The incidence of cytoplasmic fragmentation in mouse embryos in vitro is not affected by inhibition of caspase activity

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1 FERTILITY AND STERILITY VOL. 75, NO. 5, MAY 2001 Copyright 2001 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. The incidence of cytoplasmic fragmentation in mouse embryos in vitro is not affected by inhibition of caspase activity Jia-sen Xu, M.Sc., a Tak-ming Cheung, B.Sc., a Samuel Ting-hon Chan, Ph.D., b Pak-chung Ho, M.D., a and William Shu-biu Yeung, Ph.D. a University of Hong Kong, Hong Kong, China Received August 16, 2000; revised and accepted November 13, This work was supported by a grant (HKU241/95M) from the Research Grant Council, Hong Kong. Reprint requests: William Shu-biu Yeung, M.Sc., Department of Obstetrics and Gynaecology, The University of Hong Kong, Queen Mary Hospital, Hong Kong, China (FAX: ; wsbyeung@hkucc.hku.uhk). a Department of Obstetrics and Gynaecology. b Department of Zoology /01/$20.00 PII S Objective: To investigate the relationship between cytoplasmic fragmentation and caspase activity in the mouse embryo. Design: Experimental laboratory study. Setting: University gynacology unit. Animal(s): One-cell zygote of mouse (MF1 BALB/c). Intervention(s): Mouse embryos were treated with caspase inhibitors: benzyloxycarbonyl-val-ala-asp fluoromethylketone (z-vad-fmk) and benzyloxycarbonyl-asp-glu-val-asp-fluoromethyl ketone (Z-DEVDfmk). Main Outcome Measure(s): Morphological development of the embryo, proportion of fragmented embryos, caspase-3-like activity, DNA breakage, and phosphatidylserine exposure in blastomeres. Result(s): The proportion of embryo reaching two-cell, three- to four-cell, and morula stage at 48, 72, and 96 hours after hcg administration, respectively, were comparable between the control embryos and those treated with either z-vad-fmk or z-devd-fmk, at three concentrations (10 M, 50 M, and 200 M). Although the inhibitors suppressed the caspase-3-like activity in the embryo fragment before compaction and decreased DNA breakages, there was no statistically significant difference in the percentage of fragmented embryo between the control and those treated with caspase inhibitors. The inhibitors did not affect the incidence of phosphatidylserine exposure in the blastomere of the treated embryos. Conclusion(s): Cytoplasmic fragmentation in precompaction mouse embryos is not a consequence of caspase-related apoptosis. (Fertil Steril 2001;75: by American Society for Reproductive Medicine.) Key Words: Caspases, apoptosis, fragmentation, embryo Cytoplasmic fragmentation is a common phenomenon in cleavage-stage embryos cultured in vitro. The successful implantation rate of fragmented embryos decreases significantly when the fragments occupy more than 35% of the embryo volume (1). This phenomenon may be related to the quality of the sperm (2), the quality of the oocyte (3), the culture condition (4), or the actin filament distribution in the embryo (5). Jurisicova et al. (6) hypothesized that embryo fragmentation was a result of apoptosis and that the fragments were apoptotic bodies. Indeed, fragmented human embryos have three known markers of apoptosis: 1) the possession of typical apoptotic structures in fragments at electron microscopic level; 2) DNA breakage (6) as detected by the terminal deoxynucleotidyl transferase mediated dutp nick end-labeling technique (TUNEL); and 3) phosphatidylserine exposure on the outer surface of the plasma membrane as determined by annexin-v staining (7). However, Antczak and van Blerkom (8) showed there was no correlation between embryo fragmentation and apoptosis. In fact, they further suggested that fragmentation might induce apoptosis. Numerous studies have been performed on apoptosis in somatic cells such as lymphocytes and neurons. Although apoptosis can be initi- 986

2 ated by various interactions and stimuli, the final common pathway is the activation of a family of proteases known as caspases (9). These are intracellular proteases and exist in the cells as inactive precursors. After being triggered by extracellular or intracellular apoptotic signals, they are cleaved autocatalytically or by other caspases for activation in a cascade fashion, leading to proteolysis and disassembly of structural proteins, DNA degradation, and finally death of the cell with the formation of apoptotic bodies. At least 14 caspases have been reported and the family is still growing. These are categorized into three subfamilies (10): ICE-like caspase (including caspases 1, 4, and 5); CPP32-like caspase (including caspases 3, 6, 7, 8, 9, and 10), and the ICH-1 subfamily (caspase-2). Among these caspases, the CPP32-like subfamily is generally accepted as having a role in the apoptosis cascade (11). The CPP32-like caspases are further classified as initiator (caspases 8, 9, and 10) or execution (caspases 3, 6, and 7) caspases (12). The inducers of apoptosis (e.g., Fas or TNF-R1) activate the initiator caspases which in turn activate the executor caspases. Apoptotic death is irreversibly induced once the execution caspases are activated. Their activation leads to the degradation of a variety of proteins and to the activation of degrading enzymes such as nucleases. Mouse embryos express mrna of caspases 2, 3, 6, 7, and 12 (13). Activity by caspases 2, 3, and 7 can also be detected in the fragmented embryos and staurosporine-treated zygotes of mice (13 14). However, there is no direct evidence demonstrating that caspases are involved in the formation of fragments in embryos. If cytoplasmic fragmentation of embryos was a result of apoptosis, inhibition of the caspase activity would suppress fragmentation in the cleaving embryos. Our study tested this hypothesis by treating the cleavage-stage mouse embryo with two caspase inhibitors, benzyloxycarbonyl-val-ala-asp fluoromethylketone (z-vadfmk), a general inhibitor of the caspases; and benzyloxycarbonyl-asp-glu-val-asp-fluoromethylketone (Z- DEVD-fmk), an inhibitor of caspases 3, 6, 7, 8, and 10. Both inhibitors have been used extensively to inhibit apoptosis in somatic cells (15). MATERIALS AND METHODS Animals and Embryo Collection Zygotes were collected from 6- to 8-week-old MF-1 female mice mated with BALB/c mice. Superovulation was induced by a single intraperitoneal injection of 5 IU of pregnant mare s serum gonadotrophin (Sigma Chemical, St. Louis MO) followed 48 hours later by 5 IU of hcg (Sigma). The females were then placed with BALB/c males of proven fertility. Mating was confirmed by the presence of a vaginal plug the next morning. Cumulus-enclosed zygotes were collected 22 to 24 hours after administration of hcg in 20 mm HEPES-buffered CZB medium (16) containing mm of NaCl, 4.83 mm of KCl, 1.18 mm of KH 2 PO 4, 1.18 mm of MgSO 4 7H 2 O, mm of NaHCO 3, 1.70 mm of CaCl 2 2H 2 O, mm of sodium lactate, 0.27 mm of sodium pyruvate, 0.11 mm of EDTA, 1 mm of glutamine, 5 mg/ml of bovine serum albumin (BSA), 100 IU/mL of sodium penicillin, and 0.7 mm of streptomycin (CZB H). The zygotes were denuded with 0.3 mg/ml of hyaluronidase in CZB H, and were washed three times in CZB H and twice in pregassed CZB medium before they were pooled together and allocated randomly to different treatment groups. Our unpublished data showed the development of this strain of embryo in CZB medium was comparable to those cultured with KSOM, another commonly used embryo culture medium. All chemicals (cell-culture grade or embryo-culture grade) were purchased from Sigma. Embryo Culture and Inhibitors Treatment The zygotes were cultured in CZB for up to 72 hours after the hcg administration, and were then cultured in CZB supplemented with 5 mm of glucose (CZB G) for the rest of the experimental period. The embryos were transferred to fresh medium every 24 hours. Stock solutions of caspase inhibitors (20 mm in DMSO), z-vad-fmk, and z-devd-fmk (Enzyme Systems Products, Livermore, CA) were stored at 20 C until used. They were diluted to 10 M, 50 M, and 200 M with CZB or CZB G before experimentation. The same volume of DMSO was also added to CZB and CZB G, which were then used as the control. Morphology Assessment The age of the embryos was calculated with reference to the time after hcg administration. Embryo morphology was assessed under an inverted microscope at 48, 72, 96, and 120 hours after hcg administration. The proportion of embryos that had reached the two-cell, three- to four-cell, morulae, and blastocyst stage as well as the degree of fragmentation were determined. Based on the presence of two pronuclei, the fertilization rate of this mouse strain ( MF1 BALB/c) was only around 50% to 60%. This was consistent with our unpublished data that the percentage of two-cell embryos after culturing in CZB, KSOM, mtf, and G1.2 ranged from 54% to 66%. Therefore, the number of embryo at any stage of development was expressed as a normalized value against the number of two-cell embryos formed in the same group of animals after 24 hours of culture. Caspase Activity Determination Caspase activity was measured by the fluorometric method. Briefly, embryos with or without treatment were incubated in 20 L of CZB containing PhiPhiLux (OncoImmunin, Gaithersburg, MD) (1:5 v/v), a substrate of caspase-3 and caspase-3-like protease, under paraffin oil for FERTILITY & STERILITY 987

3 1 hour at 37 C in an atmosphere of 5% CO 2 in air. The embryos were then washed six times in CZB H, transferred to a 10- L drop of CZB H without phenol red on a glass slide, and observed under a Nikon epifluorescence microscope. The caspase-digested product of PhiPhiLux fluoresced at 580 nm after excitation at 552 nm. Fluorescent images of the embryos were captured by a digital camera (Photometrics SenSys, Roper Scientific, Tucson, AZ) and analyzed by a dedicated software (Metamorph, Universal Imaging Corporation, PA). The pixel intensity of the whole embryo was proportional to the caspase activity within the embryo. Propidium Iodide and Annexin-V Staining Propidium iodide (PI) and annexin-v staining were performed to determine the membrane integrity and the presence of phosphatidylserine residues on the outer surface of the plasma membrane, respectively. The presence of annexin-v staining and the absence of PI staining would exclude the possibility that the observed annexin-v staining was due to necrosis. Embryos were incubated in CZB containing 1 g/ml of PI (Sigma) and 1 g/ml of FITCconjugated annexin-v (Boerhinger Mannheim, Mannheim Germany) at 37 C for 15 minutes. Images of the stained embryos were captured after three washes in CZB H, as described previously. TUNEL TUNEL was used to detect DNA breakage in the blastomere, as previously described (17). Embryos were first incubated at 37 C for 15 minutes in CZB containing PI (1 g/ml). They were then successively washed three times in PBS, fixed in 4% formaldehyde in PBS (ph 7.4) for 30 minutes, washed six times in PBS, permeated in 0.1% Triton X-100 at 4 C for 2 to 3 seconds, washed three times in PBS, and incubated in a TUNEL reaction cocktail (In situ cell death detection system; Boehringer Mannheim) at 37 C for 1 hour. The proportion of TUNEL-positive nuclei was then counted under an inverted microscope. The total number of blastomeres per embryo was determined by staining with Hoechst (20 g/ml in PBS). Experiment Design Experiment 1 The two caspase inhibitors were applied to treat embryos individually at concentrations of 10 M, 50 M, and 200 M from 24 to 120 hours after the hcg administration. The control groups contained the same volume of DMSO as the treatment groups. Embryos were also cultured in medium alone without DMSO as a blank control. Embryo development was observed daily under an inverted microscope. Experiment 2 Embryos were treated with 10 M of z-devd-fmk or z-vad-fmk. Staining for caspase-3 activity, annexin-v, and FIGURE 1 Percentage of fragmented embryos after treatment with caspase inhibitors z-vad-fmk or DEVD-fmk for different periods (... {... Control, DMSO-10, DMSO-50,... E... DMSO-200, DEVD-10, DEVD-50, DEVD- 200, VAD-10, ƒ VAD-50, VAD-200). Xu. Embryo fragmentation and caspase activity. Fertil Steril TUNEL were performed at 48, 72, and 96 hours after hcg administration. Statistical Analysis Data from three experiments were combined for analysis. The differences between the proportion of the embryos at different stages of development, the proportion of fragmented embryos, and the proportion of embryos containing apoptotic blastomere(s) were compared by chi-square analysis. The Student t test was performed to analyze the significance of any difference in percentages of apoptotic blastomeres per embryo. RESULTS Embryo Development and Fragmentation To investigate the effect of caspase inhibitors on the incidence of cytoplasmic fragmentation, one-cell zygotes were cultured in media containing different concentrations of z-devd-fmk or z-vad-fmk for 96 hours. Figure 1 shows the proportion of fragmented embryos after treatment. There was no difference in the percentage of fragmented embryos at 48, 72, and 96 hours after hcg administration among all the groups. The proportion of embryos that reached the 988 Xu et al. Embryo fragmentation and caspase activity Vol. 75, No. 5, May 2001

4 FIGURE 2 Embryo development after treatment with caspase inhibitors z-vad-fmk or DEVD-fmk (... {... Control, DMSO-10, DMSO-50,... E... DMSO-200, DEVD-10, DEVD-50, DEVD-200, VAD-10, ƒ VAD-50, VAD- 200). FIGURE 3 Caspase activity in mouse embryos. Note that caspase activity was detected in the fragments of fragmented embryo. The corresponding bright field images are shown on the right (Arrowhead: fragments with caspase activity). Xu. Embryo fragmentation and caspase activity. Fertil Steril Xu. Embryo fragmentation and caspase activity. Fertil Steril two-cell, three- to four-cell, morula, and blastocyst stages at 48, 72, 96, and 120 hours after hcg administration, respectively, was also comparable between the treatment and the control groups (Fig. 2). These showed that z-devd-fmk and z-vad-fmk at the three concentrations used did not affect the embryo development and fragmentation. Caspases Activity Activated caspase-3-like caspases digested the nonfluorescent substrate PhiPhiLux into the fluorescent product. In the control group, caspase activity as determined by the intensity of the fluorescent product was detected in some fragments of fragmented embryo before compaction, but not in unfragmented embryos (Fig. 3). However, no caspase activity was detected in any embryos after treatment with z-devd-fmk or z-vad-fmk. Annexin-V and PI Staining The percentage of embryos that stained for annexin-v was comparable between the control embryos and those treated with z-devd-fmk or z-vad-fmk. More than 80% of the fragmented embryos (control 87%; z-devd-fmk 83%; z-vad-fmk 89%) contained annexin-v positive fragments. But less than 20% of them were PI positive (control 18%; z-devd-fmk 15%; z-vad-fmk 19%), suggesting that most of the fragments were not a product of necrosis. TUNEL Blastomeres that were TUNEL positive and PI negative were defined as apoptotic cells. In the control groups, apoptotic cells were detected only in fragmented embryos at 48 to 72 hours after hcg administration. However, at 96 hours after hcg was added, TUNEL staining was found in both fragments and some morphologically normal looking blastomeres of fragmented embryos. In contrast, embryos treated with z-devd-fmk or z-vad-fmk were TUNEL negative. DISCUSSION To investigate the relationship between embryo fragmentation and apoptosis, caspase inhibitors were used to treat mouse embryos. The inhibitors had no observable detrimental effect on the morphological development of the embryo. They suppressed the caspase activity in the cleavage-stage embryo, but did not affect the incidence of fragmentation. These results demonstrated for the first time that fragmentation was independent of caspase activity. Cytoplasmic fragmentation is a common phenomenon in cultured mammalian embryos. It significantly decreases the implantation potential of embryos when it is excessive. Based on nuclei morphology, Jurisicova et al. (18) suggested that fragments could possibly be apoptotic bodies, the final product of apoptosis. In support of this hypothesis, other markers of apoptosis, namely phosphatidylserine exposure on the outer surface of the cytoplasmic membrane (7) and DNA breakage (19), were also detected in the fragments of the human embryo. In this study, we also found TUNEL-positive fragments at different stages of embryo development in the control group; most of the embryo fragments ( 80%) were annexin-v positive and PI negative. These observations are in line with FERTILITY & STERILITY 989

5 previous suggestions that apoptosis exists in the fragmented cleavage-stage embryo. It has been commonly accepted that the activation of a caspase cascade is necessary for the execution of apoptosis. The presence of caspase-3-like activity in some fragments of cleaving embryos supports that observation. In this study, we treated the embryos with z-vad-fmk or z-devd-fmk, two inhibitors of caspases. As expected, both inhibited the caspase-3-like activity and suppressed the DNA breakage. However, they did not affect the incidence of fragmentation. These data show that the fragments in cleavage-stage embryos are not apoptotic bodies, the incidence of which would otherwise have decreased as a result of suppression of the caspase activity. This conclusion is further supported by the observation that fragmentation usually occurs in early cleaving embryos whereas DNA breakage is more often found in morulae or blastocysts. This study does not contradict the suggested association of apoptosis and fragmentation. It is possible that fragmentation is an early event before the activation of the caspase cascade in the process of apoptosis in embryos. Neither do the present data imply that fragmentation induces apoptosis, as was suggested by Antczak and van Berlerkom (8). The exact relationship between embryo fragmentation and apoptosis remains to be investigated. It has long been known that embryo fragmentation is associated with suboptimal culture conditions and that coculture improves these conditions and reduces fragmentation of the cocultured embryos (20). Van Blerkom et al. (21) proposed that embryo fragmentation was related to a shortage of energy. Compared to normal embryos, the fragmented pig embryos have a different actin filament distribution (5), which is known to be affected by the ATP supply (22). Barnett et al. (23 24) reported the redistribution of mitochondria in hamster embryos after culturing the embryos in suboptimal conditions. Thus, mitochondrial activity may be related to cytoplasmic fragmentation. On the other hand, mitochondria are also associated with apoptosis. The ability of mitochondria to oxidize substrate for energy production partly depends on its membrane potential. A decrease in mitochondrial potential not only reduces energy metabolism, but also induces the release of cytochrome-c from the mitochondria. Together with two cytosolic protein factors, Apaf-1 and procaspase-9, the released cytochrome-c will activate caspase-3 (25) and lead to irreversible apoptosis. It is possible that the suboptimal culture conditions impair the mitochondrial potential of cleaving embryos. This reduction of energy production, in turns, would affect the actin filament assembly and eventually lead to fragmentation. At the same time, cytochrome-c would be released from the impaired mitochondria, activating the apoptotic caspases. This hypothesis is currently being tested in our laboratory. The developmental outcome in this study appears low, with blastulation rates ranging from 22% to 39%. These low blastulation rates are not due to a particularly poor culture system (CZB), because the blastulation rates for these embryos in another commonly used embryo culture medium, KSOM, were similar (35%; JS Xu, WSB Yeung, unpublished data). The embryos also form a two-cell block in mouse tubal fluid medium with no blastocyst formation. Furthermore, 48 hours after hcg administration the blastulation rate for two-cell embryos obtained from the oviduct is over 90% in the same culture system (CZB). These data suggest that the one-cell embryo in this mouse strain is particularly sensitive to in vitro culture. We conclude that the formation of fragments in precompaction mouse embryos is independent of caspase activity and that embryo fragments are not apoptotic bodies. References 1. Alikani M, Cohen J, Tomkin G, Garrisi GJ, Mack C, Scott RT. Human embryo fragmentation in vitro and its implications for pregnancy and implantation. Fertil Steril 1999;71: Palermo G, Munne S, Cohen J. The human zygote inherits its mitotic potential from the male gamete. Hum Reprod 1994;9: Hardy K, Robinson FM, Paraschos T, Wicks R, Franks S, Winston RM. Normal development and metabolic activity of preimplantation embryos in vitro from patients with polycystic ovaries. Hum Reprod 1995;10: Dumoulin JC, Menheere PP, Evers JL, Kleukers AP, Pieters MH, Bras M, et al. The effects of endotoxins on gametes and preimplantation embryos cultured in vitro. Hum Reprod 1991;6: Wang WH, Abeydeera LR, Han YM, Prather RS, Day BN. 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6 human embryos in vitro by a human oviductal cell co-culture system. Hum Reprod 1992;7: Van BJ, Davis PW, Lee J. ATP content of human oocytes and developmental potential and outcome after in-vitro fertilization and embryo transfer. Hum Reprod 1995;10: Brown SS, Spudich JA. Nucleation of polar actin filament assembly by a positively charged surface. J Cell Biol 1979;80: Barnett DK, Kimura J, Bavister BD. Translocation of active mitochondria during hamster preimplantation embryo development studied by confocal laser scanning microscopy. Dev Dyn 1996;205: Barnett DK, Clayton MK, Kimura J, Bavister BD. Glucose and phosphate toxicity in hamster preimplantation embryos involves disruption of cellular organization, including distribution of active mitochondria. Mol Reprod Dev 1997;48: Li H, Yuan J. Deciphering the pathways of life and death. Curr Opin Cell Biol 1999;11: FERTILITY & STERILITY 991