Sperm Plasma Membrane Proteins Research Articles

1700029I15Rik orchestrates the biosynthesis of acrosomal membrane proteins required for sperm–egg interaction

Sperm acrosomal membrane proteins, such as Izumo sperm-egg fusion 1 (IZUMO1) and sperm acrosome-associated 6 (SPACA6), play essential roles in mammalian gamete binding or fusion. How their biosynthesis is regulated during spermiogenesis has largely remained elusive. Here, we show that 1700029I15Rik knockout male mice are severely subfertile and their spermatozoa do not fuse with eggs. 1700029I15Rik is a type-II transmembrane protein expressed in early round spermatids but not in mature spermatozoa. It interacts with proteins involved in N-linked glycosylation, disulfide isomerization, and endoplasmic reticulum (ER)-Golgi trafficking, suggesting a potential role in nascent protein processing. The ablation of 1700029I15Rik destabilizes non-catalytic subunits of the oligosaccharyltransferase (OST) complex that are pivotal for N-glycosylation. The knockout testes exhibit normal expression of sperm plasma membrane proteins, but decreased abundance of multiple acrosomal membrane proteins involved in fertilization. The knockout sperm show upregulated chaperones related to ER-associated degradation (ERAD) and elevated protein ubiquitination; strikingly, SPACA6 becomes undetectable. Our results support for a specific, 1700029I15Rik-mediated pathway underpinning the biosynthesis of acrosomal membrane proteins during spermiogenesis.

Improved quality and fertilizability of cryopreserved buffalo spermatozoa with the supplementation of methionine sulfoxide reductase A.

The excessive reactive oxygen species produced during semen-freezing and -thawing damage the macromolecules resulting in impairment of cellular functions. Proteins are the primary targets of oxidative damage, wherein methionine residues are more prone to oxidation and get converted into methionine sulfoxide, thus affecting the protein function. The methionine sulfoxide reductase A (MsrA) catalyzes the conversion of methionine sulfoxide to methionine and restores the functionality of defective proteins. To establish the expression of MsrA in male reproductive organs, including semen and its effect on quality of cryopreserved semen upon exogenous supplementation, taking buffalo semen as a model. The expression of MsrA was established by immunohistochemistry, PCR, and Western blots. Further, the effect of recombinant MsrA (rMsrA) supplementation on the quality of cryopreserved spermatozoa was assessed in three treatment groups containing 1.0, 1.5, and 2.0µg of rMsrA/50 million spermatozoa in egg yolk glycerol extender along with a control group; wherein the post-thaw progressive motility, viability, membrane integrity, and zona binding ability of cryopreserved spermatozoa were studied. The MsrA was expressed in buffalo testis, epididymis, accessory sex glands, and spermatozoa except in seminal plasma. In group 2, the supplementation has resulted in a significant (p<0.05) improvement as compared to the control group in mean progressive motility (47.50 ± 2.50 vs. 36.25 ± 2.63), viability (56.47 ± 1.85 vs. 48.05 ± 2.42), HOST (50.76 ± 1.73 vs. 44.29 ± 1.29), and zona binding ability of spermatozoa (149.50 ± 8.39 vs. 29.50 ± 2.85). In the absence of native MsrA of seminal plasma, the supplementations of rMsrA may repair the oxidatively damaged seminal plasma proteins and exposed sperm plasma membrane proteins resulting in better quality with a fivefold increase in fertilizability of frozen-thawed spermatozoa. The findings can be extended to other species to improve the semen quality with the variation in the amounts of rMsrA supplementation.

Dissecting the PRSS37 interactome and potential mechanisms leading to ADAM3 loss in PRSS37-null sperm.

A disintegrin and metalloproteinase 3 (ADAM3) is a sperm membrane protein critical for sperm migration from the uterus into the oviduct and sperm-egg binding in mice. Disruption of PRSS37 results in male infertility concurrent with the absence of mature ADAM3 from cauda epididymal sperm. However, how PRSS37 modulates ADAM3 maturation remains largely unclear. Here, we determine the PRSS37 interactome by GFP immunoprecipitation coupled with mass spectrometry in PRSS37-EGFP knock-in mice. Three molecular chaperones (CLGN, CALR3 and PDILT) and three ADAM proteins (ADAM2, ADAM6B and ADAM4) were identified to be interacting with PRSS37. Coincidently, five of them (except ADAM4) have been reported to interact with ADAM3 precursor and regulate its maturation. We further demonstrated that PRSS37 also interacts directly with ADAM3 precursor and its deficiency impedes the association between PDILT and ADAM3. This could contribute to improper translocation of ADAM3 to the germ cell surface, leading to ADAM3 loss in PRSS37-null mature sperm. The understanding of the maturation mechanisms of pivotal sperm plasma membrane proteins will pave the way toward novel strategies for contraception and the treatment of unexplained male infertility.

Research progress of the influence of sperm plasma membrane protein glycosylation on sperm maturation

Protein glycosylation is the covalent connection between monosaccharide or glycan and specific moieties of target proteins. As a ubiquitous and complex post-translational modification of proteins, glycosylation plays an important role in protein function, stability and subcellular localization. During the process of spermatogenesis in testis and maturation in epididymis, abundant glycosylation of sperm membrane proteins occurs, which plays an important role in the gradual maturation of sperm structure and function. In this paper, the research progress of glycosylation of sperm membrane protein on sperm maturation is reviewed.

Quantitative Proteomic Analysis of Seminal Plasma, Sperm Membrane Proteins, and Seminal Extracellular Vesicles Suggests Vesicular Mechanisms Aid in the Removal and Addition of Proteins to the Ram Sperm Membrane.

Quantitative proteomic studies are contributing greatly to the understanding of the spermatozoon through the provision of detailed information on the proteins spermatozoa acquire and shed in the acquisition of fertility. Extracellular vesicles (EVs) are thought to aid in the delivery of proteins to spermatozoa in the male reproductive tract. The aim of this study is to isolate, identify and quantify EV proteins isolated from ram seminal plasma. Ram sperm plasma membrane proteins are also isolated using nitrogen cavitation and identified to better understand the interplay of proteins between the sperm membrane and extracellular environment. The categorization of proteins enriched in the EV population according to their function revealed three main groupings: vesicle biogenesis, metabolism, and membrane adhesion and remodeling. The latter group contains many reproduction-specific proteins that show demonstrable links to sperm fertility. Many of these membrane-bound proteins show testicular expression and are shed from the sperm surface during epididymal maturation (e.g., testis expressed 101; TEX101 and lymphocyte Antigen 6 Family Member K; LY6K). Their association with seminal EVs suggests that EVs may not only deliver protein cargo to spermatozoa but also assist in the removal of proteins from the sperm membrane.

A comparative study of effects of different freezing methods on sperm quality, DNA integrity and membrane protein of cryopreserved boar semen*

The present study determined the effects of conventional, and controlled freezing method adopting three freezing rates, viz. 20°C, 40°C and 60°C/min on quality of sperm (motility, viability and plasma membrane integrity), DNA integrity and plasma membrane protein profile of cryopreserved boar semen. Sixty sperm-rich fractions of ejaculates from six boars were utilized for freezing of semen with different freezing methods in lactose-egg yolk glycerol extender. Semen samples were evaluated for sperm motility, viability (Propidium Iodide assay), functional integrity of plasma membrane (HOST), DNA integrity (Acridine Orange stain) and plasma membrane protein profile (SDSPAGE) after equilibration and after freezing. The results revealed that the post thaw sperm motility, sperm viability, and plasma membrane integrity (HOST-reacted) were significantly higher in all the three controlled freezing methods (20°C, 40°C and 60°C/min) as compared to that in conventional method. In addition, the number of sperm plasma membrane protein loss was less in controlled freezing methods as compared to that in conventional freezing. However, the post thaw sperm DNA integrity did not influence by difference in freezing methods. No significant difference on the post thaw sperm characteristics was recorded among the three controlled freezing rates. All the sperm parameters assessed declined significantly after freezing as compared to that after equilibration irrespective of freezing method employed. In conclusion, controlled freezing methods conferred better post thaw sperm quality as compared to conventional method, and thus the freezing rates of either 20, 40 or 60°C/min could provide better freezability of boar semen.

The roles of protein disulphide isomerase family A, member 3 (ERp57) and surface thiol/disulphide exchange in human spermatozoa–zona pellucida binding

Are multimeric sperm plasma membrane protein complexes, ERp57 and sperm surface thiol content involved in human spermatozoa-zona pellucida (ZP) interaction? ERp57 is a component of a multimeric spermatozoa-ZP receptor complex involved in regulation of human spermatozoa-ZP binding via up-regulation of sperm surface thiol content. A spermatozoon acquires its fertilization capacity within the female reproductive tract by capacitation. Spermatozoa-ZP receptor is suggested to be a composite structure that is assembled into a functional complex during capacitation. Sperm surface thiol content is elevated during capacitation. ERp57 is a protein disulphide isomerase that modulates the thiol-disulphide status of proteins. The binding ability and components of protein complexes in extracted membrane protein fractions of spermatozoa were studied. The roles of capacitation, thiol-disulphide reagent treatments and ERp57 on sperm functions and sperm surface thiol content were assessed. Spermatozoa were obtained from semen samples from normozoospermic men. Human oocytes were obtained from an assisted reproduction programme. Blue native polyacrylamide gel electrophoresis, western ligand blotting and mass spectrometry were used to identify the components of solubilized ZP/ZP3-binding complexes. The localization and expression of sperm surface thiol and ERp57 were studied by immunostaining and sperm surface protein biotinylation followed by western blotting. Sperm functions were assessed by standard assays. Several ZP-binding complexes were isolated from the cell membrane of capacitated spermatozoa. ERp57 was a component of one of these complexes. Capacitation significantly increased the sperm surface thiol content, acrosomal thiol distribution and ERp57 expression on sperm surface. Sperm surface thiol and ERp57 immunoreactivity were localized to the acrosomal region of spermatozoa, a region responsible for ZP-binding. Up-regulation of the surface thiol content or ERp57 surface expression in vitro stimulated ZP-binding capacity of human spermatozoa. Blocking of ERp57 function by specific antibody or inhibitors against ERp57 reduced the surface thiol content and ZP-binding capacity of human spermatozoa. N/A. The mechanisms by which up-regulation of surface thiol content stimulates spermatozoa-ZP binding have not been depicted. Thiol-disulphide exchange is a crucial event in capacitation. ERp57 modulates the event and the subsequent fertilization process. Modulation of the surface thiol content of the spermatozoa of subfertile men may help to increase fertilization rate in assisted reproduction. This work was supported by The Hong Kong Research Grant Council Grant HKU764611 and HKU764512M to P.C.N.C. The authors have no competing interests.

Effect of additives in semen extender on post thaw sperm quality and plasma membrane protein profile of boar semen

The objective of the study was to evaluate the effect of butylated hydroxytoluene (BHT), cholesterol-loaded methyl-ß-cyclodextrin (CLC), and their combination on sperm motility, viability, plasma membrane integrity and membrane protein profile of frozen-thawed boar semen. Ejaculates (16) from 6 boars were utilized. Each ejaculate was split into 4 fractions and cryopreserved in semen extender containing BHT (0.2 mM), CLC at 5 mg/ 200–240 × 106 sperm, BHT (0.2 mM) plus CLC at 5 mg/ 200–240 × 106 sperm or without BHT and CLC. Semen samples were evaluated for sperm motility, viability (propidium iodide assay), plasma membrane integrity (HOST) and membrane protein profile (SDS-PAGE) after equilibration and after freezing. The supplementation of BHT, CLC, and BHT plus CLC in semen extender significantly improved the viability and plasma membrane integrity after freezing as compared to control. Post thaw sperm motility was also significantly higher in BHT and BHT plus CLC. In addition, the number of sperm plasma membrane protein loss was less in additive treated sperm as compared to that in non-additive treated sperm. Among the additives, BHT plus CLC showed a significant improvement on post thaw sperm motility along with a non-significant improvement on other sperm parameters as compared to BHT and CLC alone. In conclusion, the supplementation of BHT, and CLC in extender before freezing improved the quality of cryopreserved boar semen and, the beneficial effect was more pronounced when BHT was combined with CLC than with BHT and CLC alone.

029 Rooster sperm plasma membrane protein and phospholipid organization and reorganization attributed to cooling and cryopreservation

Cholesterol to phospholipid ratio is used as a representation for membrane fluidity, and predictor of cryopreservation success but results are not consistent across species and ignore the impact of membrane proteins. Therefore, this research explored the modulation of membrane fluidity and protein organization of rooster sperm during cooling and cryopreservation using the fluorescent stains Merocyanine 540 (phospholipid order) and Rhodamine 640 (protein order). Rooster semen samples (3 roosters and breeds per pool; 4 pools total) were diluted in Lakes Low Temperature (LLT) and Sasaki diluent and cooled to 5 °C over 30 min (Step 1). The samples were then diluted with the penetrating cryoprotectants (CPA) glycerol (LLT) or methylacetamide (Sasaki), equilibrated for 30 min (Step 2), loaded into straws and cryopreserved. The cryopreservation and thawing (Step 3) was performed using the optimized procedures for each diluent. Computer automated semen analysis (CASA) was used to analyze motility characteristics and flow cytometry was used to analyze plasma membrane phospholipid, protein organization and plasma membrane integrity (PMI) at each step. Percent values from CASA and flow cytometry fluorescent medians (FM) were transformed using arcsine and Box–Cox, respectively. The GLM ANOVA was used to detect differences in motility characteristics and membrane organization and included the effects of cryopreservation step and rooster semen pool. In the total motility analyses cryopreservation diluent/CPA was a significant ( P P P P P Source of funding: USDA-ARS-NCGRP National Animal Germplasm Program. Conflict of interest: None declared. phil.purdy@ars.usda.gov

Involvement of sperm plasma membrane and cytoskeletal proteins in human male infertility

To assess the physicochemical characteristics of sperm plasma membrane and to evaluate the immunohistochemical expression of transmembrane and cytoskeletal proteins in spermatozoa isolated from normospermic fertile donors and asthenozoospermic infertile patients. Case-control study. Academic male infertility center. Twenty-five infertile patients affected by idiopathic asthenozoospermia and 21 age-matched normospermic fertile donors. Sperm parameters were evaluated; membrane fluidity and hydration studies, and immunohistochemical analysis were performed in isolated spermatozoa. Semen analyses to ascertain volume, sperm count, motility, and morphology; then membrane fluidity and hydration studies and immunohistochemical analysis were performed on isolated spermatozoa. Spermatozoa from the asthenozoospermic group exhibited a reduced fluidity at the lipid-water interface level, an increased fluidity of the deeper portion of the bilayer, and a lower plasma membrane hydration than normospermic cells. Moreover, the immunohistochemical expression of ezrin, Cdc42, CD9, F-actin, and β-tubulin was higher in normospermic samples. Our results together assume that a cytoskeletal reorganization induced by a disturbance in the physicochemical features of sperm plasma membrane, and potentially mediated by ezrin, Cdc42, and tetraspanin CD9, could have a role in idiopathic asthenozoospermia.

Activity of the Na,K‐ATPase α4 Isoform Is Regulated During Sperm Capacitation to Support Sperm Motility

The α4 polypeptide is a testis-specific isoform of the catalytic subunit of the Na,K-ATPase, which is essential for sperm motility and fertility. In the present study, we have investigated the regulation of activity of the α4 isoform and the relevance of this event for sperm capacitation. We have performed this by taking advantage of the selective high affinity of α4 for the inhibitor ouabain. Our results show that ouabain-sensitive hydrolysis of ATP and uptake of (86)Rb, corresponding to the enzymatic and ion transport activities of α4, respectively, increased during sperm capacitation in a time-dependent manner. Specific labeling of α4 with the fluorescent indicator bodipy-ouabain and immunoblot analysis of biotinylated and streptavidin-precipitated sperm plasma membrane proteins indicated a capacitation- and time-dependent rise in levels of active α4 isoform at the sperm surface. Ouabain inhibition of α4 blocked the increase in total sperm motility and the hyperactive motility pattern characteristic of sperm capacitation. Moreover, interference of α4 activity with ouabain partially prevented the intracellular decrease in Na(+) and the plasma membrane hyperpolarization that typically accompany sperm capacitation. In contrast, ouabain inhibition of α4 did not affect the spontaneous sperm acrosomal reaction following capacitation. Together, these results demonstrate that Na,K-ATPase α4 activity is up-regulated during sperm capacitation through mechanisms that involve both increases in molecular activity and levels of α4 at the sperm plasma membrane. This increase in α4 activity helps maintain the changes in motility that are associated with sperm capacitation, emphasizing the biologic relevance of the Na,K-ATPase α4 isoform in sustaining sperm function.

The Na,K-ATPase Alpha 4 Isoform Is Regulated During Sperm Capacitation.

The a4 isoform is a testis specific catalytic subunit of the Na,K-ATPase, which is essential for sperm motility and fertility. Here, we have studied the regulation of the a4 isoform and the relevance of this event in sperm capacitation. We have performed this by taking advantage of the selective high affinity of a4 to the inhibitor ouabain. Our results show that ouabain sensitive hydrolysis of ATP and uptake of 86Rb, corresponding to enzymatic and ion transport activity of a4 respectively, increased during sperm capacitation in a time-dependent manner. Specific labeling of a4 with the fluorescent indicator bodipy-ouabain and immunoblot analysis of biotinylated and streptavidin precipitated sperm plasma membrane proteins indicated a capacitation- and time-dependent rise in levels of active a4 isoform at the sperm surface. Protein phosphorylation, using anti-phosphoserine and anti-phosphotyrosine antibodies showed phosphorylation of the a4 isoform during capacitation. Ouabain inhibition of a4 blocked the increase in total sperm motility and the hyperactive motility characteristic of sperm capacitation. Moreover, interference of a4 activity with ouabain partially prevented the intracellular decrease in Na+ and the plasma membrane hyperpolarization that typically accompany sperm capacitation. In contrast, ouabain inhibition of a4, did not affect the spontaneous sperm acrosomal reaction following capacitation. Altogether, these results demonstrate that Na,K-ATPase a4 is subjected to regulation during sperm capacitation through mechanisms that involve increases in molecular activity, and phosphorylation and levels of a4 at the sperm plasma membrane. Regulation of a4 activity helps to support the changes in motility that are associated with sperm capacitation, emphasizing the biological relevance of Na,K-ATPase a4 isoform in sperm function.

Immuno-contraceptive potential of sperm specific LDHC&amp;lt;sub&amp;gt;4&amp;lt;/sub&amp;gt; and SPAM-1 (PH-20) sub units in dog

PH-20 (Spam-1) antigen appears to be a bi-functional sperm plasma membrane protein with hyaluronidase activity and its role is related to cumulus penetration as well as zona binding. (LDH-C4) is a key enzyme, distributed specifically in testis and is highly immunogenic, function of which relates to energy metabolism and capacitation of the sperm. Therefore, in this study, we observed the effect of purified native PH-20 and LDH-C4 antigens on sperm function and morphology in dog. Native PH-20 (46, 32 kDa) and LDH-C4 (36, 30, 28) sub units, purified from dog sperm SDS- extracts were used to test as immunocontraceptive candidates in stray dog. Dogs 1, 2, 3, 4, 5/6 and 7/8 were immunized with 46, 32, 36, 30/28, 46/32, 36/30/28 kDa sub units respectively by i/m route and semen was analysed at weekly intervals. High titre of 3200 - 6400 was achieved in all dogs after three boosters. Immune response in treated dogs was confirmed by increase in Ig level in blood serum and reaction of partially purified serum Ig of immunized dogs with respective antigens in double immune diffusion and immunoblots. Immunization of dogs either with individual PH-20/ LDHC sub units or pooled PH-20/LDHC4 sub units showed a significant effect on sperm count, percent motility, viability, HOST and sperm morphology. All the sperm parameters declined to a significant level (p 4 (36/30/28 kDa) sub units had an immunocontraceptive potential in dog.

Enzymatic activity of sperm proprotein convertase is important for mammalian fertilization

Proprotein convertase subtilisin/kexin 4 (PCSK4) is implicated for sperm fertilizing ability, based on studies using Pcsk4-null mice. Herein we demonstrated proprotein convertase (PC) activity in intact sperm and acrosomal vesicles. To determine whether this activity was important for sperm fertilizing ability, a peptide inhibitor was designed based on PCSK4 prodomain sequence (proPC4(75-90)), which contains its primary autocatalytic cleavage site. ProPC4(75-90) inhibited recombinant PCSK4's activity with a K(i) value of 5.4 µM, and at 500 µM, it inhibited sperm PC activity almost completely. Treatment of sperm with proPC4(75-90) inhibited their egg fertilizing ability in a dose dependent manner. Correlation between sperm PC activity and fertilizing ability showed a high co-efficient value (>0.9), indicating the importance of sperm PC activity in fertilization. In particular, sperm PC activity was important for capacitation and zona pellucida (ZP)-induced acrosome reaction, since proPC4(75-90) -treated sperm showed markedly decreased rates in these two events. These results were opposite to those observed in Pcsk4-null sperm, which contained higher PC activity than wild type sperm, possibly due to overcompensation by PCSK7, the other PCSK enzyme found in sperm. ADAM2 (45 kDa), a sperm plasma membrane protein, involved in sperm-egg plasma membrane interaction, was also processed into a smaller form (27 kDa) during capacitation at a much reduced level in proPC4(75-90) -treated sperm. This result suggested that ADAM2 may be a natural substrate of sperm PCSK4 and its cleavage by the enzyme during acrosome reaction may be relevant to the fertilization process.

Interaction of Potential Porcine Sperm Ligands with the Oocyte Plasma Membrane

We previously identified 62, 39, 27 and 7 kDa porcine sperm plasma membrane proteins that demonstrated a predominant affinity for the porcine oocyte plasma membrane by Western ligand blotting. The current experiments were designed to further investigate the potential roles of these molecules in sperm-oocyte plasma membrane interaction. Abilities of these proteins to bind to the oocyte plasma membrane and to inhibit sperm-oocyte interaction were evaluated. Plasma membrane was isolated primarily from the head of ejaculated porcine sperm by nitrogen cavitation and density gradient centrifugation. Fractions containing the 62, 39, 27 and 7 kDa proteins were electroeluted from one dimensional SDS polyacrylamide gels, dialysed and proteins biotinylated. Following incubation with zona-free porcine oocytes, bound protein was visualized with 20 μg TRITC-avidin/ml using confocal microscopy. Fractions of the dialysed, electroeluted proteins were added to porcine in vitro fertilization assays. The 62, 39, 27 and 7 kDa proteins all demonstrated binding to the oocyte plasma membrane in contrast to a biotinylated control protein. Addition of unlabelled sperm plasma membrane proteins to the biotinylated protein visibly reduced binding. Addition of each of these protein fractions to in vitro fertilization assays reduced sperm interaction with the porcine oocyte plasma membrane in a concentration-dependent manner. Binding of these sperm plasma membrane proteins to the oocyte plasma membrane and inhibition of fertilization are consistent with these proteins being involved in sperm-oocyte plasma membrane interaction.

275 EVIDENCE FOR α5 AND αV INTEGRINS ON SPERM PLASMA MEMBRANES AND THEIR ROLE IN BOVINE FERTILIZATION

Sperm-egg interaction is a complex molecular process leading to gamete fusion mediated by a series of molecular interactions. Some integrin subunits, which are adhesion molecules, are expressed on human and mouse sperm, but major questions about the roles of integrins in sperm-oocyte fusion remain unsolved. This study was conducted to determine the presence of α5 and αV integrins on cattle (Bos indicus and Bos taurus) sperm, and whether fertilization might be affected by treating sperm with antibodies to these integrin subunits. To determine if integrin subunits were present on sperm, sperm plasma membrane proteins were subjected to 1-dimensional SDS-PAGE and Western blot analysis. Frozen-thawed sperm, donated by ABS Pecplan, were centrifuged at 700 × g for 10 min, washed twice with warm PBS (Nutricell®, Campinas, Sao Paulo, Brazil), and resuspended in Jones buffer (0.4% deoxyclolic acid, 8.9 M sucrose, 0.1 M Tris, pH 8.5) for 60 min at 4°C to solubilize sperm plasma membranes. Plasma membrane proteins were then separated by SDS-PAGE and transferred to nitrocellulose. The resulting blots were probed with αV integrin antibody (Calbiochem®, San Diego, CA, USA) or α5 integrin antibody (Calbiochem) and developed using ECL. Frozen-thawed spermatozoa were washed by a 45/90% layered Percoll gradient centrifugation and incubated for 1 h in fertilization medium (FM; 1), FM with anti-integrin αV IgG (2), and FM with anti-integrin a5 IgG (3). In vitro-matured cattle oocytes were incubated (39°C, 5% CO2 in air) with 1 × 105 washed, pretreated spermatozoa per 25 oocytes for 18 h. The oocytes were fixed in acid alcohol, stained with 1% acetate-orcein, and observed to determine the presence of pronuclei. Each experiment was repeated 4 times and data from each experiment were pooled. Approximately 80 to 90 oocytes per treatment for fertilization were evaluated in each replicate. Weighted least squares means were used to analyze fertilization data (SAS software, SAS Institute, Cary, NC, USA). The significance level for all tests was P &lt; 0.05. Both antibodies for α5 (35 kDa) and αV (34 kDa) integrins showed immunoreactivity on Western blots of sperm membrane proteins. Addition of anti-integrin αV, and anti-integrin α5 decreased fertilization (P &lt; 0.05) compared with the control: (1) 94.1 ± 1.0%; (2) 18.2 ± 1.0%; (3) 12.2 ± 1.0%. These findings show that αV and α5 integrins are expressed by cattle spermatozoa and may be involved in sperm-oocyte fusion and fertilization. This study was supported by FAPESP grants (2007/00363-5 and 2006/06008-0, Brazil). We acknowledge Nutricell and ABS Pecplan for their generous contribution.

Localization and expression of a 70 kDa protein in goat spermatozoa having Na+,K+-ATPase inhibitory and arylsulphatase A activities

We have previously isolated and purified a goat sperm protein of 70 kDa molecular weight designated as P70 and characterized it as an inhibitor of Na(+),K(+)-ATPase. Our study reveals that the first 10 amino acid residues from the N-terminal end of P70 has high degree of homology with arylsulphatase A from mice, pig and human. Indirect immunofluorescence study shows the presence of the protein on goat sperm surface. Furthermore, live goat sperm and the extract of peripheral sperm plasma membrane proteins exhibit arylsulphatase A's desulphation activity. The P70 remains on the head surface of in vitro capacitated cauda epididymal sperm as shown by positive immunofluorescence staining of cauda sperm. Immunoblot and flow cytometric studies corroborate the above findings. The presence of P70 on capacitated cauda sperm surface suggest a possible role of this protein in sperm zona pellucida binding. In the present report we demonstrate arylsulphatase A like activity in P70 and describe its localization and expression in goat sperm.

Maturing the sperm: Unique mechanisms for modifying integral proteins in the sperm plasma membrane

The epididymis has been understudied, in part due to its cancer resistance and the development of effective technologies for sperm injection and in vitro fertilization. However, it is worthy of study because – absent advanced reproductive technology – its proper function is essential for conceiving children: sperm leaving the testis are immature and nonfertile. Epididymal functions can be divided into several general categories (1) concentration of sperm; (2) functional maturation; (3) storage in a quiescent state until ejaculation; (4) removal of degenerating sperm; (5) provision of appropriate conditions for survival; (6) transport by the myoid cells; (7) protection; (8) maintenance of the blood epididymal barrier. In the past decade investigators have focused on those maturational changes of the integral proteins of the sperm plasma membrane which are directly related to sperm–ova interactions. It has traditionally been thought that changes in the sperm plasma membrane proteins were limited to simple binding or removal of proteins or interactions with the proteases, glycosylases and glycotransferases present. However, the epididymis can also release secretory products in bulk through apical blebs and inject integral membrane proteins with epididymosomes which fuse with the plasma membrane. The epididymis also activates and cleaves enzymes present on the sperm surface (e.g., germ cell angiotensin converting enzyme), thus enabling them to modify proteins on the sperm membrane. Aside from the need to understand epididymal function relative to the sperm, basic science on epididymal physiology is warranted because it may help us understand the functioning of androgens, protection of tissues from oxidative damage, and resistance to cancer and benign hyperplasic growth.

Identification of an ADAM2-ADAM3 Complex on the Surface of Mouse Testicular Germ Cells and Cauda Epididymal Sperm

Male mice lacking ADAM2 (fertilin beta) or ADAM3 (cyritestin) are infertile; cauda epididymal sperm (mature sperm) from these mutant mice cannot bind to the egg zona pellucida. ADAM3 is barely present in Adam2-null sperm, despite normal levels of this protein in Adam2-null testicular germ cells (TGCs; sperm precursor cells). Here, we have explored the molecular basis for the loss of ADAM3 in Adam2-null TGCs to clarify the biosynthetic and functional linkage of ADAM2 and ADAM3. A small portion of total ADAM3 was found present on the surface of wild-type and Adam2(-/-) TGCs at similar levels. In the Adam2-null TGCs, however, surface-localized ADAM3 exhibited an increased amount of an endoglycosidase H-resistant form that may be related to instability of ADAM3. Moreover, we found a complex between ADAM2 and ADAM3 on the surface of TGCs and sperm. The intracellular chaperone calnexin was a component of the testicular ADAM2-ADAM3 complex. Our findings suggest that the association with ADAM2 is a key element for stability of ADAM3 in epididymal sperm. The presence of the ADAM2-ADAM3 complex in sperm also suggests a potential role of ADAM2 with ADAM3 in sperm binding to the egg zona pellucida.

Multiple proteins present in purified porcine sperm apical plasma membranes interact with the zona pellucida of the oocyte

An important step in fertilization is the recognition and primary binding of the sperm cell to the zona pellucida (ZP). Primary ZP binding proteins are located at the apical plasma membrane of the sperm head. In order to exclusively study primary zona binding proteins, plasma membranes of sperm heads were isolated, highly purified and subsequently solubilized with a mild or a strong solubilization procedure. Native, highly purified ZP ghosts were used as the binding substrate for solubilized sperm plasma membrane proteins, and a proteomic approach was employed to identify ZP binding proteins. Two-dimensional gel electrophoresis of ZP fragments with bound sperm proteins showed very reproducibly 24 sperm protein spots to be associated to the zona ghosts after mild plasma membrane solubilization whereas only three protein spots were detected after strong plasma membrane solubilization. This indicates the involvement of multiple sperm proteins in ZP binding. The three persistently bound proteins were identified by a tandem mass spectrometry as isoforms of AQN-3 and probably represent the main sperm protein involved in ZP binding. P47, fertilin beta and peroxiredoxin 5 were also conclusively identified. None of the identified proteins has a known acrosomal origin, which further indicated that there was no sample contamination with secondary ZP binding proteins from the acrosomal matrix. In this study, we showed and identified multiple zona binding proteins involved in primary sperm-zona binding. Although we were not able to identify all of the proteins involved, this is a first step in understanding the event of primary sperm-zona interactions and the relevance of this for fertilization is discussed.