HDAC6 deacetylates alpha tubulin in sperm and modulates sperm motility in Holtzman rat

Abstract

Histone deacetylase 6 (HDAC6) is an alpha (α)-tubulin deacetylase and its over-expression has been demonstrated to promote chemotactic cell movement. Motility in sperm is driven by the flagella, the cytoskeletal structure comprising the microtubules, which are heterodimers of α- and β-tubulins. We have hypothesized that HDAC6, by virtue of being an α-tubulin deacetylase, might modulate sperm motility. However, the presence of HDAC6 on sperm has hitherto not been reported. In this study, we have demonstrated, for the first time, the presence of HDAC6 transcript and protein in the testicular and caudal sperm of rat. We have observed a significantly overlapping expression of HDAC6 with acetyl α-tubulin (Ac α-tubulin) in the mid-piece and principal piece of sperm flagella, and the co-precipitation of α-tubulin and Ac α-tubulin together with HDAC6 and vice versa in sperm lysates. This indicates that HDAC6 interacts with α-tubulin. The HDAC6 activity of sperm, sperm motility and status of Ac α-tubulin investigated in the presence of HDAC inhibitors Trichostatin A, Tubastatin A and sodium butyrate demonstrate that HDAC6 in sperm is catalytically active and that inhibitors of HDAC6 increase acetylation and restrict sperm motility. Thus, we show that (1) active HDAC6 enzyme is present in sperm, (2) HDAC6 in sperm is able to deacetylate α-tubulin, (3) inhibition of HDAC6 results in increased Ac α-tubulin expression and (4) HDAC6 inhibition affects sperm motility. This evidence suggests that HDAC6 is involved in modulating sperm movement.

Tubulin acetylation: A novel functional avenue for CDYL in sperm

“…The overlap coefficient of CDYL and Ac a-tubulin show a significant increase in the caudal sperm as compared to testicular-and caput sperm indicating increased co-localization of the two proteins in mature sperm flagella ( Figure 2c). Similar observations were reported by us for the tubulin deacetylase HDAC6 and Ac a-tubulin (Parab et al, 2015). Whereas there HDAC6 expression was predominantly on midpiece, for CDYL we see it strongly on the principal piece.…”

“…The increase in Ac α‐tubulin observed in epididymal sperm as compared to testicular sperm was not emulated by similar changes in CDYL expression of the respective sperm (Figure b). This can be explained by the decreased levels of HDAC6 in epididymal sperm vis‐à‐vis testicular sperm that we observed earlier (Parab et al, ). Co‐localization of CDYL and Ac α‐tubulin along the length of sperm flagella suggested that the two proteins may interact.…”

Radiosynthesis and preliminary evaluation of an 18F‐labeled tubastatin A analog for PET imaging of histone deacetylase 6

“…Similarly, in some organs, the radioactivity uptake of [ 18 F]3-injected mice was significantly higher than that observed with [ 11 C]2-injected mice, including the heart, lung, liver, and muscle, at the early time points. 49 In our study, however, testis radioactivity was below blood levels at every time point for both [ 11 C]2 and [ 18 F]3. Brain uptake of [ 11 C]2 was less than 0.5% ID/g over a 60-minute period, and this observed poor BBB permeability is consistent with previous reports.…”

“…47 The development and function of the testis of HDAC6 knockout mice are normal, 48 whereas inhibition of HDAC6 restricts sperm motility in rats through increased levels of acetyl α-tubulin, which is a component of microtubules in the flagella. 49 In our study, however, testis radioactivity was below blood levels at every time point for both [ 11 C]2 and [ 18 F]3. As with the brain, the testis has a blood-testis barrier to protect spermatocytogenesis from toxicants in the systemic circulation and autoimmune responses 50 ; therefore, the barrier may inhibit testis uptake of the radioligands.…”

Developmental Testicular Expression, Cloning, and Characterization of Rat HDAC6 In Silico

“…HDAC6 and alpha-tubulin acetyl transferase 1 ( α TAT1) have been identified as α -tubulin deacetylase and α -tubulin acetyltransferase, respectively [ 1 , 8 ]. Our group has reported, for the first time, the presence of HDAC6 in sperm and demonstrated its tubulin specific deacetylase activity and its role in sperm motility in Holtzman rat [ 9 ]. Not much is known about the role of HDAC6 during different stages of spermatogenesis.…”