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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-Ć -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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NIA profile of GRP78 in rat testicular- and cauda epididymal sperm.NIA for GRP78 was performed in 20ng of the respective sperm lysates in Premix of pI range 5ā€“6 and spiked with pI standard 4 as the standard ladder. Figure shows a representative isoelectropherogram of GRP78 in rat testicular- and caudal- sperm (4 replicates represented by green, pink, red and grey peaks). Three peaks GP4.94, GP4.96 and GP5.43, are observed for testicular sperm (A). For caudal sperm, an additional peak GP4.85 is also observed (B). The pI of recombinant GRP78 is observed to be 5.32 (C) ā€˜GPā€™ represents the GRP78 peak, and the value in subscript indicates the pI of the respective peak. Experiments were performed thrice with 3 biological replicates and a minimum of 4 technical replicates for each.
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pone.0141858.g004: NIA profile of GRP78 in rat testicular- and cauda epididymal sperm.NIA for GRP78 was performed in 20ng of the respective sperm lysates in Premix of pI range 5ā€“6 and spiked with pI standard 4 as the standard ladder. Figure shows a representative isoelectropherogram of GRP78 in rat testicular- and caudal- sperm (4 replicates represented by green, pink, red and grey peaks). Three peaks GP4.94, GP4.96 and GP5.43, are observed for testicular sperm (A). For caudal sperm, an additional peak GP4.85 is also observed (B). The pI of recombinant GRP78 is observed to be 5.32 (C) ā€˜GPā€™ represents the GRP78 peak, and the value in subscript indicates the pI of the respective peak. Experiments were performed thrice with 3 biological replicates and a minimum of 4 technical replicates for each.

Mentions: The expanse of phosphorylation of GRP78 in rat- and human- sperm and its differential phosphorylation in asthenozoosperm was discerned by Nanofluidic Proteomic Immunoassay (NIA). Assays for GRP78 phospho-detection in sperm were first standardized, detailed description of which has been provided (S1 Text). The optimization data can be found in the supplemental information data (S1 to S4 Figs). Following the optimized conditions, GRP78 was analyzed from 20ngof testicular- or caudal- sperm lysates resolved in the pI range of 5ā€“6. Three peaks of GRP78 at pI 4.94 (GP4.94), 4.96 (GP4.96) and 5.43 (GP5.43) were consistently observed for testicular sperm. For caudal sperm, four peaks at pI 4.85 (GP4.85), 4.94 (GP4.94), 4.96 (GP4.96) and 5.43 (GP5.43) were observed (Fig 4A and 4B). To identify the peak representing unphosphorylated GRP78, 0.5pg recombinant GRP78 (StressMarq Biosciences, Victoria, Canada) was similarly resolved by NIA. The pI of recombinant GRP78 was observed to be 5.32 (Fig 4C).


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)

NIA profile of GRP78 in rat testicular- and cauda epididymal sperm.NIA for GRP78 was performed in 20ng of the respective sperm lysates in Premix of pI range 5ā€“6 and spiked with pI standard 4 as the standard ladder. Figure shows a representative isoelectropherogram of GRP78 in rat testicular- and caudal- sperm (4 replicates represented by green, pink, red and grey peaks). Three peaks GP4.94, GP4.96 and GP5.43, are observed for testicular sperm (A). For caudal sperm, an additional peak GP4.85 is also observed (B). The pI of recombinant GRP78 is observed to be 5.32 (C) ā€˜GPā€™ represents the GRP78 peak, and the value in subscript indicates the pI of the respective peak. Experiments were performed thrice with 3 biological replicates and a minimum of 4 technical replicates for each.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4664250&req=5

pone.0141858.g004: NIA profile of GRP78 in rat testicular- and cauda epididymal sperm.NIA for GRP78 was performed in 20ng of the respective sperm lysates in Premix of pI range 5ā€“6 and spiked with pI standard 4 as the standard ladder. Figure shows a representative isoelectropherogram of GRP78 in rat testicular- and caudal- sperm (4 replicates represented by green, pink, red and grey peaks). Three peaks GP4.94, GP4.96 and GP5.43, are observed for testicular sperm (A). For caudal sperm, an additional peak GP4.85 is also observed (B). The pI of recombinant GRP78 is observed to be 5.32 (C) ā€˜GPā€™ represents the GRP78 peak, and the value in subscript indicates the pI of the respective peak. Experiments were performed thrice with 3 biological replicates and a minimum of 4 technical replicates for each.
Mentions: The expanse of phosphorylation of GRP78 in rat- and human- sperm and its differential phosphorylation in asthenozoosperm was discerned by Nanofluidic Proteomic Immunoassay (NIA). Assays for GRP78 phospho-detection in sperm were first standardized, detailed description of which has been provided (S1 Text). The optimization data can be found in the supplemental information data (S1 to S4 Figs). Following the optimized conditions, GRP78 was analyzed from 20ngof testicular- or caudal- sperm lysates resolved in the pI range of 5ā€“6. Three peaks of GRP78 at pI 4.94 (GP4.94), 4.96 (GP4.96) and 5.43 (GP5.43) were consistently observed for testicular sperm. For caudal sperm, four peaks at pI 4.85 (GP4.85), 4.94 (GP4.94), 4.96 (GP4.96) and 5.43 (GP5.43) were observed (Fig 4A and 4B). To identify the peak representing unphosphorylated GRP78, 0.5pg recombinant GRP78 (StressMarq Biosciences, Victoria, Canada) was similarly resolved by NIA. The pI of recombinant GRP78 was observed to be 5.32 (Fig 4C).

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-Ć -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