PARENT SESSION

(W564) ESTROGEN REGULATION OF NOS ISOFORMS IN THE MOUSE PERIIMPLANTATION UTERUS.

Kwok, Willson¹, Steuerwald, Nury^{1, 2}, Huet-Hudson, Yvette ¹, ¹ UNC Charlotte, Charlotte, NC² ART Institute of New York & New Jersey, West Orange, NJ

ABSTRACT- Nitric oxide (NO) is a free radial molecule produced in vivo by nitric oxide synthase (NOS). Three isoforms of NOS exist, neuronal (NOS1), inducible (NOS2) and endothelial NOS (NOS3). As an endogenous smooth muscle relaxant and vasodilator, NO increases vascular permeability and uterine quiescence at the implantation site. NO reacts rapidly with O₂- or possibly with H₂O₂ to form ONOO- that may cause subsequent oxidative cellular and protein damage. It has been suggested that these side reactions of NO with O₂- and H₂O₂ are stabilized by superoxide dismutase (SOD) and catalase, respectively. Three isoforms of SOD exist: cytosolic antioxidant enzyme CuZn-SOD (SOD1), mitochondrial antioxidant enzyme Mn+2-SOD (SOD2), extracellular SOD (SOD3). SOD inhibits the activities of NOS, possibly by increasing the half-life of NO or by converting NO to NO to inactivate NOS in a negative feedback mechanism. We hypothesize that NO level is tightly regulated by NOS and SOD under the hormonal controls of E₂ and P₄ in the delayed implanting mouse uterus. We have previously demonstrated that NO levels increase in culture medium when ovariectomized progesterone (P₄)-treated mice were exposed to 17beta-estradiol estrogen (E₂) for 1 h in utero. However, a complete analysis of NOS and SOD isoforms mRNA in the delayed implanting mouse model has not been examined. The objectives of the present study are to quantify the amount of mRNA of the NOS isoforms as well as SOD 1 and 2 isoforms. To accomplish these objectives, ovariectomized CD-1 mice were given daily subcutaneous injections of P4 for four days and were given E₂ treatment on day 4. Uterine tissues were collected 0 hour and 1 hour after E₂ administration. RNA was isolated from these tissues, DNAse treated, reverse transcribed to cDNA, and the NOS and SOD isoforms were amplified using real-time fluorescent PCR. Our result indicates that E₂ upregulates mRNA of eNOS and nNOS but not iNOS in this model. E₂ also appears to regulate SOD expression. These results are consistent with the ability of E₂ to increase the production of NO in the uterus and with a role for NO in the implantation process.

KEY WORDS: uterus, estrogen, nitric oxide synthase, superoxide dismutase