Effect of Human Chorionic Gonadotropin and Progesterone Administration on the Developmental Potential of Mouse Somatic Cell Nuclear-Transferred Oocytes

Abstract

Somatic cell nuclear-transferred (SCNT) oocytes have a relatively high potential to develop into blastocysts in vitro, but a large proportion embryos die at various pre- and postimplantation stages after transfer to recipients. Although the reason for the high mortality of SCNT embryos at the peri- and postimplantation stages is not clear, epigenetic abnormalities of SCNT embryos are considered to be the main cause. Such abnormalities of SCNT embryos may decrease their ability to maintain the corpora lutea, which is necessary for initiating implantation and maintaining fetal development. To examine this hypothesis, human chorionic gonadotropin (hCG) and progesterone were administered at different times to recipients that received SCNT embryos. When hCG was administered daily from day 3.5 to day 6.5 of pregnancy, the implantation and fetal development rates increased significantly compared to those of controls. The potential of SCNT embryos to develop to full term, however, was not greater than that of controls, even if hCG administration was continued to day 11.5 or day 17.5 and progesterone was administered from day 7.5 to day 17.5 after hCG injection. These findings demonstrated that administering hCG to recipients protects the in vivo development of SCNT embryos until day 10.5, but other treatment is necessary to support the progression of the embryos to full-term development.

Introduction

Enucleated oocytes at the second metaphase stage transferred with somatic nuclei begin to cleave and develop to the blastocyst stage on the same time schedule as fertilized embryos. The potential of somatic cell nuclear-transferred (SCNT) oocytes to develop into blastocysts is not largely different from in vitro-fertilized (IVF) oocytes in bovine, regardless of the sex, maturity, and origin of the somatic cells (Kato et al., 1998, 2000). More than 50% of SCNT mouse oocytes receiving cumulus cells develop into blastocysts in vitro (Rybouchkin et al., 2006). The potential of SCNT embryos to implant after embryo transfer to recipients is relatively high; 30 to 50% of transferred embryos implant in the mouse (Meng et al., 2008; Rybouchkin et al., 2006). Most of the implanted embryos, however, die at various times during pregnancy, and less than 6% of the transferred embryos develop to term in the mouse (Kishigami et al., 2006; Meng et al., 2008; Rybouchkin et al., 2006). Although the morphologic appearance of SCNT embryos is not different from that of IVF embryos, the blastocyst cell number is lower (Heindryckx et al., 2002), and the expression patterns of various genes differ between SCNT and IVF embryos (e.g., see Li et al., 2005, 2006). Mouse embryos with half the normal cell number have the same potential as normal embryos to develop into fetuses (Tsunoda and McLaren, 1983); therefore, the lower cell numbers of the SCNT embryos are not the main reason for the loss of SCNT embryos after implantation. The results of many studies suggest that one of the causes of peri-implantation death of SCNT embryos is abnormal gene expression (Aston et al., 2009), but the other factors are not clear.

Embryos transferred to synchronized recipients implant into the uterine epithelium during a narrow implantation time window during which complex feto-maternal communications influence embryonic and fetal development (Cross, 2006). Because corpora lutea are not formed after natural ovulation in the nonpregnant estrous cycle in the mouse, recipient female mice must be mated with vasectomized males to induce the formation of the corpora lutea before embryo transfer. Both progesterone and estrogen are required to initiate implantation, and continuous secretion of progesterone is required to maintain the pregnancy (Paria et al., 1993). Luteinizing hormone secreted from the pituitary is a main factor required for the maintenance of the corpora lutea. If viable fetuses are not present in the mouse uterus during the second half of gestation, the corpora lutea degenerate and estrus returns (Forsyth, 1994). In bovine, the viability of frozen–thawed embryos is somewhat inferior to that of fresh embryos (Nishigai et al., 2002), suggesting that signal transmission from frozen–thawed embryos to the pituitary to stimulate hormones that support the corpora lutea is insufficient. Injection of human chorionic gonadotropin (hCG) to increase progesterone production helps to overcome the inadequate signal transmission and leads to significantly or slightly higher pregnancy rates (Chagas e Silva and Lopes da Costa, 2005; Nishigai et al., 2002).

Ewes carrying SCNT clone pregnancies have significantly lower serum progesterone levels compared to ewes carrying control pregnancies, suggesting that a low serum P4 level is one reason for the low potential of SCNT embryos to reach full term (Alexander et al., 2008). Therefore, cotransfer of fertilized embryos might be one way to overcome the inadequate signal transmission of SCNT embryos. The rates of SCNT embryos (unpublished observation) and ES cell-cloned embryos to reach full term (Amano et al., 2001; Yabuuchi et al., 2001), however, are not high after cotransfer of fertilized embryos. This might be due to the different timing of implantation between nuclear-transferred and fertilized embryos (Paria et al., 1993).

So, we examined whether hCG and progesterone administration increases the potential of SCNT mouse embryos to develop into fetuses.

Materials and Methods

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. All chemicals were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA), unless otherwise stated.

Medium

Flushing holding medium (FHM) and potassium simplex optimized medium (KSOM) (Erbach et al., 1994) were used for handling and culturing embryos, respectively. Stock solutions of cytochalasin B and the histone deacetylase inhibitor trychostatin A (TSA) were dissolved in dimethyl sulfoxide at 1 mg/mL and 1 mM, and used at 5 μg/mL and 100 nM, respectively.

Nuclear transfer, embryo culture

Cumulus cells were isolated from ovulated oocytes obtained from the oviducts of mature F1 (C57BL/6 × DBA) females 14 h after hCG injection. Cells were rinsed with FHM after dispersion with hyaluronidase and used as donor cells (Tsuji et al., 2009). Chromosomes at the second metaphase of oocytes recovered from superovulated F1 females 14 h after hCG injection were mechanically removed, and enucleated oocytes were used as the recipient cytoplasm (Tsunoda and Kato, 1995). A single cumulus cell was directly injected into the enucleated oocyte cytoplasm using a piezoelectric-actuated micromanipulator (Wakayama et al., 1998; Yabuuchi et al., 2004).

Nuclear-transferred oocytes were cultured in KSOM with 100 nM TSA for 2 h, and then activated in 10 mM SrCl2 and 5 μg/mL cytochalasin B-supplemented calcium-free KSOM with TSA for 6 h, based on findings from previous studies (Kishigami et al., 2006; Rybouchkin et al., 2006). After washing with FHM, the oocytes were cultured in KSOM without TSA for 64 h after the start of activation. The oocytes were then transferred into KSOM supplemented with a 1:200 stock solution of essential and nonessential amino acids (Invitrogen, Carlsbad, CA, USA) and 3.5 mg/mL glucose, and cultured further for 32 h.

Experimental design

Experiment 1: Effect of hCG administration

To examine the effect of hCG administration on the potential of SCNT embryos to develop into fetuses, a preliminary study was performed in which blastocysts developed from zygotes after in vitro culture for 5 days were transferred into the oviducts of day 1.0 pseudopregnant (afternoon of the day a vaginal plug was observed) ICR strain females. Daily injections of hCG (5 or 10 IU; Sankyo Co. Ltd., Tokyo, Japan) or 0.9% saline were intramuscularly administered to females on days 3.5, 4.5, or 5.5 of pregnancy. Recipients were killed on day 18.5 to examine the implantation sites and fetuses. In another experiment, 10 IU hCG was injected into recipients every day from day 3.5 to day 17.5.

Embryos that developed from SCNT oocytes to the blastocyst stage were transferred into the oviducts of day 1.0 pseudopregnant ICR strain females. The preliminary study demonstrated that the fetal development rate on day 10.5 after transfer of SCNT mouse blastocysts to oviducts on day 1.0 pseudopregnant females (17.9%) was higher than that after transfer of two-cell stage embryos to oviducts on days 0.5 and 1.0 pseudopregnant females (8.6 and 3.3%) or after transfer of blastocysts to uteri on days 2.5, 3.0, and 3.5 pseudopregnant females (6.1, 7.7, and 10.5%).

As shown in Figure 1, injection of 10 IU hCG in 0.9% saline was administered daily from day 3.5 to day 6.5 (Experiment 1-1), from day 3.5 to day 11.5 (Experiment 1-2), or from day 3.5 to day 17.5 of pregnancy (Experiment 1-3). Control females received 0.9% saline on the same schedule. Recipients were killed on days 10.5, 12.5, or 18.5, respectively, to examine the implantation sites and fetuses.

FIG. 1.

Experimental design.

Experiment 2: Effect of hCG and progesterone administration

SCNT blastocysts were transferred into the oviducts of day 1.0 pseudopregnant ICR strain females. Injection of 10 IU hCG was administered daily from day 3.5 to day 6.5, and then 1 mg progesterone in sesame oil (Ghaemi et al., 2008) was injected from day 7.5 to day 17.5 (Fig. 1, Experiment 2). Recipients were killed on day 18.5 to examine the implantation sites and fetuses. Control recipients received saline or hCG daily from day 3.5 to day 6.5, and also received sesame oil from day 7.5 to day 17.5.

Statistics

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

Results

In vitro development of SCNT oocytes

As shown in Table 1, the proportions of oocytes that were successfully enucleated, activated, and developed into blastocysts in the two experiments were 96, 79, and 58%, respectively.

Table 1.

Development of SCNT Oocytes In Vitro

No. of oocytes
Enucleated/used (%)	With pronucleus (ei)/injected (%)	Developed to blastocysts/cultured (%)
4339/4516 (96)	3408/4339 (79)	1979/3408 (58)

Experiment 1: Effect of hCG administration on in vivo development of SCNT embryos

Our preliminary study demonstrated that the proportions of blastocysts developed from zygotes that implanted and developed to full term were not significantly different among groups receiving a single injection of 5 or 10 IU hCG from days 3.5, 4.5, or 6.5; three injections from day 3.5 to day 5.5; or 15 injections from day 3.5 to day 17.5, in comparison with controls (51 to 63% vs. 55 to 75% for implantation rate; 33 to 51% vs. 30 to 53% for fetal development rate; unpublished observation). The average body weight of fetuses on day 18.5 after 15 injections with hCG, however, was significantly lower than that of controls (1.36 g vs. 1.82 g).

The proportions of SCNT blastocysts that implanted and developed into fetuses on day 10.5 after hCG administration from day 3.5 to day 6.5 were significantly higher than those in recipients after saline injection (54 vs. 40% for implantation rate; 21 vs. 10% for fetal development rate; Table 2). The number of implantation sites per female in the hCG group was larger but not significantly different from the control group (9.8 vs. 6.1 for day 10.5, 11.0 vs. 9.7 for day 18.5). Fourteen of 15 fetuses (control group) and 25 of 30 fetuses (hCG group) had a heartbeat, but the other fetuses did not have a heartbeat at the time of examination. The body length, body weight, and number of somites of live fetuses were not significantly different between the control and hCG administration groups (3.2 ± 0.8 vs. 3.1 ± 0.6 mm, 0.0071 ± 0.0053 g vs. 0.0050 ± 0.0027 g, 38.8 ± 6.2 vs. 39.2 ± 4.2).

Table 2.

Effect of hCG Administration from Day 3.5 to Day 6.5 on the Development of NT Embryos to Fetuses

					Implantation		No. of fetuses (%)				Size of live fetuses (Mean ± SD)
Examination day	Group	No. of embryos transferred	No. of recipients	No. pregnant (%)	No. (%)	Average	Live	Dead	Total	No. of placentas (%)	Length (mm)	Weight (g)	Somites	Weight of placentas (g)
10.5	control	151	10	10 (100)	61 (40)^a	6.1	14 (9)^a	1 (1)	15 (10)^a	—	3.2 ± 0.8	0.0071 ± 0.0053	38.8 ± 6.2	—
	hCG	145	9	8 (89)	78 (54)^b	9.8	25 (17)^b	5 (3)	30 (21)^b	—	3.1 ± 0.6	0.0050 ± 0.0027	39.2 ± 4.2	—
18.5	control	140	8	7 (88)	68 (49)	9.7	1 (1)	0 (0)	1 (1)	1 (1)	—	1.444	—	0.263
	hCG	153	8	8 (100)	88 (58)	11	2 (1)	0 (0)	2 (1)	1 (1)	—	1.122 ± 0.103	—	0.359 ± 0.106

a,b

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

The proportion of implantation sites observed on day 18.5 and the proportion that developed into fetuses, however, were not different between the hCG and control groups (Table 2). Although the number of full-term fetuses was small, the average body weight of fetuses with a heartbeat and the average placental weight were not different between the hCG (1.122 and 0.359 g) and control groups (1.444 and 0.263 g).

When hCG administration was continued until day 11.5, the proportions of implantations and fetuses on day 12.5 did not differ significantly from controls (Table 3). The number of implantation sites per female was similar between control and hCG groups (9.4 and 9.1). Both fetuses in the control group and 1 of 3 fetuses in the hCG group had a heart beat. The body weight of the live fetuses in the control (0.063g) and hCG groups (0.0975 g) was not different, but the body weight of the dead fetuses in the hCG group (0.0312 g and 0.0198 g) was small.

Table 3.

Effect of hCG Administration from Day 3.5 to Day 11.5 on the Development of NT Embryos to Fetuses

				Implantation		No. of fetuses on day 12.5 (%)			Size of live fetuses (Mean ± SD)
Group	No. of embryos transferred	No. of recipients	No. of pregnant (%)	No. (%)	Average	Live	Dead	Total	Length (mm)	Weight (g)
Control	130	7	7 (100)	66 (51)	9.4	2 (2)	0 (0)	2 (2)	9.0 ± 1.4	0.063 ± 0.014
hCG	156	10	10 (100)	91 (58)	9.1	1 (1)	2 (1)	3 (2)	10	0.0975

When hCG administration was continued from day 3.5 to day 17.5, the proportion of implantation sites observed on day 18.5 was significantly higher than that in controls (52 vs. 28%, Table 4). The number of implantation sites per female in the hCG group was large but not significantly different compared with the control group (9.1 vs. 7.8). Large and clear deciduas were observed in six of seven pregnant recipients in the hCG administration group, and only scant implantation residue was observed in the control group, except in recipients with fetuses. Fetuses, however, were not observed in the hCG groups.

Table 4.

Effect of hCG Administration from Day 3.5 to Day 17.5 on the Development of NT Embryos to Fetuses

				Implantation		No. of fetuses on day 18.5 (%)				Weight of live fetuses (mean ± SD)
Group	No. of embryos transferred	No. of recipients	No. of pregnant (%)	No. (%)	Average	Live	Dead	Total	No. of placentas (%) ¹	Fetuses (g)	Placentas (g)
Control	110	6	4 (67)	31 (28)^a	7.8	2 (2)	0 (0)	2 (2)	1 (1)	1.351 ± 0.162	0.386 ± 0.094
hCG	122	8	7 (88)	64 (52)^b	9.1	0 (0)	0 (0)	0 (0)	0 (0)	—	—

a,b

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

Fetus was not obtained.

Experiment 2: Effect of hCG and progesterone administration on in vivo development of SCNT embryos

The implantation rates (47 vs. 40%) and fetal development rate (2 vs. 2%) were not different between the hCG + progesterone group and hCG + sesame oil group (Table 5). However, implantation rate (31%) was low and no fetuses were obtained in saline + sesame oil group. The average number of implantation sites per female in hCG + progesterone group was large but not significantly larger than that of the saline + sesame oil and hCG + sesame oil groups (9.3 vs. 7.8 and 7.0). The average body weight and placental weight were not different between the hCG + sesame oil (1.000 and 0.417 g) and hCG + progesterone (1.364 and 0.402 g) groups. One dead fetus had a small body and placenta weight (0.669 and 0.153 g). In all three groups, placentas without fetuses were obtained. Although only a small number of placentas were examined in each group, the placental weight in the hCG + progesterone group (0.141 and 0.069 g) was low compared with the saline + sesame oil (0.316 and 0.407 g) and hCG + sesame oil (0.680 and 0.226 g) groups.

Table 5.

Effect of hCG and Progesterone Administration on the Development of NT Embryos to Fetuses

				Implantation		No. of fetuses on day 18.5 (%)				Weight of live fetuses (mean ± SD)
Group	No. of embryos transferred	No. of recipients	No. of pregnant (%)	No. (%)	Average	Live	Dead	Total	No. of placentas (%) ¹	Fetuses (g)	Placentas (g)
Saline+sesame oil	100	6	4 (67)	31 (31)^a	7.8	0 (0)	0 (0)	0 (0)	2 (2)	—	—
hCG+sesame oil	88	7	5 (71)	35 (40)	7	2 (2)	0 (0)	2 (2)	2 (2)	1 ± 0.055	0.417 ± 0.234
hCG+progesterone	158	10	8 (80)	74 (47)^b	9.3	2 (1)	1 (1)	3 (2)	2 (1)	1.364 ± 0.412	0.402 ± 0.145

a,b

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

Fetus was not obtained.

Discussion

Alexander et al. (2008) reported that ewes carrying SCNT clone pregnancies had showed significantly lower serum progesterone levels than ewes carrying control pregnancies, suggesting that the low serum P4 level is one reason for the low potential of SCNT embryos to develop to full term. Based on the hypothesis that SCNT embryos are less competent in maintaining the corpora lutea in recipients, which are necessary for the implantation and maintenance of pregnancy, we examined the enhancing effect of administering hCG and progesterone to recipients on corpora lutea function. The findings of the present study demonstrated that hCG administration at the peri-implantation stage significantly increased implantation and fetal development rates at day 10.5 of pregnancy compared with controls. Administration of hCG to recipients increases the secretion of progesterone from the corpora lutea and uterine tissues (Licht et al., 2001) through the luteinizing hormone/hCG receptor (Mukherje et al., 1994; Perrier d'Hauterive et al., 2007). The proportion of SCNT embryos that developed to full term, however, was not increased by hCG administration. The fetal development rates of SCNT embryos drastically decreased from 21% on day 10.5 to 2% on day 12.5. Because fetal loss was also observed in the control group between 10.5 and 12.5 days of pregnancy, it is reasonable to consider that feto-maternal mismatches occurred during this period. Due to the large loss of fetuses between days 10.5 and 12.5 of pregnancy, the rate of development to full term was not increased even if hCG injection was continued until day 11.5 or day 17.5. We could not, however, exclude the possibility that long-term administration of hCG to pregnant females has adverse effects on fetal development, as suggested by the lower fetal weight in recipients that received control embryos and 15 injections of hCG (unpublished observation). To exclude this possibility, progesterone instead of hCG was administered from day 7.5 to day 17.5. Full-term live fetuses were obtained after hCG and progesterone administration, but the fetal development rate was not increased compared with hCG and sesame oil administration groups (2 vs. 2%).

The reason for the large proportion of fetuses aborted after day 10.5 pregnancy is not clear. In the mouse, progesterone, which is secreted from the corpora lutea, is the main hormone necessary for the pregnancy until midgestation, but then placental lactogens become the main hormones (Forsyth, 1994). One possible reason for the drastic reduction in viable fetuses between days 10.5 to 12.5 might be the insufficient secretion of placental lactogens. A possible alternative method to maintain pregnancy following the transfer of cloned embryos is cotransfer fertilized embryos. The proportion of young after cotransfer of fertilized embryos, however, was not high after transfer of SCNT mouse embryos (unpublished observation) and embryonic stem cell cloned embryos (Amano et al., 2001; Yabuuchi et al., 2001). The precise reason is not clear, but the implantation timing of cloned and fertilized embryos might be different (Paria et al., 1993).

Amano et al. (2002) demonstrated that the low developmental potential of embryonic stem cell-cloned blastocysts to term is due to a deficiency of both the inner cell mass and trophectoderm by the reciprocal exchange of inner cell mass and trophectoderm between cloned and fertilized blastocysts. Although we observed that fetal and placental weights did not change after hCG administration, we did not examine whether the fetuses obtained were normal. It is possible that hCG administration promoted the survival of somewhat defective fetuses to day 10.5, which then died. Establishing a reliable cloning technology requires a process to select and produce SCNT embryos that will develop normally, as well as support the pregnancy.

This is the first report that endocrine enhancement in recipients increases the implantation rate and the developmental potential of SCNT embryos to fetuses.

Footnotes

Acknowledgments

This work was supported by the Ministry of Education, Science, and Culture (21028022).

Author Disclosure Statement

The authors declare that no conflicting financial interests exist.

The first two authors contributed equally to this work.

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