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Reproductive health


S. Schorderet-Slatkine and *J. Huarte
Infertility and Gynecologic Endocrinology Clinic,
Department of Obstetrics and Gynecology,
University Cantonal Hospital, 1211 Geneva 14, Switzerland
*Department of Morphology, University Medical Centre,
1211 Geneva 4, Switzerland


The sperm and the ovum are highly specialized haploid cells, that are formed through a complex set of cell divisions, differentiation and maturation steps called gametogenesis. In mammals, the life history of germ cells begins during embryonic life with the extragonadal appearance of primordial germ cells and the colonization of the genital ridges, where germ cells associate with somatic cells; it continues with their multiplication, growth and maturation, and ends at fertilization. The oocyte undergoes a tremendous growth and stockpiles a large amount of macromolecules. In contrast, the spermatozoon is an extremely streamlined, highly polarized cell, containing only elements for essential functions such as motility and a few critical enzymes to ensure efficient transmission of the paternal genome to the oocyte at fertilization. The union of sperm and egg is an extraordinary cell fusion event that gives rise to an original individual and triggers a very sophisticated developmental program.

This chapter briefly reviews some aspects of gametogenesis and fertilization in mammals.


At the stage of spermatogonia and oogonia, germ cells multiply by mitosis, subsequently, they undergo meiosis to become the fully matured gametes. Meiosis involves two consecutive divisions with only one DNA replication cycle and results in the production of haploid gametes. The pairing of homologous chromosomes is unique to meiosis. The first meiotic division enhances genetic variability by independent assortment (random distribution) of the different maternal and paternal homologs and by crossing-over between homologous non sister chromatids. The second meiotic division resembles a normal mitosis without DNA replication. Meiosis is dominated by prophase of the first meiotic division, that occupies a long period and is divided into 5 sequential stages—leptotene, zygotene, pachytene, diplotene and diakinesis—defined by morphological criteria.

Development of the sperm

Spermatogonia develop from primordial germ cells that migrate into the undifferentiated gonad early in embryogenesis. In the wall of the forming seminiferous tubules two different kinds of cells are already clearly distinguishable at this stage: the supporting Sertoli cells, thought to derive from the surface epithelium of the genital ridge, and the spermatogonia, derived from primordial germ cells. During the fetal period, spermatogonia enter a dormant or arrested phase of development, and the Sertoli cells constitute most of the seminiferous epithelium. At sexual maturity, spermatogonia begin to increase in number. It is at this time that spermatogenesis really starts since this term usually refers to the sequence of events by which spermatogonia are transformed into spermatozoa. Spermatogenesis includes three main phases: spermatogonial multiplication, meiosis, and spermiogenesis. The cells at these different stages are called spermatogonia, spermatocytes and spermatids, respectively. In men spermatogonial multiplication occurs through regular intervals of 16 days. Spermatogonia can be divided in two main types, the noncycling ones (Ao), and those that will differentiate into spermatocytes after six mitotic divisions. Type (Ao) spermatogonia are able to repopulate the seminiferous epithelium when cycling spermatogonia decrease in number. The cycling spermatogonia provide the stem cell population for meiosis, which begins when preleptotene spermatocytes start DNA replication. Each primary spermatocyte, actually the largest germ cell in the tubules, undergoes the first meiotic division, forming two secondary spermatocytes that are about half the size of the primary spermatocyte. Subsequently, these two secondary spermatocytes undergo the second meiotic division, forming four haploid spermatids that are about half the size of secondary spermatocytes. The spermatids are gradually transformed into mature sperm by an extensive process of differentiation known as spermiogenesis; finally the differentiated sperm is released from the seminiferous epithelium and becomes a free spermatozoon, a process called spermiation. In human the process of spermatogenesis extends over a period of about 60 days.

An intriguing and unique feature of spermatogenesis is that the developing male germ cells fail to complete cytoplasmic division during mitosis and meiosis, so that all the daughter cells, except for the least differentiated spermatogonia remain connected by cytoplasmic bridges. These bridges persist until the very end of sperm differentiation. It has indeed been shown that sperm nuclei are haploid but sperm cell differentiation is directed by the diploid genome.

The sperm cell consists of two morphologically and functionally distinct regions. A head containing an unusually highly condensed haploid nucleus and a tail propelling the sperm to the egg helping to enter through the egg coat. The DNA in the nucleus is inactive and extremely tightly packed as a result of its association with highly positively charged proteins, the protamines, instead of histones, which have been displaced during spermiogenesis. The head also contains a membrane-limited organelle, the acrosome, whose contents are thought to have a function in the penetration of the spermatozoon into the ovum. A variety of enzymes, including proteinases, glycosidases, phosphatases, arylsulfatases and phospholipases are present in the acrosome and in the preacrosomal membrane.

Sperm released from the seminiferous epithelium are not capable of fertilization. The long series of changes that the spermatozoa endure between casting off from the Sertoli cells, and fusing with the egg, i. e. till the fully functional state of the spermatozoa, is referred to as sperm maturation. Throughout their journey from testis to the proximity of the ovum, sperm cells are suspended in transudations and secretions of the male and female genital tracts. The chemical and physical nature of this medium progressively changes and the spermatozoa also change structurally, chemically and behaviourally. Several proteins from testicular and epididymal environment have been shown to bind to specific regions of the sperm surface that are involved in sperm maturation and in part of the gamete recognition process. Biochemical modifications of some sperm surface components are also involved, as well as an increase in interchromatin disulfide bonds for chromatin condensation during this travel which lasts several days. Sperm cells develop gradual motility and ability to bind and penetrate eggs as they progress from the caput to the cauda epididymidis.

Ejaculates contain complex secretions from the accessory glands—the Cowper’s gland, prostate, and seminal vesicles—which, contain a variety of energy substrates, hormones, nonenzymatic and enzymatic proteins and various ions. The last step of sperm cell maturation is called capacitation, which is a functional term used to indicate the changes in mammalian spermatozoa that must occur in the female genital tract, or during in vitro incubations, as preparation for the acrosome reaction (see below). Capacitation is a reversible reaction which does not involve morphological changes; it is accompanied by a hyperactivation of sperm motility: the flagellar beat pattern changes from a low amplitude favoring progressive motility to a high amplitude with little progression. Capacitation includes a lowering of the cholesterol/phospholipid ratio in the sperm membrane, a loss of sperm surface coating components (loss of the antifertility factor from human seminal plasma) probably involved with the acquisition of zona pellucida binding activity, and the phosphorylation of some plasma membrane proteins.

Development of the egg

The unfertilized egg is the end product of a discontinuous course called oogenesis, that begins during fetal development and ends in the sexually mature adult. Oogonia develop from primordial germ cells in the ovary, and multiply by mitosis only during the fetal life. By the 5th month of gestation in women, all germ cells stop proliferation and enter meiosis but pause at the prophase of the first meiotic division; arrest may last from 12 to 50 years. The spherical dictyate oocytes become enclosed within a few squamous somatic cells to form what is called primordial follicles; the oocytes are then called primordial oocytes. It is in this period of life that the ovary contains the highest number of oocytes—about one to two millions—since many of them will degenerate before puberty and through the reproductive life of a woman. At puberty only about 300’000 primary oocytes remain. They represent a stockpile from which a few are selected at any given time for development towards preovulatory follicles containing fully grown oocytes. The oocyte and its surrounding follicle grow coordinately, rather than simultaneously. Indeed, the oocyte completes its growth before the formation of the follicular antrum, i.e., the major part of follicular growth occurs after the oocyte has stopped growing. The oocyte growth results in the formation of one of the largest cells in the body. During this period its volume increases more than 300-fold; from a diameter of about 20 µm at the primordial stage, the oocyte reaches a maximal diameter of about 120 µm. Completion of growth takes approximately 2.5-3 months. The nucleus of the growing oocyte, called the germinal vesicle, is particularly apparent and contains a very refractile nucleolus. During oocyte growth an extracellular coat develops around the plasma membrane. This acellular layer, called the zona pellucida (ZP), is constituted by three major glycoproteins (ZP1, ZP2 and ZP3) that are assembled into long, interconnected filaments to form a relatively porous coat about 5 µm thick.

From the time of puberty, one developing follicle is stimulated each month to mature to complete development and to ovulate. This means that during the approximately 40 years of a woman’s reproductive life, only 400 to 500 eggs will have been released. All the rest will have degenerated. The LH surge released by the pituitary will, each month, activate one antral follicle to mature. Fully grown primary oocytes enclosed in Graafian follicles resume meiosis just prior to ovulation. This phase is called meiotic maturation. The first macroscopically observable event of meiotic maturation is the dissolution of the nuclear membrane; this process is referred to as germinal vesicle breakdown or GVBD. The oocyte then progresses through metaphase, anaphase, and telophase of the first division, emits the first polar body, and, without stopping, enters the second division up to metaphase. It is around this time that ovulation occurs, by rupture of the follicle wall at the surface of the ovary. In the oviduct, the oocyte remains at metaphase II until it is triggered by fertilization to complete the second meiotic division.

Gamete interaction

In comparison to the large number of spermatozoa laid down in the vagina at coitus, only very few sperm cells reach the ampulla and are found in the proximity of the egg. Although sperm attraction to follicular factor(s) has been claimed, sperm chemotaxis in mammalian fertilization has not been demonstrated. The leading role in the sperm-egg encounter is played by the molecular organization of their surfaces, and abundant evidence suggests that the species-specific gamete recognition and binding is mediated by receptor molecules at the gamete surface.

Initial contact between gametes occurs when the sperm attach to the unfertilized extracellular coat or zona pellucida. Capacitated, acrosome-intact sperm are capable of binding to the zona pellucida via the plasma membrane of the sperm head. Binding is an important prerequisite step for zona penetration because it initiates events that culminate in induction of the acrosome reaction.

One of the components of the zona pellucida (ZP3) representing the primary sperm receptor, is responsible for both the sperm-binding activity and the ability to induce a complete acrosome reaction. Acrosome-intact sperm bind to ZP3 in a relatively species specific manner, this gamete recognition and binding is mediated by carbohydrates and not by the polypeptide chain. Many sperm are released from the zona pellucida after undergoing the acrosome reaction, yet maintenance of sperm binding is achieved by interaction of acrosome-reacted sperm with ZP2; thus, ZP2 serves as a secondary receptor.

Putative ZP-binding-glycoproteins of spermatozoa have been recognized in various species. Several egg-binding proteins are envisaged on the sperm membrane that impart species specificity. The postulated candidates are the following: a 95kDa protein (p95 SPERM) showing a tyrosine kinase activity that is stimulated on binding and whose activation requires aggregation, a 56kDa protein (p56) of unknown function, an antigen designated p200/220 (whose monoclonal antibody is named M42) necessary for zona-induced acrosomal reaction, another related antigen the SAA-1 antigen detected on all mammalian sperm acrosomes, a ß-1,4-galactosyl-transferase mediating fertilization by binding oligosaccharide residues on zona pellucida glycoprotein. Many evidence suggest also the possible involvement of protease inhibitor sites and mannosidase sites or of other molecules called spermadhesins showing features of serine proteases having lectin-like activity.

Proteolytic enzymes appear to participate in multiple phases of mammalian fertilization, including acrosome reaction, sperm binding to zona pellucida (ZP), ZP penetration and zona reaction, however, the enzymes involved have not been completely identified. A role for sperm proacrosin and acrosin, the best characterized sperm protease, in ZP binding and penetration has been postulated. Several observations suggest that the plasminogen activator/plasmin system might play a role in mammalian fertilization. First, both mouse gametes express plasminogen-dependent proteolytic activities: ovulated eggs contain and secrete tissue-type PA (t-PA) and ejaculated spermatozoa exhibit urokinase-type PA (uPA). Second, t-PA is significantly higher in follicular fluids and granulosa cells from follicles containing oocytes that can be fertilized in vitro compared to follicles containing oocytes that fail to fertilize. Third, the addition of plasminogen to the fertilization medium increases the frequency of eggs fertilized in vitro.

Sperm cells must undergo the acrosome reaction before they can penetrate the zona pellucida and fuse with the egg plasma membrane. Acrosome reaction progresses from multiple fusion-points between the plasma and outer acrosomal membranes, which expose the inner acrosomal membrane and the acrosomal contents (enzymes), to complete vesiculation and loss of the integrity of the acrosome. The acrosome reaction bears a strong resemblance to ligand-mediated exocytotic reactions in somatic cells proceeding through an intracellular signal transduction system, it involves the participation of a Gi protein, of phospholipase C and of protein kinase C. In addition, an increase in intracellular calcium is concomitant with the induction of acrosomal loss. Acrosome reaction can be induced by biological agents such as follicular fluid (progesterone), cumulus cells or zonae pellucidae or by physiochemical agents such as calcium ionophores, lysophosphatidylcholine and electropermeabilization or by the aggregation of zona binding sites on the sperm heads.

Sperm-egg fusion

After sperm entry into the perivitelline space, the final stages of sperm-egg interaction include the binding and fusion of the sperm and egg plasma membranes, and entry of the sperm into the egg. Sperm binding to the egg surface occurs on the lateral face of the head, with the firm point of attachment between the sperm and egg plasma membranes occurring at the equatorial segment. Little is known concerning the sperm and egg surface complementary molecules (binding sites) that participate in gamete plasma membranes fusion in mammals. It has been recently shown that a sperm surface protein (PH-30, a guinea-pig sperm antigen), known to be involved in sperm-egg fusion, shares biochemical characteristics with viral fusion proteins and contains an integrin ligand domain. These results suggest that an integrin-mediated adhesion event takes place and leads to fusion.

Fusion of a single sperm sets in motion a series of egg reactions to prevent additional sperm entry, thus avoiding the lethal consequences of polyspermy. Egg cortical reaction takes place soon after fusion, causing the zona pellucida to become " hardened " and refractory to both binding and penetration of supernumerary sperm. Zona binding is prevented by the inactivation of the sperm primary receptor (and acrosome inducer) ZP3 and zona penetration is stopped through modification of the sperm secondary receptor ZP2. The cortical reaction involves the exocytosis of cortical granules and the release of their enzymatic content into the perivitelline space. The oligosaccharides of ZP3 responsible for gamete recognition and adhesion are modified by cortical granule glycosidase(s) and the glycoprotein ZP2 undergoes limited proteolysis making the zona pellucida more insoluble and " hardened ", preventing the maintenance of binding of acrosome-reacted sperm to the zona pellucida. It has been suggested that the oocyte plasminogen activator may participate in this proteolytic process although the evidence is poor.

Egg activation and pronuclei formation

Gamete fusion triggers responses within the egg that culminate in the activation of the embryonic developmental program. Activation may also be induced parthenogenetically under various physical or chemical stimuli, in all cases, calcium is an obligatory mediator. In mammals, sperm may cause both a persistent production of inositol trisphosphate (InsP3) and an increase in calcium permeability of the plasma membrane to maintain internal calcium oscillations. The early calcium increase induces cortical granule exocytosis (cortical reaction), which involves a signal transduction system that is similar to that of somatic cells, and that leads to the hardening of the zona pellucida. Activation leads to the resumption of the cell cycle: the second meiotic division is achieved, by the extrusion of the second polar body and the egg enters into interphase with formation of pronuclei. Pronuclear formation takes place a few hours after fertilization, and requires a calcium increase and a cytoplasmic alcalinization of the zygote. Following anaphase II, the egg chromosomes remaining in the cytoplasm disperse and the female pronucleus forms. Similarly, after cell fusion, the sperm nucleus is decondensed and transformed into a male pronucleus. The biochemical transitions responsible for the remodelling of the sperm nucleus consist of changes in the majority of sperm specific chromatin proteins and the acquisition of chromosomal proteins which induce a chromatin conformation compatible with fusion of male and female pronuclei. Maternal chromatin and sperm pronuclear development are regulated by common egg cytoplasmic factors involved in the regulation of the cell cycle and dependent on oocyte maturation. The pronuclear development in fertilized eggs is known to proceed through a series of transformations, which restore the transcriptional competence of the inactive gamete chromatin and re-establish the functional diploid genome of the embryo. Two stages of decondensation are observed: i) a very rapid chromatin expansion dependent on egg nucleoplasmin, and ii) a slow membrane-dependent decondensation involving protein migration into the nucleus reliant on nuclear envelope formation recruited from maternal pool.

The two pronuclei move towards the egg center, and spermaster increases in size during their migration. The end result of the migration of the pronuclei is their juxtaposition, following pronuclear envelope breakdown, giving rise to a group of chromosomes for the ensuing division. The spatial organization of microtubule arrays in a cell is largely dependent on organizing centers, the centrosomes. The proximal centriole of the sperm and its centrosomal material between apposed pronuclei are involved in the fertilization events. Human centrioles as those of other animals except the mouse are paternally derived. Eventually, there is an intermixing of the maternally and paternally derived chromosomes to establish the genome of the embryo and hence the process of fertilization can be considered as concluded.


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