Willem Dhooge

Endocrine disruptors and abnormalities of pubertal development.

Onset and development of puberty is regulated by the neuroendocrine system. Population-based studies worldwide have observed secular trends towards earlier puberty development. These changes are apparently caused by environmental factors such as improved socio-economic status, improved health care and nutrition. However, they may also partly result from endocrine-disrupting chemicals in the environment. Epidemiological studies have investigated the relationship between pubertal development and exposure to endocrine-disrupting chemicals (polychlorinated biphenyls, polybrominated biphenyls, 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane, phthalate esters, furans and the pesticide endosulfan). Associations with both perinatal and postnatal exposure have been reported. Studies in experimental animals support some of these findings and point to differential endocrine regulatory mechanisms linked to pubertal development acting in the perinatal and the pre-pubertal period. Pubertal development is naturally associated with growth and body composition. There is increasing evidence for a link between prenatal development and pubertal onset. In girls born small for gestational age (SGA), pubertal onset and age at menarche often are advanced, especially if there has been an extensive catch-up growth during the first months of life. In utero growth retardation may have multiple causes including exposure to xenobiotic substances as was suggested for some endocrine-disrupting chemicals. An abnormal perinatal environment of children born SGA may alter the endocrine status and the sensitivity of the receptors for endocrine and metabolic signalling that may have effects on maturation of brain and gonads. However, the causal pathways and the molecular mechanisms that may link the pubertal growth pattern of children born SGA, pubertal development and endocrine-disrupting chemicals need further study.

Internal exposure to pollutants and sexual maturation in Flemish adolescents.

Flanders is densely populated with much industry and intensive farming. Sexual maturation of adolescents (aged 14–15 years) was studied in relation to internal exposure to pollutants. Serum levels of pollutants and sex hormones were measured in 1679 participants selected as a random sample of the adolescents residing in the study areas. Data on sexual development were obtained from the medical school examination files. Self-assessment questionnaires provided information on health, use of medication and lifestyle factors. In boys, serum levels of hexachlorobenzene (HCB), p,p′-DDE and polychlorinated biphenyls (sum of marker PCB138, 153 and 180) were significantly and positively associated with pubertal staging (pubic hair and genital development). Higher levels of serum HCB and blood lead were associated with, respectively, a lower and a higher risk of gynecomastia. In girls, significant and negative associations were detected between blood lead and pubic hair development; higher exposure to PCBs was significantly associated with a delay in timing of menarche. Environmental exposures to pollutants at levels actually present in the Flemish population are associated with measurable effects on pubertal development. However, further understanding of toxic mode of action and sensitive windows of exposure is needed to explain the current findings.

Serum dioxin-like activity is associated with reproductive parameters in young men from the general flemish population

Background 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and some related environmental contaminants are aryl hydrocarbon receptor (AhR) ligands that exert reproductive and developmental toxicity in laboratory animals. In humans, fertility-related effects are less documented. Objective The aim of this study was to investigate the relationship between dioxin-like biological activity in serum and parameters of reproductive status in men from the general population 5 months after a polychlorinated biphenyl and dioxin food-contamination episode in Belgium. Design In the framework of the cross-sectional Flemish Environment and Health Study (FLEHS), we recruited 101 men 20–40 years of age and evaluated sperm parameters, measured sex hormones, and gathered information on a number of lifestyle factors. In addition, we determined the AhR-mediated enzymatic response elicited by individual serum samples and expressed it as TCDD equivalent concentrations (CALUX-TEQs) using an established transactivation assay. Results Age (p = 0.04) and the frequency of fish (p = 0.02) and egg (p = 0.001) consumption were independent positive determinants of serum dioxin-like activity. After correcting for possible confounders, we found that a 2-fold increase in CALUX-TEQ > 16 pg/L was associated with a 7.1% and 6.8% (both p = 0.04) decrease in total and free testosterone, respectively. We also observed a more pronounced drop in semen volume of 16.0% (p = 0.03), whereas sperm concentration rose by 25.2% (p = 0.07). No relationship was found with total sperm count or sperm morphology. Conclusions These data suggest an interaction of dioxin-like compounds with the secretory function of the seminal vesicles or prostate, possibly indirectly through an effect on testosterone secretion, at levels not affecting spermatogenesis as such.

Internal exposure to pollutants and sex hormone levels in Flemish male adolescents in a cross-sectional study: associations and dose–response relationships

Flanders is densely populated with much industry and intensive farming. Hormonal status of 14- to 15-year-old male adolescents was studied in relation to internal exposure to pollutants. A total of 887 participants were selected as a random sample of the adolescents residing in the study areas. Confounding factors and significant covariates were taken into account. Serum levels of testosterone, free testosterone and estradiol, and the aromatase index showed significant positive associations with serum levels of marker polychlorobiphenyls (sum of PCBs 138, 153, and 180) and of hexachlorobenzene (HCB) and a negative association with urinary cadmium concentration. Serum levels of estradiol also showed a positive association with serum levels of dichlorodiphenyldichloroethylene (DDE). A doubling of serum concentrations of marker PCBs and HCB and of urinary concentration of cadmium were, respectively, associated with an increase of 16.4% (P<0.00001) and 16.6% (P<0.001) and a decrease of 9.6% (P<0.001) in serum testosterone concentration. Similar findings were made after additional adjustment for concurrent exposures. Associations between biological effects and internal exposures were, in terms of the regression coefficient, often stronger at exposures below the median. Environmental exposures to pollutants resulting in “normal” levels of internal exposure were associated with quite substantial differences in hormone concentrations.

Estrogen receptor alpha (ERα) and insulin-like growth factor I receptor (IGF-IR) cross-talk in the gonadotropic αT3-1 cell line

In reproductive tissues such as the breast and the uterus, cell proliferation and differentiation is strongly regulated by complex interactions between estrogen receptor α (ERα) and growth factor receptors. In the present study, we investigated the potential occurrence of such cross‐talk in the murine, gonadotropic αT3‐1 cell line, which expresses ERα and the IGF‐I receptor (IGF‐IR). Under estrogen‐free conditions, basal cell proliferation and ER‐mediated gene transcription was strongly inhibited by the selective estrogen receptor modulator (SERM) 4‐hydroxy‐tamoxifen (4‐OH‐Tam) and by the pure anti‐estrogen ICI 182,780 (ICI). These effects can be reversed by either 17‐β‐estradiol (E2) or insulin‐like growth factor I (IGF‐I), both exerting modest mitogenic effects in the αT3‐1 cell line. Furthermore, IGF‐I enhanced both basal and E2‐induced ER‐driven gene transcription. This may be explained, at least in part, by enhanced phosphorylation of ERα at serine 118, a prerequisite for the transactivation capacity of the receptor. Finally, the IGF‐I‐induced response on cell growth and ER‐mediated transactivation can be inhibited with either ICI or 4‐OH‐Tam. In conclusion, our data indicate IGF‐IR and ER interactions in the αT3‐1 cell line, an in vitro model for the pituitary gonadotrophs, hereby suggesting a role of IGF‐I in the regulation of gonadotropin synthesis and secretion. J. Cell. Physiol. 212:583–590, 2007.

Which efforts towards conservative treatment of male infertility will be successful? Reactive oxygen species, antioxidants, and sperm phospholipids

Polymorphonuclear white blood cells in semen and, to a lesser extent, incompletely matured spermatozoa generate excess reactive oxygen species (ROS), mostly hydrogen peroxide and nitric oxide. Due to their paucity in cytoplasm and antioxidant mechanisms, spermatozoa have little defence against these ROS, which damage their membrane and DNA (Fig. 1). The former results in a lower proportion of polyunsaturated fatty acids, mainly docosohexaenoic acid (2256v3), with increased mean melting point and decreased fluidity of the membrane, particularly of the sperm head (Zalata et al., 1998). As a consequence, sperm acrosome reaction and fusogenic capacity are inhibited, causing decreased probability of conception, lower success rate of Figure 1. Overview of the effects of ROS and the alleged protective influence of antioxidants. intra-uterine insemination (IUI) (Milingos et al., 1996) and of conventional IVF (Aitken, 1989). included antibiotics in case of documented infection Damage to sperm DNA may result in transition of the accessory sex glands, spermatic venous mutagenesis whereby the oxidized product of embolization in case of varicocele, or Tamoxifen the nucleotide guanine (8-OH-2-deoxyguanosine) (Zambon) for idiopathic oligozoospermia. binds to thymine instead of cytosine (C5G conSperm concentration increased significantly verting to A5T). When spermatozoa with damaged in cases with initial oligozoospermia (<20 DNA are introduced into oocytes during ICSI, million ml−1, P<0.01), and the concentration of genetic disturbance may occur in the offspring if peroxidase-positive round cells (polymorphonuthe repair mechanisms are insufficient. clear white blood cells) remained unchanged. In Antioxidant treatment may reduce the oxidative 21 out of 22 cases ROS was decreased during damage and it may increase the in vivo and in vitro treatment to 16% of the initial value (P<0.001). fertility of spermatozoa, as well as their relative In parallel, the concentration of 8-OH-2-deoxysafety for use in ICSI. We have performed a pilot guanosine was decreased to 37% of the value study treating subfertile patients with either oral before treatment (P<0.001) and, in addition, the acetyl-cysteine (gift of Zambon, Brussels, Belgium) calcium–ionophore-induced acrosome reaction and/or a mixture of essential fatty acids (rich in increased significantly (P<0.01). docosohexaenoic acid) and natural vitamins A and Five couples out of 22 with adequate follow-up E (gift of Scotia, Stirling, Scotland) as a complement of 5.4 months (median) achieved pregnancy, correto conventional medical treatment. The latter sponding to a fecundability of 3.5% per cycle, as compared with 1.2% per cycle in historically Correspondence: F. Comhaire, Department of Endocrinology and Centre for Andrology, University Hospital Gent, Belgium. untreated or placebo-treated controls.

Male Reproductive Status and Its Relationship With Man-Made, Hormone-Disrupting Substances: Studies in Flanders, Belgium

Sperm quality seems to have declined over time in certain regions in the world. This regional trend coincides with the progressive increase in the incidence of male genital tract anomalies including testicular maldescent, hypospadias and testicular cancer. The association of these pathologies has been called the ‘testicular dysgenesis syndrome’ (TDS). The possible implications of decreased male fertility cannot be underestimated. Couples attending fertility clinics have a higher chance of multiple pregnancies, preterm delivery and consequently also of low birth weight, which is a known risk factor for a number of pathological conditions at adulthood. Many studies suggest TDS to be caused by man-made endocrine disrupters, mostly chemicals initiating estrogen actions (xenoestrogens) or inhibiting the effects of endogenous androgens (anti-androgens). Thousands of man-made chemicals have been released into the environment in vast quantities since the chemical industry began to boom in the 1950’s. Humans and wildlife are exposed to these chemicals through their nutrition, the air, the water and, more importantly, through the placenta during foetal development. In order to reliably estimate the reproductive health risks posed by these chemicals, scientific programmes should pursue the identification of the routes via which humans and wildlife are exposed to these xenobiotics using biomarkers of internal and external exposure. These are new and important tools, complementary to the traditional chemical analytical techniques as they reflect the integrated response of an individual or test organism to multiple compounds, accounting for possible additive, antagonistic or synergistic effects.

Further evaluation of the biological activity of the unique gonadotropin-releasing hormone peptide in the guinea pig brain.

In this study we compared the biological activity of a unique form of gonadotropin-releasing hormone (GnRH) in the brain of the guinea pig (gpGnRH) with mammalian GnRH (mGnRH). In gpGnRH, the highly conserved histidine in position 2 (His(2)) and leucine in position 7 (Leu(7)) are substituted by tyrosine and valine, respectively. The gpGnRH was less potent than mGnRH in stimulating the release of luteinizing hormone (LH) in vivo in the guinea pig and displayed only low activity in the rat. The gpGnRH was more rapidly degraded by serum proteolytic enzymes than mGnRH. It is concluded that gpGnRH displays lower biological activity than mGnRH in both rat and guinea pig, which may be due in part to its greater susceptibility to proteolytic degradation besides differences in receptor affinity and/or activation.

Difference in receptor-binding contributes to difference in biological activity between the unique guinea pig GnRH and mammalian GnRH

In the current study, we compared the in vitro potency of a unique form of gonadotropin-releasing hormone (GnRH) present in the brain of the guinea pig (gpGnRH) with that of mammalian GnRH (mGnRH) as well as their binding affinities to the GnRH receptor. In gpGnRH, the highly conserved histidine in position 2 (His(2)) and leucine in position 7 (Leu(7)) are substituted by tyrosine and valine, respectively. In vivo, gpGnRH was shown to be less potent than mGnRH, possibly in part because of higher susceptibility to enzymatic degradation. In the present in vitro experiments, we observed that gpGnRH was less potent than mGnRH in stimulating the release of luteinizing hormone (LH) from primary pituitary cell cultures of the rat, and at lower concentrations from primary pituitary cell culture of the guinea pig, too. These results were confirmed by radioligand-binding studies. It is concluded that the lower biological activity of gpGnRH in both rat and guinea pig may be explained by the difference in binding to the target cells, although additional factors such as proteolytic degradation may also contribute to the observed phenomenon.