Inadvertent transgenesis by conventional ICSI in mice

Pedro Nuno Moreira1, Raúl Fernández-González, Dimitrios Rizos, Miguel Ramirez, Miriam Perez-Crespo and Alfonso Gutiérrez-Adán

Departamento de Reproducción Animal, INIA, Ctra. de la Coruña Km. 5.9 Madrid 28040, Spain

1 To whom correspondence should be addressed. E-mail: pmoreira{at}inia.es


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: ICSI is a relatively new treatment for human male-related infertility, as well as an efficient method for the production of transgenic animals by injecting into the oocyte sperm previously incubated with foreign DNA. As semen samples collected in human infertility clinics are frequently contaminated with bacteria, one risk associated with the ICSI procedure is the injection of foreign, sperm-associated exogenous DNA into the oocyte, and the generation of transgenic offspring. METHODS: To analyse this possibility, ICSI was performed in mouse oocytes with frozen–thawed and Percoll-treated fresh sperm samples intentionally contaminated with plasmid EGFP-transformed E. coli bacteria or medium from which these bacteria were washed. Fertilized embryos were cultured in vitro until morula/blastocyst stage, transferred into pseudopregnant females, and at day 14, fetuses and reabsorptions were analysed by PCR for the genomic presence of integrated plasmid and/or bacterial DNA. RESULTS: Independently of the sperm pretreatment tested, transgenesis was produced. CONCLUSIONS: Inadvertent transgenesis by conventional ICSI is a possibility that should not be neglected. Particular precautions, such as full bacteriological semen examinations and effective antibiotic semen pretreatments, should be taken in human infertility clinics, in order to exclude the possibility of accidental transgenesis.

Key words: exogenous DNA integration/ICSI/sperm DNA contaminated


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The first indication that live sperm incubated with exogenous DNA could result in transgenic offspring was provided in 1989 (Lavitrano et al., 1989Go). In this method of sperm-mediated gene transfer (SMGT), sperm cells bind and internalize exogenous DNA molecules, acting as double vectors for the transmission of not only their own but also of foreign DNA (Lavitrano et al., 1992Go; Zani et al., 1995Go). Successful transgenesis occurs after fertilization by admixture of the DNA-loaded sperm cells with the oocytes in vitro or simply by in vivo artificial insemination (Lavitrano et al., 1989Go; Maione et al., 1998Go). This methodology has been confirmed in a number of laboratories that have reported successful SMGT in fish, honey bee, sea-urchin, Xenopus, mouse, chicken, sheep (Wall, 1999Go) and pig (Lavitrano et al., 2002Go). Although, in most of these studies, transgenesis was accomplished by IVF or by artificial insemination, ICSI of DNA-loaded sperm cells has also been shown to mediate mouse transgenesis at high efficiency, especially when sperm cell damage (by freeze–thaw cycles or exposure to detergents) is induced (Perry et al., 1999Go). The efficiency of ICSI-mediated transgenesis is unquestionable, being capable of mediating complete integration of DNA constructs with submegabase magnitude (Perry et al., 2001Go; Moreira et al., 2004Go) and foreign DNA molecules in minimal concentrations. Evidence of such high efficiency was previously reported by our laboratory (Moreira et al., 2004Go). In an ICSI experiment involving co-injection of frozen–thawed sperm with a 5 kb plasmid EGFP construct (pEGFP) or a large YAC construct of 250 kb, we have found respectively 45 and 35% transgenesis rate among live offspring. In addition, using the pEGFP construct at concentrations as low as 0.05 ng/µl, a concentration ~100 times smaller than the one commonly used to ensure transgenesis by pronuclear injection in mice, we observed that ~50% of developing blastocysts expressed the transgene. Similar evidence has been reported in other species. In rats, for example, a transgene concentration of 0.02 ng/µl was enough to produce >20% transgenesis by ICSI-mediated gene transfer (Hirabayashi et al., 2005Go).

The high efficiency of the ICSI procedure in introducing foreign DNA molecules into the embryonic genome, and the fact that collected semen samples for standard ICSI procedures in humans are frequently contaminated with bacteria (Krissi et al., 2004Go), made us wonder about the probability of inadvertent transgenesis caused by bacterial contamination of a cryopreserved or fresh semen sample before ICSI-mediated fertilization. In order to evaluate this probability, ICSI was performed in mouse oocytes with (i) frozen–thawed sperm samples, intentionally contaminated with pEGFP-transformed E. coli bacteria; (ii) frozen–thawed sperm samples contaminated with pEGFP-transformed E. coli washing medium (but without the presence of bacteria, excluded by centrifugation); and (iii) fresh sperm samples contaminated with pEGFP-transformed E. coli bacteria submitted to Percoll treatment. Treatments (i) and (ii) were used to evaluate the possibility of human transgenesis when male gametes cannot be washed through density gradient media before microinjection (i.e. ICSI with immotile or low motility sperm, ICSI with very few sperm cell numbers, and round spermatid nuclear injection or ROSNI). Treatment (iii) mimicked the standard human ICSI procedure with fresh motile sperm. The results of these experiments are presented and discussed further.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Reagents and media
All chemicals and media were purchased from Sigma Chemical Co. (Madrid, Spain) unless otherwise stated.

Animals
B6D2 mice (Harlan Iberica SL, Barcelona, Spain) were used as donors of oocytes and sperm for ICSI experiments. Females were 6–8 weeks old at the time of the experiments, and males ≥3 months old. CD1 females were used for surrogate mothers for embryo transfer experiments, mated with vasectomized CD1 males. Mice were fed ad libitum with a standard diet and maintained in a temperature- and light-controlled room (23°C, 14 h light:10 h dark). All animal experiments were performed in accordance with Institutional Animal Care and USE Committee guidelines and in adherence with guidelines established in the Guide for Care and Use of Laboratory Animals as adopted and promulgated by the Society for the Study of Reproduction.

Bacteria
E. coli bacteria transformed with the EGFP plasmid (5.4 kb, pEGFP-N1; Clontech Laboratories, Inc., Palo Alto, CA, USA), containing the human CMV immediate early promoter and the enhanced GFP gene, were grown during 1–3 h in 1.5 ml of LB medium to optical densities (OD) of 0.5–2.0 (~45–95x106 cells/ml respectively), and 0.1 (~15x106 cells/ml). Optical densities of bacterial growth were measured by spectrophotometry according to the instructions of the Biophotometer used (Eppendorf, Hamburg, Germany).

Gamete collection and sperm pretreatment
Metaphase II (MII) oocytes were collected 14 h post-hCG administration, from female mice superovulated with 5 IU of pregnant mare’s serum, followed 48 h later, by an equivalent dose of HCG. Cumulus cells were dispersed by 3–5 min incubation in M2 medium containing 350 IU/ml hyaluronidase, and oocytes washed and maintained in KSOM medium at 37°C in a 5% CO2 air atmosphere until use.

Epididymal sperm was collected from mature males (3–6 months old) by excising with a pair of fine scissors, and compressing with forceps, blood- and adipose tissue-free epididymal caudae into a Petri dish with sperm cell collection medium. Collected sperm was used fresh or cryopreserved for ICSI experiments.

When to be used fresh, sperm cells were collected in a minimal volume of HEPES–Tyrode (HT) medium (Shi et al., 1995Go), and mixed with 1/5 of pEGFP-transformed E. coli resuspended in HT. This mixture was layered in a 15 ml conical tube on a discontinuous 90%/45% Percoll gradient in a ratio 1:1:1 ml (cell sample: 45% Percoll:90% Percoll) and centrifuged at 500 g for 13 min. The top two layers (containing debris, non-sperm cells, and immotile cells) were discarded. The bottom pellet (containing motile sperm) was washed in HT at 350 g for 7 min. The supernatant was removed and the remaining cells were resuspended in M2 before ICSI.

When cryopreserved, collected epididymal sperm was placed in the bottom of a 1.5 ml polypropylene centrifuge tube and diluted with fresh M2 medium to a final concentration of 2.5x106 cells per ml. Equal volumes of extended sperm and pEGFP-transformed E. coli resuspended in M2, or extended sperm and pEGFP-transformed E. coli washing medium, were gently mixed. pEGFP-transformed E. coli washing medium was the supernatant collected after re-suspending and pelleting twice by centrifugation in M2, bacteria from their culture medium LB (the first supernatant was discarded). Aliquots 70–100 µl of the sperm solution generated were transferred to cryogenic 1.5 ml vials, tightly capped and directly placed into liquid nitrogen without complete immersion to avoid internalization of liquid nitrogen. Sperm samples were stored for periods ranging from 1 day to 4 weeks at –80°C. Other sources of contamination were avoided throughout the procedure. Before microinjection, an aliquot of the frozen sperm solution to be used was thawed at room temperature, and a 10 µl sample was mixed with 40–50 µl of 10% polyvinylpyrrolidone (PVP; Mr 360 000) in M2 solution and placed on the microinjection dish.

Embryo micromanipulation, culture and transfer
ICSI was performed in M2 medium at room temperature. The ICSI dish contained a manipulation drop (M2 medium), a sperm drop (sperm solution in M2/10% PVP), and an M2/10% PVP needle-cleaning drop. Injections were performed with a PMM-150 FU piezo-impact unit (Prime Tech, Japan) and Eppendorf micromanipulators (Hamburg, Germany) using a blunt-ended mercury-containing pipette with 6–7 µm of inner diameter. Individual sperm heads, decapitated by the freeze–thaw procedure (or mechanically, when fresh sperm cells were used), were co-injected into oocytes. Oocytes were injected in groups of 10. After 15 min of recovery at room temperature in M2 medium, surviving oocytes were washed three times in equilibrated KSOM, and returned to mineral oil-covered KSOM and cultured at 37°C in a 5% CO2 air atmosphere. Ninety-six hours later, morula/blastocysts were transferred to oviducts of pseudopregnant recipient females. Embryo transfer was performed as described previously (Moreira et al., 2003Go).

Analysis of genomic DNA
Genomic DNA was prepared from biopsies of day 14 embryos following standard procedures (Gutiérrez-Adán et al., 1996Go) and used for PCR of pEGFP as described (Gutiérrez-Adán and Pintado, 2000Go). Oligonucleotides used for detecting the specific 340 bp PCR product of EGFP were: GFP1F 5'-TGAACCGCATCGAGCTGAAGG-3'; GFP2R 5'-TCCAGCAGGACCATGTGATCG-3'. PCR conditions were as follows: Taq polymerase (Promega, Madison, WI, USA), 2 min at 93°C, 30 cycles of 30 s at 93°C, 45 s at 60°C and 35 s at 72°C, followed by a final extension step of 10 min at 72°C.

For bacterial DNA detection, PCR analysis of 16S ribosomal RNA was used. Oligonucleotides used for detecting the specific 195 bp PCR product were: 16SRNAF 5'-CCTACGGGAGGCAGCAGAT-3'; 16SRNA2R 5'-ATTACCGCGGCTGCTGG-3'.

PCR conditions were as follows: Taq polymerase (Promega), 2 min at 93°C, 35 cycles of 30 s at 93°C, 30 s at 65°C and 30 s at 72°C, followed by a final extension step of 10 min at 72°C.

Statistical analysis
Significant differences in the cleavage rate and proportion of transgenic embryos between groups were evaluated by {chi}2-analysis. P < 0.05 was considered significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The outcomes of our ICSI experiments with contaminated sperm samples indicated that, independently of the sperm pretreatment tested, transgenesis was always a consequence (Table I). When frozen–thawed sperm contaminated with pEGFP-transformed E. coli was used, it was observed that of 237 ICSI-produced embryos, 45 (19%) expressed the fluorescent EGFP transgene. Similar results were observed when frozen–thawed sperm cells contaminated with just pEGFP-transformed E. coli washing medium were microinjected [of 84 ICSI produced embryos, 17 (20%) expressed the transgene].


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Table I. Inadvertent transgenesis by conventional ICSI in mice

 

Fresh sperm contaminated with pEGFP-transformed E. coli bacteria produced transgenesis even after washing by Percoll gradient. Of 82 embryos fertilized by ICSI with these sperm cells, 10 (12%) became fluorescent for pEGFP. Some of these embryos became arrested very early in their development; however (and this was common for all sperm pretreatments tested), the vast majority developed to morula/blastocysts, being subsequently transferred to recipient females (Figure 1). At day 14, DNA analyses were done on biopsies collected from implanted embryos for the detection of transgenic markers. As it shown in Table I, pEGFP transgene integration was not observed among fetuses produced by ICSI with contaminated fresh sperm submitted to Percoll pretreatment, but it was detected among embryos produced with bacteria-contaminated frozen–thawed sperm samples. Nine out of 52 implanted embryos (6%) produced by ICSI with frozen–thawed sperm samples contaminated with pEGFP-transformed E. coli integrated the transgene, moreover two of these fetuses also resulted in PCR positive for bacterial DNA (Table I). In addition, the presence of bacteria in the sperm extender severely compromised embryo cleavage rate in comparison with the other two treatments (Table I).



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Figure 1. GFP expression on embryos produced by ICSI with fresh (A,B) and frozen–thawed (C,D) sperm contaminated with pEGFP-transformed bacteria or pEGFP-transformed bacteria washing medium. Independently of sperm pretreatment tested, EGFP transgene expression was always observed. Some of the EGFP-expressing embryos became arrested very early in their development; however, the vast majority developed to morphologically normal-looking morula/blastocysts, being subsequently transferred to recipient females. (A and C) Bright-field pictures of developing ICSI produced embryos; (B and D) corresponding images collected under fluorescent light exposure (magnification x200).

 


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
ICSI is becoming the most commonly used method for the treatment of male infertility factor; however, no experimental testing preceded its introduction in 1992, partly because animal models were thought to be unsuitable, and partly because of its immediate and overwhelming success (Te Velde et al., 1998Go). It has been hypothesized that one of the risks associated with the ICSI procedure is the injection of foreign sperm-bound exogenous DNA into the oocyte and the generation of transgenic offspring or assimilation of infectious particles (Bonduelle et al., 2002Go), but this possibility was never convincingly demonstrated. In this study, using the mouse as research model, we demonstrate that bacterial contamination of the sperm extender can be a potential source of exogenous DNA that may be inadvertently introduced by this technique in the embryonic genome, and produce accidental transgenesis.

We have included in our analysis an ICSI assay with fresh sperm submitted to a Percoll pretreatment that mimics the sperm-wash procedure used in human clinics. The 12% transgenesis observed among ICSI produced embryos when fresh sperm contaminated with pEGFP-transformed E. coli submitted to Percoll pretreatment was used, demonstrates that a sperm wash by a Percoll gradient is not enough to avoid the occurrence of bacteria-mediated transgenesis by ICSI. One may argue that in our experiment we have used an excessive number of contaminating cells (~15x106 cells/ml); this number was used in order to allow detection of an effect in a reasonable number of experimental assays. However, we would like to stress that, even if we have used a bacterial concentration ten times smaller (in the range of what is normally detected in a semen sample), most likely we would still be facing an undesirable transgenesis rate. In our experiment with Percoll, transgene expression was transient (since its integration was not detected on implanted fetuses), but one cannot neglect possible negative effects of an alien protein expression on subsequent embryo development. When facing these results, most emphasis should be placed on the fact that sperm cell selection procedures used in infertility clinics do not eliminate foreign DNA sources from contaminated sperm samples, which will be co-injected into the oocyte with the sperm cell during the ICSI procedure. Sperm washing procedures by swim-up or centrifugation through PureSperm or Percoll gradients do not completely eliminate contaminating DNA particles (Cottell et al., 1997Go; Levy et al., 2000Go; Nicholson et al., 2000Go; Englert et al., 2004Go).

As the incidence of transgenesis observed in our ICSI experiments with frozen–thawed sperm samples demonstrates, the possibility of co-injection of bacterial contents released during cryopreservation or centrifugation of contaminated sperm samples provides one route for host genome contamination by ICSI. This should be cause for concern, especially when male gametes cannot be passed through density gradient media (i.e. ICSI with immotile or low motility sperm, ICSI with very few sperm cell numbers, and ROSNI). However, it is also important to recall that the sperm cell immobilization required during human ICSI induces membrane fragmentation which, at least in the mouse model, seems to facilitate the incorporation and transport of exogenous DNA molecules into the embryo (Perry et al., 1999Go, 2001Go). In relation to this possibility, we want to draw the attention to the fact that in our experimental protocols, a 1–2 min period of contact between membrane-fragmented sperm cells and foreign DNA molecules is sufficient to promote transgenesis (Moreira et al., 2004Go). Our experimental results demonstrate that the possibility of permanent transgenesis (including of bacterial DNA) becomes more likely when membrane-fragmented cryopreserved sperm cells are microinjected.

In human ICSI, when semen samples from men with very low sperm cell numbers are used, cells are often morphologically poor, sometimes more susceptible to damage, presenting an extra risk of transgenesis. Microbial contamination of embryos and semen during long-term banking in liquid nitrogen has been reported (Bielanski et al., 2003Go), and there are many clinical situations where frozen–thawed sperm are used (e.g. biopsies, back-up sperm samples, donor samples), which should require specific attention. The possibility of host genome contamination also becomes a menace during assisted reproductive procedures such as ROSNI, which involves the microinjection of naked nuclei, since the lack of a cytoplasmic membrane surrounding the donor nucleus facilitates contact and adherence with foreign DNA molecules.

Knowing this, and considering that a high percentage of the collected semen samples for standard ICSI procedures in humans are contaminated with bacteria (Krissi et al., 2004Go), the results of this study strongly suggest that particular precautions, such as full bacteriological semen examinations and effective antibiotic semen processing, should be taken especially in human infertility clinics, in order to exclude any possibility of accidental transgenesis as the result of ICSI.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
This work was supported by grants No. AGL2003-05783 and OT02-008 from the Ministerio de Educación y Ciencia (Spain).


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on November 5, 2004; resubmitted on June 21, 2005; accepted on July 5, 2005.





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