Hum. Reprod. Advance Access originally published online on February 22, 2006
Human Reproduction 2006 21(7):1759-1764; doi:10.1093/humrep/del041
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Blastocyst development after assisted reproduction using spermatozoa obtained after testicular stem cell transplantation in mice
Research Laboratory for Reproduction and Genetics (EMGE), Dutch-speaking Brussels Free University, Brussels, Belgium
1 To whom correspondence should be addressed at: Research Laboratory for Reproduction and Genetics (EMGE), Dutch-speaking Brussels Free University, Laarbeeklaan 101, 1090 Brussels, Belgium. E-mail: ellen.goossens{at}az.vub.ac.be
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Abstract |
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 Top Abstract IntroductionMaterials and methodsResultsDiscussion Acknowledgements References |
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BACKGROUND: Since its introduction in 1994, testicular stem cell transplantation (TSCT) has been widely used for research. This technique may also become important for preserving fertility in pre-pubertal cancer patients. Therefore, it is necessary to investigate the safety aspects of reproduction using spermatozoa obtained after TSCT. In this study, preimplantation development of mouse embryos, using spermatozoa obtained after TSCT, was examined. METHODS: TSCT-derived spermatozoa were used for IVF and ICSI. Embryos were cultured for five days until they reached blastocyst stage and were evaluated by differential staining. RESULTS: IVF revealed significantly lower fertilization and development rates after TSCT-IVF compared to control-IVF. Blastocysts derived from TSCT-IVF had significantly lower inner cell mass numbers (ICMs) and lower ICM/trophectoderm (TE) ratios compared to control-IVF blastocysts. No differences in fertilization and development rates were observed between TSCT-ICSI and control-ICSI, and blastocyst quality in the transplanted group was similar to that of the control blastocysts. CONCLUSION: Our study showed that after TSCT-IVF, fertilization and preimplantation development were disturbed and blastocysts showed reduced ICM and ICM/TE ratio. However, after TSCT-ICSI, both fertilization and preimplantation development were normal and blastocyst formation was comparable to control-ICSI.
Key words: assisted reproduction/blastocyst/stem cell/testis/transplantation
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussion Acknowledgements References |
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The technique of testicular stem cell transplantation (TSCT) was introduced by Brinster in 1994 (Brinster and Avarbock, 1994
; Brinster and Zimmermann, 1994). In the mouse, it has been shown that injection of germ cell suspensions from a fertile donor mouse into the seminiferous tubules of an infertile recipient mouse can restore spermatogenesis from donor spermatogonial stem cells (Brinster and Avarbock, 1994; Brinster and Zimmermann, 1994). TSCT has become an established research model to study testicular stem cell biology and has been used to genetically modify the germ cell line (Brinster, 2002).
Fertility has also been restored using TSCT with cryopreserved testicular stem cells (Avarbock et al., 1996). TSCT could therefore have important clinical applications in preserving the progenitive potential of young boys who need to undergo a sterilizing chemotherapy.
Recently, some alternative methods for testicular stem cell preservation have been described (for review, see Goossens and Tournaye, in press), for example, the testicular tissue grafting of immature and adult murine and human tissue (Honaramooz et al., 2002; Geens et al., 2006; Schlatt et al., 2006), or the long-term culture of testicular stem cells (Izadyar et al., 2003; Kanatsu-Shinohara et al., 2003; Nagano et al., 2003). However, there are still ethical concerns about these alternatives, such as the risk for zoonosis after xenografting or the use of animal supplements in culture systems, which can transmit animal viruses to the human body. Therefore, TSCT is still the most promising method for preserving and using testicular stem cells for fertility restoration in pre-pubertal boys undergoing sterilizing cancer treatment.
Before accepting TSCT as an option for preserving the fertility of pre-pubertal cancer patients, all safety concerns need to be carefully evaluated. Although TSCT has been reported to produce live offspring (Brinster and Avarbock, 1994; Ogawa et al., 2000; Kanatsu-Shinohara et al., 2003), we preferred to compare fertility after TSCT to a fertile control group. In a previous study we observed that viable offspring can be obtained after TSCT; however, the litter size was significantly reduced compared to controls. We also observed a reduction in fertilization and cleavage rates (Goossens et al., 2003). Therefore, we wanted to investigate the development of blastocysts obtained after IVF and ICSI using spermatozoa from transplanted mice (TSCT-IVF and TSCT-ICSI).
The allocation of a sufficient number of cells to both trophectoderm (TE) and inner cell mass (ICM) is essential for normal preimplantation. Alterations in this process may lead to embryos that are unable to implant and further develop (Van Soom et al., 2001). Handyside and Hunter (1984) developed a differential staining technique using fluorochromes that can distinguish the cells of the TE and the cells of the ICM in the blastocyst. In this study, we evaluated the preimplantation development and assessed blastocyst formation by differential staining in order to examine the distribution of blastomeres to both lineages.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussion Acknowledgements References |
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Ethics
All experimental procedures were approved by the Animal Care and Use Committee of the Brussels Free University.
Transplantation
Donor cells were obtained from cryptorchid B6CbaF1/juco-mice. Cryptorchid testes contain a higher percentage of spermatogonia, including stem cells. As a result, the suspensions used for transplantation contain more stem cells (1/200), so increasing the chances of inducing donor spermatogenesis (Dobrinski et al., 1999). The testes were decapsulated and the testicular tissue was digested as described previously (Brinster and Avarbock, 1994). Genetically sterile W/Wv-mice, aged 4–6 weeks were used as recipient animals (Jackson Laboratory, Bar Harbor, ME, USA). Transplantation was performed through the efferent duct as previously described (Ogawa et al., 1997). Immediately after transplantation, animals were given an antibiotic at a dose of 100 µl s.c. [5% Baytril® (Bayer, Brussels, Belgium) in saline].
Histology
To analyse the histological appearance of the tubules after transplantation, the testes were fixed overnight in Bouin’s fixative at 4°C and embedded in paraffin. Four-micrometre thick sections were cut and stained with eosin and haematoxylin. The percentage of tubule cross-sections with complete spermatogenesis was recorded and expressed as median (interquartile range 1–3). Two hundred cross-sections were counted per testis. The slides were analysed under an inverted microscope with a magnification of x200.
IVF
Isolation of spermatozoa
Spermatozoa were isolated from transplanted males that had been maintained between 120 and 150 days after transplantation. The epididymides were removed and transferred into one well of a 4-well plate containing 400 µl glucose-supplemented potassium-enriched simplex optimized medium (mKSOM) (Summers et al., 1995) supplemented with 3% bovine serum albumin (BSA; Sigma, Bornem, Belgium) covered with oil. Three to four epididymal incisions were made to allow spermatozoa to move into the medium. After 5 min of incubation at 37°C, the epididymides were removed, and 100 µl of the sperm suspension was transferred to another well with 400 µl mKSOM supplemented with 3% BSA under oil. Twenty minutes later, spermatozoa were counted and, if necessary, diluted to obtain a concentration of 10 x 106 spermatozoa per ml. Capacitation was allowed to proceed for 2 h at 37°C in 5% CO2 and 5% O2.
Isolation of oocytes
Oocytes were obtained from 6- to 10-week-old B6CbaF1/juco female mice (Charles River, L’Arbresle, France), which were induced to superovulate by i.p. injection of 5 IU equine chorionic gonadotrophin (eCG; Intervet, Mechelen, Belgium) followed by 5 IU human chorionic gonadotrophin (HCG; Intervet) 48 h later.
Oviducts were collected in a test tube with 2 ml Hepes-supplemented Dulbecco’s modified Eagle’s medium (Hepes-DMEM; Invitrogen, Merelbeke, Belgium) with 0.5% BSA. Afterwards, they were transferred to a Petri dish where the cumulus-oocyte complexes (COCs) were recovered from the oviducts with a fine pincet. COCs were washed twice in 120 µl mKSOM supplemented with 3% BSA under mineral oil and transferred to a 10 µl droplet of the same medium. One big COC was placed per droplet.
IVF procedure
Ten microlitres of the sperm suspension was added to each droplet. After an incubation for 3.5 h, oocytes were washed in 120 µl mKSOM supplemented with 0.5% BSA under oil and finally transferred to 30-µl droplets of the same medium. Ten oocytes were placed per droplet.
Incubation and examination of the oocytes
The dishes were incubated at 37°C in 5% CO2 and 5% O2. Fertilization was assessed by recording the number of 2-cell embryos at 24 h after fertilization. The dishes were cultured until day 5 after fertilization. Embryos were observed at x200 magnification on the warmed stage (37°C) of an inverted microscope.
TSCT-IVF and control-IVF were performed on the same day with oocytes from the same females. Experiments were repeated five times.
ICSI
Preparation of spermatozoa
Epididymides were removed and placed in a 1-ml droplet of mKSOM supplemented with 3% BSA under mineral oil. Three cuts were made in the epididymides with a fine needle to allow the spermatozoa to be released into the medium. After 20 min of incubation at 37°C, 50 µl of the sperm suspension was transferred into a droplet of 450 µl of the same medium under mineral oil and incubated for up to 2 h at 37°C.
For the control experiments, epididymal spermatozoa from adult B6CbaF1/juco mice were used.
Preparation of oocytes
Oocytes were obtained in the same way as for IVF. The cumulus cells were removed by treating COC with 0.025% hyaluronidase (Sigma) in Hepes-DMEM for 1 min. The oocytes were rinsed thoroughly and kept in mKSOM supplemented with 0.5% crystalline BSA (Sigma) for 2 h on 37°C in 5% CO2 and 5% O2.
ICSI procedure
For micromanipulation, nine drops were prepared under mineral oil in a 50 x 9 mm Petri dish. The central drop contained 1 µl of sperm suspension in 4 µl of DMEM containing 2.5% polyvinylpirrolidone (PVP; Sigma). The eight surrounding drops contained 5 µl DMEM supplemented with 0.5% BSA and 20% foetal calf serum (FCS; Invitrogen). In each of the eight drops, one oocyte was placed. Fifteen minutes before injection, the dish was put on the cooling stage (17°C) of an inverted microscope (Zeiss Axiovert 200, Oberkochen, Germany).
Micromanipulation was performed with motor-driven micromanipulators (Eppendorf, Hamburg, Germany). A motile spermatozoon with normal morphology was injected into the oocyte (Suzuki and Yanagimachi, 1997).
Incubation and examination of the oocytes
After injection, the oocytes were kept on the cooling stage (17°C) for 15 min. They were then put at room temperature for 10 min, and finally, the oocytes were transferred into 20-µl droplets of mKSOM supplemented with 0.5% crystalline BSA under mineral oil and incubated at 37°C in 5% CO2 and 5% O2. Twenty-four hours later, the oocytes were examined for survival and fertilization under an inverted microscope with a x200 magnification. 2-cell embryos were considered fertilized. Embryos were examined every 24 h for up to 120 h.
The experiments with either spermatozoa obtained after transplantation or control spermatozoa were performed in the same time period according to an alternating schedule. Experiments were repeated five times for each condition.
Zygote culture (controls)
Six- to 10-week-old B6CbaF1/juco female mice, which were induced to superovulate by i.p. injection of 5 IU eCG followed by 5 IU HCG 48 h later, were mated overnight with B6CbaF1/juco males. Females that showed a vaginal plug the next morning were used for zygote isolation. Zygotes were cultured in mKSOM supplemented with 0.5% BSA under oil for 4.5 days.
Differential staining
Differential cell counts were performed based on the method of Hardy et al. 1989. Zona-intact day 5 blastocysts were treated with pronase (0.5% protease; Sigma) to remove the zona. Zona-free embryos were incubated in 10 mM trinitrobenzene-sulfonic acid (TNBS; Sigma) in PBS containing 3 mg/ml polyvinylpyrrolidone (PVP-40; Sigma) on ice for 10 min. They were then incubated in 30 : 70 dilution of rabbit antidinitrophenol BSA (anti-DNP-BSA; ICN; Biomedicals NV, Asse, Belgium) in TCM199 (Invitrogen) at 37°C for 30 min. After a quick wash in TCM199, the embryos were finally incubated in 1 : 4 dilution of guinea pig complement (CL5000; Cedarlane Laboratories, Uden, The Netherlands) in TCM199 supplemented with 10 µg/ml propidium iodide (PI; Sigma) for 30 min at 37°C. The embryos were fixed in ice-cold absolute ethanol and stained with 10 µg/ml bisbenzimide (Hoechst 33342; Sigma) in absolute ethanol at 4°C for 2 h. Finally, the embryos were transferred to a drop of glycerol on a microscopic slide and covered with a cover slip.
ICM nuclei labelled with bisbenzimide appeared green, and TE nuclei labelled with both bisbenzimide and propidium iodide appeared pink-to-red. Numbers of ICM and TE nuclei were examined under a fluorescence microscope (x200) and expressed as mean ± SEM.
Statistical analysis
The Student’s t-test was used for statistical analysis of spermatozoa concentration and motility. The Fisher’s exact test was used for statistical analysis of fertilization and development rates. Parameters of blastocyst quality (ICM, TE, TCN and ICM/TE) were analysed by the Mann–Whitney U-test. Only P-values <0.05 were considered significant.
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussion Acknowledgements References |
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Transplantation and histology
A testicular cell suspension was transplanted in 41 testes of 22 W/Wv-mice. Four months later, spermatogenesis was observed in 21 testes of 13 mice (21/41 or 51%), and spermatozoa were recovered from the epididymides. Successfully transplanted mice showed spermatogenesis in 53% (interquartile range 47–55%) of the testicular cross-sections, with mature spermatozoa being observed in the lumen of the seminiferous tubules.
IVF
Sperm concentration and motility in the IVF droplet from transplanted and control mice are listed in Table I.
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IVF was performed with 125 oocytes using spermatozoa obtained after transplantation. Of these, 68 (54%) were fertilized. Twenty-four (35%) embryos eventually developed into blastocysts.
In the control group, 228 (74%) oocytes out of 309 were fertilized. One hundred and sixty-seven (73%) embryos developed into blastocysts.
Both the fertilization rate (number of two-cells/number of oocytes) and the blastocyst development rate (number of blastocysts/number of two-cells) were significantly lower in the transplanted group (P < 0.001 and P < 0.0001, respectively). This difference was detected in all experiments individually and not only in the summarized results. For more details of embryo development, see Figure 1.
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ICSI
ICSI was performed with 83 oocytes using spermatozoa obtained after transplantation. Of these, 58 (70%) were fertilized. Seventeen (29%) embryos eventually developed into blastocysts.
In the control group, 62 oocytes of 103 were fertilized (60%). Fourteen (23%) embryos developed into blastocysts.
The fertilization rate and the blastocyst development rate were comparable for both groups. For more details of embryo development, see Figure 2.
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Differential staining
Differential staining was performed successfully on 94 in vivo produced blastocysts, 88 blastocysts after control-IVF and 15 blastocysts after TSCT-IVF. Blastocysts that were only partially labelled or damaged by the staining procedure were excluded from the experiments.
TCN, ICM and TE numbers and ICM/TE ratios are summarized in Table II. Blastocysts obtained after control-IVF showed an ICM/TE ratio of 0.27 ± 0.02, and blastocysts obtained after TSCT-IVF showed an ICM/TE ratio of 0.17 ± 0.04. This ratio differed significantly for TSCT-IVF and the control groups (P = 0.0016 and P = 0.0034, respectively). Also, the number of ICM cells was significantly lower after TSCT-IVF compared to the in vivo control group and control-IVF (P < 0.0001 and P = 0.0005, respectively).
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Differential staining was successfully performed on five blastocysts after control-ICSI and on four blastocysts after TSCT-ICSI. TCN, ICM and TE numbers and ICM/TE ratios are summarized in Table III. The number of ICM cells was significantly lower in the ICSI group (control- and TSCT-ICSI) compared to the in vivo control group (P = 0.0075 and P = 0.0066, respectively). Also the ICM/TE ratio differed significantly for the control-ICSI and the in vivo control group (P = 0.0115). Differences between control-ICSI and TSCT-ICSI were not observed.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussion Acknowledgements References |
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The model of testicular stem cell transplantation as introduced by Brinster and co-workers (Brinster and Zimmermann, 1994
) may have clinical applications in circumventing the sterility of young male cancer patients after chemo- or radiotherapy. In a previous study, we observed that litter sizes after in vivo conception were lower and that mouse spermatozoa obtained after transplantation were able to fertilize oocytes by IVF, but with reduced fertilization and development rates in the transplanted group (Goossens et al., 2003). These results called for further investigation of the development of blastocysts obtained after assisted reproduction with spermatozoa from transplanted mice.
The total cell number and the ICM/TE ratio are well-accepted parameters to evaluate blastocyst development. Especially the ICM population size correlates significantly with the implantation potential of the blastocyst (Leppens et al., 1996).
Again, our experiments showed a reduced fertilization rate in the TSCT-IVF group. Embryos derived from TSCT-IVF also showed a reduced development rate. Since a considerable number of embryos failed to cleave after the 2-cell stage, the poor development may be caused by genomic modifications in post-transplantation spermatozoa. Abnormalities in the paternal DNA will be reflected in the development of the embryo after zygotic activation. In the zygote, the maternal DNA of the oocyte controls the first division. From the second division onwards, the zygote controls the transcription and cleavage: early zygotic gene activation (ZGA) starts 24 h after fertilization, regardless of whether or not a 2-cell is formed. In contrast, late ZGA does not occur without formation of a 2-cell embryo and late ZGA is essential for formation of a 4-cell embryo (Nothias et al., 1995). A problem in the paternal genome may hamper or prevent correct fusion of the maternal and the paternal pronuclei, and will therefore hamper 4-cell development.
Apart from genomic modifications, spermatozoa with aneuploidy may also lead to developmental disorders during early embryonic cleavage. It would therefore be interesting to compare the proportion of spermatozoa with aneuploidy in the semen of fertile and transplanted males.
Despite the fact that TSCT-IVF showed reduced fertilization and development capacities, there were no differences in fertilization and development after TSCT-ICSI compared to control-ICSI. Moreover, TSCT-ICSI seems to improve fertilization compared to TSCT-IVF. However, a cautionary remark must be made for the ICSI experiments. The control group revealed poor fertilization and developmental rates compared to IVF. This may be due to the difficulties encountered in mouse ICSI. In humans, this technique is far easier to apply, with higher fertilization and developmental rates. The diameter of the mouse oocyte (80 µm) is almost one-half the size of the human oocyte (150 µm), and the tail of a mouse spermatozoon (140 µm) is three times longer than that of a human spermatozoon (50 µm). The volume of PVP injected into the ooplasm is therefore three times higher than for human oocytes and this factor is critical for the relatively smaller murine oocyte (Ron-El et al., 1995). Another factor is the shape of the mouse spermatozoa. The head piece shows a hook, which requires a wider diameter of the injection pipette to prevent the spermatozoon sticking to the pipette. Finally, mouse oocyte membranes present a higher elasticity, which makes it necessary to aspirate a larger amount of cytoplasm in the injection pipette before the oolemma breaks. This leads to high pressure on the membrane and will cause ruptures in the membrane at different places. Because the mouse ooplasm has a high fluidity, it can easily leak through the holes, which may cause damage of the oocyte (Rybouchkin, 1998, personal communication).
We have differentially stained embryos that were able to develop into blastocysts in order to determine blastocyst formation and implantation potential. TSCT-IVF blastocysts showed lower ICM and ICM/TE compared to control-IVF and compared to in vivo produced blastocysts. When blastocysts from TSCT-ICSI were compared to control-ICSI, no statistical differences were found. However, ICSI blastocysts had lower ICM and ICM/TE than in vivo obtained control blastocysts. The reduced developmental quality of ICSI blastocysts was also illustrated by the fact that ICSI blastocysts were more frequently damaged by the differential staining procedure than in vivo and IVF-blastocysts.
From the results of this study, we may conclude that spermatozoa obtained after TSCT are able to fertilize oocytes by IVF and ICSI, but embryonic development into blastocysts is lower compared to controls. Although, when compared to fertile mice, transplanted mice may have a lower fertility potential, these results remain very promising for future clinical applications. In the human application, transplantations will be performed autologously, which reduces the risk of rejection or inflammation. However, although donor and acceptor mice were not exactly from the same background, no signs of rejection or inflammation were noticed. Nevertheless, this limitation may be the cause for perturbed sperm maturity. Another difference with our mouse model is the use of cryptorchid instead of pre-pubertal tissue. Testicular stem cells derived from cryptorchid tissue may have undergone genetical or structural changes due to the abnormal environment and this may have caused atypical sperm maturation. In addition, the W/W recipients might have problems in the somatic testicular environment, which are responsible for impaired spermatogenesis. Considering these remarks, we believe that TSCT in the human can become a safe method for fertility preservation.
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Acknowledgements |
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TopAbstractIntroductionMaterials and methodsResultsDiscussion Acknowledgements References |
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The authors thank Mr Michael Whitburn for proofreading the manuscript.
This work was supported by grants from the Dutch-speaking Brussels Free University (OZR 318) and the Fund for Scientific Research Flanders.
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References |
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Submitted on December 23, 2005; resubmitted on January 23, 2006; accepted on January 25, 2006.
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