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Androgen receptor signalling in Vascular Endothelial cells is dispensable for spermatogenesis and male fertility.

O'Hara L, Smith LB - BMC Res Notes (2012)

Bottom Line: Immunofluorescent detection revealed expression of YFP (and therefore Cre Recombinase function) limited to VE cells and an interstitial population of cells, believed to be macrophages, that did not express AR.Ablation of AR from all VE cells was confirmed; however, no significant differences in bodyweight or reproductive tissue weights could be detected in VEARKO animals and spermatogenesis and fertility was unaffected.We demonstrate the successful generation and empirical validation of a cell-specific knockout of AR from VE cells, and conclude that AR expression in VE cells is not essential for spermatogenesis or male fertility.

View Article: PubMed Central - HTML - PubMed

Affiliation: MRC Centre for Reproductive Health, University of Edinburgh, The Queen's Medical Research Institute, 47 Little France Crescent, Edinburgh EH16 4TJ, UK. L.OHara@ed.ac.uk.

ABSTRACT

Background: Androgen signalling is essential both for male development and function of the male reproductive system in adulthood. Within the adult testis, Germ cells (GC) do not express androgen receptor (AR) suggesting androgen-mediated promotion of spermatogenesis must act via AR-expressing somatic cell-types. Several recent studies have exploited the Cre/lox system of conditional gene-targeting to ablate AR function from key somatic cell-types in order to establish the cell-specific role of AR in promotion of male fertility. In this study, we have used a similar approach to specifically ablate AR-signalling from Vascular Endothelial (VE) cells, with a view to defining the significance of androgen signalling within this cell-type on spermatogenesis.

Findings: AR expression in VE cells of the testicular vasculature was confirmed using an antibody against AR. A Cre-inducible fluorescent reporter line was used to empirically establish the utility of a mouse line expressing Cre Recombinase driven by the Tie2-Promoter, for targeting VE cells. Immunofluorescent detection revealed expression of YFP (and therefore Cre Recombinase function) limited to VE cells and an interstitial population of cells, believed to be macrophages, that did not express AR. Mating of Tie2-Cre males to females carrying a floxed AR gene produced Vascular Endothelial Androgen Receptor Knockout (VEARKO) mice and littermate controls. Ablation of AR from all VE cells was confirmed; however, no significant differences in bodyweight or reproductive tissue weights could be detected in VEARKO animals and spermatogenesis and fertility was unaffected.

Conclusions: We demonstrate the successful generation and empirical validation of a cell-specific knockout of AR from VE cells, and conclude that AR expression in VE cells is not essential for spermatogenesis or male fertility.

No MeSH data available.


Related in: MedlinePlus

A: VEARKO mice can be separated from controls through PCR interrogation of inheritance of Cre Recombinase. 324 bp amplicon = internal positive control. 102 bp amplicon = Cre Recombinase. NTC = no-template control. B: PCR amplification of AR using primers spanning the floxed exon 2 on genomic DNA isolated from VEARKO and control testes demonstrates that a proportion of cells in the testes of VEARKO mice contain a recombined AR (612 bp). No recombination is observed in testes from control animals. Testes from total-ARKO mice, which demonstrate 100% recombination of AR, are included for comparison. C: AR expression is observed in endothelial cells of testicular blood vessels from Control mice (arrows); D: Conversely, no endothelial cells in testicular blood vessels of VEARKO mice express AR (arrowheads). Scale bars are 20 μm.
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Figure 2: A: VEARKO mice can be separated from controls through PCR interrogation of inheritance of Cre Recombinase. 324 bp amplicon = internal positive control. 102 bp amplicon = Cre Recombinase. NTC = no-template control. B: PCR amplification of AR using primers spanning the floxed exon 2 on genomic DNA isolated from VEARKO and control testes demonstrates that a proportion of cells in the testes of VEARKO mice contain a recombined AR (612 bp). No recombination is observed in testes from control animals. Testes from total-ARKO mice, which demonstrate 100% recombination of AR, are included for comparison. C: AR expression is observed in endothelial cells of testicular blood vessels from Control mice (arrows); D: Conversely, no endothelial cells in testicular blood vessels of VEARKO mice express AR (arrowheads). Scale bars are 20 μm.

Mentions: To generate Vascular Endothelial Androgen Receptor Knock-Out (VEARKO) mice and littermate controls, heterozygous Tie2-Cre males were mated to homozygous ARflox [5] females. Offspring were genotyped by PCR to establish inheritance of the Tie2-Cre transgene (Figure 2a).


Androgen receptor signalling in Vascular Endothelial cells is dispensable for spermatogenesis and male fertility.

O'Hara L, Smith LB - BMC Res Notes (2012)

A: VEARKO mice can be separated from controls through PCR interrogation of inheritance of Cre Recombinase. 324 bp amplicon = internal positive control. 102 bp amplicon = Cre Recombinase. NTC = no-template control. B: PCR amplification of AR using primers spanning the floxed exon 2 on genomic DNA isolated from VEARKO and control testes demonstrates that a proportion of cells in the testes of VEARKO mice contain a recombined AR (612 bp). No recombination is observed in testes from control animals. Testes from total-ARKO mice, which demonstrate 100% recombination of AR, are included for comparison. C: AR expression is observed in endothelial cells of testicular blood vessels from Control mice (arrows); D: Conversely, no endothelial cells in testicular blood vessels of VEARKO mice express AR (arrowheads). Scale bars are 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3275443&req=5

Figure 2: A: VEARKO mice can be separated from controls through PCR interrogation of inheritance of Cre Recombinase. 324 bp amplicon = internal positive control. 102 bp amplicon = Cre Recombinase. NTC = no-template control. B: PCR amplification of AR using primers spanning the floxed exon 2 on genomic DNA isolated from VEARKO and control testes demonstrates that a proportion of cells in the testes of VEARKO mice contain a recombined AR (612 bp). No recombination is observed in testes from control animals. Testes from total-ARKO mice, which demonstrate 100% recombination of AR, are included for comparison. C: AR expression is observed in endothelial cells of testicular blood vessels from Control mice (arrows); D: Conversely, no endothelial cells in testicular blood vessels of VEARKO mice express AR (arrowheads). Scale bars are 20 μm.
Mentions: To generate Vascular Endothelial Androgen Receptor Knock-Out (VEARKO) mice and littermate controls, heterozygous Tie2-Cre males were mated to homozygous ARflox [5] females. Offspring were genotyped by PCR to establish inheritance of the Tie2-Cre transgene (Figure 2a).

Bottom Line: Immunofluorescent detection revealed expression of YFP (and therefore Cre Recombinase function) limited to VE cells and an interstitial population of cells, believed to be macrophages, that did not express AR.Ablation of AR from all VE cells was confirmed; however, no significant differences in bodyweight or reproductive tissue weights could be detected in VEARKO animals and spermatogenesis and fertility was unaffected.We demonstrate the successful generation and empirical validation of a cell-specific knockout of AR from VE cells, and conclude that AR expression in VE cells is not essential for spermatogenesis or male fertility.

View Article: PubMed Central - HTML - PubMed

Affiliation: MRC Centre for Reproductive Health, University of Edinburgh, The Queen's Medical Research Institute, 47 Little France Crescent, Edinburgh EH16 4TJ, UK. L.OHara@ed.ac.uk.

ABSTRACT

Background: Androgen signalling is essential both for male development and function of the male reproductive system in adulthood. Within the adult testis, Germ cells (GC) do not express androgen receptor (AR) suggesting androgen-mediated promotion of spermatogenesis must act via AR-expressing somatic cell-types. Several recent studies have exploited the Cre/lox system of conditional gene-targeting to ablate AR function from key somatic cell-types in order to establish the cell-specific role of AR in promotion of male fertility. In this study, we have used a similar approach to specifically ablate AR-signalling from Vascular Endothelial (VE) cells, with a view to defining the significance of androgen signalling within this cell-type on spermatogenesis.

Findings: AR expression in VE cells of the testicular vasculature was confirmed using an antibody against AR. A Cre-inducible fluorescent reporter line was used to empirically establish the utility of a mouse line expressing Cre Recombinase driven by the Tie2-Promoter, for targeting VE cells. Immunofluorescent detection revealed expression of YFP (and therefore Cre Recombinase function) limited to VE cells and an interstitial population of cells, believed to be macrophages, that did not express AR. Mating of Tie2-Cre males to females carrying a floxed AR gene produced Vascular Endothelial Androgen Receptor Knockout (VEARKO) mice and littermate controls. Ablation of AR from all VE cells was confirmed; however, no significant differences in bodyweight or reproductive tissue weights could be detected in VEARKO animals and spermatogenesis and fertility was unaffected.

Conclusions: We demonstrate the successful generation and empirical validation of a cell-specific knockout of AR from VE cells, and conclude that AR expression in VE cells is not essential for spermatogenesis or male fertility.

No MeSH data available.


Related in: MedlinePlus