Amy-Lynn Frankshun

Lactation Biology Symposium: lactocrine signaling and developmental programming.

Lactocrine signaling is defined as transmission of bioactive factors from mother to offspring as a consequence of nursing. Lactocrine transmission of signaling molecules may be an evolutionarily conserved process through which bioactive factors necessary for support of neonatal development are delivered postnatally. Dependence on maternal resources for development in eutherian mammals extends into neonatal life for at least that period of time when nutrition is obtained solely from first milk (i.e., colostrum). Data for the pig (Sus scrofa domesticus) provide evidence of lactocrine mediated effects on development of the female reproductive tract and other somatic tissues. Porcine uterine gland development, an estrogen receptor-alpha (ESR1)-dependent process, begins within 2 d of birth [postnatal day (PND) 0]. A lactocrine-driven, ESR1-mediated process was proposed as a regulatory mechanism governing onset of uterine gland development and endometrial maturation in the neonatal pig. Gilts maintained in a lactocrine-null state for 2 d from birth by milk-replacer feeding displayed altered patterns of endometrial gene expression and retarded uterine gland development by PND 14. In lactocrine-null gilts, inhibition of endometrial and cervical ESR1 and vascular endothelial growth factor (VEGFA) expression observed on PND 2 persisted to PND 14, even after gilts were returned to nursing on PND 2. Collectively, data support a role for lactocrine signaling in regulation of critical neonatal developmental events. Maternal lactocrine programming of postnatal development may help to insure healthy developmental outcomes. A systems biology approach will be required to define and understand mechanistic dynamics of lactocrine signaling events that may ultimately connect genotype to phenotype and establish the parameters of reproductive potential.

Milk-borne lactocrine-acting factors affect gene expression patterns in the developing neonatal porcine uterus

Lactocrine communication of milk-borne bioactive factors (MbFs) from mother to offspring through nursing can affect neonatal development with lasting consequences. Relaxin (RLX), a lactocrine-active peptide found in porcine colostrum, stimulates estrogen receptor-α (ESR1) expression required for uterine development shortly after birth (postnatal day=PND 0). Whether other MbFs or cooperative lactocrine mechanisms affect the neonatal uterine developmental program is unknown. To determine the effects of age, nursing, and exogenous RLX on gene expression associated with uterine development, gilts (n=4-5/group) were assigned to nurse ad libitum or to receive milk replacer, with or without exogenous RLX (20 μg/kg BW i.m./6 h for 48 h), from birth to PND 2 when uteri were collected. Body weight and uterine weight increased (P<0.05) similarly from birth to PND 2 in all gilts. However, colostrum consumption was required for normal uterine ESR1, vascular endothelial growth factor (VEGFA), matrix metalloproteinase 9 (MMP9), and RLX receptor (RXFP1) protein and/or transcript expression on PND 2. Uterine ESR1, VEGFA, and MMP9 protein levels were below (P<0.01) the assay sensitivity in replacer-fed gilts. Supplemental RLX increased (P<0.05) uterine ESR1 protein and mRNA in nursed gilts, as well as VEGFA protein in nursed and VEGFA mRNA in both nursed and replacer-fed gilts. RLX treatment did not affect uterine MMP9 mRNA levels. When compared with replacer-fed gilts on PND 2, uterine RXFP1 mRNA was reduced (P<0.05) in nursed gilts and in RLX-supplemented replacer-fed gilts. These results constitute the first evidence that establishment of the neonatal porcine uterine developmental program requires maternal lactocrine support.

Effects of relaxin and estrogens on bone remodeling markers, receptor activator of NF-kB ligand (RANKL) and osteoprotegerin (OPG), in rat adjuvant-induced arthritis

Rheumatoid arthritis (RA) is characterized by joint inflammation and bone destruction. The receptor activator of nuclear factor-kappa B ligand (RANKL)-osteoprotegerin (OPG) system is important for maintaining the balance between bone resorption and formation. Both serum RANKL/OPG protein and synovial tissue RANKL/OPG mRNA ratios are elevated in patients with RA. Studies indicate that hormones of pregnancy, estrogens and relaxin, administered in combination, reduce circulating (TNF)-α and joint inflammation in a rat adjuvant-induced arthritis (AIA) model of RA. The purpose of this study was to investigate whether relaxin (RLX), alone or in combination with estrogens, regulates the bone remodeling markers RANKL and OPG in vivo and in vitro. Results show that in AIA rats, treatment with relaxin, estradiol valerate (EV) or EV in combination with relaxin had no effect on circulating RANKL. However, EV increased systemic OPG and combined treatment with EV and relaxin further increased circulating OPG in comparison to EV alone. Importantly, the RANKL/OPG protein ratio was lower in rats treated with EV or EV+RLX when compared to arthritic controls. Relaxin in combination with EV decreased local RANKL transcripts, increased OPG mRNA and decreased the RANKL/OPG mRNA ratio in joints of arthritic rats when compared to controls. RLX family peptide receptor 1 (RXFP1) gene expression in joints of AIA rats increased in response to EV and EV+RLX. In parathyroid hormone-pretreated murine UMR 106-01 osteoblast cells, 17-β-estradiol (E) and E+RLX increased RXFP1 transcripts, while RLX reduced RANKL mRNA and protein expression. However, in vitamin D-treated primary rat osteoblast cells E+RLX increased OPG protein and reduced the RANKL/OPG protein ratio. These results suggest that modulation of the RANKL-OPG system by estrogens and RLX may contribute to their antiarthritic effects on bone during pregnancy.

Transient estrogen exposure from birth affects uterine expression of developmental markers in neonatal gilts with lasting consequences in pregnant adults.

Disruption of estrogen-sensitive, estrogen receptor (ER)-dependent events during porcine uterine development between birth (postnatal day=PND 0) and PND 14 affects patterns of uterine morphoregulatory gene expression in the neonate with lasting consequences for reproductive success. Uterine capacity for conceptus support is reduced in pregnant adult gilts exposed to estradiol valerate (EV) for 14 days from birth. Objectives here were to determine effects of EV exposure from birth through PND 13 on neonatal uterine and adult endometrial markers of growth, patterning, and remodeling. Targets included the relaxin receptor (RXFP1), estrogen receptor-alpha (ESR1) and vascular endothelial growth factor (VEGFA), morphoregulatory markers HOXA10 and WNT7A, and the matrix metalloproteinases (MMP)2 and MMP9. Gilts were treated daily with EV (50 microg/kg body weight per day, i.m.) or corn oil vehicle from birth through PND 13. Uteri were obtained from neonates on PND 14 and from adults on pregnancy day 12 (PxD 12). In neonates, EV exposure from birth increased uterine RXFP1 gene expression, and both ESR1 and VEGFA proteins. At PxD 12, endometrial RXFP1 mRNA remained elevated, while ESR1 protein was reduced. Early EV treatment decreased neonatal uterine WNT7A, but increased HOXA10 expression. WNT7A expression was reduced in EV-treated adults. Transient EV exposure increased MMP9 transcripts at PND 14, whereas both latent and active MMP9 activity was increased due to early EV treatment in adults on PxD 12. Results support the hypothesis that transient, estrogen-induced disruption of porcine uterine development from birth alters early programming events that lead to functional consequences in the adult.

Characterization and biological activity of relaxin in porcine milk.

A lactocrine mechanism for delivery of maternally derived relaxin (RLX) into the neonatal circulation as a consequence of nursing was proposed for the pig. Immunoreactive RLX was detected in colostrum and in the serum of newborn pigs only if they were allowed to nurse. Milk-borne RLX concentrations are highest during early lactation (9-19  ng/ml), declining to <2  ng/ml by postnatal day 14. Whether milk-borne RLX is bioactive is unknown. Evidence that RLX concentrations in milk are higher than in maternal circulation in several species suggests the mammary gland as a site of local RLX production. It is unknown whether the porcine mammary gland is a source of RLX. Therefore, objectives were to evaluate RLX bioactivity in porcine milk during the first 2 weeks of lactation, identify the form of RLX in porcine milk, and determine whether mammary tissue from early lactation is a source of milk-borne RLX. Milk RLX bioactivity was determined using an in vitro bioassay in which cAMP production by human embryonic kidney (HEK293T) cells transfected with the human RLX receptor (RXFP1) was measured. RLX bioactivity was highest at lactation day (LD) 0, decreasing to undetectable levels by LD 4. Immunoblot analysis of milk proteins revealed an 18  kDa band, indicating proRLX as the primary form of RLX in porcine milk. ProRLX protein and transcripts were detected in porcine mammary tissue on LD 0 and 7. Results support the lactocrine hypothesis by defining the nature and a potential source for bioactive proRLX in porcine colostrum/milk.

Biological activity of relaxin in porcine milk.

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.

Nursing During the First Two Days of Life Is Essential for the Expression of Proteins Important for Growth and Remodeling of the Neonatal Porcine Cervix

The neonatal porcine cervix is sensitive to hormones, including relaxin (RLX), from birth. Whether nursing is required to establish the cervical developmental program or to determine cervical developmental trajectory is unknown. The objective of study 1 was to determine effects of age and nursing on expression of molecular markers and mediators of porcine cervical growth and remodeling from birth to postnatal day (PND) 2 and to document effects of RLX treatment during this period on expression of targeted gene products in nursed vs. replacer-fed gilts. Study 2 was conducted to determine effects of age at first nursing and duration of nursing from birth on expression of targeted transcripts or proteins at PND 14. Nursing supported cervical estrogen receptor-α, vascular endothelial growth factor, matrix metalloproteinase (MMP)9, and antiapoptotic B-cell lymphoma-2 protein expression on PND 2. These proteins were undetectable in replacer-fed gilts. Returning replacer-fed gilts to nursing after PND 2 did not restore cervical expression of these proteins by PND 14. RLX increased (P < 0.05) cervical estrogen receptor-α, vascular endothelial growth factor, and B-cell lymphoma-2 protein in nursed gilts, MMP2 protein in nursed and replacer-fed gilts, and decreased (P < 0.05) pro-MMP9 protein in nursed gilts, and RXFP1 mRNA levels in nursed and replacer-fed gilts at PND 2. Replacer feeding for 2 wk from birth increased (P < 0.05) RXFP1 mRNA levels on PND 14. Results support the lactocrine hypothesis for maternal programming of neonatal tissues. Nursing from birth is required to establish the neonatal cervical developmental program and to maintain cervical developmental trajectory to PND 14.