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Characterization of Mammalian ADAM2 and Its Absence from Human Sperm.

Choi H, Jin S, Kwon JT, Kim J, Jeong J, Kim J, Jeon S, Park ZY, Jung KJ, Park K, Cho C - PLoS ONE (2016)

Bottom Line: Monkey ADAM2 was found to associate with chaperone proteins in testis.In humans, we identified ADAM2 as a 100-kDa protein in the testis, but failed to detect it in sperm.Taken together, our results provide new information regarding the characteristics of ADAM2 in mammalian species, including humans.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju, Korea.

ABSTRACT
The members of the ADAM (a disintegrin and metalloprotease) family are membrane-anchored multi-domain proteins that play prominent roles in male reproduction. ADAM2, which was one of the first identified ADAMs, is the best studied ADAM in reproduction. In the male germ cells of mice, ADAM2 and other ADAMs form complexes that contribute to sperm-sperm adhesion, sperm-egg interactions, and the migration of sperm in the female reproductive tract. Here, we generated specific antibodies against mouse and human ADAM2, and investigated various features of ADAM2 in mice, monkeys and humans. We found that the cytoplasmic domain of ADAM2 might enable the differential association of this protein with other ADAMs in mice. Western blot analysis with the anti-human ADAM2 antibodies showed that ADAM2 is present in the testis and sperm of monkeys. Monkey ADAM2 was found to associate with chaperone proteins in testis. In humans, we identified ADAM2 as a 100-kDa protein in the testis, but failed to detect it in sperm. This is surprising given the results in mice and monkeys, but it is consistent with the failure of ADAM2 identification in the previous proteomic analyses of human sperm. These findings suggest that the reproductive functions of ADAM2 differ between humans and mice. Our protein analysis showed the presence of potential ADAM2 complexes involving yet-unknown proteins in human testis. Taken together, our results provide new information regarding the characteristics of ADAM2 in mammalian species, including humans.

No MeSH data available.


Related in: MedlinePlus

Immunoreactivity of mouse ADAM2 antibodies.Lysates from testicular cells and sperm from wild-type and Adam2-/- mice were boiled in 3% SDS and 5% β-mercaptoethanol and subjected to Western blot analyses. A. Western blotting with the anti-mADAM2-D antibody. This antibody was raised against the ADAM2 disintegrin domain, and recognized the precursor (100-kDa) and processed (45-kDa) forms of ADAM2 in mouse testis and sperm, respectively. B and C. Blots performed using two different antibodies, which were raised against the cytoplasmic tail domain and designated B. anti-mADAM2-CyT-1 and C. anti-mADAM2-CyT-2. D. An antibody against a-tubulin (TUBA) was included as a control. Experiments were repeated three times. Reduced protein samples were subjected to SDS-PAGE using 10% resolving gels. Abbreviations: T, testicular cells; S, sperm; WT, wild-type; A2-/-, Adam2-/-; NS, non-specific. Molecular masses are presented on the left.
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pone.0158321.g001: Immunoreactivity of mouse ADAM2 antibodies.Lysates from testicular cells and sperm from wild-type and Adam2-/- mice were boiled in 3% SDS and 5% β-mercaptoethanol and subjected to Western blot analyses. A. Western blotting with the anti-mADAM2-D antibody. This antibody was raised against the ADAM2 disintegrin domain, and recognized the precursor (100-kDa) and processed (45-kDa) forms of ADAM2 in mouse testis and sperm, respectively. B and C. Blots performed using two different antibodies, which were raised against the cytoplasmic tail domain and designated B. anti-mADAM2-CyT-1 and C. anti-mADAM2-CyT-2. D. An antibody against a-tubulin (TUBA) was included as a control. Experiments were repeated three times. Reduced protein samples were subjected to SDS-PAGE using 10% resolving gels. Abbreviations: T, testicular cells; S, sperm; WT, wild-type; A2-/-, Adam2-/-; NS, non-specific. Molecular masses are presented on the left.

Mentions: It was previously suggested that ADAM2 undergoes modification in its cytoplasmic tail domain during sperm maturation in the epididymis [26]. To further characterize the ADAM2 cytoplasmic domain, we generated antibodies against amino acids 723–735 (mADAM2-CyT-1) and 721–734 (mADAM2-CyT-2) of the mouse ADAM2. Consistent with previous findings obtained using an anti-ADAM2 disintegrin domain antibody (mADAM2-D) that recognized mouse ADAM2 [21] (Fig 1A), both anti-mADAM2-CyT-1 and anti-mADAM2-CyT-2 recognized a 100-kDa band in the testis of WT but not Adam2-/- mice (Fig 1B and 1C). Anti-mADAM2-CyT-1 did not detect the processed form of ADAM2 in mature sperm (Fig 1B). The 110-kDa and 85-kDa proteins detected by anti-mADAM2CyT-1 in the testis of WT and Adam2-/- mice are non-specific (Fig 1B). The anti-mADAM2CyT-2 also detected non-specific, 40-kDa bands in both WT and Adam2-/- testes (Fig 1C). Although anti-mADAM2-CyT-2 detected a faint band between 45 and 50 kDa in WT, this band also was detected in Adam2-/- sperm (Fig 1C). However, anti-mADAM2-D clearly detected the processed form (45 kDa) of ADAM2 in mature sperm (Fig 1A). The anti-TUBA antibody, which was used as a control, showed that similar amounts of protein were loaded in each lane (Fig 1D). Our results confirm the previous report [26] that an anti-ADAM2 cytoplasmic domain antibody can detect the ADAM2 precursor but not the mature form, suggesting that the cytoplasmic domain of ADAM2 undergoes proteolysis or posttranslational modification during epididymal maturation of mouse sperm.


Characterization of Mammalian ADAM2 and Its Absence from Human Sperm.

Choi H, Jin S, Kwon JT, Kim J, Jeong J, Kim J, Jeon S, Park ZY, Jung KJ, Park K, Cho C - PLoS ONE (2016)

Immunoreactivity of mouse ADAM2 antibodies.Lysates from testicular cells and sperm from wild-type and Adam2-/- mice were boiled in 3% SDS and 5% β-mercaptoethanol and subjected to Western blot analyses. A. Western blotting with the anti-mADAM2-D antibody. This antibody was raised against the ADAM2 disintegrin domain, and recognized the precursor (100-kDa) and processed (45-kDa) forms of ADAM2 in mouse testis and sperm, respectively. B and C. Blots performed using two different antibodies, which were raised against the cytoplasmic tail domain and designated B. anti-mADAM2-CyT-1 and C. anti-mADAM2-CyT-2. D. An antibody against a-tubulin (TUBA) was included as a control. Experiments were repeated three times. Reduced protein samples were subjected to SDS-PAGE using 10% resolving gels. Abbreviations: T, testicular cells; S, sperm; WT, wild-type; A2-/-, Adam2-/-; NS, non-specific. Molecular masses are presented on the left.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4920383&req=5

pone.0158321.g001: Immunoreactivity of mouse ADAM2 antibodies.Lysates from testicular cells and sperm from wild-type and Adam2-/- mice were boiled in 3% SDS and 5% β-mercaptoethanol and subjected to Western blot analyses. A. Western blotting with the anti-mADAM2-D antibody. This antibody was raised against the ADAM2 disintegrin domain, and recognized the precursor (100-kDa) and processed (45-kDa) forms of ADAM2 in mouse testis and sperm, respectively. B and C. Blots performed using two different antibodies, which were raised against the cytoplasmic tail domain and designated B. anti-mADAM2-CyT-1 and C. anti-mADAM2-CyT-2. D. An antibody against a-tubulin (TUBA) was included as a control. Experiments were repeated three times. Reduced protein samples were subjected to SDS-PAGE using 10% resolving gels. Abbreviations: T, testicular cells; S, sperm; WT, wild-type; A2-/-, Adam2-/-; NS, non-specific. Molecular masses are presented on the left.
Mentions: It was previously suggested that ADAM2 undergoes modification in its cytoplasmic tail domain during sperm maturation in the epididymis [26]. To further characterize the ADAM2 cytoplasmic domain, we generated antibodies against amino acids 723–735 (mADAM2-CyT-1) and 721–734 (mADAM2-CyT-2) of the mouse ADAM2. Consistent with previous findings obtained using an anti-ADAM2 disintegrin domain antibody (mADAM2-D) that recognized mouse ADAM2 [21] (Fig 1A), both anti-mADAM2-CyT-1 and anti-mADAM2-CyT-2 recognized a 100-kDa band in the testis of WT but not Adam2-/- mice (Fig 1B and 1C). Anti-mADAM2-CyT-1 did not detect the processed form of ADAM2 in mature sperm (Fig 1B). The 110-kDa and 85-kDa proteins detected by anti-mADAM2CyT-1 in the testis of WT and Adam2-/- mice are non-specific (Fig 1B). The anti-mADAM2CyT-2 also detected non-specific, 40-kDa bands in both WT and Adam2-/- testes (Fig 1C). Although anti-mADAM2-CyT-2 detected a faint band between 45 and 50 kDa in WT, this band also was detected in Adam2-/- sperm (Fig 1C). However, anti-mADAM2-D clearly detected the processed form (45 kDa) of ADAM2 in mature sperm (Fig 1A). The anti-TUBA antibody, which was used as a control, showed that similar amounts of protein were loaded in each lane (Fig 1D). Our results confirm the previous report [26] that an anti-ADAM2 cytoplasmic domain antibody can detect the ADAM2 precursor but not the mature form, suggesting that the cytoplasmic domain of ADAM2 undergoes proteolysis or posttranslational modification during epididymal maturation of mouse sperm.

Bottom Line: Monkey ADAM2 was found to associate with chaperone proteins in testis.In humans, we identified ADAM2 as a 100-kDa protein in the testis, but failed to detect it in sperm.Taken together, our results provide new information regarding the characteristics of ADAM2 in mammalian species, including humans.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju, Korea.

ABSTRACT
The members of the ADAM (a disintegrin and metalloprotease) family are membrane-anchored multi-domain proteins that play prominent roles in male reproduction. ADAM2, which was one of the first identified ADAMs, is the best studied ADAM in reproduction. In the male germ cells of mice, ADAM2 and other ADAMs form complexes that contribute to sperm-sperm adhesion, sperm-egg interactions, and the migration of sperm in the female reproductive tract. Here, we generated specific antibodies against mouse and human ADAM2, and investigated various features of ADAM2 in mice, monkeys and humans. We found that the cytoplasmic domain of ADAM2 might enable the differential association of this protein with other ADAMs in mice. Western blot analysis with the anti-human ADAM2 antibodies showed that ADAM2 is present in the testis and sperm of monkeys. Monkey ADAM2 was found to associate with chaperone proteins in testis. In humans, we identified ADAM2 as a 100-kDa protein in the testis, but failed to detect it in sperm. This is surprising given the results in mice and monkeys, but it is consistent with the failure of ADAM2 identification in the previous proteomic analyses of human sperm. These findings suggest that the reproductive functions of ADAM2 differ between humans and mice. Our protein analysis showed the presence of potential ADAM2 complexes involving yet-unknown proteins in human testis. Taken together, our results provide new information regarding the characteristics of ADAM2 in mammalian species, including humans.

No MeSH data available.


Related in: MedlinePlus