Variation in the ontogeny of sex steroid levels between latitudinal populations of the medaka.

INTRODUCTION: Sex steroids mediate the expression of sexual dimorphism during ontogeny, and populations that differ in the magnitudes of sexual dimorphism may accordingly differ in the ontogenetic patterns of their sex steroid levels. The medaka, Oryzias latipes species complex, shows geographic variation in the magnitude of sexual dimorphism with respect to the lengths of their anal and dorsal fins; dimorphism is greater in low-latitude populations than in high-latitude populations. However, sexual differences in the ontogenetic dynamics of sex steroids, and its interpopulation variation, have not been examined. RESULTS: We measured testosterone (T), estradiol-17ß (E2), and 11-ketotestosterone (11-KT) concentrations throughout ontogeny of laboratory-reared fish from two latitudinal populations: Aomori (northern) and Okinawa (southern). In both populations, the levels of all three steroids were high during early ontogenetic stages and decreased with growth. After reaching about 15 mm in standard length, when sexual dimorphisms in fin lengths became apparent, steroid levels increased and tended to plateau. Sexual differences in the steroid levels were observed only in the later ontogenetic stages; T and 11-KT levels were higher in males, while E2 levels were higher in females. Accordingly, interpopulation differences also became clearer; the southern fish tended to show higher T levels and lower E2 levels than the northern fish. CONCLUSIONS: The ontogenetic patterns of sex steroid levels paralleled the ontogeny of anal and dorsal fins in the two latitudinal populations, suggesting that interpopulation variation in the degree of sexual dimorphisms in fin lengths is mediated by sex steroid-dependent regulation of fin elongation.

Immuno-based detection of extracellular vesicles in urine as diagnostic marker for prostate cancer.

Extracellular vesicles (including the subclass exosomes) secreted by cells contain specific proteins and RNA that could be of interest in determining new markers. Isolation/characterization of PCa-derived exosomes from bodily fluids enables us to discover new markers for this disease. Unfortunately, isolation with current techniques (ultracentrifugation) is labor intensive and other techniques are still under development. The goal of our study was to develop a highly sensitive time-resolved fluorescence immunoassay (TR-FIA) for capture/detection of PCa-derived exosomes. In our assay, biotinylated capture antibodies against human CD9 or CD63 were incubated on streptavidin-coated wells. After application of exosomes, Europium-labeled detection antibodies (CD9 or CD63) were added. Cell medium from 37 cell lines was taken to validate this TR-FIA. Urine was collected (after digital rectal exam) from patients with PCa (n = 67), men without PCa (n = 76). As a control, urine was collected from men after radical prostatectomy (n = 13), women (n = 16) and patients with prostate cancer without digital rectal exam (n = 16). Signal intensities were corrected for urinary PSA and creatinine. This TR-FIA can measure purified exosomes with high sensitivity and minimal background signals. Exosomes can be measured in medium from 37 cell lines and in urine. DRE resulted in a pronounced increase in CD63 signals. After DRE and correction for urinary PSA, CD9 and CD63 were significantly higher in men with PCa. This TR-FIA enabled us to measure exosomes with high sensitivity directly from urine and cell medium. This TR-FIA forms the basis for testing different antibodies directed against exosome membrane markers to generate disease-specific detection assays.

Plasma insulin levels are regulated by release, rather than transcription or translation, in barfin flounder, Verasper moseri.

We evaluated whether transcription or translation of the preproinsulin gene or insulin release into plasma is the primary regulator of plasma insulin level in barfin flounder. Three experimental groups were used: one tested 2h after feeding (Fed), one tested after fasting for 5 days (Fasted), and one tested 2 h after feeding following 5 days of fasting (Refed). No significant differences in insulin transcription, insulin concentrations in the principal islets (PI), or plasma total insulin-like growth factor-I (IGF-I) levels were observed between the three groups. In contrast, plasma insulin level in the Fasted group was significantly lower (P<0.002) than that in the other groups. These results suggest that insulin release is the primary regulator of plasma insulin level and is more sensitive to short-term changes in nutritional conditions than IGF-I level. Furthermore, we estimated the capacity for insulin release. Based on various individual measures, the average insulin stored in the PI was 82.8 µg/kg body weight (BW), and the maximum plasma content of insulin was estimated to be <1.7 µg/kg BW. The half-life of plasma insulin in diabetogenic chemically (alloxan) treated flounder injected with insulin was estimated to be 2.79 h, which is much longer than that in mammals, assuming a two-compartment model for the ß phase. These results suggest that the capacity for insulin release in fish is ensured by at least two systems, such as the ability to store excess insulin in Brockman bodies, and enhanced efficiency of insulin storage by elongating its half-life.

Design and validation of a novel immunological test for enterolactone.

Enterolactone (ENL) is produced by the gut microflora from lignans found in edible plants. ENL is estrogenic with no effect on the E-screen test and is a natural Selected Estrogen Receptor Modulator (SERM) with health interests that have to be checked in clinical studies with bioavailability assessment. Two haptens of ENL were synthesized, with a spacer arm at the C5 position having either 2 or 4 carbon atoms (ENLΔ2 and ENLΔ4, respectively). Hapten coupling to bovine serum albumin (BSA) was characterized by MALDI mass spectrometry. Polyclonal antibodies were obtained against the BSA conjugates. Additional conjugates were generated by coupling to swine thyroglobulin (Thyr). Homologous and heterologous competitive ELISAs were developed with Thyr or BSA conjugates as coating. The best assays were validated on biological samples from mice. Both antibodies exhibited the same IC50 at 1.5 ng mL(-1) with a detection limit below 0.5 ng mL(-1). Most cross-reactions with structurally related lignans were lower than 0.03%. This new assay type is faster, more specific and more reliable than existing ones.