Artificial insemination and the assisted reproductive technologies in non-human primates.

The experience with artificial insemination (AI) and the more invasive ARTs (assisted reproductive technologies) in the propagation of non-human primates (NHPs), although limited, has included representation from the Great Apes and both Old World and New World Macaques. The application of these technologies in NHPs is impacted by high cost, substantial technical requirements and the limited captive populations of available animals. A major incentive for their use would be to propagate endangered, underrepresented individuals or valuable founder animals. Detailed protocols and a substantial experience base for the ARTs are available for rhesus and cynomolgus macaques and form the basis of this review, including sperm recovery, processing and long-term storage at low temperatures, insemination techniques and timing. Controlled ovarian stimulation and subsequent oocyte recovery required for the invasive ARTs such as intracytoplasmic sperm injection (ICSI), is also described. Three recent AI reports in Old World Macaques are reviewed, along with examples of the use of the ARTs in the propagation of valuable founder animals, in the preservation of endangered macaques, and finally in the creation of neurodegenerative disease models for biomedical research purposes.

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.

Producing primate embryonic stem cells by somatic cell nuclear transfer.

Derivation of embryonic stem (ES) cells genetically identical to a patient by somatic cell nuclear transfer (SCNT) holds the potential to cure or alleviate the symptoms of many degenerative diseases while circumventing concerns regarding rejection by the host immune system. However, the concept has only been achieved in the mouse, whereas inefficient reprogramming and poor embryonic development characterizes the results obtained in primates. Here, we used a modified SCNT approach to produce rhesus macaque blastocysts from adult skin fibroblasts, and successfully isolated two ES cell lines from these embryos. DNA analysis confirmed that nuclear DNA was identical to donor somatic cells and that mitochondrial DNA originated from oocytes. Both cell lines exhibited normal ES cell morphology, expressed key stem-cell markers, were transcriptionally similar to control ES cells and differentiated into multiple cell types in vitro and in vivo. Our results represent successful nuclear reprogramming of adult somatic cells into pluripotent ES cells and demonstrate proof-of-concept for therapeutic cloning in primates.

Reprogramming following somatic cell nuclear transfer in primates is dependent upon nuclear remodeling.

BACKGROUND: Somatic cell nuclear transfer (SCNT) requires cytoplast-mediated reprogramming of the donor nucleus. Cytoplast factors such as maturation promoting factor are implicated based on their involvement in nuclear envelope breakdown (NEBD) and premature chromosome condensation (PCC). Given prior difficulties in SCNT in primates using conventional protocols, we hypothesized that the ability of cytoplasts to induce nuclear remodeling was instrumental in efficient reprogramming. METHODS: NEBD and PCC in monkey (Macaca mulatta) SCNT embryos were monitored by lamin A/C immunolabeling. RESULTS: Initially, a persistent lamin A/C signal from donor cell nuclei after fusion with cytoplasts was observed indicative of incomplete NEBD following SCNT and predictive of developmental arrest. We then identified fluorochrome-assisted enucleation and donor cell electrofusion as likely candidates for inducing premature cytoplast activation and a consequent lack of nuclear remodeling. Modified protocols designed to prevent premature cytoplast activation during SCNT showed robust NEBD and PCC. Coincidently, over 20% of SCNT embryos reconstructed with fetal fibroblasts progressed to blastocysts. Similar results were obtained with other somatic cells. Reconstructed blastocysts displayed patterns of Oct-4 expression similar to fertilized embryos reflecting successful reprogramming. CONCLUSIONS: Our results represent a significant breakthrough in elucidating the role of nuclear remodeling events in reprogramming following SCNT.

A comparative approach to somatic cell nuclear transfer in the rhesus monkey.

BACKGROUND: Despite the potential utility of primate somatic cell nuclear transfer (SCNT) to biomedical research and to the production of autologous embryonic stem (ES) cells for cell- or tissue-based therapy, a reliable method for SCNT is not yet available. Employing the rhesus monkey as a clinically relevant animal model, we have compared a conventional electrofusion method for SCNT with a one-step micromanipulation (OSM) method. METHODS: A prospective, randomized trial was conducted using only oocytes that were mature [metaphase II (MII)] at collection and a fibroblast-like cell line as nuclear donor cells (fetal fibroblasts). The embryos produced were characterized for in vitro developmental potential, cell number, karyotype and expression of nuclear mitotic apparatus (NuMA) and OCT-4. RESULTS: An in vitro blastocyst development rate of 24.4% was achieved with the OSM method, significantly higher than the 12.2% obtained following electrofusion. SCNT-produced embryos expressed normal karyotypes, cell numbers and NuMA and OCT-4 proteins in most cases. SCNT with male nuclear donor cells resulted in the production of male, SCNT blastocysts, eliminating the possibility of a parthenogenetic origin. Of the four fibroblast cell lines tested as nuclear donor cells, two supported the routine production of blastocysts following SCNT. CONCLUSIONS: The application of a modified SCNT technique (OSM) followed by embryo culture in hamster embryo culture medium-10 (HECM-10) allows, for the first time, the routine production of SCNT blastocysts, most of which appear normal by immunochemical, cytochemical and in vitro developmental criteria. These embryos will provide a resource for isolating ES cells and for studies of nuclear reprogramming by monkey cytoplasts.

Development of a monkey model for the study of primate genomic imprinting.

An understanding of the role of imprinted genes in primate development requires the identification of suitable genetic markers that allow analysis of allele-specific expression and methylation status. Four genes, NDN (Necdin), H19, SNRPN and IGF2, known to be imprinted in mice and humans, were selected for study in rhesus monkeys along with two imprinting centres (ICs) associated with the regulation of H19/IGF2, NDN and SNRPN. GAPD was employed as a non-imprinted control gene. Primers designed to amplify polymorphic regions in these genes and ICs were based on human sequences. Genomic DNA was isolated from peripheral blood leukocytes of 93 rhesus macaques of Indian or Chinese-origin. Sequence analysis of amplicons resulted in the identification of 32 unique SNPs. Country-of-origin related differences in SNP distributions were evident. Since disruptions in imprinted gene expression and associated developmental abnormalities may result from in vitro embryo manipulation, we also examined imprinting in NDN, H19, SNRPN and IGF2 in rhesus monkey infants produced by natural mating or by ICSI. Muscle biopsies followed by RT-PCR and sequence analysis were performed in four heterozygous animals produced by natural mating and all four genes were expressed monoallelically supporting the conclusion that these genes are normally imprinted in monkeys. In the case of ICSI, five informative infants were selected based on parental analysis. Allele-specific studies indicated that the expected uniparental expression patterns were retained in animals produced from manipulated embryos. Moreover, methylation analysis revealed that CpG islands within H19/IGF2 and SNURF/SNRPN ICs were differentially methylated. The approach described here will allow examination of imprinting in the embryos and embryonic stem cells of the monkey.

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.

Nuclear transfer technology in mammalian cloning.

The past several years have witnessed remarkable progress in mammalian cloning using nuclear transfer (NT). Until 1997 and the announcement of the successful cloning of sheep from adult mammary gland or fetal fibroblast cells, our working assumption was that cloning by NT could only be accomplished with relatively undifferentiated embryonic cells. Indeed, live offspring were first produced by NT over 15 years ago from totipotent, embryonic blastomeres derived from early cleavage-stage embryos. However, once begun, the progression to somatic cell cloning or NT employing differentiated cells as the source of donor nuclei was meteoric, initially involving differentiated embryonic cell cultures in sheep in 1996 and quickly thereafter, fetal or adult somatic cells in sheep, cow, mouse, goat, and pig. Several recent reviews provide a background for and discussion of these successes. Here we will focus on the potential uses of reproductive cloning along with recent activities in the field and a discussion concerning current interests in human reproductive and therapeutic cloning.

Cryoloop vitrification yields superior survival of Rhesus monkey blastocysts.

BACKGROUND: Vitrification using the cryoloop procedure was evaluated for preservation of non-human primate blastocysts by comparing survival results from two different cryoprotectant mixtures with prior results from controlled rate cooling. METHODS: Rhesus monkey blastocysts were produced by intracytoplasmic sperm injection of mature oocytes from cycling females stimulated with recombinant human hormones. Morphologically well-formed blastocysts were divided between Procedure A (2.8 mol/l dimethylsulphoxide and 3.6 mol/l ethylene glycol with 0.65 mol/l sucrose and 25 micromol/l Ficoll in TALP-HEPES with 20% fetal bovine serum (TH20)) and Procedure B (3.4 mol/l glycerol and 4.5 mol/l ethylene glycol in TH20). After >48 h in liquid nitrogen, the removal of cryoprotectants was accomplished in the presence of a 3-step series of decreasing sucrose concentrations in TH20. Surviving embryos were co-cultured on buffalo rat liver cells. RESULTS: Of 16 blastocysts vitrified via Procedure A, 38% survived with minimal lysis and only one hatched in culture; in contrast, of 33 blastocysts vitrified by Procedure B, 85% survived and 71% hatched. Of 22 blastocysts cryopreserved by conventional slow cooling, 36% survived and 6% hatched. Transfer into three recipients, each with two embryos vitrified with Procedure B, resulted in a successful twin-term pregnancy. CONCLUSION: Modified cryoloop vitrification with a final solution of 3.4 mol/l glycerol and 4.5 mol/l ethylene glycol is a promising procedure for preserving Rhesus monkey blastocysts that is simple, rapid, and inexpensive.

Human spermicidal activity of inorganic and organic oxidants.

OBJECTIVE: To identify prospective oxidants that rapidly immobilize human sperm upon contact with human semen. DESIGN: Inorganic, organic, and enzymatically-generated oxidants were mixed with human semen and spermicidal activity was tracked by a modified Sander-Cramer assay. SETTING: Commercial and university-based laboratories. PATIENT(S): Semen samples obtained through a university-based andrology laboratory. INTERVENTION(S): Not applicable. MAIN OUTCOME MEASURE(S): Quantitation of spermicidal activity of test oxidants. RESULT(S): Sperm lost motility within 20 seconds of exposure to enzymatically generated free iodine (I(2)). Toluidine blue, phenazine methosulfate, or methylene blue exhibited some, albeit much less, spermicidal activity. Oxidants formed by mixing ascorbic acid with Fe(III)-EDTA, xanthine with xanthine oxidase, or by exposing sperm to the nitric oxide generator, SIN-1 (3-morpholinosydnonimine hydrochloride), were far less potent spermicidal agents. CONCLUSION(S): Free I(2) formed in situ and presented to semen is an extremely potent spermicide. Additional studies on methods of generating de novo I(2) may be beneficial in developing a novel new class of nondetergent-based spermicides.

Intrauterine insemination-ready versus conventional semen cryopreservation for donor insemination: a comparison of retrospective results and a prospective, randomized trial.

OBJECTIVE: To compare fecundity rates following intrauterine insemination (IUI) with donor sperm frozen conventionally versus an IUI-ready preparation. DESIGN: Both retrospective results and a prospective, randomized study where recipients were assigned to one of two sperm cryopreservation methods in each cycle of intrauterine insemination are reported. SETTING: University-based infertility practice, affiliated private practices, and andrology laboratory. PATIENT(S): Women desiring therapeutic insemination in an effort to establish pregnancy. INTERVENTION(S): Intrauterine insemination with donor sperm frozen conventionally or by an IUI-ready protocol. MAIN OUTCOME MEASURE(S): Cycle fecundity in donor IUI recipients. RESULT(S): In a retrospective analysis involving 642 inseminations in 209 recipients, 79 pregnancies were recorded for an overall pregnancy rate of 12.3% per insemination (or cycle): 11.3% with IUI-ready sperm and 13.9% with conventionally preserved sperm. In a follow-up prospective, randomized study, the pregnancy rate for IUI-ready sperm preparations was 36% per cycle (14 of 39) whereas that for conventionally preserved sperm was 19.6% per cycle (9 of 46). Thirteen of the 23 pregnancies occurred in the first study cycle of insemination; only two pregnancies were observed in patients undergoing more than four cycles of insemination. CONCLUSION(S): Cycle fecundity for IUI-ready donor sperm is equivalent to conventional cryopreserved sperm based on both prospective and retrospective assessments.

Parthenogenetic activation of rhesus monkey oocytes and reconstructed embryos.

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

Developmental competence of oocytes after ICSI in the rhesus monkey.

Oocyte quantity and quality are critical to assisted reproductive technology (ART), yet few assessments beyond counting metaphase II (MII) oocytes exist. In this study, 30 +/- 2 oocytes per cycle were recovered from rhesus monkeys subjected to follicular stimulation with human gonadotrophins, of which 15 +/- 1 were MII. Oocyte quality was investigated by monitoring the developmental potential of oocytes subjected to intracytoplasmic sperm injection (ICSI). Despite uniform fertilization rates (71 +/- 4%), progression of embryos to blastocysts varied when expressed as a monthly average, from 20 to 85%, with lows from February to April and again in October, which could be attributed to developmental failure of a significant number of oocyte cohorts (14 of 55). Blastocyst rates, after elimination of failed cohorts, were uniform over time (59 +/- 4%). Neither culture conditions, the number of follicular stimulations, nor the individual sperm or oocyte donor were associated specifically with developmental failure, suggesting that intrinsic differences between stimulation cycles account for the observed variation in developmental potential. The in-vivo developmental competence of ICSI-produced embryos grown to blastocysts in vitro was also assessed. Two ongoing pregnancies and the birth of a normal female, 'Blastulina', represent landmarks in efforts to expand the use of ART in the rhesus monkey.

Nuclear transfer and cloning.

The use of nuclear transfer in human reproductive and therapeutic cloning is reviewed with attention on the origins of this technology from its evolution to the present. The successes and limitations of mammalian reproductive cloning are itemized. A case is made against the use of human reproductive cloning to reproduce an existing person, based on the unacceptable risks to the embryo, fetus, or newborn. However, support is extended for human therapeutic cloning involving the derivation and use of embryonic stem cells to treat human disease.

Mammalian cloning: possibilities and threats.

The cloning of mammals originated with the production of limited numbers of genetically identical offspring by blastomere separation or embryo splitting. In the past few years, remarkable progress has been reported in cloning by nuclear transfer (NT) with donor nuclei recovered from embryonic, fetal or adult cells. Factors that contribute to the successful reprogramming of the transferred nucleus and the normal term development of the newly reconstructed embryo include the cell cycle stage of both the donor nucleus and recipient cytoplast, the timing of fusion and cytoplast activation, and the source of donor nuclei. The possibility of producing live offspring by somatic cell NT carries potential applications in animal husbandry, biotechnology, transgenic and pharmaceutical production, biomedical research, and the preservation of endangered species. However, the low efficiencies of cloning by NT coupled with high embryonic, fetal and neonatal losses may restrict immediate commercial applications in agriculture. These limitations notwithstanding, the greatest benefits and practical implications of this new technology could be in transplantation medicine and therapeutic cloning.

Estimates of genetic variance in an F2 maize population.

Maize (Zea mays L.) breeders have used several genetic-statistical models to study the inheritance of quantitative traits. These models provide information on the importance of additive, dominance, and epistatic genetic variance for a quantitative trait. Estimates of genetic variances are useful in understanding heterosis and determining the response to selection. The objectives of this study were to estimate additive and dominance genetic variances and the average level of dominance for an F2 population derived from the B73 x Mo17 hybrid and use weighted least squares to determine the importance of digenic epistatic variances relative to additive and dominance variances. Genetic variances were estimated using Design III and weighted least squares analyses. Both analyses determined that dominance variance was more important than additive variance for grain yield. For other traits, additive genetic variance was more important than dominance variance. The average level of dominance suggests either overdominant gene effects were present for grain yield or pseudo-overdominance because of linkage disequilibrium in the F2 population. Epistatic variances generally were not significantly different from zero and therefore were relatively less important than additive and dominance variances. For several traits estimates of additive by additive epistatic variance decreased estimates of additive genetic variance, but generally the decrease in additive genetic variance was not significant.

Development and integration of an extended embryo culture program.

OBJECTIVE: To study and evaluate a sequential, extended embryo culture system. DESIGN: Prospective study. SETTING: University-affiliated IVF clinic. PATIENT(S): All couples who were treated between October 1997 and July 1998. INTERVENTION(S): A standard human tubal fluid plus 10% serum substitute supplement (SSS) culture medium was used. The embryos were transferred to extended culture medium (S2 or G2) on day 3. MAIN OUTCOME MEASURE(S): Blastocyst formation and implantation and pregnancy rates. RESULT(S): Forty percent of the 20 donated cryopreserved embryos progressed to the blastocyst stage by day 6. Clinically, 7 (5.6%) of the 125 cycles did not result in a transfer. Blastocyst formation rates ranged from 33%-63% in the five study groups. Implantation rates ranged from 15%-52% and pregnancy rates ranged from 37%-75%. CONCLUSION(S): Extended culture to day 5 or 6 results in acceptable blastocyst formation rates, implantation rates, and pregnancy rates.