Sexual behavior of male pigs

Two major characteristics of males that affect the likelihood of achieving copulation are the sexual motivation and mating competency of the male. The behavior of domestic animals, including their sexual behavior, is dependent upon a complex interaction between the organism's internal and external environment. In male pigs, as in other mammalian species, it is clear that testicular steroids are required to maintain sexual behavior and testosterone plays a critical role. Consistent with studies in other species, it is apparent in male pigs that there is a threshold circulating concentration of androgens and/or estrogens required to maintain sexual behavior and that the level of sexual motivation is unrelated to levels of sex steroids providing these are adequate for normal sexual behavior. Key aspects of the external environment that affect the sexual behavior of the male pig include genetic, seasonal, social, sexual and psychological factors.     

Phenylalanine(90) and phenylalanine(93) are crucial amino acids within the estrogen binding site of the human UDP-glucuronosyltransferase 1A10

Human UDP-glucuronosyltransferase 1A10 has been identified as the major isoform involved in the biotransformation of a wide range of phenolic substrates, including native estrogens and their oxidized metabolites. Our recent studies point to the F(90)-M(91)-V(92)-F(93) amino acid motif of UGT1A10, which was identified using photoaffinity labeling followed by LC-MS/MS analysis, as a key determinant of the binding of phenolic substrates. In this report, we have evaluated the role of F(90), V(92), and F(93) in the recognition of estrogens by UGT1A10 using site-directed mutagenesis. Kinetic studies using five mutants revealed that F(90) and F(93) are critical residues for the recognition of all estrogen substrates. The substitution of F(90) with alanine totally abolished the activity of this enzyme toward all the estrogens investigated. Overall, sequential removal for the aromatic ring (F to L) and of the hydrophobic chain (F to A and V to A) from amino acids 90, 92, and 93 effectively alters estrogen recognition. This demonstrates that individual features of the native and hydroxylated estrogens determine the specific binding properties of the compound within the binding site of the human UGT1A10 and the mutants. The resulting activities are completely abolished, unchanged, increased, or decreased depending on the structures of both the mutant and the substrate. The novel identification of UGT1A10 as the major isoform involved in the glucuronidation of all estrogens and the discovery of the importance of the FMVF motif in the binding of steroids will help to elucidate the molecular mechanism of glucuronidation, resulting in the design of more effective estrogen-based therapies.     

25 hydroxy-vitamin D(3)-1alpha hydroxylase expression and activity in cultured human osteoblasts and their modulation by parathyroid hormone, estrogenic compounds and dihydrotestosterone

Human osteoblasts (hOB) produce and respond to 1,25(OH)(2)D(3) (1,25D), suggesting an autocrine/paracrine system. We therefore examined hormonal modulation of the expression and activity of 25 hydroxy-vitamin D(3)-1alpha hydroxylase (1-Ohase) in hOB. Cells from pre- and post-menopausal women or men, were treated with estrogenic compounds and 1-OHase expression and activity were measured. 1-OHase mRNA expression was highest in pre-menopausal women hOB and was increased by all hormones tested. In post-menopausal hOB all hormones except biochainin A (BA) and genistein (G) increased 1-OHase mRNA expressions to less extent. In male-derived hOB only dihydrotestosterone (DHT) and carboxy BA (cBA) increased 1-OHase mRNA expression. 1,25D production from 25(OH)D(3) had a K(m) of approximately 769-400 ng/ml (1.92-1.07 microM) and V(max) of 31.3-17.4 ng/ml (0.078-0.044 microM/60 min/5 x 10(6)cells) respectively, and was increased by all hormones except raloxifene (Ral) with higher stimulation in pre- than in post-menopausal cells. Only BA was almost five times more potent in pre- rather than post-menopausal hOBs. In male hOB only DHT and cBA increased 1,25D production whereas estradiol-17beta (E(2)) had no effect and BA decreased it. These results provide evidence for the expression of 1-OHase mRNA and production of 1,25D in hOBs, which are age and sex dependent and are hormonally modulated. The role of this local autocrine/paracrine 1,25D system in bone physiology deserves further investigation.     

Effects of 17beta-estradiol on growth and apoptosis in human vascular endothelial cells: influence of mechanical strain and tumor necrosis factor-alpha

Vascular endothelial cell (EC) integrity is key to arterial health; endothelial dysfunction is linked to atherogenesis. Atherosclerosis shows a male preponderance, possibly related to the protective effect of estrogens in women. This study examined the effect of estrogens on growth, apoptosis and adhesion molecule expression in cultured human EC. The effects of 17beta-estradiol (E2) were studied in human umbilical vein endothelial cells (HUVEC) under normal culture conditions, and following exposure to cyclic mechanical strain or tumor necrosis factor alpha (TNFalpha). E2 enhanced HUVEC growth in serum-enriched media, in a concentration-dependent manner. This up-regulation of EC growth by E2 was associated with an increase in telomerase activity, assessed by PCR-based TRAP analysis. Cyclic strain enhanced [(3)H]-thymidine incorporation into DNA, and increased activation of mitogen-activated protein (MAP) kinase ERK1/2 and expression of early growth genes (Egr-1 and Sp-1); E2 attenuated the strain-induced ERK1/2 activation but not the early growth gene expression or DNA synthesis. TNFalpha (20 ng/mL) induced apoptosis in HUVEC, causing a decrease in DNA synthesis, increase in floating and Annexin-V-stained cell numbers, and morphological changes. TNFalpha also upregulated ERK1/2 activity and expression of adhesion molecules (ICAM-1, VCAM-1 and E-selectin). E2 significantly attenuated the effects of TNFalpha on ERK1/2 activity, apoptosis, and E-selectin expression in the cells. Thus, estradiol enhances growth and reduces TNFalpha-induced apoptosis in EC; enhanced EC growth may be mediated via upregulation of telomerase activity. These effects are possible cellular mechanisms underlying female gender-associated cardiovascular protection.     

Human models of aromatase deficiency

Estrogens exert a wide range of biological effects in both sexes also on non-reproductive systems and organs. Human congenital estrogen deficiency, due to an inactivating mutation of the aromatase gene, leads to the lack of the estrogen synthesis, with gonadotropins and circulating testosterone ranging from normal to elevated. The aromatese-deficient females show hyperandrogenism and virilization at birth with ambiguous genitalia. During childhood there are a dysfunction in the LHRH-LH/FSH axis and a progressive delay in bone age. At puberty they show primary amenorrhea, no breast development, worsening of the virilization and the absence of growth spurt. The clinical phenotype in the male affected subjects comprises tall stature, persistent linear growth and delayed bone age, osteopenia/osteoporosis, eunuchoid body proportion, different degrees of glucose-insulin and of fertility impairment. These phenotypes suggest the physiological role of estrogens on the skeleton, on pituitary function, on the reproductive system, on glucose metabolism, being the precise mechanism on each of these functions not yet known in detail. The estradiol replacement treatment leads to a complete epiphyseal closure and to the skeletal maturation. Moreover, the increasing knowledge on the role of estrogen in several metabolic pathways could be important for a better management of several metabolic diseases.     

How are pharmaceutical and personal care products (PPCPs) removed from urban wastewaters?

The presence of bioactive trace pollutants such as pharmaceuticals and ingredients of personal care products (PPCPs) in different environmental compartments (rivers, lakes, groundwaters, sediments, etc.) is an emerging issue due to the lack of existing information about the potential impact associated with their occurrence, fate and ecotoxicological effects. Due to the low PPCP concentrations reported in wastewaters (ppb or ppt) and their complex chemical structure, common technologies used in sewage and drinking water treatment plants may not be efficient enough to accomplish their complete removal. Information about physico-chemical characteristics such as acidity, lipophilicity, volatility and sorption potential is a useful tool to understand the different removal patterns observed. In order to perform an accurate overall mass balance along the different units of sewage treatment plants, it is necessary to gather information not only about the presence of micropollutants in the aqueous phase, but also on the fraction sorbed onto solids. Since only some PPCPs are very well eliminated by conventional sewage treatment configurations, new strategies such as modification of operating conditions (e.g. solids retention time), implementation of new technologies (e.g. biomembrane reactors) or additional advanced post-treatment steps (e.g. oxidation, adsorption, membranes) have been suggested for an increased efficiency.     

The role of androst-5-ene-3β,17β-diol (androstenediol) in cell proliferation in endometrium of women with polycystic ovary syndrome

Women with polycystic ovary syndrome (PCOS) show high prevalence of endometrial hyperplasia and adenocarcinoma. Endometrial proliferation is increased, evaluated by high levels of Ki67 (cell cycle marker) and low levels of p27 (negative regulator of cell cycle). Nevertheless, endometrial changes in cyclin D1 (positive regulator of cell cycle) in PCOS-women are not described. Androst-5-ene-3β,17β-diol (androstenediol), steroid with estrogenic activity present in endometria, could be related to increased endometrial cell proliferation. The objective of this study was to determine protein content of cyclin D1 and androstenediol levels in endometria from PCOS and control-women and to evaluate the possible mechanism favoring cell proliferation associated with hormonal characteristics of patients. Therefore, cyclin D1 protein content in PCOS-women and control-endometrial tissue were assessed by western blot and immunohistochemistry. The androstenediol levels were evaluated by ELISA. To further analyze the effect of steroids (androstenediol, 17β-estradiol, testosterone) in cell proliferation, levels of proteins cyclin D1, p27 and Ki67 were evaluated in an in vitro model of stromal endometrial cells T-HESC and St-T1b. An increase in cyclin D1 and androstenediol was observed in tissues from PCOS-women relative to control group (p < 0.05). In the in vitro model, androstenediol exerted increase in cyclin D1 (p < 0.05) and a decrease in p27 protein level (p < 0.05), while Ki67 in St-T1b cells increased under this stimulus (p < 0.05). Testosterone produces opposite effects in the levels of the above markers (p < 0.05). Therefore, the hormonal imbalance associated with this syndrome could alter endometrial tissue homeostasis, promoting cell proliferation. Androstenediol is a molecule that could be involved by stimulating proliferation, whereas testosterone elicits a role of cell cycle repressor.     

Steroidogenic capacity of Trypanosoma cruzi trypomastigotes

American Trypanosomiasis is caused by the hemoflagellate Trypanosoma cruzi (T. cruzi) and affects millions of persons causing variable degrees of digestive and heart disturbances. As far as we concerned, T. cruzi capacity to synthesize steroid hormones has not been investigated. Therefore, the aim of this work was to investigate the capacity of T. cruzi trypomastigotes to transform tritiated steroid precursors into androgens and estrogens. The T. cruzi Tulahuén strain was obtained from mice blood. The trypomastigotes were cultured for 6 and 24h in Dulbbeco's modified Eagle's medium plus FCS and antibiotics. Tritiated dehydroepiandrosterone or androstendione were added to the culture media and parasites were incubated for 6 or 24h. The cultures were centrifuged and ether extracted. The steroids were analyzed by thin layer chromatography (TLC) in two solvent systems. After incubation with (3)H-androstenedione, T. cruzi trypomastigotes synthesized (3)H-testosterone (T), (3)H-17beta-estradiol (E(2)) and (3)H-estrone (E(1)). Metabolism of (3)H-DHEA by the parasites yielded (3)H-androstendione and (3)H-androstendiol at 6h of incubation. The recrystallization procedure further demonstrated the (3)H-androstendiol and (3)H-17beta-estradiol syntheses. Results indicate for the first time that T. cruzi trypomastigotes produce androgens and estrogens when incubated in the presence of steroid precursors and suggest the presence of active parasite steroidogenic enzymes.     

The effect of 17-B-estradiol on the fine structure of the adrenergic axons of the rabbit myometrium

Summary The effects of 17-B-estradiol on the fine structure of the autonomic nerves of the rabbit oviduct and myometrium were studied by means of KMnO₄-fixation. The main changes due to prolonged estrogen treatment were the following: (1) the dimensions of the axons increased, (2) the amount of smooth endoplasmic reticulum in the axons increased, (3) the amounts of both large granular vesicles (LGV) and large agranular vesicles (LAV) in the axons increased and (4) electron dense grains were found within the endoplasmic tubuli of the axons. Furthermore, it seems probable that the amount of small granular vesicles was also considerably higher after two weeks of estrogen treatment.The mechanism of estrogen action on the storage of transmitter within the axon is discussed. In conclusion, the present results suggest that both the axoplasmic transport and the peripheral formation of storage sites for noradrenaline are stimulated by the increased estrogen level.This work was supported by grants from Orion, Helsinki and from Finska Läkaresällskapet.     

Differential oxidant potential of carcinogenic and weakly carcinogenic estrogens: Involvement of metabolic activation and cytochrome P450

Different estrogens vary in their carcinogenic potential despite having similar hormonal potencies; however, mechanisms of estrogen-induced carcinogenesis remain to be fully elucidated. It has been hypothesized that generation of reactive estrogen-quinones and oxidative stress, both of which result from metabolic activation of estrogens, play an essential role in estrogen-induced carcinogenesis. This hypothesis was tested using the estrogen-receptor (ER)-alpha-positive hamster kidney tumor (H301) and the human breast cancer (MCF-7) cell lines. Estrogens with differing carcinogenic potentials were compared in terms of their capacities to induce 8-iso-prostaglandin F(2alpha) (8- iso-PGF(2alpha)), a marker of oxidative stress. Tumor cells were treated with either 17beta-estradiol (E2), a carcinogenic estrogen or 17-alpha-ethinylestradiol (EE), a weakly-carcinogenic estrogen. Tumor cells were also treated with alpha-naphthoflavone, a cytochrome P450 inhibitor, or a combination of alpha-naphthoflavone and E2 to study the effect of metabolic activation of E2 on E2-induced oxidative stress. H301 cells treated with E2 displayed time- and dose-dependent increases in 8-iso-PGF(2alpha), compared to controls; treatment with 10 nM E2 resulted in a maximal 4-fold induction following 48 h of treatment. In contrast, H301 cells treated with EE did not display an increase in 8-iso-PGF(2alpha) compared with controls. In H301 cells cotreated with alpha-naphthoflavone and E2, alpha-naphthoflavone inhibited the E2-induced increase in 8-iso-PGF(2alpha). These data indicate that a carcinogenic estrogen shows strong oxidant potential, whereas a weakly-carcinogenic estrogen shows poor oxidant potential. Furthermore, inhibiting metabolic activation of a carcinogenic estrogen blocks its oxidant potential. Our data support the hypothesis that metabolic activation and subsequent generation of oxidative stress may play critical roles in estrogen-induced carcinogenesis.