Adult male sexual behavior in mammals requires the neuronal organizing effects of gonadal steroids during a sensitive perinatal period. During development, estradiol differentiates the rat preoptic area (POA), an essential brain region in the male copulatory circuit. Here we report that increases in prostaglandin-E(2) (PGE(2)), resulting from changes in cyclooxygenase-2 (COX-2) regulation induced by perinatal exposure to estradiol, are necessary and sufficient to organize the crucial neural substrate that mediates male sexual behavior. Briefly preventing prostaglandin synthesis in newborn males with the COX inhibitor indomethacin permanently downregulates markers of dendritic spines in the POA and severely impairs male sexual behavior. Developmental exposure to the COX inhibitor aspirin results in mild impairment of sexual behavior. Conversely, administration of PGE(2) to newborn females masculinizes the POA and leads to male sex behavior in adults, thereby highlighting the pathway of steroid-independent brain masculinization. Our findings show that PGE(2) functions as a downstream effector of estradiol to permanently masculinize the brain.

The mechanisms establishing and maintaining dendritic spines in the mammalian CNS remain primarily unknown. We report a novel mechanism of neuronal spine plasticity in the developing preoptic area in which estradiol induces prostaglandin-E2 (PGE2) synthesis that in turn increases the density of spine-like processes. Estradiol requires PGE2 synthesis, in vivo and in vitro, and upregulates the dendritic spine protein spinophilin, an effect attenuated by antagonism of the AMPA-kainate receptor. This signaling pathway may involve cross talk between neighboring neurons and astrocytes and appears specific to the preoptic area in that hippocampal neurons responded with an increase in spinophilin to estradiol but not PGE2. Regionally specific mechanisms of estradiol-mediated synaptic plasticity allow for epigenetic control of complex sex-typic behaviors.

Cholangiocarcinoma (CCA) presents significant diagnostic challenges, resulting in late patient diagnosis and poor survival rates. Primary Sclerosing Cholangitis (PSC) patients pose a particularly difficult clinical dilemma, since they harbor chronic biliary strictures that are difficult to distinguish from CCA. MicroRNAs (miRs) have recently emerged as a valuable class of diagnostic markers; however, thus far, neither extracellular vesicles (EVs) nor miRs within EVs have been investigated in human bile. We aimed to comprehensively characterize human biliary EVs, including their miR content. Conclusion We have established the presence of extracellular vesicles in human bile. In addition, we have demonstrated that human biliary EVs contain abundant miR species, which are stable and therefore amenable to the development of disease marker panels. Furthermore, we have characterized the protein content, size, numbers and size distribution of human biliary EVs. Utilizing Multivariate Organization of Combinatorial Alterations (MOCA), we defined a novel biliary vesicle miR-based panel for CCA diagnosis which demonstrated a sensitivity of 67% and specificity of 96%. Importantly, our control group contained 13 PSC patients, 16 patients with biliary obstruction of varying etiologies (including benign biliary stricture, papillary stenosis, choledocholithiasis, extrinsic compression from pancreatic cysts, and cholangitis), and 3 patients with bile leak syndromes. Clinically, these types of patients present with a biliary obstructive clinical picture that could be confused with CCA. These findings establish the importance of using extracellular vesicles, rather than whole bile, for developing miR-based disease markers in bile. Finally, we report the development of a novel bile-based CCA diagnostic panel that is stable, reproducible, and has potential clinical utility.

Accurate assessment of gonadal steroid levels in the developing brain is critical for understanding naturally occurring steroid-mediated sexual differentiation as well as determining the physiological relevance of exogenous steroid treatments commonly used in the study of this phenomenon. Using RIA, we measured the estradiol (E(2)) content of six regions of the developing brain immediately post partum, 1 d post partum, and after injection of exogenous estradiol benzoate, testosterone propionate, or the aromatase inhibitor formestane. We found sexually dimorphic E(2) content in several regions of the newborn brain. At 2 h of life, there was significantly higher E(2) content in males vs. females in the frontal cortex, hypothalamus and preoptic area but not in the hippocampus, brainstem, or cerebellum. Surprisingly, the female hippocampus had significantly higher E(2) content than all other female regions examined. By d 1 post partum, E(2) levels had decreased precipitously in most brain regions, and only the hypothalamus maintained a sex difference. Injection of female pups with estradiol benzoate raised tissue levels to that of the male in the hypothalamus but 2- to 3-fold higher in the other five regions. Testosterone administration increased E(2) content exclusively in the preoptic area, suggesting local variation in aromatase activity and/or substrate availability. Central administration of formestane decreased estrogen content in the male cortex, hypothalamus, and preoptic area. Formestane treatment also decreased endogenous E(2) in female cortex and hippocampus, suggesting de novo synthesis selectively in these brain regions. These data corroborate and extend previous findings of sex differences in brain E(2) levels perinatally and reveal an unexpected regional heterogeneity in E(2) synthesis and/or metabolism.