Angiogenic activity of maternal and fetal placental tissues of ewes throughout gestation

In Study 1, explants of caruncular and intercaruncular endometrium and fetal membrane were collected from ewes (5-6/day) on Days 11-13, 16-18 and 21-23 after mating and Days 10-12 after oestrus, and incubated for 24 h. Explant-conditioned media were evaluated for their effects on endothelial cell proliferation. Both caruncular and intercaruncular endometrium secreted factor(s) which stimulated endothelial cell proliferation, and which appeared to be greater than 100 x 10(3) Mr and heat-labile. In Study 2, conditioned media from explant incubations of caruncular and intercaruncular endometrium, cotyledon and intercotyledonary fetal membrane obtained from ewes (6-7/day) on Days 40, 65, 90, 115 and 140 after mating were evaluated for their effects on endothelial cell proliferation. Caruncular and intercaruncular endometrium and intercotyledonary fetal membrane secreted factor(s) which inhibited endothelial cell proliferation. Media from cotyledonary explants tended to stimulate endothelial cell proliferation on Day 115. Conditioned media from cotyledonary explants obtained from 3 additional ewes at Day 120 of gestation stimulated endothelial cell proliferation, and this activity also appeared to be greater than 100 x 10(3) Mr. Placental angiogenesis in ewes therefore appears to be modulated by both maternal and fetal placental tissues via stimulatory and inhibitory factors.     

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.     

Secretion of angiogenic activity by placental tissues of cows at several stages of gestation

Samples of maternal and fetal placental tissues were obtained from cows on Days 100 (N = 4), 150 (N = 5), 200 (N = 6) and 250 (N = 6) of gestation and incubated for 24 h. Conditioned media from caruncular explants were mitogenic for bovine aortic endothelial cells (BAEC) on all days of gestation. Media from intercaruncular endometrium were stimulatory for proliferation of BAEC on Day 100 but inhibitory on Days 150, 200 and 250. Media from cotyledonary and intercotyledonary tissues inhibited proliferation of BAEC on all days. Caruncular-conditioned media stimulated migration of BAEC on Days 150, 200 and 250. Cotyledonary-conditioned media inhibited migration of BAEC on all days. Effects of media from intercaruncular and intercotyledonary tissues on migration of BAEC varied with stage of gestation. Angiogenic activity of media from caruncular (all stages) and intercaruncular (Day 100) tissues appeared to have an Mr greater than 100,000. In cows, therefore, the maternal placentome (caruncle) appears to be the primary source of placental angiogenic activity throughout gestation. The fetal placentome (cotyledon) secretes activity which inhibits two major components of angiogenesis (proliferation and migration of endothelial cells) throughout gestation. Intercaruncular and intercotyledonary tissues may modulate placental angiogenesis throughout gestation. Placental vascular development in the cow is therefore probably controlled by an interaction between stimulatory and inhibitory factors produced by the placenta itself.     

小鼠子宫内膜LIF基因表达与雌、孕激素的关系

白血病抑制因子（LIF）是一种多功能活性的糖蛋白,LIF基因在大多数妊娠第4天的小鼠子宫内膜进行着强烈的表达,然而LIF基因表达调控的机制目前尚不清楚。本实验对168只妊娠第4~5天的小鼠LIF基因表达和血清中雌、孕激素水平分别进行了检测,发现18只小鼠无LIF基因表达,其血清中雌、孕激素水平分别极显著（P＜0.01）和显著（0.01＜P＜0.05）低于其他表达的小鼠。提示：雌、孕激素对小鼠LIF基因表达过程中起着一定的作用,将为LIF基因表达调控机制的深入研究打下基础。     

Effect of luteinizing hormone (LH), pregnancy specific protein B (PSPB), or arachidonic acid (AA) on ovine endometrium of the estrous cycle or placental secretion of prostaglandins E2 (PGE2) and F2alpha (PGF2alpha) and progesterone in vitro

The objective of this experiment was to determine the effect of AA, LH, or PSPB on secretion of PGE2, PGF2alpha, or progesterone by ovine caruncular endometrium of the estrous cycle or placental tissue of pregnancy in vitro. Ovine caruncular endometrium of the estrous cycle (days 8, 11, 13, and 15) or caruncular/placental tissue on days 8, 11, 13, 15, 20, 30, 40, 50, 60, and 90 postbreeding were incubated in vitro with vehicle, AA, LH, or PSPB in M-199 for 4 and 8 h. Secretion of PGF2alpha by caruncular endometrium of non-bred ewes on days 13 and 15 and by caruncular/placental tissue of bred ewes on days 13, 15, 20, 30, and 40 was increased (P < or = 0.05) when incubated with vehicle and declined (P < or = 0.05) after day-40 in bred ewes. Secretion of PGF2alpha by day-15 caruncular endometrium of non-bred ewes and bred ewes was increased (P < or = 0.05) by AA on days 13 and 15 and by LH on day-15. Secretion of PGF2alpha by caruncular/placental tissue from bred ewes was (P < or = 0.05) by AA on days 13, 15, 20, 30, and 40 and by LH on days 15, 20, 30, and 40, after which the response decreased (P < or = 0.05). Secretion of PGF2alpha by caruncular endometrium of non-bred ewes during the estrous cycle or by caruncular/placental tissue of bred ewes during the first trimester was not affected by PSPB (P > or = 0.05). Secretion of PGE2 by caruncular endometrium of non-bred ewes did not change (P > or = 0.05) and was increased (P < or = 0.05) by caruncular/placental tissue on days 13-90 from bred ewes when incubated with vehicle. Secretion of PGE2 by endometrium from non-bred ewes was not affected (P > or = 0.05) by AA, LH, or PSPB, but was increased (P < or = 0.05) by AA or LH on days 13-50 and by PSPB on days 60 and 90 when incubated with caruncular/placental tissue from bred ewes. Secretion of progesterone by placental tissue of bred ewes increased (P < or = 0.05) on day-50 and continued to increase through day-90. In summary, uterine/placental tissue secretion of PGF2alpha is not reduced until the end of the first trimester of pregnancy in ewes. In addition, LH appears to play a role in luteolysis of non-bred ewes by stimulating caruncular endometrial secretion of PGF2alpha and on day-5 postbreeding to prevent luteolysis during early pregnancy by stimulating caruncular/placental secretion of PGE2 throughout the first trimester of pregnancy in sheep. Secretion of PGE2 by caruncular/placental tissue after day-50 of pregnancy appears to be regulated by PSPB, not LH.     

Temporal and spatial expression of leukemia inhibitory factor in rabbit uterus during early pregnancy

Leukemia inhibitory factor (LIF) has been shown to play an important role in the implantation of mouse blastocysts. The present study was designed to document the appearance of LIF in the rabbit uterus during early pregnancy and to determine whether changes just prior to implantation, similar to those in mice, occurred. LIF was localized in endometrial epithelium, myometrium, and endometrial glands. A low level of LIF was detected in the uterus of nonestrous and estrous females. LIF expression reached its highest level on day 5 of pregnancy and declined on days 6 and 7. By day 13 of pregnancy, little endometrial LIF was apparent. The expression of LIF on day 5 of pseudopregnancy was similar to that on day 5 of pregnancy. LIF expression was much higher at implantation sites than that at nonimplantation areas on day 7 of pregnancy. It is concluded that LIF may be important for the implantation of rabbit blastocysts. © 1994 Wiley-Liss, Inc.     

Characterization of endometrial receptors for ovine trophoblast protein-1 during the estrous cycle and early pregnancy in sheep

Scatchard analysis was used to determine the distribution, number, and affinity of unoccupied receptors for ovine trophoblast protein-1 (oTP-1) in endometrium of sheep throughout the estrous cycle and early pregnancy. In Experiment I, oTP-1 receptor characteristics were determined in membrane preparations of caruncular and intercaruncular regions of endometrium collected from uterine horns ipsilateral and contralateral to the ovary bearing the corpus luteum. Receptor concentrations and affinity constants for oTP-1 were not different (p greater than 0.1) between the four endometrial regions examined, suggesting that the expression of receptors for oTP-1 occurs uniformly throughout the endometrium. Endometrial receptor characteristics for oTP-1, luteal wet weights, and progesterone contents were determined throughout the estrous cycle and early pregnancy in Experiment II. Concentration of receptors and affinity constants for oTP-1 varied throughout the estrous cycle and early pregnancy (p less than 0.01), with the pattern of change differing between cyclic and pregnant ewes (p less than 0.01). Numbers of receptors for oTP-1 were maximal on Day 4 of the estrous cycle and declined progressively to Day 12 (p less than 0.05) in both cyclic and pregnant ewes. After Day 12, the quantity of unoccupied receptors for oTP-1 increased (p less than 0.05) gradually to Day 16 in cyclic ewes, but declined (p less than 0.05) further in the endometrium of pregnant ewes. The affinity constants of endometrial receptors for oTP-1 were similar in cyclic and pregnant ewes prior to Day 12, increasing threefold from Days 4 to 12 (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of a luteolytic dose of oestradiol benzoate on uterine oxytocin receptor concentrations, phosphoinositide turnover and prostaglandin F-2 alpha secretion in sheep

Administration of oestradiol-17 beta benzoate on Days 9 and 10 of the oestrous cycle resulted in episodic secretion of PGF-2 alpha (as indicated by elevated circulating concentrations of 13,14-dihydro-15-ketoprostaglandin F-2 alpha) and a decline in circulating progesterone. Release of PGF-2 alpha began 35 +/- 3 h after first injection of oestrogen and progesterone concentrations declined from 42 +/- 3 h. Secretion of oxytocin, which was first observed 26 +/- 3 h after oestrogen treatment, preceded secretion of PGF-2 alpha; 69% of pulses of oxytocin coincided with episodes of PGF-2 alpha secretion. Uterine oxytocin receptor concentrations were raised in ewes treated with oestrogen, increases occurring in caruncular endometrium and myometrium by 12 h after treatment and in intercaruncular endometrium by 24 h. Raised receptor concentrations were followed at 24 h by increases in the incorporation of [3H]inositol into phosphatidylinositol and in the hydrolysis of labelled tissue phosphoinositides in response to oxytocin in slices of caruncular endometrium incubated in vitro. The following sequence of events is therefore suggested to occur at oestrogen-induced luteolysis: induction of the oxytocin receptor; increased turnover of phosphoinositides; onset of episodic secretion of PGF-2 alpha; and functional luteolysis.     

Leukemia inhibitory factor restores the hypertrophic response to increased loading in the LIF(-/-) mouse

Cytokines and growth factors are thought to contribute to skeletal muscle hypertrophy. Leukemia inhibitory factor (LIF), a cytokine, enhances skeletal muscle regeneration; however the role of LIF in skeletal muscle hypertrophy remains uncertain. We examined the hypertrophic ability of the plantaris and soleus muscles in wild-type mice (WT) and LIF knock-out mice [LIF(-/-)] in response to increased mechanical load. Using the functional overload model to induce increases in mechanical load on the plantaris and soleus muscle, WT mice demonstrated increases in plantaris and soleus mass after 7, 21, and 42 days of loading. However, the LIF(-/-) mice had no significant increases in plantaris muscle mass at any time point, while the soleus muscle exhibited a delayed hypertrophic response. Systemic delivery of LIF to the LIF(-/-) mice returned the hypertrophic response to the same levels as the WT mice after 21 days of functional overload. These data demonstrate for the first time that LIF expression in loaded skeletal muscle is critical for the development of skeletal muscle hypertrophy in the functional overload model.     

Effect of in vitro heat shock upon the synthesis and secretion of prostaglandins and protein by uterine and placental tissues of the sheep

The objectives of this study were to determine the secretion patterns of prostaglandins (PG) and protein during mid- (Day 100) and late- (Day 140) pregnancy in the ewe and to ascertain whether that pattern is altered by in vitro heat shock. Explant cultures were prepared from intercaruncular endometrium, caruncular endometrium, fetal cotyledon and interplacentomal placenta. Cultures were incubated at 39 or 42 degrees C for 18 h in the presence of arachidonic acid or L-[4,5(3)H]leucine. There were no effects of day of gestation or consistent effects of temperature upon de novo synthesis of tissue and secretory protein. Elevated temperature generally depressed PGE(2) secretion by maternal tissues and PGF secretion by caruncular endometrium but had little effect on PGE(2) release by fetal tissues or on PGF release by intercaruncular endometrium or fetal tissues. Day of gestation by tissue type interactions were found for PGF and PGE(2) release. At Day 100, maternal tissues secreted more PGF and PGE(2) than fetal tissues; at Day 140, PG secretion from fetal tissues was greater than at Day 100, and fetal PGE(2) release exceeded release from maternal tissues. Tissue proteins resolved by SDS-PAGE revealed the appearance in heat-shocked tissue of 93 and 72 kDa heat-shock proteins. In conclusion, elevated temperature depressed PGE(2) release, particularly from maternal tissues. Changes in PGE(2) suggest that the increase in utero-placental PGE(2) with increasing gestational age is due to changes in secretion of the fetal placenta.     

Expression of the fusion protein recombinant human granulocyte-macrophage colony stimulating factor and leukemia inhibitory factor in a baculovirus vector system

A fusion gene coding human granulocyte-macrophage colony stimulating factor (GM-CSF) and leukemia inhibitory factor (LIF) cDNAs was inserted into the transfer vector pSXIVVI⁺ X3 with the control of Syn and XIV promoters. The Sf9 cells (Spodoptera frugiperda) were co-transfected with the recombinant plasmid and TnNPV DNA (Trichoplusia ni nuclear polyhedrosis virus DNA). The fusion protein recombinant human granulocyte-macrophage colony stimulating factor (GM-CSF) and leukemia inhibitory factor (LIF) could be synthesized in cells infected with recombinant virus at a level of about 23% of their total cellular protein. Activity analysis of the fusion protein in infected cells revealed that it exhibited the dual activities of GM-CSF and LIF. Western blot analysis of the expressed fusion protein in infected larvae showed that the virus-mediated fusion protein, with a molecular weight of ∼35 kDa, is confirmed with immunoreactivity. Received 02 December 1998/ Accepted in revised form 07 May 1999     

Developmental changes in polyamine levels and synthesis in the ovine conceptus

Polyamines (putrescine, spermidine, and spermine) are essential for placental growth and angiogenesis. However, little is known about changes in polyamine synthesis associated with development of the ovine conceptus (embryo/fetus and associated placental membranes). We hypothesized that rates of placental polyamine synthesis were maximal during the rapid placental growth that occurs in the first half of pregnancy. This hypothesis was tested using ewes between Days 30 and 140 of gestation. Columbia cross-bred ewes were hysterectomized on Days 30, 40, 60, 80, 100, 120, or 140 of gestation (Day 0 = mating; n = 4 ewes/day) to obtain placentomes, intercotyledonary placenta, intercaruncular endometrium, and allantoic as well as amniotic fluids. The tissues were analyzed for ornithine decarboxylase (ODC) and arginase activities; arginine, ornithine, and polyamine concentrations; and polyamine synthesis using radiochemical and chromatographic methods. Maximal ODC and arginase activities and the highest rates of polyamine synthesis were observed in all tissues on Day 40 of gestation. Concentrations of ornithine and polyamines in placentomes and intercaruncular endometrium also peaked on Day 40 of gestation. In ovine allantoic and amniotic fluids, polyamines were most abundant during early (Days 40-60) and late (Days 100-140) gestation, respectively. Amniotic fluid spermine increased progressively with advancing gestation. Results of the present study indicate metabolic coordination among the several integrated pathways that support high rates of polyamine synthesis in the placenta and endometrium during early pregnancy. Our findings may have important implications for both intrauterine growth retardation and fetal origins of diseases in adults.     

Leukemia inhibitory factor and interleukin-11: Critical regulators in the establishment of pregnancy

Blastocyst implantation into a receptive endometrium is critical to the establishment of pregnancy and is tightly regulated by factors within the blastocyst–endometrial micro-environment. Leukemia inhibitory factor (LIF) and interleukin-11 (IL11) have key roles during implantation. Female mice with a null mutation in the LIF or IL11RA gene are infertile due to a complete failure of implantation or a defective differentiation/decidualization response to the implanting blastocyst, respectively. LIF and IL11 deficiency during pregnancy is associated with infertility and miscarriage in women. Numerous cell populations at the maternal–fetal interface are regulated by LIF/IL11 including the endometrial epithelium, decidualizing stroma, placental trophoblasts and leukocytes. This review focuses on the roles of LIF/IL11 during early pregnancy and highlights their potential as contraceptive targets and therapeutic agents for infertility.     

Leukemia inhibitory factor/differentiation-stimulating factor (LIF/D-factor): regulation of its production and possible roles in bone metabolism.

Leukemia inhibitory factor/differentiation-stimulating factor (LIF/D-factor), expression of its mRNA, and possible roles in bone metabolism were studied in murine primary and clonal osteoblast-like cells. Local bone-resorbing factors such as IL-1, TNF alpha, and LPS strongly induced expression of LIF/D-factor mRNA in both clonal MC3T3-E1 cells and primary osteoblast-like cells. Neither parathyroid hormone nor 1 alpha,25-dihydroxyvitamin D3 stimulated expression of LIF/D-factor mRNA. LIF/D-factor per se did not stimulate expression of its own mRNA. Appreciable amounts of LIF/D-factor were detected in synovial fluids from rheumatoid arthritis (RA) patients but not in those with osteoarthritis (OA). Simultaneous treatment with LIF/D-factor, IL-1, and IL-6 at the concentrations found in synovial fluids from RA patients greatly enhanced bone resorption, though these cytokines did not stimulate bone resorption when separately applied. This suggests that LIF/D-factor produced by osteoblasts is in concert with other bone-resorbing cytokines such as IL-1 and IL-6 involved in the bone resorption seen in the joints of RA patients. LIF/D-factor specifically bound to MC3T3-E1 cells with an apparent dissociation constant of 161 pM and 1,100 binding sites/cell. LIF/D-factor dose-dependently suppressed incorporation of [3H]thymidine into MC3T3-E1 cells. In addition, it potentiated the alkaline phosphatase activity induced by retinoic acid, though LIF/D-factor alone had no effect on enzyme activity. These results suggest that LIF/D-factor is involved in not only osteoclastic bone resorption but also osteoblast differentiation in conjugation with other osteotropic factors.     

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.     

Effect of leukemia inhibitory factor (LIF) on in vitro produced bovine embryos and their outgrowth colonies

In vitro produced (IVP) bovine embryos were subjected to in vitro culture with or without 1000 U/ml human recombinant leukemia inhibitory factor (LIF) added to the culture medium from Days 5 to 8 post insemination (p.i.). Resulting blastocysts were subsequently plated intact on mouse feeder cells in a medium with or without LIF. Significantly more embryos reached the hatched blastocyst stage, and the number of blastocysts with excellent morphology was significantly higher, when LIF was omitted. At Day 8 p.i., total cell count (TCC) and inner cell mass (ICM) cell count was significantly higher in embryos cultured without LIF. In embryos cultured with LIF, cytoplasmic vesicles and lipid droplets were abundant and a decreased expression of both Oct4 and laminin could be observed. Initial hypoblast formation was revealed in almost 1/3 of the LIF-cultured blastocysts whereas this feature was evident in 2/3 of the blastocysts cultured in the absence of LIF. Overall, almost 60% of the blastocysts cultured without LIF formed outgrowth colonies (OCs) when plated on feeders, whereas this phenomenon was only observed in 30% of the blastocysts cultured in the presence of LIF. A tendency for retaining a tightly packed central growth of putative ICM-derived cells was observed, when attachment to the feeder layer was initiated close to the embryonic pole of the blastocyst. At Day 8 of outgrowth culture, approximately 20% of the colonies contained a central core of putative ICM-derived cells appearing large enough for mechanical isolation and further subculture. Immunohistochemical labeling for Oct4 revealed staining of both trophectodermal and ICM-derived cells. The presence of LIF in the outgrowth culture medium did not have any apparent effect on the plating efficiency or colony type. In conclusion, LIF had an adverse effect on in vitro embryonic development when added to the culture medium in the period from Days 5 to 8 p.i., whereas it had no apparent effect on the OCs subsequently formed from such embryos.     

妊娠小鼠子宫内膜LIF基因表达的研究

本文对妊娠第4天(Ⅰ组)、第7天(Ⅱ组)、第10天(Ⅲ组)的小鼠(各20只)子宫内膜LIF基因表达进行了研究。Ⅰ组20只小鼠子宫内膜全部存在LIF基因的表达、Ⅱ组有5只小鼠表达、Ⅲ组仅有1只小鼠表达。文中对不同孕期LIF基因的表达程度与胚胎着床的关系进行了讨论。 Abstract:Leukemia inhibitory factor(LIF)is a glycoprotein with multiple activities and is essential for blastocyst implantation in mouse.We have examined LIF gene expression in mice endometrium on day 4(group Ⅰ),day 7(group Ⅱ),day 10(group Ⅲ)of pregnancy.In group Ⅰ all had LIF gene expression,5 mice had LIF gene expression in group Ⅱ,only one mouse had LIF gene expression in group Ⅲ.We discussed the relation between level of LIF gene expression and embryonic implantation.