Age and duration-dependent effects of lactocrine signaling on markers and mediators of uterine growth and remodeling in neonatal gilts

The lactocrine hypothesis describes a mechanism whereby milk-borne bioactive factors are delivered to offspring via nursing and affect development of neonatal tissues. In support of this hypothesis, data for the uterus indicate that, in comparison with gilts fed a porcine milk replacer for two days from birth (postnatal day = PND 0), nursing is essential for support of normal uterine estrogen receptor-α (ESR1), vascular endothelial growth factor (VEGF) A, and matrix metalloproteinase (MMP) 9 protein expression at PND 2. Depressed expression of these markers and mediators of uterine growth and remodeling was not restored when gilts fed milk replacer through PND 2 were switched to nursing until PND 14. These results suggest that a window of opportunity for lactocrine signaling exists within the first two days of life; however, the critical time period for such signaling within the first 48 h from birth is unknown. Here, objectives were to determine the effects of: (1) age at first nursing (0 h, 30 min, 12 h); and (2) duration of nursing (30 min, 12 h, 48 h) on uterine ESR1, VEGFA and MMP9 expression. Gilts (n=5-6/group) were randomly assigned at birth to one of six treatment groups: (1) nursed for 30 min from birth and gavage-fed milk replacer (30ml/kg BW/2 h) for 47.5 h; (2) nursed for 12 h from birth and gavage-fed milk replacer for 36 h; (3) nursed ad libitum for 48 h; (4) gavage-fed milk replacer for 30 min from birth and nursed for 47.5 h; (5) gavage-fed milk replacer for 12 h from birth and nursed for 36 h; or (6) gavage-fed milk replacer for 48 h. Uteri were collected on PND 2 (50 h). ESR1, VEGFA, and MMP9 protein expression was evaluated by immunoblotting using actin as a loading control and quantified by densitometry. Immunoreactive bands at 51 kDa for ESR1, 46 kDa for VEGFA, and 84 and 92 kDa for MMP 9 and pro-MMP9 were detected consistently in uterine tissues from gilts allowed to nurse beginning at 0 h or 30 min of age. However, as observed in gilts fed replacer for 48 h from birth, targeted proteins were undetectable in uterine tissues on PND 2 when gilts were nursed starting at 12 h after birth. Nursing for as little as 30 min from birth was sufficient to induce uterine ESR1, VEGFA and MMP9 expression at PND 2. Increasing the duration of nursing from 30 min to 12 h increased expression of targeted proteins to levels similar to those seen in gilts nursed for 48 h. Results indicate that age at first nursing is important for lactocrine signaling. Nursing initiated at 0 h or 30 min of age is sufficient to induce a normal developmental program as reflected by uterine ESR1, VEGFA, and MMP9 expression on PND 2. However, when a lactocrine-null period is imposed from birth to 12 h of age, resumption of nursing through PND 2 failed to rescue the normal uterine developmental program. Thus, the window for lactocrine signaling important for neonatal uterine development is open within 12 h of birth. Results also show that increased nursing duration correlates positively with levels of uterine ESR1, VEGFA, and MMP9 expression. This study illustrates the importance of age at first nursing and duration of nursing in support of the porcine uterine developmental program and defines the critical period for lactocrine signaling more precisely within two days of birth.