3β-HSD mRNA Research Articles

High copper levels in follicular fluid affect follicle development in polycystic ovary syndrome patients: Population-based and in vitro studies

Although the adverse effects of copper overexposure on the liver, kidney, spleen and intestinal organs are well known, information about the impact of copper toxicity on human reproduction is limited. A total of 348 infertile patients were enrolled in our present study, including 89 with polycystic ovary syndrome (PCOS), 145 with fallopian tube obstruction and 114 controls. The follicular fluid concentrations of 22 trace elements were measured by inductively coupled plasma mass spectrometry (ICP-MS). Principal component analysis was used to identify trace element profile alterations in different groups. The mRNA levels of steroidogenesis-related genes were measured by real-time PCR. Our results showed that the trace element profile in follicular fluid was obviously altered in PCOS patients. Copper concentrations were significantly (p < .05) higher in the PCOS group than in the other two groups. Increased copper levels in follicular fluid were associated with a higher number of retrievable oocytes in the PCOS group (B = 1.785, p = .001) but a lower rate of high-quality embryos (B = −6.360, p = .050). Moreover, follicular fluid copper levels were positively correlated with follicular fluid progesterone levels (r = 0.275, p = .010) and testosterone levels (r = 0.250, p = .022). Cultured human granulosa cells overexposed to copper showed significantly (p < .05) increased estradiol secretion and decreased testosterone levels. Real-time quantitative PCR revealed a significant (p < .05) increase in CYP19A1 and HSD3b mRNA expression. Our results indicate that increased copper levels in follicular fluid could affect follicle development in PCOS patients, and the mechanism may be related to copper-induced abnormalities in steroidogenesis.

Rosiglitazone increases expression of steroidogenic acute regulatory protein and progesterone production through PPARγ-EGFR-ERK1/2 in human cumulus granulosa cells.

The mechanism by which rosiglitazone (ROSI: a thiazolidinedione (TZD)) affects steroid production in undifferentiated human granulosa cells is not known. In this study, cultured human cumulus granulosa cells were exposed to ROSI and pharmacological inhibitors of the extracellular signal-regulated kinase 1/2 (ERK1/2), epidermal growth factor receptor (EGFR) and peroxisome proliferator-activated receptor gamma (PPARγ) signalling pathways. Expression of progesterone biosynthetic enzymes, PPARγ and PPARα, progesterone production and ERK1/2 activation were analysed. After 48h, 30μM ROSI increased STAR, 3βHSD and PPARγ mRNA and elevated progesterone production in human cumulus granulosa cells. Addition of ERK1/2 (U0126), EGFR (AG1478) and PPARγ (GW9662) inhibitors prevented the ROSI-induced STAR mRNA expression and progesterone production after 48h. Inhibition of PPARγ, but not EGFR or ERK1/2, decreased the PPARγ mRNA levels induced by ROSI in human cumulus granulosa cells after 48h. On the other hand, U0126 and GW9662 prevented the ROSI-induced increase in PPARγ transcripts after 6h. Western blot analysis showed that ROSI induced a rapid ERK1/2 activation, which was prevented by inhibition of ERK1/2, EGFR and PPARγ in human cumulus granulosa cells. Overall, these data suggested that PPARγ, EGFR and ERK1/2 were involved in the stimulatory effect of ROSI on STAR expression and progesterone production in undifferentiated human cumulus granulosa cells.

The effect of orexin B on steroidogenic acute regulatory protein, P450 side-chain cleavage enzyme, and 3β-hydroxysteroid dehydrogenase gene expression, and progesterone and androstenedione secretion by the porcine uterus during early pregnancy and the estrous cycle.

The aim of this study was to investigate the effect of orexin B (OXB) on progesterone (P4) and androstenedione (A4) secretion by porcine endometrial and myometrial tissue explants and on the expression of key steroidogenic proteins and enzymes involved in steroid production. The hormones secretion and the expression of steroidogenic acute regulatory protein (StAR), P450 side-chain cleavage enzyme (CYP11A1), and 3β-hydroxysteroid dehydrogenase (HSD3B1) were analyzed on days 10 to 11, 12 to 13, 15 to 16, and 27 to 28 of pregnancy and during the luteal phase of the estrous cycle (days 10 to 11). Endometrial and myometrial explants were cultured in vitro in the presence of OXB (1, 10, or 100 nM) and OXB (1, 10, or 100 nM) with 1 µM of JNJ (OX2R antagonist). Gene expression was examined by real-time PCR, and steroid secretion was determined by radioimmunoassay. Orexin B modulated StAR, CYP11A1, HSD3B1 mRNA content depending on the type of uterine tissue, the applied OXB dose, and the stage of pregnancy or the estrous cycle (P < 0.05). Orexin B increased P4 secretion in all stages of early gestation (P < 0.05). Orexin B enhanced the release of A4 on days 12 to 13, 15 to 16, and 27 to 28 of gestation, whereas on days 10 to 11 of early pregnancy, A4 secretion decreased in the endometrium and increased in the myometrium (P < 0.05). These results indicate that OXB affects the expression of key steroidogenic regulators and the secretion of steroid hormones in the porcine uterus during early pregnancy.

The impact of early-life stress on corticosteroid carrier protein levels and 11β-hydroxysteroid dehydrogenase 1 expression in adolescent rats.

Corticosteroid-binding globulin (CBG), albumin and 11β-hydroxysteroid dehydrogenase (11β-HSD) enzymes play crucial roles in the bioavailability of glucocorticoids. Downstream of the adrenal glands, these proteins affect glucocorticoid levels in target tissues. Early-life stress (ELS) is known to program glucocorticoid action at many levels. The effects of ELS on the concentrations and synthesis of CBG and albumin and on the expression of 11β-HSD remain unclear. The maternal separation (MS) procedure in rats on postnatal days 1-14 was used as a model of ELS. On postnatal day 35 (adolescence), the serum corticosterone, CBG and albumin concentrations of male rats were measured by ELISA, while the mRNA and protein levels of CBG, albumin and 11β-HSD1 in the liver and brain were examined by RT-qPCR and Western blot, respectively. Under basal conditions, MS rats displayed lower levels of serum CBG and albumin. However, MS did not affect CBG or albumin synthesis in the liver, suggesting that the half-life and/or secretion of these proteins were influenced by MS. Additionally, MS rats showed increased protein expression of 11β-HSD1, specifically in the medial prefrontal cortex. These results indicate that ELS may potentially program glucocorticoid action through its effects on glucocorticoid bioavailability in tissues.

Hydroxysteroid 11-Beta Dehydrogenase 1 Overexpression with Copy-Number Gain and Missense Mutations in Primary Gastrointestinal Stromal Tumors.

The lipid-metabolizing enzymes remain underexplored in gastrointestinal stromal tumors (GISTs). Through transcriptomic reappraisal, hydroxysteroid 11-beta dehydrogenase-1 (HSD11B1) was identified as a top-upregulated, progression-associated gene. To validate the clinical relevance of HSD11B1, the informative results of Sanger sequencing (n = 58), mRNA quantification by branched-chain DNA in situ hybridization assay (n = 70), copy number assay by fluorescent in situ hybridization (n = 350), and immunohistochemistry (n = 350) were correlated with clincopathological variables, KIT/PDGFRA/BRAF genotypes, and disease-free survival (DFS). HSD11B1 was stably silenced to explore its oncogenic function. HSD11B1 mRNA varied between high-risk and non-high-risk groups (p = 0.009) and positively correlated with HSD11B1 immunoexpression (r = 0.783, p < 0.001). HSD11B1 copy-number gain (CNG), including polysomy (5.4%) and amplification (12%), associated with HSD11B1 overexpression (p < 0.001). Predominantly involving the homodimer interface-affecting exon 6 or exon 7, missense HSD11B1 mutations (17.2%) were related to high risk (p = 0.044), age ≥70 years (p = 0.007), and shorter DFS among relapsed cases (p = 0.033). CNG was related to unfavorable KIT/PDGFRA/BRAF genotypes (p = 0.015), while HSD11B1 overexpression was preferential in non-gastric cases (p < 0.001). Both abnormalities strongly associated with risk levels (both p < 0.001) and shorter univariate DFS (both p < 0.0001), and HSD11B1 CNG remained prognostically independent (p < 0.001) with a 3-fold increased hazard ratio. In vitro, HSD11B1 knockdown significantly inhibited proliferation and caused G2/M arrest. In conclusion, HSD11B1 overexpression may occur owing to CNG, confer a pro-proliferative function, and predict a worse prognosis in GISTs.

Developmental Programming of Capuchin Monkey Adrenal Dysfunction by Gestational Chronodisruption.

In the capuchin monkey (Cebus apella), a new-world nonhuman primate, maternal exposure to constant light during last third of gestation induces precocious maturation of the fetal adrenal and increased plasma cortisol in the newborn. Here, we further explored the effects of this challenge on the developmental programming of adrenal function in newborn and infant capuchin monkeys. We measured (i) plasma dehydroepiandrosterone sulphate (DHAS) and cortisol response to ACTH in infants with suppressed endogenous ACTH, (ii) plasma DHAS and cortisol response to ACTH in vitro, and (iii) adrenal weight and expression level of key factors in steroid synthesis (StAR and 3β-HSD). In one-month-old infants from mothers subjected to constant light, plasma levels of cortisol and cortisol response to ACTH were twofold higher, whereas plasma levels of DHAS and DHAS response to ACTH were markedly reduced, compared to control conditions. At 10 months of age, DHAS levels were still lower but closer to control animals, whereas cortisol response to ACTH was similar in both experimental groups. A compensatory response was detected at the adrenal level, consisting of a 30% increase in adrenal weight and about 50% reduction of both StAR and 3β-HSD mRNA and protein expression and the magnitude of DHAS and cortisol response to ACTH in vitro. Hence, at birth and at 10 months of age, there were differential effects in DHAS, cortisol production, and their response to ACTH. However, by 10 months of age, these subsided, leading to a normal cortisol response to ACTH. These compensatory mechanisms may help to overcome the adrenal alterations induced during pregnancy to restore normal cortisol concentrations in the growing infant.

Lambda-cyhalothrin delays pubertal Leydig cell development in rats

Lambda-cyhalothrin (LCT) is a widely used broad-spectrum pyrethroid insecticide and is expected to cause deleterious effects on the male reproductive system. However, the effects of LCT on Leydig cell development during puberty are unclear. The current study addressed these effects. Twenty-eight-day-old male Sprague Dawley rats orally received LCT (0, 0.25, 0.5 or 1 mg/kg body weight/day) for 30 days. The levels of serum testosterone, luteinizing hormone, and follicle-stimulating hormone, Leydig cell number, and its specific gene and protein expression were determined. LCT exposure lowered serum testosterone levels at doses of 0.5 and 1 mg/kg and luteinizing hormone levels at a dose of 1 mg/kg, but increased follicle-stimulating hormone levels at doses of 0.5 and 1 mg/kg. LCT lowered Star and Hsd3b1 mRNA or their protein levels at a dose of 1 mg/kg. Immature Leydig cells were purified from pubertal rats and treated with different concentrations of LCT for 24 h and medium androgen levels, Leydig cell mRNA and protein levels, the mitochondrial membrane potential (△Ψm), and the apoptotic rate of immature Leydig cells were investigated. LCT inhibited androgen production at 5 μM and downregulated Scarb1 at 0.05 μM, Hsd3b1 and Hsd11b1 at 0.5 μM, and Cyp11a1 at 5 μM. LCT also decreased △Ψm at 0.5 and 50 μM. In conclusion, LCT can influence the function of Leydig cells.

Natural disaster-related prenatal maternal stress is associated with alterations in placental glucocorticoid system: The QF2011 Queensland Flood Study

We investigated the effects of a natural disaster (a sudden flood) as a source of prenatal maternal stress (PNMS) on the placental glucocorticoid system and glucose transporters. Whether the gestational age at the time of the flood moderated these effects was also evaluated. Placental samples were collected from participants in the 2011 Queensland Flood Study (QF2011) who were pregnant in the first or second trimester at the onset of the flood. Detailed questionnaire results for objective hardship and composite subjective distress were obtained to assess stress levels. Subjective distress was significantly associated with a reduction in placental NR3C1-β mRNA levels for males only (β = −0.491, p = 0.005). In female placentas, objective hardship was marginally linked with lower SLC2A1 mRNA levels while subjective distress was a marginally significant predictor of higher placental SLC2A4 mRNA levels. Gestational age at the time of the flood was a significant moderator of the effect of subjective distress on placental mRNA levels for NR3C1-α (p = 0.046) and HSD11B1 (p = 0.049) in male placentas: if the flood occurred in mid-pregnancy, lower subjective distress predicted higher HSD11B1 while higher subjective distress predicted lower NR3C1-α placental mRNA level. While results did not show any PNMS effects on placental HSD11B2 mRNA and protein levels, and activity, we showed a reduction in placental NR3C1-β mRNA level in male placentas. Our results show evidence of distinct placental glucocorticoid and glucose systems adaptations to PNMS as a function of fetal sex and gestational timing of exposure, with high subjective PNMS in mid-pregnancy associated with lower levels of expression of glucocorticoid-promoting gene in males, leaving the fetus less protected against maternal stress. The exact mechanism by which natural disaster-related PNMS acts on the placenta and the impact on fetal programming requires further investigation.

673 11β-hydroxysteroid dehydrogenase type 1 activation induced by UVB might be a possible mechanism of skin barrier dysfunction due to UV exposure

Ultraviolet light (UV) radiation to the skin causes dose- and wavelength- dependent reactions, including skin barrier dysfunction. Long-term exposure to even low dose UV induces the alteration of skin barrier presenting prominent ceramide decrease in the stratum corneum (SC) intercellular lipids. 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), which converts inactive cortisone to active cortisol in peripheral tissues and inhibits keratinocytes and fibroblasts proliferation, is known to be induced by UVB. We performed in vitro, in vivo, ex-vivo studies to elucidate whether skin barrier dysfunction induced by UV could be resulted from 11β-HSD1 activation after UV irradiation. We observed that 11β-HSD1 expression was increased by cortisol treatment itself and UV irradiation, thereby rapidly converting cortisone into cortisol. UVB irradiation increases 11β-HSD1 expression as well as inflammatory cytokines using HSD11B1 siRNA transfected normal human epidermal keratinocytes. 11β-HSD1 mRNA was prominently higher in UVB irradiated primary human keratinocytes compared with control. Keratinocytes transfected with HSD11B1 siRNA decreased the expression of 11β-HSD1 mRNA and did not increase cortisol even when the keratinocytes were irradiated by UVB. In vitro, increased cortisol converted by 11β-HSD1 inhibits keratinocyte differentiation. Cortisone decreased the mRNA and protein expression of keratinocyte differentiation markers in UVB-exposed keratinocytes. 11β-HSD1 expression remarkably increased in UV exposed skin and intrinsic aged skin. Skin barrier function presented by transpidermal water loss, SC hydration and SC integrity was impaired in long term UV exposed skin and intrinsic aged skin. Therefore, 11β-HSD1 activation by UVB might suggest another possible mechanism on skin barrier dysfunction due to UV exposure.

Growth and Stress Axis Responses to Dietary Cholesterol in Nile Tilapia (Oreochromis niloticus) in Brackish Water.

Six isonitrogenous and isocaloric diets were formulated to contain 0% (control), 0.4, 0.8, 1.2, 1.6, or 2.4% dietary cholesterol and fed to juvenile Nile tilapia (Oreochromis niloticus) (2.20 ± 0.12 g) twice daily to apparent satiation for 8 weeks in triplicate at a salinity of 16. Fish fed 0.4% cholesterol showed a higher weight gain and specific growth rate and a lower feed coefficient ratio than fish fed other diets. No difference was found in the survival of Nile tilapia fed various levels of cholesterol. Cholesterol in the serum and liver and low-density lipoprotein cholesterol in the serum increased with the increase in the dietary cholesterol content. Relative to the control, no significant difference was found in the expression of head kidney P450scc mRNA between treatment groups. The expression of head kidney 11β-HSD2 mRNA was the highest in the control group, and it decreased significantly with increasing levels of diet cholesterol. Fish fed 0.4 or 1.2% cholesterol had a higher 20β-HSD2 mRNA expression in the head kidney than those fed other diets. Fish fed 0.8% cholesterol had higher expressions of GR1 and GR2B mRNA in the liver than other groups. Fish fed 0.4% cholesterol had the highest activity of gill Na+/K+-ATPase. Fish fed 0.8 to 2.4% cholesterol had higher serum cortisol contents than the fish in the control group and the fish fed 0.4% cholesterol. This study suggests that dietary cholesterol is not essential for Nile tilapia survival in brackish water, but 0.4% cholesterol supplementation in the Nile tilapia diet contributes to the improvement of hyperosmotic adaptation and increases in gill Na+/K+-ATPase activity and serum cortisol content by regulating the hypothalamic-pituitary-interrenal stress axis.

Advanced glycation end products alter steroidogenic gene expression by granulosa cells: an effect partially reversible by vitamin D.

Does vitamin D attenuate the adverse effects of advanced glycation end products (AGEs) on steroidogenesis by human granulosa cells (GCs)? AGEs alter the expression of genes important in steroidogenesis while 1,25-dihydroxyvitamin D3 (vit D3) in vitro attenuates some of the actions of AGEs on steroidogenic gene expression, possibly by downregulating the expression of the pro-inflammatory cell membrane receptor for AGEs (RAGE). Vitamin D attenuates the pro-inflammatory effects of AGEs in non-ovarian tissues. Women who were undergoing IVF were enrolled. Follicular fluid samples (n = 71) were collected and cumulus GCs (n = 12) were treated in culture. Follicular fluid levels of the anti-inflammatory soluble RAGE (sRAGE), AGEs and 25-hydroxyvitamin D (25-OHD) were quantified for possible correlations. GCs of each participant were split equally and treated with either media alone (control) or with human glycated albumin (HGA as a precursor for AGEs) with or without vit D3 after which RT-PCR and immunofluorescence were performed and cell culture media estradiol (E2) levels were compared. In follicular fluid, sRAGE levels were positively correlated with 25-OHD levels. HGA treatment (i) increased CYP11A1 (by 48%), 3β-HSD (by 38%), StAR (by 42%), CYP17A1 (by 30%) and LHR (by 37%) mRNA expression levels (P < 0.05 for all) but did not alter CYP19A1 or FSHR mRNA expression levels; and (ii) increased E2 release in cell culture media (P = 0.02). Vit D3 treatment (i) downregulated RAGE mRNA expression by 33% and RAGE protein levels by 44% (P < 0.05); (ii) inhibited the HGA-induced increase in CYP11A1, StAR, CYP17A1 and LHR mRNA levels, but not the increase in 3β-HSD mRNA levels; and (iii) did not inhibit the HGA-induced E2 release in cell culture media. This study used luteinized GCs that were collected from women who received gonadotropins thus the results obtained may not fully extrapolate to non-luteinized GCs in vivo. This study suggests that there is a relationship between AGEs and their receptors (RAGE and sRAGE) with vitamin D. Understanding the interaction between AGEs and vitamin D in ovarian physiology could lead to a more targeted therapy for the treatment of ovarian dysfunction. Funding was received from NIH (R01 NS045940), American Society for Reproductive Medicine, Ferring Pharmaceuticals Inc., and University of Vermont College of Medicine Bridge Funds. All authors have nothing to disclose.

11β-HSD Types 1 and 2 in the Songbird Brain.

Glucocorticoid (GC) hormones act on the brain to regulate diverse functions, from behavior and homeostasis to the activity of the hypothalamic–pituitary–adrenal axis. Local regeneration and metabolism of GCs can occur in target tissues through the actions of the 11β-hydroxysteroid dehydrogenases [11 beta-hydroxysteroid dehydrogenase type 1 (11β-HSD1) and 11 beta-hydroxysteroid dehydrogenase type 2 (11β-HSD2), respectively] to regulate access to GC receptors. Songbirds have become especially important model organisms for studies of stress hormone action; however, there has been little focus on neural GC metabolism. Therefore, we tested the hypothesis that 11β-HSD1 and 11β-HSD2 are expressed in GC-sensitive regions of the songbird brain. Localization of 11β-HSD expression in these regions could provide precise temporal and spatial control over GC actions. We quantified GC sensitivity in zebra finch (Taeniopygia guttata) brain by measuring glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) expression across six regions, followed by quantification of 11β-HSD1 and 11β-HSD2 expression. We detected GR, MR, and 11β-HSD2 mRNA expression throughout the adult brain. Whereas 11β-HSD1 expression was undetectable in the adult brain, we detected low levels of expression in the brain of developing finches. Across several adult brain regions, expression of 11β-HSD2 covaried with GR and MR, with the exception of the cerebellum and hippocampus. It is possible that receptors in these latter two regions require direct access to systemic GC levels. Overall, these results suggest that 11β-HSD2 expression protects the adult songbird brain by rapid metabolism of GCs in a context and region-specific manner.

Role of 11β-HSD type 1 in abnormal HPA axis activity during immune-mediated arthritis.

Patients with chronic immune-mediated arthritis exhibit abnormal hypothalamo-pituitary-adrenal (HPA) axis activity. The basis for this abnormality is not known. Immune-mediated arthritis is associated with increased extra-adrenal synthesis of active glucocorticoids by the 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) enzyme. 11β-HSD1 is expressed in the central nervous system, including regions involved in HPA axis regulation. We examined whether altered 11β-HSD1 expression within these regions contributes to HPA axis dysregulation during arthritis. The expression of 11β-HSD1, and other components of glucocorticoid signaling, were examined in various brain regions and the pituitary gland of mice with experimentally induced arthritis. Two arthritis protocols were employed: The K/BxN spontaneous arthritis model for chronic arthritis and the K/BxN serum transfer arthritis model for acute arthritis. 11β-HSD1 mRNA (Hsd11b1) was expressed in the hippocampus, hypothalamus, cortex, cerebellum and pituitary gland. Hypothalamic Hsd11b1 expression did not change in response to arthritis in either model. Pituitary Hsd11b1 expression was however significantly increased in both chronic and acute arthritis models. Hippocampal Hsd11b1 was decreased in acute but not chronic arthritis. Chronic, but not acute, arthritis was associated with a reduction in hypothalamic corticotropin-releasing hormone and arginine vasopressin expression. In both models, serum adrenocorticotropic hormone and corticosterone levels were no different from non-inflammatory controls. These findings demonstrate inflammation-dependent regulation of Hsd11b1 expression in the pituitary gland and hippocampus. The upregulation of 11β-HSD1 expression in the pituitary during both chronic and acute arthritis, and thus, an increase in glucocorticoid negative feedback, could contribute to the abnormalities in HPA axis activity seen in immune-mediated arthritis.

Parthenogenetic bovine embryos secrete type I interferon capable of stimulating ISG15 in luteal cell culture.

Interferon tau (IFNT) is the pregnancy recognition signal in ruminants and is secreted by trophoblast cells. Paracrine action in the endometrium is well established by inhibiting luteolytic pulses of prostaglandin F2 alpha. Recently, endocrine action was documented in the corpus luteum, blood cell and liver. It was hypothesized that conditioned medium (CM) obtained from days 7, 9 and 12 parthenogenetic embryos alters luteal cell gene expression. The aim was to establish a bovine mixed luteal cell culture to evaluate cellular response associated to interferon stimulated genes, steroidogenesis and apoptosis. Conditioned medium was obtained from Days 7, 9 and 12 parthenogenetic (PA) embryos culture. Moreover, antiviral assay was performed on CM from Days 7, 9 and 12 to verify Type I interferon activity. Luteal cell culture was validated by steroidogenic and apoptotic genes (CYP11A1 , HSD3B1, BAX, BCL2, AKT and XIAP mRNA expression), and concentration of progesterone as endpoint. Luteal cell culture was treated with interferon alpha (IFNA) and CM from parthenogenetic embryos. Antiviral assay revealed Type I interferon activity on CM from embryos increasing on Days 9 and 12. ISG15 mRNA was greater in the mixed luteal cells culture treated with 1, 10 and 100ng/ml of interferon alpha (IFNA) and also on Days 7, 9 and 12 CM treatments. Concentration of progesterone was not altered in luteal cell culture regardless of treatments. Steroidogenic and apoptotic genes were similar among groups in luteal cell culture treated with different doses of IFNA or CM from PA embryos. In conclusion, parthenogenetic embryo-derived CM has antiviral activity, luteal cell culture respond to Type I interferon by expressing IGS15. These data indicate this model can be used for IFNT endocrine signaling studies.

An in vitro prototype of a porcine biomimetic testis-like cell culture system: a novel tool for the study of reassembled Sertoli and Leydig cells.

At present, there is no reliable in vitro assembled prepubertal testis-like biomimetic organ culture system designed to assess the functional effects of human gonadotropins on Sertoli and Leydig cells. Spermatogenesis is regulated by endocrine, paracrine, and juxtacrine factors (testicular cross-talk), mainly orchestrated by gonadotropins such as luteinizing hormone (LH) and follicle-stimulating hormone (FSH) that play a pivotal role by stimulating Leydig and Sertoli cells, respectively. The aim of our study was to set up an in vitro prepubertal porcine bioengineered construct as a new model for experimental studies on reassembled Sertoli and Leydig cells. We have evaluated Sertoli and Leydig cells obtained from 15- to 20-day-old neonatal pig testes in terms of purity and function. Subsequently, purified Sertoli and enriched Leydig cells were subjected to coincubation to obtain an in vitro prepubertal porcine testis-like culture system. We performed enzyme-linked immunosorbent assay (ELISA) for anti-Müllerian hormone (AMH), inhibin B, and testosterone secretion in the medium, and Real-Time PCR analysis of AMH, inhibin B, FSH-r, aromatase, LHr, and 3β-HSD mRNA expression levels. This in vitro testis-like system was highly responsive to the effects of human gonadotropins and testosterone. AMH mRNA expression and secretion declined, and inhibin-B increased, while FSH-receptor expression was downregulated upon FSH/LH exposure/treatment. Finally, the production of testosterone was increased selectively upon LH treatment. In summary, our proposed model could help to better determine the action of human gonadotropins on Sertoli and Leydig cells. The potential usefulness of the system for shedding light into male infertility-related issues is evident.

Effect of mono-(2-ethylhexyl) phthalate (MEHP) on proliferation of and steroid hormone synthesis in rat ovarian granulosa cells in vitro.

This study aimed to examine the proliferation of and secretion by rat ovarian granulosa cells (GCs) treated with mono-(2-ethylhexyl) phthalate (MEHP). Ovarian GCs were incubated with MEHP at concentration of 0, 25, 50, 100, and 200 µM for 24 hr. Cell viability was determined using the MTT Cell Proliferation Assay. Progesterone and estradiol production was evaluated by radioimmunoassay (RIA) and the expression of FSHR, PR, and ER was measured by immunocytochemistry. StAR, P450scc, 3β-HSD, 17β-HSD, and P450 arom mRNA levels were determined by RT-PCR. MEHP markedly attenuated proliferation of GCs, increased expression of sex hormone receptors and key enzymes in progesterone production, and stimulated steroid hormone secretion. The result of these analyses demonstrates that MEHP exposure of GCs may have effects on rat ovarian functions.

An in vitro prototype of a porcine biomimetic testis-like cell culture system: a novel tool for the study of reassembled Sertoli and Leydig cells

At present, there is no reliable in vitro assembled prepubertal testis-like biomimetic organ culture system designed to assess the functional effects of human gonadotropins on Sertoli and Leydig cells. Spermatogenesis is regulated by endocrine, paracrine, and juxtacrine factors (testicular cross-talk), mainly orchestrated by gonadotropins such as luteinizing hormone (LH) and follicle-stimulating hormone (FSH) that play a pivotal role by stimulating Leydig and Sertoli cells, respectively. The aim of our study was to set up an in vitro prepubertal porcine bioengineered construct as a new model for experimental studies on reassembled Sertoli and Leydig cells. We have evaluated Sertoli and Leydig cells obtained from 15- to 20-day-old neonatal pig testes in terms of purity and function. Subsequently, purified Sertoli and enriched Leydig cells were subjected to coincubation to obtain an in vitro prepubertal porcine testis-like culture system. We performed enzyme-linked immunosorbent assay (ELISA) for anti-Müllerian hormone (AMH), inhibin B, and testosterone secretion in the medium, and Real-Time PCR analysis of AMH, inhibin B, FSH-r, aromatase, LHr, and 3β-HSD mRNA expression levels. This in vitro testis-like system was highly responsive to the effects of human gonadotropins and testosterone. AMH mRNA expression and secretion declined, and inhibin-B increased, while FSH-receptor expression was downregulated upon FSH/LH exposure/treatment. Finally, the production of testosterone was increased selectively upon LH treatment. In summary, our proposed model could help to better determine the action of human gonadotropins on Sertoli and Leydig cells. The potential usefulness of the system for shedding light into male infertility-related issues is evident.

Psychosocial stress sensitizes neuroendocrine and inflammatory responses to Escherichia coli challenge in domestic piglets

Exposure to psychosocial stress can have a profound impact on immune reactivity and health mediated by hypothalamic–pituitaryadrenal (HPA) axis activation. However, current knowledge regarding the mechanisms involved in cross-sensitization between stress and the immune system is limited. Here, we investigated the effects of a single social isolation followed by repeated oral Escherichia coli (E. coli) applications on cortisol, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), haptoglobin and C-reactive protein (CRP) concentrations in the blood; on clinical signs of disease; and on mRNA expression of the glucocorticoid receptor (GR), mineralocorticoid receptor (MR), 11β-hydroxysteroid dehydrogenase 1 and 2 (11β-HSD1 and 11β-HSD2), TNF-α and IL-6 in the hypothalamus, prefrontal cortex (PFC) and spleen of 7-, 21- and 35-day-old piglets. Additionally, the protein levels of splenic TNF-α and IL-6 were analyzed. Non-isolated, E. coli-challenged piglets served as a control. Social isolation for 4 h induced a rise in the plasma cortisol concentrations immediately after social treatment and after repeated E. coli applications in isolated compared to non-isolated piglets. The circulating TNF-α concentration was not affected by social treatment. Furthermore, previously isolated piglets showed a higher frequency of signs of disease in response to E. coli challenge than non-isolated piglets, while the haptoglobin and CRP concentrations did not significantly differ between social treatments. In the brain, 11β-HSD1, 11β-HSD2 and IL-6 mRNA expression in the hypothalamus and GR, and 11β-HSD1 and 11β-HSD2 mRNA expression in the PFC were higher in isolated, E. coli-challenged piglets than in the corresponding controls. Moreover, isolated piglets also displayed higher MR, 11β-HSD1 and IL-6 mRNA expression levels and TNF-α concentrations in the spleen. The stress-induced alterations in the hypothalamus and spleen were particularly pronounced in younger piglets. The present findings may contribute to a better understanding of the complex interplay between early psychological stress and an increased risk of disease and might also have implications on aspects of the health and welfare of farm animals and humans.

Diurnal Hypothalamic-Pituitary-Adrenal Axis Measures and Inflammatory Marker Correlates in Major Depressive Disorder.

Dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis and inflammatory systems is a consistent finding in patients with Major Depressive Disorder (MDD). Cortisol is often assessed by measurement of the cortisol awakening response (CAR) and/or diurnal cortisol levels. Some methods of cortisol measurement overestimate cortisol concentration due to detection of other glucocorticoids including the relatively inert cortisone, therefore this study aimed to assess the presence of both cortisol and cortisone, and the cortisol-cortisone catalyzing enzyme 11β-hydroxysteroiddehydrogenase type 1 (11β-HSD1), in depressed patients and controls. Because the HPA axis is known to regulate the body’s immune system, relationships between measures of cytokines and cortisol were also assessed. Saliva samples were collected from 57 MDD patients and 40 healthy controls at five post-wakening time points (0, +30, +60, +720 and +750 min). Glucocorticoid concentrations were measured by liquid chromatography mass spectrometry. Whole blood mRNA expression of several inflammatory markers was measured by quantitative polymerase chain reaction. This study replicated the common finding of elevated morning cortisol and reduced CAR reactivity in MDD and found no differences in cortisone or 11β-HSD1 mRNA measures. There was a negative association between interleukin 1-β (IL-1β) mRNA and morning cortisol reactivity within the depressed group, indicating that dysregulation of the HPA axis and immune system may be interconnected.

The Role and Regulation of the 11 Beta-Hydroxysteroid Dehydrogenase Enzyme System in Patients with Inflammatory Bowel Disease.

Glucocorticoids are known to modulate a number of immunological responses including counteracting inflammation. Within tissues expressing the glucocorticoid and mineralocorticoid receptors including the colon, glucocorticoid metabolism is regulated by the isoenzymes of 11ß-hydroxysteroid dehydrogenase (11β-HSD). 11β-HSD1 acts as an oxidoreductase converting inactive cortisone into active cortisol, while 11β-HSD2 acts as a dehydrogenase converting active cortisol to inactive cortisone. Hexose-6 phosphate dehydrogenase (H6PDH) is a key regulator of 11β-HSD1 activity via its generation of NADPH. Variations in the 11β-HSD enzyme system in relation to levels of expression and regulation may have a role in IBD. The aim of this study was to investigate possible abnormalities of 11β-HSD enzyme system in the colon of patients with IBD. By using quantitative real-time PCR, we investigated the transcription levels of 11β-HSD1 and 2 in colonic tissue from IBD patients and healthy controls undergoing a colonoscopy for disease assessment. Disease activity was recorded using clinical (Mayo Score/Harvey-Bradshaw Index), Biochemical (C-reactive protein), histological, and endoscopic parameters. In addition, transcription levels of H6PDH and the glucocorticoid receptor alpha (GR-α) as well as key pro-inflammatory cytokines (TNF-α, IL-1β, IL-6, Rela (subunit for NF Kappa B)) were later examined among this group, and results were correlated with 11β-HSD2 gene expression. Results and patient demographics were expressed as a mean (and SD), and differences between IBD patients and control groups were analyzed using a Student's t test or Mann-Whitney U test as appropriate, with a p value of ≤0.05 considered significant. Results were controlled for disease activity as outlined above. Results have demonstrated a significant downregulation in 11β-HSD2 expression in IBD patients compared with controls (13.8±17.1 au vs. 318.4±521.1au, p=0.01), whereas levels of 11β-HSD1 did not appear to vary across the two groups. Among IBD patients, there was a trend toward higher 11β-HSD1 expression in inflamed tissue compared with matched non-inflamed tissue (422.1±944 au vs. 102.2±103.9, P=0.09). Levels of H6PDH and the GR-α expression did not appear to vary among active inflamed IBD tissue and controls. As a result, we examined the association between pro-inflammatory cytokines and levels of 11β-HSD2 expression. Results showed an upregulation of key pro-inflammatory cytokine mRNA expression (TNF-α, IL-1β, IL-6) during inflammation with an associated downregulation of 11β-HSD2 mRNA expression when compared to controls. Dysregulation in this pathway could have a potential role in IBD pathogenesis and may account for exogenous glucocorticoid resistance in IBD. Further work assessing the role of the 11β-HSD enzyme system in steroid-resistant subjects is warranted.