Download View Full PDF - Biochemical Society Transactions

Independent Meeting held at University of Leeds, U.K., 15–18 November 2006. Organized and Edited by M. Brinkworth (Bradford, U.K.), J. Cummins
(Murdoch University, Australia), S. Krawetz (Wayne State University, U.S.A.), D. Miller (Leeds, U.K.) and C. Spadafora (Institutio Superiore di Sanità, Italy).
The Testis as a Conduit for Genomic Plasticity:
an advanced interdisciplinary workshop
D. Miller*1 , M. Brinkworth† and D. Iles‡
*Reproduction and Early Development Group, Leeds Institute of Genetics and Health Therapeutics, University of Leeds, Leeds LS2 9JT, U.K., †Department of
Biomedical Sciences, Richmond Building, University of Bradford, Manninghan Lane, Bradford BD7 1DP, U.K., and ‡Integrative and Comparative Biology,
Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, U.K.
Abstract
The premise for this unusual amalgamation of reproductive biologists, molecular geneticists and evolutionary
biologists rested on the evidence-based assumption that reproductive tissues could be ideal environments
for the expression and transmission of transposable elements that can move into new locations in the
genome. These elements include DNA transposons and retrotransposons that, together, make up over 40%
of the human genome. The testis may be a particularly good niche for their expression because of the unique
dynamic of spermatogenesis, where the methylation–demethylation status of germ cell DNA is at its most
plastic. Hence windows of opportunity can arise that may release transposable elements from the tight
regulatory control of expression imposed on them by bulk DNA methylation. As the testis is where most
mutations become embedded in the germline, the meeting included a number of keynote presentations
that aimed to examine the potential for transposable elements to heritably alter the genome and effect
variation independently of the usual Mendelian mechanisms. In essence, could the testis be one of the
favoured sites where genomic plasticity makes its mark?
Introduction
In mid-November 2006, the University of Leeds in
West Yorkshire hosted a unique meeting that attracted
internationally recognized scientists working in the fields of
andrology and gynaecology and brought them into contact
with colleagues working on chromatin organization, DNA
transposition and RNA retrotransposition. This unconventional meeting was prompted by a strong curiosity on the part
of the organizers to see whether light could be shed on the
possibility that conditions in the testis favour the expression
or reshuffling of genetic elements that could be transmitted
to offspring (primarily, although not exclusively) via the male
germline. If this indeed proves to be so, then the testis could
be considered to be a ‘breeding ground’ for effecting rapid
genomic evolutionary change quite independently of the low
Key words: genomic plasiticity, retrotransposon, reverse transcriptase, spermatozoa, testis,
transgenesis.
Abbreviations used: LINE, long interspersed nuclear element; LTR, long terminal repeat; ORF,
open reading frame; ZGA, zygotic genome activation.
1
To whom correspondence should be addressed (email [email protected]).
The Testis as a Conduit for Genomic Plasticity
The Testis as a Conduit for Genomic
Plasticity
rates of change generated by normal mutational events and
the accepted Mendelian rules that govern the generation of
diversity in sexual reproduction. The future potential for harnessing such mechanisms for commercial and clinical exploitation cannot be overlooked, particularly since it brings with it
important ethical issues that should be addressed in advance.
The impetus for the meeting arose from several lines of
experimental evidence suggesting that the testis in general and
spermatozoa in particular may be favourable environments
or vehicles for the movement of genetic elements from
one location to another [1–3]. Spermatozoa, for example,
under certain circumstances, can adsorb exogenous DNA
and RNA and carry it into the oocyte [4]. The resulting
embryos can be transformed by the introduced gene, affecting
genotype and phenotype accordingly. This phenomenon
is the basis for sperm-mediated transgenesis that has
been successfully used to generate transgenic animals [5].
Interestingly, while full integration events do occur, they are
infrequent; instead, transforming elements usually persist and
are transmitted as extrachromosomal elements that resemble
C 2007
Biochemical Society
605
606
Biochemical Society Transactions (2007) Volume 35, part 3
proviral assemblies [retrotransposons containing LTR (long
terminal repeat) flanking regions]. This observation may
in turn be related to the observed preference of proviral
elements for reproductive tissues in males and females where
large numbers of these normally quiescent endogenous
retroviruses can be detected [6,7]. In addition, the mammalian
testis is a known site for the expression of at least one of
the ORFs (open reading frames) of the non-LTR LINE-1
(long interspersed nuclear element 1), ORF1, which encodes
a 40 kDa RNA-binding protein. The other (ORF2) encodes
an RNA-dependent DNA polymerase and endonuclease that
is essential for both the replication of LINE-1 itself and for
the replication of Alu elements derived originally from 5S
RNAs [8–10].
Although the experimental data are inconclusive, it
has been suggested that the disproportionately large
number of testis-expressed pseudogenes has arisen from
intron-containing precursors processed via retrotransposonmediated reverse transcription [11]. This phenomenon may
have been driven by the shutdown of essential X-linked
genes in spermatogenesis and the reliance on copies that
have been retrotransposed on to chromosomes in the distant
evolutionary past that remain active. However, the original
exponents of this explanation have concluded that the
phenomenon, if it does exist, is probably not related to
spermatogenesis itself [12].
Nevertheless, the testis may be an environment favouring
the activity and influence of extra- and intra-genomic
elements on the host genome for a number of reasons.
First, the process of spermatogenesis involves the epigenetic
reprogramming of the haploid genome, during which time
imprints are erased and re-established [13]. The process
involves global demethylation of spermatozoal DNA early in
spermatogenesis and hence the lifting, albeit temporarily, of
restraints that normally repress the expression of repetitive
elements, such as LINEs. More significantly, the testis has
far higher levels of the RNA polymerase II machinery than
other tissues, and one effect of this unusual abundance is
the lowering of normal promoter thresholds for driving gene
expression [14]. Therefore, in the testis, conditions exist
that could favour the expression of elements that would
otherwise be repressed in other tissues. Such an environment
is probably a by-product of the requirement to ensure
that spermatids accumulate and store sufficient RNAs for
storage and eventual translation during the condensation
stages of spermatogenesis when developing spermatids (and
also, apparently, ejaculate spermatozoa [15]) rely exclusively
on stored RNAs for protein synthesis [16]. A similar situation
may occur in the developing follicle, where large stores of
maternal RNA are required to support protein synthesis
de novo following germinal vesicle breakdown and during the
early cleavage stages of the embryo before activation of
the embryonic genome [17].
While the mechanisms described above may facilitate the
intracellular transposition of gene elements in the germline,
the intimate association between developing spermatids
and the Sertoli cells may also afford opportunities for
C 2007
Biochemical Society
the exchange of genetic information between soma and
germline. Moreover, the events leading up to fusion of
egg and sperm, as well as the fusion process itself, may
provide opportunities for intercellular gene transfer, possibly
mediated by endogenously expressed retroviral elements
or LINEs. These forms of molecular genetic cross-talk,
if they exist and become fixed in the germline, could be
transmitted vertically to offspring. However, the meeting
focused on aspects of the horizontal (lateral) transfer of
genetic information made possible by the entry of the sperm
into the egg; in essence, asking whether the generation of
transgenic embryos by sperm-mediated transfer of foreign
genes can occur naturally. The sources of foreign DNA may
be numerous and include genetic elements derived from the
normal flora of the female reproductive tract. Evidence that
events such as these have led to the acquisition of tumoursuppressor genes in the distant evolutionary past has been
essentially overlooked [18].
The workshop
The workshop itself attempted to broaden the issue of
genomic plasticity by breaking it down into separate themes.
The first of these, ‘Is the testis a permissive environment
for retroposition?’ included sessions from John Moran
(University of Michigan, Ann Arbor, MI, U.S.A.), Jim
Shapiro (University of Chicago, Chicago, IL, U.S.A.) and
Jürgen Brosius (University of Münster, Münster, Germany).
The contributors looked more closely at how the potential
for genome evolution brought about retrotransposon activity
than whether the testis itself supports such activity [19–21].
However, each talk more than reinforced the hypothesis that
retrotransposons such as LINE-1 are a major force in shaping
the genome quite independently of, but complementary
to, both spontaneous mutation and meiotic recombination.
Deborah Bourc’his (Institut Jacques Monod, Paris, France),
however, showed that retrotransposon expression is normally
under tight control in the testis and that severe disruption
of spermatogenesis is one outcome of elevated LINE-1
expression caused by engineered derepression of methylation
(Dnmt3L-knockout) [22]. This gene encodes a non-enzymic
protein that is a critical component of the methylationcontrol mechanism. Nevertheless, retrotransposon expression may be permitted in embryonic gonocytes and Atype spermatogonia where chromatin is largely demethylated.
The murine IAP (intracisternal A particle), an endogenous
retrovirus, is also expressed in these early stages [2]. Hence,
any retrotranspositional event in the testis would, under
physiological circumstances, be able to occur in the early
(pre-meiotic) phase of spermatogenesis and could be fixed
in the germline at this point. Its inheritance would then
depend on a combination of factors, such as the frequency
of transposition, whether the event did not compromise subsequent stages of spermatogenesis, whether the transposed
sperm actually fertilized the egg, and, finally, whether the
resulting zygote was developmentally viable.
Another session, ‘DNA and RNA dynamics of gametes:
new portals for lateral gene transfer?’, examined potential
The Testis as a Conduit for Genomic Plasticity
mechanisms for the generation of transformed zygotes mainly
via the introduction of exogenous DNA (or RNA) into
spermatozoa. One speaker (Barbara Knowles, The Jackson
Laboratory, Bar Harbor, ME, U.S.A.) showed that the oocyte
also is an excellent vehicle for retrotransposon-mediated
genomic rearrangements by virtue of the high levels of
(retrotransposon) RNA present in the ooplasm [23]. As
illuminated by speakers from the previous session, LINE1s have the potential to generate new gene constructs by
juxtaposing reverse transcriptase-mediated cDNAs from one
location with promoter elements to another location. They
can also create new promoters via a similar transposition and
integration mechanism [20]. Either scenario is a direct consequence of their natural mode of transmission, which is
normally hidden unless it has strong phenotypic consequences (deleterious insertions have been associated with
disease states).
This intrinsic form of genomic plasticity that may
potentially be transmitted vertically to offspring is both
different from, and related to, the form arising by the
introduction and transmission of exogenous (extrinsic) DNA
and probably occurs in both male and female germlines. On
the one hand, intrinsic plasticity may involve intracellular
(germ cell) and intercellular (soma to germ cell) transmission
of genetic information via endogenous retroelements. On
the other, extrinsic plasticity relies on the introduction and
transmission of exogenous elements in the form of DNA
or RNA into the sperm or egg and hence into the zygote,
although the sperm is the more likely vehicle. The egg as
transmitting vehicle was championed by Barbara Knowles,
while the sperm as transmitting vehicle was the topic for
discussion in the final session ‘Chromatin domains and
imprinting phenomena’ where Corrado Spadafora (Italian
National Institute of Health, Rome, Italy) showed that
spermatozoa are able to transfer exogenous DNA into the
egg and thus generate transformed zygotes. Spermatozoa can
also take up RNA and convert it into DNA, while splicing
out any introns in the process [24]. Such an activity demands
the presence of an active reverse transcriptase, which has been
demonstrated biochemically in sperm extracts and in situ by
immunoelectron microscopy.
As indicated above, sperm-mediated transgenesis is primarily an episomal event that resembles the free proviral stage
of endogenous retroviral expression; indeed, integration and
hence fixation into the genome may be a comparatively rare
event. Clearly, that imposes limitations on the development
of useful applications of the phenomena. However, such
comparisons show that they use common mechanisms that
rely on reverse transcriptase for their modus operandi.
Evidence for the central importance of reverse transcriptase in
embryonic development has been published, whereby inhibition of the enzyme with chemical-, antibody- and antisensebased strategies irreversibly prevents ZGA (zygotic genome
activation) (in murine embryos) [25]. These data augment
the discovery of high levels of retrotransposon transcripts
in murine oocytes, which in tandem with the potential for
LINE-1-mediated gene shuffling, suggests that the process of
ZGA may actually involve a reverse transcriptase-dependent
transcriptional switch. The mechanism of such a switch
could include a reverse transcriptase-mediated juxtaposition
or generation of a promoter close to a master gene that
initiates ZGA.
A number of talks, including contributions from Steve
Ward (University of Hawaii, Honolulu, HI, U.S.A.),
Andrei Zalensky (Jones Institute, Norfolk, VA, U.S.A.),
Stephen Krawetz (Wayne State University, MI, U.S.A.) and
Rod Balhorn (Lawrence Livermore National Laboratory,
CA, U.S.A.) focused on sperm chromatin packaging and
architecture [26–28]. During spermatogenesis in many species
(including all mammals), somatic histones are replaced by
transition proteins and then by protamines. These smaller
arginine-rich proteins facilitate greater compaction of sperm
chromatin and the repackaging dynamics that drive histone
substitution may open a window to the reshuffling of endogenous DNA. Another window may be opened by the endogenous endonuclease-mediated ‘breakdown’ of spermatozoal
chromatin that occurs when spermatozoa are incubated for
prolonged periods in fertilizing media or following contact
with exogenous nucleic acids [29]. In all scenarios, DNA
strand breaks must occur that could potentially allow
DNA sequences to be shuffled into novel combinations.
These mechanisms could drive genomic plasticity through
the shuffling of endogenous sequences, but interactions
with exogenous nucleic acids could extend diversity even
further by providing a route for the lateral transfer of
genetic information from almost any source. Perhaps the
autodigestion of chromatin that normally follows exposure
to foreign nucleic acids serves to prevent its deleterious
transmission by triggering apoptosis or an apoptosis-like
phenomenon that kills affected cell(s). Nevertheless, an
understanding of the regulation of such DNA strand breakage
could have potential for overcoming the fact that most
exogenous DNA carriage is episomal.
The mechanism for sperm-mediated transgenesis relies
on introduced DNA. However, Minoo Rassoulzadegan
(University of Nice-Sophia Antipolis, Nice, France) presented data showing that spermatozoal RNA can also be
introduced to the egg and generate (murine) zygotes that
show a classical epigenetic alteration to phenotype that is
more commonly observed in plants [30]. The paramutated
genotype is apparently caused by the sperm-mediated
introduction of aberrant RNA generated by disruption
to one of the c-Kit alleles and can be transmitted even
in a fully wild-type background. This is the first report
showing that spermatozoal RNA can affect phenotype and
brings us back to the original rationale behind the meeting
and the observation that the germline is a potentially
excellent environment for the transcription of RNAs that
would otherwise be repressed (in somatic cells). We can
only speculate that other, unidentified, RNAs, including
miRNAs (microRNAs), may also introduce subtle epigenetic
influences on the zygote that lead to similarly subtle
alterations to phenotype. Although there is no suggestion
in this work that the RNA is ever embedded into the zygotic
C 2007
Biochemical Society
607
608
Biochemical Society Transactions (2007) Volume 35, part 3
genome, it is possible that beneficial alterations to phenotype
generated by paramutation effects and by sperm-mediated
transgenesis could become fixed and stably heritable. Indeed,
work arising from Michael Skinner’s laboratory (Washington
State University, Seattle, WA, U.S.A.) suggests that the testis
is uniquely sensitive to environmental perturbation and that
epigenetic changes to the germline are stably heritable [31].
The mechanisms that could bring this about are possibly in
place in both sperm and egg, although they are kept under
tight regulatory control. Nevertheless, gamete generation,
and, indeed, the whole sexual reproductive process, may
be a good ‘environment’ where non-Mendelian effects
on genome organization and evolution can occur. In
mammals, genomic imprinting has evolved to ensure that
reproduction can only proceed successfully if male and female
genomes are involved. The allied mechanism governing
methylation and the repression of repetitive ‘selfish’ elements
are under dynamic control in the germline and it is here that
these elements are most likely to be activated and where the
best opportunities may lie for their proliferation.
As a final note before leaving the reader to the
following written contributions provided by speakers, we
would remind you about a somewhat overlooked and
intriguing paper that appeared in 1998 from Christine
Gosden’s group (University of Liverpool, Liverpool, U.K.).
This report provided compelling evidence for the transmission of new genetic traits in the reproductive process
by lateral gene transfer [18]. In effect, they concluded that
tumour-suppressor genes arose via this route from what were
originally yeast mating factor elements. In this context, the
benefits of co-opting a set of genes with one function in
one organism for a different function in a wholly unrelated
organism are clearly obvious. The unanswered questions
are how frequently do such things occur in Nature, how
important is their influence on shaping genomes, and what
might they tell us about how the environments in which
reproductive processes might influence their outcomes? We
think that this special advanced workshop helped us to understand these questions more fully and gave the contributors
much to think about for future experiments aimed at
answering them.
We thank everyone who contributed to the meeting and to the
Genetics Society and Society for Reproduction and Fertility for their
generous financial support. Finally, we thank the Biochemical Society
for giving us the opportunity to publish these proceedings here in
Biochemical Society Transactions and the British Andrology Society
C 2007
Biochemical Society
for their full commitment to the project by way of its integration
into their annual meeting.
References
1 Branciforte, D. and Martin, S.L. (1994) Mol. Cell. Biol. 14, 2584–2592
2 Dupressoir, A. and Heidmann, T. (1996) Mol. Cell. Biol. 16, 4495–4503
3 Pittoggi, C., Beraldi, R., Sciamanna, I., Barberi, L., Giordano, R., Magnano,
A.R., Torosantucci, L., Pesarmona, E. and Spadafora, C. (2006)
Mol. Reprod. Dev. 73, 1239–1246
4 Maione, B., Lavitrano, M., Spadafora, C. and Kiessling, A.A. (1998)
Mol. Reprod. Dev. 50, 406–409
5 Smith, K. and Spadafora, C. (2005) BioEssays 27, 551–562
6 Nilsson, B.O., Kattstrom, P.O., Sundstrom, P., Jaquemin, P. and Larsson, E.
(1992) Virus Genes 6, 221–227
7 Patience, C., Wilkinson, D.A. and Weiss, R.A. (1997) Trends Genet. 13,
116–120
8 Branciforte, D. and Martin, S.L. (1992) Mol. Biol. Cell. 3, A101
9 Singer, M.F., Krek, V., McMillan, J.P., Swergold, G.D. and Thayer, R.E.
(1993) Gene 135, 183–188
10 Sassaman, D.M., Dombroski, B.A., Moran, J.V., Kimberland, M.L., Naas,
T.P., DeBerardinis, R.J., Gabriel, A., Swergold, G.D. and Kazazian, H.H.
(1997) Nat. Genet. 16, 37–43
11 Kleene, K.C., Mulligan, E., Steiger, D., Donohue, K. and Mastrangelo, M.A.
(1998) J. Mol. Evol. 47, 275–281
12 Zhong, X.M. and Kleene, K.C. (1999) Mamm. Genome 10, 6–12
13 Trasler, J.M. (1998) Semin. Cell Dev. Biol. 9, 467–474
14 Schmidt, E.E. and Schibler, U. (1995) Development 121, 2373–2383
15 Gur, Y. and Breitbart, H. (2006) Genes Dev. 20, 411–416
16 Kleene, K.C. (1996) Mol. Reprod. Dev. 43, 268–281
17 Gosden, R., Krapez, J. and Briggs, D. (1997) BioEssays 19, 875–882
18 Gosden, C.M., Liloglou, T., Nunn, J., Gardener, D., Nickson, P., Crampton,
J.M. and Field, J.K. (1998) Int. J. Oncol. 12, 5–35
19 Brosius, J. and Tiedge, H. (1995) Virus Genes 11, 163–179
20 Moran, J.V., DeBerardinis, R.J. and Kazazian, Jr, H.H. (1999) Science 283,
1530–1534
21 Shapiro, J.A. and von Sternberg, R. (2005) Biol. Rev. Cambridge
Philos. Soc. 80, 227–250
22 Bourc’his, D. and Bestor, T.H. (2004) Nature 431, 96–99
23 Evsikov, A.V., de Vries, W.N., Peaston, A.E., Radford, E.E., Fancher, K.S.,
Chen, F.H., Blake, J.A., Bult, C.J., Latham, K.E., Solter, D. and Knowles, B.B.
(2004) Cytogenet. Genome Res. 105, 240–250
24 Sciamanna, I., Barberi, L., Martire, A., Pittoggi, C., Beraldi, R.,
Giordano, R., Magnano, A.R., Hogdson, C. and Spadafora, C. (2003)
Biochem. Biophys. Res. Commun. 312, 1039–1046
25 Beraldi, R., Pittoggi, C., Sciamanna, I., Mattei, E. and Spadafora, C. (2006)
Mol. Reprod. Dev. 73, 279–287
26 Balhorn, R., Weston, S., Thomas, C. and Wyrobek, A.J. (1984)
Exp. Cell Res. 150, 298–308
27 Zalensky, A.O., Allen, M.J., Kobayashi, A., Zalenskaya, I.A., Balhorn, R. and
Bradbury, E.M. (1995) Chromosoma 103, 577–590
28 Kramer, J.A. and Krawetz, S.A. (1996) J. Biol. Chem. 271, 11619–11622
29 Sotolongo, B., Lino, E. and Ward, W.S. (2003) Biol. Reprod. 69,
2029–2035
30 Rassoulzadegan, M., Grandjean, V., Gounon, P., Vincent, S., Gillot, I. and
Cuzin, F. (2006) Nature 441, 469–474
31 Anway, M.D., Cupp, A.S., Uzumcu, M. and Skinner, M.K. (2005) Science
308, 1466–1469
Received 12 March 2007