Orientador: Guilherme de Paula Nogueira / Banca: José Luis Moraes Vasconcelos / Banca: Ciro Moraes Barros / Resumo: Seis novilhas Nelore tiveram seus ciclos estrais acompanhados durante diferentes estações do ano (outono n=11; inverno n=8; primavera n=9; verão n=9) com exames ultrassonográficos diários para contar e mensurar folículos ≥3mm. Amostras de sangue foram colhidas a cada 12h para hormônio luteinizante (LH) e progesterona (P4), e a cada 3h do estro até a ovulação para caracterizar o pico de LH. Cinco novilhas ovariectomizadas receberam 17β-estradiol (2μg/kg/p.v.) em cada estação, e amostras de sangue foram colhidas depois disso a cada 3h para quantificação de LH. A diferença percentual mensal ( %) do peso não variou entre as estações. A concentração média de P4 no ciclo estral foi maior (p=0,001) e o número de folículos menor (p=0,001) durante o outono (2,5±0,2ng/mL; 7,8±0,1) e verão (2,9±0,3ng/mL; 6,8±0,2) comparado com o inverno (1,4±0,2ng/mL; 9,6±0,3) e primavera (1,6±0,2ng/mL; 9,7±0,3). Durante o inverno houve mais ciclos estrais com três (5 de 8) e durante o verão somente ciclos com duas ondas foliculares (p=0,009). Como a secreção de LH não variou, apesar da variação sazonal na concentração de P4, e como houve correlação negativa entre os valores máximos de P4 e a variação percentual do fotoperíodo (p=0,0056; r = -0,4465), uma variação sazonal na sensibilidade das células luteínicas ao LH precisa ser avaliada. Nas novilhas ovariectomizadas, a concentração circanual de LH sem o estímulo de estradiol foi significante (p=0,0214). A resposta de LH ao tratamento de estradiol foi menor no verão (0,8±0,2ng/mL vs 1,3±0,5ng/mL). Nós supomos que existe variação sazonal na sensibilidade hipotalâmica ao estradiol. / Abstract: Six Nelore heifers had their estrous cycle followed during different seasons of the year (autumn n=11; winter n=8; spring n=9 and summer n=9) with daily ultrasonographic exams to count and measure follicles ≥3mm. Blood was collected every 12h for luteinizing hormone (LH) and progesterone (P4), and every 3h from estrus until ovulation to characterize the LH peak. Five ovariectomized heifers were injected with 17β-estradiol (2μg/kg/LW) every season and blood samples were collected thereafter at 3h intervals for LH quantification. The monthly body weight percentile difference ( %) did not vary between seasons. Average P4 concentration for the cycle was higher (p=0.001) and follicle number lower during autumn (2.5±.2ng/ml; 7.8±.1) and summer (2.9±.3ng/ml; 6.8±.2) (p=0.001) compared to winter (1.4±.2ng/ml; 9.6±.3) and spring (1.6±.2ng/ml; 9.7±.3). During winter there were more estrous cycles with three follicle waves (5 out of 8) and during summer only cycles with two follicular waves (p=0.009). As LH secretion did not vary despite seasonal variation in P4 concentration and as there was a negative correlation between higher P4 values and daily percentile variation of photoperiod ( %, p=0.0056; r= -0.4465), a seasonal variation in luteal cell sensitivity to LH needs to be evaluated. In the ovariectomized Nelore heifers, the LH circanual concentration without estradiol stimulus was significant (p=0.0214). The LH response to estradiol treatment was lower in summer (0.8±.2ng/ml vs 1.3±.5ng/ml). We hypothesize there exists seasonal variation in hypothalamic sensitivity to estradiol. / Mestre

Cheng Fu Yip Gheorghe. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 97-116). / Abstracts in English and Chinese. / Acknowledgement --- p.I / Abstract (in English) --- p.III / Abstract (in Chinese) --- p.V / Table of Content --- p.VII / Symbols and Abbreviations --- p.XII / Scientific Names --- p.XIV / List of Tables --- p.XV / List of Figures --- p.XVI / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Gonadotropin / Chapter 1.1.1 --- Structure --- p.2 / Chapter 1.1.2 --- Function --- p.3 / Chapter 1.1.3 --- Regulation --- p.4 / Chapter 1.1.3.1 --- Neuroendocrine and endocrine regulation of GTHs --- p.4 / Chapter 1.1.3.1.1 --- Hypothalamic neuropeptides and neurotransmitters --- p.6 / Chapter 1.1.3.1.2 --- Gonadal steroids --- p.7 / Chapter 1.1.3.2 --- Paracrine regulation of GTH --- p.8 / Chapter 1.2 --- Activin / Chapter 1.2.1 --- Structure --- p.8 / Chapter 1.2.2 --- Function --- p.9 / Chapter 1.2.3 --- Regulation of activin activity --- p.12 / Chapter 1.2.3.1 --- Intracellular blockade of activin signaling by Smad7 --- p.12 / Chapter 1.2.3.2 --- Extracellular control of activin access --- p.13 / Chapter 1.2.3.2.1 --- Inhibin --- p.13 / Chapter 1.2.3.2.2 --- Activin-binding protein --- p.14 / Chapter 1.3 --- Follistatin / Chapter 1.3.1 --- Structure --- p.14 / Chapter 1.3.2 --- Function --- p.16 / Chapter 1.3.3 --- Regulation in the pituitary --- p.19 / Chapter 1.4 --- Objectives of the Present Study --- p.20 / Chapter Chapter 2 --- Cloning and Recombinant Production of Goldfish Follistatin / Chapter 2.1 --- Introduction --- p.24 / Chapter 2.2 --- Materials and Methods / Chapter 2.2.1 --- Reagents --- p.26 / Chapter 2.2.2 --- Animal --- p.26 / Chapter 2.2.3 --- Extraction of total RNA and reverse transcription --- p.27 / Chapter 2.2.4 --- Cloning of full-length cDNA encoding goldfish follistatin --- p.27 / Chapter 2.2.5 --- Sequencing of the cDNA --- p.29 / Chapter 2.2.6 --- Distribution of follistatin mRNA in different tissues --- p.29 / Chapter 2.2.7 --- Production of rgFS --- p.30 / Chapter 2.2.8 --- RT-PCR of the rgFS-positive clones --- p.34 / Chapter 2.2.9 --- Extraction of genomic DNA from rgFS-positive clones --- p.34 / Chapter 2.2.10 --- Functional analysis of rgFS --- p.35 / Chapter 2.2.11 --- Data Analysis --- p.37 / Chapter 2.3 --- Results / Chapter 2.3.1 --- Cloning and sequence analysis of goldfish follistatin --- p.37 / Chapter 2.3.2 --- Tissue distribution of follistatin mRNA in the goldfish --- p.39 / Chapter 2.3.3 --- Production and bioassay of rgFS --- p.43 / Chapter 2.4 --- Discussion --- p.47 / Chapter Chapter 3 --- Function and Regulation of Follistatin in the Goldfish Pituitary; Evidence for an Intrinsic Activin/Follistatin Regulatory Feedback Loop / Chapter 3.1 --- Introduction --- p.54 / Chapter 3.2 --- Materials and Methods / Chapter 3.2.1 --- Reagents --- p.57 / Chapter 3.2.2 --- Animals --- p.57 / Chapter 3.2.3 --- Primary culture of dispersed pituitary cells --- p.57 / Chapter 3.2.4 --- RNA extraction and reverse transcription --- p.58 / Chapter 3.2.5 --- Ovariectomy on pituitary follistatin expression --- p.5 9 / Chapter 3.2.6 --- Seasonal expression profile of follistatin --- p.59 / Chapter 3.2.7 --- Validation of semi-quantitative RT-PCR assays --- p.61 / Chapter 3.2.8 --- Real-time PCR for assay on follistatin and β-actin expression --- p.61 / Chapter 3.2.9 --- Data analysis --- p.63 / Chapter 3.3 --- Results / Chapter 3.3.1 --- Expression of follistatin in the goldfish pituitary --- p.64 / Chapter 3.3.2 --- Validation of semi-quantitative RT-PCR assay --- p.64 / Chapter 3.3.3 --- Activin regulation of pituitary follistatin --- p.64 / Chapter 3.3.4 --- Effects of sex steroids on pituitary follistatin expression --- p.69 / Chapter 3.3.5 --- Effect of GnRH on follistatin expression in the pituitary --- p.74 / Chapter 3.3.6 --- Effect of intracellular cAMP level on pituitary follistatin expression --- p.74 / Chapter 3.3.7 --- Seasonal variation profile of goldfish pituitary follistatin --- p.78 / Chapter 3.4 --- Discussion --- p.78 / Chapter Chapter 4 --- General Discussion / Chapter 4.1 --- Overview --- p.89 / Chapter 4.2 --- Contribution of the Present Study / Chapter 4.2.1 --- Cloning of full-length goldfish follistatin cDNA --- p.91 / Chapter 4.2.2 --- Establishment of stable cell line for expression of rgFS --- p.92 / Chapter 4.2.3 --- Evidence for the presence of intrinsic feedback loop of activin in the goldfish pituitary --- p.92 / Chapter 4.2.4 --- Modulation of follistatin expression in the pituitary by sex steroids --- p.93 / Chapter 4.2.5 --- Conclusions --- p.93 / Chapter 4.3 --- Future Prospects / Chapter 4.3.1 --- Production of rgFS --- p.95 / Chapter 4.3.2 --- Regulation of activin-follistatin system in the pituitary --- p.95 / Reference --- p.96

GPCRs, influencing myriads of cellular functions, are the members of the largest family of the membrane proteins. However, their structures and the signaling mechanisms still remain enigmatic. In case of the Glycoprotein Hormone Receptor (GpHR) family the structure-function relationship is less understood because of a large extra-cellular domain (ECD). This large ECD, consisting of Leucine Rich Repeats (LRRs) and membrane-proximal hinge region, is sufficient for specific binding to the hormone (Ascoli, Fanelli, & Segaloff, 2002), but for receptor activation, hormone binding is translated via a conformation wave starting at hinge region and relayed to the transmembrane domain. 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, 2005; Fox, Dias, & Van Roey, 2001; Wu, Lustbader, Liu, Canfield, & Hendrickson, 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 or regulating receptor activation in a hormone independent manner. The crystal structure of FSHR ECD (amino acids 17-366) revealed that LRRs form a semicircular palm shaped structure with the C terminus region, designated as the hinge region, protruding out like a thumb. The hinge region, rather than being a separate functional unit, was found to be an integral part of the LRR domain, having two such repeats (LRR11 &12). LRR 11 is connected to LRR12 through a hairpin loop (amino acids 280-344) harboring the invariant sulfated tyrosine residue (sTyr) in YD/EY motif (X. Jiang et al., 2012). The heterodimeric hormones consisting of a common  subunit and a hormone specific  subunit, bind to the primary hormone binding site at LRR 4-6 as reported in the FSHR-FSH co crystal (Fan & Hendrickson, 2005). This primary binding of the hormone at LRR 4-6 creates a pocket (comprising of the residues P16α, L17α, F18α, F74α, L37β, Y39β, and P45β) in the hormone for secondary binding at sTyr residue. This interaction is proposed to initiate conformation change in the hinge region which further leads to FSHR activation (X. Jiang et al., 2012). Thus, the role of hinge region in GpHR activation got evolved from a linker to a switch, which decides the fate of the receptor activity (Agrawal & Dighe, 2009; Majumdar & Dighe, 2012). sTyr residue being conserved, presents itself as a potential player in activation mechanism of all the three receptors of the family (Bonomi, Busnelli, Persani, Vassart, & Costagliola, 2006; Kreuchwig, Kleinau, & Krause, 2013). Precise involvement of sTyr in GpHR activation is yet to be explored. The previous studies from the laboratory using the hinge region specific polyclonal and monoclonal antibodies established the unequivocal role of the hinge region in FSHR and TSHR activation (Agrawal & Dighe, 2009; Majumdar & Dighe, 2012). However, its function in LHR activation has not been conclusively established. Due to the unavailability of the structural information of LHR ECD/hinge, it is more difficult to study and explain the role of hinge region in LHR activation. The hormone independent signaling by point mutants of LHR also remains poorly understood. In the present study an attempt has been made to understand the role of the hinge region in LHR signaling and modulating role of LRRs in hinge mediated LHR activation. The present study was initiated with an overall objective of understanding the molecular details of LHR activation mechanism keeping hinge at the centre of the picture. To have clarity of this picture with a holistic view of the mechanism, multi-pronged approach was adopted. Initially, ScFvs against LHR hinge region were employed as tools to probe into the hormone‐receptor interactions. Antibodies against glycoprotein hormones and their receptors have often provided insights into the mechanism of hormone‐receptor interactions and signal transduction (Agrawal & Dighe, 2009; Dighe & Moudgal, 1983; Gadkari, Sandhya, Sowdhamini, & Dighe, 2007; Gadkari et al., 2007; Kene, Nalavadi, Dighe, Iyer, & Mahale, 2004; Majumdar, Railkar, & Dighe, 2012a, 2012b). In this study, Single chain Fragment variables (ScFvs) against the hinge region of LH receptor have been employed to understand the mechanism of receptor activation. The effects of LHR ScFvs on hCG-LHR interactions have been investigated and three of the ScFvs, JE10, JE4 and JG1 could bypass the hormone and activate the receptor directly, with JE10 being the most potent one. The effect on the signaling was specific for LHR as no increase in cAMP response was observed for TSHR/FSHR in presence of these ScFvs. JE10 surprisingly was unique and could alter the hCG-LHR interaction by decreasing hormone affinity and simultaneously increasing the Bmax for the hormone. JE10 binding was decreased to the pre-formed hormone receptor complex suggesting that hCG and the stimulatory antibody show stearic hindrance at the binding sites on hinge or hormone binding induces conformational change in the epitope of JE10. The change in affinity and Bmax of the hormone by JE10 could be due to unmasking of new binding sites for hormones or an allosteric effect on the protomer interaction like explained in case of a small TMD specific allosteric modulator of FSHR (Xuliang Jiang et al., 2014). JE10 could also potentiate hCG signaling at sub-saturating concentrations of hCG, the precise mechanism of which is not clear. Through TSHR-LHR chimeric mutants, a stretch from amino acids 313-349, within the hinge region, was identified as the site recognized by JE10. In order to study structural features of the JE10 epitope, LHR ECD was modeled on the basis of FSHRED crystal structure. With most of the motifs being structurally conserved (CF3 and YPSHCCAFF); the major portion of the hinge region was found to be unstructured. This unstructured region harbored the JE10 epitope as well as the functionally important conserved sTyr residue. The CD spectra of LHR hinge in presence of ScFv JE10 suggested a ScFv induced helical conformation and stabilization of the hinge loop region, which was constrained in the homology model into helices. As loop was now constrained in the Mode 2, so was the interaction of sTyr, which was now in contact with positively charged residues, probably stabilizing its charge. The YEY motif mutants further confirmed the indirect essential role of Y331 in activation of LHR by JE10. Another approach followed to study hCG-LHR interactions was use of a series of LHR N-terminal truncation mutants and truncation mutants along with one of the LHR CAM (S277Q/D578Y). The effect of these truncations on hormone binding and receptor activation was investigated. The deletion of Cysteine box (Cb-1) of LHR (present at N-terminus of ECD) leads to abrogation of hCG binding, indicating importance of this region in maintaining ECD conformation required for hormone binding. This is the most unexplored region of the ECD. Though Cb-1 does not bind to the hormone directly (as is evident from the crystal structure) but it is indirectly essential for hormone binding. The basal activity of these truncated mutants was as low as that of the wild type LHR, reconfirming that no region of LHR ECD acts as an inverse agonist for the TMD (Karges, Gidenne, Aumas, Kelly, & Milgrom, 2005). Truncation mutants with CAM (double mutants) also showed low basal activity, suggesting that intact ECD is prerequisite for keeping LHR in a conformation, best suited for hormone binding and binding of G protein for activation. That best conformation still needs to be explored. Truncation mutants did not get stimulated by JE10 also. This observation is opposite to the previous studies in which FSHR/TSHR truncated mutants could be stimulated by hinge specific antibodies (Agrawal & Dighe, 2009; Majumdar & Dighe, 2012). This difference points out to the variations in which LHR hinge-TMD interactions prevail and lead to the receptor activation. This variation was also confirmed with a previous report in which the binding of TSHR-ECL specific antisera to wild type LHR and TSHR-LHR 6 chimeric mutant suggested that hinge of LHR does not seem to be constraining the TMD (Majumdar et al., 2012b). Thus the LHR TMD itself possesses all the inhibitory interactions, also indicated by the presence of most of the activating mutations in LHR TMD (Piersma, Verhoef-post, Berns, & Themmen, 2007). Protomer interaction is the newest aspect of GpHR activation mechanism and has not reached any conclusive, physiologically relevant explanations yet. By co-transfection of wild type LHR and ECD truncated mutants, this study suggests the LHR protomer interaction and proposes the involvement of allosteric effect of ECD on LHR protomer interaction. The effect of JE10 on activating and inactivating mutants of LHR were quite interesting. The ScFv could bind to the activating mutant D578Y (associated with precocious puberty). This mutant exhibited higher basal cAMP production, but was activated even further by the ScFv. The inactivating mutant A593P is a completely inactive receptor associated with (associated with pseudo-hermaphroditism. It does not respond to the hormone at all. The ScFv JE10 binds to this receptor and stimulates cAMP production. This observation is rather striking, as it is possible to activate a completely inactive mutant that could not be stimulated by the hormone by a binder specific for the hinge region. It is not clear how the binder that interacts with the hinge region affects the function of the inactive TMD thus providing an interesting tool to investigate the interactions between the hinge region and TMD that are probably key to understand the activation of GpHR. which has been shown to be central to the GpHR activation mechanism, (Agrawal & Dighe, 2009; Majumdar et al., 2012b; Schaarschmidt, Huth, Meier, Paschke, & Jaeschke, 2014). As per the recently suggested model by Deupi et. al., that each mutation and agonist can take a different pathway during activation (Kobilka & Deupi, 2007). The activated state induced by JE10 in D578Y and A593P seems to be different from the wild type LHR, with each activated receptor state having different capacity to bind to the G protein. The difference in G protein capacity in itself reflects the different receptor turnover or different Gs uncouplings or different Gs binding affinities, which needs to be further investigated, opening up another avenue for exploration. There is a lacuna in understanding the signal relay from the hinge to TMD. However, JE10 seems to be activating the wild type LHR and the mutants directly or indirectly by modulating the 6th helix of the TMD, known to be important for hormone independent activation of LHR (Fanelli, 2000; Latronico & Segaloff, 2007; Majumdar et al., 2012b). As evident from the absence of any hinge mediated constrain on LHR TMD and absence of uncharged residues present in LHR LRRD-TMD interface (LHR ECD Model 1), LHR hinge does not seem to be maintaining significant interactions with the TMD in absence of a ligand or in its basal state. Hormone/ agonist binding or activating mutations act as a positive regulator (inducing conformation change in hinge), required to bridge the interactions between LHR hinge and the TMD, which is supported by various studies in the past (Karges et al., 2005; Majumdar et al., 2012b; Nishi, Nakabayashi, Kobilka, & Hsueh, 2002; Osuga et al., 1997; Ryu, Gilchrist, Tung, Ji, & Ji, 1998; Zeng, Phang, Song, Ji, & Ji, 2001). This interaction bridged by the conformational change in the hinge region, seems to isomerize the closed state of LHR into an activated state. The present study supports the conformational induction model for receptor activation in which intramolecular interactions between the two domains (hinge-TMD) lead to the receptor activation. In conclusion, this study presents a possible mechanism of activation of LHR by a partial agonist ScFv, which induces the conformation change in the disordered loop region (a.a.313-349) of the hinge and stabilizes it into helical state. This conformation change is predicted to be important for relaying the activation signal to the TMD. The study also demonstrates the activation of a completely inactive mutant A593P by JE10, suggesting a distinct possibility of its use as a therapeutic tool in treating infertility caused by inactivating mutations in LHR. On a second note, the study extends the role of LRRs, apart from direct hormone binding, to an indirect allosteric role in hormone binding, LHR activation and functional stability. This functional stability does not seem to be restricted to a single LHR but also depends on its interaction with nearby protomers. Though there are evidences for and against each of the above discussed possibilities, as yet there is no accepted model that explains the precise steps of receptor activation, hence, the molecular details of these interactions needs to be investigated in future.

Made available in DSpace on 2014-06-11T19:27:18Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-08-10Bitstream added on 2014-06-13T19:35:12Z : No. of bitstreams: 1 cortejunior_ao_me_araca.pdf: 654847 bytes, checksum: bc307df312fdd42315035ada00da030f (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Seis novilhas Nelore tiveram seus ciclos estrais acompanhados durante diferentes estações do ano (outono n=11; inverno n=8; primavera n=9; verão n=9) com exames ultrassonográficos diários para contar e mensurar folículos ≥3mm. Amostras de sangue foram colhidas a cada 12h para hormônio luteinizante (LH) e progesterona (P4), e a cada 3h do estro até a ovulação para caracterizar o pico de LH. Cinco novilhas ovariectomizadas receberam 17β-estradiol (2μg/kg/p.v.) em cada estação, e amostras de sangue foram colhidas depois disso a cada 3h para quantificação de LH. A diferença percentual mensal ( %) do peso não variou entre as estações. A concentração média de P4 no ciclo estral foi maior (p=0,001) e o número de folículos menor (p=0,001) durante o outono (2,5±0,2ng/mL; 7,8±0,1) e verão (2,9±0,3ng/mL; 6,8±0,2) comparado com o inverno (1,4±0,2ng/mL; 9,6±0,3) e primavera (1,6±0,2ng/mL; 9,7±0,3). Durante o inverno houve mais ciclos estrais com três (5 de 8) e durante o verão somente ciclos com duas ondas foliculares (p=0,009). Como a secreção de LH não variou, apesar da variação sazonal na concentração de P4, e como houve correlação negativa entre os valores máximos de P4 e a variação percentual do fotoperíodo (p=0,0056; r = -0,4465), uma variação sazonal na sensibilidade das células luteínicas ao LH precisa ser avaliada. Nas novilhas ovariectomizadas, a concentração circanual de LH sem o estímulo de estradiol foi significante (p=0,0214). A resposta de LH ao tratamento de estradiol foi menor no verão (0,8±0,2ng/mL vs 1,3±0,5ng/mL). Nós supomos que existe variação sazonal na sensibilidade hipotalâmica ao estradiol. / Six Nelore heifers had their estrous cycle followed during different seasons of the year (autumn n=11; winter n=8; spring n=9 and summer n=9) with daily ultrasonographic exams to count and measure follicles ≥3mm. Blood was collected every 12h for luteinizing hormone (LH) and progesterone (P4), and every 3h from estrus until ovulation to characterize the LH peak. Five ovariectomized heifers were injected with 17β-estradiol (2μg/kg/LW) every season and blood samples were collected thereafter at 3h intervals for LH quantification. The monthly body weight percentile difference ( %) did not vary between seasons. Average P4 concentration for the cycle was higher (p=0.001) and follicle number lower during autumn (2.5±.2ng/ml; 7.8±.1) and summer (2.9±.3ng/ml; 6.8±.2) (p=0.001) compared to winter (1.4±.2ng/ml; 9.6±.3) and spring (1.6±.2ng/ml; 9.7±.3). During winter there were more estrous cycles with three follicle waves (5 out of 8) and during summer only cycles with two follicular waves (p=0.009). As LH secretion did not vary despite seasonal variation in P4 concentration and as there was a negative correlation between higher P4 values and daily percentile variation of photoperiod ( %, p=0.0056; r= -0.4465), a seasonal variation in luteal cell sensitivity to LH needs to be evaluated. In the ovariectomized Nelore heifers, the LH circanual concentration without estradiol stimulus was significant (p=0.0214). The LH response to estradiol treatment was lower in summer (0.8±.2ng/ml vs 1.3±.5ng/ml). We hypothesize there exists seasonal variation in hypothalamic sensitivity to estradiol.

Submitted by Cláudia Bueno (claudiamoura18@gmail.com) on 2016-04-01T20:41:57Z No. of bitstreams: 2 Tese - Mônica Canêdo Silva Maia - 2015.pdf: 2190840 bytes, checksum: b52ab9d3f319bc193309f78f9ef2e243 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-04-04T11:56:43Z (GMT) No. of bitstreams: 2 Tese - Mônica Canêdo Silva Maia - 2015.pdf: 2190840 bytes, checksum: b52ab9d3f319bc193309f78f9ef2e243 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2016-04-04T11:56:43Z (GMT). No. of bitstreams: 2 Tese - Mônica Canêdo Silva Maia - 2015.pdf: 2190840 bytes, checksum: b52ab9d3f319bc193309f78f9ef2e243 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2015-12-03 / Controlled ovarian stimulation has become an integral part of infertility treatment. Treatment options with recombinant gonadotrophins add more to knowledge on folliculogenesis and ovarian steroidogenesis. The role of recombinant luteinizing hormone is controversial undergoing ovarian stimulation and has been widely debated. Objective: To compare the effects of supplementation with recombinant luteinizing hormone (rLH) for controlled ovarian stimulation with recombinant follicle stimulating hormone (rFSH) in a protocol with GnRH-antagonist in cycles of IVF/ICSI. Methods: Case-control study with 113 patients attended at a university center in the city of Goiania, aged between 34-42 years, who were divided into two groups according to an ovarian stimulation scheme: Group I (n= 60): rFSH (control group) and Group II (n= 53): rFSH + rLH (treated group). These groups were comparable for age, BMI, duration of infertility, serum FSH, LH and estradiol. Numbers of oocytes collected and in metaphase II, fertilization rate, embryos rate and rates of chemical and clinical pregnancy were analyzed. Data analysis was conducted using the statistical software BioStat ® 5.3. Differences in proportions were assessed by chi-square test and means by Wilcoxon Mann- Whitney test. P < 0,05 was considered statistically significant. Results: The mean age of patients in Group I was 37.3±2.1 years and Group II 37.9±2.4 years (P > 0.05). The comparability of the other main characteristics (duration of infertility, BMI, FSH, LH and basal estradiol) were also observed between Groups I and II (P > 0.05). There was no significant difference between the two groups regarding: number of oocytes retrieved (Group I= 4.9 ± 2.1, Group II= 5.7 ± 2.6, P= 0.061), number of oocytes in metaphase II (Group I= 3.4 ± 1.6, Group II= 4.0 ± 1.9, P= 0.060), fertilization rate (Group I= 65.3%, Group II= 69.4 %, OR 1.20, 95% CI 0.85-1.70, P= 0.282), embryos rate (Group I= 85.4%, Group II= 88.5%, OR 1.31, 95% CI 0.73-2.36, P= 0.355), rate of chemical pregnancy (Group I= 20.0%, Group II= 24.5%; OR 1.30, 95% CI 0.53-3.16, P= 0.562) and clinical pregnancy rate (Group I= 20.0%, Group II= 22.6%, OR 1.17, 95% CI 0.47-2.89, P= 0.731). Conclusion: In this study it was concluded that supplementation with r-LH showed no benefit with respect to variables during controlled ovarian stimulation with GnRH antagonists. / A estimulação ovariana controlada tornou-se parte integrante no tratamento da infertilidade. As opções de tratamento com gonadotrofinas recombinantes adicionaram mais conhecimento da foliculogênese e esteroidogênese ovariana. O uso do hormônio luteinizante recombinante é controverso em pacientes que passam por estimulação ovariana e tem sido amplamente debatido. Objetivo: Comparar os efeitos da suplementação de LHr para a estimulação ovariana controlada com FSHr em um protocolo com antagonista de GnRH em ciclos de FIV/ICSI. Métodos: Estudo caso-controle com 113 pacientes atendidas em um centro universitário na cidade de Goiânia, idade entre 34 a 42 anos, as quais foram divididas em dois grupos de acordo com a estimulação ovariana: Grupo I (n= 60): FSHr (grupo controle) e Grupo II (n= 53): FSHr + LHr (grupo tratado). Estes grupos foram comparáveis para idade, IMC, duração da infertilidade, níveis séricos de FSH, LH e estradiol. Foram analisados número de ovócitos coletados e em metáfase II, taxa de fertilização, taxa de clivagem embrionária, taxas de gravidez química e clínica. A análise de dados foi realizada pelo programa estatístico Bioestat 5.3®. As diferenças de proporções foram avaliadas por teste de Qui-quadrado e as médias pelo teste Wilcoxon Mann-Whitney. p < 0,05 foi considerado estatisticamente significante. Resultados: A média de idade das pacientes do Grupo I foi 37,3 ± 2,1 anos e do Grupo II de 37,9 ± 2,4 anos (p > 0,05). A comparabilidade das outras principais características (duração da infertilidade, índice de massa corporal, FSH, LH e estradiol basal) foram também observadas entre os Grupos I e II (p > 0,05). Não houve diferença significativa entre os dois grupos em relação ao: número de ovócitos captados (Grupo I= 4,9 ± 2,1; Grupo II= 5,7 ± 2,6; p= 0,061), número de ovócitos em metáfase II (Grupo I= 3,4 ± 1,6; Grupo II= 4,0 ± 1,9; p= 0,060), taxa de fertilização (Grupo I= 65,3%; Grupo II= 69,4%; OR 1,20; IC 95% 0,85-1,70; p= 0,282), taxa de clivagem embrionária (Grupo I= 85,4%; Grupo II= 88,5%; OR 1,31; IC 95% 0,73-2,36; p= 0,355), taxa de gravidez química (Grupo I= 20,0%; Grupo II= 24,5%; OR 1,30; IC 95% 0,53-3,16; p= 0,562) e taxa de gravidez clínica (Grupo I= 20,0%; Grupo II= 22,6%, OR 1,17; IC 95% 0,47-2,89; p= 0,731). Conclusão: Neste estudo concluiu-se que a suplementação com LHr não demonstrou benefício em relação às variáveis analisadas durante a estimulação ovariana controlada com antagonistas de GnRH.

The observations that substance P (SP) is localized in the anterior pituitary gland (AP) and is regulated by the hormonal status of the animal, as well as the demonstration of SP binding sites in the AP, have led to the idea that SP may participate in the regulation of AP function. Numerous and sometimes contradictory reports of SP effects on AP hormone secretion, particularly on luteinizing hormone (LH), left the question of whether SP acts directly at the level of the AP to regulate LH secretion still unanswered. To investigate a possible physiological function of SP in the AP, the effects of exogenous SP on LH secretion from AP cells from adult and prepubertal male and female rats in short term culture were studied. It was found that SP (100nM-1μM) significantly stimulates LH release in cultured AP cells and that this effect varies as a function of age and sex. SP has no significant effect on LH release from AP cells of male and female prepubertal rats. After day 30 a sharp increase in the response to SP occurs in both sexes. This level of responsiveness continues through adulthood in AP cells from the female rat. In contrast, AP cells from male rats failed to respond during adulthood (over 50 days of age) but were highly responsive during the peripubertal period (30-35 days). The possibility that the responsiveness to SP is influenced by the endocrine status of the animal was investigated by exposing AP cells from responding animals to androgens in vivo and in vitro. It was found that AP cells from female rats treated with androgen were less responsive to 100nM SP but did respond at higher doses of SP. SP effects on AP function were further analyzed in experiments using radioligand binding assays to assess possible changes in SP receptor number or affinity as related to age and sex. In AP membranes from female rats, maximum binding is 8-fold higher (Bmax=4.2 pmo1/mg membrane protein) than in AP membranes from male rats (Bmax=560fmo1/ mg membrane protein). These studies suggest a role for SP as a secondary regulator of LH secretion with possible physiological significance for reproductive function.

No description available.

Introduction: Current health literature suggests that there has been a decline in the age of pubertal onset and that pubertal onset/duration of puberty may, besides weight status, be influenced by socioeconomic context. Objective: The goal of this study was to determine whether pubertal onset/ duration and puberty-triggering hormones luteinizing hormone (LH) and follicle-stimulating hormone (FSH) vary according to socioeconomic status (SES). Moreover, we aimed to propose cutoff values of serum LH and FSH for predicting gonadarche in boys. Methods: 2,657 apparently healthy children and adolescents between 5.5 and 18 years from the area of Leipzig were recruited from the LIFE Child study. Age at pubertal onset/end of puberty was given in 738/573 children, respectively. Anthropometric parameters of puberty, blood measurements of LH and FSH, and questionnaires assessing SES were evaluated. Results: Lower SES was associated with earlier thelarche and longer duration of puberty in overweight/obese girls, whereas age of menarche was not affected. In boys with low SES, a trend versus earlier puberty onset can be seen. Lower SES was significantly associated with boys’ age at mutation. No significant differences in boys’ and girls’ serum levels of LH and FSH during puberty according to SES were observed. Serum LH levels of 0.56 IU/L and serum FSH levels of 1.74 IU/L showed the best prediction of gonadarche in boys. Conclusion: Puberty onset/duration and boys’ age at mutation is affected by SES. The proposed cutoff levels for serum LH and FSH could provide a serological tool to determine gonadarche in boys

No description available.

Ce travail a consisté à analyser, par des approches toxicocinétiques (TK) et mécanistiques, les effets oestrogéniques du Bisphenol A (BPA) sur un biomarqueur précoce et sensible : la sécrétion de l'hormone lutéinisante (LH) chez la brebis prépubère ovariectomisée. La plus faible concentration plasmatique en BPA induisant une inhibition de LH s'est avérée proche des concentrations maximales décrites chez l'Homme. Cette inhibition de LH pourrait impliquer une inhibition des systèmes neuronaux à kisspeptine. L'approche TK comparative d'espèces a montré que la clairance du BPA est toujours élevée, proche du débit sanguin hépatique. Pour une exposition à la dose journalière admissible, cette approche permet de prédire chez l'Homme des concentrations en BPA très inférieures à celles associées à une inhibition de LH dans notre modèle. / The goal of this thesis was to analyse through toxicokinetic (TK) and mechanistic approaches the estrogeno-mimetic effects of bisphenol A (BPA) on a precocious and sensitive biomarker: LH secretion in ovariectomized female lambs. The lowest plasma BPA concentrations associated to an inhibition of LH secretion appeared to be close to the highest one reported in human. LH suppression could be mediated by an inhibition of hypothalamic kisspeptin systems. The multispecies TK approach showed that BPA clearance is always high and equivalent to the liver blood flow. For an exposure scheme corresponding to the tolerable daily intake, this approach allows to predict human BPA concentration much lower than the one associated to LH inhibition in our highly sensitive lamb model.