Novel tenascin variants with a distinctive pattern of expression in the avian embryo.

Previous studies have shown that several forms of the glycoprotein tenascin are present in the embryonic extracellular matrix. These forms are the result of alternative splicing, which generates tenascin variants with different numbers of fibronectin type III repeats. We have used degenerate primers and PCR to isolate a novel tenascin exon from an avian genomic library. Genomic clones contained a sequence encoding a fibronectin type III repeat that corresponds to repeat 'C' from the variable domain of human tenascin. To demonstrate that tenascin containing repeat 'C' is actually synthesized by avian cells, a monospecific antiserum was raised against a repeat 'C' fusion protein. This antiserum recognized a novel high-molecular-weight variant on immunoblots of tenascin isolated from chicken embryo fibroblast-conditioned medium, and stained tendons on frozen sections of chicken embryos. A cDNA probe specific for mRNA encoding repeat 'C' was used for in situ hybridization. This probe hybridized in a subset of the embryonic tissues labelled with a universal tenascin probe, including tendons, ligaments and mesenchyme at sites of epithelial-mesenchymal interactions. Finally, we provide evidence that additional fibronectin type III repeats, one corresponding to a recently discovered human repeat as well as one entirely novel sequence, also exists in chicken tenascin mRNA. These data indicate that tenascin is present in the embryonic matrix in a multitude of forms and that these forms have distinctive distributions that may reflect more than one function for tenascin in development.

Cellular origins of tenascin in the developing nervous system.

We have used in situ hybridization and reverse transcriptase polymerase chain reaction (PCR) to study the origins of the extracellular matrix glycoprotein tenascin during the development of the central and peripheral nervous systems. Previous studies have shown that neural crest cells migrate along pathways that are lined with tenascin. In situ hybridization, PCR, and western blotting reveal that these cells themselves are a major source of tenascin both in vitro and in the embryo. Thus, tenascin is probably not acting as a guidance molecule but is more likely to be promoting neural crest cell motility in a more general way. Similarly, subpopulations of proliferating and migrating glia make tenascin in the developing central nervous system, as do the radial glia that are used as a substratum for migrating neuronal cell bodies. In the adult, tenascin continues to be expressed in the cerebellum by Golgi epithelial cells. This expression, as well as the expression of tenascin in connective tissue, indicates that this molecule may also be playing a role in regulating differentiation. Finally, the distribution of tenascin transcripts in the developing brain and spinal cord is similar to the distribution of mRNAs encoding receptors for platelet-derived growth factor-AA and basic fibroblast growth factor. In vitro studies indicate that both of these factors are potential regulators of tenascin expression.

Optimization of the Hamilton-Thorn computerized sperm motility analysis system for use with rat spermatozoa in toxicological studies.

To optimize the Hamilton-Thorn Motility Analyzer (HTM; Hamilton-Thorn Research, Beverly, MA) for use in reproductive toxicology studies with rat spermatozoa, the accuracy and precision of the instrument were assessed under a variety of instrument settings. Videotapes of both fast- and slow-swimming sperm were analyzed repeatedly to obtain data across a range of sperm velocities as might be encountered as a consequence of exposure to reproductive toxicants. Acquisition rates were varied across the HTM menu choices (30, 19, 10, or 7 frames/sec) as were the number of frames analyzed (5 to 20) at each framing rate. For fast-swimming samples (mean straight-line velocity (VSL) approximately 130 microns/sec) generally good agreement between computer-assisted sperm analysis (CASA) and manually obtained data was found for percentage of motile sperm and straight-line velocity; i.e., CASA values were within 10% of manual values for most frame/rate combinations. The accuracy of these measures held true over a wide range of sperm concentrations and percentage motilities. However, CASA measures were less accurate for sperm samples of lower velocities (mean VSL approximately 50 microns/sec and mean VSL approximately 30 microns/sec) in that the velocity of very slow sperm was overestimated (particularly at 30 frames/sec). A soft-ware change (6.5R) and performing analyses at 19 instead of 30 frames/sec improved straight-line accuracy for the slow sperm and enhanced the discrimination between fast (presumably control) and slow (presumably treated) sperm samples. These data show that this motility analyzer could be successfully configured to evaluate rodent sperm samples. The use of such CASA systems in toxicology studies will provide valuable information that may improve human reproductive risk assessment.