Bioaugmentation removal and microbiome analysis of the synthetic estrogen 17α-ethynylestradiol from hostile conditions and environmental samples by Pseudomonas citronellolis SJTE-3.

Synthetic estrogens are emerging environmental contaminants with great estrogenic activities and stable structures that are widespread in various ecological systems and significantly threaten the health of organisms. Pseudomonas citronellolis SJTE-3 is reported to degrade the synthetic estrogen 17α-ethynylestradiol (EE2) efficiently in laboratory conditions. In this work, the environmental adaptability, the EE2-degrading properties, and the ecological effects of P. citronellolis SJTE-3 under different hostile conditions (heavy metals and surfactants) and various natural environment samples (solid soil, lake water, and pig manure) were studied. Strain SJTE-3 can tolerate high concentrations of Zn2+ and Cr3+, but is relatively sensitive to Cu2+. Tween 80 of low concentration can significantly promote EE2 degradation by strain SJTE-3, different from the repressing effect of Triton X-100. High concentration of Tween 80 prolonged the lagging phase of EE2-degrading process, while the final EE2 removal efficiency was improved. More importantly, strain SJTE-3 can grow normally and degrade estrogen stably in various environmental samples. Inoculation of strain SJTE-3 removed the intrinsic synthetic and natural estrogens (EE2 and estrone) in lake water samples in 4 days, and eliminated over 90% of the amended 1 mg/L EE2 in 2 days. Bioaugmentation of strain SJTE-3 in EE2-supplied solid soil and pig manure samples achieved a removal rate of over 55% and 70% of 1 mg/kg EE2 within 2 weeks. Notably, the bioaugmentation of extrinsic strain SJTE-3 had a slight influence on indigenous bacterial community in pig manure samples, and its relative abundance decreased significantly after EE2 removal. Amendment of EE2 or strain SJTE-3 in manure samples enhanced the abundance of Proteobacteria and Actinobacteria, implying their potential in utilizing EE2 or its metabolites. These findings not only shed a light on the environment adaptability and degradation efficiency of strain SJTE-3, but also provide insights for bioremediation application in complex and synthetic estrogen polluted environments.

Biodegradation of synthetic estrogen using bioelectrochemical system and degradation pathway analysis through Quadrupole-time-of-flight-mass spectrometry.

Synthetic estrogenic compounds such as 17α-ethinylestradiol (EE2) are significant environmental contaminants. This research studied the biodegradation of EE2 utilizing the EE2 adapted cells isolated from a dairy farm waste site in suspension flask vis-a-vis Bioelectrochemical System (BES) and compared the power output in the BES with and without EE2 as a co-substrate. 78% removal of EE2 was observed in the BES as against 60% removal in suspension flasks. The maximum power density in the BES increased about 53% when EE2 is used as a co-substrate. The EE2 biodegradation studied using HPLC and Q-TOF methods, also proposes a hypothetical pathway for EE2 degradation by the newly isolated strain Rhodopseudomonas palustris MDOC01 and reports the significant metabolites like nicotinic acid and oxoproline being detected during bioelectrochemical treatment process of EE2. Study also suggests that Plasma peroxide treatment of anode material enhanced the overall performance in terms of biodegradation efficiency and power output.

The adsorption, kinetics, and interaction mechanisms of various types of estrogen on electrospun polymeric nanofiber membranes.

This study focuses on the adsorption kinetics of four highly potent sex hormones (estrone (E1), 17ß-estradiol (E2), 17α-ethinylestradiol (EE2), and estriol (E3)), present in water reservoirs, which are considered a major cause of fish feminization, low sperm count in males, breast and ovarian cancer in females induced by hormonal imbalance. Herein, electrospun polymeric nanostructures were produced from cellulose acetate, polyamide, polyethersulfone, polyurethanes (918 and elastollan), and polyacrylonitrile (PAN) to simultaneously adsorbing these estrogenic hormones in a single step process and to compare their performance. These nanofibers possessed an average fiber diameter in the range 174-330 nm and their specific surface area ranged between 10.2 and 20.9 m2g-1. The adsorption-desorption process was investigated in four cycles to determine the effective reusability of the adsorption systems. A one-step high-performance liquid chromatography technique was developed to detect and quantify concurrently each hormone present in the solution. Experimental data were obtained to determine the adsorption kinetics by applying pseudo-first-order, pseudo-second-order and intraparticle diffusion models. Findings showed that E1, E2 and EE2 best fitted pseudo-second-order kinetics, while E3 followed pseudo-first-order kinetics. It was found that polyurethane Elastollan nanofibers had maximum adsorption capacities of 0.801, 0.590, 0.736 and 0.382 mg g-1for E1, E2, EE2 and E3, respectively. In addition, the results revealed that polyurethane Elastollan nanofibers had the highest percentage efficiency of estrogens removal at ∼58.9% due to its strong hydrogen bonding with estrogenic hormones, while the least removal efficiency for PAN at ∼35.1%. Consecutive adsorption-desorption cycles demonstrated that polyurethane maintained the best efficiency, even after being repeatedly used four times compared to the other polymers. Overall, the findings indicate that all the studied nanostructures have the potential to be effective adsorbents for concurrently eradicating such estrogens from the environment.

Exposure to Endocrine Disrupting Chemicals and Risk of Breast Cancer.

Breast cancer (BC) is the second most common cancer and the fifth deadliest in the world. Exposure to endocrine disrupting pollutants has been suggested to contribute to the increase in disease incidence. Indeed, a growing number of researchershave investigated the effects of widely used environmental chemicals with endocrine disrupting properties on BC development in experimental (in vitro and animal models) and epidemiological studies. The complex effects of endocrine disrupting chemicals (EDCs) on hormonal pathways, involving carcinogenic effects and an increase in mammary gland susceptibility to carcinogenesis-together with the specific characteristics of the mammary gland evolving over the course of life and the multifactorial etiology of BC-make the evaluation of these compounds a complex issue. Among the many EDCs suspected of increasing the risk of BC, strong evidence has only been provided for few EDCs including diethylstilbestrol, dichlorodiphenyltrichloroethane, dioxins and bisphenol A. However, given the ubiquitous nature and massive use of EDCs, it is essential to continue to assess their long-term health effects, particularly on carcinogenesis, to eradicate the worst of them and to sensitize the population to minimize their use.

Dual Function of a Novel Bacterium, Slackia sp. D-G6: Detoxifying Deoxynivalenol and Producing the Natural Estrogen Analogue, Equol.

Deoxynivalenol (DON) is a highly abundant mycotoxin that exerts many adverse effects on humans and animals. Much effort has been made to control DON in the past, and bio-transformation has emerged as the most promising method. However, useful and effective application of bacterial bio-transformation for the purpose of inhibiting DON remains urgently needed. The current study isolated a novel DON detoxifying bacterium, Slackia sp. D-G6 (D-G6), from chicken intestines. D-G6 is a Gram-positive, non-sporulating bacterium, which ranges in size from 0.2-0.4 µm × 0.6-1.0 µm. D-G6 de-epoxidizes DON into a non-toxic form called DOM-1. Optimum conditions required for degradation of DON are 37-47 °C and a pH of 6-10 in WCA medium containing 50% chicken intestinal extract. Besides DON detoxification, D-G6 also produces equol (EQL) from daidzein (DZN), which shows high estrogenic activity, and prevents estrogen-dependent and age-related diseases effectively. Furthermore, the genome of D-G6 was sequenced and characterized. Thirteen genes that show potential for DON de-epoxidation were identified via comparative genomics. In conclusion, a novel bacterium that exhibits the dual function of detoxifying DON and producing the beneficial natural estrogen analogue, EQL, was identified.

Biotransformation of estrone, 17ß-estradiol and 17α-ethynylestradiol by four species of microalgae.

Natural and synthetic estrogens have been widely detected in wastewater treatment plant (WWTP) influent and effluent as well as in the corresponding receiving aqueous environment and other ecosystems. Microalgae can be used to remove nitrogen and phosphorus in wastewater, but the species-dependent removal of estrogens needs further investigation. In this study we investigated estrone, 17ß-estradiol and 17α-ethynylestradiol removals and transformation products by four common microalgae Haematococcus pluvialis, Selenastrum capricornutum, Scenedesmus quadricauda, and Chlorella vulgaris. It was found that H. pluvialis, S. capricornutum and S. quadricauda could more effectively remove all three estrogens in synthetic wastewater effluent. The estrogenic activities i.e. 17ß-estradiol equivalency determined by yeast estrogenic screening assay showed substantial estrogenic activity reductions after biotransformation by H. pluvialis, S. capricornutum, and S. quadricauda. Quadrupole Time-of-flight Mass Spectrometry results identified several possible ring-cleavage metabolites as well as their metabolic pathways, which had not been reported yet, confirming the estrogen degradation rather than mere absorption or uptake by microalgae. The findings demonstrate that not only can some specific bacteria degrade estrogens, but also the widely living microalgae are able to degrade these emerging pollutants, suggesting that microalgae could be an advanced treatment of WWTPs to remove nutrients and estrogens.

Attenuation, transport, and management of estrogens: A review.

Overabundance of endocrine disruptors (EDs), such as steroid estrogens, in the natural environment disrupts hormone synthesis in aquatic organisms. Livestock and wastewater outflows contribute measurable quantities of steroid estrogens into the environment where they are picked up and transported via surface runoff and feedlot effluents into water matrices. E1, E2ß, E2α, E3 and EE2 are the most prevalent estrogens in these environmental systems. Estrogens in soils and water undergo several concurrent attenuation processes including sorption to particles, biotransformation, photo-transformation, and plant uptake. This review summarizes current studies on the attenuation and transport of steroid estrogens with a focus on estrogen attenuation and transport modeling. The authors use this information to synthesize appropriate strategies for reducing estrogenicity in the environment.

17α-Ethynylestradiol and 4-nonylphenol stimulate lung adenocarcinoma cell production in xenoestrogenic way.

Lung cancer has been one of the major cancers leading to mortalities worldwide. In addition to smoking, estrogen is considered to play an important role in the lung cancer development because women have a higher proportion of adenocarcinoma than men. In the environment, there are many metabolites and waste products that mimic human estrogen structurally and functionally. 17α-Ethynylestradiol (EE2) which is used as an oral contraceptive is released into wastewater after being utilized. Moreover, 4-nonylphenol (NP) which is found in the petrochemical products and air pollutants reveals estrogenic activity. In the present study, 17ß-estradiol (E2), EE2, and NP are administered to stimulate male lung adenocarcinoma cells (A549) and female lung adenocarcinoma cells (H1435). The results demonstrate that EE2 and NP stimulate A549 and H1435 cells proliferation in a dose- and time-dependent manner. Both estrogen receptors α and ß are simultaneously activated. In response to estrogens, up-regulation of the epidermal growth factor receptor and extracellular signal-regulated kinase expression occurs. In conclusion, this is the first study to report that EE2 and NP exert a biotoxic effect to stimulate the proliferation of both male and female lung cancer cell in a dose- and time- dependent manner. The environmental hormones posing new challenges for lung cancer deserve further investigation.

Endocrine activity in an urban river system and the biodegradation of estrogen-like endocrine disrupting chemicals through a bio-analytical approach using DRE- and ERE-CALUX bioassays.

The Zenne River, crossing the Brussels region (Belgium) is an extremely urbanized river impacted by both domestic and industrial effluents. The objective of this study was to monitor the occurrence and activity of Endocrine Active Substances (EAS) in river water and sediments in the framework of the Environmental Quality Standards Directive (2008/105/EC and 2013/39/EU). Activities were determined using Estrogen and Dioxin Responsive Elements (ERE and DRE) Chemical Activated Luciferase Gene Expression (CALUX) bioassays. A potential contamination source of estrogen active compounds was identified in the river at an industrial area downstream from Brussels with a peak value of 938 pg E2 eq./L water (above the EQS of 0.4 ng/L) and 195 pg E2 eq./g sediment. Estrogens are more abundantly present in the sediments than in the dissolved phase. Principal Component Analysis (PCA) showed high correlations between Suspended Particulate Matter (SPM), Particulate (POC) and Dissolved Organic Carbon (DOC) and estrogenic EAS. The dioxin fractions comply with previous data and all were above the United States Environmental Protection Agency (US EPA) low-level risk, with one (42 pg TCDD eq./g sediment) exceeding the high-level risk value for mammals. The self-purifying ability of the Zenne River regarding estrogens was examined with an in vitro biodegradation experiment using the bacterial community naturally present in the river. Hill coefficient and EC50 values (Effective Concentration at 50%) revealed a process of biodegradation in particulate and dissolved phase. The estrogenic activity was decreased by 80%, demonstrating the ability of self-purification of estrogenic compounds in the Zenne River.

Model-based assessment of estrogen removal by nitrifying activated sludge.

Complete removal of estrogens such as estrone (E1), estradiol (E2), estriol (E3) and ethinylestradiol (EE2) in wastewater treatment is essential since their release and accumulation in natural water bodies are giving rise to environment and health issues. To improve our understanding towards the estrogen bioremediation process, a mathematical model was proposed for describing estrogen removal by nitrifying activated sludge. Four pathways were involved in the developed model: i) biosorption by activated sludge flocs; ii) cometabolic biodegradation linked to ammonia oxidizing bacteria (AOB) growth; iii) non-growth biodegradation by AOB; and iv) biodegradation by heterotrophic bacteria (HB). The degradation kinetics was implemented into activated sludge model (ASM) framework with consideration of interactions between substrate update and microorganism growth as well as endogenous respiration. The model was calibrated and validated by fitting model predictions against two sets of batch experimental data under different conditions. The model could satisfactorily capture all the dynamics of nitrogen, organic matters (COD), and estrogens. Modeling results suggest that for E1, E2 and EE2, AOB-linked biodegradation is dominant over biodegradation by HB at all investigated COD dosing levels. However, for E3, the increase of COD dosage triggers a shift of dominant pathway from AOB biodegradation to HB biodegradation. Adsorption becomes the main contributor to estrogen removal at high biomass concentrations.

The role and impact of estrogens and xenoestrogen on the development of cervical cancer.

Throughout an individual's lifetime, the human body is exposed to many different chemical compounds, including xenoestrogens (XEs) that can be found in the environment, food, air, cosmetics and other substances, which have a positive or negative impact on their health and lifestyle. Whereas high-risk human papillomavirus (HR-HPV) is necessary but not sufficient for full malignant cervical cell transformation, other compounds such as estrogens and XEs may be risk factors for cervical cancer (CC) development. The causes and effects of some diseases such as cancer, cardiovascular, metabolic or immune system disorders are partly due to signaling pathways in response to estrogens. XEs are a vast group of natural and synthetic compounds, behaving like estrogens, that have been studied over the recent years and which may interact with estrogen receptors. The major problem with XEs is the difficulty in studying the mechanism of such complex substances as well as investigating the influences of some of the compounds (dose-dependent) over time. The impact of XEs on CC is variable, with no direct comparison between in vitro studies and in vivo XEs action.

Biomagnification of endocrine disrupting chemicals (EDCs) by Pleuronectes yokohamae: Does P. yokohamae accumulate dietary EDCs?

We evaluated the potential for biomagnification of endocrine disrupting chemicals (EDCs) such as nonylphenol (NP), octylphenol (OP), bisphenol A (BP), and natural estrogens such as estrone (E1) and 17ß-estradiol (E2) in a benthic fish, Pleuronectes yokohamae. The assimilation efficiencies (AE) of most EDCs ranged from 88 to 96% suggesting that they were efficiently incorporated and assimilated into P. yokohamae, except for NP (50%). However, the biomagnification factor (BMF) values were <1.0 suggesting that the compounds were not biomagnifying. Additionally, three of the target EDCs were not detected (BP, E1 and E2). Glucuronidation activity towards BP (11.44 ± 2.5 nmol/mg protein/min) and E2 (12.41 ± 3.2 nmol/mg protein/min) was high in the intestine suggesting that EDCs were glucuronidated prior to excretion into bile. Thus, we conclude that biomagnification of dietary EDCs is reduced in P. yokohamae because of effective glucuronidation.

Estrogens and Their Genotoxic Metabolites Are Increased in Obese Prepubertal Girls.

CONTEXT: Estrogen levels and their metabolites are higher in obese vs lean postmenopausal women, and obesity increases breast cancer risk. Quinone derivatives of 4-hydroxylated estrogen metabolites, independently of the estrogen receptor, cause depurination and impaired DNA repair (genotoxic). 16α-Hydroxy (16α-OH)-estrone (E1), eg, promotes tumor proliferation and 2-methoxy-estradiol (E2) may be chemoprotective. Childhood obesity increases breast cancer death risk in women, but levels of estrogen derivatives had not been previously studied in young children. OBJECTIVE: The objective of the study was to investigate whether total and genotoxic estrogens are increased in prepubertal obese girls compared with lean controls. DESIGN: Stored sera from 12 lean and 23 obese prepubertal girls (Tanner stage I breast and pubic hair) studied previously were assayed for E1, E2, and their multiple metabolites (12 steroids total) using highly sensitive liquid chromatography and tandem mass spectrometry. RESULTS: E2 concentrations were significantly higher in obese [3.45 (0.5, 4.65) pg/ml (median [quartile 1, quartile 3])] vs lean girls [0.5 (0.5, 2.37), P = .04], 57% of values upper quartile or greater (quartile 3) of controls. Concentrations of 16α-OH-E1 were higher in obese [7.17 (0.5, 9.64) pg/mL] vs lean girls [0.5 (0.5, 1.72, P = .007)], 65% of values quartile 3 or greater of controls. 2-Methoxy-E2 concentrations were lower in the obese group (P = .012). 16α-OH-E1 concentrations were positively correlated with body mass index, percentage fat mass, and IL-6 concentrations (P < .001). CONCLUSIONS: E2 and genotoxic metabolites were higher in obese vs lean prepubertal girls. These data suggest that obesity is associated with an increased extraglandular estrogen production and metabolism before the onset of puberty in girls. Long-term epidemiological studies are needed to assess any potential increase in breast cancer risk.

Impact of climate change and population growth on a risk assessment for endocrine disruption in fish due to steroid estrogens in England and Wales.

In England and Wales, steroid estrogens: estrone, estradiol and ethinylestradiol have previously been identified as the main chemicals causing endocrine disruption in male fish. A national risk assessment is already available for intersex in fish arising from estrogens under current flow conditions. This study presents, to our knowledge, the first set of national catchment-based risk assessments for steroid estrogen under future scenarios. The river flows and temperatures were perturbed using three climate change scenarios (ranging from relatively dry to wet). The effects of demographic changes on estrogen consumption and human population served by sewage treatment works were also included. Compared to the current situation, the results indicated increased future risk:the percentage of high risk category sites, where endocrine disruption is more likely to occur, increased. These increases were mainly caused by changes in human population. This study provides regulators with valuable information to prepare for this potential increased risk.

Analysis of estrogens and androgens in postmenopausal serum and plasma by liquid chromatography-mass spectrometry.

Liquid chromatography-selected reaction monitoring/mass spectrometry-based methodology has evolved to the point where accurate analyses of trace levels of estrogens and androgens in postmenopausal serum and plasma can be accomplished with high precision and accuracy. A suite of derivatization procedures has been developed, which together with modern mass spectrometry instrumentation provide investigators with robust and sensitive methodology. Pre-ionized derivatives are proving to be useful as they are not subject to suppression of the electrospray signal. Postmenopausal women with elevated plasma or serum estrogens are thought to be at increased risk for breast and endometrial cancer. Therefore, significant advances in risk assessment should be possible now that reliable methodology is available. It is also possible to conduct analyses of multiple estrogens in plasma or serum. Laboratories that are currently employing liquid chromatography/mass spectrometry methodology can now readily implement this strategy. This will help conserve important plasma and serum samples available in Biobanks, as it will be possible to conduct high sensitivity analyses using low initial sample volumes. Reported levels of both conjugated and non-conjugated estrogen metabolites are close to the limits of sensitivity of many assays to date, urging caution in the interpretation of these low values. The analysis of serum androgen precursors in postmenopausal women has not been conducted routinely in the past using liquid chromatography/mass spectrometry methodology. Integration of serum androgen levels into the panel of metabolites analyzed could provide additional information for assessing cancer risk and should be included in the future.

Diagnostic investigation of steroid estrogen removal by activated sludge at varying solids retention time.

The impact of solids retention time (SRT) on estrone (E1), 17ß-estradiol (E2), estriol (E3) and 17α-ethinylestradiol (EE2) removal in an activated sludge plant (ASP) was examined using a pilot plant to closely control operation. Exsitu analytical methods were simultaneously used to enable discrimination of the dominant mechanisms governing estrogen removal following transitions in SRT from short (3d) to medium (10d) and long (27d) SRTs which broadly represent those encountered at full-scale. Total estrogen (∑EST, i.e., sum of E1, E2, E3 and EE2) removals which account for aqueous and particulate concentrations were 70±8, 95±1 and 93±2% at 3, 10 and 27d SRTs respectively. The improved removal observed following an SRT increase from 3 to 10d was attributable to the augmented biodegradation of the natural estrogens E1 and E2. Interestingly, estrogen biodegradation per bacterial cell increased with SRT. These were 499, 1361 and 1750ng 10(12) viable cells(-1)d(-1). This indicated an improved efficiency of the same group or the development of a more responsive group of bacteria. In this study no improvement in absolute ∑EST removal was observed in the ASP when SRT increased from 10 to 27d. However, batch studies identified an augmented biomass sorption capacity for the more hydrophobic estrogens E2 and EE2 at 27d, equivalent to an order of magnitude. The lack of influence on estrogen removal during pilot plant operation can be ascribed to their distribution within activated sludge being under equilibrium. Consequently, lower wastage of excess sludge inherent of long SRT operation counteracts any improvement in sorption.

Determination of estrogenic steroids and microbial and photochemical degradation of 17α-ethinylestradiol (EE2) in lake surface water, a case study.

In this study, a GC-MS technique was applied to determine 17α-ethinylestradiol (EE2), an active ingredient of oral contraceptives, and its fate in Lake Quinsigamond, Massachusetts, USA. To the knowledge of the authors, this is the first study of EE2 and its microbial and photochemical degradation in a lake ecosystem. EE2 was detected at a concentration up to 11.1 ng L(-1). At this concentration EE2 may affect the reproduction of fish and other aquatic organisms in the lake due to its high estrogenic activity. EE2 was persistent to the biodegradation by the microorganisms in the lake. Under aerobic conditions a long lag phase (42 days) was observed before the biodegradation of EE2 and a half-life of 108 days was estimated. Under anaerobic conditions, EE2 experienced even a longer acclimation stage (63 days) and a slower microbial degradation in the lake water. The photodegradation of EE2 was rapid in the lake surface water under natural sunlight, with a half-life of less than 2 days in summer sunny days. Compared to biodegradation, photodegradation may represent a predominant removal mechanism for EE2 in natural surface waters.

Identification of microRNAs in human follicular fluid: characterization of microRNAs that govern steroidogenesis in vitro and are associated with polycystic ovary syndrome in vivo.

CONTEXT: Human follicular fluid is a combination of proteins, metabolites, and ionic compounds that is indicative of the general state of follicular metabolism and is associated with maturation and quality of oocytes. Deviations in these components are often associated with reproductive diseases. There has been no report of microRNAs (miRNAs) in human follicular fluids. OBJECTIVE: We hypothesized that human follicular fluid may contain miRNAs. We sought to identify cell-free miRNAs in human follicular fluid and to investigate the function of these miRNAs in vitro and any roles they play in polycystic ovary syndrome (PCOS). DESIGN: Genome-wide deep sequencing and TaqMan miRNA arrays were used to identify miRNAs, and the roles of the highly expressed miRNAs in steroidogenesis were investigated in KGN cells. Quantification of candidate miRNAs in follicular fluids of PCOS and controls was performed using TaqMan miRNA assays. RESULTS: We identified miRNAs in microvesicles and the supernatant of human follicular fluid. Bioinformatics analysis showed that the most highly expressed miRNAs targeted genes associated with reproductive, endocrine, and metabolic processes. We found that miR-132, miR-320, miR-520c-3p, miR-24, and miR-222 regulate estradiol concentrations and that miR-24, miR-193b, and miR-483-5p regulate progesterone concentrations. Finally, we showed that miR-132 and miR-320 are expressed at significantly lower levels in the follicular fluid of polycystic ovary patients than in healthy controls (P = .005 and P = .0098, respectively). CONCLUSION: These results demonstrate that there are numerous miRNAs in human follicular fluids, some of which play important roles in steroidogenesis and PCOS. This study substantially revises our understanding of the content of human follicular fluid and lays the foundation for the future investigation of the role of miRNAs in PCOS.

Implantation failure in mice with a disruption in Phospholipase C beta 1 gene: lack of embryonic attachment, aberrant steroid hormone signalling and defective endocannabinoid metabolism.

Phospholipase C beta 1 (PLCß1) is a downstream effector of G-protein-coupled receptor signalling and holds central roles in reproductive physiology. Mice with a disruption in the Plcß1 gene are infertile with pleiotropic reproductive defects, the major reproductive block in females being implantation failure. Here, PLCß1 was demonstrated at the luminal and glandular epithelia throughout the pre- and peri-implantation period, with transient stromal expression during 0.5-1.5 days post coitum (dpc). Examination of implantation sites at 4.5 dpc showed that in females lacking functional PLCß1 (knock-out (KO) females), embryos failed to establish proper contact with the uterine epithelium. Proliferating luminal epithelial cells were evident in KO implantation sites, indicating failure to establish a receptive uterus. Real-time PCR demonstrated that KO implantation sites had aberrant ovarian steroid signalling, with high levels of estrogen receptor α, lactoferrin and amphiregulin mRNA, while immunohistochemistry revealed very low levels of estrogen receptor α protein, possibly due to rapid receptor turnover. KO implantation sites expressed markedly less fatty acid amide hydrolase and monoacylglycerol lipase, indicating that endocannabinoid metabolism was also affected. Collectively, our results show that PLCß1 is essential for uterine preparation for implantation, and that defective PLCß1-mediated signalling during implantation is associated with aberrant ovarian steroid signalling and endocannabinoid metabolism.

Accurate determination of tissue steroid hormones, precursors and conjugates in adult male rat.

The actual levels of steroid hormones in organs are vital for endocrine, reproductive and neuronal health and disorders. We developed an accurate method to determine the levels of steroid hormones and steroid conjugates in various organs by an efficient preparation using a solid-phase-extraction cartridge. Each steroid was identified by the precursor ion spectra using liquid chromatography-electrospray ionization time-of-flight mass spectrometry, and the respective steroids were quantitatively analysed in the selected reaction monitoring mode by liquid chromatograph-mass spectrometry/mass spectrometry (LC-MS/MS). The data showed that significant levels of testosterone, corticosterone and precursors of both hormones were detected in all organs except liver. The glucuronide conjugates of steroid hormones and the precursors were detected in all organs except liver, but sulfate conjugates of these steroids were observed only in the target organs of the hormones and kidney. Interestingly, these steroids and the conjugates were not observed in the liver except pregnenolone. In conclusion, an accurate determination of tissue steroids was developed using LC-MS analysis. Biosynthesis of steroid hormones from the precursors was estimated even in the target organs, and the delivery of these steroid conjugates was also suggested via the circulation without any significant hepatic participation.