Réjean C. Lefebvre

In vitro culture and somatic cell nuclear transfer affect imprinting of SNRPN gene in pre- and post-implantation stages of development in cattle.

BackgroundEmbryo in vitro manipulations during early development are thought to increase mortality by altering the epigenetic regulation of some imprinted genes. Using a bovine interspecies model with a single nucleotide polymorphism, we assessed the imprinting status of the small nuclear ribonucleoprotein polypeptide N (SNRPN) gene in bovine embryos produced by artificial insemination (AI), in vitro culture (IVF) and somatic cell nuclear transfer (SCNT) and correlated allelic expression with the DNA methylation patterns of a differentially methylated region (DMR) located on the SNRPN promoter.ResultsIn the AI group, SNRPN maternal expression is silenced at day 17 and 40 of development and a third of the alleles analyzed are methylated in the DMR. In the IVF group, maternal transcripts were identified at day 17 but methylation levels were similar to the AI group. However, day-40 fetuses in the IVF group showed significantly less methylation when compared to the AI group and SNRPN expression was mostly paternal in all fetal tissues studied, except in placenta. Finally, the SCNT group presented severe loss of DMR methylation in both day-17 embryos and 40 fetuses and biallelic expression was observed in all stages and tissues analyzed.ConclusionTogether these results suggest that artificial reproductive techniques, such as prolonged in vitro culture and SCNT, lead to abnormal reprogramming of imprinting of SNRPN gene by altering methylation levels at this locus.

Loss of Methylation at H19 DMD Is Associated with Biallelic Expression and Reduced Development in Cattle Derived by Somatic Cell Nuclear Transfer

Although cloning of mammals has been achieved successfully, the percentage of live offspring is very low because of reduced fetal size and fewer implantation sites. Recent studies have attributed such pathological conditions to abnormal reprogramming of the donor cell used for cloning. The inability of the oocyte to fully restore the differentiated status of a somatic cell to its pluripotent and undifferentiated state is normally evidenced by aberrant DNA methylation patterns established throughout the genome during development to blastocyst. These aberrant methylation patterns are associated with abnormal expression of imprinted genes, which among other genes are essential for normal embryo development and gestation. We hypothesized that embryo loss and low implantation rates in cattle derived by somatic cell nuclear transfer (SCNT) are caused by abnormal epigenetic reprogramming of imprinted genes. To verify our hypothesis, we analyzed the parental expression and the differentially methylated domain (DMD) methylation status of the H19 gene. Using a parental-specific analysis, we confirmed for the first time that H19 biallelic expression is tightly associated with a severe demethylation of the paternal H19 DMD in SCNT embryos, suggesting that these epigenetic anomalies to the H19 locus could be directly responsible for the reduced size and low implantation rates of cloned embryos in cattle.

Expression of Phospholipase A2 Group IVA (PLA2G4A) Is Upregulated by Human Chorionic Gonadotropin in Bovine Granulosa Cells of Ovulatory Follicles

Abstract Prostaglandins are required for the ovulatory process, and their biosynthesis depends on the initial release of arachidonic acid from membrane phospholipids. We hypothesized that phospholipase A2 group IVA (PLA2G4A) expression is upregulated in granulosa cells (GC) at ovulation. We have characterized bovine PLA2G4A cDNA, and investigated its spatiotemporal regulation at the mRNA and protein levels in hCG-induced ovulatory follicles and in vitro, using forskolin-stimulated GC. Regulation of PLA2G4A mRNA expression was studied in GC obtained from bovine follicles collected at different developmental stages: small follicles (2–4 mm), dominant follicles at Day 5 (D5) of the estrous cycle, ovulatory follicles 24 h following injection of hCG, and corpus luteum at D5. PLA2G4A mRNA increased by 14-fold in GC of hCG-stimulated versus dominant follicles (P < 0.0001). Follicular walls obtained from ovulatory follicles recovered at 0, 6, 12, 18, and 24 h post-hCG injection showed an initial 16-fold increase in PLA2G4A transcript at 12 h that reached a 45-fold increase at 24 h, as compared to 0 h (P < 0.0001). Immunoblots of GC extracts showed an initial induction of the PLA2G4A protein at 18 h post-hCG, reaching a maximum at 24 h. Immunohistochemistry observations showed that PLA2G4A signal was mainly observed in mural GC compared to antral GC in hCG-stimulated follicles. Stimulation of cultured bovine GC with 10 μM of forskolin caused an increase in PLA2G4A mRNA and protein. Ovulation is associated with an LH/hCG-dependent induction of PLA2G4A in GC via the adenylyl cyclase/cAMP pathway.

Comparison of a leukocyte esterase test with endometrial cytology for diagnosis of subclinical endometritis in postpartum dairy cows.

The objective was to compare a leukocyte esterase (LE) test with endometrial cytology (EC) for diagnosis of subclinical endometritis in dairy cows. The relationship between subsequent fertility and the uterine (Ut) and cervical (Cx) leukocyte esterase activity was determined by the odds of pregnancy by 90 days in milk (DIM). Holstein cows (N = 218) without clinical endometritis and between 21 and 47 DIM from five commercial dairy herds were sampled for uterine and cervical leukocyte esterase activity and EC by cytobrush. To test the effect of time, cows were grouped into early (21-31 DIM) and late (32-47 DIM) animals. There was a slight agreement between UtLE and CxLE (weighted κ = 0.37). The percentage of neutrophils was correlated with esterase score either from the uterus (UtLE; P = 0.0001) or cervix (CxLE; P = 0.002). The percentage of neutrophils on EC (P < 0.001), the UtLE score (P < 0.0001), and the CxLE (P = 0.0009) diminished as DIM increased. Neither CxLE nor UtLE were statistically associated with pregnancy at 90 DIM. However, between 32 and 47 DIM, the percentage of neutrophils on EC and odds of pregnancy at 90 DIM were associated (P = 0.04). For the same interval, based on receiver/response operating characteristics analysis, the optimal cutoff was >6.7% neutrophils to classify cows with subclinical endometritis. In conclusion, uterine LE activity was correlated with percentage of neutrophils as determined by EC, but not with odds of pregnancy. Subclinical endometritis (>6.70% neutrophils) diagnosed by EC between 32 and 47 DIM was associated with reduced odds of pregnancy.

Identification of single nucleotide polymorphisms in the bovine follicle‐stimulating hormone receptor and effects of genotypes on superovulatory response traits

In dairy cows, there is evidence that failure to respond to superovulation protocols is a heritable trait. In women, genotyping for the p.N680S single nucleotide polymorphism (SNP) in the follicle-stimulating hormone receptor (FSHR) gene may help identify poor responders before ovarian stimulation is initiated. Our objectives were to identify SNPs in the coding region of the bovine FSHR gene and to investigate the effect of FSHR genotypes on superovulatory response in Holstein cattle. Sequencing of FSHR exons 1-10 revealed seven SNPs. Three were non-synonymous mutations (c.337C>G, c.871A>G and c.1973C>G). SNP c.337C>G encodes for a proline-to-alanine (p.Pro113Ala) amino acid replacement in the extracellular ligand-binding domain of the receptor. PCR-RFLP analyses showed that homozygous GG Holstein cows present a higher percentage of viable embryos, whereas GG and CG animals have less unfertilised oocytes. SNP c.871A>G results in an isoleucine-to-valine (p.Ile291Val) modification, and homozygous AA animals present lower embryo yield after superovulatory treatments. SNP c.1973C>G corresponds to a threonine-to-serine (p.The658Ser) modification in the intracellular carboxyl-terminal domain of the FSHR protein, and homozygous GG Holstein cows were associated with a lower embryo yield and a higher percentage of unfertilised oocytes. Our results suggest that specific alleles of the bovine FSHR gene are associated with variations in embryo yield and in the number of unfertilised oocytes.

Ultrasound Imaging of the Bull Reproductive Tract: An Important Field of Expertise for Veterinarians

Diagnosing male reproductive system pathologies can often be frustrating because of the challenge involved in precisely determining their site, severity, and prognosis. The introduction of complementary ultrasonographic examination enables clinicians to address these important questions. This procedure should be performed not only on bulls destined to artificial insemination, but on all farm bulls. The examination is easy to perform with a versatile ultrasonographic unit designed for bovine theriogenology. To recognize abnormal tissues, however, the operator must have an excellent knowledge of the ultrasonographic anatomy of the reproductive system. This article discusses the basis of ultrasound technique for male reproductive tract examination. Ultrasound evaluation of physiologic and pathologic conditions of external and internal reproduction organs is proposed.

Therapeutic Efficiency of Antibiotics and Prostaglandin F2α in Postpartum Dairy Cows with Clinical Endometritis: An Evidence-Based Evaluation

The occurrence of vaginal discharge in postpartum dairy cows is generally diagnosed as clinical endometritis. This uterine condition is associated with reduced fertility and economic loss for the dairy industry. Therapeutic approaches include the systemic or intrauterine application of antibiotics or the injection of prostaglandin F2α and analogues to cause luteolysis and uterine contractions to evacuate the infected content. The treatment of clinical endometritis remains a subject of considerable controversy in the literature. Better understanding of the reproductive biology of normal versus abnormal uterine involution and immune mechanisms will allow more efficient diagnostic methods and a more efficient therapeutic approach.

Low-Density Lipoprotein Receptor-Related Protein 8 (LRP8) Is Upregulated in Granulosa Cells of Bovine Dominant Follicle: Molecular Characterization and Spatio-Temporal Expression Studies

Abstract The low-density lipoprotein (LDL) receptor-related protein 8 (LRP8) is a member of the LDL receptor family that participates in endocytosis and signal transduction. We cloned the full-length bovine LRP8 cDNA in granulosa cells (GC) of the dominant follicle (DF) as well as several LRP8 mRNA splicing variants, including a variant that contains a proline-rich cytoplasmic insert (A759-K817) that is involved in intracellular signaling. Expression of the A759-K817 variant was analyzed in the GC of follicles at different developmental stages: the small follicle (SF; 2–4 mm), the DF at Day 5 (D5) of the estrus cycle, ovulatory follicles (OF) 24 h after hCG injection, and corpora lutea (CL) at D5. RT-PCR analysis showed that expression was predominant in the GC of DF compared to other follicles and CL (P < 0.0001), whereas the expression of other related receptors, such as LDLR and VLDLR, did not show differences. Temporal analyses of follicular walls from the OF following hCG treatment revealed a decrease in LRP8 mRNA expression starting 12 h post-hCG treatment (P < 0.0001). LRP8 protein was exclusively localized to the GC, with higher levels in the DF than in the SF (P < 0.05). RELN mRNA, which encodes an LRP8 ligand, was highly expressed in the theca of the DF as compared to the OF (P < 0.004), whereas MAPK8IP1 mRNA, which encodes an LRP8 intracellular interacting partner, is expressed in the GC of the DF. These results demonstrate the differential expression patterns of LRP8, RELN, and MAPK8IP1 mRNAs during final follicular growth and ovulation, and suggest that a RELN/LRP8/MAPK8IP1 paracrine interaction regulates follicular growth.

Aberrant expression of E-cadherin and β-catenin proteins in placenta of bovine embryos derived from somatic cell nuclear transfer

Abnormal placental development is common in the bovine somatic cell nuclear transfer (SCNT)-derived fetus. In the present study, we characterised the expression of E-cadherin and β-catenin, structural proteins of adherens junctions, in SCNT gestations as a model for impaired placentation. Cotyledonary tissues were separated from pregnant uteri of SCNT (n = 6) and control pregnancies (n = 8) obtained by artificial insemination. Samples were analysed by western blot, quantitative RT-PCR (qRT-PCR) and immunohistochemistry. Bovine trophectoderm cell lines derived from SCNT and control embryos were analysed to compare with the in utero condition. Although no differences in E-cadherin or β-catenin mRNA abundance were observed in fetal tissues between the two groups, proteins encoded by these genes were markedly under-expressed in SCNT trophoblast cells. Immunohistochemistry revealed a different pattern of E-cadherin and total β-catenin localisation in SCNT placentas compared with controls. No difference was observed in subcellular localisation of dephosphorylated active-β-catenin protein in SCNT tissues compared with controls. However, qRT-PCR confirmed that the wingless (WNT)/β-catenin signalling pathway target genes CCND1, CLDN1 and MSX1 were downregulated in SCNT placentas. No differences were detected between two groups of bovine trophectoderm cell lines. Our results suggest that impaired expression of E-cadherin and β-catenin proteins, along with defective β-catenin signalling during embryo attachment, specifically during placentation, is a molecular mechanism explaining insufficient placentation in the bovine SCNT-derived fetus.

Expression of steroidogenic proteins in bovine placenta during the first half of gestation

The aim of the present study was to determine the occurrence and localisation of the principal steroidogenic proteins in bovine placenta from Day 50 to Day 120 of pregnancy. Immunohistochemistry revealed that, at all stages investigated, bovine steroidogenic acute regulatory protein (StAR), cytochrome P45011A1 and hydroxy-δ-5-steroid dehydrogenase, 3β- and steroid δ-isomerase 1 proteins were found principally at the fetomaternal interdigitations: the chorionic villus and maternal septum. Moreover, caruncular epithelial cells and uninucleate trophoblast cells were the principal cells detected that were positive for the three markers. Western blot analysis showed that only caruncular tissue expressed all three steroidogenic markers; in contrast, cotyledons only expressed StAR and cytochrome P45011A1. Immunoblot results showed a complementary pattern of StAR and cytochrome P45011A1 expression between caruncles and cotyledons at different stages. These observations suggest that, in early pregnancy, the maternal compartment contributes significantly to bovine placental steroidogenesis, particularly for the synthesis of progesterone. Furthermore, the variation in StAR and cytochrome P45011A1 expression between caruncular and cotyledonary tissues across gestation suggests that placental steroidogenesis requires cell-to-cell communication between maternal and fetal cells.