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Generation of biologically active retro-genes upon interaction of mouse spermatozoa with exogenous DNA

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Mature spermatozoa of most animal species can spontaneously take up foreign DNA molecules which can be delivered to embryos upon fertilization. Following this procedure, transgenic animals of various species have been generated. We recently discovered a reverse transcriptase (RT) activity in mouse spermatozoa that can reverse-transcribe exogenous RNA molecules into cDNA copies. These cDNA copies are transferred to embryos at fertilization, mosaic propagated as non-integrated structures in tissues of founder individuals and further transmitted to F1 progeny. Reverse-transcribed sequences behave as functional genes, being correctly expressed in tissues of F0 and F1 animals. To learn more about this mechanism and further characterize the reverse transcription step, we have now incubated spermatozoa with a plasmid harboring a green fluorescent protein (EGFP) retrotransposition cassette interrupted by an intron in the opposite orientation to the EGFP gene. We found that reverse-transcribed spliced EGFP DNA sequences are generated in sperm cells and transmitted to embryos in IVF assays. After implantation in foster mothers, embryos developed into mice that expressed EGFP in the blood vessel endothelia of a variety of organs. The EGFP-encoding cDNA sequences were detected in positive tissues as extrachromosomal mosaic-propagated structures, maintained in low-copy number (<1 copy/genome), and mosaic transmitted from founders to the F1 progeny. These results indicate that an efficient machinery is present in mature spermatozoa, which can transcribe, splice, and reverse-transcribe exogenous DNA molecules. This mechanism is implicated in the genesis and non-Mendelian propagation of new genetic information besides that contained in chromosomes.
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MOLECULAR REPRODUCTION AND DEVELOPMENT 73:1239 1246 (2006)
Generation of Biologically Active Retro-genes
Upon Interaction of Mouse Spermatozoa
With Exogenous DNA
CARMINE PITTOGGI,
1
ROSANNA BERALDI,
1,2
ILARIA SCIAMANNA,
1
LAURA BARBERI,
1
ROBERTO GIORDANO,
3
ANNA ROSA MAGNANO,
2,3
LILIANA TOROSANTUCCI,
4
EDOARDO PESCARMONA,
5
AND CORRADO SPADAFORA
1
*
1
Istituto Superiore di Sanita
`(ISS), Rome, Italy
2
Department of Pediatrics, Obstetrics and Reproductive Medicine, University of Siena, Siena, Italy
3
Institute of Clinical Physiology, National Research Council (CNR), Siena, Italy
4
Institute of Molecular Biology and Pathology, CNR, Rome, Italy
5
Department of Experimental Medicine and Pathology, University ‘‘La Sapienza,’’ Rome, Italy
ABSTRACT Mature spermatozoa of most
animal species can spontaneously take up foreign
DNA molecules which can be delivered to embryos
upon fertilization. Following this procedure, transgenic
animals of various species have been generated. We
recently discovered a reverse transcriptase (RT) activity
in mouse spermatozoa that can reverse-transcribe
exogenous RNA molecules into cDNA copies. These
cDNA copies are transferred to embryos at fertilization,
mosaic propagated as non-integrated structures in
tissues of founder individuals and further transmitted
to F1 progeny. Reverse-transcribed sequences behave
as functional genes, being correctly expressed in
tissues of F0 and F1 animals. To learn more about
this mechanism and further characterize the reverse
transcription step, we have now incubated spermato-
zoa with a plasmid harboring a green fluorescent
protein (EGFP) retrotransposition cassette interrupted
by an intron in the opposite orientation to the EGFP
gene. We found that reverse-transcribed spliced EGFP
DNA sequences are generated in sperm cells and
transmitted to embryos in IVF assays. After implanta-
tion in foster mothers, embryos developed into mice
that expressed EGFP in the blood vessel endothelia of a
variety of organs. The EGFP-encoding cDNA sequ-
ences were detected in positive tissues as extrachro-
mosomal mosaic-propagated structures, maintained
in low-copy number (<1 copy/genome), and mosaic
transmitted from founders to the F1 progeny. These
results indicate that an efficient machinery is present in
mature spermatozoa, which can transcribe, splice, and
reverse-transcribe exogenous DNA molecules. This
mechanism is implicated in the genesis and non-
Mendelian propagation of new genetic information
besides that contained in chromosomes. Mol. Reprod.
Dev. 73: 1239–1246, 2006. ß2006 Wiley-Liss, Inc.
Key Words: Sperm cells; reverse transcriptase;
transgenic mice; extrachromosomal structures;
sperm-mediated gene transfer (SMGT)
INTRODUCTION
It is now well established that mature spermatozoa
of all species spontaneously take up foreign DNA
molecules and deliver them to embryos at fertilization
(for reviews see Spadafora, 1998; Chan et al., 2000;
Smith and Spadafora, 2005). Following this ‘‘sperm-
mediated gene transfer’’ (SMGT) procedure, transgenic
animals of various species have been generated. While
studying the molecular mechanisms that mediate the
process of sperm/DNA interaction, we have discovered a
variety of unsuspected metabolic functions in the nuclei
of mature spermatozoa, opposing the traditional view
that these cells are metabolically inert. These functions
include: the active uptake and intracellular transport of
exogenous nucleic acid molecules (Spadafora, 1998), a
possible mechanism of integration (Zoraqi and Spada-
fora, 1997; Spadafora, 1998), the activation of endogen-
ous nucleases (Maione et al., 1997; Pittoggi et al., 1999),
and a sperm endogenous reverse transcriptase (RT)
activity (Giordano et al., 2000), probably encoded by an
active fraction of the sperm genome enriched in LINE-1
elements (Pittoggi et al., 1999). We have shown that this
RT can reverse-transcribe exogenous poliovirus RNA
into cDNA fragments that are then transferred to
embryos upon fertilization (Giordano et al., 2000).
We recently decided to attempt the same type of
experiment using an RNA population transcribed from a
ß2006 WILEY-LISS, INC.
Grant sponsor: Italian Ministry of Health; Grant sponsor: Istituto
Superiore di Sanita
`(I. S. and C. P.); Grant sponsor: Ministry of
Education, University and Research (MIUR) (R. B.); Grant sponsor:
Istituto Superiore di Sanita
`; Grant number: R002; Grant sponsor:
Italian Ministry of Health (C. S.).
Laura Barberi’s present address is Department of Histology and
Medical Embryology and Interuniversity Institute of Myology,
University ‘‘La Sapienza,’’ Rome, Italy.
*Correspondence to: Corrado Spadafora, Istituto Superiore di Sanita
`,
Viale Regina Elena 299, 00161 Rome, Italy. E-mail: cspadaf@tin.it
Received 2 March 2006; Accepted 14 April 2006
Published online 18 July 2006 in Wiley InterScience
(www.interscience.wiley.com).
DOI 10.1002/mrd.20550
b-gal-containing construct: we found that a functional
b-gal-containing ‘retro-gene’ (i.e., generated by retro-
transcription of an RNA template) was propagated and
expressed in tissues of adult founders and further
transmitted to F1 individuals (Sciamanna et al., 2003).
Here, we have sought to determine whether reverse-
transcribed sequences competent for expression can be
generated in the offspring via the endogenous RT even
when spermatozoa are incubated with exogenous DNA,
or, in other words, whether a retrotranscription step is
an inherent step in the SMGT procedure. To answer this
question, spermatozoa were incubated with a DNA
construct harboring a EGFP-based retrotransposition
cassette interrupted by a g-globin intron cloned in the
opposite orientation to the reporter gene; we then
investigated whether processed cDNA products were
generated. To that aim, DNA-loaded spermatozoa were
used in IVF assays to produce embryos or adult animals.
We found that non-integrated spliced EGFP cDNA was
directly generated in spermatozoa, transferred to
embryos, and propagated in tissues of both founder
animals and F1 progeny, indicating that exogenous
EGFP-containing DNA construct underwent a sequen-
tial process that included: (i) transcription of the foreign
DNA, (ii) splicing of the resulting primary RNA
transcript, and (iii) reverse transcription. Moreover,
expression of the EGFP reporter gene was detected in
the endothelial lamina of blood vessels of a variety of
organs in adult animals. These results support the
conclusion that the sperm endogenous RT mediates a
novel mechanism that can generate and propagate new
genetic information.
MATERIALS AND METHODS
Preparation of Spermatozoa, Uptake of Foreign
DNA and RNA, In Vitro Fertilization (IVF)
Germ-free mice were purchased from Charles River
Italia (Calco, Italy). Epididymal spermatozoa were
obtained from CD1 murine strain, whereas oocytes were
collected from superovulated B6D2F1 females. Epidi-
dymal spermatozoa collection, IVF experiments, and
embryo cultures were as described (Zaccagnini et al.,
1998). Sperm cells were suspended in fertilization
medium (FM, Whittingham, 1971) supplemented with
4 mg/ml BSA and incubated with plasmid DNA (50 ng/
10
6
spermatozoa) for 30 min. Particular care was taken
to prepare pure sperm cell samples and avoid contam-
ination by somatic cells. To that aim, we avoided
squeezing epididymis and collected spermatozoa that
were spontaneously released through holes made by
simply puncturing the epididymis with a needle. After a
30–60 min ‘‘swim-up’’ selection step of mobile cells, the
purity of sperm cell preparations was checked by phase
contrast microscopy. Preparations containing somatic
cells were discarded.
DNA Extraction, PCR, and
Southern Blot Analysis
Genomic DNA was extracted from both spermatozoa
and tissue fragments of various organs. DNA was
purified from sperm nuclei as described (Giordano
et al., 2000). Embryos at various developmental stages
were collected in groups and lysed in buffer containing
15 mM Tris pH 7.5, 3 mM EDTA, 150 mM CaCl
2
, 0.4%
SDS, and 0.1 mg/ml Proteinase K for 45 min at 558C.
Proteinase K was inactivated by incubating at 658C for
20 min. Routinely we used 1 ml of lysis buffer for each
embryo at 2/4-cell stage and 4 ml for each morula or
blastocyst. Tissues withdrawn from born animals were
minced with a razor blade and incubated in tissue lysis
buffer (10 mM Tris pH 8.0, 2 mM EDTA, 1% SDS, and
Proteinase K 100 mg/ml) for at least 15 hr at 378C. DNA
samples were then purified by sequential phenol/chloro-
form extractions and precipitated with three volumes of
cold ethanol. Aliquots of lysed embryos, or 300 ng of DNA
from tissue samples, were subjected to direct PCR
amplification using the Platinum Taq polymerase kit
(Invitrogen, USA). EGFP-INT amplification was per-
formed usingthe following pair of forward(F) and reverse
(R) oligonucleotide primers flanking the splicing sites:
(F) 50-GCACCATCTTCTTCAAGGACGAC-30, (R) 5-
TCTTTGGCTCAGGGCGGACTG-30.
Samples were pre-incubated for 2 min at 948C, then
subjected to 35 cycles of amplification as follows: 30 sec
at 948C, 30 sec at 628C, 1 min at 728C, in a PTC-200 MJ
Research DNA cycler. PCR amplification products were
routinely fractionated through 1.4% agarose gels,
stained with ethidium bromide and Southern blotted.
Filters were hybridized using the following internal
end-labeled oligonucleotide as the probe for EGFP
amplification:
50-CAGAACACCCCCATCGGCGACGGCCCCGTG-30.
Probe labeling, filter hybridization, and washing were
essentially as described (Giordano et al., 2000). Filters
were then exposed to HR-E30 X-ray Fuji films.
Southern blot analysis of genomic DNA was per-
formed by restricting 20 mg aliquots of DNA extracted
from various tissues restricted with EcoRI restriction
nuclease and fractionated on 1% agarose gels. Blotting
and hybridization procedures were as already described
(Sciamanna et al., 2000). Filters were hybridized with a
radioactively labeled EGFP and Htf9/RanBP1 (Di
Matteo et al., 1995) probes.
Histochemistry
All tissue specimens were formalin-fixed and paraffin
embedded according to standard protocols. Immunohis-
tochemical assays were carried out using the mono-
clonal antibody GFP (B-2) raised against a recombinant
protein corresponding to amino acids 1–238 represent-
ing the full-length GFP of Aequorea victoria (Santa Cruz
Biotechnology, USA) at a working dilution of 1:250. The
reaction was visualized using Universal LSAB2/HRP kit
(Dako, Denmark).
RESULTS
Conversion of an Intron-Containing EGFP Gene
to a Processed cDNA in Spermatozoa
Our previous results indicate that sperm cells can
activate an endogenous RT activity to generate new
Molecular Reproduction and Development. DOI 10.1002/mrd
1240 C. PITTOGGI ET AL.
DNA sequences from RNA templates and propagate
them to early embryos (Giordano et al., 2000) and to F0
and F1 offspring populations (Sciamanna et al., 2003).
In order to get more insight into the mechanism(s) of
endogenous reverse transcription and identify other
molecular steps implicated in the generation of reverse-
transcribed sequences in spermatozoa, we made use of
the retrotransposition cassette pBSKS-EGFP INT
(kindly donated by E. Ostertag and H.H. Kazazian).
The map in Figure 1A shows that in this construct the
EGFP gene is interrupted by a g-globin derived intron,
cloned in the opposite orientation relative to the reporter
gene (Ostertag et al., 2000). PCR amplification using a
pair of primers flanking the splicing sites (arrowed)
enabled us to discriminate between the original,
unspliced DNA, and the spliced product.
Constant amounts of spermatozoa were incubated
with increasing amounts of pBSKS-EGFP INT DNA for
30 min; thereafter sperm cells and supernatants were
recovered by centrifugation, processed for DNA extrac-
tion, and DNA populations from both sources were
subjected to direct PCR amplification. Results in
Figure 1B show that an unspliced, 1243 bp-long
amplification product is present in all samples, as
expected; in addition, a newly generated 342 bp spliced
band is also present in variable amounts in the DNA
samples extracted from spermatozoa that had been pre-
incubated with plasmid (lanes 1–6), but not in the DNA
from plasmid samples incubated in buffer in the absence
of sperm cells (lanes 7–10).
The spliced band was more abundant in sperm cell
samples that were incubated with the highest DNA
amounts (lanes 3, 6), and predominantly accumulated in
the medium (lane 3), while being only partially retained
in nuclei (lane 6). The absence of the spliced fragment in
buffer-incubated construct samples (lanes 7– 10), or in
DNA extracted from F9 teratocarcinoma cells lipofected
with the intron-containing construct (data not shown),
rules out the possibility that its presence after incuba-
tion with sperm cells reflects contamination artifacts
and/or self-splicing events in the original construct.
Similar results were obtained when the pBSKS-EGFP
INT DNA construct was incubated with swine or
human, ejaculated and washed, spermatozoa (data not
shown). These results therefore show that exogenous
DNA templates incubated with mature sperm cells
spontaneously undergo a stepwise process of transcrip-
tion, RNA splicing, and retrotranscription.
Molecular Reproduction and Development. DOI 10.1002/mrd
Fig. 1. PCR amplification of spliced EGFP reverse-transcribed
copies in sperm cells after incubation with pBSKS-EGFP-INT DNA.
A: Map of the vector containing the EGFP gene interrupted by a g-
globin intron in opposite orientation. The CMV early promoter and the
TK poly(A) signal (pA) are indicated. SD and SA: splicing donor and
splicing acceptor sites. Arrows indicate the oligonucleotide pairs,
flanking the splicing sites, used for amplification. B: DNA was
extracted and amplified from supernatants (lanes 1–3) and nuclei
(lanes 4– 6) of sperm cells incubated with increasing amounts of
exogenous DNA construct shown in (A) and from the corresponding
amounts of DNA construct non incubated with sperm cells (lanes 7
10). PC, positive control: EGFP sequence amplification from a standard
EGFP construct not interrupted by introns. PCR amplified products
were visualized by hybridization with a specific internal oligonucleo-
tide probe.
REVERSE TRANSCRIPTASE ACTIVITY IN MURINE SPERMATOZOA 1241
Spliced EGFP cDNA is Transferred From
Spermatozoa to Early Embryos at Fertilization
and Propagated to Fetuses and Born Animals
To establish whether the newly retrotranscribed
cDNA sequences can be transferred from spermatozoa
to oocytes and further transmitted to embryos, fetuses,
and born animals, pBSKS-EGFP INT-loaded spermato-
zoa were used in IVF assays and pre-implantation
embryos were produced. Some of the two-cell embryos
were implanted into foster mothers to produce fetuses
and born animals. Pre-implantation embryos at differ-
ent stages (70 two-cell, 33 four-cell, 30 morulae, 15
blastocysts), 32 fetuses and 30 born animals were
screened by direct PCR as illustrated in Table 1. To
compensate for quantitative differences between pre-
implantation embryos with different number of cells,
PCR analysis was performed on lysate aliquots obtained
from 5 two-cell embryos, 3 four-cell embryos, 2 eight-cell
embryos, and 1 blastocyst. Embryos from each group
were lysed and analyzed by direct PCR using the same
pair of oligonucleotides flanking the splicing sites, as
shown in Figure 2. The amplification reactions from
DNA samples extracted from fetuses and adults were
calibrated so as to amplify bands of comparable intensity
to those obtained with embryo lysates. Figure 2 shows
the results from one exemplifying screening experi-
ment: the 342 bp-long retrotranscribed and spliced
product is clearly the predominant form in all analyzed
stages, and is either detected as the only product in two-
cell (panel A, lanes 1, 2, 4), four-cell (panel B, lanes 3 –4),
morulae (panel B, lanes 6 and 8), blastocysts (panel B,
lane 11), fetuses (panel C, lane 1), and adults (panel C,
lanes 3–4), or in combination with the 1243 bp-long
unspliced (intron-containing) fragment (panel A, lane 6;
B, lane 5). The 1243-bp unspliced DNA sequence alone
was detected in a proportion of pre-implantation
embryos (panel A, lane 5; B, lanes 1, 7, and 10). These
data suggest that the spliced retrotranscribed EGFP
cDNA population is preferentially propagated through-
out embryogenesis and in adults, as compared to the
original DNA sequence.
Reverse-Transcribed EGFP-Containing cDNA
Sequences Undergo Modification During
Aging of Host Animals
Reverse-transcribed sequences were further analyzed
at different times after birth in a group of six animals of
the same litter. To this end, DNA samples were extrac-
ted at different times after birth—20, 40, and 60 days—
from fragments of the tails and comparatively analyzed
through both direct PCR and Southern blot assays. The
PCR results summarized in Figure 3 show that the
reverse-transcribed sequences are characterized by
unstable patterns that undergo significant changes
according to age: 20 days after birth two animals
exhibited the spliced EGFP 342 bp product (lanes 1
and 2), two the unspliced 1243 bp form (lanes 3 and 6),
one both the spliced and the unspliced (lane 5) and one
was negative (lane 4). Forty days after birth, the
patterns were dramatically changed: five out of six
animals exhibited the spliced DNA form (lanes 1 –5),
while the unspliced sequence was only present in one
single animal (lane 6). Two months after birth, the
signal intensity of all samples was generally fading,
being clearly visible only in three animals (lanes 1–3).
Molecular Reproduction and Development. DOI 10.1002/mrd
TABLE 1. PCR Screening of EGFP cDNA Sequences in F0 Pre-implantation
Embryos, Fetuses, and Adult Mice
Developmental stage Analyzed animals
PCR-positive
Independent experimentsn%
Two-cell embryos 70 5 7.1 3
Four-cell embryos 33 3 9 2
Morulae 30 3 10 2
Blastocysts 15 2 13.3 2
14-Day fetuses 32 2 6.2 3
Adults (F0) 36 8 22.2 4
Fig. 2. PCR amplification of spliced EGFP reverse-transcribed
copies in embryos obtained in IVF assays using pBSKS-EGFP-INT
DNA-incubated spermatozoa. A:Lanes 1– 7: two-cell embryos, lanes 8
and 9: EGFP-INT and EGFP control amplification, respectively.
B:Lanes 1– 5: four-cell stage embryos; lanes 6– 9: morulae; lanes
10– 11: blastocysts; lanes 12 and 13: EGFP-INT and EGFP control
amplification, respectively. C:Lanes 1–2: 14 days fetuses; lanes 3 –8:
adults 20 days after birth; lanes 9 and 10: EGFP-INT and EGFP
control amplification, respectively.
1242 C. PITTOGGI ET AL.
In contrast, Southern blot analysis performed on the
same DNA samples using a radioactive probe indicated
that the EGFP sequence was not detectable in any of the
six founders. Under the same conditions, however, the
single-copy Htf9-RanBP1 genomic sequence harboring
the RanBP1 gene (arrowed in the right panels), used
here as an internal marker, was clearly detected,
suggesting that the EGFP sequences were markedly
under-represented in the host genome (<one copy per
genome). The low abundance of the reverse-transcribed
exogenous sequences is not a peculiar feature restricted
to the tail tissue, but is a general feature, as confirmed
by the analysis of DNA samples extracted from dif-
ferent organs of one founder animal. The positive
founder animal, analyzed in lane 1 of Figure 3, was
sacrificed and the DNA was extracted from various
organs and analyzed by PCR (Fig. 4, panel A) and
Southern blot (panel B): again, the 342 bp spliced EGFP
was clearly PCR-amplified in seven out of nine analyzed
tissues, while no corresponding EGFP signal was
detected by Southern blot in any of the analyzed DNA
samples.
EGFP cDNAs are Transferred From
Founders to the F1 Progeny
To answer the question of whether the newly reverse-
transcribed sequences can be transmitted through the
germ line from founders to the next generation, two
positive female founders, #2 and #3, were mated with
wild-type males, generating 5 and 9 F1 animals,
respectively. DNA samples were extracted from the
tails of all 14 F1 individuals and analyzed by direct PCR.
The results summarized in Table 2 show that the EGFP
sequences were amplified in one out of five animals
(20%) from litter #2, and in eight out of nine (88%)
animals from litter #3: this confirms that the reverse-
transcribed sequences are indeed transferred through
the germ line from the parents to their progeny. As
seen before in founder animals, we found that in
Molecular Reproduction and Development. DOI 10.1002/mrd
Fig. 3. Age-dependent changes in the pattern of EGFP-containing cDNAs in founder animals. Parallel
PCR and Southern blot assays were carried out using DNA samples extracted from the tails of six animals of
the same litter at 20, 40, and 60 days after birth. Numbers on the left hand side indicate the size of PCR
amplification products of the unspliced (1,243 bp) and spliced (342) EGFP sequence. On the right hand side
panels, the arrows indicate the hybridization signal of the Htf9/RanBP1 genomic probe used for Southern
blot as a single-copy gene marker. M: lambda/HindIII DNA size marker.
Fig. 4. Propagation of the EGFP-containing cDNAs in various
tissues of a founder animal. DNA samples were extracted from nine
tissues of a founder and analyzed by PCR (A) and Southern blot (B).
Lane 1: liver, lane 2: kidney, lane 3: spleen, lane 4: lung, lane 5:
ovary, lane 6: heart, lane 7: brain, lane 8: bone marrow, lane 9:
muscle. In the Southern blot panel, the arrow indicates the Htf9/
RanBP1 specific hybridization signal. Numbers on the right hand side
correspond to EGFP-INT and EGFP control amplification.
REVERSE TRANSCRIPTASE ACTIVITY IN MURINE SPERMATOZOA 1243
the F1 progeny the EGFP reverse-transcribed cDNA
molecules—predominantly represented by the spliced
342 bp product after reverse transcription—are main-
tained at a low copy number, as confirmed by the
negative results obtained in Southern blot analysis
(data not shown).
Expression of EGFP-Containing cDNA in
Different Tissues of Adult Mice
We initially attempted to detect the expression of
EGFP-containing cDNA sequences in mouse tissues,
including liver, spleen, kidney, lung, testis, and brain,
by direct fluorescence microscopy of the GFP emission:
these attempts yielded, however, essentially negative
results. Tissue fragments from PCR-positive animals
were then embedded in paraffin and sectioned for
examination of the internal structure by immunohisto-
chemical analysis using a monoclonal anti-GFP anti-
body (mAb). As shown in Figure 5, the EGFP protein was
specifically detected in the sinusoidal endothelial cells of
the liver (panel a) as well as in few scattered hepatocytes
(panel b). The EGFP protein was also specifically
expressed in endothelial cells of the glomerular capil-
laries of kidney (panel c) and in small vessels of the brain
(panel d). In contrast, no significant EGFP immuno-
reactivity was observed in spleen, lung and testis (not
shown). No EGFP immunoreactivity was detected in
organ sections withdrawn from wild-type control ani-
mals of same strain, age and sex, which were processed
in parallel (not shown). In addition, no EGFP expression
was detect in early embryos, consistent with the
reported inability of the CMV promoter to support gene
expression in mouse pre-implantation stages (Kothary
et al., 1991; Baskar et al., 1996). Together these results
support the conclusion that a transcriptionally compe-
tent reverse transcribed, spliced sequence can be
generated in spermatozoa and propagated to adult
animals when spermatozoa are incubated with exogen-
ous DNA sequences.
DISCUSSION
We previously reported that mature spermatozoa are
endowed with an endogenous RT activity capable of
retrotranscribing exogenously added RNA templates
(Giordano et al., 2000; Sciamanna et al., 2003). In the
present work we have used an intron-interrupted
EGFP-containing retrotransposition cassette in incuba-
tion assays with murine mature spermatozoa: we report
that transcriptionally competent cDNA molecules can
be generated from the exogenous DNA through sequen-
tial transcription, splicing of the full-length RNA and
further reverse transcription of the spliced product.
These molecules behave as newly acquired, biologically
active sequences, are propagated through embryogen-
esis and are expressed in various tissues of born
animals. From these studies, it emerges that sperm
endogenous RT is a key component of a novel mechanism
able to generate transcriptionally competent, reverse-
transcribed sequences in spermatozoa using an exogen-
ous template.
The reverse-transcribed cDNA molecules from the
EGFP-harboring DNA construct described here exhibit
peculiar features. These sequences are maintained at
low copy number (i.e., <one copy per genome), as
revealed by their being below the limit of resolution in
Southern blot experiments (Figs. 3 and 4); they show a
mosaic distribution, being present in many, but not in all
tissues of founder animals; they are sexually trans-
mitted from founders to the F1 progeny and are mosaic
propagated in tissues of F1 individuals, again in low
copy number. In our view, these features are consistent
with an extrachromosomal organization of these
sequences, the replication of which would be indepen-
dent from that of the host genome. In further support of
the idea that EGFP sequences remain as extrachromo-
somal structures, our attempts to investigate whether
the cDNA sequences are integrated in the host genome
yielded consistently negative results. These experi-
ments included the construction and screening of a
partial genomic library as well as a genome-wide
analysis of host genomic/construct DNA junctions by
ligation-mediated-PCR (Pfeifer et al., 1999): neither
type of assay revealed any evidence for integration
events (not shown). In further agreement with the
conclusion that retrotranscribed sequences fail to
Molecular Reproduction and Development. DOI 10.1002/mrd
TABLE 2. Transmission of EGFP cDNA to
F1 Offsprings
Founder # F1 mice (n)
PCR-positive
n%
25120
39888
Fig. 5. Expression of the EGFP protein in the vascular epithelium of
mouse tissues. EGFP was detected by immunohistochemistry on
paraffin-embedded tissues using a specific anti-GFP antibody (Santa
Cruz Biotechnology). Panels a and b: liver tissue at different
magnifications (40 and 400, respectively); arrows in panel b
indicated stained hepatocytes. Panel c: kidney, arrows indicate
positively stained glomeruli (250). Panel d: brain (250).
1244 C. PITTOGGI ET AL.
integrate in the genome is the finding that PCR
amplification signals of DNA samples extracted at
different times after birth from the same group of
animals become progressively fainter in correlation
with the age of founders (Fig. 3). At the present stage,
the mechanism through which reverse transcribed and
spliced cDNA sequences are propagated as extrachro-
mosomal structures throughout embryogenesis and
in adult animals remains elusive. Preliminary data
would, however, suggest that the reverse-transcribed
sequences assemble in a nucleoprotein complex, resem-
bling endogenous retroviral particles and are able to
propagate in permissive cell populations.
The production of extrachromosomal structures and
their propagation to the next generation have been
reported in transgenic animals obtained by SMGT in
mammals (Kuznetsov et al., 2000), birds (Rottmann
et al., 1992), fish (Khoo et al., 1992), and insects
(Robinson et al., 2000) and also, in some cases, by DNA
microinjection in mammals (Kiessling et al., 1986;
Elbrecht et al., 1987), amphibia (Etkin and Pearman,
1987), fish (Culp et al., 1991), nematodes (Mello et al.,
1991), and insects (Nikolaev et al., 1993). A significant
implication of the present work for SMGT experiments
is that the rate of positive animals may be found to vary
over time, and in particular may be higher among
younger animals, while progressively decreasing among
older ones; in other words, animals that were positive in
an early screening may be classified as negative upon
subsequent analysis. In the light of the results reported
here, we suggest that extrachromosomal sequences may
be the product of the endogenous RT activity operating
in both gametes and early embryos. We believe that this
phenomenon may be the cause of contradictory results
reported in the past years with SMGT protocols
(Brinster et al., 1989; Smith, 1999; Smith and Spada-
fora, 2005). We have obtained similar results with swine
ejaculated, seminal plasma-freed, spermatozoa (data
not shown), suggesting that these functions are not
restricted to the murine system, but rather reflect
widespread features of germ cells in mammalian
species.
The protein product synthesized from the EGFP-
containing cDNA, which was originally expressed under
the control of a CMV promoter in the original construct,
was found to be preferentially localized in the vascular
endothelium of blood vessels in liver, kidney, and brain
(Fig. 5, panels a, c, and d, respectively), and in a small
fraction of scattered hepatocytes (Fig. 5, panel b).
Though this finding may seem peculiar at first sight, it
is actually not unexpected, because the CMV promoter is
highly tissue-specific in transgenic animals and its
expression is inherently restricted to sites that correlate
with the target tissues of viral infection, such as the
vascular endothelium (Koedood et al., 1995). An addi-
tional element that may contribute to the observed
pattern, in a different perspective, may come from the
interesting observation that the vascular endothe-
lium—together with Sertoli and Leydig cells—is the
only differentiated tissue in which LINE-1 elements
encoding the cellular RT are specifically expressed
(Ergun et al., 2004). This feature may suggest the
alternative, but not necessarily mutually exclusive,
explanation that the EGFP tissue restriction is indeed
a LINE-1-related phenomenon, reflecting a tissue
permissivity for retrotransposal activity rather than,
or in addition to, the CMV promoter selectivity.
In our view, transcription, due to a RNA polymerase
activity present in sperm cells (Fuster et al., 1977),
splicing and retrotranscription are components of a
multifunctional system that may be naturally activated
in sperm nuclei at fertilization, or artificially upon
interaction of sperm cells with exogenous nucleic acids.
Recent work in our and other laboratories indicates that
endogenous RT activity and LINE-1 expression play key
roles in murine pre-implantation development (Pittoggi
et al., 2003; Beraldi et al., 2006), in cellular prolifera-
tion and differentiation (Mangiacasale et al., 2003;
Landriscina et al., 2005), and in tumor growth
(Sciamonna et al., 2005; for a recent review see
Sinibaldi-Vallebona et al., 2006). Our present results,
and previous work briefly summarized in the Introduc-
tion, also support the conclusion that a RT-mediated
mechanism has a key role in the generation and
propagation of novel genetic information during embry-
ogenesis and in tissues of adult animals. These evidence
had inspired the idea of a process that may be called
sperm-mediated ‘‘reverse’’ gene transfer, highlighting a
role of the sperm endogenous RT when these cells are
incubated with exogenous RNA molecules (Smith and
Spadafora, 2005). Here we have taken a step further and
show that the RT-mediated process is not only triggered
when spermatozoa are exposed to exogenous RNA
molecules, but is also activated when they interact with
DNA molecules. As a whole, the present results strongly
support the conclusion that the SMGT process can be
regarded as a retrotransposon-mediated phenomenon.
ACKNOWLEDGMENTS
We are grateful to Drs. Ostertag and Kazazian for the
gift of the construct pBSKS-EGFP-INT. We acknowl-
edge Carmine Nicoletti for assistance with the animal
work. I.S. and C.P. were supported by grants from
Istituto Superiore di Sanita
`and R.B. by a fellowship
from the Ministry of Education, University and
Research (MIUR). This work was supported by funds
from Istituto Superiore di Sanita
`and by grant N. R002
‘‘Role of endogenous reverse transcriptase in tumor
growth and in embryonic development’’ to C.S. from the
Italian Ministry of Health.
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1246 C. PITTOGGI ET AL.
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Exposure of spermatozoa to stress conditions causes a drastic reduction of their fertilizing ability. We report here that the decrease in fertilization can be effectively antagonized by preincubating sperm cells with the nuclease inhibitor drug aurintricarboxylic acid (ATA). Preincubation of mouse epididymal sperm cells with ATA increased the yield of 2-cell embryos produced by in vitro fertilization assays. The effect of ATA was selectively exerted via spermatozoa, since neither preincubation of eggs, nor the direct treatment of zygotes, modified the yield of 2-cell-stage embryos. Our results suggest that ATA does not directly improve the ability of sperm cells to penetrate the egg cytoplasm but instead acts by preserving sperm nuclei from induced or spontaneously occurring damage and/or favors events that trigger early embryogenesis.
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Liposomes were prepared in the presence of plasmid DNA and allowed to react with spermatozoa from roosters prior to artificial insemination of 200 hens. Twelve days old fetuses developed from fertilized eggs were analysed by Southern blot technique to detect the transfer of the foreign sequences. About 26% of all fetuses examined displayed the banding pattern of the transferred plasmid. The results indicate that the sequences are not chromosomally integrated but are present in episomic form. The method described here is easy to handle so that it may become an appropriate means to establish large numbers of transgenic animals. Liposomaler Gentransfer über Spermien in Hühnereier Mischsperma von mehreren Hähnen wurde mit Liposomen inkubiert, in denen Plasmid-DNA eingeschlossen war. Mit diesem Sperma sind 200 Hennen inseminiert worden. Nach 12 Tagen Bebrütung wurden die Föten mittels Southernblot-Technik analysiert. Rund 26% aller untersuchten Föten zeigten die typischen Bandenmuster des transferierten Plasmids. Die Befunde weisen darauf hin, daß das Plasmid nicht chromosomal integriert, sondern als Episom neben der genomischen DNA vorliegt. Das hier beschriebene Gentransfer-Verfahren ist einfach zu handhaben, so daß ohne nennenswerten Aufwand größere Zahlen transgener Tiere erstellt werden können.
Article
Mature female zebrafish (Brachydanio rerio) eggs were in-vitro fertilized with zebrafish sperm cells incubated with pUSVCAT plasmid in either circular or linearized form. Analysis of adult pectoral fins showed that 23.33% (21 out of 89) and 37.5% (3 out of 8) of the treated founder fish (P), fertilized with sperms incubated with circular and linearized plasmid DNA, respectively, showed positive hybridization with whole pUSVCAT plasmid32P radiolabelled probes. When crossbred to nont-ransgenic fish, these putative founder transgenics (P) produced transgenic F1 progenies which in turn produced transgenic F2 fry when crossbred with non-transgenics. No expression of the CAT has been detected so far. Sperm-mediated transfer of foreign genes into the zebrafish is therefore possible; however, the nature of the incorporation is not known at the moment. It would appear from the results obtained that the introduced foreign DNA exists extrachromosomally. Although it has been shown that sperms can be used to insert genes into the fish, methods to integrate the introduced gene into the fish genome need to be explored further.