Use of assisted reproductive technologies in the propagation of rhesus macaque offspring.

The assisted reproductive technologies (ARTs) as tailored to the production of rhesus monkeys at the Oregon National Primate Research Center (ONPRC) are described. Efficient fertilization of mature oocytes recovered by aspiration from females subjected to follicular stimulation was achieved with fresh or frozen sperm by intracytoplasmic sperm injection (ICSI). Embryo development to the early cleavage stage occurred at high frequency. Cryopreserved embryos showed high postthaw survival and were also transferred in efforts to establish pregnancies. Three methods of transfer were evaluated, two involving embryo placement into the oviduct, laparoscopy and minilaparotomy, and a nonsurgical, transcervical approach that resulted in uterine deposition. Early cleaving embryos (Days 1-4) were transferred into the oviducts of synchronized recipients with optimal results and pregnancy rates of up to 36%. Pregnancy rates were similar when two fresh or frozen embryos were transferred (28- 30%), although more than two embryos had to be thawed to compensate for embryo loss during freeze-thawing. Normal gestational lengths, birth weights, and growth curves were seen with ART-produced infants compared with infants produced by natural mating in the timed mated breeding (TMB) colony at the ONPRC. In 72 singleton pregnancies established following the transfer of ART-produced embryos, the live-birth rate, at 87.5%, was statistically identical to that for the TMB colony. Further development of the ARTs should result in increasing use of these techniques to augment conventional approaches to propagating monkeys, especially those of defined genotypes.

Live birth after ovarian tissue transplant.

Radiation and high-dose chemotherapy may render women with cancer prematurely sterile, a side-effect that would be avoided if ovarian tissue that had been removed before treatment could be made to function afterwards. Live offspring have been produced from transplanted ovarian tissue in mice and sheep but not in monkeys or humans, although sex steroid hormones are still secreted. Here we describe the successful transplantation of fresh ovarian tissue to a different site in a monkey, which has led to the birth of a healthy female after oocyte production, fertilization and transfer to a surrogate mother. The ectopically grafted tissue functions without surgical connection to major blood vessels and sets the stage for the transplantation of cryopreserved ovarian tissue in humans.

Circadian force and EMG activity in hindlimb muscles of rhesus monkeys.

Continuous intramuscular electromyograms (EMGs) were recorded from the soleus (Sol), medial gastrocnemius (MG), tibialis anterior (TA), and vastus lateralis (VL) muscles of Rhesus during normal cage activity throughout 24-h periods and also during treadmill locomotion. Daily levels of MG tendon force and EMG activity were obtained from five monkeys with partial datasets from three other animals. Activity levels correlated with the light-dark cycle with peak activities in most muscles occurring between 08:00 and 10:00. The lowest levels of activity generally occurred between 22:00 and 02:00. Daily EMG integrals ranged from 19 mV/s in one TA muscle to 3339 mV/s in one Sol muscle: average values were 1245 (Sol), 90 (MG), 65 (TA), and 209 (VL) mV/s. The average Sol EMG amplitude per 24-h period was 14 microV, compared with 246 microV for a short burst of locomotion. Mean EMG amplitudes for the Sol, MG, TA, and VL during active periods were 102, 18, 20, and 33 microV, respectively. EMG amplitudes that approximated recruitment of all fibers within a muscle occurred for 5-40 s/day in all muscles. The duration of daily activation was greatest in the Sol [151 +/- 45 (SE) min] and shortest in the TA (61 +/- 19 min). The results show that even a "postural" muscle such as the Sol was active for only approximately 9% of the day, whereas less active muscles were active for approximately 4% of the day. MG tendon forces were generally very low, consistent with the MG EMG data but occasionally reached levels close to estimates of the maximum force generating potential of the muscle. The Sol and TA activities were mutually exclusive, except at very low levels, suggesting very little coactivation of these antagonistic muscles. In contrast, the MG activity usually accompanied Sol activity suggesting that the MG was rarely used in the absence of Sol activation. The results clearly demonstrate a wide range of activation levels among muscles of the same animal as well as among different animals during normal cage activity.

Membrane-fusing capacity of the human immunodeficiency virus envelope proteins determines the efficiency of CD+ T-cell depletion in macaques infected by a simian-human immunodeficiency virus.

The mechanism of the progressive loss of CD4+ T lymphocytes, which underlies the development of AIDS in human immunodeficiency virus (HIV-1)-infected individuals, is unknown. Animal models, such as the infection of Old World monkeys by simian-human immunodeficiency virus (SHIV) chimerae, can assist studies of HIV-1 pathogenesis. Serial in vivo passage of the nonpathogenic SHIV-89.6 generated a virus, SHIV-89.6P, that causes rapid depletion of CD4+ T lymphocytes and AIDS-like illness in monkeys. SHIV-KB9, a molecularly cloned virus derived from SHIV-89.6P, also caused CD4+ T-cell decline and AIDS in inoculated monkeys. It has been demonstrated that changes in the envelope glycoproteins of SHIV-89.6 and SHIV-KB9 determine the degree of CD4+ T-cell loss that accompanies a given level of virus replication in the host animals (G. B. Karlsson et. al., J. Exp. Med. 188:1159-1171, 1998). The envelope glycoproteins of the pathogenic SHIV mediated membrane fusion more efficiently than those of the parental, nonpathogenic virus. Here we show that the minimal envelope glycoprotein region that specifies this increase in membrane-fusing capacity is sufficient to convert SHIV-89.6 into a virus that causes profound CD4+ T-lymphocyte depletion in monkeys. We also studied two single amino acid changes that decrease the membrane-fusing ability of the SHIV-KB9 envelope glycoproteins by different mechanisms. Each of these changes attenuated the CD4+ T-cell destruction that accompanied a given level of virus replication in SHIV-infected monkeys. Thus, the ability of the HIV-1 envelope glycoproteins to fuse membranes, which has been implicated in the induction of viral cytopathic effects in vitro, contributes to the capacity of the pathogenic SHIV to deplete CD4+ T lymphocytes in vivo.

Vaccine-elicited V3 loop-specific antibodies in rhesus monkeys and control of a simian-human immunodeficiency virus expressing a primary patient human immunodeficiency virus type 1 isolate envelope.

Vaccine-elicited antibodies specific for the third hypervariable domain of the surface gp120 of human immunodeficiency virus type 1 (HIV-1) (V3 loop) were assessed for their contribution to protection against infection in the simian-human immunodeficiency virus (SHIV)/rhesus monkey model. Peptide vaccine-elicited anti-V3 loop antibody responses were examined for their ability to contain replication of SHIV-89.6, a nonpathogenic SHIV expressing a primary patient isolate HIV-1 envelope, as well as SHIV-89.6P, a pathogenic variant of that virus. Low-titer neutralizing antibodies to SHIV-89.6 that provided partial protection against viremia following SHIV-89.6 infection were generated. A similarly low-titer neutralizing antibody response to SHIV-89.6P that did not contain viremia after infection with SHIV-89.6P was generated, but a trend toward protection against CD4+ T-lymphocyte loss was seen in these infected monkeys. These observations suggest that the V3 loop on some primary patient HIV-1 isolates may be a partially effective target for neutralizing antibodies induced by peptide immunogens.

Left ventricular, systemic arterial, and baroreflex responses to ketamine and TEE in chronically instrumented monkeys.

Effects of prescribed doses of ketamine five minutes after application and influences of transesophageal echocardiography (TEE) on left ventricular, systemic arterial, and baroreflex responses were investigated to test the hypothesis that ketamine and/or TEE probe insertion alter cardiovascular function. Seven rhesus monkeys were tested under each of four randomly selected experimental conditions: (1) intravenous bolus dose of ketamine (0.5 ml), (2) continuous infusion of ketamine (500 mg/kg/min), (3) continuous infusion of ketamine (500 mg/kg/min) with TEE, and (4) control (no ketamine or TEE). Monkeys were chronically instrumented with a high fidelity, dual-sensor micromanometer to measure left ventricular and aortic pressure and a transit-time ultrasound probe to measure aortic flow. These measures were used to calculate left ventricular function. A 4-element Windkessel lumped-parameter model was used to estimate total peripheral resistance and systemic arterial compliance. Baroreflex response was calculated as the change in R-R interval divided by the change in mean aortic pressure measured during administration of graded concentrations of nitroprusside. The results indicated that five minutes after ketamine application heart rate and left ventricular diastolic compliance decreased while TEE increased aortic systolic and diastolic pressure. We conclude that ketamine may be administered as either a bolus or continuous infusion without affecting cardiovascular function 5 minutes after application while the insertion of a TEE probe will increase aortic pressure. The results for both ketamine and TEE illustrate the classic "Hawthorne Effect," where the observed values are partly a function of the measurement process. Measures of aortic pressure, heart rate, and left ventricular diastolic pressure should be viewed as relative, as opposed to absolute, when organisms are sedated with ketamine or instrumented with a TEE probe.

Size and myonuclear domains in Rhesus soleus muscle fibers: short-term spaceflight.

The cross-sectional area (CSA), myonuclear number per mm of fiber length, and myonuclear domain (cytoplasmic volume/myonucleus) of mechanically isolated single fibers from biopsies of the soleus muscle of 5 vivarium control, 3 flight simulation and 2 flight (BION 11) Rhesus monkeys (Macaca [correction of Macacca] mulatta) were determined using confocal microscopy before and after a 14-day experimental period. Simulation monkeys were confined in chairs placed in capsules identical to those used during the flight. Fibers were classified as type I, type II or hybrid (containing both types I and II) based on myosin heavy chain (MHC) gel electrophoresis. A majority of the fibers sampled contained only type I MHC, i.e. 89, 62 and 68% for the control, simulation and flight groups, respectively. Most of the remaining fibers were hybrids, i.e. 8, 36 and 32% for the same groups. There were no significant pre-post differences in the fiber type composition for any of the experimental groups. There also were no significant pre-post differences in fiber CSA, myonuclear number or myonuclear domain. There was, however, a tendency for the fibers in the post-flight biopsies to have a smaller mean CSA and myonuclear domain (approximately 10%, p=0.07) than the fibers in the pre-flight biopsy. The combined mean cytoplasmic volume/myonucleus for all muscle fiber phenotypes in the Rhesus soleus muscle was approximately 25,000 micrometers3 and there were no differences in pre-post samples for the control and simulated groups. The cytoplasmic domains tended to be lower (p=0.08) after than before flight. No phenotype differences in cytoplasmic domains were observed. These data suggest that after a relatively short period of actual spaceflight, modest fiber atrophy occurs in the soleus muscle fibers without a concomitant change in myonuclear number.

Live rhesus offspring by artificial insemination using fresh sperm and cryopreserved sperm.

Artificial insemination (AI) and the cryopreservation of sperm with full reproductive capabilities are vital in the armamentarium of infertility clinics and reproductive laboratories. Notwithstanding the fantastic successes with AI and sperm cryopreservation in numerous species, including humans and cattle, these assisted reproductive technologies are less well developed in other species of importance for biomedical research, such as genetically modified mice and nonhuman primates. To that end, AI at high efficiency in the rhesus macaque (Macaca mullata) and the successful cryopreservation of rhesus sperm is presented here, as are the complexities of this primate model due to differences in reproductive tract anatomy and gamete physiology. Cryopreservation had no effect on the ability of sperm to fertilize oocytes in vitro or in vivo. Post-thaw progressive motility was not affected by cryopreservation; however, acrosome integrity was lower for cryopreserved (74.1%) than for fresh sperm (92.7%). Fertilization rates did not differ when fresh (58.1%; n = 32/55) or cryopreserved sperm (63.8%; n = 23/36) were used for in vitro fertilization. Similarly, pregnancy rates did not differ significantly after AI with fresh (57.1%; n = 8/14) or cryopreserved sperm (62.5%; n = 5/8). Seven live rhesus macaques were born following AI with fresh sperm, and three live offspring and two ongoing pregnancies were obtained when cryopreserved sperm were used. Cryopreservation of rhesus sperm as presented here would allow for the cost-effective storage of lineages of nonhuman primates with known genotypes. These results suggest that either national or international centers could be established as repositories to fill the global needs of sperm for nonhuman primate research and to provide the experimental foundation on which to explore and perfect the preservation of sperm from endangered nonhuman primates.

Cardiovascular responses to propofol and etomidate in long-term instrumented rhesus monkeys (Macaca mulatta).

BACKGROUND AND PURPOSE: Cardiac and arterial responses to prescribed doses of propofol and etomidate in rhesus monkeys were compared. METHODS: Intravenously administered induction doses of propofol (2 mg/kg of body weight) or etomidate (1 mg/kg) followed by continuous intravenous infusions of propofol (200 microg/kg/min) or etomidate (100 microg/kg/min) were administered. Left ventricular and right atrial access catheters were implanted for long-term use, along with a transit-time flow probe on the ascending aorta, and pericardial electrocardiogram leads. A dual sensor 3-F micromanometer was used to measure left ventricular pressure and aortic pressure, and an active redirectional transit-time probe measured aortic flow. Noordergraaf's four-element model was used to estimate total peripheral resistance and systemic arterial compliance. RESULTS: Significant (P < 0.01) decreases in mean arterial pressure, heart rate, and myocardial contractility were accompanied by an increase in systemic arterial compliance associated with propofol and etomidate. Only minimal changes in left ventricular diastolic pressure, cardiac output, stroke volume, and total peripheral resistance were found for both drugs. The changes associated with propofol are comparable to results in human beings, whereas the changes associated with etomidate did not agree with results of published human studies. CONCLUSION: The significant cardiovascular alterations associated with both agents were attributed to reductions in heart rate, although the possibility exists that negative inotropic effects may have had a role.

Fiber size and myosin phenotypes of selected rhesus lower limb muscles after a 14-day spaceflight.

Muscle biopsies were taken from the rhesus (Macaca mulatta) soleus (Sol, a slow ankle extensor), medial gastrocnemius (MG, a fast ankle extensor), tibialis anterior (TA, a fast ankle flexor), and vastus lateralis (VL, a fast knee extensor) muscles in vivarium controls (n=5) before and after either a 14-day spaceflight (Bion 11, n=2) or a 14-day ground-based flight simulation (n=3). Myosin heavy chain (MHC) composition (gel electrophoresis), fiber type distribution (immunohistochemistry), and fiber size were determined. Although there were no significant changes, each muscle showed trends towards adaptation.

Rhesus leg muscle EMG activity during a foot pedal pressing task on Bion 11.

Rhesus monkeys (Macaca mulatta) were trained to perform a foot lever pressing task for a food reward. EMG activity was recorded from selected lower limb muscles of 2 animals before, during, and after a 14-day spaceflight and from 3 animals during a ground-based simulation of the flight. Integrated EMG activity was calculated for each muscle during the 20-min test. Comparisons were made between data recorded before any experimental manipulations and during flight or flight simulation. Spaceflight reduced soleus (Sol) activity to 25% of preflight levels, whereas it was reduced to 50% of control in the flight simulation. During flight, medial gastrocnemius (MG) activity was reduced to 25% of preflight activity, whereas the simulation group showed normal activity levels throughout all tests. The change in MG activity was apparent in the first inflight recording, suggesting that some effect of microgravity on MG activity was immediate.

Bion 11 mission: primate experiments.

A summary is provided of the major operations required to conduct the wide range of primate experiments on the Bion 11 mission, which flew for 14 days beginning December 24, 1996. Information is given on preflight preparations, including flight candidate selection and training; attachment and implantation of bioinstrumentation; flight and ground experiment designs; onboard life support and test systems; ground and flight health monitoring; flight monkey selection and transport to the launch site; inflight procedures and data collection; postflight examinations and experiments; and assessment of results.

Induction of B cell hyperplasia in simian immunodeficiency virus-infected rhesus macaques with the simian homologue of Kaposi's sarcoma-associated herpesvirus.

A simian homologue of Kaposi's sarcoma-associated herpesvirus (KSHV), the eighth human herpesvirus (HHV8), was isolated from a simian immunodeficiency virus (SIV)-infected rhesus macaque (Macaca mulatta) that developed a multicentric lymphoproliferative disorder (LPD). This simian rhadinovirus is genetically similar to a recently described rhesus rhadinovirus (RRV) (Desrosiers, R.C., V.G. Sasseville, S.C. Czajak, X. Zhang, K.G. Mansfield, A. Kaur, R.P. Johnson, A.A. Lackner, and J.U. Jung. 1997. J. Virol. 71:9764-9769) and is designated RRV 17577. RRV 17577 was experimentally inoculated into rhesus macaques with and without SIV(mac239) infection to determine if RRV played a role in development of the LPD observed in the index case. In contrast to control animals inoculated with SIV(mac239) or RRV alone, two animals coinfected with SIV(mac239) and RRV 17577 developed hyperplastic LPD resembling the multicentric plasma cell variant of Castleman's disease, characterized by persistent angiofollicular lymphadenopathy, hepatomegaly, splenomegaly, and hypergammaglobulinemia. Hypergammaglobulinemia was associated with severe immune-mediated hemolytic anemia in one RRV/SIV-infected macaque. Both RRV/SIV-infected macaques exhibited persistent RRV viremia with little or no RRV-specific antibody response. The macaques inoculated with RRV alone displayed transient viremia followed by a vigorous anti-RRV antibody response and lacked evidence of LPD in peripheral blood and lymph nodes. Infectious RRV and RRV DNA were present in hyperplastic lymphoid tissues of the RRV/SIV-infected macaques, suggesting that lymphoid hyperplasia is associated with the high levels of replication. Thus, experimental RRV 17577 infection of SIV-infected rhesus macaques induces some of the hyperplastic B cell LPDs manifested in AIDS patients coinfected with KSHV.

Effects of spaceflight on rhesus quadrupedal locomotion after return to 1G.

Effects of spaceflight on Rhesus quadrupedal locomotion after return to 1G. Locomotor performance, activation patterns of the soleus (Sol), medial gastrocnemius (MG), vastus lateralis (VL), and tibialis anterior (TA) and MG tendon force during quadrupedal stepping were studied in adult Rhesus before and after 14 days of either spaceflight (n = 2) or flight simulation at 1G (n = 3). Flight simulation involved duplication of the spaceflight conditions and experimental protocol in a 1G environment. Postflight, but not postsimulation, electromyographic (EMG) recordings revealed clonus-like activity in all muscles. Compared with preflight, the cycle period and burst durations of the primary extensors (Sol, MG, and VL) tended to decrease postflight. These decreases were associated with shorter steps. The flexor (TA) EMG burst duration postflight was similar to preflight, whereas the burst amplitude was elevated. Consequently, the Sol:TA and MG:TA EMG amplitude ratios were lower following flight, reflecting a "flexor bias." Together, these alterations in mean EMG amplitudes reflect differential adaptations in motor-unit recruitment patterns of flexors and extensors as well as fast and slow motor pools. Shorter cycle period and burst durations persisted throughout the 20-day postflight testing period, whereas mean EMG returned to preflight levels by 17 days postflight. Compared with presimulation, the simulation group showed slight increases in the cycle period and burst durations of all muscles. Mean EMG amplitude decreased in the Sol, increased in the MG and VL, and was unchanged in the TA. Thus adaptations observed postsimulation were different from those observed postflight, indicating that there was a response unique to the microgravity environment, i.e., the modulations in the nervous system controlling locomotion cannot merely be attributed to restriction of movement but appear to be the result of changes in the interpretation of load-related proprioceptive feedback to the nervous system. Peak MG tendon force amplitudes were approximately two times greater post- compared with preflight or presimulation. Adaptations in tendon force and EMG amplitude ratios indicate that the nervous system undergoes a reorganization of the recruitment patterns biased toward an increased recruitment of fast versus slow motor units and flexor versus extensor muscles. Combined, these data indicate that some details of the control of motor pools during locomotion are dependent on the persistence of Earth's gravitational environment.

Unique checkpoints during the first cell cycle of fertilization after intracytoplasmic sperm injection in rhesus monkeys.

Intracytoplasmic sperm injection has begun an era of considerable improvements in treating male infertility. Despite its success, questions remain about the dangers of transmitting traits responsible for male infertility, sex and autosomal chromosome aberrations and possible mental, physical and reproductive abnormalities. We report here the first births of rhesus monkeys produced by intracytoplasmic sperm injection at rates greater or equal to those reported by clinics. Essential assumptions about this process are flawed, as shown by results with the preclinical, nonhuman primate model and with clinically discarded specimens. Dynamic imaging demonstrated the variable position of the second meiotic spindle in relation to the first polar body; consequently, microinjection targeting is imprecise and potentially lethal. Intracytoplasmic sperm injection resulted in abnormal sperm decondensation, with the unusual retention of vesicle-associated membrane protein and the perinuclear theca, and the exclusion of the nuclear mitotic apparatus from the decondensing sperm nuclear apex. Male pronuclear remodeling in the injected oocytes was required before replication of either parental genome, indicating a unique G1-to-S transition checkpoint during zygotic interphase (the first cell cycle). These irregularities indicate that the intracytoplasmic sperm injection itself might lead to the observed increased chromosome anomalies.

Changes in human immunodeficiency virus type 1 envelope glycoproteins responsible for the pathogenicity of a multiply passaged simian-human immunodeficiency virus (SHIV-HXBc2).

In vivo passage of a poorly replicating, nonpathogenic simian-human immunodeficiency virus (SHIV-HXBc2) generated an efficiently replicating virus, KU-1, that caused rapid CD4(+) T-lymphocyte depletion and AIDS-like illness in monkeys (S. V. Joag, Z. Li, L. Foresman, E. B. Stephens, L.-J. Zhao, I. Adany, D. M. Pinson, H. M. McClure, and O. Narayan, J. Virol. 70:3189-3197, 1996). The env gene of the KU-1 virus was used to create a molecularly cloned virus, SHIV-HXBc2P 3.2, that differed from a nonpathogenic SHIV-HXBc2 virus in only 12 envelope glycoprotein residues. SHIV-HXBc2P 3.2 replicated efficiently and caused rapid and persistent CD4(+) T-lymphocyte depletion in inoculated rhesus macaques. Compared with the envelope glycoproteins of the parental SHIV-HXBc2, the SHIV-HXBc2P 3.2 envelope glycoproteins supported more efficient infection of rhesus monkey peripheral blood mononuclear cells. Both the parental SHIV-HXBc2 and the pathogenic SHIV-HXBc2P 3.2 used CXCR4 but none of the other seven transmembrane segment receptors tested as a second receptor. Compared with the parental virus, viruses with the SHIV-HXBc2P 3.2 envelope glycoproteins were more resistant to neutralization by soluble CD4 and antibodies. Thus, changes in the envelope glycoproteins account for the ability of the passaged virus to deplete CD4(+) T lymphocytes rapidly and specify increased replicative capacity and resistance to neutralization.

Alterations in the volume stimulus-renal response relationship during exposure to simulated microgravity.

We measured central venous pressure (CVP), plasma volume (PV), urine volume rate (UVR), and circulating hormones (renin activity (PRA), vasopressin (AVP), atrial natriuretic peptide (ANP), and cortisol) before and after acute volume infusion (Dextran-40) to test the hypotheses that head-down tilt bedrest (HDT) caused (1) a resetting of the CVP operating point and (2) attenuated urine excretion. Six rhesus monkeys underwent two experimental conditions (HDT and control, each of 48 hour duration) with each condition separated by nine days of ambulatory activities to produce a cross-over counterbalance design. One test condition was continuous exposure to 10 degrees HDT and the second test condition was a control, defined as approximately 12-14 hours per day of 80 degrees head-up tilt and 10-12 hours prone. Following 48 hours of exposure to either test condition, 20-minute continuous infusion of Dextran-40 was administered. CVP in HDT was lower than the control condition. Similar elevations in CVP occurred 30 min post-infusion in both test conditions, and returned to pre-infusion baseline levels between 22 and 46 h post-infusion in both treatments. The UVR response during infusion was attenuated by HDT despite similar elevation in CVP. Elevation in ANP and reduction in PRA at the end of infusion were greater in Control compared to HDT. No differences between control and HDT were detected for AVP and cortisol responses to infusion. Since CVP returned to its pre-infusion levels following volume loading in HDT and control conditions, it appeared that the lower CVP may reflect a new operating point about which vascular volume is regulated. Further, attenuated ANP and PRA responses during vascular volume loading may contribute to depressed UVR in low gravity exposure.