Journal of Steroid Biochemistry & Molecular Biology 145 (2014) 13–20
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Effects of ERa-specific antagonist on mouse preimplantation embryo development and zygotic genome activation Yanqin Zhang a,1,2 , Yufei Jiang a,1, Xiuli Lian a , Songhua Xu a , Jianen Wei a,b , Chenfeng Chu a , Shie Wang a,b, * a b
Department of Human Anatomy, Histology and Embryology, School of Basic Medical Sciences, Fujian Medical University, Fuzhou,Fujian 350108, PR China Cellular and Developmental Engineering Center, School of Basic Medical Sciences, Fujian Medical University, Fuzhou, Fujian 350108, PR China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 10 December 2013 Received in revised form 5 September 2014 Accepted 23 September 2014 Available online 26 September 2014
Zygotic genome activation (ZGA) is essential for normal development of mammalian preimplantation embryos. Estrogen receptor alpha (ERa) has been implicated in early embryogenesis, and controls the expression of genes associated with proliferation, differentiation and development of cell and target organs via a genomic effect. The objective of this study was to determine whether ERa plays a role in early embryo development and affects ZGA gene expression. Toward this objective, 1-cell embryos from B6C3F1 mouse were cultured with the antiestrogen ICI182780, ERa-specific antagonist MPP, ERaspecific antibody and ERb-specific antagonist PHTPP. Development of 2-cell to 4-cell in vitro was significantly blocked by ICI182780, MPP and ERa-antibody treatment in a dose-dependent manner but not affected by PHTPP exposure. MPP decreased nuclear ERa protein levels and reduced mRNA expression levels of MuERV-L, one of the ZGA related genes. The results indicate that ERa has a functional role in early embryo development by regulation of ZGA-related genes. ã 2014 Elsevier Ltd. All rights reserved.
Keywords: Estrogen receptor alpha Estrogen receptor antagonist Preimplantation embryos Zygotic genome activation Mouse
1. Introduction Estrogen receptor alpha (ERa), a ligand-dependent transcription factor, is a member of steroid/thyroid hormone receptor superfamily and specifically binds with estrogen and regulates the estrogen responsive genes and cell proliferation [1,2]. The gene coding for ERa (ESR1) is regulated by seven different promoters that elicit different transcripts, making it one of the most complex genes in the human genome [3]. In the classic pathway ERa undergoes a conformational change in response to estradiol
Abbreviations: ERa, estrogen receptor alpha; MZT, maternal-to-zygotic transition; ZGA, zygotic genome activation; MPP, 4,40 ,400 -(4-Propyl-[1H]pyrazole-1,3,5triyl) trisphenol; PHTPP, 4-[2-Phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl] phenol; MuERV-L, murine endogenous retrovirus-like; Zscan4d, zinc finger and SCAN domain containing 4d; Hsp70.1, heat-shock protein 70.1; eIF1A, elongation initiation factor 1A. * Corresponding author at: Department of Human Anatomy, Histology and Embryology, School of Basic Medical Sciences, Fujian Medical University, Fuzhou, Fujian 350108, PR China. Tel.: +86 591 22862257; fax: +86 591 22862045. E-mail addresses: [email protected], [email protected] (S. Wang). 1 These two authors contributed equally to this work. 2 Present address: Department of Basic Medical Sciences, Quanzhou Medical College, Quanzhou, Fujian 362010, PR China. http://dx.doi.org/10.1016/j.jsbmb.2014.09.023 0960-0760/ ã 2014 Elsevier Ltd. All rights reserved.
leading to its association with ERa target genes through direct binding to regulatory elements and modulation of their expressions. As a ligand-activated transcription factor, ERa controls the expression of genes related to proliferation, differentiation and development of cell and target organs by influencing genomic transcription [4,5]. Evidence from some older studies indicates a possible functional requirement for ERa in early embryogenesis [6–8]. It has been found that during preimplantation development of mouse embryos, there was a temporal appearance of ERa mRNA and proteins. ERa transcripts appeared in the zygote, started to decline in 2-cell embryos and reappeared in blastocysts where the estrogen receptor protein was also present. These observations may suggest that environmental pollutants or diets with hormonelike activity could interact with embryonic ER receptors to alter transcriptional events thus regulating perimplantation development [9]. However, the mechanism by which ERa affects mammalian preimplantation embryos and fetal development is poorly understood. The preimplantation period of mammalian development entails formation of zygote, activation of embryonic genome and initiation of cellular differentiation. During this period, protamines are replaced by histones, the methylated haploid paterntal genomes undergo demethylation following the formation of the diploid
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zygote, and maternal control of development is succeeded by zygotic control [10]. The maternal-to-zygotic transition (MZT) serves at least three important purposes including degradation of oocyte-specific transcripts, replacement of maternal transcripts with zygotic ones, and transcriptional up-regulation of zygote-specific genes that will function to reset and reprogram gene expression patterns for the continued development of embryo [11]. MZT that occurs following fertilization is the first major transition in which zygotic transcript is predominant through activation of a new gene expression program called zygotic genome activation (ZGA) or embryonic genome activation. ZGA is essential for continued progression of embryonic development but its onset differs across species. ZGA occurs in the mouse by the 2-cell stage, in pig and human by the 4-cell stage, and in bovine by the 8- to 16-cell stage. However, more studies in mouse embryos have demonstrated that transcripts of zygotic genes could start as early as the pronuclear (i.e., 1-cell) stage [12,13]. Intriguingly, transcription begins earlier and stays higher in the male pronucleus compared to that in the female pronucleus [14,15]. Recent large-scale analyses of gene expression during preimplantation development further support the concept that ZGA constitutes one major peak of transcription, followed by another wave of gene expression for cellular differentiation leading up to blastocyst formation [14,15]. Therefore, continued embryo development in an ordered and temporally-regulated fashion is ensured by the accurate timing of ZGA and subsequent bursts of gene activity in combination with the unique nuclear environments at each developmental stage [16]. Murine endogenous retrovirus-like (MuERV-L), zinc finger and SCAN domain containing 4d (Zscan4d), heat-shock protein 70.1 (Hsp70.1) and elongation initiation factor 1A (eIF1A) are the known endogenous marker genes for ZGA [17]. MuERV-L is one of the earliest transcribed genes in mouse one-cell embryos and expresses earlier than any other genes previously reported (e.g., U2afbp-rs, 39 h after hCG; and hsp70.1, 30 h after hCG), peaking at the 2-cell stage and then gradually decreasing until the blastocyst stage [18–20]. The hsp70.1 gene is highly transcribed at the onset of ZGA. Transcription of this gene begins as early as the 1-cell stage and continued through the early 2-cell stage but is repressed before the completion of the second round of DNA replication [19]. Zscan4d is a recently identified gene that transcribes exclusively during the 2-cell stage [21]. The eIF1A gene is initially transcribed in one-cell embryos and highly expressed at the two-cell stage. Its expression profile can be used to assess embryo quality [22,23]. The objective of this study was to investigate the effects of ERa on the mouse preimplantation embryonic development and expression of developmentally important genes in mouse 1-cell embryos exposed in vitro to ER-specific antagonist, ERa-specific antagonist, ERa- specific antibody and ERb-specific antagonist. Findings from this study are expected to gain further insight into the molecular details of ERa effects on mammalian preimplantational embryo development in association with embryonic gene activation. 2. Materials and methods 2.1. Animals Female C57BL/6 mice, and male C3H/He mice were purchased from SLRC Laboratory Animal Co., Ltd. (Shanghai, China). The female mice were 5–6 weeks of age and the males were >8 weeks of age. B6C3F1 mice were obtained by mating female C57BL/ 6 mice with C3H/He males. All animal studies were conducted in accordance with guidelines established by the Institutional Animal Care and Use Committee (IACUC) of Fujian Medical University.
2.2. Reagents and experimental instruments Human chorionic gonadotropin (hCG), M2 and M16 media were purchased from Sigma (St. Louis, MO, USA); pregnant mare serum gonadotropin (PMSG) from ProSpec-Tanny TechnoGene (Rehovot, Israel); ICI 182780, MPP, PHTPP from Tocris Bioscience (Bristol, UK); Anti-ERa rabbit polyclonal antibody (sc-543) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); Alexa Fluor1 555 Goat Anti-Rabbit IgG (H + L) and DAPI from Life Technologies Corp (Molecular Probes, Inc., OR, USA); Mouse non-specific IgG and Immunol Staining Fix Solution from Beyotime (Jiangsu, China); TriPure from Roche Applied Science (Mannheim, Germany); Reverse Transcriptase kit from Fermentas (Shenzhen, China); SYBR1 Premix Ex TaqTM from TaKaRa Bio (Dalian, China); Anti-ERa mouse monoclonal antibody (04-1564) and PVDF membrane (Millipore, Bedford, MA); HRP-coupled GAPDH monoclonal antibody (Shanghai KangCheng Bioengineering Company, Shanghai, China); HRP-conjugated goat anti-rabbit IgG and ECL chemiluminescence (Supersignal Pico substrate) reagent kits (Pierce) (Thermo Fisher Scientific, Rockford, USA). The confocal images were taken with a Leica SP-5 confocal microscope (Leica Microsystems, Germany) and real time-PCR was performed with ABI 7500 from Applied Biosystems (Carlsbad, CA, USA). 2.3. Mouse embryo cultures Female B6C3F1 mice were injected intraperitoneally with 6 IU PMSG followed by 6 IU hCG 46–48 h later. The injected mice were mixed with males in the same cage at a 1:1 ratio (,B6C3F1 0.05) (Table 4). Besides, ERa-antibody also inhibited the development of B6C3F1 mouse 1-cell embryos in vitro (Table 5). 1.0 mg/ml of ERa-antibody decreased the blastocyst rate drastically (P < 0.01). 1-Cell embryos were completely arrested at the 2-cell stage at 2.5 mg/ml of ERa-antibody. To examine morphological changes after treatment with various concentrations of the three estrogen receptor antagonists and ERa-antibody, the embryonic cells at 4-cell or later stage were observed and photographed with a phase-contrast microscopy. As seen in Fig. 1, even in the presence of 50 mM PHTPP, the embryos developed to blastocyst with the similar morphology to the control as characterized by thinned zona pellucidae, well-defined inner cell masses and robust blastocoele cavities. On the contrary, the arrested 2-cell embryos developed in the presence of 25 mM MPP or 2.5 mg/ml ERa-antibody displayed lysed blastomeres and thickened zonae.
2.7. Data analyses
3.2. Determination of the optimal experimental condition for investigating the role of ERa in mouse embryonic development
2-Cell embryos were chosen as the base to calculate the development rate of 4-cell embryos and blastocysts. Las AF Lite software was used to analyze the results of confocal laser scanning microscopy. Relative mRNA levels were determined with the 2(DDCt) method. SPSS software was used for statistical analysis and differences were considered significant when P < 0.05.
The phenomenon of the developmental arrest at the 2-cell stage of 1-cell embryos during in vitro culture is known as the 2-cell block. Transcriptional activity was gradually decreased when the embryos stayed in the “2-cell block”. Many specific factors were responsible for the 2-cell block of embryos [24–26]. Simultaneously the report suggested 2-cell block phenomenon was due to
Table 1 Primer sets used for real time RT-PCR. Gene symbol
GenBank accession number
Primer sequences
ERa
NM_007956.4
F:50 -TTCTGATGATTGGTCTCGTCTG-30 R:50 -ATGCCTTCCACACATTTACCTT-30
– Eif1a
NM_010120
F:50 -CCAAAGAATAAAGGCAAAGGAG-30 R:50 -CTCACACCGTCAAAGCACATT-30
– Hsp70.1
NM_010478
F:50 -AAGAGGAAGCACAAGAAGGACA-30 R:50 -GCGTGATGGATGTGTAGAAGTC-30
– Zscan4d
NM_001100186
F:50 -CCATCTCATAGTTCTGGTGTGC-30 R:50 -GCTCCTTAGTCTGCTTTTCTGG-30
– MuERV-L
Y12713
F:50 -CGCACAGCAGCAGTCTATTATC-30 R:50 -TCTTCTCCTCTTCGGTCAGTTG-30
– H2afz
NM_016750
F:50 -GTAAAGCGTATCACCCCTCGT-30 R:50 -TCAGCGATTTGTGGATGTGT-30
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Table 2 Effects of ICI 182780 on the development of B6C3F1 mouse one-cell embryos in vitro. Group
1-Cell embryos
2-Cell embryos (post hCG 42 h)
4-Cell embryos (%) (post hCG 64 h)
Blastocysts (%) (post hCG 112 h)
M16 ICI (0.5 mM) ICI (1 mM)
163 172 157
160 167 149
154 (96.2%) 70 (41.9%)* 8 (5.37%)*
126 (78.8%) 4 (2.4%)* 0 (0%)*
%: Based on 2-cell numbers and analyzed by X2 test. * vs. M16 group, P < 0.01.
Table 3 Effects of MPP on the development of B6C3F1 mouse one-cell embryos in vitro. Group
1-Cell embryos
2-Cell embryos (post hCG 42 h)
4-Cell embryos (%) (post hCG 64 h)
Blastocysts (%) (post hCG 112 h)
M16 MPP (1 mM) MPP (10 mM) MPP (25 mM)
216 203 208 197
213 199 204 176
203 (95.3%) 190 (95.5%) 174 (85.3%)* 1 (0.6%)*
179 (84.0%) 171 (85.9%) 0 (0%)* 0 (0%)*
%: Based on 2-cell numbers and analyzed by X2 test. * vs. M16 group, P < 0.05.
Table 4 Effects of PHTPP on the development of B6C3F1 mouse one-cell embryos in vitro. Group
1-Cell embryos
2-Cell embryos (post hCG 42 h)
4-Cell embryos (%) (post hCG 64 h)
Blastocysts (%) (post hCG 112 h)
M16 PHTPP (10 mM) PHTPP (25 mM) PHTPP (50 mM)
142 148 138 148
141 146 136 148
127 140 129 129
112 112 106 106
(90.1%) (95.9%) (94.8%) (87.2%)
(79.4%) (76.7%) (77.9%) (71.6%)
%: Based on 2-cell numbers and analyzed by X2 test.
a delay of zygotic genome activation (ZGA)[27]. But the unambiguous mechanism was not uncovered completely. To better understand the role of ERa in mouse embryonic development, these viewpoints enable us to choose the favorable dose in order to ensure that 1-cell embryos could almost be arrested at the 2-cell stage. From our results, 25 mM of MPP is the optimal experimental dose. 3.3. MPP down-regulated nuclear ERa expression, but did not affect mRNA and total protein level of ERa in B6C3F1 mouse 2-cell embryos Given the larger effect of MPP, the ERa-specific antagonist, on the embryo development, we next examined whether MPP could regulate the relative expression of ERa mRNA and protein and the nuclear ERa protein expression in B6C3F1 2-cell embryos developed from 1-cell embryos exposed to 25 mM MPP. The relative abundance of ERa mRNA was quantified by qRT-PCR (Fig. 2). There was no significant difference in the expression of ERa mRNA between the MPP-treated and non-treated embryos (P > 0.05). Besides, the expression level of ERa protein was detected by Western Blot analysis (Fig. 2). The expression level of ERa protein in the MPP-treated embryos was slightly lower than that in
the non-treated embryos (0.323 0.024 vs. 0.349 0.062), but the difference was not statistically significant (P > 0.05). Also, the power of MPP to change ERa protein expression was assessed by immunostaining with an antibody specific for ERa and quantitative analysis of confocal images acquired by laser scanning confocal microscopy. As shown in Fig. 3, in the majority of the untreated embryos, diffuse and patchy staining was found in the nucleus whereas distinctly less ERa-related fluorescence was observed in the nuclei of MPP-pretreated embryos. Automated image analysis allowed quantification of the fluorescence intensity within the nucleus. Fig. 4 shows that mean fluorescence intensity per nucleus in the MPP groups was significantly lower than that in the control group (P < 0.05). These observations indicate that MPP treatment induced the inhibition of the ERa receptor translocation to nucleus. 3.4. MPP decreased the MuERV-L mRNA level To understand the molecular consequences following ERa antagonism by MPP, the relative expression of four ZGA genes (MuERV-L, Zscan4d, Hsp70.1 and Eif1a) that are known to be endogenous marker of ZGA was quantified by qRT-PCR (Fig. 5). The
Table 5 Effect of ERa monoclonal antibody on the development of B6C3F1 mouse one-cell embryos in vitro. Group
1-Cell embryos
2-Cell embryos (post hCG 42 h)
4-Cell embryos (%) (post hCG 64 h)
Blastocysts (%) (post hCG 112 h)
M16 non-specific IgG (2.5 mg/ml) Anti-ERa (0.5 mg/ml) Anti-ERa (1.0 mg/ml) Anti-ERa (2.5 mg/ml)
190 187 196 198 182
187 184 194 189 180
182 181 186 176
162 (86.63%) 148 (80.43%) 169 (87.11%) 7 (3.7%)* 0*
%: based on two-cell numbers and analyzed by X2 test. * vs. M16 group, P < 0.01.
(97.33%) (98.37%) (95.88%) (93.12%) 0*
Y. Zhang et al. / Journal of Steroid Biochemistry & Molecular Biology 145 (2014) 13–20
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Fig. 1. ER antagonists but ERb-specific antagonist PHTPP affected mouse preimplantation embryo development in vitro. (A) B6C3F1 mouse 1-cell embryos developed to 4-cell stage (post-hCG 64 h) in vitro for both control group (M16) and ER antagonists-treated group. (B) B6C3F1 mouse 1-cell embryos developed to blastocyst stage (post-hCG 112 h) in vitro for both control group (M16) and ER antagonists-treated group.
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Fig. 2. MPP had no effect on mRNA and total protein level of ERa in B6C3F1 mouse 2-cell embryos. (A) The relative expression of ERa mRNA in B6C3F1 2-cell embryos from M16 and MPP treatment group by qPCR. Columns represent the mean of five independent experiments, and bars indicate SEM. (B) Analysis for ERa protein level by Western blot. The quantitation of ERa protein was from the five independent experiments.
Fig. 3. MPP downregulated nuclear ERa expression. The B6C3F1 2-cell embryos cultured in M16 and MPP (25 mM) medium from 1-cell stage were stained with antibodies against ERa and DAPI. ERa: Red; DAPI: blue; merge: overlapping of red and blue. Bar = 25 mm. (A) 2-Cell embryos from M16 media; (B) 2-cell embryos from MPP treatment; (C) negative control without ERa antibody. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Y. Zhang et al. / Journal of Steroid Biochemistry & Molecular Biology 145 (2014) 13–20
Fig. 4. The fluorescent intensity of ERa in nucleus. The results were analyzed by a separate variance estimation T-test followed by an F-test. Deviation bars show standard deviations. Asterisks mark statistically significant difference in the intensity of fluorescence between the MPP-treated and non-treated embryos (*P < 0.05).
mRNA level of MuERV-L was 2.5-fold lower (P < 0.01) in the B6C3F1 2-cell embryos arrested by MPP relative to the untreated control, however, there was no significant difference in the expression of Zscan4d, Hsp70.1 and eIF1A between the treated and non-treated embryos (P > 0.05). 4. Discussion The maternal-to-zygotic transition is an extremely complex but fascinating process which is crucial for setting the stage for later development. Embryonic development is sensitive not only to various environmental toxicants but also to internal stimulus. Our observations that the antiestrogen ICI 182780 could block mouse 1-cell embryos at the 2-cell stage in vitro is consistent with the results of a previous study by Greenlee et al. [28] and further suggests that ER may play an important role in mouse preimplantation embryo development. ICI 182780 is termed “pure estrogen antagonist” because it blocks the action of estrogen in a dosedependent manner but does not possess estrogenic properties [29]. ER includes the two major subtypes, ERa and ERb, which are encoded by unique genes located on different chromosomes and exhibit some degrees of overlapping tissue distribution and
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function. However, growing evidence has demonstrated that they differ in binding affinity profile for different ligands and are individually involved in signaling pathways to regulate cell function [30–32], In order to differentiate the role of ERa or ERb in mouse 2-cell to 4-cell transition, B6C3F1 1-cell embryos were exposed to ERa-specific antagonist MPP, ERa-specific antibody and ERb-specific antagonist PHTPP separately. The results showed that MPP and ERa-antibody could inhibit 1-cell embryos from developing to 4-cell stage. However, PHTPP did not significantly affect the developmental competence of 1-cell embryos in vitro. This result provides evidence that ERa, rather than ERb, plays a role in preimplantation embryo development in vitro. The transcriptional machinery is silent during oocyte meiotic maturation and early embryogenesis, and thereby the early decisive events in embryo development prior to initiation of transcription from the embryonic genome are directed by the translation of pre-existing maternal mRNAs. This is critical for normal progression of early embryonic development [33]. It has been reported that recruitment of maternal mRNAs following fertilization may control the time of ZGA by managing the timing of appearance of certain transcription factors [34]. While the identity and function of many of these transcription factors remain to be determined, our finding of lowered nuclear ERa in 2-cell embryos developed from MPP-treated 1-cell embryos suggests that altered spatial distribution of ERa by MPP binding or certain genes interacting with ERa affects further development of 2-cell embryos. The results also show that the expression level of ERa protein in the MPP-treated embryos is slightly lower than non-treated embryos while the ERa mRNA expression is almost no difference. We guess ERa antagonist (MPP) does not affect ERa mRNA expression. Probably a compound forming between ERa protein and the antagonist activates the protein proteolytic system (eg., ubiquitin–proteasome degradation), leading to a slight decrease of ERa protein. Endogenous retroviruses (ERVs) constitute a substantial portion of mammalian genomes, and their retrotransposition activity helps to drive genetic variation, yet their expression is tightly regulated to prevent unchecked amplification. Ancient retroviral insertions were used to co-opt regulatory sequences targeted for epigenetic silencing of cell fate genes during early mammalian embryonic development [35]. MuERV-L has been shown to play an important role in the ZGA process during early embryogenesis. Examining the expression pattern and the function of MuERV-L in the mouse preimplantation embryos, it was observed that the expression of MuERV-L might help to clarify
Fig. 5. MPP (25 mM) decreased the MuERV-L mRNA level. The relative mRNA expression of Zscan4d, Hsp70.1, MuERV-L and eIF1A in B6C3F1 2-cell embryos for both M16 group and 25 mM MPP-treated group by qPCR. Each experiment was analyzed by a separate variance estimation T-test followed by an F-test (n = 5). Columns represent the mean of five independent experiments, and bars indicate SEM. Asterisks indicate statistical significant difference compared with control (**p < 0.01).
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the timing of the onset of ZGA [20,36]. In this study, we found that compared with the non-treated normal 2-cell embryos, MuERV-L mRNA level was significantly lower in the 2-cell embryos from 1cell embryos treated with MPP while no changes were identified in the expression levels of Hsp70.1, eIF1A and Zscan4d. We postulate that MPP may bind to the estrogen response elements of ERa and then shackle other DNA binding sites, so as to inhibit the first round DNA replication in 1-cell stage resulting in the different onset of expressions of the four ZGA genes with MuERV-L being affected earliest. It seems to be a reasonable speculation since MuERV-L is the earliest gene expression among the four ZGA markers. Thus, relative to the other three genes, the duration of its exposure to the action of MPP is relatively long and the changes occurring may be more profound. In conclusion, our study demonstrates that ERa plays an important role in the B6C3F1 mouse zygotic genome activation for early embryo development and suggests that disruption of ERa function is detrimental to continued progression of development. However, the detailed mechanism remains elusive and warrants future investigations. Conflict of interest None of the authors have any conflict of interest to declare. Acknowledgments We thank all those involved for their assistance with this project. This work was supported by grants from the National Natural Science Foundation of China (81170624) and Scientific Research Fund of Fujian Provincial Education Department (JA09104). References [1] Y. Masuhiro, Y. Mezaki, M. Sakari, K. Takeyama, T. Yoshida, K. Inoue, J. Yanagisawa, S. Hanazawa, W. O'Malley, S. Kato, Splicing potentiation by growth factor signals via estrogen receptor phosphorylation, Proc. Natl. Acad. Sci. U. S. A. 102 (23) (2005) 8126–8131. [2] J. Teng, Z.Y. Wang, D.F. Jarrard, D.E. Bjorling, Roles of estrogen receptor alpha and beta in modulating urothelial cell proliferation, Endocr.-Relat. Cancer 15 (1) (2008) 351–364. [3] M. Kos, G. Reid, S. Denger, F. Gannon, Minireview: genomic organization of the human ERalpha gene promoter region, Mol. Endocrinol. 15 (12) (2001) 2057–2063. [4] C.L. Siewit, B. Gengler, E. Vegas, R. Puckett, M.C. Louie, Cadmium promotes breast cancer cell proliferation by potentiating the interaction between ERalpha and c-Jun, Mol. Endocrinol. 24 (5) (2010) 981–992. [5] J.F. Couse, K.S. Korach, Estrogen receptor null mice: what have we learned and where will they lead us? Endocr. Rev. 20 (3) (1999) 358–417. [6] Q. Hou, J. Gorski, Estrogen receptor and progesterone receptor genes are expressed differentially in mouse embryos during preimplantation development, Proc. Natl. Acad. Sci. U. S. A. 90 (20) (1993) 9460–9464. [7] T.C. Wu, L. Wang, Y.J. Wan, Expression of estrogen receptor gene in mouse oocyte and during embryogenesis, Mol. Reprod. Dev. 33 (4) (1992) 407–412. [8] H. Hiroi, M. Momoeda, S. Inoue, F. Tsuchiya, H. Matsumi, O. Tsutsumi, M. Muramatsu, Y. Taketani, Stage-specific expression of estrogen receptor subtypes and estrogen responsive finger protein in preimplantational mouse embryos, Endocr. J. 46 (1) (1999) 153–158. [9] J. Gorski, Q. Hou, Embryonic estrogen receptors: do they have a physiological function? Environ. Health Perspect. 103 (Suppl. 7) (1995) 69–72. [10] J. Kanka, Gene expression and chromatin structure in the pre-implantation embryo, Theriogenology 59 (1) (2003) 3–19. [11] R.M. Schultz, The molecular foundations of the maternal to zygotic transition in the preimplantation embryo, Hum. Reprod. Update 8 (4) (2002) 323–331. [12] P.T. Ram, R.M. Schultz, Reporter gene expression in G2 of the 1-cell mouse embryo, Dev. Biol. 156 (2) (1993) 552–556.
[13] C. Bouniol, E. Nguyen, P. Debey, Endogenous transcription occurs at the 1-cell stage in the mouse embryo, Exp. Cell Res. 218 (1) (1995) 57–62. [14] T. Hamatani, M.G. Carter, A.A. Sharov, M.S. Ko, Dynamics of global gene expression changes during mouse preimplantation development, Dev. Cell 6 (1) (2004) 117–131. [15] Q.T. Wang, K. Piotrowska, M.A. Ciemerych, L. Milenkovic, M.P. Scott, R.W. Davis, M. Zernicka-Goetz, A genome-wide study of gene activity reveals developmental signaling pathways in the preimplantation mouse embryo, Dev. Cell 6 (1) (2004) 133–144. [16] G.N. Corry, B. Tanasijevic, E.R. Barry, W. Krueger, T.P. Rasmussen, Epigenetic regulatory mechanisms during preimplantation development, Birth Defects Res. Part C: Embryo Today: Rev. 87 (4) (2009) 297–313. [17] S.W. Shin, M. Tokoro, S. Nishikawa, H.H. Lee, Y. Hatanaka, T. Nishihara, T. Amano, M. Anzai, H. Kato, T. Mitani, S. Kishigami, K. Saeki, Y. Hosoi, A. Iritani, K. Matsumoto, Inhibition of the ubiquitin–proteasome system leads to delay of the onset of ZGA gene expression, J. Reprod. Dev. 56 (6) (2010) 655–663. [18] K.E. Latham, L. Rambhatla, Y. Hayashizaki, V.M. Chapman, Stage-specific induction and regulation by genomic imprinting of the mouse U2afbp-rs gene during preimplantation development, Dev. Biol. 168 (2) (1995) 670–676. [19] E. Christians, E. Campion, E.M. Thompson, J.P. Renard, Expression of the HSP 70.1 gene, a landmark of early zygotic activity in the mouse embryo, is restricted to the first burst of transcription, Development 121 (1) (1995) 113–122. [20] D. Kigami, N. Minami, H. Takayama, H. Imai, MuERV-L is one of the earliest transcribed genes in mouse one-cell embryos, Biol. Reprod. 68 (2) (2003) 651–654. [21] G. Falco, S.L. Lee, I. Stanghellini, U.C. Bassey, T. Hamatani, M.S. Ko, Zscan4: a novel gene expressed exclusively in late 2-cell embryos and embryonic stem cells, Dev. Biol. 307 (2) (2007) 539–550. [22] L. Magnani, C.M. Johnson, R.A. Cabot, Expression of eukaryotic elongation initiation factor 1A differentially marks zygotic genome activation in biparental and parthenogenetic porcine embryos and correlates with in vitro developmental potential, Reprod. Fertil. Dev. 20 (7) (2008) 818–825. [23] W. Davis Jr., P.A. De Sousa, R.M. Schultz, Transient expression of translation initiation factor eIF-4C during the 2-cell stage of the preimplantation mouse embryo: identification by mRNA differential display and the role of DNA replication in zygotic gene activation, Dev. Biol. 174 (2) (1996) 190–201. [24] I.O. Bogolyubova, Transcriptional activity of nuclei in 2-cell blocked mouse embryos, Tissue Cell 43 (4) (2011) 262–265. [25] S. Komatsu, S. Suzuki, H. Kitai, Y. Endo, T. Fukasawa, R. Iizuka, Studies on in vitro 2-cell block mechanism in the mouse with special reference to cytoplasmic factors, Prog. Clin. Biol. Res. 296 (1989) 322–326. [26] H. Nakano, H. Kubo, Rescue of mouse embryos from 2-cell blocks by microinjection of maturation promoting factor, Fertil. Steril. 75 (6) (2001) 1194–1197. [27] J.J. Qiu, W.W. Zhang, Z.L. Wu, Y.H. Wang, M. Qian, Y.P. Li, Delay of ZGA initiation occurred in 2-cell blocked mouse embryos, Cell Res. 13 (3) (2003) 179–185. [28] A.R. Greenlee, C.A. Quail, R.L. Berg, The antiestrogen ICI 182,780 abolishes developmental injury for murine embryos exposed in vitro to o,p0 -DDT(1), Reprod. Toxicol. 14 (3) (2000) 225–234. [29] A.E. Wakeling, Use of pure antioestrogens to elucidate the mode of action of oestrogens, Biochem. Pharmacol. 49 (11) (1995) 1545–1549. [30] A. Giddabasappa, M. Bauler, M. Yepuru, E. Chaum, J.T. Dalton, J. Eswaraka, 17-b Estradiol protects ARPE-19 cells from oxidative stress through estrogen receptor-b, Invest. Ophthalmol. Vis. Sci. 51 (10) (2010) 5278–5287. [31] J. Frasor, D.H. Barnett, J.M. Danes, R. Hess, A.F. Parlow, B.S. Katzenellenbogen, Response-specific and ligand dose-dependent modulation of estrogen receptor (ER) alpha activity by ERbeta in the uterus, Endocrinology 144 (7) (2003) 3159–3166. [32] J. Chai, K.F. Lee, E.H. Ng, W.S. Yeung, P.C. Ho, Ovarian stimulation modulates steroid receptor expression and spheroid attachment in peri-implantation endometria: studies on natural and stimulated cycles, Fertil. Steril. 96 (3) (2011) 764–768. [33] A. Bettegowda, G.W. Smith, Mechanisms of maternal mRNA regulation: implications for mammalian early embryonic development, Front. Biosci. 12 (2007) 3713–3726. [34] Q. Wang, K.E. Latham, Translation of maternal messenger ribonucleic acids encoding transcription factors during genome activation in early mouse embryos, Biol. Reprod. 62 (4) (2000) 969–978. [35] T.S. Macfarlan, W.D. Gifford, S. Agarwal, S. Driscoll, K. Lettieri, J. Wang, S.E. Andrews, L. Franco, M.G. Rosenfeld, B. Ren, S.L. Pfaff, Endogenous retroviruses and neighboring genes are coordinately repressed by LSD1/KDM1A, Genes Dev. 25 (6) (2011) 594–607. [36] T. Suzuki, N. Minami, T. Kono, H. Imai, Zygotically activated genes are suppressed in mouse nuclear transferred embryos, Cloning Stem Cells 8 (4) (2006) 295–304.
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Effects of ERa-specific antagonist on mouse preimplantation embryo development and zygotic genome activation Yanqin Zhang a,1,2 , Yufei Jiang a,1, Xiuli Lian a , Songhua Xu a , Jianen Wei a,b , Chenfeng Chu a , Shie Wang a,b, * a b
Department of Human Anatomy, Histology and Embryology, School of Basic Medical Sciences, Fujian Medical University, Fuzhou,Fujian 350108, PR China Cellular and Developmental Engineering Center, School of Basic Medical Sciences, Fujian Medical University, Fuzhou, Fujian 350108, PR China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 10 December 2013 Received in revised form 5 September 2014 Accepted 23 September 2014 Available online 26 September 2014
Zygotic genome activation (ZGA) is essential for normal development of mammalian preimplantation embryos. Estrogen receptor alpha (ERa) has been implicated in early embryogenesis, and controls the expression of genes associated with proliferation, differentiation and development of cell and target organs via a genomic effect. The objective of this study was to determine whether ERa plays a role in early embryo development and affects ZGA gene expression. Toward this objective, 1-cell embryos from B6C3F1 mouse were cultured with the antiestrogen ICI182780, ERa-specific antagonist MPP, ERaspecific antibody and ERb-specific antagonist PHTPP. Development of 2-cell to 4-cell in vitro was significantly blocked by ICI182780, MPP and ERa-antibody treatment in a dose-dependent manner but not affected by PHTPP exposure. MPP decreased nuclear ERa protein levels and reduced mRNA expression levels of MuERV-L, one of the ZGA related genes. The results indicate that ERa has a functional role in early embryo development by regulation of ZGA-related genes. ã 2014 Elsevier Ltd. All rights reserved.
Keywords: Estrogen receptor alpha Estrogen receptor antagonist Preimplantation embryos Zygotic genome activation Mouse
1. Introduction Estrogen receptor alpha (ERa), a ligand-dependent transcription factor, is a member of steroid/thyroid hormone receptor superfamily and specifically binds with estrogen and regulates the estrogen responsive genes and cell proliferation [1,2]. The gene coding for ERa (ESR1) is regulated by seven different promoters that elicit different transcripts, making it one of the most complex genes in the human genome [3]. In the classic pathway ERa undergoes a conformational change in response to estradiol
Abbreviations: ERa, estrogen receptor alpha; MZT, maternal-to-zygotic transition; ZGA, zygotic genome activation; MPP, 4,40 ,400 -(4-Propyl-[1H]pyrazole-1,3,5triyl) trisphenol; PHTPP, 4-[2-Phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl] phenol; MuERV-L, murine endogenous retrovirus-like; Zscan4d, zinc finger and SCAN domain containing 4d; Hsp70.1, heat-shock protein 70.1; eIF1A, elongation initiation factor 1A. * Corresponding author at: Department of Human Anatomy, Histology and Embryology, School of Basic Medical Sciences, Fujian Medical University, Fuzhou, Fujian 350108, PR China. Tel.: +86 591 22862257; fax: +86 591 22862045. E-mail addresses: [email protected], [email protected] (S. Wang). 1 These two authors contributed equally to this work. 2 Present address: Department of Basic Medical Sciences, Quanzhou Medical College, Quanzhou, Fujian 362010, PR China. http://dx.doi.org/10.1016/j.jsbmb.2014.09.023 0960-0760/ ã 2014 Elsevier Ltd. All rights reserved.
leading to its association with ERa target genes through direct binding to regulatory elements and modulation of their expressions. As a ligand-activated transcription factor, ERa controls the expression of genes related to proliferation, differentiation and development of cell and target organs by influencing genomic transcription [4,5]. Evidence from some older studies indicates a possible functional requirement for ERa in early embryogenesis [6–8]. It has been found that during preimplantation development of mouse embryos, there was a temporal appearance of ERa mRNA and proteins. ERa transcripts appeared in the zygote, started to decline in 2-cell embryos and reappeared in blastocysts where the estrogen receptor protein was also present. These observations may suggest that environmental pollutants or diets with hormonelike activity could interact with embryonic ER receptors to alter transcriptional events thus regulating perimplantation development [9]. However, the mechanism by which ERa affects mammalian preimplantation embryos and fetal development is poorly understood. The preimplantation period of mammalian development entails formation of zygote, activation of embryonic genome and initiation of cellular differentiation. During this period, protamines are replaced by histones, the methylated haploid paterntal genomes undergo demethylation following the formation of the diploid
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zygote, and maternal control of development is succeeded by zygotic control [10]. The maternal-to-zygotic transition (MZT) serves at least three important purposes including degradation of oocyte-specific transcripts, replacement of maternal transcripts with zygotic ones, and transcriptional up-regulation of zygote-specific genes that will function to reset and reprogram gene expression patterns for the continued development of embryo [11]. MZT that occurs following fertilization is the first major transition in which zygotic transcript is predominant through activation of a new gene expression program called zygotic genome activation (ZGA) or embryonic genome activation. ZGA is essential for continued progression of embryonic development but its onset differs across species. ZGA occurs in the mouse by the 2-cell stage, in pig and human by the 4-cell stage, and in bovine by the 8- to 16-cell stage. However, more studies in mouse embryos have demonstrated that transcripts of zygotic genes could start as early as the pronuclear (i.e., 1-cell) stage [12,13]. Intriguingly, transcription begins earlier and stays higher in the male pronucleus compared to that in the female pronucleus [14,15]. Recent large-scale analyses of gene expression during preimplantation development further support the concept that ZGA constitutes one major peak of transcription, followed by another wave of gene expression for cellular differentiation leading up to blastocyst formation [14,15]. Therefore, continued embryo development in an ordered and temporally-regulated fashion is ensured by the accurate timing of ZGA and subsequent bursts of gene activity in combination with the unique nuclear environments at each developmental stage [16]. Murine endogenous retrovirus-like (MuERV-L), zinc finger and SCAN domain containing 4d (Zscan4d), heat-shock protein 70.1 (Hsp70.1) and elongation initiation factor 1A (eIF1A) are the known endogenous marker genes for ZGA [17]. MuERV-L is one of the earliest transcribed genes in mouse one-cell embryos and expresses earlier than any other genes previously reported (e.g., U2afbp-rs, 39 h after hCG; and hsp70.1, 30 h after hCG), peaking at the 2-cell stage and then gradually decreasing until the blastocyst stage [18–20]. The hsp70.1 gene is highly transcribed at the onset of ZGA. Transcription of this gene begins as early as the 1-cell stage and continued through the early 2-cell stage but is repressed before the completion of the second round of DNA replication [19]. Zscan4d is a recently identified gene that transcribes exclusively during the 2-cell stage [21]. The eIF1A gene is initially transcribed in one-cell embryos and highly expressed at the two-cell stage. Its expression profile can be used to assess embryo quality [22,23]. The objective of this study was to investigate the effects of ERa on the mouse preimplantation embryonic development and expression of developmentally important genes in mouse 1-cell embryos exposed in vitro to ER-specific antagonist, ERa-specific antagonist, ERa- specific antibody and ERb-specific antagonist. Findings from this study are expected to gain further insight into the molecular details of ERa effects on mammalian preimplantational embryo development in association with embryonic gene activation. 2. Materials and methods 2.1. Animals Female C57BL/6 mice, and male C3H/He mice were purchased from SLRC Laboratory Animal Co., Ltd. (Shanghai, China). The female mice were 5–6 weeks of age and the males were >8 weeks of age. B6C3F1 mice were obtained by mating female C57BL/ 6 mice with C3H/He males. All animal studies were conducted in accordance with guidelines established by the Institutional Animal Care and Use Committee (IACUC) of Fujian Medical University.
2.2. Reagents and experimental instruments Human chorionic gonadotropin (hCG), M2 and M16 media were purchased from Sigma (St. Louis, MO, USA); pregnant mare serum gonadotropin (PMSG) from ProSpec-Tanny TechnoGene (Rehovot, Israel); ICI 182780, MPP, PHTPP from Tocris Bioscience (Bristol, UK); Anti-ERa rabbit polyclonal antibody (sc-543) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); Alexa Fluor1 555 Goat Anti-Rabbit IgG (H + L) and DAPI from Life Technologies Corp (Molecular Probes, Inc., OR, USA); Mouse non-specific IgG and Immunol Staining Fix Solution from Beyotime (Jiangsu, China); TriPure from Roche Applied Science (Mannheim, Germany); Reverse Transcriptase kit from Fermentas (Shenzhen, China); SYBR1 Premix Ex TaqTM from TaKaRa Bio (Dalian, China); Anti-ERa mouse monoclonal antibody (04-1564) and PVDF membrane (Millipore, Bedford, MA); HRP-coupled GAPDH monoclonal antibody (Shanghai KangCheng Bioengineering Company, Shanghai, China); HRP-conjugated goat anti-rabbit IgG and ECL chemiluminescence (Supersignal Pico substrate) reagent kits (Pierce) (Thermo Fisher Scientific, Rockford, USA). The confocal images were taken with a Leica SP-5 confocal microscope (Leica Microsystems, Germany) and real time-PCR was performed with ABI 7500 from Applied Biosystems (Carlsbad, CA, USA). 2.3. Mouse embryo cultures Female B6C3F1 mice were injected intraperitoneally with 6 IU PMSG followed by 6 IU hCG 46–48 h later. The injected mice were mixed with males in the same cage at a 1:1 ratio (,B6C3F1 0.05) (Table 4). Besides, ERa-antibody also inhibited the development of B6C3F1 mouse 1-cell embryos in vitro (Table 5). 1.0 mg/ml of ERa-antibody decreased the blastocyst rate drastically (P < 0.01). 1-Cell embryos were completely arrested at the 2-cell stage at 2.5 mg/ml of ERa-antibody. To examine morphological changes after treatment with various concentrations of the three estrogen receptor antagonists and ERa-antibody, the embryonic cells at 4-cell or later stage were observed and photographed with a phase-contrast microscopy. As seen in Fig. 1, even in the presence of 50 mM PHTPP, the embryos developed to blastocyst with the similar morphology to the control as characterized by thinned zona pellucidae, well-defined inner cell masses and robust blastocoele cavities. On the contrary, the arrested 2-cell embryos developed in the presence of 25 mM MPP or 2.5 mg/ml ERa-antibody displayed lysed blastomeres and thickened zonae.
2.7. Data analyses
3.2. Determination of the optimal experimental condition for investigating the role of ERa in mouse embryonic development
2-Cell embryos were chosen as the base to calculate the development rate of 4-cell embryos and blastocysts. Las AF Lite software was used to analyze the results of confocal laser scanning microscopy. Relative mRNA levels were determined with the 2(DDCt) method. SPSS software was used for statistical analysis and differences were considered significant when P < 0.05.
The phenomenon of the developmental arrest at the 2-cell stage of 1-cell embryos during in vitro culture is known as the 2-cell block. Transcriptional activity was gradually decreased when the embryos stayed in the “2-cell block”. Many specific factors were responsible for the 2-cell block of embryos [24–26]. Simultaneously the report suggested 2-cell block phenomenon was due to
Table 1 Primer sets used for real time RT-PCR. Gene symbol
GenBank accession number
Primer sequences
ERa
NM_007956.4
F:50 -TTCTGATGATTGGTCTCGTCTG-30 R:50 -ATGCCTTCCACACATTTACCTT-30
– Eif1a
NM_010120
F:50 -CCAAAGAATAAAGGCAAAGGAG-30 R:50 -CTCACACCGTCAAAGCACATT-30
– Hsp70.1
NM_010478
F:50 -AAGAGGAAGCACAAGAAGGACA-30 R:50 -GCGTGATGGATGTGTAGAAGTC-30
– Zscan4d
NM_001100186
F:50 -CCATCTCATAGTTCTGGTGTGC-30 R:50 -GCTCCTTAGTCTGCTTTTCTGG-30
– MuERV-L
Y12713
F:50 -CGCACAGCAGCAGTCTATTATC-30 R:50 -TCTTCTCCTCTTCGGTCAGTTG-30
– H2afz
NM_016750
F:50 -GTAAAGCGTATCACCCCTCGT-30 R:50 -TCAGCGATTTGTGGATGTGT-30
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Table 2 Effects of ICI 182780 on the development of B6C3F1 mouse one-cell embryos in vitro. Group
1-Cell embryos
2-Cell embryos (post hCG 42 h)
4-Cell embryos (%) (post hCG 64 h)
Blastocysts (%) (post hCG 112 h)
M16 ICI (0.5 mM) ICI (1 mM)
163 172 157
160 167 149
154 (96.2%) 70 (41.9%)* 8 (5.37%)*
126 (78.8%) 4 (2.4%)* 0 (0%)*
%: Based on 2-cell numbers and analyzed by X2 test. * vs. M16 group, P < 0.01.
Table 3 Effects of MPP on the development of B6C3F1 mouse one-cell embryos in vitro. Group
1-Cell embryos
2-Cell embryos (post hCG 42 h)
4-Cell embryos (%) (post hCG 64 h)
Blastocysts (%) (post hCG 112 h)
M16 MPP (1 mM) MPP (10 mM) MPP (25 mM)
216 203 208 197
213 199 204 176
203 (95.3%) 190 (95.5%) 174 (85.3%)* 1 (0.6%)*
179 (84.0%) 171 (85.9%) 0 (0%)* 0 (0%)*
%: Based on 2-cell numbers and analyzed by X2 test. * vs. M16 group, P < 0.05.
Table 4 Effects of PHTPP on the development of B6C3F1 mouse one-cell embryos in vitro. Group
1-Cell embryos
2-Cell embryos (post hCG 42 h)
4-Cell embryos (%) (post hCG 64 h)
Blastocysts (%) (post hCG 112 h)
M16 PHTPP (10 mM) PHTPP (25 mM) PHTPP (50 mM)
142 148 138 148
141 146 136 148
127 140 129 129
112 112 106 106
(90.1%) (95.9%) (94.8%) (87.2%)
(79.4%) (76.7%) (77.9%) (71.6%)
%: Based on 2-cell numbers and analyzed by X2 test.
a delay of zygotic genome activation (ZGA)[27]. But the unambiguous mechanism was not uncovered completely. To better understand the role of ERa in mouse embryonic development, these viewpoints enable us to choose the favorable dose in order to ensure that 1-cell embryos could almost be arrested at the 2-cell stage. From our results, 25 mM of MPP is the optimal experimental dose. 3.3. MPP down-regulated nuclear ERa expression, but did not affect mRNA and total protein level of ERa in B6C3F1 mouse 2-cell embryos Given the larger effect of MPP, the ERa-specific antagonist, on the embryo development, we next examined whether MPP could regulate the relative expression of ERa mRNA and protein and the nuclear ERa protein expression in B6C3F1 2-cell embryos developed from 1-cell embryos exposed to 25 mM MPP. The relative abundance of ERa mRNA was quantified by qRT-PCR (Fig. 2). There was no significant difference in the expression of ERa mRNA between the MPP-treated and non-treated embryos (P > 0.05). Besides, the expression level of ERa protein was detected by Western Blot analysis (Fig. 2). The expression level of ERa protein in the MPP-treated embryos was slightly lower than that in
the non-treated embryos (0.323 0.024 vs. 0.349 0.062), but the difference was not statistically significant (P > 0.05). Also, the power of MPP to change ERa protein expression was assessed by immunostaining with an antibody specific for ERa and quantitative analysis of confocal images acquired by laser scanning confocal microscopy. As shown in Fig. 3, in the majority of the untreated embryos, diffuse and patchy staining was found in the nucleus whereas distinctly less ERa-related fluorescence was observed in the nuclei of MPP-pretreated embryos. Automated image analysis allowed quantification of the fluorescence intensity within the nucleus. Fig. 4 shows that mean fluorescence intensity per nucleus in the MPP groups was significantly lower than that in the control group (P < 0.05). These observations indicate that MPP treatment induced the inhibition of the ERa receptor translocation to nucleus. 3.4. MPP decreased the MuERV-L mRNA level To understand the molecular consequences following ERa antagonism by MPP, the relative expression of four ZGA genes (MuERV-L, Zscan4d, Hsp70.1 and Eif1a) that are known to be endogenous marker of ZGA was quantified by qRT-PCR (Fig. 5). The
Table 5 Effect of ERa monoclonal antibody on the development of B6C3F1 mouse one-cell embryos in vitro. Group
1-Cell embryos
2-Cell embryos (post hCG 42 h)
4-Cell embryos (%) (post hCG 64 h)
Blastocysts (%) (post hCG 112 h)
M16 non-specific IgG (2.5 mg/ml) Anti-ERa (0.5 mg/ml) Anti-ERa (1.0 mg/ml) Anti-ERa (2.5 mg/ml)
190 187 196 198 182
187 184 194 189 180
182 181 186 176
162 (86.63%) 148 (80.43%) 169 (87.11%) 7 (3.7%)* 0*
%: based on two-cell numbers and analyzed by X2 test. * vs. M16 group, P < 0.01.
(97.33%) (98.37%) (95.88%) (93.12%) 0*
Y. Zhang et al. / Journal of Steroid Biochemistry & Molecular Biology 145 (2014) 13–20
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Fig. 1. ER antagonists but ERb-specific antagonist PHTPP affected mouse preimplantation embryo development in vitro. (A) B6C3F1 mouse 1-cell embryos developed to 4-cell stage (post-hCG 64 h) in vitro for both control group (M16) and ER antagonists-treated group. (B) B6C3F1 mouse 1-cell embryos developed to blastocyst stage (post-hCG 112 h) in vitro for both control group (M16) and ER antagonists-treated group.
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Fig. 2. MPP had no effect on mRNA and total protein level of ERa in B6C3F1 mouse 2-cell embryos. (A) The relative expression of ERa mRNA in B6C3F1 2-cell embryos from M16 and MPP treatment group by qPCR. Columns represent the mean of five independent experiments, and bars indicate SEM. (B) Analysis for ERa protein level by Western blot. The quantitation of ERa protein was from the five independent experiments.
Fig. 3. MPP downregulated nuclear ERa expression. The B6C3F1 2-cell embryos cultured in M16 and MPP (25 mM) medium from 1-cell stage were stained with antibodies against ERa and DAPI. ERa: Red; DAPI: blue; merge: overlapping of red and blue. Bar = 25 mm. (A) 2-Cell embryos from M16 media; (B) 2-cell embryos from MPP treatment; (C) negative control without ERa antibody. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Y. Zhang et al. / Journal of Steroid Biochemistry & Molecular Biology 145 (2014) 13–20
Fig. 4. The fluorescent intensity of ERa in nucleus. The results were analyzed by a separate variance estimation T-test followed by an F-test. Deviation bars show standard deviations. Asterisks mark statistically significant difference in the intensity of fluorescence between the MPP-treated and non-treated embryos (*P < 0.05).
mRNA level of MuERV-L was 2.5-fold lower (P < 0.01) in the B6C3F1 2-cell embryos arrested by MPP relative to the untreated control, however, there was no significant difference in the expression of Zscan4d, Hsp70.1 and eIF1A between the treated and non-treated embryos (P > 0.05). 4. Discussion The maternal-to-zygotic transition is an extremely complex but fascinating process which is crucial for setting the stage for later development. Embryonic development is sensitive not only to various environmental toxicants but also to internal stimulus. Our observations that the antiestrogen ICI 182780 could block mouse 1-cell embryos at the 2-cell stage in vitro is consistent with the results of a previous study by Greenlee et al. [28] and further suggests that ER may play an important role in mouse preimplantation embryo development. ICI 182780 is termed “pure estrogen antagonist” because it blocks the action of estrogen in a dosedependent manner but does not possess estrogenic properties [29]. ER includes the two major subtypes, ERa and ERb, which are encoded by unique genes located on different chromosomes and exhibit some degrees of overlapping tissue distribution and
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function. However, growing evidence has demonstrated that they differ in binding affinity profile for different ligands and are individually involved in signaling pathways to regulate cell function [30–32], In order to differentiate the role of ERa or ERb in mouse 2-cell to 4-cell transition, B6C3F1 1-cell embryos were exposed to ERa-specific antagonist MPP, ERa-specific antibody and ERb-specific antagonist PHTPP separately. The results showed that MPP and ERa-antibody could inhibit 1-cell embryos from developing to 4-cell stage. However, PHTPP did not significantly affect the developmental competence of 1-cell embryos in vitro. This result provides evidence that ERa, rather than ERb, plays a role in preimplantation embryo development in vitro. The transcriptional machinery is silent during oocyte meiotic maturation and early embryogenesis, and thereby the early decisive events in embryo development prior to initiation of transcription from the embryonic genome are directed by the translation of pre-existing maternal mRNAs. This is critical for normal progression of early embryonic development [33]. It has been reported that recruitment of maternal mRNAs following fertilization may control the time of ZGA by managing the timing of appearance of certain transcription factors [34]. While the identity and function of many of these transcription factors remain to be determined, our finding of lowered nuclear ERa in 2-cell embryos developed from MPP-treated 1-cell embryos suggests that altered spatial distribution of ERa by MPP binding or certain genes interacting with ERa affects further development of 2-cell embryos. The results also show that the expression level of ERa protein in the MPP-treated embryos is slightly lower than non-treated embryos while the ERa mRNA expression is almost no difference. We guess ERa antagonist (MPP) does not affect ERa mRNA expression. Probably a compound forming between ERa protein and the antagonist activates the protein proteolytic system (eg., ubiquitin–proteasome degradation), leading to a slight decrease of ERa protein. Endogenous retroviruses (ERVs) constitute a substantial portion of mammalian genomes, and their retrotransposition activity helps to drive genetic variation, yet their expression is tightly regulated to prevent unchecked amplification. Ancient retroviral insertions were used to co-opt regulatory sequences targeted for epigenetic silencing of cell fate genes during early mammalian embryonic development [35]. MuERV-L has been shown to play an important role in the ZGA process during early embryogenesis. Examining the expression pattern and the function of MuERV-L in the mouse preimplantation embryos, it was observed that the expression of MuERV-L might help to clarify
Fig. 5. MPP (25 mM) decreased the MuERV-L mRNA level. The relative mRNA expression of Zscan4d, Hsp70.1, MuERV-L and eIF1A in B6C3F1 2-cell embryos for both M16 group and 25 mM MPP-treated group by qPCR. Each experiment was analyzed by a separate variance estimation T-test followed by an F-test (n = 5). Columns represent the mean of five independent experiments, and bars indicate SEM. Asterisks indicate statistical significant difference compared with control (**p < 0.01).
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the timing of the onset of ZGA [20,36]. In this study, we found that compared with the non-treated normal 2-cell embryos, MuERV-L mRNA level was significantly lower in the 2-cell embryos from 1cell embryos treated with MPP while no changes were identified in the expression levels of Hsp70.1, eIF1A and Zscan4d. We postulate that MPP may bind to the estrogen response elements of ERa and then shackle other DNA binding sites, so as to inhibit the first round DNA replication in 1-cell stage resulting in the different onset of expressions of the four ZGA genes with MuERV-L being affected earliest. It seems to be a reasonable speculation since MuERV-L is the earliest gene expression among the four ZGA markers. Thus, relative to the other three genes, the duration of its exposure to the action of MPP is relatively long and the changes occurring may be more profound. In conclusion, our study demonstrates that ERa plays an important role in the B6C3F1 mouse zygotic genome activation for early embryo development and suggests that disruption of ERa function is detrimental to continued progression of development. However, the detailed mechanism remains elusive and warrants future investigations. Conflict of interest None of the authors have any conflict of interest to declare. Acknowledgments We thank all those involved for their assistance with this project. This work was supported by grants from the National Natural Science Foundation of China (81170624) and Scientific Research Fund of Fujian Provincial Education Department (JA09104). References [1] Y. Masuhiro, Y. Mezaki, M. Sakari, K. Takeyama, T. Yoshida, K. Inoue, J. Yanagisawa, S. Hanazawa, W. O'Malley, S. Kato, Splicing potentiation by growth factor signals via estrogen receptor phosphorylation, Proc. Natl. Acad. Sci. U. S. A. 102 (23) (2005) 8126–8131. [2] J. Teng, Z.Y. Wang, D.F. Jarrard, D.E. Bjorling, Roles of estrogen receptor alpha and beta in modulating urothelial cell proliferation, Endocr.-Relat. Cancer 15 (1) (2008) 351–364. [3] M. Kos, G. Reid, S. Denger, F. Gannon, Minireview: genomic organization of the human ERalpha gene promoter region, Mol. Endocrinol. 15 (12) (2001) 2057–2063. [4] C.L. Siewit, B. Gengler, E. Vegas, R. Puckett, M.C. Louie, Cadmium promotes breast cancer cell proliferation by potentiating the interaction between ERalpha and c-Jun, Mol. Endocrinol. 24 (5) (2010) 981–992. [5] J.F. Couse, K.S. Korach, Estrogen receptor null mice: what have we learned and where will they lead us? Endocr. Rev. 20 (3) (1999) 358–417. [6] Q. Hou, J. Gorski, Estrogen receptor and progesterone receptor genes are expressed differentially in mouse embryos during preimplantation development, Proc. Natl. Acad. Sci. U. S. A. 90 (20) (1993) 9460–9464. [7] T.C. Wu, L. Wang, Y.J. Wan, Expression of estrogen receptor gene in mouse oocyte and during embryogenesis, Mol. Reprod. Dev. 33 (4) (1992) 407–412. [8] H. Hiroi, M. Momoeda, S. Inoue, F. Tsuchiya, H. Matsumi, O. Tsutsumi, M. Muramatsu, Y. Taketani, Stage-specific expression of estrogen receptor subtypes and estrogen responsive finger protein in preimplantational mouse embryos, Endocr. J. 46 (1) (1999) 153–158. [9] J. Gorski, Q. Hou, Embryonic estrogen receptors: do they have a physiological function? Environ. Health Perspect. 103 (Suppl. 7) (1995) 69–72. [10] J. Kanka, Gene expression and chromatin structure in the pre-implantation embryo, Theriogenology 59 (1) (2003) 3–19. [11] R.M. Schultz, The molecular foundations of the maternal to zygotic transition in the preimplantation embryo, Hum. Reprod. Update 8 (4) (2002) 323–331. [12] P.T. Ram, R.M. Schultz, Reporter gene expression in G2 of the 1-cell mouse embryo, Dev. Biol. 156 (2) (1993) 552–556.
[13] C. Bouniol, E. Nguyen, P. Debey, Endogenous transcription occurs at the 1-cell stage in the mouse embryo, Exp. Cell Res. 218 (1) (1995) 57–62. [14] T. Hamatani, M.G. Carter, A.A. Sharov, M.S. Ko, Dynamics of global gene expression changes during mouse preimplantation development, Dev. Cell 6 (1) (2004) 117–131. [15] Q.T. Wang, K. Piotrowska, M.A. Ciemerych, L. Milenkovic, M.P. Scott, R.W. Davis, M. Zernicka-Goetz, A genome-wide study of gene activity reveals developmental signaling pathways in the preimplantation mouse embryo, Dev. Cell 6 (1) (2004) 133–144. [16] G.N. Corry, B. Tanasijevic, E.R. Barry, W. Krueger, T.P. Rasmussen, Epigenetic regulatory mechanisms during preimplantation development, Birth Defects Res. Part C: Embryo Today: Rev. 87 (4) (2009) 297–313. [17] S.W. Shin, M. Tokoro, S. Nishikawa, H.H. Lee, Y. Hatanaka, T. Nishihara, T. Amano, M. Anzai, H. Kato, T. Mitani, S. Kishigami, K. Saeki, Y. Hosoi, A. Iritani, K. Matsumoto, Inhibition of the ubiquitin–proteasome system leads to delay of the onset of ZGA gene expression, J. Reprod. Dev. 56 (6) (2010) 655–663. [18] K.E. Latham, L. Rambhatla, Y. Hayashizaki, V.M. Chapman, Stage-specific induction and regulation by genomic imprinting of the mouse U2afbp-rs gene during preimplantation development, Dev. Biol. 168 (2) (1995) 670–676. [19] E. Christians, E. Campion, E.M. Thompson, J.P. Renard, Expression of the HSP 70.1 gene, a landmark of early zygotic activity in the mouse embryo, is restricted to the first burst of transcription, Development 121 (1) (1995) 113–122. [20] D. Kigami, N. Minami, H. Takayama, H. Imai, MuERV-L is one of the earliest transcribed genes in mouse one-cell embryos, Biol. Reprod. 68 (2) (2003) 651–654. [21] G. Falco, S.L. Lee, I. Stanghellini, U.C. Bassey, T. Hamatani, M.S. Ko, Zscan4: a novel gene expressed exclusively in late 2-cell embryos and embryonic stem cells, Dev. Biol. 307 (2) (2007) 539–550. [22] L. Magnani, C.M. Johnson, R.A. Cabot, Expression of eukaryotic elongation initiation factor 1A differentially marks zygotic genome activation in biparental and parthenogenetic porcine embryos and correlates with in vitro developmental potential, Reprod. Fertil. Dev. 20 (7) (2008) 818–825. [23] W. Davis Jr., P.A. De Sousa, R.M. Schultz, Transient expression of translation initiation factor eIF-4C during the 2-cell stage of the preimplantation mouse embryo: identification by mRNA differential display and the role of DNA replication in zygotic gene activation, Dev. Biol. 174 (2) (1996) 190–201. [24] I.O. Bogolyubova, Transcriptional activity of nuclei in 2-cell blocked mouse embryos, Tissue Cell 43 (4) (2011) 262–265. [25] S. Komatsu, S. Suzuki, H. Kitai, Y. Endo, T. Fukasawa, R. Iizuka, Studies on in vitro 2-cell block mechanism in the mouse with special reference to cytoplasmic factors, Prog. Clin. Biol. Res. 296 (1989) 322–326. [26] H. Nakano, H. Kubo, Rescue of mouse embryos from 2-cell blocks by microinjection of maturation promoting factor, Fertil. Steril. 75 (6) (2001) 1194–1197. [27] J.J. Qiu, W.W. Zhang, Z.L. Wu, Y.H. Wang, M. Qian, Y.P. Li, Delay of ZGA initiation occurred in 2-cell blocked mouse embryos, Cell Res. 13 (3) (2003) 179–185. [28] A.R. Greenlee, C.A. Quail, R.L. Berg, The antiestrogen ICI 182,780 abolishes developmental injury for murine embryos exposed in vitro to o,p0 -DDT(1), Reprod. Toxicol. 14 (3) (2000) 225–234. [29] A.E. Wakeling, Use of pure antioestrogens to elucidate the mode of action of oestrogens, Biochem. Pharmacol. 49 (11) (1995) 1545–1549. [30] A. Giddabasappa, M. Bauler, M. Yepuru, E. Chaum, J.T. Dalton, J. Eswaraka, 17-b Estradiol protects ARPE-19 cells from oxidative stress through estrogen receptor-b, Invest. Ophthalmol. Vis. Sci. 51 (10) (2010) 5278–5287. [31] J. Frasor, D.H. Barnett, J.M. Danes, R. Hess, A.F. Parlow, B.S. Katzenellenbogen, Response-specific and ligand dose-dependent modulation of estrogen receptor (ER) alpha activity by ERbeta in the uterus, Endocrinology 144 (7) (2003) 3159–3166. [32] J. Chai, K.F. Lee, E.H. Ng, W.S. Yeung, P.C. Ho, Ovarian stimulation modulates steroid receptor expression and spheroid attachment in peri-implantation endometria: studies on natural and stimulated cycles, Fertil. Steril. 96 (3) (2011) 764–768. [33] A. Bettegowda, G.W. Smith, Mechanisms of maternal mRNA regulation: implications for mammalian early embryonic development, Front. Biosci. 12 (2007) 3713–3726. [34] Q. Wang, K.E. Latham, Translation of maternal messenger ribonucleic acids encoding transcription factors during genome activation in early mouse embryos, Biol. Reprod. 62 (4) (2000) 969–978. [35] T.S. Macfarlan, W.D. Gifford, S. Agarwal, S. Driscoll, K. Lettieri, J. Wang, S.E. Andrews, L. Franco, M.G. Rosenfeld, B. Ren, S.L. Pfaff, Endogenous retroviruses and neighboring genes are coordinately repressed by LSD1/KDM1A, Genes Dev. 25 (6) (2011) 594–607. [36] T. Suzuki, N. Minami, T. Kono, H. Imai, Zygotically activated genes are suppressed in mouse nuclear transferred embryos, Cloning Stem Cells 8 (4) (2006) 295–304.
