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Techniques for using the triploid cell marker for studying cell lineage during the development and regeneration of the axolotl limb are described. Triploid animals possess cells with three nucleoli while diploid animals possess cells with two nucleoli. We have developed a technique for isolating the limb dermis as a sheet of cells for whole-mount analysis of cellular ploidy. Whole-mount tissue preparations as well as paraffin-embedded sectioned tissues were stained specifically for nucleoli with bismuth. Cell counts from a number of triploid and diploid dermal preparations show that (1) diploid dermal cells never possess three nucleoli, (2) the frequency of trinucleolate cells in whole-mount triploid dermal preparations is not 100% but varies between animals from 30 to 76%, (3) within a single triploid animal, the frequency of trinucleolate cells in different dermal preparations is constant. These data establish the usefulness of this technique and emphasize the need for appropriate control cell counts when using the triploid cell marker in the axolotl.

Competing Interests: Author JMD was employed by company Exponent Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Introduction: Fetal skeletal assessments are routinely conducted as a part of preclinical safety studies to support the development of novel therapeutic agents. Alizarin red staining with visual inspection of fetal skeletons is the gold standard in evaluating skeletons for the presence of developmental abnormalities. X-ray based micro-computed tomography (micro-CT) imaging has been used to evaluate small skeletal structures, both in vivo and ex vivo. Recent technological advances have reduced micro-CT image acquisition time making this technology practical for routine fetal skeletal evaluations. Herein we report on the use of micro-CT imaging as a method to perform high-throughput assessment of fetal skeletons., Methods: Micro-CT imaging of rat and rabbit fetal skeletons was conducted under a variety of conditions, including, in vivo, contrast-enhanced in vivo, and ex vivo. To increase throughput, micro-CT imaging was employed using custom designed polystyrene foam fetal holders to image entire litters of ex vivo fetuses. After micro-CT imaging, fetuses were routinely stained with alizarin red to compare micro-CT imaging results with traditional alizarin red staining., Results: Fetal skeletons could be visualized using in vivo micro-CT imaging; however, due to crowding, specific identification of individual fetuses was deemed not practical. Administration of a routine contrast agent to pregnant females highlighted maternal vascular structures including the placenta, but unfortunately, did not cross the placenta and did not highlight any fetal soft tissue structures. Ex vivo fetal imaging provided the best image quality of fetal skeletons and allowed for specific fetal identification. The fetal holders allowed for micro-CT imaging of approximately 400 rat fetuses or approximately 140 rabbit fetuses per hour. Micro-CT image skeletal findings and alizarin red findings were comparable. The very few discrepancies between the two methods involved the smallest skeletal elements with minimal ossification., Discussion: In conclusion, micro-CT ex vivo imaging can provide a reliable high-throughput method to assess fetal skeletal abnormalities for developmental toxicity studies.

To assess whether ovarian histopathological examination in repeated-dose rodent toxicity study could reliably anticipate toxic effects on female reproductive function and to assess whether ovarian change could be detected in a 2-week repeated-dose toxicity study, tamoxifen was administrated orally to female rats at 0.005, 0.03, or 0.2 mg/kg/day for 2 and 4 weeks in the repeated-dose toxicity studies, and for 2 weeks prior to cohabitation, during cohabitation, and through Gestation Day 7 in a female fertility study. The relationship between ovarian histopathological findings and fertility results was investigated. Findings at 0.03 and 0.2 mg/kg/day included decreases in body weight gains associated with decreases in food consumption, in 2- and 4-week repeated-dose toxicity studies and fertility study. The ovarian histopathological findings included increases in large atretic follicles, increases in interstitium cells and absence of newly-formed corpus lutea at 0.2 mg/kg/day in the 2-week study and at 0.03 and 0.2 mg/kg/day in the 4-week study. The treatment induced estrogenic and antiestrogenic reactions in the uterus, while mucinous degeneration was detected in the vagina. Effects on female fertility consisted primarily of disturbance of estrus cycle and decreases in numbers of pregnant rats which were considered to be related to ovarian histopathological changes. Based on these findings, ovarian histopathological evaluation in the repeated-dose toxicity study could anticipate the effects of tamoxifen on female fertility via ovarian dysfunction at slightly toxic doses, and 2-week treatment of tamoxifen at appropriate dose could be sufficient to detect ovarian toxicity by microscopic examination.

The potential reproductive toxicity of the fungicide and anthelmintic thiabendazole (TBZ) was assessed in Sprague-Dawley rats for two generations. Doses of 10, 30 or 90 mg/kg/day were administered by way of the diet beginning at 8 wk of age for the F0 generation and postnatal wk 4 for the F1 generation and continuing until the animals were killed. Concentrations of TBZ in the diet were adjusted weekly, except during the gestation and lactation intervals when concentrations were held constant. There were no TBZ-related deaths or adverse physical signs during the study. TBZ-related effects consisted of decreases in average body weight gains and food consumption in the middle and high dose groups. In both the F0 and F1 generations during the premating and post-cohabitation periods, the effects in the middle-dose group were observed only in males and were generally slight in magnitude (food consumption 3-5% below control, weight gain 7-18% below control), whereas the effects in the high dose group occurred in both sexes and were slight to moderate in magnitude (food consumption 9-13% below control, weight gain 13-46% below control). During gestation of the F0 females there were slight decreases in average weight gain and food consumption (8% and 4-16% below control, respectively); a similar effect on food consumption, but not weight gain, occurred in the F1 generation. There were no effects on F0 or F1 reproductive performance (including indices of mating, fecundity, fertility, length of gestation, litter size, birth weight, and post-implantation survival), nor were any histomorphological changes observed in the reproductive tissues of animals in the high dose group. There was no evidence of developmental toxicity in the TBZ-exposed F1 or F2 generations, except for slight decreases in average pup weights between postnatal days 4 and 21 in the high dose group (5-10% below control). The NOAEL (no-observed-adverse-effect level) for all developmental, growth, survival and reproductive performance parameters assessed in this study was 10 mg/kg/day.

The thymidine analogue, 5-bromo-2'-deoxyuridine (BUdR), given at the proper dose and time to pregnant rats produces preaxial hindlimb polydactyly in a high proportion of near term foetuses. The lack of physiological cell death in an area of preaxial mesenchyme known as the foyer primaire preaxial (fpp) is thought to be important in the pathogenesis of this defect. This study addresses the question of whether BUdR's well-known antidifferentiative effects, which are due in some way to drug incorporation into DNA, are operative in this in vivo system. The dose and temporal response of BUdR for the induction of preaxial polydactyly inversely parallels the frequency of embryonic hindlimbs with an fpp. Incorporation of BUdR into degenerative fragments within the fpp of these treated limbs is demonstrated with indirect immunofluorescence using an antibody to bromouridine. Hindlimbs exposed to a threshold dose of BUdR at the optimal time for producing polydactyly have incorporated the drug into degenerative fragments within the fpp. This suggests that a higher, teratogenic dose of BUdR might likewise be incorporated. The resulting higher level of incorporation presumably alters the normal course of terminal differentiation for these cells originally destined to die. Teratogenic doses of BUdR injected at later than the optimal time are also incorporated into dead cell fragments within the fpp, suggesting that presumptive dead cells have additional rounds of DNA synthesis which are BUdR-insensitive. Approximately 12 h prior to overt death presumptive fpp cells no longer incorporate the drug. Results reported support the hypothesis that incorporation of teratogenic levels of BUdR prevent cell death in the fpp. The extra cells are thought to contribute directly or indirectly to the added digit. Contrary to other views, it is suggested that BUdR-induced teratogenesis can be a result of the drug's antidifferentiative effects on specific, 'sensitive', populations of cells.

The amino acid sequence of blastokinin, also called uteroglobin, has been determined by a combined study of both the intact native molecule and the peptide fragments resulting from tryptic and chymotryptic digestions. Sequence analyses performed by automated methods and by sequential digestion with leucine aminopeptidase and carboxypeptidase Y demonstrate that blastokinin is a dimer of identical 69-amino acid subunits held together in parallel orientation by two disulfide bridges at positions 3 and 68. The polypeptide chains are further characterized by the absence of tryptophan residues and by single residues of histidine and tyrosine at positions 8 and 21, respectively. Six of eight amino acids, positions 17--24, near the progesterone binding site of blastokinin contain a hydroxyl group. Knowledge of the chemistry of this receptor site should allow better perspectives of the chemistry of molecules in normal tissues that are dependent on progesterone for growth and development, as well as compounds that could act as cancer antagonists for endocrine therapy of hormone-dependent tumors.