Characterization of two antigenically related integral membrane proteins of the guinea pig sperm periacrosomal plasma membrane

The periacrosomal plasma membrane of mammalian spermatozoa functions both in recognition and in binding of the egg's zona pellucida and in the acrosome reaction. This study characterizes two antigenically related proteins with molecular weights of 35 kD (PM35) and 52 kD (PM52) of the guinea pig sperm periacrosomal plasma membrane. Polyclonal antisera were prepared against electrophoretically purified PM35 or PM52. Each antiserum recognized both the 35-kD and 52-kD polypeptides on Western blots, indicating that they are structurally related. This conclusion was supported by peptide mapping experiments demonstrating comparably sized fragments of both PM35 and PM52. Both PM35 and PM52 behave as integral membrane proteins during phase-separation analysis with Triton X-114. Electron microscopic immunocytochemistry and differential fractionation of sperm membranes established that both PM35 and PM52 are exclusively localized to the periacrosomal plasma membrane. Three different antisera were used for ultrastructural studies, and each specifically bound the cytoplasmic but not the extracellular membrane surface. The electrophoretic mobilities of the PM35 and PM52 polypeptides were unchanged during sperm maturation and during the ionophore-induced acrosome reaction. The localization of PM35 and PM52 suggests a potential role for these integral plasma membrane proteins in signal transduction or membrane fusion events of the acrosome reaction. © 1994 Wiley-Liss, Inc.     

Conserved ram seminal plasma proteins bind to the sperm membrane and repair cryopreservation damage

Whole seminal plasma (SP) enhances the function and fertility of frozen/thawed ram sperm. The objective of the current study was to investigate whether SP proteins capable of binding to molecules from the sperm plasma membrane were conserved among ram breeds, and whether these proteins were sufficient to overcome cryopreservation-induced reductions in sperm quality. Whole ram SP, obtained from rams of various breeds, improved progressive motility of frozen/thawed sperm at all times evaluated (P < 0.05); however, it did not improve total motility (15 min, P = 0.480; 30 min, P = 0.764; and 45 min, P = 0.795). To identify SP proteins responsible for this effect, a new method was developed to retain SP proteins that bound specifically to the sperm membrane by immobilization of sperm membrane proteins. These proteins specifically bound to the sperm surface, especially the acrosomal region. Lactotransferrin, epididymal secretory protein E1, Synaptosomal-associated protein 29, and RSVP-20 were identified (mass spectrometry) in this fraction. The retained SP proteins fraction repaired ultrastructural damage of frozen/thawed sperm and, with the addition of fructose, significantly improved motility of frozen/thawed sperm. We concluded that SP proteins that bound to the sperm membrane were conserved among ram breeds, and that when added to frozen/thawed semen (along with an energy source), they repaired ram sperm damage and enhanced sperm motility.     

Glycoprotein changes in the rat sperm plasma membrane during maturation in the epididymis.

To investigate surface glycoprotein changes during post-testicular maturation, plasma membranes were isolated from proximal caput, distal caput, and cauda epididymal rat spermatozoa. Membrane glycoproteins were identified on Western blots of SDS-PAGE fractionated samples using biotinylated lectins and Vecta-stain reagents; these were compared to glycoproteins present in cauda epididymal luminal fluid. Lens culinaris agglutinin, Pisum sativum agglutinin, peanut agglutinin, wheat germ agglutinin, Ricinus communis agglutinin, Ulaex europaeus agglutinin, and Dolichol biflorus agglutinin each bound a specific subset of the polypeptides present. Several types of glycoprotein changes were noted including their appearance, loss, alteration of staining intensity, and alteration of electrophoretic mobility. Some maturation-dependent sperm surface glycoproteins co-migrated with glycoproteins present in epididymal fluid. This approach of direct analysis of the glycoproteins in purified plasma membranes identifies a broader spectrum of maturation-related surface changes occurring within the epididymis than are noted with surface labeling procedures.     

A novel approach to identify bovine sperm membrane proteins that interact with receptors on the vitelline membrane of bovine oocytes

At fertilization, the sperm triggers intracellular calcium oscillations, which are pivotal to oocyte activation and development. A working hypothesis for the interaction between the sperm and the oocyte is that disintegrin ligands on the inner acrosomal membrane of the sperm bind to integrin receptors on the oocyte vitelline membrane. The aim of these experiments was to find and identify the sperm protein ligands involved in bovine sperm-oocyte interactions. In situ fluorescent labeling of proteins and 2-D gel electrophoresis were used to identify specific sperm membrane proteins that interact with proteins in the oocyte vitelline membrane. Sperm were labeled with a fluorescent dye and used to fertilize zona-free oocytes. Sperm-oocyte complexes were either lysed immediately, or following covalent cross-linking of proteins with dibromobimane. The cross-linking reagent serves the critical function of covalently linking proteins together so that they will remain as a unit through lysis of the cells and 2-D gel analysis, and which can be subsequently identified by mass spectrometry. Lysates were electrophoretically run on the same 2-D gel. The comparison of uncross-linked and cross-linked protein spots revealed that some proteins shifted position based on binding. These spots were picked and proteins identified by mass spectrometry. These results provide a list of specific sperm proteins that interact with oocyte membrane proteins and establish a group of candidate ligands, one or more of which may be responsible for induction of outside-in signaling resulting in oocyte activation and fusion of the gametes.     

Proteomic analysis of sperm regions that mediate sperm-egg interactions

The sperm interacts with three oocyte-associated structures during fertilization: the cumulus cell layer surrounding the oocyte, the egg extracellular matrix (the zona pellucida), and the oocyte plasma membrane. Each of these interactions is mediated by the sperm head, probably through proteins both on the sperm surface and within the acrosome, a specialized secretory granule. In this study, we have used subcellular fractionation in order to generate a proteome of the sperm head subcellular compartments that interact with oocytes. Of the proteins we identified for which a gene knockout has been tested, a third have been shown to be essential for efficient reproduction in vivo. Many of the other presently untested proteins are likely to have a similarly important role. Twenty-five percent of the cell surface fraction proteins are previously uncharacterized. We have shown that at least two of these novel proteins are localized to the sperm head. In summary, we have identified over 100 proteins that are expressed on mature sperm at the site of sperm-oocyte interactions.     

Further characterization of boar sperm plasma membrane proteins with affinity for the porcine zona pellucida

Boar sperm plasma membrane proteins (PMPs) with affinity for the zona pellucida were partially purified from columns of dextran sulfate using a linear salt gradient and a buffered detergent that retained their ability to block directly the binding of uncapacitated and capacitated sperm to isolated porcine oocytes. PMPs that bound most strongly to dextran sulfate (fraction IV) were also most effective in blocking sperm binding to porcine oocytes. These tightly bound proteins also bound to isolated zonae to a greater extent than other fractions. Monovalent antibodies to fraction IV PMPs completely blocked sperm binding to isolated eggs. Fraction IV PMPs lost the ability to inhibit directly the binding to eggs when treated with chaotropic agents and trypsin; the fraction also displayed a tendency to aggregate in the absence of high salt. This property and the affinity of proteins in this fraction for sulfated polysaccharides indicate that specific hydrophilic interactions may play a significant role in sperm-zona attachments.     

Correlation of zona-binding with oocyte maturation and sperm motility in rhesus monkeys by hemizona assay

The hemizona assay (HZA) in Rhesus monkeys was employed to study the correlation of zona-binding ability with sperm motility or with naturally developing oocytes at various maturational stages. Oocytes from unstimulated ovaries were retrieved within 2 hr from monkeys sacrificed for vaccine production (in reproductive season, but with their menstrual cycles not determined). Oocytes were divided into four groups based on their morphological maturation: 1) Oocytes surrounded by more than one cumulus layer (MC); 2) Oocytes retaining intact germinal vesicle nuclei (GV); 3) Oocytes with germinal vesicle breakdown showing distinct perivitelline space (PVS); and 4) Oocytes extruding the first polar body (PBI). The mean numbers of sperm bound to hemizona for PB1, PVS, GV, and MC groups were 132.9 ± 12.0, 71.5 ± 10.1, 36.1 ± 4.0, and 20.1 ± 2.9 (Mean ± SE), respectively. The four groups showed significant differences from each other in sperm/egg binding ability (P < 0.01). The number of bound sperm significantly increased with oocyte maturation. The present study also showed that zona-binding ability was also affected by sperm motility. For sperm with 67.7% motility and sperm with 31.2% motility, the average numbers of bound sperm were 43.5 ± 2.2 and 25.3 ± 2.9 (Mean ± SE), respectively. There was significantly higher binding ability for sperm with higher motility (P < 0.01). The results suggest that: 1) The rhesus monkey model can serve as a very sensitive model for studying sperm/egg interaction by HZA; 2) Sperm motility positively correlated with sperm/egg binding; and 3) Sperm/egg binding ability increases with oocyte maturation. The binding ability is highest when oocytes matured to the PB1 stage, which is also the best opportunity for fertilization. This is strong evidence for the “zona maturation” hypothesis. © 1994 Wiley-Liss, Inc.     

Relationship between sperm nucleus remodelling and cell cycle progression of fragments of mouse parthenogenotes

Nucleate and anucleate fragments of parthenogenetically activated mouse oocytes, as well as cybrids obtained by fusion of anucleate fragments (cytoplasts) of maturing and activated matured oocytes were fertilized at different time after activation. Remodelling of the sperm nucleus was studied by electron microscopy at 1.5 and 3 h after fertilization and, in addition, at 14 h in cybrids. Results show that (1) the nuclear envelope of the sperm nucleus can break down when the insemination takes place after the end of M-phase, but the capacity of the parthenote cytoplasm to remodel the sperm nucleus is restricted in time. (2) Male chromatin can decondense within the old, unbroken nuclear envelope, but in such cases formation of a male pronucleus, one of the two nuclei of zygote possessing inactive nucleoli, is never observed. © 1994 Wiley-Liss, Inc.     

Adsorption of oviductal fluid proteins by the bovine sperm membrane during in vitro capacitation.

125I-labeled oviductal fluid (ODF) proteins and antiserum to ODF were used to determine whether ODF proteins associate with the sperm membrane during in vitro capacitation. Luteal and nonluteal pools of ODF were obtained from oviduct catheters during the estrous cycle. Washed sperm (50 x 10(6) sperm/ml) were incubated up to 4 h in a protein-free modified Tyrode's medium (MTM), or MTM supplemented with 40% ODF, or 0.5 ng 125I-labeled ODF proteins. Solubilized sperm membrane proteins and incubation media containing ODF proteins were separated by gel electrophoresis. Membranes isolated from bovine sperm, previously incubated with ODF, adsorbed five 125I-proteins: A doublet at 85-95 kDa, and others at 24, 34, 53, and 66 kDa. The amount of 66 kDA 125I-protein associated with the sperm decreased during the incubation, whereas the amount of 85 to 95-kDa protein did not. Western blot analyses also detected the presence of ODF proteins (53, 66, 85-95, and 116 kDa) in solubilized membranes from sperm incubated in ODF. The 85 to 95-kDa protein in ODF decreased in apparent molecular weight by 5 kDa when associated with the sperm membrane. At 53 kDa, ODF proteins which associated with sperm were transformed from two to three separate proteins. These studies indicate that the surface of sperm is modified by adsorption of ODF proteins to the membrane during in vitro capacitation.     

Binding of porcine sperm plasma membrane proteins to sheep, hamster and mouse oocyte plasma membrane

Four porcine sperm plasma membrane proteins were previously identified as putative ligands for the oocyte plasma membrane. The present study examined the binding of these proteins and two additional porcine sperm membrane proteins to oocytes from sheep, mice and hamsters as a first step in assessing potential conservation of these putative sperm ligands across species and across mammalian orders. Plasma membrane vesicles were isolated from porcine sperm, solubilised, and the proteins separated by one-dimensional gel electrophoresis. The 7, 27, 39 and 62 kDa porcine sperm protein bands demonstrating predominant binding of the porcine oocyte plasma membrane on ligand blots, a 90 kDa protein band demonstrating minor binding, and a 97 kDa protein band that did not bind the oocyte plasma membrane probe were electroeluted. Proteins were biotinylated, and incubated with zona-free oocytes. Bound biotinylated protein was labelled with fluorescent avidin and the oocytes examined with a confocal microscope. The 7 kDa, 27 kDa and the 39 kDa proteins bound to the sheep oocytes but not to a majority of the hamster or mouse oocytes. The 62 kDa protein bound to sheep oocytes and mouse oocytes but not to a majority of the hamster oocytes. The 90 kDa protein bound to oocytes from all three species. The 97 kDa protein, which did not recognise the porcine oocyte probe on a Western ligand blot, did not bind to oocytes from any species and served as a negative control. These observations are consistent with significant conservation of molecule and function among species within the same mammalian order. Hence, one species may be a good model for other species from the same order. Only limited conservation of binding activity of porcine sperm plasma membrane proteins to rodent oocytes was observed, suggesting a greater divergence either in molecular structure or in function among species from different orders.     

Capacitation dependent activation of tyrosine phosphorylation generates two sperm head plasma membrane proteins with high primary binding affinity for the zona pellucida

The recognition and binding of sperm cells to the zona pellucida (the extracellular matrix of the oocyte) are essential for fertilization and are believed to be species specific. Freshly ejaculated sperm cells do not bind to the zona pellucida. Physiologically this interaction is initiated after sperm activation in the female genital tract (capacitation) via a yet unknown mechanism, resulting in the binding of a receptor in the apical sperm plasma membrane to the zona pellucida. In order to mimic this biochemically, we isolated zona pellucida fragments from gilt ovaries to prepare an affinity column with the intact zona pellucida structure and loaded this column with solubilized apical plasma membranes of boar sperm cells before and after in vitro capacitation. With this technique we demonstrated that two plasma membrane proteins of capacitated boar sperm cells showed high affinity for zona pellucida fragments. Further analysis showed that these proteins were tyrosine phosphorylated. Plasma membrane proteins from freshly ejaculated sperm cells did not exhibit any zona pellucida binding proteins, likely because these proteins were not tyrosine phosphorylated.     

Changes in exposed membrane proteins during in vitro capacitation of boar sperm

Exposed plasma membrane proteins were labeled with 125I before and after incubation of boar sperm under capacitating conditions. Labeled protein profiles were compared to the ability of the sperm to penetrate zona-free hamster ova. Quantitatively, the labeled sperm membrane proteins were primarily low Mr prior to capacitation. The majority of the labeled seminal plasma protein was also low Mr. After capacitation, two new proteins (64,000 Mr and 78,000 Mr) were labeled. Sperm did not exhibit these exposed membrane proteins when incubated under noncapacitating conditions. Appearance of these proteins was not correlated to the percentage of acrosome-reacted sperm. Although the 64,000 Mr protein was not consistently observed, the relative labeling of the 78,000 Mr protein was highly correlated with the ability of sperm to fuse with zona-free hamster ova. The 78,000 Mr protein may be a sperm protein involved in fusion with the egg plasma membrane.     

Structural requirements for the inhibitory action of the CD9 large extracellular domain in sperm/oocyte binding and fusion

CD9 has been shown to be essential for sperm/oocyte fusion in mice, the only non-redundant role found for a member of the tetraspanin family. CD9 can act in cis, reconstituting sperm/oocyte fusion when ectopically expressed in oocytes from CD9 null mice, or in trans, inhibiting sperm fusion when the large extracellular domain (LED) is added to CD9-positive oocytes as a soluble protein. In contrast to cis inhibition, the structural requirements of the trans inhibition by soluble CD9 LED are unknown. Here we show that human CD9 LED is as potent an inhibitor as mouse CD9 LED in mouse sperm/oocyte fusion assays and that CD9 LED can also inhibit sperm/oocyte binding. The two disulphide bridges that define membership of the tetraspanin family are critical for structure and function of human CD9 LED and mutation of a pentapeptide sequence in the hypervariable region further defines the critical region for trans inhibition.     

Characterization of low Mr zona pellucida binding proteins from boar spermatozoa and seminal plasma.

A group of low Mr (16 kDa-23 kDa) glycoproteins on ejaculated boar spermatozoa have been shown to have high affinity for homologous zona pellucida glycoproteins (ZPGPs). These ZPGP binding proteins are derived from seminal plasma as shown by their absence from epididymal spermatozoa and their presence in seminal plasma as identified by N-terminal amino acid sequence analysis. They bind to ZPGPs by a polysulphate recognition mechanism similar to that found for proacrosin-ZPGP interactions. The haemagglutination activity of boar seminal plasma is also associated with these low Mr glycoproteins. It is suggested that they play a role in regulating the rate of sperm capacitation and survival in the female reproductive tract.     

Requirement of sperm‐oocyte plasma membrane fusion for establishment of the plasma membrane block to polyspermy in human pronuclear oocytes

We investigated whether the incorporation of the sperm membrane into the oolemma contributes to the human plasma membrane block to polyspermy. We used zona pellucida–free oocytes fertilized by intracytoplasmic sperm injection (ICSI) or activated by parthenogenetic activation. Only two of the 35 pronuclear oocytes fertilized by spermatozoa (control) demonstrated one single penetrating spermatozoa. In contrast, the majority of ICSI and parthenogenetically activated pronuclear oocytes were penetrated with an average of three spermatozoa per oocyte. The number of fused and binding spermatozoa of ICSI and parthenogenetically activated oocytes were significantly higher than in control oocytes (3.5 ± 0.6 and 4.3 ± 0.6 for ICSI; 3.0 ± 0.3 and 3.8 ± 0.4 for activated and 0.2 ± 0.1 and 0.6 ± 0.2 for controls, respectively, P < 0.01). Furthermore, the cortical granules were released from the cortex of ICSI and calcium ionophore‐puromycin‐activated pronuclear oocytes to the same extent as that of pronuclear oocytes fertilized by spermatozoa. These results suggest that the establishment of the plasma membrane block to sperm penetration in the human oocyte may require a fusion process between sperm and oocyte plasma membranes. Mol. Reprod. Dev. 52:183–188, 1999. © 1999 Wiley‐Liss, Inc.     

哺乳动物及人精子膜离子通道的研究进展

离子的跨膜转动对精子的生理活动起重要的作用。近年,应用膜片钳及人工膜重组等研究通道有关的电生理技术,人们直接观察到哺乳动物及人精子膜上Ｋ、Ｎａ＾＋、Ｃａ＾２＋、Ｃｌ通道的存在。这些结果为揭示精子成熟、获能精卵结合反应等生理过程的某些细节提供了有有的资料,特别是对人精子膜的研究,还为临床应用提供了可能。     

The steroid-binding subunit of the Na/K-ATPase as a progesterone receptor on the amphibian oocyte plasma membrane

Progesterone acts at a plasma membrane receptor on the Rana oocyte to initiate meiosis. A cascade of lipid messengers occurs within seconds, followed by sequential changes in membrane phospholipid composition. We now show that progesterone binding to the plasma membrane increases continuously over the first 4 h. Subsequently, about 60% of the total plasma membrane and > 90% of membrane-bound progesterone, ouabain binding sites, and Na/K-ATPase activity are internalized. Until the completion of membrane internalization, oocytes must be continuously exposed to nanomolar concentrations of exogenous progesterone for meiosis to continue. The membrane-bound progesterone remains unchanged, whereas microinjected [(3)H]progesterone is rapidly metabolized. We find that progesterone and the plant steroid ouabain compete for one of two ouabain binding sites on the oocyte surface. Ouabain blocks progesterone action and inhibits subsequent meiosis if added at any time during the first 4-5 h. Western blots of SDS/PAGE extracts of isolated oocyte plasma membranes contain a -110 kDa band which binds an antibody to the steroid-binding c-terminal domain in rat and human PR. The number of binding sites and K(d) for progesterone binding to the plasma membrane is comparable to those for low-affinity ouabain binding to the alpha-subunit of the Na/K-ATPase (112 kDa). Our results suggest that progesterone binding to the ouabain binding site on the N-terminal region of the alpha-subunit of Na/K-ATPase may modulate early plasma membrane events over the first 4-6 h. Progesterone may thus act in part through the plasma membrane Na/K-ATPase signaling system.     

Proteomic analysis of rat hippocampal plasma membrane: characterization of potential neuronal-specific plasma membrane proteins

The hippocampus is a distinct brain structure that is crucial in memory storage and retrieval. To identify comprehensively proteins of hippocampal plasma membrane (PM) and detect the neuronal-specific PM proteins, we performed a proteomic analysis of rat hippocampus PM using the following three technical strategies. First, proteins of the PM were purified by differential and density-gradient centrifugation from hippocampal tissue and separated by one-dimensional electophoresis, digested with trypsin and analyzed by electrospray ionization (ESI) quadrupole time-of-flight (Q-TOF) tandem mass spectrometry (MS/MS). Second, the tryptic peptide mixture from PMs purified from hippocampal tissue using the centrifugation method was analyzed by liquid chromatography ion-trap ESI-MS/MS. Finally, the PM proteins from primary hippocampal neurons purified by a biotin-directed affinity technique were separated by one-dimensional electrophoresis, digested with trypsin and analyzed by ESI-Q-TOF-MS/MS. A total of 345, 452 and 336 non-redundant proteins were identified by each technical procedure respectively. There was a total of 867 non-redundant protein entries, of which 64.9% are integral membrane or membrane-associated proteins. One hundred and eighty-one proteins were detected only in the primary neurons and could be regarded as neuronal PM marker candidates. We also found some hypothetical proteins with no functional annotations that were first found in the hippocampal PM. This work will pave the way for further elucidation of the mechanisms of hippocampal function.