| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Regular Article |
a State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, China
b Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China
c Fuzhou Giant Panda Research Center, Fuzhou 350001, China
d China Research and Conservation Center for the Giant Panda, Wolong Nature Reserve, Wenchuan, Sichuan 623006, China
e Guangzhou Medical College, Guangzhou 510182, China
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
developmental biology, early development, embryo, implantation, pregnancy
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Several studies have shown that oocyte cytoplasm from bovines, rabbits, and sheep can support early development of embryos produced by nuclear transfer of somatic cell nuclei from various mammalian species [3–6]. Recently, the successes of cloning gaur [6, 7] and mouflon [8] have demonstrated that the technique of interspecies cloning can be practically applied to save highly endangered species, such as the giant panda.
Four issues need to be assessed in giant panda cloning. First, whether somatic cell nuclei of giant panda are able to dedifferentiate in the ooplasm from another species and support early development of the reconstructed eggs; second, whether interspecies cloned embryos are able to implant in the uterus of a species other than the giant panda; third, whether interspecies cloned embryos are able to develop normally to term in the uterus; and fourth, to examine the contribution of mitochondria from the recipient oocytes and donor cells to the nucleo-ooplasm hybrids. Previous studies in our laboratory showed that giant panda somatic cell nuclei could dedifferentiate in rabbit ooplasm and support early development of reconstructed eggs [3]. In the present work, we observed the possibility of interspecies implantation after transferring panda-rabbit cloned embryos to the uterus of rabbits or domestic cats, and examined mitochondria transformation of the panda-rabbit cloned embryos before and after implantation.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Animal care and handling were conducted in accordance with policies on the care and use of animals promulgated by the ethical committee of the State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences. Female Japan Big Eared white rabbits (purchased from the Laboratory Animal Center, Institute of Zoology, Chinese Academy of Sciences) were housed in stainless steel cages, and were fed regular rabbit fodder and water ad libitum. Domestic cats (Felis catus) (also purchased from the Laboratory Animal Center) were housed in a room with a 12L:12D light schedule at 20–26°C. Commercial cat food was given two times a day, and the animals had ad libitum access to water. The collection and use of giant panda tissues were approved by the Office of Wildlife Protection, National Forestry Bureau, China.
Culture of Donor Cells
Cell culture and assessment procedures have been described previously [9]. Briefly, abdominal muscles were collected within 30 min after the death of a 3-day-old triplet female panda. Tissues were cut into pieces and digested with 0.25% trypsin (Gibco BRL, Grand Island, NY) for 30 min at 37°C. The digested cells and tissues were cultured in Dulbecco modified Eagle medium/F12 (Gibco) supplemented with 20% fetal bovine serum (Gibco) in a 5% CO2 incubator at 37°C. Cells were passaged at 70%–80% confluence. The primary spindle-shaped cells, confirmed as fibroblasts by immunochemical staining of vimentin proteins, were isolated for further culture. Cells at passage generations 3–8 were used as donors. The serum concentration was decreased to 0.5% to starve the donor cells for 3–5 days before nuclear transfer.
Domestic cat uterus epithelium was collected from a 3-yr-old female by hysterectomy after anesthetizing the animal with an i.m. injection of 1 ml of "846," a mixture of anesthetics used for sedation, analgesia, and muscle relaxation (Changcun Agricultural and Animal Husbandry University, China). Cell culture was performed by the same method as described above.
Preparation and Enucleation of Recipient Oocytes
Female Japan Big Eared white rabbits were superovulated by administering FSH and hCG (Institute of Zoology, Academia Sinica). Each rabbit was injected i.m. with 1 mg of FSH two times daily for 3 days and with 100 IU of hCG i.v. 12 h after the last FSH injection. Rabbits were killed 14 h after hCG injection. Cumulus masses were collected by flushing the separated oviducts with M2 medium (Sigma Chemical Company, St. Louis, MO) and were treated shortly thereafter with 300 IU/ml of hyaluronidase (Sigma) in M2 medium to remove the cumulus cells. After three washings in M2 medium, the cumulus-free eggs were transferred to M2 medium containing 7.5 µg/ml of cytochalasin B (Sigma), 7.5 µg/ml of Hoechst 33342 (Sigma) and 10% FBS for 10 min, and the eggs were then used for micromanipulation. For enucleation, a small amount of cytoplasm from the area beneath the first polar body was aspirated using a 20- to 25-µm glass pipette, and then the aspirated karyoplast was exposed to UV light to confirm the presence of a nucleus. Only the oocytes from which the chromosomes were removed were used for nuclear transfer [3, 10].
Nuclear Transfer and Activation
A single cell was placed in the perivitelline space in close contact with the plasma membrane of an enucleated oocyte. The couplets were transferred to a fusion chamber containing 100 µl of fusion medium (0.25 M sorbitol, 0.5 mM Mg(CH3COO)2, 0.1 mM Ca(CH3COO)2, 0.5 mM Hepes, and 100 mg/100 ml of BSA in deionized water [11]. Fusion was induced by double DC pulses of 1.4 kV/cm for 80 µsec with an ECM2001 Electrocell Manipulator (BTX Inc., San Diego, CA). Couplets were then washed three times in M199 (Gibco) supplemented with 10% FBS, and incubated in the same medium for 30 min at 38°C in humidified air containing 5% CO2. Couplets were checked for fusion under an inverted microscope, and fused couplets were activated by double DC pulses of 1.4 kV/cm for 40 µsec, and cultured overnight in M199 supplemented with 10% FBS [3].
Synchronization of Recipients and Embryo Transfer
Healthy female domestic cats at 2–6 yr of age were selected for synchronization. Each cat was injected (i.m.) with 0.3 mg of FSH on the first day, 0.15 mg of FSH for the next three successive days, and 100 IU of hCG on the fifth day. Embryo transfer was conducted on the seventh day after the first FSH injection. Each recipient was anesthetized with 0.3–0.4 ml/kg (body weight) of "846" (Changcun Agricultural and Husbandry University, China) mixture before laparotomy. Panda-rabbit and cat-rabbit cloned embryos at the 2- to 4-cell stage were transferred into the oviduct of a recipient cat. Each recipient received an injection of 50 IU of LH (Institute of Zoology, The Chinese Academy of Sciences) after embryo transfer.
Recipient rabbits were also synchronized by injecting FSH and hCG. Each rabbit received 0.15 mg/day of FSH for three successive days, and an injection of 75 IU of hCG on the fourth day, when the vagina was stimulated using a sterile glass probe at the time of hCG injection. Embryo transfer was conducted 24 h after hCG injection. Recipient rabbits were anesthetized with 0.2–0.4 ml/kg (body weight) of 846 before laparotomy. Panda-rabbit cloned embryos at the 2-cell stage were transferred into the oviduct, and blastocysts were transferred into the uterus of synchronized rabbits [10].
Microsatellite DNA and Mitochondrial DNA Analysis
The specific primers of the giant panda microsatellite DNA, which had been previously validated [12] were p24a, 5'-ATGCATGACATTTTGGGTAGC-3' and p24b, 5'-TGAAGACCCTAGATGAAGGCA-3'. Amplification was performed at 95°C for 3 min, 94°C for 45 sec, 55°C for 45 sec, 72°C for 45 sec for 40 cycles, and at 72°C for 5 min. In order to rule out the possibility of contamination during polymerase chain reaction (PCR) processing, we designed a series of negative controls in PCR reactions. C1 was designated as the reaction system that contained all enzymes and substrates except the template, and C12 was the product of C1 in the first amplification and the template in the second amplification. C2 was the negative control in the second amplification. The final amplification products were separated via agar gel electrophoresis and sequenced by an automatic DNA sequencer (ABI 377) [12, 13].
We analyzed a region of the mitochondrial DNA (mtDNA) D-loop by a specific PCR with two sets of primers. One set was specific to panda DNA and the other set was specific to rabbit DNA. The sequences of specific primers for panda mitochondrial DNA were PDL236, 5'-ACTCATTACAAGAACTTATT-3'; and gH1142, 5'-CGGAGCGAGAAGAGGTACACGTAC-3', respectively. The rabbit-specific primers for amplifying mitochondrial DNA were HDH430, 5'-GGTAAGTTGTGAGAGGACTGG-3'; and HdL319, 5'-CATCAATTCCATAATTAAAC-3'. The final PCR products were separated via agar gel electrophoresis and were also sequenced by an automatic DNA sequencer (ABI 377).
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
We previously reported that the developmental potential of panda-rabbit cloned embryos was correlated to the donor cell types. Blastocyst rates for uterine epithelium, skeletal muscle, and mammary gland cells from a 12-yr-old panda were 9.9%, 6.8%, and 11.7%, respectively [3]. In this experiment, the blastocyst rate for panda-rabbit interspecies cloned embryos (18.5%) improved significantly when fibroblasts from a 3-day-old infant panda were used (Table 1). These panda-rabbit cloned embryos reached the blastocyst stage on the fifth day and escaped from the zona pellucida on the seventh day, when they were cultured in vitro.
|
Cat uterine epithelial cells were transferred into enucleated metaphase II rabbit oocytes. Only 48.7% of nuclear transferred oocytes could be fused, and 5.2% of these fused couplets could develop to blastocysts when they were cultured in M199 + 10% FBS (Table 1). Cat-rabbit cloned embryos normally reached the blastocyst stage 7 days after activation.
Rabbits as Recipients of Panda-Rabbit Cloned Embryos
Approximately 2300 panda-rabbit cloned embryos were transferred into 100 recipient rabbits, but none of the recipients were found to be pregnant. We examined the uteri of recipient rabbits 20–30 days after embryo transfer, but no embryo implantation site was found in any of the 30 recipients examined. Previously, we had obtained viable germinal vesicle transfer rabbits using the same embryo transfer protocol that was used in this experiment [10]. Thus, even though panda-rabbit embryos failed to implant in rabbit uteri, the cause was not an improper embryo transfer protocol.
Domestic Cats as Recipients of Panda-Rabbit Cloned Embryos
Panda-rabbit reconstructed embryos at the 2- to 4-cell stage, together with cat-rabbit interspecies cloned embryos, were transferred into oviducts of 21 cats. Each recipient cat received about 10 panda-rabbit embryos and 10 cat-rabbit embryos. Fourteen recipient cats exhibited estrus within 35 days, and five recipients exhibited estrus after 43–48 days. Two recipients died of pneumonia 21–22 days after embryo transfer; autopsy revealed that the one that died on the 21st day after embryo transfer had been pregnant, with six early fetuses, four of which were found on one side of the uterine horn, and two on the other side. Pregnant luteins were distinctly present on both ovaries. Figure 1 shows these fetuses, numbered 1 to 6. Fetuses were frozen in liquid nitrogen and then dissected. Tissues around the uterine lacuna were scratched out and microsatellite DNA was analyzed by PCR using a specific panda microsatellite primer. The panda microsatellite DNA was detected in fetuses 3 and 5 (Fig. 2), whereas the other four fetuses were negative (Fig. 3). Sequences of the PCR product for fetuses 3 and 5 were identical and matched the DNA sequences from the donor panda cells (Fig. 4).
|
|
|
|
Analysis of Mitochondrial DNA
Fetuses 3 and 5 obtained from the cat uterus and panda-rabbit cloned embryos at the blastocyst stage were used to evaluate mitochondrial DNA. Mitochondrial DNA from the panda could be detected in fetuses 3 and 5, as well as in those panda-rabbit cloned blastocysts. PCR products for fetus 3, fetus 5, and giant panda somatic cells were sequenced and shown to be identical in this 82-base pair fragment (Fig. 5). However, rabbit mitochondrial DNA was found only in blastocysts, but was not detected in fetuses 3 and 5 (Fig. 6).
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Domestic cats were selected as the recipients of panda-rabbit interspecies cloned embryos because cats and pandas are carnivores. In addition, the gestation period for domestic cats is 62–71 days [14], whereas the giant panda has a delayed implantation, in which the gestation period varies from 80 to 150 days. The gestation periods of these two species are relatively close, compared with that of rabbits, in which the gestation period is only about 30 days. Moreover, interspecies embryo transfer was successfully conducted in domestic cats [15, 16]. In a preliminary experiment, we found that the somatic cell nuclei of the domestic cat can dedifferentiate in enucleated metaphase II rabbit oocytes, and these interspecies reconstructed embryos can also implant in cat uterus. We hypothesize that giant panda embryos play an inactive role during implantation for their delaying implantation characteristics; the cat-rabbit embryos may induce or help implantation of giant panda-rabbit embryos in cat uterus. Thus, the experiment of cotransfer of panda-rabbit embryos and cat-rabbit embryos into cat oviducts was designed. The present results demonstrate that it is possible to achieve interspecies implantation for panda-rabbit cloned embryos in domestic cats. The cotransfer of interspecies embryos cloned by transferring domestic cat cells into enucleated rabbit oocytes may contribute to successful implantation of panda embryos in cat uterus. Cat-rabbit embryos may act as implantation inducers or helpers for panda-rabbit cloning embryos. During embryo implantation, the signals from embryos to a recipient uterus for implantation are controlled by the nucleus, and these signals may be species-specific. Cat-rabbit cloned embryos may give implantation signals to recipient cats and trigger the process of implantation. Nevertheless, more evidence for the benefit of embryo cotransfer in interspecies implantation are needed; further experiments will be carried out to verify this hypothesis in our laboratory.
The preimplantation embryo migration from one side of the uterine horn to the other side often occurs in domestic cats [17, 18]. This characteristic allows us to transfer all embryos into just one side of the oviduct or uterine horn. We transferred seven panda-rabbit embryos and 11 cat-rabbit embryos at the 2-cell stage from the left oviduct into this host cat, and found only four fetuses on the left side uterine horn and two fetuses on the right side. Structures such as placentae were not observed in these fetuses, and we speculate that these fetuses might be still in their very early stage and developed only about 10 days after implantation, because normal cat embryos start to implant on the 13th day after mating [17, 19].
In interspecies cloning, the fate of mitochondria from donor cells and from recipient oocytes is unclear. Mitochondria are semiautonomous organelles that contain a circular DNA (mtDNA) of about 16–17 kilobases. The mtDNA encodes only 13 proteins, 22 tRNAs, and 2 rRNAs. Up to 95% of proteins involved in biogenesis and functions of mitochondria are encoded by the nucleus [20, 21]. The number of mitochondria in a typical cell is about 2 x 103, whereas the number of mitochondria in a single oocyte is about 2 x 105 [22]. In the process of somatic nuclear transfer, mitochondria of donor somatic cells, together with the nucleus, are transferred to the recipient oocyte. Thus, the cloned embryo should harbor mitochondria from both the donor cell and the recipient oocyte. In intraspecies cloned animals, mitochondria are derived primarily from recipient oocytes [20, 21, 23–25], and mitochondria from donor cells appeared to be rapidly eliminated during the first few mitotic divisions and were hardly detectable by the blastocyst stage [23–27]. Only in some cases do mitochondria from both donor cells and recipient oocytes coexist [20, 21, 28]. In genetically close interspecies cloned animals, such as the gaur and mouflon [6, 8, 29], mitochondria were also exclusively derived from recipient oocytes. However, our results suggested a possible different pattern of mitochondrial transformation in interspecies cloning. The mitochondria from donor panda cells and those from recipient rabbit oocytes coexist in embryos before implantation, whereas mitochondria from donor panda cells remain detectable and mitochondria from the recipient rabbit oocytes are eliminated after implantation. A possible interpretation of this result is that pandas and rabbits are genetically further correlated, and nuclei from giant panda donor cells support biogenesis of mitochondria from panda cells, but they do not support those from rabbit oocytes. Anyway, this is the only report showing that mitochondria from donor cells substitute those from recipient oocytes in interspecies cloned embryos; whether this is a general phenomenon of mitochondria transformation needs further clarification.
In conclusion, our results suggest that 1) giant panda somatic cell nuclei can dedifferentiate in enucleated rabbit oocytes; 2) interspecies cloned panda-rabbit embryos can implant in the uterus of a third species, the domestic cat; and 3) mitochondria from donor cells may gradually substitute those from recipient oocytes in interspecies cloned embryos.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
1 This research was supported by Climbing special grant 08 of the Climbing Project from the Ministry of Science and Technology, China; and by grants KSCX1-05-01 and KSCX2-SW-303 from the Knowledge Innovation Project of the Chinese Academy of Sciences. 
2 Correspondence: Da-Yuan Chen, State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, 19 Zhongguancun Road, Haidian, Beijing 100080, China. FAX: 86 10 62565689; chendy{at}panda.ioz.ac.cn
3 These authors contributed equally to this work
Accepted: March 18, 2002.
Received: January 17, 2002.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  S. J. Kang, J. W. Choi, S. Y. Kim, K. J. Park, T. M. Kim, Y. M. Lee, H. Kim, J. M. Lim, and J. Y. Han Reproduction of Wild Birds via Interspecies Germ Cell Transplantation Biol Reprod, November 1, 2008; 79(5): 931 - 937. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. K Berg, C. Li, G. Asher, D. N Wells, and B. Oback Red Deer Cloned from Antler Stem Cells and Their Differentiated Progeny Biol Reprod, September 1, 2007; 77(3): 384 - 394. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. J. Bowles, J.-H. Lee, R. Alberio, R. E. I. Lloyd, D. Stekel, K. H. S. Campbell, and J. C. St. John Contrasting Effects of in Vitro Fertilization and Nuclear Transfer on the Expression of mtDNA Replication Factors Genetics, July 1, 2007; 176(3): 1511 - 1526. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E.C. Spikings, J. Alderson, and J.C.St. John Transmission of mitochondrial DNA following assisted reproduction and nuclear transfer Hum. Reprod. Update, July 1, 2006; 12(4): 401 - 415. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S.-Z. Liu, L.-J. Yao, M.-X. Jiang, Z.-L. Lei, L.-S. Zhang, Y.-L. Zhang, Q.-Y. Sun, Y.-L. Zheng, X.-F. Song, and D.-Y. Chen Apoptosis in rabbit embryos produced by fertilization or nuclear transfer with fibroblasts and cumulus cells Reproduction, September 1, 2005; 130(3): 359 - 366. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Takeda, M. Tasai, M. Iwamoto, A. Onishi, T. Tagami, K. Nirasawa, H. Hanada, and C. A. Pinkert Microinjection of Cytoplasm or Mitochondria Derived from Somatic Cells Affects Parthenogenetic Development of Murine Oocytes Biol Reprod, June 1, 2005; 72(6): 1397 - 1404. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C.-X. Yang, Z.-H. Kou, K. Wang, Y. Jiang, W.-W. Mao, Q.-Y. Sun, H.-Z. Sheng, and D.-Y. Chen Quantitative analysis of mitochondrial DNAs in macaque embryos reprogrammed by rabbit oocytes Reproduction, February 1, 2004; 127(2): 201 - 205. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. C. Gomez, J. A. Jenkins, A. Giraldo, R. F. Harris, A. King, B. L. Dresser, and C. E. Pope Nuclear Transfer of Synchronized African Wild Cat Somatic Cells into Enucleated Domestic Cat Oocytes Biol Reprod, September 1, 2003; 69(3): 1032 - 1041. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
