Human Reproduction, Vol. 17, No. 10, 2657-2664,
October 2002
© 2002 European Society of Human Reproduction and Embryology
Fate of the acrosome in ooplasm in pigs after IVF and ICSI
1 Department of Life Science, Graduate School of Science and Technology, Kobe University, Rokkodai-cho Nada-Ku, Kobe City, Hyogo and 2 Koshida Clinic, 1-12, Kakuda-cho, Kita-Ku, Osaka City, Osaka, Japan
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
BACKGROUND: ICSI bypasses the sperm–oolemma interactions that, in normal fertilization, depend on completion of the acrosome reaction. Morphological changes in the acrosomes of sperm in the ooplasm were therefore examined following IVF and ICSI using pig gametes. METHODS: In-vitro-matured porcine oocytes were used for ICSI or IVF. Oocytes were then stained with fluorescein isothiocyanate-conjugated peanut agglutinin lectin (FITC-PNA), which specifically labels the outer acrosomal membrane of boar sperm and the cortical granules (CG) in porcine oocytes. This was followed by observation under a confocal laser scanning microscope. RESULTS: In ICSI, PNA showed the presence of disintegrated acrosomes that classified into four categories. Heterogeneous chromatin decondensation was observed in the sperm with intact/disintegrated acrosome, whereas acrosomes were barely detected in oocytes which had formed a male pronucleus. Both in ICSI and IVF, PNA-positive tails were concomitantly observed with one type of disintegrated acrosome, which was considered to be acrosome-reacted. The disappearance of CG in activated oocytes after ICSI was similar to that after IVF. CONCLUSIONS: The PNA-binding properties of sperm head components introduced into the ooplasm during ICSI are different from those after IVF. The delay of sperm chromatin decondensation is associated with that of acrosomal disassembly. Acrosomes appear to disintegrate in the ooplasm whether or not the acrosome reaction has taken place. Oocytes undergoing ICSI appear normally activated in terms of meiotic resumption and CG exocytosis.
Key words: acrosome/cortical granules/ICSI/peanut agglutinin lectin/pigs
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
ICSI currently dominates assisted reproductive technology, especially in the treatment of severe male factor infertility (Palermo et al., 1992
; Van Steirteghem et al., 1993), and has been advocated as a possible technique for the conservation of endangered species where access to semen stocks is limited (Iritani, 1991).
During normal fertilization, the acrosome-reacted spermatozoon fuses with the oolemma, and the entire spermatozoon (with the exception of part of the sperm membrane, most of the outer acrosomal membrane and the acrosomal components) is incorporated into the ooplasm (Yanagimachi, 1988). Although some of the sperm structures, such as chromatin and the centriole are transformed into zygotic components, other sperm components are eliminated in the early stages of embryonic development. For example, the sperm mitochondria are eliminated by the 2- to 4-cell stage in cattle (Sutovsky et al., 2000), rats (Szollosi, 1965; Shalgi et al., 1994), mice (Shalgi et al., 1994) and hamsters (Hiraoka and Hirao, 1988). Although the sperm midpiece with paternal mitochondria is introduced into the ooplasm by ICSI methods, the mitochondria are inherited exclusively maternally in humans (Danan et al., 1999). The fibrous sheath, which is a major structure of the sperm principal piece, is reported to disappear rapidly as sperm is incorporated into the ooplasm in rats and mice (Shalgi et al., 1994) and cattle (Sutovsky et al., 1996), while the outer dense fibrous sheaths are detected until the 8-cell stage in cattle (Sutovsky et al., 1996), rats (Shalgi et al., 1994) and hamsters (Shalgi et al., 1994).
In ICSI, a spermatozoon is deposited into the ooplasm with both the acrosome and plasma membrane intact, in addition to the other sperm accessory components that are naturally eliminated in fertilized oocytes. The sperm acrosome contains a variety of hydrolytic enzymes, the release of which into the ooplasm might be harmful (Tesarik and Mendoza, 1999). Based on findings that the injection of an intact hamster spermatozoon into a mouse oocyte leads to degeneration of the ooplasm, whereas the injection of a demembraned hamster spermatozoon without acrosome forms two normal pronuclei, it was suggested that there is species-specific tolerance of the ooplasm to exotic acrosomal contents that determines the level of acrosomal toxicity (Kimura et al., 1998). However, it is unclear how an oocyte that has been injected with an acrosome-intact spermatozoon will cope with the sperm acrosome.
An acrosome introduced into the ooplasm by ICSI seems physically to disturb sperm chromatin decondensation. Sperm chromatin heterogeneously initiates decondensation under the intact acrosomal cap in mice (Kimura and Yanagimachi, 1995), rhesus monkeys (Hewitson et al., 1996) and pigs (Kim et al., 1998). DNA synthesis is delayed in both pronuclei when the paternal pronucleus is still undergoing decondensation in the apical region under the acrosomal cap, identifying a unique G1/S cell cycle checkpoint (Ramalho-Santos et al., 2000). As the X chromosome is preferentially located in the apical region of the human sperm head (Luetjens et al., 1999), it is suggested that heterogeneous sperm chromatin decondensation under the intact acrosomal cap could be the reason for increased sex-chromosome anomalies in children conceived after ICSI (Bonduelle et al., 1998; Luetjens et al., 1999).
Although the fate of the acrosome in ooplasm has been examined ultrastructurally after ICSI in humans (Sathananthan et al., 1997; Bourgain et al., 1998), further investigation would be desirable due to the quantitative limitations of using electron microscopy and human oocytes. In the present study, the morphological changes of the pig acrosome after ICSI were examined after the oocytes had been histochemically stained with fluorescein isothiocyanate conjugated peanut agglutinin (FITC-PNA). PNA, a lectin that binds specifically to the saccharide ß-D-Gal(1,3)-D-GalNAc, is known to label specifically the outer acrosomal membrane of boar (Fazeli et al., 1997; Flesch et al., 1998), mouse (Lee and Ahuja, 1987) and human sperm (Mortimer et al., 1987), and is used as a probe to examine the acrosome reaction in these species. Additionally, PNA labels cortical granules (CG) in pig oocytes, and the exocytosis of CG after fertilization or parthenogenetic activation has been demonstrated (Yoshida et al., 1993; Wang et al., 1997). Ca2+ oscillation in the ooplasm following sperm–oocyte interaction at fertilization is the pivotal regulator of oocyte activation and is responsible for post-fertilization events including meiotic resumption and CG exocytosis (Hoodbhoy and Talbot, 1994). Therefore, the behaviour of CG was also examined to assess the normality of oocyte activation after ICSI in which membrane fusion between sperm and oocytes is bypassed.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
Maturation of porcine oocytes in vitro
Prepubertal porcine ovaries were collected from local slaughterhouses and transported to the laboratory at 20°C. The collection of follicular oocytes was based on a previously published method (Kurebayashi et al., 1996). Briefly, the ovaries were rinsed in Dulbecco’s phosphate-buffered saline containing 0.1% polyvinyl alcohol (PBS-PVA), and the oocyte–cumulus–granulosa cell complexes (OCGC) were picked with forceps from the inner surface of healthy antral follicles of 4–6 mm diameter without detachment of the oocyte and granulosa cells. Groups of 30 OCGC were matured in 2 ml of TCM-199 (Earl’s salt; Nissui Pharmaceutical Co. Ltd, Tokyo, Japan) supplemented with 10% (v/v) heat-treated fetal calf serum (FCS; Biocell Inc., Carson, CA, USA), 0.1 mg/ml sodium pyruvate, 0.08 mg/ml kanamycin sulphate (Sigma Chemical Co., St Louis, MO, USA) and 0.1 IU/ml HMG (Pergonal; Teikokuzoki, Tokyo, Japan), and co-cultured with two thecal shells from healthy follicles of 4–6 mm diameter that had been freed of follicular fluid and granulosa cells. The culture was carried out in a CO2 incubator under 5% CO2 in humidified air at 38.5°C for 47–49 h with gentle agitation.
Sperm preparation for IVF and ICSI
The sperm-rich fraction of semen was collected from healthy, fertile boars by the gloved-hand method, and centrifuged at 1500g on two layers (80% and 55%) of Percoll (Amersham Pharmacia Biotech AB, Uppsala, Sweden) for 10 min. The pellets were resuspended in PBS-PVA and centrifuged twice at 1500g for 5 min. The concentration of sperm in the resultant pellets was adjusted to 2x108 cells/ml, and the suspension was stored at room temperature for 
30 min before insemination or ICSI.
IVF
After the maturation culture, OCGC were placed into 2 ml of Brackett and Oliphant solution supplemented with 5 mmol/l caffeine (modified BO solution; Brackett & Oliphant, 1975) in a 35 mm dish (Becton-Dickinson Labware, Franklin Lakes, NJ, USA) and inseminated with sperm at a concentration of 5x105 cells/ml for 6, 9 and 15 h culture.
Sperm injection into matured oocytes in vitro
Sperm were resuspended in PBS-PVA at a concentration of 1x106 cells/ml, and the sperm suspension was diluted in 10% polyvinylpyrrolidone (PVP; Irvine Scientific, Santa Ana, CA, USA) solution (1:1). Microdrops of 10 µl sperm suspension and 25 µl HEPES buffered TCM-199 (pH 7.4) containing 0.1% PVA (HEPES-199) were placed in the same culture dish (Falcon, No. 1007) on the stage of a Hoffman modulated inverted microscope equipped with micromanipulators (Narishige, Inc., Tokyo, Japan).
The microinjection of a spermatozoon into the ooplasm was performed in a microdrop of HEPES-199. After the maturation culture, oocytes were denuded of cumulus cells by a treatment with 0.01% (w/v) hyaluronidase (Sigma) and by pipetting with a small-bore pipette. Oocytes emitting a polar body were selected and transferred into the microdrop.
Each of the oocytes was held according to the position of the first polar body at 6 or 12 o’clock and injected from the 3 o’clock position. A single spermatozoon in the PVP solution was aspirated tail-first by a microinjection pipette (6–7 µm inner diameter; Humagen, Inc., Charlottesville, VA, USA) after the tail (just below the mid-piece) had been rubbed with a microinjection pipette. The pipette was pushed through the oolemma into the oocyte, and a small amount of the ooplasm was drawn into the pipette. The spermatozoon, together with the cytoplasm and a small volume of PVP solution, was then expelled into the oocyte. Some oocytes were injected in the same manner without a spermatozoon (sham injection).
After injection of a spermatozoon or sham-injection, the oocytes were cultured in TCM-199 supplemented with 10% (v/v) FCS and 0.1 mg/ml sodium pyruvate, and then fixed after 3, 6, 9 and 15 h.
Labelling of sperm and oocytes with FITC-PNA
Cumulus cells surrounding matured oocytes and fertilized oocytes in vitro were removed by pipetting with or without 0.01% (w/v) hyaluronidase. Fresh boar sperm pellets after centrifugation, oocytes matured in vitro, fertilized oocytes in vitro or oocytes receiving ICSI were fixed with 3% paraformaldehyde in PBS-PVA for 30 min at room temperature. After being rinsed twice in 10% bovine serum albumin (BSA) in PBS (BSA-PBS), the pellets were kept in BSA-PBS at least overnight. They were then treated with 0.1% Triton X-100 (Sigma) in PBS for 5 min and rinsed twice in BSA-PBS. Oocytes and sperm pellets were incubated in 20 µg/ml FITC-PNA (Sigma) in PBS for 30 min. Following rinsing in BSA-PBS, DNA was counterstained with 400 µg/ml propidium iodide (PI; Sigma) for 15–20 min. As a negative control, 10 mmol/l ß-D-Gal(1,3)-D-GalNAc (Toronto Research Chemicals Inc., North York, ON, Canada) was incubated with 20 µg/ml FITC-PNA in PBS for 30 min. After rinsing, the oocytes and sperm pellets were mounted in an anti-fade medium (Vector Laboratories, Inc., Burlingame, CA, USA) and observed under a confocal laser scanning microscope (MRC 1024 system; Bio-Rad, Hercules, CA, USA).
Statistical analyses
The data obtained from experiments with ICSI and IVF were pooled from at least four replications. Values were subjected to an arcsine transformation in each replication, and were analysed using a one-way analysis of variance (ANOVA). The significance of differences was assessed using a t-test method.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
Distribution of PNA in fresh sperm and in-vitro-matured oocytes in pigs
As a control, the binding patterns of PNA were observed in fresh sperm and oocytes before fertilization. In fresh sperm, intense PNA fluorescence was specifically and homogeneously observed on the whole acrosomal cap region (Figure 1A and A-a
). In oocytes arrested at the second metaphase, fluorescent spots of PNA were evenly scattered on the cortex of the ooplasm. Images of an equatorial series of oocytes showed that the fluorescent spots were exclusively located close to the oolemma and formed a monolayer (Figure 1B). In addition, they were incorporated into the surface of the first polar body (Figure 1B, arrowhead). The binding of PNA to oocytes was inhibited by 10 mmol/l ß-D-Gal(1,3)-D-GalNAc (negative control, data not shown).
|
Distribution of PNA in sperm during IVF
In total, 28, 20 and 26 oocytes were observed at 6, 9 and 15 h after insemination. The penetration rates were 75, 90 and 100% at 6, 9 and 15 h respectively. Multiple sperm penetration was observed in all fertilized oocytes, and the average number of penetrated sperm was 2.8, 2.3 and 4.9 per oocyte at 6, 9 and 15 h respectively. Sperm began to penetrate at 6 h, and all penetrated oocytes had male pronuclei at 15 h after insemination. Although PNA bound to both acrosomes and CG in oocytes under the conditions used, they were easily distinguished by their different distribution patterns. The most intense PNA fluorescence was observed on the surface of zona pellucida in all the oocytes (small arrows in Figure 1
). Each fluorescent spot was shaped like the acrosomal cap and painted with continuous and bright fluorescence (arrows in Figure 1C). As the sperm penetrated through the zona pellucida and reached the oolemma surface, the PNA patterns found on the sperm head altered to become characterized by the fringed fluorescent outline of the acrosomal region (Figure 1C and D). The intensity of fluorescence in the area surrounded by the fringe differed from partial (Figure 1C-a) to complete (Figure 1D-a) loss of fluorescence. The sperm found on the surface of the oolemma usually lost fluorescence in that area. Whenever membrane fusion occurred between the spermatozoon and oocytes, the fluorescence at the equatorial segment became unclear, and then the fluorescent fringes began to detach from the sperm head in the same region (Figure 1E-a). Most of the fringe usually detached completely from the sperm head, except at the apical point (Figure 1E-b). At this stage, no signs of decondensation of the sperm nucleus were observed. In the later stages, only a few (usually three) fluorescent spots were detectable in the vicinity of the sperm nucleus in most cases (Figure 1F-a and G-a), occasionally observed close to the male pronucleus (Figure 1G-b). Sperm tails showing fluorescence were also seen (Figure 1H). Such tails were consistently observed with fluorescent fringes, suggesting that fluorescence may be correlated with the incidence of the acrosome reaction.
Distribution of PNA in sperm during ICSI
After maturation culture, oocytes with a first polar body and good morphology were used for the ICSI experiments after the removal of expanded cumulus cells. Before the injection, the maturation rate of oocytes for ICSI and sham injection was 88.2% (201/228). The proportion of oocytes successfully injected with a spermatozoon was 93.2% (150/161).
Some 77% of 48 sperm-injected oocytes were activated, and 31% of sperm-injected oocytes possessed two pronuclei and two polar bodies at 15 h after the injection (Table I). No fluorescence was detected at the surface of the zona pellucida in those oocytes (Figure 2). Various binding patterns of PNA were observed in the head region of sperm injected into the ooplasm. Some of these patterns were found uniquely in the oocytes receiving ICSI; thus it was necessary to classify all the sperm into four novel categories: (i) virtually intact acrosome (Figure 2A-a); (ii) disintegrated acrosome such as partially disassembled (Figure 2B-a and C-a) and detached from sperm heads (Figure 2D-a and E); (iii) the fringed outline of the acrosomal region with the mass of fluorescence (Figure 2F-a and G-a); and (iv) dispersed fluorescence around the sperm head (Figure 2H). When acrosomes were examined 3 h after injection, disintegrated acrosomes and those with a fringed outline were observed in 56 and 16% of oocytes respectively (Table I). At this stage, chromatin decondensation was not observed. As the rate of activation increased with time, the proportions of oocytes with enlarged sperm heads or a male pronucleus also increased. Similarly, the proportions of oocytes showing no or dispersed fluorescence also increased, reaching 48% at 15 h (Table I). Intact or enlarged sperm nuclei were seen with any of the above four PNA-binding patterns. However, almost all oocytes that had developed a male pronucleus (17/18) specifically exhibited no or dispersed fluorescent spots (Table II). On the other hand, 22% of enlarged sperm heads were associated with an intact PNA-binding pattern (Table II; Figure 2A-a), which was never observed in IVF. In contrast, the several fluorescent spots usually observed in fertilized oocytes after detachment of the fluorescent fringe were hardly seen in ICSI. The patterns of PNA-binding in the tail region found after ICSI were similar to those found in IVF. Both PNA-positive and PNA-negative tails were observed after ICSI (Table III; Figure 2B-a, C-a and E). As shown in Table III, all sperm with a fringed acrosomal region had PNA-positive tails similar to those of presumably acrosome-reacting/reacted sperm in IVF. In striking contrast, sperm with intact acrosome or dispersed PNA had PNA-negative tails.
|
|
|
|
Meiotic resumption of oocytes and distribution of PNA at the cortex
In oocytes arrested at the second metaphase, the fluorescent spots were evenly dispersed in the cortex close to the oolemma (Figure 1B
). These spots had usually been observed until just after sperm–oocyte fusion (Figure 1E), though a large number of spots disappeared with the appearance of the telophase-second chromosomes or female pronucleus (Figure 1D, F, G and H). Essentially, the same patterns of disappearance of those spots were seen in oocytes receiving ICSI. Importantly, the sham injection did not activate the oocytes efficiently. Only 11% of oocytes were activated (Table I), and the oocytes still at the second metaphase retained fluorescent spots in the cortex close to the oolemma. After ICSI however, 24–77% of oocytes were activated and the female pronuclei were formed in 77% of oocytes at 15 h (Table I). Large numbers of fluorescent spots disappeared from the cortical region (Table I; Figure 2A, D and H) or clumps of fluorescence were scattered evenly on the surface of the oolemma (Table I, Figure 2E). Thus, introduction of the spermatozoon directly into the ooplasm was effective in inducing these post-fertilization events in pig oocytes. |
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
This is the first study to show the detailed morphologies of acrosomes of boar sperm injected into the ooplasm by the ICSI method. It has been shown clearly that the PNA-binding properties of sperm head components introduced into the ooplasm were different from those after IVF. Acrosomes appeared to disintegrate in the ooplasm with or without the acrosome reaction. Oocytes undergoing ICSI were activated normally in terms of the resumption of meiosis and CG exocytosis, in spite of the absence of the normal membrane fusion of sperm and oocytes, while the incidence of these events was low after the sham injection.
In order to trace the acrosome, FITC-PNA was utilized, as PNA has been reported to bind specifically to the outer acrosomal membrane of intact boar sperm (Fazeli et al., 1997; Flesch et al., 1998). In previous studies in which PNA was used, boar sperm could be categorized into three groups: (i) intense and uniform fluorescence over the acrosomal cap (acrosome-intact); (ii) varying degrees of mottled and patchy fluorescence over the acrosomal region or fluorescence confined to the equatorial segment (acrosome-reacting); and (iii) no or reduced fluorescence in the sperm head (acrosome-reacted) (Vazquez et al., 1993; Fazeli et al., 1997). In the present study, the binding patterns of PNA were first characterized for the sperm before and after fertilization. The pattern categorized as (i) was observed in the fresh sperm. As the sperm approached the oocyte surface, fluorescence was reduced in the head region. The sperm head found on the oolemma usually showed only the fluorescent fringe of the acrosomal region. In this case, PNA most likely bound to some region other than the outer acrosomal membrane. This is not surprising as many studies have reported changes in PNA-binding properties in the head region after acrosome reaction in pigs (Vazquez et al., 1993; Fazeli et al., 1997), mice (Lee and Ahuja, 1987) and humans (Mortimer et al., 1987). Moreover, with another lectin, concanavalin A (ConA), binding sites transfer from the outer acrosomal membrane to the inner acrosomal membrane after the acrosome reaction in hamsters (Koehler, 1981). Therefore, it may be considered that the sugar chains and/or protein components at the inner acrosomal membrane become PNA-positive after the acrosome reaction, or some acrosomal contents that had become PNA-positive after the acrosome reaction are left at the inner acrosomal membrane. As the acrosome reaction occurs, boar sperm lose both the plasma membrane and outer acrosomal membrane, resulting in exposure of the inner acrosomal membrane at the anterior sperm head and the formation of a fold resulting from the fusion between plasma membrane and outer acrosomal membrane at the equatorial segment (Szollosi and Hunter, 1973). In the present study, the inner acrosomal membrane—and especially the anterior and posterior sites—was most likely labelled by PNA after the acrosome reaction had occurred, and might form the fluorescent fringes together with the outer acrosomal membrane (now fused with the plasma membrane) at the equatorial segment.
After attaching to the surface of the oolemma, the fluorescent fringe of the sperm began to disappear, starting at the equatorial segment. Since the microvilli of oocytes initially associate with the boar acrosome-reacted sperm at the equatorial segment (Imai et al., 1980), this disappearance of fluorescence may suggest an initial interaction between sperm and oocytes. In the following stages, the inner acrosomal membrane detached from the sperm nucleus (Imai et al., 1980; this study), and eventually fluorescence was observed as only several spots in the vicinity of the sperm nucleus. It was improbable that the membrane itself was dismembered, because the inner acrosomal membrane can be detected during sperm chromatin decondensation (Imai et al., 1980). Occasionally, these spots remained detectable even after formation of the male pronucleus; therefore, it is suggested that the sugar chains and/or some protein components of the inner acrosomal membrane are rapidly dehydrated or altered after separation from the sperm nucleus, though some portions remained PNA-positive for many hours.
In the present study, after IVF the male pronuclei could be formed within 9 h following penetration, yet in ICSI 42% of oocytes still had an enlarged sperm nucleus at 15 h after injection. These results demonstrate that sperm nuclear decondensation is delayed in ICSI compared with IVF. Compared with the patterns of PNA binding after IVF, some unique patterns of PNA binding were observed after ICSI, suggesting that the acrosomal cap may be disassembled in an unusual manner. This seems reasonable because the sperm in IVF already showed fluorescent fringes when they attached to the oolemma, while in ICSI the sperm with intact acrosomes had to interact with the ooplasm. In IVF, whenever chromatins were evenly decondensed, only a few fluorescent spots of PNA were observed near the chromatins. In the case of ICSI, intact acrosomes and partially disintegrated acrosomes were often observed. However, even in ICSI, in the oocytes that had formed a male pronucleus, the PNA fluorescence had mostly disappeared. Therefore, the delays in the changes of PNA-binding pattern and that in the alteration of sperm chromatin morphology are most likely correlated. In humans, the acrosome reaction of injected sperm is observed before sperm chromatin decondensation (Sathananthan et al., 1997). Early signs of the acrosome reaction were observed ultrastructurally 15 min after sperm injection, and at 4 h the acrosomes were not detected in most oocytes (Bourgain et al., 1998). Importantly, the deposition of an almost intact acrosomal cap in the vicinity of the sperm nucleus was also observed in humans (Sathananthan et al., 1997). As the latter authors suggested, a full capacitation state before injection may be a prerequisite for the acrosome reaction to occur in the ooplasm, or an intact acrosome would be discarded in the ooplasm (Sathananthan et al., 1997). This might explain why, in the present study, enlarged sperm heads associated with the intact acrosome were often observed in oocytes with a female pronucleus (Table I). Similar heterogeneous decondensation of sperm chromatin under the acrosomal/perinuclear theca cap has been reported in rhesus monkeys (Ramalho-Santos et al., 2000). Moreover, when it occurred in this species, the apical portions of the sperm chromatin remained condensed even after male pronuclear formation, which could result in delayed DNA replication at the interphase of the first mitosis (Ramalho-Santos et al., 2000). Taken together, it is considered that acrosomes tend to disassemble abnormally after ICSI, which may disturb decondensation of the sperm chromatins, leading to the failure of coordinated pronucleus formation. It has been demonstrated that prior rupture of the sperm plasma membrane allowed access by oocyte decondensing factors to the sperm nucleus following ICSI in humans (Dozortsev et al., 1995). However, the continued presence of an intact acrosome might impede this process in pigs.
Another important finding in the present study was the stage-specific PNA-binding to the tail region. In the case of ICSI, all sperm associated with the fluorescent fringe in the head region had PNA-positive tails, and about half of those associated with the disintegrated acrosome showed PNA-positive tails. Essentially all of the sperm with other types of PNA-binding at the sperm head exhibited PNA-negative tails. Importantly, an examination of sperm after IVF suggested that the detection of PNA in both the head and tail regions was presumably an indication of the acrosome reaction. In mice, for example, ConA binding sites develop in the tail region during capacitation (Koehler, 1981). Sugar chains and/or protein components in the tail region may become PNA-positive after the acrosome reaction, or some acrosomal contents becoming PNA-positive after the acrosome reaction may adhere to the tail region. Therefore, it is possible that the acrosome reaction, or acrosome reaction-like events, occur in ICSI in the sperm showing PNA-positive tails and fluorescent fringes in the head. If so, in the present study almost half of the injected sperm might have undergone acrosome reaction-like events in the ooplasm without any treatment to promote capacitation prior to injection.
In the case of IVF, intense fluorescent masses of PNA were observed on the surface of the zona pellucida. As their shape was often exactly like that of the acrosomal cap, they were most likely either acrosomal ghosts or attachments of sperm (Yoshida et al., 1993). Such fluorescence was not observed after ICSI. Thus, the fluorescence on the zona pellucida was derived from the acrosome, and not from the released CG contents. CG distributions before and after IVF or ICSI observed in the present study were consistent with previous results obtained in pigs (Yoshida et al., 1993; Wang et al., 1997). Since the fusion between the sperm plasma membrane and oolemma is bypassed in ICSI, it is considered that the oocytes may be activated in an unusual manner (Tesarik and Mendoza, 1999). All the oocytes which resumed meiosis by ICSI in this study, however, released CG and the patterns of fluorescence distribution and disappearance were similar to those of fertilized oocytes. Thus, in terms of the resumption of meiosis and CG exocytosis, oocytes can achieve these post-fertilization events after ICSI.
|
Acknowledgements |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
The authors thank Dr Y. Hirao at the Department of Animal Production and Grasslands Farming, National Agricultural Research Center for Tohoku Region, for his instructive advice in this study. They are also grateful to the staff at Koshida Clinic for their generous help, and thank the staff of the Nishiharima Meat Inspection Offices of Hyogo Prefecture and the Meat Inspection Office of Kobe City for supplying porcine ovaries. This study was supported by a Grant-in-Aid from JSPS (No. RFTF97L00905), a Grant-in-Aid for Scientific Research (B-2, No. 09460129) from the Ministry of Education, Science, Sports, and Culture of Japan.
|
Notes |
|---|
3 To whom correspondence should be addressed at: Graduate School of Science and Technology, Kobe University, Rokkodai-cho Nada-Ku, Kobe City, Hyogo, 657-8501, Japan. E-mail: miyake{at}ans.kobe-u.ac.jp
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
Bonduelle, M., Aytoz, A., Assche, E.V., Devroey, P., Liebaers, I. and Van Steirteghem, A.C. (1998) Incidence of chromosomal aberrations in children born after assisted reproduction through intracytoplasmic sperm injection. Hum. Reprod., 13, 781–782.
Bourgain, C., Nagy, Z.P., Zutter, H.D., Ranst, H.V., Nogueira, D. and Van Steirteghem, A.C. (1998) Ultrastructure of gametes after intracytoplasmic sperm injection. Hum. Reprod., 13 (Suppl. 1), 107–116.
Brackett, B.G. and Oliphant, G. (1975) Capacitation of rabbit sperm in vitro. Biol. Reprod., 12, 260–274.[Abstract]
Danan, C., Sternberg, D., Van Steirteghem, A., Cazeneuve, C., Duquesnay, P., Besmond, C., Goossens, M., Lissens, W. and Amselen, S. (1999) Evaluation of parental mitochondrial inheritance in neonates born after intracytoplasmic sperm injection. Am. J. Hum. Genet., 65, 463–473.[Web of Science][Medline]
Dozortsev, D., Rybouchkin, A., De Sutter, P. and Dhont, M. (1995) Sperm plasma membrane damage prior to intracytoplasmic sperm injection: a necessary condition for sperm nucleus decondensation. Hum. Reprod., 10, 2960–2964.
Fazeli, A., Hage, W.J., Cheng, F.P., Voorhout, W.F., Marks, A., Bevers, M.M. and Colenbrander, B. (1997) Acrosome-intact boar sperm initiate binding to the homologous zona pellucida in vitro. Biol. Reprod., 56, 430–438.[Abstract]
Flesch, F.M., Voorhout, W.F., Colenbrander, B., van Golde, L.M. and Gadella, B.M. (1998) Use of lectins to characterize plasma membrane preparations from boar sperm: a novel technique for monitoring membrane purity and quantity. Biol. Reprod., 59, 1530–1539.
Hewitson, L.C., Simerly, C.R., Tengowski, M.W., Sutovsky, P., Navara, C.S., Haavisto, A.J. and Schatten, G. (1996) Microtubule and chromatin configurations during rhesus intracytoplasmic sperm injection: successes and failures. Biol. Reprod., 55, 271–280.[Abstract]
Hiraoka, J. and Hirao, Y. (1988) Fate of sperm tail components after incorporation into the hamster egg. Gamete Res., 19, 369–380.[Web of Science][Medline]
Hoodbhoy, T. and Talbot, P. (1994) Mammalian cortical granules: contents, fate, and function. Mol. Reprod. Dev., 39, 439–448.[Web of Science][Medline]
Imai, H., Niwa, K. and Iritani, A. (1980) Ultrastructural observations of boar sperm penetrating zona-free hamster egg. Biol. Reprod., 22, 481–486.
Iritani, A. (1991) Micromanipulation of gametes for in vitro assisted fertilization. Mol. Reprod. Dev., 28, 199–207.[Web of Science][Medline]
Kim, N.H., Lee, J.W., Jun, S.H., Lee, H.T. and Chung, K.S. (1998) Fertilization of porcine oocytes following intracytoplasmic spermatozoon or isolated sperm head injection. Mol. Reprod. Dev., 51, 436–444.[Web of Science][Medline]
Kimura, Y. and Yanagimachi, R. (1995) Intracytoplasmic sperm injection in the mouse. Biol. Reprod., 52, 709–720.[Abstract]
Kimura, Y., Yanagimachi, R., Kuretake, S., Bortkiewicz, H., Perry, A.C.F. and Yanagimachi, H. (1998) Analysis of mouse oocyte activation suggests the involvement of sperm perinuclear material. Biol. Reprod., 58, 1407–1415.
Koehler, J.K. (1981) Lectins as probes of the spermatozoon surface. Arch. Androl., 6, 197–217.[Web of Science][Medline]
Kure-bayashi, S., Miyake, M., Katayama, M., Miyano, T. and Kato, S. (1996) Development of porcine blastocysts from in vitro-matured and activated haploid and diploid oocytes. Theriogenology, 46, 1027–1036.
Lee, S.H. and Ahuja, K.K. (1987) An investigation using lectins of glycocomponents of mouse sperm during capacitation and sperm-zona binding. J. Reprod. Fertil., 80, 65–74.
Luetjens, C.M., Payne, C. and Schatten, G. (1999) Non-random chromosome positioning in human sperm and sex chromosome anomalies following intracytoplasmic sperm injection. Lancet, 353, 1240.[Web of Science][Medline]
Mortimer, D., Curtis, E.F. and Miller, R.G. (1987) Specific labelling by peanut agglutinin of the outer acrosomal membrane of the human spermatozoon. J. Reprod. Fertil., 81, 127–135.
Palermo, G., Joris, H., Devroey, P. and Van Steirteghem, A.C. (1992) Pregnancies after intracytoplasmic injection of single spermatozoon into an oocytes. Lancet, 340, 17–18.[Web of Science][Medline]
Ramalho-Santos, J., Sutovsky, P., Simerly, C., Oko, R., Wessel, G.M., Hewitson, L. and Schatten, G. (2000) ICSI choreography: fate of sperm structures after monospermic rhesus ICSI and first cell cycle implications. Hum. Reprod., 15, 2610–2620.
Sathananthan, A.H., Szell, A., Ng, S.C., Kausche, A., Lacham-Kalplan, O. and Trounson, A. (1997) Is the acrosome reaction a prerequisite for sperm incorporation after intra-cytoplasmic sperm injection (ICSI)? Reprod. Fertil. Dev., 9, 703–709.[Medline]
Shalgi, R., Magnus, A., Jones, R. and Phillips, D.M. (1994) Fate of sperm organelles during early embryogenesis in the rat. Mol. Reprod. Dev., 37, 264–271.[Web of Science][Medline]
Sutovsky, P., Navara, C.S. and Schatten, G. (1996) Fate of the sperm mitochondria, and the incorporation, conversion, and disassembly of the sperm tail structures during bovine fertilization. Biol. Reprod., 55, 1195–1205.[Abstract]
Sutovsky, P., Moreno, R.D., Ramalho-Santos, J., Dominko. T., Simerly, C. and Schatten, G. (2000) Ubiquitinated sperm mitochondria, selective proteolysis, and the regulation of mitochondrial inheritance in mammalian embryos. Biol. Reprod., 63, 582–590.
Szollosi, D. (1965) The fate of sperm middle piece mitochondria in the rat egg. J. Exp. Zool., 159, 367–377.[Web of Science][Medline]
Szollosi, D. and Hunter, R.H.F. (1973) Ultrastructural aspects of fertilization in the domestic pig: sperm penetration and pronucleus formation. J. Anat., 116, 181–206.[Web of Science][Medline]
Tesarik, J. and Mendoza, C. (1999) In vitro fertilization by intracytoplasmic sperm injection. BioEssays, 21, 791–801.[Web of Science][Medline]
Van Steirteghem, A.C., Nagy, Z., Joris, H., Liu, J., Staessen, C., Smitz, J., Wisanto, A. and Devroey, P. (1993) High fertilization and implantation rates after intracytoplasmic sperm injection. Hum. Reprod., 8, 1061–1066
Vazquez, J.M., Martinez, E., Roca, J., Coy, P. and Pastor, L.M. (1993) Acrosome reaction of boar sperm in homologous in vitro fertilization. Mol. Reprod. Dev., 36, 84–88.[Web of Science][Medline]
Wang, W.H., Sun, Q.Y., Hosoe, M., Shioya, Y. and Day, B.N. (1997) Quantified analysis of cortical granule distribution and exocytosis of porcine oocytes during meiotic maturation and activation. Biol. Reprod., 56, 1376–1382.[Abstract]
Yanagimachi, R. (1988) Mammalian fertilization. In: Knobil, E. and Neill, D. (eds), The Physiology of Reproduction. Raven Press, New York, pp. 135–172.
Yoshida, M., Cran, D.G. and Pursel, V.G. (1993) Confocal and fluorescence microscopic study using lectins of the distribution of cortical granules during the maturation and fertilization of pig oocytes. Mol. Reprod. Dev., 36, 462–468.[Web of Science][Medline]
Submitted on March 22, 2002; resubmitted on June 5, 2002; accepted on June 14, 2002.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  H. Tateno and Y. Kamiguchi Evaluation of Chromosomal Risk Following Intracytoplasmic Sperm Injection in the Mouse Biol Reprod, August 1, 2007; 77(2): 336 - 342. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Hao, N. Mathialagan, E. Walters, J. Mao, L. Lai, D. Becker, W. Li, J. Critser, and R. S. Prather Osteopontin Reduces Polyspermy During In Vitro Fertilization of Porcine Oocytes Biol Reprod, November 1, 2006; 75(5): 726 - 733. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Ajduk, Y. Yamauchi, and M. A Ward Sperm Chromatin Remodeling after Intracytoplasmic Sperm Injection Differs from That of In Vitro Fertilization Biol Reprod, September 1, 2006; 75(3): 442 - 451. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Nakai, N. Kashiwazaki, A. Takizawa, N. Maedomari, M. Ozawa, J. Noguchi, H. Kaneko, M. Shino, and K. Kikuchi Morphologic changes in boar sperm nuclei with reduced disulfide bonds in electrostimulated porcine oocytes. Reproduction, March 1, 2006; 131(3): 603 - 611. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Garcia-Rosello, C. Matas, S. Canovas, P. N. Moreira, J. Gadea, and P. Coy Influence of Sperm Pretreatment on the Efficiency of Intracytoplasmic Sperm Injection in Pigs J Androl, March 1, 2006; 27(2): 268 - 275. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Funahashi Effect of beta-mercaptoethanol during in vitro fertilization procedures on sperm penetration into porcine oocytes and the early development in vitro Reproduction, December 1, 2005; 130(6): 889 - 898. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Katayama, P. Sutovsky, B. S Yang, T. Cantley, A. Rieke, R. Farwell, R. Oko, and B. N Day Increased disruption of sperm plasma membrane at sperm immobilization promotes dissociation of perinuclear theca from sperm chromatin after intracytoplasmic sperm injection in pigs Reproduction, December 1, 2005; 130(6): 907 - 916. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Funahashi and R. Romar Reduction of the incidence of polyspermic penetration into porcine oocytes by pretreatment of fresh spermatozoa with adenosine and a transient co-incubation of the gametes with caffeine Reproduction, December 1, 2004; 128(6): 789 - 800. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||