Cellular, transcriptomic and methylome effects of individual and combined exposure to BPA, BPF, BPS on mouse spermatocyte GC-2 cell line.

Environmental factors, particularly xenoestrogens adversely affect reproductive health and their main mechanism is based on steroid-signaling pathway alterations. The presence of bisphenol A (BPA) in the environment has been confirmed and it is about to be replaced by analogues such as bisphenol F (BPF) and bisphenol S (BPS). Whether the BPF and BPS exert similar adverse effects to BPA has become the subject of intense scientific scrutiny. The aim of the present study was to evaluate and compare the cellular, transcriptomic and methylome effects of exposure to BPA, BPF, BPS individually and in combination on GC-2 spermatocyte cell line. The results show that all studied compounds affect cell viability, induce apoptosis and cause cellular damage. BPA, BPF and BPS also influence GC-2 cell steroid receptor and steroidogenesis related genes expressions. In addition to specific molecular mechanisms, all studied compounds also increase global DNA methylation. Exposure to a combination of all the studied compounds caused comparable effects on cell culture to each of them examined separately. These data suggest that exposure to BPA and its main substitutes- BPF and BPS induced multitude of effects and hence, BPF and BPS are not safe alternative to BPA in terms of male reproductive health.

Altered expression of ERs, aromatase, and COX2 connected to estrogen action in type 1 endometrial cancer biology.

In order to study estrogen-driven microenvironment associated with type 1 endometrial carcinoma, we evaluated estrogen receptors (ERs), aromatase, and cyclooxygenase II (COX2) molecular and immunohistochemical profiles with correlation to clinicopathological features. We investigated aromatase, ERα, ERß, and COX2 expression at the mRNA and protein levels using quantitative real-time PCR and immunohistochemical method in 51 endometrial carcinomas and 16 normal endometria. All the studied tumors, as well as normal endometria, expressed ERα, ERß, and COX2 mRNAs. Five endometrial carcinoma tissues and one normal endometrium showed no aromatase mRNA expression. The majority of tumors expressed ERα (82%), aromatase (80%), and COX2 (88%) proteins. Forty-one percent of the studied tumors were ERß-negative. ERα and ERß showed significantly decreased mRNA and protein expression levels in endometrial carcinoma as compared to normal endometrium. An opposite trend was shown for COX2 and aromatase proteins. ERα expression correlated positively with COX2 expression at both mRNA and protein levels (P < 0.005, r = 0.398; P < 0.0005, r = 0.510, respectively). There was also a positive correlation between COX2 and aromatase expression in cancer tissue (P < 0.002, r = 0.433 for transcriptional level; P < 0.0005, r = 0.614 for protein level). We observed positive correlations between ERß and ERα, as well as between ERß and COX2 at the transcriptional level only (P < 0.0005, r = 0.644; P < 0.002, r = 0.444, respectively). Negative correlations were found between pT category of primary tumor and levels of ERα and ERß transcripts (P < 0.02, r = -0.332; P < 0.02, r = -0.348, respectively). A negative association between ERß and the International Federation of Gynecology and Obstetrics (FIGO) staging was also found. The growth of EC1 with the presence of ERα and overexpression of aromatase and COX2 is dependent on estrogens. We believe that ERß may be considered as a potential marker in the progression of disease in endometrial cancer patients.

Identification and subsequent validation of transcriptomic signature associated with metabolic status in endometrial cancer.

Aberrant metabolism has been identified as a main driver of cancer. Profiling of metabolism-related pathways in cancer furthers the understanding of tumor plasticity and identification of potential metabolic vulnerabilities. In this prospective controlled study, we established transcriptomic profiles of metabolism-related pathways in endometrial cancer (EC) using a novel method, NanoString nCounter Technology. Fifty-seven ECs and 30 normal endometrial specimens were studied using the NanoString Metabolic Panel, further validated by qRT-PCR with a very high similarity. Statistical analyses were by GraphPad PRISM and Weka software. The analysis identified 11 deregulated genes (FDR ≤ 0.05; |FC|≥ 1.5) in EC: SLC7A11; SLC7A5; RUNX1; LAMA4; COL6A3; PDK1; CCNA1; ENO1; PKM; NR2F1; and NAALAD2. Gene ontology showed direct association of these genes with 'central carbon metabolism (CCM) in cancer'. Thus, 'CCM in cancer' appears to create one of the main metabolic axes in EC. Further, transcriptomic data were functionally validated with drug repurposing on three EC cell lines, with several drug candidates suggested. These results lay the foundation for personalized therapeutic strategies in this cancer. Metabolic plasticity represents a promising diagnostic and therapeutic option in EC.

Heart rate turbulence in postinfarction patients with history of malignant ventricular arrhythmias.

UNLABELLED: In the study, there has been retrospectively analyzed heart rate turbulence in postinfarction patients. The cohort of 158 patients consisted of 94 patients with documented ventricular tachycardia and/or ventricular fibrillation (VT/VF) and 64 patients without history of VT/VF. Turbulence onset and slope were calculated from Holter recordings, and left ventricle ejection fraction (LVEF) ≤35% was regarded as severe left ventricle dysfunction. Study groups were similar in age and sex. Left ventricle ejection fraction was lower in the VT/VF group (P < .005). Patients with VT/VF had higher turbulence onset (-0.22% ± 1% vs -0.8% ± 2%; P = .005) and lower turbulence slope (2.6 ± 1.9 vs 4.1 ± 3.5 milliseconds per RR interval; P = .01). These trends were observed in patients with LVEF >35% but not in subjects with LVEF ≤35%. Diabetes mellitus, previous coronary artery bypass graft, and amiodarone therapy have diminished the intergroup differences significantly. CONCLUSIONS: Heart rate turbulence is diminished in postinfarction patients with a history of malignant ventricular arrhythmias. It seems to separate subjects at arrhythmic risk among patients with relatively preserved left ventricle function, but it is diminished in patients with previous coronary artery bypass graft, diabetes, and amiodarone therapy.

The significance of the expression of ERRalpha as a potential biomarker in breast cancer.

It was shown the functional crosstalk between ERRalpha and ERalpha in breast cancer, however, the biological significance of estrogen-related receptor alpha (ERRalpha) remains largely unclear. Therefore, we examined the expression of ERRalpha in 39 primary human breast cancer tissues and 19 matched normal tissues using RT-PCR and immunohistochemistry in the context of the aromatase, ERalpha and proliferation markers (c-myc, Ki-67) expression. Compared to the normal breast tissue, breast cancer tissues showed a slightly higher expression level of ERRalpha mRNA (mean 46.2+/-S.D.42.0, 57.7+/-S.D.58.7, respectively). However, ERRalpha mRNA levels in breast cancer tissues showed greater diversity than in normal tissues. Immunohistochemical analysis of breast cancers revealed perinuclear and cytoplasmic localization of ERRalpha. Our study shows that there is no correlation between ERRalpha and ERalpha expression. We demonstrated a positive correlation between ERRalpha and c-myc at the transcriptional level and statistically significant positive correlation between aromatase and the ERRalpha at protein level. It seems that ERRalpha could play an important role in the alternative pathway to classical estrogen receptors-dependent pathway in cell signaling. Development and use of ERRs modulators might lead in the future to design new well-tolerated and individualized therapeutic agents.

Comparative evaluation of estrogen and progesterone receptor expression with connexins 26 and 43 in endometrial cancer.

Progression of numerous neoplasms could involve alterations of gap junction channels composed of connexins (Cxs). Disorders of expression and cellular displacement of Cxs were also found in endometrial cancer. Gap junctional intercellular communication can be regulated by wide array of agents, for instance, growth factors, oncogenes, and steroid hormones. Nevertheless, expressions of Cxs and progesterone receptor (PR) were not compared in human tissues. This study focused on assessment of expression of estrogen receptor alpha (ERalpha) and PRs in relation to the expression of Cx26 and Cx43 in 88 cases of endometrial cancer and analysis of these proteins' expression in comparison with anatomoclinical features. Positive ERalpha and PR nuclear staining was present in 66 (75%) and 60 (68.2%) of all studied tumors, respectively. Positive correlation was found between expression of PR and histopathologic type of tumor (P = 0.026), and negative correlation was drawn with grading (G) (P = 0.002). There were positive reactions to Cx26 and Cx43 of mainly cytoplasmic location in 60 (68.2%) and 66 (75%) of studied cancers, respectively. Progesterone receptor expression correlated negatively with Cx26 in endometrial cancers (P = 0.016, r = -0.256). Moreover, ERalpha expression positively correlated with PR expression (P < 0.001, r = 0.678). On the ground of our findings, disorders of Cx expression and altered distribution pattern occur during endometrial carcinogenesis, and it seems that PR could participate in this fact. Loss of functional gap junctions may occur because of the aberrant expression and localization of Cx26 and Cx43 in endometrial cancer.

The vitamin A family can significantly decrease the expression of ERbeta of ERs positive breast cancer cells in the presence or absence of ER ligands and paclitaxel.

Taxanes have high activity against breast cancer cells either as the single agent or in combination with other anticancer compounds. The aim of the study was to determine the effects of vitamin A compounds on the cytotoxic action of paclitaxel and on the expression of ERs in the MCF-7 breast cancer cells. Retinol and beta-carotene, but not retinoids, added to the culture exerted an effect on paclitaxel activity. However, only beta-carotene significantly reduced the percentage of proliferating cells (40.36% +/- 5.64, p < 0.01). We observed that vitamin A and its derivatives combined with paclitaxel and estradiol decreased the percentage of proliferating cells, but only in comparison to estradiol group, whereas retinol and lycopene administered together with paclitaxel and tamoxifen decrease significantly the percentage of proliferatin cells (36.85% +/- 4.71, p < 0.0001 and 37.22% +/- 1.59, p < 0.0001 respectively, compared with paclitaxel group). We have shown that paclitaxel increases the expression of ERalpha and ERbeta mRNA in MCF-7 line. The strongest effect of transcription inhibition ERalpha (2.5 times) and especially ERbeta (10 times) was observed after addition of 9-cis retinoic acid and paclitaxel. This data suggests a synergistic effect of the compounds on ERbeta down-regulation. Our results support the use of retinoid is treatment of ER positive breast cancer patients.

Features of the fetal gonad in androgen synthesis in the postpubertal testis are preserved in complete androgen insensitivity syndrome due to a novel genetic splice site donor variant in androgen receptor gene intron 1.

Mutations in the X-linked androgen receptor (AR) gene cause complete androgen insensitivity syndrome (CAIS). CAIS may cause congenital sexual development disorder, which frequently develops into testicular tumors. Here, we describe a novel splice-site intron 1 mutation in AR leading to improper splicing and AR protein absence in CAIS gonads. We characterized a patient's postpubertal gonadal steroidogenic enzyme expression profile. Localization of both CYP11A1 and CYP17A1 enzymes was restricted to both Leydig tumor cells and adjacent to tumor gonadal tissues. Sertoli cells of the CAIS gonad showed abundant HSD17B3 protein, which is an adult Leydig cell marker that enables the conversion of androstenedione to testosterone. Such HSD17B3 expression is typical for fetal-type Sertoli cells in rodents. The postpubertal CAIS gonad of our patient was completely devoid of androgen signaling pathway activity. Plausibly, the postpubertal Leydig cells consisted of two distinct cell populations: postpubertal fetal-type Leydig cells that persisted as androgen-independent cells and immature adult Leydig cells that failed to differentiate. Taken together, in this CAIS postpubertal testis, both Leydig and fetal-type Sertoli cells participated in testosterone production. Our results indicate the importance of molecular analysis as well as the characterization of steroidogenic enzyme profiling in the CAIS patient's gonad.

The Impact of OMEGA-3 Fatty Acids Supplementation on Insulin Resistance and Content of Adipocytokines and Biologically Active Lipids in Adipose Tissue of High-Fat Diet Fed Rats.

It has been established that OMEGA-3 polyunsaturated fatty acids (PUFAs) may improve lipid and glucose homeostasis and prevent the "low-grade" state of inflammation in animals. Little is known about the effect of PUFAs on adipocytokines expression and biologically active lipids accumulation under the influence of high-fat diet-induced obesity. The aim of the study was to examine the effect of fish oil supplementation on adipocytokines expression and ceramide (Cer) and diacylglycerols (DAG) content in visceral and subcutaneous adipose tissue of high-fat fed animals. The experiments were carried out on Wistar rats divided into three groups: standard diet-control (SD), high-fat diet (HFD), and high-fat diet + fish oil (HFD+FO). The fasting plasma glucose and insulin concentrations were examined. Expression of carnitine palmitoyltransferase 1 (CPT1) protein was determined using the Western blot method. Plasma adipocytokines concentration was measured using ELISA kits and mRNA expression was determined by qRT-PCR reaction. Cer, DAG, and acyl-carnitine (A-CAR) content was analyzed by UHPLC/MS/MS. The fish oil supplementation significantly decreased plasma insulin concentration and Homeostatic Model Assesment for Insulin Resistance (HOMA-IR) index and reduced content of adipose tissue biologically active lipids in comparison with HFD-fed subjects. The expression of CPT1 protein in HFD+FO in both adipose tissues was elevated, whereas the content of A-CAR was lower in both HFD groups. There was an increase of adiponectin concentration and expression in HFD+FO as compared to HFD group. OMEGA-3 fatty acids supplementation improved insulin sensitivity and decreased content of Cer and DAG in both fat depots. Our results also demonstrate that PUFAs may prevent the development of insulin resistance in response to high-fat feeding and may regulate the expression and secretion of adipocytokines in this animal model.

The preliminary association study of ADIPOQ, RBP4, and BCMO1 variants with polycystic ovary syndrome and with biochemical characteristics in a cohort of Polish women.

PURPOSE: We aimed to elucidate the frequency of the SNPs in the ADIPOQ, RBP4 and BCMO1genes in a population of Caucasian Polish women with polycystic ovary syndrome (PCOS), and to evaluate the possible associations between these variants and the susceptibility to PCOS. Additionally, the relationship of these polymorphisms to a clinical phenotype of this syndrome, and the concentrations of adipokines, were determined. MATERIALS/METHODS: Clinical and biochemical profiles, DNA isolation and genotyping, and adipokine assays were performed in 294 PCOS women and 78 controls. RESULTS: In a cohort of Polish women, for the genotype distribution and allele frequencies (minor allele frequency - MAF) proved that only the SNP rs1501299 in the gene ADIPOQ (P = 0.0010, OR = 0.41, 95% C.I.:0.24-0.70) and rs7501331 in the gene BCMO1 (P = 0.0106, OR = 0.24, 95% C.I.:0.21-0.71), are significantly associated (the latter marginally significant) with the decrease of the risk of the disease. Also for this SNPs there were significant differences in the genotypic frequencies in the study population. There was a link between rs12934922 of BCMO1 gen and serum concentration of RBP4 (P = 0.034) and adiponectin (P = 0.038) in the study group but not in the control group. The elevated mean serum concentration of cholesterol (P = 0.020) and LDL cholesterol (P = 0.005) was observed for GG rs1501299 genotype and triglycerides (P = 0.028) for TT rs2241766 genotype. CONCLUSIONS: The results of the present study revealed that the genes variants RBP4 is not associated with PCO. It seems that rs1501299 of ADIPOQ gene influences the occurrence of PCO and lipids profile in those patients.

Advances in the Molecular Pathophysiology, Genetics, and Treatment of Primary Ovarian Insufficiency.

Primary ovarian insufficiency (POI) affects ∼1% of women before 40 years of age. The recent leap in genetic knowledge obtained by next generation sequencing (NGS) together with animal models has further elucidated its molecular pathogenesis, identifying novel genes/pathways. Mutations of >60 genes emphasize high genetic heterogeneity. Genome-wide association studies have revealed a shared genetic background between POI and reproductive aging. NGS will provide a genetic diagnosis leading to genetic/therapeutic counseling: first, defects in meiosis or DNA repair genes may predispose to tumors; and second, specific gene defects may predict the risk of rapid loss of a persistent ovarian reserve, an important determinant in fertility preservation. Indeed, a recent innovative treatment of POI by in vitro activation of dormant follicles proved to be successful.

[The role of peroxisome proliferator-activated receptors (PPAR) in carcinogenesis]. / Udzial receptorów aktywujacych proliferacje peroksysomów (PPAR) w karcinogenezie.

Peroxisome proliferators-activated receptors are members of the nuclear hormone receptor superfamily of ligand-activated transcription factors. The PPAR subfamily consists of three members: PPAR-alpha, PPAR-sigma (NUC-1 or beta) and PPAR-gamma. PPARs regulate gene expression by binding, as heterodimers with retinoid X receptors (RXR), to specific response elements (PPREs) in the promoter regions of target genes. The prostaglandin 12 especially, all arachidonic acid metabolites and polyunsaturated fatty acids are naturally occuring PPAR ligands. Synthetic PPAR ligands are thiazolidinediones (TZDs--rosiglitazone, pioglitazone, troglitazone). Activation of nuclear hormone receptors has been identified as an approach to induce differentiation and inhibit proliferation of cancer lines. The anti-proliferative, pro-differentiation effects of PPAR activators (TZDs) suggest that these compounds might be useful in slowing the proliferation of un-differentiated tumour cells. TZDs inhibit proliferation of human breast, prostate and colon cancers, both in vitro and in tumours derived from these cells implanted into rodents. Furthermore, recent studies show that PPAR-gamma ligands are potent inhibitors of angiogenesis, a process essential for solid-tumour growth and metastasis. In conclusion, the evidence to date suggests that activation of PPAR should suppress tumour growth and development. This represents an exciting novel therapeutic application of TZDs. In present paper, structural features of PPARs, their gene transcription mechanisms and recent developments in the discovery of their biological functions are reviewed.

Distinct mRNA, protein expression patterns and distribution of oestrogen receptors alpha and beta in human primary breast cancer: correlation with proliferation marker Ki-67 and clinicopathological factors.

To elucidate the molecular profile of oestrogen receptors alpha and beta (ERalpha, ERbeta) we studied ERalpha and ERbeta expression at the mRNA and protein levels using real-time polymerase chain reaction (RT-PCR), Western blot analysis and immunohistochemical (IHC) methods in 41 primary breast cancers and surrounding tissues. ERalpha mRNA and ERbeta mRNA were detected in all of the breast cancer and normal matched tissues analysed. ERalpha mRNA levels showed greater diversity than ERbeta mRNA levels and the range of amount of ERbeta transcripts was far smaller than that of ERalpha. At the protein level, the percentage of ERalpha- or ERbeta-positive cases changed. Seventy percent of the tumours studied produced full-length 65 kDa ERalpha protein in Western blot analysis and 67% of assessed cases were positive in IHC. Full-length 57 kDa ERbeta protein was detected by Western blotting in 97% of analysed breast cancers, while 67% were ERbeta-positive using IHC. ERalpha was localised in the nucleus, while cytoplasmic and perinuclear localisation of ERbeta was observed in normal as well as in breast cancer cells. The amount of ERalpha (but not ERbeta) increased with age. The expression of ERalpha correlated positively with progesterone receptor and negatively with proliferation marker Ki-67. These results confirm the previous observations that the lack of ERalpha protein expression is not due to lack of ERalpha gene expression or methylation of ERalpha promoter, but due to post-transcriptional or post-translational mechanisms. Our investigation also suggests that ERalpha is more dysregulated in breast cancer, and thereby ERbeta is more tightly regulated in the tumour.

Expression of insulin-like growth factor-I receptor, estrogen receptor alpha, Bcl-2 and Bax proteins in human breast cancer.

Disturbance in expression of estrogen receptors together with changing influence of growth factor receptors and apoptosis associated proteins plays a role in breast cancer development and progression. However, immunohistochemical detection and relationships among these proteins were not often considered in relation to breast cancer and a few evaluations of expression provided mismatching results and conclusions. Consequently, we examined by immunohistochemistry the expression of the insulin-like growth factor-I receptor (IGF-IR), estrogen receptor alpha (ERalpha) and apoptosis-associated proteins, Bcl-2 and Bax, in human primary breast cancer, as well as analyzing the relationships among these proteins. The positive immunostaining for IGF-IR, ERalpha, Bcl-2 and Bax was noted in 56, 63.8, 82.8 and 50% of tumors, respectively. We observed that IGF-IR negatively correlated with ERalpha in the group of all tumors and in axillary node negative cancer (p<0.03, p<0.05, respectively), but not in the subgroup of node positive cancer. Expression of ERalpha correlated positively with Bcl-2 and negatively with Bax proteins (p<0.0001, p<0.05, respectively). We did not note significant relationships between IGF-IR and Bcl-2, or IGF-IR and Bax proteins. We found that increased Bax expression was associated with positive lymph node status, pT2 stage and G3 grade of tumors. Knowledge about alterations in the IGF-IR expression and relations of the receptor to other biological factors could help in our understanding of breast cancer biology and the importance of the IGF-IR in cancer progression as well as in effective management of breast cancer.

Immunohistochemical study of KiSS1 and KiSS1R expression in human primary breast cancer: Association with breast cancer receptor status, proliferation markers and clinicopathological features.

Recent studies have raised doubts about the protective role of KiSS1/KiSS1R in breast malignancy progression. However, the role of the KiSS1/KiSS1R system in primary breast cancer remains largely unknown. The aim of the present study was to characterize the biology and invasiveness potential of primary breast cancer through evaluation of KiSS1/KiSS1R protein expression and cellular localization with regard to lymph node metastasis status, receptor status (ERs, PR and HER-2/neu), and expression of aromatase, MMP-9, Ki-67 and Cyclin D1 in primary invasive breast cancer tissues. We showed increased protein expression of both KiSS1/KiSS1R and MMP-9 in the cancerous tissues compared with noncancerous tissue adjacent to the breast tumour. In the studied group of breast cancer samples, we observed a positive correlation between KiSS1 and MMP-9. We also showed a positive correlation between KiSS1R and aromatase expression in all studied breast cancers. We did not notice any associations between system and cell cycle regulators. KiSS1/KiSS1R did not correlate either with Cyclin D1 and Ki-67 or with receptor status. However, we showed higher levels of KiSS1R expression in ERα-negative cases than in ERα-positive cases in patients with lymph node metastasis. Present data do not confirm the protective role of KiSS1/KiSS1R in breast cancer progression, but our results do support the hypothesis that the KiSS1/KiSS1R system is activated even in primary breast cancer and sustained during invasion to local lymph nodes.

Expanding the Spectrum of Founder Mutations Causing Isolated Gonadotropin-Releasing Hormone Deficiency.

CONTEXT: Loss of function (LoF) mutations in more than 20 genes are now known to cause isolated GnRH deficiency (IGD) in humans. Most causal IGD mutations are typically private, ie, limited to a single individual/pedigree. However, somewhat paradoxically, four IGD genes (GNRH1, TAC3, PROKR2, and GNRHR) have been shown to harbor LoF founder mutations that are shared by multiple unrelated individuals. It is not known whether similar founder mutations occur in other IGD genes. OBJECTIVE: The objective of the study was to determine whether shared deleterious mutations in IGD-associated genes represent founder alleles. SETTING: This study was an international collaboration among academic medical centers. METHODS: IGD patients with shared mutations, defined as those documented in three or more unrelated probands in 14 IGD-associated genes, were identified from various academic institutions, the Human Gene Mutation Database, and literature reports by other international investigators. Haplotypes of single-nucleotide polymorphisms and short tandem repeats surrounding the mutations were constructed to assess genetic ancestry. RESULTS: A total of eight founder mutations in five genes, GNRHR (Q106R, R262Q, R139H), TACR3 (W275X), PROKR2 (R85H), FGFR1 (R250Q, G687R), and HS6ST1 (R382W) were identified. Most founder alleles were present at low frequency in the general population. The estimated age of these mutant alleles ranged from 1925 to 5600 years and corresponded to the time of rapid human population expansion. CONCLUSIONS: We have expanded the spectrum of founder alleles associated with IGD to a total of eight founder mutations. In contrast to the approximately 9000-year-old PROKR2 founder allele that may confer a heterozygote advantage, the rest of the founder alleles are relatively more recent in origin, in keeping with the timing of recent human population expansion and any selective heterozygote advantage of these alleles requires further evaluation.

A case of complete hypogonadotropic hypogonadism with a mutation in the gonadotropin-releasing hormone receptor gene.

OBJECTIVE: To screen for mutations in the GnRH receptor gene in a case of complete hypogonadotropic hypogonadism (HH) with GnRH resistance. DESIGN: Case report. SETTING: A university hospital. PATIENT(S): A male patient with the complete form of HH without anosmia. INTERVENTION(S): Physical examination and laboratory and genetic studies. MAIN OUTCOME MEASURE(S): Gonadotropins at the basal state and after GnRH administration and GnRH receptor DNA sequencing. RESULT(S): A novel missense mutation, localized in the first amino acid of the extracellular loop found in the heterozygous state, and another mutation, Arg(139)His (R139H), located in the conserved aspartate-arginine-serine motif at the junction of the third transmembrane and second intracellular loop of the GnRH receptor, were identified in the homozygous state. Pedigree studies reveal that both parents were heterozygous for R139H, while the mother carried the missense mutation at codon 1(M1T). CONCLUSION(S): GnRH receptor mutations may account for a larger proportion of cases of HH than previously thought. The phenotypic spectrum of HH seems to vary, and this heterogeneity may be related, at least in part, to the degree of impaired biological activity of the mutated GnRH receptor caused by the allelic type of mutations.

Differential effects of raloxifene and tamoxifen on the expression of estrogen receptors and antigen Ki-67 in human endometrial adenocarcinoma cell line.

Tamoxifen and raloxifene are widely used in clinical practice. It has been found that tamoxifen treatment increases the risk of development of endometrial cancer. The effects of tamoxifen and raloxifene on endometrium might be caused by different estrogen receptor expression. The aim of the present study was immunohistochemical evaluation of the effects of tamoxifen and raloxifene on estrogen receptors, and Ki-67 antigen expression in the human endometrial adenocarcinoma Ishikawa cell line. Tamoxifen in concentrations of 10 microM and 20 microM increased ERalpha expression without any effect on ERbeta. All used concentrations of tamoxifen and raloxifene (0.1 nM, 1 nM, 10 nM, 1 micro M, 10 microM and 20 microM) had no effect on expression of ERbeta. Tamoxifen, but not raloxifene, increased Ki-67 antigen expression in the Ishikawa cell line. Tamoxifen, in contrast to raloxifene, increased proliferation of endometrial adenocarcinoma cells as well as exerted the shift of ERalpha/ERbeta ratio. Thus, it could be responsible for increased carcinogenic effect during tamoxifen treatment.

Differential effects of estradiol and raloxifene on collagen biosynthesis in cultured human skin fibroblasts.

The dose-dependent effect of a 24 h treatment with estradiol (E(2)) (1, 2, 5, 10 nM) and raloxifene (Rx) (1, 5, 10, 20 microM) on ER alpha and ER beta mRNA expression, collagen bio-synthesis, prolidase activity, MMP-2, MMP-9, insulin-like growth factor I receptor expression (IGF-1R) and beta1-integrin expressions in cultured fibroblasts obtained from postmenopausal women were examined. Both ligands increased mRNA expression of ER compared to control. Rx at 5 and 10 microM concentrations had greater stimulative effect on collagen biosynthesis, prolidase activity and IGF-1R expression compared to E(2) at 2 and 5 nM concentration. Both studied ER ligands had no effect on beta1-integrin receptor expressions. MMP-2 expression was not detected in human skin fibroblast culture. In contrast to estradiol raloxifene inhibited the expression of MMP-9. Raloxifene had stronger positive stimulative effects on collagen biosynthesis, through different biochemical mechanisms, than estradiol in human skin fibroblasts and might reverse some of the postmenopausal changes in skin or connective tissue. Increase of collagen synthesis induced by raloxifene may be activated by both estrogen receptor dependent and independent pathways such as up-regulation of estrogen receptors, up-regulation of IGF receptor, transcriptional regulation of collagen genes by estrogen receptor-raloxifene complex, increasing of prolidase activity or finally by inhibition of MMP-9 expression.