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.

Expression of cytochrome P450 aromatase isoforms in female Alburnus tarichi (Guldenstaedtii, 1814)

In this study, we aimed to clone brain-derived Cyp19b and ovary-derived Cyp19a the P450 aromatase gene isoforms and to indicate the expression levels of these genes in the hypothalamus and ovary tissues from reproductively arrested ovarian development (RA) and non-arrested ovarian development (RN) Alburnus tarichi from Lake Van, Turkey. The expression levels of Cyp19b and Cyp19a genes were predominant in the hypothalamus and ovary, respectively. The level of Cyp19b mRNA was significantly lower in the hypothalamus and ovary from RA fish than in the hypothalamus and ovary from RN fish (P<0.05). The expression level of Cyp19a was significantly lower in the ovary of RA fish (P<0.05) than RN fish while no difference was found in the hypothalamus of both RA and RN fish (P<0.05). According to these findings, we suggest that the RA fish represent a segment of the population and these fish may be more sensitive to endocrine disruption compound/s than others.

Aromatase and the three little paralogs

The rapid progress of scientific knowledge is continuously built on established earlier findings. Integrating previous observations with more recent data can be challenging and occasionally well-established data is unfortunately overlooked. This may lead to unnecessary redundancy in research and, in the worst-case scenario, to misinterpretation of data. In the present contribution we would like to draw attention to such a circumstance, which has crept into several recent papers on expression and functional relevance of the porcine CYP19 gene cluster that encodes functionally distinct isoforms of aromatase cytochrome P450 (aromatase) in pigs.

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.

Electromagnetic Field (EMF) Radiation Alters Estrogen Release from the Pig Myometrium during the Peri-Implantation Period

An electromagnetic field (EMF) may affect the functions of uterine tissues. This study hypothesized that EMF changes the estrogenic activity of pig myometrium during the peri-implantation period. Tissue was collected on days 15–16 of the gestation and incubated in the presence of EMF (50 and 120 Hz, 2 and 4 h). The cytochrome P450 aromatase type 3 (CYP19A3) and hydroxysteroid 17β dehydrogenase type 4 (HSD17B4) mRNA transcript abundance, cytochrome P450arom (aromatase), and 17β hydroxysteroid dehydrogenase 17βHSD) protein abundance and estrone (E1) and estradiol-17β (E2) release were examined using Real-Time PCR, Western blot and radioimmunoassay. Selected myometrial slices were treated with progesterone (P4) to determine whether it functions as a protector against EMF. CYP19A3 mRNA transcript abundance in slices treated with EMF was less at 50 Hz (2 h) and greater at 120 Hz (2 and 4 h). HSD17B4 mRNA transcript was greater in slices treated with EMF at 120 Hz (2 h). Progesterone diminished EMF-related effects on CYP19A3 and HSD17B4. When P4 was added, EMF had suppressive (50 and 120 Hz, 2 h) or enhancing (50 Hz, 4 h) effects on aromatase abundance. The E1 release was lower after 4 h of EMF treatment at 50 Hz and P4 did not protect myometrial E1 release. In conclusion, EMF alters the synthesis and release of E1 and did not affect E2 release in the myometrium during the peri-implantation period.

Molecular and Structural Evolution of Cytochrome P450 Aromatase

Aromatase is the cytochrome P450 enzyme converting androgens into estrogen in the last phase of steroidogenesis. As estrogens are crucial in reproductive biology, aromatase is found in vertebrates and the invertebrates of the genus Branchiostoma, where it carries out the aromatization reaction of the A-ring of androgens that produces estrogens. Here, we investigate the molecular evolution of this unique and highly substrate-selective enzyme by means of structural, sequence alignment, and homology modeling, shedding light on its key role in species conservation. The alignments led to the identification of a core structure that, together with key and unique amino acids located in the active site and the substrate recognition sites, has been well conserved during evolution. Structural analysis shows what their roles are and the reason why they have been preserved. Moreover, the residues involved in the interaction with the redox partner and some phosphorylation sites appeared late during evolution. These data reveal how highly substrate-selective cytochrome P450 has evolved, indicating that the driving forces for evolution have been the optimization of the interaction with the redox partner and the introduction of phosphorylation sites that give the possibility of modulating its activity in a rapid way.

11-oxygenated estrogens are a novel class of human estrogens but do not contribute to the circulating estrogen pool

Androgens are the obligatory precursors of estrogens. In humans, classic androgen biosynthesis yields testosterone, thought to represent the predominant circulating active androgen in both men and women. However, recent work has shown that 11-ketotestosterone, derived from the newly described 11-oxygenated androgen biosynthesis pathway, makes a substantial contribution to the active androgen pool in women. Considering that classic androgens are the obligatory substrates for estrogen biosynthesis catalyzed by cytochrome P450 aromatase, we hypothesized that 11-oxygenated androgens are aromatizable. Here we utilize steroid analysis by tandem mass spectrometry to demonstrate that human aromatase generates 11-oxygenated estrogens from 11-oxygenated androgens in three different cell-based aromatase expression systems and in human ex vivo placenta explant cultures. We also show that 11-oxygenated estrogens are generated as a byproduct of the aromatization of classic androgens. We show that 11β-hydroxy-17β-estradiol binds and activates estrogen receptors α and β and that 11β-hydroxy-17β-estradiol and the classic androgen pathway-derived active estrogen, 17β-estradiol, are equipotent in stimulating breast cancer cell line proliferation and expression of estrogen-responsive genes. 11-oxygenated estrogens were, however, not detectable in serum from individuals with high aromatase levels (pregnant women) and elevated 11-oxygenated androgen levels (patients with congenital adrenal hyperplasia or adrenocortical carcinoma). Our data shows that while 11-oxygenated androgens are aromatizable in vitro and ex vivo, the resulting 11-oxygenated estrogens are not detectable in circulation, suggesting that 11-oxygenated androgens function primarily as androgens in vivo.