Final Report Summary - ECOSIGNAL (Environmental signaling and ovarian diseases susceptibility)
FINAL PUBLISHABLE SUMMARY REPORT
Environmental Signaling and Ovarian Diseases Susceptibility (EcoSignal)
Project coordinator: University of Novi Sad Faculty of Sciences (UNSPMF), Novi Sad, Serbia
Scientist in charge: Prof. emer. Radmila Kovacevic, PhD – UNSPMF radmila.kovacevic@dbe.uns.ac.rs
Principal investigator: Nebojsa Andric, PhD – UNSPMF nebojsa.andric@dbe.uns.ac.rs
EcoSignal MC-CIG project started on 1 September 2012 and ran for 48 intensive months. Reproductive dysfunction has always been a challenge for the researchers and a major puzzle to solve. EcoSignal research deals with the very current issue which caught scientific attention – investigation of the endocrine disrupting chemicals (EDCs) as a possible cause for the human and wildlife reproductive dysfunction. Project results have revealed important new results and major breakthroughs as a basis for the follow up research.
EDCs have captured scientific attention as a possible cause of human and wildlife reproductive dysfunction. EDCs are chemicals in our environment that can interfere with the normal function of human endocrine system, including reproduction. It is estimated that there are around 150,000 chemicals on the market today and many of them could potentially disrupt normal reproduction. The endpoints of EDCs’ effects in the ovary have been fairly elucidated; however, insufficient research has been done to identify the molecular mechanisms of EDCs’ action in the ovary. Identification of the molecular mechanisms by which EDCs affect ovarian and reproductive function could potentially provide a critical link between exposure and pathophysiology of reproductive diseases such as premature ovarian failure, polycystic ovary syndrome and infertility in women.
EcoSignal project was set out to investigate the effect of EDCs, including those with the estrogenic potency, on the ovarian function. Several individual EDCs and mixtures of chemicals from the environment were chosen for the project. Bisphenol A (BPA), an ubiquitous EDC with proven estrogenic properties, atrazine (ATR), a heavily used pesticide, and hexabromocyclododecane (HBCDD), a fire-protecting agent, were selected for the single EDC exposure. The environmental mixtures were obtained from the municipal wastewater from one of the discharge stations of Novi Sad, Serbia. Mixture that exerted the estrogenic property in the ER-CALUX assay was chosen for testing. The main results can be summarized according to the following:
•ATR, a widely used pesticide, disrupts production of sex steroid hormones (estradiol and progesterone) in the hormone-stimulated ovarian granulosa cells. Estradiol level decreases, whereas progesterone production increases in the ATR-exposed cells. The ratio of progesterone to estradiol during normal cycle is always lower than 1, but if is higher, than is usually considered as a pathological condition. The obtain results reveal that ATR shifts the ratio of progesterone to estradiol to the value higher than 1, thus causing the condition known as premature luteinzation in humans. This is the first time that ATR exposure was linked to one of the pathophysiological conditions of the female reproductive system.
•Also, results of this project revealed, for the first time, that ATR decreases expression of the luteinizing hormone (LH) receptor. This receptor is critical for the ovulatory process in the ovary. Insufficient number of the LH receptors prevents the ovulatory hormone LH to initiate ovulation and release of the egg from the ovary. Therefore, ATR exerts the anti-ovulatory action by preventing acquisition of this receptor on the surface of granulosa cells.
•EcoSignal results brought another discovery - at the molecular level, the project revealed the toxicity pathway responsible for the altered sex steroid hormone production and the LH receptor acquisition in the ATR-exposed granulosa cells. Atrazine enhances the cAMP signaling pathway by inhibiting the activity of an enzyme that controls the cAMP turnover (phosphodiesterase). Enhancement of cAMP further activates the cAMP-dependent downstream signaling such as activation of the protein kinase B (AKT)- and extracellular regulated kinase (ERK1/2)-dependent transcription factors in the cells. Activation of these signaling processes leads to decrease in the estradiol level and LHR expression, accompanied by increased progesterone production in the hormone-stimulated granulosa cells. Therefore, the project described the ATR toxicity pathway that changes the ratio of sex steroid hormones towards the premature luteinization phenotype and infertility.
•To support the results of in vitro experiments, a short in vivo study was performed as well. These experiments showed that ATR prevents ovulation, thus leading to the infertility phenotype in rats.
•Analysis of the HBCDD action in granulosa cells revealed that this compound activates the epidermal growth factor receptor (EGFR), thus leading to over-activation of several downstream signaling cascades in the hormone-stimulated granulosa cells, such as cAMP, AKT and ERK1/2. As a result of over-activation of the EGFR-ERK1/2 cascade by HBCDD, granulosa cells were not able to acquire a sufficient number of the LH receptors, thereby causing a diminished ovulatory response to the LH. This was the first study that showed that HBCDD could potentially jeopardize ovulation, leading to infertility.
•The project showed that BPA, a compound with estrogenic properties, interferes with the progesterone biosynthetic pathway in granulosa cells. BPA increases the expression of steroid acute regulatory protein (StAR), which is the rate-limiting enzyme in progesterone production in granulosa cells. However, BPA decreases progesterone production under the basal and hormone-stimulated conditions.. The project revealed that BPA decreases the cholesterol availability in granulosa cells preventing progesterone production. The results also revealed that the BPA-induced alterations in cholesterol level affect StAR expression.
•Finally, a mixture of environmental chemical with estrogenic properties also interferes with the progesterone biosynthetic pathways in granulosa cells. Exposure of cells to the mixture caused increased StAR expression and progesterone production in granulosa cells.
•The EcoSignal project has brought a new knowledge in the field of EDCs action on female reproduction identifying the mechanism by which chemicals alter steroidogenesis and ovulation process. The main target is scientific community and general population. The outcome of the project have a strong impact on the further research at UNSPMF in this field. The project triggered new research collaboration with the Centre for Human Reproduction, Clinical Centre of Vojvodina, Serbia with the aim to investigate association between EDCs and infertility. Knowledge transfer at the European level has been done through the new research collaboration with the Swiss Federal Institute of Aquatic Science and Toxicology-Eawag (joint work on a research project). Moreover, knowledge transfer to the UNSPMF has been achieved through offering two new courses in the ovarian physiology and the effects of EDCs on reproduction at the doctoral study level; supervising 2 PhD students and 4 MSc students.
•The popular lectures about the project were directed to raise local public awareness on reproductive effects of EDCs. To enhance the visibility and socio-economic impact of the project, MSCA NCP has invited the principal investigator on the several workshops where successful EU project have been presented to the scientific and general audience.
Environmental Signaling and Ovarian Diseases Susceptibility (EcoSignal)
Project coordinator: University of Novi Sad Faculty of Sciences (UNSPMF), Novi Sad, Serbia
Scientist in charge: Prof. emer. Radmila Kovacevic, PhD – UNSPMF radmila.kovacevic@dbe.uns.ac.rs
Principal investigator: Nebojsa Andric, PhD – UNSPMF nebojsa.andric@dbe.uns.ac.rs
EcoSignal MC-CIG project started on 1 September 2012 and ran for 48 intensive months. Reproductive dysfunction has always been a challenge for the researchers and a major puzzle to solve. EcoSignal research deals with the very current issue which caught scientific attention – investigation of the endocrine disrupting chemicals (EDCs) as a possible cause for the human and wildlife reproductive dysfunction. Project results have revealed important new results and major breakthroughs as a basis for the follow up research.
EDCs have captured scientific attention as a possible cause of human and wildlife reproductive dysfunction. EDCs are chemicals in our environment that can interfere with the normal function of human endocrine system, including reproduction. It is estimated that there are around 150,000 chemicals on the market today and many of them could potentially disrupt normal reproduction. The endpoints of EDCs’ effects in the ovary have been fairly elucidated; however, insufficient research has been done to identify the molecular mechanisms of EDCs’ action in the ovary. Identification of the molecular mechanisms by which EDCs affect ovarian and reproductive function could potentially provide a critical link between exposure and pathophysiology of reproductive diseases such as premature ovarian failure, polycystic ovary syndrome and infertility in women.
EcoSignal project was set out to investigate the effect of EDCs, including those with the estrogenic potency, on the ovarian function. Several individual EDCs and mixtures of chemicals from the environment were chosen for the project. Bisphenol A (BPA), an ubiquitous EDC with proven estrogenic properties, atrazine (ATR), a heavily used pesticide, and hexabromocyclododecane (HBCDD), a fire-protecting agent, were selected for the single EDC exposure. The environmental mixtures were obtained from the municipal wastewater from one of the discharge stations of Novi Sad, Serbia. Mixture that exerted the estrogenic property in the ER-CALUX assay was chosen for testing. The main results can be summarized according to the following:
•ATR, a widely used pesticide, disrupts production of sex steroid hormones (estradiol and progesterone) in the hormone-stimulated ovarian granulosa cells. Estradiol level decreases, whereas progesterone production increases in the ATR-exposed cells. The ratio of progesterone to estradiol during normal cycle is always lower than 1, but if is higher, than is usually considered as a pathological condition. The obtain results reveal that ATR shifts the ratio of progesterone to estradiol to the value higher than 1, thus causing the condition known as premature luteinzation in humans. This is the first time that ATR exposure was linked to one of the pathophysiological conditions of the female reproductive system.
•Also, results of this project revealed, for the first time, that ATR decreases expression of the luteinizing hormone (LH) receptor. This receptor is critical for the ovulatory process in the ovary. Insufficient number of the LH receptors prevents the ovulatory hormone LH to initiate ovulation and release of the egg from the ovary. Therefore, ATR exerts the anti-ovulatory action by preventing acquisition of this receptor on the surface of granulosa cells.
•EcoSignal results brought another discovery - at the molecular level, the project revealed the toxicity pathway responsible for the altered sex steroid hormone production and the LH receptor acquisition in the ATR-exposed granulosa cells. Atrazine enhances the cAMP signaling pathway by inhibiting the activity of an enzyme that controls the cAMP turnover (phosphodiesterase). Enhancement of cAMP further activates the cAMP-dependent downstream signaling such as activation of the protein kinase B (AKT)- and extracellular regulated kinase (ERK1/2)-dependent transcription factors in the cells. Activation of these signaling processes leads to decrease in the estradiol level and LHR expression, accompanied by increased progesterone production in the hormone-stimulated granulosa cells. Therefore, the project described the ATR toxicity pathway that changes the ratio of sex steroid hormones towards the premature luteinization phenotype and infertility.
•To support the results of in vitro experiments, a short in vivo study was performed as well. These experiments showed that ATR prevents ovulation, thus leading to the infertility phenotype in rats.
•Analysis of the HBCDD action in granulosa cells revealed that this compound activates the epidermal growth factor receptor (EGFR), thus leading to over-activation of several downstream signaling cascades in the hormone-stimulated granulosa cells, such as cAMP, AKT and ERK1/2. As a result of over-activation of the EGFR-ERK1/2 cascade by HBCDD, granulosa cells were not able to acquire a sufficient number of the LH receptors, thereby causing a diminished ovulatory response to the LH. This was the first study that showed that HBCDD could potentially jeopardize ovulation, leading to infertility.
•The project showed that BPA, a compound with estrogenic properties, interferes with the progesterone biosynthetic pathway in granulosa cells. BPA increases the expression of steroid acute regulatory protein (StAR), which is the rate-limiting enzyme in progesterone production in granulosa cells. However, BPA decreases progesterone production under the basal and hormone-stimulated conditions.. The project revealed that BPA decreases the cholesterol availability in granulosa cells preventing progesterone production. The results also revealed that the BPA-induced alterations in cholesterol level affect StAR expression.
•Finally, a mixture of environmental chemical with estrogenic properties also interferes with the progesterone biosynthetic pathways in granulosa cells. Exposure of cells to the mixture caused increased StAR expression and progesterone production in granulosa cells.
•The EcoSignal project has brought a new knowledge in the field of EDCs action on female reproduction identifying the mechanism by which chemicals alter steroidogenesis and ovulation process. The main target is scientific community and general population. The outcome of the project have a strong impact on the further research at UNSPMF in this field. The project triggered new research collaboration with the Centre for Human Reproduction, Clinical Centre of Vojvodina, Serbia with the aim to investigate association between EDCs and infertility. Knowledge transfer at the European level has been done through the new research collaboration with the Swiss Federal Institute of Aquatic Science and Toxicology-Eawag (joint work on a research project). Moreover, knowledge transfer to the UNSPMF has been achieved through offering two new courses in the ovarian physiology and the effects of EDCs on reproduction at the doctoral study level; supervising 2 PhD students and 4 MSc students.
•The popular lectures about the project were directed to raise local public awareness on reproductive effects of EDCs. To enhance the visibility and socio-economic impact of the project, MSCA NCP has invited the principal investigator on the several workshops where successful EU project have been presented to the scientific and general audience.