A Protein Extracted from Mouse Sperm That Plays an Important Role in Fertilization

ＡＰｒｏｔｅｉｎＥｘｔｒａｃｔｅｄｆｒｏｍＭｏｕｓｅＳｐｅｒｍＴｈａｔＰｌａｙｓａｎＩｍｐｏｒｔａｎｔＲｏｌｅｉｎＦｅｒｔｉｌｉｚａｔｉｏｎＺＨＵＡＮＧＤａ－ｚｈｏｎｇ;（庄大中）,ＺＨＡＮＧＴｉａｎ－ｙｉｎ（张天荫）,ＣＨＥＮＤａ－ｙｕａｎ;（陈大...     

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.     

The Ca increase by the sperm factor in physiologically polyspermic newt fertilization: Its signaling mechanism in egg cytoplasm and the species-specificity

The newt, Cynops pyrrhogaster, exhibits physiological polyspermic fertilization, in which several sperm enter an egg before egg activation. An intracellular Ca²⁺ increase occurs as a Ca²⁺ wave at each sperm entry site in the polyspermic egg. Some Ca²⁺ waves are preceded by a transient spike-like Ca²⁺ increase, probably caused by a tryptic protease in the sperm acrosome at the contact of sperm on the egg surface. The following Ca²⁺ wave was induced by a sperm factor derived from sperm cytoplasm after sperm–egg membrane fusion. The Ca²⁺ increase in the isolated, cell-free cytoplasm indicates that the endoplasmic reticulum is the major Ca²⁺ store for the Ca²⁺ wave. We previously demonstrated that citrate synthase in the sperm cytoplasm is a major sperm factor for egg activation in newt fertilization. In the present study, we found that the activation by the sperm factor as well as by fertilizing sperm was prevented by an inhibitor of citrate synthase, palmitoyl CoA, and that an injection of acetyl-CoA or oxaloacetate caused egg activation, indicating that the citrate synthase activity is necessary for egg activation at fertilization. In the frog, Xenopus laevis, which exhibits monospermic fertilization, we were unable to activate the eggs with either the homologous sperm extract or the Cynops sperm extract, indicating that Xenopus sperm lack the sperm factor for egg activation and that their eggs are insensitive to the newt sperm factor. The mechanism of egg activation in the monospermy of frog eggs is quite different from that in the physiological polyspermy of newt eggs.     

Teleost fish spermatozoa contain a cytosolic protein factor that induces calcium release in sea urchin egg homogenates and triggers calcium oscillations when injected into mouse oocytes

Established studies in a variety of organisms including amphibians, fish, ascidians, nemerteans, echinoderms, mammals, and even a species of flowering plant, clearly demonstrate that an increase in intracellular egg calcium is crucial to the process of egg activation at fertilization. In echinoderms, egg activation appears to involve an egg phospholipase C gamma (PLCgamma). However, numerous studies in mammalian species suggest that calcium is released from internal egg stores at fertilization by a sperm-derived cytosolic protein factor. Recent studies in the mouse have identified this sperm-derived factor as being a novel sperm-specific PLC isoform with distinctive properties (PLCzeta). Homologues of PLCzeta have since been isolated from human and cynomolgus monkey sperm. In addition, sperm factor activity has been detected in non-mammalian species such as chicken, Xenopus, and a flowering plant. Here we report evidence for the existence of a similar sperm-derived factor in a commercially important species of teleost fish, the Nile tilapia Oreochromis niloticus (L). Using an established bioassay for calcium release, the sea urchin egg homogenate, we demonstrate that protein extracts obtained from tilapia spermatozoa exhibit PLC activity similar to that seen in mammalian sperm extracts, and also induce calcium release when added directly to the homogenate. Further, tilapia sperm extracts induced calcium oscillations when injected into mouse oocytes.     

小鼠孤雌胚胎干细胞系的建立及生物学特性鉴定

目的:探讨建立合适的小鼠孤雌胚胎干细胞建系方法。方法:采用氯化锶联合细胞松弛素B激活B6D2F1杂交小鼠卵母细胞,所获得的囊胚与桑椹胚分别用于孤雌胚胎干细胞的建系,观察两者的建系成功率。结果:共建立了12株小鼠孤雌胚胎干细胞系,这些细胞SSEA-1抗原阳性,SSEA-4,TRA-1-81,TRA-1-60表面抗原阴性,具有AKP活性,保持正常染色体核型,体内外分化分别形成畸胎瘤和拟胚体。结论:采用囊胚和去透明带的桑葚胚建立孤雌胚胎干细胞系获得成功。该方法为人类纯合子的胚胎干细胞建系提供基础,在自体细胞治疗领域中具有潜在的应用价值。     

Characterization of Bufo arenarum oocyte plasma membrane proteins that interact with sperm

Sperm-oocyte plasma membrane interaction is an essential step in fertilization. In amphibians, the molecules involved have not been identified. Our aim was to detect and characterize oocyte molecules with binding affinity for sperm. We isolated plasma membranes free from vitelline envelope and yolk proteins from surface-biotinylated Bufo arenarum oocytes. Using binding assays we detected a biotinylated 100 kDa plasma membrane protein that consistently bound to sperm. Chromatographic studies confirmed the 100 kDa protein and detected two additional oocyte molecules of 30 and 70 kDa with affinity for sperm. Competition studies with an integrin-interacting peptide and cross-reaction with an anti-HSP70 antibody suggested that the 100 and 70 kDa proteins are members of the integrin family and HSP70, respectively. MS/MS analysis suggested extra candidates for a role in this step of fertilization. In conclusion, we provide evidence for the involvement of several proteins, including integrins and HSP70, in B. arenarum sperm-oocyte plasma membrane interactions.     

Transmembrane adenylyl cyclase regulates amphibian sperm motility through protein kinase A activation

Sperm motility is essential for achieving fertilization. In animals with external fertilization as amphibians, spermatozoa are stored in a quiescent state in the testis. Spermiation to hypotonic fertilization media triggers activation of sperm motility. Bufo arenarum sperm are immotile in artificial seminal plasma (ASP) but acquire in situ flagellar beating upon dilution. In addition to the effect of low osmolarity on sperm motility activation, we report that diffusible factors of the egg jelly coat (EW) regulate motility patterns, switching from in situ to progressive movement. The signal transduction pathway involved in amphibian sperm motility activation is mostly unknown. In the present study, we show a correlation between motility activation triggered by low osmotic pressure and activation of protein kinase A (PKA). Moreover, this is the first study to present strong evidences that point toward a role of a transmembrane adenyl-cyclase (tmAC) in the regulation of amphibian sperm motility through PKA activation.     

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.     

Inhibition of plasma membrane and mitochondrial transmembrane potentials by ethanol

The actions of ethanol and its primary oxidative metabolite, acetaldehyde, on plasma membrane and mitochondrial transmembrane potentials were examined in rat brain using fluorescence techniques. Subchronic treatment of adult rats with ethanol resulted in a significant depolarization of both the plasma and mitochondrial membranes when the mean blood ethanol level of the rats was 59±11 mM (mean±SEM, n=6). Acute dosing of animals (4.5 g/kg, i.p.) failed to show any significant alterations. Various concentrations of ethanol, added in vitro to a crude synaptosomal preparation isolated from the rat cerebrocortex (P₂) from untreated animals, depolarized both the plasma and mitochondrial transmembrane potentials in a dose-related manner. Addition of acetaldehyde in vitro did not reveal any significant effects on plasma or mitochondrial transmembrane potential.     

A general solution for optimal egg size during external fertilization, extended scope for intermediate optimal egg size and the introduction of Don Ottavio 'tango'

Egg sizes of marine invertebrates vary greatly, both within and between species. Among the proposed causes of this are a trade-off between egg size, egg number and survival probability of offspring, and a selection pressure exerted by sperm limitation during external fertilization. Although larger eggs are indeed a larger target for sperm, producing larger eggs also implies making fewer of them. There has been discussion about whether sperm limitation can (theoretically) and does (in nature) select for larger egg size than under ad libitum sperm. In one specific model, based on a particular fertilization kinetics model and an empirically derived mortality function, the theoretical possibility of a negative shift in optimal egg size with sperm concentration was demonstrated. Here we present a generalized analytical model to explore the effects of survival and fertilization probabilities on optimal egg size. It is demonstrated that incorporating fertilization kinetics greatly increases the scope for intermediate optimal egg size, as opposed to eggs of minimal or maximal size. Second, we present a general analytical qualitative solution to the question whether optimal egg size depends on sperm concentration. It is shown that, under the condition that an intermediate optimal egg size exists, this qualitative outcome of the model (positive, negative or no relation between optimal egg size and sperm limitation) depends on the structure of the fertilization kinetics part of the model. Finally, we evaluate fertilization kinetics models with respect to the general solution, using two previously published kinetics models ('Don Giovanni' and 'Don Ottavio') and a novel alteration of one of them in which sperm concentration covaries with egg concentration (Don Ottavio 'tango'). For all three models the relationship between optimal egg size and sperm concentration is shown to be always negative. This paper thus shows how biologically realistic relationships between egg size on the one hand and survival and fertilization probability on the other hand predict optimal egg size to be intermediate, and that this optimum is in general expected to increase when sperm become more limiting.     

Intracellular GABA-Activated In→Out Permeation of Chloride Across the Deiters' Neuron Membrane: Modulation by Phosphorylating Activities

The modulation of intracellular GABA activated ³⁶Cl^– inout permeation across single Deiters' neuron membranes has been studied in a microchamber system. Addition of Mg²⁺/ATP on the membrane cytoplasmic side reduces strongly the GABA effect as does ATP alone. However, the greatest inhibition of the GABA effect is given by the addition of Mg²⁺ to the intracellular side buffer: a complete block of the stimulation by GABA of ³⁶Cl^– inout permeation. This is interpreted as due to the presence in this case of a constant concentration of exogenous Mg²⁺ acting together with endogenous ATP in the small cytoplasmic layer on the membrane inner side. The addition of ADP to Mg²⁺/ATP increases the inhibitory effect of the latter. This is presumably due to an extra increase of ATP, locally under the membrane, due to phosphorylation of ADP by endogenous phosphocreatine. Overall, the data confirm that phosphorylating conditions impair the intracellular GABA action on ³⁶Cl^– inout permeation.     

精子表面凝集素受体细胞化学检测法研究──Ⅲ．标记－劈裂术及其方法学问题

本文应用标记劈裂术检测精子表面ＣｏｎＡ受体并主要从方法学上加以探讨。采用１８ｎｍ和６ｎｍ胶体金作标记物进行比较,表明６ｎｍ金标记效率高,与膜蛋白分子对应性好,并提示６ｎｍ胶体金似可对质膜ＰＦ面跨膜蛋白进行定性。文中对该方法的技术关键作了总结,表明标记－劈裂术是质膜大分子细胞化学研究的可靠的新技术。     

Sperm cell architecture, insemination, and fertilization in the model fern, Ceratopteris richardii

Motile sperm cells of land plants are released directly into the environment and encounter numerous constraints on their way to the egg. Sperm cell organization, shape, size, and plasticity are crucial to the processes associated with fertilization. We conducted an ultrastructural investigation to detail insemination (sperm release, swimming and movement within the archegonium) and fertilization in the model fern Ceratopteris richardii. Gametophytes were grown from spores using sterile culture techniques and flooded in water when sexually mature. Materials were examined at different stages post-flooding. During insemination in C. richardii, the sperm cytoskeleton and flagella rearrange, and the coils of the cell extend while entering the neck canal. In this nearly linear configuration, the dense ridge, a densely compacted band of filaments presumed to be actin, expands to surround the leading edge of the sperm cell. This ridge fuses with the receptive site on the female gamete and is the sperm component that initiates contact with the egg nuclear envelope. All cellular components, except plastids, enter the egg cytoplasm. Sperm mitochondria are distinguishable from those of the egg because they are encased by two or three additional membranes and are sequestered from the zygote cytoplasm. During karyogamy, the sperm components, including the microtubule cytoskeleton (spline) and flagella, maintain their spatial integrity. Microtubules play key roles not only in sperm cell structure but also in facilitating karyogamy in this fern. After karyogamy is completed, microtubule arrays of the sperm cell and the components of the locomotory apparatus are disassembled. We provide the first demonstration of the likely involvement of sperm actin in egg penetration in land plants and new insights into the fate of paternal organelles. This study points to the roles sperm cell structure and dynamics play in the intricate processes of insemination and fertilization in land plants.     

Membrane binding and uptake of diflubenzuron in a cell line from Manduca sexta (L.)

The binding and accumulation of the chitin synthesis inhibitor diflubenzuron (DFB) by a cell line derived from embryonic tissue of the tobacco hornworm, Manduca sexta (L.), was analyzed. A rapid and reversible binding to viable and nonviable cells suspended in the culture medium was observed at soluble concentrations of DFB for short exposure periods. Scatchard analysis gave no indication of a saturable uptake mechanism. The DFB-binding capacity of intact cells was found to be similar to that of a crude membrane preparation (70,000g pellet); however, plasma membrane-enriched fractions bound almost three times as much DFB as the homogenate. Repetitive shorttime incubations (up to 3 h) of suspended cells with DFB resulted in a stepwise intracellular accumulation of DFB. Treatment of growing cells with DFB at high concentrations (50 μM) of DFB for longer periods (up to 7 days) resulted in elevated intracellular accumulation of DFB, which exceeded the binding capacity of the cell membranes and the aqueous solubility of DFB. These results indicate that the intracellular crystals detected by transmission electron microscopy are precipitated DFB. No metabolites or other chemically modified products of intracellular DFB were detected by high pressure liquid chromatography (HPLC) after a 7-day incubation.     

Functional, biochemical, and chromatographic characterization of the complete [Ca2+]i oscillation-inducing activity of porcine sperm

A cytosolic sperm protein(s), referred to as sperm factor (SF), is delivered into eggs by the sperm during mammalian fertilization to induce repetitive increases in the intracellular concentration of free Ca2+ ([Ca2+]i) that are referred to as [Ca2+]i oscillations. [Ca2+]i oscillations are essential for egg activation and early embryonic development. Recent evidence shows that the novel sperm-specific phospholipase C (PLC), PLCzeta, may be the long sought after [Ca2+]i oscillation-inducing SF. Here, we demonstrate the complete extraction of SF from porcine sperm and show that regardless of the method of extraction a single molecule/complex appears to be responsible for the [Ca2+]i oscillation-inducing activity of these extracts. Consistent with this notion, all sperm fractions that induced [Ca2+]i oscillations, including FPLC-purified fractions, exhibited high in vitro PLC activity at basal Ca2+ levels (0.1-5 microM), a hallmark of PLCzeta. Notably, we detected immunoreactive 72-kDa PLCzeta in an inactive fraction, and several fractions capable of inducing oscillations were devoid of 72-kDa PLCzeta. Nonetheless, in the latter fractions, proteolytic fragments, presumably corresponding to cleaved forms of PLCzeta, were detected by immunoblotting. Therefore, our findings corroborate the hypothesis that a sperm-specific PLC is the main component of the [Ca2+]i oscillation-inducing activity of sperm but provide evidence that the presence of 72-kDa PLCzeta does not precisely correspond with the Ca2+ releasing activity of porcine sperm fractions.     

Intraspecific egg size variation and sperm limitation in the broadcast spawning bivalve Macoma balthica

Bottom-ice algae within Antarctic sea ice were examined using chlorophyll fluorescence imaging. The detailed structure of the bottom-ice algal community growing in the platelet and congelation layers of solid pieces of sea ice was evident for the first time in chlorophyll imaging mode. Strands of fluorescence representing algal cells were clearly visible growing upward into brine channels in a fine network. Images of effective quantum yield (Ф_PSII) revealed that the Ф_PSII of algae embedded in the sea ice was approximately 0.5. Furthermore, Ф_PSII decreased slightly with distance from the ice-water interface.The response of Antarctic sea ice algae to changes in irradiance and salinity, and the effects of slowly warming and melting the ice block sample were examined using this system. The Ф_PSII of bottom-ice algae decreased as irradiance increased and salinities decreased. Bottom-ice algae appear to be most vulnerable to changes in their environment during the melting process of the ice, and this suggests that algae from this region of the ice may not be able to cope with the stress of melting during summer.Chlorophyll fluorescence imaging provides unprecedented imagery of chlorophyll distribution in sea ice and allows measurement of the responses of sea ice algae to environmental stresses with minimal disruption to their physical habitat. The results obtained with this method are comparable to those obtained with algae that have been melted into liquid culture and this indicates that previous melting protocols reveal meaningful data. In this chlorophyll imaging study, rapid light curves did not saturate and this may prevent further use of this configuration.     

Delayed sperm injection and fertilization in parthenogenetically activated insect egg (Athalia rosae,Hymenoptera)

Summary Mature eggs dissected from the ovary of unmated females of Athalia rosae ruficornis Jakovlev (Hymenoptera, Tenthredinidae) can be activated to develop (into haploid parthenogenetic males) simply by exposing them to distilled water. These eggs, which are primary oocytes arrested at the first meiotic metaphase, resume meiosis upon activation and reach the first meiotic telophase in 20 min. Mature eggs immediately upon dissection have previously been shown to complete karyogamy and develop as fertilized diploid females if injected with sperm. We show here that the eggs activated in water for 20 min have a much higher rate of successful fertilization if injected with sperm, and that the eggs activated for 40 min, upon sperm injection, though at a reduced frequency still develop as diploid fertilized females. Eggs left in water for 60 min, however, are no longer fertilized upon sperm injection and develop as haploid males.     

Artificial fertilization for amphibian conservation: Current knowledge and future considerations

Amphibian populations in the wild are experiencing massive die-offs that have led to the extinction of an estimated 168 species in the last several decades. To address these declines, zoological institutions are playing an important role in establishing captive assurance colonies to protect species in imminent danger of extinction. Many of the threatened species recently placed into captivity are failing to reproduce before they expire, and maintaining founder populations is becoming a formidable challenge. Assisted reproductive technologies, such as hormone synchronization, gamete storage and artificial fertilization, are valuable tools for addressing reproductive failure of amphibians in captive facilities. Artificial fertilization has been commonly employed for over 60 years in several keystone laboratory species for basic studies in developmental biology and embryology. However, there are few instances of applied studies for the conservation of threatened or endangered amphibian species. In this review, we summarize valuable technological achievements in amphibian artificial fertilization, identify specific processes that need to be considered when developing artificial fertilization techniques for species conservation, and address future concerns that should be priorities for the next decade.