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.
The effect of gossypol upon organelles of rat sperm was investigated by light and electron microscopy. The drug was administered s.c. for 2 to 30 days at 20 mg/kg BW/day. Sperm from the testis, caput, corpus, and cauda epididymis were examined at regular intervals, during and after treatment, for periods extending up to 70 days. The drug induced a specific effect in the sperm tail. It consisted of segmental aplasia of the mitochondrial sheath observed in high incidence in testicular and epididymal sperm. This primary lesion, in the authors' view, predisposed a development of secondary lesions as sperm advanced along the epididymis. Secondary lesions included bulging, dislocating, fraying, or breaking of axial fibers, bending or breaking of the tail, and decapitation. A minimum of 3 days of treatment was necessary to produce an effect above control values, while 9 days or longer induced lesions in almost all sperm. Motility ceased with 30 days of treatment. Fertilizing capacity was inversely related to the increase and decline of lesions.
Fertility, spermatogenesis, and sperm phenotype were investigated in three hybrid deer (Odocoileus virginianus dacotensis × Odocoileus hemionus hemionus) and a yak × domestic cow hybrid (Bos mutus (grunniens) × Bos primigenius (taurus)) using histological techniques. All of the hybrids studied were infertile but varied in the degree of testicular differentiation, spermatogenic activity, and sperm production. The hybrid yak was the least developed and the white-tailed deer × F1 hybrid was the most advanced. F1 backcrossing improved spermatogenesis, output, and morphology of sperm, but not the fertility of the donor, indicating that normal sperm morphology alone does not assure fertility. Two deer hybrids that produced sperm differed in sperm phenotype from each other and from the parental species. Interaction of a Y chromosome from one species and autosomal sex-determining genes from the other species is suggested as a possible explanation of sterility in male hybrids.
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