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Conditional inactivation of the DNA damage response gene Hus1 in mouse testis reveals separable roles for components of the RAD9-RAD1-HUS1 complex in meiotic chromosome maintenance.

Lyndaker AM, Lim PX, Mleczko JM, Diggins CE, Holloway JK, Holmes RJ, Kan R, Schlafer DH, Freire R, Cohen PE, Weiss RS - PLoS Genet. (2013)

Bottom Line: Hus1 loss in testicular germ cells resulted in meiotic defects, germ cell depletion, and severely compromised fertility.Interestingly, RAD1 had a broader distribution that only partially overlapped with RAD9, and localization of both RAD1 and the ATR activator TOPBP1 to the XY body and to unsynapsed autosomes was intact in Hus1 conditional knockouts.We conclude that mammalian HUS1 acts as a component of the canonical 9-1-1 complex during meiotic prophase I to promote DSB repair and further propose that RAD1 and TOPBP1 respond to unsynapsed chromatin through an alternative mechanism that does not require RAD9 or HUS1.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences, Cornell University, Ithaca, New York, USA.

ABSTRACT
The RAD9-RAD1-HUS1 (9-1-1) complex is a heterotrimeric PCNA-like clamp that responds to DNA damage in somatic cells by promoting DNA repair as well as ATR-dependent DNA damage checkpoint signaling. In yeast, worms, and flies, the 9-1-1 complex is also required for meiotic checkpoint function and efficient completion of meiotic recombination; however, since Rad9, Rad1, and Hus1 are essential genes in mammals, little is known about their functions in mammalian germ cells. In this study, we assessed the meiotic functions of 9-1-1 by analyzing mice with germ cell-specific deletion of Hus1 as well as by examining the localization of RAD9 and RAD1 on meiotic chromosomes during prophase I. Hus1 loss in testicular germ cells resulted in meiotic defects, germ cell depletion, and severely compromised fertility. Hus1-deficient primary spermatocytes exhibited persistent autosomal γH2AX and RAD51 staining indicative of unrepaired meiotic DSBs, synapsis defects, an extended XY body domain often encompassing partial or whole autosomes, and an increase in structural chromosome abnormalities such as end-to-end X chromosome-autosome fusions and ruptures in the synaptonemal complex. Most of these aberrations persisted in diplotene-stage spermatocytes. Consistent with a role for the 9-1-1 complex in meiotic DSB repair, RAD9 localized to punctate, RAD51-containing foci on meiotic chromosomes in a Hus1-dependent manner. Interestingly, RAD1 had a broader distribution that only partially overlapped with RAD9, and localization of both RAD1 and the ATR activator TOPBP1 to the XY body and to unsynapsed autosomes was intact in Hus1 conditional knockouts. We conclude that mammalian HUS1 acts as a component of the canonical 9-1-1 complex during meiotic prophase I to promote DSB repair and further propose that RAD1 and TOPBP1 respond to unsynapsed chromatin through an alternative mechanism that does not require RAD9 or HUS1.

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Hus1 loss results in germ cell depletion.A. 100× images of H&E-stained histological sections from 12-week old control (left; Cre+ Hus1+/flox) and Stra8-Cre Hus1 CKO (right) testes. B. GCNA1 staining of germ cells in control and Stra8-Cre Hus1 CKO testes indicating germ cell loss. C. TUNEL staining of control and Stra8-Cre Hus1 CKO adult testes indicating germ cell apoptosis. D. Quantification of TUNEL staining shown in C, shown as the mean ± SEM. Asterisk indicates statistically significant difference between Hus1 CKO and control animals (p<0.05, Student's t-test). E. Higher magnification (400×) images of TUNEL staining in Stra8-Cre Hus1 CKO adult testes. Arrows highlight TUNEL-positive cells that appeared to be pre-meiotic (left), mid-prophase I (middle), or at meiotic metaphase (right).
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pgen-1003320-g002: Hus1 loss results in germ cell depletion.A. 100× images of H&E-stained histological sections from 12-week old control (left; Cre+ Hus1+/flox) and Stra8-Cre Hus1 CKO (right) testes. B. GCNA1 staining of germ cells in control and Stra8-Cre Hus1 CKO testes indicating germ cell loss. C. TUNEL staining of control and Stra8-Cre Hus1 CKO adult testes indicating germ cell apoptosis. D. Quantification of TUNEL staining shown in C, shown as the mean ± SEM. Asterisk indicates statistically significant difference between Hus1 CKO and control animals (p<0.05, Student's t-test). E. Higher magnification (400×) images of TUNEL staining in Stra8-Cre Hus1 CKO adult testes. Arrows highlight TUNEL-positive cells that appeared to be pre-meiotic (left), mid-prophase I (middle), or at meiotic metaphase (right).

Mentions: To determine the underlying cause of the fertility defects in Hus1-deficient mice, we analyzed testes from Stra8-Cre Hus1 CKO and control animals. As shown in Figure 2A, Stra8-Cre Hus1 CKO adult testes exhibited a marked decrease in tubule size and cellularity, with many tubules lacking spermatogenic cells within the lumen, and an abundance of both pyknotic nuclei as well as multinucleate spermatid giant cells. We additionally performed immunohistochemical staining with GCNA1 antibody to detect germ cells in testis sections. Stra8-Cre Hus1 CKO testes exhibited significant loss of germ cells, including spermatogonia (Figure 2B). To further assess germ cell loss, we utilized TUNEL staining to detect fragmented DNA in Stra8-Cre Hus1 CKO nuclei. Hus1 mutant testes exhibited a significant increase in TUNEL-positive nuclei at both 17 days (Figure S4) and 12 weeks (Figure 2C–2E). Interestingly, a significant proportion of TUNEL-positive nuclei appeared to have progressed beyond pachytene stage to the first meiotic division (arrows, Figure 2E, right panel). 21% of all TUNEL-positive tubules contained apoptotic metaphase cells, although the majority of TUNEL-positive tubules (75%) contained apoptotic cells of a morphology consistent with zygotene/pachytene spermatocytes (Figure 2E, arrows, center panel). Spo11-Cre Hus1 CKO animals, in which Hus1 inactivation occurred later in germ cell development, also exhibited a decrease in testis cellularity and an increase in both multinucleate spermatid giant cells and TUNEL-positive cells (Figure S5). Altogether, these data indicate a requirement for Hus1 and the 9-1-1 complex for germ cell maintenance and development.


Conditional inactivation of the DNA damage response gene Hus1 in mouse testis reveals separable roles for components of the RAD9-RAD1-HUS1 complex in meiotic chromosome maintenance.

Lyndaker AM, Lim PX, Mleczko JM, Diggins CE, Holloway JK, Holmes RJ, Kan R, Schlafer DH, Freire R, Cohen PE, Weiss RS - PLoS Genet. (2013)

Hus1 loss results in germ cell depletion.A. 100× images of H&E-stained histological sections from 12-week old control (left; Cre+ Hus1+/flox) and Stra8-Cre Hus1 CKO (right) testes. B. GCNA1 staining of germ cells in control and Stra8-Cre Hus1 CKO testes indicating germ cell loss. C. TUNEL staining of control and Stra8-Cre Hus1 CKO adult testes indicating germ cell apoptosis. D. Quantification of TUNEL staining shown in C, shown as the mean ± SEM. Asterisk indicates statistically significant difference between Hus1 CKO and control animals (p<0.05, Student's t-test). E. Higher magnification (400×) images of TUNEL staining in Stra8-Cre Hus1 CKO adult testes. Arrows highlight TUNEL-positive cells that appeared to be pre-meiotic (left), mid-prophase I (middle), or at meiotic metaphase (right).
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pgen-1003320-g002: Hus1 loss results in germ cell depletion.A. 100× images of H&E-stained histological sections from 12-week old control (left; Cre+ Hus1+/flox) and Stra8-Cre Hus1 CKO (right) testes. B. GCNA1 staining of germ cells in control and Stra8-Cre Hus1 CKO testes indicating germ cell loss. C. TUNEL staining of control and Stra8-Cre Hus1 CKO adult testes indicating germ cell apoptosis. D. Quantification of TUNEL staining shown in C, shown as the mean ± SEM. Asterisk indicates statistically significant difference between Hus1 CKO and control animals (p<0.05, Student's t-test). E. Higher magnification (400×) images of TUNEL staining in Stra8-Cre Hus1 CKO adult testes. Arrows highlight TUNEL-positive cells that appeared to be pre-meiotic (left), mid-prophase I (middle), or at meiotic metaphase (right).
Mentions: To determine the underlying cause of the fertility defects in Hus1-deficient mice, we analyzed testes from Stra8-Cre Hus1 CKO and control animals. As shown in Figure 2A, Stra8-Cre Hus1 CKO adult testes exhibited a marked decrease in tubule size and cellularity, with many tubules lacking spermatogenic cells within the lumen, and an abundance of both pyknotic nuclei as well as multinucleate spermatid giant cells. We additionally performed immunohistochemical staining with GCNA1 antibody to detect germ cells in testis sections. Stra8-Cre Hus1 CKO testes exhibited significant loss of germ cells, including spermatogonia (Figure 2B). To further assess germ cell loss, we utilized TUNEL staining to detect fragmented DNA in Stra8-Cre Hus1 CKO nuclei. Hus1 mutant testes exhibited a significant increase in TUNEL-positive nuclei at both 17 days (Figure S4) and 12 weeks (Figure 2C–2E). Interestingly, a significant proportion of TUNEL-positive nuclei appeared to have progressed beyond pachytene stage to the first meiotic division (arrows, Figure 2E, right panel). 21% of all TUNEL-positive tubules contained apoptotic metaphase cells, although the majority of TUNEL-positive tubules (75%) contained apoptotic cells of a morphology consistent with zygotene/pachytene spermatocytes (Figure 2E, arrows, center panel). Spo11-Cre Hus1 CKO animals, in which Hus1 inactivation occurred later in germ cell development, also exhibited a decrease in testis cellularity and an increase in both multinucleate spermatid giant cells and TUNEL-positive cells (Figure S5). Altogether, these data indicate a requirement for Hus1 and the 9-1-1 complex for germ cell maintenance and development.

Bottom Line: Hus1 loss in testicular germ cells resulted in meiotic defects, germ cell depletion, and severely compromised fertility.Interestingly, RAD1 had a broader distribution that only partially overlapped with RAD9, and localization of both RAD1 and the ATR activator TOPBP1 to the XY body and to unsynapsed autosomes was intact in Hus1 conditional knockouts.We conclude that mammalian HUS1 acts as a component of the canonical 9-1-1 complex during meiotic prophase I to promote DSB repair and further propose that RAD1 and TOPBP1 respond to unsynapsed chromatin through an alternative mechanism that does not require RAD9 or HUS1.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences, Cornell University, Ithaca, New York, USA.

ABSTRACT
The RAD9-RAD1-HUS1 (9-1-1) complex is a heterotrimeric PCNA-like clamp that responds to DNA damage in somatic cells by promoting DNA repair as well as ATR-dependent DNA damage checkpoint signaling. In yeast, worms, and flies, the 9-1-1 complex is also required for meiotic checkpoint function and efficient completion of meiotic recombination; however, since Rad9, Rad1, and Hus1 are essential genes in mammals, little is known about their functions in mammalian germ cells. In this study, we assessed the meiotic functions of 9-1-1 by analyzing mice with germ cell-specific deletion of Hus1 as well as by examining the localization of RAD9 and RAD1 on meiotic chromosomes during prophase I. Hus1 loss in testicular germ cells resulted in meiotic defects, germ cell depletion, and severely compromised fertility. Hus1-deficient primary spermatocytes exhibited persistent autosomal γH2AX and RAD51 staining indicative of unrepaired meiotic DSBs, synapsis defects, an extended XY body domain often encompassing partial or whole autosomes, and an increase in structural chromosome abnormalities such as end-to-end X chromosome-autosome fusions and ruptures in the synaptonemal complex. Most of these aberrations persisted in diplotene-stage spermatocytes. Consistent with a role for the 9-1-1 complex in meiotic DSB repair, RAD9 localized to punctate, RAD51-containing foci on meiotic chromosomes in a Hus1-dependent manner. Interestingly, RAD1 had a broader distribution that only partially overlapped with RAD9, and localization of both RAD1 and the ATR activator TOPBP1 to the XY body and to unsynapsed autosomes was intact in Hus1 conditional knockouts. We conclude that mammalian HUS1 acts as a component of the canonical 9-1-1 complex during meiotic prophase I to promote DSB repair and further propose that RAD1 and TOPBP1 respond to unsynapsed chromatin through an alternative mechanism that does not require RAD9 or HUS1.

Show MeSH
Related in: MedlinePlus