Local Regional Differences Research Articles

Fetal growth restriction is associated with increased apoptosis in the chorionic trophoblast cells of human fetal membranes

The aim of the study was to determine the incidence and spatial distribution of apoptotic cell in fetal membranes obtained from human pregnancies complicated with fetal growth restriction (FGR) for which there was no established cause. Fetal membrane samples from normal ( n = 10) and FGR-affected ( n = 10) pregnancies were collected and stored following delivery. The incidence of apoptosis and the number of apoptotic cells in normal and FGR-affected fetal membranes were determined using immunohistochemistry and a monoclonal antibody for neo-epitope of cytokeratin-18, M30. The level of apoptotic proteins in FGR-fetal membranes compared to the normal tissue was determined using Western immunoblotting analysis. Multiple labeling of trophoblast cells using immunofluorescence markers was used to investigate regional differences in localization of apoptotic cells between normal and FGR-affected fetal membranes. Apoptosis was detected in both normal and FGR-affected fetal membranes. However, quantitative analysis of apoptotic cells by immunohistochemistry showed a significant increase in FGR-affected fetal membranes compared to normal ( p < 0.005). Furthermore, it was observed that apoptotic cells were predominantly localized to chorio-decidual layer of the fetal membrane. By using semi-quantitative analysis of Western immunoblotting, a significant increase in the levels of the apoptotic marker proteins poly-ribo (ADP) polymerase (PARP) and the neo-epitope of cytokeratin-18 were observed in FGR-affected fetal membranes compared to normal ( p < 0.005). Immunofluorescence studies further confirmed the restriction of the apoptotic cells to the chorionic trophoblast cells in FGR-affected fetal membranes. Our results document for the first time an increased incidence of apoptosis in FGR-affected fetal membranes, with the apoptotic cells restricted primarily to the chorionic trophoblast layer of the fetal membranes. Increased apoptosis in FGR-affected fetal membranes may impair functions of the fetal membranes that are important for normal fetal development and growth. Elucidation of the molecular mechanisms involved in the control of apoptosis in the chorionic trophoblast layer in the FGR-affected fetal membranes may provide further insights into the etiology of FGR.

Monoclonal antibodies to epididymis-specific proteins using mice rendered immune tolerant to testicular proteins.

Monoclonal antibodies (mabs) have been used as a powerful tool for identification of newer sperm proteins. However, conventional hybridoma technology rarely provides chance to obtain mabs to epididymal proteins. To increase this chance, we have used an alternate method of neonatal tolerization. In this protocol, animals were tolerized at birth using testicular proteins followed by immunization with cauda epididymal sperm protein (which is a cocktail of proteins both from testicular and epididymal origin). This protocol induced a specific immune response to epididymal sperm proteins. Spleen from one of these animals was then used for preparation of mabs. This fusion resulted in a number of mabs reacting specifically to epididymal proteins. Although mabs identified a protein of approximately similar molecular weight on 1-dimensional Western blot analysis, there were differences in regional localization on rat sperm as seen by indirect immunofluorescence. Immunohistochemical localization of these proteins in rat epididymis showed region specific synthesis. The synthesis of proteins was seen in the distal caput epididymis, and maximum expression was seen in supranuclear region of corpus epithelium. The proteins were localized on sperm from corpus and cauda region. Epididymis specific synthesis of the proteins and agglutinating nature of the mabs to these underlines the functional importance of these proteins in sperm maturation in epididymis. These antibodies could therefore, be used as tools for understanding the physiology of maturation of sperm in epididymis and role of the epididymal protein in fertilization.

Identity and neuroanatomical localization of messenger RNAs that change expression in the neural tube of mouse embryos within 1 h after ethanol exposure

Changes in the expression of various species of messenger RNA (mRNA) are likely to be one of the initial responses of an embryo to in utero ethanol exposure. However, the identity and range of the initial species of mRNAs that change expression in an embryo after acute prenatal ethanol exposure are unknown. mRNA was isolated from embryos treated on embryonic day 9.5 and examined 1 h after treatment. Ethanol-exposed embryos were compared to similarly treated maltose/dextrin and no injection control embryos. Subtractive hybridization was used to find the species of mRNAs. The identity of the differentially expressed mRNAs was ascertained using standard sequencing techniques while in situ hybridization defined the anatomical localization of each message. Four messages from ethanol-treated embryos were differentially expressed. One message has no homology to any known gene in GenBank while three messages are homologous to known genes: the transmembrane molecule mh19, the heterologous ribonuclear protein hnRNP A3, and the mitochondrial enzyme AK2. One message (the novel gene) was turned on, while mh19, AK2 and hnRNP A3 were up-regulated in ethanol-exposed embryos. All messages were expressed in the developing neural tube of the ethanol-exposed embryos, although there were differences in regional localization and extent of expression. Thus, the initial response of the embryo is rapid with changes in expression of multiple genes observed within 1 h after ethanol exposure. Further, these differentially expressed mRNAs are candidates for modulating the type and extent of ethanol-induced damage in the developing CNS.

Chromosome mapping of genes on the short arm of human chromosome 11: parathyroid hormone gene is at 11p15 together with the genes for insulin, c-Harvey-ras 1, and beta-hemoglobin.

The human parathyroid hormone gene (PTH) was mapped to the 11p15 chromosomal band by in situ hybridization. Using the same procedures and cells, the closely linked beta-hemoglobin gene (HBB), the Harvey-ras 1 proto-oncogene (HRAS1), and the insulin gene (INS) were also mapped to this same region. Some reports have demonstrated differences in regional localization of the latter three genes, and linkage and molecular studies have not resolved how far this linkage group extends from p15 toward the centromere on the physical gene map. Our results show that all of these genes are localized at 11p15, a region of one chromosomal band that appears to comprise a genetic distance of more than 20 cM.