Isolation and characterisation of two sperm membrane proteins recognised by sperm-associated antibodies in infertile men

Abstract

Antisperm antibodies eluted from the surface of spermatozoa obtained from infertile men recognised several common epitopes. We tested whether these epitopes were relevant to fertility by isolating the immunodominant 37/36 and 19/18 protein zones. These protein zones were cut out of preparative slab gels and electro‐eluted. The isolated proteins, P36 and P18, were used for biochemical characterisation and to produce specific antibodies in rabbits. The specific reactivity of P36 and P18 with WGA and AAL lectins, respectively, indicated the presence of lactosaminyl structures with sialic acid termini in P36 and of fucosylated residues in P18. Isoelectric focusing showed that the two proteins consist of several polypeptides. Some of these polypeptides were recognised by both human and rabbit antibodies: the pl of these epitopes was around 5.5 for P36 and 8.3–10.3 for P18. Rabbit antibodies detected the corresponding proteins on the sperm heads of methanol‐fixed and of live acrosome‐reacted spermatozoa. Anti‐P36 antibodies bound mainly to the equatorial segment. They reduced the binding and, consequently, the penetration of zona‐free hamster oocytes by human spermatozoa. Anti‐P18 antibodies gave more diffuse staining of the acrosomal and post‐acrosomal regions and reduced sperm–oocyte penetration without a significant effect on sperm binding. These results suggest that P36 and P18 antigens located in different compartments of the sperm head may participate in the sperm–oolemma interaction. We are currently investigating the physiological role of these antigens by sequencing the proteins isolated from the gels. Mol. Reprod. Dev. 57:393–405, 2000. © 2000 Wiley‐Liss, Inc.

The Remarkable Role of Triosephosphate Isomerase in Diabetes Pathophysiology

“…The mTOR pathway regulates critical cellular processes, exhibiting dual roles in diabetes by both promoting β-cell proliferation (compensating for insulin resistance) and contributing to tissue damage (e.g., kidney dysfunction and metabolic immune dysregulation). In breast cancer, TIM and CDCA5 activate mTORC1/PI3K/AKT signaling, suppressing apoptosis while enhancing glycolysis through GLUT1/4 upregulation and HIF1α/c-Myc-mediated glycolytic enzyme synthesis [ 36 , 57 , 60 ]. Although TIM-sororin’s role in normal mTORC1 activation remains unconfirmed, diabetic kidney disease demonstrates DHAP-driven mTORC1/ROS pathway activation that promotes podocyte pyroptosis, potentially mitigated by reducing DHAP levels [ 70 ].…”

“…Since TIM plays an important role in the development and maturation of the male reproductive system, failures in glycolysis can lead to infertility [ 53 , 57 , 58 ]. Although not confirmed in humans, the overexpression of miR-1285-3p in diabetes could contribute to infertility by inhibiting the expression of TIM; this hypothesis deserves to be investigated in the future.…”

The Remarkable Role of Triosephosphate Isomerase in Diabetes Pathophysiology

“…The mTOR pathway regulates critical cellular processes, exhibiting dual roles in diabetes by both promoting β-cell proliferation (compensating for insulin resistance) and contributing to tissue damage (e.g., kidney dysfunction and metabolic immune dysregulation). In breast cancer, TIM and CDCA5 activate mTORC1/PI3K/AKT signaling, suppressing apoptosis while enhancing glycolysis through GLUT1/4 upregulation and HIF1α/c-Myc-mediated glycolytic enzyme synthesis [ 36 , 57 , 60 ]. Although TIM-sororin’s role in normal mTORC1 activation remains unconfirmed, diabetic kidney disease demonstrates DHAP-driven mTORC1/ROS pathway activation that promotes podocyte pyroptosis, potentially mitigated by reducing DHAP levels [ 70 ].…”

“…Since TIM plays an important role in the development and maturation of the male reproductive system, failures in glycolysis can lead to infertility [ 53 , 57 , 58 ]. Although not confirmed in humans, the overexpression of miR-1285-3p in diabetes could contribute to infertility by inhibiting the expression of TIM; this hypothesis deserves to be investigated in the future.…”

Equatorial Segment Protein (ESP) Is a Human Alloantigen Involved in Sperm‐Egg Binding and Fusion

“…Useful ASA reagents have been generated to study sperm subcompartments (Bronson and Tung, 1992; Noor and Moore, 1999; Auer et al, 2000). Monoclonal antibodies HS1A.1 (Villaroya and Scholler, 1986) and MA1–3 (Isahakia and Alexander, 1984) recognize proteins residing only in the principal segment of the human acrosome, whereas D3 (Hinrichsen‐Kohane et al, 1985) and 21D3 (Le et al, 1984) bind antigens located only in the equatorial segment.…”

“…Olson et al (1998) have demonstrated immunohistochemically that 2 major acrosomal matrix proteins, AM22 and AM29, are excluded from the equatorial segment of mouse sperm, whereas the MN9 monoclonal antibody (Toshimori et al, 1992) recognizes 38‐ and 48‐kDa proteins in mouse that are segregated specifically to the equatorial segment. Other researchers reported that the M1 (Noor and Moore, 1999) and P36 (Auer et al, 2000) monoclonal antibodies, which recognize sperm protein bands of 38 and 34 kDa in hamster and humans, respectively, localize to the equatorial segment and inhibit fertilization. However, while the identities and functions of the proteins corresponding to these immunoreagents remain unknown, there is a striking similarity in molecular weight to isoforms of ESP.…”

Immune regulatory molecules as modifiers of semen and fertility: A review

“…These included HSP70, tubulin α 1a, class 4b β‐tubulin, ATP synthase subunit β, carbonic anhydrase 2, α‐enolase, and lactate dehydrogenase (LDHC) (Nowicka‐Bauer et al, ). Western blot analysis has been used to quantify these antigens and to identify protein–protein interactions (Auer, Senechal, Desvaux, Albert, & De Almeida, ). When complemented with confocal laser microscopy the exact localization of ASA bound sperm structures, for example, to cytoplasmic droplets (Sardoy et al, ) can be resolved.…”

The Remarkable Role of Triosephosphate Isomerase in Diabetes Pathophysiology

“…The mTOR pathway regulates critical cellular processes, exhibiting dual roles in diabetes by both promoting β-cell proliferation (compensating for insulin resistance) and contributing to tissue damage (e.g., kidney dysfunction and metabolic immune dysregulation). In breast cancer, TIM and CDCA5 activate mTORC1/PI3K/AKT signaling, suppressing apoptosis while enhancing glycolysis through GLUT1/4 upregulation and HIF1α/c-Myc-mediated glycolytic enzyme synthesis [ 36 , 57 , 60 ]. Although TIM-sororin’s role in normal mTORC1 activation remains unconfirmed, diabetic kidney disease demonstrates DHAP-driven mTORC1/ROS pathway activation that promotes podocyte pyroptosis, potentially mitigated by reducing DHAP levels [ 70 ].…”

“…Since TIM plays an important role in the development and maturation of the male reproductive system, failures in glycolysis can lead to infertility [ 53 , 57 , 58 ]. Although not confirmed in humans, the overexpression of miR-1285-3p in diabetes could contribute to infertility by inhibiting the expression of TIM; this hypothesis deserves to be investigated in the future.…”

Equatorial Segment Protein (ESP) Is a Human Alloantigen Involved in Sperm‐Egg Binding and Fusion

“…Useful ASA reagents have been generated to study sperm subcompartments (Bronson and Tung, 1992; Noor and Moore, 1999; Auer et al, 2000). Monoclonal antibodies HS1A.1 (Villaroya and Scholler, 1986) and MA1–3 (Isahakia and Alexander, 1984) recognize proteins residing only in the principal segment of the human acrosome, whereas D3 (Hinrichsen‐Kohane et al, 1985) and 21D3 (Le et al, 1984) bind antigens located only in the equatorial segment.…”

“…Olson et al (1998) have demonstrated immunohistochemically that 2 major acrosomal matrix proteins, AM22 and AM29, are excluded from the equatorial segment of mouse sperm, whereas the MN9 monoclonal antibody (Toshimori et al, 1992) recognizes 38‐ and 48‐kDa proteins in mouse that are segregated specifically to the equatorial segment. Other researchers reported that the M1 (Noor and Moore, 1999) and P36 (Auer et al, 2000) monoclonal antibodies, which recognize sperm protein bands of 38 and 34 kDa in hamster and humans, respectively, localize to the equatorial segment and inhibit fertilization. However, while the identities and functions of the proteins corresponding to these immunoreagents remain unknown, there is a striking similarity in molecular weight to isoforms of ESP.…”

Immune regulatory molecules as modifiers of semen and fertility: A review

“…These included HSP70, tubulin α 1a, class 4b β‐tubulin, ATP synthase subunit β, carbonic anhydrase 2, α‐enolase, and lactate dehydrogenase (LDHC) (Nowicka‐Bauer et al, ). Western blot analysis has been used to quantify these antigens and to identify protein–protein interactions (Auer, Senechal, Desvaux, Albert, & De Almeida, ). When complemented with confocal laser microscopy the exact localization of ASA bound sperm structures, for example, to cytoplasmic droplets (Sardoy et al, ) can be resolved.…”

The Remarkable Role of Triosephosphate Isomerase in Diabetes Pathophysiology

“…The mTOR pathway regulates critical cellular processes, exhibiting dual roles in diabetes by both promoting β-cell proliferation (compensating for insulin resistance) and contributing to tissue damage (e.g., kidney dysfunction and metabolic immune dysregulation). In breast cancer, TIM and CDCA5 activate mTORC1/PI3K/AKT signaling, suppressing apoptosis while enhancing glycolysis through GLUT1/4 upregulation and HIF1α/c-Myc-mediated glycolytic enzyme synthesis [ 36 , 57 , 60 ]. Although TIM-sororin’s role in normal mTORC1 activation remains unconfirmed, diabetic kidney disease demonstrates DHAP-driven mTORC1/ROS pathway activation that promotes podocyte pyroptosis, potentially mitigated by reducing DHAP levels [ 70 ].…”

“…Since TIM plays an important role in the development and maturation of the male reproductive system, failures in glycolysis can lead to infertility [ 53 , 57 , 58 ]. Although not confirmed in humans, the overexpression of miR-1285-3p in diabetes could contribute to infertility by inhibiting the expression of TIM; this hypothesis deserves to be investigated in the future.…”

Equatorial Segment Protein (ESP) Is a Human Alloantigen Involved in Sperm‐Egg Binding and Fusion

“…Useful ASA reagents have been generated to study sperm subcompartments (Bronson and Tung, 1992; Noor and Moore, 1999; Auer et al, 2000). Monoclonal antibodies HS1A.1 (Villaroya and Scholler, 1986) and MA1–3 (Isahakia and Alexander, 1984) recognize proteins residing only in the principal segment of the human acrosome, whereas D3 (Hinrichsen‐Kohane et al, 1985) and 21D3 (Le et al, 1984) bind antigens located only in the equatorial segment.…”

“…Olson et al (1998) have demonstrated immunohistochemically that 2 major acrosomal matrix proteins, AM22 and AM29, are excluded from the equatorial segment of mouse sperm, whereas the MN9 monoclonal antibody (Toshimori et al, 1992) recognizes 38‐ and 48‐kDa proteins in mouse that are segregated specifically to the equatorial segment. Other researchers reported that the M1 (Noor and Moore, 1999) and P36 (Auer et al, 2000) monoclonal antibodies, which recognize sperm protein bands of 38 and 34 kDa in hamster and humans, respectively, localize to the equatorial segment and inhibit fertilization. However, while the identities and functions of the proteins corresponding to these immunoreagents remain unknown, there is a striking similarity in molecular weight to isoforms of ESP.…”

Immune regulatory molecules as modifiers of semen and fertility: A review

“…These included HSP70, tubulin α 1a, class 4b β‐tubulin, ATP synthase subunit β, carbonic anhydrase 2, α‐enolase, and lactate dehydrogenase (LDHC) (Nowicka‐Bauer et al, ). Western blot analysis has been used to quantify these antigens and to identify protein–protein interactions (Auer, Senechal, Desvaux, Albert, & De Almeida, ). When complemented with confocal laser microscopy the exact localization of ASA bound sperm structures, for example, to cytoplasmic droplets (Sardoy et al, ) can be resolved.…”