A simple method for the quantitative determination of sperm aggregation

Described in this paper are 2 methodological variants for photometric recording of sperm aggregation. Heparin was used to induce aggregation. One of the working principles was related to aggregation-associated alteration of turbidity in an agitated cell suspension, measured by the KZM-1 coagulation-time meter, in analogy to measurement of thrombocyte aggregation. The 2nd variant was based on variation of cloudiness due to sedimentation of aggregates in a non-agitated suspension. Both methods provided equally valid information. They characterised both intensity of aggregation as a combined effect of the number and size of aggregates as well as the rate of aggregation. Photometric recording has proved to be an objectivated method for quantitative assessment of aggregation. Its use is proposed for studies into capacitation or immunological response of spermatozoa.     

Identification of Cytokeratins in Bovine Sperm Outer Dense Fibre Fractions

Outer dense fibres (ODF) are important substructures of mammalian sperm tails that are involved in the regulation of sperm motility. In this study, we investigated the identity of several sodium dodecyl sulphate (SDS)‐insoluble ODF proteins. Bovine ODF were purified by separating sperm heads and tails using ultrasound and Percoll^® density gradient centrifugation. Sperm flagella were treated with the detergent cetyltrimethylammonium bromide (CTAB). CTAB‐insoluble material, which reportedly represents the ODF fraction, was collected, and electron microscopy confirmed a highly purified ODF fraction. We found after solubilization of this fraction with SDS that high amounts of insoluble material were retained after centrifugation. SDS‐insoluble material was collected and quantitatively dissolved in 8 M urea. SDS‐gel electrophoresis in the presence of urea revealed polypeptides with apparent molecular masses of approximately 25, 43, and 50 kDa. Subsequent immunoblotting with anti‐cytokeratin antibodies detected two urea‐soluble, SDS‐insoluble proteins with apparent molecular masses of 45 and 66 kDa. The 45‐kDa protein was identified as cytokeratin 19. An antibody reacting with a palette of cytokeratins (CK 1–18 and CK 20), KL1, was the only antibody that reacted with the 66‐kDa polypeptide. We conclude that sperm ODF fractions contain at least one each of type I and type II intermediate filaments. As keratins and intermediate filaments are described as rope‐like structures, we suggest that these intermediate filaments play an important structural or tension‐bearing role in sperm flagella.