Wisconsin Regional Primate Research Center, University of Wisconsin, Madison, Wisconsin, USA
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Abstract |
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Key words: embryo transfer/in-vitro fertilization/in-vitro maturation/oocyte/rhesus monkey infant
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Introduction |
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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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In-vitro maturation
Germinal vesicle (GV) stage oocytes enclosed by 23 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 ~1520 individual intact sheets of mural granulosa cells recovered from complementary follicular aspirates from large (56 mm) follicles (Figure 1a). After 24 h of culture, oocytes were examined at 3 h intervals until extrusion of the first polar body (2433 h). Mature oocytes were inseminated 24 h after first polar body extrusion.
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Results |
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Discussion |
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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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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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References |
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Submitted on May 15, 2000; accepted on July 21, 2000.