Genetic merit for fertility traits in Holstein cows: VI. Oocyte developmental competence and embryo development.

The hypothesis of this study was that cows with good genetic merit for fertility traits (Fert+) would produce oocytes and embryos of greater quality than cows with poor genetic merit for fertility traits (Fert-) and that mRNA expression of candidate genes would reflect the observed differences in quality. The aim of the study, therefore, was to determine the effect of genetic merit for fertility traits on morphological classification and mRNA abundance of key genes in immature oocytes and cumulus cells following ovum pick-up and in embryos following superovulation, artificial insemination (AI), and uterine flushing. In experiment 1, 17 Fert+ and 11 Fert- cows, ranging from 54 to 84 d in milk, were submitted to ovum pick-up on 4 occasions during a 2-wk period. Recovered cumulus-oocyte complexes (COC) were morphologically graded. Oocytes and cumulus cells were separated, and mRNA abundance of genes associated with oocyte developmental competence was measured. There was no effect of genotype on the distribution of COC grades or on the mRNA abundance of the candidate genes in grade 1 COC. In experiment 2, 20 Fert+ and 19 Fert- cows, ranging from 71 to 189 d in milk, were submitted to superovulation and AI. The uteri of cows that responded to the superovulation protocol (17 Fert+ and 16 Fert- cows) were nonsurgically flushed 7 d postovulation. Recovered embryos were morphologically graded, and mRNA abundance of genes associated with embryo development was measured in grade 1 blastocysts. The response to the superovulation protocol was assessed by counting the number of codominant follicles on the day of AI, which was similar for both genotypes (22.0 ± 9.7 and 19.8 ± 8.2 for Fert+ and Fert- cows, respectively). There was no effect of genotype on the proportion of transferable embryos recovered or on the mRNA abundance of the candidate genes tested in the grade 1 blastocysts. Of the total embryos classified as blastocysts, however, the Fert+ cows tended to have a greater proportion of grade 1 blastocysts compared with Fert- cows (90% vs. 64%, respectively). In conclusion, genetic merit for fertility traits had a no effect on mRNA abundance of the candidate genes that were examined in immature oocytes and cumulus cells and in embryos recovered after superovulation. The observed differences in morphological blastocyst quality following superovulation would suggest that the superior reproductive performance of Fert+ cows could arise during the later stages of embryo development from d 7 until maternal recognition of pregnancy.

Fertility and genomics: comparison of gene expression in contrasting reproductive tissues of female cattle.

To compare gene expression among bovine tissues, large bovine RNA-seq datasets were used, comprising 280 samples from 10 different bovine tissues (uterine endometrium, granulosa cells, theca cells, cervix, embryos, leucocytes, liver, hypothalamus, pituitary, muscle) and generating 260 Gbases of data. Twin approaches were used: an information-theoretic analysis of the existing annotated transcriptome to identify the most tissue-specific genes and a de-novo transcriptome annotation to evaluate general features of the transcription landscape. Expression was detected for 97% of the Ensembl transcriptome with at least one read in one sample and between 28% and 66% at a level of 10 tags per million (TPM) or greater in individual tissues. Over 95% of genes exhibited some level of tissue-specific gene expression. This was mostly due to different levels of expression in different tissues rather than exclusive expression in a single tissue. Less than 1% of annotated genes exhibited a highly restricted tissue-specific expression profile and approximately 2% exhibited classic housekeeping profiles. In conclusion, it is the combined effects of the variable expression of large numbers of genes (73%-93% of the genome) and the specific expression of a small number of genes (<1% of the transcriptome) that contribute to determining the outcome of the function of individual tissues.

Temporal regulation of fibroblast growth factors and their receptors in the endometrium and conceptus during the pre-implantation period of pregnancy in cattle.

We hypothesised that the expression pattern of members of the fibroblast growth factor (FGF) family would be altered in the endometrium as the oestrous cycle/early pregnancy progressed associated with changes in the expression pattern of their receptors in the developing embryo/conceptus. Expression of FGF1 and FGF10 transcript variants 1 and 2 increased significantly as the oestrous cycle/early pregnancy progressed. Neither progesterone (P4) supplementation nor pregnancy status significantly affected the expression of any of the FGF ligands studied. However, there was a significant interaction between day, pregnancy and P4 status on FGF2 expression (P<0.05) and a significant interaction between P4 status and day on FGF10_tv2 expression. FGF10 protein was localised in the luminal and glandular epithelium as well as the stroma but was not detected in the myometrium. By RNA sequencing, the expression of FGF ligands in the developing embryo/conceptus was found to be minimal. The expression of FGF receptor 1 (FGFR1), FGFR2, FGFR3, FGFR4, FGFRL1 and FRS3 was significantly affected by the stage of conceptus development. Interestingly, the expression of FGFR1 and FGFR4 was higher during early embryo development (days 7-13, P<0.05) but decreased on day 16 (P<0.05) while FGFR2 (P<0.001) expression was similar from day 7 through to day 13, with a significant increase by day 16 (P<0.05) that was maintained until day 19 (P>0.05). In conclusion, these data demonstrate that FGF ligands are primarily expressed by the endometrium and their modulation throughout the luteal phase of the oestrous cycle/early pregnancy are associated with alterations in the expression of their receptors in the embryo/conceptus.

Gene expression in bovine oocytes and cumulus cells after meiotic inhibition with the cyclin-dependent kinase inhibitor butyrolactone I.

The aim of this study was to determine the effect of temporary inhibition of meiosis using the cyclin-dependent kinase inhibitor butyrolactone I (BLI) on gene expression in bovine oocytes and cumulus cells. Immature bovine cumulus-oocyte complexes (COCs) were assigned to groups: (i) Control COCs collected immediately after recovery from the ovary or (ii) after in vitro maturation (IVM) for 24 h, (iii) Inhibited COCs collected 24 h after incubation with 100 µm BLI or (iv) after meiotic inhibition for 24 h followed by IVM for a further 22 h. For mRNA relative abundance analysis, pools of 10 denuded oocytes and respective cumulus cells were collected. Transcripts related to cell cycle regulation and oocyte competence were evaluated in oocytes and cumulus cells by quantitative real-time PCR (qPCR). Most of the examined transcripts were downregulated (p < 0.05) after IVM in control and inhibited oocytes (19 of 35). Nine transcripts remained stable (p > 0.05) after IVM in control oocytes; only INHBA did not show this pattern in inhibited oocytes. Seven genes were upregulated after IVM in control oocytes (p < 0.05), and only PLAT, RBP1 and INHBB were not upregulated in inhibited oocytes after IVM. In cumulus cells, six genes were upregulated (p < 0.05) after IVM and eight were downregulated (p < 0.05). Cells from inhibited oocytes showed the same pattern of expression regarding maturation profile, but were affected by the temporary meiosis inhibition of the oocyte when the same maturation stages were compared between inhibited and control groups. In conclusion, changes in transcript abundance in oocytes and cumulus cells during maturation in vitro were mostly mirrored after meiotic inhibition followed by maturation.

Regulation of non-classical major histocompatability complex class I mRNA expression in bovine embryos.

Regulation of expression of the class I major histocompatability complex (MHC class I) at the maternal fetal interface may play a critical role in embryo survival and the establishment of pregnancy in cattle. However, information concerning immunoregulation of implantation in cattle remains quite limited. Therefore, our current research is concerned with characterizing the expression and regulatory effect of a number of immune factors in the developing bovine embryo. We have analysed the effect of embryo culture in vitro (IVC) in medium supplemented with progesterone (P4): leukemia inhibitory factor (LIF), interferon gamma (IFNG), interleukin (IL)-1B, IL3, IL4, IL10 and granulocyte-colony stimulating factor (G-CSF) on in vitro embryo development and expression of the bovine non-classical MHC class I genes NC2, NC3 and N4 in blastocysts. Cytokine supplementation during IVC did not affect cleavage rate or blastocyst development. However, embryo mRNA expression of NC2, NC3 and NC4 was significantly (p≤0.05) modified in a gene- and cytokine-specific manner. Sequence analysis of the promoter regions of these genes confirmed the presence of appropriate binding sites through which the cytokine signalling could be mediated. In contrast to the lack of effect on in vitro blastocyst development, the non-classical MHC-I expression data suggests a preferential immunomodulatory role of these cytokines during preimplantation embryo development.

Effect of elevated circulating progesterone concentration on bovine blastocyst development and global transcriptome following endoscopic transfer of in vitro produced embryos to the bovine oviduct.

Elevated concentrations of circulating progesterone in the immediate postconception period have been associated with an increase in embryonic growth rate, interferon-tau production, and pregnancy rate in cattle and sheep. Much of this effect is likely mediated via downstream effects of progesterone-induced changes in gene expression in the uterine tissues. Using state-of-the-art endoscopic techniques, this study examined the effect of elevated progesterone on the development of in vitro produced bovine zygotes transferred to the oviducts of heifers with high or normal circulating progesterone concentrations and on the transcriptome of blastocysts developing under such conditions. Simmental heifers (n = 34) were synchronized using a controlled internal drug release (CIDR) device for 8 days, with a prostaglandin F(2 alpha) analogue administered 3 days before removal of the CIDR device. Only animals exhibiting a clear standing estrus (Day 0) were used. To produce animals with divergent progesterone concentrations, half of the animals received a progesterone-releasing intravaginal device (PRID) on Day 3 of the estrous cycle; the PRID was left in place until embryo recovery. All animals were sampled for blood daily from Day 0 to Day 7. Cleaved embryos were transferred by endoscopy to the ipsilateral oviduct of each recipient on Day 2 and then recovered by nonsurgically flushing the oviduct and the uterus on Day 7. The number of embryos developing to the blastocyst stage was recorded at recovery and following overnight culture in vitro. Potential effects of elevated progesterone on transcript abundance were examined using the Affymetrix GeneChip Bovine Genome Array. Insertion of a PRID on Day 3 resulted in a significant elevation of progesterone concentration (P < 0.05) from Day 3.5 until Day 6. Elevated progesterone did not affect the proportion of embryos developing to the blastocyst stage. Genomewide gene expression analysis identified 194 differentially expressed genes between embryos collected from heifers with normal or elevated progesterone, and quantitative real-time PCR validation with a subset of selected genes and an independent sample confirmed the microarray results. Interaction network analysis indicated a significant interaction between progesterone-regulated genes in the blastocyst and in the maternal endometrium. These results suggest that elevated concentrations of progesterone do not affect the ability of the early embryo to reach the blastocyst stage in vivo but do result in subtle changes to the transcriptome of the embryo that may be associated with advanced elongation posthatching.

Transcript profiles of some developmentally important genes detected in bovine oocytes and in vitro-produced blastocysts using RNA amplification and cDNA microarrays.

To study the mRNA transcript profiles of some potential candidate developmental genes during bovine oocyte and blastocyst stages, RNA amplification procedures, cDNA microarray of 82 target genes spotted onto glass slide and real-time polymerase chain reaction (PCR) were used. Messenger RNAs were isolated from in vitro-produced bovine matured oocytes and blastocysts. Using equal amounts of input mRNAs but different cycles of amplifications, cDNAs were produced and served as template for RNA amplification by the in vitro transcriptions. After amplification, the RNA yields transcribed from cDNAs of different cycles were evaluated both by hybridization on the cDNA microarrays and by using real-time PCR techniques. The analyses indicated best results from lower amplification cycle templates with consistent signals at hybridization. Generally, the RNA yield was directly proportional to the amplification cycle but inversely related with signal consistency at repeated hybridizations. Using the protocols established, equal amounts of amplified RNA from matured oocytes and blastocysts were hybridized to the array. Analyses of replicated hybridizations indicated that 35 transcripts were differentially expressed. Most of these were not described in previous bovine embryo studies. Independent analyses of 23 transcripts with real-time PCR and unamplified RNA confirmed the results of 22 genes. Moreover, the functional analyses showed various roles related to development. Hence, it is possible to conclude that the genes identified here are potential candidates for characterizing developmental competence, and that the methods established can be used for large-scale gene expression analysis with more comprehensive arrays.

Expression of retinoid X receptor transcripts and their significance for developmental competence in in vitro-produced pre-implantation-stage bovine embryos.

Retinoid X receptors are frequently implicated in their role in embryonic development. However, there is no report regarding their expression in embryos with different qualities. In this study, expression pattern and levels of retinoid X receptor transcripts in different qualities and stages of in vitro-produced pre-implantation bovine embryos were examined using real-time polymerase chain reaction (PCR) techniques. Four independent cDNA pools per sample were prepared from mRNAs isolated from different developmental-stage embryos, after classifying them to be of good and bad quality. Primers were also designed to amplify products with PCR. Based on analysis, except retinoid X receptor gamma (RXRG) transcripts, the remaining two types (alpha and beta forms) were expressed in all samples with significantly higher (p < or = 0.05) levels until the eight-cell stage compared with the stages thereafter. However, the transcript levels for bad-quality embryos were consistently lower in all cases in comparison with good-quality embryos. RXRG was identified in 39% of the samples, but all in the pre-embryonic genome activation development stage. Therefore, strong expression patterns of these transcripts in earlier stages indicates their possible role during the maternal phase of embryo development, and the variations of copy number in embryos with different qualities substantiates their possible candidacy as potential quality markers. Moreover, identifying the transcript variations in different qualities and expression of RXRG at these stages is a novel input to the pre-implantation-stage knowledge.

Mapping of ESTs derived from pre-implantation stage cattle embryos to allocate 16 new additions to the ordered comparative map of cattle and human.

Twenty expressed sequence tags (ESTs) derived from cDNA libraries of different developmental stages of embryos were mapped using a whole genome bovine hamster radiation hybrid panel. These include 14 markers representing genes, most of which have not so far been mapped in cattle, with another three being novel in both cattle and human. The markers were placed on specific chromosomes with high LOD scores and except two all localizations fit the current human and cattle comparative map. The assignment of these genes further enriches the cattle genome map and also contributes to the international effort of generating comparative maps.