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a Department of Veterinary Science, National Institute of Infectious Diseases, Tokyo 162-8640, Japan
b Gene Research Center, Tokyo Institute of Technology, Yokohama 226-8501, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Sertoli cells, the male counterparts of cumulus cells, may be used to produce cloned male mice. They are fully differentiated, quiescent G0/G1 cells and are therefore assumed to be suitable for cloning by nuclear transfer, as has been demonstrated in successful cloning using G0 cells [1, 3–6, 9]. However, only one live fetus (Day 8.5), and no live offspring, were obtained in mice after transferral of embryos derived from Sertoli cell nucleus injection [7]. Sertoli cells collected from mature males are too large to inject into mouse oocytes without damaging both the recipient oocytes and the nucleus of the Sertoli cells. This technical difficulty, rather than the biological nature of their nuclei, might have caused the poor in vivo and in vitro development of embryos reconstructed with Sertoli cell nuclei. In this study, we collected immature Sertoli cells, which are relatively small, actively proliferating cells, from 3- to 10-day-old newborn mice and injected their nuclei into enucleated oocytes to generate cloned males. We also injected nuclei from immature Sertoli cells that had been cultured in vitro, cryopreserved, or transfected with exogenous DNA in order to examine whether they are potential donors for somatic cell cloning in the mouse.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Immature Sertoli cells from [C57BL/6J Cr x DBA/2 Cr]F1 (B6D2F1) or [C57BL/6J Cr x CBA/N] hybrid F1 males (3–10 days old) were used unless otherwise stated. Testes (2–6 per experiment) were placed into PBS containing 3 mg/ml BSA, and the tunica albuginea was removed. Masses of seminiferous tubules were incubated in PBS containing BSA, 0.1 mg/ml collagenase (Sigma Chemical Co., St. Louis, MO), and 0.01 mg/ml DNase (Sigma) for 30 min at 37°C with shaking (70 oscillations/min). The tissue suspensions were then pipetted repeatedly with a 1- to 2-mm tip diameter pipette to disrupt tubule fragments. The disrupted tubules were then treated with 0.2 mg/ml trypsin (Sigma) for 5 min to loosen the cell aggregates, which were predominantly composed of Sertoli cells and germ cells. The final cell suspension for nuclear transfer was obtained after washing with BSA-containing PBS by centrifugation. Immature Sertoli cells were identified as the smallest testicular cells (about 8 µm in diameter) [11] with an irregular cell outline without villous structures [11]. The cell surface stained positive for LEA (Lycopersicon esculentum agglutinin) lectin [12] (Fig. 1b). The nucleus is not usually visible under Nomarski optics, in contrast to the situation for germ cells, which are the largest testicular cells and are also positive for LEA lectin (Fig. 1). Another important feature of immature Sertoli cells is their soft, fragile cytoplasm. At the time of microinjection, the Sertoli cell nucleus was easily isolated in the injection pipette. In contrast, testicular fibroblast cells have a harder cytoplasm and often stick inside the injection pipette. In the testicular cell suspension, approximately 60–70% of the testicular cells were identified as Sertoli cells. In our preliminary experiments using older mice (> 12 days old), we found that Sertoli cells in the cell suspension varied in size and were easily confused with other testicular cells, such as fibroblasts or differentiating germ cells (e.g., preleptotene spermatocytes). Therefore, in this study we collected immature Sertoli cells from the testes of 3- to 10-day-old mice to ensure the accuracy of cell identification.
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Preparation of Other Donor Cells
In the cow, sheep, goat, and mouse, the nuclei of cumulus cells and/or fetal fibroblast cells can support embryo development efficiently after nuclear transfer [1–3, 7]. In the first series of experiments, we compared the developmental abilities of embryos reconstructed from immature Sertoli cells, cumulus cells, and fetal fibroblast cells all having the B6D2F1 genotype. Cumulus cells were freshly isolated from recipient oocytes by hyaluronidase treatment (see below) shortly before experiments. Fetal fibroblast cells were obtained from fetuses at Day 14 of gestation, as previously reported [13], and cultured for 2–3 wk (2–4 passages) before use.
Culture of Sertoli Cells
To maintain their immaturity and mitotic activity, immature Sertoli cells isolated from the testes of 3-day-old mice were cultured in vitro in Dulbecco's modified Eagle's medium containing 3 mg/ml BSA and 1 µg/ml porcine FSH without serum [14]. The medium was changed every 48 h. After 1 wk in culture, aggregates of immature Sertoli cells on the bottom (Fig. 1c) were retrieved for treatment with trypsin and DNase for nuclear transfer. More than 70% of the cells in the suspension were identified as Sertoli cells.
Cryopreservation of Sertoli Cells
Sertoli cells cultured for 1 wk as described above were collected and suspended in a cell cryopreservation solution (Cellbanker; NZK Biochemicals, Fukushima, Japan). The masses of Sertoli cells must be completely disaggregated to maintain their viability during the freezing and thawing procedure. Approximately 1-ml aliquots of the cell suspension were transferred to 2-ml polypropylene cryotubes (Sumitomo Bakelite, Tokyo, Japan), placed in a freezing container (Bicel; Nihon Freezer Co., Osaka, Japan), and frozen at -80°C in a deep freezer. If the cells were to be cryopreserved for more than 2 wk, the cryotubes were plunged in liquid nitrogen and kept there. For thawing, the cryotubes were put into a water bath at room temperature, and about 30 sec later 1 ml BSA-containing PBS was added. After washing by centrifugation, the Sertoli cells were cultured as described above for another week and used for nuclear transfer.
Transfection of Immature Sertoli Cells
We transfected immature Sertoli cells with DNA encoding a green fluorescent protein (GFP) so that the expression of the exogenous gene could be detected in living embryos before embryo transfer. The plasmid pEGFP-C1 (Clontech, Palo Alto, CA) encodes the enhanced green fluorescent protein gene, human cytomegalovirus (immediate early) promoter, and kanamycin/neomycin resistance gene. Before transfection, this plasmid was linearized by digestion with restriction enzyme, ApaLI. After 1 wk in the culture medium described above, Sertoli cells were transfected with pEGFP-C1 using TransFast (Promega, Madison, WI) according to the manufacturer's instruction. After culturing for 3 days, the transfected cells were selected by treatment with 150 µg/ml G418 for 1 wk. The cells were used for nuclear transfer after 1–3 wk in culture.
Nuclear Transfer
Reconstruction of embryos by nuclear transfer was performed according to the method reported by Wakayama et al. [7] with a slight modification. Recipient oocytes for nuclear transfer were collected from mature B6D2F1 females superovulated by consecutive injections of 7.5 IU eCG and 7.5 IU hCG 46–52 h apart. Cumulus-enclosed oocytes retrieved 14–17 h after hCG injection were treated with 0.1% bovine testicular hyaluronidase in CZB medium [15] until the cumulus cells were completely dispersed. The cover of a plastic dish (Falcon no. 1006; Becton Dickinson, Franklin Lakes, NJ) was used as a micromanipulation chamber. Three small drops were readied for the nuclear transfer: a 3-µl drop of Hepes-buffered CZB (Hepes-CZB) containing 6 µg/ml cytochalasin D (Sigma) for enucleating the recipient oocytes, a 1-µl drop of Hepes-CZB for injecting the donor nuclei, and a 3-µl drop of Hepes-CZB containing 10% polyvinylpyrrolidone for suspending the donor cells. The micromanipulation chamber was placed on the stage of an inverted microscope (Nikon TE300; Tokyo, Japan) equipped with Nomarski differential interference optics. Oocytes were placed in the drop containing cytochalasin D for 5 min and enucleated with a 7-µm (internal diameter) glass pipette by aspirating the metaphase II plate with a small volume of surrounding cytoplasm. The enucleation pipette was attached to a Piezo micromanipulator (Prime Tech Ltd., Ibaraki, Japan) so that the zona could be penetrated easily. Enucleated oocytes were washed thoroughly to remove any cytochalasin, which may lyse oocytes at the time of microinjection. The nucleus was removed from the donor cells by gentle aspiration of the cells in and out of the injection pipette. The donor nuclei, together with a small amount of cytoplasm, were injected deep inside the ooplasm using the Piezo-driven micropipette. The inner diameters of injection pipettes were 4–5 µm for cumulus cells and immature Sertoli cells and 7–9 µm for fetal fibroblast cells. Oocytes injected with donor nuclei were incubated in CZB medium for 1–2 h under 5% CO2 in air at 37°C. At least four replicate experiments were undertaken for each experimental group.
Oocyte Activation and Embryo Culture
Oocytes injected with donor cell nuclei were activated by treatment in Ca2+-free CZB medium containing 10 mM SrCl2 for 6–7 h. The activation medium also contained 6 µg/ml cytochalasin D to prevent extrusion of the polar body. After washing, the oocytes were cultured in CZB medium for 48 or 72 h under 5% CO2 in air at 37°C.
Embryo Transfer
After 72 h in culture, embryos that had developed to the morula or blastocyst stages were transferred into the uteri of Day 3 pseudopregnant ICR females (Clea Japan, Tokyo, Japan). Some embryos that were cultured for 48 h and had reached the 4-cell stage were transferred into the oviducts of Day 1 pseudopregnant females. The number of embryos transferred into each uterine horn or oviduct was 7–10. On Days 10–13 for the first series of experiments, and on Day 20 for the second series, recipient females were killed and their uteri examined for live or dead fetuses. Some of the live fetuses found on Day 20 were raised by lactating foster ICR mothers.
Statistical Analysis
The results were evaluated using Fisher's exact probability test, with a P value less than 0.05 considered statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Approximately 70–80% of the enucleated oocytes survived microinjection with cumulus cells and immature Sertoli cells, whereas only 50% of oocytes survived injection with fetal fibroblast cells. Nuclear transfer embryos derived from cumulus cell nuclei cleaved at a significantly higher rate than those derived from fetal fibroblast or immature Sertoli cells (Table 1). Among the embryos that underwent the first cleavage, Sertoli cell embryos had the best rates of development into the morula/blastocyst stage (``%/cleaved" in Table 1, P < 0.05). They also showed the best implantation rate after embryo transfer (P < 0.05).
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Recipient females were examined at Days 10–13 of gestation. Totally, 9 living fetuses were obtained from cumulus cells and immature Sertoli cells. We found that immature Sertoli cells supported embryo development to living fetuses irrespective of the strain used. The fetuses were all morphologically normal (Fig. 2a), except for 2 developmentally retarded fetuses each from a B6 Sertoli cell and a [B6xJF1]F1 Sertoli cell. The latter 2 fetuses were obtained from Day 10 recipients, but their developmental stage corresponded to around Day 9. In all cases, the placenta was larger than that obtained from normal fertilization (Fig. 2b). In this experiment, the derivation of [B6xJF1]F1 fetuses from Sertoli cell nuclei was confirmed by DNA polymorphisms of several imprinted genes, such as Igf2, Peg3, Meg3, Igf2r, and p57kip2, between B6 (laboratory mouse: Mus musculus musculus) and JF1 (Japanese wild-type mouse: Mus musculus molossinus) mice and their parent-of-origin-specific expression patterns: paternal for the former two and maternal for the latter three genes (data not shown).
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Production of Offspring from Immature Sertoli Cells
In the second series of experiments, we examined whether the nucleus of immature Sertoli cells can support full-term development. Since Sertoli cells in the testes of newborns are proliferating and differentiating actively, their ability to support embryonic development may alter with age. We injected enucleated oocytes with fresh Sertoli cells at Days 3–5 or Days 8–10 and compared the subsequent embryo development. The embryos derived from Sertoli cells at Days 3–5 had better developmental rates into the morula/blastocyst and postimplantation stages than those from cells at Days 8–10 (Table 2). In all, 7 normal pups (3.3%, 7 of 215) were born from either group (Fig. 3a), but the efficiency was better with the Sertoli cells at Days 3–5. Then we used Sertoli cells at Days 3–5 for further experiments. Culturing Sertoli cells slightly improved the efficiency with which nuclear transfer embryos developed into morulae/blastocysts, but the difference was not significant. Two normal pups were born after nuclear transfer of cultured Sertoli cells. More than 90% of cryopreserved Sertoli cells were viable after thawing, as demonstrated by the trypan blue exclusion test. They best supported the development of reconstructed embryos into morulae/blastocysts and live offspring. Immature Sertoli cells in culture were successfully transfected with the GFP gene, and after selection with G418 all living cells showed green fluorescence (Fig. 4, a and b). The expression of GFP was also observed in embryos reconstructed with transfected cells (Fig. 4, c and d). Although some were implanted and developed until Days 12–14 (as judged by a consistent increase in the weight of the recipient females; data not shown), no pups were obtained at term. In all, 16 newborns were obtained after nuclear transfer of immature Sertoli cells (Table 2). They were all males, and each had the expected eye and coat colors. The body and placental weights at birth (mean ± SD) were 1.62 ± 0.21 and 0.40 ± 0.08, respectively. All the pups but one showed no gross abnormality or respiratory failure. One pup, derived from a cryopreserved Sertoli cell, suffered from an umbilical hernia, although it was apparently viable at birth. Of 12 pups raised by lactating foster mothers, 11 grew into normal-looking adult males. At least 7 were proven fertile by mating with ICR females (Fig. 3b).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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There are general factors that critically affect the outcome of somatic cell cloning. They include the cell cycle stage of the donor cells and reprogramming of the donor cell genome. The Honolulu method was originally developed for donor cells at the G0/G1 phase in the cell cycle. After activation of injected oocytes, the extrusion of a (pseudo-) polar body is prevented by cytochalasin treatment to keep a set of 2n diploid chromosomes within the activating oocytes. In this context, cumulus cells, which are arrested at G0/G1 after ovulation [10], were chosen as donors in the first study of somatic cell cloning in the mouse [7]. Since immature Sertoli cells are actively proliferating cells, only a fraction are thought to be in the G1 phase. In this study, we randomly selected Sertoli cells for injection because it was impossible to distinguish G1 cells from cells at other stages under a conventional microscope. In some kinds of somatic cells, cell size is a good criterion for determining the stage of the cell cycle, but the size of immature Sertoli cells varies with their maturity and therefore is not always correlated with the cell cycle. In the mouse, when the cell cycles of the donor nucleus and the recipient oocyte are not synchronized, reconstructed oocytes frequently cannot enter the first mitosis because of prolonged or complete absence of DNA synthesis [16]. This feature is compatible with the findings in our study for cumulus, fetal fibroblast, and Sertoli cells (Table 1). The cleavage rates after reconstruction of embryos were very high with cumulus cells (mostly G1/G0 cells) and relatively low with fibroblast and Sertoli cells (less than 70% G1/G0 cells [17, 18]). The G1/G0 state can be induced in culture by serum starvation [1, 3, 4]. We cultured immature Sertoli cells without serum for 1 wk and found that their speed of proliferation seemed to be lower than that in vivo, while their viability was maintained by the addition of FSH. The proportion of mitotic cells also decreased from about 5% to less than 1% (data not shown). Although still speculative, the possibility exists that the relative proportion of immature Sertoli cells at stage G0/G1 might have increased after 1 or 2 wk in culture and resulted in better cleavage rates of cloned embryos (Table 2).
Another important factor, reprogramming of the donor cell genome, which is probably achieved by unknown factor(s) present in unfertilized oocytes, is a prerequisite for the donor cell nuclei to behave as zygote nuclei. Normal zygotic gene activation and subsequent embryo development occur only when the donor genome is successfully reprogrammed. The major zygotic gene activation in the mouse occurs in the early 2-cell stage [19]. Since the majority of cleaving Sertoli cell embryos developed beyond the 2-cell stage, the genome of immature Sertoli cells might be reprogrammed at least to some extent. It is probable that mouse fetal fibroblast cells are more resistant to reprogramming than other cell types, although in domestic species they are thought to be suitable donors for somatic cell cloning [1–3].
In vitro culture and cryopreservation of cells for nuclear transfer apparently increase the chance of application of the cloning technique to basic research and commercial applications. We found that immature Sertoli cells collected from Day 3 testes could be cultured in vitro and safely cryopreserved in a solution routinely used for a wide variety of cells in our laboratory. Another purpose of this study was to generate transgenic mice using in vitro-transfected culture cells. In livestock animals, the use of somatic cell donors for nuclear transfer increases the possibility of transgenesis [2, 3, 20]. In this study, although immature Sertoli cells were successfully transfected and selected to produce transgenic embryos, no pups were obtained after embryo transfer. Some embryos seemed to develop until midgestation (Days 12–14). We do not know the cause of their death, but it is probable that they had chromosomal abnormalities, which might have been induced by prolonged in vitro culture, or exogenous gene integration, as reported in the conventional mouse transgenic procedure [21].
All but one pup born after Sertoli cell cloning were grossly normal, and their weights were within the normal range. This has also been the case in previous studies using cumulus cells [7] and tail tip cells [8]. The low rate of abnormal births is characteristic of mouse cloning using the Honolulu method. This is in sharp contrast with the cloning of domestic species, for which very high rates of peri- and postnatal death and developmental defects among cloned fetuses are reported [1, 4, 22]. In the mouse, most cloned embryos were lost shortly after implantation, as suggested by the very low number of fetuses present in the uterus at midgestation ([7, 8], and this study). Therefore, even though the majority of cloned mouse embryos develop into morulae or blastocysts, they are "abnormal" in some respects. The rejection of implanted embryos before their fetal development is probably stronger in the mouse than in domestic species, and thus only normal or nearly normal fetuses can develop further.
The fetuses and offspring obtained in this study had extraordinarily large placentas. This has also been reported after cloning with cumulus and tail tip cells [8]. Since most fetuses and offspring were healthy, these large placentas must have been functional. Histological examinations of the placentas did not reveal any apparent structural abnormalities except an irregular boundary between the layers of spongiotrophoblast and the labyrinth (unpublished results).
The development of normal fetuses and offspring after somatic cell cloning in mammals, especially mice, has important scientific implications. Mice are the most commonly used laboratory animals, and a vast amount of information is available on their biology and genetics. Therefore, the cloning of mice can be used as a new research tool to study basic biological phenomena such as reprogramming of the genome, aging, and genomic imprinting. For this, mice of both sexes should be obtained as efficiently as possible. The nuclear transfer of immature Sertoli cells, together with that of cumulus cells, may provide valuable information on basic biology.
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FOOTNOTES |
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1 Supported by grants from the Ministry of Education, Science, Sports and Culture, Japan, and the Ministry of Health and Welfare, Japan. 
2 Correspondence: Atsuo Ogura, Department of Veterinary Science, National Institute of Infectious Diseases, 1–23–1 Toyama, Shinjuku-ku, Tokyo 162–8640, Japan. FAX: 81 3 5285 1150; aogura{at}nih.go.jp
Accepted: December 31, 1999.
Received: November 9, 1999.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Y. Yamazaki, H. Makino, K. Hamaguchi-Hamada, S. Hamada, H. Sugino, E. Kawase, T. Miyata, M. Ogawa, R. Yanagimachi, and T. Yagi Assessment of the developmental totipotency of neural cells in the cerebral cortex of mouse embryo by nuclear transfer PNAS, October 31, 2001; (2001) 231489398. [Abstract] [Full Text] [PDF]
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K. Eggan, H. Akutsu, J. Loring, L. Jackson-Grusby, M. Klemm, W. M. Rideout 3rd, R. Yanagimachi, and R. Jaenisch Hybrid vigor, fetal overgrowth, and viability of mice derived by nuclear cloning and tetraploid embryo complementation PNAS, April 25, 2001; (2001) 101118898. [Abstract] [Full Text]
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K. Eggan, H. Akutsu, J. Loring, L. Jackson-Grusby, M. Klemm, W. M. Rideout 3rd, R. Yanagimachi, and R. Jaenisch Hybrid vigor, fetal overgrowth, and viability of mice derived by nuclear cloning and tetraploid embryo complementation PNAS, May 22, 2001; 98(11): 6209 - 6214. [Abstract] [Full Text] [PDF]
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Y. Yamazaki, H. Makino, K. Hamaguchi-Hamada, S. Hamada, H. Sugino, E. Kawase, T. Miyata, M. Ogawa, R. Yanagimachi, and T. Yagi Assessment of the developmental totipotency of neural cells in the cerebral cortex of mouse embryo by nuclear transfer PNAS, November 20, 2001; 98(24): 14022 - 14026. [Abstract] [Full Text] [PDF]
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