Fucosyltransferase gene expression in goat endometrium during the estrous cycle and early pregnancy.

Regulation of the expression of the alpha(1,2)fucosyltransferase (FUT) genes and their enzymatic products, including the H-type 1 antigen (HT1), on the luminal surface of the uterus is believed to be critical for establishment of pregnancy in mammals. The FUT1 gene is a marker for conception rates in dairy cows and HT1 is a marker for uterine receptivity in rodents. To determine the spatiotemporal expression patterns of FUT1 and FUT2 genes in goats, endometrial tissues were obtained on six days spanning the estrous cycle (Days 5, 11, 13, 15, 17 and 19) and seven days spanning early pregnancy (Days 5, 11, 13, 15, 17, 19 and 25). In all data, we found no effect of status (cyclic or pregnant; P > 0.1) and pooled data where appropriate. We cloned FUT1 cDNA from goat endometrium and made probes from it for Northern and slot blot analyses. The analyses indicated that FUT1 gene expression was high until Day 13, and then declined. In situ hybridization revealed a change in the cell-specificity of FUT1 gene expression over the estrous cycle and early pregnancy. In situ hybridization signal intensity scores indicated that FUT1 expression by uterine epithelium was high on Day 5, moderate on Day 11, and minimal on subsequent days. In situ hybridization signals in uterine glandular epithelial cells remained high from Day 5 to Day 13, with weaker signals thereafter. Quantitative reverse transcription-PCR (RT-qPCR) assays were used for quantitation of FUT1 and FUT2 mRNAs. Quantitative RT-qPCR data were generated from endometrium collected from cyclic and pregnant animals on Days 5, 11 and 17. Relative levels of FUT1 mRNA were high on Days 5 and 11, but then fell 5-fold by Day 17 (P < 0.01). FUT2 mRNA concentrations were below the accurate detectable limit of the assay. High levels of HT1 were observed on the apical surface of uterine luminal epithelia on Days 5, 15, 17 and 19, with much lower levels on Days 11 and 13. Thus, data suggests that FUT1 is the primary enzyme responsible for the high levels of HT1 antigen present on the uterine luminal epithelium between Days 5 and 11 of the estrous cycle and early pregnancy. But changes in the expression of the FUT1 gene does not directly correlate to HT1 staining, which increased from Day 13-15. Future studies are required to understand the regulation of the HT1 antigen on the luminal surface of endometrium.

Expression of liver fatty acid binding protein alters growth and differentiation of embryonic stem cells.

Although expression of liver fatty acid binding protein (L-FABP) modulates cell growth, it is not known if L-FABP also alters cell morphology and differentiation. Therefore, pluripotent embryonic stem cells were transfected with cDNA encoding L-FABP and a series of clones expressing increasing levels of L-FABP were isolated. Untransfected ES cells, as well as ES cells transfected only with empty vector, spontaneously differentiated from rounded adipocyte-like to fibroblast-like morphology, concomitant with marked reduction in expression of stage-specific embryonic antigen (SSEA-1). These changes in morphology and expression of SSEA-1 were greatest in ES cell clones expressing L-FABP above a threshold level. Immunofluorescence confocal microscopy revealed that L-FABP was primarily localized in a diffuse-cytosolic pattern along with a lesser degree of punctate L-FABP expression in the nucleus. Nuclear localization of L-FABP was preferentially increased in clones expressing higher levels of L-FABP. In summary, L-FABP expression altered ES cell morphology and expression of SSEA-1. Taken together with the fact that L-FABP was detected in the nucleus, these data suggested that L-FABP may play a more direct, heretofore unknown, role in regulating ES cell differentiation by acting in the nucleus as well as cytoplasm.

Spontaneous and engineered mutant mice as models for experimental and comparative pathology: history, comparison, and developmental technology.

During the last half-century pathologists have explored the biologic mechanisms associated with inherited human and veterinary diseases by using inbred and inbred mutant (spontaneous) strains of mice. The first successful gene transfer to mice by pronuclear injection of the herpes simplex virus thymidine kinase gene and rabbit and human beta-globulin genes was achieved in the early 1980s. This accomplishment was followed a few years later with the creation of a mouse bearing a disrupted hypoxanthine phosphoribosyl transferase (hrpt) gene (targeted mutation based on ES cell blastocyst injection). Since then, hundreds of genetically engineered models of biomedical importance have been created. The unprecedented scale and scope of development of engineered models present great opportunities as well as experimental challenges to the investigator. The aim of the present review is to provide a framework of information on engineered mouse models from the perspective of experimental and comparative pathology research. Sections include: 1) a brief historical account of the development of mouse models of disease, with increasing progression of genetic refinement as represented by inbred (spontaneous) and congenic (targeted) mutant strains of mice; 2) a synopsis of spontaneous and targeted mutations, with anecdotal examples of expression of individual genes and interactions between multiple mutant genes; 3) selected examples of targeted mutations of interest to developmental and cancer biologists and immunologists; 4) an overview of the technology of development of transgenic mice; and 5) an introduction to on-line database resources of current multi-species genomic information.

Conservation of germ plasm from bison infected with Brucella abortus.

Reproductive procedures for cattle were adapted to American bison (Bison bison) to evaluate the potential preservation of germ plasm from bison infected with Brucella abortus without transmission of the pathogen to the recipient or offspring. Two of four experimentally inoculated bison bulls excreted B. abortus in the semen. Four healthy calves were produced from non-infected, un-vaccinated bison cows by natural breeding with a bison bull excreting B. abortus in the semen. There was no seroconversion of the cows or their calves. Two culture negative bison calves were produced by superovulation of infected bison donor cows followed by artificial insemination and embryo transfer without transmitting B. abortus to recipient cows or calves. These limited data indicate that embryo manipulatory procedures and natural breeding in bison may facilitate preservation of valuable germ plasm from infected bison while reducing the risk of transmission of B. abortus to recipients and progeny.

Isolation and characterization of 26- and 30-kDa rat liver proteins immunoreactive to anti-sterol carrier protein-2 antibodies.

Although the existing literature suggests that the sterol carrier protein-2 (SCP-2) gene has only two initiation sites encoding for a 58- and a 15-kDa protein, respectively, this does not explain the profusion of other putative SCP-2-related proteins detectable on Western blotting. Two of these additional anti-SCP-2 immunoreactive proteins, 13.2 and 46 kDa, appear due to proteolytic processing of the two gene transcripts. However, the origin of additional immunoreactive rat liver proteins near 26 and 30 kDa is unclear. The latter proteins were consistently detected on Western blotting by three independent types of polyclonal antisera: anti-13.2-kDa SCP-2, anti-synthetic peptide from the amino-terminus of the 13.2-kDa SCP-2, and Protein A affinity-purified anti-synthetic peptide to the amino-terminus of 13.2-kDa SCP-2. To resolve whether the 26- and 30-kDa proteins are SCP-2 gene products, each protein was isolated from rat liver and purified to homogeneity as indicated by Tricine-SDS polyacrylamide gel electrophoresis, isoelectric focusing, and/or mass spectroscopy. Their masses, determined by MALDI-TOF mass spectroscopy, were 25.7 and 29.8 kDa, respectively. However, the mass spectral data were not consistent with either protein being an SCP-2 gene product. Peptide mass mapping of the 25.7-kDa protein revealed identity to the rat 25,784.79-Da glutathione-S-transferase. Furthermore, neither the mass nor the amino acid composition of the 29.8-kDa protein correlated with any SCP-2 gene product or dimerized SCP-2 gene product. A database search of the amino acid composition identified the protein as rat carbonic anhydrase. In summary, although the 26- and 29.8-kDa proteins may share some common epitopes with the 13.2-kDa SCP-2, they were not SCP-2 gene products.

Cellular differentiation and I-FABP protein expression modulate fatty acid uptake and diffusion.

The effect of cellular differentiation on fatty acid uptake and intracellular diffusion was examined in transfected pluripotent mouse embryonic stem (ES) cells stably expressing intestinal fatty acid binding protein (I-FABP). Control ES cells, whether differentiated or undifferentiated, did not express I-FABP. The initial rate and maximal uptake of the fluorescent fatty acid, 12-(N-methyl)-N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-octadec anoic acid (NBD-stearic acid), was measured in single cells by kinetic digital fluorescence imaging. I-FABP expression in undifferentiated ES cells increased the initial rate and maximal uptake of NBD-stearic acid 1.7- and 1.6-fold, respectively, as well as increased its effective intracellular diffusion constant (Deff) 1.8-fold as measured by the fluorescence recovery after photobleaching technique. In contrast, ES cell differentiation decreased I-FABP expression up to 3-fold and decreased the NBD-stearic acid initial rate of uptake, maximal uptake, and Deff by 10-, 4.7-, and 2-fold, respectively. There were no significant differences in these parameters between the differentiated control and differentiated I-FABP-expressing ES cell lines. In summary, differentiation and expression of I-FABP oppositely modulated NBD-stearic acid uptake parameters and intracellular diffusion in ES cells.

Recent advances in membrane cholesterol domain dynamics and intracellular cholesterol trafficking.

Cholesterol is distributed nonrandomly in and between biological membranes. Despite over two decades' investigation of these phenomena, the origin, regulation, and function of membrane cholesterol asymmetry are not known. Likewise, although pathways of cellular cholesterol absorption/utilization as well as de novo synthesis have been investigated in depth, parallel progress in elucidating pathways of intracellular cholesterol trafficking and final deposition of cholesterol within membranes remains undefined. Understanding the nature and regulation of these processes is essential to resolving molecular mechanisms of cholesterol uptake, reverse cholesterol transport, steroidogenesis, and modulation of membrane function. Based on the fundamental observation that cholesterol is not distributed uniformly in the cell, three key concepts have contributed to recent advances in this field: First, cholesterol is asymmetrically distributed across the cell surface plasma membrane, wherein it translocates rapidly. Second, cholesterol is distributed within the plane of biomembrane bilayers into dynamic and static domains, with the latter predominating. The exact nature and physiological functions of such cholesterol domains or pools remain an enigma. Third, regulation of the size and kinetics of biomembrane cholesterol domains may be determining factors in intracellular cholesterol trafficking, targeting, and efflux. Contributions of both cytosolic carrier proteins and vesicular processes are recognized.