This study aims to elucidate the process and mechanism of sperm penetration through eggs of Penaeus monodon. Sperm penetration of the vitelline envelopes (VEs) of P. monodon eggs were observed with the scanning electron microscope. The characteristics of sperm proteases in the sperm extracts from seminal receptacles of females were analysized. In P. monodon, mating and sperm transfer to the thelycum of female occur soon after maturity moult. Females store the sperm in the seminal receptacles. At spawning, they release stored sperm and eggs simultaneously into the water column. The outermost investment of a newly spawned egg is the VE. Sperm bind to the VE via the tip of their anterior spike. They rapidly undergo the acrosome reaction, which composes of depolymerization of the spike and exocytosis of the acrosome vesicle, pass through the VE and become bound to the egg oolemma. The isolated sperm suspended in artificial seawater were disrupted by sonication on ice. The supernatants after microcentrifuged were collected as sperm extracts. Sperm extracts were analyzed by gelatin SDS-PAGE. Sperm extract from sperm isolated from seminal receptacles of females showed clear bands of protease activity, whereas sperm extract from vas deferens and spermatophore of males did not. This results indicated that sperm of P. monodon do proceed capacitation in the seminal receptacles, and obtain sperm protease activity after capacitation. Using fluorescent peptidyl-MCA as sperm protease substrates, high trypsin-like and aminopeptidase-like activities were observed in sperm extracts. The sperm protease activity was inhibited by trypsin inhibitors aprotinin, p-aminobenzamidine (PAB), soybean trypsin inhibitor (SBTI), N-£\-p-tosyl-L-lysinechloromethyl ketone (TLCK); but was not inhibited by chymotrypsin inhibitor N-tosyl-L-phenylalaninechloromethyl ketone (TPCK) and metalloprotease inhibitor 1,10-phenanthroline (1,10-P). The results indicated that sperm undergo the acrosome reaction and release sperm proteases including trypsin-like protease, which has been implicated in facilitating sperm passage through vitelline envelope. Sperm proteases were highly active in the weak base environment, exhibiting maximum activity at pH 8.0. The protease activities were enhanced by addition of calcium chloride and magnesium chloride in the incubation medium.

Vitelline membrane strength plays a role in preventing contamination of albumen by yolk during separation and is important to food safety. Two experiments were conducted to determine if a relationship exists between vitelline membrane protein banding patterns, interior egg quality, and vitelline membrane rupture strength. Eggs were gathered from commercial egg producers from pre-molt (26 wk or 72 wk) and post-molted (74 wk or 82 wk) hens. In each of two trials twenty-one eggs were gathered and stored (4°C) per experiment. Three eggs were evaluated on days 0, 7, 14, 21, 28, 35, and 42 for eggs from pre-molted hens; and 1, 7, 14, 21, 28, 35, and 42 for eggs from molted hens for changes in SDS-PAGE protein banding patterns. The yolk from each egg was isolated and rolled on a wet paper towel to remove adhering albumen. The yolk was emptied and washed. The whole membrane was placed into double deionized water and divided into two sections. The first section was the whole membrane sample and the other was separated by forceps into inner and outer membrane samples. The three sections were dissolved separately in 1% SDS/70 mM Tris/HCl, pH 6.8. Protein concentration was determined using the Lowry method and proteins separated on 4-20% gradient gel by SDS-PAGE. Protein banding patterns were analyzed using the Bio-Rad Multi-Analyst Densitometer. Reductions of VMO I and GP II occurred along with reductions in the protein bands between 60 to 100 kDa. In each of two trials, an additional one hundred forty eggs were gathered at the same time from the same flock and stored at 4°C. Twenty eggs were evaluated for quality on days 0, 7, 14, 21, 28, 35, and 42 for eggs from pre-molted hens; and 1, 7, 14, 21, 28, 35, and 42 for eggs from molted hens. Yolk index, albumen height, albumen pH, and yolk pH were determined. Vitelline membrane strength was determined using a compression anvil. Two different treatments were used on the yolk when evaluating rupture strength: 10 egg yolks with inner thin albumen layer, and 10 egg yolks rolled on wet paper towel to remove inner thin albumen layer. Interior egg quality and vitelline membrane strength declined during storage.

No description available.

<p>In all vertebrate species, reproduction is a hormonally controlled process, important for growth and maturation of gonads and germ cells. Production of functional germ cells is of outmost importance to secure the survival of a species. Fish comprises 50% of the known vertebrates and are found in aquatic habitats all over the world. Even though fish have evolved a wide variety of morphological and physiological characteristics, due to large differences in the living environment, the growth an maturation of germ cells follows the same pattern in all species. In this thesis the focus has been directed on oocyte growth and development in Arctic char (Salvelinus alpinus), and if stress might inflict disturbances on the reproductive systems.</p><p>All sexually mature female egg laying vertebrates produces yolky eggs surrounded by an eggshell. Production of yolk and egg shell is under estrogenic control and it is known that production of egg components can be induced in male and juvenile fish by estrogenic substances. Many manmade chemicals have been found to interfere with hormonally controlled processes. Therefore production of the egg yolk precursor, vitellogenin (VTG), and the egg shell components, vitelline envelope proteins (VEP), have been used as biomarkers for estrogenic effect. Exposure to endocrine disrupting substances (EDS) does not only give rise to hormonal effects on the organism, but in addition it also gives rise to an increase in stress hormone, cortisol (F), levels. </p><p>It is evident that a wide variety of substances may affect Arctic char oocyte growth and maturation. VTG and VEP production is found to be under dose dependent estrogenic control, but the production was directly affected by F. Under natural condition it has been found that F increases towards ovulation. Even though both VTG and VTG is under estrogenic control, these studies showed that stress lead to a decrease of VTG while the VEP production increased. These effects was only observed on protein levels indicating that a post transcriptional down regulation of VTG production is mediated by F in Arctic char.</p><p>In order for an egg to become fertilizatible, it must undergo a maturation phase. This maturation phase is primarily induced by gonadotropins, which in turn induce the production of species specific maturation inducing substances (MIS). To investigate oocyte development in Arctic char a characterization of its MIS receptor was made. The MIS receptor is localized on the oocyte surface and displays a single class of high affinity and low capacity binding sites. The binding moieties displays association and dissociation kinetics typical of steroid membrane receptors.</p><p>Even though high specificity for Arctic char MIS was observed, it was found that some EDS bind to the Arctic char oocyte membrane receptor. This suggest that certain EDS might affect oocyte maturation and thereby might alter the reproductive success. Furthermore, it was found that F did not bind to the MIS receptor in Arctic char. It is therefore suggested that oocytes are more sensitive to stress during the growth phase than during maturation</p>

In all vertebrate species, reproduction is a hormonally controlled process, important for growth and maturation of gonads and germ cells. Production of functional germ cells is of outmost importance to secure the survival of a species. Fish comprises 50% of the known vertebrates and are found in aquatic habitats all over the world. Even though fish have evolved a wide variety of morphological and physiological characteristics, due to large differences in the living environment, the growth an maturation of germ cells follows the same pattern in all species. In this thesis the focus has been directed on oocyte growth and development in Arctic char (Salvelinus alpinus), and if stress might inflict disturbances on the reproductive systems. All sexually mature female egg laying vertebrates produces yolky eggs surrounded by an eggshell. Production of yolk and egg shell is under estrogenic control and it is known that production of egg components can be induced in male and juvenile fish by estrogenic substances. Many manmade chemicals have been found to interfere with hormonally controlled processes. Therefore production of the egg yolk precursor, vitellogenin (VTG), and the egg shell components, vitelline envelope proteins (VEP), have been used as biomarkers for estrogenic effect. Exposure to endocrine disrupting substances (EDS) does not only give rise to hormonal effects on the organism, but in addition it also gives rise to an increase in stress hormone, cortisol (F), levels. It is evident that a wide variety of substances may affect Arctic char oocyte growth and maturation. VTG and VEP production is found to be under dose dependent estrogenic control, but the production was directly affected by F. Under natural condition it has been found that F increases towards ovulation. Even though both VTG and VTG is under estrogenic control, these studies showed that stress lead to a decrease of VTG while the VEP production increased. These effects was only observed on protein levels indicating that a post transcriptional down regulation of VTG production is mediated by F in Arctic char. In order for an egg to become fertilizatible, it must undergo a maturation phase. This maturation phase is primarily induced by gonadotropins, which in turn induce the production of species specific maturation inducing substances (MIS). To investigate oocyte development in Arctic char a characterization of its MIS receptor was made. The MIS receptor is localized on the oocyte surface and displays a single class of high affinity and low capacity binding sites. The binding moieties displays association and dissociation kinetics typical of steroid membrane receptors. Even though high specificity for Arctic char MIS was observed, it was found that some EDS bind to the Arctic char oocyte membrane receptor. This suggest that certain EDS might affect oocyte maturation and thereby might alter the reproductive success. Furthermore, it was found that F did not bind to the MIS receptor in Arctic char. It is therefore suggested that oocytes are more sensitive to stress during the growth phase than during maturation

Submitted by (lucia.rodrigues@ufrpe.br) on 2016-06-08T12:33:25Z No. of bitstreams: 1 Jeine Emanuele Santos da Silva.pdf: 2802282 bytes, checksum: 66fe306e48bd3ebef3e793500c49baea (MD5) / Made available in DSpace on 2016-06-08T12:33:25Z (GMT). No. of bitstreams: 1 Jeine Emanuele Santos da Silva.pdf: 2802282 bytes, checksum: 66fe306e48bd3ebef3e793500c49baea (MD5) Previous issue date: 2011-02-28 / The aim of the study was to investigate the effect of different fat dietary on serum lipid profile in Japanese quails and on vascular growth in the embryos vitelline membrane (VM). These birds were fed with supplemented feed with fish oil or soybean oil. The experiment was conducted with 80 quail, distributed in five treatments named Diet Control (DC), Fish Oil (FO 2%), Fish Oil 4% (FO 4%),Soybean Oil 2% (SO 2%) and Soybean Oil 4% (SO 4%), in a ratio of 1 male to 3 females. Fertilized eggs incubated at 37.5°C for 24 hours were opened and after removal of 1 mL of albumen the window was sealed with parafilm. From 72 hours incubation images of VM vascular network were captured at intervals of 24 hours over three days. DLA were generated in order to compare their fractal dimension (D) with the VM and test whether the method affects the value of D. To obtain the average value of D of the VM vascularization and DLA were used box-counting (Dbc) and information dimension (Di) methods. An enzymatic colorimetric method was used to estimate lipid profile. The total cholesterol (TC) levels of the groups showed no difference between itself. The total triglycerides (TG) of the FO 2% and FO 4% groups were higher than the groups DC, SO 2% and SO 4%. For HDL, the group FO 4% differ in relation to other groups. The embryos MV vascularization, evaluated by fractal dimension (D), of the group FO 4% was less than the MV vascularization of the group DC, SO 2% and FO 2%. The MV vascularization of the group SO 4% was lower only when compared with group DC. The PUFA supplementation in groups FO 2% and SO 2% did not interfered with MV vascularization. The DLA fractal dimension obtained by the Dbc and Di was higher than those observed for the MV fractal dimension for all experimental groups. Differences between the values of D obtained by these methods (Dbc e Di) were statistically significant for all groups. The vitelline membrane shows an experimental model suitable for vasculogenesis study. The fractal analysis shows to be a reliable mathematical method to quantify vascular growth in vivo such as the vitelline membrane. / O objetivo deste estudo foi investigar o efeito de diferentes dietas sobre o perfil lipídico sérico de codornas japonesas e sobre o crescimento vascular na membrana vitelina (MV) dos embriões das aves suplementadas com óleo de peixe e óleo de soja. Foram utilizadas 80 codornas, distribuídas em cinco tratamentos denominados Dieta Controle (DC), Óleo de Peixe 2% (OP 2%), Óleo de Peixe 4% (OP 4%), Óleo de Soja 2% (OS 2%) e Óleo de Soja 4% (OS 4%), numa relação de 1 macho para 3 fêmeas. Ovos fertilizados incubados a 37,5ºC durante 24 horas foram abertos e após a remoção de 1 mL de albúmen a abertura foi selada com parafilme. A partir de 72 horas de incubação imagens da rede vascular da MV foram capturadas em intervalos de 24 horas durante 3 dias. DLA foram gerados a fim de comparar a sua dimensão fractal com a da MV e testar se o método usado interfere no valor da dimensão. Para obtenção do valor médio da dimensão fractal da vascularização das MV e dos DLA foram utilizados os métodos de box-counting (Dbc) e dimensão de informação (Di). Método enzimático com reação colorimétrica foi usado para estimar o perfil lipídico das aves. Os níveis de colesterol total (CT) dos grupos não apresentaram diferença entre si. Os níveis de triglicerídeos totais (TG) das aves do grupo OP 2% e OP 4% foram superiores aos dos grupos DC, OS 2% e OS 4%. Para o HDL, apenas o grupo OP 4% apresentou diferença em relação aos demais grupos. A vascularização da MV dos embriões, avaliada pela dimensão fractal D, do grupo OP 4% foi inferior a vascularização das MV dos grupos DC, OS 2% e OP 2%. A vascularização da MV do grupo OS 4% foi inferior apenas quando comparada ao grupo DC. A suplementação com PUFA nos grupos OP 2% e OS 2% não interferiu na vascularização das MV. A dimensão fractal do DLA obtida pelos métodos de Dbc e Di foi superior aquelas observadas para as dimensões das MV de todos os grupos experimentais. As diferenças entre as dimensões obtidas por estes métodos (Dbc e Di) foram estatisticamente significativas para todos os grupos avaliados. A membrana vitelina se mostra um modelo experimental adequado para o estudo da vasculogênese. A geometria fractal se mostra um método matemático confiável para a quantificação do crescimento vascular em modelos in vivo como a membrana vitelina.