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1 Division of Animal Science, 2 Departments of Obstetrics & Gynecology, University of Missouri-Columbia, 920 East Campus Drive, Columbia, MO 65211, USA, 3 National Livestock Research Institute, Korea and 4 Department of Anatomy and Cell Biology, Queens University, Kingston, Ontario K7L 3N6, Canada
Correspondence should be addressed to B N Day; Email: dayb{at}missouri.edu
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Abstract |
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Introduction |
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Many studies associated with in vitro production of pigs have been conducted. However, the progress towards efficient in vitro maturation and in vitro fertilization (IVF) has been limited by a high incidence of polyspermy (Szöllösi & Hunter 1973, Niwa 1993, Funahashi & Day 1996, Day 2000). Application of ICSI in pigs may provide a useful method for in vitro production of monospermic zygotes for embryo transfer. However, fertilization rates after ICSI vary among the reported studies and litter size after embryo transfer is reduced in pigs (Kim et al. 1999, Kolbe & Holtz 1999, 2000, Martin 2000, Lai et al. 2001, Nakai et al. 2003, Probst & Rath 2003, Kwon et al. 2004, Yong et al. 2003). The ICSI technique remains to be improved and established for the practical production of piglets.
One of the problems in pig ICSI is the failure or delay of decondensation of sperm chromatins, resulting in a low rate of male pronuclear formation (Kren et al. 2003, Lee et al. 2003). In the fertilization process, spermatozoa enter the ooplasm by sperm plasma membrane fusion with oolemma (Szöllösi & Hunter 1973, Yanagimachi 1994, Wassarman 1999, Sutovsky & Schatten 2000). Most of the sperm plasma membrane and perinuclear theca are lost during the incorporation process and sperm chromatin, especially in the postacrosomal region, is exposed to ooplasm soon after the entry into oocytes (Szöllösi and Hunter 1973, Yanagimachi 1994, Sutovsky & Schatten 2000). In the ooplasm, factors supporting chromatin decondensation, such as reduced glutathione and nucleoplasmin, interact with sperm chromatin and induce remodeling of sperm chromatin to form a male pronucleus (Perreault et al. 1984, Yanagimachi 1994, Collas and Poccia 1998, McLay & Clark 2003). On the other hand, with ICSI a spermatozoon is introduced directly into ooplasm, bypassing the penetration and demembranation process. The ICSI is performed practically in humans using spermatozoa without any pretreatments, except sperm immobilization (Palermo et al. 1992). In domestic animals, in which oocytes are usually prepared by in vitro maturation, fertilization rates and developmental ability of embryos are low after direct injection of a spermatozoon immobilized in the same manner as in humans. Actually, the retention of acrosome or subacrosomal perinuclear theca seems to prevent decondensation of sperm chromatin in ooplasm after ICSI in rhesus monkeys (Sutovsky et al. 1996, Hewitson et al. 1999), cattle (Sutovsky et al. 1997) and pigs (Katayama et al. 2002a).
Usually spermatozoa are immobilized by rubbing the sperm tail midpiece with a micropipette before injection. In humans, it is believed that the sperm plasma membrane is damaged sufficiently by rubbing the tail region during the immobilization process to cause male pronuclear formation after ICSI (Dozortsev et al. 1995), but probably such a treatment is not sufficient in porcine oocytes matured in vitro. Yanagida et al.(2001) suggested that sperm immobilization with piezo pulses would cause more severe membrane damage on human spermatozoa than rubbing with a micropipette and showed that calcium oscillations, a hallmark of oocyte activation by the factors released from the sperm perinuclear theca, start more rapidly in oocytes injected with a piezo-immobilized spermatozoon than in those injected with a rubbing-immobilized method. We considered that application of piezo pulses for sperm immobilization would also improve fertilization rates after ICSI in pigs without any additional treatments for artificial activation of oocytes.
In the present study, the same piezo pulses as those used for micropipette penetration of oolemma and zona pellucida during the ICSI process were applied for sperm immobilization. The effects of immobilization methods on male pronuclear formation, decondensation of sperm chromatin and retention of subacrosomal sperm perinuclear theca (SAR-PT) after ICSI were examined.
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Materials and methods |
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Preparation of boar spermatozoa
Freshly ejaculated sperm rich fraction was collected from fertile boars (one-half Large white x one-quarter Duloc x one-quarter Pietrain) and frozen as described previously (Abeydeera & Day 1997). A sperm pellet was thawed in 2 ml Dulbecco’s PBS (calcium- and magnesium-free) supplemented with 0.1% (w/v) PVA (PBS-PVA) at 39°C and centrifuged at 600 g on two layers (80 and 60%) of Percoll (Amersham Biosciences AB, Uppsala, Sweden) for 10 min. Spermatozoa in the resultant pellet were centrifuged once with 5 ml PBS-PVA at 1900 g for 4 min. The spermatozoa were then resuspended in modified Tris-buffered medium (mTBM; Abeydeera & Day 1997) at a sperm concentration of 1 x 106 cells/ml for IVF and (1–10) x 106 cells/ml for ICSI, and cultured at 39°C for 30 min in atmosphere of 5% CO2 in humidified air until used for IVF, ICSI or eosin Y staining.
IVF
After the completion of maturation of oocytes cultured for 42 h, cumulus cells were removed with 0.1% (w/v) hyalronidase and washed three times with mTBM. Oocytes showing clear cytoplasmic cortex and plasma membrane with a polar body were selected for IVF. After washing with mTBM, 20–30 oocytes were placed in 50 µl drops of the same medium that was covered with mineral oil in a 35 mm falcon dish. Then, a 50 µl drop of sperm suspended mTBM was added to the 50 µl drop containing oocytes and the mixture gave a final sperm concentration at 5 x 105 cells/ml. Oocytes were coincubated with spermatozoa for 6 h at 39°C in atmosphere of 5% CO2 in humidified air. The oocytes were then transferred to NCSU23 (Petters & Wells 1993) and cultured for 5–6 h at 39°C in atmosphere of 5% CO2 in humidified air.
Sperm immobilization and eosin Y staining
The influence of immobilization methods on the sperm plasma membrane integrity was assessed by eosin Y staining. Spermatozoa were cultured in mTBM for 30–90 min at 39°C in atmosphere of 5% CO2 in 95% humidified air, and 3.2 mmol·l–1 progesterone (Sigma) was added to the sperm suspension to induce acrosome reaction (Katayama et al. 2002b). For manipulation of spermatozoa, microdrops of 6 µl of 10% (w/v) polyvinylpyrrolidone (PVP; Sigma) and 1% (w/v) eosin Y solution in mTBM were placed on the inner side of the lid of the dish and covered with mineral oil. On the stage of an inverted phase-contrast microscope (Zeiss, Oberkochen, Germany) equipped with micromanipulators (Eppendorf, Hamburg, Germany) and a piezo drive unit (Prime Tech, Tsukuba, Japan), 2 µl of the sperm suspension were mixed well with a 6 µl PVP drop and sperm immobilization was performed there. Using a flat-tipped micropipette with an outer diameter of 8 µm prepared with an ultrathin glass capillary (Narishige, Tokyo, Japan), motile spermatozoa were immobilized by rubbing at the midpiece region of the tail and head region (R), by rubbing at the same region while giving a low intensity (at level 2 or 3 at speed 2) of piezo pulses used for penetration of oolemma (L) or by rubbing at the same region while giving a high intensity (at level of 6–8 at speed 5) of piezo pulses used for penetration of the zona pellucida (H). After each immobilization method, some immobilized spermatozoa were aspirated into the micro-pipette and piezo L and H pulses were applied to the spermatozoa located in the micropipette in the same manner as to when the micropipette penetrated into zona pellucida and oolemma during the ICSI process (+shake). This was done to determine the effect of a set of piezo pulses applied at the zona and oolemma penetration on damage of sperm plasma membrane. Immobilized spermatozoa were then immediately transferred to the drop of 1% eosin Y. As the center of the eosin Y drop had a strong pink color due to the thickness of the drop, a spermatozoon was placed close to the edge of the drop to observe changes in the color of the head region. The time required for staining of the entire sperm head region was recorded.
ICSI driven with piezo pulses
After the completion of maturation culture for 42 h, cumulus cells were removed from oocytes as described above and oocytes showing an acceptable morphology with a polar body were selected for ICSI. For the manipulation, microdrops of 6 µl 10% PVP and 20 µl HEPES-buffered NCSU23 with adjusted osmolarity (HEPES-NCSU23) were put on the inner side of the lid of the dish and covered with mineral oil. ICSI was performed using the Eppendorf Cell Tram microinjection system equipped with a Prime Tech piezo drill. After sperm immobilization by one of the three different methods described above, a spermatozoon was aspirated into a micropipette and transferred to the microdrop of HEPES-NCSU23. There an oocyte was held by a holding pipette so that the first polar body was located at 12 or 6 o’clock. The micropipette was driven from the 3 o’clock area with a piezo-pulse force at intensity 6–8 and speed 5 to penetrate the zona pellucida, and the spermatozoon was then pushed to the tip of the micropipette when the tip of the pipette advanced into the perivitelline space. The micropipette was carefully placed against the oocyte without breaching the oolemma and piezo pulses at the intensity 2 or 3 and speed 2 were given to the tensed oolemma through the micropipette. The oolemma was then released from tension at the same time as the pipette penetrated the oolemma. To confirm the penetration of the pipette into the ooplasm, a small amount of ooplasm was aspirated. The micropipette was inserted more deeply to the 9 o’clock area of the oocyte, and then the spermatozoon was released into the oocyte with small amount of ooplasm and PVP solution.
Examination of pronuclear formation after IVF and ICSI
After sperm injection, oocytes were washed twice with NCSU23 and cultured in a 10 µl drop of the same medium for 10–12 h at 39°C in an atmosphere of 5% CO2 in humidified air. Presumed zygotes were mounted on a slide and fixed for 72 h in 25% (v/v) acetic acid in ethanol at room temperature. After being stained with 1% (w/v) orcein in 45% (v/v) acetic acid, presumed zygotes were observed under a phase-contrast microscope at a magnification of x 400. Oocytes released from arrest at the MII stage were recorded as activated oocytes.
Simultaneous detection of SAR-PT, nuclear pore complexes (NPCs) and DNA after IVF and ICSI
At 4–10 h after sperm insemination (IVF), or at 10 h after injection of spermatozoa immobilized by R, L or H (ICSI), oocytes were treated as described previously (Sutovsky et al. 1997) to examine the configurations of sperm peri-nuclear theca, NPCs and DNA. Briefly, the zona pellucida was removed by 0.5% (w/v) protease and oocytes were fixed with 2% (v/v) formaldehyde for 40 min. Zona-free, fixed oocytes were treated in PBS supplemented with 5% normal goat serum and 0.1% Triton X-100 for 30 min at room temperature. To label NPCs and SAR-PT, a mouse monoclonal antibody of mAB414 (BabCo/Covance, Berkeley, CA, USA) and a rabbit antibody against SAR-PT/inner acrosomal membrane protein IAM32 (Oko & Maravei 1994; kindly provided by R O) were diluted at 1/200 and 1/20, respectively in PBS supplemented with 1% (v/v) normal goat serum and 0.1% (v/v) Triton X-100 (labeling medium) and the oocytes were incubated for 1 h at room temperature in the medium. Then, the oocytes were washed and incubated in labeling medium with a 1/80 (v/v) dilution of fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG, a 1/80 (v/v) dilution of tetramethylrhodamine ßisothiocyanate (TRITC)-conjugated anti-rabbit IgG and 0.71 µmol·l–1 DAPI for 40 min. Labeled oocytes were mounted on glass slides with VectaShield medium (Vector Laboratories, Burlingame, CA, USA) and observed under a Nikon Eclipse 800 epifluorescent microscope (Nikon, Tokyo, Japan) equipped with a CoolSnap CCD HQ camera (Roper Scientific, Tucson, AZ, USA) and MetaMorph image-acquisition software (Universal Imaging Corp., Downington, PA, USA). Figure plates were edited using Adobe Photoshop 5.0.
Statistical analyses
The data obtained from observation of oocytes stained with aceto-orcein were pooled from five replicates. Values in each replicate were analyzed using one-way analysis of variance (ANOVA). Significance of differences was assessed by Student’s t test. The time required for staining a sperm head after each immobilization method was analyzed using one-way ANOVA and significance of differences was assessed by t-test. The data from immunocytochemical study were pooled from three replications, and any significant differences in the values were determined by a 
2 test.
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). Sperm immobilization by treatment H significantly (P < 0.05) reduced the time required for complete staining compared with treatments R and L (Table 1).
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Application of piezo pulses of L or H for sperm immobilization significantly (P < 0.05) increased rates of oocyte activation, fertilization and male pronuclear formation compared with R, and there were no differences between L and H (Table 2).
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). Detailed observations of SAR-PT revealed the remnants of SAR-PT signal around the postacrosomal segment of the sperm head and near the implantation fossa (arrowheads in Fig. 1C and D) prior to sperm chromatin decondensation. SAR-PT signals were last detected in anterior part of sperm heads concomitantly with the onset of chromatin decondensation in the postacrosomal region (Fig. 1F). In this case, remnants of SAR-PT were observed around the anterior part of sperm heads when NPCs started to be assembled around sperm chromatin (Fig. 1G and H). No signal of SAR-PT was observed around male pronuclei whereas SAR-PT was detected on the sperm heads that were still being decondensed or did not decondense in polyspermic oocytes (Fig. 1I).
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). In oocyte cytoplasm, SAR-PT was either (1) positioned close to sperm chromatin on apical surface of the sperm nucleus (associated; Fig. 2A–D), (2) present at the equatorial segment in sperm heads (equatorial; Fig. 2E), in which case sperm heads were kept almost intact, (3) detached from sperm chromatin (dissociated; Fig. 2F and G) or (4) not present at all (missing; Fig. 2H). The immunodetection of SAR-PT closely adjacent to sperm chromatin coincided with sperm heads/male pronuclei containing an area of condensed chromatin under SAR-PT (Fig. 2B) or an atypical, small male pronucleus (Fig. 2C). The H immobilization significantly increased the percentage of oocytes showing SAR-PT dissociated from sperm chromatin (Table 3) and reduced the percentage of male pronuclei with the area of condensed chromatin to 35% (7/20), which was significantly lower (P < 0.05) than those in R and L (87% (13/15) and 60% (9/15), respectively). Also, H immobilization decreased the incidence of oocytes that had spermatozoa labeled at the equatorial segment (Table 3).
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), but most of male pronuclei formed after ICSI contained NPCs (80% (12/15), 80% (12/15) and 95% (19/20) of male pronuclei in the R, L and H groups, respectively), even when the male pronucleus had an area of condensed chromatin (Fig. 2B). |
Discussion |
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Even though the injection of an intact spermatozoon permits sperm head decondensation in humans, non-human primates and rodents, the decondensation of sperm chromatin does not occur in cattle after injection of intact bull spermatozoa, possibly due to the higher rigidity of their perinuclear theca (Sutovsky et al. 1997). The perinuclear theca of sperm heads is composed of three parts: the subacrosomal region (SAR-PT), the outer layer at the equatorial segment and the postacrosomal region. Ultrastructural studies show that the perinuclear theca at postacrosomal region becomes solubilized and disappears soon after sperm entry into oocytes (Yanagimachi 1994, Sutovsky & Schatten 2000, Sutovsky et al. 2003). On the other hand, SAR-PT remains around sperm chromatin temporarily after IVF (Sutovsky & Schatten 2000, Sutovsky et al. 2003). In rhesus monkeys, a male pronucleus is formed after ICSI but the area of condensed chromatin in male pronculei is observed in the anterior part of the sperm nucleus because SAR-PT is retained there and remains closely associated with male chromatin for several hours (Sutovsky et al. 1996, Hewitson et al. 2000). Heterogeneous decondensation of male chromatin could be considered as one of reasons for the delay of the initiation of DNA synthesis prior to the first mitosis and low developmental capability of embryos derived from ICSI in rhesus monkeys (Hewitson et al. 1999). These studies suggest that disassembly of SAR-PT is one of the key requirements for successful fertilization after ICSI. Our immunocytochemical study with the antibody against SAR-PT revealed that H immobilization promoted the dissociation of SAR-PT from sperm chromatin, which also decreased the frequency of male pronuclei containing an area of condensed chromatin. Recently it was shown that disruption of sperm tail midpiece in human sperm caused mechanical damage along the plasma membrane to the postacrosomal perinuclear theca and the outer acrosomal membrane (Takeuchi et al. 2004). There are several studies to have suggested the beneficial effects of membrane damage of spermatozoa on fertilization in humans (Palermo et al. 1992, Swann et al. 1994, Dozortsev et al. 1994, 1995, Tesarik & Mendoza 1999, Yanagida et al. 2001). The present results indicate that a similar mechanism of membrane damage at sperm immobilization improves fertilization and sperm chromatin decondensation after ICSI in pigs.
The removal and disassembly of SAR-PT has been studied during bovine IVF and suggests that the interaction of SAR-PT with microvilli on the oolemma at sperm–oocyte fusion is necessary for the removal of SAR-PT from sperm chromatin at early stages of fertilization (Sutovsky et al. 1997 Sutovsky et al. 2003). Disassembly and/or solubilization of SAR-PT after IVF in pigs was also observed in the cortical area of ooplasm in the present study. The interaction of SAR-PT with microvilli and sperm–oocyte fusion are bypassed in the ICSI procedure where spermatozoa are injected directly into the ooplasm after sperm immobilization. It is considered that some spermatozoa were kept intact in the ooplasm although spermatozoa were damaged by immobilization before injection. As the increased damage on the sperm plasma membrane by application of piezo high pulses decreased the frequency of spermatozoa with a SAR-PT signal at the equatorial segment and promoted the dissociation of SAR-PT from sperm chromatin, the direct interaction of SAR-PT with the ooplasm after breakdown of sperm plasma membrane might be involved in the dissociation process in ICSI oocytes.
The IVF results in the present study showed that SAR-PT was disassembled as it was distributed in the area in which sperm chromatin became decondensed and gradually disappeared from the anterior area of enlarged sperm heads or early male pronuclei. In contrast, most of oocytes after ICSI had detectable remnants of SAR-PT as late as the pronuclear apposition stage of zygotic development. The signal of SAR-PT observed in association with male pronuclei at apposition was relatively strong and appeared to be just separated from sperm chromatins in ICSI oocytes. It is considered that the removal of SAR-PT after ICSI was delayed and/or its mechanism was not the same as in IVF oocytes, where SAR-PT disassembled coordinately with the sperm incorporation into ooplasm and decondensation of sperm chromatin. Acrosome reaction and interaction of SAR-PT with microvilli, which are bypassed in the ICSI procedure, may cause the different behavior of SAR-PT after IVF.
Recently it has been reported that SAR-PT contains unique proteins, such as a variant of histone protein SubH2Bv (Aul & Oko 2002) and core somatic histones (Tovich & Oko 2001), which implies an involvement of perinuclear theca or its proteins in fertilization and zygotic development. Although little is known about the role of SAR-PT, disassembly of SAR-PT coordinated with decondensation of sperm chromatin after IVF as observed in the present study also suggest a contribution of sperm SAR-PT to an early stage of the fertilization process. If so, the altered characteristics of male pronuclei formed after ICSI as compared with those after IVF may be due to the lack of SAR-PT association with sperm chromatin during decondensation.
An IVF study in cattle showed that the demembranation of the sperm chromatin soon after sperm penetration into ooplasm is almost immediately followed by the de novo assembly of pronuclear envelope and
NPCs. The assembly of NPCs from recruited, ooplasmic NPC proteins is required for normal development of pronuclei (Sutovsky et al. 1998, Payne et al. 2003). The failure of male pronucleus to attract nucleoporins from the cytoplasm of an activated oocyte was observed after ICSI in rhesus monkeys (Sutovsky et al. 1996). In the present study, some male pronuclei were not decorated with NPCs whereas female pronuclei in the corresponding ICSI ova did contain NPCs at 10 h after ICSI. The sperm immobilization methods did not seem to affect the association of NPCs with male pronucleus. Even when male pronuclei had an area of condensed chromatin, NPCs were observed around the male pronucleus in the activated ova with a female pronucleus. As almost all pronuclei formed at 10 h after IVF in this study were surrounded with NPCs, the lack of such a pattern could be considered an abnormality after ICSI. The nucleocytoplasmic transport through NPCs is required for many cellular activities, such as DNA replication (Hanover 1992, Harel et al. 2003). Thus, the formation of male pronuclei without NPC association may cause failure or delay of the first cleavage of zygotes after ICSI.
In conclusion, application of piezo pulses for sperm immobilization increased sperm membrane damage and improved fertilization rates following ICSI compared with simple sperm rubbing during the piezo-driven ICSI procedure in pigs. Increased membrane damage caused by high-intensity piezo pulses promoted dissociation of perinuclear theca of spermatozoa which allowed uniform decondensation of chromatin in male pronuclei. Also, the behavior of SAR-PT after ICSI deviated from that observed in oocytes after IVF by the virtue of prolonged SAR-PT association with the male pronuclei.
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Footnotes |
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References |
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