Menstrual cycle dependent expression of CD44 in normal human endometrium

Menstrual Cycle Dependent Expression of CD44 in Normal Human Endometrium NOBUO YAEGASHI, MD, NOBUHIRO FUJITA, MD, AKIRA YAJtMA, MD, AND MASATAKA NAKAMURA, PHD CD44 is a cell surface glycoprotein known to be a hyaluronate receptor, which functions in lymphocyte homing and cancer metastasis. We have previously shown that CD44 molecules are expressed in normal endometrial tissue. In this study we immunohistochemically examined expression of CD44 glycoproteins in normal human endometrial tissue to gain insight into roles of CD44 molecules. No expression of the CD44 standard form was observed with 11 endometria in the proliferative phase. On the conlrary, 13 of 19 secretory phase endometria were stained intensely by anti-CD44 standard form antibody; all CD44-positive cases were in the mid and late secretory phases. Among the 13 CD44-positive endometria, four also were positive for the CD44 variant with exon v6. CD44 molecules were present at all but the luminal cell surfaces. These results indicate that expression of CD44 depends on menstrual cycle, suggesting that CD44 molecules might be involved in implantation of fertilized ovum in endometrinm. HUM PATHOL 26:862--865. Copyright © 1995 by W.B. Saunders Company Key words: CD44, endomeWinm, menslruation. Abbreviations: PFA, paraformaldehyde; PBS, phosphate-buffered saline; DAB, diaminobenzidine. CD44 is postulated to function t h r o u g h cell-cell and cell-matrix interaction, such as binding to hyaluronate and lymphocyte homing, l'2 and is widely expressed n o t only on lymphocytes but also o n many types of epithelium. ~5 Physiological roles of CD44 on epithelium, however, are as yet not well d o c u m e n t e d . CD44 is a glycoprotein with polymorphism, which is mainly generated by alternative splicing; the CD44 gene has 20 exons, of which 10 (exon vl to exon vl0) at the middle are alternatively spliced to p r o d u c e variable isoforms carrying different inserts. 5~ The standard (hematopoietic) form does n o t include any variant exons. The CD44 molecules with variant inserts, especially the v6 exon, have been shown to be closely related to metastasis of cancer cells. In animal models this was shown by conversion of a nonmetastatic carcinoma cell line into a metastatic state by transfection with a variant form carrying the v6 exon, 9-1a and by inhibition of the ability o f the transfected cell to metastasize by using a m o n o c l o n a l antibody specific for the v6 variant region. 12'1~ Furthermore, clinical investigations have showed that alternatively spliced variants of the CD44 molecules are associated with iuvasive and metastatic potential of cancer cells as well as p o o r prognosis in several types o f carcinoma, such as in colon cancer 1cells, 3 ' 14-16 breast cancer cells, 14 ' 17 gastric cancer, 18 ' 19 and lung cancer, 2° supporting the above experimental resuits. Little is known about involvement of CD44 in the reproduction field. We have already demonstrated that endometrial tissue expresses variant CD44 forms as well as the standard form. 21 Additionally, CD44 expression in the h u m a n placenta has been reported. ~ These re- suits tempt us to speculate that CD44 may play some role in the implantation process. We report here the expression of CD44 glycoprotein, n o t only the standard form but also variant forms with the v6 exon, in normal h u m a n endometrial tissues. The CD44 expression was only seen in the mid to late secretory phases, and n o t in the proliferative phase of the menstrual cycle. From the Departments of Microbiology and Obstetrics and Gynecology, Tohoku University School of Medicine, Sendai, Japan. Accepted for publication November 28, 1994. Supported by a grant from the Ministry of Education of Japan and by a grant from the Ministry of Health and Welfare of Japan. Address correspondence and reprint requests to Masataka Natamura, PhD, Tohoku University School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-77, Japan. Copyright © 1995 by W.B. Saunders Company 0046-8177/95/2608-000755.00/0 862 MATERIALS AND METHODS Tissue Samples Normal endometria were obtained from surgically removed uteri diagnosed as myoma uteri; Tissues were fixed in 10% formalin (formalin solution, neutral buffered) or in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS), and embedded in paraffin. Tissues also were cryoprotected by incubation with 20% sucrose in PBS prior to embedding in OCT compound (Tissue Teck, Miles, IN) and frozen in liquid nitrogen until use. The manufacturer's instructions (Bender MedSystems, Austria) recommend the use of cryostat sections for staining procedures with monoclonal antibodies; however, no comment on samples prepared by other fixation methods was provided. We first examined samples fixed by three different methods: formalin-fixed, PFA-fixed, and freshfrozen cryostat sections. Paraformaldehyde-fixed sections showed immunohistochemical staining as fine as that on cryostat sections, whereas formalin-fixed sections gave poor stainingv(data not shown). Thus, we used PFA-fixed tissues instead of cryostat sections in this study. Immunohistochemical Staining Six-micron sections were pretreated in 3% hydrogen peroxide to block intrinsic peroxidase activity. Each slide was sequentially treated with normal goat serum, mouse monoclonal antibody against CD44, biotinylated rabbit antimouse IgG, and avidin-peroxidase. The peroxidase reaction was visualized in the presence of hydrogen peroxide by adding 3,3diaminobenzidine tetrahydrochloride (DAB). Mouse monoclonal antibodies (Bender MedSystems) against human CD44 were SFF-2 for an epitope in the CD44 standard form that recognizes both standard and variant forms, VFF-7 for an epitope encoded by exon v6, VFF-17 for an epitope encoded by exons v7 and v8, and VFF-14 for an epitope encoded by MENSTRUAL CYCLE AND CD44 IN ENDOMETRIUM (Yaegashi et al) exons v8 through vl0. The optimal dilution (1:50) of the antibodies was determined from serial dilutions of the antibodies by staining normal skin squamous epithelium, which is known to express CD44 intensely. 4'23 Mouse monoclonal antibody specific for human parvovirus B19 was used as a control. 24 Endometrial Dating Precise endometrial dating was determined according to the criteria of Hendrickson and Kempson. 25 The secretory phase of endometrium was divided into three subgroups: early (postovulatory day 1 to 5), mid (postovulatory day 6 to 7), and late (postovulatory day 9 to 14). Statistical Analysis Statistical analysis was performed according to Fisher's exact test. RESULTS Eleven sections f r o m 33 n o r m a l e n d o m e t r i a were f r o m the proliferative phase, 19 were f r o m the secretory phase, a n d three were f r o m the atrophic state. All were fixed in PFA a n d thin sections were prepared. T h e sections were treated with m o n o c l o n a l antibodies specific for CD44. No staining with SFF-2 against the standard f r o m of CD44 was observed on the sections f r o m the proliferative phase (Fig 1A) or atrophic state (data not shown). T h e o t h e r antibodies specific for the variant forms o f CD44 did not show any positive color on the same sections. These results show that e n d o m e t r i a in the proliferative phase do n o t express CD44 glycoproteins. O n the o t h e r hand, 13 sections f r o m the 19 secretory e n d o m e t r i a were strongly stained with SFF-2 (Fig 1B). T h e control antibody, PAR3, gave no staining signal, indicating that m o r e than two thirds of e n d o m e t r i a in the secretory phase express CD44. A difference in frequency between proliferative and secretory phases was highly significant ( P < .05) (Table 1). F u r t h e r m o r e , e n d o m e t r i a l dating analysis showed that the four early secretory e n d o m e t r i a were negative for CD44, but all four o f the mid secretory e n d o m e t r i a and almost all (nine of 11) of the late secretory e n d o m e t r i a were positive. Staining analysis of the 13 e n d o m e t r i a positive for CD44 with VFF-7 specific for v6 showed that four (two of the four mid secretory a n d two of the nine late secretory) expressed variant forms carrying the v6 exon (Fig 1C). No positive signal by staining with VFF-17 (antibody to v7 and v8) or VFF-14 (antibody to v8 t h r o u g h vl0) was seen with any of the specimens. These results are s u m m a r i z e d in Table 1. Intense signals for CD44 staining with SFF-2 were observed on the surface of e n d o m e t r i a l gland cells at the b a s e m e n t m e m b r a n e side a n d between the gland cells, and little or no signal was observed at the l u m e n side (Fig 1B). E n d o m e t r i a l stroma did not show appreciable signal for staining. T h e same results were observed by staining with VFF-7 (Fig 1C). 863 FIGURE 1, Immunohistochemical detection of CD44 in human endometrial tissue. (A) Normal human endometria in the proliferative phase were not stained with monoclonal antibody (SFF-2) for CD44 molecules. (B) Endometria in the secretory phase were stained with monoclonal antibodies to the standard form of CD44 and (C) to a variant exon, v6. The letters, G, S, and L on the figures represent gland epithelial cells, stroma, and lumens, respectively. The arrows indicate a monolayer of gland epithelial cells stained with monoclonal antibodies. HUMAN PATHOLOGY Volume 26, No. 8 (August 1995) TABLE 1. Summary of Immunohistochemical Staining of Human Endometrium With Anti-CD44 Antibodies Anti-CD44 SFF-2 VFF-7 VFF-17 Exon VFF-14 Endometrial Dating (phase) (No. o f specimens tested) Standard v6 v7 and v8 v8-vl0 Proliferative (n = 11) Secretory (n = 19) Early (n = 4) Mid (n = 4) Late (n = 11) Atrophic (n = 3) 0* 13" 0 4 9 0 0 4 0 2 2 0 0 0 0 0 0 0 0 0 0 0 0 0 NOTE: N u m b e r o f positively stained specimens were shown. * Fisher's exact test: P < .05. DISCUSSION Our study clearly illustrates that normal endometria in the secretory phase exclusively express not only the standard form of CD44 but also variant form(s) with the v6 exon, whereas e n d o m e t r i a in the proliferative phase and atrophic state do not. Thus, the results indicate that expression o f CD44 molecules is modulated during the menstrual cycle and presumably suggest a functional role of CD44 in the normal endometrium. Two groups, including ours, have reported CD44 expression in h u m a n endometrium, 4'21 but this study is the first r e p o r t describing the modulation of CD44 expression d e p e n d i n g on the menstrual cycle. As expression of molecules in e n d o m e t r i u m is highly influenced by sex steroids, such as estrogens and progesterone, CD44 expression may be modulated by these hormones. It has been r e p o r t e d that the CD44 p r o m o t e r is upregulated by the activated o n c o g e n e cHa-ras, which also influences splicing mRNA for CD44. 26 T h e ras activation is involved in carcinogenesis o f endometrial cancer. 27 It is thus of interest to see relationship between ras activation and CD44 expression in the endometrial gland cells. T h e ras induced activation of the CD44 p r o m o t e r is mediated by AP-126 and interestingly, expression o f the c-jun protooncogene, a c o m p o n e n t of AP-1, is inhibited by estrogen and induced by tamoxifen, an antiestrogen agent. 28"29 Furthermore, AP-1 and the steroid receptor superfamily (receptors for glucocorticoid, retinoic acid, thyroid hormone, and vitamin D) are shown to function antagonistically in their binding to e n h a n c e r elements or in direct protein-protein interaction. 3° Thus CD44 may be suppressed in the proliferative phase, during which secretion of sex steroid, estrogens, is dominant. We also detected variant form(s) o f CD44 in some endometria in the secretory phase. They expressed variant form(s), including the v6 exon but not the v7 to vl0 exons. T h e v6 exon is closely related to cell mobility, as is the case with cancer metastasis and lymphocyte homing. In this context it is interesting to note that expression of CD44 in endometrial gland cells is restricted to the cell surface in contact with other cells and matrix: the basement m e m b r a n e side and between 864 gland cells. Endometrial stroma in any of the phases was not stained with antibodies for CD44. Restrictions of location and exon in CD44 expression imply that CD44 molecules in endometrial gland cells may function in a monolayer three dimensional tissue constitution of the gland cells through adhesion to adjacent cells and matrix as well as in proliferation and differentiation during the menstrual cycle. T h e e n d o m e t r i u m in the mid to late secretory phase, exclusively expressing CD44, is functionally involved in the implantation of a fertilized ovum. 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