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. 2017 Apr;34(4):495-503.
doi: 10.1007/s10815-017-0879-5. Epub 2017 Feb 24.

Progesterone requires heat shock protein 90 (HSP90) in human sperm to regulate motility and acrosome reaction

Vrushali Sagare-Patil  1 , Rashmi Bhilawadikar  2 , Mosami Galvankar  1 , Kusum Zaveri  2 , Indira Hinduja  2 , Deepak Modi  3
Affiliations

Progesterone requires heat shock protein 90 (HSP90) in human sperm to regulate motility and acrosome reaction

Vrushali Sagare-Patil et al. J Assist Reprod Genet. 2017 Apr.

Erratum in

Abstract

Purpose: The aims of this paper were to study whether heat shock protein 90 (HSP90) is a regulator of sperm functions and to determine its association with oligoasthenozoospermia.

Methods: The levels of HSP90 in sperm lysates were measured by ELISA. Localization of HSP90 and its isoforms was evaluated by immunofluorescence. Sperm motility and kinetics were assessed by computer-assisted sperm analysis. Acrosome reaction was determined by lectin staining.

Results: The levels of HSP90 were lower in oligoasthenozoospermic men and correlated positively with the number of motile spermatozoa. In capacitated human spermatozoa, HSP90α was mostly found in residual nuclear envelope, and the HSP90β isoform was higher in the flagella. Inhibition of HSP90 by geldanamycin or 17-AAG did not affect basal motility, but suppressed progesterone-mediated forward progressive motility, hyperactivation and acrosome reaction. Progesterone treatment dephosphorylated both HSP90α and HSP90β at Ser/Thr-Pro residues, but not Tyr residues.

Conclusion: HSP90 levels are downregulated in oligoasthenozoospermia, and its functional inhibition attenuates progesterone-mediated sperm motility and acrosome reaction.

Keywords: Acrosome reaction; HSP90; Heat shock protein; Human sperm; Motility; Oligoasthenozoospermia; Phosphorylation; Progesterone.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1
Fig. 1
Localization of HSP90 and its isoforms in human spermatozoa. a Representative immunofluorescence images for HSP90 and its isoforms in human spermatozoa. Capacitated spermatozoa were probed with antibodies against HSP90 (that recognizes both the isoforms) or those that recognize specifically the α- and β-isoforms. The antibodies were detected using an Alexa 595-labelled secondary antibody. Red fluorescence is staining for HSP90; the nuclei are counterstained with DAPI (blue fluorescence). The negative controls are cells incubated without primary antibody. Upper panel are lower magnification images and lower panel are higher magnification images. Appropriate scale bars are shown in the negative control. The experiment was repeated three times on different pools of semen samples from different individuals. b Percentages of spermatozoa showing staining in residual nuclear envelope (RNE) and the flagella. Data are the mean ± SD for three biological replicates. Asterisk indicates value significantly different as compared to that observed for HSP90
Fig. 2
Fig. 2
Association of HSP90 levels and sperm motility. HSP90 levels were measured by ELISA in spermatozoa from normozoospermic and oligoasthenozoospermic men. a Levels of HSP90 in spermatozoa of normozoospermic and oligoasthenozoospermic men. Values on the Y-axis are the mean ± SE (n = 30 in each group) of the ratios of OD at 450 nm for HSP90/β-actin. *Significantly different as compared to normozoospermic men (p < 0.05). b Scatter plot for percentage of motile spermatozoa and levels of HSP90 protein. Values on the Y-axis are the ratios of OD at 450 nm of HSP90/actin, and values on the X-axis are the percentages of motile spermatozoa. Each spot represents data from one individual
Fig. 3
Fig. 3
Effect of HSP90 inhibitors on sperm motility and acrosome reaction. Capacitated spermatozoa were incubated with HSP90 inhibitors (geldanamycin and 17-AAG) with or without progesterone (P4), and motility parameters were assessed by CASA. a Effect of HSP90 inhibitors on basal sperm motility. b Motility pattern in the presence of progesterone. c Percentage of acrosome-reacted spermatozoa as measured by lectin staining. In (a) and (b), values on the Y-axis are the mean + SEM of fold change where the value obtained for a normozoospermic sample was taken as 1. In (c), values on the Y-axis are the mean + SE of the percentages of acrosome-reacted spermatozoa. Data are derived from three independent pools of samples. *Value significantly different as compared to the control. #Value significantly different as compared to progesterone treatment (p < 0.05)
Fig. 4
Fig. 4
Progesterone alters the phosphorylation of HSP90. Spermatozoa were incubated with or without progesterone for 30 min and phosphorylation of HSP90 and its isoforms at the tyrosine (Tyr) and serine/threonine-proline (Ser/Thr-Pro) residues was determined by ELISA. Values on the Y-axis are the mean + SE of per cent reactivity with respect to a control (untreated) sample. Data are derived from four independent replicates. *Value significantly different as compared to the control (p < 0.05)
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