Gary L. Nieder

Localization of NADPH diaphorase in the mouse uterus during the first half of pregnancy and during an artificially induced decidual cell reaction.

Uteri from non-pregnant and pregnant (Days 1-10) mice were examined for the presence of NADPH diaphorase (NADPH-d) activity by histochemical techniques. Macrophages positive for NADPH-d were observed in all uterine sections but appeared to migrate out of the implantation site and cluster in the mesometrium and interimplantation zones beginning on Day 4. NADPH-d activity was seen in the luminal and glandular epithelium and in several isolated fibers coursing through the myometrium. Many branches of the uterine artery also expressed activity, with the most intense staining in the vessels of the mesometrium. However, the most remarkable staining began on Day 6 within the primary decidual zone. When the stromal cells underwent decidualization, they began to show NADPH-d activity, with the pattern of activity matching the expanding area of decidualization. By Day 9 most of the decidual cell reaction had occurred and the mesometrial decidual staining began to decrease. However, the blood vessels and the cells surrounding the developing blood spaces continued to express activity, and heavy staining was evident within the antimesometrial decidua. No NADPH-d activity was seen in any of the trophoblast cells at any time, or in embryonic tissue, except on Day 8. NADPH-d has been used to identify nitric oxide (NO) synthase. Therefore, it may represent an NO-mediated paracrine control over decidual blood flow, myometrial quiescence, or immune response during pregnancy.

The Effect of Vanadate on Human Kidney Potassium Dependent Phosphatase

SummaryThis study examined the effects of vanadate on the potassium dependent phosphatase activity present in purified human kidney microsomal (Na++K+)-adenosine triphosphatase. Vanadate anion inhibited the K+-dependent phosphatase at a KI of 35 nM. This inhibition, was noncompetitive with the substrate, p-nitrophenylphosphate. The inhibition by vanadate at 1 mM K+ was only 45% of the inhibition that was observed at 10 mM K+. Neither preincubation of the enzyme with vanadate, nor changing the pH of the assay from 8.2 to 7.2 had any effect on the KI for vanadate. The inclusion of 2.5 mM isoproterenol, to complex the yanadate, reversed the inhibition, as did diluting the enzymatic reaction. Vanadate also inhibited the overall (Na++K+)-ATPase reaction at a KI of 1.91 μM. This inhibition was also reversible upon inclusion of isoproterenol in the assay. Increasing the level of magnesium from 6 mM to 30 mM lowered the KI of vanadate to 0.25 μM. The possible role of vanadate as a physiological mediator of (Na++K+)-ATPase activity is discussed.

Quantitative Histochemical Measurement of Pyruvate and Lactate in Mouse Oviduct During the Estrous Cycle

Pyruvate and lactate are important energy sources for preimplantation embryos cultured in vitro. The purpose of this study was to determine in vivo levels of these substances in mouse oviductal tissues throughout the estrous cycle. Quantitative histochemical assays were developed to analyze these metabolites in submicrogram samples of freeze-dried ampullar and isthmic oviduct. The potential for other alpha-keto acids to interfere with the pyruvate assay was assessed and found to be minimal with this procedure. The importance of the collection method in maintaining in vivo metabolite levels was demonstrated by the marked changes observed with extended anoxia or pentobarbital anesthesia. Pyruvate levels at 3 hr postovulation (2.6 mmol X kg-1 dry weight) were higher than 12 hr before ovulation or 12 to 72 hr after ovulation (1.6 to 2.2 mol X kg-1) in both ampulla and isthmus. Lactate levels were significantly increased at 12 to 24 hr in the isthmus (28 mmol X kg-1) compared to other times during the cycle (9-19 mmol X kg-1). The observed levels of these metabolites may reflect changes in oviductal metabolism, induced by the hormone pattern of the estrous cycle, that promote the availability of needed energy substrates for the early embryo.