Representative reverse transcription–polymerase chain reaction (RT–PCR) analysis of NOS isoenzyme expression in human term placenta (lane 1). Multiplex PCR was performed as described in the text. PCR was carried out in the absence of reverse transcription (lane 2). DNA molecular weight markers (M) are indicated in bp. 

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... cDNA was amplified from the developing mouse placentae on days 12, 14, 16, 18 and 20 post coitum ( Figure 1A). Direct DNA sequence analysis of the PCR products confirmed they were identical to the published sequence for the murine macrophage cDNA (Lyons et al ., 1992). Positive controls consisted of mRNA from J774 murine macrophages treated with interferon- γ and bacterial LPS. While a small amount of iNOS mRNA was detected by PCR in untreated J774 cells, it was undetectable by Northern blotting even after prolonged exposure (data not shown). The mRNA for eNOS was also detected by RNA–PCR in the placenta at all the times examined (Figure 1B). β -Actin expression was used as a control for loading and RNA integrity of all samples (Figure 1C). Northern blot analysis of murine placental mRNA revealed that the quantity of the 4 kb iNOS transcript remained largely unchanged between days 12 and 20 of pregnancy (Figure 2). The quantity of placental iNOS message present was less than that observed in J774 murine macrophages treated with interferon- γ and bacterial LPS. Probing for β -actin confirmed the loading of approximately equivalent amounts of mRNA. Although we previously reported that eNOS was the only NOS isoform present in human placenta at term based on Northern blot studies (Garvey et al ., 1994), we now find that iNOS -specific mRNA is also present in the human placenta at term using a multiplex PCR assay and conditions modified to enhance sensitivity (Figure 3). The authenticity of these PCR products was confirmed by restriction enzyme digestion. nNOS (expected size of 220 bp) was not detected in any of the samples analysed. iNOS-specific mRNA was also identified by RT–PCR in murine embryonic stem cells (derived from the inner cell mass of the blastocyst) (Figure 4) but not the 2-cell embryo or the whole blastocyst embryo (not shown). Calcium-dependent NOS activity of murine placenta did not change with advancing gestational age, averaging 1.79 pmol/ min/mg protein. In contrast, the calcium-independent NOS 281 activity decreased with time from days 14–20 gestation (day of delivery). A statistically significant decrease was observed by day 18. However, earlier in gestation (day 12) calcium- independent NOS activity was not measurable (Figure 5). The level of calcium-independent NOS specific activity from day 14 post-coitum placental tissue was similar to that found in the spleens of mice treated with LPS. iNOS mRNA was not seen in either the developing placenta or the underlying myometrium of the day 7 mouse (Figure 6A). It was found by day 9.5 post coitum where there was extensive staining for iNOS in the spongiotrophoblast and trophoblast giant cells (Figure 6B and C). Subsequently (days 14 and 19), staining was observed in both uterine epithelium and stroma (Figure 7A–D). Human placenta at weeks 16–18 also contained iNOS mRNA, but predominantly within the syncitiotrophoblast and less in the cytotrophoblastic cells (Figure 8A and B). In addition, extensive iNOS-specific staining was observed in the placental arteriolar smooth muscle (Figure 8C and D). 282 Serial sections from human placenta that were probed for iNOS were either stained for the presence of macrophages (Figure 9A) or for alkaline phosphatase activity (Figure 9B). Macrophages were found both within and outside the microvasculature and in the cytotrophoblast. They clearly did not coincide with the iNOS mRNA in either the trophoblast or vascular smooth muscle. This study demonstrates for the first time that: (i) iNOS specific mRNA is present in embryonic stem cells derived from the inner cell mass of the blastocyst but not detectable in the two cell embryos, whole blastocysts and mouse placenta on day 6 but becomes present by day 9.5 post coitum ; (ii) iNOS gene is expressed in both spongiotrophoblast and trophoblast giant cells of the developing and mature murine placenta; (iii) although the levels of iNOS mRNA appear to be constant from days 12 to 20, calcium-independent NOS activity becomes detectable around day 12, is maximal on day 14, and falls with advancing gestation; and (iv) iNOS mRNA is also present in human placenta during midtrimester within the wall of the placental vessels and predominantly in syncytio- but also in cytotrophoblast; it does not co-localize with macrophages. In 1993, Myatt et al . (1993a) and Conrad et al. (1993b) reported that human placenta at term expresses only the eNOS isoform. Both studies identified a minor calcium-independent NOS activity (only 5–6% of the total NOS activity) without being able to identify iNOS mRNA (Conrad et al ., 1993b) or protein (Myatt et al ., 1993b). However, recently, Myatt et al . (1997) showed that iNOS immunostaining could be detected in human term placenta mainly in the villous tissue and the Hofbauer cells of the villous stroma, and less in the syncytiotrophoblast and vascular endothelium (Myatt et al ., 1997). This is in contrast to another recent report that identified iNOS mRNA only in term placentae from pregnancies complic- ated with gestational diabetes (Schonfelder et al ., 1996). Our present study suggests that iNOS is expressed in the normal developing human placenta and at term. This finding is consistent with Ramsay et al . (1996) who describe significantly higher calcium-independent NOS activity early in gestation than at term. However, our study used two different methods (ISH and PCR) with different sensitivities to detect iNOS 283 mRNA in early and term human placenta respectively. At this time, we have not attempted to quantify gestational changes between these two points. In contrast, we used PCR to detect iNOS mRNA as well as Northern blotting to quantify changes in murine placentae, since both methods taken alone have limitations, e.g. quantitative errors due to plateau effects (for PCR) or low sensitivity (for the Northern blots). Our ISH results in human developing placenta show that iNOS is also expressed in cytotrophoblastic cells. This suggests that iNOS is expressed earlier in placentation than eNOS, since most authors agree that eNOS is found exclusively in the syncytiotrophoblast (Conrad et al ., 1993b; Myatt et al ., 1993a; Buttery et al ., 1994; Eis et al ., 1995). Our results are consistent with Eis et al . (1995) who observed NADPH diaphorase activity in the trophoblast of the human first trimester placenta that did not correlate with eNOS expression. It is possible the NADPH diaphorase ...