Sperm-Mediated Gene Transfer: Concepts and Controversies

The concept of Sperm-Mediated Gene Transfer (SMGT) describes the ability of spermatozoa to deliver to embryos not only their own genome during fertilization, but also foreign genetic information with which they may come in contact. The concept was established in 1989, when it was first shown that exogenous DNA incubated with mouse spermatozoa can be detected in tissues of born offspring. That initial finding has promoted a wealth of studies, progressively extended to a variety of species and still ongoing, with the dual aim to a] clarify the basic molecular mechanisms underlying SMGT on the one hand, and, b] on the other hand, develop biotechnology applications to generate genetically modified animals. Progress in any scientific field is often achieved in a non-linear manner and re-examining its historical unfolding serves to formalise conceptual advance as well as any remaining gaps. In this chapter I recapitulate the research progress and conceptual evolution within the SMGT field since its initial discovery. I focus on basic studies and particularly discuss findings that I consider as the distinctive milestones of the field. This historical journey spans more than twenty years, from the earliest observations that first suggested the idea that sperm cells are permeable to exogenous molecules to the recent finding that a reverse transcriptase (RT)-dependent mechanism is active in sperm cells and can regarded as a continuous source of novel genetic traits.

The finding that spermatozoa of virtually all animal species can spontaneously bind exogenous DNA molecules and deliver it to oocytes at fertilisation first suggested that these cells can be used as vectors for introducing new genetic and phenotypic traits in animals. That has led to the development of a novel approach to animal transgenesis, namely Sperm Mediated Gene Transfer (SMGT). Here we review findings obtained using this experimental approach. A critical examination of published evidence indicates that the alternative between direct binding to the plasma membrane of sperm cells, or its bypass, represents a crucial parameter for the fate of exogenous nucleic acid molecules: in the former case, episomal structures are mainly generated; in the latter, integration in the host genome is more frequent. The original protocol was based on the direct interaction between sperm cells and foreign DNA. Several alternative variants have been developed thereafter to improve the efficacy of the method. Improved protocols include the combination of SMGT with: i) ICSI (intracytoplasmic sperm injection) technology, ii) restriction enzymes favoring DNA integration (REMI), or iii) linker-based (LB), in which the DNA binding is mediated by an antibody recognizing a membrane antigen. In another approach, the aim is to produce “transgenic spermatozoa”: for example, in testis-mediated gene transfer (TMGT) the foreign DNA is microinjected directly into testis; in virus-mediated transgenesis, new genes are delivered to spermatogonal stem cells by viral vectors. The recent finding that mature spermatozoa are the source of non-integrated, transcriptionally competent retrogenes also suggests a potential use of SMGT for embryonic gene therapy.

Strong natural barriers exist against SMGT. However, such barriers are unlikely to be absolutely inviolable. If sperm cells can indeed act as vectors for exogenous DNA, it follows that the genome of sexually reproducing animals may be subject to alteration by exogenous DNA sequences carried by sperm cells. At present there are insufficient data to permit quantification of the rate at which SMGT may occur in nature. Nevertheless, the implications of such ‘natural’ SMGT are significant, and include evolutionary effects on the mammalian genome and pathologies in humans from de novo mutations.

Sperm mediated gene transfer (SMGT) was developed as an alternative technique for the production of transgenic animals. This technique is based on the ability of spermatozoa to take up exogenous genes of interest in the form of DNA molecules in vitro and deliver them to the oocyte during fertilisation. Thus, novel genetic information could be integrated into the embryo genome in order to alter the expression of specific genes of the offspring and subsequent generations. DNA uptake by spermatozoa is a very specific and well regulated mechanism. Although SMGT has been shown to be efficient, protocols for animal transgenesis are still under optimisation. Recent modifications of SMGT protocols, including intracytoplasmic sperm injection derived transgenesis (ICSI-Tr) and testis mediated gene transfer (TMGT), have been reported. Further understanding of the mechanisms involved in SMGT will enhance our understanding of the biology of fertilisation. Although not yet perfect, the technique of SMGT is of high biotechnological and medical potential. The use of SMGT to generate transgenic domestic animals could enhance their performance, and could also enable the production of proteins and pharmaceuticals within the milk of farm mammals. In addition, it could be used to generate animals as models for human diseases or to produce multitransgenic animals for xenotransplantation purpose. Finally, SMGT also holds promise in the context of human gene therapy in future.

The capture of exogenous DNA and its transfer to eggs by sperm cells are the two key processes in SMGT. During the past two decades, the efficiency of the capture of exogenous DNA molecules by sperm cells has been increased by various approaches, such as use of liposomes, electroporation, Triton-X or DMSO-treated spermatozoa, viral vectors, and magnetic nanoparticles. Testis-mediated gene transfer (TMGT) is a novel alternative variant of SMGT used to produce transgenic animals in vivo. This chapter presents the background and features of both non-viral and viral forms of SMGT, together with TMGT, as developed in recent years. Additionally, modes of fertilisation by transgene-bearing sperm are discussed.

Sperm-mediated gene transfer (SMGT) in mice has undergone rapid developments over the past twenty years, because of the obvious advantages in operation: it is inexpensive and convenient, and requires minimal specialist training and equipment. The typical method of SMGT technology is to use the mature spermatozoa as vehicles for DNA delivery in vitro. However, the low efficiency associated with this approach has resulted in many negative results, with consequent controversy over its biological basis. By contrast, in vivo gene transfer through direct introduction of foreign DNA into the male reproductive tract is developing apace. The mouse as a major model animal has made an important contribution to SMGT progress.

Transgenic research has been developed for several purposes including genetic improvement of aquacultured species and production of experimental models for biomedical research. Microinjection of DNA into pronuclei/nuclei of fertilised eggs is the technique that has been most used and applied successfully in transgenic animal production. However, this technique is laborious, time-consuming and it is limited by egg characteristics of some species. In aquatic animals such as marine finfish and shellfish the obstacles are even higher since these organisms have, in general, small and fragile eggs associated with high mortality during the first stages of development. Thus, en masse transgenesis methods such as Sperm Mediated Gene Transfer (SMGT) or Testis Mediated Gene Transfer (TMGT) could be useful for the production of genetically modified aquatic species. Although SMTG has been proved to be a good alternative for en masse transgenesis, this technique will only be routinely applicable in the near future if further investigations are focused on the factors that influence the interaction between spermatozoa and exogenous DNA as well as the processes that regulate transgene integration and expression.

The importance of transgenic farm animals resides in their usefulness for very different objectives: for instance in human medicine (to obtain pharmaceutical products, organs for xenotransplantation, or as models for research in gene therapy ) or for agricultural applications such as improved output of the carcass or milk production and composition, increased growth rate, improved feed utilisation, enhanced reproductive performance, increased prolificacy, as well as to enhance disease resistance or reduce environmental impact. Pronuclear DNA microinjection has long been the most reliable method to produce transgenic animals. However, although transgenic animals have been generated using this approach, it has many limitations. Sperm Mediated Gene Transfer (SMGT) is based on the ability of sperm to bind, internalise, and transport exogenous DNA into an oocyte during fertilisation. In this chapter we review the state of art of SMGT in farm animals with a special emphasis on porcine and bovine animals, with additional information related to other ruminants and horses. We evaluate the possible applications of transgenic pigs and cattle and review the factors related to the success of SGMT in these species and offer our own experience based on studies analyzing the main factors in porcine and bovine SMGT.

The current method of micromanipulation used for domestic animals results in less than 1% transgenic animals. This makes it extremely difficult to produce transgenic cows and is not feasible for producing transgenic chickens. The purpose of this work was to find a more efficient method for producing transgenic calves and chicks using a combination of two techniques, lipofection and restriction enzyme mediated insertion (REMI). Previously investigators were unable to produce transgenic chickens using lipofection alone. On the other hand, injection of isolated sperm nucleus incubated with restriction enzyme into oocytes has only been shown to be effective in frogs. In this study, we demonstrated for the first time, that lipofection of both DNA and restriction enzyme could be used to successfully integrate DNA into the sperm genome DNA and then used for routine AI to produce transgenic calves and chicks. First it was demonstrated using needle pricking and southern blot analysis of genomic DNA that the restriction enzyme opens up “hot” spots in the sperm genomic DNA. This produces sticky ends by which foreign DNA can be inserted and integrated into the sperm genomic DNA. The “transgenic sperm” thus made were used in IVF and AI to produce embryos expressing a foreign DNA, EGFP (enhanced green fluorescent protein). Using Not I and linearized pEGFP lipofected to sperm for AI resulted with two calves which expressed the exogenous DNA in their lymphocytes as determined by (a) PCR and RT-PCR; (b) specific emission of green fluorescence by the EGFP protein; (c) homology analysis between EGFP DNA and PCR product DNA sequences and (d) Southern blot analysis. Similarly in the chicken, linearized plasmid EGFP sequences with the corresponding restriction enzyme (REMI) were lipofected into the sperm. The transfected sperm were then used for AI in hens and 90% (17/19) of the resultant chicks expressed the exogenous DNA in their lymphocytes as determined by: (a) PCR and RT-PCR; (b) specific emission of green fluorescence by the EGFP; and (c) Southern blot analysis. A complete homology was found between the Jellyfish EGFP DNA and a 313 bp PCR product of DNA from chick blood cells. The procedure was then tested with an additional construct, hFSH. The construct of hFSH consisted of both subunits, α and β and the PCR product used primers for both α and β subunit resulted with a PCR product of 584 bp which was unique to transgenic chickens. The procedure was then used to lipofect a construct of hFSH (Human Follicular Stimulating Hormone) into chicken sperm and used for AI. The resultant offspring were transgenic for at least three generations as determined by: (1) measurement of hFSH protein in chicken blood using enzyme immunoassay and RIA; (2) RT-PCR and PCR; and (3) copy number.

We conclude: (1) that lipofection of both DNA and restriction enzyme into sperm (bovine and chicken) induces the integration of the DNA into the sperm genomic DNA; (2) lipofected sperm can be used in AI to produce a high percentage of transgenic calves and chicks; (3) The integrated gene is expressed in the first, second and third generation; and (4) the method is not limited to specific genes. The technique of lipofection of DNA combined with REMI is therefore an efficient and stable method of producing transgenic domestic animals. Efficient production of transgenic domestic animals could have major impact on gene therapy, improving livestock breeds and the production of valuable pharmaceuticals, e.g. hFSH, which could be extracted from eggs and milk.

Using the mouse model, intracytoplasmic sperm injection (ICSI)-mediated transgenesis has been shown to be a valuable tool for the production of transgenic animals, an essential instrument for basic and applied research in bioscience. This method of transgenesis consists of the microinjection of spermatozoa preincubated with foreign DNA. ICSI of DNA-loaded sperm cells has been shown to mediate mouse transgenesis at high efficiency, especially when sperm cell damage (by freeze-thaw cycles or exposure to detergents) is induced. The greatest advantage of ICSI-mediated transgenesis is that it allows introduction of very large DNA transgenes (e.g., yeast artificial chromosomes), with relatively high efficiency into the genome of hosts, as compared to pronuclear microinjection. In addition, ICSI-mediated transgenesis is associated with (a) low frequencies of embryo mosaicism, one of the major limitations of pronuclear microinjection for the production of transgenic livestock, and (b) a high frequency of Mendelian germline transmission of transgenic sequences among founder animals. In this chapter we will review some factors that can increase the efficiency of the ICSI-mediated transgenesis and we will describe a new active form of ICSImediated transgenesis employing fresh sperm in conjunction with recombinase or transposase molecules.

Nanoparticles are being incorporated into many products of daily use, e.g. fillers, pacifiers, catalysts, pharmaceuticals, lubricants, cosmetics, pharmaceuticals, electronic devices or other domestic appliances. These approaches demonstrate the high potential of nanocomposites in cellular biotechnology as well as for the development of more efficient methods to introduce foreign DNA to sperm cells and consequently improve the techniques for transgenic animal technology. This chapter will address the main approaches for the use of nanocomposites for gene delivery and the potential uses in nanoparticle spermmediated gene transfer (Nano-SMGT).

The discovery that spermatozoa of essentially all species can spontaneously take up exogenous DNA and deliver it to oocytes at fertilization suggested their use as vectors of foreign information for the generation of transgenic animals. Hence a variety of protocols of sperm-mediated gene transfer (SMGT) have been developed in numerous species. The outcomes in stability and transmission of the exogenous sequences are highly heterogeneous, casting doubt on whether the generated animals are truly transgenic and leaving an open question as to the final fate of the DNA molecules delivered by sperm cells. Two findings have contributed to clarify the underlying molecular mechanism of SMGT: i) the discovery that DNA-loaded demembranated spermatozoa used in ICSI assays increase the yield of genuine transgenic mice, and ii) the identification of a reverse transcriptase (RT) activity in mature spermatozoa. When membrane-disrupted sperm cells are incubated with DNA prior to microinjection in oocytes, the foreign sequences are integrated in the host genome and a high proportion of the offspring are genuinely transgenic. In intact spermatozoa, instead, the foreign DNA binds to the plasma membrane, hence is internalised and undergoes a two-step reaction, first of transcription in RNA and then of reverse-transcription in cDNA copies; these cDNAs behave as transcriptionally competent retrogenes, are propagated as non-integrated extrachromosomal structures and are transmitted to the progeny in a non-Mendelian fashion. We have called this phenomenon sperm-mediated “reverse” gene transfer (SMRGT). These results point out the central role of the plasma membrane in the final fate of exogenous sequences as either integrated transgenes or extrachromosomal retrogenes. The latter underscore a previously unrecognised transgenerational genetics, and a form of non-Mendelian inheritance, mediated by an RT-dependent mechanism.

Several experts in the various fields of SMGT have contributed valuable knowledge to this eBook. This chapter offers a synthesis of the key concepts covered throughout the book, and critically evaluates the current status and controversial aspects of SMGT.