Wakayama Medical College, Wakayama, Japan

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1 FERTILITY AND STERILITY VOL. 74, NO. 2, AUGUST 2000 Copyright 2000 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. REPRODUCTIVE BIOLOGY Type VI collagen expression during growth of human ovarian follicles Masaaki Iwahashi, M.D., a Yasuteru Muragaki, M.D., b Akira Ooshima, M.D., b and Ryosuke Nakano, M.D. a Wakayama Medical College, Wakayama, Japan Objective: To identify type VI collagen expression in human ovarian follicles during follicular growth. Design: In vitro experiment. Setting: Department of Obstetrics and Gynecology, Wakayama Medical College, Japan. Patient(s): Regularly cycling women who underwent adnexectomy. Intervention(s): Immunohistochemistry and in situ hybridization for human type VI collagen. Main Outcome Measure(s): Expression of type VI collagen. Result(s): Expression of type VI collagen was observed in the theca cell layers during folliculogenesis, whereas no expression of type VI collagen was observed in the granulosa cell layers at the mrna and protein levels. As the follicles grew, immunostaining for type VI collagen became intense in the theca cell layers, especially the theca externa. In preovulatory follicles, however, weak, fragmented, or discontinuous immunostaining of the theca cell layers was observed. This fragmented or discontinuous immunostaining was evident predominantly in the apical area of preovulatory follicles rather than in the basal area. Conclusion(s): Type VI collagen is present in the theca cell layers of follicles during folliculogenesis and plays an important role in interactions between the theca cells and extracellular matrix. These interactions may lead to changes in the shape, proliferation, migration, or differentiation of follicular cells during follicular development, maturation, and ovulation. (Fertil Steril 2000;74: by American Society for Reproductive Medicine.) Key Words: Type VI collagen, ovarian follicle, immunohistochemistry, in situ hybridization Received October 1, 1999; revised and accepted March 7, Reprint requests: Ryosuke Nakano, M.D., Department of Obstetrics and Gynecology, Wakayama Medical College, Kimiidera, Wakayama , Japan (FAX: ; a Department of Obstetrics and Gynecology. b Department of Pathology /00/$20.00 PII S (00)00618-X Follicular development and ovulation are preceded by extensive remodeling of the ovarian extracellular matrix (1 4). This process is complicated and involves various proteinases such as collagenases, gelatinases, and plasminogen activators (5, 6). The extracellular matrix is considered to play an important role in stabilizing tissues and in regulating the growth and differentiation of cells (7, 8). Synthesis, accumulation, and catabolism of the extracellular matrix are involved in wound healing and in the initiation and progression of numerous diseases (9). Collagens are major components of the extracellular matrix. Among the various collagens, type VI collagen is a structural glycoprotein composed of three chains: 1(VI), 2(VI), and 3(VI) (10, 11). Type VI collagen has been suggested to play a role in determining the organization and architecture of the extracellular matrix (12), and it has been shown to be abundant in a variety of tissues (13, 14). However, little is known about alterations in the expression of type VI collagen in the human ovarian follicle during follicular growth. In the present study, we investigated changes in the distribution of this glycoprotein by an indirect immunofluorescence (IIF) method using a specific monoclonal antibody (mab) for human type VI collagen. We also studied type VI collagen mrna expression in ovarian follicular tissues by in situ hybridization. MATERIALS AND METHODS The project was approved by the Committee on Investigations Involving Human Subjects of Wakayama Medical College. Informed consent was obtained from each patient after the purpose and nature of the study had been fully explained. 343

2 Tissues Human ovarian tissue, including follicles, was obtained from 10 patients with regular menstrual cycles. The patients were between 39 and 46 years old and were undergoing abdominal hysterectomy for cervical cancer. None of the women had received any hormone therapy or had any ovarian or endocrine disorder before the operation. The menstrual history and BBT record were used to determine the day of the cycle. The menstrual intervals of all patients were days. The follicular phase was divided into the early follicular phase (cycle days 1 5; 4 ovaries), the midfollicular phase (cycle days 6 9; 3 ovaries), and the preovulatory phase (cycle days 10 14; 5 ovaries). We did not detect cancer metastases in any ovarian samples. All ovarian samples were processed at the same time. Primary Antibodies An mab against human type VI collagen was used for this study. Preparation of the antibodies has been described previously (15). In brief, BALB/C mice were immunized with human type VI collagen, which had been extracted from human placentas. The spleen cells were hybridized with myeloma cells. After hypoxanthine-aminopterine-thymidine selection, positive hybrids were screened by ELISA. The specificity of each antibody was determined by an immunoblot or inhibition ELISA. No cross-reaction was recognized among the antibodies against human types I, III, and IV collagen. Immunohistochemistry Immunostaining was performed by the IIF method. In brief, 3- m frozen sections were rehydrated in phosphatebuffered saline (PBS) at room temperature and then incubated with the primary antibody diluted 1:100 in PBS at 4 C for 12 hours in a humidified chamber. After incubation, the sections were washed twice in PBS for 3 minutes. Each slide was then incubated at room temperature for 1 hour with human plasma preabsorbed, fluorescein isothiocyanate conjugated goat antibodies against mouse immunoglobulins (Igs) that had been diluted 1:100 (Organon Teknik Co., West Chester, PA). The sections were washed again in PBS, mounted in buffered glycerol, and examined under a fluorescence microscope (Olympus Co., Tokyo, Japan). Control sections were stained with goat antibodies against mouse IgG (secondary antibodies) without previous application of the primary antibody. When the mab was first absorbed with an excess of human type VI collagen, no immunostaining was observed (data not shown). cdna Probe Plasmid P 18 with a 2.1-kb insert specific for the 1(VI) collagen (11) was used in this study. The cdna probe was labeled radioactively with [ 35 S]dCTP by random priming to a specific activity of cpm/ g. In Situ Hybridization Tissue sections (5 m) were deparaffinized, rehydrated, and treated with 0.2 N HCl for 20 minutes at room temperature, followed by 15 minutes in proteinase K (1 g/ml in Tris EDTA NaCl at 37 C; Sigma Chemical Co., St. Louis, MO) to remove basic proteins. The sections were washed in 2 SSC, acetylated in 0.25% acetic anhydride in 0.1 M triethanolamine for 10 minutes, and hybridized overnight with 35 S-labeled antisense cdna. Hybridizations were followed by four stringent washes (2 SSC and 1 SSC for 15 minutes at room temperature) before autoradiography using D 19 emulsion (Kodak Co., Rochester, NY). Filters were exposed for 21 days before development. The controls were performed under the same conditions with 35 S-labeled sense cdna. All slides were counterstained with toluidine blue, mounted, and examined under an Axiophot microscope (Carl Zeiss Co., Jena, Germany). RESULTS Immunohistochemical Findings Control sections of follicles were stained with goat antibodies to mouse IgG without previous application of the appropriate primary antibody. When the mabs were first allowed to react with an excess of each specific type of collagen, no immunostaining was observed. No immunostaining with the mab against type VI collagen was observed in primordial and primary follicles obtained during the early follicular phase (data not shown). Immunostaining began to appear in the theca cell layers of secondary follicles obtained during the midfollicular phase (data not shown). In tertiary follicles obtained during the late follicular phase, intense immunostaining for type VI collagen showed a fibrillary pattern in the theca cell layers, being most marked in the theca externa (Fig. 1A). In contrast, there was weak, fragmented, or discontinuous immunostaining in the theca cell layers of the preovulatory follicle (diameter of follicles, mm) obtained during the late follicular phase (Fig. 1B and 1C). This change was predominant in the apical area (Fig. 1C) of preovulatory follicles rather than in the basal area (Fig. 1B). Immunostaining of the surrounding ovarian stroma was very faint, whereas staining was present in the blood vessels. No immunostaining with the mab against type VI collagen was observed in the granulosa cell layer during follicular development. In Situ Hybridization Findings The results of in situ hybridization for 1(VI) collagen mrna with the antisense 1(VI) probe are shown in Figure 2. The antisense probe hybridized strongly to the theca interna layers of developing follicles obtained during the midfollicular phase as assessed by bright-field (Fig. 2B) and dark-field (Fig. 2C) examinations, whereas no hybridization signal was seen in the granulosa cells. No hybridization 344 Iwahashi et al. Type VI collagen in human ovarian follicles Vol. 74, No. 2, August 2000

3 FIGURE 1 Immunofluorescence micrographs of human ovarian follicles immunostained with an mab specific for type VI collagen during follicular growth. No immunofluorescence was recognized in the control section. In addition, no immunofluorescence with the mabs against type VI collagen was observed in a primordial follicle and a primary follicle obtained during the early follicular phase (data not shown). The primordial and primary follicles from three different patients were examined. Immunofluorescence began to appear in the theca cell layers of a secondary follicle obtained during the midfollicular phase (data not shown). The secondary follicles from two different patients were examined. In a tertiary follicle obtained during the late follicular phase, intense immunostaining for type VI collagen was seen in a fibrillary pattern in the theca cell layers, especially the theca externa (A). The tertiary follicles from three different patients were examined. Weak, fragmented, and discontinuous immunostaining was observed in the theca cell layers of a preovulatory follicle (diameter of follicles, mm) obtained during the late follicular phase (B, C). This fragmented or discontinuous immunostaining was more evident in the apical portion (C) of the preovulatory follicle than in the basal region (B). The preovulatory follicles from three different patients were examined. No immunostaining was observed in the granulosa cells. A antrum; G granulosa cell layer; TI theca interna layer; TE theca externa layer. (Original magnification, 250.) Iwahashi. Type IV collagen expression. Fertil Steril signal was seen in primordial or primary follicles obtained during the early follicular phase (data not shown). The distribution of mrna signals for 1(VI) was similar during follicular development. DISCUSSION In view of the extensive changes in follicular collagen, it appears that other types of collagens, including types I, III, and IV, should play a role in follicular development and ovulation (2 4, 16). These types of collagens may be associated with the regulation of proliferation, differentiation, and apoptosis of follicular cells (3, 4). In the present study, the protein and mrna for type VI collagen, microfibrillary collagen, were found to be localized diffusely in the theca cell layers of the human ovarian follicles during follicular development, but not in the granulosa cell layers. Although immunostaining intensity was remarkable in the theca externa layer, hybridization intensity appeared greatest in the theca interna layer. This suggests that the theca interna layer may be responsible for synthesis of type VI collagen, and the collagen may accumulate in the theca externa layer of the ovarian follicles. Follicular accumulation of type VI collagen is probably associated with folliculogenesis, as demonstrated in the present study by IIF and in situ hybridization. Type VI collagen is unusual and its functions remain relatively unknown. Immunologic studies have revealed that type VI collagen is concentrated around the basement membrane and cell surface, suggesting that it may help to anchor tissues and cells to the connective tissue extracellular matrix (12, 14). Indeed, it has been suggested that type VI collagen may be capable of interacting with numerous extracellular matrix components, including type I collagen (17) and hyaluronic acid (18, 19), as well as with cell surface receptors such as NG2 proteoglycan (20 22) and integrins (23, 24). Thus, an increase in type VI collagen synthesis may result in close interactions between ovarian theca cells and the extracellular matrix, which in turn may lead to changes in cell shape, proliferation, migration, or differentiation during follicular development. FERTILITY & STERILITY 345

4 FIGURE 2 In situ hybridization for 1(VI) collagen mrna in tertiary follicles. In situ hybridization of a section with the antisense 1(VI) probe is shown under bright-field (B) and dark-field (C) conditions. An adjacent section hybridized with the sense 1(VI) probe is shown under bright-field conditions (A). The antisense probe hybridized strongly to the theca interna layers of a developing follicle (B, C) obtained during the midfollicular phase, whereas no hybridization signal was seen in the granulosa cell layer. G granulosa cell layer; TI theca interna layer. (Original magnification, 125.) Iwahashi. Type IV collagen expression. Fertil Steril The preovulatory follicles showed degradation of follicular type VI collagen that was more marked in the apical area than in the basal area. This finding is in agreement with previous reports (16) and suggests that a decrease of type VI collagen in the follicular walls may be associated with ovulation. It has been suggested that type VI collagen microfibrils are susceptible to degradation by serine proteinases but are resistant to matrix metalloproteinases (25), the family of enzymes that is generally believed to be involved in physiologic degradation of the matrix. The susceptibility of type VI collagen to degradation by the serine proteinases, enzymes characteristically secreted by neutrophils (26) and granulocytes (27), indicates that it may be vulnerable to degradation primarily at the time of ovulation with inflammatory cell involvement (28 30) as well as in early inflammatory lesions (31 33). In conclusion, our observations suggest that type VI collagen is present in the theca cell layers of follicles during follicular development and that this collagen may play an important role in follicular development, maturation, and ovulation. Further detailed work on the extracellular matrix in the human ovary will increase our knowledge of ovarian physiology and pathophysiology. References 1. Espey LL. Ultrastructure of the apex of the rabbit graaffian follicle during the ovulatory process. Endocrinology 1967;81: Palotie A, Peltonen L, Foidart JM, Rajaniemi H. Immunohistochemical localization of basement membrane components and interstitial collagen types in preovulatory rat ovarian follicles. Collagen Relat Res 1984;4: Zhao Y, Luck MR. Gene expression and protein distribution of collagen, fibronectin and laminin in bovine follicles and corpora lutea. J Reprod Fertil 1995;104: Huet C, Monget P, Pisselet C, Monniaux D. Changes in extracellular matrix components and steroidogenic enzymes during growth and atresia of antral ovarian follicles in the sheep. Biol Reprod 1997;56: Espey LL, Coons PJ. Factors which influence ovulatory degradation of rabbit ovarian follicles. Biol Reprod 1976;14: Reich R, Tsafriri A, Mechanic GL. The involvement of collagenolysis in ovulation in the rat. Endocrinology 1985;116: Lin CQ, Bissell MJ. Multi-faceted regulation of cell differentiation by extracellular matrix. FASEB J 1993;7: Madri JA, Basson MD. Extracellular matrix-cell interactions: dynamic modulators of cell, tissue and organism structure and function. Lab Invest 1992;66: Haralson MA. Extracellular matrix and growth factors: an integrated interplay controlling tissue repair and progression to disease. Lab Invest 1993;69: Trueb B, Winterhalter K. Type VI collagen is composed of a 200 kd subunit and two 140 kd subunits. EMBO J 1986;5: Chu M-L, Mann K, Deutzmann K, Pribola-Conway D, Hsu-Chen CC, Bernard MP, et al. Characterization of three constituent chains of collagen type VI by peptide sequences and cdna clones. Eur J Biochem 1987;168: Keene DR, Engvall E, Glanville RW. Ultrastructure of type VI collagen in human skin and cartilage suggests an anchoring function for this filamentous network. J Cell Biol 1988;107: Trueb B, Schreier T, Bruckner P, Winterhalter KH. Type VI collagen 346 Iwahashi et al. Type VI collagen in human ovarian follicles Vol. 74, No. 2, August 2000

5 represents a major fraction of connective tissue collagens. Eur J Biochem 1987;166: Hessle H, Engvall E. Type VI collagen. Studies on its localization, structure, and biosynthetic form with monoclonal antibodies. J Biol Chem 1984;259: Ooshima A, Muragaki Y. Collagen metabolism in atherogenesis. Ann N Y Acad Sci 1990;598: Murdoch WJ, McCormick RJ. Enhanced degradation of collagen within apical vs. basal wall of ovulatory ovine follicle. Am J Physiol 1992; 263:E Hedbom E, Heinegard D. Binding of fibromodulin and decorin to separate sites on fibrillar collagens. J Biol Chem 1993;268: McDevitt CA, Marcelino J, Tucker L. Interaction of intact type VI collagen with hyaluronan. FEBS Lett 1991;294: Kielty CM, Whittaker SP, Grant ME, Shuttleworth CA. Type VI collagen microfibrils: evidence for a structural association with hyaluronan. J Cell Biol 1991;118: Stallcup WB, Dahlin K, Healy P. Interaction of the NG2 chondroitin sulfate proteoglycan with type VI collagen. J Cell Biol 1990;111: Nishiyama A, Stallcup WB. Expression of NG2 proteoglycan causes retention of type VI collagen on the cell surface. Mol Biol Cell 1993;4: Burg MA, Tillet E, Timpl R, Stallcup WB. Binding of the NG2 proteoglycan to type VI collagen and other extracellular matrix. J Biol Chem 1996;271: Donae KJ, Yang G, Birk DE. Corneal cell-matrix interactions: type VI collagen promotes adhesion and spreading of corneal fibroblasts. Exp Cell Res 1992;200: Pfaff M, Aumailley M, Specks U, Knolle J, Zerwes HG, Timpl R. Integrin and Arg-Gly-Asp dependence of cell adhesion to the native and unfolded triple helix of collagen type VI. Exp Cell Res 1993;206: Kielty CM, Lees M, Shuttleworth CA, Woolley D. Catabolism of intact type VI collagen microfibrils: susceptibility to degradation by serine proteinases. Biochem Biophys Res Commun 1993;191: Salvesen G, Enghild JJ. An unusual specificity in the activation of neutrophil serine proteinase zymogens. Biochemistry 1990;29: Morgan JG, Sukiennicki T, Pereira HA, Spitznagel JK, Guerra ME, Larrick JW. Cloning of the cdna for the serine protease homolog CAP37/azurocidin, a microbicidal and chemotactic protein from human granulocytes. J Immunol 1991;147: Hellberg P, Thomsen P, Janson PO, Brannstrom M. Leukocyte supplementation increases the luteinizing hormone-induced ovulation rate in the in vitro-perfused rat ovary. Biol Reprod 1991;44: Brannstrom M, Mayrhofer G, Robertson SA. Localization of leukocyte subsets in the rat ovary during the periovulatory period. Biol Reprod 1993;48: Bonello N, McKie K, Jasper M, Andrew L, Ross N, Braybon E, et al. Inhibition of nitric oxide: effects on interleukin-1beta enhanced ovulation rate, steroid hormones, and ovarian leukocyte distribution at ovulation in the rat. Biol Reprod 1996;54: Brannstrom M, Wang L, Norman RJ. Effects of cytokines on prostaglandin production and steroidogenesis of incubated preovulatory follicles of the rat. Biol Reprod 1993;48: Brannstrom M, Norman RJ, Seamark RF, Robertson SA. Rat ovary produces cytokines during ovulation. Biol Reprod 1994;50: Arici A, Oral E, Bukulmez O, Buradagunta S, Engin O, Olive DL. Interleukin-8 expression and modulation in human preovulatory follicles and ovarian cells. Endocrinology 1996;137: FERTILITY & STERILITY 347