CLONING AND STEM CELLS Volume 10, Number 4, 2008 Mary Ann Liebert, Inc. DOI: 10.1089/clo.2008.

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Cotransfer of Parthenogenetic Embryos Improves the Pregnancy and Implantation of Nuclear Transfer Embryos in Mouse
Qinggang Meng,1 Minkang Wang,1 Claudia Ana Stanca,1 Szilard Bodo,1 and Andras Dinnyes1,2

Abstract

The majority of somatic cell nuclear transfer (SCNT) clones dies in the peri- or postimplantation period. Improvement of the full-term healthy pregnancy rates is a key issue for the economical viability and animal welfare profile of SCNT technology. In this study the effects of cotransfer of parthenogenetic or fertilized embryos on the pregnancy and implantation of SCNT mouse embryos have been investigated. SCNT embryos were produced by transferring cumulus cell nuclei into enucleated B6D2F1 mouse oocytes, whereas parthenogenetically activated (PA) and fertilized embryos were derived from ICR mice by artificial activation with strontium and in vivo fertilization, respectively. SCNT embryos were inferior in their developmental capacity to blastocyst compared to either PA or fertilized embryos. SCNT embryos were transferred alone (SCNT), or cotransferred with two to three PA (SCNT PA) or fertilized (SCNT Fert) embryos into the oviducts of an ICR recipient. Both pregnancy and implantation rates originating from clones in the SCNT PA group were significantly higher than those of SCNT group (p 0.05). The weight of placentas of clones derived from SCNT, SCNT PA, or SCNT Fert was in all cases significantly higher than that of fertilized controls (p 0.001). Most of the clones derived from SCNT embryos cotransferred with PA or fertilized embryos survived to adulthood and were fertile and healthy according to histopathological observations. Our results demonstrate in mouse that cotransfer of PA embryos improves the pregnancy and implantation of SCNT embryos without compromising the overall health of the resulting clones.

Introduction

NIMAL CLONING by somatic cell nuclear transfer (SCNT) technology has been reported successfully in many species over the last 10 years, including mouse (Wakayama et al., 1998). However, the efficiency of producing SCNT progeny is still low, which seriously limits its application to the reproduction in farm, laboratory, or endangered species. Despite the fact that half of SCNT embryos or even more can develop to blastocyst stage or implant after transfer into recipient females (Gao et al., 2003; Jouneau et al., 2006; Wakayama et al., 1998; Wakayama and Yamagimachi, 2001), only a few percent of transferred embryos give rise to live pups. The common finding of placental defects in SCNT mice, cows, and sheep suggests placental pathology as a major cause of pregnancy complications. This hypothesis has been supported by chimera studies in which tetraploid blastomeres were mixed with preimplantation SCNT embryos

(Cross, 2006). In these chimeras, the tetraploid cells contributed to trophoblast derivatives, but not the embryos/fetus proper (Cross, 2001). Tetraploid chimeras in both mice (Eggan et al., 2001; Jouneau et al., 2006) and cattle (Iwasaki et al., 2000) do better than regular clones, implying that trophoblast lineage defects in clones account for at least some of the pregnancy complications. Moreover, aberrant hypermethylation at the Spalt-like gene3 (Sall3) locus is associated with abnormal placental development caused by nuclear transfer of somatic cells (Ohgane et al., 2004). It is possible that many of the defects in SCNT embryos occur in the later reprogramming stage, which may be attributable to problems with placenta development and function (Yang et al., 2007). Many efforts have been taken to improve the cloning efficiency. Numerous factors that could affect the nuclear transfer cloning efficiency have been reviewed recently, including the timing or protocol of oocyte activation, use of

1Genetic

2Molecular

Reprogramming Group, Agricultural Biotechnology Center, Godollo, Hungary. Animal Biotechnology Laboratory, Szent Istvan University, Godollo, Hungary

2 cytokinesis inhibitor, timing of the removal of the oocyte spindle, in vitro culture period, effect of dimethyl sulfoxide, volume of recipient oocyte cytoplasm, use of trichostatin A, origin of donor cell, cell type, cell cycle, genotype of donor cell, effect of recloning, effect of somatic cell cytoplasm transferred into the oocyte (Wakayama, 2007). Modifications of technical steps, such as serial nuclear transfer (Kato et al., 1999), culture conditions of the SCNT embryos (Gao et al., 2003; Heindryckx et al., 2001), 5-aza-20-deoxycytidine treatment (Shi et al., 2003), and cloneclone aggregation (Boiani et al., 2003) have been investigated before, aiming to increase the efficiency of this technology. Relatively few approaches focused on the improvement of the implantation. The postblastocyst stage mouse embryo development relies on close relationships between the conceptus and the maternal environment, and within the conceptus, between the embryo proper and its surrounding extra-embryonic tissues (Camus et al., 2004). The function of the placenta during early gestation is primarily to mediate implantation of the embryo into the uterus and, secondarily, to produce hormones that induce maternal recognition of pregnancy (Cross, 2006). The pregnancy establishment and maintenance depend on both the embryos and the uterus environment of the recipient. It is not clear why the recipients receiving SCNT embryos have poor pregnancy establishment and maintenance, but possibly related to the disturbed gene expression profile of cloned placenta compared to fertilized ones (Yang et al., 2007). In multipara animals, the pregnancy needs a minimum number of healthy embryos to trigger and maintain it; the presence of parthenogenetically activated (PA) or fertilized embryos might assist the implantation and subsequent development of SCNT embryos. In the mouse, the pregnancy rate, the total number of pups born, and the number and percentage of pups from microinjected embryos were improved by cotransfer of uninjected embryos (Canesco et al., 1994). In pigs, the pregnancies were established with a mixture of a low number (three) of fertilized and 5560 parthenote embryos and the fertilized embryos developped to full term (King et al., 2002). Previous reports in the pig indicated that cotransfer of PA embryos served better for the purpose of SCNT embryo pregnancy maintenance than using timed administration of estradiol or eCG, with the former yielding a cloned piglet (De Sousa et al., 2002). Cotransfer of low number of PA (15) and SCNT embryos (22) triggered the pregnancy establishment in porcine, but in that case the pregnancy terminated at day 69 (Lai et al., 2002); and cotransfer of SCNT embryos with large number of fertilized ones only resulted in the births of fertilized piglets (Onishi et al., 2000). Recently, this strategy was also used to maintain pregnancies of rat embryonic nuclear transfer embryos by cotransfering fertilized embryos (Popova et al., 2006). However, the number of cotransfer with cloned embryos studies is still low, and few in mice. The effects of embryo cotransfer on the implantation and term development of SCNT embryos still remains unclear, despite the fact that this approach has the potential to the efficiency of SCNT technology. We conducted this study to improve the overall efficiency of SCNT progeny production in mouse using cotransfer of PA or fertilized embryos, and evaluating its effects on the implantation, pregnancy, and full-term development of SCNT mouse embryos. Materials and Methods Animals

MENG ET AL.

All experiments were conducted under the Guidelines for the Care and Use of Laboratory Animals, from Agricultural Biotechnology Center, Hungary. B6D2F1 mice (C57BL/6 DBA/2) were used as oocyte and somatic cell (cumulus) donors for production of SCNT embryos. ICR mice were used for production of PA and in vivo fertilized embryos. Surrogate females were ICR females mated with vasectomized males of the same strain. Collection of oocytes Eight12-week-old B6D2F1 females were induced to superovulate with 7.5 IU PMSG, followed by 7.5 IU hCG 48 h later. Cumulusoocytecomplexes were collected from oviducts 1415 h post-hCG injection, placed in Hepesbuffered CZB (Chatot et al., 1989) medium, and treated with 0.1% hyaluronidase until the cumulus cells dispersed. Prior to their enucleation the oocytes were placed for 30 min to 2 h into CZB medium supplemented with 5.5 mM glucose (CZBG) at 37C with 5% CO2 in air. Preparation of donor cells for nuclear transfer B6D2F1 mouse cumulus cells were removed from oocytes using hyaluronidase as described above, and transferred and maintained in HEPES-buffered CZB medium. Just before use, they were resuspended with 10% polyvinyl pyrrolidine (PVP) in HEPES-buffered CZB medium. Production of cloned embryos by using adult somatic cells Nuclear transfer was performed as described (Kishigami et al., 2006b). All micromanipulations were performed by using Narishige micromanipulation system installed on Olympus IX 71 microscope. After 10 min of pretreatment in HEPESbuffered CZB medium supplemented with 5 g/mL cytochalasin B (CB), meiotic spindles of B6D2F1 mouse oocytes were removed using a Piezo-driven (Prime Tech, Japan) pipette with an outer diameter of 10 m. Enucleated oocytes were then washed extensively and returned to culture as above, for 12 h before injection. Single nuclei were isolated from cumulus cells by pipetting the cells with a 67 m pipette, and injected individually into enucleated B6D2F1 oocytes. One hour after nuclear transfer, the reconstructed oocytes were activated by 10 mM SrCl2 in Ca2 -free CZB medium supplemented with 5 g/mL CB and 5 nM trichostatin A (TSA) for 6 h. The reconstructed oocytes were then incubated in 5nM TSA in synthetic oviductal medium enriched with potassium (KSOM) supplemented with NEAA and EAA and 1 mg/mL BSA for 4 h at 37C with 5% CO2 in air. TSA is a histone deacetylase inhibitor; treating the reconstructed oocytes with TSA could enhance the embryo development (Kishigami et al., 2006a; Rybouchkin et al., 2006). Finally, the reconstructed embryos were rinsed with KSOM and cultured in this medium for 20 h until the two-cell stage or for 4 days until the blastocyst stage. Collection of in vivo fertilized pronuclear (PN) stage embryos Eight12-week-old female ICR mice were induced to superovulate as described above, and mated with male ICR mice

EMBRYO COTRANSFER IN MOUSE SCNT CLONING TABLE 1. COMPARISON OF IN VITRO DEVELOPMENT OF SCNT, PARTHENOGENETIC (PA) AND FERTILIZED EMBRYOS Embryos SCNT PA Fertilized No. embryos cultured 62 70 51 No. cleaved (%) 56 (90.3) 67 (95.7) 49 (96.0) No. blastocyst (%) 18 (32.1)a 37 (65.7)b 40 (82.0)b

3 fully implanted (which most likely was not the case). Therefore, our data presented is probably slightly underestimating the SCNT embryo implantation rates. The cloned pups could be distinguished from the fertilized ones by the color of the eyes at birth and by fur color 7 days later. The eyes of cloned pups were black while the fertilized ones were red; the fur of cloned pups was black while the fertilized ones was white. Fertilized ICR embryos were transferred to recipients as control. The controls were allowed to give birth naturally or collected by cesarean section at 19.5 dpc. At least four embryo transfers were performed in each group. Statistical analysis Statistical analyses were performed with SSPS software. Data were analyzed by Anova. Significance level was considered at p 0.05. Results In vitro development of SCNT, PA, and fertilized embryos Developmental capacity of SCNT, PA and fertilized embryos during early stages was assessed and compared during culture. Cleavage rates were not different; however, the percentage of zygotes developing to blastocyst from SCNT embryos was significantly lower than that of the PA or fertilized ones (32.1 vs. 65.7%, p 0.05; 32.1 vs .82.0%, p 0.05) (Table 1). Embryo transfer of in vivo fertilized embryos A total of 186 two-cell embryos were transferred into 15 recipients. Thirteen (86.7%) recipients became pregnant and gave birth to 94 (58.1%) pups. Effects of unilateral embryo cotransfer on the implantation and term development of SCNT embryos in the same individual In the first cotransfer experiment using SCNT and fertilized embryos 8 to 11 two-cell stage SCNT embryos were transferred to one side of the oviducts (SCNT), while seven to eight SCNT embryos were cotransferred with two fertilized embryos to the other side of the same individual (NT Fert). The implantation rates of the total embryos transferred or cotransferred were not different (28.3 vs. 34.8%). All the live births of the SCNT embryos were found in the cotransfer group (5.6%). All the fertilized pups were found in the original side where they were transferred to, indicating that these embryos did not migrate from one side to the other (Table 2).

a,b:Values in the same column with different superscript are significantly different.

after hCG injection. PN stage embryos were collected from the mated female 20 h postcoitum and cultured in KSOM for 20 h until the two-cell stage or for 4 days until the blastocyst stage. Production of PA embryos Mature oocytes were collected from the superovulated ICR female mice at 1516 h post-hCG injection. The oocytes were incubated in CZBG medium at 37C with 5% CO2 in air for 34 h prior to parthenogenetic activation by 10 mM SrCl2 in Ca2 -free CZB medium supplemented with 5 g/ml CB for 6 h. After activation the oocytes were cultured in KSOM for 20 h until the two-cell stage or for 4 days until the blastocyst stage. Embryo transfer and cotransfer ICR females mated with vasectomized males of the same strain were used as recipients. All the embryos were transferred at the two-cell stage to the oviducts of 0.5 day postcoitum (dpc) recipients. SCNT embryos were divided randomly into control embryo transfer and cotransfer groups. In the control transfer group (SCNT), 1521 SCNT embryos were transferred into the two oviducts of a recipient. In the cotransfer group, 1518 SCNT embryos were cotransferred with 23 PA (SCNT PA) or fertilized (SCNT Fert) embryos into the two oviducts (811 embryos/oviduct) of a recipient. In some recipients only one side of oviducts received cotransferred embryos, while the other side received only control SCNT embryos (unilateral embryo cotransfer). The recipients were sacrificed 19.5 dpc. The implantation sites in the uteri were inspected, and the live pups were fostered to an ICR mother. In the case of implantation sites, which were visually nondistinguishable by the origin of the embryos, the calculation of SCNT embryo implantation was conservative by presuming that all of the fertilized or PA embryos successTABLE 2.

COMPARATIVE DEVELOPMENT OF SCNT EMBRYOS TRANSFERRED ALONE VS. COTRANSFERRED WITH FERTILIZED EMBRYOS TO THE OPPOSITE OVIDUCT OF THE SAME RECIPIENT No. pregnant recipients (%) 3 (60) No. implanted embryos (%) 13 (28.3) 16 (34.8) No. SCNT implanted Embryos a(%) 13 (28.3) 10 (27.8) No. clone live offspring (%) 0 (0) 2 (5.6) No. Fertilized offspring (%) n/a 3 (30.0)

Embryos SCNT SCNT Fert

No. embryos transferred 46 36 10

No. recipient 5

aCalculated

by deducting the No. of fertilized embryos transferred to the pregnant recipients from the No. of implantation sites.

4 TABLE 3.
WITH

MENG ET AL. COMPARATIVE DEVELOPMENT OF SCNT EMBRYOS TRANSFERRED ALONE VS. COTRANSFERRED PARTHENOGENETIC (PA) EMBRYOS TO THE OPPOSITE OVIDUCT OF THE SAME RECIPIENT No. pregnant recipients (%) 3 (75) No. implantation sites (%) 12 (33.3) 18 (47.4) SCNT Embryo implantation sites (%)a 12 (33.3) 12 (40.0) No. cloned live offspring (%) 1 (2.8) 2 (6.7)

Embryos SCNT SCNT PA

No. embryos transferred 36 30 8

No. recipient 4

aCalculated

by deducting the No. PA embryos transferred to the pregnant recipients from the No. of implantation sites.

Similarly, 810 two-cell SCNT embryos were transferred to one side of the oviducts, while seven to eight SCNT embryos were cotransferred with two PA embryos to the other side of the same individual. There was no significant difference between the SCNT and SCNT PA groups in the implantation rates (33.3 vs. 47.4%) and in the birth rates (2.8 vs. 6.7%) (Table 3). None of the parthenotes developed to term. Effects of embryo cotransfer on the implantation and term development of SCNT embryos Two-cell stage SCNT embryos were transferred or cotransferred with fertilized embryos to the recipients. The pregnancy, implantation, and birth rates increased in the SCNT Fert group compared to the SCNT group (Table 4). Although the implantation rate, after deducting the number of fertilized embryos cotransferred, was higher in the cotransfer group than in the normal SCNT group (22.1 vs. 11.4%), this difference was not significant statistically (p 0.05). Similarly to the experiment above, two-cell stage SCNT embryos were transferred or cotransferred with PA embryos into the recipients. The pregnancy rate increased significantly in the SCNT PA group compared to the SCNT group (80.0 vs. 22.2%, p 0.05). The implantation rate also increased significantly in the SCNT PA group compared to the SCNT group (38.7 vs. 8.6%; p 0.05); also, after deducting the number of PA embryos cotransferred, it was still significantly higher in the cotransfer group than in the normal SCNT embryo transfer group (33.3 vs. 8.6%; p 0.05). The birth rate from cotransferred SCNT embryos was much higher than that of normal SCNT embryo transfer group (Table 5). Placental and birth weights of neonatal mice derived from SCNT embryos The placental weight of live clones derived from SCNT, SCNT Fert and SCNT PA groups was 0.32 0.03 (mean SE, gram), 0.38 0.01 and 0.41 0.03, respectively, TABLE 4. TERM DEVELOPMENT
OF

and there was no significant difference between these groups. The average placental weight of these three groups of clones was 0.38 0.06, which was significantly higher than that of fertilized controls (0.38 0.06 vs. 0.15 0.04, p 0.001). There was no significant difference in the birth weight between the clones or fertilized controls (1.56 0.08 vs. 1.44 0.03). One clone derived from the normal SCNT embryo transfer and one from SCNT cotransferred with fertilized embryos died within 48 h after birth due to unknown reasons; all the other clones survived to adulthood, and gave births to progeny after mating with adult male mice. Discussion The efficiency of SCNT cloning in mouse is still low, and most losses of SCNT embryos occure during the pregnancy and in the peri- or postimplantation periods. Our results revealed that cotransfer of parthenogenetic, and to a lesser extent fertilized embryos, can improve the pregnancy and implantation of SCNT mouse embryos. The mechanism of developmental improvement of SCNT embryos by cotransfer is unclear. In the normal in vivo development, establishment and maintenance of pregnancy requires signaling by the conceptus (embryo/fetus and associated extraembryonic membranes) and reciprocal interactions between the conceptus and endometrium (Spencer et al., 2004). Trophoblast giant cells can secrete protein factors (Lee et al., 1988; Linzer and Fisher, 1999) acting as paracrine regulators within the endometrium to maintain pregnancy. A recent study showed that the postimplantation mouse conceptus, at least in part, regulates the expression of specific genes (Angpt1/2, Dtprp, G1p2, and Prlpa) in the endometrium undergoing decidualization. Moreover, certain genes expression in the uterus is dependent on the presence of a healthy conceptus (Bany and Cross, 2006). SCNT clones commonly have a high occurrence of placental defects (Yang et al., 2007), and our in vitro experimental results indicate that the SCNT mouse embryos have
OR

SCNT EMBRYOS TRANSFERRED ALONE No. pregnant recipients (%) 3 (37.5) 3 (42.9)

CO-TRANSFERRED No. SCNT implanted embryos (%)a 18 (11.4) 23 (22.1)

WITH

FERTILIZED EMBRYOS No. cloned live offspring (%) 1 (0.6) 2 (1.9) No. fertilized offspring (%) n/a 3 (20.0)

Embryos SCNT SCNT Fert

No. embryos transferred 158 104 15

No. recipients 8 7

No. implanted embryos (%) 18 (11.4) 29 (24.4)

aCalculated

by deducting the No. of fertilized embryos transferred to the pregnant recipients from the No. of implantation sites.

EMBRYO COTRANSFER IN MOUSE SCNT CLONING TABLE 5. TERM DEVELOPMENT OF SCNT EMBRYOS TRANSFERRED ALONE CO-TRANSFERRED WITH PARTHENOGENETIC (PA) EMBRYOS No. pregnant recipient (%) 2 (22.2)a 4 (80.0)b No. implanted embryos (%) 13 (8.6)a 36 (38.7)b
OR

Embryos SCNT SCNT PA

No. embryos transferred 152 81 12

No. recipient 9 5

No. SCNT implanted embryos (%)c 13 (8.6)a 27 (33.3)b

No. live offspring (%) 1 (0.7) 4 (4.9)

a,b:Values

cCalculated

in the same column with different superscript are significantly different. by deducting the No. of PA embryos transferred to the pregnant recipients from the No. of implantation sites.

lower developmental capacity at least until the blastocyst stage compared with parthenogenetic or fertilized ones. Thus, it is possible that the parthenogenetic or fertilized embryos could implant in the endometrium more efficiently and trigger a more receptive status to other embryos to implant and maintain pregnancy, possibly including the SCNT ones. We also found that the implantation rates are always better in the individuals that gave birth to live pups (data no shown), implying one or two obust individual embryos could be helpful for implantation and maintaining pregnancy of other embryos. Previous reports also indicated that pregnancy of SCNT porcine embryos could be maintained by cotransfer of parthenogenetic embryos (De Sousa et al., 2002). In our experiment, cotransferred PA embryos were more efficient in the improvement of implantation and pregnancy of SCNT embryos compared to fertilized embryos. In porcine studies cotransferred fertilized embryos might have outcompeted developmentally inferior SCNT embryos, and among the several hundreds of SCNT embryos cotransferred with fertilized embryos only one was derived from the SCNT embryos transferred (Onishi et al., 2000; Verma et al., 2000). In the rat, cotransfer of 76 embryo blastomer NT cloned and 36 fertilized embryos into two recipients resulted in full-term pregnancy maintenance in both recipients; however, from the 19 pups born only one was from cloning (Popova et al., 2006). These results indicate that the cotransferred embryos not only help the SCNT embryos to implant but also might compete with them. To minimize the competition effects from the helper embryos in our experiments, only two to three fertilized or parthenogenetic embryos (accounting for 1015% of the total embryos transferred) were cotransferred with SCNT ones. While the parthenotes could only develop until about 10 dpc (Surani and Barton, 1983; Surani et al., 1984), the fertilized embryos developed full term, and might have been more vigorous than SCNT embryos during the entire gestation. As expected, none of the parthenotes developed to term in our experiment, and the better overall results suggest that their reduced viability might have been beneficial to maintain pregnancy of SCNT embryos. In our experiment, the placentas of all SCNT derived pups were significantly heavier than that of fertilized controls, in accordance with earlier reports (Eggan et al., 2001; Miki et al., 2005; Tanaka et al., 2001) depicting this as typical feature of mouse cloning. Cotransfer therefore did not normalize the SCNT placental overgrowth. All of the clones surviving to adulthood were proven fertile, and the six clone females mated with regular males gave birth to 130 progeny before being euthanized for further examinations. Only one clone female died early in 6

months of age due to unknown reasons. The surviving five clones and 10 of their progeny were examined by a detailed histopathological examination, which did not reveal any unusual pathological findings compared to the five control animals (data not shown). Although one progeny of a clone had a bronchioloalveolar adenoma, which was unusual for a young animal, this was most likely an accidental observation, as no other clone or progeny manifested any similar pathology. Overall, our results demonstrated that the clones derived from the SCNT embryos cotransferred with PA or fertilized embryos are healthy and fertile, and according to morphological examination standards, not different from the clones from normally transferred SCNT embryos. We have described an improved implantation in the recipients receiving cotransferred embryos compared to those with SCNT embryos only. However, there was no difference in implantation rates between the SCNT embryo alone and PA or fertilized cotransfer side of uterus in the same individual. Earlier studies have shown that trans-uterine migration of fertilized ova is not a common event in mice, and is prevented mainly by the separation of the uterine cavity by a median septum (Rulicke et al., 2006). In our experiments, the fetuses were all located in the side of uteri corresponding to the oviduct where the embryos were transferred. This result indicated that none of these embryos migrated and the potential alteration of implantation in these unilateral cotransfer individuals was not attributable to embryo migration. Our data indicates that the hormones and factors produced by the concepti, endometrium, or ovaries could have been affecting the other side of uterus through the circulation system and helped to establish the pregnancies and increase the number of implantation sites from SCNT embryos. In summary, our results demonstrated for the first time that cotransfer of parthenogenetic embryos can improve the implantation and pregnancy of SCNT mouse embryos, and the resulting clones were healthy and fertile. The underlying biology and optimization of the number and developmental stage of cotransferred embryos require further studies as this might improve further the poor in vivo development of SCNT embryos, which is currently the main limiting factor of the entire SCNT technology. Acknowledgments This study was supported by Wellcome Trust (Grant No. 070246), EU FP6 (MEXT-CT-2003-509582, LSHG-CT-2006518240, MRTN-CT-2006-035468), and Chinese-Hungarian Bilateral Scientific and Technology Collaboration projects (TET CHN-28/04, CHN-41/05).

6 Author Disclosure Statement The authors declare no financial conflicts exist. References
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Address requests for reprints to: Andras Dinnyes Agricultural Biotechnology Center Szent-Gyorgyi Albert 4. Godollo 2100 Hungary E-mail: Dinnyes@abc.hu