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From the * Departments of Endocrinology, Faculty
of Biology, and Cell Biology, University
Medical Center, Utrecht, The Netherlands; and the
Institute for Animal Science and Health
(ID-Lelystad), Lelystad, The Netherlands.
| Correspondence to: F. Izadyar DVM, PhD, University Medical Center, Department of Cell Biology, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands (e-mail: fizadyar{at}lab.azu.nl ). |
| Received for publication October 16, 2001; accepted for publication February 7, 2002. |
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Abstract |
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Key words: Freezing, spermatogonial stem cells, transplantation
Isolation of pure type A spermatogonia (among them spermatogonial stem cells) from various species including rodents (Bellve et al, 1977; Morena et al, 1996; Van Pelt et al, 1996) and domestic animals (Dirami et al, 1999; Izadyar et al, 2000) has been described. In principle, these spermatogonia can be preserved in 2 ways, long-term culture or cryopreservation. So far it has not been possible to preserve pure populations of spermatogonia for longer than 1 week in culture (Dirami et al, 1999). However, when spermatogonia are cultured in the presence of serum and a feeder layer, some spermatogonial stem cells survive long-term culture and repopulate recipient testes after transplantation (Nagano et al, 1998).
An alternative and probably the best method for longterm preservation of spermatogonial stem cells is cryopreservation. To date, no attempt has been made to develop an optimal protocol to cryopreserve these cells. The method employed in spermatogonial stem cell transplantation experiments is one generally used for somatic cells and cell lines. This method results in the survival of approximately 40% of cells in isolated germ cell mixtures after the freeze/thaw procedure (Avarbock et al, 1996; Lovell-Badge, 1996; Brinster, 1998). The cell suspensions used in these studies were not enriched for type A spermatogonia. Hence, the survival rate of type A spermatogonia in this procedure is not known. Nevertheless, at least some spermatogonial stem cells survive freezing/thawing with this method.
In the present study, a method was developed to cryopreserve pure populations of bovine type A spermatogonia by testing different freezing protocols and cryoprotectants. The survival and proliferation of type A spermatogonia after cryopreservation was studied in culture, and the functionality of spermatogonial stem cells among them was assessed by transplantation of these cells into recipient mouse testes.
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Materials and Methods |
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Cryopreservation
Immediately after cell isolation, viability was assessed (see below) and
the cells were transferred on ice to a cold room (4°C) for further
preparations. Cell suspensions in 0.5-ml aliquots (6 x 106
cells per mL) were prepared. Then, an equal volume of 2x concentrated
freezing medium was added dropwise to the Eppendorf vial containing the cell
suspension during a period of 10-15 minutes, and after gently mixing by
inverting the vial, a sample was taken for viability assessment. The freezing
media were based on MEM supplemented with 1 g of bovine serum albumin (BSA)
per 100 mL (MEM/BSA) and contained (final concentrations): 1) 10 % (v/v) FCS;
or 2) 10 % (v/v) FCS plus 1.4 M glycerol; or 3-6) 10% (v/v) FCS and 1.4M DMSO;
and either 0, 0.07, 0.14 or 0.21 M sucrose; or 7) 20% (v/v) FCS and 1.4 M
DMSO.
Different freezing protocols were compared using media 2 (glycerol) and 3 (DMSO). For the controlled-rate freezing protocols, the cell suspensions were packed in "French" straws (inside diameter, 1.6 mm, IMV, L'Aigle, France), which were heat-sealed. The volume of the straws was approximately 220 µL. All straws were completely filled, except for a small air bubble of approximately 10 µL. The straws were placed inside a programmable freezing cabinet (Planer 10, Cryotech Benelux, Schagen, The Netherlands), which had been set to a starting temperature of +5°C, and was then cooled with a linear rate of either -1°C/min or -5°C/min to -80°C, followed by cooling at a rate of -50°C/min to -120°C. Straws were then plunged into liquid nitrogen (-196°C).
For noncontrolled-rate freezing, 1.8-mL cryovials vials (Nunc, Life Technologies, Roskilde, Denmark) containing 1.0 mL of cell suspension in freezing medium were placed in an insulated (polystyrene) container at -80°C for at least 1 day and then plunged into liquid nitrogen. To monitor the temperature during the freezing protocol, one straw or vial containing the respective freezing medium was fitted with a copper/Constantan thermo-couple (wires of 0.15 mm diameter). Typical runs are shown in Figure 1. The cells were thawed by swirling in 38°C water bath for 30 seconds (straws) or 2 minutes (vials). The contents of the straw or vial was transferred to a tube and diluted slowly by adding two volumes, dropwise, of MEM supplemented with 10% FCS. Then, the cells were pooled and centrifuged at 2000 x g for 5 minutes, the supernatant was removed, and the pellet was resuspended in MEM/BSA. A sample was taken for viability assessment, and the remainder of the cells were used for culture or transplantation experiments.
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Evaluation of Spermatogonial Survival and Proliferation
Cultures were performed at 37°C in a humidified atmosphere with 5%
CO2, and medium was changed twice per week. To investigate the
survival of spermatogonia after cryopreservation, cells (5 x
104) were cultured in 96-well plates containing 200 µL of MEM
medium supplemented with 2.5% FCS for 1 week. Viability of cells during the
isolation and cryopreservation steps and after 1 week of culture was
determined using a mixture of Calcein-AM and ethidium homodimer (1 µM per
each; the so-called live and dead kit, Molecular Probes, Eugene, Ore). During
culture, a colorimetric assay was used to quantify proliferative activity,
based on the cleavage of the tetrazolium salt
4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate
by mitochondrial dehydrogenases in viable cells (WST-1;
Boehringer-Mannheim).
To study the proliferative activity of spermatogonia after cryopreservation, dolichos biflorus agglutinin (DBA), a marker for type A spermatogonia (Ertl and Wrobel, 1992) and bromode-oxyuridine (BrdU) incorporation was used. Therefore, cells (the same concentration) were cultured in 250 µL of medium in 8-well chamber slides (Nunc), and at days 4 and 7 of culture BrdU (0.3 mg/mL) was added to the medium, and after an incubation of 2 hours the slides were washed in PBS, fixed in Bouin solution, and the number of type A spermatogonia in the S phase of the cell cycle was determined using DBA-BrdU immunolabeling (see "Immunohistochemistry").
Preparation of Donor Cells, Recipient Testes, and
Transplantation
Approximately 2-4 months after cryopreservation, the spermatogonia were
thawed as described above and resuspended at a density of 20 x
106 cells/mL and kept on ice until transplantation. Adult NMRI mice
HsdCpb (nu/nu, Harlan-Netherlands, Horst, Netherlands) were used as
recipients. NMRI mice lack T cells and are immunodeficient; therefore, they
were housed in specific pathogen-free conditions with all the food, water, and
bedding autoclaved before use. The mice were housed in a 12-hour light:
12-hour dark cycle at constant temperature, and were provided with food and
water ad libitum. Recipient mice were given a fractionated dose of 1.5 and 12
Gy of x-rays in 24-hour intervals to destroy endogenous spermatogenesis. In a
previous study we found that this irradiation protocol is enough to remove
virtually all endogenous spermatogenesis and does not have any apparent
harmful effect on supporting Sertoli cells (Creemers et al, 2002).
One month after irradiation, the recipient mice were anesthetized by intraperitoneal administration of a mixture of Fentanyl, Fluanison, and Midazolam (10 mg/kg). Testes were exteriorized through a midline abdominal incision, and donor cells (approximately 25 µL) were injected through a micropipette (Clark Electromedical Instruments, Reading, UK) via the efferent duct into the rete testis as described by Ogawa et al (1997). The contralateral testis was used as the negative control. Testes were harvested at 2-3 months after transplantation, were fixed in Bouin solution, and the colonization efficiency was assessed using DBA immunohistochemistry. The experimental protocol of this study followed the National Institutes of Health Guidelines of the Care and Use of Laboratory Animals and was approved by the animal care and use committee of Utrecht University.
Immunohistochmistry
Type A spermatogonia were distinguished using DBA immunohistochemistry as
described by Ertl and Wrobel
(1992). Bouin-fixed,
paraffin-embedded testes (positive control) and cells cultured in glass
chamber slides were used for immunohistochemistry. Briefly, after paraffin was
removed (only for tissue sections) and sections were rehydrated, they were
treated with 3% H2O2 (Merck, Darmstadt, Germany) for 10
minutes to inhibit endogenous peroxidase, and rinsed in phosphate-buffered
saline (PBS). Incubation in 5% BSA in PBS for 15 minutes before lectin
incubation was advantageous to block nonspecific adhesion sites. The sections
were then incubated in DBA conjugated with horseradish peroxidase (DBA-HRP; EY
Laboratories, San Mateo, Cal) 1:100 in PBS and 1% BSA for 1 hour at 37°C
in a moist chamber. Following the lectin incubation the sections were rinsed
in PBS 3 times. Staining of the DBA-HRP was obtained by treating the sections
for 5-15 minutes with PBS containing 25 mg 3,3'-diaminobenzidine
tetrahydrochloride (DAB; Sigma), 1 mL of 1% nickel ammoniumsulfate solution,
1.25 mL of a 1% cobalt chloride solution, and 17 µL of 35%
H2O2 per 50 mL. Then the slides were thoroughly rinsed
in distilled water and, if necessary, counterstained with hematoxylin and
Mayer for 1-2 minutes. The sections were dehydrated in a graded alcohol
series, cleared in xylol, and mounted with Pertex (Cell Path; Compulink,
United Kingdom). Negative control sections were incubated in 1% BSA in PBS
without lectin.
To study the proliferative activity of the cultured spermatogonia more accurately, BrdU and DBA double fluorescence immunolabeling was employed. After rinsing in distilled water, the slides were transferred to 1% warm (60°C) periodic acid and incubated at 60°C for 30 minutes. After thoroughly rinsing in plain water and distilled water, the slides were washed in PBS for 15 minutes. To block nonspecific binding, the slides were incubated in 5% BSA in PBS for 10 minutes at room temperature, and subsequently overnight with anti-BrdU (Becton Dickinson, San Jose, Cal) 1:80 in PBS supplemented with 1% BSA at room temperature. After thoroughly rinsing in PBS, the slides were incubated in goat anti-mouse—Texas red (Jackson ImmunoResearch Laboratories, West Grove, Pa) 1:150 in PBS for 1 hour at room temperature in a dark chamber. After rinsing in PBS, the slides were incubated in DBA-fluorescein isothiocyanate (EY Laboratories) in PBS 1:50 for 1 hour at 37°C. After a long rinse in PBS, they were mounted in VECTAshield (Vector Laboratories, Burlingame, Cal), sealed with nail polish, and evaluated under a Nikon inverted light microscope equipped with an epifluorescence mercury lamp. Micrographs were made using Kodak Ektachrome EL 400 ASA film.
Statistical Analysis
The results are presented as means ± SEM. Statistical analysis was
performed by two-sample t-test, and the difference was considered
significant when P was <.05.
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Results |
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Effect of Cooling Rate on Viability After Thaw
Controlled-rate slow freezing (1°C/min) resulted in significantly
(P <.05) more viable cells than fast (5°C/min) freezing
(Table 2). Noncontrolled-rate
freezing with a comparably low cooling rate resulted in an even higher
percentage of living cells. Registration of temperature during freezing with
different programs showed that ice nucleation occurs around -13°C in all
programs; however, the noncontrolled method had a longer plateau after ice
nucleation compared to the controlled methods, in which the plateau was much
shorter, and the rate of cooling after ice nucleation was clearly higher
(Figure 1a). Cooling rate as a
function of time or as a function of temperature is separately demonstrated in
Figure 1, b and c,
respectively. The cooling rate remains higher in the 1°C/min method up to
the point at which the temperature has decreased to -15°C. At lower
temperatures, the cooling rate of the noncontrolled-rate method starts to
increase and becomes higher than that of the 1°C/min method, but the
cooling rate still remains lower than that of 5°C/min method.
Survival and Proliferation of Frozen-Thawed Spermatogonia in
Culture
After 1 week of culture in MEM supplemented with 2.5% FCS, more than 50% of
the frozen/thawed cells were viable as determined by live and dead staining
(data not shown). In addition, the WST-1 value of the frozen/thawed cells was
increased significantly (P <.001) during culture, indicating
survival and proliferation of cells in culture
(Figure 2). Survival and
proliferation in culture, as determined by WST-1, was significantly
(P <.001) higher in cultures of spermatogonia that had been frozen
in the media containing 0.07, 0.14, or 0.21 M sucrose than those without
sucrose (Figure 3).
Immunolocalization of DBA and BrdU showed that after 1 week of culture, about
30% of the frozen/thawed cells were positive for the spermatogonial marker DBA
and from those, about 5%-10% were positive for BrdU (data not shown). This is
similar to that of the freshly cultured cells, indicating that the
proliferative activity of spermatogonia in culture was not influenced by the
freeze/thaw procedure.
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Transplantation
To investigate the functionality of the spermatogonial stem cells among the
purified A spermatogonia after cryopreservation, cells were transplanted into
recipient immunodeficient nude mouse testes. Colonization efficiency was
established by determining the proportion of tubule cross-sections containing
DBA-positive cells and the number of DBA-positive cells per seminiferous
tubule. Three months after transplantation of either fresh or frozen/thawed
cells, groups of bovine type A spermatogonia, as detected by DBA staining,
were found in the tubule cross-sections of the recipient mouse testes
(Figure 4). Repopulation
efficiency of the fresh cells was higher (P <.01) than that of
cells frozen in the MEM/FCS/DMSO freezing medium. The repopulation efficiency
of frozen/thawed spermatogonia was significantly (P <.05) higher
for cells frozen in the MEM/FCS/DMSO freezing medium containing 0.07 M sucrose
compared with cells frozen in the same medium but without sucrose. After
transplantation of both freshly isolated and cryopreserved spermatogonial stem
cells, no differentiated bovine germ cells were observed in the recipient
mouse testes. No DBA-positive cells were found in the control testes,
confirming the specificity of this identification method
(Figure 5).
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Discussion |
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-glucose, had
the same effect on survival of spermatogonia (data not shown). Sugars are
nonpenetrating cryoprotectants, which because of their positive influence on
preserving cell membrane integrity, have been used successfully in
cryopreservation protocols of many cell types, including embryos
(Lazar et al, 2000; Oberstein et al, 2001) and
gametes (Yildiz et al, 2000;
Fabbri et al, 2001; Park et al, 2001;
Sztein et al, 2001). In
cryopreservation of hematopoietic stem cells, the polysaccharide hydroxy ethyl
starch (HES) has been shown to improve cell survival
(Donaldson et al, 1996; Walter et al, 1999). The cooling rate during freezing is an important parameter for cell survival. Because much of the extracellular water is transformed into ice, the concentration of liquid water progressively decreases, leading to an extensive dehydration of the cells. Slow-cooling damage has been attributed to such phenomena as an increase of the external and internal solute (salt) concentration (Lovelock, 1953; Daw et al, 1973; Griffiths et al, 1979), the small size of the channels of unfrozen solution (Mazur and Rigopoulos, 1983), the mechanical stress of cell shrinkage (Mazur and Rigopoulos, 1983), and destabilization of membranes and proteins at low water potential (Crowe and Crowe, 1984; Rudolph and Crowe, 1985; Carpenter and Crowe, 1988). Increasing the cooling rate would reduce the time during which the cells remain vulnerable to the unfavorable conditions resulting from ice formation. Moreover, at higher cooling rates, intracellular dehydration, intracellular solute concentration, and cell shrinkage may become less severe. However, when cooling rates are increased too much, dehydration may not be fast enough to prevent lethal intracellular ice formation (Mazur, 1963, 1977; Mazur et al, 1972). This mechanism is believed to be responsible for fast cooling damage, at least in a number of systems (Mazur et al, 1972; Bank, 1974; Mazur, 1977).
Different cell types may differ in optimal cooling rate, depending on the water content, size and shape of cells, and water permeability coefficient (Lp) of their cytoplasmic membrane. Small cells, such as red blood cells or spermatozoa, are known to have high permeabilities to water compared to other cells, and to tolerate ultrafast freezing in the range of 15-60°C/min, depending on species (McColm and Latter, 1986; Liu et al, 1998). Somatic cells, such as cumulus cells, blastocysts, and many cell lines (Dong and Xia, 1996; Nguyen et al, 2000; Saeed et al, 2000) can also resist rapid freezing but in relatively lower cooling rates. Hematopoietic stem cells have been successfully preserved at cooling rates between 1-5°C/min (Donaldson et al, 1996; Walter et al, 1999). There is scant information on the optimal cooling rate for spermatogenic stem cells. Two reports describe the use of a noncontrolled-rate freezing method for nonpurified spermatogonial populations, which were assumed to contain a small percentage of spermatogonial stem cells (Avarbock et al, 1996; Brinster, 1998). In our study, pure populations of bovine type A spermatogonia containing more spermatogonial stem cells were frozen successfully using a linear cooling rate of 1°C/min. The cell survival rate using a noncontrolled-rate freezing method was even better. In this method the cooling rate is variable, depending among other things, on the heat capacity of the sample and the liberation of heat of fusion. Up to the ice nucleation point, the cooling rate of the controlled slow-freezing rate method was higher than that of the noncontrolled-rate method. After ice nucleation, the cooling rate of the noncontrolled-rate method starts to increase and becomes higher than that of the slow freezing method, but it is still lower than with the fast-freezing method. The only range of temperatures in which the cooling rate of the noncontrolled-rate freezing protocol approaches that of the controlled fast-rate freezing protocol is between -30°C and -55°C (see Figure 1). The risk of "fast cooling damage" in this range is very small (Liu et al, 2000; Viveiros et al, 2002), which may explain why the linear cooling rate of 5°C/min appeared to be less suited for freezing bovine spermatogonia than the noncontrolled-rate freezing program. Because we have no data on the efficiency of controlled-rate freezing with rates lower than 1°C/min, the optimal cooling rate for bovine spermatogonia may be as low as or lower than 1°C/min, which is at the lower end of the range found to be optimal for hematopoietic stem cells (Donaldson et al, 1996; Walter et al, 1999).
We also demonstrated that frozen/thawed spermatogonia survive and proliferate in culture. Both the viability and proliferation of freshly isolated spermatogonia, as determined by the WST-1 colorimetric assay, were higher than that of frozen/thawed cells. This was mainly due to the diminished cell recovery following the freeze/thaw procedure and not to a lower proliferative activity after cryopreservation, because BrdU incorporation of frozen/thawed spermatogonia was similar to that of fresh cells. Reduced cell recovery following the freeze/thaw procedure was also reported by other investigators studying cryopreservation of nonpure spermatogonia from other species, including rodents (Avarbock et al, 1996; Brinster, 1998) and domestic animals (Dobrinski et al, 2000).
Purified spermatogonia consisted of a mixture of a few spermatogonial stem cells and A spermatogonia that were already destined to develop into spermatozoa. To test the functionality of spermatogonial stem cells among these cells after cryopreservation, cells were transplanted into immunodeficient mouse testes. Three months after transplantation, frozen/thawed spermatogonia were found to have colonized recipient mouse testes, and groups of bovine spermatogonial stem cells, as detected by DBA staining, were found on the basal membrane of the tubule cross-sections of the recipient testes. Hence, spermatogonial stem cells maintain their functionality after cryopreservation. Both freshly isolated as well as cryopreserved bovine spermatogonial stem cells did not produce more advanced germ cells in the recipient mouse testes. This is probably due to the phylogenetic distance between the donor and the recipient species, as has been reported by other investigators working on xenogeneic spermatogonial transplantation (Dobrinski et al, 1999, 2000).
In summary, we developed an appropriate protocol for the cryopreservation of purified bovine type A spermatogonia, in which 70% survive the protocol and remain able to survive and proliferate in culture. The spermatogonial stem cells among these cells were found to remain functional and able to colonize recipient mouse testes after transplantation. These findings demonstrate that spermatogonial stem cells from a large domestic animal can be successfully cryopreserved, and are able to recover with full functional capability after freezing and thawing procedures. Cryopreservation of the male germ line effectively establishes the potential of generating, at any time, clones of the original male following spermatogonial stem cell transplantation to multiple recipients, and it has valuable implications in veterinary medicine as well as human medicine.
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Acknowledgments |
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Footnotes |
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 M. Geens, E. Goossens, G. De Block, L. Ning, D. Van Saen, and H. Tournaye Autologous spermatogonial stem cell transplantation in man: current obstacles for a future clinical application Hum. Reprod. Update, March 1, 2008; 14(2): 121 - 130. [Abstract] [Full Text] [PDF]
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C. Wyns, M. Curaba, B. Martinez-Madrid, A. Van Langendonckt, W. Francois-Xavier, and J. Donnez Spermatogonial survival after cryopreservation and short-term orthotopic immature human cryptorchid testicular tissue grafting to immunodeficient mice Hum. Reprod., June 1, 2007; 22(6): 1603 - 1611. [Abstract] [Full Text] [PDF]
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K. Jahnukainen, J. Ehmcke, S. D. Hergenrother, and S. Schlatt Effect of cold storage and cryopreservation of immature non-human primate testicular tissue on spermatogonial stem cell potential in xenografts Hum. Reprod., April 1, 2007; 22(4): 1060 - 1067. [Abstract] [Full Text] [PDF]
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M. Geens, H. Van de Velde, G. De Block, E. Goossens, A. Van Steirteghem, and H. Tournaye The efficiency of magnetic-activated cell sorting and fluorescence-activated cell sorting in the decontamination of testicular cell suspensions in cancer patients Hum. Reprod., March 1, 2007; 22(3): 733 - 742. [Abstract] [Full Text] [PDF]
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V. Keros, B. Rosenlund, K. Hultenby, L. Aghajanova, L. Levkov, and O. Hovatta Optimizing cryopreservation of human testicular tissue: comparison of protocols with glycerol, propanediol and dimethylsulphoxide as cryoprotectants Hum. Reprod., June 1, 2005; 20(6): 1676 - 1687. [Abstract] [Full Text] [PDF]
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 D. Shin, K. C. Lo, and L. I. Lipshultz Treatment Options for the Infertile Male With Cancer J Natl Cancer Inst Monographs, March 1, 2005; 2005(34): 48 - 50. [Abstract] [Full Text] [PDF]
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H. Tournaye, E. Goossens, G. Verheyen, V. Frederickx, G. De Block, P. Devroey, and A. Van Steirteghem Preserving the reproductive potential of men and boys with cancer: current concepts and future prospects Hum. Reprod. Update, November 1, 2004; 10(6): 525 - 532. [Abstract] [Full Text] [PDF]
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 J. M. Oatley, J. J. Reeves, and D. J. McLean Biological Activity of Cryopreserved Bovine Spermatogonial Stem Cells During In Vitro Culture Biol Reprod, September 1, 2004; 71(3): 942 - 947. [Abstract] [Full Text] [PDF]
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V. Frederickx, A. Michiels, E. Goossens, G. De Block, A.C. Van Steirteghem, and H. Tournaye Recovery, survival and functional evaluation by transplantation of frozen-thawed mouse germ cells Hum. Reprod., April 1, 2004; 19(4): 948 - 953. [Abstract] [Full Text] [PDF]
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M. Kanatsu-Shinohara, N. Ogonuki, K. Inoue, A. Ogura, S. Toyokuni, and T. Shinohara Restoration of fertility in infertile mice by transplantation of cryopreserved male germline stem cells Hum. Reprod., December 1, 2003; 18(12): 2660 - 2667. [Abstract] [Full Text] [PDF]
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