Effects of prolonged exposure of mouse embryos to elevated temperatures on embryonic developmental competence

Reproductive BioMedicine Online (2015) 31, 171–179

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ARTICLE

Effects of prolonged exposure of mouse embryos to elevated temperatures on embryonic developmental competence Inchul Choi a,b,1,*, Amarnath Dasari a,c,1, Nam-Hyung Kim d, Keith HS Campbell a a

Animal Development and Biotechnology Group, School of Biosciences, The University of Nottingham, Sutton Bonington Campus, Loughborough, UK; b Department of Animal Biosystem Sciences, College of Agriculture and Life Sciences, Chungnam National University 305-764, Republic of Korea; c Taconic Farms Inc., Five University Place Rensselaer, NY 12144-3439, USA; d Department of Animal Sciences, Chungbuk National University, Gaesin-dong, Cheongju, Chungbuk. 361-763, Republic of Korea * Corresponding author.

E-mail address: [email protected] (I Choi). 1 These authors contibuted equally to this work. Inchul Choi obtained his PhD degree under the tuition of Professor Keith Campbell from the University of Nottingham, UK in 2008, and worked as a research fellow until 2010 in the same lab. He is currently an assistant professor in the Department of Animal Biosystem Sciences at Chungnam National University, Korea. His research interests are in the area of gene expression and particularly gametogenesis and preimplantational embryogenesis, including somatic cell nuclear transfer (SCNT) embryos. He currently focuses on the role of transcription factors during blastocyst formation in mouse and domestic animals.

To investigate effects of heat stress on developmental competence, in-vitro fertilized zygotes were incubated at different temperatures until 96 h post human chorionic gonadotrophin (HCG). Under severe and moderate conditions (41°C and 40°C), most embryos did not overcome the 2-cell block. In long-term mild heat stress (39°C until 96 h post HCG), cleavage and blastocyst formation were comparable to non-heat-stress control, but the number of live pups per transferred embryo and mean litter size were significantly affected (P < 0.05) in the mild-heat-stress group (19.4%, and 5.1 ± 0.4, respectively), compared with control (41.7% and 8.3 ± 0.3, respectively). To elucidate the different competence, gene expression was examined and the numbers of inner cell mass (ICM) and trophectoderm (TE) cells were counted. Aberrant expression of genes for embryonic viability and trophoblast differentiation in the mild-heat-stressed blastocysts was found. Moreover, the expanded blastocysts in the heat-stressed group and the control had a ICM:TE ratio of 1:2.47 and 1:2.96 with average total cell numbers of 59.21 ± 2.38 and 72.79 ± 2.40, respectively (P < 0.05), indicating lower cell numbers in TE. These findings underscore that prevention of heat stress in early embryos is important for maintaining embryo viability embryos during pregnancy.

Abstract

© 2015 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. KEYWORDS: cell number, developmental competence, embryo development, gene expression, heat stress

http://dx.doi.org/10.1016/j.rbmo.2015.04.017 1472-6483/© 2015 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

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Introduction It is well known that competence of embryo development is affected by various culture conditions, including temperature and composition of medium and oxygen tension, which are optimized or formulated to have similar physiological and biochemical conditions in reproductive tracts (Cooke et al., 2002; Fischer and Bavister, 1993; Summers and Biggers, 2003). In particular, heat-induced stress adversely affects reproductive efficiency in domestic animals and humans (Ivell, 2007; Jordan, 2003; Rensis and Scaramuzzi, 2003). Although the need for research had been raised regarding effects of hot climates on fertility many years ago (Meleney, 1964), most studies so far have examined preimplantation embryonic development, effects of in-vitro culture conditions, genotype resistance to elevated temperature, spermatogenesis and genes associated with heat shock under relatively mild temperatures (approximately 2°C higher than normal body temperatures) or short-term heat-shock conditions (i.e. 12 to 24 h), (Barros et al., 2006; Hansen, 2009; Paul et al., 2008; Paula-Lopes et al., 2013; Sharpe, 2010). In domestic animals and mouse studies, elevated temperature affects oocyte maturation and embryonic development because oocytes/early cleaving embryos prior to zygotic gene activation are more vulnerable to heat stress, and the heat-stressed oocytes/ embryos show impaired capacity to implant and to carry a pregnancy to term (Ealy and Hansen, 1994; Edwards and Hansen, 1997; Elliott and Ulberg, 1971; Monty Jr and Racowsky, 1987; Ulberg and Burfenin, 1967). More interestingly, early cleaving embryos exposed to a first short-term mild heat stress can develop to the blastocyst after the second more severe heat stress that was relatively longer; however, if not exposed to the first heat stress, the majority of embryos die, suggesting that preimplantation embryos can acquire thermotolerance by a heat shock memory that induces defence systems for embryo viability (Jia et al., 2010; Mirkes et al., 1999). In the present study, we examined the association between in-vitro culture of zygotes at different temperatures and preimplantation development, and also compared the developmental competence of embryos exposed to heat stress with control embryos after the embryos were transferred to surrogates. In addition, in order to elucidate differences in developmental potential between heat-stressed and control embryos, we analysed relative transcript expression levels of genes responsible for embryo viability by using quantitative real time PCR (qRT-PCR), and trophectoderm (TE) and inner cell mass (ICM) cell numbers and the ICM:TE ratio by differential staining of blastocysts.

Materials and methods Reagents All chemicals were purchased from Sigma-Aldrich unless otherwise stated.

Collection of oocyte and IVF All animal procedures were performed under Home Office project licence, according to UK Home Office and institutional

I Choi et al. guidelines and with prior approval from the University of Nottingham Animal Welfare and Ethical Review Body(Licence No. PPL40/3443, 24 Aug 2010). For collection of in-vivomatured (IVO) oocytes and IVF, B6D2/F1 female mice aged 6–8 weeks were superovulated by an i.p. injection of 5 IU of pregnant mare serum gonadotrophin (PMSG), followed by an i.p. injection of 5 IU of HCG 48 h later. Cumulus–oocyte– complexes (COC) were collected 14–15 h later from ampullar regions of the oviducts. IVF was conducted as previously described (Vasudevan et al., 2010). In brief, capacitated spermatozoa that were collected from 10- to 12-week-old B6D2/F1 males and incubated for 60 min at 37°C with 5% CO2 in a humidified incubator were added to the IVF medium (MEM-alpha supplemented with 0.4% BSA) containing COC. The inseminated oocytes were further incubated for 5 h at 37°C with 5% CO2 in a humidified incubator. Subsequently cumulus cells and adhered spermatozoa were removed by repeating pipetting prior to transferring to culture medium.

Embryo culture, induced heat stress and embryo transfer The in-vitro fertilized zygotes were cultured in modified potassium simplex optimized medium (KSOM; EMD Millipore, Billerica, MA, USA) under mineral oil at 37°C in a humidified atmosphere of 5% O2, 5% CO2, 90% N2. For a long-term mild condition, the 1-cell zygotes were incubated in modified KSOM medium at 39°C in a humidified atmosphere of 5% O2, 5% CO2 and 90% N2 until 96 h post HCG for blastocysts, and for a shortterm mild heat stress, the fertilized embryos were incubated under the same condition for 8 h until 24 h post HCG. In addition, the fertilized zygotes were incubated under the moderate and severe condition, 40°C and 41°C, respectively (summarized in Figure 1A). Long-term mild-heat-stressed blastocysts, control blastocysts (at 96 h post HCG), or short-term mild-heat-stressed 2-cell embryos were transferred to the oviduct of Day 1 pseudo-pregnant females mated to vasectomized males of proven sterility. Twelve to twenty embryos were equally transferred to the both oviducts of each female.

Quantitative real-time PCR Total RNA isolated from three pools of 10 blastocysts per treatment were directly converted to cDNA using the FastLane cell cDNA kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Blastocyst embryos were selected on the basis of their morphological appearance (diameter and cavitation) at 96 h post HCG and then were lysed with 8 µl FCP buffer, followed by elimination of genomic DNA by gDNA Wipeout. The whole amount of treated lysate was directly used for reverse transcription at 42°C for 45 min. The resulting cDNA was then diluted 1:5 in PCR grade water (Roche Diagnostics, Mannheim, Germany) before analysis. Quantitative real-time PCR (qRT-PCR) was conducted as previously described (Amarnath et al., 2011a). qRT-PCR was carried out on LightCycler 480 (Roche Diagnostics) using

Heat-stressed mouse embryo development

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Figure 1 A schematic diagram of the experimental design. (A) Experimental design. Cumulus–oocyte–complexes were retrieved from female mice at 14–15 h post HCG, in-vitro fertilized at 16 h post HCG, and incubated at 37°C (green), 39°C (yellow), 40°C (orange) and 41°C (red). IVF Blastocyst or 4-cell embryos were transferred to surrogates. EB = early blastocyst; EX/H = expanding/hatching blastocyst. (B) Representative images of non-heat-stressed (control) and long-term mild-heat-stressed embryo development. Scale bars: 30 µm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

384-well plates. Each15 µl of volume reaction contained 2 µl of diluted DNA or cDNA, 7.5 µl of 2 × LightCycler 480 SYBR Green I Master Mix (Roche Diagnostics), 330 nmol/l of primers (Amarnath et al., 2011a) and PCR grade water (Roche Diagnostics). The thermal programme included a 10-min incubation at 95°C to activate FastStart DNA polymerase, followed by 45 cycles of 95°C for 10 s, the appropriate annealing temperature for 15 s and 72°C for the appropriate extension time with single fluorescence acquisition. Melting curves were carried out to ensure gene specific amplifications. The experiments were performed twice in triplicates from each pool and threshold cycle (Ct) values of the target genes were normalized to an internal standard Gapdh for gene expression analysis. Quantification of different gene expression was calculated by using the 2–ΔΔCT method (Amarnath et al., 2011a; Schmittgen and Livak, 2008).

Immunocytochemistry and counting total cell numbers Early blastocysts were selected as described above, further incubated for 24 h at 37°C or 39°C to allow them to expand or hatch, and then collected at 120 h post HCG. The blastocysts were fixed with 3.7% paraformaldehyde for 20 min, permeabilized and blocked with PBS containing 0.1% Tween 20 and 0.1% BSA for 1 h at room temperature, and incubated with Tcfap2c and Oct4 antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz. CA, USA) in the blocking solution overnight at 4°C. After twice washing for 15 min, the embryos were incubated with Alexa Fluor 488 and 594 (Molecular Probes, Eugene, OR, USA) antibodies. Finally, the embryos were mounted in Vectashield containing DAPI (4,6-diamidino2-phenylindole; Vector Laboratories, Burlingame, CA, USA)

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I Choi et al. 84.8%) and the long-term mild-heat-stressed group (39°C; 98.2%, 97.3%, 92.4% and 81.3%) when they were incubated until 96 h post HCG in a humidified atmosphere of 5% O2, 5% CO2 and 90% N2. In moderate-heat-stressed conditions (40°C), first cleavage rate did not differ significantly (96.2%), compared with the control group and long-term mild-heatstressed group (39°C), but no embryos overcame the 2-cell block. Moreover, very few embryos exposed to long severe heat stress (41°C) underwent a 2-cell division (10.4%). Interestingly, control embryos were slightly larger and clearly more developmentally advanced, for example early cavitation at 96 h post HCG and degree of expansion and hatching status at 120 h post HCG (Figure 1B). To estimate total cell numbers and lineage-specific allocation in blastocyst at 120 h post HCG, we also counted nuclei of total cells, the ICM, TE using DAPI, anti-Oct4, and anti-Tcfap2c antibodies, respectively. Table 2 shows the distribution of the number of cells in the ICM and TE, the total number of cells, portion of ICM and the ratio of ICM to TE cells of blastocyst. The mean number of ICM cells was 17.50 ± 0.94 and 18.71 ± 0.84 for blastocysts at 120 h post HCG in the long-term mild heat stress and the control group, respectively. The mean number of TE cells was 41.71 ± 2.00 and 54.07 ± 2.27 for blastocysts in the long-term mild heat stress and the control, respectively (P < 0.05), revealing a statistically significantly reduction in TE cells in the heat-stress group. Consequently, the heatstress group had lower ICM:TE ratio (1:2.47) compared with the ratio of the control group (1:2.96).

and imaged using a Nikon Eclipse Ti-U microscope equipped with a colour cooled digital camera DS-Ri 1 (Nikon, Tokyo, Japan). Counting the cell numbers was performed as previously described with slight modification (Ono et al., 2010; Su et al., 2012). Total cell numbers in the blastocysts were counted by using DAPI staining, and the cell numbers of the ICM and TE were estimated by counting the number of nuclei immunostained for Oct4 and Tcfap2c that are detected only in the ICM and TE, respectively (Choi et al., 2012).

Statistics Statistical analysis was performed using the Sigma-Stat software (Jandel Scientific Software, Chicago, IL, USA). Developmental rates of pre-and post-implantation were compared by a contingency table analysis followed by a chi-squared or Fisher’s exact test. ICM and TE cell number and ICM/TE ratio were compared using the Mann–Whitney U-test or one-way analysis of variance (ANOVA) combined with the Tukey posthoc test. One-way ANOVA (followed by multiple pairwise comparisons using Student–Newman–Kleus methods) was used for the analysis of differences in relative transcription levels assayed by qRT-PCR. P-values < 0.05 were considered statistically significant unless otherwise stated.

Results Long-term mild heat stress did not affect preimplantation embryonic development

Long-term mild-heat-stress-induced severe developmental retardation in mouse post-implantation development

The developmental competence of heat-stressed embryos is summarized in Table 1. The percentage of embryos that developed to the 2-cell, 4-cell, and the morula or blastocyst stage was not significantly different between the nonheat-stressed control group (37°C; 98.6%, 98.6%, 95.7% and

Table 1

To assess post-implantation development (summarized in Table 3), the blastocysts were obtained under a long-term mild heat stress or control condition, and then the 160 heatstressed and 60 control blastocysts were transferred to eight

Effects of chronic heat stress on preimplantation embryo development.

Temperature (°C) 37 39 40 41

No. of oocytes

No. of 2-cells (%)

210 225 130 115

207 221 125 12

(98.6)a (98.2)a (96.2)a (10.4)b

No. of 4-cells (%) 207 219 0 0

(98.6)a (97.3)a (0)b (0)b

No. of morulae (%) 201 208 0 0

(95.7)a (92.4)a (0)b (0)b

No. of blastocysts (%) 178 183 0 0

(84.8)a (81.3)a (0)b (0)b

Different superscripts within the same column indicate significant differences (P < 0.05).

a,b

Table 2

ICM, TE and total cell numbers of 37°C control and 39°C long-term mild-heat-stressed blastocysts (120 h post HCG).

Treatment

37°C control 39°C long-term mild heat

No. of blastocysts

30 30

Cell numbers (mean ± s.e.m.) ICM

TE

Total

18.71 ± 0.84 17.50 ± 0.94

54.07 ± 2.27a 41.71 ± 2.00 b

72.79 ± 2.40 a 59.21 ± 2.38 b

a,b Different superscripts within the same column indicate significant differences (P < 0.05). ICM = inner cell mass; TE = trophectoderm.

% ICM of total cell numbers

ICM:TE ratio

25.89 ± 1.15 a 29.83 ± 1.58 b

1:2.96 a 1:2.47 b

Heat-stressed mouse embryo development Table 3

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Effects of mild heat stress on post-implantation embryo development.

37°C control blastocysts 39°C long-term mild-heatstressed blastocysts 39°C short-term heat-stressed 2-cell embryos

No. of recipients

No. of transferred embryos/recipient

No. of recipients carrying to term (%)

No. of pups/transferred embryos (%)

Mean litter size, n ± s.e.m

3 8

20 20

3 (100) 7 (87.5)

25/60 (41.7)a 31/160 (19.4)b

8.3 ± 0.3a 5.1 ± 0.4

6

12

6 (100)

29/72 (40.3)a

5.1 ± 0.3b

Different superscripts within the same column indicate significant differences (P < 0.05).

a,b

and three recipients, respectively. The rates of recipients that carried to term were not significantly different between the two groups (100% versus 87.5%), and 25 live-pups were obtained from the control group (pups/embryo transferred; 41.7%) and 31 pups from the long-term mild-heat-stressed group (pups/ embryo transferred; 19.4%), indicating that the mean litter size of surrogates receiving the heat-stressed blastocysts (5.1 ± 0.4) was lower than that of the control surrogates (8.3 ± 0.3) (P < 0.05). In addition, we also transferred 72 embryos at 2-cell stage to six recipients in order to examine effects of heat stress prior to zygotic gene activation on embryonic developmental competence. One-cell zygotes were incubated at 39°C for 8 h (short-term mild heat stress) and were then transferred into the oviduct of recipient mice. The surrogate mothers delivered 29 live pups and the approximate overall yields (the number of pups per embryos transferred) were not significantly different from those of the control (40.3%) although mean litter size decreased, probably due to a lower number of transferred embryos per recipient (Table 3).

Transcript level of genes responsible for embryo viability and differentiation were altered in heatstressed blastocyst embryos Because of perturbations of term development and a decrease in the number of TE cells in the long-term mild-heatstress group, this study postulated that key genes involved in trophoblast development and embryo viability are altered in the blastocysts. We examined the expression of genes necessary for blastocyst formation, anti-apoptotic response to heat shock, and mitochondrial function by using qRT-PCR (summarized in Figure 2A and B). We observed a significant reduction in the expression levels of TE lineage-specific transcription factor such as Gata3 (GATA binding protein 3), Tead4 (TEA domain family member 4), Cdx2 (caudal-related homeobox 2) and Tcfap2c (transcription factor AP-2, gamma) in the long-term mild-heat-stressed blastocyst (P < 0.05). By contrast, the levels of Oct4 (POU domain, class5, transcription factor; Pou5fl) and Nanog exclusively expressed in the ICM at blastocyst were not changed, and similar expression patterns of Oct4 and Tcfap2c were observed in both qRTPCR and ICC experiments (Figures 2 and 3). We also found that Glut1 (glucose transporter 1, also known as Slc2a1; solute carrier family 2), and Glut3 (glucose transporter3, also known as Slc2a3; solute carrier family 3) transcripts were very significantly reduced in the heat-stress group (P < 0.01). Likewise, the expressions of anti-apoptotic genes, Bcl2 (B-cell CLL/lymphoma 2), and Hspb1 (heat shock 27 kDa protein1) were significantly decreased (P < 0.01 and P < 0.05,

respectively). Finally, we examined the expression of genes involved in mitochondrial function, including nuclear-encoded mitochondrial transcription factor A (Tfam), mitochondrialspecific DNA polymerase gamma catalytic subunit (PloG) and accessory subunit (PloG2), mitochondrial nuclear encoded cytochrome c oxidase I (mt-Cox1) and 2 (mt-Cox2). Interestingly, all of the mitochondria related genes examined were unchanged, except for PloG2.

Discussion In this study, we demonstrated that prolonged exposure of mouse embryos to elevated temperatures severely affects embryo viability during pregnancy, and the decreased developmental competency in the heat-stressed embryos is more likely to be caused by poor TE development, particularly TE cell number and TE lineage-specific gene expression. It is well documented that exposure of early cleaving embryos to high temperatures has an adverse effect on cleavage, blastocyst yield or early fetal development in domestic animals and mice (Alliston et al., 1965; Barros et al., 2006;Elliott et al., 1968; Gwazdauskas et al., 1992; Rivera and Hansen, 2001; Sakatani et al., 2012), and the heat stress effects varied depending upon experimental conditions, including species, strain, sex, duration of heat stress, timing of heat stress and culture medium composition (Gwazdauskas et al., 1992; Ozawa et al., 2002; Pérez-Crespo et al., 2005; Sakatani et al., 2012). A previous study reported that in-vivo fertilized embryos cultured in M16 medium at 39°C in air for 96 h failed to develop to blastocyst stage (Gwazdauskas et al., 1992). However, we demonstrated here that the majority of in-vitro fertilized embryos in modified KSOM reached the blastocyst stage when the zygotes were exposed to 39°C and 5% O2 for 80 h. This discrepancy could be due to the different culture media and oxygen tensions because embryos cultured in M16 displayed lower developmental competence including pre- and postimplantation development (Amarnath et al., 2011b; Ho et al., 1995; Kamjoo et al., 2002; Summers and Biggers, 2003) and lower oxygen (5% vol/vol) similar to partial oxygen pressure of the uterus (1.5% to 8.7%) enhanced embryonic development in humans, cattle and mice (Bontekoe et al., 2012; Pabon et al., 1989; Sakatani et al., 2012). In addition, we found that embryos treated at 39°C during preimplantation development (long-term mild-heat stress) had compromised fetal development and pregnancies, indicating that 39°C is a pivotal temperature of developmental competence. Reportedly, a culture temperature of 39°C is approximately equal to a room temperature of 35°C and 60% relative humidity in which average mouse rectal temperatures

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Figure 2 (A) Relative expression levels of genes in long-term mild-heat-stressed blastocyst. Relative gene expression compared with the control mouse embryos was evaluated by qRT-PCR. Bars represent the value (mean ± SD). * and ** indicate that the expression levels are significantly different with P < 0.05 and P < 0.01, respectively. (B) Summary of gene expression changes in long-term mildheat-stressed blastocyst, and biological functions of genes examined above. Expression levels are coloured to represent different levels of reduction: green (less than 10%), yellow (less than 20%), orange (20 to 40%), red (more than 40%). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

ranged from 39.4°C to 39.7°C, a physiological mild heat stress, whilst a room temperature of 25°C and 50% relative humidity provides a normal rectal temperature, 37.7°C in the mouse (Ozawa et al., 2002). Moreover, mitotic division defects in embryos treated at ≥40°C is consistent with previous studies (Ozawa et al., 2002; Pérez-Crespo et al., 2005; Sakatani et al., 2012), suggesting that early zygotic embryos exposed to supraphysiological temperatures (an increase in temperature by 3°C above normal body temperature) are very vulnerable to heat stress. Acquiring thermotolerance in response to short-term and mild heat stress coincided with the expression of genes responsible for heat shock protein or apoptosis (Edwards et al., 2001; Hartshorn et al., 2007; Jia et al., 2010). Thus, it can be speculated that the loss of developmental competence in mild-heat-stress embryos is due to altered expression levels of genes responsible for establishment of cell lineages as well

as heat-induced apoptosis, because a variety of cellular events including cell division, proliferation, differentiation and programmed cell death occurs during preimplantation embryonic development as a dynamic process. We first examined the transcript levels of genes for mitochondria replication and mitochondria encoded proteins because mitochondria are susceptible to heat stress. In particular, hyperthermia has been shown to increase free radicals such as superoxide anion or hydroperoxide in embryos by uncoupling mitochondria (Flanagan et al., 1998; Ozawa et al., 2002). In addition, oxidative stress caused by heat stress has been reported to induce mitochondria swelling by loss of membrane potential, and apoptosis in preimplantation embryos (Esfandiari et al., 2007; Rivera et al., 2003) by impairing electron transport and decreasing adenosine-5′triphosphate (ATP) synthesizing and oxidative phosphorylation (Findly et al., 1983; Halestrap et al., 2002; Lemasters, 1999).

Heat-stressed mouse embryo development

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Figure 3 Differential staining of mouse blastocyst: control non-heat stressed and long-term mild-heat-stressed blastocyst (at 120 h post HCG) were immunostained using anti-Oct4 and anti-Tcfap2c for the inner cell mass (ICM) and trophectoderm (TE), respectively. Scale bars: 20 µm.

However, no differences were found in the transcript levels of genes for mitochondria replication and mitochondria encoded genes because mtDNA replication does not occur during cleavage stages, and the mitochondrial content generated during oogenesis is sufficient to maintain preimplantation development (Ebert et al., 1988). Interestingly, reduced transcript expression of Plog2 seems to affect embryo postimplantation development because Polg2 knockdown leads to reductions in mtDNA copy number and in ATP content, subsequently impaired mitochondrial function perturbs fetal and placental development (Lee et al., 2014; Wakefield et al., 2011). Moreover, heat stress or heat-induced oxygen stress may affect the amount of ATP production required for further embryo viability because GLUT3, localized on the apical membrane in the TE, is the main glucose transporter providing the ICM with glucose, together with GLUT1 (Moley et al., 1998; Pantaleon and Kaye, 1998; Pantaleon et al., 1997). Therefore, lower expression of Glut-1 and Glut-3 in heat-stressed blastocysts might be associated with formation of the TE. In addition, apoptosis is considered one of the important processes for normal embryo development by eliminating damaged or abnormal cells (Beere and Green, 2001; Jacobson et al., 1997; Meier et al., 2000). It is possible therefore that aberrant expressions of genes in response to heat stress lead to necrosis, retardation of development or embryonic lethality. Expression of anti-apoptotic genes such as Hspb1 and Bcl-2 are elevated during heat stress for thermotolerance (Geum et al., 2002; Guay et al., 1997; Kim et al., 2002; Lavoie et al., 1995; Yang and Rajamahendran, 2002), but those were significantly decreased in the long-term mild-heat-stressed embryos. Considering the retarded post-implantation development, we also investigated regulatory networks of genes involved in the establishment of the TE and the ICM in the blastocyst. To create the first two lineages, lineage-specific transcription factors should activate genes within the same lineage, or reciprocally inhibit genes between two different lineages. In the regulatory networks, downstream genes of Tead4, Cdx2 and Gata3 activate Eomes, and Cdx2 and Tcfap2c

are reciprocally activated for the establishment or survival of TE, in return, activated Cdx2 and Tcfap2c affect Oct4 and Nanog, respectively (Chen et al., 2002; Choi et al., 2012, 2013; Home et al., 2009; Kuckenberg et al., 2010; Nishioka et al., 2008, 2009; Niwa et al., 2005; Ralston et al., 2010; Yagi et al., 2007). Thus, disruption of the network or insufficient expression of genes including Cdx2, Gata3, Tead4 and Tcfap2c for the TE can cause adverse effects on trophoblast differentiation. Moreover, a recent study supports our finding that blastocysts in the long-term heat-stressed embryos showed late cavitation and a decrease in hatching rate because Tcfap2c knock-down inhibits cell proliferation and tight junction biogenesis in mouse embryos (Choi et al., 2012). More evidently, the time of blastocyst development is important, but the blastocyst formation rate is not strongly correlated with pregnancy outcome. However, in human blastocysts with a full blastocoel cavity many cells showed the highest pregnancy and implantation (Balaban et al., 2000; Gardner et al., 2000; Lane and Gardner, 1997). Therefore, a decrease in the total cell numbers at the blastocyst is likely to lead to reduced competence to establish pregnancy after embryo transfer. In this context, aberrant expression patterns of genes involved in the TE lineage formation could be observed in qRT-PCR experiments although the underlying mechanism of gene expression and its association with heat stress are not known. In the present study, we reported that mouse embryos exposed to a relatively mild elevated temperature (39°C) developed to the blastocyst stage without any perturbation caused by heat stress, but the resultant blastocyst showed aberrant gene expression patterns, a decrease in TE cell numbers and compromised developmental competence after embryo transfer.

Acknowledgements I would like to thank Professor Keith Campbell for his guidance and contribution before he passed away suddenly. I also

178 thank the following funding bodies for supporting this study. This work was supported by iPET (Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries; Ministry of Agriculture, Food and Rural Affairs, Republic of Korea Project No. 313020042HD020, and was carried out with the support of ‘ Next-Generation BioGreen21 Program’ (CABX, Project title: Enhancement of reprogramming mediated by cellular polarization and MET, Project No. PJ 011213), Rural Development Administration (RDA), Republic of Korea.

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Declaration: The author reports no financial or commercial conflicts of interest.

Received 5 August 2014; refereed 30 April 2015; accepted 30 April 2015.