Dynamics of maternal androgens and its metabolites during early embryonic development: embryonic modification of a maternal effect.

In birds, exposure to maternal (yolk) testosterone affects a diversity of offspring post-hatching traits, which eventually affect offspring competitiveness. However, maternal testosterone is heavily metabolized at very early embryonic developmental stages to hydrophilic metabolites that are often assumed to be much less biologically potent. Either the rapid metabolism could either keep the maternal testosterone from reaching the embryos, opening the possibility for a mother-offspring conflict or the metabolites may facilitate the uptake of the lipophilic testosterone from the yolk into the embryonic circulation after which they are either converted back to the testosterone or functioning directly as metabolites. To test these possibilities, we injected isotope-labeled testosterone (T-[D5]) into the yolk of freshly laid Rock pigeon (Columba livia) eggs and determined the concentration and distribution of T-[D5] and its labeled metabolites within different egg fractions by liquid chromatography combined with tandem mass spectrometry at day 2, 5 and 10 of incubation. Although under a supraphysiological dosage injection, yolk testosterone decreased within 2 days and was metabolized into androstenedione, conjugated testosterone, etiocholanolone and other components that were unidentifiable due to methodological limitation. We show for the first time that testosterone, androstenedione and conjugated testosterone, but not etiocholanolone, reached the embryo including its brain. Their high concentrations in the yolk and extraembryonic membranes suggest that conversion takes place here. We also found no sex-specific metabolism, explaining why maternal testosterone does not affect sexual differentiation. Our findings showed that maternal testosterone is quickly converted by the embryo, with several but not all metabolites reaching the embryo providing evidence for both hypotheses.

Plasticity in metabolism of maternal androgens in avian embryos.

Mothers can influence offspring phenotypes by transferring non-genetic information to the young, which provides them with a flexible tool to adjust the developmental trajectory of the young in fluctuating environments. Mothers can differentially deposit their resources in the same reproductive attempt in relation to the offspring position in the sibling hierarchy. However, whether embryos from different positions can be plastic in their response to the maternal signals, potentially leading to a mother-offspring conflict, is yet unclear. We used Rock pigeons (Columba livia), that lay two egg clutches where maternal androgen levels in second laid eggs at oviposition are higher than in first laid eggs, and investigated the plasticity of embryonic metabolism of maternal androgens. We experimentally elevated androstenedione and testosterone levels in first eggs to that present in second eggs and measured the change in androgen levels and its main metabolites (etiocholanolone and conjugated testosterone) after 3.5 days of incubation. We found that eggs with increased androgens show a different degree of androgen metabolism depending either on the egg laying sequence or initial androgen levels or both. Our findings indicate that embryos have certain plasticity in response to maternal androgen levels depending on maternal signals.

T cell cholesterol efflux suppresses apoptosis and senescence and increases atherosclerosis in middle aged mice.

Atherosclerosis is a chronic inflammatory disease driven by hypercholesterolemia. During aging, T cells accumulate cholesterol, potentially affecting inflammation. However, the effect of cholesterol efflux pathways mediated by ATP-binding cassette A1 and G1 (ABCA1/ABCG1) on T cell-dependent age-related inflammation and atherosclerosis remains poorly understood. In this study, we generate mice with T cell-specific Abca1/Abcg1-deficiency on the low-density-lipoprotein-receptor deficient (Ldlr-/-) background. T cell Abca1/Abcg1-deficiency decreases blood, lymph node, and splenic T cells, and increases T cell activation and apoptosis. T cell Abca1/Abcg1-deficiency induces a premature T cell aging phenotype in middle-aged (12-13 months) Ldlr-/- mice, reflected by upregulation of senescence markers. Despite T cell senescence and enhanced T cell activation, T cell Abca1/Abcg1-deficiency decreases atherosclerosis and aortic inflammation in middle-aged Ldlr-/- mice, accompanied by decreased T cells in atherosclerotic plaques. We attribute these effects to T cell apoptosis downstream of T cell activation, compromising T cell functionality. Collectively, we show that T cell cholesterol efflux pathways suppress T cell apoptosis and senescence, and induce atherosclerosis in middle-aged Ldlr-/- mice.

Elevated granulocyte-colony stimulating factor and hematopoietic stem cell mobilization in Niemann-Pick type C1 disease.

Niemann-Pick type C1 (NPC1) disease is a progressive lysosomal storage disorder caused by mutations of the NPC1 gene. While neurodegeneration is the most severe symptom, a large proportion of NPC1 patients also present with splenomegaly, which has been attributed to cholesterol and glycosphingolipid accumulation in late endosomes and lysosomes. However, recent data also reveal an increase in the inflammatory monocyte subset in the Npc1nih mouse model expressing an Npc1 null allele. We evaluated the contribution of hematopoietic cells to splenomegaly in NPC1 disease under conditions of hypercholesterolemia. We transplanted Npc1nih (Npc1 null mutation) or Npc1wt bone marrow (BM) into Ldlr-/- mice and fed these mice a cholesterol-rich Western-type diet. At 9 weeks after BM transplant, on a chow diet, the Npc1 null mutation increased plasma granulocyte-colony stimulating factor (G-CSF) by 2-fold and caused mild neutrophilia. At 18 weeks after BM transplant, including 9 weeks of Western-type diet feeding, the Npc1 mutation increased G-csf mRNA levels by ∼5-fold in splenic monocytes/macrophages accompanied by a ∼4-fold increase in splenic neutrophils compared with controls. We also observed ∼5-fold increased long-term and short-term hematopoietic stem cells (HSCs) in the spleen, and a ∼30-75% decrease of these populations in BM, reflecting HSC mobilization, presumably downstream of elevated G-CSF. In line with these data, four patients with NPC1 disease showed higher plasma G-CSF compared with age-matched and gender-matched healthy controls. In conclusion, we show elevated G-CSF levels and HSC mobilization in the setting of an Npc1 null mutation and propose that this contributes to splenomegaly in patients with NPC1 disease.

Determination of reference intervals for urinary steroid profiling using a newly validated GC-MS/MS method.

BACKGROUND: Urinary steroid profiling (USP) is a powerful diagnostic tool to asses disorders of steroidogenesis. Pre-analytical factors such as age, sex and use of oral contraceptive pills (OCP) may affect steroid hormone synthesis and metabolism. In general, USP reference intervals are not adjusted for these variables. In this study we aimed to establish such reference intervals using a newly-developed and validated gas chromatography with tandem mass spectrometry detection method (GC-MS/MS). METHODS: Two hundred and forty healthy subjects aged 20-79 years, stratified into six consecutive decade groups each containing 20 males and 20 females, were included. None of the subjects used medications. In addition, 40 women aged 20-39 years using OCP were selected. A GC-MS/MS assay, using hydrolysis, solid phase extraction and double derivatization, was extensively validated and applied for determining USP reference intervals. RESULTS: Androgen metabolite excretion declined with age in both men and women. Cortisol metabolite excretion remained constant during life in both sexes but increased in women 70-79 years of age. Progesterone metabolite excretion peaked in 30-39-year-old women and declined afterwards. Women using OCP had lower excretions of androgen metabolites, progesterone metabolites and cortisol metabolites. Method validation results met prerequisites and revealed the robustness of the GC-MS/MS method. CONCLUSIONS: We developed a new GC-MS/MS method for USP which is applicable for high throughput analysis. Widely applicable age and sex specific reference intervals for 33 metabolites and their diagnostic ratios have been defined. In addition to age and gender, USP reference intervals should be adjusted for OCP use.