Slight Improvement in Full-Term Development of Mouse Somatic Cell Nuclear-Transferred Embryos by Cotransfer of Fertilized Embryos

Abstract

In our previous study (Tsuji et al., 2010), administration of hCG to recipients around the timing of implantation significantly increased the in vivo development of mouse embryos after somatic cell nuclear transfer (SCNT) until day 10.5, but did not increase the development to full term. The present study was undertaken to examine whether cotransfer of fertilized embryos or parthenogenetic embryos prevents the embryonic loss of SCNT embryos after day 10.5, allowing them to develop to full term. We found that compared with SCNT embryo transfer alone, full-term development of SCNT embryos slightly, but not significantly, increased by cotransfer of mouse hybrid blastocysts derived from BDF1 (C57BL/6×DBA) female×ICR male into the oviducts of recipients administered hCG (2.0% vs. 5.5%). This was not the case with the cotransfer of blastocysts from an ICR female×ICR male (2.5% vs. 2.2%) or parthenogenetic blastocysts from BDF1 female (3.0% vs. 2.0%). Furthernore, when SCNT blastocysts were transferred into the uteri of recipients, full-term development did not increase even with the cotransfer of hybrid blastocysts. The mechanisms of the effect of cotransfer of fertilized and parthenogenetic embryos on the full-term development of SCNT mouse embryos are discussed.

Introduction

The current somatic cell nuclear transfer (SCNT) technique in mammals includes the transfer of somatic cell nuclei into enucleated M II oocytes, parthenogenetic activation and in vitro culture of SCNT oocytes, and embryo transfer to recipients (Kato and Tsunoda, 2010). Thus far, various efforts to facilitate and improve the first step of reprogramming of introduced nuclei have been reported; these studies have examined the effects of the condition of oocytes, treatment with deacetylation inhibitors, and types of donor nuclei (Kato and Tsunoda, 2010; Wakayama and Perry, 2002). Various methods to enhance the second step of reprogramming have been reported; these studies have examined the methods of parthenogenetic activation and in vitro culture condition (Heindryckx et al., 2006; Wakayama and Perry, 2002). Thus far, the potential of SCNT mouse oocytes to develop into blastocysts in vitro has been found to be not largely different from that of in vitro fertilized oocytes (Rybouchkin et al., 2006). Although more than 40% of SCNT mouse blastocysts implant after transfer to recipients (Tsuji et al., 2010), the proportion of SCNT embryos to achieve full-term development remains low because most of the SCNT embryos implanted die at the early stage of pregnancy (Joneau et al., 2006; Rybouchkin et al., 2006; Tsuji et al., 2010; Wakayama et al., 1998). Although the precise mechanisms for the high abortion rate after implantation are not clear, abnormal placental development is thought to be one factor, because high pregnancy loss of SCNT embryos is often associated with functional defects of placentae (Chae et al., 2008; Cibelli et al., 2002; Yang et al., 2007).

Although the precise interaction between the placenta and maternal uterine epithelium is important for fetal development, few studies have been focused on the fetomaternal interaction in recipients of SCNT embryos (Tsuji et al., 2010). The corpora lutea producing progesterone are necessary for maintaining pregnancy until parturition in the mouse (Mulac-Jericevic et al., 2000; Paria et al., 1993). The function of the corpora lutea is largely regulated by luteotropic hormone complexes. During the first 10 days of gestation, luteinizing hormone secreted from the pituitary is a main factor in the maintenance of the corpora lutea. Thereafter, lactogens secreted from placentae take over the role of the pituitary gland for maintenance of corpora luteal function (Heap, 1972; Richard, 1994). Pituitary prolactin (PPL) is the predominant lactogen during the first 8 to 9 days of gestation and placental lactogens then begin to be secreted until the end of the pregnancy (Galosy and Talamantes, 1995; Linzer and Fisher, 1999).

The authors (Tsuji et al., 2010) have previously reported that the injection of human chorionic gonadotropin (hCG) into recipient mice, which is known to increase the secretion of progesterone from the copora lutea and uterine tissues (Licht et al., 2001), significantly improves the in vivo development of SCNT embryos until day 10.5 of pregnancy. This result suggests that endocrine enhancement in recipients increases the potential of mouse SCNT embryos to develop into fetuses at midgestation. However, the proportion of SCNT embryos that developed to full term was not increased by hCG or progesterone administration. The proportion of fetuses that developed from SCNT embryos drastically decreased from 21 to 2% between days 10.5 and 12.5 of gestation (Tsuji et al., 2010). Considering the high frequencies of placental abnormalities observed in SCNT clones (Wilmut, 2006), it is speculated that placentae of SCNT embryos have an inferior potential to maintain the copora lutea.

One idea for enhancing the potential of developmental inferior embryos to develop into young is cotransfer with fertilized embryos or parthenogenetic embryos (De Sousa et al., 2002; Meng et al., 2008, 2009; Popova et al., 2006; Tsunoda and McLaren, 1983). So far, only one study (Meng et al., 2008) has precisely compared the effectiveness of cotransfer of fertilized and parthenogenetic embryos with regard to the development of SCNT mouse embryos; this study demonstrated that the cotransfer of parthenogenetic embryos resulted in a greater improvement in achieving implantation and pregnancy with SCNT embryos than cotransfer of fertilized embryos.

In the present study we examined whether the high fetal rate of SCNT embryos observed on day 10.5 of pregnancy, which was induced by hCG administration, could be maintained until full term by cotransfer of fertilized or parthenogenetic embryos.

Materials and Methods

Animals

BDF1 (C57BL/6×DBA) female mice were used to prepare oocytes and somatic cells (cumulus cells). BDF1 and albino ICR females, and ICR males were used to produce the in vivo fertilized embryos. Recipient females were ICR females mated with vasectomized males of the same strain. All experiments and protocols were performed in strict accordance with the Guiding Principles for the Care and Use of Research Animals adopted by the Kinki University Committee on Animal Research and Bioethics.

Collection of oocytes and embryo culture

Mature oocytes were collected from the oviducts of BDF1 females that had been induced to superovulate with 5 IU pregnant mare serum gonadotrophin (PMSG) injection followed by 5 IU hCG injection 48 h later. Oocytes collected 14–15 h after hCG injection were rinsed with M2 medium (Fulton and Whittingham, 1978) after dispersion with 300 IU/mL hyaluronidase. Cumulus cells isolated from oocytes were used as donor cells. Prior to enucleation of oocytes, the oocytes were placed in KSOM medium (Erbach et al., 1994) at 37°C with 5% CO₂ in air.

Production of cloned embryos

Nuclear transfer was performed as described previously (Tsuji et al., 2009). Chromosomes at the second metaphase of oocytes were mechanically removed and used as recipient cytoplasm (Tsunoda and Kato, 1995). A single cumulus cell was directly injected into the enucleated oocyte. After nuclear transfer, the reconstructed oocytes were cultured in KSOM containing 100 nM Trichostatin A (TSA) for 2 h, and then activated in 10 mM SrCl₂ and 5 μg/mL CB supplemented in Ca2-free KSOM with 100 nM TSA for 6 h. The activated oocytes were then washed with M2 medium and cultured in KSOM. After 64 h, from the start of activation, the oocytes were transferred into KSOM supplemented with a 1:200 stock solution of essential and nonessential amino acids and 3.5 mg/mL glucose, and cultured further for 32 h.

Collection of fertilized embryos

To compare the effectiveness of cotransfer of fertilized and parthenogenetic embryos on the development of SCNT embryos, blastocysts developed in vitro, not in vivo, were used. BDF1 or ICR females, induced to superovulate as described above, were mated with ICR males after hCG injection. Zygotes from BDF1 females or four-cell stage embryos from ICR females were recovered 20 h or 44 h after hCG injection, respectively, and then cultured in KSOM until the blastocyst stage.

Production of parthenogenetically activated embryos

Oocytes recovered from BDF1 females were activated by the same procedures as for nuclear-transferred embryos. After activation, oocytes were cultured in KSOM to the blastocyst stage for 96 h.

Embryo transfer

Because it has been reported that the transuterine migration of mouse embryos is not observed after embryo transfer (Rulicke et al., 2006), in the present study, SCNT and fertilized or parthenogenetic embryos were both transferred into either the oviduct or uterus of the same recipient. All recipients were sacrificed on day 18.5 of pregnancy before parturition to examine the implantation sites and the full-term fetus rate in each uterus.

Experiment 1

SCNT blastocysts were divided into three groups. In groups A and C, 10 SCNT blastocysts were transferred to one oviduct and 5 fertilized BDF1 blastocysts were transferred to the other oviduct of the same pseudopregnant female on day 1.0. In group B, 10 SCNT blastocysts were transferred to each oviduct of pseudopregnant females on day 1.0. According to a previous report (Tsuji et al., 2010), daily injections of 0.9% saline in group A, or 10 IU hCG in groups B and C were intramuscularly administered to females on days 3.5, 4.5, 5.5, and 6.5 of pregnancy. When ICR blastocysts were cotransferred, the protocols for groups B and C were carried out.

Experiment 2

It has been reported that developmentally inferior mouse embryos such as half embryos have a greater chance of developing into young when they are transferred into oviducts on day 1 than into uteri (Tsunoda and McLaren, 1983), but this issue has not been studied for SCNT embryos. Therefore, SCNT and fertilized blastocysts in this experiment were transferred to uteri of pseudopregnant females on day 3.5 of pregnancy for the comparison to Experiment 1. As in Experiment 1, groups A and C received 10 SCNT blastocysts and 5 fertilized blastocysts in different uteri, respectively. Group B received 10 SCNT blastocysts in each uterus. Saline (for group A) or hCG (for groups B and C) were administered according to the same schedule as in Experiment 1.

Experiment 3

In groups A and C, 10 SCNT blastocysts and 5 parthenogenetic blastocysts were transferred to different oviducts of pseudopregnant females on day 1.0 of pregnancy, respectively. In group B, 10 SCNT blastocysts were transferred to each oviduct of pseudopregnant females on day 1.0 of pregnancy. Saline (for group A) or hCG (for groups B and C) were administered according to the same schedule as in Experiment 1.

Statistics

Data on embryo development were analyzed using a chi-square test, and the body weights of fetuses or placentae were compared using the Student's t-test. A p-value of less than 0.05 was considered to be statistically significant.

Results

In vitro development of fertilized, parthenogenetic, and SCNT embryos

Table 1 shows the in vitro developmental potential of fertilized BDF1 zygotes and ICR four-cell stage embryos, parthenogenetic, and SCNT oocytes. Although the proportion of four-cell embryos that developed to blastocysts was significantly higher than that of fertilized BDF1 zygotes (99 vs. 93%), the potential of parthenogenetic oocytes to develop into blastocysts was not different from that of fertilized zygotes (89 vs. 93%). The proportion of SCNT oocytes to develop into blastocysts was significantly lower than those of fertilized and parthenogenetic oocytes (62 vs. 93% and 89%).

Table 1.

In Vitro Development of Fertilized, Parthenogenetic, and SCNT Oocytes and Embryos

	No. of oocytes			No. of oocytes developed to (%)
Oocytes or embryos	Enucleated/used (%)	With pronucleus(ei) (%)	Cultured	Two-cell	Four- to eight-cell	Morulae	Blastocysts
Fertilized (ICR)	—	—	151	—	—	150(99)	149(99)^a
Fertilized (BDF1)	—	—	1237	1229(99)	1215(98)	1197(97)	1155(93)^b
Parthenogenetic	—	—	196	195(99)	191(97)	187(95)	175(89)^b
SCNT	5284/5494((96)	4197/5234(80)	4197	4085(97)	3820(91)	3496(83)	2590(62)^c

a–c

Values with different superscripts in the same column differ significantly (p<0.05).

Experiment 1; Effect of cotransfer of fertilized embryos on the in vivo development of SCNT oocytes transferred into oviducts

Table 2 shows the effect of c-transfer of fertilized embryos into oviducts of pseudopregnant recipients with or without hCG administration on the potential of SCNT embryos to develop into fetuses on day 18.5 of pregnancy. The proportions of implantations and fetuses obtained after cotransfer of BDF1 fertilized embryos did not differ between with (group C) and without hCG (group A) (72% vs. 62 % for the implantation rate, 53% vs. 50% for the fetus rate). However, the ratio of live fetuses against implantation sites in group C was significantly higher than group A (80.6 vs. 73.6%). The implantation rate of SCNT embryos in group C (with hCG and cotransfer) was significantly higher than that of group B (with hCG and without cotransfer) (47 vs. 35%). In group C, the proportions of fetuses in relation to the number of blastocysts transferred and the number of implantations on day 18.5 were slightly, but not significantly, higher (5.5% and 11.8%) than those in groups A (2.5 and 5.7%) and B (2.0 and 5.7%). The average placental weights in the three SCNT groups (0.26, 0.29, and 0.27 g for groups A, B, and C) were significantly higher than placentae from fertilized embryos (0.19 g and 0.18 g for groups A and C). On the other hand, the average weights of SCNT fetuses (1.29, 1.24, and 1.10 g) were slightly or significantly lower than those of fetuses obtained from fertilized embryos (1.55 and 1.45 g).

Table 2.

Effect of Cotransfer of Fertilized Embryos on the In Vivo Development of SCNT Embrryos Transferred Into Oviducts of Pseudopregnat Females

			No. of									Weight (g) (Mean±SD) of
Group	Origin of fertilized embryos	With (+) or without (−) hCG injection	Embryos transferred		Recipients	Pregnant (%)	implantations (%)	Live fetuses in total recipients (%)	% of live fetuses in pregnant	No.of live fetuses/implantations (%)	No.of placentae (%)	Fetuses	Placentae
B	ICR X ICR	+	SCNT	198	10	8(80)	85(43)^ab	5(2.5)^a	3.1^a	5/85(5.9)^a	2(1.0)	1.23±0.13^ab	0.25±0.12^a
C		+	fertilized	45	9	8(89)	35(78)^c	21(46.7)^b	52.5^b	21/35(60.0)^b	1(2.2)	1.79±0.15^c	0.16±0.04^b
			SCNT	90			45(50)^b	2(2.2)^a	2.5^a	2/45(4.4)^a	0(0)	1.07±0.06^ab	0.24±0.01^a
A	BDF1 X ICR	−	fertilized	100	20	16(80)	62(62)^c	50(50.0)^b	62.5^b	50/62(80.6)^c	0(0)	1.55±0.24^d	0.19±0.06^c
			SCNT	200			87(44)^ab	5(2.5)^a	3.1^a	5/87(5.7)^a	3(2)	1.29±0.28^ab	0.26±0.06^a
B		+	SCNT	200	10	8(80)	70(35)^a	4(2.0)^a	2.7^a	4/70(5.7)^a	5(3)	1.24±0.33^ab	0.29±0.04^a
C		+	fertilized	100	20	17(85)	72(72)^c	53(53.0)^b	58.9^b	53/72(73.6)^bd	0(0)	1.45±0.24^a	0.18±0.04^c
			SCNT	200			93(47)^b	11(5.5)^a	6.5^a	11/93(11.8)^a	1(1)	1.10±0.21^b	0.27±0.09^a

a–d

Values with different superscripts in the same column and the same origin of fertilized embryos differ significantly (p<0.05).

Recipients in groups A and C received SCNT and fertilized embryos in one oviduct.

Recipient in group B received SCNT embryos in both oviducts.

In contrast to the results obtained after cotransfer of BDF1 embryos, cotransfer of ICR embryos did not increase the potential of SCNT embryos to develop into fetuses on day 18.5 and the ratio of fetuses against implantation sites (2.2 and 4.4% vs. 2.5 and 5.9%) (Table 2). It is not strictly reasonable to examine the statistical difference between cotransfer data with different origins of fertilized embryos because the embryo transfer was carried out on different experimental days. Nonetheless, although the implantation rates of ICR×ICR and BDF1×ICR embryos were not different, the ratio of live fetuses against number of implantations in ICR×ICR embryos was low compared with BDF1×ICR embryos (60.0 vs. 73.6%). Placenta and fetus weights were also high and low in SCNT embryos, respectively, compared with those obtained with fertilized embryos.

Experiment 2; Effect of cotransfer of fertilized embryos on the in vivo development of SCNT oocytes transferred into uteri

The pregnancy rates after cotransfer of BDF1 fertilized embryos into the uteri of recipients were low compared with cotransfer into oviducts (55 and 70% in Table 3 vs. 80 and 85% in Table 2). When the implantation and fetal rates of BDF1 fertilized embryos on day 18.5 were calculated based on the number of embryos transferred, the rates of fertilized embryos were found to be low after transfer to uteri (43 and 50% for implantation rate, and 39.0 and 43.0% for fetus rate in Table 3) than after cotransfer to oviducts (62 and 72 % for implantation rate, 50.0 and 53.0% for fetus rate in Table 2). In group C, the proportions of fetuses in relation to the number of SCNT embryos transferred and to the number of implantations on day 18.5 (2.5 and 6.6%) were not different from those in groups A (1.5 and 5.5%) and B (2.0 and 5.0%). The average placental weights from SCNT embryos in three groups (0.25, 0.39, and 0.24 g) were significantly higher than those from fertilized embryos (0.15 and 0.15 g). As with the results in Experiment 1, the weights of SCNT fetuses (1.03, 1.16, and 1.31 g) were slightly or significantly lower than those from fertilized embryos (1.52 and 1.44 g).

Table 3.

Effect of Cotransfer of Fertilized Embryos on the In Vivo Development of SCNT Embrryos Transferred Into Uteri of Pseudopregnat Females

		No. of									Weight (g) (Mean±SD) of
Group	With (+) or without (−)hCG injection	Embryos transferred		Recipients	Pregnant (%)	Implantations (%)	Live fetuses in total recipients (%)	% of live fetuses in pregnant	No.of live fetuses/implantations (%)	No. of placentae (%)	Fetuses	Placentae
A	−	fertilized	100	20	11(55)	43(43)^ab	39(39.0)^a	70.9^a	39/43(90.1)^a	0(0)	1.52±0.24^a	0.15±0.04^a
		SCNT	195			55(28)^c	3(1.5)^b	2.7^b	3/55(5.5)^b	2(1)	1.03±0.19^b	0.25±0.07^b
B	+	SCNT	200	10	9(90)	80(40)^ab	4(2.0)^b	2.4^b	4/80(5.0)^b	2(1)	1.16±0.15^b	0.39±0.18^b
C	+	fertilized	100	20	14(70)	50(50)^a	43(43.0)^a	66.2^a	43/50(86.0)^a	0(0)	1.44±0.24^a	0.15±0.04^a
		SCNT	200			76(38)^b	5(2.5)^b	3.1^b	5/76(6.6)^b	2(1)	1.31±0.23^ab	0.24±0.07^b

a–c

Values with different superscripts in the same column differ significantly (p<0.05).

Recipients in groups A and C received SCNT and fertilized embryos in one uterus.

Recipient in group B received SCNT embryos in both uteri.

Experiment 3: Effect of cotransfer of parthenogenetic embryos on the in vivo development of SCNT oocytes transferred into oviducts

The implantation rates of parthenogenetic embryos on day 18.5 did not differ between with hCG (group C) and without hCG (group A) administration groups (46 vs. 50%) (Table 4). The proportions of SCNT embryos implanted also did not differ among the three groups (41, 56, and 44%). Cotransfer of parthenogenetic embryos into recipient oviducts following hCG injection did not increase the proportions of fetuses in relation to the number of SCNT blastocysts transferred and to the number of implantations on day 18.5 (2.0 and 4.5% in group C) compared with those obtained without cotransfer (3.0 and 5.4% in group B).

Table 4.

Effect of Cotransfer of Parthenogenetic Embryos on the In Vivo Development of SCNT Embrryos Transferred Into Oviducts of Pseudopregnat Females

		No. of									weight (g) (Mean±SD) of
Group	With (+) or without (−) hCG injection	Embryos transferred		Recipients	Pregnant (%)	Implantations (%)	Live fetuses in total recipients (%)	% of live fetuses in pregnant	No.of live fetuses/implantations (%)	No.of placentae (%)	Fetuses	Placentae
A	−	partheno-genone	50	10	7(70)	25(50)	—	—	—	—	—	—
		SCNT	100			41(41)	2(2.0)	2.9	2/41(4.9)	2(2)	1.22±0.13	0.23±0.09
B	+	SCNT	100	5	5(100)	56(56)	3(3.0)	3	3/56(5.4)	4(4)	1.11±0.42	0.22±0.05
C	+	partheno-genone	50	10	7(70)	23(46)	—	—	—	—	—	—
		SCNT	100			44(44)	2(2.0)	3.3	2/44(4.5)	1(1)	1.17±0.21	0.22±0.08

Recipients in groups A and C received SCNT and parthenogenetic embryos in one oviduct.

Recipient in group B received SCNT embryos in both oviducts.

Discussion

Several studies have demonstrated that the potential of SCNT mouse embryos to develop into blastocysts is high, but that most SCNT embryos die during various periods after embryo transfer, with only a few embryos developing to full term (Kishigami et al., 2006; Meng et al., 2008; Rybouchkin et al., 2006; Tsuji et al., 2009).

The precisely controlled fetomaternal relationship is important for the maintenance of pregnancy, and the placenta plays an important role in fetal development in the mouse after midgestation (Forsyth, 1994). The improper reprogramming of somatic cell nuclei in SCNT embryos induces abnormal placenta formation after transfer to recipients (Palmieri et al., 2008). Morphologically and functionally abnormal placentae also induce abnormal gene expression in uterine epithelium (Bauersachs et al., 2009), and might be inefficient in maintaining functional corpus lutea. We previously reported that the injection into recipients of hCG, to enhance the maintenance of the corpus lutea, increases the development of SCNT embryos until day 10.5, but not to full term, and that other treatment is necessary to enhance the progression of SCNT embryos to full-term development (Tsuji et al., 2010).

Several methods have been developed to enhance the developmental potential of developmentally inferior preimplantation embryos to term: (1) increase the number of embryos transferred to each recipient (Yin et al., 2002); (2) transfer the inferior embryos into pregnant females (Richa and Lo, 1990; Tsunoda and McLaren, 1983); and (3) transfer the inferior embryos with fertilized embryos that have high developmental potential or parthenogenetic embryos (Tsunoda and McLaren, 1983). Considering the small size of the mouse genital tract, it is not practical to transfer more than 200 SCNT embryos into each recipient, as in the case of pig (Yin et al., 2002). The transfer of inferior embryos into pregnant recipients is expected to increase the developmental potential, but the developmental potential of the inferior embryos might be inhibited due to competition with the high developmental embryos. Meng et al. (2008) have demonstrated that both pregnancy and implantation rates originating from clones in the SCNT with parthenogenetic embryos are significantly higher than those of the SCNT group. Meng et al. (2008) have also demonstrated that the proportions of SCNT embryos in the cotransfer group with parthenogenones developing into young were slightly, but not significantly, higher than those after transfer of SCNT embryos (2.8% vs. 6.7%).

The present study has demonstrated that the potential of SCNT embryos to develop into full-term fetuses was not significantly different among all the groups examined. However, the proportion of SCNT embryos that developed to fetuses in recipients injected with hCG and with cotransfer of BDF1 fertilized embryos to oviducts (group C), was slightly, but not significantly, higher than that in recipients injected with hCG but without cotransfer (group B) (5.5 vs. 2.0% in Table 2) and recipients without hCG injection but with cotransfer of BDF1 fertilized embryos (5.5 vs. 2.5% in Table 2). Such an increase in the fetus rate, however, was not observed when SCNT embryos were cotransferred with ICR fertilized embryos. The reason for this difference observed between BDF1 and ICR fertilized embryos was not clear. One reason might be the different developmental potential of both embryos to develop into full-term fetuses, because the implantation rates between BDF1 and ICR fertilized embryos did not differ but the proportion of full-term fetuses in relation to the number of implantations in BDF1 embryos was higher than that in ICR embryos (73.6 vs. 60.0% in group C). The same reason is also considered for the low effectiveness of cotransfer of fertilized BDF1 embryos into uteri of recipients with regard to the potential of SCNT embryos to develop into full-term fetuses.

In contrast to the report of Meng et al. (2008), who demonstrated that cotransfer of parthenogenetic embryos more efficiently improved pregnancy and implantation rates than cotransfer of fertilized embryos, in the present study, cotransfer of parthenogenetic embryos did not increase the pregnancy and implantation rates of SCNT embryos. The most feasible reason for the difference between the report of Meng et al. (2008) and the present study is considered to be the fact that in the present study hCG-administered recipients were used. Our previous study demonstrated that hCG injection to recipients around the timing of implantation significantly increased the implantation and fetus rates of SCNT mouse embryos on day 10.5 of pregnancy, but not on day 18.5 (Tsuji et al., 2010). The present study also demonstrated that hCG injection slightly increased the implantation rate in BDF1 fertilized embryos (Table 2). Because in the present study the pregnancy rate (80% in Table 2 and 100% in Table 4) and implantation rate (35% and 43% in Table 2, and 56% in Table 4) after transfer of SCNT blastocysts into oviducts were higher than those (37.5 and 22.2% for pregnancy rate, 11.4 and 8.6% for implantation rate) reported by Meng et al. (2008), the effectiveness of cotransfer with parthenogenetic embryos might not be observed.

The other reason might be the differences between the developmental fate of fertilized and parthenogenetic embryos after midgestation. In the present study, we expected the secretion of placental lactogens from placentae originating from fertilized or parthenogenetic embryos, which support corpora lutea function (Galosy and Talamantes, 1995; Linzer and Fisher, 1999). Although we did not measure the secretion of placenta lactogens, because the parthenogenetic mouse embryos die around day 10.5 of pregnancy (Surani et al., 1984) due to the lack of paternally expressed genes (Ono et al., 2006), the secretion of placental lactogens might be not expected.

The increase in fetal development of SCNT embryos to full term, however, was not as great as expected. Several reasons for the relatively low effectiveness in enhancing the development of SCNT embryos from day 10.5 to full term are considered. The abnormalities of fetuses might be one reason, although in the present study we did not examine the morphology of fetuses, because 5 of 30 fetuse obtained on day 10.5 had no heartbeat (Tsuji et al., 2010). The other reason might be a contribution of immunological rejection of SCNT fetuses by recipients. We demonstrated that administration of cyclosporine A, an immunoprotectant, increases the fetus rate of SCNT embruyos on day 10.5 of pregnancy (Tsuji et al., 2011). To increase the overall potential of SCNT mouse embryos to develop to full term, studies of a method to produce and select SCNT embryos with high potential to develop into live young, and at the same time methods to enhance pregnancy such as cotransfer of fertilized two-cell embryos into oviducts should be further examined.

Footnotes

Acknowledgments

This work was supported by grant from the Ministry of Education, Culture, Sports, Science, and Technology, Japan to Y. Kato (23013020), and a Research Fellowship of the Japan Society for the Promotion of Science for Young scientists to Y. Tsuji.

Author Disclosure Statement

The authors declare that no conflicting financial interests exist.

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