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Regular Article |
c Laboratory of Reproductive Physiology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
d Maebashi Institute of Animal Science, Livestock Improvement Association, JAPAN, Inc., Maebashi 371-0121, Japan
ABSTRACT
The effect of the oviductal environment on gene expression in 2-cell mouse embryos was examined with mRNA differential display. Embryos used for experiments were cultured in modified Whitten medium with or without oviductal tissue until late 2-cell stage. The results of sequencing indicated that the genes for ATP synthase (ATPase 6), S-adenosylmethionine decarboxylase (S-AMDC) and nuclear autoantigenic sperm protein (NASP) were differentially expressed in embryos cultured in the oviductal environment (nonblocking culture condition). The ATPase 6 gene is encoded by mitochondrial DNA and is essential for the production of ATP. This indicates that the expression of ATP synthesis-related genes at the 2-cell stage may be required to maintain normal development in vitro. S-Adenosylmethionine decarboxylase decarboxylates adenosylmethionine, which is a substrate of DNA methylation. The expression of S-AMDC may be responsible for the low level of methylation of preimplantation development. As NASP is a histone-binding protein that is thought to be testis and sperm specific, its function in embryos remains unclear. On the other hand, the Tcl1 gene and a novel gene, the c-1 gene, were strongly expressed in embryos cultured without oviductal tissue (blocking culture condition). The expression patterns of these genes are quite similar. However, the detailed functions of these genes in embryos remain to be determined.
gametogenesis, implantation/early development
INTRODUCTION
The development of 1-cell mouse embryos, except for embryos of some inbred strains and their F1 hybrids, is blocked at the 2-cell stage, a phenomenon that has been termed the 2-cell block [1–3]. Cross-breeding experiments have revealed that maternally inherited developmental information plays an important role in controlling early cleavage of the mouse embryo [2]. In addition, the transfer of cytoplasm from nonblocked embryos into blocked embryo recovers the developmental competence of 2-cell embryos in vitro [4]. The developmental arrest in vitro can also be overcome by modifying the culture conditions: addition of EDTA [5, 6] and deletion of phosphate [7] can eliminate the developmental arrest in vitro. Biggers et al. [8] demonstrated that isolated mouse ampulla maintained in organ culture can overcome the 2-cell block in the mouse. Thereafter, various culture media and several culture systems have been exploited. Mouse 1-cell embryos can develop to the blastocyst stage by coculturing them with an isolated mouse ampulla, and the effect is restricted to the G2 phase of the second cell cycle [9]. In many mammalian species, development of 1-cell embryos is also blocked at various early stages in vitro. It has been reported that the time of developmental arrest in vitro coincides with the time of zygotic gene activation [10], suggesting that there may be a relationship between the developmental arrest and transcriptional activity of embryos.
In mammals, zygotic gene activation has been shown to be a two-step process consisting of minor and major phases [11]. In the mouse, the minor zygotic gene activation phase is initiated at the late 1-cell stage (G2 phase) with a very weak transcriptional activity [12–18]. Consequently, some of the proteins are synthesized at the early 2-cell stage (G1/S) for the next phase of zygotic gene activation, the major phase [19–24]. Recently, it has been reported that reporter genes microinjected into the pronuclei of 1-cell mouse embryos are transcribed during the minor zygotic gene activation phase [17, 25–28]. However, transcription of the reporter genes is repressed after the first mitosis unless the reporter genes contain the appropriate enhancers [26]. At the G2 phase of the second cell cycle, the major zygotic gene activation phase, which is characterized by an increase of transcriptional and translational activity, occurs and results in a dramatic change in the pattern of protein synthesis [11, 19, 29–32]. Furthermore, embryos arrested at the 2-cell stage are tetraploid [2], the embryos are able to incorporate bromodeoxyuridine before arrest [33], and in addition, there is no sign of nuclear envelope breakdown or chromosome condensation in a 2-cell blocked embryo [33]. These results indicate that embryos that are blocked at the 2-cell stage are arrested at the G2 phase of the second cell cycle.
One-cell mouse embryos cultured in medium containing micromolar concentrations of EDTA or in medium without phosphate can develop beyond the 2-cell stage ([5, 34]; unpublished data). However, these culture conditions are far from physiological because oviductal fluid does not contain EDTA and does contain phosphate.
Although several attempts have been made to overcome the developmental arrest in vitro, few studies have attempted to elucidate the genetic mechanism of this arrest. As it has become possible to overcome the 2-cell block by changing the culture environment, clues to this mechanism may be provided by identifying differentially expressed genes of preimplantation embryos cultured under different conditions. Although several reports have investigated gene expression in preimplantation embryos, none of them addressed the relationship between gene expression and developmental arrest.
In the present study, we examined differences in gene expression in mouse embryos that develop normally and mouse embryos that exhibit the 2-cell block in vitro using the fluorescein differential display method. We also used a coculture method to overcome the 2-cell block in vitro.
MATERIALS AND METHODS
In Vitro Fertilization and Embryo Culture
Three- to five-week-old female Crj:CD-1(ICR) mice (Charles River Japan Inc., Tokyo, Japan) were superovulated with i.p. injections of 5 IU eCG (Teikoku Hormone M.F.G. Co., Ltd., Tokyo, Japan) followed in 48 h by 5 IU hCG (Sankyo Zoki Co., Ltd., Tokyo, Japan). Ovulated oocyte-cumulus complexes were collected from ampullae of the oviduct 15 h after hCG injection and placed in a 200-µl droplet of fertilization medium. Spermatozoa were collected from the cauda epididymis of male mice of the same strain and cultured for 1 h in 400 µl of preincubation medium. The medium used for in vitro fertilization (IVF) was modified Whitten medium (m-WM) [9] containing 4 mg/ml BSA (A7638; Sigma Chemical Co., St. Louis, MO). After preincubation, sperm were introduced into fertilization droplets at a final concentration of 2 x 104 cells/ml.
Oviducts for coculture were flushed with m-WM containing 3 mg/ml polyvinylpyrrolidone (PVP; K-30, Nacalai tesque, Kyoto, Japan) at the time of oocyte collection and the isthmic and fimbrial regions of oviducts were removed. The ampullae were dissected longitudinally with microscissors and washed two times with m-WM containing PVP and then transferred to 100 µl of culture medium (m-WM containing 3 mg/ml PVP, 1 ampulla/100 µl).
Five hours after insemination, fertilized eggs were washed three times with m-WM containing PVP and transferred to 100 µl of culture medium with an ampulla (nonblocking culture condition) or without an ampulla (blocking culture condition).
All incubations were performed in a humidified atmosphere containing 5% CO2 and 95% air at 37°C.
All animal experiments were performed in accordance with Institutional Animal Care and Use Committee and in adherence with guidelines established in the Guide for Care and Use of Laboratory Animals as adopted and promulgated by the Society for the Study of Reproduction.
Embryo Collection
In our preliminary experiments, the first cleavage of oocytes fertilized in vitro was observed 16–18 h after insemination and the second cleavage was observed approximately 24 h later. Twenty-one hours after the first cleavage, corresponding to the late G2 phase of the second cell cycle of the embryos, 280 2-cell embryos from each culture condition were collected and used for the extraction of total RNA and synthesis of cDNA for fluorescein differential display reverse transcriptase polymerase chain reaction (FDD-RT-PCR). For semiquantitative RT-PCR, 50 embryos were collected at the same time. The collected embryos in <5 µl of culture medium were stored in liquid nitrogen until use (for 1 mo at most).
RNA Isolation and Purification
Total RNA was isolated from each group of 280 2-cell embryos (cultured with or without ampullae) using an RNA isolation kit (Stratagene, La Jolla, CA) according to the manufacturer's instructions. Briefly, embryos were suspended in 100 µl of denaturing solution and vortexed for 2 min at room temperature. After phenol-chloroform extraction, the supernatant was transferred to a fresh microfuge tube containing 2.5 µl glycogen (20 mg/ml) and 100 µl of isopropyl alcohol, and then stored at -80°C for 30 min. Total RNA was obtained as a pellet with glycogen after centrifugation at 12 000 x g for 30 min at 4°C. The pellet was resuspended with 50 µl of 0.1% diethyl pyrocarbonate (DEPC)-treated water. The isolated RNA was then treated with RNase-free DNase (Life Technologies, Inc., Rockville, MD) for 30 min at 37°C. After treatment with DNase, the solution was extracted with phenol-chloroform, ethanol precipitated with 2.5 µl of glycogen (20 mg/ml), and then resuspended with 28.2 µl DEPC water (9.4 µl x3).
Fluorescein Differential Display RT-PCR
Reverse transcription was performed on RNA obtained from an equivalent of 93 2-cell embryos using Moloney murine leukemia virus (M-MLV) reverse transcriptase (Superscript II, Life Technologies) and the three one-base-anchored oligo dT(15) primers with an additional HindIII site on its 5' end in each 20 µl of reaction mixture. The samples containing RNA and each primer were heated to 70°C for 10 min and then cooled to 42°C. After cooling, the reaction buffer was added and the mixture was allowed to stand for 2 min. One microliter of Superscript II (200 U, Life Technologies) was added, and the samples were incubated at 42°C for 50 min.
The differential display (DD)-PCR reaction was carried out on each cDNA in 20-µl reaction mixtures containing 50 mM dNTPs, 1.5 mM magnesium chloride, single strength buffer (provided with the enzyme), 1 U AmpliTaq (Applied Biosystems, Foster City, CA), 0.5 µM arbitrary primer (HAP-1: 5'-AAgCTTgATTgCC-3', GenHunter, Nashville, TN) and 0.25 µM of each one base-anchored oligo dT(15) primer (HT15M) labeled with fluorescein isothiocyanate (FITC). The samples were subjected to PCR on a thermal cycler (PTC-100; MJ Research, Watertown, MA) under the following conditions: (94°C for 2 min, 40°C for 5 min, 72°C for 5 min) x 1 cycle and (94°C for 15 sec, 40°C for 2 min, 72°C for 1 min) x 39 cycles; the last cycles was followed by a 5-min extension at 72°C. The fluorescein-labeled amplified cDNA fragments were resolved on a 6% sequencing gel and visualized by a fluorescent image analyzer (FMBIO II; Hitachi Software Engineering Inc., Yokohama, Japan).
Recovery and Reamplification of cDNA Fragments
After making an image of the gel with the analyzer, the gel plate was placed on a printed image in layers, and the area of a band of interest was cut out from the gel. The gel slice was transferred to a 1.5-ml polypropylene tube and stored at -20°C until use. The gel slice was soaked in 100 µl of sterile water and stored for 10 min at room temperature, and then boiled for 5 min for extraction. After boiling, the solution was ethanol precipitated with 2.5 µl of glycogen (20 mg/ml). The pellet was resuspended with 10 µl of sterile water, and 4 µl of the resuspension was used for amplification. Amplification was performed in a 20-µl reaction mixture containing 200 µM dNTPs, 1.5 mM MgCl2, single-strength buffer (provided with the enzyme), 1 U AmpliTaq, 0.2 µM HAP-1, and 0.2 µM each HT15M primer under the same conditions of DD-PCR. The amplified products were confirmed to have the appropriate sizes by electrophoresis in a 2% agarose gel.
Cloning of Amplified cDNA Fragment and DNA Sequencing
The amplified cDNA was cloned using a TOPO TA cloning kit (Invitrogen Corp., Carlsbad, CA) according to the manufacturer's instructions. Cloned DNA was sequenced with an ABI Prism 377 automatic sequencer using an ABI DNA sequencing kit (Dye Terminator Cycle Sequencing FS Ready Reaction Kit; Applied Biosystems). Sequence analysis was carried out with the DNA sequence assembly software package AutoAssembler (Applied Biosystems). The DNA sequences were compared with the sequences in the GenBank, EMBL, and dbEST databases using the BLAST program.
Semiquantitative RT-PCR
Total RNA was isolated from each group of 50 embryos 21 h after cleavage (cultured with or without oviductal tissue) using an RNA isolation kit (Stratagene), and first-strand cDNA was synthesized with M-MLV reverse transcriptase (Superscript II) and random primers (Life Technologies) in 20-µl reaction mixtures as described above. The PCR reactions were performed in 20-µl reaction mixtures containing 0.01–1 µl of cDNA, 200 µM dNTPs, 0.5 U Taq polymerase (Takara), single-strength reaction buffer (provided with the enzyme), and a pair of primers specific for each gene or ß-actin (20–50 pmol each). The PCR products were subjected to electrophoresis. Relative band intensities were determined with a model 4.0 Atto densitograph (Atto Inc., Tokyo, Japan). Intensities were expressed relative to the intensity of the band for ß-actin expression, and the averages of three trials were compared and analyzed in each gene (Fig. 2).
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RESULTS
As shown in Table 1, 95% of the IVF embryos developed to the 4-cell stage and 68% developed to the blastocyst stage when they were cultured with oviductal tissue. On the other hand, only 18% developed beyond the 2-cell stage and only 6% reached the blastocyst stage without oviductal tissue (Table 1). This result indicates that the oviductal tissue plays an important role in the development of mouse IVF embryos in vitro.
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As shown by electrophoresis of FDD-RT-PCR products (Fig. 1), several genes were found to be differentially expressed under different culture conditions. The differentially expressed bands were excised and DNAs were extracted from the gels for sequencing. After sequencing, homology searches were performed using the GenBank and EMBL databases. In order to confirm the differential expression of the genes that were observed with FDD-RT-PCR, gene-specific primers were designed and used for RT-PCR. As shown in Figure 2, several differentially expressed genes at 21 h after the first cleavage were identified using comparative quantitation with the ß-actin gene. As summarized in Figure 2, ATP synthase 6 (ATPase 6), S-adenosylmethionine decarboxylase (S-AMDC), eukaryotic translation initiation factor 6 (eIF6), and nuclear autoantigenic sperm protein (NASP) were strongly expressed in embryos developed under nonblocking culture conditions (with oviductal tissue) in which embryos do not exhibit developmental arrest at the 2-cell stage. On the other hand, Tcl1 and a novel gene, c-1, were strongly expressed in the embryos developed under blocking culture conditions (without oviductal tissue) in which embryos exhibit developmental arrest at the 2-cell stage. Expression of the novel gene c-1 was not observed in liver, kidney, spleen, heart, lung, or brain, but it was observed in oocytes and the ovary. However, an alternative-splicing product was amplified when the testis cDNA was used (data not shown). In addition, a difference in the expression in 2-cell embryos was already observed at 1 h after the first cleavage, and no expression was observed after the 4-cell stage (data not shown).
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DISCUSSION
When mouse embryos are cultured in vitro from the 1-cell stage, development beyond the 2-cell stage is arrested. As shown in Table 1, the presence of oviductal tissue was effective in overcoming the developmental arrest in vitro.
As shown in Figure 1, several genes were found to be more highly expressed under nonblocking conditions. One of these genes is a mitochondrial gene that encodes ATPase 6. ATPase 6 is one of the subunits of F1Fo-ATP synthase, which is complex V of the respiratory chain and is encoded by mitochondrial DNA. The F1Fo-ATP synthase is composed of 14 polypeptide subunits in mammals; two of the subunits are encoded in mitochondrial DNA (subunits a and A6L), and the remainder are encoded by nuclear genes. Mitochondrial DNA does not undergo replication during early development through the blastocyst stage [35, 36], although it is transcribed actively from the 2-cell stage onward [35]. Expressions of the genes for subunit alpha (encoded by the nuclear genome) and subunit A6L (encoded by mitochondrial DNA) were also more prominent in nonblocked embryos than in blocked embryos (data not shown). The development of nonblocked embryos in vitro may be due to the higher transcriptional activity of these ATP synthase-related genes. Taylor and Piko [37] showed that the activation and transcription of the mitochondrial and nuclear genes for the components of the respiratory apparatus are coordinated and that these events begin with the 2-cell stage and increase dramatically during the cleavage stage. They also suggest that this rapid buildup of the mitochondrial oxidative phosphorylation system is mostly preparatory for postimplantation development. This new transcription plays an essential role in mitochondrial differentiation during cleavage [38]. These results indicate that the transcriptional activity of the components of the respiratory apparatus at the late 2-cell stage play important roles in the further development of embryos. This transcriptional activity may also affect the cytoplasmic localization of mitochondria. In developmentally arrested embryos in vitro, the mitochondria become progressively aggregated and localized in the perinuclear region and in the area of the cytocortex immediately adjacent to the plasma membrane [39], while in normally cleaving embryos in vitro and in vivo they are found to be homogeneously distributed throughout the cytoplasm of the blastomeres during interphase [39].
S-Adenosylmethionine decarboxylase, through its substrate, S-adenosylmethionine (AdoMet), is involved in DNA methylation and polyamine synthesis. Changes in S-AMDC expression could cause changes in cellular polyamine levels that, in turn, may affect the degree of DNA methylation. The DNA methylation patterns of the male and female pronuclei are erased in the morula and early blastocyst, and when the blastocyst forms, most of the DNA has become demethylated. Although the decarboxylated AdoMet molecule contains a methyl group, it does not act as a methyl group donor in DNA methylation. Instead it acts as a competitive inhibitor of DNA (cytosine-5)methyltransferase (DNA MTase). Therefore, a consequence of polyamine depletion is genomewide loss of DNA methylation due to insufficient maintenance methylation during successive rounds of DNA replication [40, 41]. Thus, activation of S-AMDC may be needed to provide the required level of methylation during normal preimplantation development. In Xenopus embryos, the expression of S-AMDC greatly increased at the time of zygotic gene activation [42], suggesting that the increase of S-AMDC in nonblocked mouse 2-cell embryos is due to the normal occurrence of zygotic gene activation.
Functional analysis of mammalian eIF6 has not yet been determined, however, deletion of the yeast eIF6 is lethal, indicating that eIF6 is an essential gene [43]. Furthermore, the study of the yeast homolog of mammalian translation initiation factor revealed that eIF6 plays a role in translation of mRNA by maintaining the steady-state level of a pool of 60S ribosomal subunit [43, 44]. In the mouse, as translational activity dramatically increases during the 2-cell stage, the increased transcription of eIF6 may play an important role in the development of early preimplantation development.
Nuclear autoantigenic sperm protein was first identified as a nuclear-associated protein in rabbit testis [45]. Nuclear autoantigenic sperm protein has high homology with Xenopus histone-binding protein, N1/N2, which is expressed in oocytes [46, 47]. However, because NASP is thought to be a testis- and sperm-specific protein [45, 48], the function of the transcript in mouse embryos remains unknown.
Tcl1 is an oncogene that is involved in chromosome translocations that are observed mostly in mature T-cell proliferations and chronic lymphocytic leukemias (CLL) [49]. It has been reported that murine Tcl1 mRNAs are abundant in mouse oocytes and 2-cell embryos but rare in various adult tissues and lymphoid cell lines [50]. As shown in Figure 2, the expression of Tcl1 in 2-cell embryos is prominent when the embryos are cultured under blocking conditions. However, the detailed functions of the gene remain to be determined.
In the present study, we also sequenced a novel gene, named c-1, whose expression pattern is quite similar to that of Tcl1. The gene may be germ cell specific because its expression was observed in oocytes, embryos, and ovary but not in liver, kidney, spleen, heart, lung, or brain (data not shown). The fragment that was amplified in testis cDNA was shown by sequencing to be a product of alternative splicing (data not shown).
In the mouse, zygotic gene activation has been shown to be a two-step process composed of a minor zygotic gene activation phase at the late 1-cell stage (G2 phase) with a very weak transcriptional activity and a major zygotic gene activation phase followed by the synthesis of some proteins at the early 2-cell stage (G1/S) [11]. The inhibitory effect of phosphate on mouse embryo development suggests that suppression of initial zygotic gene activation induction at the late 1-cell stage is involved in the 2-cell block in vitro [7]. Also, the influence of oviductal tissue that overcomes the 2-cell block of mouse embryos is critical at the G2 phase of the second cell cycle [9]. These results, together with the present results, indicate that the normal progress of both phases of zygotic gene activation is probably needed to maintain the developmental competence of mouse 2-cell embryos cultured in vitro. Clarification of the key events of normal zygotic gene activation should be of great value in understanding the mechanism of the developmental arrest in vitro.
The two techniques used, mRNA DD and semiquantitative RT-PCR are very sensitive methods. There were several genes that were expressed differentially by DD-RT-PCR but were not confirmed by semiquantitative RT-PCR. Accordingly, there may be the case that the differences obtained by mRNA DD could be due to noise or may not be real. However, these techniques are useful to study genetic mechanisms of early embryogenesis.
In conclusion, several genes that are differentially expressed in 2-cell embryos were identified using FDD. Although the detailed function of the genes and their products remains to be determined, it is likely that the mechanisms of the 2-cell block in vitro can be elucidated genetically by the analysis of differentially expressed genes in the future.
FOOTNOTES
1 Part of this work was supported by a grant from the Ministry of Education, Science and Culture (no. 10660270 to N.M.) and a grant from the Japan Society for the Promotion of Science (JPS-RFTF 97L00905 to N.M.). 
2 Correspondence. FAX: 81 75 753 6329; naojiro{at}kais.kyoto-u.ac.jp
3 Current address: Drug Safety Research Laboratories, Pharmaceutical Research Division, Takeda Chemical Industries, Ltd., 17-85, Jusohonmachi 2-choume Yodogawa-ku, Osaka 532-8686, Japan.
Accepted: August 8, 2000.
Received: June 13, 2000.
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