A dependable source of quality fish spawn is a fundamental prerequisite for fish culture development. This is especially important inasmuch as most of the cultivable species do not breed in confined waters. Also, sport fisheries are gaining greater popularity, and subsequently the fish supply is being taxed. Still another need for fish spawn is in the ever increasing demand for bait minnows. Also, the construction of more and more dams has resulted in insurmountable obstacles for ascending and descending fish, which may ultimately result in complete destruction of some fisheries. Thus some measure of artificial propagation will have to be taken to safegaurd our valuable fishery resources. A partial solution to this problem of supplementing natural propagation is that of inducing the fish to spawn artificially in the hatchery. A method of doing this is by stimulating fish to breed by the use of pituitary hormones. Those pituitary hormone-containing glands are often collected under a variety of field conditions which may involve considerable effort, time, and money. Therefore,, it was my objective in this study to develop a practical refined assay on hormones using as small an amount as possible of the crude extract of pituitary suspension, and to make it simple enough that every lay fisheries man ,dll be able to apply it, thus meeting his demand for quality fish eggs in his own hatchery when he needs it most.

The focus of this dissertation was leptin and the leptin receptor, and the role of these genes (OB and OB-R) in the development of obesity and type 2 diabetes in humans and Psammomys obesus, a polygenic rodent model of obesity and type 2 diabetes. Studies in humans showed that circulating leptin concentrations were positively associated with adiposity, and independently associated with circulating insulin and triglyceride concentrations. Analysis of two leptin receptor sequence polymorphisms in a Caucasian Australian population and a population of Nauruan males, with very high prevalence rates of obesity, showed no associations between sequence variation within the OB-R gene and obesity- or diabetes-related phenotypic measures. In addition, these two OB-R polymorphisms were not associated with longitudinal changes in body mass or composition in either of the populations examined. A unique analysis of the effects of multiple gene defects in the Nauruan population, demonstrated that the presence of sequence alterations in both the OB and OB-R genes were associated with insulin resistance. Psammomys obesus is regarded as an excellent rodent model in which to study the development of obesity and type 2 diabetes in humans. Examination of circulating leptin concentrations in Psammomys revealed that, as in humans, leptin concentrations were associated with adiposity, and independently associated with circulating insulin concentrations. This animal model was utilised to examine expression of OB-R, and the regulation of expression of this gene after dietary manipulation. OB-R is known to have several isoforms, and in particular, OB-RA and OB-RB gene expression were examined. OB-RB is the main signalling isoform of the leptin receptors. It has a long intracellular domain and has previously been shown to play an important role in energy balance and body weight regulation in rodents and humans. OB-RA is a much shorter isoform of OB-R, and although it lacks the long intracellular domain necessary to activate the JAK/STAT pathway, OB-RA is also capable of signalling, although to a lesser degree than OB-RB. OB-RA is found to be expressed almost ubiquitously throughout the body, and this isoform may be involved in transport of leptin into the cell, although its role remains unclear. OB-RA and OB-RB were both found to be expressed in a large number of tissues in Psammomys obesus. Interestingly, obese Psammomys were found to have lower levels of expression of OB-RA and OB-RB in the hypothalamus, compared to lean animals. This finding raises the possibility that decreased leptin signalling in the brain of obese, hyperleptinemic Psammomys obesus may contribute to the leptin resistance previously described in this animal model. However, the primary defect is unclear, as alternatively, increased circulating leptin concentrations may lead to down-regulation of leptin receptors. The effect of fasting on leptin concentrations and gene expression of OB-RA and OB-RB was also examined. A 24-hour fast resulted in no change in body weight, but a reduction in circulating leptin concentrations, and an increase in hypothalamic OB-RB gene expression in lean Psammomys. In obese animals, fasting again did not alter body weight, but resulted in an increase in both circulating leptin concentrations and hypothalamic OB-RB gene expression. In the liver, fasting resulted in a large increase in OB-RA gene expression in both lean and obese animals. These results highlighted the fact that regulation of leptin receptor gene expression in polygenic models of obesity and type 2 diabetes is complex, and not solely under the control of circulating leptin concentrations. Sucrose-feeding is an established method of inducing obesity and type 2 diabetes in rodents, and this experimental paradigm was utilised to examine the effects of longer term perturbations of energy balance on the leptin signalling pathway in Psammomys obesus. Addition of a 5% sucrose solution to the diet of lean and obese Psammomys resulted in increased body weight in both groups of animals, however only obese Psammomys showed increased fat mass and the development of type 2 diabetes. The changes in body mass and composition with sucrose-feeding were accompanied by decreased circulating leptin concentrations in both groups of animals, as well as a range of changes in leptin receptor gene expression. Sucrose-feeding increased hypothalamic OB-RB gene expression in obese Psammomys only, while in the liver there was evidence of a reduction in OB-RA and OB-RB gene expression in both lean and obese animals. The direct effects of sucrose on the leptin signalling pathway are unclear, however it is possible to speculate that the effect of sucrose to decrease leptin concentrations may have been involved in the exacerbation of obesity and the development of type 2 diabetes in obese Psammomys, From these studies, it appears that sequence variation in the OB and OB-R genes is unlikely to be a major factor in the etiology of obesity in human populations. The ability to examine regulation of expression of these genes in Psammomys obesus, however, has demonstrated that the effects of nutritional modifications on leptin receptor gene expression need closer attention. The role of the OB and OB-R genes in metabolism and the development of type 2 diabetes also warrants further examination, with particular attention on the differential effects of dietary modifications on leptin receptor gene expression across a range of tissues.

The neurohypophysial peptide system is involved in modulating a variety of physiological, neurological, and behavioral responses in vertebrates. The principal forms of these peptides in non-mammalian tetrapods are vasotocin (VT) and mesotocin (MT). The studies described in this thesis used pharmacological, molecular, and biochemical techniques, along with phylogenetic analyses, to identify and characterize the mRNA sequences encoding the neurohypophysial peptide precursor proteins and their receptors in urodele amphibians. The cDNAs encoding preproVT and preproMT were amplified by PCR from the brains of two salamander species; the rough-skinned newt, Taricha granulosa, and the red-legged salamander, Plethodon shermani. The neurohypophysial peptides encoded by the identified Taricha cDNAs were VT and MT; the Plethodon cDNAs encoded VT and a novel MT-like peptide, [Val⁴]-MT. Phylogenetic analyses grouped both the Taricha and Plethodon preproVT and preproMT-like sequences with previously identified tetrapod preproVT-like and preproMT-like sequences, respectively. Additional analysis of the preproneurohypophysial sequences indicated that gene conversion (non-homologous crossing over of DNA sequences) appears to have occurred more frequently in mammals than in other tetrapod classes. The cDNAs encoding the VT receptor (VTR) and MT receptor (MTR) were amplified from the brain of T. granulosa by PCR. Sequence identity, and phylogenetic analysis, indicated that the Taricha MTR and VTR were most similar to MTR/OTRs and V[subscript 1a]-like VTRs, respectively. Distribution of PCR amplicons specific to the Taricha MTR and VTR matched previously reported tissue distributions of MTRs and VTRs in other vertebrates in every tissue but kidney, from which the Taricha primers were unable to amplify a cDNA product. Binding experiments of transiently expressed Taricha MTR indicated two binding states, and allowed the determination of ligand binding affinities for this receptor. Inositol phosphate accumulation assays demonstrated that the expressed Taricha MTR and VTR cDNA produced functional receptors, and allowed calculation of ligand potencies of activation and inhibition. Surprisingly, an antagonist frequently used in behavioral experiments to specifically block VTR activity, inhibited inositol phosphate accumulation in cells transfected with either the Taricha MTR or VTR. In conclusion, these studies report the first identified cDNA sequences encoding the preproVT, preproMT, MTR, and V[subscript 1a]-like VTR proteins from urodele amphibians. / Graduation date: 2005

Graduation date: 1996

Thesis (M.S.)--West Virginia University, 2006. / Title from document title page. Document formatted into pages; contains vi, 86 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 75-85).

The glycoprotein hormone family comprising of Luteinizing Hormone (LH), Chorionic Gonadotropin (hCG), Follicle Stimulating Hormone (FSH) and Thyroid Stimulating Hormone (TSH) plays important role in reproduction and overall physiology of the organism. These hormones are heterodimeric molecules consisting of an identical α subunit non-covalently associated with the hormone-specific β subunit. Both subunits of all these hormones are N-glycosylated. In addition, hCGβ subunit also has four O-linked oligosaccharides located at the C-terminus of the polypeptide(1). The α and β subunits of all these hormones contain five and six disulfide bonds respectively and the crystal structures of hCG and hFSH indicate that both subunits of the hormones belong to the cystine knot family of proteins(2-4). Although the β subunits are hormone specific, there are distinct similarities in these subunits with the 12 cysteines conserved in all these subunits (1). These hormones, because of their unique structural features have proved to be important models for structure–function relationship studies of complex dimeric glycoproteins. Folding of subunits during biosynthesis, role of glycosylation in folding pathways and in vitro and in vivo bioactivity of the hormone, as well as, identification of domains important for subunit association, receptor binding and subsequent signal transduction have been topics of active investigations. The receptors of these hormones belong to the family of G-protein coupled receptors (GPCR) and have unique hormone specific exodomain not present in other members of the GPCR family and characteristic seven transmembrane domains followed by a C terminal domain(5). Primary structure analysis of Glycoprotein hormone receptors family revealed sequence conservation, maximum homology being observed in the transmembrane domain (TMD)(6). Significant homologies could be observed in the hormone specific extracellular domains (ECD) also (7). Despite these homologies, the receptors exhibit exquisite specificity with very low cross reactivity with other members of the hormone family (8). Elucidation of the molecular details of the contacts between the hormone and the receptors has not been achieved so far. Various approaches have been employed to delineate the residues or domains of both hormone and receptors involved in interaction. These include testing of chimeras or mutants of hormones or receptors for changes in activity (9-12), chemical modifications(13) and competition with peptides from either hormones (14) or receptors (15). Polyclonal and monoclonal antibodies against glycoprotein hormones and various fragments of their receptors have been used to determine the role of different domains of both in binding and response (6, 16, 17). However, till date there is no consensus on the specific mechanisms by which the glycoprotein hormone docks onto its receptor. It was proposed that the initial contact between the hormone and the receptor occurs through high affinity binding of the hormone specific β subunit to the Leucine rich regions of the ECD that results in conformational changes in both hormone, as well as, the receptor and brings hormone/ECD complex closer to the TMD of the receptor. The secondary, relatively lower affinity interactions between the hormone and the receptor then take place through common α subunit and exoloops of TMD of the receptor resulting in signal generation (18, 19). Recently a different kind of model has been proposed which suggests that the hormone does not make any direct contacts with the TMD of the receptor. The signal is transduced by the change in contacts between ECD and TMD brought about by hormone’s interaction with ECD(8, 20). The present study was initiated with an overall objective of understanding the molecular details of the hormone receptor interactions of this family, particularly hCG- LH receptor interactions. Two different approaches were employed for this purpose; the first, direct approach being structure elucidation of the members of the glycoprotein hormone family while the second approach uses antibodies against hCG as tools to probe into hormone-receptor interactions. The results obtained using these two approaches have been consolidated in the present thesis and are organized as follows. Chapter 1 is an extensive review of the literature and it builds background for the present work while the exact aim and scope of the present work have been defined in Chapter 2. Chapter 3 describes cloning, expression and purification of recombinant glycoprotein hormones hLH, hCG and single chain derivative of hCG. The Chapter 4 gives details of the molecular aspects of hCG-LH receptor interaction dissected using hCG monoclonal antibodies (MAbs). Chapter 5 discusses implications of the observations made in the present study and states the future directions envisaged. There are a number of endocrinopathies associated with abnormal levels of glycoprotein hormones and treatments of such disorders often demand large quantities of either agonists or antagonists of the hormones. The structure-function relationship studies should help in identifying domains/residues important for subunit interaction, receptor binding, and signal transduction, which would also allow engineering of agonists and antagonists of hormone action. However, structure determination of the glycoprotein hormone family using X-ray crystallography has proved to be a difficult task and it is believed that the heterogeneity in glycosylation is the primary reason for this low success rate in the process of crystallization. The first crystal structure of hCG was that of completely deglycosylated hCG but such a molecule displays antagonistic behavior(2, 3). Use of NMR spectroscopy, the alternate method commonly used for structure determination is often limited by the availability of large quantities of biologically active hormones free of any contaminants. Large quantities of LH, hCG and FSH are also required for treatment of infertile patients suffering from gonadotropin deficiency. The first goal of the present study was thus to produce and purify biologically active recombinant hCG and hLH. Owing to the inherent features of glycoprotein hormones and their potential therapeutic applications, the recombinant expression of these hormones is an important goal from both basic research, as well as, commercial point of view. Considering the above mentioned features it is clear that the expression system used for the hyperexpression of these glycoprotein hormones should also serve as a model system for investigating structure–function relationships and folding of subunits during biosynthesis, in addition to providing sufficient quantities of the hormones for clinical applications. It has been demonstrated that N-linked glycosylation during biosynthesis facilitates protein folding and conformational maturation of glycoprotein hormone subunits into an assembly-competent, biologically active form (21). Therefore, the ideal recombinant expression system should also be able to glycosylate the protein during biosynthesis. The Pichia pastoris yeast expression system was chosen for hyperexpression of glycoprotein hormones as it blends the advantages of both bacterial and mammalian expression systems. Earlier, expression of biologically active hCG and the subunits of hCG and bovine FSH using Pichia pastoris expression system has been reported from the laboratory (22, 23). Chapter 3 (section 3.3.1) of the thesis describes hyperexpression of hLH. The expression of these heterologous proteins was scaled up using fermentation procedures to fulfill the requirements of large quantities of hormones for various applications. Purification of Pichia expressed hormones turned out to be a complex task as large quantity of the hormone was secreted out in the fermentation medium (10litre volume) that was of high ionic strength. Of several different strategies attempted for concentration and partial purification of recombinant hCG, hydrophobic interaction chromatography (HIC) using Phenyl Sepharose matrix emerged as the most efficient technique as a first step of purification. Subsequently, cation exchange chromatography using SP- Sepharose matrix yielded completely purified biologically active recombinant hCG (section 3.3.2). The preliminary data also suggested that Pichia cells express a biologically active form of hCG which appeared to be less glycosylated and of lower molecular weight. Using the same protocol purification of hLH, as well as, single chain derivative of hCG, phCGαβ was achieved (section 3.3.3). These recombinant proteins were characterized extensively using various biochemical, as well as, immunological criteria and were shown to be similar to their natural counterparts with respect to their ability to bind LH receptor and to transduce signal as judged by radioreceptorassays and in vitro bioassays respectively. The hydrophobic interaction chromatography proved an important starting point for purification of all the other members of the glycoprotein hormone family expressed using Pichia pastoris expression system. With the availability of purified, biologically active recombinant hCG in large quantities it was now possible to make attempts towards structure elucidation using NMR spectroscopy. The structure determination of such complex proteins by NMR spectroscopy is made relatively easier by labeling the proteins with magnetically more active, stable isotopes of carbon and nitrogen, 13C and 15N respectively however the cost is often prohibitively high. The Pichia pastoris expression system offers simple means of labeling the proteins as the cells can be grown on simple salts of carbon and nitrogen such as 13C labeled methanol, 15N labeled ammonium chloride or ammonium sulphate. The Chapter 3 also gives a brief account of the preliminary attempts made to label the recombinant hCG with 15N and the structural studies carried out with the carbohydrate moieties of the recombinant hCG using solution NMR spectroscopy. This work was carried out in collaboration with the laboratory of Prof. J.P Kamerling of the University of Utrecht, Netherlands and the efforts are currently underway to elucidate the complete structure of the Pichia expressed hCG. The common feature of receptors and antibodies against the ligand is that both display very specific, high affinity binding towards the ligand. Hence, it is logical to speculate that the antigen binding regions of the antibodies that inhibit hormone binding and/or response, exhibit homology with distinct domains of the receptor. By identifying the epitopes recognized by such antibodies, it should be possible to predict contact points between the hormone and the receptor. In the present study, this hypothesis has been tested using monoclonal antibodies (MAbs) against hCG recognizing different epitopes in the hormone molecule and having different effects on hormone binding and response (Chapter 4). These MAbs were classified as α subunit specific, β subunit specific or heterodimer specific depending on their abilities to bind either subunit in addition to the hormone itself. Interestingly, it was observed that the hCGβ subunit specific MAbs, as well as, heterodimer specific MAbs inhibited hCG receptor binding and hence the response generated by hCG, while the hCGα subunit specific MAbs inhibited only response to the hormone without interfering in binding (Section 4.3.1). To dissect out these interactions further the epitopes recognized by these antibodies on hCG molecule were determined (Section 4.3.2), single chain fragment variable (ScFv) were generated from each of these antibodies and it was shown that these ScFv retain the functionality of the original antibody (Section 4.3.3). Further, the amino acid sequence of each antibody was determined (Section 4.3.4) and finally shown that the antigen binding domains of antibodies show homology to the distinct regions of the LH receptor on sequence alignments between the two using three different programs (4.3.5). The hCGβ subunit specific MAb 52/28' displayed distinct homology with the ECD of LH receptor while the α subunit specific MAb C10 showed regions homologous to TMD of the receptor and the heterodimer specific MAb E12 was found to be similar to the hinge region of the receptor. This clearly indicates that the β subunit of hCG is in close contact with ECD of the receptor while the α subunit makes contacts with the TMD of receptor. The present study thus supports the existing model of hormone receptor interactions, which states that the hormone first binds to the exodomain of the receptor mainly through its β subunit while the integrity of the α subunit is critical for signaling. (24, 25). Also, the observations made in the present study exhibit an interesting possibility of antigen antibody complexes being used as surrogate models for gaining insights into hormone receptor complex. Further, it has been reported that hCG has immunocontraceptive potential(26). Active and passive immunization studies with hCG in primates and humans have demonstrated the possibility of controlling fertility by the antibodies capable of neutralizing hCG. This forms the basis for female contraceptive vaccine that has undergone Phase II clinical trials in India. The MAb E12 characterized in the present study displayed highly specific binding to heterodimeric hCG exclusively without showing any cross reactivity with hLH (Section 4.3.1). The epitope mapping analysis revealed that this antibody recognizes a unique discontinuous epitope present only in the heterodimeric hCG and is distinct from the unique C-terminal extension of hCGβ absent in hLHβ (Section 4.3.2). The MAb, IgG or its recombinant single chain fragment variable (ScFv), inhibited response to hCG, but not to hLH (4.3.3). Thus, the epitope recognized by this MAb is an ideal candidate antigen for immunocontraception. The MAb E12 can also be used for passive immunization in case of emergency contraception. Another potential application of hCG specific antibodies is in homing and the treatment of tumors that secrete hCGβ subunit. The hCGβ subunit specific MAbs used in the present study 52/12 and 52/28' that inhibited hCG receptor binding as well as response generated by hCG can be used in treating such tumors. The functional ScFvs generated from these MAbs in the present study can be made use of on humanization. Thus, the present study has yielded some important molecules for therapeutic applications besides providing a new platform for structure-function relationship studies of the complex glycoprotein hormones.

The focus of the research described in this section of the thesis is the synthesis of compounds expected to bind strongly to both the estrogen β and α receptors and act as estrogen agonists. Based on earlier results in our group and docking studies we prepared a series of A-CD analogs, compounds 1, in which the usual 13-methyl group was replaced by an ethyl group. Docking studies also indicated that substituents at C8 could lead to enhancement of binding to the estrogen receptor. With this in mind two such derivatives, compounds 2 were prepared. A major concern in the use of estradiol in hormone replacement therapy is its potential metabolism of dangerous ortho-quinones. The 1,2-naphthalenediol derivatives 3 avoid this possibility. They were predicted to be potent binders to the estrogen receptors with the naphthalene diol portion serving as rings A and B and the hydroxyl group taking the place of the 17-OH group of estradiol. The preparation of several derivatives of 2 is reported. The estrogen receptor binding [ERB] relative to estradiol as standard has been determined at the University of Illinois for a number of the compounds prepared in this thesis. Unfortunately, the results were not as encouraging as expected. Importantly, all of the 13-ethyl derivatives tested showed lower binding affinity compared to the 13-methyl analogs. Similarly, the derivatives with substituents at C8 do not show higher activity than those having only hydrogens at C8. Finally, the situation with the naphthalene derivatives is, at this stage, still not completely resolved. The binding for the compounds thus tested is quite low, but it must be admitted that the structures thus far synthesized have a much lower LogP than estradiol, a factor known to greatly decrease the binding constants to the estrogen receptors.

No description available.

In the ewe, one to three antral follicles emerge or grow from a pool of small antral follicles (1 to 3 mm in diameter) every 3 to 5 days and reach diameters of ¡Ý5 mm before regression or ovulation. Each follicular wave is triggered by a peak in serum concentrations of FSH. It is not clear what characteristics of an FSH peak cause follicular wave emergence and what aspects of development of a follicular wave are regulated by its preceding FSH peak.<p> In Experiment 1, we found that the amplitude of FSH peaks decreased, while basal serum FSH concentrations increased across the inter-ovulatory interval (P < 0.05). However, there were no associated changes in the growth, static or regression phases of follicular waves or the number and size of follicles in a wave. In Experiment 2, using computer-assisted quantitative echotextural analysis, we found that the numerical pixel value (NPV) for the wall of anovulatory follicles emerging in the third wave of the cycle was significantly higher than for waves 1 and 2 at the time of wave emergence but it decreased as follicles reached maximum follicular diameter (P < 0.05). A tendency for a similar pattern for the wall of follicles in the last wave of the cycle was also observed (P = 0.07).<p> In Experiment 3, treatment with ovine FSH (oFSH) increased the amplitude of an FSH peak by 5 to 6 fold. This treatment increased estradiol production (P < 0.05) but had little effect on other characteristics of the subsequent follicular wave. Daily injections of oFSH (Experiment 4) for four days, resulted in the occurrence of 4 discrete peaks in serum FSH concentrations. Each injection of oFSH resulted in the emergence of a new follicular wave.<p> In Experiment 5, six cyclic ewes received oFSH (0.1 ¦Ìg/kg, sc) every 6 h for 42 h, to try to give a gradual increase in the leading slope of an FSH peak. Serum FSH concentrations increased in oFSH treated ewes (P < 0.05) resulting in an additional peak between two endogenously driven FSH peaks and therefore, did not give the planned gradual leading slope to an FSH peak. Ovine FSH treatment occurred in the early growth phase of wave 1 of the inter-ovulatory interval and increased the growth rate of growing follicles in that wave, compared to control ewes (P < 0.05). This apparently induced dominance in follicles in wave 1, causing them to suppress wave emergence in response to the injected FSH. In Experiment 6, oFSH was infused constantly (1.98 ¦Ìg/ewe/h, iv, n = 6) for 60 h. Infusion of oFSH maintained serum FSH concentrations at a level similar to the zenith of a peak. This resulted in a superstimulatory effect with a peak in the mean number of large follicles on Day 2 after the start of FSH infusion (P < 0.001).<p> A hormonal milieu similar to low serum progesterone concentrations was created by treatment of ewes with prostaglandin and medroxyprogesterone acetate (MAP) sponges (Experiment 7). This treatment delayed regression of the penultimate follicular wave of a cycle. However, the delayed follicular atresia was accompanied by a greater degree of apoptosis in somatic cells of follicles growing in the penultimate wave compared to those in the final wave of the cycle, when collected one day before expected ovulation.<p> In conclusion, trends in basal serum concentrations of FSH and peaks in serum FSH concentrations, across the estrous cycle, are associated with changes in the image attributes of follicles emerging later in the estrous cycle, perhaps reflecting a greater readiness of those follicles for ovulation and formation of CL. The ovine ovary can respond to discrete peaks in serum FSH concentrations with the emergence of new follicular waves on a daily basis. This led us to conclude that follicular dominance is not evident in the ewe and peaks in serum FSH concentrations are likely to be driven by some endogenous rhythm that is unrelated to ovarian follicular secretory products. However, direct dominance can be induced by giving supplemented FSH during the growth phase of a follicle. Extended exposure of ovine ovaries to the serum concentrations of FSH found at the zenith of a peak overrides the mechanisms that recruit follicles into a wave and induces a superovulatory response in cyclic ewes. Finally, an increase in the incidence of apoptosis occurs in antral follicles in sheep that have an extended lifespan, prior to any morphological changes detectable by ultrasonography. This would seem to cause decreased follicular viability and lowered fertility of the oocytes that the follicles contain.

The focus of the research described in this section of the thesis is the synthesis of compounds expected to bind strongly to both the estrogen β and α receptors and act as estrogen agonists. Based on earlier results in our group and docking studies we prepared a series of A-CD analogs, compounds 1, in which the usual 13-methyl group was replaced by an ethyl group. Docking studies also indicated that substituents at C8 could lead to enhancement of binding to the estrogen receptor. With this in mind two such derivatives, compounds 2 were prepared. A major concern in the use of estradiol in hormone replacement therapy is its potential metabolism of dangerous ortho-quinones. The 1,2-naphthalenediol derivatives 3 avoid this possibility. They were predicted to be potent binders to the estrogen receptors with the naphthalene diol portion serving as rings A and B and the hydroxyl group taking the place of the 17-OH group of estradiol. The preparation of several derivatives of 2 is reported. The estrogen receptor binding [ERB] relative to estradiol as standard has been determined at the University of Illinois for a number of the compounds prepared in this thesis. Unfortunately, the results were not as encouraging as expected. Importantly, all of the 13-ethyl derivatives tested showed lower binding affinity compared to the 13-methyl analogs. Similarly, the derivatives with substituents at C8 do not show higher activity than those having only hydrogens at C8. Finally, the situation with the naphthalene derivatives is, at this stage, still not completely resolved. The binding for the compounds thus tested is quite low, but it must be admitted that the structures thus far synthesized have a much lower LogP than estradiol, a factor known to greatly decrease the binding constants to the estrogen receptors.