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Androgens and spermatogenesis: lessons from transgenic mouse models.

Verhoeven G, Willems A, Denolet E, Swinnen JV, De Gendt K - Philos. Trans. R. Soc. Lond., B, Biol. Sci. (2010)

Bottom Line: Cell-selective ablation of the androgen receptor (AR) in Sertoli cells (SC) results in a complete block in meiosis and unambiguously identifies the SC as the main cellular mediator of the effects of androgens on spermatogenesis.Genes related to tubular restructuring, cell junction dynamics, the cytoskeleton, solute transportation and vitamin A metabolism are prominently present.Further research will be needed to decide which of these genes are physiologically relevant and to identify genes that can be used as diagnostic tools or targets to modulate the effects of androgens in spermatogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Medicine, Laboratory for Experimental Medicine and Endocrinology, Katholieke Universiteit Leuven, Gasthuisberg, Herestraat 49, 3000 Leuven, Belgium. guido.verhoeven@med.kuleuven.be

ABSTRACT
Transgenic mouse models have contributed considerably to our understanding of the cellular and molecular mechanisms by which androgens control spermatogenesis. Cell-selective ablation of the androgen receptor (AR) in Sertoli cells (SC) results in a complete block in meiosis and unambiguously identifies the SC as the main cellular mediator of the effects of androgens on spermatogenesis. This conclusion is corroborated by similar knockouts in other potential testicular target cells. Mutations resulting in diminished expression of the AR or in alleles with increased length of the CAG repeat mimick specific human forms of disturbed fertility that are not accompanied by defects in male sexual development. Transcriptional profiling studies in mice with cell-selective and general knockouts of the AR, searching for androgen-regulated genes relevant to the control of spermatogenesis, have identified many candidate target genes. However, with the exception of Rhox5, the identified subsets of genes show little overlap. Genes related to tubular restructuring, cell junction dynamics, the cytoskeleton, solute transportation and vitamin A metabolism are prominently present. Further research will be needed to decide which of these genes are physiologically relevant and to identify genes that can be used as diagnostic tools or targets to modulate the effects of androgens in spermatogenesis.

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Related in: MedlinePlus

Expression pattern (from days 8 to 20) for a subset of genes originally identified as differentially expressed in SCARKO and control mice on day 10. The genes studied are Rhox5 (Pem), Eppin, Galgt1 (β-1,4-acetylgalactosaminyltransferase), Drd4 (dopamine receptor D4), Tsx (testis-specific X-linked), Gpd1 (glycerol-3-phosphate dehydrogenase 1), Tubb3 (tubulin β3), PCI (protein C inhibitor) and Tpd52l1 (tumour protein D52-like 1). Expression was assessed by microarray analysis and quantitative RT-PCR (qPCR). Left axis (bars): expression levels measured by qPCR in testes of control and SCARKO mice of the indicated ages (n = 3). Data were normalized to an external luciferase standard. All values are expressed as a percentage of the highest value measured for the corresponding gene arbitrarily set at 100. Values represent the mean ± s.e.m. of three measurements. Right axis (lines): gene expression measured by microarray analysis on a pool of mRNA from three testes of three control or SCARKO mice of the indicated ages. Data were expressed as a percentage of the highest signal observed for the studied gene, arbitrarily set at 100. Notice that qPCR confirms differential expression between SCARKO and control on day 10 for all the genes identified by microarray analysis. While the microarray data (reflecting the number of transcripts in a given amount of RNA) suggest a decrease in the transcript levels for most of the studied genes, and for some of them a loss of differential expression, the qPCR measurements (corrected for exogenously added luciferase and accordingly reflecting transcript levels per testis) show that this is an artefact caused by the increased contribution of developing germ cells to the total amount of RNA selectively in the control. The experiment illustrates that genes differentially expressed in SC may be missed by microarray analysis on samples with different degrees of germ cell maturation. Unfilled bar, control (qPCR); filled bar, SCARKO (qPCR); unfilled circle, control (microarray); filled circle, SCARKO (microarray).
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RSTB20090117F2: Expression pattern (from days 8 to 20) for a subset of genes originally identified as differentially expressed in SCARKO and control mice on day 10. The genes studied are Rhox5 (Pem), Eppin, Galgt1 (β-1,4-acetylgalactosaminyltransferase), Drd4 (dopamine receptor D4), Tsx (testis-specific X-linked), Gpd1 (glycerol-3-phosphate dehydrogenase 1), Tubb3 (tubulin β3), PCI (protein C inhibitor) and Tpd52l1 (tumour protein D52-like 1). Expression was assessed by microarray analysis and quantitative RT-PCR (qPCR). Left axis (bars): expression levels measured by qPCR in testes of control and SCARKO mice of the indicated ages (n = 3). Data were normalized to an external luciferase standard. All values are expressed as a percentage of the highest value measured for the corresponding gene arbitrarily set at 100. Values represent the mean ± s.e.m. of three measurements. Right axis (lines): gene expression measured by microarray analysis on a pool of mRNA from three testes of three control or SCARKO mice of the indicated ages. Data were expressed as a percentage of the highest signal observed for the studied gene, arbitrarily set at 100. Notice that qPCR confirms differential expression between SCARKO and control on day 10 for all the genes identified by microarray analysis. While the microarray data (reflecting the number of transcripts in a given amount of RNA) suggest a decrease in the transcript levels for most of the studied genes, and for some of them a loss of differential expression, the qPCR measurements (corrected for exogenously added luciferase and accordingly reflecting transcript levels per testis) show that this is an artefact caused by the increased contribution of developing germ cells to the total amount of RNA selectively in the control. The experiment illustrates that genes differentially expressed in SC may be missed by microarray analysis on samples with different degrees of germ cell maturation. Unfilled bar, control (qPCR); filled bar, SCARKO (qPCR); unfilled circle, control (microarray); filled circle, SCARKO (microarray).

Mentions: A first AR-knockout study compared transcript levels in testes derived from 10-day-old SCARKO and control mice (Denolet et al. 2006a). Theoretically, SCARKO mice represent an ideal experimental paradigm to search for genes that depend on the AR in SC for their expression. The age of 10 days was selected as up to that time, at the onset of meiosis, the composition of the testis in SCARKO and control mice is indistinguishable and as effects of androgens on Rhox5 expression were previously observed from day 9 on. Microarray analysis revealed that a surprisingly high number of genes (at least 692) were already differentially expressed on day 10. Forty of them (further referred to as ‘strongly regulated’) displayed a difference of at least twofold (28 downregulated and 12 upregulated in the SCARKO). The physiological relevance of the identified set of genes was supported by the observation that Rhox5 displayed the highest degree of differential expression, and that the subset of strongly regulated genes included at least three genes (Galgt1 (β-1,4-acetylgalactosaminyltransferase), PCI (protein C inhibitor) and Eppin (a serine protease inhibitor)) that were already known to cause male infertility when inactivated, and at least six genes (Rhox5, Eppin, Gpd1 (glycerol-3-phosphate dehydrogenase 1), Tubb3 (tubulin-β3), Tpd52l1 (tumour protein D52-like 1) and PCI) for which there was previous evidence for androgen regulation in the testis or other tissues. Nine of the strongly regulated genes (Rhox5, Eppin, Galgt1, Drd4 (dopamine receptor D4), Tsx (testis-specific X-linked gene), Gpd1, Tubb3, PCI, Tpd52l1) were studied in more detail. Decreased expression in the SCARKO from day 8 up to day 20 (figure 2) and preferential expression in the tubular compartment could be confirmed by quantitative RT-PCR (qPCR). Androgen regulation could also be confirmed in independent systems such as prepubertal mice treated with antiandrogens and organotypic cultures of 8-day-old testes (De Gendt et al. 2009). A functional analysis of the 692 differentially expressed genes pointed to an over-representation of genes involved in signal transduction, mitogen-activated protein kinase (MAPK) activity, cell adhesion, calcium binding, insulin-like growth factor (IGF) binding and, perhaps most strikingly, serine protease inhibition. In fact, five of these SERPINS were downregulated more than twofold in the SCARKO. A microarray time study (days 8–20) indicated that, apart from serine protease inhibitors, also serine proteases, cell adhesion molecules, cytoskeletal elements and extracellular matrix components displayed early and important differences in expression, supporting the hypothesis that tubular restructuring and changes in cell junction dynamics may be processes targeted by androgens during early puberty. As the AR in mouse SC appears from day 5 on, a complementary microarray study was performed on day 6 to search for genes that might display a very early (maybe transient) primary response to androgens and that might subsequently activate secondary cascades of androgen effects (Willems et al. 2009). Unfortunately, no such genes could be identified but the study confirmed that several of the genes identified in the day 10 study already display responses at or before day 6.


Androgens and spermatogenesis: lessons from transgenic mouse models.

Verhoeven G, Willems A, Denolet E, Swinnen JV, De Gendt K - Philos. Trans. R. Soc. Lond., B, Biol. Sci. (2010)

Expression pattern (from days 8 to 20) for a subset of genes originally identified as differentially expressed in SCARKO and control mice on day 10. The genes studied are Rhox5 (Pem), Eppin, Galgt1 (β-1,4-acetylgalactosaminyltransferase), Drd4 (dopamine receptor D4), Tsx (testis-specific X-linked), Gpd1 (glycerol-3-phosphate dehydrogenase 1), Tubb3 (tubulin β3), PCI (protein C inhibitor) and Tpd52l1 (tumour protein D52-like 1). Expression was assessed by microarray analysis and quantitative RT-PCR (qPCR). Left axis (bars): expression levels measured by qPCR in testes of control and SCARKO mice of the indicated ages (n = 3). Data were normalized to an external luciferase standard. All values are expressed as a percentage of the highest value measured for the corresponding gene arbitrarily set at 100. Values represent the mean ± s.e.m. of three measurements. Right axis (lines): gene expression measured by microarray analysis on a pool of mRNA from three testes of three control or SCARKO mice of the indicated ages. Data were expressed as a percentage of the highest signal observed for the studied gene, arbitrarily set at 100. Notice that qPCR confirms differential expression between SCARKO and control on day 10 for all the genes identified by microarray analysis. While the microarray data (reflecting the number of transcripts in a given amount of RNA) suggest a decrease in the transcript levels for most of the studied genes, and for some of them a loss of differential expression, the qPCR measurements (corrected for exogenously added luciferase and accordingly reflecting transcript levels per testis) show that this is an artefact caused by the increased contribution of developing germ cells to the total amount of RNA selectively in the control. The experiment illustrates that genes differentially expressed in SC may be missed by microarray analysis on samples with different degrees of germ cell maturation. Unfilled bar, control (qPCR); filled bar, SCARKO (qPCR); unfilled circle, control (microarray); filled circle, SCARKO (microarray).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2871915&req=5

RSTB20090117F2: Expression pattern (from days 8 to 20) for a subset of genes originally identified as differentially expressed in SCARKO and control mice on day 10. The genes studied are Rhox5 (Pem), Eppin, Galgt1 (β-1,4-acetylgalactosaminyltransferase), Drd4 (dopamine receptor D4), Tsx (testis-specific X-linked), Gpd1 (glycerol-3-phosphate dehydrogenase 1), Tubb3 (tubulin β3), PCI (protein C inhibitor) and Tpd52l1 (tumour protein D52-like 1). Expression was assessed by microarray analysis and quantitative RT-PCR (qPCR). Left axis (bars): expression levels measured by qPCR in testes of control and SCARKO mice of the indicated ages (n = 3). Data were normalized to an external luciferase standard. All values are expressed as a percentage of the highest value measured for the corresponding gene arbitrarily set at 100. Values represent the mean ± s.e.m. of three measurements. Right axis (lines): gene expression measured by microarray analysis on a pool of mRNA from three testes of three control or SCARKO mice of the indicated ages. Data were expressed as a percentage of the highest signal observed for the studied gene, arbitrarily set at 100. Notice that qPCR confirms differential expression between SCARKO and control on day 10 for all the genes identified by microarray analysis. While the microarray data (reflecting the number of transcripts in a given amount of RNA) suggest a decrease in the transcript levels for most of the studied genes, and for some of them a loss of differential expression, the qPCR measurements (corrected for exogenously added luciferase and accordingly reflecting transcript levels per testis) show that this is an artefact caused by the increased contribution of developing germ cells to the total amount of RNA selectively in the control. The experiment illustrates that genes differentially expressed in SC may be missed by microarray analysis on samples with different degrees of germ cell maturation. Unfilled bar, control (qPCR); filled bar, SCARKO (qPCR); unfilled circle, control (microarray); filled circle, SCARKO (microarray).
Mentions: A first AR-knockout study compared transcript levels in testes derived from 10-day-old SCARKO and control mice (Denolet et al. 2006a). Theoretically, SCARKO mice represent an ideal experimental paradigm to search for genes that depend on the AR in SC for their expression. The age of 10 days was selected as up to that time, at the onset of meiosis, the composition of the testis in SCARKO and control mice is indistinguishable and as effects of androgens on Rhox5 expression were previously observed from day 9 on. Microarray analysis revealed that a surprisingly high number of genes (at least 692) were already differentially expressed on day 10. Forty of them (further referred to as ‘strongly regulated’) displayed a difference of at least twofold (28 downregulated and 12 upregulated in the SCARKO). The physiological relevance of the identified set of genes was supported by the observation that Rhox5 displayed the highest degree of differential expression, and that the subset of strongly regulated genes included at least three genes (Galgt1 (β-1,4-acetylgalactosaminyltransferase), PCI (protein C inhibitor) and Eppin (a serine protease inhibitor)) that were already known to cause male infertility when inactivated, and at least six genes (Rhox5, Eppin, Gpd1 (glycerol-3-phosphate dehydrogenase 1), Tubb3 (tubulin-β3), Tpd52l1 (tumour protein D52-like 1) and PCI) for which there was previous evidence for androgen regulation in the testis or other tissues. Nine of the strongly regulated genes (Rhox5, Eppin, Galgt1, Drd4 (dopamine receptor D4), Tsx (testis-specific X-linked gene), Gpd1, Tubb3, PCI, Tpd52l1) were studied in more detail. Decreased expression in the SCARKO from day 8 up to day 20 (figure 2) and preferential expression in the tubular compartment could be confirmed by quantitative RT-PCR (qPCR). Androgen regulation could also be confirmed in independent systems such as prepubertal mice treated with antiandrogens and organotypic cultures of 8-day-old testes (De Gendt et al. 2009). A functional analysis of the 692 differentially expressed genes pointed to an over-representation of genes involved in signal transduction, mitogen-activated protein kinase (MAPK) activity, cell adhesion, calcium binding, insulin-like growth factor (IGF) binding and, perhaps most strikingly, serine protease inhibition. In fact, five of these SERPINS were downregulated more than twofold in the SCARKO. A microarray time study (days 8–20) indicated that, apart from serine protease inhibitors, also serine proteases, cell adhesion molecules, cytoskeletal elements and extracellular matrix components displayed early and important differences in expression, supporting the hypothesis that tubular restructuring and changes in cell junction dynamics may be processes targeted by androgens during early puberty. As the AR in mouse SC appears from day 5 on, a complementary microarray study was performed on day 6 to search for genes that might display a very early (maybe transient) primary response to androgens and that might subsequently activate secondary cascades of androgen effects (Willems et al. 2009). Unfortunately, no such genes could be identified but the study confirmed that several of the genes identified in the day 10 study already display responses at or before day 6.

Bottom Line: Cell-selective ablation of the androgen receptor (AR) in Sertoli cells (SC) results in a complete block in meiosis and unambiguously identifies the SC as the main cellular mediator of the effects of androgens on spermatogenesis.Genes related to tubular restructuring, cell junction dynamics, the cytoskeleton, solute transportation and vitamin A metabolism are prominently present.Further research will be needed to decide which of these genes are physiologically relevant and to identify genes that can be used as diagnostic tools or targets to modulate the effects of androgens in spermatogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Medicine, Laboratory for Experimental Medicine and Endocrinology, Katholieke Universiteit Leuven, Gasthuisberg, Herestraat 49, 3000 Leuven, Belgium. guido.verhoeven@med.kuleuven.be

ABSTRACT
Transgenic mouse models have contributed considerably to our understanding of the cellular and molecular mechanisms by which androgens control spermatogenesis. Cell-selective ablation of the androgen receptor (AR) in Sertoli cells (SC) results in a complete block in meiosis and unambiguously identifies the SC as the main cellular mediator of the effects of androgens on spermatogenesis. This conclusion is corroborated by similar knockouts in other potential testicular target cells. Mutations resulting in diminished expression of the AR or in alleles with increased length of the CAG repeat mimick specific human forms of disturbed fertility that are not accompanied by defects in male sexual development. Transcriptional profiling studies in mice with cell-selective and general knockouts of the AR, searching for androgen-regulated genes relevant to the control of spermatogenesis, have identified many candidate target genes. However, with the exception of Rhox5, the identified subsets of genes show little overlap. Genes related to tubular restructuring, cell junction dynamics, the cytoskeleton, solute transportation and vitamin A metabolism are prominently present. Further research will be needed to decide which of these genes are physiologically relevant and to identify genes that can be used as diagnostic tools or targets to modulate the effects of androgens in spermatogenesis.

Show MeSH
Related in: MedlinePlus