Comparative in vitro responses of fetal, pregnant and non-pregnant rabbits' adrenal glands to steroidogenic agents

The in vitro secretion of aldosterone and corticosterone by the adrenal glands of fetal (day 30), pregnant and non-pregnant rabbits was examined under basal and stimulated conditions. In general, non-pregnant animals basally secreted less aldosterone than either pregnant or fetal rabbits, whereas basal corticosterone secretion by pregnant animals exceeded that of either fetal or non-pregnant animals. At similar doses of adrenocorticotropin (ACTH), fetal and pregnant adrenal glands produced comparatively more aldosterone than non-pregnant animals, while corticosterone secretion was accelerated to a greater degree in fetal rabbits than in the other groups. Angiotensin II had its greatest effect on the aldosterone secretory rates of fetal and non-pregnant animals without affecting corticosterone secretion in any group. Elevated potassium (K+) enhanced the secretory rates of aldosterone and corticosterone in fetal animals, while increasing only aldosterone secretion in non-pregnant rabbits. Serotonin accelerated aldosterone secretion in all animals, whereas it increased corticosterone secretion only in non-pregnant animals. These results suggest that (1) in fetal rabbits, the secretory rates of both aldosterone and corticosterone are regulated primarily by ACTH and to a much lesser extent by angiotensin II and K+, (2) the corticosterone secretory rates of pregnant and non-pregnant rabbits are controlled mainly by ACTH, and (3) aldosterone secretion by non-pregnant animals is regulated primarily by angiotensin II and secondarily by ACTH and K+, while in pregnant animals ACTH may be the primary regulator of aldosterone secretion as it is in the fetus.     

Reaction of the leukocytes of melanoma patients and control donors,including pregnant women,with melanoma- and fetus-derived materials

Summary Using a one-stage capillary leukocyte migration inhibition assay, we examined peripheral blood leukocytes from 97 patients with malignant melanoma, 194 pregnant women, and 123 non-pregnant donors for their reactivity with materials from fetal and melanomatous tissues. As in previous studies, we found melanoma extracts selectively to inhibit melanoma patients' leukocytes. First-trimester fetal extracts also selectively inhibited melanoma patients' leukocytes, suggesting their cross-reactivity with melanoma-derived antigens. We could not localize the source of the inhibitory materials within the fetuses. We found no evidence that pregnant women were selectively immunized to fetal or melanoma extracts, regardless of their parity or the stage of their pregnancy.     

The concentrations of soluble HLA-G protein are elevated during mid-gestation and decreased in pre-eclampsia

The aim of our study was to investigate the dynamics of the alterations of soluble human leukocyte antigen-G (sHLA-G) concentrations in sera of healthy non-pregnant women, as well as healthy pregnant women and patients with pre-eclampsia. Thirty five patients with pre-eclampsia, 52 healthy pregnant women, and 24 healthy non-pregnant women were included in the study. Sera concentrations of sHLA-G protein were determined using the immunoenzymatic ELISA method. Statistical analysis was performed using ANOVA and Mann-Whitney U tests. The concentrations of sHLA-G protein in sera of pregnant women in the first, as well as the second and third, trimesters of normal pregnancy were significantly higher in comparison with healthy nonpregnant women. The sera concentrations of sHLA-G in pregnant women in the second trimester of pregnancy were significantly higher compared to the first and third trimesters. The concentrations of sHLA-G in sera of patients with pre-eclampsia were significantly lower than in pregnant women in the third trimester of physiological pregnancy. The results of our study suggest that normal physiological pregnancy is associated with elevated sera concentrations of sHLA-G molecule. The increased concentrations of sHLA-G molecule in mid-gestation could suggest a role for the protein in the second phase of a physiological invasion of extravillous cytotrophoblast to spiral arteries. Furthermore, the results could suggest a role for the decreased sera concentrations of sHLA-G in the pathogenesis of pre-eclampsia.     

Proteolysis of insulin-like growth factor binding protein-3 in serum from pregnant, non-pregnant and fetal rats by matrix metalloproteinases and serine proteases.

The insulin-like growth factors (IGFs) in adult mammalian plasma circulate predominantly in 150-kDa complexes that also contain IGF-binding protein-3 (IGFBP-3) and an acid-labile subunit. Proteolysis of IGFBP-3 within the 150-kDa complex decreases its affinity for IGFs, facilitating their release to the tissues. By contrast, 150-kDa complexes are not detected in serum from fetal or pregnant adult rats. Decreased complex formation results from insufficient availability of IGFBP-3 due to increased IGFBP-3 proteolysis. The present study characterizes IGFBP-3 protease activity in serum from fetal, pregnant and non-pregnant adult rats by comparing the effect of different protease inhibitors. Proteolysis of exogenous recombinant human IGFBP-3 (for fetal and pregnancy serum) or endogenous IGFBP-3 (for non-pregnant adult rat serum) following incubation at 37 degrees C was measured by ligand blotting. In all three sera, IGFBP-3 proteolysis was inhibited completely by: (i) EDTA, a chelator of divalent cations. Inhibition was reversed by zinc, but not by calcium ions; (ii) 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF), an inhibitor of serine proteases; and (iii) a specific tissue inhibitor of matrix metalloproteinases (TIMP-1). Recombinant human matrix metalloproteinase-3 (MMP-3) proteolyzed recombinant human IGFBP-3 or endogenous rat IGFBP-3 in non-pregnancy serum pretreated with AEBSF to inactivate endogenous serine proteases. These results suggest that serine proteases initiate the activation of latent MMP precursor, and that the activated MMP directly degrades IGFBP-3.     

Different isoforms of the soluble leptin receptor in non-pregnant and pregnant mice

Leptin circulates in murine serum in a free and a bound form. As shown in humans, a soluble leptin receptor (sOB-R), which modulates the effects of its ligand, circulates in murine blood. The aim of our study was to determine abundance and biochemical nature of this protein. For the quantification of sOB-R we developed a ligand-immunofunctional assay (LIFA) which is based on both, leptin binding and immunological recognition. The use of this LIFA revealed that during late gestation sera of pregnant mice had a approximately 290-fold higher level of sOB-R than non-pregnant animals. As investigated by size exclusion chromatography these mice sera demonstrated a co-elution of their leptin binding activity with leptin immunoreactivity and levels of sOB-R measured by LIFA. Therefore, it has to be concluded that sOB-R represents the major leptin binding activity in murine circulation. The molecular analysis of sOB-R by Western blot and by cross-linking with 125I-leptin in sera of pregnant and non-pregnant mice demonstrated two different isoforms of sOB-R, which were capable of leptin binding. The sOB-R in serum migrated at a molecular weight of 150kDa in pregnant and only of 120kDa in non-pregnant animals. Deglycosylation of these isoforms led to sOB-R molecules which were found at the same molecular weight in SDS-PAGE. This finding indicates that both isoforms differ only in the degree of their glycosylation. In conclusion, the non-pregnant and the pregnant states are accompanied by differently glycosylated isoforms of sOB-R whose physiological relevance remains to be determined.     

Antibodies to rubellavirus, herpes simplex virus and Toxoplasma gondii in serial serum samples of pregnant and non-pregnant women.

By testing serial serum samples of 213 pregnant women for rubellavirus, of 196 for herpes simplex virus and of 134 for Toxoplasma gondii, it was found that during pregnancy there was a fall in the humoral antibody level. Presence and titre of antibodies were lower in sera of pregnant than of non-pregnant women. Alteration of the humoral antibody level during pregnancy may influence serological studies aimed at clarifying the role of infections in fetal malformations. Serial serum samples (4 samples from each pregnant woman involved) should be tested for obtaining reliable data regarding the frequency of infections during pregnancy.     

Two neuroactive steroids in midpregnancy as measured in maternal and fetal sera and in amniotic fluid

Allopregnanolone and pregnenolone sulfate, which are neuroactive steroids that differentially modulate the sensitivity of GABA(A) and NMDA receptors, were measured simultaneously in maternal and fetal sera and in amniotic fluid for the first time during the second trimester of gravidity. The study included 33 pregnant women, who underwent cordocentesis due to suspicion of fetal abnormality or alloimmunization. Allopregnanolone concentrations in maternal and fetal sera were similar and close to the previously found levels in healthy controls at 20 weeks of gestation The levels of pregnenolone sulfate in maternal serum were 2-3 times higher and in fetal serum, as much as 10-25 times higher than those found by others in the serum of healthy, non-pregnant women. A positive correlation between maternal and fetal allopregnanolone indicate similar 5alpha-reductase activities or the efficient transport of allopregnanolone between the two subjects. No correlation of pregnenolone sulfate levels between mother and fetus was found. This finding suggests the autonomous production of pregnenolone sulfate in mother and fetus.     

An immunochemical demonstration of a pregnancy-specific protein in the horse and its use in the serological detection of early pregnancy

Two-dimensional crossed immunoelectrophoresis of sera from pregnant and non-pregnant horses, using antisera developed against early pregnant mare serum, revealed the presence of two immunologically related proteins one of which appeared to be specific to the pregnant state. This pregnancy-specific protein had beta 2-electrophoretic mobility and was first detectable at Day 6 after successful mating with a stallion. The second protein had gamma 2-electrophoretic mobility and was present in sera from pregnant and non-pregnant horses. The proteins were termed beta 2-horse pregnancy protein and gamma 2-horse protein respectively. The latter appeared to be immunologically related to the former in that the precipitin lines of the 2 proteins showed continuity. Samples from 16 mares mated with a stallion were investigated for the beta 2-protein during the first 3 weeks after mating. Of the 11 successful matings, confirmed by ultrasonic scanning at 90 days or by a successful outcome, 10 mares showed the presence of the protein. In all of 14 non-pregnant sera taken from mares not recently mated, the protein was not detectable. The validity of detection of beta 2-protein as an indication of pregnancy was clinically significant at the 10% level. The presence of the protein in 2 out of the 5 recently mated mares that did not become pregnant may be indicative of a biochemical pregnancy that failed at a later stage of gestation.     

Epidermal growth factor stimulate cell proliferation and inhibits prolactin secretion of the human decidual cells in culture

Epidermal growth factor (EGF) has been found to be mitogenic in a variety of tissues. We investigated the biological effect of EGF on early pregnant human decidua and the non-pregnant decidualized human endometrium in the primary cell culture. EGF had a stimulatory action on cell proliferation in the early pregnant decidual cells and an inhibitory effect on prolactin (PRL) secretion from the decidual cells. The addition of progesterone into culture medium suppressed cell proliferation of decidual cells, whereas it enhanced PRL secretion from decidua. The analysis of the specific receptor for EGF in the early pregnant decidua and non-pregnant decidualized endometrium revealed that both tissues had a single component EGF receptor with a dissociation constant of nM order. These results suggest that EGF may play a role in the growth and function of endometrial stromal cells.     

Ontogeny of hematological cell and biochemical profiles in maternal and fetal baboons (Papio species)

The normal ranges of hematological cell profiles and biochemistry are documented in adult non-pregnant, pregnant, juvenile, and neonatal baboons. Despite the extensive use of the baboon as a model for the study of various aspects of pregnancy, there is no data from paired mothers and their fetuses at different stages of gestation. Hematologic and biochemical profile data were obtained from eight non-pregnant female baboons, 37 mothers and 38 fetal baboons at 30 +/- 2, 90 +/- 2, 125 +/- 2, and 175 +/- 2 days of gestation (mean +/- range; dGA; term, 180 dGA). Changes observed in fetal and maternal blood during normal baboon pregnancy were similar to those reported in human pregnancy. The level of alkaline phosphatase was two times higher in fetal blood circulation than that reported in human pregnancy.     

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.     

Inhibitory effect of GnRH on isolated rat uterine muscle contractility

The effect of GnRH upon uterine contractions of both non-pregnant and pregnant rats was examined in vitro. In the non-pregnant rat uterus, GnRH inhibited in a concentration-and-time dependent manner the contractions induced by acetylcholine and oxytocin, but not those caused by bradykinin and angiotensin II. GnRH also inhibited the rhythmic contractions induced by oxytocin in uterine strips from late pregnant rats. These findings show that GnRH has a direct inhibitory effect on the rat uterine contractions, suggesting that GnRH-like substances may exert modulatory influences upon rat uterine contractility.     

Prostaglandins and oxytocin: Their effects on uterine smooth muscle

A subcellular fraction was isolated from uteri of non-pregnant and pregnant cows. ATP-dependent calcium binding was shown to take place in this fraction. This calcium binding was inhibited in a dose related fashion when increasing amounts of prostaglandin (PG) E₂ or F_2α were added to the in vitro experimental medium. The physiologically inactive PGF_1β had no inhibitory effect. Oxytocin caused inhibition of calcium binding in preparations from both pregnant and non-pregnant cows. The response to PGE₂ and PGF_2α was somewhat greater in preparations from pregnant uteri than from non-pregnant uteri. The response to oxytocin was very much greater in pregnant uteri. Because of the high PG sensitivity of calcium binding in preparations from the non-pregnant uterus, it is concluded that the PGs may be the more suitable agent in the control of reproduction.     

Distribution of Methylene Blue after Injection into the Epidural Space of Anaesthetized Pregnant and Non-Pregnant Sheep

The aim of the study was to determine the distribution of different volumes of methylene blue solution injected into the epidural space in anaesthetized pregnant and non-pregnant sheep, to evaluate its cranial distribution and to compare between them. Fifteen pregnant and fifteen non-pregnant sheep were included in the study. Sheep were anaesthetized and received 0.05, 0.1, or 0.2 mL/kg of a lumbosacral epidural solution containing 0.12% methylene blue in 0.9% saline. Thirty minutes after the epidural injection, the ewes were euthanized. The extension of the dye within the epidural space was measured, and the correlation between the volume of the dye injected and the number of stained vertebrae was evaluated. The cranial migration of the dye between pregnant and non-pregnant sheep was also compared. The results show that the volume of methylene blue injected epidurally into pregnant and non-pregnant sheep correlated directly with its cephalic distribution into the epidural space; and a volume of 0.1 mL/kg or 0.2 mL/kg stained up to the first lumbar segment in pregnant and non-pregnant sheep, respectively. Also, the results suggest that the volume of drugs administered into the epidural space of pregnant sheep should be half the volume that would be used in non-pregnant sheep.     

Acid phosphatase and leucine aminopeptidase activity in the uterine flushings of non-pregnant and pregnant gilts

The activities of uteroferrin, measured as acid phosphatase (AP), and an aminoacylpeptidase (AA) were measured in uterine flushings collected from gilts on Days 6, 8, 10, 12, 14, 15, 16 and 18 of the oestrous cycle and pregnancy (N = 37). Changes in AP (P less than 0.05) were associated with day for both specific and total AP in non-pregnant and pregnant gilts. For pregnant and non-pregnant gilts, AP activity was greatest between Days 14 and 16 and then decreased to Day 18. The AA specific activity increased (P less than 0.01) between Days 10 and 12 of the oestrous cycle and pregnancy, but neither effects of pregnancy nor day by pregnancy status interaction were detected. The AA total activity was greater for pregnant gilts (P less than 0.01). These data suggest an inhibitory effect of oestrogens of blastocyst origin on synthesis and/or secretion of uteroferrin, but not AA.     

The inhibition and reactivation of human maternal and fetal plasma cholinesterase following exposure to the organophosphate, dichlorvos.

Spontaneous and chemically-induced reactivation of organophosphate-inhibited cholinesterase were studied using as an enzyme source plasma obtained from non-pregnant females, pregnant females at term and their respective neonates, sampled immediately following delivery. Aliquots of plasma were incubated with dichlorvos (10^?6M) for 5 min at 37°C resulting in a 96 percent inhibition of cholinesterase activity in all three groups at which time either pralidoxime chloride (10^?3M) or an equivalent volume of saline was added to the reaction flask and the restoration of cholinesterase activity was monitored over the next 120 min. Pralidoxime-mediated cholinesterase reactivation in ‘non-pregnancy’ plasma was significantly greater than that observed in either ‘maternal’ or ‘fetal’ plasma, however, no significant difference was noted in reactivation rates for these latter two groups. Significant differences were also observed in the rates of spontaneous reactivation, however, after correcting for this, there were still significant differences in the rates of pralidoxime-mediated reactivation (non-pregnant > pregnant ≥ fetal).     

Differences in the effects of vasopressin and oxytocin on rabbit myometrial activity and a possible mediation of prostaglandins

Uterine responses to vasopressin and oxytocin were monitored in non-pregnant and 3- or 6-8-day-pregnant rabbits by recording the intrauterine pressure. Oxytocin stimulated uterine activity in all groups, but the effect of vasopressin was stimulatory in non-pregnant animals, inhibitory in those 3 days post coitum and weakly stimulatory in those later in pregnancy. Inhibition of prostaglandin (PG) synthesis, by the administration of indomethacin, reduced the spontaneous uterine activity as well as the responses to oxytocin and vasopressin in the non-pregnant rabbits, but had little effect in the pregnant animals. During infusion of PGF-2alpha, PGE-1 or PGE-2 in 6-8-day-pregnant rabbits, the stimulatory response to vasopressin, although slight before the infusion, was inhibited whereas the stimulatory response to oxytocin remained virtually unchanged. The results suggest that vasopressin and oxytocin under certain hormonal conditions, are able to activated the uterine contractions by mechanisms in which the involvement of PG is not obligatory.     

Aberrant Pregnancy Adaptations in the Peripheral Immune Response in Type 1 Diabetes: A Rat Model

Introduction

Despite tight glycemic control, pregnancy complication rate in type 1 diabetes patients is higher than in normal pregnancy. Other etiological factors may be responsible for the development of adverse pregnancy outcome. Acceptance of the semi-allogeneic fetus is accompanied by adaptations in the maternal immune-response. Maladaptations of the immune-response has been shown to contribute to pregnancy complications. We hypothesized that type 1 diabetes, as an autoimmune disease, may be associated with maladaptations of the immune-response to pregnancy, possibly resulting in pregnancy complications.

Methods

We studied pregnancy outcome and pregnancy-induced immunological adaptations in a normoglycemic rat-model of type 1 diabetes, i.e. biobreeding diabetes-prone rats (BBDP; 5 non-pregnant rats, 7 pregnant day 10 rats and 6 pregnant day 18 rats) , versus non-diabetic control rats (i.e. congenic non-diabetic biobreeding diabetes-resistant (BBDR; 6 non-pregnant rats, 6 pregnant day 10 rats and 6 pregnant day 18 rats) and Wistar-rats (6 non-pregnant, 6 pregnant day 10 rats and 5 pregnant day 18 rats)).

Results

We observed reduced litter size, lower fetal weight of viable fetuses and increased numbers of resorptions versus control rats. These complications are accompanied by various differences in the immune-response between BBDP and control rats in both pregnant and non-pregnant animals. The immune-response in non-pregnant BBDP-rats was characterized by decreased percentages of lymphocytes, increased percentages of effector T-cells, regulatory T-cells and natural killer cells, an increased Th1/Th2-ratio and activated monocytes versus Wistar and BBDR-rats. Furthermore, pregnancy-induced adaptations in BBDP-rats coincided with an increased Th1/Th2-ratio, a decreased mean fluorescence intensity CD161a/NKR-P1b ratio and no further activation of monocytes versus non-diabetic control rats.

Conclusion

This study suggests that even in the face of strict normoglycemia, pregnancy complications still occur in type 1 diabetic pregnancies. This adverse pregnancy outcome may be related to the aberrant immunological adaptations to pregnancy in diabetic rats.     

Prostaglandin-induced luteolysis in pregnant and pseudopregnant rabbits and the resultant effects on the myometrial activity.

The effect of prostaglandin (PG)-induced luteolysis on the myometrial activity in 20--21-day-pregnant and 11--12-day-pseudopregnant rabbits was studied by intrauterine pressure (IUP) recording during PG infusions. The same dose of PG (10 micrograms/h during 8h) was also given to 7 non-pregnant (untreated) does that were used as controls. Peripheral plasma concentration of progesterone and oestradiol-17 beta were measured at 2-h intervals during the infusion. Plasma progesterone level decreased significantly within 2 h or the start of infusion in pregnant and pseudopregnant does and continued to decrease; at the end of 8 h, the concentrations were 31 and 41%, respectively, of the pre-infusion levels. The amplitude of uterine contractions increased significantly after 4 h in pseudopregnant does, increased slightly but insignificantly in the pregnant does and showed no significant change in the non-pregnant does during PG infusion. The amplitudes developed in the pregnant and pseudopregnant does were significantly different. The direct effect of progesterone (1--3 micrograms/h during 4 h) was also studied in 7 non-pregnant rabbits. After 2 h the amplitude of contractions had decreased markedly and the pattern of activity had become irregular. The results support the concept of a myometrial inhibitory factor other than progesterone in rabbit pregnancy and suggest that this factor(s) originates in conceptus.