Academic literature on the topic 'Prenatal testosterone'

Androgens play important roles during the first trimester of intrauterine life, coinciding with genital tract differentiation, during virilization and maintenance of secondary male characteristics, and during initiation of spermatogenesis. Little is known about the impact of inappropriate exposure to excess androgens during fetal development on male sexual maturation and reproduction. The objectives of this study were to determine the effects of prenatal 5α-dihydrotestosterone (DHT) and testosterone treatment during ovine sexual differentiation on post-pubertal testicular formation and subsequent potential for fertility as assessed by epididymal sperm characteristics. Rams prenatally treated with testosterone exhibited increased testicular weight relative to age-matched controls and prenatal DHT-treated rams (P<0.05), as well as elevated total and free testosterone concentrations compared with DHT-treated rams (P=0.07 and P<0.05 respectively). The percentage of progressively motile sperm from the epididymis was significantly reduced in prenatal DHT-treated but not testosterone-treated rams compared with control rams (P<0.05). The testosterone-treated rams had a greater number of germ cell layers than DHT-treated rams, but comparable to the controls. Prenatal testosterone-treated rams had significantly larger seminiferous tubule diameter and lumen diameter compared with prenatal DHT-treated (P<0.05). Significantly, more prenatal DHT- and testosterone-treated rams (P<0.05) had occluded tubule lumen than control rams. Findings from this study demonstrate that exposure to excess testosterone/DHT during male fetal sexual differentiation have differential effects on post-pubertal testicular size, seminiferous tubule size and function, sperm motility, and testosterone concentrations.

The prenatal environment is a source of phenotypic variability influencing the animal's characteristics. Prenatal stress affects not only the development of offspring, but also that of the following generation. Such effects have been best documented in mammals but can also be observed in birds, suggesting common processes across phylogenetic orders. We found previously that Japanese quail females stressed during laying produced offspring with higher fearfulness, probably related to modulation of testosterone levels in their eggs. Here, we evaluated long-term effects of prenatal stress by analysing reproductive traits of these F 1 offspring and, then, the development of their subsequent (F 2 ) offspring. The sexual behaviour of F 1 prenatally stressed (F1PS) males was impaired. F1PS females' eggs contained less yolk and more albumen, and higher yolk testosterone and progesterone levels than did F 1 prenatal control females. The fearfulness of F 2 prenatally stressed quail was greater than that of F 2 prenatal control quail. These F 2 behavioural differences paralleled those evidenced by their parents, suggesting trans-generational transmission of prenatal stress effects, probably mediated by egg compositions of F1PS females.

Prenatal androgen exposure induces metabolic disorders in female offspring. However, the long-term effect of maternal testosterone excess on glucose metabolism, especially on pancreatic beta-cell function, is rarely investigated. Our current study mainly focused on the effects of prenatal testosterone exposure on glucose metabolism and pancreatic beta- cell function in aged female offspring. By using maternal mice and their female offspring as animal models, we found that prenatal androgen treatment induced obesity and glucose intolerance in aged offspring. These influences were accompanied by decreased fasting serum insulin concentration, elevated serum triglyceride, and testosterone concentrations. Glucose stimulated insulin secretion in pancreatic beta cells of aged female offspring was also affected by prenatal testosterone exposure. We further confirmed that increased serum testosterone contributed to downregulation of sirtuin 3 expression, activated oxidative stress, and impaired pancreatic beta-cell function in aged female offspring. Moreover, over-expression of sirtuin 3 in islets isolated from female offspring treated with prenatal testosterone normalized the oxidative stress level, restored cyclic AMP, and ATP generation, which finally improved glucose-stimulated insulin secretion in beta cells. Taken together, these results demonstrated that prenatal testosterone exposure caused a metabolic disturbance in aged female offspring via suppression of sirtuin 3 expression and activation of oxidative stress in pancreatic beta cells.

Testosterone plays an important role in mammalian brain development. In neural regions with appropriate receptors testosterone, or its metabolites, influences patterns of cell death and survival, neural connectivity and neurochemical characterization. Consequently, testosterone exposure during critical periods of early development produces permanent behavioural changes. In humans, affected behaviours include childhood play behaviour, sexual orientation, core gender identity and other characteristics that show sex differences (i.e. differ on average between males and females). These influences have been demonstrated primarily in individuals who experienced marked prenatal hormone abnormalities and associated ambiguities of genital development (e.g. congenital adrenal hyperplasia). However, there is also evidence that testosterone works within the normal range to make some individuals within each sex more sex-typical than others. The size of testosterone-related influences, and perhaps even their existence, varies from one sex-typed characteristic to another. For instance: prenatal exposure to high levels of testosterone has a substantial influence on sex-typical play behaviour, including sex-typed toy preferences, whereas influences on core gender identify and sexual orientation are less dramatic. In addition: there appears to be little or no influence of prenatal testosterone on mental rotations ability, although mental rotations ability shows a marked sex difference. These findings have implications for basic understanding of the role of testosterone in normative gender development, as well as for the clinical management of individuals with disorders of sex development (formerly called intersex syndromes).

BackgroundTestosterone may be a biological factor that protects males against eating disorders. Elevated prenatal testosterone exposure is linked to lower levels of disordered eating symptoms, but effects emerge only after mid-puberty. Whether circulating levels of testosterone account for decreased risk for disordered eating in boys after mid-puberty is currently unknown; however, animal data support this possibility. In rodents, prenatal testosterone's masculinizing effects on sex-differentiated behaviors emerge during puberty when circulating levels of testosterone increase and ‘activate’ the expression of masculinized phenotypes. This study investigated whether higher levels of circulating testosterone predict lower levels of disordered eating symptoms in adolescent boys, and in particular whether effects are associated with advancing pubertal maturation.MethodParticipants were 213 male twins from the Michigan State University Twin Registry. The Minnesota Eating Behavior Survey and Eating Disorder Examination Questionnaire assessed several disordered eating symptoms. The Pubertal Development Scale assessed pubertal status. Afternoon saliva samples were assayed for testosterone using enzyme immunoassays.ResultsConsistent with animal data, higher levels of circulating testosterone predicted lower levels of disordered eating symptoms in adolescent boys and effects emerged with advancing puberty. Results were not accounted for by several important covariates, including age, adiposity, or mood/anxiety symptoms.ConclusionsFindings suggest that elevated circulating testosterone may be protective and underlie decreased risk for eating pathology in males during/after puberty, whereas lower levels of testosterone may increase risk and explain why some, albeit relatively few, males develop eating disorders.

Prenatal exposure to excess testosterone induces reproductive disturbances in both female and male sheep. In females, it alters the hypothalamus–pituitary–ovarian axis. In males, prenatal testosterone excess reduces sperm count and motility. Focusing on males, this study tested whether pituitary LH responsiveness to GNRH is increased in prenatal testosterone-exposed males and whether testicular function is compromised in the testosterone-exposed males. Control males (n=6) and males born to ewes exposed to twice weekly injections of 30 mg testosterone propionate from days 30 to 90 and of 40 mg testosterone propionate from days 90 to 120 of gestation (n=6) were studied at 20 and 30 weeks of age. Pituitary and testicular responsiveness was tested by administering a GNRH analog (leuprolide acetate). To complement the analyses, the mRNA expression of LH receptor (LHR) and that of steroidogenic enzymes were determined in testicular tissue. Basal LH and testosterone concentrations were higher in the testosterone-exposed-males. While LH response to the GNRH analog was higher in the testosterone-exposed males than in the control males, testosterone responses did not differ between the treatment groups. The testosterone:LH ratio was higher in the control males than in the testosterone-exposed males of 30 weeks of age, suggestive of reduced Leydig cell sensitivity to LH in the testosterone-exposed males. The expression of LHR mRNA was lower in the testosterone-exposed males, but the mRNA expression of steroidogenic enzymes did not differ between the groups. These findings indicate that prenatal testosterone excess has opposing effects at the pituitary and testicular levels, namely increased pituitary sensitivity to GNRH at the level of pituitary and decreased sensitivity of the testes to LH.

ObjectiveWe investigated how different levels of prenatal exposure to testosterone influence physiological reactions to dyadic interactions, hypothesising that higher levels of prenatal testosterone are linked to greater physiological responses.MethodAutonomic nervous system responses to dyadic interactions focussed on social or physical norms were measured. Physiological assessment of excitability (heart rate, facial temperature) and a behavioural assessment (Likert items judgements) were run on 25 neurotypical participants who had distinct testosterone exposure levels in utero. In utero exposure to testosterone was assessed measuring 2D : 4D (ratio between the lengths of the index and the ring fingers).ResultsHigher testosterone exposure participants showed greater physiological arousal: a greater heart rate decrease, independent from scenario type (p<0.05), and opposite facial temperature changes in response to social (increase) (vs.) physical scenarios (decrease) were found (Left-cheek: p<0.05; Right-cheek: p<0.05).ConclusionThese findings suggest a long-term influence of prenatal environment on adults’ physiological responses during social situations.

Abstract Prenatally testosterone (T)-treated sheep present metabolic disruptions similar to those seen in women with polycystic ovary syndrome. These females exhibit an increased ratio of small to large adipocytes, which may be the earliest event in the development of adult insulin resistance. Additionally, our longitudinal studies suggest the existence of a period of compensatory adaptation during development. This study tested whether 1) in utero cotreatment of prenatally T-treated sheep with androgen antagonist (flutamide) or insulin sensitizer (rosiglitazone) prevents juvenile insulin resistance and adult changes in adipocyte size; and 2) visceral adiposity and insulin sensitivity are both unaltered during early adulthood, confirming the predicted developmental trajectory in this animal model. Insulin sensitivity was tested during juvenile development and adipose tissue distribution, adipocyte size, and concentrations of adipokines were determined during early adulthood. Prenatal T-treated females manifested juvenile insulin resistance, which was prevented by prenatal rosiglitazone cotreatment. Neither visceral adiposity nor insulin sensitivity differed between groups during early adulthood. Prenatal T-treated sheep presented an increase in the relative proportion of small adipocytes, which was not substantially prevented by either prenatal intervention. A large effect size was observed for increased leptin concentrations in prenatal T-treated sheep compared with controls, which was prevented by prenatal rosiglitazone. In conclusion, gestational alterations in insulin-glucose homeostasis likely play a role in programming insulin resistance, but not adipocyte size distribution, in prenatal T-treated sheep. Furthermore, these results support the notion that a period of compensatory adaptation of the metabolic system to prenatal T exposure occurs between puberty and adulthood.

The present thesis sought to investigate the potential relationship between the second to fourth finger ratio (2D:4D), as a somatic marker of prenatal testosterone exposure, and basic numerical skills in children and adults. Chapter 1 presents a basic overview of the nature and effects of sex steroids followed by a more comprehensive consideration of literature regarding the reported effects of prenatal testosterone (PT) on the brain and cognition. The chapter then more specifically considers the possible influence of PT on numerical and mathematical competencies. Experiment 1 attempted to replicate evidence for a relationship between 2D:4D and basic numerical skills in children. The results revealed only one significant correlation, namely a significant positive correlation between right hand 2D:4D and number comparison scores in females. Chapter 3 discussed research regarding the nature and characteristics of so called ‗core‘ numerical competencies. Experiments 2-4 then attempted to explore any relationship between 2D:4D and performance on tasks designed to assess such skills in adults. The results of all three studies revealed an association between 2D:4D and lateralization for the process of subitizing relative to a comparable control task. The nature of this observed effect however varied across the three experiments. Experiment 4 also identified significant positive correlations between left hand 2D:4D and counting reaction times in females and a series of two way interaction effects between 2D:4D and task (numerical vs. control) for subitizing, counting and number comparison performance. The revealed interactions predominantly suggested faster task reaction times/higher accuracy in high 2D:4D (low PT) participants as compared to low 2D:4D (high PT) participants on the numerical tasks and the opposite pattern of results (i.e. high 2D:4D associated with poorer performance) on the control tasks. Experiment 5 investigated the association between 2D:4D and core numerical skills in children. Significant correlations were observed between; left hand 2D:4D and subitizing reaction times to the left visual field in males (negative direction), right hand 2D:4D and subitizing reaction times the right visual field in females (positive direction) and left hand 2D:4D and subitizing percentage error scores to the right visual field in females (negative direction). A possible relationship between 2D:4D and lateralization for both subitizing and number comparison relative to control was also found. For both numerical tasks low 2D:4D participants showed a right visual field advantage and high 2D:4D participants showed a left visual field advantage while different patterns of results were shown on the control task. Experiment 6 re-considered the relationship between 2D:4D and basic and core numerical skills in children using a standardised assessment of numerical competencies (the Dyscalculia Screener). No significant correlations however between 2D:4D and performance were identified. Finally, experiment 7 re-examined evidence for a link between 2D:4D and Key Stage 1 Standardised Assessment Test (SAT) scores. While the findings did not replicate evidence for a direct link between 2D:4D and SAT numeracy scores the results did demonstrate a significant negative relationship between right hand 2D:4D minus left hand 2D:4D (Dr-1; higher scores thought to indicate lower exposure to PT) and SAT numeracy scores in females. Such findings may potentially suggest a facilitative influence of PT on numeracy in women. Overall, while a number of interesting findings were revealed, limited consistency was identified across the results of the experiments conducted in the present thesis. The findings therefore offer no concrete support for a possible association between 2D:4D and basic numerical skills in either children or adults. The final chapter summarises the findings of each experiment and considers the results in the context of previous literature. General limitations of the research and suggestions for future research are discussed.

Fetal testosterone (FT) exposure influences sexual differentiation and may promote well-established sex differences in externalizing (EXT) behavior. Although puberty may be a critical period for these effects, it is unknown how FT exposure influences EXT as a function of pubertal development. We used a longitudinal, multi-sample design to test the relationships between two proxy indices of FT exposure and EXT as a function of age and pubertal development (approximately ages 6, 9, 11, 14, and 16). Twin data were used to approximate FT exposure (TT-FT) because testosterone is thought to cross the intrauterine membrane and cause variability in co-twin gonadal hormone exposure, with increasing exposure for males and participants with male co-twins. Increasing number of older siblings may also approximate increasing FT exposure (SI-FT), although existing research has yet to disentangle possible postnatal socialization effects from potential FT exposure using this variable. Given that biologically related siblings share a fetal and social environment while non-biologically related siblings simply share a social environment, we tested the independent effect of SI-FT on EXT using a sibling adoption design. Across four independent samples, SI-FT and TT-FT predicted externalizing for males alone. SI-FT predicted EXT over-and-above socialization influences and interacted with pubertal development in two independent samples, with elevated EXT for those in mid-late puberty that were exposed to increased FT. TT-FT predicted EXT differentially as a function of developmental period. Our data are consistent with the notion that exposure to FT promotes sexually differentiated, sexually selected behavior during reproductively relevant periods.

Prenatal ethanol (E) exposure has marked effects on development of the hypothalamic-pituitary-adrenal (HPA) and -gonadal (HPG) axes. E rats show HPA hyperresponsiveness to stressors and altered reproductive function in adulthood. Importantly, prenatal ethanol differentially alters stress responsiveness in adult males and females, raising the possibility that gonadal hormones play a role in mediating ethanol effects on HPA function. To address this possibility, two studies were conducted to test the hypothesis that the differential alterations in HPA activity observed in E compared to control males are mediated, at least in part, by ethanol-induced changes in HPG effects on HPA regulation. The first study compared the effects of gonadectomy (GDX) on HPA and HPG activity in adult male offspring from prenatal E, pair-fed (PF) and ad libitum-fed control (C) dams. There were no differences among groups in basal testosterone levels under intact conditions. However, E males showed increased adrenocorticotropin but blunted testosterone and luteinizing hormone (LH) responses to restraint stress compared to PF and/or C rats, and no stress-induced elevation in arginine vasopressin (AVP) mRNA levels. GDX eliminated these differences among groups. The second study explored dose-related effects of testosterone on HPA regulation. Testosterone had less of an inhibitory effect on stress-induced CORT and LH increases in E than in PF and C males. Furthermore, testosterone had a reduced effect on central corticotropin-releasing hormone pathways, but an increased effect on central AVP pathways in E compared to PF and/or C males. Importantly, reduced androgen receptor (AR) mRNA levels, possibly reflecting downregulation of AR in key brain areas, may counteract the increased inhibitory AVP signals upstream from the paraventricular nucleus, and thus contribute to the HPA hyperresponsiveness seen in E males. Together these findings suggest that central regulation of both the HPA and HPG axes are altered by prenatal ethanol exposure. The capacity of testosterone to regulate HPA activity is altered in E males, with some effects mediated by the nutritional effects of ethanol. These changes would impair the ability to maintain homeostasis in E animals and have implications for the development of secondary disabilities in children with Fetal Alcohol Spectrum Disorder.

Background: Exposure to perfluorooctane sulfonic acid (PFOS) or to perfluorooctanoic acid (PFOA) increases mouse and human peroxisome proliferator–activated receptor alpha (PPARα) subtype activity, which influences lipid metabolism. Because cholesterol is the substrate from which testosterone is synthesized, exposure to these substances has the potential to alter testosterone concentrations. Objectives: We explored associations of total testosterone and sex hormone–binding globulin (SHBG) concentrations at age 15 years with prenatal exposures to PFOS, PFOA, perfluorohexane sulfonic acid (PFHxS), and perfluoronanoic acid (PFNA) in females. Methods: Prenatal concentrations of the perfluoroalkyl acids (PFAAs) were measured in serum collected from pregnant mothers at enrollment (1991–1992) in the Avon Longitudinal Study of Parents and Children (ALSPAC). The median gestational age when the maternal blood sample was obtained was 16 weeks (interquartile range, 11–28 weeks). Total testosterone and SHBG concentrations were measured in serum obtained from their daughters at 15 years of age. Associations between prenatal PFAAs concentrations and reproductive outcomes were estimated using linear regression models (n = 72). Results: Adjusted total testosterone concentrations were on average 0.18-nmol/L (95% CI: 0.01, 0.35) higher in daughters with prenatal PFOS in the upper concentration tertile compared with daughters with prenatal PFOS in the lower tertile. Adjusted total testosterone concentrations were also higher in daughters with prenatal concentrations of PFOA (β = 0.24; 95% CI: 0.05, 0.43) and PFHxS (β = 0.18; 95% CI: 0.00, 0.35) in the upper tertile compared with daughters with concentrations in the lower tertile. We did not find evidence of associations between PFNA and total testosterone or between any of the PFAAs and SHBG. Conclusions: Our findings were based on a small study sample and should be interpreted with caution. However, they suggest that prenatal exposure to some PFAAs may alter testosterone concentrations in females.

The second (index finger) to fourth (ring finger) digit length ratio (2D:4D) is known to be a putative marker of prenatal exposure to the testosterone. It has been reported that fetal testosterone may be critical for development of morphological and psychological traits such as quality of the cardiovascular system, visuo-spatial ability, risk-taking behavior and behavioral masculinity. Testosterone-driven attributes are associated with success in male-to- male physical competition, which may be proxied by ability in sports. Many studies have found that 2D:4D is sexually dimorphic and low (male-typical) 2D:4D ratio is associated with athletic performance. This study aims to investigate possible associations of performance in sport with 2D:4D ratio, personality characteristics (Big Five model), willing to take risks and training habits, in a sample of 57 top and 57 recreational snowboard racers. We did not find any associations between 2D:4D ratio and sport performance, no significant differences were found in 2D:4D ratio between samples. We found negative associations between agreeableness and sport performance; individuals with low agreeableness achieved higher results in the real competition of freestyle snowboarding. Low agreeableness used to be associated with aggressiveness, emulation and...

Einleitung: Der Einfluss von Stress während der Schwangerschaft auf den Fetus lässt sich auch postnatal bis in das Erwachsenenalter und teilweise sogar bis in nachfolgende Generationen nachweisen. Eine Vielzahl an Störgrößen erschwert es enorm, beim Menschen konkrete Kausalitäten auszuarbeiten. Mit dem Weißbüschelaffen steht ein humanrelevanter Modellorganismus zur Verfügung, der eine dem Menschen sehr ähnliche Situation in Bezug auf die Physiologie der Trächtigkeit sowie der männlichen Reproduktion bietet, gleichzeitig jedoch einen hohen Grad der Standardisierung ermöglicht. Ziele der Untersuchungen: Im Rahmen der vorliegenden Arbeit sollten ausgewählte Faktoren des Reproduktionssystems in den Hoden adulter Weißbüschelaffen mittels qPCR und Immunhistochemie nachgewiesen und auf Zellebene lokalisiert werden. Weiterhin sollte ermittelt werden, ob die nachgewiesenen Proteine durch pränatalen Stress beeinflusst werden. Ziel des zweiten Teils der vorliegenden Arbeit war es herauszufinden, ob und in welcher Form sich ein standardisierter pränataler Stressreiz postnatal auf ausgewählte Parameter der männlichen Reproduktion des Weißbüschelaffen bis hin zur F3-Generation auswirken kann. Tiere, Material und Methoden: Trächtige Weißbüschelaffen wurden von Trächtigkeitstag 42 bis 48 (EDEX) bzw. Trächtigkeitstag 90 bis 96 (LDEX) täglich mit 5 mg Dexamethason (DEX) pro kg Körpergewicht per os behandelt. Eine Kontrollgruppe (C) erhielt während der gesamten Trächtigkeit keine Glucocorticoide. Von den adulten männlichen Nachkommen der F1-Generation wurden 9 (C), 8 (EDEX) und 9 (LDEX) jeweils tiefgefrorene Hoden auf 12 Transkripte aus den Gruppen der Enzyme der Steroidbiosynthese, der Steroidrezeptoren, des Relaxinsystems und der Proliferationsmarker mittels qPCR quantitativ untersucht. Die jeweils kontralateralen Hoden waren in Paraformaldehyd fixiert worden und wurden parallel auf Proteinebene mittels Immunhistochemie (IHC) untersucht. Für den zweiten Teil der vorliegenden Arbeit wurden die jeweils in maternaler Linie weitergezüchteten Männchen der DEX F2 (n = 2) und DEX F3 (n = 3) auf relevante Parameter der Reproduktionsfähigkeit hin untersucht: Die Größe der Hoden wurde im Frühling, im Sommer und im Winter gemessen, Blutplasma¬proben an einem Tag im Sommer um 8 Uhr, 12 Uhr und 16 Uhr sowie an einem Tag im Winter um 8 Uhr entnommen und daraus mittels ELISA die Konzentration von Testosteron sowie teilweise von 17β Östradiol ermittelt; Ejakulate wurden durch penile Vibrostimulation (PVS) gewonnen und computergestützt untersucht. Alle Ergebnisse wurden jeweils mit mindestens elf unbehandelten männlichen Tieren derselben Kolonie verglichen. Ergebnisse: Alle untersuchten für die männliche Reproduktion relevanten Proteine konnten in den Hoden der F1 nachgewiesen werden. In den Hoden der EDEX F1 und der LDEX F1 war auf Proteinebene jeweils einzig die Steroid-5α-Reduktase 1 (SRD5A1) gegenüber C erhöht. Auf Genebene waren von den untersuchten Transkripten nur in den Hoden der EDEX F1 die SRD5A1, SRD5A2 und Ki 67 jeweils signifikant gegenüber C aufreguliert. Die Hodengröße änderte sich nicht signifikant im Jahresverlauf. Im Tagesverlauf konnte ein signifikanter Anstieg der Testosteronkonzentration im Blutplasma zwischen 8 Uhr und 12 Uhr ermittelt werden. Jedoch konnte kein signifikanter Unterschied zwischen Testosteronkonzentration im weiteren Tageszeitenvergleich, im Sommer und Winter und in Relation zum Alter festgestellt werden. Darüber hinaus bestand keine Korrelation zwischen Testosteron- und 17β Östradiol¬konzentration aus denselben Blutproben. Der Vergleich aller im Zusammenhang mit der PVS erhobenen Parameter zwischen DEX F2/F3 und C ergab, dass einzig die Erfolgsrate der PVS in DEX F2/F3 niedriger lag als in C. Der versuchsgruppen-übergreifende Vergleich aller Ejakulate zwischen den Altersgruppen ergab bei jung adulten Tieren einen signifikant höheren Anteil an motilen Spermien sowie eine signifikant schlechtere Erfolgsrate der PVS gegenüber adulten Tieren. Schlussfolgerungen: Die Ergebnisse der vorliegenden Arbeit geben Hinweise darauf, wie sich Stress während der Trächtigkeit beim Weißbüschelaffen auf die Nachkommen auswirken könnte: In DEX F1 sind wichtige Voraussetzungen für eine funktionierende Testosteron-Biosynthese sowie die Vermittlung von Testosteron- und Östrogen-mediierten Signalen im Hoden gegeben, möglicherweise sogar in verstärkter Weise. Darüber hinaus bestätigen diese Ergebnisse die Humanrelevanz des Weißbüschelaffen als Modell¬organismus zum männlichen Reproduktionssystem. In DEX F2/F3 könnte die niedrige Erfolgsrate der PVS ein Hinweis auf verminderte Konzentrationsfähigkeit in den auf intra¬uterinen Stress folgenden Generationen sein. Jedoch unterscheiden sich die meisten erhobenen reproduktions-physiologisch bedeutsamen Parameter nicht zwischen DEX F2/F3 und C.:1 EINLEITUNG 2 LITERATURÜBERSICHT 2.1 Glucocorticoide und Stress während der Trächtigkeit 2.2 Relevante Hormone der Reproduktion und ihre Biosynthese 2.2.1 3β-Hydroxysteroid-Dehydrogenase 2 2.2.2 Testosteron und Androgenrezeptor 2.2.3 Steroid-5α-Reduktasen 2.2.4 Aromatase 2.2.5 17β-Hydroxysteroid-Dehydrogenase 7 2.2.6 Östradiol und Östrogenrezeptoren 2.2.7 Relaxinsystem 2.2.8 Proliferationsfaktoren 2.3 Weißbüschelaffen 2.3.1 Allgemeines zu Weißbüschelaffen 2.3.2 Reproduktionsbiologie der männlichen Weißbüschelaffen 3 TIERE, MATERIAL UND METHODEN 3.1 Versuchsteil I – ex vivo Hoden DEX F1 3.1.1 Versuchsaufbau (DEX) im DPZ 3.1.2 qPCR an DEX F1 Hoden 3.1.3 Immunhistochemie an Hoden der DEX F1 3.2 Versuchsteil II – in vivo DEX F2/DEX F3 3.2.1 Herkunft der Weißbüschelaffen 3.2.2 Haltung der Weißbüschelaffen 3.2.3 Bestimmung der Hodengrößen und des Körpergewichtes 3.2.4 Blutentnahmen und Hormonbestimmungen mittels ELISA 3.2.5 Penile Vibrostimulation 3.3 Statistische Auswertung 4 ERGEBNISSE 4.1 Versuchsteil I – ex vivo Hoden DEX F1 4.1.1 qPCR der Hoden der F1 4.1.2 Immunhistochemie der Hoden der DEX F1 4.1.2.1 Enzyme der Steroidbiosynthese 4.1.2.2 Steroidrezeptoren 4.1.2.3 Relaxinsystem 4.1.2.4 Proliferationsmarker 4.1.3 Zusammenfassung der Ergebnisse des Versuchsteils I – ex vivo DEX F1 4.2 Versuchsteil II – in vivo DEX F2/F3 4.2.1 Hodengrößen 4.2.2 Testosteron 4.2.3 Ejakulatanalyse 4.2.4 Zusammenfassung der Ergebnisse des Versuchsteils II – in vivo DEX F2/F3 5 DISKUSSION 5.1 Versuchsteil I – ex vivo Hoden DEX F1 5.1.1 Vergleichende Diskussion der Ergebnisse auf Gen- und Proteinebene in den Hoden der F1 5.1.1.1 Relevante Enzyme der Testosteronbiosynthese 5.1.1.2 Steroidrezeptoren 5.1.1.3 Das Relaxinsystem 5.1.1.4 Proliferationsfaktoren 5.2 Versuchsteil II – in vivo DEX F2/F3 5.2.1 Hodengrößen 5.2.2 Testosteron 5.2.3 Ejakulatanalyse 5.3 Schlussbetrachtung 6 ZUSAMMENFASSUNG 7 SUMMARY 8 Literaturverzeichnis 9 ANHANG 10 DANKSAGUNG
Introduction: The impact of prenatal stress can also be detected in the grown-up offspring and partly even in following generations. There are many interfering variables that make it nearly impossible to establish precise correlations in humans. However, the common marmoset is a model organism very similar to humans with respect to the physiology of pregnancy as well as to male reproduction. In addition to that, the possibility of a high-grade standardisation is extremely advantageous. Objective: The first part of this work aimed at detecting selected factors of the reproductive system in testes of adult common marmosets by qPCR and immunohistochemistry, and localising them at the cellular level; both were directed at clarifying whether or not the target proteins respond to prenatal stress. The second part aimed at determining in which form and to what extent standardised prenatal stress affects selected parameters of male reproduction up to the F3-generation in the common marmoset. Animals, materials and methods: Pregnant common marmosets were treated daily with a dose of 5 mg dexamethasone (DEX) per kg body weight per os on gestational days 42 to 48 (EDEX) and gestational days 90 to 96 (LDEX). A control group (C) received no glucocorticoid treatment during pregnancy at all. By qPCR, 12 transcripts from the groups of steroidogenic enzymes, steroid receptors, the relaxin system and the proliferation markers were quantitatively analysed in 9 (C), 8 (EDEX) und 9 (LDEX) frozen testes of F1 generation adult male offspring. Respective contralateral testes had been fixed in paraformaldehyde and were analysed on protein level by immunohistochemistry (IHC). In the second part of this work, males of DEX F2 (n = 2) and DEX F3 (n = 3), each bred in the maternal line, were analysed for relevant parameters of reproduction ability: testes were measured in spring, summer, and winter; blood plasma samples were taken on one day in summer at 8 a.m., 12 noon and 4 p.m. as well as on a day in winter at 8 a.m. and subsequently analysed by ELISA for testosterone and partly 17β oestradiol; ejaculates have been gathered by penile vibrostimulation (PVS) and were tested by computer-assisted sperm analysis. All results were compared with those on at least 11 untreated male common marmosets of the same colony. Results: Each of the targeted proteins relevant for male reproduction was detected in testes of F1. At the protein level only steroid 5α-reductase 1 (SRD5A1) was enhanced expressed in testes of EDEX F1, and LDEX F1 compared to C. At the gene level, SRD5A1, SRD5A2 and Ki 67 were each enhanced expressed compared to C, but in testes of EDEX F1 only. Testis size did not vary significantly during the course of the year. During the course of the day there was a significant rise of the testosterone concentration in blood plasma between 8 a.m. and 12 noon. However, there was no significant difference in the testosterone concentration during other times of the day, nor between summer and winter or in relation to the age of the monkeys. Furthermore there was no correlation between the testosterone and the 17β oestradiol concentrations in the same blood samples. Comparisons of all measured PVS parameters between DEX F2/F3 and C gave a lower success rate of PVS in DEX F2/F3 as the only difference. Comparisons of the ejaculates between age groups irrespective of the DEX classification revealed that young adult common marmosets possess a significantly higher percentage of motile sperms as well as a significantly lower success rate of PVS compared to adult monkeys. Conclusions: The results of this study suggest how stress during pregnancy could influence subsequent generations in the common marmoset: in DEX F1, important requirements of active testosterone biosynthesis as well as of mediation of testosterone and oestrogen signals in testis are met, possibly even enhanced. In addition, these results confirm the relevance of the common marmoset as a model organism for human male reproduction. In DEX F2/F3, the low success rate of PVS might indicate a reduced capability to concentrate in generations following intrauterine stress on F1. Yet, most of the tested parameters pertinent to reproduction physiology do not differ between DEX F2/F3 and C.:1 EINLEITUNG 2 LITERATURÜBERSICHT 2.1 Glucocorticoide und Stress während der Trächtigkeit 2.2 Relevante Hormone der Reproduktion und ihre Biosynthese 2.2.1 3β-Hydroxysteroid-Dehydrogenase 2 2.2.2 Testosteron und Androgenrezeptor 2.2.3 Steroid-5α-Reduktasen 2.2.4 Aromatase 2.2.5 17β-Hydroxysteroid-Dehydrogenase 7 2.2.6 Östradiol und Östrogenrezeptoren 2.2.7 Relaxinsystem 2.2.8 Proliferationsfaktoren 2.3 Weißbüschelaffen 2.3.1 Allgemeines zu Weißbüschelaffen 2.3.2 Reproduktionsbiologie der männlichen Weißbüschelaffen 3 TIERE, MATERIAL UND METHODEN 3.1 Versuchsteil I – ex vivo Hoden DEX F1 3.1.1 Versuchsaufbau (DEX) im DPZ 3.1.2 qPCR an DEX F1 Hoden 3.1.3 Immunhistochemie an Hoden der DEX F1 3.2 Versuchsteil II – in vivo DEX F2/DEX F3 3.2.1 Herkunft der Weißbüschelaffen 3.2.2 Haltung der Weißbüschelaffen 3.2.3 Bestimmung der Hodengrößen und des Körpergewichtes 3.2.4 Blutentnahmen und Hormonbestimmungen mittels ELISA 3.2.5 Penile Vibrostimulation 3.3 Statistische Auswertung 4 ERGEBNISSE 4.1 Versuchsteil I – ex vivo Hoden DEX F1 4.1.1 qPCR der Hoden der F1 4.1.2 Immunhistochemie der Hoden der DEX F1 4.1.2.1 Enzyme der Steroidbiosynthese 4.1.2.2 Steroidrezeptoren 4.1.2.3 Relaxinsystem 4.1.2.4 Proliferationsmarker 4.1.3 Zusammenfassung der Ergebnisse des Versuchsteils I – ex vivo DEX F1 4.2 Versuchsteil II – in vivo DEX F2/F3 4.2.1 Hodengrößen 4.2.2 Testosteron 4.2.3 Ejakulatanalyse 4.2.4 Zusammenfassung der Ergebnisse des Versuchsteils II – in vivo DEX F2/F3 5 DISKUSSION 5.1 Versuchsteil I – ex vivo Hoden DEX F1 5.1.1 Vergleichende Diskussion der Ergebnisse auf Gen- und Proteinebene in den Hoden der F1 5.1.1.1 Relevante Enzyme der Testosteronbiosynthese 5.1.1.2 Steroidrezeptoren 5.1.1.3 Das Relaxinsystem 5.1.1.4 Proliferationsfaktoren 5.2 Versuchsteil II – in vivo DEX F2/F3 5.2.1 Hodengrößen 5.2.2 Testosteron 5.2.3 Ejakulatanalyse 5.3 Schlussbetrachtung 6 ZUSAMMENFASSUNG 7 SUMMARY 8 Literaturverzeichnis 9 ANHANG 10 DANKSAGUNG

Dissertação de Mestrado em Investigação Biomédica apresentada à Faculdade de Medicina
Neuropsychiatric disorders are the most common mental illnesses in the world and present a sex dimorphism in prevalence, symptoms and treatment. Sex differences arise since neurodevelopment in which males experience a peak in testosterone that is responsible for brain masculinization, meaning there is an organization in density, connectivity and morphology of cells, including microglia, in several brain regions. This remodeling of the brain will have long term consequences in behavior.Microglia are the immune cells of the central nervous system that are responsible for sculpting neuronal circuits during neurodevelopment, but also ensuring the homeostasis through screening the parenchyma with their highly dynamic ramifications. The morphology and function of microglia are under the influence of the A2A receptor (A2AR), which blockade is known to have anxiolytic effects. Our group has demonstrated that upon prenatal stress, induced by dexamethasone (DEX) exposure on gestational days 18 and 19, males and females have an anxious-like behavior at adulthood (postnatal day 90 – PND90) and present a sex-specific remodeling of microglia morphology in the prefrontal cortex (PFC), a brain region essential for emotional regulation. Furthermore, when these animals are treated with an anxiolytic (SCH58261, an antagonist of the A2AR), only male behavior and microglia morphology return to physiological conditions. The sex dimorphism observed in this study lead to the hypothesis that testosterone could have a protective effect in males.The aim of this thesis is to assess A2AR ability to retrieve microglia morphology and behavior upon brain masculinization of females prenatally exposed to DEX. In other words, through female masculinization (by mimicking the peak of testosterone in males on PND0), we aim to evaluate if testosterone has a permissive effect for the action of the antagonist of the A2AR in females exposed to DEX. Neurodevelopmental behavior is assessed between PND5 and 17, through a battery of tests. Regarding sex differences, males and females have a similar performance in physiology therefore, neonatal administration of testosterone did not have major effects on behavior. When DEX is administered, no effect is observed on behavior however, females have reduced strength and an anticipation of eye opening day. In DEX groups, no sex dimorphism is observed, but testosterone increases strength and delays eye opening day in females. Furthermore, testosterone induces a delay in cliff avoidance, which is reverted by DEX exposure. Previous results from our group show that males and females prenatally exposed to DEX have changes in neurodevelopmental tests performance and anxious-like behavior at adulthood. In this cohort of animals DEX did not induce changes in the progeny performance in neurodevelopmental tests nor an anxious-like behavior, suggesting that alterations in neurodevelopmental behavior might be used as predictive factors for the development of neuropsychiatric disorders.When DEX females are neonatally masculinized with testosterone, no anxious-like behavior is observed with or without SCH58261 administration, suggesting a protective effect of testosterone in behavior. However, when we analyze masculinized females’ microglia from the PFC at adulthood, we observe testosterone was not able to revert DEX-induced atrophy even with anxiolytic treatment. Furthermore, when comparing CTRL+T and DEX+T females we observe no association between behavior and microglia morphology. These results suggest that testosterone was not permissive to the action of SCH58261, and also that testosterone has a mechanism to modulate behavior that is independent of microglia morphology. Neonatal testosterone administration did not induce changes in peripheral levels of corticosterone at adulthood. Hence, when evaluating testosterone levels from serum we observe a tendency for SCH58261 to reduce levels of this hormone in CTRL+T females, but when this anxiolytic is administered in DEX+T females the pattern of action changes, and there is a tendency for an increase in testosterone levels. These results lead us to suggest that the blockage of A2AR modulates testosterone levels, peripherally. In vitro studies were conducted aiming to unravel a possible interaction between androgen receptors (AR) and A2AR specifically in microglial cells. We show that testosterone has an impact on A2AR density that is dependent of time of exposure and concentration. On the other hand, activating or blocking A2AR does not have an impact on AR density. This work highlights the potential anxiolytic properties of testosterone, and also its role in modulating A2AR, namely in microglia, which could account for the sex dimorphism observed in microglia morphology and in behavior. Nonetheless, additional studies are needed to further unveil the possible protective and organizational effect of testosterone in this model of chronic anxiety.
As doenças neuropsiquiátricas, são as enfermidades mentais mais comuns no mundo e apresentam dimorfismo de sexo na sua prevalência, sintomatologia e tratamento. Estas diferenças iniciam-se no neurodesenvolvimento, pois os machos têm um pico de testosterona que induz masculinização cerebral, ou seja, reorganização na densidade, conectividade e morfologia das células, como a microglia, em várias regiões cerebrais. A remodelação cerebral irá ter repercussões a longo-prazo no comportamento. A microglia é a célula imune do sistema nervoso central que é responsável por esculpir os circuitos neuronais durante o desenvolvimento cerebral, mas também por manter a homeostasia no parênquima, através das suas ramificações altamente dinâmicas. A morfologia e função da microglia estão sob a influência dos recetores A2A (A2AR), cujo bloqueio foi demonstrado como tendo efeito ansiolítico.O nosso grupo mostrou que numa situação de stress pré-natal, induzido por dexametasona (DEX) nos dias gestacionais 18 e 19, machos e fêmeas têm um comportamento do tipo ansioso na idade adulta (dia pós-natal 90 – PND90) e remodelação da morfologia da microglia dependente do sexo no córtex pré-frontal (PFC), uma região cerebral essencial para a regulação emocional. Adicionalmente, quando os descendentes são tratados com um ansiolítico (SCH58261; antagonista dos A2AR), apenas o comportamento e a morfologia da microglia dos machos regressa à fisiologia. O dimorfismo de sexo visto neste estudo levou à hipótese de que a testosterona poderia ter um efeito protetor nos machos.O objetivo deste estudo é perceber a capacidade dos A2AR recuperarem a morfologia da microglia e comportamento após masculinização cerebral de fêmeas prenatalmente expostas a DEX. Noutras palavras, através da masculinização feminina (mimetização do pico de testosterona que ocorre a PND0 em machos), objetivámos avaliar se a testosterona tem um efeito permissivo para a ação do antagonista dos A2AR em fêmeas expostas a DEX. O comportamento no neurodesenvolvimento foi estudado entre PND5 e 17 através de uma bateria de testes. Relativamente às diferenças de sexo, machos e fêmeas têm uma performance semelhante fisiologicamente, como tal a administração neonatal de testosterona não teve efeitos major no comportamento. A exposição a DEX não teve efeito no comportamento, contudo, as fêmeas têm uma redução na força e uma antecipação do dia de abertura do olho. Nos grupos DEX, não se observou dimorfismo de sexo, mas a testosterona aumentou a força e atrasou o dia de abertura do olho em fêmeas. Além disso, a testosterona induziu um atraso na aversão ao precipício, que foi revertida pela DEX. Estudos prévios do grupo mostraram que machos e fêmeas prenatalmente expostos a DEX têm mudanças no comportamento do neurodesenvolvimento e um comportamento do tipo ansioso a PND90. Nestes animais a DEX não induziu mudanças comportamentais em nenhuma idade, sugerindo que as alterações no comportamento do neurodesenvolvimento poderão ser usadas como fatores preditivos para o desenvolvimento de doenças psiquiátricas.Quando as fêmeas DEX são masculinizadas neonatalmente com testosterona, não se observa comportamento do tipo ansioso, com ou sem administração de SCH58261, sugerindo um efeito protetor da testosterona no comportamento. No entanto, quando analisamos a microglia do PFC de fêmeas a PND90, observamos que a testosterona não é capaz de reverter a atrofia induzida pela DEX mesmo com a administração do ansiolítico. Adicionalmente, quando comparamos as fêmeas CTRL+T e DEX+T vimos que não há associação entre o comportamento e a morfologia da microglia. Estes resultados sugerem que a testosterona não teve um efeito permissivo à ação do SCH58261, mas também que o mecanismo de ação da testosterona no comportamento é independente da morfologia da microglia.A administração neonatal de testosterona não induziu alterações nos níveis periféricos de corticosterona a PND90. Todavia, quando avaliamos os níveis de testosterona vemos uma tendência para o SCH58261 reduzir os níveis desta hormona em fêmeas CTRL+T, mas em fêmeas DEX+T o ansiolítico induz uma tendência para o aumento de testosterona. Estes resultados levam a sugerir que o bloqueio dos A2AR modula os níveis de testosterona periféricos. Estudos in vitro tiveram o objetivo de perceber a possível interação entre recetores de androgénios (AR) e A2AR na microglia. Mostramos que a testosterona modula a densidade dos A2AR, de modo dependente do tempo de exposição e da concentração. Por outro lado, a ativação ou bloqueio dos A2AR não teve impacto na densidade dos AR. Este trabalho mostra as propriedades ansiolíticas da testosterona, mas também o seu papel modulador dos A2AR, nomeadamente na microglia, o que poderá explicar o dimorfismo de sexo na morfologia da microglia e comportamento. No entanto, estudos adicionais são necessários para melhor perceber o possível efeito protetor e organizacional da testosterona neste modelo de ansiedade crónica.
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