Fertilization and embryo development in a mouse ICSI model using human and mouse sperm after immobilization in polyvinylpyrrolidone

Kaoruko Mizuno1,2,3, Kazuhiko Hoshi2 and Thomas Huang1

1 Kapiolani Medical Center, Department of Obstetrics and Gynecology, University of Hawaii School of Medicine, Honolulu, HI 96826, USA and 2 Department of Obstetrics and Gynecology, Yamanashi Medical University, Shimokato 1110, Tamaho, Nakakoma, Yamanashi, 409-3898, Japan


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: For human ICSI, sperm are normally immobilized immediately prior to injection. However, there are some situations when only sperm of questionable viability are available. There are few evaluations of fertilization or developmental problems in human or animal models using sperm having known intervals between immobilization and injection. METHODS: Immobilized human sperm were maintained for 1–24 h in 10% polyvinylpyrrolidone (PVP) before injection into mouse oocytes. Mouse sperm heads were similarly maintained in either PVP or a high potassium-containing `nucleus isolation medium' (NIM) before ICSI and embryo development to the blastocyst stage. RESULTS: Immobilized human sperm activated mouse oocytes comparably to controls even 24 h after immobilization. However, mouse sperm heads showed a decrease in activating ability 6 h after isolation, either in PVP or NIM. A significant reduction in blastocyst development occurred if mouse sperm heads were maintained for even 1 h in PVP. After 6 h, no blastocysts formed, with arrest occurring at the morula stage. NIM provided partial protection for up to 3 h. CONCLUSIONS: Immobilized human sperm maintained oocyte activating activity for 24 h. However, mouse sperm are susceptible to alterations that affect both fertilization and development.

Key words: embryo development/fertilization/ICSI/PVP/sperm immobilization


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The development of ICSI has revolutionized the treatment of male factor infertility (Palermo et al., 1992Go). The success rates using ICSI are increased by sperm immobilization immediately before injection (Bergh et al., 1995Go; Catt and O'Neill, 1995Go; Fishel et al., 1995Go; Gerris et al., 1995Go; Palermo et al., 1996Go). Sperm immobilization prior to ICSI can be accomplished by compressing the sperm tail with the injection pipette (Hoshi et al., 1995Go; Palermo et al., 1996Go), freezing (Goto et al., 1990Go; Goto, 1993Go; Rybouchkin et al., 1996Go), sonication (Martin et al., 1988Go; Kuretake et al., 1996Go; Tateno et al., 2000Go), detergent treatment (Kuretake et al., 1996Go; Ahmadi and Ng, 1997Go, 1999Go), and piezo-actuated pulses (Kimura and Yanagimachi, 1995Go; Yanagida et al., 1998Go). Plasma membrane disruption after immobilization is thought to facilitate the release of sperm-associated oocyte-activating factors (SOAF) (Dozortsev et al., 1997Go) once the sperm is inside the oocyte.

Clinically, problems relating to the use of immotile sperm remain unclear since their membranes may have degraded prior to injection. This situation can arise with severely asthenospermic specimens, with testicular biopsies from non-obstructive azoospermic patients, with frozen–thawed sperm, or in cases where sperm are held in polyvinylpyrrolidone (PVP) for logistical reasons before use. It was recently reported that mouse and human sperm immobilized by sonication, and kept for up to 2 h in culture, showed an increase in structural chromosome aberrations (Tateno et al., 2000Go). They concluded that prolonged exposure of sperm nuclei to culture medium, rather than sonication, caused the aberrations. They further showed that use of a K+-rich nuclear isolation medium (NIM) partially protected against chromosome damage (Suzuki et al., 1998Go; Tateno et al., 2000Go). Sperm disruption in isotonic NaCl for 2 h after sonication also impaired normal embryo development (Kuretake et al., 1996Go), consistent with a genetic cause for developmental damage. PVP is thought to have a sperm membrane-stabilizing effect, even at low concentrations (Dozortsev et al., 1995Go). In vitro, this may help to protect the sperm from degradative damage. Therefore, the purpose of the present study was to systematically evaluate the effects of immobilizing sperm for defined intervals in PVP or NIM buffers on oocyte activation and development to the blastocyst stage.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Reagents
All inorganic and organic reagents were purchased from Sigma Chemical Co. (St Louis, MO, USA) unless otherwise stated.

Media
The medium for the culture of mouse oocytes was Chatot–Tasca–Ziomek (CZB) medium supplemented with 5.56 mmol/l D-glucose and 5 mg/ml bovine serum albumin (BSA) (Chatot et al., 1989Go, 1990Go). The medium for collection of oocytes from oviducts and for micromanipulation was a modified CZB (HEPES–CZB) containing 20 mmol/l HEPES, 5 mmol/l NaHCO3 and 0.1 mg/ml polyvinyl alcohol (PVA, cold water soluble) instead of BSA. The pH value of both media was 7.4 under 5% CO2 in air (37°C) or room air respectively.

The medium used for isolation of sperm heads (nuclei) was modified nucleus isolation medium (NIM) (Kuretake et al., 1996Go). This consisted of 123.0 mmol/l KCl, 2.6 mmol/l NaCl, 7.8 mmol/l NaH2PO4, 1.4 mmol/l KH2PO4 and 3 mmol/l EDTA (disodium salt). We used phenylmethylsulphonyl fluoride (protease inhibitor)-free NIM. Its pH value was adjusted to 7.2 by addition of a small quantity of 1 mol/l KOH.

Preparation of mouse oocytes
B6D2F1 female mice, 8–12 weeks old, underwent ovulation induction by i.p. injection of 5 IU pregnant mare's serum gonadotrophin followed by 5 IU hCG 48 h later. Oocytes were collected from oviducts ~14 h after hCG injection. The oocytes were freed from cumulus cells by exposure to 0.1% hyaluronidase in HEPES–CZB for a few minutes. They were rinsed and incubated in CZB for up to 4 h at 37°C under 5% CO2 in air before sperm injection.

Preparation of human and mouse sperm
Human semen samples were obtained from fertile volunteers. The semen parameters were normal by published standards (World Health Organization, 1992Go). Semen samples were allowed to liquefy for 1h, mixed with 10 ml of CZB, and centrifuged at 500 g for 5 min. The sperm pellet was resuspended in 0.5 ml of CZB, then layered beneath 1 ml of CZB for 1 h at 37°C. Motile swim-up sperm were retrieved and transferred to another tube.

The cauda epididymides of a B6D2F1 male mouse were cut with a pair of scissors, and a dense sperm mass squeezed out into a 5 ml tube containing 1.5 ml of CZB. After incubation at 37°C under 5% CO2 in air for ~1 h, the upper portion of the medium was collected and examined. Over 80% of sperm displayed high motility.

Immobilization of sperm and ICSI
A small amount of human sperm suspension was suspended in 5 µl HEPES–CZB containing a 10% PVP (mol. wt 360 000) (CZB–PVP). Human sperm were immobilized by drawing a single motile spermatozoon, tail first, midway into the injection pipette. At that point, several piezo-pulses were applied to the tail until the spermatozoon became completely immotile (Kimura and Yanagimachi, 1995Go). For the mouse, a small amount of epididymal sperm suspension was first suspended in drops of CZB–PVP and a single spermatozoon was drawn, tail first, into the injection pipette just to the head–midpiece junction. Application of a few piezo-pulses separated the head from the tail (Kuretake et al., 1996Go).

Control human immobilized sperm were injected immediately into mouse oocytes as previously described (Kimura and Yanagimachi, 1995Go). Immobilized human sperm in experimental groups were transferred into another drop of CZB–PVP and kept for 1–24 h at room temperature before injection. Similarly, isolated mouse heads in experimental groups were transferred into either CZB–PVP or PVP-free NIM buffer and maintained there for 1–24 h at room temperature before injection. Sperm incubation intervals in all cases were staggered so that all retrieved fresh mouse oocytes were injected within a 2–4 h time interval.

Examination of oocytes and embryos
After injection with human sperm or mouse sperm heads, mouse oocytes were incubated in CZB for 6–8 h and examined with an Olympus IM-6 microscope with Hoffman differential interference optics for evidence of activation. Two types of activation were scored. First, a `normal' group exhibited two pronuclei and a second polar body (2PN + PB2). Second, a `total' number of activated oocytes was tallied by including oocytes exhibiting either 1PN + PB2 or 3PN without PB2.

Oocytes fertilized with mouse sperm heads were cultured in CZB medium. Embryos were examined at 24 h intervals for up to 120 h to evaluate development in vitro to the blastocyst stage.

Statistical analysis
Results were compared using the {chi}2-test with the Yate's correction. Differences at P < 0.05 were considered significant.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Activation by human sperm
Table IGo summarizes activation of mouse oocytes by human sperm after varying maintenance intervals between immobilization and ICSI. When scoring normally activated oocytes (2PN + PB2), there were no differences between controls (84%) and sperm maintained for any interval tested up to 24 h (88%).


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Table I. Oocyte-activating ability of human sperm with increasing time after immobilization and storage in Chatot–Tasca–Ziomek medium (CZB)–polyvinylpyrrolidone (PVP)*
 
Activation and development by mouse sperm heads
Compared with the human sperm, isolated mouse sperm heads showed a slight decrease in activation rate over time. Controls showed a 98% activation rate (Table IIGo). After maintenance in CZB–PVP, a significant decline in total activation rate was seen after 6 h (79%, P < 0.005) and 24 h (82%, P < 0.005) in CZB–PVP. Maintenance in NIM buffer did not show a detectable deterioration after 6 h.


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Table II. Comparison of oocyte activation and embryo development using mouse sperm heads stored in C-Z-B medium (CZB)-polyvinylpyrrolidone or nucleus isolation medium (NIM) with increasing time intervals prior to ICSI*
 
The rate of development to blastocyst was more sensitive than activation as an indicator of potential biological damage following immobilization (Table IIGo). In controls where injection immediately followed mouse sperm head isolation, 75% successfully developed to the blastocyst stage. However, this rate declined significantly even after a 1 h maintenance interval in CZB–PVP (39%, P < 0.005). This decline continued with time, and no blastocysts developed if the sperm heads were maintained for 6 h prior to injection. Maintaining the sperm heads in NIM buffer provided partial protection from developmental damage over a 1–2 h time interval. A significant decline in blastocyst development could be seen at 3 h (31%, P < 0.005), and by 24 h, only 3% of activated oocytes developed to the blastocyst stage.

The effects of maintaining mouse sperm in CZB–PVP buffer or NIM on developmental arrest are shown in Figures 1 and 2GoGo respectively. In CZB–PVP there was a time-dependent decline in blastocyst development beginning with the 1 h time interval, where the majority of embryos arrested at the morula stage. This pattern of arrest was similar during 4 h. However, developmental arrest occurred even earlier than the 4-cell stage when utilizing sperm maintained for 6 or 24 h prior to injection. Figure 2Go shows that NIM partially protected embryos from arrest only for 1–2 h. By 3 h, arrest at the morula stage was similar to that seen in the CZB–PVP group.



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Figure 1. In-vitro development of mouse oocytes fertilized by injection of mouse sperm heads stored in Chatot–Tasca–Ziomek medium (CZB)–polyvinylpyrrolidone.

 


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Figure 2. In-vitro development of mouse oocytes fertilized by injection of mouse sperm heads stored in nucleus isolation medium (NIM).

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The fertilizing mammalian spermatozoon becomes immobilized the moment it fuses with the oolemma after passing through the zona pellucida (Yanagimachi, 1981Go). Under typical ICSI conditions, the spermatozoon is immobilized just prior to injection, which results in optimal fertilization rates (Palermo et al., 1995Go, 1996Go). However, there are circumstances where only immotile sperm (fresh or frozen) are available and the integrity of individual sperm membranes is unknown. Additionally, there may be circumstances requiring some holding time of a spermatozoon between its capture and collection from the male reproductive tract and its injection. The fertilization or developmental risks of such time intervals have not been evaluated. Data presented here utilize an experimental mouse ICSI model to approach this question using fertilization and preimplantation development as endpoints.

Results indicate that fertilization rates are relatively unaffected with increasing time intervals from immobilization to injection using either entire human or detached mouse sperm heads. There were no significant differences in fertilization rates using human sperm over any interval tested up to 24 h after immobilization in CZB–PVP medium (Table IGo). SOAF are not very species-specific, which allows mouse oocytes to be activated by human sperm (Rybouchkin et al., 1995Go). SOAF appears to be localized within the perinuclear theca of the sperm head (Kimura et al., 1998Go; Perry et al., 1999Go). While there was a reduction in oocyte activation using isolated mouse sperm heads after a 6 h maintenance period, the decline over controls never fell below 82% (Table IIGo) even after 24 h. This could be explained by a more rapid degradative process compared with the human sperm given that the mouse sperm head is completely dissociated from its tail. (Human sperm heads cannot be easily dissociated from tails using piezo, making a direct comparison difficult in these experiments). Since human sperm membranes may be more stable than those of mouse sperm (Kasai et al., 1999Go), one cannot conclude that there are obvious species differences in this regard. Immotile ejaculated human sperm can fertilize oocytes but often at a lower rate (Nagy et al., 1995Go). This could be explained by more extensive degradative damage with sperm immotile for >24 h. Finally, the results do not address whether qualitative aspects of oocyte activation (e.g. the frequency of calcium transients) might also be different over the different time intervals tested.

Embryo development was a more sensitive indicator that time-dependent damage occurs following sperm immobilization (Table IIGo). Using CZB–PVP buffer as the holding medium, blastocyst development was impaired at the earliest interval assayed (1 h) for mouse sperm. This showed a further time-dependent effect such that virtually no blastocyst development occurred by 4–6 h. Additional experiments would be needed to rule out that developmental effects might occur within several minutes of sperm head separation. Holding the sperm heads in the K+-rich NIM buffer protects from this developmental effect only for ~2 h. Thereafter, the time-dependent decline in development occurred as seen in the CZB–PVP buffer.

Using either CZB–PVP or NIM buffer, the clearest block in development occurred at the morula stage (Table IIGo). However, with increasing time intervals after immobilization, earlier cleavage stages were arrested as well. The mouse embryonic genome becomes increasingly active by the 2-cell stage, including paternal markers (Wudl and Chapman, 1976Go; Sawicki et al., 1981Go; Latham, 1999Go). Thus, the developmental data are consistent with some effect on appropriate genome activation or expression. This is further supported by the protective effect in NIM buffer (Table IIGo). Using cytogenetic analysis, NIM has been shown to protect the mouse sperm from genetic damage from the extracellular environment after membrane disruption (Tateno et al., 2000Go). The Na+ and K+ concentrations in bovine sperm have been estimated at 14 ± 2 and 120 ± 5 mmol/l respectively (Babcock, 1983Go), and NIM is formulated to maintain a K+-rich ionic environment around the disrupted sperm. Sperm chromatin may be susceptible to ion-dependent genetic damage. This protection, however, is limited and the data do not rule out additional epigenetic developmental mechanisms that have been described through nuclear transfer experiments and involving cytoplasmic interactions (Reik et al., 1993Go; Roemer et al., 1997Go).

The mouse data described cannot yet be extrapolated to the human since isolated human sperm heads were not evaluated; however, the data support the hypothesis that prolonged exposure to the extracellular environment could pose a developmental risk for the membrane-compromised human sperm heads. First, structural damage to human chromosomes after injection into mouse oocytes has been described, although mouse chromosomes may be affected more rapidly (Tateno et al., 2000Go). Second, it has been reported that injection of isolated sperm heads may lead to chromosomal errors in the resulting embryos (Colombero et al., 1999Go). However, it remains to be ruled out that this is secondary to damage to the sperm centrosome, which is of paternal origin in the human. In the mouse, the sperm centriole is lost during testicular and epididymal transit and is not required for normal fertilization and development (Manandhar et al., 1998Go). Third, there is evidence that human sperm chromatin is sensitive to some stresses such as sonication (Kuretake et al., 1996Go). Human sperm have less disulphide bonding within protamines (Perreault et al., 1988Go), and ~15% of human sperm DNA remains bound to nucleohistones (Gatewood et al., 1987Go; Choudhary et al., 1995Go).

On the other hand, human sperm also have some measure of protection from the types of conditions reported here using isolated mouse epididymal sperm heads. Ejaculated human sperm chromatin takes up considerable amounts of zinc from the seminal fluid which acts to stabilize nuclear proteins (Kvist et al., 1985Go). In addition, human sperm heads are rarely used for ICSI as described here for the mouse. The mouse sperm tail is routinely removed prior to ICSI to improve oocyte survival, and this does not appear to pose a developmental risk as long as the heads are injected in a timely manner (Ron-El et al., 1995Go; Kuretake et al., 1996Go). Nevertheless, the data are consistent with the hypothesis that sperm-immobilizing conditions could pose developmental risks which would not be apparent from fertilization rate or normal activation rate (2PN) endpoints alone.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
We thank Dr Yukiko Yamazaki and Dr Ryuzo Yanagimachi, Department of Anatomy and Reproductive Biology and Institute for Biogenesis Research, University of Hawaii School of Medicine, for discussions and generously providing the mice used for experiments.


    Notes
 
3 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, Yamanashi Medical University, Shimokato 1110, Tamaho, Nakakoma, Yamanashi, 409-3898, Japan. E-mail: kmizuno{at}res.yamanashi-med.ac.jp Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on October 1, 2001; resubmitted on March 13, 2002; accepted on May 17, 2002.