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

7. FACTORS IMPAIRING SPERM-OOCYTE FUSION

Based on hamster or human oocyte penetration assays it has been suggested that defects in sperm-oocyte fusion may account for some form of human infertility. These defects may arise from failure of the sperm to respond to Ca influx and proton efflux and to undergo acrosome reaction. They may also be due to structural abnormalities in the sperm particularly at the equatorial region of the sperm head where fusion with the oocyte is initiated. In addition, biochemical defects in the plasma membrane of sperm and oolemma can inhibit the initiation of exocytosis, acrosome reaction and fusogenic changes required in fertilization of egg.

7.1. Free radical-mediated lipid peroxidation

Oxidative stress, commonly defined as a disturbance in the prooxidant and antioxidant levels, may be involved in the defective sperm-oocyte fusion. Human sperm are highly susceptible to peroxidative damage because they contain a high levels of long-chain polyunsaturated fatty acids and because unlike somatic cells, they lack the mechanism to institute membrane repair (112). Numerous studies have shown that human sperm exhibit the capacity to generate reactive oxygen species (ROS) such as superoxide anion, hydrogen peroxide, and hydroxyl radicals (112-117). Most of the hydrogen peroxide appears to be generated by the action of sperm superoxide dismutase on the superoxide radical produced by sperm. The production of ROS by human sperm is due to a membrane-bound NADPH oxidase system (112).

Under normal circumstances, this NADPH oxidase complex is quiescent. However, in certain cases of male infertility this system is extremely active, and results in a chronic elevation in the rate of production of ROS (117). In such cases, the capacity of sperm for sperm-oocyte fusion is diminished. Aitkin et al. suggest that high failure rate of sperm-oocyte fusion bioassay can be related to increased generation of lipoperoxides (115). In a subset of infertile patients sperm are refractory to the second messenger signal generated by Ca ionophore, excessively generate ROS and exhibit a high failure rate in sperm-oocyte fusion bioassays (115).

The major consequence of generation of reactive oxygen species is to induce lipid peroxidation in sperm. As a consequence, the plasma membrane of sperm loses the integrity and fluidity necessary for acrosome reaction and fusion with the oolemma. Superoxide appears to be the main inducer of lipid peroxidation (114). The initiation of lipid peroxidation is thought to involve the generation of hydroxyl radicals from the hydrogen peroxide and superoxide anion by the sperm and the contaminating leukocytes in the ejaculate (113). Iron catalyzes the peroxidation of constituents of plasma membranes of sperm and results in a loss of sperm-function. This is reflected in a failure of the sperm to exhibit sperm-oocyte fusion in response to Ca ionophore.

Additional evidence for the adverse effects of lipid-peroxidation on sperm-oocyte fusion has also come from studies in which several antioxidants, (-tocopherol, superoxide dismutase, xanthine oxidase, catalase), partially rescue the function of sperm exposed to inducers of ROD (118-121).

7.2. Immature sperm surface proteins

SUZI or partial zona dissection are being employed for treatment of severe male factor infertility or standard IVF failure. These techniques allow sperm to directly access to the oolemma, by bypassing the ZP. Similar to the physiological fertilization process, only acrosome-reacted sperm bind to ZP-free oocytes. These studies indeed confirm that oolemma is strictly selective for acrosome-reacted sperm. Interestingly, direct cytoplasmic injection of sperm produces higher fertilization rates than SUZI and partial zona dissection when the same sperm preparation is used (122). Therefore, it appears that the acrosome reaction may be only required for sperm to penetrate the ZP and fuse with the oolemma. The acrosome status does not influence the ability of sperm heads to decondense in the ooplasm and form into a male pronucleus. The use of SUZI in IVF has shown that one cause of human infertility is due to incomplete processing of sperm surface proteins or immature surface proteins resulting in defective sperm-oocyte fusion.

7.3. Abnormal nuclear condensation

SUZI and ICSI have provided evidence that some fertility defects may be related to intrinsic sperm anomalies at the sperm membrane and/or chromatin (123). Such anomalies could lead to abnormal appearance of the pronuclei. Fertilizing sperm is known to trigger oocyte activation as a prelude to pronuclear formation. This is thought to occur by binding to a G-protein coupled receptor located in the oolemma. Activation of G-protein would induce the production of the second messenger, inositol triphosphate, which in turn would lead to increased level of intracellular Ca and lead to oocyte activation. A decrease of the amplitude and frequency of Ca oscillation can slow down the time course of pronuclear formation. A relationship between the organization of the sperm chromatin and the success of fertilization has been reported (123). Sperm with abnormal nuclear chromatin are more frequent in infertile men than in fertile men and may lead to the reduced capacity of sperm to fertilize an oocyte in vitro. One cause of this is the decrease in protamine P2 content in sperm chromatin (123). Thus, decondensation of sperm head may be hampered when chromatin organization is abnormal.

7.4. Antisperm antibodies (ASA)

Presence of ASA in the male or female have been shown to interfere with sperm-oocyte interaction (124-130). ASA are directed to multiple sperm antigens and can interfere with normal fertility by preventing the sperm transport or possibly by interfering with sperm-oocyte fusion and fertilization of oocyte (125-127). Hendry and coworkers studied infertile couples who underwent treatment by IVF (128). In their study, the fertilization rate in men with ASA was significantly reduced when compared to men without ASA. However, the pregnancy rate per embryo transfer was not significantly different in the two groups. This indicated that ASA in the male interfere with sperm-egg fusion and subsequent fertilization but once fertilization has occurred, the pregnancy rate remains the same. Sperm-associated ASA interfered with sperm binding to the ZP but not to the oolemma of the oocytes that had failed to fertilize in vitro, or the oolemma of the human ZP-free oocyte (129). Based on this evidence, it was suggested that ASA blockade of fertilization may be due to significant impairment of sperm binding to the ZP. These findings contrasts with studies in which ASA did impair sperm penetration and decondensation in ZP-free hamster oocytes (124). The success of IVF using SUZI of antibody-coated sperm was inversely related to the density of sperm-bound antibodies (130). Thus, ASA reduce fertilization by interfering with the regulatory mechanisms of events leading to pronuclear formation.