A congenic strain of rat for investigation of control of estrogen-induced growth.

The rat pituitary has been used for several decades as a model system to investigate the role of estrogen in tumor growth, cell proliferation, and angiogenesis. A major reason for using the rat pituitary is the great difference between laboratory rat strains in the effect of chronic estrogen treatment on pituitary tumor growth (Wiklund and Gorski 1982; Wendell et al. 1996; Spady et al. 1999). For example, when rats of the inbred strain Fischer 344 (F344) are given a chronic estrogen treatment, the pituitary grows continuously to become over 10 times normal mass in 8 weeks (Wiklund and Gorski 1982). Whereas the F344 strain is deficient in growth control, the Brown Norway (BN) rat strain is proficient in the control of estrogen-stimulated pituitary growth and does not form a pituitary tumor, nor even any significant change in pituitary mass, upon chronic estrogen treatment (Wendell et al. 1996). This estrogen-induced pituitary tumor (so-called due to its excessive growth) exhibits uncontrolled cell proliferation and consists primarily of lactotrophs (Phelps and Hymer 1983). When rats are administered estrogen for several days, cells in the pituitary of tumor-resistant Holtzman rats are first stimulated to proliferate, but then become refractory to the hormone (Wiklund and Gorski 1982). This refractory response is not due to insensitivity to hormone, because the pituitary continues to synthesize and release prolactin (Wiklund et al. 1981). Thus, there must be an active negative feedback mechanism specific to growth. Uncovering the basis of the difference between tumor-susceptible and tumor-resistant strains can shed light on the means by which normal cells subsequently restrain growth. The difference in growth control between F344 and BN is polygenic and owing to at least six quantitative trait loci (QTL) that were mapped in two-generation genetic crosses between BN and F344 (Wendell and Gorski 1997). Edpm3 (estrogendependent pituitary mass QTL on rat Chr3) was the QTL with largest effect (16.6% of total phenotypic variance) identified in an F2 intercross between F344 and BN (Wendell and Gorski 1997) and has been selected for further study. The BN allele of Edpm3 correlates with control of pituitary growth in estrogentreated rats, and inheritance of the F344 allele of Edpm3 correlates with uncontrolled estrogen-dependent growth (Wendell and Gorski 1997). To investigate the contribution of Edpm3 to the control of estrogen-dependent growth, we constructed a congenic strain in which the segment of rat Chr3 containing the Edpm3 QTL from BN was introgressed into the F344 strain. Thus, this strain is homozygous for the BN allele of Edpm3, but homozygous for the F344 alleles of all other QTL. The congenic strain was bred by what is now the standard ‘‘speed congenic’’ strategy (Markel et al. 1997) (Fig. 1). At each generation, animals were selected that bore the BN allele of microsatellites from the markers across the 2-LOD support interval of Edpm3. The resulting congenic strain, named F344/NHsdEdpm3 in accordance with the guidelines of the Committee on Rat Nomenclature (1992), is homozygous for BN alleles over the majority of rat Chr3. The animals were homozygous for F344 alleles of all markers tested on other chromosomes (at least two per chromosome). The F344/NHsd-Edpm3 strain does develop a pituitary tumor after chronic estrogen treatment. However, the estrogen-dependent pituitary tumors formed by F344/NHsdEdpm3 rats are on average smaller than those developed by the F344 strain (Fig. 2). This is as expected because Edpm3 is one member of a group of additively acting QTL for estrogen-dependent pituitary growth. The difference in mean estrogen-induced pituitary tumor mass between the F344/ NHsd-Edpm3 and F344 strains is equivalent to the massreducing effect of two BN alleles of Edpm3 that was previously estimated in an F2 intercross of F344 and BN (Wendell and Gorski 1997). Phelps and Hymer reported that estrogen treatment increases the rate of cell proliferation in the F344 pituitary and that the fraction of cells in S-phase increases with duration of treatment (Phelps and Hymer 1983). Our S-phase fraction data for the F344 follow the same pattern as reported by Phelps and Hymer (Fig. 3), though the values are not as high. This may reflect a difference in the age of animals used. Although the duration of estrogen treatment is similar, Phelps and Hymer administered chronic estrogen treatment to ovariectomized adult females, whereas we began the estrogen treatment with 21day-old (prepubertal, weanling) females (Wendell et al. 1996). Analysis of the fraction of cells in S-phase in the pituitary of estrogen-treated F344/NHsd-Edpm3 congenic strain rats indicates that the BN allele of Edpm3 has a significant suppressive effect on estrogen-induced cell proliferation. Although there is no difference in S-phase fraction between untreated F344 and untreated F344/NsRd-Edpm3 congenic rats, estrogen-treated F344/NHsd-Edpm3 rats exhibit a significantly lower S-phase fraction than estrogen-treated F344, and this difference increases with duration of estrogen treatment (Fig. 3). Thus, comparison of F344 and the F344/NHsdEdpm3 congenic strain makes it clear that the presence of BN alleles of Edpm3 reduces entry of estrogen-stimulated pituitary cells into S-phase. Estrogen is known to stimulate cells to progress from G1 to S phase (Foster et al. 2001). The effect of the BN allele of Edpm3 could occur by either restraint of the G1-S phase transition, or by triggering apoptosis before entry into S-phase. F344 and the F344/NRsd-Edpm3 congenic strain as a pair make an experimental system to investigate the process of Mammalian Genome 13, 664–666 (2002). DOI: 10.1007/s00335-002-2183-6