Relaxin and the 'Milky Way': The lactocrine hypothesis and maternal programming of development.

Maternal effects on early postnatal development in mammals are mediated, in part, by milk-borne bioactive factors transmitted from mother to nursing offspring. The term 'lactocrine' was coined to describe this mode of signaling. Relaxin (RLX), one of a family of neohormones found in mammals, is detectable in milk from multiple species. In the pig, evidence of bioactive proRLX in colostrum/milk, immunoreactive RLX in the circulation of nursed neonates, and RLX receptor expression in RLX-sensitive neonatal female reproductive tract tissues, established RLX as a prototypical lactocrine-active factor. Observations provided the foundation for the lactocrine hypothesis for maternal programming of postnatal development. Studies designed to test the lactocrine hypothesis provided insights into both short-term effects of milk-borne bioactive factors in the neonate, and long-term consequences of maternal lactocrine programming of endometrial function and fecundity in adults. Thus, RLX led to the 'Milky Way'.

Neonatal lactocrine deficiency affects the adult porcine endometrial transcriptome at pregnancy day 13.

Reproductive performance of female pigs that do not receive sufficient colostrum from birth is permanently impaired. Whether lactocrine deficiency, reflected by low serum immunoglobulin immunocrit (iCrit), affects patterns of endometrial gene expression during the periattachment period of early pregnancy is unknown. Here, objectives were to determine effects of low iCrit at birth on the adult endometrial transcriptome on pregnancy day (PxD) 13. On the first day of postnatal life, gilts were assigned to high or low iCrit groups. Adult high (n = 8) and low (n = 7) iCrit gilts were bred (PxD 0), and humanely slaughtered on PxD 13 when tissues and fluids were collected. The endometrial transcriptome was defined for each group using mRNAseq and microRNAseq. Reads were mapped to the Sus scrofa 11.1 genome build. Mature microRNAs were annotated using miRBase 21. Differential expression was defined based on fold change (≥ ±1.5). Lactocrine deficiency did not affect corpora lutea number, uterine horn length, uterine wet weight, conceptus recovery, or uterine luminal fluid estrogen content on PxD 13. However, mRNAseq revealed 1157 differentially expressed endometrial mRNAs in high versus low iCrit gilts. Differentially expressed genes had functions related to solute transport, endometrial receptivity, and immune response. Six differentially expressed endometrial microRNAs included five predicted to target 62 differentially expressed mRNAs, affecting similar biological processes. Thus, lactocrine deficiency on the first day of postnatal life can alter uterine developmental trajectory with lasting effects on endometrial responses to pregnancy as reflected at the level of the transcriptome on PxD 13.

Effects of colostrum, feeding method and oral IGF1 on porcine uterine development.

Nursing ensures lactocrine delivery of maternally derived, milk-borne bioactive factors to offspring, which affects postnatal development of female reproductive tract tissues. Disruption of lactocrine communication for two days from birth (postnatal day (PND) 0) by feeding milk replacer in lieu of nursing or consumption of colostrum alters porcine uterine gene expression globally by PND 2 and inhibits uterine gland genesis by PND 14. Here, objectives were to determine effects of: (1) nursing or milk replacer feeding from birth; (2) a single dose of colostrum or milk replacer and method of feeding and (3) a single feeding of colostrum or milk replacer, with or without oral supplementation of IGF1, administered at birth on aspects of porcine uterine development at 12-h postnatally. Results indicate nursing for 12 h from birth supports rapid establishment of a uterine developmental program, illustrated by patterns of endometrial cell proliferation, expression of genes associated with uterine wall development and entry into mitosis and establishment of a uterine MMP9/TIMP1 system. A single feeding of colostrum at birth increased endometrial cell proliferation at 12 h, regardless of method of feeding. Oral supplementation of IGF1 was sufficient to support endometrial cell proliferation at 12 h in replacer-fed gilts, and supplementation of colostrum with IGF1 further increased endometrial cell proliferation. Results indicate that lactocrine regulation of postnatal uterine development is initiated with the first ingestion of colostrum. Further, results suggest IGF1 may be lactocrine-active and support a 12-h bioassay, which can be used to identify uterotrophic lactocrine activity.

Maternal lactocrine programming of porcine reproductive tract development.

The lactocrine hypothesis for maternal programming of female reproductive tract development is based on the idea that non-nutritive, milk-borne bioactive factors (MbFs), delivered from mother to offspring during nursing, play a role in determining the trajectory of development with long-term consequences in the adult. Porcine female reproductive tract development is completed postnatally, and the period during which maternal support of neonatal growth derives exclusively from colostrum/milk defines a window of opportunity for lactocrine programming of reproductive tissues. Beyond nutrition, milk serves as a delivery system for a variety of bioactive factors. Porcine relaxin is a prototypical MbF. Present in colostrum at highest concentrations at birth, relaxin is transmitted into the circulation of nursing piglets where it can act on Relaxin receptors found in neonatal female reproductive tract tissues. This process is facilitated by the physiology of the maternal-neonatal dyad and the fact that the neonatal gastrointestinal tract is open to absorb macromolecules for a period of time postnatally. Age at first nursing and duration of nursing from birth are also important for porcine female reproductive tract development. These parameters affect both the quality and quantity of colostrum consumed. Disruption of lactocrine signaling by feeding milk replacer from birth altered porcine uterine, cervical, and testicular development by postnatal Day 2. Moreover, insufficient colostrum consumption in nursing piglets can impair uterine capacity to support viable litters of optimal size in adulthood. In the pig, lactocrine signaling supports neonatal organizational events associated with normal reproductive development and may program adult uterine capacity.

Defining age- and lactocrine-sensitive elements of the neonatal porcine uterine microRNA-mRNA interactome.

Factors delivered to offspring in colostrum within 2 days of birth support neonatal porcine uterine development. The uterine mRNA transcriptome is affected by age and nursing during this period. Whether uterine microRNA (miRNA) expression is affected similarly is unknown. Objectives were to (1) determine effects of age and nursing on porcine uterine miRNA expression between birth and postnatal day (PND) 2 using miRNA sequencing (miRNAseq) and; (2) define affected miRNA­mRNA interactions and associated biological processes using integrated target prediction analysis. At birth (PND 0), gilts were euthanized, nursed ad libitum, or gavage-fed milk replacer for 48 h. Uteri were collected at birth or 50 h postnatal. MicroRNAseq data were validated using quantitative real-time PCR. Targets were predicted using an established mRNA database generated from the same tissues. For PND 2 versus PND 0 comparisons, 31 differentially expressed (DE) miRNAs were identified for nursed, and 42 DE miRNAs were identified for replacer-fed gilts. Six DE miRNAs were identified for nursed versus replacer-fed gilts on PND 2. Target prediction for inversely correlated DE miRNA­mRNA pairings indicated 20 miRNAs targeting 251 mRNAs in nursed, versus 29 miRNAs targeting 585 mRNAs in replacer-fed gilts for PND 2 versus PND 0 comparisons, and 5 miRNAs targeting 81 mRNAs for nursed versus replacer-fed gilts on PND 2. Biological processes predicted to be affected by age and nursing included cell-to-cell signaling, cell morphology, and tissue morphology. Results indicate novel age- and lactocrine-sensitive miRNA­mRNA relationships associated with porcine neonatal uterine development between birth and PND 2

Age and Nursing Affect the Neonatal Porcine Uterine Transcriptome.

The lactocrine hypothesis for maternal programming of neonatal development was proposed to describe a mechanism through which milk-borne bioactive factors, delivered from mother to nursing offspring, could affect development of tissues, including the uterus. Porcine uterine development, initiated before birth, is completed postnatally. However, age- and lactocrine-sensitive elements of the neonatal porcine uterine developmental program are undefined. Here, effects of age and nursing on the uterine transcriptome for 48 h from birth (Postnatal Day [PND] = 0) were identified using RNA sequencing (RNAseq). Uterine tissues were obtained from neonatal gilts (n = 4 per group) within 1 h of birth and before feeding (PND 0), or 48 h after nursing ad libitum (PND 2N) or feeding a commercial milk replacer (PND 2R). RNAseq analysis revealed differentially expressed genes (DEGs) associated with both age (PND 2N vs. PND 0; 3283 DEGs) and nursing on PND 2 (PND 2N vs PND 2R; 896 DEGs). Expression of selected uterine genes was validated using quantitative real-time PCR. Bioinformatic analyses revealed multiple biological processes enriched in response to both age and nursing, including cell adhesion, morphogenesis, and cell-cell signaling. Age-sensitive pathways also included estrogen receptor-alpha and hedgehog signaling cascades. Lactocrine-sensitive processes in nursed gilts included those involved in response to wounding, the plasminogen activator network and coagulation. Overall, RNAseq analysis revealed comprehensive age- and nursing-related transcriptomic differences in the neonatal porcine uterus and identified novel pathways and biological processes regulating uterine development.

Methoxychlor and its metabolite HPTE inhibit cAMP production and expression of estrogen receptors α and ß in the rat granulosa cell in vitro.

The major metabolite of the estrogenic pesticide methoxychlor (MXC) HPTE is a stronger ESR1 agonist than MXC and acts also as an ESR2 antagonist. In granulosa cells (GCs), FSH stimulates estradiol via the second messenger cAMP. HPTE inhibits estradiol biosynthesis, and this effect is greater in FSH-treated GCs than in cAMP-treated GCs. Therefore; we examined the effect of MXC/HPTE on FSH-stimulated cAMP production in cultured GCs. To test involvement of ESR-signaling, we used the ESR1 and ESR2 antagonist ICI 182,780, ESR2 selective antagonist PHTPP, and ESR2 selective agonist DPN. ESR1 and ESR2 mRNA and protein levels were quantified. Both HPTE and MXC inhibited the FSH-induced cAMP production. ICI 182,780 and PHTPP mimicked the inhibitory action of HPTE. MXC/HPTE reduced FSH-stimulated Esr2 mRNA and protein to basal levels. MXC/HPTE also inhibited FSH-stimulated Esr1. The greater inhibition on FSH-stimulated GCs is likely due to reduced cAMP level that involves ESR-signaling, through ESR2.

Effects of age, nursing, and oral IGF1 supplementation on neonatal porcine cervical development.

Nursing supports neonatal porcine uterine and testicular development, however, lactocrine effects on cervical development are undefined. Studies were conducted to determine the effects of i) age and the imposition of the lactocrine-null state from birth (postnatal day 0 (PND0)) by milk replacer feeding on cervical histology; ii) imposition of the lactocrine-null state for 2 days from birth on cervical cell proliferation, as reflected by proliferating cell nuclear antigen immunostaining; and iii) a single feeding of colostrum or milk replacer, administered at birth, with or without oral IGF1, on cervical cell proliferation and phosphorylated AKT (pAKT) and B-cell lymphoma 2 (BCL2) protein levels at 12 h postnatal. Cervical crypt depth and height of luminal epithelium (LE) increased with age by PND14, when both responses were reduced in replacer-fed gilts. Cell proliferation was reduced in LE at PND2, and in crypt epithelium and stroma by PND14 in replacer-fed gilts. Returning replacer-fed gilts to nursing on PND2 did not rescue the cervical phenotype by PND14. A single feeding of colostrum, but not milk replacer, was sufficient to support cervical cell proliferation at 12 h postnatal. IGF1 supplementation induced cell proliferation in replacer-fed gilts, and increased cervical pAKT and BCL2 levels in colostrum-fed gilts and replacer-fed gilts at 12 h postnatal. Results indicate that age and nursing support porcine cervical development, support is initiated at first ingestion of colostrum, IGF1 may be lactocrine-active, and identification of lactocrine-active factors can be accomplished by 12 h postnatal using this bioassay system.

Nursing for 48 hours from birth supports porcine uterine gland development and endometrial cell compartment-specific gene expression.

The first 2 wk of neonatal life constitute a critical period for estrogen receptor alpha (ESR1)-dependent uterine adenogenesis in the pig. A relaxin receptor (RXFP1)-mediated, lactocrine-driven mechanism was proposed to explain how nursing could regulate endometrial ESR1 and related gene expression events associated with adenogenesis in the porcine neonate during this period. To determine effects of nursing on endometrial morphogenesis and cell compartment-specific gene expression, gilts (n = 6-8/group) were assigned at birth to be either 1) nursed ad libitum for 48 h, 2) gavage fed milk replacer for 48 h, 3) nursed ad libitum to Postnatal Day (PND) 14, or 4) gavage fed milk replacer for 48 h followed by ad libitum nursing to PND 14. Uteri were collected on PND 2 or PND 14. Endometrial histoarchitecture and both ESR1 and proliferating cell nuclear antigen (PCNA) labeling indexes (LIs) were evaluated. Laser microdissection was used to capture epithelium and stroma to evaluate treatment effects on cell compartment-specific ESR1, VEGFA, and RXFP1 expression. Imposition of a lactocrine-null state by milk replacer feeding for 48 h from birth retarded endometrial development and adenogenesis. Effects of replacer feeding, evident by PND 2, were marked by PND 14 when endometrial thickness, glandularity, and gland depth were reduced. Consistently, in lactocrine-null gilts, PCNA LI was reduced in glandular epithelium (GE) and stroma on PND 14, when epithelial ESR1 expression and ESR1 LI in GE were reduced and stromal VEGFA and RXFP1 expression increased. Results establish that lactocrine signaling effects morphogenetic changes in developing uterine tissues that may determine reproductive capacity later in life.

Milk-borne relaxin and reproductive system development.

A window of opportunity for maternal programming of neonatal development is open in the first few days of life as a consequence of nursing. Colostrum (first milk) supports neonatal development by providing a conduit for delivery of milk-borne bioactive factors, exemplified by relaxin, from mother to offspring as proposed in the lactocrine hypothesis. Relaxin, a prototypical milk-borne bioactive factor, is detectable in colostrum from multiple species, including the pig. Thus, relaxin serves as a model for understanding lactocrine signals that support development of neonatal tissues.

Anti-arthritic actions of relaxin, in combination with estrogens, in joint and bone tissue.

The incidence and severity of rheumatoid arthritis decline during pregnancy. However, the role of hormones of pregnancy, including estrogens and relaxin, in attenuating the symptoms of rheumatoid arthritis, including joint inflammation and bone destruction is unknown. In rat adjuvant-induced arthritis, a model for rheumatoid arthritis, relaxin in combination with estrogens, reduced joint inflammation and circulating levels of pro-inflammatory, tumor necrosis factor alpha. In addition, relaxin together with estrogens, altered systemic levels of bone remodeling markers receptor activator of nuclear factor-kappa B, its ligand and osteoprotegerin to improve bone health when compared with arthritic controls. In vitro studies using primary rat osteoblasts and an osteoblast cell line showed a similar bon-saving response to treatment with estrogens in combination with relaxin.

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.

Lactocrine programming of female reproductive tract development: environmental connections to the reproductive continuum.

For eutherian mammals a continuum of maternal support insures that development of progeny follows an optimal program. Beginning in utero, such support extends into the early neonatal period when bioactive factors are communicated from mother to offspring in colostrum/milk. Defined as lactocrine signaling, communication of milk-borne bioactive factors from mother to offspring as a consequence of nursing is important for development of somatic tissues, including the female reproductive tract (FRT). Data for the domestic pig indicate that lactocrine signaling contributes to the maternal continuum of factors that define the developmental program and determine the developmental trajectory of FRT tissues during early neonatal life. Both naturally occurring and manmade factors of environmental origin can be communicated to neonates in milk and affect development with lasting consequences. Here, evidence for lactocrine programming of FRT development and the potential for environmental endocrine disruption of this process are reviewed.

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-ß-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.

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.

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.

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.

Milk-borne relaxin and the lactocrine hypothesis for maternal programming of neonatal tissues.

The fact that all newborn mammals drink milk extends the time frame of maternal influence on development into neonatal life. While the nutritional and immunological benefits of milk are clear, the role of milk as a conduit for bioactive factors with the potential to affect neonatal development is less well defined. Porcine and canine milk contain immunoreactive relaxin (RLX) that is transmitted into the circulation of nursing offspring. In the pig, a window of opportunity for transmission of milk-borne RLX is open at birth and remains so for about the first 3 days of neonatal life. Recent studies have shown that pro RLX is the major form of RLX in milk and that milk-borne porcine pro RLX is biologically active. Moreover, RLX receptor (RXFP1) expression is detectable in the porcine female reproductive tract and other somatic tissues at birth. The lactocrine hypothesis for maternal programming of neonatal development was proposed as a mechanism whereby RLX, a prototypical milk-borne growth factor in the pig, is delivered to nursing offspring, where it can affect development of RXFP1-positive target tissues. Data indicating that treatment of newborn gilts with RLX increased estrogen receptor-alpha expression in the uterus and cervix by postnatal day 2 support a role for RLX in lactocrine programming of the female reproductive tract. Effects of RLX on Wnt/beta-catenin expression in neonatal porcine cardiac tissue support a role for RLX in developmental programming of nonreproductive target tissues as well. Ongoing studies will test the lactocrine hypothesis for maternal programming of development by RLX and related milk-borne factors.

Relaxin and maternal lactocrine programming of neonatal uterine development.

In mammals, including the pig (Sus scrofa domesticus), structural patterning and functional programming of uterine tissues involve events that occur shortly after birth. Porcine endometrial development between birth (postnatal day 0 [PND 0]) and PND 15 is estrogen receptor (ER) dependent and estrogen sensitive. The endometrium is relaxin (RLX) receptor (RXFP1) positive and ERalpha negative at birth. Uterine expression of RXFP1 and ERalpha, detectable by PND 2, increases with age from PND 0 to 14. Estrogen exposure during this period sufficient to affect uterine developmental trajectory and adult uterine phenotype also alters uterine RXFP1 gene expression patterns in the neonatal uterus. Data implicate RXFP1 as an element of the uterine developmental program. Uterotrophic effects documented for both estrogen and RLX in the neonatal pig are age-specific and most pronounced after onset of ERalpha expression. Patterns of inhibition of RLX effects on uterine development induced with ICI 182,780, an ER antagonist, indicate that cross talk between RLX and estrogen signaling systems evolve with age in the porcine uterus. Given that RLX administered from birth stimulates uterine ERalpha expression and that estrogen administered from birth stimulates RXFP1 expression by PND 2, a feed-forward relationship between these signaling systems is envisioned. Evidence that RLX is present in porcine milk and in the circulation of nursing offspring supports the lactocrine hypothesis for maternal programming of uterine tissues in which milk-borne RLX, acting via RXFP1, sustains ERalpha expression and porcine endometrial development in the neonate.