Despite massive research efforts, the molecular etiology of bovine polledness and the developmental pathways involved in horn ontogenesis are still poorly understood. In a recent article, we provided evidence for the existence of at least two different alleles at the Polled locus and identified candidate mutations for each of them. None of these mutations was located in known coding or regulatory regions, thus adding to the complexity of understanding the molecular basis of polledness. We confirm previous results here and exhaustively identify the causative mutation for the Celtic allele (PC) and four candidate mutations for the Friesian allele (PF). We describe a previously unreported eyelash-and-eyelid phenotype associated with regular polledness, and present unique histological and gene expression data on bovine horn bud differentiation in fetuses affected by three different horn defect syndromes, as well as in wild-type controls. We propose the ectopic expression of a lincRNA in PC/p horn buds as a probable cause of horn bud agenesis. In addition, we provide evidence for an involvement of OLIG2, FOXL2 and RXFP2 in horn bud differentiation, and draw a first link between bovine, ovine and caprine Polled loci. Our results represent a first and important step in understanding the genetic pathways and key process involved in horn bud differentiation in Bovidae.

During the periovulatory period, the induction of prostaglandin G/H synthase-2 (PTGS2) expression in cumulus cells and associated prostaglandin E2 (PGE2) production are implicated in the terminal differentiation of the cumulus-oocyte complex. During the present study, the effects of the PTGS2/PGE2 pathway on the developmental competence of bovine oocytes were investigated using an in vitro model of maturation, fertilization, and early embryonic development. The specific inhibition of PTGS2 activity with NS-398 during in vitro maturation (IVM) significantly restricted mitogen-activated protein kinase (MAPK) activation in oocytes at the germinal vesicle breakdown stage and reduced both cumulus expansion and the maturation rate after 22 h of culture. In addition, significantly higher rates of abnormal meiotic spindle organization were observed after 26 h of culture. Periconceptional PTGS2 inhibition did not affect fertilization but significantly reduced the speed of embryo development. Embryo output rates were significantly decreased on Day 6 postfertilization but not on Day 7. However, total blastomere number was significantly lower in embryos obtained after PTGS2 inhibition. The addition of PGE2 to IVM and in vitro fertilization cultures containing NS-398 overrode oocyte maturation and early embryonic developmental defects. Protein and mRNA expression for the prostaglandin E receptor PTGER2 were found in oocytes, whereas the PTGER2, PTGER3, and PTGER4 subtypes were expressed in cumulus cells. This study is the first to report the involvement of PGE2 in oocyte MAPK activation during the maturation process. Taken together, these results indicate that PGE2-mediated interactions between somatic and germ cells during the periconceptional period promote both in vitro oocyte maturation and preimplantation embryonic development in cattle.

Prostaglandin E 2 (PGE 2 ) and progesterone appear to be critical mediators of cumulus expansion and the resumption of oocyte meiosis. The aim of this study was to identify the types of prostaglandin E synthase (PTGES) expressed in the bovine cumulus-oocyte complex (COC), to characterize their temporal expression during the periconceptional interval using an in vitro model of maturation (IVM) and fertilization (IVF), and to compare their expression with the level of steroidogenic gene expression. Real-time RT-PCR analysis revealed that enzymes related to the PGE 2 biosynthesis pathway were mainly expressed during IVM. Transcripts encoding PTGES1-3 were detected in bovine COCs. Only the expression of PTGES1 significantly increased during IVM whereas that of PTGES2 and PTGES3 remained unchanged. The induction of PTGES1 expression paralleled the induction of prostaglandin G/H synthase-2 (PTGS2) expression and the amounts of PGE 2 secreted by maturing COCs. Concomitantly, cholesterol side chain cleavage cytochrome P450 expression was significantly upregulated in maturing COCs and the high level of expression persisted in fertilized COCs. The expression of the StAR protein remained constant during IVM and then decreased significantly during IVF. Expression of the progesterone catabolic-related enzyme, 20a-hydroxysteroid dehydrogenase significantly decreased throughout the periconceptional interval. This was associated with a rising level of progesterone released by COCs in the culture media. In conclusion, our results suggest that the periconceptional differentiation of the bovine COC includes the transient induction of PGE 2 biosynthetic activity via the PTGS2/PTGES1 pathway during the maturation period and the increasing ability to produce progesterone from the immature to the fertilized stages. Reproduction (2008) 135 593-603

Polled and Multisystemic Syndrome (PMS) is a novel developmental disorder occurring in the progeny of a single bull. Its clinical spectrum includes polledness (complete agenesis of horns), facial dysmorphism, growth delay, chronic diarrhea, premature ovarian failure, and variable neurological and cardiac anomalies. PMS is also characterized by a deviation of the sex-ratio, suggesting male lethality during pregnancy. Using Mendelian error mapping and whole-genome sequencing, we identified a 3.7 Mb deletion on the paternal bovine chromosome 2 encompassing ARHGAP15, GTDC1 and ZEB2 genes. We then produced control and affected 90-day old fetuses to characterize this syndrome by histological and expression analyses. Compared to wild type individuals, affected animals showed a decreased expression of the three deleted genes. Based on a comparison with human Mowat-Wilson syndrome, we suggest that deletion of ZEB2, is responsible for most of the effects of the mutation. Finally sperm-FISH, embryo genotyping and analysis of reproduction records confirmed somatic mosaicism in the founder bull and male-specific lethality during the first third of gestation. In conclusion, we identified a novel locus involved in bovid horn ontogenesis and suggest that epithelial-to-mesenchymal transition plays a critical role in horn bud differentiation. We also provide new insights into the pathogenicity of ZEB2 loss of heterozygosity in bovine and humans and describe the first case of male-specific lethality associated with an autosomal locus in a non-murine mammalian species. This result sets PMS as a unique model to study sex-specific gene expression/regulation.

The polled and multisystemic syndrome (PMS) is a genetic abnormality observed in the progeny of a unique bull affected by a large chromosomal deletion and cellular mosaicism. Hemizygous females, representing 15% of total progeny, are hornless and show organ malformations, including of the ovaries. Hemizygous males are assumed to die in early fetal development. This study was initiated to produce sufficiently affected fetuses using the semen of the sire as a unique genetic resource. Oocytes from slaughterhouse ovaries were in vitro matured, fertilized with the same bull and cultured in SOF medium using standard in vitro procedures currently performed in the laboratory. On Day 7, grade 1 to 3 embryos were biopsied (5 to 10 cells) and frozen using a conventional glycerol procedure. Whole-biopsy amplification of genomic DNA was performed using a Qiagen Repli-g® Mini Kit (Qiagen, Valencia, CA, USA). Sex determination was done by PCR (UNCEIA Sexing Kit, UNCEIA, Paris, France). The PMS status was indirectly determined using both tetra-primer amplification refractory mutation system-PCR and PCR-restriction fragment length polymorphism procedures to genotype a single SNP located within the deleted region. Because the sire was both homo- and hemizygous for this SNP, 3 categories of progeny were defined according to their genotypes: unaffected (heterozygous), potentially affected (homo- or hemizygous for the same allele as their sire) and affected (hemizygous for the alternate allele). Thirteen affected and 21 potentially affected female embryos were thawed and transferred (1 ≤ n ≤ 4) into 17 Day 7 recipients. Pregnant females were slaughtered on Day 9 and fetuses were recovered; sex and PMS status were then verified. From 2133 inseminated oocytes (7 replicates), 64% cleaved and 10% (n = 216) developed to the blastocyst stage on Day 7. This was significantly lower than the 87% cleavage and 25% blastocyst development rates observed with a control bull used to inseminate 368 oocytes from the same batches. Finally, 174 embryos were biopsied and 169 were frozen. Fifty percent were sexed as female (n = 87). Among them, 15% (n = 13) were affected and 29% (n = 25) were potentially affected. The 24-h survival rate averaged 67% from 54 thawed, unaffected embryos, whereas the hatching rate at 72 h was 59%. Seven female fetuses were recovered from 6 recipient cows. Clinical examination revealed an absence of horn buds for 3 of them. This status was subsequently confirmed by a non-Mendelian inheritance study based on Illumina BovineSNP50 BeadChip (Illumina Inc., San Diego, CA, USA) genotyping data. This combination of in vitro procedures allowed us to increase the number of affected fetuses from 15 to 43% (3/7) in the mosaic bull progeny. In addition, it enabled us to produce valuable material from a very limited resource to perform clinical and functional studies. Finally, it demonstrates the feasibility of a pre-implantation genetic diagnosis combined with freezing and transfer of IVP embryos.