IMMUNOCYTOCHEMICAL LOCALIZATION OF ZP3 IN PRIMORDIAL FOLLICLES OF RABBIT, MARMOSET, RHESUS-MONKEY AND HUMAN OVARIES USING ANTIBODIES AGAINST HUMAN ZP3

Active immunization with zona pellucida (ZP) proteins leads to depleti on of primordial and recruited follicles in the ovary by a yet unknown mechanism. Small amounts of ZP present on primordial follicles or gra nulosa cells may be one of the reasons for this ovarian pathology. Thi s study was undertaken in an attempt to identify the presence of ZP in or on primordial follicles and granulosa cells in ovaries of rabbits, common marmosets (Callithrix jacchus), rhesus monkeys (Macacas mulatt a) and humans. Therefore, monoclonal antibodies (mAbs) and rabbit and mouse antisera against recombinant human ZP3 (hZP3) were produced. All these antibodies bound to the ZP of native intact human oocytes. Seve ral fixatives have been tested on pieces of rabbit ovaries. With 4% (w /v) paraformaldehyde in PBS (4% PFA) followed by paraffin embedding an d cryostat sections postfixed with 4% PFA, the most intense staining o f ZP with one of the mAbs was obtained and the morphology was well pre served. In humans, besides the ZP, the oocyte cytoplasm and granulosa cells of most primordial and recruited follicles were also stained wit h these antibodies. In rhesus monkey ovaries, all primordial oocytes w ere also stained, in addition to some granulosa cells of primary folli cles. In marmosets, small dots of immunoreactive ZP were found on 60% of the primordial follicles but granulosa cells were not stained. In r abbits, only minor staining of primordial follicles was observed. Afte r passive immunization of rabbits with mAb 4, antibodies were found on both primordial and recruited follicles. These results clearly show t he presence of ZP3 on primordial follicles and in granulosa cells, and this could explain the ZP-induced ovarian pathology after active immu nization with ZP3.