H.A. de Boer

Structure and expression of the mouse casein gene locus

The analysis of yeast artificial chromosomes (YACs) containing the complete mouse casein gene locus revealed the presence of five casein genes, α -, ß-, γ -, δ-, and κ-casein, in this order, in the locus. The a- and ß-casein genes are only 10 kb apart and have convergent transcriptional orientations. The distance between the ß-casein gene and the αs2-like γ-casein gene is about 70 kb, and these genes have divergent transcriptional orientations. The γ - and δ-casein genes, both encoding a αs2-like casein, are linked within 60 kb and convergently transcribed. The κ-casein gene is located about 100 kb from the δ-gene. Except for the presence of the δ-casein gene, the organization of the mouse casein locus resembles that of the bovine locus, including the transcriptional orientation of the genes. In contrast to the other casein genes, which are strongly induced at mid-lactation, expression of the 8-casein gene is abruptly induced upon parturition. Comparative analysis of αs2-like sequences from various species suggests that the ancestral αs2-like gene duplicated around the time of radiation of the rodent and artiodactylid ancestors.

Lymphocyte Homing and Ig Secretion in the Murine Mammary Gland

In mice the majority of the immunoglobulins (Ig) in milk belongs to the IgA class. Prior to its transepithelial transportation into the milk, dimeric IgA (dIgA) is bound to the transmembrane form of the secretory component or polymeric Ig receptor (SC/pIgR). The latter is synthesized in the epithelial cells lining the ducts and alveoli of the mammary gland. A candidate for playing the role of adhesion molecule to primed lymphocytes present in the murine mammary gland might be the mucosal addressin cell adhesion molecule‐1 (MAdCAM‐1). We studied the correlation between the levels of IgA in colostrum and milk, the number of IgA producing plasma cells in the mammary gland and the expression of MAdCAM‐1 in mammary gland endothelial cells during pregnancy and lactation. The relation between the IgA levels in the milk and the expression levels of pIgR in mammary gland epithelial cells was also investigated. We found that the expression of MAdCAM‐1 and pIgR starts in early–mid pregnancy; the number of IgA‐producing plasma cells and the IgA concentration in milk increase from early lactation onwards. The MAdCAM‐1 expression declines during lactation whereas the pIgR levels and IgA‐producing plasma cell numbers rise until the end of lactation. Because the MAdCAM‐1 level starts to rise several days before the rise of the IgA‐producing plasma cell level, MAdCAM‐1 cannot be the rate determining factor governing extravasation of primed B cells to the mammary gland. We also conclude that the pIgR is present in sufficient amounts to enable increasing S‐IgA secretion into the milk during lactation.

Increased immunoglobulin A levels in milk by over‐expressing the murine polymeric immunoglobulin receptor gene in the mammary gland epithelial cells of transgenic mice

The polymeric immunoglobulin receptor (pIgR) transports dimeric immunoglobulin A (dIgA) across the epithelial cell layers into the secretions of various mucosal and glandular surfaces of mammals. At these mucosal sites, such as the gastrointestinal tract, respiratory tract, urogenital tract and the mammary glands, dIgA protects the body against pathogens. The pIgR binds dIgA at the basolateral side and transports it via the complex mechanism of transcytosis to the apical side of the epithelial cells lining the mucosa. Here, the extracellular part of the receptor is cleaved to form the secretory component (SC), which remains associated to dIgA, thereby protecting it from degradation in the secretions. One pIgR molecule transports only one dIgA molecule (1 : 1 ratio) and the pIgR is not recycled after each round of transport. This implies that the amount of available receptor could be a rate‐limiting factor determining both the rate and amount of IgA transported per cell and therefore determining the total IgA output into the lumen or, in case of the mammary gland, into the milk. In order to test this hypothesis, we set up an in vivo model system. We generated transgenic mice over‐expressing the murine pIgR gene under lactogenic control, by using a milk gene promoter, rather than under immunological control. Mice over‐expressing the pIgR protein, in mammary gland epithelial cells, from 60‐ up to 270‐fold above normal pIgR protein levels showed total IgA levels in the milk to be 1·5–2‐fold higher, respectively, compared with the IgA levels in the milk of non‐transgenic mice. This indicates that the amount of pIgR produced is indeed a limiting factor in the transport of dIgA into the milk under normal non‐inflammatory circumstances.

Kinetics of first polar body extrusion and the effect of time of stripping of the cumulus and time of insemination on developmental competence of bovine oocytes.

Kinetics of extrusion of the first polar body was examined as well as the effect of the time of stripping of the cumulus cells on this kinetics. In addition, the effects of time of stripping and time of insemination on developmental competence of the oocytes, as evaluated by the percentage of morulae and blastocysts, were studied. Polar body extrusion occurred in 80% of the oocytes between 12 and 18 h after the onset of maturation. The remainder of the oocytes did not extrude a polar body at all. Stripping of the cumulus at 12 h after the onset of maturation delayed polar body extrusion significantly by about 1 h. No significant differences were found in the percentage of oocytes that could be fertilized, and the percentage of oocytes that cleaved and developed to the morula and blastocyst stages, between oocytes that were stripped free of cumulus and inseminated at either 16 or 20 h after onset maturation. Oocytes that had extruded a polar body at either 16 or 20 h after onset maturation showed significantly higher percentages of cleavage and development than oocytes that had not extruded a polar body at those time points. However, the percentage of oocytes that could be fertilized was not affected.