In the biochemical evaluation of hirsutism, 50% or less of patients have an elevated total serum testosterone. Recent work has suggested that measuring salivary testosterone or a derived serum ‘free testosterone index’ may be of use in the evaluation of hyperandrogenism. We have measured serum total, derived serum free indices and salivary concentrations of testosterone and 5α-dihydro-testosterone in an unselected group of hirsute patients in order to assess their value in the routine evaluation of hirsutism. The assays were performed using a novel oxidation procedure to overcome the need for chromatographic separation. The ‘free testosterone index’ gave the best discrimination. Salivary androgen concentrations were comparatively poor and cannot be recommended for routine use.
Mouse embryonic stem cells are derived from in vitro explantation of blastocyst epiblasts 1,2 and contribute to both the somatic lineage and germline when returned to the blastocyst 3 but are normally excluded from the trophoblast lineage and primitive endoderm [4][5][6] . Here, we report that cultures of expanded potential stem cells (EPSCs) We sought to establish cultures of new stem cells from cleavage stage mouse embryos. Under such a culture condition, we speculated that the self renewing stem cell population might have expanded potential as the cells of 4-cell (4C) or 8-cell (8C) embryos or the individual blastomeres retain the potential to differentiate to both the trophoectoderm (TE) and the inner cell mass (ICM) [7][8][9][10] . In order to prevent blastomeres from further differentiation and to derive stem cell lines from these cells, we speculated that modulation of the key signaling pathways implicated in the earliest stages of embryonic development might be a rate-limiting step. Genetic and developmental studies have revealed the key roles of conserved mitogen-activated protein kinases (MAPKs), Src, and Hippo pathways in the segregation of the TE and peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.canThe copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/124479 doi: bioRxiv preprint first posted online Apr. 6, 2017; 3 ICM lineages; and furthermore, how disrupting them causes developmental arrest [11][12][13][14][15][16][17] .In addition, Wnt signaling is known to be a key orchestrator of the earliest development stages of vertebrates 18 , and to be involved in the development of mouse preimplantation embryos and trophoblasts [19][20][21][22] . Recent advances have uncovered important functional interactions between Wnt and MAPK pathways via Yap, the key Hippo pathway downstream effector [23][24][25] . We therefore selected inhibitors to simultaneously target these pathways or kinases to block development for the derivation of novel stem cell lines.In order to target MAPKs, we used Mek1 inhibitor PD0325901, JNK Inhibitor VIII (for Jun N-Terminal Kinase) and p38 inhibitor SB203580. We chose A-419259, a potent pyrrolopyrimidine inhibitor, to block activities of Src family kinases 26 . To modulate Wnt signaling, XAV939 was used to stabilize AXIN1, the concentrationlimiting component of the β -catenin and Yap destruction complex 27,28 . XAV939 may also suppress Yap activities via angiomotin 25 , and has been shown to improve culturing pluripotent stem cells 29,30 . Finally, we included a GSK3 inhibitor, CHIR99021, and leukemia inhibitory factor (LIF). Although, the pro-pluripotency role of GSK3 inhibition (higher Wnt activities) appears to be partially redundant when PD0325901 and LIF are present [31][32][33][34] , CHIR99021 improves metabolic and biosynthetic processes and therefore cell culture robustness 35 . LIF may promote the rare totipotent cells in mouse ESC culture 36 . We hereafter named the medium that ...
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