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A zygote is the diploid cell that results from the fusion of two haploid gametes, usually a spermatozoon and an oocyte, in sexual reproduction. It is the first cell of a new organism of an individual that develops by sexual reproduction, and it contains a combination of the genetic material of both parents. The zygote is totipotent, which means that it and the cells of the earliest cleavage stages can give rise to all of the cell types of the embryo and of the extraembryonic tissues, such as the placenta and the yolk sac. The term is also used for the single-celled stage of fungi, algae, and plants after fusion of gametes, and in organisms that are not animals the zygote may be a resting stage. The study of how the zygote forms and develops is part of developmental biology and reproductive biology.

The zygote is not simply a passive container of two genomes. The oocyte supplies most of the cytoplasm, the mitochondria, and a large store of maternal mRNAs and proteins that direct the first divisions. The sperm provides the paternal genome, a centriole in many species (not in mice), and factors that activate the egg. In mammals, the zygote exists only for about 12 to 24 hours after fertilization, before the first cleavage division.

Gamete contact and fusion

In mammals, fertilization takes place in the ampulla of the oviduct. Sperm that are deposited in the female tract undergo capacitation, a set of changes (including changes in the lipid composition of the membrane, in intracellular pH and calcium, and in protein phosphorylation) that prepare them to fertilize. The oocyte is surrounded by the cumulus cells and by the zona pellucida, an extracellular matrix of glycoproteins (ZP1, ZP2, ZP3, and ZP4 in humans). Sperm bind to the zona pellucida, and this triggers the acrosome reaction, an exocytosis in which the contents of the acrosome are released. In mice, it is now thought that the acrosome reaction often begins before the sperm contact the zona, and that the sperm that fertilize have started the reaction on their way through the cumulus, so that earlier models, in which it is triggered only on the zona, are simplified. The acrosomal enzymes, together with the motility of the sperm, help the sperm to penetrate the zona.

At the oocyte membrane, the sperm protein IZUMO1 binds to the oocyte receptor JUNO (also called folate receptor 4), which is required for adhesion in mice and humans. Other proteins, including the oocyte tetraspanin CD9 and the sperm proteins SPACA6, TMEM81, and DCST1/2, are also needed for fusion. The fusion of the plasma membranes of the two gametes (plasmogamy) permits the sperm nucleus, and a sperm factor, to enter the oocyte cytoplasm. In the human, the entire sperm, including the tail, enters the oocyte, whereas in other species, only part of the sperm may be taken up. Paternal mitochondria are generally eliminated or diluted during early embryonic development, and in most animals mitochondrial DNA is inherited from the mother.

Egg activation and calcium oscillations

Fusion triggers egg activation. The sperm delivers a soluble factor, phospholipase C zeta (PLC_ζ_), which is the main trigger of activation in mammals. PLC_ζ_ hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP₂) to produce inositol 1,4,5-trisphosphate (IP₃) and diacylglycerol. IP₃ binds the IP₃ receptors (ligand-gated calcium channels) on the membrane of the endoplasmic reticulum, which releases calcium (Ca²⁺) into the cytoplasm. In mammals, the calcium signal consists of repeated spikes (oscillations) that last for several hours, whereas in many other animals, such as sea urchins and frogs, there is a single wave. The oscillations are produced by a feedback loop in which calcium sensitizes the IP₃ receptor, and by the pumping of calcium back into the ER. Mutations of PLC_ζ_ in men have been associated with failure of fertilization, and in some cases of infertility, the activation of oocytes is assisted by calcium ionophores. A resting cytosolic calcium concentration of about 100 nM can rise to about 1 μM in a spike, which is a tenfold change.

The calcium signal activates the enzymes that release the oocyte from its arrest at metaphase of the second meiotic division (metaphase II). The arrest is maintained by cytostatic factor (CSF), which includes the proteins Emi2 and Mos. Calcium activates calmodulin-dependent protein kinase II (CaMKII), which leads to degradation of Emi2, and the anaphase-promoting complex then degrades cyclin B and securin, so that the oocyte completes meiosis II and extrudes the second polar body. The second polar body contains a set of the maternal chromatids, and the remaining haploid set forms the female pronucleus.

Block to polyspermy

Fertilization by more than one sperm (polyspermy) leads to an abnormal ploidy and usually to embryonic death in humans. Several mechanisms prevent it. In many species (including sea urchins and frogs) there is a fast, electrical block to polyspermy, which is a depolarization of the membrane of the egg after fusion with the first sperm. Mammalian oocytes do not have a clear fast electrical block, and the mechanism there is mainly the slower block in the zona pellucida (the zona reaction). Cortical granules, which are secretory vesicles in the cortex of the oocyte, undergo exocytosis in response to the calcium rise, and release enzymes, including ovastacin, into the perivitelline space. Ovastacin cleaves the zona protein ZP2, which changes the properties of the zona and prevents the binding of other sperm. It is also reported that the oocyte membrane's ability to bind and fuse with sperm decreases after fertilization, and that JUNO is shed from the oocyte surface in vesicles, which contributes to the block. The statement that the zona "cross-links" and that Juno is cleaved by the cortical granule enzymes is therefore an oversimplification, and the cleavage of ZP2 is the best-established step. The block at the zona is not absolute, and polyspermy occurs in some cases, in particular in assisted reproduction.

Remodeling of the paternal genome

In the sperm, most histones are replaced during spermiogenesis by protamines, which are small, arginine-rich proteins that package the DNA in a very compact form. In humans, about 5 to 15 percent of the histones are retained. Protamines are cross-linked by disulfide bonds between cysteine residues, which are formed in the epididymis. After the sperm enters the oocyte, the disulfide bonds are reduced (the oocyte cytoplasm is rich in glutathione and other reducing agents), the protamines are removed, and maternal histones (including the variant H3.3 and histone chaperones, such as HIRA) are assembled on the paternal DNA. The male pronucleus swells as the chromatin decondenses. Nucleoplasmin and related proteins have roles in the removal of sperm proteins in some species. The paternal chromatin is therefore remodeled to a nucleosomal form. The description of the sperm DNA as a "crystalline state" is inaccurate. It is a highly condensed, toroidal structure that is not a crystal in the physical sense.

The two pronuclei, one of paternal and one of maternal origin, are visible in the zygote about 6 to 12 hours after fertilization in the human, and their appearance (the number of pronuclei, which should be two, and the arrangement of nucleoli) is used in assisted reproduction to evaluate the zygote.

Pronuclear migration and the first division

The male pronucleus is associated with a centrosome that is derived from the sperm, which is a source of microtubules in most mammals except rodents (in mice, the centrosome is of maternal origin and is formed from many acentriolar sites in the cytoplasm). The pronuclei migrate toward each other. In most mammals, the pronuclei do not fuse into a single nucleus. Instead, the nuclear envelopes of both pronuclei break down at the beginning of the first mitosis, and the two sets of chromosomes (the maternal and paternal chromosomes) align on a common spindle. This is a difference from many invertebrates (and from the sea urchin), in which the pronuclei fuse before the first mitosis (karyogamy). The statement that the pronuclei "meet at the exact center of the zygote" is also a simplification. They meet in the central region, and the maternal and paternal genomes remain in separate domains in the early embryo, but they are combined on the spindle, which makes syngamy occur at the first mitosis (the paternal and maternal chromosomes can be spatially distinct through the first few divisions in mice, and in some human zygotes, a dual spindle has been observed, which can result in errors). The first mitosis is prone to errors of chromosome segregation, which may contribute to mosaicism and to aneuploidy in human embryos.

Quantitative Comparison of Zygote Development and Maternal-to-Zygotic Transition Across Model Organisms

Asymmetric Paternal vs. Maternal Demethylation

Differentiated gametes carry epigenetic marks that are different from those of the totipotent cells of the embryo, and the zygote is the place of a large-scale reorganization of the epigenome (epigenetic reprogramming). A feature is a change in DNA methylation. The DNA of the sperm is highly methylated, and that of the oocyte is methylated to a lower degree. After fertilization, the paternal genome loses much of its methylation (active demethylation) within hours, which can be seen in immunostaining of the male pronucleus for 5-methylcytosine. The enzymes TET3 (a ten-eleven translocation dioxygenase) oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine, and 5-carboxylcytosine. These derivatives are then lost by dilution during replication, in a passive process, and some by base excision repair with thymine DNA glycosylase. The maternal genome loses methylation more slowly and passively, by replication in the absence of maintenance methylation, as DNMT1 is excluded from the nucleus in the early cleavage stages, and it is protected in part by the factor STELLA (DPPA3). The loss of methylation is global but not complete, and it differs among regions.

Genomic Imprinting Protection and Non-Canonical Histone Marks

The reprogramming does not completely erase the epigenetic marks of the gametes. Regions that are called imprinting control regions retain their parent-specific methylation, which is protected by factors that include ZFP57 and DPPA3, and the imprinted genes are expressed from only one parental allele. Several classes of repeats, including some retrotransposons (for example IAP elements in mice), are protected from demethylation. Histone modifications are also reorganized, and the genome-wide pattern of the histone marks of the sperm and oocyte is replaced by a pattern of the embryo in a gradual way. Some regions of the paternal and maternal genome keep some histone marks, and studies have reported that in mice, transmission of some histone marks from the oocyte (such as H3K27me3 at imprinted genes) occurs, which is called non-canonical imprinting. Whether there is a transgenerational inheritance of environmentally induced epigenetic states through the gametes in mammals is a subject of research, with limited evidence in mammals. The description of the process as resetting the chromatin "to a blank, foundational state" is therefore a simplification.

Maternal control and zygotic genome activation

The development of the early embryo depends on maternal products that were made in the growing oocyte, including mRNAs and proteins, with the "maternal-effect genes". The oocyte is transcriptionally silent in its late stage, and the zygote is also mostly silent initially, although it is not strictly "functionally silent", because a low level of transcription (the minor wave of zygotic genome activation) is found in the pronuclei in mice, and in the human at the one-cell stage a small number of genes is expressed. The main (major) activation of the embryonic genome occurs later. The transition from control by maternal products to control by the zygotic genome is the maternal-to-zygotic transition (MZT), and it has two parts: the degradation of maternal mRNAs and proteins and the zygotic genome activation (ZGA).

• Maternal clearance. Many maternal transcripts are destroyed after fertilization. The degradation is carried out by maternal factors, which include RNA-binding proteins and deadenylation by the CCR4-NOT complex, and in some species the zygotically expressed microRNAs (as in the zebrafish, with miR-430) and the N⁶-methyladenosine (m⁶A) machinery contribute. Maternal proteins are also degraded.
• Zygotic genome activation. In the mouse, minor ZGA begins in the one-cell stage (the zygote) and major ZGA occurs at the two-cell stage. In the human, major ZGA takes place at about the four- to eight-cell stage. In the frog and zebrafish, ZGA takes place at the mid-blastula transition after several rapid cell divisions, and it is determined by the ratio of nucleus to cytoplasm. In bovine and pig embryos, it is at the 8- to 16-cell stage. Pioneer transcription factors, such as DUX in the mouse and DUX4 in the human, activate the first genes, and the chromatin becomes accessible. Failure of ZGA results in arrest of the development of the embryo, and it is a cause of early embryonic loss. The statement that "absolute embryonic lethality" follows from any failure is an overstatement.

Holoblastic Rotational Cleavage and Compaction

The first mitotic divisions after fertilization, in which the cells divide without a growth in the total size of the embryo, are called cleavage. The daughter cells are blastomeres. In the human, the first cleavage occurs about 24 to 30 hours after fertilization, and cleavage divisions are relatively slow and asynchronous (about every 12 to 24 hours), compared with many other animals. The zygote divides into 2, 4, 8, and then 16 cells, and the embryo at the 16-cell stage is the morula. Compaction occurs at the 8- to 16-cell stage, in which the blastomeres flatten against each other and form tight junctions and polarity. The cells of the morula are then segregated into the outer cells (the trophectoderm) and the inner cell mass, which form the blastocyst, in which the first lineage decisions are made. The mammalian embryo is free in the oviduct and uterus, and it is enclosed within the zona pellucida until it hatches, at the blastocyst stage, before implantation (at about day 6 to 7 in the human).

Nuclear-to-Cytoplasmic Volume Scaling

The volume of the cytoplasm is divided among the blastomeres, so that the cell volume decreases with each division and the nuclear-to-cytoplasmic ratio increases. For example, a cell volume V₀ for a zygote gives a blastomere volume of V₀ / 2ⁿ after n rounds of cleavage, so that after four rounds (16 cells), each blastomere has about 6 percent of the original volume, and the number of cells is 2ⁿ. The ratio of nucleus to cytoplasm is the basis of the timing of ZGA in some species (for example frogs and fish). The pattern of cleavage differs among animals. In species whose eggs have large amounts of yolk, such as birds, reptiles, and many fish, cleavage is partial and not complete, and in insects, nuclear divisions occur without cell division to give a syncytium. In mammals, cleavage is rotational and holoblastic.

Lineage Segregation and Hippo-YAP Signaling

The zygote is totipotent. In mammals, the blastomeres of the two-cell and probably of the four-cell embryo are also able to give rise to a complete organism in at least some cases, which is the basis for the natural origin of monozygotic twins and for experimental embryo splitting. Mouse blastomeres from the 8-cell stage are no longer individually totipotent in the strict sense, although they are able to contribute to all lineages in chimeras. The two cell fates, trophectoderm and inner cell mass, arise at the morula and blastocyst stage under the influence of cell position and signaling (the Hippo pathway), and the expression of the transcription factors CDX2, OCT4, and NANOG. It is also not true that the zygote "is the only totipotent cell". Totipotency is a property of the zygote and the earliest blastomeres, and cells that have properties of totipotency have been described in cultures of embryonic stem cells (2-cell-like cells), but whether they are equivalent to the zygote is debated.

Somatic Cell Nuclear Transfer (SCNT) Reprogramming

The zygote is also the cell that is the product of the transfer of a somatic nucleus into an enucleated oocyte, in cloning (somatic cell nuclear transfer), in which the cytoplasm of the oocyte reprograms the nucleus, and of the artificial activation of oocytes in parthenogenesis. These show that the oocyte cytoplasm is able to reprogram a differentiated nucleus, although with low efficiency, and that the reprogramming that occurs in the normal zygote is not entirely replicated.

In in vitro fertilization (IVF), oocytes are fertilized outside the body, either by mixing with sperm or by intracytoplasmic sperm injection (ICSI), and the zygotes are cultured for 3 to 6 days. Zygotes with an abnormal number of pronuclei are usually not transferred. Zygote intrafallopian transfer is a rarely used variant. Fertilization failure, polyspermy, and errors in the first mitosis are causes of infertility and of early pregnancy loss. A large part of human embryos have chromosomal abnormalities, mostly aneuploidy that arises in the oocyte at meiosis (the rate increases with maternal age) and some from mitotic errors, and many do not develop to birth. Estimates of the loss of pregnancy before it is recognized are of the order of 30 percent or more of conceptions, although the figures are uncertain. Ethical and legal questions about the status of the zygote, and about research on human embryos, differ among countries and are the subject of debate, and many jurisdictions limit research on human embryos to the first 14 days.

How does totipotency in the zygote differ from pluripotency in embryonic stem cells?

A totipotent cell—such as the fertilized zygote and individual 2-cell blastomeres in mammals—possesses the developmental potential to generate an entire autonomous organism, including both the embryo proper and all extraembryonic tissues (specifically the trophoblast/placenta and primitive endoderm/yolk sac). Conversely, pluripotent inner cell mass (ICM) cells can differentiate into all three embryonic germ layers (ectoderm, mesoderm, endoderm) but cannot form the placenta.

Why are paternal mitochondria eliminated from the mammalian zygote after fertilization?

During spermatogenesis and fertilization, paternal sperm mitochondria experience elevated oxidative damage and are tagged with ubiquitin on their outer membranes. Upon entering the ooplasm, the zygote selectively degrades paternal mitochondria via autophagosomal mitophagy (mediated by Parkin, MUL1, and p62/SQSTM1) and endonuclease G cleavage of paternal mtDNA, ensuring homoplasmic maternal inheritance and preventing competition between divergent mitochondrial genomes.

Why is the first mitotic division of the human zygote unusually prone to chromosomal aneuploidy?

Recent live-imaging studies demonstrate that following nuclear envelope breakdown in mammalian zygotes, the maternal and paternal chromosome complements initially assemble two separate bipolar microtubule spindles that must coalesce into a single common metaphase spindle prior to anaphase. Incomplete alignment or asynchronous kinetochore capture between the two parental spindles frequently induces merotelic attachments, lagging chromosomes, and mosaic blastomere aneuploidy.

• Fertilization
• Gamete
• Embryogenesis
• Blastocyst
• Zygotic genome activation
• Totipotency
• Cleavage (embryo)