Morphological changes induced by testosterone in the mammary glands of female Wistar rats

Increased levels of androgens in postmenopausal women are considered to be a risk factor for breast cancer. Testosterone, alone or in combination with estrogen, induces epithelial dysplasia and mammary tumors in Noble rats. Since this model of hormone-induced neoplasia has not been reported in other rat strains, we studied the effect of testosterone on the mammary gland morphology of female Wistar rats. Sixty adult, non-castrated, female Wistar rats were implanted in the dorsum midline with a silicone tube containing 50 mg testosterone (testosterone propionate in 30 animals and non-esterified testosterone in the remaining 30 animals) and 20 additional animals were implanted with empty tubes and used as control. Five animals per group were killed 30, 60, 90, 120, 150, and 180 days after implantation, and the mammary glands were dissected, fixed and embedded in paraffin. Histological sections were then stained with hematoxylin and eosin and picrosyrius red for collagen visualization. Morphological and morphometric analysis demonstrated ductal proliferation and acinotubular differentiation with secretory activity in all treated animals, peaking at 90 days of androgen exposure. After 90 days the proliferation of acinar epithelial cells was evident, but there was a progressive reduction of secretory differentiation and an increase in intralobular collagen fibers. There was no morphological evidence of dysplastic changes or other pre-neoplastic lesions. Testosterone treatment applied to adult, non-castrated female Wistar rats induced a mammary gland hyperplasia resembling the lactating differentiation, with progressive reduction in secretory differentiation.

A connection between extracellular matrix and hormonal signals during the development of the human fetal adrenal gland

The human adrenal cortex, involved in adaptive responses to stress, body homeostasis and secondary sexual characters, emerges from a tightly regulated development of a zone-specific secretion pattern during fetal life. Its development during fetal life is critical for the well being of pregnancy, the initiation of delivery, and even for an adequate adaptation to extra-uterine life. As early as from the sixth week of pregnancy, the fetal adrenal gland is characterized by a highly proliferative zone at the periphery, a concentric migration accompanied by cell differentiation (cortisol secretion) and apoptosis in the central androgen-secreting fetal zone. After birth, a strong reorganization occurs in the adrenal gland so that it better fulfills the newborn's needs, with aldosterone production in the external zona glomerulosa, cortisol secretion in the zona fasciculata and androgens in the central zona reticularis. In addition to the major hormonal stimuli provided by angiotensin II and adrenocorticotropin, we have tested for some years the hypotheses that such plasticity may be under the control of the extracellular matrix. A growing number of data have been harvested during the last years, in particular about extracellular matrix expression and its putative role in the development of the human adrenal cortex. Laminin, collagen and fibronectin have been shown to play important roles not only in the plasticity of the adrenal cortex, but also in cell responsiveness to hormones, thus clarifying some of the unexplained observations that used to feed controversies.

Allogeneic hematopoietic stem cell transplantation from an alternative stem cell source in Fanconi anemia patients: analysis of 47 patients from a single institution

We transplanted 47 patients with Fanconi anemia using an alternative source of hematopoietic cells. The patients were assigned to the following groups: group 1, unrelated bone marrow (N = 15); group 2, unrelated cord blood (N = 17), and group 3, related non-sibling bone marrow (N = 15). Twenty-four patients (51%) had complete engraftment, which was not influenced by gender (P = 0.87), age (P = 0.45), dose of cyclophosphamide (P = 0.80), nucleated cell dose infused (P = 0.60), or use of anti-T serotherapy (P = 0.20). Favorable factors for superior engraftment were full HLA compatibility (independent of the source of cells; P = 0.007) and use of a fludarabine-based conditioning regimen (P = 0.046). Unfavorable factors were > or = 25 transfusions pre-transplant (P = 0.011) and degree of HLA disparity (P = 0.007). Intensity of mucositis (P = 0.50) and use of androgen prior to transplant had no influence on survival (P = 0.80). Acute graft-versus-host disease (GVHD) grade II-IV and chronic GVHD were diagnosed in 47 and 23% of available patients, respectively, and infections prevailed as the main cause of death, associated or not with GVHD. Eighteen patients are alive, the Kaplan-Meyer overall survival is 38% at ~8 years, and the best results were obtained with related non-sibling bone marrow patients. Three recommendations emerged from the present study: fludarabine as part of conditioning, transplant in patients with <25 transfusions and avoidance of HLA disparity. In addition, an extended family search (even when consanguinity is not present) seeking for a related non-sibling donor is highly recommended.

Sex differences in angiotensin II- induced hypertension

Sex differences in the development of hypertension and cardiovascular disease have been described in humans and in animal models. In this paper we will review some of our studies which have as their emphasis the examination of the role of sex differences and sex steroids in modulating the central actions of angiotensin II (ANG II) via interactions with free radicals and nitric oxide, generating pathways within brain circumventricular organs and in central sympathomodulatory systems. Our studies indicate that low-dose infusions of ANG II result in hypertension in wild-type male mice but not in intact wild-type females. Furthermore, we have demonstrated that ANG II-induced hypertension in males is blocked by central infusions of the androgen receptor antagonist, flutamide, and by central infusions of the superoxide dismutase mimetic, tempol. We have also found that, in comparison to females, males show greater levels of intracellular reactive oxygen species in circumventricular organ neurons following long-term ANG II infusions. In female mice, ovariectomy, central blockade of estrogen receptors or total knockout of estrogen a receptors augments the pressor effects of ANG II. Finally, in females but not in males, central blockade of nitric oxide synthase increases the pressor effects of ANG II. Taken together, these results suggest that sex differences and estrogen and testosterone play important roles in the development of ANG II-induced hypertension.

To divide or differentiate? : nestin-mediated regulation of Cdk5 in cell fate decisions

The molecular functions of the non-cell cycle-related Cyclin-dependent kinase 5 (Cdk5) have been of primary interest within the neuroscience field, but novel undertakings are constantly emerging for the kinase in tissue homeostasis, as well as in diseases such as diabetes and cancer. Although Cdk5 activation is predominantly regulated by specific non-cyclin activator protein binding, additional mechanisms have proved to orchestrate Cdk5 signaling in cells. For example, the interaction between the intermediate filament protein nestin and Cdk5 has been proposed to determine cellular fate during neuronal apoptosis through nestin-dependent adjustment of the sensitive balance and turnover of Cdk5 activators. While nestin constitutes a crucial regulatory scaffold for appropriate Cdk5 activation in apoptosis, Cdk5 itself phosphorylates nestin with the consequence of filament reorganization in both neuronal progenitors and differentiating muscle cells. Interestingly, the two proteins are often found coexpressed in various tissues and cell types, proposing that nestin-mediated scaffolding of Cdk5 and its activators may be applicable to other tissue systems as well. In the literature, the molecular functions of nestin have remained in the shade, as it is mostly exploited as a marker protein for progenitor cells. In light of these studies, the aim of this thesis was to assess the importance of the nestin scaffold in regulation of Cdk5 actions in cell fate decisions. This thesis can be subdivided into two major projects: one that studied the nature of the Cdk5-nestin interplay in muscle, and one that assessed their role in prostate cancer. During differentiation of a myoblast cell line, the filament formation properties of nestin was found to be crucial in directing Cdk5 activity, with direct consequences on the process of differentiation. Also the genetic knockout of nestin was found to influence Cdk5 activity, although differentiation per se was not affected. Instead, the genetic ablation of nestin had broad consequences on muscle homeostasis and regeneration. While the nestin-mediated regulation of Cdk5 in muscle was found to act in multiple ways, the connection remained more elusive in cancer models. Cdk5 was, however, established as a significant determinant of prostate cancer proliferation; a behavior uncharacteristic for this differentiation-associated kinase. Through complex and simultaneous regulation of two major prostate cancer pathways, Cdk5 was placed upstream of both Akt kinase and the androgen receptor. Its action on proliferation was nonetheless mainly exerted through the Akt signaling pathway in various cancer models. In summary, this thesis contributed to the knowledge of Cdk5 regulation and functions in two atypical settings; proliferation (in a cancer framework) and muscle differentiation, which is a poorly understood model system in the Cdk5 field. This balance between proliferation and differentiation implemented by Cdk5 is ultimately regulated (where present) by the dynamics of the cytoskeletal nestin scaffold.

Cryptic mosaicism involving a second chromosome X in patients with Turner syndrome

The high abortion rate of 45,X embryos indicates that patients with Turner syndrome and 45,X karyotype could be mosaics, in at least one phase of embryo development or cellular lineage, due to the need for the other sex chromosome presence for conceptus to be compatible with life. In cases of structural chromosomal aberrations or hidden mosaicism, conventional cytogenetic techniques can be ineffective and molecular investigation is indicated. Two hundred and fifty patients with Turner syndrome stigmata were studied and 36 who had female genitalia and had been cytogenetically diagnosed as having "pure" 45,X karyotype were selected after 100 metaphases were analyzed in order to exclude mosaicism and the presence of genomic Y-specific sequences (SRY, TSPY, and DAZ) was excluded by PCR. Genomic DNA was extracted from peripheral blood and screened by the human androgen receptor (HUMARA) assay. The HUMARA gene has a polymorphic CAG repeat and, in the presence of a second chromosome with a different HUMARA allele, a second band will be amplified by PCR. Additionally, the CAG repeats contain two methylation-sensitive HpaII enzyme restriction sites, which can be used to verify skewed inactivation. Twenty-five percent (9/36) of the cases showed a cryptic mosaicism involving a second X and approximately 14% (5/36), or 55% (5/9) of the patients with cryptic mosaicism, also presented skewed inactivation. The laboratory identification of the second X chromosome and its inactivation pattern are important for the clinical management (hormone replacement therapy, and inclusion in an oocyte donation program) and prognostic counseling of patients with Turner syndrome.

The effect of microgravity on tissue structure and function of rat testis

To explore whether an environment of weightlessness will cause damage to the reproductive system of animals, we used the tail-suspension model to simulate microgravity, and investigated the effect of microgravity on the tissue structure and function of the testis in sexually mature male rats. Forty-eight male Wistar rats weighing 200-250 g were randomly assigned to three groups (N = 16 each): control, tail traction, and tail suspension. After the rats were suspended for 7 or 14 days, morphological changes of testis were evaluated by histological and electron microscopic methods. The expression of HSP70, bax/bcl-2 and AR (androgen receptor) in testis was measured by immunohistochemistry. Obvious pathological lesions were present in the testis after the rats were suspended for 7 or 14 days. We detected overexpression of HSP70 and an increase of apoptotic cells, which may have contributed to the injury to the testis. The expression of AR, as an effector molecule in the testis, was significantly decreased in the suspended groups compared to control (P < 0.01). We also observed that, with a longer time of suspension, the aforementioned pathological damage became more serious and some pathological injury to the testis was irreversible. The results demonstrated that a short- or medium-term microgravity environment could lead to severe irreversible damage to the structure of rat testis.

Bone properties and skeletal maturity in adolescent males, as assessed by quantitative ultrasound

ABSTRACT Background: Previous studies have implied that weight-bearing, intense and prolonged physical activities optimize bone accretion during the grow^ing years. The majority of past inquiries have used dual-energy X-ray absorptiometry (DXA) to examine bone strength and hand-wrist radiography to determine skeletal maturity in children. Recently, quantitative ultrasound (QUS) technologies have been developed to examine bone properties and skeletal maturity in a safe, noninvasive and cost-effective manner. Objective: The purpose of this study was to compare bone properties and skeletal maturity in competitive male child and adolescent athletes with minimallyactive, age-matched controls, using QUS technology. >. Methods: In total, 224 males were included in the study. The 115 pre-pubertal boys aged 10-12 years consisted of control, minimally-active children (n=34), soccer players (n=26), gymnasts (n=25) and hockey players (n=30). In addition, the 109 late-pubertal boys aged 14-16 years consisted of control, minimally-active adolescents (n=31), soccer players (n=30), gymnasts (n=17) and hockey players (n=31). The athletic groups were elite level players that predominantly trained year-round. Physical activity, nutrition and sports participation were assessed with various questionnaires. Anthropometries, such as height, weight and relative body fat percentage (BF%) were assessed using standard measures. Skeletal strength and age were evaluated using bone QUS. Lastly, salivary testosterone (sT) concentration was measured using Radioimmunoassay (RIA). Results: Within each age group, there were no significant differences between the activity groups in age and pubertal stage. An age effect was apparent in all variables, as expected. A sport effect was noted in all physical characteristics: the child and adolescent gymnasts were shorter and lighter than other sports groups. Adiposity was greater in the controls and in the hockey players. All child subjects were pubertal stage (fanner) I or II, while adolescent subjects were pubertal stage IV or V. There were no differences in daily energy and mineral intakes between sports groups. In both age groups, gymnasts had a higher training volume than other athletic groups. Bone speed of sound (50s) was higher in adolescents compared with the children. Gymnasts had signifieantly higher radial 50S than controls, hockey and soccer players in both age cohorts. Hockey athletes also had higher radial 50S than controls and soccer players in the child and adolescent groups, respectiyely. Child gymnasts and soccer players had greater tibial 50S compared with the hockey players and control groups. Likewise, adolescent gymnasts and soccer players had higher tibial SoS compared with the control group. No interaction was apparent between age and type of activity in any of the bone measures. » Lastly, maturity as assessed by sT and secondary sex characteristics (Tanner stage) was not different between sports group within each age group. Despite the similarity in chronological age, androgen levels and sexual maturity, differences between activity groups were noted in skeletal maturity. In the younger group, hockey players had the highest bone age while the soccer players had the lowest bone age. In the adolescent group, gymnasts and hockey players were characterized by higher skeletal maturity compared with controls. An interaction between the age and sport type effects was apparent in skeletal maturity, reflecting the fact that among the children, the soccer players were significantly less mature than the rest of the groups, while in the adolescents, the controls were the least skeletally mature. Summary and Conclusions: In summary, radial and tibial SOS are enhanced by the unique loading pattern in each sport (i.e, upper and lower extremities in gymnastics, lower extremities in soccer), with no cumulative effect between childhood and adolescence. That is, the effect of sport participation on bone SOS was apparent already among the young athletes. Enhanced bone properties among athletes of specific sports suggest that participation in these sports can improve bone strength and potential bone health.

Élucidation des mécanismes moléculaires impliqués dans l’expression aberrante du récepteur au peptide insulinotropique glucose-dépendant (GIP) dans les tumeurs du cortex de la glande surrénale

Les tumeurs du cortex surrénalien sont variées et fréquentes dans la population. Bien que des mutations aient été identifiées dans certains syndromes familiaux, les causes génétiques menant à la formation de tumeur du cortex surrénalien ne sont encore que peu connues. Un sous-type de ces tumeurs incluent les hyperplasies macronodulaires et sont pressenties comme la voie d’entrée de la tumorigenèse du cortex surrénalien. L’événement génétique le plus fréquemment observé dans ces tumeurs est l’expression aberrante d’un ou plusieurs récepteurs couplés aux protéines G qui contrôle la production de stéroïdes ainsi que la prolifération cellulaire. L’événement génétique menant à l’expression aberrante de ces récepteurs est encore inconnu. En utilisant le récepteur au peptide insulinotropique dépendant du glucose (GIP) comme modèle, cette étude se propose d’identifier les mécanismes moléculaires impliqués dans l’expression aberrante du récepteur au GIP (GIPR) dans les tumeurs du cortex surrénalien. Une partie clinique de cette étude se penchera sur l’identification de nouveaux cas de tumeurs surrénaliennes exprimant le GIPR de façon aberrante. Les patients étudiés seront soumis à un protocole d’investigation in vivo complet et les tumeurs prélevées seront étudiées extensivement in vitro par RT-PCR en temps réel, culture primaire des tumeurs, immunohistochimie et biopuces. Le lien entre le GIP et la physiologie normal sera également étudiée de cette façon. Une autre partie de l’étude utilisera les nouvelles techniques d’investigation à grande échelle en identifiant le transcriptome de différents cas de tumeurs exprimant le GIPR de façon aberrante. L’importance fonctionnelle des gènes identifiée par ces techniques sera confirmée dans des modèles cellulaires. Cette étude présente pour la première des cas de tumeurs productrices d’aldostérone présentant des réponses aberrantes, auparavant confinées aux tumeurs productrice de cortisol ou d’androgènes surrénaliens. Le cas probant présenté avait une production d’aldostérone sensible au GIP, le GIPR était surexprimé au niveau de l’ARNm et un fort marquage a été identifié dans la tumeur spécifiquement. Dans les surrénales normales, cette étude démontre que le GIP est impliqué dans le contrôle de la production d’aldostérone. Ces résultats ont été confirmés in vitro. Finalement, le profilage à grande échelle des niveaux d’expression de tous les gènes du génome a permis d’isoler une liste de gènes spécifiquement liés à la présence du GIPR dans des hyperplasies du cortex surrénalien. Cette liste inclus la périlipine, une protéine de stockage des lipides dans les adipocytes et la glande surrénale, dont l’expression est fortement réprimée dans les cas GIP-dépendant. Des études dans un modèle cellulaire démontrent que la répression de ce gène par siRNA est suffisante pour induire l’expression du récepteur au GIP et que cette protéine est impliquée dans la stimulation de la stéroïdogénèse par le GIP. En alliant des méthodes d’investigation in vivo de pointe à des techniques in vitro avancée, cette étude offre de nouveaux regards sur les liens entre le GIP et la physiologie de la glande surrénale, que ce soit dans des conditions normales ou pathologiques.

Influence de la testostérone sur les paramètres électrocardiographiques des hommes et des femmes

Les hommes et les femmes diffèrent devant la survenue de plusieurs types d’arythmies cardiaques. Ainsi, la prévalence des fibrillations auriculaires, ou celle du syndrome de Brugada est plus élevée chez les hommes, tandis que les femmes sont plus susceptibles de développer des torsades de pointe. La survenue de ces arythmies pourrait être affectée par l’existence de disparités entre les sexes au niveau de l’activité électrique cardiaque normale. Des différences ont d’ailleurs été décrites dans les paramètres électrocardiographiques des hommes et des femmes, et notamment au niveau des paramètres traduisant la phase de repolarisation ventriculaire. Par ailleurs, plusieurs études cliniques et expérimentales ont permis d’avancer que les androgènes pourraient contribuer à expliquer les différences homme-femme observées dans la repolarisation ventriculaire. Leur influence sur les autres paramètres électrocardiographiques a toutefois reçu peu d’attention jusqu’à aujourd’hui. Dans ce contexte, l’objectif de ce travail était de mieux comprendre l’implication de la testostérone dans les différences sexuelles observées au niveau de l’électrocardiogramme. En travaillant à partir d’une cohorte de volontaires sains, nous avons tout d’abord confirmé l’existence de différences électrocardiographiques rapportées précédemment dans la littérature. Nous avons de plus noté un segment PR plus long chez les hommes, bien que l’onde P et l’intervalle PR n’étaient pas significativement différents entre les sexes. Par la suite, nous avons évalué l’association de la testostérone avec les différents paramètres de l’électrocardiogramme humain. La testostérone était le plus fortement associée au complexe QRS, au segment ST et à l’intervalle QTc. Nous avons donc examiné la contribution de la testostérone dans les mécanismes responsables des différences sexuelles pour ces trois paramètres. D’après les résultats obtenus, la testostérone ne semblait toutefois pas expliquer ces différences homme-femme. En conclusion, les résultats de cette étude suggèrent que la testostérone n’est pas responsable des différences observées au niveau du complexe QRS, du segment ST et de l’intervalle QTc des hommes et des femmes de notre cohorte. D’autres médiateurs, seuls ou en association avec la testostérone, pourraient jouer un rôle dans l’établissement de ces différences électrocardiographiques associées au sexe.

Biomarqueurs pronostiques dans le cancer de la prostate : mieux prédire pour mieux traiter

Le cancer de la prostate (CP) est le cancer le plus fréquemment diagnostiqué en Amérique du Nord et est au troisième rang en termes de létalité chez les hommes. Suite aux traitements de première ligne, 20 à 30% des patients diagnostiqués avec un cancer localisé auront une récidive biochimique de la maladie. La déplétion androgénique mène fréquemment au développement du stade de résistance à la castration (RC). Ce dernier est associé avec une augmentation de la morbidité (métastases osseuses) et de la mortalité avec une survie moyenne inférieure à deux ans. L’évolution du CP est très hétérogène dans la population et il n’existe actuellement aucun biomarqueur pronostique permettant d’identifier les patients à risque de récurrence biochimique, de métastases osseuses et de développement d’une résistance à la castration. De nombreuses études ont démontré que les cytokines inflammatoires IL-6 et IL-8 jouent un rôle dans la pathogénèse du CP, notamment dans le développement de la résistance à la castration. Par ailleurs, les niveaux sériques élevés de ces cytokines ont été associés à un mauvais pronostic. Précédemment, notre laboratoire a démontré in vitro que la protéine IKKε entraîne une augmentation de la sécrétion de ces cytokines dans les cellules du CP et qu’elle est exprimée davantage dans les tissus de cancers plus avancés. Le premier objectif du présent mémoire fut d’évaluer dans des tissus humains la corrélation d’IKKε, IL-6 et IL-8 avec des paramètres cliniques. Nos résultats soulignent le potentiel d’IKKε comme biomarqueur tissulaire pronostique de récurrence biochimique et de métastases osseuses. Nous n’avons trouvé aucune association entre IL-6/IL-8 et les paramètres cliniques inclus dans l’étude. Le second objectif de ce projet fut d’évaluer la coexpression de ces trois molécules dans l’épithélium du CP. Nos résultats confirment les observations in vitro en mettant en évidence une forte association entre l’expression d’IKKε, IL-6 et IL-8. Le troisième objectif fut d’évaluer la relation entre les niveaux sériques et tissulaires d’IL-6 et d’IL-8. Aucune relation significative n’a été établie, suggérant que les cytokines sériques ne sont pas uniquement d’origine prostatique. En conclusion, mon projet de maîtrise aura permis de préciser le potentiel d’IKKε comme biomarqueur tissulaire pronostique et de valider pour la première fois dans des tissus humains sa co-expression avec les cytokines IL-6 et IL-8, dont le rôle dans la pathogénèse de la maladie est bien établi. Une étude plus exhaustive des voies de signalisation d’IKKε reste d’intérêt pour élucider notamment les mécanismes par lesquels IKKε stimule la production de cytokines et par quels moyens cette protéine pourrait être impliquée dans le développement d’un état résistant à la castration.

Evidence for an inhibitory role of bone morphogenetic protein(s) in the follicular-luteal transition in cattle

Bone morphogenetic proteins (BMPs) and their receptors are expressed in ovarian theca cells (TC) and granulosa cells (GC) and BMPs have been implicated in the regulation of several aspects of follicle development including thecal androgen production and granulosal oestrogen production. Their potential involvement in luteal function has received less attention. in this study, we first compared relative abundance of mRNA transcripts for BMPs, activin-beta A and BMP/activin receptors in bovine corpus luteum (CL) and follicular theca and granulosa layers before undertaking functional in vitro experiments to test the effect of selected ligands (BMP6 and activin A) on luteinizing bovine TC and GC. Relative to P-actin transcript abundance, CL tissue contained more BMP4 and -6 mRNA than granulosa, more BMP2 mRNA than theca but much less activin-beta A mRNA than both granulosa and theca. Transcripts for all seven BMP/activin receptors were readily detected in each tissue and two transcripts (BMPRII, ActRIIA) were more abundant in CL than either theca or granulosa, consistent with tissue responsiveness. In vitro luteinization of TC and GC from antral follicles (4-6 mm) was achieved by culturing with 5% serum for 6 days. Treatment with BMP6 (0, 2, 10, and 50 ng/ml) and activin A (0, 2, 10 and 50 ng/ml) under these conditions dose-dependently suppressed forskolin-induced progesterone (P-4) secretion from both cell types without affecting cell number. BMP6 reduced forskolin-stimulated upregulation of STAR mRNA and raised 'basal' CYP17A1 mRNA level in theca-lutein cells without affecting expression of CYP11A1 or hydroxy-Delta-5-steroid dehydrogenase, 3 beta- and steroid Delta-isomerase 1 (HSD3B1). In granulosa-lutein cells, STAR transcript abundance was not affected by BMP6, whereas forskolin-induced expression of CYP11A1, HSD3B1, CYP19A1 and oxytocin transcripts was reduced. In both cell types, follistatin attenuated the suppressive effect of activin A and BMP6 on forskolin-induced P4 secretion but had no effect alone. Granulosa-lutein cells secreted low levels of endogenous activin A (similar to 1 ng/ml); BMP6 reduced this, while raising follistatin secretion thus decreasing activin A:follistatin ratio. Collectively, these findings support inhibitory roles for BMP/activin signalling in luteinization and steroidogenesis in both TC and GC.

Paraben esters: review of recent studies of endocrine toxicity, absorption, esterase and human exposure, and discussion of potential human health risks

This toxicology update reviews research over the past four years since publication in 2004 of the first measurement of intact esters of p-hydroxybenzoic acid (parabens) in human breast cancer tissues, and the suggestion that their presence in the human body might originate from topical application of bodycare cosmetics. The presence of intact paraben esters in human body tissues has now been confirmed by independent measurements in human urine, and the ability of parabens to penetrate human skin intact without breakdown by esterases and to be absorbed systemically has been demonstrated through studies not only in vitro but also in vivo using healthy human subjects. Using a wide variety of assay systems in vitro and in vivo, the oestrogen agonist properties of parabens together with their common metabolite (p-hydroxybenzoic acid) have been extensively documented, and, in addition, the parabens have now also been shown to possess androgen antagonist activity, to act as inhibitors of sulfotransferase enzymes and to possess genotoxic activity. With the continued use of parabens in the majority of bodycare cosmetics, there is a need to carry out detailed evaluation of the potential for parabens, together with other oestrogenic and genotoxic co-formulants of bodycare cosmetics, to increase female breast cancer incidence, to interfere with male reproductive functions and to influence development of malignant melanoma which has also recently been shown to be influenced by oestrogenic stimulation. Copyright (C) 2008 John Wiley & Sons, Ltd.

Oestrogenic and androgenic activity of triclosan in breast cancer cells

As a consequence of its widespread use as an antimicrobial agent in consumer goods, triclosan has become distributed ubiquitously across the ecosystem, and recent reports that it can cause endocrine disruption in aquatic species has increased concern. It is reported here that triclosan possesses intrinsic oestrogenic and androgenic activity in a range of assays in vitro which could provide some explanation for the endocrine disrupting properties described in aquatic populations. In terms of oestrogenic activity, triclosan displaced [H-3]oestradiol from oestrogen receptors (ER) of MCF7 human breast cancer cells and from recombinant human ER alpha/ER beta. Triclosan at 10(-5) M completely inhibited the induction of the oestrogen-responsive ERE-CAT reporter gene in MCF7 cells by 10(-10) M 17 beta-oestradiol and the stimulation of growth of MCF7 human breast cancer cells by 10(-10) M 17 beta-oestradiol. On its own, 1 mu M triclosan increased the growth of MCF7 cells over 21 days. Triclosan also had androgenic activity. It displaced [H-3]testosterone from binding to the ligand binding domain of the rat androgen receptor (AR). Triclosan was able to inhibit the induction of the androgen-responsive LTR-CAT reporter gene in S115 mouse mammary tumour cells by 10(-9) M testosterone and in T47D human breast cancer cells by 10(-8) M testosterone at concentrations of 10(-7) M and 10(-6) M, respectively. Triclosan at 2 x 10(-5) M antagonized the stimulation of the growth of S115+A mouse mammary tumour cells by 10(-9) M testosterone. The finding that triclosan has oestrogenic and androgenic activity warrants further investigation in relation to both endocrine disruption of aquatic wildlife and any possible impact on human health. Copyright (C) 2007 John Wiley & Sons, Ltd.

TGF-beta superfamily members and ovarian follicle development

In recent years, exciting progress has been made towards unravelling the complex intraovarian control mechanisms that, in concert with systemic signals, coordinate the recruitment, selection and growth of follicles from the primordial stage through to ovulation and corpus luteum formation. A plethora of growth factors, many belonging to the transforming growth factor-beta (TGF-beta) superfamily, are expressed by ovarian somatic cells and oocytes in a developmental, stage-related manner and function as intraovarian regulators of folliculogenesis. Two such factors, bone morphogenetic proteins, RMP-4 and BMP-7, are expressed by ovarian stromal cells and/or theca cells and have recently been implicated as positive regulators of the primordial-to-primary follicle transition. In contrast, evidence indicates a negative role for anti-Mullerian hormone (AMH, also known as Mullerian-inhibiting substance) of pre-granulosa/granulosa cell origin in this key event and subsequent progression to the antral stage. Two other TGF-beta superfamily members, growth and differentiation factor-9 (GDF-9) and BMP-15 (also known as GDF-9B) are expressed in an oocyte-specific manner from a very early stage and play key roles in promoting follicle growth beyond the primary stage; mice with null mutations in the gdf-9 gene or ewes with inactivating mutations in gdf-9 or bmp-15 genes are infertile with follicle development arrested at the primary stage. Studies on later stages of follicle development indicate positive roles for granulosa cell-derived activin, BMP-2, -5 and -6, theca cell-derived BMP-2, -4 and -7 and oocyte-derived BMP-6 in promoting granulosa cell proliferation, follicle survival and prevention of premature luteinization and/or atresia. Concomitantly, activin, TGF-beta and several BMPs may exert paracrine actions on theca cells to attenuate LH-dependent androgen production in small to medium-size antral follicles. Dominant follicle selection in monovular species may depend on differential FSH sensitivity amongst a growing cohort of small antral follicles. Changes in intrafollicular activins, GDF-9, AMH and several BMPs may contribute to this selection process by modulating both FSH- and IGF-dependent signalling pathways in granulosa cells. Activin may also play a positive role in oocyte maturation and acquisition of developmental competence. in addition to its endocrine role to suppress FSH secretion, increased output of inhibin by the selected dominant follicle(s) may upregulate LH-induced androgen secretion that is required to sustain a high level of oestradiol secretion during the pre-ovulatory phase. Advances in our understanding of intraovarian regulatory mechanisms should facilitate the development of new approaches for monitoring and manipulating ovarian function and improving fertility in domesticated livestock, endangered species and man.