In vivo dynamics of pro-inflammatory factors, mucins, and polymorph nuclear neutrophils in the bovine oviduct during the follicular and luteal phase

Dynamic functional changes in the oviductal microenvironment are the prerequisite for the establishment of pregnancy. The objective of this study was to gain the first insights into oestrous cycle-dependent dynamics of polymorph nuclear neutrophils (PMN) and the mRNA abundance of selected genes and their correlations in the oviduct of living cows. Mini-cytobrush samples were taken from the oviducts of healthy heifers (n = 6) and cows (n = 7) during the follicular (FOL) and luteal phase (LUT) by transvaginal endoscopy. Total RNA was isolated from the samples and subjected to reverse transcription-quantitative PCR for selected pro-inflammatory factors, glycoproteins, and a metabolic marker. The percentage of PMN was determined by cytological examination. The mean PMN percentage was 2.8-fold greater during LUT than FOL. During LUT, significantly greater mRNA abundance of the pro-inflammatory factors IL1B, CXCL1, CXCL3, and CXCL8 was observed. The OVGP1 mRNA abundance was twice as high during FOL than in LUT. Pearson correlation, principal component analysis and heatmap analyses indicated characteristic functional patterns with strong correlations among investigated factors. Using this novel approach, we illustrate complex physiological dynamics and interactions of the mRNA expression of pro-inflammatory factors, mucins, OVGP1, and PMN in the oviduct during the oestrous cycle

Transcriptome profiling of rabbit parthenogenetic blastocysts developed under in vivo conditions

Parthenogenetic embryos are one attractive alternative as a source of embryonic stem cells, although many aspects related to the biology of parthenogenetic embryos and parthenogenetically derived cell lines still need to be elucidated. The present work was conducted to investigate the gene expression profile of rabbit parthenote embryos cultured under in vivo conditions using microarray analysis. Transcriptomic profiles indicate 2541 differentially expressed genes between parthenotes and normal in vivo fertilised blastocysts, of which 76 genes were upregulated and 16 genes downregulated in in vivo cultured parthenote blastocyst, using 3 fold-changes as a cut-off. While differentially upregulated expressed genes are related to transport and protein metabolic process, downregulated expressed genes are related to DNA and RNA binding. Using microarray data, 6 imprinted genes were identified as conserved among rabbits, humans and mice: GRB10, ATP10A, ZNF215, NDN, IMPACT and SFMBT2. We also found that 26 putative genes have at least one member of that gene family imprinted in other species. These data strengthen the view that a large fraction of genes is differentially expressed between parthenogenetic and normal embryos cultured under the same conditions and offer a new approach to the identification of imprinted genes in rabbit. © 2012 Naturil-Alfonso et al.This work was supported by Generalitat Valenciana research programme (Prometeo 2009/125). Carmen Naturil was supported by Generalitat Valenciana research programme (Prometeo 2009/125). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Naturil Alfonso, C.; Saenz De Juano Ribes, MDLD.; Peñaranda, D.; Vicente Antón, JS.; Marco Jiménez, F. (2012). Transcriptome profiling of rabbit parthenogenetic blastocysts developed under in vivo conditions. PLoS ONE. 7(12):1-11. https://doi.org/10.1371/journal.pone.0051271S111712Harness, J. 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Proceedings of the National Academy of Sciences, 100(Supplement 1), 11911-11916. doi:10.1073/pnas.2034195100Chen, Z., Liu, Z., Huang, J., Amano, T., Li, C., Cao, S., … Liu, L. (2009). Birth of Parthenote Mice Directly from Parthenogenetic Embryonic Stem Cells. Stem Cells, 27(9), 2136-2145. doi:10.1002/stem.158Sritanaudomchai, H., Ma, H., Clepper, L., Gokhale, S., Bogan, R., Hennebold, J., … Mitalipov, S. (2010). Discovery of a novel imprinted gene by transcriptional analysis of parthenogenetic embryonic stem cells. Human Reproduction, 25(8), 1927-1941. doi:10.1093/humrep/deq144Fang, Z. F., Gai, H., Huang, Y. Z., Li, S. G., Chen, X. J., Shi, J. J., … Sheng, H. Z. (2006). Rabbit embryonic stem cell lines derived from fertilized, parthenogenetic or somatic cell nuclear transfer embryos. Experimental Cell Research, 312(18), 3669-3682. doi:10.1016/j.yexcr.2006.08.013Wang, S., Tang, X., Niu, Y., Chen, H., Li, B., Li, T., … Ji, W. (2007). Generation and Characterization of Rabbit Embryonic Stem Cells. Stem Cells, 25(2), 481-489. doi:10.1634/stemcells.2006-0226Piedrahita, J. A., Anderson, G. B., & BonDurant, R. H. (1990). On the isolation of embryonic stem cells: Comparative behavior of murine, porcine and ovine embryos. Theriogenology, 34(5), 879-901. doi:10.1016/0093-691x(90)90559-cNaturil-Alfonso, C., Saenz-de-Juano, M. D., Peñaranda, D. S., Vicente, J. S., & Marco-Jiménez, F. (2011). Parthenogenic blastocysts cultured under in vivo conditions exhibit proliferation and differentiation expression genes similar to those of normal embryos. Animal Reproduction Science, 127(3-4), 222-228. doi:10.1016/j.anireprosci.2011.08.005Besenfelder, U., Strouhal, C., & Brem, G. (1998). A Method for Endoscopic Embryo Collection and Transfer in the Rabbit. Journal of Veterinary Medicine Series A, 45(1-10), 577-579. doi:10.1111/j.1439-0442.1998.tb00861.xMehaisen, G. M. K., Viudes-de-Castro, M. P., Vicente, J. S., & Lavara, R. (2006). 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General and Comparative Endocrinology, 142(1-2), 134-142. doi:10.1016/j.ygcen.2004.12.019Llobat, L., Marco-Jiménez, F., Peñaranda, D., Saenz-de-Juano, M., & Vicente, J. (2011). Effect of Embryonic Genotype on Reference Gene Selection for RT-qPCR Normalization. Reproduction in Domestic Animals, 47(4), 629-634. doi:10.1111/j.1439-0531.2011.01934.xLiu, N., Enkemann, S. A., Liang, P., Hersmus, R., Zanazzi, C., Huang, J., … Liu, L. (2010). Genome-wide Gene Expression Profiling Reveals Aberrant MAPK and Wnt Signaling Pathways Associated with Early Parthenogenesis. Journal of Molecular Cell Biology, 2(6), 333-344. doi:10.1093/jmcb/mjq029Abdoon, A. S., Ghanem, N., Kandil, O. M., Gad, A., Schellander, K., & Tesfaye, D. (2012). cDNA microarray analysis of gene expression in parthenotes and in vitro produced buffalo embryos. Theriogenology, 77(6), 1240-1251. doi:10.1016/j.theriogenology.2011.11.004Labrecque, R., & Sirard, M.-A. (2011). Gene expression analysis of bovine blastocysts produced by parthenogenic activation or fertilisation. Reproduction, Fertility and Development, 23(4), 591. doi:10.1071/rd10243Rizos, D., Clemente, M., Bermejo-Alvarez, P., de La Fuente, J., Lonergan, P., & Gutiérrez-Adán, A. (2008). Consequences ofIn VitroCulture Conditions on Embryo Development and Quality. Reproduction in Domestic Animals, 43, 44-50. doi:10.1111/j.1439-0531.2008.01230.xLonergan, P., Rizos, D., Kanka, J., Nemcova, L., Mbaye, A., Kingston, M., … Boland, M. (2003). Temporal sensitivity of bovine embryos to culture environment after fertilization and the implications for blastocyst quality. Reproduction, 337-346. doi:10.1530/rep.0.1260337Memili, E., & First, N. L. (2000). Zygotic and embryonic gene expression in cow: a review of timing and mechanisms of early gene expression as compared with other species. Zygote, 8(1), 87-96. doi:10.1017/s0967199400000861Latham, K. E. (2001). Embryonic genome activation. Frontiers in Bioscience, 6(3), d748-759. doi:10.2741/a639Niemann, H., & Wrenzycki, C. (2000). Alterations of expression of developmentally important genes in preimplantation bovine embryos by in vitro culture conditions: Implications for subsequent development. Theriogenology, 53(1), 21-34. doi:10.1016/s0093-691x(99)00237-xCorcoran, D., Fair, T., Park, S., Rizos, D., Patel, O. V., Smith, G. W., … Lonergan, P. (2006). Suppressed expression of genes involved in transcription and translation in in vitro compared with in vivo cultured bovine embryos. Reproduction, 131(4), 651-660. doi:10.1530/rep.1.01015Morison, I. M., Ramsay, J. P., & Spencer, H. G. (2005). A census of mammalian imprinting. Trends in Genetics, 21(8), 457-465. doi:10.1016/j.tig.2005.06.008Bischoff, S. R., Tsai, S., Hardison, N., Motsinger-Reif, A. A., Freking, B. A., Nonneman, D., … Piedrahita, J. A. (2009). Characterization of Conserved and Nonconserved Imprinted Genes in Swine1. 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Development of Cheaper Embryo Vitrification Device Using the Minimum Volume Method

[EN] This study was designed to compare the efficiency of the Cryotop and Calibrated plastic inoculation loop (CPIL) devices for vitrification of rabbit embryos on in vitro development and implantation rate, offspring rate at birth and embryonic and fetal losses. CPIL is a simple tool used mainly by microbiologists to retrieve an inoculum from a culture of microorganisms. In experiment 1, embryos were vitrified using a Cryotop device and a CPIL device. There were no significant differences in hatched/hatching blastocyst stage rates after 48 h of culture among the vitrified groups (62±4.7% and 62±4.9%, respectively); however, the rates were significantly lower (P<0.05) than those of the fresh group (95±3.4%). In experiment 2, vitrified embryos were transferred using laparoscopic technique. The number of implanted embryos was estimated by laparoscopy as number of implantation sites at day 14 of gestation. At birth, total offspring were recorded. Embryonic and fetal losses were calculated as the difference between implanted embryos and embryos transferred and total born at birth and implanted embryos, respectively. The rate of implantation and development to term was similar between both vitrification devices (56±7.2% and 50±6.8% for implantation rate and 40±7.1% and 35±6.5% for offspring rate at birth); but significantly lower than in the fresh group (78±6.6% for implantation rate and 70±7.2% for offspring rate at birth, P<0.05). Likewise, embryonic losses were similar between both vitrification devices (44±7.2% and 50±6.8%), but significantly higher than in the fresh group (23±6.6%, P < 0.05). However, fetal losses were similar between groups (10±4.4%, 15±4.8% and 8±4.2%, for vitrified, Cryotop or CPIL and fresh, respectively). These results indicate that the CPIL device is as effective as the Cryotop device for vitrification of rabbit embryos, but at a cost of 0.05 per device.This research was supported by the projects Spanish Research project AGL2014-53405-C2-1-P Comision Interministerial de Ciencia y Tecnologia (FMJ, JSV) and Generalitat Valenciana research program (Prometeo II 2014/036, JSV, FMJ).Marco Jiménez, F.; Jiménez Trigos, ME.; Almela-Miralles, V.; Vicente Antón, JS. (2016). Development of Cheaper Embryo Vitrification Device Using the Minimum Volume Method. PLoS ONE. 11(2):1-9. https://doi.org/10.1371/journal.pone.0148661S1911

Effect of Embryo Vitrification on Rabbit Foetal Placenta Proteome during Pregnancy

Very limited information on the post-implantatory effects of vitrification has been published till now. We observed in a previous study that the vitrification procedure for the cryopreservation of embryos introduced transcriptomic and proteomic modifications in the rabbit foetal placenta at the middle of gestation. Now, we have conducted a proteomic study to determine whether protein alterations in the foetal placenta induced by the vitrification procedure remain during pregnancy. In this study, we used 2D-DIGE and mass spectrometry (MALDI-TOF-TOF and LC-MS/MS analysis) to identify the protein changes during middle and late stages of gestation (Day 14 and Day 24, respectively) in rabbit foetal placenta. We identified 11 differentially expressed proteins at Day 14 and 13 proteins at Day 24. Data are available via ProteomeXchange with identifiers PXD001840 and PXD001836. In addition, we demonstrate the presence of three proteins, serum albumin, isocitrate dehydrogenase 1 [NADP+], and phosphoglycerate mutase 1, which were altered during pregnancy. We demonstrate the existence of changes in foetal placental protein during pregnancy induced by the vitrification procedure, which brings into question whether vitrification effects observed during foetal development could lead to physiological and metabolic disorders in adulthood. This effect, taken together with other effects reported in the literature, suggests that embryo cryopreservation is not neutral.This work was supported by the Generalitat Valenciana research program (Prometeo 2014/036) and the Spanish Research Projects (CICYT AGL2011-29831-C03-01). M. D. Saenz-de-Juano was supported by a research grant from Generalitat Valenciana (Programa VALI+d, ACIF/2011/254). Nofima AS provided support in the form of salaries for author KH, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the Author Contributions section.Saenz De Juano Ribes, MDLD.; Vicente Antón, JS.; Hollung, K.; Marco Jiménez, F. (2015). Effect of Embryo Vitrification on Rabbit Foetal Placenta Proteome during Pregnancy. PLoS ONE. 10(4):e0125157-e0125157. https://doi.org/10.1371/journal.pone.0125157Se0125157e012515710

Contribution of the female reproductive tract to low fertility in postpartum lactating dairy cows

Infertility in dairy cattle is a multifactorial problem that may be linked to follicle development and the quality of the ovulated oocyte, to sperm transport and fertilization, to the reproductive tract environment, or to a combination of these factors. Using a state-of-the-art endoscopic embryo transfer technique, the aim of this study was to compare the ability of the reproductive tract of postpartum dairy cows and nulliparous heifers to support the development of early embryos to the blastocyst stage. Bovine embryos of 2 to 4 cells (n=1,800) were produced by in vitro maturation and fertilization of oocytes derived from the ovaries of slaughtered cattle. The estrus cycles of nulliparous Holstein heifers (n=10) and postpartum Holstein cows (n=8, approximately 60 d postpartum) were synchronized using an 8-d controlled internal drug release device coupled with prostaglandin injection. On d 2, one hundred 2- to 4-cell embryos were endoscopically transferred to the oviduct ipsilateral to the corpus luteum. Five days later, on d 7, the oviduct and uterus were flushed nonsurgically to recover the embryos. The number of embryos developing to the blastocyst stage was recorded immediately at recovery and following overnight culture in vitro. A representative number of blastocysts from heifers and cows were stained to assess cell number. Progesterone concentrations were lower in cows than in heifers on d 5, 6, and 7 (d 7=2.39±0.33 vs. 5.34±0.77. ng/mL, respectively). More embryos were recovered from heifers than cows (79.0±7.0 vs. 57.2±11.4%). Of the embryos recovered, 33.9±3.6% had developed to the blastocyst stage in the heifer oviduct compared with 18.3±7.9% in the postpartum cow oviduct. There was no evidence of a difference in blastocyst quality as evidenced by total cell number in the blastocysts (71.2±5.7 vs. 67.0±5.3, respectively). In conclusion, the reproductive tract of the postpartum lactating dairy cow may be less capable of supporting early embryo development than that of the nonlactating heifer, and this may contribute to the lower conception rates observed in such animals. © 2010 American Dairy Science Association