Biochemical alterations in sex hormone-induced hyperplasia and dysplasia of the dorsolateral prostates of Noble rats.

Simultaneous implantation of intact Noble (Nb) rats with testosterone and 17 beta-estradiol (E2)-filled silastic capsules for 16 weeks caused atypical hyperplasia (dysplasia) and striking enlargement exclusively in the dorsolateral prostates (DLPs) of all animals. The dysplastic lesion may be preneoplastic since long-term administration of these steroids to Nb rats is known to induce a high incidence of adenocarcinoma in the DLP. Treatment of rats with nonaromatizable 5 alpha-dihydrotestosterone (DHT) for 16 weeks caused enlargement but not dysplasia, implicating estrogen as a key factor in the genesis of the proliferative lesion. Compared with controls, the testosterone plus E2 treatment caused a 2.5-fold increase in nuclear type II estrogen binding sites which were confined to the DLP. Neither treatment significantly altered androgen content or levels of androgen receptor in the ventral prostate or DLP. Organ cultures of enlarged DLP containing foci of dysplasia metabolized more [3H]DHT than control tissue, which resulted in increased formation of the 5 alpha-androstane-3 beta, 17 beta-diol (3 beta-androstanediol) metabolite by these explants. Because 3 beta-androstanediol has previously been shown to displace [3H]E2 from cytosolic type I estrogen binding sites, the dysplasia may be caused by hyperstimulation of the DLP by the hormones and their normal metabolites produced in abnormal amounts.

Testosterone-mediated increase in 5 alpha-dihydrotestosterone content, nuclear androgen receptor levels, and cell division in an androgen-independent prostate carcinoma of Noble rats.

An androgen-independent, transplantable prostate carcinoma line (AIT), originally derived from the dorsolateral prostate (DLP) of Noble rat, was implanted into orchiectomized Noble rats and its response to androgen stimulation was studied and compared to that of the regenerating DLP tissue in sexually ablated rats. AIT tumors carried in castrated hosts displayed a high basal level of proliferative activity (mitotic index (MI), 15.0 +/- 0.5) while DLP tissue in untreated castrates exhibited no proliferative activity. Following androgen stimulation by testosterone capsule implantation into host rats, the AIT responded with a marked increase in cell proliferation; MI values doubled to 30.0 +/- 2.9 on Day 5 following androgen stimulation. This androgen-induced increase in MI values was coincident with elevations in nuclear androgen receptor (20-fold increase) and 5 alpha-dihydrotestosterone content (3-fold increase) in the tumor. However, by Day 10 following androgen treatment, indices of cell proliferation in the AIT declined to pre-androgen-stimulated levels (MI, 14.8 +/- 1.9) despite the continued elevations in nuclear androgen receptor and tissue 5 alpha-dihydrotestosterone contents. Parallel changes in MI were also observed in the normal regenerating DLP following androgen stimulation. MI values in this tissue increased from nondetectable levels to 38.1 +/- 4.7 on Day 5 but declined to relatively low levels (4.5 +/- 0.9) by Day 10 following androgen replacement. Taken together these findings led us to conclude that the AIT carried in castrates is capable of responding to testosterone in a manner similar to that observed for androgen-stimulated DLP of sexually ablated rats. Thus, in both the neoplastic and regenerating tissues, the initial response to androgen is characterized by a marked enhancement of cell proliferation which was correlated with an increase in androgen receptor and 5 alpha-dihydrotestosterone content. However, like its tissue of origin, the AIT possesses mechanisms which act to limit androgen-induced cell division despite continued elevations in key parameters of androgen activation.

Sex hormone-binding globulin and male sexual development.

The masculinization of the brain, reproductive tract and many other structures is critically dependent on the testicular hormone, testosterone (T). In many species, T circulates bound with high affinity to sex hormone-binding globulin (SHBG). This protein has a wide phylogenetic distribution and SHBG or SHBG-like proteins are produced by the liver, testes, placenta, brain and other tissues. SHBG activity is detectable during gestation and its expression is both stage- and tissue-dependent. Although SHBG binds circulating androgens, it is argued that the trapping of steroids in the circulation is not the principal function of this protein. The specific binding and uptake of SHBG by various tissues has been observed and suggests that SHBG may directly affect the delivery of androgen signals to target tissues. Effects of SHBG on androgen metabolism, tissue retention, cellular targeting, and action are reviewed. Evidence to date indicates that SHBG is able to enhance or inhibit the uptake of androgens in a cell- and tissue-specific manner. Future work will be necessary to demonstrate whether such actions of SHBG are important for normal male reproductive development.

Effects of chronic infusions of sex steroid-binding protein on the testosterone-mediated inhibition of gonadotropin secretion and maintenance of sex accessory glands in male rats.

Sex steroid-binding protein (SBP) is a plasma glycoprotein which, in man and many other mammals, binds significant amounts of circulating testosterone (T) with high affinity. To evaluate the effects of T binding by SBP on the control of reproductive function in males, SBP was purified from human plasma and administered by chronic infusion to castrated T-treated rats, animals that lack endogenous SBP. Plasma T concentrations in males implanted with 20-mm T capsules and infused with vehicle for 48 h were maintained at 1.37 +/- 0.14 ng/ml, and plasma LH and FSH concentrations remained at intact levels. Castrates implanted with identical T capsules but infused with human (h) SBP at a rate that produced a steady state plasma hSBP concentration of 75 nM, had markedly higher plasma T concentrations (3.44 +/- 0.32 ng/ml); however, circulating LH and FSH concentrations were not significantly different from those of the vehicle-infused controls. Similarly, the pituitary LH response to a bolus injection of LHRH was not affected by hSBP infusions. Measurements of tissue concentrations of T, dihydrotestosterone, and 3 alpha-androstanediol in the ventral prostate and seminal vesicles, as well as the weights of these organs, failed to reveal any effects of hSBP infusions on the uptake, metabolism, or action of T. Analysis of T distribution in plasma showed that the hSBP infusions produced marked increases in total plasma T levels but resulted in only marginal reduction in the concentration of T not bound to hSBP. These results are consistent with the hypothesis that T not bound to SBP is the major feedback-effective moiety and that under conditions of constant release of T into the circulation, an increase in plasma T binding by SBP increases the total plasma T concentration but does not appear to significantly alter the feedback-effective levels of T in the circulation. Therefore, it would appear that moderate changes in the amount of high affinity binding of T in the circulation would have little, if any, effect on androgen balance in adult males.

Perinatal and postnatal patterns of plasma sex steroid-binding protein and testosterone in relation to puberty in the male little brown bat.

The little brown bat (Myotis lucifugus lucifugus) possesses a specific plasma sex steroid-binding protein (SBP) which exhibits high affinity for both testosterone (T) and estradiol. To examine the peri- and postnatal patterns of plasma androgen binding in relation to puberty, concentrations of SBP and total T levels were measured in males at various intervals throughout the first year of life. In addition, reproductive organs were examined histologically over this period. SBP levels were low (less than 7 nM) in near-term fetuses (mid-June) but increased more than 30-fold by 1 week of age (range, 245-602 nM). Elevated titers of this protein were present until 3 weeks of age. By 4 weeks of age (onset of puberty, initiation of the first spermatogenesis), SBP concentrations had fallen significantly, and by 12 weeks, just before the first winter hibernation period, plasma SBP levels had returned to low prenatal-like values (3-12 nM). These levels were maintained throughout winter dormancy. The total plasma T concentrations were also elevated during the early postnatal period and were comparable to or even exceeded those previously measured in spermatogenically active adults. Plasma T levels then declined in parallel to those of SBP and reached a nadir (less than 0.6 ng/ml) by 12 weeks of age. As with SBP, T also remained low throughout hibernation. Although a complete spermatogenic cycle was found to occur in postnatal bats during the summer of their birth, this cycle was greatly reduced compared to that which occurs in sexually mature adults during the same period. Maximal testicular weights from the postnatal males were only one fifth of those previously measured in adult males. In addition, the epididymides in the young bats remained virtually devoid of sperm, and the accessory glands showed little evidence of stimulation. Thus, this first spermatogenic cycle in Myotis does not result in the attainment of sexual maturity and, therefore, appears to be "silent." Since sexual maturity is reached during the following summer when the bats are approximately 1 yr of age, the pubertal process in Myotis is prolonged and appears to be biphasic. Nevertheless, the existence of a perinatal rise in SBP levels (which is repeated in this species when the pubertal process is reinitiated during the second summer) as well as a decline in SBP levels during the onset of puberty resembles that previously described for other mammals.

Control of plasma sex steroid-binding protein (SBP) in the little brown bat: effects of photoperiod and orchiectomy on the induction of SBP in immature males.

Immature male little brown bats (Myotis lucifugus lucifugus) exhibit striking increases in plasma levels of sex steroid-binding protein (SBP) when aroused prematurely from the latter half of hibernation. To identify some of the factors that control this induction, the effects of castration, photoperiod manipulation, and season (early vs. late hibernation) were studied. During the latter half of hibernation, the elevation of plasma SBP levels induced by premature arousal was found to be similar in intact and castrated males. Since castration completely abolished the postarousal rise in plasma testosterone levels, and estradiol concentrations remained low (less than 10 pg/ml) in both groups, changes in gonadal steroids are apparently not responsible for these increases in SBP. The induction of SBP in males aroused late in hibernation also appeared to be independent of whether animals were maintained under long [16 h of light, 8 h of darkness (16L:8D)] or short (8L:16D) photoperiods. Three weeks after arousal, plasma SBP concentrations in the intact groups were 228 +/- 17 nM (mean +/- SE; 16L:8D) and 271 +/- 5 (8L:16D), while in the castrated groups, SBP levels were 256 +/- 43 (16L:8D) and 231 +/- 19 (8L:16D). In addition, comparable degrees of gonadal activation, including increases in testicular weight, rises in circulating testosterone, and initiation of spermatogenesis, were observed in both the long and the short photoperiod groups. During the first half of hibernation, the induction of SBP was similarly unaffected by castration. However, at this time, photoperiod had a marked effect on SBP induction. Only males maintained under a long photoperiod after arousal from early hibernation exhibited increased levels of SBP (158 +/- 51 nM) and evidence of gonadal activity. The animals housed under a short photoperiod had lower levels of SBP (44 +/- 22 nM) and exhibited no gonadal activation. In general, responses seen in animals aroused during the first half of hibernation were reduced compared to those observed after arousal during the second half of hibernation. Thus, the postarousal increase in SBP appears to be independent of gonadal secretions, but can be influenced by photoperiod during early hibernation.

Negative feedback control of LH by testosterone: a quantitative study in male rats.

Adult male rats were castrated and implanted sc with testosterone (T)-filled Silastic capsules of different sizes which produce stable plasma T levels proportional to capsule length. T and LH levels were determined in blood samples obtained at 3-day intervals for 21 days. Plasma T values greater than 1.8 ng/ml suppressed plasma LH to levels below the intact range, while a plasma T of less than 0.5 ng/ml yeilded no significant inhibition of the post-castration LH rise. Animals with a plasma T of about 0.7 ng/ml, however, showed intact, castrate, or immediate LH levels. The feedback-effective range for all animals was between 0.5 ngT/mg (castrate-like LH response) and 1.8 ng/ml (LH at or below intact levels), but individual rats seemed to have a more restricted feedback range than that indicated by these group data. To test this hypothesis further, rats were implanted at the time of castration with five 5-mm T-filled capsules. One capsule was removed each week, producing step function drops in plasma T, and allowing examination of the LH responses of individual animals. Plasma t levels greater than 1.6 ng/ml maintained LH values at or below intact levels. However, when plasma T dropped to 1.1 ng/ml, some rats showed castration-like LH increase and others did not respond until after a further drop to 0.6 ng/ml. It was concluded that the feedback-effective range of plasma T is very restricted and differs in individual rats.

Biological effects of sex hormone-binding globulin on androgen-induced proliferation and androgen metabolism in LNCaP prostate cells.

The effects of purified human sex hormone-binding globulin (SHBG) on androgen-sensitive cell proliferation were examined using a human prostatic cell line (LNCaP-FGC). Cells were grown for 5 days in medium supplemented with 10% charcoal-dextran-stripped human serum (10% CDHuS) and various concentrations of 5 alpha-dihydrotestosterone (DHT). In 10% CDHuS, without SHBG, the proliferative response of these cells to androgens was typically biphasic. At low androgen concentrations, cell yields were increased in a dose-dependent manner, reaching maximal levels at 0.3 nM DHT. However, at high androgen concentrations, cell proliferation was inhibited. Addition of purified human SHBG to the medium reduced the effectiveness of DHT on both phases of the proliferative response in a dose-dependent manner. These effects of SHBG appeared to be due primarily to the high affinity binding of DHT by SHBG. Proliferative responses induced by the synthetic androgen methyltrienolone (R1881), which binds poorly to SHBG, were not affected by added SHBG. Furthermore, analysis of the protein binding of DHT revealed that cell proliferation correlated best with the concentration of DHT not bound to SHBG. The presence of SHBG in the medium also altered the uptake and metabolism of DHT. LNCaP-FGC cells rapidly metabolized DHT to a polar glucuronidase-sensitive conjugate of DHT. In 10% CDHuS, LNCaP-FGC cells conjugated virtually all of the added DHT during the 5-day experiment. However, in medium containing SHBG, the SHBG-bound DHT remained unconjugated; more than 90% of the DHT initially bound to SHBG was present in the medium at the end of the experiment as unconjugated DHT. Uptake of radiolabeled DHT by cells was also inhibited by SHBG. In summary, these experiments provide evidence that 1) SHBG-bound DHT is not a signal for DHT-induced cell proliferation and 2) SHBG inhibits the uptake and metabolism of DHT by LNCaP-FGC cells.

Effects of a non-steroidal antiandrogen on sexual behavior and pituitary-gonadal function in the male rat.

An investigation was conducted on the effects of the non-steroidal antiandrogen flutamide (F; alpha-alpha-alpha-tri-fluoro-2-methyl-4-nitro-m-propionotoluidide) on two neuroendocrine mechanisms in the male rat, androgen-dependent sexual behavior, and LH regulation. F was administered in the dose of 50 mg/kg/day SC. In intact, sexually experienced adult males, no quantitative or qualitative behavioral effects were noted. In long-term castrates, F completely suppressed the effects of 100 mug testosterone propionate (TP) per day on accessory sexual glands and penes, but only partially inhibited the marked stimulatory effects of this moderate TP dose on mating. Although the incidence of testosterone (T)-activated ejaculatory behavior was markedly diminished, there was no statistically significant effect on occurrence of mount and intromission behavior. The rapid and profound elevations of circulating LH and T in intact males indicate an effective antagonism of the negative feedback effect of endogenous androgen, and suggest the usefulness of F as a provocative test of pituitary-testicular function. Pituitary LH response to exogenous LHRH was markedly enhanced, as previously found in castrated rats. The administration of F did not affect circulating T levels in T-treated or untreated castrates, indicating lack of interference of circulating F in the T assay. It was concluded that, like the steroidal antiandrogen cyproterone, non-steroidal F shows a divergence between its effects on peripheral androgen-dependent and central feedback mechanisms on the one hand, and sexual behavior on the other. It was not determined whether the inhibition of ejaculatory behavior following F treatment is centrally mediated or results from failure of the peripheral, androgen-dependent structural or functional elements.

Effects of testosterone, dihydrotestosterone, or estradiol administered neonatally on sexual behavior of female ferrets.

Groups of female ferrets born in the laboratory received sc Silastic capsules containing testosterone (T), 5 alpha-dihydrotestosterone (DHT), 17 beta-estradiol (E), or no steroid for 15 days beginning on the day of birth; an additional group of male ferrets received empty sc capsules neonatally. All ferrets were gonadectomized at 11 weeks of age and were subsequently tested for masculine and feminine sexual behaviors while being treated consecutively over an 8-month period with several different gonadal steroids. The ability to display masculine sexual behavior was studied in the absence of replacement hormones and during a counterbalanced sequence of treatments with Silastic capsules containing T, E, or DHT. The maximal amount of neck grip, mount, and pelvic thrusting behavior displayed, regardless of adult endocrine treatment, was significantly greater in control male and neonatally T-treated females than in females that had received no hormone, E, or DHT neonatally. Animals in all five groups displayed equivalent increments in sexual receptivity in response to daily sc injections of increasing dosages of estradiol benzoate. Polyacrylamide gel electrophoresis of plasma collected from newborn female ferrets revealed no binding of either [3H]E or [3H]T, whereas two binding peaks were found for [3H]DHT. After the administration of androgen in adulthood, equivalent clitoral growth and ossification occurred in females given either T or DHT neonatally. These results suggest that in ferrets, behavioral masculinization occurs in response to neonatal exposure to T itself and not to its major neural metabolites, E and DHT. They also show that behavioral defeminization fails to occur in ferrets even after neonatal exposure to pharmacological amounts of E, T, or DHT.

Testosterone regulation of proopiomelanocortin messenger ribonucleic acid in the arcuate nucleus of the male rat.

GnRH regulates the secretion of LH and FSH, which stimulate the secretion of testicular hormones. Acting in a reciprocal fashion, these hormones, including testosterone and inhibin, exert a negative feedback effect on GnRH and gonadotropin secretion. Endogenous opioid peptides (EOPs) have been implicated to play a role in steroid-mediated regulation of gonadotropin secretion. In this context, certain steroid hormones (e.g. testosterone) increase EOP activity and ultimately inhibit GnRH secretion; however, the cellular mechanism by which this occurs is unknown. beta-Endorphin is one of these EOPs, and it is derived from a larger precursor molecule, POMC. We tested the hypothesis that testicular hormones and testosterone, in particular, stimulate POMC gene expression in the arcuate nucleus of the male rat brain. First, we compared POMC mRNA levels between intact and castrated male rats. Adult male rats were killed 4 days (n = 4) and 21 days (n = 5) after castration. Intact animals (sham-operated; n = 6) were used as controls. Using in situ hybridization and a computerized image analysis system, we measured the POMC mRNA content in individual cells of the arcuate nucleus. POMC mRNA signal was significantly lower (P less than 0.0003) in both 4-day (126 +/- 2 grains/cell) and 21-day (117 +/- 5 grains/cell) castrates than in controls (142 +/- 2 grains/cell). In a second experiment we tested whether testosterone would reverse the castration-induced loss of POMC message. Again, we castrated animals and immediately implanted them with either empty (sham; n = 6) or testosterone-containing Silastic implants (n = 5) of a size that would deliver physiological levels of testosterone (3.6 +/- 1.5 ng/ml). We observed that testosterone-treated animals had significantly higher levels of POMC mRNA signal (121.8 +/- 3.8 grains/cell) than sham-treated castrates (111.4 +/- 3.6 grains/cell; P less than 0.03) and that the testosterone-treated castrates had POMC mRNA signal levels indistinguishable from those of intact controls (122.0 +/- 1.1 grains/cell). These observations lend credence to the theory that one mechanism by which testosterone may regulate GnRH secretion is by increasing the synthesis of POMC in the arcuate nucleus.

Control of sex steroid-binding protein (SBP) in the male little brown bat: relationship of plasma thyroxine levels to the induction of plasma SBP in immature males.

The seasonally reproductive male little brown bat (Myotis lucifugus lucifugus) exhibits marked increases in plasma concentrations of sex steroid-binding protein (SBP) in the spring following arousal from hibernation. In this species an increase in SBP levels is induced prematurely in male bats aroused during the first half of hibernation and housed under long photoperiods; however, this rise is inhibited in bats housed under short photoperiods. In order to investigate the physiological role of the thyroid gland in the regulation of plasma SBP activity, plasma total thyroxine (T4) and SBP concentrations were determined in immature male little brown bats prematurely aroused from the first half of hibernation and maintained on either a short or long photoperiod. For this purpose a radioimmunoassay for the measurement of total T4 in bat plasma was established and validated. The results showed that immature male little brown bats aroused prematurely from hibernation and housed under a long spring-like photoperiod exhibited marked increases in plasma T4 and SBP concentrations, while animals housed under a short photoperiod showed only marginal increases in SBP, and plasma T4 remained undetectable. These results suggest that the thyroid gland, through the action of T4, may normally play an important role in the control of the post-arousal rise in plasma SBP concentrations in the little brown bat.

Patterns of plasma sex hormone-binding globulin, thyroxine and thyroxine-binding globulin in relation to reproductive state and hibernation in female little brown bats.

A sex hormone-binding globulin (SHBG), which bound both oestradiol and dihydrotestosterone, was studied in the plasma of adult female little brown bats throughout the annual reproductive cycle. This protein was present at low baseline levels from September to May inclusive, months which correspond to the periods of hibernation, ovulation and early pregnancy. During the second half of pregnancy in June, SHBG levels increased 15- to 30-fold and remained increased throughout lactation and anoestrus/prooestrus (July-August). Although SHBG was increased during late pregnancy, the fact that levels were also high during and after lactation indicates that this protein is not specific to pregnancy. Plasma concentrations of thyroxine (T4) and the percentage binding of T4 to thyroxine-binding globulin (TBG) also showed marked seasonal variations, with T4 levels exhibiting a biphasic seasonal pattern. A major peak in plasma concentrations of T4 occurred around the time of spring arousal from hibernation and subsequent ovulation, while a second peak of lesser magnitude was measured in August, corresponding to the time of pro-oestrus and the onset of mating. The percentage binding of T4 by TBG was increased during the summer months in parallel with the increase in SHBG concentrations. Electrophoretic analysis of plasma T4 binding revealed a single peak of TBG activity throughout most of the year; however, during the early lactational period TBG was resolved as a double peak, suggesting the presence of a molecular variant during this reproductive stage.

Control of plasma sex steroid-binding protein in the bat Myotis lucifugus lucifugus: induction of steroid-binding activity in immature males.

Concentrations of sex steroid-binding protein (SBP) in plasma of immature male little brown bats (Myotis lucifugus lucifugus) were found to be low (15 +/- 2.8 (S.E.M) nmol/l) and stable throughout the period of hibernation which immediately precedes the onset of the first spermatogenic cycle (sexual maturity). In contrast, plasma SBP titres in spermatogenically active adults (summer) were markedly raised (238 +/- 26 nmol/l). When immature males were removed from hibernation and maintained on a long photoperiod of 16 h light: 8 h darkness at a constant temperature of 25 degrees C to accelerate the onset of sexual maturity, these animals exhibited a rapid rise in circulating SBP. Plasma levels of SBP in immature males maintained on a long photoperiod increased significantly after 2 weeks, and by 3 weeks reached values (234 +/- 20 nmol/l) that were indistinguishable from those seen in spermatogenically active adults. These high plasma SBP titres at 3 weeks were accompanied by increases in testicular and epididymal weights and the onset of spermatogenesis. However, no stimulation of the sex accessory glands was observed, possibly reflecting the effects of increased plasma SBP on the biological activity of circulating androgens. The ability of immature male Myotis to exhibit a pubertal increase in plasma SBP following simple environmental manipulation should provide a valuable model for the study of the control and action of sex steroid-binding protein.

Perinatal exposure to low doses of bisphenol A affects body weight, patterns of estrous cyclicity, and plasma LH levels.

The nonsteroidal estrogenic compound bisphenol A (BPA) is a monomer used in the manufacture of polycarbonate plastics and resins. BPA may be ingested by humans as it reportedly leaches from the lining of tin cans into foods, from dental sealants into saliva, and from polycarbonate bottles into their contents. Because BPA is weakly estrogenic--approximately 10,000-fold less potent than 17beta-estradiol--current environmental exposure levels have been considered orders of magnitude below the dose required for adverse effects on health. Herein we demonstrate measurable effects on the offspring of Sprague-Dawley female rats that were exposed, via their drinking water, to approximately 0.1 mg BPA/kg body weight (bw)/day (low dose) or 1.2 mg BPA/kg bw/day (high dose) from day 6 of pregnancy through the period of lactation. Offspring exposed to BPA exhibited an increase in body weight that was apparent soon after birth and continued into adulthood. In addition, female offspring exposed perinatally to the high dose of BPA exhibited altered patterns of estrous cyclicity and decreased levels of plasma luteinizing hormone (LH) in adulthood. Administration of neither the doses of BPA that caused effects during perinatal exposure nor a 10-fold higher dose was able to evoke a uterotropic response in ovariectomized postpubertal females. These data indicate an increased sensitivity to BPA during the perinatal period and suggest the need for careful evaluation of the current levels of exposure to this compound.

Hepatic expression of sex hormone-binding globulin associated with the postnatal surge of serum androgen-binding activity in the Djungarian hamster.

Serum androgen-binding capacity in Djungarian hamsters, as in many other mammals, increases within days after birth and remains elevated until puberty. This increased activity has been attributed to a hepatic glycoprotein, sex hormone-binding globulin (SHBG), but expression of SHBG by the postnatal liver has not been demonstrated. Therefore, a full-length SHBG cDNA was cloned from the liver of neonatal hamsters and the expression of SHBG during development was examined. Hepatic SHBG RNA levels, as measured by both competitive RT-PCR and Northern analysis, were very low in fetal animals but increased significantly within 24 h of birth. Maximal values were maintained for 1 week after parturition, and then declined to basal adult levels. The developmental pattern in hepatic SHBG immunoactivity, as determined by Western analysis, mirrored that of hepatic SHBG mRNA. However, changes in serum SHBG immunoactivity and steroid-binding activity occurred approximately 1 week later. There were no sex differences in the levels of hepatic SHBG mRNA or protein during development, but serum immunoactivity tended to be higher in females at puberty. Sex- and age-related differences in the relative abundance of SHBG isoforms were also noted. Results of these studies demonstrate that Djungarian hamsters express an authentic SHBG and indicate that the postnatal surge in serum androgen-binding activity is due to perinatal up-regulation of SHBG expression.

Changes in populations of LHRH-immunopositive cell bodies following gonadectomy.

One day after castration of male rats, plasma LH rose and the number of LHRH immunopositive neuronal perikarya decreased. As plasma LH continued to rise six days and three weeks post-castration, the number of LHRH immunopositive neurons also increased. The largest population of LHRH immunopositive neurons was detected three weeks post-castration and the cell group that showed the greatest increase was in the rostral preoptic area. In females, the largest population of LHRH immunopositive neurons was observed one day post-ovariectomy; at this time plasma LH levels were not significantly elevated above diestrous levels. Six days post-ovariectomy, LH levels were elevated and the number of LHRH immunopositive cells decreased. As LH levels continued to rise three weeks post-ovariectomy, the population increased in size. In males, primarily LHRH cells of the rostral preoptic area increased in in number; in females, the cell groups that increased were scattered over the diagonal band of Broca, preoptic and anterior hypothalamic areas. Although LHRH neurons demonstrated these variations following gonadectomy, there was no evidence of alteration(s) in molecular processing of precursor hormone.

Luteinizing hormone-releasing hormone (LH-RH) cells and their projections in the forebrain of the bat Myotis lucifugus lucifugus.

Luteinizing hormone-releasing hormone (LH-RH) neurons and their projections were studied by immunocytochemistry in the brains of little brown bats (Myotis lucifugus lucifugus: Chiroptera: Vespertilionidae ) as a first step in the study of relationships between these neurons and the seasonal reproductive events characteristic of this species. The majority of immunoreactive neurons in adult male, adult female, and fetal bats were ovoid bipolar cells with one thin and one thicker process, both of which gave rise to fine varicose fibers. LH-RH-immunoreactive perikarya were concentrated in the region of the arcuate nuclei in all bats examined. Perikarya were also consistently found dispersed in the mammillary region, anterior hypothalamus, preoptic areas, septum, diagonal band of Broca, and olfactory tracts; they were occasionally observed in the dorsal hypothalamus, organum vasculosum of the lamina terminalis (OVLT), habenula, amygdala, and cingulate gyrus. LH-RH-immunoreactive fibers projected heavily to the median eminence, infundibular stalk, and posterior pituitary. In extrahypothalamic areas, these fibers were especially abundant in the stria medullaris/habenula and stria terminalis/amygdala, but also contributed to the diagonal band of Broca and the olfactory tracts. Immunoreactive fibers that may be components of many different pathways clustered in the rostral septum and permeated the medial hypothalamus. LH-RH-containing fibers frequently entered the subfornical organ, but were observed less often in the OVLT and only occasionally in the pineal. The organization of the LH-RH system in the little brown bat resembles that of primates, but differs considerably from that in the rat. Anatomical characteristics of the LH-RH system in bats thus suggest that this animal may be a particularly suitable species for further study of neuroendocrine control of reproductive function as it may relate to primates, including humans.

Androgen receptor levels and androgen contents in the prostate lobes of intact and testosterone-treated Noble rats.

Plasma testosterone (T) levels were correlated with androgen receptors, tissue content of T, and 5 alpha-dihydrotestosterone (DHT) in the three anatomically-discrete prostate lobes of intact and castrated Noble (Nb) rats bearing T-filled silastic capsules. Differences in androgen receptor content and tissue androgen levels were observed among the three prostatic lobes of intact Nb rats. Total (cytosolic and nuclear) androgen receptor levels were highest in the ventral prostate followed by the dorsolateral and anterior prostate lobes. In the ventral and anterior prostate, androgen receptors were found to be equally distributed between cytosols and nuclear extracts, whereas in the dorsolateral prostate, androgen receptors were predominantly nuclear (cytosolic: nuclear = 1.5). The ventral prostate had the highest total androgen content and DHT was the major tissue androgen in all three lobes. The ratio of tissue DHT:T varied among the lobes; the highest value was observed in the dorsolateral prostate. The higher proportions of nuclear androgen receptor, as well as the elevated tissue DHT:T found in the dorsolateral prostate compared to other lobes, suggest that differences in the androgen activation process may exist between the dorsolateral prostate and other prostatic lobes. Despite lower plasma and tissue T levels, the DHT content, weight and cytodifferentiation in all lobes of T-treated castrated rats closely approximated the situation found in intact animals. Total androgen receptor levels were, however, elevated in all prostatic lobes of T-treated, castrated rats as compared to intact controls. These increases were primarily attributed to the augmented levels of androgen receptor in the nuclear extracts of the three prostate lobes. Exposure of the prostate to a constant level of T, produced by silastic implantation, might be responsible for the higher total androgen receptor levels and enhanced nuclear androgen receptor retention found in the prostates of T-treated, castrated rats.

Variants of the human prostate LNCaP cell line as tools to study discrete components of the androgen-mediated proliferative response.

Androgens regulate the proliferation of their target cells through a sequential two-step mechanism. In the first step, androgens increase proliferation rates; following this proliferative response, a second step of proliferative shutoff ensues. Human prostate LNCaP cell variants were used to assess whether the proliferative and shutoff effects of androgens could be segregated selectively by manipulating the hormonal milieu. The LNCaP-FGC and LNCaP-LNO cell lines were derived from the same biopsy specimen but exhibited different proliferative responses. Cell proliferation was inhibited by treatment with 10% charcoal-dextran stripped human serum in the LNCaP-FGC variant but not in LNCaP-LNO cells. Physiological androgen concentrations induced a proliferative shutoff in LNCaP-LNO cells (G1 arrest), an effect also expressed by LNCaP-FGC cells. This G1 arrest was irreversible in the LNCaP-FGC variant but was reversed upon androgen withdrawal in LNCaP-LNO cells. A new variant (LNCaP-TJA) was selected from LNCaP-LNO cells treated with 30 nM methyltrienolone for 4 months; these cells proliferated maximally regardless of the presence of androgens. All three cell variants had functional androgen receptors, and androgens induced prostate-specific antigen secretion. The androgen-induced proliferative shutoff in LNCaP-FGC and LNO cells was partially antagonized by antiandrogens and was not mediated by autocrine factors. Finally, the variant LNCaP cell lines described herein are probably representative of phenotypes present in prostate cancer patients during the course of this disease and may arise from selective pressure imposed by therapeutic protocols aimed at modifying the hormonal milieu of the host.