Molecular Human Reproduction, Vol. 9, No. 9, 523-533,
September 2003
© 2003 European Society of Human Reproduction and Embryology
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ICSI-generated mouse zygotes exhibit altered calcium oscillations, inositol 1,4,5-trisphosphate receptor-1 down-regulation, and embryo development
Submitted on April 22, 2003; accepted on May 19, 2003
Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, MA 01003, USA
1 To whom correspondence should be addressed. e-mail: rfissore{at}vasci.umass.edu
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ICSI bypasses not only fusion of the gametes but also a series of signalling events that occur in the sperm prior to and during interaction with the oocyte’s vestments. The effect of this altered encounter of the gametes on the fertilization-associated intracellular calcium ([Ca2+]i) oscillations has not been thoroughly investigated. Here, ICSI and IVF were performed using gametes from two mouse strains, B6D2F1 and CD1, and in-vitro development, pattern of [Ca2+]i oscillations and down-regulation of inositol 1,4,5-trisphosphate receptor-1 (IP3R-1) in the produced embryos were compared. ICSI and IVF resulted in comparable rates of activation and pre-implantation development. However, ICSI-generated zygotes cleaved at a slower rate, had lower cell numbers and lower hatching rates. The deleterious effects of ICSI could not be exclusively attributed to the injury by the injection since sham-injected IVF zygotes only exhibited delayed progression to the blastocyst stage. ICSI and IVF induced similar initial [Ca2+]i responses, although ICSI zygotes exhibited shorter durations of [Ca2+]i oscillations and showed diminished degradation of IP3R-1. Importantly, sperm manipulation affected the pattern of oscillations, which further decreased pre-implantation developmental rates. Our results demonstrate that ICSI-induced [Ca2+]i responses are not equivalent to those initiated by IVF and that this may have developmental consequences.
Key words: fertilization/IP3/mouse/oocytes
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Introduction |
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ICSI has been widely used in human clinics for treatment of severe male infertility (Palermo et al., 1992; Van Steirteghem et al., 1993) and in other mammalian species, including the mouse, for research and generation of offspring (Kimura and Yanagimachi, 1995). In the human, however, concerns have been raised regarding the potential safety of ICSI because the technique bypasses natural selection of the fertilizing sperm as well as physiological interactions between the oocyte and sperm (Schultz and Williams, 2002). Overall, the clinical outcome in ICSI-conceived offspring has been reassuring (Bonduelle et al., 1996; Tarlatzis and Grimbizis, 1999; Sutcliffe et al., 2001). Nevertheless, there are several reports that show decreased developmental potential of ICSI embryos (Shoukir et al., 1998; Dumoulin et al., 2000; Bhattacharya et al., 2001), slightly higher incidences of karyotype abnormalities (Macas et al., 2001), as well as other congenital anomalies in ICSI-conceived offspring (Bowen et al., 1998; Wennerholm et al., 2000). However, it is unclear whether these defects are related to the ICSI procedure itself or rather to abnormalities of the sperm obtained from infertile males.
During mammalian fertilization, the fertilizing sperm evokes a series of intracellular calcium ([Ca2+]i) oscillations that are responsible for inducing oocyte activation and initiation of embryonic development (Miyazaki et al., 1993; Schultz and Kopf, 1995). Accumulating evidence suggests that a factor(s) delivered by the fertilizing sperm, sperm factor (SF), is responsible for initiating these [Ca2+]i oscillations (Dale et al., 1985; Stice and Robl, 1990; Swann, 1990; Swann and Lai, 1997). In support of this notion, direct injection of sperm, ICSI (Nakano et al., 1997) as well as of sperm extracts into oocytes has been shown to trigger [Ca2+]i oscillations that closely resemble those induced by fertilization (Wu et al., 1997, 1998; Swann and Parrington, 1999).
Although the nature of the [Ca2+]i oscillation-inducing SF has not been fully characterized and is still under investigation, accumulating evidence suggests that it may stimulate phosphoinositide turnover and production of inositol 1,4,5-trisphosphate (IP3) (Jones et al., 1998; Wu et al., 2001; Saunders et al., 2002). IP3 triggers Ca2+ release by binding to the IP3 receptor (IP3R-1), which is widely present in the endoplasmic reticulum (ER). IP3 binding also causes a conformational change of the receptor (Mignery and Sudhof, 1990; Zhu et al., 1999) that in turn is responsible for inducing ubiquitination and proteolysis of IP3R-1 (Bokkala and Joseph, 1997; Oberdorf et al., 1999). Therefore, in addition to [Ca2+]i oscillations, mammalian fertilization is characteristically accompanied by down-regulation of the IP3R-1 (Parrington et al., 1998; He et al., 1999; Brind et al., 2000; Jellerette et al., 2000).
The location of the putative SF molecule(s) in the sperm head and its physiological mode of release remain unknown. ICSI could negatively impact embryo development by affecting the release of SF, which may result in the delivery of an abnormal activation stimulus. It is worth noting that ICSI studies in the human have revealed that sperm immobilization, which presumably results in the breakdown of the sperm plasma membrane, thereby facilitating the release of SF, is required to obtain high rates of fertilization (Palermo et al., 1996; Vanderzwalmen et al., 1996; Yanagimachi, 1998). However, the consequences of this probable unnatural release of the factor on the persistence of the fertilization-associated [Ca2+]i oscillations and on pre-implantation development have not been determined.
In somatic cells a variety of cellular functions are, at least in part, regulated by the pattern of [Ca2+]i oscillations. For instance, it is well documented that the frequency of [Ca2+]i rises modulates kinase activity (De Koninck and Schulman, 1998). Likewise, it has been shown that different oscillation patterns can lead to induction of different patterns of gene expression (Dolmetsch et al., 1997, 1998; Li et al., 1998) and cell differentiation (Gu and Spitzer, 1995). Previous studies have also suggested that the pattern of [Ca2+]i rises during the early zygotic stage can have a significant impact on cleavage rates (Ozil, 1990; Vitullo and Ozil, 1992), cell numbers in the inner cell mass (Bos-Mikich et al., 1997), and post-implantation development (Ozil, 1990; Ozil and Huneau, 2001). Therefore, the present study was undertaken to investigate whether or not ICSI-initiated fertilization causes alterations in [Ca2+]i responses and affects pre-implantation development, and whether or not manipulations of the sperm prior to injection change the pattern/persistence of the subsequent [Ca2+]i responses.
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Materials and methods |
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Gamete collection, preparation and IVF
Metaphase II (MII) oocytes were obtained from B6D2F1 (C57BL/6JxDBA/2J) and CD1 female mice (8–12 weeks old) superovulated by injection of 5 IU of pregnant mare serum gonadotrophin (PMSG; Sigma, USA) followed 48 h later by 5 IU of hCG (Sigma). Oocytes were recovered 14 h post-hCG into a HEPES-buffered Tyrode–lactate solution (TL-HEPES; Parrish et al., 1988) supplemented with 5% heat-treated calf serum (Gibco, USA). Cumulus cells were removed by brief treatment with 0.1% bovine testes hyaluronidase (Sigma).
Sperm from B6D2F1 and CD1 male mice (12–24 weeks old) were used for ICSI. Mouse sperm collected from the cauda epididymis and boar sperm collected from freshly ejaculated semen were washed two times with injection buffer (75 mmol/l KCl and 20 mmol/l HEPES, pH 7.0), after which the sperm were used for ICSI.
Conventional IVF was conducted using human tubal fluid (HTF) medium (Quinn et al., 1985). Sperm from epididymides of B6D2F1 or CD1 mice were directly collected into HTF medium, which had been previously equilibrated at 36°C under a 5.5% CO2 atmosphere. Collected sperm were further incubated for 1–2 h prior to insemination to induce sperm capacitation and were added into medium containing the cumulus-intact oocytes to final concentrations of 1–3x105 sperm/ml. Sperm and oocytes were co-cultured for 4 h in insemination medium, after which oocytes were cultured in potassium simplex optimized medium (KSOM; Specialty Media, USA) to assess oocyte activation and development. In some experiments, to synchronize fertilization, oocytes were recovered from HTF medium and transferred to KSOM within 1 h of the addition of sperm. Extrusion of the second polar body (PB2) was used to estimate the time of sperm entry, which in our conditions occurred 1.5–2.0 h post-sperm entry (data not shown). All procedures involving live animal handling and euthanasia were performed according to standard animal protocols approved by the University of Massachusetts Animal Care Committee.
ICSI
ICSI was carried out as previously described (Kimura and Yanagimachi, 1995; Fukami et al., 2001) using Narishige manipulators (Medical System Corp., USA) mounted on a Nikon diaphot microscope (Nikon Inc., USA). ICSI was performed in flushing and holding medium (FHM; Specialty Media) either at room temperature for B6D2F1 oocytes, or at 19–20°C on a cooling stage (BC-100, Bionomic Controller Technology Inc.) for CD1 oocytes to increase survival rates (Kimura and Yanagimachi, 1995). One part sperm suspension was mixed with one part injection buffer containing 12% polyvinylpyrrolidone (PVP, Mr = 360 kDa; Sigma). Sperm were delivered into the oocytes’ cytosol using a piezo micropipette-driving unit (Piezodrill; Burleigh Instruments Inc., USA) as described elsewhere (Kimura and Yanagimachi, 1995); a few piezo-pulses were applied to puncture the oocyte plasma membrane following penetration of the zona pellucida. When a whole live sperm was injected, a motile sperm was gently aspirated into the injection pipette from the tail and released into the oocyte with minimal damage to the sperm; the sperm exhibited flagellar movement inside the oocyte for a few minutes after ICSI. Mouse sperm heads were separated from tails by applying a few piezo-pulses at the mid-piece of the sperm immediately prior to injection into the oocyte. On occasions sonication was used (Kimura et al., 1998); sonication (XL2020; Heat Systems Inc., USA) was carried out for 5 s at 4°C with or without 0.05% (v/v) Triton-X 100 (TX; Sigma), which resulted in >90% of sperm heads separated from their tails. After sonication, sperm were washed three times with injection buffer. In some experiments, sperm heads were co-injected with porcine sperm extracts (pSE) prepared as previously described (Wu et al., 1997, 1998). To accomplish this, sonicated sperm heads were mixed with PVP and pSE, both of which were dissolved in injection buffer, to obtain final concentrations of 6% PVP and 0.1 µg protein/µl pSE. Following ICSI, oocytes were either used for [Ca2+]i monitoring or cultured in KSOM to evaluate activation and development at 36.5°C in a humidified atmosphere containing 5.5% CO2. Activation was assessed from the number of zygotes with extrusion of PB2 and two pronuclei (PN) at 6 h post-ICSI or -IVF, and cleavage to the 2-cell stage after 24 h. Development was assessed by monitoring progression to the blastocyst on days 4 and 5, and hatching on day 5. The number of cells per blastocyst was estimated from the number of nuclei following staining with Hoechst 33342 (Sigma).
[Ca2+]i measurements
[Ca2+]i measurements were carried out as previously described (Wu et al., 1998; Gordo et al., 2002). Oocytes were injected with the fluorescent dye fura-2 dextran (fura-2 D; Molecular Probes, USA) or loaded with 1 µmol/l fura-2 acetoxymethylester (fura-2 AM; Molecular Probes) supplemented with 0.02% pluronic acid (Molecular Probes) for 20 min at room temperature. Ca2+ values were monitored using a Nikon Diaphot microscope fitted for fluorescence measurements. [Ca2+]i concentrations, Rmin and Rmax, were calculated according to Grynkiewicz et al. (1985) and as described by Wu et al. (1997). Oocytes were individually monitored in 50 µl drops of TL-HEPES supplemented with BSA (1 mg/ml) placed on a glass coverslip sealed over an opening in the bottom of a culture dish and covered with mineral oil. Single oocytes were monitored using a modified Phoscan 3.0 software and fluorescence was averaged for the whole oocyte. For certain experiments, 2–10 oocytes were measured simultaneously using the software Image 1/FL (Universal Imaging, USA). Images were acquired using a SIT camera (Dage-MTI, USA) coupled to an amplifier (Video Scope International Ltd, USA). [Ca2+]i values were not calibrated in this system and are therefore reported as the ratios of 340/380 nm fluorescence. Fluorescence ratios were obtained every 4–15 s depending on the experiments.
Unless otherwise stated, the time course for [Ca2+]i measurements was set as shown in Figure 1. For measurements of initial [Ca2+]i patterns (Figure 1A), ICSI-fertilized oocytes were preloaded with fura-2 prior to ICSI, while IVF-fertilized oocytes were loaded with fura-2 after insemination. To synchronize the time of fertilization, ICSI and IVF were performed for 0.5 h respectively, thus the first [Ca2+]i rise was not always detected. For long-term [Ca2+]i measurements (Figure 1B), ICSI and IVF were performed for 1 h, and then the oocytes were cultured for 1 h to allow extrusion of PB2. Only oocytes that started PB2 extrusion (observed as a clear protrusion of the oocyte plasma membrane) at 2 h post-ICSI or -insemination were selected for long-term [Ca2+]i measurements (time = 0). We estimated that initiation of [Ca2+]i oscillations was synchronous in both groups because although in ICSI the sperm is directly delivered into the cytoplasm, there is a 15–30 min time lag until [Ca2+]i oscillations begin (Nakano et al., 1997). Our unpublished results also show a lag time from sperm injection until the first [Ca2+]i rise of 13.0 ± 16.0 min (mean ± SD, range = 0 to 37 min; n = 26). In addition, under our IVF conditions 
30% and 60% of the oocytes were penetrated by 0.5 and 1.0 h post-insemination respectively, and it has been reported that [Ca2+]i oscillations start immediately after gamete fusion (Lawrence et al., 1997). [Ca2+]i was monitored at different time points, 0–1.5, 1–2.5, and 2–3.5 h post-PB2 extrusion using different oocyte groups to minimize the effects of UV light exposure since our preliminary experiments revealed that prolonged exposure to UV light may delay cell cycle progression (data not shown).
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Following monitoring of [Ca2+]i, IVF oocytes were stained with Hoechst 33342 and only oocytes which had been fertilized by a single sperm were included in the data.
Western blot analysis
Western blotting was carried out as previously described (He et al., 1997; Jellerette et al., 2000). Protein samples from ICSI and IVF were prepared using 15 zygotes per sample synchronized according to the time of PB2 extrusion (only oocytes extruding PB2 within 1.5–2 h post-ICSI or post-insemination were collected). Following collection, the zygotes were washed with Dulbecco’s phosphate-buffered solution, and stored in sample buffer at –80°C until use. Samples were subjected to 4% sodium dodecyl sulphate–polyacrylamide gel electrophoresis and then transferred onto nitrocellulose membranes (Micron Separation, USA) for 2 h at 4°C and probed with a rabbit polyclonal antibody raised against a 15 amino acid sequence of the C-terminal end of the IP3R-1 subtype (Rbt-03; a generous gift of Dr J.B.Parys, Katholieke Universiteit of Leuven, Belgium) (Parys et al., 1995). The membranes were probed with a conjugated horse-radish peroxidase secondary antibody. Immunoreactivity was detected using chemiluminescence reagents (NEN Life Science Products, USA) and maximum sensitivity film (Kodak, Fisher Scientific, USA). The mean pixel intensity of the IP3R-1 bands was determined using Adobe Photoshop (USA). Western blotting procedures were repeated at least three times. Data are presented as mean ± SD of three to five separate experiments.
Statistical analysis
Statistical comparisons of oocyte activation parameters and development data (Tables I and IV) were performed using 
2-test. Fisher’s exact test and one-way analysis of variance followed by Tukey’s multiple comparison test were used in Tables II and III respectively. Frequency of [Ca2+]i oscillations, cell numbers, and down-regulation of IP3R-1 following ICSI and IVF were compared by Student’s t-test. Statistical significance was assumed at P < 0.05.
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Results |
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ICSI embryos exhibit lower in-vitro developmental rates than IVF embryos
The advent of ICSI has provided an important new tool to investigate the mechanism of mammalian fertilization; however, the impact of this technique on pre-implantation embryo development, and whether or not the success of this procedure in the mouse extends to many different strains, have not been thoroughly investigated (Kimura and Yanagimachi, 1995; Kawase et al., 2001). To address these issues, we have performed ICSI and IVF using gamete combinations from two mouse strains, B6D2F1 and CD1. ICSI was initially accomplished by injecting a sperm head, rather than a whole sperm, because it produced the highest developmental rates among the sperm treatments that we tested (see below). In addition, it was also used as a form of immobilization, since in humans sperm immobilization prior to ICSI is required to obtain high rates of fertilization and development. ICSI and IVF using homologous gametes (B6D2F1xB6D2F1 and CD1xCD1) resulted in activation and cleavage rates to the 2-cell stage that were comparable between the two methods (Table I). Likewise, when the number of embryos that developed to the blastocyst stage was evaluated at day 5 of culture, no differences were observed between the two methods of fertilization (Table II). Nevertheless, upon closer scrutiny, the rates of cleavage and development were slower in ICSI-generated embryos than in those generated from IVF (blastocyst-day 4, Table I; P < 0.05). In addition, the percentage of hatching/hatched blastocyst and the total number of cells per blastocyst at day 5 were lower in ICSI embryos than in IVF embryos (Table I; P < 0.05).
To rule out the possibility that the detrimental effects of ICSI could be attributed to one of the gametes, or to gametes from one of the strains, ICSI and IVF were conducted using heterologous combinations of gametes (B6D2F1xCD1 and CD1xB6D2F1). Oocyte activation (PB2 and 2PN formation) and cleavage rates to the 2-cell stage were not different between the two fertilization methods (Table I), whereas all the other development parameters evaluated were significantly, and adversely, affected by the ICSI procedure (Table I; P < 0.05).
Since the injection procedure was common to all ICSI zygotes, we evaluated whether the trauma caused by the injection itself could be responsible for some of the detrimental effects observed after ICSI. To test this, we performed mock-ICSI on B6D2F1 oocytes that had been inseminated for 0.5 h; and 2 h later, oocytes that had extruded PB2, which were assumed fertilized and later confirmed to be so by the presence of two PN, were selected for evaluation of embryonic development. A cohort group of inseminated oocytes was not sham-injected and served as a fertilization and development control. Regardless of the sham-injection, all oocytes with PB2 cleaved to the 2-cell stage. Nonetheless, when evaluated at day 4, fewer sham-injected IVF zygotes reached the blastocyst stage (47.8%, n = 39) than non-injected IVF zygotes (72.3%, n = 47; P < 0.05, 2-test). Importantly, these differences in developmental rates disappeared when ascertained at day 5 (82.1% for sham-injected and 85.1% for non-injected IVF zygotes). Likewise, differences in hatching rates (43.6 versus 46.8%) and total cell numbers (59.3 ± 24.4 versus 60.5 ± 19.0) also became negligible.
Fertilization by ICSI and IVF initiate comparable [Ca2+]i oscillations
A factor that could compromise the developmental competence of ICSI-generated embryos is the modification of the initiation and/or persistence of the fertilization-associated [Ca2+]i oscillations. The initiation of [Ca2+]i oscillations evoked by ICSI has been previously monitored in mouse and human oocytes, although in previous studies only oocytes injected with whole immobilized sperm were monitored (Tesarik et al., 1994; Nakano et al., 1997; Sato et al., 1999; Yanagida et al., 2001). In our study, [Ca2+]i oscillations were monitored for the initial 1 h following injection of homologous and heterologous sperm heads and the [Ca2+]i responses compared with those induced by the same gamete combinations after IVF. Fertilization by ICSI and IVF using homologous gametes induced [Ca2+]i responses that were comparable between the two methods (Figure 2A, A', B and B'). Likewise, IVF and ICSI using the heterologous gamete combination of B6D2F1 sperm heads and CD1 oocytes resulted in [Ca2+]i oscillations (Figure 2D and D') comparable to those observed using homologous gametes. However, when fertilization was performed using CD1 sperm and B6D2F1 oocytes, the mean frequency of [Ca2+]i oscillations following ICSI was significantly higher than that following IVF (P < 0.05; Figure 2C and C'), although even after IVF this gamete combination induced the highest frequency of any of the gamete combinations tested (Figure 2).
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) and oocytes (
) from CD1 and B6D2F1 mice in homologous (A, A', B, and B') or heterologous (C, C', D, and D') gamete combinations. The initiation of [Ca2+]i oscillations was comparable between the two methods except that, upon fertilization of CD1 sperm and B6D2F1 oocytes, ICSI zygotes exhibited a significantly higher frequency of [Ca2+]i rises than IVF zygotes (C–C'; P < 0.05). Mock-ICSI (F) did not influence the initial pattern of [Ca2+]i oscillations triggered by IVF (E; B6D2F1 gametes).Although with most of the gamete combinations used ICSI did not modify the initial Ca2+ patterns, we assessed whether or not the injection procedure itself had an impact on IVF-induced oscillations. To investigate this, a group of oocytes was sham-injected, followed immediately by removal of the zona pellucida and insemination with 0.5–1.0x105 sperm/ml. A group of control oocytes was identically treated but not sham-injected (all B6D2F1 gametes). [Ca2+]i monitoring, which started immediately after insemination, revealed no differences between the two groups under investigation (Figure 2E and F).
Disruption of sperm membranes affects the pattern of [Ca2+]i oscillations
Because different immobilization techniques are used in human ICSI, we sought to ascertain whether or not handling of the sperm prior to injection could influence the pattern of oscillations following ICSI. To accomplish this, we monitored [Ca2+]i responses initiated by injection of sperm heads, whole live sperm, or of sperm heads treated with TX. Injection of B6D2F1 sperm heads or of whole intact B6D2F1 sperm into B6D2F1 oocytes initiated oscillations with comparable frequencies (Figure 3A and B). Nevertheless, injection of membrane-depleted sperm heads by exposure to 0.05% TX and sonication markedly accelerated the frequency of oscillations (Figure 3C). Interestingly, the solubility/release of SF may have some degree of variability between strains of mice, and may be highly optimized within strains, because injection of whole intact CD1 sperm into B6D2F1 oocytes induced lower frequency oscillations than those induced by injection of CD1 sperm heads (Figure 3D and E). As seen with B6D2F1 sperm, brief treatment of CD1 sperm with TX induced [Ca2+]i oscillations with higher frequency (Figure 3F).
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ICSI-initiated [Ca2+]i oscillations cease prematurely
Inasmuch as fertilization by ICSI initiated normal oscillations, we investigated whether or not ICSI and treatments of the sperm modified the duration of [Ca2+]i oscillations. Using B6D2F1 gametes, [Ca2+]i responses were analysed at different time points (0–1.5, 1–2.5 and 2–3.5 h) after extrusion of PB2, which was used to approximate the time of sperm entry in ICSI and IVF oocytes; only oocytes that were starting to extrude the PB2 at 2 h post-ICSI or post-insemination were used. We reasoned that since re-entry of oocytes into the cell cycle has been reported to depend on both the total number and frequency of [Ca2+]i rises (Ozil and Swann, 1995), and since the frequency and amplitude of the initial oscillations between ICSI and IVF zygotes were similar (Figure 2), then oocytes extruding PB2 at the same time should represent oocytes that initiated oscillations synchronously. As shown in Table II, oocytes injected with intact live sperm were the least able to support long-term [Ca2+]i responses, and [Ca2+]i oscillations ceased earlier compared with oocytes injected with TX-treated and untreated sperm heads; [Ca2+]i oscillations in IVF oocytes persisted the longest, up to
4 h after insemination. Furthermore, [Ca2+]i oscillations induced by IVF continued to occur at normal intervals for a longer period of time than those initiated by ICSI, regardless of sperm treatment (P < 0.05; Table III). [Ca2+]i rises were not detected for any of the fertilization procedures at 2–3.5 h post-PB2 extrusion, the time at which initial PN formation was observed in both ICSI and IVF zygotes (Figure 4).
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IP3R-1 down-regulation in IVF and ICSI zygotes
Recent studies have revealed that steady down-regulation of IP3R-1 occurs following fertilization in mammalian oocytes (Parrington et al., 1998; He et al., 1999). We therefore investigated whether or not IP3R-1 degradation in ICSI zygotes is comparable to that in IVF embryos. IVF or ICSI were performed using B6D2F1 mouse oocytes and CD1 or B6D2F1 sperm heads, and fertilized oocytes were collected at 2 h post-PB2 extrusion, which is
3.5–4.0 h post-fertilization. As shown in Figure 5, regardless of the type of sperm and fertilization method, fertilized zygotes exhibited significant down-regulation of IP3R-1. However, ICSI zygotes showed a markedly reduced down-regulation of IP3R-1 compared with that observed in IVF zygotes (P < 0.05).
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Altered [Ca2+]i oscillation patterns affect pre-implantation embryo development
We next investigated whether or not altered [Ca2+]i oscillation patterns could be responsible, at least in part, for the observed impaired embryonic development of ICSI-derived zygotes. ICSI was performed with homologous combinations of B6D2F1 gametes using whole intact sperm, sperm heads, or TX-treated sperm heads. As shown in Table IV, although no differences were observed in activation rates, cleavage rates to the 2-cell stage were lower in oocytes injected with whole live sperm than in oocytes injected with sperm heads (P < 0.05). Nonetheless, development to the blastocyst stage was comparable between these two groups (P > 0.05). Importantly, development to the blastocyst stage was severely impaired in zygotes generated by injection of TX-treated sperm heads (Table IV; P < 0.05) indicating, possibly, that high frequency [Ca2+]i oscillations may negatively impact embryonic development. To evaluate this possibility, a single sperm head was co-injected with 0.1 µg/µl pSE. This treatment resulted in high frequency [Ca2+]i oscillations (Figure 3G), and although activation and cleavage rates were comparable to those of control zygotes, development to the blastocyst stage was compromised (Table IV; P < 0.05).
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Diminished embryonic competence of ICSI-generated embryos
The present study compared pre-implantation development and [Ca2+]i responses in mouse zygotes generated by ICSI and IVF. It was our intention to ascertain whether or not defects that may occur early in the generation of ICSI zygotes could explain some of the alleged developmental abnormalities of these embryos. In addition, we investigated whether the success of ICSI extended to non-hybrid strains, and we also performed cross-strain fertilization studies to ascertain whether the source of the gametes affected the success of ICSI. Our results show that >80% of ICSI-generated zygotes develop to the blastocyst stage when using homologous gamete combinations, and these rates are comparable to those reported by others (Kimura and Yanagimachi, 1995; Kuretake et al., 1996; Kimura et al., 1998). However, ICSI embryos generated using heterologous gametes exhibited lower rates of development, although these gamete combinations did not alter development after IVF. More importantly, regardless of the gamete combination, progression to the blastocyst stage, hatching rates, and total cell number in blastocysts were all negatively affected in ICSI-derived embryos. Together, these results suggest that the developmental defects of embryos generated by ICSI may arise, at least in part, from the abnormal interaction of oocytes and sperm rather than from an ‘oocyte factor’ alone, as recently noted to explain a post-implantation defect of ICSI-generated embryos using an inbred strain of mice (Kawase et al., 2001). Moreover, our observations are in line with a recent report that demonstrated subtle developmental defects in ICSI-generated embryos when compared with zygotes generated by IVF (Szczygiel et al., 2002).
The cumulative effect of several insults during fertilization may negatively impact the development potential of ICSI-produced embryos, with perhaps the most obvious being the traumatic injury inflicted to oocytes by the microinjection pipette. Our results show that indeed the trauma caused by the injection procedure increases the time that sham-injected IVF embryos require to reach the blastocyst stage, despite the fact that the piezo-electric method used to penetrate oocytes in our study greatly decreases the damage inflicted to the membranes (Kimura and Yanagimachi, 1995), and that hybrid oocytes are reportedly more resistant to traumatic injury (Kawase et al., 2001). Nonetheless, the detrimental effect of the injection injury appeared short-lived, since development to the blastocyst stage, hatching rates, and total cell number per blastocyst were comparable between sham-injected and control IVF embryos at day 5 post-insemination, which was not the case for ICSI-generated zygotes. Therefore, other events such as sperm head decondensation, shedding of the acrosome and perinuclear theca, and DNA synthesis, all of which appear to be delayed in ICSI fertilization in human and primates (Bourgain et al., 1998; Hewitson et al., 1999; Ramalho-Santos et al., 2000), may further compromise the development by ICSI-generated zygotes. Nonetheless, our recent observations using transmission electron microscopy show that within 2 h after ICSI, mouse sperm have lost the acrosome and have completely shed the perinuclear theca, suggesting that defects in sperm head decondensation and male pronuclear formation may not be as significant in the mouse as reported in primates (data not shown).
ICSI alters the pattern of [Ca2+]i oscillations
Defects in the initiation and persistence of [Ca2+]i oscillations may also impact the success of ICSI. Although reports in the mouse and human show that fertilization by ICSI initiates fertilization-like oscillations, some significant differences have been noted. For instance, the time from sperm injection to initiation of oscillations appears significantly delayed in human ICSI (Tesarik et al., 1994). Notably, this delay is dramatically shortened by sperm immobilization (Yanagida et al., 2001). In mouse ICSI, despite a disparity in the spatial progression of the first rise, subsequent [Ca2+]i oscillations reportedly exhibit patterns similar to those following IVF (Nakano et al., 1997; Sato et al., 1999), although exhaustive comparisons were not conducted. In our study, the initial [Ca2+]i oscillations induced by injection of sperm heads were similar to those induced by IVF, except in the case of injection of CD1 sperm heads into B6D2F1 oocytes, which induced higher frequency oscillations than in the homologous gamete combinations. Moreover, sperm handling procedures, especially permeabilization with TX, resulted in higher frequency oscillations. These results suggest that initiation of [Ca2+]i oscillations may be facilitated by exposure of the SF to the oocyte’s cytosol. Furthermore, the demonstration that injection of CD1 sperm heads induced [Ca2+]i oscillations with higher frequency than B6D2F1 sperm when injected into B6D2F1 oocytes suggests that an optimization has occurred between gametes within strains (species) to produce the physiological pattern of [Ca2+]i oscillations. It is worth noting that CD1 and B6D2F1 sperm heads elicited [Ca2+]i oscillations with similar frequency in CD1 oocytes, suggesting that the frequency of [Ca2+]i oscillations may not only be regulated by the amount and/or activity of the SF, but also by an oocyte cytosolic factor, the nature of which is presently unknown.
Although fertilization by ICSI can modify the initiation of [Ca2+]i oscillations, the greatest impact of this technique may be on the persistence and duration of the oscillations. It has been shown that fertilization-associated [Ca2+]i oscillations in mouse oocytes last approximately until the time of PN formation (Jones et al., 1995; Day et al., 2000) and, in accordance with this, it was reported that ICSI-initiated oscillations exhibit similar persistence (Nakano et al., 1997). We extended those studies by monitoring the effects of different sperm treatments on the duration of oscillations by ICSI and by recording [Ca2+]i responses for 1.5 h intervals to decrease the detrimental effects of prolonged UV exposure. ICSI-induced [Ca2+]i oscillations occurred more infrequently and ceased significantly earlier compared with those initiated by IVF, especially after the injection of whole live sperm. Several factors may account for the early termination of oscillations in ICSI-generated embryos. First, particularly in the case of whole live sperm, which were presumably injected with an intact plasma membrane, the release and activation of the [Ca2+]i oscillation-inducing SF may be impaired. In the case of TX-treated as well as for separated sperm heads, it is possible that premature or excessive release of SF may facilitate inactivation/degradation of the factor, thereby causing earlier than anticipated termination of oscillations. We cannot rule out the possibility that rupture of the membranes may result in the release of proteolytic enzymes from the acrosome, which may degrade SF. Finally, it is possible that capacitation/acrosome reaction and/or fusion, which are bypassed in ICSI, may impact the release/activity of the factor or availability of a substrate(s) in the oocyte, thus limiting the persistence of oscillations.
We have also ascertained whether these disturbed patterns of oscillations may be responsible, at least in part, for the pre-implantation development defects observed in ICSI-generated embryos. In our study, fewer oocytes injected with TX-treated sperm heads, which exhibited initial [Ca2+]i oscillations with high frequency, reached the blastocyst stage than oocytes injected with untreated sperm heads. The same negative effects could be reproduced by co-injection of an untreated sperm head with pSE, which also accelerated the frequency of the initial [Ca2+]i oscillations. Collectively, these results demonstrate an association between altered sperm-induced [Ca2+]i oscillation patterns and impaired pre-implantation embryo development.
Can altered Ca2+ signalling affect embryonic development?
Mouse zygotes generated by ICSI are likely activated by an altered number of [Ca2+]i rises under our conditions. Pre- and post-implantation developmental studies have demonstrated that the activating Ca2+ signal may impact the developmental outcome of mouse embryos (Ozil, 1990; Vitullo and Ozil, 1992; Bos-Mikich et al., 1997; Ozil and Huneau, 2001). However, it remains unknown how [Ca2+]i rises may positively affect embryonic development. [Ca2+]i oscillations have been shown to modify kinase activity (De Koninck and Schulman, 1998) and gene expression (Dolmetsch et al., 1997, 1998; Li et al., 1998) although, in mammalian embryos, transcriptional activity is not significant during the time that fertilization-associated oscillations occur. It is possible, however, that [Ca2+]i rises may have a residual/long-term effect on gene expression by producing modifications of the chromatin structure that may facilitate epigenetic modifications and/or expression of developmentally important genes (Ozil and Huneau, 2001). What is more, [Ca2+]i oscillations may modify gene expression by affecting protein synthesis and degradation during the first cell cycle. Fertilization has been associated with specific recruitment of maternal RNA (Cascio and Wassarman, 1982) as well as with post-translational modifications of proteins, thereby producing a specific pattern of protein synthesis at the zygotic stage (Van Blerkom, 1981; Endo et al., 1986; Howlett, 1986; Xu et al., 1994). It is probable, therefore, that some functional aspect of the protein synthesis machinery may be modulated by the frequency and/or persistence of [Ca2+]i oscillations, both of which are altered during ICSI. In this regard, a recent report showed that the translation of specific proteins at the zygote stage is directly determined by the number of [Ca2+]i rises (Ducibella et al., 2002). Moreover, our results showing that IP3R-1 is down-regulated to a lesser extent in ICSI- compared with IVF-produced zygotes provides evidence that altered activation of the signalling pathway involved in the generation of [Ca2+]i oscillations can modify the post-fertilization protein profile. Therefore, it would be of particular interest to compare the protein expression profile of ICSI-generated embryos with that of IVF-generated embryos.
Collectively, our results suggest that ICSI-induced [Ca2+]i responses are not equivalent to those initiated by IVF, especially in regards to persistence, and that handling of the sperm prior to injection can significantly impact the pattern of oscillations with developmental consequences. Future studies should investigate the molecular changes and post-implantation developmental consequences of these non-physiological [Ca2+]i patterns.
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Acknowledgements |
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We would like to acknowledge valuable discussions with Drs Ken-ichi Sato and Tom Ducibella, and Mr Jeremy Smyth. We also wish to thank the technical support of Ms Changli He. This work was supported in part by grants from the USDA to R.A.F. (99-2371; 02-2078) and by a grant from the Lalor Foundation (M.K.).
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