Altering HIF-1α through 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure affects coronary vessel development.

Differential tissue hypoxia drives normal cardiogenic events including coronary vessel development. This requirement renders cardiogenic processes potentially susceptible to teratogens that activate a transcriptional pathway that intersects with the hypoxia-inducible factor (HIF-1) pathway. The potent toxin 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is known to cause cardiovascular defects by way of reduced myocardial hypoxia, inhibition of angiogenic stimuli, and alterations in responsiveness of endothelial cells to those stimuli. Our working hypothesis is that HIF-1 levels and thus HIF-1 signaling in the developing myocardium will be reduced by TCDD treatment in vivo during a critical stage and in particularly sensitive sites during heart morphogenesis. This inadequate HIF-1 signaling will subsequently result in outflow tract (OFT) and coronary vasculature defects. Our current data using the chicken embryo model showed a marked decrease in the intensity of immunostaining for HIF-1α nuclear expression in the OFT myocardium of TCDD-treated embryos. This area at the base of the OFT is particularly hypoxic during normal development; where endothelial cells initially form a concentrated anastomosing network known as the peritruncal ring; and where the left and right coronary arteries eventually connect to the aortic lumen. Consistent with this finding, anomalies of the proximal coronaries were detected after TCDD treatment and HIF-1α protein levels decreased in a TCDD dose-dependent manner

The effects of age on the chromosomal and physiological integrity of human sperm.

Integrated biomarkers of physiologic and genetic damage in sperm are needed to identify factors that affect male fertility and influence a father\u27s risk of producing abnormal progeny. Offspring studies show a preferential paternal origin for heritable structural chromosomal abnormalities, presenting the need for methods to efficiently detect structural chromosomal abnormalities in human sperm. This dissertation breaks new conceptual ground toward the understanding of paternally transmitted genetic disease by adapting FISH technology to detect human sperm that carry chromosomal breaks and partial chromosomal duplications and deletions and by integrating state-of-the-art biomarkers of semen quality including computer-assisted sperm analysis (CASA). These sperm biomarkers of genetic and physiologic damage were applied in a large-scale epidemiological study that investigated the effects of age in a population of about 100 healthy men, 22 to 80 years of age. The results revealed age-related increases in the frequency of sperm carrying de novo breaks and partial chromosomal duplications and deletions and a continuous decline in the kinetics of sperm motion. The paternal-age effect on motility has important implications for men who choose to delay fatherhood, since age may reduce their chance for success. The paternal-age effect on sperm chromosomal abnormalities is important because changes in the number of sperm carrying structural chromosomal abnormalities would be expected to have a corresponding effect on the number of affected newborns. Understanding the effects of host factors, such as age, on baseline frequencies of abnormal sperm will be extremely important for identifying potential confounders or effect modifiers in future epidemiological studies evaluating the effects of chemicals and genotoxic agents on male reproductive health. The CASA and FISH technologies applied in this research promise to provide an efficient approach for identifying paternal factors that affect male fertility and increase the risk of producing a chromosomally abnormal baby

Multicolor FISH Analysis of Chromosomal Breaks, Duplications, Deletions, and Numerical Abnormalities in the Sperm of Healthy Men

Transmitted de novo structural chromosomal abnormalities, the majority of which are paternally derived, can lead to abnormal reproductive outcomes as well as genetic diseases in offspring. We developed and validated a new multicolor FISH procedure (sperm ACM, which utilizes DNA probes specific for the alpha [1cen], classical, [1q12], and midi [1p36.3] satellites of chromosome 1) which utilizes DNA probes specific for three regions of chromosome 1 to detect human sperm that carry numerical abnormalities plus two categories of structural aberrations: (1) duplications and deletions of 1pter and 1cen, and (2) chromosomal breaks within the 1cen-1q12 region. In healthy men, the average frequencies of sperm with duplications and deletions were (a) 4.5 ± 0.5 and 4.1 ± 1.3 per 10(4) involving 1pter and (b) 0.9 ± 0.4 and 0.8 ± 0.3 per 10(4) involving 1cen, respectively. The frequency of sperm exhibiting breaks within the 1cen-1q12 region was 14.1 ± 1.2 per 10(4). Structural aberrations accounted for 71% of the abnormalities detected by sperm ACM, which was significantly higher than numerical abnormalities (P=2×10(-8)). Our findings also suggest that, for healthy men, (a) sperm carrying postmeiotic chromosomal breaks appear to be more prevalent than those carrying products of premeiotic or meiotic breakage or rearrangements, (b) the high frequency of chromosome breaks measured after “fertilization” by the hamster-egg cytogenetic method already appear to be present and detectable within human sperm by FISH, and (c) there are nonrandom and donor-specific distributions of breakpoint locations within 1q12 in sperm. FISH facilitates the analysis of much larger numbers of sperm than was possible when the hamster-egg method was used. Therefore, FISH-based procedures for simultaneously detecting chromosomal breaks, rearrangements, and numerical abnormalities in sperm may have widespread applications in human genetics, genetic toxicology, and reproductive medicine