The Major Soluble Tubulins Are Found In Mega Dalton (MDa) Fractions ...

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Abstract

During progesterone-induced oocyte maturation, the cell is converted from prophase I to metaphase II, which is known to involve microtubule changes. We hypothesize that progesterone affects the polymeric state of tubulins, isoform dynamics, as well as tubulin synthesis and degradation. To test the former, leopard frog (Rana pipiens) oocytes were treated with progesterone, homogenized and centrifuged at 16,000 x g. The resulting pellet and supernatant fractions were analyzed using immunoblots probed with anti-alpha-tubulin antibody DM1A and anti-beta-tubulin antibody DM1B. The results indicate that the majority of tubulins are in supernatant fractions (soluble cellular component) and further indicate changes in size of pelletable tubulins with time post-progesterone treatment. To characterize the supernatant tubulins, Superose-6b columns were used to fractionate 16,000 x g supernatant materials. The results indicate tubulins in fractions of up to 5 MDa (equivalent in size to c. 50 tubulin dimers or 5,000 kDa) predominate, while tubulin dimers and monomers of 110 kDa and 55 kDa, respectively, are relatively minor components. The tubulin in MDa fraction pools in the oocyte soluble component seem to be tissue specific, since the reverse patterns were found in either frog brain or rat (Rattus norvegicus) brain tissue under the same conditions. Microtubule poisons, taxol and nocodazole, were used to test the dynamics of tubulin in MDa fractions and the results were opposite to that expected, i.e., taxol, a microtubule stabilizer, decreased tubulin in large MDa fractions while nocodazole, a microtubule destabilizer, increased it. Preliminary data also indicated that progesterone treatment alters the tubulin size classes (size-shift) in comparison with that in immature oocytes. These results, in combination with other research reports, suggest that tubulin in MDa fractions may either be associated with other cellular components or act as intermediates in microtubule dynamics, perhaps via oligomerization, during oocyte maturation and early development.

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