Chapter 7:  Structure of the Gametes

Sperm

It is only within the past century that the sperm’s role in fertilization has been known. Anton van Leeuwenhoek, the Dutch microscopist who co-discovered sperm in 1678, first believed them to be parasitic animals living within the semen (hence the term spermatozoa, meaning “sperm animals”). He originally assumed that they had nothing at all to do with reproducing the organism in which they were found, but he later came to believe that each sperm contained a preformed embryo. Leeuwenhoek (1685) wrote that sperm were seeds (both sperma and semen mean “seed”) and that the female merely provided the nutrient soil in which the seeds were planted. In this, he was returning to a notion of procreation promulgated by Aristotle 2000 years earlier. Try as he might, Leeuwenhoek was continually disappointed in his attempts to find the preformed embryo within the spermatozoa. Nicolas Hartsoeker, the other co-discoverer of sperm, drew a picture of what he hoped to find: a preformed human (“homunculus”) within the human sperm (Figure 7.1). This belief that the sperm contained the entire embryonic organism never gained much acceptance, as it implied an enormous waste of potential life. Most investigators regarded the sperm as unimportant. (See Pinto-Correia 1997 for details of this remarkable story.)

The first evidence suggesting the importance of sperm in reproduction came from a series of experiments performed by Lazzaro Spallanzani in the late 1700s. Spallanzani demonstrated that filtered toad semen devoid of sperm would not fertilize eggs. He concluded, however, that the viscous fluid retained by the filter paper, and not the sperm, was the agent of fertilization. He, like many others, felt that the spermatic “animals” were parasites.

The combination of better microscopic lenses and the cell theory led to a new appreciation of spermatic function. In 1824, J. L. Prevost and J. B. Dumas claimed that sperm were not parasites, but rather the active agents of fertilization. They noted the universal existence of sperm in sexually mature males and their absence in immature and aged individuals. These observations, coupled with the known absence of spermatozoa in the sterile mule, convinced them that “there exists an intimate relation between their presence in the organs and the fecundating capacity of the animal.” They proposed that the sperm entered the egg and contributed materially to the next generation.

These claims were largely disregarded until the 1840s, when A. von Kolliker described the formation of sperm from cells within the adult testes. He ridiculed the idea that the semen could be normal and yet support such an enormous number of parasites. Even so, von Kolliker denied that there was any physical contact between sperm and egg. He believed that the sperm excited the egg to develop, much as a magnet communicates its presence to iron. It was only in 1876 that Oscar Hertwig and Herman Fol independently demonstrated sperm entry into the egg and the union of the two cells’ nuclei. Hertwig had sought an organism suitable for detailed microscopic observations, and he found that the Mediterranean sea urchin, Toxopneustes lividus, was perfect. Not only was it common throughout the region and sexually mature throughout most of the year, but its eggs were available in large numbers and were transparent even at high magnifications. After mixing sperm and egg suspensions together, Hertwig repeatedly observed a sperm entering an egg and saw the two nuclei unite. He also noted that only one sperm was seen to enter each egg, and that all the nuclei of the embryo were derived from the fused nucleus created at fertilization. Fol made similar observations and detailed the mechanism of sperm entry. Fertilization was at last recognized as the union of sperm and egg, and the union of sea urchin gametes remains one of the best-studied examples of fertilization.

Each sperm 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. Most of the sperm’s cytoplasm is eliminated during maturation, leaving only certain organelles that are modified for spermatic function (Figure 7.2). During the course of sperm maturation, the haploid nucleus becomes very streamlined, and its DNA becomes tightly compressed. In front of this compressed haploid nucleus lies the acrosomal vesicle, or acrosome, which is derived from the Golgi apparatus and contains enzymes that digest proteins and complex sugars; thus, it can be considered a modified secretory vesicle. These stored enzymes are used to lyse the outer coverings of the egg. In many species, such as sea urchins, a region of globular actin molecules lies between the 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 several other species, recognition between sperm and egg involves molecules on the acrosomal process. Together, the acrosome and nucleus constitute the head of the sperm.

The means by which sperm are propelled vary according to how the species has adapted to environmental conditions. In some species (such as the parasitic roundworm Ascaris), the sperm travel by the amoeboid motion of lamellipodial extensions of the cell membrane. In most species, however, each sperm is able to travel long distances by whipping its flagellum. Flagella are complex structures. The major motor portion of the flagellum is called the axoneme. It is formed by microtubules emanating from the centriole at the base of the sperm nucleus (Figures 7.2 and 7.3). The core of the axoneme consists of two central microtubules surrounded by a row of nine doublet microtubules. Actually, only one microtubule of each doublet is complete, having 13 protofilaments; the other is C-shaped and has only 11 protofilaments (Figure 7.3B). A three-dimensional model of a complete microtubule is shown in Figure 7.3C. Here we can see the 13 interconnected protofilaments, which 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 that is attached to the microtubules (Figure 7.3B). Dynein hydrolyzes molecules of ATP and can convert the released chemical energy into the mechanical energy that propels the sperm. This energy allows the active sliding of the outer doublet microtubules, causing the flagellum to bend (Ogawa et al. 1977; Shinyoji et al. 1998). The importance of dynein can be seen in individuals with the genetic syndrome called the Kartagener triad. These individuals lack dynein on all their ciliated and flagellated cells, rendering these structures immotile. Males with this disease are sterile (immotile sperm), are susceptible to bronchial infections (immotile respiratory cilia), and have a 50% chance of having the heart on the right side of the body (Afzelius 1976). Another important flagellar protein appears to be histone H1. This protein is usually found inside the nucleus, where it folds the chromatin into tight clusters. However, Multigner and colleagues (1992) found that this same protein stabilizes the flagellar microtubules so that they do not disassemble.

The “9 + 2” microtubule arrangement with the dynein arms has been conserved in axonemes throughout the eukaryotic kingdoms, suggesting that this arrangement is extremely well suited for transmitting energy for movement. The ATP needed to whip the flagellum and propel the sperm comes from rings of mitochondria located in the neck region of the sperm (see Figure 7.2). In many species (notably mammals), a layer of dense fibers has interposed itself between the mitochondrial sheath and the axoneme. 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 has undergone extensive modification for the transport of its nucleus to the egg.

The differentiation of mammalian sperm is not completed in the testes. After being expelled into the lumen of the seminiferous tubules, the sperm are stored in the epididymis, where they acquire the ability to move. Motility is achieved through changes in the ATP-generating system (possibly through modification of dynein) as well as changes in the plasma membrane that make it more fluid (Yanagimachi 1994). The sperm released during ejaculation are able to move, yet they do not yet have the capacity to bind to and fertilize an egg. These final stages of sperm maturation (called capacitation) do not occur until the sperm has been inside the female reproductive tract for a certain period of time.

The egg

All the material necessary for the beginning of growth and development must be stored in the mature egg (the ovum). Whereas the sperm has eliminated most of its cytoplasm, the developing egg (called the oocyte before it reaches the stage of meiosis at which it is fertilized) not only conserves its material, but is actively involved in accumulating more. The meiotic divisions that form the oocyte conserve its cytoplasm (rather than giving half of it away), and the oocyte either synthesizes or absorbs proteins, such as yolk, that act as food reservoirs for the developing embryo. Thus, birds’ eggs are enormous single cells, swollen with their accumulated yolk. Even eggs with relatively sparse yolk are comparatively large. The volume of a sea urchin egg is about 200 picoliters (2 × 10^–4 mm³, more than 10,000 times the volume of the sperm) (Figure 7.4). So, while sperm and egg have equal haploid nuclear components, the egg also has a remarkable cytoplasmic storehouse that it has accumulated during its maturation. This cytoplasmic trove includes the following:^*

• Proteins. It will be a long while before the embryo is able to feed itself or obtain food from its mother. The early embryonic cells need a supply of energy and amino acids. In many species, this is accomplished by accumulating yolk proteins in the egg. Many of the yolk proteins are made in other organs (liver, fat body) and travel through the maternal blood to the egg.

• Ribosomes and tRNA. The early embryo needs to make many of its own proteins, and in some species, there is a burst of protein synthesis soon after fertilization. Protein synthesis is accomplished by ribosomes and tRNA, which exist in the egg. The developing egg has special mechanisms to synthesize ribosomes, and certain amphibian oocytes produce as many as 10¹² ribosomes during their meiotic prophase.

• Messenger RNA. In most organisms, the instructions for proteins made during early development are already packaged in the oocyte. It is estimated that the eggs of sea urchins contain 25,000 to 50,000 different types of mRNA. This mRNA, however, remains dormant until after fertilization (see Chapter 5).

• Morphogenetic factors. Molecules that direct the differentiation of cells into certain cell types are present in the egg. They appear to be localized in different regions of the egg and become segregated into different cells during cleavage (see Chapter 8).

• Protective chemicals. The embryo cannot run away from predators or move to a safer environment, so it must come equipped to deal with threats. Many eggs contain ultraviolet filters and DNA repair enzymes that protect them from sunlight; some eggs contain molecules that potential predators find distasteful; and the yolk of bird eggs even contains antibodies.

Within this enormous volume of cytoplasm resides a large nucleus. In some species (e.g., sea urchins), the nucleus is already haploid at the time of fertilization. In other species (including many worms and most mammals), the egg nucleus is still diploid, and the sperm enters before the meiotic divisions are completed. The stage of the egg nucleus at the time of sperm entry in different species is illustrated in Figure 7.5.

Enclosing the cytoplasm is the egg plasma membrane. This membrane must regulate the flow of certain ions during fertilization and must be capable of fusing with the sperm plasma membrane. Outside the plasma membrane is the vitelline envelope (Figure 7.6), which forms a fibrous mat around the egg. This envelope contains at least eight different glycoproteins and is often involved in sperm-egg recognition (Correia and Carroll 1997). It is supplemented by extensions of membrane glycoproteins from the plasma membrane and by proteinaceous vitelline posts that adhere the vitelline envelope to the membrane (Mozingo and Chandler 1991). The vitelline envelope is essential for the species-specific binding of sperm. In mammals, the vitelline envelope is a separate and thick extracellular matrix called the zona pellucida. The mammalian egg is also surrounded by a layer of cells called the cumulus (Figure 7.7), which is made up of the ovarian follicular cells that were nurturing the egg at the time of its release from the ovary. Mammalian sperm have to get past these cells to fertilize the egg. The innermost layer of cumulus cells, immediately adjacent to the zona pellucida, is called the corona radiata.

Lying immediately beneath the plasma membrane of the egg is a thin shell (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 known as microfilaments. Microfilaments are necessary for cell division, and they also are used to extend the egg surface into small projections called microvilli, which may aid sperm entry into the cell (see Figure 7.6B; also see Figure 7.19). Also within the cortex are the cortical granules (see Figures 7.4 and 7.6B). These membrane-bound structures, which are homologous to the acrosomal vesicle of the sperm, are Golgi-derived organelles containing proteolytic enzymes. However, whereas each sperm contains one acrosomal vesicle, each sea urchin egg contains approximately 15,000 cortical granules. Moreover, in addition to digestive enzymes, the cortical granules contain mucopolysaccharides, adhesive glycoproteins, and hyalin protein. The enzymes and mucopolysaccharides are active in preventing other sperm from entering the egg after the first sperm has entered, and the hyalin and adhesive glycoproteins surround the early embryo and provide support for the cleavage-stage blastomeres.

Many types of eggs also have an egg jelly outside the vitelline envelope (see Figure 7.4). This glycoprotein meshwork can have numerous functions, but most commonly is used either to attract or to activate sperm. The egg, then, is a cell specialized for receiving sperm and initiating development.