RESUMO
The possible epigenomic effect of oviductal fluid on expression of DNA methyltransferase (DNMT) genes was examined in early bovine embryos (4-cell stage). Real-time quantitative PCR was performed to determine the relative expression of DNMT1, DNMT3a and DNMT3b transcripts in embryos cultured in vitro in the presence or absence of oviductal fluid. Expression of DNMT1 significantly increased when cultured with oviductal fluid, whereas DNMT3a and DNMT3b transcripts were unaffected by the addition of oviductal fluid. These results may help reveal the role of oviductal factors in the regulation of DNMT expression.
Assuntos
Bovinos/embriologia , Meios de Cultura/química , Metilases de Modificação do DNA/metabolismo , Técnicas de Cultura Embrionária/veterinária , Regulação Enzimológica da Expressão Gênica/fisiologia , Animais , Metilases de Modificação do DNA/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , RNA/genética , RNA/metabolismoRESUMO
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
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
Protein kinase activity of intact, motile sperm was assessed by measuring the transfer of the terminal phosphate from [32P]ATP to tricholoroacetic acid (TCA)-precipitable casein. The action of TEST (TES and Tris) yolk buffer (TYB) treatment on phosphorylation of sperm and TYB proteins was studied by detecting labelled phosphoproteins by autoradiography of polyacrylamide gel electrophoresis (PAGE). Results demonstrate that intact, forward-motile sperm have cell surface protein kinase activities. Although the difference between the kinase activity of freshly ejaculated sperm incubated in TYB was not significant, the protein phosphorylation during incubation in TYB showed that: (i) specific sperm surface proteins were phosphorylated to different degrees during the course of treatment; (ii) TYB proteins were phosphorylated by sperm during incubation; (iii) solubilised [32P]-labelled surface proteins were similar in molecular weight to TYB-labelled proteins. Taking into account that specific proteins on the human sperm surface undergo phosphorylation during incubation in TYB and that the sperm enzyme also acts specifically on some TYB proteins that become attached to the surface of the sperm, working hypotheses are proposed that suggest some correlation between the preservation of semen in TYB and the phosphorylation of proteins by intact human sperm.