Preprint of a chapter that appeared in Sperm Cell Research in the 21st Century:
Historical Discoveries to New Horizons, ed. by M. Morisawa (Adthree Publishing Co.,
Ltd., Tokoyo), 2012
Sperm Topoisomerase II May be Inherited by the Paternal Pronucleus
by
Joanna Gawecka and W. Steven Ward
Institute for Biogenesis Research (IBR), John A. Burns School of Medicine, University
of Hawaii at Manoa
Address correspondence to:
W. Steven Ward, Ph.D.
Professor and Director
Institute for Biogenesis Research
John A. Burns School of Medicine
University of Hawaii at Manoa
1960 East-West Rd.
Honolulu, HI 96822
Tel: 808-956-5189
Fax: 808-956-7316
E-mail: [email protected]
Introduction
Our laboratory has focused on one major hypothesis, that the sperm nucleus
provides heritable molecular information to the developing embryo in addition to the
genetic information contained within the DNA sequence of the paternal genome. We
include all these molecular components within the term “epigenetic contributions”. In
our view, most epigenetic contributions of the sperm to the zygote provide key signals
for the proper function of the paternal DNA. Here, we present an extension of our
major hypothesis, that sperm topoisomerase 2B (TOP2B) in incorporated into the
newly formed paternal pronucleus. TOP2B is a key component of the sperm nuclear
matrix and the DNA loop domain organization that the nuclear matrix orchestrates,
both of which we believe becomes part of the paternal pronucleus after fertilization
(see Figure).
Epigenetic Contributions of the Sperm Nucleus to the Embryo
The function of the sperm cell is to deliver the paternal genetic information to the
oocyte to contribute half of the DNA to the newly formed embryo. Researchers are
becoming increasingly aware that this genetic information requires a series of heritable,
epigenetic modifications that accompany the DNA to ensure its proper function. We
have recently proposed that at least six different types of epigenetic instructions
accompany the paternal chromatin that are all required for proper embryonic
development after fertilization1. DNA methylation is the best characterized of these2,3.
The histones that remain in the condensed sperm chromatin at the end of
spermiogenesis have been shown by two groups to bind specific DNA sites4,5. More
recently, it was shown that histone H3.3 is essential for development in the paternal
DNA6. We have demonstrated that the DNA loop domain formation by the sperm
nuclear matrix is essential for paternal DNA replication in the one cell embryo7. Two
other types of proteins in the sperm cell that we predict are important for
embryogenesis are the nuclear matrix and perinuclear theca proteins. Finally, several
groups have provided evidence that sperm born RNA also contribute to
embryogenesis8-10.
DNA Loop Domain Organization is Constant during Spermiogenesis and is
Required for Embryogenesis
Eukaryotic DNA is organized into loop domains ranging from 20 kb to 120 kb in
size, that are attached at their bases to a proteinaceous structure termed the nuclear
matrix11. Sperm chromatin has a similar organization12,13, and this level of chromatin
structure is not affected by the condensation of DNA by protamines during
spermiogenesis14 (see Figure). We previously hypothesized that sperm DNA loop
domain structure was inherited by the paternal pronucleus, and that this chromatin
organization was important for embryogenesis. While our attempts to visualize DNA
loop domain structure in paternal pronuclei failed because of technical limitations, we
did provide evidence that the sperm DNA loop domain structure was necessary for
embryogenesis7,15.
Topoisomerase II is a Nuclear Matrix Protein Associated with DNA Loop Domains
Several years ago, topoisomerase II (TOP2) was shown to be part the nuclear
matrix in somatic cells and part of the mitotic chromosome scaffold16. TOP2 is an
enzyme that unravels DNA by creating a double-strand break in the DNA, passing
another strand of DNA between the cleaved strands, and resealing the doublestranded break17. TOP2 is required for DNA replication because it helps to keep the
newly replicated DNA from becoming entanlged18,19. It later became apparent that
TOP2 was also an important part of the initial degradation of DNA during apoptosis.
DNA degradation occurs when TOP2 creates the double-stranded DNA break, but
does not repair it20-22. Shortly after the TOP2 break, nucleases irreversibly digest the
DNA20,23-25.
We have recently demonstrated that mouse spermatozoa can be induced to
cleave all the DNA into loop-sized fragments in a manner similar to that of TOP2
mediated DNA degradation during somatic cell apoptosis26,27. This fragmentation can
be reversed by treatment with EDTA, a hallmark of TOP2 DNA breakage. At least four
separate laboratories have reported that fully mature spermatozoa contain TOP228-31.
The fact that this degradation occurs in fully mature spermatozoa isolated from the vas
deferens or cauda epididymus, indicates that TOP2 is active in fully condensed sperm
chromatin. To our knowledge, it is the only functional nuclear enzyme that is active in
fully condensed spermatozoa27,29.
TOP2B as a Candidate Sperm Nuclear Matrix Protein that is Inherited by the
Paternal Pronucleus
Three attributes of TOP2 make it an interesting candidate for a sperm protein
that becomes incorporated into the paternal pronucleus. First, it is localized at the sites
of DNA attachment to the nuclear matrix, which is also the site of DNA replication32,33.
The replisome, the group of enzymes and DNA binding proteins that copy the genome,
is located at this fixed site34. As mentioned above, TOP2 is part of the replication
machinery18,19. Second, TOP2 is a structural component of the nuclear matrix and may
help to organize the DNA into loops. The loop organization of different cell types
varies13,35, and sperm TOP2 may be important for the sperm specific DNA organization
that is required for paternal DNA replication7. Third, TOP2 is an enzyme associated
with sperm chromatin that is fully functional in mature spermatozoa27,29. These
features suggest that TOP2 is an integral component of functioning sperm chromatin.
We propose that sperm TOP2 is inherited by the paternal pronucleus at the
bases of DNA loop domains (see Figure). It is possible that all the sperm TOP2
enzymes are replaced by TOP2 proteins supplied by the oocyte after fertilization, but
we view this as unlikely, largely because it would seem unnecessary. We are currently
developing methods to test this hypothesis, directly. If true, TOP2 would represent a
functional enzyme that is inherited by the paternal pronucleus, where it may play an
important role in DNA replication. The most important aspect of this hypothesis,
however, is that not only is the enzyme, itself, passed on from the sperm to the zygote,
but that its positioning along the sperm genome may include information as to which
sites in the paternal genome are to be utilized by the developing embryo, first.
Acknowledgements
This work was supported by NIH Grant HD060722 to W. S. W.
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Figure Legend
DNA Loop Domain Organization in Spermatozoa. Top; Round spermatid
nucleus, sperm nucleus, and paternal zygote pronucleus showing nuclear matrices
(grey), DNA loops (red) and TOP2B (green). Only three of the estimated 50,000 DNA
loops are shown for clarity. In spermatids and paternal pronuclei, DNA is condensed
by histones. In spermatozoa, DNA loops are condensed much more by protamines.
Bottom; Diagrams of DNA loop domains in nuclei that have been extracted with salt.
DNA loop domain organization is identical in round spermatids and spermatozoa,
indicating DNA loop formation is independent of protamine binding (ref. 14). Paternal
pronuclear DNA loop organization is unknown, but presumed to be the same as in
spermatozoa.