Molecular Human Reproduction, Vol. 7, No. 5, 431-436,
May 2001
© 2001 European Society of Human Reproduction and Embryology
Embryology |
Expression of Fas and Fas ligand mRNA in rat and human preimplantation embryos
1 Department of Obstetrics and Gynecology, Akita University School of Medicine and 2 Akita University College of Allied Medical Science, Akita, 010-0041 Japan
Abstract
The Fas-Fas ligand (L) system is one of the major signalling pathways to induce apoptosis in various cells and tissues. The aim of this study was to investigate the expression of the Fas-Fas L system in rat and human oocytes and preimplantation embryos. We determined the expression of Fas and Fas L mRNA of rat oocytes and embryos up to the blastocyst stage, and of human embryos at the 2- or 4-cell stage, using reverse transcription polymerase chain reaction (PCR) and nested PCR techniques. Moreover, we investigated the expression of Fas mRNA in human fragmented embryos. In rat embryos, Fas mRNA was expressed at the 2-cell stage only, whereas Fas L mRNA was expressed in oocytes, and at the pronuclear (1-cell) and 2-cell stages. In human embryos, Fas mRNA was expressed at the 4-cell stage only, whereas Fas L mRNA was expressed at both 2- and 4-cell stages. Human fragmented embryos expressed both Fas and Fas L mRNA. Because simultaneous expression of Fas and Fas L mRNA occurred in 2-cell rat embryos and in 4-cell human embryos, the Fas–Fas L system might be involved in the apoptotic pathway in the early embryos of these species.
apoptosis/embryo/Fas/Fas ligand/RT-PCR
Introduction
Apoptosis or programmed cell death is an essential physiological process in the normal development of embryos either for eliminating abnormal embryonic cells or for controlling the number of developing embryos in various species (Kerr et al., 1972
; Allen, 1987; Schwartzman and Cidlowski, 1993). Recent studies have focused on the role of apoptosis in degenerative early embryos during in-vitro culture. Some possible causes of apoptosis in the preimplantation embryos; e.g. suboptimal culture condition and/or lack of growth factors, have been proposed (Hardy, 1997, 1999), but the mechanisms whereby apoptosis is induced in preimplantation embryos are not clear.
Diverse signals and molecular pathways lead to apoptosis, and these apoptotic pathways are thought to terminate in the activation of the caspase family of proteases. Recently, Exley et al. demonstrated that murine preimplantation embryos express various members of the caspase proteins (Exley et al., 1999). Caspase activity is regulated on the basis of the balance of apoptotic or survival gene products (Cohen, 1997; Salveson and Dixit, 1997; Cryns and Yuan, 1998), and murine preimplantation embryos have been reported to express some of these genes (Jurisicova et al., 1998a; Exley et al., 1999). However, the signals that induce apoptosis in preimplantation embryos are still unknown.
The Fas–Fas L system is a major pathway in the induction of apoptosis in various cells and tissues. Fas is a 45 kDa type I membrane protein that belongs to the tumour necrosis factor (TNF) receptor family which includes TNF receptor p55 (TNFR p55). In contrast, Fas L, a 37 kDa type II membrane protein, belongs to the TNF and CD40 ligand family of proteins. Upon contact with Fas L, cells expressing Fas undergo apoptosis rapidly by way of an intracellular signalling pathway dependent on a distinct cytoplasmic motif called `death domain' (Itoh and Nagata, 1993; Nagata and Golstein, 1995). Although the expression of Fas and Fas L proteins in human gametes and embryos has been examined by Zaninovic et al. (1997), there have been no known reports concerning the expression of the Fas and Fas L mRNA in human and rodent preimplantation embryos.
In human embryos, the fragmentation of one or more blastomeres is thought to be associated with decreased developmental potential. Recent studies in mice and human have reported that apoptosis is responsible for fragmentation of oocytes and preimplantation embryos assessed by morphological criteria, terminal transferase-mediated DNA end labelling (TUNEL) analysis of DNA cleavage products (Jurisicova et al., 1995, 1996Jurisicova et al., 1998; Takase et al., 1995; Fujino et al., 1996; Yang, 1998) or annexin V staining of externalization of phosphatidylserine (Levy et al., 1998; Perez et al., 1999). However, the correlation between fragmentation and apoptosis is still controversial, because it has found (Antczak and Van Blerkom et al., 1999) that the majority of human fragmented embryos are not labelled with annexin V staining and TUNEL analysis.
In the present study, we sought to determine (i) the temporal expression of Fas and Fas L mRNA in rat oocytes and preimplantation embryos up to the blastocyst stage by reverse transcription-polymerase chain reaction (RT–PCR) and nested PCR techniques, (ii) the expression of Fas, Fas L and TNFR p55 mRNA in human embryos at the 2-cell and 4-cell stages, and (iii) the expression of Fas, Fas L and TNFR p55 mRNA in fragmented human embryos.
Materials and methods
Rat oocytes and embryos
Female Wistar-Imamichi rats, aged 9 weeks, were purchased from the Institute for Animal Reproduction, Ibaragi, Japan. Rats were killed by cervical dislocation at different intervals after mating, and oocytes or embryos at pronuclear (1-cell), 2-cell (late 2-cell), 4-cell, 8-cell, morula or blastocyst stages were collected by flushing oviducts or uterine horns with human tubal fluid (HTF) medium (Ansai et al., 1994). Oocytes and 1-cell stage embryos were immediately incubated in HTF medium containing 1 mg/ml hyaluronidase (Sigma, St Louis, MO, USA) for 5 min at 37°C to remove cumulus cells. Oocytes and embryos without fragmentation were used for this study. Each of 15 cells recovered was suspended in 20 µl of HTF medium, and stored frozen at –80°C until mRNA extraction. All procedures involving the care and use of animals were approved by the Animal Research Committee at Akita University School of Medicine.
Human embryos
We used surplus embryos at the 2- or 4-cell stages from couples who did not wish to use the embryo for their IVF treatments at the Department of Obstetrics and Gynecology, Akita University School of Medicine (Kodama et al., 1995), and after they agreed to provide us with their embryos for this research. Among 41 surplus embryos, five were at the 2-cell stage and 18 were at the 4-cell stage. All the remaining 18 embryos at the 4-cell stage were classified as type IV fragments, which were equal or close to the size of whole blastomeres according to published criteria (Warner et al., 1998), and these embryos were characterized as fragmented embryos. The human embryos were used for mRNA extraction without storage. This research was approved by the Human Ethics Committee of the Akita University School of Medicine.
RT–PCR and nested PCR
The poly (A)+ mRNA was isolated by a mRNA isolation kit (Micro-FastTrackTM mRNA Isolation Kit; Invitrogen, San Diego, CA, USA) with slight modifications (Lu et al., 1993). Twenty picograms of rabbit 
-globin mRNA (Gibco BRL, Rockville, USA) was added to each sample before mRNA extraction in order to evaluate the efficiency of mRNA extraction and the RT procedure.
Primers used for RT–PCR and (hemi-) nested PCR of Fas, Fas L, TNFR p55, ß-actin and -globin were synthesized by Sawady Technology (Tokyo, Japan). The sequences of the primers for PCR (listed in Table I) were designed according to published cDNA sequences (Loetscher et al., 1990; Itoh et al., 1991; Suda et al., 1993; Kimura et al., 1994; Mita et al., 1994; Takahashi et al., 1994; Alderson et al., 1995). To ensure that the PCR products were from cDNA, and not from contaminating genomic DNA, the primers were designed to amplify sequences spanning introns.
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The mRNA extracted from oocytes or embryos at different stages was used for each RT reaction. For each reaction, 2 µl of mRNA was suspended in 17.5 µl of RT mixture [4 µl of 5xcloned Moloney Murine Leukemia Virus (M-MLV) RT buffer (Toyobo, Osaka, Japan), 4 µl of 2.5 mmol/l dNTP mixture, 1 µl of oligo (dT)-Latex (Takara, Tokyo, Japan), and 8.5 µl of distilled water] in a thin wall PCR tube (0.2 ml, Perkin-Elmer, Norwalk, CT, USA). The RT reaction was initiated by adding 0.5 µl of M-MLV reverse transcriptase (Toyobo) with a program of 37°C for 60 min, and 95°C for 10 min, in a thermal cycler (GeneAmp® PCR System 2400; Perkin Elmer).
The amounts of cDNA subjected to each PCR reaction were equivalent to the number of genomes (e.g. one 2-cell stage embryo or one-quarter of an 8-cell stage embryo) according to a published method (Wu et al., 1998), so that each PCR product was derived from the same number of transcribing genomes. For the first PCR, each 2 µl of RT products was suspended in 48 µl of PCR mixture [5 µl of 10xEx-TaqTM buffer, 4 µl of 2.5 mmol/l dNTP mixture, 1.25 U/µl TaKaRa Ex-TaqTM DNA polymerase (all Takara), 37 µl of distilled water, and 2 µl of 10 µmol/l sense and antisense primers]. After mixing all reagents in a PCR tube, the PCR reaction was performed in a thermal cycler according to the programmes described in Table I
. For positive controls, human placenta cDNA and rat granulosa cell cDNA were amplified simultaneously. For negative controls, a specimen in which water was substituted for mRNA was amplified. Due to small amounts of Fas, Fas L and TNFR p55 mRNA in embryos, nested PCR was needed to obtain optimal results. For the nested PCR, 2 µl of the first PCR product was suspended in the PCR mixture containing the second sense and antisense primers (Table I). The nested PCR was performed according to the programme described in Table I.
The PCR products were separated by 2% agarose gel electrophoresis (Agarose-LE, Nacalai Tesque Inc., Kyoto, Japan) in the presence of ethidium bromide solution (Sigma), and visualized with UV transilluminator (Funakoshi, Tokyo, Japan).
Assay for apoptosis
We confirmed that the present treatment of oocytes and embryos with hyaluronidase did not induce apoptosis by TUNEL analysis and annexin V staining (data not shown). These studies were performed according to a published method (Levy et al., 1998). Positive controls were prepared by treating embryos with DNase I (Sigma, St Louis, MO, USA) for TUNEL analysis and with staurosporine at 10 µmol/l (Sigma, St Louis, MO, USA) for annexin V staining (Weil et al., 1996; Matwee et al., 2000).
Results
Expression of Fas and Fas L mRNA in rat oocytes and preimplantation embryos
The expression of Fas and Fas L mRNA in the rat oocytes and embryos at different stages (1-cell, 2-cell, 4-cell, 8-cell, morula and blastocyst) is shown in Figure 1 and summarized in Table II. Fas mRNA was detected in 2-cell stage embryos only, whereas Fas L mRNA was expressed in oocytes, 1-cell and 2-cell stage embryos, but disappeared in embryos at and after the 4-cell stage. All PCR products were validated by diagnostic restriction enzyme digestion (data not shown). The expression of ß-actin and -globin mRNA was detected in the first PCR amplification, and no significant differences were observed in the band intensities of -globin and ß-actin amplification products among oocytes and embryos at different stages. Experiments in the present study were performed three times on five separate pools of 15 oocytes and embryos at each stage, and the results were found to be reproducible.
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x 174-Hae III digest (Takara); posi = positive control for Fas, Fas L, p55 and ß-actin (rat granulosa cell cDNA); nega = negative control (distilled water); bp = base pair.
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Expression of Fas, Fas L and TNFR p55 mRNA in human preimplantation embryos
The expression of Fas, Fas L and TNFR p55 mRNA in the human 2-cell and 4-cell stage embryos is shown in Figure 2
and summarized in Table II. Fas mRNA was detected only in 4-cell stage embryos, whereas Fas L mRNA was expressed in both 2-cell and 4-cell stage embryos. TNFR p55 mRNA was expressed in 2-cell stage embryos only. Fragmented embryos expressed both Fas and Fas L mRNA, but did not express TNFR p55 mRNA. All PCR products were validated by the method described above. All assays were repeated three times with the same results.
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Discussion
In rat oocytes and preimplantation embryos, Fas mRNA was expressed in late 2-cell stage embryos only, whereas Fas L mRNA was expressed in oocytes, and in pronuclear (1-cell) and late 2-cell stage embryos. During rat embryo development, embryonic genome activation, which is an event of transition from maternally inherited RNA and proteins to newly translated embryonic ones, is known to take place between the middle 2-cell and early 4-cell stages (Zernicka-Goetz, 1994). Thus, transcripts from embryonic genes are detectable after the late 2-cell stage. In this experiment, Fas mRNA was detected at late 2-cell stage only, suggesting that this transcript may be originating from an embryonic gene. On the other hand, Fas L mRNA of rat embryos probably is maternally inherited RNA, because it was detected in the oocytes and embryos at the stages prior to embryonic genome activation.
In human embryos, Fas mRNA was expressed in 4-cell stage embryos only, whereas Fas L mRNA was expressed in both 2-cell and 4-cell stage embryos (Table II). In the human, embryonic genome activation is reported to take place between the 4-cell and 8-cell stages (Braude et al., 1988), and, similar to rat embryos, co-expression of Fas and Fas L mRNA occurred at the stage when activation of the embryonic genome was initiated. However, the population of 4-cell embryos examined in this study was heterogeneous because of different timing post insemination, and therefore it may be difficult to estimate the precise period in which both Fas and Fas L mRNA are expressed. It has been demonstrated (Zaninovic et al., 1997) that the Fas protein was detected in human oocytes and preimplantation embryos up to the blastocyst stage, whereas the Fas L protein was not detected in either oocytes or embryos using immunofluorescence and immunoblotting. The differences of the stages of Fas and Fas L expression based on the mRNA analysis in our study and the protein determination in their study cannot be clearly explained, but may be attributed to differential translational or post-translational regulation.
The simultaneous expression of Fas and Fas mRNA occurred in 2-cell stage rat embryos. The blastomeres of 2-cell stage embryos could undergo apoptosis, when the Fas L-bearing blastomere interacts with membrane bound Fas. Because co-expression of Fas and Fas L mRNA could induce apoptosis during the process of normal embryonic development, additional mechanisms may need to be activated to inhibit Fas receptor signalling in the early embryos. Alternatively, it is possible that the five separate pools of 15 rat embryos at 2-cell stage used for this RT–PCR study may contain abnormal embryos that would eventually undergo apoptosis, and the expression of both Fas and Fas L mRNA may be associated with pre-apoptotic embryos.
Numerous studies have been reported on apoptosis in mammalian blastocysts (Hardy, 1997, 1999). Apoptosis has also been shown to occur in bovine embryos of normal morphology at 8–16 cells up to blastocyst stage (Byrne et al., 1999; Matwee et al., 2000). During blastocyst development, apoptosis is thought to have a protective role by eliminating abnormal cells or redundant inner cell mass cell with the potential to differentiate in trophectoderm (Pierce et al., 1989). Recent examinations have revealed that some growth factors regulate apoptosis in blastocyst. Supplementation of culture medium with transforming growth factor alpha or insulin-like growth factor-I (IGF-I) reduce the incidence of apoptosis in blastocyst (Brison and Schultz, 1997; Herrler et al., 1998; Spanos et al., 2000). In this study, the lack of expression of both Fas and Fas L mRNA in rat blastocysts suggests that the Fas and Fas ligand system is not operational in embryos at this stage.
Over the past few years, several studies, as mentioned earlier, have focused on the correlation between apoptosis and fragmentation of oocytes and preimplantation embryos assessed by morphological criteria, TUNEL analysis of DNA cleavage and annexin V staining of externalization of phosphatidylserine. However, it is not clear whether apoptosis is responsible for fragmentation of oocytes and preimplantation embryos, because it was found (Antczak and Van Blerkom et al., 1999) that the majority of human fragmented embryos did not label with annexin V staining and TUNEL analysis. These authors postulated that fragmentation was not part of, or a consequence of, apoptosis. Little is known about the molecular mechanisms, especially ligand–receptor-mediated signal transduction systems that induce apoptosis in preimplantation embryos. It has been demonstrated (Jurisicova et al., 1998a) that the expression of apoptotic (MA-3, p53, bad and bcl-xS) genes are increased, whereas the expression of a survival (bcl-2) gene is reduced in fragmented murine embryos. Exley et al. also showed that levels of bcl-2 immunofluorescence in the fragmented murine blastocysts are lower than that of normal blastocysts (Exley et al., 1999). Both bax and bcl-2 mRNA are expressed between the 2- and 12-cell stages of human embryos (Warner et al., 1998). A high level of bcl-2 and low level of Bax immunostaining has been observed in human blastocysts with normal morphology, but in fragmented blastocyst, the level of Bax immunostaining was increased (Hardy, 1999). In this study, human fragmented embryos expressed both Fas and Fas L mRNA which may be involved in the initiation of apoptosis. However, whether the Fas and Fas L system plays a significant role in the formation of fragmented embryos remains to be determined.
Similar to Fas, TNF receptor p55 (TNFR p55) is a death domain-containing receptor known to mediate apoptosis in various cells and tissues (Greenblatt and Elias, 1992; Tartagria et al., 1993). We also examined the expression of TNFR p55 mRNA in human embryos, and found that the TNFR p55 mRNA was expressed in 2-cell stage embryos, but not at the 4-cell stage. The expression of TNFR p55 was not detected in fragmented embryos. In human embryos, TNF-, a ligand of TNFR p55, was reported to be expressed in the 4-cell and morula stages, but there was no expression in the 2-cell stage (Sharkey et. al., 1995). Thus, TNFR p55 is unlikely to be involved in the signal transduction pathway of apoptosis in human embryos.
In summary, we used RT–PCR and nested PCR to investigate the expression of Fas and Fas L in human and rat preimplantation embryos, and have shown that the simultaneous expression of Fas and Fas L mRNA in preimplantation embryos occurred at the 2-cell stage in rat and at the 4-cell stage in human. Human fragmented embryos also expressed both Fas and Fas mRNA. These data suggest that the Fas–Fas L system may be involved in apoptosis regulation in rat and human early embryos.
Acknowledgements
We thank Dr Aaron J.Hsueh, Stanford University School of Medicine (Stanford, CA, USA), for reading this manuscript. This work was supported by a Grant-in Aid for Scientific Research (C: 30195747) from the Japanese Ministry of Education, Science, Sports and Culture.
Notes
3 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, Akita University School of Medicine, Akita, 010-0041 Japan. E-mail: kawamurak{at}obgyn.med.akita-u.ac.jp 
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Submitted on October 2, 2000; accepted on February 12, 2001.
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