Smooth Muscle Myosin II and Alpha Smooth Muscle Actin Expression ...

BIOLOGY OF REPRODUCTION 53, 598-608 (1995)

Smooth Muscle Myosin II and Alpha Smooth Muscle Actin Expression in the Baboon (Papio anubis) Uterus Is Associated with Glandular Secretory Activity and Stromal Cell Transformation' S. Christensen,36 H.G. Verhage,6 G. Nowak, 7 P. de Lanerolle, 7 S. Fleming, 4 8 S.C. Bell, 8 A.T. Fazleabas,2 6 and S. Hild-Petito 5' 6

Departments of Obstetrics and Gynecology 6 and Physiology and Biophysics 7 University of Illinois College of Medicine, Chicago, Illinois Department of Obstetricsand Gynecology,8 University of Leicester, Leicester, United Kingdom ABSTRACT The objective of this study was to investigate the localization and hormonal regulation of smooth muscle myosin II (SMM II)and a smooth muscle actin (aSMA) in the baboon uterus, since cytoskeletal proteins are involved in secretory function and morphological transformation. Uterine tissue was obtained from baboons 1)during the menstrual cycle, 2)following steroid treatment of ovariectomized baboons, 3) during pregnancy (Days 14-60 postovulation [PO]), and 4)during simulated pregnancy (Days 18-32 PO). Tissues were processed for immunocytochemical localization of SMM II or aSMA with specific polyclonal or monoclonal antibodies, respectively. SMM II stained all smooth muscle cells of blood vessels and myometrium regardless of treatment. Glandular epithelial staining was present only in endometrium obtained during the luteal phase or following estrogen and progesterone treatment. Staining intensity was greater inthe basalis than in the functionalis. The number of glands staining positive for SMM II on Days 18-32 of pregnancy and simulated pregnancy was variable. Glandular stain was absent after Day 32 PO. These immunocytochemical data were confirmed by immunoblot analysis of glandular cytosolic extracts. Stromal staining for SMM IIwas present underthe luminal epithelium during simulated pregnancy (Days 1832), on Day 25 of steroid treatment in the simulated-pregnant controls, and in nonimplantation sites during pregnancy. In contrast, SMA staining was low or absent in all uterine cell types inovariectomized baboons. Under estrogen-dominated conditions (follicular phase and estrogen treatment), aSMA staining was present in smooth muscle cells, and this staining persisted throughout the remaining treatment periods. Glandular epithelial staining for aSMA was absent in all treatment groups. However, aSMA staining in stromal fibroblasts underneath the luminal epithelium was evident as early as Day 14 of pregnancy and Day 18 of simulated pregnancy. The number of stromal fibroblasts that stained positive increased in the surface region of the functionalis between Days 18 and 32 PO, and the staining extended throughout the upper functionalis region. There was a decrease in the number of positively stained stromal fibroblasts, particularly at the implantation site, between Days 32 and 40 of pregnancy. By Days 50-60 of pregnancy, this staining was almost absent. The induction of aSMA instromal fibroblasts in the functionalis region inpregnant baboons was confirmed by immunoblot analysis of stromal cell cytosol extracts. We conclude that the progesterone-induced glandular expression of SMM II may be involved in uterine secretory function and that aSMA expression in stromal fibroblasts during pregnancy and after long-term steroid treatment is associated with the decidualization process.

INTRODUCTION

mone regulating the appearance and function of the endometrium during the luteal phase [1]. The stroma is less compact, and an increase in proliferation is apparent. The glandular epithelium exhibits decreased mitosis and differentiates into a columnar epithelium. The glands become coiled and their lumens become dilated with glycogen [1]. In addition, progesterone induces the production of two specific proteins-insulin-like growth factor binding protein-1 (IGFBP-1) [2] and retinol-binding protein (RBP) [3]in the glands of the baboon endometrium. These progesterone-induced proteins have been proposed to be important factors in fetal/maternal interactions during implantation, possibly regulating trophoblast growth, invasion, and differentiation [4]. There is an increase in the production of IGFBP-1 and RBP in the endometrial glands of the baboon during early pregnancy [4]. However, the production of these glandular proteins decreases as the glands regress by the end of the first third of pregnancy [5]. During early pregnancy the stroma undergoes decidualization, a process best described

Dramatic morphological and physiological changes occur in the endometrium during the primate menstrual cycle. During the follicular phase, the appearance of the glandular epithelial cells changes from cuboidal to more pseudostratified while the stroma is composed of compact cells with very little cytoplasm [1]. Estrogen induces mitosis in the glands and regulates the morphological appearance of the endometrium. Progesterone becomes the dominant horAccepted April 23, 1995. Received January 26, 1995. 'Supported by NIH grants HD 21991 and HD 29964 (A.T.F.) and HL 35808 and HL 02411 (P. de L.). ZCorrespondence: Asgi T. Fazleabas, Ph.D., The University of Illinois at Chicago, Department of Obstetrics and Gynecology, 820 South Wood Street (M/C 808), Chicago, IL 60612-7313. FAX: (312) 996-4238. 3 Current address: Department of Obstetrics and Gynecology, Merit Care Medical Group, 737 Broadway Street, Fargo, ND 58123. 4Current address: Department of Obstetrics and Gynecology, University of Nottingham, Nottingham NG7 2UH, UK. 5Current address: Bioqual Inc., 9600 Medical Center Drive, Rockville, MD 20850.

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CYTOSKELETAL PROTEINS IN THE BABOON UTERUS

as a change in cell shape from that of a spindle-shaped fibroblast to that of a rounded epithelial-like cell with increased cytoplasm [6]. In addition to the morphological change, these decidualized cells synthesize specific proteins. In particular there is an induction of IGFBP-1 and prolactin [4, 7, 8]. Decidualization is complete by the end of the first third of pregnancy in the baboon [5]. The role of cytoskeletal proteins in cell function has become increasingly evident. The cytoskeletal proteins are important not only for mitosis, cell growth, and changes in cell shape, but also for the regulation of protein secretion [9]. The major elements of the cytoskeleton network are microtubules, microfilaments, intermediate filaments, and the microtubular lattice. Important filamentous proteins involved in the cytoskeleton are actin and myosin [9]. We hypothesized that the actin and myosin cytoskeletal network is intimately involved in the dynamic morphological and functional changes that occur in the endometrium. Since these events are hormonally regulated, we also postulated that the cytoskeletal proteins are regulated by steroid hormones. Therefore, the focus of these studies was to determine changes in a smooth muscle actin (aSMA) and smooth muscle myosin II (SMM II) expression in the baboon endometrium during the menstrual cycle and pregnancy and to confirm their hormonal regulation in steroid-treated baboons.

TREATMENT GROUPS Group 1- Cycling M I

Tissue Collection Uterine tissue was obtained at laparotomy from adult female baboons (Papio anubis). All procedures were approved by the Animal Care Committee of the University of Illinois at Chicago. The baboons used in this study were divided into four major groups: 1) cycling, 2) ovariectomized, steroid-treated, 3) pregnant, and 4) simulated-pregnant baboons (Fig. 1). Animals in group 2 were used to confirm whether changes observed during the menstrual cycle (group 1) were hormonally modulated. Animals in group 4 were utilized to determine whether or not changes observed during pregnancy (group 3) required the presence of a conceptus. Group 1. Tissue samples were obtained during the early and late follicular and mid- to late luteal phases of the menstrual cycle [10]. The follicular phase was identified on the basis of menstrual records and confirmed by measurement of peripheral serum levels of estrogen. For luteal phase tissue, ovulation was assumed to occur 2 days after the estradiol surge [11], and tissue was obtained between Days 5 and 10 postovulation (PO) (midluteal) and Days 12 and 14 PO (late luteal). A minimum of three baboons were analyzed at each stage of the cycle, and peripheral serum levels of estrogen and progesterone on the day of tissue collection were determined.

LF

ML

+ fI III~~~~--I I I I 8 12 14 post-menses

LL

f

f 10 12 14

s

post-ovulation

Days Group 2 - Steroid treated ovx

E2 implants

E2 + P implants

4,4

,

I

I

-60

0

I 7d E 2

4 I 14dE 2

I 7d E 2 +P

4d E 1 2 +P

Days of Treatment Group 3 - Pregnant

~I

-I Ovulationand

i'

I 18

14

32

25

I

I 40

I 50

60

Days Post-ovulation

mating

Group 4 - Simulated pregnant E2+P implants 6

16

21

+

+

+ + +

I

..

Ovulation

18

I

MATERIALS AND METHODS

EF

24

25

27

32

Days Post-ovulation FIG. 1. Diagram illustrating treatment groups and sampling time points; n = 15 (group 1), n = 14 (group 2), n = 12 (group 3), n = 12 (group 4). The days indicated below the lines are the times at which tissue was obtained. The arrows and days above the lines in groups 2 and 4 represent the times at which steroid-containing silastic implants were inserted s.c. and the period of hCG treatment. M = menses; EF = early follicular; LF = late follicular; ML = mid-luteal; LL = late luteal, ovx = ovariectomy.

Group 2. Ovariectomized baboons were treated for 7 (n = 2) or 14 (n = 5) days with estradiol (E2) alone (2 X 6-cm silastic implants (Dow-Corning, Midland, MI) [12]) or for 14 days with E2 followed by 7 (n = 3) or 14 (n = 4) days of E2 plus progesterone (E2 + P; 1 X 6-cm E2 implants, and 3 X 6-cm P implants, Dow-Corning [12]). The mean peripheral serum steroid levels in these animals were 57.8 pg/ml E2 and 0.12 ng/ml P (14-day E2-treated), 20 pg/ml E2 and 6.7 ng/ml P (7-day E2 + P-treated), and 17.5 pg/ml E2 and 5.4 ng/ml P (14-day E2 + P-treated). These levels are comparable to our previously published data [12]. Group 3. Mature cycling baboons were mated with fertile males during the periovulatory period as determined by sex skin tumescence. Uterine samples were obtained on Days 14-18 (n = 3), 22-32 (n = 5), and 40-60 (n = 8) of pregnancy [5]. The stage of pregnancy was confirmed by ultrasound and circulating levels of chorionic gonadotropin, E2, and P [13, 14].

600

CHRISTENSEN ET AL.

Group 4. Normal cycling baboons received increasing dosages of hCG (Profasi; Serono Laboratories, Inc., Norwell, MA) twice daily for 10 days beginning on Day 6 PO. Tissues were obtained on Day 18 PO (n = 3) from baboons receiving 2 additional days of hCG injections. Since the nonpregnant CL becomes refractory to hCG after 10 days of treatment [15], the animals were given steroids via s.c. silastic implants (Dow-Corning) [12] to achieve peripheral levels comparable to those of pregnancy [14]. Endometrium was obtained on Day 25 (hCG and E2 + P, n = 4), and Day 32 PO (hCG and E2 + P, n = 3). A control group of baboons were treated with E2 and P implants only, and endometrial tissues were obtained on Day 25 PO (E2 + P only, n = 2). Immunocytochemistry Tissue was immersion-fixed in Bouin's solution, dehydrated in ethanol, cleared in xylene, and embedded in paraffin [16]. Tissues obtained from pregnant baboons were divided into those from implantation and nonimplantation sites prior to fixation. Sections (6 jim) were cut on a rotary microtome and placed on acid-cleaned glass slides coated with 0.1% poly-L-lysine. SMM II was localized by means of a rabbit polyclonal antibody generated against SMM II purified from tracheal smooth muscle [17]. The antibody was affinity-purified through use of a tracheal SMM II heavy chain-Sepharose 4B column (Pharmacia, Uppsala, Sweden). The purified antibody was used at a dilution of 1:1000. The aSMA was localized through use of a monoclonal antibody (Dako Corp., Capenteria, CA) at a dilution of 1:1000. Immunoreactive product was visualized through use of ABC Vectastain kits (Vector Laboratories, Inc., Burlingame, CA) as previously described [18]. Negative controls consisted of preimmune rabbit serum and mouse ascites fluid (Sigma, St. Louis, MO). The specificity of the stain for SMM II and aSMA was confirmed by incubating adjacent sections with an antibody generated against purified nonmuscle myosin II [19] at a 1:60 dilution and an antibody that cross-reacts with all actin isoforms (Sigma) at a 1:50 dilution. To determine whether IGFBP-1 (a decidual cell marker) and aSMA were colocalized in the same cells, tissue sections were double-stained through use of Vectastain ABC and peroxidase substrate kits. IGFBP-1 was immunolocalized with a specific monoclonal antibody (C4H11 [2]) that was reacted with diaminobenzidine tetrahydrochloride as the chromogen. Sections were then incubated with antibody against aSMA and reacted with the chromogen VIP (Vector Laboratories). With this procedure, cells containing IGFBP1 appear brown whereas cells containing aSMA are purple. The distribution of positively stained cells within the uterine endometrium in each of the treatment groups was microscopically evaluated by two independent observers. The presence or absence of staining in various cell types throughout the entire section was determined.

Extraction of Protein Endometrial and myometrial tissues obtained from cycling (group 1) and steroid-treated (group 2) baboons were frozen in liquid nitrogen. Protein was prepared according to the method of Hofig and colleagues [20]. Briefly, tissues were homogenized with a tissuemizer (Tissue Tek, Cincinnati, OH) in 50 mM Tris buffer, pH 7.4, containing 0.1 mM PMSF and 0.25 M sucrose. The homogenate was centrifuged at 15 000 X g for 20 min. The supernatant was transferred to a clean ultracentrifuge tube, centrifuged at 100 000 X g 0 for 90 min, and aliquoted and stored at - 70 C until assayed. Protein content was determined by the method of Lowry et al. [21]. To confirm the presence of SMM II and aSMA in the two major cell types of the endometrium, glandular fragments and stromal fibroblasts were isolated from baboons during the menstrual cycle (Day 10 PO) and early pregnancy (Days 22-25). The isolation procedure was essentially as described by Osteen et al. [22] with minor modifications. Briefly, minced endometrial tissue was digested at 37°C for 1 h with 0.75% collagenase (Worthington Biochemicals, Freehold, NJ), 0.02% DNase (Boehringer-Mannheim, India+napolis, IN), and 2% chicken serum (Sigma) in Ca+Mg free Hanks' buffered salt solution (HBSS; dispersion medium) with repeated pipetting. After filtration through an 88-Lgm filter (Millipore, Bedford, MA), the retentate was further digested for 15 min at 37°C with dispersion medium plus 0.1% pronase (Boehringer-Mannheim) and 0.1% hyaluronidase (Sigma) and then filtered (88-pm filter). The retentate containing the glandular fragments was frozen in liquid nitrogen and stored at -70°C. After centrifugation (200 X g, 5 min) of the filtrate, the stromal cell digest was resuspended in 0.8% ammonium chloride in HBSS and incubated for 5 min at 37°C to lyse the red blood cells. The stromal cells were then filtered through a 20-plm filter, and the filtrate was layered onto a 30-60% Percoll gradient. After centrifugation (670 X g, 30 min), stromal cells with > 95% purity were recovered at the 30% Percoll interface and frozen. For protein extraction, the isolated glandular fragments and stromal cells were homogenized with a Dounce

FIG. 2. Localization of SMM II in the endometrium of baboons. Staining for SMM II was absent in glands and stroma of endometrium in ovariectomized (A)and E2treated baboons (B). Glandular staining was evident in the endometrium during the mid-luteal stage and following 7 days of E2 +P treatment (C). The staining intensity was greatest in the glands of the basalis (C)as compared to the functionalis. However, strong staining was present in the superficial glands and luminal epithelium on Day 8 PO (D). The number of glands staining positive for SMM II decreased during early pregnancy (E,Day 25 PO), and glandular staining was absent by Day 60 of pregnancy (F). SMM IIstaining was present in stromal fibroblasts only on Day 25 PO following E2+P treatment (G)and in the nonimplantation site of pregnant baboons at Days 25 and 60 PO (I and J, respectively). In contrast, specific staining for SMM II was not apparent in the stromal fibroblasts at the implantation site at Day 25 PO (E). H = negative control of G. e = luminal epithelium; g = gland; s = stroma; v = blood vessels. A-D and F-J, bar = 10 pm. E, bar = 100 pm.


601

602

CHRISTENSEN ET AL. TABLE 1. Summary of SMM type II localization inthe baboon uterus.* Pregnancyb Days 18-32 PO

Hormone Treatmentsa Cell type

Ovx

E2

E2 + P

IS

Days 40-60 PO

Simulated Pregnancy

NIS

IS

NIS

Days 18-32 PO

-

+

Luminal epithelium

0

0

+

-

+

-

Glandular epithelium (functionalis)

0

0

+

+

+

0

O

+

Glandular epithelium (basalis)

0

0

+

+

0

0

+

Stroma (upper functionalis)

0

0

0

0

0

+

+

+

aChanges during the follicular and luteal phase of the menstrual cycle (group 1)are similar to those in the E2 -treated and E2 +Ptreated animals (group 2). blS = implantation site, NIS = nonimplantation site. *Smooth muscle cells always stained positive and stromal cells in the basalis always stained negative in all treatment groups; - = no luminal epithelium present, + = many positive cells, + = some positive and negative cells, 0 = no positive stain.

homogenizer (Fisher Scientific, Itasca, IL). Cellular protein was extracted as described above. Immunoblotting Extracted protein from endometrium, myometrium, and isolated glandular fragments and stromal fibroblasts was separated by one-dimensional SDS-PAGE under reducing conditions on a 5-20% gradient or 12.5% acrylamide gels. The proteins were then transferred to nitrocellulose membranes as described by Towbin et al. [23]. After electroblotting, the blots were incubated with the antibody against either SMM II (0.4 pg/ml), non-muscle myosin II (0.4 jg/ml), or aSMA (1:1000) overnight at room temperature. The immunoreactive products in cytosol extracts of whole tissues were detected with an alkaline phosphatase-conjugated second antibody (Bio-Rad Laboratories, Richmond, CA). To detect SMM II, non-muscle myosin II, and aSMA in cytosol extracts of isolated glands and stromal fibroblasts, the immunoreactive products were visualized by the more sensitive Enhanced Chemiluminescence procedure (ECL; Amersham, Arlington Heights, IL). Molecular weight standards were visualized with Ponceau red stain. RESULTS Animals in groups 2 (ovariectomized, steroid-treated) and 4 (simulated-pregnant) were treated with hormones such that the peripheral serum levels were comparable to those of cycling (group 1) and early-pregnant (group 3) baboons, respectively. Therefore, staining patterns were compared between groups 1 and 2 and between groups 3 and 4. SMM II

Specific immunocytochemical stain for SMM II was present in smooth muscle cells of the myometrium and vascular smooth muscle of all blood vessels, including the spiral arteries, in all treatment groups. No staining was present

when the primary antibody was replaced with normal rabbit serum (Fig. 2H). The pattern of staining for SMM II changed only in the endometrium during different reproductive stages (see Table 1 for summary). In the absence of hormones (ovariectomy, Fig. 2A), in follicular phase, and in ovariectomized E2-treated baboons (Fig. 2B), specific stain for SMM II was absent in the stroma, glands, and luminal epithelium. In contrast, in the luteal phase and after E, + P treatment of ovariectomized animals (Fig. 2C), staining for SMM II was present in the glandular epithelium. The staining intensity was greater in the glands of the basalis (Fig. 2C) than in those in the functionalis (data not shown). The number of glands staining positive for SMM II was greater in endometrium from baboons on Days 5-8 PO and following 7 days of E2 + P treatment; this value decreased by Days 12-14 PO and following 14 days of E2 + P treatment. In addition, staining of the luminal epithelium was primarily evident on Day 8 PO (Fig. 2D) and in the baboons treated with E2 + P for 7 days. There was variability in the number of SMM II-positive glands between Days 18 and 32 of pregnancy and simulated pregnancy (see Table 1 for summary). Some glands were positive for SMM II (Fig. 2E) while others were negative. In addition, some cells in a gland stained positive while others in the same gland were negative. Staining for SMM II was absent in the glands between Days 40 and 60 of pregnancy (Fig. 2F). Stromal staining for SMM II was present in the functionalis just under the luminal epithelium during Days 18-32 of simulated pregnancy, at Day 25 of E2 + P treatment in the simulated-pregnant control animals (Fig. 2G), and in nonimplantation sites (Fig. 2, I and J) during pregnancy. This staining of the stroma persisted through the first third of pregnancy (Day 60 PO, Fig. 2J). However, stromal stain for SMM II was absent at the implantation site in pregnant animals (Fig. 2E). An antibody specific for non-muscle myosin II lightly stained the glandular epithelium and smooth muscle cells (data not shown). However, this stain did not vary between

603


the different treatment groups, suggesting that the staining observed for SMM II was not attributable to cross-reactivity with non-muscle myosin II. A band corresponding to the molecular weight of SMM II (-200 000; Fig. 3A) and non-muscle myosin II (-200 000; Fig. 3B) was present in the protein extracts of endometrium and myometrium in all treatment groups. The amount of SMM II in myometrial extracts did not differ between hormone treatments (Fig. 3A; lanes 3-5); however, SMM II levels were lower in endometrium from a baboon treated with E2 + P for 14 days than in the myometrium (Fig. 3A; lane 2). The immunoreactive product present in endometrial extracts from the follicular stage (Fig. 3A; lane 1) probably represents SMM II present in the smooth muscle cells of the blood vessels (Fig. 2, A and D). SMM II was detected in protein extracted from isolated endometrial glands obtained at Day 10 PO (mid-luteal; Fig. 3C, lane 2). The levels of SMM II in endometrial glands were higher during the mid-luteal phase than in glands from pregnant animals (Fig. 3C; lanes 2 and 3); this correlates with the immunocytochemical data. In contrast, the amount of non-muscle myosin II in the endometrial and myometrial (Fig. 3B) extracts did not differ between the treatment groups.

FIG. 3. Immunoblot of cytosolic protein extracts from myometrium, endometrium, and isolated endometrial glands stained for SMM II(Aand C)and non-muscle myosin II(Band D). A strong band at M,200 000, corresponding to SMM II,was present in myometrial extracts from ovariectomized, E2 -treated, and 7-day-E 2 +P-treated baboons (A, lanes 3, 4, and 5, respectively; 25 gg protein). SMM II was also detected in protein extracts from the endometrium of follicular and 14-day-E2 + P-treated baboons (A, lanes 1 and 2, respectively; 50 pg protein). A band at M,r200 000, corresponding to non-muscle myosin II,was also present in the same samples (lanes 15 in Bare the same as in A). No difference in the levels of non-muscle myosin II was apparent between the treatment groups. The amount of SMM II in glandular extracts was higher during the mid-luteal phase (C,lane 2; Day 10 PO, 25 g protein) than during early pregnancy (C; lane 3; Day 22 PO, 25 g protein). In contrast, the levels of non-muscle myosin IIdid not differ between these same two groups (lanes 2-3 in D are the same as in C; lane 1 in both panels contain 5 pg of myometrial extract as control). A and Bwere detected through use of the alkaline phosphatase procedure, whereas Cand Dwere detected by ECL.

Alpha SMA Specific immunocytochemical staining for aSMA was absent in both the endometrium and myometrium (Fig. 4A) in ovariectomized baboons. However, during the follicular phase and following E2 treatment of ovariectomized animals (Fig. 4B), intense staining for aSMA was evident in smooth muscle cells of the myometrium and blood vessels, including arteries, arterioles, and veins. This stain was not detected in an adjacent section in which the primary antibody was replaced with mouse ascites fluid (Fig. 4B, inset). Alpha SMA was present in smooth muscle cells of the myometrium and blood vessels in all treatment groups represented in this

study with the exception of tissues obtained from untreated, ovariectomized animals (Figs. 4 and 5; see Table 2 for summary). Alpha SMA was not detected in epithelial cells of the endometrium at any reproductive stage or in any treatment group included in this study (Figs. 4 and 5; Table 2).

TABLE 2. Summary of aSMA localization in the baboon uterus.* Pregnancyb Hormone Treatmentsa Cell type

Days 18-32 PO

Days 40-60 PO

Simulated Pregnancy

Ovx

E2

E2 + P

IS

NIS

IS

NIS

Days 18-32 PO

SMCC

0

+

+

+

+

+

+

+

Stroma (upper functionalis)

0

0

0

+

+

0

0

+

Stroma (lower functionalis)

0

0

0

+

±0

+

+

Stroma (basalis)

0

0

0

0

0

+

0

+

aChanges during the follicular and luteal phases of the menstrual cycle (group 1)are similar to those in the E -treated and E +P2 2 treated animals (group 2). blS = implantation site, NIS = nonimplantation site. CSMC = Smooth muscle cells of the myometrium and blood vessels. *Luminal and glandular epithelium stained negative for aSMA inall treatment groups; + = many positive cells, + = some positive and negative cells, 0 = no positive stain.

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CHRISTENSEN ET AL.

FIG. 4. Immunocytochemical localization of aSMA in the baboon uterus. Specific staining for aSMA was absent in myometrium from an ovariectomized baboon (A) but was present in the smooth muscle cells of the myometrium from an estrogen-treated baboon (B; inset = negative control). Alpha SMA was present only in the smooth muscle cells of blood vessels in endometrium from a 7-day-E2+ P-treated baboon (C). Stromal fibroblasts showed positive staining for ctSMA at the implantation (D)and nonimplantation (E)sites of pregnant baboons at Day 25 PO. In all simulated-pregnancy groups, stromal fibroblasts also displayed positive staining for aSMA (F). cts = cytotrophoblastic shell; e = luminal epithelium; g = gland; s = stroma; v = blood vessels. A-C and E-F, bar = 10 gm. D, bar = 100 g.m.


605

FIG. 5. Colocalization of IGFBP-1 and aSMA protein in decidualizing endometrium of pregnancy. IGFBP-1 (brown stain) was detected in the decidualized stromal fibroblasts in the upper functionalis region of the nonimplantation site at Day 50 of pregnancy (A). Alpha SMA (purple stain) was detected in adjacent stromal fibroblasts in this one baboon. A clearly demonstrates that these two proteins are not expressed simultaneously in the same cell. IGFBP-1 stain was also present in decidualized stromal fibroblasts surrounding blood vessels (B). Stain for IGFBP-1 was present in the decidualized stroma directly under the luminal epithelium of the nonimplantation site (C), whereas staining for aSMA appeared only in the smooth muscle cells of blood vessels and the stromal fibroblasts of the basalis (D) of this baboon at Day 50 of pregnancy. The staining pattern observed in C and D was more typical of pregnant baboon endometrium. e = luminal epithelium; g = gland; s = stroma; v = blood vessels. A-D, x360; bar = 10p m.

Alpha SMA was absent in stromal fibroblasts during the menstrual cycle and after steroid treatment. It was first observed in the stromal fibroblasts below the luminal epithelium of the functionalis on Day 14 PO in a pregnant baboon (data not shown). The presence of aSMA was associated with an epithelioid or rounded appearance of the cells. As pregnancy proceeded, the number of aSMA-positive stromal fibroblasts increased between Days 18 and 32, with staining apparent throughout the functionalis of implantation and nonimplantation site endometrium (Fig. 4E; Table 2). There was a decrease in the number of aSMA-poSitive stromal fibroblasts in the functionalis, particularly at the implantation site and below the luminal epithelium at the non-

implantation site, by Day 40 of pregnancy. However, positive staining for aSMA was present in the stroma of the basalis region. At Days 50 and 60 of pregnancy, stromal staining for ctSMA was virtually absent with only a few stromal fibroblasts staining positive in the basalis (Fig. 5, C and D). Stromal staining for aSMA was also detected below the luminal epithelium by Day 18 PO in simulated-pregnant baboons and by Day 25 PO in the E2 + P-treated simulatedpregnant controls (Fig. 4F; see Table 2 for summary). The number of aSMA-positive stromal fibroblasts increased throughout the functionalis in the later stages of simulated pregnancy; however, stromal staining was absent in the bas-

606

CHRISTENSEN ET AL.

from the nonimplantation site of pregnant baboons were analyzed, aSMA was readily detectable (Fig. 6B; lanes 4 and 5). Alpha SMA was absent in isolated stromal fibroblasts from a nonpregnant baboon (Fig. 6B, lane 3), thus confirming the immunocytochemical data. DISCUSSION

FIG. 6. Immunoblot of protein extracts from myometrium, endometrium (A), and isolated stromal fibroblasts (B)stained for aSMA. A distinct band at M, 44 000, corresponding to aSMA, was present in myometrial extracts from E2-treated and 7day-E, + P-treated baboons (A, lanes 1 and 2,respectively; 25 pg protein). The aSMA detected in protein extracts from the endometrium of follicular and 14-day-E2+Ptreated baboons presumably represents contribution from the smooth muscle cells of the blood vessels (A,lanes 3 and 4, respectively; 50 g protein). Alpha SMA was detected in cytosolic protein extracts from isolated stromal fibroblasts obtained from the nonimplantation site of Day 25 PO pregnant baboons (B,lanes 4 and 5; 50 pgprotein), but not in isolated stromal fibroblasts from the luteal phase of the menstrual cycle (B, lane 3; 50 pg protein). Lanes 1 and 2 in B are 5 and 10 g, respectively, of myometrial and nonpregnant endometrial cytosolic extracts. The alkaline phosphatase procedure was used in A,whereas ECL was used in B.

alis. Unlike the situation for pregnancy, staining for aSMA persisted in the functionalis throughout simulated pregnancy and in the controls treated with steroid only. An antibody that recognizes the various other isoforms of actin stained the smooth muscle cells of the myometrium and blood vessels (data not shown). No staining of stromal fibroblasts or epithelium was apparent (data not shown). This suggests that the stain for aSMA observed in stromal fibroblasts was not attributable to cross-reactivity with other actin isoforms. Colocalization of IGFBP-1 (a protein produced by fully differentiated decidual cells) and aSMA demonstrated that these two proteins are not expressed in the same endometrial cell (Fig. 5). As pregnancy proceeded, staining for IGFBP-1 became more evident, particularly around spiral arteries (Fig. 5B), at the implantation site, and in the functionalis at the nonimplantation site (Fig. 5, A and C). In contrast, aSMA staining was absent in the functionalis by Day 50 of pregnancy (Fig. 5C) except in one baboon (Fig. 5A). Staining for aSMA was typically present only in the basalis (Fig. 5D) by the end of the first third of pregnancy. A band corresponding to the molecular weight of aSMA (-44 000) was present in the protein extracts of myometrium from ovariectomized, E2-treated, or E2 + P-treated baboons (Fig. 6A; lanes 1 and 2). Less aSMA was present in endometrial protein extracts obtained from nonpregnant baboons (Fig. 6A: lanes 3 and 4) and presumably represents aSMA present in smooth muscle cells of the uterine blood vessels (Fig. 4C). In addition, the hormonal treatment or stage of the cycle did not affect the amount of aSMA present in these whole endometrial or myometrial extracts. In contrast, when cytosol extracts from isolated stromal fibroblasts

This is the first report to correlate the localization of SMM II and aSMA with glandular and stromal differentiation in the primate uterus during the menstrual cycle, during pregnancy, and after hormonal treatments. Our data suggest that steroid hormones are involved in the regulation of SMM II and aSMA in specific cell types and that these cytoskeletal proteins may be important in cellular differentiation and regulation of protein synthesis. Besides showing the constitutive expression of SMM II in smooth muscle cells of the myometrium and endometrium, these data demonstrate the presence of this cytoskeletal protein in endometrial glands during the luteal phase of the menstrual cycle and early pregnancy (Days 18-32 PO). Expression of SMM II in glandular epithelium coincided with the increased secretory activity of these cells [4, 101. In contrast, aSMA was first detected in stromal fibroblasts during pregnancy and after hormonal treatments in simulated-pregnant baboons. However, expression of aSMA in stromal fibroblasts was transient during early pregnancy. The expression of aSMA in stromal cells was associated with changes in cell shape and preceded the induction of IGFBP-1, a marker of a fully differentiated decidual cell [2], suggesting that aSMA may be associated with the initial transformation of a stromal fibroblast to a decidual cell. The expression of SMM II in the glandular epithelium during the luteal phase correlates with the progestational epithelial response [1] and an increased synthesis of specific proteins particularly in the basal glands [4, 10]. A decrease in SMM II is associated with glandular regression and decreased protein production during both the late luteal phase and pregnancy (Days 32-60 of pregnancy [1, 5]). The variability in glandular SMM II staining during pregnancy may be reflective of this process. We hypothesize that the loss of cytoskeletal elements disrupts cellular metabolism and results in glandular regression. SMM II expression in glandular epithelium during the luteal phase also correlates with development of extensive elongated microvilli in these cells of the baboon (Verhage and Fazleabas, unpublished results) and human endometrium [24]. Since myosin microfilaments are important for microvilli formation in other epithelial cells [25-27], we suggest that SMM II may be involved in the development of microvilli in the epithelium of the endometrium. SMM II was also localized in the luminal epithelium during the mid-luteal stage, a period that coincides with uterine receptivity. Prior to implantation, the shape of microvilli of

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the luminal epithelium changes from long, thin, and regular, and the microvilli become irregular projections in the rat uterus [25-27]. This unique surface transformation appears to be critical for successful implantation in this species [28]. Thus, regulation of SMM II in the endometrial epithelium may play a critical role not only in the differentiation of the progestational cell type, but also in blastocyst attachment and implantation. Although SMM II does not appear to be hormonally regulated in smooth muscle cells of the myometrium and blood vessels, its expression in endometrial epithelium was markedly up-regulated by short-term progesterone exposure (78 days). However, longer exposure of the endometrium to progesterone resulted in decreased epithelial SMM II. Expression of SMM II in the epithelium corresponds to the expression of progesterone receptor in these cell types [1, 29]. Thus, progesterone regulation of epithelial SMM II expression occurs presumably via a receptor-mediated mechanism. The increasing levels of estrogen and progesterone associated with pregnancy, or in simulated-pregnant animals, induced SMM II expression in stromal cells below the luminal epithelium. We hypothesize that the transient expression of aSMA in stromal fibroblasts during pregnancy represents cytoskeletal changes involved in decidualization. Changes in the actin cytoskeleton or in actin-binding proteins appear to be important for cellular differentiation of granulosa cells [301, chondrocytes [31], and HL-60 leukemia cells [32]. These cytoskeletal changes not only are important for the cell phenotype, but also play a role in biochemical processes such as coordination of cellular organelles involved in protein synthesis and steroidogenesis. Tsukada and colleagues [33] described a particular aSMA-positive fibroblast cell present in various tumors. This "myofibroblast" contained well-developed cellular organelles associated with protein synthesis [331 and has been detected in epithelial hyperplasia and invasive tumors of the breast [34, 35], cervix [36], and ovary [37]. These myofibroblasts produce increased amounts of extracellular matrix including collagen types I, III, and V, fibronectin, vimentin, and oncofetal fibronectin [34]. In addition, reorganization of the actin network results in a loss of chondroitin sulfate proteoglycan and type II collagen gene expression and an increase in type I collagen production in differentiating chondrocytes [31]. Ronnov-Jessen and Pettersen [38] hypothesized that the presence of aSMA-positive myofibroblasts in breast tumors is involved in tissue remodeling. We postulate that the initial changes in decidualization involve the induction of aSMA and that expression of aSMA is important for cellular transformation, including changes in cell shape and protein synthetic capacity. In contrast to SMM II, aSMA appears to be regulated by steroid hormones in the smooth muscle cells of the myometrium and blood vessels. Our data suggest that estrogen

up-regulates the expression of this protein in uterine smooth muscle cells. This agrees with previous findings in the rat uterus [39, 40]. The presence of aSMA corresponds with the localization of estrogen receptors in the smooth muscle cells of the uterus and the continued presence of these receptors in smooth muscle cells throughout the menstrual cycle and early pregnancy [29]. This supports the hypothesis that estrogen regulates aSMA expression via a receptor-mediated mechanism. A study by Cicatello and colleagues [39] suggests that estrogen regulation may require the synthesis of an intermediate protein. A potential candidate is TGFO,, which stimulates aSMA mRNA and protein in breast myofibroblasts [38]. Our data also suggest that the induction of aSMA in stromal fibroblasts is regulated by long-term and increasing levels of estrogen and progesterone associated with pregnancy. During pregnancy, aSMA expression was transitory, exhibiting a decrease first at the implantation site and subsequently in the nonimplantation site. In contrast, aSMA was not down-regulated in the endometrium from hormone-treated baboons. These data imply that the implanting trophoblast down-regulates the expression of aSMA in endometrial stromal fibroblasts and support our hypothesis that the baboon conceptus plays an obligatory role in the decidualization process [4, 8]. In summary, this study is the first to demonstrate the regulation of two cytoskeletal proteins, SMM II and aSMA, in the primate endometrium. These two cytoskeletal proteins are associated with endometrial cellular differentiation and the synthesis of specific proteins. SMM II may be important for the development of secretory epithelial cellG and for the formation of microvilli, whereas aSMA appears to be important for decidual transformation and the production of specific proteins. ACKNOWLEDGMENTS These studies were done as part of the National Cooperative Program for Markers of Uterine Receptivity for Non-human Blastocyst Implantation and were supported by Cooperative Agreement #HD 29964. We would also like to acknowledge the surgical assistance of Dr. Jeffrey Fortman and the secretarial skills of Ms. Margarita Guerrero. Primal de Lanerolle is the Florence and Arthur Brock Established Investigator of the Chicago Lung Association.

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