The Ultrastructural Study of the Cytoskeleton of the Human Oocytes Subjected to Micromanipulation

Abstract

Objectives The objectives of the study are :- 1. To establish a technique for preparing the human oocytes for transmission electron microscopy 2. To develop an immunogold method for the localisation of cytoskeletal elements of the human oocytes 3. To study the ultrastructure of the human oocytes and the cytoskeleton, subjected to micromanipulation.

Subjects and methods The oocytes, which underwent in vitro fertilization (IVF) or microas sisted fertilization (MAF) e.g. Subzonal Injection of Sperms (SUZI), Direct Injection of Sperm to Cytoplasm of the Oocyte (DISCO), were collected and assigned to the control and study groups respectively at NUTURE, Queen's medical centre from 25th October to 24th December 1993.

Results The cytoskeleton (microtubules and microfilaments) has been examined by electron microscopy and immunocytochemistry. Due to their small size and number of the human oocytes, a new method for handling and preparation for transmission electron microscopy (TEM) has been developed. This method combined protein embedding with centrifugation to locate the specimens on the face of a Beem capsule mould. Therefore, it facilitated both the processing of human oocytes with minimal loss and rapid location of the specimens within the block of serial sectioning, staining and examination. The effects of microassisted (SUZI ; Subzonal Injection of Sperm, DISCO ; Direct Injection of Sperm to Cytoplasm of the Oocyte), sucrose and sonic sword on the microfilamentous and microtubular systems of the human oocytes were studied.  Microfilaments of the human oocytes could be found at the core of microvilli and at the periphery of the cytocortex of all the groups studied by electron microscopy and electron microscopic immunocytochemistry. Most of the oocytes had a uniform microfilament distribution by light microscopic immunocytochemistry except those oocytes exposed to multiple risk factors (SUZI, sucrose, sonic sword) or the direct disturbance of the cytoskeleton system produced by DISCO. Microtubular system of the human oocytes could be detected in all the groups by light microscopic immunocytochemistry. This showed that only two out of six oocytes from normal in vitro fertilization (IVF) (control group) and the manual microinjection of spermatozoa without sucrose and sonic sword had the normal barrel shape spindles, whilst the others had abnormal spindles. By TEM study, microtubules could be found in only one section which was cut through the chromosome and microtubule level transversely. The microtubular system could not be detected in any groups by electron microscopic immunocytochemistry. However, there was no difference in the character and distribution of other organelles in both control and study groups. Conclusion Microassisted fertilization (SUZI, DISCO), sucrose, and sonic sword may be the risk factors of the human oocyte cytoskeleton abnormality, especially those exposed to combined risk factors or the direct disturbance of the cytoskeleton system produced by DISCO.