Fine structural studies of rat seminal vesicle in castrated and intact animals following estrogen treatment

The effect of estradiol and/or testosterone upon secretion by seminal vesicle in castrated and intact rats was assessed in young adult Sprague-Dawley rats, using light microscopy (LM), transmission (TEM) and scanning (SEM) electron microscopy. Hormones were injected daily for ten days beginning ten days after castrations were performed. The normal rat seminal vesicle, as revealed by SEM, was characterized by a large saccular lumen with highly folded walls. Cell surfaces were covered with microvilli, or occasionally displayed a protruding, ruffled surface, sparsely covered with short microvilli. Cytology was normal in testosterone-treated animals. Estradiol treatment of castrated animals stimulated secretion by seminal vesicle epithelial cells as evidenced by the presence of normal secretory bodies, the presence of RER, and moderately hypertrophied Golgi complexes. These glands were not heavier than were glands from castrated, untreated animals, although the epithelial cells were significantly taller. Secretion was maintained in intact animals treated with estradiol, although glands were smaller and epithelial height was reduced. Estradiol and testosterone treatment in combination did not appear to have an additive effect on secretion, weight of the gland, or epithelial height. The following results support the hypothesis that estrogen-induced prolactin synthesis and release may be involved in the mechanism by which estradiol effected stimulation of seminal vesicle epithelium. Prolactin-treated, castrated animals exhibited focal areas of stimulated epithelium. In hypophysectomized animals (untreated controls), the seminal vesicle epithelium retained some secretory bodies and secretory fluid in the glandular lumen; epithelial height was taller than that in castrated controls. Estrogen treatment reduced the epithelial height to that of castrated controls; there was no evidence of secretion. This suggests that in the absence of anterior pituitary hormones, including prolactin, the stimulatory effect of estradiol on seminal vesicle epithelium was nullified. In adrenalectomized/castrated animals, estradiol treatment stimulated secretion in seminal vesicle epithelium just as in non-adrenalectomized/castrated animals. This indicates that the adrenal gland plays a non-essential role in the action of estrogen on seminal vesicle epithelium.     

Testosterone elevates pituitary prolactin content of long-term castrated rats under constant but not under periodic light

Intact and castrated male rats were kept for 1 week under constant or periodic light, during which time they received daily injections of either vehicle or testosterone propionate (125 or 250 micrograms day-1). Immediately after the experiment the rats were decapitated and serum and pituitary prolactin were measured radioimmunologically. The pituitary prolactin content was decreased after castration in both lighting conditions. Daily testosterone injections elevated the pituitary prolactin content of the castrated rats to the level of the intact rats in constant-light conditions but were ineffective under periodic light. Serum prolactin concentrations were not affected by constant light. We have previously shown that testosterone decreases the serum luteinizing hormone concentration of castrated rats more effectively under constant than under periodic light. In the present study we showed that serum prolactin was not elevated in constant light and thus could not be the cause of increased testosterone sensitivity. We also showed that testosterone elevates the pituitary prolactin content of castrated rats under constant but not under periodic light. This finding gives further support to the hypothesis that constant light sensitizes the hypothalamo-pituitary axis of castrated male rats to the effects of testosterone.     

Lack of an inhibitory effect of hyperprolactinemia on androgen-dependent marking.

An experiment was performed to determine if hyperprolactinemia (chronically elevated serum prolactin levels), which inhibits testosterone-activated male sexual activity, also affects other androgen-dependent behaviors. Thus defecation and urine marking in response to a novel environment were examined in sham-operated and pituitary-grafted (hyperprolactinemic) male rats that had been castrated or castrated and given subcutaneous testosterone implants. Both castration and pituitary grafting significantly inhibited defecation, with the inhibitory effects of hyperprolactinemia being most pronounced in the castrated non-testosterone-treated animals. In contrast, castration significantly reduced the amount of urine marking observed, but pituitary grafting was without effect on this behavior. Thus, although hyperprolactinemia may inhibit sexual activity through an antagonism of the activational effects of testosterone, these results suggest that this effect is specific to sexual behavior and does not involve a more generalized inhibition of the effects of testosterone on androgen-dependent behaviors.     

Hypothalamic LHRH concentration decreases in intact but increases in castrated rats in constant light

In order to evaluate the effects of environmental lighting on the hypothalamic secretion of luteinizing hormone-releasing hormone (LHRH), intact and castrated male Wistar rats were kept either in periodic (LD) or in constant light (LL) for 1 week, and the hypothalamic LHRH concentration was measured by radioimmunoassay. In the intact rats the LHRH concentration was lower in constant than in periodic light (30 +/- 3 vs 61 +/- 3 pg mg-1, P less than 0.05), whereas in the castrated rats it was higher (16 +/- 2 vs 5 +/- 1 pg mg-1, P less than 0.05). The intact and castrated rats received melatonin (50 micrograms) or saline injections daily at either 09.00 h or 16.00 h in LD and LL for 1 week. In intact or castrated rats the melatonin injections were not able to prevent the change of hypothalamic LHRH in constant light. In castrated rats testosterone (125 or 250 micrograms) had no effect on the LHRH concentration under either lighting condition. The present results suggest that constant light reduces the synthesis of hypothalamic LHRH in intact rats but increases it in castrated rats. These effects of constant light do not appear to be related to melatonin.     

Synergistic effects of prolactin and testosterone in the restoration of rat prostatic epithelium following castration

Prolactin is known to enhance the uptake and metabolism of testosterone in male accessory sex organs and to increase the weight of accessory sex organs from castrated rats over those from controls treated with testosterone alone. The present study was directed toward defining fine structural changes detectable with scanning and transmission electron microscopy which might accompany such responses. Accordingly, rat ventral prostate gland was examined from castrated animals which had received testosterone propionate and ovine prolactin singly or together, or which had received vehicle only. Unoperated animals served as additional controls. Post-castration glandular atrophy was not influenced by prolactin treatment alone. Testosterone restored epithelial height, secretory product, Golgi complexes and rough endoplasmic reticulum, such that cellular and tissue morphology was generally indistinguishable from that of unoperated controls. Prostatic tissue from animals given testosterone and prolactin simultaneously exhibited pleomorphic, cytoplasmic apical projections which extended into the acinar lumen. Transmission electron microscopy demonstrated that these blebs were devoid of organelles and microvilli; scanning electron microscopy revealed that the blebs were highly wrinkled and more numerous than were the projections observed in tissue from animals treated with testosterone alone, or in tissue from unoperated controls. It is suggested that such blebbing may reflect enhanced apocrine secretion in prolactin/testosterone stimulated restoration of the prostate gland in castrated rats.     

Role of insulin and testosterone in prostatic growth: Who is doing what?

Previous studies have demonstrated increased incidence of benign prostatic hyperplasia in insulin-resistant individuals. In addition to androgens, prostatic growth is sensitive to the peptide growth factors including insulin. Experimental studies employing intervention of selective β-cell toxin streptozotocin and castration suggest that depletion of either insulin or testosterone results in the severe prostatic atrophy (>80%). Exogenous testosterone and diet-induced experimental hyperinsulinemia induces prostatic enlargement in rats. Further, hyperinsulinemia sensitizes prostate towards the growth promoting effect of testosterone, and testosterone augments prostatic growth even in the hypoinsulinemic rats. However, in castrated rats diet-induced hyperinsulinemia fails to promote prostatic growth. Based on these evidences it is hypothesized that in the presence of testosterone insulin plays an important role in the prostatic growth. The epidemiological reports witnessing increased incidences of prostatic enlargement in men with metabolic syndrome, which are known to have increased level of insulin, provides a validating clue to the hypothesis. Further, the hypothesis suggests that targeting insulin signaling pathway could be a new objective for the treatment of prostatic enlargement.     

Effect of testosterone administration on the epiphyseal cartilage of hypophysectomized rats.

For the study of the mechanism of action of testosterone histological, carbohydrate-, and enzyme histochemical investigations were carried out on the epiphyseal cartilage of (1) hypophysectomized rats treated with testosterone [(THX group); (2)] hypophysectomized rats without hormonal treatment (HX group), and (3) intact, untreated control rats. The results were compared with the data obtained in a previous experiment in which intact rats were treated with testosterone (T group). The experiments showed that testosterone exerts a peripheral direct effect on the enzyme system of the epiphyseal cartilage cells by changing their metabolism in the direction of early ageing. This effect elicits characteristic changes in enzyme activity of the cartilage cells if normal hypophyseal activity is present. Impairment and ossification of the epiphyseal cartilage ensue also in hypophysectomized animals treated with testosterone (THX), even faster than in animals subjected only to hypophysectomy (HX), but this process has lost its characteristic feature: the initial increase of enzyme activity in the cartilage cells. Thus, the presence or absence of the hypophysis only modifies the effect of testosterone. Presumably, the latter effect - though not asserting itself through the hypophysis - is highly dependent on hypophyseal activity, that is, on the presence of the hormones produced or mediated by the hypophysis. If testosterone does influence the secretion of these hormones and the activity of the hypothalamus, a central effect of testosterone seems also to be involved in the changes of the epiphyseal cartilage disc.     

Pituitary modulation of sulpiride action in the central nervous system of the rat

To verify whether the stimulation by sulpiride of hypothalamic adenylate cyclase was direct or mediated by release of pituitary hormones, the effect of sulpiride on female hypophysectomized rats was studied. In these animals sulpiride does not substantially modify hypothalamic adenylate cyclase, brain 3,4-dihydroxyphenylacetic acid (DOPAC) levels and serum prolactin concentrations. Chlorpromazine on the contrary inhibits hypothalamic adenylate cyclase activity and increases DOPAC levels both in intact and in hypophysectomized rats.

As sulpiride hardly crosses the blood-brain barrier, its action on pituitary with prolactin release seems to be essential to start the neurochemical phenomena in the central nervous system.     

Dependence of tonin activity in rat submaxillary gland on growth hormone and testosterone.

Tonin, an enzyme present in rat submaxillary gland, converts angiotensin I to angiotensin II and is able to form angiotensin II directly from renin substrates. This enzyme was previously shown to be different from renin, tissue isorenins, and angiotensin I converting enzyme. The specific activity of tonin in rat submaxillary gland increases with the age of the animal and is much higher in male than in female rats; this sex difference is apparent from 60 to 70 days of age. There is a sharp drop of tonin activity in hypophysectomized animals, whereas adrenalectomy, thyroidectomy, and gonadectomy have have little effect. The marked increase in tonin activity was observed in animals bearing MtT-F4 transplantable tumors known to produce ACTH, prolactin, and growth hormone. Tonin specific activity in hypophysectomized male rats is restored to control levels by combined treatment with growth hormone and testosterone. Prolactin alone or in combination with testosterone, as well as transplanted pituitaries, has no effect in hypophysectomized animals. There is a significant specific binding of 125I-labeled growth hormone to isolated membranes of rat submaxillary gland.     

Urinary testosterone levels in the male blind mole rat (Spalax ehrenbergi) affect female preference

This study investigated the sexual attraction of female blind mole rats to four groups of male mole rats: (a) intact males raised in captivity; (b) intact males trapped in the field; (c) captive males injected with testosterone; (d) captive castrated males. In the first part we measured blood testosterone, androstenedione, and dihydrotestosterone (DHT) levels, by radioimmunoassay; and urine testosterone levels, measured by GC-MS. The second part examined the relationship between urine testosterone levels in males and their attractiveness to females. Higher blood and urine testosterone levels were found in the field animals and in those injected with testosterone compared to captive intact or castrated animals: urine testosterone levels in the two other groups were not detectable. Blood androstenedione levels were also higher in the field animals and in those injected with testosterone compared to captive intact or castrated mole rats. Blood dihydrotestosterone levels were not detectable in all four experimental groups. Female mole rats chose to spend a longer period of time next to males with high blood and urine testosterone levels and high blood androstenedione levels than next to those with lower levels of these hormones. Because courtship and sexual behavior are influenced both by high levels of blood and urine testosterone and high levels of blood androstenedione, we suggest that the low levels of courtship and other sexual behavior in captive mole rats may be related to the lack of female attraction to these males, which display low levels of all three parameters.     

Influence of castration on isoprenaline-induced amylase release in parotid gland from male rats

The purpose of this study was to determine the influence of testosterone, the male sex hormone, on beta-adrenergic agonist-induced amylase secretion from rat parotid glands. Isoprenaline (isoproterenol)-induced amylase secretion was measured in vitro from the parotid glands of control and castrated rats with and without testosterone replacement. The isoprenaline-induced amylase release was reduced in parotid glands from castrated rats compared to controls. The reduction of amylase release by isoprenaline in parotid glands of castrated rats, could be reversed by administration of testosterone. Furthermore, beta-adrenergic receptor density and the level of isoprenaline-evoked cAMP in parotid glands from castrated rats was lower compared to intact rats. Using SQ-22536 (an adenylyl cyclase inhibitor), dibutyryl cAMP (a cAMP analogue) and verapamil (a calcium channel blocker), we conclude that the impairment of amylase release from parotid glands after castration was not related to either adenylyl cyclase activity or cAMP accumulation. Amylase release from the parotid glands of castrated rats appears to be mediated by an increase in calcium ion influx.     

Failure of ACTH to prolong extinction of a conditioned taste aversion in the absence of the testes

Both corticotropin (ACTH) and testosterone prolong the extinction of a conditioned taste aversion in water-deprived intact male rats. An investigation was made to determine whether ACTH affects extinction in the absence of the testes and also to determine the effect of ACTH on serum testosterone levels. Water-deprived intact males showed prolonged extinction after ACTH injections; water-deprived gonadectomized males and intact females did not. All three of these groups showed elevated testosterone levels after ACTH administration, but testosterone levels were higher in the intact males than in the gonadectomized males or intact females. These results clearly show that in the absence of the testes ACTH is unable to prolong extinction. It is proposed that the increased level of testosterone following ACTH injection in water-deprived intact males is responsible for the prolonged extinction of a conditioned taste aversion. Although testosterone levels may increase in females and castrated males following ACTH injection, the increase is not sufficient to prolong extinction in these water-deprived animals.     

The role of 5alpha-reductase activity in sexual behaviors of the green anole lizard

Both testosterone (T) and its metabolite, 5alpha-dihydrotestosterone (DHT), can facilitate male sexual behavior in the lizard Anolis carolinensis. The present study addresses the role of DHT synthesis in regulating male sexual behavior by inhibiting 5alpha-reductase, the enzyme that converts T into DHT. In two separate experiments (one replacement and one maintenance paradigm), breeding adult males were castrated and implanted with capsules of T, DHT, or a control capsule (blank, BL). The animals were then injected with the 5alpha-reductase inhibitor, FCE, or with steroid suspending vehicle (SSV) as a control. Both experiments produced similar results. Overall, T was most effective in eliciting courtship and copulatory behaviors above control levels. In both experiments, treatment with FCE attenuated the T-induced effects on courtship behavior, whereas the inhibition of 5alpha-reductase activity resulted in modest and inconsistent effects on the latency to intromission and the proportion of copulating males. DHT treatment did not significantly increase courtship or copulatory behaviors above control levels. These results suggest that (a) 5alpha-reductase activity is necessary but that DHT alone is not sufficient for stimulating courtship in male A. carolinensis; and (b) courtship behavior is more sensitive than copulatory behavior to the activity of the androgen metabolizing enzyme.     

Effect of Thymomodulin on luteinizing hormone, prolactin and testosterone in male rats.

The effect of Thymomodulin (TMD), a calf thymus derivative, on luteinizing hormone, prolactin and testosterone was studied in male rats after acute and chronic treatment. The results showed that the stimulatory action on prolactin and testosterone secretion after acute (prolactin) or one month chronic (testosterone) treatments completely vanished during six month chronic administration. No effect was observed on luteinizing hormone after acute or chronic treatment.     

Androgen-mediated sex differences of cardiovascular responses in rats

Sex differences in the systemic depressor response to arachidonic acid (50 or 150 microgram/kg iv) were observed in intact and castrated anesthetized Sprague-Dawley rats. The rank order of responsiveness was: castrate males, castrate females, females, males; all four groups were significantly different (P less than 0.05) at the higher dose. Castrated males pretreated with testosterone (1 mg/kg sc) 5 or 7 days previously gave a response at the higher arachidonate dose levels that was of the same order as that obtained with intact males. Similar treatment of castrate males with androgen potentiated (P less than 0.05) the vasopressor action of norepinephrine (0.25 microgram/kg) on day 7 after the testosterone pretreatment. In contrast, treatment with depot estradiol (100 microgram/kg sc) in castrate males produced no significant change in the response to either of the vasoactive compounds on both days 5 and 7 after pretreatment. These data suggest that testosterone may be a significant factor in the development of sex differences in the cardiovascular systems of rats.     

Effects of testrosterone replacement on the recovery from increased emotionality,produced by septal lesions in prepubertal castrated male rats

One hundred forty-seven male rats were studied in an attempt to relate testosterone replacement therapy to the recovery process from increased emotionality following septal lesions. The following groups were used: (a) animals castrated at each of the ages 22–31 days and either testosterone replaced or vehicle injected or not injected; (b) intact animals which were either testosterone injected or vehicle injected or not injected. All animals underwent bilateral septal lesions at about the age of 65 days, and their emotionality was rated 1 day, 3 days and 7 days postoperatively. Recovery from increased emotionality was found to be dependent on age of castration. Testosterone replacement, although efficient in restoring sexual functions, did not have an effect on the recovery process.     

Testosterone modulation of seizure susceptibility is mediated by neurosteroids 3alpha-androstanediol and 17beta-estradiol

Testosterone modulates seizure susceptibility in animals and humans, but the underlying mechanisms are obscure. Here, testosterone modulation of seizure susceptibility is hypothesized to occur through its conversion to neurosteroids with "anticonvulsant" and "proconvulsant" actions, and hence the net effect of testosterone on neural excitability and seizure activity depends on the levels of distinct testosterone metabolites. Testosterone undergoes metabolism to neurosteroids via two distinct pathways. Aromatization of the A-ring converts testosterone into 17beta-estradiol. Reduction of testosterone by 5alpha-reductase generates 5alpha-dihydrotestosterone (DHT), which is then converted to 3alpha-androstanediol (3alpha-Diol), a powerful GABA(A) receptor-modulating neurosteroid with anticonvulsant properties. Systemic doses of testosterone decreased seizure threshold in rats and increased the incidence and severity of pentylenetetrazol (PTZ)-induced seizures in mice. These proconvulsant effects of testosterone were associated with increases in plasma 17beta-estradiol and 3alpha-Diol concentrations. Pretreatment with letrozole, an aromatase inhibitor that blocks the conversion of testosterone to 17beta-estradiol, significantly inhibited testosterone-induced exacerbation of seizures. The 5alpha-reductase inhibitor finasteride significantly reduced 3alpha-Diol levels and also blocked letrozole's ability to inhibit the proconvulsant effects of testosterone. The 5alpha-reduced metabolites of testosterone, DHT and 3alpha-Diol, had powerful anticonvulsant activity in the PTZ test. Letrozole or finasteride had no effect on seizure protection by DHT and 3alpha-Diol, but indomethacin partially reversed DHT actions. 3alpha-Diol but not 3beta-androstanediol, a GABA(A) receptor-inactive stereoisomer, suppressed 4-aminopyridine-induced spontaneous epileptiform bursting in rat hippocampal slices. Thus, testosterone-derived neurosteroids 3alpha-Diol and 17beta-estradiol could contribute to the net cellular actions of testosterone on neural excitability and seizure susceptibility.     

Impact of testosterone and oestradiol on region specificity of skeletal muscle-ATP, creatine phosphokinase and myokinase in male and female Wistar rats.

The main aim of the present study was to test the hypothesis that skeletal muscle ATP concentration, creatine phosphokinase and myokinase enzyme activities are stimulated by the sex steroids in both male and female rats (animals were not subjected to any kind of exercise or any training). To test the hypothesis healthy mature (90-120 days old, weighing about 160-180 g) male and female rats were gonadectomized. Gonadectomized male and female rats were administered with testosterone (Sigma Chemical, St Louis, MO, USA) at a dose of 100 microg (100 g body weight)-1 day-1 for males and 5 microg (100 g body weight)-1 day-1 for females for 30 days from day 31 post-castration onwards; and oestradiol at a dose of 5 microg (100 g body weight)-1 day-1 for 30 days from day 31 post-castration onwards for both males and females (17beta oestradiol, Sigma Chemical Company, St Louis, MO, USA). The ATP content, creatine phosphokinase and myokinase enzyme activities of skeletal muscles were significantly higher than that of skeletal muscles of female control rats. Gonadectomy resulted in a significant decrease in ATP content and creatine phosphokinase myokinase enzyme activities in both male and female rats. Testosterone treatment to gonadectomized male rats brought back the parameters to normalcy whereas the same to the female rats enhanced the enzyme activities and ATP contents to the level of control male rats. Oestradiol treatment to castrated male rats did not bring about any significant alterations whereas the same in gonadectomized female rats brought them back to normalcy. Therefore from the present study it is concluded that testosterone is effective in both males and females whereas oestradiol was effective only in the females in enhancing skeletal muscle energy metabolism.     

Intercellular communication within the rat anterior pituitary gland. II. Castration effects and changes after injection of luteinizing hormone-releasing hormone (LH-RH) or testosterone

This study investigated the relationship between gap junction formation and sex steroids in the male rat anterior pituitary gland. Animals were castrated at 5 days of age and separated into the following three groups: 1) oil-treated controls, 2) those injected with LH-RH, and 3) those given testosterone. On days 10, 20, 30, and 40, five rats in each group were sacrificed and their hypophyses removed for ultrastructural examination. When compared with age-matched, intact animals, there was a marked suppression in follicular development and in the number of gap junctions present in the pituitary glands of both the castrated controls as well as the castrates given luteinizing hormone releasing hormone (LH-RH). In contrast, the morphology of these structures in the animals given testosterone was indistinguishable from that observed in the intact controls. These observations provide more definitive evidence that in the male rat pituitary gland maturation of the structural organization of the follicles, including gap junction formation, requires an intact hypophyseal-gonadal axis and is highly dependent on the hormone testosterone.