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
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Abstract |
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Key words: exogenous DNA integration/ICSI/sperm DNA contaminated
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Introduction |
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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., 2004), 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) frozenthawed sperm samples, intentionally contaminated with pEGFP-transformed E. coli bacteria; (ii) frozenthawed 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.
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Materials and methods |
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Animals
B6D2 mice (Harlan Iberica SL, Barcelona, Spain) were used as donors of oocytes and sperm for ICSI experiments. Females were 68 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 13 h in 1.5 ml of LB medium to optical densities (OD) of 0.52.0 (4595x106 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 mares serum, followed 48 h later, by an equivalent dose of HCG. Cumulus cells were dispersed by 35 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 (36 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 HEPESTyrode (HT) medium (Shi et al., 1995), 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 70100 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 4050 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 67 µm of inner diameter. Individual sperm heads, decapitated by the freezethaw 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., 2003).
Analysis of genomic DNA
Genomic DNA was prepared from biopsies of day 14 embryos following standard procedures (Gutiérrez-Adán et al., 1996) and used for PCR of pEGFP as described (Gutiérrez-Adán and Pintado, 2000
). 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 2-analysis. P < 0.05 was considered significant.
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Results |
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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 frozenthawed sperm samples. Nine out of 52 implanted embryos (6%) produced by ICSI with frozenthawed 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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Discussion |
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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., 1997
; Levy et al., 2000
; Nicholson et al., 2000
; Englert et al., 2004
).
As the incidence of transgenesis observed in our ICSI experiments with frozenthawed 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., 1999, 2001
). In relation to this possibility, we want to draw the attention to the fact that in our experimental protocols, a 12 min period of contact between membrane-fragmented sperm cells and foreign DNA molecules is sufficient to promote transgenesis (Moreira et al., 2004
). 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., 2003), and there are many clinical situations where frozenthawed 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., 2004), 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.
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Acknowledgements |
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References |
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Submitted on November 5, 2004; resubmitted on June 21, 2005; accepted on July 5, 2005.
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