Abstract
BackgroundPost-meiotically expressed genes in the testis are essential for the proper progression of spermatogenesis, and yet, aside from the construction of individual transgenic mice using specific promoters to drive reporter plasmids, there are only very limited possibilities for relevant and quantitative analysis of gene promoters. This is due to the special nature of post-meiotic haploid cells, which to date are not represented in any appropriate cell-lines. This article reports the development of novel methodology using isolated and cultured rat seminiferous tubules in a multiwell format, into which promoter-reporter constructs can be introduced by a combination of microinjection and electroporation.MethodsCulture conditions were developed which allowed the continued incubation of isolated rat seminiferous tubules for up to 48 h without obvious cell death and loss of post-meiotic cells. Transfection of intact seminiferous tubules by microinjection and electroporation was optimized to achieve high expression efficiencies of control plasmids, using either fluorescent protein or luciferase as reporters, thereby allowing both morphological as well as quantitative assessment.ResultsSuccessful transfection was achieved into all cell types except for mature spermatozoa. However, there appeared to be only limited cell-type specificity for the promoters used, even though these had appeared to be specific when used in transgenic animals.ConclusionWe have devised a methodology which allows relatively high throughput analysis of post-meiotic gene promoters into primary cells of intact seminiferous tubules. An apparent lack of cell-type specificity suggests that the gene fragments used do not contain sufficient targeting information, or that the transient episomal expression of the constructs does not encourage appropriate expression specificity. The results also highlight the doubtful interpretation of many studies using heterologous transfection systems to analyse post-meiotically expressed genes.
Highlights
Post-meiotically expressed genes in the testis are essential for the proper progression of spermatogenesis, and yet, aside from the construction of individual transgenic mice using specific promoters to drive reporter plasmids, there are only very limited possibilities for relevant and quantitative analysis of gene promoters
There are reports of direct in vivo transfection of gene constructs into the exposed seminiferous tubules of rodent testes, again with the limitation that a single testis is required for each construct, and that there are no appropriate means of quantification of the specific gene expression [12,13,14,15,16]
In order to verify the persistent presence of late post-meiotic germ cells, specific antibodies for the spermatid marker endozepine-like peptide (ELP) [19] were used (Fig. 2)
Summary
Post-meiotically expressed genes in the testis are essential for the proper progression of spermatogenesis, and yet, aside from the construction of individual transgenic mice using specific promoters to drive reporter plasmids, there are only very limited possibilities for relevant and quantitative analysis of gene promoters. Conventional gain-offunction transgenesis has been used to assess promoter specificity for post-meiotic genes [7,8,9,10] This approach is largely limited to mice, and the fact that only a single construct can be used per individual animal has severely restricted both statistical analysis and a more detailed molecular dissection of promoter regions. Another procedure, which has permitted the analysis of post-meiotic promoters, is to transfect isolated spermatogonia in vitro, and to transplant these into the testes of prepubertal or azoospermic animals [11]. There are reports of direct in vivo transfection of gene constructs into the exposed seminiferous tubules of rodent testes, again with the limitation that a single testis is required for each construct, and that there are no appropriate means of quantification of the specific gene expression [12,13,14,15,16]
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