Hum. Reprod. Advance Access originally published online on February 11, 2008
Human Reproduction 2008 23(4):735-740; doi:10.1093/humrep/dem425
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Sperm-mediated ‘reverse’ gene transfer: a role of reverse transcriptase in the generation of new genetic information
Istituto Superiore di Sanità, Viale Regina Elena 299, Rome 00161, Italy
Tel: + 39-06-49903117; Fax: +39-06-49903672; E-mail: cspadaf{at}tin.it
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Abstract |
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 Top Abstract BackgroundReverse transcriptase activity...SMRGT: a modelGenesis and inheritance of...ConclusionsFundingReferences |
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Sperm-mediated gene transfer (SMGT) is a procedure through which new genetic traits are introduced in animals by exploiting the ability of spermatozoa to take up exogenous DNA molecules and deliver them to oocytes at fertilization. The interaction of exogenous DNA with sperm cells is a regulated process mediated by specific factors; among those, a reverse transcriptase (RT) activity plays a central role in SMGT. ‘Retro-genes’ are generated either through reverse transcription of exogenous RNA internalized in spermatozoa, or through sequential transcription, splicing and reverse transcription of exogenous DNA. The resulting retro-genes are delivered to oocytes and transmitted to embryos and born animals as low-copy, transcriptionally competent, extrachromosomal structures capable of determining new phenotypic traits. Retro-genes can be further transmitted through sexual reproduction from founders to their F1 progeny: new genetic and phenotypic features, unlinked to chromosomes, can thus be generated and inherited in a non-Mendelian ratio. We have called this phenomenon sperm-mediated ‘reverse’ gene transfer (SMRGT). Thus, a RT-mediated machinery operates in sperm cells and is responsible for the genesis and non-Mendelian propagation of new genetic information. The features of RT-generated traits elicited in SMRGT resemble those characterized in recent studies of RNA-mediated inheritance of extra-genomic information.
Key words: sperm-mediated gene transfer/reverse transcriptase/transgenesis/extrachromosomal inheritance
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Background |
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TopAbstractBackgroundReverse transcriptase activity...SMRGT: a modelGenesis and inheritance of...ConclusionsFundingReferences |
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It is now widely accepted that spermatozoa of virtually all animal species have the spontaneous ability to take up exogenous DNA molecules and to deliver them to oocytes at fertilization. This feature has been exploited to generate genetically modified animals, with variable efficiency, according to a protocol called sperm-mediated gene transfer (SMGT) (reviewed by Smith and Spadafora, 2005
).
SMGT has been a highly controversial issue since its first appearance (Lavitrano et al., 1989), because shortly after the earliest report several groups reported their failure to reproduce the original protocol (Brinster et al., 1989). Since then, however, SMGT protocols have been extended to virtually all animal species: thus, a wealth of studies have generally confirmed the original report and provide a sound foundation to the notion that sperm cells can indeed act as vectors of foreign genetic information (Smith and Spadafora, 2005). These studies, however, have yielded conflicting conclusions as to the final fate of the foreign nucleic acid sequences, and inconsistencies concerning the reproducibility associated with this method remain unsolved. Actually, it is still hard to predict whether, and with what efficiency, transgenic animals will be obtained from any given experiment: this reflects an as yet incomplete understanding of the underlying basic mechanisms of SMGT, with a still partial identification of all experimental parameters required for full control of the process. Studies in the field, in effect, have mostly highlighted the practical aspects of SMGT—regarded as a simple, low-cost procedure to generate transgenic animals, and therefore worth optimizing—but have generally neglected its underlying molecular basis. There is currently a general consensus that only two steps in the SMGT process are well-established and fully reproducible: (i) the spontaneous interaction between sperm cells and foreign DNA molecules, and (ii) the delivery of sperm-bound DNA to oocytes at fertilization.
The subsequent fate of sperm-bound DNA, after delivery in the oocyte, is still a contradictory issue; in particular, the question of whether foreign molecules of nucleic acids become integrated into the host genome or remain as extrachromosomal structures is still unsolved. Available data indicate that the fate of the exogenous DNA depends on the procedures through which sperm cells and DNA come together: the generation of non-integrated episomal structures is a highly probable event when foreign DNA molecules are directly incubated with intact spermatozoa that are then used in fertilization assays (Khoo et al., 1992; Khoo, 2000; Kuznetsov et al., 2000; Robinson et al., 2000; Tsai, 2000). Integration in the host genome is rare under these conditions, and so far has only been claimed by one single group in swine (Lavitrano et al., 2002; Webster et al., 2005); the same group, however, also reported later the transmission of non-integrated sequences (Manzini et al., 2006). In contrast, integration seems to be the favored outcome when using protocols that avoid a direct interaction between the exogenous nucleic acid molecules and the sperm membrane. A number of procedures have been optimized in order to achieve this:
- incubating demembranated spermatozoa with exogenous DNA molecules, followed by microinjection of the sperm/DNA complex in oocytes, in what is now known as the ICSI procedure (Perry et al., 1999; Naruse et al., 2005);
- bypassing the plasma membrane by lipofection (Shemesh et al., 2000; Wang et al., 2001) and
- incubating sperm cells with antibody directed against specific proteins expressed on the sperm surface; the antibody will then mediate the interaction of exogenous nucleic acid molecules with the sperm membrane (linker-based mediated transgenesis) (Chang et al., 2002).
Taken as a whole, the results from these works suggest that the final fate of the exogenous DNA molecules depends on whether they interact directly with, or bypass, the sperm membrane: in the former case, as illustrated in Fig. 1, non-integrated extrachromosomal structures will mostly be generated, whereas integration of the transgene in the genome of sperm cells will be favored in the latter. This conclusion underscores the importance of what appears to be the earliest and the most fundamental event of the entire SMGT process, namely, the interaction between spermatozoa and exogenous DNA or RNA molecules.
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Work in our and other laboratories has shown that the interaction of exogenous nucleic acid sequences with sperm cells, and their ensuing internalization in nuclei, are not random events, but are well-regulated processes mediated by specific factors. Relevant steps in the interaction, as well as the nuclear internalization of foreign molecules in sperm cells, are schematically represented in Fig. 1. Although the precise mechanisms that mediate nuclear internalization are not fully clarified as yet, most of the necessary factors have been identified, as extensively described elsewhere (Spadafora, 1998
) and will not be re-examined here. For the scope of the subject of the ‘unusual’ inheritance discussed here, it is useful to recapitulate the SMGT steps that follow the internalization of the foreign DNA. Briefly, the internalized exogenous sequences reach the nuclear scaffold of sperm cells; therein, they are subjected to rearrangement(s) mediated by endogenous nucleases and undergo recombination events that eventually cause their integration in the sperm genome. To elucidate the mechanism through which this occurs, we constructed a genomic library from murine sperm cells incubated with a reporter plasmid and screened it extensively in order to identify the integration sites: the results suggest that integration occurs in one, or only very few, preferential site(s) in the sperm genome and is a very infrequent event, likely occurring in a small subpopulation of mouse sperm cells (Zoraqi and Spadafora, 1997). The interaction of sperm cells with exogenous DNA molecules activates one or more endogenous nuclease activities in a DNA dose-dependent manner (Maione et al., 1997). These nucleases heavily degrade the foreign DNA; eventually, they also locally cleave a minor chromatin component of sperm nuclei that retains a nucleohistone organization (Gatewood et al., 1987,1990; Pittoggi et al., 1999). These results therefore suggest that discrete sites of nuclease sensitivity exist within nucleohistone domains of the otherwise tightly packed chromatin of mature sperm cells (Sotolongo et al., 2005; Shaman et al., 2006); these sites are preferential targets for the integration of exogenous sequences (Pittoggi et al., 2000). |
Reverse transcriptase activity in spermatozoa and the generation of biologically active ‘retro’ genes |
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TopAbstractBackgroundReverse transcriptase activity...SMRGT: a modelGenesis and inheritance of...ConclusionsFundingReferences |
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The nucleohistone fraction of mouse sperm chromatin has features that closely resemble those of the active chromatin of somatic cells: it is nuclease-sensitive, organized in nucleosomes and its DNA component has a very low level of methylation (Uscheva et al., 1982
; Gatewood et al., 1987,1990; Banerjee et al., 1995; Pittoggi et al., 1999). Sequence analysis of randomly isolated clones from that fraction suggested an unexpected enrichment in DNA sequences of retrotransposon origin, among which the most abundant elements are represented by reverse transcriptase (RT)-encoding LINE-1 ORF2 sequences (Pittoggi et al., 1999, 2000).
These features emerged in the search for factors implicated in SMGT, but their existence, as a reflection of an endogenous machinery present in sperm cells, was highly intriguing and prompted us to investigate if a functional RT activity is present in mature spermatozoa. To this end, we planned a straightforward experiment in which mouse spermatozoa were incubated with exogenous RNA molecules; possible complementary DNA (cDNA) products were then searched by direct PCR amplification. The underlying idea was that if spermatozoa are actually endowed with a RT activity, then the internalized exogenous RNA might be used as a substrate and reverse-transcribed to cDNA copies. In a first set of experiments, we used human poliovirus chromosomal RNA in order to rule out any possible artifact due to contaminating endogenous DNA, since the poliovirus RNA chromosome replicates through a RNA (–) strand with no DNA intermediate (as is the case for retroviruses). That experiment showed that poliovirus RNA was indeed taken up by sperm cells; actually, the nucleic acid-binding molecules present on the sperm cell surface did not show any obvious preference in their interaction with DNA or RNA. The poliovirus RNA was then reverse-transcribed to cDNA copies, which were transferred to oocytes during IVF and further transmitted to two-cell embryos (Giordano et al., 2000). Moreover, immunoelectronmicroscopic analysis using anti-RT antibody showed that RT molecules were stably associated with the sperm nuclear scaffold.
We next asked whether the newly synthesized cDNAs in spermatozoa behave as biologically active retrogenes, or, on the contrary, as non-functional products. We incubated sperm cells with RNA populations transcribed from a construct expressing a β-galactosidase (β-gal) reporter gene; we next used these cells in IVF assays. We produced a F0 founder progeny and, subsequently, a F1 progeny by normal breeding. Direct PCR analysis of DNA samples from both F0 and F1 animal populations confirmed that β-gal-containing cDNAs were generated in sperm, delivered to oocytes, mosaic propagated throughout embryogenesis in various tissues of adult animals and mosaic transmitted from one generation to the next.
Some remarkable features of these sequences are worth noting: (i) the stable maintenance at low-copy number (<1 copy per genome), and thus below the resolving power of Southern blot but detectable by PCR amplification, (ii) their mosaic distribution, (iii) their maintenance as non-integrated extrachromosomal structures and (iv) their non-Mendelian inheritance.
Most importantly, expression of the X-gal protein was detected in a variety of positive tissues, in both F0 and F1 animal populations (Sciamanna et al., 2003). These results showed for the first time that ‘transgenic’ mice can be obtained using RNA, instead of DNA molecules, through a spontaneous reverse transcription-mediated process which we have called sperm-mediated ‘reverse’ gene transfer (SMRGT). Given the features of the foreign sequences transmitted via this process, the animals obtained by this procedure do express a new trait, yet can hardly be regarded as transgenics: transgenic animals are characterized by well-defined features, including (i) a stable integration of at least one copy of the transgene in the germ line, (ii) its Mendelian inheritance in the progeny, (iii) an ubiquitous distribution in the genome of somatic cells and (iv) a correctly modulated expression. With the exception of the latter, none of the other features are observed in mice generated by SMRGT (Smith and Spadafora, 2005).
In more recent work, surprisingly, we have found that an RT-dependent process is triggered not only when spermatozoa are incubated with RNA, but also when sperm cells are exposed to exogenous DNA (Pittoggi et al., 2006). That unexpected phenomenon emerged form experiments in which sperm cells were incubated with a retrotransposition cassette-containing DNA construct, constituted by an enhanced green fluorescence protein (EGFP) reporter gene interrupted by a 
-globin intron placed in the opposite orientation to that of EGFP transcription (Ostertag et al., 2000). In order to be expressed, the reporter gene needs to go through a reverse transcription step. The experiment was carried out following the same outline described above: first, the exogenous DNA interacts with the sperm and is internalized into nuclei; a sequential process then occurs, in which the foreign DNA is transcribed, the primary RNA transcript is spliced and finally reverse-transcribed to EGFP-containing cDNA copies (Pittoggi et al., 2006). Interestingly, only a small proportion of the newly synthesized cDNAs is retained within the sperm heads, with most of the reverse transcribed molecules being released from spermatozoa into the incubation medium where their concentration gradually increases in a time-dependent manner. These released molecules are then available for further interaction with sperm cells: this leads to a steady-state situation in which the vast majority of the sperm cell population is associated with ‘foreign’ cDNAs. These reverse-transcribed cDNAs, delivered to oocytes at fertilization, exhibit the same features of those obtained when sperm cells were incubated with exogenous RNA, essentially as extrachromosomal, low-copy structures. These cDNAs are transcriptionally competent: indeed, the EGFP reporter gene was found to be expressed in the vascular epithelium of various positive tissues of adult animals (Pittoggi et al., 2006).
In summary, transcriptionally competent ‘retro-genes’ can be generated in a reverse transcription-mediated process, regardless of whether intact, viable spermatozoa are incubated with exogenous DNA or RNA molecules. These reverse-transcribed sequences are: transferred to embryos at fertilization, mosaic propagated in the tissues of founder animals, again mosaic transmitted in a non-Mendelian fashion from founders to F1 progeny, maintained as non-integrated, low-copy number structures identified by direct PCR (but not by Southern blot) in tissues of both F0 and F1 animal populations, transcriptionally competent and expressed in various tissues of F0 and F1 animals. Based on these results, we have come to realize that reverse-transcribed copies, rather than the DNA sequences originally incubated with spermatozoa, are preferentially propagated and expressed in the tissues of positive animals. This conclusion represents a very important element in re-evaluating the original SMGT experiments. What follows is a proposed unifying mechanism for SMGT process.
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SMRGT: a model |
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TopAbstractBackgroundReverse transcriptase activity...SMRGT: a modelGenesis and inheritance of...ConclusionsFundingReferences |
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As schematically represented in Fig. 1, exogenous DNA or RNA molecules can bind to nucleic acids-binding proteins (in blue) located on the sperm plasma membrane and are subsequently internalized in the sperm nucleus, in close contact with the nuclear scaffold (see Spadafora, 1998
, for details). A sperm endogenous RNA polymerase (Fuster et al., 1977) then transcribes the internalized exogenous DNA (black) to RNA molecules (blue). An interesting scenario emerges at this point: the RNA molecules are captured by a LINE 1-encoded RT-containing machinery (represented by the brown oval associated with the sperm nuclear scaffold), spliced and further reverse-transcribed to cDNA copies (red circles). Exogenous RNA molecules internalized in sperm cells are instead directly reverse-transcribed to cDNAs in a single-step process. Based on immunoelectronmicroscopy localization (Giordano et al., 2000), the RT molecules can be regarded as integral components of the sperm nuclear scaffold. Most newly synthesized cDNA molecules are extrachromosomal, autonomously replicating structures that are partly stored in sperm heads and partly released from spermatozoa into the incubation medium (Pittoggi et al., 2006). Rare integration events, however, may occur in the matrix-bound chromosomal DNA that connects adjacent nucleoprotamine loop domains (Zoraqi and Spadafora, 1997; Pittoggi et al., 2000). In contrast with the latter, in which the DNA is tightly packed and virtually unaccessible to foreign molecules, the matrix-bound, inter-loop domains are regarded as accessible, nuclease-hypersensitive chromatin regions, in which the nucleosomal organisation is preserved and histones were not replaced by protamines during spermatogenesis, which makes them integration-prone (McCarthy and Ward, 1999; Pittoggi et al., 1999, 2000). The integrated foreign sequences can in turn contribute to generate extrachromosomal cDNA structures via reverse-transcription of their own transcribed RNA, reminiscent of a provirus-type model. It must be stressed that integration is an extremely infrequent event occurring in a minor proportion of sperm cells; this conclusion suggests, therefore, that the probability that a truly ‘transgenic’ spermatozoon (i.e. a spermatozoon carrying an integrated foreign sequence in its own genome) fertilizes an oocyte is extremely low. Autonomously replicating, extrachromosomal structures, abundantly produced and released in the medium, are instead easily taken up by spermatozoa swimming in the medium, delivered to oocytes at fertilization and propagated in embryos and in tissues of adult animals. These structures are also present in the germ line and stored in mature gametes of F0 individuals to be transmitted to F1 progeny in a non-Mendelian fashion. The mechanism through which these extrachromosomal structures are replicated and propagated has still to be elucidated.
A model emerges from these results, suggesting that the expression of some phenotypic traits in supposedly transgenic animals does not depend uniquely on chromosomal genes, but may derive from a more subtle flow of RNA-mediated genetic information generated by the retrotransposon system. As a whole, these findings confirm and further expand the view that spermatozoa do much more than simply delivering the male genome at fertilization, e.g. by contributing transcription factors (Pittoggi et al., 2001) and, most importantly, a variety of RNA populations (Krawetz, 2005). As will be briefly discussed below, the RNA population carried by sperm cells has been recently identified as the key determinant in an emerging mechanism of non-Mendelian inheritance of genetic traits.
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Genesis and inheritance of non-mendelian genetic traits |
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TopAbstractBackgroundReverse transcriptase activity...SMRGT: a modelGenesis and inheritance of...ConclusionsFundingReferences |
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A recent article reports on instances of non-Mendelian, RNA-mediated inheritance of extra-genomic information in mice (Rasoulzadegan et al., 2006
). The authors reported that mutations (caused by a Lac Z insertion) in the Kit gene generate white patches on the tail and the feet, which are maintained in heterozygous individuals. The surprising finding is that some of the offspring of such mice, who inherited two wild-type Kit alleles, still exhibited the white patches—though to a variable extent—characteristic of Kit mutant animals. The mutant phenotype is due to reduced expression of wild-type Kit, concomitant with an accumulation of non-polyadenylated RNA molecules of abnormal size in spermatozoa. Microinjection of this RNA population in zygotes induced a heritable white tail phenotype. This report shows, therefore, that phenotypic features are not exclusively linked to the expression of chromosomal genes, but can depend on information stored in a stable class of RNA molecules. More specifically, the work clearly demonstrates that RNA molecules stored in mature spermatozoa are responsible for the extra-genomic mode of inheritance of the mutant phenotype under study, which is transmissible either through fertilization or by direct microinjection of the RNA population into zygotes. These results have been interpreted as a para-mutation (Chandler, 2007) phenomenon and the RNA molecules are supposed to be propagated throughout generations by virtue of an RNA-dependent RNA polymerase (RDRP) (Alleman et al., 2006). The phenomenon described by Rasoulzadegan and coworkers (Rasoulzadegan et al., 2006) shares surprising analogies with the model described for SMRGT. The RNA-mediated inheritance and SMRGT do indeed describe paradoxical situations, in which the expression of a specific gene—an ‘artificial’ reporter gene—is phenotypically preserved and transmitted in a non-Mendelian fashion to the progeny, even when that gene is in fact virtually absent from the host genome.
The mechanism of RNA-mediated inheritance is not fully clarified as yet. A crucial outstanding question concerns the mechanism through which RNA molecules responsible for the mutant phenotypes are maintained throughout embryogenesis, propagated in adult F0 animals and transmitted to the F1 progeny. It has been suggested that a RDRP activity is implicated in this process (Alleman et al., 2006). Based on the analogies between the two systems, it is possible that RNA molecules replicate through DNA intermediates generated during a reverse transcription step, essentially as schematized in Fig. 1. Such a RT-mediated replication expansion process may not only take place in sperm cells, but perhaps also in embryos and in differentiated somatic cells, given that RT activities operate throughout embryogenesis (Pittoggi et al., 2003,2006), as well as in adult tissues (Kiessling and Goulian, 1979; Salganik et al., 1985; Medstrand and Blomberg, 1993; Banerjee and Thampan, 2000). In principle, therefore, this mechanism may allow the maintenance, propagation and inheritance of that particular class of RNA molecules throughout the whole life cycle of the animal. In conclusion, I would like to suggest the possibility that the RT-dependent mechanism that underlies the SMRGT process also offers a plausible scenario for an RNA-mediated inheritance phenomenon.
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Conclusions |
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TopAbstractBackgroundReverse transcriptase activity...SMRGT: a modelGenesis and inheritance of...ConclusionsFundingReferences |
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The data discussed in this review support the idea that the interaction of exogenous sequences, both DNA and RNA, with the sperm plasma membrane triggers a previously unknown RT-dependent mechanism: such a mechanism would lead to the generation and non-Mendelian propagation of new genetic information in mature spermatozoa, independent from the information carried in the genome. This mechanism, called SMRGT, generates new inheritable phenotypic traits in founders, in the virtual absence of corresponding chromosomal genes. Furthermore, in agreement with a ‘functionalist’ interpretation (Shapiro and von Sternberg, 2005
; von Sternberg and Shapiro, 2005), the SMRGT model implies a key functional role for endogenous RT activity and for the genomic retroelements that harbor the RT-encoding genes; collectively, these elements account for 
45% of the human genome (International Human Genome Consortium, 2001). Based on the close analogy between SMRGT and the recently discovered RNA-mediated inheritance, both processes can be viewed as part of a more global phenomenon which is progressively disclosing the complex network of a genetic machinery controlling gene expression and inheritance, within which retroelements play non-irrelevant roles (Whitelaw and Martin, 2001; Song et al., 2004; Han and Boeke, 2005). Many aspects of the basic SMRGT mechanism still remain elusive, but there are sufficient clues to regard it as the source of a subtle flow of genetic information that parallels, and complements, that associated with chromosomal genes. Recent work in our laboratory has identified a specific LINE-1-encoded RT as a key element indispensable for preimplantation embryonic development in the mouse (Pittoggi et al., 2003; Beraldi et al., 2006). While the mechanistic role of this embryonic RT is not fully understood, emerging evidence suggests that it may be involved in formation of the embryonic diploid genome and in control of embryonic gene activation. In this perspective, the sperm RT may play a role in the reorganization of the male genome soon after fertilization; athough completely speculative at this stage, an attractive possibility is that the RNA population stored in sperm cells (Krawetz, 2005) contains target molecules for reverse transcription. |
Funding |
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TopAbstractBackgroundReverse transcriptase activity...SMRGT: a modelGenesis and inheritance of...ConclusionsFundingReferences |
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Istituto Superiore di Sanità and the Italian Ministry of Health grants (No: 501/ and 501/2 and 530/F17 to C.S.).
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Submitted on October 17, 2007; resubmitted on November 21, 2007; accepted on December 13, 2007.
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