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LD5655.V855 1987.H664.Pdf (6.917Mb) IN VITR0 CHARACTERIZATION 0F AROMATASE, ESTRONE SULFOTRANSFERASE AND ESTRONE SULFATASE ACTIVITIES IN THE RORCINE RLACENTA AND ENDOMETRIUM AT 30, 60 AND 90 DAYS 0F GESTATION bv Katherine Lee Hopkins Thesis submitted to the Faculty of the Virginia Rolytechnic Institute and State University in partial fulfullment of the requirements for the degree of MASTER 0F SCIENCE in Animal Science (Reproductive Rhysiology) APPROVED: i J. W. Knight hairman W. E. Beal D.R. Notter R. R. Frahm, Dept. Head July, l987 Blacksburg, Virginia I IN \/ITI?20 CHARACTERIZATION 0F AROMATASE, ESTRONE SULFOTRANSFERASE AND ESTRONE SULFATASE ACTIVITES IN THE PORCINE PILACENTA AND ENDOIVIETRIUIYI AT 30, 60 AND 90 DAYS 0F GESTATION bv Katherine I.ee Hopkins (ABSTRACT) The purpose of this investigation was to characterize the activities of three enzymes, aromatase, estrone (E1) sulfotransferase and E] sulfatase, in the porcine placenta and endometrium on d 30, 60 and 90 of gestation. These enzymes play key roles in determining in utero concentrations of estrogens. Days 30, 60 and 90 were chosen because previous investigations had determined that these were times of substantial changes in in vitro estrogen production by the porcine placenta and endometrium. Each enzyme assay was performed in separate tissue culture systems, and the level of enzyme was determined by measuring product formation from tritium—Iabelled (3H) substrates provided in the incubation medium. Aromatase activity was determined by the formation of 3H20 and estradiol from 3H—testosterone (T). E] sulfotransferase activity was determined by the formation of 3H-estrone sulfate (EISOA) from 3H-E]. Estrone sulfatase activity was 1 determined by the formation of 3H—E] from 3H-E]504 Concentrations of T, E]504 and E] in plasma collected from the anterior vena cava (AVC), uterine artery (UA) and uterine vein (UV) were determined py radioimmunoassay. There were no significant differences in mean enzyme activity of the placenta or the endometrium among the three days of gestation examined. Endometrial aromatase activity was twice that of placental aromatase on all three days that were examined. The ratio of placental to endometrial sulfotransferase activity underwent a reversal from 1:1.3 on d 30 to 2.3:1 on d 60 and 90. The ratio of placental to endometrial sulfatase activity changed from 1:1.6 on d 30 to 1:2.2 on d 60 and 1:3.0 on d 90. There were significant differences in response of enzyme activity to extended periods (3 h) in incubation which varied with tissue type, gestational age and enzyme of interest. Overall, placental enzymes did not respond consistently to increased time in incubation with increased product formation. Endometrial aromatase activity on all three days and endometrial sulfatase activity on d 90 responded to increased time in incubation with increased product formation. In vivo, the uterus appears to oe involved in T, E] and E]504 production since UV steroid concentrations were greater than those of the UA. The E1504 to E] ratio of the UV decreased from 15:1 on d 30 to 4:1 on d 60 and 1:2 on d 90. I ACKNOWLEDGEMENTS I would like to take this opportunity to recognize the contributions of several individuals who have assisted me in the construction of this thesis, promoted my education and who have become my firm friends during my three years at \/irginia Tech. First, I would like to thank Dr. J. W. Knight, committee chairman, Dr. W. E. Beal and Dr. D. R. Notter for agreeing to serve on my committee, oversee my career as a Masters candidate and for assisting me in getting my thesis transformed into the final product. Their willingness to offer advice and direction was greatly appreciated. Almost last, by certainly not least, I would like to recognize the contributions iv I TABLE 0F CONTENTS ABSTPACT ............................................................................................ ii ACKNDWLEDGEMENTB.......................................................................... iv QQQQ INTPODUCTION..................................................................................... I PE\/IEW 0F LITEPATUPE...................................................................... 4 Porcine Placental Development and Function........................... 4 Placental Development................................................. 4 Placental Function....................................................... I7 Estrogens......,..........,..................................................,.............. IQ Aromatase............................................................,..................... 30 Bulfotransferase........................................................................ 36 Sulfatase.............................,................,..................................... 42 Steroidogenic and Enzymatic Profile of the Pig Durihg Gestation........................................................................ 46 MATEPIAL5 AND PIETHODS.................................................................. BB Tissue Collection Procedures.................................................... BB Tissue Incupation Procedures................................................... 6I Assay of Aromatase Activity.........................„...........,................ 62 Assay of Estrone Sulfotransferase and Estrone Bulfatase Activity...................................................................................... 64 Soluble Protein Determination................................,.......„......... 6Q Assays of Blood Steroid Concentrations..................................... 70 Testosterone................................................................ 70 vi I i Estrone and Estrone Sulfate....................................... 71 Statistical Analysis.................................................................... 73 PESULTS............................................................................................... 77 Protein Content of Incubated Tissue Samples........................... 77 In vitro Aromatase Activity on d 30, 60 and 90 of Gestation........................................................................,............ 82 In vitro Sulfotransferase Activity on d 30, 60 and 90 of Gestation................................................................................. 91 In vitro Sulfatase Activity on d 30, 60 and 90 of Gestation..... 97 Steroid Concentrations in Maternal Plasma on d 30, 60 and 90 of Gestation.................................................................... 103 Testosterone............................................................. 103 Estrone....................................................,,............... 106 Estrone Sulfate..............................,.......................„. 106 Comparison of Ivlaternal Plasma Testosterone, Estrone and Estrone Sulfate........................................... 111 DISCUSSION......................................................................................... 114 CONCLUSION......................................................................................... 134 LITEPATUPE CITED...................................,........................................... 136 APPENDICES......................................................................................... 149 vITA...................................................................................................... 161 vii I I LIST 0F TABLES TabIe page I. Spontaneous suifation of estrone................................................... 67 2. Spontaneous nydroIysis of estrone sulfate..................................... 68 3. Regression of coefficients and mean protein content.................... 74 4. Aromatization of testosterone py microwaved pIacentaI tissue over a 3 n incubation period at 30, 60 and 90 d of gestation....... 83 5. Aromatization of testosterone py microwaved endometriaI tissue over a 3 n incubation period at 30, 60 and 90 d of gestation....... 84 6. Mean aromatase activity of pIacentaI, endometriaI, microwaved pIacentaI and microwaved endometriaI tissue at 30, 60 and 90 d of gestation................................................................................. 85 7. Aromatization of testosterone py pIacentaI tissue over a 3 n incubation period at 30, 60 and 90 d of gestation...................... 88 8. Aromatization of testosterone by endometriaI tissue over a 3 n incuoation period at 30, 60 and 90 d of gestation....................... 89 9. Riacentai suIfotransferase activity over a 3 n incubation period I at 30, 60 and 90 d of gestation........................................................................................ 92 I0. EndometriaI suifotransferase activity over a 3 n incubation period at 30, 60 and 90 d of gestation.......................................... 93 I I. Mean suIfotransferse activity of pIacentaI and endometriaI tissue at 30, 60 and 90 d of gestation.......................................... 94 I2. RIacentaI suIfatase activity over a 3 n incuoation period at 30, 60 and 90 day of gestation........................................................... 98 I3. Endometrial suIfatase activity over a 3 n incubation period at 30, 60 and 90 d of gestation........................................................ 99 viii 14. Mean sulfatase activity of placental endometrial tissue at 30, 60 and 90 d of gestation.............................................................. 100 15. Plasma testosterone concentrations in the anterior vena cava, uterine artery and uterine vein at 30, 60, and 90 d of gestation.......................................................................................
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