Human Reproduction, Vol. 15, No. 11, 2411-2414,
November 2000
© 2000 European Society of Human Reproduction and Embryology
Birth of rhesus monkey infant after transfer of embryos derived from in-vitro matured oocytes: Short communication
Wisconsin Regional Primate Research Center, University of Wisconsin, Madison, Wisconsin, USA
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Abstract |
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Although strategies for in-vitro maturation of oocytes from rodents and domestic species have been relatively successful, application of these techniques to primates has not met with similar success. Currently, evaluation of the developmental capacity of oocytes following fertilization is the only reliable means to assess cytoplasmic maturation. Although rhesus monkey blastocysts have previously been produced from in-vitro matured oocytes, full developmental competence has not been demonstrated by term development. Here we report the birth of the first non-human primate infant derived from in-vitro matured oocytes.
Key words: embryo transfer/in-vitro fertilization/in-vitro maturation/oocyte/rhesus monkey infant
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Over 60 years ago, Pincus and Enzmann (1935) demonstrated that mammalian oocytes undergo meiotic maturation in-vitro when removed from their follicular environment. However, with the advent of in-vitro fertilization (IVF) techniques, it became evident that, in many species, although these in-vitro matured oocytes had completed meiosis, they were not all competent to be fertilized or undergo normal embryonic development (Thibault, 1987). The acquisition of developmental competence by oocytes, which is now commonly referred to as `cytoplasmic maturation', is poorly understood.
Development of a consistently successful in-vitro maturation (IVM) procedure for the production of developmentally competent human oocytes would have important clinical implications for assisted reproductive technology, and would be a particularly valuable infertility treatment for women with polycystic ovarian syndrome (Trounson et al., 1994
). Application of this technology to non-human primates would potentially increase numbers of viable oocytes and embryos for biomedical research, including cloning and production of transgenic monkeys as models for human disease.
Although a limited number of in-vitro matured/in-vitro fertilized human oocytes have resulted in the birth of normal offspring following transfer (Cha et al., 1991; Trounson et al., 1994; Barnes et al., 1995; Russell et al., 1997), the developmental competence of primate oocytes matured in vitro remains markedly inferior to that of their in-vivo matured counterparts (Bavister et al., 1983; Boatman, 1987; Wolf et al., 1989; Lanzendorf et al., 1990; Cha et al., 1991, 1992; Morgan et al., 1991; Zhang et al., 1993; Schramm and Bavister, 1994, 1995, 1996a, Schramm and Bavister, b; Trounson et al., 1994; Barnes et al., 1995), and to that of in-vitro matured oocytes from rodents and domestic species (Staigmiller and Moor, 1984; Leibfried-Rutledge et al., 1987; Mattioli et al., 1988; Eppig and Schroeder, 1989; Frei et al., 1989; Hirao et al., 1990; Vanderhyden and Armstrong, 1990; Eppig et al., 1992; Funahashi et al., 1994; Hirao et al., 1994; Kobayahi et al., 1994; Keskintepe and Brackett, 1996). This is primarily due to our poor understanding of the molecular processes involved in the acquisition of developmental competence in primate oocytes. Furthermore, there are substantial differences in the physiology of the regulation of oocyte maturation and embryogenesis between rodents and primates, such that direct extrapolation of information is not reliable (Bavister, 1987; Winston and Johnson, 1992). Because many reproductive studies with human oocytes or embryos cannot presently be done in a controlled experimental setting, and the types of experiments that are feasible are limited due to legal constraints on the study of human fertilization and embryonic development, a non-human primate model is essential for understanding the regulation of primate oocyte maturation and developing successful techniques for the routine production of developmentally competent in-vitro matured primate oocytes.
Currently, evaluation of the developmental capacity of oocytes following fertilization is the only reliable means to assess cytoplasmic maturation. For comparative purposes, development to the blastocyst stage in vitro is commonly used as a developmental endpoint for assessing cytoplasmic maturation. However, data that rely solely on this criterion must be interpreted with caution because subtle imperfections incurred during maturation are often not expressed until after implantation. Complete normality of oocytes can be unequivocally demonstrated only by birth of normal offspring following embryo transfer. Although this approach is far too inefficient to be used routinely as an experimental endpoint, demonstration of term development is essential for complete validation of IVM procedures. Although rhesus monkey blastocysts have previously been produced from in-vitro matured oocytes (Schramm and Bavister, 1994, 1996a), full developmental competence has not been demonstrated by term development. Here we report the birth of the first non-human primate infant derived from in-vitro matured oocytes.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Animals and oocyte recovery
Two rhesus macaques (Macaca mulatta) received twice daily injections of recombinant human follicle stimulating hormone (FSH) (Organon Inc., West Orange, NJ, USA) for 8 days, beginning on days 1–3 of the menstrual cycle, as described previously (Schramm and Bavister, 1994
, 1996b). Oocytes were aspirated laparoscopically (Bavister et al., 1983) 1 day after the final day of FSH treatment into TL-Hepes medium (37°C) containing 5% bovine calf serum (BCS; Hyclone Laboratories Inc., Logan, UH, USA) and 10 IU/ml heparin (Elkins-Sinn, Cherry Hill, NJ, USA).
In-vitro maturation
Germinal vesicle (GV) stage oocytes enclosed by 2–3 layers of condensed cumulus cells were selected for IVM and placed into culture within 1 h of retrieval. Denuded oocytes were not used in the present study. Oocytes (5/drop) were cultured at 37°C in 25 µl drops of modified CMRL-1066 medium (CMRL; Gibco Life Technologies, Grand Island, NY, USA; Boatman, 1987), containing 20% BCS overlaid with mineral oil. Culture drops included human gonadotrophins (5 µg/ml FSH, bioactivity 1683 IU/mg and 10 µg/ml luteinizing hormone (LH), bioactivity 4015 IU/mg, provided by the National Institute of Diabetes and Digestive and Kidney Diseases), and ~15–20 individual intact sheets of mural granulosa cells recovered from complementary follicular aspirates from large (5–6 mm) follicles (Figure 1a). After 24 h of culture, oocytes were examined at 3 h intervals until extrusion of the first polar body (24–33 h). Mature oocytes were inseminated 2–4 h after first polar body extrusion.
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IVF/embryo culture/embryo transfer
Sperm capacitation and IVF were done as described previously (Bavister et al., 1983
; Boatman and Bavister, 1984). Spermatozoa (2x105/ml) and oocytes (5/drop) were co-incubated for 12–14 h at 37°C in 100 µl drops of TALP medium overlaid with mineral oil, and then transferred into 50 µl drops of G1/G2 culture medium (Gardner and Lane, 1997) overlaid with mineral oil and examined for presence of pronuclei. Embryos having two pronuclei were cultured for 24–40 h post-insemination in G1 medium, and were then surgically transferred to the oviducts of synchronized recipient monkeys 2 days after the day of the LH peak (Seshagiri et al., 1993, 1994). Embryos were selected for transfer based upon morphology (round, equally-sized blastomeres and absence of fragmentation). Blood samples were collected twice weekly from day 16 post-transfer to day 34 post-transfer and assayed for chorionic gonadotrophin. Pregnancy was assessed by ultrasonography on days 20 (yolk sac) and 28 (fetal heartbeat) post-transfer. |
Results |
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Numbers of immature cumulus enclosed oocytes (CEO) recovered, and the maturation, fertilization and implantation rates for each of the donor monkeys are shown in Table I
. All aspirated oocytes were GV stage at recovery. A total of 50 oocytes was recovered from two animals, although only 16 of these oocytes were considered cumulus-enclosed. The time interval to first polar body extrusion ranged from 23–30 h.
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Each of two synchronized recipient monkeys received four embryos (2-cell or 5- to 8-cell stage) on the side ipsilateral to the ovulatory stigma. In each of the recipients, a single yolk sac was revealed by ultrasonography on day 20 following embryo transfer, and a fetal heartbeat was detected on day 28 (Figure 1b
). The first pregnancy was lost 2 weeks prior to delivery from unknown causes. Concentrations of CG on days 18, 27, 31 and 34 post-transfer were 26, 2528, 69 and 17 ng/ml respectively. Necropsy of the aborted fetus and placenta did not reveal any apparent abnormalities. In the second pregnancy, concentrations of CG on days 16, 19, 23 and 26 post transfer were 37, 663, 3493 and 3308 ng/ml respectively. A single healthy male rhesus monkey infant weighing 0.5 kg (Figure 1c) was recovered by Caesarean section 158 days after embryo transfer. The remaining 2-cell stage embryo from animal 92098, that was not transferred, developed into a blastocyst in culture. |
Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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In this paper, we report the birth of the first non-human primate derived from in-vitro matured oocytes, demonstrating the full developmental competence of some in-vitro matured macaque oocytes. Although strategies for IVM of oocytes from rodents and domestic species have been relatively successful, application of these techniques to primates has not met with similar success. In the present study, immature oocytes were recovered from FSH-primed rather than non-stimulated monkeys. Priming of monkeys with FSH before collection of immature oocytes allows for recovery of GV oocytes from large follicles that have developed under the same conditions as those from which in-vivo matured oocytes are obtained, but have not been exposed to human chorionic gonadotrophin (HCG). This enables us to study oocyte maturation per se, rather than oocyte growth and development. We previously demonstrated that FSH-priming of monkeys improved the developmental competence of in-vitro matured oocytes, resulting in production of the first primate blastocysts derived from IVM oocytes (Schramm and Bavister, 1994
). In this report, we have demonstrated that some in-vitro matured oocytes obtained from FSH-primed monkeys are capable of term development. Concentrations of CG followed a pattern similar to that of a normal pregnancy, indicating timely implantation and trophectoderm function. Ultrasonographs on days 20 and 28 of pregnancy appeared normal with respect to the stage of gestation. Gestation length was similar to that of a natural pregnancy, and birthweight was in the normal range. The validation of the normality of in-vitro matured oocytes derived from FSH-primed monkeys reported here will facilitate our efforts to develop a successful procedure for IVM of human oocytes, and enable us to develop further strategies for growth and maturation of oocytes from non-stimulated monkeys.
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Acknowledgments |
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The authors gratefully acknowledge Steve Eisele, Lisa Knowles, Melissa Brown and Michele Shotzko of Reproductive Services for menstrual cycle monitoring, hormone injections, blood sampling, sperm collection, and ultrasonography. We are grateful to Denny Mohr and Michele Shotzko for surgical assistance, Fritz Wegner for hormone assay services and Dr Iris Bolton for performing the c-sections. We further thank Bob Becker for photographic services and Dr Catherine VandeVoort for critical review of the manuscript.
This work was supported by research grant NIH RR00167. This is publication number 40-010 of the WRPRC.
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Notes |
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1 To whom correspondence should be addressed at: Wisconsin Regional Primate Research Center, 1223 Capitol Court, Madison, WI 53715, USA. E-mail: schramm{at}primate.wisc.edu
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Submitted on May 15, 2000; accepted on July 21, 2000.
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