Castrated Male Mice Research Articles

Castration of male mice by a synthetic LH-RH-MDP conjugate administered in saline

Castration of male mice by a synthetic LH-RH-MDP conjugate administered in saline

Testosterone acts as a prohormone to stimulate male copulatory behavior in male deer mice (peromyscus maniculatus bairdi).

In order to determine the importance of reduced and aromatized metabolites of testosterone for male sexual behavior in Peromyscus maniculatus bairdi, castrated males were treated with 5 alpha-reductase and aromatase inhibitors. In the first experiment, testosterone propionate (TP) activation of male copulatory behavior was blocked by the administration of the 5 alpha-reductase inhibitor 4-androsten-3-one-17-beta carboxylic acid (17 beta C). These treatments also prevented TP stimulation of seminal vesicles and ventral prostate gland weight. The inhibitory effects of 17 beta C were specific to testosterone, since 17 beta C did not prevent dihydrotestosterone propionate (DHTP) induction of male sexual behavior or seminal vesicles and ventral prostate gland weight increases. In the second experiment, TP activation of male copulatory behavior was prevented by the administration of the aromatase inhibitor 1,4,6-androstatriene-3,17-dione (ATD). The ATD did not interfere with DHTP activation of male reproductive behavior. Also, TP and DHTP stimulation of accessory sex organ weight was not blocked by ATD. On the basis of these data, it is suggested that metabolism of testosterone to both 5 alpha-reduced androgens and estrogens is obligatory for testosterone to reliably stimulate male sexual behavior in castrated male deer mice.

Androgens and the thymus.

Tritiated dihydrotestosterone ([3H]DHT) binds with high affinity to a single class of limited capacity sites in cytoplasmic preparations from rat and mouse thymus glands. The affinity of these bindings sites for [3H]DHT (Kd at 4 C, 1 nM) and their specificity [methyltrienolone (R1881) greater than DHT greater than testosterone much greater than estradiol = progesterone greater than promegesterone (R5020) much greater than aldosterone = cortisol] are characteristic of androgen receptors in classical target tissues. The affinity of thymic androgen receptors for [3H]DHT did not differ with age, sex, or species; the concentration of androgen receptors was higher in males than females and higher in older compared with younger animals. Castration of male mice was followed by marked (approximately 2-fold) thymic hypertrophy; the concentration of androgen receptors fell to approximately one third the control value as a function of organ weight, but did not change significantly as a function of thymic DNA. Chronic administration of DHT (200 micrograms/day) did not alter thymic weight or androgen receptor levels in intact male mice. In castrate mice, however, the administration of 200 micrograms DHT completely abolished the thymic hypertrophy otherwise seen post castration and significantly increased the total thymic content of androgen receptor. The administration of dexamethasone (0.6-60 micrograms/day for 5 days) caused dose-related thymolysis; on a weight basis, the concentration of androgen receptor rose markedly, suggesting that androgen receptors are confined to the subpopulation of thymocytes relatively resistant to glucocorticoids.

Reduction of fertilizing capacity of epididymal spermatozoa by 5 alpha-steroid reductase inhibitors.

Antifertility effects on epididymal spermatozoa were tested by administration of 5α-reductase inhibitors to androgen substituted adult castrated male mice. The inhibitors depressed the in vitro fertilizing ability and in vitro blastocyst development in testosterone substituted castrated male mice.

Mammary tumors from gr mice contain more than one population of mouse mammary tumor virus-infected cells

Spontaneous mammary tumors in the GR mouse strain contain several acquired copies of mouse mammary tumor virus (MMTV) DNA that are not present in normal organ DNA and that are detectable by restriction endonuclease analysis and the Southern blotting procedure. Hormone-responsive and -independent cell populations were selected from spontaneous GR mammary tumors by grafting the tumors in castrated male GR mice in the presence and absence of female sex hormones. Analysis of the acquired MMTV DNA copies revealed differences between hormone-responsive and -independent cells derived from the same tumor; however, specific MMTV DNA fragments could not necessarily be correlated with the hormone responsiveness of the tumor. In some cases more than one proviral pattern could be detected for both hormone-responsive and hormone-independent cells. These results suggest that spontaneous GR mammary tumors are made up of more than one population of hormone-responsive and -independent cells which can be distinguished by their MMTV-specific proviral restriction fragments.

Learned aversion to the odors of male mice: Effects on agonistic behavior

Intact and castrate male mice were subjected to avoidance training using the urine of intact and castrate donors as stimuli. Both intacts and castrates learned to discriminate between the odors, with such learning modifying their spontaneous responsiveness to such odors in an open field in the predicted direction. This result suggests that the previously reported lack of responsiveness in castrates [15] was not due to a simple sensory deficit. A second experiment determined that increasing the relative aversion exhibited by intact subjects to the odors of intact donors also increased the subject's aggression directed at a castrate opponent swabbed with such odors. Modification of castrates' responsivity had no such effect on their aggression. This indicated that replacing the responsivity to aggression-related odors which is lost following castration, is not equivalent to replacing the hormone. The results are discussed with regard to possible hormonal effects on the social significance of olfactory cues to the recipient animal.

Effect of chronic castration on testosterone and estrogen binding to mouse kidney.

The effects of prolonged castration on cytosol and nuclear androgen binding were studied in an attempt to explain the apparent resistance to androgen action observed in chronic castrates of various species. The mouse kidney was chosen for study because it is an androgen responsive tissue, extensively characterized, which does not atrophy following castration. Presence of estradiol receptors in the same tissue permitted us to compare the effects of castration on androgen and estrogen binding. Specific nuclear uptake of [3H]-testosterone was not different in kidney minces from 24 h, 1 week and 2 week castrate male mice. Four and 6 weeks after castration, however, a 2-fold increase in the number of available cytosol testosterone binding sites was observed. No changes in estrogen binding were found in chronically castrated male mice. Treatment with the nonaromatizable androgen, dihydrotestosterone, prevented these changes in androgen binding, whereas treatment with estradiol did not. These data indicate that androgen exposure is important in the regulation of androgen receptor activity in male mouse kidney. Whether the differences observed in prolonged castrates result from changes in receptor synthesis, processing or metabolism remains to be determined.

The effect of the non-steroidal antiandrogen flutamide on neural receptor binding of testosterone and intermale aggressive behavior in mice

(1) The effect of the potent non-steroidal antiandrogen, flutamide, on neural receptor binding of testosterone and intermale aggressive behavior was studied. (2) The main findings were: (i) flutamide exhibited a high affinity in vivo for androgen binding macromolecules within the hypothalamus and preoptic-septal region of the brain. This finding was in contrast with the negligible testosterone receptor antagonist activity of flutamide in vitro; (ii) flutamide, given in conjunction with testosterone to castrated male mice, had no suppressive effect on the restoration of intermale aggressive behavior. (3) The relative effectiveness of flutamide in suppressing [ 3H]-testosterone binding in vivo and in vitro is interpreted to mean that flutamide exerts its antiandrogenic effect within the central nervous system, at least in part, by means of its conversion in vivo to an active metabolite. (4) The results of the study on the effects of flutamide on aggressive behavior are interpreted in support of the aromatization hypothesis for the mechanism of action of testosterone on the central nervous system.

A single injection of 17β-estradiol facilitates sexual behavior in castrated male mice

Sexual behavior was assessed in castrated adult CD-1 male mice given exogenous steroids under various treatment regimens. Castrated mice maintained on 20 μg testosterone (T) daily for 1 week, but given 250 μg testosterone propionate (TP) on the day of testing showed higher levels of copulatory activity than intact mice or the males receiving an additional dose of 20 μg T on the test day, although plasma testosterone levels were not different at the time of behavioral testing. Castrated males given 50, 125, or 250 μg TP for 1 week including the day of testing showed higher levels of sexual behavior than males receiving the same doses of TP only once, on the test day. A single injection of 17β-estradiol (E 2) completely restored the male copulatory pattern, including ejaculation, in castrated mice under every condition examined. Testosterone and dihydrotestosterone (DHT) were less effective than E 2, as was the combination of E 2 and DHT. The relative efficacy of a single dose of T, DHT, and E 2 plus DHT was dependent upon factors such as the delay between steroid administration and testing, as well as whether or not the castrated mice received androgen replacement prior to testing. Estradiol benzoate (E 2B) was not capable of restoring sexual behavior in castrated mice in this study. The comparison of results obtained with TP, T, E 2, and E 2B suggests that an appreciable, but not necessarily sustained, elevation of E 2 levels in the brain may be critical in the facilitation of male copulatory behavior in mice.

Effects of treatments with testosterone and oestradiol on the attack directed by groups of gonadectomized male and female mice towards lactating intruders

Both intact female and castrated male group-housed mice direct high incidences of attack to lactating female intruders. This type of attack is not shown by intact, group-housed males, suggesting that androgens inhibit this behaviour. It was consequently thought to be of interest to assess the influences of testosterone (T) and oestradiol (O) on the attack directed by gonadectomized groups of male and female mice towards lactating female intruders. Compared with cholesterol implanted counterparts, T implanted females and both T and O implanted males evidenced reduced attack on lactating intruders. These results confirm that sex steroids inhibit this form of attack. It should also be noted that fighting was evident between members of groups when the intruders were present but increased intra-group fighting does not appear to account for the steroid-mediated suppression of attack on lactating intruders.

The effect of the antiestrogen CI-628 on androgen-induced aggressive behavior in castrated male mice

This study was conducted to determine whether or not the antiestrogen CI-628 would block testosterone-maintained fighting in castrated male mice. TO strain adult male mice were castrated and injected s.c. every day for 14 days with either (1) 75 μg testosterone or (2) 75 μg testosterone and 1 mg CI-628, and in addition 1 mg of CI-628 6 hr prior to each injection of antiestrogen and androgen. Vehicle-injected, castrated, and CI-628-injected animals were employed as controls. Testosterone-maintained intermale aggressive behavior was blocked by the antiestrogen CI-628. This study provides support for the hypothesis that testosterone exerts its effects on the central nervous elements involved in the control of aggressive behavior by its aromatization to estrogenic metabolites.

Failure of induced functional activity and cell deficit to restore preoperative cell number in paired accessory sex glands following unilateral ablation in castrated male mice

AbstractThe proliferative response to testosterone propionate in the accessory sex glands of castrated male Balb/c mice has been studied when the glands were intact, and when the glands were unilaterally ablated. The response was analysed by serial measurements of DNA and weight, and by 3H‐thymidine labelling techniques. When stimulation is started at three days after castration, most seminal vesicle cells of intact glands carry out DNA synthesis to restore preoperative cell number, whilst the smaller reduction in coagulating gland DNA prior to stimulation results in only about 30% of cells carrying out DNA synthesis to restore former levels. In the unilaterally ablated glands of the 3‐day castrates, the cell kinetics of stimulated DNA synthesis and the net accumulation of DNA were similar to the intact glands. A much larger deficit in DNA was apparent in all glands of unstimulated 14‐day castrates, resulting in a much larger net accumulation of DNA following testosterone stimulation. No difference between intact and unilaterally ablated glands was detected, and despite prolonged stimulation DNA levels did not exceed preoperative values. Unlike gland DNA levels, gland weights exceeded those of non‐castrated mice following prolonged stimulation. However, unilateral ablation did not result in further weight increases in the remaining contralateral glands. It is concluded that the absence of the contralateral sex gland is not detected by the remaining partner during the response to the combined stimuli of a reduction in cell number (resulting from androgen deprivation), and testosterone propionate administration.

A Mechanism for the Antiandrogenic Action of Medrogestone1

The antiandrogenic action of the progestin, medrogestone, in rat and man was ascribed initially to an alteration in testosterone biosynthesis and metabolism rather than to a direct effect via the androgen receptor. However in previous studies we had found that many progestins bind directly to the androgen receptor in mouse kidney and have androgenic, antiandrogenic or synandrogenic activity. It was thus of interest to characterize the effect of medrogestone on androgen action in mouse kidney and to determine if this progestin was bound by the androgen receptor. Medrogestone inhibited the growth response and increase in the activity of β‐glucuronidase and alcohol dehydrogenase induced by exogenous testosterone in the kidney of castrate male mice. It also inhibited the growth response of preputial gland and seminal vesicle. This progestin suppressed nuclear uptake of [3H]androgen in kidney, seminal vesicle and submaxillary gland following the in vivo administration of [3H] testosterone. In vitro, medrogestone directly inhibited [3H]testosterone binding by the androgen receptor in mouse kidney cytosol. To insure that these effects were not unique to the mouse, similar studies were repeated in the rat. In this species, medrogestone also inhibited nuclear androgen accumulation and receptor binding in prostate and seminal vesicle. Thus medrogestone can affect androgen action directly via binding to the androgen receptor in the mouse and rat.

Sex difference in L-glutamine D-fructose-6-phosphate aminotransferase activity of mouse submandibular gland

L-Glutamine D-fructose-6-phosphate aminotransferase (2-amino-2-deoxy- D-glucose-6-phosphate ketol-isomerase (amino transferring), EC 5.3.1.19) activities in the three main salivary glands of male and female mice were measured. It was found that the activity in the submandibular gland was about 10 times more in females than in males, whereas the activities in the sublingual and parotid glands of males and females were similar. The activity in the submandibular gland of female mice was not affected appreciably by ovariectomy but it decreased to the level in males on injection of testosterone. The activity in males was not affected appreciably by injection of progesterone or 17β-estradiol, but it increased to the level in females after castration. The increased acitivity in castrated male mice was decreased again to the normal level by testosterone injection. Thus, this sex difference is caused by androgen, not by female hormones. On the basis of in vivo experiments using actinomycin D, it was suggested that testosterone produced an “enzyme inhibitor”, which suppressed the enzyme activity in the submandibular glands of androgen-rich animals.

Physical properties of androgen receptors in brain cytosol from normal and testicular feminized (Tfm/y hermaphrodite) mice.

Our previous results had shown that brain cytosol from androgen-insensitive, testicular feminized (Tfm/y) mice contains a reduced amount (20-25%) of androgen receptors, compared to normal female or castrated male mice, with unchanged affinity for dihydrotestosterone (DHT). We have now used various physicochemical techniques to ask whether there is a qualitative difference between these residual receptors and those in normal brain. When androgen receptors labeled with [3H]DHT in concentrated crude brain cytosol were analyzed by density gradient centrifugation and Agarose gel filtration in buffers containing 0.4-0.5 M KCl, Tfm/y receptors appeared to be smaller and more symmetrical than those from their normal siblings; however, if wild-type receptors were partially purified or prepared from slightly more dilute homogenates, their properties approached those of Tfm/y receptors in crude cytosol. This suggested that receptor molecules in concentrated cytosol from normal mice were aggregated to a greater extent than were those from the mutant. The molecular weight (54,000) and axial ratio (3:1 for prolate or oblate ellipsoid) calculated for Tfm/y receptors, therefore, may provide estimates for normal receptors as well. By DNA-cellulose chromatography, Tfm/y and female cytosol receptors were both resolved into two components, eluting at about 0.13-0.15 and 0.22-0.24 M NaCl. On DEAE-cellulose columns, both were eluted as a single major species at approximately 0.08-0.10 M KCl. Thus, excluding their state of aggregation, presumably resulting from their different concentrations in crude cytosol, Tfm/y and normal receptors were substantially identical as concerns the physical parameters examined in this study.

Effects of testosterone upon feeding in male mice

Food intake and pattern of feeding were recorded in intact and castrate male mice. Intact adults are larger and eat more than pre-pubertal castrates, due to rapid growth at puberty. Castration in adulthood has little effect upon food intake, but feeding becomes concentrated into large, infrequent meals. The change in meal size appears primary and is reversed by testosterone. During rapid growth at puberty, both meal size and frequency are high. It is suggested that testosterone affects feeding in two ways. Rapid weight gain at puberty leads to high meal size and frequency, but in adulthood meal size is depressed. Meal size in adults is further depressed by stimuli from females, and it is argued in this case that testosterone affects a general mechanism for altering feeding when other activities have a high priority.

Progesterone: Effects on investigatory preferences, aggression, and olfaction in orchidectomized, testosterone-treated mice

Effects of testosterone alone and in combination with progesterone on investigatory preferences, fighting, and olfaction in castrated male CD-1 mice were assessed. Testosterone stimulated both preferences for intact male mice and aggression, while progesterone reduced attention to intact male mice and reduced aggression. Accessory sex tissue weights and olfactory ability were unaffected by progesterone treatment. The possibility of a centrally located antagonism of testosterone by progesterone exclusive of the olfactory system is discussed.

Inhibition of testosterone 5alpha-reductase activity and growth of accessory sex tissues in castrated male mice.

The steroid 4-androsten-3-one-17beta-carboxylic acid (17betaC) reduced the growth-promoting actions of testosterone, but not those of DHT in accessory sex tissues of castrated mice. The 5alpha-reduction of testosterone to DHT in these tissues was also reduced by 17betaC treatment, suggesting that DHT formation is a required step in the mechanism of action of testosterone.

Dominant-subordinate relationships in castrated male mice bearing testosterone implants.

This study tested the hypothesis that subordination in male mice is directly related to reduced levels of circulating testosterone. Several measures of aggressiveness and submissiveness were quantified during or after daily encounters between pairs of intact males, castrated males, or castrated males bearing testosterone implants that were designed to maintain plasma testosterone titers at levels slightly above those of intact males. Behavioral measures included the total amount of fighting that resulted from daily pairing, the tendency for subordinates to fight back, and the relative frequency of urine marking. In all these regards, pairs of testosterone-implanted, castrated males formed dominant-subordinate relationships that involved apparently normal submissive behavior (compared to intact control pairs). Thus we found no evidence that submissive behavior was directly related to a reduction in circulating testosterone titers.

Effect of Testosterone on the Amount of Serous-like Granules in Convoluted Tubular Cells of Mouse Submandibular Glands

The effect of testosterone on the amount of granules present in convoluted tubular cells of the submadibular glands of mice was studied by the following two methods; 1) an immunochemical method using antiserum specific to the granular components, and 2) a histographic method. The results obtained by these two methods were in agreement. The amounts of the granules in normal female and castrated male mice were one-tenth to one-twentieth of that in normal male mice. When male mice were castrated, the amount of granules decreased, reaching a minimum 20 days after the aperation the injection of the male hormone, testosterone, into castrated male mice caused an increase in the amount of granules; this increase reached a maximum 15 days after the injection. The increase of granules caused by testosterone injection was almost completely prevented by inhibitors of protein synthesis, actinomycin D and puromycin. This suggests that protein synthesis was indispensable to the increase in the amount of granules. In male mice, the injection of female hormones scarcely affected the amount of granules. Kinetic analysis of the decrease and increase of granules on castration and testosterone injection suggested that the male hormone stimulated granule synthesis, but it hardly influenced the loss of granules.