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.

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

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.

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.

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.

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.

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.

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.

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'.

Relaxin (RLX) and estrogen affect estrogen receptor alpha, vascular endothelial growth factor, and RLX receptor expression in the neonatal porcine uterus and cervix.

The porcine female reproductive tract undergoes estrogen receptor (ER) alpha-dependent development after birth (postnatal day=PND 0), the course of which can determine adult uterine function. Uterotrophic effects of relaxin (RLX) in the porcine neonate are age specific and may involve ER activation. Here, objectives were to determine effects of RLX and estrogen administered from birth on uterine and cervical growth and expression of ERalpha, vascular endothelial growth factor (VEGF), and the RLX receptor (RXFP1). On PND 0, gilts were treated with the antiestrogen ICI 182 780 (ICI) or vehicle alone and, 2 h later, were given estradiol-17beta (E) or porcine RLX for 2 days. Neither RLX nor E affected uterine wet weight or protein content on PND 2. However, RLX, but not E, increased cervical wet weight and protein content when compared with controls. Pretreatment with ICI did not inhibit RLX-stimulated cervical growth. Uterine and cervical ERalpha increased in response to RLX, but not E. Both RLX and E increased VEGF in the uterus and cervix on PND 2. Pretreatment with ICI increased VEGF in both tissues and increased RLX-induced cervical VEGF. In the uterus E, but not RLX, increased RXFP1 mRNA. In the cervix, E increased RXFP1 gene expression whereas RLX decreased it. Results indicate that the neonatal uterus and cervix are sensitive to E and RLX and that growth responses to RLX in these tissues differ by PND 2. Effects of RLX on uterine and cervical ERalpha and VEGF expression may be important for neonatal reproductive tract development.

Relaxin-induced matrix metalloproteinase-9 expression is associated with activation of the NF-kappaB pathway in human THP-1 cells.

Matrix metalloproteinases (MMPs) and relaxin (RLX) are reported to play an important role in tissue remodeling and wound repair. When macrophages populate wound sites, they secrete biologically active substances, including MMPs. The transcription factor NF-kappaB is important in MMP gene regulation in macrophage cells. Thus, a monocyte/macrophage cell line, THP-1, was used to study the molecular mechanism of RLX action on MMP-2 and MMP-9 expression. After 24 h incubation with porcine RLX (100 ng/ml), conditioned media (CM) and THP-1 cells were collected. Gelatin zymography demonstrated an increase in pro-MMP-9 activity in response to RLX in CM, and no significant change in pro-MMP-2 expression was observed. Immunoblot analysis also revealed an increase in pro-MMP-9 in CM from RLX-treated THP-1 cells. Gel EMSA showed that NF-kappaB DNA-binding activity was elevated in THP-1 cells treated with RLX for 10 min and reached a peak at 30 min. The NF-kappaB DNA complex was supershifted using antibodies against NF-kappaB subunits p50 and p65. Increased expression of the p50 and p65 NF-kappaB subunits was also detected in THP-1 cells after RLX treatment. Incubation with RLX (90 min) reduced THP-1 expression of the NF-kappaB inhibitor protein, IkappaB-alpha. Using a specific NF-kappaB inhibitor, pyrrolidine dithiocarmate (PDTC) inhibited nuclear binding of NF-kappaB. Pre-exposure to PDTC suppressed pro-MMP-9 activity and protein levels in RLX-treated THP-1 cells. In conclusion, these data suggest that RLX-induced tissue remodeling through increasing MMP-9 expression is dependent on NF-kappaB activation.

Neonatal porcine endometrial development and epithelial proliferation affected by age and exposure to estrogen and relaxin.

In the pig, temporospatially regulated proliferation of uterine luminal (LE) and glandular (GE) epithelium between birth (postnatal day=PND 0) and PND 15 is essential for success of endometrial development. Exposure of gilts to estrogen (E) or relaxin (RLX) during this period disrupts uterine development, and neonatal E exposure can compromise adult uterine function. Neonatal uterotrophic effects of E and RLX, administered for 2 days beginning on PND 12, can be inhibited with the antiestrogen ICI 182,780 (ICI) indicating crosstalk between RLX and E signaling systems. Here, objectives were to determine effects of: (study 1) neonatal age and (study 2) exposure to E, RLX, and ICI on porcine neonatal uterine histoarchitecture and patterns of epithelial cell proliferation as reflected by proliferating cell nuclear antigen labeling index. In study 1, uteri were obtained on PND 0, 3, 6, 9, 12 and 15. Glandular epithelium, absent at birth, was observed by PND 3. Overall, epithelial labeling index increased from birth to PND 3, declined from PND 6-9 in LE and GE, and increased to PND 15 in GE. In study 2, uteri were collected on PND 14 after administration of vehicle, E, or RLX for 2 days, or following pretreatment with ICI. Alone, E was uterotrophic and adenogenic and increased labeling index in both LE and GE. Both RLX and ICI increased proliferation in GE. Effects of E and RLX were attenuated by ICI, providing further support for crosstalk between these signaling systems in the developing neonatal porcine endometrium.

Expression of LGR7 and LGR8 by neonatal porcine uterine tissues and transmission of milk-borne relaxin into the neonatal circulation by suckling.

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 microg/kg body weight x 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.

Production and characterization of recombinant equine prorelaxin.

Relaxin is a peptide hormone produced by a wide variety of mammals. In the horse, the placenta is the major source of relaxin. Since pure equine relaxin is difficult to obtain to study its role in the pregnant mare, the objectives of this study were to produce recombinant equine prorelaxin and characterize its immunological and biological activity. First, an equine relaxin gene cassette was transfected into immortalized bovine mammary epithelial (MAC-T) cells. Second, immunological activity of media conditioned by transfected MAC-T cells was tested by Western blotting and quantified using a homologous equine radioimmunoassay. Finally, bioactivity of the conditioned media was tested using the human monocyte cell line, THP-1, which exhibits a rapid and dose-dependent increase in the accumulation of cAMP upon binding relaxin. The results showed that conditioned media, concentrated 5x, yielded 4.11 +/- 0.81 ng/ml recombinant equine prorelaxin. In addition, a 19 kDa immunoreactive band, corresponding to the expected size of equine prorelaxin, was visualized by SDS-PAGE. THP-1 cells incubated with conditioned media (5x) from transfected cells, in the presence of forskolin (1 microM) and isobutylmethylxanthine (50 microM), showed an increase in cAMP production over media from mock-transfected cells alone. In conclusion, recombinant equine prorelaxin secreted by MAC-T cells was both immunologically and biologically active. This study demonstrates the first attempt to produce recombinant equine prorelaxin, important for further study of the role of relaxin in the mare.

Relaxin induces matrix metalloproteinase-9 through activation of nuclear factor kappa B in human THP-1 cells.

Matrix metalloproteinase (MMP) and relaxin are important for tissue remodeling and wound repair. Macrophages populate wound sites and secrete MMPs. Nuclear factor kappa B (NF-kappaB) is linked to MMP gene regulation. Thus, a monocyte/macrophage cell line, THP-1, was used to study the mechanism of relaxin's action on MMPs. Relaxin increased MMP-9 protein and activity in THP-1 cell-conditioned media, with no significant change in MMP-2 activity. NF-kappaB DNA binding activity was elevated in response to relaxin, and supershift assay showed activation of both NF-kappaB subunits p50 and p65. Relaxin also reduced NF-kappaB inhibitor protein, IkappaB-alpha. In conclusion, these data suggest that relaxin-induced MMP-9 expression in THP-1 cells involves NF-kappaB activation.

Tissue-specific effects of relaxin on the reproductive tract of neonatal gilts.

In the pig, relaxin treatment for 2 d from birth (postnatal day 0, PND 0) was not uterotrophic, but it increased cervical size. To understand the mechanism underlying this tissue-specific response, relaxin receptor (LGR7) expression in the neonatal uterus and cervix was investigated. At PND 2, quantitative RT-PCR analysis showed that LGR7 levels were higher in the cervix than in the uterus. Interestingly, relaxin decreased cervical LGR7 expression when compared to that of the controls. Differential expression of LGR7 between uterine and cervical tissues may contribute to tissue-specific relaxin responsiveness in the neonatal porcine reproductive tract.

Effects of relaxin on neonatal porcine uterine growth and development.

Relaxin (RLX), a key reproductive hormone in pigs, stimulates uterine growth in pregnant and prepubertal gilts and in neonates 2 weeks after birth. The neonatal uterotrophic response to RLX is developmentally regulated and estrogen receptor dependent because RLX fails to increase uterine weight in the absence of estrogen receptor (ER)-alpha or when the ER is chemically inactivated. However, the role of RLX and insulin-like peptide-3 receptors, LGR7 and LGR8, respectively, in the neonatal uterotrophic response is unknown. Current studies focus on direct (LGR7/8-mediated) and indirect (ER-mediated) effects of RLX in the neonatal porcine uterus. Porcine LGR7 and LGR8 cDNAs were cloned and used as probes to identify uterine transcripts for LGR7 and LGR8, which increased from birth (postnatal day [PND] 0) to PND 14, a critical period for porcine uterine development. In situ hybridization showed that endometrial signals for both LGR7 and LGR8 are predominantly stromal during this period. Administration of RLX on PND 0, before onset of uterine ER expression, increased uterine luminal epithelial height (P < .05) but not uterine weight in the LGR7/8-positive uterus on PND 2. However, RLX increased both uterine weight and luminal epithelial height by PND 14 (P < .05), after overt endometrial ER expression. Aberrant ER activation between PND 0 and 14 alters the uterine organizational program and affects the function of adult porcine uterine tissues. Present data suggest that crosstalk between LGR7/8 and ER may be involved in estrogen-sensitive morphoregulatory events that are central to the development of an optimally functional adult uterus in the pig.

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.