Rhesus Monkey Oocytes Research Articles

Effect of age and breeding season on the developmental capacity of oocytes from unstimulated and follicle-stimulating hormone-stimulated rhesus monkeys.

Effects of age and season on the developmental capacity of oocytes from unstimulated and FSH-stimulated rhesus monkeys were examined. Immature cumulus-oocyte complexes were matured in vitro in modified CMRL-1066 medium containing 20% bovine calf serum and subjected to in vitro fertilization followed by embryo culture. After fertilization, ova from unstimulated prepubertal monkeys displayed lower development to morula (4%) than those from unstimulated adult females (18% in breeding season and 22% in nonbreeding season). No developmental difference was found between ova from unstimulated adult monkeys in breeding and nonbreeding seasons. However, ova from FSH-primed prepubertal monkeys displayed greater development to blastocyst stage (54%) than those from adult monkeys in the breeding season (16%) and nonbreeding season (0%); and ova from FSH-primed adult females in the breeding season had significantly (P < 0.05) greater developmental competence than those obtained in the nonbreeding season (> or = morula stage, 54% vs. 3%; blastocyst stage, 16% vs. 0%). These data indicate that 1) rhesus monkey oocytes acquire developmental competence in a donor age-dependent manner, and 2) animal age and breeding season modulate the effect of FSH on oocyte developmental competence in the rhesus monkey.

Transgenic monkeys produced by retroviral gene transfer into mature oocytes.

Transgenic rhesus monkeys carrying the green fluorescent protein (GFP) gene were produced by injecting pseudotyped replication-defective retroviral vector into the perivitelline space of 224 mature rhesus oocytes, later fertilized by intracytoplasmic sperm injection. Of the three males born from 20 embryo transfers, one was transgenic when accessible tissues were assayed for transgene DNA and messenger RNA. All tissues that were studied from a fraternal set of twins, miscarried at 73 days, carried the transgene, as confirmed by Southern analyses, and the GFP transgene reporter was detected by both direct and indirect fluorescence imaging.

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.

Elongated Spermatid Injection (ELSI) in the Rhesus Monkey

Objectives: The objectives of this study were to: 1) isolate testicular sperm and elongated spermatids from testicular biopsies retrieved from fertile male rhesus monkeys; 2) fertilize rhesus oocytes using testicular sperm and elongated spermatids and 3) produce live offspring by these novel methods of assisted reproduction.

Mitochondrial distribution and function in oocytes and early embryos.

Active mitochondria relocate during oocyte maturation or fertilization in several species. Detailed studies with hamster oocytes and early embryos reveal a pattern of active mitochondria migrating to surround the pronuclei to form a pattern that persists through the early cleavage stages. Although the functional significance of this relocation is unknown, it appears to be an important part of normal development in hamsters. Treatments that disrupt embryo development in vitro (such as the presence of inorganic phosphate or alteration of intracellular pH) also disrupt the normal pattern of mitochondrial distribution. Active mitochondria also reorganize during maturation in bovine oocytes and during fertilization in rhesus monkey oocytes. Examination of these changes in mitochondrial organization may provide insights into the regulation of normal embryo development and might serve as predictors of oocyte or embryo developmental competence.

Optimization Strategies for Production of Mammalian Embryos by Nuclear Transfer

In order to optimize each of the individual steps in the nuclear transfer procedure, we report alternative protocols useful for producing recipient cytoplasts and for improving the success rate of nuclear transfer embryos in cattle, rhesus monkey, and hamster. Vital labeling of maternal chromatin/spindle is accomplished by long wavelength fluorochromes Sybr14 and rhodamine labeled tubulin allowing constant monitoring and verification during enucleation. The use of Chinese hamster ovary (CHO) donor cells expressing the viral influenza hemagglutinin fusion protein (HA-300a+), to adhere and induce fusion between the donor cells and enucleated cow, rhesus and hamster oocytes was examined. Cell surface hemagglutinin was activated with trypsin prior to nuclear transfer and fusion was induced by a short incubation of a newly created nuclear transfer couplet at pH 5.2 at room temperature. Donor cell cytoplasm was dynamically labeled with CMFDA, or further transfected with the green fluorescence protein (GFP) gene, so that fusion could be directly monitored using live imaging. High rates of fusion were observed between CHO donor cells and hamster (100%), rhesus (100%), and cow recipient cytoplasts (81.6%). Live imaging during fusion revealed rapid intermixing of cytoplasmic components between a recipient and a donor cell. Prelabeled donor cytoplasmic components were uniformly distributed throughout the recipient cytoplast, within minutes of fusion, while the newly introduced nucleus remained at the periphery. The fusion process did not induce activation as evidenced by unchanged distribution and density of cortical granules in the recipient cytoplasts. After artificial activation, the nuclear transfer embryos created in this manner were capable of completing several embryonic cell divisions. These procedures hold promise for enhancing the efficiency of nuclear transfer in mammals of importance for biomedical research, agriculture, biotechnology, and preserving unique, rare, and endangered species.

The implications of a paternally derived centrosome during human fertilization: consequences for reproduction and the treatment of male factor infertility.

Successful fertilization in humans follows a complex series of events, including the completion of meiotic maturation of the oocyte with the extrusion of the second polar body, the decondensation of the sperm nucleus and the maternal chromosomes into male and female pronuclei, the restoration of the sperm centrosome, and the nucleation of microtubule-mediated motility necessary to bring the male and female pronuclei into close apposition. These events occur after both fertilization in vitro and after intracytoplasmic sperm injection (ICSI), a new technique which is currently being applied in many clinics to overcome severe male infertility. Defects in any of the events leading to fertilization can be lethal to the zygote and may prove to be causes of infertility. Imaging of inseminated human and rhesus oocytes using immunohistochemical techniques reveals several phases at which fertilization arrests. Oocytes from some infertile patients failed to complete fertilization due to failure of the sperm aster microtubules in uniting the sperm and egg nuclei. The rate of sperm aster formation, size, and organization during fertilization has been used as a measurement of bovine sperm quality. The development of an assay using Xenopus laevis oocyte extract can also be used to test sperm from various species for their ability to form esters and perform other centrosomal functions in vitro, as well as another indicator of sperm quality. Semen from men with questionable fertility was found to contain sperm which are generally incapable of producing sperm asters. In addition, the activity of centrosomal proteins such as gamma-tubulin and centrin have been detected in mammalian eggs and sperm. The levels of gamma-tubulin increase markedly after exposure to X. laevis egg extract. Defects in either male or female nucleus decondensation also resulted in the arrest of fertilization and was found to occur in both inseminated human oocytes and in rhesus oocytes fertilized by ICSI. These discoveries on the molecular basis of infertility in humans have important implications for infertility diagnosis and managing reproduction.

Effects of EGTA and cytochalasin-B during freezing and vitrification of immature and mature bovine and rhesus monkEY ( M. mulatta) oocytes

Effects of EGTA and cytochalasin-B during freezing and vitrification of immature and mature bovine and rhesus monkEY ( M. mulatta) oocytes

Enhancement of primate oocyte maturation and fertilization in vitro by inhibin A and activin A

A role for inhibin and activin in primate oocyte maturation was investigated. The maturation and fertilization of rhesus monkey oocytes recovered from the excised ovaries of nine regularly cycling animals was compared for untreated germinal vesicle (GV)-intact controls versus oocytes cultured in the presence of inhibin, activin, inhibin + activin, or in a combination with follistatin. Nonhuman primates in a research institute environment. Bilateral oophorectomy. Oocyte maturation from germinal vesicle breakdown (GVBD) to metaphase II (MII) and fertilization. Germinal vesicle breakdown, progression to MII and fertilization was monitored in oocytes cultured for 48 hours. Activin alone, at an optimum concentration of 100 ng/mL, stimulated GVBD whereas both GVBD and MII development was enhanced in the presence of inhibin + activin. The latter also accelerated the rate of maturation to MII. All treatment groups exhibited a higher incidence of GVBD compared with controls. When follistatin was added, the stimulatory effect of activin or activin + inhibin was abolished. Exposure to medium containing inhibin + activin significantly increased the percentage of MII oocytes that fertilized compared with controls (68% versus 25%, respectively). Inhibin and activin are potent stimulators of primate oocyte maturation, producing mature oocytes in vitro that fertilize with high efficiency.

Microtubule and chromatin configurations during rhesus intracytoplasmic sperm injection: successes and failures.

Intracytoplasmic sperm injection (ICSI) was performed on rhesus monkey oocytes, and the resultant microtubule and DNA configurations were imaged by laser-scanning confocal microscopy. In addition, polyspermic oocytes fertilized by ICSI were examined by transmission electron microscopy (TEM). Successful rhesus fertilization by ICSI revealed microtubule and DNA configurations similar to those observed during in vitro fertilization of human and rhesus monkey oocytes, including sperm aster formation, pronuclei decondensation, spindle formation, and cell division. Several abnormalities, however, were also observed: 1) inability to complete meiosis; 2) inability to undergo male or female pronucleus formation; 3) separation of the sperm tail from the sperm nucleus; 4) premature chromosome condensation with the formation of a paternal meiotic spindle; and 5) formation of multiple female pronuclei (karyomeres) during chromosome decondensation. TEM analysis revealed that sperm can undergo decondensation in the presence of an intact acrosome at least 18 h after sperm injection. These results demonstrate the utility of rhesus ICSI in pre-clinical applications as well as with endangered species. However, the different types of fertilization failures observed here indicate that although ICSI may be a readily accepted means of fertilization of human oocytes in many clinics, we should further characterize the cellular and genetic abnormalities associated with ICSI in both human and nonhuman primates.

Intracytoplasmic sperm injection for Rhesus monkey fertilization results in unusual chromatin, cytoskeletal, and membrane events, but eventually leads to pronuclear development and sperm aster assembly.

The disassembly and reorganization of sperm-derived structures are landmarks for the onset of embryonic development. Since complete information on these events is not yet available, we examined the disassembly of the sperm axoneme, the formation of the sperm aster, and the decondensation and development of the male and female pronuclei in inseminated Rhesus monkey oocytes conceived by in-vitro fertilization (IVF) or by intracytoplasmic sperm injection. During IVF, the spermatozoa lose their acrosomes after contacting the zona pellucida, and the plasma membrane and nuclear envelope disappear after fusion with the oolemma. Subsequently, a sperm aster of microtubules forms around the proximal centriole, which is bound to the sperm connecting piece. This process is then followed by the formation of both pronuclei, which single sperm centriole later duplicates and the bipolar mitotic apparatus is observed. Following sperm injection, the spermatozoa have both an intact plasma membrane and acrosome. Although the microtubules form the sperm aster in a fashion identical to that seen during IVF, the presence of an intact acrosome appears to be associated with a heterogeneity in the decondensation of sperm chromatin. While this may indicate an abnormal pattern of chromatin decondensation during the formation of the male pronucleus following sperm injection, the male pronucleus eventually fully decondenses, as during IVF. Sperm mitochondria are displaced as the sperm centriole is exposed. Annulate lamellae and a previously undescribed organelle which seems to contain annulate lamellae precursors, as well as maternal mitochondria, are found in association with the developing pronuclear envelopes. This information increases understanding of fertilization in primates, and may also be of significance for use in assisted human reproduction as well as in the preservation of endangered mammalian species. In addition, these results demonstrates the similarities between fertilization in Rhesus monkeys and humans, providing additional evidence for the use of this non-human primate as a model system in which to investigate the cellular and molecular biological basis of human reproduction.

Cryobiology of non-human primate oocytes

The responses to various stresses involved with cryopreservation protocols were investigated using non-human primate oocytes. Fluorescence microscopy was used to assess the status of the F-actin microfilament system of rhesus monkey oocytes after exposure to different concentrations of glycerol. The F-actin organization around the cortex and in the transzonal processes was modified by exposure to 1.0 ot 2.0 M glycerol at ambient temperature. These effects were reduced significantly when exposure to glycerol was combined with cooling to O degrees C. Cynomolgus monkey oocytes were also subjected to hyperosmotic stress and observed for morphological changes. An irregular shrinkage phenomenon was observed with germinal vesicle or metaphase I but not metaphase II (MII) oocytes. The irregular shrinkage became uniform and spherical when the oocytes were pretreated with ethyleneglycol-bis-(beta-aminoethyl ether)N,N,N'N' tetraacetic acid (EGTA) before exposure to hypertonic solution. Also, in-vitro-matured MII oocytes from cynomolgus monkeys were used to determine crucial biophysical parameters for freezing primate oocytes. The permeability of oocyte plasma membrane to water, Lpg, and its activation energy, ELp, were determined between 0 and -12 degrees C in the absence of cryoprotective additives. The Lpg was found to be 3.8x10(-14) m3N/s and the ELp was 141.5 kJ/mol. the pre-exponential kinetic and exponential thermodynamic parameters of intracellular ice formation were determined to be 8x108 m2/S and 2. 2x10(9) K5 respectively. By combining models of water transport and intracellular ice formation, the cumulative fraction of oocytes with intracellular ice as a function of the cooling rate was also predicted, and it was shown to correlate reasonably with experimental observations.

Maturity and fertility of rhesus monkey oocytes collected at different intervals after an ovulatory stimulus (human chorionic gonadotropin) in in vitro fertilization cycles.

In rhesus monkeys undergoing ovarian stimulation for in vitro fertilization (IVF), a midcycle injection of human chorionic gonadotropin (hCG) substitutes for the LH surge and induces preovulatory oocyte maturation. The time interval between injection and oocyte collection, ideally, allows for the completion of oocyte maturation without ovulation, which would reduce the number of oocytes available for harvest. To evaluate the influence of this time interval on oocyte parameters following hCG administration, we conducted a series of gonadotropin treatment protocols in 51 animals in which the interval from hCG administration to follicular aspiration was systematically varied from 27 to 36 hr. Follicle number and size, evaluated prior to hCG administration by sonography, did not vary significantly or consistently with preovulatory maturation time. Oocytes were harvested by laparotomy or laparoscopy, and scored for maturity before insemination. The percentage of mature, metaphase II (MII) oocytes at recovery increased significantly with increasing preovulatory time and was inversely proportional to that of metaphase I (MI) oocytes. However, oocyte yield tended toward a progressive decrease with increasing preovulatory maturation times from a high of 27 oocytes at 27 hr to a low of 17 oocytes/animal at the 36 hr time interval. Fertilization levels declined significantly from a high of 50% at 27 hr to a low of 30% at 36 hr. Thus, although higher percentages of mature oocytes were recovered at the longer time intervals, optimal oocytes/embryo harvests were realized after the shorter time intervals (27 and 32 hr) and are most compatible with the goal of achieving high yields of fertile oocytes and embryos following gonadotropin stimulation in rhesus monkeys.

Effects of human FSH, LH or TSH on meiotic maturation of rhesus monkey oocytes isolated from antral follicles

A solid-phase immunometric assay of human lutropin (hLH) is described. Two different anti-hLH antibodies were utilized as capture antibodies, and anti-IgG antibodies covalently coupled to magnetic particles and horseradish peroxidase, respectively, served as ‘universal’ detection reagents.An anti-hLH antibody raised in rabbits was incubated with a goat anti-rabbit IgG covalently bound to magnetic particles. The resulting complex was added to a separately incubated mixture of hLH and monoclonal anti-hLH antibody. Following incubation, the immunocomplex was sedimented in a magnetic field and the supernatant discarded. Finally a sheep anti-mouse antibody (F(ab′)2 fragment) conjugated to horseradish peroxidase as label was added. Following a further incubation, the particles were sedimented in the magnetic field and washed. The hLH content of the sample was quantitated by measuring ’enhanced chemiluminescence’.The sensitivity of the assay was 2.5 ± 0.9 IU/l (mean ± SD), the within-run variation ranged from 7.9 to 11%, the between-run variation from 12.9 to 19.8%. Cross-section with hFSH or hTSH could not be detected, but was approximately 0.1% with hCG. The results correlated well with those obtained by radioimmunoasay (r = 0.84).

Collection and fertilization potential of sperm from the Sulawesi crested black macaque (Macaca nigra).

In this study, semen was obtained by rectal probe electrostimulation (RPE) from the Sulawesi crested black macaque (Macaca nigra). Three experimental series were conducted. First, semen was collected from four animals anesthetized with either tiletamine-zolazepam (Telazol®) or ket-amine-HCl (Vetalar®) (five collections from each animal with each drug). Because of greater muscle relaxation and analgesia, we found tiletaminezolazepam to be an attractive alternative to ketamine-HCl as an anesthetic agent for RPE in M. nigra. Second, semen was collected from another four animals at stimulation frequencies of either 30 Hz or 60 Hz (five collections from each animal at each frequency). There were no significant differences in sperm number, in percentage of sperm with progressive motility, in the current required for sample recovery between tiletamine-zolazepam or ketamine-HCl anesthesia, or between a 30 Hz or 60 Hz stimulation frequency. Third, to check for retrograde sperm loss, the bladders of four animals were emptied, flushed with sterile saline, and then infused with TALP-Hepes medium. After RPE, sperm numbers in the bladder were compared with those in the ejaculate. Although sperm were recovered from the bladder [1.6 (± 0.9) × 106] (mean ± SEM), the numbers were significantly less (P < 0.05) than those in the ejaculate [49 (± 18) × 106]. The percentage of sperm with normal morphology in these samples was high (96.8 ± 1.0%). The average sperm number in the 84 samples collected for this study was 33.8 (± 4.1) × 106. In preliminary experiments, we found that M. nigra sperm will fertilize rhesus monkey oocytes (Macaca mulatta) in vitro. © 1992 Wiley-Liss, Inc.

Quality of oocytes from superovulated rhesus monkeys

The parameters, serum oestradiol (E2) response, follicle size and cumulus morphology, which are commonly used to determine in-vivo oocyte maturation in human in-vitro fertilization and embryo transfer (IVF-ET) programmes, were shown to be unreliable predictors of maturation of rhesus oocytes. In two groups of rhesus, one stimulated with pregnant mares' serum gonadotrophin (PMSG) and human chorionic gonadotrophin (HCG) and the other with human menopausal gonadotrophin (HMG) and HCG, the three parameters varied widely within and between protocols. Triple fluorochrome staining (TFS) for chromatin, microtubules and filamentous actin (f-actin) in oocytes at the time of collection and following 24 h in culture showed major differences in their maturation both in vivo and in vitro following priming with PMSG and HMG. In evaluating IVF protocols, TFS provides a valuable assay for the meiotic status of fixed oocytes of non-human primates.

Maturity at Collection and the Developmental Potential of Rhesus Monkey Oocytes1

The purpose of this study was to evaluate the in vitro fertilizability of rhesus monkey oocytes and the developmental capacity of the resulting embryos as they relate to oocyte maturation at the time of follicular aspiration. Animals were hyperstimulated with human follicle-stimulating hormone (hFSH) and human luteinizing hormone (hLH), with follicular aspiration performed 27 h after administration of an ovulatory stimulus (1000 IU human chorionic gonadotropin [hCG] or 3 x 100 micrograms gonadotropin-releasing hormone [GnRH]). In 7 animals exhibiting a continuously rising pattern of serum estradiol through Day 10 of hyperstimulation, 45 germinal vesicle-intact (GV), 106 metaphase I (MI), and 24 metaphase II (MII) oocytes were collected and cultured in vitro. Upon reaching MII, oocytes were inseminated with 5 x 10(4) motile sperm/ml. Twenty-four percent of GV oocytes cultured in vitro matured to MII with 11 inseminated and none fertilized. Seventy-three percent of MI oocytes matured to MII in vitro with 50% inseminated and 32% fertilized. Oocytes collected at MII stage and inseminated underwent fertilization at a high rate of efficiency (93%). Pronuclear to 8-cell stage embryos were frozen and, upon thawing, 67% (10/15) survived with all blastomeres intact. Frozen-thawed embryos (2- to 6-cell) were transferred to the oviducts of 4 recipients (2 embryos/recipient) during the early luteal phase (1-3 days post LH surge) of natural menstrual cycles. Three twin pregnancies resulted. Thus, a positive correlation exists between the degree of nuclear maturation of rhesus monkey oocytes at collection and their potential for fertilization in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Hypoxanthine and cyclic adenosine 5’-monophosphate maintain meiotic arrest of rhesus monkey oocytes in vitro

In the present study, hypoxanthine (HX) and dibutyryl cyclic adenosine monophosphate (dbcAMP) were tested as inhibitors of rhesus monkey oocyte maturation in vitro, because of their similar effect in mice. A significantly larger proportion (72%) of oocytes underwent germinal vesicle breakdown (GVB) in group I (control) after 40 hours in culture, as compared with 21% in treatment group II with HX plus dbcAMP (P less than 0.001). Seventy-five percent of oocytes had GVB by 80 hours after being initially inhibited for 40 hours with HX and dbcAMP; however, only 34% of oocytes had GVB when treated with HX and dbcAMP continuously for 80 hours (P less than 0.005). These results demonstrate that a significant proportion of rhesus monkey oocytes can be maintained in meiotic arrest for 40 hours by treatment with HX and dbcAMP, and that this effect can be overcome by removal of inhibitors from the culture medium.

Stimulation of rhesus monkey sperm capacitation by cyclic nucleotide mediators.

Capacitation of rhesus monkey spermatozoa was assessed by monitoring sperm flagellar beat and trajectory changes during incubation in vitro and by determining sperm penetration into rhesus oocytes and hamster zona-free ova. Rhesus sperm capacitation in vitro depended on the addition to the culture medium of the cyclic nucleotide mediators, caffeine and dibutyryl cyclic AMP. Capacitation was correlated with the development of hyperactivated motility. Spermatozoa treated with the cyclic nucleotide mediators, and showing hyperactivated motility, penetrated 57.4% of all rhesus oocytes and fertilized 88.9% of mature rhesus oocytes that were morphologically normal. Control spermatozoa did not penetrate any of the eggs. Some sperm penetration into hamster ova occurred but was not statistically significant. These data provide a basis for achieving in-vitro fertilization in the rhesus monkey and information on specific sperm motility characteristics associated with fertilizing ability.

Fertilization and cleavage of rhesus monkey oocytes in vitro.

Fertilization in vitro of rhesus oocytes has been performed in order to provide a model for the study of primate embryonic development. Rhesus monkeys were superstimulated by daily injections of pregnant mare’s serum gonadotropin (PMSG) for 12 days beginning Days 3-5 of menses. An injection of human chorionic gonadotropin (hCG) was given 24 h after the last PMSG injection to induce oocyte maturation. Laparoscopic aspiration of 106 follicles approximately 30 h after hCG injection yielded 87 oocytes (82% recovery). Fifty-three of these oocytes (62%) were enclosed in loose (expanded) cumulus oophorus. Washed, ejaculated rhesus spermatozoa (20 X 106 /ml) were preincubated for 4-6 h in a modified Tyrode’s solution, then diluted (1/10) into 100-M1 culture drops containing oocytes, and caffeine and dibutyryl cAMP (1 mM each). In later experiments, these chemicals were added to the sperm suspensions during the last 1.5 h of preincubation, so that they became diluted 1/10 (final concentrations 0.1 mM each) in the fertilization drops. Sperm and oocytes were coincubated for 18-24 h, then oocytes were either fixed or transferred to fresh culture medium. In 9 experiments a total of 70 rhesus oocytes were inseminated in vitro. Total results for 5 experiments were 13/30 oocytes (43%) showing signs consistent with fertilization (sperm in vitellus and/or more than 1 pronucleus or early stages of cleavage). Four more experiments yielded confirmatory evidence of fertilization. When oocytes were inseminated with live sperm that had been preincubated with caffeine and dibutyryl cAMP, 23/29 oocytes (79%) cleaved at least once; 20 of these developed to the 8-cell stage or further. A further 11 oocytes, randomly selected from the pool of available eggs in the 4 experiments, were inseminated under identical conditions with killed sperm. None of these oocytes showed activation, cleavage or fragmentation during culture for 48-73 h. Activation of oocytes and cleavage resulted only from interaction with live sperm, indicating that fertilization was in progress or had been completed.