The biological steps of fertilization are principally the same across varying organisms yet differences at the cellular and molecular level are evident. In mammals millions of sperm are deposited within the female reproductive tract and, yet, are challenged to reach the site of fertilization, the oviduct, where one sperm will successfully penetrate the egg. During transit to the oviduct, sperm undergo a necessary change in cellular composition, called capacitation, which enables sperm to become activated and fertilize the egg. Capacitation is reported to include changes in the outer plasma membrane wherein cholesterol is removed and proteins and lipid domains on the sperm surface are exposed and/or remodeled. Capacitation results in changes in sperm motility, respiration, intracellular tyrosine phosphorylation and swimming pattern. In vitro, capacitation for mammalian sperm typically takes about three hours and in vivo, it can take shorter or longer times.
Once inside the zona pellucida, a reacted sperm can interact directly with the egg plasma membrane. Proteins on the equatorial segment of the sperm plasma membrane are believed to be direct players in sperm-egg binding and fusion. The egg plasma membrane, or oolemma, is a heterogeneous surface with tiny microvillar projections over approximately 80% of the cell surface. Gamete fusion predominantly occurs within the microvillar region. Perhaps fusion occurs there because of increased surface area to capture sperm and the existence of localized regions of proteins and/or lipid domains required for gamete binding and fusion. The other 20% of the egg is a microvillar-free zone that overlies the inactivated meiotic spindle. The microvillar-free region may contain the same or similar proteins, but at much lower concentrations or they may be completely absent. Thus the microvillar-free region’s role may be to give polarity to a mammalian egg; it is the region that is lost in the form of a polar body after fertilization.
Candidates for sperm proteins involved in gamete fusion
DE. The sperm epididymal protein DE, originally identified in rat, has been implicated in gamete membrane fusion. Additionally, studies of recombinant forms of protein DE (recDE) have been beneficial for immuno-contraceptive development in which rats immunized with recDE show a marked decrease in sperm fusion ability. DE, a CRISP family member (cysteine rich secretory protein), is secreted by the rat epididymis and bound to the dorsal region of the sperm acrosomal region during epididymal transit. During the acrosome reaction, a tightly bound population of DE remains attached to the sperm and then migrates to the equatorial segment, where sperm-egg interaction and fusion occur. In vitro, it was shown that both native and recDE decrease sperm penetration of mouse and rat eggs in heterologous assays. By binding to the egg microvillar region, as detected by immunofluorescence, exogenous DE competes with sperm DE and prevents fusion. The active site of the protein involved has not been determined. Further studies are being generated to ascertain its biochemical activity and to understand where it fits in the model for gamete fusion. Since it is unlike the ADAM family of sperm proteins, it could possibly function at a step different from the proposed hypothesis for the sperm-specific ADAMs.
ADAMs. On the sperm surface there exists a subset of the ADAM (A Disintegrin And Metalloprotease domain) family of proteins suggested to be involved in sperm-egg plasma membrane adhesion and fusion by studies initially performed in guinea pig. The prototype of an ADAM consists of multiple domains: pro-, metalloprotease, disintegrin, cysteine-rich (with putative viral fusion peptide), epidermal growth factor -like, typical transmembrane, and a short cytoplasmic tail. Of the 37 ADAMs known, about 16 are found expressed in testis. For several ADAMs, during transit of sperm through the epididymis the pro-domain and metalloprotease domain are proteolytically released, exposing the disintegrin and cysteine-rich domain. Based on its molecular structure and sperm specific ex
Candidates for egg proteins involved in gamete fusion
Integrins. In the model that suggested the sperm ADAM disintegrin domain acts in gamete adhesion and fusion, it was proposed that an integrin on the egg would be a complimentary receptor for gamete binding, if not fusion. Integrins are a family of adhesion proteins found on the surface of cells as α and β heterodimers. They can be divided into several groups based on the particular β subunit present. Various integrins on somatic cells have been shown to bind ADAMs and several integrins have been identified on the surface of mouse eggs.
The tetraspanin CD9. Mammalian eggs express the tetraspanin CD9. The tetraspanin family has more than 30 members in mammals and these proteins share a common structural plan: each tetraspanin spans the membrane four times forming two extracellular loops, one small and one large, and short intracellular N- and C-termini. Proteins from the tetraspanin family are involved in cell interaction and fusion, intracellular signaling, viral fusion and other activities. Chen and co-workers originally announced the presence of CD9 on the surface of mouse eggs and its potential role in sperm-egg binding and fusion by showing that the CD9 function blocking antibody KMC 8 could inhibit sperm-egg binding and fusion. In 2000 three independent knockout studies concluded that CD9 is necessary for fertilization. In CD9 null females, infertility was found after short periods of mating and even after extended mating (ie. 30 days) only low rates of pregnancy occurred: litter size was less than 2% of wild type matings. In vitro the step impaired during fertilization was sperm-egg membrane fusion and not adhesion, since many sperm were able to penetrate the zona pellucida, bind the oolemma, but none were able to penetrate the egg plasma membrane.
CD9 and other tetraspanins are proposed to function via intermolecular membrane networks or “webs”. Miyado and coworkers had shown by co-immunoprecipitation that the α6β1 integrin interacted with CD9 in eggs, thus suggesting a similar web found in egg. Since studies mentioned above showed α6β1 is not required for sperm-egg fusion, the physiological role of such an association may be in oogenesis or post-fertilization.