Mechanism of action of a repressor of dioxin-dependent induction of Cyp1a1 gene transcription.

A dominant mutant of Hepa-1 cells, c31, expresses a repressor that prevents 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-dependent stimulation of Cyp1a1 transcription. The repressor acts via the xenobiotic-responsive elements (XREs), which are the DNA-binding sites for the aryl hydrocarbon (Ah) receptor-TCDD complex during transcriptional activation of the gene. High-salt nuclear extracts prepared from c31 cells grown with TCDD contained normal levels of the Ah receptor which bound the XRE with normal affinity, as judged by in vitro gel mobility shift assays. Furthermore, extracts prepared from these cells, grown either with or without TCDD, contained no novel XRE-binding proteins compared with extracts from wild-type Hepa-1 cells. However, in vivo genomic footprinting demonstrated that TCDD treatment leads to binding of the Ah receptor to the XREs in Hepa-1 but not mutant cells. This finding suggests that the repressor associates with the Ah receptor to prevent its binding to the XREs and that high-salt treatment either causes dissociation of the receptor/repressor complex or fails to extract the repressor from nuclei. The results underscore the importance of using both in vivo and in vitro assays for analyzing DNA-protein interactions.

Genetic and molecular analysis of the Ah receptor and of Cyp1a1 gene expression.

The Ah receptor is a soluble protein complex that mediates carcinogenesis by a wide range of environmental pollutants, including polycyclic aromatic hydrocarbons, heterocyclic amines, and polychlorinated aromatic compounds. The best understood activity of the receptor concerns its role in the induction of cytochrome P450IA1. We undertook a somatic cell genetic analysis of P450IA1 induction using the mouse hepatoma cell line, Hepa-1. Clones of Hepa-1 were isolated that are defective in induction of P450IA1. Evidence was obtained that the clones are mutational in origin. Cell fusion experiments demonstrated that a few of the mutants are dominant, while the majority are recessive. The dominant mutants were shown to synthesize a repressor of P450IA1 transcription. The recessive mutants were assigned to 4 complementation groups (probably corresponding to 4 different genes). Complementation group A corresponds to the P450IA1 structural gene. Mutations in the B, C and D genes all affect functioning of the Ah receptor. A 'reverse selection procedure', whereby cells that express P450IA1 inducibility can be selected from a majority population of cells lacking inducibility, was developed. The reverse selection procedure was used to isolate transfectants of representative recessive mutants in which the mutational defects are complemented by exogenously applied genomic DNA. A human DNA-derived transfectant of a C- mutant was used to clone the human C gene. The C gene is not the ligand-binding subunit of the Ah receptor but is a protein that is required for translocation of Ah receptor-ligand complexes from cytoplasm to nucleus. In analogous experiments the dominant gene from one of the dominant mutants was transfected into wild-type Hepa-1 cells. Success in transfecting the dominant gene should provide the means for cloning it.

Influence of genetic increases in egg production on traits associated with broodiness in turkeys.

Egg production, nesting frequency, and serum levels of prolactin, estradiol, and total phosphorus were monitored in relatively nonbroody (egg) and relatively broody (RBC1) strains of turkey hens during a reproductive period. In the egg strain, prolactin levels were increased in a group with a relatively high frequency of nesting in comparison to a group with a relatively low frequency of nesting. No differences between these two groups were detected for serum estradiol, total phosphorus, or egg production. In the RBC1 strain, prolactin levels did not differ between a group of hens that did not exhibit broodiness and a group that exhibited one or more bouts of broodiness. A broodiness treatment was used for the latter group. The broody group exhibited extremely variable levels of prolactin. In individual broody hens, the levels of prolactin were relatively high. After broodiness in 12 of 13 hens, the level of prolactin fell to relatively low levels. In the one hen not responding to broodiness treatment, the level of prolactin became low levels. In the one hen not responding to broodiness treatment, the level of prolactin became extremely high. Nesting frequency, total serum phosphorus, and egg production were generally not different between the two groups. The level of prolactin showed seasonal changes in both strains of hens, starting low, increasing to maximal levels between 40 and 80 days of production, and then declining to low levels late in the reproductive period. Laying hens always had higher levels of prolactin than nonlaying, nonbroody hens.

Changes in plasma calcium, phosphorus, lipids, and estrogens in turkey hens with reproductive state.

Plasma calcium increased during the two weeks prior to first egg. The increase was in the protein bound (CaB) but not the ultrafilterable (CaU) fraction. Plasma neutral lipids (NL) and low density lipoprotein fraction (LDF, d < 1.006) increased similarly to CaB. Free fatty acids (FFA) had a transitory increase at first egg but then returned to levels not different from those when the hens were immature or in a reproductive pause. Plasma total phosphorus (Pt) increased similarly to CaB. This increase was associated with the protein (Pp) and lipid (Pl) phosphorus fractions. No change was noted for inorganic (Pi) phosphorus. Total plasma estrogen (Et), as estimated with radioimmunoassay utilizing an estriol antiserum, increaed similarly to CaB. Most of the increase was in an unidentified estrogen (Eu) fraction, but a sustained increase was also noted in the estrone (El) fraction. Estradiol (E2) levels did not differ with reproductive period. Simple correlations were calculated between all factors within the stimulatory and laying periods. In general, all correlations except those with diffusible calcium (CaD), FFA, and E2 were positive, relatively high (> .6) and significant (P < .01). The correlations tended to be greater in the stimulatory period than in the laying period. Partial correlations of the three estrogens with the other factors were calculated for the stimulatory and laying periods. The partial correlations of Eu were significant for all factors except CaD during the stimulatory period, but only with FFA, Pp and Pi in the laying period. The partial correlation of El with LDF was significant in the stimulatory period, and for LDF, NL, and Pi in the laying period. Partial correlations of E2 within the stimulatory period were not significant, but in the laying period a significant partial correlation with NL was noted.

Persistence of semen quality during the reproductive period in two strains of turkeys differing in semen yield.

Semen yield, sperm concentration and frequency of bent sperm were measured during the ninth week following stimulatory lighting (in January) and correlated with similar measurements obtained 1-1/2 and 4-1/2 months later in the reproduction period for two strains of medium bodied turkeys differing in semen yield. These correlations, based on measurements made early in the reproductive period, were not large enough so that the male's subsequent performance could accurately be predicted. Strain differences were also apparent in correlation coefficients for semen yield. The correlation coefficients were larger for the strain having the smallest yeild. In general, the significant correlation coefficients between semen yield and sperm concentration were positive and those between semen yield and percent bent sperm and between sperm concentration and percent bent sperm were negative. However, the correlation coefficients tended to be low in magnitude.

The association of semen traits and fertility in the turkey.

There were no significant correlation coefficients between semen traits (yield, concentration and frequency of bent sperm) measured prior to the first insemination and fertility over a 12-week hatching period when the amount of semen inseminated per female was greater than the minimum (.025 cc.) usually recommended. Although there were some statistically significant correlations between semen measurements made later in the reproduction period and fertility for the entire period, they were very small ranging from -- .29 to +.20.

Activity of eight enzymes of chicken seminal plasma in the eluent from agarose gel chromatography.

Chromatography of seminal plasma from fresh, untreated chicken semen on Bio-Rad A1.5m agarose gel yielded five major peaks of ultraviolet absorbancy at 280 nm. Two peaks with 280/260 absorbancy ratios less than unity suggested the presence of free nucleotides. Enzyme assays on the eluent fractions resulted in substantial single peaks of lactic dehydrogenase, glutamic oxaloacetic transaminase, phosphohexose isomerase, acid phosphatase and alkaline phosphatase activity. Acetylcholinesterase and aminopeptidase assays produced multiple peaks of activity. No trypsin-like enzyme activity was detected, suggesting the presence of a seminal plasma trypsin-like enzyme inhibitor. Molecular weight estimates were obtained for all enzyme activity peaks.