Abstract
During murine oogenesis, the zona pellucida proteins (ZP1, ZP2, and ZP3) are synthesized and secreted to form an extracellular matrix that surrounds the oocyte and mediates specific biological functions essential to mammalian fertilization and early development. To investigate the relationship among the zona proteins during zona matrix assembly, we have undertaken to inhibit de novo biosynthesis of specific zona proteins with antisense oligonucleotides complementary to the 5'-ends of ZP2 (nucleotide position 19-42) and ZP3 (nucleotide 21-44) mRNAs. When injected into the cytoplasm of growing mouse oocytes, the antisense oligonucleotides targeted specific zona mRNAs for degradation, as confirmed by a RNase protection assay. Individual zona pellucida protein synthesis was followed by immunoprecipitation with ZP2- and ZP3-specific monoclonal antibodies. New zona protein synthesis from the targeted mRNA was abolished, but nontargeted zona protein continued to be synthesized. Interestingly, abolishment of either ZP2 or ZP3 protein synthesis prevented the incorporation of the other protein into the extracellular zona matrix. These results suggest that ZP2 and ZP3 proteins are independent of each other in their biosynthesis but are dependent upon each other for their incorporation into the zona pellucida matrix. This study provides an experimental system in which destruction of a targeted mRNA generates a transient loss-of-expression phenotype during mouse oocyte growth.
Highlights
The three zona proteins are assembled into a matrix that first appears in the early stages of oocyte growth and eventually forms a 7-8-/Lm-thick coat surrounding fully grown 00cytes, which is distinct from the plasma membrane
There are no reported null mutations in the mouse zona pellucida genes that would allow an analysis of the biosynthesis of one zona protein in the absence of another
The specific degradation of either mRNA effectively abolishes the biosynthesis of the corresponding protein
Summary
Vol 270, No., Issue of January 13, pp. 849-853, 1995 Printed in U.S.A. Inhibition of Zona Pellucida Gene Expression by Antisense Oligonucleotides Injected Into Mouse Oocytes*. The zona pellucida proteins (ZP1, ZP2, and ZP3) are synthesized and secreted to form an extracellular matrix that surrounds the oocyte and mediates specific biological functions essential to mammalian fertilization and early development. Abolishment of either ZP2 or ZP3 protein synthesis prevented the incorporation of the other protein into the extracellular zona matrix. We find that the inhibition of either ZP2 or ZP3 protein synthesis prevents the incorporation of the other protein into the extracellular zona matrix of growing mouse oocytes. The zona pellucida is an extracellular matrix that surrounds mammalian oocytes and mediates initial sperm-egg interactions at fertilization, The mouse zona is composed of three sulfated glycoproteins, ZPll (180-200 kDa), ZP2 (120-140 kDa), and ZP3 (83 kDa) [1, 2].
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