RESUMO
Sperm reservoirs in South American Camelids would be crucial for successful fertilization. Since ovulation occurs approximately 36 h after mating, the maintenance of the sperm viability in the oviduct waiting for the ovum is a critical reproductive event. Our study aimed at determining whether the isthmus or the utero tubal junction (UTJ) could function as a sperm reservoir in llama by means of in vivo and in vitro experiments. For the in vivo experiments, the oviducts of adult females with a dominant follicle bigger than 7 mm were examined for the presence of sperm at 6, 18, 24, 28 and 35 h after mating. The results using scanning and transmission electron microscopy showed ultrastructural differences between isthmus and UTJ with respect to (1) predominance of secretory cells in the UTJ and ciliated cells in the isthmus epithelium and (2) cytoplasmic bulbous projection of the secretory cells in the UTJ. Sperm adhered by a mucus-like substance were seen only in the UTJ at 6, 18, 24 and 28 h postmating. Lack of sperm adhered to oviductal mucosa was observed around ovulation (35 h). In vitro experiments demonstrated higher ability of UTJ epithelial cell explants with respect to isthmus explants to bind sperm in a co-cultured system. The anatomical features and the presence of a sperm bonding agent in the UTJ together with the in vitro differential binding of sperm to UTJ explants strongly suggest that both may be feasible mechanisms that facilitate sperm storage in this oviductal region in llama.
Assuntos
Camelídeos Americanos/fisiologia , Células Epiteliais/fisiologia , Células Epiteliais/ultraestrutura , Tubas Uterinas/fisiologia , Espermatozoides/fisiologia , Espermatozoides/ultraestrutura , Animais , Tubas Uterinas/ultraestrutura , Feminino , Masculino , Fatores de TempoRESUMO
Denuded Bufo arenarum oocytes matured in vitro by progesterone treatment exhibited abnormal segmentation due to the penetration of more than one sperm. These oocytes were able to respond to activation stimuli and exhibited the external signs characteristic of activation. However, the prevention of polyspermy was not effective in these oocytes, which exhibited numerous sperm in their cytoplasm. The aim of this work was to analyse the cortical reaction in polyspermic Bufo arenarum oocytes matured in vitro. The result indicate that the cortical reaction of these oocytes seems to occur with a chronological sequence similar to that described for ovoposited oocytes of this species. In addition, when, 1 min after pricking, cortical granule exocytosis occurred, the oocytes became refractory to sperm entry, suggesting that they are able to establish a slow block to polyspermy.
Assuntos
Oócitos/citologia , Oócitos/metabolismo , Interações Espermatozoide-Óvulo , Animais , Bufo arenarum , Citoplasma/metabolismo , Eletroforese em Gel de Poliacrilamida , Exocitose , Feminino , Fertilização , Técnicas In Vitro , Inseminação Artificial , Masculino , Microscopia Eletrônica , Oócitos/ultraestrutura , Ovário/metabolismo , Progesterona/farmacologia , Fatores de TempoRESUMO
At present the physiological role of most oviductal proteins remains unknown. In this work, we present evidence that the oviductal secretion as well as the crude oviductal tissue-extract show proteolytic-like esterase and amidase activity. The proteolytic activity of the oviductal enzymes was higher in the oviducts of superovulated hamster females than in those of normal ones, indicating that gonadotrophic hormones would stimulate the synthesis and secretion of these enzymes. Some of their properties were analyzed in the 15,600-g supernatant of both oviductal tissue extracts (OE) and oviductal fluid (OF). The enzymatic activity toward the synthetic substrates p-tosyl-l-arginine methyl ester-HCl (TAME) and alpha-N-benzoyl-dl-arginine-p-nitroanilide HCl (BAPNA) was activated by calcium ions, reached a maximum at pH 7.5, and was inhibited by soybean trypsin inhibitor (SBTI), N-alpha-p-tosyl-l-lysine chloromethyl ketone HCl (TLCK), phenyl methyl sulfonyl fluoride (PMSF), and benzamidine. The OE glycoprotein fraction recognized by WGA-Sepharose affinity columns (37% total proteins) showed proteolytic activity with properties similar to the OE and OF enzymes. The protease activity could be ascribed to a plasminogen activator (PA) detected in the Triton X-100 treated tissue crude membrane fraction (Triton-CMF) and in the oviductal secretion of the superovulated females. In the Triton-CMF fraction, 100% of the proteolytic activity was plasminogen-dependent. The use of amiloride, a selective urokinase-type plasminogen activator (uPA) inhibitor, shows that 90% of this activity was due to a tissue-type plasminogen activator (tPA) and 10% to uPA whereas in the uterus 100% of the activity was tPA. Only a small percentage of the OF proteolytic activity was plasminogen-dependent, probably due to the presence of PA inhibitors in this medium.
Assuntos
Tubas Uterinas/enzimologia , Mesocricetus/metabolismo , Ativadores de Plasminogênio/metabolismo , Amidoidrolases/metabolismo , Animais , Benzoilarginina Nitroanilida/metabolismo , Cálcio/farmacologia , Cloreto de Cálcio/farmacologia , Compostos Cromogênicos/farmacologia , Cricetinae , Relação Dose-Resposta a Droga , Epitélio/efeitos dos fármacos , Epitélio/enzimologia , Esterases/metabolismo , Tubas Uterinas/efeitos dos fármacos , Feminino , Concentração de Íons de Hidrogênio , Inibidores de Serina Proteinase/farmacologia , Tosilarginina Metil Éster/metabolismo , Útero/efeitos dos fármacos , Útero/metabolismoRESUMO
Because of the need for antibodies in our studies involving the third component of complement in Bufo arenarum, we performed a simple procedure to purify C3 from B. arenarum serum to use as antigen in the preparation of the antiserum. The strategy was based on the well-known ability of C3 to bind to zymosan (Zy), a yeast cell wall extract comprised of polysaccharides. The Zy-bound fraction showed cross reactivity with a commercial antibody to human C3 as well as a similar electrophoretic profile (SDS-PAGE) to C3 from other species. The Zy-C3 complex resulting from binding Zy to B. arenarum serum was injected into rabbits and the antiserum against this C3-like fraction was purified by protein A-Sepharose chromatography. The purified C3 antibody was found to be suitable for immunochemical studies.
Assuntos
Bufonidae/imunologia , Complemento C3/imunologia , Complemento C3/isolamento & purificação , Soros Imunes , Animais , Especificidade de Anticorpos , Fracionamento Químico , Complemento C3/metabolismo , Complemento C3b/química , Complemento C3b/isolamento & purificação , Reações Cruzadas , Humanos , Soros Imunes/imunologia , Soros Imunes/isolamento & purificação , Immunoblotting , Epitopos Imunodominantes , Coelhos , Zimosan/imunologiaRESUMO
When spermatozoa from Bufo arenarum are incubated with molecules extracted from the vitelline envelopes of homologous oocytes, they lose their fertilizing capacity. Those molecules are glycoproteins, and the elimination of mannoside residues from them results in activity loss, while digestion of the proteic moiety did not alter their biological effect. Sepharose-concanavalin A columns were used to purify the glycoproteins, since the active fraction binds to the column. The fertility-impairing effect observed does not seem to be mediated by an acrosome reaction-inducing effect.