A novel method for chromosome analysis of human sperm using enucleated mouse oocytes

Yasuyuki Araki1,2,3,4, Midori Yoshizawa1,2 and Yasuhisa Araki3

1 Science of Plant and Animal Production, United Graduated School of Agricultural Science, Tokyo University of Agriculture and Technology, Fuchuu-city, Tokyo, 183-8509, 2 Department of Animal Breeding and Reproduction, Faculty of Agriculture, Utsunomiya University, 350, Mine-machi, Utsunomiya, Tochigi, 321-8505 and 3 Institute for Advanced Reproductive Medical Technology, 909-21, Ishii, Fujimi, Seta-gun, Gunma, 371-0105, Japan

4 To whom correspondence should be addressed at: Institute for Advanced Reproductive Medical Technology, 909-21, Ishii, Fujimi, Setagun, Gunma, 371-0105, Japan. Email: yaaraki{at}nifty.com


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: Mouse oocytes can be used in conjunction with intracytoplasmic sperm injection (ICSI) as a technique to permit chromosomal analysis of human sperm. However, chromosomes derived both from the human sperm and the mouse oocyte appear simultaneously following ICSI. The present study focused on evaluating whether or not previously enucleated mouse oocytes are usable for the analysis of human sperm chromosomes. METHODS: The metaphase chromosome–spindle complex was removed from a mouse oocyte. Human sperm from a donor with proven fertility were injected into mouse enucleated oocytes or intact oocytes. The presence of pronuclei in the oocytes was confirmed ~7–11 h after ICSI, and the oocytes were then fixed so that the nuclei could be observed as chromosome samples at 15–16 h after ICSI. RESULTS: The formation rate of one pronucleus in enucleated oocytes after ICSI was 93.9% (186/198) while that of two pronuclei in intact oocytes after ICSI was 85.4% (88/103). The appearance rate of metaphase chromosomes of human sperm in the enucleated oocytes, 89.4% (160/179), was significantly higher than that in intact oocytes, 78.7% (74/94) (P=0.017). CONCLUSIONS: An efficient ICSI method using enucleated mouse oocytes was established to allow the visualization of the human sperm chromosome complement without the risk of confusion with mouse oocyte chromosomes.

Key words: chromosomes/enucleated mouse oocytes/human sperm/ICSI


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
It was revealed that chromosome samples of human sperm could be obtained using the hamster oocyte–human sperm fusion system (Rudak et al., 1978Go). However, this method required motile sperm and did not permit analysis of morphologically abnormal sperm.

Martin et al. (1988)Go analysed the chromosomes of human sperm injected into hamster oocytes by intracytoplasmic sperm injection (ICSI), and ICSI enabled us to use sperm from oligospermic patients and sperm with low-motility for chromosomal analysis. Moreover, Rybouchkin et al. (1995)Go proved that human sperm chromosomes can be analysed by sperm injection into mouse oocytes. Since an efficient and stable ICSI method had been developed in mouse oocytes by Kimura and Yanagimachi (1995)Go, the use of mouse oocytes in the human sperm chromosome assay prevailed (Lee et al., 1996Go; Araki et al., 1999Go, Araki et al., 2004Go; Ogawa et al., 2000Go; Tsuchiya et al., 2002Go; Watanabe, 2003Go).

In general karyotyping, it is desirable that the chromosomes are dispersed moderately without overlap. However, if samples with chromosomes that are dispersed sufficiently are produced, it is sometimes difficult to distinguish human sperm chromosomes from mouse chromosomes if their metaphase figures are confused with each other. In such cases, it is convenient for the analysis of human sperm chromosomes if the chromosomes from mouse oocytes do not appear.

Kishikawa et al. (1999)Go, who used enucleated mouse oocytes for the analysis of mouse sperm chromosomes, suggested that the oocyte nucleus was not needed for the sperm nucleus to develop to the metaphase stage of the first cleavage after sperm injection.

Therefore, we examined whether or not it is possible to produce samples of human sperm chromosomes without the appearance of mouse chromosomes by removing the chromosomes of the mouse oocyte before injecting a human sperm.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Preparation of mouse oocytes
Mature B6D2F1 female mice were superovulated by an intraperitoneal injection of 5IU pregnant mares serum gonadotrophin (PMSG) followed after 48 h by the administration of 5IU hCG. Mouse oocytes were collected from the oviducts of females 14–16 h after the hCG injection. The oocytes were freed from the cumulus cells by pipetting in a Hepes–human follicular fluid medium (Hepes–HFF99; Fuso Pharmaceutical Industries, Osaka, Japan) supplemented with 10% synthetic serum substance (SSS; Irvine Scientific, Santa Ana, CA) and 60 IU/ml bovine testicular hyaluronidase (Sigma Chemical Co., St Louis, MO) at 37 °C. These oocytes were rinsed and kept in a human follicle fluid medium (HFF99; Fuso Pharmaceutical Industries) at 37 °C under 5% CO2 in air until sperm injection.

Preparation of human sperm
Human sperm were obtained from a donor with proven fertility after informed consent was given. Semen samples were centrifuged at 730 g for 20 min with 80% Percoll and then washed with HFF99+10% SSS. After removal of the supernatant, motile sperm were collected by a swim-up method at 37 °C under 5% CO2 in air. Although the swum-up sperm were used for ICSI in the present study, this swim-up step would not be appropriate in cases of patients with oligoastheno- or oligoasthenoteratozoospermic samples.

Enucleation of oocytes
Enucleation of oocytes was carried out according to the method of Wakayama et al. (1998)Go. The lid of a plastic dish (Falcon No. 1001 or 1006; Becton Dickinson, Franklin Lakes, NJ) was used as a micromanipulation chamber. The oocytes were transferred into a droplet of Hepes-HFF99+10% SSS containing 5 µg/ml cytochalasin B (Wako Chemical Co., Osaka, Japan) for 10 min at 37 °C. The metaphase II chromosome–spindle complex was aspirated into an enucleated pipette with a minimal volume of oocyte cytoplasm. Enucleated oocytes were transferred into HFF99+10% SSS and kept there until sperm injection.

Intracytoplasmic sperm injection
Human sperms were injected into mouse enucleated oocytes or intact oocytes by a Piezo-driven unit according to the procedure of Araki et al. (2004)Go. Each sperm tail was injured at mid-length by applying 5–10 Piezo pulses, and then the sperm was drawn up into an injection pipette headfirst. An oocyte was held on a holding pipette and its zona pellucida was drilled by applying 2–3 Piezo pulses. The injection pipette was advanced mechanically until the tip almost reached the opposite side of the oocyte's cortex. The sperm kept at the back of the injection pipette was transferred to the top of the injection pipette, and injected into the ooplasm after the oocyte membrane was broken by applying one or two faint Piezo-pulses. Approximately 7–11 h after the injection, an inverted microscope was used to determine whether or not a second polar body and pronuclei were present in the oocytes. After confirmation of their presence, the oocytes were cultured in HFF99 medium with 10% SSS containing 30 ng/ml vinblastine (Sigma Chemical Co.) in order to disturb the spindle formation (Yoshizawa et al., 1997Go).

Fixation and staining of chromosome
The oocytes were made into chromosome samples according to the methods of Yoshizawa et al. (1997Go, 1999)Go at 15–16 h after sperm injection. Before the preparation, the zona pellucida was removed using acidic Tyrode solution (Sigma Chemical Co.). The oocytes were briefly rinsed in 1% hypotonic sodium citrate solution supplemented with 10% SSS and placed in 0.4 ml of this solution for several minutes. For mouse oocytes whose pipette-penetrated zona pellucidae were still attached, the short-term hypotonic treatment for chromosome preparation is useful (Yoshizawa et al., 1990Go). A very small volume (10–20 µl) of fixative (methanol:acetic acid, 3:1) was poured into the hypotonic solution. Each oocyte was placed individually on a glass slide, and several droplets of fixative were placed on the oocyte. After drying, some of the samples were staine by the G-band staining method of Ogawa et al. (2000)Go modified slightly by the authors. The slides were treated with 0.125% trypsin solution in phosphate buffer (PB) solution for a few minutes at room temperature, washed with water, 50% methanol in PB, and stained with 2% Giemsa solution for 10 min.

Statistical analysis
The obtained data were analysed by the chi-square test.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The rates of pronuclear formation in the oocytes after sperm injection are presented in Table I. The survival rate of oocytes after enucleation was 91.0% (232/255). The survival rates of enucleated oocytes and intact oocytes after sperm injection were 86.1% (198/230) and 80.5% (103/128), respectively. The formation rate of one pronucleus in enucleated oocytes was 93.9% (186/198), while that of two pronuclei with the second polar body (2nd PB) in intact oocytes was 85.4 % (88/103). All of the enucleated oocytes injected with human sperm did not eject the 2nd PB justifiably (Figure 1A). When those nuclear membranes had disappeared (Figure 1B), the oocytes were fixed as chromosome samples.


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Table I. Rates of pronuclear formation in enucleated mouse oocytes injected with human sperm

 


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Figure 1. Enucleated mouse oocytes after injection of human sperm. (A) One pronucleus appeared in each oocyte, 7 h after injection of sperm. (B) The pronuclear membranes disappeared at 15 h after injection. Original magnification x100.

 
Table II shows the rates of appearance of metaphase chromosome sets of human sperm in the oocytes. The success rates of the chromosome preparations were 90.4% (179/198) in enucleated oocytes and 91.3% (94/103) in intact oocytes. The appearance rate of the metaphase chromosomes of human sperm in the enucleated oocytes, 89.4% (160/179), was significantly higher than that in intact oocytes, 78.7 % (74/94) (P=0.017). In the intact oocytes, human sperm chromosomes appeared simultaneously with mouse chromosomes at significantly high rates, 98.6% (73/74). In contrast, all of the human sperm chromosomes appeared independently without mouse chromosomes in the enucleated oocytes, 0.0% (0/160) (Figure 2).


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Table II. Rates of appearance of first mitotic chromosomes of human sperm in enucleated mouse oocytes

 


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Figure 2. Human sperm chromosomes appeared independently without mouse oocyte chromosomes (G-band staining). (A) Original magnification x200. (B) The same figure shown by high magnification; original magnification x1000.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The ICSI technique has enabled successful fertilization with the sperm of patients who have extremely low numbers of viable sperm; therefore, these patients show a very poor probability of achieving fertilization in vitro.

However, it has been shown that the patients undergoing ICSI have high incidences of chromosomal abnormalities in their spermatozoa (Calogero et al., 2001Go; Macas et al., 2001Go; Rodrigo et al., 2004Go; Tang et al., 2004Go), and these high incidences remain the issue that must be resolved for the sequential development and birth of normal infants to occur. To resolve this issue, high analysable rates in the diagnosis of samples using limited gametes must be obtained; therefore, improvement of the chromosome preparation method for human sperm must be achieved.

In producing samples of human sperm chromosomes, the success rate is relatively high when mouse oocytes are used (Lee et al., 1996Go). In the present study also, the appearance rate of human sperm chromosomes from the intact mouse oocytes injected with human sperm was 57.8% (74/128), which was similar to the rates obtained by other researchers, including 71.4% (200/279) obtained by Lee et al.(1996)Go, 56.5% (108/191) by Tsuchiya et al. (2002)Go and 52.2% (504/965) by Watanabe (2003)Go.

Kishikawa et al. (1999)Go produced chromosome samples of mouse sperm without the appearance of oocyte chromosomes by using enucleated mouse oocytes. The present study also revealed that it is possible to produce chromosome samples of human sperm without the appearance of chromosomes from mouse oocytes. The enucleated mouse oocytes injected with human sperm formed a single nucleus at a high rate (93.9%, 186/198), and it was found that the sperm could develop to the metaphase stage (80.8%, 160/198) even if the oocyte nucleus did not exist. Moreover, the appearance rate of the metaphase chromosomes in enucleated oocytes was higher than that in the intact oocytes. We can not speculate about the reason for the higher appearance rate of metaphase chromosomes of human sperms in enucleated oocytes. However, this result is very interesting and may be an important factor in our understanding of the processes involved with the appearance of sperm chromosomes.

It is generally accepted that chromosomes of mouse oocytes appear simultaneously when human sperm chromosomes appear (Watanabe, 2003Go). In the intact oocytes of the present study, the chromosomes of mouse oocytes appeared simultaneously with the human sperm chromosomes in almost all of the samples observed (98.6%, 73/74) although the mouse oocyte chromosomes never appeared in the enucleated oocytes (0.0%, 0/160).

This high rate of simultaneous appearance was considered to be the reason that the rate of enucleation in the present study was extremely high (as in the results of Wakayama et al. 1998Go), because the enucleation method used was suitable to mouse oocytes.

An effective assay for human sperm chromosomes was established by the present study because the appearance of human sperm chromosomes alone was made possible by the injection of sperm into enucleated mouse oocytes. A morphologically abnormal acrosome rate in sperm of patients who fail to fertilize oocytes by ICSI is high (Araki et al., 2004Go), but chromosomal analysis of these sperm has not been reported. It is necessary to inform patients about the risks of chromosomal abnormalities of sperm before the ICSI treatment.

The present paper describes a reliable methodology for producing chromosome samples of human sperm using enucleated mouse oocytes.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Araki Y, Ogawa S, Ohno M, Yoshizawa M, Motoyama M, Araki S, Aslam I and Fishel S (1999) Successful metaphase chromosome analysis of human elongated spermatids using mouse oocytes. Mol Hum Reprod 5, 784–787.[Abstract/Free Full Text]

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Kishikawa H, Tateno H and Yanagimachi R (1999) Chromosome analysis of BALB/c mouse spermatozoa with normal and abnormal head morphology. Biol Reprod 61, 809–812.[Abstract/Free Full Text]

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Submitted on October 18, 2004; resubmitted on December 9, 2004; accepted on December 14, 2004.





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