Pregnancy by the tubal transfer of embryos developed after injection of round spermatids into oocyte cytoplasm of the cynomolgus monkey (Macaca fascicularis)

Narumi Ogonuki1,2,3, Hideaki Tsuchiya1,4, Yoshihiro Hirose1, Hironori Okada1,5, Atsuo Ogura2,3 and Tadashi Sankai1,6

1 Tsukuba Primate Center for Medical Science, National Institute of Infectious Diseases, Hachimandai-1, Tsukuba, Ibaraki, 305–0843, 2 Bioresource Engineering Division, Bioresource Center, RIKEN Tsukuba Institute, Ibaraki, 3 Department of Veterinary Science, National Institute of Infectious Diseases, Tokyo, 4 National Institute for Minamata Disease, Kumamoto and 5 Tokyo University of Agriculture, Hokkaido, Japan

6 To whom correspondence should be addressed. e-mail: sankai{at}nih.go.jp


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: Round spermatids have been used as substitute gametes in basic reproductive research and in infertility clinics. In humans, however, the efficiency of fertilization and pregnancy is generally much lower after round spermatid injection (ROSI) than after injection with mature sperm. We examined the ability of round spermatids to support embryonic development using a non-human primate as a model. We chose cynomolgus monkeys because, as in humans, their round spermatids have the oocyte-activating capacity of mature sperm. METHODS: We examined fertilization and subsequent development of embryos after ROSI and then transferred the embryos into the oviducts of female monkeys. RESULTS: Seventy-seven per cent of survived oocytes were activated and had formed pronuclei or the second polar body; 79% of the oocytes cultured developed to the 2-cell stage, and 23% developed to the blastocyst stage. Ultrasonography showed a normal-sized fetus in the uterus of a recipient, but the fetus spontaneously aborted at day 103. CONCLUSIONS: The round spermatids of cynomolgus monkeys can be used as substitute gametes to support embryonic development at least to mid-gestation. This non-human primate is a suitable animal model for round spermatid conception in mammals, especially humans, and for biological and genetic characterization of events following ROSI.

Key words: cynomolgus monkey/embryo transfer/oocyte activation/ROSI/round spermatid


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Since the first success of ICSI in humans, it has been used for treatment of male factor infertility. However, idiopathic azoospermia caused by spermatogenic arrest cannot be remedied by this technique, and therefore many attempts have been made to develop new techniques of assisted fertilization using immature male germ cells. It is now accepted that elongated spermatids can be efficiently used (Antinori et al., 1997Go; Sofikitis et al., 1998aGo) as substitute gametes because of their high fertilizing ability comparable with that of mature sperm. In contrast, many controversial issues have been raised concerning the use of round spermatids. While several normal pregnancies following round spermatid injection (ROSI) have been reported (Fishel et al., 1995Go; 1996; Tesarik et al., 1995Go; 1996; Antinori et al., 1997Go), disappointingly poor fertilization and subsequent embryonic development are the common outcome in some clinics (Yamanaka et al., 1997Go; Levran et al., 2000Go; Vicdan et al., 2001Go). Round spermatids differ biologically from mature sperm in some important respects, such as immaturity of certain cytoplasmic and nuclear proteins (Ziyyat and Lefevre, 2001Go). Therefore, it is very likely that this immaturity is one of the causes of the poor pregnancy rates following ROSI in humans (Ghazzawi et al., 1999Go). Consistent with this assumption is the finding from mouse experiments that the overall efficiency of ROSI is relatively poor compared with that of ICSI. However, the circumstances in human ROSI are more complex because the fertilizing ability of patients’ round spermatids might be congenitally impaired, or reduced to some extent by an inadequate testicular environment (Vanderzwalmen et al., 1998Go). Thus, to assess the fertilizing ability of round spermatids, it is essential to recover them from fertile individuals. Non-human primates are best suited for this end. To our knowledge, however, there are only a few reports of successful ICSI in non-human primates and no reports of successful ROSI.

We have previously confirmed the technical basis for embryo culture, embryo transfer (Sankai et al., 1994Go; Sankai, 2000Go), and ICSI (Ogonuki et al., 1998Go) in non-human primates, especially the cynomolgus monkey (Macaca fascicularis). By using the ICSI and ROSI techniques, we found that not only mature sperm but also round spermatids from cynomolgus monkeys can activate mouse oocytes by inducing repetitive intracellular Ca2+ increases (Ca2+ oscillations) (Ogonuki et al., 2001Go). In this regard, cynomolgus monkey round spermatids are very similar to human round spermatids (Yazawa et al., 2000Go). Although, in the rhesus monkey, an infant was born from embryos after injection of elongated spermatids (Hewitson et al., 2002Go), the round spermatids of this species do not have oocyte-activating capacity (Hewitson et al., 2000Go). Therefore, the cynomolgus monkey may be a potential experimental model for a better understanding of human ROSI. In this study, we injected round spermatids from cynomolgus monkeys into homologous mature oocytes and examined their ability to support embryo development in vitro and in vivo.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Collection of spermatogenic cells
Spermatogenic cells were collected from two sexually mature male cynomolgus monkeys (M. fascicularis) that were bred and maintained in the Tsukuba Primate Center for Medical Science, National Institute of Infectious Diseases, Japan (Honjo, 1985Go). Testes were removed from the animals, which had been euthanized by deep injection of ketamine hydrochloride (Ketalar; Sankyo, Japan) (Cho et al., 1969Go), for use in other experiments. Spermatogenic cells were collected from the testes and then frozen by the method developed for the golden hamster (Ogura and Yanagimachi, 1993Go) and the mouse (Ogura et al., 1996Go). In brief, testes were placed in erythrocyte-lysing buffer (155 mmol/l NH4Cl, 10 mmol/l KHCO3, 2 mmol/l EDTA, pH 7.2). All of the following operations were carried out at 4°C on crushed ice, unless otherwise stated. After the testes were washed and the tunica albuginea was removed, seminiferous tubules were transferred into Dulbecco’s phosphate-buffered saline (PBS) supplemented with 5.6 mmol/l glucose, 5.4 mmol/l sodium lactate, 5 mg/ml of bovine serum albumin, and 0.01% polyvinylpyrrolidone (PVP; mol. wt 360 000; Wako Pure Chemical Industries, Japan) (GL-PBS) and cut into small pieces with fine scissors. The pieces were pipetted gently into GL-PBS to release spermatogenic cells. The cell suspension was filtered through a nylon mesh (mesh size 38 µm) and then centrifuged at 200 g for 5 min at 4°C. The cells were resuspended in GL-PBS and washed twice by further centrifugation (as above). After centrifugation, the pellet was resuspended in GL-PBS containing 7.5% glycerol and 7.5% fetal bovine serum. 1 ml of cell suspension was placed in 2 ml cryotubes and frozen to –80°C at –1°C/min. Over the next day, the cryotubes were transferred into liquid nitrogen and kept until use.

On the day of the experiment, a cryotube was placed in a water bath at room temperature. When the suspension began to thaw, 1 ml of GL-PBS was added. The mixture was then poured into a 15 ml tube containing 5 ml of GL-PBS. After complete thawing, the suspension was pipetted gently and washed twice by centrifugation using GL-PBS. To prevent the cells from losing their viability, the cell suspension was kept at 4°C until just before use.

Preparation of oocytes
Eight sexually mature female cynomolgus monkeys, whose menstrual cycles were confirmed to be normal (28–32 days) by examining vaginal bleeding (Honjo et al., 1984Go) for at least three cycles, were used for oocyte collection. Females received multiple i.m. injections (two times per day; 12–14 times total) of hMG (Pergonal; Teikoku Hormone, Japan). hCG (Profasi; Ares Serono, USA) was injected i.m. on the morning after the last hMG injection. In four of the eight females, to inhibit spontaneous release of LH or FSH from the pituitary gland, GnRH agonist (Leuplin; Takeda, Japan) was injected on the first day of menses; 2 weeks later, the four females received the first of multiple injections of hMG. Between 28 and 43 h after the hCG injection, all females were anaesthetized with a combination of 10 mg of ketamine hydrochloride per kg of body mass (Ketalar) and 1 mg of xylazine hydrochloride per kg of body weight (Seraktarl; Bayer, Germany). Ovaries were exposed through an abdominal incision (Figure 1a), and the contents of enlarged follicles (1–5 mm in diameter) were aspirated through a 25 gauge needle connected to a 2.5 ml syringe. The collected follicular contents were diluted immediately with HEPES-TYH medium (Toyoda et al., 1971Go) containing 2.5 IU/ml of heparin (Novo Nordisk, Denmark), and the oocytes were rinsed with TYH medium consisting of 119.37 mmol/l NaCl, 4.78 mmol/l KCl, 12.6 mmol/l CaCl22H2O, 1.19 mmol/l MgSO47H2O, 1.19 mmol/l KH2PO4, 25.07 mmol/l NaHCO3, 5.56 mmol/l glucose, 1.0 mmol/l pyruvic acid (sodium salt), 5 mg/ml BSA, penicillin-G (sodium salt), and streptomycin sulphate. HEPES-TYH was made by substituting HEPES for 20 mmol/l NaHCO3. Heparin was added to the HEPES-TYH medium to prevent coagulation of contaminating blood.



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Figure 1. (a) Cynomolgus monkey ovary treated with hMG and hCG, showing numerous developed follicles (arrow). Arrowhead indicates a uterus. Bar = 20 mm. (b) Cynomolgus monkey oocytes. (b-1) Mature oocyte with the first polar body (arrow) surrounded by cumulus cells. (b-2) Immature oocyte at the germinal vesicle stage (arrow). Bar = 20 µm.

 
The oocytes were examined under a dissection microscope and rinsed with HEPES -TYH medium without heparin. The oocytes were freed from cumulus cells by treating them with 0.1% hyaluronidase in HEPES-TYH medium and then pipetting. Oocytes were examined for meiotic maturity under an inverted microscope, and those that had extruded the first polar body (Figure 1b-1) were selected for ROSI. The selected oocytes were transferred to fresh TYH medium and kept at 38°C in a humidified 5% CO2 and 5% O2 atmosphere with N2 gas until micromanipulation. Immature oocytes in either a germinal vesicle or a germinal-vesicle-breakdown formation (Figure 1b-2) were cultured in a 50 µl drop of [Tissue culture medium-199 (TCM-199); Flow Laboratories, USA] containing 10% fetal calf serum (FCS), 10 IU/ml of equine chorionic gonadotrophin, and 10 IU/ml of hCG. Maturation culturing was done in an incubator at 38°C in a humidified 5% CO2 and 5% O2 atmosphere with N2 gas. When the oocytes had extruded the first polar body, they were selected for ROSI.

Microinjection of round spermatids
Microinjection was done using a micromanipulation system equipped with a piezo-micropipette-driving unit (Prime Tech, Japan) combined with a Nomarski interference-contrast microscope (Nikon Optical Co., Japan) (Figure 2). This procedure was reported previously (Kimura and Yanagimachi, 1995aGo,b; Ogonuki et al., 1998Go). The cover of a plastic dish (Falcon no. 1006; Becton Dickinson, USA) was used as the microinjection chamber. We remodelled the covers to use a Nomarski interference-contrast microscope by hollowing out a 2 cm diameter section and then placing over this section a glass coverslip with silicon-based adhesive around its circumference. Several small drops (2–5 µl), each containing HEPES-TYH (for oocytes) and 12% PVP in HEPES-TYH, were placed on the coverslip. In one of these drops, spermatogenic cells were suspended to a final PVP concentration of 6%. The drops were then covered with silicone oil (Aldrich Chemical Co., USA). The monkey round spermatids were ~10–12 µm in diameter (Figure 2a) and were thus easily distinguished from primary spermatocytes (17–20 µm in diameter). A round spermatid was drawn into the injection pipette (Figure 2b-1). After the plasma membrane was broken by pipetting, the nucleus with a small amount of the cytoplasm of a round spermatid was injected into the ooplasm (Figure 2b-2–4). Oocytes injected with round spermatids were transferred into TYH medium, covered with silicone oil, and then kept at 38°C in a humidified 5% CO2 and 5% O2 atmosphere with N2 gas.



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Figure 2. (a) Cynomolgus monkey spermatogenic cells. Arrows and arrowheads indicate round spermatids and spermatocytes respectively. Bar = 20 µm. (b) ROSI procedure. (b-1) Round spermatids are captured in an injection pipette. (b-2) The pipette is easily inserted into the perivitelline space by using piezo pulses, and thus no pressure is exerted on the zona pellucida. (b-3) The pipette is inserted deep into the ooplasm, the oolemma is pierced by using piezo pulses, and an entire round spermatid is injected. (b-4) The pipette is carefully removed. Arrow indicates a round spermatid. (c) Cynomolgus monkey embryos obtained by ROSI. Pronuclei are in focus (arrows), and the first and second polar bodies are out of focus (arrowheads). Bar = 20 µm. (c-1) Female and male pronuclei. (c-2) Separated pronuclei. After several hours, the pronuclei were fused.

 
Examination of oocytes
Between 8 and 17 h post-ROSI, the oocytes were examined with an inverted microscope. Oocytes with two distinct pronuclei and the second polar body were considered normally fertilized. All living oocytes judged normally fertilized were transferred to developmental culture medium (CMRL-1066; Connaught Medical Research Laboratory) (Boatman, 1987Go).

Embryos obtained by ROSI were co-cultured with buffalo rat liver cells (Zhang et al., 1994Go) at 2–3x104 cells per well of a dish for IVF (Falcon no. 353653; Becton Dickinson) in CMRL-1066 medium supplemented with 10% FCS. The progression of embryo growth was examined daily by using a Hoffman modulation contrast microscope (Leica; Heerbrugg, Switzerland).

Embryo transfer
Five females were used for tubal embryo transfer. Ovulation in the menstrual cycle of the females was estimated by measuring the serum concentrations of estradiol using a commercially available enzyme immunoassay kit for human hormones (IMx system; Dainabot Co., Ltd) (Ogonuki et al., 1997Go). Blood samples were taken in the morning, and sera separated by centrifuge were frozen until the measurements were taken. The day of ovulation was estimated as the day that the estradiol concentration significantly decreased. Approximately 46–67 h post-ROSI, embryos at the 6–16-cell stage were selected for tubal transfer. These criteria were based on our previous experience. Embryos were picked from the culture dishes into a micro-glass capillary containing 3–5 µl of culture medium. Synchronizing the developmental stage of each embryo with the menstrual cycle of the recipient, tubal embryo transfer was done 2–3 days after ovulation. Females were anaesthetized with a combination of ketamine hydrochloride and xylazine hydrochloride. Fallopian tubes and ovaries were exposed through an abdominal incision, and the micro-glass capillary containing the embryos was introduced through the fimbriated end of the Fallopian tube; one embryo per tube was transferred into the mid-ampullary portion of the oviduct.

At 28 days post-ROSI, the presence of a gestational sac and the heartbeat of a fetus were confirmed by ultrasonography. Subsequently, the growth of the fetus was examined at 56 and 84 days post-ROSI.

Statistical analysis
Results were evaluated using Fisher’s exact test; P < 0.05 was considered significant.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Table I shows the results of pronucleus formation after ROSI in cynomolgus monkeys. We tried ROSI eight times using two types of oocytes: one in which the first polar body had already been extruded at collection time (in vivo), and another in which the first polar body was extruded after in-vitro maturation. In-vitro maturation took between 9 and 30 h. No oocytes extruded the first polar body after 30 h. There was no difference in the rates of survival and fertilization between oocytes that matured in vitro or in vivo. After ROSI, 104 oocytes (91.2%) survived, and 72 (76.6%) of these either formed pronuclei or released the second polar body, 51 (54.3%) oocytes showed normal fertilization (two distinct pronuclei and the second polar body) between 8 and 17 h post-ROSI (Figure 2c-1, -2). Table II shows the developmental stages of oocytes after ROSI of oocytes that matured in vivo or in vitro (Figure 3a-1–5). Of the 53 oocytes, 42 (79.2%) subsequently developed to the 2-cell stage between 24 and 50 h post-ROSI, and 47.7 and 22.7% of oocytes developed into morulae and blastocysts respectively by 199 h post-ROSI.


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Table I. Pronucleus formation in cynomolgus monkey oocytes post-round spermatid injection (ROSI)
 

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Table II. In-vitro development of cynomolgus monkey oocytes post-round spermatid injection (ROSI)
 


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Figure 3. (a) Cynomolgus monkey embryos were obtained by ROSI and developed in vitro to the 2-cell (a-1), 4-cell (a-2), 8-cell (a-3), 16-cell (a-4), and blastocyst (a-5) stages. Then 6–16-cell-stage embryos were transferred into the oviduct of a female cynomolgus monkey through the tubal fimbria. Bar = 20 µm. (b) Ultrasonograms of a cynomolgus monkey fetus in a recipient female (animal no. 1010011182). (b-1) Heart and gestation sac at 28 days post-ROSI. A beating heart was confirmed. (b-2) Skeletal formation of the head of the fetus at 84 days post-ROSI. The head size was 27 mm (distance between arrows), indicating normal growth. Again, a beating heart was confirmed, and hands, feet, and tail of the fetus were also confirmed.

 
Nine embryos at the 6–16-cell stage were transferred into the oviducts of five recipient females (Table III). At 28, 56, and 84 days post-ROSI, a normal-sized fetus was detected by ultrasonography in the uterus of one of the recipients (Figure 3b-1, -2); the gestational sac and heartbeat were detected by ultrasonography. However, the fetus was spontaneously aborted on day 103 post-ROSI.


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Table III. Embryo transfer of cynomolgus monkey oocytes fertilized by round spermatid injection (ROSI)
 

    Discussion
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
We report the first known pregnancy following ROSI in non-human primates. We previously reported that 61% (19/31) of embryos were fertilized and developed to the morula stage after ICSI of cynomolgus monkeys (Ogonuki et al., 1998Go). Hewitson et al. (1999Go) succeeded in delivery of an infant by ICSI of rhesus monkeys. Recently, ICSI in Macaca monkeys was established. Furthermore, we reported that cynomolgus monkey round spermatids readily activated mouse oocytes and formed pronuclei at a rate similar to that after injection with monkey sperm (Ogonuki et al., 2001Go). Round spermatids also had the ability to induce Ca2+ oscillations, the repetitive intracellular Ca2+ increases that are induced by fertilizing sperm in all mammalian species examined to date. In the present experiments, we extended the previous study to homologous fertilization and found that, as in heterologous fertilization, the monkey round spermatids normally fertilized monkey oocytes without any artificial activation stimuli. This finding clearly indicates that monkey round spermatids are capable of activating oocytes. Meng and Wolf (1997Go) reported that sham-injected oocytes of rhesus monkeys did not activate readily (2/16; 13%). Dozortsev et al. (1995Go) reported that human oocytes treated by sham injection were also not activated (13/98; 13%). Thus, human and monkey oocytes are not sensitive to mechanical stimulation. When oocytes injected with round spermatids are activated by parthenogenesis, development of the embryo stops at the morula stage. Because the embryos we obtained by ROSI developed to the blastocyst stage, we believe that they did not originate from parthenogenetic activation.

The first fertilization using round spermatids was reported in hamsters (Ogura and Yanagimachi, 1993Go). It is now generally accepted that mouse round spermatids can support full-term embryo development following ROSI with an efficiency comparable with that of mature sperm (Ogura et al., 1994Go; 2001; Kimura and Yanagimachi, 1995bGo). ROSI in other animals, including rabbit (Sofikitis et al., 1994Go) and rat (Hirabayashi et al., 2002Go), has also been reported to result in the birth of normal offspring. However, the use of round spermatids as substitute gametes in humans has been controversial. Some infertility clinics have achieved successful birth (Fishel et al., 1995Go; 1996; Tesarik et al., 1995Go; 1996; Antinori et al., 1997Go), but many clinics have experienced very low fertilization rates following ROSI (Yamanaka et al., 1997Go; Levran et al., 2000Go; Vicdan et al., 2001Go). This discrepancy between humans and other animals in round spermatid conceptions may be attributable either to species-specific differences in round spermatids or to circumstances in which round spermatids are collected from infertile humans with spermiogenic failure. According to Yazawa et al. (2000Go), human round spermatids essentially have oocyte-activating capacity while inducing intracellular Ca2+ oscillations, as demonstrated by the assay system using mouse mature oocytes. The present study indicates that, once oocytes of cynomolgus monkeys are successfully activated by round spermatids, many of them undergo normal preimplantation development, and some continue their development at least until mid-gestation. Thus, it is very probable that the poor outcome in human ROSI is caused by the impaired oocyte-fertilizing ability, or, more specifically, the oocyte-activating capacity, of round spermatids collected from individual patients. However, this assumption is incompatible with a finding by Hewitson et al. (2000Go), who reported an extremely low efficiency of ROSI in the rhesus monkey (Macaca mulatta) whereas injection of elongated spermatids was successful in the same species. Because the stage at which spermatogenic cells acquire the oocyte-activating capacity is species-dependent (Fishel et al., 1997Go; Sofikitis et al., 1997Go; Yazawa et al., 2000Go; Ogonuki et al., 2001Go), the detailed examination of rhesus monkey round spermatids for oocyte-activating capacity is important for resolving the discrepancy between the two macaque species.

We performed ROSI using oocytes that matured in vitro as well as in vivo. No significant differences in percentages of pronucleus formation, cleavage, morula, and blastocyst formation were noted between the two groups. However, only a small percentage of oocytes matured from the germinal vesicle stage during this culture period, which varied from 9 to 30 h. The most serious problem we encountered was a fluctuation in the quality of oocytes, which varied greatly among different donor females. At present, it is very difficult to obtain monkey oocytes of good quality. Many oocytes at the time of collection are either overmatured or are too immature (at the germinal vesicle stage), and only a few immature oocytes undergo maturation in vitro. More reliable methods for ovarian stimulation or for in-vitro maturation are needed to obtain oocytes of good quality that will ensure consistent experimental outcomes. Such technical improvements may further increase the rates of normal fertilization and embryo development following ROSI and will make ROSI a common artificially assisted reproduction technique in non-human primates.

In cynomolgus monkeys, the fertilization rate by conventional IVF (assessed by pronucleus formation) is 57% (Sankai et al., 1994Go), whereas the fertilization rate by ROSI is 69% (the present study). This is because sperm with a high motility are necessary for successful IVF, while ICSI and ROSI do not require motility of male germ cells. It is also advantageous that cells which had been frozen–thawed by a simple method can also be used for ICSI into oocytes. Thus, in cynomolgus monkeys we obtained preimplantation embryos more easily by ROSI than by IVF.

This does not necessarily mean that offspring can be obtained easily by ROSI, because ours is the first known case of pregnancy of a cynomolgus monkey by ROSI; there have been no other reports of pregnancy following ROSI in a non-human primate. Sofikitis et al. (1996Go) reported that electrical stimulation of rabbit oocytes before round spermatid nuclei injection (ROSNI) had beneficial effects on oocyte activation, fertilization and development. Yazawa et al. (2000Go) reported that rabbit round spermatids activated mouse oocytes but could not induce Ca2+ oscillation; normal Ca2+ oscillation patterns were seen in only three of nine oocytes. In contrast, our earlier report (Ogonuki et al., 2001Go) showed that cynomolgus monkey round spermatids could activate mouse oocytes (57/60; 95%) with normal Ca2+ oscillation (10/14; 71%). From these two reports, we conclude that monkey round spermatids have a higher concentration of oocyte-activating factor or have a more mature form than rabbit round spermatids. If we use spermatids that have little or no oocyte-activating factor, such as those from mice or rabbits, electrical stimulation has a beneficial effect. We cannot predict whether electrical stimulation would produce good results for ICSI of cynomolgus monkey spermatids that have already matured and acquired oocyte-activating capacity.

Thus, although the ROSI technique has been established in many animals, the species in which ROSI is successful need chemical or electrical stimulation to activate the oocyte. Cynomolgus monkeys are the only species other than humans in which the round spermatids like human round spermatids have oocyte-activating factor. We must have an animal model that is similar to the infertile human with spermiogenic failure. We therefore believe that the cynomolgus monkey is a suitable model for humans.

In the colony of cynomolgus monkeys at our institute, spontaneous abortion rarely occurs, and at present we do not know the reason for the spontaneous abortion of the ROSI fetus. It may have been due to either technical or maternal factors. Nevertheless, in this study, we succeeded in the first post-implantation development of a ROSI fetus that lasted to day 103 of the 156 day pregnancy period of the cynomolgus monkey. Because the safety and effectiveness of round spermatid conception in humans are still far from certain because of the lack of substantial scientific evidence (Aslam et al., 1998Go; Sofikitis et al., 1998bGo), cynomolgus monkeys are valuable experimental models for assessing the application of this technique to treatment for human infertility. In the future, the ROSI technique will also be applicable to rescue of endangered wild animals in which IVF and ICSI have never been successful.


    Acknowledgements
 
This study was supported in part by grants from the Ministry of Health, Labour and Welfare of Japan and by special coordination funds for promoting Science and Technology of Japan.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on April 12, 2002; resubmitted on November 4, 2002; accepted on January 21, 2003.