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BOR - Papers in Press, published online ahead of print May 4, 2005.
Biol Reprod 2005, 10.1095/biolreprod.105.041673
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BIOLOGY OF REPRODUCTION 73, 390–395 (2005)
DOI: 10.1095/biolreprod.105.041673
© 2005 by the Society for the Study of Reproduction, Inc.

Generation of Normal Progeny by Intracytoplasmic Sperm Injection Following Grafting of Testicular Tissue from Cloned Mice That Died Postnatally1

Hiroshi Ohta 2 , and Teruhiko Wakayama 

Laboratory for Genomic Reprogramming, Center for Developmental Biology, RIKEN Kobe Institute, Kobe, Hyogo 650-0047, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animal cloning by nuclear transfer has been successful in several species and was expected to become an alternative reproductive technique. Among the problems associated with this cloning technique, however, are its low success rate and high mortality of cloned animals even if they develop to term. Nuclear transfer has thus come to be considered too difficult to apply as a reproductive technique. The transplantation of male germ cells or pieces of testicular tissue has enabled the induction of spermatogenesis from fetal or postnatal male mice. In the present study, we examined whether functional male gametes could be obtained by the transplantation of pieces of testicular tissue from cloned mice that died immediately after birth with typical aberrant phenotypes, such as large offspring syndrome. Donor testicular tissues were retrieved from cloned mice that died postnatally and were transplanted into the testes of recipient nude mice. Two to three months after transplantation, the grafted donor testicular tissue had grown in the host testis, and histological analysis showed that spermatogenesis occurred within the graft. Intracytoplasmic sperm injection demonstrated that the testicular sperm generated in the grafted donor tissue were able to support full-term development of progeny. These results clearly showed that functional spermatogenesis could be induced by transplanting testicular tissue from cloned mice that died postnatally into recipient mice. The strategy presented here will be applicable to cloned animals of other species, because the xenografting of testicular tissue into mice has been demonstrated previously to be possible.

assisted reproductive technology, developmental biology, sperm, testis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animal cloning is the asexual reproduction of an individual such that the offspring have an essentially identical nuclear genome. Nuclear transfer and cloning have been achieved in a number of species, including sheep [1], mice [2], pigs [3, 4], cattle (both domestic and endangered) [5– 8], goats [9], and rabbits [10]. Successes also have been reported in the cloning of a cat [11], rat [12], mule [13], and horse [14]. Thus, cloning techniques are available for domestic as well as laboratory animals and have created new opportunities in research, medicine, and agriculture. However, the usefulness of these techniques is limited by their low success rates; the birth of cloned offspring is quite rare, with average cloning efficiency rates of just 1–3%, regardless of the species [15]. In addition, cloned animals often die before sexual maturity as a result of unknown abnormalities [16]. Two strategies have been considered to overcome this obstacle and apply cloning techniques as a means of artificial reproduction: improving the success rate of the cloning techniques themselves, and obtaining functional gametes from cloned animals even if they die before sexual maturity. In the present study, we focused on the latter approach to determine whether functional germ cell differentiation can be achieved from the germ cells of cloned mice that die postnatally.

Although several techniques are potentially available to induce gametogenesis from immature germ cells [17, 18], recent progress in transplantation techniques for male germ cells provides a promising option [18, 19]. A technique for the transplantation of spermatogonial stem cells into the mouse testis has been developed by injecting donor germ cells into the seminiferous tubules of recipient mice [18, 20]. In this system, the transplanted spermatogonial stem cells undergo normal spermatogenesis [20], and the recipients are capable of transmitting the donor haplotype to progeny [18]. Although the transplantation of spermatogonial stem cells into seminiferous tubules has become a powerful method in basic research as well as in the agricultural and medical fields [21–23], its technical limitations have been reported. For example, cross-species transplantation of spermatogonial stem cells from donor rats or hamsters resulted in spermatogenesis in the recipient mouse testis [24, 25], but the transplantation of germ cells from phylogenetically more distant species, including rabbit, dog, pig, bull, horse, and primate, into the mouse testis did not result in spermatogenesis beyond the stage of spermatogonial proliferation [26–28]. Our previous report also indicated that the transplantation of mouse fetal male germ cells into recipient seminiferous tubules was not effective in producing functional male gametes in the recipient testes [29].

Recently, a new method of generating male gametes through artificial techniques has been developed that involves grafting testicular tissue under the skin [19] or into the testis [30]. Grafting of testicular tissue from neonatal mouse, pig, goat, hamster, and marmoset into recipient mice revealed that the testicular grafts were able to maintain spermatogenesis [19, 30–32]. In addition, we previously demonstrated that functional male gametes can be obtained from fetal male gonadal tissue grafted into the testis [29] or under the skin [33]. Thus, the transplantation of testicular tissue provides a new approach to achieving maturation of mammalian male germ cells.

In the present study, we examined whether functional male gametes can be obtained from cloned mice that die postnatally by transplanting their testicular tissue into recipient testes and assessing the resulting male gametes using intracytoplasmic sperm injection (ICSI).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice

Immature male B6C3F1, adult female ICR, and adult female BDF1 mice were purchased from Shizuoka Laboratory Animal Center (Hamamatsu, Japan). Female 129/Sv-ter and male ICR nude mice were purchased from CLEA Japan, Inc., and Charles River Japan, Inc., respectively. The green fluorescent protein (GFP) transgenic mice carrying both the acrosin/eGFP (Acr3-EGFP) [34] and pCX-eGFP [35] transgenes (C57BL/ 6TgN(acro/act-EGFP)OsbC3-N01-FJ002 [36]) were kindly provided by Dr. M. Okabe (Osaka University, Osaka, Japan; all strain designations are those of the original studies). To generate 129B6F1 mice carrying the GFP transgene, female 129/Sv-ter mice were mated with male C57BL/6 GFP transgenic mice, and the offspring of these matings, which were hemizygous for the GFP transgene, were used as donors for nuclear transplantation. All animal experiments conformed to the Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Committee of Laboratory Animal Experimentation of the RIKEN Kobe Institute.

Preparation of Donor Cells for Nuclear Transfer

To obtain cloned mice, immature Sertoli cells or embryonic stem (ES) cells were used as donors for nuclear transplantation [37, 38]. Briefly, the testes from immature 3- to 10-day-old male GFP transgenic 129B6F1 mice or B6C3F1 mice were decapsulated, and the seminiferous tubules were placed in Hanks solution containing 0.25% trypsin and 1 mM EDTA (Gibco, Grand Island, NY) at 37°C for 10 min with manual agitation at 5-min intervals. After adding a sufficient volume of Dulbecco modified Eagle medium (DMEM) containing 10% fetal bovine serum to neutralize the trypsin, the cell suspension was washed twice with PBS and then resuspended in DMEM. The cell suspension was stored at 4°C until nuclear transplantation.

The GR14 ES cell line was previously established in our laboratory from the tail-tip cells of male 129/Sv x BDF1 mice via nuclear transfer and was used in the present study as donors for producing cloned mice derived from ES cells.

Nuclear Transfer

The reconstruction of embryos by nuclear transfer was performed according to the method described by Wakayama et al. [2] with slight modifications. Briefly, mature BDF1 female mice were induced to superovulate by the intraperitoneal injection of 5 IU of eCG (Teikokuzoki Co., Tokyo, Japan), followed 48–52 h later by injection of 5 IU of hCG (Teikokuzoki). Mature oocytes were collected from the ampullary region of the oviducts 13–15 h after the hCG injection. The oocytes were freed from the cumulus cells by treatment with 0.1% bovine testicular hyaluronidase (Sigma, St. Louis, MO) in Hepes-CZB medium, rinsed, and kept in fresh CZB medium at 37°C under 5% CO2 until injection.

A group of oocytes (usually 20) was transferred to a droplet (~10 µl) of Hepes-CZB containing 5 µg/ml of cytochalasin B, which previously had been placed under mineral oil in the operation chamber on the microscope stage. While an oocyte was held by a pipette, its zona pellucida was drilled by applying several piezo pulses to the tip of an enucleation pipette (inner diameter, 9–10 µm) ([2]). The metaphase II chromosome-spindle complex, distinguished as a translucent spot in the ooplasm, was drawn into the pipette with a small amount of accompanying ooplasm and then pulled gently away from the oocyte until the stretched cytoplasmic bridge was pinched off. After the oocytes in one group were enucleated, which took approximately 10 min, they were transferred into cytochalasin B-free CZB and held for as long as 2 h at 37°C.

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 of individual enucleated oocytes using a piezo-driven micropipette. The injected oocytes were incubated in CZB medium for 0.5–2.5 h and were activated by treatment in Ca2+-free CZB medium containing 10 mM SrCl2 and 5 mg/ml of cytochalasin B for 6 h. After washing, the oocytes were cultured in CZB medium for 24 h. All incubations were at 37°C under 5% CO2 in air.

The 2-cell embryos obtained were transferred into the oviducts of pseudopregnant ICR females at 0.5 days postcoitus (dpc). Seven to ten embryos were transferred into each oviduct. The recipient females were killed on Day 18.5 to obtain the cloned mice by cesarean section. A cloned mouse was considered to be dead at birth if it did not initiate or sustain respiration after birth. In some cases, cloned mice were found dead on the day following the cesarean procedure even if respiration had been normal at delivery; these mice also were used as donors for testicular grafting.

Testicular Tissue Transplantation

Donor testes were retrieved from cloned mice that exhibited postnatal death. The tunica albuginea was removed, and the testis was cut in half with fine scissors and stored in DMEM at 4°C until transplantation.

To avoid immunological rejection and to remove endogenous germ cells, 8-wk-old male ICR nude mice, which had been treated with busulfan (40 mg/kg) at 4 wk of age, were used as recipients. To increase the survival probability of the recipient mice, they were given bone marrow transplantations from untreated nude mice 4–5 days after the busulfan treatment. For the testicular tissue transplantation, the recipient mouse was anesthetized, and the testis was exteriorized through a midline abdominal incision. Using fine scissors, a small cut was made in the tunica albuginea, and a single piece of donor testicular tissue was inserted through the incision. The grafts were analyzed at 2–3 mo after transplantation.

Cryopreservation of Donor Testicular Tissue

Donor testes were retrieved from cloned mice that died postnatally. The tunica albuginea was removed, and the testis was cut into quarters with fine scissors, then placed into a cryogenic vial containing cryopreservation solution containing 10% dimethyl sulfoxide (BANBANKER; Japan Genetics, Tokyo, Japan). The vial was held at 4°C for 10 min and stored at –80°C for 7–10 days. For transplantation, the vials were incubated at 37°C, and the testicular tissue was washed three times with DMEM and stored at 4°C until transplantation.

Intracytoplasmic Sperm Injection

The microinsemination of ICR oocytes collected from superovulated females was performed using ICSI [39]. A donor testicular cell suspension was prepared from the grafted testicular tissue in the recipient mice 2–3 mo after the transplantation. Briefly, the grafts were dissected from the recipient testes, minced with fine scissors, and resuspended in DMEM to obtain testicular sperm suspensions. A single testicular sperm head was injected into each oocyte using a piezo-driven micropipette. After ICSI, the oocytes were incubated in CZB medium at 37°C under 5% CO2 in air. The 2-cell embryos that developed were transferred into the oviducts of 0.5-dpc pseudopregnant ICR females.

Histological Analysis

For histological analysis, some recipient testes were dissected from the recipient mice and fixed in 4% paraformaldehyde overnight at 4°C. After washing with PBS for 4 h, the tissues were placed in acetone for 1 h and then embedded in glycol methacrylate (Technovit 8100; Kulzer, Wehrheim, Germany). Serial cross-sections (thickness, 5 µm) were cut at 50-µm intervals and stained with hematoxylin.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Mortality of Mice Cloned from Somatic or ES Cells

Cloned mice were generated by nuclear transfer using immature Sertoli cells from B6C3F1 or 129B6F1 mice or GR14 ES cells as nuclear donors, and 3–7% of the transferred blastocysts developed to term, which was similar to the success rates reported in previous studies [2, 38, 40]. Of the 36 cloned mice generated from somatic or ES cells, 15 died immediately after cesarean birth or were found dead the next day (Table 1). The cloned mice generated from somatic cells (B6C3F1 or 129B6F1 Sertoli cells) that died at birth or shortly thereafter tended to have lower body weights compared to their live counterparts (Table 1), suggesting that mortality was associated with growth retardation. In contrast, postnatal death in cloned mice derived from ES cells was associated with large offspring syndrome (Fig. 1A and Table 1), which is caused by unknown epigenetic errors. No obvious differences were seen in the placentas of the cloned mice regardless of postnatal death or survival, but the placentas of all cloned mice were much larger than those of control mice (Table 1). Histological analysis indicated that undifferentiated germ cells were present in the testes of cloned mice, including those that exhibited large offspring syndrome (Fig. 1B).


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  		TABLE 1. Generation of cloned mice via nuclear transfer from embryonic stem cells or somatic cells



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  		FIG. 1. Cloned mice with large offspring syndrome generated by nuclear transfer from GR14 ES cells. A) Cloned mice with large offspring phenotype (left, center) and a live offspring (right) derived from the GR14 ES cell line. B) A cross-section of the testis of a newborn cloned mouse. Bar = 50 µm

Induction of Spermatogenesis by Testicular Tissue Transplantation from Cloned Mice that Died Postnatally and Generation of Progeny via ICSI

To examine whether functional spermatogenesis could be induced by transplantation of testicular tissue from cloned mice that died postnatally, one mouse clone derived from immature Sertoli cells and four derived from ES cells (all four with large offspring syndrome) were used for transplantation (Table 2). All the cloned mice died immediately after birth because of respiratory failure. Testicular tissues were retrieved from the mice after death, and four pieces of donor testicular tissue from each mouse were transplanted under the tunica of the testes of recipient ICR nude mice. Two to three months after the transplantation, the grafts had grown within the recipient testes (Fig. 2A). Differentiated testicular sperm were found in at least half of the four grafted tissue specimens from each donor mouse, although some grafts had no testicular sperm (Fig. 2, B and C, and Table 2).


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  		TABLE 2. Induction of spermatogenesis from cloned mice that died postnatally by transplantation of testicular tissue and production of progeny via ICSI using testicular sperm recovered from the grafts.a



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  		FIG. 2. Transplantation of pieces of testicular tissue from cloned mice that died postnatally into the testes of nude mice. A) A stereomicroscopic picture of the recipient testis at 2 mo after transplantation of testicular tissue. An arrowhead indicates the grafted tissue. B) A cross-section of the recipient testis shown in A. The grafted tissue is indicated with a dashed line. C) A higher magnification of the cross-section shown in B. A seminiferous tubule from the area in the square in B is shown. Bar = 1 mm (A and B) and 100 µm (C)

The functionality of the differentiated testicular sperm in the grafts (Fig. 3A) was investigated by ICSI. Of the resulting 2-cell embryos, 304 were transferred into the oviducts of pseudopregnant females, and 99 normal pups (33%) were born (Fig. 3B and Table 2). The coat color of the progeny indicated that 98% were derived from donor testicular sperm and 2% from endogenous testicular sperm from the transplant hosts. The progeny did not exhibit signs of the typical phenotypes of cloned mice; the placentas were of normal size (± SD; 0.17 ± 0.03 g, n = 37), and the body weights also were normal (1.55 ± 0.2 g, n = 37). Normal fertility was confirmed by matings between some of the ICSI-derived progeny (data not shown). These results indicated that functional spermatogenesis from cloned mice that died at birth was inducible by means of testicular tissue grafting even if the cloned mouse exhibited one of the typical abnormal phenotypes of clones, such as large offspring syndrome.



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  		FIG. 3. Generation of progeny by ICSI of the differentiated testicular sperm in the grafted tissue. A) Testicular sperm prepared from the grafted tissue used for ICSI. B) Normal progeny obtained via ICSI. Bar = 50 µm

Induction of Spermatogenesis from Cryopreserved Testicular Tissue

To investigate further the possibilities of testicular tissue grafting, cryopreserved testicular tissues from six cloned mice that died postnatally were stored at –80°C for 7–10 days before being thawed and transplanted into recipient testes. For each donor mouse, eight pieces of testicular tissue were transplanted into the eight testes of four nude recipient mice. Two months after the transplantation, the testicular tissue had grown within the recipient testes, but the induction of spermatogenesis was inefficient compared to that achieved with fresh donor tissues. Differentiated testicular sperm were obtained from the testicular grafts derived from three of the six donors (Table 3). Using ICSI, we succeeded in producing normal progeny from two of the three donors from which differentiated testicular sperm were obtained after transplantation. These results demonstrated that functional spermatogenesis could be induced by transplanting cryopreserved testicular tissue, although the recovery of functional sperm production from frozen testicular tissues was less than that from fresh testicular tissue.


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  		TABLE 3. Transplantation of cryopreserved testicular tissue and production of progeny via ICSI using testicular sperm recovered from the grafts.a


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The production of cloned animals by nuclear transfer from somatic cells is a promising technique in animal biotechnology. Despite numerous reports of the successful production of viable offspring in various species, the efficiency of the technique remains very low [15]. In addition, live-born cloned offspring exhibit high mortality [16]. Thus, although cloning is expected to become an alternative technique for artificial reproduction, the problem of high pre- and postnatal mortality needs to be resolved or bypassed before cloning can be applied routinely for animal reproduction. In the present study, we demonstrated that functional spermatogenesis could be induced by transplanting pieces of testicular tissue from cloned mice that died postnatally into the testes of recipient nude mice even if the donor exhibited an aberrant phenotype typical of cloned animals, such as large offspring syndrome. The technique presented here likely is usable for other animal species as well, because xenografting testicular tissue into recipient nude mice has been demonstrated to be possible.

Recently, transplantation techniques for the induction of male germ cell differentiation have been established in the mouse [18, 19]. The reported transplantation systems can be classified into two types: the transplantation of germ cells into the seminiferous tubules of the recipient mice [18, 20], and the transplantation of pieces of testicular tissue into the testes [30] or under the skin [19] of the recipients. Using the former technique, the transplanted spermatogonia can proliferate and differentiate, along with normal spermatogenesis in the testis, and continuous production of mature sperm from the transplanted spermatogonia can be achieved [18, 20]. However, this technique is not appropriate for xenogeneic transplantation; spermatogonia prepared from phylogenetically distant species cannot differentiate into mature sperm after transplantation into recipient mouse testes because of defects in the interactions between the Sertoli cells of the recipient mouse and the transplanted xenogeneic germ cells [26–28]. On the other hand, the transplantation of pieces of testicular tissue can induce functional spermatogenesis from phylogenetically distant species in the recipient mouse [19, 30–32], but achieving continuous production of sperm in the graft appears to be difficult [19]. In addition, we previously reported that functional spermatogenesis from fetal male germ cells could be induced using this technique [29, 33], suggesting that the strategy in the present study also may be feasible for cloned animals that exhibit prenatal mortality. Furthermore, it has been reported that pieces of cryopreserved testicular tissue could initiate spermatogenesis after transplantation into recipient testes [30], indicating that long-term preservation of donor tissue is possible. Thus, testicular tissue grafting into mice is a possible method for the induction of spermatogenesis from different species and for enabling functional sperm to be obtained from donors even if they die before sexual maturity.

In the present study, cloned mice derived from ES cells that developed large offspring syndrome were used as a model for a cloned animal that exhibits postnatal mortality. Several obvious abnormalities, such as large placentas, obesity, and large offspring, have been reported to occur in cloned mice (for review, see [15]). Interestingly, in the present study, large offspring syndrome often was observed in clones produced by nuclear transfer from ES cell donors, whereas this phenotype was not observed when somatic cells were used as nuclear donors (Table 1). Cloned mice with large offspring syndrome usually died just after birth as a result of respiratory failure. Our results demonstrated that spermatogenesis occurred in the grafted tissue and that normal progeny could be obtained by ICSI using the recovered testicular sperm. The progeny thus generated did not exhibit large offspring syndrome or have large placentas, suggesting that the epigenetic errors observed in the donor cloned mice were not transmitted to their progeny, at least not as a dominant phenotype. The epigenetic errors in the cloned mice probably were corrected during germ cell differentiation, as has been reported previously [41, 42], even if spermatogenesis was induced artificially by transplantation, although further investigation is needed to confirm this hypothesis. Thus, we succeeded in obtaining normal progeny via ICSI using the testicular sperm obtained from cloned mice by transplantation even when the original clones died because of severe epigenetic errors.

Although we succeeded in inducing spermatogenesis from pieces of cryopreserved testis (Table 3), the rate of recovery of testicular sperm after transplantation was lower than that in the previous report [30]. This inconsistency probably is attributable to differences in the preservation procedures, such as the conditions for tissue freezing or the cryopreservation solution used.

The findings of the present study indicate that functional male gametes from cloned mice that died postnatally can be obtained by testicular tissue grafting and that normal progeny can be obtained by ICSI. To the best of our knowledge, this is the first report to demonstrate that testicular tissue grafting is feasible for generating progeny from mice that die postnatally.


   FOOTNOTES
 
1 Supported by a Grant-in-Aid for Creative Scientific Research (13GS0008) and grants from the Scientific Research in Priority Areas (15080211) and Young Scientists A (15681014) programs as well as from the Project for the Realization of Regenerative Medicine (research filed for technical development of stem cell manipulation) to T.W. from the Ministry of Education, Science, Sports, Culture, and Technology of Japan. H.O. is the recipient of a research fellowship from the Special Postdoctoral Researchers Program. Back

2 Correspondence: Hiroshi Ohta, Laboratory for Genomic Reprogramming, Center for Developmental Biology, RIKEN Kobe Institute, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan. FAX: 81 78 306 3095; ohta{at}cdb.riken.go.jp Back

Received: 8 March 2005.

First decision: 29 March 2005.

Accepted: 26 April 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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