Developmental Expression of the Rabbit 55-kDa Zona Pellucida Protein and Messenger RNA in Ovarian Follicles

Abstract

The events associated with the early stages of oocyte activation and granulosa cell (GC) differentiation during ovarian follicular development have been investigated using immunological and cDNA probes. Because zona pellucida (ZP) proteins provide excellent markers for oocyte activation during early stages of ovarian development, a cDNA encoding a full-length ZP protein with calculated molecular weight of 57,185 (rc55) and a specific antibody to the corresponding native rabbit ZP protein (R55) were developed. Immunohistochemical analysis of ovaries demonstrated that R55 was localized in follicles of animals 2 weeks and older. R55 was localized in the oocytes of primordial follicles and the oocytes and GCs of primary follicles but was undetectable in the GCs of large antral follicles. GCs were isolated from 6-week-old rabbit ovaries and cultured in defined media to demonstrate expression of rabbit ZP proteins by GCs in vitro. Rabbit ZP proteins were localized in the cytoplasm of cultured GCs if these cells were treated with monensin, a compound that blocks glycoprotein secretion. Northern blot analysis with the cDNA probe rc55 revealed that cultured GCs contained a mRNA for R55 and that there is stage-specific expression of the R55 mRNA in developing rabbit ovaries. Immunoblot analysis demonstrated that R55 is detectable at 4 weeks and accumulates in 6-, 8-, and 12-week ovaries which contain developing primary and secondary follicles. Collectively, these data illustrate developmental expression of R55 in oocytes and GCs of rabbit ovarian follicles.