Activin-inhibitory action on lactotrophs is decreased in lactotroph hyperplasia.

Among all the hormone-secreting pituitary tumours, prolactinomas are the most frequently found in the clinic. Since dopamine is the primary inhibitor of lactotroph function, dopamine agonists represent the first-line therapy. However, a subset of patients exhibits resistance to these drugs, and therefore, alternative treatments are desired. As activins inhibit prolactin gene expression through the inhibition of Pit-1 involving the p38MAPK pathway, in the present work, we studied the local activin system as an alternative inhibitory system for lactotroph hyperplasia treatment. We used two different mouse models of prolactinoma: transgenic mice with overexpression of the human chorionic gonadotropin ß-subunit (hCGß) and mice lacking dopamine receptor type 2. In both models, females, but not males, develop lactotroph hyperplasia from the fourth month of life. We found reduced expression of pituitary activin subunits and activin receptors in hyperplastic pituitaries from both models compared with wild-type counterparts. Consequently, hyperplastic pituitaries presented a reduced activin-inhibitory action on prolactin secretion. Additionally, while female wild-type lactotrophs presented high levels of phospho-p38MAPK, it was lost in prolactinomas, concomitant with decreased activin expression, increased Pit-1 expression and tumour development. In contrast, male pituitaries express higher mRNA levels of activin subunits ßA and ßB, which would suggest a stronger activin inhibitory function on lactotrophs, protecting this sex from tumour development, despite genotype. The present results highlight the importance of the activin inhibitory action on lactotroph function and place the local activin system as a new target for the treatment of dopamine agonist-resistant prolactinomas.

Hormonal regulation of pituitary FSH sialylation in male rats.

Sialic acid content in FSH is modulated by GnRH and sexual steroids. Galbeta1,3GlcNAcalpha2,3-sialyltransferase (ST3Gal III) and Galbeta1,4GlcNAcalpha2,6-sialyltransferase (ST6Gal I) incorporate sialic acid residues into FSH oligosaccharides. The aim of the present study was to assess pituitary FSH molecular microheterogeneity and ST3Gal III/ST6Gal I expression during sexual development and after castration in male rats. Preparative isoelectric focusing and lectin chromatography were used to isolate FSH glycosylation variants according to charge and complexity of their oligosaccharides; RT-PCR and immunohistochemistry were employed to analyse sialyltransferase expression. Sexual development was associated with a progressive shift towards more acidic/sialylated FSH glycoforms concomitantly with an increment in ST6Gal I gene and protein expression. After castration, a transient decrease followed by a marked increase in ST6Gal I expression were observed. Less acidic/sialylated FSH glycoforms bearing incomplete oligosaccharides increased after castration, despite high ST6Gal I expression. ST3Gal III expression remained unchanged in all the experimental conditions examined. These results show that the synthesis of FSH isoforms possessing alpha2,6-linked sialic acid is hormonally regulated in male rats.

Luteinizing hormone receptor knockout (LuRKO) mice and transgenic human chorionic gonadotropin (hCG)-overexpressing mice (hCG alphabeta+) have bone phenotypes.

Considerable attention has been paid to the role of sex steroids during periods of major skeletal turnover, but the interaction of the gonadotropic hormones, which include LH, FSH, and human chorionic gonadotropin (hCG), within bone tissue have been overlooked. The question is pertinent due to the recent detection of extragonadal expression of gonadotropin receptors. Western blotting, immunolocalization, and RT-PCR supported the presence of osteoblast LH receptors. However, osteoblast cells failed to bind [(125)I]hCG and treatment with hCG failed to generate either cAMP or phosphorylated ERK 1/2. Bone mineral density (BMD) and bone histomorphometry were examined in the following models: 1) LH receptor null mutant (LuRKO) mice; 2) transgenic mice overexpressing hCG (hCG alphabeta+); and 3) ovariectomized (OVX) hCG alphabeta+ model. Male LuRKO mice showed a decrease in BMD after 5 months, apparently secondary to suppressed gonadal steroid production. Similarly, 9- to 10-wk-old female LuRKO mice exhibited decreases in histomorphometric parameters tested. The data indicate that loss of LH signaling results in a reduction in bone formation or an increase in bone resorption. By contrast, there were significant increases in BMD and histomorphometric indices for female, but not male, hCG alphabeta+ mice, indicating that chronic exposure to hCG results in bone formation or a decrease in bone resorption. However, OVX of the hCG alphabeta+ mice resulted in a significant reduction in BMD comparable to OVX WT controls. Although gonadotropin levels are tightly linked to sex steroid titers, it appears that their effects on the skeleton are indirect.

Ultrastructural and quantitative immunohistochemical changes induced by nonsteroid antiandrogens on pituitary gonadotroph population of prepubertal male rats.

Specific blockade of the androgen receptor by the nonsteroid antiandrogens flutamide and Casodex has proven to be a useful tool for studying androgens in vivo. The aim of the present study was to investigate the effect of antiandrogen administration at the pituitary level by evaluating the ultrastructural changes in gonadotrophs, in correlation with the quantitative immunohistochemical findings, and by comparing these alterations with the effect of androgen deprivation by castration either with or without subsequent androgen replacement. Male Sprague-Dawley rats (23 days old) were grouped as follows: (1) controls, (2) flutamide-injected (10 mg/rat/day), (3) Casodex-injected (10 mg/rat/day), (4) castrated, and (5) castrated plus androgen-replaced (dihydrotestosterone propionate; 40 microg/rat/day). Groups were sacrificed after 10 days of maintenance under each condition. Pituitaries were processed for both light and electron microscopy. Serial sections (4 microm) were obtained at different levels and immunostained by means of the primary murine monoclonal antibodies anti-FSH and anti-LH and a peroxidase-mediated EnVision System (Dako). Volume density, cell density and mean cell area were measured with an image analysis system (Imaging Technology, Software Optimas 5.2). The mean cell area (p < 0.001) and the volume density (p < 0.05) increased significantly in the flutamide- and Casodex-treated groups as well as the castrated group of FSH and LH cells. On the other hand, androgen replacement in the castrated rats, however, reduced in both parameters related to control animals. The cell density of FSH-secreting cells was increased (p < 0.05) in the Casodex and flutamide treatment as well as castrated group. The cell density of LH-secreting cells was augmented (p < 0.05) in the Casodex-treated group, while there was no increase in such parameter with flutamide and castration. The ultrastructure of all groups showed two types of gonadotrophs. Type I cells contained large (300-500 nm) and small (150-200 nm) secretory granules, while type II cells were smaller, and exhibited only small granules (100-200 nm). Flutamide-treated, Casodex-treated and castrated groups presented a decreased number of secretory granules with some exocytotic profiles, well-developed rough endoplasmic reticulum and an expanded Golgi complex of both types of cells. The gonadotrophs from the castrated group exhibited numerous mitochondria with electron-dense ring-shaped laminar figures, while in the castrated plus androgen-replaced rats only a few mitochondria had similar changes to those observed in castrated animals, as a possible residual alteration. Finally, the gonadotrophs from flutamide-treated rats showed mitochondrial alterations with clear areas and isolated electron-dense laminar figures. In summary, we conclude that lack of androgen reaction through the effects of nonsteroid antiandrogens and castration on prepubertal rats produced a hypertrophia-hyperplasia of the FSH cells, and hypertrophia of LH-secreting cells, with marked alterations at the ultrastructural level suggestive of a hyperstimulation stage.

Androgen regulation of immunological and biological activities of pituitary follicle-stimulating hormone isoforms in male rats.

Follicle-stimulating hormone (FSH) is involved in the regulation and maintenance of gametogenesis. It exists in multiple molecular forms with different oligosaccharide structures which in turn are influenced by the hormonal milieu. Previous studies from our laboratory demonstrated that antiandrogen administration to immature male rats altered the biological activity and the distribution profile of pituitary FSH isoforms. The aim of this study was to examine possible modifications in pituitary FSH polymorphism throughout sexual development (10-, 32- and 75-day-old rats). In addition, the effect of androgen deprivation by castration (32-day-old rats) and its replacement with a nonaromatizable androgen - dihydrotestosterone - on pituitary FSH polymorphism was determined. Concanavalin A affinity chromatography was used to isolate groups of FSH isoforms according to their carbohydrate inner structure. Radioimmunoassay and Sertoli cell bioassay were used to evaluate FSH immuno- and bioactivities. Androgen rise in serum was accompanied by a marked increase in pituitary bio- and immuno-FSH content in 32- and 75-day-old rats. However, FSH pituitary content did not vary despite the significant increment observed in serum FSH levels after castration and decrease to control levels after androgen replacement. The distribution profile of immuno- and bioactive FSH changed throughout sexual maturation. The proportion of pituitary FSH isoforms bearing complex oligosaccharide structures (triantennary, bisecting, complete and truncated biantennary) increased with age, with a concomitant decrease in the proportion of isoforms bearing incomplete carbohydrate chains. The distribution profile observed in castrated 32-day-old rats was similar to that determined in 10-day-old animals. Androgen replacement restored the distribution profile to normal. These results suggest that androgens regulate the incorporation of sugar residues to the carbohydrate chains of pituitary FSH favoring the biosynthesis of complex-type oligosaccharide structures.

Effects of androgens and antiandrogens on the quantitative immunohistochemistry of gonadotrope cells in prepubertal male rats.

In the male rat, androgens are involved in the feedback regulation of gonadotropin synthesis and secretion. Specific androgen-receptor blockade by the nonsteroidal antiandrogens, flutamide and Casodex, has proven to be a valid tool for studying androgen effects in vivo. The aim of the present study was to investigate the effect of antiandrogen administration at the pituitary level by evaluating the changes in gonadotropes through quantitative immunohistochemistry, and by comparing these alterations with the effect of androgen deprivation by castration either with or without subsequent androgen replacement. Male Sprague-Dawley rats (23 days old) were randomly divided into 5 groups for the following treatments: (a) controls; (b) flutamide-injected (10 mg/rat/day in a gelatin vehicle); (c) Casodex-injected (10 mg/rat/day in an oil vehicle); (d) castrated, and (e) castrated and dihydrotestosterone propionate-replaced (40 microg/rat/day in an oil vehicle). Groups were then sacrificed after 10 days of maintenance under each condition. Pituitaries were fixed in Bouin's fluid and embedded in paraffin. Serial sections (4 micrometer) were obtained at different levels and immunostained by means of the primary murine monoclonal antibodies anti-FSH and anti-LH and a peroxidase-mediated EnVision System (Dako). Measurements of volume density (VD) and individual mean cell area were made by means of an image-analysis system (Imaging Technology, Optimas). Serum FSH and LH levels were determined by radioimmunoassay (RIA). Serum gonadotropin levels, VD, and mean cell area increased significantly in the flutamide-treated, Casodex-treated, and castrated groups (p < 0.05). Androgen replacement in the castrated rats, however, reduced VD, mean cell area, and serum gonadotropins to levels comparable to those of controls. We conclude that either androgen blockade by antiandrogens or castration produce an enhancement in the gonadotrope cell population in prepubertal rats, as shown by an increase in both VD and mean cell area, as well as an elevation in FSH- and LH-immunoreactive cells. These observations correlate well with the changes found in the levels of circulating gonadotropins as measured by RIA.

Ornithine decarboxylase activity and polyamine levels in the epididymis of prepubertal rat after antiandrogen administration.

The antiandrogens flutamide and casodex have been administered subcutaneously (vehicle, 1, 5 or 10 mg per day) to prepubertal male rats for 10 days. A significant change of epididymal weight has been observed after both treatments, from the lowest dose used. Epididymal dihydrotestosterone concentrations were significantly increased in flutamide- or casodex-treated rats, while epididymal 3 alpha-androstanediol concentrations were affected only after flutamide administration, suggesting a differential effect on androgen metabolism between both antiandrogens. Ornithine decarboxylase (ODC) activity was significantly decreased by flutamide, and to a lesser extent by casodex. Antiandrogen administration resulted in a significant decrease in epididymal content of the polyamines putrescine, spermidine, and spermine. A slight but significant decrease in putrescine and spermidine concentrations, but not in spermine, was observed after flutamide treatment. However, casodex had no effects on polyamines levels. A decrease in putrescine concentration was detected only when ODC activity fell to rather low levels. Interference of the antiandrogens with the biological action of androgens on ODC activity was clearly seen in immature male rats. Therefore, both epididymal growth and differentiation, in correlation to ODC activity, would be severely affected at an early period of sexual development, such as prepuberty.

Immunological and biological activities of pituitary FSH isoforms in prepubertal male rats: effect of antiandrogens.

In male rats androgens are involved in the regulation of follicle-stimulating hormone (FSH) synthesis and secretion. Two nonsteroidal antiandrogens, flutamide and Casodex, were used to study the influence of androgens on the carbohydrate structure of FSH isoforms and the relationship with their bioactivity in prepubertal male rats. Different doses of flutamide or Casodex (vehicle, 1, 5, or 10 mg/rat/day) were administered subcutaneously for 10 days to 23-day-old rats. Immunological FSH was determined by radioimmunoassay and the bioactivity by in vitro Sertoli cell bioassay. Concanavalin A affinity chromatography was used to study the distribution of immunoactive and bioactive pituitary FSH isoforms. A significant depletion of immunological and biological pituitary FSH contents was observed even at the lowest dose of flutamide or Casodex used. The bioactive/immunoactive ratio of pituitary FSH was reduced at the highest dose of flutamide; however, no change was observed in Casodex-treated rats, suggesting a differential effect of the antiandrogens on the FSH bioactivity. Flutamide treatment provoked a significant decrease in proportion and bioactivity of FSH isoforms bearing biantennary and truncated hybrid oligosaccharide side chains and an increase in the proportion but a decrease in bioactivity of FSH isoforms bearing high-mannose oligosaccharides. Conversely, Casodex administration did not modify the proportions of FSH isoforms, although those bearing biantennary and truncated hybrid structures were less bioactive, while those bearing high-mannose oligosaccharides were more bioactive. The highest dose of flutamide decreased the bioactive/immunoactive ratio of FSH isoforms with a high degree of branching in their carbohydrate chains. Our results suggest that androgens, acting directly and indirectly at the pituitary, regulate the selective incorporation of sugar residues to the FSH molecule, thus modulating its biological activity.

Polyamines in the male reproductive system.

This review covers some common aspects of the biosynthesis, interconversion pathways and biochemical functions of polyamines. A particular emphasis is given in experimental models as well as humans, to their presence in the male gonad, prostate gland, seminal vesicles, epididymis and semen. The interaction between hormones (androgens, LH, FSH and PRL) and the main enzymes involved on the polyamine biosynthesis, and the relationship of these compounds on cell growth and differentiation, are also discussed. In this regard, an attention is offered to the potential role of polyamines during early spermatogenesis stages and the use of some enzymes involved in their biosynthesis as sensitive and specific markers of the action of androgens and antiandrogens in the epididymis. Finally, a special issue is addressed to the controversial information documented on polyamines, their oxidation products and the relationship with male fertility.

Polyamines in the male reproductive system

This review covers some common aspects of the biosynthesis, interconversion pathways and biochemical functions of polyamines. A particular emphasis is given in experitemtal models as well as humans, to their presence in the male gonad, postate gland, seminal vesicles, epididymis and semen. The interaction between hormones (androgens, LH, FSH and PRL) and the main enzymes involved on the polymine biosynthesis, and the relationship of these compounds on cell growth and differentation, are also discussed. In this regard, an attention is offered to the potential role of polymines during early spermatogenesis stages and the use of some enzymed involved in their biosynthesis as sensitive and specific markers of the action of androgens and antiandrogens in the epididymis. Finally, a special issue is addressed to the controversial information documented on polymines, their oxidation products and the relationship with male fertility.

Polyamines in the male reproductive system

This review covers some common aspects of the biosynthesis, interconversion pathways and biochemical functions of polyamines. A particular emphasis is given in experitemtal models as well as humans, to their presence in the male gonad, postate gland, seminal vesicles, epididymis and semen. The interaction between hormones (androgens, LH, FSH and PRL) and the main enzymes involved on the polymine biosynthesis, and the relationship of these compounds on cell growth and differentation, are also discussed. In this regard, an attention is offered to the potential role of polymines during early spermatogenesis stages and the use of some enzymed involved in their biosynthesis as sensitive and specific markers of the action of androgens and antiandrogens in the epididymis. Finally, a special issue is addressed to the controversial information documented on polymines, their oxidation products and the relationship with male fertility. (AU)

Effects of two non-steroidal antiandrogens on testicular function in prepubertal rats.

The effects of two non-steroidal antiandrogens, flutamide and casodex, were evaluated in prepubertal male rats. Animals (23 days old) were subcutaneously administered vehicle or 1, 2, 5, or 10 mg/day of flutamide or casodex for 10 days. Testis weights were diminished at the 10 mg/day dose of both antiandrogens. A significant increase in serum luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels was detected. Notwithstanding, flutamide influenced LH/FSH levels more severely than casodex. No changes were observed in serum prolactin. Serum testosterone, dihydrotestosterone, and 3 alpha-androstanediol levels were increased in flutamide-treated rats from the 2 mg/day dose, whereas only 3 alpha-androstanediol was modified at 10 mg/day of casodex, suggesting a differential effect on androgen metabolism. An elevation of testicular concentration and basal production of androgens was found, indicating that flutamide and casodex administration are capable of stimulating testicular steroidogenesis, as well as 5 alpha-reduction. However, the in vitro maximal responsiveness of the gonads to human chorionic gonadotropin was preserved. Antiandrogen administration did not modify testicular androgen binding protein concentration. In conclusion, the blockade of androgen action during sexual maturation caused profound changes on the pituitary-gonadal axis in male rats.

Modulation of ornithine decarboxylase activity by prolactin in seminal vesicles of the rat.

The stimulatory effect of prolactin on sexual accessory glands is well established, though the mechanism of trophic action remains poorly understood. We have therefore assessed the effect of high levels of prolactin on ornithine decarboxylase activity and polyamine content in seminal vesicles (SV) of adult rats. Hyperprolactinaemia was induced by implantation of tissue fragments of a prolactin-secreting tumour 7315a, and the rats killed at 35 days post-inoculation. Serum levels of prolactin were increased significantly (p < 0.001) in tumour-bearing rats. Levels of testosterone in serum were reduced markedly, whereas LH levels remained unchanged. SV weight in the experimental group was not significantly different from that in the corresponding control group. A clear increase in ornithine decarboxylase activity of SV was observed (p < 0.001) in the hyperprolactinaemic rats. However, there was no change in the polyamine content of SV in tumour-bearing rats, compared to the control group. These results indicate that ornithine decarboxylase activity in SV of adult rats is regulated mainly by prolactin. This experimental model may be useful for identification of some of the events involved in the trophic action of prolactin.