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.     

Prp-c and Prp-Sc at the fetal-maternal interface

Scrapie is a naturally occurring prion (PrP) disease causing a fatal neurodegenerative disorder in sheep and goats. Previous studies suggest that scrapie is transmitted naturally through exposure to the scrapie agent in wasted placentas of infected ewes. This study determined the distribution and biochemical properties of PrP cellular (PrP-C) and the distribution of PrP scrapie (PrP-Sc) in reproductive, placental, and selected fetal tissues and fetal fluids in sheep. Glycosylated, N-terminally truncated, proteinase K-sensitive PrP-C with apparent molecular masses of 23-37 kDa was present in reproductive, placental, and fetal tissues and fetal fluids. PrP-C was low or undetectable in intercotyledonary chorioallantois, amnion, urachus, amniotic fluid, and fetal urine. In pregnant ewes, cotyledonary chorioallantois, allantoic fluid, and caruncular endometrium contained higher levels of PrP-C than did intercaruncular endometrium, myometrium, oviduct, ovary, fetal bladder, or fetal kidney. Caruncular endometrial PrP-C was up-regulated during pregnancy. Despite the wide distribution of PrP-C in reproductive, placental, and selected fetal tissues and fetal fluid, PrP-Sc was detected only in caruncular endometrium and cotyledonary chorioallantois of pregnant scrapie-infected ewes. The embryo/fetus may not be exposed to scrapie in utero because it is separated physically from PrP-positive allantois and chorioallantois by PrP-negative amnion.     

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.     

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)     

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.     

Developmental changes in nitric oxide synthesis in the ovine placenta

Nitric oxide (NO), synthesized from l-arginine by NO synthase (NOS), is a key regulator of placental angiogenesis and growth during pregnancy. However, little is known about placental NO synthesis associated with ovine conceptus development. This study was conducted to test the hypothesis that placental NO synthesis is greatest during early gestation. Columbia cross-bred ewes were hysterectomized on Days 30, 40, 60, 80, 100, 120, or 140 of gestation (n = 4 per day) to obtain placentomes, intercotyledonary placenta, and intercaruncular endometrium. Tissues were analyzed for constitutive NOS (cNOS) and inducible NOS (iNOS) activities, NO synthesis, tetrahydrobiopterin (BH4) and NADPH (essential cofactors for NOS), and GTP-cyclohydrolase I (GTP-CH, a rate-controlling enzyme in de novo synthesis of BH4) activity using radiochemical and chromatographic methods. Marked changes in NO synthesis, cNOS and iNOS activities, GTP-CH activity, and concentrations of BH4 and NADPH occurred in all placental and endometrial tissues between Days 30 and 140 of gestation. NO synthesis peaked on Day 60 of gestation in both intercotyledonary placenta and placentomes and on Days 40-60 in intercaruncular endometrium. NO synthesis in placentomes increased 100% between Days 80 and 100 of gestation, when placental and uterine blood flows increase continuously. In all placental and endometrial tissues, NO synthesis was positively correlated with total NOS activity, GTP-CH activity, and concentrations of BH4 and NADPH. Importantly, these results indicate a high degree of metabolic coordination among the several integrated pathways that support high rates of NO synthesis in the conceptus and uterus and establish a new base of information for future studies to define the roles of NO in fetal-placental growth and development.     

Morphometric analysis of collagen in gestational and retained bovine placentomes

Bovine placentome collagen was quantified (P<0.01) at four gestational stages (90, 150, 210 and 270 d, n = 8 d ), at 2 h post partum without (n = 4) and at 2 and 12 h post partum with (n = 8) experimentally-induced placental retention. Placentome sections were fixed and stained for collagen. Fetal cotyledonary (FC) collagen volume fraction (V(V)) increased over days of gestation studied (V(V)=0.03+/-0.01, 0.06+/-0.01, 0.13+/-0.01 and 0.19+/-0.01). Fetal cotyledonary hydroxyproline (3.15+/-0.41, 4.55+/-0.41 and 7.04+/-0.41 mg/g) and FC protein (432.0+/-17.1, 479.9+/-17.1, 585.4+/-17.1 mg/g) increased over Days 90, 150 and 210 and were similar on Days 210 and 270. Fetal cotyledonary collagen V(V) and hydroxyproline did not differ between Day 270, retained and nonretained cotyledons. Protein concentration was higher in 2 h (578.1+/-18.5 mg/g) and 12 h (526.0+/-18.5 mg/g) retained versus nonretained (400.4+/-36.2 mg/g) cotyledons. Maternal caruncular (MC) collagen V(V) and protein concentration were higher on Days 90 and 150 than on Days 210 and 270. Maternal caruncular hydroxyproline was similar from Day 90 to 210 and increased from Day 210 to 270. Maternal caruncular collagen V(V), hydroxyproline and protein concentrations were similar on Day 270 and in 2 h and 12 h retained membrane caruncles. Gestational increases in placentome collagen occurred from FC sources. No difference in FC or MC collagen V(V) existed between Day 270, retained and nonretained placentomes.     

Gene expression of leukemia inhibitory factor (LIF) and macrophage colony stimulating factor (M-CSF) in bovine endometrium during early pregnancy

Leukemia inhibitory factor (LIF) and macrophage colony stimulating factor (M-CSF), members of the group of hemopoietic cytokines, play a primary role in the control of embryo development and implantation and in the growth of the placenta in humans and mice. Gene expressions of LIF and M-CSF were investigated using quantitative RT-PCR in bovine endometrial tissues during early and mid-pregnancy (Days 16-17, 20-21, 30-36, 48-49 and 74-140) and during the estrous cycle (Days 13-14). Leukemia inhibitory factor and M-CSF genes were expressed in all samples examined. Significant differences were found between the gene expression patterns of LIF and M-CSF. Leukemia inhibitory factor expression level at Days 48-49 was the highest in caruncular endometrium, however, the large variability negated any significant differences. Leukemia inhibitory factor expression levels in intercaruncular endometrium at Days 48-49 and 74-140 of pregnancy were greater than at Days 13-14 of the estrous cycle and at other days of pregnancy. No significant change was recognized in M-CSF expression levels in caruncular endometrium. Macrophage colony stimulating factor expression level in intercaruncular endometrium at Days 74-140 was greater than those of the other samples. These results suggest that LIF and M-CSF are produced in the endometrium and may play different roles in early and mid-pregnancy.     

Evidence that platelet-activating factor suppresses uterine oxytocin-induced 13,14-dihydro-15-keto-prostaglandin F2 alpha release and phosphatidylinositol hydrolysis in the ewe.

This study was conducted to determine whether platelet-activating factor (PAF) (1) attenuated oxytocin-induced secretion of the prostaglandin (PG) F2 alpha metabolite, PGFM, by the ovine uterus in situ and (2) inhibited the generation of the inositol phosphate secondary messengers by endometrial tissue in response to oxytocin challenge in vitro. Ovariectomized ewes received steroid replacement to mimic the luteal phase. Six ewes received intrauterine injections of 200 micrograms PAF/uterine horn/day on Days 11-15, and 6 ewes were treated with vehicle. All ewes received 1 microgram oxytocin i.v. on Days 13-16. Pretreatment of ewes with PAF significantly suppressed PGFM release in response to oxytocin on Days 14 and 15 (p less than 0.005) compared to vehicle-treated ewes. PAF was not administered on Day 16, and the PGFM response to oxytocin was not different between groups. In a second experiment, ewes were given intrauterine injections of 200 micrograms PAF/uterine horn/day (n = 8) or vehicle (n = 7) on Days 11-15, and all ewes received 1 microgram oxytocin i.v. on Days 13 and 14. On Day 15 the uterus was removed, and the incorporation of 3H-inositol into inositol phosphates was determined in caruncular endometrium. Treatment of ewes with PAF in vivo reduced inositol monophosphate (IP1) generated by oxytocin (10(-6) M) by 56.4%, compared to that in endometrium from vehicle-treated controls, and also inhibited the incorporation of 3H-inositol into glycerophosphoinositol (GPI). If PAF was added to the endometrium during the incubation in vitro, the attenuation of inositol phosphate generation did not occur.(ABSTRACT TRUNCATED AT 250 WORDS)     

Endometrial protein secretion during early pregnancy in entire and ovariectomized ewes

The secretion and synthesis of protein in vitro by explants of endometrium were examined in entire ewes during the first 10 days of the oestrous cycle and during an equivalent interval in ovariectomized ewes which received injections of oestradiol and progesterone. The schedule of steroid injections given was designed to simulate endogenous ovarian secretion of progesterone during the luteal phase before oestrus, of oestradiol around oestrus and of progesterone during the luteal phase after oestrus. The rate of protein synthesis and tissue RNA:DNA and protein:DNA ratios in intercaruncular and caruncular endometrium were generally higher in entire than in ovariectomized ewes. In ovariectomized ewes oestradiol increased these activities at 2-4 days after oestrus, whereas progesterone preceding oestradiol caused increases at oestrus, but not thereafter. In entire ewes and in ovariectomized ewes receiving the full steroid treatment regimen, protein secretion was high at oestrus and declined markedly during the next 4-6 days. In ovariectomized ewes not receiving progesterone before oestradiol, secretion increased between 4 and 6 days after oestrus, or during the equivalent stage of treatment in ewes which did not show oestrus. The omission of this progesterone did not modify secretion by caruncular endometrium. Oestradiol increased protein secretion by both tissues. The data suggest that progesterone given before oestradiol (or its equivalent in entire ewes) inhibits the secretion, at about 4-7 days after oestrus, of uterine proteins which may impair embryo development in ovariectomized ewes which do not receive this progesterone.     

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.     

Prostaglandin F and E levels in the conceptus, uterus and plasma during early pregnancy in the ewe

Concentrations of prostaglandins E and F (PGE and PGF) were measured in the embryo or fetus, extra embryonic or fetal membranes (membranes), intercaruncular and caruncular endometrium and plasma collected from uterine and ovarian arterial and venous vessels from separate groups of ewes laparotomized at 5 day intervals from day 10 to day 55 of pregnancy. Our purpose was to investigate the role of prostaglandins E and F in the maternal recognition of pregnancy, implantation and early placental function. Our data suggest that the initial maintenance of the corpus luteum in the pregnant ewe does not involve a reduction in PGF production, compared to pregnant ewes; but a change in the pattern of PGF secretion. This is accompanied by an elevation in PGE production of similar magnitude to that observed in non pregnant ewes. The extra embryonic/fetal membranes appear to be the major source of elevated PGF levels in the maternal circulation prior to day 30 of pregnancy. Between days 35 and 55 of gestation the rising PGF levels in maternal serum probably come from the fetus. Over the same period PGE levels rise in the fetus and intercaruncular endometrium, but PGE secretion into the maternal circulation is not enhanced. A role for PGF and PGE in fetal, placental and uterine growth is suggested; placental and uterine endocrine function may also be targets.     

Prostaglandin F and E levels in the conceptus, uterus and plasma during early pregnancy in the ewe

Concentrations of prostaglandins E and F (PGE and PGF) were measured in the embryo or fetus, extra embryonic or fetal membranes (membranes), intercaruncular and caruncular endometrium and plasma collected from uterine and ovarian arterial and venous vessels from separate groups of ewes laparotomized at 5 day intervals from day 10 to day 55 of pregnancy. Our purpose was to investigate the role of prostaglandins E and F in the maternal recognition of pregnancy, implantation and early placental function. Our data suggest that the initial maintenance of the corpus luteum in the pregnant ewe does not involve a reduction in PGF production, compared to pregnant ewes; but a change in the pattern of PGF secretion. This is accompanied by an elevation in PGE production of similar magnitude to that observed in non pregnant ewes. The extra embryonic/fetal membranes appear to be the major source of elevated PGF levels in the maternal circulation prior to day 30 of pregnancy. Between days 35 and 55 of gestation the rising PGF levels in maternal serum probably come from the fetus. Over the same period PGE levels rise in the fetus and intercaruncular endometrium, but PGE secretion into the maternal circulation is not enhanced. A role for PGF and PGE in fetal, placental and uterine growth is suggested; placental and uterine endocrine function may also be targets.     

Role of progesterone and oestradiol in the regulation of uterine oxytocin receptors in ewes.

The interaction between oestrogen and progesterone in the regulation of the uterine oxytocin receptor in sheep was evaluated by measuring the binding of oxytocin to membrane preparations of caruncular and intercaruncular endometrium and myometrium. Ovariectomized ewes were assigned in groups of five to each cell of a 4 x 2 factorial design. The four treatments were (a) vehicle (maize oil) for 12 days, (b) progesterone (10 mg day-1) for 9 days, (c) progesterone for 9 days followed by maize oil until day 12 and (d) progesterone for 12 days. The two oestradiol treatments consisted of the administration of implants in the presence or absence of oestradiol. The ewes were killed on day 10 (group b) or day 13 (groups a, c and d) for collection of uterine tissues. The response of the caruncular and intercaruncular endometrium to the treatments was similar. In the absence of oestradiol, treatment with progesterone continuously for either 9 or 12 days reduced the concentration of the oxytocin receptor in comparison with both the control and the progesterone withdrawal group (in which values were similar). The presence of oestradiol reduced the receptor concentrations in control and both 9- and 12-day continuous progesterone treatment groups, but enhanced the concentration in the progesterone withdrawal group. The myometrial oxytocin receptors responded in a similar way to those in the endometrium to progesterone treatment alone, but the addition of oestradiol produced no further effect. In conclusion, progesterone and oestradiol caused downregulation of the endometrial oxytocin receptor. On the other hand, progesterone withdrawal, similar to that which occurs during luteolysis, increased receptor density in the presence of oestradiol. Progesterone may influence the response of the myometrium to oxytocin by causing a reduction in receptor density.     

Continuous infusion of oxytocin prevents induction of uterine oxytocin receptor and blocks luteal regression in cyclic ewes

Continuous intravenous infusion of oxytocin (3 micrograms/h) between Days 13 and 21 after oestrus delayed return to oestrus by 7 days (length of cycle 23.3 +/- 0.6 days compared to 16.6 +/- 0.2 days in control ewes). At a lower infusion rate (0.3 micrograms/h) oxytocin delayed luteolysis in only 2 of 5 ewes. Treatment from Day 14, when luteolysis had already begun, was ineffective. Delay of luteal regression by oxytocin had no effect on the length of subsequent cycles. Measurement of circulating progesterone concentrations and luteal weight showed that prolongation of the oestrous cycle was due to prevention of luteal regression. Luteal regression and behavioural oestrus were induced during continuous oxytocin administration begun on Day 13 when cloprostenol was given on Day 15 (mean cycle length, 17.3 +/- 0.21 days). Continuous oxytocin infusion from Day 13 blocked the rise in uterine oxytocin receptor concentrations which normally precedes oestrus. Mean receptor concentrations in caruncular and intercaruncular endometrium and in myometrium were 76, 36 and 9 fmol/mg protein on Day 17 in ewes receiving continuous oxytocin (3 micrograms/h); in control ewes these values were 675, 638 and 130 fmol/mg protein respectively at oestrus. Receptor concentrations on the day of oestrus in ewes receiving oxytocin and cloprostenol were not significantly different from those in control ewes (649, 852, and 109 fmol/mg protein respectively). Since cloprostenol, a PGF-2 alpha analogue, overcame the antiluteolytic action of oxytocin, it is suggested that continuous oxytocin treatment may inhibit uterine production of PGF-2 alpha, possibly by down regulating the uterine oxytocin receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Growth and in-vitro metabolism of placental tissues of cows from day 100 to day 250 of gestation.

Weight of placental tissues of cows increased exponentially from Day 100 to Day 250 of gestation, but at much slower relative and absolute rates than fetal weight. In addition, growth rate of fetal placental tissues was less than that of maternal placental tissues. Concentrations of DNA, RNA and protein, however, increased in fetal placental but not in maternal placental tissues. Fetal placental tissues therefore exhibited hyperplasia, which probably contributes to increased functional capacity of the placenta during late gestation. The rate of O2 uptake in vitro was greatest for maternal placental tissues, suggesting that the maternal portion of the placenta accounts for most of the large rate of placental O2 utilization in vivo. Compared with other placental tissues, rate of secretion of macromolecules by intercaruncular endometrium was high, but decreased from Day 100 to 250, suggesting that uterine glandular secretory activity may decrease as gestation advances. Rate of secretion of macromolecules also was high for intercotyledonary tissues and increased with day of gestation, suggesting a role for secretory products of chorioallantois in gravid uterine function.