Transcription factors, POU5F1/OCT4 and NANOG, whose expression is restricted to the inner cell mass (ICM) in mouse and human blastocysts, are used to characterize undifferentiated embryonic stem cells (ESC) in vitro. However, POU5F1 may not be a useful marker in domestic animals due to its expression in both ICM and trophectoderm (TE), while NANOG mRNA and protein expression have only been described fully in mice. In an effort to identify ESC markers for domestic animals, expression patterns of NANOG, POU5F1, and the cell surface markers (SSEA1, SSEA4, TRA-1-60, TRA-1-81) were examined in preimplantation goat embryos, a species that has proven to be a superior choice for the production of transgenic proteins in milk (biopharming). Our results indicate that while goat embryos express POU5F1, SSEA1, and SSEA4 proteins, their expression is not strictly restricted to the ICM. In a unique staining pattern, NANOG protein was localized to the nucleoplasm and nucleoli in ICM cells, but was localized strictly to nucleoli in TE. This pattern may reflect down-regulation of protein by sequestration/degradation utilizing a nucleolar mechanism known to operate in stem cells. Furthermore, NANOG mRNA in TE was also significantly down-regulated as compared with that in ICM. Taken together, this novel expression pattern of NANOG in goat preimplantation embryos suggests that NANOG could serve as marker of pluripotency in goats and may be useful in derivation and characterization of caprine ESC. This study is the first to characterize both NANOG mRNA and protein expression in any species other than the mouse.

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Embryonic stem (ES) cells can differentiate into all three embryonic germ layers but rarely into trophectoderm (TE) lineages that contribute to the placenta, although TE differentiation can be initiated by genetic manipulation of key genes involved in TE development. We demonstrate that Wnt signaling can initiate TE lineage differentiation by triggering an appropriate cue, caudal-related homeobox 2 (Cdx2). Overexpression and RNA interference knockdown studies indicate that Cdx2 induction in response to Wnt3a is mediated by lymphoid enhancer factor 1, whose expression is regulated by leukemia inhibitory factor (LIF) and bone morphogenetic protein. Removal of LIF, along with addition of Wnt3a, stimulated Cdx2 expression and induced formation of trophoblast stem (TS) cells. These TS cells were able to differentiate into cells with characteristics of spongiotrophoblast and trophoblast giant cells. This is, to our knowledge, the first evidence that TE lineage differentiation can be induced by Wnt signaling in mouse ES cells. STEM CELLS 2008;26:842-849 Disclosure of potential conflicts of interest is found at the end of this article.

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Market-sized 2-year-old striped bass Morone saxatilis (average weight, 645 g) were evaluated in three different concentrations of Aqui-S at one of three different temperatures. Time to stage 4 of anesthesia, recovery time, and plasma cortisol level as an indicator of acute stress response, were determined. We found a significant difference in the time to stage 4 between 25 mg/L and 35 or 45 mg/L. This difference was present at all three temperatures tested. There were also significant differences in recovery time and blood cortisol level, but these differences were not consistent across all temperatures. There was a significant interaction between temperature and dose when analyzing cortisol levels. For the range of concentrations evaluated, we recommend an Aqui-S dose of 35 mg/L when anesthetizing market-sized striped bass.

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Four experiments were designed to evaluate the effects of osmolality, cryoprotectant, and equilibration time on striped bass sperm motility. In the first experiment, solutions of NaCI or KCI with osmolalities ranging from 0 to 700 mmol/kg were tested on sperm activation. Over 60% of the sperm were activated by isotonic NaCI and KCI solutions with a treatment osmolality of 350 mmol/kg. Sperm remained motile until osmolality increased to 600 mmol/ kg. In the second and third experiments, Extenders 1, 2 and 3 with osmolalities of 350, 500, and 600 mmol/kg, respectively, were tested. Sperm samples stored in Extender 2 showed significantly higher (P 0.01) sperm motility after 10 min of exposure as well as greater (P < 0.01) post‐thaw motility when compared to samples stored in Extenders 1 and 3. In the fourth experiment, two trials were carried out to evaluate the effects of cryoprotectant and equilibration time. In the first trial, methanol with a concentration of 5% and 10% yielded the highest (P < 0.05) sperm motility prior to freezing at all equilibration times examined. However, 5% DMSO yielded the highest (P < 0.01) post‐thaw motility (38 ± 3.6%). DMSO with concentrations of 10% and 15% resulted in 17 ± 2.3% and 6 ± 1.0% post‐thaw motility, respectively. Both methanol and DMA, at all concentrations tested, resulted in less than 10% post‐thaw motility. In the second trial, four DMSO concentrations with three different equilibration times were examined. We observed a significant (P < 0.001) interaction effect between DMSO concentration and equilibration time. Post‐thaw motility was significantly greater (P < 0.01) with a concentration of 5% DMSO at all equilibration times examined, compared to 1.25, 2.5, and 10% DMSO. An average post‐thaw motility of 40 ± 2.9% was achieved after 10 min equilibration using 5% DMSO.