Intracytoplasmic sperm injection of mouse oocytes with 5 mm Ca 2+ at different intervals

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1 Human Reproduction vol. no. pp Intracytoplasmic sperm injection of mouse oocytes with mm Ca + at different intervals A.Ahmadi, S.C.Ng, S.L.Liow, J.Ali, A.Bongso and S.S.Ratnam Department of Obstetrics and Gynaecology, National University of Singapore, Lower Kent Ridge Road, Singapore 'To whom correspondence should be addressed The objective of this investigation was to determine whether intracytoplasmic sperm injection (ICSI) can be performed in the mouse. Metaphase II oocytes were obtained from Fl hybrid mice (C7BLXCBA) by i.p. injections of IU pregnant mare's serum gonadotrophin (PMSG) and human chorionic gonadotrophin (HCG) administered h apart. Oocytes with cumulus oophorus were retrieved -4 h post HCG. Cumulus was dispersed with.% hyaluronidase. Mouse spermatozoa were obtained from the cauda epididymides of males of the same strain. The spermatozoa were processed by the standard swim-up procedure. The harvested spermatozoa were then incubated for. h to allow capacitation. Healthy oocytes were injected with - 4 pi mm Ca +, followed by one live morphologically normal spermatozoon into the cytoplasm at intervals of,.,, and h. The proportion of -cell embryos that developed from oocytes injected with Ca + and spermatozoa ranged between 9. and 6.% in all groups, with no statistical difference between treatments. Chromosomal analysis showed that two-thirds of the ICSI-derived -cell embryos were diploid. The proportion of parthenogenetically activated embryos in the ICSI groups was similar to that in the control group (8-%) which was injected with Ca + and polyvinyl pyrrolidone only. The proportion of blastocysts that developed in culture from the ICSIderived -cell embryos was of the order of 6-4%. Some blastocysts were used for cell number counts. There was a significant increase in total and inner cell mass counts of blastocysts in which the spermatozoon was injected at and h following Ca +. The remaining blastocysts were transferred to day pseudopregnant mice, of which % subsequently became pregnant. Of the blastocysts transferred, 6-% developed to term in vivo. No deformities were observed in the pups. We believe this is the first report of live-birth following mouse ICSI. Key words: Ca + injection/intracytoplasmic sperm injection/ micro-injection/mouse oocyte/spermatozoa Introduction Early investigations into the use of micromanipulation technology for assisted fertilization in animals began in the 96s. This technique was used to investigate the early events of fertilization such as membrane fusion between homologous and heterologous gametes, activation of the ooplasm and formation of male and female pronuclei. The pioneering work in the mammalian system was carried out by Uehara and Yanagimachi (976, 977). They injected human and hamster spermatozoa into hamster oocytes to determine whether they could develop into male pronuclei (Iritani, 99; Ng et ai, 99). In the human, it was reported that pronuclear formation could be achieved following intracytoplasmic sperm injection (ICSI; Lanzendorf et ai, 988); however, it was not until 6 years after this report that human offspring following ICSI were born (Palermo et ai, 99). In the mouse, fertilization has been achieved after ICSI (Markert, 98; Gomibuchi et ai, 98) but the development potential beyond early cleavage stages, and in particular invivo development following implantation of transferred ICSI embryos, has not been well documented. The objective of this study was to determine whether fertilization and subsequent development in the mouse could be achieved following ICSI. Materials and methods Preparation of oocytes and spermatozoa Fl hybrid female mice (C7BLXCBA) 4- weeks old were used as oocyte donors. Superovulation was induced by i.p. injections of IU pregnant mare's serum gonadotrophin (PMSG, Folligon; Intervet International BV, Boxmeer, The Netherlands) followed h later with IU human chorionic gonadotrophin (HCG, Chorulon; Intervet International BV). Oocytes with cumulus oophorus were retrieved from the oviducts -4 h post-hcg injection and cumulus cells were dispersed by treatment with.% hyaluronidase for - min. The cumulus-free oocytes were washed and transferred in Whittingham's T6 medium (Whittingham, 97) supplemented with 4 mg/ml bovine serum albumin (BSA Fraction V; Sigma Chemical Co., St Louis, MO, USA) and cultured at 7 C in % CO in air prior to micro-injection. The oocytes were injected with?>-$ pi of mm Ca + (CaCl -H O in milli-q processed water) followed by injection of one live morphologically normal spermatozoon directly into the cytoplasm. Spermatozoa were injected at intervals of,.,, and h following Ca + injection. Downloaded from at Pennsylvania State University on September 6, 6 Oxford University Press 4

2 A.Ahmadi et al. Both the cauda epididymides of - week old Fl hybrid males (C7BLXCBA) were excised and the contents expressed into. ml of Whittingham's T6 medium supplemented with mg/ml BSA. The standard swim-up procedure was performed to harvest motile spermatozoa. The harvested spermatozoa were incubated for. h to allow capacitation. To reduce the velocity and facilitate the capture of spermatozoa during ICSI, one part of sperm suspension was mixed with three to four parts of a viscous solution of % polyvinyl pyrrolidone (PVP; 6 mol. wt, dialysed and lyophilized; Sigma Chemical Co.) in HEPES-buffered T6 medium. Micro-injection procedure The holding pipette was fashioned out of a drawn-out Pasteur pipette. The fire-polished tip of the Pasteur pipette had an opening diameter of between and 4 (im. The microinjection pipette was prepared with the horizontal microelectrode puller (P-87; Sutter Instruments, CA, USA), the Narishige microgrinder (EG-4; Narishige, Tokyo, Japan) and de Fonbrune microforge (MF-; Technical Products International Inc., St Louis, MO, USA). The internal diameter of the microinjection pipette was 7-9 Jim and the external diameter was - n,m. The angle of the bevel was. To facilitate the injection procedure, both holding and micro-injection pipettes were bent to an angle of -4 to allow horizontal displacement. One droplet of sperm PVP suspension (- (xl) and one droplet of mm Ca + ( fi.) preparation were placed on the left side of a Petri dish ( mm diameter; Becton Dickinson, NJ, USA) and droplets ( il) of HEPES-buffered medium on the right side. These droplets were covered by light-weight paraffin oil (embryo tested; Sigma Chemical Co.). The Ca + solution was injected with the micro-injection pipette used for the spermatozoa as described above. ICSI was carried out on the heated stage (7 C) using a Zeiss motorized micromanipulator and an Axiovert M inverted microscope (Carl Zeiss, Oberkochen, Germany). The injection pipette was fitted to an Alcatel micro-injector (oil pump, Alcatel, France). A single spermatozoon was selected from the edge of the sperm PVP droplet and aspirated tail first into the tip of the injection pipette. Metaphase II oocytes were held under negative pressure by the holding pipette with the polar body at the 6 or o'clock position. Then the micro-injection pipette was pushed through the zona pellucida and into the cytoplasm of the oocyte at o'clock. After puncturing and breaking the oolemma by sucking in a minute amount of cytoplasm, a single spermatozoon was injected into the ooplasm with a minimal volume of sperm PVP medium (- pi). The injection pipette was removed slowly and the oocyte was released from the holding pipette. There were two control groups. In the first control group the oocytes were injected with PVP and medium only. After micro-injection, the oocytes were washed three times in T6 medium before incubation at 7 C and in % CO in air. In the second control group in-vitro fertilization (IVF) was performed. The oocytes were transferred into ul droplets of medium containing X 6 spermatozoa/ml and incubated for 4-6 h. Thereafter, the oocytes were washed and cultured. 4 Culture of embryos The oocytes were checked for cleavage to the -cell stage 8- h later. Some of the cleaved embryos were allowed to develop to the blastocyst stage in Whittingham's T6 medium supplemented with 4 mg/ml BSA. Some blastocysts were transferred to day pseudopregnant mice and the remaining ones were used for cell number counts as described previously (Handyside and Hunter, 984; Papaioannou and Ebert, 988; Vasuthevan et al., 99). Some -cell embryos obtained following ICSI were also karyotyped as described by Bongso et al. (99). The -cell embryos were transferred to ug/ml colcemid solution and incubated for 8- h at 7 C in % CO in air. The embryos were then transferred to chilled % sodium citrate and kept for - min at room temperature. Each embryo was transferred to a precleaned grease-free glass slide and excess sodium citrate removed. After fixing with fixative (three parts methanol and one part glacial acetic acid) and staining with Giemsa, chromosomes of the embryos were examined under X magnification. One way analysis of variance was used for statistical analysis of the results. Results Between 9. and 6.% of oocytes developed to -cell embryos following ICSI at different intervals after the Ca + injection (Table I); there was no significant difference in the -cell embryo development between the different time intervals. ICSI of spermatozoa without additional Ca + resulted in four -cell embryos out of 4 oocytes (8.8%). Control ICSI (injected PVP without spermatozoa) revealed parthenogenetic activation of between 8 and % (P < " 6 ). The IVF control group consistently resulted in higher fertilization and cleavage rates (.9-6.4%) as compared with the ICSI groups (9.- 6.%; P < " 6 ). Of the fertilized oocytes that cleaved to the -cell stage, -6% of the -cell embryos from the IVF control group developed to blastocysts as compared with 6-4% following ICSI but this difference was not significant. During the micro-injection procedure, % of oocytes were damaged. Karyotype studies showed that two-thirds of all embryos that cleaved in all ICSI groups were diploid (Table II). Very few blastocysts were available for total and inner cell mass (ICM) cell counts (Table III). However there appears to be a significant increase in total cell numbers and ICM in the groups in which spermatozoa were injected into the cytoplasm at and h following Ca + injection. Embryo transfer in pseudopregnant mice resulted in a live-birth rate of 6-% in all ICSI groups, with no significant difference between treatments (Table IV). Discussion There have been reports of live-birth in mammals following injection of spermatozoa into the perivitelline space in mouse (Mann, 988) and in the human (Ng et al., 989). Successful Downloaded from at Pennsylvania State University on September 6, 6

3 Intracytoplasmic injections of calcium and spermatozoa into mouse oocytes Table I. Results of intracytoplasmic sperm injection (ICSI) in mouse oocytes Procedure Time /u\b No. oocytes Injected Cultured -cell embryos (% of oocytes cultured) Blastocysts c fey rt.-c ^\ *-*,-..»»!»- - m*a yio or z-cell ICSI. Con-ICSI. Con-IVF (9.) 4 (.) 8 (.) 6 (.) 8 (6.) 8 (8.) (9.8) (9.6) 9 (9.) (9.9) 4 (.9) 6 (.) 6(7.) (6.4) 7 (4.8) 6 (6.4) (6.6) 4 (6.8) (4.6) 6(4.) (6.) 7 (6.7) (.) (6.6) 6(6.) Con-ICSI, injection of oocytes with PVP only; Con-IVF, oocytes inseminated with spermatozoa. a There was no significant difference for -cell and blastocyst development between all ICSI procedures. "Time interval between Ca + and sperm injection. c Blastocyst and -cell embryo development between ICSI and control groups (IVF and ICSI) were significantly different, P <. and P <. respectively. Table II. Results of karyotyping of mouse embryos obtained following intracytoplasmic sperm injection (ICSI) Procedure" Time b ICSI. Con-ICSI. No. oocytes Injected Cultured -cell embryos (% of -cell (9.4) 6 (.) 6 (.6) 8 (4.6) 9 (.8) (.) 6(.) (.4) (.4) 6(.) Con-ICSI, injection of oocytes with PVP only "There was no significant difference for -cell embryo development between the ICSI and Con-ICSI groups. 'Time interval between Ca + and sperm injection. Two-cell embryo development between ICSI and Con-ICSI groups was significantly different (/> =.4). births have also resulted from injection of spermatozoa into the cytoplasm of oocytes in the rabbit (Hosoi et al., 988), in the cow (Goto et al, 99) and in the human (Palermo et al., 99; Van Steirteghem et al., 99a). Here, we report the successful generation of mouse embryos following ICSI which resulted in live-births subsequent to transfer to pseudopregnant mice. To our knowledge this is the first report in the mouse. There were no obvious deformities in the pups. Pronuclear formation and normal development to the blastocyst stage following ICSI in the mouse had been described but no attempt was made to obtain post-implantation development because of the low yield of embryos (Markert, 98; Gomibuchi et al., 98). Our study shows that the frequency of blastocyst development from oocytes fertilized by ICSI (6-4%) was not as high as in oocytes fertilized in vitro (-6%, Table I). The cleavage rate following ICSI reported here for apparently fertile mice was between 9. and 6.% (Table I) compared Haploid (% of -cell (.) (.) (.) (7.8) (6.) 6 6 Diploid (% of -cell (.7) (68.7) (68.7) (7.) 4 (7.7) Table III. Cell counts in mouse blastocysts [inner cell mass (ICM) and total cells) Time". Total no. blastocysts Cell numbers (mean ± SD) Inner cell mass b 4.7 ±..7 ±.. ±. 9. ±. 9. ±. Total cells 8.7 ± ±.7 9. ±.8 9. ± ±.7 Time interval between Ca + and sperm injection. b ICM cell numbers were significantly different for the different intervals of Ca + and sperm introduction, P <.. with sub-fertile men which was reported to be as high as 6% (Van Steirteghem et al., 99b). The low fertilization rate in the mouse could also be attributed to the shape and size of Downloaded from at Pennsylvania State University on September 6, 6 4

4 A.Ahmadi et al. Table IV. Results of embryo transfer Time. No. blastocysts b No. live pups c (% of blastocysts) () (6) (8) () () a Interval between Ca + and sperm injection. b For each experimental group, the stated number of blastocysts was transferred to three surrogate mice. There was no significant difference between treatments. the mouse spermatozoon. The mouse spermatozoon is large ( im) and the head is sickle-shaped, as compared with the human spermatozoon which is shorter (6 Jim) with an ovalshaped head. It is obvious that micro-injection with human spermatozoa is likely to be much simpler to perform with minimal damage to the spermatozoon as compared with the mouse spermatozoon. Furthermore, the mouse oocyte is significantly smaller (8 u.m) than the human oocyte ( im). The needle for mouse sperm injection has to be slightly larger to accommodate the large mouse spermatozoon. This poses a technical problem which resulted in damage to % of the mouse oocytes. In addition, because of the large size of the spermatozoa, more medium was inevitably injected into the oocyte during mouse micro-injection. All these factors could possibly play a role in reducing the number of viable embryos obtained following ICSI. The damage to the mitotic spindle is likely to be higher in the mouse than the human for the same technical reasons. All these factors may have contributed to the zero fertilization reported by Ron-El et al. (99) for ICSI in the mouse. The role of intracellular Ca + in oocyte activation is well documented. Recent reports suggested that in human oocytes, ICSI triggers release of intracellular Ca + which then activates the oocyte. Edwards and Van Steirteghem (99) suggested that in the human oocyte, the Ca *" in the medium (-.78 mm) with which spermatozoa were injected into the cytoplasm triggered oocyte activation by a calcium-induced calciumrelease function, which was known to occur in oocytes of some species. Subsequent work by Tesarik et al. (994), however, showed that Ca + injection into the human oocyte under conditions of ICSI did not activate it but intracellular Ca + was marginally increased. However, it is obvious from previous reports on human ICSI that injection of Ca + other than that present in the medium was not required to achieve high fertilization rates (Van Steirteghem et al., 99a). On the other hand, in the mouse we observed that additional Ca + injection of the order of mm resulted in a better fertilization rate (9.-6.%) as compared with injection with.78 mm Ca + which was present in the medium (8.8%). The injection of mm Ca + at different time intervals following ICSI resulted in similar fertilization rates, although fertilization was slightly but not significantly higher (6.%) in oocytes injected after h as compared with h (9.%). Fulton and Whittingham (978) were successful in parthenogenetically activating 9% 44 of mouse oocytes by iontophoretic injection of mm Ca +. Iontophoretic injection allowed the use of micropipettes with a narrower tip (<<. \im). In view of this finding, it would be of interest to investigate whether higher fertilization rates in the mouse could be obtained following injection of amounts of Ca + > mm. It was reported that the presence of a spermatozoon is necessary for oocyte activation (Dale et al., 98; Stice and Robl, 99). This view is supported by our data, as 8-% of oocytes undergoing control ICSI (injected Ca + and PVP only) were fertilized as compared with 9.-6.% of oocytes injected with Ca + and spermatozoa (Tables I and II). Spermatozoa induce activation of the oocyte by releasing Ca + from intracellular stores. Two different hypotheses have been suggested for this mechanism. One suggestion is that the spermatozoon binds to an extracellular membrane receptor, through acting on G-protein, which stimulates the phosphoinositide pathway, causing activation (Kline et at, 988). The other hypothesis depends on a factor within the spermatozoon which is released into the cytoplasm and which interacts with the ooplasm or inner membrane, thus stimulating the system to activate the oocyte (Longo et al., 986). In conditions of ICSI, it appears that the second hypothesis may be responsible for oocyte activation. It is apparent from our study that the time at which the spermatozoon is introduced into the cytoplasm following injection of Ca + may not have a significant effect on the subsequent events of fertilization and male pronuclei formation. It was interesting to note that the total and ICM numbers were higher in blastocysts that developed from oocytes in which the spermatozoon was injected at and h after Ca + injection (Table III). It is also tempting to speculate that the embryos formed following these two treatments were more robust than the others; however, the number of blastocysts in which the cell counts were performed was too small to be conclusive. More work needs to be carried out to ascertain whether the injection of spermatozoa - h after Ca + injection into the oocyte would result in better quality embryos. In conclusion, our data support the use of mm Ca + in ICSI for mouse oocytes, although the time interval between the introduction of Ca + and spermatozoon does not appear to be important. The use of a higher Ca + concentration may improve embryo yield. Acknowledgements A.A. is presently a postgraduate student at the National University of Singapore from Shahid Sadoughi University of Medical Science, Yazd, Iran. This work was supported by the National University of Singapore and the Iranian Ministry of Health and Medical Education. References Bongso.A., Ng,S.C, Lim,J., Fong,C.Y. and Ratnam.S. (99) Preimplantation genetics: chromosomes of fragmented human embryos. Fertil. Steril., 6, -7. Dale,B., De Felice.L.J. and Ehrenstein.G. (98) Injection of a soluble sperm extract into sea urchin eggs triggers the cortical reaction. Experientia, 4, Edwards.R.G. and Van Steirteghem,A.C. (99) Intracytoplasmic Downloaded from at Pennsylvania State University on September 6, 6

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