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Mammary Gland Evaluation in Juvenile Toxicity Studies: Temporal Developmental Patterns in the Male and Female Harlan Sprague-Dawley Rat

Filgo AJ, Foley JF, Puvanesarajah S, Borde AR, Midkiff BR, Reed CE, Chappell VA, Alexander LB, Borde PR, Troester MA, Hayes Bouknight SA, Fenton SE.
Toxicol Pathol (2016), DOI: https://doi.org/10.1177/0192623316663864 PMID: 27613106


Publication


Abstract

There are currently no reports describing mammary gland development in the Harlan Sprague-Dawley (HSD) rat, the current strain of choice for National Toxicology Program (NTP) testing. Our goals were to empower the NTP, contract labs, and other researchers in understanding and interpreting chemical effects in this rat strain. To delineate similarities/differences between the female and male mammary gland, data were compiled starting on embryonic day 15.5 through postnatal day 70. Mammary gland whole mounts, histology sections, and immunohistochemically stained tissues for estrogen, progesterone, and androgen receptors were evaluated in both sexes; qualitative and quantitative differences are highlighted using a comprehensive visual timeline. Research on endocrine disrupting chemicals in animal models has highlighted chemically induced mammary gland anomalies that may potentially impact human health. In order to investigate these effects within the HSD strain, 2,3,7,8-tetrachlorodibenzo-p-dioxin, diethylstilbestrol, or vehicle control was gavage dosed on gestation day 15 and 18 to demonstrate delayed, accelerated, and control mammary gland growth in offspring, respectively. We provide illustrations of normal and chemically altered mammary gland development in HSD male and female rats to help inform researchers unfamiliar with the tissue and may facilitate enhanced evaluation of both male and female mammary glands in juvenile toxicity studies.

Figures


Figure 1. Representative hematoxylin and eosin images of female Harlan Sprague-Dawley rat fetal mamm

(A and B) Mammary bud formation, E15.5. (C and D) Mammary stalk formation, E16.5. (D) Mammary mesenchyme formation (arrow). (E and F) Elongation of mammary stalk, E17.5. (A, C, E) Original magnification 16×. (B, D, F) Original magnification 40×.

Figure 2. H&E images of female SD rat fetal mammary gland development from E18.5 to E21.5

Representative hematoxylin and eosin images of female Harlan Sprague-Dawley rat fetal mammary gland development from embryonic day (E) 18.5 to E21.5. (A and B) Invagination of the skin above the mammary gland, E18.5. (C and D) Elongation of the mammary gland stalk, E19.5. (E and F) Formation of the nipple sheath, E20.5. (F) Nipple sheath forms (arrow). (G and H) Primary duct begins lumen formation, E21.5 (A, C, E, G) Original magnification 16×. (B, D, F, H) Original magnification 40×.

Figure 3. H&E images of male SD rat fetal mammary gland development from E15.5 to E17.5

Representative hematoxylin and eosin images of male Harlan Sprague-Dawley rat fetal mammary gland development from embryonic day (E) 15.5 to E17.5. (A and B) Mammary bud formation, E15.5. (C and D) Mammary stalk formation, E16.5. (D) Mammary mesenchyme formation (arrow). (E and F) Elongation of mammary stalk, E17.5. (A, C, E) Original magnification 16×. (B, D, F) Original magnification 40×.

Figure 4. H&E images of male SD rat fetal mammary gland development from E18.5 to E21.5

Representative hematoxylin and eosin images of male Harlan Sprague-Dawley rat fetal mammary gland development from embryonic day (E) 18.5 to E21.5. (A and B) Atrophy of the mammary gland stalk, E18.5. (C and D) Further atrophy of the mammary gland stalk, E19.5. (E and F) Primary duct begins lumen formation, E20.5. (G and H) Elongation of the primary duct with apparent lumen, E21.5. (A, C, E, G) Original magnification 16×. (B, D, F, H) Original magnification 40×.

Figure 5. H&E images of female mammary gland development from birth to sexual maurity

Hematoxylin and eosin images of female mammary gland development in Harlan Sprague-Dawley rat offspring from birth to sexual maturity. (A) At postnatal day (PND) 4, mammary gland ducts are lined by a single or double layer of cuboidal epithelial cells. (B) At PND 21, there is early branching of the mammary epithelium. (C) By PND 46, the terminal end buds have cleaved to form new ducts. (D) By PND 70, female mammary gland is tubuloalveolar. The lobule is composed primarily of ducts and clusters of 3 to 5 alveolar buds, each with a centrally located lumen surrounded by a layer of cuboidal epithelial cells. Terminal ductal lobular unit (TDLU) forming (arrow), (A–D) original magnification 40×.

Figure 6. H&E images of male mammary gland development from birth to sexual maurity

Hematoxylin and eosin images of male mammary gland development in Harlan Sprague-Dawley rat offspring from birth to sexual maturity. (A) At postnatal day (PND) 4, the mammary gland ducts are lined by simple cuboidal epithelial cells. (B) At PND 21, there is early branching of the mammary gland epithelium. (C) At PND 46, the terminal end buds have cleaved to form new ducts. (D) By PND 70, the male mammary gland is predominantly lobuloalveolar. Alveoli are prominent and ducts are infrequent. Alveoli and ducts are lined by stratified epithelium that consists of tall vacuolated cuboidal to short columnar epithelial cells, (A–D) original magnification 40×.

Figure 7. H&E sections of SD dam mammary gland during gestation and lactation

Hematoxylin and eosin-stained sections of Harlan Sprague-Dawley dam mammary gland during gestation and lactation. Control glands from (A) gestational day (GD) 15, (B) GD 17, (C) GD 21 and (D) postnatal day 4. Original magnification 40×.

Figure 8. ERα, PR, and AR IHC staining of female mammary gland epithelium

Estrogen receptor alpha (ERα), progesterone receptor (PR), and androgen receptor (AR) immunohistochemistry staining of Harlan Sprague-Dawley female mammary gland epithelium. (A–C) ERα, (D–F) PR, and (G–I) AR. (A, D, and G) Postnatal day (PND) 8, (B, E, and H) PND 33, and (C, F, and I) PND 70. Original magnification 40×. Nuclear staining of ERα became apparent at PND 21, peaked by PND 33, and slightly decreased by PND 70. Nuclear epithelial staining of PR was evident by PND 33 and peaked by PND 46, while AR was consistently low and variable over time in the female mammary gland. There was nonspecific staining of the mast cells in the surrounding stroma (arrow).

Figure 9. ERα, PR, and AR IHC staining of male mammary gland epithelium

Estrogen receptor alpha (ERα), progesterone receptor (PR), and androgen receptor (AR) immunohistochemistry staining of Harlan Sprague-Dawley male mammary gland epithelium. (A–C) ERα, (D–F) PR, and (G–I) AR. (A, D, and G) Postnatal day (PND) 8, (B, E, and H) PND 33, and (C, F, and I) PND 70. Original magnification 40×. Male mammary gland sections contained low numbers of weakly staining epithelia for ERα. It was evident at PND 33 and remained low over time. AR immunohistochemical staining was evident in the male early, peaked by PND 46, and was found at lower levels in the differentiated adult gland. There was no nuclear staining of PR at any time point. There was nonspecific staining of the mast cells, not a part of the mammary gland epithelium (arrow).

Figure 10. Whole-mounted female mammary gland developmental progression

Whole-mounted female mammary gland developmental progression from birth to sexual maturity. Representative carmine stained 4th mammary gland whole-mount images of Harlan Sprague-Dawley rat, picturing the mammary epithelium, inguinal lymph node, and surrounding stroma and fat pad. (A) Mammary gland present at birth on postnatal day (PND) 1. (B) Mammary gland on PND 8 showing increased budding on ducts. (C) Prepubertal mammary gland on PND 21; beginning of terminal end bud (TEB) presence (arrow). (D) Peripubertal mammary gland on PND 33 demonstrating abundant TEB presence, exponential mammary epithelium growth, and convergence of the 4th and 5th gland (arrow). (E) Continued postpubertal mammary gland growth on PND 46 with terminal ends beginning to differentiate at the edges of the fat pad. (F) The mammary gland has filled the fat pad and image shows density of mammary gland epithelium yet ducts are still visible on PND 70. Sizes are shown in figures.

Figure 11. Whole-mounted male mammary gland developmental progression

Whole-mounted male mammary gland developmental progression from birth to sexual maturity. Representative carmine stained 4th mammary gland whole-mount images of Harlan Sprague-Dawley rat, picturing the mammary epithelium, inguinal lymph node, and surrounding stroma and fat pad. (A) Mammary gland present at birth on postnatal day (PND). (B) Mammary gland on PND 8 showing increased budding on ducts. (C) Mammary gland on PND 21 demonstrating the beginning of terminal end bud (TEB) presence. (D) Peripubertal mammary gland on PND 33 demonstrating mammary epithelium growth and connection of the 4th and 5th gland (arrow). (E) Continued postpubertal mammary gland growth on PND 46 with terminal ends beginning to differentiate before reaching the edges of the fat pad. (F) The mammary gland fails to fill the fat pad and image shows the enlarged mammary gland lobules and decreased visibility of mammary gland ducts indicative of the sexually dimorphic lobuloalveolar mammary epithelium on PND 70.

Figure 12. Mammary gland development of prenatally TCDD-exposed male rats

Whole-mount and histology comparison of mammary gland development of control and prenatally 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-exposed Harlan Sprague-Dawley (HSD) males at postnatal day 33. Representative carmine stained 4th mammary gland whole-mount images (A and C) and H&E-stained histology sections (B and D) of control (A and B) and TCDD (C and D) prenatally exposed offspring. (A) Mammary whole mount of control HSD male demonstrating normal lateral growth of the 4th gland past the lymph node and the presences of the 5th gland and its connection to the 4th gland (arrow). (B) H&E section has normal tubuloalveolar morphology characterized by numerous branching from the major duct. (C) Mammary whole mount of HSD male gestationally treated with TCDD demonstrating stunted lateral mammary gland branching and absence of the 5th gland, which was not specific to TCDD treatment, (A, C) original magnification 0.8×. (D) H&E section has normal tubuloalveolar morphology but with reduced side branching of ducts, (B, D) original magnification 40×.

Figure 13. Mammary gland development of prenatally TCDD-exposed female rats

Whole-mount and histology comparison of mammary gland development of control and prenatally 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-exposed Harlan Sprague-Dawley (HSD) females at postnatal day 21. Representative carmine-stained 4th mammary gland whole-mount images (A and C) and H&E-stained histology sections (B and D) of control (A and B) and TCDD (C and D) prenatally exposed offspring. (A) Mammary whole mount of the 4th gland of the control HSD female demonstrating normal lateral growth just past the lymph node and extensive side budding of the ducts. (B) H&E section of normal tubuloalveolar morphology and terminal end bud (TEB) development. (C) Mammary whole mount of the 4th gland of the HSD female gestationally treated with TCDD demonstrating stunted mammary gland growth, less extensive side budding of the ducts, and failure to form complex ends, (A, C) original magnification 2×. (D) H&E section characterized by normal tubuloalveolar morphology with small ductal buds, (B, D) original magnification 40×.

Figure 14. Mammary gland development of prenatally DES-exposed male rats

Whole-mount and histology comparison of mammary gland development of control and prenatally diethylstilbestrol-exposed males at postnatal day 70. Representative carmine-stained mammary gland whole-mount images (A and C) and H&E-stained histology sections (B and D) of control (A and B) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (C and D) prenatally exposed offspring. (A) Mammary whole mount of control Harlan Sprague-Dawley (HSD) male demonstrating increased density of the mammary epithelium with indiscernible ducts. (B) H&E section of normal lobuloalveolar morphology. (C) Mammary whole mount of control HSD male demonstrating dense mammary epithelium with discernable ducts, (A, C) original magnification 2.5×. (D) H&E section of normal lobuloalveolar morphology of the mammary epithelium located at the center of the mammary gland (left) and ducts and tubules with morphologic characteristics similar to those observed in female rats (tubuloalveolar morphology) at the periphery of the mammary gland (right), (B, D) original magnification 40×.

Figure 15. ERα, PR, and AR quantification in female and male offspring mammary glands

Estrogen receptor alpha (ERα), progesterone receptor (PR), and androgen receptor (AR) quantification in female and male offspring mammary glands. Quick Scores were determined for the nuclear receptor staining. (A) Nuclear ERα first appeared at postnatal day (PND) 15 in the diethylstilbestrol (DES)-exposed females and PND 21 in control and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-exposed females, peaking at PND21 in DES-treated females and PND33 in control females. (B) Nuclear ERα first appeared at PND 33 in the males. (C) There was very little AR staining in the females and was sporadic in the chemically exposed animals and more consistent in the control females. (D) AR-stained male mammary epithelia at low levels until puberty in control and TCDD-exposed animals. DES had a less intense nuclear staining. (E) PR was only expressed in the females and first appeared at PND 33 in control and DES-exposed animals and PND 46 in TCDD-exposed animals. Values from this figure are shown in Supplementary Tables 2, 3 and 4.

Figure 16. Compiled literary data on the female rat

The developmental events, receptor expression of the mammary gland, and the range of hormone levels in the serum of female rats (largely Charles River CD1 origin), from neonates to adults. Darkest bar intensity represents strongest expression/presence. 1(Emanuele et al. 2001), 2(Biegel et al. 1998), 3(Kim et al. 2002), 4(Saji et al. 2000), 5(Kariagina, Aupperlee, and Haslam 2008), 6(Miousse et al. 2013), 7(Cheung et al. 2001), 8(Cotroneo et al. 2002), 9(Tan et al. 2004), 10(Thordarson et al. 1995), 11(Hvid et al. 2011), 12(Kao, Hiipakka, and Liao 2000), 13(Dohler and Wuttke 1975), 14(Darcy et al. 1999), 15(Weisz and Ward 1980), 16(Wilson and Handa 1997), and 17(Frawley and Henricks 1979).

Tables


Table 1. Age-adjusted Treatment Associations with Digitally Quantified Epithelial Measures

Age-adjusted Treatment Associations with Digitally Quantified Epithelial Measures, among Female Rats Less than 70 Days.

Supplemental Materials


Supplemental Material