The effects of oestradiol-17beta on the synthesis of ribosomal proteins in the immature rat uterus

Muller, Robert E (1983) The effects of oestradiol-17beta on the synthesis of ribosomal proteins in the immature rat uterus. PhD thesis, University of Glasgow.

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Abstract

The effects of oestrogen on the immature rat uterus may be summarised as follows. The hormone, circulating in the bloodstream enters uterine cells and binds to a receptor. The hormone receptor complex binds to chromatin and this leads to quantitative and qualitative changes in transcription. Newly synthesised hnRNA matures to mRNA and is translated by pre-existing ribosomes in the cytoplasm. A small number of new proteins are synthesised which are required for the subsequent stimulation of RNA and protein synthesis. These proteins are required for the transcription of new rRNA and mRNA encoding ribosomal proteins. New polysomes are then assembled and are responsible for protein synthesis in the developing uterus. Changes in RNA and protein bring about tissue hypertrophy which is followed by DNA replication and cell division. The resulting growth and development of the tissue initiates its preparation for the possible implantation of a fertilised ovum. Studies of protein synthesis in the uterus responding to oestrogen have revealed that major changes in the gross protein population were not to be found. The changes oestrogens induced were altogether too subtle to be revealed by the crude analysis of total protein. Therefore, one group of oestrogen-induced proteins was selected for detailed study, namely the ribosomal proteins. Previous work had shown that the synthesis of these proteins coincided with the appearance of new polysomes in vivo. However, the small quantity of ribosomal proteins in the uterus relative to the whole body of the rat severely limited these studies, and thus it was decided to pursue further studies by the use of two different procedures in vitro. The first of these procedures required the incubation, with radio-labelled precursors, of whole uteri in vitro after oestrogen stimulation vivo. The uteri, removed from immature rats after various periods of oestrogen treatment, were incubated with either [3H]-leucine, [35S]-methionine or [32P]-phosphate. In this way, the synthesis of total ribosomal proteins, and the synthesis and post-translational modification of individual proteins could be analysed. It was found that oestrogen stimulated ribosomal protein synthesis vitro in the same way as in vivo and that the maximal incorporation of new ribosomal proteins into ribosomes coincided with maximal polysome assembly. Incorporation of radioactivity into ribosomal proteins of the unstimulated rat uterus was insignificant, except in the case of the large-subunit protein L10. Oestradiol treatment of rats at various times before death was followed by stimulated incorporation of radio-labelled precursor into individual ribosomal proteins in vitro. This stimulation was maximal 12 hours after hormone administration and could not be accounted for by increased pool sizes. It was possible to quantify the effect of oestradiol on 17 small subunit proteins and 16 large subunit proteins. Response to the hormone was not uniform and varied from 3-fold stimulation of synthesis of L10 to an almost 5-fold increase in the synthesis of protein L19. Incubation of uteri with [32P]-inorganic phosphate resulted in the labelling of 2 large subunit proteins and 1 protein of the small subunit. However, an apparent increase in the incorporation of label after oestrogen stimulation could be accounted for by changes in the size of precursor pools. A second analytical approach involved the isolation of active uterine polysomes and their subsequent translation in a cell free protein synthesisiing system. The synthesis of total proteins increased from control levels until 8-12 hours after oestrogen treatment and then declined to control levels once more. The proportion of ribosomal proteins synthesised in this system became nearly maximal at 2 hours and their synthesis remained a constant proportion of the total activity of uterine polysomes until polysome disassembly after 12 hours. An anti-ribosomal protein antibody was raised in rabbits and used to determine the antigenicity of uterine homogenate after oestrogen treatment of rats. The results produced by this analysis were unexpected in that they revealed an apparent decrease in antigenicity after 12 hours. This conflicted with the current idea of ribosome turnover requiring many days, but could be rationalised in terms of a pool of free ribosomal proteins becoming less antigenic as they were incorporated into ribonucleoprotein particles. These results were compared to other relevant work and discussed with a view to future studies.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: John Knowler
Keywords: Endocrinology
Date of Award: 1983
Depositing User: Enlighten Team
Unique ID: glathesis:1983-71628
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 10 May 2019 14:03
Last Modified: 10 May 2019 14:03
URI: http://theses.gla.ac.uk/id/eprint/71628

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