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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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Generation of Biologically Active Retro-genes
Upon Interaction of Mouse Spermatozoa
With Exogenous DNA
Istituto Superiore di Sanita
`(ISS), Rome, Italy
Department of Pediatrics, Obstetrics and Reproductive Medicine, University of Siena, Siena, Italy
Institute of Clinical Physiology, National Research Council (CNR), Siena, Italy
Institute of Molecular Biology and Pathology, CNR, Rome, Italy
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)
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
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:
Received 2 March 2006; Accepted 14 April 2006
Published online 18 July 2006 in Wiley InterScience
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.
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/
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
, 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:
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
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.
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).
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
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.
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
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.
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
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.
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.
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)
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).
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
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.
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
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Molecular Reproduction and Development. DOI 10.1002/mrd
... Indeed, converging conclusions from in vitro fertilization assays in mice, artificial insemination in large farm animals and external fertilization in fish and amphibia, indicate that seminal fluid-free spermatozoa are permeable to exogenous RNA and DNA molecules which they carry through and deliver to oocytes at fertilization. The foreign molecules propagate as non-integrated structures in developing embryos and are eventually mosaic-inherited, with variable efficacy, in tissues of born animals, where they are expressed as novel traits ( [41,43,44], reviewed in Ref. [45]). This process, called spermmediated gene transfer, reveals the ability of mature sperm cells to take up and deliver foreign nucleic acids as a common feature in virtually all animal species, from echinoids to mammals, including humans [45]. ...
... LINE-1 retroelements encode their RT enzyme, that is active in preimplantation embryos [95,96] and in epididymal spermatozoa ( [42,44,45], reviewed in Ref. [41]). Earlier work showed that mouse zygotes incorporate 5 ' -bromodeoxyuridine (BrdU) cultured even in the presence of aphidicolin, a strong inhibitor of DNA replication, but this incorporation is abolished in the presence of the nucleoside RT inhibitor abacavir [96] which inhibits LINE-1-encoded RT [97]: thus, reverse transcription is ongoing in both paternal and maternal pronuclei (more pronouncedly in the former, see Fig. 2), suggesting a role in the targeted retrotransposition events (see above). ...
... Sperm cells are endowed with both RT and RNA polymerase activities [100] and can host such a process. That was shown in Transgenerational epigenetic reprogramming of early embryos | 5 in vitro fertilization assays using epididymal spermatozoa that were pre-incubated with either b-gal or EGFP-encoding RNA and DNA molecules: the retro-transcribed/transcribed molecules were mosaic propagated as extrachromosomal sequences throughout embryogenesis and, later, in tissues of adult individuals, where they induced phenotypic variations [43,44]. The reporter genes were further expressed in the next generation obtained from founders. ...
Full-text available
The notion that epigenetic information can be transmitted across generations is supported by mounting waves of data, but the underlying mechanisms remain elusive. Here, a model is proposed which combines different lines of experimental evidence. First, it has been shown that somatic tissues exposed to stressing stimuli release circulating RNA-containing extra-cellular vesicles; second, epididymal spermatozoa can take up, internalize and deliver the RNA-containing extracellular vesicles to oocytes at fertilization; third, early embryos can process RNA-based information. These elements constitute the building blocks upon which the model is built. The model proposes that a continuous stream of epigenetic information flows from parental somatic tissues to the developing embryos. The flow can cross the Weismann barrier, is mediated by circulating vesicles and epididymal spermatozoa, and has the potential to generate epigenetic traits that are then stably acquired in the offspring. In a broader perspective, it emerges that a natural 'assembly line' operates continuously, aiming at passing the parental epigenetic blueprint in growing embryos.
... They can even be passed on to future generations. These plasmids can remain autonomous and are able to clone their DNA independently of the human genome [18]. It is therefore conceivable that such a process could take place following mRNA vaccination, which would result in an infant whose cells would have the capability of synthesizing spike protein and whose immune system would view the spike protein as a self-protein, with unknown consequences. ...
... RNA molecules have the ability to spontaneously modify their sequences and, even when fragmented, to direct the synthesis of their respective copies [44]. RNA recombination [45] and transmissibility via sperm [18] or via metathesis reactions to the next generation of cells is one of the major obstacles to overcome in mRNA technology application for infectious disease vaccination [1]. ...
Full-text available
Background The findings of a sequence embedded in Human DNA that was almost identical to a sequence in the SARS-CoV-2 genome, and the identification of plausible integration of SARS-CoV-2 RNA into human DNA by endogenous reverse transcriptase activity expressed by Long Interspersed Nuclear Element (LINE)-1 (17% of Human DNA) have raised concerns about the long-term safety of messenger-RNA (mRNA) based vaccination. Recent data demonstrate that SARS-CoV-2 RNA sequences can be transcribed into DNA and may be actively integrated into the genome of affected human cells, mediated by retrotransposons. Complementarily, in some SARS-CoV-2 infected patient specimens, there is evidence for a large fraction SARS-CoV-2 sequence integration and subsequent generation of SARS-CoV-2-human chimeric transcripts. 2 Results In this review, the potential role of mobile genetic elements in the etiopathogenesis of cardiovascular, neurological, immunological, and oncological disease and the possibilities of human DNA interference by SARS-CoV-2 vaccination are repositioned. Vulnerable human stem cells as well as gametocytes can presumably be the first targets for unwanted RNA interference. Given the many genetic manipulations of the RNA coding for the SARS-CoV-2 spike glycoprotein in the vaccines, manipulations designed to increase stability and efficiency of spike protein translation, much remains uncertain about the potential disruptions to cellular physiology and homeostasis that could ensue. The predicted consequences pose serious risks to human health that are in need of clarification. Conclusion Further toxicity evaluations are urgently needed to quantify potential emergence of interference with canonical DNA processes that could detrimentally impact the mRNA-vaccinated population.
... In fact, they survive into adulthood as extrachromosomal structures and are capable of being passed on to progeny. These plasmids are transcriptionally competent, meaning that they can be used to synthesize proteins encoded by the DNA they contain (Pittoggi et al., 2006). ...
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Operation Warp Speed brought to market in the United States two mRNA vaccines, produced by Pfizer and Moderna. Interim data suggested high efficacy for both of these vaccines, which helped legitimize Emergency Use Authorization (EUA) by the FDA. However, the exceptionally rapid movement of these vaccines through controlled trials and into mass deployment raises multiple safety concerns. In this review we first describe the technology underlying these vaccines in detail. We then review both components of and the intended biological response to these vaccines, including production of the spike protein itself, and their potential relationship to a wide range of both acute and long-term induced pathologies, such as blood disorders, neurodegenerative diseases and autoimmune diseases. Among these potential induced pathologies, we discuss the relevance of prion-protein-related amino acid sequences within the spike protein. We also present a brief review of studies supporting the potential for spike protein "shedding", transmission of the protein from a vaccinated to an unvaccinated person, resulting in symptoms induced in the latter. We finish by addressing a common point of debate, namely, whether or not these vaccines could modify the DNA of those receiving the vaccination. While there are no studies demonstrating definitively that this is happening, we provide a plausible scenario, supported by previously established pathways for transformation and transport of genetic material, whereby injected mRNA could ultimately be incorporated into germ cell DNA for transgenerational transmission. We conclude with our recommendations regarding surveillance that will help to clarify the long-term effects of these experimental drugs and allow us to better assess the true risk/benefit ratio of these novel technologies.
... Giordano and Magnano discovered that reverse transcriptase activity is present in mature spermatozoa (Giordano et al., 2000). Subsequently, it was found that the transcription and splicing activities are initiated in mature sperm (Pittoggi et al., 2006). Moreover, a comparative analysis of the transcripts between bovine spermatids and mature sperm showed that 4.5% were specific to mature sperm (Gilbert et al., 2007). ...
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Seminal plasma contains a large number of extracellular vesicles (EVs). However, the roles of these EVs and their interactions with sperm are not clear. To identify the important molecules affecting sperm motility in EVs, we analyzed RNA from seminal plasma EVs of boars with different sperm motility using whole-transcriptome sequencing and proteomic analysis. In total, 7 miRNAs, 67 lncRNAs, 126 mRNAs and 76 proteins were differentially expressed between the two groups. We observed that EV-miR-222 can obviously improve sperm motility. In addition, the results suggested that miR-222 was transferred into sperm by the EVs and that miR-222 affected sperm apoptosis by inhibiting the expression of EGFR, BCL2L11, BAX, CYCs, CASP9 and CASP3 . The results of electron microscopy also showed that overexpression of miR-222 in EVs could reduce sperm apoptosis. The study of the whole transcriptomes and proteomes of EVs in boar semen revealed some miRNAs may play an important role in these EVs interactions with Duroc sperm, and the findings suggest that the release of miR-222 by semen EVs is an important mechanism by which sperm viability is maintained and sperm apoptosis is reduced. Our studies provide a new insight of miR-222 in EVs regulation for sperm motility and sperm apoptosis.
... The discovery of functional RT in sperm cells provides the basis for SMRGT: in this process, the exogenous RNA is probably ''captured'' by the retrotransposon-mediated mechanism active in sperm cells, reverse-transcribed, further propagated through the embryo as non-integrated structures in tissues of founder individuals and transmitted to F1 progeny. It is demonstrated that reverse-transcribed sequences behave as functional genes, being correctly expressed in tissues of F0 and F1 animals (Pittoggi et al., 2006). ...
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In the field of animal transgenesis, many attempts have been made recently to simplify facilitate, and reduce the cost and labor required to do such tasks. Although several transgenesis techniques (such as DNA microinjection and somatic cell nuclear transfer) have been applied successfully to produce transgenic animals, these traditional techniques are so tedious and have several disadvantages. Retroviral mediated gene transfer has solved some of these usual problems but has, however, inevitable disadvantages represented most prominently by its biological hazard. Many researchers found that the most simple and non-cost effective way to produce transgenic animals is to focus on the natural ability of the sperm to "carry" the foreign DNA and to "fertilize" the oocyte. The most important breakthrough obtained in this aspect is the accumulated information that demonstrated the ability of foreign DNA to be internalized into the sperm head after simple incubation step. Accordingly, the only manipulation step is restricted into the head of the sperm. Then, nature will be allowed to fulfill its scheduled task of reproduction. This method known as sperm mediated gene transfer or SMGT. However, simple incubation of naked DNA with sperm head is not efficient enough to integrate the foreign DNA into the genome of the sperm. Thus, this review aims to pave the way for every effort to enable the researchers to undergo the transgenesis experiments in the routine laboratories. This is potentially can be done by testing the validity of the most modern enhancement approaches suggested on the original SMGT.
... The age of artificial insemination new born babies at which blood withdraw taken place was only two days since it was demonstrated that PCR amplification bands from gWizGFP DNA was found to become progressively less intense as the animal aged. Thus, an animal that tested gWizGFP DNA -positive at a young age might be tested gWizGFP DNA negative at later screenings, making the rate of PCR -positive animals vary over time (Pittoggi, 2006). The most critical step toward REMI-SMGT approach was not just to incorporate the exogenous DNA into the head of sperm, but to force it to be integrated into the genome of the sperm. ...
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The present study tends to test the validity of REMI-SMGT and to evaluate the efficiency of REMI-SMGT on generating other mammalian species rather than the sheep that made only by one group and to observe the possibility of doing so by using rabbits as a model for this approach and also to reduce the cost of REMI-SMGT by substituting liposomes and highly cost effective media with a high efficient, non-cost effective substitute. Direct protective relationship of liposome with DNA and seminal fluid was identified compared with DMSO. While different treatments (linearized DNA-restriction enzyme-liposome complex, DNA-restriction enzyme-DMSO complex, DNA-DMSO complex, DNA-liposome complex, and even naked DNA) were all found to be successful to internalize inside the head of the sperm according to PCR results, only three (one by restriction enzyme-liposome treatment and two by restriction enzyme-DMSO treatment) out of fourteen new born babies were found to be transgenic by PCR. Despite the absolute ability of exogenous DNA to be internalized inside rabbit's sperm head only few percent of transgenic babies were obtained. This may not reflect the weakness of restriction enzyme mediated transgenesis technique itself but it reflects the inability of recombinant sperm to fertilize superovulated oocyte compared with their normal counterparts. Comparable results were found between liposome and DMSO treatment which may reflect direct relationship of DMSO with the cell membrane instead of with the exogenous DNA itself as what is found with liposome.
Epigenetic marks in gametes, which both respond to the parental environmental factors and shape offspring phenotypes, are usually positioned to mediate intergenerational or transgenerational epigenetic inheritance. Nonetheless, the mechanisms through which gametic epigenetic signatures encode parental acquired phenotypes, and further initiate a cascade of molecular events to affect offspring phenotypes during early embryonic development, remain unclear. Retrotransposons are mobile DNA elements that could resist to genomic epigenetic reprogramming at specific loci and rewire the core regulatory networks of embryogenesis. Increasing evidences show that retrotransposons in the embryonic genome could interact with gametic epigenetic marks, which provides a tentative possibility that retrotransposons may serve as a relay of gametic epigenetic marks to transmit parental acquired traits. Here, we summarize the recent progress in exploring the crosstalk between gametic epigenetic marks and retrotransposons, and the regulation of gene expression and early embryonic development by retrotransposons. Accordingly, deciphering the mystery of interactions between gametic epigenetic marks and retrotransposons during early embryonic development will provide valuable insights into the intergenerational or transgenerational transmission of acquired traits.
The common belief that the neo-Darwinian Modern Synthesis (MS) was buttressed by the discoveries of molecular biology is incorrect. On the contrary those discoveries have undermined the MS. This article discusses the many processes revealed by molecular studies and genome sequencing that contribute to evolution but nonetheless lie beyond the strict confines of the MS formulated in the 1940s. The core assumptions of the MS that molecular studies have discredited include the idea that DNA is intrinsically a faithful self-replicator, the one-way transfer of heritable information from nucleic acids to other cell molecules, the myth of “selfish DNA,” and the existence of an impenetrable Weismann Barrier separating somatic and germ line cells. Processes fundamental to modern evolutionary theory include symbiogenesis, biosphere interactions between distant taxa (including viruses), horizontal DNA transfers, natural genetic engineering, organismal stress responses that activate intrinsic genome change operators, and macroevolution by genome restructuring (distinct from the gradual accumulation of local microevolutionary changes in the MS). These 21st Century concepts treat the evolving genome as a highly formatted and integrated Read-Write (RW) database rather than a Read-Only Memory (ROM) collection of independent gene units that change by random copying errors. Most of the discoverers of these macroevolutionary processes have been ignored in mainstream textbooks and popularizations of evolutionary biology, as we document in some detail. Ironically, we show that the active view of evolution that emerges from genomics and molecular biology is much closer to the 19th century ideas of both Darwin and Lamarck. The capacity of cells to activate evolutionary genome change under stress can account for some of the most negative clinical results in oncology, especially the sudden appearance of treatment-resistant and more aggressive tumors following therapies intended to eradicate all cancer cells. Knowing that extreme stress can be a trigger for punctuated macroevolutionary change suggests that less lethal therapies may result in longer survival times.
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Sperm contributes diverse RNAs to the zygote. While sperm small RNAs have been shown to impact offspring phenotypes, our knowledge of the sperm transcriptome, especially the composition of long RNAs, has been limited by the lack of sensitive, high-throughput experimental techniques that can distinguish intact RNAs from fragmented RNAs, known to abound in sperm. Here, we integrate single-molecule long-read sequencing with short-read sequencing to detect sperm intact RNAs (spiRNAs). We identify 3440 spiRNA species in mice and 4100 in humans. The spiRNA profile consists of both mRNAs and long non-coding RNAs, is evolutionarily conserved between mice and humans, and displays an enrichment in mRNAs encoding for ribosome. In sum, we characterize the landscape of intact long RNAs in sperm, paving the way for future studies on their biogenesis and functions. Our experimental and bioinformatics approaches can be applied to other tissues and organisms to detect intact transcripts.
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We have tested three parameters in sperm-mediated gene transfer assays with mice and pigs: (i) the epididymal versus ejaculated origin of sperm cells, (ii) the primary structure, and (iii) the amount of the challenging foreign DNA. We have found that the pVLCNhGH construct, of retrotransposon origin, causes a massive embryo lethality and yet increases the yield of genetic transformation among born animals of both species compared to viral constructs. Arrest of embryonic development is a DNA dose-dependent effect, which is observed with high DNA doses, while lower doses are compatible with development. Finally, the overall efficiency of sperm-mediated gene transfer is higher when ejaculated, versus epididymal, spermatozoa are used. We suggest that this difference is related to the highly efficient apoptotic response in epididymal compared to ejaculated spermatozoa, triggered by the interaction of exogenous DNA molecules with the sperm membrane. Mol. Reprod. Dev. 56:301–305, 2000. © 2000 Wiley-Liss, Inc.
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Recent developments in studies of sperm-mediated gene transfer (SMGT) now provide solid ground for the notion that sperm cells can act as vectors for exogenous genetic sequences. A substantive body of evidence indicates that SMGT is potentially useable in animal transgenesis, but also suggests that the final fate of the exogenous sequences transferred by sperm is not always predictable. The analysis of SMGT-derived offspring has shown the existence of integrated foreign sequences in some cases, while in others stable modifications of the genome are difficult to detect. The appearance of SMGT-derived modified offspring on the one hand and, on the other hand, the rarity of actual modification of the genome, suggest inheritance as extrachromosomal structures. Several specific factors have been identified that mediate distinct steps in SMGT. Among those, a prominent role is played by an endogenous reverse transcriptase of retro-transposon origin. Mature spermatozoa are naturally protected against the intrusion of foreign nucleic acid molecules; however, particular environmental conditions , such as those occurring during human assisted reproduction, can abolish this protection. The possibility that sperm cells under these conditions carry genetic sequences affecting the integrity or identity of the host genome should be critically considered. These considerations further suggest the possibility that SMGT events may occasionally take place in nature, with profound implications for evolutionary processes.
We describe a dominant behavioral marker, rol‐6(su‐1006), and an efficient microinjection procedure which facilitate the recovery of Caenorhabditis elegans transformants. We use these tools to study the mechanism of C.elegans DNA transformation. By injecting mixtures of genetically marked DNA molecules, we show that large extrachromosomal arrays assemble directly from the injected molecules and that homologous recombination drives array assembly. Appropriately placed double‐strand breaks stimulated homologous recombination during array formation. Our data indicate that the size of the assembled transgenic structures determines whether or not they will be maintained extrachromosomally or lost. We show that low copy number extrachromosomal transformation can be achieved by adjusting the relative concentration of DNA molecules in the injection mixture. Integration of the injected DNA, though relatively rare, was reproducibly achieved when single‐stranded oligonucleotide was co‐injected with the double‐stranded DNA.
We show here that a reverse transcriptase (RT) activity is present in murine epididymal spermatozoa. Sperm cells incubated with human poliovirus RNA can take up exogenous RNA molecules and internalize them in nuclei. Direct PCR amplification of DNA extracted from RNA-incubated spermatozoa indicate that poliovirus RNA is reverse-transcribed in cDNA fragments. PCR analysis of two-cell embryos shows that poliovirus RNA-challenged spermatozoa transfer retrotranscribed cDNA molecules into eggs during in vitro fertilization. Finally, RT molecules can be visualized on sperm nuclear scaffolds by immunogold electron microscopy. These results, therefore, reveal a novel metabolic function in spermatozoa, which may play a role during early embryonic development.
Washed mature spermatozoa from bulls incorporate ribonucleoside triphosphates into RNA using an endogenous template. Maximum incorporation was observed at 31 degrees C in the presence of MgCl2, all four ribonucleoside triphosphates, beta-mercaptoethanol, and glycine sodium hydroxide buffer at pH 9.0. The amount of synthesis was linearly dependent upon the concentration of spermatozoa and continued for at least 4 h. Digestion studies revealed the RNA to be present in a protected (intracellular?) location in the spermatozoa. The RNA synthesis was inhibited by ethidium bromide, rifampicin, acriflavine, actinomycin D, and caffeine, but not by alpha-amanitine or rifamycin SV. Fractionation of the spermatozoa by sonication and separation of the heads and tails by centrifugation through a discontinuous gradient revealed that more than half of the total RNA polymerase activity was associated with the tail fraction.
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.
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.
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.