Study on in vitro sperm capacitation and egg-penetration of giant panda

A preliminary report on in vitro sperm capacitation and egg-penetration of giant panda is briefly presented. The panda spermatozoon consists of head, neck and tail, just like the spermatozoa of other animals. Before capacitation sperm heads clustered together and dispersed after capacitation. They were then able to swim straight forward. During the time of in vitro capacitation the plasma membrane of the sperm head was first expanded to various degrees, then disintegrated, and finally became detached. The electro-dense material in the acrosome appeared in small clumps with high density. Extensive vesiculation occurred between the bi-layered acrosome membranes and thus led to disintegration. Vesiculation in panda sperm differs from that reported in hamsters. When the capacitated panda spermatozoa came into contact with the hamster eggs, the region between the acrosome collar and postacrosome cap first fused with the egg membrane followed by the penetration of the nucleus into the cortex of the egg. Some of the penetrating sperm nuclei became decondensed and some did not. The success of in vitro sperm capacitation and egg-penetration of giant panda is of great significance, suggesting that it is possible to carry out in vitro fertilization and embryo transplantation in this endangered species.     

Studies on relationship between Na,K-ATPase activity and sperm capacitation in guinea pig

In order to elucidate the biochemical mechanism of sperm capacitation, the relationship between plasmalemma Na,K-ATPase, capacitation and acrosome reaction was investigated. Plasmalemma of guinea pig spermatozoa was isolated by sucrose gradient centrifugation for the determination of Na,K-ATPase activity. As far as the authors are aware, the Na,K-ATPase activity in plasmalemma of the guinea pig has never been detected. By treating sperm plasmalemma with 0.05% DOC (deoxycholate), enzyme activity could be quantitatively measured. After spermatozoa were incubated in capacitation medium for 8 h, Na,K-ATPase activity was found to be decreased to 35.6% as compared with that before incubation. The spermatozoa incubated for 10.5 h in capacitation medium containing 1 and 5 mumol/L ouabain showed 46.5% and 64.4% acrosome reactions respectively, while the acrosome reaction of the control group was only 27.4%. The above results suggest that the decrease in the Na,K-ATPase activity of guinea pig spermatozoa may be a prerequisite for capacitation. Experimental results demonstrated for the first time that Na,K-ATPase activity exists in the sperm plasmalemma of the guinea pig. It was further found that the decrease of Na,K-ATPase activity of plasmalemma enhances sperm capacitation. It is suggested that sperm capacitation in the guinea pig is possibly induced by the decrease in plasmalemma Na,K-ATPase and, as a consequence, the intracellular Na+ is increased, which would benefit the exchange of Na+out/Ca++in and the onset of acrosome reaction.     

Biological effects of helium-neon (HeNe) laser irradiation on acrosome reaction in bull sperm cells

The purpose of this study was to determine the effect of He---Ne laser irradiation (632.8 nm, 10 mW) on the induction of acrosome reaction and mortality in bull sperm cells in comparison with two important capacitation agents; calcium and heparin. Frozen-thawed bull sperm cells were washed in percoll gradient and suspended at a concentration of 1 × 10⁶ ml⁻¹ in sp-TALP medium, capacitated in the presence of 2 mM CaCl₂, 10 μg ml⁻¹ heparin, or irradiated at fluences from 2 to 16 J cm⁻², and incubated for 0, 30, 60 and 90 minutes. At the end of the incubation period, the percentage of sperm that were acrosome-reacted and dead was determined. The results obtained indicated that laser irradiation at all fluences produced a significant increase (p < 0.001) in the percentage of sperm cells that were acrosome reacted, and a significant decrease (p < 0.001) in the percentage of dead sperm at 90 minutes of incubation in comparison to other capacitation agents and the control group. The percentage of sperm cells with acrosome reaction was increased with increasing fluences of laser irradiation and time of incubation. It is conclude that the application of He---Ne laser irradiation at fluences from 2 to 16 J cm⁻² induced the acrosome reaction and decreased the sperm mortality percentage in vitro of bull sperm cells.     

Comparative Spermatology of Protura and Its Significance on Proturan Systematics

A comparative study on the ultrastructure of the spermatozoa of 20 species of Protura belonging to 16 genera and 8 families was conducted. The most remarkable characteristics are the unexpected diversity of the spermatozoa, both in shape and fine structure. In general they can be grouped into two main categories: long flagellate spermatozoa and short aflagellate ones. Accordingly two sperm evolutionary lines of the spermatozoa were obtained: one trends towards the complicated acrosome structure and the increased number of axonemal doublets, and the other line is to shorten cell form and simplify sperm structure. The results indicate that the spermatozoa of Hesperentomon may be the primitive type, and the spermatozoa of Eosentomids probably represent the most evolved type. A sperm evolutionary tree was drawn according to the apomorphies of proturan spermatozoa. The phylogenetic position of Protura was also discussed.     

Loading magnetic nanoparticles into sperm cells does not affect their functionality

The spontaneous loading of magnetite nanoparticles into sperm cell was carried out by mixing an aqueous colloidal solution of Fe3O4-PVA with sperm cells (10(8) cells/ml) for 2 h at 37 degrees C suspended in glucose-free modified Tyrode solution. The penetration of the magnetite nanoparticles into the sperm cells was monitored by conventional analytical chemistry. We have demonstrated that the motility and the ability to undergo the acrosome reaction (i.e., the ability to fertilize the egg) were not affected by the presence of the magnetite nanoparticles.     

Kinetic Study of 17α-Estradiol Mechanism during Rat Sperm Capacitation

17α-Estradiol (αE2) is a natural diastereoisomer of 17β-estradiol (E2). It is well known that αE2 can bind to estrogen receptors. However, its biological activity is less than that of E2 and is species and tissue specific. The goal of our study was to propose the mechanism of αE2 hormonal response in rat sperm during their capacitation in vitro and compare it with a previously studied mouse model. Concentration changes in externally added αE2 during capacitation of rat sperm were monitored by the high-performance liquid chromatographic method with tandem mass spectrometric detection (HPLC-MS/MS). The calculated values of relative concentrations B_t were subjected to kinetic analysis. The findings indicated that αE2 in rat sperm did not trigger autocatalytic reaction, in contrast to the mouse sperm, and that the initiation of the hormone penetration through the sperm plasma membrane was substantially faster in rats.     

Effect of light on calcium transport in bull sperm cells.

The effect of light on calcium transport was studied. Bull sperm cells were irradiated with an He-Ne (630 mm) laser and a 780 nm diode laser at various energy doses, and 45Ca2+ uptake was measured by the filtration technique. It was found that there is an accelerated Ca2+ transport in the irradiated cells, which means that laser light can stimulate Ca2+ exchange through the cell membrane. This may cause transient changes in the cytoplasmic Ca2+ concentration which, in spermatozoa, has a regulatory role in control of motility and acrosome reaction, and in other cells can trigger mitosis.     

The surface events of fertilization: the movements of the spermatozoon through the sea urchin egg surface and the roles of the surface layers.

The sea urchin egg surface at fertilization has been examined with the scanning electron microscope to reveal the movements of the spermatozoon from the exterior, through the surface layers, and into the egg cytoplasm. The layers that the spermatozoon encounter have been studied to determine their physical and chemical natures and their role in early development. By studying the outside of whole eggs and the inner face of surfaces isolated shortly after fertilization, it has been possible to compile data on the movements of the spermatozoon through the egg surface. The spermatozoon initially contacts the egg with the elongated acrosomal process. The vitelline sheet, the outermost layer of the egg, separates slightly next to the attached spermatozoon. As membrane fusion between the gametes occurs, the plasma membrane from the egg engulfs the spermhead, the cortical granules start to discharge their contents, and a spreading surface deformation, concommitant with a distortion of the fibrous cortex, is initiated. A cluster of elongate microville surround the perpendicularly fusing spermatozoon. These microvilli interidigitate as the spermatozoon is forced to lie upon the egg surface between the plasma membrane and the matrix of cortical fibers. The spermatozoon then rotates additionally to enter the egg cytoplasm with the posterior end first; it has rotated 180 degrees through the cell surface. Finally, it detaches into the egg cytoplasm, leaving a scar in the cortex through which it penetrated. The egg cortex, previously unobserved by electron microscopy, is revealed to be composed of 50-200 nm fibers. At fertilization they are uniformly organized but during later development this order is lost. The cortex is from 0.2-0.5 micronm thick and is a contractile structure. The role of the outer surface in releasing the cell from the metabolic constraints of the unfertilized egg is shown, and the apparent differences in the mobilities of the membranes derived from the sperm and from the egg are demonstrated. The relation of these layers to the movements of the spermatozoon, to the activation of the egg, to the block to polyspermy, and to each other are discussed.     

The RING-finger protein haprin: domains and function in the acrosome reaction

The RBCC (RING finger, B-box type zinc finger, coiled-coil domain) motif family contains a large number of proteins implicated in many cellular processes, including vesicle exocytosis. The acrosome reaction, the sperm exocytotic event that is required for fertilization, involves essentially the same process of intracellular membrane fusions as vesicular exocytosis in somatic cells. We have previously isolated a haploid-germ-cell-specific gene designated haprin, which encodes a RBCC motif protein that plays a role in the acrosome reaction of sperm by mediating protein complex formation via the RBCC motif. In this review, we describe the potential role of Haprin in the molecular mechanisms of acrosome reaction, as compared with some other RBCC proteins. The conserved structure and localization of the Haprin protein in human and mouse suggest an indispensable role for Haprin in the functioning of mammalian sperm.     

Dimyristoylated peptides incorporated into liposomes are polyvalent fertilin beta mimics

[structure: see text]. Fertilin beta is an integral membrane sperm protein that is involved in sperm binding to the egg plasma membrane. We synthesized a dimyristoylated fertilin beta peptide and incorporated it into POPC liposomes at 1 mol %. The concentration of fertilin beta peptide required for 50% inhibition is reduced 100-fold to 5.2 +/- 1.6 microM relative to a monomeric control. Moreover, in contrast to the inhibition observed with monomeric peptides, we obtain complete inhibition with the peptidic liposomes.     

Effects of tributyltin(IV) chloride on fertilization of Styela plicata (Ascidiacea: Tunicata): II. Scanning and transmission electron microscopy studies

The morphological aspects of Styela plicata fertilization after treatment with tributyltin(IV) chloride are described by means of scanning and transmission electron microscopy investigations. Alterations have been shown both on female and male gametes; spermatozoa, all the egg envelopes and the mitochondria of the egg cortical cytoplasm are modified in relation to incubation time. As a consequence, the damage to gametes blocks sperm–egg interaction and fertilization does not occur. Copyright © 2003 John Wiley & Sons, Ltd.     

Activation of Sperm EGFR by Light Irradiation is Mediated by Reactive Oxygen Species

To acquire fertilization competence, spermatozoa must undergo several biochemical and motility changes in the female reproductive tract, collectively called capacitation. Actin polymerization and the development of hyperactivated motility (HAM) are part of the capacitation process. In a recent study, we showed that irradiation of human sperm with visible light stimulates HAM through a mechanism involving reactive‐oxygen‐species (ROS), Ca²⁺ influx, protein kinases A (PKA), and sarcoma protein kinase (Src). Here, we showed that this effect of light on HAM is mediated by ROS‐dependent activation of the epidermal growth factor receptor (EGFR). Interestingly, ROS‐mediated HAM even when the EGFR was activated by EGF, the physiological ligand of EGFR. Light irradiation stimulated ROS‐dependent actin polymerization, and this effect was abrogated by PBP10, a peptide which activates the actin‐severing protein, gelsolin, and causes actin‐depolymerization in human sperm. Light‐stimulated tyrosine phosphorylation of Src‐dependent gelsolin, resulting in enhanced HAM. Thus, light irradiation stimulates HAM through a mechanism involving Src‐mediated actin polymerization. Light‐stimulated HAM and in vitro‐fertilization (IVF) rate in mouse sperm, and these effects were mediated by ROS and EGFR. In conclusion, we show here that irradiation of sperm with visible light, enhances their fertilization capacity via a mechanism requiring ROS, EGFR and HAM.     

Model systems for membrane fusion

Membrane fusion has an overarching influence on living organisms. The fusion of sperm and egg membranes initiates the life of a sexually reproducing organism. Intracellular membrane fusion facilitates molecular trafficking within every cell of the organism during its entire lifetime, and virus-cell membrane fusion may signal the end of the organism's life. Considering its importance, surprisingly little is known about the molecular-level mechanism of membrane fusion. Due to the complexity of a living cell, observations often leave room for ambiguity in interpretation. Therefore artificial model systems composed of only a few components are being used to further our understanding of controlled fusion processes. In this critical review we first give an overview of the hypothesized mechanism of membrane fusion and the techniques that are used to investigate it, and then present a selection of non-targeted and targeted model systems, finishing with current applications and predictions on future developments (85 references).     

Allurin and egg jelly coat impact on in-vitro fertilization success of endangered Albanian water frog,Pelophylax shqipericus

Abstract

Amphibian egg-jelly coat plays an important role in successful fertilization and development. Here, we ask whether proteins like allurin in the jelly coats of frog eggs might influence fertilization rate success. Using in vitro fertilization of Albanian water frog, Pelophylax shqipericus, we found that body cavity eggs or eggs deprived of jelly coat were not fertilized, compromising the success of in vitro fertilization procedure. When de-jellied eggs were inseminated with sperm suspension, the fertilization efficiency is dramatically decreased even inhibited, suggesting that the gel structure is one of the major factors in the achievement of fertilization in the frogs. Fertilization of de-jellied eggs with sperm pre-treated with egg jelly coat, restored the fertilization competency. Such a result suggests that egg jelly coat probably guides the sperm to the egg surface while maintaining the fertilization ability, contributing to a successful in vitro fertilization of Pelophylax shqipericus.     

Molecular identification of Ca2+ channels in human sperm

The acrosome reaction is a Ca(2+)-dependent exocytotic process that is a prerequisite step for fertilization. External calcium entry through voltage-activated Ca(2+) channels is known to be essential in inducing the acrosome reaction of mammalian spermatozoa. Due to their complex geometry, however, electrophysiological identification of sperm Ca(2+) channels has been limited. Here we identified Ca(2+) channel mRNAs expressed in motile human sperm using RT-PCR and their levels were compared using RNase protection assays. L-type, non- L-type, and T-type Ca(2+) channel mRNAs were detected by RT-PCR using degenerate primers. Cloning and sequencing of the PCR products revealed alpha1B, alpha1C, alpha1E, alpha1G, and alpha1H sequences. RT-PCR using specific primers repeatedly detected alpha1B, alpha1C, alpha1E, alpha1G, and alpha1H mRNAs, and additionally alpha1I mRNA. But alpha1A and alpha1D messages were not detected. Relative expression levels of the detected Ca(2+) channel subtypes were compared by RNase protection assays. The abundance of detected mRNA messages was in the following order: alpha1H alpha1G alpha1E alpha1B alpha1C alpha1I. These findings indicated that human motile sperm express multiple voltage-activated Ca(2+) channel RNAs among which T-type and non-L-type channel messages are likely to be predominantly expressed. Based on their relative expression levels, we propose that not only T-type but also non-L-type calcium channels may be major gates for the external calcium influx, required for the acrosome reaction.     

Distinct and independent dielectrophoretic behavior of the head and tail of sperm and its potential for the safe sorting and isolation of rare spermatozoa

Often, in semen samples with minute amounts of sperm, even the single spermatozoon required to fertilize an oocyte cannot be found in the ejaculate. This is primarily because currently, sperm is generally searched for manually under a microscope. In this study, dielectrophoresis (DEP) was investigated as an alternative automated technique for sorting sperm cells. Using a quadrupolar electrode array it was shown that the head and tail of the sperm had independent and unique crossover frequencies corresponding to the transition of the DEP force from repulsive (negative) to attractive (positive). These surprising results were further analyzed, showing that the head and tail have their own distinct electrical properties. This significant result allows for the sperm's head, which contains the DNA, to be distanced from potentially damaging high electric fields using negative DEP while simultaneously manipulating and sorting the sperm using the positive DEP response of the tail. A proof of concept sorting chip was designed and tested. The low crossover frequency of the tail also allows for the use of a higher conductivity, and thus more physiological, medium than the conventional DEP solutions. Although more research is required to design and optimize an efficient, user‐friendly, and high‐throughput device, this research is a proof of concept that DEP has the potential to automate and improve the processing of semen samples, especially those containing only rare spermatozoa.     

Electron spin resonance studies of phosphatidylcholine interacted with cholesterol and with a hopanoid in liposomal membrane.

The effects of bacteriohopane-32-ol (Monol) on liposomal membrane composed of dipalmitoylphosphatidylcholine (DPPC) or egg yolk phosphatidylcholine (egg PC) were compared with those of cholesterol (Chol) in the change of fluidity using a spin label. The fluidity change close to the polar head groups caused by temperature increase in the DPPC membrane containing Monol was different from that of Chol. Chol had a condensing effect on DPPC membrane, whereas Monol had only a slight effect except when used at 20 mol%. Near the hydrophobic end, Chol incorporation into DPPC led to fluidization below transition temperature (Tm) and condensation above Tm. Monol incorporation into DPPC had only a fluidizing effect below Tm. On the other hand, in egg PC membrane Chol had the condensing effect at any temperature, whereas Monol had only slight effect. These results suggest that Monol may have a role in supporting constant membrane fluidity under drastic conditions.     

The Effects of Salt and pH on the Spermatozoa Agglutinating Activity of Carp Ovum Cystatin by Molecular Dynamics Simulations

In order to elucidate how the spermatozoa agglutinating activity of carp ovum cystatin is significantly affected by salt and pH in the atomic scale, an homology model of carp ovum cystatin was constructed based on the crystal structure of chicken egg white cystatin in this study. Although these two proteins exhibit only 36.11% of sequence identity, they share similar folds. Our homology model also shows that 16 positively charged residues are equally distributed on the surface of carp ovum cystatin, leading to agglutinating spermatozoa via electrostatic interaction. The structure of carp ovum cystatin is not significantly affected by salt and pH during 400 ps molecular dynamics simulations. However, the total accessible surface area (ASA) of the positively charged residues on the surface of carp ovum cystatin decreased with increasing salt concentration, leading to the abolishment of its spermatozoa agglutinating activity. Moreover, 13 Lys residues became deprotonated at pH 11, resulting in the decrease of the net positive charges on the surface of carp ovum cystatin and thus losing its spermatozoa agglutinating activity.     

80 kDa mouse sperm protein as a substrate of protein kinase C.

Calcium and phospholipid-dependent protein kinase (PKC) activity was detected mainly in the cytosol of the mouse sperm. The PKC in the cytosol fraction was partially purified by ion-exchange chromatography. Using the partially purified PKC, the phosphorylation of PKC substrates was examined in vitro. The phosphorylation of the 80 kDa protein was enhanced by phorbol ester treatment in vitro as well as in vivo. The partial amino acid sequence of this protein was homologous with that of guanosine 5'-cyclic monophosphate (cGMP)-dependent protein kinase and myosin light chain kinase, both of which are related to ligand-receptor-transduction. The present data suggest that the activation of PKC and subsequent specific protein phosphorylation might be involved in the regulation of the zona pellucida-induced acrosome reaction.