Proteomic and transcriptomic analysis reveals an intracellular role for zona pellucida protein 2 in mouse morula-blastocyst transition
Abstract
Background: Since the first description in 1827, the zona pellucida (ZP) has been viewed as an extracellular structure surrounding mammalian oocytes, with three canonical roles: encasement of the oocyte in the follicle, sperm binding at fertilization, and physical protection of the early embryo. However, over the past 10 years, mass spectrometry analyses in this laboratory have repeatedly and consistently detected abundant ZP proteins inside mouse embryos. Therefore, this study investigated whether ZP2, the most abundant of the ZP proteins, might also serve non-canonical functions that operate from inside the embryo rather than from the outside.
Results: Using mass spectrometry and monoclonal immunofluorescence, an intracellular fraction of ZP2 was found to persist throughout preimplantation mouse development, being among the most abundant proteins of the embryo. Intracellular ZP2 underwent an apparent increase at the 8-cell to morula stage rather than the monotonic decline expected of a passive maternal legacy. Basic colocalization analysis showed that part of this intracellular pool overlapped neither the cis-Golgi marker GM130 nor the endoplasmic reticulum marker Calreticulin, identifying a non-Golgi, non-ER intracellular ZP2 fraction. Consistent with this, ZP2 was susceptible to Trim-Away mediated depletion, which typically can act on a protein only if this is cytosolically accessible. Although ZP2 depletion stalled at approximately 50% due to the enormous abundance of ZP2, this partial knockdown delayed the morula-to-blastocyst transition and produced blastocysts with reduced trophectoderm and primitive endoderm and impaired postimplantation development in vitro and in vivo. These effects were specific to ZP2, as they did not occur when Trim-Away was redirected to an irrelevant target. Proteome and transcriptome analyses of ZP2 knockdown embryos returned overrepresented gene ontology terms related to cytoplasmic translation in the proteome, which was confirmed by an O-propargyl-puromycin incorporation assay; and to endoderm development and gastrulation in the transcriptome, which provide a coherent molecular correlate of the morula retardation phenotype.
Conclusions: These results confer ZP2 with hitherto unsuspected intracellular roles during embryogenesis in the mouse model of human reproduction. ZP2 can therefore no longer be referred to as a solely extracellular-operating, oocyte-specific gene product.
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BibTeX
@article{nolte2026proteomic,
title = {{Proteomic and transcriptomic analysis reveals an intracellular role for zona pellucida protein 2 in mouse morula-blastocyst transition}},
author = {Thomas Nolte and Steffen Israel and Hannes C. A. Drexler and Georg Fuellen and Julina Wiese and Michele Boiani},
journal = {BMC Genomics},
year = {2026},
month = oct,
doi = {10.1186/s12864-026-13487-z},
url = {https://link.springer.com/article/10.1186/s12864-026-13487-z},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Proteomic and transcriptomic analysis reveals an intracellular role for zona pellucida protein 2 in mouse morula-blastocyst transition
AU - Thomas Nolte
AU - Steffen Israel
AU - Hannes C. A. Drexler
AU - Georg Fuellen
AU - Julina Wiese
AU - Michele Boiani
PY - 2026
DA - 2026/10/01
JO - BMC Genomics
DO - 10.1186/s12864-026-13487-z
UR - https://link.springer.com/article/10.1186/s12864-026-13487-z
AB - Background: Since the first description in 1827, the zona pellucida (ZP) has been viewed as an extracellular structure surrounding mammalian oocytes, with three canonical roles: encasement of the oocyte in the follicle, sperm binding at fertilization, and physical protection of the early embryo. However, over the past 10 years, mass spectrometry analyses in this laboratory have repeatedly and consistently detected abundant ZP proteins inside mouse embryos. Therefore, this study investigated whether ZP2, the most abundant of the ZP proteins, might also serve non-canonical functions that operate from inside the embryo rather than from the outside. Results: Using mass spectrometry and monoclonal immunofluorescence, an intracellular fraction of ZP2 was found to persist throughout preimplantation mouse development, being among the most abundant proteins of the embryo. Intracellular ZP2 underwent an apparent increase at the 8-cell to morula stage rather than the monotonic decline expected of a passive maternal legacy. Basic colocalization analysis showed that part of this intracellular pool overlapped neither the cis-Golgi marker GM130 nor the endoplasmic reticulum marker Calreticulin, identifying a non-Golgi, non-ER intracellular ZP2 fraction. Consistent with this, ZP2 was susceptible to Trim-Away mediated depletion, which typically can act on a protein only if this is cytosolically accessible. Although ZP2 depletion stalled at approximately 50% due to the enormous abundance of ZP2, this partial knockdown delayed the morula-to-blastocyst transition and produced blastocysts with reduced trophectoderm and primitive endoderm and impaired postimplantation development in vitro and in vivo. These effects were specific to ZP2, as they did not occur when Trim-Away was redirected to an irrelevant target. Proteome and transcriptome analyses of ZP2 knockdown embryos returned overrepresented gene ontology terms related to cytoplasmic translation in the proteome, which was confirmed by an O-propargyl-puromycin incorporation assay; and to endoderm development and gastrulation in the transcriptome, which provide a coherent molecular correlate of the morula retardation phenotype. Conclusions: These results confer ZP2 with hitherto unsuspected intracellular roles during embryogenesis in the mouse model of human reproduction. ZP2 can therefore no longer be referred to as a solely extracellular-operating, oocyte-specific gene product.
SN - 1471-2164
ER -