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1.  The primate preimplantation embryo is a target for relaxin during early pregnancy 
Fertility and sterility  2011;96(1):203-207.
OBJECTIVE
To determine if preimplantation embryos are targets for relaxin secreted from the corpus luteum of the menstrual cycle.
DESIGN
Rhesus monkey oocytes obtained from females undergoing controlled ovarian hyperstimulation were inseminated and the resulting embryos were cultured in medium with or without recombinant human relaxin (20 ng/ml) for 8 days.
SETTING
Research laboratory.
ANIMALS
Rhesus monkey.
INTERVENTIONS
Controlled ovarian stimulation to obtain oocytes for in vitro produced embryos that were cultured with or without human recombinant relaxin.
MAIN OUTCOME MEASURES
The rate of blastocyst development and the percentage of blastocysts and ICM/TE ratio were measured on Day 8 of culture. The presence of relaxin receptor (RXFP1) mRNA in 8 cell embryos was observed by array hybridization.
RESULTS
RXFP1 receptor expression was localized to the inner cell mass of blastocysts as shown by immunohistochemistry. The percentage of embryos that developed to blastocyst and the inner cell mass/ trophectoderm cell ratio was unchanged with relaxin supplementation, however the relaxin-treated embryos developed into blastocysts significantly sooner than untreated embryos.
CONCLUSIONS
These results are the first evidence that the preimplantation primate embryo is a target for relaxin and that the addition of relaxin to in vitro culture medium enhances rhesus monkey embryo development.
doi:10.1016/j.fertnstert.2011.05.016
PMCID: PMC3129389  PMID: 21645893
gene expression; granulosa cells; blastocyst; rhesus macaque
2.  Ontological aspects of pluripotency and stemness gene expression pattern in the rhesus monkey 
Gene expression patterns : GEP  2011;11(3-4):285-298.
Two essential aspects of mammalian development are the progressive specialization of cells toward different lineages, and the maintenance of progenitor cells that will give rise to the differentiated components of each tissue and also contribute new cells as older cells die or become injured. The transition from totipotentiality to pluripotentiality, to multipotentiality, to monopotentiality, and then to differentiation is a continuous process during development. The ontological relationship between these different stages is not well understood. We report for the first time an ontological survey of expression of 45 putative “stemness” and “pluripotency” genes in rhesus monkey oocytes and preimplantation stage embryos, and comparison to the expression in the inner cell mass, trophoblast stem cells, and a rhesus monkey (ORMES6) embryonic stem cell line. Our results reveal that some of these genes are not highly expressed in all totipotent or pluripotent cell types. Some are predominantly maternal mRNAs present in oocytes and embryos before transcriptional activation, and diminishing before the blastocyst stage. Others are well expressed in morulae or early blastocysts, but are poorly expressed in later blastocysts or ICMs. Also, some of the genes employed to induce pluripotent stem cells from somatic cells (iPS genes) appear unlikely to play major roles as stemness or pluripotency genes in normal embryos.
doi:10.1016/j.gep.2011.02.001
PMCID: PMC3109727  PMID: 21329766
stem cell; cell lineage; embryo; trophoblast
3.  Differential Effects of Follistatin on Nonhuman Primate Oocyte Maturation and Pre-Implantation Embryo Development In Vitro1 
Biology of Reproduction  2009;81(6):1139-1146.
There is a vital need to identify factors that enhance human and nonhuman primate in vitro embryo culture and outcome, and to identify the factors that facilitate that objective. Granulosa and cumulus cells were obtained from rhesus monkeys that had either been FSH-primed (in vitro maturation [IVM]) or FSH and hCG-primed (in vivo maturation [VVM]) and compared for the expression of mRNAs encoding follistatin (FST), inhibin, and activin receptors. The FST mRNA displayed marginally decreased expression (P = 0.05) in association with IVM in the granulosa cells. The ACVR1B mRNA was more highly expressed in cumulus cells with IVM compared with VVM. Cumulus-oocyte complexes from FSH-primed monkeys exposed to exogenous FST during the 24-h IVM period exhibited no differences in the percentage of oocytes maturing to the metaphase II stage of meiosis compared to controls. However, embryos from these oocytes had significantly decreased development to the blastocyst stage. The effect of FST on early embryo culture was determined by exposing fertilized VVM oocytes to exogenous FST from 12 to 60 h postinsemination. FST significantly improved time to first cleavage and embryo development to the blastocyst stage compared with controls. The differential effects of exogenous FST on embryo development, when administered before and after oocyte maturation, may depend on the endogenous concentration in cumulus cells and oocytes. These results reveal evolutionary conservation of a positive effect of FST on embryogenesis that may be broadly applicable to enhance in vitro embryogenesis, with potential application to human clinical outcome and livestock and conservation biology.
Follistatin supplementation of culture medium after fertilization improves primate embryo development, while addition during in vitro maturation decreases oocyte developmental potential.
doi:10.1095/biolreprod.109.077198
PMCID: PMC2802231  PMID: 19641179
embryo; gene expression; gene regulation; granulosa cells; oocyte development; ovary; rhesus macaque
4.  Expression of microRNA processing machinery genes in rhesus monkey oocytes and embryos of different developmental potentials 
MicroRNAs (miRNAs) are a class of small RNAs that silence gene expression. In animal cells, miRNAs bind to the 3′ untranslated regions of specific mRNAs and inhibit their translation. The correct regulation of mRNA expression by miRNAs is believed to be important for oocyte maturation, early development and implantation. We examined the expression of 25 mRNAs involved in the microRNA processing pathway in a non human primate oocyte and embryo model. We observed that mRNAs related to miRNA splicing are downregulated during oocyte maturation while those related to miRNA processing are upregulated, indicating that there may exist a temporal difference in their activities related to transcriptional activity in germinal vesicle stage oocytes. We also observed that the vast majority of mRNAs examined were insensitive to α-amanitin at the 8-16 cell stage. The expression data did not reveal a major impact of embryo culture, and hormonal stimulation protocol affected only a small number of mRNAs, suggesting that the components of the pathway may be accumulated in the oocyte during oogenesis and resistant to exogenous insults. In comparison to published mouse array data, we observed species differences and similarities in the temporal expression patterns of some genes, suggesting that miRNA processing may be regulated differently. These data extend our understanding of the potential roles of miRNA during primate embryogenesis.
doi:10.1002/mrd.20950
PMCID: PMC2631102  PMID: 18646051
miRNA; embryo; oocyte; gene regulation; oocyte quality; in vitro maturation; cleavage; preimplantation embryo
5.  Effects of Ooplasm Manipulation on DNA Methylation and Growth of Progeny in Mice1 
Biology of Reproduction  2009;80(3):464-472.
New techniques to boost male and female fertility are being pioneered at a rapid pace in fertility clinics to increase the efficiency of assisted reproduction methods in couples in which natural conception has not been achieved. This study investigates the possible epigenetic effects of ooplasm manipulation methods on postnatal growth and development using a mouse genetic model, with particular emphasis on the possible effects of intergenotype manipulations. We performed interstrain and control intrastrain maternal pronuclear transfers, metaphase-II spindle transfers, and ooplasm transfer between C57BL/6 and DBA/2 mice, and found no major, long-term growth defects or epigenetic abnormalities, in either males or females, associated with intergenotype transfers. Ooplasm transfer itself was associated with reduced viability, and additional subtle effects of ooplasm strain of origin were observed. Both inter- and intrastrain ooplasm transfer were associated with subtle, transient effects on growth early in life. We also performed inter- and intrastrain germinal vesicle transfers (GVTs). Interstrain GVT females, but not males, had significantly lower body weights at birth and thereafter compared with the intrastrain GVT and non-GVT controls. No GVT-associated changes were observed in DNA methylation of the Mup1, Rasgrf1, H19, Snrpn, or Peg3 genes, nor any difference in expression of the imprinted Rasgrf1, Igf2r, or Mest genes. These results indicate that some ooplasm manipulation procedures may exert subtle effects on growth early in life, while intergenotype GVT can result in significant growth deficiencies after birth.
Ooplasm manipulation studies in mice reveal effects of cytoplasm transfer and germinal vesicle transfer on growth.
doi:10.1095/biolreprod.108.073593
PMCID: PMC2805397  PMID: 19073997
assisted reproduction; assisted reproductive technology; DNA methylation; embryo; germinal vesicle transfer; nuclear transfer; oocyte; ovum
6.  Hybrid Vigor and Transgenerational Epigenetic Effects on Early Mouse Embryo Phenotype1  
Biology of Reproduction  2008;79(4):638-648.
Mouse embryos display a strain-dependent propensity for blastomere cytofragmentation at the two-cell stage. The maternal pronucleus exerts a predominant, transcription-dependent effect on this phenotype, with lesser effects of the ooplasm and the paternal pronucleus. A parental origin effect has been observed as an inequality in the cytofragmentation rate of embryos produced through genetic crosses of reciprocal F1 hybrid females. To understand the basis for this, we conducted an extensive series of pronuclear transfer studies employing different combinations of inbred and F1 hybrid maternal and paternal genotypes. We find that the parental origin effect is the result of a transgenerational epigenetic modification, whereby the inherited maternal grandpaternal contribution interacts with the fertilizing paternal genome and the ooplasm. This result indicates that some epigenetic information related to grandparental origins of chromosomes (i.e., imprinting of chromosomes in the mother) is retained through oogenesis and transmitted to progeny, where it affects gene expression from the maternal pronucleus and subsequent embryo phenotype. These results reveal for the first time that mammalian embryonic development can be affected by the epigenotype of at least three individuals. Additionally, we observe a significant suppression of fragmentation by F1 hybrid ooplasm when it is separated from the F1 hybrid maternal pronucleus. This latter effect is a striking example of heterosis in the early mammalian embryo, and it provides a new opportunity for examining the molecular mechanisms of heterosis. These results are relevant to our understanding of the mechanisms of epigenetic effects on development and the possible fertility effects of genetic and epigenetic interactions in reproductive medicine.
Summary: Cytofragmentation in two-cell mouse embryos is controlled by strain-specific factors, epigenetic information from the maternal grandfather, and a striking hybrid vigor effect mediated by the ooplasm
doi:10.1095/biolreprod.108.069096
PMCID: PMC2844494  PMID: 18562704
apoptosis; cytofragmentation; embryo; gene regulation; genomic imprinting; heterosis; mitochondria; nuclear transfer; oocyte development; parental origin effects; superovulation; transgenerational effect

Results 1-6 (6)