Research Centre for Reproduction and Genetics, Faculty of Medicine and Pharmacy, Dutch-speaking Brussels Free University, Laarbeeklaan 101, 1090 Brussels, Belgium
1 To whom correspondence should be addressed. e-mail: ellen.goossens{at}az.vub.ac.be
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Abstract |
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Key words: early development/fertilization/spermatogenesis/testis/transplantation
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Introduction |
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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 Zimmerman, 1994
). The first meiotic germ cells appear after 1 month and their number gradually increases thereafter (Parreira et al., 1998
). Successful spermatogonial transplantation has also been described in rats (Ogawa et al., 1999a
), in the cynomolgus monkey (Schlatt et al., 1999
), in pigs (Honaramooz et al., 2002
) and in goats (Honaramooz et al., 2003
). Recolonization of mouse testes was also observed with stem cells obtained from rabbits, dogs, pigs, bulls, stallions and baboons, but without further differentiation taking place (Ogawa et al., 1999b
; Dobrinski et al., 1999a
; 2000; Nagano et al., 2001
). Recently, Nagano et al. (2002
) even reported survival and proliferation of human stem cells in the mouse testes until 6 months after transplantation.
Before considering stem cell transplantation for preserving the fertility of pre-pubertal boys, there are concerns that need careful evaluation (Aslam et al., 2000). The testis biopsy taken from the cancer patient may contain malignant cells. These cells should be removed from the cell suspension since it has been shown in rats that one single malignant cell may reintroduce the disease (Jahnukainen et al., 2001
). Furthermore, the cell suspension consists of all testicular cells. The proportion of spermatogonial stem cells is very low (estimated at 1/5000) (Meistrich and van Beek, 1993
). Use of an in-vitro culture system to enrich the stem cell population may provide a solution to this problem. Purification of stem cells may also increase the efficiency of transplantation. Shinohara et al. (1999
) used
1- and
6-integrin as markers for selecting mouse spermatogonial stem cells, but unfortunately these markers were also present in other progenitor cells. Furthermore, there is the need to develop a safe and efficient cryopreservation protocol. Avarbock et al. (1996
) has cryopreserved murine spermatogonial stem cells with dimethylsulphoxide (DMSO) as a cryoprotectant. This cryoprotectant may not be suitable for human use, because of its carcinogenic properties. On the other hand, DMSO does not differ from other cryoprotectants with respect to survival rates after freezing and thawing (Brook et al., 2001
).
Finally, it remains unknown whether meiosis occurs normally after transplantation and whether the fertilizing and developmental potential of sperm obtained after transplantation is normally preserved. Only a few live offspring have been reported after testicular germ cell transplantation since the first report on this technique (Brinster and Avarbock, 1994). At present, it remains unclear whether a human application will be feasible in the future and whether techniques of assisted reproduction would be required for reproduction after testicular germ cell transplantation.
This study therefore aimed at evaluating the fertilizing and developmental abilities of sperm obtained after stem cell transplantation. Transplanted animals were mated with females in order to evaluate in-vivo conception. The functionality of sperm obtained after testicular germ cell transplantation was further investigated by assisted reproductive techniques. We performed both IVF and ICSI in order to determine whether such sperm have normal fertilizing capacity and whether they can support normal preimplantation embryonic development in vitro.
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Materials and methods |
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Transplantation procedure
Donor cells were obtained from C57Bl xCba F1 mice (Iffa Credo, France) that were made cryptorchid at the age of 6 weeks, 3 months before transplantation. 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., 1999b). The testes were decapsulated and the testicular tissue was digested according to a modified technique as described by Brinster and Avarbock (1994
). First, the tissue was digested with 380 IU/ml collagenase Ia (Sigma, Belgium) in Dulbeccos modified Eagles medium (DMEM; Invitrogen, Belgium) for 1 h in a 37°C water bath. The digested tissue was then centrifuged at 400 g for 6 min. The supernatant was discarded, the cells were resuspended in DMEM with 2.5% trypsin (Sigma) and 10 µg/ml DNase (Sigma) and put in a water bath at 37°C for 15 min. Afterwards, 4% fetal calf serum (FCS; Life Technologies) was added and the cell suspension was again centrifuged at 400 g for 10 min. Finally, the cells were resuspended in DMEM supplemented with 5% penicillin (Sigma), 5% streptomycin (Sigma) and 10% FCS, so as to obtain a concentration of 107 cells/ml. Trypan Blue dye, used to visualize the entry of the injected cells in the seminiferous tubules, was not added to the injection medium, but was aspirated only in the tip of the pipette to minimize its possible toxic effects.
As recipient animals, 46 week old W/Wv mice (Jackson Labs, USA) were used, because these mice lack c-kit receptor and do not present endogenous spermatogenesis (Handel and Eppig, 1979). Transplantation was performed under a stereomicroscope. The transplantation pipette was introduced into the rete testis via the efferent duct (Ogawa et al., 1997
). A volume of 310 µl, equivalent to
310x104 cells, was injected into the tubules.
Immediately after transplantation the mice were injected s.c. with 100 µl of antibiotics [1900 µl phosphate-buffered saline + 100 µl Baytril® 2.5% (Bayer, Belgium)].
All experimental procedures were approved by the Animal Care and Use Committee at the Brussels Free University.
Histology
In order to analyse the histological appearance of the tubules after transplantation; the testes were fixed overnight in 2.5% paraformaldehyde (Sigma) at 4°C and embedded in paraffin. Sections 5 µm thick were cut and stained with eosin and haematoxylin. The percentage of tubule cross-sections with complete spermatogenesis was recorded. One hundred cross-sections were counted per testis. The slides were analysed under an inverted microscope with a magnification of x200. An adult W/Wv mouse was used as a negative control.
In-vivo conception
Four months after transplantation, animals were mated with 6 week old C57BlxCba F1 hybrid females to evaluate in-vivo conception. Control experiments were also performed using fertile males. The animals were housed together at 15:00 and evaluated for the presence of a vaginal plug on the next morning. Females with a positive plug were selected and killed on day 17 of gestation. The uterine horns were dissected and examined for the presence of fetuses.
IVF
Preparation of sperm
Recipient males were maintained between 120 and 150 days before IVF was performed. The animals were killed by cervical dislocation. The epididymides were removed and transferred into one well of a 4-well plate containing 400 µl glucose-supplemented sodium-enriched synthetic oviductal medium [(modified potassium simplex optimised medium) mKSOM] (Summers et al., 1995) supplemented with 3% bovine serum albumin (BSA; Sigma) covered with oil. Three to four incisions were made to allow sperm to move into the medium. After 5 min of incubation at 37°C, the epididymides were discarded 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, cells were counted and, if necessary, diluted to obtain a concentration of 10x106 cells per ml. Capacitation was allowed to proceed for 2 h at 37°C, in a 5% CO2 and 5% O2 incubator.
Preparation of oocytes
Six to ten week old C57BlxCba F1 hybrid female mice (Iffa Credo, France) were induced to superovulate by i.p. injection of 5 IU pregnant mares serum gonadotrophin (PMSG; Intervet, Belgium) followed by 5 IU hCG (Intervet) 48 h later. Animals were killed by cervical dislocation 16 h after hCG injection. Oviducts were collected in a test tube with 2 ml HEPESDMEM supplemented with 0.5% BSA. Afterwards, they were transferred to a Petri dish where the oocytes were recovered from the oviducts with a fine pincet. Cumulusoocyte complexes (COC) 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 COC (± 10 oocytes) was placed per droplet.
IVF procedure
Ten µl of the sperm suspension were added to each of the three droplets with the COC and placed in the incubator for 3.5 h. Oocytes were then 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 in a humidified incubator. Fertilization was assessed by recording the number of 2-cell embryos 24 h after fertilization. The dishes were further kept in culture until day 5 after fertilization. Embryos were observed at x200 magnification on the warmed stage (37°C) of an inverted microscope.
IVF experiments with either sperm obtained after transplantation or control sperm were performed in the same time period according to an alternating schedule. Experiments were repeated five times for each condition.
ICSI
Preparation of sperm
The transplantation procedure was similar to that used for IVF. 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 sperm cells 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 sperm from adult C57BlxCba F1 hybrid mice were used.
Preparation of oocytes
Oocytes were obtained as for IVF. The cumulus cells were removed by treating COC with 0.025% hyaluronidase (Sigma) in HEPESDMEM 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 a 5% CO2 and 5% O2 incubator.
ICSI procedure
For micromanipulation, nine drops were prepared under mineral oil in a 50x9 mm Petri dish. The central drop contained 1 µl of sperm suspension in 4 µl of DMEM containing 2.5% polyvinylpyrrolidone (PVP; Sigma). The eight surrounding drops contained 5 µl DMEM supplemented with 0.5% BSA and 20% FCS. A high concentration of FCS was used in order to increase the survival and fertilization rates (Suzuki and Yanagimachi, 1997). 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, Germany).
Micromanipulation was performed with motor-driven micromanipulators (Eppendorf, Germany). A motile spermatozoon with normal morphology was immobilized by touching its tail and it was then aspirated tail-first into the injection pipette. The oocyte was held by the holding pipette with the polar body in the 6 or 12 oclock position. The injection pipette was introduced through the zona pellucida and the oolemma until it almost reached the opposite side. If the membrane did not break immediately, it was sucked into the injection pipette until it broke and was then released. At the same time, the spermatozoon was injected into the oocyte (Palermo et al., 1992; 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 under 5% CO2 and 5% O2. Twenty-four hours later, the oocytes were examined for survival and fertilization under an inverted microscope at x200 magnification. Two-cell embryos were considered fertilized. Embryos were examined every 24 h for up to 120 h.
The experiments with either sperm obtained after transplantation or control sperm were performed in the same time period according to an alternating schedule. Experiments were repeated five times for each condition.
Statistical analysis
Students t-test was used for statistical analysis of concentration and motility of sperm. Fishers exact test was used for statistical analysis of fertilization and developmental rates. P < 0.05 was considered significant.
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Results |
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Fertile mice showed spermatogenesis in 55% (interquartile range of 4857%) of the testicular cross-sections. Mature sperm were seen in the lumen of the seminiferous tubules (Figure 1A). However, 60% of the cross-sections showed stage C: VIVII; or stage D: VIIIIX; an observation also reported by Ventelä et al. (2002). The testes of a negative control W/Wv mouse did not show any spermatogenesis (Figure 1B).
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The fertilization rate and the blastocyst development rate were significantly lower in the transplanted group (both P < 0.00001).
ICSI
Motile epididymal sperm obtained after transplantation were used to microinject a total of 187 oocytes. Eighty-three oocytes survived the injection procedure (44%) and 57 of these became fertilized (69%). Seventeen 2-cell embryos became blastocysts (30%) (Table III) (Figure 2B).
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Discussion |
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In our study we used sperm from animals that were recovered after an average of 5 months (4 months) post-transplantation. This time period covers more than three spermatogenic cycles (Brinster and Avarbock, 1994
), i.e. long enough to omit the first waves of spermatogenesis which may include more apoptotic sperm or sperm with a limited reproductive capacity. However, in a human situation, transplantation may be performed at puberty leaving
10 years for spermatogenic recovery. In the case of human application, autologous transplantation of cryopreserved testicular cells will be performed to a testicular environment that has been exposed to chemotherapy. This exposure may induce functional changes to the Leydig cell population (Howell and Shalet, 2002
) and may eventually interfere with the outcome after transplantation. In our study we used W/Wv mice as recipient animals rather than mice treated with busulphan because W/Wv mice do not show endogenous spermatogenesis, but have the potential to support donor spermatogenesis (Ogawa et al., 2000
). Russell et al. (1996
) observed some morphological abnormalities in germ cells obtained after stem cell transplantation. Whether this implies that after transplantation in a recipient animal spermatogonial stem cells do not differentiate correctly and may generate sperm with limited functional capacities remains unclear. In order to explore this functional capacity, we examined both in-vivo and in-vitro reproduction with sperm obtained after transplantation.
First, we evaluated in-vivo conception by mating transplanted animals with females. Whereas in the control group 90% of mice with a copulating plug were impregnated, only 35% of mice became pregnant in the experimental group. Furthermore an average of 7.6 fetuses was observed in control mice with a copulatory plug versus only 1.0 in the experimental mice having a copulatory plug. This difference in fecundity may be due to the lower motility observed in the epididymal sperm of transplanted animals. Although it cannot be excluded that this difference is caused by deficiencies in spermatogenesis after transplantation as described by Russell et al. (1996), an insufficient recovery period post-transplantation may also be at the origin of the observed reduction in motility. The lower motility may also be the result of local reactions in the testes. Although all animals had an antibiotic treatment after transplantation, an non-specific inflammatory reaction to the transplanted cell suspensions or the minute concentrations of Trypan Blue used, may have caused this response.
The testis is assumed to be an immunologically privileged organ (Barker and Billingham, 1977), yet an adverse immune response may also be involved. In the human, where autotransplantation instead of allogeneic transplantation would be performed, an adverse immune reaction is less likely.
In order to further examine the functional capacities of sperm obtained after transplantation, we also performed assisted reproductive techniques. We observed that sperm obtained after transplantation are able to fertilize oocytes both after IVF and ICSI and that fertilized oocytes can develop into blastocysts in vitro.
The results of IVF showed that fertilization rates were lower in the experimental group (27 versus 79%, P < 0.00001). In contrast, fertilization after ICSI was not different between controls and transplanted animals (69 versus 68%). The lower fertilization rate after IVF may again be due to the lower motility of sperm obtained from transplanted animals, although functional deficiencies cannot be excluded. Russel et al. (1996) reported more elongation abnormalities after transplantation. ICSI may correct for both motility and morphological deficiencies that may interfere with gamete interaction during fertilization. Once fertilization was achieved, the development of fertilized oocytes was comparable between experimental and control oocytes when ICSI was applied, but not when IVF was applied. After IVF, a lower developmental rate was observed in the experimental group. Because the results after ICSI are not different from controls, we may assume that the meiotic process is undisturbed in transplanted germ cells. The lower fertility rates after IVF and after in-vivo mating are probably related to the lower number of sperm observed after transplantation in our experiments.
Our study shows that in-vivo conception after testicular stem cell transplantation is realistic in mice. Differences between control and transplanted animals may be attributed to deficiencies in the animal model used. Whenever applied in a human situation, most of these deficiencies would probably not exist. Our results confirm again the potential of the testicular stem cell transplantation technique for circumventing sterility in male childhood cancer survivors.
Although based on a limited series, inherent with the complexity of the transplantation IVF and ICSI model, we also showed that sperm obtained after testicular stem cell transplantation are able to fertilize oocytes in vitro by means of techniques of assisted reproduction. Albeit fertilization and blastocyst developmental rates after IVF were lower when post-transplantation epididymal sperm were used, again limitations of the experimental model used may account for this finding.
Although additional study, including post-implantation development, is needed, our data strengthen the hope for future application of this approach in the human.
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Acknowledgements |
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References |
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Submitted on October 22, 2002; resubmitted on March 27, 2003; accepted on May 16, 2003.