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Laser zona drilling does not induce hsp70i transcription in blastomeres of eight-cell mouse embryos

2005; Elsevier BV; Volume: 84; Issue: 5 Linguagem: Inglês

10.1016/j.fertnstert.2005.05.023

1556-5653

Cristina Hartshorn, Aleksandra Anshelevich, Lawrence J. Wangh,

Reproductive Biology and Fertility

To assess whether zona drilling with a 1,480-nm laser induces heat shock in eight-cell embryos, we measured hsp70i RNA levels in sets of single blastomeres isolated after laser treatment of mouse embryos that had or had not been heated at 43°C. Unlike heating, laser zona drilling did not stimulate hsp70i expression, even in the blastomeres closest to the laser beam, corroborating the safety of this procedure for assisted reproduction. To assess whether zona drilling with a 1,480-nm laser induces heat shock in eight-cell embryos, we measured hsp70i RNA levels in sets of single blastomeres isolated after laser treatment of mouse embryos that had or had not been heated at 43°C. Unlike heating, laser zona drilling did not stimulate hsp70i expression, even in the blastomeres closest to the laser beam, corroborating the safety of this procedure for assisted reproduction. The perforation of the zona pellucida (ZP) of embryos fertilized in vitro is known to enhance hatching and facilitate implantation (1Liu H.C. Cohen J. Alikani M. Noyes N. Rosenwaks Z. Assisted hatching facilitates earlier implantation.Fertil Steril. 1993; 60: 871-875Abstract Full Text PDF PubMed Google Scholar) and is a necessary step for the isolation of single blastomeres or polar bodies for genetic analysis (2De Vos A. Van Steirteghem A. Aspects of biopsy procedures prior to preimplantation genetic diagnosis.Prenat Diagn. 2001; 21: 767-780Crossref PubMed Scopus (118) Google Scholar). The recent availability of lasers suitable for zona drilling has spurred numerous studies aimed at establishing the validity of this procedure. The mode and intensity of heating generated by the laser have been scrutinized with particular attention because hyperthermia is a known cause of teratogenesis and embryonic death (3Graham Jr, J.M. Edwards M.J. Edwards M.J. Teratogen update gestational effects of maternal hyperthermia due to febrile illnesses and resultant patterns of defects in humans.Teratology. 1998; 58: 209-221Crossref PubMed Scopus (180) Google Scholar).Diode 1,480-nm lasers (4Germond M. Nocera D. Senn A. Rink K. Delacretaz G. Fakan S. Microdissection of mouse and human zona pellucida using a 1.48-microns diode laser beam efficacy and safety of the procedure.Fertil Steril. 1995; 64: 604-611Abstract Full Text PDF PubMed Google Scholar) present ideal characteristics to avoid blastomere damage. They emit a long-wavelength, nonmutagenic radiation that is deliverable in noncontact mode directly through a microscope lens. Quantification of the heat produced by a 1,480-nm laser beam has shown that short pulses at high power minimize thermal excursion in the blastomeres' proximity while allowing precise zona cutting (5Douglas-Hamilton D.H. Conia J. Thermal effects in laser-assisted pre-embryo zona drilling.J Biomed Opt. 2001; 6: 205-213Crossref PubMed Scopus (38) Google Scholar). Several studies employing this type of laser on human embryos have addressed the issue of treatment safety by looking at outcomes such as blastocyst development (6Wong B.C. Boyd C.A. Lanzendorf S.E. Randomized controlled study of human zona pellucida dissection using the zona infrared laser optical system evaluation of blastomere damage, embryo development, and subsequent hatching.Fertil Steril. 2003; 80: 1249-1254Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 7Caswell W.A. Bochicchio J.A. Tucker M. San Roman G.A. Stelling J.R. Comparison of laser assisted hatching to chemical assisted hatching for embryo biopsy.Fertil Steril. 2003; 80 ([abstract no. P-216]): S193Abstract Full Text Full Text PDF PubMed Google Scholar, 8Carter J. Graham J. Han T. Davis A. Kearns W.G. Tucker M. Embryo development post laser biopsy for PGD.Fertil Steril. 2003; 80 ([abstract no. P-227]): S197Abstract Full Text Full Text PDF Google Scholar), implantation rate (9Montag M. van der Ven H. Laser-assisted hatching in assisted reproduction.Croat Med J. 1999; 40: 398-403PubMed Google Scholar), or pregnancy success (10Han T.S. Sagoskin A.W. Graham J.R. Tucker M.J. Liebermann J. Laser-assisted human embryo biopsy on the third day of development for preimplantation genetic diagnosis two successful case reports.Fertil Steril. 2003; 80: 453-455Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar, 11Levron J. Ferber-Meiri B. Bider D. Shulman A. Levin T. Shporn E. A prospective randomized study comparing laser and Tyrode's mediated methods of assisted hatching.Fertil Steril. 2003; 80 ([abstract no. P-241]): S202Abstract Full Text Full Text PDF Google Scholar) and have concluded that laser ablation of the zona is as good or better than any other available method. The effects of laser treatment, however, have not been investigated at the molecular level.We have recently have demonstrated that embryos at the eight-cell stage are able to respond to thermal shock by activating heat shock proteins (HSPs) production, as shown by the sharp increase in hsp70i transcription that follows embryo exposure to elevated temperature (12Hartshorn C. Anshelevich A. Wangh L.J. Rapid, single-tube method for quantitative preparation and analysis of RNA and DNA in samples as small as one cell.BMC Biotechnol. 2005; 5: 2Crossref PubMed Scopus (31) Google Scholar). In the present study, we measured hsp70i RNA levels in individual blastomeres isolated from eight-cell mouse embryos after zona drilling with a 1,480 nm laser and we have compared them to those found in cells of embryos heated at 43°C, using a direct and highly sensitive molecular method in order to evaluate the possibility of heat response in laser-treated embryos.The designation hsp70i includes the two heat-inducible members of the hsp70 family of genes, hsp70.1 and hsp70.3, that share genomic sequence in the translated region and whose individual expression patterns are still largely unknown but are probably overlapping (12Hartshorn C. Anshelevich A. Wangh L.J. Rapid, single-tube method for quantitative preparation and analysis of RNA and DNA in samples as small as one cell.BMC Biotechnol. 2005; 5: 2Crossref PubMed Scopus (31) Google Scholar, 13Huang L. Mivechi N.F. Moskophidis D. Insights into regulation and function of the major stress-induced hsp70 molecular chaperone in vivo analysis of mice with targeted gene disruption of the hsp70.1 or hsp70.3 gene.Mol Cell Biol. 2001; 21: 8575-8591Crossref PubMed Scopus (117) Google Scholar, 14Hunt C.R. Dix D.J. Sharma G.G. Pandita R.K. Gupta A. Funk M. et al.Genomic instability and enhanced radiosensitivity in Hsp70.1- and Hsp70.3-deficient mice.Mol Cell Biol. 2004; 24: 899-911Crossref PubMed Scopus (198) Google Scholar).Mouse embryos (B6C3F1 females bred with B6D2F1 males) were purchased frozen at the two-cell stage from Embryotech Laboratories, Inc. (Wilmington, MA) and cultured in GEM-PS medium (Duncan Holly, Bedford, MA) to the precompaction eight-cell stage, as described elsewhere (15Hartshorn C. Rice J.E. Wangh L.J. Differential pattern of Xist RNA accumulation in single blastomeres isolated from 8-cell stage mouse embryos following laser zona drilling.Mol Reprod Dev. 2003; 64: 41-51Crossref PubMed Scopus (25) Google Scholar). To induce heat shock, embryos were placed at 43°C for 30 minutes and allowed to recover at 37°C for 2 or 3 hours, during which RNA synthesis took place (12Hartshorn C. Anshelevich A. Wangh L.J. Rapid, single-tube method for quantitative preparation and analysis of RNA and DNA in samples as small as one cell.BMC Biotechnol. 2005; 5: 2Crossref PubMed Scopus (31) Google Scholar). Because of cell cycle arrest, cell division did not occur in these embryos (henceforth designated as heat-shocked) during recovery. Control eight-cell embryos were not heated. Embryos were then collected intact or were dissociated in single cells after laser zona drilling according to our standard protocol (12Hartshorn C. Anshelevich A. Wangh L.J. Rapid, single-tube method for quantitative preparation and analysis of RNA and DNA in samples as small as one cell.BMC Biotechnol. 2005; 5: 2Crossref PubMed Scopus (31) Google Scholar, 15Hartshorn C. Rice J.E. Wangh L.J. Differential pattern of Xist RNA accumulation in single blastomeres isolated from 8-cell stage mouse embryos following laser zona drilling.Mol Reprod Dev. 2003; 64: 41-51Crossref PubMed Scopus (25) Google Scholar), which requires three 1-ms pulses with a noncontact 1,480-nm diode laser beam (ZILOS-tk™ zona infrared laser optical system; Hamilton Thorne Biosciences, Inc., Beverly, MA). Blastomeres were harvested immediately after zona drilling (12Hartshorn C. Anshelevich A. Wangh L.J. Rapid, single-tube method for quantitative preparation and analysis of RNA and DNA in samples as small as one cell.BMC Biotechnol. 2005; 5: 2Crossref PubMed Scopus (31) Google Scholar, 15Hartshorn C. Rice J.E. Wangh L.J. Differential pattern of Xist RNA accumulation in single blastomeres isolated from 8-cell stage mouse embryos following laser zona drilling.Mol Reprod Dev. 2003; 64: 41-51Crossref PubMed Scopus (25) Google Scholar) so that hsp70i RNA transcription had time to take place after the heating step, but not as a result of laser treatment.An alternative protocol was used for embryos designated as laser-treated, which were not incubated at 43°C. Samples underwent laser zona drilling and were then placed back at 37°C for 2 hours before either cell dissociation or harvesting of whole embryos, to permit hsp70i RNA accumulation and investigate a possible heat shock effect generated by the laser beam itself. In contrast to heat-shocked embryos, cell division took place in several of these specimens during recovery time, resulting in 9- to 10-cell embryos at collection time.Nucleic acids preparation, reverse transcription, and real-time polymerase chain reaction (PCR) were performed in a single tube by the PurAmp method (12Hartshorn C. Anshelevich A. Wangh L.J. Rapid, single-tube method for quantitative preparation and analysis of RNA and DNA in samples as small as one cell.BMC Biotechnol. 2005; 5: 2Crossref PubMed Scopus (31) Google Scholar). Briefly, samples were lysed on a PCR tube lid, releasing both RNA and DNA, and reverse transcription and real-time PCR were then carried out in the same tube. The hsp70i PCR assay and the strategy used to calculate hsp70i RNA levels were as described elsewhere (12Hartshorn C. Anshelevich A. Wangh L.J. Rapid, single-tube method for quantitative preparation and analysis of RNA and DNA in samples as small as one cell.BMC Biotechnol. 2005; 5: 2Crossref PubMed Scopus (31) Google Scholar). The hsp70i cDNA and genomic DNA templates (total hsp70i templates) were identical because the hsp70i genes are intronless, and they were coamplified in each sample. Standard curves were obtained from serial dilutions of genomic DNA (12Hartshorn C. Anshelevich A. Wangh L.J. Rapid, single-tube method for quantitative preparation and analysis of RNA and DNA in samples as small as one cell.BMC Biotechnol. 2005; 5: 2Crossref PubMed Scopus (31) Google Scholar, 15Hartshorn C. Rice J.E. Wangh L.J. Differential pattern of Xist RNA accumulation in single blastomeres isolated from 8-cell stage mouse embryos following laser zona drilling.Mol Reprod Dev. 2003; 64: 41-51Crossref PubMed Scopus (25) Google Scholar). Mouse DNA contains four hsp70i copies per genome, one copy of hsp70.1 and one copy of hsp70.3 on each chromosome 17. Four copies per cell were thus subtracted from total hsp70i template numbers to calculate hsp70i RNA levels.Heat shock produced a sharp increase in the hsp70i RNA levels quantified in embryos at the eight-cell stage, as illustrated in Figure 1A. In that figure, blue bars indicate the hsp70i RNA + DNA contents of all single blastomeres that could be recovered from each of three embryos (a, b, and c) after incubation at 43°C. The contribution of hsp70i DNA (four copies per cell) is negligible on the scale used, but its presence confirmed that every cell had been delivered to the assay tube even when RNA was not present. (See cell 4a [top panel], which contained only four hsp70i templates.)The data in Figure 1A are plotted according to the cell-picking order for each embryo. Cell 1 was the first to exit through the laser-drilled hole and thus was the closest to the laser beam because embryos do not rotate freely inside the ZP (16Gardner R.L. Specification of embryonic axes begins before cleavage in normal mouse development.Development. 2001; 128: 839-847PubMed Google Scholar). The asterisks on some of the bars in Figure 1 mark pairs of blastomeres, stemming from recent cell division, that required separation with a micropipette. It is evident from the bar graphs in Figure 1A that all but one of the cells recovered from heat-shocked embryos contained hundreds of hsp70i RNA copies. The one nonresponsive cell derived from a pair, and it is possible that its transcription machinery was slow in restarting after cell division. The proximity of a cell to the laser radiation did not affect its hsp70i RNA content, because the hsp70i RNA levels of the first-picked blastomeres were not consistently lower than those of the others. Hence, laser treatment does not damage the RNA present in the cells. Conversely, an increase in hsp70i transcription caused by the heat generated by the laser would not have the time to occur in this type of experiment because the cells were collected right after laser treatment.To exclude the possibility that some RNA was degraded during cell biopsy, resulting in artifactually variable response levels, we compared our single-blastomere results with hsp70i RNA measurements performed on whole embryos. The average hsp70i RNA + DNA contents of the heat-shocked blastomeres recovered from each of the three embryos (green bars in Fig. 1A) are juxtaposed to average hsp70i RNA + DNA copy numbers per cell calculated from analyses of whole heat-shocked embryos (red bars in Fig. 1A). The two sets of data are in all cases remarkably similar, demonstrating that the cell isolation procedure does not affect RNA recovery. In the absence of heat shock, hsp70i RNA levels were minimal, as indicated by the mean hsp70i RNA + DNA content of four untreated whole embryos, corresponding to 19 ± 7 hsp70i total templates, or 15 copies of hsp70i RNA, per cell (black bars in Fig. 1A).For our second set of experiments, we collected series of individual blastomeres from embryos that had not been purposely exposed to high temperature but had undergone the laser zona drilling procedure necessary for cell harvesting. To investigate the possible occurrence of thermal stress, we allowed enough time for transcript synthesis and accumulation to take place before the embryos were dissociated in individual blastomeres or collected whole.As shown in Figure 1B, the number of hsp70i RNA copies measured in the first blastomere recovered from each of four eight-cell embryos after laser zona drilling averaged 25 ± 22 (blue bar 1), consistent with the average number of hsp70i RNA copies per cell (24 ± 16) calculated from whole embryos. Similarly, the average number of hsp70i transcripts in the other blastomeres of the same embryos varied from 16 ± 22 to 37 ± 48 copies (blue bars 2–9). As shown above for non-heated embryos, these low levels of hsp70i transcription are normally present in embryonic blastomeres. More pulses were used for this experiment (four to ten 1-ms pulses) than were required by our standard protocol, strengthening the conclusion that treatment with this type of laser does not generate thermal shock in embryonic cells. Analysis of a whole eight-cell embryo whose zona was removed with eight 1.5-ms laser pulses (pulses more numerous and longer than usual) gave comparable results (36 copies of hsp70i RNA per cell, yellow bar). In contrast, embryos' exposure to 43°C again induced a marked surge in the number of hsp70i RNA copies per cell (1,331 ± 238; red bar). Our results, thus, overall prove that laser treatment under the conditions described in this report does not produce a heat shock–like effect on hsp70i transcription.To corroborate this conclusion, we compared the rates of development to the blastocyst stage of laser-treated and untreated eight-cell embryos. Forty-six hours after treatment, all 24 embryos used for this experiment had become expanded blastocysts and had begun hatching, although hatching was more advanced in the 12 laser-treated embryos. The unchanged rate of development strongly indicates that laser treatment did not induce cell cycle arrest, a well-known effect of hyperthermia (17Kuhl N.M. Rensing L. Heat shock effects on cell cycle progression.Cell Mol Life Sci. 2000; 57: 450-463Crossref PubMed Scopus (103) Google Scholar).Comparatively longer treatments with lasers of different wavelengths and powers are known to activate the hsp70 promoter in a number of different systems (18Halloran M.C. Sato-Maeda M. Warren J.T. Su F. Lele Z. Krone P.H. et al.Laser-induced gene expression in specific cells of transgenic zebrafish.Development. 2000; 127: 1953-1960PubMed Google Scholar, 19Souil E. Capon A. Mordon S. Dinh-Xuan A.T. Polla B.S. Bachelet M. Treatment with 815-nm diode laser induces long-lasting expression of 72-kDa heat shock protein in normal rat skin.Br J Dermatol. 2001; 144: 260-266Crossref PubMed Scopus (97) Google Scholar). In contrast, our findings that laser zona drilling does not trigger hsp70i expression in eight-cell embryos nor affect the rate of preimplantation development, unlike heating at 43°C, strongly support the conclusion that embryonic cells were not thermally stressed by the procedure employed for this study.These results have important implications, because an increase in hsp70i transcription signals the occurrence of cell damage and cell cycle arrest even in the absence, or well in advance, of identifiable morphological changes. Such events, even if they are transient, could affect specific blastomeres' survival and developmental fate, as recent data overwhelmingly indicate that the destiny of mammalian blastomeres at the cleavage stages already may be determined and may be linked to the timing of the cell cycle (20Pearson H. Your destiny, from day one.Nature. 2002; 418: 14-15Crossref PubMed Scopus (36) Google Scholar, 21Piotrowska-Nitsche K. Zernicka-Goetz M. Spatial arrangement of individual 4-cell stage blastomeres and the order in which they are generated correlate with blastocyst pattern in the mouse embryo.Mech Dev. 2005; 122: 487-500Crossref PubMed Scopus (107) Google Scholar).In conclusion, this study thus provides the first evidence at the molecular level for the safety of ZP perforation with a 1,480-nm diode laser under carefully chosen conditions. The perforation of the zona pellucida (ZP) of embryos fertilized in vitro is known to enhance hatching and facilitate implantation (1Liu H.C. Cohen J. Alikani M. Noyes N. Rosenwaks Z. Assisted hatching facilitates earlier implantation.Fertil Steril. 1993; 60: 871-875Abstract Full Text PDF PubMed Google Scholar) and is a necessary step for the isolation of single blastomeres or polar bodies for genetic analysis (2De Vos A. Van Steirteghem A. Aspects of biopsy procedures prior to preimplantation genetic diagnosis.Prenat Diagn. 2001; 21: 767-780Crossref PubMed Scopus (118) Google Scholar). The recent availability of lasers suitable for zona drilling has spurred numerous studies aimed at establishing the validity of this procedure. The mode and intensity of heating generated by the laser have been scrutinized with particular attention because hyperthermia is a known cause of teratogenesis and embryonic death (3Graham Jr, J.M. Edwards M.J. Edwards M.J. Teratogen update gestational effects of maternal hyperthermia due to febrile illnesses and resultant patterns of defects in humans.Teratology. 1998; 58: 209-221Crossref PubMed Scopus (180) Google Scholar). Diode 1,480-nm lasers (4Germond M. Nocera D. Senn A. Rink K. Delacretaz G. Fakan S. Microdissection of mouse and human zona pellucida using a 1.48-microns diode laser beam efficacy and safety of the procedure.Fertil Steril. 1995; 64: 604-611Abstract Full Text PDF PubMed Google Scholar) present ideal characteristics to avoid blastomere damage. They emit a long-wavelength, nonmutagenic radiation that is deliverable in noncontact mode directly through a microscope lens. Quantification of the heat produced by a 1,480-nm laser beam has shown that short pulses at high power minimize thermal excursion in the blastomeres' proximity while allowing precise zona cutting (5Douglas-Hamilton D.H. Conia J. Thermal effects in laser-assisted pre-embryo zona drilling.J Biomed Opt. 2001; 6: 205-213Crossref PubMed Scopus (38) Google Scholar). Several studies employing this type of laser on human embryos have addressed the issue of treatment safety by looking at outcomes such as blastocyst development (6Wong B.C. Boyd C.A. Lanzendorf S.E. Randomized controlled study of human zona pellucida dissection using the zona infrared laser optical system evaluation of blastomere damage, embryo development, and subsequent hatching.Fertil Steril. 2003; 80: 1249-1254Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 7Caswell W.A. Bochicchio J.A. Tucker M. San Roman G.A. Stelling J.R. Comparison of laser assisted hatching to chemical assisted hatching for embryo biopsy.Fertil Steril. 2003; 80 ([abstract no. P-216]): S193Abstract Full Text Full Text PDF PubMed Google Scholar, 8Carter J. Graham J. Han T. Davis A. Kearns W.G. Tucker M. Embryo development post laser biopsy for PGD.Fertil Steril. 2003; 80 ([abstract no. P-227]): S197Abstract Full Text Full Text PDF Google Scholar), implantation rate (9Montag M. van der Ven H. Laser-assisted hatching in assisted reproduction.Croat Med J. 1999; 40: 398-403PubMed Google Scholar), or pregnancy success (10Han T.S. Sagoskin A.W. Graham J.R. Tucker M.J. Liebermann J. Laser-assisted human embryo biopsy on the third day of development for preimplantation genetic diagnosis two successful case reports.Fertil Steril. 2003; 80: 453-455Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar, 11Levron J. Ferber-Meiri B. Bider D. Shulman A. Levin T. Shporn E. A prospective randomized study comparing laser and Tyrode's mediated methods of assisted hatching.Fertil Steril. 2003; 80 ([abstract no. P-241]): S202Abstract Full Text Full Text PDF Google Scholar) and have concluded that laser ablation of the zona is as good or better than any other available method. The effects of laser treatment, however, have not been investigated at the molecular level. We have recently have demonstrated that embryos at the eight-cell stage are able to respond to thermal shock by activating heat shock proteins (HSPs) production, as shown by the sharp increase in hsp70i transcription that follows embryo exposure to elevated temperature (12Hartshorn C. Anshelevich A. Wangh L.J. Rapid, single-tube method for quantitative preparation and analysis of RNA and DNA in samples as small as one cell.BMC Biotechnol. 2005; 5: 2Crossref PubMed Scopus (31) Google Scholar). In the present study, we measured hsp70i RNA levels in individual blastomeres isolated from eight-cell mouse embryos after zona drilling with a 1,480 nm laser and we have compared them to those found in cells of embryos heated at 43°C, using a direct and highly sensitive molecular method in order to evaluate the possibility of heat response in laser-treated embryos. The designation hsp70i includes the two heat-inducible members of the hsp70 family of genes, hsp70.1 and hsp70.3, that share genomic sequence in the translated region and whose individual expression patterns are still largely unknown but are probably overlapping (12Hartshorn C. Anshelevich A. Wangh L.J. Rapid, single-tube method for quantitative preparation and analysis of RNA and DNA in samples as small as one cell.BMC Biotechnol. 2005; 5: 2Crossref PubMed Scopus (31) Google Scholar, 13Huang L. Mivechi N.F. Moskophidis D. Insights into regulation and function of the major stress-induced hsp70 molecular chaperone in vivo analysis of mice with targeted gene disruption of the hsp70.1 or hsp70.3 gene.Mol Cell Biol. 2001; 21: 8575-8591Crossref PubMed Scopus (117) Google Scholar, 14Hunt C.R. Dix D.J. Sharma G.G. Pandita R.K. Gupta A. Funk M. et al.Genomic instability and enhanced radiosensitivity in Hsp70.1- and Hsp70.3-deficient mice.Mol Cell Biol. 2004; 24: 899-911Crossref PubMed Scopus (198) Google Scholar). Mouse embryos (B6C3F1 females bred with B6D2F1 males) were purchased frozen at the two-cell stage from Embryotech Laboratories, Inc. (Wilmington, MA) and cultured in GEM-PS medium (Duncan Holly, Bedford, MA) to the precompaction eight-cell stage, as described elsewhere (15Hartshorn C. Rice J.E. Wangh L.J. Differential pattern of Xist RNA accumulation in single blastomeres isolated from 8-cell stage mouse embryos following laser zona drilling.Mol Reprod Dev. 2003; 64: 41-51Crossref PubMed Scopus (25) Google Scholar). To induce heat shock, embryos were placed at 43°C for 30 minutes and allowed to recover at 37°C for 2 or 3 hours, during which RNA synthesis took place (12Hartshorn C. Anshelevich A. Wangh L.J. Rapid, single-tube method for quantitative preparation and analysis of RNA and DNA in samples as small as one cell.BMC Biotechnol. 2005; 5: 2Crossref PubMed Scopus (31) Google Scholar). Because of cell cycle arrest, cell division did not occur in these embryos (henceforth designated as heat-shocked) during recovery. Control eight-cell embryos were not heated. Embryos were then collected intact or were dissociated in single cells after laser zona drilling according to our standard protocol (12Hartshorn C. Anshelevich A. Wangh L.J. Rapid, single-tube method for quantitative preparation and analysis of RNA and DNA in samples as small as one cell.BMC Biotechnol. 2005; 5: 2Crossref PubMed Scopus (31) Google Scholar, 15Hartshorn C. Rice J.E. Wangh L.J. Differential pattern of Xist RNA accumulation in single blastomeres isolated from 8-cell stage mouse embryos following laser zona drilling.Mol Reprod Dev. 2003; 64: 41-51Crossref PubMed Scopus (25) Google Scholar), which requires three 1-ms pulses with a noncontact 1,480-nm diode laser beam (ZILOS-tk™ zona infrared laser optical system; Hamilton Thorne Biosciences, Inc., Beverly, MA). Blastomeres were harvested immediately after zona drilling (12Hartshorn C. Anshelevich A. Wangh L.J. Rapid, single-tube method for quantitative preparation and analysis of RNA and DNA in samples as small as one cell.BMC Biotechnol. 2005; 5: 2Crossref PubMed Scopus (31) Google Scholar, 15Hartshorn C. Rice J.E. Wangh L.J. Differential pattern of Xist RNA accumulation in single blastomeres isolated from 8-cell stage mouse embryos following laser zona drilling.Mol Reprod Dev. 2003; 64: 41-51Crossref PubMed Scopus (25) Google Scholar) so that hsp70i RNA transcription had time to take place after the heating step, but not as a result of laser treatment. An alternative protocol was used for embryos designated as laser-treated, which were not incubated at 43°C. Samples underwent laser zona drilling and were then placed back at 37°C for 2 hours before either cell dissociation or harvesting of whole embryos, to permit hsp70i RNA accumulation and investigate a possible heat shock effect generated by the laser beam itself. In contrast to heat-shocked embryos, cell division took place in several of these specimens during recovery time, resulting in 9- to 10-cell embryos at collection time. Nucleic acids preparation, reverse transcription, and real-time polymerase chain reaction (PCR) were performed in a single tube by the PurAmp method (12Hartshorn C. Anshelevich A. Wangh L.J. Rapid, single-tube method for quantitative preparation and analysis of RNA and DNA in samples as small as one cell.BMC Biotechnol. 2005; 5: 2Crossref PubMed Scopus (31) Google Scholar). Briefly, samples were lysed on a PCR tube lid, releasing both RNA and DNA, and reverse transcription and real-time PCR were then carried out in the same tube. The hsp70i PCR assay and the strategy used to calculate hsp70i RNA levels were as described elsewhere (12Hartshorn C. Anshelevich A. Wangh L.J. Rapid, single-tube method for quantitative preparation and analysis of RNA and DNA in samples as small as one cell.BMC Biotechnol. 2005; 5: 2Crossref PubMed Scopus (31) Google Scholar). The hsp70i cDNA and genomic DNA templates (total hsp70i templates) were identical because the hsp70i genes are intronless, and they were coamplified in each sample. Standard curves were obtained from serial dilutions of genomic DNA (12Hartshorn C. Anshelevich A. Wangh L.J. Rapid, single-tube method for quantitative preparation and analysis of RNA and DNA in samples as small as one cell.BMC Biotechnol. 2005; 5: 2Crossref PubMed Scopus (31) Google Scholar, 15Hartshorn C. Rice J.E. Wangh L.J. Differential pattern of Xist RNA accumulation in single blastomeres isolated from 8-cell stage mouse embryos following laser zona drilling.Mol Reprod Dev. 2003; 64: 41-51Crossref PubMed Scopus (25) Google Scholar). Mouse DNA contains four hsp70i copies per genome, one copy of hsp70.1 and one copy of hsp70.3 on each chromosome 17. Four copies per cell were thus subtracted from total hsp70i template numbers to calculate hsp70i RNA levels. Heat shock produced a sharp increase in the hsp70i RNA levels quantified in embryos at the eight-cell stage, as illustrated in Figure 1A. In that figure, blue bars indicate the hsp70i RNA + DNA contents of all single blastomeres that could be recovered from each of three embryos (a, b, and c) after incubation at 43°C. The contribution of hsp70i DNA (four copies per cell) is negligible on the scale used, but its presence confirmed that every cell had been delivered to the assay tube even when RNA was not present. (See cell 4a [top panel], which contained only four hsp70i templates.) The data in Figure 1A are plotted according to the cell-picking order for each embryo. Cell 1 was the first to exit through the laser-drilled hole and thus was the closest to the laser beam because embryos do not rotate freely inside the ZP (16Gardner R.L. Specification of embryonic axes begins before cleavage in normal mouse development.Development. 2001; 128: 839-847PubMed Google Scholar). The asterisks on some of the bars in Figure 1 mark pairs of blastomeres, stemming from recent cell division, that required separation with a micropipette. It is evident from the bar graphs in Figure 1A that all but one of the cells recovered from heat-shocked embryos contained hundreds of hsp70i RNA copies. The one nonresponsive cell derived from a pair, and it is possible that its transcription machinery was slow in restarting after cell division. The proximity of a cell to the laser radiation did not affect its hsp70i RNA content, because the hsp70i RNA levels of the first-picked blastomeres were not consistently lower than those of the others. Hence, laser treatment does not damage the RNA present in the cells. Conversely, an increase in hsp70i transcription caused by the heat generated by the laser would not have the time to occur in this type of experiment because the cells were collected right after laser treatment. To exclude the possibility that some RNA was degraded during cell biopsy, resulting in artifactually variable response levels, we compared our single-blastomere results with hsp70i RNA measurements performed on whole embryos. The average hsp70i RNA + DNA contents of the heat-shocked blastomeres recovered from each of the three embryos (green bars in Fig. 1A) are juxtaposed to average hsp70i RNA + DNA copy numbers per cell calculated from analyses of whole heat-shocked embryos (red bars in Fig. 1A). The two sets of data are in all cases remarkably similar, demonstrating that the cell isolation procedure does not affect RNA recovery. In the absence of heat shock, hsp70i RNA levels were minimal, as indicated by the mean hsp70i RNA + DNA content of four untreated whole embryos, corresponding to 19 ± 7 hsp70i total templates, or 15 copies of hsp70i RNA, per cell (black bars in Fig. 1A). For our second set of experiments, we collected series of individual blastomeres from embryos that had not been purposely exposed to high temperature but had undergone the laser zona drilling procedure necessary for cell harvesting. To investigate the possible occurrence of thermal stress, we allowed enough time for transcript synthesis and accumulation to take place before the embryos were dissociated in individual blastomeres or collected whole. As shown in Figure 1B, the number of hsp70i RNA copies measured in the first blastomere recovered from each of four eight-cell embryos after laser zona drilling averaged 25 ± 22 (blue bar 1), consistent with the average number of hsp70i RNA copies per cell (24 ± 16) calculated from whole embryos. Similarly, the average number of hsp70i transcripts in the other blastomeres of the same embryos varied from 16 ± 22 to 37 ± 48 copies (blue bars 2–9). As shown above for non-heated embryos, these low levels of hsp70i transcription are normally present in embryonic blastomeres. More pulses were used for this experiment (four to ten 1-ms pulses) than were required by our standard protocol, strengthening the conclusion that treatment with this type of laser does not generate thermal shock in embryonic cells. Analysis of a whole eight-cell embryo whose zona was removed with eight 1.5-ms laser pulses (pulses more numerous and longer than usual) gave comparable results (36 copies of hsp70i RNA per cell, yellow bar). In contrast, embryos' exposure to 43°C again induced a marked surge in the number of hsp70i RNA copies per cell (1,331 ± 238; red bar). Our results, thus, overall prove that laser treatment under the conditions described in this report does not produce a heat shock–like effect on hsp70i transcription. To corroborate this conclusion, we compared the rates of development to the blastocyst stage of laser-treated and untreated eight-cell embryos. Forty-six hours after treatment, all 24 embryos used for this experiment had become expanded blastocysts and had begun hatching, although hatching was more advanced in the 12 laser-treated embryos. The unchanged rate of development strongly indicates that laser treatment did not induce cell cycle arrest, a well-known effect of hyperthermia (17Kuhl N.M. Rensing L. Heat shock effects on cell cycle progression.Cell Mol Life Sci. 2000; 57: 450-463Crossref PubMed Scopus (103) Google Scholar). Comparatively longer treatments with lasers of different wavelengths and powers are known to activate the hsp70 promoter in a number of different systems (18Halloran M.C. Sato-Maeda M. Warren J.T. Su F. Lele Z. Krone P.H. et al.Laser-induced gene expression in specific cells of transgenic zebrafish.Development. 2000; 127: 1953-1960PubMed Google Scholar, 19Souil E. Capon A. Mordon S. Dinh-Xuan A.T. Polla B.S. Bachelet M. Treatment with 815-nm diode laser induces long-lasting expression of 72-kDa heat shock protein in normal rat skin.Br J Dermatol. 2001; 144: 260-266Crossref PubMed Scopus (97) Google Scholar). In contrast, our findings that laser zona drilling does not trigger hsp70i expression in eight-cell embryos nor affect the rate of preimplantation development, unlike heating at 43°C, strongly support the conclusion that embryonic cells were not thermally stressed by the procedure employed for this study. These results have important implications, because an increase in hsp70i transcription signals the occurrence of cell damage and cell cycle arrest even in the absence, or well in advance, of identifiable morphological changes. Such events, even if they are transient, could affect specific blastomeres' survival and developmental fate, as recent data overwhelmingly indicate that the destiny of mammalian blastomeres at the cleavage stages already may be determined and may be linked to the timing of the cell cycle (20Pearson H. Your destiny, from day one.Nature. 2002; 418: 14-15Crossref PubMed Scopus (36) Google Scholar, 21Piotrowska-Nitsche K. Zernicka-Goetz M. Spatial arrangement of individual 4-cell stage blastomeres and the order in which they are generated correlate with blastocyst pattern in the mouse embryo.Mech Dev. 2005; 122: 487-500Crossref PubMed Scopus (107) Google Scholar). In conclusion, this study thus provides the first evidence at the molecular level for the safety of ZP perforation with a 1,480-nm diode laser under carefully chosen conditions. The laser used in this study was kindly provided by Hamilton Thorne Biosciences, Inc., Beverly, Massachusetts.