Interacting quantitative trait loci control phenotypic variation in murine estradiol-regulated responses.

The steroid hormone estradiol (E2) elicits a spectrum of systemic and uterotropic responses in vivo. For example, E2 treatment of ovariectomized adult and sexually immature rodents leads to uterine leukocytic infiltration, cell proliferation, and organ growth. E2-regulated growth is also associated with a variety of normal and pathological phenotypes. Historically, the uterine growth response has been used as the key model to understand the molecular and biochemical mechanisms underlying E2-dependent growth. In this study, genome exclusion mapping identified two quantitative trait loci (QTL) in the mouse, Est2 and Est3 on chromosomes 5 and 11, respectively, that control the phenotypic variation in uterine wet weight. Both QTL are linked to a variety of E2-regulated genes, suggesting that they may represent loci within conserved gene complexes that play fundamental roles in mediating the effects of E2. Interaction and multiple trait analyses using the uterine leukocyte response and wet weight suggest that Est4, a QTL on chromosome 10, may encode an interacting factor that influences the quantitative variation in both responses. Our results show that E2-dependent responses can be genetically controlled and that a genetic basis may underlie the variation observed in many E2-dependent phenotypes.

Experimental allergic orchitis in mice. VII. Preliminary characterization of the aspermatogenic autoantigens responsible for eliciting actively and passively induced disease.

Experimental allergic orchitis (EAO) can be induced actively and passively in mice by either immunization with mouse testicular homogenate (MTH) in conjunction with the appropriate adjuvants or by transferring CD4+ T cells isolated from sensitized donors into non-immunized, naive recipients. The distribution of inflammatory lesions seen in active and passive EAO are markedly different. In active EAO maximal disease is observed in the seminiferous tubules, whereas in passive EAO lesions occur primarily in the straight tubules, rete testis, and ductus efferentes. These observations suggest that different immunopathogenic mechanisms and/or aspermatogenic autoantigens may be responsible for the distinct histopathologic profiles. Two murine testis-specific aspermatogenic autoantigens (mAP1 and mAP2) were partially purified from MT acetone powder by extraction in 7-M urea under reducing conditions, gel filtration, ion-exchange chromatography, and preparative isoelectric focusing from pH 3 to 10. In gel filtration on Sephacryl S-400 in 7-M urea, mAP1 is confined to the V0 peak, while mAP2 is in the major included peak. mAP1 has an isoelectric point of 4.4-4.9, is sensitive to both pronase and DNase but not RNase, and is active at a minimal dose of 250-500 micrograms (dry wt). Dose-response bioassays for active and passive EAO revealed that mAP1 preferentially elicits active disease, whereas mAP2 is most effective at eliciting passive disease. These results support the concept that the different histopathologic profiles seen in active and passive EAO are, in part, the result of different immunopathologic responses elicited by separate aspermatogenic autoantigens.

Apomorphine-induced vomiting in rainbow trout (Salmo gairdneri).

1. The LD50 for a 7-day period following intraperitoneal injection of apomorphine-HCl was calculated to be 158 mg/kg in rainbow trout. 2. Intraperitoneal injection of apomorphine at doses of 60 mg/kg or greater caused vomiting of plastic balls which had been placed in the stomachs of rainbow trout. 3. Apomorphine-induced effects included vomiting, vomiting behavior, toxicity, increased respiration, impaired motor control and equilibrium, and increased aggression. 4. The vomiting control mechanism of trout may be similar to that described in mammals.

Culturing Culex quinquefasciatus mosquitoes with a blood substitute diet for the females.

The tropical house mosquito Culex quinquefasciatus was cultured by feeding the females on an artificial diet, not on live animals or whole blood. This anautogenous strain has been maintained for more than fifty generations. The blood replacement diet for female mosquitoes, designed to simulate the tonicity and density of host blood, was based on ovalbumin, soya infant formula, globulins and adenosine triphosphate. Female adults of Cx quinquefasciatus were fed the artificial blood formula from "Parafilm' wax membrane-covered beakers. The diet was heated by radiant heat from a chamber containing an exothermic chemical reaction. This maintained the diet at a temperature of 33-37 degrees C for a period of up to 6 h, sufficient time to enable all the female mosquitoes to imbibe to satiation. After six generations on the artificial diet, female fecundity stabilized to a satisfactory level: the number of eggs per gonotrophic cycle averaged c. 85% of the "control' strain fed on whole blood from live anaesthetized guineapigs, i.e. 156 eggs per female from nine feeds on the artificial diet compared with 183 from six feeds of whole blood. Adult weight of Cx quinquefasciatus females was not significantly different, from generation 6 onwards, for strains fed on artificial diet or whole blood. Sex ratio and the rate of egg viability were also equivalent for the two strains.

An analysis of spontaneous impulse activity of units in the striate cortex of unrestrained cats.

1. Recordings were made from sixty-five cells in the visual cortex of unanaesthetized, dark-adapted cats and transferred automatically to computer input paper tape.2. The activity of each cell was measured as a function of time (the running mean). The unit of time used was inversely proportional to the mean firing rate, in order to give comparable results for different cells.3. For sixty-nine sections of discharge from fifty-two cells, the length of time recorded was sufficient to test for the long-term stability of the running mean. In twenty-six sections, various kinds of trend and long-term irregularity were found.4. The interspike interval histogram was computed for the forty-one sections (from thirty-one cells) in which there were more than 2000 discharges. Only eight histograms approximated closely to the exponential form. A test was also made of the distribution of the longer intervals alone and in twenty-five sections they did not deviate significantly from the exponential form.5. There was no significant correlation between the behaviour of the longer interspike intervals and the long-term stability or otherwise of the running mean.

Multiple loci govern the bone marrow-derived immunoregulatory mechanism controlling dominant resistance to autoimmune orchitis.

The existence of immunoregulatory genes conferring dominant resistance to autoimmunity is well documented. In an effort to better understand the nature and mechanisms of action of these genes, we utilized the murine model of autoimmune orchitis as a prototype. When the orchitis-resistant strain DBA/2J is crossed with the orchitis-susceptible strain BALB/cByJ, the F1 hybrid is completely resistant to the disease. By using reciprocal radiation bone marrow chimeras, the functional component mediating this resistance was mapped to the bone marrow-derived compartment. Resistance is not a function of either low-dose irradiation- or cyclophosphamide (20 mg/kg)-sensitive immunoregulatory cells, but can be adoptively transferred by primed splenocytes. Genome exclusion mapping identified three loci controlling the resistant phenotype. Orch3 maps to chromosome 11, whereas Orch4 and Orch5 map to the telomeric and centromeric regions of chromosome 1, respectively. All three genes are linked to a number of immunologically relevant candidate loci. Most significant, however, is the linkage of Orch3 to Idd4 and Orch5 to Idd5, two susceptibility genes which play a role in autoimmune insulin-dependent type 1 diabetes mellitus in the nonobese diabetic mouse.

Evidence for the genetic control of estradiol-regulated responses. Implications for variation in normal and pathological hormone-dependent phenotypes.

The ovarian steroid hormone estrogen (E2) elicits a multiplicity of both systemic and uterotropic responses in vivo. For example, the administration of E2 to ovariectomized (Ovx) and sexually immature rodents leads to uterine-specific inflammatory infiltrates. In this study, we quantitated the number of eosinophils and BM8+, Ia+, and CD4+ cells in uteri obtained from adult Ovx control and E2-treated C57BL/6J, C3H/HeJ, and (C57BL/6J x C3H/HeJ) (B6C3) F1 hybrid mice. All three strains exhibited a significant increase in the number of uterine eosinophils and BM8+ macrophages after E2 treatment. However, C57BL/6J and B6C3 F1 hybrid mice responded with a greater number of infiltrating eosinophils and macrophages as compared with C3H/HeJ. A similar analysis of Ia+ and CD4+ cells showed that E2 treatment either down-regulates or does not affect the number of such cells in all three strains. Genome exclusion mapping using a (C57BL/6J x C3H/HeJ) x C3H/HeJ backcross population localized Est1, the major locus controlling the number of eosinophils infiltrating the uterus after E2 treatment, to chromosome 4. In addition, suggestive linkage to marker loci on chromosomes 10 and 16 was detected and evidence for locus interaction is presented. Our results conclusively demonstrate that E2-regulated/ dependent responses can be genetically controlled, indicating that the phenotypic variation observed in both the normal and pathological effects of E2 may, in part, be due to a genetic component.

Aod1, the immunoregulatory locus controlling abrogation of tolerance in neonatal thymectomy-induced autoimmune ovarian dysgenesis, maps to mouse chromosome 16.

Mice thymectomized at three days of age (D3Tx) develop during adulthood a variety of organ-specific autoimmune diseases, including autoimmune ovarian dysgenesis (AOD). The phenotypic spectrum of AOD is characterized by the development of anti-ovarian autoantibodies, oophoritis, and atrophy. The D3Tx model of AOD is unique in that disease induction depends exclusively on perturbation of the normal developing immune system, is T-cell-mediated, and is strain specific. For example, D3Tx A/J mice are highly susceptible to AOD, whereas C57BL/6J mice are resistant. After D3Tx, self ovarian antigens, expressed at physiological levels, trigger an autoimmune response capable of eliciting disease. The D3Tx model provides, therefore, the opportunity to focus on the mechanisms of self-tolerance that are relevant to disease pathogenesis. Previous studies indicate that the principal mechanisms involved in AOD susceptibility are genetically controlled and govern developmental processes associated with the induction and maintenance of peripheral tolerance. We report here the mapping of the Aod1 locus to mouse chromosome 16 within a region encoding several loci of immunologic relevance, including scid, Igl1, VpreB, Igll, Igl1r, Mtv6 (Mls-3), Ly-7, Ifnar, and Ifgt.