Gamete membrane dynamics during double fertilization in Arabidopsis

In the double fertilization of angiosperms, one sperm cell fertilizes an egg cell to produce a zygote, whereas the other sperm cell fertilizes a central cell to give rise to an endosperm. There is little information on gamete membrane dynamics during double fertilization even though the cell surface structure is critical for male and female gamete interactions. In a recent study, we analyzed gamete membrane behavior during double fertilization by live-cell imaging with Arabidopsis gamete membrane marker lines. We observed that the sperm membrane signals occasionally remained at the boundary of the female gametes after gamete fusion. In addition, sperm membrane signals entering the fertilized female gametes were detected. These findings suggested that plasma membrane fusion between male and female gametes occurred with the sperm internal membrane components entering the female gametes, and this was followed by plasmogamy.     

Female control of male gamete delivery during fertilization in Arabidopsis thaliana

Fertilization in both animals and plants relies on the correct targeting of the male gametes to the female gametes. In flowering plants, the pollen tube carries two male gametes through the maternal reproductive tissues to the embryo sac, which contains two female gametes. The pollen tube then releases its two male gametes into a specialized receptor cell of the embryo sac, the synergid cell. The mechanisms controlling this critical step of gamete delivery are unknown. Here, data based on the new sirène (srn) mutant of Arabidopsis thaliana provide the first evidence for female control over male gamete delivery. Live imaging of fertilization shows that wild-type pollen tubes do not stop their growth and do not deliver their contents in srn embryo sacs.     

Calcium and other ion dynamics during gamete maturation and fertilization

Ion currents and cytosolic free calcium ([Ca(2+)](i)) elevations are crucial events in triggering the complex machinery involved in both gamete maturation and fertilization. Oocyte maturation is triggered by hormone signaling which causes ion currents and [Ca(2+)](i) increase. Extracellular calcium seems to be required for meiosis progression since: (i) calcium depletion in the maturation medium severely affects oocyte developmental competence; (ii) the activity of plasma membrane L-type Ca(2+) currents decreases during maturation; (iii) the exposure to verapamil, a specific Ca(2+) channel blocker, decreases in vitro maturation efficiency. In spermatozoa, maturation initiates inside the epididymis and ends in the female genital tract. During their journey through the female reproductive tract, sperm undergo a dramatic selection and capacitation achieving fertilization competence. Adhesion to the tubal epithelium extends sperm life through depression of [Ca(2+)](i) until capacitation signals trigger an [Ca(2+)](i) elevation followed by sperm release. At fertilization, egg-sperm interaction evokes well-described transient and almost simultaneous events: i.e., fertilization current, a change in resting potential, and an increase in free [Ca(2+)](i) concentration. These events, termed oocyte activation, are the direct consequence of sperm interaction via either activation of a receptor or entry of a sperm factor. The latter hypothesis has been recently supported by the discovery of PCLzeta, a sperm-specific isozyme triggering a dramatic [Ca(2+)](i) increase via inositol 1,4,5-trisphosphate (IP(3)) production. The course of ion currents and [Ca(2+)](i) transients during maturation and fertilization plays a pivotal role in correct embryo development.     

Live-cell imaging reveals the dynamics of two sperm cells during double fertilization in Arabidopsis thaliana

Flowering plants have evolved a unique reproductive process called double fertilization, whereby two dimorphic female gametes are fertilized by two immotile sperm cells conveyed by the pollen tube. The two sperm cells are arranged in tandem with a leading pollen tube nucleus to form the male germ unit and are placed under the same genetic controls. Genes controlling double fertilization have been identified, but whether each sperm cell is able to fertilize either female gamete is still unclear. The dynamics of individual sperm cells after their release in the female tissue remain largely unknown. In this study, we photolabeled individual isomorphic sperm cells before their release and analyzed their fate during double fertilization in Arabidopsis thaliana. We found that sperm delivery was composed of three steps. Sperm cells were projected together to the boundary between the two female gametes. After a long period of immobility, each sperm cell fused with either female gamete in no particular order, and no preference was observed for either female gamete. Our results suggest that the two sperm cells at the front and back of the male germ unit are functionally equivalent and suggest unexpected cell-cell communications required for sperm cells to coordinate double fertilization of the two female gametes.     

Cell-cell communication during double fertilization

Double fertilization in flowering seed plants requires intercellular signaling events between many interacting partners. The four cell types of the seven-celled female gametophyte communicate with each other to establish and maintain their identity. They secrete signaling molecules to guide the male gametophyte and to mediate sperm cell discharge and transport towards the two female gametes (the egg and central cell). After fusion of the gametes, guidance signals have to be removed to prevent polyspermy, embryo and endosperm development is induced generating daughter cells or nuclear regions of a different fate, and cell death is induced in the surrounding ovular cells. Until recently, little was known about the molecular nature of the signaling molecules that are involved in these processes. Now, small secreted proteins and peptides have been identified as prime candidates mediating several of these communication events.     

Cadherins expression during gamete maturation and fertilization in the rat

A role for adhesion molecules in gamete fusion, preceding fertilization, has been previously suggested. We investigated the presence of cadherins, Ca(2+) dependent cell-cell adhesion molecules, in rat oocytes and spermatozoa using an anti-pan-cadherin antibody and specific antibodies against the 3 classical cadherins: E- (epithelial), P- (placental), and N- (neural) cadherins. Electrophoretic separation was performed on samples of lysed oocytes of different stages: germinal vesicle oocytes, metaphase II eggs, newly fertilized and 2-cell embryos, as well as spermatozoa from testes, caput and cauda epididymis and ejaculate. Localization of cadherins was determined on intact, gametes by immunocytochemistry, using confocal microscopy. Immunoblotting with the pan-cadherin antibody revealed a major band of approximately 120 kD in all oocyte and sperm extracts. Oocytes presented E-cadherin at appropriate molecular weight but N-cadherin only as a specific 40 kD band. In sperm lysate, at all stages, both E- and N-cadherin were demonstrated as major protein bands but a series of lower molecular weight proteins (that may represent protein degradation) were also detected. Immunohistochemical evaluation showed that E- and N-cadherins are already present on the plasma membrane of immature unfertilized oocytes, although their concentration increases after fertilization in early cleavage stage embryos. Cadherin localization on spermatozoa changed during maturation from a dispersed pattern over the entire head plasma membrane of testicular spermatozoa to a restricted equatorial and post-acrosomal plasma membrane staining in ejaculated spermatozoa. These findings suggest a specific cadherin organization at the fusogenic domains of both gametes.     

Contribution of mouse egg zona pellucida glycoproteins to gamete recognition during fertilization

For sperm to fertilize eggs, they must first bind to the thick zona pellucida (ZP) that surrounds the plasma membrane of all unfertilized mammalian eggs. An extensive literature suggests that mouse sperm recognize and bind to a specific ZP glycoprotein called mZP3. However, the role of individual ZP glycoproteins in binding of mouse sperm to eggs has been called into question by recent transgenic experiments with null mice. Results of such experiments have been interpreted to mean that binding of sperm depends on the supramolecular structure of the ZP, not on an individual ZP glycoprotein. Here, it is argued that results of these transgenic experiments actually are consistent with the prevailing view of gamete recognition that implicates a specific ZP glycoprotein in both binding of mouse sperm to eggs and induction of the acrosome reaction.     

The egg cell is preferentially fertilized in Arabidopsis double fertilization

Each cyclin-dependent kinase a;1 mutant pollen grain contains a single sperm-like cell that can fertilize egg cells, similar to sperm cells. Pollination assays with mutant pollen demonstrated that the egg cell is preferentially fertilized in Arabidopsis.     

Role of the sperm proteasome during fertilization and gamete interaction in the mouse

In this work, we have investigated the role of the sperm proteasome during in vitro fertilization (IVF) and gamete interaction in the mouse. Proteasome activity was measured in extract and intact sperm using a specific substrate. In addition, sperm were treated with specific proteasome inhibitors and evaluated during IVF, binding to the zona pellucida, and progesterone- and zona pellucida-induced acrosome reactions. In other experiments, sperm membrane proteins were obtained resuspending them in Triton X-114, shaking vigorously and let standing by 4 hr. Soluble sperm proteins were partitioned in the aqueous phase and sperm membrane proteins in the detergent phase. In both phases, proteasome activity was measured. Labeling of cell surface sperm proteins was carried out with the cell-impermeable NHS-LC biotin, extracted with Triton X-114, and mixing with avidin-agarose beads. Nonpermeabilized sperm were incubated with an anti-proteasome monoclonal antibody and evaluated by indirect immunofluorescence. The results indicate that sperm extracts as well as intact sperm had proteasome activity; the sperm proteasome was involved in IVF, specifically during sperm-zona pellucida binding and the acrosome reaction; soluble sperm membrane proteins exhibited proteasome activity; biotin experiments indicated the presence of proteasomes on the sperm surface, which was corroborated by indirect immunofluorescence experiments. All these observations indicate that the mouse sperm proteasome participates in the binding to the zona pellucida and the acrosome reaction and that there is a pool of proteasomes located on the sperm head.     

Cell-cell membrane fusion during mammalian fertilization

The mechanism of sperm-egg fusion in mammals is a research area that has greatly benefited from the use of gene deletion technology. Because fertilization is internal in mammals and the gametes (particularly the eggs) are sparse in number, in vitro studies have considerable limitations. Using gene deletions, a few cell surface proteins in both gametes have been identified as essential for gamete fusion. Ongoing studies are directed at analysis of the function of these proteins and the search for additional proteins that may be involved in this process. So far, no mammalian proteins have been found that also function in sperm-egg fusion of non-mammalian species or in other types of cell-cell fusion.     

Calcium dynamics during fertilization in C. elegans

Background

Of the animals typically used to study fertilization-induced calcium dynamics, none is as accessible to genetics and molecular biology as the model organism Caenorhabditis elegans. Motivated by the experimental possibilities inherent in using such a well-established model organism, we have characterized fertilization-induced calcium dynamics in C. elegans.

Results

Owing to the transparency of the nematode, we have been able to study the calcium signal in C. elegans fertilization in vivo by monitoring the fluorescence of calcium indicator dyes that we introduce into the cytosol of oocytes. In C. elegans, fertilization induces a single calcium transient that is initiated soon after oocyte entry into the spermatheca, the compartment that contains sperm. Therefore, it is likely that the calcium transient is initiated by contact with sperm. This calcium elevation spreads throughout the oocyte, and decays monotonically after which the cytosolic calcium concentration returns to that preceding fertilization. Only this single calcium transient is observed.

Conclusion

Development of a technique to study fertilization induced calcium transients opens several experimental possibilities, e.g., identification of the signaling events intervening sperm binding and calcium elevation, identifying the possible roles of the calcium elevation such as the completion of meiosis, the formation of the eggshell, and the establishing of the embryo's axis of symmetry.     

Structural features of the abalone egg extracellular matrix and its role in gamete interaction during fertilization

Abalone eggs are surrounded by a complex extracellular coat that contains three distinct elements: the jelly layer, the vitelline envelope, and the egg surface coat. In this study we used light and electron microscopy to describe these three elements in the red abalone (Haliotis rufescens) and ascribe function to each based on their interactions with sperm. The jelly coat is a spongy matrix that lies at the outermost margin of the egg and consists of variably sized fibers. Sperm pass through this layer with their acrosomes intact and then go on to bind to the vitelline envelope. The vitelline envelope is a multilamellar fibrous layer that appears to trigger the acrosome reaction after sperm binding. Next, sperm release lysin from their acrosomal granules, a nonenzymatic protein that dissolves a hole in the vitelline envelope through which the sperm swims. Sperm then contact the egg surface coat, a network of uniformly sized filaments lying directly above the egg plasma membrane. This layer mediates attachment of sperm, via their acrosomal process, to the egg surface. © 1995 Wiley-Liss, Inc.     

In vitro double fertilization in Nicotiana tabacum (L.): fusion behavior and gamete interaction traced by video-enhanced microscopy

In vitro double fertilization in tobacco was carried out with attention to fusion behavior and gamete interaction. Structural and cytological events indicating possible reaction to the fusion of sperm-egg and especially sperm-central cell were recorded by video-enhanced microscopy. Generative cells were fused with the egg cell or central cell as a control system to better understand gamete interaction. As early as adherence of the male cell, the female cell showed response by means of cytoplasm strand formation. After gamete fusion, cytoplasm activation in the egg cell was observed as long distance movement of organelles. In fertilized central cells, however, fusion did not result in notable cytological change within 30 min. Male nuclear movement recorded in the female cell illustrated two different patterns of movement which showed similarity to organelle movement. The dynamics of male and female nuclear fusion after in vitro fertilization was also recorded in the central cell. It revealed that the fusion process requires only a few seconds and is similar to that of gamete fusion in vitro. This may offer a new clue for understanding how female and male nuclei attract, adhere and finally fuse each other. Received: 13 October 1999 / Revision accepted: 6 December 1999     

Coupling actin dynamics and membrane dynamics during endocytosis.

A convergence of cellular, genetic and biochemical studies supports the hypothesis that the actin cytoskeleton is coupled to endocytic processes, but the roles played by actin filaments during endocytosis are not yet clear. Recent studies have identified several proteins that may functionally link the endocytic machinery with actin filament dynamics. Three of these proteins, Abp1p, Pan1p and cortactin, are activators of actin assembly nucleated by the Arp2/3 complex, a key regulator of actin assembly in vivo. Two others, intersectin and syndapin, bind N-WASp, a potent activator of actin assembly via the Arp2/3 complex. All of these proteins also bind components of the endocytic machinery, and thus, could coordinately regulate actin assembly and trafficking events. Hip1R, an F-actin-binding protein that associates with clathrin-coated vesicles, may physically link endocytic vesicles to actin filaments. The GTPase dynamin is implicated in modulating actin filaments at specialized actin-rich structures of the cell cortex, suggesting that dynamin may regulate the organization of cortical actin filaments as well as regulate actin dynamics during endocytosis. Finally, myosin VI may generate actin-dependent forces for membrane invagination or vesicle movement during the early stages of endocytosis.     

Thylakoid membrane remodeling during state transitions in Arabidopsis

Adaptability of oxygenic photosynthetic organisms to fluctuations in light spectral composition and intensity is conferred by state transitions, short-term regulatory processes that enable the photosynthetic apparatus to rapidly adjust to variations in light quality. In green algae and higher plants, these processes are accompanied by reversible structural rearrangements in the thylakoid membranes. We studied these structural changes in the thylakoid membranes of Arabidopsis thaliana chloroplasts using atomic force microscopy, scanning and transmission electron microscopy, and confocal imaging. Based on our results and on the recently determined three-dimensional structure of higher-plant thylakoids trapped in one of the two major light-adapted states, we propose a model for the transitions in membrane architecture. The model suggests that reorganization of the membranes involves fission and fusion events that occur at the interface between the appressed (granal) and nonappressed (stroma lamellar) domains of the thylakoid membranes. Vertical and lateral displacements of the grana layers presumably follow these localized events, eventually leading to macroscopic rearrangements of the entire membrane network.     

Sperm-oocyte membrane fusion in the human during monospermic fertilization

Sperm-oocyte membrane fusion has been observed during monospermic fertilization of a human oocyte in vitro. Women were stimulated with both clomiphene citrate and human menopausal gonadotropin and were given human chorionic gonadotropin before a LH-surge. Twelve oocytes, collected at laparoscopy from six women who became pregnant by IVF, were allowed to mature for 7–14 hours in vitro and inseminated with preincubated sperm, fixed between 1–3 hours after insemination, and examined by transmission electron microscopy. Membrane fusion had occurred in one ovum 2 hours after insemination, and the oocyte had resumed maturation and was at anaphase II of meiosis. Cortical granules had been exocytosed, and some of their contents were visible at the surface close to the oolemma all around the oocyte. The sperm that fused with this oocyte was acrosome-reacted and had been partly incorporated into the ooplasm, while the anterior two-thirds of its head was phagocytosed by a tongue of cortical ooplasm. Membrane fusion had occurred between the oolemma and the plasma membrane overlying the postacrosomal segment of the sperm head, posterior to the equatorial vestige. Sperm chromatin had not decondensed, and serial sections revealed a midpiece attached to the basal plate and a tail located deeper in the ooplasm, all devoid of plasma membrane. Supplementary sperm penetrating the inner zona, approaching the perivitelline space, had undergone the acrosome reaction but had a persistent vestige of the equatorial segment of the acrosome with intact plasma membrane. Evidence of sperm chromatin decondensation was seen in other oocytes, 3 hours after insemination, which were at telophase II of meiosis. Eight oocytes penetrated by sperm were monospermic, while four were unfertilized. The general pattern of sperm fusion and incorporation appears to conform to that seen in most other mammals. The study also reveals that sperm have to complete the acrosome reaction before fusing with the egg.     

Protein dynamics in sperm membranes: implications for sperm function during gamete interaction.

A number of mammalian sperm plasma membrane antigens have been implicated as playing a functional role in sperm-egg interaction, by virtue of the fact that antibodies against these antigens interfere with fertilization. Two such mouse sperm plasma membrane antigens are M42, a 200/220 kD glycoprotein doublet, and M5, a 150-160 kD glycoprotein. We show that both of these antigens are concentrated on the posterior region of caudal epididymal and capacitated mouse sperm heads and are relatively diffusible, as determined by fluorescence recovery after photobleaching measurements (D = 3-8 x 10(-9) cm2/s with approximately 23% diffusing). Crosslinking of these antigens with bivalent antibodies causes them to redistribute into the anterior region (acrosomal crescent) of the sperm head. In contrast, we describe a third antigen, P220, which is also localized to the posterior region of the sperm head on caudal epididymal sperm but which exhibits very little diffusion and does not redistribute upon crosslinking. Bivalent anti-M42 blocks the ZP3-induced acrosome reaction. We have found that monovalent Fab fragments of anti-M42 do not block the ZP3-induced acrosome reaction, but that inhibition is restored by addition of a second antibody which crosslinks the Fabs. Thus, crosslinking is required for both inhibition of the acrosome reaction and redistribution. This suggests that redistribution of antigen away from the posterior region of the head may be part of the mechanism of inhibition of the ZP3-induced acrosome reaction.     

Effects of ultrashort gamete co-incubation time on porcine in vitro fertilization

A reduction in co-incubation time has been suggested as an alternative method to reduce polyspermic fertilization. The aim of this study was to evaluate the effect of short periods of gamete co-incubation during pig in vitro fertilization. A total of 2833 in vitro matured oocytes were inseminated with thawed spermatozoa and coincubated for 0.25, 1, 2, 3, 7, 10 min and 6 h. The oocytes from the 0.25–10 min groups were washed three times in modified Tris-buffered medium (mTBM) medium to remove spermatozoa not bound to the zona and transferred to the same medium (containing no spermatozoa) until 6 h of co-incubation time were completed. After 6 h, presumptive zygotes from each group were cultured in NCSU-23 medium for 12–15 h to assess fertilization parameters. After each period of co-incubation, 45–50 oocytes from each group were stained with Hoechst-33342 and the number of spermatozoa bound to the zona was counted. Although the number of zona bound spermatozoa increased (p < 0.05) with the co-incubation time, no increase was observed in penetration rates among groups from 2 min to 6 h of co-incubation time (ranging from 53.5 ± 2.8 to 61.3 ± 2.6%). Similarly, the efficiency of fertilization reached a maximum for the 2 min of co-incubation group with values ranging between 32.3 ± 2.4 and 41.9 ± 2.5%. The reduction of co-incubation time did not affect the monospermy rate (range: 71.3 ± 3.4–80.2 ± 3.8%) and the mean number of spermatozoa/oocyte (range: 1.2 ± 0.4–1.4 ± 0.5). These results show that, under our in vitro conditions, high penetration rate can be obtained with co-incubation times as short as 2 min, although monospermy could not be improved using this strategy.