Ronald K. Christenson

A Chemokine, Interferon (IFN)-γ-Inducible Protein 10 kDa, Is Stimulated by IFN-τ and Recruits Immune Cells in the Ovine Endometrium

Abstract Proper distribution of immune cells in the uterus is a prerequisite for successful implantation and subsequent placentation, but biochemical signals that govern such events have not been well characterized. In the present study, the cDNA of a chemokine, interferon (IFN)-γ-inducible protein 10 kDa (IP-10), was identified from a cDNA subtraction study between uterine endometrial tissues from Day 17 pregnant and Day 15 cyclic ewes. The effect of IFN-τ on IP-10 expression and the involvement of IP-10 in the recruitment of immune cells were then investigated. Northern blot analysis revealed that large amounts of IP-10 mRNA were present during conceptus attachment to maternal endometrium and early placentation. IP-10 mRNA was localized to monocytes distributed in the subepithelial stroma of pregnant but not cyclic uteri. This finding was supported by the discovery of IP-10 mRNA expression in monocytes but not in lymphocytes, uterine epithelial cells, or stromal cells. Moreover, the expression of IP-10 mRNA by the monocytes was stimulated by IFN-α, IFN-γ, and IFN-τ in a dose-dependent manner, but the expression of IP-10 mRNA by the endometrial explants was most stimulated by IFN-τ. In a chemotaxis assay, migration of peripheral blood mononuclear cells was stimulated by the addition of IFN-τ stimulated-endometrial culture medium, and the effect was significantly reduced by neutralization with an anti-IP-10 antibody. These results suggest that endometrial IP-10 regulated by conceptus IFN-τ regulates recruitment and/or distribution of immune cells seen in the early pregnant uterus.

Effect of Progesterone, Mifepristone, and Estrogen Treatment During Early Pregnancy on Conceptus Development and Uterine Capacity in Swine

Abstract A series of experiments was performed to investigate the influence of progesterone at Days 2 and 3 of pregnancy on conceptus development and uterine capacity. In experiment 1, unilaterally hysterectomized-ovariectomized (UHO) white crossbred gilts were given no treatment, estradiol valerate (5 mg given on Days 11 and 12), or progesterone (200 mg/day on Days 2 and 3 after mating). On Day 105 of pregnancy, each fetus and its associated placenta were weighed, and the number of live and dead fetuses was recorded for each litter. Early progesterone treatment reduced (P < 0.05) litter size (a measure of uterine capacity in UHO gilts). In experiment 2, intact white crossbred gilts were mated, given no treatment or progesterone treatment on Days 2 and 3 of pregnancy, and farrowed. Progesterone treatment decreased (P < 0.05) pregnancy rates. In pregnant gilts, progesterone had no effect on the number of live or stillborn piglets at birth, and gestation length was decreased (P < 0.05). Progesterone treatment did not affect the number of large or small piglets. In experiment 3, intact gilts were mated at estrus and then received 1) no treatment or treatment with 2) 100 mg, 3) 200 mg, or 4) 400 mg mifepristone (also known as RU486) on Day 2 of pregnancy. On Day 11 of pregnancy, both uterine horns were flushed, the number and diameter of each conceptus was recorded, and the flushed material was assayed for total protein and acid phosphatase. The 400 mg mifepristone treatment decreased conceptus diameter (P < 0.05) and total protein (P = 0.06) in the uterine flushings. In experiment 4, UHO gilts were mated at estrus, injected with either corn oil (control) or mifepristone (400 mg) on Day 2 of pregnancy, and killed on Day 105 of pregnancy, and the number and weight of live fetuses and placentas was recorded. In contrast to the effect of progesterone treatment, mifepristone decreased uterine capacity by decreasing the number of small conceptuses. These data suggest that progesterone concentrations on Days 2 and 3 of pregnancy in swine influence the rate of conceptus development during early pregnancy and uterine capacity during later pregnancy.

Changes in Immune Cell Distribution and IL‐10 Production are Regulated through Endometrial IP‐10 Expression in the Goat Uterus

Problem:  Changes in distribution or redistribution of immune cells are required for the establishment and maintenance of pregnancy, but these changes during early pregnancy have been poorly understood in the ruminant ungulates. Expression of a chemokine, interferon‐γ (IFN‐γ)‐inducible protein 10 kDa (IP‐10, CXCL10), was identified in the endometrium of pregnant goats. Population and/or distribution of endometrial immune cells and their cytokine productions could be regulated by IP‐10 during the period of pregnancy establishment.

Characterization of uterine epidermal growth factor during early pregnancy in pigs

Genomic research has identified a quantitative trait locus for uterine capacity, a component trait contributing to litter size, on porcine chromosome 8. The epidermal growth factor (EGF) gene, on porcine chromosome 8, may influence uterine capacity because of its growth-promoting activities. Using reverse transcription-polymerase chain reaction (PCR) and iterative screening of a porcine reproductive tissue cDNA library, 4932 bp cDNA sequence coding for porcine EGF precursor was obtained. The predicted protein sequence of the EGF precursor contained 1214 amino acids, similar to human EGF precursor (1207 amino acids, 81% identity). Curiously, the sequence of the mature peptide was less homologous between species than other regions of EGF precursor. The presence of conserved regions outside the mature peptide may suggest that these regions are functionally important. Expression of EGF mRNA in the endometrium of White crossbred gilts (n = 3 to 5 each) was determined by Northern blotting using 20 microg of total RNA from endometrium of D 10, 13, and 15 cyclic, and D 10, 13, 15, 20, 30, and 40 of pregnant gilts. A 3342 bp probe from EGF precursor was used. The bands corresponding to EGF mRNA were quantified by densitometry and results were analyzed by ANOVA. EGF mRNA expression decreased significantly from D 13 to 15 of the cycle and pregnancy (P = 0.04), and from D 30 to 40 of pregnancy (P = 0.01). These findings show that EGF mRNA expression is temporally regulated during the cycle and early pregnancy, and this pattern of gene expression may be important during early conceptus development.

11β-Hydroxysteroid Dehydrogenase and Glucocorticoid Receptor Messenger RNA Expression in Porcine Placentae: Effects of Stage of Gestation, Breed, and Uterine Environment

Abstract Glucocorticoids are known to influence many aspects of prenatal development. Three important regulators of glucocorticoid actions at the cellular level are the enzymes 11β-hydroxysteroid dehydrogenase type 1 (11βHSD-1), 11β-hydroxysteroid dehydrogenase type 2 (11βHSD-2), and glucocorticoid receptors (GR). The present study was conducted to determine the presence of these regulators in porcine placentae during early gestation (Days 24–40; term = 114 days) and to examine the influence of breed and uterine environment. Three pig models differing in uterine environment as reflected by embryonic survival from Days 24 to 40 were used: intact white cross-bred gilts (WC-INT); white cross-bred gilts that had been unilaterally hysterectomized-ovariectomized before puberty (WC-UHO); and intact Meishan gilts (ME). Porcine-specific partial cDNAs for 11βHSD-1 and 11βHSD-2 and a cRNA for GRα were developed and used to produce 32P-labeled probes for Northern blot analyses. The 11βHSD dehydrogenase activity was measured in vitro at saturating concentrations of substrate and coenzyme. At Day 24 of gestation, 11βHSD-2 mRNA, dehydrogenase activity, and GR mRNA were present, but 11βHSD-1 mRNA was absent. All three mRNAs and dehydrogenase activity increased (P < 0.01) by Day 40. On Day 30, placental 11βHSD-2 mRNA was decreased (P = 0.03) by 47% in WC-UHO versus WC-INT. Placental 11βHSD dehydrogenase activity was 2-fold greater (P < 0.01) in ME versus WC-INT on Day 24 of gestation. These results demonstrate, to our knowledge for the first time, the presence of 11βHSD-1, 11βHSD-2, and GR mRNA as well as 11βHSD dehydrogenase activity in the porcine placenta during early pregnancy. Moreover, a role for glucocorticoids in porcine embryonic development is suggested.

Regulation of endometrial granulocyte macrophage-colony stimulating factor (GM-CSF) in the ewe

Granulocyte macrophage-colony stimulating factor (GM-CSF) increases ovine interferon-tau (oIFNtau) secretion by ovine conceptuses, but endometrial production of GM-CSF has not been characterized. Endometrial GM-CSF expression was evaluated in ovariectomized ewes implanted with estradiol-17beta (E(2)) and/or progesterone (P(4)) for 14 days, in day 14 cyclic and day 14 pregnant ewes. Relative levels of endometrial GM-CSF mRNA were 3-fold higher in E(2)- and E(2)/P(4)-treated ewes than that of control or P(4)-treated ovariectomized ewes. Levels of endometrial GM-CSF mRNA for cyclic ewes were similar to E(2)- and E(2)/P(4)-treated ewes, but amounts of GM-CSF mRNA in pregnant ewes were 2-fold higher. GM-CSF concentrations in endometrial culture media, determined by GM-CSF bioassay, for cyclic and E(2)/P(4)-treated ovariectomized ewes were 3-fold higher than those of control, E(2)- and P(4)-treated ovariectomized ewes; however, amounts of GM-CSF in pregnant ewes were 2-fold higher. Immunoreactive GM-CSF, examined by western blot, was detected in the culture medium from E(2)/P(4)-treated ovariectomized, cyclic and pregnant ewes. Luminal and glandular epithelia and stromal regions were determined to be sites of GM-CSF expression by immunohistochemistry and in situ hybridization techniques. Data indicate that combined E(2) and P(4) treatment of ovariectomized ewes is sufficient to restore GM-CSF expression to the level found in cyclic ewes; however, GM-CSF mRNA and protein in pregnant ewes is 2-fold greater than in ovariectomized or cyclic ewes. These data suggest that the conceptus, in addition to steroids, may play a role in the regulation of endometrial production of GM-CSF.

Characterization of porcine uterine estrogen sulfotransferase

A quantitative trait locus (QTL) for uterine capacity is located on chromosome 8. Comparison of porcine and human genetic maps suggested that the estrogen sulfotransferase (STE) gene may be located near this region. The objectives of this study were to clone the full coding region for STE, compare endometrial STE gene expression between Meishan and White composite pigs during early pregnancy, and map the STE gene. We obtained a clone (1886 bp) containing the full coding region of STE by iterative screening of an expressed sequence tag library. Endometrial STE mRNA expression in White composite gilts was determined by Northern blotting on days 10, 13, and 15 of the estrous cycle; and on days 10, 13, 15, 20, 30, and 40 of pregnancy. STE mRNA expression was elevated (P < 0.01) on days 20 and 30 of pregnancy compared to other days of the cycle or pregnancy. Endometrial STE mRNA expression during early pregnancy, determined using real-time RT-PCR, was elevated (P < 0.01) on day 20 compared to day 15, decreased (P = 0.02) between days 20 and 30, and decreased further (P < 0.01) between days 30 and 40 in both Meishan and White composite pigs. Expression of STE mRNA was greater (P = 0.01) in White composite pigs compared to Meishan pigs. Using a microsatellite from an STE containing BAC genomic clone, the STE gene was mapped to 65 centimorgans on chromosome 8. Because STE mRNA expression differs between Meishan and White composite pigs, the STE gene may be a candidate for the uterine capacity QTL.

Erythropoietin mRNA expression in pig embryos

To address whether altered erythropoietin (EPO) synthesis might be involved in prenatal pig mortality, studies were conducted to measure porcine embryonic EPO mRNA expression during early gestation (days 24-40). Three pig models differing in embryonic survival from days 24-40 were investigated: intact white crossbred gilts (INT), white crossbred gilts that were unilaterally hysterectomized-ovariectomized before puberty and whose pregnant uterus constituted a crowded environment (UHO), and prolific, intact Meishan gilts (ME). A partial cDNA for porcine EPO, developed via reverse transcription and polymerase chain reaction procedures was used to generate a 32P-labeled probe for use in Northern analyses. In an initial study, embryonic liver EPO mRNA was greatest on day 24, decreased by day 30 (P<0.01), and was barely detectable by day 40. EPO mRNA expression was not influenced by pig model. Placental EPO mRNA expression was detectable in only 4 of 53 placentae examined. In a second study at day 35 of gestation, embryonic liver EPO mRNA expression was measured in the same three pig models and in two embryos of divergent weights from each gilt. Meishan embryos had lower (P<0.01) plasma immunoassayable EPO concentrations (P=0.04) and higher survival rates (87+/-2.7%) at day 35 than did white crossbred embryos (75+/-5%). Liver EPO mRNA expression did not differ among animal models, nor did plasma EPO or tissue EPO mRNA expression differ between large and small embryos. There was no apparent relationship between embryonic development, measured as embryonic and placental size, and plasma EPO concentrations or liver EPO mRNA expression. These results indicate that at the gestational ages examined, the embryonic liver is one source of plasma erythropoietin and suggest that at the ages sampled, EPO is not a limiting factor in embryonic development.

CORTISOL CONCENTRATIONS IN EARLY PORCINE EMBRYOS

A study was conducted to determine the presence of cortisol in body tissue of porcine embryos at days 25 and 35 of gestation. Cortisol concentrations (ng/mg DNA) were low but measurable at day 25 and increased eightfold by day 35 during a time when body weight increased 6.4-fold. At day 35, there was a highly significant positive linear regression of body weight on cortisol concentrations. The source of this embryonic cortisol is not known, but its presence suggests the opportunity for cortisol to influence porcine embryonic development at these early gestational stages.

Hemopoietic Cytokine Regulation of Trophoblast Interferon, Ovine Trophoblast Protein-1

In early pregnancy, local signals derived from the uterine epithelium regulate extensive differentiation and reorganization of stroma cells. However, molecular mechanisms associated with rapid cellular reorganization of the uterus, particularly biochemical events associated with uterine receptivity, are poorly understood. Only a number of potential mediators that are involved in embryo-maternal, and in inter-uterine cellular communication have been identified, but their physiological significance in vivo remains to be determined. Recently, it has become apparent that lympho-hemopoietic growth factors derived from maternal endometrial cells are required for proper placental cell growth and differentiation.