Progress 09/01/03 to 08/31/06
Outputs
Results are summarized for each Specific Aim. Aim 1a: Determine effects of age from birth (PND 0) through PND 15 on porcine uterine mRNA expression patterns for the tissue patterning genes Wnt7a, Wnt4, Wnt5a, Hoxa10 and Hoxa11, the relaxin (RLX) receptors RXFP1 and RXFP2, and ER-alpha (ER). - Results showed that: (1) the neonatal porcine endometrium is ER-negative and, Wnt7a-, Wnt4-, Wnt5a-, Hoxa10, Hoxa11-, RXFP1- and RXFP2- positive at birth; (2) temporospatial expression patterns of these genes are developmentally regulated; and (3) the neonatal porcine endometrium undergoes a morphogenetic transition from an infantile, quiescence state at birth to a morphogenetically active state by PND 3, when nascent, ER-positive glandular epithelium is proliferating rapidly. Aim 1b: Determine effects of estradiol-17beta valerate (EV), administered from birth (PND 0-14), on expression patterns of specific elements of the uterine morphoregulatory transcriptome/proteome identified
in Aim 1a, on PND 15. - Administration of EV for two weeks from birth increased uterine, cervical and vaginal weight when compared to controls at PND 14. Additionally, EV exposure altered morphoregulatory gene expression and markers of RLX action. Uterine Wnt7a gene expression decreased on PND 14 in response to EV, whereas Hoxa10 signal increased. Data were corroborated by in situ hybridization evidence indicating that chronic EV exposure from birth reduced Wnt7a signal in luminal epithelium and Wnt-4 in stroma, but increased stromal Hoxa10 signal. While there was no effect of EV on MMP-2 expression, uterine LGR7 expression increased on PND 14 in response to EV as evaluated by both ISH and quantitative RT-PCR. Aim 2a: Determine effects of RLX, administered (i.m.) to gilts for two days from birth, on specific uterine growth responses and markers of connective tissue remodeling and differentiation in naturally ER-negative/RXFP1-positive uterine tissues on PND 2. - Results showed that
uterine ER and vascular endothelial growth factor expression increased, while LGR7 expression was unaffected on PND 2 following RLX administration from birth. Aim 2b: Determine effects of RLX and E, in the presence or absence of ICI 182,780 (ICI), on uterine growth and development, connective tissue remodeling and markers of differentiation in naturally ER-positive/LGR7-positive neonatal uterine tissues at PND 14. - Results indicate that the developing uterine Hoxa/Wnt expression axis is sensitive to disruption by estrogen exposure. Neonatal uterine epithelial proliferation increased after administration of either E or RLX in week two of neonatal life and effects were inhibited by ICI 182, 780 in ER-positive cells. Data support the hypothesis that ER activation between birth and PND 15 affects the porcine uterine developmental program and that RLX affects organizational processes, in part, through cross-talk with the ER signaling system. Identification of colostrum as a source of RLX
in the neonatal environment provided the first evidence for a maternally-driven lactocrine mechanism by which RLX can reach the porcine neonatal circulation within 48 h of birth.
Impacts
Data generated through this project indicate that the essential elements of a maternally lactocrine driven mechanism for relaxin-mediated programming of the porcine uterus during the early postnatal period are in place. Included is evidence that: (1) the window of opportunity for postnatal maternal programming of uterine tissues is open during the first three days of neonatal life as a critical morphoregulatory gene expression axis is developing; (2) neonatal uterine and cervical tissues are RXFP1-positive at birth, prior to onset of uterine ER expression; (3) exogenous porcine relaxin has trophic effects on uterine and cervical tissues that are evident by PND 2 when exposure begins at birth; (4) relaxin administered from birth induces ER expression in developing uterine and cervical tissues by PND 2; (5) a natural source of relaxin is found in porcine milk, where relaxin concentrations are highest within 48h of parturition; and (6) relaxin is not detectable in the
systemic circulation of newborn pigs prior to nursing or in neonatal pigs fed hormone-free milk replacer, but is detectable at serum concentrations approaching 200 pg/ml on PND 0-1 in neonatal pigs allowed to nurse. Using uterine data as a reference, observations suggest that relaxin, delivered via a lactocrine mechanism, is available to support levels of ER expression required to insure that critical estrogen-sensitive, ER-dependent elements of the postnatal organizational program are optimized. Data provide a new perspective on perinatal factors affecting reproductive development in swine and the potential for maternal factors to affect patterns of development in neonatal mammals.
Publications
- Bartol FF, Wiley AA, Bagnell CA. 2006. Uterine development and endometrial programming. In: (Ashworth CJ, Kraeling RR, eds) Control of Pig Reproduction VII. Reproduction Supplement 62:113-130.
- Masters RA, Crean BD, Yan W, Moss A, Wiley AA, Bagnell CA, Bartol FF. 2006. Neonatal porcine endometrial development and epithelial proliferation affected by age and exposure to estrogen and relaxin. Domestic Animal Endocrinology. 33:335-346.
- Yan W, Wiley AA, Bathgate RAD, Frankshun AL, Lasano S, Crean BD, Steinetz BG, Bagnell CA, Bartol FF. 2006. Expression of LGR7 and LGR8 by neonatal porcine uterine tissues and transmission of milk-borne relaxin into the neonatal circulation by suckling. Endocrinology 147:4303-10.
- Yan W, Ryan PL, Bartol FF, Bagnell CA. 2006. Uterotrophic effects of relaxin related to age and estrogen receptor activation in neonatal pigs. Reproduction 131:943-950.
- Yan W, Wiley, AA, Bartol FF and Bagnell CA. 2005. Tissue-specific effects of relaxin on the reproductive tract of neonatal gilts. Ann N Y Acad Sci. 1041:132-5.
- Bagnell CA, Yan W, Wiley AA, Bartol FF. 2005. Effects of relaxin on neonatal porcine uterine growth and development. Ann N Y Acad Sci. 1041:248-55.
- Ho TY and Bagnell CA. 2005. Relaxin induces matrix metalloproteinase-9 through activation of nuclear factor kappa B in human THP-1 cells. Ann N Y Acad Sci. 1041:314-6.
- Chen J, Frankshun A-L, Wiley AA, Ho TH, Bartol FF, Bagnell CA. 2007. Estrogen exposure from birth affects uterine gene expression in neonatal gilts that persists in pregnant adults. Biology of Reproduction 2007. (Special Issue) 152.
- Crean BD, Masters RA, Yan W, Wiley AA, Bagnell CA, Bartol FF. 2006. Porcine uterine expression of Msx1 and Msx2: Effects of relaxin, estrogen and ICI 182,780 in the neonatal endometrium. Biology of Reproduction. (Special Issue) 92.
- Ho TY, Yan W, Wiley AA, Bartol FF, Bagnell CA. 2005 Evolution of the Hoxa/Wnt axis and estrogen regulation of Wnt7a expression in the neonatal porcine uterus. Biology of Reproduction. (Special Issue) 138.
- Masters RA, Crean BD, Yan W, Wiley AA, Bagnell CA, Bartol FF. 2005. Distribution of Active beta-catenin in neonatal porcine uterine tissues affected by age and exposure to estrogen and relaxin. Biology of Reproduction. (Special Issue) 132.
- Yan W, Lasano S, Steinetz B, Bartol FF, Bagnell CA. 2005. Presence of relaxin in the milk of lactating sows and its transmission to neonatal pigs via suckling. Biology of Reproduction.Special Issue: 94.
- Bartol FF, Crean BD, Wiley AA. Coordinate expression of Indian Hedgehog and the Hedgehog receptor Patched in neonatal and adult porcine uterine tissues. 2004. Biology of Reproduction. 2004 (Special Issue) 278.
- Crean BD, Wiley AA, Bartol FF. Transient neonatal estrogen exposure from birth affects the adult porcine endometrial proteome on Day 12 of pregnancy. 2004. Biology of Reproduction. (Special Issue) 158.
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Progress 09/01/03 to 12/31/03
Outputs
The central hypothesis of ongoing research is that gene products for which uterine expression is altered during the first weeks of neonatal life by factors that affect estrogen receptor-alpha (ER) activation, either directly or indirectly via cross-talk with peptide receptor systems, are likely to have mechanistic roles in organizational processes that determine uterine function in adult gilts. Aim 1 objectives are to determine effects of age from birth (PND 0) through PND 15 (a critical period for porcine uterine development) on uterine mRNA expression patterns for targeted morphoregulatory genes, as well as relaxin (Rlx) family receptors (LGR7 and LGR8) and ER in normal tissues. Additionally, effects of estradiol-17beta valerate (EV), a developmentally disruptive agent, on the uterine transcriptome and proteome will be evaluated at the end of the second week of neonatal life following exposure of gilts to EV from birth. Tissue collection for Aim 1(a) began in Fall,
2003. New data indicate that Indian hedgehog (Ihh), a secreted morphoregulatory protein that acts via the patched (Ptc) receptor to affect developmentally critical cell behaviors, could be involved in uterine tissue patterning and function. Using total uterine RNA from PND 14, a 221 bp porcine cDNA that displayed 94% sequence identity with the human Ihh transcript was generated by RT-PCR, cloned and the sequence submitted to the NCBI (Accession No.: CK172438). In situ localization of Ihh mRNA using a [35S]-UTP-labeled antisense cRNA probe, as well as immunohistochemical localization of Ihh and Ptc proteins in neonatal uterine tissues from PND 0, 7 and 14 revealed both Ihh and Ptc expression that increased from birth and was predominant in luminal and glandular epithelium by PND 14. Signal was also detected in endometrial stroma. Data constitute the first evidence of Ihh and Ptc expression in the porcine uterus and indicate that organization of this important morphoregulatory system is
regulated developmentally in the pig. These efforts add the Ihh/Ptc system to the list of primary morphoregulatory genes, including Hoxa-10 and Hoxa-11, as well as Wnt4, Wnt5a and Wnt7a that were shown by this laboratory to be involved in normal and EV-disrupted uterine organizational events in early postnatal life. Other technical accomplishments include establishment of image analysis procedures for evaluation of the uterine proteome, initiation of efforts to validate quantitative RT-PCR procedures for LGR7 and LGR8, and development of procedures for DNA microarray analysis of the neonatal uterine transcriptome. Initial microarray analyses will employ a new porcine array generated from normalized uterine and hypothalamic cDNA libraries by a collaborator at the University of Illinois, Urbana-Champaign. This array contains approximately 5100 unique clones. Preliminary microarray data, in which the PND 14 uterine transcriptome of gilts exposed to EV from birth was compared with the
normal (control) transcriptome, indicate substantial EV-induced alterations in gene expression patterns, details of which are now being characterized.
Impacts
Data now being generated through this project will provide the first systematic evalution of normal and aberrant expression patterns for morphoregulatory genes and signaling systems that regulate development and functional programming of uterine tissues in the pig.
Publications
- Bartol FF, Crean BD, Thacker SS, Wiley AA. 2003. NCBI Submission. pIHH Sus scrofa neonatal uterus RT-PCR Sus scrofa cDNA similar to Indian Hedgehog, MRNA sequence gi 39026781 gb CK172438.1 [39026781]
- Bartol FF, Crean BD, Wiley AA. 2004. Uterine patterning, endometrial programming and reproductive performance. Proceedings of the Annual Conference of the Society for Theriogenology/American College of Theriogenology. In Press [Invited Review].
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