Fig. 3. Stage-specific redistribution of HSPA2.

A) Stage-specific micrococcal nuclease-dependent release of HSPA2 from spermatogenic cell nuclei. Nuclei were prepared from pooled fractions of spermatocytes (Spc) and elongating and condensing spermatids (EC) and submitted to extensive micrococcal nuclease digestion. Material released through the action of the enzyme was visualized after silver staining of a gel. The two indicated bands were cut on another gel and identified by mass spectrometry. Band N°1 contained HSPA2 and HSPA1L/HSC70t and band N°2 contained disulfide isomerase A3 (PDA3).

B) Stage-specific intranuclear redistribution of HSPA2 during spermiogenesis.

Suspension of spermatogenic cells were used to prepare nuclei which were then subjected to co-immunolocalization of HSPA2 and acetylated lysines (monoclonal antibody detecting essentially histones in the nucleus). Images were acquired using a confocal microscope. Four representative situations are shown, R, round spermatids; E, elongating spermatids and C, condensing spermatids.

These experiments were performed on purified nuclear suspensions, because the cytoplasmic HSPA2 signal interfered with a clear detection of nuclear HSPA2, which was the object of this study. Some of the remaining cytoplasmic HSPA2 can be observed in the C panel (arrow head). Scale bar represents 2 μm.