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The Possibility of Organic Anion Transporter Affecting the Fetal Hypothalamic Pituitary Adrenal Axis

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Title:
The Possibility of Organic Anion Transporter Affecting the Fetal Hypothalamic Pituitary Adrenal Axis
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COUSINS, RODERICK DIMITRI ( Author, Primary )
Copyright Date:
2008

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Subjects / Keywords:
Anions ( jstor )
Antibodies ( jstor )
Cerebellum ( jstor )
Estrogens ( jstor )
Hypothalamus ( jstor )
Immunohistochemistry ( jstor )
Messenger RNA ( jstor )
Oats ( jstor )
Pregnancy ( jstor )
Sheep ( jstor )

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University of Florida
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University of Florida
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Copyright Roderick Dimitri Cousins. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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12/31/2016

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1 THE POSSIBILITY OF OR GANIC ANION TRANSPORTER AFFECTING THE FETAL HYPOTHALAMIC PITUITARY ADRENAL AXIS By RODERICK DIMITRI COUSINS A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2006

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2 Copyright 2006 by Roderick Dimitri Cousins

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3 ACKNOWLEDGMENTS I thank my parents for all their morale s upport and Dr. Charles Wood for giving me the opportunity to work in his lab as well as suppor t my ideas, schooling, and theories. I also would like to thank my lab mates Christine Schaub, Me lanie Powers, Jason Ge rsting, and Dr. Maureen Keller-Wood and her lab for helping me with e xperiments, writing, conferences, and helping me with classes.

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4 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................3 LIST OF FIGURES................................................................................................................ .........5 ABSTRACT....................................................................................................................... ..............7 CHAPTER 1 INTRODUCTION................................................................................................................. ...9 2 MATERIALS AND METHODS...........................................................................................18 3 RESULTS...................................................................................................................... .........23 4 DISSCUSSION.................................................................................................................. .....39 LIST OF REFERENCES............................................................................................................. ..45 BIOGRAPHICAL SKETCH.........................................................................................................48

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5 LIST OF FIGURES Figure page 1-1 A diagram of the fetal HPA axis. CY450 is 17-alpha-hydoxylase....................................16 1-2 Shows how organic anion tran sporters work on the basolate ral side of cells. Organic anions are brought into the cell by exchangi ng alpha-ketogluterate in the cell with organic anion in the blood..................................................................................................16 1-3 Pavlova’s data. OAT1 expression was determ ined by In-situ hybridization in pictures A through J in the fetal kidney and brain. Nort hern blot analysis was also done on the kidney which is shown by K. A and C shows dark-field X-ray film images of the murine where OAT1-specific riboprobes hybridiz ed to embryo histological sections.....17 2-1 An example of RT-PCR data generate d by the ABI Prism 7000 for OAT1 ontogeny in the pituitary............................................................................................................... .....22 3-1 Fold change in OAT1 expression in Hypot halamus with legend that shows statistical difference between groups. An upward tre nd is shown in the graph as gestation progresses..................................................................................................................... ......26 3-2 Fold change in OAT3 expression in Hypot halamus continues to increase even after birth. There is an unusual reading in fold ch ange at 130 days of ge stational age (dga)....27 3-3 Fold change in OAT1 expression in P ituitary with legend showing statistical difference between groups. No apparent trend found as gestation progresses but statistical significance is s een between the age groups......................................................27 3-4 Fold change in OAT3 expression in P ituitary with legend showing statistical difference between groups. Apparent down regulation is seen as gestation is progressing.................................................................................................................... .....28 3-5 Fold change in OAT1 (A) and OAT3 expression (B) in the brainstem shows no apparent trend through out gestation and no statistical signif icance between age groups......................................................................................................................... ........29 3-6 Fold change in OAT1 expression in cerebellum throughout gestation. There is no overall statistical significance but there is statistical signifi cance between certain gestational age groups........................................................................................................3 0 3-7 Fold change in OAT3 expression in Ce rebellum with legend showing statistical difference between groups. No apparent tr end is seen throughout gestation but at 130dga there is significant increase in fold change and is the only gestational age showing statistical significance..........................................................................................30

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6 3-8 Immunohistochemistry of OAT1 Cerebell um showing layers of the cerebellum. OAT1 is mostly found in the granular layer of the cerebellum and the layer closest to the cerebrospinal fluid........................................................................................................ 31 3-9 Immunohistochemistry of OAT1 Cerebell um showing ventricular membrane staining for OAT1. OAT1 staining also found in the surrounding tissue of the 4th ventricle in the cerebellum................................................................................................................. ...31 3-10 Control for Cerebellum Imunohistohemistry that is stained with only the secondary antibody to determine any non-specific binding. There is no apparent binding of the secondary antibody to cerebellular tissue..........................................................................32 3-11 (A) OAT1 hypothalamus Immunohistochemi stry shown by making picture black and white to better highlight the staining around blood vesse ls. There is significant staining shown around the blood vessels of th e hypothalamus as well as endothelial cells of the hypothalamus...................................................................................................33 3-12 Hypothalamus control which is stained only with secondary antibody in order to detect any anti-specific binding of the sec ondary. There is no non-specific binding of the secondary to hypothalamic tissue................................................................................34 3-13. OAT1 pituitary Immunohistochemistry s hown by conversion of picture to Black and white. OAT1 staining in the pituitary is seen in the membranes of cells and the vasculature of the pituitary. Nuclei are al so staining for OAT1 in the pituitary as well........................................................................................................................... ..........34 3-14 OAT3 pituitary immunohistochemistry shown by making picture black and white shows OAT3 in the nuclei of pituitary cells......................................................................35 3-15 (A) Hemotoxylin staining with OAT1 antibody staining. (B) He motoxylin staining with only secondary antibody to test for non-specific binding in the pituitary. The above figure shows that there is no non-sp ecific binding of the secondary antibody in the pituitary.................................................................................................................. ......36 3-16 Western blot for OAT1 ontogeny of the fetal cerebellum shows the75kda band gets darker as gestation progre sses but as the 60Kda band ge ts lighter as gestation progresses..................................................................................................................... ......37 3-17 Densitometry graph showing statistical significance for 75Kda band as gestation progresses. There is an upwar d trend in the intensity of the 75Kda band as gestation progresses showing possible influence by gestation..........................................................38 3-18 Densitometry graph showing statistical significance for 60Kda band as gestation progresses. A downward trend is seen as gestation progresses showing possible influence of gestation......................................................................................................... 38

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7 Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science THE POSSIBILITY OF OR GANIC ANION TRANSPORTERS AFFECTING THE FETAL HYPOTHALAMIC PITUITARY ADRENAL AXIS By Roderick Dimitri Cousins December 2006 Chair: Charles Wood Major Department: Medical Sciences Physiology and Functional Genomics Preterm labor is a major problem in our soci ety and has been the number one killer of newborns. Little is known about the triggering of labor causing birth but wh at is known is that birth cannot occur with out the fe tal hypothalamus, pituitary, or ad renal gland also known as the fetal hypothalamic pituitary adrenal axis (fetal HP A axis). As gestation progresses the fetal HPA axis continues to increase its output until righ t before birth where the end product of the fetal HPA axis is released in one large amount right before birth. The end product of the fetal HPA axis in humans is dehydroepiandrosterone (DHEA) but our animal model is sheep and its fetal HPA axis end product is cortisol. Both end pr oducts cause the production of estrogens in the placenta by different mechanisms and it has been s hown in the literature that estrogens produced by cortisol in fetal sheep stimulate the fetal HPA axis to increase its output. This could be the reason for the positive feedback loop of the fetal HPA axis in sheep. The estrogen made by fetal sheep influenced by cortisol is 17-beta-estradi ol-sulfate. The estrogen is inactivated by the enzyme Estrogen Sulfotransferase (EST) in th e placenta by conjugating a sulfate group to the estrogen which is then made active by Steroi d Sulfatase (STS) by re moving the sulfate group. STS has been found in fetal sheep brain cells sh edding light on possible feedback mechanism. The hurdle is how does 17-beta-estra diol sulfate get into the feta l brain? We propose that this

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8 achieved by organic anion transporters (OAT) 1 and 3 which are both efficient in moving 17beta-estradiol sulfate. Based on Reverse-Tran scriptase Polymerase Chain Reaction (RT-PCR) experiments OAT1 is increasingly up regulated in the cerebellum and hypothalamus throughout gestation until birth where only OAT1 is dramatically down regulated in the cerebellum. OAT1 mRNA expression is variable in the pituitary showing no correlat ion to gestational age and the brainstem shows no change in mRNA expressi on levels throughout ge station for OAT1 or OAT3. OAT3 RT-PCR data shows that OAT3 is up regulated in the hypothalamus through out gestation and maintains mRNA expression levels after birth. The cerebellum at 130 days of gestation for OAT3 is the only age where OAT3 is up regulated dramatically during gestation. OAT3 is down regulated however in the pituit ary throughout gestation and after birth it is dramatically up regulated back to the mRNA e xpression levels at 80 da ys gestation. Western blots were performed only on the cerebellum b ecause the cerebellum was the only part of the brain that gave us enough tissu e to run a western blot. A uniqu e pattern was seen and proven by densitometry and that is that the 75kda band got darker as the 60kda band got lighter as gestational age increased which could be caused by glycosylati on. Immunohistochemistry (IHC) shows in the various tissues that OAT1 is f ound in hypothalamic endothelial cells, membranes of pituitary cells, and in the outermo st layers of the cerebellum that are exposed to cerebrospinal fluid (CSF). IHC for OAT3 is seen only in the nuclei of pituitary cells . OAT IHC supports the possibility that OAT1 is bringing in 17-beta-estradiol sulfate from the blood and CSF into or out of the fetal brain cells. The results on RT -PCR shows that the OAT1 and 3 mRNA expression levels correlate with gestational age and could play a possible role in moving 17-beta-estradiolsulfate into the brain.

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9 CHAPTER 1 INTRODUCTION The incidence of premature birth has increased 29% between 1981 and 2004 and is still on the rise. Premature birth is the num ber-one killer of newborns and is a persistent problem in our society. About 12% of newborns in America toda y will be born prematurely. Babies that are born prematurely are also at hi gh risk of getting other conditi ons such as cerebral palsy, lung diseases, blindness, learning disabi lities and development disabilities . Care of a premature infant also costs 15 times as much as the care for a healthy baby and that does not include the money needed to care for future problems that might have been caused by premature birth. To prevent premature birth there first must be a way to moni tor fetal development in order to make sure the baby will be born healthy and on time and secondly to prevent the event of labor (parturition) of the pregnant woman carrying the potentially premature baby. There is a poor understanding of the mechanism initiating labor or the cause of premature birth for that matter. The Hypothalamus-Pituitary-Adrenal gland Axis (HPA axis) in the fetus plays a crucial part in parturition (Am J Obstet Gynecol. 1967 Liggins et al.). The HPA axis is known to be important in the process of partur ition because if any one of the organs in the HPA axis were to be removed, parturition would not take place. Th is was determined by Liggins and colleagues by either removing any of the organs involved in the HPA axis or creating lesions in them through electro coagulation (Am J Ob stet Gynecol. 1967 Liggins et al.) on fetal sheep. The HPA axis works by first having the hypotha lamus release cortic otrophin releasing hormone (CRH), which causes the anterior pitu itary to release adrenocorticotropin (ACTH), which then stimulates the adrena l gland to release cortisol in sheep and Dehydroepiandrosterone (DHEA) in humans which is used by the placen ta to make estrogen in humans. In the sheep model for estrogen production determined by Liggi ns (Figure 1-1) the only difference from

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10 human estrogen production is that cortisol is released by the adrenal gland, which activates enzymes (ex. 17-alpha-hydroxylase) to convert pr ogesterone to estrogen (Basic Life Sci. 1974 G.C. Liggins). Liggins used sheep as an animal model because it is the animal which most closely mimic’s human gestation where experiment ation can be done effec tively. It was also the sheep model used to extrapolate on the hum an reproductive model to determine human reproductive function initially (Bio of Repro. 1977 Liggins et al.) We have proposed that the trigger to partur ition is in the fetal brain either at the hypothalamus, pituitary or possibly some other brain region that c ould influence a change at the hypothalamus or pituitary. The secr etion of ACTH during gestati on causes the adrenal gland to release cortisol in sheep and DHEA in humans. As gestation progresses, plas ma levels of cortisol and 17-beta-estradiol increase in fetal sheep until the end of gestation until there is an exponential increase in cortisol ri ght before birth (there is also an increase in estrogen as well); this is called the cortisol surge. These hormones are crucially important to parturition. We have shown that 17-beta-estradiol increase basal AC TH levels in the fetus (Am J Physiol.1997 Saoud and Wood) suggesting that 17-beta -estradiol might stimulate the HPA axis to further increase cortisol in secretion. Therefore, we will focus on 17-beta-estradiol as a possible hormone trigger of parturition. The estrogen 17-beta-estradiol is rendered inactive by the placenta by conjugating it to sulfate. The enzyme in the placenta that does this is called Estrogen Sulfotransferase (EST). This enzyme conjugates the sulfate group to the 3 carbon in 17-beta-estra diol making 17-betaestradiol-3-sulfate (FASEB.1997 Fala ny). The sulfation of 17-beta-est adiol renders it inactive so that the estrogen cannot diffuse through the bi-lipid membranes of cells or interact with estrogen receptors to transcribe genes. It needs another enzyme to activate it again by removing the sulfate

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11 group. This enzyme is called Steroid Sulfatase (STS) and it deconjugate s the steroid from the sulfate group which then renders the inactive steroid active by removing the sulfate conjugate. STS is found in the cytosol in ce rtain cells in the fetal brain and in cells that make up the blood brain barrier (FASEB.1997 Falany). EST is found in the placenta and was shown to rise with gestational length in pregnant mice (Biochem.1983 Hobkirk). We have found in our lab that STS is highly expressed in the cereb ellum and hypothalamus and that expression increases in these tissues at late gestation (unpublishe d data generated by Jared Winikor). It is not known how 17-beta-estradiol-3-sulfate gets into the brain to possibly stimulate the HPA axis. Organic anion transporters (OATs) could quite possibly do th is. OATs are proteins found in the membrane of cells that work off th e electrochemical gradient made by the sodium potassium pump (Figure 2). This allows an exchange of an anion in the ce ll with an anion outside the cell through a tertia ry transport system which brings the an ion originally in the cell used in the exchange back in the cell (Phys iol Rev. 2004 Dantzler and Wright). There are a number of protei ns that belong to the OAT fa mily and they are OAT 1, 2, 3, and 4 (Pflugers Arch. 2000 Sekine et al.). Organic anion transporte rs are initially found in the kidney and are believed to have significance ther e because they help the body get rid of wastes by taking harmful anions out of th e blood and into the lumen of the kidney proximal tubules. It is known that OATs can transport a wide variety of di fferent things and some of these things are steroids especially estrogens that are conjugated to sulfates such as estrone-3-sulphate and 17beta-estradiol-3-sulphate. OAT1 an d 3 are the most efficient at transporting 17-beta-estradiol when sulfoconjugated. OAT3 is the better of th e two (Rev Physiol Biochem Pharmacol. 2003 Buckhardt and Buckhardt) (Pflugers Arch. 2000 Se kine et al.). Therefore we will focus on OAT1 and OAT3 in the fetal brain.

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12 Genetic regulation of OAT1 and 3 is still no t determined. Some xenobiotics have caused upregulation. The loop diuretic Furosemide and the thiazide hydrochlor othiazide also have increased protein levels of the OAT1 protein (Nephrol. Dial. Transp lant 2003 Kim et al). It also has been shown that the drug dexamethasone can upregulate the OAT1 gene (Exp Toxicol Pathol.2003 Bahn et al.) (M ed Bio 1986 Braunlich). Braunlich has also shown that there is a difference in genetic regulation of OAT1 between the adult and fetus (as well as the newborn). When Braunlich infused dexamethasone (a synthe tic glucocorticoid) in premature (5, 10, and 15day-old rats) rats, Braunlich found an increase in p-aminohippurate secret ion in the premature rats when dexamethasone was infused. The re garded p-aminohippurate transporter is OAT1 (Med Bio.1986 Braulich). Adult rats were also infused with dexa mthasone but did not show the dramatic change in p-aminohippurate secretion that the immature rats show ed. In the literature I have also found that prostaglandin E2 (PGE2) cau ses an increase in the basolateral uptake of organic anions by OAT3 (PGE also does the sa me thing to OAT1) (J Am Soc Nephrol. 2003 Sauvant). Maybe increased expression doesn’t have to be a trigger to incr eased uptake of 17-beta -estradiol-3-sulfate. Estrogen acti on in the brain does cause the pr oduction of PGE2 (Brain Res. 2003 Wood) so there might also be a possible influence from PGE2. It has been shown by Walker and Pratt that when th e drug probenecid (inhibits OAT1 a nd OAT3 transport) is infused into the sheep, PGE2 concentrations in the cere brospinal fluid in the fe tal brain increases (J Physiol. 1998. Walker DW, Pratt N.). This s hows that there is a possible mechanism for transporting PGE2 out of the CSF and possibly into the blood or cells in the brain that is related to OAT1 and OAT3 transport. Jones et al. also found out that it was the brain that was producing PGE2 and that there is a possible mechanism for transport across the blood brain barrier or cells in the brain (Biol Neonate. 1994. Jones et al.). Jone s et al. put Indomethacin, which is a drug that

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13 inhibits the production of PGE2 into the feta l brain intracerebroventri cularly (ICV) and found out that PGE2 concentration levels in the feta l brain and fetal plasma decreased (Biol Neonate. 1994. Jones et al.). This proves th at PGE2 is present and can influence OAT transport in the brain. Another interesting fact is that OAT1 can transport PGE2 very efficiently (Biochem Biophys Res Commun. 2000 Nishio et al.) and that PGE2 can also stimulate the HPA axis to release cortisol (J Endocrinol. 1992. Brooks AN, Gibson F.) (J Neuroendocrinol. 1996. Young et al.). Kis and colleagues have also found that there is a prostagl andin transporte r called PGT at the third ventricle where the hypotha lamus and the third ventricle ar e in close proximity (J Appl Physiol. 2005 Kis et al.). Prostaglandin transporte r are also in the cells of the paraventriculuar nuclei (J Appl Physiol. 2005 Kis et al.) show ing a possible indirect mechanism for PGE2 to stimulate the HPA axis via CSF. Even though OAT1 and OAT3 function and possible expression mechanisms might support our theory, where exactly is OAT1 and OAT 3 in the brain and does the localization of OAT1 and OAT3 support our theory as well? In situ hybridization on fetal mice for OAT1 performed by Pavlova and colleagues demonstrat ed that OAT1 is found more predominantly in the fetal brain (dura matter, epithelial lining of the ventricles and choroi d plexus) than the fetal kidney (Am J Physiol Renal Physiol. 2000 Pavlova et al.). Pavlova proposed that OAT1 is playing some role in development of the brai n (Am J Physiol Renal Physiol. 2000 Pavlova et al.). The most predominant expression was seen around the cerebellum and hindbrain of the fetal mouse (Figure 1-3). Palova found that OAT1 expre ssion declined dramatically in the postnatal mouse as well (OAT1 expression was significantly higher during fetal and ea rly postnatal life of the mice) showing some hint of developmen tal properties during ge station. Nishio and

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14 colleagues also did in situ hybridization for mOAT1 and showed also that is was very predominant in the cerebellum of mice (Bio chem Biophys Res Commun. 2000 Nishio et al.). The expression of OAT1 in the fetal brain is consistent with the theory that OAT1 might play some part in moving something into the brai n during gestation (or out) that is relevant to parturition. It is possible that 17beta-estradiol-3-sulfate is let into the cell or that another molecule made due to estrogen action is moved out of the cell. Eraly and coworkers suggests that because OAT1 and OAT3 have close phylogenetic relations they have similar tissue distributions, they transport the same substrates and they could quite pos sibly co-regulate each other’s gene expression (Biochem Biophys Res Co mmun. 2003 Eraly et al.) . This could probably give reason as into why OAT1 a nd OAT3 are found in the same places within the fetal brain. To see if OAT1 or OAT3 is necessary for fetal development, OAT3 and OAT1 knockout mice have been made. Neither were embryonic lethal knockouts. This could be due to the overlapping of substrates that OAT1 and 3 moves across membranes and the fact that they have similar tissue distributio n as well. A double knockout might be a better approach to determine OAT1 and OAT3 relevancy to fetal development. Fetal kidney development was looked at only for OAT1 knockout mice which showed no tissue abnormalities in the kidney (J Biol Chem. 2006 Eraly et al.) and in the OAT3 knock out mice there were no tissue abnormalities overall (J Biol Chem. 2002 by Sweet et al.) . OAT1 knock out mice had more anions in the blood than the control and the OAT3 knock out mice (J Biol Chem. 2006 Eraly et al.). We propose that OAT1 and OAT3 mRNA expressi on levels in the fetal sheep brain will increase as gestation progresse s thus creating more of the OAT1 and OAT3 protein allowing more estrogen action in the brain which would cause a greater output of the HPA axis. We are going to prove this by doing reverse transcri ptase polymerase chain reaction (RT-PCR) to

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15 determine mRNA expression levels at different gestational lengths. We also propose that the OAT1 and OAT3 protein will be found on the cells of the blood brain barrier and cells within the fetal sheep brain. Immunohistochemistry on th e pituitary, hypothalamus, brainstem, and cerebellum will show us where exactly the OAT1 and OAT3 proteins are in the fetal brain. The reason for looking at the pituitar y and hypothalamus is to see if 17-beta-estradiol-3-sulfate directly get into cells at the pituitary and the hypothalamus and stimulate the HPA axis directly. We decided to look at the brainstem because the brainstem can also stimulate the HPA axis when the fetus is under particular stresses such as hypoxia by making prostaglandin E2 which can also stimulate the fetal HPA axis (Neuroendocrinology 1996 Young et al .). We also decided to look at the cerebellum even though th ere is no connection between th e cerebellum and the fetal HPA axis found in the literature but it has been found in th e literature that estrog en action is present in the fetal cerebellum. There also could be a possibl e indirect stimulation of the HPA axis via the cerebellum. When an infusion of 17-beta-estradiol is put into the fetal ci rculation Fos expression in the cerebellum increases dramatically (Bra in Research 2003 Wood, Gridley, and Giroux). Fos is a transcription activator for cyclooxygenase 2, which produces PGE2. PGE2 has also been shown to stimulate the HPA axis as well (N euroendocrinology 1996 Young et al.) this might account for a possible connection between the ce rebellum and HPA axis. Western blots will be done as well to determine that protein leve ls coincide with RT-PCR data but only on the cerebellum due to it being the only brain regi on with enough tissue to perform this task.

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16 Figure 1-1. A diagram of the fetal HPA axis. CY450 is 17-alpha-hydoxylase. Figure 1-2. Shows how organi c anion transporters work on the ba solateral side of cells. Organic anions are brought into the cell by exchangi ng alpha-ketogluterate in the cell with organic anion in the blood. Alpha-ketogluter ate is an important metabolite so it is brought back in the cell via symport with sodium. The sodium needed for symport is provided by the electrochemical gradient cr eated by ATPase. Permission was given to use this figure (Physiol Rev. 2004 William H. Dantzler, and Stephen H. Wright)

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17 Figure 1-3. Pavlova’s data. OAT1 expression was de termined by In-situ hybrid ization in pictures A through J in the fetal kidney and brain. Nort hern blot analysis was also done on the kidney which is shown by K. A and C shows dark-field X-ray film images of the murine where OAT1-specific riboprobes hybrid ized to embryo histological sections. A and C shows sagittal sections through whole embryos. OAT1 specific-riboprobes were found to hybridize mainly in the dur a matter, choroid plexus, and kidneys. Permission was given to use this figure. ( Am J Physiol Renal Physiol. 2000 Pavlova et al)

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18 CHAPTER 2 MATERIALS AND METHODS Fetal sheep where extracted from the uterus of the pregnant ewe after an overdose of sodium pentobarbital was injected into the moth er intravenously which e ffectively kills mother and fetus. Fetal sheep were extracted at gest ational length ages of 80 (n=5), 100 (n=4), 120 (n=4), 130 (n=4), and 145 (n=5) days of gesta tion and newborn. After extraction of fetus from the mother’s womb we harvested brain samples from the cerebellum, pituitary, brainstem and hypothalamus of the fetus. They were snap-froze n in liquid nitrogen and stored at -80C. Messenger RNA was isolated by Trizol (Gibc o, Invitrogen Corp., Carsland, Ca) reagent and was stored at -80C in RNA secure (Ambion Corp., Austin Texas). Samples containing 4ug of mR NA was converted to cDNA by using the High Capacity cDNA archive Kit and following direction indi cated by manufacturer (Applied Biosystems, Foster City, Ca). RT-PCR was performed looking for OAT1 and OAT3 mRNA expression in the different regions of the fetal brain using AmpliTaq Gold DNA polymerase (Applied Biosystems) and primers (using primer software from Applied Biosystems) and probes (GenoMechanix, Alachua, Fl). Primers and probes fo r OAT1 and 3 were derived from the bovine mRNA sequences (accession number AJ549816 fo r OAT1 and accession number AJ627254 for OAT3) and TaqMan probes (Applied Biosystems) were used (Table 2-1). Primers and probes for Eukaryotic ribosomal 18s (Applied Biosyste ms) provided by Applied Biosystems were used to generate controls. 100ug of cDNA was used along with Forward primer at 900nanomole and reverse primer at 900nan omole were used as well as the probe at 250 micromole. The probes used for detecting OAT1 and OAT3 had 6-carbo xyfluoresceine (6-FAM) at the 5’ position and carboxytetramethyl rhodamine (TAMRA) at the 3’ position. Every sample that we ran RT-PCR on for OAT1 and OAT3 we also ran RT-PCR for th e18s ribosomal subunit to normalize the data.

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19 The ABI Prism 7000 detection sequence system (A pplied Biosystems) was used to perform RTPCR. Reactions done by the ABI Prism 7000 were run at: 48C for 30 minutes then 95C for 10 minutes, then 40 cycles of 95C for 15 seconds followed by 60C for 1 min (example of out put of ABI Prism 7000 in figure 2-1). We also ran controls with one well having only mRNA and another with just water. No pr oducts generated in the controls. Statistical analysis was done on the ct values calculated by using the Ct method. The Ct method is calculated by finding the difference between the mRNA OAT1 and OAT3 expres sion to that of the 18s ribosomal subunit relative to the mean ct valu e at 80 days of gestation. Immunohistochemistry was also performed on the fetal brain tissue samples looking for OAT1 and OAT3 mRNA expression in the brain tissue samples with antibodies for OAT1 and OAT3 (cat# OAT11-S and OAT311-S, Alpha Diagnosti cs, San Antonio, TX). After killing the mother and harvesting the fetus, pieces of the di fferent brain regions wher e then fixed overnight with 4% paraformaldahyde in test tubes and th en embedded in paraffin the next day. Then 5 micrometer thick section were prepared on s lides and treated with antibodies for OAT1 and OAT3. The protocol for antibody stai ning was as follows with 2minut e intervals: xylene, xylene, 100% alcohol, 90% alcohol, 70% alcohol, 50% alc ohol, 25% alcohol, water, and slide were than put in PBS, all at 2 min intervals. Then bloc king serum (10% goat serum in PBS) was than put on the slides for 10 min after usi ng the Barrier Pap Pen (Scientific Device Lab Inc, Des Plaines, Ill) to isolate reagents to th e tissue of the slide. Than the primary antibody (diluted at 1:1000 for OAT1 and 1:500 OAT3) was added and allowed to sit for about an hour (rabbit anti-rat for OAT1 and 3). After the primary antibody the slid es are washed in PBS for 2 min than the secondary antibody is added (goa t anti-rabbit) and left on the slide for an hour. After the secondary antibody there another wash with PBS fo r 2 min and then the streptavidin conjugate

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20 with the horseradish peroxidase (Zymed Inc., South San Francisco, Ca) is then added to the slides for 10 min with another PBS wash and then DAB (Pierce Co., Rockford,IL) is added to the slides for 10 min. After this we dehydrated the specimens using increasing concentrations of alcohol, then xylene, then finally cover slipped with Permount (Fischer Scientific, Fair Lawn, New Jersey) and allowed to sit overnight before observation under a microscope. All controls followed same protocol except no primary antib ody was used. In the Immunohistochemistry protocol for the pituitary slides was similar to all the others except Hemotoxylin (PolySciences Inc, Warrington, PA) was used for background stai ning to better show OAT1 and OAT3 staining with the horseradish peroxida se after dehydrating slides. Western blot analysis was also done using 7.5% Tris gels from Bio-Rad. There was 40ug of protein per well. The gel ran for 2 hours at 75 volts in Tris-HCL running buffer and was transferred to nitrocellulose membrane (Bio-R ad laboratories, Hercul es, CA) overnight in transfer buffer at 22 volts. The membranes was then blocked with 5% milk in PBS for 3 hours and then treated with OAT1 (1:1000) or OAT 3 (1:500) antibody and sat overnight. Then the membrane was washed 5 times with a wash period of 5 min. The nitrocellulose membranes were than put into a container where ECL reagen t (Amersham Pharmacia Biotech) was added and allowed to sit for a couple of minutes. Then the ECL treated membrane was exposed to the film (Blurate Automated film from Bioexpress) and was developed with exposure times between 3 to 10 minutes in an Autoradiography cassette (Fisher Scientific, Pittsburgh, PA). Densitometry was also done on western blots using the ChemiDoc XRS system (Bio-Rad ) to take the images of the western blot film and the Quantity One software (Bio-Rad) to quantify the intensity of the bands of different gestational ages on the western blot s. Densitometry was quantified as arbitrary units of optical density per mm2. Then one way analysis of variance (one way ANOVA) was done on

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21 the Ct values at different gestational lengths and densitometry of cerebellum western blots using the SigmaStat program (SPSS, Chicago, IL) and pair-wise comparisons were made using the Duncan multiple range tests. The null hypothesi s was rejected if the p value was less than 0.05. A correlation was also done on the Ct values of the cerebellu m to gestational age using the SigmaStat program (SPSS, Chicago, IL). The Pearson Test was used when doing the correlation. The Bonferonni test was also done on Ct values of the cerebellum to determine significance based on the small sample sizes and th e fact that the Duncan multiple range test wasn’t adequate in finding signi ficance therefor we did a non-para matric test (Bonferonni test). The Bonferonni test was calculated by th e SPSS program version 14.0 (SPSS, Chicago,IL).

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22 Table 2-1.Probe and primer sequences for re al-time RT-PCR analys is of OAT1 and OAT3 mRNA Gene of interest Forward primer Reverse primer Probe oOAT1 CATCTACCTAATCCA GGTGATCTTTG TGTTGATGACAAG GAAGCTCACA TGCTGTGGACCTG CCTGCCAAG bOAT3 CTGTGTGGCTTCGGC ATCT GGACACCCACTCG ACATTCAA AGGCATTACCCTG AGCACCGTCA Figure 2-1. An example of RT-PCR data ge nerated by the ABI Prism 7000 for OAT1 ontogeny in the pituitary.

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23 CHAPTER3 RESULTS In the hypothalamus OAT1 mRNA expression levels increased as gestational age increased until birth. This is when expression of OAT 1 starts to decline. The highest jump in fold change was between 145 days of gestation and birth. There was an upwar d trend in the mRNA expression level as gestation progressed. This con tinued until term where mR NA levels started to decline. The data is statistically significant and groups that are statistica lly different from one another are mentioned above each bar in the ba r graph according to the legend (Figure 3-1). OAT3 mRNA expression levels also appears to increase with gesta tional age but does not decrease after birth. mRNA expression levels con tinued to rise even after term. The apparent changes are not statistically significant (Figure 3-2). OAT1 mRNA expression was not consistent in the pituitary. There is no apparent expression pattern but there is statistical significan ce between certain gestational age groups (Figure 3-3). OAT3 mRNA e xpression level in the pituitary st eadily decreased until after birth where it dramatically increased back to fold cha nge level at 80 days of gestation. The data is statistically significant and groups that are statistically different from one another are mentioned above each bar in the bar gra ph according to the legend (Figure 3-4). OAT1 and OAT3 mRNA expression were constant in th e brainstem and no trend can be seen according to the graphs (Figure 3-5). The fold change of OAT1 expression in the cerebellum was the most dramatic of all the brain regions (Figure 3-6). As gestation pr ogressed mRNA expressi on levels increased dramatically. The highest jump in fold change was between 130 and 145 da ys of gestation right before birth. After birth fold change decrease s dramatically from a fold change of 600 to 10. There is no overall statistical significance as analyzed by ANOVA, but there is statistical

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24 significance as analyzed by Bonferr oni pairwise comparisons, as hi ghlighted in the graph (Figure 3-6). OAT3 expression in the cerebellum showed no apparent trend thr ough out gestation but there is a dramatic increase in mRNA for OA T3 at 130 days of gest ation (Figure 3-7). OAT1 staining is very abundant in the cerebellum as shown by immunohistochemistry in figures 3-8 and 3-9. It is found to be very abundant in the ventricle lining (F igure 3-9) as previously shown by Pavlova et al. (Am J Physiol Re nal Physiol. 2000 Pavlova et al .). OAT1 is also abundant in the outermost layers of the cerebellum that are e xposed to the cerebrospinal fluid as well as the granular layer of the cerebellum (Figure 3-8). OAT3 is not that abundant at all in the cerebellum but most OAT3 that showed up on slides were found in the granular layer and fiber tracts of the cerebellum. Immunohistochemistry of the hypothalamus s hown in black and white showed that OAT1 is mainly found around the blood vessels (Figure 3-11). OAT3 showed no significant staining in the hypothalamus. OAT 1 and OAT3 staining was very abundant in the pituitary. We made the pictures of IHC black and white to he lp show the contrast in the location of the different membrane proteins. As you can see OAT 1 is seen predominately on the membrane of pituitary cells (Figure 3-13) and OAT3 is found pr edominately on what seems to be the nuclei of the pituitary cells (Figure 3-14). Controls for all of IHC showed no nonspecific binding of the secondary antibody. Results from the western blots of the cerebe llum showed that OAT1 is found at molecular weights of 75kda and around 60kda (Figure 3-16 ). As gestation progresses the 75kda band becomes darker and the 60kda band gets lighter (Figure 3-16). Densitometry was also done on the western blot to confirm this event and it sh ows that the intensity for the 75Kda band increase

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25 as gestation progresses and the intensity for the 60Kda band decr eases as gestation progresses (Figures 3-17 and 3-18).

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26 Fold Change in OAT1 Expression in HypothalamusAge 80 dga100 dga120 dga130 dga145 dgaNew Born1 week Fold Change 0 5 10 15 20 25 LEGEND a-80dga b-100dga c-120dga d-130dga e-145dga f-Newborn g-1week bcdefg fg g g Figure 3-1. Fold change in OAT1 expression in Hypothalamus with legend that shows statistical difference between groups. An upward tre nd is shown in the graph as gestation progresses. Statistical significance is seen between 80 days of ge stational age (dga) and all other time periods suppor ting up regulation of gene.

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27 Fold Change in OAT3 Expression in HypothalamusAge 80 dga100 dga120 dga130 dga145 dgaNew Born1 Week Fold Change 0 2 4 6 8 10 12 14 Figure 3-2. Fold change in OAT3 expression in Hypothalamus continues to increase even after birth. There is an unusual reading in fold ch ange at 130 days of ge stational age (dga). There is however no statisti cal significance supp orting that the notion that the OAT3 gene in the hypothalamus is up regulated. Fold Change in Oat1 Expression in PituitaryAge 80 dga100 dga120 dga130 dga145 dgaNew Born1 week Fold Change 0.01 0.1 1 10 100 Statistical Signifigance a-80dga b-100dga c-120dga d-130dga e-145dga f-New Born g-1week bcg ade ade bcg bcg ade Figure 3-3. Fold change in OAT1 expression in Pituitary with lege nd showing statistical difference between groups. No apparent trend found as gestation progresses but statistical significance is s een between the age groups.

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28 Oat 3 mRNA expression Ontogeny in fetal pituitaryAge 80 dga100 dga120 dga130 dga145 dgaNew Born1 week Fold change 0.1 1 10 100 1000 Statistical Signifigance a-80dga b-100dga c-130dga d-145dga e-Newborn f-1week acd b b b Figure 3-4.Fold change in OAT3 expression in Pituitary with legend showing statistical difference between groups. Apparent down regulation is seen as gestation is progressing. There seems to be sign ificance between 100dga and 130dga and 145dga.

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29 A Fold Change in OAT 1 Expression in BrainstemAge 80dga100dga120dga130dga145dganewborn1week Fold Change 0.001 0.01 0.1 1 10 B Fold Change in OAT3 Expression in BrainstemAge 80dga100dga120dga130dga145dganewborn1week Fold Change 0.0001 0.001 0.01 0.1 1 10 Figure 3-5.Fold change in OAT1 (A) and OAT3 expression (B) in the brainstem shows no apparent trend through out gestation and no statistical signif icance between age groups.

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30 Fold Change in OAT1 Ex pression in CerebellumAge 80 dga100 dga120 dga130 dga145 dgaNew Born1 Week Fold Change 1 10 100 1000 10000 g,e e a,b a Statistical Significance a-80dga b-100dga c-120dga d-140dga e-145dga f-NewBorn g-1week Figure 3-6.Fold change in OAT1 expression in cerebellum throughout gestation. There is no overall statistical significance but there is statistical significance between certain gestational age groups. An upward trend is s een as gestation is progressing until birth where fold change decreases dramatically after birth. Fold Change in OAT3 Expression in CerebellumAge 80dga100dga120dga130dga145dganewborn1week Fold Change 0 20 40 60 80 100 120 140 160 a d Statistical Signifigance a80dga b-100dga c-120dga d-130dga e-145dga f-New Born g-1week Figure 3-7.Fold change in OAT3 expression in Cerebellum with legend showing statistical difference between groups. No apparent tr end is seen throughout gestation but at 130dga there is significant increase in fold change and is the only gestational age showing statistical significance.

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31 Figure 3-8.Immunohistochemistry of OAT1 Cere bellum showing layers of the cerebellum. OAT1 is mostly found in the granular layer of the cerebellum and the layer closest to the cerebrospinal fluid. Figure 3-9.Immunohistochemistry of OAT1 Cerebe llum showing ventricular membrane staining for OAT1. OAT1 staining also found in the surrounding tissue of the 4th ventricle in the cerebellum.

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32 Figure 3-10.Control for Cerebellum Imunohistohemist ry that is stained with only the secondary antibody to determine any non-specific binding. There is no apparent binding of the secondary antibody to cerebellular tissue

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33 A B Figure 3-11.(A) OAT1 hypothalamus Immunohistoc hemistry shown by making picture black and white to better highlight the staini ng around blood vessels. There is significant staining shown around the blood vessels of th e hypothalamus as well as endothelial cells of the hypothalamus(B) Same picture as the top picture but without being made black and white.

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34 Figure 3-12.Hypothalamus control which is stai ned only with secondary antibody in order to detect any anti-specific binding of the sec ondary. There is no non-specific binding of the secondary to hypothalamic tissue. Figure 3-13.OAT1 pituitary Immunohistochemistry shown by conversion of picture to Black and white. OAT1 staining in the pituitary is seen in the membranes of cells and the vasculature of the pituitary. Nu clei are also staining for OAT1 in the pituitary as well.

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35 Figure 3-14.OAT3 pituitary immunohistochemist ry shown by making picture black and white shows OAT3 in the nuclei of pituitary cells.

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36 A B Figure 3-15.(A) Hemotoxylin staining with OAT1 antibody staining. (B) Hemotoxylin staining with only secondary antibody to test for non-specific binding in the pituitary. The above figure shows that there is no non-sp ecific binding of the secondary antibody in the pituitary.

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37 Figure 3-16.Western blot for OAT 1 ontogeny of the fetal cerebe llum shows the75kda band gets darker as gestation progre sses but as the 60Kda band ge ts lighter as gestation progresses. The 60Kda band is where the darker band is seen below the 75Kda marker.

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38 Densitometry Graph 75KdaAge 80dga100dga120dga130dga145dganewborn1 weekmat INT*mm2 8000 9000 10000 11000 12000 13000 f,g,h h,f,g h a a,b,h a,b a,b,c Figure 3-17.Densitometry graph showing statisti cal significance for 75Kda band as gestation progresses. There is an upwar d trend in the intensity of the 75Kda band as gestation progresses showing possibl e influence by gestation. Figure 3-18.Densitometry graph showing statisti cal significance for 60Kda band as gestation progresses. A downward trend is seen as gestation progresses showing possible influence of gestation. Densitometry Graph for 60KdaAge 80dga100dga120dga130dga145dganewborn1 weekmat INT*mm2 10000 11000 12000 13000 14000 15000 16000 g,h g,h g,h g,h g,h g,h Statistical Significance to a-80dga b-100dga c-120dga d-130dga e-145dga f-Newborn g 1Week Statistical Significance to a-80dga b-100dga c-120dga d-130dga e-145dga f-Newborn g 1Week

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39 CHAPTER4 DISSCUSSION Our data shows that mRNA expression le vels of OAT1 and OAT3 do increase as gestation progresses in the feta l brain. The RT-PCR data supports the possibility that OAT1 and OAT3 might be the cause of a con tinual increase of HPA axis output in fetal sheep as gestation progresses. Immunohistochemistry also shows th at OAT1 and OAT3 protein also has been found in all the brain regions that have been stained some more than others have. Immunohistochemistry supports the theory that OAT1 and OAT3 is present in the tissue and could be transporting substances in or out of the brain durin g gestation. Here is a breakdown according to brain region based on the results from each region. In the hypothalamus, OAT1 and OAT3 expr ession patterns incr eased as gestation progressed except that OAT1 decreased after bi rth. The expression pattern could mean that OAT1 is playing some part in moving a cer tain substrate into hypothalamic cells during gestation. The OAT3 mRNA expressi on pattern continually increas ed showing that OAT3 could play a regulatory role in the ne w born sheep’s day to day functions or it could be a strategy for neonate survival until the body fully develops. Immunohistochemistry showed that the hypothalamus has a lot more OAT1 protein th an OAT3 protein. OAT1 was seen around blood vessels in the hypothalamus and the staining was much darker than OAT3. OAT3 staining was very faint and you could hardly see it under a micros cope. This is peculiar seeing the fold change reading was around the same except after birth. No western blots can be attained from the hypothalamus because there was not enough tissue to be collected to create enough protein to run on a gel. In the Pituitary mRNA expression pattern for OAT1 shows no pattern that would signify a possible role or function of OAT1 in the pituitary. The OAT3 expr ession pattern in the pituitary

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40 however decreased as gestation pr ogressed then increased right af ter birth. This could signify that whatever OAT3 is transporting in the fetus, it is being prevented fr om entering the pituitary cells during gestation by down regulating the OAT3 gene. Our lab proposes that OAT1 and OAT3 are transporting 17-beta-estradiol-3-sulpha te into cells and in a paper by Saoud and Wood they did an experiment showing that 17-beta-estra diol treatment of the p ituitary decreased active ACTH in the pituitary (Peptides 1996 Saoud and Wood). This coul d be a strategy to increase ACTH output of the pituitary to perpetuate the positive feedback loop. Immunohistochemistry for OAT1 and OAT3 were seen on both membranes and nuclei. Which type of pituitary cells they were on we do not know but it looks as if mo stly all of the pituitary cells on the slide were stained for OAT1 and OAT3. This c ould probably mean that 17-beta-e strdiol-3-sulphate is being pumped directly into pituitary cells such as corticotrophs based on results from Wood’s paper showing that infused 17-beta es tradiol increased basal ACTH levels (J Soc Gynecol Investig. 1997 Wood and Saoud). There is also a difference in their localization. OAT3 was found to be predominately on nuclei while OAT1 was predom inately found on the membranes of pituitary cells. OAT3 is the better transporter of sulfu r-conjugated estrogens out of the OAT family and therefore might play a bigger role in estrogen ac tion via 17-beta-estradiol at the pituitary. No western blots can be attained from the pituita ry because there wasn’t enough tissue to be collected to create enough protein to run on a gel. The brainstem showed no significant expression pattern for OAT1 or OAT3, Immunohistochemistry showed nothing significant except uniform staini ng throughout the slide which was faint. Data supports the notion that OA T1 and OAT3 have no significant role at the brain stem in regards to a possi ble effect of HPA axis output. No western blot data was done on

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41 the brain stem due to the fact that no patterns were seen in mRNA expression levels through RTPCR or immunohistochemistry. The cerebellum showed novel results. OAT1’ s expression pattern positively correlated (with raw data r =.476 and with the means of each gestational age r = .80 ) with gestation and birth, not to mention it had a very high fold change . At 80 days of gestational age its fold change was around 2. At 145 days of gestation (right before birth) the fold chan ge reading was about 600. Then right after birth the fold change dropped right back to 10. This data supports the idea that OAT1 at the cerebellum might play some role during gestation that is related to the timing of parturition since its fold change increases during gestation and then returns dramatically back to around its initial reading after bi rth. Immunohistochemistry showed that OAT1 was seen in the ventricular lining and membranes exposed to the cer ebrospinal fluid (CSF) in the cerebellum. It was also seen in the granular layer of the ce rebellum where OAT3 is also seen. OAT3 was also found in the fiber tracts in the cerebellum as we ll. This type of localization could mean that OAT1 might be transporting substrates in to the cells of the cerebellum or out of the cells of the cerebellum into the CSF. This shows that the ce rebellum could indirectly stimulate the HPA axis via the CSF in the brain. What was also interes ting is that as gestati on age increases the 75kda band gets darker and the 60kda band gets lighter on the western blot. Based on the amount of amino acids in the protein the molecular weight of the protein should be 60kda but as gestation progresses the 75kda band gets darker . It is believed by Tanaka et al that glycosylation might be activating the OAT1 protein and qu ite possibly altering OAT1 affinity for certain substrates (J Biol Chem. 2004. Tanaka et al.). Based on Tanaka’s results it c ould be glycosylation that is activating the OAT1 protein in th e cerebellum causing the 75kda OAT1 protein to become more predominant as gestation continues possibly cont ributing to the positive feedback loop of the

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42 fetal HPA axis. Glycosylated activation of the OAT 1 protein could also mean that there is the possibility that OAT1 found in the fetal brain coul d function differently or function at the brain for a different reason than OAT1 in the fetal kidn ey. The western blot pattern could be indicative of this because it is proposed by Nakajima and colleagues that OAT1 protein levels remain low in the kidney during gestation a nd doesn’t increase until after bi rth in rats (Kidney Int. 2000 Nakajima et al.). Nakajima did western blot s showing the protein levels for OAT1 during gestation was low until after birth in the rat. This western blot pa ttern might also be found in the other brain regions as well but th at will be determined in futu re experiments. The cerebellum might also play some role in increasing HPA axis output by bringing in 17-beta-estradiol into the CSF from the blood as well. Even though the data in our paper supports th e HPA axis output being influenced via the brain, data from another paper shows that OAT1 is found in the adrenal gland and has its action there. Beery and colleagues found out that OAT1 is found in the adrenal gland and is regulated by ACTH (Endocrinology 2003 Beery et al.). The mo re ACTH secreted the more OAT1 protein made in the adrenal gland (Endocrinology 2003 Beer y et al.). Beery and colleagues also did nonradioactive in situ hybrid ization of rats and found out that OAT1 was found mainly in the zona fasciculata, where glucocortico ids are synthesized and suggest s that OAT1 as well as other organic anion transporters are responsible for cortisol release (Endocrinology 2003 Beery et al.). OAT1 might influence the HPA axis at the adrenal gland and not at the br ain in fetal physiology or there is the possibility that it might affect the HPA axis at both places. This is a new perspective to look at when doing future experiments. The major strength of this work is the demonstration of OAT1 and OAT3 in the fetal brain using mRNA expression levels, OAT1 ontogeny western blot, and immunohistochemistry.

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43 Another strength is the demonstr ation that OAT1 and OAT3 are expr essed in patterns that are consistent with fetal neuroendocrine development. The mRNA expression levels of the pituitary and hypothalamus for OAT3 and the mRNA ex pression levels of the hypothalamus and cerebellum for OAT1 support the theory that orga nic anion transporter might affect the fetal HPA axis. The western blot for the ontogeny of the OAT1 protein in the cerebellum also supports the theory based on there being a lot of the OAT1 protein being present and the unique pattern involving the 75Kda band getting dark er and the 60Kda ba nd getting lighter as gestational age increases. Immunohistochemistry also shows the localization for OAT1 at the membranes of pituitary, cerebe llar, and hypothalamic cells possi bly allowing 17-beta-estradiolsulfate into or out of fetal brain cells. The major limitation of this work is that it was performed entirely at the mRNA and protei n expression levels and no experi ments were performed on intact animals. This limited our ability to test the phys iological function of thes e transporters in the fetal brain. Specific limitations in these experime nts include the fact that there were no ontogeny western blots done on the pituita ry or hypothalamus to support RT-PCR data at those brain regions. RT-PCR data does not always reflect prot ein levels and western blots must be done in the future to see if the RT-PCR data does. Anothe r limitation of this study is that we aren’t certain that OAT1 and OAT3 are m oving only 17-beta-estradiol into the brain since they have a wide substrate selectivity. We also don’t know the direction of movement of sulfoconjugated steroids either into or out of the brain. We are the first to look at the possibil ity of OAT1 and OAT3 moving sulfoconjugated estrogens into the brain to stimulate the HPA ax is. The functional role of OAT1 and OAT3 in the fetal brain is still unknown and th is study is to help reveal a po ssibility of OAT1 and OAT3 and its relation to fetal development. Data so far supp orts that gestational ag e does affect the mRNA

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44 expression level of OAT1 and OAT3 in different fetal brain tissues. OAT1 and OAT3 protein is also found in strategic places within the feta l brain, which support the theory of 17-betaestradiol-3-sulfate transport to specific regions of the brain. Future experiments will be done to help shine some light on the possible developm ental function of OAT1 and OAT3 in the fetal brain by inhibition of transpor t by using Probenecid in fetal sheep and monitoring HPA axis output. Other methods of inhibition of OAT1 will also be looked for as well since Probenecid blocks the whole organic anion transporter fa mily and we want to look at OAT1 and OAT3 specifically.

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45 LIST OF REFERENCES Beery E, Middel P, Bahn A, Willenberg HS, Hagos Y, Koepsell H, Bornstein SR, Muller GA,Burckhardt , Steffgen J. Molecular eviden ce of organic ion tran sporters in the rat adrenal cortex with adrenocorticotropin-re gulated zonal expre ssion. Endocrinology. 2003 Oct;144(10):4519-26. Epub 2003 Jun 26. Brooks AN, Gibson F. Prostaglandin E2 enha nces AVP-stimulated but not CRF-stimulated ACTH secretion from cultured fetal sh eep pituitary cells. J Endocrinol. 1992 Jan;132(1):33-8. Burckhardt BC, Burckhardt G. Transport of orga nic anions across the basolateral membrane of proximal tubule cells. Rev Physiol Biochem Pharmacol. 2003;146:95-158.. Dantzler WH, Wright SH. Th e molecular and cellular physio logy of basolateral organic aniontransport in mammalian renal t ubules. Biochim Biophys Acta. 2003 Dec 30;1618(2):185-93. Review. Eraly SA, Hamilton BA, Nigam SK . Organic anion and cation tr ansporters occur in pairs ofsimilar and similarly expressed gene s. Biochem Biophys Res Commun. 2003 Jan 10;300(2):333-42. Eraly SA, Vallon V, Vaughn DA, Gangoiti JA, Rich ter K, Nagle M, Monte JC, Rieg T, Truong DM, Long JM, Barshop BA, Kaler G, Nigam SK. Decreased renal orga nic anion secretion and plasma accumulation of endogenous organi c anions in OAT1 knockout mice. J Biol Chem. 2005 Dec 14. Hashimoto H, Noto T, Nakajima T. Effects of prostaglandin E2 and D2 on the release of vasopressin and oxytocin. Pros taglandins Leukot Essent Fa tty Acids. 1989 Apr;36(1):9-14. Jones SA, Adamson SL, Bishai I, Engelberts D, Norton JL, Coceani F. Prostaglandin E2 in cerebrospinal fluid of fetal and newborn sh eep: central versus pe ripheral source. Biol Neonate. 1994;66(6):339-51. Kim GH, Na KY, Kim SY, Joo KW, Oh YK, Ch ae SW, Endou H, Han JS. Upregulation of organic anion transporter 1 protein induced by chronic furosemide or hydrochlorothiazide infusion in rat kidney. Nephro. Dial. Transplant. 2003 Aug; 18(8): 1505-11 Kimura H, Takeda M, Narikawa S, Enomoto A, Ichida K, Endou H. Human organic anion transporters and human organic cation tr ansporters mediate renal transport of prostaglandins. J Pharmacol Exp Ther. 2002 Apr;301(1):293-8. Kis B, Isse T, Snipes JA, Chen L, Yamashita H, Ueta Y, Busija DW. Effects of LPS stimulation on the expression of prostaglandin carriers in the cells of th e blood-brain and bloodcerebrospinal fluid barriers. J Appl Physiol. 2005 Dec 1 Krunic N, Adamson SL, Bishai I, Coceani F. Pros taglandin uptake and catabolism by the choroid plexus during development in sheep. Brai n Res Dev Brain Res. 1997 May 20;100(1):82-9.

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46 Liggins GC. Parturition in the sheep and th e human. Basic Life Sci. 1974;4(PT. B):423-43. Review Liggins GC, Kennedy PC, Holm LW. Failure of in itiation of parturition after electrocoagulation of the pituitary of the fetal lamb. American Journal of Obstetrics and Gynecology, volume 98, app. 1080-1086, 1967. Am J Obstet Gynecol. 2000 Feb;182(2):473-4. Lu R, Kanai N, Bao Y, Schuster VL. Cloning, in vitro expression, and tiss ue distribution of a human prostaglandin transporter cDNA(hPGT ) .J Clin Invest. 1996 Sep 1;98(5):1142-9. Nakajima N, Sekine T, Cha SH, Tojo A, Hosoya mada M, Kanai Y, Yan K, Awa S, Endou H. Developmental changes in multispecific organic anion transporter 1 expression in the rat kidney. Kidney Int. 2000 Apr;57(4):1608-16 Nishio T, Adachi H, Nakagomi R, Tokui T, Sato E, Tanemoto M, Fujiwara K, Okabe M, Onogawa T, Suzuki T, Nakai D, Shiiba K, Su zuki M, Ohtani H, Kondo Y, Unno M, Ito S, Iinuma K, Nunoki K, Matsuno S, Abe T. Mol ecular identification of a rat novel organic anion transporter moat1, which transports prostaglandin D(2), le ukotriene C(4), and taurocholate. Biochem Biophys Re s Commun. 2000 Sep 7;275(3):831-8. Pavlova A, Sakurai H, Leclercq B, Beier DR , Yu AS, Nigam SK. Developmentally regulated expression of organic ion transporters NKT (OAT1), OCT1, NLT (OAT2), and Roct. Am J Physiol Renal Physiol. 2000 Apr;278(4):F635-43. Puurunen J, Leppaluoto J. Centrally administered PGE2 inhibits gastric secretion in the rat by releasing vasopressin. Eur J Pharmacol. 1984 Sep 3;104(1-2):145-50. Reimsnider SK, Wood CE. Does reduction of circulating prostagla ndin E2 reduce fetal hypothalamic-pituitary-adrenal axis activity? J Soc Gynecol Investig. 2005 May;12(4):e139. Saoud CJ, Wood CE. Ontogeny of proopiomelanocor tin postranslational processing in ovine fetal pituitary. Peptides. 1996; 17(4): 649-653 Sekine T, Cha SH, Endou H. The multispecific or ganic anion transporter (OAT) family. Pflugers Arch. 2000 Jul;440(3):337-50. Review. Sweet DH, Miller DS, Pritchard JB, Fujiwara Y, Beier DR, Nigam SK. Impaired organic anion transport in kidney and choroid plexus of organic anion transporter 3 (Oat3 (Slc22a8)) knockout mice. J Biol Chem. 2002 Jul 26;277(30):26934-43. Tamai I, Nezu J, Uchino H, Sai Y, Oku A, Sh imane M, Tsuji A. Molecular identification and characterization of novel memb ers of the human organic anion transporter (OATP) family.Biochem Biophys Res Commun. 2000 Jun 24;273(1):251-60.

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47 Walker DW, Pratt N. Effect of probenecid on breathing movements and cerebral clearance of prostaglandin E2 in fetal sheep. J Ph ysiol. 1998 Jan 1;506 ( Pt 1):253-62. Wood CE, Giroux D, Gridley K. Fetal brain re gional responses to ce rebral hypoperfusion: modulation by estrogen. Brain Res. 2003 Dec 12;993(1-2):84-9. Wood CE, Saoud CJ Influence of estradiol and an drostenedione on ACTH and cortisol secretion in the ovine fetus. J Soc Gyneco l Investig. 1997 Nov-Dec;4(6):279-83 Young IR, Loose JM, Kleftogiannis F, Canny BJ. Pr ostaglandin E2 acts via the hypothalamus to stimulate ACTH secretion in the fetal sheep. J Neuroendocrinol. 1996 Sep;8(9):713-20.

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48 BIOGRAPHICAL SKETCH Roderick Dimitri Cousins was born on Decembe r 6, 1980 in Miami, Florida. Roderick is the oldest of three children and son to Donnetta Cousins and Roderick Rufus Cousins. He grew up in Miami, Florida until eighth grade when he th en left the United States of America to go to school in Manchester, Jamaica at Belair High Sch ool. Then in his tenth grade year of school, he returned to the United States of America to finish High School at Barbara Goleman High School in Hialeah, Florida in the spri ng of 1999. After completion of hi gh school, Roderick attended the University of Florida where he received his B.S. degree in animal sciences form the College of Agriculture and Life Sciences in May of 2005. After graduating with his bachelors of science, Roderick decided to become a candidate for an M.S. in the Biomedical Sciences to do rese arch in Dr. Charles Wood’s lab. Roderick plans on using this degree to further enhance the co mmunication between medical doctors and basic scientists, in order to enhance the application of research done in the lab to research done in the clinic. Roderick plans on applying to medical school upon graduation to aid in moving basic science research in the lab into a clinical research setting.