A colostrum trypsin inhibitor gene expressed in the Cape fur seal mammary gland during lactation.

The colostrum trypsin inhibitor (CTI) gene and transcript were cloned from the Cape fur seal mammary gland and CTI identified by in silico analysis of the Pacific walrus and polar bear genomes (Order Carnivora), and in marine and terrestrial mammals of the Orders Cetartiodactyla (yak, whales, camel) and Perissodactyla (white rhinoceros). Unexpectedly, Weddell seal CTI was predicted to be a pseudogene. Cape fur seal CTI was expressed in the mammary gland of a pregnant multiparous seal, but not in a seal in its first pregnancy. While bovine CTI is expressed for 24-48 h postpartum (pp) and secreted in colostrum only, Cape fur seal CTI was detected for at least 2-3 months pp while the mother was suckling its young on-shore. Furthermore, CTI was expressed in the mammary gland of only one of the lactating seals that was foraging at-sea. The expression of ß-casein (CSN2) and ß-lactoglobulin II (LGB2), but not CTI in the second lactating seal foraging at-sea suggested that CTI may be intermittently expressed during lactation. Cape fur seal and walrus CTI encode putative small, secreted, N-glycosylated proteins with a single Kunitz/bovine pancreatic trypsin inhibitor (BPTI) domain indicative of serine protease inhibition. Mature Cape fur seal CTI shares 92% sequence identity with Pacific walrus CTI, but only 35% identity with BPTI. Structural homology modelling of Cape fur seal CTI and Pacific walrus trypsin based on the model of the second Kunitz domain of human tissue factor pathway inhibitor (TFPI) and porcine trypsin (Protein Data Bank: 1TFX) confirmed that CTI inhibits trypsin in a canonical fashion. Therefore, pinniped CTI may be critical for preventing the proteolytic degradation of immunoglobulins that are passively transferred from mother to young via colostrum and milk.

Expression of the mammary gland-specific tammar wallaby early lactation protein gene is maintained in vitro in the absence of prolactin.

Marsupial ELP (early lactation protein) and its eutherian orthologue, CTI (colostrum trypsin inhibitor) are expressed in the mammary gland only for the first 100 days postpartum (Phase 2A) in the tammar wallaby and during the bovine and canine colostrogenesis period 24-36h postpartum respectively. The factors which regulate temporal ELP and CTI expression are unknown. A tammar mammary gland explant culture model was used to investigate ELP gene regulation during pregnancy and early- and mid-lactation (Phase 1, 2A and 2B respectively). Tammar ELP expression could only be manipulated in explants in vitro if the gene was already expressed in vivo. ELP expression was maximal in Phase 1 explants treated with lactogenic hormones (insulin, hydrocortisone and prolactin), but unlike LGB (ß-lactoglobulin), ELP expression was maintained in insulin or insulin and hydrocortisone over a 12-day culture period. In contrast, ELP was down-regulated when cultured without hormones. ELP could not be induced in explants cultured from mid-lactation which suggested that transcriptional repressors may prevent ELP expression during this period.