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Biology of Mammalian Fertilization: Role of the Zona Pellucida

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Biology of Mammalian Fertilization: Role of the Zona Pellucida Perspectives Biology of Mammalian Fertilization: Role of the Zona Pellucida Jurrien Dean Laboratory of Cellular and Developmental Biology, National Institute ofDiabetes and Digestive and Kidney Diseases, National Institutes ofHealth, Bethesda, Maryland 20892 Introduction before approaching the ovulated egg in the oviduct (Fig. 1 A). In mammals, a series of carefully orchestrated events culmi- Motile sperm pass through the enveloping cumulus oophorus, nate in the fusion of a sperm and egg to form a one-cell zygote, which is composed of a glycosylaminoglycan matrix and cu- the obligatory precursor of all cells in the embryo. The overall mulus cells. They then bind to the zona pellucida that rate of fertilization in humans has contributed to sustained surrounds the mammalian egg (1, 2). The mouse and human increases in the world population and added urgency to the zonae pellucidae are composed of three major glycoproteins, need to develop new, effective contraceptive agents. For some, ZP1, ZP2, and ZP3. Solubilized zonae pellucidae from unfertil- however, the success rate is considerably lower, and there are ized mouse eggs (but not from two-cell embryos) can inhibit millions of infertile couples in the United States. Dramatic ad- sperm binding to ovulated eggs (3). This sperm-receptor activ- vances in reproductive biology have provided some relief for ity of the zona has been ascribed to a class of 3.9-kD 0-linked these individuals through the development of in vitro fertiliza- oligosaccharides on ZP3 (4). ZP2 has been implicated as a sec- tion techniques. Further progress requires additional under- ondary sperm receptor that binds sperm only after the induc- standing ofthe molecular basis for normal fertilization and our tion of the sperm acrosome reaction (5). willingness to exploit this knowledge to facilitate desired preg- The acrosome is a membrane-bound organelle anteriorly nancies or prevent unwanted ones. These joint desires to have located in the head of the sperm. It has been proposed that the greater control over our reproductive destiny has led to an in- binding of sperm to ZP3 induces a signal transduction across creased interest in the macromolecules involved in fertiliza- the sperm membrane by aggregating a sperm-specific 95-kD tion. protein with tyrosine kinase activity (6, 7). This leads to the The zona pellucida, an extracellular matrix surrounding acrosome reaction (8) in which the sperm plasma membrane the female gamete, contains the primary and secondary sperm fuses with the outer acrosomal membrane, resulting in the exo- receptors and plays a pivotal role in sperm-egg interactions. cytosis of the acrosomal contents (Fig. 1 B). The lytic enzymes Although millions of sperm are deposited in the female repro- (e.g., acrosin, glycosidases) that are released, as well as some ductive tract, fewer than 100 approach the ovulated egg in the that remain associated with the inner acrosomal membrane, oviduct, and normally only one successfully penetrates such as acrosin, appear to facilitate passage ofthe motile sperm through the zona pellucida to fuse with the plasma membrane through the zona pellucida. of the ovum. Immediately following fertilization, the plasma A number ofsperm surface macromolecules have been pro- membrane and zona pellucida are modified to prevent poly- posed as zona adhesion molecules, e.g., serine proteinases and spermy and, perhaps, to provide additional resiliency to the ,B-l1,4-galactosyltransferase. More recent candidates include a zona so that it can protect the preimplantation embryo. Inter- 95,000-D protein with tyrosine kinase activity and a 56,000-D actions between the sperm and egg appear to be relatively spe- protein isolated by its ability to bind ZP3 (for review see [9]). It cies-specific and research has focused on the male and female is likely that more than one interaction is responsible for the macromolecules involved. This perspective will concentrate on binding ofsperm to the zona, both initially and during penetra- recent advances in our knowledge of the zona pellucida genes tion. Some of the sperm ligands present on the surface of the and proteins of the mouse, an experimental animal that has inner acrosomal membrane (e.g., proacrosin and PH-20) may provided much of our current understanding of the molecular become available for zona binding only after the sperm acro- details of fertilization. some reaction. These latter molecules may be particularly im- portant for successful penetration of the zona pellucida. Fertilization: a precis Immediately after fertilization there are two major changes After ejaculation into the female reproductive tract, sperm un- that prevent polyspermy: a rapid electrical depolarization of dergo a poorly understood maturation process (capacitation) the egg plasma membrane that blocks additional sperm in the perivitelline space from fusing with the egg (10), and biochemi- cal modifications of the zona pellucida. These latter changes Address reprint requests to Dr. Jurrien Dean, Laboratory of Cellular occur secondarily to the fusion ofcytoplasmic cortical granules and Developmental Biology, National Institute of Diabetes and Diges- tive and Kidney Diseases, Building 6, Room B 1-26, National Institutes with the egg plasma membrane, and the subsequent discharge of Health, Bethesda, MD 20892. ofthe granules' enzymatic contents into the perivitelline space. Receivedfor publication 31 December 1991 and in revisedform 21 The release of proteinases and glycosidases modifies the zona January 1992. pellucida (zona reaction), resulting in a block to additional sperm binding and inhibition of zona-bound sperm penetra- The Journal of Clinical Investigation, Inc. tion (1, 10). Both ZP2 and ZP3 are modified by the zona reac- Volume 89, April 1992, 1055-1059 tion: ZP2 undergoes a proteolytic cleavage associated with the Zona Pellucida 1055 A Figure 1. Mammalian fertilization. (A) The Zona Pellucida binding of sperm to the zona pellucida surrounding the ovulated egg induces the sperm acrosome reaction. The release of Sperm lytic enzymes from the acrosome and the forward motility of the sperm permit pene- Sperm tration of the zona pellucida. After fusion Binding with the egg's plasma membrane, the sperm enters the cytoplasm and forms the male pronucleus of the one cell zygote. The fe- male pronucleus, formed at the same time, B contains the female haploid genome. Fol- lowing fertilization, the zona pellucida is Acrosome Intact Sperm biochemically modified to prevent addi- tional sperm from binding or penetrating Tail Acrosome Reacted Sperm the zona. (B) The acrosome, a lysosomal- like structure on the anterior head of sperm, contains an inner and outer mem- brane that fuse during the acrosome reac- tion. This results in the release of lytic en- zymes that are important for the penetra- tion of the zona pellucida by the sperm. block to polyspermy (11), and ZP3 loses both its ability to The biosynthetic pathways of ZP2 and ZP3 have been in- induce the acrosome reaction and its sperm receptor activity vestigated using radioactive precursors and in vitro culture. (8, 12). The ZP2 core protein has a molecular weight of 76,373, de- The zona pellucida proteins duced from its nucleic acid sequence (15). Six complex-type N-linked side chains and an undetermined number of zona 0-linked The pellucida proteins are synthesized, glycosylated, and oligosaccharides are attached to the core protein and the ma- sulfated in oocytes before their secretion to form the zona ma- ture 120-140-kD glycoprotein is secreted and incorporated trix that surrounds the growing oocyte, ovulated egg and early into the extracellular zona pellucida (21). The ZP3 protein is embryo. first detected in the oocyte as a 44,000-D protein to which three Structure. Mouse ZP1 (185-200 kD) is a disulfide-linked or four complex-type N-linked carbohydrate side chains are dimer which has yet to be characterized in molecular detail. added. The further attachment of0-linked sugars results in the The primary amino acid sequences of mouse ZP2 (120-140 production of the mature secreted 83-kD ZP3 glycoprotein kD) and ZP3 (83 kD) have been deduced from the nucleic acid (18). The intracellular transport patterns and mechanisms of sequence of their cognate genes (see below). Although the pep- secretion for these proteins have yet to be delineated. tide backbones ofthe human ZP2 and ZP3 are similar in length to those of their mouse homologues (see below), the mature Zona pellucida genes human proteins are smaller, ZP1 (90-1 10 kD), ZP2 (64-76 Structure ofthe mouse genes. Mouse Zp-2 and Zp-3, each sin- kD), and ZP3 (57-73 kD) (13), presumably due to differences gle copy genes, are located on chromosomes 7 and 5, respec- in glycosylation. Recent experiments with immunological tively (22). Mouse Zp-2 contains 18 exons (Fig. 2) that range in probes have identified two isoforms of human ZP3, ZP3H and size from 45 to 190 bp and are separated by 17 introns (81 to ZP3L (14), but whether these represent different posttranscrip- 1,490 bp). The gene spans 12.1 kbp of DNA. Zp-2 is tran- tional modifications of a single ZP3 gene product or the pres- scribed and processed into a 2,201-nt mRNA with very short 5' ence of an additional gene remains to be determined. (30 nt) and 3' (32 nt) untranslated regions. The detection of a Biosynthesis. There is no zona pellucida surrounding rest- 2.4-kb transcript in oocyte RNA suggests that ZP2 mRNA con- ing mouse oocytes, and zona protein synthesis is first detected tains a poly(A) tail of 200 nt. ZP2 mRNA has a single open when oocytes enter their two week growth phase. Investigations reading frame initiated at an ATG that encodes a polypeptide in a number of species have concluded that the zona pellucida of 713 amino acids with a molecular weight of 80,217. The first proteins are synthesized in oocytes and secreted into the zona 34 amino acids represent a signal peptide that directs secretion matrix, although some studies have also identified zona pro- and, following cleavage, the resultant core polypeptide (76,373 teins in granulosa cells.
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