Biological Activity of Relaxin in Porcine Milk

Abstract

A lactocrine mechanism for delivery of maternally derived relaxin (RLX) into the neonatal circulation as a consequence of nursing has been proposed for the pig. Consistently, immunoreactive porcine RLX was detected in colostrum as well as in the serum of nursing pigs. Concentrations of porcine RLX in milk are highest during early lactation (9-19 ng/mL) and decline to less than 2 ng/mL by postnatal day 14. However, RLX bioactivity has not been described in porcine milk. Therefore, this study was designed to establish an assay for RLX bioactivity in porcine milk and to determine if milk RLX bioactivity was related to RLX concentrations in milk collected at parturition (lactation day 0) and on lactation day 14. To assess milk RLX bioactivity, an in vitro bioassay using human embryonic kidney (HEK293T) cells transfected with the human RLX receptor (LGR7) was developed. Milk RLX bioactivity was confirmed by documentation of a systematic increase in cAMP production by HEK293T-LGR7 cells in response to increasing volumes of day 0 milk. Addition of lactation day 14 milk, porcine insulin, or human insulin-like growth factor 1 to HEK293T-LGR7 cells, or porcine RLX treatment of nontransfected HEK293T cells, failed to elicit a cAMP response. Western blot analysis of milk proteins revealed an 18-kDa protein band, indicating that pro RLX is the primary form of bioactive RLX in porcine milk. Data support the lactocrine hypothesis and suggest a role for milk-borne pro RLX in porcine neonatal development.