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Glucose Regulated Protein 78 Phosphorylation in Sperm Undergoes Dynamic Changes during Maturation.

Lobo V, Rao P, Gajbhiye R, Kulkarni V, Parte P - PLoS ONE (2015)

Bottom Line: Immunoprecipitation studies using antibodies specific to GRP78, serine-, threonine-, and tyrosine phosphorylation and Pan phospho antibody demonstrates GRP78 to be phosphorylated at all three residues in rat spermatozoa.GP4.94[P = 0.014]andGP5.04 [P = 0.02] are significantly reduced in asthenozoosperm.Ours is the first report indicating GRP78 in sperm to be phosphorylated at serine, threonine and tyrosine residues contrary to published literature reporting GRP78 not to be tyrosine phosphorylated.

View Article: PubMed Central - PubMed

Affiliation: Department of Gamete Immunobiology, National Institute for Research in Reproductive Health (ICMR), Mumbai, 400012, India.

ABSTRACT
GRP78, a resident endoplasmic reticulum (ER) chaperone involved in protein transport, folding and assembly, has been reported in sperm. It is shown to be localized in the neck region of human sperm. We have previously reported GRP78 to be less phosphorylated in asthenozoosperm.The present study aimed to determine whether sperm GRP78 undergoes phosphorylation changes during epididymal maturation and whether there are any differences in GRP78 phosphoforms in asthenozoosperm vis-à-vis normozoosperm. Testicular- and cauda epididymal- sperm from adult male Holtzman rats, and semen ejaculates collected from normal and asthenozoospermic individuals were investigated. DIGE carried out to determine phosphorylation of GRP78 in asthenozoosperm and normal sperm reveals a shift in the location of GRP78 of asthenozoosperm towards the alkaline pH, indicative of reduced GRP78 phosphorylation. Immunoprecipitation studies using antibodies specific to GRP78, serine-, threonine-, and tyrosine phosphorylation and Pan phospho antibody demonstrates GRP78 to be phosphorylated at all three residues in rat spermatozoa. Phosphatase assays using Calf intestinal alkaline phosphatase and Lambda protein phosphatase followed by nanofluidic proteomic immunoassay (NIA) show that in rat, GP4.96, GP4.94 and GP4.85 are the three phosphoforms in mature (caudal) sperm as against two phosphoforms GP4.96and GP4.94in immature (testicular) sperm. In mature human sperm GP5.04, GP4.96, and GP4.94were the 3 phosphoforms observed. GP4.94[P = 0.014]andGP5.04 [P = 0.02] are significantly reduced in asthenozoosperm. Ours is the first report indicating GRP78 in sperm to be phosphorylated at serine, threonine and tyrosine residues contrary to published literature reporting GRP78 not to be tyrosine phosphorylated. We report the presence of GRP78 phosphoforms in rat- and human- sperm and our data suggest that GRP78 phosphorylation in sperm undergoes spatial reorganization during epididymal maturation. Significant differences observed in 2 out of 3 phosphoforms in asthenozoosperm suggest that GRP78 phosphorylation may have functional relevance in sperm with consequent clinical implications.

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GRP78 expression in sperm.A) Western blot analysis of GRP78 in human sperm, rat testis, and rat testicular- and caudal- sperm. GRP78 expression in human sperm (a) [Lane 1: negative control], and rat sperm and testis (b) [-ve: Negative control, C: Caudal sperm, T: Testicular sperm, Ts: Testicular tissue] B) IIF localization of GRP78. In normal human sperm, GRP78 expression seen in the equatorial region of sperm head, neck and midpiece when non-permeabilized (a) and on neck and midpiece when permeabilized (b). In caudal (mature) rat sperm, GRP78 localization is observed in the equatorial region of sperm head when non-permeabilized (c) and on neck and anterior tip of sperm head on permeabilization (d) (Left panel). Inset shows the negative control for the respective images. DIC images of the respective sperm (Right panel)
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pone.0141858.g002: GRP78 expression in sperm.A) Western blot analysis of GRP78 in human sperm, rat testis, and rat testicular- and caudal- sperm. GRP78 expression in human sperm (a) [Lane 1: negative control], and rat sperm and testis (b) [-ve: Negative control, C: Caudal sperm, T: Testicular sperm, Ts: Testicular tissue] B) IIF localization of GRP78. In normal human sperm, GRP78 expression seen in the equatorial region of sperm head, neck and midpiece when non-permeabilized (a) and on neck and midpiece when permeabilized (b). In caudal (mature) rat sperm, GRP78 localization is observed in the equatorial region of sperm head when non-permeabilized (c) and on neck and anterior tip of sperm head on permeabilization (d) (Left panel). Inset shows the negative control for the respective images. DIC images of the respective sperm (Right panel)

Mentions: Western blot analysis using GRP78 antibody demonstrated a band of ~78kDa in human sperm [Fig 2A (a)]. It was also detected in rat testicular tissue, and in testicular-, and caudal- sperm [Fig 2A (b)]. By Indirect Immunofluorescence using non permeabilized normal human sperm, GRP78 expression was seen in the equatorial region of sperm head, neck and mid- and on neck and mid-piece when permeabilized using 1% Triton X- 100 and 0.5% glacial acetic acid[Fig 2B (a, b)]. In rat caudal (mature) sperm, GRP78 localization is observed in the equatorial region of sperm head when non-permeabilized and on neck and anterior tip of sperm head on permeabilization [Fig 2B (c, d)].


Glucose Regulated Protein 78 Phosphorylation in Sperm Undergoes Dynamic Changes during Maturation.

Lobo V, Rao P, Gajbhiye R, Kulkarni V, Parte P - PLoS ONE (2015)

GRP78 expression in sperm.A) Western blot analysis of GRP78 in human sperm, rat testis, and rat testicular- and caudal- sperm. GRP78 expression in human sperm (a) [Lane 1: negative control], and rat sperm and testis (b) [-ve: Negative control, C: Caudal sperm, T: Testicular sperm, Ts: Testicular tissue] B) IIF localization of GRP78. In normal human sperm, GRP78 expression seen in the equatorial region of sperm head, neck and midpiece when non-permeabilized (a) and on neck and midpiece when permeabilized (b). In caudal (mature) rat sperm, GRP78 localization is observed in the equatorial region of sperm head when non-permeabilized (c) and on neck and anterior tip of sperm head on permeabilization (d) (Left panel). Inset shows the negative control for the respective images. DIC images of the respective sperm (Right panel)
© Copyright Policy
Related In: Results  -  Collection

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pone.0141858.g002: GRP78 expression in sperm.A) Western blot analysis of GRP78 in human sperm, rat testis, and rat testicular- and caudal- sperm. GRP78 expression in human sperm (a) [Lane 1: negative control], and rat sperm and testis (b) [-ve: Negative control, C: Caudal sperm, T: Testicular sperm, Ts: Testicular tissue] B) IIF localization of GRP78. In normal human sperm, GRP78 expression seen in the equatorial region of sperm head, neck and midpiece when non-permeabilized (a) and on neck and midpiece when permeabilized (b). In caudal (mature) rat sperm, GRP78 localization is observed in the equatorial region of sperm head when non-permeabilized (c) and on neck and anterior tip of sperm head on permeabilization (d) (Left panel). Inset shows the negative control for the respective images. DIC images of the respective sperm (Right panel)
Mentions: Western blot analysis using GRP78 antibody demonstrated a band of ~78kDa in human sperm [Fig 2A (a)]. It was also detected in rat testicular tissue, and in testicular-, and caudal- sperm [Fig 2A (b)]. By Indirect Immunofluorescence using non permeabilized normal human sperm, GRP78 expression was seen in the equatorial region of sperm head, neck and mid- and on neck and mid-piece when permeabilized using 1% Triton X- 100 and 0.5% glacial acetic acid[Fig 2B (a, b)]. In rat caudal (mature) sperm, GRP78 localization is observed in the equatorial region of sperm head when non-permeabilized and on neck and anterior tip of sperm head on permeabilization [Fig 2B (c, d)].

Bottom Line: Immunoprecipitation studies using antibodies specific to GRP78, serine-, threonine-, and tyrosine phosphorylation and Pan phospho antibody demonstrates GRP78 to be phosphorylated at all three residues in rat spermatozoa.GP4.94[P = 0.014]andGP5.04 [P = 0.02] are significantly reduced in asthenozoosperm.Ours is the first report indicating GRP78 in sperm to be phosphorylated at serine, threonine and tyrosine residues contrary to published literature reporting GRP78 not to be tyrosine phosphorylated.

View Article: PubMed Central - PubMed

Affiliation: Department of Gamete Immunobiology, National Institute for Research in Reproductive Health (ICMR), Mumbai, 400012, India.

ABSTRACT
GRP78, a resident endoplasmic reticulum (ER) chaperone involved in protein transport, folding and assembly, has been reported in sperm. It is shown to be localized in the neck region of human sperm. We have previously reported GRP78 to be less phosphorylated in asthenozoosperm.The present study aimed to determine whether sperm GRP78 undergoes phosphorylation changes during epididymal maturation and whether there are any differences in GRP78 phosphoforms in asthenozoosperm vis-à-vis normozoosperm. Testicular- and cauda epididymal- sperm from adult male Holtzman rats, and semen ejaculates collected from normal and asthenozoospermic individuals were investigated. DIGE carried out to determine phosphorylation of GRP78 in asthenozoosperm and normal sperm reveals a shift in the location of GRP78 of asthenozoosperm towards the alkaline pH, indicative of reduced GRP78 phosphorylation. Immunoprecipitation studies using antibodies specific to GRP78, serine-, threonine-, and tyrosine phosphorylation and Pan phospho antibody demonstrates GRP78 to be phosphorylated at all three residues in rat spermatozoa. Phosphatase assays using Calf intestinal alkaline phosphatase and Lambda protein phosphatase followed by nanofluidic proteomic immunoassay (NIA) show that in rat, GP4.96, GP4.94 and GP4.85 are the three phosphoforms in mature (caudal) sperm as against two phosphoforms GP4.96and GP4.94in immature (testicular) sperm. In mature human sperm GP5.04, GP4.96, and GP4.94were the 3 phosphoforms observed. GP4.94[P = 0.014]andGP5.04 [P = 0.02] are significantly reduced in asthenozoosperm. Ours is the first report indicating GRP78 in sperm to be phosphorylated at serine, threonine and tyrosine residues contrary to published literature reporting GRP78 not to be tyrosine phosphorylated. We report the presence of GRP78 phosphoforms in rat- and human- sperm and our data suggest that GRP78 phosphorylation in sperm undergoes spatial reorganization during epididymal maturation. Significant differences observed in 2 out of 3 phosphoforms in asthenozoosperm suggest that GRP78 phosphorylation may have functional relevance in sperm with consequent clinical implications.

Show MeSH
Related in: MedlinePlus