The Rat Mammary Gland: Morphologic Changes as an Indicator of Systemic Hormonal Perturbations Induced by Xenobiotics

Abstract

The development and morphology of the rat mammary gland are dependent upon several hormones including estrogens, androgens, progesterone, growth hormone and prolactin. In toxicology studies, treatment with xenobiotics may alter these hormones resulting in changes in the morphology of reproductive tissues such as the mammary gland. In the rat, male and female mammary glands exhibit striking morphologic differences that can be altered secondary to hormonal perturbations. Recognizing these morphologic changes can help the pathologist predict potential xenobiotic-induced perturbations in the systemic hormonal milieu. This review examines the development of the rat mammary gland and the influence of sex hormones on the morphology of the adult male and female rat mammary gland. Specific case examples from the literature and data from our laboratory highlight the dynamic nature of the rat mammary gland in response to hormonal changes.

Development of the Rat Mammary Gland

Embryologic development of the rat mammary gland has been investigated extensively and will only be briefly considered here (for reviews see Ceriani, 1970; Knight and Peaker, 1982; Russo et al., 1989c). The male and female rat mammary gland develops from a single layer of cuboidal epithelium, progressing in a cephalo-caudal sequence (Myers, 1917). Islands of epithelium thicken into hillocks of cuboidal cells resting on an indistinct basal membrane; simultaneously there is atrophy of the epithelial cells between the hillocks (Myers, 1917). In the male or female fetus, mammary glands remain as rudimentary buds of epithelium without development into distinct lobules or alveoli (Knight and Peaker, 1982).

In the rat fetus the importance of androgens in mammary gland development is well documented. Androgens initiate differentiation of the male phenotype by promoting atrophy of the rudimentary buds in the male (Goldman et al., 1976; Sourla et al., 1998a, 1998b). This atrophy of the buds is triggered by testosterone-induced condensation of the stroma (Topper and Freeman, 1980). Mammary gland morphology in the male rat is altered (feminization) to the morphology of the female rat if androgens are eliminated and androgens administered to female rat fetuses cause mammary glands to appear male-like (virilization) (Goldman et al., 1976). In contrast, the roles of estrogens (E) and progesterone (P4) in the rat fetus are less clear and their proposed function has been extrapolated from studies in mice and ex vivo mammary gland explants. Progesterone does not appear critical to embryonic development of mammary glands in mice and absence in utero does not affect the potential for maturation in adulthood (Freeman and Topper, 1978). Estrogens promote mammary gland development in the mouse fetus; however E inhibits development in the rat fetus (Ceriani, 1970).

The neonatal male and female rat has 6 pairs of glands with a central lactiferous duct, several branching secondary ducts and numerous tertiary ducts (Ceriani, 1970). The glands are distributed in pairs along the milk line, with 1 pair cervical, 2 thoracic, 1 abdominal and 2 inguinal (Astwood et al., 1937). Mammary tissue is embedded in a mass of adipocytes, pre-adipocytes and fibroblasts, referred to as the fat pad, with a thin layer of stroma separating the epithelial cells from the fat pad (Hovey et al., 1999; Silberstein, 2001; Imagawa et al., 2002). In the pre-pubescent female rat, mammary growth is mainly influenced by growth hormone (GH) and prolactin (PRL) with minimal influences of estradiol (E2) and P4 (Knight and Peaker, 1982).

Mammary growth in the male and female rat during puberty is dependent on normal gonadal function, as indicated by the absence of development in ovariectomized or gonadectomized rats (Cowie and Folley, 1961). To our knowledge, a detailed description of pubescent mammary gland development in males is not published. In females, growth of the mammary gland during puberty is characterized by differentiation of the epithelium into terminal end bud units (TEBs); rapid expansion by elongation and branching of the ducts; and hypertrophy of the fat pad (Cowie and Folley, 1961; Russo et al., 1989c; Knight and Peaker, 1982).

Study Designs and Collection and Evaluation of Rat Mammary Glands

In the evaluation of the rat mammary gland, a systematic approach to study design and sampling and processing of the mammary gland for histology is critical. The morphologic characteristics of the rat mammary gland are both site and age-specific (Russo et al., 1986, 1989a). For this reason, when designing studies it is important to use rats that are sexually mature at study start and to collect the same mammary gland pair at necropsy. In our laboratory, we examine the inguinal mammary gland pair and collect the gland with the overlying skin. Included in the trimmed section is the inguinal lymph node that can be used by the histologist as a landmark for slide preparation. Consistent sectioning is very helpful in the histologic evaluation of the rat mammary gland especially when evaluating mammary gland atrophy.

Unique Sex-Specific Morphology of Rat Mammary Gland

The mammary gland from preclinical species such as the mouse, beagle dog, and nonhuman primate do not exhibit significantly different microscopic morphology between the nonlactating adult female and male, only the quantity of glandular tissue is different (Richert et al., 2000). However, in the sexually mature rat there is a striking histologic divergence of male and female mammary glands (Table 1 and Figure 1). While these differences in female and male mammary gland morphology were first noted approximately 50 years ago (Ahren and Etienne, 1957), the observations and their potential mechanism have been given little attention in the literature except for a review by Cardy (1991) and a recent publication by Rudmann et al. (2005). For this reason, these differences are reviewed briefly next.

The virgin female rat mammary gland is described as tubuloalveolar and dominated by a scattering of branched tubular ducts and fewer alveoli in the hypodermis while in the male the mammary gland is described as lobuloalveolar, composed of large contiguous lobules of alveoli with fewer ducts (Figure 1; Cardy, 1991; Rudmann et al., 2005) and in whole mount preparations (Russo et al., 1989c; Wang et al., 2005). Not only does the frequency of duct and alveolar structures differ for males and females, but the morphologic characteristics of the ducts and alveoli also have a contrasting morphologic appearance.

Ducts in females have a distinct lumina lined by a single layer of cuboidal epithelial cells that is surrounded by a myopepitheial tunic and thick basement membrane (Cardy, 1991; Masso-Welch et al., 2000). The alveolar buds appear as clusters of tubules with a central lumen, which is lined by cuboidal epithelium. Epithelial cells in ducts and alveoli of females have less cytoplasm than males that is basophilic. Mitoses and apoptotic cells are not observed. Terminal ducts are small, finger-shaped, with thin walls and wide lumens (Russo et al., 1989c). Male ducts and alveoli have a pseuodstratified or stratified epithelium and cells are larger with more abundant cytoplasm that is eosinophilic and usually vacuolated (Cardy, 1991; Rudmann et al., 2005). Apoptotic cells are observed in the epithelial lining as well as in alveolar and ductal lumina. In many cases alveolar and ductal epithelial cells fill the lumina of the male structures.

There are also other normal variations in the morphology of the male and female rat mammary gland that are important for interpreting experimental results. Terminal end buds as described earlier are normal structures that are commonly observed in sexually immature rats but are also observed in sexually mature rats (Figure 1). These structures should not be confused with foci of ductular or alveolar hyperplasia. Mammary gland morphology in the female is also influenced by the stage of estrus (Lotz and Krause, 1978) and if the stage of the female is not considered it is possible that the variation could be interpreted as a treatment-related change. For the male, tubular and ductal profiles with female-like characteristics are observed occasionally adjacent to normal male structures (Figure 1).

Implications for Toxicology Studies

It is important to recognize the morphologic differences between the mammary gland of normal adult male and female rats because, by virtue of its unique sexual dimorphism, the rat mammary gland may undergo morphologic changes indicating endocrine disruption (Table 2). The purpose of this section is to familiarize the pathologist with the different morphologic changes in the rat mammary gland that can occur with altered systemic hormone levels. Because of the complex interplay involved with maintaining hormonal homeostasis, effects on one hormone or its receptor will usually result in changes in multiple hormones. Therefore, the case studies provided below include examples of mammary gland changes observed with perturbation of single and multiple hormones. For the purpose of this review, the discussion and case studies will be limited to non-neoplastic mammary gland alterations observed in studies ≤6 months in duration.

Estrogens

Estrogens play an important role in adult rat mammary gland development and function (Russo et al., 1989b). While there is considerable literature evaluating the effects of estrogens in rats, many of the studies used experimental designs or rat strains not standard for toxicology laboratories today. A recent comprehensive study examined the effect of 3 months of dietary administration of 17 β-estradiol in Crl:CD male and female rats (Biegel et al., 1998b). In this study, mammary glands from both male and female rats had altered mammary gland morphology. Male rat mammary glands were feminized, characterized by the conversion of the normal lobuloalveolar morphology to the tubuloalveolar morphology expected for the normal female (Biegel et al., 1998b). There was also secretory material in the ducts and alveoli of the feminized gland and less frequently focal lobular hyperplasia.

Female rats from this study had multifocal or diffuse lobuloalveolar hyperplasia. Glands had an increased density of alveoli centered on ducts. Morphologic changes in both male and female rats were accompanied by elevated serum prolactin and estrogen levels (Biegel et al., 1998a; Cook et al., 1998), the former expected due to the positive regulation of pituitary prolactin secretion by estrogen (Nolin et al., 1977) (Table 3).

Similar morphologic and hormonal findings have been reported in SD male rats treated with phytoestrogens and endocrine active compounds (Wang et al., 2005) and observed in our laboratory in both male and female F344 rats treated with compounds that have ER agonist activity (Table 3, Figure 2A–B). As discussed later, mammary glands from male and female rats treated with compounds that only cause hyperprolactinemia also cause comparable mammary gland changes in males (feminization) and females (lobuloalveolar hyperplasia with increased secretions). Therefore, when these changes are observed in male and female mammae, elevations of E and/or PRL should be suspected (Table 2).

Hypoestrogenism or ER antagonism in rats unaccompanied by other hormonal changes causes atrophy of the female mammary gland (Table 2). The atrophy in the female mammary gland affects both the ductular and alveolar components and the fat pad appears prominent because of the loss of the gland parenchyma. In the affected ducts and alveolae, epithelial cells are low cuboidal and have high nuclear to cytoplasmic ratios. The selective estrogen receptor modulators (SERM) Tamoxifen and toremifene are examples of potent estrogen receptor antagonists in the rat mammary gland that cause decreases in ductal ectasia and mammary gland atrophy in female rats (Greaves et al., 1993; Karlsson et al., 1996) and acinar atrophy in male rats (Kennel et al., 2003) (Table 3).

Androgens

The male and female rat mammary gland, in contrast to the mouse mammary gland, develops and proliferates after puberty in response to androgens, GH, and IGF-1 (Topper et al., 1972; Kleinberg et al., 1990; Ruan et al., 1992; Feldman et al., 1993; Ruan et al., 1995, 1999). When T is administered to adult ovary intact female rats, the tubuloalveolar morphology of the mammary gland is altered to a more male-like lobuloalveolar appearance with ductal and alveolar dilatation and secretory activity (Selye et al., 1936; Laqueur and Fluhmann, 1942). Hypophysectomy ablates the mammary gland changes suggesting that pituitary hormones like PRL or GH are necessary for the effects (Laquer and Fluhmann, 1942). While PRL was not measured in these early studies, adult ovariectomized female rats and intact male rats given T have elevated circulating T, DHT, and PRL (O’Connor et al., 2000) (Table 3). When rats are treated with T, PRL secretion is likely stimulated because T is aromatized to E, a positive regulator of pituitary prolactin secretion (Nolin et al., 1977). Intact or ovariectomized female rats given the androgen DHT which is not aromatized to E do not have increases in serum prolactin but have changes in other hormones comparable to rats treated with T (Nolin et al., 1977) (Table 3).

Androgens in the absence of hyperprolactinemia cause virilization of the normal female gland. The tubuloalveolar morphology of the normal female mammary gland is converted to the lobuloalveolar morphology of the male mammary gland (Table 1, Figure 1). This alteration has been observed in ovariectomized female rats given non-aromatizable androgens (Sourla et al., 1998) and recently described in female ovary intact rats treated with a SERM (Rudmann et al., 2005) (Figure 3). For the SERM studies, female rats had hormonal changes including elevated E2 and T without changes in PRL or P4. While the mammary gland effects of increased E2 were blocked by the SERM, the effects of hyperandrogenemia were unopposed resulting in mammary gland virilization that could be blocked by the AR antagonist flutamide (Rudmann et al., 2005). SERM-treated rats did not have the increased ductal and alveolar secretions observed with T or DHT treatment presumably due to the lack of PRL elevation in SERM-treated rats (Rudmann et al., 2005).

The mammary gland effects of blocking androgens in the rat have been investigated in experiments using the androgen receptor antagonist flutamide (Toyoda et al., 2000; Rudmann et al., 2005) (Table 3). Male rat mammary glands demonstrate lobular atrophy despite elevated serum levels of T and E (Toyoda et al., 2000). Female rats treated with flutamide do not have morphologic changes in the mammary glands or changes in systemic levels of other mammotrophic hormones (Toyoda et al., 2000; Rudmann et al., 2005). Comparable effects would be expected for compounds that decrease androgen levels like testosterone biosynthesis inhibitors (O’Connor et al., 2002a, 2002b) (Table 3). Increased E2 and decreased T bioactivity due to AR antagonism are both observed with flutamide treatment in male rats but PRL is unaffected and the mammary gland is atrophic (Toyoda et al., 2000). In contrast, in male rats treated with E, T is decreased and E and PRL are increased and there is mammary gland feminization (Table 3). Based on these combined data sets, it is likely that male mammary gland atrophy can be driven by AR antagonism or decreased T while male mammary gland feminization is caused by elevated PRL and not increased E or decreased T (Table 2).

Prolactin

PRL is synthesized in the pituitary and at a number of extra-pituitary sites including the rat mammary gland (Ben-Jonathon et al., 1996). PRL of pituitary origin promotes lobuloalveolar development and initiates milk production during pregnancy (Likhider et al., 1997; Harris et al., 2004). PRL is also produced locally in the mammary gland and is thought to have a role in lactogenesis i.e., in sustaining milk production (Harris et al., 2004). The action of PRL on the rat and mouse duct is inferred from evidence in mice in which genetic deletion of PRL in mice causes an absence of secondary branching of the ducts (Brisken C et al., 1999). PRL probably promotes ductal branching independently of E and synergistically with P4 by inducing local factors in the fat pad (Oka and Topper, 1972; Brisken et al., 1999; Daniel and Silberstein, 1987; Imagawa et al., 2002). While PRL may have effects on duct branching, several other hormones can promote duct development independently, including androgens, GH and EGF (Carsol et al., 2002; Gallego et al., 2001).

Dopaminergic receptor antagonists cause elevated PRL and mammary lobuloalveolar hyperplasia in female rats (Lotz and Krausse, 1978; O’Connor, 1996) (Table 3). Cardy (1991) described feminization of male rat mammary gland in males treated with a dopamine antagonist for 52 weeks. In these male rats, the lobuloalveolar appearance changed to the tubuloalveolar appearance of the female and there was increased secretory activity and duct ectasia (Cardy, 1991). There was no hormone data reported; however, we also recently observed mammary gland feminization in male rats with compounds that cause hyperprolactinemia and lobuloalveolar mammary gland hyperplasia in female rats (Figure 2C–D).

Feminization of the male gland consisted of loss of the normal male lobuloalveolar arrangement and development of a tubuloalveolar arrangement with an increased number of ducts and more abundant small alveoli lined by a low cuboidal epithelium surrounding small lumina. As reported by Cardy, at lower doses males had a blend of male and female mammary gland morphology. These data suggest that hyperprolactinemia by itself can produce mammary gland feminization in males and lobuloalveolar hyperplasia in females (Table 2). To our knowledge there are no reports of the effects of decreased prolactin on the mammary gland morphology of male and female rats.

Progesterone

Progesterone is important in the ductular and lobuloalveolar changes that occur in prepubescent development and pregnancy as indicated by studies in transgenic mice and P4 knockout animals. Ablation of PR in the female mouse causes a decrease in pregnancy-associated development and branching of the ductal epithelium, failure of alveologeneis and absence of ductal side-branching proteins, absence of terminal end buds, and inhibition of lobuloalveolar differentiation in response to E or P4 (Atwood et al., 2000; Conneely et al., 2000; Fernandez-Valdivia et al., 2005). However, P4 is not essential for the pubescent development of the mammary gland in the PR knockout mouse (Fernandez-Valdivia R et al., 2005). Transgenic mice with an excess of P4 receptor develop thick ducts with extensive lateral branching and large numbers of terminal buds, lined by cells that are multilayered and disorganized (Shyamala, 1999; Shyamala et al., 1999). The effects of progesterone on ductal development is synergistic with other hormones such as estrogen and IGF-1 (Ruan et al., 2005) and in the rat P4 acts synergistically with E2, PRL, and GH to promote duct growth (Kamiya et al., 1998).

In rats, the effects of blocking progesterone activity have been examined in adult ovary intact females given RU486 (Mifepristone), a potent PR antagonist (van der Schoot et al., 1987) (Table 3). In female rats administered RU486 for 4 weeks, there was mammary gland lobuloalveolar hyperplasia with increased secretions and the formation of large cysts filled with milk (van der Schoot et al., 1987). Serum concentrations of E2 and P4 were elevated and the pituitary gland was enlarged. These morphologic changes were consistent with those caused by hyperprolactinemia in female rats. While PRL was not measured in the van der Schoot study, Ruiz (1996) demonstrated that female rats given RU486 had approximately 4-fold increases in serum prolactin. Of note is that lobuloalveolar hyperplasia was observed despite complete antagonism of the PR receptor. The effects of RU486 on male rat mammary gland morphology were not described. Intact male rats treated with RU486 have increased serum FSH and LH concentrations (O’Connor et al., 2000).

Growth Hormone

The pituitary is required for mammary glandular development (Reece et al., 1936; Lewis et al., 1942). Pituitary secretion of GH stimulates TEB proliferation, IGF-1 mRNA production, and acts via GH receptors in the mammary gland stroma and fat pad (Kleinberg, 1997). The fat pad becomes hypertrophic and synthesizes growth regulatory molecules like IGF-1 and TEBs proliferate resulting in lengthening and increased branching of ducts in rats during puberty (Knight and Peaker, 1982; Russo et al., 1989c; Hovey et al., 1999).

Treatment of rats with hGH for 1 or 3 months causes mammary gland lobuloalveolar hyperplasia with secretory activity in male and female rats (Jorgensen et al., 1988). These changes are similar to those observed with hyperprolactinemia and GH is known to have prolactin-like activity in the rat (Peckham et al., 1967). There are no reports of the mammary gland effects of GH depletion in rats.

Conclusion

The normal male and female rat mammary gland has striking species specific differences in morphology. Morphologic changes in the normal mammary gland morphology provide important clues to the endocrine effects of xenobiotics (Table 3). Recognizing these morphologic patterns not only helps the toxicologic pathologist predict specific changes in the hormonal milieu but also helps the pathologist partner with the toxicologist and endocrinologist to define follow-up studies that directly test hypotheses around endocrine dysfunction.