Zona-binding inhibitory factor-1 from human follicular fluid is an isoform of glycodelin

Zona-binding inhibitory factor-1 (ZIF-1), a glycoprotein in human follicular fluid, reduces the binding of spermatozoa to the zona pellucida. ZIF-1 has a number of properties similar to those of glycodelin-A from human follicular fluid. The objective of this study was to compare the biochemical characteristics of these two glycoproteins. N-terminal sequencing and protease-digested peptide mapping showed that ZIF-1 and glycodelin-A have the same protein core. However, these glycoproteins differ in their oligosaccharide chains, as demonstrated by fluorophore-assisted carbohydrate electrophoresis, lectin-binding ability, and isoelectric focusing. ZIF-1 inhibited spermatozoa-zona pellucida binding slightly more than did glycodelin-A and significantly suppressed progesterone-induced acrosome reaction of human spermatozoa. Indirect immunofluorescence staining revealed specific binding of glycodelin-A and ZIF-1 to the acrosome region of human spermatozoa, where ZIF-1 produced a stronger signal than did glycodelin-A at the same protein concentration. These data suggest that ZIF-1 is a differentially glycosylated isoform of glycodelin that potently inhibits human sperm-egg interaction. Future study on the function role of ZIF-1 would provide a better understanding of the regulation of fertilization in humans.     

The Parkes Lecture. Zona pellucida glycoprotein mZP3: a versatile player during mammalian fertilization

All mammalian eggs are surrounded by a relatively thick extracellular coat, the zona pellucida (ZP), which facilitates fertilization of eggs by a single spermatozoon. The mouse egg ZP is constructed of only three glycoproteins, termed mZP1-3. Each of these glycoproteins consists of a unique polypeptide that is heterogeneously glycosylated with both asparagine-(N-)linked and serine/threonine-(O-)linked oligosaccharides. Polypeptides of ZP glycoproteins are highly conserved among mammalian species and are similar to polypeptides of egg vitelline envelope glycoproteins of fish, birds and amphibians. One of the mouse ZP glycoproteins, mZP3, serves as both a receptor for spermatozoa and an inducer of the acrosome reaction during fertilization. Free-swimming acrosome-intact spermatozoa recognize and bind to certain serine-(O-)linked oligosaccharides located close to the carboxy terminus of mZP3 polypeptide and, after binding, undergo the acrosome reaction (cellular exocytosis). In this review, in addition to the background information presented, results of recent experiments using homologous recombination to produce mZP3 null mice and site-directed mutagenesis to inactivate mZP3 as a sperm receptor and inducer of the acrosome reaction are presented and discussed.     

Mammalian zona pellucida glycoproteins: structure and function during fertilization

Zona pellucida (ZP) is a glycoproteinaceous translucent matrix that surrounds the mammalian oocyte and plays a critical role in the accomplishment of fertilization. In humans, it is composed of 4 glycoproteins designated as ZP1, ZP2, ZP3 and ZP4, whereas mouse ZP is composed of ZP1, ZP2 and ZP3 (Zp4 being a pseudogene). In addition to a variable sequence identity of a given zona protein among various species, human ZP1 and ZP4 are paralogs and mature polypeptide chains share an identity of 47%. Employing either affinity purified native or recombinant human zona proteins, it has been demonstrated that ZP1, ZP3 and ZP4 bind to the capacitated human spermatozoa and induce an acrosome reaction, whereas in mice, ZP3 acts as the putative primary sperm receptor. Human ZP2 only binds to acrosome-reacted spermatozoa and thus may be acting as a secondary sperm receptor. In contrast to O-linked glycans of ZP3 in mice, N-linked glycans of human ZP3 and ZP4 are more relevant for induction of the acrosome reaction. Recent studies suggest that Sialyl-Lewis^x sequence present on both N- and O-glycans of human ZP play an important role in human sperm?Cegg binding. There are subtle differences in the downstream signaling events associated with ZP3 versus ZP1/ZP4-mediated induction of the acrosome reaction. For example, ZP3 but not ZP1/ZP4-mediated induction of the acrosome reaction is dependent on the activation of the G_i protein-coupled receptor. Thus, various studies suggest that, in contrast to mice, in humans more than one zona protein binds to spermatozoa and induces an acrosome reaction.     

The use of anti-VDAC2 antibody for the combined assessment of human sperm acrosome integrity and ionophore A23187-induced acrosome reaction

Voltage-dependent anion channel (VDAC) is mainly located in the mitochondrial outer membrane and participates in many biological processes. In mammals, three VDAC subtypes (VDAC1, 2 and 3) have been identified. Although VDAC has been extensively studied in various tissues and cells, there is little knowledge about the distribution and function of VDAC in male mammalian reproductive system. Several studies have demonstrated that VDAC exists in mammalian spermatozoa and is implicated in spermatogenesis, sperm maturation, motility and fertilization. However, there is no knowledge about the respective localization and function of three VDAC subtypes in human spermatozoa. In this study, we focused on the presence of VDAC2 in human spermatozoa and its possible role in the acrosomal integrity and acrosome reaction using specific anti-VDAC2 monoclonal antibody for the first time. The results exhibited that native VDAC2 existed in the membrane components of human spermatozoa. The co-incubation of spermatozoa with anti-VDAC2 antibody did not affect the acrosomal integrity and acrosome reaction, but inhibited ionophore A23187-induced intracellular Ca(2+) increase. Our study suggested that VDAC2 was located in the acrosomal membrane or plasma membrane of human spermatozoa, and played putative roles in sperm functions through mediating Ca(2+) transmembrane transport.     

Mouse gamete interactions: The zona pellucida is the site of the acrosome reaction leading to fertilization in vitro

The question of whether the acrosome reaction, which leads to fertilization, occurs in intact sperm bound to the zona pellucida of the egg or in intact sperm before contact with the egg, was addressed by assessing the effect of 3-quinuclidinyl benzilate (QNB) on the two types of acrosome reaction. QNB is a specific inhibitor of the fertilization of zona-intact mouse eggs by mouse sperm. Mouse spermatozoa in suspension underwent acrosome reactions at a low rate, which could be accelerated by addition of 5 μM divalent cation ionophore A23187; the occurrence of such acrosome reactions was not inhibited by QNB. The rate at which acrosome reactions occurred in sperm bound to the zona pellucida of cumulus-free eggs, bound to isolated zonae, or exposed to acid-solubilized zona components, was greatly accelerated relative to that observed in the absence of zonae. These acrosome reactions were strongly inhibited by QNB at concentrations which inhibit the fertilization of zona-intact mouse eggs in vitro. These data suggest that the zona pellucida can induce acrosome reactions in mouse spermatozoa and that these acrosome reactions are the ones which lead to the fertilization of zona-intact eggs. In contrast, the acrosome rection in sperm which are not in contact with the zona is not associated with fertilization of zona-intact eggs.     

Expression and localization of the spermatogenesis-related gene, Znf230, in mouse testis and spermatozoa during postnatal development

Znf230, the mouse homologue of the human spermatogenesis-related gene, ZNF230, has been cloned by rapid amplification of cDNA ends (RACE). This gene is expressed predominantly in testis, but its expression in different testicular cells and spermatogenic stages has not been previously analyzed in detail. In the present study, the cellular localization of the Znf230 protein in mouse testis and epididymal spermatozoa was determined by RT-PCR, immunoblotting, immunohistochemistry and immunofluorescence. It is primarily expressed in the nuclei of spermatogonia and subsequently in the acrosome system and the entire tail of developing spermatids and spermatozoa. The results indicate that Znf230 may play an important role in mouse spermatogenesis, including spermatogenic cell proliferation and sperm maturation, as well as motility and fertilization.     

Mammalian sperm acrosome: formation, contents, and function

Sperm-egg interaction is a carbohydrate-mediated species-specific event which initiates a signal transduction cascade resulting in the exocytosis of sperm acrosomal contents (i.e., the acrosome reaction). This step is believed to be a prerequisite which enables the acrosome-reacted spermatozoa to penetrate the zona pellucida (ZP) and fertilize the egg. Successful fertilization in the mouse and several other species, including man, involves several sequential steps. These are (1) sperm capacitation in the female genital tract; (2) binding of capacitated spermatozoa to the egg's extracellular coat, the ZP; (3) induction of acrosome reaction (i.e., sperm activation); (4) penetration of the ZP; and (5) fusion of spermatozoon with the egg vitelline membrane. This minireview focuses on the most important aspects of the sperm acrosome, from its formation during sperm development in the testis (spermatogenesis) to its modification in the epididymis and function following sperm-egg interaction. Special emphasis has been given to spermatogenesis, a complex process involving multiple molecular events during mitotic cell division, meiosis, and the process of spermiogenesis. The last event is the final phase when a nondividing round spermatid is transformed into the complex structure of the spermatozoon containing a well-developed acrosome. Our intention is also to briefly discuss the functional significance of the contents of the sperm acrosome during fertilization. It is important to mention that only the carbohydrate-recognizing receptor molecules (glycohydrolases, glycosyltransferases, and/or lectin-like molecules) present on the surface of capacitated spermatozoa are capable of binding to their complementary glycan chains on the ZP. The species-specific binding event starts a calcium-dependent signal transduction pathway resulting in sperm activation. The hydrolytic and proteolytic enzymes released at the site of sperm-zona interaction along with the enhanced thrust of the hyperactivated beat pattern of the bound spermatozoon, are important factors in regulating the penetration of the zona-intact egg.     

Ultrastructural cytochemistry of human spermatozoa]

Enzymatic activities and repartition of glycoproteins were studied with electron microscopy in human ejaculated spermatozoa. Enzymatic activities are localised in the head of spermatozoon: arylsulfatase in the acrosome, acid phosphatase in the periacrosomal cytoplasm. Phosphotungstic acid at low pH and collo?dal iron allow detection of glycoproteins and acid groups on the sperm cell surface. Glycoproteins are present in the acrosome. These results are slightly different to those obtained in other species.     

哺乳动物透明带糖蛋白及其生物活性

透明带在哺乳动物的受精过程中发挥着重要作用,透明带化学成分及其生物活性的研究对于了解精卵识别、顶体反应的诱导等过程的分子机理具有重要意义．近10年来的研究表明透明带主要由糖蛋白（ZPGPs）组成,是在细胞质内合成后转移至透明带的.大多数动物的ZPGPs为4～5种,不同种类动物的ZPGPs的氨基酸序列存在高度同源性.在受精过程中,ZPGPs具有精子受体和诱导顶体反应的双重作用,ZPGPs含有N－连接和O－连接寡聚糖,这些寡糖链是ZPGPs行使生物活性不可缺少的部分．     

Dynamin Regulates Specific Membrane Fusion Events Necessary for Acrosomal Exocytosis in Mouse Spermatozoa

Mammalian spermatozoa must complete an acrosome reaction prior to fertilizing an oocyte. The acrosome reaction is a unique exocytotic event involving a series of prolonged membrane fusions that ultimately result in the production of membrane vesicles and release of the acrosomal contents. This event requires the concerted action of a large number of fusion-competent signaling and scaffolding proteins. Here we show that two different members of the dynamin GTPase family localize to the developing acrosome of maturing mouse germ cells. Both dynamin 1 and 2 also remain within the periacrosomal region of mature mouse spermatozoa and are thus well positioned to regulate the acrosome reaction. Two pharmacological inhibitors of dynamin, dynasore and Dyngo-4a, blocked the in vitro induction of acrosomal exocytosis by progesterone, but not by the calcium ionophore A23187, and elicited a concomitant reduction of in vitro fertilization. In vivo treatment with these inhibitors also resulted in spermatozoa displaying reduced acrosome reaction potential. Dynamin 1 and 2 phosphorylation increased on progesterone treatment, and this was also selectively blocked by dynasore. On the basis of our collective data, we propose that dynamin could regulate specific membrane fusion events necessary for acrosomal exocytosis in mouse spermatozoa.     

External calcium concentration and fertilization in the sea urchin, Strongylocentrotusfranciscansus (A. Agassiz) (Echinodermata)

The role of external calcium ions in the fertilization of the sea urchin, Strongylocentrotusfranciscansus (A. Agassiz), was studied. When eggs were inseminated with jelly-treated sperm in artificial sea waters containing various concentrations of calcium, the percentage of fertilization decreased with decreasing concentrations of calcium. A small amount of external calcium ions was also found to be essential for fertilization by spermatozoa with reacted acrosomes. The binding capacity of jelly-treated spermatozoa to eggs was, however, not adversely affected by a calcium deficiency. These results suggest that calcium ions are required not only for the initiation of the acrosome reaction, but also for successful levels of fertilization following sperm-binding to eggs even after the acrosome reaction has occurred.     

Molecules involved in sperm-zona pellucida interaction in mammals. Role in human fertility

Fertilization in mammals requires an initial interaction of sperm with the oocyte envelope, the zona pellucida (ZP), before it reaches the oocyte. ZP is a highly glycosylated structure, composed of three (mouse) or four (rabbit, boar, bovine, humans...) glycoproteins. The presence of ZP around the oocyte does not allow heterospecific fertilization. This barrier is principally due to the presence of species-specific glycosylations on ZP proteins. Sperm bind ZP by means of membrane receptors which recognize carbohydrate moieties on ZP glycoproteins according to a well-precised sequential process. Upon initial attachment, spermatozoa bind ZP3/ZP4 which induces the sperm acrosome exocytosis followed by a secondary binding of acrosome reacted spermatozoa to ZP2 and by ZP penetration. The sperm receptors are adhesive proteins or integral plasma membrane proteins linked to intraspermatic signalling pathways activating the acrosome reaction. Over the last twenty years, numerous studies have been carried out to identify sperm receptors to ZP in several species, but the data in humans are still incomplete. Work initiated in our research group has identified several proteins interacting with recombinant human ZP2, ZP3 and ZP4, among which are glycolytic enzymes. These enzymes are involved in the gamete interaction by means of their affinity to sugars and not by their catalytic properties. From a clinical point of view, an observed lack or weak expression of some sperm receptors to ZP3 in cases of idiopathic infertility associated with in vitro fertilization failure suggests that knowing the molecular mechanism driving the gamete recognition can be important at the diagnostic level. Furthermore, it has been shown that proteins that mediate gamete recognition diverge rapidly, as a result of positive darwinian selection. A sexual conflict can drive co-evolution of reproductive molecules in both sexes resulting in reproductive isolation and species emergence.     

EARLY STEPS OF SPERM—EGG INTERACTIONS DURING MAMMALIAN FERTILIZATION

Mammalian eggs are surrounded by two egg coats: the cumulus oophorus and the zona pellucida, which is an extracellular matrix composed of sulfated glycoproteins. The first association of the spermatozoon with the zona pellucida occurs between the zona glycoprotein, ZP3 and sperm receptors, located at the sperm plasma membrane, such as the 95kDa tyrosine kinase-protein. This association induces the acrosome reaction and exposes the proacrosin/acrosin system. Proacrosin transforms itself, by autoactivation, into the proteolytical active form: acrosin. This is a serine protease that has been shown to be involved in secondary binding of spermatozoa to the zona pellucida and in the penetration of mammalian spermatozoa through it. The zona pellucida is a specific and natural substrate for acrosin and its hydrolysis and fertilization can be inhibited by antiacrosin monoclonal antibodies. Moreover, inin vitrofertilization experiments, trypsin inhibitors significantly inhibits fertilization. The use of the silver-enhanced immunogold technique has allowed immunolocalization of the proacrosin/acrosin system in spermatozoa after the occurrence of the acrosome reaction. This system remains associated to the surface of the inner acrosomal membrane for several hours in human, rabbit and guinea-pig spermatozoa while in the hamster it is rapidly lost. In the hamster, the loss of acrosin parallels the capability of the sperm to cross the zona pellucida. Rabbit perivitelline spermatozoa can fertilize freshly ovulated rabbit eggs and retain acrosin in the equatorial and postacrosomal region. These spermatozoa also show digestion halos on gelatin plates that can be inhibited by trypsin inhibitors. This evidence strongly suggests the involvement of acrosin in sperm penetration through the mammalian zona. Recently it was shown, however, that acrosin would not be essential for fertilization. It is likely, then, that such an important phenomenon in the mammalian reproductive cycle would be ensured though several alternative mechanisms.     

Achievements in the study of the development and ultrastructure of spermatozoa (on the 300th anniversary of the discovery of spermatozoa)]

A review of principal advances in studying spermiogenesis and fine structure of spermatozoa is given on the occasion of the 3rd centennary of the discovery of spermatozoa by Loevenhuk. The most important achievements are the discovery of acrosome and its role in fertilization, the discovery of centriole and its role in the formation of flagellum, the discovery of axial complex and the role of its components in the flagellum movement. A study of spermatogenesis has revealed the syncytial nature of spermatogenic epithelium, synaptinemal complexes and their role in chromosome conjugation. The role of nuclear membrane in chromosome conjugation was established, the ultrastructural and biochemical transformation of chromatin was investigated, as well as that of microtubules in spermatogenesis. Some other discoveries were made which are of great importance for diagnosis of sterility.     

External calcium ions are requisite for fertilization of sea urchin eggs by spermatozoa with reacted acrosomes

That a small amount of external calcium ions is requisite for the fertilization by spermatozoa with reacted acrosomes was found by some simple experiments using jelly-treated sperm of the sea urchin, Hemicentrotus pulcherrimus. When eggs were inseminated with the jelly-treated sperm in artificial seawaters containing calcium at various concentrations, the percentage of fertilization decreased concomitant with the reduction in the amount of external calcium ions, 50% at 40 μM calcium and almost 0% at less than 10 μM. On the other hand, it was observed that both the morphology of the reacted acrosome and the binding capacity of the jelly-treated spermatozoa to eggs were not influenced by the calcium deficiency. These results suggest that external calcium ions are indispensable even for the fertilization processes following sperm binding to eggs after the acrosome reaction, such as penetration of reacted spermatozoa through vitelline layer and/or membrane fusion between egg and spermatozoon.     

Nutlin-3a Decreases Male Fertility via UQCRC2

Ubiquinol-cytochrome-c reductase core protein 2 (UQCRC2) is a component of ubiquinol-cytochrome c reductase complex that is known to correlate with male fertility via spermatogenesis. Simultaneously, nutlin-3a is a small molecule antagonist of mouse double minute 2 repressor (MDM2), activate p53 and induce apoptosis responsible for spermatogenesis. To date, however there are no known effects of nutlin-3a on reproduction. Therefore, present study was designed to investigate the effect of nutlin-3a on male fertility via UQCRC2. In this in vitro trial with mice spermatozoa, we utilized CASA, CTC staining, ATP assay, western blotting, and IVF to measure the main study outcome. The short-term exposure of spermatozoa in nutlin-3a decreases sperm motion kinematics, intracellular ATP production, capacitation, the acrosome reaction, UQCRC2, and tyrosine phosphorylation (TYP) of sperm proteins in a dose-dependent manner. Notably, the decreased UQCRC2 and TYP were associated with reduced sperm kinematics, ATP production, and capacitation, which ultimately led to adverse effects on male fertility such as poor fertilization rates and embryo development. Thus, nutlin-3a may be considered as a potential male contraceptive agent due to its ability to decrease fertility secondary to changes in overall sperm physiology and embryonic development. However, the results of this preliminary study have to be confirmed by additional independent trial.     

Vesicular traffic and golgi apparatus dynamics during mammalian spermatogenesis: implications for acrosome architecture

Vesicular membrane trafficking during acrosome biogenesis in bull and rhesus monkey spermatogenesis differs from the somatic cell paradigm as imaged dynamically using the Golgi apparatus probes beta-COP, giantin, Golgin-97, and Golgin-95/GM130. In particular, sorting and delivery of proteins seemed less precise during spermatogenesis. In early stages of spermiogenesis, many Golgi resident proteins and specific acrosomal markers were present in the acrosome. Trafficking in both round and elongating spermatids was similar to what has been described for somatic cells, as judged by the kinetics of Golgi protein incorporation into endoplasmic reticulum-like structures after brefeldin A treatment. These Golgi components were retrieved from the acrosome at later stages of differentiation and were completely devoid of immature spermatozoa. Our data suggest that active anterograde and retrograde vesicular transport trafficking pathways, involving both beta-COP- and clathrin-coated vesicles, are involved in retrieving Golgi proteins missorted to the acrosome and in controlling the growth and shape of this organelle.     

Spermatogenesis of the spider crab Maja brachydactyla (Decapoda: Brachyura)

This study describes spermatogenesis in a majid crab (Maja brachydactyla) using electron microscopy and reports the origin of the different organelles present in the spermatozoa. Spermatogenesis in M. brachydactyla follows the general pattern observed in other brachyuran species but with several peculiarities. Annulate lamellae have been reported in brachyuran spermatogenesis during the diplotene stage of first spermatocytes, the early and mid‐spermatids. Unlike previous observations, a Golgi complex has been found in mid‐spermatids and is involved in the development of the acrosome. The Golgi complex produces two types of vesicles: light vesicles and electron‐dense vesicles. The light vesicles merge into the cytoplasm, giving rise to the proacrosomal vesicle. The electron‐dense vesicles are implicated in the formation of an electron‐dense granule, which later merges with the proacrosomal vesicle. In the late spermatid, the endoplasmic reticulum and the Golgi complex degenerate and form the structures–organelles complex found in the spermatozoa. At the end of spermatogenesis, the materials in the proacrosomal vesicle aggregate in a two‐step process, forming the characteristic concentric three‐layered structure of the spermatozoon acrosome. The newly formed spermatozoa from testis show the typical brachyuran morphology. J. Morphol., 2010. © 2009 Wiley‐Liss, Inc.     

Analysis of Site-specific Glycosylation of Renal and Hepatic γ-Glutamyl Transpeptidase from Normal Human Tissue

The cell surface glycoprotein γ-glutamyl transpeptidase (GGT) was isolated from healthy human kidney and liver to characterize its glycosylation in normal human tissue in vivo. GGT is expressed by a single cell type in the kidney. The spectrum of N-glycans released from kidney GGT constituted a subset of the N-glycans identified from renal membrane glycoproteins. Recent advances in mass spectrometry enabled us to identify the microheterogeneity and relative abundance of glycans on specific glycopeptides and revealed a broader spectrum of glycans than was observed among glycans enzymatically released from isolated GGT. A total of 36 glycan compositions, with 40 unique structures, were identified by site-specific glycan analysis. Up to 15 different glycans were observed at a single site, with site-specific variation in glycan composition. N-Glycans released from liver membrane glycoproteins included many glycans also identified in the kidney. However, analysis of hepatic GGT glycopeptides revealed 11 glycan compositions, with 12 unique structures, none of which were observed on kidney GGT. No variation in glycosylation was observed among multiple kidney and liver donors. Two glycosylation sites on renal GGT were modified exclusively by neutral glycans. In silico modeling of GGT predicts that these two glycans are located in clefts on the surface of the protein facing the cell membrane, and their synthesis may be subject to steric constraints. This is the first analysis at the level of individual glycopeptides of a human glycoprotein produced by two different tissues in vivo and provides novel insights into tissue-specific and site-specific glycosylation in normal human tissues.