Non-castrated Rats Research Articles

Effect of testosterone on TSH and the development of thyroid tumors in rats induced by N-bis(2-Hydroxypropyl)-nitrosamine.

Effects of testosterone(17α-methyl testosterone) on the serum concentration of TSH(thyroid stimulating hormone) and on the development of thyroid tumors were studied in castrated rats pretreated with N-bis(2-hydroxypropyl) nitrosamine(DHPN). In addition, the dose dependency of testosterone on serum TSH concentration was assessed in castrated animals. The incidences of thyroid tumors induced by DHPN were 69% in non-castrated rats, 40% in castrated rats, 85% in castrated rats treated with 300 ppm testosterone diet, 21% in castrated rats treated with 1500 ppm testosterone diet, 20% and 6% in non-castrated rats treated with 300 ppm or 1500 ppm testosterone diet, respectively. The mean serum TSH concentration in castrated rats treated with DHPN and 1500 ppm testosterone diet showed a tendency of higher than the other group values, but not significantly, and the incidences of thyroid tumors did not demonstrate any clear relation to serum TSH concentration values. The serum TSH concentrations were 2.86±1.86 ng/ml in non-castrated rats and 2.80±0.16, 2.57±0.21, 2.43±0.92, 2.10±0.59, 3.09±1.03, 2.73±0.80 and 3.58±0.16 ng/ml in castrated rats fed on diets containing 0, 100, 300, 600, 900, 1200 and 1500 ppm testosterone, respectively. A dose dependent influence of testosterone on mean seminal vesicle and prostate weights but not on serum TSH concentration as well as thyroid weights was observed. The results, thus, suggest that higher concentration of testosterone without higher concentration of TSH inhibits the development of thyroid tumors in rats treated with DHPN.

Regional studies of brain biogenic amines in castrated muricidal and non-muricidal wistar rats

Castrated Wistar rats were isolated for 8 months and their muricidal behavior was investigated. Significantly fewer (35%) of such rats became muricidal (CM) compared to controls. The steady-state levels of 5-HT, 5-HIAA, DA, DOPAC, and NE, as well as the changes in synthesis or utilization rats of 5-HT and DA, were analyzed in 15 brain areas derived from CM rats and non-muricidal (CNM) control subjects. In CM rats, higher 5-HT levels were recorded in 5 areas considered to be involved in muricidal behavior: raphe, amygdala, olfactory tubercles, olfactory bulbs, and striatum. The alterations of serotonergic neurotransmission in castrated muricidal rats differ strikingly from those observed in non-castrated muricidal rats. An increase of 5-HT level and in the 5-HT synthesis index as well as a lower 5-HT utilization index were recorded in the raphe of CM rats. Our data suggest that the decrease of 5-HT levels generally said to be the main alteration in the muricidal rat's brain has to be reconsidered. Increased DA levels were observed in CM rats: raphe (50%), amygdala, olfactory tubercles, striatum, and septum (40%), while DA was decreased in cortical areas. There were slight increases of DA synthesis indices in the septum, olfactory tubercles and striatum with a decreased utilization index in the olfactory tubercles. Few alterations in NE levels were observed in CM rats: a decrease in the olfactory tubercles, superior colliculus, and striatum and an increase in temporal cortex. The monoaminergic alterations correlate with the modulation of muricidal behavior. Some areas (the olfactory tubercles, raphe, striatum, and temporal cortex) seem to be particularly involved. © 1992 Wiley-Liss, Inc.

Characterization of castration‐induced cell death in the rat prostate by immunohistochemical localization of cathepsin D

Activities of cathepsin D (EC 3.4.23.5) were determined in three lobes of the prostate during their involution by both biochemical and immunohistochemical procedures. The activity of cathepsin D in noncastrated rats was 0.9 +/- 0.2 (mean +/- SE) 5.7 +/- 0.6, and 13.1 +/- 0.8 units/mg protein for the ventral, lateral, dorsal lobes, respectively. Following castration, there was a significant increase in enzymatic activity in all three lobes within 2-3 days. In the ventral lobe, the activity peaked in 5 days to 6.2 +/- 0.9 units/mg protein and declined slightly thereafter. In the lateral and dorsal lobes, the activity remained elevated (14-20 units/mg protein) throughout the postcastration period studied. Immunohistochemical staining of cathepsin D was localized in the cytoplasm of prostatic epithelial cells as fine discrete lysosomal granules. These granules were larger and more abundant in the dorsal and lateral lobes than in the ventral lobe and were not detected in prostatic stromal cells and seldom in the luminal fluid. Castration resulted in an immediate increase in the size and number of these granules in the epithelial cells, followed by a sudden further increase in cathepsin D staining in some but not all epithelial cells. Lysosomal granules gradually coalesced in these cells to form large vacuoles that fit the characteristic description of apoptotic bodies. Finally, after day 7 postcastration, collapse and disintegration of the entire glandular structure was noted. Using this procedure to localize cathepsin D as a tool, we were able to follow the morphological events of prostatic cell death during castration-induced involution in the rat at the light microscopic level.

A possible explanation for the peripheral selectivity of a novel non-steroidal pure antiandrogen, Casodex (ICI 176,334).

The in vivo antiandrogenicity of Casodex has been confirmed and characterised. Androgen receptor (AR) binding assays of rat ventral prostate gland cytosols revealed a relative binding affinity (RBA) for the AR of 0.267 and a k1 of 1.25 x 10(-7) M for Casodex. In addition, the peripheral selectivity of Casodex relative to other non-steroidal antiandrogens was confirmed in that daily treatment of non-castrated rats with Casodex (25 mg kg-1) did not elicit any changes in serum LH and testosterone concentrations relative to vehicle-treated controls, whereas elevated serum LH and testosterone were observed in rats treated with flutamide (25 mg kg-1). The peripheral selectivity of Casodex in the intact male rat was related to the distribution of radiolabelled antiandrogen following intravenous injection. All tissues with the exception of the hypothalamus and cerebral cortex (CC) sequestered radioactivity such that the tissue:serum ratio (TSR) for the drug was greater than unity. In the testis, the TSR was less than unity 1 h after injection but approached unity 5 h after injection and was greater than unity 10 h after injection. This may be explained by the presence of a blood-testis barrier for the drug, resulting in delayed equilibration between the blood and testis tissue. By comparison, an order of magnitude lower amounts of radioactivity in the hypothalamus and CC were maintained for the 10 h period after injection. These data, together with known physicochemical properties of Casodex suggest that a blood-brain barrier exists for the drug which results in exclusion of this antiandrogen from central sites of androgen negative feedback and that this accounts for its peripherally selective antihormonal profile.

Proliferative Response of Rat Accessory Sex Organs to Dietary Sex Hormones After Castration or Initial Dietary Administration of Estrogen

The hormone dependent proliferative response of five accessory sex organs was examined subsequent to castration-induced or estrogen-induced atrophy using male six-week-old F344 rats. Three weeks after castration, rats were treated with dietary methyltestosterone (300ppm), ethinyl estradiol (0.75ppm) or methyltestosterone plus ethinyl estradiol for up to 17 days. Cell proliferation was assessed by 3H-thymidine incorporation as determined from labelling indices using autoradiography. The maximum labelling indices which appeared on days 2 or 3 of administration of methyltestosterone were 24.3, 8.4, 9.6, 21.6 and 13.7% for the ventral, lateral and dorsal prostate, seminal vesicle and coagulating glands, respectively. Combined methyltestosterone and ethinyl estradiol induced mild proliferative responses whereas ethinyl estradiol did not. In a further group of non-castrated rats which were pretreated with dietary ethinyl estradiol for three weeks and then given methyltestosterone, the maximum labelling indices were about 70 to 80% of that in castrated rats.

Rapid eye movement sleep deprivation affects sleep similarly in castrated and noncastrated rats

Twenty-four-hour recordings of electrophysiological correlates of the sleep-waking cycle in castrated and noncastrated Wistar rats were performed to validate the cuff pedestal technique in the deprivation of rapid eye movement sleep. An undisturbed pattern of sleep was found in both castrated and noncastrated rats when the cuffs were in the raised position. The lowering of the cuff for 4 days virtually abolished REMs in both groups of rats. During neither the dark nor the light period was there any difference between the castrated and noncastrated rats in the total amount of REMs rebound. The results accord with the data obtained by the conventional flowerpot procedure and show that castration does not influence the amount of REMs before, during, and after REMs deprivation in the rat. It is suggested that testicular testosterone, contrary to growth hormone, is not essential for the triggering of REMs sleep, although both have anabolic actions.

Effects of castration on adipose tissue growth and regrowth in the male rat

Adipose tissue has been found to regrow in the male rat following surgical removal (lipectomy) of inguinal subcutaneous depots, but the degree of regrowth has varied widely across experiments. It is possible that at least part of the disparity of previous findings occurred because of differences among the experiments in the testicular integrity of experimental animals. To address this possibility, the present study examined effects of castration on adipose tissue regrowth in rats treated either as weanlings or as young adults. Male Sprague-Dawley rats, at either 4 or 15 weeks of age, were subjected to one of four surgical procedures: bilateral lipectomy of the inguinal subcutaneous depots; castration; lipectomy and castration; or sham surgery. Adipose tissue mass and cellularity were examined 6 months later. Castration reduced body weight gain, but castrated rats achieved a higher ratio of adipose weight to body weight than noncastrated rats. In rats lipectomized but not castrated at 15 weeks of age, partial regeneration and a small increase in growth of noninguinal subcutaneous adipose tissue combined to produce substantial restoration of adipose mass. The same surgery in 4-week-old rats did not result in significant restoration because growth of noninguinal subcutaneous adipose tissue was reduced. In rats that were both castrated and lipectomized, regrowth of adipose tissue was substantial regardless of age at time of surgery. Thus, castration is seen to impede body weight gain while sparing ordinary growth of adipose tissue and facilitating regrowth of adipose tissue following lipectomy. Since adipose tissue regrowth varied with age only in noncastrated rats, it appears to be facilitated as well by testicular maturation.

Increased activity of plasminogen activators during involution of the rat ventral prostate.

Plasminogen activator was measured in the ventral prostates of non-castrated, castrated, and androgen-treated rats to determine whether changes in this activity correlated with the process of glandular involution. While the activity was very low in cytosolic extracts from the prostates of non-castrated rats, 2 days following castration the plasminogen activator activity increased in a near-linear fashion such that by day 7 it was 10-fold higher in terms of specific activity (per mg of protein) and cellular concentration (per mg of DNA). During this interval there was a rapid decrease in the cell population of the prostates. Treatment of the 7-day castrated rats with the potent androgen, dihydrotestosterone, both reduced the plasminogen activator activity and restored the cell number in a dose-related manner. Gel electrophoretic analysis revealed two major bands of plasminogen activator activity in the cytosolic extracts from 4- and 7-day castrated rats, plus additional minor bands in samples from 10- and 14-day castrated rats. Approx. 10% of the cellular concentration of plasminogen activator activity was recovered in association with an 18000g pellet fraction from the prostates; this fraction showed less heterogeneity of the plasminogen activator forms as observed by gel electrophoresis. Inhibitor studies indicated that the 18000g pellet fraction from the prostates of non-castrated rats possessed some plasminogen activator inhibitor activity, but the relative concentration of the inhibitor activity was small. We conclude that the involution of the prostate is probably associated with increased synthesis of plasminogen activators through a de-repression process which may involve loss of androgen receptors.

Acid ribonuclease in rat prostate during castration-induced involution.

The activity of acid ribonuclease (RNase) in the ventral prostate of rats was measured at different intervals following castration. The specific activity (units/mg protein) in the prostate of noncastrated rats was relatively low and increased gradually, though not significantly, through the first 3 days after castration. The enzyme activity reached a maximal level between Days 5 and 7 (P<0.01), then decreased gradually, and returned to the precastration level by Day 14. Subcutaneous injection of actinomycin D at 50 μg/day to castrated rats for 4 or 5 consecutive days retarded the rate of prostatic regression and was accompanied by a significant reduction in RNase activity in the prostate. These results indicate that elevated specific activity of RNase coincided with the period of rapid prostatic regression and that this elevation in enzyme activity can be suppressed by actinomycin D.

The oxytocin content of the hypothalamus and hypophysis in rats (author's transl)

One of the neurohypophyseal hormones, oxytocin, is known to have the biological activities of uterine contraction and milk ejection. Recently, serum levels of this hormone have been studied at several laboratories using the radioimmunoassay method, but reports dealing with the hypothalamic and pituitary regions where biosynthesis, storage and secretion take place, are rare. In this report, we will present the oxytocin content of the hypothalamus and hypophysis in Sprague-Dawley rats under various conditions, using a specific radioimmunoassay as previously reported.In brief, an anti-oxytocin serum was made by immunizing New Zealand white rabbits with an oxytocin-bovine serum albumin complex, using Freunds' complete adjuvant. The antiserum was diluted to 1 : 9,000 for use in the radioimmunoassay. The chrolamine T method was used with some modifications for labelling 125I to synthetic oxytocin, and the double antibody technique was used for separation of B/F hormones. In this assay system, the cross-reactivities of Arg-vasopressin and Lys-vasopressin were negligible, and the recovery rate was 102.5 ± 18.4% (mean ± S.D.). The values of the inter-assay coefficient of variation were less than 13.6%, and those of the intra-assay coefficient of variation were less than 7.5%.Under ether anesthesia, the rats were decapitated, and the hypothalamus and hypophysis were removed. Isolated tissues were homogenized immediately with 2 ml of a 0.05 M phosphate buffer pH 7.5 containing 0.1M EDTA and orthophenanthroline under ice cold conditions. After overnight extraction at 4°C, the homogenate was centrifuged at 4°C for 20 minutes at 3,000 rpm. A sample of the supernatant was measured by radioimmunoassay, and the following results were obtained : 1) Dilution curves of the hypothalamic and pituitary extracts showed a parallel relation with the standard curve of synthetic oxytocin, and the same elution profiles were observed between these extracts and the synthetic oxytocin on a Sephadex G-25 column chromatography (1.2 X 90cm). From these results, the immunoreactive substance presented in the hypothalamic and pituitary extracts was considered to be the same molecule as synthetic oxytocin.2) Oxytocin contents of the hypothalamus in male and female adult rats were 2.14 ± 0.59, 1.57 ± 0.32 mU/rat, respectively (mean ± S.D., n=10), and those of the hypophysis were 197.4 ± 30.7,169.2 ±18.2 mU/rat, respectively (mean ± S.D., n=10). There were no statistically significant differences between male and female rats in either tissue.3) Pituitary oxytocin was measurable as early as the 4th day after birth; it then showed a marked increase with growth. Sexuality caused no difference at any age tested.4) Hypothalamic oxytocin contents in pregnant rats showed a significant increase on the 3rd day of gestation (3.59 ± 0.79 mU/rat, n=10, p<0.001), as compared with nonpregnant levels, then gradually decreased towards the delivery date and reached minimum levels at the 20th day of gestation (1.29 ± 1.04 mU/rat, n=10). On the 4th day of postpartum, they increased again to the level of 2.75 ± 0.72 mU/rat (mean ± S.D., n=6), which was significantly higher than the nonpregnant levels (p<0.01).5) Pituitary oxytocin contents in pregnant rats increased continuously from the 3rd day of gestation (377 ± 47.5 mU/rat, n=10) to the delivery date and reached maximum levels at the 20th day of gestation (492 ± 95.7 mU/rat, n=10). On the 4th day of postpartum, they remained at still higher levels (269 ± 62.9 mU/rat, n=6) than the nonpregnant levels.6) Exogenously administered estradiol (50,100μg) or estradiol (100μg) with progesterone (25 mg) could not change the oxytocin content of either the hypothalamus or hypophysis in castrated or noncastrated rats.

Effect of sex hormones on plasma cholesterol in castrated and noncastrated male rats.

The administration of estradiol to both castrated and noncastrated male rats was associated with significantly increased plasma cholesterol levels as compared to controls, the estradiol in the noncastrated rats overriding the tendency of testosterone to lower plasma cholesterol.

Effects of sex hormones on the passive mechanical properties of rat carotid artery.

The effects of chronic administration of estradiol and testosterone on the passive mechanical properties of carotid arteries were determined using castrated and noncastrated male rats. Blood vessel segments were removed from the animals and mounted in a bath at &lt;i&gt;in vivo&lt;/i&gt; length. Measurements of external diameter and transmural pressure were made in response to inflation in the presence of smooth muscle inhibitors. These data were used to determine stress-strain relations and values of incremental elastic moduli for the various arteries. Relative to arteries from control animals, passive tangential stress-strain curves for arteries from estradiol treated rats were found to be shifted significantly to the right, i.e., to larger strains. Similar data for testosterone-treated arteries were shifted to the left, but the effect was smaller than the former one and only significant for values of tangential stress greater than 2 × 10&lt;sup&gt;–6&lt;/sup&gt; dyn/cm&lt;sup&gt;2&lt;/sup&gt;. At specific values of wall strain, estradiol produced a decrease in incremental elastic modulus relative to the arteries from control animals, while testosterone produced an increase in elastic modulus which was only significant for strains &gt; 0.7. Quantitatively, similar results were found in castrated and noncastrated rats. These results are consistent with the concepts that estradiol decreases the stiffness of arteries while testosterone to a smaller degree increases the latter through the effects of these steroids on connective tissue accumulation in the arterial wall.

Effect of sex hormones on blood pressure and vascular connective tissue in castrated and noncastrated male rats.

Aortic collagen and elastin were quantitated in three groups of castrated and two groups of noncastrated male rats treated by intramuscular injection for 3 wk with oil, testosterone, or estradiol. The greatest differences were found between the castrated rats receiving testosterone and those receiving estradiol, the estradiol-treated rats having significantly lower total collagen, percent collagen, total elastin, and collagen/elastin (C/E), and higher percent elastin than those rats receiving testosterone. In noncastrated rats, administration of estradiol resulted in significantly lower total collagen, percent collagen, total elastin, and C/E. Systolic blood pressure was highest in rats receiving testerone and lowest in rats receiving estradiol. It is concluded that 1) estradiol in the presence or absence of testosterone decreases total accumulation of vascular connective tissue and alters the proportions of collagen and elastin so that the vessel is more distensible, 2) testosterone has an opposite but less marked effect than estradiol on vascular connective tissue, and 3) estradiol and testosterone alter blood pressure in opposite directions in the male rat.

Anabolic effect of testosterone on the levator ani muscle of the rat.

1. The effect of testosterone on the weight and on the contractile, histochemical and electrophysiological properties of the levator ani (LA) muscle and other skeletal muscles of non-castrated rats was studied. 2. Administration of testosterone for 12 days resulted in a more than two-fold increase of the weight of LA muscle and in increased muscle fibre diameter. Contraction time and other parameters of the twitch were markedly shortened. Twitch tension as well as maximum tetanic tension increased, in the latter case up to 250% of the control values. A similar, but less pronounced anabolic effect on the LA muscle was also observed after 30 days of testosterone administration. 3. No change in the muscle fibre pattern of ATP-ase activity was found in the LA muscle after long-term testosterone treatment. 4. Resting membrane potential of LA muscle fibres increased after 30 days of testosterone administration from 74–83 mV. Increase of resting potential was blocked only partially by 2×10−5 M ouabain in vitro. 5. LA muscle fibres from testosterone treated rats showed a 60% increase of input membrane resistance and a lower rate of sodium permeability activation during action potential generation. 6. Frequency of miniature end-plate potentials in LA muscles of rats treated for 12 and 30 days decreased to about one third of control values. 7. No similar changes of weight, contraction and electrophysiological properties were found in the soleus, extensor digitorum longus and diaphragm muscles of rats treated with testosterone for 12 and 30 days respectively.

Acid phosphatases: androgen dependent markers of rat prostate.

Our investigations on acid phosphatase (AP) were aimed at finding a biochemical assay marker for androgen actions in the rat prostrate. We quantitatively examined the effects of l-tartrate or formaldehyde on AP activity in tissue filtrates from nine adult male rat tissues, plasma and hemolysed red blood cells (HRBC). There was significant inhibition of AP activity in all instances with the exception of HRBC with tartrate. The prostate inhibition results were not different from those for seminal vesicles and adrenals but were different from the other tissues studied. Ten days following castration the inhibition by tartrate was less in all tissues studied except plasma and HRBC; the formaldehyde inhibition percentages were not altered.

Effects of castration, testosterone and immobilization on the activities of choline acetyltransferase and cholinesterase in rat limb muscles

Thirteen months after castration of male rats the weight of their soleus muscles was lowered to 82% and their choline acetyltransferase (ChAc) activity to 83% of control values. The administration of testosterone lasting 5 weeks increased the weight of the soleus muscles of castrated animals by 19% and their ChAc activity by 37%. Changes in the activity of cholinesterase occurring after castration and testosterone treatment were not statistically significant. It is assumed that the effect of testosterone on the activity of ChAc was mainly due to an increase in the functional activity of the motoneurones innervating the muscle. Rapid developmental increase of ChAc activity was observed in the muscles of intact rats between the age of 48 and 82 days. During this period of development the activity of ChAc rose faster than the weight of the muscles. Testosterone had no effect on the weight and ChAc activity of the soleus and extensor digitorum longus muscles of non-castrated rats after 1 week's administration; after 5 weeks' administration the weight of the muscles and their ChAc activity were diminished. After the soleus muscles of non-castrated rats had been immobilized for 10 days, their ChAc activity was 56% and their weight 51% of control values. The administration of testosterone did not alter the effect of immobilization on the ChAc and weight of the muscle.

Testosterone-induced changes of choline acetyl-transferase and cholinesterase activities in rat levator ani muscle

One year after castration the activities of choline acetyltransferase (ChAc) and of cholinesterase (ChE) in the levator ani (LA) muscle of male rats were lowered by 42 and 79% respectively. The weight of the muscle corresponded to 15% of control values. These changes were not accompanied by a decrease in the number of the muscle fibres. Treatment with testosterone rapidly increased the activity of ChAc and the weight of the muscle near to control values; the restoration of ChE was less complete. Testosterone produced an increase in the size of the muscle fibres and increased the histochemically observed activity of ChE in the postsynaptic part of the motor end-plates. In non-castrated rats the administration of testosterone increased the weight of the LA muscle, but was not accompanied by an increase of ChAc above control values. Changes in the total activity of ChAc in the muscle induced by castration and by testosterone probably reflect changes in the content of enzyme in individual nerve terminals or in the number of nerve terminals per muscle end-plate. Whereas the testosterone-induced changes in the weight and ChE activity of the muscle are due to a direct action of testosterone on the muscle, the increase in the activity of ChAc is interpreted as a consequence of a retrograde effect of the LA muscle fibres endowed with specific receptors to androgens on their motoneurons and possibly also of increased impulse activity of the corresponding motoneurons.

Propagation and hormone production by human normal and malignant trophoblast in rats: II. Choriocarcinoma in Millipore diffusion chambers in rats

Human choriocarcinoma, hamster cheek pouch transplants from the WO line, was transferable subcutaneously to Millipore filter (MF) chambers in rats. Independent of sex and age, the choriocarcinoma cells grew on the inner surface of the filters and produced human chorionic gonadotropin hormone as evidenced by gonadotropic stimulation of adult and immature rats of both sexes. In ovariectomized females, there was no alteration of the oviducts and uteri, but the growth pattern and morphology of the cells in the MF chambers was the same as in noncastrated rats. This transplantation method provides the possibility of a combined tissue-culture and hormone-assay method.

SOME HORMONAL PROPERTIES OFMORUS ALBALEAVES AS EXAMINED BY FEEDING EXPERIMENTS

Mulberry leaves fed to rats accelerated the growth of immature rats and significantly increased their body weight. In immature noncastrated rats they increased the seminal vesicle weights to a great extent. Moreover mulberry leaves fed to experimentally hyperglycemie rats, lowered the blood sugar. It seems possible therefore that Morus alba leaves are of. growth–hormone like, testosterone–like, and insulin like activities.

Mitotic activity of mammary gland tissues after trauma in noncastrated and castrated rats

Dynamics of mitotic activity of the tissue cells of the mammary gland was studied in the process of posttraumatic regeneration in the parous albino rats (44 noncastrated and 37 castrated ones). These experimental studies demonstrated that the influence of ovarian hormones, which had a distinct effect on the mitotic regime of the epithelial cells in the uninjured mammary gland, became of secondary importance in the course of the changes developing after its injury. Local tissue are prominent here.