Estrogen receptors alpha and beta (ERalpha and ERbeta) and androgen receptor (AR) in human sperm: localization of ERbeta and AR in mitochondria of the midpiece

BACKGROUND: The central role of estrogens and androgens in the male reproductive system has focused attention on the presence and distribution of their cognate receptors [estrogen receptor (ER) alpha, ERbeta and androgen receptor (AR)] in male reproductive tissues and cells. Since the presence of steroid hormone receptors in mitochondria of mammalian cells has been well documented, we investigated the possibility of mitochondrial localization of sex steroid hormone receptors in sperm. METHODS AND RESULTS: Applying immunofluorescence labelling and confocal laser scanning microscopy we show that the estrogen receptor beta and the AR of human sperm are specifically enriched in the midpiece, at the site of the mitochondria, which were visualized by labelling with the vital dye CMX. Nuclear and mitochondrial localization of AR was also detected in LnCap human prostate cancer cells. Differentially, most of the ERalpha immunostaining is in the form of a compact zone at a region corresponding to the equatorial segment of the upper post-acrosomal region of the sperm head. Immunoblotting experiments using sperm extracts revealed the presence of a 66 and a 45 kDa protein reacting with the ERalpha antibody, one 64 kDa protein reacting with the ERbeta antibody and a 110 and a 90 kDa protein reacting with the antibody against AR. CONCLUSIONS: Our findings suggest that the differential localization of AR and ER isoforms in human sperm reveals distinct roles of these receptors in the physiology of sperm cells and, perhaps, also in the process of fertilization.     

Phytoestrogens inhibit aromatase but not 17beta-hydroxysteroid dehydrogenase (HSD) type 1 in human granulosa-luteal cells: evidence for FSH induction of 17beta-HSD

BACKGROUND: Studies using purified enzyme preparations, placental microsomes or cell lines have shown that certain phytoestrogens can inhibit the enzymes that convert androgens to estrogens, namely aromatase and 17beta-hydroxysteroid dehydrogenase (HSD) type 1 and type 5. The study aim was to investigate the effects of selected phytoestrogens on aromatase and 17beta-HSD type 1 activity in primary cultures of human granulosa-luteal (GL) cells. METHODS AND RESULTS: GL cells, cultured for 48 h in medium containing 5% fetal calf serum and for a further 24 h in serum-free medium with or without hFSH or hCG, were exposed to steroid substrates during the last 1-4 h of the experiment. The production of progesterone in the presence of pregnenolone or estradiol synthesis from androstenedione, estrone or testosterone showed dose- and time-dependent increases. Whilst hCG priming had no effect on progesterone production, FSH priming induced mean 68 and 56% increases in the production of estradiol from androstenedione (A-dione) and estrone respectively, but had no significant effect on the metabolism of testosterone to estradiol. None of the phytoestrogens investigated had any acute effects on enzyme activity. In contrast, when GL cells were exposed to the compounds for 24 h prior to exposure to steroid substrates for 4 h, 10 micro mol/l apigenin and zearalenone significantly inhibited aromatase activity, whilst biochanin A and quercetin had no effect. None of the phytoestrogens inhibited FSH-induced 17beta-HSD type 1 activity, and only quercetin significantly inhibited progesterone production. CONCLUSIONS: The inability of phytoestrogens to acutely inhibit steroidogenic enzymes in human GL cells (as has been shown in cell-free models) suggests that they are either rapidly metabolized to relatively inactive compounds or that the high enzyme activity in human GL cells masks any inhibitory effects of the compounds at the concentration tested.     

Gestational development of human placental 17{beta}-hydroxysteroid oxidoreductase types 1 and 2

Human placenta is a rich source of 17-hydroxysteroid oxidoreductase(17-HOR) type 1, a cytosolic enzyme highly specific for 17-oestradiol,and type 2, a microsomal form reactive with both oestradioland testosterone. Although a number of studies have establishedthat 17-HOR activity is present in placenta as early as weeks4–5 of gestation, more specific data on the pattern ofdevelopment of these two enzyme forms are lacking. In this study,samples of villous tissue from weeks 7–20 of gestationwere fractionated into cytosol and microsomes and 17-HOR activityassayed under conditions which differentiate between the twoenzyme types. Type 1 activity with oestradiol of cytosol andmicrosomal type 2 activity with oestradiol and testosteroneincreased from week 7 to week 20. Activities at 17–20weeks approximated those at 38dash;40 weeks. The high, cytosolicoestradiol/T activity ratio (160 ± 20), characteristicof 17-HOR type 1, was constant between weeks 7 and 20, as wasthe low microsomal ratio (3.4 ± 0.4) characteristic ofthe type 2 activity. There was a relationship between cytosolictype 1 activity and microsomal type 2 activity between weeks7 and 20 (r equals; 0.59, P equals; 0.0055). These results indicateboth activities increase coincident with the luteal-placentalshift and that their temporal patterns of development are relatedbetween weeks 7 and 20 of gestation.     

Effects of progesterone and oestradiol-17{beta} on 17{beta}-ol-dehydrogenase activity in stromal cells of human endometrium under in-vitro conditions

A 17-ol-dehydrogenase activity could be demonstrated in fibroblastmonolayer cell cultures of proliferative human endometrium.After 24 h incubation 100 nCi [6, 7–3H] oestradiol-17was completely oxidized to oestrone. Progesterone was not ableto enhance the metabolizing velocity. In contrast, progesteroneincubation revealed a decreasing oxidation rate with increasingmolarity. Histological changes after transformation of the endometriumare discussed to explain in-vivo results showing an increased17-ol-dehydrogenase activity in the secretory phase of the cycle     

Ovarian modulators of type 1 11beta-hydroxysteroid dehydrogenase (11betaHSD) activity and intra-follicular cortisol:cortisone ratios correlate with the clinical outcome of IVF

BACKGROUND: Follicular fluid (FF) contains compounds that can modulate NADP(+)-dependent oxidation of cortisol by type 1 11beta-hydroxysteroid dehydrogenase (11betaHSD). The objective of this study was to investigate the relationships between levels of the ovarian modulators of type 1 11betaHSD, intra-follicular cortisol:cortisone ratios and the clinical outcome of IVF cycles. METHODS: A single random sample of FF was aspirated from each of 132 patients undergoing gonadotrophin-stimulated IVF. Components of FF, resolved using C18 column chromatography, were evaluated for effects on NADP(+)-dependent cortisol oxidation in rat kidney homogenates. Intra- follicular steroid concentrations were measured by radioimmunoassays. Clinical pregnancies were confirmed by ultrasonography at 6 weeks post-embryo transfer. RESULTS: Levels of the hydrophilic ovarian 11betaHSD stimuli were significantly lower (P<0.0001) and levels of the hydrophobic ovarian 11betaHSD inhibitors were significantly higher (P<0.002) in conception versus non-conception cycles. Intra-follicular cortisol:cortisone ratios increased with the degree of inhibition of 11betaHSD by the hydrophobic FF fractions. FF obtained from conception cycles had significantly higher cortisol:cortisone ratios than samples from non-conception cycles (12.9+/-0.3 versus 8.5+/-0.2, respectively; P<0.0001). CONCLUSIONS: Conception by IVF is associated with elevated intra-follicular cortisol:cortisone ratios, which reflect low levels of ovarian stimuli and/or high levels of ovarian inhibitors of type 1 11betaHSD.     

Combined estrogen receptor alpha and estrogen receptor beta genotypes influence the age of menarche

BACKGROUND: Age at menarche has a strong genetic influence. We reported recently an association between the XbaI (351A-->C)and PvuII (397T-->C) polymorphisms of the estrogen receptor (ER)alpha gene with the age of menarche in Greek adolescents. In the present study, we examined whether ERbeta genotypes alone, or in combination with ERalpha genotypes, may also influence onset of menarche. METHODS: We performed genotyping for the single nucleotide polymorphisms 1730A-->G and 1082G-->A of the ERbeta gene and examined their association with the age of menarche in the same cohort of 145 Greek girls. We then looked for a possible effect of combined ERalpha and beta genotypes on the age of menarche. RESULTS: Menarche occurred 7 months later in girls with the AA genotype of the 1730A-->G polymorphism than in girls with the AG genotype (mean +/- SD: 13.23 +/- 1.24 versus 12.66 +/- 1.26 years, respectively; P = 0.005). The 1082G-->A polymorphism was not detected in any of the girls examined. A significant effect of combined ERalpha and beta genotypes was also apparent. Menarche occurred 11 months later in girls bearing the AA/TT,AA (ERalpha, ERbeta) genotypes compared with girls with the CC/CC,AG genotype (13.30 +/- 1.27 nersus 12.41 +/- 1.28 years; P = 0.042). The difference remained significant after adjusting for body mass index (P = 0.034). CONCLUSION: Combined ERalpha and ERbeta polymorphisms may influence the age of menarche.     

Mechanisms regulating the expression of indoleamine 2,3-dioxygenase during decidualization of human endometrium

BACKGROUND: Expression of the tryptophan catabolizing enzyme, indoleamine 2,3-dioxygenase, in the mouse placenta has been shown to be critical in preventing immunological rejection of the fetal allograft. To clarify the physiological importance of indoleamine 2,3-dioxygenase in human pregnancy, we have studied how the expression of this enzyme changes during decidualization of human endometrium at both the cell and tissue level. METHODS AND RESULTS: The level of indoleamine 2,3-dioxygenase mRNA expression (determined by RT-PCR) was higher in decidual than in endometrial tissue. Uterine decidual tissue in ectopic pregnancy similarly showed increased mRNA expression. Immunohistochemistry demonstrated that indoleamine 2,3-dioxygenase protein immunoreactivity was found in glandular epithelium and in stromal cells. The intensity of this immunoreactivity was increased in decidualized tissue. In a cell culture model, the level of indoleamine 2,3-dioxygenase mRNA was suppressed specifically by progesterone-induced decidualization of isolated endometrial stromal cells. Indoleamine 2,3-dioxygenase protein abundance (determined by Western blot) was also decreased by progesterone-induced decidualization. However interferon-gamma, a potent stimulator of indoleamine 2,3-dioxygenase gene expression, increased the level of indoleamine 2,3-dioxygenase mRNA and protein in both non-decidualized and in decidualized cells. Indoleamine 2,3-dioxygenase activity (determined by measuring the concentration of tryptophan and its indoleamine 2,3-dioxygenase catabolite, kynurenine) was also decreased by progesterone-induced decidualization but enhanced following interferon-gamma treatment. Expression of other interferon-gamma inducible genes (STAT1 and tryptophanyl-tRNA synthetase) showed the same pattern as that of indoleamine 2,3-dioxygenase in tissue samples, but was not changed by decidualization in the cell culture model. CONCLUSIONS: These data suggest that despite suppression by progesterone, indoleamine 2,3-dioxygenase expression in endometrial stromal cells may increase during decidualization due to stimulation by interferon-gamma secreted by infiltrating leukocytes.     

Induction of artificial endometrial cycles with oestradiol implants and injectable progesterone: establishment of a viable pregnancy in a woman with 17-a-hydroxylase deficiency

Repeated attempts with oral oestrogens and injectable progesteronefailed to induce secretory endometrium in a woman with 17--hydroxylasedeficiency. The insertion of s.c. 17--oestradiol implants dramaticallyimproved the endometrial response and enabled the establishmentof endometrial maturation. A viable pregnancy was achieved afterthe uterine transfer of in-vitro fertilized donated eggs.     

Expression and function of interleukin-11 and its receptor alpha in the human endometrium

The interleukin-11 (IL-11) receptor alpha has an important function in decidualization of mouse endometrial stroma but the function of IL-11 and its receptor in the human endometrium remains unknown. The mRNA for IL-11 and its receptor alpha in human endometrial tissue samples were analysed by semi-quantitative RT-PCR and RNase protection assays respectively. The proteins were detected in frozen endometrial tissue samples by immunofluorescence. The effect of heparin-binding epidermal growth factor (HB-EGF) on secretion of IL-11 by cultured endometrial stromal cells was assessed by enzyme-linked immunosorbent assay. The proliferative potential of IL-11 in endometrial stromal cells was assessed by [(3)H]thymidine uptake. IL-11 and its receptor alpha mRNAs and proteins were detected in the endometrium throughout the cycle. Distinct patterns of localization of the ligand and receptor were observed. HB-EGF induced IL-11 secretion by cultured stromal cells, and IL-11 induced [(3)H]thymidine uptake by these cells. Our data suggest that IL-11-receptor interactions may perform different functions in the human endometrium at different stages of the cycle, and that secretion of IL-11 is modulated by local growth factors.     

Maternal serum levels of interferon-gamma and interleukin-2 soluble receptor-alpha predict the outcome of early IVF pregnancies

BACKGROUND: Elevated maternal serum levels of interleukin-2 soluble receptor-alpha (IL-2 sRalpha), tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) have been associated with pregnancy loss. The aim of our study was to evaluate the predictive value of these cytokines in the outcome of early IVF pregnancies. METHODS: One hundred and fifty-nine consecutive IVF patients who were subsequently diagnosed to have a biochemical pregnancy (n = 23), a first-trimester miscarriage (n = 19) or a normal term delivery (n = 117) were included in this study. Serum was collected from the initial pregnancy test, 11 days after a day 3 embryo transfer, and all samples were analysed for IL-2 sRalpha, TNF-alpha and IFN-gamma by commercially available enzyme-linked immunosorbent assay (ELISA) kits. RESULTS: IL-2 sRalpha levels were significantly higher in patients with an early pregnancy loss compared with patients with a normal term delivery (849.5 +/- 69.6 versus 693.5 +/- 31.2 pg/ml, P = 0.02), and a cut-off point of IL-2 sRalpha >1000 pg/ml predicted a poor pregnancy outcome (44.4 versus 22.7% pregnancy loss, IL-2 sRalpha >or=1000 versus IL-2 sRalpha <1000 pg/ml; P = 0.02). IFN-gamma-positive patients had twice the risk for poor IVF pregnancy outcome compared with IFN-gamma-negative subjects (40.8 versus 20.0%, respectively; P < 0.02), including a significantly lower implantation rate (37.6 +/- 0.05 versus 50.0 +/- 0.03%, respectively; P = 0.02). There was no difference in pregnancy outcome based upon serum levels, or the ability to detect the presence of TNF-alpha. No differences in levels of these cytokines were found based on the aetiology of the patients' infertility. CONCLUSIONS: Elevated maternal serum levels of IL-2 sRalpha and IFN-gamma as early as 11 days after embryo transfer are associated with poor IVF pregnancy outcome.     

Stimulation of prostaglandin (PG) F2{alpha} and PGE2 release by tumour necrosis factor-{alpha} and interleukin-1{alpha} in cultured human luteal phase endometrial cells

Tumour necrosis factor- (TNF-) and interleukin-1 (IL-1) areimportant mediators of cell signalling in the uterus. Prostaglandins(PG) have been implicated in the increase of endometrial vascularpermeability which occurs during the implantation process. Thisstudy evaluates the effect of these two pleiotropic cytokineson PGF₂ and PGE₂ release from human luteal phase endometrialglandular epithelial cells (GEC) and stromal cells (STC) inculture. Basal PGF and PGE release did not differ significantlyfrom each other or among cell types, and declined significantlywith increasing number of days in culture. On day 3, basal PGrelease had decreased to half of that on day 1 of culture. However,both cell types were still able to respond to the addition ofexogenous arachidonic acid (5 µM) on day 3 of culture,with PG release by GEC being elevated 7- to 10-fold and by STCmoderately, but still significantly, on day 4. The permissiveeffect of arachidonic acid on the stimulation of PG releasemay indicate the down-regulation of phospholipase A₂ with continuedtime in culture. However, the addition of arachidonic acid (5µM) on day 0 of culture, while able to cause significantlyincreased PG release from GEC, had no effect on STC. In contrast,the addition of a combination of arachidonic acid (5 µM),and either recombinant human TNF- (10 µg rhTNF-/I) or10 µg rhlL-1/I, had a synergistic action and caused thesignificantly increased release of PGF and PGE from both celltypes, compared with that achieved with either arachidonic acidor the cytokine alone (although GEC responded more than STC).During the first 24 h after the addition of rhTNF- or rhlL-1,both cytokines stimulated PG release from both cell types ina dose- and time-dependent fashion. Neither cycloheximide (10µM) nor actinomycin D (10 µM) affected basal PGrelease, but both blocked cytokine-induced PG release from bothcell types. These results suggest that there is a differentialcontrol of human endometrial cell PG biosynthesis, and thatPG release may be regulated through gene activation.     

Independent regulation of prostaglandins and monocyte chemoattractant protein-1 by interleukin-1beta and hCG in human endometrial cells

BACKGROUND: Inflammatory mediators such as prostaglandins (PG), chemokines, cytokines and their interactions regulate reproductive functions. The relationship between PG and monocyte chemoattractant protein-1 (MCP-1) has not been elucidated in human endometrium. The presence of hCG receptors in the human endometrium suggests that this embryonic signal may exert a local function in this tissue. Our objectives were to investigate the possible association between PG and MCP-1 and to examine the role of hCG in interleukin-1beta (IL-1beta)-regulated PG and MCP-1 production in human endometrium. METHODS: Primary cell cultures isolated from endometrial biopsies were used as an in vitro model. PG and MCP-1 levels were measured in the culture medium. RESULTS: IL-1beta stimulates the production of both PG and MCP-1. Neither COX inhibitors nor direct addition of PG affects MCP-1 production. By contrast, MCP-1 is able to induce PGE2 and PGF2alpha in a concentration-dependent manner but it does not appear to contribute to the increase in PG accumulation following IL-1beta stimulation. hCG inhibits IL-1beta-induced PG level. However, hCG has no effect on either basal or IL-1beta-mediated MCP-1 level. CONCLUSIONS: PG are not involved in the regulation of MCP-1 production in endometrial cells. hCG appears to play a local function in the endometrium.     

Sharing of the IL-2 receptor {gamma} chain with the functional IL-9 receptor complex

The third subunit, the so-called common (_c) chain, of the IL-2receptor is shared among the receptors for IL-2, IL-4, IL-7and IL-15, and dysfunction of the _c chain is thought to causeX-linked severe combined immunodeficiency (XSCID) ascribed toimpairment of early T cell development. However, cytokines linkedto XSCID are as yet unidentified. A mAb specific for the _c chain,TUGm2, profoundly inhibited cell proliferation in response toIL-9. Another mAb, TUGm3, immunoprecipltated [¹²⁵I]IL-9 cross-linkedwith either the IL-9 receptor or the _c chain. These resultsdemonstrate that the _c chain is included in the functional receptorcomplex for IL-9, which was initially characterized as a T cellgrowth factor and is essential for IL-9-dependent growth signaltransductlon.     

Transient hypogonadotrophic hypogonadism in males with acute toxoplasmosis: suppressive effect of interleukin-1 beta on the secretion of GnRH

BACKGROUND: In September 2002, an outbreak of toxoplasmosis was noted in a male boarding high school on the Aegean coast of Turkey. We have focused our efforts to investigate the sex hormones in this population. METHODS: Blood samples were collected from 40 male patients, 17-18 years old, who also had positive titres of antibody to Toxoplasma gondii. Serum FSH, LH, free testosterone (FT), total testosterone (TT), interferon-gamma (IFN-gamma), interleukin-1beta (IL-1beta) and macrophage-inflammatory protein-1alpha (MIP-1alpha) concentrations were measured in all patients and 20 control subjects. Initially, the patients were divided on the basis of the levels of sex hormones into the following groups: patients who had normal sex hormone levels (n = 31) as group A and patients with low sex hormone levels (n = 9) as group B. RESULTS: IL-1beta levels were found to be higher in group B patients than group A. The levels of IL-1beta correlated significantly in a negative manner with FSH, LH, FT and TT in all patients with acute toxoplasmosis (n = 40). CONCLUSIONS: Acute toxoplasma infection may cause temporary hypogonadotrophic gonadal insufficiency regardless of the course of the disease.     

Activation of Vgamma9Vdelta2 T cells by non-peptidic antigens induces the inhibition of subgenomic HCV replication

Hepatitis C virus (HCV) has evolved complex strategies to evade host immune responses and establish chronic infection. Since human Vgamma9Vdelta2 T lymphocytes play a critical role in the immune response against viruses, we analyzed their antiviral functions on Huh7 hepatoma cells carrying the subgenomic HCV replicon (Rep60 cells). In a transwell culture system, Rep60 cells were co-cultured with either PBMCs or highly purified gammadelta T cells stimulated by non-peptidic antigens. Vgamma9Vdelta2 T cell activation was associated with a dramatic reduction of HCV RNA levels. Neutralizing antibodies targeting IFN-gamma revealed a critical role for this cytokine in the inhibition of HCV replication. Interestingly, drugs already in clinical use, such as Phosphostim and Zoledronate, known to activate gammadelta T cells, were shown to induce the inhibition of HCV replication mediated by Vgamma9Vdelta2 T cells of HCV patients. Our data suggest that the therapeutic activation of Vgamma9Vdelta2 T lymphocytes may represent an additional strategy to inhibit HCV replication and to restore a Th1-oriented immune response in HCV-infected patients.     

Monitoring of clomiphene citrate stimulation by means of plasma 17{beta}-oestradiol determinations and ultrasonographic follicle measurements in in-vitro fertilization treatment cycles

In 57 in-vitro fertilization (IVF) cycles stimulated with clomiphenecitrate the relationship between plasma 17-oestradiol (E₂) andultrasonographic measurements of follicle diameter was assessed.Under both monofollicular and multifollicular conditions a widerange in plasma E₂ values was observed in the late follicularphase. No significant correlation could be established betweenthe dimensions of the dominant preovulatory follicle and plasmaE₂ values, in mono-follicular or multi-follicular cycles. Pregnanciesand conceptions occurred in cycles with both low and high circulatingE₂ levels. In pregnancy cycles a slight increase in plasma E₂values was found on the day following administration of humanchorionic gonadotrophin (HCG). In conceptional cycles not leadingto a clinical pregnancy, plasma E₂ profiles varied considerably,whereas in cycles in which no oocytes were fertilized, plateauingor a distinct decrease occurred during this particular period.The present study suggests that the relative daily increasein plasma E₂ values may be the most relevant aspect of plasmaE₂ monitoring.