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Endocrinology Vol. 147, No. 9 4303-4310
Copyright © 2006 by The Endocrine Society

Expression of LGR7 and LGR8 by Neonatal Porcine Uterine Tissues and Transmission of Milk-Borne Relaxin into the Neonatal Circulation by Suckling

Wenbo Yan, Anne A. Wiley, Ross A. D. Bathgate, Amy-Lynn Frankshun, Sally Lasano, Bethany D. Crean, Bernard G. Steinetz, Carol A. Bagnell and Frank F. Bartol

Department of Animal Sciences (W.Y., A.-L.F., C.A.B.), Rutgers University, New Brunswick, New Jersey 08901; Department of Animal Sciences, Cellular and Molecular Biosciences Program (A.A.W., B.D.C., F.F.B.), Auburn University, Auburn, Alabama 26849; Howard Florey Institute (R.A.D.B.), University of Melbourne, Victoria 3010, Australia; and Department of Environmental Medicine (S.L., B.G.S.), Nelson Institute of Environmental Medicine, New York University School of Medicine, Tuxedo, New York 10987

Address all correspondence and requests for reprints to: Dr. Frank F. Bartol, Department of Animal Sciences, Upchurch Hall, Auburn University, Auburn, Alabama 36849-5415. E-mail: bartoff{at}auburn.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Estrogen receptor-dependent organizational events between birth [postnatal day (PND) 0] and PND 14 affect development and function of porcine uterine tissues. Observations that uterotrophic effects of relaxin (RLX) in neonatal gilts were inhibited by the antiestrogen ICI 182,780 suggested that a RLX signaling system, capable of cross-talk with the estrogen receptor, evolves during a critical period for uterine programming (PND 0–14). Objectives were to determine 1) effects of age and estrogen exposure from birth on porcine uterine RLX/insulin-like 3 receptor (LGR7/LGR8) expression and 2) whether milk serves as a natural source of RLX in neonatal pigs. Uterine LGR7/LGR8 expression, detected by RT-PCR and in situ hybridization on PND 0, 7, and 14, was predominantly stromal for LGR7, myometrial for LGR8, and increased with age and after treatment with estradiol valerate (50 µg/kg body weight·d) from birth. Stromal expression of LGR7 was also detected immunohistochemically. Milk RLX concentrations declined (P < 0.001) from 17.3 ± 1.4 ng/ml (lactation d 0) to 1.7 ± 0.3 ng/ml (lactation d 14). RLX, present in the serum of nursing pigs on PND 0 and 1, was undetectable before nursing and in neonates fed RLX-free milk replacer for 12 h. Thus, a developmentally regulated, estrogen-sensitive LGR7 and LGR8 receptor system is present in the porcine uterus at birth and may be activated by milk-borne RLX delivered into the circulation during the first 48 h of postnatal life. Maternal lactocrine contributions to the neonatal hormonal milieu could affect the developmental programming of uterine and other somatic tissues.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
EPIGENETIC PROGRAMS GOVERNING morphogenesis and cytodifferentiation of reproductive tissues and organs are defined, in part, by the long-term effects of endocrine-active agents to which developing tissues are exposed during organizationally critical periods of fetal and perinatal life (1). Both normal and aberrant programming of immature reproductive tissues, including the female reproductive tract (FRT), are likely to involve coordinated or dysregulated effects of both steroid and peptide hormone signaling events (2, 3). Where the FRT is concerned, recent studies showed that, when administered to neonatal gilts, porcine relaxin (RLX) stimulated endometrial development and uterine growth at or very shortly after birth [postnatal day (PND) 0] and that observed uterotrophic effects of RLX became more pronounced with age between PND 0 and 14 (4). In the pig, this neonatal period is associated with the onset of uterine gland genesis (5), an estrogen-sensitive, estrogen receptor-{alpha} (ER)-dependent process (6).

The fact that both uterine gland genesis (6) and specific uterotrophic effects of RLX administered during this period (4) were inhibited by administration of the type-II antiestrogen ICI 182,780 indicated that these events involve activation of the ER system. Neonatal estrogen exposure during this period (and associated hyperactivation of the developing uterine ER system) altered the normal uterine organizational program as reflected by patterns of morphoregulatory gene expression (3) and reduced functional uterine capacity in adult gilts (5, 7). Data reinforced the importance of identifying factors that affect ER activation during this critical uterine developmental period in the pig. Given that the porcine endometrium is RLX sensitive at or very shortly after birth, and that uterotrophic effects of RLX administered neonatally can be inhibited with ICI 182,780, it is reasonable to expect that a RLX signaling system is present or evolving in the neonatal porcine uterine wall. If so, such an active RLX signaling system could be involved, directly as well as indirectly via cross-talk with the ER system (8), in critical organizational events necessary to ensure optimal adult uterine function. Moreover, factors that affect the functional dynamics of this system could alter the developmental trajectory of uterine tissues with negative consequences for reproductive performance (3, 4).

RLX and insulin-like factor 3 (INSL3), two members of the insulin family of hormones, are important for growth and remodeling of reproductive tissues. RLX (9) and INSL3 (10) were identified in pigs, and binding sites for RLX were localized in porcine uterine tissues (11, 12). Studies show that estrogen increased RLX binding in both myometrium and endometrium (13, 14). However, RLX and INSL3 receptors were identified and described in detail only recently. In 2002, two human leucine-rich repeat-containing G protein-coupled receptors, LGR7 and LGR8, were identified as the cognate receptors for RLX (15) and INSL3 (15, 16), respectively. Although RLX can activate LGR8 at high concentrations in vitro (15), studies in rodents indicated that RLX/LGR7 and INSL3/LGR8 pathways are nonoverlapping in vivo (17, 18). Whether RLX can activate LGR8 in vivo in other species is not known. Using the human LGR7 coding sequence as a template, mouse and rat LGR7 orthologs were characterized (19), and human, rat (20), and equine (21) LGR8 orthologs have also been identified. Both LGR7 and LGR8 are expressed in a wide range of human (15, 22) and rodent (19, 20, 23) tissues. However, information on LGR7 and LGR8 expression in the pig has not been reported.

Evidence that neonatal porcine uterine tissues respond to exogenous RLX raises the question of whether there is a natural source of RLX in the neonatal pig. Studies in other species indicate that RLX of maternal origin could be transmitted to the neonate by way of ingestion of colostrum and milk. RLX is detectable in human (24) and canine (25) colostrum and milk, and mammary tissue was suggested as a local source of RLX (26). In dogs, milk-borne RLX is absorbed into the systemic circulation of neonates through the gut (25). Whether RLX is present in porcine colostrum or milk, hereafter referred to as milk, and can reach the neonatal circulation as a consequence of nursing remains to be determined.

As an extension of work reported earlier by this laboratory (27), the objectives of this study were to determine 1) whether receptors for RLX (LGR7) and/or INSL3 (LGR8) are expressed in uterine tissues obtained at birth or on PND 7 or 14; 2) whether the temporospatial patterns of LGR7 or LGR8 uterine gene expression are affected by age or treatment with estradiol valerate (EV) during a critical period for estrogen-sensitive porcine uterine development between birth and PND 14; and 3) whether milk could serve as a natural exogenous source of RLX in the neonatal pig.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and experimental procedures
To study LGR7/8 expression in neonatal porcine tissues, 30 white crossbred gilts were assigned randomly at birth (PND 0) to one of three treatment groups (n = 8–12 gilts per group) to receive no daily injection or daily injection of either corn oil vehicle (CO; 50 µl /kg body weight·d, im) or EV (50 µg /kg body weight·d, im) from PND 0 through PND 14. Uteri were obtained from gilts under halothane anesthesia on PND 0 for animals that were not injected and on PND 7 and 14 for animals treated with corn oil or EV. Adult uterine tissues from pregnancy d 42–45 were also collected for use as positive controls in analyses of LGR7 and LGR8 expression.

Porcine milk RLX concentrations were determined in samples obtained from Yorkshire-Landrace sows. Milk (1–2 ml/sample) was collected daily during the first week of lactation on d 0–7 and on lactation d 14 (n = 3 sows). During the periparturient period, milk (1–2 ml/sample) was collected 12 h before parturition (n = 4 sows) and on lactation d 0 (n = 10 sows) and 1 (n = 7 sows). The day of parturition was defined as lactation d 0. All milk samples were collected by hand milking of nursing sows and were stored at –20 C until assay.

Effects of nursing and neonatal age on RLX serum concentrations in the neonate were evaluated in male and female pigs chosen randomly from five litters at birth. Blood (~2 ml/sample) was collected daily from six to 12 neonatal pigs on PND 0–7 and on PND 14 by venipuncture of the external iliac vein. To collect prenursing serum samples, blood was collected from newborn pigs (n = 11) before they had a chance to suckle. Additionally, nursing pigs at PND 0 (n = 4 pigs from two litters) were removed from sows and fed RLX-free milk replacer (Land O’Lakes Animal Milk Products Co., Shoreview, MN) for 12 h before blood samples were collected. All blood samples were centrifuged at approximately 3000 x g at 4 C for 30 min to obtain serum and stored at –20 C until assay.

All procedures involving animals were approved by the Auburn University Institutional Animal Care and Use Committee and the Rutgers University Animal Care and Facilities Committee (Protocol no. 88-079) and were performed in accordance with the Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching.

Tissue processing and RNA isolation
Uterine tissues were kept on ice throughout processing and trimmed free of connective tissue. A segment from the middle of each uterine horn was fixed in 4% paraformaldehyde, embedded in Paraplast-Plus (Sherwood Medical, St. Louis, MO), and sectioned at 5–7 µm for histological evaluations. The rest of the uterus was frozen immediately in liquid nitrogen and stored at –80 C. Total RNA was isolated from frozen tissues using Trizol reagent as directed by the manufacturer (Invitrogen, Carlsbad, CA). Each RNA sample was evaluated spectrophotometrically, and integrity of ethidium-bromide-stained RNA was assessed after electrophoresis through 1.5% agarose gels.

Primer sequence for conventional PCR and cloning of the pLGR7/8
Porcine cDNA amplicons were generated using HotStar (QIAGEN, Valencia, CA) Touchdown PCR procedures with total RNA from uterine tissues obtained on PND 0, 7, and 14. Human LGR7 and LGR8 sequences were used in BLAST searches to identify pig orthologs. Both porcine LGR7 and LGR8 orthologs were identified in the EST database and subsequently used to design primers for RT-PCR amplification of pig LGR7- and LGR8-specific products. For LGR7, the forward primer sequence is 5'-ggaaactattttgctcctggg-3', and the reverse primer sequence is 5'-aggcacgcttcgaagatca-3' with expected amplicon size of 431 bp. For LGR8, the forward primer sequence is 5'-gttctcgatgccatctgctg-3', and the reverse primer sequence is 5'-cgctggttgtgcagcagctg-3' with expected amplicon size of 198 bp. Porcine cDNAs generated from tissues obtained on PND 14 were cloned into plasmid pDrive (QIAGEN) vectors, sequenced using an ABI Prism 3100 genetic analyzer (Applied Biosystems, Foster City, CA), and sequences were submitted for BLAST analyses [www.ncbi.nlm.nih.gov (28)] to confirm identity.

In situ hybridization (ISH)
ISH was used to localize LGR7 and LGR8 mRNAs in uterine tissues as described previously (27). Antisense and sense [35S]UTP-labeled (1000 Ci/mmol; ICN, Costa Mesa, CA) cRNA probes were produced by in vitro transcription using a MaxiScript kit (Ambion, Austin, TX). Either T7 or SP6 RNA polymerases and appropriately linearized LGR7 or LGR8 cDNA-containing plasmid vectors were used as templates in in vitro transcription reactions. On each slide, sections were incubated overnight at 55 C with either sense (negative control) or antisense cRNA probe, and all slides were processed together. After posthybridization processing, slides were immersed in NTB-2 emulsion (Eastman Kodak, Rochester, NY) and exposed for 4 wk at 4 C. Images representative of ISH results were captured digitally by dark-field microscopy using constant conditions to ensure accurate comparisons between images, as described previously (29).

Immunohistochemistry
Uterine LGR7 was localized using rabbit-antihuman LGR7 antibody (L7–3) (30) kindly provided by Dr. Richard Ivell, University of Adelaide. Briefly, uterine tissue sections were passed through xylene and descending ethanols and washed in Tris-buffered saline (TBS; 0.05 M Tris-HCl, 0.15 M NaCl, pH 7.4). Sections were then blocked with 10% (vol/vol) normal goat serum for 1 h. After brief rinsing in TBS, sections were incubated with normal rabbit serum or the primary antiserum, diluted 1:5000 (L7–3) in antibody dilution buffer [TBS containing 2% (vol/vol) normal goat serum plus 0.05% (wt/vol) BSA], overnight at 4 C. After rinsing in TBS (three times for 5 min each) and application of the secondary antibody, endogenous peroxidase was suppressed by incubation of sections in 3% (vol/vol) H2O2 for 45 min at room temperature. Otherwise, uterine LGR7 protein was visualized using Vectastain Elite ABC kit procedures (Vector Laboratories, Burlingame, CA). Specific signals were detected using diaminobenzidine (Sigma Chemical Co., St. Louis, MO) as a chromogen, and no counterstaining was applied.

Porcine RLX RIA
RLX in milk and serum was measured using a homologous porcine RLX RIA as previously described (31, 32) with modifications. Purified porcine RLX was used as the standard and 125I-labeled tyrosyl porcine RLX was used as the radioligand. Antiserum R6 was used at a concentration of 1:20,000. All standards (31–2000 pg/tube) and unknown samples (5–100 µl for milk and 300 µl for serum) were run in triplicate in final volumes of 500 µl PBS containing 1% BSA (pH 7.0) before addition of antiserum (100 µl) and trace (100 µl = 30,000 cpm/tube). The minimal detectable concentration of porcine RLX in these assays ranged from 32–64 pg/ml. The within- and between-assay coefficients of variation were 12.5 and 2.2%, respectively.

To validate the porcine RLX RIA for detecting RLX in whole-milk samples recovery, parallelism and specificity were checked. When known amounts of porcine RLX (0–125 pg) were added to a fixed amount of pig milk collected on lactation d 6, the data (not shown) were additive, indicating good recovery. Also, dilution curves of porcine milk collected on lactation d 0 and pure porcine RLX standards were parallel (data not shown). When porcine insulin, rat prolactin, or human IGF-I (1 µg/ml each) were added to pig milk collected on lactation d 14, the readings were all below assay sensitivity, indicating the absence of cross-reactivity.

Statistical analyses
Quantitative data generated by RIA for RLX in milk and neonatal serum were subjected to analyses of variance using general linear models programs (33). Analyses considered variation caused by the main effects of day of lactation (milk) or day of life (serum), pig, nursing status (serum data only), and their interactions as appropriate. Tests of significance were determined for a mixed model according to expectations of the error mean squares in each case. Data are presented as individual observations or least-squares means (LSM) with SEM as indicated.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PCR cloning and characterization of the porcine LGR7 and LGR8 cDNAs
Results of RT-PCR analyses indicated that receptors for RLX and INSL3, LGR7 (Fig. 1AGo) and LGR8 (Fig. 1BGo), are expressed by the neonatal porcine uterus on PND 0, 7, and 14 and by porcine endometrium obtained from pregnant adults. Amplicons of expected sizes were generated in all cases (Fig. 1Go).


Figure 1
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  		FIG. 1. Identification of LGR7 and LGR8 expression in neonatal uterine and adult endometrial tissues by RT-PCR. Results of RT-PCR designed to amplify cDNAs indicative of LGR7 (A) and LGR8 (B) expression in neonatal uterine and adult endometrial tissues are shown. Endometrial tissues from pregnancy (Px) d 45 served as a positive control. RT-PCR products were analyzed by 1% (wt/vol) agarose gel electrophoresis (ethidium bromide staining). Ld, 100-bp ladder; NT, no RNA template.

 
Both LGR7 (431 bp) and LGR8 (198 bp) cDNAs, generated from PND 14 uterine RNA, were cloned, sequenced, and registered with the NCBI dbEST database. At the nucleotide level, these porcine LGR7 (GenBank accession no. CA994862) and LGR8 (GenBank accession no. CA997681) cDNA sequences were found to share 88 and 85% identity with corresponding human LGR7 and LGR8 sequences and similarly high homologies with corresponding sequences at the amino acid level (27).

In situ expression of LGR7and LGR8 in neonatal uterine tissues
Results of ISH studies for both LGR7 and LGR8 expression in neonatal uterine tissues are shown in Fig. 2Go, A and B. Both transcripts were detected in all neonatal tissues. Signal for LGR7 (Fig. 2AGo) was evident on PND 0 and increased thereafter, with more pronounced stromal signal observed in tissues from PND 7 and PND 14. Signal above background for LGR7 was not observed consistently in either luminal or glandular epithelium. Estrogen exposure increased the apparent intensity of LGR7 signal observed for endometrial tissues on both PND 7 and 14. Signal indicative of uterine LGR8 expression (Fig. 2BGo) became visibly more pronounced over time and in response to EV in the developing myometrium, where LGR8 signal was most intense.


Figure 2
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  		FIG. 2. Temporospatial expression of LGR7 (A) and LGR8 (B) mRNA in neonatal porcine uterine tissues: effects of age and EV. Representative dark-field photomicrographs show signal (white grains) for mRNA. Images are from tissues incubated with antisense probes unless specified otherwise. Sense means incubated with sense probe. Arrows indicate luminal epithelium, and arrowheads glandular epithelium. M, Myometrium; s, stroma. Magnification bars, 100 µm.

 
Results of immunohistochemical localization of LGR7 protein in neonatal uterine tissues from control and EV-treated gilts on PND 14 and from pregnant adult gilts are shown in Fig. 3Go. Immunostaining for LGR7 in all tissues was observed in the endometrial stroma. In neonatal tissues, LGR7 staining was most pronounced in the shallow stroma for control gilts on PND 14, whereas staining was more generalized throughout the stromal compartment in tissues obtained from EV-exposed gilts on PND 14. Epithelial staining, observed in some neonatal tissues, was an inconsistent observation. Myometrial staining was not pronounced. Intense staining for LGR7 was observed in the shallow subepithelial stroma of uterine tissues from pregnant gilts, included as a positive control.


Figure 3
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  		FIG. 3. FIG. 3Go. Immunolocalization of LGR7 in porcine neonatal (PND 14) endometrium after treatment from birth with either corn oil (CO) vehicle (A) or EV (C) and in an adult gilt on d 42 of pregnancy (Px) (E). Tissue sections were incubated with either LGR7 antibody (A, C, and E) or normal rabbit serum at 1:5000 dilution (B, D, and F). Arrows indicate luminal epithelium, and arrowheads glandular epithelium. s, Stroma. Magnification bars, 50 µm.

 
RLX in the milk of lactating sows
Immunoreactive RLX was detected in all milk samples. In those samples obtained at 12- to 24-h intervals for the first 7 d of lactation and on lactation d 14, RLX concentrations were highest during the first 24 h of lactation, ranging from approximately 9–19 ng/ml. Milk RLX concentrations declined linearly from d 0 (P < 0.001) to d 7 and remained low on lactation d 14 (1.7 ± 0.3 ng/ml; Fig. 4AGo). Evaluation of peripartum milk RLX levels revealed concentrations of RLX ranging from 10–30 ng/ml in milk collected before delivery (Fig. 4BGo) that decreased (P < 0.001) by the time of parturition to an average concentration of 17.3 ± 1.4 ng/ml and averaged 9.8 ± 1.1 ng/ml on lactation d 1 (Fig. 4BGo).


Figure 4
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  		FIG. 4. A, Immunoreactive RLX in porcine milk during the first 2 wk of lactation. Data are shown for three sows. Each line represents one animal. B, Periparturient period in hours before and after delivery (0 h). Milk was collected 12 h before delivery (n = 4 sows), on lactation d 0 (n = 10 sows), and lactation d 1 (n = 7 sows).

 
Effects of nursing on serum RLX levels in neonatal pigs
In pigs allowed to nurse naturally at birth, systemic RLX levels approached 200 pg/ml in serum samples collected on PND 0 and 1 (Fig. 5Go). In contrast, RLX concentrations in serum collected on PND 2–7 and on PND 14 were below RIA sensitivity (32 pg/ml; Fig. 5Go). When neonates were removed from sows immediately after birth and before they had a chance to nurse, serum RLX concentrations were undetectable (Fig. 5Go). When neonatal pigs were removed from the sow and fed RLX-free milk replacer for 12 h, serum RLX concentrations fell below RIA sensitivity (Fig. 6Go).


Figure 5
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  		FIG. 5. Effects of nursing on serum RLX levels (pg/ml) in neonatal pigs on PND 0–7 and PND 14: {square}, nursing (n = 6–12/d); {blacksquare}, prenursing (n = 11). Data from PND 0 and PND 1 samples are expressed as LSM ± SEM. ND, Not detectable.

 

Figure 6
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  		FIG. 6. Effects of neonatal age and RLX-free milk replacer on RLX concentrations in milk (A) and neonatal porcine serum (B): {square}, nursing (n = 12); {blacksquare}, milk replacer fed (n = 6). Data are expressed as LSM ± SEM. ND, Not detectable. *, P < 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The recent demonstration that RLX stimulates luminal epithelial development within 2 d from the time of birth (4) suggests that RLX-mediated signaling could be a component of the mechanism through which critical early events in postnatal uterine endometrial programming are initiated in the pig (3). If so, then basic elements of such a signaling system, including 1) uterine expression of RLX receptors, 2) a natural source of RLX, and 3) detectable concentrations of bioactive RLX in the neonatal circulation, should be present or develop during the first few days of neonatal life. Pending evidence of bioactivity in milk-borne RLX, results of this study provide compelling evidence that these elements and conditions are present in the pig at birth. Moreover, normal developmentally regulated patterns of uterine LGR7 and LGR8 expression were visibly altered by exposure of neonatal tissues to estrogen during a critical period for estrogen-sensitive uterine programming.

Results of RT-PCR analyses indicated that receptors for both RLX (LGR7) and INSL3 (LGR8) are expressed by the neonatal porcine uterus and by porcine endometrium obtained from adult gilts on d 45 of pregnancy (3). Porcine LGR7 and LGR8 cDNA sequences shared high homology with both mouse (82% for LGR7 and 84% for LGR8) and human (88%) sequences (27). Evidence for expression of LGR7/LGR8 in the neonatal uterus from birth through PND 14 is consistent with the fact that RLX has uterotrophic effects during this same period (4). In addition, the developmental increase in uterine stromal LGR7 and myometrial LGR8 expression apparent by PND 14 may explain why neonatal uterotrophic responses to RLX are more robust when treatment begins on PND 12 than when it begins on PND 0 (4).

ISH studies confirmed that transcripts for both LGR7 and LGR8 are present in the porcine uterine wall at birth and indicated that the relative levels of both transcripts increase during the first 2 wk of neonatal life. Although LGR7 expression was predominant in endometrial stroma, LGR8 expression, also evident in this endometrial compartment, was highly expressed in developing myometrium. Localization of LGR7 transcripts to porcine endometrial stroma was confirmed immunohistochemically. Earlier reports provide different perspectives with regard to the cellular localization of RLX-binding sites and/or LGR7 expression in uterine tissues. For example, whereas binding sites for biotinylated RLX were identified in uterine luminal and glandular epithelium in pregnant gilts (12) and women (34), only endometrial stroma bound biotinylated RLX in the marmoset (35). In another study, human uterine luminal and glandular epithelium bound radiolabeled RLX, whereas stromal cells did not (36). However, using carefully validated LGR7 antibodies, studies in humans and primates showed specific LGR7 immunostaining in uterine stroma surrounding endometrial glands (30). Here, using the same LGR7 antibodies on porcine uterine tissues, a similar pattern of LGR7 protein localization was observed. Differences in species, reproductive status, and methodological sensitivity may explain some of the variations reported for uterine localization of RLX-binding sites and LGR7 in situ. In any event, evidence presented here for LGR7 and LGR8 expression in the uterine stroma suggests that trophic actions of RLX on uterine epithelium (4) may involve paracrine signaling through stromal-epithelial interactions.

Evidence that RLX induced both LGR7 and LGR8 to stimulate cAMP production in cultured cells (15, 22) suggested that circulating RLX could elicit physiological actions through both of these receptors in vivo. This led us to investigate the expression of both LGR7 and LGR8 in the neonatal uterus. However, subsequent molecular targeting studies in rodents showed that LGR8 does not contribute to RLX signaling pathways and that INSL3/LGR8 and RLX/LGR7 actions do not overlap in vivo (17). Although porcine RLX can activate human LGR8 in vitro (37), the role of LGR8 in neonatal porcine uterine development and the extent, if any, to which RLX might exert effects through LGR8 in pigs remain to be determined. Additionally, a natural source of INSL3 in the neonatal gilt has yet to be identified.

When treated with EV from birth to PND 14, signal intensity for both endometrial LGR7 and myometrial LGR8 transcripts appeared to increase in situ. Data support and extend previous studies indicating that estrogen can sensitize reproductive tissues to RLX (14, 38, 39) and may explain why estrogen priming is either required (40, 41) or will enhance responsiveness to RLX in female reproductive tract tissues (42). Given that 1) both estrogen and RLX receptor systems are present (LGR7) and/or evolving (ER) in the uterine wall between birth and PND 14 (29), 2) ER activation during this period is required for normal uterine development and aberrant ER activation during this period can compromise adult uterine phenotype (3, 7), and 3) RLX can affect patterns of ER activation (4, 43), the idea that neonatal RLX exposure may affect critical uterine tissue programming events must be considered.

RLX is present in porcine milk during lactation in concentrations higher than those found in the maternal circulation. In the first 24 h after parturition, porcine milk RLX ranges from 15–20 ng/ml, whereas circulating maternal RLX is in the 1 ng/ml range (44). A similar phenomenon was reported in paired samples of human milk and maternal serum obtained 3 days after delivery (24). Likewise, RLX levels in canine milk are much greater than in maternal serum during lactation, with highest milk RLX evident within 2 d of delivery (25). Overall, results are consistent with the fact that many hormones in milk exist in concentrations that exceed those found in maternal plasma (45). The higher RLX content of human, canine, and porcine milk could be the result of a concentrating mechanism and/or local production in the mammary gland first proposed more than 20 yr ago (46). Evidence in support of the mammary gland as a source of RLX comes from studies in guinea pigs and humans. RLX has been immunolocalized in guinea pig mammary gland (47), and the human RLX gene was detected in both normal and neoplastic breast tissue (26). In pigs, there is evidence that RLX plays a role in promoting development and differentiation of the mammary gland (48, 49, 50). Thus, RLX in milk may also serve as a paracrine and/or autocrine factor affecting the growth and function of the mammary gland during lactation (45).

There is evidence for placental transfer of RLX from mother to fetus in primates (51, 52) and in dogs (25). Although pregnant sows have relatively high concentrations of circulating RLX around the time of parturition (50–150 ng/ml) (53, 54), results of the present study provided no evidence for maternal-fetal exchange of RLX before parturition. RLX was undetectable in the circulation of newborn pigs before nursing. This may be explained, in part, by the epitheliochorial nature of the porcine placenta in which six layers of cells separate the maternal and fetal circulations (55). When neonates were fed artificial milk replacer for 12 h, serum RLX levels were below the sensitivity of the assay in newborn pigs, strongly suggesting that circulating RLX in neonatal pigs on PND 0–1 is from the ingestion of milk. Whether RLX is produced endogenously in the neonate is unknown. However, data presented here can be interpreted to indicate that any such endogenous source does not contribute tangibly to RLX levels in the neonatal circulation.

Detection of RLX in both milk and in the neonatal circulation during early lactation correlates with gut closure time in pigs, estimated to occur at 24–48 h after birth (56). In neonatal pigs, milk intake was estimated to approach 30% of body weight (57). Therefore, neonatal pigs can ingest significant quantities (micrograms) of milk-borne RLX before gut closure. Present results are consistent with similar studies in dogs showing that milk-borne RLX is transmitted from lactating bitches to pups via suckling and is then absorbed into the systemic circulation (25). Noteworthy is the fact that the porcine RLX RIA used here detects only intact RLX with A and B chains linked by disulfide bonds, not isolated peptide chains or reduced-alkylated RLX (32). Therefore, although definitive evidence of the bioactivity of milk-borne porcine RLX remains to be generated, data indicate that intact, immunoreactive RLX is present in both porcine milk and the neonatal circulation. In this light, we hypothesize a maternally driven lactocrine mechanism in which milk-borne RLX, absorbed into the neonatal circulation shortly after birth, acts directly through its own receptor, LGR7, and/or indirectly through the developing uterine ER system, to affect critical uterine programming events that can have long-term consequences for uterine function and reproductive efficiency (Fig. 7Go).


Figure 7
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  		FIG. 7. Schematic presentation of the putative role of milk-borne RLX in neonatal FRT development. Exposure of neonatal pigs to milk-borne relaxin (PND 0–1) may contribute to the organizational program necessary to ensure optimal development of uterine tissues in the pig.

 
In addition to targeting the female reproductive tract, RLX can have diverse actions in nonreproductive tissues (58). RLX receptor transcripts have been identified in immature rat (23), adult mouse (17), and human (22) tissues in males and females including brain, heart, kidney, lung, and liver. This evidence supports the possibility that milk-borne RLX may have a functional role in neonatal development of other vital organs and tissues in pigs of both sexes. In this context, the RLX-dependent lactocrine mechanism envisioned here may have broad developmental and somatotrophic implications.

In summary, evidence was obtained to indicate that 1) neonatal porcine uterine expression of the cognate RLX receptor, LGR7, as well as the INSL3 receptor, LGR8, is detectable at birth, is regulated developmentally during an established critical period for estrogen-sensitive uterine programming, and is stimulated by estrogen exposure during this period; 2) porcine milk contains RLX and, therefore, can serve as a natural source of this peptide hormone; and 3) RLX is detectable in the peripheral circulation of neonatal pigs within 48 h of birth only if they are allowed to nurse. Taken together with data indicating that neonatal uterine tissues are sensitive to exogenous RLX from the time of birth (4), data can be interpreted to indicate that maternal contributions to the neonatal hormonal milieu may contribute to conditions necessary to ensure that an optimal uterine developmental program is realized.


   Acknowledgments
 
We thank Dr. Richard Ivell, University of Adelaide, for kindly providing the rabbit-antihuman LGR7 antibody (L7–3) and Dr. Anthony G. Moss of the Department of Biological Sciences at Auburn University for assistance with photomicrography.


   Footnotes
 
This work was supported by USDA-NRI-2003-35203-13572 (to F.F.B. and C.A.B.), NSF EPS-0447675 (F.F.B.), and National Health and Medical Research Council 300012 (to R.A.D.B.).

Disclosure statement: All authors have nothing to declare.

First Published Online June 1, 2006

Abbreviations: ER, Estrogen receptor-{alpha}; EV, estradiol valerate; FRT, female reproductive tract; INSL3, insulin-like factor 3; ISH, in situ hybridization; LSM, least-squares means; PND, postnatal day; RLX, relaxin; TBS, Tris-buffered saline.

Received March 28, 2006.

Accepted for publication May 24, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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W. Yan, J. Chen, A. A Wiley, B. D Crean-Harris, F. F Bartol, and C. A Bagnell
Relaxin (RLX) and estrogen affect estrogen receptor {alpha}, vascular endothelial growth factor, and RLX receptor expression in the neonatal porcine uterus and cervix
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