[show abstract][hide abstract] ABSTRACT: The bovine endometrium recognizes early embryos and reacts differently depending on the developmental potential of the embryo. However, it is unknown whether the endometrium can distinguish embryonic sex. Our objective was to analyze sexual dimorphism in the uterus in response to male and female embryos. Differentially expressed (DE) proteins, different levels of hexoses and other embryotrophic differences were analyzed in uterine fluid (UF). Proteomic analysis of day-8 UF recovered from heifers after the transfer of day-5 male or female embryos identified 23 DE proteins. Regulated proteasome/immunoproteasome protein subunits indicated differences in antigen processing between UF carrying male embryos (male-UF) and female-UF. Several enzymes involved in glycolysis/gluconeogenesis and antioxidative/antistress responses were up-regulated in female-UF. Fructose concentration was increased in female-UF versus male-UF, while glucose levels were similar. In vitro cultures with molecules isolated from male-UF were found to improve male embryo development compared to female embryos cultured with molecules isolated from female-UF. We postulated that, in vivo, male embryos induce changes in the endometrium to help ensure their survival. In contrast, female embryos do not appear to induce these changes.
Journal of Proteome Research 02/2013; · 5.06 Impact Factor
[show abstract][hide abstract] ABSTRACT: Pluripotency can be captured in vitro, providing that the culture environment meets the requirements that avoid differentiation while stimulating self-renewal. From studies in the mouse embryo, two kinds of pluripotent stem cells have been obtained from the early and late epiblast, embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs), representing the naive and primed states, respectively. All attempts to derive convincing ESCs in ungulates have been unsuccessful, although all attempts were based on the assumption that the conditions used to derive mouse ESCs or human ESC could be applied in other species. Pluripotent cells derived in primates, rabbit, and pig strongly indicate that the state of pluripotency of these cells is, in fact, closer to EpiSCs than to ESCs, and thus depend on fibroblast growth factor (FGF) and Activin signaling pathways. Based on this observation, we have tried to derive EpiSC from the epiblast of bovine elongated embryos as well as ESCs from Day-8 blastocysts. We here show that the core transcription factors Oct4/Sox2/Nanog can be used as markers of pluripotency in the bovine since their expression was restricted to the developing epiblast after Day 8, and disappeared following differentiation of both the ESC-like and EpiSC-like cultures. Although FGF and Activin pathways are indeed present and active in the bovine, it is not sufficient/enough to maintain a long-term pluripotency ex vivo, as was reported for mouse and pig EpiSCs.
Molecular Reproduction and Development 05/2012; 79(7):461-77. · 2.81 Impact Factor
[show abstract][hide abstract] ABSTRACT: We analyzed embryo-maternal interactions in the bovine uterus on day 8 of development. Proteomic profiles were obtained by two-dimensional difference gel electrophoresis from 8 paired samples of uterine fluid (UF) from the same animal with and without embryos in the uterus. Results were contrasted with UF obtained after artificial insemination. We detected 50 differential protein spots (t test, p < 0.05). Subsequent protein characterization by nano-LC-ESI-MS/MS enabled us to identify 38 proteins, obtaining for first time the earliest evidence of involvement of the down-regulated NFkB system in cattle as a pregnancy signature pathway. Embryos enhanced the embryotrophic ability of UF and decreased uterine protein, while blood progesterone was unaltered. Twinfilin, hepatoma-derived growth factor, and synaptotagmin-binding cytoplasmic RNA interacting protein have not previously been identified in the mammalian uterus. TNFα and IL-1B were localized to embryos by immunocytochemistry, and other proteins were validated by Western blot in UF. Glycosylated-TNFα, IL-1B, insulin, lactotransferrin, nonphosphorylated-peroxiredoxin, albumin, purine nucleoside phosphorylase, HSPA5, and NFkB were down-regulated, while phosphorylated-peroxiredoxin, annexin A4, and nonglycosylated-TNFα were up-regulated. The embryonic signaling agents involved could be TNFα and IL-1B, either alone or in a collective dialogue with other proteins. Such molecules might explain the immune privilege during early bovine development.
Journal of Proteome Research 12/2011; 11(2):751-66. · 5.06 Impact Factor
[show abstract][hide abstract] ABSTRACT: Mammalian oocytes can undergo artificial parthenogenesis in vitro and develop to the blastocyst stage. In this study, using real-time PCR, we analyzed the expression of genes representative of essential events in development. In vitro matured oocytes were either fertilized or activated with ionomycin + 6-DMAP and cultured in simple medium. The pluripotency-related gene Oct3/4 was downregulated in parthenotes, while the de novo methylation DNMT3A gene was unchanged. Among the pregnancy recognition genes, IFN-t was upregulated, PGRMC1 was downregulated and PLAC8 was unchanged in parthenotes. Among the metabolism genes, SLC2A1 was downregulated, while AKR1B1, COX2, H6PD and TXN were upregulated in parthenotes; there was no difference in SLC2A5. Among the genes involved in compaction/blastulation, GJA1 expression increased in parthenotes, but no differences were detected within ATP1A1 and CDH1. Expression of p66(shc) and the Bax/Bcl2 ratio were higher in parthenotes, and there was no difference in p53. Parthenotes and embryos may differ in the way they stimulate apoptosis, with a preponderant role for p66(shc) within parthenotes. Differentially affected functions may also include pluripotency, de novo methylation and early embryonic signalling.
Journal of Reproduction and Development 09/2009; 55(6):607-14. · 1.76 Impact Factor
[show abstract][hide abstract] ABSTRACT: High follicular testosterone levels have been associated with a skew in the sex ratio in favor of males following in vitro fertilization, whereas egg incubation temperature has been found to influence sex ratio in some reptiles. The incubation temperature interferes with the aromatase activity, resulting in a sex determination mechanism thought to be lost in mammals. In this work we aimed to test the effects of testosterone on sex ratio of bovine embryos produced in vitro and to determine whether effects of sex and temperature are effectively decoupled in mammals. Bovine oocytes were in vitro matured for 22 hr in TCM199, PVA, FSH and LH after a 22 hr meiotic arrest in TCM199, PVA and roscovitine 25 microM. Matured oocytes were in vitro fertilized and cultured up to Day 3, and embryos having three or more cells were sexed. In the first experiment, testosterone (0, 30, 300 and 1,500 nM), present both during meiotic inhibition and subsequent in vitro maturation (IVM), did not affect development rates or embryonic sex ratio. In the second experiment, increasing incubation temperatures (38, 39 or 40 degrees C) during meiotic inhibition and subsequent IVM, reduced embryo development, but did not change the sex ratio. Under our experimental conditions, testosterone does not promote a preferential selection of Y-chromosome bearing spermatozoa by the oocyte, and temperature and sex ratio seems to be decoupled in mammals.
Journal of Experimental Zoology Part A Ecological Genetics and Physiology 05/2009; 311(6):448-52. · 1.61 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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 +6-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 p53 respectively, and in their mechanisms to control pluripotency and de novo methylation.
[show abstract][hide abstract] ABSTRACT: Retinoids regulate development and differentiation of the bovine blastocyst in vitro, although the underlying mechanisms remain to be clarified. A challenge in reproductive biotechnology is the identification of pathways that regulate early embryonic development and their influence on blastocyst differentiation, apoptosis and survival to cryopreservation as traits of embryo quality. The present paper analyses the effects of short-term exposure (24 h) to retinoids on in vitro-produced bovine morulae. Immature cumulus oocyte complexes were in vitro matured and fertilised. Presumptive zygotes were subsequently cultured in modified synthetic oviduct fluid up to Day 6, in which morulae were randomly allocated to the different experimental groups. The treatments consisted of 0.1 microM LG100268 (LG; a retinoid X receptor agonist), 0.7 microM all-trans retinoic acid (ATRA; a retinoic acid receptor agonist) or no additives. Day 8 blastocyst development was increased in the ATRA-treated group compared with the LG and untreated embryos. In Day 7 embryos, the number of total cells and cells allocated to the trophectoderm were higher in the ATRA-treated group compared with untreated embryos. Apoptosis in the inner cell mass increased after LG treatment, whereas ATRA had no effect. After vitrification and warming, survival and hatching rates of Day 7 blastocysts did not change with retinoid treatment. Within the LG-treated and untreated blastocyst groups, survival and hatching rates were higher for Day 7 than Day 8 embryos; however, Day 8 blastocysts treated with ATRA showed improved hatching rates. In conclusion, treatment of morulae with ATRA in serum-free medium improves embryo development and quality without increasing the incidence of apoptosis and necrosis.
Reproduction Fertility and Development 02/2008; 20(8):884-91. · 2.58 Impact Factor