In this thesis a detailed study of the kinetics of GnRH-stimulated LH release was made. GnRH stimulated LH release in a biphasic manner. During the first 3 minutes of stimulation, there was a transient spike phase of release followed by plateau phase of lower amplitude. Both phases of release are largely dependent on extracellular Ca²⁺. The spike phase of release is dependent on Ca²⁺ entry via a receptor-operated Ca²⁺ channel (ROCC) (about 90%) and on the mobilization of intracellular Ca²⁺ stores. The role of ROCC were examined by using ruthenium red which inhibits both ROCC and voltage-sensitive Ca²⁺ channels (VSCC). VSCC are not involved in the spike phase of GnRH-stimulated LH release since D600 and nifedipine, inhibitors of VSCC, have no effect on the spike phase. The plateau phase of release is dependent on Ca²⁺ entry via VSCC (about 50%) and ROCC (about 50%). Forskolin, an activator of adenylate cyclase, was used to investigate the role of cAMP in LH release. Forskolin stimulated an increase in both LH release and cellular cAMP levels. GnRH was also able to elevate the cellular CAMP concentration. GnRH interacted synergistically with forskolin to stimulate LH release. The synergism between GnRH and forskolin was not due to an interaction at (1) the GnRH receptor, (2) the level of intracellular Ca²⁺ mobilization, or (3) inositol phosphate metabolism. However, forskolin was able to synergistically interact with secretagogues that increase the cytosolic Ca²⁺ concentration and activators of protein kinase C. This suggested that forskolin was interacting with GnRH at a site distal to the activation of the Ca²⁺ second messenger system and protein kinase C. The data suggest that the initial response to GnRH is largely Ca²⁺-dependent and that other second messengers, if active, play a minor role. cAMP is thought to play a modulatory role and may be involved in the maintenance of secretion.

OBJECTIVE: To evaluate the effectiveness of offering double intrauterine insemination (IUI) to clients in our fertility program. STUDY DESIGN: In this prospective, nonrandomized study, 595 couples with ovulatory dysfunction, endometriosis, male factor, unexplained, tubal factor and combined diagnoses utilizing clomiphene citrate-hCG (CC-hCG), CC-gonadotropin-hCG (CC-Gn-hCG), Gn-hCG, lupron-Gn-hCG (L-Gn-hCG) or luteinizing hormone (LH) surge monitoring of natural cycles were offered single or double IUI in a total of 1,276 cycles. Single IUIs were performed at 36 hours following hCG or the day following LH surge; double IUIs were performed 18 and 36 hours following hCG or the day of and day following LH surge. Single versus double IUI clinical pregnancy outcomes were compared between ovarian stimulation protocols and diagnostic categories. RESULTS: One hundred ten clinical pregnancies occurred for 508 couples in 999 single IUI cycles (fecundity, 11.0%); 45 clinical pregnancies for 174 couples occurred in 277 double IUI cycles (16.2%, p < 0.004). The single IUI group was younger than the double IUI group (32.8 vs 33.7, p < 0.004). Differences for fecundity were noted regarding diagnostic categories between single and double IUI groups (ovulation dysfunction, 12.9% vs 19.5%, p < 0.048, and male factor, 7.9% vs 17.5%, p < 0.030) and ovulation protocols (CC-Gn-hCG, 13.0% vs 21.3%, p < 0.031, and L-Gn-hCG, 4.2% vs 25.0%, p < 0.002). CONCLUSION: Double IUI is superior to single IUI overall, especially when comparing Gn-containing ovarian stimulation protocols or within the ovulatory dysfunction and malefactor diagnostic categories.

In this study, the structure and function of gonadotropin-releasing hormone (GnRH) in different vertebrate species, in the classes Aves, Reptilia and Pisces was investigated. Acetic acid extracts were subjected to gel filtration chromatography and semipreparative high performance liquid chromatography (HPLC) to partially purify the GnRHs. The GnRH immunoreactivity was then characterized by analytical HPLC, and by assaying HPLC fractions by radioimmunoassay with region-specific antisera generated against mammalian GnRH, Gln⁸-GnRH and Trp⁷,Leu⁸-GnRH and assessing luteinizing hormone (LH)-releasing activity of fractions in a chicken dispersed anterior pituitary cell bioassay. Five GnRH molecular forms have thusfar been structurally characterized in vertebrate brain. In mammals a GnRH with the structure pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH₂ has been demonstrated in the hypothalamus (Matsuo et al., 1971; Burgus et al., 1972). Gln⁸-GnRH and His⁵,Trp⁷,Tyr⁸-GnRH were present in chicken hypothalamus (King and Millar, 1982a, 1982c; Miyamoto et al., 1983, 1984), Trp⁷,Leu⁸-GnRH in salmon brain (Sherwood et al., 1983) and Tyr³,Leu⁵,Glu⁶,Trp⁷,Lys⁸-GnRH in lamprey brain (Sherwood et al., 1986). In ostrich (Struthio camelus) hypothalamus two GnRHs with identical properties to Gln⁸-GnRH and His⁵,Trp⁷,Tyr⁸-GnRH have been demonstrated, as well as four other LR-releasing factors with different chromatographic and immunological properties to any of the known naturally-occurring GnRHs. Since Gln⁸-GnRH and His⁵,Trp⁷,Tyr⁸-GnRH were also present in chicken hypothalamus it appears likely that these two GnRHs occur in all birds. In alligator (Alligator mississippiensis) brain only two GnRHs were detected. These forms co-eluted with Gln⁸-GnRH and His⁵,Trp⁷,Tyr⁸-GnRH in two HPLC systems. They cross-reacted similarly to the two synthetic peptides with antisera directed against mammalian GnRH and Gln⁸-GnRH and released LH from chicken dispersed anterior pituitary cells in a similar manner to the synthetic peptides. The Archosaurs (alligators and crocodiles) are believed to be closely related to birds and therefore it seems likely that they should have identical GnRHs. In skink (Calcides ocellatus tiligugu) brain one GnRH, which co-eluted with His⁵,Trp⁷,Tyr⁸-GnRH, was demonstrated. Two other lizards (Cordylis nigra and Pordarcis s. sicula) have been studied (Powell et al., 1985; R.C. Powell, G. Ciarcia, V. Lance, R.P. Millar and J.A. King, submitted). In c. nigra four immunoreactive GnRHs were detected, two of which co-eluted released chicken LH similarly to, Trp⁷,Leu⁸-GnRH and with, and His⁵,Trp⁷,Tyr⁸-GnRH. In P. s. sicula a GnRH molecular form similar to Trp⁷,Leu⁸-GnRH occurred as well as two novel GnRHs. It thus appears that Gln⁸-GnRH does not occur in lower reptiles, but His⁵,Trp⁷,Tyr⁸-GnRH and/or Trp⁷,Leu⁸-GnRH do. His⁵,Trp⁷,Tyr⁸-GnRH appears to he a widespread GnRH, occurring in vertebrates as diverse as birds and elasmobranch fish. In dogfish (Poroderma africanum) brain seven factors, which stimulated release of LH from chicken dispersed anterior pituitary cells, were separated on analytical HPLC. Two of these factors were partially characterized as Trp⁷,Leu⁸-GnRH and His⁵,Trp⁷,Tyr⁸-GnRH. Three of the other forms cross-reacted with GnRH antisera, but appear to be novel GnRHs. In teleost (Coris julis) brain two GnRHs similar to Trp⁷,Leu⁸-GnRH and His⁵,Trp⁷,Tyr⁸-GnRH were present. These two GnRHs therefore appear to occur in both fish species studied. Trp⁷,Leu⁸-GnRH is widespread amongst teleost fish (Jackson and Pan, 1983; Sherwood et al., 1983; Breton et al., 1984; Sherwood et al., 1984; King and Millar, 1985). From these data it seems evident that the mammalian GnRH molecular form occurs only in mammals and amphibians, Gln⁸-GnRH in birds and higher reptiles, and Trp⁷,Leu⁸-GnRH in gnathostomes. His⁵,Trp⁷, Tyr⁸-GnRH appears to he present in numerous different vertebrates. Tyr³,Leu⁵,Glu⁶,Trp⁷,Lys⁸-GnRH has thus far only been detected in lamprey brain. A number of novel GnRHs, whose structures have not been elucidated are present.

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

Contraception through a vaccine has been a very attractive proposition and several attempts were made in the past. To achieve contraception through immunological means, several points need to be considered. First, the targeted antigen should be an important component of reproduction and interference in its actions should lead to infertility. Second, the antigen must be highly immunogenic and the antibodies elicited should be able to block the functions of the antigen. Third, the antibody titres should be effective and must sustain for longer periods. Gonadotropins fulfill all the above criteria and therefore, have been attractive targets for developing human contraceptive vaccines. The pituitary gonadotropins- Luteinizing hormone (LH) and the Follicle stimulating hormone (FSH) are the principal regulators of the reproduction process in all the mammalian species (McLachlan et al., 1995c; Moudgal et al., 1992b; Murty et al., 1979a; Selvaraj and Moudgal, 1994a; Weinbauer et al., 1991). In males, LH binds to its specific receptor-LHR, expressed on the Leydig cells and regulates the production of testosterone. This testosterone binds to the androgen receptors expressed in the Sertoli cells and along with FSH, which binds to the specific receptors present on the Sertoli cell membranes, regulate the testicular functions and the spermatogenesis (Simoni et al., 1997; Themmen and Huhtaniemi, 2000; Ulloa-Aguirre and Timossi, 1998). The well documented studies have unequivocally established that the specific immunoneutralization of either hormone by active or passive immunization, leads to disruption of the gonadal functions (Fraser et al., 1986a; Marathe et al., 1995; Moudgal et al., 1992b; Murty et al., 1979b; Shetty et al., 1996; Srinath et al., 1983b) and consequent infertility and this observation formed the basis of the human contraceptive vaccines (Moudgal et al., 1997b; Talwar et al., 2011a; Talwar et al., 2009a). Several studies using testosterone as the main male hormonal contraception method (Matsumoto et al., 1986; Matsumoto et al., 1983a) and anti-hCG vaccine as the female hormonal contraceptive vaccine reached Phase I and II clinical trials (Talwar, 1997; Talwar et al., 1994; Talwar et al., 1997) . However, these human contraceptive vaccines faced several limitations. There was a need to inhibit only particular segments of the entire reproduction process whereas others needed to remain completely unaffected. For example, in males, the FSH regulated functions, the sperm production and spermatogenesis needed to be inhibited whereas the LH/testosterone associated functions should be unaffected. Similarly in females, the functions of hCG alone, elaborated by the conceptus should be blocked without affecting either LH or FSH regulated functions, thus, maintaining the normal reproductive cycle. This however is a difficult task especially when the antigens share a large degree of homology and common subunits (Pierce and Parsons, 1981). Moreover, the issues relating to the development and sustenance of high titres of the bioneutralizing antibodies were major limitations of these human contraceptive vaccines. Therefore, despite reaching Phase I and II clinical trials, these studies did not progress further. However, the same concept of an immunocontraceptive vaccine involving the neutralization of the functions of the gonadotropins is an extremely attractive strategy for controlling the animal populations where the reproduction process could be inhibited in its entirety. The overgrowing populations of the stray animals such as dogs and cats pose problems unlike those experienced with the human overpopulation. Thus, there is an immediate need to develop the methods of controlling the populations of these animals both in the developed and the developing countries. Whereas, in countries like the US, the major emphasis is on the domestic animals, in countries like India, the populations of the stray animals need to be controlled. The current methods employed for reducing the numbers of these animals include either castration or culling of the animals. These methods are however, traumatic, unsafe and not widely accepted by the society. The animal contraceptive vaccines currently available are mostly GnRH vaccines which have high cost of production, are not safe for animal use and elicit unwanted side effects. Apart from these, the animals need multiple administrations of these vaccines to elicit high and effective antibody titres, mostly with the use of conventional but non-approved adjuvants (Boedeker et al., 2009; McCoy, 1994). As mentioned above, the gonadotropins, by virtue of their ability to control the mammalian reproduction process, are attractive targets for achieving contraception. Moreover, the ease of administration of this vaccine to neutralize the functions of the endogenous circulating hormones makes them ideal targets for developing animal immunocontraceptive vaccines. This method of neutralizing the functions of the gonadotropins is also humane and safe for the animals as opposed to the current methods which are employed to reduce their numbers. However, in case of animal contraception, particularly for strays such as dogs, where large numbers of animals need to be treated, the challenge is to develop a method to sustain the high levels of the bioneutralizing antibodies for prolonged periods preferably with a single administration of the immunogen and without the use of conventional adjuvants such as the Freund’s adjuvant. In the present study, an attempt has been made develop a strategy to achieve a sustained immune response to small quantities of the hormonal antigens, preferably with a single administration of the immunogen resulting in complete disruption of the gonadal function for prolonged periods. To achieve this goal, recent developments in the field of immunology and vaccinology have been employed. This involves targeting of the hormonal antigens to the dendritic cells. Targeting the antigens to the dendritic cells for vaccination is becoming an extremely fascinating strategy and is being used extensively to target the antigens involved in several diseases (Escudier et al., 2005; Frankel et al., 1998; Garcia et al., 2005; Nouri-Shirazi et al., 2000a; Nouri-Shirazi et al., 2000b; Steinman and Germain, 1998). Most antigens are targeted to the dendritic cells by coupling them to the antibodies specific for the receptors expressed on the dendritic cell surface. One such receptor is the DEC205, which is expressed on most of the dendritic cells (Jiang et al., 1995) and is being widely used to develop vaccines and vaccination strategies. Targeting the antigens to the dendritic cells provides advantages such as ability to induce hundred fold higher immune response to very low doses of antigen without the use of any conventional adjuvant (Bonifaz et al., 2004a). Therefore, in the present study, these features of the dendritic cells have been harnessed to target the hormonal antigens (hCG and hFSH) to the canine DEC205 receptor to induce a long-term immune response capable of disrupting the gonadal functions. Towards this goal of delivering hormonal antigens to the dendritic cells, a fragment of the canine DEC205 corresponding to the Cysteine Rich Fibronectin II domain (CR/FNII) was expressed and used to isolate several canine DEC205 specific recombinant antibodies in the form of single chain fragment variable (ScFvs) from the Tomlinson’s and the yeast human ScFv display libraries. From a pool of eight unique ScFvs screened from the Tomlinson’s libraries, three ScFvs namely B3, G10 and H4 were characterized. All these ScFvs could bind to the human DEC205 receptor but not to the mouse DEC205. Their inability to recognise the mouse DEC205 suggested that mouse could not be used as the model system for these studies and therefore, a surrogate model system was needed. As the canine CR/FNII shared a high degree of homology with the rabbit counterpart, adult rabbits have been used as the surrogate model for immunization studies after confirming the binding of the ScFvs to the rabbit dendritic cells. Since the goal of the study was to deliver the hormonal antigens to the dendritic cells, each ScFv was translationally fused to a core streptavidin fragment, thus creating bi-functional agents (ScFv-CS) capable of binding to the dendritic cells and also to any biotin-tagged antigen, thus delivering the antigen to the dendritic cells. Of the three ScFvs, the ScFv-CS-H4 which could bind to the canine CR/FNII with the KD of 25nM was used for demonstrating the ability of the ScFv-hormone complex to elicit the bioneutralizing antibody response. The ScFv-CS-H4-biotin-hCG or hFSH or both were administered to adult male rabbits along with poly IC: LC, a Toll-like receptor agonist and the antibody titres were monitored. It was possible to maintain high titres of the bioneutralizing antibodies for more than one year with a single administration of the immunogen. Testicular histology of the immunized animals showed extensive disruption of spermatogenesis with most of the germ cells being TUNEL positive undergoing apoptosis. There was complete absence of elongated spermatids and sperms in the testis indicating infertility caused by immunization with the gonadotropins. These data show that targeting the hormonal antigens to the dendritic cells leads to long-term infertility with minimal immunization. Although the ScFvs from the Tomlinson’s libraries were able to deliver the hormonal antigens to the dendritic cells and produce robust and sustained antibody response capable of disrupting the gonadal functions, the affinities of these ScFvs to DEC205 were moderate. It was felt that increasing the affinities of the ScFvs could enhance the effect with respect to the dose of the antigen that needs to be administered and the duration until which the high antibody titres could be maintained. Therefore, the yeast human ScFv display library offering higher diversity of the human ScFvs displayed, was screened for high affinity DEC205 specific binders. From a pool of several ScFvs, six unique ScFvs were characterized. The amino acid sequences of all ScFvs followed the Kabat's rules for identifying the complimentarity determining regions of the heavy and the light chains of the antibodies. All these ScFvs were unique in their amino acid sequences. The dissociation constants of all these antibodies for the canine CR/ FNII ranged from 10-9 to 10-11 M which was 20-300 fold higher than the ScFvs obtained from the Tomlinson’s libraries. The best ScFv obtained from this library was ScFv-92 with a KD value of 8 x10-11 M. All these ScFvs were able to deliver the payload antigen to both, the mouse DEC205 over-expressing cells and the bone marrow derived dendritic cells. Mice immunized with yeast display ScFvs also yielded antibody response to very small quantities of the immunogen with the highest antibody titres obtained with the ScFv-92. It was further demonstrated that all ScFvs also activated the cell-mediated immunity with significant increase in the antigen stimulated T cell proliferation. These ScFvs could also deliver the antigen to the human dendritic cells differentiated from the human monocytes in vitro, thus emphasising their utility in human vaccine development. An attempt was also made to develop nanoparticle (NP) based strategies of delivering the antigen to the dendritic cells. The PLGA-NPs, encapsulating hCG and coated with the DEC205 ScFv-92 was able to elicit high antibody response to very low doses of the antigen. This response could be sustained for 120 days and was higher than the response obtained with similar doses of hCG encapsulated NPs or hCG complexed to ScFv-92 alone. Targeting of the NPs also elicited antigen specific T cell response thus, potentiating their use in cell mediated immunity along with humoral immune responses. In conclusion, this approach of delivering the gonadotropins to the dendritic cells resulted in the production of bioneutralizing antibodies that could disrupt the gonadal functions for a prolonged period and can be effectively used in the fields for controlling the animal populations. This method fulfils all the criteria for any animal contraception. This strategy also elicits both T cell mediated and humoral immunity and can thus be used for producing vaccine against viral and parasitic infections. It can also be used for cancer immunotherapy. Another exciting feature of the strategy used in this study is the usage of ScFv-CS which allows the delivery of any biotin tagged antigen to the rodent and human dendritic cells. As discussed above, the methods for controlling the animal populations are expected to be effective, humane, safe, simple, non-surgical, single shot with long lasting effects, cheap, applicable in the fields and widely accepted by different societies. The methods presented in this study fulfill all these criteria and should be effective in controlling populations of different animal species.

Chez les vertébrés, le processus de la spermatogénèse est directement contrôlé par deux hormones gonadotropes hypophysaires, Fsh et Lh. Chez les salmonidés, les profils de sécrétion des 2 hormones diffèrent et présentent des variations significatives au cours du cycle de développement spermatogénétique, suggérant que Fsh et Lh interviennent à des étapes différentes du processus. A la différence des mammifères, chez les poissons les 2 gonadotropines exercent une forte activité stéroïdogène, et par ailleurs il a été rapporté par plusieurs auteurs que leurs récepteurs seraient moins sélectifs vis-à-vis des 2 ligands. Ainsi, le périmètre des actions respectives de Fsh et de Lh n'est pas défini chez les poissons. D'autre part, les mécanismes de l'action de Fsh qui ne seraient pas relayés par les stéroïdes sont très mal connus. Chez la truite, nous avons déterminé que chaque gonadotropine agit essentiellement par l'intermédiaire de son récepteur respectif. L'analyse des variations du transcriptome testiculaire après un traitement in vitro par les hormones de la reproduction (Fsh, Lh, androgènes) a permis 1- de révéler des actions distinctes de Fsh et de Lh sur l'expression des gènes, 2- de mettre en évidence deux mécanismes d'action de la Fsh, l'un dépendant et l'autre indépendant de la production de stéroïdes et 3- d'identifier plusieurs acteurs d'interaction cellulaire régulés par Fsh, et probablement impliqués dans les étapes précoces de prolifération ou de différenciation des cellules germinales, tels que l'hormone antimüllérienne, la midkine, l'insulin-like growth factor1b, la follistatine-like 3 et l'activine, 4-de proposer une implication de Fsh dans les évènements tardifs de maturation et d'excrétion du sperme. Au-delà des acquis concernant les régulations endocriniennes et moléculaires chez la truite, ces travaux constituent un apport de connaissances qui peut être étendu à d'autres téléostéens pour décrypter l'action propre à Fsh dans le déclenchement de la maturation pubertaire. Enfin, nous montrons qu'une vaccination contre les récepteurs des gonadotropines constitue une voie potentielle de contrôle des maturations précoces en élevage. / In vertebrates, spermatogenesis is under the direct control of two pituitary gonadotropic hormones named Fsh and Lh. In salmonids, the 2 hormones are differentially secreted in the plasma through the reproductive cycle, suggesting that Fsh and Lh are involved in the regulation of different steps of the process. Unlike in mammals, both fish gonadotropins exert a strong steroidogenic activity and, besides, some authors reported for their receptors a much lower selectivity towards the two ligands. Yet, their respective roles are not established in fish. Furthermore, the mechanisms of Fsh action that would not be mediated by steroids are poorly investigated. In trout, we showed that each gonadotropin mainly acts through its cognate receptor. The analysis of the variations of testicular transcriptome following an in vitro treatment with reproductive hormones (Fsh, Lh and androgens) permitted 1- to reveal that Fsh and Lh have distinct effects on gene expression, 2- to highlight two mechanisms of Fsh action, one dependent on the steroid production and the second one independent of that production, 3- to identify several Fsh regulated factors involved in cellular interactions and particularly in the control of germ cell proliferation / differentiation (anti mullerian hormone, midkine, insulin-like growth factor 1b, follistatin-like 3 and activin), 4- to propose an involvement of Fsh in late events of sperm maturation and release. In addition to knowledge on endocrine and molecular regulations in trout, this work provides a fund of knowledge useful in other teleosts to decipher the action of Fsh in triggering puberty onset. Finally, we showed that an immunization against the gonadotropin receptors is a potential method to delay sexual maturation in farmed fish.

Sendo o GnRH a molécula capaz de iniciar a cascata hormonal reprodutiva, realizamos a clonagem, analisamos os efeitos da indução à reprodução e as funções dos GnRHs de A. altiparanae. Como resultados, obtivemos as sequências dos cDNAs do GnRH2 e GnRH3. Quando induzidos à reprodução, a desova foi às 20 h pós-estímulo (hpe) em fêmeas e às 16 hpe em machos, o aumento da expressão do mRNA de GnRH3 ocorreu às 8 hpe em fêmeas, e o aumento da expressão do mRNA de GnRH2 foi às 0 hpe em machos. Com relação ao efeito dos GnRHs, todos estimularam a expressão do mRNA de &beta;LH, mas não de &beta;FSH, e só o GnRH2 foi capaz de elevar o MIS e causar a desova. Como conclusão, temos que as sequências dos cDNAs dos GnRHs se mostraram conservadas; a indução à reprodução por redução do nível da água foi eficaz; em cativeiro, a espécie teve desenvolvimento assincrônico; o GnRH2 provavelmente está ligado ao comportamento reprodutivo, e o GnRH3 é a possível forma hipofisiotrópica. Por fim, só o GnRH2 desencadeou toda a cascata hormonal. / The study on reproduction in fish has been acquired great importance in last years, mainly for the benefit of threatened species. During the reproductive process, the hypothalamic neurons synthesize and release GnRH that stimulates the pituitary cells to release FSH and LH, which, in turn, promote the gonadal maturation. In fact, the morphological changes in gonads are the result of the endocrine action of the reproductive axis, in which the GnRH is the key molecule to starting the reproductive axis control. Thus, the knowledge about the GnRH, as well as about the gonadal morphological changes in the spawning might contribute to effectiveness of reproduction. Therefore, in this work, with Astyanax altiparanae as a model, we made the molecular characterization of the GnRHs, and we analyzed the gonadal morphological changes during the reproductive stimulus. In addition, we evaluated the role of injected GnRHs in vivo. As results, we obtain the cDNA complete sequence of preproGnRH2 (612bp) and preproGnRH3 (407bp) of A. altiparanae. Regarding the induction of reproduction by water level drawdown, the released of gametes occurred at 20 hours after stimulus in female and at 16 hours after stimulus in males, the mRNA expression of GnRH3 increased at 8 hours after stimulus in female and the mRNA expression of GnRH2 increased at 0 hours in males. Regarding the effects of injected GnRH, all of them stimulated the &beta;LH but not &beta;FSH mRNA expression, and only the GnRH2 was able to rise the MIS and stimulate the released of gametes. We conclude that the cDNAs sequences of preproGnRH2 and preproGnRH3 were conserved, although there is a change in the amino acid at the position 8 of the GnRH3 decapeptide of A. altiparanae. Furthermore, the induced reproduction by water level drawdown was effective, and in captivity, the A. altiparanae has an asynchronous development with splitted spawning during the breeding season. The analysis of the animals submitted to the reproductive stimulus allowed us to suggest that in A. altiparanae, the GnRH2 probably has a role in sexual behavior and the GnRH3 possibly is the hypophysiotropic form. Finally, analyzing the GnRH effects, we observed that only the GnRH2 was able to start the entire reproductive hormonal cascade, leading the animal to spawning.

A qualidade do oócito é um fator limitante na fertilidade das fêmeas e reflete seu intrínseco potencial ao desenvolvimento embrionário subsequente. As alterações moleculares e bioquímicas no processo de maturação dos oócitos são necessárias para permitir a fecundação destes. Sob influência das gonadotrofinas, uma cascata de eventos é desencadeada, alterando a expressão gênica e a estrutura dos folículos. A maturação ocorre pelo intercâmbio entre o oócito e as células do cumulus que irão fornecer fatores para o desenvolvimento do oócito e criar o microambiente necessário para garantir o sucesso na maturação. A ação do FSH sobre a retomada da meiose ocorre, possivelmente, por ativação da proteína quinase C (PKC). A via de sinalização desta proteína parece estar envolvida na ativação da quinase ativada por mitógeno (MAPK) em oócitos e células do cumulus, na maturação induzida por FSH e LH, além de regular a síntese do Fator de Crescimento Epidermal (EGF). Deste modo, o objetivo do presente trabalho foi avaliar a ação da PKC na maturação de oócitos bovinos e se esta ativação envolve o EGF. Para tal foram realizados dois experimentos. Em ambos, a progressão do ciclo celular foi avaliada utilizando a sonda fluorescente Hoechst 33342. A expansão das células do cumulus foi avaliada utilizando-se o software Image Pro Plus 5.1 para análise das imagens dos oócitos geradas em microscópio Olympus IX81. O maior diâmetro de cada complexo cumulus oócito foi adotado como parâmetro de mensuração da expansão. A dosagem de progesterona do meio de cultivo foi realizada pela técnica de RIA. A ativação da PKC e da MAPK foi avaliada pela técnica de Western blot. Os dados foram avaliados pelo software SigmaPlot versão 12.2 e submetidos ao teste de normalidade (Shapiro-Wilk). Quando necessário, os dados foram transformados. Para comparação entre dois tratamentos, utilizou-se o teste t-student. Para mais de dois tratamentos foi realizada análise de variância e teste de comparação de médias (TUKEY), considerando-se 0,05 para rejeitar a hipótese de nulidade. No experimento 1 foi avaliado se a ativação da PKC foi estimulada por gonadotrofinas. Os oócitos foram maturados in vitro tratados com gonadotrofinas, na presença ou ausência do inibidor de PKC. A presença do inibidor de PKC diminuiu as taxas de quebra de vesícula germinativa e a expansão das células do cumulus, sem alterar a esteroidogênese. Estes resultados demonstram que a PKC participa da via de sinalização da retomada da meiose. No experimento 2 foi avaliado se o EGF está envolvido na via regulada pela PKC. Os oócitos foram maturados in vitro, na presença ou ausência de LH e FSH, do inibidor de PKC e do EGF. O EGF foi capaz de reverter os efeitos do inibidor de PKC, aumentando as taxas de quebra de vesícula germinativa e a expansão de células do cumulus. Não foi possível detectar, nas condições deste experimento, a ativação das proteínas PKC e MAPK através do Western Blot. Este trabalho permite concluir que a via de sinalização da maturação de oócitos bovinos envolve a PKC e sugere a participação do EGF nesta via. / Oocyte quality is a limiting factor in female fertility and reflects its potential to the subsequent embryonic development. Molecular and biochemical alterations during the oocyte maturation process are needed to allow fecundation. Under gonadotropin influence, cascade of events occurs changing gene expression and follicle structure. Maturation depends on the interaction between oocyte and cumulus cells interaction, which provides factors for oocyte development and create the ideal microenvironment for the success of the maturation process. The FSH stimulation of meiosis resumption probably occurs through PKC activation. The signaling pathway of PKC might be involved by the mitogen activated protein kinase (MAPK) in oocytes and cumulus cells during FSH-LH induced maturation. Furthermore, MAPK regulates the epidermal growth factor (EGF) synthesis. The aim of the present study was to evaluate PKC function during bovine oocyte maturation and if its activity involves EGF. Two experiments were performed. In both experiments, the cell cycle progression was analyzed by Hoechst 33342 fluorescent dye. The cumulus cells expansion was performed using software Image Pro Plus 5.1 by the analysis of oocyte images taken in Olympus IX81 microscope. The highest diameter of each cumulus oocyte complex was recorded as the expansion value. The RIA and Western Blot techniques were used to measure progesterone concentration in the culture media and the PKC and MAPK activity, respectively. Data was analyzed by SigmaPlot software, version 12.2. The Shapiro-Wilk test was used to assess for normality and, when needed, the data was transformed. Student t tests were carried out to compare two treatments. Differences between more than two means were assessed by analysis of variance followed by Tukey test, considering P-value lower than 0.05 as statistically significant. Experiment 1 studied whether PKC function was stimulated by gonadotropins. FSH and LH were used for oocyte maturation in vitro, with or without PKC inhibitor. The presence of PKC inhibitor decreased germinal vesicle breakdown and the cumulus cells expansion, but did not alter the steroidogenesis. These results show that PKC participates in the signaling pathway of meiosis resumption. The Experiment 2 evaluated whether EGF influences PKC signaling pathway. The oocytes were matured in vitro, in the presence or absence of LH and FSH, PKC inhibitor and EGF. Epidermal Growth Factor was able to reverse PKC inhibitor effects, increasing germinal vesicle breakdown rates and cumulus cells expansion. The Western Blot technique was not able to detect PKC and MAPK activity, considering the conditions of this study. In conclusion, PKC is involved in the signaling pathway of bovine oocytes maturation and its pathway is mediated by EGF.

O estradiol secretado pelo folículo dominante (DOM) desempenha importante papel na luteólise da vaca. Em adição, o reconhecimento materno da prenhez (MRP) requer um ambiente uterino otimizado, o qual por sua vez depende da função luteínica e de adequadas concentrações de progesterona circulante. Os objetivos do presente estudo foi testar diferentes estratégias para otimizar a função luteínica e prevenir a influência de um DOM durante o período crítico (CP) para o MRP (de D13 a D20 após o estro o estro). Diferentes abordagens foram testadas. No exp.1, 23 vacas Nelore foram tratadas com o protocolo ovsynch para induzir um cio sincronizado (D0). As vacas receberam: Gc (n=7) - nada mais; ThCG (n=5) - 3000 IU de hCG no D5; TE2 (n=6) - 5mg de 17b-estradiol (E2) no D12; ThCG/E2 (n=5) - hCG/D5 + E2/D12. Ultra-sonografias e dosagens de progesterona plasmática durante o ciclo estral permitiram determinar que o E2 reprogramou o ciclo ovariano ao prevenir a presença (P&it;0,05) de um DOM durante quase todo CP (0,6&plusmn;0,7 dias entre D15 e D20), porém o E2 induziu a luteólise. As vacas que receberam a hCG desenvolveram corpo lúteo acessório e tiveram [P4] mais alta até o D13 (P&it;0,05). Portanto, a fase luteínica não foi prolongada No exp.2, os mesmos tratamentos foram impostos a 220 vacas Nelore (55 por grupo) após uma inseminação artificial em tempo fixo (TAI). As taxas de prenhez (PR) à TAI ou às inseminais de repasse durante uma estação com 64 dias de duração foram diminuídas (P&it;0,05) quando o E2 foi usado e a hCG não foi capaz de aumentar aquelas PR. No exp.3, vacas Red Angus no pós-parto tiveram seu estro sincronizado (D0) e receberam: nada mais (GCT, n=5) ou 200mg de gonadorrelina no D5 mais 3000 IU hCG no D13 (GRF, n=5); ou ablação de todos folículos &sup3;7mm através de aspiração folicular em D14, D17 e D20 (GRM, n=5). GRF teve luteólise retardada (18,2&plusmn;1,0b dias, 23,6&plusmn1,0a dias e 18,7&plusmn1,2b dias para GCT, GRF e GRM, respectivamente) e maior [P4] que os outros grupos. Folículos maiores que 7mm foram observados quando das aspirações. Foi possível concluir que: a) E2 permitiu consistentemente reprogramar o desenvolvimento folicular, porém causou luteólise e o seu uso trouxe efeitos negativos sobre as PR; b) a hCG melhorou a função luteínica mas não aumentou as PR; c) a ablação dos folículos &sup3;7mm não preveniu a presença do DOM no CP para o MRP e d) a associação GnRH/hCG otimizou a função luteínica, retardou a luteólise e prolongou a fase luteínica de modo que todo o CP esteve sob influência da progesterona / Estradiol secreted from the dominant follicle (DOM) plays a key role in triggering luteolysis in the cow. In addition, maternal recognition of pregnancy (MRP) requires an optimum uterine environment, which directly depends on luteal function and adequate levels of circulating progesterone. The aim of this study was to test different strategies to optimize luteal function and prevent the influence of a DOM throughout the critical period (CP) for MRP (from D13 to D20 after estrus). Different approaches were tested. In exp.1, 23 Nelore cows were treated with the ovsync protocol to induce a synchronized estrus (D0). Cows received: Gc (n=7) - nothing else; ThCG (n=5) - 3000 IU of hCG five days (D5) after estrus; TE2 (n=6) - 5mg of 17b-estradiol (E2) on D12; ThCG/E2 (n=5) - hCG/D5 + E2/D12. Ultrasound evaluation and plasmatic progesterone concentration ([P4]) throughout estrous cycle allowed to conclude: E2 reprogrammed ovarian cycle by preventing the presence (P&it;.05) of a DOM during almost all CP (0.6±.7 days within the D15 to D20 interval) but induced luteolysis; cows receiving hCG developed accessory corpus luteum and had higher [P4] up to D13 (P&it;.05). Therefore, luteolysis was not delayed and luteal phase was not prolonged. In exp.2, same treatments were imposed to 220 Nelore cows (55 per group) after a timed artificial insemination (TAI). Pregnancy rates (PR) at TAI or at AIs thereafter over a 64-day period were reduced (P&it;0.05) by using E2 and hCG was not capable to improve those PR. In exp.3, postpartum Red Angus cows were estrus synchronized (D0) and received: nothing else (GCT, n=5) or 200mg of gonadorrelin on D5 plus 3000 IU hCG on D13 (GRF, n=5); or ablation of all follicles &sup3;7mm through follicular aspiration on D14, D17 and D20 (GRM, n=5). GRF had delayed luteolysis (18.2&plusmn;1.0b days, 23.6&plusmn;1.0a days, 18.7&plusmn;1.2b days for GCT, GRF, GRM, respectively) and higher [P4] than other groups. Follicles larger than 7mm were observed in all occasions of aspiration. In could be concluded that: a) E2 allowed to consistently reschedule follicular development but caused luteolysis and its use was detrimental to PR; b) hCG improved luteal function but did not increase PR; c) ablation of 7mm follicles did not prevent a DOM throughout CP for MRP and d) GnRH/hCG association optimized luteal function, delayed luteolysis and prolonged luteal phase in such a way that all CP was under progesterone influence

O objetivo do estudo realizado no Laboratório de Pesquisa em Suínos, da Faculdade de Medicina Veterinária e Zootecnia da Universidade de São Paulo, Pirassununga – SP, foi verificar os efeitos combinados de gonadotrofinas exógenas, (eCG e LH) e flushing sobre a duração e dispersão da manifestação do primeiro e segundo estros, ciclicidade das fêmeas, número de ovulações, viabilidade embrionária e freqüência de cistos ovarianos. Foram utilizadas 72 marrãs pré-púberes da linhagem Pen Ar Lan NAIMA®, com idade média de 157,49±5,01 dias, e 96,65±7,70kgs de peso, distribuidas em quatro tratamentos, em arranjo fatorial, com e sem flushing , e com e sem hormônio. A ração utilizada continha 16% de PB e 3.286,73 kcal/kg ME. A partir do 7º dia do primeiro estro, induzido com hormônio, as fêmeas do tratamento com flushing receberam 50% de incremento da mesma ração. No 16º dia, após o primeiro estro, as marrãs no tratamento hormonal receberam aplicação de 600 UI de eCG (Novormon®) e 2,5 mg de LH (Lutropin®), 72 horas após. Todas as marrãs foram expostas ao macho duas vezes ao dia, a partir do 10º dia e inseminadas ao segundo estro com sêmem heterospérmico contendo 4 x 109 espermatozóides. Ao abate, realizado no 5º dia após a inseminação, os genitais foram colhidos para exame dos ovários e coleta dos embriões. Não se observou interação entre os fatores. Os efeitos analisados em separado, não evidenciaram diferença significativa no Número de ovulações (14,28±5,06 vs 13,47±5,57 com e sem Flushing e 14,60±5,78 vs 13,23±4,83 com e sem Hormônio, respectivamente, P>0,05), na freqüência de cistos ovarianos (22,86% vs 11,11%, com e sem Flushing e 15,15% vs 18,42%, com e sem Hormônio, respectivamente,. P>0,05), na Manifestação do 2º estro (85,71% vs 91,43% com e sem Flushing e 90,63% vs 86,84% com e sem Hormônio, respectivamente, P>0,05), no Peso Final (115,35±8,23 vs 111,56±8,30kg, com e sem Hormônio, respectivamente, P>0,05), na Espessura de toucinho (10,0±2,0 vs 10,2±1,8mm com e sem Hormônio, respectivamente, P>0,05), e na Duração dos estros (57,68±20,02 vs 59,68±14,76 horas, com e sem Hormônio e 57,75±14,99 vs 59,62±19,43 horas, com e sem Flushing, respectivamente, P>0,05). Constatou-se diferença significativa na Viabilidade embrionária (11,55±4,81 vs 8,50±5,32 embriões viáveis, com e sem Flushing e 8,39±5,66 vs 11,25±4,58 embriões viáveis, com e sem Hormônio, respectivamente P=0,02), indicando valor superior para o tratamento com Flushing e inferior no grupo com Hormônio. No Peso final (117,48±9,82 vs 109,28±7,57kg, com e sem Flushing, respectivamente, P=0,0001) e na Espessura de toucinho (10,7±2,2 vs 9,5±1,9kg, com e sem Flushing, respectivamente, P=0,03) o valor superior ficou para o grupo com Flushing. Concluiu-se no estudo a não interação dos efeitos, entre os fatores Hormônio e Flushing . O uso das gonadotrofinas (eCG e LH) reduziu a dispersão de manifestação do segundo estro, sendo efetiva a sua sincronização. O emprego do Flushing no primeiro ciclo estral em marrãs influenciou positivamente a viabilidade embrionária e na associação do eCG e LH mais Flushing não notou-se efeito interativo, identificando-se diminuição da viabilidade embrionária, pela ação hormonal. / The objective of this study, held at the Swine Research Laboratory of the School of Veterinary Medicine and Zootechnology of the Univ. of São Paulo in Pirassununga, São Paulo, was to verify the combined effects of exogenic gonadotropin (eCG and LH) and flushing on the duration and dispersion of the manifestation of initial and second estruses, cyclicity of females, number of ovulations, embryonic viability, and frequency of ovarian cysts. Used for the experiment were 72 pubertal gilts of Pen Ar Lan NAIMA® lineage, with an average age of 157.49±5.01 days, and weight of 96.65±7.70kg. They were distributed in four treatments in a factorial arrangement, with and without flushing, and with and without hormone treatment. The feed used contained 16% CP and 3,286.73 kcal/kg ME. Starting on the seven day of the first estrus, induced with homone, the females with flushing treament received a 50% increase in the amount of the same type feed that they had been receiving. On the sixteenth day of the first estrus the gilts that received hormone treatment were given 600 UI of eCG (Novormon®) and 2.5 mg of LH (Lutopin®), 72 hours later. All the gilts were exposed to boar twice at day, starting on the tenth day and inseminated during the second estrus with heterospermic semen containing 4 x 109 spermatozoon. At slaughter on the fifth day after insemination, the genitals were removed to examine the ovaries and collect the embryos. Interaction between the factors were not observed. The effects that were analyzed separately did not show significant differences in the number of ovulations (14.28±5.06 vs 13.47±5.57 with and without flushing, and 14.60±5.78 vs 13.23±4.83 with and without hormones, respectively, P>0.05), in the frequency of ovarian cysts (22.86% vs 11.11%, with and without flushing and 15.15% vs 18.42% with and without hormones, respectively, P>0.05), during the second estrus (85.71% vs 91.43% with and without flushing and 90.63% vs 86.84% with and without hormones respectively, P>0.05), at final weight (115.35 ± 8.23 vs 111.56 ± 8.30kg, with and without hormones, respectively, P>0.05), in the backfat (10.0±2.0 vs 10.2±1.8mm with and without hormones, respectively, P>0.05), and in the duration of the estruses (57.68±20.02 vs 59.68±14.76 hours, with and without hormones and 57.75±14.99 vs 59.62±19.43 hours, with and without flushing, respectively, P>0.05). A significant difference was found in embryonic viability (11.55±4.81 vs 8.50±5.32 viable embryos, with and without flushing and 8.39±5.66 vs 11.25±4.58 viable embryos with and without hormones, respectively P=0.02), which indicated higher numbers when treated with flushing and lower numbers when treated with hormones. At the final weight (117.48±9.82kg vs. 109.28±7.57kg with and without flushing respectively, P=0.0001) and in the backfat (10.7±12.2 vs 9.5±1.9kg, with and without flushing, respectively, P=0.03) flushing resulted in a higher number. It is concluded in this study that there is a lack of interactive effects between treaments with hormones and flushing. Utilization of gonadotropins (eCG e LH) reduced the dispersion of the second estrus within an effective synchronization. The utilization of flushing in the first estrous cycle in gilts gave positive results as regards to embryonic viability. The association of eCG and LH, plus flushing did not show an interactive effect, and a reduction was identified in embryonic viability based the action of hormones.