Search

The biological activity of methanolic and aqueous extracts from dehydrated hypocotyls of Lepidium meyenii (Brassicaceae, vernacular name "maca"), was studied on rat hepatocytes and human breast cancer MCF-7 cells. The extracts did not exhibit cytotoxicity in hepatocyte primary cultures up to 10 mg/ml as measured by the MTT viability test, and lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) leakage. Moreover, after 72 h, extracts inhibited LDH and AST leakage from the hepatocytes. When hepatocytes were intoxicated by t-butyl hydroperoxide, neither extract prevented oxidative damage. Both extracts showed weak antioxidant activity in the DPPH radical scavenging test with IC(50) values of 3.46 +/- 0.16 and 0.71 +/- 0.10 mg/ml, for aqueous and methanolic extracts, respectively. Thus, the observed effect on spontaneous enzyme leakage is probably mediated through mechanisms other than antioxidant activity. Both methanolic and aqueous extracts have shown estrogenic activity comparable with that of silymarin in MCF-7 cell line. Maca estrogenicity was exhibited in the range from 100 to 200 mug of extract per ml. The findings in the present study show that maca does not display in vitro hepatotoxicity. In contrast, a slight cytoprotective effect, probably not mediated by antioxidant capacity, was noted. Maca extracts exhibited estrogenic activity comparably to the effect of silymarin in MCF-7 cells.

In previous studies a series of Mabs against boar capacitated sperm have been produced. One of these Mabs--4B12--was found to recognize a surface membrane-associated protein located in the acrosome portion of the spermatozoa that became accessible to antibody after capacitation. In biological experiments it was shown that Mab 4B12 significantly inhibited boar sperm-porcine ZP binding. In attempts to investigate the mechanisms by which Mab 4B12 affected sperm-ZP binding, the role of the cognate protein on some functional parameters such as sperm motility and ability of the capacitated spermatozoa to undergo AR was studied. Experimental models of premature AR and AR physiologically induced with ZP were applied to study the effect of Mab 4B12 on boar sperm AR using PSA staining to estimate the acrosome-reacted state of spermatozoa. Sperm motility characteristics were determined by the time-exposure photokinesigraphic method. The results obtained in the present study, together with previously established inhibition of sperm-ZP binding by Mab 4B12, documented the participation of the 4B12 protein in primary sperm-ZP binding. The protein is not connected with sperm motility and secondary sperm-ZP binding.

Certain morphological changes such as rearrangement of cytoskeletal proteins of the mammalian spermatozoa are detectable during the AR. The type of changes differs according to the studied sperm species and follows the course of AR. Relocation of cytoskeletal structures was previously observed especially in the case of actin-, alpha-, gamma-tubulin- and spectrin-containing structures. To prove these findings we used specific inhibitors of cytoskeletal proteins (eg. colcemide, cytochalasine B, nocodazole and vinblastine). It has been shown that the AR is influenced by cytoskeletal inhibitors, but the obtained results also document that cytoskeletal proteins actin, tubulin and spectrin play a significant role in the course of AR in vitro. Our results of confocal and electron microscopy also demonstrate visible changes of actin-, tubulin- and spectrin-containing structures after the AR. Our data indicate that specific cytoskeletal inhibitors influence the AR and they prove the role of cytoskeletal proteins in this process.

Several cytoskeletal proteins (alpha-tubulin, beta-tubulin, actin, spectrin, tropomyosin, vimentin and cytokeratin) were studied in human and boar spermatozoa. Their localization was observed by means of specific antibodies using indirect immunofluorescence technique. Immunocytochemical results were confirmed by the Western blot technique. Cytoskeletal proteins were examined in ejaculated spermatozoa before and after acrosome reaction induced by the ionophore A23187. The immunofluorescence assay revealed that localization of the studied cytoskeletal proteins in human and boar spermatozoa differ remarkably. In human spermatozoa, the localization of actin and spectrin changed after acrosome reaction; on the other hand, in boar spermatozoa, the changes of localization concerned alpha-tubulin, beta-tubulin, actin and spectrin. The type of changes differs in the studied species and follows probably the course of acrosome reaction. The results suggest that cytoskeleton participates in the process of acrosome reaction of mammalian spermatozoa.