Characterization of the first cell cycle in human zygotes: implications for cryopreservation*

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1 FERTILITY AND STERILITY Copyright 993 The American Fertility Society Printed on acid-free paper in U.S.A. Characterization of the first cell cycle in human zygotes: implications for cryopreservation* Hanna Balakier, Ph.D.t:!: Neil J. MacLusky, Ph.D.t Robert F. Casper, M.D.tll Division of Reproductive Sciences, University of Toronto, Toronto, Ontario, Canada Objective: To study the temporal pattern of deoxyribonucleic acid (DNA) synthesis leading up to the first mitotic division in human one-cell stage zygotes. Setting: In vitro fertilization program of a university hospital. Patients: Couples donating spare embryos before the existence of an embryo freezing program. Design: Incorporation of 3H -thymidine was examined between 9 to 27 hours after insemination in 72 untransferred human zygotes containing two pronuclei. Microscopic observations on an additional 978 transferred zygotes extended the 3H-thymidine incorporation data. Results: The first thymidine incorporation was seen 9 to 0 hours after insemination, and the heaviest incorporation occurred between and 3 hours. Subsequently, thymidine labeling declined, and chromosomal condensation and cell division first occurred approximately 9 to 20 hours. At 20 hours after insemination, 89% of the zygotes had two visible pronuclei (PN). In contrast, by 24 hours, 4 % had no visible PN, whereas 5% had cleaved to the two-cell stage. By 27 hours, 38% had cleaved to the two-cell stage, and only 25% still had two visible PN. Conclusions: These results suggest that the DNA-synthetic, S-phase of the human zygote is initiated by approximately 9 to 0 hours after insemination and is completed approximately 3 to 5 hours later. The duration of G2 phase and mitosis is in the range of 4 to 6 and 3 to 3.5 hours, respectively. Because zygotes may be particularly susceptible to damage during the S phase of the cell cycle, these findings suggest that the optimal time for freezing of human zygotes may be approximately 20 to 22 hours after insemination when the majority of zygotes should have entered the G2 phase, before pronuclear dissolution and chromosome condensation. Fertil Steril 993;59: Key Words: Human zygotes, DNA synthesis, cell cycle, cryopreservation In contrast to several animal species in which the first cell cycle of fertilized oocytes has been described (-7), similar data are not available for the human. Received June 2, 992; revised and accepted October 5, 992. * Supported by grant 0428 from the Medical Research Council of Canada, Ottawa, and the Royal Bank of Canada, Toronto, Ontario, Canada. t Department of Obstetrics and Gynecology. :\: Present address: IVF Clinic, Toronto, Ontario, Canada. Department of Physiology. II Reprint requests: Robert F. Casper, M.D., Division of Reproductive Sciences, Department of Obstetrics and Gynecology, The Toronto Hospital, Toronto General Division, EN-246, 200 Elizabeth Street, Toronto, Ontario M5G 2C4 Canada. Detailed knowledge of the cell cycle may have practical importance in relation to human assisted reproductive technologies, especially in cryopreservation of human embryos. We recently demonstrated significantly improved survival and increased developmental capability of murine embryos frozen in the G2 phase of the cell cycle compared with those frozen during deoxyribonucleic acid (DNA) synthesis (8 phase) (8). Our observations also suggested that multicell embryos may not be optimal for cryopreservation because of the considerable asynchrony in the second and third cell cycles that results in difficulty in determining the appropriate time for freezing. The objective of the present study was to Balakier et al. Cell cycle in human zygotes 359

2 r determine the period of DNA synthesis in human zygotes by examining 3H-thymidine incorporation by the pronuclei at various times after insemination. We studied spare zygotes that could not be transferred into the uterus during in vitro fertilization (IVF). Particular care was taken to define the time when the majority of zygotes were finishing DNA synthesis and were entering the G2 phase of their cycle. In conjunction with determination of the time of the first cell division of living zygotes that were subsequently transferred to the patient, we were able to estimate the approximate length of the S, G2, and M phases of the first cell cycle in the human. MATERIALS AND METHODS In Vitro Fertilization Protocol The women underwent gonadotropin suppression with an oral contraceptive (Demulen 50; Searle, Oakville, Ontario, Canada) for 8 to 35 days before ovarian stimulation, as we have previously described (9). Follicular development was induced with clomiphene citrate (Serophene; Serono, Toronto, Ontario, Canada), 00 mg/d on cycle days 5 to 9 and human menopausal gonadotropins (hmg, Pergonal; Serono) 50 IU starting on day 5 or 6. The concentration of serum estradiol (E2) and luteinizing hormone and the sonographic determination of the number and diameter of ovarian follicles were monitored daily. The dose of hmg was sometimes increased to 225 or 300 IU / d if serum E2 did not at least double each day in response to the stimulation protocol. Human chorionic gonadotropin (hcg, Profasi; Serono), 5,000 IU, was administered for the final stage of follicular maturation. Oocytes were collected by ultrasound-guided follicle aspiration at 34 to 35 hours after hcg injection. The oocytes were cultured for 5 to 9 hours before insemination in human tubal fluid medium (0) supplemented with 0% human albumin at 37 C in an atmosphere of 5% CO 2, 5% O2, and 90% N 2' At 9 to 8 hours after insemination (00,000 motile sperm/0.8 ml of culture medium containing from to 4 oocytes), oocytes were examined for the presence of pronuclei (PN). At 43 to 45 hours after insemination up to four two- to sixcell stage embryos were transferred to the uterus. Human Zygotes Seventy-two human zygotes containing 2PN were donated for this research project that was approved by the Committee for Human Research of The To- 360 Balakier et ai. Cell cycle in human zygotes ronto Hospital and the University of Toronto. At the time this study was performed, the IVF program did not have the capability for embryo cryopreservation, and a maximum of four embryos was transferred to the uterus in an attempt to avoid high multiple pregnancies. All patients enrolled in the program were given information concerning the study, including a copy of the consent form, before superovulation and cycle monitoring were performed. After oocyte retrieval, those couples who were anticipated to have more than seven embryos were asked to donate one or two zygotes for this project and signed the consent at that time. At the time of embryo transfer, other spare two- to fourcell embryos were either donated for research involving karyotyping or else were destroyed by being transferred into the vagina, according to the wishes of the couple. Labeling of DNA and Zygote Preparation The one-cell stage zygotes were exposed to 3H_ thymidine (specific activity 25 Cijmmol; Amersham International, Amersham, United Kingdom) at a concentration of 2 mci/ml for hour at different times, from 9 to 27 hours, after insemination. After incubation with nucleic acid precursor, the zygotes were extensively washed in fresh culture medium to remove unincorporated radioactivity. Air-dried preparations () were made of28 zygotes. The other 44 zygotes were fixed with 2.5% glutaraldehyde, postfixed with 2% aqueous osmium tetroxide, dehydrated in serial dilutions of alcohol and propylene oxide, and embedded in Araldite 502 (CIBA-Geigy Canada Ltd, Dorval, Quebec, Canada). Thin sections, from to 5,um thick, were cut with a LKB III ultramicrotome (LKB-Produkter AB, Bromma, Sweden). Autoradiography The air-dried preparations that had been washed in 5 % trichloroacetic acid and the thin sections were coated with Kodak NTB-2 emulsion (Kodak, Rochester, New York) and stored for 3 or 2 days, respectively, in a lightproof box at -4 C before development in D-9 (Kodak). The air-dried preparations were stained with 2% Giemsa in 0.02 M phosphate buffer, ph 6.8, and the thin sections were stained with % toluidine blue. Determination of the Onset of Mitosis Microscopic observations of 978 living zygotes, which were subsequently transferred in the IVF Fertility and Sterility

3 program, were performed first between 9 to 8 hours after insemination for identification of fertilization and the number of PN and then re-examined between 20 and 27 hours after insemination. We determined the number of zygotes in which the 2PN were well visualized between 20 and 27 hours, those which had entered mitosis as indicated by the disappearance of the PN, and those which had cleaved into two-cell stage embryos. RESULTS Very clear localization of radioactive grains over the PN, with extremely low background radioactivity in the cytoplasm of the human zygotes (Fig. ), indicated that the labeled thymidine was incorporated into the DNA and that washing the cells after exposure to thymidine removed unbound label as previously demonstrated in fertilized hamster 00- cytes (2). Autoradiography of the zygotes showed that thymidine incorporation could clearly be separated into three different patterns. In the sections of plastic-embedded zygotes (approximately 50 to 200 serial sections were examined from each zygote), the PN considered to be heavily labeled were almost uniformly saturated with silver grains in the majority of their sections (Fig. A). In those PN that we defined as having moderate labeling, the intensity of thymidine incorporation was relatively lower, and the PN were usually observed to have radioactivity a9cumulating near the nuclear membrane and nucleoli, whereas the interior of the PN remained unlabeled (Fig. B). In some cases of moderate incorporation, the entire surface ofthe nuclear membrane was occupied by silver grains, whereas in others 30% to 50% of the surface was labeled. In the cases that we defined as weak incorporation, only a few silver grains were present within both PN (Fig. C) or in 4 of 4 zygotes only in a single pronucleus. Often the PN exhibited distinct asynchrony in thymidine incorporation, with a higher intensity of label observed in one of the 2PN (Fig. C and D). The assessment of the degree of labeling depended on the pronucleus with the heavier labeling. This was especially evident in the PN exhibiting weak (asynchrony in approximately 80%) or moderate (asynchrony in approximately 50%) thymidine incorporation. Asynchrony between the PN exhibiting heavy incorporation of thymidine was not apparent. In 25 zygotes with moderate or weak incorporation showing asynchronous labeling, 5 had heavier labeling in the smaller pronucleus, and 5 had heavier labeling in the larger pronucleus. In the remaining 5 zygotes, no difference could be distinguished between the size of the 2PN. In the air-dried preparations, whole pronuclear spreadf'l were examined, and 7 of 28 zygotes were seen to have thymidine incorporation (Fig. 2). Two zygotes with heavy labeling and one with moderate labeling were fixed at 3 hours after insemination, and 4 zygotes with weak labeling were fixed at 8 to 9 hours. The other zygotes had no thymidine incorporation and exhibited interphase nuclei (2 zygotes at 9 hours and 9 zygotes at 24 to 26 hours) or condensing chromosomes (0 zygotes at 9 to 27 hours). Combined results from the air-dried preparations and thin sections are shown in Table. All human zygotes examined in the period 9 to 3 hours after insemination exhibited pronuclear thymidine incorporation (Fig. A to C and Fig. 2). The two earliest zygotes, fixed at 0 hours, showed weak incorporation. Fifteen zygotes were fixed between and 3 hours and exhibited mostly moderate (6 zygotes) or heavy (7 zygotes) thymidine incorporation, with only two cases having weak incorporation of 3H_ thymidine. The first zygotes showing no incorporation of 3H -thymidine were seen at 4 and 7 hours. By 20 to 27 hours after insemination, only 2 of 33 zygotes were observed with weak thymidine incorporation, whereas 3 zygotes showed either no incorporation (20 zygotes; Fig. E) or chromosomal condensation ( zygotes), indicating the beginning of their first mitotic division. The first zygotes in mitosis were seen 9 to 20 hours after insemination. Besides radioactive labeling within the PN of the zygotes, thymidine incorporation was also found in occasional cumulus cells that were attached to the zona pellucida as well as in several second polar bodies. The second polar bodies were completely visualized in the thin sections of 2 zygotes. In 7 cases, radioactivity was seen in the polar bodies, whereas the PN of these 7 zygotes demonstrated weak (5 zygotes fixed at 3, 7, and 2 to 23 hours; Fig. C) or no thymidine incorporation (2 zygotes fixed at 8 and 22 hours). In the other 4 cases, 9 second polar bodies had no evidence of 3H-thymidine incorporation, whereas the PN ofthe zygotes exhibited weak (2 zygotes fixed at 0 hours), moderate (4 zygotes fixed at 0, 5, and 8 hours), or heavy (3 zygotes fixed at 3 hours) incorporation. In an additional 5 zygotes, fixed at 4, 7, and 20 to 22 hours, no incorporation of thymidine was seen in the PN or in the second polar body. The results of microscopic observations of 978 living zygotes that were subsequently transferred in Balakier et al. Cell cycle in human zygotes 36

4 Figure Thin sections from human zygotes (fixed at different times after insemination). (A), Pronuclei exhibiting heavy incorporation are saturated with radioactive grains (fixed at hours; magnification was Xl,400). (B), Pronuclei showing moderate incor poration with accumulation of thymidine along the nuclear membranes and nucleoli (fixed at hours; magnification was X2,300). (C), Weak thymidine incorporation in the PN. One pronucleus exhibits higher radioactive label than the other. The second polar body also contains thymidine incorporation (fixed at 3 hours; magnification was Xl,400). (D), Weak asynchronous 3H-thymidine incorporation within the PN (fixed at 8 hours; magnification was X2,OOO). (E), Large, adhering PN exhibit no detectable 3H-thymidine incorporation (fixed at 8 hours; magnification was X2,OOO). the IVF program are shown in Table 2. At 20 hours after insemination, 89% of the zygotes had two visible PN. Only of 40 zygotes observed at this time had cleaved to the two-cell stage. Similarly, at 22 hours, only 3 of 68 zygotes had cleaved to the twocell embryo stage. However, by 24 hours after insemination, 4 % had no visible PN, and 5% of the embryos had cleaved to the two-cell stage, whereas only 54% of the zygotes had two visible PN. By 27 hours, 38% had cleaved to the two-cell stage, and only 25% of the zygotes still had 2PN visible. We also observed that by 9 hours after insemination, separate, growing PN were present in some zygotes, whereas in others, the PN were just becoming visible in the cytoplasm. Usually, in living zygotes, the 2PN varied in size, and the smaller pronucleus was localized more closely to the second polar body than the larger one (Fig. e and Fig. 3). The fully grown PN appeared to flatten and become adherent to each other immediately before the first division. When the nuclear membranes appeared to be hazy, the PN disappeared within 30 minutes, and the first mitotic division occurred in approximately 3 to 3.5 hours. approximately 9 to 0 hours after insemination when the PN enlarge and move toward the center of the cell. We cannot exclude the possibility that some zygotes may start DNA replication at an even earlier time because younger zygotes were not examined in this study. The first zygotes exhibiting thymidine incorporation within the PN were observed 9 to 0 hours after insemination, and the first zygotes to complete DNA synthesis appeared at 4 to 7 hours (incubated with labeled thymidine from 3 hours on). Therefore, the length of S phase in human zygotes can be estimated to be approximately 3 to 5 hours. Based on our observation that the earliest evidence of chromosomal condensation was seen at DISCUSSION Figure 2 Two PN from one human zygote fixed by the airdried technique. The whole pronuclear spreads exhibit heavy thymidine incorporation over the nuclear chromatin threads (magnification was Xl,OOO). The present data suggest that DNA synthesis or the S phase of the first cell cycle in the human begins 362 Balakier et al. Cell cycle in human zygotes Fertility and Sterility

5 Table Pronuclear DNA Synthesis in Human Zygotes Exposure time after insemination to 7 8 to 9 20 to to 27 Total no. of zygotes Thymidine incorporation Heavy Moderate Weak No incorporation Chromosome condensation t :j: * Hours of zygote fixation. t Two zygotes fixed at 4 hours, one zygote at 7 hours. :j: Three zygotes fixed at 2 hours, three zygotes at 22 hours, one zygote at 24 hours. 8 to 9 hours, the duration of G2 is likely in the range of 4 to 6 hours. The length of the G phase, which begins with the formation of the second polar body and completion of meiosis II, could not be determined from this study. However, based on previous data (3, 4) indicating that the second meiotic division ends approximately 3 hours after insemination and our present observation that DNA synthesis starts approximately 9 hours after insemination, we estimate that the G phase in human zygotes lasts approximately 6 hours. Our observations of living zygotes indicated that the duration of the full cell cycle seems to be 20 to 22 hours after insemination because by that time a few zygotes have already finished cleavage to two-cell embryos. However, it remains unresolved whether the length of the first cell cycle in human zygotes is relatively stable or varies, either intrinsically or because of culture conditions related to the IVF procedure. Considerable variation in the time of DNA synthesis and of mitotic division seen in the zygotes in this study could be explained by variability in the time of oocyte fertilization. The precise time of sperm penetration could not be determined, but the variable size of PN within different zygotes at 9 to 8 hours after insemination suggests that fertilization in some oocytes may have been delayed. The time of sperm penetration probably depends on the maturity of the oocyte. It is likely that mature oocytes undergo fertilization within the first 5 hours after insemination and complete the first cell cycle approximately 24 to 26 hours after insemination (5% to 2% of zygotes). Less mature oocytes with delayed fertilization may undergo delayed cleavage as suggested by the observation that approximately 25% of zygotes were still in the pronuclear stage at 27 hours after insemination. The duration of the S phase in human zygotes determined from the present study seems to be similar to that reported for the rabbit (3 to 4 hours) (2) and shorter than that determined for the mouse (6 to 8 hours) (4, 5, 7). In contrast, the estimated G2 phase in human zygotes appears to differ from the rabbit in which the G2 phase may be quite long (0 to 2 hours) (2) but seems to be similar to the G2 phase observed in the mouse (4 to 5 hours). The Table 2 Cell Division in Human Zygotes After Insemination 2PN 2PN Two-cell Time Total visible not visible stage embryos h (89)* 4 (0) () (83) 26 (5) 3 (2) (54) 79 (4) 0 (5) (42) 67 (43) 25 (5) (40) 62 (35) 45 (25) (25) 53 (37) 55 (38) * Values in parentheses are percents. Figure 3 A living zygote with 2PN in the center and two polar bodies in the perivitelline space. The PN vary in size. The smaller pronucleus is localized more closely to the polar bodies than the larger one (20 hours after insemination; magnification was X600). Balakier et ai. Cell cycle in human zygotes 363

6 r length of the first mitotic division in the human, determined to be 3 to 3.5 hours in this study, is similar to the reported length of mitosis in mouse zygotes (6). Deoxyribonucleic acid duplication appears to occur shortly after pronuclear formation in many species (-3, 6). Studies in the hamster (2) and in the human (5-7) demonstrated that initiation of DNA synthesis correlates closely with the stage of development of the PN. Mature, stage II male PN in the hamster consistently incorporated 3H-thymidine, whereas early (stage I) male PN or decondensed sperm nuclei showed no evidence of DNA synthesis (2). In the human, incorporation of labeled thymidine was also detected only in fully developed male PN (5-7). Our data suggest that DNA synthesis starts in human zygotes at the beginning of the pronuclear growth phase, which may occur relatively early after insemination of the oocytes. Our observation of the initiation of DNA synthesis by 9 hours after insemination differs from the previously reported human data. Tesarik and Kopecny (5) found that 3H-thymidine incorporation did not occur until 2 hours after fertilization in re-inseminated oocytes from an IVF program. However, the failure of the oocytes to fertilize initially or the advanced age (8+ hours) of the oocytes in that study may have resulted in a delay in the development of intracellular mechanisms necessary for initiation of DNA replication. In addition, it is possible that the significant degree of polyspermy seen in the study of Tesarik and Kopecny (5) could also delay sperm transformation and initiation of DNA synthesis. However, 29 polyspermic human triploid zygotes examined by us (Balakier H, Casper RF, unpublished data) appeared to be similar to normal zygotes with respect to time of pronuclear DNA synthesis as determined by 3H_ thymidine incorporation. Our finding of much earlier initiation of DNA synthesis may be a reflection of primary fertilization of fresh oocytes, with resultant production of presumably normal zygotes. From the present observations, it seems likely that the male pronucleus in human zygotes, similar to mouse zygotes (3, 4, 7), is larger than the female pronucleus and forms further from the second polar body. One of the PN often appears to begin and end DNA synthesis earlier than the other, but at present, there is not enough data to know if this occurs randomly or preferentially in either the male or female pronucleus. Deoxyribonucleic acid synthesis was also observed in the second polar body of human zygotes. Thymidine incorporation appeared to be asynchronous in the second polar body, occurring in most cases just before completion of, or shortly after, pronuclear DNA replication. The zygotes showing heavy or moderate patterns of pronuclear thymidine incorporation did not have any labeling in the second polar body, whereas 3H-thymidine labeling in the second polar body was seen later when the PN exhibited weak or absent incorporation. Similar observations have been described in mouse zygotes (4, 5). The results of this study may have clinical relevance for cryopreservation of human embryos. Based on our previous results from cryopreservation of murine embryos (8), we believe it is optimal to avoid the phase of DNA synthesis when freezing human zygotes and embryos. Our data from observations of 3H -thymidine incorporation and ofthe morphology of viable zygotes in the present study suggest that human zygotes should be frozen between 20 to 22 hours after insemination, as long as 2PN are still visible. At this time, the zygotes are most likely to be in the G2 phase of the first cell cycle. If the borders of the PN are hazy or if they have disappeared, suggesting the beginning of mitosis, cryopreservation would likely best be postponed until embryo cleavage to the two- or four-cell stage has been completed. Acknowledgments. We thank all members of our IVF team who participated in collecting zygotes and Mr. Daniel Hunter for help with the preparation of thin sections. REFERENCES. Simmel E, Karnofsky DA. Observations on the uptake of tritiated thymidine in the pronuclei of fertilized sand dollar embryos. J Biochem Cytol 96;0: Szollosi D. Time and duration of DNA synthesis in rabbit eggs after sperm penetration. Anat Rec 966;54: Luthardt FW, Donahue RP. Pronuclear DNA synthesis in mouse eggs. An autoradiographic study. Exp Cell Res 973;82: Siracusa G, Coletta M, Monesi V. Duplication of DNA during the first cell cycle in the mouse embryo. J Reprod Fertil 975;42: Kirshna M, Generoso WM. 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