Cytochemical and ultracytochemical demonstration of cytochrome c oxidase in spermatozoa and dynamics of its changes accompanying ageing or induced by stress

Abstract

Cytochrome c oxidase (CcO) plays a key role in cellular respiration and energetic metabolism. The latter is a prerequisite for osmotic and synthetic function, motility and the maintenance of cell structure. The objective of this study was to develop and assess a facile microscopic technique that would demonstrate CcO activity insitu, both at the microscopic and sub-microscopic levels. The cytochemical technique proposed is based on oxidation of 3,3'-diaminobenzidine (DAB) by the cytochrome c complex (including CcO), in a chain reaction in which the reagent is polymerized and deposited at the reaction sites. The deposit can be identified by its colour under the light microscope and by osmiophilia under the electron microscope. The reaction is restricted to mitochondria at the light microscope level, and to the outer face of the inner mitochondrial membrane at the ultrastructural level. The activity is inhibited promptly by greater than or equal to 0.5 mM KCN, a specific inhibitor of CcO, and by sodium azide or heat (70 degrees C/5 min). The technique was validated on a number of domestic and laboratory animals, using sperm that were ejaculated or epididymal in origin, cryopreserved or treated. The data obtained displayed activity profiles of individual cells, ejaculates or donors and emphasized differences among species. This technique depicts spontaneous CcO decline during ageing and changes induced by various physical or chemical treatments.

Effect of leukocytospermia and processing by discontinuous density gradient on sperm nuclear DNA fragmentation and mitochondrial activity

“…To determine sperm mitochondrial activity, the method proposed by Hrudka [26] was used, based on the oxidation, polymerization, and deposition of 3,3′-diaminobenzidine (DAB) by mitochondrial cytochrome c-oxidase. Briefly, semen was diluted 1:1 to 1:3 in a solution containing 1 mg/mL of 3-3′-diaminobenzidine (DAB) in PBS (phosphate-buffered saline-137 mM NaCl, 2.7 mM KCl, 4.3 mM Na 2 HPO 4 , 1.4 mM KH 2 PO 4 , pH=7.4) and incubated at 37°C for 1 h in the dark.…”

Efeito de diferentes concentrações de melatonina em espermatozoides de carneiros sobre estresse oxidativo após criopreservação

“…A atividade mitocondrial foi determinada conforme Hrudka (1987), onde 25μL de cada tratamento foi incubado com 25μL de DAB (1mg/mL de PBS), a 37°C, por uma hora, na ausência de luz. Foram contados 200 espermatozoides por lâmina em microscópio óptico (DM 750 ® , Leica Microsystems, Heerbrugg, Suiça), em objetiva de 100x, obedecendo à escala de quatro classes propostas por Hrudka (1987), onde: Classe I: células espermáticas com peça intermediária totalmente corada, alta atividade mitocondrial (DAB I); Classe II: células espermáticas com segmentos corados (ativos) e não corados (inativos), havendo predominância dos ativos (DAB II); Classe III: células espermáticas com segmentos corados (ativos) e não corados (inativos), havendo predominância dos inativos (DAB III); Classe IV: células espermáticas com peça intermediária totalmente descorada, sem atividade mitocondrial (DAB IV).…”

Viabilidade do sêmen congelado obtido do epidídimo de touros post-mortem

“…A integridade funcional da membrana foi avaliada pelo teste hiposmótico segundo a técnica descrita por Jeyendran et al (1984), que consistiu em misturar 0,1 mL do sêmen com 0,9 mL da solução hiposmótica de 150 mOsmol (7,35 g citrato de sódio.2H 2 O, 13,51g frutose em 1000 mL de água destilada). Uma solução isosmótica de 300 mOsmol (14,7 g citrato de sódio.2H 2 O, 27,02 g frutose em 1000 mL de água destilada) foi utilizada como controle.…”