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BOR - Papers in Press, published online ahead of print June 14, 2006.
Biol Reprod 2006, 10.1095/biolreprod.106.053223
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BIOLOGY OF REPRODUCTION 75, 442–451 (2006)
DOI: 10.1095/biolreprod.106.053223
© 2006 by the Society for the Study of Reproduction, Inc.

Research Article

Sperm Chromatin Remodeling after Intracytoplasmic Sperm Injection Differs from That of In Vitro Fertilization1

Anna Ajduk 45 , Yasuhiro Yamauchi 4, and Monika A Ward 2 3 4

Institute for Biogenesis Research,4 John A. Burns School of Medicine, University of Hawaii, Honolulu, Hawaii 96822 Department of Embryology,5 Institute of Zoology, Warsaw University, 02–096 Warsaw, Poland

ABSTRACT

Intracytoplasmic sperm injection (ICSI) is a popular method used in assisted conception, and live offspring have been born from a variety of species, including humans. In ICSI, sperm chromatin is introduced into the oocyte together with the acrosome, a structure that does not enter the oocyte during normal fertilization. We compared sperm chromatin remodeling, the potential of embryos to develop in vitro, and DNA synthesis in mouse embryos obtained from in vitro fertilization (IVF) and ICSI. We also tested whether sperm pretreatment prior to ICSI (i.e., capacitation, acrosome reaction, membrane removal, and reduction of disulfide bonds in protamines) facilitates chromatin remodeling and affects embryo development. Sperm chromatin was examined on air-dried, Giemsa-stained preparations at 30-min intervals for up to 4.5 h postfertilization. In all experimental groups, the oocytes underwent activation and formed pronuclei with similar rates. However, the dynamics of sperm chromatin remodeling in ICSI and IVF embryos varied. In ICSI, chromatin remodeling was more asynchronous than in IVF. Sperm capacitation prior to injection enhanced remodeling asynchrony and resulted in delayed pronuclei formation and DNA synthesis. The removal of the acrosome prior to injection with calcium ionophore A23187 but not with detergent Triton X-100 allowed more synchronous chromatin remodeling, timely DNA synthesis, and good embryo development. Our data have significance for the refinement of the molecular and biologic mechanisms associated with ICSI for current and future applications.

assisted reproductive technology, embryo, fertilization, in vitro fertilization, sperm

INTRODUCTION

Mammalian spermatozoa reach competence for fertilization and fuse with the oocytes in the female reproductive tract [1]. First, spermatozoa undergo a process called capacitation (from "capacity to fertilize eggs"), during which numerous molecular changes in the sperm take place, resulting in sperm becoming competent to undergo the acrosome reaction. During acrosome reaction the membrane surrounding the acrosome fuses with the plasma membrane of the sperm, releasing acrosome contents and rendering sperm capable of penetrating zona pellucida. The result of acrosome reaction is that the equatorial region of the sperm membrane is exposed and fuses with the oolemma, facilitating introduction of the sperm nucleus into the oocyte. Once in the ooplasm, the sperm nucleus undergoes several structural changes, known as chromatin remodeling. One component of global remodeling is the assembly of histones; others involve chromatin maturation, positioning of nucleosomes, and addition of other DNA-binding proteins [2]. In mice, paternal chromatin remodeling consists of three phases: decondensation, recondensation, and male pronuclei formation [3]. Decondensation overlaps with the anaphase of the second meiotic division. During this stage disulfide bonds in protamines are reduced due to the activity of glutathione, a reducing factor present in ooplasm [4, 5]. Reduction of the disulfide bridges enables protamine removal from sperm chromatin [6]. Protamine–histone exchange is thought to be mediated by specific proteins. In Xenopus this role is played by nucleoplasmin [7]. In mammals these proteins are not yet fully defined, but the strongest candidates are nucleoplasmin 3 (NPM3) and nuclesome assembly proteins [2]. The exact time during which protamines are exchanged with histones is not known, and the existing data are contradictory [8–12]. It is thought that at the end of chromatin recondensation, which overlaps with the telophase of the second meiotic division, sperm DNA already is bound to histones and has a nucleosome-based structure [2, 12]. The last stage of sperm chromatin remodeling is the decondensation of tightly recondensed sperm chromatin, leading to the formation of male pronuclei. During this phase sperm chromatin interacts with proteins involved in kinetochore formation [13] and/or proteins involved in DNA replication [2].

When fertilization does not occur in a natural way, various approaches of assisted fertilization can be used. The most popular are in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). In IVF, capacitation and acrosome reaction are achieved through maintaining specific culture conditions during gamete co-incubation [14–16]. The sperm fuses with the oocyte in a similar manner, as it takes place during fertilization in vivo. In ICSI, capacitation, acrosome reaction, and membrane fusion are bypassed. Sperm chromatin enters ooplasm together with the perinuclear material, acrosome, and cell membrane. Although these components eventually disintegrate inside the oocyte, it was suggested that they might interfere with sperm chromatin remodeling [17–23]. Live offspring were born with ICSI in a variety of species, including humans, and the technique is considered successful. However, existing reports on a higher incidence of chromosome aberrations [24, 25], lower developmental potentials [26–29], and abnormal calcium oscillations [29, 30] in embryos produced with ICSI indicate that further evaluations on ICSI effects are needed.

In this study we examined sperm chromatin remodeling in embryos obtained by IVF and ICSI. We also tested whether sperm pretreatment prior to ICSI (i.e., capacitation, acrosome reaction, membrane removal, and reduction of disulfide bonds in protamines) facilitates chromatin remodeling and affects embryo development.

MATERIALS AND METHODS

Chemicals

Mineral oil was purchased from Squibb and Sons (Princeton, NJ); eCG and hCG were from Calbiochem (San Diego, CA). All other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO) unless otherwise stated.

Animals

B6D2F1 (C57BL/6J x DBA/2) mice were obtained at 6 wk of age from the National Cancer Institute (Raleigh, NC). The mice were fed ad libitum with a standard diet and maintained in a temperature- and light-controlled room (22°C, 14L:10D) in accordance with the guidelines of the Laboratory Animal Services at the University of Hawaii and guidelines presented in National Research Council's Guide for Care and Use of Laboratory Animals published in 1996 by Institute for Laboratory Animal Research (ILAR) of the National Academy of Science (Bethesda, MD).

Media

Medium T6 [31] was used for IVF, and HEPES-buffered CZB medium (HEPES-CZB [32]) was used for gamete handling and ICSI. Medium CZB [33] was used for embryo culture. Both CZB and T6 were maintained in an atmosphere of 5% CO2 in air, and HEPES-CZB was maintained in air.

Sperm Collection and Preparation

Epididymal spermatozoa were obtained from males aged 8 to 16 wk. Spermatozoa were used for IVF and for ICSI. For IVF, the caudae epididymides were removed from each animal and placed in a 0.4-ml drop of T6 medium (capacitation drop) under oil. The epididymal contents were expressed from the caudae epididymides with needles, and the tissue was discarded. Spermatozoa were allowed to disperse for 2 to 3 min at room temperature and were then capacitated for 1.5 h at 37°C in a humidified atmosphere of 5% CO2.

For ICSI, spermatozoa were prepared in following groups: 1) fresh spermatozoa; 2) capacitated spermatozoa; 3) capacitated spermatozoa treated with calcium ionophore; 4) spermatozoa treated with Triton X-100; and 5) spermatozoa treated with Triton X-100 + dithiothreitol (DTT). For fresh sperm ICSI, caudae epididymides were removed from one male, and the epididymal fluid was squeezed out and placed on the bottom the 1.5-ml tube containing 0.4 ml HEPES-CZB. Spermatozoa were allowed to swim up for 5 min at room temperature and were then taken for injections. Capacitated sperm for ICSI were prepared as described for IVF. To obtain acrosome-reacted spermatozoa after treatment with calcium ionophore, spermatozoa were first capacitated as for IVF. Capacitated sperm were then incubated in the presence of 20 µm calcium ionophore A23187 for 5 min at 37°C, 5% CO2. The ionophore was washed out (720 x g, 5 min, 25°C), and the sperm pellet was resuspended in 200 µl HEPES-CZB and used for ICSI. To obtain detergent-treated spermatozoa, caudae epididymides were dissected, and the epididymal fluid was squeezed out and placed in 1 ml chilled HEPES-CZB supplemented with 0.5% Triton X-100 ± 2 mM DTT. Spermatozoa were vigorously pipetted to disperse evenly in the solution and were incubated for 15 min on ice. To remove detergent and DTT, 1 ml sperm suspension was carefully layered over a 0.5-ml cushion of 1 M sucrose, 25 mM Tris, pH 7.4, in a centrifuge test tube. This step gradient was centrifuged at 3000 x g for 10 min at 4°C. The sperm pellet was resuspended in 200 µl HEPES-CZB and used for ICSI.

Oocyte Collection

Mice aged 8 to 12 wk were induced to superovulate with injections of 5 IU eCG and 5 IU hCG given 48 h apart. Oviducts were removed 14 to 15 h after the injection of hCG and placed in PBS in a Petri dish. For IVF, oviducts were transferred beneath the mineral in the plastic dish (catalogue number 351007; Falcon, Bedford, MA) close to the fertilization drop (T6 medium plus spermatozoa). The cumulus–oocyte complex was released from the ampullary region of each oviduct into the oil by rupturing the oviduct with the aid of a 25-gauge needle. The oviduct was discarded, and the cumulus oocyte–complex was moved into the fertilization drop. For ICSI, the cumulus–oocyte complexes were released from the oviducts into 0.1% of bovine testicular hyaluronidase (300 USP U/mg) in HEPES-CZB medium to disperse cumulus cells. The cumulus-free oocytes were washed with HEPES-CZB medium and used immediately for ICSI.

Mating

B6D2F1 females were mated with B6D2F1 males 14 h after hCG injection. The females were examined for the presence of vaginal plugs as an indication of successful mating. The first examination was done 30 min after pairing. If there was no plug at this time, a second examination was done 1 h after pairing. Positive females were killed 4 h after finding the plugs. Cumulus-free oocytes were obtained as described for ICSI. In vivo-fertilized oocytes were used as a quality control for all chromatin remodeling stages.

In Vitro Fertilization

The method for sperm capacitation and IVF using T6 medium has been described elsewhere [31]. Briefly, 200-µl drops of T6 medium (fertilization drops) were overlaid with mineral oil in a plastic culture dish (60-mm diameter) and equilibrated overnight at 37°C in a humidified atmosphere of 5% CO2 in air. The volume of sperm suspension added to the fertilization drop was dependent on the concentration of spermatozoa after dispersion in capacitation drop. Generally, 10 µl of sperm suspension from capacitation drop was added to each fertilization drop to give final sperm concentrations of approximately 2 x 106/ml. The contents of four oviducts were released into each fertilization drop. Gametes were co-incubated for 60 min. Preliminary experiments demonstrated that this was a minimal time to maintain high fertilization rates. When gametes were co-incubated for shorter time (30 min), only ~20% of oocytes were fertilized, and in those fertilized oocytes sperm nuclei remained unchanged, indicating that the sperm had just fused with the oocytes. After gamete co-incubation, the oocytes were washed several times with HEPES-CZB medium, followed by at least one wash with CZB medium. Only morphologically normal oocytes were selected for culture and analysis.

Intracytoplasmic Sperm Injection

ICSI was carried out as described recently by Szczygiel and Yanagimachi [34]. Briefly, a small drop of incubated sperm suspension was mixed thoroughly with an equal volume of HEPES-CZB containing 12% (w/v) polyvinyl pyrrolidone (PVP; Mr 360 kDa) immediately before ICSI. ICSI was performed using Eppendorf Micromanipulators (Micromanipulator TransferMan; Eppendorf, Germany) with a Piezo-electric actuator (PMM Controller, model PMAS-CT150; Prima Tech, Tsukuba, Japan). A single spermatozoon was drawn tail first into the injection pipette and moved back and forth until the head-midpiece junction (the neck) was at the opening of the injection pipette. The head was separated from the midpiece by applying one or more piezo pulses. After discarding the midpiece and tail, the head was redrawn into the pipette and injected immediately into an oocyte.

ICSI was done in HEPES-CZB within 1 to 2 h after oocyte collection. The oocytes were injected in groups of 5 to 10 eggs, and the time of injection per group was less than 10 min. Sperm-injected oocytes were transferred into CZB medium and cultured at 37°C, 5% CO2 in air. The oocytes were fixed at specific timepoints after injection.

Embryo Culture

After IVF and ICSI, the oocytes were placed in 50-µl drops of CZB medium pre-equilibrated overnight with humidified 5% CO2 in air. The culture drops were contained in plastic culture dishes (Falcon) and overlaid with mineral oil. Cultured embryos were evaluated for developmental progress at 24, 48, 72, and 96 h postfertilization.

Air-Dried, Giemsa-Stained Sperm Preparations

Fertilized oocytes were washed in Dulbecco PBS, pH 7.3 (Gibco, Grand Island, NY), and air dried on microscope slides starting from Time 0 (reflecting the approximate time of sperm–oocyte fusion) at 30-min intervals for up to 4.5 h (10 timepoints). The preparations were fixed in absolute ethanol and glacial acetic acid (3:1) for 10 min and stained in 2% Giemsa (Merck, Darmstadt, Germany) in buffered saline solution, pH 6.8, for 10 min. The slides were examined using light microscope with magnification 1000x. At least 10 oocytes (range: 10–20) were examined at each timepoint in each group. In contrast with ICSI, in IVF the exact fertilization time could not be clearly defined. Gametes were co-incubated for 60 min. The "0-min" timepoint in the IVF group was defined as 30 min after initiation of gamete co-incubation. In ICSI, fertilization was achieved within 10 min (ICSI duration: 4.76 ± 3.3 min; mean ± SD of all injections). The "0-min" timepoint in ICSI was defined as the end of injection of one group of oocytes. The following sequential remodeling stages were differentiated: unchanged sperm head, chromatin decondensation, chromatin recondensation, beginning of pronuclei formation, and developed pronuclei, as well as their frequency at specific timepoints was noted. In the chromatin decondensation and recondensation groups both partially and fully decondensed or recondensed sperm, respectively, were included. The developed pronuclei group contained early and fully developed pronuclei. Remodeling stages examined in oocytes fertilized in vivo were the quality control for remodeling analysis in IVF- and ICSI-produced embryos. The efficiency of decondensation and recondensation was examined by measuring areas of fully decondensed and recondensed sperm heads. Sperm chromatin was photographed with a digital camera (DSC-F717; Sony, Tokyo, Japan). Sperm head areas were measured using Image J software (developed by Wayne Rasband and available online at http://rsb.info.nih.gov/ij/). A line around each sperm head was drawn, and the area inside was automatically measured. The measurements were repeated three times for each sperm head, and the mean was calculated.

Acrosome Reaction Assay

Acrosome reaction assay [35] was performed to test for the presence of intact acrosome in spermatozoa from all examined groups. Sperm suspension (in HEPES-CZB or T6) was centrifuged at 720 x g for 5 min at 25°C. Pelleted spermatozoa were resuspended in 200 µl of 4% paraformaldehyde (PFA) and fixed for 10 min at room temperature. Fixed sperm were centrifuged at 720 x g for 5 min at 25°C, and the pellet was gently resuspended in 200 µl of 0.1 M ammonium acetate, pH 9.0. Ten microliters of sperm suspension was smeared on a microscope slide and air dried. Slides were stained in Coomassie Blue G-250 (Bio-Rad, Hercules, CA) for 1 h. Spermatozoa with intact acrosomes had intensely stained acrosomal ridges. To define the proportion of acrosome-reacted (acrosome-less) spermatozoa, 100 sperm were scored on each slide, and at least three slides were analyzed in each group.

DNA Replication Assessment

DNA replication analysis was performed according to the procedure described previously [36], with modifications. Fertilized oocytes were incubated in CZB with 10 µM 5-bromo-2-deoxyuridine (BrdU) for 30 min. Following incubation the oocytes were fixed in 2.5% PFA, 0.5 M NaOH, Dulbecco PBS, pH 7.3, at room temperature for 15 min. Fixed oocytes were washed in 10% fetal bovine serum (FBS), 0.2% TX-100, Dulbecco PBS, pH 7.3, and blocked in the same solution for 30 min at 37°C. The oocytes were then washed in 2% FBS, 0.1% TX-100, Dulbecco PBS, pH 7.3; incubated in drops of anti-BrdU antibody conjugated with Alexa Fluor 488 (Molecular Probes, Eugene, OR); diluted 1:19 in 2% FBS, 0.1% TX-100, Dulbecco PBS, pH 7.3, for 1 h at 37°C; and washed again in 2% FBS, 0.1% TX-100, Dulbecco PBS, pH 7.3. The oocytes were placed on the poly-L-lysine-coated (1 mg/ml) microscope slides. The preparations were covered with VectaShield mounting media (Vector Laboratories, Burlingame, CA) and cover glasses, and they were examined using fluorescence.

Statistics

Chi-square, likelihood ratio, Fisher exact probability, and Student t-tests were used for analyzing all responses. Lack of statistical significance was reported when all tests gave P > 0.05. Presence of statistical significance was noted when at least one of the three tests showed P ≤ 0.01 or P ≤ 0.05. The computations were done using KyPlot version 2.0 beta 13 software.

RESULTS

Sperm Chromatin Remodeling in IVF and ICSI

The major difference in chromatin remodeling between IVF and ICSI was the synchrony. We defined each remodeling stage as synchronous when ≥80% of the oocytes with the chromatin in this stage were accumulated in at least one single timepoint. In IVF almost all remodeling stages (four of five) were synchronous (Fig. 1A). This was remarkable because the fertilization window for this group was 60 min. In contrast, in ICSI with fresh spermatozoa, which had a fertilization window of 10 min maximum, only three stages (unchanged sperm head, chromatin recondensation, and formed pronuclei) were synchronous (Fig. 1B). Chromatin decondensation was impaired; it was less prominent than in IVF and did not prevail over the other remodeling stages at any timepoint. Mean areas of fully decondensed sperm heads in ICSI and IVF groups were not significantly different (Table 1). Recondensation of sperm chromatin was accelerated (reaching its peak at 1.5 h after fertilization; 94.7% of oocytes) but its efficiency (determined by the intensity of chromatin compaction; e.g., the lower the mean sperm head area, the more efficient recondensation) was weaker than in IVF (42.05 ± 6.34 µm2 vs. 35.32 ± 5.79 µm2, P < 0.05) (Table 1).


Figure 1
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  		FIG. 1. Sperm chromatin remodeling. Paternal chromatin remodeling was examined in oocytes fertilized by IVF (A) and by ICSI (B–F) at 10 different timepoints. At least 10 oocytes (range: 10–20) were examined at each timepoint in each group


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  		TABLE 1. Efficiency of chromatin decondensation and recondensation (mean ± SD)

Capacitation is the first step in preparing sperm for natural fertilization. Thus, it might have some beneficial effects on sperm quality. Moreover, in assisted reproduction technology (ART) clinics the same sperm sample often is used for both ICSI and IVF, and the capacitated rather than the freshly obtained sperm are injected. When we injected capacitated sperm, we found that chromatin remodeling was even less synchronous than in ICSI with fresh sperm (Fig. 1C): only two stages (unchanged sperm head and formed pronucleus) were synchronous. Moreover, pronuclei formation and maturation were significantly delayed compared with IVF and fresh sperm ICSI (P < 0.05). At 4.5 h postinjection some capacitated sperm were still at the stage of forming pronuclei rather than formed pronuclei. There was no difference in decondensation efficiency between fresh and capacitated sperm, but capacitated sperm recondensed stronger than fresh sperm (31.51 ± 8.99 µm2 vs. 42.05 ± 6.34 µm2, P < 0.05), similar to sperm from IVF (Table 1). Overall, injecting capacitated sperm rather than fresh sperm did not improve chromatin remodeling in ICSI. On the contrary, it resulted in higher asynchrony and delays in final remodeling stages.

It was suggested previously that the presence of the acrosome is hazardous for ICSI outcome in some species, and its removal prior to injection is preferable [37]. To test whether it was indeed the acrosome that caused asynchrony in chromatin remodeling, we performed ICSI with sperm from which the acrosome was removed. To remove the acrosome sperm were treated with calcium ionophore A23187 or with TX-100. Acrosome removal with TX-100 was more effective than treatment with calcium ionophore, as judged by the acrosome reaction assay (58.7% ± 7.02% vs. 42.7% ± 5.86% of sperm without acrosome, respectively). Spermatozoa were scored as without acrosome when no Coomassie blue staining of acrosome ridge was observed. Although less than 60% of sperm were scored as acrosomeless in TX-100 and calcium ionophore groups, in many cases when spermatozoa had some staining and therefore had to be classified as acrosome positive, the staining was much weaker than in other groups (and/or did not cover the entire acrosome ridge). In these spermatozoa the acrosome might have been partially removed.

When sperm treated with calcium ionophore were used for ICSI, most remodeling stages were synchronous (four of five), and the remodeling pattern was the closest to that observed in IVF (Fig. 1D). There was a slight delay in pronuclei formation and maturation, but the difference was not statistically significant compared with IVF or fresh sperm ICSI (P > 0.05).

Remodeling in oocytes injected with TX-100-treated sperm resulted in the same synchrony as in ICSI with fresh sperm (Fig. 1E). Interestingly, acrosome removal with TX-100 did not reset the decondensation stage to resemble that seen in IVF. In both calcium ionophore and TX-100-treated sperm chromatin, decondensation was slightly delayed compared with the other groups, but it did not affect its efficiency. In the TX-100-treated sperm group the first recondensing sperm were observed at the same timepoint as the first decondensing sperm, indicating that the decondensation stage was short. Mean areas of fully decondensed and recondensed sperm in TX-100 and calcium ionophore-treated sperm were not significantly different from each other or from other experimental groups (Table 1). It seems that partial or complete removal of the acrosome prior to ICSI with calcium ionophore but not with TX-100, synchronizes sperm chromatin remodeling while maintaining normal intensity of chromatin decondensation or recondensation.

Dithiotreitol (DTT) was used for sperm pretreatment prior to ICSI in the bovine [38, 39] and minke whales [40], which implies that this reagent may be considered for ICSI in other species. It was suggested that DTT facilitates chromatin decondensation and consequently leads to more successful ICSI outcome [38]. Here we injected sperm treated with DTT to elucidate the effects of this treatment on all stages of chromatin remodeling. DTT was used in conjunction with TX-100, as described previously [41]. This approach results in complete reduction of disulfide bonds in all treated sperm. When such sperm were injected into the oocytes, three of five remodeling stages were synchronous according to our definition (Fig. 1F). However, one stage (decondensation) barely made the 80% cutoff, and the overall remodeling pattern was significantly affected. Decondensation was highly accelerated. At 30 min postfertilization 80% (12 of 15) of sperm were at various decondensation stages, whereas in all other ICSI groups decondensation peak was observed 1 h later (at 1.5 h postfertilization). Decondensation efficiency was the highest when compared with other groups, but the difference reached statistical significance only when compared with sperm treated with TX-100 (Table 1). One sperm in the TX-100 + DTT group decondensed very strongly (1373.0 µm2) and was excluded from the analysis. Chromatin recondensation and pronuclei formation/maturation were highly asynchronous. The recondensation efficiency was higher than that of fresh sperm (35.77 ± 6.09 vs. 42.05 ± 6.34, P < 0.05) but did not differ from sperm from all other experimental groups (Table 1). Reduction of disulfide bonds with DTT synchronized chromatin decondensation but caused significant asynchrony in the subsequent remodeling stages. It also increased overall recondensation efficiency.

Two Patterns of Decondensation

To examine chromatin decondensation in more detail, partially decondensed sperm from all examined groups were selected for analysis. Two different decondensation patterns were differentiated. The first was typical for IVF (100%, 14 of 14), fresh sperm (100%, 7 of 7), and most capacitated sperm (67%, 4 of 6) and calcium ionophore-treated sperm (75%, 3 of 4). In this pattern chromatin started to decondense in the basal (caudal) side of the sperm head, and then decondensation expanded gradually toward the apical sperm side (Fig. 2, A–E). The second decondensation pattern was typical for sperm treated with TX-100 + DTT (100%, 8 of 8) and, presumably, also for sperm treated with TX-100 (only one sperm was classified as "partially decondensed" in this group, and it represented the second type of decondensation pattern). In this pattern chromatin started to decondense simultaneously along the entire length of the dorsal side of the sperm head and expanded toward the ventral side sperm of the sperm head (Fig. 2, F–J). Recondensation and pronuclei formation were the same in all experimental groups (Fig. 3).


Figure 2
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  		FIG. 2. Two patterns of paternal chromatin decondensation. A–E) Pattern typical for IVF, ICSI with fresh sperm, and most capacitated and calcium ionophore-treated sperm. Note initial decondensation at the basal side of the sperm head. F–J) Pattern typical for sperm treated with Triton X-100 and DTT. Note decondensation starting simultaneously along the entire length of the dorsal side of the sperm head. Bar = 10 µm


Figure 3
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  		FIG. 3. Paternal chromatin recondensation and pronuclei formation. A) Completely decondensed chromatin. It has a large size and is evenly stained and lightly colored. B, C) Partially recondensed chromatin. It is stained darker than in its fully decondensed form but lighter compared with the fully recondensed stage. The tightness of packaging reflected by the size is between the fully decondensed and recondensed stages. D) Fully recondensed chromatin. This stage is characterized by a small size, tight packaging, and dark staining of chromatin. The shape is still asymmetric, not ovoid. E, F) Beginning of pronucleus formation. The chromatin is less condensed than in the fully recondensed stage, and it has a granular structure. Dark spots of condensed chromatin can be observed on a lighter background. The shape is close to oval or oval. G) Early pronucleus. Chromatin begins to swell and gains more spherical shape. There is more lightly colored decondensed chromatin, but some dark spots are still visible. H) Fully developed pronucleus. It is the biggest in size of all pronuclei stages and has lightly stained chromatin and a characteristic net-like structure: chromatin threads are interspaced with free space. Bar = 10 µm

Developmental Potentials of Embryos

In all ICSI groups the same proportions of oocytes were fertilized (observed as the presence of two well-developed pronuclei and an extruded second polar body) after sperm injection. Embryo development up to the morula stage was similar in all groups. However, at 96 h postfertilization, fewer blastocyst stage embryos were observed when spermatozoa were treated with TX-100 or TX-100 + DTT (50% and 46%, respectively) compared with IVF (91%, P < 0.01) and fresh sperm ICSI (78%, P < 0.05) (Table 2). The development of blastocysts produced with TX-100 and TX-100 + DTT at 96 h was delayed. More than 20% of the blastocysts were classified as early blastocyst stage at this time, compared with 13% or fewer in all other groups.


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  		TABLE 2. Development of embryos obtained by IVF and ICSI.*

DNA Replication

DNA replication was first observed 5 h after fertilization (Table 3). In all experimental groups except for ICSI with capacitated sperm, DNA replication was detected in the majority of zygotes (68%–81%) 6 h after fertilization. The highest number of oocytes with DNA replication was observed in the IVF group (Fig. 4, A and B). At the same timepoint, DNA replication was noted in less than one fourth (23%, 6 of 26) of zygotes produced by ICSI with capacitated sperm (statistically significant difference when compared with all other groups) (Fig. 4, C and D). DNA replication assessment in the capacitated sperm group at 6 h postfertilization was performed in four independent trials, and the results were consistent (range: 14%–29%). The result was in agreement with chromatin remodeling data—embryos produced by ICSI with capacitated sperm exhibited slower and the most asynchronous pronuclei formation. At 7 and 8 h after fertilization the proportion of zygotes, in which the replication took place, was similar in all groups.


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  		TABLE 3. DNA synthesis in embryos produced by IVF and ICSI.*


Figure 4
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  		FIG. 4. DNA replication in one-cell embryos 6 h after fertilization. A, C) Phase contrast. B, D) Fluorescence. Oocytes fertilized in IVF (A, B) and injected with capacitated sperm (C, D). Bar = 50 µm

DISCUSSION

In this study we examined sperm chromatin remodeling in embryos produced by IVF and ICSI with various sperm treatments prior to injection. Chromatin remodeling in IVF embryos resembled that described earlier by Adenot et al. [3]. The absolute areas of sperm heads at different remodeling stages are higher in our work, probably because of chromatin expansion during drying. However, the overall timing of remodeling and the ratio of mean chromatin areas of the decondensation versus recondensation stages were similar to those reported earlier [3].

The most striking difference between chromatin remodeling in embryos produced by IVF and ICSI was the synchrony. When normal fresh sperm were injected into the oocytes by ICSI, chromatin remodeling became more asynchronous than in IVF, with the decondensation stage being affected most. It was suggested before that a full capacitation state prior to injection may be a prerequisite for acrosome reaction to take place in the ooplasm [42]. Here, surprisingly, sperm capacitation prior to injection not only did not improve chromatin remodeling but caused more prominent asynchrony, affecting most remodeling stages. The most important changes that sperm undergo during capacitation involve acquiring a specific pattern of movement, known as sperm hyperactivation, and ability to undergo the acrosome reaction. Both of these events are crucial for active sperm penetration into the oocytes but are redundant when sperm are injected [30, 43]. During capacitation various membrane modifications take place as well. One of the processes is a polymerization of globular (G) actin to filamentous (F) actin. F-actin formation is important for the translocation of phospholipase C from cytosol to the sperm plasma membrane during capacitation. During the acrosome reaction, however, the F-actin must undergo depolymerization to enable fusion of outer acrosome membrane and the overlying plasma membrane [44]. It is possible that capacitated spermatozoa injected into the oocytes had F rather than G actin, which caused the asynchrony of chromatin remodeling.

It was suggested that the presence of structures surrounding the sperm chromatin in the oocyte after injection impairs nuclear remodeling [45], and this can be overcome by acrosome removal [37]. Our study demonstrates that the method used to remove the acrosome is not without significance. Removal of sperm membranes and/or acrosome with calcium ionophore but not with TX-100 resulted in chromatin remodeling becoming more synchronous. All remodeling stages except for the beginning of pronuclei formation were highly synchronous and resembled those of IVF embryos. As revealed by electron microscopy studies, TX-100 removes all membranes (acrosome and plasma membranes) as well as the acrosomal content but leaves perinuclear material around sperm nucleus [46]. Calcium ionophore induces the acrosome reaction by equilibrating intracellular and extracellular calcium concentrations and requires sperm to be capacitated prior to reaction [47]. Thus, the action mode of these two reagents is completely different, with calcium ionophore being more physiologic than TX-100. During normal fertilization the sperm enters the ooplasm after undergoing both capacitation and the acrosome reaction, and this may explain why the remodeling pattern after ICSI with calcium ionophore-treated, acrosome-reacted sperm is the closest to that observed in IVF.

It was demonstrated in rhesus monkeys [18, 20, 21, 48], pigs [23], and humans [22] that the presence of the acrosome, perinuclear theca, and membrane around sperm chromatin during injection interferes with chromatin decondensation in the oocyte. In all previous works the authors indicated delayed and heterogeneous decondensation in ICSI, with the apical region of the sperm head remaining more condensed compared with the rest of decondensing head. Our results show that in the mouse the decondensation pattern is similar in IVF and ICSI with fresh sperm (chromatin decondenses, expanding initially from the basal and ending in the apical region of the sperm head), there is no delay in achieving the fully decondensed stage, and upon completion of the decondensation process all chromatin is decondensed evenly. Perhaps the observed difference between our results and those of other studies is caused by the fact that, previously, sperm were injected into the oocytes with tails and were from species that had morphologies different than that of rodent sperm. Hook-shaped and protruding, prominent acrosomes make mouse spermatozoa more vulnerable to mechanical damage [49–52]. It was reported that tail removal prior to ICSI may disrupt the acrosome [53]. It is possible that in our study minute acrosome changes taking place during sperm head removal were sufficient to prevent prolonged delay in decondensation of the apical region of the sperm head. The delay in decondensation of the apical region of sperm head reported after ICSI was called aberrant [22]. However, in most studies proper IVF (or in vivo) controls were missing. Because it is not easy to define the exact fertilization time in IVF due to long gamete co-incubation, it is reasonable to suspect that early decondensation stages escaped the analysis. Our results suggest that the delay in decondensation of the apical region of sperm head may be a normal stage of the decondensation process.

When spermatozoa were subjected to treatment with TX-100 and DTT prior to ICSI, a different pattern of decondensation was observed. Decondensation initiated from the dorsal side of the sperm head and expanded toward its ventral side. Perhaps this peculiar way of decondensing was caused by some membrane changes resulting from treatment with TX-100. This can be clarified in the future by evaluating sperm decondensation after sperm treatment with various detergents and other insults that damage sperm membrane. The implications of an abnormal decondensation pattern are unclear but may have some effect on embryo development.

The pattern of recondensation, the stage during which sperm chromatin acquires a nucleosome-type structure [2, 12], was the same for all groups. This suggests that ICSI does not interfere with basic chromatin packaging. We have shown previously that when sperm treated with TX-100 + DTT were injected into the oocytes, the paternal chromosomes were broken in the majority of resulting zygotes [41]. Here, except for the lack of synchrony we did not note abnormal recondensation in sperm treated with TX-100 + DTT. Thus, it seems that the chromosome damage observed before was not related to problems with chromatin packaging.

It was shown that although M-phase cytoplasm is sufficient for chromatin decondensation, recondensation and assembly of histones [2], the later stages of remodeling (i.e., pronuclei formation and import of nuclear proteins into the pronucleus) require transition from M phase to interphase. This transition is triggered by Ca2+ oscillations [54] normally induced by phospholipase C in the fertilizing spermatozoon [55]. The first description of Ca2+ signals after ICSI came from studies on human oocytes [56, 57]. These studies demonstrated that ICSI resulted in delayed, truncated Ca2+ response that nevertheless maintained the form of Ca2+ waves and Ca2+ oscillations typical for normal fertilization. It was reported recently in the mouse that ICSI-induced calcium oscillations are not equivalent to those initiated by IVF [29]. Thus, the delay in pronuclei formation observed in this study might have been caused by suboptimal M-phase-to-interphase transition. Lee at al. [58] showed that pronuclei formation was accelerated when sperm with removed acrosomes were used for ICSI, compared with acrosome-intact sperm. Here, we did not observe such acceleration when sperm were treated with TX-100 or calcium ionophore. On the contrary, we noted a tendency toward delay in the calcium ionophore-treated group and a significant delay in capacitated sperm group.

Delayed pronuclei formation did not affect the potential for an embryo to develop in vitro. The proportion of blastocysts obtained after ICSI with fresh, capacitated, and calcium ionophore-treated sperm did not differ from that of blastocysts obtained after IVF. However, when compared with other groups, significantly fewer embryos were capable of developing to the blastocyst stage if sperm were treated with TX-100 or TX-100 + DTT. Kurokawa and Fissore [29] reported accelerated frequency of calcium oscillations after ICSI with sperm treated with TX-100 that was accompanied by impairment in embryo development to the blastocyst stage. Our results of embryo development are close to those published previously [29], both in control (fresh sperm ICSI) and after treatment with TX-100. Thus, we believe that poorer embryo development and a slight delay in blastocyst maturation originated from suboptimal calcium oscillations. This is in agreement with previous findings demonstrating that the pattern of Ca2+ increases have an impact on the developmental outcome of the mouse embryos [59–61]. Because it was also shown that live offspring can be obtained after ICSI with TX-100-treated sperm [46], we believe that the embryos that do develop are normal. However, we cannot exclude the possibility that ICSI with detergent-treated sperm have hidden effects that have escaped analyses thus far. We and others continue searching for the effects of various gamete manipulations on fertilization and offspring well-being.

It was reported that in rhesus monkeys a DNA synthesis was delayed in ICSI embryos [21]. In the mouse we did not observe differences in DNA synthesis between ICSI and IVF. The delay was only noted when capacitated sperm were injected; this presumably was due to the presence of polymerized actin, as explained earlier.

In this study we present for the first time paternal chromatin remodeling in mouse embryos produced by ICSI. We demonstrated that ICSI resulted in less synchronous chromatin remodeling than in IVF. Sperm capacitation prior to injection enhanced remodeling asynchrony and resulted in delayed pronuclei formation and DNA synthesis. The removal of the acrosome with calcium ionophore prior to injection restored the synchrony of chromatin remodeling to resemble that observed in IVF embryos. Although detergent was recommended previously for acrosome removal [37, 62], our results suggest that calcium ionophore is better. Sperm treatment with TX-100 did not improve chromatin remodeling but resulted in an unusual decondensation pattern and impaired embryo development.

The results from our study are relevant for ART in humans. In ART clinics the same ejaculate often is used for IVF and ICSI, and capacitated sperm rather than freshly obtained sperm are injected. Here we show that using capacitated sperm for ICSI results in stronger remodeling asynchrony than in ICSI with fresh sperm. If our observations in the mouse are valid also in humans, then our results provide a clear message for ART clinicians to avoid using capacitated sperm for ICSI. Our study also demonstrates that sperm pretreatment with calcium ionophore allows for the remodeling of paternal chromatin to be reset to resemble that of IVF, timely DNA synthesis, and efficient embryo development in vitro. Calcium ionophore already is used in ART clinics to help overcome infertility in cases of repeated failed fertilization after ICSI by means of assisted oocyte activation [63–66]. It is also used as an assay to predict to the outcomes of assisted reproduction treatments by providing information about sperm ability to undergo acrosome reaction [67, 68]. Our results supplement previously published data and support the use of calcium ionophore in human ART.

Much still is unknown about possible ICSI effects, and studies in this area are ongoing. We do not know exactly at this point what the lack of synchrony of chromatin remodeling in ICSI means, nor how it can affect ICSI offspring. However, high synchrony observed in IVF suggests that this is a highly regulated process and therefore may have biologic significance. Although IVF involves a variety of gamete manipulations, it is more physiologic than ICSI. Thus, resetting early postfertilization events after ICSI to resemble those of IVF may be closer to what happens in nature and, therefore, beneficial for ART success.

ICSI in the mouse is very successful and yields live offspring with high efficiency [69, 70]. Thus, skewed chromatin remodeling does not prevent the ultimate outcome of fertilization—birth of a new individual. However, it is likely that ICSI will be modified further for an even wider variety of applications, such as improvements in ICSI-mediated transgenesis techniques or achievement of fertilization with genetically deficient gametes. The early events in fertilization, such as chromatin remodeling and DNA synthesis, are likely to be more sensitive to these modifications than to routine ICSI. Our data will be useful in refining the molecular and biologic mechanisms associated with ICSI for current and future applications.

FOOTNOTES

1 Supported by grant R21 HD048845 to M.A.W. Presented in part at the 39th Annual Meeting for the Society for the Study of Reproduction, July 29–August 1, 2006, Omaha, Nebraska. Back

2 Correspondence. FAX: 808 956 7316; mward{at}hawaii.edu Back

3 Monika A. Ward previously published manuscripts under the name Monika A. Szczygiel. Back

Received: 16 April 2006.

First decision: 18 May 2006.

Accepted: 13 June 2006.

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