Down Regulation of Catsper1 Expression by Calmodulin Inhibitor (Calmidazolium): Possible Implications for Fertility.

The CatSper channel localizes exclusively in the flagella of sperm cells. The Catsper1 protein, together with three pore units, is essential for the CatSper Channel formation, which produces flagellum hyperactivation and confers sperm fertility. Catsper1 expression is dependent on Sox transcription factors, which can recognize in vitro at least three Sox binding sites on the promoter. Sox transcription factors have calmodulin-binding domains for nuclear importation. Calmodulin (CaM) is affected by the specific inhibitor calmidazolium (CMZ), which prevents the nuclear transport of Sox factors. In this work, we assess the regulation of the Catsper1 promoter in vivo by Sox factors in the murine testis and evaluate the effects of the inhibitor calmidazolium on the expression of the Casper genes, and the motility and fertility of the sperm. Catsper1 promoter has significant transcriptional activity in vivo; on the contrary, three Sox site mutants in the Catsper1 promoter reduced transcriptional activity in the testis. CaM inhibition affects Sox factor nuclear transport and has notable implications in the expression and production of Catsper1, as well as in the motility and fertility capability of sperm. The molecular mechanism described here might conform to the basis of a male contraceptive strategy acting at the transcriptional level by affecting the production of the CatSper channel, a fundamental piece of male fertility.

Effects of the lifestyle habits in breast cancer transcriptional regulation.

Through research carried out in the last 25 years about the breast cancer etiology, it has been possible to estimate that less than 10 % of patients who are diagnosed with the condition are carriers of some germline or somatic mutation. The clinical reports of breast cancer patients with healthy twins and the development of disease in women without high penetrance mutations detected, warn the participation more factors in the transformation process. The high incidence of mammary adenocarcinoma in the modern woman and the urgent need for new methods of prevention and early detection have demanded more information about the role that environment and lifestyle have on the transformation of mammary gland epithelial cells. Obesity, alcoholism and smoking are factors that have shown a close correlation with the risk of developing breast cancer. And although these conditions affect different cell regulation levels, the study of its effects in the mechanisms of transcriptional and epigenetic regulation is considered critical for a better understanding of the loss of identity of epithelial cells during carcinogenesis of this tissue. The main objective of this review was to establish the importance of changes occurring to transcriptional level in the mammary gland as a consequence of acute or chronic exposure to harmful products such as obesity-causing foods, ethanol and cigarette smoke components. At analyze the main studies related to topic, it has concluded that the understanding of effects caused by the lifestyle factors in performance of the transcriptional mechanisms that determine gene expression of the mammary gland epithelial cells, may help explain the development of this disease in women without genetic propensity and different phenotypic manifestations of this cancer type.

A través de la investigación realizada en los últimos 25 años en torno a la etiología del cáncer de mama, ha sido posible estimar que menos del 10 % de las pacientes que son diagnosticadas con la enfermedad son portadoras de alguna mutación de línea germinal o somática. Los informes clínicos de pacientes de cáncer de mama con gemelas saludables y el desarrollo de la enfermedad en las mujeres sin mutaciones de alta penetrancia detectadas, advierten la participación de otros factores en el proceso de transformación. La alta incidencia de adenocarcinoma de mama en la mujer moderna y la necesidad urgente de nuevos métodos para la prevención y detección temprana han exigido una mayor información en relación al papel que el medio ambiente y el estilo de vida tienen en la transformación de las células epiteliales de la glándula mamaria. La obesidad, el alcoholismo y el tabaquismo son factores que han demostrado una estrecha correlación con el riesgo de desarrollar cáncer de mama. Y aunque estas condiciones pueden afectar distintos niveles de regulación celular, el estudio de sus efectos en los mecanismos de regulación transcripcional y epigenética, es considerado fundamental para un mayor entendimiento de la pérdida de identidad de las células epiteliales durante la carcinogénesis de este tejido. El objetivo principal de esta revisión fue establecer la importancia de los cambios que ocurren a nivel transcripcional en la glándula mamaria, como consecuencia de una exposición aguda o crónica a productos nocivos, tales como los alimentos que favorecen la obesidad, el etanol y los componentes del humo del cigarro. Al analizar los principales estudios relacionados con el tema, se ha llegado a la conclusión de que la comprensión de los efectos causados por los factores de estilo de vida sobre el desempeño de los mecanismos de regulación transcripcional responsables de la expresión génica de las células epiteliales de la glándula mamaria, pueden ayudar a explicar el desarrollo de esta enfermedad en las mujeres que no son genéticamente propensas así como las diferentes manifestaciones fenotípicas de este tipo de cáncer.

Differential effect of DDT, DDE, and DDD on COX-2 expression in the human trophoblast derived HTR-8/SVneo cells.

The purpose of this study was to investigate the effect of 1,1,1-trichloro-2,2-bis-(chlorophenyl)ethane (DDT), 1,1-bis-(chlorophenyl)-2,2-dichloroethene (DDE), and 1,1-dichloro-2,2-bis(chlorophenyl)ethane (DDD) isomers on COX-2 expression in a human trophoblast-derived cell line. Cultured HTR-8/SVneo trophoblast cells were exposed to DDT isomers and its metabolites for 24 h, and COX-2 mRNA and protein expression were assessed by RT-PCR, Western blotting, and ELISA. Prostaglandin E2 production was also measured by ELISA. Both COX-2 mRNA and protein were detected under control (unexposed) conditions in the HTR-8/SVneo cell line. COX-2 protein expression and prostaglandin E2 production but not COX-2 mRNA levels increased only after DDE and DDD isomers exposure. It is concluded that DDE and DDD exposure induce the expression of COX-2 protein, leading to increased prostaglandin E2 production. Interestingly, the regulation of COX-2 by these organochlorines pesticides appears to be at the translational level.

Reproductive axis function and gonadotropin microheterogeneity in a male rat model of diet-induced obesity.

Obesity causes complex metabolic and endocrine changes that may lead to adverse outcomes, including hypogonadism. We herein studied the reproductive axis function in male rats under a high-fat diet and analyzed the impact of changes in glycosylation of pituitary LH on the bioactivity of this gonadotropin. Rats were fed with a diet enriched in saturated fat (20% of total calories) and euthanized on days 90 or 180 of diet. Long-term (180 days), high-fat feeding rats exhibited a metabolic profile compatible with insulin resistance and metabolic syndrome; they concomitantly showed decreased intrapituitary and serum LH concentrations, low serum testosterone levels, and elevated serum 17beta-estradiol concentrations. A fall in biological to immunological ratio of intrapituitary LH was detected in 180 days control diet-treated rats but not in high-fat-fed animals, as assessed by a homologous in vitro bioassay. Chromatofocusing of pituitary extracts yielded multiple LH charge isoforms; a trend towards decreased abundance of more basic isoforms (pH 9.99-9.0) was apparent in rats fed with the control diet for 180 days but not in those that were fed the diet enriched in saturated fat. It is concluded that long-term high-fat feeding alters the function of the pituitary-testicular axis, resulting in hypogonadotropic hypogonadism. The alterations in LH function found in these animals might be subserved by changes in hypothalamic GnRH output and/or sustained gonadotrope exposure to an altered sex steroid hormone milieu, representing a distinctly different regulatory mechanism whereby the pituitary attempts to counterbalance the effects of long-term obesity on reproductive function.

GPR30 expression and function in breast cancer cells are induced through a cis­acting element targeted by ETS factors.

The capacity that G protein­coupled receptor 30 (GPR30) has demonstrated for triggering estrogen­dependent signaling pathways has attracted the interest of breast cancer researchers; however, the reported expression profiles and functions of GPR30 in breast cancer are inconsistent. The main purpose of the present investigation was to identify transcriptional mechanisms underlying the expression of GPR30 that allow a better understanding of its role in breast cancer progression. In the cell lines used as different polarity models in the present study, it was determined immunologically that GPR30 is expressed in normal mammary gland cells and that this expression decreased considerably during breast cancer development, where cell identity is lost. However, it was also determined that, in spite of low GPR30 expression levels in breast cancer cells with little differentiation, this membrane estrogen receptor (ER) is able to increase cell viability and suppress migration in cells that have acquired metastatic capacity. In addition, through transient expression assays in breast cancer cells, it was revealed that a transcriptional mechanism dependent on protein kinase A and susceptible to retinoic acid in ER­positive cells induces GPR30 expression through a cis­regulatory element for E26 transformation­specific transcription factors, located between ­631 and ­625 bp from the GPR30 translation start codon. Overall, these results suggested that in vitro transcriptional regulation of GPR30 expression in breast cancer cells may serve a relevant role in the conservation of an epithelial phenotype, and also may be important to avoid the transition to metastasis.

Pituitary-testicular axis function, biological to immunological ratio and charge isoform distribution of pituitary LH in male rats with experimental diabetes.

Men with insulinopenic diabetes mellitus frequently present hypogonadism and exhibit circulating luteinizing hormone (LH) molecules with increased biological activity. To further study this latter issue, we analyzed the pattern of isoform distribution and the impact of changes in terminal glycosylation of pituitary LH on the bioactivity of this gonadotropin in experimental diabetes. Adult male rats were treated with streptozotocin or vehicle and euthanized on days 30, 60, or 90 posttreatment. All diabetic groups exhibited a significant decrease in serum insulin and testosterone levels as well as in sperm count; serum gonadotropins and 17beta-estradiol decreased only after 90 days of insulinopenia. Both the immunoreactive concentrations and the biological to immunological ratio of intrapituitary LH significantly increased in all experimental groups, as assessed by an in vitro homologous bioassay in HEK-293 cells expressing a recombinant LH receptor. Chromatofocusing of pituitary extracts revealed the presence of multiple LH charge isoforms; the pH distribution profile of LH in diabetic and control rats was indistinguishable on days 30 and 60 posttreatment. By contrast, the abundance of more basic isoforms (pH 9.99-9.0) decreased and that of isoforms with pH values 8.99-8.0 increased in rats with long-standing diabetes compared to controls. It is concluded that experimental diabetes alters the function of the pituitary-testicular axis, resulting in reduced sex steroids levels and hypogonadotropism. Long-standing insulinopenia leads to a paradoxical accumulation of intrapituitary LH molecules enriched in bioactivity with altered terminal glycosylation, which are apparently subserved by distinct mechanisms involving altered hypothalamic and/or gonadal inputs on the gonadotrope.

Expression profiling of lipocalin-2 and 24p3 receptor in murine gonads at different developmental stages.

Numerous clinical studies have reported the association between high circulating levels of lipocalin-2 (LCN2) and metabolic diseases. However, only few studies have addressed sexually dimorphic, either in its circulating concentration or in its expression in other organs. To the best of our knowledge, LCN2 and the 24p3 receptor (24p3R), have not been identified in gonads; therefore, the present study analyzed their mRNA expression profile and cellular localization in gonads collected from fetal rats at 21 days post coitum, as well as from neonatal rats at 0, 2, 4, 6, 12, 20 and 30 postnatal days. Semiquantitative polymerase chain reaction and immunohistochemical assays revealed that the LCN2 mRNA during perinatal and pre-pubertal stages presented a sex-specific expression pattern, being higher in ovaries than in testes collected at these stages. Furthermore, the mRNA levels of the long and short isoforms of the 24p3R (507 and 350 bp, respectively), were lower in female gonads from postnatal day 0 onwards in comparison with the levels observed in males, but before birth, the short isoform of the 24p3R was higher in ovaries than in testes. In addition, in females, the abundance of mRNA of this isoform was drastically diminished at 24 h after birth. Furthermore, this specific expression profile of LCN2 and 24p3R at perinatal and prepubertal stages coincides with events of cellular proliferation and apoptosis within both gonads. Immunohistochemical assays revealed that in ovaries, LCN2 and 24p3R are present in germinal and somatic cells of follicles, while in testes, this adipokine and its receptor are only located in germinal cells. These findings suggest that in murine gonads, LCN2/24p3R signaling may be involved either in cell proliferation or cell death driven by gonadotropin-independent or -dependent mechanisms.

Regulation of CATSPER1 expression by the testis-determining gene SRY.

CATSPER1 gene encodes a pore-forming and pH-sensing subunit of the CatSper Ca2+- permeable channel, a protein in the flagellum essential for sperm hyperactivation. Previous studies have shown that the murine Catsper1 gene promoter is regulated by different Sox proteins. Likewise, it is acknowledged that the human CATSPER1 gene promoter sequence is enriched in potential interaction sites for the sex-determining region Y gene (SRY), which suggest a novel regulatory transcriptional mechanism for CatSper1 channel expression. Therefore, in this work, we sought to determine whether the human CATSPER1 gene expression is regulated by the SRY transcription factor. To this end, a series of deletions and mutations were introduced in the wild- type CATSPER1 gene promoter to eliminate the SRY sites, and the different constructs were tested for their ability to activate transcription in human embryonic kidney and murine spermatogonial germ cell lines (HEK-293 and GC1-spg, respectively) using luciferase assays. In addition, by using a strategy that combines electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation (ChIP) we investigated whether the CATSPER1 gene expression is regulated by the SRY transcription factor both in vitro and in vivo. Our results show that the transcriptional factor SRY specifically binds to different sites in the promoter sequence and has the ability to control CATSPER1 gene transcription.

Correlation of Kupperman's index with estrogen and androgen levels, according to weight and body fat distribution in postmenopausal women from Mexico City.

OBJECTIVES: To establish the differences in Kupperman's index (KI) and hormone levels according to weight and body fat distribution in postmenopausal women, since obesity and fat distribution affect hormone levels. MATERIAL AND METHODS: One hundred and twenty-five postmenopausal women were studied and divided according to body mass index (BMI) and waist-hip ratio (WHR): normal weight (BMI < or = 27), obesity (BMI > 27); lower-level body fat distribution (WHR < or = 0.85) and upper-level body fat distribution (WHR >0.85). Afterwards four subgroups were created: (I) BMI < or = 27 and WHR < or = 0.85, (II) BMI < or = 27 and WHR > 0.85, (III) BMI > 27 and WHR < or = 0.85, and (IV) BMI >27 and WHR > 0.85. Climacteric symptoms were analyzed with Kupperman's index. Estrone, estradiol, testosterone, androstenedione, and dehydroepiandrosterone sulfate determinations were done by radioimmunoassay and verified by chemoluminescence. The androstenedione-estrone and testosterone-estradiol ratios were calculated. Statistical analysis was by Student's t test for independent samples, plus Pearson's correlation analysis. RESULTS: Average age was 53.0 +/- 6.5 years, time since menopause 74.2 +/- 64.3 months. When comparing those with lower-level body fat distribution and those with upper-level body fat distribution, the A levels were significantly lower (P < 0.04) in those with upper-level distribution. Kupperman's index was significantly lower in subgroup I when compared with subgroups III and IV. The androstenedione level was lower in subgroup IV compared with subgroup III. In the whole sample, there was a correlation of the WHR with testosterone (0.297, P < .004) and the testosterone-estradiol ratio (0.209, P < .04). CONCLUSION: It was shown that the testosterone-estradiol ratio has a better correlation with the symptoms, so it can be used to evaluate climacteric patients when they complain of menopausal symptoms.

Ribosomal S6 kinase 4 (RSK4) expression in ovarian tumors and its regulation by antineoplastic drugs in ovarian cancer cell lines.

Survival rate in ovarian cancer depends on the stage of the disease. RSK4, which has been considered as a tumor suppressor factor, controls cells invasion due to its antiinvasive and antimetastatic properties. Modulation of RSK4 expression could be an important event to increase the survival rate in ovarian cancer patients. Thus, the goal of the present study was to establish the differences in RSK4 expression among normal, benign and malignant ovarian tissues and to determine whether antineoplastic drugs regulate its expression in SKOV3 and TOV-112D cells. RSK4 levels in 30 malignant ovarian tumors, 64 benign tumors and 36 normal ovary tissues were determined by reverse transcription polymerase chain reaction and Western blot. Modulation of RSK4 expression by two antineoplastic drugs (cisplatin and vorinostat) was also studied in the SKOV3 and TOV-112D ovarian cancer cell lines using the same techniques. RSK4 mRNA and protein levels were decreased in malignant ovarian tumors as compared to benign tumors and normal tissue. These low-RSK4 levels were significantly associated with advanced stages of ovarian cancer. RSK4 expression was increased after incubation of SKOV3 and TOV-112D cell lines with cisplatin and vorinostat for 24 h. The combination of these antineoplastic drugs did not produce a synergistic or additive effect. These results suggest that RSK4 is expressed at low levels in malignant ovarian tumors, which correlates with advanced stages of the disease. Additionally, RSK4 expression is regulated by cisplatin and vorinostat in two ovarian cancer cell lines.

Evidence of sexual dimorphism in placental vitamin D metabolism: Testosterone inhibits calcitriol-dependent cathelicidin expression.

Male fetus and neonates show increased immune vulnerability compared to females, which results in a higher risk of perinatal infections. These differences could partially be due to sex steroids differential modulation of vitamin D metabolism; since calcitriol, the most active vitamin D metabolite, regulates immune responses and transcriptionally induces the antimicrobial peptide cathelicidin in the human placenta. Calcitriol availability depends on CYP27B1 and CYP24A1 expression, the cytochromes involved in its synthesis and degradation, respectively. However, the effects of testosterone upon these enzymes and the final biological outcome upon the calcitriol-dependent immune-target cathelicidin in the placenta have not been studied. In this study we show that testosterone significantly inhibited CYP27B1 while stimulated CYP24A1 gene expression in cultured trophoblasts. These effects were accompanied by CREB activation through cAMP-independent and androgen receptor-dependent mechanisms. Male placental cotyledons showed reduced basal CYP27B1 and cathelicidin gene expression compared to females (P<0.05). Testosterone concentration was higher in the cord blood of male neonates (P=0.007), whereas cathelicidin levels were lesser compared to females (P=0.002). Altogether our results suggest that male placentas produce less cathelicidin due to decreased calcitriol bioavailability. We propose that the observed sex-dependent differences in placental vitamin D metabolism contribute in fetal responses to infections and could partially explain why the increased male fetuses immune vulnerability. Moreover, gestational hyperandrogenemia could adversely affect placental vitamin D metabolism independently of fetal sex.

Effects of FSH and 17beta-estradiol on the transactivation of estrogen-regulated promoters and cell proliferation in L cells.

In the present study, we analyzed human follicle-stimulating hormone (FSH)-induced cell proliferation and transactivation of estrogen-sensitive reporter genes-in L cells stably expressing the human FSH receptor [L-(hFSHR(+)) cells]. In order to dissect the signaling pathways involved in this process, L-(hFSHR(+)) cells were transiently transfected with either the 3X-ERE-TAT-Luc or the ERE-VitA2-TK-CAT reporter genes and treated with FSH or PKA activators (cholera toxin, forskolin and 8-Br-cAMP) in the presence or absence of various kinase inhibitors. We found that FSH and all PKA activators, specifically induced transactivation of both reporter genes. Transactivation of estrogen-sensitive genes by FSH or PKA activators were blocked (approximately 90%) by H89 (PKA inhibitor) and LY294002 but not by Wortmannin (PI3-K inhibitors), 4-OH-tamoxifen, ICI182,780 or SB203580 (p38 MAPK inhibitor); PD98059 (ERK1/2 inhibitor) partially (approximately 30%) blocked the FSH-mediated effect. The combination of FSH and estradiol resulted in a synergistic effect on transactivation as well as on cell proliferation, and this enhancement was attenuated by antiestrogens. We additionally analyzed the participation of the coactivators SRC-1 and cAMP response element binding protein (CREB)-binding protein (CBP) in FSH-evoked estrogen receptor (ER)-dependent transactivation; we found that CBP but not SRC-1 potentiated FSH-induced transcriptional activation of both ER-sensitive reporters, being this effect stronger on the ERE-VitA2-TK-CAT than on the 3X-ERE-TAT-Luc reporter. Thus, in L-(hFSHR(+)) cells FSH induces transcriptional activation of estrogen-sensitive genes through an A-kinase-triggered signaling pathway, using also to a lesser extent the ERK1/2 and p38 pathways. PI3-K is not apparently involved in this FSH-mediated process since LY294002, but not Wortmannin, specifically binds ERs and completely blocks estrogen action. Presumably, CBP cooperates with the ER on genes that contain estrogen responsive elements through mechanisms involving the participation of other proteins and/or basal transcription factors (e.g. CREB), which in turn mediate the transcriptional response of estrogen-sensitive reporter genes to FSH stimulation.

[Modification of C3 and C4 levels after the administration of conjugated estrogens plus medroxiprogesterone or raloxifene in postmenopausal women]. / Modificación de las concentraciones de C3 y C4 luego de la administración de estrógenos conjugados más medroxiprogesterona o raloxifeno en mujeres posmenopáusicas.

OBJECTIVE: To evaluate the effect of conjugated equine estrogens (CEE) plus medroxyprogesterone acetate (MPA) and raloxifene on serum levels of complement C3 and C4 fractions in postmenopausal women. PATIENTS AND METHOD: Twenty healthy postmenopausal women were studied. In all weight, height and body mass index (BMI) were documented. FSH and estradiol levels were measured. They were randomly divided into two groups, according to the treatment they received: group I, CEE 0.625 mg/day plus MPA 2.5 mg/day (n = 7); group II, raloxifene 60 mg/day (n = 13), both treatments were continuous. Serum levels of C3 and C4 complement fractions were measured by immunonephelometry at baseline and six months after start of treatment. Differences among groups of baseline and final C3 and C4 levels were measured with Student's t test for independent and paired samples, respectively. RESULTS: There were no differences among groups in age, weight, height and body mass index, neither in C3 and C4 levels among baseline and final levels when comparing each group separately. CONCLUSIONS: Complement may not intervene significantly in the atherosclerotic inflammatory process in women receiving CEE plus MPA or raloxifene.

Effect of ovine luteinizing hormone (oLH) charge isoforms on VEGF and cAMP production.

Although an increase in VEGF expression and synthesis in association with LH has been established; it is unknown if all LH isoforms act similarly. This study evaluated the production of cAMP and VEGF among LH isoforms in two in vitro bioassays. The LH was obtained from hypophyses and the group of isoforms was isolated by chromatofocusing. cAMP production was assessed using the in vitro bioassay of HEK-293 cells and VEGF production was evaluated in granulosa cells. Immunological activity was measured with a homologous RIA. Immunoactivity and bioactivity for each isoform were compared against a standard, by estimating the IC50 and the EC50. The basic isoforms were more immunoactive than the standard. The neutral and the moderately acidic had an immunological activity similar to the standard. The acidic isoform was the least immunoreactive. cAMP production at the EC50 dose was similar among the basic isoforms, the moderately acidic and the standard; for the neutral and the acidic, the EC50 dose was higher. It was observed that compared with the control, VEGF production at the lowest LH dose was no different in the standard and each isoform. In the intermediate dose, a positive response was caused in the standard and the neutral and basic isoforms. Although the acidic isoform showed a dose-dependent response, it was not significant relative to the control. In conclusion, the basic isoform generated the greatest cAMP and VEGF production, similar to the reference standard, and the acidic the smallest.

Poorly controlled type I diabetes mellitus in young men selectively suppresses luteinizing hormone secretory burst mass.

Alterations in the reproductive axis function are present to a variable extent in patients with type 1 diabetes mellitus (IDDM). Results from studies in IDDM men have yielded discrepant findings, which may reflect nonuniform patient selection criteria, age, diabetic status, duration of the disease and differences in sampling protocols. To more clearly define the impact of early diabetic alterations in the male reproductive axis, we applied a combined strategy of patient selection restricted to young men with relatively short duration of IDDM, dual control groups, multiparameter deconvolution analysis to assess LH secretory activity, and assessment of time-dependent changes in human chorionic gonadotropin (hCG)-stimulated serum testosterone concentrations. Three groups of subjects were studied: 11 young men with poorly controlled IDDM, 9 well controlled diabetics, and 9 healthy men. All volunteers underwent blood sampling at 10-min intervals before and after 2 consecutive iv pulses of 10 micro g GnRH. On a separate day, 40 IU/kg hCG were given im, and blood samples were collected before hCG administration, every 60 min thereafter for 6 h, and then 24, 48, and 72 h after the injection. Mean serum LH concentrations across the basal 6-h sampling period were significantly (P < 0.05) decreased in men with poorly controlled IDDM (11 +/- 1.6 IU/liter) compared with those in well controlled diabetics (19 +/- 1.8 IU/liter) and healthy controls (19 +/- 1.5 IU/liter). Multiple parameter deconvolution analysis revealed a 50% reduction in the mass of LH secreted per burst and the pulsatile LH secretion rate in poorly controlled IDDM (mass of LH secreted/burst, 7 +/- 1.1 vs. 12 +/- 2.1 and 13 +/- 1.5 IU/liter; LH secretion rate, 47 +/- 6.3 vs. 78 +/- 10 and 87 +/- 11 IU/liter.6 h; poorly controlled vs. well controlled IDDM and healthy controls, respectively; P < 0.05 for both parameters). Uncontrolled IDDM patients had significantly (P < 0.05) lower integrated serum LH concentrations after the first and second GnRH pulses (first GnRH pulse, 4460 +/- 770 vs. 7250 +/- 1200 and 5120 +/- 910 IU/liter; second pulse, 4700 +/- 615 vs. 7640 +/- 881 and 7100 +/- 1230 IU/liter; poorly controlled vs. well controlled IDDM and healthy men, respectively) and markedly attenuated LH secretory burst mass after the second GnRH stimulus (49 +/- 8.8 vs. 90 +/- 13 and 83 +/- 19 IU/liter; poorly controlled vs. well controlled IDDM and healthy controls, respectively). The biological to immunological ratio of LH released in baseline conditions was higher in uncontrolled IDDM patients (0.81 +/- 0.10) than in controlled IDDM (0.37 +/- 0.08) and healthy controls (0.48 +/- 0.06; P < 0.01), whereas LH released in response to exogenous GnRH exhibited comparable ratios among the three study cohorts. Baseline serum testosterone levels as well as absolute and incremental responses to exogenous hCG did not differ by degree of metabolic control. Collectively, these results indicate that the function of the hypothalamic-gonadotrope axis is compromised in young men with poorly controlled IDDM, such that the amplitude of spontaneous pulsatile and exogenous GnRH-stimulated LH secretion is attenuated. This central hypogonadotropism is paradoxically associated with the presence in the circulation of gonadotropin molecules with enriched biological activity, which is evidently sufficient to temporarily maintain normal total testosterone concentrations in the earlier stages of IDDM.

Regulation of Clara cell secretory protein gene expression by the CCAAT-binding factor NF-Y.

Analysis of the transcriptional regulation of the Clara cell secretory protein (CCSP) gene has resulted in the characterization of several trans-acting factors that regulate the activity of this gene. However, little is known about negative regulatory elements involved in CCSP gene transcription. Using transient transfections of luciferase reporter constructs driven by various fragments of the Neotomodon CCSP (nCCSP) promoter, we identified an inhibitory region that contains an inverted CCAAT box located -225 to -221 bp upstream of the transcriptional start site. Sequence analysis in a broad region of the nCCSP promoter (-744/+33) identified another potentially important CCAAT motif (-459/-455). Gel shift and supershift assays indicated that the transcription factor NF-Y binds to both CCAAT boxes. Mutation of the CCAAT motif prevented the in vitro binding of NF-Y and led to a significant increase of CCSP promoter activity in both pulmonary (H441) and non-pulmonary (HeLa and MCF-7) cells, suggesting that NF-Y is involved in a negative transcriptional regulation that may potentially contribute to the highly cell-specific expression of the anti-inflammatory CCSP gene.

Effects of metformin on inappropriate LH release in women with polycystic ovarian syndrome and insulin resistance.

The role of hyperinsulinaemia in neuroendocrine abnormalities in polycystic ovarian syndrome (PCOS) is controversial. The present study applied frequent blood sampling to assess the response of LH to metformin treatment in insulin-resistant women with PCOS. Thirteen predominantly overweight women with PCOS were studied before and after treatment with 1.5 g/day metformin for 3 months. Serum LH and testosterone were measured every 10 min for 10 h; LH was measured for an additional 2 h after gonadotrophin-releasing hormone (GnRH) administration. LH pulses were characterized by cluster analysis, secretory LH episodes by a deconvolution procedure, and synchronicity of paired LH-testosterone concentrations by lag-specific cross-correlation. After treatment, basal LH concentrations, amplitude of LH pulses, LH secretory amplitude, response to exogenous GnRH, and basal testosterone concentrations significantly decreased in seven patients, whereas in the remaining women these parameters remained unaltered. Before treatment, decreased coordinate LH and testosterone release was manifested by all patients; metformin treatment led to re-establishment of the feed-back control of testosterone on LH secretory rates by -20 to 0 min. Treatment did not modify the glucose:insulin ratio or serum insulin concentrations. In conclusion, administration of metformin allowed the identification of two subsets of PCOS women in whom neuroendocrine abnormalities may improve independently of the presence of insulin resistance or hyperinsulinaemia.