A macaque model for studying mechanisms controlling oocyte development and maturation in human and non-human primates

R.D. Schramm1,3 and B.D. Bavister1,2

1 Wisconsin Regional Primate Research Center, 1223 Capitol Court, Madison, WI 53715 and 2 Department of Animal Health and Biomedical Sciences, University of Wisconsin, Madison, USA


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A model to study mechanisms controlling nuclear and cytoplasmic maturation of primate oocytes is being developed in our laboratory. The high incidence of pregnancy failure in women following in-vitro fertilization (IVF) may be partly attributed to inadequate cytoplasmic maturation of oocytes. Advancement of knowledge of mechanisms controlling primate oocyte maturation would have important implications for treatment of human infertility, and would potentially increase numbers of viable non-human primate embryos for biomedical research. Use of a non-human primate model to study oocyte and embryo biology avoids legal, ethical and experimental limitations encountered in a clinical situation. Using this model, the meiotic and developmental capacity of oocytes from three sources have been compared: (i) in-vivo matured oocytes from monkeys stimulated with follicle-stimulating hormone (FSH) and human chorionic gonadotrophin, (ii) in-vitro matured oocytes from monkeys primed with FSH, and (iii) in-vitro matured oocytes from non-stimulated monkeys. This work demonstrates that oocyte developmental competence is likely acquired both during follicle development, before meiotic resumption, and during meiotic progression, concurrent with nuclear maturation. Potential causes of developmental failure of in-vitro matured oocytes, implications for human infertility, and future strategies to study the regulation of primate oocyte maturation are discussed.

Key words: cytoplasmic maturation/in-vitro maturation/in-vivo maturation/macaque oocyte/nuclear maturation


    Introduction
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 Abstract
 Introduction
 Conclusions and implications for...
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Over 60 years ago, it was demonstrated that mammalian oocytes undergo meiotic maturation in vitro when removed from their follicular environment (Pincus and Enzmann, 1935Go). Extrusion of the first polar body has commonly been used as morphological evidence of the completion of oocyte maturation. 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. In addition to the nuclear changes that accompany meiotic maturation, important cytoplasmic changes must occur during maturation that render the oocyte competent to be fertilized and undergo normal embryonic development (Thibault et al., 1987Go). Thus, extrusion of the first polar body is not an adequate indicator of the completion of oocyte maturation, because it is indicative only of nuclear maturation. The acquisition of developmental competence by oocytes, which is now commonly referred to as `cytoplasmic maturation', is poorly understood.

Although clinical human IVF has been practised successfully since 1978, live births per cycle are still relatively low, approximately 20%. Since the majority of in-vitro fertilized human embryos fail to implant and maintain secretion of human chorionic gonadotrophin (HCG), inadequate or incomplete cytoplasmic maturation of oocytes, leading to developmentally incompetent embryos, likely contributes significantly to the high incidence of pregnancy failure. The development of a consistently successful in-vitro maturation (IVM) procedure for the production of developmentally competent human oocytes would have important implications for assisted reproduction for infertile women. 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 collected from non-stimulated women have resulted in the birth of normal offspring following transfer (Cha et al., 1991Go; Trounson et al., 1994Go; Barnes et al., 1995Go; Russell et al., 1997Go), and a few immature oocytes collected from non-stimulated women (Barnes et al., 1995Go) and monkeys (Schramm and Bavister, 1996aGo) have developed into blastocysts in vitro, IVM is far from being a successful procedure for the production of developmentally competent primate oocytes. Both human and non-human primate oocytes were first matured in vitro over 30 years ago (Edwards, 1965Go). However, the developmental competence of primate oocytes matured in vitro is markedly inferior to that of their in-vivo (stimulated) matured counterparts (Bavister et al., 1983Go; Boatman, 1987Go; Wolf et al., 1989Go; Lanzendorf et al., 1990Go; Cha et al., 1991Go, 1992Go; Morgan et al., 1991Go; Zhang et al., 1993Go; Trounson et al., 1994Go; Barnes et al., 1995Go) and to that of in-vitro matured oocytes from other species, including mice (Eppig and Schroeder, 1989Go; Hirao et al., 1990Go; van de Sandt et al., 1990Go; Eppig et al., 1992Go), rats (Vanderhyden and Armstrong, 1990Go), sheep (Staigmiller and Moor, 1984Go), cattle (Liebfried-Rutledge et al., 1987Go; Frei et al., 1989Go; Mochizuki et al., 1991Go; Rose and Bavister, 1992Go; Kobayahi et al., 1994Go; Keskintepe and Brackett, 1996Go) and pigs (Mattioli et al., 1988Go; Funahashi et al., 1994Go; Hirao et al., 1994Go). This is primarily due to our poor understanding of the molecular processes involved in cytoplasmic maturation of primate oocytes and the lack of tools to study this process. Over the past decade, some progress has been made in developing culture conditions that support cytoplasmic maturation of oocytes from rodents (Eppig and Schroeder, 1989Go; Hirao et al., 1990Go; Vanderhyden and Armstrong, 1990Go; Eppig et al., 1992Go) and domestic species (Liebfried-Rutledge et al., 1987Go; Mattioli et al., 1988Go; Frei et al., 1989Go; Mochizuki et al., 1991Go; Funahashi et al., 1994Go; Hirao et al., 1994Go; Kobayahi et al., 1994Go; Keskintepe and Brackett, 1996Go; Rose-Hellekant et al., 1998Go). However, information on the molecular, biochemical and physiological processes involved in the acquisition of developmental competence by oocytes is sparse, particularly in primates. This situation is complicated by uncertainty about whether in-vitro matured oocytes from primates are intrinsically compromised, or whether current culture conditions per se are inadequate to support cytoplasmic maturation of oocytes.

Currently, evaluation of the developmental capacity of oocytes following fertilization is the most useful means to assess cytoplasmic maturation. Unfortunately, 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 ethical or legal constraints on the study of human fertilization and embryonic development. Furthermore, results obtained from studies conducted using rejected human oocytes or oocytes that have failed to fertilize or cleave are unreliable. 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, 1987Go; Winston and Johnson, 1992Go). Thus, 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.

A non-human primate model would also be a valuable asset for studies on maternal age-related developmental impairments and post-ovulatory ageing of oocytes. Several case studies indicate that infertility in older women can be reversed by oocyte donation from younger women (Navot et al., 1991Go; Rotsztein and Asch, 1991Go; Sauer et al., 1992Go), indicating that their oocytes are inherently defective. In women, the frequency of post-implantation spontaneous abortion increases with age of oocyte donor (Levran et al., 1991Go). The incidence of chromosomal anomalies in spontaneous aborti increases with maternal age (Hassold et al., 1984Go; Hassold and Jacobs, 1984Go; Stein, 1985Go). However, some chromosome errors may not be detected in spontaneous abortions or live births because they are lethal prior to implantation, but may be present at a relatively high frequency in oocytes (Angell et al., 1983Go) and possibly in preimplantation embryos (Angell et al., 1983Go; Plachot et al., 1988Go). It has been reported that 50% of human IVF cleavage stage preimplantation embryos, over all age groups of women, exhibit chromosomal abnormalities, although little is known about chromosome features in human embryos later than 2 days post-fertilization (Benkhalifa et al., 1993Go; Almeida and Bolton, 1996Go). Post-ovulatory ageing of the egg may also contribute to developmental failure (Fugo and Butcher, 1966Go), particularly in humans as the frequency of intercourse declines with advancing age (Hassold and Jacobs, 1984Go; Jongbloet, 1986Go) or during prolonged co-incubation with sperm during IVF (Wramsby et al., 1987Go; Tarin et al., 1991Go). However, controlled studies on post-maturation ageing of oocytes in primates are scarce (Edwards, 1980Go). Development of strategies for treatment of several human diseases that compromise fertility, such as polycystic ovarian syndrome, would also benefit from studies using non-human primates.

Development of a non-human primate model for oocyte maturation
Rhesus monkeys (Macaca mulatta) and other macaque species are often the animal model of choice for biomedical research, particularly developmental biology, due to their genetic and physiological similarity to humans (VandeBurg and Williams-Blangero, 1996Go). A macaque model to study the mechanisms regulating nuclear and cytoplasmic maturation of primate oocytes in vivo and in vitro is being developed in our laboratory. The strategy behind the development of this model is based upon varying the proportion of maturation occurring in vivo or in vitro. Theoretically, the most accurate baseline data on `normal' oocyte maturation would be obtained from mature (metaphase II; MII) oocytes aspirated from the dominant pre-ovulatory follicle of a spontaneous cycle. However, these oocytes are extremely difficult to obtain without knowing the precise time of the initiation of the luteinizing hormone (LH) surge, and the limited amount of data obtained from single oocytes makes it difficult to justify the expense and effort to collect them. Thus, normal baseline data for in-vivo matured oocytes has been obtained from gonadotrophin-stimulated monkeys using various follicle-stimulating hormone (FSH) preparations to recruit multiple ovarian follicles followed by treatment with HCG to induce oocyte maturation. Over the past several years, our laboratory has used purified porcine FSH (Vetrepharm Inc, London, ON, Canada), human urinary FSH (Metrodin®; Serono Laboratories Inc., Norwell, MA, USA), and most recently, recombinant human FSH (Organon Inc., West Orange, NJ, USA). In vivo (stimulated) matured oocytes exhibit a relatively high developmental capacity in vitro, and when transferred to recipients, have resulted in birth of normal offspring (Bavister et al., 1984Go; Wolf et al., 1989Go; Lanzendorf et al., 1990Go; Weston et al., 1996Go). Although complete normality of oocytes can be unequivocally demonstrated only by birth of normal offspring following embryo transfer, this approach is far too inefficient in monkeys to be useful as an experimental endpoint. Therefore, for comparative purposes, development to the blastocyst stage in vitro is typically used as a developmental endpoint, although data that rely solely on this criterion must be interpreted with caution.

In contrast to in-vivo matured oocytes, use of immature (germinal vesicle stage; GV) oocytes obtained from excised ovaries of non-stimulated monkeys enables us to study oocyte maturation entirely in vitro under completely controlled conditions, and provides us with the opportunity to study the growth and acquisition of meiotic and developmental competence in oocytes from small antral or even pre-antral follicles. Recovery of immature oocytes from monkeys primed with FSH, but not treated with HCG, also allows us to study maturation in vitro, but has the advantage that oocytes are fully grown and obtained from larger, more developed follicles that are not potentially in any stage of atresia. This enables us to study oocyte maturation per se, rather than oocyte growth and development. Oocytes derived from FSH-treated monkeys and matured in vitro are valuable for comparison to results from oocytes obtained from small undeveloped follicles of non-stimulated monkeys, as well as those matured in vivo (stimulated). For experimental purposes, monkeys can be primed with FSH for progressively shorter time intervals before collection of GV oocytes, in order to study the role of follicular development on acquisition of developmental competence.

To illustrate the utility of this model, Table IGo depicts the meiotic and developmental competence of oocytes obtained from the three sources described above. Group I: in-vivo matured oocytes from gonadotrophin-stimulated (FSH and HCG) monkeys; group II: in-vitro matured oocytes from FSH-primed monkeys and group III: in-vitro matured oocytes from non-stimulated monkeys. Representative photographs of oocytes from each of the three groups are shown in Figure 1Go. Mature oocytes collected from monkeys treated with HCG (group I;a) typically exhibit an expanded cumulus oophorus and radiating corona cells. Immature oocytes collected from FSH-primed monkeys (group II;b) are similar in appearance to in-vivo matured oocytes, having a uniform colour and texture to the cytoplasm and being generally free of vacuoles or vesicles, although less developmentally competent. Immature oocytes obtained from excized ovaries of non-stimulated monkeys (group III;c) typically have cytoplasm that is more coarse or granular in appearance and they often exhibit a high incidence of vacuoles (~70%; Schramm et al., 1993) and reduced developmental potential, compared to in-vivo (stimulated) matured oocytes or oocytes obtained from FSH-primed monkeys.


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Table I. Meiotic and developmental capacity of rhesus macaque oocytes matured in vivo (stimulated) or in vitroa
 


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Figure 1. Representative oocytes recovered from (a) monkeys treated with follicle stimulating hormone and human chorionic gonadotrophin (metaphase II stage), (b) monkeys treated with follicle stimulating hormone alone (germinal vesicle stage), and (c) excised ovaries of non-stimulated monkeys (germinal vesicle stage). Note vacuoles (arrowheads) present in oocytes from excised ovaries of non-stimulated monkeys (c).

 
In-vivo matured oocytes (gonadotrophin-stimulated monkeys)
Currently, the most developmentally competent oocytes that can be efficiently obtained from macaques are in-vivo (stimulated) matured oocytes recovered from animals stimulated with gonadotrophins (FSH) for development of multiple antral follicles, followed by treatment with HCG to induce oocyte maturation. Oocytes such as these complete meiosis, fertilize and develop in vitro at a relatively high rate, with 61% and 37% or more of MII oocytes reaching the morula and blastocyst stages respectively (Table IGo). When transferred to synchronized recipients, these oocytes have resulted in birth of normal offspring (Bavister et al., 1984Go; Wolf et al., 1989Go; Lanzendorf et al., 1990Go; Weston et al., 1996Go). Relatively little cleavage arrest occurs before the 8-cell stage, similar to previous findings for human embryos derived from in-vivo (stimulated) matured oocytes (Hardy et al., 1989aGo, bGo; Artley et al., 1992Go; Handyside, 1992Go). This has limited our ability to evaluate the developmental competence of human oocytes matured in vitro, since embryos are typically transferred at the 4- to 8-cell stage (Cha et al., 1991Go, 1992Go; Trounson et al., 1994Go), although this practice may ultimately change in favour of blastocyst transfer (Barnes et al., 1995Go; Gardner and Lane, 1997Go). It is conceivable that primate embryos derived from in-vivo (stimulated) matured oocytes are less vulnerable to developmental arrest before the major activation of the embryonic genome (6- to 8-cell stage; Tesarik et al., 1986a, b, 1988; Braude et al., 1988; Schramm and Bavister, 1999) or before they enter the uterus in vivo (Bavister, 1995Go), compared to later developmental stages.

Approximately one quarter of the oocytes from FSH- and HCG-treated monkeys have completed nuclear maturation at the time of follicular aspiration (27–32 h post-HCG), and another 50% extrude their first polar body within 12 h post-collection. The time of first polar body extrusion varies considerably among and within rhesus monkeys, ranging from approximately 24–40 h following HCG injection (R.D.Schramm, unpublished data). The degree of maturity of rhesus oocytes at collection is positively correlated with their potential for fertilization and embryonic development (Lanzendorf et al., 1990Go; Morgan et al., 1990Go), indicating either that slower maturing oocytes are less developmentally competent, as found for bovine oocytes (Dominko and First, 1997Go), or that completion of the final hour of maturation in vitro may compromise their subsequent fertilization and developmental potential (Schramm and Bavister, 1994Go). The time window between the completion of maturation and insemination may also be critical for normal chromosome segregation, fertilization, cleavage and preimplantation development, as shown for rodent and cattle oocytes (Yanagimachi and Chang, 1961Go; Fugo and Butcher, 1966Go; Hunter, 1980Go, 1989Go; Juetten and Bavister, 1983Go). Wolf et al. (1996) have shown that although greater proportions of MII oocytes can be recovered when the collection time is delayed until 27–36 h following HCG, fertilization of these MII oocytes is reduced at 36 h. This may indicate that either post-maturity ageing or increased exposure to HCG in vivo may alter some aspect of cytoplasmic maturation that is subsequently detrimental to fertilization. In summary, evaluation of in-vivo (stimulated) matured oocytes can thus provide normal baseline data for comparison to results from in-vitro matured oocytes, which is essential for understanding the normal mechanisms regulating oocyte maturation and the potential impairments in this process incurred by maturing oocytes in vitro.

In-vitro matured oocytes (FSH-primed 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 (stimulated) matured oocytes are obtained, but have not been exposed to HCG. When compared to oocytes from non-stimulated monkeys, FSH-priming of monkeys enhanced the incidence of nuclear maturation and fertilization, and improved the competence of in-vitro matured oocytes to develop through the 8-cell (49%), morula (29%) and blastocyst (7%) stages following fertilization (Table IGo), resulting in production of the first in-vitro matured/in-vitro fertilized primate blastocysts in vitro (Schramm and Bavister, 1994Go). Ongoing pregnancies have recently been established using this strategy in rhesus monkeys (R.D.Schramm, unpublished data). FSH priming similarly enhanced the meiotic competence of squirrel monkey (Yeoman et al., 1994Go) and human (Wynn et al., 1998Go) oocytes matured in vitro, although subsequent embryonic development was not evaluated. This indicates that in primates, cytoplasmic maturation of oocytes may require more time to complete than nuclear maturation and may commence during follicle growth and development, well in advance of meiotic resumption. Protein synthesis in human oocytes occurs throughout the follicular phase until and beyond the time of germinal vesicle breakdown (Schultz et al., 1988Go). However, although FSH priming enhances the developmental potential of in-vitro matured oocytes, competence to develop to blastula stages remains substantially greater for oocytes matured in vivo (stimulated) (Boatman, 1987Go; Wolf et al., 1989Go; Schramm and Bavister, 1996bGo). This indicates that the intrafollicular environment may be critically important during the last hour of pre-ovulatory development, while current IVM conditions are inadequate to replace the natural milieu. This conclusion may have implications for human IVF practice, in which follicular oocytes may often be aspirated before completion of cytoplasmic maturation. Both nuclear and cytoplasmic maturation were greater for cumulus-enclosed oocytes than for oocytes denuded of cumulus cells before IVM (Schramm and Bavister, 1994Go), further illustrating that developmental competence is partially acquired during meiotic progression. Interestingly, the meiotic and developmental competence of denuded oocytes from FSH-primed monkeys were greater than those of cumulus-enclosed oocytes from non-stimulated monkeys (Schramm and Bavister, 1994Go), demonstrating the importance of the oocyte's developmental history before IVM. However, it is unclear whether FSH-priming improves the meiotic and developmental competence of in-vitro matured oocytes by improving cytoplasmic maturation or by rescuing follicles from atresia.

In-vitro matured oocytes (non-stimulated monkeys)
Collection of immature oocytes from non-stimulated monkeys enables us to study maturation under well-defined conditions in vitro. Comparison of these oocytes to those from FSH-primed monkeys matured under identical conditions in vitro allows us to investigate the effects of follicle and oocyte development on meiotic and developmental competence of oocytes. Immature oocytes from non-stimulated monkeys are obtained by dissection of follicles from excised ovaries, and are cultured approximately 36 h in modified CMRL-1066 culture medium (Boatman, 1987Go) containing 20% bovine calf serum, under mineral oil (Schramm and Bavister, 1994Go). The mean time required to complete meiosis in vitro is 34 h (Schramm et al., 1994Go), and can range from 24–48 h (Edwards, 1965Go; Suzuki and Mastroianni, 1966Go; Morgan et al., 1991Go; Alak and Wolf, 1994Go). Both the meiotic and developmental competence of in-vitro matured oocytes from non-stimulated monkeys are relatively poor, with less than 50% completing nuclear maturation and <4% and 0% reaching the morula and blastocyst stages respectively (low controls; Table IGo). There are several likely explanations for the poor meiotic and developmental capacity of these oocytes. One explanation may be that the oocytes themselves may be defective. These oocytes are often dark and granular in appearance, and approximately 70% of them contain one or more cytoplasmic vacuoles or vesicles. It is unknown what causes these vacuoles, or to what degree they may contribute to the poor developmental capacity of oocytes from non-stimulated monkeys. There is some evidence that removal of ovaries before collection of oocytes may increase the incidence of vacuoles (R.D.Schramm, unpublished data), perhaps due to loss of blood flow to the follicles. However, vacuolated oocytes are often recovered inadvertently, perhaps from atretic follicles, during aspiration of oocytes from gonadotrophin stimulated monkeys (R.D.Schramm, unpublished data), indicating that this phenomenon may be a normal occurrence in macaques. Immature human oocytes obtained from non-stimulated women frequently exhibit degenerate chromatin configurations (Racowski and Kaufman, 1992Go), which can also be associated with follicular atresia. Because only ~50% of granulosa cells from follicles of non-stimulated monkeys are viable, compared to ~90% of those from FSH-primed monkeys (Schramm and Bavister, 1996aGo), the presence of vacuoles may be associated with some degree of follicular atresia, thus reducing the viability of the oocyte. However, healthy-appearing, non-vacuolated oocytes from non-stimulated monkeys are generally also developmentally incompetent. Thus, inadequate cytoplasmic maturation of oocytes from small undeveloped antral follicles may be an alternative explanation for the poor developmental competence of oocytes from non-stimulated monkeys, perhaps resulting from insufficient accumulation of maternally-derived transcripts and proteins.

The relationships of follicular and oocyte diameters to meiotic and developmental competence of primate oocytes have been reported previously (Adachi et al., 1982Go; Tsuji et al., 1985Go; Lefevre et al., 1989Go; Schramm et al., 1993Go; Schramm and Bavister, 1995Go). In rhesus monkeys, antrum formation begins when follicles reach approximately 200 µm in diameter. Oocytes within follicles of this size are not fully grown and less than 9% of them are meiotically competent (Schramm et al., 1993Go). Previous studies in rodents (Iwamatsu and Yanagimachi, 1975Go; Bar-Ami and Tsafriri, 1981Go; Mattson and Albertini, 1989Go) and domestic species (Tsafriri and Channing, 1975Go; Motlik et al., 1984Go; Motlik and Fulka, 1986Go) indicate that acquisition of meiotic competence occurs in fully grown oocytes and is associated with the condensation of chromatin around the nucleolus, commonly referred to as nucleolar encapsulation or rimming (Schramm et al., 1993Go). Rimming is associated with the appearance of other M-phase characteristics in oocytes, such as a diminution of cytoplasmic microtubules in conjunction with the formation of multiple microtubule organizing centres and expression of M-phase-specific phosphoproteins (Wickramasinghe et al., 1991Go). In rhesus monkeys, oocytes obtain maximum size when follicles reach approximately 500 µm in diameter, but only 50% of them exhibit nucleolar encapsulation, and only 18% are competent to complete meiotic maturation. In follicles greater than 1 mm, however, ~85% of oocytes exhibit nucleolar encapsulation and 55% of them are meiotically competent (Schramm et al., 1993Go). Thus, meiotic competence increases with follicle size and is not strictly associated with maximum oocyte diameter or antrum formation, but may be associated with nucleolar encapsulation or rimming, which increases with follicle size, in fully grown oocytes. Nevertheless, rimming alone is not a sufficient indicator of meiotic competence as 30% of rimmed oocytes fail to mature (Schramm et al., 1993Go). With respect to developmental competence, while ~30% of oocytes from antral follicles 1.0–2.5 mm in diameter developed to the 5- to 8-cell stage, only 7% of those from follicles 0.7–0.99 mm in diameter reached the 5- to 8-cell stage (Schramm and Bavister, 1995Go). Similarly, human embryos derived from immature oocytes obtained from small antral follicles had a slower rate of development and a higher incidence of cleavage arrest than those obtained from larger follicles (Barnes et al., 1996Go). FSH-priming of monkeys, which enhances antral follicle growth (3–6 mm follicles), improves the meiotic and developmental competence of oocytes (Schramm and Bavister, 1994Go). Therefore, fully grown primate oocytes from antral follicles are not all equally competent to mature and develop completely, but acquire both meiotic and developmental competence with increasing antral follicle size, similar to data reported for oocytes from cattle (Pavlok et al., 1992Go), pigs (Motlik et al., 1984Go; Motlik and Fulka, 1986Go) and gonadotrophin-stimulated women (Simmonetti et al., 1985; Zhang et al., 1993Go). It is important to emphasize that although oocytes obtained from non-stimulated and FSH-primed monkeys have been compared under identical IVM conditions, it is probable that culture conditions that support cytoplasmic maturation of oocytes from FSH-primed monkeys may be inappropriate or inadequate to support cytoplasmic maturation of oocytes from non-stimulated monkeys.

Environmental factors affecting nuclear and cytoplasmic maturation of primate oocytes in vitro
In rodents and domestic species, the developmental competence of in-vitro matured oocytes has been improved by culture with gonadotrophins, steroids, growth factors and granulosa cells. These strategies have improved the developmental capacity of oocytes from non-stimulated macaques as well, but in general, have not been as successful as they have been with other species. It remains to be determined whether this is due to species differences in the regulation of oocyte maturation or to a reduced ability of primate oocytes obtained from relatively small undeveloped follicles to respond to hormones, growth factors or other signals.

Gonadotrophins and granulosa cells
Unlike non-primate species (Moor and Trounson, 1977Go; Younis et al., 1989Go; Johnston et al., 1989Go; Wiemer et al., 1991Go; Mattioli et al., 1991Go; Galli and Moore, 1991), gonadotrophins alone did not enhance the incidence of nuclear maturation of in-vitro matured oocytes from non-stimulated monkeys (Morgan et al., 1991Go; Schramm et al., 1994Go; Schramm and Bavister, 1995Go). However, addition of granulosa cells from non-stimulated macaques to the culture containing gonadotrophins improved the meiotic capacity of oocytes (Schramm and Bavister, 1995Go), indicating that gonadotrophin enhancement of nuclear maturation during IVM of macaque oocytes may be mediated largely via membrana granulosa cells, as found for bovine oocytes (Sirard and Bilodeau, 1990aGo,bGo). Paracrine factors from granulosa cells may elevate cyclic adenosine monophosphate in oocytes, via cumulus cells, thereby promoting acquisition of meiotic competence (Eppig, 1996Go).

In contrast to their synergistic effects upon nuclear maturation, both gonadotrophins and granulosa cells alone had separate effects upon cytoplasmic maturation. Gonadotrophins improved activation and early cleavage events through the 8-cell stage in oocytes from non-stimulated monkeys (Morgan et al., 1991Go; Schramm and Bavister, 1995Go), similar to results for human oocytes (Prins et al., 1987Go; Zhang et al., 1993Go). Unlike gonadotrophins, culture of oocytes from non-stimulated monkeys with their own granulosa cell complements in the absence of gonadotrophins (not shown in Table IGo) during IVM had no effects on development through the early cleavage stages, but improved their subsequent development to the morula stage (Schramm and Bavister, 1995Go). Furthermore, culture of oocytes with granulosa cells from FSH-primed, but not non-stimulated, monkeys in the presence of gonadotrophins improved development to the blastocyst stage, resulting in production of the first blastocysts obtained from in-vitro matured oocytes from non-stimulated rhesus monkeys (Schramm and Bavister, 1996bGo). In contrast, cytoplasmic maturation of human oocytes collected from women during the early follicular phase of normal menstrual cycles was not improved by culture with granulosa cells obtained from superstimulated women before HCG administration (Trounson et al., 1994Go). However, in this study, development was not evaluated beyond the 8-cell stage. Although culture of oocytes with gonadotrophins and granulosa cells mimics, to some degree, the follicular environment during maturation in vivo, their meiotic and developmental capacity remains inferior to that of oocytes matured in vivo (Bavister et al., 1983Go; Boatman, 1987Go; Wolf et al., 1989Go; Lanzendorf et al., 1990Go). It is plausible that specialized functions of cumulus cells on oocyte competence are lost in vitro, or that cell-to-cell contact between granulosa and cumulus cells may be required to further improve the developmental capacity of in-vitro matured primate oocytes.

Alternatively, although the source of granulosa cells may be important, macaque oocytes from small unstimulated follicles obtained relatively early in their growth process may respond poorly to granulosa cell signals during IVM. Accordingly, neither gonadotrophins nor granulosa cells had any effects upon oocytes from antral follicles less than 1 mm in diameter (Schramm and Bavister, 1995Go). A relative lack of granulosa cell gonadotrophin receptors in these early stages of follicular development (Richards, 1980Go) may explain the differential responsiveness of oocytes from these two size classes of follicles. Thus, it is plausible that oocytes from larger, more developed follicles of FSH-primed females may be more responsive to stimulatory signals from granulosa cells than oocytes from non-stimulated females. It has been reported that GV oocytes collected from gonadotrophin-stimulated women, then cultured with their accompanying granulosa cells during IVM, showed improved fertilization over controls (Dandekar et al., 1991Go). However, granulosa cells obtained from both stimulated women following HCG and from non-stimulated women with polycystic ovarian syndrome enhanced blastulation of mouse oocytes obtained from gonadotrophin-stimulated donors (Anderiesz and Trounson, 1995Go). A major problem with interpreting these data, as well as improving the developmental competence of in-vitro matured oocytes, is that it is largely unknown how granulosa cells support cytoplasmic maturation in vivo or in vitro. Factors emanating from granulosa cells include steroids (Moor and Trounson, 1977Go; McNatty et al., 1979Go; Osborn and Moor, 1983Go; Erickson et al., 1991Go; Anderiesz and Trounson, 1995Go; Tornell et al., 1995Go), growth factors (Hammond et al., 1985Go; Maruo et al., 1993Go), and other compounds in vitro that potentially enhance cytoplasmic maturation of oocytes. Identification of somatic signals that affect cytoplasmic maturation may be a key to future improvements in the quality of oocytes matured in vitro (Moor et al., 1998Go). Development of chemically-defined culture media that can support nuclear and cytoplasmic maturation of primate oocytes would facilitate studies on the regulation of these processes in primates.

Steroids
Although primate granulosa cells secrete a variety of steroid hormones, specific roles of steroids in primate oocyte maturation are unclear (Lobo et al., 1985Go; Morgan et al., 1990Go; Zelinski-Wooten et al., 1993Go; Schramm and Bavister, 1995Go). Nuclear and cytoplasmic maturation of oocytes has been related to higher follicular fluid concentrations of both oestradiol and progesterone in gonadotrophin-stimulated monkeys (Morgan et al., 1990Go) and women (Botero-Ruiz et al., 1984Go; Lobo et al., 1985Go; Kreiner et al., 1987Go). Oestradiol is well known for its effects upon granulosa cell proliferation and function in rodents (Richards, 1980Go). Oestrogen receptors (ERs) have been localized to granulosa cells in rodents and domestic species (Richards, 1975Go; Glass et al., 1984Go), and have recently been classified as ERß (Kuiper et al., 1996Go). Although ERs were not detected in pre-antral, pre-ovulatory or peri-ovulatory follicles of rhesus monkeys (Hild-Petito et al., 1988Go), possible effects of oestradiol via ERß have not yet been investigated in macaque follicles. In contrast, human and baboon granulosa cells were positive for ERs (Iwai et al., 1990Go; Billiar et al., 1992Go). Moreover, ER mRNA has been detected in mature human oocytes (Wu et al., 1993Go), and both P450C17 (Tamura et al., 1992Go), and 3ß-HSD activity (Suzuki et al., 1983Go) have been detected in immature human oocytes, suggesting potential autocrine–paracrine roles for steroids in primate oocyte maturation. Treatment of monkeys with trilostane, a 3ß-HSD inhibitor, which reduces progesterone and oestrogen levels, did not affect meiosis, but impaired fertilization (Hibbert et al., 1996Go; Zelinski-Wooten et al., 1994Go). Likewise, fertilization, but not oocyte nuclear maturation, was hindered in a patient with very low intrafollicular oestradiol levels resulting from a partial 17, 20-desmolase deficiency (Pellicer et al., 1991Go). However, exposure of oocytes to prolonged periods of exogenous oestradiol in vivo reduced subsequent quality and viability of human oocytes (Russell et al., 1997Go). Thus, neither oestradiol nor progesterone seem to be required for nuclear maturation, but may play a role in cytoplasmic maturation of primate oocytes. In contrast, androgens are detrimental to nuclear maturation in primate oocytes (McNatty et al., 1979Go; Zelinski-Wooten et al., 1993Go), and reduce the developmental competence of mouse oocytes in vitro (Anderiesz and Trounson, 1995Go). Taken together, these findings indicate that primate oocytes are highly sensitive to incorrect steroid signals.

Growth factors
Granulosa cell production of growth factors, such as epidermal growth factor or insulin-like growth factor-I, is thought to be important for cytoplasmic maturation of oocytes (Coskun et al., 1991Go; Harper and Brackett, 1993Go; Maruo et al., 1993Go; Xia et al., 1994Go; Eppig and O'Brien, 1996Go), and may enhance granulosa cell metabolism (Wynn et al., 1998Go). However, studies on effects of growth factors on primate oocyte maturation are scant, and experiments have not been designed adequately (Das et al., 1991Go; Gomez et al., 1993Go). Other autocrine or paracrine factors, such as inhibins and activins, may also be involved in primate oocyte maturation. In primates, inhibins are produced by granulosa cells during the follicular phase (Hillier et al., 1989Go; Brannian et al., 1992Go), and their concentrations increase with follicle size during normal and stimulated cycles (McLachlan et al., 1987; Fraser et al., 1989Go; Hughes et al., 1990Go). Although inhibin has been reported to block meiotic resumption in rat oocytes (O et al., 1989Go), culture of immature macaque oocytes with inhibin had no effect upon meiotic resumption, but the combination of inhibin and activin improved nuclear maturation and fertilization (Alak et al., 1996Go).

Stage of cycle
The role of the stage of the reproductive cycle from which oocytes are obtained on their meiotic and developmental competence is not clear and may differ among species. In cattle, the stage of the oestrous cycle during which oocytes are obtained has no effect upon meiotic or developmental competence of oocytes in vitro (Leibfried and First, 1979; First and Parrish, 1987Go). However, in domestic cats, meiotic competence is greater for oocytes obtained from follicular versus luteal phase donors (Johnston et al., 1989Go). In primates, conflicting results have been obtained. Some studies (Barnes et al., 1996Go) have indicated that the developmental capacity of human primary oocytes is higher if recovered from women with regular menstrual cycles relative to those with irregular cycles or anovulation associated with polycystic ovarian disease. Other studies in primates (Alak and Wolf, 1994Go) reported that oocytes (>=0.3 mm follicles) obtained from non-stimulated monkeys during the early follicular phase were more competent to complete nuclear maturation than those from the later follicular phase or from the luteal phase. In contrast, human oocytes obtained from small (1–4 mm) follicles were more competent to complete nuclear maturation (Tsuji et al., 1985Go) and cleave (Cha et al., 1992Go) if obtained during the luteal phase, although this may not be true of oocytes from large follicles. Similarly, results from our laboratory indicate that meiotic and developmental competence was greater for oocytes (0.7–1.0 mm follicles) obtained from luteal versus follicular phase monkeys, although monkeys were not classified as early or late follicular phase (Schramm and Bavister, 1995Go). The reasons for these possible conflicting results are unknown, but may be related to sizes of follicles selected and degrees of follicular atresia encountered, as well as differences in the dynamics of follicle growth and development among species.

Potential molecular causes of developmental failure of in-vitro matured primate oocytes
There are many potential causes of developmental failure of in-vitro matured oocytes. Exposure to inappropriate hormonal regimes in vivo or to suboptimal culture conditions during maturation in vitro may result in gross abnormalities or subtle imperfections in oocytes that can lead to developmental failure during pre- or post-implantation embryogenesis (Moor et al., 1998Go). It has been speculated that developmental failure of many embryos before `genome activation' may be the result of premature exhaustion of some maternally-derived and developmentally relevant molecules before they can be replaced with embryo-derived products following the onset of embryonic transcription (Tesarik, 1987Go; Winston et al., 1991Go; Hardy et al., 1993Go; Tesarik, 1994Go). Likewise, because multinucleate blastomeres occur in human embryos as early as the 2- to 4-cell stage and primarily during the third cleavage division, it has been suggested that deficiencies of maternal proteins derived from the oocyte may lead to blocked cytokinesis (Winston et al., 1991Go; Hardy et al., 1993Go). Therefore, a deficiency of epigenetic programming of the maternal genome during IVM may lead to developmental failure during the maternally-controlled period of embryogenesis (Latham, 1999Go).

Because activation of the embryonic genome is also thought to be under the control of maternally inherited molecules (Tesarik et al., 1987Go; Tesarik, 1994Go; Wang and Latham, 1997Go), incomplete or inadequate oocyte maturation may lead to a delay or failure in the onset of embryonic transcription (Tesarik, 1987Go, 1994Go; Winston et al., 1991Go; Hardy et al., 1993Go), which may be a direct cause of developmental failure. Autoradiographic studies of 3H-uridine incorporation into in-vivo matured/in-vitro fertilized human embryos obtained from gonadotrophin-stimulated women have shown that nucleolar synthesis of rRNA, indicative of genome activation, can be absent in up to 30% of blastomeres within many 8-cell and some early morula stage embryos (Tesarik et al., 1986bGo, 1988Go). Furthermore, nucleolar incorporation of 3H-uridine was observed in 8-cell bovine embryos derived from oocytes from large antral follicles, but was totally absent in embryos derived from oocytes from small antral follicles, indicating a slower onset or even failure of genomic transcription (Pavlok et al., 1993Go). These results were highly correlated with the reduced developmental capacity of embryos derived from oocytes from small antral follicles (Pavlok et al., 1992Go). Similar results have been obtained in rhesus monkey embryos derived from in-vitro matured oocytes (R.D.Schramm, unpublished data), suggesting that inadequate cytoplasmic maturation of oocytes may subsequently lead to delayed or asynchronous activation of nucleolar transcription among blastomeres. In addition, some maternally-inherited messages may be involved in the control of cellular events in relatively late stages of human preimplantation development, after genome activation has occurred (Tesarik, 1989Go).

Alternatively, although failure of genome activation may contribute to developmental failure, some in-vitro fertilized human embryos are impaired in their ability to transcribe some, but not all, embryonically-encoded genes (Artley et al., 1992Go). Therefore, cleavage arrest may not necessarily be due to complete failure of onset of transcriptional activity, but failure only to transcribe specific genes. Latham (1999) further suggested that abnormal programming of the nuclei by the ooplasm, before and after fertilization, is likely to contribute to developmental failure at later stages. Therefore, it is reasonable to speculate that developmental failure may often result from insufficient accumulation of developmentally important maternally-derived molecules during either oocyte development or maturation, subsequently leading to impairments in the transition from maternal to embryonic control of development, or to abnormal programming of the embryonic nuclei.


    Conclusions and implications for assisted reproductive technology
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 Abstract
 Introduction
 Conclusions and implications for...
 References
 
Although acquisition of meiotic and developmental competence occurs in fully grown oocytes at or shortly after antrum formation in non-primate species, this is clearly not the case for macaque oocytes. Thus, it may be more meaningful to describe the relationship between acquisition of oocyte competence and follicular diameter with respect to the species-specific size of a dominant follicle at the time of ovulation. For example, bovine oocytes used for IVM are typically selected from follicles 2–8 mm in diameter, ~18–47% of ovulatory size. Oocytes from follicles in this size range were the most competent to develop into blastocysts in vitro (Pavlok et al., 1992Go). Similarly, oocytes from non-stimulated marmoset monkeys, which are more competent to mature and develop in vitro than those from rhesus monkeys, are obtained from follicles 0.6–2.0 mm in diameter (Gilchrist et al., 1995Go, 1997Go), ~25–80% of ovulatory size. In contrast, oocytes obtained from non-stimulated rhesus monkeys are from follicles that generally range from 0.7–2.0 mm in diameter, ~8–22% of ovulatory size. Therefore, oocytes from non-stimulated rhesus monkeys may be less developmentally advanced than their counterparts from larger follicles and those from other species, including marmoset monkeys.

Based upon this supposition, perhaps a mere 36 h of in-vitro culture, which is sufficient for completion of nuclear maturation, is not adequate time for primate oocytes, particularly those from small antral follicles, to complete cytoplasmic maturation, which may be initiated during follicle growth and development. This seems reasonable in light of the fact that the primate follicular phase, entailing the final development of the pre-ovulatory follicle and oocyte, is approximately 12–14 days long compared to 1–5 days in rodents and most domestic species. Thus, it would not be surprising if primate oocytes required a much longer period of time to acquire full developmental competence compared to those of rodents and many domestic species. In this respect, the large dominant follicle of the spontaneous menstrual cycle in non-stimulated monkeys may contain an oocyte whose developmental capacity is similar to that of oocytes from FSH-primed monkeys. Perhaps oocytes from larger, more developed follicles have acquired a more sufficient store of maternally-derived, developmentally important mRNAs and proteins than those from smaller, less developed follicles (Howe and Solter, 1979Go; Latham et al., 1991Go), which may be critical during the maternally-driven period of embryogenesis (Bachvarova and De Leon, 1980Go; McLaren, 1981Go; Tesarik, 1987Go, 1994Go; Winston et al., 1991Go; Hardy et al., 1993Go), as well as for proper activation of genomic transcription (Tesarik, 1987Go, 1994Go; Wang and Latham, 1997Go). If true, perhaps culture for several days with gonadotrophins, and other potential factors involved in cytoplasmic maturation, in the presence of hypoxanthine and dbcAMP, which inhibit meiotic resumption (Warikoo and Bavister, 1989Go; Tornell and Hillensjo, 1993Go), would be reasonable strategies for enhancing cytoplasmic maturation in primates. This approach is currently being pursued (R.D.Schramm and B.D.Bavister, unpublished data).

Although IVF and embryo transfer have become routine procedures for women seeking infertility therapy, women exhibiting premature ovarian failure may not respond to gonadotrophin stimulation, while women with polycystic ovarian syndrome may be extremely sensitive to exogenous gonadotrophins and at risk of hyperstimulation syndrome (Trounson et al., 1994Go). Thus, in many clinical situations, collection of immature oocytes during natural cycles may be a reliable alternative to ovarian stimulation if production of developmentally competent oocytes by IVM could be accomplished routinely. Additionally, due perhaps to asynchronous follicular development, immature oocytes, as well as oocytes that have not completed nuclear maturation at the time of retrieval, are frequently collected along with mature oocytes from gonadotrophin-stimulated women. Conditions supporting IVM of these oocytes would potentially increase the numbers of embryos available for transfer. Perfection of IVM technology could eliminate use of expensive hormones and unpleasant daily injections, with additional benefits such as avoidance of potential disturbances to the uterine environment and possible chromosomal aberrations of oocytes resulting from administration of exogenous hormones (Wramsby et al., 1987Go; Wramsby and Fredga, 1987Go). IVM in association with cryopreservation of oocytes would be valuable for rescuing genetic material from cancer patients prior to chemo- or radio-therapy and for banking donated oocytes for establishing pregnancy in older women or for potential use in cytoplasmic transfers (Cohen et al., 1997Go, 1998Go). Thus, development of culture conditions that support nuclear and cytoplasmic maturation of immature primate oocytes will have important applications for human IVF programmes, increase the numbers of viable non-human primate oocytes/embryos for biomedical research and enable the rescue of valuable genetic material from rare species of non-human primates. The non-human primate model that our laboratory is developing is invaluable and essential to the formulation of successful strategies for improvements in the treatment of human infertility and advancement of assisted reproductive technologies, particularly IVM of primate oocytes.


    Acknowledgments
 
The work from our laboratory included in this review was done as part of the National Cooperative Program on Non-Human In vitro Fertilization and Preimplantation Development, and was funded by the National Institute of Child Health and Human Development, NIH, through cooperative agreement HD-22023. We thank Drs Mary Zelinski-Wooten, Cathi Vandevoort, Randy Prather, Keith Latham, Richard Tasca, and John Eppig for their helpful comments on this manuscript.


    Notes
 
3 To whom correspondence should be addressed Back


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Submitted on February 23, 1999; accepted on July 9, 1999.