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.     

Analysis of gamete membrane dynamics during double fertilization of Arabidopsis

Angiosperms have a unique sexual reproduction system called “double fertilization.” One sperm cell fertilizes the egg and another sperm cell fertilizes the central cell. To date, plant gamete membrane dynamics during fertilization has been poorly understood. To analyze this unrevealed gamete subcellular behavior, live cell imaging analyses of Arabidopsis double fertilization were performed. We produced female gamete membrane marker lines in which fluorescent proteins conjugated with PIP2a finely visualized egg cell and central cell surfaces. Using those lines together with a sperm cell membrane marker line expressing GCS1-GFP, the double fertilization process was observed. As a result, after gamete fusion, putative sperm plasma membrane GFP signals were occasionally detected on the egg cell surface adjacent to the central cell. In addition, time-lapse imaging revealed that GCS1-GFP signals entered both the egg cell and the central cell in parallel with the sperm cell movement toward the female gametes during double fertilization. These findings suggested that the gamete fusion process based on membrane dynamics was composed of (1) plasma membrane fusion on male and female gamete surfaces, (2) entry of sperm internal membrane components into the female gametes, and (3) plasmogamy.     

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.     

Calcium ion currents mediating oocyte maturation events

During maturation, the last phase of oogenesis, the oocyte undergoes several changes which prepare it to be ovulated and fertilized. Immature oocytes are arrested in the first meiotic process prophase, that is morphologically identified by a germinal vesicle. The removal of the first meiotic block marks the initiation of maturation. Although a large number of molecules are involved in complex sequences of events, there is evidence that a calcium increase plays a pivotal role in meiosis re-initiation. It is well established that, during this process, calcium is released from the intracellular stores, whereas less is known on the role of external calcium entering the cell through the plasma membrane ion channels. This review is focused on the functional role of calcium currents during oocyte maturation in all the species, from invertebrates to mammals. The emerging role of specific L-type calcium channels will be discussed.     

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 function and distribution during fertilization in angiosperms

Calcium has an essential signaling, physiological, and regulatory role during sexual reproduction in flowering plants; elevation of calcium amounts is an accurate predictor of plant fertility. Calcium is present in three forms: (1) covalently bound calcium, (2) loosely bound calcium typically associated with fixed and mobile anions (ionic bonding); and (3) cytosolic free calcium-an important secondary messenger in cell signaling. Pollen often requires calcium for germination. Pollen tube elongation typically relies on external calcium stores in the pistil. Calcium establishes polarity of the pollen tube and forms a basis for pulsatory growth. Applying calcium on the tip may alter the axis; thus calcium may have a role in determining the directionality of tube elongation. In the ovary and ovule, an abundance of calcium signals receptivity, provides essential mineral nutrition, and guides the pollen tube in some plants. Calcium patterns in the embryo sac also correspond to synergid receptivity, reflecting programmed cell death in one synergid cell that triggers degeneration and prepares this cell to receive the pollen tube. Male gametes are released in the synergid, and fusion of the gametes requires calcium, according to in vitro fertilization studies. Fusion of plant gametes in vitro triggers calcium oscillations evident in both the zygote and primary endosperm during double fertilization that are similar to those in animals.     

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.     

Calcium in sea urchin egg during fertilization

Calcium plays a strikingly important role in two of the major events in developmental biology: cell activation and differentiation. In this review we begin with the location and quantity of intracellular calcium in sea urchin oocytes, and then discuss the changes that occur during fertilization and egg activation, placing special emphasis on the mobilization and redistribution of intracellular calcium. We also discuss the propagation of the calcium wave and the role of the burst of calcium on the process of reorganizing the egg cortex at fertilization.     

Calcium signaling differentiation during Xenopus oocyte maturation

Ca(2+) is the universal signal for egg activation at fertilization in all sexually reproducing species. The Ca(2+) signal at fertilization is necessary for egg activation and exhibits specialized spatial and temporal dynamics. Eggs acquire the ability to produce the fertilization-specific Ca(2+) signal during oocyte maturation. However, the mechanisms regulating Ca(2+) signaling differentiation during oocyte maturation remain largely unknown. At fertilization, Xenopus eggs produce a cytoplasmic Ca(2+) (Ca(2+)(cyt)) rise that lasts for several minutes, and is required for egg activation. Here, we show that during oocyte maturation Ca(2+) transport effectors are tightly modulated. The plasma membrane Ca(2+) ATPase (PMCA) is completely internalized during maturation, and is therefore unable to extrude Ca(2+) out of the cell. Furthermore, IP(3)-dependent Ca(2+) release is required for the sustained Ca(2+)(cyt) rise in eggs, showing that Ca(2+) that is pumped into the ER leaks back out through IP(3) receptors. This apparent futile cycle allows eggs to maintain elevated cytoplasmic Ca(2+) despite the limited available Ca(2+) in intracellular stores. Therefore, Ca(2+) signaling differentiates in a highly orchestrated fashion during Xenopus oocyte maturation endowing the egg with the capacity to produce a sustained Ca(2+)(cyt) transient at fertilization, which defines the egg's competence to activate and initiate embryonic development.     

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.     

MADS-complexes regulate transcriptome dynamics during pollen maturation

Background  

Differentiation processes are responsible for the diversity and functional specialization of the cell types that compose an organism. The outcome of these processes can be studied at molecular, physiologic, and biochemical levels by comparing different cell types, but the complexity and dynamics of the regulatory processes that specify the differentiation are largely unexplored.     

Ultrastructural study of cat zona pellucida during oocyte maturation and fertilization

The mammalian zona pellucida (ZP) is an extracellular glycoprotein structure formed around growing oocytes, ovulated eggs and preimplantation embryos. The specific functions of ZP are highly determined by its morphological structure. Studies on cat oocytes during maturation and after fertilization were undertaken, using routine transmission (TEM) and scanning electron microscopy (SEM). Two basic ZP layers – outer with rough spongy appearance and inner with smaller fenestrations and smooth fibrous network – were visible. Deposits, secreted by oviductal cells formed new layer, the so called oviductal ZP. After fertilization outer ZP showed rougher meshed network due to fusion between filaments as a consequence from sperm penetration while the inner was smoother with melted appearance. The presented data on the SEM and TEM characteristics of cat oocytes, together with our previous studies on carbohydrate distribution suggest that during oocyte maturation and fertilization ZP undergoes structural and functional rearrangements related to sperm binding and penetration.     

Structural reorganizations of the endoplasmic reticulum during egg maturation and fertilization

The endoplasmic reticulum (ER) of eggs is a major internal store of calcium ions that must be properly mobilized at fertilization for development to proceed. In most species, the ER develops distinct clusters in the cortical ooplasm as the oocyte matures into a fertilizable egg. Following fertilization, the structure of the ER rapidly reorganizes in eggs that produce a single fertilization-induced calcium wave, whereas ER clusters persist for relatively long periods in eggs that generate multiple calcium oscillations. This review considers such pre- and post-fertilization reorganizations of the ER and what effects these changes might have on calcium signaling patterns.     

Maternal chromatin remodeling during maturation and after fertilization in mouse oocytes

Immunofluorescence staining with antibodies against acetylated histone H4 and 5-methylcytosine was carried out to investigate female chromatin remodeling throughout oocyte maturation and chromatin rearrangement involving both male and female genomes after fertilization. Oocyte cytoplasm remodels female chromatin in preparation of the fertilizing event and the subsequent chromatin rearrangement. Histone H4 are in fact progressively deacetylated whereas demethylating enzymes do not seem to be active over this period. The acetylase/deacetylase balance seems to be cell cycle dependent as female chromatin is deacetylated during maturation and reacetylated at telophase II stage both after fertilization and activation. On the contrary, DNA demethylation seems to be strictly selective. It is in fact confined to the remodeling of paternal genome after fertilization of mature oocytes as the ooplasm is not effective in demethylating either paternal chromatin in germinal vesicle breakdown (GVBD) fertilized oocytes or maternal genome of partenogenetically activated oocytes. Surprisingly, we induced maternal chromatin demethylation after fertilization by treating oocytes with a combination of a methyltransferase inhibitor, 5-azacytidine (5-AzaC), and a reversible and specific inhibitor of histone deacetylase, trichostatin A (TSA). This treatment likely induces a hyperacetylation of histones (thus favoring the access to demethylating enzymes by opening female chromatin structure) associated with a block of reparative methylation by inhibiting methytransferases. This manipulation of chromatin remodeling may have applications regarding the biological significance of aberrant DNA methylation.     

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.     

Calcium ion influx during mitogenic stimulation of lymphocytes

The uptake of free calcium ion (Ca2+) in PHA- or A23187-stimulated lymphocytes was measured using 45CaCl2 and 3H-water. Augmentation of Ca2+ uptake by both mitogens was observed, but the enhanced uptake occurred transiently, sometime within 30 min of the stimulation. The total amount of calcium in quiescent lymphocytes as determined by atomic absorption spectroscopy was about 2.9 X 10(-15) g/cell. When stimulated with PHA, more calcium gradually accumulated in the cells. The maximum amount of accumulation occurred at around 40 h, and was about 2-fold higher than that of control cells. In A23187-stimulated cells, the calcium content increased within 1 h by about 4-fold, reached a maximum at about 6 h (6-fold) and thereafter, surplus calcium was pumped out. The cytosolic free calcium ion concentration (the [Ca2+]i) within single cells was measured using quin 2 or fura-2. The [Ca2+]i was about 1 X 10(-7) M, and a transient increase in the [Ca2+]i was observed in some cells within 1 min after Con A-stimulation. Another rise in the [Ca2+]i was observed around the 40th h, and the maximum expression of the IL-2 receptor was observed at about this time. Therefore the results may indicate that the IL-2-mediated lymphocyte transformation is dependent on the rise in the [Ca2+]i.     

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.     

Protein synthesis during hormonally induced meiotic maturation and fertilization in starfish oocytes

Uptake of radioactive amino acids and their incorporation into protein were examined during 1-methyladenine-induced maturation and subsequent fertilization of oocytes of the starfish Patiria miniata. The initial response to the hormone was a nearly immediate decrease in permeability to amino acids, indicating that the site of action of the hormone is on the cell surface. Protein synthesis began to increase starting about 12 min after 1-methyladenine stimulation and prior to germinal vesicle breakdown. It continued to rise throughout the first meiotic division. This protein synthesis was not required for assembly or initial functioning of the meiotic apparatus, although it was necessary for the completion of meiosis. Fertilization had no effect on the rate of protein synthesis.Oocytes of P. miniata provide an example of hormonal stimulation of protein synthesis in an invertebrate system.