Annulate lamellae assembly in non-inseminated and failed fertilized human oocytes

Abstract

Objective: Functional nuclear pore complexes (NPCs) and their cytoplasmic stacks, annulate lamellae (AL), are engaged in nucleo-cytoplasmic transport, in mammalian zygotes. However, the distribution and dynamics of NPCs in human oocytes is not known. Previous studies showed that a percentage of human eggs fertilized in vitro were defective in the development of male and/or female pronuclei, and failed to cleave and develop into normal embryos. We speculated that a failure of nucleoporin recruitment and/or pre-assembly of NPCs into AL might had been involved in this type of fertilization arrest. Design: Prospective study Materials/Methods: A total of 75 non-inseminated and arrested-fertilized human oocytes were studied for nucleoporins and chromatin labeling. Nuclear pore components were recognized using a monoclonal antibody (mAb414) and a fluorescein-conjugated secondary antibody. DNA was stained with Hoescht 33258. Eleven arrested-fertilized (2PN stage) eggs were individually studied using Transmission Electron Microscopy (TEM). Supernumerary fertilized eggs, discarded upon the request of the consenting donors, were used as controls. Results: As expected, nucleoporin assembly did not occur before fertilization in humans. Once fertilization took place, formation of AL was observed throughout the cytoplasm and near the forming pronuclei. On the contrary, immunofluorescence and TEM showed that nucleoporins assembly in arrested fertilized oocytes was disrupted. NPCs inside the pronuclei, and the lack of their incorporation into nuclear envelopes were some of the characteristics observed. Conclusions: The results presented here seem to indicate that the normal assembly of AL is a crucial event during human fertilization. Although not detected in non inseminated oocytes, AL are rapidly assembled in the oocyte cytoplasm after sperm penetration. This event was evidenced by the presence of a diffuse background-like stain in the cytoplasm of Metaphase I and II oocytes that rapidly developed into a brilliant, punctuate labelling of cytoplasmic, AL-associated pool of nuclear pore components after fertilization. After fertilization arrest at 2PN stage, AL started to form large patches clumped near the decondensed pronuclei. This observation led us to hypothesize that the nucleo-cytoplasmic transport was altered and, as a consequence, the embryonic cleavage had not taken place. Supported by: CEGYR foundation.