Inter-laboratory assessment of a harmonized zebrafish developmental toxicology assay - progress report on phase I.

This report provides a progress update of a consortium effort to develop a harmonized zebrafish developmental toxicity assay. Twenty non-proprietary compounds (10 animal teratogens and 10 animal non-teratogens) were evaluated blinded in 4 laboratories. Zebrafish embryos from pond-derived and cultivated strain wild types were exposed to the test compounds for 5 days and subsequently evaluated for lethality and morphological changes. Each of the testing laboratories achieved similar overall concordance to the animal data (60-70%). Subsequent optimization procedures to improve the overall concordance focused on compound formulation and test concentration adjustments, chorion permeation and number of replicates. These optimized procedures were integrated into a revised protocol and all compounds were retested in one lab using embryos from pond-derived zebrafish and achieved 85% total concordance. To further assess assay performance, a study of additional compounds is currently in progress at two laboratories using embryos from pond-derived and cultivated-strain wild type zebrafish.

Estradiol up-regulates estrogen receptor and progesterone receptor gene expression in specific ovine uterine cells.

The regulation of estrogen receptor (ER) and progesterone receptor (PR) genes is critical to estrogen and progesterone responsiveness of the uterus during the estrous cycle. A low dose of estradiol, given to ovariectomized ewes to mimic the preovulatory estrogen surge, acutely enhanced ER and PR gene expression in most uterine cells. Estradiol effects were measured at 12, 24, and 48 h post-injection (n = 6 ewes per time) with immunohistochemistry and in situ hybridization. Whereas vehicle-treated ovariectomized ewes demonstrated low to moderate ER and PR mRNA and protein expression, estradiol enhanced PR mRNA and protein expression (at 12 h and 24 h, respectively) more rapidly than ER mRNA and protein expression (at 24 h and 48 h, respectively) in most uterine cells. However, the timing and extent of the estradiol response depended partly upon cell type (epithelial, stromal, or myometrial), cell region (luminal, superficial, middle, or deep endometrial or myometrial), adjacent cells, and prior progesterone treatment. For example, PR mRNA up-regulation was prolonged in middle and deep endometrial stroma, but increases in PR protein expression were highest in superficial and middle endometrial compartments, including the luminal epithelium. The luminal epithelium and myometrium were unique in that estradiol failed to up-regulate ER gene expression within them. ER mRNA levels rose within these compartments only when estradiol followed steroid hormone treatment designed to induce an artificial estrous cycle (estradiol-progesterone-estradiol [EPE] treatment). The EPE treatment also augmented the rise in ER mRNA concentrations within stromal cells compared to estradiol treatment alone. Within uterine cell compartments, subpopulations of adjacent cells showed distinct estradiol responses, e.g., very high levels of ER and PR gene expression within stromal cells directly underlying glandular epithelial cells. Because the estradiol response did not always correlate with initial ER protein levels and was partly dependent upon cell compartment and adjacent cells, we must conclude that direct transcriptional and/or posttranscriptional actions of estradiol cooperate with other cellular and paracrine regulatory factors to regulate ER and PR gene expression and, thus, the steroid responsiveness of uterine cells.

Media alternatives for the collection, culture and freezing of mouse and cattle embryos.

The replacement of biological products in media for the collection, culture and freezing of mammalian embryos was studied. To test the hypothesis that chemically defined surfactants can replace bovine serum albumin (BSA) or serum in embryo media, morula-stage mouse and cattle embryos were collected, cultured, and/or frozen in the surfactant compound, VF5. Collection efficiency of mouse and cattle embryos did not differ whether the medium contained serum or surfactant. In addition, morula-stage mouse and cattle embryos developed and hatched at similar rates in culture media containing either BSA or surfactant. Although the freeze/thaw survival and development in culture of bovine embryos was not significantly different in any of the media, there was a significantly lower hatching rate of mouse embryos frozen with serum or surfactant than with cryoprotectant alone or with cryoprotectant plus albumin-free serum. However, the absence of serum or surfactant in embryo freezing media resulted in embryo loss, presumably due to stickiness. The data suggest that serum can be replaced by a chemically defined surfactant in mouse and cattle embryo transfer systems for the collection, culturing and freezing of embryos. It is likely that the beneficial effects of serum are due to its surfactant properties.