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HP1gamma function is required for male germ cell survival and spermatogenesis.

Brown JP, Bullwinkel J, Baron-Lühr B, Billur M, Schneider P, Winking H, Singh PB - Epigenetics Chromatin (2010)

Bottom Line: While targeting the Cbx3 gene (encoding the HP1gamma protein) with a conditional targeting vector, we generated a hypomorphic allele (Cbx3hypo), which resulted in much reduced (barely detectable) levels of HP1gamma protein.Adult males exhibit a severe hypogonadism that is associated with a loss of germ cells, with some seminiferous tubules retaining only the supporting Sertoli cells (Sertoli cell-only phenotype).The Cbx3hypo/hypo spermatogenesis defect is similar to that found in Miwi2 and Dnmt3L mutants.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Immunoepigenetics, Department of Immunology and Cell Biology, Research Center Borstel, D-23845 Borstel, Germany. psingh@fz-borstel.de.

ABSTRACT

Background: HP1 proteins are conserved components of eukaryotic constitutive heterochromatin. In mammals, there are three genes that encode HP1-like proteins, termed HP1alpha, HP1beta and HP1gamma, which have a high degree of homology This paper describes for the first time, to our knowledge, the physiological function of HP1gamma using a gene-targeted mouse.

Results: While targeting the Cbx3 gene (encoding the HP1gamma protein) with a conditional targeting vector, we generated a hypomorphic allele (Cbx3hypo), which resulted in much reduced (barely detectable) levels of HP1gamma protein. Homozygotes for the hypomorphic allele (Cbx3hypo/hypo) are rare, with only 1% of Cbx3hypo/hypo animals reaching adulthood. Adult males exhibit a severe hypogonadism that is associated with a loss of germ cells, with some seminiferous tubules retaining only the supporting Sertoli cells (Sertoli cell-only phenotype). The percentage of seminiferous tubules that are positive for L1 ORF1 protein (ORF1p) in Cbx3hypo/hypo testes is greater than that for wild-type testes, indicating that L1 retrotransposon silencing is reversed, leading to ectopic expression of ORF1p in Cbx3hypo/hypo germ cells.

Conclusions: The Cbx3 gene product (the HP1gamma protein) has a non-redundant function during spermatogenesis that cannot be compensated for by the other two HP1 isotypes. The Cbx3hypo/hypo spermatogenesis defect is similar to that found in Miwi2 and Dnmt3L mutants. The Cbx3 gene-targeted mice generated in this study provide an appropriate model for the study of HP1gamma in transposon silencing and parental imprinting.

No MeSH data available.


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Cbx3hypo/hypo testes showed a dramatic loss in the number of germ cells. (a,b) Typical germ cell nuclear antigen (GCNA) staining of germ cells within wild-type testes. (c,d) In Cbx3hypo/hypo testes, there was a dramatic reduction in the numbers of GCNA-positive germ cells. Some tubules contained no GCNA-positive germ cells and presented a Sertolin cells-only phenotype (asterisk). Bar = 100 μm.
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Figure 5: Cbx3hypo/hypo testes showed a dramatic loss in the number of germ cells. (a,b) Typical germ cell nuclear antigen (GCNA) staining of germ cells within wild-type testes. (c,d) In Cbx3hypo/hypo testes, there was a dramatic reduction in the numbers of GCNA-positive germ cells. Some tubules contained no GCNA-positive germ cells and presented a Sertolin cells-only phenotype (asterisk). Bar = 100 μm.

Mentions: By contrast, HP1γ staining was almost undetectable in sections taken from Cbx3hypo/hypo testes (Figure 4e, f). Histological examination revealed a severe impairment of spermatogenesis in Cbx3hypo/hypo testes. The diameter of the tubules in Cbx3hypo/hypo testes was smaller (0.13 mm) than that in wild-type testes (0.2 mm). Of 70 tubules examined, 22 were almost completely devoid of germ cells (for example, Figure 4e, upper right tubule) and 48 tubules had impaired spermatogenesis. Tubules in which mature sperm could be observed were rare (Figure 4f, especially the magnified inset). The loss of germ cells was confirmed using a germ-specific antibody (anti-germ cell nuclear antigen (GCNA)) [20], which revealed a dramatic reduction in germ cells (GCNA-positive cells) in Cbx3hypo/hypo testes (Figure 5c, Figure 5d) compared with wild-type mice (Figure 5, Figure 5b). Some of the tubules in Cbx3hypo/hypo testes exhibited a Sertoli cell-only (SCO) phenotype, reminiscent of the tubules seen in the Dnmt3L and Miwi2 mutants [21,22]. Thus, the Cbx3hypo/hypo mutation results in the general suppression of spermatogenesis, which can vary from tubule to tubule; in some tubules suppression is complete, resulting in a SCO phenotype, whereas in others, spermatogenesis can proceed and give rise to some mature sperms. This variation across the tubules might reflect the fact that the Cbx3hypo mutation is 'leaky', that is, the interference of Cbx3 by the neo-tk is variable giving rise to leaky expression of Cbx3. Some Cbx3hypo/hypo germ cells and their progeny might have closer to wild-type levels of HP1γ, thus enabling greater likelihood of survival with more normal development.


HP1gamma function is required for male germ cell survival and spermatogenesis.

Brown JP, Bullwinkel J, Baron-Lühr B, Billur M, Schneider P, Winking H, Singh PB - Epigenetics Chromatin (2010)

Cbx3hypo/hypo testes showed a dramatic loss in the number of germ cells. (a,b) Typical germ cell nuclear antigen (GCNA) staining of germ cells within wild-type testes. (c,d) In Cbx3hypo/hypo testes, there was a dramatic reduction in the numbers of GCNA-positive germ cells. Some tubules contained no GCNA-positive germ cells and presented a Sertolin cells-only phenotype (asterisk). Bar = 100 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2877046&req=5

Figure 5: Cbx3hypo/hypo testes showed a dramatic loss in the number of germ cells. (a,b) Typical germ cell nuclear antigen (GCNA) staining of germ cells within wild-type testes. (c,d) In Cbx3hypo/hypo testes, there was a dramatic reduction in the numbers of GCNA-positive germ cells. Some tubules contained no GCNA-positive germ cells and presented a Sertolin cells-only phenotype (asterisk). Bar = 100 μm.
Mentions: By contrast, HP1γ staining was almost undetectable in sections taken from Cbx3hypo/hypo testes (Figure 4e, f). Histological examination revealed a severe impairment of spermatogenesis in Cbx3hypo/hypo testes. The diameter of the tubules in Cbx3hypo/hypo testes was smaller (0.13 mm) than that in wild-type testes (0.2 mm). Of 70 tubules examined, 22 were almost completely devoid of germ cells (for example, Figure 4e, upper right tubule) and 48 tubules had impaired spermatogenesis. Tubules in which mature sperm could be observed were rare (Figure 4f, especially the magnified inset). The loss of germ cells was confirmed using a germ-specific antibody (anti-germ cell nuclear antigen (GCNA)) [20], which revealed a dramatic reduction in germ cells (GCNA-positive cells) in Cbx3hypo/hypo testes (Figure 5c, Figure 5d) compared with wild-type mice (Figure 5, Figure 5b). Some of the tubules in Cbx3hypo/hypo testes exhibited a Sertoli cell-only (SCO) phenotype, reminiscent of the tubules seen in the Dnmt3L and Miwi2 mutants [21,22]. Thus, the Cbx3hypo/hypo mutation results in the general suppression of spermatogenesis, which can vary from tubule to tubule; in some tubules suppression is complete, resulting in a SCO phenotype, whereas in others, spermatogenesis can proceed and give rise to some mature sperms. This variation across the tubules might reflect the fact that the Cbx3hypo mutation is 'leaky', that is, the interference of Cbx3 by the neo-tk is variable giving rise to leaky expression of Cbx3. Some Cbx3hypo/hypo germ cells and their progeny might have closer to wild-type levels of HP1γ, thus enabling greater likelihood of survival with more normal development.

Bottom Line: While targeting the Cbx3 gene (encoding the HP1gamma protein) with a conditional targeting vector, we generated a hypomorphic allele (Cbx3hypo), which resulted in much reduced (barely detectable) levels of HP1gamma protein.Adult males exhibit a severe hypogonadism that is associated with a loss of germ cells, with some seminiferous tubules retaining only the supporting Sertoli cells (Sertoli cell-only phenotype).The Cbx3hypo/hypo spermatogenesis defect is similar to that found in Miwi2 and Dnmt3L mutants.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Immunoepigenetics, Department of Immunology and Cell Biology, Research Center Borstel, D-23845 Borstel, Germany. psingh@fz-borstel.de.

ABSTRACT

Background: HP1 proteins are conserved components of eukaryotic constitutive heterochromatin. In mammals, there are three genes that encode HP1-like proteins, termed HP1alpha, HP1beta and HP1gamma, which have a high degree of homology This paper describes for the first time, to our knowledge, the physiological function of HP1gamma using a gene-targeted mouse.

Results: While targeting the Cbx3 gene (encoding the HP1gamma protein) with a conditional targeting vector, we generated a hypomorphic allele (Cbx3hypo), which resulted in much reduced (barely detectable) levels of HP1gamma protein. Homozygotes for the hypomorphic allele (Cbx3hypo/hypo) are rare, with only 1% of Cbx3hypo/hypo animals reaching adulthood. Adult males exhibit a severe hypogonadism that is associated with a loss of germ cells, with some seminiferous tubules retaining only the supporting Sertoli cells (Sertoli cell-only phenotype). The percentage of seminiferous tubules that are positive for L1 ORF1 protein (ORF1p) in Cbx3hypo/hypo testes is greater than that for wild-type testes, indicating that L1 retrotransposon silencing is reversed, leading to ectopic expression of ORF1p in Cbx3hypo/hypo germ cells.

Conclusions: The Cbx3 gene product (the HP1gamma protein) has a non-redundant function during spermatogenesis that cannot be compensated for by the other two HP1 isotypes. The Cbx3hypo/hypo spermatogenesis defect is similar to that found in Miwi2 and Dnmt3L mutants. The Cbx3 gene-targeted mice generated in this study provide an appropriate model for the study of HP1gamma in transposon silencing and parental imprinting.

No MeSH data available.


Related in: MedlinePlus