Strategies for the production of genetically identical monkeys by embryo splitting.

Genetically identical rhesus monkeys would have tremendous utility as models for the study of human disease and would be particularly valuable for vaccine trials and tissue transplantation studies where immune function is important. While advances in nuclear transfer technology may someday enable monkeys to be cloned with some efficiency, embryo splitting may be a more realistic approach to creating pairs of genetically identical monkeys. Although several different approaches to embryo splitting, including blastocyst bisection and blastomere separation, have been used successfully in rodents and domestic species for production of pairs and sets of identical offspring, efforts to create monozygotic twins in rhesus monkeys using these approaches have not met with similar success. Aggregation of split embryos with other types of blastomeres, such as tetraploid and developmentally asynchronous blastomeres, that could potentially increase their cell numbers and developmental competence without contributing to term development has been investigated as an alternative approach to creating monozygotic twin monkeys. The major challenges encountered with respect to the efficient production of monozygotic twins in rhesus monkeys and potential strategies to overcome these challenges are discussed.

In vitro development and cell allocation following aggregation of split embryos with tetraploid or developmentally asynchronous blastomeres in rhesus monkeys.

Production of genetically identical pairs of monkeys would have tremendous implications for biomedical research, particularly immunological studies and vaccine trials. Specific aims of this study were to (1) determine whether aggregation of embryos split into halves or quarters with equal numbers of either developmentally asynchronous or tetraploid blastomeres would enhance their developmental potential in vitro and increase total cell numbers in resulting blastocysts, and (2) determine the allocation of tetraploid and developmentally asynchronous blastomeres in resulting blastocysts. Results demonstrated that development into blastocysts was greater (p < 0.05) for embryos split into pairs (39.8%) than for those split into quadruplet sets (17.4%) and similar (p > 0.05) to that of nonmanipulated controls (59.6%). Creation of chimeras from aggregation of a single 4-cell and four 16-cell stage blastomeres resulted in blastocyst formation (69.2%) similar to that of nonmanipulated control embryos (66.9%). However, neither development nor total cell numbers in resulting blastocysts differed between aggregate chimeras and those split into quadruplet sets at the 16-cell stage. Blastocysts resulting from the aggregate chimeras were derived strictly from the 16-cell stage blastomeres, with complete exclusion of the 4-cell stage blastomeres. Aggregation of split embryos with equal numbers of tetraploid blastomeres doubled (p < 0.05) both the proportion developing into blastocysts and the total cell numbers in resulting blastocysts. Tetraploid blastomeres were allocated to both the inner cell mass and trophectoderm of resulting blastocysts. In conclusion, due to exclusion of the less advanced cells, aggregation of developmentally asynchronous blastomeres did not improve the developmental competence or cell numbers of split rhesus embryos. Reconstitution of split embryos with equal numbers of tetraploid blastomeres enhanced their developmental potential and cell numbers in resulting blastocysts. However, tetraploid blastomeres were allocated to both the inner cell mass and trophectoderm.

Imaging mitochondrial organization in living primate oocytes and embryos using multiphoton microscopy.

We employed multiphoton laser scanning microscopy (MPLSM) to image changes in mitochondrial distribution in living rhesus monkey embryos. This method of imaging does not impair development; thus, the same specimen can be visualized multiple times at various developmental stages. Not only does this increase the amount of information that can be gathered on a single specimen but it permits the correlation of early events with subsequent development in the same specimen. Here we demonstrate the utility of MPLSM for determining changes in mitochondrial organization at various developmental stages and show that rhesus zygotes possess a distinct accumulation of mitochondria between the pronuclei prior to syngamy. We present evidence that suggests that this pronuclear accumulation may be positively correlated with development to the blastocyst stage-in the same embryo-thereby illustrating how MPLSM can be used to correlate cellular dynamics of primate oocytes and early embryos with their developmental potential. Understanding the relationship between mitochondrial distribution and the subsequent development of mammalian embryos, particularly primates, will increase our ability to improve embryo culture technologies, including those used for human assisted reproduction.

Birth of MHC-defined rhesus monkeys produced by assisted reproductive technology.

One of the best animal approaches for testing HIV vaccines is the challenge of vaccinated rhesus macaques with SHIV or SIV. Production of rhesus macaques in which all of the MHC class I and II alleles are known represents an opportunity to characterize the entire immune response to SIV and should be an invaluable resource for understanding pathogenesis and vaccine-induced immune responses. Unfortunately, there are few MHC-defined rhesus macaques available for vaccine research. Selective breeding supports the production of limited numbers of macaques that express particular MHC class I alleles. If both parents express the allele of interest, only three quarters of the offspring will express the same allele. However, assisted reproductive technologies, such as in vitro fertilization (IVF) and embryo transfer, can be used for production of MHC-defined macaques, expressing multiple MHC class I and II molecules for which SIV peptides, tetramers and ELISPOT assays exist. Here, we report the birth of MHC-defined rhesus monkeys produced by assisted reproductive technology. Continued improvements in assisted reproductive technologies in rhesus monkeys will enable us to develop a unique prototypic animal production program for the creation of MHC-defined and genetically-identical monkeys for vaccine research.

Birth of rhesus monkey infant after transfer of embryos derived from in-vitro matured oocytes: short communication.

Although strategies for in-vitro maturation of oocytes from rodents and domestic species have been relatively successful, application of these techniques to primates has not met with similar success. Currently, evaluation of the developmental capacity of oocytes following fertilization is the only reliable means to assess cytoplasmic maturation. Although rhesus monkey blastocysts have previously been produced from in-vitro matured oocytes, full developmental competence has not been demonstrated by term development. Here we report the birth of the first non-human primate infant derived from in-vitro matured oocytes.

Expression of fibrillarin in bovine oocytes and preimplantation embryos developing in vitro.

Studies in somatic cells have demonstrated the participation of some defined nucleolar specific proteins in pre-rRNA synthesis and processing. Fibrillarin, a 34 kd protein localized to the dense fibrillar component of the nucleolus, is involved in the earliest cleavage step of pre-rRNA processing. Expression of fibrillarin has not been characterized during the course of bovine preimplantation development. Specific aims of this study were to characterize the expression of fibrillarin in bovine oocytes and preimplantation embryos developing in vitro. Oocytes and embryos were labeled with an anti-fibrillarin primary antibody and an FITC-conjugated goat antihuman secondary antibody and dual stained with Hoechst 33342 DNA dye. In germinal vesicle (GV) stage oocytes, obtained from 2 to 6 mm follicles, fibrillarin appeared as several large distinct fluorescent spheres within the nucleoplasm, but was not strictly localized to the nucleolus. Fibrillarin was not detected in metaphase oocytes. Expression of fibrillarin was first detected in embryos at the pronucleate stage, appearing as small spots or distinct fluorescent spheres randomly distributed within the nucleoplasm; this pattern persisted in 2- and 4-cell stage embryos. Beginning at the 8-cell stage, fibrillarin signals appeared as distinct fluorescent rings encapsulating each nucleolus. From the 16-cell through the blastocyst stage, a branching immunofluorescent pattern was observed in association with fully reticulated nucleoli. Expression of fibrillarin was absent in 8-cell, but not pronucleate stage embryos cultured with alpha-amanitin, indicating that fibrillarin is initially recruited from maternal stores in pronucleate stage embryos, but then subsequently transcribed beginning at the 8-cell stage, coincident with the onset of nucleolar, transcription. Results from this study provide further insight into the organization and development of the embryonic nucleolus during bovine preimplantation development, and may have important implications for assisted reproductive technologies, including nucleus transfer.

In vitro culture of bovine IVM-IVF embryos: Cooperative interaction among embryos and the role of growth factors.

The objective of this study was to determine whether there is a cooperative interaction among bovine embryos during in vitro culture. Furthermore, culture medium was supplemented with the growth factors, epidermal growth factor (EGF) and transforming growth factor-beta1 (TGF-beta1), to determine if these factors had a stimulatory effect on bovine embryo development similar to that seen in mouse development. In vitro matured - in vitro fertilized bovine embryos (2- to 8-cell) were cultured singly and in groups of five in 25 mul of medium (CR1 + amino acids + fatty acid-free bovine serum albumin) with or without EGF and TGF-beta1. Bovine embryos cultured in groups had a significantly higher rate of development to the blastocyst stage than embryos cultured singly. Neither EGF (10 ng/ml) nor TGF-beta1 (2 ng/ml) affected blastocyst development, hatching or the cell number of the embryos cultured in groups. Epidermal growth factor stimulated hatching of embryos cultured singly from the 8-cell stage, but did not significantly affect blastocyst development.