T H E ANATOMICAL RECORD 223:384-386 (1989) S-100 Protein-lmmunoreactive Cells in the Bovine Ovary SHINJI KAMIYA, MASUMI TSUKUSHI, SHUJI YAMANO, AND MASAYUKI DAIGO Department of Veterinary Anatomy, Nippon Veterinary and Zootechnical College, Tokyo 180, Japan ABSTRACT The present study deals with an immunohistochemical localization of 5-100 protein in the bovine ovary. Immunoreactivity for 5-100 was observed in various types of cells, as well as in cells of the nervous system. The endothelial cells of arterial vessels, blood capillaries, and lymph vessels; the epithelial cells of ovarian cysts; and the oocytes of normal and atretic follicles showed a n S-100 protein positivity. The immunoreactivity also was found in the epithelial cells of the rete ovarii. No cells other than these showed immunoreactivity to the anti-S-100 serum. S-100 protein can be a useful marker for providing information on ovarian function. In 1965, Moore described for the first time the presence of s-100 protein in the nervous system of various animals. This protein is localized mainly in glial elements in the central and peripheral nervous systems (Nakajima et al., 1984) and has been regarded until recently as a nervous system-specific protein. However, it now has been demonstrated that S-100 protein is present also in various cells of non-nervous tissues, such as myoepithelial cells, melanocytes, adipocytes, macrophages, Langerhans cells, follicular dendritic cells, chondrocytes, and tumor cells derived from these cells (Suzuki et al., 1982; Nakajima et al., 1984; Donato, 1986; Vanstapel et aI., 1986; Sugimura et al., 1987). However, the biological role of S-100 protein in these cells has not been elucidated. Recently, it has been demonstrated that in fat cells S-100 protein might serve as one of the carrier proteins of free fatty acids (Haimoto et al., 1985; Iwanaga et al., 1987). 5-100 protein has been reported to induce hormone secretion in cultured prolactin cells (Ishikawa et al., 1983). In addition, it was shown to interact with 5-100 protein with the microtubule system (Donato, 1986). There have been many reports on S-100 protein in various non-nervous tissues but only a few descriptions of its occurrence in the ovary (Michetti et al., 1985; Haimoto et al., 1987). To our knowledge, however, there is no report describing the localization of S-100 protein in the bovine ovary. The present study deals with a peculiar localization of S-100-immunoreactive cells in the bovine ovary. MATERIALS AND METHODS Ovaries of 50 Holstein cows were obtained a t a slaughterhouse. The tissue blocks were fixed in 10%buffered neutral formalin, dehydrated in ethanol, and embedded in paraffin according to conventional procedures. Paraffin sections 3-6-pm thick were cut serially. Dewaxed paraffin sections were submitted to the peroxidase-antiperoxidase (PAP) method (Polak and Noorden, 1986). The antiserum used in this study was a polyclonal rabbit anti-S-100 protein antibody obtained commercially (Dakopatts, Denmark and Advance, Japan). The sec01989 ALAN R. LISS, INC tions were treated with 0.3% H202 in methanol for 30 min to block endogenous peroxidase activity and then soaked in non-immune swine serum (diluted 1:20, Dakopatts) to inhibit non-specific binding of immunoglobulins. The sections were incubated with anti-S-100 protein serum (1:1,000) for 1 hr or more. After rinsing, treatment followed with anti-rabbit IgG swine serum (190, Dakopatts) and PAP solution (1:50, Dakopatts) for 30 mins. Finally, the sections were treated with diaminobenzidine and then counterstained with hematoxylin or Fig. 1. Sequential sections of bovine ovary. a: Endothelial cells of the artery (A) are immunoreactive for S-100 protein, while those of the vein (V) are negative. Arrowheads indicate immunoreactive nerve bundles. h: A control section exhibits low-level, nonspecific staining. x75. Fig. 2. Endothelial cells of both artery (A) and lymph vessel (L) react to the S-100antiserum. ~ 7 5 . Fig, 3. Endothelial cells of arteriol (A) and lymph vessel (L) are immunoreactive for S-100 protein. ~ 1 5 0 . Fig. 4. Oocytes in the primordial follicles are stained with S-100 protein antiserum. ~ 7 5 Insert: . Oocyte in the growing follicle reacts to the S-100antiserum. x 150. Fig. 5. Oocyte i n the growing secondary follicle is positive for S-100, while other elements in t h e follicle a r e negative. ~ 7 5 . Fig. 6. Endothelial cells of capillaries and oocyte in t h e atretic follicle are immunoreactive for S-100 protein. ~ 7 5 . Fig. 7. Luteal cells of the corpus luteum (CL) are stained negatively with S-100 protein antiserum, but the endothelium of arteries (A) is stained positively. ~ 7 5 . Fig. 8. Epithelium of the ovarian cysts reacts to the S-100antiserum. x75. Fig. 9. Epithelial cells of the rete ovarii a r e stained positively with S-100protein antiserum. ~ 7 5 . Received April 1, 1988; accepted July 20, 1988. Address reprint requests to Shinji Kamiya, Department of Veterinary Anatomy, Nippon Veterinary and Zootechnical College, 1-7-1 Kyonan-cho, Musashino, Tokyo 180, Japan. S-100PROTEIN IN BOVINE OVARIES 385 386 S.KAMIYA E T AL methyl green. All reactions were done at room temperature. Normal rabbit serum and antisera extensively absorbed with bovine S-100 protein were used instead of the primary antibody as a control (Polak and Noorden, 1986; Sugimura et al., 1987). The sections adjacent to immunostained sections were stained with hematoxylin-eosin for histological observations. protein would detect positive immunoreactivity in the follicular and theca cells of the bovine ovary. The present study demonstrated for the first time the immunoreactivity of the epithelial cells of the rete ovarii and of ovarian cysts to the anti-S-100 serum. Although it is difficult to correlate these cells with other S-100containing cells in functional and embryological aspects, it can be pointed out that cultured prolactin cells share some biological properties with epithelial cells of the rete ovarii. S-100 protein has been reported to induce RESULTS hormone secretion in cultured prolactin cells (Ishikawa In the bovine ovary, immunoreactivity for S-100 pro- et al., 1983). Odend’hal et al. (1986) reported that the tein was observed in the endothelial cells of arterial rete ovarii might respond to hormones and possibly even vessels distributed throughout the ovary (Figs. 1-3). No produce hormones. Thus, the immunostaining with Sother cellular elements in the artery were immunola- 100 protein may be a useful method for identifying the beled, except for those of the nervous system adjacent to function of the rete ovarii. An ovarian cyst may be formed from the follicle, corand supplying the wall of the vessels (Fig. la). Control sections showed negative staining for S-100protein (Fig. pus luteum, rete ovarii, or the cortical invaginations of lb). The immunoreactivity for S-100 also was found in the surface epithelium (Odend’hal et al., 1986; Clement, the endothelial cells of blood capillaries (Fig. 6) and 1987). It is suspected that if the epithelium of ovarian lymph vessels (Figs. 2,3). In contrast, the endothelial cysts, immunopositive for S-100 protein in this study, cells of venous vessels did not react to the S-100 anti- arises from the rete ovarii, immunostaining with S-100 serum (Fig. la). protein could provide a useful method for distinguishing Oocytes of normal and atretic follicles showed a posi- the rete cysts from the other cysts. At any rate, further tive reaction to the anti-S-100 serum (Figs. 4-6). No study will be needed to clarify the correlation between other elements in the follicle such as follicular epithelial 5-100 protein and intraovarian structures. cells, were immunoreactive, except for the endothelial ACKNOWLEDGMENTS cells of the blood capillaries (Fig. 6). The luteal cells of The authors wish to express their thanks to Prof. M. the corpus luteum did not react to the S-100protein (Fig. 7), but the epithelial cells of the ovarian cyst were posi- Sugimura, Department of Veterinary Anatomy, Faculty tive for S-100 (Fig. 8). In addition, it was noticed that of Veterinary Medicine, Hokkaido University, for his the epithelial cells of the rete ovarii were immunoreac- valuable suggestions, including criticism of the manuscript, during this study. tive for S-100 protein (Fig. 9). DISCUSSION LITERATURE CITED To now, there have been many reports indicating that S-100 protein is present in various types of cells in the intra- and extraneuronal tissues (Nakajima et al., 1984; Donato, 1986; Vanstapel et al., 1986; Haimoto et al., 1987; Sugimura et al., 19871, although the physiological role of S-100 protein is still obscure. Recently, Iwanaga et al. (1987) reported that immunoreactivity for S-100 protein was seen in the endothelial cells of arteries and blood capillaries, but not in those of veins. In the present study, similar results were obtained. Moreover, it has been postulated that S-100 protein in the endothelial cells may be involved in the mechanism of transcytosis of fatty acid andior other substances (Haimoto et al., 1985). However, there is no inclusive explanation for this variety of cells sharing S-100 protein. In the present study, immunoreactivity for S-100 protein was observed in the oocytes, in the epithelial cells of rete ovarii, and in ovarian cysts. On the other hand, contradictory results have been reported; for example, no immunoreactivity was observed in any cell type of the rat ovary (Michetti et al., 1985). Haimoto et al. (1987),however, recently examined the localization of S100 protein in various human tissues by use of monoclonal antibodies. They stated that in the ovary, oocytes and follicular cells of the primary folIicIe and the theca cells of the corpus luteum were immunoreactive. The reasons for these discrepancies are unclear. Vanstapel et al. (1987)reported that monoclonal antibodies specific for S-100 protein gave results different from those obtained using polyclonal antisera of the same specificity. It is reasonable to assume that the use of antisera that can recognize different portions in the sequence of S-100 Clement, P.B. 1987 Histology of the ovary. Am. J. Surg. Pathol., 11:277303. Donato, R. 1986 S-100 proteins. Cell Calcium, 7,123-145. Haimoto, H., S. Hosoda, and K. Kato 1987 Differential distribution of immunoreactive S100-a and S100-0 proteins i n normal nonnervous human tissues. Lab. Invest., 57:488-498. Haimoto, H., K. Kato, F. Suzuki, and H. Nagura 1985 The ultrastructural changes of 5-100 protein localization during lipolysis in adipocytes: An immunoelectron-microscopic study. Am. J. Pathol., 12Ir185-191. Ishikawa, H., H. Nogami, and N. Shirasawa 1983 Novel clonal strains from adult r a t anterior pituitary producing S-100 protein. Nature, 30.?:711-713. Iwanaga, T., T. Fujita, Y. Takahashi, and T. Isobe 1987 Immunohistochemical demonstration of S-100 protein in the endothelial cells of blood vessels in the pig and cattle. Biomed. Res., 8r329-334. Michetti, F., L. Lauriola, M. Rende, V.M. Stolfi, F. Battaglia, and D. Cocchia 1985 S-100 protein in the testis: An immunochemical and immunohistochemical study. Cell Tissue Res., 240:137-142. Moore, B.W. 1965 A soluble protein characteristic of the nervous system. Biochem. Biophys. Res. Commun., 19:739-744. Nakajima, T., T. Kameya, S. Watanabe, T. Hirota, Y. Shimosato, and T. lsobe 1984 S-100 protein distribution in normal and neoplastic tissues. In: Advances in Immunohistochernistry. R.A. DeLellis, ed. Masson, New York, pp. 141-158. Odend’hal, S., J.G.W. Wenzel, and E.C. Player 1986 The rete ovarii of cattle and deer communicates with the uterine tube. Anat. Rec., 216t40-43. Polak, J.M., and S.V. Noorden 1986 Immunocytochemistry: Modern Methods and Applications, 2nd ed. Wright, Bristol. Sugimura, M., H. Ishimaru, Y. Atoji, and Y. Suzuki 1987 S-100protein N subunit immunoreactivity of follicular dendritic cells in germinal centers of canine and caprine lymph nodes. J p n . J. Vet. Sci., 49: 1183-1185. Suzuki, F., T. Nakajima, and K. Kato 1982 Peripheral distribution of nervous system-specific 5.100 protein in rat. J. Biochem., 92335838. Vanstapel, M.-J., K.C. Gatter, C.D. Wolf-Peetcm, D.Y. Mason, and V.D. Desmet 1986 New site of human S-100 immunoreactivity detected with monoclonal antibodies. Am. J. Clin. Pathol., 85,160-168.