Panel of monoclonal antibodies to sperm surface proteins as a tool for monitoring localization and identification of sperm–zona pellucida receptors

Abstract

Primary binding of the sperm to the zona pellucida (ZP) is one of the many steps necessary for successful fertilization. Sperm bind ZP by means of membrane receptors which recognize carbohydrate moieties on ZP glycoproteins according to a well-defined sequential process. Primary binding receptors, many of which have been disclosed in various mammals, are localized throughout the acrosomal region of the sperm surface. A panel of monoclonal antibodies against proteins from the sperm surface was prepared. Antibodies were screened by immunofluorescence for protein localization and Western blotting. Proteins localized on the sperm head and simultaneously detected by Western blotting were further studied in terms of immunolocalization in reproductive tissues and fluids, binding to ZP, immunoprecipitation and sequencing. Of 17 prepared antibodies, 8 recognized proteins localized on the sperm head and also detected proteins of interest by Western blotting. Only three other antibodies recognized proteins that also coincided in binding to ZP. These three antibodies were used for immunoprecipitation, and further protein sequencing of immunoprecipitates revealed that these antibodies distinguished acrosin precursor, RAB-2A protein, and lactadherin P47. This is not the first time we have detected acrosin on the surface of ejaculated and capacitated sperm. However, to our knowledge, this is the first time RAB-2A has been detected on the sperm surface. Lactadherin P47 has already been characterized and its physiological function in reproduction has been proposed.

Compartmentalization of the proteasome-interacting proteins during sperm capacitation

“…To determine whether and in what manner the proteins previously reported to co-purify with the 26S proteasome were processed during sperm IVC, we implemented IBFC and WB analysis after IVC under the proteasome permissive and inhibiting conditions (Figs 3–6). The redistribution of lactadherin MFGE8 (aliases p47, SED1) from the apical ridge region of the ejaculated spermatozoa to the entire acrosomal region of capacitated spermatozoa was observed in accordance with previously reported results as well as the presence of two different forms of lactadherin in spermatozoa 34,44–47 . Furthermore, MFGE8 can be isolated from OAM-matrix complex of bovine spermatozoa after IVC 45 .…”

“…Rabbit polyclonal anti-ADAM5 antibody (cat # ab101143) was bought from Abcam, Cambridge, United Kingdom. Rabbit polyclonal anti-sperm associated, acrosin inhibitor (SPINK2) antibody 53,61 and anti-P47 lactadherin antibody, clone 1H9 47 were raised in-house. Goat anti-mouse IgG (cat # 31430) and goat anti-rabbit IgG (cat # 31460), HRP secondary antibodies were purchased from ThermoFisher Scientific, Rockford, IL, USA.…”

“…All the antibodies used for flow cytometric studies were previously characterized. Primary antibodies used were as follows: anti-acrosin binding protein (ACRBP, 1:100 dilution; PA5-38735, Invitrogen), anti ADAM5 (1:100 dilution; ab101143, Abcam), anti-sperm associated, acrosin inhibitor (SPINK2; 1:200 dilution; [27]), anti-P47 lactadherin, clone 1H9 (1:200 dilution) 47 , and anti-phosphotyrosine, clone 4G10 (1:100 dilution; 05-321, Millipore Sigma); all diluted in PBST supplemented with 1% NGS. Primary antibodies were added to sperm sample tubes and incubated overnight at 4 °C.…”

“…Residual gels after electro-transfer were stained with CBB R-250 for protein load control. The PVDF membranes (Millipore Sigma) with the transferred proteins were blocked with 10% (w/v) non-fat milk in TBS with 0.1% (v/v) Tween 20 (TBST) and incubated with the following primary antibodies: anti-acrosin binding protein (ACRBP, 1:2,000 dilution; PA5-38735, Invitrogen), anti-ADAM5 (1:8,000 dilution; ab101143, Abcam), anti-sperm associated, acrosin inhibitor (1:8,000 dilution; SPINK2 53,61 ), anti-P47 lactadherin (1:500 dilution; clone 1H9 47 ), anti-phosphotyrosine, clone 4G10 (1:2000 dilution; 05-321, Millipore Sigma), and anti-acrosin Acr-2 (1:250 dilution) 60 , overnight. The membranes were then incubated with an appropriate species-specific secondary antibody, such as the HRP-conjugated goat anti-rabbit (GAR-IgG-HRP) or anti-mouse (GAM-IgG-HRP) antibodies (1:10,000 dilution; Invitrogen).…”

Effects of Obesity and Diabetes on Sperm Cell Proteomics in Rats

“…144 In somatic cells, Rab2A function is modulated by G3PDH, and in hyperglycemia associated with oxidative stress, a higher abundance of Rab2A induces lower levels of degradation of unfolded proteins, which accumulate near the endoplasmic reticulum (ER) and result in ER stress. 145,146 In bovines, Rab2A is located on the sperm superface 147 and is associated with vesicular transport in spermatids during acrosomal formation. 148 In our study, Rab2A expression was increased in the diabetic group and correlated with decreased G3PDH expression, 149 which indicates reduced fertility associated with oxidative stress, although no difference in lipid peroxidation was observed between our groups.…”