PC4/PCSK4 is the major Proprotein Convertase (PC) enzyme that plays a key role in mammalian fertilisation. It is detected in the acrosomal granules of round spermatids, acrosomal ridges of elongated spermatids and sperm plasma membrane overlying the acrosome with K-X-K/X-R as its preferred cleavage motif. Such motifs are present in male germ cell proteins ADAMs, proPACAP and proIGF-1/2 and these precursor proteins are processed most likely by PC4 during spermatogenesis, sperm maturation and sperm-egg interaction. For fertilization to occur, the mature sperm must penetrate the Zona Pelucida (ZP) and bind to the egg. Previously, PC4 null mouse sperm and wild type sperm treated with a specific PC4-inhibitor have shown to reduced abilities to penetrate the cumulus mass, bind to ZP and fertilize eggs. These findings suggest that sperm-PC4 plays an important role in fertilization and hence regulation of its activity is crucial for successful fertilization. But how PC4 activity is regulated in vivo is not yet clear. Recently, in epididymal fluid a serpin (serine protease inhibitor) called CRES has been described but the protease linked to this serpin in epididymis has not been identified. However in endocrine cells where CRES is also expressed, it inhibits PC2 enzyme. Thus based on localization and preliminary study, we propose that PC4 is the target enzyme for CRES in the reproductive tract. During sperm migration and storage in epididymis, sperm PC4 activity may be modulated by CRES so that premature sperm activation may not occur. Our data showed that CRES inhibits PC4 both in vitro (with IC50 in µM range) as well as ex vivo in human placenta trophoblast cell lines. Moreover CRES was found to be cleaved by PC4 suggesting a Serpin-Protease binding type of mechanism in the inhibition of protease activity. Taken together, we conclude that CRES regulates PC4 activity in reproductive tract crucial for mammalian fertilization.

Beyond the haploid genome, mammalian sperm carry a payload of epigenetic information with the potential to modulate offspring phenotype. Morphologically mature sperm exit the testes, but cannot swim or interact with the oocyte without extensive remodeling during epididymal transit; this includes modifications to the lipid composition of the sperm membrane, gain of necessary proteins, and a dramatic shift in sperm RNA content. Epididymal maturation has also been linked to changes in the sperm methylome suggesting that the epididymis might play a broader role in shaping the sperm epigenome. First, we characterized the genome-wide methylation landscape in seven germ cell populations from throughout the male reproductive tract. Our data emphasize the stability of cytosine methylation in mammalian sperm, and identify a surprising, albeit transient, period during which sperm are associated with extracellular DNA. Second, given our interest in the small RNA repertoire of sperm we set out to address known bias in sequencing protocols by comparing several small RNA cloning protocols. We found a protocol recently developed by Kathleen Collins’ lab (OTTR) to be superior to commercially available kits in providing an accurate representation of tRNA fragment levels as compared to Northern blotting. These results not only provide a more accurate representation of tRNA fragments, but also more complexity than previously seen allowing us to reassess the true sperm small RNA content. Taken together, these results provide significant insight into the mechanisms and factors modulating sperm epigenomics during post-testicular sperm maturation.

Cocaine- and amphetamine-regulated transcript (CART) is a novel family of peptides, of which CART peptide fragments 55-102 and 62-102 are reported to be the endogenous, physiologically active peptides. Immunohistochemical studies with an antiserum directed against the CART peptide fragment 55-102 revealed CART-like immunoreactive (CART-LI) nerve fibers in the rat epididymis. The number was highest in the cauda epididymis and became progressively fewer toward the caput epididymis; the vas deferens exhibited an abundance of CART-LI fibers. Injection of the retrograde tracer Fluorogold (Fluorochrome, Inc., Englewood, CO) to the junction between the vas deferens and cauda epididymis labeled a large number of neurons in the major pelvic ganglion, some of which were CART-positive. Double-labeling the ganglion sections with tyrosine hydroxylase (TH) and CART antisera revealed that CART-LI and TH-LI were expressed in two distinct populations of ganglion cells. Some of the TH-LI cells in the ganglia, however, were covered with web-like CART-LI endings. The effects of CART peptide 55-102, referred to herein as CART, on anion secretion in the form of short circuit currents (Isc) were assessed in cultured epithelia. The CART (1 to 5 μM) applied to the basolateral or apical side of the cultured epithelia caused no significant responses on Isc, whereas lys-bradykinin (1 μM) produced a large Isc response in the same preparations. Our results show that CART-LI is present in a population of rat pelvic ganglion cells, which may give rise to CART-LI nerve fibers as observed in the vas deferens and the epididymis. The biological function of CART in the rat epididymis is not known, but it apparently is not involved in ion secretion across the epithelium.

Les récepteurs couplés aux protéines G (RCPG) sont impliqués dans la majeure partie des communications intercellulaires. Un grand nombre de RCPG ont été découverts en comparant la séquence des récepteurs connus avec les données fournies par le séquençage du génome humain. Pour plus d'une centaine de ces récepteurs, le ligand activateur ou agoniste est inconnu. Ces récepteurs sont dès lors qualifiés d'orphelins. Les LGR forment une sous-famille de RCPG structurellement proches de la rhodopsine qui comprend les récepteurs aux hormones glycoprotéiques (TSH, LH, hCG, FSH) et à la relaxine. LGR4 est un membre de cette famille dont ni la fonction précise, ni l'agoniste ne sont connus. Dans un premier temps, une cartographie détaillée de l'expression de Lgr4 chez la souris a été obtenue. Nous avons tiré parti de l'existence d'une lignée de souris transgéniques dont le gène Lgr4 a été interrompu par l'introduction d'une cassette comportant deux marqueurs histologiques. L'activité beta-galactosidase d'un de ces marqueurs a été analysée chez les souris hétérozygotes. Ces dernières ne présentent pas de phénotype particulier, ce qui permet d'estimer que l'expression des marqueurs rend effectivement compte de l'expression normale du gène Lgr4. Lgr4 est exprimé dans un grand nombre de structures, notamment dans le cartilage, le rein, les appareils reproducteurs mâle et femelle et certaines cellules du système nerveux. Ensuite, le phénotype des souris homozygotes pour l'inactivation de Lgr4 (LGR4KO) a été exploré. Ces souris présentent à la naissance un poids inférieur à leurs congénères des autres phénotypes. Les mâles sont stériles à cause d'une malformation des tubules efférents et de l'épididyme. Un blocage au niveau des tubules efférents reliant le testicule à l'épididyme contraint les spermatozoïdes à s'accumuler à la sortie du testicule, dans la région du rete testis. De plus, les tubes de l'épididyme, pourtant normaux à la naissance, ne s'allongent pas pour former la structure convolutée habituelle. L'épithélium de ces tubes est aplati et est entouré d'une quantité anormalement élevée de mésenchyme. Dans un troisième temps, des outils nécessaires aux futures tentatives d'identification de l'agoniste naturel de LGR4 ont été réalisés. Il s'agit : (1) d'anticorps monoclonaux dirigés contre la partie extracellulaire du récepteur humain. (2) d'un appât moléculaire pour la ‘pêche au ligand’. Cet appât est constitué du domaine extracellulaire du récepteur humain couplé à un marqueur histologique. (3) d'une construction peptidique constituée du domaine extracellulaire du récepteur humain couplé à une queue poly-histidine. Cette construction est destinée à servir de greffon lors de chromatographies d'affinités devant permettre de purifier le ligand. (4) de lignées cellulaires exprimant le récepteur LGR4 humain ainsi que le système æquorine devant permettre de détecter l'activation de ce récepteur. Les données apportées par ce travail montrent un rôle important du récepteur LGR4 au cours du développement et permettent de circonscrire le champ des recherches futures. Ceci, ainsi que les outils moléculaires développés, constitue une base pour l'identification future de l'agoniste et la détermination précise de la fonction de LGR4.

A hipótese desta pesquisa foi que a maturação epididimária dos espermatozoides caninos envolve modificações nas proteínas e ácidos graxos do fluido sintetizado pelos distintos segmentos epididimários, além de alterações no perfil de ácidos graxos da membrana plasmática, organização celular e morfológica dos espermatozoides. Para tanto, este projeto foi realizado com 20 cães em idade reprodutiva e negativos para Brucella canis. Após a orquiectomia bilateral, os testículos e epidídimos foram acondicionados a 5°C por no máximo 24 horas, em seguida, as amostras foram colhidas por incisões na cauda, corpo e cabeça do epidídimo. As amostras foram imediatamente processadas por avaliação subjetiva e automática da motilidade e vigor espermático, concentração, morfologia espermática, integridade da membrana plasmática, acrossomal e atividade mitocondrial. Foi possível observar determinar maior número de espermatozoides dentro da normalidade na cauda do epidídimo, especialmente, referindo-se à motilidade, integridade de membrana e atividade mitocondrial. A avaliação das modificações ultraestruturais dos espermatozoides permitiu observar a migração da gota citoplasmática e alterações acrossomais. No perfil de ácidos graxos observaram-se variações na quantidade e presença dos ácidos durante o traje epididimário, destacando o acréscimo do DHA na região da cauda epididimária. Ainda, no perfil protéico do plasma epididimário canino, foi possível identificar um padrão regional de secreção de proteínas, maior nas regiões da cabeça e corpo em relação à cauda do epidídimo. Apesar das importantes informações geradas a partir do presente trabalho, mais estudos são necessários para a compreensão da fisiologia reprodutiva, especialmente da maturação espermática epididimária na espécie canina. / The hypothesis of this research is that the canine maturation of epididymal spermatozoa involves progressive changes in the protein and fatty acid content of the fluid synthesized by the different epididymal segments, as well as changes in the fatty acid profile of the sperm plasma membrane, cell organization and morphology of the canine sperm. Therefore, this experiment was conducted with 20 dogs in reproductive age and free of Brucella canis. After bilateral orchiectomy, testes and epididymis were stored at 5°C for up to 24 hours and then the samples were harvested through incisions of the tail, corpus and caput of the epididymis. The samples were immediately processed for subjective and automatic motility and sperm vigor, sperm count, morphology, plasma membrane integrity, acrosomal and mitochondrial activity. It was possible to observe a greater number of mature sperm in the epididymal tail compared to the corpus and caput, especially referring to motility, membrane integrity and mitochondrial activity. The evaluation of ultrastructural changes of the spermatozoa allowed to observed the migration of the cytoplasmic droplets and acrosomal changes. In the fatty acid profile was observed variations in the amount and presence of acids during epididymal maturation, highlighting the addition of DHA in the epididymal tail region. Moreover, in the protein profile of the canine epididymal plasma, it was possible to identify a regional pattern of protein secretion, higher in the caput and corpus in relation to the tail epididymis. In spite of the important data generated from this study, further studies are needed for understand the reproductive physiology, especially the epididymal sperm maturation in dogs.

A ema (Rhea americana americana) é uma ave que pertence ao grupo das Ratitas, ordem Rheiforme e a família Rheidae. Neste trabalho foram analisadas as características morfológicas, macroscópicas e microscópicas, dos órgãos do aparelho reprodutor masculino (testículos, epidídimos, ductos deferentes e falo) e a cloaca em 23 emas, quatro filhotes com duas semanas, sete jovens (de três a oito meses) e doze adultas (três anos), provenientes do abatedouro da Cooperativa Emas do Brasil, RS e do CEMAS, Mossoró, RN. Fragmentos de cada órgão foram fixados por imersão em formoldeído 10%, tampão fosfato pH 7,4, 0,1M ou em solução de Karnovsky (paraformoldeído 4% e glutaraldeído 2,5%, tampão fosfato pH 7,4, 0,1M) e processados na rotina para microscopia de luz e eletrônica de varredura, respectivamente. Os testículos da ema possuem formato alongado e localizam-se na cavidade celomática, na região intra-abdominal dorsal, com comprimento e largura médias de 7,6 ± 1,2 cm e 2,6 ± 0,7 cm nos adultos; 4,5 ± 1,5 cm e 0,9 ± 0,4 cm nos jovens; e 0,8 ± 0,3 cm, e 0,2 ± 0,1 cm nos filhotes. O testículo está envolto pela túnica albugínea e seu parênquima possui túbulos seminíferos, compostos por epitélio espermatogênico e por células de sustentação, e pelo tecido intersticial, com as células endócrinas intersticiais, tecido conjuntivo frouxo e vasos. Nos adultos observaram-se todas as células da linhagem espermatogênica, enquanto nos jovens com 3 meses os testículos apresentaram túbulos seminíferos com luz reduzidas, espermatogônia e células de sustentação indiferenciadas. O epidídimo apresentou-se alongado e fusiforme junto a margem medial do testículo. Os ductos eferentes possuem um epitélio pseudoestratificado colunar ciliado baixo, enquanto no ducto epididimário o epitélio é alto. O ducto deferentes apresentou trajeto sinuoso nos adultos, retilíneo nos jovens, convoluto na sua porção média, diminuindo seu formato sigmóide em sua porção caudal, próximo à cloaca. O epitélio é pseudoestratificado reveste a luz irregular nos adultos, e circular nos jovens, mantendo proximidade com o ureter. A cloaca dividiu-se em três segmentos: o coprodeu, o urodeo e o proctodeo. No urodeu os ductos deferentes desembocaram em papilas na parede ventro-lateral, próximo a inserção do falo fibroso. O falo é um órgão fibroso linfático, localizado na parede ventral, no assoalho da cloaca, e apresentou duas porções: uma rígida bifurcada e contorcida, e outra simples espiralada e flexível, a qual normalmente esteve invertida. De forma geral os órgãos reprodutores das emas compartilharam da morfologia de outras aves, principalmente aquelas descritas para os avestruzes. / The ema (Rhea american american) is a bird that belongs to the group of the Ratitas, order Rheiforme and family Rheidae. At this study were analyzed the morphological characteristics, macroscopic and microscopic, of the male genital organ (testes, epididymis, deferent ducts, and fhallus) and the cloaca in 23 emas, four chicken (two weeks old), young witer three to ten months old and twelve adult animals (three years old), originating from Cooperativa Emas do Brasil, RS and also from CEMAS, Mossoró, RN. Slices from each organ were fixed by immersion in phorformaldehyde 10%, or in Karnovsky solution (phorformaldehyde 10%, buffer phosphate pH 7.4, 0.1M and glutaraldehyde 2.5% buffer phosphate pH 7.4, 0.1M) and processed for light and scanning microscopy, respectively. The testis of rhea had elongated shape and were located inside coelomatic cavity, in dorsal region of abdominal cavity, with medium length and width of 7.6 ± 1.2 cm and 2.6 ± 0.7 cm at adult animals; 4.5 ± 1.5 cm and 0.9 ± 0.4 cm at young animals; and 0.8 ± 0.3 cm, and 0.2 ±0.1 cm at chicken. The testis were recovered by the tunica albuginea and its parenchyma had seminiferous tubules composed by spermatogenic epithelium and by sustentation cells, and also interstitial tissue, with interstitial endocrine cells, connective tissue and vessels. At the adult animals were observed all the cells from spermatogenic lineage, whilst at the youngs with 3 months the seminiferous tubules had a smale lumen with spermatogonia and undifferentiated sustentacular cells. The epididymis was elongated and fusiform closely to medial testis board. The efferents ductus were composed by a low ciliated pseudoestratified epithelium, while the epididimydis duct had a light epithelium. The deferent duct had sinuous stretch at adult animals, rectilineae at young animal, convolute at its medium portion, decreasing its sigmoid shape at caudal portion, next to cloaca. The epithelium was pseudostratified ciliated, irregular lumen at adult animal, and circular at young animal, closely with urether. The cloaca was divided into three segments: coprodeum, urodeum and proctodeum. At urodeum the deferent ducts discharged into papillas at the ventral side wall, next to fibrous phallus`s insertion. The phallus was a lymphatic fibrous organ, located at ventral wall, at the cloaca floor, and was composed by two portions: one rigid forked and twisted, and another simple spiraled and flexible, which normally was inverted. In a general way the Rhea genital organs shared the morphology from others birds, mainly those described to the ostrich.

Afin de mettre en évidence les principaux éléments de la voie androgénique et œstrogénique dans l’épididyme du rat des sables adulte, capturé dans la région de Beni Abbès, en Algérie, l’aromatase, l’œstradiol, les récepteurs des androgènes (RA) et des œstrogènes (REα, REβ, GPR30) ont été recherchés chez des animaux en saison d’activité, en saison de repos sexuel, chez des animaux castrés, castrés puis traités par la testostérone et chez des animaux ayant subi la ligature des canaux efférents. En saison d’activité, les RA sont ubiquitaires, l’aromatase est cytoplasmique par contre l’œstradiol est nucléaire et cytoplasmique. Les REα et le GPR30 sont principalement dans le cytoplasme apical par contre les REβ sont nucléaires. En saison de repos sexuel, les RA, l’aromatase, l’œstradiol, les REα et le GPR30 persistent, cependant, les REβ subissent une translocation cytoplasmique. Chez les animaux castrés, les RA, l’aromatase et l’œstradiol sont réduits par contre les REα persistent avec une faible intensité. Le GPR30 est cytoplasmique et nucléaire. Chez les animaux castrés puis traités, les RA, l’aromatase, l’œstradiol, les REα, les REβ et le GPR30 sont restaurés. Chez les animaux ligaturés, le RA est faiblement conservé uniquement dans l’épididyme proximal. L’aromatase et l’œstradiol sont conservés. Le signal des REα, des REβ et du GPR30 est fortement exprimé dans le noyau et le cytoplasme dans l’épididyme proximal par contre il est fortement exprimé uniquement pour les REα dans l’épididyme distal. Par western blot, les RA, REα, REβ et GPR30 sont de 122, 64, 55 et 55 kDa respectivement. / In order to highlight the main elements of androgen and estrogen pathway in the epididymis of sand rat, captured in Beni Abbès area, in Algeria, androgen receptor (AR), aromatase, estradiol, estrogen receptors (ERα, ERβ and GPR30) were explored in breeding season, in resting season and in animals underwent castration, castration then testosterone treatment and ligation of efferent ducts. In breeding season, AR has a ubiquitous distribution, aromatase is exclusively cytoplasmic and estradiol is nuclear and cytoplasmic. The ERα and GPR30 were distributed with a high intensity in the apical cytoplasm contrarily to ERβ which were nuclear. In resting season, AR, aromatase, estradiol, ERα persist with lower staining. However, ERβ undergo cytoplasmic translocation and GPR30 persist in cytoplasm. In castrated animals, AR, aromatase and estradiol are reduced. ERα persist with low intensity in the apical cytoplasm. GPR30 is distributed in the cytoplasm and the nucleus. In castrated then treated animals, AR is restored; aromatase and estradiol reappear with a cytoplasmic localization for aromatase, nuclear and apical for ERα. ERβ and GPR30 are restored and have a cytoplasmic localization. In ligatured, RA is preserved in the caput, aromatase and estradiol persist caput and cauda. The signal of ERα, ERβ and GPR30 is highly expressed in the nucleus and cytoplasm of caput epididymis and highly expressed of ERα exclusively in cauda. By Western blot, RA, ERα, ERβ and GPR30 are found with molecular weights of 122, 64, 55 and 55 kDa respectively.

Lun Samantha Wei Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 142-174). / Abstracts in English and Chinese. / Contents --- p.ii / Acknowledgements --- p.vii / Abstract --- p.viii / 摘要 --- p.xi / Chapter Section 1. --- Introduction / Chapter 1.1 --- General review of the epididymis --- p.1 / Chapter 1.1.1 --- Structure of the epididymis --- p.1 / Chapter 1.1.2 --- Function of the epididymis --- p.3 / Chapter 1.1.3 --- Regulation of the epididymal function --- p.5 / Chapter 1.2 --- Vascular endothelial growth factor (VEGF) --- p.8 / Chapter 1.2.1 --- VEGF peptides --- p.8 / Chapter 1.2.2 --- Biological activities of VEGF --- p.10 / Chapter 1.2.3 --- Hormonal regulation of VEGF --- p.11 / Chapter 1.3 --- VEGF receptors --- p.12 / Chapter 1.3.1 --- Flt-1 or VEGFR1 --- p.12 / Chapter 1.3.2 --- Flk-1 or VEGFR2 --- p.13 / Chapter 1.4 --- Caveolae --- p.15 / Chapter 1.4.1 --- Overview of caveolae --- p.15 / Chapter 1.4.2 --- Caveolins/caveolin-1 --- p.16 / Chapter 1.4.3 --- Caveolae and VEGF --- p.18 / Chapter 1.4.4 --- Caveolae and the epididymis --- p.20 / Chapter 1.5 --- VEGF/ VEGF receptors in the epididymis --- p.20 / Chapter 1.6 --- Aims of study --- p.22 / Chapter Section 2. --- Materials and Methods / Chapter 2.1 --- Materials --- p.24 / Chapter 2.2 --- Animal surgery --- p.35 / Chapter 2.2.1 --- Animals --- p.35 / Chapter 2.2.2 --- Castration and hemi-castration --- p.35 / Chapter 2.2.3 --- Efferent duct ligation (EDL) --- p.36 / Chapter 2.2.4 --- Tissue collection --- p.37 / Chapter 2.3 --- Epididymal cell culture --- p.38 / Chapter 2.4 --- Sample preparation --- p.40 / Chapter 2.4.1 --- Collection of epididymal plasma and sperm --- p.40 / Chapter 2.4.2 --- Purification of caveolae fraction --- p.41 / Chapter 2.5 --- Reverse-transcription polymerase chain reaction (RT-PCR) and semi-quantitative RT-PCR --- p.43 / Chapter 2.5.1 --- Preparation of RNA from epididymal tissues --- p.43 / Chapter 2.5.2 --- Quantitation of total RNA --- p.44 / Chapter 2.5.3 --- Reverse transcription (RT) and polymerase chain reaction (PCR) --- p.44 / Chapter 2.5.4 --- Purification and authenticity confirmation of PCR products --- p.50 / Chapter 2.6 --- Western immunoblotting --- p.53 / Chapter 2.6.1 --- Preparation of protein --- p.53 / Chapter 2.6.2 --- SDS-PAGE --- p.53 / Chapter 2.6.3 --- Western immunoblotting --- p.55 / Chapter 2.7 --- Immunohistochemistry --- p.56 / Chapter 2.7.1 --- Preparation of tissue sections --- p.56 / Chapter 2.7.2 --- Immunohistochemical staining of tissue sections --- p.57 / Chapter 2.7.3 --- Immunostaining of cultured cells --- p.59 / Chapter 2.8 --- Enzyme Linked Immunosorbant Assay (ELISA) --- p.59 / Chapter 2.9 --- Statistical analyses --- p.60 / Chapter Section 3. --- Results / Chapter 3.1 --- Expression and localization of VEGF in the rat epididymis --- p.62 / Chapter 3.1.1 --- RT-PCR of VEGF in the rat epididymis --- p.62 / Chapter 3.1.2 --- Western immunoblot of VEGF in the rat epididymis --- p.63 / Chapter 3.1.3 --- Developmental changes in VEGF expression in the rat epididymis --- p.66 / Chapter 3.1.4 --- Immunolocalization of VEGF in the rat epididymis --- p.66 / Chapter 3.1.5 --- Summary of the localization and expression of VEGF in the rat epididymis --- p.72 / Chapter 3.2 --- Expression and localization of VEGF receptors in the rat epididymis --- p.74 / Chapter 3.2.1 --- RT-PCR of Flt-1 and sFlt-1 in the rat epididymis --- p.74 / Chapter 3.2.2 --- Western immunoblot of Flt-1 in the rat epididymis --- p.74 / Chapter 3.2.3 --- Immunolocalization of Flt-1 in the rat epididymis --- p.75 / Chapter 3.2.4 --- RT-PCR of Flk-1 in the rat epididymis --- p.75 / Chapter 3.2.5 --- Western immunoblot of Flk-1 in the rat epididymis --- p.79 / Chapter 3.2.6 --- Immunolocalization of Flk-1 in the rat epididymis --- p.79 / Chapter 3.2.7 --- Summary on the localization and expression of VEGF receptors in the rat epididymis --- p.83 / Chapter 3.3 --- Detection of VEGF immunoreactivity in epididymal plasma and sperm lysate collected from cauda epididymidis --- p.83 / Chapter 3.4 --- Effect of castration on VEGF and VEGF receptor expression in the rat epididymis --- p.84 / Chapter 3.4.1 --- Effect of castration with or without testosterone replacement on VEGF expression in the rat epididymis --- p.84 / Chapter 3.4.2 --- Effect of castration with or without testosterone replacement on Flt-1 expression in the rat epididymis --- p.94 / Chapter 3.4.3 --- Effect of castration with or without testosterone replacement on Flk-1 expression in the rat epididymis --- p.98 / Chapter 3.5 --- Effect of efferent duct ligation and hemicastration on VEGF peptide levels in the rat epididymis --- p.102 / Chapter 3.5.1 --- Effect of efferent duct ligation on VEGF expression in the rat epididymis --- p.102 / Chapter 3.5.2 --- Effect of hemi-castration on VEGF expression in the rat epididymis --- p.106 / Chapter 3.6 --- "Summary on the effects of castration, efferent duct ligation, and hemicastration on the epididymal weight, and VEGF/VEGF receptor expression in the rat epididymis" --- p.107 / Chapter 3.6.1 --- "Summary on the effects of castration, efferent duct ligation and hemicastration on the epididymal weight" --- p.107 / Chapter 3.6.2 --- "Summary on the effects of castration, efferent duct ligation and hemicastration on VEGF and VEGF receptor expression in the rat epididymis" --- p.112 / Chapter 3.7 --- Localization and expression of caveolin-1 and 226}0ؤ2 in the rat epididymis --- p.113 / Chapter 3.7.1 --- RT-PCR of caveolin-1 and caveolin-2 in the rat epididymis --- p.113 / Chapter 3.7.2 --- Western immunoblot of caveolin-1 and caveolin-2 in the rat epididymis --- p.114 / Chapter 3.7.3 --- Immunolocalization of caveolin-1 in the rat epididymis --- p.117 / Chapter 3.7.4 --- Summary on the localization and expression of caveolin-1 and -2 in the rat epididymis --- p.119 / Chapter 3.8 --- Co-localization of VEGF receptors with caveolae in the rat epididymis --- p.119 / Chapter Section 4. --- Discussion --- p.124 / Chapter 4.1 --- VEGF expression and localization --- p.124 / Chapter 4.2 --- VEGF receptors expression and localization --- p.129 / Chapter 4.3 --- Possible VEGF action in the rat epididymis --- p.133 / Chapter 4.4 --- Regulation of VEGF and its receptor expression by androgen and/or other testicular factors --- p.136 / References

Foram utilizados dez jacarés - do - Pantanal (Caiman crocodylus yacare), machos de faixa etária juvenil; sete espécimes foram destinados ao estudo macroscópico e os outros três foram utilizados para o estudo microscópico de luz. Após a abertura da cavidade pleuroperitoneal foi observado e identificado que os testículos são em pares e apresentam-se aderidos a parede dorsal da cavidade pleuroperitoneal. O epidídimo é alongado e muito enovelado, encontra-se na extremidade cranial do testículo, seguindo medialmente até sua extremidade caudal, onde inicia-se com o ducto deferente e posterior abertura na cloaca. O falo tem um formato tubular de aspecto cônico, mostrando ser um órgão com breve resistência, porém é flexível e possuem características de tecido cartilaginoso. Na microscopia de luz foi observada que, o testículo é limitado pela túnica albugínea. Os túbulos seminíferos são extremamente contorcidos e em seus espaços estão preenchidos por tecido intersticial e células de Leydig. Encontram-se na rede testicular túbulos revestidos por epitélio cubóide. Os ductos do epidídimo são revestidos pelo epitélio pseudo-estratificado não ciliado que variam entre células cúbicas e prismáticas. Os ductos deferentes caracterizam-se por apresentar uma luz estreita revestida pelo epitélio pseudo-estratificado prismático não ciliado. O falo é revestido pelo epitélio estratificado escamoso não queratinizado e envolto por tecido conjuntivo. / Ten Young-aged male Pantanal alligators (Caiman crocodylus yacare) were used; seven specimes were destinated to macroscopic study and the other three were used in the study of light microscopy. After the opening of the pleuroperitoneal cavity, we observed and identified that testes are in pairs and adhered to the dorsal wall of the pleuroperitoneal cavity. The epididymis is long and very convoluted. It is found in the cranial extremity of the testis, following medially until its caudal extremity, where it is initiated with ductus deferens and posterior opening in the phallus. Phallus has a tubular shape in conic aspect showing to be an organ with little resistence, but it is flexible and has features of cartilaginous tissue. In the light microscopy we observed that the testis is limited by the tunica albuginea. The seminiferous tubules are extremely convoluted and its spaces are filled with intersticial tissue and Leydig cells. Tubule lined with cuboidal epithelium are found in the rete testis. The ductus epididymus are lined with by non-ciliated pseudostratified epithelium to columnar cells. Ductus deferens show a very narrow light lined by the non-ciliated columnar pseudostratified epithelium. Phallus is lined by the non-keratinized stratified squamous epithelium and connective tissue.

Submitted by (lucia.rodrigues@ufrpe.br) on 2017-02-08T14:39:09Z No. of bitstreams: 1 Felipe Costa Almeida.pdf: 906567 bytes, checksum: 56e2db3d9385785462dcff3582c0c339 (MD5) / Made available in DSpace on 2017-02-08T14:39:09Z (GMT). No. of bitstreams: 1 Felipe Costa Almeida.pdf: 906567 bytes, checksum: 56e2db3d9385785462dcff3582c0c339 (MD5) Previous issue date: 2013-02-26 / This work aimed to study fertility of sperm obtained from epididymis tail of Nelore bulls and subjected to cryopreservation in extenders with three different concentrations of glycerol, and stabilization times at 5 °C and antioxidants addition to semen extender. Epididymides were obtained from slaughterhouse up to one hour after the death of the animal, placed in individual plastic bags and transported in box at room temperature (28 °C). At the laboratory, spermatozoa were recovered from epididymis tail using flotation technique and then diluted in Tris-yolk egg. In experiment 1 three concentrations of glycerol (3%, 5% and 7%) was evaluated at different stabilization times (0h, 2h and 4h). In the second experiment was evaluated the effect of adding enzymatic antioxidants in epididymal spermatozoa cryopreservation, resulting in following experimental groups: (Control; CAT 50 and 100 U/mL, and SOD 50 and 100 U/mL) at a concentration of 60 x 106 sperm/mL, packaged in straws (0.25 mL) and frozen in automated system. In both experiments, semen samples were thawed at 37 °C/30 sec was evaluated for plasma membrane integrity (PMi), acrosomal integrity (Aci), mitochondrial membrane potential (MMP) and sperm kinematics. In Experiment 1, PMi and Aci analyzes demonstrated values lower (P<0.05) for glycerol 3%, at stabilization time at 4h compared to other times. In kinematic evaluation, sperm values were lower (P<0.05) for BCF parameter in glycerol 3% and 7% groups at 2h of stabilization time compared to glycerol 5%; for ALH, glycerol 3% group in stabilization time at 4h demonstrated lowest (P<0.05) compared to other groups. Glycerol 5% group, settling time 0h, showed lower values (P <0.05) for the hPMM and Aci parameters, when compared to other groups. For IVF experiment, only glycerol 5% at stabilization time at 4h was used. Forty percents of embryonic viability were obtained from oocytes used. In experiment 2, parameters of MT, MP, VCL, VSL, VAP, STR, ALH and BFC did not differ (P> 0.05) between control and treated groups. However, significant differences (P <0.05) were observed between groups for Linearity (LIN) and oscillation index (WOB), being less than 100 SOD treatment (P<0.05) than other groups. Addition of SOD 100 determined lower (P<0.001) percentages of blastocysts post-cleavage. Based on results of kinematic and in vitro fertilization, it is possible to conclude that spermatozoa cryopreservation obtained from epididymis tail of bulls should be performed using extender with glycerol at 5% of concentration and stabilization time at 5 °C during 4h. Addition of CAT concentrations of 50 and 100 U/mL and SOD at concentration of 50 U/mL did not affect the sperm quality. Addition of SOD at a concentration of 100 U/mL reduces fertility of epididymal sperm after cryopreservation. However, it is noteworthy that sperm obtained from epididymis tail of bulls can be successfully used in IVF programs. / Este trabalho teve como objetivo estudar a fertilidade de espermatozoides obtidos da cauda do epidídimo de touros da raça Nelore e submetidos à criopreservação em diluentes com três diferentes concentrações de glicerol, tempos de estabilização a 5 °C e adição de antioxidantes ao diluidor de sêmen. Os epidídimos foram obtidos em matadouro, até uma hora após a morte do animal, colocados em sacos plásticos individuais e transportados em caixa térmica em temperatura ambiente (28 ºC). No laboratório, os espermatozoides foram recuperados da cauda do epidídimo utilizando a técnica de flutuação e, a seguir, diluídos em Tris-gema. No experimento 1 foi avaliado diferentes concentrações de glicerol (3%, 5% e 7%) em diferentes tempos de estabilização (0h, 2h e 4h). No experimento 2 foi avaliado o efeito da adição de antioxidantes enzimáticos na criopreservação de espermatozoides epididimários, constituindo os seguintes grupos experimentais: (Controle; CAT 50 e 100 U/mL; SOD 50 e 100 U/mL), na concentração de 60 x 106 espermatozoides/mL, acondicionados em palhetas (0,25 mL) e congelados em método automatizado. Em ambos os experimentos, as amostras de sêmen foram descongeladas a 37 °C/ 30 s e avaliadas quanto a integridade de membrana plasmática (iMP), integridade acrossomal (iAc), potencial de membrana mitocondrial (PMM) e cinética espermática. No experimento 1, as análises de iMP e iAc apresentaram valores inferiores (P<0,05) para o glicerol 3%, no tempo de 4h em comparação aos demais tempos de estabilização. Na avaliação da cinética espermática, foram encontrados valores inferiores (P<0,05) para o parâmetro BCF nos tratamentos glicerol 3% e 7%, no tempo de 2h em comparação ao do glicerol 5%; para ALH, o grupo glicerol 3%, no tempo 4h de estabilização apresentou menor valor (P<0,05) em relação aos demais grupos. O grupo glicerol 5%, no tempo de estabilização 0h, apresentou valores inferiores (P<0,05) para os parâmetros de aPMM e iAc, quando comparado aos demais grupos. Para a FIV foi utilizado o grupo glicerol 5%, no tempo de estabilização 4h. Foram obtidos 40% de viabilidade embrionária dos oócitos utilizados. No experimento 2, os parâmetros de MT, MP, VCL, VSL, VAP, STR, ALH e BFC não diferiram (P>0,05) entre o grupo controle e os grupos tratados. Todavia, diferenças significativas (P<0,05) foram observadas entre os grupos para Linearidade (LIN) e no índice de oscilação (WOB), sendo o tratamento SOD 100 inferior (P<0,05) aos demais grupos. A adição de SOD 100 determinou ainda menores (P<0,001) porcentagens de blastocistos pós-clivagem. Com base nos resultados de cinética e fertilização in vitro, é possível concluir que a criopreservação de espermatozoides obtidos da cauda do epidídimo de touros deve ser realizada utilizando diluidor com glicerol na concentração de 5% e tempo de estabilização a 5 °C de 4h. A adição de CAT nas concentrações de 50 e 100 U/mL e SOD na concentração de 50 U/mL não influencia na qualidade espermática. A adição de SOD na concentração de 100 U/mL reduz a fertilidade dos espermatozoides epididimários pós-criopreservação. Todavia, ressalta-se que espermatozoides obtidos da cauda do