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Spermatogenesis in the rat: Isolation of post-meiotic germ cells and characterisation of stage-specific gene expression

Pelengaris, Stella Anne; (1995) Spermatogenesis in the rat: Isolation of post-meiotic germ cells and characterisation of stage-specific gene expression. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

Spermatogenesis is largely dependent on stage-specific gene expression that takes place within the developing germ cell, and requires the close association between each spermatogenic cell stage and the supporting Sertoli cell. The aim of my thesis was to isolate purified populations of the post-meiotic germ cell (round spermatids) from the rat, and to characterise genes expressed at this cell stage which might encode proteins targeted to the acrosome A novel panning technique was devised which allowed the isolation of 95% pure round spermatids from adult rat testis, exceeding purities obtained by sedimentation through a BSA gradient. Subsequently, two experimental strategies were devised which included the use of antiserum raised against mammalian acrosomal membranes to 1) immunologically isolate specific polysomal mRNA or 2) to screen a human testis cDNA expression library. Although limitations were encountered using the polysome approach, a 2 kb cDNA clone (352) was isolated following library screening, which appeared to derive from a highly conserved, novel gene Low levels of 352 mRNA expression were evident in a range of human tissues, but were clearly up-regulated in the testis Before 15 days of age, 352 mRNA was not apparently expressed in the rat testis. Between 15 and 22 days of age, expression was at a very low level, coinciding with the initial accumulation of pachytene spermatocytes within the seminiferous epithelium. However, in males of 27 days or older, 352 mRNA was significantly elevated. This expression was concomitant with the appearance and subsequent differentiation of stage 1-5 spermatids. Surprisingly, Northern blot analysis indicated that round spermatids were not the cell type responsible for the elevated levels of expression of 352 mRNA in 27 day old rats. It was hypothesized that this up-regulation might be the result of predominant expression in Sertoli cells, such that, the appearance of round spermatids could induce Sertoli cells to synthesize 352 mRNA, and subsequently transfer the protein product to round spermatids where it may be targeted to the acrosome.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Spermatogenesis in the rat: Isolation of post-meiotic germ cells and characterisation of stage-specific gene expression
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
URI: https://discovery.ucl.ac.uk/id/eprint/10103243
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