Comparative Expression and Distribution of c - fos,Estrogen Receptorα (ERα),and p38α in the Uterus of Rats,Monkeys,and Humans

Abstract

The uterine cellular expression and distribution of c-fos, ERα and p38α was compared in humans, nonhuman primates, and rats using immunohistochemistry. ERα and c-fos were present in the glandular (GE) and luminal epithelial cells (LE) of humans and nonhuman primates, with differing expression patterns evident between proliferative and secretory cycle phases. In rats, the highest and lowest expression of c-fos was present during proestrus and estrus, respectively, in the LE and GE. The most intense ERα staining in rats was observed during proestrus in the GE, while the least intense staining was seen in the LE during proestrus. Strong LE and GE expression of p38α as present in rats in all stages of the estrous cycle and during the proliferative phase in both humans and nonhuman primates. No p38α expression was observed during the secretory phase in either humans or nonhuman primates. Our work suggests that c-fos, ERα and p38α (a) are primarily expressed during the proliferative phase, but not the secretory phase and exhibit interspecies expression variability, and (b) rats exhibit cyclic changes in the expression of c-fos and ERα.

Introduction

A variety of intracellular signals, including those involving Estrogen Receptorα (ERα), c-fos, and p38α, orchestrate physiological events in the uterus during the secretory and proliferative phases of menstrual cycle in humans and non-human primates. Emerging data suggests that uterine function effects may be attributable to the close interrelationships of these signaling pathways and their modulation.

The uterus is composed of a heterogeneous population of cells, including epithelium, stroma, and myometrium, which all respond to steroid hormones (i.e., estrogen). Estrogen stimulates DNA synthesis, and cellular proliferation and differentiation in the uterus of mammals (Quarmby and Korach, 1984; Mendoza-Rodriguez et al., 2003). Binding of estrogen to its receptor (ER) contributes to uterine cellular proliferation via increased expression of immediate early response genes, as seen in the uterus following treatment with 17β estradiol (E₂) (Quarmby and Korach, 1984; Weisz and Bresciani, 1988; Papa et al., 1991; Bigsby and Li, 1994; Nephew et al., 1995; Mendoza-Rodriguez et al., 2003).

c-fos is an immediate early proto-oncogene and signal transducer that forms a heterodimer with c-jun and translocates to the nucleus (Muller et al., 1983, 1984, 1986). c-fos and c-jun interact with the transcription factor activator protein-1 (AP-1) in the nucleus to initiate a cascade of gene induction events that lead to cell proliferation (Yoshino et al., 2003). Constitutive basal expression of the c-fos gene in mice is typically low, but can be rapidly induced by a variety of agents (Cohen and Curran, 1989). Rapid and marked induction of c-fos mRNA in the uterus of ovariectomized rats, mice, and guinea pigs treated with estradiol (Loose-Mitchell et al., 1988; Weisz and Bresciani, 1988; Jouvenot et al., 1990) suggests that the c-fos gene plays a role in proliferation of uterine epithelial cells (Nephew et al., 1995; Mendoza-Rodriguez et al., 2003). In addition, the uterine c-fos gene isolated from mice and humans contains a functional estrogen response element, which implies that estrogen has a direct stimulatory effect on this gene (Hyder et al., 1991; Oldenhof et al., 2002). The expression of c-fos in the uterus has been previously investigated in humans, pigs, mice, ewes, and rats (Papa et al., 1991; Dubois et al., 1993; Nephew et al., 1995; Salmi and Rutanen, 1996; Yamashita et al., 1996; Johnson et al., 1997; Maldonado et al., 2003; Mendoza-Rodriguez et al., 2003). Low levels of c-fos are found in the liver, heart, kidney, gonads, muscle, and fibroblasts (Thompson et al., 1986; Cohen and Curran, 1989; Teofoli et al., 1999), while higher levels are found in the spleen, thymus, lungs, salivary glands, amnion, yolk sac, bone marrow cells, macrophages, and mast cells (Muller et al., 1983; Muller, 1986; Caubet et al., 1989; Cohen and Curran, 1989; Bottazzi et al., 1990; Lee et al., 2004). However, the comparative expression of c-fos and microanatomic location in normal uteri of humans, nonhuman primates and rats has not been previously examined.

The mitogen-activated protein kinases (MAPKs) transduce a variety of extracellular signals to the transcription machinery and include three distinct mammalian types, extracellular signal-regulated kinases (ERKs), c-Jun NH₂-terminal kinases (JNKs), and p38 MAPKs (p38), the latter having four isoforms of its own (α, β, γ, δ) (Takanami-Ohnishi et al., 2001). MAP kinases regulate AP-1 transcriptional activity and c-fos expression in the uterus and mediate stretch-induced c-fos expression in myometrial smooth muscle cells (Papa et al., 1991; Whitmarsh and Davis, 1996; Oldenhof et al., 2002; Roux and Blenis, 2004). Recent studies also suggest that p38 may play a role in implantation in the human endometrium (Yoshino et al., 2003).

Collectively, these observations imply a close interrelationship between ERα, c-fos, and p38α in modulating uterine functions during the estrous cycle and early embryonic processes. Therefore, pharmacologic modulation of one or more of these molecules may have an effect on the uterus. There are currently no published reports examining the comparative expression and microanatomic location of these molecules in humans or animals. Therefore, using immunohistochemistry, we investigated the microanatomic location and expression of c-fos, ERα and p38α in normal uterine tissues from humans, nonhuman primates and rats during various stages of the uterine cycle. This study is the first comparative report on interspecies expression of these molecules.

Material and Methods

Study Uterine Tissue Samples

Uterine tissues obtained from humans, nonhuman primates (Cynomolgus monkeys), and Sprague-Dawley rats were examined. Subjects ranged in age from 25 to 44 years (n = 10), 6 to 9 years (n = 6), and 8–12 weeks (n = 15) for humans, nonhuman primates, and rats, respectively. Human uterine tissues were surgically obtained from patients undergoing hysterectomy for benign gynecological conditions (St. Louis Tissue Bank, St. Louis, MO). All subjects had regular menstrual cycles and none had received hormonal treatment prior to surgery. Uterine tissues from untreated control nonhuman primates and rats were obtained at the time of necropsy (Pfizer Pathology Archives). All tissue samples were obtained in compliance with federal and all other applicable guidelines. Tissues were fixed in 10% neutral buffered formalin for 24 hours and embedded in paraffin wax. Then 3-μm-thick sections were cut and stained with hematoxylin and eosin (H&E) for histological examination, or stained immunohistochemically with antibodies to c-fos, ERα or p38α.

Microscopic Features For Assigning Uterine Stages

The histologic appearance of the uterus varies with the stage of the reproductive cycle. There are four phases (proestrus, estrus, metestrus (diestrus I), and diestrus II) in the estrous cycle in rodents. Proestrus is characterized by distention of the uterine lumen with clear fluid and the lumen is usually lined by large low columnar cells (Yuan and Foley, 2002). Smooth muscle hypertrophy, endometrial stromal cell proliferation, stromal edema, few mitotic figures, and proliferation of luminal and glandular epithelium are present during proestrus. In estrus, the uterine lumen is lined by very large, tall columnar epithelium, there is myometrial hypertrophy, and many of the luminal and glandular epithelial cells undergo vacuolar degeneration and necrosis (Yuan and Foley, 2002). In metestrus (diestrus I), the luminal lining epithelial cells are reduced in height, the stroma becomes denser, and mitotic activity decreases (Yuan and Foley, 2002). During diestrus II, the uterus is quiescent with dense endometrial stroma, atrophied myometrium, and cuboidal glandular and luminal epithelium.

There are 2 phases (proliferative and secretory) in the menstrual cycle in nonhuman primates and humans (Crum, 1999). During the proliferative phase the endometrium begins to thicken and growth of all endometrial cells (endothelium, myometrium, and stroma) takes place. Mitoses are present in the endometrial glandular epithelium during the follicular (“proliferative”) phase of the cycle, and in the stroma during the early luteal (“secretory”) phase. No evidence of mucus secretion or vacuolation is present during the proliferative phase. In the secretory phase, the endometrium slows its growth, stromal edema is evident, basal subnuclear secretory vacuoles are present in the glandular epithelium, and there is secretory exhaustion. Stromal edema is usually present at 2 times in the menstrual cycle, once in the mid-follicular/proliferative phase and once in mid-luteal/secretory.

Immunohistochemistry

The 3-μm sections were cut from formalin-fixed, paraffin-embedded blocks, mounted on positively charged glass slides, dried, deparaffinized in 3 changes of xylene for 2 minutes each, and then rehydrated through graded ETOH rinses to inactivate endogenous peroxidase activity, including a 0.3% solution of hydrogen peroxide. Sections were then subjected to heat-induced epitope retrieval (HIER). HIER was accomplished by immersing the sections in a prewarmed pressure cooker (Biocare Medical, Walnut Creek, CA) containing EDTA solution (Biocare Medical) at pH 9.5 for ERα staining sections, and citrate buffer solution (Biocare Medical) at pH 6 for c-fos and p38α staining sections for 3 minutes at 20 psi. The chamber was then cooled for 10 minutes with the chamber closed, followed by an additional 10 minutes of cooling with the chamber opened. Sections were then rinsed in tap water and immunostains performed at room temperature using a Nexus automated immunostainer (Ventana Medical Systems, Tucson, AZ). The automation included exposure to 300 μL of primary anti-c-fos rabbit polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) diluted at 1:500 at room temperature for 32 minutes, anti-p38α rabbit polyclonal antibody (Covance, St. Louis, MO) diluted at 1:1000 at room temperature for 32 minutes or anti-ERα mouse monoclonal antibody diluted at 1:20 (Vector Laboratories, Burlingame, CA) at approximately 37°C for 30 minutes with reagent diluent (Ventana Medical Systems). After rinsing, the sections were incubated for 4 minutes with Avidin-Biotin blocking solution (Ventana Medical Systems). Then, 300 μL of the appropriate biotinylated IgG (H+L) linking solution (Vector Laboratories) was applied to each section at 1:200 dilution for 32 minutes at room temperature. Sections were again rinsed and allowed to react with 300 μL of diaminobenzidine (DAB Detection Kit) substrate solution (Ventana Medical Systems) for 8 minutes, followed by counterstaining with hematoxylin and then Bluing Reagent for 4 minutes each, removed from the autostainer, washed in warm water, dehydrated through graded alcohol, cleared in xylene, and cover slipped. Control reactions included: (1) sections incubated with the omission of primary antibody and processed as mentioned before, and (2) sections incubated with normal rabbit or mouse serum instead of the primary antibody and processed as above.

c-fos, ERα, and p38α expression in uterine tissues was semiquantitatively evaluated. Staining scores with regard to the percentage of immunostained cells were scored: (−) = no staining, (+) = mild staining and less than 30% of the cell population stained; (++) = moderate staining and less than 60% of the cell population stained; (+++) = strong staining with greater than 60% of the cells stained.

Results

The comparative uterine cellular expression and distribution of c-fos, ERα, and p38α are summarized in Table 1. ERα, c-fos, and p38 were present in the uterine tissues of all three species, with mild differences in the expression pattern between proliferative and secretory phases in humans and nonhuman primates. Staining intensity ranged from mild to strong.

In human uterine tissue, moderate (++) to strong (+++) luminal epithelial nuclear c-fos localization and mild (+) to strong (+++) glandular epithelial nuclear c-fos localization was seen during the proliferative phase (Figure 1A). In nonhuman primates, moderate (++) glandular and moderate (++) to strong (+++) luminal epithelial nuclear c-fos localization was seen during the proliferative phase (Figure 1C). In addition, moderate (++) to strong (+++) nuclear staining of endometrial stromal cells and mild (+) nuclear staining of myometrial smooth muscle cells was present in both species. No epithelial c-fos expression was present during the secretory phase of the uterus, or in myometrial smooth muscle or endometrial stromal cells of either species (Figures 1B, 1D). In rat uterine sections, c-fos expression varied with estrous cycle stage. Moderate (++) to strong (+++) luminal and glandular epithelial nuclear staining was present during the proestrus stage (Figure 1E). Mild (+) luminal and glandular epithelial nuclear staining was present during estrus stage (Figure 1F). Moderate (++) glandular and luminal epithelial nuclear staining were present during the metestrus stage (Figure 1G). In all stages of estrous cycle, mild (+) nuclear staining was present in both myometrial smooth muscle cells and endometrial stromal cell (Figure 1E). Staining was absent in all negative controls.

ERα nuclear staining was moderate (++) to strong (+++) in glandular and luminal epithelial cells during the uterine proliferative phase in humans (Figure 2A). Endometrial stromal cell had strong (+++) staining during this phase, while myometrial smooth muscle cell staining was moderate (++). Uterine cells had no (−) staining during the secretory phase (Figure 2B). In nonhuman primate uterine sections, strong (+++) endometrial stromal cell nuclear staining, moderate (++) to strong (+++) glandular epithelial nuclear staining, mild (+) luminal epithelial and myometrial smooth muscle cell nuclear staining were seen during the proliferative phase (Figure 2C). During the secretory phase, mild (+) glandular epithelial nuclear staining, negative (−) myometrial smooth muscle cell and luminal epithelial nuclear staining, and strong (+++) endometrial stromal cellstaining were observed (Figure 2D). In rat uterine sections, no staining (−) to mild (+) staining was observed in luminal glandular epithelium, while moderate (++) to strong (+++) staining was observed in the glandular nuclear epithelium during the proestrus stage (Figure 2E). During the estrus stage, moderate (++) glandular and luminal epithelial nuclear staining was present (Figure 2F). In the metestrus stage, luminal epithelial cells had mild (+) nuclear staining and glandular epithelial cells had moderate (++) nuclear staining (Figure 2G). In all stages of estrus cycle, endometrial stromal cell had mild (+) to moderate (++) staining, while myometrial smooth muscle cells had mild (+) nuclear staining. Staining was absent in all negative controls.

For p38α, strong (+++) glandular and luminal epithelial cytoplasmic expression was present during the proliferative phase in both human and nonhuman primate uterine sections (Figure 3B). Myometrial smooth muscle cells had mild (+) to moderate (++) cytoplasmic staining, while endometrial stromal cell had strong (+++) cytoplasmic staining in both species. No epithelial p38α expression was present during the secretory phase of the uterus of either species (Figures 3A, 3C). Myometrial smooth muscle and endometrial stromal cells were also negative. In rat uterine sections, strong (+++) glandular and luminal epithelial cytoplasmic staining was present during proestrus, estrus, and metestrus stages (Figures 3D, 3E, 3F). Myometrial smooth muscle cells and endometrial stromal cell had strong (+++) cytoplasmic staining.

Discussion

Review of literature indicates that ERα, c-fos, and p38α orchestrate uterine functions during the estrous cycle and early embryonic processes. Therefore, chemical or biochemical modulation of one or more of these molecules coupled with interspecies differences in uterine cyclicity may influence the uterine functions. There are currently no published reports examining the comparative expression of these molecules in humans or animals. In this study, we characterized the microanatomic location and expression of c-fos, ERα and p38α in normal uterine tissues from humans, nonhuman primates and rats during various stages of the uterine cycle.

Results of the study show that c-fos is expressed in the luminal epithelia (LE) and glandular epithelia (GE) during the proliferative phase of endometria in humans and nonhuman primates. These findings are consistent with previous studies that demonstrated the highest levels and strong c-fos expression in non–pregnant endometria during the proliferative phase, under high estrogen levels, of the menstrual cycle in humans (Fujimoto et al., 1994; Salmi et al., 1996; Salmi and Rutanen, 1996; Maldonado et al., 2003). Uterine tissue is characterized by its proliferative potential and the biologic significance of c-fos expression in the uterus is related to endometrial proliferative response (Salmi et al., 1996; Mendoza-Rodriguez et al., 2003). No expression of c-fos was seen during the secretory phase of endometria, under high progesterone levels, in either humans or nonhuman primates. This result is consistent with other studies that demonstrated a sharp reduction in c-fos protein (Reis et al., 1999) and nearly undetectable levels of c-fos during the secretory phase of human endometria (Fujimoto et al., 1994). Contrastingly, Salmi and Rutanen (1996) observed strong c-fos mRNA expression in the secretory phase of endometria in humans, indicating the possibility of a post-transcriptional regulation of c-fos in the uterus. These discrepancies may reflect variability in methods or tissue preparation.

Mild myometrial smooth muscle cell and moderate to strong endometrial stromal cell c-fos expression was present during the proliferative phase in our study in humans and nonhuman primates. Similar results were seen with weaker c-fos expression in the adjacent myometrium in humans (Salmi and Rutanen, 1996), and strong c-fos in interstitial cells (Maldonado et al., 2003). In rats, the LE and GE had comparable quantities of c-fos expression in each stage of the cycle. However, the expression of c-fos was highest in the LE and GE during proestrus and lowest during estrus, with modest expression in metestrus, as previously demonstrated by Mendoza-Rodriguez et al. (2003). In the same study, the highest and lowest levels of estrogen were found during proestrus and estrus in rats, respectively (Mendoza-Rodriguez et al., 2003). In addition, the myometrial smooth muscle and endometrial stromal cell had mild staining during all stages of the estrous cycle in our study. The expression of c-fos in these nonepithelial cells suggests a paracrine signaling role from endometrial stroma to epithelia in the uterus.

Estrogen induces DNA replication and cellular proliferation in the mammalian uterus via modification of gene expression, including c-fos, and interaction with its specific nuclear receptor (ER) (Mendoza-Rodriguez et al., 2003). In most mammalian species, cycling hormone levels regulate ER uterine activity. Of the 2 estrogen receptors isoforms (α and β), ERα is dominant in the uterus (Mendoza-Rodriguez et al., 2003). This data corroborates with the infertility and lack of uterotrophic response in ERα knockout mice (Lubahn et al., 1993).

During our study, human uterine tissue showed moderate to strong ERα nuclear localization in the LE and GE during the proliferative phase, with no expression during the secretory phase. This finding is consistent with demonstrated maximal ER concentration in the proliferative phase of menstrual cycle and declined concentration in the secretory phase of published studies (Press et al., 1984; Lessey et al., 1988). Myometrial smooth muscle cells had moderate nuclear staining, while strong nuclear staining was observed in endometrial stromal cell during the proliferative phase in humans in all studies described (Press et al., 1984; Lessey et al., 1988). In nonhuman primates, moderate-to-strong GE ERα nuclear staining and mild LE ERα nuclear staining were seen in our study during the proliferative phase. LE lacked staining, while GE had mild ERα nuclear staining during the secretory phase in our study in nonhuman primates. These results concur with other studies in nonhuman primates demonstrating the presence of strong nuclear ER in endometrium during the proliferative phase of menstrual cycle, with some weak immunoreactive ER staining detectable during the secretory phase (Okulicz et al., 1990). In baboons, ER has been noted in the GE and endometrial stroma during the proliferative phase, with no detectable counterpart during the secretory phase (Hild-Petito and Fazleabas, 1997). Strong endometrial stromal cell and mild myometrial smooth muscle cell staining was present in the proliferative phase, but no immunoreactivity was present during the secretory phase in our study. Endometrial stroma, and myometrial smooth muscle cells showed positive immunoreactivity for ER on day 9 of an artificial menstrual cycle, while on day 23, a marked loss of staining in stromal cells was seen in nonhuman primates (Okulicz et al., 1990).

The changing pattern of estradiol (E) and progesterone (P) secretion during the nonhuman primate menstrual cycle is essential for the hormonal regulation of endometrial growth and differentiation and P action is essential for the proper maturation of the endometrium (Okulicz et al., 1996). The transition from a proliferative (E-dominated) to secretory (P-dominated) endometrium results in the appropriate differentiation that permits implantation (Okulicz et al., 1996). The numbers of ER in nonhuman primate endometrium are low when serum P levels are elevated during the secretory phase of menstrual cycle, but rise 2 to 3 fold when P levels decline during the proliferative phase (West and Brenner, 1983). In rats, preovulatory E produced by ovarian follicles peaks in concentration during proestrus and mitotic activity can be seen in uterine glandular cells as well as those cells lining the endometrium. When E levels drop during estrus, there is a corresponding lack of glandular and luminal epithelial growth and increased apoptosis in these cells (Mendoza-Rodriguez et al., 2003). The uterine lumen is dilated during both proestrus and estrus. As a result of the preovulatory E surge, ovulation and mating behavior occur at the beginning of metestrus (M). In our study, nuclear ERα expression in rats was highest in the GE during proestrus, while mild to moderate staining intensity was observed in both LE and GE during estrus and M. LE either lacked ERα staining or had mild staining during proestrus. A possible explanation of this finding is the temporal pattern of stimulation of estrogen in rodent uterine tissues, with the GE responding before LE (Martin and Finn, 1968; Korach and Lamb, 1981; Quarmby and Korach, 1984). In comparison, intense ER nuclear staining during metestrus in both the GE and LE and a decrease in immunoreactivity during proestrus and estrus days has been observed (Mendoza-Rodriguez et al., 2003).

Cycling hormones, including estrogen, are widely shown to regulate ERα activity in the uterus (Mendoza-Rodriguez et al., 2003). For example, estrogen has been reported to stimulate DNA synthesis via ER-mediated events in rodent uterus and estrogen stimulated cell proliferation in endometrial epithelia in adult rodents (Martin et al., 1973; Papa et al., 1991). It has also been suggested that the initial steps in the mechanism of mitogenesis by estrogen involve activation of c-fos gene expression in the rat uterus, emphasizing the orchestrated effort of these molecules in directing uterine function (Weisz and Bresciani, 1988). Moderate to strong endometrial stromal cell and mild myometrial smooth muscle cell ERα nuclear staining was seen at all stages of the estrous cycle in rats consistent with these studies (Mendoza-Rodriguez et al., 2003).

Studies have suggested the role of the p38 signaling pathway in relation to uterine function. P38 is thought to contribute to parturition (Takanami-Ohnishi et al., 2001; Oldenhof et al., 2002; Otun et al., 2005). Marked increase in the kinase activity of p38 in the human uterus was observed on day 19 of gestation and during labor, and declined to the control level post-delivery (Takanami-Ohnishi et al., 2001). It is also shown to be present in endometriotic cells from humans and activated by pro-inflammatory agents (Yoshino et al., 2004). However, there are currently no reports on the microanatomic localization of p38α in the normal mammalian uterus. In our study, the GE and LE had strong cytoplasmic expression of p38α in human and nonhuman primates during the proliferative phase of endometria only. At all stages of the estrous cycle in rats, p38α was expressed in both the GE and LE. This interspecies discrepancy in p38 expression may be related to variations in hormone concentrations among these species. Less extreme peaks and troughs in the rodents might be one explanation for the continued strong staining for p38 throughout the estrous cycle when primates have a more cyclic pattern of expression. In addition, myometrial smooth muscle cells and endometrial stromal cell expressed p38α in this species. These findings are consistent with other reports that have shown uniform expression of p38 in the myometrium of the nonpregnant uterus (Otun et al., 2005). It is known that MAPKs play a role in regulating cellular hypertrophy and hyperplasia via mechanical stretch of the uterus (Yamazaki et al., 1998; Oldenhof et al., 2002). Therefore, the high myometrial expression of p38α may be important in preparing the uterus for labor.

In conclusion, our work demonstrate, for the first time, that c-fos, ERα and p38α (a) are primarily expressed during the proliferative phase, but not the secretory phase, of the human and nonhuman primate uterus, and exhibit interspecies expression variability, and b) rats exhibit cyclic changes in the expression of c-fos and ERα.