It is now well established that testosterone can be converted intracellularly into 5α-androstan-17β-ol-3-one (dihydrotestosterone, DHT), 5α-androstan-3β,17β-diol (3β-diol) and 5α-androstan-3α,17β-diol (3α-diol) in order to become fully active on androgen-dependent peripheral structures (Robel, 1971). Massa, Stupnicka, Kniewald & Martini (1972) have shown that testosterone is also converted into DHT, 3α-diol and 3β-diol in the anterior pituitary and in the hypothalamus, i.e. in those structures on which androgens exert their feedback control on gonadotrophin secretion. The experiments to be described here were designed to discover whether: (a) the three 5α-reduced metabolites of testosterone mentioned above might exert an inhibitory action on gonadotrophin secretion; (b) the feedback activity of these steroids might be correlated with their androgenic potencies.

Adult male Sprague—Dawley rats, castrated 3 weeks before use, were each given one single subcutaneous injection of 2 mg of testosterone, DHT, 3α-diol or 3β-diol. All steroids were used in the free-alcohol form. Animals

ABSTRACT

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 5α-reduced or oestrogenic metabolites, testosterone, 5α-androstan-17β-ol-3-one (DHT), 5α-androstane-3α, 17β-diol (3α-diol) and 5α-androstane-3β,17β-diol (3β-diol) (2 mg/rat per day for 6 days) and oestradiol (0·1, 0·5, 1·0 and 5·0 μg/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 5α-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-17β or in the benzoate form.

Treatment with testosterone, DHT, 3α-diol and 3β-diol resulted in a significant decrease in serum LH levels; all the 5α-reduced testosterone derivatives were more effective than testosterone in this respect. Testosterone and DHT propionates suppressed LH release following orchidectomy totally; 3α-diol and 3β-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 5α-reduced steroids was capable of modifying the low intrahypothalamic levels of LHRH found following orchidectomy; only 3α-diol dipropionate exhibited some activity, but this was much lower than that of testosterone propionate.

Oestradiol-17β was totally ineffective in decreasing serum LH in orchidectomized animals; in contrast, oestradiol benzoate progressively decreased serum LH. Oestradiol in the free form was unable to increase LHRH stores, as was oestradiol benzoate except at the highest dose.

The results suggest that the effect exerted by testosterone on hypothalamic LHRH is due to the hormone as such and does not involve its conversion into either 5α-reduced or oestrogenic metabolites.

J. Endocr. (1986) 109, 291–296

ABSTRACT

Studies were undertaken using the opiate receptor antagonist naloxone to examine the hypothesis that endogenous opiates may have a restraining effect on prepubertal gonadotrophin secretion and may be involved in the maturation of the central nervous system mechanisms regulating the onset of puberty in the female rat. Naloxone (2·5 mg/kg) administered intraperitoneally every 6 h to female rats from day 1 to day 10 of postnatal life significantly (P <0·001) advanced the age of onset of puberty assessed in terms of the day of vaginal opening and first oestrus (32·3 ± 0·2 vs 40·8 ± 0·4 days in control saline-treated animals). Animals so treated with naloxone showed significantly (P < 0·001) higher levels of FSH (761·4 ± 87·6 vs 483·8± 57·2 μg/l in control animals) and LH (562·8 ± 57·4 vs 351·3 ± 43·3 μg/l in control animals) at the first late pro-oestrus and a significantly (P < 0·001) higher number of ova released at first oestrus (12·4 ± 0·4 vs 8·1±0·3 in controls). Body weight at first oestrus was significantly (P <0·001) lower in the naloxone-treated animals, an indication that these animals were much younger. The weights (per 100 g body wt) of the ovaries and uteri at the first oestrus were significantly (P <0·01) higher in the naloxone-treated rats than in the controls. However, there were no significant differences in the weights of the adrenals and anterior pituitary glands between the two groups of animals. A study of the cyclic patterns of the neonatally naloxone-treated animals performed for 15 consecutive cycles after the first oestrus showed normal 4- or 5-day cycles similar to those occurring in the saline-treated animals. The lengths of the first and second cycles in the naloxone-treated animals were not significantly different from controls. No significant differences in body weight or in organ weights at oestrus or in the levels of LH and FSH determined during the various stages of the oestrous cycle were found between naloxone- and saline-treated animals when these parameters were examined at 3 months of age. Naloxone had no effect on onset of puberty when administered during the other stages of prepubertal life. The mechanisms by which naloxone acts specifically during the neonatal period to induce precocious puberty are at present not known but are being investigated; they may be related to naloxone-induced alterations in the inhibitory synaptic arrangements between opiatergic and gonadotrophin-releasing hormone (GnRH) neurones, with a resulting decrease in the inhibitory influence exerted by endogenous opioids on GnRH neurones during this period of intense neurological development. The results suggest that the endogenous opiate peptides could play a key role in the central mechanisms which trigger the onset of puberty in the female rat.

J. Endocr. (1985) 104, 299–307

SUMMARY

The neurohaemal part of the median eminence of the rat hypothalamus is characterized by numerous nerve terminals which end near a rich network of fenestrated capillaries. An attempt was made to isolate different types of nerve terminals by means of sucrose density-gradient centrifugation. The subcellular fractions obtained were assayed for dopamine, noradrenaline and 5-hydroxytryptamine. In addition FSH- and GH-releasing activities were determined. A sample of each fraction obtained was taken for electron microscopical observations.

Dopamine, noradrenaline, 5-hydroxytryptamine, GH- and FSH-releasing factors were present in higher concentration in the nerve endings. A further fractionation showed that noradrenaline was present in the lightest synaptosomal band, dopamine in the middle one, and 5-hydroxytryptamine in the heaviest. GH-RF and FSH-RF were recovered mainly from the band containing dopamine. The relevance of this localization to the physiological role of the median eminence is discussed.

Androgens have been found in mammary epithelium and in milk throughout the cycle of the mammary gland in vivo. The aim of this study was to investigate the possible role of these substances in mammary epithelial growth and differentiation in the mouse HC11 cell line. Cells were stimulated with testosterone, dihydrotestosterone, androstenedione and 5alpha-androstane-3alpha,17beta-diol at concentrations ranging between 0.3 nM and 30 nM. Cyproterone acetate or flutamide, androgen receptor antagonists, (3 microM) were used to block specific androgen effects. Proliferative effects were measured by an MTT (tetrazolium blue) conversion test and [(3)H]thymidine uptake. HC11 cells were transfected with pbetacCAT, a chimeric rat beta-casein gene promoter-chloramphenicol acetyl transferase (CAT) gene construct and CAT ELISA was used to determine gene expression. RT-PCR was performed to detect androgen receptor expression. After 24, 48 and 72 h androgens significantly (P<0.05) increased proliferation. Androgen antagonists significantly (P<0.05) reduced the proliferative effects. Furthermore androgens potentiated the lactogenic effect of prolactin, insulin and dexamethasone (P<0.05). Finally, the androgen receptor gene was expressed in both proliferating and differentiated HC11 cells. These observations lead us to hypothesize an activity of this class of steroids in mammary physiology. In particular, androgens stimulate cell proliferation and beta-casein gene expression; this influence appears to be mediated by androgen receptors.

Somatostatin analogues inhibit in vitro cell proliferation via specific membrane receptors (SSTRs). Recent studies on transfected cell lines have shown a ligand-induced formation of receptor dimers. The aim of this study is 1) to evaluate the role of specific ligands in modulating receptor interactions in the androgen-dependent prostate cancer cell line, LNCaP, and in the non-small cell lung cancer line, Calu-6, by co-immunoprecipitation and immunoblot; and 2) to correlate the antiproliferative effect of these compounds with their ability in modulating receptor interactions. In LNCaP, we have demonstrated the constitutive presence of sstr₁/sstr₂, sstr₂/sstr₅, sstr₅/dopamine (DA) type 2 receptor (D₂R), and sstr₂/D₂R dimers. BIM-23704 (sstr₁- and sstr₂-preferential compound) increased the co-immunoprecipitation of sstr₁/sstr₂ and significantly inhibited proliferation (−30.98%). BIM-23244 (sstr₂–sstr₅ selective agonist) significantly increased the co-immunoprecipitation of sstr₂/sstr₅, and induced a −41.36% inhibition of proliferation. BIM-23A760, a new somatostatin/DA chimeric agonist with a high affinity for sstr₂ and D₂R and a moderate affinity for sstr₅, significantly increased the sstr₅/D₂R and sstr₂/D₂R complexes and was the most powerful in inhibiting proliferation (−42.30%). The chimeric compound was also the most efficient in modulating receptor interaction in Calu-6, increasing the co-immunoprecipitation of D₂R/sstr₅ and inhibiting cell proliferation (−30.54%). However, behind BIM-23A760, BIM-53097 (D₂R-preferential compound) also significantly inhibited Calu-6 proliferation (−17.71%), suggesting a key role for D₂R in receptor cross talk and in controlling cell growth. Indeed, activation of monomeric receptors did not affect receptor co-immunoprecipitation, whereas cell proliferation was significantly inhibited when the receptors were synergistically activated. In conclusion, our data show a dynamic ligand-induced somatostatin and DA receptor interaction, which may be crucial for the antiproliferative effects of the new analogues.