346 ULTRASTRUCTURE OF A HUMAN GIANT MII OOCYTE: CASE REPORT

Abstract

The occurrence of giant oocytes has been reported with considerable frequency during IVF procedures in women of all ages and was associated with an increased response to gonadotrophin therapy. Previous studies suggested that giant oocytes, larger in diameter than normal oocytes and either at the GV, MI, or MII stage, might be a source of human digynic triploidy. However, to our knowledge there are no previous reports concerning the ultrastructure of human giant oocytes, and therefore the objective of this study was to characterize a human giant mature metaphase II oocyte from the ultrastuctural point of view. A giant oocyte with a visible polar body was used under informed consent, after controlled superovulation during a IVF treatment cycle. The oocyte was fixed with Karnovsky’s fixative, post-fixed in 2% OsO4 in 0.15 M cacodylate buffer, pH 7.2, dehydrated and embedded in Epon Semithin and ultrathin sections were cut with a diamond Diatome knife (Diatome, Hatfield, PA, USA) in a LKB ultramicrotome. Semithin sections were stained with aqueous azur II and methylene blue (1:1). Ultrathin sections were collected on 200 mesh copper grids (Taab Laboratories Equipment Ltd, Aldermaston, UK) and stained with 3% aqueous uranyl acetate (20 min) and Reynolds lead citrate (10 min). Contrast ultrathin sections were observed in a JEOL 100XII transmission electron microscope (JEOL Ltd., Tokyo, Japan) operated at 60 Kv. Cytogenetic analysis was not performed in this oocyte. At the time of collection, the giant oocyte was 1.4-fold larger than average size oocytes and contained a fragmented polar body. The zona pellucida had a normal loosely fibrillar structure and the perivitelline space was normal containing remnants of follicular cells. The cytoplasmic organelles were no uniformly dispersed with a large organelle-free zone (lake) observed in the periphery of the oocyte cortex and smaller focal lakes visible in the subcortex. Within the cortex the dense cortical vesicles were reduced in number and did not form one or two continuous rows beneath the oolemma. Smooth endoplasmic reticulum (SER) aggregates of tubules were also scarce but underdeveloped SER aggregates of tubules were present in the cortex. The distribution of the SER large and middle vesicles was abnormal: these organelles were nearly absent in the cortex but present in the inner cytoplasm. The content of some vesicles was denser than normal. We also observed the presence of several large secondary lysosomes filled with multiple small and medium lipid droplets (lipofuscin bodies), corresponding to retractile bodies located in the cortex. Large dense vesicles of different densities and volumes were found in the cortex and subcortex. The polar body showed very few cortical vesicles and did not contain mitochondria and SER large or small vesicles. The condensed chromosomes were aligned on the metaphase II plate, in the cortex of the oocyte. The ultrastructural alterations observed in this oocyte corroborated the conclusions of previous genetic studies and confirmed that giant oocytes are immature cytoplasmatically and would be associated with a higher frequency of abnormal development following fertilization. Thus, giant oocytes should not be used in IVF treatments.

ASL was supported by FCT, Portugal.