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Publications (28)35.92 Total impact

  • Enrique Gomez, Marta Muñoz
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    ABSTRACT: In this paper, we highlight the need to study the very early maternal-embryo interactions and discuss how these interactions can be addressed. Bovine species normally carry one or, less frequently, two embryos to term; there are very rare cases of triplets or higher-order multiple pregnancies in which all the offspring are born alive. Multiple embryo transfer in cattle allows for detecting endometrial responses in a scenario where single embryo transfer would not. Although multiple embryo transfer is non-physiological, our study shows that at the very early embryonic stages, the uterus carrying zona-enclosed embryos does not exhibit non-physiological reactions, but rather a sum of multiple individual effects triggered by developing embryos, probably in their particular response to the uterine environment. We provide arguments that support our hypothesis describing a rationale for current work with multiple embryo transfer and discuss alternative hypotheses. Using cattle as a model species, we describe how technical approaches to analyzing zona-enclosed early embryo maternal interactions (i.e., transcriptomics, proteomics, and endometrial cell culture) can help identify molecular changes that may be difficult to observe when only a single embryo is present. We conclude that multiple embryo transfer can be used for studying very early maternal-embryo interactions in vivo in monotocous species.
    Reproduction 04/2015; DOI:10.1530/REP-14-0465
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    ABSTRACT: The interleukin-1 (IL1) system likely mediates mammalian embryo-maternal communication. In cattle, we have reported that the uterine fluid of heifers carrying early embryos shows downregulated IL1 beta (IL1B), which could lead to reduced NFkB expression and dampening of maternal innate immune responses. In this work, we assessed the expression of IL 1 beta (IL1B) and its receptor, interleukin 1 receptor type I (IL1R1) in the bovine endometrium and embryos by RT-PCR, immunohistochemistry and Western blot at the time of blastocyst development. Day 8 endometrium, both collected from animals after transfer of day 5 embryos (ET) and sham transferred (ST), showed IL1B and IL1R1 mRNA transcription and protein co-localization. Similarly, day 8 blastocyst, from ET animals and entirely produced in vitro, showed IL1R1 mRNA transcription and IL1B and IL1R1 protein co-localization. IL1B mRNA was detected in the analyzed blastocysts, but at very low levels that precluded its quantification. IL1B and IL1R1 immunostaining was observed in luminal epithelial cells, glandular epithelium and stromal cells. The presence of embryos increased endometrial IL1B protein locally, while no differences regarding IL1R1 protein and IL1B and IL1R1 mRNA were detected. These results suggest that the early preimplantation bovine embryo in the maternal tract might interact with the maternal immune system through the IL1 system. Such a mechanism may allow the embryo to elicit local endometrial responses at early stages, which are required for the development of a receptive endometrium. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.
    Journal of Reproductive Immunology 04/2015; 110:1-13. DOI:10.1016/j.jri.2015.03.006 · 2.37 Impact Factor
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    ABSTRACT: Early in cow embryo development, hepatoma-derived growth factor (HDGF) is detectable in uterine fluid. The origin of HDGF in maternal tissues is unknown, as is the effect of the induction on developing embryos. Here we analyze HDGF expression in day-8 endometrium exposed to embryos, as well as the effects of recombinant HDGF (rHDGF) on embryo growth. Exposure to embryos did not alter endometrial levels of HDGF mRNA or protein. HDGF protein localized to cell nuclei in the luminal epithelium and superficial glands and to the apical cytoplasm in deep glands. After uterine passage, levels of embryonic HDGF mRNA decreased and HDGF protein was detected only in the trophectoderm. In fetal fibroblast cultures, addition of rHDGF promoted cell proliferation. In experiments with group cultures of morulae in protein free medium containing polyvinyalcohol, adding rHDGF inhibited blastocyst development and did not affect cell counts when the morulae were early (day 5), whereas it enhanced blastocyst development and increased cell counts when the morulae were compact (day 6). In cultures of individual day 6 morulae, adding rHDGF promoted blastocyst development and increased cell counts. Our experiments with rHDGF indicate that the growth factor stimulates embryonic development and cell proliferation. The HDGF is synthesized by the endometrium and embryo alike, and it may exert embryotrophic effects by autocrine and/or paracrine mechanisms.
    Reproduction (Cambridge, England) 07/2014; 148(4). DOI:10.1530/REP-14-0304 · 3.26 Impact Factor
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    ABSTRACT: Implications Using an in vitro-in vivo model we showed that the IL-1 system could play a role in the bovine embryo-maternal crosstalk during the period of blastocyst formation. Furthermore we detected for the first time Interleukin-1 receptor type I (IL-1R tI) in bovine blastocysts. This information might contribute to improve the in vitro culture of bovine embryos. Introduction The IL-1 system, detected in mammalian embryos and in the reproductive tract, has been suggested as the initiator of conceptus-uterine cross talk during pregnancy (Lindhard et al. 2002; Ross et al. 2003). In cattle, IL-1ß has been detected in the endometrium throughout the estrous cycle (Paula-Lopes et al. 1999) and in embryos and uterine fluid during early preimplantation development (Muñoz et al. 2012) but no information is available about the Interleukin-1 receptor type I (IL-1R tI). In this work we analysed the expression of IL-1ß and IL-1R tI as well as their co-localization in bovine embryos and endometrium during the early development (between day 5 and day 8). Material and methods Synchronized recipients were transferred with multiple in vitro produced morulae on day 5 after in vitro fertilization (ET; N=5). On day 8, animals were slaughtered and their embryos recovered by uterine flushing. Control animals were sham transferred (ST; N=6), and control embryos were entirely cultured in vitro until day 8. Localization of IL-1ß and IL-1R tI in endometrium and embryos were performed by immunohistochemistry and confocal microscopy. Endometrial protein expression differences were evaluated by western blot (WB). Data were analyzed using the GLM procedure of SAS Version 9.2 and REGWQ test for means. Results In the endometrium, IL-1ß and IL-1R tI were highly co-localized in the glandular and luminal epithelial cells, whereas IL-1R tI localized only to blood vessel walls and myometrium. No differences in staining patterns were found between ET and ST animals. WB analyses of IL-1ß showed a major band of 35kDa and a smaller band of 17 kDa corresponding to the reported pro-form and matured forms of IL- 1ß. IL-1ß pro-form was significantly higher in the ET versus ST animals (p0.001) whereas no significant differences were found for mature IL-1ß. WB of IL-1R tI showed one band at 65kDa which correspond to the reported molecular weight of IL-1R tI. No significant differences were found for IL-1R tI expression. In the bovine blastocyst, IL-1ß and IL-1R tI were predominately co-localized in the cytoplasm of trophectoderm cells. No differences in signal intensity were found between in vivo and control embryos. Conclusion The expression of IL-1 beta in the apical cytoplasm adjacent to the glandular lumen and the higher expression of IL-1 beta proform found in the endometrium of ET animals points out to a higher protein translation which will be readily secreted into the uterine lumen in ET vs. ST animals. Our findings suggest that the IL-1 system might exert an important role in the early embryo-maternal dialogue in bovine Acknowledgements Spanish Ministry of Science and Innovation -MICINN–, projects AGL2012-37772. MM and EC and BT are supported by MICINN-RYC08-03454 and MEC-FPU-AP2009-5265 The authors are members of the COST Action FA1201 Epiconcept: Epigenetics and Periconception environment
    International Cow Fertility Conference, Westport, Ireland; 05/2014
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    ABSTRACT: Tumour necrosis factor α (TNF) is expressed in reproductive tissues of several mammals. In cattle, we identified TNF protein present in uterine fluid and expressed in endometrium and blastocysts (Correia et al. 2012, Reproduction, Fertility and Development 25(1) 207-207; Muñoz et al. 2012 J. Proteome Res. 11(2), 751-766). Therefore, TNF may be involved in embryo-maternal communication. In this work we have investigated the RNA expression of TNF and its receptor TNFR2 in bovine endometrium and blastocysts. On day 5 after estrus, cows were sham transferred (N=6) or transferred (N=5) with multiple in vitro produced (IVP) embryos (N=50). All cows were slaughtered on day 8. Embryos were flushed and endometrial samples were collected from caruncular and intercaruncular areas in the middle and cranial horn regions. Blastocysts that developed entirely in vitro were also collected. Endometrial samples and blastocysts were subjected to RT-qPCR and values were normalized by GeNorm using three reference genes (SLC30A6, C20RF29, and RPL19 for endometrium, and SDHA, GAPD, YWHAZ for embryos). Data were analyzed using the GLM procedure of SAS Version 9.2 and REGWQ test. Endometrial TNF and TNFR2 mRNA levels were not significantly affected by the presence of embryos. However, expression of TNF and TNFR2 transcripts was higher in IVP blastocysts than in blastocysts developed in the uterine tract (p<0.05). TNF transcript level increased in middle vs. the cranial uterine region (p=0.03). Our results suggest that TNF and TNFR2 mediate early embryo-maternal interactions in cattle. Grant support: Spanish Ministry of Science and Innovation (MINECO, project AGL2012-37772 and FEDER). ANR-08-GENM-037 (France). MM was supported by grant MICINN-RYC08-03454; ECA, by grants MEC-FPU-AP2009-5265 and COST-STSM-FA1201-12950. The authors are members of the COST Action FA1201 Epiconcept: Epigenetics and Periconception Environment.
    EPICONCEPT, Epigenetics and Periconception Environment, COST Action FA1201, Las Palmas, Spain; 05/2014
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    ABSTRACT: . doi:10.1155/2014/608579.
    BioMed Research International 04/2014; DOI:10.1155/2014/608579. · 2.71 Impact Factor
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    ABSTRACT: We analyzed embryo culture medium (CM) and recipient blood plasma using Fourier transform infrared spectroscopy (FTIR) metabolomics to identify spectral models predictive of pregnancy outcome. Embryos collected on Day 6 from superovulated cows in 2 countries were individually cultured in synthetic oviduct fluid medium with BSA for 24 h before embryo transfer. Spent CM, blank controls, and plasma samples (Day 0 and Day 7) were evaluated using FTIR. The spectra obtained were analyzed. The discrimination capability of the classifiers was assessed for accuracy, sensitivity (pregnancy), specificity (nonpregnancy), and area under the ROC curve (AUC). Endpoints considered were Day 60 pregnancy and birth. High AUC was obtained for Day 60 pregnancy in CM within individual laboratories (France AUC = 0.751 ± 0.039, Spain AUC = 0.718 ± 0.024), while cumulative data decreased the AUC (AUC = 0.604 ± 0.029). Predictions for CM at birth were lower than Day 60 pregnancy. Predictions with plasma at birth improved cumulative over individual results (Day 0: France AUC = 0.690 ± 0.044; Spain AUC < 0.55; cumulative AUC = 0.747 ± 0.032). Plasma generally predicted pregnancy and birth better than CM. These first results show that FTIR metabolomics could allow the identification of embryos and recipients with improved pregnancy viability, which may contribute to increasing the efficiency of selection schemes based on ET.
    04/2014; 2014:608579. DOI:10.1155/2014/608579
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    ABSTRACT: Abstract The objective of this work was to determine whether metabolic fingerprinting of spent bovine embryo culture media using Fourier transform infrared spectroscopy (FTIR) correlates with embryonic sex. Embryos were produced in vitro from oocytes collected from cows slaughtered in an abattoir. Day-6 embryos were individually cultured in synthetic oviduct fluid for 24 h, prior to the time (Day-7) intended for embryo transfer or cryopreservation. Culture medium was analyzed by FTIR. Embryos were sexed by a PCR procedure based on amelogenin gene amplification or transferred to a recipient and sex observed at birth. Media samples from embryos diagnosed as male (n = 47) or female (n = 70) were individually collected and evaluated using FTIR. The spectra obtained were analyzed according to metabolomic profile of embryo culture media and embryonic sex. The discrimination capability of the classifiers was assessed for accuracy, sensitivity (female), sensitivity (male) and area under the ROC curve (AUC). Performance of sex prediction (%) was high within early blastocysts ? blastocysts (74.4 ± 10.2, accuracy; 0.749 ± 0.099, AUC) and excellent for expanded blastocysts (86.0 ± 12.6, accuracy; 0.898 ± 0.094, AUC). A combination of metabolomic and bioinformatic analysis provides a non-invasive mean of embryonic sex analysis.
    Metabolomics 01/2014; 10:443-451. · 3.97 Impact Factor
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    ABSTRACT: The objective of this work was to determine whether metabolic fingerprinting of spent bovine embryo culture media using Fourier transform infrared spectroscopy (FTIR) correlates with embryonic sex. Embryos were produced in vitro from oocytes collected from cows slaughtered in an abattoir. Day-6 embryos were individually cultured in synthetic oviduct fluid for 24 h, prior to the time (Day-7) intended for embryo transfer or cryopreservation. Culture medium was analyzed by FTIR. Embryos were sexed by a PCR procedure based on amelogenin gene amplification or transferred to a recipient and sex observed at birth. Media samples from embryos diagnosed as male (n = 47) or female (n = 70) were individually collected and evaluated using FTIR. The spectra obtained were analyzed according to metabolomic profile of embryo culture media and embryonic sex. The discrimination capability of the classifiers was assessed for accuracy, sensitivity (female), sensitivity (male) and area under the ROC curve (AUC). Performance of sex prediction (%) was high within early blastocysts + blastocysts (74.4 ± 10.2, accuracy; 0.749 ± 0.099, AUC) and excellent for expanded blastocysts (86.0 ± 12.6, accuracy; 0.898 ± 0.094, AUC). A combination of metabolomic and bioinformatic analysis provides a non-invasive mean of embryonic sex analysis.
    Metabolomics 09/2013; 10(3). DOI:10.1007/s11306-013-0587-9 · 3.97 Impact Factor
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    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; 12(3). DOI:10.1021/pr300845e · 5.00 Impact Factor
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    ABSTRACT: Tumor necrosis factor alpha (TNF), a pleiotropic cytokine that could be involved in early embryo-maternal interactions (Muñoz et al. 2012 J. Proteome Res. 11, 751-766), binds to receptors TNFR1 and TNFR2. The TNFR2 mediates apoptotic and survival processes (Fischer et al. 2011, Cell. Signal. 23, 161-170) and its expression is hormonally regulated (Okuda et al. 2010, Mol. Cell. Endocrinol. 330, 41-48). In this work we analyzed the expression of TNFRII by western blot (WB) and immunocytochemistry (ICQ) and its co-localization with TNF by ICQ in bovine embryos and endometrium. Heifers that were transferred with multiple in vitro produced (IVP) embryos (N=3) or sham transferred (N=3) on Day-5 to horn ipsilateral to the corpus luteum were slaughtered on Day-8. Embryos were flushed and endometrial samples were collected from caruncular and intercaruncular regions in the middle and cranial horn thirds. Endometrial samples and Day-8 IVP embryos were subjected to ICQ, and the immunostaining pattern of TNFR2 and TNF was examined by confocal microscopy. Endometrial samples were also subjected to WB. TNRF2 expression was quantified by densitometry (immunoblots) and blind assessment (immunostaining). Data were analyzed using the GLM procedure of SAS Version 9.2 and REGWQ test for means. Trophectoderm (TF) cells from blastocysts and uterine epithelial and stromal cells showed TNFR2 expression. TNF and TNRF2 were predominantly co-localized in embryos and endometrial samples, which showed few unbound ligands and receptors. The presence of embryos increased TNFR2 in the basal glandular epithelia (p≤0.05). Moreover, TNFR2 was higher in the intercaruncular than in the caruncular luminal epithelium (p=0.07). The presence of embryos did not affect TNFR2 expression between cranial and middle horn thirds. However, the TNFR2 low molecular weight isoform (Lmw) in the caruncles and in the middle third of the uterine horn tended to increase in the presence of embryos (p≤0.1). Interestingly, TNFR2 Lmw was more abundant in the middle caruncular region than in other endometrial regions (p0.05). Our findings suggest that TNF can mediate embryo-maternal communication in the uterus acting both in the embryonic and maternal sides. In addition, although implantation does not begin in ruminants until elongation is complete, early bovine embryos seem to show an ability to interact with caruncles. Acknowledgements: Project AGL2009-10059 (MICINN). Muñoz M, Balseiro A, Trigal B and Correia E are sponsored by RYC08-03454, “Contrato de Investigación para Doctores” grant from INIA, Cajastur and FPU (AP2009-5265), respectively
    Internacional Embryo Transfer Society (IETS), Hannover, Germany; 01/2013
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    ABSTRACT: Bilateral asimmetry in the cow affects ovarian function, uterine horn morphology, pregnancy and embryonic sex. However, many aspects and molecular mechanisms of such laterality remain obscure. The objective of this work was identifying new traits of ovarian and uterine asimmetry, as based on oestrus and ovarian monitoring, P4 concentrations, early embryo development, flushing performance, and pregnancy rates. In addition, proteins identified in previous work by DIGE-MS in uterine fluid (UF) were re-analyzed on a horn-of-origin basis (n=9 and n=7, and n=7 and n=7, right and left horn flushes from Muñoz et al, 2012, J Proteome Res 11:751 and Gómez et al Reprod Fert Dev 24(1):152, respectively). Studies were performed in experimental herd and on field farms under an embryo transfer (ET) program. Data were analyzed by Proc GLM of SAS/STAT (Version 9.2; SAS Institute Inc., Cary, NC) and REGWQ test for means. In experimental herd, we analyzed ovarian and uterine asymmetry within animals (n=25) monitored through different reproductive cycles (n=109). Animals synchronized with progestagen+PGF2α were alternatively transferred with IVP embryos (n=30-60) or vehicle (sham transfer) on Day-5 to ipsilateral horn. On Day-8, embryos and/or diluted UF were recovered by flushing. Non significant differences (p>0.7) were obtained within ovulatory follicle diameters measured 48h after PGF2α injection, standing oestrus time and total recoverable protein by flushing between left and right uterine horns. However, Day-8 P4 concentration was higher (p=0.03) in the right (26.3±1.5) than in the left (21.6±1.8) ovary. Fluid recovery (%) was lower in the left (47.0±6.3) than in the right (64.4±5.0%) horn when 30mL were infused (p=0.035); in contrast, 45 mL infused did not significantly differ between horns (61.6±4.1 vs. 67.9±4.1). Less total embryos were recovered from the left (14.6±4.7) than the right (31.0±3.7) horn (p<0.02), although the relative proportions of viable embryos were conserved. Among 76 proteins analyzed, n=9 (VLCAD, TWF1, ENO1, KPYM, CFB, ALB, FGG, EZR and ACTB) differed (P≤0.05) in concentration between left and right horns. On field ET experiments (n=286 synchronized animals; n=39 farms, with at least 3 ETs per farm) confirmed on Day-7 the above differences in P4 (right: 8.3±0.43 vs. left: 6.1±0.55; P=0.0058). The origin of embryos transferred (n=33 IVP fresh; n=42 IVP vitrified; and n=109 in vivo frozen) did not have significant effects. Interestingly, between pregnant and open animals, P4 concentrations differ (P=0.018) in the left horn (15.9±1.7 vs. 8.3±1.2) but not in the right horn (12.4±1.3 vs. 12.4±1.2). Genital asimmetry in the cow has physical concerns (flushing and recoveries), while changes in P4 and/or proteins could influence the pregnancy outcomes after ET. Acknowledgements: Project AGL2009-10059 (MICINN). MM, BT and EC are sponsored by RYC08-03454, Cajastur and AP2009-5265, respectively.
    Internacional Embryo Transfer Society (IETS), Hannover, Germany; 01/2013
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    ABSTRACT: High hydrostatic pressure (HHP) treatment of immature porcine oocytes improves embryo development rates and cell numbers (Pribenszky et al. 2008 Anim. Reprod. Sci. 106, 200–207). However, it is unknown if similar effects can be obtained with bovine oocytes, and how HHP affects cryopreservation of the developed blastocysts. In this work, we analyzed the effect of a HHP treatment (Cryo–Innovation Ltd, Budapest, Hungary) in bovine cumulus oocyte complex (COCs) on their developmental ability and embryo quality. Immature COCs were submitted to a pressure treatment (200 bar, 1h at 37°C; HHP group) (n=643) in Hepes buffered TCM199 (HM). Simultaneously, a group of COCs was held at 37°C for 1h (T group; n=304) in HM, while other COCs were untreated (n=1182). After IVM, COCs were in vitro fertilized (IVF) and cultured in mSOF + 6 gL–1 BSA (Holm et al. 1999 Theriogenology 52, 683–700), and embryo development was recorded (5 replicates). Day 7 and 8 excellent and good quality embryos were selected for vitrification (Cryologic Vitrification Method; Trigal et al. 2012 Theriogenology 10.1016/j.theriogenology.2012.06.018). After warming, vitrified blastocysts were cultured in mSOF + 6 gL–1 BSA + 10% FCS for 48 h (3 replicates). Those blastocysts hatching after warming (at 24 and 48h) were fixed and stained for differential cell counts. Data were analyzed by ANOVA and REGWQ test, and are presented as LSM±SE. HHP treated oocytes showed increased development rates on Day 3 (Day 3 ≥ 5–cell embryos: 64.5±2.9a, 53.4±3.9b, 56.7±2.2b for HHP, T and untreated groups, respectively; a vs b: P<0.05); however, D8 blastocyst rates were not affected by the pressure treatment (28.5±1.6, 26.4±2.2 and 27.8±1.3 for HHP, T and untreated groups respectively). Treatment did not affect survival rates to vitrification (2h re–expansion rates: 100±6.7, 100±6.7 and 95.4±6.7; 48h hatching rates: 58.1±9.4, 71.2±9.4 and 62.3±9.4, for HHP, T and untreated, respectively). Embryos that hatched after warming did not differ in ICM and TE cell counts (ICM: 15.0±1.9, 12.7±3.0 and 13.0±2.0; TE: 133.6±8.4, 137.3±12.8 and 138.4±8.6 for HHP, T and untreated groups, respectively; P>0.05). Complementary studies are needed to analyze the effects of a sublethal stress in bovine oocytes on the subsequent embryo production and quality. Species–specific mechanisms could underlie the differences in results obtained in bovine and porcine. Acknowledgements: RTA2011–00090 (FEDER–INIA). Muñoz, Trigal and Correia are sponsored by RYC08–03454, Cajastur and FPU2009–5265, respectively.
    Internacional Embryo Transfer Society (IETS), Hannover, Germany; 01/2013
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    ABSTRACT: Vitrification allows cryopreserving cells without forming detrimental intracellular ice. However, the warming procedure limits its on field application, as several rehydration steps are usually required. Contrary to vitrification, freezing allows direct transfer (DT) of embryos, although we recently developed a successful vitrification procedure allowing an one–step warming (Trigal et al. 2012, Reprod. Domest. Anim. 47, 94). The aim of this work was to analyze survival to cryopreservation of vitrified or frozen in vitro produced (IVP) embryos after warming or thawing, respectively, by one–step procedures. Bovine blastocysts were produced in vitro as previously reported (Gómez et al. 2009 Reproduction 137, 285–295). Day 7 and 8 excellent and good quality expanded blastocysts were selected for freezing (n=175) or vitrification (n=176) in 4 replicates. Freezing was performed in phosphate buffered saline containing ethylene–glycol 1.5 M and sucrose 0.1 M. Embryos were placed in a Biocool chamber (Biocool II FTS®Systems, Inc) at –7°C for 5 min and seeded. After 5 min embryos were cooled at –0.3°C/min until –32°C and plunged in LN2. Embryos were thawed in a water bath at 37°C for 30 s. Vitrification was performed in fibreplugs as previously described (Trigal et al. 2012 Theriogenology 10.1016/j.theriogenology.2012.06.018). For warming, embryos were incubated for 5 min in TCM199–HEPES, 20% FCS and sucrose 0.25 M. Thawed and warmed embryos were washed and subsequently cultured in mSOFaaci + 6 gL–1 BSA + 10% FCS (Holm et al. 1999 Theriogenology 52, 683–700) for 48 h. Re–expansion (RE) (at 2, 24 and 48 h in culture) and hatching rates (HR) (at 24 and 48 h in culture) were recorded. Total cells were counted in blastocysts that hatched at 24 and 48 h after fixation and bisbenzimide staining. Data were analyzed by ANOVA and are presented as LSM±SE. No differences were found within RE at 2 and 24 h, and HR at 24 h (2 h RE: 94.1±4.9 vs 95.4±4.9; 24 h RE: 92.6±4.9 vs 94.5±4.9; HR: 21.0±7.0 vs 20.6±7.0, for frozen and vitrified embryos respectively; P>0.05). However, at 48 h, vitrified embryos hatched at higher rates than frozen embryos (53.6±10.2 vs 32.5±10.2; P<0.05). Vitrified embryos (143.5±12.7) had higher (P<0.05) cell numbers than frozen embryos (106.1±9.6) after hatching. Our results show that vitrification of IVP embryos in fibreplugs followed by a one–step warming is a promising candidate procedure to replace freezing for DT on field. These results must be completed with embryo transfers to analyze pregnancy rates. Acknowledgements: RTA2011–00090 (FEDER–INIA). Muñoz, Trigal and Correia are sponsored by RYC08–03454, Cajastur and FPU2009–5265 respectively.
    Internacional Embryo Transfer Society (IETS), Hannover, Germany; 01/2013
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    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 12/2012; 79(7):461-77. DOI:10.1002/mrd.22051 · 2.68 Impact Factor
  • 11 Congreso Internacional de la Asociación Española de Reproducción Animal (AERA), Córdoba, Spain; 06/2012
  • 11 Congreso Internacional de la Asociación Española de Reproducción Animal (AERA), Córdoba, Spain; 06/2012
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    ABSTRACT: TNF and IL may be important local mediators in bovine embryo-maternal interactions during early development. In this study we analyzed TNF and IL expression by western blot and immunocytochemistry in the bovine endometrium. Samples from caruncular and intercaruncular endometrium of middle and cranial uterine horns that carried multiple embryos transferred from Day-5 to Day-8 (ET) or sham transferred (ST) were taken. TNF and IL staining was very strong on the apical glandular epithelia (GE), endothelium and stroma, whereas it was weak in luminal epithelia and caruncles. TNF isoforms, 17 kDa, 34 kDa and 37 kDa, showed higher expression in ET than in ST endometrium (p<0.05). The 26 kDa TNF isoform was more abundant in the middle than in the cranial region (p<0.01) and tended to be higher in caruncular than in intercaruncular areas (p=0.06). No significant differences were detected for IL. In summary, TNF secreted by endometrial GE may locally play a role in regulating the early embryo development. That IL might be a mediator in the embryo-maternal dialogue should not be discarded, but the complexity of the IL signalling could make it difficult to detect differences. Further research on endometrial TNF and IL signalling is necessary. Acknowledgements: Project AGL2009-10059 (MICINN). GEMINI, COST Action FA0702. Muñoz M, Trigal B. and Correia E. are sponsored by RYC08-03454, Cajastur and AP2009-5265.
    The 1st Biomarker Meeting in Reproductive Medicine: Emergence of a New Field., Valencia, Spain; 03/2012
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    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. DOI:10.1021/pr200969a · 5.00 Impact Factor
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    ABSTRACT: Individual culture of single embryos is an essential requirement for biomarker technology. In this work we described how a simple culture step allows individual follow-up without reducing embryonic development and viability. In a first experiment, IVM-IVF oocytes were cultured in groups in SOFaaci+BSA (Spain) or SOFaaci+FCS (France) up to Day-6. Morulae and early blastocysts were picked-up and cultured in SOFaaci+BSA droplets under mineral oil for 48h individually or in group culture. Blastocyst development rates did not differ between single and group culture on Day-7 and Day-8 for total blastocysts and expansion rates (p=0.78). However, individually cultured embryos showed improved (p<0.02) Day-8 blastocyst hatching rates in BSA (16.7±0.8 vs. 7.5±0.8, respectively), while no differences were observed in FCS. In BSA, cell counts in the ICM and TE did not differ between single- and group-culture (36.0±2.2 vs. 32.5±2.5 and 143.6±7.6 vs. 138.5±6.7, respectively [p>0.10]); however, single embryos grown with FCS showed increased ICM counts (52.2±1.0 vs. 39.3±1.0; p<0.01). When embryos were grown in BSA, TUNEL staining did not show differences between groups (not performed with FCS). Subsequently, IVP single-cultured embryos were transferred to recipients as fresh (n=22, BSA, 64% pregnancy at Day-60; n=5, FCS, 100% pregnancy) or vitrified/warmed (n=24, BSA, 46% pregnancy) as described in Trigal et al, 2011 (AETE-2011, accepted). In a second experiment, Day-6 morulae (n=50) were obtained from superovulated donors. Morulae were individually cultured for 24h, and only one morula degenerated in culture. Following transfer of fresh embryos, pregnancy rates were 61% (France, n=28) and 57% (Spain, n=21), respectively. Our results show that an individual culture step allows embryonic monitoring, improves embryo development and quality in vitro and provides high pregnancy rates with IVP and in vivo recovered embryos. Projects: AGL2009-10059; RTA2008-0082; M. Muñoz, B. Trigal and E. Correia are sponsored by RYC08-03454, Cajastur and AP2009-5265.
    COST-GEMINI 4th Meeting: Maternal Communication with Gametes and embryo, Gijón, Spain; 09/2011