Synthesis of zona pellucida proteins by denuded and follicle-enclosed mouse oocytes during culture in vitro

Abstract

ABSTRACTlucida proteins during culture in vitro, whereas zonae pellucidae removed from fully-grown oocytes (85 gm) were not radiolabeled to a significant extent. After culture of denuded or follicleenclosed oocytes for 12 hr, more than 95% of the [3Hlfucose incorporated into oocyte proteins was found in ZP1, ZP2, and ZP3, indicating that the zona pellucida proteins are the major class of proteins glycosylated during oocyte growth. These results provide biochemical evidence supporting the idea that the zona pellucida originates from the mammalian oocyte itself, rather than from the surrounding follicle cells.The zona pellucida is a relatively thick, translucent, acellular coat that surrounds the plasma membrane of fully grown mammalian oocytes and performs a variety of vital biological functions during early mammalian development. The zona pellucida is laid down during growth of the ovarian follicle, remains throughout preimplantation development, and is finally shed as the blastocyst readies for implantation (1-3). Several lines of evidence suggest that the zona pellucida possesses a receptor for sperm, that it acts as a barrier to further sperm penetration after fertilization, and that its presence is necessary for normal early development in vivo (3-5). In certain respects the zona pellucida is morphologically and functionally analogous to the vitelline envelope that surrounds oocytes of lower animal species (6, 7).The site of origin of the zona pellucida has been the subject of considerable interest for more than a century (8-10). DespiteThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 1029 numerous morphological, ultrastructural, histological, and autoradiographic studies, it has remained an unresolved issue as to whether the zona pellucida originates from the oocyte, follicle cells, or both (11)(12)(13)(14).Recently, we identified and characterized those proteins that compose the mouse oocyte's zona pellucida (15,16). It has been known for some time, primarily on the basis of its staining properties, that the zona pellucida contains protein and carbohydrate (3, 5), and our recent work demonstrated that the zona pellucida is composed of three different protein species, which we have designated ZP1, ZP2, and ZP3. Having identified the proteins that compose the zona pellucida has permitted us to examine the site of synthesis of these proteins during follicular development. In this report we provide biochemical evidence supporting the idea that the zona pellucida originates from the oocyte itself. MATERIALS AND METHODS Collection and Culture of Denuded and Follicle-EnclosedOocytes. Ovaries were excised from 8 to 15-day-old Swiss albino mice (CD-1, Charles River Breeding Laboratories), placed into standard egg culture medium (SECM) (17), and then punctured with fine steel needles in order to obtain denuded oocytes (i.e., free of follicle cells by morph...

Monoclonal antibodies as probes of the distribution of ZP-2, the major sulfated glycoprotein of the murine zona pellucida.

“…During oogenesis the size ofthe oocyte increases dramatically . The presence of the zona pellucida has been observed in oocytes with a diameter of 30 ttm (26) and its production continues in oocytes as large as 70 pro (3,25). This growth represents at least a fivefold increase in the surface area ofthe oocyte .…”

“…Following fertilization the zona is modified in the "hardening reaction" to prevent polyspermy and may also play a role in protecting the embryo as it passes down the oviduct (29). The murine zona is comprised ofthree sulfated glycoproteins designated ZP-1, ZP-2, and ZP-3 with average molecular weights of 185,000, 140,000, and 83,000, respectively (1,3,25). ZP-2 appears to be the major protein of the murine zona pellucida and is biochemically modified following fertilization (1,4,25).…”

Molecular characterization of egg envelope glycoprotein ZPD in the ovary of Japanese quail (Coturnix japonica)

“…Although no ZPD homolog has been identified in mammalian species, the expression of ZP glycoproteins appears to be regulated differently in different species. There is clear evidence showing that ZP3 is synthesized by the oocytes themselves in mice (Bleil & Wassarman 1980) and in Xenopus (Yamaguchi et al 1989), whereas the granulosa cells in the follicles also participate in the formation of ZP3 in rabbits (Lee & Dunbar 1993) and in cynomolgus monkeys (Martinez et al 1996). On the other hand, a glycoprotein homologous to ZP3 in chorion, the fish egg envelope, is produced in the liver and transported to the ovary by the blood circulation, like vitellogenin (Hamazaki et al 1985(Hamazaki et al , 1989.…”

Synthesis of fucosyl-containing glycoproteins of the vitelline coat in oocytes of Ciona intestinalis (Ascidia).

“…Recently, Bleil and Wassarman (8) have provided convincing evidence that the zona pellucida is synthesized by the oocyte.…”