Primate Oocytes Research Articles

The non-human primate oocyte and embryo as a model for women, or is it vice versa?

The role of the non-human primate (NHP) oocyte and embryo in translational research is considered here including both in vitro activities directly involving oocytes or embryos as well as animal studies that impact reproductive function. Reasons to consider NHPs as animal research models along with their limitations are summarized. A case is made that in limited instances, such as in the development and application of the assisted reproductive technologies or in the study of embryonic stem cells, the human oocyte and embryo have acted as models for the monkey. The development of strategies for the preservation of fertility is used as an example of ongoing research in the non-human primate that cannot be conducted in women for ethical reasons. In animal studies, monitoring reproductive potential, responses to embryonic stem cell transplantation, along with translational research in the field of contraceptive development for women are considered as subjects that benefit from the availability of a NHP model.

Effects of ovarian stimulation, with and without human chorionic gonadotrophin, on oocyte meiotic and developmental competence in the marmoset monkey ( Callithrix jacchus)

A reliable ovarian stimulation protocol for marmosets is needed to enhance their use as a model for studying human and non-human primate oocyte biology. In this species, a standard dose of hCG did not effectively induce oocyte maturation in vivo. The objectives of this study were to characterize ovarian response to an FSH priming regimen in marmosets, given without or with a high dose of hCG, and to determine the meiotic and developmental competence of the oocytes isolated. Ovaries were removed from synchronized marmosets treated with FSH alone (50 IU/d for 6 d) or the same FSH treatment combined with a single injection of hCG (500 IU). Cumulus–oocyte complexes (COCs) were isolated from large (>1.5 mm) and small (0.7–1.5 mm) antral follicles. In vivo-matured oocytes were subsequently activated parthenogenetically or fertilized in vitro. Immature oocytes were subjected to in vitro maturation and then activated parthenogenetically. Treatment with FSH and hCG combined increased the number of expanded COCs from large antral follicles compared with FSH alone (23.5 ± 9.3 versus 6.4 ± 2.7, mean ± S.E.M.). Approximately 90% of oocytes surrounded by expanded cumulus cells at the time of isolation were meiotically mature. A blastocyst formation rate of 47% was achieved following fertilization of in vivo-matured oocytes, whereas parthenogenetic activation failed to induce development to the blastocyst stage. The capacity of oocytes to complete meiosis in vitro and cleave was positively correlated with follicle diameter. A dramatic effect of follicle size on spindle formation was observed in oocytes that failed to complete meiosis in vitro. Using the combined FSH and hCG regimen described in this study, large numbers of in vivo matured marmoset oocytes could be reliably collected in a single cycle, making the marmoset a valuable model for studying oocyte maturation in human and non-human primates.

Prevention of zona hardening in non-human primate oocytes cultured in protein-free medium

Development of a protein-free medium for in vitro maturation of oocytes that prevents zona hardening is essential for the study of components that affect the maturation process. Immature macaque oocytes were cultured in modified CMRL medium with serum protein or without protein [with or without polyvinyl alcohol (PVA)] for 24 hours. Sperm penetration of oocytes cultured for 24 hours prior to insemination was poor in CMRL medium alone, but was dramatically improved in CMRL medium with the addition of either PVA or BCS. In the second experiment, in vivo matured oocytes were cultured in CMRL with PVA or serum for 6 hours prior to insemination. The incidence of fertilization and embryo development to the blastocyst stage were similar in CMRL with PVA. These results indicate that fertilization failure occurs when macaque oocytes are cultured in medium without protein, but can be prevented with PVA.

O-98: The phosphodiesterase 3 inhibitor ORG 9935 inhibits oocyte maturation in the naturally-selected dominant follicle in rhesus macaques

Background: The study was conducted to determine whether the phosphodiesterase (PDE) 3 inhibitor ORG 9935 prevents the resumption of meiosis in primate oocytes during natural menstrual cycles. Study Design: Regularly cycling adult female macaques (n=8) were followed during the follicular phase and then started on a 2-day treatment regimen of human recombinant gonadotropins to control the timing of ovulation. Monkeys received no further treatment (controls) or ORG 9935. Oocytes were recovered by laparoscopic follicle aspiration 27 h after an ovulatory stimulus, cultured in vitro in the absence of inhibitor and inseminated. The primary outcome was the meiotic stage of the oocyte. Results: In six ORG 9935 cycles, five of the recovered oocytes were germinal vesicle (GV)-intact, and one exhibited GV breakdown (GVBD). In contrast, all three oocytes that recovered during control cycles were GVBD (pb.05). None of the ORG 9935-treated oocytes underwent fertilization compared with 2/3 (67%) from controls. Conclusions: These results demonstrate that ORG 9935 blocks resumption of meiosis in the naturally selected dominant follicle in primates and suggest that PDE3 inhibitors have potential clinical use as contraceptives in women. © 2008 Elsevier Inc. All rights reserved.

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.

Developmental competence of transported in-vitro matured macaque oocytes

This study examines in-vitro maturation (IVM) in a non-human primate model, Macaca fascicularis. The animals had hormonal injections and laparoscopic oocyte retrieval (OR)) at 12- and 24- h after human chorionic gonadotrophin (HCG). The immature oocytes were placed in tightly capped tubes containing pre-equilibrated IVM medium and transported for 5 h in a dry portable 37°C incubator without CO 2 supplement. Meiotic spindle was observed at 36–38- h post-HCG by polarized microscopy in 72 and 84.5% of mature oocytes collected at 12- and 24- h post-HCG oocyte retrieval intervals respectively. However, abnormal spindle formations were detected in some IVM oocytes by confocal microscopy. The IVM oocytes were also randomly selected for (i) intracytoplasmic injection with frozen–thawed epididymal M. fascicularis spermatozoa and (ii) nuclear transfer (NT) with fresh M. fascicularis cumulus cells. Embryonic development of sperm-injected embryos was not affected by the 12- and 24- h post-HCG oocyte retrieval intervals (22.5 versus 27.9% respectively). However, embryonic development of NT embryos was significantly affected by the 12- h post-HCG oocyte retrieval interval (4.5 versus 31.7% respectively; P < 0.01). In conclusion, IVM of monkey oocytes in a dry portable incubator for 5 h did not affect the maturation rate. However, the ability of primate oocytes to develop after somatic cell nuclear transfer was affected by oocyte retrieval time post-HCG.

Sphingosine-1-Phosphate Inhibits Apoptosis in Rhesus Monkey Ovarian Tissue In Vitro and May Improve Reproductive Function in Xenografts

Cryopreservation of ovarian tissue with subsequent transplantation carries promise as a treatment for chemotherapy-induced sterility in women. Xenografting ovarian cortical tissue into an intermediate mouse host could alleviate the risk of cancer transmission. Follicular development after xenografting has been reported, but fertilization of recovered primate oocytes has been limited. We hypothesize that sphingosine-1-phosphate (S1P), a caspase inhibitor, may minimize apoptosis in monkey ovarian tissue prepared for grafting, and may enhance graft survival and function.

The phosphodiesterase 3 inhibitor ORG 9935 inhibits oocyte maturation during gonadotropin-stimulated ovarian cycles in rhesus macaques

To determine whether phosphodiesterase (PDE) 3 inhibitors prevent the resumption of meiosis by primate oocytes in vivo, rhesus macaques were stimulated to develop multiple preovulatory follicles by administering human recombinant gonadotropins, and follicles were aspirated 34 h after an ovulatory stimulus (human chorionic gonadotropin [hCG]). Monkeys received no further treatment (controls) or the PDE3 inhibitor ORG 9935 (a) exclusively in the periovulatory interval beginning 6–12 h prior to receiving hCG at 200 mg/kg every 12 h orally (PER200) or a 200 mg/kg oral loading dose followed by 50 mg/kg sc every 6 h (PER50) or (b) throughout the ovarian stimulation protocol with daily increases until a dose of 200 mg/kg bid was administered onward from the eighth day of ovarian stimulation (EXT200). The primary outcome was the number of oocytes that had resumed meiosis (germinal vesicle breakdown [GVBD]) at collection. At initial aspiration, 85% of oocytes recovered from control animals ( n=4) had progressed to GVBD compared with 53% (p<.01), 23% (p<.01), and 13% (p<.01) recovered from animals in the PER200 ( n=2), PER50 ( n=1) and EXT200 ( n=3) groups, respectively. Although spontaneous maturation of oocytes was observed during follow-up culture in the absence of ORG 9935, none of the oocytes in the PER50 or EXT200 underwent normal fertilization in vitro. These results demonstrate that the PDE3 inhibitor ORG 9935 blocks oocyte maturation during gonadotropin-stimulated ovarian cycles in rhesus macaques and suggest that PDE3 inhibitors have potential clinical use as contraceptives in women.

128 MITOCHONDRIAL DNA DELETIONS IN RHESUS MACAQUE OOCYTES, EMBRYOS, AND ADULT AND EMBRYONIC STEM CELLS

Mitochondria are the most abundant organelles in mammalian oocytes and early embryos. Previous data have shown that mitochondrial DNA (mtDNA) deletions are present both in human oocytes and in embryos from in vitro fertilization (IVF) patients and suggest that accumulation of these deletions may contribute to mitochondrial dysfunction and impaired ATP production. In addition, high levels of mitochondrial mutations are present in skeletal muscle fibers from aged rhesus macaques. The specific aims of this study were to determine whether the mitochondrial common deletion is present in non-human primate oocytes and embryos generated by IVF and to determine whether mtDNA mutations are already present in immature oocytes from rhesus ovaries. Using a nested primer polymerase chain reaction (PCR) strategy, we determined the frequency of the rhesus common deletion in immature oocytes compared with stimulated oocytes and embryos. There was a low incidence (21%) of the rhesus common deletion present in immature, unstimulated oocytes derived from necropsied ovaries of 2 to 10-yr-old rhesus macaques. However, there was &gt;3-fold increase (71.4%) in the frequency of deleted mtDNA in stimulated oocytes and IVF embryos from age-matched fertile monkeys. We postulated that, in addition to skeletal muscle, a similar time-dependent accumulation of mtDNA deletions occurs in fertile rhesus macaque oocytes and embryos. We are now investigating the effects of culture and passage number on mtDNA deletions in primate adult and embryonic stem cells. We propose the rhesus monkey to be an excellent model to assess the quality of gametes and embryos, as well as stem cells, and their developmental competence in human and non-human primates. This study was supported by National Institutes of Health grants RR15395 and HD045966.

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 Phosphodiesterase 3 inhibitor org 9935 inhibits oocyte maturation during gonadotropin-stimulated ovarian cycles in Rhesus macaques

Abstract To determine whether phosphodiesterase (PDE) 3 inhibitors prevent the resumption of meiosis by primate oocytes in vivo, rhesus macaques were stimulated to develop multiple preovulatory follicles by administering human recombinant gonadotropins, and follicles were aspirated 34 h after an ovulatory stimulus (human chorionic gonadotropin [hCG]). Monkeys received no further treatment (controls) or the PDE3 inhibitor ORG 9935 (a) exclusively in the periovulatory interval beginning 6���12 h prior to receiving hCG at 200 mg/kg every 12 h orally (PER200) or a 200 mg/kg oral loading dose followed by 50 mg/kg sc every 6 h (PER50) or (b) throughout the ovarian stimulation protocol with daily increases until a dose of 200 mg/kg bid was administered onward from the eighth day of ovarian stimulation (EXT200). The primary outcome was the number of oocytes that had resumed meiosis (germinal vesicle breakdown [GVBD]) at collection. At initial aspiration, 85% of oocytes recovered from control animals ( n =4) had progressed to GVBD compared with 53% (p n =2), PER50 ( n =1) and EXT200 ( n =3) groups, respectively. Although spontaneous maturation of oocytes was observed during follow-up culture in the absence of ORG 9935, none of the oocytes in the PER50 or EXT200 underwent normal fertilization in vitro. These results demonstrate that the PDE3 inhibitor ORG 9935 blocks oocyte maturation during gonadotropin-stimulated ovarian cycles in rhesus macaques and suggest that PDE3 inhibitors have potential clinical use as contraceptives in women.

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.

313IN VITRO MATURATION AND FERTILIZATION OF OOCYTES COLLECTED FROM UNSTIMULATED MACACA NEMISTRINA OVARIES

Reports describing the IVM of Macaca nemestrina (Mn) oocytes are limited (Cranfield MR et al. 1989 Zoo. Biol. (Supp. 1), 33). The use of gonadotrophins (Gnt) for IVM of non-human primate (NHP) oocytes is common but the concentrations used are often high (8–40IUmL−1) and the species of origin and biological activity of Gnt varies (Schramm RD and Paprocki AM, 2000 Hum. Reprod. 15, 2411). We have compared two different IVM systems with human Gnt on maturation and fertilization of oocytes collected from unstimulated Mn ovaries (n=6–10 animals). Oocytes were subjected to IVM in modified (minus PVA and pantothenic acid, plus 20 amino acids) HECM−10+15% FCS (Zheng P et al., 2001 Mol. Reprod. Dev. 58, 348) for a) 36h in the presence (mHECM+36, n=322) or absence (mHECM−36, n=99) of FSH and LH applied sequentially (FSH 1IUmL−1 0–24h; 10IUmL−1 FSH and LH 24–36h) or b) 24h in the presence (mHECM+24, n=119) or absence (mHECM−24, n=56) of static concentrations of Gnt (FSH and LH 1IUmL−1 0–24h; no Gnt 24–30h). Oocytes exhibiting first polar body extrusion at 36 and 30h were recorded as mature (MII) and subjected to IVF in HTF+BSA (3mg mL−1) with Mn sperm pretreated with 1.0mM caffeine and 0.1mM dbcAMP. Fertilized oocytes (pronuclei and/or 2nd polar body extrusion) were cultured in sequential culture medium for 48h, assessed for cleavage and either fixed or frozen. Proportional data (mature/total, fertilized/mature or cleaved/fertilized) were compared by chi-square analysis and are reported as percentages. Oocytes cultured in mHECM+36 and mHECM−36 exhibited similar rates of GVBD (58.7% v. 53.5%) but the percentage of MII oocytes was significantly higher (P=0.0244) in mHECM+36 (41.3%) v. mHECM−36 (28.3%). Fertilization rates were comparable between mHECM+36 (61.5%) and mHECM−36 (60.9%), whereas cleavage rates were significantly higher (P=0.0004) in mHECM+36 (74.6%) v. HECM−36 (21.4%). Oocytes cultured in mHECM+24 and mHECM−24 exhibited similar rates of GVBD (76.5% v. 62.5%) but the proportion of MII oocytes was significantly higher (P=0.0159) in mHECM+24 (55.5%) v. mHECM−24 (35.7%). Fertilization and cleavage rates were comparable between mHECM+24 (58.8% v. 63.3%) and mHECM−24 (50.0% v. 42.8%). A comparison between mHECM+36 and mHECM+24 indicated a significantly lower (P=0.0005) percentage of GV oocytes and a significantly higher (P=0.0096) percentage of MII oocytes in mHECM+24 (23.5% v. 55.5%) compared to mHECM+36 (41.3% v. 41.3%). Fertilization and cleavage rates were not significantly different between mHECM+36 and mHECM+24. Oocyte maturation and fertilization and embryo cleavage were not different for mHECM−36 and mHECM−24 (P=0.3138–0.8202). Mn oocytes exhibit high rates of Gnt-independent GVBD (52.5%–53.5%) and maturation (28.3%–35.7%) in vitro, and maturation rates were improved in Gnt supplemented maturation medium. However, reduced exposure to lower concentrations of FSH and increased exposure to lower concentrations of LH was associated with higher rates of oocyte maturation in vitro. The use of lower concentrations of FSH and LH for reduced periods may improve IVM of NHP oocytes. This work was supported by the Tissue Distribution Program of the WaNPRC (NIH grant # R00166).

High quality sperm for nonhuman primate ART: Production and assessment

Factors that affect sperm quality can include method of semen collection, conditions for capacitation and whether or not agglutination is present. Media and procedures for sperm washing can also impair or improve sperm function in assisted reproductive technologies. For example, the removal of seminal fluid through large volume washing is required to eliminate decapacitation activity of seminal plasma. The forces involved with centrifugation and the metabolic stress of tightly pelleting sperm during washing procedures can have deleterious results. In contrast to human sperm, sperm from the most commonly used species of nonhuman primates, rhesus and cynomolgus macaques, do not spontaneously capacitate in vitro; rather, chemical activation with dibutryl cyclic AMP and caffeine is required. Recognizing motility patterns of non-activated and activated sperm can be accomplished with simple observation. After activation, sperm agglutination sometimes occurs and can interfere with sperm binding to the zona pellucida. Because nonhuman primate oocytes require a large investment to produce and currently, each animal can be hormonally stimulated a limited number of times, it is important to have means to evaluate quality prior to using sperm from a new male for in vitro fertilization. Methods for producing live, acrosome reacted sperm may also have application for ICSI. Because many genetically valuable males are now being identified, it may be necessary to individualize sperm preparation to accommodate male-to-male variation.

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&amp;gt;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.

In vitro exposure to inhibitors of phosphodiesterase (PDE) 3, but not PDE 4, prevent spontaneous resumption of meiosis in rhesus macaque oocytes.

Objective: To determine whether Phosphodiesterase (PDE) 3 inhibitors selectively prevent the resumption of meiosis in primate oocytes. Design: Nonhuman primate, in vitro experiment. Materials/Methods: Oocytes were obtained surgically from rhesus macaques stimulated to develop multiple preovulatory follicles by administering human recombinant gonadotropins (r-hFSH, r-hLH, Ares Advanced Technologies, Inc), plus a gonadotropin releasing hormone antagonist (Antide, AAT) to prevent a spontaneous gonadotropin surge. Oocytes with intact germinal vesicles were incubated under oil in TALP with bovine serum albumin and pyruvate with or without various doses of one of three PDE 3 inhibitors; Cilostamide (Sigma), Milrinone (Sigma) or ORG 9935 (Organon), or a selective PDE 4 inhibitor Rolipram (Sigma). An inverted microscope at 200× was used to assess the meiotic maturation of oocytes at 24 and 48 hours. Results: At 24 hours, 72 of 121 (59.5%) control oocytes progressed to germinal vesicle breakdown (GVBD) compared with 0/28 (0%, p < 0.01), 4/36 (11.1%, p < 0.01) and 9/34 (26.5%, p < 0.01) oocytes incubated with Org 9935 at 1.0 μM, 0.5 μM, and 0.1 μM, respectively. Whereas the inhibition of GVBD at 24 hours produced by 1.0 μM Cilostamide (2/24 oocytes, 8.3%, p < 0.01) was similar to 1.0 μM ORG 9935, a higher concentration of Milrinone (100 μM) was necessary to achieve comparable results (2/32, 6.3%, p < 0.01). By 48 hours, additional control (82/121, 67.8%) and 1.0 μM Cilostamide (4/24, 16.7%, p < 0.01) treated oocytes progressed to GVBD, but not oocytes in 1.0 μM ORG 9935 or 100 μM Milrinone. In contrast, no significant difference in GVBD was noted between control oocytes and those incubated with up to 100 μM Rolipram for 24 hours (43/58, 74.1%) or 48 hours (44/58, 75.9%). Conclusions: These experiments establish the specificity and dose-dependent ability of PDE 3, but not PDE 4, inhibitors to block resumption of meiosis in macaque oocytes in vitro. Since different forms of PDE are present in the oocyte (PDE 3) and follicular cells (PDE 4), PDE inhibitors have potential as oocyte specific contraceptives without altering other events in the ovarian cycle. Supported By: (NICHD/NIH SCCPRR HD18185-17, HD01243, RR00163).

Energy substrate requirement for in vitro maturation of oocytes from unstimulated adult rhesus monkeys.

The energy substrates lactate, pyruvate, and glucose were evaluated for supporting in vitro cytoplasmic maturation of rhesus monkey oocytes. A total of 321 cumulus-oocyte complexes (COCs) aspirated from > or = 1000 microm diameter follicles of unstimulated adult monkeys were matured in one of six media with various individual or combinations of energy substrates: (1) mCMRL-1066 (control); (2) HECM-10 (containing 4.5 mM lactate); (3) HECM-10+0.2 mM pyruvate; (4) HECM-10 + 5.0 mM glucose; (5) HECM-10+ 0.2 mM pyruvate + 5.0 mM glucose; and (6) HECM-10 minus lactate + 5.0 mM glucose. All media contained gonadotropins, oestradiol, and progesterone. Following maturation, all mature oocytes were subjected to the same in vitro fertilization and embryo culture procedures. Oocytes matured in control medium or in treatment groups 4 and 6 had the best morulae+ blastocysts developmental responses (35, 36, and 32%, respectively, P < 0.05). HECM-10 + 0.2 mM pyruvate + 5.0 mM glucose for COC maturation supported intermediate embryonic development (16% morulae + blastocysts). The lowest (P < 0.05) morula + blastocyst developmental responses were obtained after maturation of COCs in HECM-t10 and HECM-10 + 0.2 mM pyruvate (4 and 6%, respectively). The COCs matured in glucose-containing medium showed greater levels of cumulus expansion than those in glucose-free medium. These results indicate that (a) glucose is both necessary and sufficient as the energy substrate for supporting optimal cytoplasmic maturation in vitro of oocytes from unstimulated rhesus monkeys; (b) pyruvate suppresses the stimulatory effect of glucose on oocyte maturation; (c) glucose is involved in cumulus expansion; (d) cumulus expansion is not a reliable indicator of primate oocyte competence.

Is Round Spermatid Injection (ROSI) a Therapy for Male Infertility?: ROSI in the Rhesus Monkey is Unsuccessful

Objectives: The use of round spermatids for human fertilization has met with limited success perhaps because of the immaturity of the spermatids, the difficulty in their identification and the poor rates of oocyte activation with these cells. The objectives of this study were to: 1) develop an animal model for round spermatid injection (ROSI) using non-human primate oocytes; 2) to examine the cytoskeletal events after rhesus ROSI and 3) to perform ICSI ‘rescue’ on failed ROSI fertilizations with subsequent embryo development and transfer.

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

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.