A computational approach to characterization of bovine sperm chromatin alterations

We describe here a computational morphology-based approach to the investigation of possible causes of chromatin alterations in sperm. A comprehensive set of state-of-the-art and geometric measures are computationally extracted from toluidine blue stained images and analyzed to infer the possible processes leading to normal and abnormal chromatin formation while seeking a possible taxonomy of chromatin alterations and their influence on sperm head morphology. Using this methodology, we have identified higher chromatin fragility at some specific points of the sperm head. Despite the lack of correlation between morphologies of sperm head and chromatin structure, four main morphological types of chromatin alterations in bull spermatozoa have been identified and their possible causes discussed.     

Canine prostaglandin F2alpha receptor (FP) and prostaglandin F2alpha synthase (PGFS): molecular cloning and expression in the corpus luteum

In the dog luteolysis is not affected by hysterectomy. This observation led to the hypothesis that paracrine/autocrine rather than endocrine mechanisms of PGF2alpha are responsible for luteal regression in the dioestric bitch. The present experiments tested for the capacity of canine CL to produce and respond to PGF2alpha by qualitatively and quantitatively determining the expressions of PGFS, the enzyme converting PGH2 into PGF2alpha, and the PGF2alpha-receptor (FP) in CL of non-pregnant dogs during dioestrus. Canine PGFS and FP were isolated and cloned; both genes show a high homology (82-94%) when compared to those of other species. Relatively weak FP mRNA expression was detected on day 5 of dioestrus. It had increased by day 25 and remained constant thereafter. In situ hybridization (ISH) localized FP solely to the cytoplasm of the luteal cells, suggesting that these cells are the only luteal targets of PGF2alpha in this species. Only negative results were obtained for the expression of PGFS in canine CL by routine qualitative RT-PCR. When Real Time (TaqMan) PCR was applied, repetitively more negative than positive results were obtained at all timepoints. Any positive measurements observed at any point were neither repeatable nor related to the stage of dioestrus. This led us to conclude that expression of PGFS is either absent or present at very low level only. These data suggest that luteal regression in non-pregnant bitches is not modulated by PGF2alpha. However, the FP seems to be constitutionally expressed, explaining the receptivity of canine CL to exogenous PGF2alpha.     

A tetraantennary glycan with bisecting N-acetylglucosamine and the Sd(a) antigen is the predominant N-glycan on bovine pregnancy-associated glycoproteins

Pregnancy-associated glycoproteins (PAGs) are major secretory proteins of trophoblast cells in ruminants. Binucleate trophoblast giant cells (BNCs) store these proteins in secretory granules and release them into the maternal organism after fusion with maternal uterine epithelial cells. By matrix assisted laser desorption ionisation-mass spectrometry (MALDI-MS) analysis and linkage analysis, we show that by far, the most abundant N-glycan of PAGs in midpregnancy is a tetraantennary core-fucosylated structure with a bisecting N-acetylglucosamine (GlcNAc). All four antennae consist of the Sd(a)-antigen (NeuAcalpha2-3[GalNAcbeta1-4]Galbeta1-4GlcNAc-). Immunohistochemistry with the mono- clonal antibody CT1, which recognizes the Sd(a)-antigen, shows that BNC granules contain the Sd(a)-antigen from gestation day (gd) 32 until a few days before parturition. Lectin histochemistry with Maackia amurensis lectin (MAL), which binds to alpha2-3sialylated lactosamine, shows that BNC granules are MAL-positive prior to gd 32 and also at parturition. The observed tetraantennary glycan is a highly unusual structure, since during the synthesis of N-glycans, the insertion of a bisecting GlcNAc inhibits the activity of the GlcNAc-transferases that leads to tri- and tetraantennary glycans. The study defines the substantial changes of PAG N-glycosylation in the course of pregnancy. This promotes the hypothesis that PAGs may have different carbohydrate-mediated functions at different stages of pregnancy.     

Epigenetic regulation of insulin-like growth factor binding protein-3 (IGFBP-3) in cancer

Epigenetics refers to heritable changes in gene expression that are independent of alterations in DNA sequence. It is now accepted that disruption of epigenetic mechanisms plays a key role in the pathogenesis of cancer: culminating in altered gene function and malignant cellular transformation. DNA methylation and histone modifications are the most widely studied changes but non-coding RNAs such as miRNAs are also considered part of the epigenetic machinery. The insulin-like growth factor (IGF) axis is composed of two ligands, IGF-I and –II, their receptors and six high affinity IGF binding proteins (IGFBPs). The IGF axis plays a key role in cancer development and progression. As IGFBP genes have consistently been identified among the most common to be aberrantly altered in tumours, this review will focus on epigenetic regulation of IGFBP-3 in cancer for which the majority of evidence has been obtained.