Assisted reproductive technologies in rhesus macaques

Abstract

The assisted reproductive technologies (ARTs) have been used in the production of rhesus monkey offspring at the Oregon National Primate Research Center (ONPRC) and that experience is summarized here. Additionally these technologies serve as a source of oocytes/embryos for monozygotic twinning, embryonic stem (ES) cell derivation and cloning. High fertilization efficiencies were realized with conventional insemination or following the use of intracytoplasmic sperm injection (ICSI) and approximately 50% of the resulting embryos grew in vitro to blastocysts. Both fresh and frozen sperm were employed in fertilization by ICSI and the resulting embryos could be low temperature stored for subsequent thawing and transfer when a synchronized recipient female was available or after shipment to another facility. Following the transfer of up to 3 embryos, an overall pregnancy rate of 30% was achieved with increasing rates dependent upon the number of embryos transferred. Singleton pregnancy outcomes following the transfer of ART produced embryos were similar to those observed in a control group of animals in the timed mated breeding colony at ONPRC. ICSI produced embryos were used in efforts to create monozygotic twins by blastomere separation or blastocyst splitting. While pregnancies were achieved following the transfer of demi-embryos, only one was a twin and it was lost to spontaneous abortion. ICSI produced embryos have also served as the source of blastocysts for the derivation of embryonic stem cells. These pluripotent cells hold potential for cell based therapies and we consider the monkey an important translational model in which to evaluate safety, efficacy and feasibility of regenerative medicine approaches based on the transplantation of stem cell-derived progeny. Finally, efforts to produce genetically-identical monkeys by nuclear transfer have been briefly summarized.

Figure 1

Schematic presentation of our approach to producing mamu A*01 positive rhesus monkey offspring. Sperm were acquired from an A*01-positive, homozygous male housed at the New England National Primate Research Center, frozen and shipped to ONPRC. Subsequent fertilization by ICSI produced heterozygous mamu A*01 offspring if the oocyte donor was mamu A*01 negative (over 80% of the colony at ONPRC) or 50% homozygous and 50% heterozygous if the oocyte donor was heterozygous for the mamu A*01 allele. We have now produced in excess of 30 offspring using this approach. Infants have also resulted from transfers of frozen-thawed embryos produced in Oregon and shipped to New England.

Figure 2

Average time in days for ART-produced embryos to reach a given developmental stage. Embryos were cultured in CMRL/BRL (yellow triangles), KSOM-AA supplemented with 10% fetal calf serum (pink squares) or HECM-9 with albumin (blue diamonds). M, morula; B, blastocyst; XB, expanded blastocyst. The results for CMRL/BRL coculture and KSOM-AA came from Weston and Wolf [14] and the embryos were produced by IVF while for culture in HECM-9, the results are from Wolf and coauthors [7] with the embryos produced by ISCI. Estimated in vivo developmental rates (open circles) have been included for reference purposes [12].

Figure 3

Illustrations of the progressive stages in laparoscopic embryo transfer in rhesus monkeys. In Panel A, the fimbrium is grasped with a Patton retractor and placed in traction. A guide cannula is then introduced into the oviduct (Panel B). Finally the loaded transfer catheter is inserted transabdominally and advanced into the oviduct 1–3 cm in preparation for embryo deposition (Panel C).

Figure 4

Schematic representation of the derivation of monkey embryonic stem cells. ART-produced embryos are cultured to the expanded blastocyst stage before the trophectoderm is selectively lysed by immunosurgery. The ICM is recovered and cultured on mitotically-inactivated, mouse fetal fibroblasts. Epithelial outgrowths are harvested and dissociated before replating on the coculture. Eventually, ES cell colonies emerge that are selected, expanded and eventually subcloned before characterization [29].

Figure 5

Growth rates for ART-produced offspring. The average weight in kilograms for singleton (closed diamonds) and twin (closed square) offspring are plotted versus time in years. Results are compared to a group of singleton infants produced in the timed mated breeding colony (closed triangle) at ONPRC. Significant differences were observed between the singleton and twin offspring at birth and up until 1 year of age. Results are from Wolf and coauthors [7].