Lower birthweight and left-/mixed-handedness are associated with intensified age-related sex steroid decline in men. Findings from the Men's Health 40+ Study.

In males, age-related decline in free testosterone (T) and dehydroepiandrosterone (DHEA) by 2-3% per year has been reported. Estradiol (E2) and progesterone (P) seem to decrease as well, but to a lesser extent. Lower sex steroid levels in men have been related to physical and mental symptoms. Low birthweight and left-/mixed-handedness (L/MH) are indicators of an adverse fetal environment during pregnancy, and both have been linked to morbidity in later life. The aim of this study is to examine the relationship between lower birthweight as well as L/MH and age-related sex steroid decline. In a cross-sectional study design, saliva samples were collected under standardized conditions from healthy men for subsequent steroid hormone analysis using standard luminescence immunoassays. T (M = 67.57 pg/mL), DHEA (M = 247.91 pg/mL), E2 (M = 1.29 pg/mL), and P (M = 28.20 pg/mL) have been quantified leading to a final sample of 256 men providing complete data on sex hormones (MAge =57.8; SDAge  = 10.8). Information on participants' birthweight was obtained from birth reports (N = 134), and participants were asked about their handedness (right-handed, left-handed, mixed-handed) (N = 256). Multivariate-adjusted linear regression models relating each sex hormone individually and the principal component of declining steroid hormones (DSH)-an integrated hormonal parameter-with handedness and birthweight did not identify significant associations except for handedness and E2. Moderation analysis using robust regression accounting for bias due to influential data points detected a significant association between age and DSH for handedness (ß = -0.0314, p = 0.040) but only a trend for birthweight (ß = 0.0309, p = 0.073). For lower birthweight, a trend toward intensified age-related sex steroid decline in men was observed, while for L/MH, a significant association with intensified age-related sex steroid decline was identified. These results indicate that L/MH and potentially also lower birthweight might be considered as early risk factors for endocrine health in later life.

Impairment of T helper and T regulatory cell responses at birth.

BACKGROUND: There is strong evidence that reduced exposures to microbial compounds triggering innate immune responses early in life are critical for the development of allergic illnesses. The underlying mechanisms remain unknown, but will include T-cell responses either along T helper type 1 (Th1)/Th2 pathways or via T regulatory and Th17 cells. Yet, little is known about innate immune responses and the function of T regulatory/Th17 cells at birth. The aim of this study was to investigate T-cell responses to innate (Lipid A/LpA, peptidoglycan/Ppg) and adaptive (phytohemagglutinin) stimuli at birth and to compare these findings with adult immune responses. METHODS: Cord and peripheral blood mononuclear cells including T regulatory and Th17 cells from 25 neonates and 25 adults were examined for proliferation, cytokine secretion, surface, mRNA expression and functional suppression assays. RESULTS: Proliferation and cytokine responses to innate stimuli were less mature at birth than in adulthood. T regulatory and Th17 cells were less expressed in cord than in adult blood (Ppg-induced Foxp3, P = 0.001, LpA-induced CD4(+) CD25(+) high, P = 0.02; Th17 : P < 0.0001). Mitogen-induced suppression of T-regulatory cells on T-effector cell function was less efficient in cord than in adult blood (P = 0.01). At both ages, Th17 cells were correlated with Th1/Th2 cells (P < 0.01), but not with interleukin-10 secretion following innate-stimulation. CONCLUSION: Innate immune responses are immature at birth. Furthermore, the function of T regulatory and Th17 cells is impaired. Th17 cells in association with Th1/Th2 cells may be involved in early immuno-modulation. Potent innate immune stimulation early in life can potentially contribute to protection from allergic diseases.

Difference in XTcf-3 dependency accounts for change in response to beta-catenin-mediated Wnt signalling in Xenopus blastula.

Wnt signalling functions in many tissues and during different stages of animal development to produce very specific responses. In early Xenopus embryos there is a dramatic change in response to Wnt signalling within only a few hours of development. Wnt signalling in very early embryos leads to a dorsalising response, which establishes the endogenous dorsal axis. Only a few hours later in development, almost the opposite happens: Xwnt-8 functions to pattern the embryonic mesoderm by promoting ventral and lateral mesoderm. The specificity of the response could conceivably be carried out by differential use of different signal transduction pathways, many of which have recently been described. We have found, however, that this dramatic shift in response to Wnt signalling in early Xenopus is not brought about by differential use of distinct signal transduction pathways. In fact beta-catenin, a downstream component of the canonical Wnt signal transduction pathway, functions not only in the early dorsalising response but also in the later ventrolateral-promoting response. Interaction of beta-catenin with the XTcf-3 transcription factor is required for the early dorsalising activity. In contrast, our experiments suggest that late Wnt signalling in the ventrolateral mesoderm does not require a similar dependency of beta-catenin function on XTcf-3. Our results highlight the potential versatility of the canonical Wnt pathway to interact with tissue-specific factors downstream of beta-catenin, in order to achieve tissue-specific effects.

Inducible gene expression in transgenic Xenopus embryos.

The amphibian Xenopus laevis has been successfully used for many years as a model system for studying vertebrate development. Because of technical limitations, however, molecular investigations have mainly concentrated on early stages. We have developed a straightforward method for stage-specific induction of gene expression in transgenic Xenopus embryos [1] [2]. This method is based on the Xenopus heat shock protein 70 (Xhsp70 [3]) promoter driving the expression of desired gene products. We found that ubiquitous expression of the transgene is induced upon relatively mild heat treatment. Green fluorescent protein (GFP) was used as a marker to monitor successful induction of gene expression in transgenic embryos. We used this method to study the stage specificity of Wnt signalling function. Transient ectopic Wnt-8 expression during early neurulation was sufficient to repress anterior head development and this capacity was restricted to early stages of neurulation. By transient over-expression at different stages of development, we show that frizzled-7 disrupted morphogenesis sequentially from anterior to posterior along the dorsal axis as development proceeds. These results demonstrate that this method for inducible gene expression in transgenic Xenopus embryos will be a very powerful tool for temporal analysis of gene function and for studying molecular mechanisms of vertebrate organogenesis.

Two novel Xenopus frizzled genes expressed in developing heart and brain.

A family of genes related to the Drosophila wingless receptor frizzled have been found in vertebrates. We have cloned full length cDNAs of two novel frizzled genes from embryonic Xenopus tissue. We are calling them Xfz7 and Xfz9 (for Xenopus frizzled) because their deduced peptide sequences show extensive similarity to other vertebrate frizzled molecules. Xfz7 is closely related to human, chick and mouse frz-7 and Xfz9 is most related to human FZD9 and mouse fzd9. Xfz7 is expressed in a broad, complex and dynamic pattern beginning at gastrulation. At later stages Xfz7 expression is found in neural crest, neural tube, eye, pronephric duct and the heart. Xfz9 expression in contrast is more restricted to the neuroectoderm and, at later stages of development, to the dorsal regions of the mid- and hindbrain.

BMP-2/-4 and Wnt-8 cooperatively pattern the Xenopus mesoderm.

Establishment of the dorsoventral axis is central to animal embryonic organization. In Xenopus two different classes of signaling molecules function in the dorsoventral patterning of the mesoderm. Both the TGF-beta-related products of the BMP-2 and BMP-4 genes and the Wnt molecule encoded by Xenopus Wnt-8 specify ventral fate and appear to inhibit dorsal mesodermal development. The similar functions of these molecularly very different classes of signaling molecules prompted us to study their mutual regulation and to closely compare their roles in mesoderm patterning. We find that Wnt-8 and BMP-4 are indistinguishable in their abilities to induce expression of ventral genes. Although BMP-2/-4 signaling regulates Wnt-8 expression, these genes do not function in a linear pathway because Wnt-8 overexpression cannot compensate for an inhibition of BMP-2/-4 function, but rather BMP-4 overexpression rescues ventral gene expression in embryos with inhibited Wnt-8 function. We further find that Wnt-8 and BMP-2/-4 differ in their abilities to regulate dorsal gene expression. While BMP-4 appears to generally inhibit the expression of dorsal genes, XenopusWnt-8 only inhibits the expression of the notochord marker Xnot. Whereas the inhibitory effect of BMP-2/-4 localizes dorsal mesodermal fate, our results suggest that Xenopus Wnt-8 functions in the further patterning of the dorsal mesoderm into the most dorsal sector from which the notochord develops and the dorsolateral sector from where the somites differentiate.

Wnt and FGF pathways cooperatively pattern anteroposterior neural ectoderm in Xenopus.

Previous gain-of-function assays in Xenopus have demonstrated that Xwnt-3a can pattern neural tissue by reducing the expression of anterior neural genes, and elevating the expression of posterior neural genes. To date, no loss-of-function studies have been conducted in Xenopus to show a requirement of endogenous Wnt signaling for patterning of the neural ectoderm along the anteroposterior axis. We report that expression of a dominant negative Wnt in Xenopus embryos causes a reduction in the expression of posterior neural genes, and an elevation in the expression of anterior neural genes, thereby confirming the involvement of endogenous Wnt signaling in patterning the neural axis. We further demonstrate that the ability of Xwnt-3a to decrease expression of anterior neural genes in noggin-treated explants is dependent upon a functional FGF signaling pathway, while the elevation of expression of posterior neural genes does not require FGF signaling. The previously reported ability of FGF to elevate the expression of posterior neural genes in noggin-treated explants was found to be dependent on endogenous Wnt signaling. We conclude that neural induction occurs initially in a Wnt-independent manner, but that generation of complete anteroposterior neural pattern requires the cooperative actions of Wnt and FGF pathways.

Expression of a dominant-negative Wnt blocks induction of MyoD in Xenopus embryos.

During gastrulation of Xenopus embryos the prospective mesoderm is induced initially with domains of dorsal and ventral fate, then further patterned to generate somitic mesoderm by signals from the gastrula organizer. Although Xwnt-8 is expressed in future ventrolateral mesoderm and induces prospective epidermis to differentiate in vitro as ventral mesoderm, no loss-of-function studies have demonstrated a requirement for Wnt signaling for the normal expression of mesodermal genes in the gastrula. We report development of a dominant-negative Wnt (dnXwnt-8) that inhibits embryonic responses to Wnt signaling in a cell-nonautonomous fashion. By expressing dnXwnt-8 in Xenopus embryos, we uncover a requirement of Wnt signaling for localized expression in prospective mesoderm of XMyoDa and Xenopus-posterior (Xpo). Because ectopic expression of functional Xwnt-8 in the dorsal marginal zone of the gastrula induces ectopic XMyoDa and Xpo, both gain-of-function and loss-of-function experiments support a model in which endogenous Xwnt-8 functions to induce expression of genes involved in specification of ventral and somitic mesoderm.

decapentaplegic, a target gene of the wingless signalling pathway in the Drosophila midgut.

dishevelled, shaggy/zeste-white 3 and armadillo are required for transmission of the wingless signal in the Drosophila epidermis. We show that these genes act in the same epistatic order in the embryonic midgut to transmit the wingless signal. In addition to mediating transcriptional stimulation of the homeotic genes Ultrabithorax and labial, they are also required for transcriptional repression of labial by high wingless levels. Efficient labial expression thus only occurs within a window of intermediate wingless pathway activity. Finally, the shaggy/zeste-white 3 mutants revealed that wingless signalling can stimulate decapentaplegic transcription in the absence of Ultrabithorax, identifying decapentaplegic as a target gene of wingless. As decapentaplegic itself is required for wingless expression in the midgut, this represents a positive feed-back loop between two cell groups signalling to each other to stimulate each other's signal production.

Two different thresholds of wingless signalling with distinct developmental consequences in the Drosophila midgut.

Drosophila wingless encodes a Wnt protein which mediates communication between cells. Although wingless protein is secreted from cells, there is debate as to what is the range of wingless action. We examined the function of wingless in the larval midgut, and found that wingless acts at two different thresholds to pattern this tissue. Low wingless levels are required to promote the development of copper cells, highly differentiated midgut cells of the larval midgut that are specified by the homeotic gene labial. High wingless levels repress copper cell development and allow differentiation of an alternative cell type, called large flat cells. These two developmental outcomes reflect labial expression, which is stimulated at low levels and repressed at high levels of wingless signalling. Thus, midgut cells respond differentially to distinct wingless thresholds in terms of both gene control and cellular differentiation.

Specification of a single cell type by a Drosophila homeotic gene.

Homeotic genes function in blocks of cells along the body axis to specify diverse developmental pathways. Among the Drosophila homeotic genes, only one is known to be expressed in the endoderm. We show here that expression of this gene, labial (lab), coincides with copper cells, highly specialized cells of the larval midgut. lab is strictly required for copper cell formation, a requirement that starts in the embryo and extends through larval stages. This implies a function of lab not only in the determination and differentiation of copper cells, but also in the maintenance of their differentiated state. Ectopic expression of lab during embryogenesis reprograms other midgut cells within and outside the lab domain to become copper cell-like. Thus, lab functions in the larval midgut to specify a single cell type.