (4, 5, 10, 20 and 40) of weekly applications of DMBA to the dorsal skin of intact and castrate male and female rats on the induction of basal and squamous celled epitheliomas and of sarcomas has been investigated.Basal celled tumours originate mainly in hair follicles and squamous celled neoplasms in the interfollicular regions of the epidermis and differ in their progression to malignancy. Penetration of the panniculus carnosus is neither a sufficient nor necessary criterion of malignancy since growing hair follicles pass through the muscle layer and carcinomas and sarcomas which are still confined to the dermis, spread along the perineural lymphatics and metastasise to the lungs.Sex and castration do not affect carcinogenesis of epitheliomas in the dorsal skin at any dose level. Significantly more sarcomas result from 20 weekly paintings in male than in female or castrate rats.The induction period for all tumour types is shortened in sensitive individuals only by an increase from 5 to 10 weekly applications. For less sensitive animals the rate of oncogenesis is accelerated with number of administrations up to 20, but slowed down from this level by 40 paintings. The optimal dose for speed of induction of all tumour types, for maximal yield of basal celled epitheliomas and for that of sarcomas in male rats is 20 weekly applications.THE PROGRESSION TO MALIGNANCY VARIES WITH TUMOUR TYPE: it is fast for sarcomas and slow for basal celled neoplasms. Of the 336 rats at risk only 1% have fibromas or other precursor lesions, while 40% have sarcomas; animals with squamous celled papillomas account for 12%, but those with carcinomas for 66%; there are, however, 64% of rats with basal celled papillomas and only 9% with carcinomas.The optimal dose phenomenon in carcinogenesis is discussed.

Effect of estrogen on bone mineral turnover in mature female rats as measured by strontium-85.

The hypothesis that daily physiologic doses of estrogen enhance bone mineral retention was tested with strontium-85 in male female and ovariectomized rats. Rats received 25 mc Ci strontium-85 chloride ip and then 5 mcg 17beta-estradiol benzoate in oil daily for 45 days. After the 45 days of estrogen whole body counts and femur counts showed that strontium retention was greater in males (p<.01) than in intact or ovariectomized females but when expressed by body weight retention was lower in the ovariectomized rats. A second experiment included untreated controls and ovariectomized rats restricted to 15 gm food per day. No difference appeared between ovariectomized rats on free or restricted diet. Retention was 12-15% greater (p<.05) in estrogen-treated normal and ovariectomized rats than in untreated controls; 24% greater in estrogen-treated ovariectomized rats on restricted diet than in untreated unrestricted rats. In femurs retention was higher in restricted castrated and unrestricted intact female rats given estrogen than in untreated controls but not increased in the unrestricted ovariectomized group. Holtzman females retained more strontium-85 in experiment 2 than did Sprague-Dawley females in experiment 1

Dietary regulation of glycolytic enzymes X. The effect of oral, intramuscular and conjugated sex steroids on jejunal folate-metabolizing enzyme activities in normal and castrated male and female rats

1. 1. The effects of oral, intramuscular and conjugated sex hormones (progesterone, testosterone and estradiol-17β) upon the activities of four enzymes involved in folate metabolism, (glutamate formiminotransferase (N-formimino- L-glutamate:tetrahydrofolate 5-formiminotransferase, EC 2.1.2.5), serine hydroxymethyltransferase ( L-serine : tetrahydrofolate 5,10-hydroxymethyltransferase, EC 2.1.2.1), methylenetetrahydrofolate dehydrogenase (5,10-methylenetetrahydrofolate : NADP oxidoreductase, EC 1.5.1.5) and formyltetrahydrofolate synthetase (formate : tetrahydrofolate ligase (ADP), EC 6.3.4.3)) were studied in the jejunum of normal and castrated male and female rats. 2. 2. These studies demonstrate that oral sex hormones (estradiol in females, testosterone in males) produce significant adaptive increases (P < 0.01) in the activities of jejunal glutamate formiminotransferase, serine hydroxymethyltransferase and methylenetetrahydrofolate dehydrogenase in both normal and castrated rats, while intramuscular sex hormones produced no adaptive increases in the activities of these same enzymes in normal rats. Oral conjugated sex hormones also produce adaptive increases in the activities of serine hydroxymethyltransferase and methylenetetrahydrofolate dehydrogenase in normal rat jejunum.

Dietary regulation of glycolytic enzymes IX. The effect of oral, intramuscular and conjugated sex steroids on jejunal glycolytic enzyme activities in normal and castrated male and female rats

1. 1. The effects of oral, intramuscular and conjugated sex hormones (estradiol, progesterone and testosterone) on the activities of three glycolytic enzymes (phosphofructokinase (ATP : D-fructose-6-phosphate 6-phosphotransferase, EC 2.7.1.11), pyruvate kinase (ATP : pyruvate phosphotransferase, EC 2.7.1.40) and fructose diphosphate aldolase (fructose-1,6-diphosphate D-glyceraldehyde-lyse, EC 4.1.2.13) were studied in the jejunum of both non-castrate and castrate male and female rats. 2. 2. These studies demonstrate that the oral sex hormones (estradiol in females, testosterone in males) produce adaptive increases in the activity of jejunal pyruvate kinase in both non-castrate and castrate rats, while intramuscular sex hormones are effective only in castrate rats. Sex hormones also produced adaptive increases in phosphofructokinase activities in castrate rats. Oral conjugated sex hormones are nearly as effective as the non-conjugated sex hormones in producing adaptive increases in the activity of jejunal pyruvate kinase. No significant changes were seen in fructose-1,6-phosphate alsolase activities. 3. 3. We postulate that a sex hormone-dependent binding site in jejunum accounts for the differences in action between intramuscular and oral sex steroids in castrate and non-castrate rats.

Ontogeny of the estrogen receptor during early uterine development.

The number of estrogen binding sites in uterine cytoplasm on a per cell basis reaches a maximum by day 10 of life in both intact and castrate female rats. After this peak is reached, the number of binding sites per cell decreases, and the ratio remains constant until days 22 to 23 of life. Thus, the ontogeny of the estrogen binding protein is not dependent upon estrogen from the ovary and is probably an autonomous property of uterine cells. Sedimentation values and dissociation constants of the protein when the animals are 5 to 10 days of age are similar to those of the 22-day-old animal, indicating that the same protein is present throughout postnatal development.

31 Lack of Effect of Calcitonin on Skeletal Mass in Osteoporotic Castrate Female Rats

31 Lack of Effect of Calcitonin on Skeletal Mass in Osteoporotic Castrate Female Rats

Testosterone inhibition of estradiol-induced stimulation of adrenal 11-beta- and 18-hydroxylation.

SummaryAdrenal homogenates from intact and castrated male and female rats treated with 1 μg 17β-estradiol iv were tested for ability to convert exogenous DOC to B and 18-OH DOC. Estradiol, which causes a significant increase in hydroxylating capacity in intact female rats, produced no such increase in intact males. Orchiectomized males, however, responded to estradiol with increased ability to hydroxylate. The estradiol response was lost after administration of testosterone to both orchiectomized males and intact females.

EFFECTS OF MORPHINE TREATMENT AND STARVATION ON THE SUBSTRATE INTERACTION WITH P-450 IN THE OXIDATION OF DRUGS BY LIVER MICROSOMES

In previous papers (1, 2), it has been reported that in male rats the administration of morphine and also starvation caused a marked decrease in the activities of drug-metabolizing enzymes of liver microsomes for hexobarbital hydroxylation and aminopyrine N-demethylation, but this was not the case in female rats. On the other hand, the activities of drugmetabolizing enzymes for aniline hydroxylation and zoxazolamine hydroxylation were not significantly altered in both sexes or even increased by the same treatments. The castration of male rats markedly decreased the activities of hexobarbital hydroxylation and aminopyrine N-demethylation, while the activities were not altered by the castration of female rats (1, 2). The administration of methyltestosterone to the castrated male and female rats increased the activities of hexobarbital hydroxylation and aminopyrine N-demethylation in liver microsomes and these activities again decreased by the administration of morphine and by starvation in the androgen-treated rats (1, 2). These results indicate that both morphine and starvation decrease the activities of drug-metabolizing enzymes which are stimulated by androgens, probably through the impairment of an androgen-dependent stimulating mechanism (1-4). Recent studies have established that an unique hemoprotein clled P-450 (5) is involved as the oxygen activating component in a number of NADPH-dependent monooxygenase reactions, such as hydroxylations of drugs and steroid hormones (6-10). Imai and Sato (11) and Schenkman et al. (12) have recently reported that a number of drugs, substrates of hepatic microsomal hydroxylases, react with the microsomal cytochrome to give two characteristic types of spectral change. These results suggest that the spectral changes observed are indicative of substrate interaction with cytochrome P-450 presumably representing the primary binding of substrate for enzymic hydroxylation (13, 14). Moreover, Schenkman et al. (15) demonstrated that the magnitude of the hexobarbital-induced spectral change was greater in liver microsomes isolated from male rats than in those from female rats and castration of male rats abolished this sex difference. These observations suggested that the sex difference in the hydroxylation of hexobarbital may be due to the difference in the substrate interaction with P-450. The purpose of the present communication is to investigate whether the decrease in the hexobarbital hydroxylation and aminopyrine N-demethylation in liver microsomes from morphine-treated and fasted male rats is related to an impairment of androgeninduced stimulation of the substrate-P-450 interaction.

Estrogen: direct effects on hypothalamus or pituitary in relation to pituitary weight changes.

The effects of implanted estrogen in the hypothalamus or pituitary on the weight of the pituitary gland was studied in intact and castrate female rats. Uterine weight increased as the amount of estrogen entering the circulation increased. Pituitary weight increase was a function of the closeness of the estrogen source to the pituitary. The greatest weight increase of the pituitary occurred when the estrogen was implanted directly into the gland.

Influence of reserpine on the pituitary content of melanocyte-stimulating hormone and on hypothalamic factors which affect its release.

SUMMARY The melanocyte-stimulating hormone (MSH) content of whole toad pituitary glands decreased upon treatment with reserpine. With daily injections the values remained low for 1 week but regained normal levels after 2 weeks in spite of an increased secretion of MSH as indicated by darkening of the skin. In rats a drop in pituitary MSH content also occurred after reserpine injection but normal values were found after 7 and 14 days of treatment. MSH-releasing factor found in stalk-median eminence tissue of normal male rats was not present in the reserpine-injected animals, but after 7 days of treatment an increase in MSH-release-inhibiting factor (MSH-R-IF) was demonstrated. MSH-R-IF was also found to have increased in female castrated rats after 2 days of treatment with reserpine. It is concluded that reserpine permits the secretion of pituitary MSH by blocking the release of MSH-R-IF, which accumulates in the hypothalamic neurones.