Expression of ovarian tumour suppressor OPCML in the female CD-1 mouse reproductive tract

Abstract

Opioid binding protein/cell adhesion molecule-like gene (OPCML) is frequently inactivated in epithelial ovarian cancer, but the role of this membrane protein in normal reproductive function is unclear. The ovarian surface epithelium (OSE) is thought to be the cell of origin of most epithelial ovarian cancers, some of which arise after transformation of OSE cells lining ovarian inclusion cysts, formed during ovulation. We used immunohistochemistry, immunoblotting and quantitative RT-PCR (qRT-PCR) to investigate OPCML expression in the uteri and ovaries of cycling 3-month CD-1 mice, as well as in ovaries from older mice containing inclusion cysts derived from rete ovarii tubules. Immunoblotting showed OPCML bands in uterine, but not whole ovarian or muscle extracts. Strong OPCML immunoreactivity was observed in oviduct, rete ovarii and uterus, whereas in ovary more immunoreactivity was seen in granulosa cells than OSE. No staining was observed in OSE around ovulation sites, where OSE cells divide to cover the site. OPCML immunoreactivity was also weaker in more dysplastic cells lining large ovarian inclusion cysts, compared with normal rete ovarii. No significant changes in Opcml mRNA expression were observed in whole ovarian and uterine extracts at different stages of the cycle. We conclude that murine OPCML is more consistently expressed in cells lining the uterus, oviduct and rete ovarii than in ovary and is not expressed in OSE associated with ovulation sites. This observation supports the hypothesis that a proportion of epithelial ovarian cancers arise from ductal cells and other epithelia of the secondary Mullerian system, rather than the OSE.

Figure 1

Western blot of OPCML immunoreactivity in extracts of ovary (O), uterus (U) and skeletal muscle (M) from 3-month CD-1 mice. Two immunoreactive bands at approximately 42 KDa and 50 KDa were observed in uterine extracts only, even when 20-μg protein was loaded onto the polyacrylamide gel.

Figure 2

OPCML immunohistochemistry in 4μm sections of CD-1 mouse uterus (a), ovaries (b-d) and oviducts (e and f) obtained in the morning of (b and c, e and f) oestrus (Oe) and (d) metoestrus (Met). A fresh ovulation site (Ov site) is seen in (c), with the granulosa cells spilling over and covering the OSE, which is immuno-negative in comparison with the granulosa cells of the ovulated follicle. Negative controls, without primary antibody, are shown in the insets of (a) and (e). The inset of (b) shows an example of positive OPCML immunoreactivity in the OSE. (a) OPCML staining was observed on the luminal surface of the uterine glands (G) and endometrium (E), in granulosa cells (GC; b and c), in the extra-ovarian rete ovarii tubules (RO; d), in parts of the surface epithelium (OSE; b and c) and on the surface of epithelial cells of the oviduct (ovi; e and f). Ciliated cells are indicated by an asterisk (*). Bars = 100 μm.

Figure 3

OPCML immunohistochemistry in 4 μm sections of cystic ovaries from 9-month (b-c) and 12-month CD-1 mice (d-f) subjected to incessant ovulation from weaning. OPCML immunoreactivity was observed in the cells of normal and moderately dilated extra-ovarian rete ovarii (RO) tubules (a) and in the cells lining a cortical inclusion cyst with no connection to the ovarian hilus and therefore to the rete ovarii (b). Variable immunoreactivity was noted in flattened or ciliated cells (*) lining inclusion cysts with a hilar origin, most likely derived from dilation of the rete ovarii (c) - (f). The epithelium of papillary hilar inclusion cysts was weakly stained, especially on cells with a “signet ring” appearance (arrowhead) or when the cells contained lipid droplets (f). The granulosa cells and zona pellucida of follicles were positively stained (foll). Bars in (a-c) and (e) = 100 μm and in (d) and (f) = 20 μm.