Expression Of P450 Aromatase Research Articles

Unique features of KGN granulosa-like tumour cells in the regulation of steroidogenic and antioxidant genes.

The ovarian KGN granulosa-like tumour cell line is commonly used as a model for human granulosa cells, especially since it produces steroid hormones. To explore this further, we identified genes that were differentially expressed by KGN cells compared to primary human granulosa cells using three public RNA sequence datasets. Of significance, we identified that the expression of the antioxidant gene TXNRD1 (thioredoxin reductase 1) was extremely high in KGN cells. This is ominous since cytochrome P450 enzymes leak electrons and produce reactive oxygen species during the biosynthesis of steroid hormones. Gene Ontology (GO) analysis identified steroid biosynthetic and cholesterol metabolic processes were more active in primary granulosa cells, whilst in KGN cells, DNA processing, chromosome segregation and kinetochore pathways were more prominent. Expression of cytochrome P450 cholesterol side-chain cleavage (CYP11A1) and cytochrome P450 aromatase (CYP19A1), which are important for the biosynthesis of the steroid hormones progesterone and oestrogen, plus their electron transport chain members (FDXR, FDX1, POR) were measured in cultured KGN cells. KGN cells were treated with 1 mM dibutyryl cAMP (dbcAMP) or 10 μM forskolin, with or without siRNA knockdown of TXNRD1. We also examined expression of antioxidant genes, H2O2 production by Amplex Red assay and DNA damage by γH2Ax staining. Significant increases in CYP11A1 and CYP19A1 were observed by either dbcAMP or forskolin treatments. However, no significant changes in H2O2 levels or DNA damage were found. Knockdown of expression of TXNRD1 by siRNA blocked the stimulation of expression of CYP11A1 and CYP19A1 by dbcAMP. Thus, with TXNRD1 playing such a pivotal role in steroidogenesis in the KGN cells and it being so highly overexpressed, we conclude that KGN cells might not be the most appropriate model of primary granulosa cells for studying the interplay between ovarian steroidogenesis, reactive oxygen species and antioxidants.

Expression and clinical significance of HER2/neu, aromatase P450 and adhesion molecule CD24 in endometrial cancer.

This study aimed at exploring the expression and clinical significance of aromatase P450, adhesion molecule CD24 and HER2/neu in endometrial cancer. The expression of aromatase P450, adhesion molecule CD24 and HER2/neu was detected by immunohistochemistry in 15 cases of endometrial hyperplasia group, 50 cases of endometrial adenocarcinoma and 3 cases of uterine papillary adenocarcinoma, with 15 cases of normal endometrium as control group. We detected no expression of aromatase P450, adhesion molecule CD24 or HER2/neu in control group. Aromatase P450 positive expression rate was 66.7% in endometrial hyperplasia group and 70.3% in endometrial carcinoma group, without significant difference (p>0.05). There was no significant difference (p>0.05) in the positive expression rate of aromatase P450 between different myometrial invasion groups of endometrial adenocarcinomas. CD24 positive expression rate was 40.0% in endometrial hyperplasia group and 79.6% in endometrial carcinoma group, with significant difference (p<0.05). HER2/neu positive expression rate was 26.7% in the endometrial hyperplasia group and 57% in endometrial carcinoma group, with significant difference (p<0.05). In conclusion, aromatase P450 may be one factor associated with endometrial cancer cell proliferation, while CD24 and HER2/neu may be important factors associated with the invasion and metastasis of endometrial cancer.

Kisspeptin promotes follicular development through its effects on modulation of P450 aromatase expression and steroidogenesis in sheep ovarian follicles.

The present study was conducted to ascertain whether the role of kisspeptin in promoting in vitro development of preantral follicles was through the regulation of P450 aromatase gene expression and steroidogenesis in sheep. Accordingly, the cumulus cells and oocytes were collected from different development stages of preantral follicles grown in vivo and cultured in vitro in TCM199B (Group I), TCM199B + KP (10 μg/mL) (Group II) and Standard medium + KP (10 μg/mL). To measure the steroid (Estradiol-17β; E2 and Progesterone; P4 ) synthesis through ELISA, spent culture medium was collected separately from the same in vitro groups. E2 synthesis in the spent medium collected from all the three groups showed an increasing trend from PFs' exposed to respective culture media for 3 min to 2-day culture stage but decreased thereafter till 6-day culture stage. This is followed by a sharp increase in E2 concentration in the spent medium collected after in vitro maturation. However, P4 synthesis in group III followed increased pattern as the development progressed from PFs' exposed to culture medium for 3 min to in vitro maturation stage. The steroid production was observed at all stages of in vitro development in altered supplemented conditions. The steroid synthesis in the spent medium was highest in the 6 day cultured PFs' in Standard medium + KP matured in vitro for 24 h. Therefore, supplementation of kisspeptin along with other growth factors promoted steroid production in cultured preantral follicles far better than in other media.

Letrozole administration effects on the P450aromatase expression and the reproductive parameters in male sheep (Ovis aries)

Letrozole administration effects on the P450aromatase expression and the reproductive parameters in male sheep (Ovis aries)

Evaluation of Expression of Cytochrome P450 Aromatase and Inflammatory, Oxidative, and Apoptotic Markers in Testicular Tissue of Obese Rats (Pre)Treated with Garlic Powder.

Today, adolescent obesity is recognized as an epidemic and a cause of reproductive disorders. Decreased testosterone levels occur due to functional defects in the hypothalamus-pituitary axis, excessive activity of cytochrome P450 aromatase enzyme, and testicular dysfunction in these people. Oxidative damage, inflammation, and apoptosis are also the main mechanisms of testicular damage during obesity. The use of herbal products such as garlic can improve this disorder due to its anti-inflammatory and antioxidant properties. Therefore, the aim of this study is to investigate the effect of pretreatment and treatment of garlic powder on the expression of cytochrome P450 aromatase enzyme and the expression of genes involved in testosterone synthesis, inflammation, oxidative damage, apoptosis in testicular tissue, and metabolic function of liver tissue in young male obese rats. Eighty male Wistar rats were divided into the controlled and treated groups. Serum levels of lipid, glucose, and insulin as metabolic factors were measured along with the testicular antioxidant and inflammation markers. The expression of Bcl2, Bax, and caspase-3 along with NF-κB, SREBP-1c, CPT-1beta, Nrf-2, CD36, FAS, CYP19A1, P450scc, StAR, 17βHSD, PPARα, and aromatase (CYP19, P450arom) was also measured. Testicular histological evaluation and spermatogenic process was also performed. The results showed that oxidative, inflammatory, and metabolic factors significantly increased in obese rats. The testicular expression of aromatase, NF-κB, Bax, and caspase 3 increased and Nrf2 expression decreased in obese rats, while (pre) treatment with garlic powder significantly decreased the expression of these genes in obese rats. These results were also confirmed by the findings of the histological evaluation and sperm analysis. It can be concluded that garlic powder could improve reproductive dysfunction in obese rats.

Expression and localization of apelin and apelin receptor (APJ) in buffalo ovarian follicles and corpus luteum and the in-vitro effect of apelin on steroidogenesis and survival of granulosa cells

Apelin is an adipose tissue-derived hormone with many physiological functions, including the regulation of female reproduction. It acts through an orphan G protein-coupled receptor APJ/APLNR. The present study aimed to investigate the expression of apelin and its receptor APJ in the ovarian follicles and corpus luteum (CL) and the role of apelin on steroidogenesis and cell survival. Ovarian follicles were classified into four groups based on size and estradiol (E2) level in the follicular fluid as follows: (i) F1 (4–6 mm; <0.5 ng/mL) (ii) F2 (7–9 mm; 0.5–5 ng/mL) (iii) F3 (10–13 mm; 5–40 ng/mL) and (iv) F4 (dominant/pre-ovulatory follicle) (>13 mm; >180 ng/mL). The corpora lutea (CL) were categorized into early (CL1), mid (CL2), late luteal (CL3), and regressing (CL4) CL stages. Expression of apelin increased with follicle size, with significantly greatest in the dominant or pre-ovulatory follicle (P < 0.05). Expression of APJ was greater in large and dominant follicles than in small and medium follicles (P < 0.05). In CL, the mRNA and protein abundance of apelin and apelin receptor was greater during mid (CL2) and late luteal (CL3) stages as compared to early (CL1) and regressing (CL4) stages (P < 0.05). Both the factors were localized in granulosa and theca cells of follicles and small and large luteal cells of CL. The pattern of the intensity of immunofluorescence was similar to mRNA and protein expression. Granulosa cells were cultured in vitro and treated at 1, 10, and 10 ng/mL apelin-13 either alone or in the presence of the follicle-stimulating hormone (FSH) (30 ng/mL) or insulin-like growth factor-I (IGF-I) (10 ng/mL) for 48 h. The luteal cells were treated with apelin-13 at 1, 10, and 100 ng/mL doses for 48 h. Apelin treatment at 10 and 100 ng/ml significantly (P < 0.05) increased E2 secretion, cytochrome P450 aromatase or CYP19A1 expression in GC. In luteal cells, apelin at 10 ng/mL and 100 ng/mL significantly (P < 0.05) increased progesterone (P4) secretion and HSD3B1 expression. In GCs, apelin, either alone or in combination, increased PCNA expression and inhibited CASPASE3 expression suggesting its role in cell survival. In conclusion, this study provides novel evidence for the presence of apelin and receptor APJ in ovarian follicles and corpora lutea and the stimulatory effect on E2 and P4 production and promotes GC survival in buffalo, suggesting the role of apelin in follicular and luteal functions in buffalo.

Maternal Protein Restriction Alters the Expression of Proteins Related to the Structure and Functioning of the Rat Offspring Epididymis in an Age-Dependent Manner.

Nutrition is an environmental factor able to activate physiological interactions between fetus and mother. Maternal protein restriction is able to alter sperm parameters associated with epididymal functions. Since correct development and functioning of the epididymides are fundamental for mammalian reproductive success, this study investigated the effects of maternal protein restriction on epididymal morphology and morphometry in rat offspring as well as on the expression of Src, Cldn-1, AR, ER, aromatase p450, and 5α-reductase in different stages of postnatal epididymal development. For this purpose, pregnant females were allocated to normal-protein (NP—17% protein) and low-protein (LP—6% protein) groups that received specific diets during gestation and lactation. After weaning, male offspring was provided only normal-protein diet until the ages of 21, 44, and 120 days, when they were euthanized and their epididymides collected. Maternal protein restriction decreased genital organs weight as well as crown-rump length and anogenital distance at all ages. Although the low-protein diet did not change the integrity of the epididymal epithelium, we observed decreases in tubular diameter, epithelial height and luminal diameter of the epididymal duct in 21-day-old LP animals. The maternal low-protein diet changed AR, ERα, ERβ, Src 416, and Src 527 expression in offspring epididymides in an age-dependent manner. Finally, maternal protein restriction increased Cldn-1 expression throughout the epididymides at all analyzed ages. Although some of these changes did not remain until adulthood, the insufficient supply of proteins in early life altered the structure and functioning of the epididymis in important periods of postnatal development.

Assessment of testicular steroidogenic enzymes expression in experimental animal model following withdrawal of nandrolone decanoate.

Anabolic steroids are frequently used to increase the growth rate of meat-producing animals. Exposure to an anabolic-androgenic steroid, nandrolone decanoate (ND), is associated with expressional reduction of testicular steroidogenic enzymes. However, the effect of withdrawal of ND exposure on the expression of these testicular molecules has not been thoroughly explored. The current research investigated expression changes of testicular steroidogenic enzymes in rats at several recovery periods (2, 6, and 12 weeks) after the stop of ND treatment with different doses (2 and 10 mg/kg body weight) for 12 weeks. Body and testis weights were recorded, and transcript levels of molecules were determined by quantitative real-time polymerase chain reaction (PCR). The immunohistochemistry was used to examine the changes of immuno-intensities of molecules. At 6 and 12 weeks of the recovery period, the 10 mg/kg ND-treated rats were lighter than other experimental groups. The interstitial compartment vanished by ND treatment filled up as the recovery period became longer. The expression of steroidogenic acute regulatory protein was returned to the control level at 12 weeks of the recovery period. Expression levels of cytochrome P450 side-chain cleavage and 17a-hydroxylase were increased in 2 mg/kg ND-treated group at 6 weeks of the recovery period, and transcript levels of these molecules in 2 and 10 mg/kg ND-treated groups at 12 weeks of the recovery period were significantly lower than the control. Expression levels of 3β-hydroxysteroid dehydrogenase (HSD) type I and 17β-HSD type 3 in 2 mg/kg ND-treated group were comparable with those of control at 12 weeks of the recovery period, but not in 10 mg/kg ND-treated group. Expression of cytochrome P450 aromatase (Cyp19) was reverted to the control level at 2 weeks of the recovery period. Except for Cyp19, there was a visible increase of immuno-staining intensity of other testicular steroidogenic enzymes in the Leydig cells as the recovery period progressed. This research has demonstrated that the cease of ND administration could restore the expression of testicular steroidogenic enzymes close to the normal level. Nevertheless, a relatively long recovery period, compared to the ND-exposure period would be required to retrieve normal expression levels of testicular steroidogenic enzymes.

Regulation of uterine function during estrous cycle, anestrus phase and pregnancy by steroids in red deer (Cervus elaphus L.)

Steroid synthesis and production in ruminant uterus is not obvious, especially in seasonally reproduced. We compared steroid production by investigating enzymes involved in red deer uterine steroid metabolism in reproductive seasons. Blood and uteri (endometrium and myometrium) were collected post mortem from hinds on 4th day (N = 8), 13th day of the cycle (N = 8), anestrus (N = 8) and pregnancy (N = 8). The expression of cytochrome P450 aromatase (P450), 3 -beta-hydroxysteroid dehydrogenase (3β-HSD), 17 -beta-hydroxysteroid dehydrogenase (17β-HSD), aldo–keto reductase family 1 C1 (AKR1C1), estrogen receptor alpha (ERα), and progesterone receptors (PRs), were analyzed using real-time-PCR and Western Blotting. Plasma samples were assayed for 17-beta-estradiol (E2), progesterone (P4), luteinizing hormone (LH), follicle-stimulating hormone (FSH), and testosterone (T4) concentrations by EIA. Hinds at the beginning of the estrous cycle, mainly in endometrium, were characterized by a high mRNA expression of 3β-HSD, AKR1C1, PRs and ERα, contrary to the expression in myometrium during pregnancy (P < 0.05). For P4, E2, and FSH, concentration was the highest during the 13th day of the estrous cycle (P < 0.05). Uterine steroid production and output in hinds as a representative seasonally reproduced ruminant occurred mainly during the estrous cycle and sustained in anestrus.

Molecular Characterization and Expression of Cytochrome P450 Aromatase in Atlantic Croaker Brain: Regulation by Antioxidant Status and Nitric Oxide Synthase During Hypoxia Stress.

We have previously shown that nitric oxide synthase (NOS, an enzyme) is significantly increased during hypoxic stress in Atlantic croaker brains and modulated by an antioxidant (AOX). However, the influence of NOS and AOX on cytochrome P450 aromatase (AROM, CYP19a1, an enzyme) activity on vertebrate brains during hypoxic stress is largely unknown. In this study, we characterized brain AROM (bAROM, CYP19a1b) cDNA in croaker and examined the interactive effects of hypoxia and a NOS-inhibitor or AOX on AROM activity. The amino acid sequence of croaker bAROM cDNA is highly homologous (76–80%) to other marine teleost bAROM cDNAs. Both real-time PCR and Northern blot analyses showed that bAROM transcript (size: ∼2.8 kb) is highly expressed in the preoptic-anterior hypothalamus (POAH). Hypoxia exposure (dissolved oxygen, DO: 1.7 mg/L for 4 weeks) caused significant decreases in hypothalamic AROM activity, bAROM mRNA and protein expressions. Hypothalamic AROM activity and mRNA levels were also decreased by pharmacological treatment with N-ethylmaleimide (NEM, an alkylating drug that modifies sulfhydryl groups) of fish exposed to normoxic (DO: ∼6.5 mg/L) conditions. On the other hand, treatments with Nω-nitro-L-arginine methyl ester (NAME, a competitive NOS-inhibitor) or vitamin-E (Vit-E, a powerful AOX) prevented the downregulation of hypothalamic AROM activity and mRNA levels in hypoxic fish. Moreover, NAME and Vit-E treatments also restored gonadal growth in hypoxic fish. Double-labeled immunohistochemistry results showed that AROM and NOS proteins are co-expressed with NADPH oxidase (generates superoxide anion) in the POAH. Collectively, these results suggest that the hypoxia-induced downregulation of AROM activity in teleost brains is influenced by neuronal NOS activity and AOX status. The present study provides, to the best of our knowledge, the first evidence of restoration of AROM levels in vertebrate brains by a competitive NOS-inhibitor and potent AOX during hypoxic stress.

The lack of Irs2 induces changes in the immunocytochemical expression of aromatase in the mouse retina

Insulin receptor substrate (Irs) belongs to a family of proteins that mediate the intracellular signaling of insulin and IGF-1. Insulin receptor substrate 2 (Irs2) is necessary for retinal function, since its failure in Irs2-deficient mice in hyperglycemic situation promotes photoreceptor degeneration and visual dysfunction, like in diabetic retinopathy. The expression of P450 aromatase, which catalyzes androgen aromatization to form 17ß-estradiol, increases in some neurodegenerative diseases thus promoting the local synthesis of neuroestrogens that exert relevant neuroprotective functions. Aromatase is also expressed in neurons and glial cells of the central nervous system (CNS), including the retina. To further understand the role of Irs2 at the retinal level, we performed an immunocytochemical study in adult normoglycemic Irs2-deficient mice. For this aim, the retinal immunoexpression of neuromodulators, such as aromatase, glutamine synthetase (GS), and tyrosine hydroxylase (TH) was analyzed, joint to a morphometric and planimetric study of the retinal layers. Comparing with wild-type (WT) control mice, the Irs2-knockout (Irs2-KO) animals showed a significant increase in the immunopositivity to aromatase in almost all of the retinal layers. Besides, Irs2-KO mice exhibited a decreased immunopositive reaction for GS and TH, in Müller and amacrine cells, respectively; morphological variations were also found in these retinal cell types. Furthermore, the retina of Irs2-KO mice displayed alterations in the structural organization, and a generalized decrease in the retinal thickness was observed in each of the layers, except for the inner nuclear layer. Our findings suggest that the absence of Irs2 induces retinal neurodegenerative changes in Müller and amacrine cells that are unrelated to hyperglycemia. Accordingly, in the Irs2-KO mice, the increased retinal immunocytochemical reactivity of aromatase could be associated with an attempt to repair such neural retina injuries by promoting local neuroprotective mediators.

Characterization of Estrogenic Activity and Site-Specific Accumulation of Bisphenol-A in Epididymal Fat Pad: Interfering Effects on the Endocannabinoid System and Temporal Progression of Germ Cells

The objective of this work has been to characterize the estrogenic activity of bisphenol-A (BPA) and the adverse effects on the endocannabinoid system (ECS) in modulating germ cell progression. Male offspring exposed to BPA during the foetal-perinatal period at doses below the no-observed-adverse-effect-level were used to investigate the exposure effects in adulthood. Results showed that BPA accumulates specifically in epididymal fat rather than in abdominal fat and targets testicular expression of 3β-hydroxysteroid dehydrogenase and cytochrome P450 aromatase, thus promoting sustained increase of estrogens and a decrease of testosterone. The exposure to BPA affects the expression levels of some ECS components, namely type-1 (CB1) and type-2 cannabinoid (CB2) receptor and monoacylglycerol-lipase (MAGL). Furthermore, it affects the temporal progression of germ cells reported to be responsive to ECS and promotes epithelial germ cell exfoliation. In particular, it increases the germ cell content (i.e., spermatogonia while reducing spermatocytes and spermatids), accelerates progression of spermatocytes and spermatids, promotes epithelial detachment of round and condensed spermatids and interferes with expression of cell–cell junction genes (i.e., zonula occcludens protein-1, vimentin and β-catenin). Altogether, our study provides evidence that early exposure to BPA produces in adulthood sustained and site-specific BPA accumulation in epididymal fat, becoming a risk factor for the reproductive endocrine pathways associated to ECS.

MiRNA320a-3p/RUNX2 signal programming mediates the transgenerational inheritance of inhibited ovarian estrogen synthesis in female offspring rats induced by prenatal dexamethasone exposure

Our previous studies found that prenatal dexamethasone exposure could cause abnormal follicular development in fetal rats. This study intends to observe the transgenerational inheritance effects of ovarian estrogen inhibition in offspring exposed to dexamethasone (0.2 mg/kg • d) from gestational day 9 (GD9) to GD20 in Wistar rats, and explore the intrauterine programming mechanisms. Prenatal dexamethasone exposure reduced the expression of ovarian cytochrome P450 aromatase (P450arom), the level of serum estradiol (E2) and the number of primordial follicles, while increased the number of atresia follicles before and after birth in F1 offspring rats. At the same time, the expression of miRNA320a-3p in F1 ovaries was down-regulated, and RUNX2 expression increased significantly. These changes were continued to F2 and F3 generations, accompanied by consistently down-regulated miRNA320a-3p expression in oocyte of F1 and F2 adult offspring. In vitro, fetal rat ovaries and KGN human ovarian granulosa cells were treated with dexamethasone. It showed that dexamethasone decreased miRNA320a-3p and P450arom expression, as well as E2 synthesis, and increased RUNX2 expression. All these effects could be reversed by the GR antagonist RU486. The overexpression of miRNA320a-3p in vitro could also reverse the effects of dexamethasone on RUNX2, P450arom, and E2 levels. The dual-luciferase reporter gene experiment further confirmed the direct targeted regulation of miRNA320a-3p on RUNX2. These results indicate that prenatal dexamethasone exposure induces ovarian E2 synthesis inhibition mediated by the GR/miRNA320a-3p/RUNX2/P450arom cascade signal in fetal rat ovary, which has transgenerational inheritance effects and may related to the inhibited miRNA320a-3p expression in oocyte.

Dihydrotestosterone regulates oestrogen secretion, oestrogen receptor expression, and apoptosis in granulosa cells during antral follicle development

Dihydrotestosterone (DHT) is involved in the development of preantral follicles. However, the effect of DHT on the development of antral follicles has yet to be fully investigated. Herein, we used enzyme-linked immunosorbent assays, immunofluorescence assays, quantitative real time-polymerase chain reaction, immunohistochemical staining, and western blotting to investigate the effect of DHT on antral follicle development. First, we detected the concentration of DHT and the expression of the androgen receptor (AR) in different antral follicles. Second, multiple DHT concentration (10−10–10-7 M) were added to granulosa cells cultured in vitro to examine the influence of DHT on AR expression. Third, to study changes in the expression of oestrogen (E2) synthase and receptors during the development of antral follicles, we divided them according to their diameters into small (≤ 2 mm), medium (2–5 mm), and large (≥ 5 mm) groups. Fourth, we added DHT (10-8 M) and flutamide (Flu, 10-7 M) to granulosa cells to determine whether DHT regulates the expression of cytochrome P450 aromatase (CYP19A1) and the associated receptors through the AR pathway. Fifth, we tested the effect of DHT and Flu on the expression of apoptotic genes and proteins in granulosa cells. We found that AR was expressed in sheep antral follicle granulosa cells and was regulated by DHT. During antral follicle development, the concentration of E2 and the expression of CYP19A1 and E2 receptors significantly increased in granulosa cells. DHT influenced this increase, at least partially, through the AR. Moreover, DHT regulated the expression of apoptotic genes and proteins through the AR. Our study expands our knowledge on the regulatory mechanism of DHT in antral follicle development and guides further research on the androgen regulation of ovarian function.

BMP2 increases the production of BDNF through the upregulation of proBDNF and furin expression in human granulosa-lutein cells.

Locally produced in human granulosa cells of the developing follicle, bone morphogenetic protein 2 (BMP2) plays a crucial role in the regulation of ovarian folliculogenesis and luteal formation. Brain-derived neurotrophic factor (BDNF) is an intraovarian neurotrophic factor that has been shown to promote oocyte maturation and subsequent fertilization competency. At present, little is known regarding the intracellular regulation, assembly and secretion of endogenous BDNF in human granulosa cells. The aim of this study was to explore the effect of BMP2 on the expression and production of BDNF in human granulosa cells and the molecular mechanisms underlying this effect. An immortalized human granulosa cell line (SVOG) and primary human granulosa-lutein (hGL) cells were utilized as in vitro study models. Our results showed that BMP2 significantly increased the mRNA and secreted levels of BDNF. Additionally, BMP2 upregulated the expression of furin at the transcriptional and translational levels. Knockdown of endogenous furin partially attenuated the BMP2-induced increase in BDNF production, indicating that furin is involved in the maturation process of BDNF. Using pharmacological (kinase receptor inhibitors) and siRNA-mediated inhibition approaches, we demonstrated that BMP2-induced upregulation of BDNF and furin expression is most likely mediated by the activin receptor-like kinase (ALK)2/ALK3-SMAD4 signaling pathway. Notably, analysis using clinical samples revealed that there was a positive correlation between follicular fluid concentrations of BMP2 and those of BDNF. These results indicate that BMP2 increases the production of mature BDNF by upregulating the precursor BDNF and promoting the proteolytic processing of mature BDNF. Finally, we also investigated the effects of BMP2 on ovarian steroidogenesis and the results showed that BMP2 treatment significantly increased the accumulated level of estradiol (by upregulating the expression of FSH receptor and cytochrome P450 aromatase), whereas it decreased the accumulated level of progesterone (by downregulating the expression of LH receptors and steroidogenic acute regulatory protein) in primary hGL cells. Our findings provide a novel paracrine mechanism underlying the regulation of an intraovarian growth factor in human granulosa cells.

Mammalian gonadal differentiation: the pig model

In mammals, testicular differentiation is initiated by SRY (the sex-determining region of the Y chromosome) gene expression in Sertoli cell precursors, followed by upregulation of the SOX9 gene (SRY-related HMG box gene 9). Subsequently, differentiated testis produces two hormones that induce sexual differentiation of the internal and external genital tract. Knowledge of the molecular mechanisms involved in gonadal differentiation has increased greatly over the past decade. Several genes are involved in genital ridge formation in both sexes, and others act specifically in testicular or ovarian developmental pathways. As for other mammals, relatively few data are available on the first steps of ovarian differentiation in pigs. In this review, the expression profiles of most genes known to be involved in gonadal differentiation in pigs will be presented and compared with those observed in mice. The main feature of gonadal differentiation in the pig is fetal steroidogenesis, especially cytochrome P450 aromatase gene organization and expression. Another specific feature of gonadal differentiation in pigs is the appearance of numerous cases of XX sex-reversed animals. This intersex condition occurs as early as day 50 after coitus, during embryogenesis, and appears to be triggered genetically. It leads to a wide range of phenotypes, strikingly similar to those observed in humans. Identification of the genes involved in this pathology will improve our knowledge of mammalian gonadal differentiation and may allow the eradication of this genetic disease in pigs.

Quantitative comparative analysis uncovered the role of E2 in Nile tilapia GSD + TE

Nile tilapia is a GSD + TE (genotypic sex determination plus temperature effect) fish species. However, the role of 17β-estradiol (E2) during high-temperature-induced masculinization is unknown in fish species exhibiting GSD + TE. Because letrozole can specifically downregulate E2, the differences of the E2 level and sex ratio between the high-temperature-treated groups and various doses of letrozole treated groups could help quantitatively analyze the role of E2 in Nile tilapia GSD + TE. The results indicated that high-temperature could also significantly downregulate E2, and the level of E2 in the XX + HT (9 days postfertilization (dpf) larvae cultured in 36 °C water for 12 days) group at 21 dpf was only approximately half that in the XX group. Comparison of E2 and the sex reversal ratio between the high-temperature treated group and various letrozole treated groups showed that the level of E2 in the XX + HT group at 21 dpf was almost the same as that in the XX + L1.5 (9 dpf XX larvae cultured in 28 °C water with1.5 μg/L letrozole for 12 days) group, but the 70.00% male ratio in the XX + HT group was significantly higher than that in the XX + L1.5 (58.33%) group and was similar to that in the XX + L4.5 groups (66.25%) and the XX + L13.5 group (77.08%). Comparison of E2 and the female ratio between the high-temperature treated group and the HT + E2 compensation groups further indicated that the E2 level in the XX + HT + E:4 (9 dpf fry cultured in 36 °C water containing 4 μg/L E2 for 12 days) group was similar to that in the XX group at 21 dpf, and the female ratio was close between the two groups. qRT-PCR and western blot analysis showed that the high-temperature treatment significantly downregulated the expression of ovary-type P450 aromatase (cyp19a1a) and upregulated the expression of the male sex differentiation genes doublesex and mab-3 related transcription factor 1 (Dmrt1) at 21 dpf. Additionally, the high-temperature treatment also down-regulated Nile tilapia gonad aromatase activity. Collectively, the present study uncovered the role of E2 during high-temperature–induced Nile tilapia female sex reversal into pseudomales (phenotypic males) and the regulatory effect of high-temperature on the E2 pathway, providing an important foundation for understanding the mechanisms of high-temperature-induced Nile tilapia female sex reversal.

A grape seed extract maternal dietary supplementation improves egg quality and reduces ovarian steroidogenesis without affecting fertility parameters in reproductive hens.

In broiler hens, the genetic selection increased susceptibility to metabolic disorders and reproductive dysfunctions. In human ovarian cells, grape seed extracts (GSE) improved steroid production. Here, we investigated the effects of a GSE dietary supplementation on egg production and quality, fertility parameters, Reactive Oxygen Species (ROS) and steroid content in yolk egg associated to plasma adipokines in broiler hens. For this, we designed two in vivo experiments, the first one included three groups of hens: A (control), B and C (supplemented with GSE at 0.5% and 1% of the total diet composition, respectively, since week 4), and the second one used two groups of hens: A (control) and D (supplemented with GSE at 1% of the total diet composition since hatching). We assessed the egg production from 23th to 40th weeks and quality at 33th week. After artificial inseminations, the fertility parameters were calculated. In egg yolk, Reactive Oxygen Species (ROS) level and steroid production were evaluated by Ros-Glo H202 and ELISA assay, respectively. Expression of steroidogenic enzymes and adipokines and their receptors was determined by RT-qPCR in ovarian cells and plasma adipokines (RARRES2, ADIPOQ and NAMPT) were evaluated by specific ELISA assays. The fertility parameters and egg production were unaffected by GSE supplementation whatever the experiment (exp.). However, the rate of double-yolk eggs decreased for all GSE supplemented groups (exp. 1 P <0.01, exp.2, P<0.02). In exp.1, C group eggs were bigger and larger (P<0.0001) and the shell elasticity was higher for both B and C (P<0.0003) as compared to control. In the egg yolk, GSE supplementation in both exp. reduced ROS content and steroidogenesis consistent with a decrease in P450 aromatase and StAR mRNA expression and basal in vitro progesterone secretion in granulosa cells (P<0.001). Interestingly, in both exp. RARRES2 plasma levels were positively correlated while ADIPOQ and NAMPT plasma levels were negatively correlated, with steroids and ROS in yolk (P<0.0001). Taken together, maternal dietary GSE supplementation did not affect egg production and fertility parameters whereas it reduced ROS content and steroidogenesis in yolk egg. Furthermore, it ameliorated egg quality by decreasing the number of double-yolk eggs and by improving the size of normal eggs and the elasticity of the shell. Taken together, our data suggest the possibility of using dietary maternal GSE to improve egg quality.

Celecoxib, a selective COX-2 inhibitor, markedly reduced the severity of tamoxifen-induced adenomyosis in a murine model.

The aim of the present study was to evaluate the effects of the selective cyclooxygenase (COX)-2 inhibitor celecoxib on the development of uterine adenomyosis in mice. ICR neonatal mice were first exposed to tamoxifen to establish a mouse model of adenomyosis. Following 60 days of celecoxib treatment, pathological formation of adenomyosis lesions and the depth of myometrial infiltration were evaluated using hematoxylin and eosin staining. To examine thermal pain modulation in mice, a hotplate test was conducted every 15 days from postnatal day 30 onwards. Immunohistochemistry was performed to assess the expression of aromatase P450, N-cadherin, E-cadherin, COX-2 and cluster of differentiation 31, whereas the levels of estrogen were analyzed in uterine tissue homogenates using ELISA. Masson trichrome staining was performed to assess the extent of fibrosis in the uterus. Celecoxib treatment significantly inhibited the depth of infiltration into the myometrium, resulting in significantly reduced disease severity. Treatment with high doses of celecoxib significantly prolonged thermal response latency. Following celecoxib treatment, the expression of E-cadherin was significantly increased whereas the expression of N-cadherin was significantly decreased. Concomitantly, the extent of fibrosis was also reduced following celecoxib treatment. Uterine tissue homogenates isolated from mice treated with both high and low doses of celecoxib exhibited lower concentrations of estrogen and decreased expression of aromatase P450. These observations suggest that celecoxib reduces adenomyosis severity by suppressing estrogen production in the uterus, reversing epithelial-mesenchymal transition and relieving fibrosis. Taken together, the results of the present study support the potential use of celecoxib, a selective COX-2 inhibitor, for the treatment of adenomyosis.

Estrogens and Glucocorticoids in Mammary Adipose Tissue: Relationships with Body Mass Index and Breast Cancer Features

ContextAdipose tissue is an important site for extragonadal steroid hormone biosynthesis through the expression and activity of P450 aromatase, 11β-hydroxysteroid dehydrogenase (HSD) 1, and 17β-HSDs. The contribution of steroid hormones produced by adjacent adipose tissue for the progression and survival of breast tumors is unknown.ObjectiveTo quantify estrogens (estradiol, estrone) and glucocorticoids (cortisol, cortisone) in breast adipose tissue from both healthy and diseased women and their relationships with adiposity indices and breast cancer prognostic markers.Design and settingBreast adipose tissue was collected at time of surgery.PatientsPre- and postmenopausal women undergoing partial mastectomy for treatment of breast cancer (n = 17) or reduction mammoplasty (n = 6) were studied.InterventionsRelative estrogen and glucocorticoid amounts were determined by liquid chromatography tandem mass spectrometry.ResultsThe targeted steroids were reliably detected and quantified in mammary adipose tissues. Women with ER+/PR+ tumor had higher relative estradiol amount than women with ER–/PR– tumor (P < .05). The ratio of estradiol-to-estrone was higher in lean women than in women with a body mass index (BMI) ≥ 25 kg/m2 (P < .05). Mixed-model analyses showed that estradiol, cortisone, and cortisol were negatively associated with tumor size (P < .05). Relationships between glucocorticoids and tumor size remained significant after adjustment for BMI. The cortisol-to-cortisone ratio was negatively associated with tumor stage (P < .05) independently of BMI.ConclusionsWe reliably quantified estrogens and glucocorticoids in breast adipose tissue from healthy women and women suffering from breast cancer. Our findings suggest that smaller breast tumors are associated with higher relative amounts of estradiol and cortisol in adipose tissue.