The start of the menopausal transition involves the introduction of various somatic, urogenital, and psychological symptoms; of the symptoms, irritability is one of the main complaints reported by women. The use of bioidentical hormone replacement therapy has become more prevalent in society, specifically treating the somatic and urogenital symptoms of the menopausal transition. This study aims to determine the effects of bioidentical hormone replacement therapy (BHRT) on irritability in menopausal women. To test the hypotheses, an online survey was distributed to women via social media and word-of-mouth. Participants were asked to respond to various questions, which were then analyzed based on BHRT use. An independent samples t-test was used to analyze the data. The results exemplified no significant relationship between BHRT and irritability; using BHRT does not significantly reduce irritability scores.

The role of endogenous dopamine, utilizing a dopamine antagonist (fluphenazine; FLU), in modulation of gonadotropin, growth hormone (GH) and prolactin (PRL) secretion during the early postpartum period and various stages of the estrous cycle was investigated in Holstein cows. Experiment 1 was conducted in anovulatory early postpartum cows. Fluphenazine caused a decrease (P < .05) in mean serum LH concentration and LH pulse frequency. Likewise, FLU caused a (P < .05) decrease in mean GH concentration. These results suggest that endogenous dopamine, at least in part, is responsible for regulation of LH and GH secretion in anovulatory Holstein cows. Experiment 2 was conducted in cyclic lactating Holstein cows during the mid-luteal phase of the estrous cycle. Mean serum LH and FSH concentrations, pulse frequencies, and peak amplitudes did not change in response to FLU. FLU did not affect mean serum GH concentration. These results suggest that a dopamine-mediated mechanism for modulation of gonadotropin and GH secretion is absent or perhaps overridden by high progesterone concentration during the luteal phase of the estrous cycle in lactating dairy cows. Experiment 3 was conducted during the early follicular phase of the estrous cycle in Holstein cows. During the follicular phase, FLU caused a decrease (P < .05) in mean serum LH concentration and LH pulse frequency. However, FLU had no effect on mean serum FSH concentration or pulse frequency. Further, FLU increased (P < .05) GH concentrations during the follicular phase. Experiment 4 was conducted during the early metestrus phase of the estrous cycle. During the metestrus phase, FLU tended to decrease (P < .1) mean LH concentration and suppressed (P < .05) LH pulse frequency but had no effect on FSH secretion. Fluphenazine caused a transient increase (P < .05) in mean serum GH concentration. The results of the third and fourth experiments suggest that, during the early follicular and metestrus phases of the estrous cycle, when progesterone concentration is low, modulation of LH and GH secretion, at least in part, is modulated by endogenous dopamine. However, a dopamine mediated mechanism for FSH secretion is absent during both phases of the estrous cycle in lactating Holstein cows. In all experiments FLU increased (P < .01) PRL secretion indicating that endogenous dopamine suppresses PRL secretion in cattle regardless of ovarian status. It is concluded that: 1) endogenous dopamine plays a stimulatory role in LH secretion during the anovulatory postpartum period and during the estrous cycle only when serum progesterone is low. 2) FLU decreased GH secretion in anovulatory postpartum Holstein cows but it increased GH secretion during the follicular and metestrus phases of the estrous cycle. However FLU had no effect on GH secretion during the luteal phase of the estrous cycle. Thus it appears that, modulation of GH secretion is dependent upon reproductive status and ovarian hormones secretion. / Ph. D.

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京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13362号 / 論医博第2204号 / 新制||医||1045(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 稲垣 暢也, 教授 渡邉 大, 教授 影山 龍一郎 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

The glycoprotein hormones, Luteinizing Hormone (LH), human Chorionic Gonadotropin (hCG), Follicle Stimulating Hormone (FSH) and Thyroid Stimulating Hormone (TSH) are heterodimeric proteins with an identical α-subunit associated noncovalently with the hormone specific β-subunit and play important roles in reproduction and overall physiology of the organism (Pierce & Parsons, 1981). The receptors of these hormones belong to the family of G-protein coupled receptors (GPCR) and have a large extracellular domain (ECD)comprising of 9-10 leucine rich repeats (LRR) followed by a flexible hinge region, a seven helical transmembrane domain (TMD) and a C terminal cytoplasmic tail (Vassart et al, 2004). Despite significant sequence and structural homologies observed between the ECDs of the receptors and the specific β-subunits of the hormones, the hormone-receptor pairs exhibit exquisite specificity with very low cross-reactivity with other members of the family. Several biochemical, immunological and molecular biological tools have been employed to elucidate the structure– function relationship of the hormones and their receptors. These studies also helped in deciphering some of the regions present in both the hormones and the receptors involved in maintaining the specificity of their interaction (Fan & Hendrickson, 2005b; Fox et al, 2001; Wu et al, 1994). However, the complete understanding of the hormone-receptor contact sites and mechanism of receptor activation are still an enigma. Understanding the molecular details of these phenomena can lead to the development of novel strategies of regulating hormone action. Binding of FSH to FSHR occurs in the large extracellular NH2-terminal domain where the participation of the LRRs (amino acids 18-259) is essential to determine the ligand selectivity (Dias & Van Roey, 2001; Fan & Hendrickson, 2005a; Szkudlinski et al, 2002). In fact, mutations in these regions lead to reduction in binding of the agonist to the receptor. It is not known how the signal from the large extracellular domain liganded complex is transmitted to the TMD (amino acids 367-695). It is envisioned that hormone binding to the LRRs leads to series of conformational changes leading activation of the TMD resulting in signal transduction. The recently reported crystal structure of the single chain form of FSH in complex with the leucine rich repeats of the FSHR (amino acids 1-268) (Fan & Hendrickson, 2005b), although provides detailed understanding of the molecular interactions of the LRRs with the hormone, fails to provide any insights into mechanism of receptor activation as the information regarding critical interaction of the hormone with TMD. This structure also did not provide any information on the role of the hinge region (amino acids 259-366) that connects the LRRs to the TMD in hormone binding and activation of the receptor. In the present study an attempt has been made to understand the role of the hinge region in hormone binding and signal transduction. The overall objective of the study is to elucidate the molecular details of the hormone receptor interactions, particularly FSH-FSHR interaction. Antibodies to glycoprotein hormones and their receptors have often provided insights into the mechanism of hormone-receptor interactions and signal transduction. While the TSH receptor antibodies and their effects on the overall physiology have been well documented (Khoo & Bahn, 2007; Rapoport & McLachlan, 2007), reports of such antibodies against FSHR or LHR and their possible effects on the reproductive functions are not available. In the present study, effects of FSHR antibodies with different specificities on FSH-FSHR interactions have been investigated. Antibodies to different regions of rat FSHR, were raised and extensively characterized and their effects of FSH-FSHR interactions and signaling were investigated. It was found that a polyclonal antibody against the hinge of the receptor (RF2 antiserum, amino acids 218-336), while having no significant effect on hormone binding and response could stimulate the receptor by itself bypassing the hormone. This stimulation of FSHR was very specific as this antiserum could not stimulate LHR or TSHR and could be blocked by preincubating the antibody with the antigen. Through competition experiments with different synthetic peptides of human FSHR, a stretch of hinge region corresponding to amino acids 296-331 was identified as the site recognized by the stimulatory antibody. This antibody did not interfere in hormone binding and could also bind to the pre-formed hormone-receptor complex suggesting that the binding site of the antibody may not participate directly in hormone binding. Subsequently the antibody was extensively characterized for its effect of hormone receptor interactions (Chapter 2). Previous studies considered the hinge region to be an inert linker connecting the LRRs to the TMD, a structural entity without any known functional significance (Vlaeminck-Guillem et al, 2002). However, the data with RF2 antibody suggested a direct role of the hinge region in signal transduction. Therefore, a systematic study to dissect the role to hinge region in hormone binding and signal transduction was conducted. Several truncations, deletions, activating and inactivating point mutations in the FSHR were generated to understand the mechanism of receptor activation. Firstly, these mutant receptors were characterized for their ability to translocate to the cell surface when transfected in the cultured mammalian cells. Secondly, affinity of all the mutant receptors for the hormone was determined in order to understand the effect of mutations on hormone binding. Finally, the cAMP response of these mutant receptors to the hormone and the stimulatory antibody was investigated to understand the effects of mutations on signal transduction. The results are described in Chapter 3. The hormone binding analysis and the affinity measurement of the mutant receptors showed that the LRRs are involved in high affinity hormone binding while the hinge region may not contribute to the process. This is in agreement with the crystal structure data which showed that the hormone was bound to the truncated receptor fragment representing only the LRRs (Fan & Hendrickson, 2005b). These binding data also corroborated the earlier data indicating that the antibodies against the hinge region do not interfere in hormone-receptor interactions. Further, the analysis of different N-terminally truncated receptor mutants provided strong evidence indicating that the constraining intramolecular interactions between the extracellular and the transmembrane domains are required to maintain the FSHR in an inactive conformation in the absence of an agonist. The analysis of the constitutive basal activity of the mutant receptors in absence of hormone suggested that certain regions of the extracellular domain had an attenuating effect over the TMDs that prevented constitutive activation of the receptor. This was demonstrated by a marked increase in the basal constitutive activity of the receptor upon the complete removal of its extracellular domain. Detailed analysis of the mutants suggested that LRR portion does not contribute to this attenuating effect, but it is the hinge region that perhaps interacts with the TMDs and dampens its basal constitutive activity. This attenuating effect was further narrowed down to a small stretch of 35 amino acids (296-331) within the hinge region. It was striking that the similar stretch was identified as the binding site of the stimulatory receptor antibody. In pharmacology, an ‘inverse agonist’ is an agent which binds to the receptor and reverses the constitutive activity of receptors. Thus the hinge region of the receptor could be termed as a ‘tethered inverse agonist’ of the TMD, since it is covalently associated with the TMD and their interactions dampen the basal constitutive activity of the receptor. However, careful comparison of the activities of the mutants (receptors harboring deletions and gain-of-function mutations) with maximally stimulated wild-type FSHR indicated that these mutations of the receptor resulted only in partial activation of the serpentine domain suggesting that only the ECD in complex with the hormone is the full agonist of the receptor. Moreover, the hinge region stabilizes the TMD in an inactive conformation and the activating mutations disengage the inhibitory ECD–TMD interactions bringing about partial activation of the receptor. Most interestingly, the deletion of amino acids 296-331 from hFSHR resulted in no further response to the hormone indicating that this part of the receptor is also critical for hormonal activation, perhaps playing a dual role in the attenuation of the basal activity and a direct involvement in the hormonal activation of the receptor. Progressive sequential deletions of ten amino acids from 290 to 329 yielded similar results (high basal cAMP production with concomitant loss of hormone and antibody response) clearly demonstrating that the integrity of this region is absolutely essential for hormonal activation. In conclusion, the study provides a conclusive evidence to show that the hinge region of FSHR, although not involved in primary high affinity hormone binding, plays a critical role in the modulation of the receptor activity in absence, as well as, presence of the hormone. A large array of reproductive abnormalities is associated with malfunctioning of FSHR. To explore the possibility of using the stimulatory antibodies for therapeutic purpose, three inactivating mutations of hFSHR were analyzed. In corroboration with the earlier reports (Doherty et al, 2002; Touraine et al, 1999), the mutants A419T and L601V are incapable of transducing the signal, despite having adequate cell surface expression and wild type affinities for the hormone, mainly because of defective TMD. The RF2 antibody failed to elicit any response from these mutants suggesting that its ability to activate the receptor depends on the status of the TMD. Interestingly, the activating mutant D576G, which showed very high basal cAMP production, could be stimulated by both antibody and the hormone to the nearly wild type levels suggesting that in this mutant the interactions between the hinge region and TMD are similar to that of wild type and higher basal cAMP production could be due to different interactions of the TMD with the G-Proteins. Structure-function studies of glycoprotein hormones and their receptors have been hampered due to low levels of expression of the properly folded proteins in heterologous systems (Chazenbalk & Rapoport, 1995; Hong et al, 1999b; Peterson et al, 2000; Sharma & Catterall, 1995; Thomas & Segaloff, 1994). Previous studies from the laboratory have shown that the Pichiapastoris,which blends the advantages of both bacterial and mammalian expression systems, can be used to hyper-express biologically active hormones (Blanchard et al, 2008; Gadkari et al, 2003; Samaddar et al, 1997). In addition, the same expression system has been used to produce single chain hormone analogs (Roy et al, 2007; Setlur & Dighe, 2007). Further, methodologies for Pichiafermentation and purification of recombinant hormones from the fermentation media have been wellestablished in the laboratory. Chapter 4 describes the work carried out to express, purify and characterize a fully functional hFSHR extracellular domain. Thus a stage is now set to attempt structural studies with the receptor. The results are discussed at the end of each of these chapters and future directions have been discussed at the end of this thesis.

In a HEK 293 cell line stably expressing seabream GHS-R1a (sbGHS-R1a), we found that a synthetic growth hormone secretagogue (GHS) increased [ 3H]-inositol phosphate production, clearly indicating coupling of this receptor to Gq/11-proteins. Using Western blotting, we found that GHS could also stimulate extracellular signal-regulated kinases 1 and 2 (ERK1/2), and that this response was inhibited by the MEK inhibitor U0126. For both the [3H]-inositol phosphate and ERK1/2 assays, the presence of the GHS-R antagonist D-Lys(3)-GHRP-6 significantly inhibited the GHS-stimulated activities, and in addition inhibited basal activities by 50% and 40%, respectively. These results showed that sbGHS-R1a is a constitutively active receptor and the antagonist D-Lys(3)-GHRP-6 is an inverse agonist. We also proposed that the expression of sbGHS-Rs was involved in the regulation of cell apoptosis. / Oligomerization of the human GHS-Rs (hGHS-Rs) was explored by transient transfection of the hGHS-Rs in HEK 293 cells followed by co-immunoprecipitation of differentially epitope-tagged forms of the receptors and bioluminescence resonance energy transfer 2 (BRET2) studies. (Abstract shortened by UMI.) / The concept that G protein-coupled receptors (GPCRs) exist and potentially function as dimers and/or higher oligomers has progressed from hypothesis to being widely accepted recently. Oligomerization of GPCRs has been increasingly noted in the regulation of the biological activity of the receptors. The growth hormone secretagogue receptor 1a (GHS-R1a) is a GPCR which principally regulates the pulsatile release of growth hormone from the pituitary gland. The GHS-R exists in two forms: GHS-R1a being a constitutively-active GPCR with 7 transmembrane (TM) domains, and GHS-R1b being a truncated version of type 1a but having only 5 TM domains. The endogenous agonist for GHS-R1a is ghrelin which exerts a wide range of physiological actions, but the function of GHS-R1b is still unclear. Since the tissue distribution patterns of the two isoforms of GHS-R are different, the objective of the present study is to explore the mechanisms of cell signalling of GHS-R1a and to determine the extent and importance of interactions between these two receptor isoforms. / Leung Po Ki. / "July 2005." / Adviser: Helen Wise. / Source: Dissertation Abstracts International, Volume: 67-07, Section: B, page: 3728. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 189-210). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / School code: 1307.

The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from PDF of title page (University of Missouri--Columbia, viewed on January 6, 2010). Thesis advisor: M. Cathleen Kuehl-Kovarik. Includes bibliographical references.

Thesis (M.A.)--University of Hawaii at Manoa, 2005. / Includes bibliographical references (leaves 34-45). / 52 leaves, bound ill. 29 cm

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