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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

Ablation of either RXR or RAR in spermatogonia does not alter the first round of spermatogenesis.(A) Percentages of seminiferous tubule cross-sections in which preleptotene/leptotene (PR+L), zygotene and early pachytene (Z+eP), late pachytene (P) and diplotene (D) spermatocytes or round spermatids (RS) represent the most advanced germ cell-types in control (white bars), Rxra/b/gSpg–/–(black bars) and Rara/b/gSpg–/–(grey bars) testes at post-natal day 20 (PN20). The bars represent mean ± s.e.m. (n = 4–5). (B-D) Histological sections of seminiferous from post-natal day 25 (PN25) control, Rara/b/gSpg–/– and RaraSer–/–/Rarg–/– mice stained with hematoxylin and eosin. Note the absence of spermatocytes (C) or of all meiotic and post meiotic germ cells (D) in the mutant testes. (E-J) Detection of spermatogonia expressing KIT (red signal in E, F, H and J) and RARG (red signal in G and I) in control and Rara/b/gSpg–/– testes at 6 weeks of age. ZBTB16 (green nuclear signal in E, F, H and J) identifies spermatogonia. Alexa Fluor 488-conjugated peanut agglutinin (in E and F) labels the acrosomal system of spermatids, allowing precise identification of the stage of the seminiferous epithelium cycle. (G and H) and (I and J) represent consecutive histological sections. All sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) to label nuclei (blue signal). A1, A1 spermatogonia, based on their presence in seminiferous tubule sections that contain both preleptotene spermatocytes (KIT-positive and ZBTB16-negative) and step8 spermatids. Int, intermediate spermatogonia, based on cell density co-expression of KIT and ZBTB16, and peanut hemagglutinin staining of acrosomes on consecutive sections. PR, P, preleptotene and pachytene spermatocytes, respectively; SG, spermatogonia, St7 and St8, step 7 and step8 round spermatids. Scale bars: 30 μm (B-D), 20 μm (E and F) and 55 μm (G-J).
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pgen.1005501.g005: Ablation of either RXR or RAR in spermatogonia does not alter the first round of spermatogenesis.(A) Percentages of seminiferous tubule cross-sections in which preleptotene/leptotene (PR+L), zygotene and early pachytene (Z+eP), late pachytene (P) and diplotene (D) spermatocytes or round spermatids (RS) represent the most advanced germ cell-types in control (white bars), Rxra/b/gSpg–/–(black bars) and Rara/b/gSpg–/–(grey bars) testes at post-natal day 20 (PN20). The bars represent mean ± s.e.m. (n = 4–5). (B-D) Histological sections of seminiferous from post-natal day 25 (PN25) control, Rara/b/gSpg–/– and RaraSer–/–/Rarg–/– mice stained with hematoxylin and eosin. Note the absence of spermatocytes (C) or of all meiotic and post meiotic germ cells (D) in the mutant testes. (E-J) Detection of spermatogonia expressing KIT (red signal in E, F, H and J) and RARG (red signal in G and I) in control and Rara/b/gSpg–/– testes at 6 weeks of age. ZBTB16 (green nuclear signal in E, F, H and J) identifies spermatogonia. Alexa Fluor 488-conjugated peanut agglutinin (in E and F) labels the acrosomal system of spermatids, allowing precise identification of the stage of the seminiferous epithelium cycle. (G and H) and (I and J) represent consecutive histological sections. All sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) to label nuclei (blue signal). A1, A1 spermatogonia, based on their presence in seminiferous tubule sections that contain both preleptotene spermatocytes (KIT-positive and ZBTB16-negative) and step8 spermatids. Int, intermediate spermatogonia, based on cell density co-expression of KIT and ZBTB16, and peanut hemagglutinin staining of acrosomes on consecutive sections. PR, P, preleptotene and pachytene spermatocytes, respectively; SG, spermatogonia, St7 and St8, step 7 and step8 round spermatids. Scale bars: 30 μm (B-D), 20 μm (E and F) and 55 μm (G-J).

Mentions: Assuming that the excision of Rar and Rxr genes was complete from PN5 onwards, the impact of RAR or RXR loss-of-functions during the pubertal development of the testis was evaluated at PN20, i.e., when the first post-meiotic cells appear. At this developmental stage, control, Rxra;b;gSpg–/– and Rara;b;gSpg–/– mutant testes (n = 4 for each genotype) were indistinguishable: in both situations, late pachytene and diplotene spermatocytes represented the most advanced germ cell in the vast majority of tubule sections (Fig 5A). These results indicate that the spermatocytes present at PN20 in the mutants testes, appeared in due time, likely because the spermatogonia from which they derived started to differentiate before PN3, at a time when Rar or Rxr genes were not yet knocked out. More importantly, they also indicate that all preleptotene spermatocytes initiated meiosis normally (around PN8), at a time when they were devoid of RAR or RXR since ablation was obvious from PN5 in their precursors (see above). Analyzing the seminiferous epithelium later during pubertal development revealed the occurrence of abnormal cellular associations at PN25: few tubule sections in mutant testes displayed spermatogonia associated with round spermatids but without the intervening layers of preleptotene and pachytene spermatocytes (Fig 5B and 5C). This observation confirms the initial wave of A1 spermatogonia differentiation was not affected (yielding step 7 spermatids at PN25), and suggests the second wave was arrested (or delayed) in few tubules at some point before meiosis, leading to the absence of spermatocytes at PN25. However, the presence of normal cellular associations in the majority of tubule sections indicates that both A1 spermatogonia differentiation and meiosis occurred, despite absence of RAR or RXR in germ cells (see above). In keeping with this, KIT-positive A1 spermatogonia were found at stages VII-VIII of the seminiferous epithelium cycle in Rara;b;gSpg–/– mutants at PN60, similarly to the situation in control mice (Fig 5E and 5F).


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)

Ablation of either RXR or RAR in spermatogonia does not alter the first round of spermatogenesis.(A) Percentages of seminiferous tubule cross-sections in which preleptotene/leptotene (PR+L), zygotene and early pachytene (Z+eP), late pachytene (P) and diplotene (D) spermatocytes or round spermatids (RS) represent the most advanced germ cell-types in control (white bars), Rxra/b/gSpg–/–(black bars) and Rara/b/gSpg–/–(grey bars) testes at post-natal day 20 (PN20). The bars represent mean ± s.e.m. (n = 4–5). (B-D) Histological sections of seminiferous from post-natal day 25 (PN25) control, Rara/b/gSpg–/– and RaraSer–/–/Rarg–/– mice stained with hematoxylin and eosin. Note the absence of spermatocytes (C) or of all meiotic and post meiotic germ cells (D) in the mutant testes. (E-J) Detection of spermatogonia expressing KIT (red signal in E, F, H and J) and RARG (red signal in G and I) in control and Rara/b/gSpg–/– testes at 6 weeks of age. ZBTB16 (green nuclear signal in E, F, H and J) identifies spermatogonia. Alexa Fluor 488-conjugated peanut agglutinin (in E and F) labels the acrosomal system of spermatids, allowing precise identification of the stage of the seminiferous epithelium cycle. (G and H) and (I and J) represent consecutive histological sections. All sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) to label nuclei (blue signal). A1, A1 spermatogonia, based on their presence in seminiferous tubule sections that contain both preleptotene spermatocytes (KIT-positive and ZBTB16-negative) and step8 spermatids. Int, intermediate spermatogonia, based on cell density co-expression of KIT and ZBTB16, and peanut hemagglutinin staining of acrosomes on consecutive sections. PR, P, preleptotene and pachytene spermatocytes, respectively; SG, spermatogonia, St7 and St8, step 7 and step8 round spermatids. Scale bars: 30 μm (B-D), 20 μm (E and F) and 55 μm (G-J).
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pgen.1005501.g005: Ablation of either RXR or RAR in spermatogonia does not alter the first round of spermatogenesis.(A) Percentages of seminiferous tubule cross-sections in which preleptotene/leptotene (PR+L), zygotene and early pachytene (Z+eP), late pachytene (P) and diplotene (D) spermatocytes or round spermatids (RS) represent the most advanced germ cell-types in control (white bars), Rxra/b/gSpg–/–(black bars) and Rara/b/gSpg–/–(grey bars) testes at post-natal day 20 (PN20). The bars represent mean ± s.e.m. (n = 4–5). (B-D) Histological sections of seminiferous from post-natal day 25 (PN25) control, Rara/b/gSpg–/– and RaraSer–/–/Rarg–/– mice stained with hematoxylin and eosin. Note the absence of spermatocytes (C) or of all meiotic and post meiotic germ cells (D) in the mutant testes. (E-J) Detection of spermatogonia expressing KIT (red signal in E, F, H and J) and RARG (red signal in G and I) in control and Rara/b/gSpg–/– testes at 6 weeks of age. ZBTB16 (green nuclear signal in E, F, H and J) identifies spermatogonia. Alexa Fluor 488-conjugated peanut agglutinin (in E and F) labels the acrosomal system of spermatids, allowing precise identification of the stage of the seminiferous epithelium cycle. (G and H) and (I and J) represent consecutive histological sections. All sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) to label nuclei (blue signal). A1, A1 spermatogonia, based on their presence in seminiferous tubule sections that contain both preleptotene spermatocytes (KIT-positive and ZBTB16-negative) and step8 spermatids. Int, intermediate spermatogonia, based on cell density co-expression of KIT and ZBTB16, and peanut hemagglutinin staining of acrosomes on consecutive sections. PR, P, preleptotene and pachytene spermatocytes, respectively; SG, spermatogonia, St7 and St8, step 7 and step8 round spermatids. Scale bars: 30 μm (B-D), 20 μm (E and F) and 55 μm (G-J).
Mentions: Assuming that the excision of Rar and Rxr genes was complete from PN5 onwards, the impact of RAR or RXR loss-of-functions during the pubertal development of the testis was evaluated at PN20, i.e., when the first post-meiotic cells appear. At this developmental stage, control, Rxra;b;gSpg–/– and Rara;b;gSpg–/– mutant testes (n = 4 for each genotype) were indistinguishable: in both situations, late pachytene and diplotene spermatocytes represented the most advanced germ cell in the vast majority of tubule sections (Fig 5A). These results indicate that the spermatocytes present at PN20 in the mutants testes, appeared in due time, likely because the spermatogonia from which they derived started to differentiate before PN3, at a time when Rar or Rxr genes were not yet knocked out. More importantly, they also indicate that all preleptotene spermatocytes initiated meiosis normally (around PN8), at a time when they were devoid of RAR or RXR since ablation was obvious from PN5 in their precursors (see above). Analyzing the seminiferous epithelium later during pubertal development revealed the occurrence of abnormal cellular associations at PN25: few tubule sections in mutant testes displayed spermatogonia associated with round spermatids but without the intervening layers of preleptotene and pachytene spermatocytes (Fig 5B and 5C). This observation confirms the initial wave of A1 spermatogonia differentiation was not affected (yielding step 7 spermatids at PN25), and suggests the second wave was arrested (or delayed) in few tubules at some point before meiosis, leading to the absence of spermatocytes at PN25. However, the presence of normal cellular associations in the majority of tubule sections indicates that both A1 spermatogonia differentiation and meiosis occurred, despite absence of RAR or RXR in germ cells (see above). In keeping with this, KIT-positive A1 spermatogonia were found at stages VII-VIII of the seminiferous epithelium cycle in Rara;b;gSpg–/– mutants at PN60, similarly to the situation in control mice (Fig 5E and 5F).

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