Male reproductive effects induced by in utero and lactational exposure to TCDD were analyzed in three rat lines that are differently sensitive to TCDD. Rats from lines A, B, and C were selectively bred from TCDD-resistant Han/Wistar (Kuopio, H/W) and TCDD-sensitive Long-Evans (Turku/AB, L-E) rats and exhibited very different LD50 values for TCDD: >10,000, 830, and 40 microg/kg in males, respectively. The resistance in line A rats was linked to a mutated H/W-type aryl hydrocarbon receptor (Ahr(hw)) and in line B rats to a H/W-type unknown allele B (B(hw)). Line C rats had no resistance alleles. Influence of the resistance alleles on developmentally induced male reproductive effects of TCDD was studied by exposing pregnant females to TCDD (0.03, 0.1, 0.3, or 1 microg/kg) on gestation day (GD) 15. Male progeny were sacrificed on postnatal day (PND) 70. Next, the dams were given 1 microg/kg TCDD on GD 15 and male progeny were sacrificed on PND 14, 21, 28, 35, or 49. Serum testosterone concentration, male sex organ weights, and testicular and cauda epididymal sperm numbers were analyzed; the most sensitive end point was decreased sperm numbers. The dose of 1 microg/kg TCDD reduced daily sperm production by 9.3, 25, and 36%, and cauda epididymal sperm reserves by 18, 42, and 49% in rat lines A, B, and C when measured on PND 70, respectively. The most consistent and significant effect was decreased weight of prostate lobes. The growth of the male reproductive organs was not markedly affected by the resistance alleles Ahr(hw) and B(hw). In contrast, the effects on sperm parameters appeared to be slightly modified by the resistance alleles. Thus, the intraspecies genetic differences in C-terminal transactivation domain of AHR appear to modify the sensitivity to only certain dioxin-induced male reproductive effects.

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We describe the site-specific enzymatic biotinylation of recombinant anti-estradiol Fab fragments through a 13 amino acid acceptor peptide translationally fused to the C-terminus of the Fd chain. The Fab-peptide fusion proteins were secreted to the periplasm of Escherichia coli, purified, and biotinylated in vitro using biotin ligase, biotin, and ATP. The E. coli biotin ligase (the BirA protein) was produced as a novel N-terminal fusion protein with glutathione S-transferase (GST) and purified in one step from bacterial cell lysate using a Glutathione Sepharose affinity column. The purified fusion protein worked as such (without cleavage of the GST part) for the in vitro biotinylation of the Fab fragments. After the removal of nonbiotinylated Fab fragments by monomeric avidin chromatography, the overall yield of biotinylated Fab was 40%. The site-specifically biotinylated Fab fragments (BioFab) were tested in streptavidin-coated microtitration wells, to which they were shown to bind linearly with respect to the amount of BioFab added, specifically as indicated by biotin inhibition, and tightly with a half-life of several days. Moreover, the enzymatic BioFab exhibited uniform antigen binding affinity unlike the same recombinant Fab fragments biotinylated through random chemical conjugation to surface lysines. Finally, the BioFab demonstrated its potential as a well-behaving immunoassay reagent in a model competitive assay for estradiol.