Relationship of estrogen synthesis capacity in the brain with obesity and self-control in men and women.

Gonadal hormones are linked to mechanisms that govern appetitive behavior and its suppression. Estrogens are synthesized from androgens by the enzyme aromatase, highly expressed in the ovaries of reproductive-aged women and in the brains of men and women of all ages. We measured aromatase availability in the amygdala using positron emission tomography (PET) with the aromatase inhibitor [11C]vorozole in a sample of 43 adult, normal-weight, overweight, or obese men and women. A subsample of 27 also completed personality measures to examine the relationship between aromatase and personality traits related to self-regulation and inhibitory control. Results indicated that aromatase availability in the amygdala was negatively associated with body mass index (BMI) (in kilograms per square meter) and positively correlated with scores of the personality trait constraint independent of sex or age. Individual variations in the brain's capacity to synthesize estrogen may influence the risk of obesity and self-control in men and women.

Expression and localization of aromatase in human gastric mucosa : Immunohistochemical study using biopsy materials.

Parietal cells in the gastric mucosa are known not only as cells playing major roles in food digestion but also as cells bearing endocrine function. In addition to their production of gastrin and ghrelin, it has been recently revealed that these cells are also involved in the synthesis and secretion of estrogens with their expression of aromatase in experimental animals. Although aromatase activity has been detected in human gastric cancer cells and related cell lines, much less study has been done to ascertain the expression of the enzymatic activity in normal gastric mucosa. It has not been established which cell type is responsible for estrogen production in human gastric glands consisting of epithelial cells of several types. The aim of this study is to define the expression of aromatase by parietal cells in human gastric glands using immunohistochemical techniques. We retrieved formalin-fixed paraffin embedded materials of gastric biopsies from 16 patients (nine men, seven women). Colocalization of aromatase and H+/K+-ATPase ß-subunit indicated that positive cells are parietal cells, but not chief cells and mucous cells. Furthermore, immunoreactivity of aromatase was detected within gastric glands irrespective of age or sex. These results suggest that human parietal cells synthesize estrogens within gastric mucosa and subsequently secrete them to the portal vein via gastric vein, as they do in rats. These estrogens might influence liver functions in humans. The estrogenic effects related to liver dysfunction might also be attributed to them.

Human chorionic gonadotropin-induced amphiregulin stimulates aromatase expression in human granulosa-lutein cells: a mechanism for estradiol production in the luteal phase.

STUDY QUESTION: Does amphiregulin (AREG), the most abundant and important epidermal growth factor receptor (EGFR) ligand in the follicular fluid, regulate aromatase expression in human granulosa-lutein (hGL) cells? SUMMARY ANSWER: AREG mediates the hCG-induced up-regulation of aromatase expression and estradiol (E2) production in hGL cells. WHAT IS KNOWN ALREADY: AREG expression and secretion are rapidly induced by hCG in hGL cells and mediate physiological functions of LH/hCG in the ovary. EGFR protein is expressed in follicles not only in the pre-ovulatory phase but also throughout the luteal phase of the menstrual cycle. After the LH surge, the human corpus luteum secretes high levels of E2, which regulates various luteal cell functions. Aromatase is an enzyme responsible for a key step in the biosynthesis of E2. However, whether AREG regulates aromatase expression and E2 production in hGL cells remains unexplored. STUDY DESIGN, SIZE, DURATION: This study is an experimental study performed over a 1-year period. In vitro investigations examined the role of AREG in the regulation of aromatase expression and E2 production in primary hGL cells. PARTICIPANTS/MATERIALS, SETTING, METHODS: Primary hGL cells were obtained from women undergoing IVF treatment in an academic research center. Aromatase mRNA and protein levels were examined after exposure of hGL cells to recombinant human AREG, hCG or LH. The EGFR tyrosine kinase inhibitor AG1478, PI3K inhibitor LY294002 and siRNAs targeting EGFR, LH receptor, StAR and AREG were used to verify the specificity of the effects and to investigate the underlying molecular mechanisms. Reverse transcription quantitative real-time PCR (RT-qPCR) and western blot were used to measure the specific mRNA and protein levels, respectively. Follicular fluid and serum were collected from 65 infertile women during IVF treatment. Pearson's correlation analysis was performed to examine the correlation coefficient between two values. MAIN RESULTS AND THE ROLE OF CHANCE: Treatment of hGL cells with AREG-stimulated aromatase expression and E2 production. Using pharmacological inhibitors and specific siRNAs, we revealed that AREG-stimulated aromatase expression and E2 production via EGFR-mediated activation of the protein kinase B (AKT) signaling pathway. In addition, inhibition of EGFR activity and AREG knockdown attenuated hCG-induced up-regulation of aromatase expression and E2 production. Importantly, the protein levels of AREG in the follicular fluid were positively correlated with the E2 levels in serum after 2 days of oocyte pick-up and in the follicular fluid of IVF patients. LARGE-SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: The in vitro setting of this study is a limitation that may not reflect the real intra-ovarian microenvironment. Clinical data were obtained from a small sample size. WIDER IMPLICATIONS OF THE FINDINGS: Our results provide the first evidence that hCG-induced AREG contributes to aromatase expression and E2 production in the luteal phase of the menstrual cycle. A better understanding of the hormonal regulation of female reproductive function may help to develop new strategies for the treatment of clinical infertility. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the National Natural Science Foundation of China for Young Scientists (81601253), the specific fund of clinical medical research of Chinese Medical Association (16020160632) and the Foundation from the First Affiliated Hospital of Zhengzhou University for Young Scientists to Lanlan Fang. This work was also supported by an operating grant from the National Natural Science Foundation of China (81820108016) to Ying-Pu Sun. All authors declare no conflict of interest.

Expression and characterization of the cyp19a gene and its responses to estradiol/letrozole exposure in Chinese soft-shelled turtle (Pelodiscus sinensis).

Cytochrome P450 aromatase (CYP19) catalyzes the conversion of androgens to estrogens and is critical in sex differentiation. CYP19 exists as the ovarian type and brain type. Herein, we cloned the full-length ovarian cyp19a gene from the Chinese soft-shelled turtle, Pelodiscus sinensis (pscyp19a). We determined the distribution of pscyp19a in adult tissue and evaluated its expression during embryonic development, following treatment with 17ß-estradiol (E2) or letrozole (LE). The pscyp19a complementary DNA is 2,285 bp in length and comprises a 1,512 bp open reading frame that encodes a protein of 503 AA. The nucleotide sequence and amino acid of pscyp19a shared significant identity with other vertebrate sequences. Expression of pscyp19a was high in the ovary (p < 0.01), and exhibited modest expression in the female brain and intestine. Expression of pscyp19a displayed significant differences between sexes during early embryo development stages; expression increased gradually during embryonic development in females, but the opposite trend was observed in males. Female embryos treated with different concentrations of E2 and LE displayed altered pscyp19a expression compared with untreated individuals, and E2 clearly induced pscyp19a expression. These results indicate that pscyp19a gene plays important roles in early developmental stages in Chinese soft-shelled turtle, and may assist future studies on sex differentiation and sex control in this and similar species.

An assessment of the multifactorial profile of steroid-metabolizing enzymes and steroid receptors in the eutopic endometrium during moderate to severe ovarian endometriosis.

BACKGROUND: Previous studies of expression profiles of major endometrial effectors of steroid physiology in endometriosis have yielded markedly conflicting conclusions, presumably because the relative effects of type of endometriosis, fertility history and menstrual cycle phases on the measured variables were not considered. In the present study, endometrial mRNA and protein levels of several effectors of steroid biosynthesis and action in patients with stage III-IV ovarian endometriosis (OE) with known fertility and menstrual cycle histories were compared with the levels in control endometrium to test this concept. METHODS: Endometrial samples were collected from patients without endometriosis (n = 32) or OE stages III-IV (n = 52) with known fertility and cycle histories. qRT-PCR and immunoblotting experiments were performed to measure levels of NR5A1, STAR, CYP19A1, HSD17Bs, ESRs and PGR transcripts and proteins, respectively. Tissue concentrations of steroids (P4, T, E1 and E2) were measured using ELISAs. RESULTS: The levels of expression of aromatase and ERß were lower (P < 0.0001) and 17ß-HSD1 (P < 0.0001) and PRA (P < 0.01) were higher in OE endometrium. Lower aromatase levels and higher 17ß-HSD1 levels were detected in fertile (aromatase: P < 0.05; 17ß-HSD1: P < 0.0001) and infertile (aromatase: P < 0.0001; 17ß-HSD1: P < 0.0001) OE endometrium than in the matched control tissues. Both proliferative (PP) and secretory (SP) phase OE samples expressed aromatase (P < 0.0001) and ERß (PP: P < 0.001; SP: P < 0.01) at lower levels and 17ß-HSD1 (P < 0.0001) and PRA (PP: P < 0.01; SP: P < 0.0001) at higher levels than matched controls. Higher 17ß-HSD1 (P < 0.01) and E2 (P < 0.05) levels and a lower (P < 0.01) PRB/PRA ratio was observed in infertile secretory phase OE endometrium than in control. CONCLUSIONS: We report that dysregulated expression of 17ß-HSD1 and PGR resulting in hyperestrogenism and progesterone resistance during the secretory phase of the menstrual cycle, rather than an anomaly in aromatase expression, was the hallmark of eutopic endometrium from infertile OE patients. Furthermore, the results provide proof of concept that the fertility and menstrual cycle histories exerted relatively different effects on steroid physiology in the endometrium from OE patients compared with the control subjects.

A novel GC-MS methodology to evaluate aromatase activity in human placental microsomes: a comparative study with the standard radiometric assay.

Estrogens are key factors in the development of the estrogen receptor-positive (ER+) breast cancer. Estrogens, estrone (E1), and estradiol (E2) production is achieved by aromatase, a cytochrome P450 enzyme that has androgens, androstenedione (AD), and testosterone (T) as substrates. Nowadays, third-generation aromatase inhibitors (AIs) are considered the gold-standard treatment for ER+ breast cancer in postmenopausal women as well as in premenopausal women with ovary ablation. Aromatase activity assessment still relies on radiometric assays that are expensive, hazardous, and non-environmentally friendly. Thus, in order to overcome these disadvantages, a new methodology was developed to evaluate aromatase activity, based on dispersive liquid-liquid microextraction (DLLME) followed by gas chromatography-mass spectrometry (GC-MS). The enzymatic reaction was carried out in human placental microsomes, using AD as substrate, and the anti-aromatase activity was measured by determining the conversion percentage of AD into E1 (ratio E1/AD) using isotopic analogues as internal standards. The method showed good linearity (r2 = 0.9908 for AD and 0.9944 for E1), high accuracy (more than 74% for AD and more than 66% for E1), high extraction efficiency, and good intra-day and inter-day precision (below 14%, 4 levels). In this work, the IC50 values of the third-generation AIs, anastrozole, letrozole, and exemestane, obtained from the radiometric assay are also compared, and similar IC50 values are described. This method is a good alternative to the current radiometric assay, being fast and sensitive with a good extraction efficiency, accuracy, and recovery. In addition, it may be applied for the evaluation of the anti-aromatase activity of new potential AIs. Graphical abstract.

In vitro screening and isolation of human aromatase inhibitors from Cicer arietinum by a novel continuous online method combining chromatographic techniques.

Ultrafiltration liquid chromatography with mass spectrometry can efficiently and rapidly screen and identify ligands from the seeds of Cicer arietinum for human aromatase. Using this method, we identified 11 major compounds, including organic acids, organic acid glycosides, flavone glycosides, isoflavones, and isoflavone glycosides, as potent human aromatase inhibitors. A continuous online method, including pressurized liquid extraction, countercurrent chromatography, and preparative liquid chromatography, was developed for scaling up the production of these compounds with high purity and efficiency. The bioactivity of the separated compounds was assessed by an in vitro enzyme inhibition assay. This novel approach using a combination of ultrafiltration liquid chromatography with mass spectrometry and pressurized liquid extraction with countercurrent chromatography and preparative liquid chromatography as well as an in vitro enzyme inhibition assay could be applied to efficiently screen and isolate human aromatase inhibitors from complex samples and to the large-scale production of functional food and nutraceutical ingredients.

Differences in aromatase expression and steroid hormone concentrations in the reproductive tissues of male domestic turkeys (Meleagris gallopavo) with white and yellow semen.

1. To show hormonal differences between male turkeys with yellow semen syndrome (YSS) and white, normal semen (WNS), the expression of aromatase, oestrogen receptor α (ERα), and oestrogen receptor ß (ERß) as well as testosterone and oestradiol concentrations in YSS and WNS testes, epididymis, and ductus deferens were examined. 2. To measure gene expression levels of aromatase and oestrogen receptors (ERs), three complementary techniques (real-time PCR, Western blot, and immunohistochemistry) were used, whereas steroid hormone levels were determined radio-immunologically. 3. Upregulation of aromatase and ERα mRNAs in YSS testes (P < 0.05; P < 0.01), epididymis (P < 0.001; P < 0.001), and ductus deferens (P < 0.05; P < 0.01) compared to those of WNS tissues was detected. Significant increases in the levels of aromatase and ERα proteins were detected in YSS testes (P < 0.001; P < 0.05), epididymis (P < 0.001; P < 0.001), and ductus deferens (P < 0.001; P < 0.05). The expression of ERß mRNA and protein level was upregulated in the testes (P < 0.05; P < 0.01) and epididymis (P < 0.001; P < 0.01) but not in ductus deferens where it was downregulated (P < 0.01; P < 0.01). Increased intensity of immunoreactive proteins in YSS versus WNS reproductive tissues corroborated gene expression results. 4. Testosterone concentration diminished in YSS epididymis (P < 0.05) and ductus deferens (P < 0.05), but not in the testes, remaining at high level (P < 0.05) compared to WNS values. Concomitantly, increased oestradiol concentration was found in YSS testes (P < 0.05) and epididymis (P < 0.05) but decreased in the ductus deferens (P < 0.05). 5. From the published literature, this study is the first to demonstrate the ability for androgen aromatisation in the turkey reproductive tissues and to show the cellular targets for locally produced oestrogens. The data suggested that the androgen/oestrogen ratio is a mechanistic basis for amplification of differences between turkeys with white and yellow semen and that these results can have a relevance in applied sciences to widen the knowledge on domestic bird reproduction.

Abnormal lipid/lipoprotein metabolism and high plasma testosterone levels in male but not female aromatase-knockout mice.

Sex steroid hormones, such as estrogen and testosterone, are believed to play important roles in lipid metabolism. To elucidate the effects of estrogen depletion on lipid metabolism in male and female mice, we used aromatase-knockout (ArKO) mice, in which Cyp19 gene disruption prevented estrogen synthesis in vivo. These mice were divided into the following 4 groups: male and female ArKO mice and male and female wild-type (WT) mice. These mice were fed a normal-fat diet (13.6% fat) ad libitum. At 159 days after birth, the mice were tested for liver and plasma lipid content and hepatic hormone receptor- and lipid/lipoprotein metabolism-related gene expression. Interestingly, we found that hepatic steatosis was accompanied by markedly elevated plasma testosterone levels in male ArKO mice but not in female ArKO mice. Plasma lipoprotein profiles exhibited concurrent decreases in LDL- and small dense LDL-triglyceride (TG) levels in male ArKO mice. Moreover, male mice, but not female mice, exhibited marked elevations in androgen receptor (AR), sterol regulatory element-binding protein 1 (SREBP1), and CD36 expression. These results strongly suggest that Cyp19 gene disruption, which induces a sexually dimorphic response and high plasma testosterone levels in male mice, also induces hepatic steatosis.

Combination of the non-invasive tests for the diagnosis of endometriosis.

BACKGROUND: About 10% of women of reproductive age suffer from endometriosis, a costly chronic disease causing pelvic pain and subfertility. Laparoscopy is the gold standard diagnostic test for endometriosis, but is expensive and carries surgical risks. Currently, there are no non-invasive tests available in clinical practice to accurately diagnose endometriosis. This review assessed the diagnostic accuracy of combinations of different non-invasive testing modalities for endometriosis and provided a summary of all the reviews in the non-invasive tests for endometriosis series. OBJECTIVES: To estimate the diagnostic accuracy of any combination of non-invasive tests for the diagnosis of pelvic endometriosis (peritoneal and/or ovarian or deep infiltrating) compared to surgical diagnosis as a reference standard. The combined tests were evaluated as replacement tests for diagnostic surgery and triage tests to assist decision-making to undertake diagnostic surgery for endometriosis. SEARCH METHODS: We did not restrict the searches to particular study designs, language or publication dates. We searched CENTRAL to July 2015, MEDLINE and EMBASE to May 2015, as well as the following databases to April 2015: CINAHL, PsycINFO, Web of Science, LILACS, OAIster, TRIP, ClinicalTrials.gov, DARE and PubMed. SELECTION CRITERIA: We considered published, peer-reviewed, randomised controlled or cross-sectional studies of any size, including prospectively collected samples from any population of women of reproductive age suspected of having one or more of the following target conditions: ovarian, peritoneal or deep infiltrating endometriosis (DIE). We included studies comparing the diagnostic test accuracy of a combination of several testing modalities with the findings of surgical visualisation of endometriotic lesions. DATA COLLECTION AND ANALYSIS: Three review authors independently collected and performed a quality assessment of the data from each study by using the QUADAS-2 tool. For each test, the data were classified as positive or negative for the surgical detection of endometriosis and sensitivity and specificity estimates were calculated. The bivariate model was planned to obtain pooled estimates of sensitivity and specificity whenever sufficient data were available. The predetermined criteria for a clinically useful test to replace diagnostic surgery were a sensitivity of 0.94 and a specificity of 0.79 to detect endometriosis. We set the criteria for triage tests at a sensitivity of 0.95 and above and a specificity of 0.50 and above, which 'rules out' the diagnosis with high accuracy if there is a negative test result (SnOUT test), or a sensitivity of 0.50 and above and a specificity of 0.95 and above, which 'rules in' the diagnosis with high accuracy if there is a positive result (SpIN test). MAIN RESULTS: Eleven eligible studies included 1339 participants. All the studies were of poor methodological quality. Seven studies evaluated pelvic endometriosis, one study considered DIE and/or ovarian endometrioma, two studies differentiated endometrioma from other ovarian cysts and one study addressed mapping DIE at specific anatomical sites. Fifteen different diagnostic combinations were assessed, including blood, urinary or endometrial biomarkers, transvaginal ultrasound (TVUS) and clinical history or examination. We did not pool estimates of sensitivity and specificity, as each study analysed independent combinations of the non-invasive tests.Tests that met the criteria for a replacement test were: a combination of serum IL-6 (cut-off >15.4 pg/ml) and endometrial PGP 9.5 for pelvic endometriosis (sensitivity 1.00 (95% confidence interval (CI) 0.91 to 1.00), specificity 0.93 (95% CI, 0.80, 0.98) and the combination of vaginal examination and transvaginal ultrasound (TVUS) for rectal endometriosis (sensitivity 0.96 (95% CI 0.86 to 0.99), specificity 0.98 (95% CI 0.94 to 1.00)). Tests that met the criteria for SpIN triage tests for pelvic endometriosis were: 1. a multiplication of urine vitamin-D-binding protein (VDBP) and serum CA-125 (cut-off >2755) (sensitivity 0.74 (95% CI 0.60 to 0.84), specificity 0.97 (95% CI 0.86 to 1.00)) and 2. a combination of history (length of menses), serum CA-125 (cut-off >35 U/ml) and endometrial leukocytes (sensitivity 0.61 (95% CI 0.54 to 0.69), specificity 0.95 (95% CI 0.91 to 0.98)). For endometrioma, the following combinations qualified as SpIN test: 1. TVUS and either serum CA-125 (cut-off ≥25 U/ml) or CA 19.9 (cut-off ≥12 U/ml) (sensitivity 0.79 (95% CI 0.64 to 0.91), specificity 0.97 (95% CI 0.91 to 1.00)); 2. TVUS and serum CA 19.9 (cut-off ≥12 U/ml) (sensitivity 0.54 (95% CI 0.37 to 0.70), specificity 0.97 (95% CI 0.91 to 1.0)); 3-4. TVUS and serum CA-125 (cut-off ≥20 U/ml or cut-off ≥25 U/ml) (sensitivity 0.69 (95% CI 0.49 to 0.85), specificity 0.96 (95% CI 0.88 to 0.99)); 5. TVUS and serum CA-125 (cut-off ≥35 U/ml) (sensitivity 0.52 (95% CI 0.33 to 0.71), specificity 0.97 (95% CI 0.90 to 1.00)). A combination of vaginal examination and TVUS reached the threshold for a SpIN test for obliterated pouch of Douglas (sensitivity 0.87 (95% CI 0.69 to 0.96), specificity 0.98 (95% CI 0.95 to 1.00)), vaginal wall endometriosis (sensitivity 0.82 (95% CI 0.60 to 0.95), specificity 0.99 (95% CI 0.97 to 1.0)) and rectovaginal septum endometriosis (sensitivity 0.88 (95% CI 0.47 to 1.00), specificity 0.99 (95% CI 0.96 to 1.00)).All the tests were evaluated in individual studies and displayed wide CIs. Due to the heterogeneity and high risk of bias of the included studies, the clinical utility of the studied combination diagnostic tests for endometriosis remains unclear. AUTHORS' CONCLUSIONS: None of the biomarkers evaluated in this review could be evaluated in a meaningful way and there was insufficient or poor-quality evidence. Laparoscopy remains the gold standard for the diagnosis of endometriosis and using any non-invasive tests should only be undertaken in a research setting.

Differential expression patterns of three aromatase genes and of four estrogen receptors genes in the testes of trout (Oncorhynchus mykiss).

Estrogens are implicated in male gonad function, although their physiological roles remain uncertain. In the present study, we take advantage of the original model of spatio-temporal organization of trout spermatogenesis to revisit the synthesis and action sites of estrogens in fish testis. Within this system, somatic cell and germ cell development are synchronized due to a strict seasonal spermatogenetic cycle and the cystic organization of gonads. We evaluated the expression patterns and regulation of three aromatase isoforms (cyp19a, cyp19b-I, and cyp19b-II) and four estrogen receptors (esr1a, esr1b, esr2a, and esr2b) by quantitative reverse-transcriptase PCR during testicular maturation and in isolated germ cell populations. Our data demonstrated a reciprocal relationship between cyp19a and cyp19b (I and II) expression during testicular development (cyp19a decreased while cyp19b increased with maturation). Furthermore, cyp19b is significantly expressed in late germ cells. At the protein level, aromatase was immunohistochemically identified in interstitial tissue and in germ cells. Remarkable elevation of esr1a and esr2a was observed during the final stage of spermiation, while esr1b was expressed in an early stage of spermatogenetic development. Estrogen implants reduced testicular cyp19a transcript abundance while up-regulating cyp19b levels, whereas androgens up-regulated testicular esr1a, esr2a, and esr2b. Together, the distinct spatio-temporal expression profiles and regulation of aromatases and estrogen receptors suggest that estrogens have discrete physiological functions during an early step of spermatogenesis and in the final stages of germ cell maturation and/or excretion.

Long-Term Outcome of Aromatase Inhibitor Therapy With Letrozole in Patients With Advanced Low-Grade Endometrial Stromal Sarcoma.

BACKGROUND: There has been no consensus on the indications for the treatment of advanced low-grade endometrial stromal sarcoma (LGESS), and the possible effects of hormonal treatment including progestins and aromatase inhibitors have been reported. The aim of this study was to investigate the efficacy of aromatase inhibitor therapy with letrozole for patients with residual or recurrent LGESS. METHODS: We retrospectively reviewed the clinical response of patients with advanced LGESS who had been treated with letrozole. We also analyzed the adverse effects after the administration of letrozole. The expression levels of estrogen receptor and aromatase in the tumors were immunohistochemically examined. RESULTS: In 5 patients who had been treated for unresectable LGESS lesions after initial or repeat surgical procedures, residual lesions in 3 patients and recurrence lesions in 2 patients were the indications for hormonal therapy with letrozole. The median duration of letrozole exposure at retrospective analysis was 53 (10-96) months. The clinical outcomes were classified as complete response in 2 patients, partial response in 1 patient, and stable disease in 2 patients. Myalgias, hot flashes, and arthralgias were not observed during the follow-up period in any patients. The median serum levels of estradiol were <5.0 (cutoff value, <0.5-11.8) pg/mL. The median age-matched bone mineral densities were 92% (79%-123%). The LGESS tissues in all 5 patients were positive for estrogen receptor and aromatase expression. CONCLUSIONS: Letrozole as well as progestins could be the first choice of treatment for patients with recurrent or residual LGESS, which is difficult to resect surgically because of its efficacy and minimal adverse effects.

Alginate hydrogel matrix stiffness influences the in vitro development of caprine preantral follicles.

This study examined caprine follicular development in different concentrations of alginate matrix to determine the optimal conditions for culture. Caprine preantral follicles were cultured in a two-dimensional system (control) or a three-dimensional encapsulated system in 0.25%, 0.5%, or 1% alginate (ALG 0.25, ALG 0.5, and ALG 1, respectively). A higher percentage of morphologically normal follicles developed in ALG 0.5 and ALG 1 than in ALG 0.25 or the control (P < 0.05). The rate of antrum formation, however, was higher in ALG 0.25 than in ALG 0.5 and ALG 1 conditions (P < 0.05), but similar to the control. Follicles cultured in ALG 0.25 had higher growth rates and meiotic resumption than those cultured in ALG 0.5, ALG 1, or the control (P < 0.05). Moreover, follicles cultured in ALG 0.25 had higher levels of estradiol and progesterone than those cultured in ALG 0.5, ALG 1, or the control, as well as higher levels of CYP19A1 and HSD3B mRNA. In conclusion, a three-dimensional system that uses ALG 0.25 fosters the in vitro development of caprine preantral follicles and increases the rate of meiotic resumption.

Gestational methyl donor deficiency alters key proteins involved in neurosteroidogenesis in the olfactory bulbs of newborn female rats and is associated with impaired olfactory performance.

Gestational methyl donor deficiency (MDD) leads to growth retardation as well as to cognitive and motor disorders in 21-d-old rat pups. These disorders are related to impaired neurogenesis in the cerebral neurogenic areas. Olfactory bulbs (OB), the main target of neuronal progenitors originating from the subventricular zone, play a critical role during the postnatal period by allowing the pups to identify maternal odour. We hypothesised that growth retardation could result from impaired suckling due to impaired olfactory discrimination through imbalanced apoptosis/neurogenesis in the OB. Since neurosteroidogenesis modulates neurogenesis in OB, in the present study, we investigated whether altered neurosteroidogenesis could explain some these effects. Pups born to dams fed a normal diet (n 24) and a MDD diet (n 27) were subjected to olfactory tests during the lactation and weaning periods (n 24 and 20, respectively). We studied the markers of apoptosis/neurogenesis and the expression levels of the key neurosteroidogenic enzyme aromatase, the cholesterol-transfer protein StAR (steroidogenic acute regulatory protein) and the ERα oestrogen receptor and the content of oestradiol in OB. The 21-d-old MDD female pups displayed lower body weight and impaired olfactory discrimination when compared with the control pups. MDD led to greater homocysteine accumulation and more pronounced apoptosis, along with impaired cell proliferation in the OB of female pups. The expression levels of aromatase, StAR and ERα as well as the content of oestradiol were lower in the OB of the MDD female pups than in those of the control female pups. In conclusion, gestational MDD may alter olfactory discrimination performances by affecting neurogenesis, apoptosis and neurosteroidogenesis in OB in a sex-dependent manner. It may be involved in growth retardation through impaired suckling.

Correlations among antral follicular echotexture, apoptosis and expression of key steroidogenic enzymes in sheep.

Nineteen cycling ewes underwent transrectal ultrasonography of ovaries followed by ovariectomies during the growth phase of the first follicular wave of the interovulatory interval or the proestrus/estrus phase of the cycle. Quantitative ultrasonographic characteristics of the antrum and follicular wall in a total of forty-three ovine antral follicles were examined for correlations with the protein expression of three steroidogenic enzymes (cytochrome P450 17α-hydroxylase, CYP17; cytochrome P450 aromatase, CYP19; and 3ß-hydroxysteroid dehydrogenase, 3ß-HSD) determined by densitometric analysis of immunohistochemical slides, follicular dimensions, granulosa layer thickness and the percentage of apoptotic granulosa cells. Significant correlations were found between echotextural attributes of ovine antral follicles and the percentage of apoptotic granulosa cells, CYP17 expression (theca), CYP19 expression (granulosa) and 3ß-HSD expression (theca cells). Computer-aided analyses of ultrasonographic images can be beneficial to the development of assisted reproductive technologies and diagnosis of hormonal imbalances without the need for ovarian biopsies or hormone assays.

Endocrine and inflammatory factors and endometriosis-associated infertility in assisted reproduction techniques.

PURPOSE: Our research aimed to evaluate the effect of endometriosis on folliculogenesis and pregnancy, and to assess the involvement of inflammatory factors (IL1b, PGE2, PGF2α, and TGFß2) in follicular fluid. METHODS: A total of 65 follicular fluid aspirates were collected. Concentrations of inflammatory factors (IL1b, PGE2, PGF2α, and TGFß2) and steroid hormones (E2, progesterone, FSH, and LH) within follicular fluid as well as serum E2 and LH concentrations were measured. The mRNA expression of IL1b, Ptgs2, aromatase, and PPARγ in granulosa cells was determined. The outcome of ART was monitored and recorded. RESULTS: The oocyte retrieval, rate of metaphase II oocyte, cleavage rate, effective embryo rate, and pregnancy rates of patients with endometriosis were all significantly lower than those of the control patients. In those with endometriosis, serum E2 concentrations were lower than those observed in controls. Aromatase levels in the granulosa cells of the endometriosis group were lower while concentrations of PGE2 in follicular fluid were higher than in the control group. Concentrations of PGE2, PGF2α, TGFß2, and IL1b were significantly correlated with each other. CONCLUSIONS: These results suggest that the outcomes of ART, in relation to serum E2 concentration, were adversely affected by the presence of endometriosis. Furthermore, the results supported that, among the endocrine and inflammatory factors, PGE2 within the follicular fluid impairs the number and quality of oocytes.

The effects of insulin and follicle-simulating hormone (FSH) during in vitro development of ovarian goat preantral follicles and the relative mRNA expression for insulin and FSH receptors and cytochrome P450 aromatase in cultured follicles.

The actions of different concentrations of insulin alone or in combination with follicle-stimulating hormone (FSH) were evaluated by in vitro follicular development and mRNA expression of cytochrome P450 aromatase (CYP19A1) and as receptors for insulin (INSR) and FSH (FSHR) from isolated, cultured goat preantral follicles. Goat preantral follicles were microdissected and cultured for 18 days in the absence or presence of insulin (5 and 10 ng/ml or 10 µg/ml) alone or in combination with FSH. After 18 days, the addition of the maximum concentration of insulin to the culture medium reduced follicular survival and antrum formation rates significantly compared to the other treatments. However, when FSH was added to the culture medium, no differences between these two parameters were observed. Preantral and antral follicles from the fresh control as well as from all cultured follicles still presented a normal ultrastructural pattern. In medium supplemented with FSH, only insulin at 10 ng/ml presented oocytes with higher rates of meiosis resumption compared to control, as well as oocytes in metaphase II. Treatment with insulin (10 ng/ml) plus FSH resulted in significantly increased levels of INSR and CYP19A1 mRNA compared to that with other treatments. In conclusion, 10 ng/ml insulin associated with FSH was more efficient in promoting resumption of oocyte meiosis, maintaining survival, stimulating follicular development, and increasing expression of the INSR and CYP19A1 genes in goat preantral follicles.

Aromatase expression in human peripheral blood leucocytes (PBLs) and in various tissues in primates: studies in elderly humans and cynomolgus monkeys.

BACKGROUND: Previous analysis of aromatase gene and protein expression in peripheral blood leucocytes (PBLs), studied in children and adults, was extended to elderly subjects. In addition, we assessed whether aromatase expression in PBLs could be used as a parameter of aromatase expression in other tissues, using the cynomolgus monkey as model. METHODS: Real-time PCR analysis of aromatase gene expression and protein evaluation by Western blot was performed in PBLs of human elderly subjects and in various tissues from cynomolgus monkeys. RESULTS: No gender-related difference in CYP19A1 mRNA and protein expression in PBLs from human elderly women and men was found. In elderly male cynomolgus monkeys, CYP19A1 mRNA and protein were expressed in all cells and tissues analysed, with the lowest levels in PBLs but no clear-cut correlation with other tissues. CONCLUSIONS: Aromatase expression in PBLs in elderly human subjects is not gender-related and cannot be a surrogate of aromatase expression for other tissues.

Aromatase immunoreactivity is increased in mammographically dense regions of the breast.

Mammographic breast density (MBD) is one of the strongest risk factors for breast cancer. Unfortunately, the biologic basis underlying this association is unknown. This study compared aromatase expression or immunoreactivity (IR) in core biopsies from mammographically dense versus non-dense regions of the breast to examine whether estrogen synthesis in the breast is associated with MBD and one possible mechanism through which MBD may influence breast cancer. Eligible participants were 40+ years, had a screening mammogram with visible MBD and no prior cancer or current endocrine therapy. Mammograms were used to identify dense and non-dense regions and ultrasound-guided core biopsies were performed to obtain tissue from these regions. Immunostaining for aromatase employed the streptavidin-biotin amplification method and #677 mouse monoclonal antibody. Aromatase IR was scored in terms of extent and intensity of staining for each cell type (stroma, epithelium, adipocytes) on histologic sections. A modified histological H-score provided quantitation of aromatase IR in each cell type and overall. Repeated measure analyses evaluated average differences (ß(H)) in H-score in dense versus non-dense tissue within and across cell types. Forty-nine women with mean age 50 years (range: 40-82), participated. Aromatase IR was increased in dense (vs. non-dense) tissue in both the stroma (ß(H) = 0.58) and epithelium (ß(H) = 0.12) (P < 0.01). Adipocytes from non-dense tissue, however, had a greater IR compared to those from dense tissue (ß(H) = -0.24, P < 0.01). An overall H-score which integrated results from all cell types demonstrated that aromatase IR was twice as great for dense (mean H-score = 0.90, SD = 0.53) versus non-dense (mean H-score = 0.45, SD = 0.39) breast tissue (ß(H) = 0.45; P < 0.001). Overall, aromatase IR was greater for mammographically dense versus non-dense tissue and may partly explain how MBD influences breast cancer.

Methoxychlor reduces estradiol levels by altering steroidogenesis and metabolism in mouse antral follicles in vitro.

The organochlorine pesticide methoxychlor (MXC) is a known endocrine disruptor that affects adult rodent females by causing reduced fertility, persistent estrus, and ovarian atrophy. Since MXC is also known to target antral follicles, the major producer of sex steroids in the ovary, the present study was designed to test the hypothesis that MXC decreases estradiol (E2) levels by altering steroidogenic and metabolic enzymes in the antral follicles. To test this hypothesis, antral follicles were isolated from CD-1 mouse ovaries and cultured with either dimethylsulfoxide (DMSO) or MXC. Follicle growth was measured every 24 h for 96 h. In addition, sex steroid hormone levels were measured using enzyme-linked immunosorbent assays (ELISA) and mRNA expression levels of steroidogenic enzymes as well as the E2 metabolic enzyme Cyp1b1 were measured using qPCR. The results indicate that MXC decreased E2, testosterone, androstenedione, and progesterone (P4) levels compared to DMSO. In addition, MXC decreased expression of aromatase (Cyp19a1), 17ß-hydroxysteroid dehydrogenase 1 (Hsd17b1), 17α-hydroxylase/17,20-lyase (Cyp17a1), 3ß hydroxysteroid dehydrogenase 1 (Hsd3b1), cholesterol side-chain cleavage (Cyp11a1), steroid acute regulatory protein (Star), and increased expression of Cyp1b1 enzyme levels. Thus, these data suggest that MXC decreases steroidogenic enzyme levels, increases metabolic enzyme expression and this in turn leads to decreased sex steroid hormone levels.