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a Department of Obstetrics&Gynecology, The University of Texas Medical Branch, Galveston, Texas 77555
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(PGF2; 11.6% vs. 100% in control); 5) treatment with progesterone prevented PGF2-induced inhibition in NOS II mRNA expression; 6) ICI 164384, an antiestrogen, significantly increased serum progesterone concentration and stimulated NOS II expression by the uterus in a time-dependent manner; 7) as shown by immunofluorescent studies, cells stained by NOS II antibodies were apparent in the decidual compartment as well as in areas between myometrial cell bundles in the pregnant rat uterus. The density of staining decreased in the specimens at labor and postpartum. We conclude that NOS II gene expression in the rat uterus was enhanced during pregnancy and decreased during labor and postpartum. NOS II in rat uterus is up-regulated by progesterone and down-regulated by estrogens and prostaglandins, consistent with their role in uterine activity regulation during pregnancy and labor.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Three isoforms of NOS have been identified to date: two calcium-dependent, constitutive isoforms, originally isolated from endothelial (NOS III) and neuronal (NOS I) cells, and one calcium-independent inducible form (NOS II) present in many cell types [10]. Constitutive NOS isoforms release small amounts of NO, while the inducible isoform synthesizes large quantities of NO. Immunoblotting studies and NOS activity assays have indicated that in the rat and rabbit uteri, expression of NOS II proteins is elevated during mid to late pregnancy and is reduced during term and preterm labor [7–9]. Furthermore, the expression of NOS II was also highest in the uterus of preterm, not-in-labor patients [11]. At term, NOS II expression fell by 75% and was barely detectable in preterm in-labor or term in-labor specimens. In contrast, no significant changes in NOS III protein were noted, and NOS I was undetectable in the rat uterus during pregnancy. Thus, NOS II protein in the rat uterus was increased during pregnancy and decreased during labor with a concomitant alteration in NO generation. However, the mRNA expression of NOS II in the rat uterus during various reproductive states remains unclear. In addition, the expression and localization of NOS II in rat uterus during pregnancy and its regulation by steroid hormones during pregnancy are not well understood. Therefore, the present studies were designed to determine 1) whether mRNA for NOS II are differentially expressed in the rat uterus during pregnancy and labor and 2) whether progesterone and estrogen regulate NOS II mRNA expression in the rat uterus during pregnancy.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Adult nonpregnant (180–200 g, BW) and pregnant Sprague-Dawley rats were purchased from Harlan Sprague Dawley (Houston, TX) and maintained on a 12L:12D schedule. Animals received an ad libitum supply of rat chow and water. Two experiments were included in this study. In experiment 1, we examined the changes in NOS II mRNA levels and NOS II protein localization in the uterus during pregnancy and labor. In experiment 2, we examined the regulation of uterine NOS II mRNA levels during pregnancy by progesterone, antiprogesterone, prostaglandins, and antiestrogen. These investigations were conducted in accordance with the Guiding Principles for the Care and Use of Research Animals promulgated by the Society for the Study of Reproduction.
Experiment 1 Uterine tissues were obtained from six rats in each group of nonpregnant animals at diestrus and in each group of time-mated rats on Day 18 of gestation (Day 1 of gestation is day of a sperm-positive vaginal smear, and normal duration of gestation for rats is 22 days), at the time of spontaneous delivery at term (one to three pups delivered), or on Day 1 postpartum. Animals were killed in a CO2 inhalation chamber; the uteri were removed immediately and cleaned, and the full-thickness uterus from antimesometrial area was quickly frozen in liquid nitrogen and stored at -70°C until used. For immunofluorescent studies, tissues were frozen in Cyroform (International Equipment Company, Needham, MA) medium and processed as described below. All procedures were approved by the Animal Care and Use Committee of the University of Texas Medical Branch.
Experiment 2
Time-mated rats were divided into eight groups (6–18 rats in each group). 1) Rats on Day 17 of gestation were injected s.c. with an antagonist of progesterone, RU-486 (Biomol, Plymouth Meeting, PA; 10 mg/rat) and killed at 6, 12, and 24 h after injection. 2) Progesterone (Sigma, St. Louis, MO; 4 mg/rat per day) was injected s.c. from Day 20 to Day 22 of gestation, and rats were killed on Day 22. 3) Prostaglandin F2 (PGF2; Sigma; 300 µg/rat) was injected s.c. on Day 18 of gestation, and rats were killed on Day 20. 4) PGF2 (dose as above injected on Day 18) plus progesterone (4 mg/rat from Day 18 to Day 20) was injected s.c., and animals were killed on Day 20. 5) ICI 164384, an antiestrogen (ICI Pharmaceuticals, Cheshire, UK; 0.3 µg/rat), was injected s.c. on Day 17, and animals were killed 24 and 48 h after injection. The blood was drawn from the heart. The serum was saved for steroid analysis. The full-thickness uteri were cleaned, and the antimesometrial tissues were frozen in liquid nitrogen and stored at -70°C until used. The animals that were injected s.c. with either saline (0.2 ml/rat), PGF2, or PGF2 plus progesterone were closely monitored for labor and delivery of the pups. This was judged by the birth of the first pup, and delivery rate within 48 h after treatment was calculated.
Relative Levels of NOS II mRNA Measured by Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
Total cellular RNA was extracted from the rat uterus by a single-step guanidine thiocyanate method [12] using the reagent Trizol (Gibco/BRL Life Technologies, Gaithersburg, MD). First-strand cDNA synthesis was primed with oligodeoxythymidine using 2 µg of total extranuclear RNA with 10 units of reverse transcriptase and oligodeoxythymidine12–18 (Gibco/BRL Life Technologies) as primer in a total volume of 20 µl at 42°C for 40 min, as described previously [13–15]. Ten percent of the cDNA made from 2 µg total RNA was used for amplification by PCR with 35 cycles as previously reported [15]. PCR primers used in the current study were derived from the published sequences of murine macrophage inducible nitric oxide synthase cDNA [16]. The primers used for amplification of the housekeeping gene, ß-actin, were derived from the rat ß-actin cDNA sequence [17]. The specific primers used for amplification are as follows: 1) inducible nitric oxide synthase (NOS II): forward primer 5'-ATGGCTTGCCCCTGGAACTTTCTC-3', reverse primer 5'-GCCGACCTGAT-GTTGCCACTGTT-3' (with an expected amplified length of 718 base pairs [bp]) and 2) ß-actin: forward primer 5'-GTCGACAACGGCTCCGGCA-3', reverse primer 5'-GTCAGGTCCCGGCCAGCCA-3' (with an expected length of 530 bp). PCR products, with the use of these primers, were identical to those of published sequences of inducible NOS [16] as confirmed by direct double-strand sequencing (Fmol DNA sequencing kit; Promega, Madison, WI; data not shown). The relative concentrations of NOS II mRNA were determined by densitometric analysis of photographs of the ethidium bromide-stained reaction products with use of the Discovery Series densitometer (Quality One, Sparc, NY). The results are expressed as the ratio of the densitometric readings for NOS II mRNA to ß-actin mRNA, and therefore the results indicate relative changes in uterine NOS II mRNA levels.
Progesterone Assay
Rat serum progesterone was assayed in duplicate with use of [125I]progesterone RIA kit (Diagnostic Systems Laboratories, Webster, TX), using an antiserum that is highly specific for progesterone. Cross-reactivity to other naturally occurring steroids is low (e.g., 5-pregnane-3,20-dione 5.0%, 20-dihydroprogesterone 0.35%, and 11-deoxycortisol 0.27%). The interassay and intraassay variations were less than 10%.
Immunofluorescent Localization of NOS II
The immunofluorescent staining was performed by a modified immunofluorescence protocol [18, 19]. On Day 18 of gestation, 5-µm cryosections from the uterus of nonpregnant and pregnant rats were cut and fixed with 70% acetone. Five percent normal goat serum and avidin:biotin blocking buffer was applied to slides to reduce nonspecific binding. Primary (NOS II) polyclonal antibody (Upstate Biotechnology, Lake Placid, NY) in PBS buffer was added to slides and incubated for 90 min at 22°C and then washed in PBS. Slides were then incubated with biotinylated goat antirabbit IgG (Vector Labs., Burlingame, CA) for 45 min at 22°C. After slides were washed in PBS, the detection step was performed by incubation of slides with fluorescein avidin-D (Vector Labs.) for 1 h at 22°C. After this incubation, slides were washed at least four times in PBS buffer. Then propidium iodine (Boehringer-Mannheim, Indianapolis, IN) in PBS was applied as counterstaining to the slides to visualize cellular nuclei. After washing in PBS buffer, slides were mounted with Vectashield mounting medium (Vector Labs.) and viewed under a Nikon fluorescent microscope (Nikon Corporation, Chiyoda-ku, Tokyo, Japan).
Statistics
Results are expressed as mean ± SEM. Data were analyzed for statistical differences with the use of one-way ANOVA followed by Bonferroni t-test or Student's t-test. Data of delivery rates were analyzed with the chi-square test. Differences were considered significant if p < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To examine the effects of progesterone on NOS II mRNA expression in the rat uterus, we measured the levels of NOS II mRNA in progesterone-treated rats during spontaneous labor or preterm labor induced by PGF2. As shown in Figure 2, treatment of rats with progesterone from Day 20 of gestation prevented the decrease in NOS II mRNA expression that occurred during spontaneous labor on Day 22 (374% vs. 100%, p < 0.01). Further, as shown in Figure 3, relative levels of mRNA for NOS II in the uterus were significantly reduced by PGF2 (11% vs. 100% in control, p < 0.01), and preterm labor occurred in all animals of this group within 48 h (Fig. 4). Cotreatment with progesterone arrested the PGF2-induced preterm labor in all animals and prevented PGF2-induced inhibition in NOS II mRNA expression (988% vs. 100% in control, p < 0.01). These data provide further evidence that progesterone is necessary for maintaining NOS II gene expression during pregnancy.
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We measured the levels of NOS II mRNA in the uterus from the rats treated with an antagonist of progesterone, RU-486, to further ascertain the role of progesterone in NOS II gene regulation during pregnancy. All rats given RU-486 delivered pups within 24 h after injections (data not shown). As shown in Figure 5A, RU-486-induced preterm labor was associated with a time-dependent decrease in NOS II mRNA expression. The relative levels of NOS II mRNA in the uterus of rats treated with RU-486 were 60.9%, 20.3%, and 2.9% at 6, 12, and 24 h after injection, respectively, compared with control levels (100%) on Day 19 of gestation (Fig. 5B). This confirms that progesterone is required for maintaining NOS II mRNA expression during pregnancy.
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To assess whether endogenous estradiol plays a role in NOS II mRNA expression, we measured the levels of NOS II mRNA in the uterus from the rats treated with the antiestrogen, ICI 164384 (Fig. 6). Treatment with ICI 164384 on Day 18 of gestation significantly increased NOS II mRNA levels in the uterus in a time-dependent manner, with maximal effects observed at 48 h after injections (318% vs. 100% in control, p < 0.01). This suggests that estradiol may also modulate uterine NOS II mRNA expression in the pregnant rat uterus.
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To determine the effects of ICI 164384 and PGF2 on progesterone secretion in the rat, we measured the concentration of serum progesterone from the rats treated with ICI 164384 and PGF2. As shown in Figure 7, serum progesterone levels were 48.9 ± 3.3 ng/ml on Day 19 of gestation and were 6.3 ± 0.8 ng/ml at the time of spontaneous labor on Day 22. Treatment with antiestrogen ICI 164384 significantly increased serum progesterone concentration compared with the control value (66.4 ± 5.1 ng/ml vs. 48.9 ± 3.3 ng/ml, p < 0.01), indicating the possible involvement of estradiol in the regulation of serum levels of progesterone. In addition, administration of PGF2 markedly reduced the concentration of serum progesterone (6.3 ± 0.7 vs. 48.9 ± 3.3 ng/ml, p < 0.01), confirming the inhibitory effects of PGF2 on progesterone secretion.
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Using immunofluorescent methods, we found that immunoreactive NOS II was primarily localized to the cells in the decidual compartment as well as cells between smooth muscle cell bundles of the uterus in the pregnant rat (Day 19) (Fig. 8b). However, the staining was not detectable in epithelial cells and smooth muscle cells. These positively stained cells histologically appeared to be blood-derived cells or connective tissue cells between smooth muscle bundles. Staining was mostly strong and uniform, and was in the extranuclear cytoplasm (Fig. 8b, arrow). Control sections without the primary antibody showed no specific staining to these cells in pregnant uteri (Fig. 9b).
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In contrast to the pregnant uterine sections, the nonpregnant uterine sections had no detectable staining in any cell type (Fig. 8a). Uterine sections from labor and postpartum groups showed significantly fewer NOS II-positive cells and exhibited reduced staining intensity within individual cells as well (Fig. 8, c and d). Again, control sections without primary antibody showed no specific staining in the uteri of nonpregnant rats or of pregnant rats during labor and postpartum (Fig. 9, a, c, and d).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, was prevented by progesterone administration. RU-486-induced preterm labor was also associated with a marked decrease in uterine NOS II mRNA. Further, uterine NOS II mRNA expression increased with the administration of an antiestrogen, ICI 164384, to pregnant rats. These results with RT-PCR and immunolocalization methods further substantiate our prior observations that NOS II protein is increased with pregnancy and decreased with labor either at term or preterm. Furthermore, progesterone is essential for maintaining the expression of NOS II in the uterus, while estrogens appear to inhibit NOS II expression during pregnancy. Thus, we suggest that decreased NO production at term may be due to decreased progesterone and increased estradiol resulting in reduced expression of NOS II in the uterus. It has been demonstrated that an L-arginine-nitric oxide-cGMP pathway is present in the rat uterus and modulates uterine contractility [4–7]. In the rat, both the production of NO and its relaxation effects on the uterus were elevated during pregnancy and declined during labor. Further investigations have demonstrated that all three NOS isoforms are present in the rat uterus [9]. NOS I was present in the rat uterus during the nonpregnant stage but not during pregnancy and delivery [9]. NOS II proteins in the uterus were substantially increased during pregnancy and decreased during delivery. NOS III was present at all stages examined, but the concentrations were unchanged [9]. Present studies demonstrate that NOS II mRNA expression in the rat uterus is substantially increased during pregnancy and decreased during labor, consistent with the changes in uterine NO production and NOS protein. Therefore, regulation of NOS II at the mRNA level may play a role in modulating uterine activity during pregnancy.
This study demonstrates the presence of NOS II in the decidual compartment of the pregnant rat uterus as well as in between muscle cells. These cells histologically appeared to be macrophage-like cells or connective cells between smooth muscle bundles. In addition, uterine NOS II expression is highest during gestation when the uterus undergoes massive distention by the growing fetus. At term, NOS II-expressing cells were dramatically decreased. Further, NOS II expression was almost undetectable from the uterus of nonpregnant and postpartum animals, suggesting that pregnancy and perhaps hormonal changes may regulate NOS II gene expression. Previous studies have indicated several possible sites of NO production in the rat uterus. Using NADPH diaphorase staining, we demonstrated the presence of NO synthetic activity localized to the myometrium, endometrium, blood vessels, and the uterus during pregnancy [20], indicating that these tissues possess the NOS necessary for producing NO. As NADPH diaphorase staining identifies all isoforms of NOS and several other enzymes [21], it was not clear which isoforms of NOS were responsible for the increased NO production or which cell types it was localized in. The present study reveals specific NOS II expression in the macrophage-like cells within the uterus. In addition, we recently demonstrated that NOS III expression was localized to epithelial and myometrial cells in the uterus [22].
Although experimentally one can induce NOS II in a variety of cell types with cytokine treatment [23], the mechanisms regulating uterine NOS II expression during pregnancy have not been fully elucidated. We proposed that progesterone up-regulates NO synthesis in the uterus [9]. This suggestion is supported by our previous studies demonstrating that administration of antiprogestin decreases NOS II protein in the uterus prematurely before term and that this is accompanied by preterm birth, suggesting that progesterone up-regulates NO synthesis in the rat uterus [9]. Though RU-486 is a potent synthetic antagonist of progesterone, it also exhibits a weak affinity for glucocorticoid receptors both in vivo and in vitro [24]. Since glucocorticoids inhibit the expression of NOS II [25], the possibility that the effects of RU-486 on NOS II expression may involve glucocorticoid receptor blockage cannot be excluded. However, this is unlikely given the overwhelming evidence for RU-486 regulation of NOS II expression through its progesterone receptor blockade. RU-486-induced preterm labor was associated with a marked decrease in NOS II mRNA (current studies) and NOS protein [9], and progesterone administration prevented the decline of NOS II mRNA that occurred during term labor. Therefore, we suggest that progesterone up-regulates NOS II gene expression and increases NO production in uterus, and this may play a role in the maintenance of uterine quiescence during pregnancy.
Several studies have demonstrated that estradiol-17ß improves endothelium-dependent vascular responses [26], stimulates endothelial NO production [27], and enhances the activity of NOS III in endothelial cells of cultured human umbilical vein [28]. However, the effect of estradiol-17ß on NOS II expression in rat uterus has not been investigated. Estradiol-17ß and progesterone have been shown to inhibit basal nitrite production by unactivated hepatocytes in culture [29]. More recently, we demonstrated that total nitrites were significantly inhibited in rat uterine tissues from prepubertal animals treated with estradiol and estradiol plus progesterone as compared with levels in tissues from animals receiving vehicle or progesterone alone [6]. These studies are consistent with the reports that estradiol-17ß reduces interleukin-1ß-induced excessive NO production in isolated rat thoracic aortic rings [30]. We hypothesized that estradiol-17ß inhibits NO production in rat uterus via inhibition of NOS II mRNA expression. In support of this hypothesis we found that the antiestrogen ICI 164384 significantly stimulated NOS II expression by the uterus in a time-dependent manner, with maximal effects observed at 48 h after injection. Further, we also found that NOS II mRNA expression in the rat uterus was substantially decreased at term coincident with the well-documented significant increases in serum estrogens [31]. We suggest that elevated levels of progesterone during pregnancy may up-regulate NO production via increasing NOS II gene expression and that decreases in progesterone levels with an elevation of estradiol-17ß at term would inhibit NOS II mRNA expression. Therefore, the decrease in NOS II mRNA and NO production at term in rats may be a reflection of changes in the estrogen:progesterone ratio. In addition, the current experiments demonstrated that treatment of pregnant rats with ICI 164384 significantly increased serum progesterone concentration, indicating that estrogen may indirectly regulate NOS II gene expression through modulating progesterone levels as well. Further studies are required to clarify these interactions of estradiol and progesterone and their regulation on uterine NOS II.
Endogenous prostaglandins, particularly PGF2 and PGE2, play a role in preparing the uterus and the cervix for delivery. It has been shown that all manipulations of the cervix, including membrane stripping and artificial rupture of the membranes, produce these changes by stimulating the synthesis of prostaglandins (PG) [32]. Additionally, the administration of PG to pregnant women at any stage of gestation caused uterine contractions. Therefore, PG are widely used to induce abortion [33]. In the rat uterus, interactions between NO and PG have been reported during implantation [34]. During mid to late pregnancy, NO stimulated the uterine PG production in a dose-dependent manner, and the inhibition of endogenous NO caused a reduction in PG production [35]. Further, PG inhibited cytokine-induced increases in uterine NO production [35], suggesting that increased PG production at term may down-regulate uterine NO production and therefore facilitate labor. To identify the isoforms responsible for the changes in NO synthesis after PGF2 administration, we examined NOS II mRNA levels in the rat uterus by RT-PCR. As shown in this study, the relative levels of NOS II mRNA in the uterus were reduced in animals treated with PGF2, indicating that a decrease in NOS II mRNA levels may be responsible for reduced NO production after PGF2 injection. Further, treatment with progesterone prevented the PGF2-induced inhibition in NOS II mRNA expression, confirming the requirement of progesterone for uterine NOS II expression. On the basis of these studies as models for NO and PG interactions, we propose that NO is uniquely involved in the maintenance of uterine relaxation during pregnancy and in the initiation of labor. Steroid hormone and PG are involved in NO modulation in the rat uterus through their regulation of NOS II mRNA expression.
In summary, we found that uterine NOS II gene expression increases during pregnancy and decreases during labor and postpartum. Together with our previous studies showing that exogenous NO inhibits uterine contractility [19], these data suggest that an increase in NOS II gene expression in the uterus may be important in the maintenance of pregnancy and that a decrease of NOS II mRNA in the uterus at term may be involved in the initiation of parturition. In addition, NOS II expression is up-regulated by progesterone and down-regulated by estrogens and PG. Therefore, regulation of uterine contractile activity during pregnancy by the ovarian steroid hormones may involve the changes in uterine NOS II gene expression and, therefore, NO generation.
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FOOTNOTES |
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2 Correspondence: Chandrasekhar Yallampalli, Department of Obstetrics&Gynecology, 301 University Blvd., Medical Research Bldg., Rm. 11.138, Galveston, TX 77555–1062. FAX: 409 747 0475; chyallam{at}utmb.edu
Accepted: June 1, 1998.
Received: April 2, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-induced preterm labor in rats. Am J Obstet Gynecol 1997; 177:907–918.[CrossRef][Medline] production and the nitridergic system during embryonic implantation in the rat. Prostaglandins 1996; 51:363–376.[CrossRef][Medline]This article has been cited by other articles:
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  A. M. Mackler, T. C. Ducsay, C. A. Ducsay, and S. M. Yellon Effects of Endotoxin and Macrophage-Related Cytokines on the Contractile Activity of the Gravid Murine Uterus Biol Reprod, October 1, 2003; 69(4): 1165 - 1169. [Abstract] [Full Text] [PDF]
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H.-C. Keith Ho, K. E. McGrath, K. C. Brodbeck, J. Palis, and B. P. Schick Serglycin Proteoglycan Synthesis in the Murine Uterine Decidua and Early Embryo Biol Reprod, June 1, 2001; 64(6): 1667 - 1676. [Abstract] [Full Text]
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 L. Fang, B. Nowicki, and C. Yallampalli Differential expression of uterine NO in pregnant and nonpregnant rats with intrauterine bacterial infection Am J Physiol Regulatory Integrative Comp Physiol, May 1, 2001; 280(5): R1356 - R1363. [Abstract] [Full Text] [PDF]
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C. Yallampalli and Y.-L. Dong Estradiol-17{beta} Inhibits Nitric Oxide Synthase (NOS)-II and Stimulates NOS-III Gene Expression in the Rat Uterus Biol Reprod, July 1, 2000; 63(1): 34 - 41. [Abstract] [Full Text]
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 I. A. Buhimschi, C. Yallampalli, C. S. Buhimschi, G. R. Saade, and R. E. Garfield Distinct regulation of nitric oxide and cyclic guanosine monophosphate production by steroid hormones in the rat uterus Mol. Hum. Reprod., May 1, 2000; 6(5): 404 - 414. [Abstract] [Full Text] [PDF]
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A.M. Mackler, L.M. Green, P.J. McMillan, and S.M. Yellon Distribution and Activation of Uterine Mononuclear Phagocytes in Peripartum Endometrium and Myometrium of the Mouse Biol Reprod, May 1, 2000; 62(5): 1193 - 1200. [Abstract] [Full Text]
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Y.-L. Dong and C. Yallampalli Pregnancy and Exogenous Steroid Treatments Modulate the Expression of Relaxant EP2 and Contractile FP Receptors in the Rat Uterus Biol Reprod, March 1, 2000; 62(3): 533 - 539. [Abstract] [Full Text]
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 P. R. R. Gangula, H. Zhao, S. C. Supowit, S. J. Wimalawansa, D. J. Dipette, K. N. Westlund, R. F. Gagel, and C. Yallampalli Increased Blood Pressure in {alpha}-Calcitonin Gene-Related Peptide/Calcitonin Gene Knockout Mice Hypertension, January 1, 2000; 35(1): 470 - 475. [Abstract] [Full Text] [PDF]
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C. Yallampalli, P.R.R. Gangula, S. Kondapaka, L. Fang, and S. Wimalawansa Regulation of Calcitonin Gene-Related Peptide Receptors in the Rat Uterus During Pregnancy and Labor and by Progesterone Biol Reprod, October 1, 1999; 61(4): 1023 - 1030. [Abstract] [Full Text]
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 A. Manabe, T. Hata, T. Yanagihara, M. Hashimoto, Y. Yamada, S. Irikoma, S. Aoki, S. Masumura, and K. Miyazaki Nitric oxide synthesis is increased after dehydroepiandrosterone sulphate administration in term human pregnancy Hum. Reprod., August 1, 1999; 14(8): 2116 - 2119. [Abstract] [Full Text] [PDF]
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lane: positive control from mouse macrophages. B) Relative levels of NOS II mRNA. Densitometric readings of NOS II samples are presented as ratio of readings of corresponding ß-actin samples. Each bar graph represents mean ± SEM of six replicate animals. Groups with different letters (a, b) at the top of bars vary significantly (p < 0.01, ANOVA).
