Redistribution of the sheep neonatal Fc receptor in the mammary gland around the time of parturition in ewes and its localization in the small intestine of neonatal lambs

Abstract

Maternal immunity is mediated exclusively by colostral immunoglobulins in ruminants. As the neonatal Fc receptor (FcRn) is suggested to be involved in the transport of immunoglobulin G (IgG) in the mammary gland, we cloned this receptor from sheep and analysed its expression in the mammary gland around the time of parturition and also in the small intestine from the newborn lamb. FcRn heavy-chain mRNA was detected (by using in situ hybridization) exclusively in the acinar and ductal epithelial cells in mammary gland biopsies both before and after parturition. Immunohistochemistry revealed that the cytoplasm of the epithelial cells of the acini and ducts in the mammary gland biopsies stained homogeneously before parturition. A remarkable difference was observed in the pattern after lambing, where the apical side of the cells was strongly stained. The presence of the FcRn in the acinar and ductal epithelial cells of the mammary gland, and the obvious change in distribution before and after parturition, indicate that the FcRn plays an important role in the transport of IgG during colostrum formation in ruminants. Immunohistochemical analysis detected a strong apical and a weak basal FcRn signal in the duodenal crypt cells of a neonatal lamb, which have been previously demonstrated to secrete IgG1 in newborn ruminants. The FcRn was not detected in the duodenal enterocytes, which absorb intact IgG from the colostrum in a non-specific manner. These data suggest that FcRn is involved in IgG1 secretion in ruminant epithelial cells.

Figure 1

The nucleotide sequence and deduced amino acid sequence of the sheep neonatal Fc receptor (FcRn) α-chain. The potential ATG start is marked in bold type, while the consensus initiation site is underlined. The predicted N-terminal after signal peptide cleavage is indicated by ‘+1’ under Ala. The hydrophobic membrane-spanning segment is shown by italic characters while the polyadenylation signal ‘AATAAA’ in the 3′ untranslated region is underlined. CYT, cytoplasmic; TM transmembrane.

Figure 2

Domain-by-domain alignment of the predicted amino acid sequences for cow (b), sheep (o), human (h) and rat (r) neonatal Fc receptor (FcRn) α-chains. The N-linked glycosylation site, which is found in all the sequences, is shown by a black triangle, while white triangles indicate additional sites in the rat sequence. The grey bar indicates the hydrophobic transmembrane region. Consensus residues are assigned based on the number of occurrences of the character in the column, emphasizing the degree of conservation. The higher the conservation in a column, the darker the background of the character. CYT, cytoplasmic; TM transmembrane.

Figure 3

In situ hybridization on a typical sheep mammary gland biopsy (5 days postpartum). (a) Biopsy tissue was hybridized with an anti-sense sheep neonatal Fc receptor (FcRn)-specific polymerase chain reaction (PCR)-generated, digoxigenin (DIG)-labelled probe; (b) the biopsy was hybridized with a sense probe derived from the same fragment, as a negative control [same area as (a)]. Shown are low power ( × 40). (c) and (d) A single acinus at higher power (× 100) as positive and negative samples, respectively.

Figure 4

Western blot analysis of the neonatal Fc receptor (FcRn) specific antibody against an oligopeptide (CLEWKEPPSMRLKAR representing amino acids 173–186 of the α2 and α3 domains). (a) Sera, 500× dilution; (b) affinity-purified sera, 50× dilution. A clone (B1) of IMCD cells transfected with cDNA encoding the bovine FcRn heavy chain and untransfected IMCD cells were extracted in 1% sodium dodecyl sulphate (SDS). Arrowhead indicates the bovine FcRn α-chain (≈ 40 kDa), while numbers indicate the apparent molecular weight of the Benchmark Prestained Protein Ladder (Invitrogen).

Figure 5

Immunohistochemical analyses of the sheep mammary gland biopsies around parturition. Strong and diffuse neonatal Fc receptor (FcRn) expression was detected 24 (a) and 10 days (b) prepartum in the acinar and ductal cells. On samples derived postpartum day 1 (c), day 5 (d) and day 14 (e), the FcRn appeared mainly at the apical side of these cells. At 75 days postpartum (f), diffuse localization was observed in the cytoplasmic region. L, lumen of the acini. The main panels are shown at low power ( × 40) and the inserts at higher power ( × 100).

Figure 6

Immunohistochemical analysis detected strong apical and weak basal (arrows) neonatal Fc receptor (FcRn) in the duodenal crypt cells of a neonatal lamb. However, FcRn was not detected in the duodenal enterocytes. Magnification, × 20.