An integrated approach to bovine oocyte quality: from phenotype to genes.

In cattle, early embryonic failure plays a major role in the limitation of reproductive performance and is influenced by genetic effects. Suboptimal oocyte quality, including an inadequate store of maternal factors, is suspected to contribute to this phenomenon. In the present study, 13 Montbeliarde cows were phenotyped on oocyte quality, based on their ability to produce viable embryos after in vitro maturation, fertilisation and culture for 7 days. This discriminated two groups of animals, exhibiting developmental rates below 18.8% or above 40.9% (relative to cleaved embryos). Using microarrays, transcriptomic profiles were compared between oocytes collected in vivo from these two groups of animals. The difference in oocyte development potential was associated with changes in transcripts from 60 genes in immature oocytes and 135 genes in mature oocytes (following Bonferroni 5% correction). Of these, 16 and 32 genes were located in previously identified fertility quantitative trait loci. A subset of differential genes was investigated on distinct samples by reverse transcription-quantitative polymerase chain reaction. For SLC25A16, PPP1R14C, ROBO1, AMDHD1 and MEAF6 transcripts, differential expression was confirmed between high and low oocyte potential animals. Further sequencing and searches for polymorphisms will pave the way for implementing their use in genomic selection.

Metabolomic prediction of pregnancy viability in superovulated cattle embryos and recipients with fourier transform infrared spectroscopy.

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.

Specific expression patterns and cell distribution of ancient and modern PAG in bovine placenta during pregnancy.

Pregnancy-associated glycoproteins (PAGs) constitute a multigenic family of aspartic proteinases expressed in the trophoblast of the ruminant placenta. In Bos taurus, this family comprises 21 members segregated into ancient and modern phylogenetic groups. Ancient PAGs have been reported to be synthesized throughout the trophoblastic cell layer whereas modern PAGs are produced by binucleate cells of cotyledons. The aim of this study was to investigate modern and ancient PAGs during gestation in cotyledonary and intercotyledonary tissues. To obtain convincing and innovative results despite the high sequence identity shared between PAGs, we designed specific tools such as amplification primers and antibodies. Using real-time RT-PCR, we described the transcript expression of 16 bovine PAGs. Overall, PAGs are characterized by an increase in their expression during gestation. However, we demonstrated a segregation of modern PAGs in cotyledons and of ancient PAGs in the intercotyledonary chorion, except for the ancient PAG2 expressed in cotyledons. By raising specific antibodies against the modern PAG1 and ancient PAG11 and PAG2, we established the expression kinetics of the proteins using western blotting. Immunohistochemistry showed that PAGs were produced by specific cellular populations: PAG1 by binucleate cells in the whole trophoblastic layer, PAG11 was localized in binucleate cells of the intercotyledonary trophoblast and the chorionic plate of the cotyledon, while PAG2 was produced in mononucleate cells of the internal villi of the cotyledon. These results revealed a highly specific regulation of PAG expression and cell localization as a function of their phylogenetic status, suggesting distinct biological functions within placental tissues.

In vitro maturation of oocytes alters gene expression and signaling pathways in bovine cumulus cells.

In vitro maturation (IVM) of immature oocytes is widely used in assisted reproduction technologies in cattle, and is increasingly used to treat human infertility. The development competence of IVM oocytes, however, is lower than preovulatory, in vivo-matured oocytes. During maturation, cumulus cells (CC) are metabolically coupled with an oocyte and support the acquisition of its developmental potential. Our objective was to identify genes and pathways that were affected by IVM in bovine CC. Microarray transcriptomic analysis of CC enclosing in vitro- or in vivo-mature oocytes revealed 472 differentially expressed genes, including 28% related to apoptosis, correlating with twofold higher cell death after IVM than in vivo, as detected by TUNEL. Genes overexpressed after IVM were significantly enriched in functions involved in cell movement, focal adhesion, extracellular matrix function, and TGF-beta signaling, whereas under-expressed genes were enriched in regulating gene expression, energy metabolism, stress response, and MAP kinases pathway functions. Differential expression of 15 genes, including PAG11 (increased) and TXNIP (decreased), which were never detected in CC before, was validated by real-time RT-PCR. Moreover, protein quantification confirmed the lower abundance of glutathione S-transferase A1 and prostaglandin G/H synthase 2, and the higher abundance of hyaluronan synthase 2 and SMAD4, a member of TGF-beta pathway, in CC after IVM. Phosphorylation levels of SMAD2, MAPK3/1, and MAPK14, but not MAPK8, were higher after IVM that in vivo. In conclusion, IVM provokes the hyper-activation of TGF-beta and MAPK signaling components, modifies gene expression, leads to increased apoptosis in CC, and thus affects oocyte quality.

Expression of enzymes involved in the synthesis of prostaglandin E2 in bovine in vitro-produced embryos.

Prostaglandin E2 (PGE2) may play a major role in embryo development and the establishment of pregnancy in cattle. The biosynthesis of PGE2 implies the sequential transformation of arachidonic acid to PGH2 by cyclooxygenases (COXs), then the conversion of PGH2 to PGE2 by prostaglandin E synthases (PGESs). Quantitative RT-PCR was used to examine the expression of COX-1, COX-2, microsomal PGES-1 (mPGES-1), microsomal PGES-2 (mPGES-2) and cytosolic PGES (cPGES) mRNAs in day 7 in vitro-produced (IVP) embryos from oocytes collected by ovum pick-up in Holstein heifers. Transcripts for COX-2 and mPGES-1 were detected in all embryos, whereas transcripts for COX-1 and mPGES-2 were not detected and cPGESs were at the limit of detection in 40% of embryos. Levels of COX-2 and mPGES-1 mRNAs were significantly higher in blastocysts and expanded blastocysts than in morulae and early blastocysts. Furthermore, excellent-quality embryos (grade 1) displayed higher levels of both COX-2 and mPGES-1 than did embryos of good and medium qualities (grades 2-3). Our results suggest that bovine IVP embryos at the morula and blastocyst stages use exclusively the COX-2/mPGES-1 pathway for PGE2 biosynthesis, and that PGE2 is potentially involved in blastocyst expansion and developmental competence.

Reproductive technologies and genomic selection in cattle.

The recent development of genomic selection induces dramatic changes in the way genetic selection schemes are to be conducted. This review describes the new context and corresponding needs for genomic based selection schemes and how reproductive technologies can be used to meet those needs. Information brought by reproductive physiology will provide new markers and new improved phenotypes that will increase the efficiency of selection schemes for reproductive traits. In this context, the value of the reproductive techniques including assisted embryo based reproductive technologies (Multiple Ovaluation Embryo Transfer and Ovum pick up associated to in vitro Fertilization) is also revisited. The interest of embryo typing is discussed. The recent results obtained with this emerging technology which are compatible with the use of the last generation of chips for genotype analysis may lead to very promising applications for the breeding industry. The combined use of several embryo based reproductive technologies will probably be more important in the near future to satisfy the needs of genomic selection for increasing the number of candidates and to preserve at the same time genetic variability.

Thymosins ß-4 and ß-10 are expressed in bovine ovarian follicles and upregulated in cumulus cells during meiotic maturation.

ß-Thymosins are small proteins that regulate the actin cytoskeleton and are involved in cell motility, differentiation, the induction of metalloproteinases, in anti-inflammatory processes and tumourigenesis. However, their roles in the ovary have not yet been elucidated. Using transcriptomics and real time reverse transcription-polymerase chain reaction validation, the present study demonstrates that thymosin ß-4 (TMSB4) and thymosin ß-10 (TMSB10) are upregulated in bovine cumulus cells (CCs) during in vitro maturation of cumulus-oocyte complexes (COCs) in parallel with an increase in mRNA expression of HAS2, COX2 and PGR genes. Using immunocytochemistry, both proteins were found to be localised mainly in granulosa cells, CCs and oocytes, in both the cytoplasm and nucleus, as well as being colocalised with F-actin stress fibres in CCs. Using different maturation mediums, we showed that the expression of TMSB10, but not TMSB4, was positively correlated with COC expansion and progesterone secretion and negatively correlated with apoptosis. Immunofluorescence, coupled with terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling (TUNEL), demonstrated the absence of TMSB4 and/or TMSB10 in apoptotic cells. TMSB10 expression was higher in COCs matured in vivo than in vitro, and differences related to the age of the animal were observed. TMSB4 and/or TMSB10 expression was unchanged, whereas HAS2 overexpressed in CCs from oocytes that developed to the blastocyst stage in vitro compared with those that did not. Thus, TMSB4 and/or TMSB10 ovarian expression patterns suggest that these two thymosins may be involved in cumulus modifications during maturation.

Genes preferentially expressed in bovine oocytes revealed by subtractive and suppressive hybridization.

To isolate bovine oocyte marker genes, we performed suppressive and subtractive hybridization between oocytes and somatic tissues (i.e., intestine, lung, muscle, and cumulus cells). The subtracted library was characterized by sequencing 185 random clone inserts, representing 146 nonredundant genes. After Blast analysis within GenBank, 64% could be identified, 21% were homologous to unannotated expressed sequence tag (EST) or genomic sequences, and 15% were novel. Of 768 clone inserts submitted for differential screening by macroarray hybridization, 83% displayed a fourfold overexpression in the oocyte. The 40 most preferential nonredundant ESTs were submitted to GenBank analysis. Several well-known oocyte-specific genes were represented, including growth differentiation factor 9, bone morphogenetic protein 15, or the zona pellucida glycoprotein genes. Other ESTs were not identified. We investigated the expression profile of several candidates in the oocyte and a panel of gonadal and somatic tissues by reverse transcription-polymerase chain reaction. B-cell translocation gene 4, cullin 1, MCF.2 transforming sequence, a locus similar to snail soma ferritin, and three unidentified genes were, indeed, preferentially expressed in the oocyte, even though most were also highly expressed in testis. The transcripts were degraded throughout preimplantation development and were not compensated for by embryonic transcription after the morula stage. These profiles suggest a role in gametogenesis, fertilization, or early embryonic development.