Changes in the temporal and spatial expression of H beta 58 during formation and maturation of the chorioallantoic placenta in the Rat

Cloning and sequencing of a cDNA amplified by RNA fingerprinting at the implantation site of pregnant rats revealed 80% similarity with H beta 58, previously shown to be essential for formation of the chorioallantoic placenta in the mouse. H beta 58 mRNA was detected in the endometrium of hormonally sensitized rats stimulated to undergo decidualization and in the contralateral uterine horns lacking a decidual stimulus, indicating that uterine expression of H beta 58 mRNA did not require decidualization or the presence of a blastocyst. Immunodetection in the early postimplantation uterus (Days 6-8 of pregnancy) showed H beta 58 localized in the luminal and glandular epithelia and some stromal cells. Decidual cells at Day 6 of pregnancy expressed H beta 58, and by Day 9 of pregnancy, the protein localized throughout the maternal decidua. The temporal and spatial distribution of H beta 58 in the developing chorioallantoic placenta was assessed at Days 10, 12, and 14 of pregnancy. Immunoreactive H beta 58 localized to erythroid cells within the developing fetal vasculature of the chorioallantoic primordia at Day 10 of pregnancy. By Day 12, the fetal vasculature extended into the placental labyrinth, and the erythroid stem cells continued to strongly express H beta 58. At Day 14 of pregnancy, immunoreactivity became evident in the trophoblast giant cells and syncytiotrophoblast of the fetal placenta. As the chorioallantoic placenta matured (Day 18), H beta 58 mRNA was 3.6-fold higher in the labyrinth compared with the junctional region. Stable cell lines (HRP/LRP) isolated from the rat labyrinthine placenta expressed H beta 58 mRNA and protein. The expression pattern of H beta maternal and fetal placental tissues and its early expression in fetal erythroid stem cells during formation and maturation of the chorioallantoic placenta suggest that H beta 58 plays key roles in the regulatory networks that control hematopoietic development and placentation.     

Estrogen induces expression of secretory leukocyte protease inhibitor in rat uterus

In rodents, the steroid hormone estrogen (E) profoundly influences the early events in the uterus leading to embryo implantation. It is thought that E triggers the expression of a unique set of genes in the endometrium that in turn control implantation. To identify these E-induced genes, we employed a delayed implantation model system in which embryo attachment to rat endometrium is dependent upon E administration. Using a gene expression screen method, we isolated a number of cDNAs representing mRNAs whose expression is either turned on or turned off in response to an implantation-inducing dose of E. We identified one of these cDNAs as that encoding secretory leukocyte protease inhibitor (SLPI), an inhibitor of serine proteases. The expression of SLPI mRNA was induced in the uteri of ovariectomized rats in response to E, confirming the hormonal regulation of this molecule. Spatiotemporal analysis revealed a biphasic pattern of expression of SLPI mRNA during early pregnancy. A considerable amount of SLPI mRNA was detected in the uterine epithelium on Day 1 of pregnancy. The level of this mRNA, however, declined sharply on Days 2 and 3 of gestation. Interestingly, on Day 4 of gestation, there was a marked resurgence in SLPI mRNA expression in the uterine epithelium. This second burst of SLPI expression diminished by Day 6 of pregnancy. The transient induction of SLPI mRNA during Days 4 and 5 overlapped with the window of implantation in the rat. Although the precise function of SLPI in the uterus eludes us presently, its known effects as a serine protease inhibitor in other tissues and its hormone-induced expression in the rat uterus immediately preceding implantation lead us to propose that this gene plays an important role in controlling excessive proteolysis and inflammation during a critical phase of early pregnancy.     

Regulated expression of osteopontin in the peri-implantation rabbit uterus

Blastocyst attachment to the lining of the mammalian uterus during early implantation involves the initial apposition of the trophoblast to the uterine epithelial surface. Osteopontin (OPN) is a glycoprotein component of the extracellular matrix that is secreted by the glandular epithelium of mammalian uteri at the time of implantation. This protein is recognized by several members of the integrin family and promotes cell-cell attachment and adhesion. In the present study, rabbit uteri were examined using Northern and in situ hybridization to evaluate the temporal and spatial distribution of OPN mRNA during early pregnancy. Northern blot analysis demonstrated a dramatic increase in OPN expression on Days 4-7 of pregnancy, corresponding to the rise in circulating progesterone and the time of initial embryo attachment in this species. In situ hybridization analysis revealed OPN mRNA expression on Day 6.75 of pregnancy, which was most prominent on endometrial epithelium. Using immunofluorescence, OPN protein was present on the glandular epithelium on Day 6.75 of pregnancy, but was absent on blastocysts. Further, no expression of OPN mRNA or protein was found in the nonpregnant endometrium. Induction of endometrial OPN expression was observed in unmated rabbits treated with progesterone alone and was prevented by cotreatment with the antiprogestin ZK137.316. Estradiol-17beta had no effect on OPN expression by itself, and estrogen priming was not necessary to demonstrate the stimulatory effect of progesterone. In The rabbit uterus, as in other mammalian species studied, OPN is expressed in a stage-specific manner by the endometrial glands during the peri-implantation period and is regulated by progesterone.     

Regulation of expression and role of leukemia inhibitory factor and interleukin-6 in the uterus of early pregnant pigs

Cytokines, which are generally involved in the process of inflammation, may also play a critical role in conceptus implantation. We examined: (1) the expression profiles of leukemia inhibitory factor (LIF) and interleukin (IL)-6 mRNA and their protein content in the endometrium of cyclic and pregnant gilts on Days 10 to 18 after estrus; (2) the effect of conceptus-exposed medium on LIF and IL-6 synthesis in the endometrium; (3) the profiles of IL6R and LIFR mRNA expression in pig conceptuses collected on Days 10 to 18 of pregnancy; and (4) the effect of LIF and IL-6 on the attachment and proliferation of porcine trophoblast cells. The expression of LIF mRNA in the endometrium increased between Days 10 and 12 in both cyclic and pregnant gilts, and tended to be higher in Day 12 pregnant animals compared with nonpregnant ones. The LIF protein content in the uterine lumen peaked on Day 12 of pregnancy, and was higher than on Day 12 of the estrous cycle. Endometrial IL-6 mRNA expression was upregulated on Day 12 in pregnant gilts compared with nonpregnant animals. Moreover, a higher content of IL-6 protein was observed in pregnant than in cyclic gilts. The addition of conceptus-exposed medium resulted in up-regulation of LIF and IL6 mRNA expression, and increased IL-6 content in endometrial slices. In conceptuses, increased mRNA expression was detected on Days 10 to 14 for IL6R and on Day 14 for LIFR, when compared with other days studied. LIF and IL-6 stimulated the attachment and proliferation of trophoblast cells in vitro. In summary, LIF and IL-6 are important components of embryo-uterine interactions during early pregnancy in the pig, and may contribute to successful conceptus implantation.     

Implantation-associated changes in bovine uterine expression of integrins and extracellular matrix

Appropriate integrin expression appears to be necessary for successful implantation of human embryos and varies considerably among species. The present study was undertaken to determine the distributions of integrin subunits alpha(1), alpha(3), and alpha(6) as well as the extracellular matrix (ECM) components collagen IV and laminin in implanting bovine trophoblast and endometrium. Immunohistochemical staining of cryostat sections prepared from nonpregnant endometrium, of preattachment through to early villus development pregnant endometrium (Days 18, 21, 24, and 30), and of isolated trophoblast binucleate cells was performed. Trophoblast down-regulated the integrin alpha(1) subunit as attachment proceeded, whereas reactivity scores for alpha(6) antibody tended to increase from Day 18 through 24 and remained high. A subpopulation of trophoblast binucleate cells expressed the alpha(3) integrin subunit. Uterine epithelium constitutively expressed alpha(3) and alpha(6) integrin subunits, but the alpha(1) subunit was down-regulated as the luminal epithelium was modified. Collagen IV and laminin reactivity increased in the basal lamina and underlying subepithelial stroma as pregnancy proceeded. The results suggest that binucleate cell fusion with the maternal epithelium initiates integrin and ECM changes in the subepithelial stroma.     

Regulation of prostacyclin synthase expression and prostacyclin content in the pig endometrium

Prostaglandins (PGs) are critical regulators of a number of reproductive processes, including embryo development and implantation. In the present study, prostacyclin (PGI₂) synthase (PGIS) mRNA and protein expression, as well as 6-keto PGF_1α (a PGI₂ metabolite) concentration, were investigated in the pig uterus. Endometrial tissue and uterine luminal flushings were obtained on Days 4 to 18 of the estrous cycle and pregnancy. Additionally, conceptuses were collected and examined for PGIS mRNA expression and 6-keto PGF_1α concentration. Regulation of PGI₂ synthesis in the porcine endometrium by steroids, conceptus products, and cytokines was studied in vitro and/or in vivo. Endometrial PGIS protein level increased on Days 12 and 16 in pregnant but not in cyclic gilts. Moreover, higher PGIS protein expression on Day 12 of pregnancy was accompanied by a greater content of 6-keto PGF_1α in the endometrium. The concentration of 6-keto PGF_1α in uterine luminal flushings increased substantially on Days 16 and 18 in pregnant gilts and was higher than in cyclic animals. Greater PGIS mRNA expression and PGI₂ metabolite concentration were detected in Day 12 and 14 conceptuses, respectively. Incubation of endometrial explants with conceptus-conditioned medium resulted in upregulation of PGIS protein expression and increased PGI₂ secretion. Moreover, PGIS mRNA and protein expression were upregulated in the endometrium collected from gravid uterine horn on Day 14 of pregnancy. In summary, PGIS is differentially expressed in the endometrium of cyclic and pregnant gilts resulting in higher PGI₂ synthesis in pregnant animals. Porcine conceptuses are important regulators of endometrial PGIS expression and PGI₂ release during the implantation period.     

Expression of the vascular endothelial growth factor-receptor system in the porcine endometrium throughout the estrous cycle and early pregnancy

The objective of this study was to investigate the protein and mRNA expression of vascular endothelial growth factor (VEGF), VEGFR-1 (fms-like tyrosine kinase, Flt-1) and VEGFR-2 (fetal liver kinase-1/kinase insert domain-containing receptor, Flk-1/KDR) in the endometrium during the estrous cycle and early pregnancy in pigs. The VEGF-receptor system was localized in epithelial and stromal cells, blood vessels, and myometrium. Western blot analysis showed higher levels of VEGF protein during the periovulatory and periimplantation periods (P < 0.001, and P < 0.05, respectively). Constant expression of VEGF mRNA during the cycle and significant upregulation on Days 22-25 of gestation (vs. Days 9-17; P < 0.001) was observed. Stable levels of VEGFR-1 mRNA and protein were detected in the endometrium of cyclic animals. However, higher VEGFR-1 protein expression was found on Days 16-17 of the estrous cycle (P < 0.01) and Days 13-15 of gestation (P < 0.05). Protein expression of VEGFR-2 was elevated on Days 2-4 of the estrous cycle (P < 0.001), but mRNA levels were constant during the cycle. In pregnancy, VEGFR-2 protein expression started to increase after Day 15 (vs. Days 9-12; P < 0.05), but induction of VEGFR-2 mRNA expression occurred earlier on Days 13-15. It appears from the present study that the VEGF-receptor system is regulated in a temporal and spatial manner during the estrous cycle and early pregnancy in pigs. The results suggest that VEGF-A family members are probably involved in appropriate preparation of endometrium for implantation and in vascular events during implantation in pigs.     

Stanniocalcin (STC) in the endometrial glands of the ovine uterus: regulation by progesterone and placental hormones

Stanniocalcin (STC) is a hormone in fish that regulates calcium levels. Mammals have two orthologs of STC with roles in calcium and phosphate metabolism and perhaps cell differentiation. In the kidney and gut, STC regulates calcium and phosphate homeostasis. In the mouse uterus, Stc1 increases in the mesometrial decidua during implantation. These studies determined the effects of pregnancy and related hormones on STC expression in the ovine uterus. In Days 10-16 cyclic and pregnant ewes, STC1 mRNA was not detected in the uterus. Intriguingly, STC1 mRNA appeared on Day 18 of pregnancy, specifically in the endometrial glands, increased from Day 18 to Day 80, and remained abundant to Day 120 of gestation. STC1 mRNA was not detected in the placenta, whereas STC2 mRNA was detected at low abundance in conceptus trophectoderm and endometrial glands during later pregnancy. Immunoreactive STC1 protein was detected predominantly in the endometrial glands after Day 16 of pregnancy and in areolae that transport uterine gland secretions across the placenta. In ovariectomized ewes, long-term progesterone therapy induced STC1 mRNA. Although interferon tau had no effect on endometrial STC1, intrauterine infusions of ovine placental lactogen (PL) increased endometrial gland STC1 mRNA abundance in progestinized ewes. These studies demonstrate that STC1 is induced by progesterone and increased by a placental hormone (PL) in endometrial glands of the ovine uterus during conceptus (embryo/fetus and extraembryonic membranes) implantation and placentation. Western blot analyses revealed the presence of a 25-kDa STC1 protein in the endometrium, uterine luminal fluid, and allantoic fluid. The data suggest that STC1 secreted by the endometrial glands is transported into the fetal circulation and allantoic fluid, where it is hypothesized to regulate growth and differentiation of the fetus and placenta, by placental areolae.     

Expression profile and role of prostacyclin receptor (PTGIR) in peri-implantation porcine conceptuses

Prostacyclin (prostaglandin I₂ [PGI₂]) signaling system not only plays a pivotal role in vascular function in many species but is also important during early pregnancy in rodents and ruminants. Recently, abundant concentrations of PGI₂ were found in the endometrium and uterine lumen of gilts at the time of implantation. In the present study, conceptuses collected on Days 10, 12, 14, 16, and 18 of pregnancy were examined for the expression of PGI₂ receptors, PTGIR. Moreover, the effect of iloprost (a PGI₂ analogue) on attachment, proliferation, and apoptosis in conceptus trophoblast (Tr) cells was investigated in vitro. Increased PTGIR mRNA expression was observed in Day 16 trophoblasts compared with Days 10, 12, and 14 conceptuses (P < 0.001) and Day 18 trophoblast tissue (P < 0.01). Embryos from Day 18 of gestation revealed greater PTGIR mRNA expression compared with Day 16 embryos (P < 0.01). In contrast to mRNA, PTGIR protein level in conceptus and trophoblast tissue was high on Days 12 and 14, followed by a decrease observed on Day 16. On Day 18 of pregnancy, PTGIR protein was detected in both trophoblast and embryonic tissue. Iloprost stimulated attachment and proliferation of Tr cells, but this effect was abolished by the addition of the PTGIR-specific antagonist, CAY10441, into culture medium. Addition of iloprost neither did affect the ratio of BAX/BCL-2 gene expression in cultured Tr cells nor did protect these cells from staurosporine-induced apoptosis. In summary, PTGIR is expressed in porcine conceptuses, and PGI₂ acting through this receptor may promote the attachment and proliferation of Tr cells, thereby facilitating conceptus implantation.     

Expression of estrogen receptors alpha and beta in the corpus luteum and uterus from non-pregnant and pregnant llamas

Because estrogen may be involved in maternal recognition of pregnancy and embryonic migration in llamas, expression of estrogen receptor subtypes alpha (ERalpha) and beta (ERbeta) was evaluated in corpus luteum (CL), endometrium, and uterus using relative RT-PCR. Tissues were recovered from sterile-mated (SM) and pregnant (PG) females during Days 7-11 and 7-13 (Day 0 = day of mating), respectively, and follicular phase and juvenile females. Luteal expression of ERalpha and beta was similar (P > 0.10) in SM and PG females and within Days 7-11, however, expression of ERalpha in ovarian tissue from follicular phase females was greater (P < 0.05) than Days 7 and 9 CL. Uterus expressed less ERalpha and beta compared to endometrium (P = 0.07 and P < 0.01, respectively). Expression of ERalpha was greater (P < 0.05) in Day 7 and follicular phase uteri than Days 9 and 11, Day 13 PG and juvenile uteri. Uterine ERbeta expression was greater (P = 0.09) in PG versus SM females and in mated compared to follicular phase females (P < 0.05). Endometrial expression of ERalpha and beta did not differ (P > 0.10) between SM and PG females or by day. The presence of luteal ER during this period may mean a role for estradiol in maternal recognition of pregnancy. Observed increases in uterine ER expression with no changes in endometrium suggest expression increased in myometrium and/or perimetrium. Upregulation of myometrial ERbeta in PG females may be involved in supporting uterine migration of the embryo.     

Expression of alphav, alpha4, alpha5 and beta3 integrin subunits, fibronectin and vitronectin in goat peri-implantation

Integrins are glycoprotein heterodimers located in the cell membranes that stimulate intercellular adhesion and act as extracellular matrix (ECM) protein receptors. Although integrins have been detected in the implantation sites of various species, little is known about their participation in ruminant non-invasive placentation. The objective of this study was the detection of alphav, alpha4, alpha5, beta1 and beta3 integrin subunits and of two of their ligands, fibronectin and vitronectin, to determine their participation in the caprine peri-implantation process. On Day 21 post-coitum (pc), endometrial epithelium and trophoblastic cells showed an intense alphav and beta3 integrin subunits expression and moderate staining for alpha4 and alpha5. On Day 23 pc, integrin expression decreased noticeably and only a weak staining of alpha4 and beta3 integrin subunits were observed. No beta1 integrin subunit expression was detected on either of the days studied. Fibronectin (FN) expression in trophectodermic and endometrial epithelium was weak or moderate on the days studied while vitronectin (VN) expression in the same tissues was moderate or strong on Day 21 pc but decreased on Day 23 pc. These results suggest that alphavbeta3 integrin, alpha4 and alpha5 subunits, VN and FN are expressed in caprine endometrium and blastocyst and may play a role in the cascade of the implantation process.     

Characterization of the interleukin-1beta system during porcine trophoblastic elongation and early placental attachment

Conceptus-uterine communication is established during trophoblastic elongation when the conceptus synthesizes and releases estrogen, the maternal recognition signal in the pig. Interleukin-1beta (IL-1beta) is a differentially expressed gene during rapid trophoblastic elongation in the pig. The current investigation determined conceptus and endometrial changes in gene expression for IL-1beta, IL-1 receptor antagonist (IL-1Rant), IL-1 receptor type 1 (IL-1RT1), and IL-1 receptor accessory protein (IL-1RAP) in developing peri- and postimplantation conceptuses as well as uterine endometrium collected from cyclic and pregnant gilts. Conceptus IL-1beta gene expression was enhanced during the period of rapid trophoblastic elongation compared with earlier spherical conceptuses, followed by a dramatic decrease in elongated Day 15 conceptuses. IL-1RT1 and IL-1RAP gene expression was greater in Day 12 and 15 filamentous conceptuses compared with earlier morphologies while IL-1Rant gene expression was unchanged by conceptus development. The uterine lumenal content of IL-1beta increased during the process of trophoblastic elongation on Day 12. Uterine IL-1beta content declined on Day 15, reaching a nadir by Day 18 of pregnancy. IL-1beta gene expression in porcine conceptuses was temporally associated with an increase in endometrial IL-1RT1 and IL-1RAP gene expression in pregnant gilts. Endometrial IL-1beta and IL-1Rant gene expression were lowest during Days 10-15 of the estrous cycle and pregnancy. The temporal expression of IL-1beta during conceptus development and the initiation of conceptus-uterine communication suggests conceptus IL-1beta synthesis plays an important role in porcine conceptus elongation and the establishment of pregnancy in the pig.     

Analysis of osteopontin at the maternal-placental interface in pigs

Noninvasive, epitheliochorial placentation in the pig follows a prolonged preimplantation period characterized by migration, spacing and elongation of conceptuses, and secretion of estrogen for maternal recognition of pregnancy. Osteopontin (OPN) is an extracellular matrix protein that binds integrins to promote cell-cell attachment and communication. OPN appears to play a key role in conceptus implantation and maintenance of pregnancy in sheep; however, a role for OPN in the porcine uterus has not been established. Therefore, this study examined OPN expression and function in the porcine uterus and conceptus (embryo/fetus and associated extraembryonic membranes). Northern and slot blot hybridization detected an increase in endometrial OPN expression between Days 25 and 30, and levels remained elevated through Day 85 of pregnancy. In situ hybridization localized OPN mRNA to discrete regions of the uterine luminal epithelium (LE) on Day 15 of pregnancy and to the entire LE thereafter. Glandular epithelial (GE) expression of OPN mRNA was first detected on Day 35 of pregnancy and increased through Day 85. Both 70- and 45-kDa forms of OPN protein were detected in cyclic and pregnant endometrium by Western blotting. OPN protein was localized to the LE and GE by immunofluorescence; however, only the 70-kDa OPN was detected in uterine flushings. OPN protein was present along the entire uterine-placental interface after Day 30 of pregnancy. In addition, OPN mRNA and protein were localized to immune-like cells within the stratum compactum of the endometrium in both Day 9 cyclic and pregnant gilts. Incubation of OPN-coated microbeads with porcine trophectoderm and uterine luminal epithelial cells induced Arg-Gly-Asp (RGD)-dependent integrin activation and transmembrane accumulation of cytoskeletal molecules at the apical cell surface as assessed by immunofluorescence detection of talin or alpha-actinin as markers for focal adhesions. These results suggest that OPN, expressed by uterine epithelium and immune cells, may interact with receptors (i.e., integrins) on conceptus and uterus to promote conceptus development and signaling between these tissues as key contributors to attachment and placentation in the pig.     

Gastrin-releasing peptide (GRP) in the ovine uterus: regulation by interferon tau and progesterone

Gastrin-releasing peptide (GRP) is abundantly expressed by endometrial glands of the ovine uterus and processed into different bioactive peptides, including GRP1-27, GRP18-27, and a C-terminus, that affect cell proliferation and migration. However, little information is available concerning the hormonal regulation of endometrial GRP and expression of GRP receptors in the ovine endometrium and conceptus. These studies determined the effects of pregnancy, progesterone (P4), interferon tau (IFNT), placental lactogen (CSH1), and growth hormone (GH) on expression of GRP in the endometrium and GRP receptors (GRPR, NMBR, BRS3) in the endometrium, conceptus, and placenta. In pregnant ewes, GRP mRNA and protein were first detected predominantly in endometrial glands after Day 10 and were abundant from Days 18 through 120 of gestation. Treatment with IFNT and progesterone but not CSH1 or GH stimulated GRP expression in the endometrial glands. Western blot analyses identified proGRP in uterine luminal fluid and allantoic fluid from Day 80 unilateral pregnant ewes but not in uterine luminal fluid of either cyclic or early pregnant ewes. GRPR mRNA was very low in the Day 18 conceptus and undetectable in the endometrium and placenta; NMBR and BRS3 mRNAs were undetectable in ovine uteroplacental tissues. Collectively, the present studies validate GRP as a novel IFNT-stimulated gene in the glands of the ovine uterus, revealed that IFNT induction of GRP is dependent on P4, and found that exposure of the ovine uterus to P4 for 20 days induces GRP expression in endometrial glands.     

Temporal and spatial expression of integrins and their extracellular matrix ligands at the maternal-fetal interface in the rhesus monkey during pregnancy

The integrin and extracellular matrix protein (ECM)-mediated adhesion and invasion of the receptive maternal uterine endometrium by trophoblasts is a critical event in the complex physiological process of pregnancy. Although the process has been largely characterized in mice, the relevant mechanism in primates remains unclear. We investigated the expression patterns and dynamic alterations of integrin subunits (alpha1, alpha5, alpha6, beta1, and beta4) and their ECM ligands, such as laminin (LN), type IV collagen (Col IV), and fibronectin (FN), at the maternal-fetal interface during Gestational Days 15, 25, 50, and 100 and at full term in 20 pregnant rhesus monkeys. Immunohistochemistry and in situ hybridization revealed that a relatively high expression of integrins occurred in trophoblast cells at Gestational Day 15, with the peak level occurring at Day 25. The expression level decreased from Day 50 to term. Along the invasive pathway, expression levels of integrin alpha1, alpha5, and beta1 subunits were gradually elevated from the proximal to distal column, reaching peak level in the trophoblast shell, but were reduced in those invasive extravillous cytotrophoblast (EVCT) cells in contact with the decidua. Integrin alpha1, alpha5, beta1, and beta4 subunits were also highly expressed in decidual stromal cells and moderately expressed in the maternal epithelium and endothelium. Immunoreactive FN, LN, and Col IV were distributed in EVCT and decidual stromal cells and part of the uterine epithelial and endothelial cells. These data suggest that the correlated expression of integrins and their ECM ligands at the maternal-fetal interface might be involved in regulation of cell proliferation and differentiation and the counterbalanced invasion-accelerating and invasion-restraining processes in trophoblast cells during the early stage of pregnancy.     

Presence of the acute phase protein, bikunin, in the endometrium of gilts during estrous cycle and early pregnancy

Noninvasive, epitheliochorial placental attachment in the pig is regulated through endometrial production of protease inhibitors. The objective of the present study was to determine if the light-chain serine protease inhibitor of the inter-alpha-trypsin inhibitor family, bikunin, is produced by the porcine endometrium during the estrous cycle and early pregnancy. Western blot analysis revealed the presence of bikunin in uterine flushings of gilts collected during the luteal phase of the estrous cycle and early pregnancy (Days 12-18). However, bikunin unbound to the inter-alpha-trypsin heavy chains was detected only in endometrial explant culture medium obtained from estrus and pregnant (Days 12, 15, and 18) gilts. Endometrial bikunin gene expression was lowest on Day 10 of the estrous cycle and pregnancy, followed by a 30- to 77-fold increase on Day 15 of the estrous cycle and pregnancy. Bikunin gene expression decreased on Day 18 of the estrous cycle, whereas endometrial bikunin gene expression continued to increase in pregnant gilts. Bikunin mRNA was localized to the uterine glands between Days 15 and 18 of the estrous cycle and pregnancy. In addition to its role as a protease inhibitor, bikunin functions in stabilization of the extracellular matrix, which suggests that bikunin could be involved with facilitating placental attachment to the uterine epithelial surface in the pig.     

Insulin-like growth factor-I expression during early conceptus development in the pig

Porcine conceptuses (embryo and associated membranes) in utero undergo developmental morphological transformations coincident with structural and biochemical changes in the uterine endometrium during early gestation. To elucidate a possible role for insulin-like growth factor-I (IGF-I) in these events, porcine endometrial (Days 8, 10, 11, 12, 14, and 30) and conceptus (Days 12, 14, and 16) tissues were characterized for the presence of IGF-I peptide and mRNAs. The corresponding uterine luminal fluids (ULF) at these stages of pregnancy were also analyzed for immunoreactive IGF-I concentration. ULF IGF-I was lowest on Day 8, highest on Day 12, and declined by Day 14. In contrast, endometrial tissue IGF-I content remained constant during this period. Conceptus tissues contained less IGF-I than endometrial tissues; however, conceptus IGF-I values were maximum on Day 12 coincident with peak values for ULF IGF-I. Dot-blot hybridization analyses revealed temporal variation in steady-state levels of IGF-I mRNAs in endometrium. Highest levels of endometrial IGF-I mRNA were detected on Day 12 and were about 4-fold greater than on Day 30 of pregnancy. IGF-I mRNA expression in conceptus tissues on Days 12, 14, and 16 was the same and was significantly less than that in endometrium on Day 12. These results demonstrate the temporal variation of IGF-I mRNA abundance in uterine endometrium and of immunoreactive IGF-I in ULF and in conceptus tissues, with the developmental processes occurring in the conceptuses at early pregnancy.(ABSTRACT TRUNCATED AT 250 WORDS)