The ovarian surface epithelium (OSE) is a modified peritoneal mesothelium. It is composed of a single-layered sheet of flat to cuboidal cells that rest on a basal lamina and express microvillus toward the peritoneal cavity. The OSE is distinguished from extraovarian mesothelium by its capacity of steroid synthesis and expression of keratin and mucin. Epithelial-derived ovarian tumors are commonly believed to originate from OSE-lining inclusion cysts located in the ovarian stroma just beneath the surface. These cyst cells express E-cadherin and hence are more epithelial-like than the E-cadherin-negative OSE. It has also been shown that SV40-immortalized OSE expressing stably transfected E-cadherin have an increased tumor-igenic capacity, resembling ovarian adenocarcinoma compared with nontransfected OSE. Therefore, unlike carcinomas in most other organs in which loss of E-cadherin are common, the normal ovarian tumor precursor cells, OSE, seem to transdifferentiate and acquire characteristics of Mullerian duct-derived epithelium when present in the ovarian stroma. The mechanism by which this occurs is unknown, largely due to the fact that very little information is available on the normal biology of OSE and its relationship to the development of ovarian cancer. In the present study, we show that the normal human OSE in situ expresses tight junction (TJ) proteins, i.e., ZO-1, occludin and claudin-1. Moreover, normal OSE cells in primary and secondary cultures form a confluent monolayer that establishes a transepithelial barrier typical of low-resistance epithelium.
Tight Junction Proteins in Human Ovarian Biopsies
By immunofluorescence microscopic analysis, we could determine that the main localization of the TJ-associated protein ZO-1 and the integral membrane proteins occludin and claudin-1 was at the cell borders of normal OSE (Fig. 1, A through C). The underlying stroma were without staining, indicated by stars. TJ protein immunofluorescent staining was the same in sections from both pre- and postmenopausal women. The histological appearance of the ovarian surface and the underlying stroma is shown with hematoxylin and eosin staining (Fig. 1D). In the negative control, where primary antibody was replaced by nonfat milk, no staining was seen (Fig. 1E).
Characterization of Normal Ovarian Surface Epithelium in Culture
To further investigate TJ in human, OSE cells, isolated by the brush technique from ovaries in pre- and postmenopausal women (n = 32), were cultured and used for im-munohistochemistry, immunoblotting, and measurement of TER in Transwell experiments (Table 1). Primary cultured OSE cells typically formed a monolayer with a characteristic cobblestone-like appearance, and this growth pattern was maintained during the first three passages in approximately 45% of the patient samples (Fig. 2A).
OSE cells and tissue biopsies from normal ovaries were obtained from women operated on for benign nonovarian diseases at the Gynecology Unit, Sahlgrenska University Hospital, Goteborg, Sweden. OSE cells for culture were obtained from a total of 32 women. Normal ovarian tissue biopsies were collected from a total of nine women. A description of age at time for operation, reproductive status, and parity are found in Table 1. The study was approved by the Ethical Committee of Goteborg University and the patients had given their informed consent.
Harvest of Cells and Culture Conditions
To obtain OSE, cytobrushes (Cytobrush Plus; Medscand AB, Malmo, Sweden) were used as the first step after entering the peritoneal cavity at laparotomy or laparoscopy. The brushes were simultaneously rotated and moved 2-3 times over the ovarian surface, exerting only a slight pressure to minimize the risk of damaging or disrupting the underlying basal membrane and tunica albuginea. The brushes were withdrawn and immediately placed in culture medium, taken to the laboratory, and gently rubbed against each other to release the cells.
The ovary is covered by a single layer of mesothelial-type epithelial cells, generally referred to as the ovarian surface epithelium (OSE). This single layer of cells is considered to be the origin of approximately 90% of all ovarian cancers, thus referred to as epithelial ovarian cancer. OSE is a simple, rather primitive epithelium with some stromal features, such as vimentin expression. However, when it progresses toward malignancy, it loses its stromal appearance and acquires the characteristics of any of the Mullerian duct-derived epithelia, i.e., those of the oviductal epithelium, endometrium, and the endocervical epithelium. This will thus give rise to a complex and heterogeneous tumor type with regard to histopathologic diagnosis, expression of tumor markers, and response to chemotherapeutic agents. The paradoxical differentiation of cells during ovarian tumorigenesis is also reflected in tissue culture studies of human OSE. Stable transfections with E-cadherin give the OSE cells a more epithelial-like phenotype and they will develop tumor-like features. Even though OSE cells have got more attention in research during the last couple of years, mainly because of their role in ovarian tumor development, the epithelial features of these cells in normal conditions are not fully elucidated.