Immunolocalization of Vasa, PIWI, and TDRKH proteins in male germ cells during spermatogenesis of the teleost fish Poecilia reticulata.

Vasa, PIWI and TDRKH are conserved components of germ granules that in metazoans are involved in germline specification and differentiation, as documented by mutational experiments in some model animals. So far, investigations on PIWI during spermatogenesis of fish has been limited to a few species, and no information is available for TDRKH, another protein involved in the piRNA pathway. In this study, the immunolocalization of these three germline determinants was analyzed in male gonads of the teleost fish Poecilia reticulata to document their localization pattern in the different stages of germ cell differentiation. To analyze their distribution pattern during the different stages of spermatogenesis we performed immunohistochemistry (IHC) and immunofluorescence (IF) assays using primary polyclonal antibodies after testing their specificity with Western Blot. Moreover, sections of testis stained with haematoxylin and eosin clarified the structural organization of P. reticulata testis, while the use of the confocal microscope and the nuclear staining clarified the different stages of germ cell differentiation during spermatogenesis. The results showed that Vasa, PIWI and TDRKH were specifically immunolocalized in the germ cells of P. reticulata, with no specific signal detected in Sertoli cells and in other somatic cells of the gonad. These markers were detected in all stages of differentiation from early spermatogonia to advanced spermatids. Vasa staining was the strongest in spermatogonia, and then decreases throughout differentiation. Instead, both PIWI and TDRKH staining increases during differentiation, and their distribution pattern, similar to what observed in the mouse, suggests their concerted participation in the piRNA pathway also in this fish.

Determination of free and total cholesterol in human bile samples using an enzyme electrode.

A reliable cholesterol electrode was assembled with a Clark type oxygen electrode and cholesterol oxidase immobilised on a nylon net fixed on the electrode surface. Determination of free (unesterified) and total (esterified and unesterified) cholesterol in human bile samples was performed. Results were compared with the enzymatic-spectrophotometric procedure based on the Roeschlau method. The electrode has a usable life of more than two weeks, and more than 300 determinations could be performed. The CV values for free and total cholesterol in bile samples is 5-6%. The comparison between the proposed procedure and the spectrophotometric procedure is fair for the half of the results (delta less than 5%) and acceptable in the other half (delta less than 25%).