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Published in final edited form as: Toxicol Pathol. 2015 Dec 20;44(3):450–457. doi: 10.1177/0192623315621414

Immunoexpression of Steroid Hormone Receptors and Proliferation Markers in Uterine Leiomyoma and Normal Myometrial Tissues from the Miniature Pig, Sus scrofa

Kristie Mozzachio 1, Alicia B Moore 2, Grace E Kissling 3, Darlene Dixon 2
PMCID: PMC4805434  NIHMSID: NIHMS739620  PMID: 26692562

Abstract

Uterine leiomyomas in miniature pet pigs occur similarly to that in women with regards to frequency, age, parity and cycling. Clinical signs, gross and histologic features of the porcine tumors closely resemble uterine leiomyomas (fibroids) in women. Although fibroids are hormonally responsive in women, the role of estrogen and progesterone has not been elucidated. Current animal models present challenges in data extrapolation to humans. In this study, immunohistochemistry was used to assess the expression of the steroid hormone receptors, ER-α, ER-β and PR, and cell proliferation markers, PCNA and Ki-67, in tumor and matched myometrial tissues sampled from miniature pigs. A “quickscore” method was used to determine receptor expression, and percent labeling indices were calculated for the markers. ER-α/β and PR were localized to nuclei of smooth muscle cells in both tissues. PR expression was intense and diffuse throughout all tissues, with correlation between tumors and matched myometria. Conversely, ER-α expression was more variable between the tissues and animals. ER-β expression was low. PCNA and Ki-67 were localized to the nucleus and expression varied among tumors; however, normal tissues were overall negative. These findings support further investigation into the use of the miniature pig as a model of fibroids in women.

Keywords: miniature pig, uterine leiomyoma, fibroid, steroid hormone receptors, proliferation markers, immunoexpression

Introduction

Fibroids are an extremely common benign neoplasm affecting the female reproductive tract, with an estimated cumulative incidence of 70–85% in American women and leading to clinical problems such as pain, abnormal bleeding and infertility (Segars et al., 2014, Cardozo et al., 2012, Buttram and Reiter, 1981, Cramer and Patel, 1990, Ellenson, 2010, Baird et al., 2003). Despite an abundance of research that has well-characterized these tumors, determined risk factors, and allowed advances in treatment and prognosis, the pathogenesis of fibroids is not fully understood and efforts to prevent development or cure existing disease are elusive. Although the Eker rat is an established animal model with a high spontaneous occurrence of uterine leiomyomas (~65%) (Walker, 2003), complicating factors include atypical epithelioid phenotypes and malignant smooth muscle tumor development as well as a high incidence of concurrent splenic and renal tumors (Everitt et al., 1995). Other animal models, including mice and guinea pigs, require genetic manipulation, tumor induction, or xenograft implantation as the incidence of spontaneous development is low as compared to women (Tsuiji et al., 2010, Romagnolo et al., 1996, Porter et al., 1995, Newbold et al., 2002). While nonhuman primates have the greatest similarity to the human female reproductive system and spontaneous occurrence of uterine leiomyomas has been reported in the Rhesus macaque (Simmons and Mattison, 2011), neoplasms of the reproductive tract are infrequent (Cline, 2004) and practical and ethical considerations have prevented extensive evaluation of this species. Overall, current animal models have inherent challenges to the extrapolation of data to humans.

Miniature, or potbellied, pigs attained popular pet status in the mid-1980’s and an aging pet population has enabled veterinarians to identify the uterine leiomyoma as the most common tumor in intact females (Ilha et al., 2010). These tumors occur spontaneously at a similar frequency to that observed in humans, affecting approximately 65% of pigs aged 5 yr or older. Both gross and histologic features of the porcine tumor are similar to those seen in women (Mozzachio et al., 2004), and the pig has a 21-day, year-round estrous cycle (Swindle, 2007) that more closely approximates that of women as compared to current animal models of this tumor. Additionally, porcine uterine leiomyomas are more common in nulliparous animals as parity appears to be protective, just as it is in women. Although the complex signaling pathways involved in fibroid pathogenesis remain the focus of ongoing research, there is clear evidence that estrogen and progesterone play a significant role in the growth of these tumors in women, through interaction with their respective receptors (Kim and Sefton, 2012, Borahay et al., 2015). However, the receptors for these critical steroid hormones have not yet been evaluated in the minipig. Furthermore, cell proliferation is an important indicator of growth of these tumors. Increased cell proliferation has been reported to be a significant contributor to growth and is autonomous for each tumor in a given woman (Dixon et al., 2002).

In order to gain a better understanding of the pathology of the tumors in women, animal models are necessary. Earlier we reported the potbellied, or miniature, pig (Sus scrofa) as a potential animal model because their uterine smooth muscle tumors grossly and histomorphologically resemble human fibroids (Mozzachio et al., 2004). The goal of this study was to assess the immunoexpression of the steroid hormone receptors, estrogen receptor alpha (ER-α), estrogen receptor beta (ER-β) and progesterone receptor (PR) as well as the endogenous cell proliferation markers, proliferating cell nuclear antigen (PCNA) and Ki-67, in miniature pig leiomyomas and matched normal myometrial tissues.

Materials and Methods

Tissue Collection, Fixation and Histological Stains

Tissue samples of all gross masses and matched normal myometrium, as well as a myometrial sample from an ovariectomized female, were obtained at surgery or necropsy from client-owned or rescue minipigs presenting to the North Carolina State University College of Veterinary Medicine (NCSU-CVM); pigs ranged from approximately 6 to 12 yr of age. Tissues were placed in 10% neutral buffered formalin at room temperature for 24-72 hr, then processed and paraffin-embedded using routine methods. Samples were sectioned at 4–5µm and stained with hematoxylin and eosin (H&E) and Masson’s Trichrome. Unstained tissue sections were cut at 4–5µm and used for immunohistochemical studies.

Immunohistochemical Stains

In the initial retrospective study, immunohistochemical staining for α-smooth muscle actin was performed to confirm the smooth muscle origin of the tumors; results have been previously published. In the current study, immunohistochemical staining for the steroid hormone receptors, ER-α, ER-β, and PR, as well as the cell proliferation markers, PCNA and Ki-67, was performed on leiomyoma and animal-matched normal myometrial tissues from a subset of minipigs from the original study. In addition, the atrophied uterus of a single ovariectomized minipig was included to determine the extent of steroid hormone receptor expression in the myometrium in the absence of ovarian hormones. All tissues were evaluated by routine light microscopy and a total of 22 tumors and animal-matched normal myometrium from 7 minipigs were assessed for the sex steroid hormone receptors. A total of 10 tumors and animal-matched normal myometrium from 6 minipigs were evaluated for the proliferation markers.

Following deparaffinization in xylene and rehydration through graded alcohols, endogenous peroxidase was blocked using 3% H2O2. Heat-induced antigen retrieval was performed using either a microwave or the Decloaker (Biocare Medical, Concord, CA). Non-specific sites were blocked using normal horse or goat serum at room temperature for 20 min, respective to the secondary antibody used. Tissues were then incubated with each of the primary antibodies listed in Table 1 for 1 hr at room temperature, followed by incubation with the appropriate secondary antibody for 30 min at room temperature. Negative controls consisted of normal mouse IgG at a concentration the same as the primary antibody. Immunoreactive complexes were detected by an avidin-biotin affinity system (Vectastain Elite ABC Kit, Vector Laboratories, Burlingame, CA) and visualized with 3,3'-diaminobenzidine (DAB) chromogen (DakoCytomation, Carpenteria, CA). Finally, the sections were counterstained with hematoxylin, dehydrated through graded ethanol, cleared in xylene and coverslipped.

Table 1.

Immunohistochemical Stains

Stain Primary Antibody Dilution Secondary Antibody Dilution
ER-α mouse anti-human ER (ER1D5)a 1:300 horse anti-mouse IgGb Manufacturer’s recommendation (Vectastain Elite ABC kit)b
ER-β mouse anti-human ER-beta (PPG5/10)c 1:25 horse anti-mouse IgGb 1:1500
PR mouse anti-human PR (PR10A9)a 1:600 horse anti-mouse IgGb Manufacturer’s recommendation (Vectastain Elite ABC kit)b
PCNA mouse anti-human PCNA (131–11912)d 1:1500 goat anti-mouse IgMe 1:400
Ki-67 mouse anti-rat Ki-67 (MIB-5)f 1:25 horse anti-mouse IgGb 1:500
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a

- Immunotech, Inc., Marseille, France

b

- Vector Laboratories, Burlingame, CA

c

- AbD Serotec, Raleigh, NC

d

- Millipore, Billerica, MA

e

- Jackson Immunoresearch Laboratories, West Grove, PA

f

- Dakocytomation Corporation, Carpinteria CA

Microscopic Evaluation

All tissues were evaluated via routine light microscopy. For the sex steroid hormone receptors, the semiquantitative method described by Detre et. al. (Detre et al., 1995) was utilized to calculate a “quickscore” based on immunostain average intensity and overall percent of positive staining (Table 2). The tissues that were stained for the steroid hormones were independently scored by two co-authors. For the proliferation markers, labeling indices were calculated by dividing the number of positive (brown-staining) nuclei by a total count of 1000 cells, multiplied by 100.

Table 2.

Quickscore Criteria

Proportion of brown-staining nuclei Staining Intensity Quickscore
1 – 0–4% 0 - negative Proportion of brown-staining nuclei × staining intensity (range: 0–18)
2 – 5–19% 1 - weak
3 – 20–39% 2 - intermediate
4 – 40–59% 3 - strong
5 – 60–79%
6 – 80–100%
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Statistical Analysis

Because normal tissue was sampled near tumor tissue, the statistical analysis accounted for this pairing of the data. Furthermore, quickscores were not normally distributed, so a nonparametric test, the Wilcoxon signed ranks test, was used to compare tumor to normal tissues. P-values are two-sided and considered statistically significant if less than 0.05 (Conover 1971).

Results

Tissue samples of all gross masses and matched normal myometrium from minipigs ranging in age from approximately 6 to 12 yr, as well as a myometrial sample from an ovariectomized minipig, were evaluated for sex hormone receptors and proliferation markers using immunohistochemistry. Immunoexpression of the sex hormone receptors was localized to nuclei of normal and neoplastic smooth muscle cells and positive staining was indicated by a variable brown color. ER-α expression (Figure 1A and 1B) was highly variable, even among different tumors in the same animal. Quickscores ranging from 1–18 were assigned to the tumors evaluated, and matched normal myometrium was higher, lower or equivalent to that of the tumor tissue, with a quickscore range of 5–18. The ER-α quickscore in the ovariectomized female was 11. ER-β was weakly expressed (Figure 1C and 1D), averaging a quickscore <6 in the majority of tumor tissues and in all normal myometrial tissues. The ER-β quickscore for the ovariectomized female was 1. In contrast, PR expression was typically intense and diffuse throughout both leiomyoma (Figure 1E) and normal myometrial (Figure 1F) tissue, often attaining the maximum quickscore value of 18 (tumor range 10–18; normal range 12–18). PR expression was high but slightly less intense in the ovariectomized female, with a resultant quickscore of 12.

Figure 1.

Figure 1

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Immunohistochemical stains for sex steroid hormone receptors in porcine leiomyoma and matched normal myometrium. (A, B) ER-α immunoreactivity in tumor (A) versus normal myometrium (B); (C, D) ER-β immunoreactivity in tumor (C) versus normal myometrium (D); (E, F) PR immunoreactivity in tumor (E) versus normal myometrium (F); insets = negative controls.

There was no significant difference in sex steroid hormone receptor expression between normal and neoplastic tissues for all receptors (Figure 2). However, PR expression levels were overall significantly higher (p<0.05) in both leiomyoma and matched myometrial samples compared to expression of ER-α and ER-β in these tissues. Expression of ER-α was significantly (p<0.05) increased compared to ER-β in both tumors and normal myometrial samples. A summary of quickscore values as well as individual quickscores for the sex steroid hormone receptors in normal and tumor tissues is presented in Figure 2 and Table 3, respectively.

Figure 2.

Figure 2

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Graph shows overall average (+/− standard error of mean) quickscores for steroid receptors in uterine leiomyoma and matched myometrial tissues from miniature pigs. ER-α and PR expression levels were significantly higher in both leiomyoma and matched myometrial samples compared to expression of ER-β. Expression of PR was significantly higher than that of ER-α in both tumors and normal myometrial samples. There were no significant differences between tumor and normal tissues for each steroid receptor evaluated. a, statistically different from ER-β (p<0.05); b, statistically different from ER-α (p<0.05).

Table 3.

Individual Steroid Receptor Quickscores in Miniature Pig Tissues

Animal & Tumor Nos. ER-α ER-β PR
Pig Tumor Normal Tumor Normal Tumor Normal Tumor
1 1 8 3 6 2 18 10
2 6 6 5 1.5 15 18
3 12 12 5 7.5 18 18
4 12 12 5 0 18 18
2 1 6 18 4.5 2 18 18
2 10 12 5 6 18 18
3 11 18 5.5 0 18 18
4 6 5 5 4.5 18 18
5 5 10 3.5 4.5 18 18
3 1 15 10 6 1.5 18 18
4 1 10 12 5 6 18 18
2 10 12 5 12 12 18
3 1 15 1 5.5 1 18
5 1 1 18 1 12 1 18
2 12 11 6 6 18 18
3 8 5 6 5.5 18 18
4 1 1 1 3 1 8
6 1 15 10 5 5.5 18 18
7 1 18 18 3 6 18 18
2 18 18 5 5.5 18 18
3 18 18 10 5.5 18 18
4 18 18 11 2 18 18
Ovariectomized female 11 NA 1 NA 12 NA
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1

Myometrium specifically matched to tumor not available.

2

Small tumor lost to sectioning.

NA = Not applicable.

PCNA expression (Figure 3A) was greater than that of Ki-67 (Figure 3B), and both markers were present within the majority of tumors but absent or minimally expressed in normal myometrium. Labeling indices in neoplastic tissue ranged from 0–52.2% for PCNA and from 0–6.7% for Ki-67. Expression was autonomous for individual tumors in a given animal, although large tumors (>30cm) typically demonstrated greater expression than small tumors (<2cm).

Figure 3.

Figure 3

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Proliferation marker immunohistochemical staining in a porcine leiomyoma, PCNA (A) and Ki-67 (B).

Despite the absence of historical information on clinical signs of estrus, ovarian and uterine evaluation suggested that all pigs were actively cycling. However, specific estrous cycle stage data could not be ascertained.

Conclusions

The miniature pig has been identified as a promising animal model of fibroids in women (Mozzachio et al., 2004). High spontaneous occurrence, clinical presentation, effects of parity, and gross (Figure 4) and histologic (Figure 5) features are remarkably similar to observations in humans (Parazzini et al., 1996, Segars et al., 2014). Although the role of estrogen and progesterone in uterine leiomyoma development and growth is complex, the effects are elicited through their respective receptors, and the expression of these receptors has been described as a key feature of any potential animal model (Segars et al., 2014).

Figure 4.

Figure 4

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Minipig uterine leiomyoma: A firm, round, well-circumscribed mass (*) extends from the serosa of the uterine horn into the broad ligament. On cut surface (inset), the mass is white to tan and whorled. Arrow = uterine bifurcation; Circle = ovary.

Figure 5.

Figure 5

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Minipig uterine leiomyoma: A) Hematoxylin and eosin (H&E) stain showing well-differentiated smooth muscle cells arranged in interlacing fascicles. B) Cytoplasm of tumor cells stains positively for α-smooth muscle actin.

In this study, immunohistochemistry was used to assess the expression of the steroid hormone receptors, ER-α, ER-β and PR, and cell proliferation markers, PCNA and Ki-67, in tumor and matched myometrial tissues sampled from miniature pigs. Reports of immunoexpression of the sex steroid hormone receptors in fibroids in women are contradictory, although the majority indicate increased expression of steroid receptors in tumor tissue compared to normal myometrium (Bakas et al., 2008, Flake et al., 2003, Jakimiuk et al., 2004, Roan et al., 2005, Tamaya et al., 1985, Wei et al., 2005, Wilson et al., 1980). Although, in this study, there was no significant difference in steroid hormone receptor expression between normal and tumor tissues in minipigs, PR expression was greater than that of ER-α, and ER-α expression was greater than that of ER-β in both normal myometrium and leiomyomas. This has also been reported in women (Plewka et al., 2014, Zaslawski et al., 2001). For the proliferation markers, PCNA was more highly expressed than Ki-67, and leiomyomas frequently exhibited an increased proliferative rate over matched myometrium. The subset of porcine tumors evaluated for proliferation markers was small, but demonstrated proliferation indices were autonomous for individual tumors in a given animal. In women, the proliferative state has been reported to be autonomous for each tumor in a given individual although independent of tumor size (Dixon et al., 2002).

It is evident that fibroids are dependent on the steroid hormones, estrogen and progesterone, which play a role in the proliferation of these tumors. Steroid hormones are also thought to be important in regulating growth factors and cytokines, which can influence uterine leiomyoma cell growth in addition to contributing to the expansion of an abundance of disorganized extracellular matrix (ECM) that consists predominantly of collagen and is believed to also play a role in tumor growth (Leppert et al., 2014, Ciarmela et al., 2011). Contrarily, many of the current animal models do not share this characteristic overproduction of ECM, which is a consistent feature of many fibroids in women; however, the authors have found that, over the years of characterizing minipig tumors, excessive ECM is common and appears to expand with increasing tumor size. This phenomenon of increased tumor size and enhanced collagen production has been observed in fibroids in women (Flake et al., 2013). Growth factors that appear to be important in influencing ECM production as well as tumor cell proliferation are transforming growth factor-β, basic fibroblast growth factor, and insulin-like growth factor-I, among others, which have all been reported in human leiomyomas (Flake et al., 2003, Yu et al., 2008).

Recently, there has been a shift of focus to not only include steroid hormones but to explore the role of genetics and stem cells in fibroid development and growth (Moravek and Bulun, 2015). Genetic abnormalities, recurrent genetic aberrations, and mutations may also contribute to the development of uterine leiomyomas (Hodge et al., 2009, Makinen et al., 2011, Parker, 2007, Velagaleti et al., 2010) and are considered to play pivotal roles in tumorigenesis. Although the cellular origin of uterine fibroids remains unknown, fibroids are thought to be monoclonal tumors that originate from the transformation of a single somatic, or adult, stem cell (Canevari et al., 2005, Zhang et al., 2006), although the initiating event causing the neoplastic transformation is currently unknown. Recently, a few significant advances have been made to pave the way to understanding the pathophysiology of fibroids. Somatic stem cells have been isolated and appear to be necessary for steroid hormone-dependent growth (Mas et al., 2012, Ono et al., 2012). In particular, one study identified a somatic single-gene defect encoding Mediator Complex Subunit 12 (MED 12) gene in a majority (~70%–75%) of uterine fibroid tumors (Makinen et al., 2011). MED12 is a subunit of the Mediator complex, which is thought to regulate transcription (Taatjes, 2010). Another study showed that the growth of human fibroid tumors, which are dependent on estrogen and progesterone, also require the presence of these stem cells, which make up just one percent of the tumor; moreover, the fibroid stem cells, but not the stem cells from the surrounding uterine myometrial tissue, carried MED12 mutations. Interestingly, one lab summarizes that tumors will not develop without sex hormones, especially progesterone, as well as stem cells in the environment that carry the most common mutation, MED12 (Bulun et al., 2015). Although the regulation of estrogen and progesterone on the growth of leiomyoma stem cells is unknown, it has been demonstrated that other factors such as the wingless-type (WNT)/β-catenin pathway plays a critical role in uterine leiomyoma tumorigenesis in a paracrine manner (Ono et al., 2013).

Conventionally, estradiol has been considered the primary stimulus for uterine leiomyoma growth, and studies with cell cultures (Barbarisi et al., 2001) and animal models (Newbold et al., 2002) support this concept. However, there is emerging evidence that progesterone and the progesterone receptor play key roles in the growth and development of uterine leiomyomas (Kim et al., 2013; Ishikawa et al., 2010). The minipig tumors express both ER and PR and could, therefore, allow study of the interaction of these steroid hormone receptor pathways, in addition to their interaction with growth factors and other cytokines in regulating fibroid cell growth and expansion of the ECM. Additionally, the minipig could serve as an excellent model for future studies to further explore the role of genetic alterations involving MED12 and /or the role of stem cells in uterine leiomyoma development and growth.

Acknowledgments

The authors would like to thank the NIEHS Histology and Immunohistochemistry laboratories for their technical support, and Norris Flagler, Eli Ney and Beth Mahler for their expertise with digital imaging. The authors also thank Cynthia Swanson, of WIL Research-Hillsborough, and Experimental Pathology Laboratories, Inc. for support with imaging. Lastly, the authors kindly thank Drs. Xiaohua Gao and Mark Cesta for their critical review of this manuscript. This research was supported, in part, by the Intramural Research Program of the NIH, NIEHS and DNTP.

Abbreviations

CEH

Cystic endometrial hyperplasia

DAB

3,3'-diaminobenzidine

ECM

Extracellular matrix

ER-α

Estrogen receptor alpha

ER-β

Estrogen receptor beta

H&E

Hematoxylin and eosin

H2O2

Hydrogen peroxide

IgG

Immunoglobulin G

MED 12

Mediator complex subunit 12

PR

Progesterone receptor

PCNA

Proliferating cell nuclear antigen

WNT

Wingless-type

Footnotes

Competing financial interests

The authors declare no conflicts of interest, financial or otherwise.

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