Εισαγωγή: Το σύνδρομο της μεμονωμένης ανεπάρκειας της εκλυτικής ορμόνης των γοναδοτροπινών (IGD) χαρακτηρίζεται από μεμονωμένη λειτουργική ανεπάρκεια της υποθαλαμικής παραγωγής ή/και έκκρισης της GnRH οδηγώντας σε μεμονωμένη ανεπάρκεια των γοναδοτροπινών με φυσιολογική λειτουργικότητα των υπολοίπων υποφυσιακών ορμονών. Η συνύπαρξη IGD και ανοσμίας αναφέρεται ως σύνδρομο Kallmann (ΣΚ), ενώ η απουσία οσφρητικής διαταραχής αναφέρεται ως ιδιοπαθής υπογοναδοτροφικός υπογοναδισμός (ΙΥΥ). Σκοπός: Σκοπός της μελέτης είναι η περιγραφή των φαινοτυπικών χαρακτηριστικών ασθενών με μεμονωμένη ανεπάρκεια GnRH (ΙΥΥ και ΣΚ), η διερεύνηση της ύπαρξης μεταλλάξεων στα γονίδια KAL1, FGFR1 (υποδοχέας του αυξητικού παράγοντα των ινοβλαστών 1), PROK2 (προκινετισίνη 2), PROKR2 (υποδοχέας της προκινετισίνης 2) και GPR54 (KISS1R: υποδοχέας της κισσπεπτίνης) στους ασθενείς αυτούς, καθώς και η συσχέτιση μεταξύ του γονότυπου των ασθενών και ειδικών κλινικών φαινοτύπων. Επίσης διερευνείται η παρουσία μεταλλάξεων στο γονίδιο KAL1 σε ομάδα φαινομενικά υγιών παιδιών με ετερόπλευρη αγενεσία/δυσγενεσία νεφρού (ΕΝΑ), σε μια προσπάθεια καθορισμού της συχνότητας των μεταλλάξεων του γονιδίου KAL1 στην ΕΝΑ. Τέλος πραγματοποιείται in-vitro λειτουργικός έλεγχος δύο σημειακών μεταλλάξεων του γονιδίου FGFR1 που επηρεάζουν το ίδιο αμινοξύ της πρωτεΐνης (R254W και R254Q), με στόχο τη συσχέτιση των in-vitro ευρημάτων με τον κλινικό φαινότυπο των ασθενών. Ασθενείς: Μελετήθηκαν συνολικά εξήντα έξι (66) ασθενείς με μεμονωμένη ανεπάρκεια GnRH (26 με ΣΚ και 40 με ΙΥΥ), στους οποίους πραγματοποιήθηκε μοριακός έλεγχος των γονιδίων KAL1, FGFR1, PROK2, PROKR2 και GPR54. Επίσης μελετήθηκαν 13 παιδιά (ηλικίας κάτω των 15 ετών) στα οποία υπήρχε απεικονιστικά επιβεβαιωμένη συγγενής ετερόπλευρη νεφρική αγενεσία/δυσγενεσία, η οποία δεν παρατηρήθηκε στα πλαίσια γνωστού συνδρόμου και τα οποία ελέχθησαν για την παρουσία μεταλλάξεων στο γονίδιο KAL1. Μέθοδοι: Η μεθοδολογία του μοριακού γονιδιακού ελέγχου περιλάμβανε την απομόνωση DNA γονιδιώματος από δείγμα ολικού αίματος, τον εκλεκτικό πολλαπλασιασμό των εξονίων των υπό μελέτη γονιδίων με την αλυσιδωτή αντίδραση της πολυμεράσης (PCR amplification) και τον προσδιορισμό της αλληλουχίας του DNA στα προϊόντα της PCR (DNA sequencing). Ο in-vitro λειτουργικός έλεγχος των δύο μεταλλαγμένων μορφών του υποδοχέα FGFR1 (R254W και R254Q) περιλάμβανε την μελέτη της σηματοδοτικής δραστηριότητας του υποδοχέα κατόπιν διέγερσής του από τον προσδέτη FGF2, καθώς και τον προσδιορισμό των επιπέδων έκφρασης των μεταλλαγμένων μορφών του υποδοχέα FGFR1, τα οποία συγκρίθηκαν με τα αντίστοιχα του φυσιολογικού υποδοχέα (WT=wild type). Η μετάδοση σήματος του υποδοχέα FGFR1 αξιολογήθηκε με την τεχνική ανίχνευσης δραστηριότητας του γονιδίου αναφοράς της λουσιφεράσης. Η μέτρηση των επιπέδων της ολικής έκφρασης του FGFR1 πραγματοποιήθηκε με την τεχνική της ανάλυσης πρωτεϊνών με ηλεκτροφόρηση σε πήκτωμα πολυακριλαμιδίου, ακολουθούμενη από την τεχνική της ανάλυσης κατά Western. Η εκτίμηση της ενδοκυττάριας ωρίμανσης του πρωτεϊνικού μορίου του υποδοχέα FGFR1 έγινε μέσω ενζυμικής πέψης της γλυκοπρωτεΐνης με ενδογλυκοσιδάσες, ενώ ο υπολογισμός των επιπέδων έκφρασης του FGFR1 στην κυτταροπλασματική μεμβράνη έγινε με την πρόσδεση ραδιοσημασμένου αντισώματος (radiolabelled antibody binding assay). Αποτελέσματα: Εκ του μοριακού γονιδιακού ελέγχου που πραγματοποιήθηκε στους ασθενείς με IGD εντοπίσθηκαν πέντε διαφορετικές μεταλλάξεις στο γονίδιο KAL1 σε τρεις άρρενες ασθενείς με σύνδρομο Kallmann (Ε514Κ, Α660Τ, Ε37Κ, T235S, έλλειψη εξονίων 5-10), καθώς και μια σημειακή μετάλλαξη στο γονίδιο FGFR1 (R254W) σε έναν άρρενα ασθενή με ιδιοπαθή υπογοναδοτροφικό υπογοναδισμό. Εκ του in-vitro λειτουργικού ελέγχου των δύο σημειακών μεταλλάξεων του γονιδίου FGFR1 (R254W και R254Q) που μελετήθηκαν, προέκυψε ότι η μέγιστη σηματοδοτική δραστηριότητα για τη μεταλλαγμένη μορφή του υποδοχέα R254W παρουσιάζει μείωση κατά 45% σε σύγκριση με τον wild-type υποδοχέα (p<0.01), ενώ η μέγιστη απάντηση της μεταλλαγμένης μορφής R254Q μειώνεται κατά 15% σε σχέση με το wild-type υποδοχέα, διαφορά που δεν αναδείχθηκε στατιστικά σημαντική. Ωστόσο και οι δυο μεταλλαγμένες μορφές R254W και R254Q εμφανίζουν ελαττωμένα επίπεδα ολικής έκφρασης (40% και 30% μείωση σε σχέση με τον wild-type, αντίστοιχα), ενώ η πρωτεϊνική ωρίμανση δεν φαίνεται να επηρεάζεται. Τέλος η έκφραση των μεταλλαγμένων μορφών R254W και R254Q επί της κυτταρικής επιφάνειας παρουσιάζεται σημαντικά ελαττωμένη (35%, p<0.01 και 15%, p<0.05, αντιστοίχως). Εκ του μοριακού γονιδιακού ελέγχου που πραγματοποιήθηκε στους ασθενείς με ΕΝΑ βρέθηκε μετάλλαξη του KAL1 σε έναν ασθενή 12 ετών, ο οποίος εμφάνιζε συνοδό ανοσμία, συγκινησία άνω άκρων και κρυψορχία. Στον ασθενή αυτόν τέθηκε η γενετική διάγνωση του ΣΚ και αργότερα υποβλήθηκε σε έγκαιρη έναρξη θεραπευτικής αγωγής. Συμπεράσματα: Το ποσοστό των γνωστών μεταλλάξεων που έχουν εντοπισθεί στους ασθενείς με IGD του Ελλαδικού χώρου είναι πολύ μικρό και επομένως παραμένει πρόσφορο το πεδίο για περαιτέρω έρευνα προς την κατεύθυνση της διευκρίνησης της μοριακής αιτιοπαθογένειας της νόσου. Η σύγκριση φαινότυπου-γονότυπου των ασθενών με σύνδρομο Kallmann υποδεικνύει ότι η παρουσία συνοδού ετερόπλευρης αγενεσίας νεφρού αποτελεί ισχυρή ένδειξη για την ύπαρξη μεταλλαγών στο γονίδιο KAL1. Η ανεύρεση μεταλλάξεων του γονιδίου KAL1 σε παιδιά με ΕΝΑ έχει διττή σημασία· αφενός επιβεβαιώνει την εμπλοκή της ανοσμίνης-1 (του προϊόντος του γονιδίου KAL1) στην οργανογένεση του νεφρού και αφετέρου οδηγεί στην πρώιμη διάγνωση του ΣΚ. Ο μοριακός έλεγχος του γονιδίου KAL1 σε παιδιά με ΕΝΑ συστήνεται επί συνύπαρξης και άλλων κλινικών σημείων του ΣΚ (ανοσμία, κινήσεις καθρέπτη, κρυψορχία, μικροφαλλία) ή ανάδειξης οικογενειακού ιστορικού υπογοναδισμού και ανοσμίας. Ο συγκριτικός λειτουργικός έλεγχος δύο μεταλλάξεων του FGFR1 που επηρεάζουν το ίδιο αμινοξύ (R254W, R254Q) αναδεικνύει την απώλεια της λειτουργικότητας των μεταλλαγμένων μορφών του υποδοχέα in-vitro. Αν και από τον in-vitro λειτουργικό έλεγχο προκύπτει ότι η μετάλλαξη R254W είναι πιο σοβαρή από τη μετάλλαξη R254Q, ωστόσο δεν παρατηρείται συσχέτιση του βαθμού της απώλειας της in-vitro λειτουργικότητας των μεταλλαγμένων μορφών του υποδοχέα με τον κλινικό φαινότυπο των ασθενών που φέρουν αυτές τις μεταλλάξεις. / Background: Isolated GnRH deficiency (IGD) is characterized by a functional deficit of GnRH production or secretion in the hypothalamus resulting in the loss of pulsatile secretion of GnRH and in impaired gonadotropin release, in the setting of otherwise normal anterior pituitary anatomy and function and in the absence of secondary causes of hypogonadotropic hypogonadism (HH). Kallmann syndrome (KS) is characterized by the association of IGD and anosmia, whereas patients with normal olfactory function are referred as having normosmic Idiopathic Hypogonadotropic Hypogonadism (nIHH). Objective: The objective of the study was to describe the different patients phenotypes with IGD (KS and nIHH), to identify mutations in the KAL1, FGFR1, PROK2, PROKR2 and GPR54 genes and to correlate specific phenotypes with the patients genotypes. We also studied the presence of KAL1 mutations in young children with unilateral renal agenesis/dysgenesis, in order to determine the incidence of KAL1 gene mutations in this population. In addition, we attempted to define the in vitro functionality of two FGFR1 mutants (R254W and R254Q), resulting from two different amino acid substitutions of the same residue, and to correlate the in vitro findings to the patients phenotypes. Patients: A total of 66 patients with IGD (26 with KS and 40 with nIHH) were included in this study and mutation analysis of KAL1, FGFR1, PROK2, PROKR2 and GPR54 genes was performed for this group of patients. We also studied 13 children (up to the age of 15) with unilateral renal agenesis/dysgenesis, confirmed by imaging studies. Mutation analysis of KAL1 gene was performed for the later group of patients. Methods: Gene mutation analysis methodology included DNA extraction, polymerase chain reaction amplification, and DNA sequence analysis of all exons of the KAL1, FGFR1, PROK2, PROKR2 and GPR54 genes. The in-vitro functional studies of two FGFR1 mutants (R254W and R254Q) included evaluation of the mutant signaling activity and the expression levels, which were compared to the wild type (WT) receptor signaling activity and expression. Signaling activity was determined by a FGF2/FGFR1dependent transcription reporter assay. Receptor total expression levels were assessed by Western blot assay and receptor cell surface expression was measured by radiolabelled antibody binding assay. Results: We identified 5 different mutations in KAL1 gene in three unrelated male patients with KS (Ε514Κ, Α660Τ, Ε37Κ, T235S, deletion of exons 5-10 of KAL1 gene with STS gene deletion) and one point mutation in FGFR1 gene (R254W) in a male patient with nIHH. The in-vitro functional studies of the two FGFR1 mutants (R254W and R254Q) showed that R254W maximal receptor signaling capacity was reduced by 45% (p<0.01), while maximal signaling of R254Q was also reduced (−15%) but the reduction was not statistically significant relative to WT. However, both mutants displayed diminished total protein expression levels (40% and 30% reduction relative to WT, respectively), while protein maturation was unaffected. Accordingly, cell surface expression levels of the mutant receptors were also significantly reduced (35% p<0.01 and 15% p<0.05, respectively). Sequence analysis of KAL1 gene in the group of patients with unilateral renal agenesis/dysgenesis revealed genetic defects in KAL1 gene in a 12 year old child with associated anosmia, bimanual synkinesis of upper limbs and cryptorchidism. The genetic diagnosis of Kallmann syndrome was established in this case, enabling a prompt therapeutic intervention for puberty induction at a later stage. Conclusions: Up to date very few mutations have been described in patients with IGD in the Greek population and the genetic causes of IGD still remains unclear in the majority of cases, pointing out the importance of further studies for determining the molecular pathogenesis of the disease. The phenotype of renal agenesis/dysgenesis strongly indicates the existence of KAL1 gene defects in the genotype of patients with sporadic KS, providing evidence for the X-linked mode of inheritance and offering the opportunity for genetic counseling. The detection of KAL1 gene mutations in children with unilateral renal agenesis (URA) not only confirms the involvement of anosmin-1 (the product of KAL1 gene) in kidney organogenesis, but it can also lead to an early prepubertal diagnosis of KS. Sequence analysis of KAL1 gene in patients with URA is recommended in those cases with associated clinical signs of KS (e.g. anosmia, mirror movements, cryptorchidism, microphallus) or with familial history of hypogonadism or/and anosmia. The comparative functional analysis of two FGFR1 mutations affecting the same residue (R254W, R254Q) in two unrelated patients with IGD showed that both are loss-of-function mutations and that a tryptophan substitution at R254 (R254W) is more disruptive to receptor structure than the more conserved glutamine substitution (R254Q). However, no clear correlation between the severity of in vitro loss-of-function and phenotypic presentation could be assigned.

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Das triple-negative Mammakarzinom exprimiert weder Östrogen- noch Progesteronrezeptoren und es kommt zu keiner Überexprimierung des HER2-neu Gens. Daher haben bei diesem Subtyp des Mammakarzinoms spezifische Therapien, welche gezielt an diesen Rezeptoren wirken, keinerlei Nutzen. Etwa 60% aller Mammakarzinome exprimieren GnRH-Rezeptoren, welche als Ziel genutzt werden könnten. Der GnRH-Rezeptor kann für eine gezielte Chemotherapie mit zytotoxischen GnRH-Agonisten wie AN-152, bei welchem Doxorubicin an [D-Lys6]GnRH gebunden ist, genutzt werden. In der vorliegenden Arbeit habe ich in vitro als auch in vivo analysiert ob der zytotoxische GnRH-Agonist AN-152 Apoptose in triple-negativen Mammakarzinomzellen, welche GnRH-Rezeptoren exprimieren, induziert. Die GnRH-Rezeptorexpression in Tumorbiopsien triple-negativer Mammakarzinome wurde immunhistochemisch getestet. Die Zellproliferation wurde unter Verwendung des AlamarBlue®-Proliferationsassays analysiert. Die Apoptoseinduktion wurde durch die Bestimmung des mitochondrialen Membranpotentialverlustes quantifiziert. Die in vivo Experimente wurden mit Nackmäusen nach Xenotransplantation von humanen Brustkrebszellen durchgeführt. Wir konnten zeigen das die Behandlung triple-negativer aber GnRH-positiver MDA¬ MB 231, HCC 1806 und HCC 1937 Mammakarzinomzellen mit AN-152 in vitro zum apoptotischen Zelltod durch Aktivierung der Caspase-3 führt. Diese Antitumoreffekte konnten im Nacktmausmodell bestätigt werden. AN-152 inhibierte das Wachstum triple-negativer Mammakarzinomxenotransplantate in den Nacktmäusen komplett, ohne offensichtliche Nebenwirkungen. Der zytotoxische GnRH-Agonist AN-152 scheint ein passendes Medikament mit niedriger Toxizität für eine effiziente Therapie des triple-negativen Mammakarzinoms zu sein. (Föst C, Duwe F, Hellriegel M, Schweyer S, Emons G, Gründker C (2011). Targeted chemotherapy for triple-negative breast cancers via LHRH receptor. Oncol Rep, 25, 1481-7.)

Das Mammakarzinom ist weltweit eine der häufigsten Malignomerkrankung der Frau. Im fortgeschrittenen Stadium der Erkrankung entwickeln bis zu 75 % der Patientinnen ossäre Metastasen. Basierend auf der Erkenntnis, dass GnRH-Analoga in vitro die Migration und Invasion von Mammakarzinomzellen während der Kokultur mit humanen Osteoblasten bzw. der Osteoblasten-ähnlichen Zellreihe MG-63 hemmen, sollten in dieser Arbeit die dem zugrundeliegenden molekularen Mechanismen näher betrachtet werden. Es ist bekannt, dass GnRH-Analoga in vielen verschiedenen GnRH-Rezeptor exprimierenden Tumorzelllinen die Wachstumsfaktor- und Wachstumsfaktorrezeptor-Expression beeinflussen und somit z.B. antiproliferative oder Apoptose-induzierende Effekte vermitteln. In dieser Arbeit wurde die humane Mammakarzinomzelllinie MCF-7 mit der osteoblasten-ähnlichen Zellreihe MG-63 kokultiviert. Zu bestimmten Zeitpunkten erfolgte die Behandlung der Mammakarzinomzellen mit drei verschiedenen GnRH-Analoga. Dem GnRH-I-Agonisten Triptorelin, dem GnRH-I-Antagonisten Cetrorelix und dem GnRH-II-Agonisten [D-Lysin]-GnRH-II. Neben einer nicht-kokultivierten Kontrolle jeder Zelllinie wurde eine nicht behandelte Kokulturkontrolle mitgeführt. Nach 48 h (bzw. nach 72 h od. 96 h) wurde der Versuch beendet und die mRNA-Expression von EGF, EGFR, TGF-beta und TGFBRI und -II in MCF-7 und MG-63 mittels PCR bestimmt. Zudem wurde die mRNA-Expression von EGF, EGFR und TGF-beta auch nach längerer Kokultur- und Behandlungszeit sowie unter Stressbedingungen beobachtet. Es hat sich gezeigt, dass es weder in MG-63 noch in MCF-7 signifikante Expressionsunterschiede von EGF, EGFR und TGF-beta im Vergleich zur Kokulturkontrolle gab. Dies hat sich auch weder im Zeitverlauf noch unter Stressbedingungen geändert. In MG-63 konnte man ebenfalls keine Expressionsunterschiede der TGFB-Rezeptoren beobachten. Dagegen konnte erstmals gezeigt werden, dass alle drei GnRH-Analoga die mRNA-Expression des TGFBR-I und -II während der Kokultur mit der osteoblasten-ähnlichen Zellreihe MG-63 signifikant hemmen. Während man in anderen Gewebetypen bereits eine verminderte Expression von TGFBR-I und -II nach Behandlung mit GnRH-Analoga nachweisen konnte, ist dies erstmals in einer Mammakarzinomzelllinie gelungen. Desweiteren kann man aus den vorliegenden Ergebnissen den Schluss ziehen, dass die verminderte Expression der TGFB-Rezeptoren eine Rolle in der bereits nachgewiesene GnRH-Analoga-vermittelten Migrations- und Invasionshemmung spielt.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Progestin associated to estradiol benzoate (EB) became the most used treatment for estrous induction in beef cow. However, those programs that use this association still can be enhanced to produce better pregnancy rates and easily the management. Two experiments were designed with this aim. In the first, it was investigated if progestin-estradiol association is able to induce follicular regression in different stages of ovarian follicular waves in cattle. For this, the follicular dynamics of 36 suckling cows, whose received 3 progestin treatments associated to EB before (follicles between 5-7mm), during (8-10mm) or after (>10mm) expected follicular deviation moment. Since, all cows presented regression of the largest follicles, it was concluded that independent of follicular size, cows bearing emerged growing follicles presented similar responses on follicular wave dynamics when treated with EB and different progestin protocols. The second experiment was design to compare two estrus induction protocols for cows in postpartum period, using GnRH and two-day artificial insemination (AI) or EB and fixed-time artificial insemination (TAI). A total of 250 suckled beef cows received a vaginal device containing 250mg of medroxyprogesterone acetate (MPA) and an injection of 5mg of EB intramuscularly (IM) on day 0. The vaginal device was removed on day 7. On day 6, the cows were injected with 400IU eCG (IM) and 5mg Dinoprost (into vulvar submucosa) and calves were removed for 96h. After removing the vaginal devices (day 7), the cows were divided in two groups. In BioRep group (n=150), estrus detection was carried out twice a day during 48h and the animals were inseminated 12h after detection. Cows not detected in estrus received 100μg of GnRH and were inseminated after 16 to 18h. In EB group, cows received im 1mg of EB on day 8 (24h after removing MPA) and were inseminated on day 9 without estrus detection (54h after removing MPA). The diagnosis of pregnancy was performed 40 days after AI with ultrasound. The data were analyzed by Chi-square test. The pregnancy rate was higher (p<0.01) in BioRep group (54.7%) than EB group (33.3%). To obtain high pregnancy rate, the estrus induction protocols for postpartum beef cows should use two-day AI and GnRH to induce ovulation instead of EB with TAI. / A associação de progestágenos ao benzoato de estradiol (BE) tornou-se o tratamento mais utilizado para indução de estro em vacas de corte. No entanto, os programas que utilizam esta associação hormonal ainda precisam ser aperfeiçoados, buscando aumentar as taxas de prenhez e facilitar o manejo. Dois experimentos foram delineados com este intuito: o primeiro determinou se a capacidade da associação progestágeno-estradiol em induzir a regressão folicular é dependente da fase de crescimento folicular no momento do tratamento. Para isso, avaliou-se a dinâmica folicular de 36 vacas amamentando, que receberam 3 tratamentos com progestágenos associados ao BE antes (folículos de 5-7mm), durante (8-10mm) ou depois (>10mm) do momento esperado da divergência folicular. Foi observado que todas as fêmeas tratadas apresentaram regressão dos maiores folículos, concluindo-se que independentemente do tamanho folicular, vacas com folículos em crescimento apresentam respostas semelhantes quando tratadas com progestágenos associados ao BE. O segundo experimento objetivou comparar dois protocolos de indução de estro para vacas no período pós-parto, utilizando hormônio liberador de gonadotrofinas (GnRH) e inseminação artificial (IA) em dois dias ou BE e inseminação artificial em tempo fixo (IATF). Para isso, foram utilizadas 250 vacas, amamentado, que receberam um pessário vaginal, contendo 250mg de acetato de medroxi-progesterona (MAP) e uma injeção intramuscular (IM) de 5mg de BE no dia 0 (início do tratamento). O pessário vaginal permaneceu por sete dias. No dia 6, foram aplicadas 400UI de gonadotrofina coriônica eqüina por via IM e 5mg de Dinoprost na submucosa vulvar, realizando nesse momento o início do desmame por 96 h. Após a retirada dos pessários (dia 7), as vacas foram distribuídas em dois grupos. No grupo BioRep (n=150), as fêmeas foram observadas duas vezes por dia para detecção de estro por 48h e IA 12h após sua manifestação. Os animais que não foram detectados em estro nesse período receberam uma injeção IM de 100μg de GnRH, sendo IATF, nas 16 às 18h seguintes. No grupo BE (n=100), as vacas receberam uma injeção de 1mg de BE IM no dia 8 (24h após a retirada do MAP) e foram IATF no dia 9 (54h após a retirada do MAP). O diagnóstico de gestação foi realizado por exame ultra-sonográfico 40 dias após IA. A percentagem de prenhez no grupo BioRep (54,7%) foi maior (p<0,01) do que no grupo BE (33,3%). Sendo assim, a fim de obter melhores taxas de prenhez, os protocolos de indução de estro de eleição para o período pós-parto poderão empregar observação de estro por dois dias e utilização de IATF com GnRH ao contrário de BE associado exclusivamente a IATF.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The aim of this study was to develop a timed artificial insemination system (TAI) in suckled beef cows. For this, in 227 cows 60-80 days postpartum, received estradiol benzoate (5mg) and a vaginal device containing 250mg of medroxyprogesterone acetate (MAP; day 0). On day six, prostaglandin analogous (125μg), eCG (400IU) was administered and calves were removed for 88h. The device was removed on day seven (BioRep group) or on day eight (TAI group) and the cows of both groups received GnRH (100μg; day 9) 48h or 24h after device withdrawal, respectively. Experiment I: the follicular growth was daily monitored, from day 6 to day 9 (36h after GnRH), in 14 cows. The average of dominant follicle size on day nine was 11.1±0.99mm (BioRep, n=7) and 11.5±0.65mm (TAI, n=7) and all animals ovulated. Experiment II: in the BioRep group (n=106), the cows was observed for estrus behavior after withdrawal the device, twice a day during 48h and inseminated at 12h after detection; In the TAI group (n=107), the devices were withdrawn on day eight and after 24h these cows and those from the BioRep group, which were not stand in estrus, received 100μg of GnRH and TAI 16h later. The pregnancy rates were 57.6% (BioRep) and 52.3% (TAI). The time of MAP exposure and the period from MAP to GnRH did not affect the follicular dynamic and pregnancy rates. Furthermore, the treatment for eight days allows an efficient TAI system in suckled beef cows. / O objetivo deste estudo foi desenvolver um protocolo de inseminação artificial com tempo fixo (IATF) em vacas de corte amamentando, avaliando o intervalo entre a retirada do progestágeno e a aplicação de GnRH sobre a dinâmica folicular e a prenhez. Para isto, 227 vacas 60-80 dias pós-parto receberam benzoato de estradiol (5mg) e um pessário vaginal de acetato de medroxiprogesterona (250mg MAP; dia 0). No dia seis, cloprostenol sódico (125μg), gonadotrofina coriônica eqüina (400UI) e desmame temporário (88h). O MAP foi retirado no dia sete (Grupo BioRep) ou no dia oito (Grupo IATF) e, 48h ou 24h após os animais receberam GnRH (100μg; dia 9), respectivamente. No experimento I, o monitoramento das estruturas ovarianas de 14 vacas foi realizado a cada 24h, desde o dia seis até 36h após a aplicação de GnRH em ambos os grupos. O tamanho médio do folículo dominante no dia nove foi de 11,1±0,99mm (BioRep n=7) e 11,5±0,65mm (IATF n=7) e todos os animais ovularam. No experimento II, no grupo BioRep (n=106), após a retirada do MAP, as fêmeas foram inseminadas com detecção de estro durante 48 horas. O restante dos animais do grupo BioRep e todos do grupo IATF (n=107) receberam 100μg de GnRH (dia 9) e, após 16h, IATF. Os índices de prenhez foram de 57,6% (BioRep) e de 52,3% (IATF). O intervalo de 24h entre a retirada do MAP, mantido por 8 dias, e a aplicação de GnRH não interfere na dinâmica folicular e prenhez, viabilizando inseminar vacas de corte amamentando sem observação de estro.

The study summarizes methods of artificiale and semiartificiale reproduction of pikeperch. The aim of the present study is to study ovulation rate by injection of different hormones: carp pituitary, analogs of GnRH Supergestran, Ovopel (containing dopamine inhibitor), Dagin (containing too dopamine inhibitor) in zander (Sander lucioperca) and determination of stripped eggs to broodstock weight, relative and absolute fertility and period of latency. On the basis of results, zander can be considered to be usefull species for semiartificiale and artificiale propagation.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The objective of this study was to investigate the role of GnRH on the preimplantation development of mouse embryos in vitro. GnRH-I, GnRH-II, and GnRH agonists: Des-Gly, Des-Trp and histrelin did not improve embryo development. However, treatment with the specific GnRH antagonist SB-75 blocked embryo development at morula stage. The inhibition of embryo development by SB-75 could be rescued by the addition of histrelin. To determine which intracellular signaling cascade is involved following binding of GnRH to the GnRHR, embryos were cultured in the presence of specific PKC (GFX) or PKA (SQ22536) inhibitors. The PKC inhibitor blocked embryo development at a similar stage as SB-75, whereas SQ22536 had an inhibitory effect, diminishing blastocyst formation and hatched rates. There are evidences that GnRH has an essential autocrine effect on mouse embryonic development via GnRHR, probably by activating PKC signaling cascade while the inhibition of the GnRH signaling does not activate apoptotic mechanisms involving caspase-3. In another experiment, development in vitro of embryos from Chinese Meishan (M) and occidental white crossbred (WC) females were investigated after improving the vitrification protocol for pig embryos. Efficient cryopreservation of zona pellucida-intact porcine embryos and studies of the difference among breeds could greatly impact the swine industry. The percentage of embryos surviving 24 h after cryopreservation without lysis or degeneration was higher for M (72%) than WC (44%). However, in vitro development of embryos that survived cryopreservation was not different between M and WC at the expanded (64%) or hatched (22%) blastocyst stages. Developmental rates were significantly higher for control embryos than frozen embryos from both breeds at expanded blastocyst stage, but not at hatched blastocyst stage. Rates of expanded blastocyst formation did not differ between M and WC control embryos (98 and 95%, respectively). With a new procedure to warm vitrified pig embryos, the survival rates may be improved. The optimal stages to vitrify pig embryos using the microdroplet method ranges from late compact morula to early expanded blastocyst. The results suggest that M embryos have a higher capacity to survive the vitrification process than WC embryos. / O objetivo do presente estudo foi investigar a importância do GnRH no desenvolvimento embrionário precoce em camundongos. GnRH-I, GnRH-II e os GnRH agonistas: Des-Gly, Des-Trp e histrelina não incrementaram o desenvolvimento embrionário. Entretanto, o tratamento com SB-75, um antagonista específico do GnRH, bloqueou o desenvolvimento embrionário no estádio de mórula. A inibição do desenvolvimento embrionário pelo SB-75 pôde ser revertida com a adição de histrelina. Para determinar a cascata do sinal intracelular desencadeada pela ligação do GnRH com o seu receptor, embriões foram cultivados na presença de inibidores específicos da PKC (GFX) e da PKA (SQ22536). O inibidor da PKC bloqueou o desenvolvimento embrionário em estádio similar ao bloqueio mediado pelo SB-75, enquanto o SQ22536 teve efeito inibitório diminuindo a formação de blastocisto e taxas de eclosão. Os resultados sugerem que o GnRH tem um efeito autócrino essencial no desenvolvimento embrionário através do GnRHR, provavelmente, ativando a cascata da PKC. Por outro lado, a inibição do sinal do GnRH não ativa mecanismos apoptóticos que involvam caspase-3. Em outro experimento, foi investigado o desenvolvimento in vitro de embriões da raça Meishan (M) e branco cruzado (WC) após vitrificação pelo método microgota. O desenvolvimento de protocolos eficientes para criopreservação de embriões suínos com a zona pelúcida intacta e a avaliação das diferenças entre raças pode ter um significativo impacto na suinocultura. A percentagem de embriões que sobreviveram à criopreservação depois de 24 h foi maior na M (72%) do que na WC (44%). No entanto, o desenvolvimento in vitro dos embriões que sobreviveram à criopreservação não foi diferente entre M e WC nos estádios de blastocisto expandido (64%) ou eclodido (22%). Os índices de desenvolvimento foram significativamente mais altos para os embriões controle do que para os embriões vitrificados nas duas raças no estádio de blastocisto expandido, porém não foram diferentes para o estádio de blastocisto eclodido. A formação de blastocisto expandido não diferiu entre os embriões controle M e WC (98 e 95%, respectivamente). Com o novo procedimento ( hot warm ) para descongelar embriões vitrificados pelo método de microgota, pode-se aumentar dos índices de sobrevivência. Os melhores estádios embrionários para a vitrificação de embriões suínos variam de mórula compacta tardia até blastocisto expandido inicial. Os resultados sugerem que embriões M têm mais capacidade de sobreviver ao processo de vitrificação do que embriões WC.

Made available in DSpace on 2016-01-26T18:55:29Z (GMT). No. of bitstreams: 1 Dissertacao.pdf: 349806 bytes, checksum: 1fd0f5db382ba931252ba16e3de9a860 (MD5) Previous issue date: 2010-03-03 / The technique of timed artificial insemination (TAI) can be used as a tool to optimize reproductive efficiency. Among the alternative protocols hormone, prostaglandin (PGF2&#945;) and estradiol benzoate (EB) have been used frequently, or combined with progesterone and GnRH analogues. This work aimed to evaluate the influence of follicular diameter on the pregnancy rate using BE or GnRH on the placement of the implant of progesterone (D0) in beef cows divided into two groups: G-BE (n = 32) and G-GnRH (n = 29). The D0 was placed implant P4 (CIDR ®) and applied 2 ml of BE (G-BE) or 2.5 mL of GnRH (GnRH-G). In D9 the implant was removed, concomitant administration of 2.5 mL of 0.25 mL PGF2&#945; and estradiol cypionate (ECP ®) followed by removal of calves. After 48 h all the cows were inseminated and the calves returned. At D0 and D9 was held ultrasound to measure the dominant follicle (DF) present in the ovary. There was no difference (p&gt; 0.05) in pregnancy rate between treatments, BE (55%) and GnRH (41%), but the follicular diameter was significantly higher (p &lt;0.05) in pregnant cows treated with EB (10.7 mm vs. 8.5 mm) and in cows treated with GnRH there was no difference (p&gt; 0.05) between pregnant and empty (11.6 mm vs. 10.2 mm). We also evaluate the postpartum period (&gt; or &lt;45 days), the application of eCG or temporary removal of the calves (RTB) and the influence of genetic group on TAI. We used the 678 cows being: Nelore (n = 234), Nelore, Brahman (n = 159) and ¾ Nellore-Red Angus (n = 285) divided into G-Early (GP, n = 151) and G-Late (GT, n = 527). Again divided into GP-RTB (n = 93) and GT-RTB (n = 299), GP-eCG (n = 58) and GT-eCG (n = 228). The animals received CIDR ® + 2 mL of BE (D0). In D8, the device was removed and all groups received 2.5 mL PGF2&#945; &#61472;, and 1.5 mL eCG (GP-GT-eCG and eCG) or removal of calves (GP-GT-RTB and RTB). In D9 the animals received 1 mL BE and 24 hours after held IATF. The pregnancy rate did not change (p&gt; 0.05), and 40% (GP) and 48% (GT). Also did not change (p&gt; 0.05) in groups: GP-eCG (37.9%), GP-RTB (41.9%), GT-eCG (51.7%) and GT-RTB (45.1 %). The pregnancy rate in Nelore cows - Brahman (37%) was lower (p &lt;0.05) than the Nellore (52%) and ¾ Nellore Red - Angus (45%). It was concluded that the use of GnRH in D0 does not improve pregnancy rate in cows in the postpartum period, even increasing follicular diameter, which females with less than 45 days postpartum are able to IATF, regardless of the use of eCG or RTB and race influence of mothers on pregnancy rate. / A técnica da inseminação artificial em tempo fixo (IATF) pode ser utilizada como ferramenta para otimização da eficiência reprodutiva. Entre as alternativas de protocolos hormonais, a prostaglandina F2&#945; (PGF2&#945;), seus análogos e o benzoato de estradiol (BE) têm sido utilizados com freqüência, combinados à progesterona (P4) ou análogos e GnRH ou análogos. Neste trabalho objetivou-se avaliar a influência do diâmetro folicular sobre a taxa de prenhez, utilizando BE ou GnRH no dia da colocação do implante de progesterona (D0) em vacas de corte divididas em dois grupos: G-BE (n=32) e G-GnRH (n=29). No D0 foi colocado um dispositivo intravaginal de P4 (CIDR®) e aplicado 2mL de BE (G-BE) ou 2,5 mL de GnRH (G-GnRH). No D9 foi retirado o implante, concomitante à administração de 2,5 mL de PGF2&#945; e 0,25 mL de cipionato de estradiol (E.C.P.®) seguido de remoção dos bezerros. Após 48h todas as vacas foram inseminadas e os bezerros retornados. No D0 e D9 foi realizada ultrassonografia para medir o folículo dominante (FD) presente no ovário. Não houve diferença (p>0,05) na taxa de prenhez entre os tratamentos, BE (55%) e GnRH (41%). O diâmetro folicular foi significativamente maior (p<0,05) nas vacas prenhes tratadas com BE (10,7mm vs 8,5mm) e nas vacas tratadas com GnRH não houve diferença (p>0,05) entre as prenhes e vazias (11,6mm vs 10,2mm). Em outro experimento, avaliou-se o período pós-parto (> ou <45 dias), a aplicação de eCG ou remoção temporária dos bezerros (RTB) e influência do grupo genético na IATF. Utilizou-se 678 vacas sendo: Nelore (n=234), ½ Nelore-Brahman (n=159) e ¾ Nelore-Red Angus (n=285) divididas em G-Precoce (G-P, n=151) e G-Tardio (G-T, n=527). Novamente divididas em G-P-RTB (n=93) e G-T-RTB (n=299); G-P-eCG (n=58) e G-T-eCG (n=228). Os animais receberam CIDR® + 2 mL de BE (D0). No D8, o dispositivo foi retirado e todos os grupos receberam 2,5 mL PGF2&#945;&#61472;, e 1,5 mL eCG (G-P-eCG e G-T-eCG) ou remoção dos bezerros (G-P-RTB e G-T-RTB). No D9 os animais receberam 1 mL BE e 24h após realizou-se IATF. A taxa de prenhez não variou (p>0,05), sendo 40% (G-P) e 48% (G-T). Também não variou (p>0,05) nos grupos: G-P-eCG (37,9%), G-P-RTB (41,9%), G-T-eCG (51,7%) e G-T-RTB (45,1%). A taxa de prenhez nas vacas ½ Nelore Brahman (37%) foi inferior (p<0,05) que os da raça Nelore (52%) e ¾ Nelore Red Angus (45%). Foi concluído que o uso de GnRH no D0 não melhorou a taxa de prenhez em vacas no pós-parto, mesmo aumentando o diâmetro folicular, que fêmeas com menos de 45 dias pós-parto estão aptas para IATF, independente do uso de eCG ou RTB e a raça das matrizes influencia nas taxas de prenhez, sendo a raça Nelore a que apresentou a maior taxa.