Grootenhuis A J, Philipsen H L, de Breet-Grijsbach J T, van Duin M
Scientific Development Group, N.V. Organon, Oss, Netherlands.
J Reprod Fertil Suppl. 1996;50:43-54.
Active immunization with zona pellucida (ZP) proteins leads to depletion of primordial and recruited follicles in the ovary by a yet unknown mechanism. Small amounts of ZP present on primordial follicles or granulosa cells may be one of the reasons for this ovarian pathology. This 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 mulatta) 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. Several fixatives have been tested on pieces of rabbit ovaries. With 4% (w/v) paraformaldehyde in PBS (4% PFA) followed by paraffin embedding and cryostat sections postfixed with 4% PFA, the most intense staining of ZP with one of the mAbs was obtained and the morphology was well preserved. In humans, besides the ZP, the oocyte cytoplasm and granulosa cells of most primordial and recruited follicles were also stained with these antibodies. In rhesus monkey ovaries, all primordial oocytes were also stained, in addition to some granulosa cells of primary follicles. In marmosets, small dots of immunoreactive ZP were found on 60% of the primordial follicles but granulosa cells were not stained. In rabbits, only minor staining of primordial follicles was observed. After passive immunization of rabbits with mAb 4, antibodies were found on both primordial and recruited follicles. These results clearly show the presence of ZP3 on primordial follicles and in granulosa cells, and this could explain the ZP-induced ovarian pathology after active immunization with ZP3.
用透明带（ZP）蛋白进行主动免疫会通过一种尚不清楚的机制导致卵巢中原始卵泡和募集卵泡的耗竭。原始卵泡或颗粒细胞上存在的少量ZP可能是这种卵巢病理状态的原因之一。本研究旨在确定兔、普通狨猴（Callithrix jacchus）、恒河猴（Macacas mulatta）和人类卵巢中原始卵泡和颗粒细胞内或表面是否存在ZP。因此，制备了针对重组人ZP3（hZP3）的单克隆抗体（mAb）以及兔和小鼠抗血清。所有这些抗体都能与天然完整人卵母细胞的ZP结合。对兔卵巢组织块测试了几种固定剂。用PBS中的4%（w/v）多聚甲醛（4% PFA）固定，然后进行石蜡包埋，并用4% PFA进行低温恒温切片后固定，用其中一种mAb对ZP的染色最为强烈，且形态保存良好。在人类中，除了ZP外，大多数原始卵泡和募集卵泡的卵母细胞胞质和颗粒细胞也被这些抗体染色。在恒河猴卵巢中，所有原始卵母细胞以及一些初级卵泡的颗粒细胞也被染色。在狨猴中，60%的原始卵泡上发现了免疫反应性ZP的小点，但颗粒细胞未被染色。在兔中，仅观察到原始卵泡有轻微染色。用mAb 4对兔进行被动免疫后，在原始卵泡和募集卵泡上均发现了抗体。这些结果清楚地表明原始卵泡和颗粒细胞上存在ZP3，这可以解释用ZP3进行主动免疫后ZP诱导的卵巢病理状态。
