Pseudopregnant Rats Research Articles

Prostaglandin F2α (PGF2α) stimulates PTGS2 expression and PGF2α synthesis through NFKB activation via reactive oxygen species in the corpus luteum of pseudopregnant rats

This study was undertaken to investigate how prostaglandin F(2α) (PGF(2α)) increases PGF(2α) synthesis and PTGS2 expression in the corpus luteum of pseudopregnant rats. We further investigated the molecular mechanism by which PGF(2α) stimulates PTGS2 expression. PGF(2α) (3 mg/kg) or phosphate buffer as a control was injected s.c. on day 7 of pseudopregnancy. Ptgs2 mRNA expression and PGF(2α) concentrations in the corpus luteum were measured at 2, 6, and 24 h after PGF(2α) injection. PGF(2α) significantly increased Ptgs2 mRNA expression at 2 h and luteal PGF(2α) concentrations at 24 h. PGF(2α) significantly decreased serum progesterone levels at all of the times studied. Simultaneous administration of a selective PTGS2 inhibitor (NS-398, 10 mg/kg) completely abolished the increase in luteal PGF(2α) concentrations induced by PGF(2α). PGF(2α) increased NFKB p65 protein expression in the nucleus of luteal cells 30 min after PGF(2α) injection, and electrophoretic mobility shift assay revealed that PGF(2α) increased binding activities of NFKB to the NFKB consensus sequence of the Ptgs2 gene promoter. Simultaneous administration of both superoxide dismutase and catalase to scavenge reactive oxygen species (ROS) inhibited the increases of nuclear NFKB p65 protein expression, lipid peroxide levels, and Ptgs2 mRNA expression induced by PGF(2α). In conclusion, PGF(2α) stimulates Ptgs2 mRNA expression and PGF(2α) synthesis through NFKB activation via ROS in the corpus luteum of pseudopregnant rats.

Expression of COX-1 and COX-2 in the endometrium of cyclic, pregnant and in a model of pseudopregnant rats and their regulation by sex steroids

BackgroundCyclooxygenases (COXs) are the rate limiting enzymes in the process of prostaglandins (PGs) synthesis, which are critical regulators of a number of reproductive processes, including ovulation, implantation, decidualization and parturition. The aim of the present study was to investigate the expression and regulation of COX-1 and COX-2 and levels of prostaglandins during rat pregnancy, in a model of pseudopregnancy and estrous cycle.MethodsUteri were collected from the cyclic rats on each day of estrous cycle, after every two days for pregnant (days 2 to 22) and pseudopregnant rats (days 1 to 9). In vitro primary endometrial stromal cells were cultured in the presence of steroid hormones and their respective inhibitors for the possible modulation of COX-1 and COX-2. Endometrial protein extracts were used for western blot analysis and tissue sections were prepared for protein localization using immunofluorescence. Measurements of PGF2alpha and PGE2 metabolites in serum were performed by enzyme immunoassay (EIA).ResultsCOX-1 expression was found to be elevated during implantation and parturition, however, the levels of COX-1 decreased during decidualization periods. COX-2 was detected during early pregnancy from day 2 to 5, increased during decidual regression, and was also expressed at the time of parturition. COX-2 protein expression was found to be increased at estrus phase in cyclic rats. Both enzymes were found to be modulated in the endometrium of pseudopregnant rats, suggesting that they are regulated by 17beta-estradiol and progesterone. A significant increase in PGE2 metabolite levels was observed on day 10, 12 and 14 of pregnancy. However, an increase in PGF2alpha metabolite levels was observed only on day 14. The concentration of both these metabolites changed during pseudopregnancy and maximum levels were observed at day 7. Significant increase in PGE2 metabolite was observed at proestrus phase, on the other hand, PGF2alpha metabolite was significantly increased at proestrus and metestrus phase. COX-2 protein was regulated by 17beta-estradiol in cultured endometrial stromal cells which was blocked in the presence of ICI-182,780.ConclusionsTaken together, these results suggest that COX-1 and COX-2 could be differentially regulated by steroid hormones and might be the key factors involved in embryo implantation, decidualization, decidua basalis regression and parturition in rats.

Evidence suggesting multiple promoting roles of luteal group IVA phospholipase A 2 in prostaglandin F 2α-induced regression in pseudopregnant rats

We evaluated effects of local administration of selective inhibitors of group IVA phospholipase A 2 (GIVA PLA 2) and cyclooxygenase (COX) on exogenous prostaglandin (PG) F 2α-induced luteal regression in pseudopregnant rats. Intra-bursal treatment with a GIVA PLA 2 inhibitor AACOCF 3 just prior to PGF 2α (30 μg, subcutaneously) on day 6 of pseudopregnancy (PSP6) prevented a decline in circulating progesterone and inhibited TUNEL-positive reactions of steroidogenic cell. Its treatment on PSP9 failed to inhibit functional regression, but reduced significantly apoptosis of steroidogenic cells and vascular endothelial cells, and suppressed the infiltration of macrophages. A COX-2-selective inhibitor NS398 inhibited the decline of progesterone and apoptosis of steroidogenic cells on PSP6 but not on PSP9. A COX-1 inhibitor SC560 exerted insignificant anti-luteolytic effects. Overall data suggest that luteal GIVA PLA 2 plays multiple promoting roles in PGF 2α-induced luteal regression at least partly by a COX-2 activity-related mechanism in pseudopregnant rats.

Hormone Interactions Regulating Energy Balance During Pregnancy

Appetite and food intake are increased during pregnancy, comprising an adaptive response that facilitates energy storage in preparation for the high metabolic demands of pregnancy and subsequent lactation. To maintain the increased energy intake in the face of increased adiposity and rising leptin levels, pregnant females become resistant to the central anorectic actions of leptin. In rats, pregnancy-induced leptin resistance is characterised by elevated neuropeptide Y and reduced pro-opiomelanocortin expression in the arcuate nucleus, reduced leptin receptor mRNA levels and suppression of leptin-induced phosphorylated signal transducer and activator of transcription-3 protein in the ventromedial hypothalamic nucleus, as well as a loss of anorectic responses to both leptin and alpha-melantocyte-stimulating hormone. Our recent data suggest that this leptin-resistance may also cause central insulin resistance and an altered peripheral glucose homeostasis. The specific hormone changes during pregnancy that might mediate these effects on leptin signalling are a current focus of investigation. In pseudopregnant rats, chronic i.c.v. infusion of ovine prolactin to mimic patterns of placental lactogen secretion that occur during pregnancy completely blocked the ability of leptin to suppress food intake. These data suggest that placental lactogen secretion may mediate the hormone-induced loss of response to leptin during pregnancy. This action of prolactin/placental lactogen appears to be mediated downstream of the primary leptin-responsive neurones in the mediobasal hypothalamus, possibly in the paraventricular nucleus. Our studies show complex hormone-induced adaptations in the normal hypothalamic pathways regulating body weight homeostasis during pregnancy.

Pregnancy and Pseudopregnancy: Physiological States with High Endogenous Ouabain, Coexistent Suppression of Maternal Arterial Myocyte NCX and α2 Na+ Pumps, and Resistance to Ouabain‐Induced Hypertension

Endogenous ouabain (EO) is elevated and correlates with blood pressure (BP) in ~50% of patients with essential hypertension. “Ouabain‐like” inhibitors are increased in pre‐eclampsia (PE) and correlate with BP. EO and ouabain inhibit the α2‐Na+ pump isoform in arterial myocytes, enhance Ca2+ entry via NCX1.3, and raise BP. We explored whether EO per se is elevated in pregnancy and pseudopregnancy (PSP) and determined whether exogenous ouabain induces PE in otherwise normal pregnant rats. We implanted mini‐osmotic pumps containing ouabain in pregnant (E10), non‐pregnant, and PSP rats and measured BP by tail cuff. At E20 or equivalent, blood, adrenals, and aortas were collected to determine ouabain (RIA), ouabain‐like materials (RRA), NCX, and α2‐Na+ pump expression (Western blot). Plasma EO increased in pregnant (4.7 ± 1.9 nM plasma equiv) vs non‐pregnant (0.3 ± 0.02 nM plasma equiv, p<0.001) rats and was confirmed by HPLC‐RIA and mass spectrometry. Adrenal EO content was lower in pregnant (12.3 ± 5.0 nmoles/kg) vs non‐pregnant rats (32.5 ± 15.3 nmoles/kg, p<0.01). NCX and α2‐Na+ pump expression decreased in pregnancy. Ouabain infusion raised BP in non‐pregnant rats (+21.3 mmHg, p<0.01), but had no effect on BP in their pregnant or PSP counterparts. These data show: 1) that the pregnant and PSP conditions are physiologically high EO states; 2) both conditions are associated with the development of a physiological resistance to ouabain that prevents BP from increasing. 3) The mechanism of the ouabain resistance is exclusively maternal, and likely related to decreased expression of, and reduced Ca2+ entry by, vascular NCX. Failure of the maternal circulation to develop resistance to EO may underlie hypertension in PE.

Decidualization results in the polarization of Ang‐(1‐7), Ang II, and ACE2 to the antimesometrial pole

The two regions of the decidualized or pregnant uterine horn are programmed for different roles with the antimesometrial (AM) pole participating in implantation (embryo, oil), while the mesometrial (M) pole is fundamental for placenta blood flow. The present study was undertaken in order to understand the immunocytochemical (ICC) distribution of Ang II, Ang‐(1‐7), ACE and ACE2 in the non‐decidualized (ND) as compared to oil‐induced decidualized (OI) uterine horn of pseudopregnant rats. Ovariectomized adult rats were sensitized for the decidual cell reaction with steroid treatment (progesterone and 17‐β estradiol) and oil was injected into the left horn; the right horn served as control. For ICC, antibodies used were against Ang‐(1‐7) (1:25), Ang II (1:750), ACE2 (1:150), and ACE (1:25). In the ND horn, Ang‐(1‐7), ACE2, Ang II and ACE showed high expression in the glandular and luminal epithelium. Both Ang II and ACE were distributed throughout the ND horn, whereas Ang‐(1‐7) and ACE2 were predominately localized in the M end. In the decidualized horn, Ang II, Ang‐(1‐7) and ACE2 were localized more prominently at the antimesometrial (AM) pole; ACE was localized more at the M end. In conclusion, our studies show that the distribution of Ang II, Ang‐(1‐7) and ACE2 shifted to the AM pole with decidualization, with ACE shifting to the M. The differential expression of Ang II, Ang‐(1‐7) with ACE2 and not ACE in the AM pole of the decidualized horn favors the formation and action of Ang‐(1‐7) in the AM, an area which plays a prominent role in the implantation process. This research was supported by NHLBI PO1‐HL051952.

Group IVA phospholipase A 2 activity may mediate prostaglandin F 2α-induced luteal regression in pseudopregnant rats

We investigated role(s) of luteal group IVA phospholipase A 2 (GIVA PLA 2) in prostaglandin (PG) F 2α-induced regression in pseudopregnant rats. Prostaglandin F 2α (PGF 2α) treatment of day 6 pseudopregnant rats stimulated luteal PLA 2 activity, which was sensitive to inhibitors and associated with increased GIVA PLA 2 immunoreactivity. Intra-bursal treatment with the enzyme inhibitor (AACOCF 3) prior to PGF 2α failed to prevent the initial decline in progesterone but induced subsequently a persistent rise that was significantly higher than that of vehicle-treated group. TUNEL-positive signals in luteal cells of control group were reduced by AACOCF 3 treatment. TUNEL-positive reaction induced in luteal cells in vitro by combined cytokines and agonistic anti-Fas were both reduced by AACOCF 3 and another inhibitor pyrrophenone. Overall data show that luteal GIVA PLA 2 activity and expression increased following PGF 2α administration and that acute chemical inhibition of this activity could reverse, at least partly, PGF 2α-induced functional regression and prevent apoptosis induced by PGF 2α in vivo and by cytokines in vitro.

Increased expression of heat shock protein 105 in rat uterus of early pregnancy and its significance in embryo implantation

BackgroundHeat shock proteins (Hsps) are a set of highly conserved proteins, Hsp105, has been suggested to play a role in reproduction.MethodsSpatio-temporal expression of Hsp105 in rat uterus during peri-implantation period was examined by immunohistochemistry and Western blot, pseudopregnant uterus was used as control. Injection of antisense oligodeoxynucleotides to Hsp105 into pregnant rat uteri was carried out to look at effect of Hsp105 on embryo implantation.ResultsExpression of Hsp105 was mainly in the luminal epithelium on day 1 of pregnancy, and reached a peak level on day 5, whereas in stroma cells, adjacent to the implanting embryo, the strongest expression of Hsp105 was observed on day 6. The immunostaining profile in the uterus was consistent with that obtained by Western blot in the early pregnancy. In contrast, no obvious peak level of Hsp105 was observed in the uterus of pseudopregnant rat on day 5 or day 6. Furthermore, injection of antisense oligodeoxynucleotides to Hsp105 into the rat uterine horn on day 3 of pregnancy obviously suppressed the protein expression as expected and reduced number of the implanted embryos as compared with the control.ConclusionTemporal and spatial changes in Hsp105 expression in pregnant rat uterus may play a physiological role in regulating embryo implantation.

156. EFFECTS OF ALBENDAZOLE ON OVARIAN OESTROGEN SYNTHESIS IN THE RAT

Albendazole is a drug commonly used for treatment of helminth infestation in human and livestock populations. Recent studies show that albendazole reduces ovarian follicular fluid oestrogen levels in sheep1, but the mechanism involved is unknown. The aims of this study were to determine whether albendazole exerts similar effects on ovarian oestrogen levels in the rat, and to assess the effects of albendazole on expression of key steroidogenic genes in the rat ovary. Oestrus cycles were continuously monitored in Wistar rats by vaginal smears. Commencing at proestrus, albendazole was administered for 12 days in drinking water (approximate dose 15 mg/kg/day). Plasma and whole ovaries were collected on the fourth proestrus (1500–1600h). A second group of rats were treated similarly except that pseudopregnancy (PSP) was induced by mating with a vasectomised male at the second proestrus. Plasma and the non-luteal ovary were collected on day 8 of PSP. Oestradiol was extracted from plasma and ovaries with ethyl acetate and concentrations measured by a chemiluminescent assay. Expression of steroidogenic acute regulatory protein (StAR), P450 side chain cleavage (P450scc), 3β-hydroxysteroid dehydrogenase (3β-HSD), aromatase and 20α-hydroxysteroid dehydrogenase (20α-HSD) mRNAs were measured by RT-PCR. Oestrus cyclicity, ovarian weight and mating behaviour were all unaffected by albendazole in cycling and PSP rats, although as expected levels of oestradiol were lower in PSP. In ovaries of cycling rats albendazole did not affect oestradiol concentrations but reduced ovarian P450scc mRNA expression (by 65%; P=0.024) and there was a trend for an increase in 3β-HSD (P=0.09) and aromatase expression (P=0.12). Expression of the other steroidogenic genes was unaffected and no changes in gene expression were observed in PSP rats. In conclusion, albendazole treatment reduced ovarian P450scc in cycling rats but did not inhibit ovarian oestradiol synthesis or reproductive function.

Mating‐Induced Neuroendocrine Responses During Pseudopregnancy in the Female Mouse

Pseudopregnancy (PSP) is a neuroendocrine reflex triggered by vaginocervical stimulation similar to the neuroendocrine response of early pregnancy and is characterised by short-term neural activity, resulting in long-term neuroendocrine responses that cause repeated release of pituitary prolactin (PRL) over many days. PSP is a useful model to study how somatosensory input is transduced in the brain into neuroendocrine responses, and has been extensively characterised in rats. With increasing use of mice as an experimental model, however, and to allow use of transgenic mice to investigate mechanisms of this sensory response, it is important to characterise the principal neuroendocrine response of pseudopregnancy in this species. The present study aimed to examine the induction and neuroendocrine responses of PSP in mice using vasectomised stud males, to investigate mating-induced changes in vaginal cytology, uterine growth, and PRL secretion, and to map certain aspects of somatosensory transduction by assessing the neural activity marker FOS. Unlike the induction of pseudopregnancy in rats, which can be induced simply by multiple intromissions from a male or artificial mechanical stimulation of the cervix, PSP induction in mice required the receipt of an ejaculation from a male. In mice that received PSP-inducible mating stimuli, FOS expression was observed in a slightly different range of brain regions than has been observed in rats, with increases in the bed nucleus of the stria terminalis, medial preoptic area, and ventromedial hypothalamus, but not in limbic areas examined. Moreover, PSP mice expressed a single diurnal PRL surge on day 6 of PSP. Thus, the data demonstrate important species differences in the neuroendocrine mechanisms activated in response to a mating stimulus in mice compared with rats. A clear understanding of the species-specific response will be required in interpreting research into the reproductive biology of this species.

Clomiphene citrate modulates the expression of endometrial carbohydrates (especially N-acetyl-d-glucosamine and sialic acid) in pseudopregnant rats

This study investigated the presence of carbohydrates N-acetyl-d-glucosamine and sialic acid oligosaccharides, in the uterine epithelium of pseudopregnant rats treated with ovarian hormones and Clomiphene citrate (CC) a commonly used fertility drug associated with low pregnancy rates. Ovariectomized sexually mature rats were given 0.25mg CC prior to the implantation-priming hormone sequence of 5mg progesterone for 3 days and a single dose of 0.5microg estradiol-17beta (E(2)) on day 3 (PP(PE)) and sacrificed 24h after the last hormone treatment. Uterine tissue was incubated with the lectin Triticum vulgare (wheat germ agglutinin, WGA), associated with avidin and subsequently labelled with biotinylated-ferritin for electron microscopy, a combined alcian blue/PAS technique for light microscopy and RT-PCR was run for progesterone-associated endometrial protein (PAEP) gene, a pregnancy related endometrial gene that is associated with the protein thought to express carbohydrates in the uterus and suppress immune function. The results indicate that CC administration decreases the expression of these carbohydrates both at a cellular level and genetic level when compared to the PP(PE) group. However, the lowest expression of N-acetyl-d-glucosamine and sialic acid was seen in the placebo group. The ovarian hormones were therefore shown to be important for the synthesis of these carbohydrates that are important in the implantation period and the maintenance of pregnancy. The results suggest that the effect of CC on the expression of N-acetyl-d-glucosamine and sialic acid is a significant reason why there are low pregnancy rates with CC use.

Effects of Caffeine Intake on the Development of Deciduoma in Pseudopregnant and on Embryo Implantation in Pregnant Rats.

Some epidemiologic studies have suggested that ingestion of caffeine increase the risk of spontaneous abortion, but most studies have used maternal self-reported caffeine consumption to estimate fetal exposure. This study was specially designed to test the direct effects of different doses of caffeine exposure on the decidualization of pseudopregnant and high doses used during implantation periods of pregnant rats. Uterine decidualization was induced bilaterally by twice of endometrial scratching on the morning of day 4 pseudopregnant rats. At the time of stimulation, one uterine horn installed histamine antagonist drug or different doses of caffeine; the contra lateral uterine horn was treated with saline solution as control. After 72 hours, the uterine deciduoma was significantly inhibited by histamine antagonist (p<0.05); contrast to histamine antagonist the caffeine-treated horn did not show the same inhibit results. We further estimated caffeine effect on the nature pregnant rats; they were feed with caffeine (60mg/kg/day) or saline by gastric instillation from gestation day 2 to gestation day7. At the end of this period, all of them were autopsied. There are no significant different in the total number of embryo counts or conceptus weight between these two groups. Our results indicate that caffeine has no direct effect on decidual formation in pseudopregnant rats and no direct influence on embryo implantation or conceptus weight in pregnant rats. Further work to confirm the distinct mechanisms of caffeine to influence early pregnant outcomes are required.

From feeding one to feeding many: hormone‐induced changes in bodyweight homeostasis during pregnancy

Pregnancy is associated with hyperphagia, increased fat mass, hyperleptinaemia and hyperprolactinaemia. The neuroendocrine control of bodyweight involves appetite-regulating centres in the hypothalamus, containing both orexigenic and anorexigenic neurons that express leptin receptors (LepR). In the rat, central leptin resistance develops during mid pregnancy, well after hyperphagia becomes apparent, to negate the appetite suppressing effects of leptin. We have investigated the hypothalamic response to leptin during pregnancy and examined the role of pregnancy hormones in inducing these changes. We have shown that there are multiple levels of leptin resistance during pregnancy. Despite elevated serum leptin, neuropeptide Y and agouti related peptide mRNA in the arcuate nucleus are not suppressed and may even be increased during pregnancy. LepR mRNA and leptin-induced pSTAT3 expression, however, are relatively normal in the arcuate nucleus. In contrast, both LepR and leptin-induced pSTAT3 are reduced in the ventromedial hypothalamic nucleus. Injecting alpha-melanocyte-stimulating hormone (alpha-MSH) into the brain, to bypass the first-order leptin-responsive neurons in the arcuate nucleus, also fails to suppress food intake during pregnancy, suggesting that pregnancy is also a melanocortin-resistant state. Using a pseudopregnant rat model, we have demonstrated that in addition to the changes in maternal ovarian steroid secretion, placental lactogen production is essential for the induction of leptin resistance in pregnancy. Thus, hormonal changes associated with pregnancy induce adaptive changes in the maternal hypothalamus, stimulating food intake and then allowing elevated food intake to be maintained in the face of elevated leptin levels, resulting in fat deposition to provide energy stores in preparation for the high metabolic demands of late pregnancy and lactation.

Changes in Uterine Receptor mRNAs for Oxytocin and Estrogen in the Pseudopregnant Rat

This study examined the uterine oxytocin- (OTR) and estrogen- (ER) receptor mRNA levels during and after pseudopregnancy (PSP) in rats. An increased OTR mRNA level was observed from day 14 of PSP, and the maximal level was attained during the following proestrus. The levels of ERalpha mRNA were low during PSP and significantly increased during the following estrus. The level of ERbeta mRNA was significantly decreased during proestrus and then returned to the values observed during days 7-14 of PSP by estrus. These results suggest i) suppression of ERalpha mRNA during the luteal phase and that ii) the changes in OTR, ERalpha and ERbeta mRNA levels during proestrus and estrus following PSP are similar to those during the normal estrous cycle.

Induction of Central Leptin Resistance in Hyperphagic Pseudopregnant Rats by Chronic Prolactin Infusion

Pregnancy in rats is associated with hyperphagia, increased fat deposition, and elevated plasma leptin concentrations. Elevated leptin would be expected to inhibit food intake, but hypothalamic leptin resistance develops around midpregnancy, allowing hyperphagia to be maintained and excess energy to be stored as fat in preparation for future metabolic demands of lactation. To investigate the hormonal mechanisms inducing leptin resistance during pregnancy, the anorectic response to leptin was examined during pseudopregnancy. Pseudopregnant rats have identical hormonal profiles to early pregnancy, but no placenta formation, allowing differentiation of maternal and placental hormone effects on appetite. To investigate the effect of leptin on food intake, d-9 pseudopregnant rats were injected with leptin (4 microg) via an intracerebroventricular (icv) cannula, and then food intake was measured 24 h later. Pseudopregnant rats were hyperphagic but had normal anorectic responses to leptin. We therefore hypothesized that a longer exposure time to high concentrations of progesterone might be required to mimic the leptin resistance that occurs on d 14 of pregnancy. Pseudopregnant rats were given progesterone to prolong pseudopregnancy beyond the time that leptin resistance develops during pregnancy. However, rats remained responsive to icv leptin. To model the placental lactogen secretion that occurs during pregnancy, pseudopregnant rats were given progesterone and chronic icv ovine prolactin infusion. Central icv injection of leptin had no effect on food intake in pseudopregnant rats receiving chronic ovine prolactin. These results suggest that chronically high lactogen levels, secreted by the placenta during the second half of pregnancy, induce central leptin resistance.

Regulation of prostaglandin D synthase and prostacyclin synthase in the endometrium of cyclic, pregnant, and pseudopregnant rats and their regulation by sex steroids

Prostaglandins (PGs) are critical regulators of a number of reproductive processes. To date, the presence and regulation of PGS in the rat endometrium have not yet been described. The objective of the present study was to investigate the expression of PGD synthase (PGDS) and prostacyclin synthase (PGIS) in the endometrium. Endometrial proteins and tissues were collected from cyclic non-pregnant, pregnant, and steroid-induced pseudopregnant rats. PGIS and PGDS were detected in the endometrium of cyclic, pregnant, and pseudopregnant rats but were not influenced by the estrous cycle. During early pregnancy, PGIS was significantly higher at day 5 and was gradually decreased from day 5.5 to 6.5. Later during pregnancy, PGIS was maximal on day 12 and gradually decreased to the end of pregnancy. PGDS expression was high during early and was maximal at the end of pregnancy. During pseudopregnancy, PGDS and PGIS were increased in a time-dependent manner and were maximal at day 5. Immunohistochemical analysis revealed that PGDS and PGIS were found in luminal as well as glandular epithelial cells and in stroma during late pregnancy. We also found a significant increase of PGD(2) serum metabolite at days 21 and 22 of pregnancy. During steroid-induced pseudopregnancy, PGI(2) serum metabolite was increased in a time-dependent manner and was maximal at day 7. These results suggest that PGDS and PGIS are present and could be regulated by steroids in the rat uterus during pregnancy, and that the endometrium could be a significant source of PGD(2) and PGI(2) at specific times during pregnancy.

Hormonal induction of leptin resistance during pregnancy

Despite elevated plasma leptin, food intake is increased during pregnancy leading to fat deposition. We have demonstrated that intracerebroventricular (icv) leptin is unable to suppress food intake in pregnant rats, as it does in non-pregnant animals. Hence, central leptin resistance develops during pregnancy. These changes are physiologically appropriate, providing increased energy reserves to help meet the high metabolic demands of fetal development and lactation. To characterise this central leptin resistance, we have measured levels of leptin receptor (Ob-Rb) mRNA in the hypothalamus, and examined leptin-induced phosphorylation of STAT3 (pSTAT3) in specific regions of the hypothalamus. In addition, to investigate the mechanism underlying pregnancy-induced leptin resistance, we have investigated effects of hormone treatments on hypothalamic responses to leptin in a pseudopregnant rat model. We observed a significant reduction of Ob-Rb mRNA levels in the ventromedial hypothalamic nucleus (VMH) during pregnancy, with no changes detected in other hypothalamic nuclei. Levels of leptin-induced pSTAT3 were specifically suppressed in the VMH and arcuate nucleus of pregnant rats compared to non-pregnant rats. Pseudopregnant rats were hyperphagic but did not become leptin resistant, suggesting that fetal or placental factors are required for the induction of leptin resistance. These data implicate the VMH as a key hypothalamic site involved in hormone-induced leptin resistance during pregnancy, and suggest that placental hormone secretion may mediate the hormone-induced loss of response to leptin.

Uterotrophic, Fetotoxic and Abortifacient Effect of a Malaysian Variety of Plumbago rosea L. on Isolated Rat Uterus and Pregnant Mice

Traditionally Plumbago rosea L. is used as an abortifacient in the Southeast Asian region. Methanolic root extract of a local species of Plumbago rosea L. was studied to evaluate its traditional antifertility claim. Interestingly, it was found to possess dose related inhibitory effect on uterine contractile responses elicited by oxytocic agents on isolated uteri of pregnant and pseudo-pregnant rats. Furthermore, it was found to possess significant (p < 0.05) fetotoxic activity along with mild abortive potential in pregnant mice when given orally at high doses (400 and 800 mg kg(-1)) once daily for ten days starting from day 10 of gestation. The results derived indicated possible presence of utero-active compound (s) in this plant that inhibited oxytocic agents induced uterine motility. Moreover, pronounced fetotoxic and mild abortifacient potentials observed at higher doses in pregnant mice might support its accredited traditional use to avoid unwanted pregnancy.

Potential role for peroxisome proliferator-activated receptor γ in regulating luteal lifespan in the rat

Peroxisome proliferator-activated receptor gamma (PPARgamma) has been shown to stimulate progesterone production by bovine luteal cells. We previously reported higher expression of PPARgamma in old compared with new luteal tissue in the rat. The following studies were conducted to determine the role of PPARgamma in rat corpora lutea (CL) and test the hypothesis that PPARgamma plays a role in the metabolism of progesterone and/or luteal lifespan. Ovaries were removed from naturally cycling rats throughout the estrous cycle, and pseudopregnant rats. mRNA for PPARgamma and P450 side-chain cleavage (SCC) was localized in luteal tissue by in situ hybridization, and protein corresponding to PPARgamma and macrophages identified by immunohistochemistry. Luteal tissue was cultured with agonists (ciglitazone, prostaglandin J2) or an antagonist (GW-9662) of PPARgamma. Progesterone was measured in media by RIA and levels of mRNA for 20alpha-hydroxysteriod dehydrogenase (HSD) and bcl-2 were measured in luteal tissue after culture by RT-PCR. An inverse relationship existed between the expression of mRNA for SCC and PPARgamma. There was no effect of PPARgamma agonists or the antagonist on luteal progesterone production in vitro, or levels of mRNA for 20alpha-HSD. PPARgamma protein was localized to the nuclei of luteal cells and did not correspond with the presence of macrophages. In new CL, ciglitazone decreased mRNA for bcl-2 on proestrus, estrus, and metestrus. Interestingly, GW-9662 also decreased mRNA for bcl-2 on proestrus and diestrus in old and new CL, and on metestrus in new CL. These data indicate that PPARgamma is not a major player in luteal progesterone production or metabolism but may be involved in regulating luteal lifespan.

Decidualization of pseduopregnant uterus is associated with marked reduction in Ang II and Ang‐(1‐7) content

Previous studies showed that Ang II and Ang-(1–7) concentrations are reduced in the implantation site at day 7 of pregnancy in Sprague-Dawley rats as compared to the interimplantation site. To understand whether the regulation of the Ang peptide content at early pregnancy depends on the presence of a conceptus, the uterine content of Ang I, Ang II, and Ang-(1–7) was examined in the oil-induced decidualized uterus of pseudopregnant rats. Ovariectomized, adult female rats were sensitized for the decidual cell reaction with steroid treatments; decidualization was induced by oil-injection of the left horn, the right horn served as a control. Five days after oil injection uterine horn weight increased 12-fold as compared to the non-infused horn (1543 ± 133 vs 171 ± 7 mg, p<0.00001). The Figure illustrates the profile of angiotensin peptides in the oil-infused vs non-infused horn. Both Ang-(1–7) and Ang II were significantly reduced in the oil-infused horn as compared to the non-infused horn. Ang-(1–7) was the predominate peptide in the non-infused horn [Ang-(1–7)> Ang II> Ang I p<0.05], but in the infused horn Ang-(1–7)=Ang II>Ang I (p<0.05). In conclusion, the decidualization process elicits marked reduction in uterine Ang II and Ang-(1–7) content independent of the presence of the conceptus. These findings suggest that down-regulation of both active peptides of the RAS are required for successful decidualization. Supported by NHLBI51952; AHA0565390U Ang I (A-I), Ang II (A-II), and Ang-(1–7)(A-1–7) content in non-infused (N) and oil-infused pseudopregnant horns of uterus.