Demonstration of a beta-casomorphin immunoreactive material in the plasma of newborn calves after milk intake

Blood was collected from newborn calves before and after their first milk intake after birth; extracts of plasma were assayed by radioimmunoassay for the presence of beta-casomorphin-7 immunoreactive materials. No beta-casomorphin immunoreactivity was found in samples collected before milk ingestion; however, in samples collected after milk ingestion a beta-casomorphin-7 immunoreactive material was detected. Chromatographic characterization showed that this material was not identical with beta-casomorphin-7 but might rather represent a precursor thereof. The material proved resistant to enzymatic attack during a 30-min incubation period at 37 degrees C in the plasma of newborn calves, whereas beta-casomorphin-7 was degraded under these conditions. A physiological significance of beta-casomorphin-7 eventually cleaved from such a precursor material at any site in the newborn mammal is suggested.     

Urocortin in human placenta and maternal plasma.

Plasma immunoreactive (IR-) urocortin (Ucn) and corticotropin-releasing factor (CRF) levels in pregnant women were measured by their specific radioimmunoassays after extraction. Although plasma IR-CRF levels were increased in pregnant women as compared to men and non-pregnant women, there was no difference of plasma IR-Ucn levels among groups. Ucn mRNA was detected in cytotrophoblasts and syncytiotrophoblasts by in situ hybridization. A reverse-phase high-performance liquid chromatography (HPLC) showed the major peak of IR-Ucn in placenta and plasma that had similar chromatographic mobility to synthetic Ucn1-40. These data suggest that Ucn is produced and processed into the same form of synthetic Ucn in placenta, but not secreted into maternal blood.     

Effects of growth hormone and hypophysectomy of pregnant rats on serum concentrations of pregnancy-associated murine protein-1

Continuous infusion of bovine GH to hypophysectomized non-pregnant rats increased serum concentrations of pregnancy-associated murine protein-1 (PAMP-1) to the levels of adult female rats and pregnant rats. Serum concentrations of PAMP-1 were followed from Day 16 of gestation until 3 days after parturition in hypophysectomized (on Day 14 of gestation) and intact pregnant rats. In the intact pregnant rat there was a decrease in PAMP-1 values from Day 16 until delivery. The serum concentrations of PAMP-1 in hypophysectomized pregnant rats were similar to those in intact pregnant rats before parturition, but PAMP-1 concentrations decreased markedly after parturition in the hypophysectomized rats. We suggest that the serum concentrations of PAMP-1 can be maintained without pituitary GH in late pregnancy, while serum values of PAMP-1 in non-pregnant rats is dependent upon a continuous secretion of pituitary GH.     

Levonorgestrel as a new radioligand for determination of sex hormone binding globulin in human plasma

Levonorgestrel which binds with high affinity to SHBG, is suggested as a new radioligand for estimation of SHBG in human plasma. Using 3H-levonorgestrel as a ligand, a number of samples from men, women pregnant and non-pregnant were analysed. The SHBG content was lowest in men, low in women and rose to higher level during pregnancy. The results of the present study suggest that levonorgestrel appears to be a better ligand than dihydrotestosterone for measuring SHBG.     

Demonstration of beta-casomorphin immunoreactive materials in in vitro digests of bovine milk and in small intestine contents after bovine milk ingestion in adult humans

Healthy young volunteers ingested one liter of cows' milk; then the contents of the small intestine were aspirated through an intestinal tube at various times and assayed for the presence of bovine beta-casomorphin immunoreactive materials. Considerable amounts of beta-casomorphin-7, but no beta-casomorphin-5 and only small amounts of beta-casomorphin-4 or -6 immunoreactive materials were found. Chromatographical characterization showed that most of the beta-casomorphin-7 immunoreactive material was not identical with beta-casomorphin-7, whereas the major part of the beta-casomorphin-4 or -6 immunoreactive materials might be identical with their corresponding beta-casomorphins. Analogous results were obtained for in vitro digestion of bovine milk which had been designed as a rough imitation of the gastrointestinal digestion process. A regulatory influence of beta-casomorphins as "food hormones" on intestinal functions is suggested.     

Fetal role in the control of parturition in the tammar, Macropus eugenii

When female tammars carrying dormant blastocysts were injected with progesterone at the time of removal of their pouch young the development of the fetus was advanced and parturition occurred 5 days earlier than in the control tammars. In these tammars the prolactin pulse was also advanced by 5 days but the usually concomitant fall in progesterone was not. In non-pregnant tammars similar injections of progesterone did not advance the subsequent fall in progesterone, oestrus, or the LH pulse. In non-pregnant tammars injected with ovine prolactin on Day 26, to mimic the prolactin pulse, plasma progesterone was reduced to basal levels within 12 h, significantly earlier than in controls. Conversely, in 5 pregnant and 1 non-pregnant tammar injected with ovine prolactin on Day 23, to mimic the condition induced by advancing the time of parturition with progesterone, the decline in plasma progesterone was not advanced and the endogenous prolactin pulse, parturition, post-partum ovulation and the LH pulse all occurred after intervals similar to those of controls. The results support the view that the fetus is associated with the pre-partum prolactin pulse in maternal plasma and that a prolactin pulse at this stage is luteolytic in non-pregnant tammars.     

Circadian Periodicity of Serum Prolactin Concentration in Man

Immunoreactive human serum prolactin of pituitary origin has been measured by a radioimmunoassay developed for ovine prolactin. Blood samples were collected at four-hour intervals during a 24-hour period from 12 non-pregnant women, three pregnant women, and seven adult men. A circadian periodicity was found in serum prolactin concentration, with peak values during the night, between 1 a.m. and 5 a.m. for the non-pregnant women, and at 5 a.m. for the adult men. Mean serum levels of prolactin were 1·5 times higher in non-pregnant women than in men. In women investigated during the last month of their pregnancy the mean serum prolactin levels were 2·3 times higher than in the non-pregnant women, but there was no circadian periodicity.     

Morphology of the porcine myometrium during parturition

The ultrastructure of the porcine myometrium collected at well-defined stages during parturition was investigated by transmission electron microscopy. The morphology of the parturient myometrium resembled in general that in pregnant and non-pregnant pigs. The diameter of the smooth muscle cells was, however, about twice that of non-pregnant myometrium. Thick myofilaments were numerous. The number of caveolae seemed to be higher in parturient compared with non-pregnant and pregnant cells. Gap junctions occurred richly and were large, while the intrinsic innervation was very scanty. To conclude, the endocrinological changes in the pig taking place just prior to parturition, are translated into morphological changes by stimulating the formation of uterine gap junctions. This provides low-resistance pathways between the muscle cells and activates the myometrium for the delivery process.     

Oxytocin in the cerebrospinal fluid and plasma of pregnant and nonpregnant subjects

The oxytocin concentration in the cerebrospinal fluid (CSF) and plasma of pregnant women at term with and without labor pain were measured by radioimmunoassay and compared with those of non-pregnant women of matched age. The oxytocin concentrations in the CSF were 4.9 +/- 4.1 microU/ml (mean +/- S.D.) in pregnant women with labor pain, 4.1 +/- 2.4 microU/ml in those without labor pain and 4.0 +/- 2.8 microU/ml in nonpregnant women, and the oxytocin concentrations in the plasma of these subjects were 45.2 +/- 19.6, 17.1 +/- 22.2 and 7.0 +/- 5.3 microU/ml, respectively. Thus the oxytocin level in the CSF did not change appreciably even when the level in the plasma was raised in the pregnant women with labor pain. These findings suggest that oxytocin does not penetrate the blood-brain barrier, and that oxytocin in the CFS has little or no central role in parturition in women.     

Cortisol and susceptibility to malaria during pregnancy

We measured cortisol and prolactin concentrations in the peripheral venous blood of 23 non-pregnant and 59 pregnant Gabonese women from the second trimester of pregnancy until delivery. Cortisol concentrations were significantly higher in primigravidae women than in multigravidae women between 20 and 25 weeks' gestational age (166 vs. 132 ng/ml, respectively), between 28 and 37 weeks (226 vs. 161 ng/ml) and at delivery (287 vs. 188 ng/ml). Conversely, plasma prolactin levels were highest in multigravidae women. Cortisol and prolactin concentrations both increased with the period of pregnancy (P = 0.01 and P < 0.01, respectively), suggesting that a sustained increase in cortisol level underlies the increased susceptibility of pregnant women, particularly primigravidae women, to malaria. In support of this hypothesis, we found a significant association between cortisol concentration and Plasmodium falciparum infection, on the one hand, and strong correlations with parasite load in P. falciparum-infected primigravidae women, on the other hand (rho between 0.35 and 0.45 with P < 0.01).     

Structural characterization of calcitonin gene-related peptide purified from rabbit intestine

Calcitonin gene-related peptide (CGRP) immunoreactive material has been found in extracts of the intestine, however, the structure of intestinal CGRP is not known. Analytical reverse phase HPLC and ion-exchange FPLC revealed one predominant immunoreactive CGRP peak in rabbit intestinal extracts. This material was purified from rabbit intestine by sequential steps of reverse phase HPLC and ion-exchange FPLC. Microsequence and mass spectral analysis of the purified peptide and its chymotryptic fragments were consistent with the structure: GCNTATCVTHRLAGLLSRSGGMVKSNFVPTNVGSEAF-amide. Rabbit intestinal CGRP is identical to human CGRP-II in 35 of 37 amino acid residues. Two amino acid differences were detected at position 1, with Gly in rabbit CGRP instead of Ala in human CGRP-II, and at position 35, with Glu instead of Lys, respectively. Rabbit CGRP differed from human CGRP-I by three additional amino acids at positions 3, 22, and 25. This report shows that a CGRP form which closely resembles human CGRP-II, by means of chemical characterization, is the predominant form in rabbit intestine. Rabbit CGRP is the only CGRP form which has Gly as the amino terminal amino acid. Since the amino terminus of CGRP seems to be important for expression of bioactivity, the biological activity of rabbit CGRP may differ from human, rat and porcine CGRP.     

Atrial natriuretic peptide in the sheep

To study atrial natriuretic peptide (ANP) physiology in the chronically catheterized pregnant sheep model we developed a heterologous radioimmunoassay for ovine ANP using an antiserum raised against 1-28 human ANP. This antiserum (Tor I) is specific for the aminoterminus of the human ANP molecule and shows little cross reaction with any carboxyterminus ANP fragments. Ovine ANP immunoreactivity was characterized using this antiserum and a commercially available carboxyterminus ANP antiserum obtained from Peninsula Laboratories. Each antiserum detected 2 peaks of immunoreactivity in ovine atrial extracts chromatographed on a Biogel P-10 column. The minor peak migrated at a position close to 125I-human ANP whereas the major peak represented a larger molecular weight species of ANP. Examination of gel filtration eluates of ovine plasma extracts showed one immunoreactive ANP peak using the Tor I assay system and 2 peaks with the Peninsula Laboratories assay. Plasma immunoreactive ANP levels were determined in 9 sheep using both radioimmunoassay systems. Mean (+/- SEM) levels were similar using the Peninsula Laboratories and the Tor I assay systems (57 +/- 8 pg/ml versus 43 +/- 4 pg/ml, P greater than 0.05). Using the Tor I antiserum, fetal plasma immunoreactive ANP levels were found to be significantly higher than maternal levels (188 +/- 17 versus 48 +/- 8 pg/ml, P less than 0.01) whereas pregnant and nonpregnant adult sheep had similar plasma immunoreactive ANP levels (48 +/- 8 versus 43 +/- 4 pg/ml, P greater than 0.05). Disappearance curves of synthetic human ANP from the plasma of maternal and fetal sheep were assessed using both immunoassay systems and found to be similar.     

Plasma levels of the immunomodulatory cytokine interleukin-10 during normal human pregnancy: a longitudinal study

Pregnancy is proposed to be a Th2 phenomenon, where Th2 cytokines inhibit Th1 responses to improve foetal survival. The importance of interleukin-10 (IL-10), an immunomodulatory cytokine produced by Th2 cells, in the maintenance of normal pregnancy is becoming increasingly apparent. In a longitudinal case-control study, the physiological effect of pregnancy on plasma IL-10 was investigated. The plasma concentration of IL-10 was determined using an ELISA technique in 99 pregnant women sampled at 12, 20 and 35 weeks of gestation, 38 non-pregnant control subjects sampled in parallel and in a subgroup of women sampled at 3 days post-partum (n, pregnant 21, non-pregnant 21). Plasma IL-10 was significantly higher in pregnant women at 12, 20 and 35 weeks of gestation (p<0.05, p<0.01 and p<0.0001, respectively), and in mothers post-delivery (p<0.01) when compared to non-pregnant control subjects. Furthermore, there was no significant effect of gestational time on IL-10 concentration. Results from the current study suggest that elevated IL-10 is a physiological consequence of normal healthy pregnancy. These findings help clarify previous conflicting results and establish a range for plasma levels of IL-10 in normal healthy pregnancy.     

Elevated plasma beta-endorphin levels in pregnant women and their neonates.

Using radioimmunoassay technique β-endorphin levels were measured in the plasma of women undergoing labour and partirition and in the plasma of their neonates. The level of immunoreactive β-endorphin in the plasma of women undergoing labour was found to be significantly elevated (mean values: 38–135 fmoles/ml) above the levels found in non-pregnant women (mean values: 5–10 fmoles/ml). After birth, the level of β-endorphin-like immunoreactivity in maternal venous plasma was significantly higher than that in the umbilical vein and artery plasma of the new-borns, but there was no arterio-venous difference in the neonatal plasma. Since the antiserum used displayed the same avidity for human β-endorphin and β-lipotropin chromatographic separation of the immunoreactive components was performed by gelfiltration. Both peptides were found in the plasma of non-pregnant women, in maternal plasma and in the plasma of the neonates. In addition, high amounts of both peptides were found in the fetal pituitary gland showing that the fetus can probably produce its own peptides.     

Transplacental transfer of medetomidine and ketamine in pregnant ewes

The extent of placental transfer of medetomidine and ketamine is unknown in pregnant ewes. Date-mated singleton (n = 8) and twin (n = 8) pregnant merino cross ewes were anaesthetized for Caesarean delivery of preterm lamb fetuses. A combination of medetomidine (20 μg/kg) and ketamine (10 mg/kg) was administered by intravenous injection and surgery performed immediately thereafter. Blood samples were collected from the ewe at one, five and 10 min after intravenous injection and from the umbilical vein of the fetus at delivery. Non-pregnant ewes were also anaesthetized (n = 8). There was no difference in the plasma concentration of medetomidine or ketamine when comparing singleton and twin ewes or pregnant and non-pregnant ewes for the short duration of the study. Fetal plasma concentrations of each drug were comparable to the maternal concentrations at the same time. We conclude that both drugs cross the placenta readily and provide anaesthesia and analgesia for the fetus when it is delivered.     

Prostaglandin F2 alpha-induced prolactin release and luteolysis in the goat.

Injection of prostaglandin F2 alpha (PGF2 alpha) initiated a significant increase in plasma prolactin levels in all goats except those in anoestrus. Luteolysis occurred in non-pregnant goats during the mid luteal phase when the goats were given PGF2 alpha either with or without the suppression of prolactin release by bromocryptine (CB154). Luteolysis and subsequent parturition also occurred in pregnant goats in mid and late gestation after PGF2 alpha injection, with an associated release of prolactin and decrease in plasma progesterone. Acute prolactin release in response to injection of thyrotrophin releasing factor may have had a transient effect on plasma progesterone levels, but did not appear to be luteolytic in either pregnant or non-pregnant goats.     

Development of the hypothalamic-pituitary-axis in the ovine fetus: ontogeny of action of ovine corticotropin-releasing factor

In samples from twenty chronically cannulated ovine fetuses the plasma immunoreactive adrenocorticotrophin (ACTH) concentrations were 12.5 +/- 3.2(8), 15.2 +/- 4.1(9) and 21.2 +/- 5.6(8) pg/ml at periods, prior to parturition, of -30 to -35, -25 to -29 and -20 to -24 days respectively. Values are mean +/- SEM (number of samples). These values were not significantly different from each other but were significantly lower (P less than 0.02) than values in the next two age groups -36.0 +/- 4.9(7) pg/ml at -19 to -15 days, and 39.6 +/- 6.6(11) pg/ml at -14 to -9 days. A further significant increase (P less than 0.05) occurred in the -8 to -3 day period, ACTH being 53.9 +/- 5.4(12) pg/ml. On day of delivery two samples had values of 325 and 360 pg/ml. A single injection, intravenously of 1.0 microgram ovine corticotrophin-releasing factor (O-CRF), caused a significant increase in fetal plasma ACTH concentrations in fetuses of -6 to -23 days prior to delivery but not in fetuses -24 to -35 days prior to parturition. The maximum values of ACTH after O-CRF were significantly greater in fetuses -2 to 0 days prior to parturition than in younger fetuses (P less than 0.01). In 6 experiments in 4 fetuses (parturition -1 to -13 days) the effect of 1.0 microgram O-CRF persisted for at least 2.5 h. The results support the hypothesis that the pituitary release of ACTH changes sensitivity to hypothalamic O-CRF at least twice during the last fifth of gestation; an increasing sensitivity is seen as term approaches.     

Protein profiling underscores immunological functions of uterine cervical mucus plug in human pregnancy

The cervical mucus plug (CMP) differs from the cervical secretions of non-pregnant women, and is the ultimate sealant of the uterine cavity during pregnancy. Although several studies have analyzed biochemical properties of large glycoproteins in the CMP, comprehensive information about its protein composition is yet unavailable. We hypothesized that protein profiling of the CMP could provide key clues to its physiological functions in pregnancy. For this purpose, five CMPs obtained from women in labor at term were analyzed by LC-MS/MS. Out of 291 total proteins identified, 137 were detected in two or more samples, which included S100A8, S100A9, and complement proteins (C3, C4a, C4b, C6, and C8g). Several proteins, which have not been described in the cervical mucus of non-pregnant women or in cervicovaginal fluids, such as CD81 antigen and pregnancy zone protein, were also identified. Gene ontology analysis of identified proteins showed significant enrichment of 28 biological processes such as 'activation of plasma proteins involved in acute inflammatory response' and 'positive regulation of cholesterol esterification'. We report the proteome of CMPs from pregnant women at term for the first time, and the overall findings strongly suggest an important role for the CMP in the maintenance of pregnancy and parturition.     

Rate of clearance of immunoreactive hCG from the serum of the pregnant bonnet monkey (Macaca radiata)

The rate of clearance of immunoreactive hCG (human chorionic gonadotrophin) from serum in six pregnant and three non-pregnant monkeys was determined by radioimmunoassay, following the i.v. injection of 2 mg of highly purified hCG. The study revealed that the disappearance of hCG takes place in two phases in both groups, an initial fast (197.5 ± 14.5 min for pregnant and 94 ± 4.8 min for non-pregnant monkeys) phase, followed by a slow (1230 ± 62 min for pregnant and 966 ± 43 min for non-pregnant monkeys) phase. The rate of clearance appears to be faster in non-pregnant than in pregnant monkeys.     

Prostaglandin Release at Dexamethasone Induced Parturitions in Cows

Three cows of the Swedish Red and White Breed (SRB) were used in the experiment. Cow no. 1 pregnant for 247 days, was given 20 mg dexamethasone twice with an interval of 48 hrs. Cows nos. 2 and 3, each pregnant for 254 days, received 20 mg of dexamethasone twice with an interval of 24 hrs. The cows delivered normal living calves 153, 138 and 137 hrs., respectively, after the second injection of dexamethasone. Blood samples were drawn from the jugular vein every third hour during the experimental period and the samples were analyzed for estrone, progesterone and 15-keto-13,14-dihydroprostaglandin F2α. Following the dexamethasone injections there was a continuous increase in the blood plasma levels of estrone followed by a sharp decrease in conjunction with parturition. The blood plasma level of progesterone showed a slow but continuous decrease until about 24 hrs. before delivery when a marked drop occurred. The levels of the prostaglandin metabolite increased gradually until about 24 hrs. prior to delivery. This was followed by an abrupt rise, and high levels of the prostaglandin metabolite were recorded for up to four days following parturition. It is concluded that the estrone increase preceded that of the prostaglandin metabolite and that the final drop in the progesterone was synchronous with the final rise of the prostaglandin metabolite level.