After ejaculation, sperm are transported through the female reproductive tract. Most sperm cells are completely matured in the epididymis, while a few may still have a cytoplasmic droplet, particularly in subfertile men (Figures 4.1-4.3).
Sperm undergo the acrosome reaction before fertilization in order to penetrate the zona pellucida (ZP) of the oocyte. This process occurs following binding to the ZP only if the sperm have previously undergone a maturation process called capacitation. Binding of the capacitated sperm to the oocyte ZP activates a low-voltage Ca2+ channel and a store- operated Ca2+ channel, which causes a sustained elevation in the concentration of intracellular Ca2+ leading to the acrosome reaction (Tables 4.1 and 4.2). The priming of sperm to such calcium signals during capacitation involves many changes, including cholesterol efflux from the plasma membrane and increases in intracellular free Ca2+ cyclic adenosine monophosphate (cAMP), pH, protein tyrosine phosphorylation and actin polymerization (Aitken et al., 1998;Arnoult et al., 1999; de Lamirande et al., 1997; Galantino-Homer et al., 1997; Yanagamachi, 1994).
Reactive oxygen species (ROS) such as hydrogen peroxide and superoxide anions are involved in the regulation of sperm capacitation and protein tyrosine phosphorylation. Hydrogen peroxide is also involved in actin polymerization in bovine sperm and in the elevation of intracellular Ca2+ levels and fertilizing potential of sperm. cAMP and protein kinase A are regulators of protein tyrosine phosphorylation. ROS generation must lie upstream from cAMP in the reaction cascade, because ROS are located downstream from cAMP in the reaction sequence. The soluble adenylyl cyclase (AC) present in sperm is activated by HCO3. Relatively low concentrations of hydrogen peroxide are beneficial for sperm capacitation; however, too high a concentration inhibits the process. Hydrogen peroxide activates adenylyl cyclase to produce cAMP, leading to protein kinase A-dependent protein tyrosine phosphorylation (Aitken et al., 1998; Chen et al., 2000; Lecleere et al., 1998; O’Toole et al., 2000; Pariaud and Milhet, 1996; Rivlin et al., 2004).
Figure 4.1 Ejaculated semen is composed of, in addition to a small amount of testicular fluid, contributions from several accessory organs including the epididymis (Caput-corpus epid and Cauda epid), ampullary glands (AMP), vesicular glands, prostate and bulbourethral glands
Figure 4.2 Sperm are suspended in various luminal fluids from the time of sperm production in the seminiferous tubules until fertilization in the oviduct
Figure 4.3 Relationships between the retention of excess residual cytoplasm, oxidative stress and defective sperm function are shown. The presence of glucose-6-phosphate dehydrogenase in excess cytoplasm generates NADPH, which acts as a substrate to produce superoxide anions, which dismutate to hydrogen peroxide causing cell damage. From Aitken et al., 1994, with permission
Sperm capacitation
Sperm acquire fertilizability after residing in the female tract for some time. Capacitation involves several physiological, biochemical and molecular changes involved in the removal or alteration of a stabilizer or protective coat from the sperm plasma membrane; this is under autonomic nerve and hormonal controls. Capacitation involves various parameters:
(1) Sperm are found in close contact with epithelial cells of the isthmus and uterotubal junction.
(2) Most capacitation occurs in the lower segment of the isthmus where fertilizing sperm are stored; capacitation within the isthmus progresses faster when females mate after ovulation rather than before ovulation.
(3) Capacitation is facilitated by ‘rubbing off’ sperm surface-absorbed materials (including seminal plasma proteins) against cervical mucus.
(4) Specificity/specialty of female tract: sperm are fully capacitated in the uterus without ascending the oviduct. Successful pregnancies occur following gamete intrafallopian transfer (GIFT) or direct intraperitoneal insemination (DIPI); capacitation is possible in the peritoneal cavity and/or ampulla. Capacitation is not strictly organ specific. Sperm adhering to the epithelium of the isthmus undergo capacitation slowly.
(5) In vitro sperm capacitation (IVC) and in vitro fertilization (IVF) are routinely applied using several commercially available tissue culture media or in modified Tyrode’s/Krebs-Ringer solutions supplemented with energy sources (e.g. glucose, lactate and pyruvate) and albumin (e.g. Ham’s F10) supplemented with blood serum. Serum is also commonly used, particularly for human IVF.
Sperm capacitation is associated with modifications in plasma membrane composition and fluidity, changes in intracellular ion concentrations, an increase in protein tyrosine phosphorylation and alterations in the oxidative metabolism. Changes at the flagellum enable sperm to develop hyperactivated motility. Modifications on the sperm head allow the initiation of the acrosome reaction (AR) in response to adequate stimuli such as the ZP or follicular fluid components. Different steps of the fertilization process have distinctive Ca2+ requirements. Whereas 0.22 mmol/l of Ca2+ is sufficient for the development of some capacitation-related events, human follicular fluid-induced AR and sperm-ZP interaction require 0.58 mmol/l of this cation (Marin-Briggiler et al., 2003).
Ultrastructural and biochemical parameters
Several ultrastructural and biochemical changes occur during capacitation of mammalian sperm:
(1) Antigens change their distribution, particularly fibronectin-like molecules, on the sperm surface;
(2) Some glycoproteins or proteins are released from sperm surfaces;
(3) Changes occur in the lectin-binding ability of the sperm plasma membrane;
(4) Intramembranous particles (IMPs), intrinsic proteins within lipid bilayers, change their distribution in both the head and tail regions. IMPs are evenly distributed in the plasma membrane over the acrosome. The membrane of capacitated sperm has many small patches free of IMPs.
Uncapacitated sperm treated with filipin contain densely and evenly distributed complexes in the plasma membranes of the acrosomal region. The sperm plasma membrane undergoes several complex changes in preparation for fertilization. Sperm plasma membrane is ‘biologically frozen’ when sperm leave the male’s body and its ‘defrosting’ represents capacitation.
Sperm hyperactivation
Sperm hyperactivation is a necessary step in the capacitation sequence. Sperm motility is measured by videotape to evaluate the straight line velocity (μm/s) (VSL). Fertilization, and subsequent embryonic development, result from single-sperm transfer (S-MIST) and multiple-sperm transfer.
Fertilization, and subsequent embryo development, is achieved by injection of immotile sperm into the perivitelline space. To assess sperm viability, a survival stain suitable for use in combination with immunofluorescent assay, Hoechst 33258, is used. The dye fluoresces an intense blue when bound to DNA. To assess sperm plasma membrane integrity, a hypo-osmotic swelling test (HOST) is performed, using fluoresceinated D-mannose- enriched albumin (FITC-DMA). The ability of sperm to swell under hypo-osmotic conditions indicates an intact membrane.
Biochemical markers of sperm quality
Several biochemical markers have been used to evaluate human sperm maturity and function including sperm creatine-N-phosphotransferase or creatine kinase (CK), CK enzymatic active-site labeling and CK immunocytochemistry of individual sperm; men with low sperm concentrations who have increased numbers of individual sperm, and men with low sperm concentrations who have increased incidence of infertility, also show increased levels of sperm CK activity. This may be related to a defect of sperm development in the last phase of spermatogenesis, when excess cytoplasm is normally extruded and left in the adluminal area as residual bodies. Thus, sperm with a high CK content, an indication of surplus cytoplasm, have not completed cellular maturation (Huszar et al., 2003). There is also a second sperm maturation marker, which was initially thought to be a sperm-specific creatine kinase M-isoform and identified as the testis-expressed chaperone protein HspA2 (a member of the HSP70-2 family), that, along with the homologous hsc70, is synthesized in two waves of expression. The first wave occurs during meiosis, as HspA2 is part of the synaptonemal complex. The second wave of major expression occurs simultaneously with cytoplasm extrusion, in terminal spermatogenesis.
THE ACROSOME AND ACROSOME REACTION
The acrosome consists of an anteriorly located acrosomal cap and posteriorly located equatorial segment. The acrosomal cap is endowed with a variety of hydrolyzing enzymes, whereas the equatorial segment is enzymatically ‘empty’. Hyaluronidase and acrosin are acrosomal enzymes that have been extensively studied and characterized. The acrosome contains several powerful hydrolyzing enzymes. Structural proteins maintain acrosome shape and the positioning of different functional components within the acrosomal matrix; acrosome organization is required to ensure release of acrosome components in a precise sequence so that sperm can traverse the egg vestments (Figures 4.4-4.12).
Figure 4.4 Fertilization sequence: interaction of the zona pel- lucida (ZP) and its receptor on the sperm head leads to the acrosomal reaction (AR) and to the subsequent enzymatic attack of the ZP. The sperm penetrates the ZP and reaches the perivitelline space (PS). The oocyte and sperm membranes fuse and fertilization takes place. The cortical reaction (CR) prevents polyspermia.The second meiotic division of the oocyte is completed and the second polar body (II PB) is extruded. Upon fusion, the two pronuclei (Pn) give rise to the formation of the mitotic spindle (MS) and to the first cleavage. I PB,first polar body
Figure 4.5 Sperm-egg interaction
Figure 4.6 Signaling proteins expressed in sperm. Several proteins involved in signal transduction processes in somatic cells are also expressed in sperm.Their differential expression in the acrosomal compartment, equatorial region, postacrosomal region, and middle, main and end pieces of the sperm tail is shown.Those signaling components the expression of which is postulated but not proven or the exact localization of which is not known are labeled by a question mark. AT1R, AT1 angiotensin receptor;AT2R, AT2 angiotensin receptor;CNG channels, cyclic nucleotide-gated channels;EGFR, epidermal growth factor receptor; Ga, a subunit of heterotrimeric guanine nucleotide- binding protein;Gp, β subunit of heterotrimeric guanine nucleotide-binding protein;GnRH-R, gonadotropin-releasing hormone receptor; GRK, G-protein-coupled receptor kinase;IFN-Y-R, interferon-Y-receptor; IGF-1-R, insulin-like growth factor-1 receptor; IP3R, inositol triphosphate receptor; Kir3.2,a G-protein-gated inwardly rectifying K+ channel subunit;PGE, prostaglandin E;PKC, protein kinase C;PLA2, phospholipase A2; PLC, phospholipase C;TRP channels, transient receptor potential channels; ZP3, zona pellucida protein 3
The acrosome reaction involves the following parameters:
(1) To fertilize eggs, sperm must be highly motile as well as capable of undergoing the acrosome reaction (AR), penetrating through egg investments and fusing with the egg.
(2) AR acts as in indicator of completed capaci- tation because sperm do not undergo AR either spontaneously or mediated by ligands (e.g. zona pellucida) unless they become capacitated.
(3) Massive influx of Ca2+ occurs during AR. Concentration of intracellular CA2+ in sperm is low, in both head and tail regions, because of the presence of ATPase-mediated Ca2+ antiporter, and a Ca2+/H+ exchange system in plasma membrane.
(4) Carbohydrate is a distinct component of the acrosomal matrix. A glycoprotein layer covering the inner surface of the outer acrosomal membrane holds vesiculated (fenestrated) plasma and outer acrosomal membranes together during the acrosome reaction.
(5) The form of acrosome does not change during capacitation; enzymatically inactive proacrosin in the sperm acrosome is converted to enzymatically active acrosin by glycosaminoglycans in uterine fluid.
(6) Function of AR: eggs are surrounded by lipoprotein coats through which sperm must
Figure 4.7 Acrosome reaction in mammalian sperm. (a) Intact plasma and acrosomal membrane of an unreacted spermatozoon. (b) Initiation of the acrosome reaction showing multiple fusion points between the plasma membrane and outer acrosomal membrane. Fusion leaves the appearance of numerous vesicles over the cell surface. Note that fusion is absent in the equatorial and postacrosomal regions. Acrosomal enzymes involved with ovum penetration are released and exposed on the inner acrosomal membrane. (c) The acrosome swells and is eventually lost during penetration of the zona pellucida, leaving only the inner acrosomal membrane exposed on the upper portion of the sperm head. From Saacke and White (1972), with permission
Figure 4.8 The protein ZP3 in the zona pellucida is the sperm receptor. Exposure of sperm to unfertilized ova results in sperm binding to ZP3 (a). In the competition assay (b), sperm are incubated with ZP3 first and then exposed to ova; however,the sperm are unable to bind to the ZP3 proteins on the zona pellucida because their P3 binding proteins are already occupied (blocked) by the free ZP3 to which the sperm were exposed before introduction of ova. From Wassarman (1988), with permission
Figure 4.9 Interaction between sperm receptors and metabolite from oviduct epithelium during fertilization. S, sperm; C, kinocilia which mix sperm with oviduct secretions; Bb, basal bodies of kinocilia for locomotion; N, nucleus; G, Golgi apparatus; Bm, basal membrane of epithelium; Mr, microvilli; Sq, secretory granules; Mi, mitochondria
Figure 4.10 Scanning electron microscopy of capacitated sperm at site of fertilization
Figure 4.11 Sequence of events of sperm-oocyte interaction: penetration of cumulus and zona pellucida, ion exchange, pronuclei formation, DNA synthesis, merging haploid sets of chrosomes and cleavage
Figure 4.12 Time sequence of ejaculation, sperm migration, sperm release from reservoirs, penetration of zona pellucida and fertile lives of sperm and egg
pass before reaching the egg plasma membrane. Acrosome-reacted sperm dissolve the coat locally to produce a ‘hole’ through which the sperm swim. The outer acrosomal membrane overlying the plasma membrane is partially or totally destroyed, or becomes detached from the main body of the sperm.
Oocyte-cumulus complex
The oocyte-cumulus complex (OCC) in the mature, ovulated oocyte is made up of corona cells loosely attached to each other around the zona pellucida, and radially expanded cumulus cells with an extracellular matrix of glycosaminoglycans. This extracellular matrix is composed of granules that are trypsin sensitive and filaments of mostly hyaluronic acid (Acosta, 1996).
Table 4.3 Sperm and egg physiology related to fertilization
|
Parameter |
Oogenesis and characteristics of ova |
Spermatogenesis and characteristics of sperm |
|
First maturational division in gamete |
First maturational division is completed in preovulatory follicle |
First meiotic division results in two cells of equal size |
|
Second maturational division |
Second maturational division is completed only when the egg is penetrated by sperm |
Not comparable |
|
Number of gametes produced during reproductive life |
Thousands of oogonia are found in the neonatal ovary |
Millions of sperm are produced in each ejaculate from puberty, with reduced numbers during senility |
|
Sex chromosome in gamete |
X |
X or Y |
|
Amount of cytoplasm in gamete |
As oocyte matures, the amount of its cytoplasm increases |
As spermatid develops into sperm, the amount of cytoplasm decreases, acrosome and tail develop in late spermatid |
|
Motility of gamete |
Oocytes, surrounded by follicular cells, are immotile |
Sperm motility develops gradually in various parts of epididymis and increases at ejaculation |
|
Plasma membrane at fertilization |
Egg acquires plasma membranes from sperm |
Sperm loses its plasma membranes to egg |
|
Survival in female reproductive tract |
12-24 h after ovulation |
Fertilizability is maintained 24 h after ejaculation |
Table 4.5 Chromosomal abnormalities and different developmental stages (Rosnina et al., 2000)
|
Stage |
Cytogenetic anomalies |
|
Maternal gametogenesis |
Somatic division of oogonia before onset of meiosis or during first or second meiotic division |
|
Paternal gametogenesis |
Mitotic divisions while male is in utero, or during post-puberty first or second meiotic division after puberty |
|
Fertilization |
Normal chromosomes with errors during fertilization; fertilization of a normal egg by two normal sperm |
|
Zygote |
Improper division of one or more chromosomes, anaphase lag or no disjunction; failure of cleavage of blastomeres or mosaic embryo |
Figure 4.13 Physiological processes of human fertilization. (a) Sperm (Sp)-oocyte (O) membrane fusion: plasma membrane (PM) of midsegment of sperm head fuses. (b) Sperm-egg fusion: the second polar body (PB2) is extruded into the perivitelline space (PVS) and the fertilized ovum completes the second meiotic maturation division (the anaphase 2 spindle). (c) Cortical reaction (CR): sperm fuses with egg cortical granules (CG) beneath oolemma and they release their contents into PVS by exocytosis. (d) Zona reaction (ZR): interaction of CG contents with lipoproteins of the zona pellucida (ZP) establishes a primary block to polyspermy at the level of the ZP. (Chemical hardening prevents supernumerary sperm from entering PVS after fertilization.) (e) Female and male pronuclei (FPN and MPN) migrate to the center of egg and become closely associated with one another but do not fuse
Gamete interaction
The bulk of semen enters the uterus directly or is forced through the cervical canal during momentary relaxation of the cervix assisted by the vaginal contraction resulting from copulatory stimuli. Most sperm are eliminated from the female reproductive tract, leaving a few sperm to be transported to the site of fertilization in the ampulla and/or ampulla- isthmic junction (Tables 4.2-4.4).
The sperm-to-egg ratio in the ampulla during fertilization is 1:1 or less. The female reproductive tract prevents morphologically abnormal sperm from reaching the site of fertilization. Not all fertilizing sperm are genetically normal. Normal embryonic development is maximal when eggs in the oviduct are fertilized soon after ovulation.
The gametes undergo a complex ultrastruc- tural process of proliferation, chromosomal exchange and reduction at meiosis, differentiation, maturation and transport. Membrane fusion occurs between the sperm and oocyte. Chromosomal abnormalities occur at different developmental stages (Table 4.5).
Figure 4.14 Male/female pronuclei at syngamy: (a) phasecontrast microscopy and (b) light microscopy. (c) Morulae with 6-8 blastomeres. (d) Two pronuclei in the perivitelline space underneath the zona pellucida. (e) Scanning electron microscopy of sperm-egg interaction; note the polar body. From Zaneveld, with permission
Figure 4.15 Prefertilization, fertilization and postfertilization in farm animals
Figure 4.16 Relationship between sperm characteristics and sperm-zona pellucida (ZP) binding and penetration
FERTILIZATION
The interaction between the protein ZP3 of the zona pellucida (ZP) and its receptor on the sperm head leads to the AR and to the subsequent enzymatic attack of the ZP (Figures 4.4 and 4.13-4.17). The sperm penetrates the ZP and reaches the perivitelline space. The oocyte and sperm membranes fuse and fertilization takes place. The cortical reaction prevents polyspermia. The second meiotic division of the oocyte is completed and the second polar body is extruded. Upon fusion, the two pronuclei give rise to the formation of the mitotic spindle and to the first cleavage (Revelli et al., 2003a).
CAPACITATION, AROSOME REACTION AND FERTILIZATION
Figure 4.17 Stepwise progressive diagnosis in cases of recurrent failed in vitro fertilization (IVF). Sequential analysis of specific sperm defects in cases of recurrent failed fertilization. FSH, follicle stimulating hormone; ICSI, intracytoplasmic sperm injection; SUZI, subzonal insemination
IMPLANTATION
The uterine endometrium is an extremely dynamic tissue, undergoing sequential developmental changes in preparation for implantation during each estrous cycle. Successful implantation of the blastocyst requires successful development of uterine endometrial receptivity. Cyclic endometrial development in the adult can be considered to be analogous to embryonic development.