The aim of this study was to examine the presence and regulation of glycogen synthase kinase-3α (GSK3A) and GSK-3β (GSK3B) in bovine embryos and their possible roles in embryo development. Our results show that GSK3A and GSK3B are present in bovine embryos at the two-cell stage to the hatched blastocyst stage. Bovine embryo development was associated with an increase in the phosphorylation of both isoforms, being statistically significant at blastocyst and hatched blastocyst stages, compared with earlier stages. Inhibition of GSK3 with CT99021 (3 μM) resulted in a significant increase in the percentage and quality of blastocysts, while inhibition of GSK3 with lithium chloride (LiCl; 20 mM) significantly reduced at the proportion of eight-cell embryos on day 3 and inhibited blastocyst formation. The use of LY294002 (10 μM), a specific inhibitor of phosphatidylinositol-3 kinase, also produced a significant decrease in embryo development. In addition, treatment with LiCl and LY294002 produced a significant decrease in the serine phosphorylation of both isoforms of GSK3. Finally, CT99021 and LiCl reduced the phosphorylation of β-catenin on Ser45 in two-cell embryos, while LY294002 increased it. Despite the fact that LiCl inhibited GSK3 activity, as demonstrated by β-catenin phosphorylation, its effects on the bovine embryo could be mediated through other signaling pathways leading finally to a decrease in the phosphorylation of GSK3 and a reduction in embryo development. Therefore, in conclusion, GSK3A/B serine phosphorylation was positively correlated with embryo development, indicating the importance of an accurate regulation of GSK3 activity during developmental stages to achieve normal bovine embryo development.
I M Aparicio, M Garcia-Herreros, T Fair and P Lonergan
K Bender, S Walsh, A C O Evans, T Fair and L Brennan
There has been a marked decline in the fertility of dairy cows over the past decades, and metabolomic analysis offers a potential to investigate the underlying causes. Metabolite composition of the follicular fluid, which presents the intrafollicular environment, may be an important factor affecting oocyte maturation and subsequent early embryo development. The aim of the present study was to investigate the metabolic differences between follicular fluid from the dominant follicle of lactating cows and heifers using gas chromatography mass spectrometry (GC–MS)-based metabolomics. Follicular fluid and serum were collected from cows and heifers over three phases of follicle development: newly selected dominant follicles, preovulatory follicles prior to oestrus and post-LH surge follicles. Analysis of the fatty acids revealed that there were 24 fatty acids and 9 aqueous metabolites significantly different between cows and heifers. Of particular interest were the higher concentrations of saturated fatty acids (palmitic acid, P=0.001; stearic acid, P=0.005) in follicular fluid from cows and higher docosahexaenoic acid levels (P=0.022) in follicular fluid from heifers. Analysis of the metabolite composition of serum revealed that follicular fluid had a unique lipid composition. The higher concentrations of detrimental saturated fatty in cows will have a negative impact on oocyte maturation and early embryo development. Overall, the results suggest that the follicle microenvironment in cows potentially places their oocytes at a developmental disadvantage compared with heifers, and that this may contribute to well-characterised differences in fertility.
P Hyttel, D Viuff, T Fair, J Laurincik, PD Thomsen, H Callesen, PL Vos, PJ Hendriksen, SJ Dieleman, K Schellander, U Besenfelder and T Greve
This review focuses on the key features of development of the bovine oocyte and embryo, with comparisons of the developmental characteristics of embryos produced in vivo and in vitro. The oocyte is transcriptionally quiescent in the primordial and primary follicle. In the secondary follicle transcription is initiated in the oocyte and a ribosome-synthesizing nucleolus is established in this cell. Transcription and nucleolar activity are enhanced in the tertiary follicle during oocyte growth. When the oocyte reaches approximately 110 microm in diameter, corresponding to a follicle of about 3 mm in diameter, transcription ceases and the nucleolus is inactivated, forming a dense spherical remnant. During the final phase of follicular dominance this remnant becomes vacuolated and, in conjunction with resumption of meiosis, it disperses. The rRNA genes are apparently re-activated during the four-cell stage, that is, the third cell cycle after fertilization, but a nucleolus is not formed. During the subsequent cell cycle, that is, during the eight-cell stage, ribosome-synthesizing nucleoli are again established. Bovine embryos produced in vitro apparently display the same pattern of nucleolus development as that in embryos developed in vivo. Examination of the ploidy of embryonic cells using fluorescence in situ hybridization has revealed that the production of bovine embryos in vitro is associated with increased chromosome aberrations in the embryos. Blastocysts produced in vitro display a significantly higher rate of mixoploidy, that is, when the embryo consists of both normal diploid and abnormal polyploid cells, than that in embryos developed in vivo. The rate of mixoploidy among embryos produced in vitro increases with increasing developmental stage. Moreover, after fertilization in vitro, initially there is a high rate of 'true' polyploidy, that is, when all cells of the embryos are polyploid. However, the polyploid embryos are eliminated before they cleave beyond the eight-cell stage, the stage at which major activation of the embryonic genome occurs in cattle.
M Clemente, J de La Fuente, T Fair, A Al Naib, A Gutierrez-Adan, J F Roche, D Rizos and P Lonergan
The steroid hormone progesterone (P4) plays a key role in the reproductive events associated with pregnancy establishment and maintenance. High concentrations of circulating P4 in the immediate post-conception period have been associated with an advancement of conceptus elongation, an associated increase in interferon-τ production and higher pregnancy rates in cattle. Using in vitro and in vivo models and ∼8500 bovine oocytes across six experiments, the aim of this study was to establish the route through which P4 affects bovine embryo development in vitro and in vivo. mRNA for P4 receptors was present at all stages of embryo development raising the possibility of a direct effect of P4 on the embryo. Exposure to P4 in vitro in the absence or presence of oviduct epithelial cells did not affect the proportion of embryos developing to the blastocyst stage, blastocyst cell number or the relative abundance of selected transcripts in the blastocyst. Furthermore, exposure to P4 in vitro did not affect post-hatching elongation of the embryo following transfer to synchronized recipients and recovery on Day 14. By contrast, transfer of in vitro derived blastocysts to a uterine environment previously primed by elevated P4 resulted in a fourfold increase in conceptus length on Day 14. These data provide clear evidence to support the hypothesis that P4-induced changes in the uterine environment are responsible for the advancement in conceptus elongation reported previously in cattle and that, interestingly, the embryo does not need to be present during the period of high P4 in order to exhibit advanced elongation.
C Passaro, D Tutt, D J Mathew, J M Sanchez, J A Browne, G B Boe-Hansen, T Fair and P Lonergan
The objectives of this study were (i) to determine whether blastocyst-induced responses in endometrial explants were detectable after 6- or 24-h co-culture in vitro; (ii) to test if direct contact is required between embryos and the endometrial surface in order to stimulate endometrial gene expression; (iii) to establish the number of blastocysts required to elicit a detectable endometrial response; (iv) to investigate if upregulation of five interferon-stimulated genes (ISGs) in the endometrium was specific to the blastocyst stage and (v) to test if alterations in endometrial gene expression can be induced by blastocyst-conditioned medium. Exposure of endometrial explants to Day 8 blastocysts in vitro for 6 or 24 h induced the expression of ISGs (MX1, MX2, OAS1, ISG15, RSAD2); expression of IFNAR1, IFNAR2, NFKB1, IL1B, STAT1, LGALS3BP, LGALS9, HPGD, PTGES, ITGB1, AKR1C4, AMD1 and AQP4 was not affected. Culture of explants in the presence of more than five blastocysts was sufficient to induce the effect, with maximum expression of ISGs occurring in the presence of 20 blastocysts. This effect was exclusive to blastocyst stage embryos; oocytes, 2-cell embryos or Day 5 morulae did not alter the relative abundance of any of the transcripts examined. Direct contact between blastocysts and the endometrial surface was not required in order to alter the abundance of these transcripts and blastocyst-conditioned medium alone was sufficient to stimulate a response. Results support the notion that local embryo–maternal interaction may occur as early as Day 8 of pregnancy in cattle.
D Corcoran, T Fair, S Park, D Rizos, O V Patel, G W Smith, P M Coussens, J J Ireland, M P Boland, A C O Evans and P Lonergan
In vivo-derived bovine embryos are of higher quality than those derived in vitro. Many of the differences in quality can be related to culture environment-induced changes in mRNA abundance. The aim of this study was to identify a range of mRNA transcripts that are differentially expressed between bovine blastocysts derived from in vitro versus in vivo culture. Microarray (BOTL5) comparison between in vivo- and in vitro-cultured bovine blastocysts identified 384 genes and expressed sequence tags (ESTs) that were differentially expressed; 85% of these were down-regulated in in vitro cultured blastocysts, showing a much reduced overall level of mRNA expression in in vitro- compared with in vivo-cultured blastocysts. Relative expression of 16 out of 23 (70%) differentially expressed genes (according to P value) were verified in new pools of in vivo- and in vitro-cultured blastocysts, using quantitative real-time PCR. Most (10 out of 16) are involved in transcription and translation events, suggesting that the reason why in vitro-derived embryos are of inferior quality compared with in vivo-derived embryos is due to a deficiency of the machinery associated with transcription and translation.