Fertilization is the process where by THE GAMETES—sperm and egg—fuse together to begin the creation of a new organism. Fertilization accomplishes two separate ends: sex (the combining of genes derived from two parents) and reproduction (the generation of a new organism). Thus, the first function of fertilization is to transmit genes from parent to offspring, and the second is to initiate in the egg cytoplasm those reactions that permit development to proceed. Although the details of fertilization vary from species to species, it generally consists of four major events:
1. Contact and recognition between sperm and egg. In most cases, this ensures that the sperm and egg are of the same species.
2. Regulation of sperm entry into the egg. Only one sperm nucleus can ultimately unite with the egg nucleus. This is usually accomplished by allowing only one sperm to enter the egg and actively inhibiting any others from entering.
3. Fusion of the genetic material of sperm and egg.
4. Activation of egg metabolism to start development.
During fertilization, the egg and sperm must meet, the genetic material of the sperm must enter the egg, and the fertilized egg must initiate cell division and the other processes of development. Sperm and egg must travel toward each other, and chemicals from the eggs can attract the sperm. Gamete recognition occurs when proteins on the sperm cell membrane meet proteins on the extracellular coating of the egg. In preparation for this meeting, the sperm cell membrane is altered significantly by exocytotic events. The sperm activates development by releasing calcium ions (Ca2+) from within the egg. These ions stimulate the enzymes needed for DNA synthesis, RNA synthesis, protein synthesis, and cell division. The sperm and egg pronuclei travel toward one another and the genetic material of the gametes combines to form the diploid chromosome content carrying the genetic information for the development of a new organism.Sperm anatomy
Each sperm cell consists of a haploid nucleus, a propulsion system to move the nucleus, and a sac
of enzymes that enable the nucleus to enter the egg. In most species, almost all of the cell’s cytoplasm is eliminated during sperm maturation, leaving only certain organelles that are modified for spermatic function (Figure A,B below). During the course of maturation, the sperm’s haploid nucleus becomes very streamlined and its DNA becomes tightly compressed. In front or to the side of this compressed haploid nucleus lies the acrosomal vesicle
, or acrosome (Figure C). The acrosome is derived from the cell’s Golgi apparatus and contains enzymes that digest proteins and complex sugars. Enzymes stored in the acrosome create a path through the outer coverings of the egg. In many species, a region of globular actin proteins lies between the sperm nucleus and the acrosomal vesicle. These proteins are used to extend a fingerlike acrosomal process from the sperm during the early stages of fertilization. In sea urchins and numerous other species, recognition between sperm and egg involves molecules on the acrosomal process. Together, the acrosome and nucleus constitute the sperm head
. The means by which sperm are propelled vary according to how the species is adapted to environmental conditions. In most species, an individual sperm is able to travel by whipping its flagellum. The major motor portion of the flagellum is the axoneme, a structure formed by microtubules emanating from the centriole at the base of the sperm nucleus. The core of the axoneme
consists of two central microtubules surrounded by a row of nine doublet microtubules. These microtubules are made exclusively of the dimeric protein tubulin
. Although tubulin is the basis for the structure of the flagellum, other proteins are also critical for flagellar function. The force for sperm propulsion is provided by dynein, a protein attached to the microtubules. Dynein is an ATPase—an enzyme that hydrolyzes ATP, converting the released chemical energy into mechanical energy that propels the sperm. This energy allows the active sliding of the outer doublet microtubules, causing the flagellum to bend. The ATP needed to move the flagellum and propel the sperm comes from rings of mitochondria located in the midpiece of the sperm (see Figure B). In many species (notably mammals), a layer of dense fibers has interposed itself between the mitochondrial sheath and the cell membrane. This fiber layer stiffens the sperm tail. Because the thickness of this layer decreases toward the tip, the fibers probably prevent the sperm head from being whipped around too suddenly. Thus, the sperm cell has undergone extensive modification for the transport of its nucleus to the egg
. In mammals, the differentiation of sperm is not completed in the testes. Although they are able to move, the sperm released during ejaculation do not yet have the capacity to bind to and fertilize an egg. The final stages of sperm maturation, cumulatively referred to as capacitation, do not occur in mammals until the sperm has been inside the female reproductive tract for a certain period of time. The eggcytoplasm and nucleus.
All the material necessary to begin growth and development must be stored in the egg, or ovum. Whereas the sperm eliminates most of its cytoplasm as it matures, the developing egg (called the oocyte before it reaches the stage of meiosis at which it is fertilized) not only conserves the material it has, but actively accumulates more. The meiotic divisions that form the oocyte conserve its cytoplasm rather than giving half of it away; at the same time, the oocyte either synthesizes or absorbs proteins such as yolk that act as food reservoirs for the developing embryo. Birds’ eggs are enormous single cells, swollen with accumulated yolk. Even eggs with relatively sparse yolk are large compared to sperm. The volume of a sea urchin egg is more than 10,000 times the volume of sea urchin sperm. So even though sperm and egg have equal haploid nuclear components, the egg accumulates a remarkable cytoplasmic storehouse during its maturation. Discoidal meroblastic cleavage in a chick egg. (A)
Avian eggs include some of the largest cells known (inches across), but cleavage takes place in only a small region. The yolk fills up the entire cytoplasm of the egg cell, with the exception of a small blastodisc in which
cleavage and development will take place. The chalaza are protein strings that keep the yolky egg cell centered
in the shell. The albumin (egg white) is secreted onto the egg in its passage out of the oviduct. (B)
Early cleavage stages viewed from the animal pole (the future dorsal side of the embryo). In the micrographs, the tightly apposed cell membranes have been stained with phalloidin (green). (C)
Schematic view of cellularization in the chick egg during the day it is fertilized and still inside the hen. The numbers refer to the layers of cells.
This cytoplasmic trove includes the following:
• Nutritive proteins.
The early embryonic cells must have a supply of energy and amino acids. In many species, this is accomplished by accumulating yolk proteins in the egg. Many of these yolk proteins are made in other organs
(e.g., liver, fat bodies) and travel through the maternal blood to the oocyte.
• Ribosomes and tRNA.
The early embryo must make many of its own structural proteins and enzymes, and in some species
there is a burst of protein synthesis soon after fertilization. Protein synthesis is accomplished by ribosomes and tRNA that exist in the egg. The developing egg has special mechanisms for synthesizing ribosomes; certain amphibian oocytes produce as many as 10^12 ribosomes during their meiotic prophase.
• Messenger RNAs.
The oocyte not only accumulates proteins, it also accumulates mRNAs that encode proteins for the early stages of development. It is estimated that sea urchin eggs contain thousands of different types of mRNA
that remain repressed until after fertilization.
• Morphogenetic factors.
Molecules that direct the differentiation of cells into certain cell types are present in the egg. These include transcription factors and paracrine factors. In many species, they are localized in different regions of the egg and become segregated into different cells during cleavage.
• Protective chemicals.
The embryo cannot run away from predators or move to a safer environment, so it must be equipped to deal with threats. Many eggs contain ultraviolet filters and DNA repair enzymes that protect them from sunlight
, and some eggs contain molecules that potential predators find distasteful
. The yolk of bird eggs contains antibodies that protect the embryo against microbes
Within the enormous volume of egg cytoplasm resides a large nucleus. In a few species (such as sea urchins), this female pronucleus is already haploid at the time of fertilization. In other species (including many worms and most mammals), the egg nucleus is still diploid—the sperm enters before the egg’s meiotic divisions are completed. Stages of egg maturation at the time of sperm entry in different animal species. Note that in most species, sperm entry occurs before the egg nucleus has completed meiosis. The germinal vesicle is the name given to the large diploid nucleus of the primary oocyte. The polar bodies are nonfunctional cellsproduced by meiosis
In these species, the final stages of egg meiosis will take place after the sperm’s nuclear material—the male pronucleus—is already inside the egg cytoplasm. Cell membrane and extracellular envelope
The membrane enclosing the egg cytoplasm regulates the flow of specific ions during fertilization and must be capable of fusing with the sperm cell membrane. Outside this egg cell membrane is an extracellular matrix that forms a fibrous mat around the egg and is often involved in sperm-egg recognition. In invertebrates, this structure is usually called the vitelline envelope (Figure A). Sea urchin egg cell surfaces(A) Scanning electron micrograph of an egg before fertilization. The cell membrane is exposed where the vitelline envelope has been torn. (B) Transmission electron micrograph of an unfertilized egg, showing microvilli and cell membrane, which are closely covered by the vitelline envelope. A cortical granule lies directly beneath the cell membrane.
The vitelline envelope contains several different glycoproteins. It is supplemented by extensions of membrane glycoproteins from the cell membrane and by proteinaceous “posts” that adhere the vitelline envelope to the cell membrane. The vitelline envelope is essential for the species-specific binding of sperm.
Many types of eggs also have a layer of egg jelly outside the vitelline envelope. This glycoprotein meshwork can have numerous
functions, but most commonly it is used either to attract or to activate sperm. The egg, then, is a cell specialized for receiving sperm and initiating development. Lying immediately beneath the cell membrane of most eggs is a thin layer (about 5 μm) of gel-like cytoplasm called the cortex. The cytoplasm in this region is stiffer than the internal cytoplasm and contains high concentrations of globular actin molecules. During fertilization, these actin molecules polymerize to form long cables of actin microfilaments. Microfilaments are necessary for cell division. They are also used to extend the egg surface into small projections called microvilli, which may aid sperm entry into the cell (Fig ure B above). Also within the cortex are the cortical granules (see B). These membrane-bound, Golgi-derived structures contain proteolytic enzymes and are thus homologous to the acrosomal vesicle of the sperm. However,
whereas a sea urchin sperm contains just one acrosomal vesicle, each sea urchin egg contains approximately 15,000 cortical granules. In addition to digestive enzymes, the cortical granules contain mucopolysaccharides, adhesive glycoproteins, and hyalin protein. As we will soon describe, the enzymes and mucopolysaccharides help prevent polyspermy—that is, they prevent additional sperm from entering the egg after the first sperm has entered—while hyalin and the adhesive glycoproteins surround the early embryo, providing support for cleavage-stage blastomeres. In mammalian eggs, the extracellular envelope is a separate, thick matrix called the zona pellucida. The mammalian egg is also surrounded by a layer of cells called the cumulus.Spermiogenesis: the differentiation of the sperm
The production of mature and motile sperm is a detailed process that utilizes many molecular players to ensure the faithful execution of spermatogenesis. Spermatogenesis begins with a single cell that undergoes dramatic transformation
, culminating with the hypercompaction of DNA into the sperm head by replacing histones with protamines
. Precise execution of the stages of spermatogenesis results in the production of motile sperm. Spermatogenesis is a highly orchestrated process that requires the correct interplay and timing of all molecular constituents to produce fully functional and motile sperm. Defects in spermatogenesis can impact a male’s overall fitness, which encompasses the ability to both survive and reproduce successfully. Aberrations during any stage within spermatogenesis can have profound effects on sperm quantity, motility, morphology and ability to fertilize an egg. In addition, poor packaging of chromatin within sperm nuclei can reduce the protection of DNA against chemical and physical damage, potentially leading to mutations and unfit offspring. Modifications to sperm chromatin require the use of specialized DNA-binding proteins, referred to as protamines, which are capable of achieving the level of organization and compression necessary to fit the haploid genome into the compact sperm head.
Sperm were discovered in the 1670s, but their role in fertilization was not discovered until the mid-1800s. It was only in the 1840s,
after Albert von Kölliker described the formation of sperm from cells in the adult testes that fertilization research could really begin. Even so, von Kölliker denied that there was any physical contact between sperm and egg. He believed that the sperm excited the egg to develop in much the same way a magnet communicates its presence to iron. The first description of fertilization was published in 1847 by Karl Ernst von Baer, who showed the union of sperm and egg in sea urchins and tunicates. He described the fertilization envelope, the migration of the sperm nucleus to the center of the egg, and the subsequent early cell divisions of development. In the 1870s, Oscar Hertwig and Herman Fol repeated this work and detailed the union of the two cells’ nuclei.A complex dialogue exists between egg and sperm. The egg activates the sperm metabolism that is essential for fertilization, and the sperm reciprocates by activating the egg metabolism needed for the onset of development.
In male humans, the process of spermatogenesis supports a production rate of approximately 120 million mature spermatozoa per day by the human testis (approximately 1000 per heartbeat!). Spermatogenesis, the process by
which stem cells (spermatogonia) differentiate into mature spermatozoa, proceeds in three functionally distinct phases:
(1) the mitotic or proliferative phase, during which the majority of spermatogonia undergo mitosis to renew the stem cell pool and a minority become committed to further differentiation to produce spermatocytes;
(2) the meiotic phase, during which spermatocytes undergo successive meiotic divisions to produce haploid germ cells (spermatids); and
(3) spermiogenesis, during which immature, round spermatids differentiate into mature spermatozoa Schematic diagram of human spermatogenesis. Spermatogonial stem cells undergo self-renewal by mitotic division. At the initiation of spermatogenesis, some spermatogonia undergo differentiation into primary spermatocytes, which contain a diploid number of chromosomes (2N = 46 chromosomes). The primary spermatocytes then undergo two successive meiotic divisions to form spermatids, which contain a haploid number of chromosomes (1N = 23 chromosomes). Spermatids undergo spermiogenesis to form mature spermatozoa, which also contain a haploid number of chromosomes.
The mammalian haploid a
spermatid is a round
cell that looks nothing like the mature vertebrate sperm. The next step in sperm maturation, then, is spermiogenesis, the differentiation of the sperm cell. For fertilization to occur, the sperm has to meet and bind with an egg, and spermiogenesis prepares the sperm for these functions of motility and interaction
. In many organisms (e.g., humans, mice, Drosophila), male germ cells undergo a series of morphological transformations during spermiogenesis to build a sperm
with its typical species-specific shape from an initially round cell Modification of a germ cell to form a mammalian sperm. (A) The centriole produces a long flagellum at what will be the posterior end of the sperm. The Golgi apparatus forms the acrosomal vesicle at the future anterior end. Mitochondria collect around the flagellum near the base of the haploid nucleus and become incorporated into the midpiece (“neck”) of the sperm. The remaining cytoplasm is jettisoned, and the nucleus condenses. The size of the mature sperm has been enlarged relative to the other stages. (B) Mature bull sperm. The DNA is stained blue, mitochondria are stained green, and the tubulin of the flagellum is stained red. (C) The acrosomal vesicle of this mouse sperm is stained green by the fusion of proacrosin with green fluorescent protein (GFP). The eight steps of spermatid differentiation during spermiogenesisA: acrosome, An: annulus, Ax: axoneme, C: centriole, F: flowerlike structures, Fs: flagellar substructures, M: mitochondria, Mp: middle piece, Mt: manchette microtubules, N: nucleus, Ne: neck (connecting piece), PP: principal piece, R: ribs of the fibrous sheath, Sb: spindle-shaped body. The acrosome of STEP 1 spermatids is oriented toward the seminiferous tubule lumen. When progressing to step 2, spermatids rotate 180° so that the acrosome faces the seminiferous tubule basement membrane.
The first step is the construction of the acrosomal vesicle b
from the Golgi apparatus, a process about which we know very little. The acrosome forms a cap that covers the sperm nucleus. As the acrosomal cap is formed, the nucleus rotates so that the cap faces the basal lamina of the seminiferous tubule c
. This rotation is necessary because the flagellum, which is beginning to form from the centriole on the other side of the nucleus, will extend into the lumen of the seminiferous tubule. During the last stage of spermiogenesis, the nucleus flattens and condenses, the remaining cytoplasm (the residual body, or cytoplasmic droplet) is jettisoned, and the mitochondria form a ring around the base of the flagellum.
During spermiogenesis, the histones of the spermatogonia are often replaced by sperm-specific histone variants, and widespread nucleosome dissociation takes place. This remodelling
of nucleosomes might also be the point at which the Primordial germ cells (PGC) pattern of methylation is removed and the male genome-specific pattern of methylation is established on the sperm DNA. As spermiogenesis ends, the histones of the haploid nucleus are eventually replaced by protamines. This replacement results in the complete shutdown of transcription in the nucleus and facilitates the nucleus assuming an almost crystalline structure. The resulting sperm then enter the lumen of the seminiferous tubule. Unexpectedly, the sperm continue to develop after they leave the testes. When being transported from the testes, sperm reside in the epididymis. During this residence, the epididymal cells release exosomes that fuse with the sperm. These exosomes have been shown to contain small ncRNAs and other factors that can activate and repress certain genes, and the sperm will bring these agents into the egg. And the sperm still isn’t fully mature, even when it exits the urethra. The final differentiation of the sperm occurs in the reproductive tract of the female. Here, secretions from the oviducts will change the sperm cell membrane so that it can fuse with the membrane of the egg cell. Thus, the full differentiation of the sperm take place in two different organisms.
Each day, some 100 million sperm are made in each human testicle, and each ejaculation releases 200 million sperm. Unused sperm are
either resorbed or passed out of the body in urine. During his lifetime, a human male can produce 10^12 to 10^13 sperm.
Spermatozoa arise within testicular seminiferous tubules by spermatogenesis, the series of cell processes by which undifferentiated germ cells give rise to mature spermatozoa. It comprises three sequential phases:1)
Mitotic multiplication/differentiation of spermatogonia, resulting in a geometric numerical expansion, loss of their stem cell properties and further commitment to advance in spermatogenesis. This phase yields a large
number of spermatogonia ready to undergo meiotic division.(2)
Meiosis, a series of two coupled cell divisions leading to genomic recombination and haploidization. These processes occur during the prophase of Meiosis I by pairing and genetic recombination between homologous
chromosomes and are followed by reduction of chromosomal numbers. Each resulting secondary spermatocyte, a unique combination of the male paternal and maternal genomes, goes into Meiosis II and generates two haploid spermatids ready to go through the last phase of spermatogenesis.(3)
Spermiogenesis, through which newly formed postmeiotic round spermatids undergo a complex cell differentiation leading to the production of elongated spermatids that leave the germinal epithelium to become free mature spermatozoa in the lumen of seminiferous tubules.Human spermiogenesis depicts particular features related to the uniqueness of human spermatozoa,
including specific modifications of nuclear shape and chromatin structure, acrosome development, flagellar
growth, and formation of the mitochondrial sheath. Round euchromatic nuclei of early spermatids elongate while their chromatin progressively condenses, the Golgi-derived acrosome locates at the cephalic pole and spreads flat over the nucleus, the proximal centriole migrates and lodges at the caudal nuclear pole and the axoneme of the future sperm cell grows from the distal centriole and emerges into the extracellular space. These changes have been summarized in a series of eight steps.Steps 1–2:
Spermatid nuclei have finely granular chromatin with small non-prominent nucleoli. The Golgi complex, in a juxtanuclear location, has given rise to pro-acrosomic vesicles that contain dense granules and fuse into a large acrosomic
vesicle that attaches to the nuclear membrane at the spermatid cranial pole. This vesicle flattens and spreads as a dense acrosome that will eventually cover between half and two-thirds of the nuclear surface. At these stages the centrioles migrate first to the cell membrane (where the axoneme grows from the distal centriole toward the extracellular space), and later to the caudal nuclear pole, where the proximal centriole lodges in a shallow concavity (the implantation fossa) defining the bipolar nature of the spermatozoon. Mitochondria, first located at the cell periphery, become randomly distributed on the caudal spermatid cytoplasm in step 2. At the neck region, around the proximal centriole, a dense striated structure develops that will become the connecting piece, a structure that fastens the tail to the sperm head.Steps 3–5:
The spermatid nucleus progressively elongates and flattens at the area covered by the acrosome while chromatin starts to condense by increasing the size and density of its granular components. Chromatin condensation starts at the tip of the head and progresses caudally, accompanied by a significant reduction of nuclear size and further flattening of the acrosome-covered area. The axoneme elongates and thickens by the addition of periaxonemal
structures: the outer dense fibres and the fibrous sheath. The spermatid cytoplasm now occupies a distal position that anticipates the formation of the residual cytoplasm that will eventually disengage from the main sperm cell body at spermiation
Nuclear elongation/flattening and chromatin condensation proceed to their mature stage. Mitochondria assemble around the first part of the axoneme to form the sperm midpiece. The residual cytoplasm further separates from the main cell body but remains attached to it by a slender stalk until mature spermatids are ready to be released into the lumen of seminiferous tubules as free spermatozoa.The spermatid nucleus and the golgi complex; nuclear remodelling, chromatin compaction and acrosome developmentDuring spermiogenesis, the organization of germ cell d DNA undergoes remarkable modifications.
In early round spermatids e
, DNA is associated with nuclear histones, forming a protein complex that organizes into
globular octamers by the association of a pair of each of the four histone components [H2A, H2B, H3 and H4]. These core particles are encircled by 146 DNA base pair filaments, forming nucleosomes, the basic structure of loosely bound DNA–histone complexes linearly assembled as beads in a string. This loose association is characteristic of euchromatin, prevalent in transcriptionally active somatic cells and germ cells up to early spermatids, and results in a dispersed microgranular and filamentous nuclear substructure. During spermiogenesis chromatin condensation starts when histones are removed and replaced by intermediate proteins and ultimately by protamines, smaller and structurally very different proteins that accommodate into minor DNA grooves and establish a strong bond that is further stabilized by cross-linking of disulphide bonds, resulting in very stable, highly compacted DNA. This spatial macromolecular organization renders DNA transcriptionally silent, but at the same time shields it and ensures its stability and resiliency to external influences during sperm transit.
The process of DNA protamination
–condensation is visualized through spermiogenesis as the evolution of dispersed chromatin into a progressively denser granular structure that leads to the highly compact chromatin of mature spermatozoa. Chromatin dynamics during spermiogenesis
The function of sperm is to safely transport the haploid paternal genome ( haploid = one copy of each chromosome ) to the egg containing the maternal genome. The subsequent fertilization leads to transmission of a new unique diploid genome to the next generation. Before the sperm can set out on its adventurous journey, remarkable arrangements need to be made. Haploid spermatids undergo extensive morphological changes, including a striking reorganization and compaction of their chromatin. Thereby, the nucleosomal, histone-based structure is nearly completely substituted by a protamine-based structure. This replacement is likely facilitated by incorporation of histone variants, post-translational histone modifications, chromatin-remodelling complexes, as well as transient DNA strand breaks.
Protamines are necessary to protect the paternal genome. Hyperacetylation of histones just before their displacement is vital for progress in chromatin reorganization
but is clearly not the sole inducer. In this review, we highlight the current knowledge on post-meiotic chromatin reorganization and reveal for the first time intriguing parallels in this process in Drosophila and mammals. 2 We now know that in many organisms spermiogenesis is accompanied by a dramatic reorganization of chromatin from a nucleosomal histone-based structure to a structure largely based on protamines
. The replacement of histones by protamines is gradual. First, some of the canonical histones are replaced by testis-specific histone variants. Subsequently, so-called transition proteins are incorporated as nucleosomes are removed, and finally, protamines generate the tightly packaged sperm nucleus. In both flies and mammals, specific histone modifications and transient formation of DNA breaks precede or accompany protamine deposition. Over the past two decades, many chromatin components that specifically function during spermiogenesis have been described.Germ cells mediate the transfer of genetic information from generation to generation and are thus pivotal for maintenance of life.
Spermatogenesis is a continuous and precisely controlled process
that leads to the formation of haploid sperm capable of fertilization. The process of spermatogenesis is highly conserved among many organisms
and can be subdivided into three crucial phases: a mitotic amplification phase, a meiotic phase, and a post-meiotic phase also known as spermiogenesis. Germ cells in the post-meiotic phase can be subdivided into early spermatids with round nuclei, intermediate spermatids with elongating nuclei, and spermatids with condensed nuclei. During spermiogenesis in both flies and mammals, round spermatids differentiate into mature sperm. During this process, the nuclear volume dramatically reduces, and histones are gradually replaced by protamines Key chromatin remodeling events during spermiogenesis in mice and flies. Shortly after meiosis, spermatids are characterized by a round nucleus that elongates and reshapes during spermiogenesis. In parallel, the nucleosomal histone-based chromatin configuration is replaced by a mainly protamine-based tightly compacted structure. This chromatin reorganization is accompanied by hyperacetylation of histone H4 as well as the transient appearance of DNA breaks.From histones to protamines
The process of spermatogenesis in mammals and in Drosophila species is similar, although the size and shape of mature sperm, as well as the time span for spermatogenesis, differ considerably. The testes of Drosophila and all mammalian species contain all stages of spermatogenesis, from stem cells to mature sperm, and spermatogenesis occurs within a tubular structure. Germ cells develop in close contact with the surrounding somatic cells, known as cyst cells in Drosophila and Sertoli cells in mammals. In mammals, the number of stages that can be observed varies among species.
Protamines stabilize sperm chromatin by their assembly in the minor groove of DNA into densely packed arrays linked by intermolecular and intramolecular disulfide bonds. In addition, protamine deficiencies are associated with infertility in men, and the frequency of human sperm with DNA damage correlates with failure of embryonic development.
3 A reduction in the amount of protamine would change not only the stoichiometry of the major components of the chromatin but also the net charge in the sperm nucleus, thereby affecting chromatin condensation and stability.
Fertilization involves a direct interaction amongst spermatozoa and oocytes, a merger of the cell membrane, and a union of male and female gamete genome. The process can thoroughly take place when supported by the compact spermatozoa DNA integrity. Spermatozoa's DNA integrity plays a significant role in delivering accurate genetic information.
6 The compactness of spermatozoa chromatin is due to the bonding between DNA and proteins of core spermatozoa, particularly the protamine
. A number of causes why spermatozoa DNA damages are the protamine deficiency. Protamine plays an important role in male's normal fertility. The deficiency in P1 and P2 causes subfertile or severe infertile condition.
The human sperm nucleus contains two types of protamine: protamine 1 (P1) encoded by a single-copy gene and the family of protamine 2 (P2) proteins (P2, P3 and P4), all also encoded by a single gene that is transcribed and translated into a precursor protein. 7 Their function goes from condensation of the sperm nucleus into a compact hydrodynamic shape, protection of the genetic message delivered by the spermatozoa, involvement in the processes maintaining the integrity and repair of DNA during or after the nucleohistone–nucleoprotamine transition and involvement in the epigenetic imprinting of the spermatozoa. The protamine family of sperm nuclear proteins
The protamines are a diverse family of small proteins that are synthesized in the late-stage spermatids of many animals and plants and bind to DNA, condensing the spermatid genome into a genetically inactive state. The two protamines found in mammals, P1 and P2, are the most widely studied. P1 packages sperm DNA in all mammals, whereas protamine P2 is present only in the sperm of primates, many rodents and a subset of other placental mammals. P2, but not P1, is synthesized as a precursor that undergoes proteolytic processing after binding to DNA. Both P1 and P2 have been shown to be required for normal sperm function in primates and many rodents.
Comparisons of the amino-acid sequences of vertebrate and invertebrate protamines show that the protamines from all animals do not constitute a true familyMy comment: Of course, thats a major headage for evolutionary biologists. To overcome the problem, the "ad-hoc" explanation is:
the sequence, structure, and possibly function of protamines evolved independently in vertebrates and various invertebrate groups (mollusks
, cephalopods and tunicates).Hum... Really?!!
Two structural elements have been identified in all vertebrate protamines. One is a series of small 'anchoring' domains containing multiple arginine or lysine amino acids (three or more per domain, highlighted in red in the figures in Additional data file 1) that are used to bind the protein to DNA. The protamines present in eutherian mammals all contain multiple cysteine residues that are oxidized to form disulfide bridges that link the protamines together and stabilize the chromatin complex during the final stages of sperm maturation.
Upon binding to DNA, P1 wraps around the DNA helix in the major groove with one protamine molecule being bound per turn of DNA helix. Protamine molecules bind in the major groove of DNA, neutralizing the phosphodiester backbone of DNA and causing the DNA molecules to coil into toroidal structures.(a) Model showing how two adjacent salmon protamine molecules (blue atoms) wrap around the DNA helix (white atoms) and bind within the major groove of DNA.(b) Scanning-probe images of toroidal DNA-protamine complexes prepared in vitro on a graphite surface by adding protamine to DNA attached loosely to the surface. The toroids formed in vitro are similar in size and shape to those isolated from human sperm chromatin(c). (c) Scanning-probe microscope images of native DNA-protamine toroids obtained from human sperm chromatin. These toroids, which comprise the basic subunit structure of protamine-bound DNA, contain approximately 50,000 bp of DNA coiled into each donut-shaped structure.Transition proteins: transiently expressed non-histone chromatin components in spermatids
Between histone removal and protamine deposition in mammalian spermatids, about 90% of the basic chromatin components consists of transition proteins. Mouse transition protein 1 (TP1) and 2 (TP2), encoded by Tnp1 and Tnp2, are arginine- and lysine-rich proteins that bind strongly to DNA. TP1 decreases the melting temperature of DNA, relaxes the DNA in nucleosomal core particles, and stimulates the DNA-relaxing activity of topoisomerase I, which indicates that TPs could help chromatin remodelling by making the DNA more flexible. However, others have reported that neither TP1 nor TP2 is able to cause topological changes in supercoiled DNA. In addition, it has been proposed that the TPs can mediate DNA and chromatin condensation. Finally, TP1 has been found to stimulate repair of single-strand DNA breaks. Tnp1 knockout in mice leads to severely reduced sperm motility and to elevated levels of TP2 and of some protamine 2 precursors in spermatid nuclei that might partially compensate for the lack of TP1. About 60% of Tnp1-deficient males is sterile. In contrast, Tnp2- deficient mice are fertile and display only mild sperm abnormalities. In mice lacking both TPs, histone displacement and protamine deposition proceed relatively normally, while chromatin condensation is irregular in all spermatids. Furthermore, many late spermatids show DNA breaks, the number of epididymal sperm is drastically reduced, and mice are sterile. There is some functional redundancy between TP1 and TP2, and that they are not required for histone removal and protamine loading, yet are important for the proper regulation of chromatin structure. Protamines and chromatin components of mature sperm chromatin
Transition proteins are subsequently replaced by the highly basic protamines in late spermatids. Mammalian protamines are arginine- and cysteine-rich proteins with a low molecular mass and are associated with DNA in late spermatids and mature sperm. Most mammalian species have only one type of protamine present in sperm, while the genomes of mice and humans encode two protamine types: Protamine 1 (PRM1 or P1) and Protamine 2 (PRM2 or P2). Whereas protamines of the P2 family (P2, P3, and P4), which differ only in the N-terminal extension of 1–4 residues, are generated by proteolysis from a precursor encoded by a single gene, protamine 1 is synthesized as a mature protein. Just as in mice and humans, also the genome of Drosophila encodes two different protamines. These protamines, ProtA and ProtB (encoded by Mst35Ba and Mst35Bb, respectively), are cysteine-rich but less arginine-rich than mammalian P1 and P2. In mice, PRM1 and PRM2 are essential for male fertility, and even deletion of a single copy of either Prm1 or Prm2 leads to infertility. In humans, nuclei of normal sperm cells contain similar amounts of protamine 1 and 2 (P1/P2 ratio of 1); deviations from this ratio correlate with male infertility.
Surprisingly, in Drosophila, ProtA and ProtB are not essential for fertility; when both protamines are missing, only about 20% of the sperm nuclei displays abnormal morphology. However, one has to consider that these two protamines are not the only chromatin components of sperm in flies. The HILS1- like protein Mst77F marks distinct chromatin regions within sperm nuclei, and its deposition is independent of the incorporation of protamines. It needs to be clarified whether further chromatin components exist in mature sperm that might be functionally redundant with protamines.In spermiogenesis, the majority of the core histones are replaced sequentially, first by transition proteins and then protamines, facilitating chromatin hyper-compaction.
1 In this histone-to-protamine transition process
epigenetic regulators interact with each other to remodel chromatin architecture. Testis-specific transcription factors play a critical role in controlling the haploid-specific developmental program, recent studies underscore the essential functions of epigenetic players involved in the dramatic genome remodeling that takes place during wholesale histone replacement.
Histone variants and histone writers/readers/erasers, rewire the haploid spermatid genome to facilitate histone substitution by protamines in mammals. During spermatid development, the paternal genome is re-organized and packaged into highly condensed nuclei of the spermatozoa. One of the dramatic changes that occurs lies in the transition from nucleosome-based chromatin to protamine-based chromatin arrays, which facilitates the condensation of sperm heads and protects the paternal DNA from damage and mutagenesis.
Epigenetics, referring to the phenotypic inheritance of traits in the progeny without altering the genetic DNA code, is involved in a wide range of biological processes, including germ cell development. They undergo global genome-wide de novo reprogramming mainly orchestrated by the DNA demethylases and methyltransferases
, such as ten-eleven translocation proteins (TET1/2)
, which induce the active DNA de-methylation and methylation, respectively, in both the male and female primordial germ cell populations.Histone modifications that precede histone removal during spermiogenesisDuring spermiogenesis, most histones are replaced by transition proteins and subsequently by protamines. This drastic alteration in chromatin configuration is expected to require mechanisms to promote eviction of nucleosomes in favor of incorporation of transition proteins, followed by a subsequent exchange of transition proteins for protamines.
In addition to the incorporation of histone variants, specific histone modifications also affect the higher-order chromatin structure and play important roles in gene regulation and maintenance of genome integrity. Histones are commonly post-translationally modified at the amino-terminal tails. The most-studied histone modifications include acetylation, methylation, phosphorylation, ubiquitination, and SUMOylation. These modifications can be recognized by proteins and facilitate downstream events on chromatin, resulting in a more open or closed chromatin configuration. In both flies and mammals, hyperacetylation of histone H4 has been observed in elongating spermatids just prior to histone removal. H4 hyperacetylation in mammalian spermatids leads to an open chromatin structure that facilitates and induces histone displacement. In mice and humans, reduced levels of histone H4 hyperacetylation in sperm correlates with impaired fertility. Previously established testis organ cultures have enabled us to address the functional importance of histone hyperacetylation in Drosophila. Indeed, inhibition of histone acetyltransferases (HATs) in in vitro cultures of Drosophila testes leads to spermiogenesis arrest
, and the chromatin remains in a histone-based configuration. Thus, hyperacetylation appears to be a prerequisite for proper chromatin reorganization
. However, premature acetylation does not induce premature chromatin reorganization in Drosophila, which suggests that H4 hyperacetylation is not the sole inducer of chromatin remodelling.
Expression of different histone methyltransferases and demethylases has been observed during spermatid elongation in flies and mammals. This coexistence of both types of enzymes might be crucial to balance regions of “opened”
and “closed” chromatin. Insights into the mechanisms of chromatin reorganization during spermiogenesis
The histone-to-protamine transition, require mechanisms to access DNA. These mechanisms often require one or more covalent histone modifications and histone replacement by histone variants. In addition, ATP-dependent chromatin remodelling complexes often participate in gaining DNA accessibility.The mechanisms that potentially lead to the mainly protamine-based DNA packaging in mature sperm are schematically summarized within a model. Model of the regulatory mechanisms that may lead from a nucleosomal-based to a mainly protamine-based chromatin configuration. (A) Increased levels of histone acetyltransferases mediate hyperacetylation of histones to obtain a more open chromatin structure. This process might be supported by specific degradation of histone deacetylases. In parallel, chaperones participate in incorporation of histone variants to loosen the nucleosomal structure. Exchanged canonical histones, in turn, may undergo poly-ubiquitination and become degraded through proteasomes. (B) Binding of bromodomain proteins to acetyl-residues may facilitate recruitment of chromatin-remodelling complexes to further relax chromatin. This is accompanied by the induction of DNA strand breaks. The relaxed chromatin configuration allows replacement of histones by transition proteins, possibly with the help of molecular chaperones. Subsequently, DNA damage repair mechanisms must act to ensure chromatin integrity. (C) Finally, molecular chaperones most likely aid in replacement of transition proteins by protamines to allow tight packaging of DNA into a higher-order structure. Transition proteins may become degraded through poly-ubiquitination and proteasome activity. Note that residual histones are not depicted.
To date, no ATP-dependent chromatin-remodelling complexes have been described in post-meiotic male germ cells. Nevertheless, in rat, the ATPase subunits of the ATP-dependent remodelling complex SWI/ SNF, Smarca2/Brm and Smarca4/Brg-1, are highly expressed in round spermatids. In addition, high expression levels of the SWI/SNF subunit Smarce1/BAF57 have been observed in round spermatids. The protein Spermatogenic cell HDAC-interacting protein 1 (SHIP1) is expressed in the nuclei of spermatocytes and round spermatids before hyperacetylation of histone H4, and SHIP1 is a component of a complex with chromatin-remodelling activity. Thus, SHIP1 and SWI/SNF chromatin-remodelling complexes might assist in chromatin reorganization during spermiogenesis. In addition, transition proteins themselves might help to remodel chromatin. However, in mice, apart from a putative role in histone removal, these remodelers may also aid in the global repression of gene transcription that occurs during this phase of spermiogenesis. Still, based on the currently available data, it appears most likely that a chromatin-remodelling complex might guide the replacement of histones by transition proteins during spermatid differentiation.The process of chromatin reorganization in spermatids most likely also requires mechanisms to incorporate histone variants, transition proteins, and protamines. In addition, mechanisms to remove histones and transition proteins have to exist.
Chromatin assembly is mediated by histone chaperones and ATP-utilizing motor proteins. Histone chaperones function in histone removal as well as histone exchange and that they team up with ATP-dependent chromatin-remodelling factors. Molecular chaperones are required to remove histones and at the same time aid in incorporating transition proteins and protamines into spermatid chromatin.After fertilization: back to histones
The fusion of the oocyte and the sperm entails resumption ofmeiosis of the oocyte and formation of the male pronucleus. Directly after the release of the sperm nucleus into the oocyte cytoplasm, protamines are quickly replaced by maternally supplied histones.
In addition, testis-specific histone variants rapidly disappear from the paternal genome after fertilization. The treatment of hamster eggs with antimycin A prevents paternal chromatin remodeling, which suggests that ATP-dependent processes are required for protamine replacement. It has been reported that in Xenopus laevis
, the chaperone nucleoplasmin (NPM) is involved in histone assembly onto the paternal chromatin. In mammals, NPM1, NPM2, and NPM3 members of the nucleoplasmin/nucleophosmin (NPM)
family are expressed in oocytes, and they regulate sperm chromatin decondensation.
Moreover, in NPM2-deficient mice, early embryogenesis is severely impaired. Additionally, the Drosophila proteins nucleosome assembly protein-1 (dNAP-1) and DF31 as well as the human Template activating factor I (TAF-I) have been shown to promote decondensation of sperm chromatin. In Drosophila, the maternal effect embryonic lethal mutation sésame (ssm) affects male pronucleus formation but not protamine removal. ssm is a point mutation in the Hira gene that encodes the H3.3 histone chaperone HIRA. HIRA is essential for decondensation of the sperm nucleus and nucleosome assembly
. Eggs laid by Hiradeficient females lose the paternal chromosomes and produce gynogenic haploid embryos.
Recently, it has been proposed that HIRA cooperates with Yemanuclein to establish the H3.3-containing nucleosome in the male nucleus at fertilization. Also in mouse zygotes, a strong enrichment of H3.3 as well as HIRA expression has been observed, which indicates that the function of HIRA in the protamine-to-histone transition may be conserved from flies to mammals
. Summary of events leading to the fusion of egg and sperm cell membranes in sea urchin fertilization, which is external.(1) The sperm is chemotactically attracted to and activated by the egg. (2, 3) Contact with the egg jelly triggers the acrosome reaction, allowing the acrosomal process to form and release proteolytic enzymes. (4) The sperm adheres to the vitelline envelope and lyses a hole in it. (5) The sperm adheres to the egg cell membrane and fuses with it. The sperm pronucleus can now enter the egg cytoplasm.
Two major mechanisms use species-specific sperm attraction and species-specific sperm activation.The acrosome reactionThe acrosome plays a crucial role in the recognition and penetration of the egg at the time of fertilization.
Globozoospermia is diagnosed when all spermatozoa are round-headed and lack acrosomes
. This defect is genetic in origin and originates in spermiogenesis, specifically during acrosome formation and sperm head elongation. The chromatin compaction appears to be disturbed. Affected males suffer from reduced fertility or even infertility.
In most marine invertebrates, the acrosome reaction has two components: the fusion of the acrosomal vesicle with the sperm cell membrane (an exocytosis that results in the release of the contents of the acrosomal vesicle), and the extension of the acrosomal process. The acrosome reaction in sea urchins is initiated by contact of the sperm with the egg jelly. Contact causes the exocytosis of the sperm’s acrosomal vesicle. The proteolytic enzymes and proteasomes (protein-digesting complexes) thus released digest a path through the jelly coat to the egg cell surface. Once the sperm reaches the egg surface, the acrosomal process adheres to the vitelline envelope and tethers the sperm to the egg. It is possible that proteasomes from the acrosome coat the acrosomal process, allowing it to digest the vitelline envelope at the point of attachment and proceed toward the egg. In sea urchins, the acrosome reaction is initiated by sulfate-containing polysaccharides in the egg jelly that bind to specific receptors located directly above the acrosomal vesicle on the sperm cell membrane. These polysaccharides are often highly speciesspecific, and egg jelly factors from one species of sea urchin generally fail to activate the acrosome reaction even in closely related species. Thus, activation of the acrosome reaction serves as a barrier to interspecies (and thus unviable) fertilizations. This is important when numerous species inhabit the same habitat and when their spawning seasons overlap.Sperm attraction: Action at a distance
Species-specific sperm attraction has been documented in numerous species, including cnidarians, mollusks, echinoderms, amphibians, and urochordates. In many species, sperm are attracted toward eggs of their species by chemotaxis—that is, by following a gradient of a chemical secreted by the egg. These oocytes control not only the type of sperm they attract, but also the time at which they attract them, releasing the chemotactic factor only after they reach maturation. The mechanisms of chemotaxis differ among species, and chemotactic molecules are different even in closely related species. In sea urchins, sperm motility is acquired only after the sperm are spawned. As long as sperm cells are in the testes, they cannot move because their internal pH is kept low (about pH 7.2) by the high concentrations of CO2 in the gonad. However, once sperm are spawned into seawater, their pH is elevated to about 7.6, resulting in the activation of the dynein ATPase. The splitting of ATP provides the energy for the flagella to wave, and the sperm begin swimming vigorously.a b
The acrosome is an organelle that develops over the anterior half of the head in the spermatozoa (sperm cells) of many animals including humans. It is a cap-like structure derived from the Golgi apparatus. Acrosome formation is fully completed 5–10 years after testicular maturation. In Eutherian mammals the acrosome contains digestive enzymes. These enzymes break down the outer membrane of the ovum, called the zona pellucida, allowing the haploid nucleus in the sperm cell to join with the haploid nucleus in the ovum.c
A germ cell is any biological cell that gives rise to the gametes of an organism that reproduces sexually. In many animals, the germ cells originate in the primitive streak and migrate via the gut of an embryo to the developing gonads. There, they undergo meiosis, followed by cellular differentiation into mature gametes, either eggs or sperm. 4e The spermatid is the haploid male gametid that results from division of secondary spermatocytes. As a result of meiosis, each spermatid contains only half of the genetic material present in the original primary spermatocyte. 5