Suppression of the development of female hamster behaviour by implants of testosterone and non-aromatizable androgens administered neonatally.

Testosterone in its free form, and dihydrotestosterone (DHT) and androsterone, both androgens which are not aromatizable to oestrogen, injected in oil during the neonatal period have been reported not to modify the development of female sexual behaviour. This failure might be due to the short period of activity of these substances when injected in liquid vehicles. In the current study, a Silastic pellet containing 9% of its weight of testosterone, androsterone, or DHT was implanted subcutaneously in 42 female and 38 neonatally castrated male hamsters on day 2 of life and removed on day 10. Pellets of pure Silastic were implanted in 36 control animals. Males were gonadectomized on day 5 and females on day 45. Female sexual behaviour induced by oestradiol benzoate and progesterone was measured in a series of 10-min mating tests with vigorous males, starting at 55 days of age. The duration of lordosis was consistently reduced below control levels in females implanted with testosterone, DHT, and androsterone, and in males, with testosterone and DHT. Thus the free form of testosterone, and some non-aromatizable androgens, when present for a sufficiently long period after birth, can permanently suppress development of female reproductive behaviour.     

Hormonal correlates of polyandry in the spotted sandpiper, Actitis macularia

Plasma samples from naturally breeding populations of spotted sandpipers, Actitis macularia, were analyzed by radioimmunoassay for circulating levels of immunoreactive luteinizing hormone (irLH), and four steroid hormones. Plasma levels of irLH, corticosterone (C), and estradiol-17 beta (E) did not differ between males and females. Males had curiously high levels of E (0.64 pg/ml in nonnesting males; 0.35 pg/ml in nesting males) which are similar to those of nonlaying females (0.23 pg/ml). Testosterone (T) and dihydrotestosterone (DHT) were higher in nonnesting males (0.95 ng/ml for T; 0.20 ng/ml for DHT) than in females (0.18 ng/ml for T; 0.12 ng/ml for DHT) or nesting males (0.17 ng/ml for T; 0.09 ng/ml for DHT). These data do not support earlier suggestions that in polyandrous mating systems, females have higher circulating levels of androgens than males.     

Enzymatic oxidation and reduction of C19-delta5-3beta-hydroxysteroids by hepatic microsomes. IV. Induction of DHA hydroxylases and aminopyrine N-demethylase in immature male rats by androgens.

The capacities of various C19 steroids for prematurely inducing the normal metabolic patterns of rat liver in adulthood (70 days old) have been studied with hepatic microsomes of 42-day-old males castrated on day 24, 30, 32, or 34 of life. Dehydroepiandrosterone 16alpha-hydroxylase activity was significantly reduced (P less than 0.05) by castration during this 10-day interval but the 7alpha- and 7beta-hydroxylases, the N-demethylation of aminopyrine, and cytochrome P-450 concentration were unaffected. Daily administration of testosterone stimulated the DHA 16-alpha- and 17beta-hydroxylases, aminopyrine N-demethylation, and increased the P-450 content, but suppressed the 7alpha-hydroxylase. These effects only appeared with more than 1 week of the continuous treatment. Testosterone was the most active of the androgens studied; dihydrotestosterone (DHT) increased the DHA 16alpha-hydroxylase to a lesser extent, but this steroid and etiocholanolone stimulated DHA 7alpha-hydroxylation; androsterone was totally ineffective. These data suggest that testosterone rather than DHT from pubertal testes plays a significant role in control of hepatic oxidative enzyme activities during puberty.     

Preponderance of serum and intra-hepatic 5 alpha-dihydrotestosterone in males with hepatocellular carcinoma despite low circulating androgen levels

To investigate possible influences of the sex-steroid milieu on hepatocellular carcinoma (HCC) and vice versa, circulating and intra-hepatic sex-steroid levels were investigated and compared with the levels in cirrhosis alone. In cirrhotic men with HCC, serum 17 beta-oestradiol levels were normal, unlike the elevated levels in men with cirrhosis alone. Total and free levels of testosterone and 5 alpha-dihydrotestosterone (DHT) were lower in patients with HCC than in cirrhotic or normal men; the greater decrease in testosterone levels caused an elevated DHT: testosterone ratio. Hypothalamic-pituitary axis dysfunction demonstrated for HCC and cirrhotic groups could not explain the differences in sex-steroids between them. Compared with normal tissue, HCC cytosol had lower testosterone and similar DHT levels; both androgens were higher than in cirrhotic tissue, and the intracellular DHT: testosterone ratio in the tumour was much higher than in control tissue. Results suggest alterations in sex-steroid metabolism in HCC favouring hepatic accumulation of 5 alpha-reduced metabolites aided by the elevated intracellular sex-hormone binding globulin levels shown in HCC tissue.     

Correlation between LH secretion in castrated rats with cellular proliferation and synthesis of DNA in the anterior pituitary gland

Testosterone metabolism in the brain and pituitary and cloacal glands of male and female Japanese quail was studied in vitro during sexual maturation (from 1 day to 5 weeks after hatching). The production of 5 alpha-dihydrotestosterone in the hyperstriatum and cloacal gland and that of androstenedione in the cloacal gland of males was highest at 1 day after hatching, which could be related to the peak of plasma androgens previously demonstrated in neonatal quail. 5 beta-Reductase activity was very high in the brain, but not the pituitary or cloacal glands of young chicks and decreased markedly, especially in the hypothalamus, during sexual maturation. As 5 beta-reduced metabolites of testosterone are inactive androgens, it is suggested that the decrease of 5 beta-reductase activity with age corresponds to a potentiation of the effects of testosterone at the level of the brain.     

Gonadal source of testosterone metabolites in urine of male cotton-top tamarin monkeys (Saguinus oedipus)

Examining gonadal function in the small excitable cotton-top tamarin monkey (Saguinus oedipus) requires noninvasive sampling techniques. Two studies were performed to identify the quantifiable urinary metabolites of testosterone in cotton-top tamarins and which of the measurable metabolites would best reflect a gonadal source of testosterone secretion. In the first study, we injected unlabeled testosterone i.m. in males at either 500-ng or 1-microg levels. Urine samples were analyzed for androgens and estrogens. Testosterone and dihydrotestosterone (DHT) increased significantly following the injections in test males but not in control males. No significant increases in androstenedione occurred. Mean levels of estradiol and estrone did not consistently increase during the 5 days following injection. In the second study, a gonadotropin-releasing hormone antagonist, Antide, was used to block LH stimulation of gonadal steroidogenesis. Males given Antide at either a 6 mg/kg dose or an 18 mg/kg dose showed significantly lower levels of urinary LH than controls. At the higher Antide dose, testosterone levels were significantly reduced during weeks 1 and 2 posttreatment, whereas DHT levels significantly declined during the 2nd week posttreatment. Estradiol levels were highly variable prior to treatment but decreased significantly following treatment, whereas estrone levels remained variable throughout. These results indicate that measurement of urinary testosterone and possibly DHT reflect gonadal function in male cotton-top tamarins. Other sources of urinary estrogens may occur for the male cotton-top tamarin, but these data suggest that a substantial part of urinary estradiol is from gonadal sources, whereas urinary estrone appears to be mainly from extragonadal sources.     

Sex steroid levels in developing and adult male and female zebra finches (Poephila guttata)

Serum samples from male and female zebra finches ranging in age from 1 day before hatch to 54 days posthatch were assayed for 17 beta-estradiol (E), androgen, testosterone (T), or 5 alpha-dihydrotestosterone (DHT). Additional samples were assayed from intact and gonadectomized adults, gonadectomized adults with intraperitoneal implants of testosterone propionate (TP) or estradiol benzoate (EB), gonadectomized nestlings, and nestlings injected subcutaneously with EB. DHT levels of developing birds did not vary as a function of either sex or age. During development, average androgen and T levels were highest during the nestling period, prior to sexual maturation, and were higher in females than in males. Endogenous androgen levels of most subjects that were sampled repeatedly rose and then declined between 24 and 49 days. TP implants produced higher T levels in adult females than in adult males. Levels of E were higher in both sexes during the hatching period (Days -1 through 0) than during the nestling period (Days 2 through 14). A greater number of males than females had relatively high E levels on Days 12 and 14 and during the second week overall. There was no sex difference in levels of E in adults, and gonadectomized adults had markedly higher E levels than intact adults. Gonadectomized nestlings had the same androgen and E levels as intact nestlings of the same age; EB injected nestlings had elevated E levels. The present results indicate specific endocrine changes that mirror events crucial to sexual differentiation of endocrine and behavioral components of reproduction, and have important implications for models of sexual differentiation in zebra finches.     

Metabolism and binding of androgens by mouse mammary tumour cells in culture

Both testosterone and 5α-dihydrotestosterone (DHT) increased the growth rate of androgendependent mouse mammary tumour cells (S115) in culture. DHT was effective at lower concentrations and increased the thymidine labelling index with a shorter lag-period than testosterone. The S115 cells converted testosterone to DHT, 5α-androstane-3α-17β-diol, androsterone and androstanedione. DHT was not converted to testosterone. All this data was consistent with the view that testosterone exerted its biological effect in this system by conversion to DHT. However, the high affinity, 8–9 cytoplasmic receptor bound testosterone as well as DHT. Testosterone competed with DHT for binding sites and vice versa; oestradiol 17β blocked the binding of both androgens but no binding of ³H-oestradiol-17β to an 8–9 component was detected. After incubation of cells with ³H-testosterone at 37 °C a 5S receptor could be extracted from nuclei. The major radioactive steroid in the nucleus under these conditions was testosterone. This binding data suggests that testosterone might be biologically active without metabolic conversion to DHT.     

Testosterone sensitivity of the seminal sacs of tree sparrows (Spizella arborea) in different reproductive states

Testosterone sensitivity of the seminal sacs of castrated tree sparrows from each of three reproductive states was evaluated by measuring the change in seminal-sac mass per unit change in the logarithm of replacement or plasma testosterone. Birds were exposed to exogenous testosterone for 38 days. Replacement doses less than 0.17 mumol or plasma concentrations less than about 0.7 nmol/l did not induce seminal-sac growth in photosensitive castrated birds held on short days, in photosensitive castrated birds transferred from short to long days, or in photorefractory castrated birds retained on long days. Higher replacement doses or plasma concentrations, however, stimulated log dose-dependent growth of the seminal sacs in castrated birds from all three reproductive states. The change in seminal-sac mass per unit change in the logarithm of the dose of replacement testosterone was less (P = 0.0495) in photosensitive castrated birds held on short days than in photosensitive castrated birds transferred to long days. A more critical test of sensitivity (i.e. the change in seminal-sac mass per unit change in the logarithm of mean plasma testosterone concentration) indicated, however, that sensitivity of the seminal sacs to testosterone is independent of reproductive state. That result, when considered in the context of the plasma testosterone profile of intact males during a simulated reproductive cycle, argues that the seminal sacs of sexually quiescent (photosensitive or photorefractory) tree sparrows are small not because of their insensitivity to androgens, but because of a deficiency of circulating androgens.     

Role of testosterone and its metabolites in the differentiation of the mammary gland in rats.

The capacity of various androgens to virilize the differentiating mammary gland in the female rat fetus has been determined. Testosterone, 5alpha-androstane-3alpha, 17beta-diol (3alpha-diol), and dihydrotestosterone (DHT) virilize the anlagen of the mammary gland by suppressing nipple formation but 5alpha-androstane-3beta, 17beta-diol, androsterone, and dehydroepiandrosterone sulfate do not affect female mammary differentiation. However, unlike the genitalia and wolffian ducts of the female rat fetus in which the masculinizing potency of DHT and 3alpha-diol is greater than that of testosterone, testosterone is more potent than its metabolites DHT and 3alpha-diol, in virilizing the mammary gland. The results suggest that testosterone is the fetal androgen mediating masculine development of the mammary gland.     

Annual cycles of steroid hormone production, gonad development, and reproductive behavior in the Atlantic stingray

The mating season of the Atlantic stingray (Dasyatis sabina), which begins in August and continues through April, is the longest documented for any elasmobranch fish. Despite this protracted mating period, female stingrays ovulate synchronously at the end of the mating season and there is no evidence for sperm storage by females. Thus, the proximate causal factors and ultimate function of this extended preovulatory mating are unknown. Annual cycles of the gonadal steroids testosterone (T), dihydrotestosterone (DHT), 17beta-estradiol (E2), and progesterone (P4) were measured for 26 months in a wild estuarine population of Atlantic stingrays to test for associations with their reproductive biology, gametogenesis, and sexual behavior. Serum androgen levels in males showed four phases within an annual cycle: (1) androgen suppression between reproductive seasons (April-July), (2) primary androgen increase during the onset of spermatocyte development (August-October), (3) androgen decrease following maximum testis growth and spermatocyte development (November-December), and (4) secondary androgen increase during the peak of sperm maturation (January-March). Increases in male E2 and P4 were correlated with spermatocyte/spermatocyst formation, maximum testis weight, and the primary (but not secondary) androgen surge. We propose that the production of male androgens across the full seven-month preovulatory mating period promotes their aggressive reproductive behavior and drives the protracted mating season of this species. In females, serum T and DHT showed relatively brief increases near ovulation, whereas E2 and P4 showed brief increases near both ovulation and parturition. The increase in female androgens near ovulation may increase female aggression when they are impregnable by courting males and enhance their choice of mates. This estuary sample population shows higher absolute steroid levels and distinct differences in temporal cycles compared to another Florida fresh water lake population, but the cause and significance of these differences are unknown. Experiments are needed to confirm that the aggressive and protracted mating behavior is the result of prolonged male androgen production and to determine whether the sustained preovulatory mating serves some function related to female reproduction.     

Preponderance of serum and intra-hepatic 5α-dihydrotestosterone in males with hepatocellular carcinoma despite low circulating androgen levels

To investigate possible influences of the sex-steroid milieu on hepatocellular carcinoma (HCC) and vice versa, circulating and intra-hepatic sex-steroid levels were investigated and compared with the levels in cirrhosis alone. In cirrhotic men with HCC, serum 17β-oestradiol levels were normal, unlike the elevated levels in men with cirrhosis alone. Total and free levels of testosterone and 5α-dihydrotestosterone (DHT) were lower in patients with HCC than in cirrhotic or normal men; the greater decrease in testosterone levels caused an elevated DHT:testosterone ratio. Hypothalamic-pituitary axis dysfunction demonstrated for HCC and cirrhotic groups could not explain the differences in sex-steroids between them. Compared with normal tissue, HCC cytosol had lower testosterone and similar DHT levels; both androgens were higher than in cirrhotic tissue, and the intracellular DHT:testosterone ratio in the tumour was much higher than in control tissue. Results suggest alterations in sex-steroid metabolism in HCC favouring hepatic accumulation of 5α-reduced metabolites aided by the elevated intracellular sex-hormone binding globulin levels shown in HCC tissue.     

Gonadotropin dynamics in XX males

Hypothalamic-pituitary gonadotropin function was evaluated in two postpubertal XX males. Serum levels of LH and FSH were moderately elevated, and the serum testosterone level was low. A subnormal response by testicular Leydig cells to hCG was observed. The LH and FSH responses to LRH were normal. A significant LH increase was observed after castration. Weekly administration of testosterone enanthate (250 mg) for 10 consecutive weeks caused a reduction (greater than 75%) in gonadotropins and abolishment of the LRH pituitary response. No differences were observed in terms of gonadotropin dynamics compared with other forms of hypergonadotropic hypogonadism. These results indicate that XX males exhibit hypergonadotropic hypogonadism secondary to testicular failure with a preserved androgen responsiveness of the hypothalamic-pituitary unit.     

Dihydrotestosterone formation (5 alpha-reductase activity) in cultured human skin fibroblasts

Testosterone secreted from the testis is converted to dihydrotestosterone (DHT) in target tissues by 5 alpha-reductase and DHT exerts its action on the nucleus. Since skin is one of the target tissues of androgen we established a system for culturing human skin fibroblasts, and we investigated the conversion of testosterone to DHT in genital and nongenital skin fibroblasts obtained from males as well as from females. In our system, fibroblasts were incubated with 0.1 microM 1, 2-3H testosterone, and labeled metabolites were separated by thin layer chromatography. DHT formation was linear for 6 hours and the rate of DHT formation correlated with the growth phase of the fibroblasts. In the course of serial subculture, DHT formation did not change between the 3rd and 10th subculture in four cell strains. DHT formation in nongenital skin fibroblasts did not differ significantly between males and females. Fibroblasts obtained from male genital skin produced significantly greater amounts of DHT than those from male or female nongenital skin. Furthermore, DHT formation in genital skin fibroblasts did not differ between normal males and hypogonadal males. These data suggest that genital skin is the target tissue where testosterone strongly converts to DHT and the difference of male secondary sexual characteristic appearance between males and females may not depend on the sensitivity of the target tissues to androgen but on the secretion rate of androgen.     

Testosterone metabolites do not participate in the control of hypothalamic LH-releasing hormone

To determine whether the ability of testosterone to increase intrahypothalamic LH-releasing hormone (LHRH) in orchidectomized rats might be explained by the conversion of the hormone into either its 5 alpha-reduced or oestrogenic metabolites, testosterone, 5 alpha-androstan-17 beta-ol-3-one (DHT), 5 alpha-androstane-3 alpha,17 beta-diol (3 alpha-diol) and 5 alpha-androstane-3 beta,17 beta-diol (3 beta-diol) (2 mg/rat per day for 6 days) and oestradiol (0.1, 0.5, 1.0 and 5.0 micrograms/rat per day for 6 days) were injected into castrated male rats. After 6 days the rats were killed and serum LH levels and intrahypothalamic LHRH stores measured using specific radioimmunoassay procedures. Testosterone and its 5 alpha-reduced metabolites were used in either the free alcohol or the propionate form (dipropionates in the case of the diols); oestradiol was used as oestradiol-17 beta or in the benzoate form. Treatment with testosterone, DHT, 3 alpha-diol and 3 beta-diol resulted in a significant decrease in serum LH levels; all the 5 alpha-reduced testosterone derivatives were more effective than testosterone in this respect. Testosterone and DHT propionates suppressed LH release following orchidectomy totally; 3 alpha-diol and 3 beta-diol dipropionates were less effective. Testosterone increased intrahypothalamic LHRH stores, this effect being much higher after testosterone propionate, i.e. when intrahypothalamic LHRH stores were restored to pre-castration levels. None of the 5 alpha-reduced steroids was capable of modifying the low intrahypothalamic levels of LHRH found following orchidectomy; only 3 alpha-diol dipropionate exhibited some activity, but this was much lower than that of testosterone propionate. Oestradiol-17 beta was totally ineffective in decreasing serum LH in orchidectomized animals; in contrast, oestradiol benzoate progressively decreased serum LH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Seasonal and population variation in male testosterone levels in breeding orange-crowned warblers (Vermivora celata)

Comparative hormone studies can reveal how physiology underlies life history variation. Here, we examined seasonal variation in plasma testosterone concentration between populations of male orange-crowned warblers (Vermivora celata) breeding in Fairbanks, Alaska (V. c. celata) and on Santa Catalina Island, California (V. c. sordida). These populations face different ecological constraints and exhibit different life histories. Alaska birds have a short breeding season, low annual adult survival, and high reproductive rates. In contrast, Catalina Island birds exhibit high adult survival and low reproductive rates despite having a long breeding season. We examined seasonal variation in male testosterone concentrations as a potential mechanism underlying differences in male reproductive strategies between populations. From 2006 to 2008, we sampled males during the pre-incubation, incubation, and nestling stages. Alaska males exhibited a seasonal testosterone pattern typical of northern passerines: testosterone levels were high during pre-incubation and declined during incubation to low levels during nestling provisioning. Testosterone concentrations in Catalina Island males, however, did not vary consistently with breeding stage, remained elevated throughout the breeding season, and were higher than in Alaska males during the nestling stage. We hypothesize that in Alaska, where short seasons and high adult mortality limit breeding opportunities, the seasonal testosterone pattern facilitates high mating effort prior to incubation, but high parental investment during the nestling stage. On Catalina Island, elevated testosterone levels may reflect the extended mating opportunities and high population density facing males in this population. Our results suggest that population variation in seasonal testosterone patterns in orange-crowned warblers may be a function of differences in life history strategy and the social environment.     

Role of androgens in mediating renal injury in aging SHR

Men have an increased risk of cardiovascular and renal diseases and develop greater renal injury despite similar levels of blood pressure when compared with women. The mechanisms responsible for this predisposition are unknown. Using the spontaneously hypertensive rat (SHR), we have found that androgens play an important role in the development of hypertension in young male SHR. However, the role that androgens play in age-related renal injury and dysfunction in SHR is unknown. Our hypothesis was that despite reductions in serum testosterone with age, androgens mediate renal injury and dysfunction in male SHR. Male SHR were castrated at 8 months of age, studied at 18 months of age, and compared with age-matched, intact males and young intact males (4 months). Serum testosterone was reduced by 30% in aging males compared with young SHR. With castration, blood pressure (mean arterial pressure [MAP]) was decreased by >20 mm Hg compared with old males, glomerular filtration rate (GFR) was increased by >35%, and renal vascular resistance (RVR) was reduced by >40%. MAP, GFR, and RVR in castrated, old males were similar to values in young males. With castration, glomerular sclerosis was reversed and proteinuria was also decreased by >80% when compared with old intact males. In addition, in castrated old males, plasma renin activity was decreased by 30% compared with old males and by 60% compared with young rats. The data support the hypothesis that despite a reduction in testosterone with age, androgens play an important role in age-related renal injury and dysfunction in SHR.     

A longitudinal study of LH, gonadal and adrenal steroids in four intact Asian bull elephants (Elephas maximus) and one castrate African bull (Loxodonta africana) during musth and non-musth periods

During their annual musth cycle, adult African and Asian bull elephants have increased gonadal androgens (testosterone [T], dihydrotestosterone [DHT], androstenedione [A4]). Because musth is a physiologically and psychologically stressful time, this study was conducted to investigate whether the adrenal glands (stimulated by stress) increase production of both glucocorticoids and androgens during musth. Weekly serum samples were taken for 11-15 months from four intact adult Asian bull elephants, and from a castrate African bull elephant who exhibits musth. Testosterone, androstenediol (A5), A4, luteinizing hormone (LH), cortisol, and dehydroepiandrosterone (DHEA) were measured in each sample. In three of the four intact bulls, all hormones measured increased during musth. Adrenal androgens were strongly correlated with LH and testicular androgens, though not to cortisol. None of the hormones measured in the castrate bull increased during his musth cycles. While the significance of adrenal activity in the elephant during musth has yet to be determined, this study provides evidence that the adrenal gland actively produces both glucocorticoids and androgens during musth in the Asian elephant.     

Action of sex steroid hormones on temperature-induced sex determination in the snapping turtle (Chelydra serpentina)

Administration of exogenous estradiol benzoate (EB) or an estrogen agonist, R2858, into eggs of snapping turtles caused all embryos incubated at male-producing temperatures to develop as females, whereas testosterone propionate (TP) caused 42% of the embryos to develop as females. Some of the embryos treated with EB, R2858, or TP also had hypertrophied oviducts. Neither dihydrotestosterone (DHT) nor cholesterol had any apparent effect on the sex determination of embryos incubated at male-producing temperatures. Injections of TP, DHT, the androgen agonist R1881, or cholesterol had no apparent effect on sex determination of embryos incubated at female-producing temperatures. Administration of estradiol antiserum or testosterone antiserum resulted in some individuals having undifferentiated or ambiguous gonads. Although both exogenous estrogens and androgens can induce embryonic gonads to develop as ovaries, the findings of this study indicate that estrogen is the female-inducing hormone and that androgens may feminize gonads via aromatization to estrogen. Furthermore, the results of the antisera injection suggest that endogenous steroid hormones may have a natural role in gonadal differentiation of reptiles with environmental sex determination.