Inhibition of polyadenylation of mRNA by gonadotropin releasing hormone

The mechanism of extra pituitary inhibitory action of gonadotropin releasing hormone (GnRH) was investigated. Simultaneous injection of GnRH caused dose dependent inhibition of dihydrotestosterone (DHT) induced poly(A) polymerase in the ventral prostate of rat. In addition injection of GnRH to DHT treated animals caused reduced incorporation of 3H-uridine into poly(A)+ mRNA. Since poly(A) segment is known to help in translation of mRNA, it is possible that the inhibitory effect of GnRH is due to the inhibition of polyadenylation of mRNA.     

Metabolism of testosterone by tes epididymis and ventral prostate of rat and its inhibition by cyproterone acetate

The metabolism of ³H-testosterone by the epididymis and accessory organs of adult male rats exposed continuously to microdoses of cyproterone acetate from subcutaneous capsules were studied. The major metabolite of ³M-testosterone in the epididymis, vas deferens and ventral prostate of control rat was dihydrotestosterone while the formation of androstanediol by these tissues was low. The highest percentage of DHT was formed by the ventral prostate and cauda epididymis. In rats exposed to cyproterone acetate for four months, the conversion of testosterone to DHT was inhibited in all the tissues but maximally in the ventral prostate and cauda epididymis. In these rats, the secretory function of the ventral prostate was normal while that of the epididymis was markedly decreased. These data are discussed based on the differential thresholds of androgens required to regulate the functions of the accessory organs.     

Differential effects of 2-difluoromethylornithine and methylglyoxal bis(guanylhydrazone) on the testosterone-induced growth of ventral prostate and seminal vesicles of castrated rats

2-Difluoromethylornithine totally prevented any increases in putrescine and spermidine concentrations in the ventral prostate of castrated rats during a 6-day testosterone treatment. Prostatic ornithine decarboxylase activity was inhibited by 80%, whereas S-adenosylmethionine decarboxylase was stimulated by more than 9-fold. In seminal vesicle, the inhibition of putrescine and spermidine accumulation, as well as of ornithine decarboxylase activity, was only minimal, and no stimulation of S-adenosylmethionine decarboxylase was observed. Administration of methylglyoxal bis(guanylhydrazone) to castrated androgen-treated rats resulted in a marked increase in concentrations of all prostatic polyamines. Prostatic ornithine decarboxylase activity was nearly 2 times and adenosylmethionine decarboxylase activity 9 times higher than that of the testosterone-treated animals. In contrast with ventral prostate, methylglyoxal bis(guanylhydrazone) treatment inhibited moderately the accumulation of spermidine and spermine in seminal vesicle, although both ornithine decarboxylase and S-adenosylmethionine decarboxylase activities were stimulated. Difluoromethylornithine inhibited significantly the weight gain of ventral prostate, but methylglyoxal bis(guanylhydrazone) produced a substantial increase in prostatic weight. These changes were largely due to the fact that the volume of prostatic secretion was greatly decreased by difluoromethylornithine, whereas methylglyoxal bis(guanylhydrazone) increased the amount of secretion. Treatment with difluoromethylornithine strikingly increased the methylglyoxal bis(guanylhydrazone) content of both ventral prostate and seminal vesicle, but even under these conditions the drug concentration remained low in comparison with other tissues. The results indicate that a combined use of these two polyamine anti-metabolites does not necessarily result in a synergistic growth inhibition of the androgen-induced growth of male accessory sexual glands.     

Action of a novel nonsteroidal antiandrogen, AA560

The antiandrogenic properties of a new nonsteroidal antiandrogens, AA560 (N-2-chloromethyl-2-hydroxypropionyl)-3, 4, 5-trichloroaniline) were investigated. The ventral prostate, dorselateral prostate and coagulating gland weights in rats given AA560 at 1-9 mg/head were significantly less than those in the intact rats. The seminal vesicle weights in rats given 3-9 mg/head were significantly less than those of the intact group. In intact animals given daily 3 or 9 mg of AA560 there were significantly increases of serum FSH, LH and testosterone concentrations. In the in vivo experiment, the pretreatment with AA560 decreased the uptake of 3H-androgens in the nuclear fraction of the ventral prostate. On the other hand, a significant increase in the uptake of 3H-radioactivity in the cytosol fraction was observed. It was proved by the in vitro displacement study that AA560 inhibited the binding of 5 alpha-dihydrotestosterone with a receptor protein in the prostatic cytosol.     

Modifications of the gonadal function in the adult rat after fetal exposure to spironolactone

The effects of fetal exposure to spironolactone (SPL), an aldosterone antagonist with weak antiandrogen and gestagen properties, upon the pituitary-gonadal axis were studied in the offspring of rats that had been treated daily from gestation day 14 to day 20 with 10 or 20 mg SPL or the solvent vehicle (for controls). At 70-80 days of age, SPL-exposed rats showed no alterations in external genitalia or in body weight. However, males displayed a dose-dependent decrease in the weights of the ventral prostate and seminal vesicles. Whereas basal and gonadotropin-releasing hormone (GnRH)-induced plasma luteinizing hormone (LH), follicle-stimulating hormone (FSH), testosterone, and 5 alpha-dihydrotestosterone levels were similar to controls, basal plasma and pituitary prolactin (Prl) levels were reduced (SPL-exposed 6.8 +/- 1.0 vs. controls 15.8 +/- 2.8 ng/ml and 6.1 +/- 1.2 vs. 11.6 +/- 1.8 microgram/anterior pituitary gland; mean +/- SEM). Cytosolic androgen receptors in ventral prostate were nonsignificantly decreased, but they increased after GnRH in contrast to controls. Nuclear androgen receptors were normal. Females displayed normal estrous cycles. Basal and GnRH-induced plasma FSH, Prl, estradiol, and progesterone concentrations were similar to controls, whereas plasma LH was elevated. Estrogen receptors in uterine cytosol were low and increased after GnRH. Ovaries and uteri were enlarged. The present study demonstrates that in utero exposure to SPL leads to endocrine dysfunctions that persist into adulthood. They are characterized in males by hypoprolactinemia, reduced weights of accessory sex organs, and a suggestion of functional modifications of androgen receptors. In females they are characterized by increased LH secretion, increased ovarian and uterine weights, and decreased uterine cytosol estrogen receptors, suggesting enhanced estrogenic action.     

Enzymic properties of a Ca2+-dependent protease in rat ventral prostate: differences in distribution between lobes of the prostatic complex

Soluble extracts of rat ventral prostate contain a calcium-dependent, neutral thiol protease which is separated from an endogenous inhibitor by DEAE-cellulose chromatography. The Ca2+-dependent protease had a high calcium requirement (half maximal activation at 0.19 mM CaCl2), a pH optimum in the neutral range (pH 7-8), and it was inhibited by increased ionic strength (30% inhibition at 0.2 M NaCl). Leupeptin and antipain were strong inhibitors of the enzyme. Ca2+-activated protease activities of the coagulating gland (anterior prostate) were about 40% of those of the ventral prostate and were not detectable in the dorsolateral prostatic lobe. There was no difference in specific activities of this enzyme in chromatographed extracts of prostatic lobes from young sexually mature adults and 12 month old retired breeders. In addition, Ca2+-dependent protease activity was not detectable in chromatograms of rat ventral prostate and coagulating gland secretions. Therefore, the Ca2+-activated protease does not appear to be a secretory protein and probably acts at some intracellular site(s).     

Differential response of testis and serum gonadotropins to testosterone in rats treated with a gonadotropin releasing hormone antagonist or estradiol 17 beta.

Adult rats treated with a GnRH antagonist (Ac D2Nal1, D4Cl Phe2, DTrp3, DArg6, DAla10 GnRH; code: 103-289-10, National Institutes of Health, USA) for 5 weeks (250 micrograms/kg b.w) showed multiple degrees of impairment and atrophy of the genital organs concomitant with decreased serum levels of testosterone, LH and FSH. Inhibition of spermatogenesis was characterized by germ cell degeneration and overall decline in different cell numbers and in particular, spermatids of any kind were completely absent. Testosterone supplementation (60 micrograms/rat/day, sc) to GnRH antagonist treated rats, for the same period, significantly elevated the weights of the sex organs, and the serum levels of hormones. Spermatogenesis was improved both qualitatively and quantitatively; albeit failed to be restored back to control levels. Treatment with estradiol 17 beta (1 microgram/rat/day) for 5 weeks had insignificant effect on spermatogenesis but the weights of the genital organs (seminal vesicles by 19% and ventral prostate by 40%) and the levels of serum hormones (LH by 24%, FSH 22% and testosterone by 25%) were otherwise reduced. Administration of testosterone either alone or in combination with estradiol 17 beta had only a marginal effect on spermatogenesis or on other reproductive parameters. The results indicate a positive shift in the response of the testis and serum levels of gonadotropins to testosterone supplementation in rats treated with either GnRH antagonist or estradiol 17 beta.     

GnRH systems of <Emphasis Type="Italic">Cichlasoma dimerus</Emphasis> (Perciformes,Cichlidae) revisited: a localization study with antibodies and riboprobes to GnRH-associated peptides

The distribution of cells that express three prepro-gonadotropin-releasing hormones (GnRH), corresponding to salmon GnRH, sea bream GnRH (sbGnRH), and chicken II GnRH, was studied in the brain and pituitary of the South American cichlid fish, Cichlasoma dimerus. Although the ontogeny and distribution of GnRH neuronal systems have previously been examined immunohistochemically with antibodies and antisera against the various GnRH decapeptides, we have used antisera against various perciform GnRH-associated peptides (GAPs) and riboprobes to various perciform GnRH+GAPs. The results demonstrate that: (1) the GnRH neuronal populations in the forebrain (salmon and sea bream GAPs; sGAP and sbGAP, respectively) show an overlapping pattern along the olfactory bulbs, nucleus olfacto-retinalis, ventral telencephalon, and preoptic area; (2) projections with sGAP are mainly located in the forebrain and contribute to the pituitary innervation, with projections containing chicken GAP II being mainly distributed along the mid and hindbrain and not contributing to pituitary innervation, whereas sbGAP projections are restricted to the ventral forebrain, being the most important molecular form in relation to pituitary innervation; (3) sbGnRH (GnRH I) neurons have an olfactory origin; (4) GAP antibodies and GAP riboprobes are valuable tools for the study of various GnRH systems, by avoiding the cross-reactivity problems that occur when using GnRH antibodies and GnRH riboprobes alone.This work was supported by grants UBACYT X-217, Conicet PIP 0539/R188; NIH-HD-29186 and Fogarty International Fellowship TW00086.     

Melengestrol acetate and megestrol acetate are prostatic tumor inhibiting agents

We had previously reported that 6-methylene progesterone, an inhibitor of 5 alpha-reductase, the enzyme which converts testosterone to dihydrotestosterone, markedly inhibited growth of the androgen-dependent Dunning R3327-H rat prostatic tumors. We now find that the progesterone derivatives melengestrol acetate (MGA) and megestrol acetate (MA) inhibit both the androgen-dependent (Dunning R3327-H) and the androgen-independent (Dunning R3327-AT3) prostatic tumors. Growth of the AT3 tumors was suppressed by approximately 53% after 9 days of daily s.c. injections with MGA at 10 mg/kg body weight. MGA also caused a 54% weight reduction of the ventral prostate and a 53% reduction of the seminal vesicles. Adrenal weights were reduced by 42%. A 24-day oral treatment with MGA (at approximately 15-17 mg/(kg.day)) inhibited AT3 tumor growth by 59% and caused a weight reduction in the following tissues: prostate (46%), seminal vesicles (19%), testes (12%), and adrenals (52%). Under the same protocol, MA inhibited AT3 tumor growth by 32% and reduced the weight of the ventral prostate by 49% and the weight of the adrenals by 18%, but had no effect on the seminal vesicles and testes. The extent of the MGA-induced prostatic regression was accompanied by cytological changes similar to those effected by 6-methylene progesterone, i.e., shrinking of the acinar epithelium. The AT3 tumors in MGA-treated rats displayed a limited degree of apoptosis. Atrophy of the adrenal cortex and lowered plasma levels of corticosterone and dihydroepiandrosterone were also observed. A therapeutic role for MGA and MA against androgen-independent prostatic neoplasms in man is forecast by these observations.     

Peptides and antitumor activity. Development and investigation of some peptides with antitumor activity

We developed a group of synthetic analogs of GnRH and Somatostatin to inhibit the tumor growth of different kind. The GnRH analogs decreasing the gonadotroph and steroid hormone levels act on the hormone dependent tumors and influence their growth. One of the most effective antitumor analog was patented under the name FOLLIGEN which inhibited the breast cancer caused by DMBA in rats without any side-effects. Other inhibitory analogs of GnRH with long-lasting effect were effective in the treatment of breast, ovary and prostate tumors. Another analog [alpha-Asp(DEA)]6,Gln8-hGnRH showed a very low endocrine but high antitumor effect in both in vitro and in vivo experiments. Its tritium labeled derivative exhibited specific binding sites on human tumor cell lines. We synthesized the analogs of GnRH-III with effective selective antitumor activity which does not alter the ovarian cycle of rats but inhibits the colony-formation of human breast cancer cell lines and has a significant antiproliferative effect. We also synthesized conjugates of potent GnRH analogs with a branched chain polylysine backbone which induce a 33-35% decrease of cell numbers of MCF-7 and MDA-MB-231 human breast cancer cell lines and 45-50% inhibition of cell proliferation. Another conjugate decreased the tumor growth of MDA-MB-231 xenografts by 80% in a treatment of 9 weeks and even tumor free animals could be found among the ones treated. Using these radiolabeled peptide hormone analogs we found that human tumor cell lines and xenografts specifically bind the GnRH conjugates. We also synthesized a series of Somatostatin analogs which inhibit tyrosine kinases and the growth of several breast, prostate and colon tumor cell lines. One of our best analogs was a heptapeptide, TT-232, which strongly inhibited the tyrosine kinase activity and the cell-proliferation in different colon tumor cells. However, it did not inhibit the growth hormone release either in vitro or in vivo from rat pituitary cells. The TT-232 was found to be effective on 60 human tumor cell lines, it significantly inhibited the tumor growth on different animal tumor models, and induced apoptosis, as a result of which some animals became tumor free. The TT-232 inhibited the tumor growth of PC3 prostate xenografts with 60% and caused a 100% survival of mice 60 days after the transplantation. It is being preclinically tested at present. We have shown that the new GnRH analogs acting without any hormonal effect and the Somatostatin analogs with strong antitumor and tyrosine kinase inhibitory activity but no hormonal effect may represent a breakthrough in the research of the antitumor peptides, having direct effect on tumor cells.     

Axl and Tyro3 modulate female reproduction by influencing gonadotropin-releasing hormone neuron survival and migration

GnRH neurons must undergo a complex and precise pattern of neuronal migration to appropriately target their projections to the median eminence to trigger gonadotropin secretion and thereby control reproduction. Using NLT GnRH cells as a model of early GnRH neuronal development, we identified the potential importance of Axl and Tyro3, members of the TAM (Tyro3, Axl, and Mer) family of receptor tyrosine kinases in GnRH neuronal cell survival and migration. Silencing studies evaluated the role of Tyro3 and Axl in NLT GnRH neuronal cells and suggest that both play a role in Gas6 stimulation of GnRH neuronal survival and migration. Analysis of mice null for both Axl and Tyro3 showed normal onset of vaginal opening but delayed first estrus and persistently abnormal estrous cyclicity compared with wild-type controls. Analysis of GnRH neuronal numbers and positioning in the adult revealed a total loss of 24% of the neuronal network that was more striking (34%) when considered within specific anatomical compartments, with the largest deficit surrounding the organum vasculosum of the lamina terminalis. Analysis of GnRH neurons during embryogenesis identified a striking loss of immunoreactive cells within the context of the ventral forebrain compartment (36%) and not more rostrally. Studies using caspase 3 cleavage as a marker of apoptosis showed that Axl(-/-), Tyro3(-/-) double-knockout mice had increased cell death in the nose and dorsal forebrain, supporting the underlying mechanism of cell loss. Together these data suggest that Axl and Tyro3 mediate the survival and appropriate targeting of GnRH neurons to the ventral forebrain, thereby contributing to normal reproductive function and cyclicity in the female.     

Localization of protein phosphokinase activities in the nucleolus distinct from extra-nucleolar regions in rat ventral prostate nuclei

Rat ventral prostate nucleoli contain protein phosphokinase(s) which have distinctly different characteristics from protein phosphokinase(s) localized in the extra-nucleolar regions of the nucleus. The differences pertain to pH vs activity profiles and activation by divalent cations, utilizing dephospho-phosvitin as substrate. Lysine-rich and arginine-rich F3 histones are also phosphorylated by nucleolar protein phosphokinase(s). Phosphorylation of histones by either nucleolar or extra-nucleolar fractions was not stimulated by cAMP, whereas that of phosvitin was slightly inhibited.     

Studies on the mechanism of action of Win 49596: a steroidal androgen receptor antagonist

Win 49596 is an orally active antiandrogen in the rat. This report describes a series of in vitro and in vivo studies which were performed to characterize the mechanism of action of this compound. In vitro competition and Lineweaver-Burk analyses indicate that Win 49596 binds competitively to the rat ventral prostate androgen receptor with a Ki of 2.2 +/- 0.4 microM. Similar to other androgen antagonists, the relative binding affinity (RBA) of Win 49596 was greater after 1 h of incubation with androgen receptor than after an 18 h incubation (RBA of 2.2 versus 0.05, respectively). Win 49596 did not bind to rat cytosolic uterine estrogen or progesterone receptors or thymus glucocorticoid receptors. Furthermore, Win 49596 did not inhibit rat ventral prostate 5 alpha-reductase or 3 alpha-oxidoreductase, rat adrenal 3 beta-hydroxysteroid dehydrogenase or human placental aromatase activity in vitro at concentrations as high as 10 microM. A series of in vivo studies demonstrated that Win 49596 inhibited the uptake of [3H]testosterone as well as testosterone-induced nuclear accumulation of androgen receptor in the rat ventral prostate. Collectively, these results support direct androgen receptor antagonism as the mechanism for the antiandrogenic effects of Win 49596.     

ADP-ribosylation of nuclear proteins in rat ventral prostate during ageing

Poly(ADPR)polymerase activity and poly(ADP-ribosyl)ation of nuclear proteins have been investigated in ventral prostate nuclei of different aged rats (14, 28, 60, 180, 360 day old animals), by reverse-phase HPLC and acetic acid-urea polyacrylamide gel electrophoresis. The major ADP-ribose acceptor proteins were identified as histone H1 and H2b. It is concluded that concomitant with major changes to chromatin organization, poly(ADP-ribosyl)ation reaction is progressively inhibited during aging of rat ventral prostate. These results support the hypothesis that prostatic dysfunction in senescent animals is related to a failure of DNA repair mechanisms and deregulated template activity.     

Immunohistochemical localization of gonadotropin-releasing hormone (GnRH) in the fetal and early postnatal mouse brain.

The objectives were to (a) determine the age in development when GnRH is first detectable in the brain and (b) observe the distribution of GnRH throughout the fetal and early postnatal period. GnRH was localized immunohistochemically in fetal (15, 16, 17 and 19 days of gestation) and early postnatal (1- and 7-day-old) mice with the peroxidase-antiperoxidase (PAP) method of Sternberger. In the organum vasculosum of the lamina terminalis (OVLT) and in the median eminence of the fetus, GnRH was first detected at 17 days of gestation. In the OVLT, GnRH was found ventral to the preoptic recess of the third ventricle near the ventral surface of the brain. In addition, GnRH was located adjacent to the superficial portal capillaries near the surface of the median eminence. At 19 days of gestation, the distribution of GnRH was similar to that observed at 17 days and there was a marked increase in amount. In the newborn mouse, GnRH was undetectable in the OVLT and its content in the median eminence was decreased as compared to that observed in the fetus. By the seventh postnatal day, a considerable accumulation of GnRH had occurred in the OVLT and median eminence. In the OVLT, it was associated with capillaries ventral to the preoptic recess, and its distribution in the median eminence was similar to that in the adult mouse. In both the OVLT and median eminence of the fetal and early postnatal mouse GnRH appeared to be stored in axons and axon endings, but was not detectable in nerve cell bodies or ependymal cells. These observations suggest that the potential for neuroendocrine control of gonadotropin secretion exists in the fetal mouse early as 17 days of gestation.