The timing of pronuclear formation, DNA synthesis and cleavage in the human 1-cell embryo

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1 Molecular Human Reproduction vol. no. pp., The timing of pronuclear formation, DNA synthesis and cleavage in the human cell embryo Gemma Capmany, Alison Taylor, Peter R.Braude and Virginia N.Bolton Assisted Conception Unit, Department of Obstetrics & Gynaecology, King's College School of Medicine & Dentistry, Bessemer Road, London SE RX and Assisted Conception Research Unit, UMDS Department of Obstetrics & Gynaecology, Guy's and St Thomas' Hospital Trust, Lambeth Palace Road, London SE EH, UK 'To whom correspondence should be addressed The timing of pronuclear formation and breakdown, DNA synthesis and cleavage during the first cell cycle of human embryogenesis are described. Pronuclei formed between and h postinsemination (hpi; median hpi). Sphase commenced between and hpi, and was completed between and hpi. Mphase was observed between and hpi (median duration h), and cleavage to the cell stage took place between and hpi. The timing of the same events was determined in cell embryos derived from reinseminated human oocytes that had failed to fertilize during therapeutic invitro fertilization (IVF). In these embryos, pronuclei formed between and h postreinsemination (hpri), coinciding with the beginning of Sphase. While Sphase was completed as early as hpri in some embryos, it extended until at least hpri in others. Pronuclear breakdown and cleavage occurred from and hpri respectively; however, they did not occur in some embryos until after hpri. The results demonstrate a markedly greater degree of variation in the timing of these events in embryos derived from reinseminated oocytes compared with embryos derived from conventional IVF, and thus throw into question the validity of using the former as models for studies of the first cell cycle of human embryogenesis. Key words: DNA synthesis/first cell cycle/human oocytes/pronuclear formation Introduction The precise timing of events during the first cell cycle of human embryogenesis has not yet been established. Two conflicting studies have reported that, following insemination in vitro, pronuclear DNA synthesis commences at either h (Tesarik and Kopecny, a) or between and h (Balakier et al, ). Although both studies examined the incorporation of [ H]thymidine into the pronuclei, the difference in the two sets of findings may be explained by the fact that the first study used reinseminated, zonafree oocytes prepared from oocytes that had failed to fertilize during conventional therapeutic invitro fertilization (TVF), whereas the second study used normally fertilized pronucleatestage embryos. We have determined the timing of DNA replication, pronucleus formation and breakdown, and the first cleavage division in pronucleatestage human embryos derived from conventional IVF, using light microscopy and fluorodensitometry. In addition, we have extended the two earlier studies (Tesarik and Kopecny, a; Balakier et al., ) by examining the time course of the same events in embryos derived from reinsemination of zonafree unfertilized oocytes. Comparison of the results obtained from the two groups of embryos has enabled an evaluation of the use of reinseminated unfertilized oocytes in studies of events taking place during normal human embryogenesis. European Society for Human Reproduction and Embryology Materials and methods Source of gametes Human gametes were donated to research by patients undergoing therapeutic IVF at the Assisted Conception Unit of King's College Hospital, London, UK. Hypothalamic pituitary suppression and ovarian stimulation, using luteinizing hormonereleasing hormone analogue and human menopausal gonadotrophin, and oocyte retrieval were all carried out as described by Waterstone and Parsons (). Following retrieval, oocytes were cultured at C in an atmosphere of % CO in air in Earle's balanced salt solution (EBSS) supplemented with.% (v/v) synthetic serum replacement and % (w/v) human serum albumin (Medicult; Imperial Laboratories, Andover, UK), overlaid with a thin layer of light paraffin oil (Sigma, Poole, UK). Semen samples were prepared by isopyenic centrifugan'on on discontinuous Percoll gradients ( and % in EBSS; Pharmacia, Andover, UK), and insemination was carried out h after a superovulatory injection of human chorionic gonadotrophin (HCG) using motile spermatozoa/ml. Freshly inseminated oocytes At denned times between. and h posthcg (hphcg;. and h post insemination; hpi), oocytes were cleaned of cumulus and corona cells using a pulled Pasteur pipette and scored for the presence of pronuclei. Those which showed two or more pronuclei were either returned to culture for defined intervals up to hphcg ( hpi), or fixed immediately in % (w/v) formaldehyde in phosphatebuffered saline (PBS; Sigma) for min. Fixed embryos were rinsed three times in ml drops of PBS and stored at C in. ml drops of Downloaded from on March

2 G.Capmany et al. Table I. Numbers of freshly inseminated and reinseminated oocytes showing sperm penetration and pronuclear formation Stage of oocytes analysed Time fixed (hours postinsemination oir post reinsemination) Total no. of. Freshly inseminated oocytes Inseminated Penetrated With PN precursors/pn using DAPI With PN using light microscope Reinseminated oocytes Inseminated Penetrated With PN precursors/pn using DAPI With PN using light microscope PN = pronuclei; DAPI =,diamidinophenylindole. PBS containing % (w/v) sodium azide (PBSazidc) under a thin layer of light paraffin oil. Embryos in culture were scored hourly between and hphcg ( and hpi) for pronuclear breakdown and cleavage to the cell stage, with care taken to minimize the length of time embryos were exposed to air and room temperature. Reinseminated oocytes Oocytes that had failed to develop pronuclei by hphcg ( hpi) had their zonae removed by brief exposure ( s) to acid Tyrode's solution (ph.) at C; they were then rinsed twice in ml drops of PBS and returned to culture in ml drops of EBSS until hphcg, when they were reinseminated with motile spermatozoa/ml. Thirty minutes after reinsemination, the oocytes were transferred to fresh drops of EBSS for further culture. Reinseminated oocytes were scored for the presence of pronuclei and fixed at defined times between. and hphcg (. and h postreinsemination; hpri) as described above, or cultured to hphcg ( hpri) and scored hourly thereafter until hphcg ( hpri) for pronuclear breakdown and cleavage to the cell stage. Controls Controls for delayed pronuclear formation in freshly inseminated oocytes that failed to develop pronuclei by hpi, and for acid Tyrode'sstimulated parthenogenetic activation, and a reference value for the C DNA content of zonafree oocytes were provided by unfertilized oocytes that were zonafree, cultured until hphcg and fixed. A reference value for the C DNA content of zonaintact freshly inseminated oocytes was provided by unfertilized oocytes fixed at hphcg in % (w/v) formaldehyde in PBS. Analysis of sperm penetration and DNA content Fixed oocytes and embryos were transferred to u. drops of PBS containing Hg/ml of,diamidinophenylindole (DAPI; Sigma). After min, oocytes and embryos were rinsed four times in il drops of PBS and transferred to a final u. drop of PBS. The number of oocytes that had been penetrated by spermatozoa was scored by fluorescence microscopy under UV excitation. Analysis of DNA content was performed min later using a computerized image analysis system (Seescan, Cambridge, UK) and UV excitation as described by Taylor and Braude (). The min delay between removal from the DAPI stain and analysis of the DNA content was necessary because there is an exponential drop in the measurements obtained for a given nucleus during this interval; after min, the measurements reach a plateau (Taylor and Braude, ). Sphase was considered to have begun if at least one of the pronuclei had a DNA content >C, and to have been completed if both pronuclei had a DNA content of at least C. Statistical analysis Statistical analysis was performed using the x tes t> and P *=. was taken to be significant Results Formation of pronuclei A total of freshly inseminated and reinseminated oocytes were analysed. In the freshly inseminated group, (%) oocytes developed two pronuclei, and (%) developed three pronuclei; no oocytes developed more than three pronuclei. In the reinseminated group, a total of (%) oocytes developed two or more pronuclei (two pronuclei:, %; three pronuclei:, %; four pronuclei:, %; five pronuclei:, %; > pronuclei:, %). Parthenogenetic activation, as indicated by the presence of one pronucleus, occurred in seven (%) freshly inseminated oocytes and in (%) reinseminated oocytes. This difference is statistically significant (P <.). There was no difference in the incidence of sperm penetration, as demonstrated by DAPI stain, in the remaining freshly inseminated oocytes (/, %), compared with / (%) in the reinseminated group. However, as expected, the incidence of polyspermy did differ significantly between the two groups. Thus, of the freshly inseminated oocytes that were penetrated, (%) were penetrated by a single spermatozoon and (%) by two spermatozoa. This compares with (%) reinseminated oocytes penetrated by one spermatozoon, (%) by two, (%) by three, (%) by four, and (%) by more than four spermatozoa (P <.). The number of oocytes in each group that were fixed and analysed for sperm penetration and pronuclear development is shown in Table I. Among the freshly inseminated oocytes, four of the five oocytes analysed at. hpi showed evidence of sperm penetration using DAPI stain, and although none had developed pronuclei that were visible using the light microscope, all four showed pronuclear precursors using DAPI. Of the seven oocytes fixed at hpi, none had been penetrated Downloaded from on March

3 Timing of events in the cell embryo Figure. Freshly inseminated oocytes fixed at different times (hours postinsemination, hpi) and viewed by phasecontrast microscopy (a, d) or after staining with the fiuorochrome,diamidinophenylindole and viewed under ultraviolet excitation (b, c, e, f) ( a and b) threepronucleate embryo fixed at hpi, (c) threepronucleate embryo fixed at hpi, (d and e) twopronucleate embryo fixed at hpi, ( onepronucleate oocyte fixed at hpi demonstrating evidence of sperm penetration but failure of decondensation of the sperm head, pn = pronucleus; csh = condensed sperm head by spermatozoa. At hpi, all six oocytes were penetrated by spermatozoa and showed pronuclei or pronuclear precursors using DAPI (Figure lb); in four cases pronuclei were visible using the light microscope (Figure la). From hpi, pronuclei were visible using the light microscope in all embryos where pronuclei were visible using DAPI (Figure lc, d, e). However, in seven (%) of the embryos analysed from hpi, the presence of a single, condensed sperm head in the cytoplasm was demonstrated by DAPI stain, but decondensation of sperm heads did not take place (Figure If)Among the reinseminated oocytes, of the oocytes analysed at. hpri had been penetrated by spermatozoa and showed pronucleus precursors using DAPI stain, although only two had developed small pronuclei that were just visible using the light microscope but that contained no nucleoli (Figure ). Of the oocytes fixed at hpri, had been penetrated and showed pronuclei or pronuclear precursors using DAPI; five Downloaded from on March

4 G.Capmany et al. Figure. A reinseminated oocyte fixed at. h post reinsemination (hpri) and viewed under UV excitation after,diamidinophenylindole staining. Multiple decondensing sperm heads (dsh; pronuclear precursors) and two condensed sperm heads (csh) can be seen. Table II. Number of embryos analysed for DNA content at different times following insemination or reinsemination in vitro Oocytes that developed >PN Freshly inseminated oocytes with PN PN Reinseminated oocytes with PN PN >PN Total no. of Hours post insemination/reinsemination. oocytes PN = pronuclei. showed large, obvious pronuclei in which nucleoli were visible using the light microscope. From hpri, where pronuclei were visible using DAPI, they were also visible using the light microscope. However, as with the freshly inseminated oocytes, decondensation of sperm heads did not always take place following sperm penetration, in this case in of (%) of embryos analysed from hpri, seven of which had been penetrated by one spermatozoon, two by two, three by three, one by five, and three by more than five spermatozoa. The difference between the numbers of freshly inseminated and reinseminated oocytes that were penetrated but failed to develop pronuclei is statistically significant (P <.). DNA synthesis The number of fertilized freshly inseminated and reinseminated oocytes analysed for DNA content at different times postinsemination is shown in Table n. Of the oocytes that were fixed for analysis, one freshjy inseminated and reinseminated oocytes could not be analysed for DNA content Downloaded from on March either because the stain was too faint (n = ), or because the pronuclei were overlapping (n = ). The number of embryos analysed in each group, and the DNA content of their pronuclei at each time point are shown in Table HI; the time course of DNA synthesis in each group is illustrated in Figures and. Sixteen of the freshly inseminated embryos analysed showed a DNA content of >C in one or both of their pronuclei (two embryos at hpi, four at hpi, one at hpi, three at hpi, three at hpi, and three at hpi). Three of the reinseminated embryos analysed showed at least one pronucleus with a DNA content of ^C (one embryo at hpri, one at hpri, and one at hpri). The timing of DNA synthesis and the percentage of embryos with a DNA content >C in at least one of their pronuclei were not significantly different in embryos derived from freshly inseminated oocytes that developed two (n = ) or three pronuclei (n = ; data not shown). In embryos derived from reinseminated oocytes, the same comparison could not be made because of the degree of variation observed between embryos within each group.

5 Timing of events in the cell embryo Table HI. Numbers of freshly inseminated and reinseminated oocytes analysed content of individual pronuclei at different times after insemination in vitro grouped according to the DNA Pronuclear DNA content (C) in embryos Hours post insemination/reinsemination. Embryos derived from freshly inseminated oocytes <C C CX>C C >C No. of pronuclei No. of embryos Embryos derived from reinseminated oocytes <C C CX>C C >C No. of pronuclei No. of embryos standardized Seescan measurement of DNA content [C] standardized => Seescan measurement of DNA content [C] Metaphase hp! II oocytes [n=] [n=] hpi [n=) hpl [n=z] hpl [n=] Tune: hours post Insemination [hp{] hpi [n=j hpi [n=] Figure. Box plots of standardized corrected nuclear intensity measurements of DNA content in individual pronuclei for, diamidinophenylindolestained human embryos derived from freshly inseminated oocytes, fixed at specified times between and hpi. In view of the small number of pronuclei analysed, measurements were pooled for embryos fixed between the time intervals indicated. The upper, middle and lower bars of the box represent the th, th and th centiles respectively. The th and th centiles are marked by the whiskers, with any values falling outside this range shown as individual points, n = number of pronuclei measured. The first cleavage division The timing of pronuclear breakdown and of the first cleavage division of embryos derived from freshly inseminated oocytes that showed two pronuclei at hpi is illustrated in Figure. The earliest time at which pronuclear breakdown had taken place was hpi, and the latest time was between and hpi, with a median time for pronuclear breakdown of hpi. Embryos completed the first cleavage division between and hpi (median hpi), and only one embryo Metaphase hpi II oocytes (n=) [n=] hpi [n= hpi (n=l hpi [n=] Time: hours post Insemination [hpri] hpi [n=] hpi [n=] Figure. Box plots of standardized corrected nuclear intensity measurements of DNA content in individual pronuclei for, diamidinophenylindolestained human embryos derived from reinseminated oocytes, fixed at specified time points between and hpri. In view of the small number of pronuclei analysed, measurements were pooled for embryos fixed between the time intervals indicated. The upper, middle and lower bars of the box represent the th, th and th centiles respectively. The th and th centiles are marked by the whiskers, with any values falling outside this range shown as individual points, n = number of pronuclei measured. had failed to undergo cleavage by hpi; this embryo was considered to have arrested. The shortest and longest intervals between pronuclear breakdown and cleavage (Mphase) were and h respectively (median h). The timing of the same events in embryos that developed two (n = ), three (n = ), four (n = ) or more than four pronuclei («= ) after reinsemination is illustrated in Figure. The earliest time at which pronuclear breakdown had taken place was hpi, and the latest between and hpi (Xtwopronucleate, Xthreepronucleate, lxfourpronucle Downloaded from on March

6 G.Capmany et al. PN vmhr D PN bre«kdown Zcell embryo Figure. Diagram summarizing the findings of the present study, illustrating the timing of the main events taking place during the first cell cycle of human embryogenesis. See text for more details. TIME (hpi) Figure. Time of pronuclear (PN) breakdown and cleavage in pronucleatestage human embryos derived from freshly inseminated oocytes, scored at hourly intervals between and h postinsemination (hpi). GJ P!N w * developed PM >U<Me rn breakdown ceil embryo O at Figure. Time of pronuclear (PN) breakdown and cleavage in pronucleatestage human embryos derived from reinseminated oocytes, scored at h intervals between and h post reinsemination (hpri) and at hpri. ate). However, two embryos (lxtwopronucleate and X>pronucleate) still had pronuclei visible at hpi. Of the seven embryos that underwent the first cleavage division by hpi, three had done so within h of reinsemination, with the earliest one to cleave having done so by hpi. Thus, the shortest interval between pronuclear breakdown and cleavage (Mphase) was h, while the longest Mphase lasted at least h. Controls Of control oocytes that had their zonae removed at hpi and were cultured until hphcg ( hpi), five (%) developed one pronucleus and, following staining with DAPI, two (%) were found to have been penetrated by a single spermatozoon. None of the oocytes developed two or more pronuclei. Downloaded from on March Discussion This study describes the timing of pronuclear formation, DNA synthesis, pronuclear breakdown and cleavage during the first cell cycle of human embryogenesis following fertilization in vitro. The findings are summarized in Figure. These data were obtained from a limited number of oocytes, donated to research by patients undergoing therapeutic IVF. Unfortunately, none of the oocytes fixed at hpi were penetrated by spermatozoa, which meant that it was not possible to define as precisely as desirable the timing of pronuclear formation, and of the beginning of DNA synthesis. Nonetheless, it can be stated that pronuclear formation takes place between and hpi. The only other study which has described the dynamics of pronuclear formation in cell embryos derived from conventional IVF (Balakier et al, ) did not examine embryos for the presence of pronuclei before hpi, and found the earliest pronuclear formation to take place around hpi. However, two studies have described the dynamics of pronuclear formation in cell embryos derived following intracytoplasmic sperm injection (ICSI) and subzonal insemination (SUZI) respectively, and both report similar findings to those described here (Nagy et al, ; Van Wissen et al, ). Thus, pronuclei were first observed h posticsi, and % of embryos had developed pronuclei by h posticsi (Nagy et al, ), compared with being observed between. and h, and % by. h following SUZI (Van Wissen et al, ). DNA synthesis begins between and hpi and is completed in the majority of embryos between and hpi. Gl lasts ~ h, based on the observations that all four embryos fixed at hpi had already completed meiosis, and only one of four embryos fixed at hpi had entered Sphase. These results are generally in agreement with those reported by Balakier et al (), who estimated that Sphase begins at hpi and is completed by hpi, with a range from to hpi. A small number of the embryos derived from freshly inseminated oocytes that were analysed between and hpi showed DNA values of s=c in one or both of their pronuclei (/ twcpronucleate embryos, / threepronucleate embryos). The reasons for this elevated DNA content are unclear, but are clearly not associated with ageing, since high DNA contents were found more frequently in inseminated than in reinseminated oocytes. It could be postulated that

7 Timing of events in the cell embryo these embryos have arrested at the cell stage, but have nonetheless commenced the second cycle of DNA synthesis. This seems unlikely, since while it might explain the four cases where embryos were analysed at hpi, it would explain neither the case where the DNA content was >C, nor the seven cases where the DNA content was >C before hpi. Seven embryos that arrested at the cell stage were analysed for DNA content after fixation at (n = ) or hpi (n = ; G.Capmany, unpublished observations). Of these, two embryos fixed at hpi appeared to have undergone two rounds of DNA replication (.C and.c) in the absence of karyokinesis, one embryo fixed at hpi that showed five small nuclei had a total DNA content equivalent to a cell embryo (C), and one embryo fixed at hpi which showed multiple nuclei that appeared to have undergone three rounds of replication (C). The remaining three embryos had failed to replicate their DNA. Of the embryos analysed, had developed three pronuclei. There appear to be no differences between these embryos and those which developed two pronuclei in the timing of pronuclei formation and DNA synthesis. This confirmed the findings of Balakier (). The duration of Mphase was relatively constant (median h, range h), whereas the time of pronuclear breakdown varied considerably between embryos (from to hpi), as did the time of the first cleavage division (from to hpi). Despite this range, the majority of embryos underwent pronuclear breakdown and cleavage between and hpi and and hpi respectively. These findings support those of the earlier studies of embryos derived by conventional FVF (Balakier et al, ; Van Wissen et al, ), although following microinjection, pronuclear breakdown and cleavage to the cell stage both appear to proceed between and h earlier (Nagy et al, ; Van Wissen et al, ). The only other study which describes the time of onset of DNA synthesis in the human cell embryo used embryos derived from reinseminated human oocytes (Tesarik and Kopecny, a), and the results conflict with those described for freshly inseminated oocytes both in the present study and in that of Balakier et al. (). This throws into question the validity of using reinseminated oocytes in any investigations of normal human embryogenesis (Tesarik and Kopecny, a,b). The detailed description of events during the first cell cycle of reinseminated oocytes presented here means that it is now possible to compare them with those of freshly inseminated oocytes, and thus to determine whether or not reinseminated oocytes provide a valid model of normal human embryogenesis. The incidence of development of one pronucleus was the same (%) in both reinseminated and control oocytes, so it can be assumed that exposure to acid Tyrode's solution in order to dissolve the zona pellucida does not increase the activation rate of reinseminated oocytes. The rate of sperm penetration is not significantly different in the two groups of oocytes. However, the rate of pronuclear formation is significantly lower in reinseminated than in freshly inseminated oocytes and, where pronuclei did develop, they formed more rapidly than in freshly inseminated oocytes, being visible in the majority of embryos between and hpri, and appearing as early as hpri. Sphase commences and is completed approximately and h sooner respectively in embryos derived from reinseminated oocytes than in those derived from freshly inseminated oocytes. This finding conflicts with that of Tesarik and Kopecny (a), who reported that DNA synthesis commences in reinseminated oocytes at hpri. The reason for this discrepancy is unclear, particularly since the technique used by Tesarik and Kopecny (a) to determine the onset of DNA synthesis, although different from ours, is the same as that used by Balakier et al. (), whose results are in agreement with our own. In both groups of embryos analysed, pronuclei formation coincides with the beginning of Sphase, supporting the suggestion that the onset of DNA synthesis is related to pronuclear development (Tesarik and Kopecny, a,b). Likewise, both groups of embryos display variation in the lengths of Sphase and G. This variation may simply reflect normal asynchrony between embryos during the first cell cycle. Alternatively, it may be due to delayed or arrested development, or even be related to variation in the time at which sperm penetration occurs in each oocyte (Balakier et al, ). It is unlikely that the presence of pronuclei and the detection of DNA synthesis by hpri could have resulted from delayed pronuclear formation following the penetration of spermatozoa after the initial insemination at hphcg, since (i) only % of embryos derived from freshly inseminated oocytes failed to develop pronuclei within hpi, (ii) five or more decondensing sperm heads were observed at and hpri in % of the embryos which showed DNA synthesis and (iii) none of the control oocytes developed more than one pronucleus. Pronuclear breakdown took place later in embryos derived from reinseminated oocytes than in those derived from freshly inseminated oocytes; thus, of the embryos that underwent pronuclear breakdown, six (%) did so between and hpi. A high percentage (/, %) of reinseminated oocytes which developed pronuclei failed to undergo cleavage by hpri, compared with only one of (%) embryos derived from freshly inseminated oocytes. Finally, only % (/) of embryos derived from reinseminated oocytes that underwent cleavage did so before hpi, whereas all embryos derived from freshly inseminated oocytes which cleaved did so within this time. Two factors may account for the differences observed between embryos derived from freshly inseminated and reinseminated oocytes. First, the oocytes used for reinsemination had failed to fertilize either because they had been freshly inseminated with dysfunctional spermatozoa, or because they themselves could not achieve fertilization, as indicated by the fertilization of sibling oocytes. It is possible that the latter group of oocytes has a reduced capacity to support normal development Second, removal of the zona pellucida prior to reinsemination allows the penetration of more than one spermatozoon, and it has been shown that, following reinsemination, the rates of both pronuclear development and DNA synthesis are altered when oocytes are penetrated by more than five spermatozoa (Tesarik and Kopecny, b). These results suggest that the use of embryos derived from reinseminated oocytes as a model for the first cell cycle of Downloaded from on March

8 G.Capmany at al. human embryogenesis is limited. However, in view of the scarcity of pronucleatestage human embryos for research, it may prove useful to use such material, provided any results obtained from them are confirmed using relatively small numbers of freshly inseminated oocytes. Acknowledgements We would like to thank the staff of the Assisted Conception Unit at King's College Hospital for their assistance in providing material. G.C. is supported by a grant from the EC Science Programme. References Balakier, H. () Tripronuclear human zygotes: the first cell cycle and subsequent development. Hum. Reprod.,,. Balakier, H., Zenzes, M., Wang, P., MacLusky, NJ. and Casper, R.F. () The effect of cryopreservation on the development of S and Gphase mouse embryos. J. In Vitro Fertil. Embryo Transfer,,. Balakier, H., MacLusky, NJ. and Casper, R.F. () Characterisation of the first cell cycle in human zygotes: implications for cryopreservation. Fertil. Steril.,,. Nagy, Z.P., Liu, J., Joris, H., Devroey, P. and Van Steirteghem, A. () Timecourse of oocyte activation, pronucleus formation and cleavage in human oocytes fertilized by intracytoplasmic sperm injection. Hum. Reprod.,,. Taylor, A.S. and Braude, P.R. () The early development and DNA content of activated human oocytes and parthenogenetic human embryos. Hum. Reprod..,. Taylor, A.S. and Braude, P.R. () Evaluation of a simple method of measuring the cellular content of mouse oocytes and embryos, human fibroblasts, and parthenogenctically activated human oocytes using a computerized image analysis system (Seescan). Zygote,,. Tesarik, J. and Kopecny, V. (a) Nucleic acid synthesis and development of human male pronucleus. J. Reprod. Fertil.,,. Tesarik, J. and Kopecny, V. (b) Developmental control of the human male pronucleus by ooplasmic factors. Hum. Reprod.,,. Van Wissen, B., Wolf, J.P., BomselHelmreich, O., Frydman, R. and Jouannet, P. () Timing of pronuclear development and first cleavages in human embryos after subzonal insemination: influence of sperm phcnotypc. Hum. Reprod.,,. Waterstone, JJ. and Parsons, J.H. () A prospective study to investigate the value of flushing follicles during transvaginal ultrasounddirected follicle aspiration. Fertil Steril.,,. Received on November, ; accepted on March, Downloaded from on March