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Bat3 deficiency accelerates the degradation of Hsp70-2/HspA2 during spermatogenesis.

Sasaki T, Marcon E, McQuire T, Arai Y, Moens PB, Okada H - J. Cell Biol. (2008)

Bottom Line: Further investigation revealed that a testis-specific protein, Hsp70-2/HspA2, is absent in Bat3-deficient male GCs at any stage of spermatogenesis; however, Hsp70-2 transcripts are expressed at normal levels.We found that Bat3 deficiency induces polyubiquitylation and subsequent degradation of Hsp70-2.Inhibition of proteasomal degradation restores Hsp70-2 protein levels.

View Article: PubMed Central - PubMed

Affiliation: The Campbell Family Institute for Breast Cancer Research, Ontario Cancer Institute, University Health Network, University of Toronto, Toronto, Ontario M5G2C1, Canada.

ABSTRACT
Meiosis is critical for sexual reproduction. During meiosis, the dynamics and integrity of homologous chromosomes are tightly regulated. The genetic and molecular mechanisms governing these processes in vivo, however, remain largely unknown. In this study, we demonstrate that Bat3/Scythe is essential for survival and maintenance of male germ cells (GCs). Targeted inactivation of Bat3/Scythe in mice results in widespread apoptosis of meiotic male GCs and complete male infertility. Pachytene spermatocytes exhibit abnormal assembly and disassembly of synaptonemal complexes as demonstrated by abnormal SYCP3 staining and sustained gamma-H2AX and Rad51/replication protein A foci. Further investigation revealed that a testis-specific protein, Hsp70-2/HspA2, is absent in Bat3-deficient male GCs at any stage of spermatogenesis; however, Hsp70-2 transcripts are expressed at normal levels. We found that Bat3 deficiency induces polyubiquitylation and subsequent degradation of Hsp70-2. Inhibition of proteasomal degradation restores Hsp70-2 protein levels. Our findings identify Bat3 as a critical regulator of Hsp70-2 in spermatogenesis, thereby providing a possible molecular target in idiopathic male infertility.

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Related in: MedlinePlus

Bat3 is required for Hsp70-2 protein stability in multiple tissues. (A–C) Bat3, Hsp70-2, and Hsp70 protein levels were evaluated in Bat3+/+ and Bat3−/− MEFs (A), a human teratocarcinoma cell line, NTERA2 (B), and a mouse spermatocyte cell line, GC-2 (C), in the presence or absence of Bat3 with cycloheximide treatment for the indicated times (in hours). (D) Decreased Hsp70-2 protein stability induced by Bat3 KD was rescued by the RNAi-resistant Bat3 mutant (Bat3ΔRNAi). siCNT, control siRNA. (E) Hsp70-2 levels in primary Bat3-deficient male GCs were restored by proteasome inhibitor (MG132) treatment.
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fig4: Bat3 is required for Hsp70-2 protein stability in multiple tissues. (A–C) Bat3, Hsp70-2, and Hsp70 protein levels were evaluated in Bat3+/+ and Bat3−/− MEFs (A), a human teratocarcinoma cell line, NTERA2 (B), and a mouse spermatocyte cell line, GC-2 (C), in the presence or absence of Bat3 with cycloheximide treatment for the indicated times (in hours). (D) Decreased Hsp70-2 protein stability induced by Bat3 KD was rescued by the RNAi-resistant Bat3 mutant (Bat3ΔRNAi). siCNT, control siRNA. (E) Hsp70-2 levels in primary Bat3-deficient male GCs were restored by proteasome inhibitor (MG132) treatment.

Mentions: We first examined whether Bat3 knockout or knockdown (KD) could reduce Hsp70-2 protein levels. Hsp70-2 levels were significantly lower in both Bat3−/− MEFs (Fig. 4 A) and multiple cell lines depleted for Bat3 (Fig. 4, B and C). We excluded potential off-target effects by introducing a Bat3 mutant resistant to RNAi (ΔRNAi-B3; Fig. 4 D; Sasaki et al., 2007). These data demonstrate that Bat3 is required for maintaining Hsp70-2 protein levels. We also found that Hsp70-2 protein levels were restored in Bat3−/− mouse primary GCs after treatment with MG132 (Fig. 4 E). Because Bat3 regulates the chaperone function of Hsp70 in vitro (Thress et al., 2001), we also examined Hsp70 protein levels. Notably, we found that Hsp70 protein levels were not significantly affected by Bat3 status (Fig. 4, A–C).


Bat3 deficiency accelerates the degradation of Hsp70-2/HspA2 during spermatogenesis.

Sasaki T, Marcon E, McQuire T, Arai Y, Moens PB, Okada H - J. Cell Biol. (2008)

Bat3 is required for Hsp70-2 protein stability in multiple tissues. (A–C) Bat3, Hsp70-2, and Hsp70 protein levels were evaluated in Bat3+/+ and Bat3−/− MEFs (A), a human teratocarcinoma cell line, NTERA2 (B), and a mouse spermatocyte cell line, GC-2 (C), in the presence or absence of Bat3 with cycloheximide treatment for the indicated times (in hours). (D) Decreased Hsp70-2 protein stability induced by Bat3 KD was rescued by the RNAi-resistant Bat3 mutant (Bat3ΔRNAi). siCNT, control siRNA. (E) Hsp70-2 levels in primary Bat3-deficient male GCs were restored by proteasome inhibitor (MG132) treatment.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2500131&req=5

fig4: Bat3 is required for Hsp70-2 protein stability in multiple tissues. (A–C) Bat3, Hsp70-2, and Hsp70 protein levels were evaluated in Bat3+/+ and Bat3−/− MEFs (A), a human teratocarcinoma cell line, NTERA2 (B), and a mouse spermatocyte cell line, GC-2 (C), in the presence or absence of Bat3 with cycloheximide treatment for the indicated times (in hours). (D) Decreased Hsp70-2 protein stability induced by Bat3 KD was rescued by the RNAi-resistant Bat3 mutant (Bat3ΔRNAi). siCNT, control siRNA. (E) Hsp70-2 levels in primary Bat3-deficient male GCs were restored by proteasome inhibitor (MG132) treatment.
Mentions: We first examined whether Bat3 knockout or knockdown (KD) could reduce Hsp70-2 protein levels. Hsp70-2 levels were significantly lower in both Bat3−/− MEFs (Fig. 4 A) and multiple cell lines depleted for Bat3 (Fig. 4, B and C). We excluded potential off-target effects by introducing a Bat3 mutant resistant to RNAi (ΔRNAi-B3; Fig. 4 D; Sasaki et al., 2007). These data demonstrate that Bat3 is required for maintaining Hsp70-2 protein levels. We also found that Hsp70-2 protein levels were restored in Bat3−/− mouse primary GCs after treatment with MG132 (Fig. 4 E). Because Bat3 regulates the chaperone function of Hsp70 in vitro (Thress et al., 2001), we also examined Hsp70 protein levels. Notably, we found that Hsp70 protein levels were not significantly affected by Bat3 status (Fig. 4, A–C).

Bottom Line: Further investigation revealed that a testis-specific protein, Hsp70-2/HspA2, is absent in Bat3-deficient male GCs at any stage of spermatogenesis; however, Hsp70-2 transcripts are expressed at normal levels.We found that Bat3 deficiency induces polyubiquitylation and subsequent degradation of Hsp70-2.Inhibition of proteasomal degradation restores Hsp70-2 protein levels.

View Article: PubMed Central - PubMed

Affiliation: The Campbell Family Institute for Breast Cancer Research, Ontario Cancer Institute, University Health Network, University of Toronto, Toronto, Ontario M5G2C1, Canada.

ABSTRACT
Meiosis is critical for sexual reproduction. During meiosis, the dynamics and integrity of homologous chromosomes are tightly regulated. The genetic and molecular mechanisms governing these processes in vivo, however, remain largely unknown. In this study, we demonstrate that Bat3/Scythe is essential for survival and maintenance of male germ cells (GCs). Targeted inactivation of Bat3/Scythe in mice results in widespread apoptosis of meiotic male GCs and complete male infertility. Pachytene spermatocytes exhibit abnormal assembly and disassembly of synaptonemal complexes as demonstrated by abnormal SYCP3 staining and sustained gamma-H2AX and Rad51/replication protein A foci. Further investigation revealed that a testis-specific protein, Hsp70-2/HspA2, is absent in Bat3-deficient male GCs at any stage of spermatogenesis; however, Hsp70-2 transcripts are expressed at normal levels. We found that Bat3 deficiency induces polyubiquitylation and subsequent degradation of Hsp70-2. Inhibition of proteasomal degradation restores Hsp70-2 protein levels. Our findings identify Bat3 as a critical regulator of Hsp70-2 in spermatogenesis, thereby providing a possible molecular target in idiopathic male infertility.

Show MeSH
Related in: MedlinePlus