The luteinizing hormone / choriogonadotropin receptor (LH/CG-R) is a member of theG protein-coupled receptor family. The LH/CG-R has seven transmembrane helices, threeexoloops, three cytoloops, a C-terminal tail, and an extensive N-terminal exodomain. Theexodomain is capable of binding hormone with high affinity without hormone action. Previousstudies have shown that the amino-terminal region of the LH/CG receptor contacts both subunitsof human chorionic gonadotropin (hCG). In particular, three residues (Leu20, Cys22, and Gly24)were found to be crucial for hormone binding. In this thesis work, benzoylphenylalanine (Bpa),a photoactivatable reagent, was used to continue investigating the interactions of the N-terminalregion of the LH/CG-R with hCG. Bpa has been directly incorporated at a defined position intopeptides representing amino acids 17-36 of the LH/CG-R. These peptides were radiolabeledwith 125I and used in photoaffinity labeling studies to identify and characterize the contact site(s)between the N-terminal region of the LH/CG-R and hCG. Results suggest that Cys22 is theprimary contact residue in this region. Peptide and hormone concentration dependent as well asUV duration dependent photoaffinity labeling experiments confirm that the photolabeling ofhCG by hLHR17-36(C22Bpa) is specific. Competition of labeling studies indicate that the hLHR17-36(C22Bpa) peptide is a good mimic of the wild type N-terminal portion of the receptor. In-geldigestions of photolabeled hCG ?? and photolabeled hCG ?? with CNBr indicate that the Nterminalregions of both hCG ?? and hCG ?? were photoaffinity labeled by hLHR17-36(C22Bpa).Based on the fact that the N-terminal regions of each subunit are located on the convex side ofthe heterodimer, these results provide evidence that the N-terminal portion of the receptor wrapsaround the back of hCG, contacting the convex face of the hormone.

Os genes dos receptores do hormônio luteinizante (LHR) e folículo estimulante (FSHR), conhecidos por sua influência na manifestação da puberdade, foram avaliados por análise PCR-RFLP em uma população de 370 novilhas de corte compostas, de diferentes composições raciais Europeu-Zebu. Os objetivos foram caracterizar geneticamente a população investigada, utilizando-se de freqüências genotípicas e alélicas e estimativas de variabilidade e diversidade gênica; avaliar o efeito dos marcadores sobre a precocidade sexual, caracterizada pela probabilidade de prenhez por ocasião da primeira estação de monta (PP), e estimar a proporção da variância genética total da variável PP atribuída aos marcadores investigados, bem como a herdabilidade da característica pelo método de Máxima Verossimilhança Restrita (REML) sob modelo animal e sob modelo touro. Contatou-se elevada freqüência de animais heterozigotos em quase todas as composições raciais investigadas, para ambos os genes, com um valor médio de heterozigosidade de 57%, resultados estes que refletem a elevada variabilidade genética desta população híbrida. As novilhas heterozigotas apresentaram maiores taxas de prenhez (67 e 66% respectivamente, para os genes do LHR e FSHR), entretanto não se constataram efeito dos polimorfismos RFLP LHR (P=0,9188) e FSHR (P=0,8831) sobre a manifestação deste evento. As estimativas de herdabilidade obtidas para a PP foram de 0,21 e 0,41, respectivamente sob modelo animal e sob modelo touro. A magnitude das estimativas dos componentes de (co)variância atribuídas aos efeitos dos marcadores se mostrou muito baixa, constatando-se a pequena contribuição destes marcadores na proporção da variância total da prenhez, indicando ser esta uma característica de herança poligênica. Os resultados aqui demonstrados indicam que a seleção de novilhas para a precocidade sexual, com base em sua informação genotípica para os marcadores RFLP LHR e FSHR, não se justifica em programas de melhoramento genético animal, sugerindo-se a investigação de outros genes igualmente importantes, envolvidos na manifestação deste evento. Contudo, os marcadores avaliados se mostraram informativos, sendo indicados em estudos de caracterização genética em outras populações bovinas. A elevada heterozigosidade verificada na população composta estudada viabiliza a exploração destes animais em cruzamentos / The luteinizing hormone receptor (LHR) and follicle-stimulating receptor (FSHR) genes, known for their influence on the onset of puberty were evaluated by PCR-RFLP analysis in a population of 370 European-Zebu composite beef heifers from different breed contributions. The objectives were to genetically characterize the investigated population using genotype and allelic frequencies values besides on variability and gene diversity estimates; to evaluate the effect of markers on sexual precocity, characterized as the probability of pregnancy during the first breeding season (PP); and to estimate the proportion of total genetic variance in the PP related to the investigated markers, as well as the heritability estimate for PP by the Restricted Maximum Likelihood (REML) method for animal and sire models. The high number of heterozygous animals observed in almost all breed compositions studied for both loci, with average heterozigosity values of 57%, showed the high genetic variability on this hybrid population. Higher pregnancy rates were observed in heterozygous heifers (67% and 66% for LHR and FSHR genes, respectively), however, no effect of RFLP polymorphism for LHR (P=0.9188) and FSHR (P=0.8831) on pregnancy rate was observed. The heritabily estimates for PP were 0.21 and 0.41, using the animal and sire model, respectively. The small magnitude of the (co)variance components estimates related to random effects of LHR and FSHR, showed their small contribution in the proportion of total variance of pregnancy, indicating a polygenic inheritance for this trait. The results found in this work indicate that selection of beef heifers in animal genetic breeding programs for sexual precocity based on the inclusion of genotype information on RFLP for LHR e FSHR markers is not recommended. The investigation of other important genes related to puberty onset in heifers is necessary. However, the evaluated markers were informative and may be indicated for genetic characterization studies in other bovine populations. The high heterozigosity observed on the studied composite population maximizes the exploration of these animals in crossbreeding

Os genes dos receptores do hormônio luteinizante (LHR) e folículo estimulante (FSHR), conhecidos por sua influência na manifestação da puberdade, foram avaliados por análise PCR-RFLP em uma população de 370 novilhas de corte compostas, de diferentes composições raciais Europeu-Zebu. Os objetivos foram caracterizar geneticamente a população investigada, utilizando-se de freqüências genotípicas e alélicas e estimativas de variabilidade e diversidade gênica; avaliar o efeito dos marcadores sobre a precocidade sexual, caracterizada pela probabilidade de prenhez por ocasião da primeira estação de monta (PP), e estimar a proporção da variância genética total da variável PP atribuída aos marcadores investigados, bem como a herdabilidade da característica pelo método de Máxima Verossimilhança Restrita (REML) sob modelo animal e sob modelo touro. Contatou-se elevada freqüência de animais heterozigotos em quase todas as composições raciais investigadas, para ambos os genes, com um valor médio de heterozigosidade de 57%, resultados estes que refletem a elevada variabilidade genética desta população híbrida. As novilhas heterozigotas apresentaram maiores taxas de prenhez (67 e 66% respectivamente, para os genes do LHR e FSHR), entretanto não se constataram efeito dos polimorfismos RFLP LHR (P=0,9188) e FSHR (P=0,8831) sobre a manifestação deste evento. As estimativas de herdabilidade obtidas para a PP foram de 0,21 e 0,41, respectivamente sob modelo animal e sob modelo touro. A magnitude das estimativas dos componentes de (co)variância atribuídas aos efeitos dos marcadores se mostrou muito baixa, constatando-se a pequena contribuição destes marcadores na proporção da variância total da prenhez, indicando ser esta uma característica de herança poligênica. Os resultados aqui demonstrados indicam que a seleção de novilhas para a precocidade sexual, com base em sua informação genotípica para os marcadores RFLP LHR e FSHR, não se justifica em programas de melhoramento genético animal, sugerindo-se a investigação de outros genes igualmente importantes, envolvidos na manifestação deste evento. Contudo, os marcadores avaliados se mostraram informativos, sendo indicados em estudos de caracterização genética em outras populações bovinas. A elevada heterozigosidade verificada na população composta estudada viabiliza a exploração destes animais em cruzamentos / The luteinizing hormone receptor (LHR) and follicle-stimulating receptor (FSHR) genes, known for their influence on the onset of puberty were evaluated by PCR-RFLP analysis in a population of 370 European-Zebu composite beef heifers from different breed contributions. The objectives were to genetically characterize the investigated population using genotype and allelic frequencies values besides on variability and gene diversity estimates; to evaluate the effect of markers on sexual precocity, characterized as the probability of pregnancy during the first breeding season (PP); and to estimate the proportion of total genetic variance in the PP related to the investigated markers, as well as the heritability estimate for PP by the Restricted Maximum Likelihood (REML) method for animal and sire models. The high number of heterozygous animals observed in almost all breed compositions studied for both loci, with average heterozigosity values of 57%, showed the high genetic variability on this hybrid population. Higher pregnancy rates were observed in heterozygous heifers (67% and 66% for LHR and FSHR genes, respectively), however, no effect of RFLP polymorphism for LHR (P=0.9188) and FSHR (P=0.8831) on pregnancy rate was observed. The heritabily estimates for PP were 0.21 and 0.41, using the animal and sire model, respectively. The small magnitude of the (co)variance components estimates related to random effects of LHR and FSHR, showed their small contribution in the proportion of total variance of pregnancy, indicating a polygenic inheritance for this trait. The results found in this work indicate that selection of beef heifers in animal genetic breeding programs for sexual precocity based on the inclusion of genotype information on RFLP for LHR e FSHR markers is not recommended. The investigation of other important genes related to puberty onset in heifers is necessary. However, the evaluated markers were informative and may be indicated for genetic characterization studies in other bovine populations. The high heterozigosity observed on the studied composite population maximizes the exploration of these animals in crossbreeding

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.

<p>An up-regulation of gene expression of the gonadotropin receptors FSHR and LHR, and a down-regulation of anti-Müllerian hormone (AMH), in the gonads of early maturing male Atlantic salmon parr (<em>Salmo salar</em>) have been shown to take place during the process of sexual maturation. It has however not been determined if this happens prior to or after the onset of spermatogenesis. The aim of this study was to see if such an up-regulation of the gene expression of FSHR and LHR mRNA, and down-regulation of AMH in the gonad tissue could be seen prior to the onset of spermatogenesis in early maturing salmon. For this study gonad tissues were sampled before and after the onset of spermatogenesis. Small pieces of testis tissue were removed using surgery from individually tagged one year old male Atlantic salmon parr in April prior to the onset of spermatogenesis. The same salmon were sampled again in July and maturing and non-maturing fish could be distinguished by differences in GSI. Each tissue sample from April could be matched with the July sample from the same individual by pit-tag numbers. This made it possible to separate the April samples into a maturing and non-maturing group. Gene expression levels were analysed using real-time PCR. The findings of this study showed that all three genes were expressed in the gonads in April but no significant difference in expression levels between maturing and non-maturing salmon was seen for any of the genes, which indicate that no up- or down-regulations had taken place in early maturing fish at this time. In July however, total FSHR and LHR expression levels/testis were significantly higher in maturing salmon which is in accordance with previous studies. AMH expression levels/unit RNA in July were found to be on average 25 times higher in the non-maturing group. A 100-fold drop in AMH from April through July was seen in the maturing fish, while only a 4-fold drop was seen in the non-maturing group which may indicate that a down-regulation of AMH expression took place as spermatogenesis was initiated in the maturing males.</p> / <p>En uppreglering av genuttrycket av gonadotropinreceptorerna (FSHR och LHR) samt en nedreglering av anti-Müllerian hormon (AMH) har observerats i gonaderna under spermatogenesen hos tidigt mognande atlantlaxhanar (<em>Salmo salar</em>). Man har dock inte kunnat visa huruvida detta sker före eller efter spermatogenesen inletts. Syftet med denna studie var att undersöka om en uppreglering av FSHR och LHR samt en nedreglering av AMH kunde ses hos tidigt mognande laxar redan före spermatogenesen inletts. I den här studien användes gonadprover tagna före och efter spermatogenesen inletts. Små gonadvävnadsprover togs från individuellt märkta ettåriga atlantlaxhanar i april, före spermatogenesen inletts, genom ett operativt ingrepp. I juli togs sedan gonadprover från samma individer igen och mognande och icke-mognande individer kunde nu särskiljas genom deras GSI-värden. Varje gonadprov från april kunde matchas med proverna från juli för samtliga individer genom att jämföra pit-tag nummer och möjliggjorde att även aprilproverna kunde sorteras i en mognande och en icke-mognande grupp. Nivåerna av genuttryck av mRNA analyserades med real-time PCR. Resultaten från denna studie visade att alla tre generna var uttryckta i de omogna gonaderna i april men det var ingen signifikant skillnad mellan de mognande och icke-mognande fiskarna för någon av generna, vilket betyder att det ännu inte skett någon upp- eller nedreglering vid denna tidpunkt hos de tidigt mognande fiskarna. I juli var dock de totala FSHR och LHR mRNA nivåerna/testikel signifikant högre hos mognande fiskar vilket överrensstämmer med tidigare studier. AMH mRNA/enhet RNA var uttryckt 25 gånger högre i de omogna gonaderna jämfört med de mognande. Mellan april och juli föll AMH nivåerna hos mognande fiskar nästan 100 gånger, medan de hos de omogna minskade endast 4 gånger vilket kan indikera att en nedreglering av genuttrycket för AMH skett i de individer där spermatogenesen inletts.</p>

An up-regulation of gene expression of the gonadotropin receptors FSHR and LHR, and a down-regulation of anti-Müllerian hormone (AMH), in the gonads of early maturing male Atlantic salmon parr (Salmo salar) have been shown to take place during the process of sexual maturation. It has however not been determined if this happens prior to or after the onset of spermatogenesis. The aim of this study was to see if such an up-regulation of the gene expression of FSHR and LHR mRNA, and down-regulation of AMH in the gonad tissue could be seen prior to the onset of spermatogenesis in early maturing salmon. For this study gonad tissues were sampled before and after the onset of spermatogenesis. Small pieces of testis tissue were removed using surgery from individually tagged one year old male Atlantic salmon parr in April prior to the onset of spermatogenesis. The same salmon were sampled again in July and maturing and non-maturing fish could be distinguished by differences in GSI. Each tissue sample from April could be matched with the July sample from the same individual by pit-tag numbers. This made it possible to separate the April samples into a maturing and non-maturing group. Gene expression levels were analysed using real-time PCR. The findings of this study showed that all three genes were expressed in the gonads in April but no significant difference in expression levels between maturing and non-maturing salmon was seen for any of the genes, which indicate that no up- or down-regulations had taken place in early maturing fish at this time. In July however, total FSHR and LHR expression levels/testis were significantly higher in maturing salmon which is in accordance with previous studies. AMH expression levels/unit RNA in July were found to be on average 25 times higher in the non-maturing group. A 100-fold drop in AMH from April through July was seen in the maturing fish, while only a 4-fold drop was seen in the non-maturing group which may indicate that a down-regulation of AMH expression took place as spermatogenesis was initiated in the maturing males. / En uppreglering av genuttrycket av gonadotropinreceptorerna (FSHR och LHR) samt en nedreglering av anti-Müllerian hormon (AMH) har observerats i gonaderna under spermatogenesen hos tidigt mognande atlantlaxhanar (Salmo salar). Man har dock inte kunnat visa huruvida detta sker före eller efter spermatogenesen inletts. Syftet med denna studie var att undersöka om en uppreglering av FSHR och LHR samt en nedreglering av AMH kunde ses hos tidigt mognande laxar redan före spermatogenesen inletts. I den här studien användes gonadprover tagna före och efter spermatogenesen inletts. Små gonadvävnadsprover togs från individuellt märkta ettåriga atlantlaxhanar i april, före spermatogenesen inletts, genom ett operativt ingrepp. I juli togs sedan gonadprover från samma individer igen och mognande och icke-mognande individer kunde nu särskiljas genom deras GSI-värden. Varje gonadprov från april kunde matchas med proverna från juli för samtliga individer genom att jämföra pit-tag nummer och möjliggjorde att även aprilproverna kunde sorteras i en mognande och en icke-mognande grupp. Nivåerna av genuttryck av mRNA analyserades med real-time PCR. Resultaten från denna studie visade att alla tre generna var uttryckta i de omogna gonaderna i april men det var ingen signifikant skillnad mellan de mognande och icke-mognande fiskarna för någon av generna, vilket betyder att det ännu inte skett någon upp- eller nedreglering vid denna tidpunkt hos de tidigt mognande fiskarna. I juli var dock de totala FSHR och LHR mRNA nivåerna/testikel signifikant högre hos mognande fiskar vilket överrensstämmer med tidigare studier. AMH mRNA/enhet RNA var uttryckt 25 gånger högre i de omogna gonaderna jämfört med de mognande. Mellan april och juli föll AMH nivåerna hos mognande fiskar nästan 100 gånger, medan de hos de omogna minskade endast 4 gånger vilket kan indikera att en nedreglering av genuttrycket för AMH skett i de individer där spermatogenesen inletts.

The luteinizing hormone receptor (LHR) belongs to the G protein-coupled receptorfamily. It consists of two distinct domains; the N-terminal extracellular exodomain and themembrane associated endodomain which includes 7 transmembrane domains, 3 exoloops, 3cytoloops and a C-terminal tail. Sequence alignment and computer modeling suggest thepresence of Leu Rich Repeat (LRR) motifs in the exodomain. Although their structuralsimilarity is high, each LRR is not equally important for hormone binding. Ala-scanning andtruncation studies performed in our laboratory suggest that LRR2 and LRR4 appear to be themost crucial. The Ala-scanning data suggest that Leu103 and Ile105 in LRR4 are important forhormone binding. However, it is not clear whether these two residues make direct contact withhuman chorionic gonadotropin (hCG) or if they are necessary for the overall structural integrityof LRR4. In this work, the LHR peptide mimics of LRR4 were used for photoaffinity labeling todetermine whether Leu103 and Ile105 directly interact with hormone. Furthermore, LRR4peptides containing the photoactivable benzoylphenylalanine (Bpa) were used to determinewhether the LRR structure really exists in the LHR exodomain, whether LRR 4 interact withhCG, and which residues of LRR4 interact with hCG. Bpa was directly incorporated intodifferent positions of the LRR4 peptide sequence to examine the labeling ability of individualamino acids. The results suggest that LRR4, in particular the sequence of Lys101-Cys106,makes direct contact with hCG. However Leu103 and Ile105 do not interact with hCG but mayform the hydrophobic core of the LRR4 loop, which appears to be crucial for the LRR structure.Existing data suggest that glycoprotein hormones initially bind the exodomain. Thehormone/exodomain complex undergoes conformational adjustments and stimulates theendodomain of the receptor to generate hormone signals. The exoloops modulate hormonebinding and signaling; however, little is known about whether the hormone/exodomain complexcontacts the endodomain. To address this issue, we investigated whether the exoloops interactwith the hormone. First, we examined exoloop 3 that connects transmembrane domains 6 and 7which are important for signal generation. We present the first physical evidence that LHRexoloop 3 interacts with hCG.

Com o objetivo de semi-quantificar a expressão gênica para receptores de gonadotrofinas nas células de folículos dominantes em animais pré-púberes (bezerras) das raças Nelore e Marchigiana e novilhas púberes da raça Nelore, buscando diferenças que possibilitem a procura de respostas para a deficiência da competência de desenvolvimento dos oócitos de animais pré-púberes que permitam a utilização destes em programas de melhoramento genético animal, foram utilizados 8 animais, sendo três bezerras Nelore, três bezerras Marchigiana, com 7 meses de idade, e duas novilhas Nelore, com 25 meses de idade. Foi realizado acompanhamento da onda folicular através de ultra-sonografia por dois meses, e os animais foram abatidos no momento que o folículo dominante apresentava diâmetro máximo (8, 10 e 14 mm, respectivamente). Folículos dominantes foram dissecados e isolados, e foi feita extração de RNA total. Foram feitas quatro reações de síntese de cDNA a partir de cada amostra. Para análise da expressão gênica semi-quantitativa, foram realizadas reações de Duplex-PCR, utilizando-se dois oligonucleotídeos iniciadores em cada reação, um para gene controle, o gapdh, e outro para o gene alvo, receptor do hormônio folículo estimulante ou receptor do hormônio luteinizante. Estas reações foram realizadas em triplicata a partir de cada fonte de cDNA de cada amostra, e os produtos foram aplicados em gel de poliacrilamida, e fotografados. Os resultados foram obtidos a partir da razão entre a intensidade da banda do gene alvo e a intensidade da banda do gene controle, e os valores médios foram analisados estatisticamente. Foi encontrada menor expressão relativa do gene do receptor de LH em bezerras da raça Nelore quando comparadas com as novilhas da raça Nelore e as bezerras da raça Marchigiana. Foi detectada a presença de um splicing alternativo para o gene do LHr, que apresentou comportamento semelhante ao gene padrão. Não foi encontrada diferença significativa na expressão do gene do FSHr entre os grupos estudados. Os resultados sugerem uma maior importância do LH nos estágios finais de desenvolvimento folicular, demonstrado aqui pela menor expressão relativa de lhr nos folículos dominantes de bezerras Nelore em relação às novilhas púberes, que pode ser um dos motivos para o baixo potencial de desenvolvimento dos oócitos destes animais após a fertilização in vitro / With the objective to semiquantify the gene expression for gonadotrophins receptors in dominant follicles in Nelore and Marchigiana prepubertal heifers and Nelore adult cows, being searched differences that make possible the search of answers for the low developmental potential of oocytes from prepubertal heifers that allow the use of these in programs of animal genetic improvement, had been used 8 animals, being three Nelore heifers, three Marchigiana heifers, with 7 months of age, and two Nelore heifers, with 25 months of age. Accompaniment of the follicular wave was carried through daily ultrasonografic exams for two months, and the animals had been slaughtered at the moment that dominant follicle presented maximum diameter (8, 10 and 14 mm, respectively). Dominant follicles had been dissected and isolated, and extraction of total RNA was performed. Four reactions of synthesis of cDNA from each sample had been made. For analysis of the semi-quantitative gene expression, reactions of Duplex-PCR had been carried through, using themselves two initiating oligonucleotídeos in each reaction, one for housekeeping gene, gapdh, and another one for the target gene, fshr and lhr. These reactions had been carried through in third copy from each source of cDNA of each sample, and the products had been applied in poliacrilamida gel electrophoresis, and photographed. The results had been gotten from the reason enter the intensity of the band of the target gene and the intensity of the band of the housekeeping gene, and the average values had been analyzed in a statistical software. Lower relative expression of lhr in Nelore heifers was found when compared with the Nelore adult cows and the Marchigiana heifers. An alternative splicing for lhr was detected, that presented similar behavior to the gene standard. Significant difference in the expression of the gene of the FSHr between the studied groups was not found. The results suggest a importance of the LH in the final periods of follicular development, demonstrated for the lower relative expression of lhr in the dominant follicles of Nelore heifers in relation to the adult cows in this study, that can after be the one of the reasons for the low developmental capacity of oocytes of these animals after in vitro fertilization

Com o objetivo de semi-quantificar a expressão gênica para receptores de gonadotrofinas nas células de folículos dominantes em animais pré-púberes (bezerras) das raças Nelore e Marchigiana e novilhas púberes da raça Nelore, buscando diferenças que possibilitem a procura de respostas para a deficiência da competência de desenvolvimento dos oócitos de animais pré-púberes que permitam a utilização destes em programas de melhoramento genético animal, foram utilizados 8 animais, sendo três bezerras Nelore, três bezerras Marchigiana, com 7 meses de idade, e duas novilhas Nelore, com 25 meses de idade. Foi realizado acompanhamento da onda folicular através de ultra-sonografia por dois meses, e os animais foram abatidos no momento que o folículo dominante apresentava diâmetro máximo (8, 10 e 14 mm, respectivamente). Folículos dominantes foram dissecados e isolados, e foi feita extração de RNA total. Foram feitas quatro reações de síntese de cDNA a partir de cada amostra. Para análise da expressão gênica semi-quantitativa, foram realizadas reações de Duplex-PCR, utilizando-se dois oligonucleotídeos iniciadores em cada reação, um para gene controle, o gapdh, e outro para o gene alvo, receptor do hormônio folículo estimulante ou receptor do hormônio luteinizante. Estas reações foram realizadas em triplicata a partir de cada fonte de cDNA de cada amostra, e os produtos foram aplicados em gel de poliacrilamida, e fotografados. Os resultados foram obtidos a partir da razão entre a intensidade da banda do gene alvo e a intensidade da banda do gene controle, e os valores médios foram analisados estatisticamente. Foi encontrada menor expressão relativa do gene do receptor de LH em bezerras da raça Nelore quando comparadas com as novilhas da raça Nelore e as bezerras da raça Marchigiana. Foi detectada a presença de um splicing alternativo para o gene do LHr, que apresentou comportamento semelhante ao gene padrão. Não foi encontrada diferença significativa na expressão do gene do FSHr entre os grupos estudados. Os resultados sugerem uma maior importância do LH nos estágios finais de desenvolvimento folicular, demonstrado aqui pela menor expressão relativa de lhr nos folículos dominantes de bezerras Nelore em relação às novilhas púberes, que pode ser um dos motivos para o baixo potencial de desenvolvimento dos oócitos destes animais após a fertilização in vitro / With the objective to semiquantify the gene expression for gonadotrophins receptors in dominant follicles in Nelore and Marchigiana prepubertal heifers and Nelore adult cows, being searched differences that make possible the search of answers for the low developmental potential of oocytes from prepubertal heifers that allow the use of these in programs of animal genetic improvement, had been used 8 animals, being three Nelore heifers, three Marchigiana heifers, with 7 months of age, and two Nelore heifers, with 25 months of age. Accompaniment of the follicular wave was carried through daily ultrasonografic exams for two months, and the animals had been slaughtered at the moment that dominant follicle presented maximum diameter (8, 10 and 14 mm, respectively). Dominant follicles had been dissected and isolated, and extraction of total RNA was performed. Four reactions of synthesis of cDNA from each sample had been made. For analysis of the semi-quantitative gene expression, reactions of Duplex-PCR had been carried through, using themselves two initiating oligonucleotídeos in each reaction, one for housekeeping gene, gapdh, and another one for the target gene, fshr and lhr. These reactions had been carried through in third copy from each source of cDNA of each sample, and the products had been applied in poliacrilamida gel electrophoresis, and photographed. The results had been gotten from the reason enter the intensity of the band of the target gene and the intensity of the band of the housekeeping gene, and the average values had been analyzed in a statistical software. Lower relative expression of lhr in Nelore heifers was found when compared with the Nelore adult cows and the Marchigiana heifers. An alternative splicing for lhr was detected, that presented similar behavior to the gene standard. Significant difference in the expression of the gene of the FSHr between the studied groups was not found. The results suggest a importance of the LH in the final periods of follicular development, demonstrated for the lower relative expression of lhr in the dominant follicles of Nelore heifers in relation to the adult cows in this study, that can after be the one of the reasons for the low developmental capacity of oocytes of these animals after in vitro fertilization

BPS/IC (Bladder Pain Syndrome/Interstitial Cystitis) ist ein sehr schweres und noch weitgehend unverstandenes Krankheitsbild in der Urologie. Viele Frauen sind im Alltag durch den ständigen Harndrang und die Schmerzen stark eingeschränkt und von Depressionen betroffen. Die Aufklärung der Pathogenese ist deshalb sehr wichtig, um eine adäquate Therapie für die Betroffenen zu entwickeln und die Krankheit möglichst frühzeitig diagnostizieren zu können. Das Schwangerschaftshormon hCG (humanes Choriongonadotropin) besitzt differenzierende und wachstumsfördernde Eigenschaften und eine Rolle in der Urothelregeneration und – stabilisierung scheint möglich. Daher ist das Ziel dieser Arbeit seinen Rezeptor, den LHCGR (Luteinizing-Hormone/Choriogonadotropin Rezeptor), in der Harnblase nachzuweisen und die urothelialen und muskulären Charakteristika zwischen gesunden und an BPS/IC erkrankten Harnblasen zu vergleichen. Die Darstellung des LHCGR erfolgte auf Proteinebene mittels indirekter Immunfluoreszenz und auf mRNA-Ebene durch Standard-PCR. Es zeigten sich im Urothel von Harnblase und Ureter 5 unterschiedliche Verteilungsmuster des Rezeptors hinsichtlich seiner Expression in verschiedenen Zellschichten und seiner subzellulären Lokalisation. Je nach Urothelzustand und zwischen den Entitäten Kontroll- bzw. BPS/IC-Harnblase variierten diese Muster in ihrer Häufigkeit. In anderen Epithelien, wie dem Vaginalepithel, änderte sich die zelluläre Verteilung des LHCGR in Abhängigkeit vom Differenzierungsgrad der Zellen. Es scheint möglich, dass auch die Rezeptorexpression in Urothelzellen deren verschiedene Differenzierungszustände widerspiegelt. Dies unterstützt den für hCG vermuteten Einfluss auf die Epithelregeneration. Ein Vergleich der urothelialen Fluoreszenzintensitäten zwischen weiblichen Kontroll – und BPS/IC-Harnblasen zeigte eine signifikant stärkere Expression des Rezeptors bei erkrankten Patienten. Dem gegenüber war kein Unterschied im Detrusor, weder zwischen Kontroll – und BPS/IC-Harnblasen noch im geschlechtsspezifischen Vergleich, festzustellen. Damit scheint der Rezeptor seine Hauptaufgabe vorrangig im Urothel zu entfalten. Die Korrelationsanalysen ergaben keinen signifikanten Zusammenhang zwischen dem Erkrankungsalter (Zeitpunkt der Diagnosestellung und Biopsieentnahme) und der LHCGR-Immunfluoreszenz. Ein endokrinologischer Einfluss auf die Rezeptorexpression wurde dadurch unwahrscheinlich und unterstützt die immer akzeptiertere Auffassung, dass BPS/IC nicht mehr mit der Menopause assoziiert ist. Neben dem Urothel und Detrusor zeigten auch Lamina propria und Gefäße von Harnblase und Ureter die Expression des LHCGR in der Immunhistochemie. Unterschiedliche Clustermuster des Rezeptors im Detrusor ließen auf die Oligomerisierung des Rezeptors schließen. Die Bedeutung dieser Zusammenschlüsse ist jedoch noch unklar, wobei unterschiedliche funktionelle Zustände des Rezeptors vermutet werden. Orientierung bieten andere Rezeptoren, die durch Dimerisierung verschiedener Rezeptorvarianten ihre Funktionalität verbessern oder verschlechtern konnten. Obwohl für keine bisher entdeckte Variante des LHCGR eine definitive Aufgabe ermittelt werden konnte, scheinen doch viele Varianten auch unterschiedliche Funktionen wahrnehmen zu können. Besonders auf der Regulierbarkeit des Rezeptors mittels interagierender Splicevarianten sollte das Augenmerk zukünftiger Studien liegen. Ob durch Komplexbildung verschiedener Varianten oder Bildung nichtfunktioneller trunkierter Rezeptoren, die Kontrollmöglichkeiten sind vielfältig und können auch auf Liganden wirken. Letztlich ließ der Nachweis des LHCGR in allen Schichten von Harnblase und Ureter eher eine globale Rolle des Rezeptors im Harntrakt des Menschen vermuten. Dazu passten auch die bereits nachgewiesenen Einflüsse seiner Liganden auf die Blasenfunktion von Hunden. Die hier vorgelegte Arbeit untersuchte zum ersten Mal die Expression des LHCGR mittels PCR und Immunhistochemie in humanen Harnblasen und Ureteren. Dabei löste sie sich von den sonst üblichen Vorstellungen einer Beziehung des Rezeptors zu Blasentumoren, Schwangerschaft oder Inkontinenz. Diagnose und Therapie von BPS/IC sind zur Zeit noch ständigen Wandlungen unterworfen und dabei entgehen viele Patienten der (frühen) Diagnosestellung und einer adäquaten Behandlung. Diese Studie sollte dazu beitragen neue Einblicke in die Pathophysiologie der Erkrankung zu erlangen, um eine kausale Therapie entwickeln zu können. Zukünftig könnten diese Ergebnisse dabei helfen die Anwendung einer sensitiven und vor allem spezifischen Diagnostik auf molekularer Ebene (mRNA - oder Proteinnachweis) zu ermöglichen.