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The impacts of endocrine disrupters on wildlife, people and their environments - The Weybridge+15 (1996–2011) report
No full textRates of endocrine diseases and disorders, such
as some reproductive and developmental harm in
human populations, have changed in line with the
growth of the chemical industry, leading to concerns
that these factors may be linked. For example, the
current status of semen quality in the few European
countries where studies have been systematically
conducted, is very poor: fertility in approximately
40 % of men is impaired. There is also evidence of
reproductive and developmental harm linked to
impairments in endocrine function in a number
of wildlife species, particularly in environments
that are contaminated by cocktails of chemicals
that are in everyday use. Based on the human and
wildlife evidence, many scientists are concerned
about chemical pollutants being able to interfere
with the normal functioning of hormones, so-called
endocrine-disrupting chemicals (EDCs), that could
play a causative role in these diseases and disorders.
If this holds true, then these 'early warnings' signal
a failure in environmental protection that should be
addressed.
(In this report...) Several key conclusions, challenges and recommendations have been drawn from the
research over the last 14 years, not least of which
is the conclusion that that chemically induced ED
likely affects human and wildlife endocrine health
the world over. A much better understanding of the
role of exposure to environmental contaminants in
the prevalence and risk of endocrine disease in both
humans and wildlife is needed if we are to protect
ourselves and wildlife from any harm caused by
these chemicals
Fetal but not adult Leydig cells are susceptible to adenoma formation in response to persistently high hCG level; a study on hCG overexpressing transgenic mice
Full text linkWe have previously demonstrated that male transgenic (TG) mice overexpressing human chorionic gonadotropin (hCG+) develop reproductive organ defects, but no tumors, in adult age. In this study, the effects of persistently elevated hCG were followed in TG males between day 5 postpartum and adulthood. Leydig cell (LC) adenomas were found in prepubertal mice, most prominently at the age of 10 days, but not in adult age. Serum testosterone concentrations were significantly increased in TG males at all ages studied. The phenotype of the prepubertal hCG+ males resembled that found in boys upon expression of constitutively activating luteinizing hormone (LH) receptor mutations. The temporal expression patterns of the fetal LC marker gene, thrombospondin 2, and those of adult LCs, hydroxysteroid dehydrogenase-6, delta5-3-beta and prostaglandin D synthase, were similar in wild-type and hCG+ males. Hence, the postnatal adenomas resemble functionally fetal LCs, and only these cells are susceptible to hCG-induced tumorigenesis. Our findings demonstrate a novel intriguing difference between the fetal and adult LC populations and provide further insight into the potential tumorigenic effects of gonadotropins.Fil: Ahtianen, Petteri. University of Turku; FinlandiaFil: Rulli, Susana Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Shariatmadari, Ramin. University of Turku; FinlandiaFil: Pelliniemi, Lauri J.. University of Turku; FinlandiaFil: Toppari, Jorma. University of Turku; FinlandiaFil: Poutanen, Matti. University of Turku; FinlandiaFil: Huhtaniemi, Ilpo T.. University of Turku; Finlandia. Imperial College London; Reino Unid
Endocrine function and growth in young patients with childhood- or adolescence-onset epilepsy
Full text linkAbstractEndocrine disorders are common in adults with epilepsy on antiepileptic drugs (AEDs). This study aimed to evaluate endocrine function, lipid metabolism and growth in patients with childhood- or adolescence-onset epilepsy.Altogether 148 patients with epilepsy on carbamazepine (CBZ), oxcarbazepine (OXC), valproate (VPA) or lamotrigine monotherapy during pubertal maturation and 124 healthy controls participated in this population-based cohort study. Boys and young men (n = 140) underwent cross-sectional evaluation once, and girls and young women (n = 132) twice at an approximate interval of 6 years. Gonadal structure and serum reproductive and thyroid hormone and lipid concentrations were evaluated and growth data were gathered.Elevated serum testosterone and androstenedione levels and polycystic ovary syndrome (PCOS) were common in female subjects whose medication, especially VPA, continued into young adulthood. Serum reproductive hormone concentrations and ovarian structure were similar in patients off medication and controls in young adulthood. CBZ was associated with elevated serum sex hormone binding globulin and decreased dehydroepiandrosterone sulfate levels and VPA with elevated serum androstenedione concentrations in male patients. Testicular structure was similar in patients and controls. CBZ, OXC and VPA were associated with changes in serum thyroid hormone, thyrotropin and lipid levels during pubertal maturation in female patients, but these levels returned to normal after withdrawal of medication. Linear growth and final height were normal in female patients, but overweight was common if they had been obese and on VPA medication during pubertal maturation.Elevated serum androgen levels, PCOS and overweight are common if epilepsy and AED use, especially VPA, continue into young adulthood. These untoward changes or alterations in serum thyroid hormone or lipid concentrations are not present in young women with medication withdrawn. CBZ and VPA are associated with changes in serum sex hormone levels in boys and young men with epilepsy. Epilepsy and AED use during pubertal maturation does not seem to affect growth.Academic Dissertation to be presented with the assent of the Faculty of Medicine, University of Oulu, for public discussion in the Auditorium L12 of the Department of Paediatrics and Adolescence, Oulu University Hospital,
on September 3rd, 2004, at 12 noon.Abstract
Endocrine disorders are common in adults with epilepsy on antiepileptic drugs (AEDs). This study aimed to evaluate endocrine function, lipid metabolism and growth in patients with childhood- or adolescence-onset epilepsy.
Altogether 148 patients with epilepsy on carbamazepine (CBZ), oxcarbazepine (OXC), valproate (VPA) or lamotrigine monotherapy during pubertal maturation and 124 healthy controls participated in this population-based cohort study. Boys and young men (n = 140) underwent cross-sectional evaluation once, and girls and young women (n = 132) twice at an approximate interval of 6 years. Gonadal structure and serum reproductive and thyroid hormone and lipid concentrations were evaluated and growth data were gathered.
Elevated serum testosterone and androstenedione levels and polycystic ovary syndrome (PCOS) were common in female subjects whose medication, especially VPA, continued into young adulthood. Serum reproductive hormone concentrations and ovarian structure were similar in patients off medication and controls in young adulthood. CBZ was associated with elevated serum sex hormone binding globulin and decreased dehydroepiandrosterone sulfate levels and VPA with elevated serum androstenedione concentrations in male patients. Testicular structure was similar in patients and controls. CBZ, OXC and VPA were associated with changes in serum thyroid hormone, thyrotropin and lipid levels during pubertal maturation in female patients, but these levels returned to normal after withdrawal of medication. Linear growth and final height were normal in female patients, but overweight was common if they had been obese and on VPA medication during pubertal maturation.
Elevated serum androgen levels, PCOS and overweight are common if epilepsy and AED use, especially VPA, continue into young adulthood. These untoward changes or alterations in serum thyroid hormone or lipid concentrations are not present in young women with medication withdrawn. CBZ and VPA are associated with changes in serum sex hormone levels in boys and young men with epilepsy. Epilepsy and AED use during pubertal maturation does not seem to affect growth
Proinflammatory cytokines modify the expression of surfactant proteins : Study in perinatal rabbit lung
Full text linkAbstractDeficiency of pulmonary surfactant is the main cause of respiratory distress syndrome (RDS) in premature newborn infants, which is often complicated by chronic lung disease (CLD). Preterm birth is often associated with intra-amniotic infection (IUI), which is characterized by increased proinflammatory cytokines, such as interleukin-1 (IL-1) and tumor necrosis factor-α (TNF-α), in the amniotic fluid. In very preterm birth due to IUI, the incidence of RDS is decreased, while the incidence of CLD is increased. Maternal glucocorticoids are used in imminent preterm birth to prevent RDS. This study was designed to clarify the contrasting association of these perinatal pulmonary diseases with IUI and the pathogenesis of these lung diseases using an in vitro rabbit model.IL-1 increased the expression of surfactant protein (SP)-A and SP-B in very immature lung. Contrariwise, in transitional and mature fetal lung as well as in newborn lung, IL-1 additively with TNF-α decreased the expression of SP-B and SP-C. Bacterial lipopolysaccharide (LPS) decreased SP-A, -B and -C mRNAs in mature fetal and newborn lung, but had no effect on SP expression in immature lung. Interferon-γ (IFN-γ) had no effect on SP expression at any gestational age, but it modified the effects of the other cytokines. Dexamethasone (Dx) and IL-1 in combination additively increased SP-A and SP-B mRNAs in immature lung. Dx abolished the inhibitory effect of IL-1 on SP-B and SP-C in mature lung. Dx and IL-1 together tended to stabilize SP mRNAs. The present findings provide additional evidence of the role of the transcription factors nuclear factor-κB (NF-κB) and C/CAAT enhancer-binding protein δ (C/EBPδ) in the upregulation of SP-A by IL-1 in immature lung.Proinflammatory cytokines profoundly influence the expression of surfactant proteins in a manner that is strictly dependent on the length of gestation. The present findings help to explain the differences in the incidence of RDS and CLD in preterm births caused by IUI, and they may clarify further the role of surfactant in the pathogenesesis of lung diseases in neonatal infants. TiivistelmäKeuhkosurfaktantin puute aiheuttaa ennenaikaisesti syntyville keskosille vastasyntyneen hengitysvaikeusoireyhtymää eli RDS-tautia (Respiratory Distress Syndrome). Toinen keskosilla esiintyvä keuhkosairaus on krooninen keuhkosairaus eli CLD (Chronic Lung Disease). Glukokortikoideja käytetään hoitona ennenaikaisen synnytyksen uhatessa, koska niiden tiedetään vähentävän RDS-taudin riskiä. Kohdunsisäinen infektio on huomattava ennenaikaisen synnytyksen aiheuttaja. Infektiossa tulehduksen välittäjäaineet, kuten sytokiinit interleukiini-1 (IL-1) ja tuumorinekroositekijä alfa (TNF-α) lisääntyvät lapsivedessä. Infektiosta aiheutunut ennenaikainen synnytys vähentää RDS-taudin ilmaantumista pienille keskosille ja toisaalta lisää kroonisen keuhkosairauden riskiä. Tutkimuksen oli tavoitteena selvittää, miksi RDS ja CLD ilmaantuvat eriävästi infektion vuoksi ennenaikaisesti syntyneille vauvoille.Viljelemällä eri-ikäisten kanin sikiöiden sekä vastasyntyneiden kanin poikasten keuhkon kappaleita tutkittiin tulehduksen välittäjäaineiden sekä anti-inflammatorisen glukokortikoidin (deksametasonin) vaikutusta surfaktantin toiminnalle tarpeellisten surfaktanttiproteiinien (SP) ilmentymiseen. IL-1 lisäsi SP-A:n ja SP-B:n ilmentymistä erittäin epäkypsässä kanin sikiön keuhkossa. Toisaalta IL-1 ja TNF-α vähensivät SP-B:n ja SP-C:n ilmentymistä kypsemmässä sikiön sekä vastasyntyneen kanin keuhkossa. Interferoni-gamma (IFN-γ) ei vaikuttanut surfaktanttiproteiinien ilmentymiseen missään gestaatioiässä, mutta se muunsi muiden sytokiinien surfaktanttivaikutusta. Gram-negatiivisten bakteerien soluseinän tuote, lipopolysakkaridi (LPS) vähensi SP-A:n, SP-B:n ja SP-C:n ilmentymistä kypsässä kanin sikiön ja vastasyntyneen kanin keuhkossa. IL-1:llä ja deksametasonilla oli positiivinen yhteisvaikutus surfaktanttiproteiinien ilmentymiseen. Tämän surfaktanttiproteiineja lisäävän vaikutuksen mekanismiksi havaittiin pääasiallisesti lisääntynyt mRNA:n stabiliteetti. Lisäksi tutkimus antaa lisätietoa kahden transkriptiofaktorin, NF-κB:n (nuclear factor kappa B) ja C/EBPγ:n (C/CAAT enhancer binding protein delta), osuudesta IL-1:n aiheuttamassa SP-A:n ilmentymisen lisääntymisessä.Sytokiinien vaikutukset surfaktanttiproteiinien ilmentymiseen ovat riippuvaisia gestaatioiästä. Tutkimuksen löydökset auttavat ymmärtämään RDS:n ja CLD:n vastakohtaista esiintymismäärää keskosilla, joiden ennenaikainen synnytys on aiheutunut kohdunsisäisestä tulehduksesta. Edelleen tutkimus selittää glukokortikoidien positiivista vaikutusta hengitysvajaukseen johtavassa keuhkotulehduksessa.Academic Dissertation to be presented with the assent of the Faculty of Medicine, University of Oulu, for public discussion in the Auditorium L12 of the Department of Paediatrics, University Hospital of Oulu, on June 18th, 2003, at 12 noon.Abstract
Deficiency of pulmonary surfactant is the main cause of respiratory distress syndrome (RDS) in premature newborn infants, which is often complicated by chronic lung disease (CLD). Preterm birth is often associated with intra-amniotic infection (IUI), which is characterized by increased proinflammatory cytokines, such as interleukin-1 (IL-1) and tumor necrosis factor-α (TNF-α), in the amniotic fluid. In very preterm birth due to IUI, the incidence of RDS is decreased, while the incidence of CLD is increased. Maternal glucocorticoids are used in imminent preterm birth to prevent RDS. This study was designed to clarify the contrasting association of these perinatal pulmonary diseases with IUI and the pathogenesis of these lung diseases using an in vitro rabbit model.
IL-1 increased the expression of surfactant protein (SP)-A and SP-B in very immature lung. Contrariwise, in transitional and mature fetal lung as well as in newborn lung, IL-1 additively with TNF-α decreased the expression of SP-B and SP-C. Bacterial lipopolysaccharide (LPS) decreased SP-A, -B and -C mRNAs in mature fetal and newborn lung, but had no effect on SP expression in immature lung. Interferon-γ (IFN-γ) had no effect on SP expression at any gestational age, but it modified the effects of the other cytokines. Dexamethasone (Dx) and IL-1 in combination additively increased SP-A and SP-B mRNAs in immature lung. Dx abolished the inhibitory effect of IL-1 on SP-B and SP-C in mature lung. Dx and IL-1 together tended to stabilize SP mRNAs. The present findings provide additional evidence of the role of the transcription factors nuclear factor-κB (NF-κB) and C/CAAT enhancer-binding protein δ (C/EBPδ) in the upregulation of SP-A by IL-1 in immature lung.
Proinflammatory cytokines profoundly influence the expression of surfactant proteins in a manner that is strictly dependent on the length of gestation. The present findings help to explain the differences in the incidence of RDS and CLD in preterm births caused by IUI, and they may clarify further the role of surfactant in the pathogenesesis of lung diseases in neonatal infants.Tiivistelmä
Keuhkosurfaktantin puute aiheuttaa ennenaikaisesti syntyville keskosille vastasyntyneen hengitysvaikeusoireyhtymää eli RDS-tautia (Respiratory Distress Syndrome). Toinen keskosilla esiintyvä keuhkosairaus on krooninen keuhkosairaus eli CLD (Chronic Lung Disease). Glukokortikoideja käytetään hoitona ennenaikaisen synnytyksen uhatessa, koska niiden tiedetään vähentävän RDS-taudin riskiä. Kohdunsisäinen infektio on huomattava ennenaikaisen synnytyksen aiheuttaja. Infektiossa tulehduksen välittäjäaineet, kuten sytokiinit interleukiini-1 (IL-1) ja tuumorinekroositekijä alfa (TNF-α) lisääntyvät lapsivedessä. Infektiosta aiheutunut ennenaikainen synnytys vähentää RDS-taudin ilmaantumista pienille keskosille ja toisaalta lisää kroonisen keuhkosairauden riskiä. Tutkimuksen oli tavoitteena selvittää, miksi RDS ja CLD ilmaantuvat eriävästi infektion vuoksi ennenaikaisesti syntyneille vauvoille.
Viljelemällä eri-ikäisten kanin sikiöiden sekä vastasyntyneiden kanin poikasten keuhkon kappaleita tutkittiin tulehduksen välittäjäaineiden sekä anti-inflammatorisen glukokortikoidin (deksametasonin) vaikutusta surfaktantin toiminnalle tarpeellisten surfaktanttiproteiinien (SP) ilmentymiseen. IL-1 lisäsi SP-A:n ja SP-B:n ilmentymistä erittäin epäkypsässä kanin sikiön keuhkossa. Toisaalta IL-1 ja TNF-α vähensivät SP-B:n ja SP-C:n ilmentymistä kypsemmässä sikiön sekä vastasyntyneen kanin keuhkossa. Interferoni-gamma (IFN-γ) ei vaikuttanut surfaktanttiproteiinien ilmentymiseen missään gestaatioiässä, mutta se muunsi muiden sytokiinien surfaktanttivaikutusta. Gram-negatiivisten bakteerien soluseinän tuote, lipopolysakkaridi (LPS) vähensi SP-A:n, SP-B:n ja SP-C:n ilmentymistä kypsässä kanin sikiön ja vastasyntyneen kanin keuhkossa. IL-1:llä ja deksametasonilla oli positiivinen yhteisvaikutus surfaktanttiproteiinien ilmentymiseen. Tämän surfaktanttiproteiineja lisäävän vaikutuksen mekanismiksi havaittiin pääasiallisesti lisääntynyt mRNA:n stabiliteetti. Lisäksi tutkimus antaa lisätietoa kahden transkriptiofaktorin, NF-κB:n (nuclear factor kappa B) ja C/EBPγ:n (C/CAAT enhancer binding protein delta), osuudesta IL-1:n aiheuttamassa SP-A:n ilmentymisen lisääntymisessä.
Sytokiinien vaikutukset surfaktanttiproteiinien ilmentymiseen ovat riippuvaisia gestaatioiästä. Tutkimuksen löydökset auttavat ymmärtämään RDS:n ja CLD:n vastakohtaista esiintymismäärää keskosilla, joiden ennenaikainen synnytys on aiheutunut kohdunsisäisestä tulehduksesta. Edelleen tutkimus selittää glukokortikoidien positiivista vaikutusta hengitysvajaukseen johtavassa keuhkotulehduksessa
Carbonic anhydrases in the reproductive system : With special emphasis on isoenzymes VI, IX, XII, and a novel nuclear nonclassical form
Full text linkAbstractCarbonic anhydrases (CAs) are a group of zinc-containing metalloenzymes that catalyze the interconversion of carbon dioxide and bicarbonate (CO2 + H2O ⇔ HCO3- + H+). They are present in almost all organs and are implicated in various biological functions, the most important of which is participation in the regulation of ion, water, and acid-base balance. Recently, some members of the CA gene family have been suggested to promote cell proliferation and to act as trophic growth factors. The present study was undertaken to examine the distribution of CA isoenzymes in the reproductive system, to attain a more detailed view on their linkage to the reproductive processes and to neonatal development. The expression of membrane-bound CA IX and CA XII was studied in the female and male reproductive tracts by immunohistochemistry and western blotting. CA XII was found to be expressed in the basolateral plasma membrane of luminal and glandular epithelia in human uterus. In human efferent ducts, it was located in the basolateral plasma membrane of luminal epithelium, where it coexpressed with Aquaporin-1. In epididymal duct, CA XII was only expressed in occasional epithelial cells. These cells coexpressed CA II, suggesting that they represent apical mitochondria-rich cells (AMRC). CA IX was also expressed in the basolateral plasma membrane of luminal epithelium in human efferent ducts, but its expression was not uniform among the tubules. These findings suggest that basolateral plasma membrane-associated CA IX and CA XII contribute, along with CA II and CA IV, to the regulation of acid-base balance and water transport in the reproductive tract.Western blotting of rat Leydig tumor cells and testis for CA II revealed an unidentified 66-kDa polypeptide band. The polypeptide was successfully purified from several rat tissues using CA inhibitor affinity chromatography. The amino acid sequence of the polypeptide showed it to be identical to NonO/p54nrb, a non-POU domain-containing octamer-binding protein previously implicated in transcriptional regulation. The recombinant NonO/p54nrb was shown to display CA activity, and the antibody to it predominantly immunostained the nuclei in lymphocytes, where CA activity was also detected histochemically. Accordingly, the nuclear Leydig cell CA immunoreactivity represents NonO/p54nrb. It is classified as a novel, nonclassical CA, and it may participate in pH-related events in the nucleus.Human and rat milk was found to contain CA VI by immunohistochemistry and western blotting. The enzyme purified from human milk by CA inhibitor affinity chromatography was confirmed by PNGase F digestion and amino acid sequence as CA VI. The CA VI concentrations in human colostral milk were approximately eight times higher than those in mature milk (34.7 mg/l vs. 4.5 mg/l). Secretion of CA VI into milk is suggested by its localization in the alveolar epithelium of the rat mammary gland. The structural and functional stability of CA VI in an acidic milieu, its suggested growth-supporting function in taste bud stem cells, and its high concentration in colostrum suggest that it is an essential factor for the growth and development of the newborn alimentary canal.Academic Dissertation to be presented with the assent of the Faculty of Medicine, University of Oulu, for public discussion in the Auditorium of the Department of Anatomy and Cell Biology, on May 17th, 2002, at 12 noon.Abstract
Carbonic anhydrases (CAs) are a group of zinc-containing metalloenzymes that catalyze the interconversion of carbon dioxide and bicarbonate (CO2 + H2O ⇔ HCO3- + H+). They are present in almost all organs and are implicated in various biological functions, the most important of which is participation in the regulation of ion, water, and acid-base balance. Recently, some members of the CA gene family have been suggested to promote cell proliferation and to act as trophic growth factors.
The present study was undertaken to examine the distribution of CA isoenzymes in the reproductive system, to attain a more detailed view on their linkage to the reproductive processes and to neonatal development.
The expression of membrane-bound CA IX and CA XII was studied in the female and male reproductive tracts by immunohistochemistry and western blotting. CA XII was found to be expressed in the basolateral plasma membrane of luminal and glandular epithelia in human uterus. In human efferent ducts, it was located in the basolateral plasma membrane of luminal epithelium, where it coexpressed with Aquaporin-1. In epididymal duct, CA XII was only expressed in occasional epithelial cells. These cells coexpressed CA II, suggesting that they represent apical mitochondria-rich cells (AMRC). CA IX was also expressed in the basolateral plasma membrane of luminal epithelium in human efferent ducts, but its expression was not uniform among the tubules. These findings suggest that basolateral plasma membrane-associated CA IX and CA XII contribute, along with CA II and CA IV, to the regulation of acid-base balance and water transport in the reproductive tract.
Western blotting of rat Leydig tumor cells and testis for CA II revealed an unidentified 66-kDa polypeptide band. The polypeptide was successfully purified from several rat tissues using CA inhibitor affinity chromatography. The amino acid sequence of the polypeptide showed it to be identical to NonO/p54nrb, a non-POU domain-containing octamer-binding protein previously implicated in transcriptional regulation. The recombinant NonO/p54nrb was shown to display CA activity, and the antibody to it predominantly immunostained the nuclei in lymphocytes, where CA activity was also detected histochemically. Accordingly, the nuclear Leydig cell CA immunoreactivity represents NonO/p54nrb. It is classified as a novel, nonclassical CA, and it may participate in pH-related events in the nucleus.
Human and rat milk was found to contain CA VI by immunohistochemistry and western blotting. The enzyme purified from human milk by CA inhibitor affinity chromatography was confirmed by PNGase F digestion and amino acid sequence as CA VI. The CA VI concentrations in human colostral milk were approximately eight times higher than those in mature milk (34.7 mg/l vs. 4.5 mg/l). Secretion of CA VI into milk is suggested by its localization in the alveolar epithelium of the rat mammary gland. The structural and functional stability of CA VI in an acidic milieu, its suggested growth-supporting function in taste bud stem cells, and its high concentration in colostrum suggest that it is an essential factor for the growth and development of the newborn alimentary canal
Luteinizing hormone receptor : Expression and post-translational regulation of the rat receptor and its ectodomain splice variant
Full text linkAbstractThe luteinizing hormone receptor (LHR) is a G protein-coupled receptor (GPCR) that has a large N-terminal ligand binding ectodomain. The LHR ectodomain splice variant, expressed concomitantly with the full-length LHR in tissues, has an unknown biological function. GPCRs are a major pharmacological target, however, very little is known about the intracellular regulation of these receptors. In the present work, expression and maturation of the rat LHR and its variant were elucidated using both tissues and heterologous expression systems. A special effort was made to identify the role of developmental stage and tissue type on the LHR maturation and to find out about the molecular role of the ectodomain splice variant.We found two sites of localization for the receptor, namely the sensory system and urogenital tissues. This was demonstrated at mRNA and protein level and by rat LHR promoter-driven β-galactosidase (β-Gal) expression in the mice. In neurons, the β-Gal co-localized with the cytochrome P450 side chain cleavage enzyme, which may indicate a novel role in the neurosteroid synthesis.The neuronal LHR was expressed in the mature and immature protein forms in both developing and adult tissues, being able to bind hormone with similar high-affinity as gonadal receptors. In contrast, only immature receptors were detected in the fetal rat urogenital structures. A significant novel finding was substantial upregulation of the LHR in pregnant female rat adrenal glands and kidneys at a time that coincides with the differentiation of the fetal urogenital tissues.The mice overexpressing the ectodomain splice variant showed interference in pituitary-gonadal functions and morphological changes in the urogenital tissues. The studies showed that the variant was an endoplasmic reticulum (ER)-retained soluble protein. It accumulated in juxtanuclear regions of the ER together with ER folding chaperones and was a substrate for ER associated degradation (ERAD). The co-expression of the variant with the full-length receptor decreased the amount of receptors and misrouted them to the juxtanuclear ER subcompartment.Taken together, we suggest that the maturation of the LHR protein is developmentally and physiologically regulated at the post-translational level in tissues. The LHR ectodomain splice variant possibly modulates post-translationally the number of full-length receptors through physiological signals. Our observation of the chaperone and protein accumulation into a specific ER subcompartment may represent a protein quality control holding compartment for inefficiently/misfolded ERAD substrates.Academic Dissertation to be presented with the assent of the Faculty of Medicine, University of Oulu, for public discussion in Auditorium 5 of the Institute of Dentistry, on November 25th, 2005, at 12 noonAbstract
The luteinizing hormone receptor (LHR) is a G protein-coupled receptor (GPCR) that has a large N-terminal ligand binding ectodomain. The LHR ectodomain splice variant, expressed concomitantly with the full-length LHR in tissues, has an unknown biological function. GPCRs are a major pharmacological target, however, very little is known about the intracellular regulation of these receptors. In the present work, expression and maturation of the rat LHR and its variant were elucidated using both tissues and heterologous expression systems. A special effort was made to identify the role of developmental stage and tissue type on the LHR maturation and to find out about the molecular role of the ectodomain splice variant.
We found two sites of localization for the receptor, namely the sensory system and urogenital tissues. This was demonstrated at mRNA and protein level and by rat LHR promoter-driven β-galactosidase (β-Gal) expression in the mice. In neurons, the β-Gal co-localized with the cytochrome P450 side chain cleavage enzyme, which may indicate a novel role in the neurosteroid synthesis.
The neuronal LHR was expressed in the mature and immature protein forms in both developing and adult tissues, being able to bind hormone with similar high-affinity as gonadal receptors. In contrast, only immature receptors were detected in the fetal rat urogenital structures. A significant novel finding was substantial upregulation of the LHR in pregnant female rat adrenal glands and kidneys at a time that coincides with the differentiation of the fetal urogenital tissues.
The mice overexpressing the ectodomain splice variant showed interference in pituitary-gonadal functions and morphological changes in the urogenital tissues. The studies showed that the variant was an endoplasmic reticulum (ER)-retained soluble protein. It accumulated in juxtanuclear regions of the ER together with ER folding chaperones and was a substrate for ER associated degradation (ERAD). The co-expression of the variant with the full-length receptor decreased the amount of receptors and misrouted them to the juxtanuclear ER subcompartment.
Taken together, we suggest that the maturation of the LHR protein is developmentally and physiologically regulated at the post-translational level in tissues. The LHR ectodomain splice variant possibly modulates post-translationally the number of full-length receptors through physiological signals. Our observation of the chaperone and protein accumulation into a specific ER subcompartment may represent a protein quality control holding compartment for inefficiently/misfolded ERAD substrates
Sex steroid metabolism in the placenta and the breast
Full text linkAbstractThe biosynthesis and metabolism of sex steroids are controlled by a series of steroidogenic enzymes. In the placenta and the breast, 3β-hydroxysteroid dehydrogenase type 1 (3β-HSD1) is essential for the synthesis of all steroid hormones by catalyzing pregnenolone to progesterone (P) or dehydroepiandrosterone (DHEA) to androstenedione (A-dione). P450 aromatase (P450arom) converts androgens to estrogens and is therefore critical for estrogen production. 17β-hydroxysteroid dehydrogenases (17HSDs) are a group of enzymes responsible for the interconversion between low-activity 17-ketosteroids and high-activity 17β-hydroxysteroids, thus acting as key enzymes modulating the biosynthesis and metabolism of both estrogens and androgens. In situ hybridization assays showed that 3β-HSD1, P450arom and 17HSD1, 2, 5 and 7 are expressed in early and mid-gestation placentas. Abundant expressions of 3β-HSD1, P450arom and 17HSD1 were seen in syncytiotrophoblast (ST) cells. Signals of these three enzymes were also detected in some column cytotrophoblast (CCT) cells. 17HSD2 and 5 were located in intravillous stromal (IS) cells, whereas 17HSD7 mRNA was present in all types of placental cells. This suggests that the human placenta produces not only P and estrogens, but also androgens. Moreover, the placenta possesses a function, by the action of 17HSD2, to protect the fetus and the maternal body from excessive sex steroid influence. In tubal pregnancy, P450arom and 17HSD1 were found in ST cells, implying an estrogen biosynthesis mechanism similar to that in normal intrauterine pregnancy. In both JEG-3 choriocarcinoma cell line and cultured normal human cytotrophoblast (CTB) cells, retinoic acids were shown to promote the enzyme activity as well as mRNA expression of P450arom and 17HSD1, and hence their action on the biosynthesis of E2. The mRNA expressions of 17HSD1, 2 and 5 in 794 breast carcinoma specimens were analyzed and correlated with ERα, ERβ, PR, Ki67, c-erbB2 and clinical parameters. 17HSD1, 2 and 5 were detected in epithelial cells in normal and malignant breast tissues. In breast cancer specimens, the positive cases for 17HSD1, 2 and 5 were 16%, 25% and 65%, respectively. 17HSD1 was found to be an independent prognostic marker of the progression of breast cancer.Academic Dissertation to be presented with the assent of the Faculty of Medicine, University of Oulu, for public discussion in the Auditorium 9 of Oulu University Hospital, on February 20th, 2004, at 12 noon.Abstract
The biosynthesis and metabolism of sex steroids are controlled by a series of steroidogenic enzymes. In the placenta and the breast, 3β-hydroxysteroid dehydrogenase type 1 (3β-HSD1) is essential for the synthesis of all steroid hormones by catalyzing pregnenolone to progesterone (P) or dehydroepiandrosterone (DHEA) to androstenedione (A-dione). P450 aromatase (P450arom) converts androgens to estrogens and is therefore critical for estrogen production. 17β-hydroxysteroid dehydrogenases (17HSDs) are a group of enzymes responsible for the interconversion between low-activity 17-ketosteroids and high-activity 17β-hydroxysteroids, thus acting as key enzymes modulating the biosynthesis and metabolism of both estrogens and androgens.
In situ hybridization assays showed that 3β-HSD1, P450arom and 17HSD1, 2, 5 and 7 are expressed in early and mid-gestation placentas. Abundant expressions of 3β-HSD1, P450arom and 17HSD1 were seen in syncytiotrophoblast (ST) cells. Signals of these three enzymes were also detected in some column cytotrophoblast (CCT) cells. 17HSD2 and 5 were located in intravillous stromal (IS) cells, whereas 17HSD7 mRNA was present in all types of placental cells. This suggests that the human placenta produces not only P and estrogens, but also androgens. Moreover, the placenta possesses a function, by the action of 17HSD2, to protect the fetus and the maternal body from excessive sex steroid influence.
In tubal pregnancy, P450arom and 17HSD1 were found in ST cells, implying an estrogen biosynthesis mechanism similar to that in normal intrauterine pregnancy. In both JEG-3 choriocarcinoma cell line and cultured normal human cytotrophoblast (CTB) cells, retinoic acids were shown to promote the enzyme activity as well as mRNA expression of P450arom and 17HSD1, and hence their action on the biosynthesis of E2.
The mRNA expressions of 17HSD1, 2 and 5 in 794 breast carcinoma specimens were analyzed and correlated with ERα, ERβ, PR, Ki67, c-erbB2 and clinical parameters. 17HSD1, 2 and 5 were detected in epithelial cells in normal and malignant breast tissues. In breast cancer specimens, the positive cases for 17HSD1, 2 and 5 were 16%, 25% and 65%, respectively. 17HSD1 was found to be an independent prognostic marker of the progression of breast cancer
17β-Hydroxysteroid dehydrogenases/17-ketosteroid reductases (17HSD/KSRs) in prostate cancer : The role of 17HSD/KSR types 2, 5, and 7 in steroid hormone action and loss of heterozygosity at chromosome region 16q
Full text linkAbstractProstate cancer is the most frequently diagnosed cancer in men in industrialized countries. Despite the substantial clinical importance of the disease, the mechanisms underlying the development and progression of prostate cancer are poorly understood. In the present study, fragment analysis of chromosome arm 16q was carried out with the aim of searching for sites of consistent chromosomal deletion, possibly uncovering the location of target genes that become inactivated in prostate carcinogenesis. The highest percentage of loss of heterozygosity (LOH) was found at chromosomal region 16q24.1-q24.2, including the gene for 17β-hydroxysteroid dehydrogenase/17-ketosteroid reductase (17HSD/KSR) type 2, HSD17B2. The data further indicated an association between loss of the most commonly deleted region and clinically aggressive features of the disease. A fragment analysis performed using sequential primary and locally recurrent prostate cancer specimens suggested the location of the genes related to prostate cancer progression to be at 16q24.3 and, further, gave rise to a hypothesis of the potential role of locus HSD17B2 as a prognostic marker for prostate cancer progression. Quantitative real-time polymerase chain reaction (PCR) revealed a decreased HSD17B2 gene copy number in prostate cancer specimens compared to their normal counterparts. A diminished HSD17B2 gene copy number was significantly associated with poor differentiation of the tumor. The progression of prostate cancer during androgen deprivation is a serious clinical problem, the molecular mechanisms of which largely remain to be clarified. The present data of enzyme activity measurements performed using high-performance liquid chromatography (HPLC) provided evidence of a substantial decrease in oxidative and an increase in reductive 17HSD/KSR activity during the transition of prostate cancer LNCaP cells into an androgen-independent state. Further, the changes detected in the activities largely coincided with the changes in the relative expression levels of genes for the potential 17HSD/KSR isoenzymes; 17HSD/KSR types 2, 5, and 7, as evidenced by relative quantitative reverse transcription PCR (RT-PCR). The data on the expression analysis of mRNA for 17HSD/KSR types 5 and 7 in prostate tissue specimens performed using in situ hybridization showed a moderately low but constitutive level for 17HSD/KSR7 mRNA in tissues of cancerous as well as hyperplastic origin. The expression of mRNA for 17HSD/KSR type 5, instead, varied considerably between different specimens, the highest expressions being strongly associated with aggressive and metastatic prostate cancer. Interestingly, furthermore, the intense expression of 17HSD/KSR5 was significantly associated with the androgen deprivation achieved either surgically or medically.Since 17HSD/KSRs critically contribute to the control of the bioavailability of active sex steroid hormones locally in the prostate, the variation in intraprostatic 17HSD/KSR activity might be crucially involved in the regulation of the growth and function of the organ.Academic Dissertation to be presented with the assent of the Faculty of Medicine, University of Oulu, for public discussion in the Auditorium 8 of Oulu University Hospital, on December 2nd, 2005, at 11 a.m.Abstract
Prostate cancer is the most frequently diagnosed cancer in men in industrialized countries. Despite the substantial clinical importance of the disease, the mechanisms underlying the development and progression of prostate cancer are poorly understood.
In the present study, fragment analysis of chromosome arm 16q was carried out with the aim of searching for sites of consistent chromosomal deletion, possibly uncovering the location of target genes that become inactivated in prostate carcinogenesis. The highest percentage of loss of heterozygosity (LOH) was found at chromosomal region 16q24.1-q24.2, including the gene for 17β-hydroxysteroid dehydrogenase/17-ketosteroid reductase (17HSD/KSR) type 2, HSD17B2. The data further indicated an association between loss of the most commonly deleted region and clinically aggressive features of the disease. A fragment analysis performed using sequential primary and locally recurrent prostate cancer specimens suggested the location of the genes related to prostate cancer progression to be at 16q24.3 and, further, gave rise to a hypothesis of the potential role of locus HSD17B2 as a prognostic marker for prostate cancer progression. Quantitative real-time polymerase chain reaction (PCR) revealed a decreased HSD17B2 gene copy number in prostate cancer specimens compared to their normal counterparts. A diminished HSD17B2 gene copy number was significantly associated with poor differentiation of the tumor.
The progression of prostate cancer during androgen deprivation is a serious clinical problem, the molecular mechanisms of which largely remain to be clarified. The present data of enzyme activity measurements performed using high-performance liquid chromatography (HPLC) provided evidence of a substantial decrease in oxidative and an increase in reductive 17HSD/KSR activity during the transition of prostate cancer LNCaP cells into an androgen-independent state. Further, the changes detected in the activities largely coincided with the changes in the relative expression levels of genes for the potential 17HSD/KSR isoenzymes; 17HSD/KSR types 2, 5, and 7, as evidenced by relative quantitative reverse transcription PCR (RT-PCR). The data on the expression analysis of mRNA for 17HSD/KSR types 5 and 7 in prostate tissue specimens performed using in situ hybridization showed a moderately low but constitutive level for 17HSD/KSR7 mRNA in tissues of cancerous as well as hyperplastic origin. The expression of mRNA for 17HSD/KSR type 5, instead, varied considerably between different specimens, the highest expressions being strongly associated with aggressive and metastatic prostate cancer. Interestingly, furthermore, the intense expression of 17HSD/KSR5 was significantly associated with the androgen deprivation achieved either surgically or medically.
Since 17HSD/KSRs critically contribute to the control of the bioavailability of active sex steroid hormones locally in the prostate, the variation in intraprostatic 17HSD/KSR activity might be crucially involved in the regulation of the growth and function of the organ
Regulation of apoptosis in the female reproductive system
Full text linkAbstractApoptosis is a genetically programmed mechanism for a multicellular organism to remove cells that are unnecessary, or potentially harmful. The female reproductive system is characterised by a high rate of cellular proliferation. At the same time, apoptosis is also abundant during the normal physiological function of the ovary and endometrium. More than half of the 7 million oocytes that are produced during human ovarian development are deleted before birth and only about 400 oocytes reach the stage of ovulation during the female fertile lifespan. The fate of the non-ovulatory follicles is atresia, occurring through the mechanism of apoptosis. The endometrium goes through radical renewal processes during each menstrual cycle. Apoptosis has been suggested to participate in the regulation of endometrial cellular homeostasis. Errors in this mechanism can result in endometrial diseases such as hyperplasia and cancer. In this work, apoptosis and its regulation were studied in the human fetal and adult ovary, normal endometrium and endometrial pathologies.In fetal ovaries, apoptosis was already abundantly present in oocytes at 13 weeks of gestation. The maximum rate of apoptosis was seen between the 14th and 20th weeks, after which apoptosis decreased towards term. Ovarian Bcl-2 expression was detected in early fetal life during weeks 13 and 14. Bax expression was observed throughout the studied period, from week 13 to 40. The expression of transcription factor GATA-4, which is linked to follicular survival, was localised to the granulosa cells and was high in early fetal life and decreased somewhat towards term. In adult life apoptosis was located in the granulosa cells of the growing follicles. In ovarian biopsies from women homozygous for the inactivating C566T mutation of the FSH receptor, apoptosis or GATA-4 expression was not detected. During corpus luteum regression a peak in apoptosis was detected 10–12 days after the LH surge, and was preceded by an increase in 17HSD type 1 and TNF-α expression. During normal menstrual cycles, the highest rate of apoptosis was observed in the menstrual endometrium. This increase in apoptosis was preceded by a decreased Bcl-2/Bax ratio. In endometrial hyperplasia, the rate of apoptosis was similar to that seen during normal proliferation of the endometrium, but an apparent increase was observed in grade II endometrial carcinoma. In grade III carcinoma, the rate of apoptosis was lower than in grade II carcinoma but higher than in hyperplasia.These results indicate that apoptosis is the mechanism behind the substantial oocyte demise during ovarian development. During adult life, apoptosis was mainly localised to the granulosa cells of the growing follicles which do not reach the stage of a dominant follicle. In ovaries where FSH action is abolished, folliculogenesis was impaired and ovarian apoptosis was negligible. Apoptosis is also the underlying mechanism of corpus luteum regression. In the endometrium, apoptosis has a role in rejuvenating the endometrium for growth during the next endometrial cycle and in regulating cellular homeostasis.Academic Dissertation to be presented with the assent of the Faculty of Medicine, University of Oulu, for public discussion in the Auditorium 4 of the University Hospital of Oulu, on May 8th, 2002, at 12 noon.Abstract
Apoptosis is a genetically programmed mechanism for a multicellular organism to remove cells that are unnecessary, or potentially harmful. The female reproductive system is characterised by a high rate of cellular proliferation. At the same time, apoptosis is also abundant during the normal physiological function of the ovary and endometrium. More than half of the 7 million oocytes that are produced during human ovarian development are deleted before birth and only about 400 oocytes reach the stage of ovulation during the female fertile lifespan. The fate of the non-ovulatory follicles is atresia, occurring through the mechanism of apoptosis. The endometrium goes through radical renewal processes during each menstrual cycle. Apoptosis has been suggested to participate in the regulation of endometrial cellular homeostasis. Errors in this mechanism can result in endometrial diseases such as hyperplasia and cancer. In this work, apoptosis and its regulation were studied in the human fetal and adult ovary, normal endometrium and endometrial pathologies.
In fetal ovaries, apoptosis was already abundantly present in oocytes at 13 weeks of gestation. The maximum rate of apoptosis was seen between the 14th and 20th weeks, after which apoptosis decreased towards term. Ovarian Bcl-2 expression was detected in early fetal life during weeks 13 and 14. Bax expression was observed throughout the studied period, from week 13 to 40. The expression of transcription factor GATA-4, which is linked to follicular survival, was localised to the granulosa cells and was high in early fetal life and decreased somewhat towards term. In adult life apoptosis was located in the granulosa cells of the growing follicles. In ovarian biopsies from women homozygous for the inactivating C566T mutation of the FSH receptor, apoptosis or GATA-4 expression was not detected. During corpus luteum regression a peak in apoptosis was detected 10–12 days after the LH surge, and was preceded by an increase in 17HSD type 1 and TNF-α expression. During normal menstrual cycles, the highest rate of apoptosis was observed in the menstrual endometrium. This increase in apoptosis was preceded by a decreased Bcl-2/Bax ratio. In endometrial hyperplasia, the rate of apoptosis was similar to that seen during normal proliferation of the endometrium, but an apparent increase was observed in grade II endometrial carcinoma. In grade III carcinoma, the rate of apoptosis was lower than in grade II carcinoma but higher than in hyperplasia.
These results indicate that apoptosis is the mechanism behind the substantial oocyte demise during ovarian development. During adult life, apoptosis was mainly localised to the granulosa cells of the growing follicles which do not reach the stage of a dominant follicle. In ovaries where FSH action is abolished, folliculogenesis was impaired and ovarian apoptosis was negligible. Apoptosis is also the underlying mechanism of corpus luteum regression. In the endometrium, apoptosis has a role in rejuvenating the endometrium for growth during the next endometrial cycle and in regulating cellular homeostasis
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