SEAMEO Jasper Fellowship Monograph 1993 Series 1

SPERM-MEDIATED GENE TRANSFER:
A REVIEW

Hong-Woo Khoo¹, Jawahar G Patil¹ and Ee-Sin Chen²

¹ Fisheries Biology Laboratory, Department of Zoology;
² Institute of Molecular and Cell Biology
National University of Singapore

Abstract

This paper describes the progress of studies on sperm as vectors for gene transfer for production of transgenic fish at the Fisheries Biology Laboratory, Department of Zoology, National University of Singapore. The application of this method in other vertebrate groups is also described. Its potential for transgenic fish production is discussed.

Introduction

Lavitrano et al (1989) a stir when they first demonstrated that transgenic mice could be produced efficiently by in vitro fertilization with sperm which had simply been incubated with DNA. However, a number of other researchers were unable to produce transgenic individuals (Brinster et al, 1989). If sperm could be used gene transfer would become dramatically simpler (Archibald, 1991).

This paper will review the evidences, obtained to date, to answer the question: Can sperm be used as vector for gene transfer?

This idea of using sperms for exogenous gene transfer in fish was first conceived during the first author's sabbatical leave with E.M. Donaldson at the West Vancouver Laboratory, Department of Fisheries and Oceans in November 1984 (Khoo et al, unpublished). At that time, micropylar microinjection was developed and conducted in Flycatcher's laboratory in Eastern Canada where attempts were already on their way to introduce the antifreeze gene into the Atlantic Salmon. Micropylar microinjection has by then been attempted in the West Vancouver Laboratory by a summer school student and microinjection equipment were already available. However, there were no molecular biology facilities. A collaboration with Jurgen Vielkind of the Environmental Carcinogenesis Unit, British Columbia Cancer Research Centre, Vancouver was then established. He was interested in the fate of microinjected DNA into the yolk sac of fish embryos after hatching (Schwab et al, 1976). It was there that the molecular biological and recombinant DNA techniques were were available. The awareness of the usefulness of molecular biology in fisheries and aquaculture was already primed through a one month attachment in Choy L. Hew's laboratory at the Banting Institute, University of Toronto in October 1984 to study the use of HPLC for steroid bioassay, a month prior to going to the West Vancouver Laboratory. His laboratory was already steeped in molecular biology at that time and he had been working on the anti-freeze gene and already collaborating with Fletcher.

After several trials at micropylar microinjection into Coho salmon eggs obtained from a nearly hatchery together with Tilmann Benfey, the first author realised that the method was too tedious and that the problem would be much greater with the smaller eggs of many tropical fish available in his home country. Tilmann was conducting routine in vitro fertilization at that time and was producing coho triploids using pressure. The paper that helped to suggest that spermatozoa is a possible vehicle for introducing exogenous genes into the fish embryo was that by Mulcahy and Pascho (1984). They demonstrated that 99 percent of a vertically transmitted fish rhabdovirus, infectious haematopoietic necrosis virus, was adsorbed to the surface membrane of sperm from two genera of salmonoid fishes and suggested that such adsorption may be involved in vertical transition of these viruses.

A check in the literature then showed that the earliest study by Brackett et al (1971) showed that simian virus 40 (SV 40) DNA adsorbed on to rabbit spermatozoa and was successfully carried into the ovum during the process of normal fertilization.

That spermatozoa are able to seek out the female pronucleus with such precision seems reasonable enough therefore to think that they may be utilised to deliver foreign DNA to the target ovum and also at the same time resulting in the successful integration into the genome of the introduced gene. This strategy, thus overcomes the problems of pinpointing the nuclear target, the sperm does this naturally.

Experiments have been conducted in this laboratory using zebrafish as the model fish to show that plasmid DNA is easily attached to the spermatozoa and that a proportion of the embryos resulting from fertilization by such sperms carried the DNA maker and that some of these embryos showed germ-line transmission of the inserted DNA to their progeny.

Sperm Uptake of DNA

The aim is to establish the uptake of DNA by the sperm using the Southern Hybridization Method for monitoring.

Sperm was incubated with the pGEM-luc plasmid DNA (4933 bp) for 10 and 30 minutes. Non-motile formaldehyde treated sperm was also incubated with the plasmid for 30 min. A positive control consisting of all reagents was added to the plasmid but without sperms. The negative control contained all reagents except the plasmid DNA. Untreated sperm control was also included. The experimental protocols for preparing the sperms and extraction of DNA are described in Khoo et al (1992). The probe used in the Southern Blot was labeled by multiprime labeling kit (Amersham) and the sperm DNA extracts were linearised with Hind III.

Positive hybridization signals were observed for the treated samples indicating that DNA had been taken up by the sperm. A clear band each with slightly higher molecular size than the linearised pGEM-luc size of 4.9 kb is seen in the autoradiograph (Fig 1) both for the samples incubated for 10 min (lanes 11-12) and for 30 min (lanes 13-14). Similar bands are also obtained from the formalin treated sperms (lanes 9-10).

The results showed that zebrafish sperms do pick up DNA in less than 10 min and that live as well as formalin treated sperms can take up the DNA.

Internalization of DNA into Sperm

The aim is to determine whether the DNA taken up is internalized into the sperm or not .

Zebrafish spermatozoa were obtained as described in Khoo et al (1992). Aliquots of about 10⁷ spermatozoa washed in PBS free from Ca²⁺ and Mg²⁺ were incubated with 1 mg/ml ³H-radiolabelled or unlabelled pMTL plasmid for 30 min at room temperature, washed in PBS, fixed and sections prepared for film emulsion autoradiography to be observed under light microscopy as well as for ultrastructural-in-situ-hybridization (UISH) and autoradiography. Details are described by Patil and Khoo (in press).

Light microscopic autoradiograms showed that radioactive DNA was associated with a number of sperm cells but not all the sperm cells were labeled indicating that some sperm cells were unable to pick up DNA.

Evidence from electron microscope examination of the autoradigraphs obtained after in-situ hybridization showed that foreign DNA was present within the sperm nucleus (Fig 2). This clear showed that DNA had been internalised into the sperm head. DNA on the sperm mid-piece have also been observed but none was observed on the tail.

Sperm-Mediated DNA Transfer

The aim is to demonstrate that embryos produced from oocytes fertilized by DNA soaked sperms carry these exogenous DNA markers. A schematic representation of the same is shown in Fig 3.

Sperm from mature adult male zebrafish were incubated in plasmid DNA solution for 30 min and then used to fertilize eggs stripped from gravid females. The embryos were reared to fry stage and their genomic DNA extracted and screened for the presence of the exogenous DNA by Southern analysis and PCR methods. Expression of the inserts were also monitored. Three recombinant DNA plasmids were tested as reporters (Fig 4): pUSVCAT, pXGH5 and pMTL and their use and monitoring details are described by Chong et al (1989), Khoo et al (in press) and Patil et al (1994), respectively. All three reporters and their assays kits are commercially available.

About 37.5% and 23.3% of the embryos produced from sperm soaked in linearised and circular pUSVCAT DNA, respectively, showed the presence of the exogenous maker. About 33% of the embryos produced from circular pMTL DNA soaked sperm were positive as determined by PCR.

Expression was not observed for the pUSVCAT and pMTL treatments but 50% and 30% of embryos treated with the linearised and circular forms of pXGH5, respectively, showed positive expression of the growth hormone gene.

These results indicated that sperm mediated transfer occurs irrespective of the type of plasmids used and that in all three cases about one third of the treated eggs were affected. The DNA inserts were probably not integrated with the genomic DNA because only small fragments were observed in the Southern Blot autoradiographs (Fig 5). Episomal integration rather than genomic integration was probably the main outcome.

The above results also showed that expression products may be produced as shown by the growth hormone reporter (Khoo et al, in press). The apparent absence of expression products from the other two reporters, pUSVCAT and pMTL was probably due to the low level of expression since microinjection of both the CAT and the LUC reporters into the zebrafish embryos had been shown to result in positive expressions (Khoo, 1993; Patil et al, 1994). The latter was probably due to the greater amount of DNA injected.

Germ-Line Transmission

The aim is to demonstrate germ-line transmission and persistence of the introduced genes.

Zebrafish founders which were the outcome of sperm mediated transfer using the pUSVCAT as reporter were reared to adulthood and their genomic DNA extracted from portions of their pectoral fins and monitored for the presence of the reporter. Positive individuals were then cross-bred with untreated adults to produce F¹ progeny which were then assayed both for the presence of reporter DNA as well as for expression. Putative transgenic F¹ adults were also crossed with untreated individuals and their F₂ progeny monitored for the presence of the exogenous gene insert.

Khoo et al (1992) showed that germ-line transmission of the pUSVCAT insert down to the F₂ generation was possible. CAT expression, in both the generations were not detected. Tests with pXGH5 plasmids, however, did not result in any germ-line transmission (Khoo et al, in press).

Southern blot data showed that only a few progeny showed genomic integration of the gene insert as shown by the high molecular weight bands, however the majority of them showed episomal persistence of the pUSVCAT plasmid insert. The plasmid DNA appears to persist extrachromosomally and to replicate in the embryo.

Discussion

Information obtained from the above experiments showed that DNA is easily attached to zebrafish fish sperm and conveyed to the embryo bin in vitro fertilization. Most of the fish produced were mosaic and true genomic integration of exogenous DNA has not been shown so far because high molecular weight presence of the gene insert is not evidence enough to assume integration because concatemers of the gene insert could also result in these high molecular weight forms (Chong and Vielkind, 1989). Germ-line transmission is also no guarantee that the gene has been integrated because episomal integration and extrachromosomal DNA transmission has been shown and commonly observed in other species such as Caenorhabditis elegans, zebrafish and mice (Patil et al, 1994) as well as in birds (Freeman and Bumstead, 1987).

Sperm-Mediated Transfer in Species Other than Fish

The uptake of DNA by sperm cells has been documented in many species (Lauria and Gandolfi, 1993). The species listed include rabbit, mouse, boar, bull, buffalo, ram, goat, man, rooster, carp, honeybee and blowfly. Castro et al (1990) reported that avian sperm associated with exogenous DNA within the first hour of incubation. Nuclear internalization of exogenous DNA was also demonstrated for mouse (Francolini et al, 1993).

Lauria and Gandolfi (1993) also gave a list of species in which successful sperm-mediated DNA transfer has been described. Their list included rabbit, mouse, sea urchin, honeybee, pig, chicken and bovine. The chicken sperm cells can be used to deliver exogenous genes to the ovum which was demonstrated by Gruenbaum et al (1991). Sperm cells were incubated in a buffer containing lacZ or cat genes and then used to inseminate hens. PCR and Southern blot analysis showed 30-60% of the birds produced contained the genes and some of these were germ-line transmittable (Shuman, 1991). Cat and beta-galactosidase assays showed that the transfected birds were mosaic. Nakanishi and Iritani (1993) showed that exogenous DNA was transferred from the sperm to the eggs following fertilization in vivo. Low level of DNA was incorporated in the sperm incubated with DNA but almost one-half of the eggs monitored were found to incorporate the exogenous DNA. They also observed only episomal integration. There was no genomic integration.

In the fourth European Congress of Cell Biology in Prague, 26 June - 1 July 1994, further evidence for successful sperm mediated transfers were demonstrated in mouse, swine and bovine embryos (Lavitrano et al, 1994); Xenopus laevis (Jonak et al, 1994); rabbits (Rottmann et al, 1994); and sheep (Xin et al, 1994).

Unsuccessful attempts, however, have also been reported in a few cases in mouse (Brinster et al, 1989; Al Shawi et al, 1990; Hochi et al, 1990). Brister et al (1989) had listed at least 7 investigators who had failed to obtain positive results. Interestingly Brinster and Zimmermann (1994) subsequently developed and patented another approach to sperm mediated gene transfer by culturing spermatogonia and using spermatogonial cell transplantation (Brinster and Avarbock, 1994; Coghland, 1994)

Gavora et al (1991) also obtained negative results for mice as well as in chicken.

Further Evidence for Sperm-Mediated Gene Transfer in Fish

A number of studies in fish using sperm as the vector have been published. Ber et al (1992) obtained two positive results out of 38 tilapias (Oreochromis niloticus) produced from sperm exposed to RSV-CAT DNA. The sperm treated for 7-9 min with a CaPO₄/DNA precipitate at a ratio of 5 x 10³ copies of RSV-CAT plasmid DNA per sperm cell. Southern blot hybridization was used. Sheela and Pandian (1994) incubated Oreochromis mossambicus sperms in 0.2-4.0 mg/ml of circular and linearised pRSV rtGH plasmid DNA. The presumptive transgenic individuals were growing relatively faster than their sibling controls. Symonds et al (1994b) observed that 15% of the chinook salmon fry developed from embryos fertilized by sperm, treated in circular pSV2CAT DNA, were positive.

A number of negative reports, however, have been obtained using this simple incubation method. Muller et al (1992) reported no success in common carp, African catfish and tilapia. About 10 ug of circular or linearised pRSV/lacZ, pHSVtk/neo or pGM3H4/CAT plasmids in 100-500 ul of incubation solution (1.8% w/v sodium citrate or 2.8% w/v potassium citrate for carp) was used. Incubation time was from 40-60 min at RT. Dot blot and the respective expression assays were used. Chourrout and Perrot (1992) also obtained negative results for rainbow trout. The sperms were incubated for 60 min in 4.4 ug/ml of linearised pBGH7 plasmid in MMSF with or without DMSO and 240 ug/ml of pCMVCAT for 90 min. Slot blot and cat assays were conducted.

Modified Sperm-Mediated DNA Transfer

Gagne et al (1991) showed that foreign nucleic sequences can be introduced into bovine oocyte by electroporated spermatozoa. Muller et al (1992) also showed that electroporation of sperm in a medium of DNA improved the transfer efficiency of the DNA in carp, African catfish and tilapia. The pulse amplitude used was 750-2,250 V/cm and 175 or 270 microfarads capacitance. Sin et al (1993) also obtained gene transfer in chinook salmon using electroporated DNA-soaked sperm and Symonds et al. (1994b) showed that electroporation enhanced the transfer of pRSV-lacZ and pSV2CAT DNA. Field strengths of 250-1150 V/cm with pulse length of 7.7-27.4 ms and 10 ug/ml plasmid DNA in HEPES buffer were investigated. Electroporation improved the efficiency of DNA transfer to 30-85% by manipulating pulse length and field strength, pulse number and ionic strength of the buffer. Tseng et al (1994) electroporated loach sperm in opAFPGHc DNA and obtained 50% gene transfer. Dot blot, Southern blot and PCR were employed for monitoring. Enhanced growth was observed in the experimental group.

Increased sperm/DNA association was observed when 2 pulses were applied (Symonds et al (1994a). Nakanishi and Iritani (1993) also found that a second pulse enhanced DNA uptake by chicken sperm. Uptake of DNA by mature sperm cells of zebrafish was also enhanced by electroporation and this enhancement was due probably to the increase in the amount of exogenous DNA taken into the sperm nucleus and not due to the increase in the number of DNA bearing sperm (Patil and Khoo, unpublished).

Nakanishi and Iritani (1993), on the other hand, demonstrated that although chicken sperm incorporation of DNA was enhanced by electroporation its DNA transfer efficiency into the embryo is much reduced and this was attributed to the damaged acrosomes which affects the fertilising ability of the sperm. They, however, showed that lipofectin-treated sperm exhibited the best transfer efficiency. Bachiller et al (1991) also reported efficient uptake into mouse sperm using lipofectin.

Another reported modified sperm-mediated approach to transfer genes in chicken is by irradiating sperm. Hens were initially inseminated with irradiated semen obtained from donor males carrying marker genes. 24 h later the hens were reinseminated with nonirradiated semen obtained from makes that were of the same strain as the hen. 3-5% of the resulting progeny had feather and egg colour characteristics of the maker male (Pandy and Patchell, 1982; Tomita et al, 1988). Bumstead et al (1987) used the same technique to introduce disease resistance from one strain to another.

Potential of Sperm Vector for Transgenic Fish Production

The main observations obtained from using the sperm-mediated method are that the transferred DNA exist extrachromosomally and that the level of DNA transferred is low. The transfected products are also often mosaic. Expressions of inserted genes have been negative in many past studies but in more recent reports positive expressions have been obtained. The obvious advantage of using sperm is its natural capability to target and unite with the female germinal vesicle to form the zygotic nucleus. Simple incubation of sperms in DNA has been shown to be an effective means of gene transfer. Its effectiveness may be enhanced if greater amount of exogenous DNA can be introduced into the sperm for genomic integration. Methods such as electroporation and lipofection seem to enhance sperm uptake of DNA.

The sperm-mediated method like that of microinjection, however, suffers from a disadvantage in that DNA integration may be random and thus will not be useful to transfer genes for improving the genetic qualities of livestocks and fish. More studies have to be conducted to facilitate gene targeting. Unlike mammalian fertilization where only the sperm nucleus penetrates the egg, the whole sperm head and sometimes the tail enters the ova in most finfish species. This allows for greater entrapment and transfer of foreign genes bound to the surface of the sperm cells, as well as those that have been internalised. The chances for transformation is excepted to be better with fish (Muller et al, 1992).

This method is a mass method and is more advantage as, compared to the microinjection method which is tedious, having to inject each embryo individually.

One of the possible reasons why there are so much variations in the results using sperms as vectors is that different fish species were studied. There may be species differences, similarly the amount of DNA used were also quite different in the various studies. Another possibility is that the amount of DNA that is transferred by a single sperm is so little that existing techniques might have not been able to detect it in the embryo, especially if the plasmid insert is not replicated in the embryo. The structure of the plasmid construct would thus have a profound effect and may be one of the major causes of the discrepancy in the results obtained so far.

The mechanism in which DNA enters the sperm head is at the moment, not well understood. Such understanding is required if techniques to increase the DNA uptake of the sperm is to be successful. What happens to the DNA after it has entered the egg is also not known. Many studies have shown that the transferred DNA remains extra chromosomal and a few have observed that they have been rearranged or modified.

PCR has increased the sensitivity of detecting the presence of the inserted DNA but the assay for the expression product has still been at a very low sensitivity level. Sinai et al (1994) showed that luciferase recombinant is the most sensitive of the reporter genes used here in this paper. Its detection limit in number of molecules is 10⁵, whilst that of CAT and GH are around 10⁸. Further studies should be conducted with pSEAP and pFGP, which have expression products detectable at 10⁴ molecules. Recently we have used a GEP plasmid and were able to observe an expression under the microscope.

Other aspects of the sperm-mediated procedure also require further elucidation. For example, oocytes and fertilized eggs have been observed to take up exogenous DNA too. In the sperm-mediated experiments conducted so far, the oocytes were usually fertilised in a solution containing the DNA. DNA could have entered the embryo through the micropyle in fish or it could have entered through the chorion during water hardening. Sperms per se, may only be one of the many possible routes by which DNA enters the embryo. The eventual success of the sperm-mediated transfer technique, and its application in aquaculture together with the other transfer techniques (Khoo, 1995) is dependent on our understanding of the molecular and cell biology of the sperm and on the molecular mechanism of fertilization.

Acknowledgement

The authors wish to acknowledge the following: David Chin, Lay-Hong Ang, Hueh-Bin Lim, Andrew Tan and Hee-Peng Ang, who were either students or research assistants at one time or another in the Fisheries Biology Laboratory and have helped in some of the above studies as well as to K Y Wong and Saffuan Jasmawi in the Zoology Department of the National University of Singapore for technical and fish maintenance assistance, respectively. The first author wishes to express his gratitude to Dr J R Vielkind and Barbara Schmidt of the B C Cancer Research Centre, Vancouver, B C Canada and for the plasmid pUSVCAT as well as to Dr Ed Donaldson of the West of the West Vancouver Laboratory. Acknowledgement is also due to Dr T Ishikawa of Tokyo University for the pMTL plasmid. This study was supported by a grant from the National University of Singapore (RP 332/87).

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