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Inhibitory phosphorylation of separase is essential for genome stability and viability of murine embryonic germ cells.

Huang X, Andreu-Vieyra CV, York JP, Hatcher R, Lu T, Matzuk MM, Zhang P - PLoS Biol. (2008)

Bottom Line: We show that germ cells in the mutants are depleted during development.We further demonstrate that S1121A causes chromosome misalignment during proliferation of the postmigratory primordial germ cells, resulting in mitotic arrest, aneuploidy, and eventual cell death.Our results indicate that inhibitory phosphorylation of separase plays a critical role in the maintenance of sister chromatid cohesion and genome stability in proliferating postmigratory primordial germ cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, Texas, United States of America.

ABSTRACT
Activity of separase, a cysteine protease that cleaves sister chromatid cohesin at the onset of anaphase, is tightly regulated to ensure faithful chromosome segregation and genome stability. Two mechanisms negatively regulate separase: inhibition by securin and phosphorylation on serine 1121. To gauge the physiological significance of the inhibitory phosphorylation, we created a mouse strain in which Ser1121 was mutated to Ala (S1121A). Here we report that this S1121A point mutation causes infertility in mice. We show that germ cells in the mutants are depleted during development. We further demonstrate that S1121A causes chromosome misalignment during proliferation of the postmigratory primordial germ cells, resulting in mitotic arrest, aneuploidy, and eventual cell death. Our results indicate that inhibitory phosphorylation of separase plays a critical role in the maintenance of sister chromatid cohesion and genome stability in proliferating postmigratory primordial germ cells.

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Mitotic Arrest of separase S1121A Mutant Germ Cells(A) Mitotic indices of germ cells at different stages of development.(B) Mitotic indices of non–germ cells at different stages of development.(C) Western blot analysis of phosphorylated histone H3 in extracts of 13.5 dpc genital ridges.(D) Abnormal metaphase configuration of mutant germ cells at 13.5 dpc. Arrows indicate misaligned chromosomes.(E) Chromosome spreads of cultured PGCs after 6-h nocodazole treatment.
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pbio-0060015-g003: Mitotic Arrest of separase S1121A Mutant Germ Cells(A) Mitotic indices of germ cells at different stages of development.(B) Mitotic indices of non–germ cells at different stages of development.(C) Western blot analysis of phosphorylated histone H3 in extracts of 13.5 dpc genital ridges.(D) Abnormal metaphase configuration of mutant germ cells at 13.5 dpc. Arrows indicate misaligned chromosomes.(E) Chromosome spreads of cultured PGCs after 6-h nocodazole treatment.

Mentions: At 6 wk of age, mutant testes are much smaller than the controls (Figure 1A). Histological examination revealed that the mutant seminiferous tubules are devoid of spermatocytes and spermatids (Figure 1B), suggesting spermatogenesis failure in separase S1121A mutant mice. Spermatogenesis in mice starts around 10 d post-partum (dpp) when spermatocytes are first produced, followed by spermatids around 20 dpp and spermatozoa around 35 dpp [25]. We examined testes isolated from mice ranging from 4 dpp up to 2 wk old. We could not detect histologically any differences between the control and the mutant before the age of 2 wk (Figure 1C). However, at 2 wk when the first wave of spermatogenesis had started in the control, we found no sign of spermatogenesis in the mutant (Figure 1C). The most likely reason for this result is the lack of spermatogonia in the mutant testes. To determine if that was the case, we carried out immunostainings for Tra98, a germ cell marker [26], and Sox9, a marker for Sertoli cells [27,28]. As shown in Figure 1D, we could not detect Tra98 in the mutant testes at 4 d after birth, whereas Sox-9 was readily detected. Absence of Tra98 was confirmed by western blot analysis (Figure 1E). Three additional germ cell markers, Plzf [29], Sohlh1 [30], and GCNA [31] were also absent in the mutant testes (Figures 1–3 and S1–S3), indicating that the loss of inhibitory phosphorylation of separase leads to spermatogonia cell depletion.


Inhibitory phosphorylation of separase is essential for genome stability and viability of murine embryonic germ cells.

Huang X, Andreu-Vieyra CV, York JP, Hatcher R, Lu T, Matzuk MM, Zhang P - PLoS Biol. (2008)

Mitotic Arrest of separase S1121A Mutant Germ Cells(A) Mitotic indices of germ cells at different stages of development.(B) Mitotic indices of non–germ cells at different stages of development.(C) Western blot analysis of phosphorylated histone H3 in extracts of 13.5 dpc genital ridges.(D) Abnormal metaphase configuration of mutant germ cells at 13.5 dpc. Arrows indicate misaligned chromosomes.(E) Chromosome spreads of cultured PGCs after 6-h nocodazole treatment.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2214812&req=5

pbio-0060015-g003: Mitotic Arrest of separase S1121A Mutant Germ Cells(A) Mitotic indices of germ cells at different stages of development.(B) Mitotic indices of non–germ cells at different stages of development.(C) Western blot analysis of phosphorylated histone H3 in extracts of 13.5 dpc genital ridges.(D) Abnormal metaphase configuration of mutant germ cells at 13.5 dpc. Arrows indicate misaligned chromosomes.(E) Chromosome spreads of cultured PGCs after 6-h nocodazole treatment.
Mentions: At 6 wk of age, mutant testes are much smaller than the controls (Figure 1A). Histological examination revealed that the mutant seminiferous tubules are devoid of spermatocytes and spermatids (Figure 1B), suggesting spermatogenesis failure in separase S1121A mutant mice. Spermatogenesis in mice starts around 10 d post-partum (dpp) when spermatocytes are first produced, followed by spermatids around 20 dpp and spermatozoa around 35 dpp [25]. We examined testes isolated from mice ranging from 4 dpp up to 2 wk old. We could not detect histologically any differences between the control and the mutant before the age of 2 wk (Figure 1C). However, at 2 wk when the first wave of spermatogenesis had started in the control, we found no sign of spermatogenesis in the mutant (Figure 1C). The most likely reason for this result is the lack of spermatogonia in the mutant testes. To determine if that was the case, we carried out immunostainings for Tra98, a germ cell marker [26], and Sox9, a marker for Sertoli cells [27,28]. As shown in Figure 1D, we could not detect Tra98 in the mutant testes at 4 d after birth, whereas Sox-9 was readily detected. Absence of Tra98 was confirmed by western blot analysis (Figure 1E). Three additional germ cell markers, Plzf [29], Sohlh1 [30], and GCNA [31] were also absent in the mutant testes (Figures 1–3 and S1–S3), indicating that the loss of inhibitory phosphorylation of separase leads to spermatogonia cell depletion.

Bottom Line: We show that germ cells in the mutants are depleted during development.We further demonstrate that S1121A causes chromosome misalignment during proliferation of the postmigratory primordial germ cells, resulting in mitotic arrest, aneuploidy, and eventual cell death.Our results indicate that inhibitory phosphorylation of separase plays a critical role in the maintenance of sister chromatid cohesion and genome stability in proliferating postmigratory primordial germ cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, Texas, United States of America.

ABSTRACT
Activity of separase, a cysteine protease that cleaves sister chromatid cohesin at the onset of anaphase, is tightly regulated to ensure faithful chromosome segregation and genome stability. Two mechanisms negatively regulate separase: inhibition by securin and phosphorylation on serine 1121. To gauge the physiological significance of the inhibitory phosphorylation, we created a mouse strain in which Ser1121 was mutated to Ala (S1121A). Here we report that this S1121A point mutation causes infertility in mice. We show that germ cells in the mutants are depleted during development. We further demonstrate that S1121A causes chromosome misalignment during proliferation of the postmigratory primordial germ cells, resulting in mitotic arrest, aneuploidy, and eventual cell death. Our results indicate that inhibitory phosphorylation of separase plays a critical role in the maintenance of sister chromatid cohesion and genome stability in proliferating postmigratory primordial germ cells.

Show MeSH
Related in: MedlinePlus