The mammalian sperm cell offers a unique opportunity to understand the principles of molecular inheritance beyond the genetic code, and this knowledge has a direct bearing on human assisted reproduction practices. The compact sperm chromatin is devoid of virtually all enzymatic machinery associated with normal cellular processes, and only maintains the epigenetic and genetic components that are necessary to mediate inheritance. The hypothesis which we propose to test in this application is that in addition to the father's DNA the sperm nucleus provides molecular components that are essential for the proper initiation and regulation of paternal DNA replication. Without these elements, our published data suggest that the paternal DNA would never be replicated, and embryonic development would not be possible. Significance: In this era of increased ART, more and more human embryos that will become children undergo this first, defining round of DNA synthesis in vitro after clinical manipulation of the gametes. We understand how to keep the DNA intact during gamete storage, but we do not know what parameters are necessary to maintain proper origin recognition sites. There are also important biological implications as the mammalian one-cell embryo is a particularly suitable model for understanding how cells demarcate and license DNA replication origins. DNA replication mechanisms are also important targets for many human diseases. The molecular machinery for the initiation, control, and completion of the complicated process by which the 6 billion base pairs of mammalian DNA is replicated is now well understood. Each origin of replication is """"""""licensed"""""""" by the binding of series of proteins, beginning with the origin recognition complex (ORC) made up of six proteins, ORC1L - ORC6L. When licensed origins enter S-phase, another host of proteins associate in a specific manner to eventually recruit DNA polymerases. Licensing ensures that each component of the entire genome is replicated only once per cycle. In mammalian cells ORC2L-5L remain bound to replication origins throughout the cell cycle, while ORC1L is recruited to the origin in G1, and degraded during S-phase (the fate of ORC6L through the cell cycle is unknown). In the mammalian one cell the paternal and maternal genomes are replicated independently, and asynchronously, and our preliminary data suggest that initiation of DNA synthesis of two pronuclei in the same oocyte cytoplasm can vary by as much as three hours.
In Specific Aim 1 we will determine the timing of licensing in the mouse one cell embryo using pronuclear transfer experiments.
In Specific Aim 2 we will test the prediction of our hypothesis that ORC2L-5L are already present on the maternal chromatin before fertilization, but load onto the male chromatin after fertilization.
In Specific Aim 3 we will test whether ORC2L-5L or ORC1L binds directly to the sperm nuclear matrix, using ORC-GFP fusion proteins. Future studies will identify the epigenetic component in sperm chromatin to which ORC1L-5L proteins bind.

Public Health Relevance

The 30,000 or so genes that make up the human genome physically reside in DNA, a long tape-like molecule. Each cell in the body has the entire DNA sequence, and it takes roughly 6 feet of DNA to encode all 30,000 genes. At fertilization, there are only two copies of this DNA, one from the mother and one from the father, and both need to be replicated flawlessly. This application proposes to test the idea that the sperm cell brings to the egg more than only the DNA, itself, but instructions on how to replicate it properly in the first embryonic cell division.

National Institute of Health (NIH)
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Research Project (R01)
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Cellular, Molecular and Integrative Reproduction Study Section (CMIR)
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Ravindranath, Neelakanta
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University of Hawaii
Anatomy/Cell Biology
Schools of Medicine
United States
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Ward, W Steven (2017) Eight tests for sperm DNA fragmentation and their roles in the clinic. Transl Androl Urol 6:S468-S470
Nguyen, Hieu; James, Nicholas G; Nguyen, Lynn et al. (2017) Higher Order Oligomerization of the Licensing ORC4 Protein Is Required for Polar Body Extrusion in Murine Meiosis. J Cell Biochem 118:2941-2949
Ortega, Michael A; Ko, Myungjun; Marh, Joel et al. (2016) Presence of the Paternal Pronucleus Assists Embryo in Overcoming Cycloheximide Induced Abnormalities in Zygotic Mitosis. J Cell Biochem 117:1806-12
Ortega, Michael A; Nguyen, Hieu; Ward, W Steven (2016) ORC proteins in the mammalian zygote. Cell Tissue Res 363:195-200
Gawecka, Joanna E; Ribas-Maynou, Jordi; Benet, Jordi et al. (2015) A model for the control of DNA integrity by the sperm nuclear matrix. Asian J Androl 17:610-5
Nguyen, Hieu; Ortega, Michael A; Ko, Myungjun et al. (2015) ORC4 surrounds extruded chromatin in female meiosis. J Cell Biochem 116:778-86
Ribas-Maynou, J; Gawecka, J E; Benet, J et al. (2014) Double-stranded DNA breaks hidden in the neutral Comet assay suggest a role of the sperm nuclear matrix in DNA integrity maintenance. Mol Hum Reprod 20:330-40
Gawecka, Joanna E; Marh, Joel; Ortega, Michael et al. (2013) Mouse zygotes respond to severe sperm DNA damage by delaying paternal DNA replication and embryonic development. PLoS One 8:e56385
Ward, W Steven (2013) Isolation of sperm nuclei and nuclear matrices from the mouse, and other rodents. Methods Mol Biol 927:437-44
Ortega, Michael A; Marh, Joel; Alarcon, Vernadeth B et al. (2012) Unique pattern of ORC2 and MCM7 localization during DNA replication licensing in the mouse zygote. Biol Reprod 87:62

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