Rhesus Monkey Oocytes Research Articles

Molecular control of mitochondrial function in developing rhesus monkey oocytes and preimplantation-stage embryos

The mitochondrion undergoes significant functional and structural changes, as well as an increase in number, during preimplantation embryonic development. The mitochondrion generates ATP and regulates a range of cellular processes, such as signal transduction and apoptosis. Therefore, mitochondria contribute to overall oocyte quality and embryo developmental competence. The present study identified, for the first time, the detailed temporal expression of mRNAs related to mitochondrial biogenesis in rhesus monkey oocytes and embryos. Persistent expression of maternally encoded mRNAs was observed, in combination with transcriptional activation and mRNA accumulation at the eight-cell stage, around the time of embryonic genome activation. The expression of these transcripts was significantly altered in oocytes and embryos with reduced developmental potential. In these embryos, most maternally encoded transcripts were precociously depleted. Embryo culture and specific culture media affected the expression of some of these transcripts, including a deficiency in the expression of key transcriptional regulators. Several genes involved in regulating mitochondrial transcription and replication are similarly affected by in vitro conditions and their downregulation may be instrumental in maintaining the mRNA profiles of mitochondrially encoded genes observed in the present study. These data support the hypothesis that the molecular control of mitochondrial biogenesis, and therefore mitochondrial function, is impaired in in vitro-cultured embryos. These results highlight the need for additional studies in human and non-human primate model species to determine how mitochondrial biogenesis can be altered by oocyte and embryo manipulation protocols and whether this affects physiological function in progeny.

Differential Expression of Cell Cycle Genes in Rhesus Monkey Oocytes and Embryos of Different Developmental Potentials1

Correct cell cycle regulation is especially challenging at the start of life. Ovulated oocytes must maintain meiotic arrest until fertilization, and then complete meiosis and initiate a series of modified cell divisions without growth. Moreover, myriad key developmental events, such as chromatin remodeling and transcriptional activation of the genome, are coordinated with each other via the cell cycle, particularly passage through the DNA synthesis phase (S Phase). We examined here the expression of more than 30 mRNAs related to cell cycle regulation in rhesus monkey oocytes and embryos and compared the expression of these mRNAs between oocytes and embryos of different developmental potentials. We find that the maternally inherited stores of cell cycle regulatory mRNAs are especially susceptible to disruption in cases of diminished oocyte and embryo quality in the rhesus monkey. In comparison to published mouse array data, we also observed striking species differences in the temporal expression patterns of many of these genes, suggesting that mechanisms of cell cycle control may differ and that the responses of oocytes and embryos to external insults may likewise differ.

Ubiquitin proteasome pathway gene expression varies in rhesus monkey oocytes and embryos of different developmental potential

Protein degradation via the ubiquitin-proteasome pathway (UPP) plays a key role in diverse aspects of cell physiology and development. In the early embryo, the UPP may play an important role in the transition from maternal to embryonic control of development. Disruptions in the UPP could thus compromise embryo developmental potential. Additionally, species-specific requirements may dictate diverse patterns of regulation of the UPP components. To investigate the expression of UPP components in a nonhuman primate embryo model, to compare expression between a primate and nonprimate species, and to determine whether disruption of this pathway may contribute to reduced developmental potential, we examined the expression of >50 mRNAs encoding UPP components in rhesus monkey oocytes and embryos. We compared this expression between the rhesus monkey and mouse embryo and between rhesus monkey oocytes and embryos of high, intermediate, and low developmental potential. We report here the temporal patterns of UPP gene expression in oocytes and during preimplantation development, including striking differences between the rhesus monkey and mouse. We also report significant differences in UPP gene expression correlating with oocyte and embryo developmental competence and associated with altered regulation of maternally inherited mRNAs encoding these proteins.

Effects of in vitro oocyte maturation and embryo culture on the expression of glucose transporters, glucose metabolism and insulin signaling genes in rhesus monkey oocytes and preimplantation embryos

Glucose plays a fundamental role during oogenesis and embryogenesis, satisfying the metabolic demands of oocytes and embryos, providing for stored energy reserves in the form of glycogen and supporting nucleotide biosynthesis via the pentose phosphate pathway. Glucose also contributes to the production of amino acids, glycosylated proteins and extracellular components. A detailed understanding of the molecular mechanisms that mediate and regulate glucose uptake and metabolism at different stages of oogenesis and preimplantation embryogenesis could greatly benefit the development of improved methods for in vitro oocyte maturation and in vitro embryo production. Although these processes have been examined in a variety of rodent and agricultural species, detailed information has not yet been described for non-human primates. In this study, we examined the expression of the genes encoding glucose transporters, glucose metabolism enzymes and potential regulators of glucose metabolism in rhesus monkey oocytes and embryos. The data reveal stage-specific regulation of expression of specific types of glucose transporters, stage-specific changes in expression of genes related to different pathways of glucose metabolism and temporal changes in the expression of mRNAs related to insulin signaling. Additionally, the data reveal significant differences in expression of some of these genes in cultured embryos as compared with flushed embryos and between oocytes and embryos obtained following different hormonal stimulation and oocyte maturation protocols.

Effects of in vitro maturation of monkey oocytes on their developmental capacity

The study of in vitro maturation (IVM) of rhesus monkey oocytes has important implications for biomedical research and human infertility treatment. In vitro-matured rhesus monkey oocytes show much less developmental potential than IVM oocytes of other species. Since about 1980 when rhesus monkey IVM, in vitro fertilization (IVF) and in vitro embryo culture (IVC) systems were established, numerous efforts have been made to improve the developmental competence of oocytes and to understand the mechanisms regulating oocyte maturation. This review describes recent progress in this area, particularly the effects of factors such as steroid hormones, energy substrates, amino acids, ovarian follicle status, maternal age and breeding season on the developmental competence, gene expression patterns and genome integrity of rhesus IVM oocytes.

Expression and downregulation of WNT signaling pathway genes in rhesus monkey oocytes and embryos

Mammalian WNT genes encode secreted glycoproteins that are conserved homologues of the Drosophila Wingless gene, which plays a crucial role in Drosophila development. Recently, WNT pathway signaling has been implicated in ovarian development, oogenesis, and early development. We sought to evaluate whether these genes may contribute to the formation of healthy human oocytes or embryos, and whether the expression of these genes could provide informative markers of human oocyte and embryo quality. To do this, we employed the primate embryo gene expression resource (PREGER; www.preger.org) to examine expression of mRNAs encoding 38 components of the WNT signaling pathway in rhesus monkey oocytes and embryos as a nonhuman primate model. We observed considerable conservation between rhesus monkey and mouse of expression of WNT, FZD, and effector gene mRNAs, and a generalized downregulation of genes encoding key components of the WNT signaling pathway during preimplantation development. Our results support a role for WNT signaling during oocyte growth or maturation, but not during preimplantation development. Additionally, we observed differences between in vitro cultured and in vivo developing blastocysts, indicating possible effects of culture on WNT signaling during the peri-implantation period.

The Primate Embryo Gene Expression Resource in embryology and stem cell biology

The analysis of temporal patterns of gene expression in embryos is an essential component of any research program seeking to understand molecular mechanisms that control development. Little is known of early regulatory mechanisms that operate in primate oocytes and preimplantation-stage embryos. Such studies have been hindered by the cost of obtaining, and limited availability of, non-human primate oocytes and embryos, and by ethical and legal constraints on studies of human embryos. Over the past 4 years we have established the Primate Embryo Gene Expression Resource (PREGER) to circumvent these limitations. A set of over 200 samples of rhesus monkey oocytes and embryos has been converted to cDNA libraries, which are, in turn, used for a variety of molecular analyses. Both the libraries and cDNA dot blots can be distributed free of charge to anyone wishing to study gene expression at these stages. This includes providing an inexpensive and rapid method for confirming and extending results of gene discovery approaches such as microarray analysis. PREGER includes an on-line resource with a database and other useful tools for embryologists. The resource is being expanded to incorporate samples from other species and from embryonic stem cells.

Mitochondrial DNA deletions in rhesus macaque oocytes and embryos

Mitochondria are the most abundant organelles in mammalian oocytes and early embryos. Mitochondrial DNA (mtDNA) mutations, including the common deletion, have been found in skeletal muscle fibres from aged rhesus macaques. The specific aims of this study were to determine whether the mitochondrial common deletion is present in rhesus oocytes after hormonal stimulation and in embryos generated by in vitro production, or whether this deletion is already present in the immature oocyte. Using a nested primer PCR strategy, we found a significant increase in the proportion of mtDNA deletions in stimulated oocytes and embryos from rhesus macaques, compared with mtDNA deletions in immature, unstimulated oocytes derived from necropsied ovaries of age-matched monkeys. The common deletion is larger in the rhesus (5704 bp) than in humans (4977 bp). Accumulation of mtDNA deletions in oocytes may contribute to mitochondrial dysfunction and impaired ATP production. We propose the rhesus to be an excellent model to assess the quality of gametes and embryos and their developmental competence in primates, including humans.

Developmental Regulation and In Vitro Culture Effects on Expression of DNA Repair and Cell Cycle Checkpoint Control Genes in Rhesus Monkey Oocytes and Embryos1

DNA repair is essential for maintaining genomic integrity, and may be required in the early embryo to correct damage inherited via the gametes, damage that arises during DNA replication, or damage that arises in response to exposure to genotoxic agents. The capacity of preimplantation stage mammalian embryos to repair damaged DNA has not been well characterized, particularly in primate embryos. In this study, we examined the expression of 48 mRNAs related to sensing different kinds of DNA damage, repairing that DNA damage, and controlling the cell cycle to provide an opportunity for DNA repair. The expression data reveal dynamic temporal changes, indicating a changing ability of the rhesus embryo to detect and repair different kinds of DNA damage. Low expression or overexpression of specific DNA repair genes may limit the ability of the embryo to respond to DNA damage at certain stages. Additionally, our data reveal that in vitro culture may lead to dysregulation of many such genes and a potentially impaired ability to repair DNA damage, thus affecting cellular viability and long-term embryo viability via effects on genome integrity. This effect of in vitro culture on nonhuman primate embryos may be relevant to assessing the potential advantages and disadvantages of prolonged in vitro culture of human embryos.

Effects of Follicle Size and Oocyte Maturation Conditions on Maternal Messenger RNA Regulation and Gene Expression in Rhesus Monkey Oocytes and Embryos1

The relationship between alterations in gene expression and differences in developmental potential in primate oocytes and embryos was examined. Oocytes from 3 sources were used for these studies: 1) in vivo-matured oocytes from monkeys stimulated with FSH and hCG, 2) in vitro-matured oocytes from large follicles of monkeys primed with FSH, and 3) in vitro-matured oocytes from small follicles from nonstimulated (NS) monkeys. Following in vitro fertilization, embryos from these oocytes displayed high, moderate, and low developmental competence, respectively. Oocytes from NS females displayed aberrant accumulation of a number of maternal mRNAs, followed by precocious loss of many maternal mRNAs by the 2-cell stage. Embryos from NS oocytes displayed alterations in expression of key transcription factors after the 8-cell stage. Oocytes and embryos from FSH-stimulated females also displayed alterations in gene expression relative to hCG-stimulated females, but these alterations were much less severe than those observed for NS oocytes and embryos. Our data are consistent with the hypothesis that continued development and maturation of the oocyte within the ovarian follicle in vivo facilitates the production of oocytes of the highest developmental potential, and that in vitro conditions may not support this process as effectively due to differences in the extracellular milieu. These observations are relevant to understanding the role of the in vivo environment on oocyte maturation, and the potential effects of in vitro maturation on human assisted reproduction methods.

The primate embryo gene expression resource: a novel resource to facilitate rapid analysis of gene expression patterns in non-human primate oocytes and preimplantation stage embryos.

Detailed molecular studies of preimplantation stage development in a suitable nonhuman primate model organism have been inhibited due to the cost and scarcity of embryos. To circumvent these limitations, we have created a new resource for the research community, designated as the Primate Embryo Gene Expression Resource (PREGER). The PREGER sample collection currently contains over 160 informative samples of oocytes, obtained from various sized antral follicles, and embryos obtained through a variety of different protocols. The PREGER makes it possible to undertake quantitative gene-expression studies in rhesus monkey oocytes and embryos through simple and cost-effective hybridization-based methods. The PREGER also makes available other molecular tools to facilitate nonhuman primate embryology. We used PREGER here to compare the temporal expression patterns of five housekeeping mRNAs and three transcription factor mRNAs between mouse and rhesus monkey. We observed noticeable differences in temporal expression patterns between species for some mRNAs, but clear similarities for others. Our results also provide new information related to genome activation and the effects of embryo culture conditions on gene expression in primate embryos. These results provide one illustration of how the PREGER can be employed to obtain novel insight into primate embryogenesis.

Expression of genes encoding chromatin regulatory factors in developing rhesus monkey oocytes and preimplantation stage embryos: possible roles in genome activation.

One of the most critical events of preimplantation development is the successful activation of gene transcription. Both the timing and the array of genes activated must be controlled. The ability to regulate gene transcription appears to be reduced just prior to the time of the major genome activation event, and changes in chromatin structure appear essential for establishing this ability. Major molecules that modulate chromatin structure are the linker and core histones, enzymes that modify histones, and a wide variety of other factors that associate with DNA and mediate either repressive or activating changes. Among the latter are chromatin accessibility complexes, SWI/SNF complexes, and the YY1 protein and its associated factors. Detailed information about the expression and regulation of these factors in preimplantation stage embryos has not been published for any species. In order to ascertain which of these factors may participate in chromatin remodeling, genome activation, and DNA replication during early primate embryogenesis, we determined the temporal expression patterns of mRNA encoding these factors. Our data identify the predominant members of these different functional classes of factors expressed in oocytes and embryos, and reveal patterns of expression distinct from those patterns seen in somatic cells. Among each of four classes of mRNAs examined, some mRNAs were expressed predominantly in the oocyte, with these largely giving way to others expressed stage specifically in the embryo. This transition may be part of a global mechanism underlying the transition from maternal to embryonic control of development, wherein the oocyte program is silenced and an embryonic pattern of gene expression becomes established. Possible roles for these mRNAs in chromatin remodeling, genome activation, DNA replication, cell lineage determination, and nuclear reprogramming are discussed.

95FIRST REPORT ON CLEAVAGE DEVELOPMENT FOLLOWING CRYOPRESERVATION OF ADULT AND PREPUBERTAL RHESUS MONKEY (MACACA MULATTA) OOCYTES

Cryopreservation of non-human primate oocytes would allow a more efficient management of biological resources for medical research and endangered species preservation. Despite previous attempts, no cleavage-stage development has been reported following cryopreservation of rhesus monkey (Macaca mulatta) or other non-human primate oocytes. The extreme chilling sensitivity of rhesus monkey oocytes (Songsasen N et al., 2002 Fertil. Steril. 77, 818–825) might be overcomed by vitrification methods. Our aim was to test the Solid Surface Vitrification (SSV, Dinnyes A et al., 2000 Biol. Reprod. 63, 513–518) method on metaphase II (MII)-stage rhesus monkey oocytes and to achieve successful fertilization of warmed oocytes. Oocytes were obtained from hormonally stimulated adult and unstimulated prepubertal females and matured in vitro for 36 to 48h as described by Zhang P et al. (2001 Biol. Reprod. 64, 1417–1421). The vitrification solution contained 35% ethylene glycol (EG), 5% polyvinyl pyrrolidone, and 0.4M trehalose in Tyrode-lactate (TL)-HEPES medium with 3mgmL−1 BSA added. Oocytes at MII-stage were equilibrated in 4% EG in TL-HEPES at 37°C for 10 to 12min and then exposed to the vitrification solution at 37°C for about 20s. Oocytes in 2-μL droplets of vitrification solution were dropped onto a metal surface at about −180°C where they were vitrified instantaneously. Warming was performed by moving the vitrified droplets into 0.3M trehalose at 37°C. Recovered oocytes were exposed to 0.15M and then 0.075M trehalose for 2min each and then rinsed three times in TL-HEPES. Warmed oocytes were fertilized in vitro and then cultured for 96h in 50-μL drops of mCMRL-1066 containing 20% FCS at 37°C in a humidified atmosphere of 5% CO2, 5% O2, and 90% N2, as described in details by Zhang (see above). A total of 21 MII oocytes were collected from 2 adult and 55 MII oocytes from 14 prepubertal animals. No difference has been found between the rate of adult-origin (15/19; 79%) v. prepubertal-origin (37/52; 71%) oocytes surviving the vitrification process without lysis (P>0.05, chi-square analyses). Following subsequent IVF, 1/15 (7%) adult-origin and 4/23 (17%) prepubertal-origin oocytes cleaved further, which was lower than that of the corresponding controls (1/2 (50%) and 1/3 (33%), respectively). The furthest development observed following cryopreservation was 16-cell stage, verified by counting of stained nuclei. This is the first report on cleavage-stage development of cryopreserved rhesus monkey oocytes, demonstrating the feasibility of vitrification and the potential for gene banking of non-human primate oocytes, even from prepubertal animals. Further experiments are needed to achieve higher rates of cryosurvival and progeny development. This research was supported by a Chinese-Hungarian Bilateral Technological and Scientific Collaboration Project (No. CHN14/02).

Causes of developmental failure of in-vitro matured rhesus monkey oocytes: impairments in embryonic genome activation.

Understanding the causes of developmental failure of in-vitro matured primate oocytes may lead to viable strategies for improving their developmental competence. The aims of this study were to determine whether the timely onset of embryonic genome activation among individual blastomeres of preimplantation macaque embryos is impaired by in-vitro maturation (IVM) of oocytes and whether these impairments are associated with developmental failure during the embryonically controlled period of preimplantation development. Genome activation among individual blastomeres was assessed using expression of fibrillarin as a marker of nucleolar transcription. Immature oocytes were obtained from rhesus monkeys following treatment with recombinant human FSH and matured in-vitro in one of two IVM media (CMRLa or CMRLb). In-vivo matured oocytes were obtained from FSH treated monkeys following administration of hCG. Oocytes were fertilized in vitro and either cultured for developmental studies or processed at the 8-12-cell stage for expression of fibrillarin. Developmental competence of embryos derived from in-vitro matured CMRLa oocytes was markedly (P < 0.05) impaired compared with those derived from in-vivo matured or in-vitro matured CMRLb oocytes. Developmental profiles were similar among the groups prior to the 8-cell stage. However, in embryos derived from in-vitro matured CMRLa oocytes, developmental failure increased significantly (P < 0.05) after the time of genome activation compared with those derived from in-vivo matured or in-vitro matured CMRLb oocytes. The mean percentages of non-activated blastomeres per embryo, as well as the proportions of embryos with at least one non-activated blastomere, or with no activated blastomeres, were all significantly (P < 0.05) greater in embryos derived from in-vitro matured CMRLa oocytes than in those derived from in-vivo matured or CMRLb oocytes. The relatively poor developmental competence of in-vitro matured primate oocytes is likely caused in part by failure in the timely onset of embryonic genome activation resulting from incomplete cytoplasmic maturation.

ICSI, male pronuclear remodeling and cell cycle checkpoints.

Intracytoplasmic sperm injection (ICSI; Palermo et al., 1992; Van Steirteghem et al., 1993) has heralded an era of enormous improvements in treating male infertility; it has resulted in the births of thousands of babies. In fact its success has led some clinicians to claim that male infertility is now “cured”, while others raise concerns over the global application of ICSI for all cases of in vitro fertilization (Tucker et al., 1995). These concerns may be, in part, based on the lack of supporting pre-clinical studies in relevant animal models (Ng et al., 1995; Yanagimachi et al, 1995). Others are worried about the possible long-term effects of ICSI on the offspring (Hamberger et al., 1998]; Moutel et al., 1999). While the current view of ICSI is one of cautious optimism, the development of a preclinical, non-human primate model has been established to address the safety of ICSI and other methods of assisted reproduction (Hewitson et al., 1998; Hewitson et al., 1999; Hewitson et al., 2001). Using advanced fluorescent imaging techniques, the cytoskeletal events that occur during rhesus fertilization have been examined after both in vitro fertilization (IVF) and ICSI. It was found that rhesus monkeys share many similarities with humans in terms of cytoskeletal and chromatin dynamics after fertilization by IVF (Simerly et al., 1995; Wu et al., 1996). However, rhesus oocytes fertilized by ICSI resulted in abnormal nuclear remodeling leading to asynchronous chromatin decondensation in the apical region of the sperm head, delaying the onset of DNA synthesis. The persistence of the acrosome and perinuclear theca on the apex of sperm introduced into the oocyte by ICSI may constrict the DNA in this region (Hewitson et al., 1999; Ramalho-Santos et al., 2000a). Despite these differentces, normal rhesus ICSI embryos have been produced and, after transfer to surrogate females, have resulted in several births (Hewitson et al., 1999; Hewitson et al., 2001)

Amino acid requirements for maturation of rhesus monkey (Macacca mulatta) oocytes in culture

This study evaluated the effects of different amino acid formulations on supporting meiotic and cytoplasmic maturation of rhesus monkey (Macacca mulatta) oocytes in vitro. Five hundred and forty-six cumulus-oocyte complexes (COCs) aspirated from unstimulated adult monkey follicles (> or = 1000 microm in diameter) were cultured in either modified Connaught Medical Research Laboratories 1066 medium (mCMRL-1066) or in one of eight chemically defined media (modified basic medium 5 supplemented with 5.5 mmol glucose l(-1), 0.003 mmol pantothenic acid l(-1) and different amino acid formulations) as below: (1) modified basic medium 5 (mBM5) containing no amino acid; (2) mBM5 + 0.2 mmol glutamine l(-1); (3) mBM5 + 11 amino acids from hamster embryo culture medium 6 (HECM-6) (11 AA); (4) mBM5 + Eagle's non-essential amino acids (NEA); (5) mBM5 + NEA + 0.2 mmol glutamine l(-1); (6) mBM5 + Eagle's essential amino acids (EA) without glutamine; (7) mBM5 + EA + 0.2 mmol glutamine l(-1); (8) mBM5 + Eagle's 20 amino acids (20 AA) + 0.2 mmol glutamine l(-1); and (9) mCMRL-1066 (control). All media contained FSH, LH, oestradiol and progesterone. After maturation, mature oocytes were subjected to the same fertilization and embryo culture procedures. COCs matured in treatment 5 had greater potential to progress to metaphase II (66%; P < 0.05) than did those in treatments 1 (37.3%), 2 (48.3%), 3 (41%), 6 (41%) and 9 (43%). Oocytes matured in treatment 8 had the best morula (53%) and blastocyst (18%) developmental responses (P < 0.05). The lowest (P < 0.05) morula and blastocyst developmental responses were obtained from COCs matured in treatments 1 (0%) and 6 (8%). The other media supported intermediate embryonic development (range 11-38% of morula and blastocyst). These results indicate that the choice of amino acids affects the competence of oocyte maturation and that Eagle's 20 AA with 0.2 mmol glutamine l(-1) is more efficient than the other amino acid formulations for maturation of rhesus monkey oocytes.

Amino acid requirements for maturation of rhesus monkey (Macacca mulatta) oocytes in culture.

This study evaluated the effects of different amino acid formulations on supporting meiotic and cytoplasmic maturation of rhesus monkey (Macacca mulatta) oocytes in vitro. Five hundred and forty-six cumulus-oocyte complexes (COCs) aspirated from unstimulated adult monkey follicles (> or = 1000 microm in diameter) were cultured in either modified Connaught Medical Research Laboratories 1066 medium (mCMRL-1066) or in one of eight chemically defined media (modified basic medium 5 supplemented with 5.5 mmol glucose l(-1), 0.003 mmol pantothenic acid l(-1) and different amino acid formulations) as below: (1) modified basic medium 5 (mBM5) containing no amino acid; (2) mBM5 + 0.2 mmol glutamine l(-1); (3) mBM5 + 11 amino acids from hamster embryo culture medium 6 (HECM-6) (11 AA); (4) mBM5 + Eagle's non-essential amino acids (NEA); (5) mBM5 + NEA + 0.2 mmol glutamine l(-1); (6) mBM5 + Eagle's essential amino acids (EA) without glutamine; (7) mBM5 + EA + 0.2 mmol glutamine l(-1); (8) mBM5 + Eagle's 20 amino acids (20 AA) + 0.2 mmol glutamine l(-1); and (9) mCMRL-1066 (control). All media contained FSH, LH, oestradiol and progesterone. After maturation, mature oocytes were subjected to the same fertilization and embryo culture procedures. COCs matured in treatment 5 had greater potential to progress to metaphase II (66%; P < 0.05) than did those in treatments 1 (37.3%), 2 (48.3%), 3 (41%), 6 (41%) and 9 (43%). Oocytes matured in treatment 8 had the best morula (53%) and blastocyst (18%) developmental responses (P < 0.05). The lowest (P < 0.05) morula and blastocyst developmental responses were obtained from COCs matured in treatments 1 (0%) and 6 (8%). The other media supported intermediate embryonic development (range 11-38% of morula and blastocyst). These results indicate that the choice of amino acids affects the competence of oocyte maturation and that Eagle's 20 AA with 0.2 mmol glutamine l(-1) is more efficient than the other amino acid formulations for maturation of rhesus monkey oocytes.

Permeability characteristics and osmotic sensitivity of rhesus monkey (Macaca mulatta) oocytes.

Permeability characteristics and sensitivity to osmotic shock are principal parameters that are important to derive procedures for the successful cryopreservation of mammalian oocytes. The osmotically inactive volume of rhesus monkey oocytes was determined by measuring their volumes in the presence of hypertonic solutions of sucrose from 0.2 to 1.5 mol/l, compared with their volume in isotonic TALP-HEPES solution. Boyle-van't Hoff plots at infinite osmolality indicated that the non-osmotic volumes of immature and mature oocytes were 20 and 17% respectively. Osmotic responses of oocytes exposed to 1.0 mol/l solutions of glycerol, dimethylsulphoxide (DMSO) and ethylene glycol (EG) were determined. Rhesus monkey oocytes appeared to be less permeable to glycerol than to DMSO or to EG. Sensitivity of oocytes to osmotic shock was determined by exposing them to various solutions of EG (0.1 to 5.0 mol/l) and then abruptly diluting them into isotonic medium. Morphological survival, as measured by membrane integrity, of oocytes diluted out of EG depended significantly on the concentration of EG (P < 0.01). Determination of permeability characteristics and sensitivity to osmotic shock of rhesus oocytes will aid in the derivation of procedures for their cryopreservation.

Effect of chilling on the organization of tubulin and chromosomes in rhesus monkey oocytes

Objective: To determine the effects of chilling on the organization and distribution of tubulin and chromosomes in rhesus monkey oocytes. Design: Comparative laboratory study. Setting: Academic research laboratory. Animal(s): Eight adult female rhesus monkeys (Macaca mulatta) aged 6–16 years. Intervention(s): A total of 171 oocytes retrieved from eight rhesus monkeys were separated into nine groups. One group of control oocytes was held at 37°C during the experiment. Four groups of oocytes were rapidly cooled to 0°C and held for 1, 5, 10, or 30 minutes and then fixed and stained. Four other groups of oocytes were cooled to 0°C, held for 1, 5, 10, or 30 minutes, warmed and incubated at 37°C for 60 minutes, and then fixed and stained. Main Outcome Measure(s): Organization of cytoskeleton and chromosomes. Result(s): Exposure of rhesus oocytes to 0°C for only 1 minute resulted in complete depolymerization of tubulin. Incubation of chilled oocytes at 37°C for 60 minutes caused partial restoration of tubulin, although most oocytes exhibited abnormal alignment of chromosomes and disorganized meiotic spindles. Conclusion(s): We conclude that rhesus monkey oocytes are extremely sensitive to chilling injury. Their successful cryopreservation may require rapid cooling to outpace this injury.

Parthenogenetic activation of rhesus monkey oocytes and reconstructed embryos.

This study determines the efficiency of sequential calcium treatments (electroporation or ionomycin) combined with protein synthesis (cycloheximide) or phosphorylation inhibitors (6-dimethylaminopurine) or the specific maturation promoting factor (MPF) inhibitor, roscovitine, in inducing artificial activation and development of rhesus macaque parthenotes or nuclear transfer embryos. Exposure of oocytes arrested at metaphase II (MII) to ionomycin followed by 6-dimethylaminopurine or to electroporation followed by cycloheximide and cytochalasin B induced pronuclear formation and development to the blastocyst stage at a rate similar to control embryos produced by intracytoplasmic sperm injection. Parthenotes did not complete meiosis or extrude a second polar body, consistent with their presumed diploid status. In contrast, oocytes treated sequentially with ionomycin and roscovitine extruded the second polar body and formed a pronucleus at a rate higher than that observed in controls. Following reconstruction by nuclear transfer, activation with ionomycin/6-dimethylaminopurine resulted in embryos that contained a single pronucleus and no polar bodies. All nuclear transfer embryos activated with ionomycin/roscovitine contained one large pronucleus. However, a third of these embryos emitted one or two polar bodies, clearly containing chromatin material. In summary, we have identified simple yet effective methods of oocyte or cytoplast activation in the monkey, ionomycin/6-dimethylaminopurine, electroporation/cycloheximide/cytochalasin B, and ionomycin/roscovitine, which are applicable to parthenote or nuclear transfer embryo production.