Parthenotes may represent an alternate ethical source of stem cells, once biological differences between parthenotes and embryos can be understood. In this study, we analyzed development, trophectoderm (TE) differentiation, apoptosis/necrosis, and ploidy in parthenotes and in vitro produced bovine embryos. Subsequently, using real-time PCR, we analyzed the expression of genes expected to underlie the observed differences at the blastocyst stage. In vitro matured oocytes were either fertilized or activated with ionomycin C6-DMAP and cultured in simple medium. Parthenotes showed enhanced blastocyst development and diploidy and reduced TE cell counts. Apoptotic and necrotic indexes did not vary, but parthenotes evidenced a higher relative proportion of apoptotic cells between inner cell mass and TE. The pluripotence-related POU5F1 and the methylation DNMT3A genes were downregulated in parthenotes. Among pregnancy recognition genes, TP-1 was upregulated in parthenotes, while PGRMC1 and PLAC8 did not change. Expression of p66 shc and BAX/BCL2 ratio were higher, and p53 lower, in parthenotes. Among metabolism genes, SLC2A1 was downregulated, while AKR1B1, PTGS2, H6PD, and TXN were upregulated in parthenotes, and SLC2A5 did not differ. Among genes involved in compaction/blastulation, GJA1 was downregulated in parthenotes, but no differences were detected within ATP1A1 and CDH1. Within parthenotes, the expression levels of SLC2A1, TP-1, and H6PD, and possibly AKR1B1, resemble patterns described in female embryos. The pro-apoptotic profile is more pronounced in parthenotes than in embryos, which may differ in their way to channel apoptotic stimuli, through p66 shc and p53respectively, and in their mechanisms to control pluripotency and de novo methylation.Reproduction (2009) 137 285-295

A major goal in reproductive biotechnology is the identification of pathways that regulate early embryonic development and the allocation of cells to the inner cell mass (ICM) and trophectoderm (TE). Retinoids regulate the development and differentiation of the bovine blastocyst in vitro, although the involvement of the retinoid X receptors (RXRs) remains to be clarified. This paper compares the effect of a synthetic RXR agonist (LG100268; LG) with that of the retinoic acid receptor (RAR) agonist all-trans retinoic acid (ATRA) on blastulation. In vitro-produced morulae were treated for 48 h with LG (0.1 mM, 1 mM and 10 mM), ATRA 0.7 mM, or no additives. Treatment with ATRA did not increase the rate of development; however, the LG 0.1 mM treatment increased both the blastocyst development and hatching rate. Cell numbers increased in the ICM with LG 10 mM, while a dosedependent reduction was observed in the TE in the presence of LG. Gene expression levels of p53 and p66 did not vary with LG but increased with ATRA. Both LG and ATRA activated bax, a pro-apoptotic gene and H2A.Z, a cell cycle-related gene. The above effects suggest the existence of active p53-dependent and -independent apoptotic pathways for ATRA and LG, respectively, in the bovine embryo. The expression of p53 and H2A.Z showed a strong, positive correlation (r = 0.93; p < 0.0001) in all experimental groups; both proteins are linked through the cell cycle. Agonists of RXR could be used to control blastocyst development and differentiation. #

Parthenogenetic embryos allow study of the roles of paternal and maternal genomes in early mammalian development. Nevertheless, pregnancies established with parthenotes arrest around 48 days. Genomic imprinting alterations, fewer cells, and apoptotic index are higher in parthenotes than in IVF embryos and are likely to contribute to the failure to reach full-term development. Neurotrophins are a family of anti-apoptotic cytokines that mediate survival, growth, and differentiation by binding to two types of cell surface receptors, tyrosine kinase (Trk) and the low affinity p75 neurotrophin receptor (p75). Trk and p75 receptors have been localized in early bovine in vitro-produced embryos. At present there are no available data on expression of Trk and numbers of cells in the inner cell mass (ICM) and the trophectoderm (TE) of parthenogenetic embryos. The aim of this study was to evaluate the quality of bovine parthenotes in terms of cell allocation and blastocyst development, and to analyze TrkA, TrkB, and TrKC expression in the ICM and TE. Starting from in vitro-matured slaughterhouse oocytes, embryos were produced by conventional IVF, while parthenotes resulted from ionomycin activation followed by 6-dimethylaminopurine. Zygotes were cultured in SOF + 6 gL– 1 BSA. In vitro development was assessed for IVF embryos on (and referred to on) Days 3, 6, 7, and 8 after fertilization, and 24 h before these time points for parthenotes. Data were analyzed by the GLM procedure of SAS SAS Institute, Inc., Cary, NC, USA). Parthenotes cleaved at rates similar to IVF embryos (80.8 � 3.9 v. 85.8 � 3.9, respectively), but percentages of 5–8 and 8–16 cell stages were lower in parthenotes (40.4 � 4.3 v. 67.9 � 4.3, P < 0.005, and 9.7 � 3.5 v. 25.3 � 3.5, P < 0.01, respectively). However, parthenogenetic blastocyst rates were higher than those in IVF embryos (Day 6: 33.6 � 2.6 v. 11.0 � 2.6, P < 0.005; Day 7: 49.2 � 4.1 v. 30.0 � 4.1, P < 0.02). Double staining showed fewer TE cells in parthenotes (78.7 � 8.5) than in IVF embryos (111.0 � 8.6, P < 0.02). This reduction accounted for a reduced number of total cells in parthenotes (105.3 � 9.9) v. controls (144.0 � 9.8, P < 0.01), while numbers of cells in the ICM were comparable (27.9 � 3.5 v. 31.1 � 3.5, in parthenotes and controls, respectively). As in the case of IVF embryos, immunocytochemical analysis showed positive staining for Trk receptors in parthenotes. Although parthenotes showed blastocyst development rates higher than in IVF embryos, the reduced amount of TE cells in parthenotes could negatively affect implantation. Interestingly, parthenotes do not contain abnormally reduced cell numbers in their ICM, and they express Trks. Therefore, specific stimulation of these receptors with appropriate cytokines could improve blastocyst development and embryonic stem cell derivation. This work was supported by the Spanish Ministry of Science and Education (AGL2005-04479). Dr. Muñoz was supported by FICYT.