Gregor Majdic

Sex differences in brain developing in the presence or absence of gonads

Brain sexual differentiation results from the interaction of genetic and hormonal influences. This study used a unique agonadal mouse model to determine relative contributions of genetic and gonadal hormone influences in the differentiation of selected brain regions. SF‐1 knockout (SF‐1 KO) mice are born without gonads and adrenal glands and are not exposed to endogenous sex steroids during fetal/neonatal development. Consequently, male and female SF‐1 KO mice are born with female external genitalia and if left on their own, die shortly after birth due to adrenal insufficiency. In this study, SF‐1 KO mice were rescued by neonatal adrenal transplantation to examine their brain morphology in adult life. To determine potential brain loci that might mediate functional sex differences, we examined the area and distribution of immunoreactive calbindin and neuronal nitric oxide synthase in the preoptic area (POA) and ventromedial nucleus of the hypothalamus, two areas previously reported to be sexually dimorphic in the mammalian brain. A sex difference in the positioning of cells containing immunoreactive calbindin in a group within the POA was clearly gonad dependent based on the elimination of the sex difference in SF‐1 KO mice. Several other differences in the area of ventromedial hypothalamus and in POA were maintained in male and female SF‐1 KO mice, suggesting gonad‐independent genetic influences on sexually dimorphic brain development.

Determination of reference genes for circadian studies in different tissues and mouse strains

BackgroundCircadian rhythms have a profound effect on human health. Their disruption can lead to serious pathologies, such as cancer and obesity. Gene expression studies in these pathologies are often studied in different mouse strains by quantitative real time polymerase chain reaction (qPCR). Selection of reference genes is a crucial step of qPCR experiments. Recent studies show that reference gene stability can vary between species and tissues, but none has taken circadian experiments into consideration.ResultsIn the present study the expression of ten candidate reference genes (Actb, Eif2a, Gapdh, Hmbs, Hprt1, Ppib, Rn18s, Rplp0, Tbcc and Utp6c) was measured in 131 liver and 97 adrenal gland samples taken from three mouse strains (C57BL/6JOlaHsd, 129Pas plus C57BL/6J and Crem KO on 129Pas plus C57BL/6J background) every 4 h in a 24 h period. Expression stability was evaluated by geNorm and NormFinder programs. Differences in ranking of the most stable reference genes were observed both between individual mouse strains as well as between tissues within each mouse strain. We show that selection of reference gene (Actb) that is often used for analyses in individual mouse strains leads to errors if used for normalization when different mouse strains are compared. We identified alternative reference genes that are stable in these comparisons.ConclusionsGenetic background and circadian time influence the expression stability of reference genes. Differences between mouse strains and tissues should be taken into consideration to avoid false interpretations. We show that the use of a single reference gene can lead to false biological conclusions. This manuscript provides a useful reference point for researchers that search for stable reference genes in the field of circadian biology.

Cooperation of sex chromosomal genes and endocrine influences for hypothalamic sexual differentiation.

There is little debate that mammalian sexual differentiation starts from the perspective of two primary sexes that correspond to differential sex chromosomes (X versus Y) that lead to individuals with sex typical characteristics. Sex steroid hormones account for most aspects of brain sexual differentiation, however, a growing literature has raised important questions about the role of sex chromosomal genes separate from sex steroid actions. Several important model animals are being used to address these issues and, in particular, they are taking advantage of molecular genetic approaches using different mouse strains. The current review examines the cooperation of genetic and endocrine influences from the perspective of behavioral and morphological hypothalamic sexual differentiation, first in adults and then in development. In the final analysis, there is an ongoing need to account for the influence of hormones in the context of underlying genetic circumstances and null hormone conditions.

Aggressive Behaviors in Adult SF-1 Knockout Mice That Are Not Exposed to Gonadal Steroids During Development

Sex hormones are a major factor responsible for the development of sex differences. Steroidogenic factor 1 (SF-1) is a key regulator of gonadal and adrenal development, and SF-1 knockout mice (SF-1 KO) are born without gonads and adrenal glands. Consequently, these mice are not exposed to gonadal sex steroids. SF-1 KO pups die shortly after birth due to adrenal deficiency. In the present study, SF-1 KO mice were rescued by neonatal corticosteroid injections followed by adrenal transplantations on day 7-8 postnatally. Control mice received corticosteroid injections and were gonadectomized prior to puberty. Mice were observed interacting with ovariectomized hormone primed females and gonad-intact males. In the absence of sex steroid replacement, adult SF-1 KO mice were significantly more aggressive than control mice in tests with stimulus females. After testosterone treatment, control males displayed significantly more aggression towards male intruders than control female mice, or male and female SF-1 KO mice, suggesting a developmental role of gonadal hormones in the expression of aggressive behavior and affirming SF-1 KO mice as a behavioral model to investigate affects of fetal gonad deficiency.

Novel Insights into the Downstream Pathways and Targets Controlled by Transcription Factors CREM in the Testis

The essential role of the Crem gene in normal sperm development is widely accepted and is confirmed by azoospermia in male mice lacking the Crem gene. The exact number of genes affected by Crem absence is not known, however a large difference has been observed recently between the estimated number of differentially expressed genes found in Crem knock-out (KO) mice compared to the number of gene loci bound by CREM. We therefore re-examined global gene expression in male mice lacking the Crem gene using whole genome transcriptome analysis with Affymetrix microarrays and compared the lists of differentially expressed genes from Crem−/− mice to a dataset of genes where binding of CREM was determined by Chip-seq. We determined the global effect of CREM on spermatogenesis as well as distinguished between primary and secondary effects of the CREM absence. We demonstrated that the absence of Crem deregulates over 4700 genes in KO testis. Among them are 101 genes associated with spermatogenesis 41 of which are bound by CREM and are deregulated in Crem KO testis. Absence of several of these genes in mouse models has proven their importance for normal spermatogenesis and male fertility. Our study showed that the absence of Crem plays a more important role on different aspects of spermatogenesis as estimated previously, with its impact ranging from apoptosis induction to deregulation of major circadian clock genes, steroidogenesis and the cell-cell junction dynamics. Several new genes important for normal spermatogenesis and fertility are down-regulated in KO testis and are therefore possible novel targets of CREM.

Steroidogenic Factor 1 and the Central Nervous System

Steroidogenic factor 1 (SF‐1; officially designated NR5a1) is a member of a nuclear receptor superfamily with important roles in the development of endocrine systems. Studies with global and tissue‐specific (i.e. central nervous system) knockout mice have revealed several roles of SF‐1 in brain. These include morphological effects on the development of the ventromedial nucleus of the hypothalamus and functional effects on body weight regulation through modulation of physical activity, anxiety‐like behaviours and female sexual behaviours. Although such defects are almost certainly a result of the absence of SF‐1 acting as a transcription factor in the hypothalamus, global SF‐1 knockout mice also represent a model for studying the sex differences in the brain that develop in the absence of exposure to foetal sex steroid hormones as a result of the absence of gonads. In the present review, current knowledge of the roles of SF‐1 protein in the central nervous system is discussed.

Evidence that Sex Chromosome Genes Affect Sexual Differentiation of Female Sexual Behavior

Female receptivity including the immobile hormone-dependent lordosis posture is essential for successful reproduction in rodents. It is well documented that lordosis is organized during the perinatal period when the actions of androgens decrease the males ability to display this behavior in adulthood. Conversely the absence of androgens, and the presence of low levels of prepubertal estrogens, preserve circuitry that regulates this behavior in females. The current study set out to determine whether sex chromosomal genes are involved in the differentiation of this behavior. An agonadal mouse model was used to test this hypothesis. The SF-1 gene (Nr5a1) is required for development of gonads and adrenal glands, and knockout mice are consequently not exposed to endogenous gonadal steroids. Thus contributions of sex chromosome genes can be disassociated from the actions of estrogens. Use of this model reveals a direct genetic contribution from sex chromosomes in the display of lordosis and other female-typical sexual behavior patterns. It is likely that the concentrations of gonadal steroids present during normal male development modify the actions of sex chromosome genes on the potential to display female sexual behavior.

Circadian expression of steroidogenic cytochromes P450 in the mouse adrenal gland – involvement of cAMP‐responsive element modulator in epigenetic regulation of Cyp17a1

The cytochromeu2003P450 (CYP) genes Cyp51, Cyp11a1, Cyp17a1, Cyb11b1, Cyp11b2 and Cyp21a1 are involved in the adrenal production of corticosteroids, whose circulating levels are circadian. cAMP signaling plays an important role in adrenal steroidogenesis. By using cAMP responsive element modulator (Crem) knockout mice, we show that CREM isoforms contribute to circadian expression of steroidogenic CYPs in the mouse adrenal gland. Most striking was the CREM‐dependent hypomethylation of the Cyp17a1 promoter at zeitgeber timeu200312, which resulted in higher Cyp17a1 mRNA and protein expression in the knockout adrenal glands. The data indicate that products of the Crem gene control the epigenetic repression of Cyp17 in mouse adrenal glands.

Covalent immobilization of antimicrobial agents on titanium prevents Staphylococcus aureus and Candida albicans colonization and biofilm formation

OBJECTIVESnBiofilm-associated implant infections represent a serious public health problem. Covalent immobilization of antimicrobial agents on titanium (Ti), thereby inhibiting biofilm formation of microbial pathogens, is a solution to this problem.nnnMETHODSnVancomycin (VAN) and caspofungin (CAS) were covalently bound on Ti substrates using an improved processing technique adapted to large-scale coating of implants. Resistance of the VAN-coated Ti (VAN-Ti) and CAS-coated Ti (CAS-Ti) substrates against in vitro biofilm formation of the bacterium Staphylococcus aureus and the fungal pathogen Candida albicans was determined by plate counting and visualized by confocal laser scanning microscopy. The efficacy of the coated Ti substrates was also tested in vivo using an adapted biomaterial-associated murine infection model in which control-Ti, VAN-Ti or CAS-Ti substrates were implanted subcutaneously and subsequently challenged with the respective pathogens. The osseointegration potential of VAN-Ti and CAS-Ti was examined in vitro using human bone marrow-derived stromal cells, and for VAN-Ti also in a rat osseointegration model.nnnRESULTSnIn vitro biofilm formation of S. aureus and C. albicans on VAN-Ti and CAS-Ti substrates, respectively, was significantly reduced compared with biofilm formation on control-Ti. In vivo, we observed over 99.9% reduction in biofilm formation of S. aureus on VAN-Ti substrates and 89% reduction in biofilm formation of C. albicans on CAS-Ti substrates, compared with control-Ti substrates. The coated substrates supported osseointegration in vitro and in vivo.nnnCONCLUSIONSnThese data demonstrate the clinical potential of covalently bound VAN and CAS on Ti to reduce microbial biofilm formation without jeopardizing osseointegration.

Relationship between genome and epigenome--challenges and requirements for future research.

Understanding the links between genetic, epigenetic and non-genetic factors throughout the lifespan and across generations and their role in disease susceptibility and disease progression offer entirely new avenues and solutions to major problems in our society. To overcome the numerous challenges, we have come up with nine major conclusions to set the vision for future policies and research agendas at the European level.