Sperm Entry Point Research Articles

The Importance of the Position of the Nucleus in Drosophila Oocyte Development.

Oogenesis is a developmental process leading to the formation of an oocyte, a haploid gamete, which upon fertilisation and sperm entry allows the male and the female pronuclei to fuse and give rise to a zygote. In addition to forming a haploid gamete, oogenesis builds up a store of proteins, mRNAs, and organelles in the oocyte needed for the development of the future embryo. In several species, such as Drosophila, the polarity axes determinants of the future embryo must be asymmetrically distributed prior to fertilisation. In the Drosophila oocyte, the correct positioning of the nucleus is essential for establishing the dorsoventral polarity axis of the future embryo and allowing the meiotic spindles to be positioned in close vicinity to the unique sperm entry point into the oocyte.

Analytic solutions for calcium ion fertilisation waves on the surface of eggs.

The evolution of calcium fertilisation waves on the cortex of amphibian eggs can be described by a nonlinear reaction-diffusion process on the surface of a sphere. Here, we use the nonclassical symmetry technique to find an exact analytic solution that describes the evolution of the calcium concentration. The solutions presented compare well with published experimental results. The analytic solution can be used to give insight into the processes governing the fertilisation wave, such as the flow of calcium ions from the sperm entry point. By finding a spiral solution to an approximate equation linearised near saturation, we also demonstrate how solutions with other properties may be constructed using this technique.

Retrospective analysis: reproducibility of interblastomere differences of mRNA expression in 2-cell stage mouse embryos is remarkably poor due to combinatorial mechanisms of blastomere diversification.

Retrospective analysis: reproducibility of interblastomere differences of mRNA expression in 2-cell stage mouse embryos is remarkably poor due to combinatorial mechanisms of blastomere diversification.

Evidence of Oocyte Polarity in Bovine; Implications for Intracytoplasmic Sperm Injection and Somatic Cell Nuclear Transfer.

We recently demonstrated spatial regionalization of maternal transcripts and proteins within unfertilized ovine oocyte. Here, we investigated the likelihood of oocyte polarity for the first time in bovine. In this experimental study, in vitro matured bovine oocytes were used for manual bisection [into oocyte halve that were near-to (HNS) and far-from (FS) spindle] or trisection [into MII-spindle (S), the spindle-side half (NS), and the distal half unassociated with the spindle (FS)]. Prepared pools of oocyte substructures were used for comparative quantitative real-time polymerase chain reaction (RT-qPCR). To map the possible preferential sperm entry point (SEP), the spatial relationship between SEP and MII-spindle was measured 5 hours post-fertilization. The proportional amount of maternal mRNA in S oocyte fragment was estimated to be 6 to 11-fold higher than NS and FS counterparts. The relative abundances of Nanog, Oct4, Fgf4 and Tead4 were significantly higher in HNS oocyte fragment compared t0 FS. The relative abundances of Ctnb, Carm1, Rex1, Sox2 and Cdx2 were comparable between HNS and NS oocyte fragments. FS oocyte fragment possessed significantly higher transcripts of Gata4 compared to HNS. The distribution of certain transcripts related to pluripotency and lineage commitment were different depending upon the region of the oocyte; either enriched at S (Tead4, Nanog, Ctnb and Sox2), NS (Oct4), or FS (Gata6). The SEP in almost (90%) fertilized oocytes was located in MII-hemisphere. The observation of spatial restriction of mRNAs and SEP within MII-oocyte may indicate that the principal forces of oocyte polarity are evolutionary conserved. This may in turn highlight the need for refinements in the methodology of intracytoplasmic sperm injection (where a sperm is injected far from the MII-spindle) and somatic cell nuclear transfer (where a major amount of regulative mRNAs that are associated with MIIspindle is removed during enucleation).

Totipotency segregates between the sister blastomeres of two-cell stage mouse embryos

Following fertilization in mammals, it is generally accepted that totipotent cells are exclusive to the zygote and to each of the two blastomeres originating from the first mitotic division. This model of totipotency was inferred from a minority of cases in which blastomeres produced monozygotic twins in mice. Was this due to experimental limitation or biological constraint? Here we removed experimental obstacles and achieved reliable quantification of the prevalence of dual totipotency among mouse two-cell stage blastomeres. We separated the blastomeres of 1,252 two-cell embryos, preserving 1,210 of the pairs. Two classes of monozygotic twins became apparent at the blastocyst stage: 27% formed a functional epiblast in both members (concordant), and 73% did so in only one member of the pair (discordant) – a partition that proved insensitive to oocyte quality, sperm-entry point, culture environment and pattern of cleavage. In intact two-cell embryos, the ability of sister blastomeres to generate epiblast was also skewed. Class discovery clustering of the individual blastomeres’ and blastocysts’ transcriptomes points to an innate origin of concordance and discordance rather than developmental acquisition. Our data place constraints on the commonly accepted idea that totipotency is allocated equally between the two-cell stage blastomeres in mice.

The Principal Forces of Oocyte Polarity Are Evolutionary Conserved but May Not Affect the Contribution of the First Two Blastomeres to the Blastocyst Development in Mammals.

Oocyte polarity and embryonic patterning are well-established features of development in lower species. Whether a similar form of pre-patterning exists in mammals is currently under hot debate in mice. This study investigated this issue for the first time in ovine as a large mammal model. Microsurgical trisection of unfertilized MII-oocytes revealed that cortical cytoplasm around spindle (S) contained significant amounts of total maternal mRNAs and proteins compared to matched cytoplast hemispheres that were located either near (NS) or far (FS) -to-spindle. RT-qPCR provided striking examples of maternal mRNA localized to subcellular substructures S (NPM2, GMNN, H19, PCAF, DNMT3A, DNMT1, and STELLA), NS (SOX2, NANOG, POU5F1, and TET1), and FS (GCN) of MII oocyte. Immunoblotting revealed that specific maternal proteins DNMT3A and NANOG were asymmetrically enriched in MII-spindle-half of the oocytes. Topological analysis of sperm entry point (SEP) revealed that sperm preferentially entered via the MII-spindle-half of the oocytes. Even though, the topological position of first cleavage plane with regard to SEP was quite stochastic. Spatial comparison of lipid content revealed symmetrical distribution of lipids between 2-cell blastomeres. Lineage tracing using Dil, a fluorescent dye, revealed that while the progeny of leading blastomere of 2-cell embryos contributed to more cells in the developed blastocysts compared to lagging counterpart, the contributions of leading and lagging blastomeres to the embryonic-abembryonic parts of the developed blastocysts were almost unbiased. And finally, separated sister blastomeres of 2-cell embryos had an overall similar probability to arrest at any stage before the blastocyst (2-cell, 4-cell, 8-cell, and morula) or to achieve the blastocyst stage. It was concluded that the localization of maternal mRNAs and proteins at the spindle are evolutionarily conserved between mammals unfertilized ovine oocyte could be considered polar with respect to the spatial regionalization of maternal transcripts and proteins. Even though, the principal forces of this definitive oocyte polarity may not persist during embryonic cleavages.

A Critical Pull to Polarize the Cell

A Critical Pull to Polarize the Cell

Analysis of the dynamic process of sperm penetration of the human oocyte using time-lapse cinematography

OBJECTIVE: Using time-lapse cinematography (TLC), we analyzed the dynamic process of human sperm penetration to investigate the mechanisms blocking polyspermy after sperm attachment. DESIGN: Retrospective visual analysis. MATERIALS AND METHODS: Between July 2004 and April 2008, TLC was performed on single, randomly selected oocytes after insemination, as previously described (Mio, 2008). The study focused on oocytes where the movements of the leading sperm (the one that penetrated the zona pellucida (ZP) most deeply) and the following sperm were captured in the same field. Sperm movements were then analyzed from the time of entry into the ZP to the incorporation of the leading sperm. RESULTS: We observed and recorded the process of sperm penetration and the sperm entry point in 22 embryos. In 3 of these embryos (target embryos), we were able to capture the movements of the leading and following sperm. Sperm attached to the oocyte membrane approx 96 minutes after insemination and 37 minutes later, the sperm head decondensed and disappeared. There was no difference in the time-course of the fertilization process of the target embryos and the other embryos observed. In the 3 target embryos, both leading and following sperm proceeded forward until the leading sperm attached on the ooplasmic membrane. The following sperm penetrated 12.9 μm (68.9%) of the ZP on average and, despite their active tail movements, stopped their progression within 10 seconds of the leading sperm attaching on the membrane. CONCLUSIONS: To date, the primary block to polyspermy in the mammalian oocyte is described as a zona reaction induced approximately 5 to 8 minutes after oocyte activation by sperm incorporation. In this study, 10 seconds after sperm-oocyte attachment, the following sperm were unable to continue further despite their sustained tail motility. This suggests that an unknown process has prevented the progression of the following sperm. Further investigations using TLC are underway to elucidate this mechanism of polyspermy prevention.

Assisted fertilization and embryonic axis formation in higher primates

In naturally fertilized embryos of various organisms, the spermatozoon provides a localized cue to initiate early embryonic patterning. In mice, the sperm entry point (SEP) may reorient the first cleavage division, which separates the zygote into two halves that follow distinct fates. However, it is unknown whether the mechanical injection of spermatozoa into an oocyte by intracytoplasmic sperm injection (ICSI), a technique commonly used in human assisted reproduction, possesses such a role. Rhesus macaque embryos fertilized by ICSI were examined in order to determine the consequences of placing the spermatozoon at specific positions in the ooplasm and whether this can provide new information about patterning in mammalian eggs. The SEP specified by the injected spermatozoa was most often localized near the first cleavage plane and was mainly distributed along the boundary zone that separates the embryonic and abembryonic parts of the monkey blastocyst. Moreover, the ICSI data, when compared with naturally fertilized mouse embryos, showed a similar outcome in terms of cleavage axes and first embryonic axis specification. As there are no studies to date regarding sperm entry in human oocytes and its influence on embryonic development, this investigation using the rhesus macaque as a clinical model is noteworthy.

Symmetry Breaking in C. elegans: Another Gift from the Sperm

Polarization of the C. elegans embryo depends on the sperm-contributed centrosome, which cues a retraction of the actomyosin cortex to the opposite end of the embryo by an unknown mechanism. New evidence reveals that the sperm donates a second polarizing cue that may locally relax the actomyosin cortex near the point of sperm entry.

Does prepatterning occur in the mouse egg?

A recurring question in developmental biology has been whether localized determinants play any role in mammalian preimplantation development. This is a controversial issue that brings back the idea of prepatterning and is explored further by Plusa et al., who claim it is the first cleavage of the mouse zygote that predicts the blastocyst axis, rather than the animal pole or sperm entry point, as previously suggested. However, other evidence indicates that the blasotcyst axis is not predetermined and there is no prepatterning in the mouse egg. Here we investigate the origin of these different views and conclude that they arise from differences in the data themselves and in their interpretation.

Fertilization triggers localized activation of Src-family protein kinases in the zebrafish egg

Fertilization triggers activation of Src-family kinases in eggs of various species including marine invertebrates and lower vertebrates. While immunofluorescence studies have localized Src-family kinases to the plasma membrane or cortical cytoplasm, no information is available regarding the extent to which these kinases are activated in different regions of the zygote. The objective of the present study was to detect the subcellular distribution of activated Src-family kinases in the fertilized zebrafish egg. An antibody specific for the active, non-phosphorylated form of Src-family PTKs was used to detect these activated kinases by immunofluorescence. The results demonstrate that Fyn, and possibly other Src family members are activated by dephosphorylation of the C-terminal tyrosine at fertilization. The activated Src-family kinases are asymmetrically distributed around the egg cortex with an area of higher kinase activity localized adjacent to the micropyle near the presumptive animal pole. Fertilization initially caused elevation of kinase activity in the cytoplasm underlying the micropyle, but this quickly spread to involve the entire zygote cortex. Later, during egg activation, formation of the blastodisc involved concentration of active Src-family kinase in the blastodisc cortex. As cytokinesis began, activated Src-family kinases were no longer limited to the cortex, but became more evenly distributed in the clear apical cytoplasm of the blastomeres. The results demonstrate that the cortex of the zebrafish egg is functionally differentiated and that fertilization triggers localized activation of Src-family kinases at the point of sperm entry, which subsequently progresses through the entire egg cortex.

CD9 controls the formation of clusters that contain tetraspanins and the integrin α6β1, which are involved in human and mouse gamete fusion

The process of gamete fusion has been largely studied in the mouse and has revealed the crucial role of the tetraspanin CD9. By contrast, human gamete fusion remains largely unknown. We now show that an anti-alpha 6 integrin mAb (GoH3) strongly inhibited human sperm-egg fusion in human zona-free eggs. Furthermore, a mAb directed against CD151, a tetraspanin known to associate with alpha 6 beta1, partially inhibited sperm-egg fusion. By contrast, the addition of an anti-CD9 mAb to zona free eggs had no effect. The integrin alpha 6 beta1, CD151 and CD9 tetraspanins were evenly distributed on human zona-intact oocytes. On zona-free eggs, the integrin alpha 6 beta1 and tetraspanin CD151 patched and co-localized but the tetraspanin CD9 remained unchanged. CD9 mAb prevented alpha 6 beta1 integrin clustering and gamete fusion when added prior to, but not after, zona removal. Antibody-mediated aggregation of integrin alpha 6 beta1 yielded patches that were bigger and more heterogeneous in mouse oocytes lacking CD9. Moreover, a strong labelling of alpha 6 beta1 could be observed at the sperm entry point. Altogether, these data show that CD9 controls the redistribution of some membrane proteins including the alpha 6 beta1 integrin into clusters that may be necessary for gamete fusion.

Specific location of sperm mitochondria in mussel Mytilus galloprovincialis zygotes stained by MitoTracker

In Mytilidae, mitochondrial DNA (mtDNA) in the offspring is inherited from male and female parents. Sperm mitochondria are only incorporated into the testes. This phenomenon is called doubly uniparental inheritance (DUI). Sperm mitochondria should locate in the primordial germ cell during development to maintain DUI. However, the mechanism of sperm mitochondria localization is still unknown. To reveal the mechanism, we followed the location of sperm mitochondria in Mytilus galloprovincialis zygotes fertilized with sperm stained by MitoTracker. Just after fertilization, sperm mitochondria, which were found to enter eggs from various sites, remained at sperm entry point. Five sperm mitochondria located at the male pronucleus. After pronuclear expansion, sperm mitochondria migrated to the center of the egg together with the male pronucleus. At anaphase of cleavage-I, the distribution pattern of sperm mitochondria was divided into two patterns. In pattern A, sperm mitochondria located in the equatorial region of the eggs. In pattern B, sperm mitochondria migrated and divided into two groups with chromosomes. From observations of colchicine-treated eggs, we suggest that sperm mitochondria migration from fertilization to anaphase of cleavage-I depends on the microtubules. The difference between pattern A and pattern B may be caused by whether sperm mitochondria migrated or not by the microtubules at cleavage-I.

Mitotic Spindles and Cleavage Planes Are Oriented Randomly in the Two-Cell Mouse Embryo

Most experimental embryological studies performed on the early mouse embryo have led to the conclusion that there are no mosaically distributed developmental determinants in the zygote and early embryo (for example see [1-6]). It has been suggested recently that "the cleavage pattern of the early mouse embryo is not random and that the three-dimensional body plan is pre-patterned in the egg" (in [7] for review see [8-10]). Two major spatial cues influencing the pattern of cleavage divisions have been proposed: the site of the second meiotic division [11, 12] and the sperm entry point [13-14], although the latter is controversial [15-17]. An implication of this hypothesis is that the orientations of the first few cleavage divisions are stereotyped. Such a define cleavage pattern, leading to the segregation of developmental determinants, is observed in many species [18]. Recently, it was shown that the first cleavage plane is not predetermined but defined by the topology of the two apposing pronuclei [19]. Because the position of the female pronucleus is dependent upon the site of polar body extrusion and the position of the male pronuclei is dependent upon the sperm entry point [19-20], this observation leaves open the possibility that the sperm may provide some kind of directionality [7]. But, even if asymmetries were set up only after fertilization, a stereotyped cleavage pattern should take place during the following cleavage divisions. Thus, we studied the cleavage pattern of two-cell embryos by videomicroscopy to distinguish between the two hypotheses. After the mitotic spindle formed, its orientation did not change until cleavage. During late metaphase and anaphase, the spindle poles appear to be anchored to the cortex through astral microtubules and PARD6a. Only at the time of cleavage, during late anaphase, do the forming daughter cells change their relative positions. These studies show that cleavage planes are oriented randomly in two-cell embryos. This argues against a prepatterning of the mouse embryo before compaction.

Xenopus Egg Wnt/β-Catenin Pathway

In embryonic development in vertebrates, β-catenin signaling promotes polarization of the embryo to establish the dorsoventral axis, and it is this process that is highlighted by the Xenopus Egg Wnt/β-Catenin Pathway. In the amphibian Xenopus , fertilization of the egg results in the establishment of a parallel array of microtubules with the plus end pointing away from the sperm entry point. Concurrent with a process of cortical rotation, in which the cortex of the egg utilizes these microtubules to rotate relative to the inner cytoplasm, there is a movement of small vesicles toward the plus end of the microtubules. Cells inheriting these vesicles are destined to give rise to dorsal and anterior structures of the embryo. Three components of the Wnt/β-catenin pathway—Dishevelled, glycogen synthase kinase 3 (GSK-3)-binding protein (GBP), and β-catenin—accumulate on the side of the egg and early embryo that receive these small vesicles. It is likely that Dishevelled and GBP are associated with the vesicles that move along the microtubules and that they promote the stabilization and dorsal accumulation of β-catenin. Although Wnts and Frizzled are present in the egg, their roles in axis specification remain unclear. Dorsal β-catenin then activates direct target genes, including the homeobox genes siamois and twinned . Activation of these genes on the prospective dorsal side is necessary and sufficient for formation of the gastrula organizer, which organizes the embryonic germ layers and axes.

Cortical Flows Powered by Asymmetrical Contraction Transport PAR Proteins to Establish and Maintain Anterior-Posterior Polarity in the Early C. elegans Embryo

The C. elegans PAR proteins PAR-3, PAR-6, and PKC-3 are asymmetrically localized and have essential roles in cell polarity. We show that the one-cell C. elegans embryo contains a dynamic and contractile actomyosin network that appears to be destabilized near the point of sperm entry. This asymmetry initiates a flow of cortical nonmuscle myosin (NMY-2) and F-actin toward the opposite, future anterior, pole. PAR-3, PAR-6, and PKC-3, as well as non-PAR proteins that associate with the cytoskeleton, appear to be transported to the anterior by this cortical flow. In turn, PAR-3, PAR-6, and PKC-3 modulate cortical actomyosin dynamics and promote cortical flow. PAR-2, which localizes to the posterior cortex, inhibits NMY-2 from accumulating at the posterior cortex during flow, thus maintaining asymmetry by preventing inappropriate, posterior-directed flows. Similar actomyosin flows accompany the establishment of PAR asymmetries that form after the one-cell stage, suggesting that actomyosin-mediated cortical flows have a general role in PAR asymmetry.

A wave of IP 3 production accompanies the fertilization Ca 2+ wave in the egg of the frog, Xenopus laevis: theoretical and experimental support

The fertilization Ca 2+ wave in Xenopus laevis is a single, large wave of elevated free Ca 2+ that is initiated at the point of sperm–egg fusion and traverses the entire width of the egg. This Ca 2+ wave involves an increase in inositol-1,4,5-trisphosphate (IP 3) resulting from the interaction of the sperm and egg, which then results in the activation of the endoplasmic reticulum Ca 2+ release machinery. The extraordinarily large size of this cell (1.2 mm diameter) together with the small surface region of sperm-receptor activation makes special demands on the IP 3-dependent Ca 2+ mobilizing machinery. We propose a detailed model of the fertilization Ca 2+ wave in Xenopus eggs that requires an accompanying wave of IP 3 production. While the Ca 2+ wave is initiated by a localized increase of IP 3 near the site of sperm–egg fusion, the Ca 2+ wave propagates via IP 3 production correlated with the Ca 2+ wave—possibly via Ca 2+-mediated PLC activation. Such a Ca 2+-mediated IP 3 production wave has not been required previously to explain the fertilization Ca 2+ wave in eggs; we argue this is necessary to explain the observed IP 3 dynamics in Xenopus eggs. To test our hypothesis, we have measured the IP 3 levels from 20 nl “sips” of the egg cortex during wave propagation. We were unable to detect the low IP 3 levels in unfertilized eggs, but after fertilization, [IP 3] ranged from 175 to 430 nM at the sperm entry point and from 120 to 700 nM 90° away once the Ca 2+ wave passed that region about 2 min after fertilization. Prior to the Ca 2+ wave reaching that region the IP 3 levels were undetectable. Since significant IP 3 could not diffuse to this region from the sperm entry point within 2 min, this observation is consistent with a regenerative wave of IP 3 production.

The Xenopus Egg Wnt/β-catenin Pathway

In embryonic development in vertebrates, β-catenin signaling promotes polarization of the embryo to establish the dorsoventral axis and it is this process that is highlighted by the Xenopus Egg Wnt/β-catenin Pathway. In the amphibian Xenopus , fertilization of the egg results in the establishment of a parallel array of microtubules with the plus end pointing away from the sperm entry point. Concurrent with a process of cortical rotation, in which the cortex of the egg utilizes these microtubules to rotate relative to the inner cytoplasm, there is a movement of small vesicles toward the plus end of the microtubules. Cells inheriting these vesicles are destined to give rise to dorsal and anterior structures of the embryo. Two components of the Wnt/β-catenin pathway, Dishevelled and β-catenin, accumulate on the side of the egg and early embryo that receive these small vesicles. It is likely that Dishevelled is associated with the vesicles that move along the microtubules, and that it promotes the stabilization and dorsal accumulation of β-catenin. Although Wnts and Frizzled are present in the egg, their roles in axis specification remain unclear. Dorsal β-catenin then activates direct target genes, including the homeobox genes siamois and twinned . Activation of these genes on the prospective dorsal side is necessary and sufficient for formation of the gastrula organizer, which organizes the embryonic germ layers and axes. Science Viewpoint R. T. Moon, B. Bowerman, M. Boutros, and N. Perrimon, The promise and perils of Wnt signaling through β-catenin. Science 296 , 1644-1646 (2002). [ Abstract ] [ Full Text ]

The Xenopus Egg Wnt/ -catenin Pathway

In embryonic development in vertebrates, β-catenin signaling promotes polarization of the embryo to establish the dorsoventral axis and it is this process that is highlighted by the Xenopus Egg Wnt/β-catenin Pathway. In the amphibian Xenopus, fertilization of the egg results in the establishment of a parallel array of microtubules with the plus end pointing away from the sperm entry point. Concurrent with a process of cortical rotation, in which the cortex of the egg utilizes these microtubules to rotate relative to the inner cytoplasm, there is a movement of small vesicles toward the plus end of the microtubules. Cells inheriting these vesicles are destined to give rise to dorsal and anterior structures of the embryo. Two components of the Wnt/β-catenin pathway, Dishevelled and β-catenin, accumulate on the side of the egg and early embryo that receive these small vesicles. It is likely that Dishevelled is associated with the vesicles that move along the microtubules, and that it promotes the stabilization and dorsal accumulation of β-catenin. Although Wnts and Frizzled are present in the egg, their roles in axis specification remain unclear. Dorsal β-catenin then activates direct target genes, including the homeobox genes siamois and twinned. Activation of these genes on the prospective dorsal side is necessary and sufficient for formation of the gastrula organizer, which organizes the embryonic germ layers and axes.