Molecular Human Reproduction, Vol. 5, No. 8, 784-787,
August 1999
© 1999 European Society of Human Reproduction and Embryology
Successful metaphase chromosome analysis of human elongated spermatids using mouse oocytes
1 The Institute of Advanced Medical Technology Central Clinic, Yakushiji 3154, Minamikawachi, Kawachi, Tochigi 329-0431, 2 Department of Obstetrics and Gynecology, Jichi Medical University, Yakushiji 3153, Minamikawachi, Kawachi, Tochigi 329-0431, 3 Department of Animal Breeding and Reproduction, Faculty of Agriculture, Utsunomiya University, Utsunomiya 321-8505, Japan, and 4 CARE (Centres for Assisted Reproduction), The Park Hospital, Sherwood Lodge Drive, Burntstump Country Park, Arnold, Nottingham NG5 8RX, UK
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Abstract |
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Human elongated spermatids from azoospermic patients were inserted into mouse oocytes by intracytoplasmic sperm injection (ICSI). The injection resulted in survival rates of 46.5% (180 out of 387) and activation rates of 36.1% (65 out of 180). The rate of two pronuclear (2PN) formation was 35.4% (23 out of 65). Only 34.8% (eight out of 23) metaphase chromosome spreads from 2PN zygotes could be analysed; however, all were of normal karyotype. Cytogenetic analysis at the first metaphase revealed that human elongated spermatid chromosomes were able to undergo replication in a heterogeneous environment.
azoospermia/chromosome karyotype/elongated spermatid/G-banding/ICSI
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Introduction |
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Intracytoplasmic spermatid/sperm injection (ICSI) is currently used as a treatment for patients with severe male factor in fertility in some countries. Intact round spermatid injection (ROSI) has resulted in the delivery of healthy infants (Tesarik and Mendoza, 1996a
; Tesarik et al., 1996b; Vanderzwalmen et al., 1997; Barak et al., 1998; Bernabeu et al., 1998), as has treatment with elongated spermatid injection (ELSI) (Fishel et al., 1995, 1997; Mansour et al, 1996; ; Araki et al., 1997; Vanderzwalmen et al., 1997; Sofikitis et al., 1998).
However, the incidence of successful pregnancy after ELSI is lower than after conventional ICSI. In spite of these preliminary human data and studies using animal models (Ogura et al., 1994; Sofikitis et al., 1994; Kimura and Yanagimachi, 1995b; Sasagawa et al., 1998; Sato et al., 1998), much is still unknown about the mechanism of fertilization with these spermatids. Processes such as genomic imprinting, changes in DNA association with nuclear proteins, oocyte activation, cell-cycle synchronization of the gametes and karyotype of spermatid in azoospermia need further study to elucidate this mechanism (Fishel et al., 1996; Aslam et al., 1998).
It is now known that the rate of abnormal karyotypes among infertile men is higher than among fertile men (Edwards and Brody, 1995; Yoshida et al., 1996). However, utilizing spermatids for procreation is the only possible approach for some couples. Questions which remain to be answered are on the efficacy and, more importantly, the safety of using spermatids. This is the first report to analyse the karyotype of human elongated spermatid from azoospermic males after ICSI into mouse oocytes.
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Materials and methods |
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Reagents
Pregnant mare's serum gonadotrophin (PMSG) was purchased from TeikokuZouki, Tokyo, Japan and human chorionic gonadotrophin (HCG) from Mochida, Tokyo, Japan. M2 and M16 media, hyaluronidase (type VIII), sodium pyruvate, penicillin-G, bovine serum albumin (BSA; fraction V), mineral oil, nocodazole, EDTA and polyvinylpyrrolidone (PVP; M360000) were all purchased from Sigma Chemical Co (St Louis, MO, USA). Glass capillaries for microtool preparation were acquired from Sutter Instrument Co (Novato, CA, USA).
Oocyte collection
Oocytes were obtained by ovulation stimulation of hybrid female mice BDF1 (Shizuoka, Japan). Ovulation was induced by s.c. injection of 10 IU of PMSG followed by i.p. administration of 10 IU HCG 48 h later. The animals were killed by cervical dislocation 14 h after HCG administration. Their oviducts were removed and placed on a warming stage in a Petri dish with M2 medium supplemented with 100 IU/ml hyaluronidase. Cumulus–oocyte complexes were released by dissection of the ampullae of the oviducts. After incubation with hyaluronidase for 3–4 min, the oocytes were washed in M2 medium and transferred to M16 medium containing 100 µmol/l EDTA, and then they were incubated in a humidified atmosphere of 5% CO2 in air at 37°C. Oocytes were cultured for at least 2 h before injection. Those oocytes which extruded a second polar body during this time were considered to be activated by the hyaluronidase treatment and were discarded. Only oocytes with a small perivitelline space, a homogeneously granulated cytoplasm and remainder of the polar body were used for the experiments.
Testicular elongated spermatid preparation
This study involves eight men suffering from non-obstructive azoospermia. Seminiferous tissue was extracted by testicular open biopsy. The extracted tissue (each mass was ~6–9 mm3) was minced using a sterile slide glass and was then rinsed with modified human tubal fluid (m-HTF) and centrifuged for 10 min at 750 g. The resulting pellet was used after dilution. One droplet (~10 µl) of this diluted suspension was placed on a dish, covered with mineral oil. Elongated spermatids were easily recognized by their morphology (Vanderzwalmen et al., 1998). In cases in which this process was initially unsuccessful, it was repeated a second time using a continuous Percoll density gradient (Araki et al., 1997), followed by washing twice with m-HTF.
Microinjection
Micromanipulation was performed under an inverted microscope (Olympus IX70, Tokyo, Japan) with mounted Narishige micromanipulators (Narishige Co Ltd, Tokyo, Japan).
Microinjection was performed on the cover of a Petri dish containing seven 10 µl drops of M2 medium under mineral oil. A central drop contained spermatids resuspended in injection medium (10% PVP/M2). The oocytes were placed for injection in seven drops of M2 medium. For sham ICSI, oocytes were injected with a small amount of injection medium (10% PVP/M2) devoid of a spermatid. The oocytes were held by a holding pipette so that the cone of the metaphase chromosomes was at six o'clock, to avoid destruction of the metaphase plate by the injection. The injection pipette was inserted in such a way that a deep invagination of the vitelline membrane was created. Thereafter, the membrane was broken by gentle suction and the cytoplasm was returned to the oocyte together with the spermatid.
Cytological and cytogenetic analysis
After injection, oocytes were kept in M16 medium in the incubator. They were continuously (every 2 h) checked for second polar body extrusion 10–16 h after injection. Those which formed a pronucleus (PN) and a second polar body were recorded as fertilized. Some of the fertilized oocytes (zygotes) were fixed and analysed at the PN stage, whereas others were transferred to 0.1 µg/ml nocodazole in M16 medium and fixed 1 h after the disappearance of the pronuclei, according to the technique described previously (Rybouchkin et al., 1995, 1997). Before fixation, the zona pellucida was removed using 0.5% pronase. They were treated with a hypotonic solution (75 mmol/l KCl) for 8 min at room temperature, and fixed with methanol:acetic acid (3:1) and air-dried. After 2–3 days in room temperature, they were additionally treated with 0.025% trypsin solution in phosphate-buffered saline (PBS), pH 6.8, for 3–4 s at room temperature. The slides were washed with PBS, treated with 5% methanol–PBS for 30 s, and finally washed again with PBS. When the slide was completely dry, it was stained with a 2.5% Giemsa solution in PBS for 10 min.
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Results |
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Elongated spermatids were recovered from all eight patients. The mouse oocyte survival, activation and fertilization rates are summarized in Table I
. Survival rates were 46.5% (180 out of 387), with 53.5% (207 out of 387) degenerating after ICSI. The activation rate was 36.1% (65 out of 180) and of those, 1PN formation was 27.7% (18 out of 65), 2PN formation 36.9% (24 out of 65) and 3PN formation was 4.6% (three out of 65). Cleaved oocytes in which no PN were observed occurred at a rate of 18.5% (12 out of 65). The incidence of second polar body extrusion without PN formation was 12.3% (eight out of 65). Only 33.3% (eight out of 24) metaphase chromosome spreads could be analysed from the fertilized 2PN oocytes. All have a normal karyotype (Figures 1 and 2). Figure 3 shows a G-banding karyotype pattern from human elongated spermatid. In oocytes in which 1PN was found (five out of 18; 27.8%) there was invariably a swollen sperm head and with the appearance of prematurely condensed sperm chromosomes (PCC). Of eight oocytes with a second polar body, only three had chromosomes, but these three could not be analysed for a detailed karyotype. The remaining five oocytes did not yield any data due to inadequate fixation using this technique; nor could the 3PN oocytes be analysed, due to the same problem with fixation.
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Discussion |
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The chromosome constitution of human spermatozoa has been studied following fusion with zona-free hamster oocytes (Rudak et al., 1978
; Kamiguchi and Mikamo, 1986; Martin et al., 1991). However, that system requires millions of motile and acrosome-reacted spermatozoa. To observe the karyotype of immotile spermatozoa, testicular spermatids/spermatozoa of non-obstructive azoospermic males or biologically infertile spermatozoa (e.g. spermatozoa incapable of fusion with the zona pellucida or oolemma) the hamster test is inappropriate. These spermatozoa need to be injected into an oocyte to permit development of the sperm chromosomes to the metaphase state.
It has been reported that after injecting individual spermatozoa into mouse oocytes, of 105 oocytes which survived the injection with initially motile spermatozoa, 102 (97%) became activated (Rybouchkin et al., 1995). Most of the activated oocytes formed 2PN. C-banding confirmed the presence, in the common cytoplasm, of chromosomes of both human and mouse origin. Both chromosomal sets had an appearance typical of normally replicated chromosomes. Also, after ICSI of a mouse, of a total of 279 eggs arrested at first cleavage metaphase, 200 (71.7%) were suitable for the analysis of sperm chromosome (Lee et al., 1996). These authors concluded that mouse oocytes are suitable for this purpose and that they are perhaps more `hospitable' to human sperm chromosomes.
Chromosomes were analysed by G-banding and recently by fluorescent in-situ hybridization (FISH) (data not shown). FISH is suitable for specific site detection only, and it is difficult to examine the detail of an abnormal site without a specific probe.
In this study we found that about half of the mouse oocytes survived ICSI with human spermatids. Only 33.3% of metaphase chromosomes from 2PN oocytes could be analysed, but all of them had a normal karyotype (Figure 2
). Although only eight elongated spermatids were successfully karyotyped, comparison of the current data with previously obtained karyotypes of mature spermatozoa (Araki et al., 1997b) indicate that the karyotypes of elongated spermatids are indistinguishable from those of mature spermatozoa obtained with mouse oocytes. The 2PN formation rate was low by mechanical ICSI and, based on both unpublished research (S.Ogawa et al.) and other studies (Kimura and Yanagimachi, 1995a; Lee et al., 1996; Sasagawa et al., 1998; Sato et al., 1998), we believe that Piezo ICSI (PICSI) will yield much greater survival and activation rates in the future. In these experiments, spermatids were collected from a normal fertile mouse strain. More recently, it has also been shown that normal embryo development could be obtained from a sterile mouse strain after ROSI (Sasagawa et al., 1998). In addition, in the human, babies have been delivered from round and elongated spermatids (Fishel et al., 1995, 1997; Mansour et al., 1996; Tesarik and Mendoza, 1996a; Tesarik et al., 1996b; Antinori., 1997; Araki et al., 1997; Vanderwalmen et al., 1997; Barak et al., 1998, Bernabeu et al., 1998; Sofikitis et al., 1998), but the efficacy of the process is not yet established. Although the underlying mechanism of this technique is not fully understood, the fact that fertilization, implantation, and birth of healthy infants can occur after the injection of spermatids into oocytes has led us to use this technique for infertile couples in those cases in which the technique represented their only chance of pregnancy. We are in the process of examining the chromosome complement of ROSI into mouse oocytes.
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Conclusions |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionsReferences |
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From our data we concluded that human elongated spermatids can be injected successfully into mouse oocytes resulting in a reasonable survival rate by mechanical ICSI, and the mouse oocytes provide a useful model for the assessment of spermatid karyotype. In our data on elongated spermatids all karyotypes which could be analysed had a normal profile. However, it must be emphasized that only eight karyotypes were obtained out of 387 procedures. Despite the paucity of analysable karyotypes and gross inefficiency of this method, it is clear from the quality of the karyotypes obtained that this technique holds promise. Research should continue in order to remedy the efficiency and quantity of analysable data.
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Notes |
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5 To whom correspondence should be addressed
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References |
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Submitted on January 6, 1999; accepted on April 15, 1999.
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