Osmond J. D'Cruz, Ph.D

Section of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73190

4. ASSESSMENT OF SPERM-OOCYTE INTERACTION

The process of sperm-oocyte interaction and defects manifested at the level of sperm membrane-oolemma fusion, pronuclear decondensation, and oocyte anomalies that lead to IVF failure are studied by indirect and direct bioassays using light microscopic approach. These bioassays include, hamster oocyte-human sperm penetration assay (26), human oocyte-human sperm fusion test (27), subzonal sperm insertion technique (28), and intracytoplasmic sperm injection assay (29). These methods are currently used to predict fertilization as well to delineate functional defects at the level of sperm membrane-oolemma interaction observed in a subset of infertile couples.

4.1. Human sperm-zona-free hamster oocyte penetration assay (HPA)

The fusion of zona-free hamster oocytes with acrosome-reacted human sperm is the most widely used bioassay for physiological sperm-oocyte fusion. HPA has been shown to exhibit excellent correlation with the fertility of human sperm in vivo and in vitro (30). In this procedure, the hamster oocytes are stripped of their ZP by treatment with 0.1% trypsin and then exposed to suspensions of capacitated human sperm. The criterion for penetration is conventionally the percentage of oocytes possessing decondensed sperm nucleus (-ei) after a 3 hour incubation period and 25-50 oocytes are normally used to assess each sperm sample. This test is useful to predict fertilizability of human sperm or intrinsic oocyte problems in patients with total fertilization failure after IVF.

4.2. Human sperm-zona-free human oocyte fusion test

This test is used to evaluate the fusion potential of nonfertilizing human sperm. In this procedure, unfertilized human oocytes are treated with 0.5% pronase to remove ZP, loaded with DNA-specific fluorochrome, and then exposed to suspensions of capacitated human sperm. The criterion for fertilization is the presence of pronuclei (2 or more) after 15 to 20 hour incubation period and 48 hour later for cleavage. Direct examination of fertilization rates of unfertilized human oocytes inseminated with fertile donor sperm have suggested that one cause of fertilization failure may be due to intrinsic oocyte problem confined to the oolemma.

4.3. The subzonal sperm insertion (SUZI) technique

This technique is used to assess the potential of motile sperm to form pronuclei. In this method, the frequency of sperm fusion, is calculated based on the total number of male pronuclei formed in relation to the total number of sperm inserted subzonally. At least 30% to 60% of normal sperm inserted subzonally are able to fuse with the oolemma and decondense within the ooplasm. Subzonal insertion of sperm from men with abnormal semen analyses show significantly low sperm fusion with oolemma and with variable ability to form a pronucleus. Thus, SUZI allows one to distinguish defects on ZP from those of oolemma fusion.

4.4. Intracytoplasmic sperm injection (ICSI)

ICSI of unfertilized human eggs after IVF are used to evaluate the ability of sperm to activate human oocyte. In this method, oocytes are harvested after superovulation with GnRH agonist and gonadotropins. After removing the cumulus cells, a single sperm is injected directly into the cytoplasm of metaphase II oocytes, and sperm decondensation and the number of pronuclei formed are enumerated.

4.5. Hybridoma/molecular cloning approach

Murine monoclonal antibody (mAb) library raised against human sperm head proteins and zona-free human oocytes are valuable tools to investigate the various steps in sperm-oocyte interactions. The acrosome is known to contain more than 20 hydrolyzing enzymes, such as hyaluronidase, acid phosphatase, acrosin, and a number of antigens. Several mAbs directed against the acrosomal antigens (FA-1, HS-63, SP-10, CD46, acrosin) of human sperm or to oolemma have been shown to inhibit heterologous and/or homologous gamete-interaction in vitro (31-34). Therefore, antigens localized on the acrosomal region and equatorial band that interfere with sperm-oocyte fusion have been extensively investigated for their contraceptive potential (4, 35). However, some of these mAbs also display extensive crossreactivity with antigens of somatic cells and placental cells.

Molecular cloning offers an alternative approach for identifying the putative antigens involved in sperm-oocyte interactions. Antisperm antibodies that block gamete interaction in vitro have been used to screen human testis lambda gt11 cDNA expression library. By this method, several recombinant proteins have been identified (36-38). Fusion proteins characterized thus far, include a serine protease inhibitor (serpin), and cytokeratins localized to the acrosomal region of sperm head (39-40).