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Title: Inducible gene targeting in the male : tamoxifen adversely impacts postnatal testicular development and function
Author: Patel, Saloni Hiten
ISNI:       0000 0004 6499 9925
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2016
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Normal development and function of the male reproductive tract relies on the crucial balance between androgen and estrogen signalling, furthermore estrogens play an important role in the regulation of spermatogenesis and steroidogenesis. Tamoxifen (TAM) inducible Cre-loxP systems are widely used to study testicular function. TAM is a selective estrogen receptor modulator (SERM), thereby exerting anti- and pro-estrogenic effects. Therefore, it was hypothesised that acute (single dose) TAM administration to the postnatal testis has significant long-term effects on testicular development and adult testis function, questioning its utility in inducible transgenic systems. A suitable Cre line was first validated as a tool to target the postnatal adult Leydig cell (ALC) population. The Nestin-Cre expressing stem Leydig cells (LCs) were demonstrated as a source of a subset of the ALCs. Hence, a TAM inducible Nestin- Cre line was one of the mouse lines employed for further studies. A comprehensive investigation was carried out to assess any short-and long-term testicular phenotype upon administration of high (3mg) and low (1mg, 500ug and 250ug) doses of TAM. These studies were carried out in TAM-inducible Nestin- Cre/ERT2 and PDGFRA-Cre/ERT2 mouse lines as well as C57Bl/6 mice, to ensure that the observations made were independent of transgene effects. High dose TAM treatment resulted in transgene induction, however this also caused short-term spermatogenic arrest, alterations to steroidogenesis and LC number. Spermatogenesis recovers in young adults, but LCs show delayed maturation, suggesting changes in developmental programming of the ALC population. Thus it was concluded that a single dose of TAM in early postnatal life disrupts testicular function in adulthood. Single low doses of TAM did not induce the transgene, but surprisingly also had a long-term impact on ALC development, steroidogenesis and spermatogenesis. Severity of the phenotype worsened with dose concentration, indicating dose dependent impacts of TAM on the testis. Therefore, TAM has adverse impacts on the testis at doses below the threshold of Cre induction. In order to find a substitute for TAM in transgene induction studies, Raloxifene (RAL), another SERM, was hypothesised to induce transgenes with minimal disruption of testicular function. A 3mg dose of RAL did not show the adverse impacts of TAM. However, different dose regimens were assayed to induce the transgene without success, hence ruling out RAL as a substitute for TAM. Given the severity of previously undocumented TAM-induced phenotypes elucidated in these studies, it is evident that the off-target effects of TAM are severely underappreciated and can cause long-term programming effects. These off-target effects are likely to be present in other estrogen responsive tissues. Hence TAM-inducible Cre systems should be used with rigorous controls, to ensure correct conclusions are drawn from results obtained.
Supervisor: Smith, Lee ; Sharpe, Richard Sponsor: Medical Research Council (MRC)
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Tamoxifen ; testis ; Leydig cells