Progesterone dose during synchronization treatment alters luteinizing hormone receptor and steroidogenic enzyme mRNA abundances in granulosa cells of Nellore heifers.

The objective was to investigate effects of progesterone (P4) dose on abundance of luteinizing hormone receptor (LHCGR), aromatase (CYP19A1), 3ß-hydroxysteroid dehydrogenase (HSD3B1), and other steroidogenic mRNA transcripts in granulosa cells from dominant follicles. Nellore heifers were assigned to one of six groups: new, first-use controlled internal drug release device (CIDR1) inserted for 5 days (Large-P4-dose-D5; n = 7) or 6 days (Large-P4-dose-D6; n = 8), prostaglandin (PG)F2α administered on D0 and 1 previously-used CIDR (CIDR3) inserted for 5 days (Small- P4-dose-D5; n = 8) or 6 days (Small-P4-dose-D6; n = 8), CIDR1 inserted on D0 and removed plus PGF2α on D5 (Large-P4-dose-proestrus (PE); n = 7), and CIDR3 and PGF2α on D0 and 1, CIDR3 removed plus PGF2α on D5 (Small-P4-dose-PE; n = 7). Duration of P4 treatment (D5 compared to D6) affected abundances of CYP19A1 mRNA transcripts, with there being greater abundances on D6 than D5 (P ≤ 0.05). Heifers treated with the large dose of P4 had a smaller dominant follicle, less serum and intra-follicular estradiol (E2) concentrations (P ≤ 0.05) and lesser LHCGR, CYP19A1, and HSD3B1 transcript abundances (P ≤ 0.05). Heifers treated to induce PE had a larger follicle diameter (P = 0.09), greater intra-follicular E2 concentrations and larger abundances of CYP19A1 mRNA transcript (P ≤ 0.05) than heifers of the D6 group. Overall, treatment with larger doses of P4 resulted in lesser abundances of LHCGR, HSD3B1, and CYP19A1 mRNA transcripts; thus, potentially leading to development of smaller dominant follicles and lesser E2 concentrations.

Mutation of HSD3B2 Gene and Fate of Dehydroepiandrosterone.

3ßHSD2 enzyme is crucial for adrenal and gonad steroid biosynthesis. In enzyme deficiency states, due to recessive loss-of-function HSD3B2 mutations, steroid flux is altered and clinical manifestations result. Deficiency of 3ßHSD2 activity in the adrenals precludes normal aldosterone and cortisol synthesis and the alternative backdoor and 11-oxygenated C19 steroid pathways and the flooding of cortisol precursors along the Δ5 pathway with a marked rise in DHEA and DHEAS production. In gonads, it precludes normal T and estrogen synthesis. Here, we review androgen-dependent male differentiation of the external genitalia in humans and link this to female development and steroidogenesis in the developing adrenal cortex. The molecular mechanisms governing postnatal adrenal cortex zonation and ZR development were also revised. This chapter will review relevant clinical, hormonal, and genetic aspects of 3ßHSD2 deficiency with emphasis on the significance of alternate fates encountered by steroid hormone precursors in the adrenal gland and gonads. Our current knowledge of the process of steroidogenesis and steroid action is derived from pathological conditions. In humans the 3ßHSD2 deficiency represents a model of nature that reinforces our knowledge about the role of the steroidogenic alternative pathway in sex differentiation in both sexes. However, the physiological role of the high serum DHEAS levels in fetal life as well as after adrenarche remains to be elucidated.

The role of ARMC5 in human cell cultures from nodules of primary macronodular adrenocortical hyperplasia (PMAH).

The participation of aberrant receptors and intra-adrenal ACTH in hyperplastic tissue are considered mechanisms that regulate hypercortisolism in PMAH. Additionally, germline ARMC5 mutations have been described as the most frequent genetic abnormality found in patients diagnosed with PMAH. Previous functional studies analyzed ARMC5 role using H295R cells. Therefore, we investigated the role of ARMC5 in cell cultures obtained from PMAH nodules containing steroidogenic cells, aberrant receptors and intra-adrenal ACTH. ARMC5 silencing in non-mutated PMAH cell cultures decreased steroidogenesis-related genes and increased CCNE1 mRNA expression and proliferative capacity without affecting cell viability. Additionally, ARMC5 overexpression induced cell death in PMAH mutated cell cultures, thereby decreasing cell viability. We confirmed the role of ARMC5 as an important pro-apoptotic protein involved in PMAH-related steroidogenesis. We also report for the first time the involvement of ARMC5 in controlling proliferation and regulating cell cycle in PMAH cell cultures; these effects need to be explored further.

Respuesta ultrarrapida de tecnologia sanitária: ácido cólico / Ultrarapid response of sanitary technology: colic acid

INTRODUCCIÓN: El término colestasis comprende todas las situaciones en las cuales existe un impedimento en el flujo normal de bilis desde el polo canalicular del hepatocito hasta el duodeno, lo que produce alteraciones morfológicas, fisiológicas y clínicas. De acuerdo a su mecanismo de producción se clasifican en colestasis intra y extrahepáticas, la cual es universalmente aceptada y fue establecida por la Asociación Internacional para el Estudio del Hígado (1994), ya que provee un esquema práctico, con importantes implicaciones diagnósticas y terapéuticas. Los mecanismos involucrados en las colestasis intrahepáticas familiares suceden por: 1. alteración del transporte canalicular de los componentes normales de la bilis: debido a mutaciones en los genes que codifican estos transportadores. 2. defectos en la síntesis de ácidos biliares: a consecuencia de diferentes deficiencias enzimáticas específicas, debidas a mutaciones1 . El trastorno de la síntesis de ácidos biliares (en inglés

Estrogen and progesterone synthesis with cellular response in a C57BL/6 mouse model of cuprizone-induced demyelination

Background: Demyelination refers to the degradation or loss of myelin sheath. In demyelination model studies, it has been reported that demyelination is regressed by giving steroid hormones such as estrogen and progesterone. However, there are not many studies investigating the synthesis of these two hormones by the brain during demyelination and remyelination. Neurosteroids are steroid hormones synthesized by the brain independently from peripheral tissues. In this study, it was aimed to have knowledge about the synthesis of these two hormones by the brain in experimentally formed demyelination process in brains of C57BL/6 mice and their role in the cellular response formed in the region.Materials, Methods & Results: In the study, 36 C57BL/6 mice were used: 12 mice were fed normal diet for 12 weeks as control group (Group I); 12 of them were fed 0.2% cuprizone diet for 8 weeks (Group II) and 12 mice were fed normal diet for 4 weeks after feeding cuprizone diet for 8 weeks (Group III). At the end of the experiment, mice were perfused with 4% paraformaldehyde and brain tissues were blocked in paraffin. 6 μm-thick section was taken from each block. Sections were stained histologically with LFB staining and immunohistochemically with MBP staining in order to determine the demyelination in sections. All sections were also immunohistochemically stained with GFAP to detect astrocytes, with NG2 to detect young OPCs, with aromatase for estrogen synthesis and with 3βHSD antibodies for progesterone synthesis. At the end of the study, complete myelination was observed in group I, while severe demyelination was determined in group II as a result of blind evaluation of LFB and MBP staining by two pathologists. In group III, demyelination was found to be mild. In immunostaining with GFAP and NG2 antibodies, the number of GFAP and NG2 positive cells in Group II was found to be increased compared to the control group.[...](AU)

Estrogen and progesterone synthesis with cellular response in a C57BL/6 mouse model of cuprizone-induced demyelination

Background: Demyelination refers to the degradation or loss of myelin sheath. In demyelination model studies, it has been reported that demyelination is regressed by giving steroid hormones such as estrogen and progesterone. However, there are not many studies investigating the synthesis of these two hormones by the brain during demyelination and remyelination. Neurosteroids are steroid hormones synthesized by the brain independently from peripheral tissues. In this study, it was aimed to have knowledge about the synthesis of these two hormones by the brain in experimentally formed demyelination process in brains of C57BL/6 mice and their role in the cellular response formed in the region.Materials, Methods & Results: In the study, 36 C57BL/6 mice were used: 12 mice were fed normal diet for 12 weeks as control group (Group I); 12 of them were fed 0.2% cuprizone diet for 8 weeks (Group II) and 12 mice were fed normal diet for 4 weeks after feeding cuprizone diet for 8 weeks (Group III). At the end of the experiment, mice were perfused with 4% paraformaldehyde and brain tissues were blocked in paraffin. 6 μm-thick section was taken from each block. Sections were stained histologically with LFB staining and immunohistochemically with MBP staining in order to determine the demyelination in sections. All sections were also immunohistochemically stained with GFAP to detect astrocytes, with NG2 to detect young OPCs, with aromatase for estrogen synthesis and with 3βHSD antibodies for progesterone synthesis. At the end of the study, complete myelination was observed in group I, while severe demyelination was determined in group II as a result of blind evaluation of LFB and MBP staining by two pathologists. In group III, demyelination was found to be mild. In immunostaining with GFAP and NG2 antibodies, the number of GFAP and NG2 positive cells in Group II was found to be increased compared to the control group.[...]

Comparison of steroidogenic pathways among normoandrogenic and hyperandrogenic polycystic ovary syndrome patients and normal cycling women.

AIM: To compare the corticosteroidogenic enzyme activities between normal cycling non-polycystic ovary syndrome (PCOS), and normoandrogenic PCOS (NA-PCOS) and hyperandrogenic PCOS (HA-PCOS) patients. METHODS: This cohort study was conducted at Julio Muller University Hospital and Tropical Institute of Reproductive Medicine and Menopause, and enrolled 114 non-PCOS women and 355 PCOS patients. The steroidogenic enzyme activities were measured using the serum steroid product/precursor molar ratio. RESULTS: In the Δ5 pathway the 17,20 lyase activity was equally low in the NA-PCOS and HA-PCOS women compared with the non-PCOS women (P < 0.01 and P < 0.001, respectively). In the Δ4 pathway, the 17,20 lyase activity was higher only in the HA-PCOS group (P < 0.001). The 17-hydroxylase activity was the same in PCOS and non-PCOS subjects (P > 0.05). The 3ß-hydroxysteroid dehydrogenase II (3ß-HSDII) activity was higher in the conversion of dehydroepiandrosterone into androstenedione in the HA-PCOS than in the NA-PCOS (P < 0.05) and the non-PCOS patients (P < 0.01). The aromatase activity was lower in the HA-PCOS than in the NA-PCOS (P < 0.05) patients and non-PCOS subjects (P < 0.01). In HA-PCOS subjects, the 17,20 lyase activity was related to insulin, estradiol, total testosterone concentrations and free androgen index in the Δ5 pathway. 3ß-HSDII showed weak correlation with estradiol in the HA-PCOS group. Anthropometric parameters had little impact, if any, on the steroidogenic enzyme activities. CONCLUSION: The NA-PCOS and HA-PCOS patients demonstrated different enzyme activities, and the results provided new directions for future studies including PCOS patients with different phenotypes.

A novel missense mutation in the HSD3B2 gene, underlying nonsalt-wasting congenital adrenal hyperplasia. new insight into the structure-function relationships of 3ß-hydroxysteroid dehidrogenase type II.

CONTEXT: 3ßHSD2 is a bifunctional microsomal NAD+-dependent enzyme crucial for adrenal and gonad steroid biosynthesis, converting Δ5-steroids to Δ4-steroids. 3ßHSD2 deficiency is a rare cause of congenital adrenal hyperplasia caused by recessive loss-of-function HSD3B2 mutations. OBJECTIVE: The aim was to define the pathogenic consequences of a novel missense mutation in the HSD3B2 gene. PATIENT: We report a 7-month-old 46,XX girl referred because of precocious pubarche and postnatal clitoromegaly. Hormonal profile showed inadequate glucocorticoid levels, increased 17OHP and renin levels, and very high DHEAS levels, suggestive of compensated nonsalt-losing 3ßHSD2 deficiency. DESIGN AND RESULTS: Direct sequencing revealed a novel, homozygous, pG250V HSD3B2 mutation. In vitro analysis in intact COS-7 cells showed impaired enzymatic activity for the conversion of pregnenolone to progesterone and dehydroepiandrosterone to androstenedione (20% and 27% of WT at 6 h, respectively). G250V-3ßHSD2 decreased the Vmax for progesterone synthesis without affecting the Km for pregnenolone. Western blot and immunofluorescence suggested that p.G250V mutation has no effect on the expression and intracellular localization of the mutant protein. Molecular homology modeling predicted that mutant V250 affected an L239-Q251 loop next to a ß-sheet structure in the NAD+-binding domain. CONCLUSIONS: We identified a novel p.G250V mutation of HSD3B2 which causes an incomplete loss of enzymatic activity, explaining the compensated nonsalt loss phenotype. In vitro and in silico experiments provided insight into the structure-function relationship of the 3ßHSD2 protein suggesting the importance of the L239-Q251 loop for the catalytic activity of the otherwise stable 3ßHSD2 enzyme.

3ß-hydroxysteroid dehydrogenase type II deficiency on newborn screening test.

3ß-hydroxysteroid dehydrogenase II (3ß-HSD) deficiency represents a rare CAH variant. Newborns affected with its classic form have salt wasting in early infancy and genital ambiguity in both sexes. High levels of 17-hydroxypregnenolone (Δ517OHP) are characteristic, but extra-adrenal conversion to 17-hydroxyprogesterone (17OHP) may lead to positive results on newborn screening tests. Filter paper 17OHP on newborn screening test was performed by immunofluorometric assay, and serum determinations of 17OHP and Δ517OHP, by radioimmunoassay. A 46,XY infant with genital ambiguity and adrenal crisis at three months of age presented a positive result on newborn screening for CAH. Serum determinations of 17OHP and Δ517OHP were elevated, and a high Δ517OHP/cortisol relation was compatible with the diagnosis of 3ß-HSD deficiency. Molecular analysis of the HSD3B2 gene from the affected case revealed the presence of the homozygous p.P222Q mutation, whereas his parents were heterozygous for it. We present the first report of 3ß-HSD type II deficiency genotype-proven detected at the Newborn Screening Program in Brazil. The case described herein corroborates the strong genotype-phenotype correlation associated with the HSD3B2 p.P222Q mutation, which leads to a classic salt-wasting 3ß-HSD deficiency. Further evaluation of 17OHP assays used in newborn screening tests would aid in determining their reproducibility, as well as the potential significance of moderately elevated 17OHP levels as an early indicator to the diagnosis of other forms of classic CAH, beyond 21-hydroxylase deficiency.

3?-hydroxysteroid dehydrogenase type II deficiency on newborn screening test / Deficiência de 3?-hidroxiesteroide desidrogenase tipo 2 em teste de triagem neonatal

3b-hydroxysteroid dehydrogenase II (3β-HSD) deficiency represents a rare CAH variant. Newborns affected with its classic form have salt wasting in early infancy and genital ambiguity in both sexes. High levels of 17-hydroxypregnenolone (Δ517OHP) are characteristic, but extra-adrenal conversion to 17-hydroxyprogesterone (17OHP) may lead to positive results on newborn screening tests. Filter paper 17OHP on newborn screening test was performed by immunofluorometric assay, and serum determinations of 17OHP and Δ517OHP, by radioimmunoassay. A 46,XY infant with genital ambiguity and adrenal crisis at three months of age presented a positive result on newborn screening for CAH. Serum determinations of 17OHP and Δ517OHP were elevated, and a high Δ517OHP/cortisol relation was compatible with the diagnosis of 3β-HSD deficiency. Molecular analysis of the HSD3B2 gene from the affected case revealed the presence of the homozygous p.P222Q mutation, whereas his parents were heterozygous for it. We present the first report of 3β-HSD type II deficiency genotype-proven detected at the Newborn Screening Program in Brazil. The case described herein corroborates the strong genotype-phenotype correlation associated with the HSD3B2 p.P222Q mutation, which leads to a classic salt-wasting 3β-HSD deficiency. Further evaluation of 17OHP assays used in newborn screening tests would aid in determining their reproducibility, as well as the potential significance of moderately elevated 17OHP levels as an early indicator to the diagnosis of other forms of classic CAH, beyond 21-hydroxylase deficiency.

A deficiência da enzima 3β-hidroxiesteroide desidrogenase tipo 2 (3β-HSD) representa variante rara de hiperplasia adrenal congenital (HAC). Recém-nascidos afetados com a forma clássica apresentam perda de sal nas primeiras semanas de vida e ambiguidade genital em ambos os sexos. Concentrações elevadas de 17-hidroxipregnenolona (Δ517OHP) são características, porém sua conversão extra-adrenal a 17-hidroxiprogesterona (17OHP) pode resultar em resultados positivos no teste de triagem neonatal. A determinação da concentração de 17OHP obtida em amostra de sangue colhida em papel-filtro para triagem neonatal foi realizada por ensaio imunofluorimétrico, e as concentrações séricas de 17OHP and Δ517OHP, por radioimunoensaio. Um menino, 46,XY, com ambiguidade genital e crise adrenal aos 3 meses de vida, apresentou teste positivo na triagem neonatal para HAC. As concentrações séricas de 17OHP e Δ517OHP estavam aumentadas, bem como a relação Δ517OHP/cortisol, o que foi compatível com o diagnóstico de deficiência de 3β-HSD. A análise molecular do gene HSD3B2 revelou a mutação p.P222Q em homozigose na criança afetada e em heterozigose em seus pais, o que confirmou a deficiência de 3β-HSD com resultado moderadamente elevado na dosagem de 17OHP no “Teste do Pezinho” (Programa de Triagem Neonatal do Distrito Federal, Brasil). Esse caso corrobora a forte correlação genótipo-fenótipo associada à mutação p.P222Q no gene HSD3B2. Estudos futuros para avaliação dos ensaios utilizados na triagem neonatal para determinação de 17OHP poderão auxiliar na determinação do significado potencial de concentrações moderadamente elevadas de 17OHP como um indicador precoce para o diagnóstico de outras formas de HAC clássicas, além da deficiência de 21-hidroxilase.

Culture of bovine ovarian follicle wall sections maintained the highly estrogenic profile under basal and chemically defined conditions.

Follicle cultures reproduce in vitro the functional features observed in vivo. In a search for an ideal model, we cultured bovine antral follicle wall sections (FWS) in a serum-free defined medium (DM) known to induce 17ß-estradiol (E2) production, and in a nondefined medium (NDM) containing serum. Follicles were sectioned and cultured in NDM or DM for 24 or 48 h. Morphological features were determined by light microscopy. Gene expression of steroidogenic enzymes and follicle-stimulating hormone (FSH) receptor were determined by RT-PCR; progesterone (P4) and E2 concentrations in the media were measured by radioimmunoassay. DM, but not NDM, maintained an FWS morphology in vitro that was similar to fresh tissue. DM also induced an increase in the expression of all steroidogenic enzymes, except FSH receptor, but NDM did not. In both DM and NDM, there was a gradual increase in P4 throughout the culture period; however, P4 concentration was significantly higher in NDM. In both media, E2 concentration was increased at 24 h, followed by a decrease at 48 h. The E2:P4 ratio was higher in DM than in NDM. These results suggest that DM maintains morphological structure, upregulates the expression of steroidogenic enzyme genes, and maintains steroid production with a high E2:P4 ratio in FWS cultures.

Culture of bovine ovarian follicle wall sections maintained the highly estrogenic profile under basal and chemically defined conditions

Follicle cultures reproduce in vitro the functional features observed in vivo. In a search for an ideal model, we cultured bovine antral follicle wall sections (FWS) in a serum-free defined medium (DM) known to induce 17β-estradiol (E2) production, and in a nondefined medium (NDM) containing serum. Follicles were sectioned and cultured in NDM or DM for 24 or 48 h. Morphological features were determined by light microscopy. Gene expression of steroidogenic enzymes and follicle-stimulating hormone (FSH) receptor were determined by RT-PCR; progesterone (P4) and E2 concentrations in the media were measured by radioimmunoassay. DM, but not NDM, maintained an FWS morphology in vitro that was similar to fresh tissue. DM also induced an increase in the expression of all steroidogenic enzymes, except FSH receptor, but NDM did not. In both DM and NDM, there was a gradual increase in P4 throughout the culture period; however, P4 concentration was significantly higher in NDM. In both media, E2 concentration was increased at 24 h, followed by a decrease at 48 h. The E2:P4 ratio was higher in DM than in NDM. These results suggest that DM maintains morphological structure, upregulates the expression of steroidogenic enzyme genes, and maintains steroid production with a high E2:P4 ratio in FWS cultures.

Structural aspects of the p.P222Q homozygous mutation of HSD3B2 gene in a patient with congenital adrenal hyperplasia / Aspectos estruturais da mutação homozigótica p.P222Q do gene HSD3B2 em um paciente com hiperplasia congênita da adrenal

Type II 3β-hydroxysteroid dehydrogenase/Δ5-Δ4-isomerase (3β-HSD2), encoded by the HSD3B2 gene, is a key enzyme involved in the biosynthesis of all the classes of steroid hormones. Deleterious mutations in the HSD3B2 gene cause the classical deficiency of 3β-HSD2, which is a rare autosomal recessive disease that leads to congenital adrenal hyperplasia (CAH). CAH is the most frequent cause of ambiguous genitalia and adrenal insufficiency in newborn infants with variable degrees of salt losing. Here we report the molecular and structural analysis of the HSD3B2 gene in a 46,XY child, who was born from consanguineous parents, and presented with ambiguous genitalia and salt losing. The patient carries a homozygous nucleotide c.665C>A change in exon 4 that putatively substitutes the proline at codon 222 for glutamine. Molecular homology modeling of normal and mutant 3β-HSD2 enzymes emphasizes codon 222 as an important residue for the folding pattern of the enzyme and validates a suitable model for analysis of new mutations.

A enzima 3β-hydroxysteroid dehydrogenase/Δ5-Δ4-isomerase do tipo 2 (3β-HSD2), codificada pelo gene HSD3B2, é importante na biossíntese de todas as classes de hormônios esteroides. As mutações no gene HSD3B2 podem causar deficiência da 3β-HSD2 da forma clássica. É de herança autossômica recessiva e uma das causas mais raras de hiperplasia congênita da adrenal (HCA). A deficiência dessa enzima leva frequentemente à ambiguidade genital e à insuficiência da adrenal em recém-nascidos com vários níveis de perda de sal. Neste trabalho, foi feito o estudo estrutural e molecular do gene HSD3B2 gene em um paciente 46,XY, filho de pais consanguíneos, com ambiguidade genital e perda de sal. O paciente é homozigoto para a troca nucleotídica c.665C>A no éxon 4, que putativamente leva à substituição de uma prolina do códon 222 por uma glutamina. A modelagem molecular por homologia das enzimas 3β-HSD2 normal e mutantes ressaltou que a prolina no códon 222 é um resíduo importante no enovelamento da enzima e validou um modelo adequado para avaliações de novas mutações.

Structural aspects of the p.P222Q homozygous mutation of HSD3B2 gene in a patient with congenital adrenal hyperplasia.

Type II 3ß-hydroxysteroid dehydrogenase/Δ(5)-Δ(4)-isomerase (3ß-HSD2), encoded by the HSD3B2 gene, is a key enzyme involved in the biosynthesis of all the classes of steroid hormones. Deleterious mutations in the HSD3B2 gene cause the classical deficiency of 3ß-HSD2, which is a rare autosomal recessive disease that leads to congenital adrenal hyperplasia (CAH). CAH is the most frequent cause of ambiguous genitalia and adrenal insufficiency in newborn infants with variable degrees of salt losing. Here we report the molecular and structural analysis of the HSD3B2 gene in a 46,XY child, who was born from consanguineous parents, and presented with ambiguous genitalia and salt losing. The patient carries a homozygous nucleotide c.665C>A change in exon 4 that putatively substitutes the proline at codon 222 for glutamine. Molecular homology modeling of normal and mutant 3ß-HSD2 enzymes emphasizes codon 222 as an important residue for the folding pattern of the enzyme and validates a suitable model for analysis of new mutations.

Calcitriol inhibits TNF-alpha-induced inflammatory cytokines in human trophoblasts.

Elevated placental proinflammatory cytokine release is associated with miscarriage, preterm labor and preeclampsia. Specifically, tumor necrosis factor-alpha (TNF-alpha)-induced cytokines may threaten pregnancy outcome. Since trophoblasts produce calcitriol, a hormone with strong immunosuppressive properties, we assessed the effects of this secosteroid on inflammatory cytokines induced in trophoblasts by challenge with TNF-alpha. The effects of calcitriol on synthesis of mRNAs encoding interleukin-6 (IL-6), interferon-gamma (IFN-gamma), and TNF-alpha were measured by real time RT-PCR. Secreted cytokines were quantified by ELISA. The effects of TNF-alpha on CYP24A1, chorionic gonadotropin (hCG), 3beta-hydroxysteroid dehydrogenase (HSD3B1) and P(450)-aromatase (CYP19) mRNA expression were also studied. TNF-alpha stimulated IL-6, IFN-gamma and its own expression more than 3-fold over controls (P<0.05). Calcitriol inhibited the expression profile of inflammatory cytokine genes in a dose-response manner (P<0.05). This effect was prevented by addition of the vitamin D receptor antagonist TEI-9647. TNF-alpha also significantly inhibited expression of hCG, HSD3B1 and CYP19 genes, and stimulated CYP24A1 gene expression. These data show that calcitriol prevents TNF-alpha induction of inflammatory cytokines through a process likely to be mediated by the vitamin D receptor. We conclude that TNF-alpha inhibits placental hormone synthesis and stimulates calcitriol catabolism by regulating enzymes involved in these processes.

Luteal development and progesterone levels during pregnancy of the viviparous temperate lizard Barisia imbricata imbricata (Reptilia: Anguidae).

The relationship between plasma progesterone (P(4)) levels and the formation and degeneration of the corpus luteum (CL) was assessed monthly during gestation of the viviparous lizard Barisia imbricata imbricata. Histochemical activity of the delta(5-4) isomerase 3 beta-hydroxysteroide dehydrogenase (delta(5-4)3beta-HSD) in the luteal tissue and embryonic development were also observed. Females were gravid throughout winter and great part of spring (late November or early December until late May or early June). Corpus luteum development occurred in the first third of gestation (December and January) when the embryo reached developmental stage 27. Four sequential stages were identified during development and three stages during regression of the CL. The follicular and thecal tissue participated in the formation of the luteal cell mass. According to Xavier's classification, the CL of B. i. imbricata is a subtype from Type III. The activity of delta(5-4)3beta-HSD was observed mainly in the luteal cell mass. The first degenerative changes in the CL were observed in the early second third of the gestation and continued gradually until parturition. Progesterone levels increased in early pregnancy and reached its highest level during January (3.07+/-1.04 ng/ml) when mature corpora lutea were present. Gradual diminution in progesterone concentrations occurred in the second and last third of pregnancy and coincided with advanced degenerative changes and diminution in histochemical activity of delta(5-4)3beta-HSD in the luteal tissue. These observations suggest that the CL is the major source of progesterone during pregnancy of B. i. imbricata.

In vitro hCG and human recombinant FSH actions on testicular steroidogenesis in the toad Bufo arenarum.

In order to study the regulation of testicular steroidogenesis in the toad Bufo arenarum, the effect of gonadotropins (hCG and hrFSH) on steroidogenic enzymes was determined using an in vitro system. 3beta-Hydroxysteroid dehydrogenase/isomerase activity was not affected by any of the gonadotropins, at any of the concentrations used. In contrast, 5alpha-reductase activity was strongly reduced by both hCG and hrFSH. Human chorionic gonadotropin inhibited the activity of cytochrome P450 17alpha-hydroxylase-C(17-20) lyase (P450(c17)), only at the highest concentration used, while hrFSH strongly reduced P450(c17) activity at all the doses assayed. In conclusion, these data suggest that LH (hCG) and FSH regulate steroidogenic enzymes such as 5alphaRed and P450(c17). The results also suggest that FSH could be involved in the regulation of the change in steroidogenesis undergone by the testis during the breeding season. In turn, the inhibition of P450(c17) activity could result in a reduction of androgen production and an increment of C21 steroids.

Kinetic properties of microsomal 3beta hydroxysteroid dehydrogenase-isomerase from the testis of Bufo arenarum H.

3beta-hydroxysteroid dehydrogenase 5-ene isomerase (3betaHSD/I) activity is necessary for the biosynthesis of hormonally active steroids. A dual distribution of the enzyme was described in toad testes. The present study demonstrates that in testicular tissue of Bufo arenarum H., microsomal 3betaHSD/I has more affinity for dehydroepiandrosterone (DHEA) than for pregnenolone (K(m)=0.17+/-0. 03 and 1.02 microM, respectively). The Hill coefficient for the conversion of DHEA and pregnenolone were 1.04 and 1.01, respectively. The inclusion of DHEA in the kinetic analysis of pregnenolone conversion affected V(max) while K(m) was not modified, suggesting a non-competitive inhibition of the conversion of pregnenolone. K(i) was calculated from replot of Dixon's slope for each substrate concentration. K(i) from the intercept and the slope of this replot were similar (0.276+/-0.01 and 0.263+/-0.02 microM) and higher than the K(m) for DHEA. The K(m) and K(i) values suggest the presence of two different binding sites. When pregnenolone was present in the assays with DHEA as substrate, no effect was observed on the V(max) while K(m) values slightly increased with pregnenolone concentration. Consequently, pregnenolone inhibited the transformation of DHEA in a competitive fashion. These studies suggest that, in this species, the microsomal biosyntheses of androgens and progesterone are catalysed by different active sites.

Regulatory role of angiotensin II on progesterone production by cultured human granulosa cells. Expression of angiotensin II type-2 receptor.

The role of angiotensin II (AngII) in ovarian steroidogenesis is not clearly understood. In order to study its action on progesterone synthesis and to determine which receptor subtype is involve, granulosa cells obtained from women undergoing in-vitro fertilization were cultured for 2 or 4 days and then incubated in the presence of AngII (10(-7) M) with or without human chorionic gonadotrophin (HCG, 10 IU/ml) for 3 or 18 h. In cells cultured for 2 days, incubation with AngII decreased progesterone secretion by 36%, and inhibited activity of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) by 87% (P < 0.05), although its expression was not significantly reduced. However, in cells cultured for 4 days, progesterone production was enhanced by incubation with AngII (38%), and no change was observed in 3 beta-HSD expression. Both inhibitory and stimulatory effects were dose-dependent. Progesterone secretion was increased (93%) by incubation with HCG of cells cultured for 4, but not for 2 days, and no potentiation was observed with AngII. Treatment with PD123177 completely blocked the action of AngII and decreased the HCG-stimulated secretion of progesterone by 27%. Angiotensin type-2 (AT2) receptor mRNA was expressed in cells cultured for 4 days. In conclusion, AngII showed a regulatory role in in-vitro progesterone production by human granulosa luteinized cells, modulating the activity of 3 beta-HSD. It is likely that these actions may be mediated via membrane receptors, possibly of the AT2 receptor family.