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Retinoic Acid Receptors Control Spermatogonia Cell-Fate and Induce Expression of the SALL4A Transcription Factor.

Gely-Pernot A, Raverdeau M, Teletin M, Vernet N, Féret B, Klopfenstein M, Dennefeld C, Davidson I, Benoit G, Mark M, Ghyselinck NB - PLoS Genet. (2015)

Bottom Line: We also show that ATRA activates RAR and RXR bound to a conserved regulatory region to increase expression of the SALL4A transcription factor in spermatogonia.Our results reveal that this major pluripotency gene is a target of ATRA signaling and that RAR/RXR heterodimers are the functional units driving its expression in spermatogonia.They add to the mechanisms through which ATRA promote expression of the KIT tyrosine kinase receptor to trigger a critical step in spermatogonia differentiation.

View Article: PubMed Central - PubMed

Affiliation: Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Département de Génétique Fonctionnelle et Cancer, Illkirch, France; Centre National de la Recherche Scientifique (CNRS), UMR7104, Illkirch, France; Institut National de la Santé et de la Recherche Médicale (INSERM), U964, Illkirch, France; Université de Strasbourg (UNISTRA), Illkirch Cedex, France.

ABSTRACT
All-trans retinoic acid (ATRA) is instrumental to male germ cell differentiation, but its mechanism of action remains elusive. To address this question, we have analyzed the phenotypes of mice lacking, in spermatogonia, all rexinoid receptors (RXRA, RXRB and RXRG) or all ATRA receptors (RARA, RARB and RARG). We demonstrate that the combined ablation of RXRA and RXRB in spermatogonia recapitulates the set of defects observed both upon ablation of RAR in spermatogonia. We also show that ATRA activates RAR and RXR bound to a conserved regulatory region to increase expression of the SALL4A transcription factor in spermatogonia. Our results reveal that this major pluripotency gene is a target of ATRA signaling and that RAR/RXR heterodimers are the functional units driving its expression in spermatogonia. They add to the mechanisms through which ATRA promote expression of the KIT tyrosine kinase receptor to trigger a critical step in spermatogonia differentiation. Importantly, they indicate also that meiosis eventually occurs in the absence of a RAR/RXR pathway within germ cells and suggest that instructing this process is either ATRA-independent or requires an ATRA signal originating from Sertoli cells.

No MeSH data available.


Related in: MedlinePlus

Sall4a expression in undifferentiated spermatogonia is controlled by ligand-activated RARG.(A) Relative expression of Sall4a mRNA quantified by RT-qPCR in Aldh1a1-3Ser−/− testes cultured in the absence (−) or in the presence (+) of cycloheximide (CHX) and treated for 6 hours with vehicle (white bar) and BM961 (grey bars). Error bars represent s.e.m. (n = 5); * p < 0.05. (B) Western blot analysis of protein extracts from testes of mutants as indicated treated with BMS961 (+) or with vehicle (−), using anti-SALL4 or anti-ACTIN antibodies. NS points to an unspecific signal. (C) Relative expression of Sall4a, Sall4b and Zbtb16 mRNA quantified by RT-qPCR in whole testes from control (white bars), Rara;b;gSpg−/− (grey bars) and Rxra;b;gSpg−/− (black bars) mice at PN60. Error bars represent s.e.m. (n = 5); * p < 0.05.
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pgen.1005501.g006: Sall4a expression in undifferentiated spermatogonia is controlled by ligand-activated RARG.(A) Relative expression of Sall4a mRNA quantified by RT-qPCR in Aldh1a1-3Ser−/− testes cultured in the absence (−) or in the presence (+) of cycloheximide (CHX) and treated for 6 hours with vehicle (white bar) and BM961 (grey bars). Error bars represent s.e.m. (n = 5); * p < 0.05. (B) Western blot analysis of protein extracts from testes of mutants as indicated treated with BMS961 (+) or with vehicle (−), using anti-SALL4 or anti-ACTIN antibodies. NS points to an unspecific signal. (C) Relative expression of Sall4a, Sall4b and Zbtb16 mRNA quantified by RT-qPCR in whole testes from control (white bars), Rara;b;gSpg−/− (grey bars) and Rxra;b;gSpg−/− (black bars) mice at PN60. Error bars represent s.e.m. (n = 5); * p < 0.05.

Mentions: We confirmed by RT-qPCR that Sall4a mRNA steady state level was increased upon BMS961 administration in Aldh1a1-3Ser−/− testes, without the need for intermediate protein synthesis as this increase occurred in the presence of cycloheximide (Fig 6A). Western-blot analysis of protein extracts from Aldh1a1-3Ser−/− testes revealed that SALL4A protein level was increased by BMS961-activated RARG (Fig 6B, compare lane 1 to 2); this increase was prevented in mice additionally carrying a Rarg knock-out (Fig 6B, compare lane 3 to 4) and was not observed in BMS961-treated Rara;b;gSpg–/– mutants (Fig 6B, compare lane 5 to 6). In addition, Sall4a mRNA levels were significantly decreased in whole testis of Rara;b;gSpg–/– and Rxra;b;gSpg–/– mutants at PN60, while Sall4b and Zbtb16 mRNA levels were unchanged (Fig 6C). The finding that Sall4b mRNA level was not altered is in keeping with previous reports showing that SALL4B is expressed at a constant level in spermatogonia [31,32]. Altogether our results indicate that (i) Sall4a expression is decreased in testes of mice lacking RAR or RXR in spermatogonia (Rara;b;gSpg–/– and Rxra;b;gSpg–/– testes); (ii) SALL4A is detected at a low level in the seminiferous epithelium of mice deficient in ATRA (Aldh1a1-3Ser−/− testes), but at a high level when RARG is activated by BMS961 in these mice (Aldh1a1-3Ser−/− testes treated with BMS961), except when RARG is lacking (Aldh1a1-3Ser−/−;Rarg−/− testes, treated with BMS961); and (iii) SALL4A is not detected in testes of adult mice lacking RAR in spermatogonia even in the presence of the RARG agonist (Rara;b;gSpg–/– testes, treated with BMS961). Altogether, these data indicate that Sall4a expression is controlled by ATRA-activated RARG in spermatogonia.


Retinoic Acid Receptors Control Spermatogonia Cell-Fate and Induce Expression of the SALL4A Transcription Factor.

Gely-Pernot A, Raverdeau M, Teletin M, Vernet N, Féret B, Klopfenstein M, Dennefeld C, Davidson I, Benoit G, Mark M, Ghyselinck NB - PLoS Genet. (2015)

Sall4a expression in undifferentiated spermatogonia is controlled by ligand-activated RARG.(A) Relative expression of Sall4a mRNA quantified by RT-qPCR in Aldh1a1-3Ser−/− testes cultured in the absence (−) or in the presence (+) of cycloheximide (CHX) and treated for 6 hours with vehicle (white bar) and BM961 (grey bars). Error bars represent s.e.m. (n = 5); * p < 0.05. (B) Western blot analysis of protein extracts from testes of mutants as indicated treated with BMS961 (+) or with vehicle (−), using anti-SALL4 or anti-ACTIN antibodies. NS points to an unspecific signal. (C) Relative expression of Sall4a, Sall4b and Zbtb16 mRNA quantified by RT-qPCR in whole testes from control (white bars), Rara;b;gSpg−/− (grey bars) and Rxra;b;gSpg−/− (black bars) mice at PN60. Error bars represent s.e.m. (n = 5); * p < 0.05.
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Related In: Results  -  Collection

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pgen.1005501.g006: Sall4a expression in undifferentiated spermatogonia is controlled by ligand-activated RARG.(A) Relative expression of Sall4a mRNA quantified by RT-qPCR in Aldh1a1-3Ser−/− testes cultured in the absence (−) or in the presence (+) of cycloheximide (CHX) and treated for 6 hours with vehicle (white bar) and BM961 (grey bars). Error bars represent s.e.m. (n = 5); * p < 0.05. (B) Western blot analysis of protein extracts from testes of mutants as indicated treated with BMS961 (+) or with vehicle (−), using anti-SALL4 or anti-ACTIN antibodies. NS points to an unspecific signal. (C) Relative expression of Sall4a, Sall4b and Zbtb16 mRNA quantified by RT-qPCR in whole testes from control (white bars), Rara;b;gSpg−/− (grey bars) and Rxra;b;gSpg−/− (black bars) mice at PN60. Error bars represent s.e.m. (n = 5); * p < 0.05.
Mentions: We confirmed by RT-qPCR that Sall4a mRNA steady state level was increased upon BMS961 administration in Aldh1a1-3Ser−/− testes, without the need for intermediate protein synthesis as this increase occurred in the presence of cycloheximide (Fig 6A). Western-blot analysis of protein extracts from Aldh1a1-3Ser−/− testes revealed that SALL4A protein level was increased by BMS961-activated RARG (Fig 6B, compare lane 1 to 2); this increase was prevented in mice additionally carrying a Rarg knock-out (Fig 6B, compare lane 3 to 4) and was not observed in BMS961-treated Rara;b;gSpg–/– mutants (Fig 6B, compare lane 5 to 6). In addition, Sall4a mRNA levels were significantly decreased in whole testis of Rara;b;gSpg–/– and Rxra;b;gSpg–/– mutants at PN60, while Sall4b and Zbtb16 mRNA levels were unchanged (Fig 6C). The finding that Sall4b mRNA level was not altered is in keeping with previous reports showing that SALL4B is expressed at a constant level in spermatogonia [31,32]. Altogether our results indicate that (i) Sall4a expression is decreased in testes of mice lacking RAR or RXR in spermatogonia (Rara;b;gSpg–/– and Rxra;b;gSpg–/– testes); (ii) SALL4A is detected at a low level in the seminiferous epithelium of mice deficient in ATRA (Aldh1a1-3Ser−/− testes), but at a high level when RARG is activated by BMS961 in these mice (Aldh1a1-3Ser−/− testes treated with BMS961), except when RARG is lacking (Aldh1a1-3Ser−/−;Rarg−/− testes, treated with BMS961); and (iii) SALL4A is not detected in testes of adult mice lacking RAR in spermatogonia even in the presence of the RARG agonist (Rara;b;gSpg–/– testes, treated with BMS961). Altogether, these data indicate that Sall4a expression is controlled by ATRA-activated RARG in spermatogonia.

Bottom Line: We also show that ATRA activates RAR and RXR bound to a conserved regulatory region to increase expression of the SALL4A transcription factor in spermatogonia.Our results reveal that this major pluripotency gene is a target of ATRA signaling and that RAR/RXR heterodimers are the functional units driving its expression in spermatogonia.They add to the mechanisms through which ATRA promote expression of the KIT tyrosine kinase receptor to trigger a critical step in spermatogonia differentiation.

View Article: PubMed Central - PubMed

Affiliation: Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Département de Génétique Fonctionnelle et Cancer, Illkirch, France; Centre National de la Recherche Scientifique (CNRS), UMR7104, Illkirch, France; Institut National de la Santé et de la Recherche Médicale (INSERM), U964, Illkirch, France; Université de Strasbourg (UNISTRA), Illkirch Cedex, France.

ABSTRACT
All-trans retinoic acid (ATRA) is instrumental to male germ cell differentiation, but its mechanism of action remains elusive. To address this question, we have analyzed the phenotypes of mice lacking, in spermatogonia, all rexinoid receptors (RXRA, RXRB and RXRG) or all ATRA receptors (RARA, RARB and RARG). We demonstrate that the combined ablation of RXRA and RXRB in spermatogonia recapitulates the set of defects observed both upon ablation of RAR in spermatogonia. We also show that ATRA activates RAR and RXR bound to a conserved regulatory region to increase expression of the SALL4A transcription factor in spermatogonia. Our results reveal that this major pluripotency gene is a target of ATRA signaling and that RAR/RXR heterodimers are the functional units driving its expression in spermatogonia. They add to the mechanisms through which ATRA promote expression of the KIT tyrosine kinase receptor to trigger a critical step in spermatogonia differentiation. Importantly, they indicate also that meiosis eventually occurs in the absence of a RAR/RXR pathway within germ cells and suggest that instructing this process is either ATRA-independent or requires an ATRA signal originating from Sertoli cells.

No MeSH data available.


Related in: MedlinePlus