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Differences in the Rate of in Situ Mammary Gland Development and Other Developmental Endpoints in Three Strains of Female Rat Commonly Used in Mammary Carcinogenesis Studies: Implications for Timing of Carcinogen Exposure

Stanko JP, Kissling GE, Chappell VA, Fenton SE.
Toxicol Pathol (2016), DOI: http:/dx.doi.org/10.1177/0192623316655222 PMID: 27613105


Publication


Abstract

The potential of chemicals to alter susceptibility to mammary tumor formation is often assessed using a carcinogen-induced study design in various rat strains. The rate of mammary gland (MG) development must be considered so that the timing of carcinogen administration is impactful. In this study, in situ MG development was assessed in females of the Harlan Sprague-Dawley (Hsd:SD), Charles River Sprague-Dawley (Crl:SD), and Charles River Long-Evans (Crl:LE) rat strains at postnatal days 25, 33, and 45. Development was evaluated by physical assessment of growth parameters, developmental scoring, and quantitative morphometric analysis. Although body weight (BW) was consistently lower and day of vaginal opening (VO) occurred latest in female Hsd:SD rats, they exhibited accelerated pre- and peripubertal MG development compared to other strains. Glands of Crl:SD and Crl:LE rats exhibited significantly more terminal end buds (TEBs) and TEB/mm than Hsd:SD rats around the time of VO. These data suggest a considerable difference in the rate of MG development across commonly used strains, which is independent of BW and timing of VO. In mammary tumor induction studies employing these strains, administration of the carcinogen should be timed appropriately, based on strain, to specifically target the peak of TEB occurrence.

Figures


Figure 1. Body weight data.

ap < .01 from Charles River Long-Evans (Crl:LE); bp < .0001 from Harlan Sprague-Dawley (Hsd:SD); cp < .01 from Hsd:SD by analysis of variance with Tukey multiple comparison post hoc test. n = 24, 24, 13 (Crl:LE); 22, 24, 14 (Charles River Sprague-Dawley); and 22, 22, 14 (Hsd:SD) by postnatal day, respectively.

Figure 2. Female vaginal opening (VO) timing.

(A) Least squares mean for day of VO and (B) true mean for body weight (BW) on the day of VO. There was a significant effect of BW on the day of VO. ap < .0001 from Charles River Long-Evans (Crl:LE) and Charles River Sprague-Dawley (Crl:SD); bp < .001 from Crl:LE; cp < .0001 from Harlan Sprague-Dawley (Hsd:SD) by analysis of variance with Tukey multiple comparison post hoc test. n = 28, 24, 21 (Crl:LE, Crl:SD, and Hsd:SD).

Figure 3. Mammary gland (MG) whole mount images at postnatal days 25 and 33.

Images are of MG4 and representative of the intrastrain developmental scores at each time point. Distance between MG4 and MG5 is denoted by arrows. Scale bars are 1 mm.

Figure 4. Mammary gland (MG) developmental scores.

(A) No differences in MG development were observed when glands were compared within strain. (B) When adjusted for across strain comparison, at postnatal days (PNDs) 25 and 33. MG scores for Harlan Sprague-Dawley (Hsd:SD) rats were significantly greater than MG scores for Charles River Long-Evans (Crl:LE) and Charles River Sprague-Dawley (Crl:SD) rats and MG scores of Crl:SD rats were significantly greater than MG scores for Crl:LE rats. ap < .0001 from Crl:LE; bp < .001 from Crl:SD; cp < .005 from Crl:SD; dp < .001 from Crl:LE; ep < .05 from Crl:LE by nonparametric one-way analysis of variance using the Kruskal–Wallis test on Wilcoxon rank sums. n = 24, 22, 12 (Crl:LE); 22, 23, 14 (Crl:SD); and 22, 22, 13 (Hsd:SD) by PND, respectively.

Figure 5. Relative estrogen receptor-α (ER-α) and progesterone receptor (PR) expression quickscore.

(A) Nuclear ER-α quickscore was significantly greater in mammary glands of Harlan Sprague-Dawley rats than that of Charles River Long-Evans (Crl:LE) and Charles River Sprague-Dawley (Crl:SD) rats at postnatal days (PNDs) 25 and 33. ap < .0001 from Crl:LE; bp < .0005 from Crl:SD; cp < .005 from Crl:LE; dp = .05 from Crl:SD by analysis of variance (ANOVA) with Tukey post hoc comparison. (B) Though the quickscore for nuclear PR increased across time points, the only significant difference observed was between Crl:LE and Crl:SD at PND33 (p = .05 by ANOVA with Tukey post hoc comparison). n = 10, for all strains and time points.

Tables


Table 1. Physical Parameters of MG Growth.

Table 2. Quantitative Morphometric Analysis.