Tissue-specific actions of the Ept1, Ept2, Ept6, and Ept9 genetic determinants of responsiveness to estrogens in the female rat

Abstract

Ept1, Ept2, Ept6, and Ept9 are quantitative trait loci mapped in crosses between the ACI and Copenhagen (COP) rat strains as genetic determinants of responsiveness of the pituitary gland to estrogens. We have developed four congenic rat strains, each of which carries, on the genetic background of the ACI rat strain, alleles from the COP rat strain that span one of these quantitative trait loci. Relative to the female ACI rats, female ACI.COP-Ept1 rats exhibited reduced responsiveness to 17beta-estradiol (E2) in the pituitary gland, as evidenced by quantification of pituitary mass and circulating prolactin, and in the mammary gland, as evidenced by reduced susceptibility to E2-induced mammary cancer. The ACI.COP-Ept2 rat strain exhibited reduced responsiveness to E2 in the pituitary gland but did not differ from the ACI strain in regard to susceptibility to E2-induced mammary cancer. Interestingly, female Ept2 congenic rats exhibited increased responsiveness to E2 in the thymus, as evidenced by enhanced thymic atrophy. The ACI.COP-Ept6 rat strain exhibited increased responsiveness to E2 in the pituitary gland, which was associated with a qualitative phenotype suggestive of enhanced pituitary vascularization. The ACI.COP-Ept9 rat strain exhibited reduced responsiveness to E2 in the anterior pituitary gland, relative to the ACI rat strain. Neither Ept6 nor Ept9 impacted responsiveness to E2 in the mammary gland or thymus. These data indicate that each of these Ept genetic determinants of estrogen action is unique in regard to the tissues in which it exerts its effects and/or the direction of its effect on estrogen responsiveness.

Figure 1

Ept congenic intervals. The vertical line in each panel represents the physical distance along the indicated rat chromosome. The location of each polymorphic microsatellite marker at which genotype was defined is indicated to the right of the line, and the genome coordinate of each marker (Mb) is indicated to the left of the line. The solid black rectangles represent the 95% CI for each Ept locus, as defined previously by composite interval mapping (12). The stippled gray rectangles represent the regions of each chromosome known to be homozygous for COP alleles. The white extensions to these rectangles represent regions of undefined genotype. All remaining segments of each chromosome are homozygous for ACI alleles. A, RNO3 and locations of Ept2 and Ept6. B, RNO6 and location of Ept1. C, RNO10 and location of Ept9.

Figure 2

Impact of Ept loci on pituitary mass after 12-wk E2 treatment. Female rats of each strain were treated with E2, released from sc SILASTIC brand implants, beginning at 9 wk of age. Control rats received empty implants. The rats were euthanized 12 wk later, and the pituitary glands were removed and weighed. Each bar represents the mean pituitary mass ± sd of the mean. The untreated control groups included eight to 12 rats. The E2-treated groups included 10–25 rats. Numeral 1 indicates a statistically significant (P < 0.05) difference relative to the corresponding untreated control group. Numeral 2 indicates a statistically significant difference relative to ACI rats receiving the same treatment. Numeral 3 indicates a statistically significant difference relative to COP rats receiving the same treatment.

Figure 3

Impact of Ept loci on pituitary mass after 28-wk E2 treatment. Female rats of each strain were treated with E2, released from sc SILASTIC brand implants, beginning at 9 wk of age. Control rats received empty implants. The rats were euthanized 28 wk later, and the pituitary glands were removed and weighed. Each bar represents the mean pituitary mass ± sd of the mean. The untreated control groups included three to five rats. The E2-treated groups included seven to 21 rats. Numeral 1 indicates a statistically significant (P < 0.05) difference relative to the corresponding untreated control group. Numeral 2 indicates a statistically significant difference relative to ACI rats receiving the same treatment. Numeral 3 indicates a statistically significant difference relative to COP rats receiving the same treatment.

Figure 4

Impact of Ept loci on pituitary gross morphology. Each pituitary gland was evaluated visually at necropsy. Photographs of pituitary glands from representative untreated and E2-treated rats illustrate the impact of E2 on gland enlargement, morphology, and coloration. The bar graph to the right of each photograph indicates the percentage of animals in each group exhibiting the dark-red discoloration indicative of increased vascularization and/or hemorrhage. The absence of a red bar is indicative of a 0% incidence of discolored pituitary glands in that experimental group. Grossly evident discoloration was observed in a fraction of Ept6 rats treated with E2 for 12 wk, as well as in a fraction of the ACI, Ept1, Ept6, and Ept9 rats treated with E2 for 28 wk. Discoloration of the pituitary gland was not observed in E2-treated COP or Ept2 rats at either time point.

Figure 5

Impact of Ept loci on circulating PRL after 12-wk E2 treatment. Female rats of each strain were treated as described in Fig. 2. Trunk blood was collected after decapitation, serum was isolated, and PRL was assayed by radioimmunochemistry. Each bar represents the mean PRL level ± sd of the mean. The untreated control groups included eight to 12 rats. The E2-treated groups included 10–25 rats. Numeral 1 indicates a statistically significant (P < 0.05) difference relative to the corresponding untreated control group. Numeral 2 indicates a statistically significant difference relative to ACI rats receiving the same treatment. Numeral 3 indicates a statistically significant difference relative to COP rats receiving the same treatment.

Figure 6

Impact of Ept loci on thymic atrophy after 12-wk E2 treatment. Female rats of each strain were treated with E2, released from sc SILASTIC brand implants, beginning at 9 wk of age. Control rats received empty implants. The rats were euthanized 12 wk later, and the thymus was removed and weighed. A, Each bar represents the mean thymus mass ± sd of the mean. The untreated control groups included four to 12 rats. The E2-treated groups included four to 25 rats. Numeral 1 indicates a statistically significant (P < 0.05) difference relative to the corresponding untreated control group. Numeral 2 indicates a statistically significant difference relative to ACI rats receiving the same treatment. Numeral 3 indicates a statistically significant difference relative to COP rats receiving the same treatment. B, Each bar indicates the decrease in average thymus mass in E2-treated rats relative to untreated control rats of the same strain.

Figure 7

Impact of Ept loci on E2-induced mammary cancer. Female rats of each strain were treated with E2, released from sc SILASTIC brand implants, beginning at 9 wk of age. Control rats received empty implants. The rats were euthanized 28 wk later or as necessitated due to treatment-related morbidity. Each data point represents the time at which an animal in the population at risk exhibited the first palpable mammary cancer. Mammary cancer did not develop in untreated, ovary intact, female rats during the course of this experiment. A, Data from the Ept1 and Ept2 congenic strains are illustrated relative to data from the ACI and COP strains. B, Data from the Ept6 and Ept9 congenic strains are illustrated relative to data from the ACI and COP strains.