Sequential expression of zona pellucida protein genes during the oogenesis of domestic cats.

Abstract

The aim of the present study was to determine the earliest developmental stage of zona pellucida (ZP) protein gene expression during oogenesis in domestic cats (Felis catus) by means of immunohistochemical and molecular biological methods. Semi-thin sections (1 microm) from domestic cat ovaries were treated with anti-cat ZP serum raised in guinea pig, and then incubated with silver-labeled anti-guinea pig IgG. To distinguish between the three ZP proteins, total RNA was extracted from freshly isolated cat primordial, primary, and secondary follicles as well as from cumulus-oocyte complexes (COCs) and subjected to reverse transcription (RT). The generated cDNAs were used for polymerase chain reaction (PCR) with specific feline ZPA, ZPB, and ZPC gene primers. All amplified products were sequenced to confirm their identity. Neither ZP mRNAs nor ZP proteins were detectable in primordial and early primary follicles. The immunohistological approach indicated the expression of ZP proteins in some of the primary follicles as well as in secondary follicles and COCs. Follow-up by RT-PCR revealed that only one ZP (ZPB) was expressed in growing primary follicles (70-80 microm), whereas all three ZP mRNAs were detectable in secondary follicles and COCs. We therefore assume a sequential synthesis of zona proteins in the cat ovary.