Fertilization Flashcards
(499 cards)
1
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A complex cascade of maturational events during the process of follicular development confers on th
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o0cyte the capacity to undergo fertilization and subsequent development (see Chapter 1). Complete
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physiologic maturation requires both nuclear and cytoplasmic changes that must be coordinated to ensure
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optimal cellular function.
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NOTE: Nuclear maturity alone is insufficient to determine the competency of an oocyte.
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Fertilization: The processes involving the union of male and female germ cells that result in formation of a
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pronuclear zygote or one-cell embryo are known as fertilization. This process is essential in the generation
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of new progeny who display all the characteristics of the species. If fertilization does not occur
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both oocyte
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and sperm will degenerate in the female reproductive tract. For normal fertilization to proceed
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several steps
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must occur in a sequential manner:
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a. ovulation and oviductal collection of the oocyte
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b. deposition of sperm of sufficient number and motility within the vagina
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C.
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- Fertilization
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d. sperm traversing the cumulus oophorus
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f.
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sperm capacitation and location of the oocyte
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e. sperm interaction with
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and penetration of
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sperm-oocyte fusion
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g. oocyte activation
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i.
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61/154
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h. male pronucleus formation
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syngamy
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Compromising any one of these steps will result in failed fertilization. Each of these events will be discussed
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in greater detail
below.
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A. Ovulation
the Oviduct and the Oocyte: The final stages of mammalian oogenesis prepare the oocyte
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for fertilization. The preovulatory surge of pituitary gonadotrophins act upon large antral (graafian)
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follicles to induce the resumption of meiosis in fully-grown oocytes arrested in prophase of meiosis I
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(see Chapter 1)
bringing about detailed changes in gene expression and follicular structure. The
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cumulus oophorus of the oocyte destined to ovulate is stimulated to begin expansion such that the
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ovulated secondary oocyte is surrounded by a mass of "sticky" cumulus oophorus cells and is ready
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for interaction with sperm. n vivo
an expanded
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in oocyte uptake by fimbriae of the human oviduct. The follicle must also initiate tissue restructuring to
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form the corpus luteum (Russell and Robker
2007).
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B. Sperm Number
Motility
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that the concentration of sperm within the ejaculate can influence fertility. On average between 10 and
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108 sperm are deposited in the anterior vagina near the cervical os. Sperm will begin to swim into the
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cervical canal within minutes of deposition. Human semen coagulates within about a minute of coitus
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forming a loose gel composed of structural proteins semenogelin l and ll and a glycosylated form of
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semenogelin II
which are secreted primarily by the seminal vesicles. The gel is degraded 30 to 60
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minutes later (liquefaction) by prostate-specific antigen (PSA) an enzyme secreted by the prostate
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gland. The coagulum may serve to hold the sperm at the cervical os
as well as protecting sperm against
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the harsh environment of the vagina. The vagina is equipped with antimicrobial defenses; an acidic pH
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maintained through lactic acid production by anaerobic lactobacilli and immunological responses that
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can damage sperm as well as infectious organisms. The defensive mechanisms of seminal plasma
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include pH buffers and immune response inhibitors and are important because sperm are terminally
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differentiated cells that must survive in the female reproductive tract without reparative mechanisms.
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Sperm may also undergo oxidative damage (such as DNA fragmentation) due to physical stresses
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during ejaculation and contractions of the female reproductive tract (Suarez and Pacey
2006).
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Cervical mucus facilitates sperm penetrability under the influence of estrogen
when it is highly
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hydrated
but may also provide a means of sperm selection
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abnormal sperm that cannot swim properly. Cervical mucus is mostly made up of mucins
the
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composition and organization of which give mucus its viscosity and elasticity. Sperm may traverse the
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cervical canal by following mucosal grooves. Little is known about how long sperm spend traversing
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the cervix or whether sperm are stored there
but large numbers are lost and it has been estimated that
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only a few thousand reach the oviducts (fallopian tubes) and only about 100 to 150 sperm make it to
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the site of fertilization in the ampulla region.
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Uterine transportation of sperm is likely aided by contractions of the uterine myometrium that may be
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stimulated by seminal components. How long sperm take to pass through the uterus is difficult to
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determine
although more rapid transport may benefit sperm by propelling them past the immunological
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defenses of the female. While contractions of the fermale reproductive tract are esserntial for aiding
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sperm passage from the vagina to the oviduct
the sperm's forward progressive motility is essential for
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its transport. It is easy to appreciate how oligo- and asthenozoospermia contribute to male factor
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infertility.
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Sperm in the oviduct: Sperm exit the uterine cavity into the isthmus of the oviduct via the uterotubal
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junction. In addition to normal morphology and motility
sperm require a factor
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surface protein or proteins
to enable them to pass through the junction. The oviduct free of
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immunological substrates and maintains the fertility of sperm for several hours
or even a few days
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until ovulation. Detaining sperm at the tubal isthmus and allowing only a few at a time to reach the site
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of fertilization in the ampulla may help to preventpolyspermic fertilization. The heads of motile sperm
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have been observed to interact with the apical surface of the isthmic but not ampullary epithelium
and
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this contact may somehow preserve sperm during storage
possibly by preventing capacitation (see
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below). Interestingly
there is evidence in vitro to suggest that poor sperm interaction with the tubal
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epithelium might be associated with reduced fertility in women with a previous diagnosis of
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endometriosis. Human tubal fluid may also regulate sperm fertilizing capacity (Zumoffen et al.
2013).
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After fertilization
any sperm remaining in the female reproductive tract may be phagocytosed by isthmic
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epithelial cells or eliminated into the peritoneal cavity where they are phagocytosed (Suarez and Pacey
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2006).
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NOTE: Many thousands of sperm are routinely cultured with the oocyte to achieve fertilization in vitro
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compared to the situation in vivo where only a few sperm are thought to be present in the oviduct at
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the site of fertilization. The reproductive epithelium may therefore have an important influence on
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the physiology and function of gametes and embryos that cannot be completely mimicked by sperm
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preparation processes and culture conditions used in the assisted reproductive technology (ART)
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laboratory.
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Sperm integrity: The mature spermatozoa arrive in the female reproductive tract with highiy
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condensed chromatin in the sperm nucleus that is transcriptionally quiescent (no replication occurs).
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However
there are now reports that propose the sperm delivers a unique set of fully processed
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messenger RNAs (mRNAs) to the oocyte and
in specific instances sperm RNAs do have a biological
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role (Nanassy and Carrell
2008
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means of protecting DNA integrity during the sperm's journey to the oocyte. DNA damage is a
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relatively common feature of human sperm and has been associated with impaired fertilization
poor
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embryonic development
high rates of miscarriage
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significant reduction in sperm concentration has been associated with increasing paternal age
sperm
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motility and semen volume are observed to decline and DNA damage levels increase. However
this is
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exhibited as a higher mutational load
with no apparent age-related impact on the incidence of
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aneuploidy (Wyrobek et al.
2006). The live birth rate following IVF with donor oocytes decreased
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significantly in couples with a male partner greater than 50 years of age (Fratterelli et al.
2008).
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C. Sperm Capacitation and Locating the Oocyte: More than sixty years ago
Austin and Chang
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independently discovered that sperm must reside within the female reproductive tract for a period of
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time before gaining fertilization competence (Chang
1951; Austin
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competence was termed capacitation
defined as a functional maturation involving processes that
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does not alter the cell structure but changes its potential for fertilization." These processes bring about
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changes in the plasma membrane that prepare sperm to undergo the acrosome reaction are essential
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for normal fertilization and include the following:
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> shedding of proteins and cholesterol
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post-translational and structural modification of proteins
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a change in flagellar (tail) beating that typically involves an increase in the flagellar bend amplitude
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concomitant with a decrease in progressive movement
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At a cellular level
capacitation is believed to involve the removal of an inhibitory factor from sperm
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accompanied by alterations in membrane proteins and lipids.
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At a molecular level
most of the modifications that collectively represent capacitation appear to be
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regulated by changes in intracellular pH
calcium concentration [Ca1
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tyrosine phosphorylation/ dephosphorylation.
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Characteristics of capacitated sperm include the ability to:
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> display hyperactivated motility
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bind to the zona pellucida
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> undergo the acrosome reaction
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Hyperactivation occurs at some point in the female tract
most likely in the oviduct and may be
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regulated by a different pathway from that regulating acrosomal responsiveness. Other evidence
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indicates that hyperactivation is required by sperm to progress towards the oocyte and penetrate its
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vestments
although the functional importance of hyperactivated motility is not completely understood.
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One theory suggests that it allows a greater surface area to be covered within the oviduct
increasing
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the opportunity to contact the oocyte-cumulus complex. Hyperactivation also enhances the sperm's
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capacity to swim through viscoelastic substances such as mucus in the tubal lumen and the
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extracellular matrix of the cumulus oophorus.
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Capacitation in vitro can be accomplished following removal of sperm from the seminal plasma by a
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short-term incubation (2-4 hours) in a bicarbonate-buffered balanced salt solution with energy
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sources and serum albumin.
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Locating the oocyte and the way in which this is achieved by the sperm remains under investigation.
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There is evidence for the existence of two complementary guidance mechanisms operating within
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the oviduct;
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A long-range guidance mechanism involving thermotaxis along a temperature gradient that is
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lower in the isthmus and higher in the ampulary region of the oviduct to guide capacitated sperm
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towards the site of fertilization.
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A short-range guidance mechanism using a chemotactic method that may operate when the
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sperm are in closer proximity to the oocyte
as well as potential assistance from oviductal
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contractions (Eisenbach and Giojalas
2006). There is mounting evidence that human sperm use
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chemical cues in the female reproductive tract to locate the o0cyte using a repertoire of
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chemoreceptors expressed on the surface of the sperm. Olfactory receptors (ORs) are the largest
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group of human chemoreceptors and OR protein expression has been detected in the midpiece
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flagella
equatorial segment
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indicate that as yet to be identified sperm chemoattractants are secreted within the follicle prior to
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ovulation as well as by the mature oocyte and surrounding cumulus cells outside the follicle (Sun
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et al.
2005).
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D. Traversing the Cumulus Oophorus: The cumulus oophorus is composed of cels and a matrix of
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polymerized hyaluronic acid. Current dogma suggests that sperm must be capacitated in order to
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penetrate the cumulus oophorus. However
if sperm undergo a premature acrosome reaction prior to
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contact with the cumulus mass
they do not have the ability to penetrate the cumulus oophorus
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(Cummins and Yanagimachi
1986). Hyaluronidase
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(Talbot
1985)
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digesting the hyaluronic acid matrix between cells of the cumulus oophorus. Under normal
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circumstances sperm are theorized to utilize both mechanical (motility) and enzymatic forces to traverse
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the cumulus mass.
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The acrosome reaction (AR): AR
an exocytotic event that occurs in response to specific stimuli
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must be completed prior to fusion wth the oocyte and is induced following sperm binding to the zona
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pellucida (ZP). The acrosome is a membrane-bound organelle that originates from the Golgi apparatus.
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During the AR
the outer acrosomal membrane fuses with the overlying sperm plasma membrane
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allowing release of the acrosomal contents. At least two different receptor-mediated signaling
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pathways in the sperm plasma membrane have been shown to be involved in acrosomal exocytosis
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induced by the ZP:
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) a guanine nucleotide-binding regulatory protein (Gi protein)-coupled receptor that activates the
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phospholipase CB1 (PLCB1-mediated signaling pathway
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i) a tyrosine kinase (TK) receptor coupled to PLCY
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The identification of the substrates that stimulate onset of the AR and the downstream pathways has
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proved elusive. However
two principle physiological agonists have been identifed in mammals;
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progesterone (produced by the oocyte and the steroidogenic cumulus cells that surround it) and the
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ZP proteins. The downstream pathways activated by these agonists both involve an increase in
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intracellular calcium levels that is an absolute requirement for the completion of the AR. Overall
a high
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proportion of motile human sperm have been shown to respond to AR stimuli
but their response differs
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likely due to significant variations observed in their acrosomal status and competence for acrosomal
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reactivity. The cause of these variations in AR competence between sperm is unclear but may stem
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from differences in degrees of capacitation
which will impact intracellular calcium concentrations or
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other factors affecing sperm viability (Harper et al.
2006). Inhibition of this calcium increase blocks
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progression of the AR. Other studies have demonstrated a role for phospholipase A2 whose
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contribution to the AR depends on progesterone levels and the production of lysophospholipids and/or
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fatty acids that are required for fusion of the acrosomal and plasma membranes (Nahed et al
2016).
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Additional events that occur upon ZP-sperm binding are:
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> depolarization of sperm membrane potential
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increased intracellular pH through Na /H* exchanger activation
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increased intracellular calcium concentration ([Ca*) via voltage-operated calcium channels.
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These changes lead to fusion of the sperm plasma membrane with the outer acrosomal membrane and
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release of the acrosomal contents.
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E. Sperm-ZP Binding and Zona Penetration: The ZP is an acellular glycoprotein matrix that forms a
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shell-like structure around o0cytes from the human and other mammals. Biochemical and molecular
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biology tools have enabled significant advances in our understanding of ZP structure and function in
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recent years. These data show some similarities as well as major differences between species and
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care must be taken to ensure the origin of information is specific to fertilization events in the human.
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Mature human oocytes have been observed to developa 'spongy" less compact outer ZP layer
while
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maintaining a more compact inner layer. Indeed
the human ZP is now recognized to be a highly
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organized dynamic structure that is indispensable for effective oogenesis
fertilization
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development steps. A current understanding of human ZP structure and function is reviewed in detail
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by Gupta (2018).
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Acrosome-Intact
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Plasma Membrane (PM)
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+Spen Binding Protein
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Outer Acrosomal
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Membrane (0AM)
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Key:
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Inner Acrosomal
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Membrane
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+ Proacrosin
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A-Acrosome
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N-Nucleus
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N
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Acrosome-Reacted
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Figure 1: The acrosome reaction
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Fenestration:
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PM +OAM fusion
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Acrosin&
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Hyaluronidase
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released
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The human ZP is primarily composed of four glycoproteins: ZP1
ZP2
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common structural elements and are encoded by genes on chromosomes 11
16
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(Lefièvre et al.
2004). All four ZP proteins share a motif known as the "ZP domain" (itself composed of
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two sub-domains
ZP-N and ZP-C) located in the C-terminus that likely plays an important role in their
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polymerization into a filamentous structure. ZP gene mRNA expression has been positively related
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to oocyte maturity
zona inner layer retardance
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Additional studies indicate that
in humans
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ZP proteins play a role in sperm-egg binding and more than one ZP protein induces the
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acrosome reaction. The data available suggest that each ZP protein mediates the acrosome reaction
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by similar but different downstream signaling pathways that likely converge at a common downstream
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signaling event to induce the AR. ZP protein glycosylation does not appear to be essential for sperm
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binding. ZP1
ZP3
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to the acrosome-reacted sperm.
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Roles of the ZP in the fertilization process include:
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sperm binding
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induction of the sperm AR
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acrosomal exocytosis
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> the block to polyspermy
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Sperm penetrate the ZP
likely using a combination of actual mechanical forces due to sperm motility
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combined with acrosomal proteases that hydrolyze ZP glycoproteins and facilitate passage through
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the ZP (Salvidar-Hernández et al.
2015). ZP glycoproteins are also involved in mechanisms to prevent
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binding of additional sperm subsequent to fertilization as part of the block to polyspermy
preventing
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the formation of polyploid non-viable embryos. The mechanisms responsible for the process in humans
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is poorly understood due to the events occurring inside the female reproductive tract and lack of suitable
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in vitro models. The data available suggest two mechanisms: a "fast block" to polyspermy attributed
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to an "oocyte membrane block" achieved primarily by depolarization of the oocyte membrane
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following binding of the first sperm
and a "slow block" or "zona reaction/cortical reaction" involving
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modification of the ZP glycoproteins that "harden" the ZP. This is achieved by enzymes (e.g.
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hydrolases
proteinases
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binding and prevernt binding/penetration of additional sperm. Sperm that have commenced the AR
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may stilH be able to bind to the ZP
consistent with observations from transition electron microscopy of
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human sperm
showing that acrosomal exocytosis is not an al-or-nothing event (Stock and Fraser
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1987). Sperm interaction with the ZP seems to be a very redundant process
one that involves several
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proteins
since the deletion of individual proteins does not necessarily result in a complete block to
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sperm-ZP binding. Many sperm binding proteins
either associated with the surface of acrosome
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intact sperm (3-1
4-galactosyltransferase
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been demonstrated to have an affinity for the ZP and to be involved in sperm attachment
binding and
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penetration of the zona (Kohn et al
2010). Multimeric protein complexes have also been proposed
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to be involved in sperm-ZP binding (Redgrove et al
2011; Dean
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underline the complexity of the fertilization process.
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Note: Tight binding between the sperm and zona pellucida is species specific.
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Reactive oxygen species (ROS) and the signaling pathways upon which they act may
surprisingly
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have a role in sperm capacitation
hyperactivation
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the action of ROS
modulate capacitation. In sperm cells that lack protein synthesis and hence have
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limited repair mechanisms
the presence of multiple pathways may ensure that these processes are
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achieved. Less is known currently regarding the involvement of ROS-related events or the presence of
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parallel signaling pathways in the acrosome reaction
although redundancy to ensure its completion
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is also likely (de Lamirande et al.
2008).
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F. Sperm-Oocyte Fusion: Sperm that enter into the periviteline space can fuse with the oolemma. Fusion
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occurs between the post-acrosomal region of the sperm's plasma membrane and the oocyte's
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Roles of the ZP in the fertilization process include:
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sperm binding
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induction of the sperm AR
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acrosomal exocytosis
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> the block to polyspermy
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Sperm penetrate the ZP
likely using a combination of actual mechanical forces due to sperm motility
269
combined with acrosomal proteases that hydrolyze ZP glycoproteins and facilitate passage through
270
the ZP (Salvidar-Hernández et al.
2015). ZP glycoproteins are also involved in mechanisms to prevent
271
binding of additional sperm subsequent to fertilization as part of the block to polyspermy
preventing
272
the formation of polyploid non-viable embryos. The mechanisms responsible for the process in humans
273
is poorly understood due to the events occurring inside the female reproductive tract and lack of suitable
274
in vitro models. The data available suggest two mechanisms: a "fast block" to polyspermy attributed
275
to an "oocyte membrane block" achieved primarily by depolarization of the oocyte membrane
276
following binding of the first sperm
and a "slow block" or "zona reaction/cortical reaction" involving
277
modification of the ZP glycoproteins that "harden" the ZP. This is achieved by enzymes (e.g.
278
hydrolases
proteinases
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binding and prevernt binding/penetration of additional sperm. Sperm that have commenced the AR
280
may stilH be able to bind to the ZP
consistent with observations from transition electron microscopy of
281
human sperm
showing that acrosomal exocytosis is not an al-or-nothing event (Stock and Fraser
282
1987). Sperm interaction with the ZP seems to be a very redundant process
one that involves several
283
proteins
since the deletion of individual proteins does not necessarily result in a complete block to
284
sperm-ZP binding. Many sperm binding proteins
either associated with the surface of acrosome
285
intact sperm (3-1
4-galactosyltransferase
286
been demonstrated to have an affinity for the ZP and to be involved in sperm attachment
binding and
287
penetration of the zona (Kohn et al
2010). Multimeric protein complexes have also been proposed
288
to be involved in sperm-ZP binding (Redgrove et al
2011; Dean
289
underline the complexity of the fertilization process.
290
Note: Tight binding between the sperm and zona pellucida is species specific.
291
Reactive oxygen species (ROS) and the signaling pathways upon which they act may
surprisingly
292
have a role in sperm capacitation
hyperactivation
293
the action of ROS
modulate capacitation. In sperm cells that lack protein synthesis and hence have
294
limited repair mechanisms
the presence of multiple pathways may ensure that these processes are
295
achieved. Less is known currently regarding the involvement of ROS-related events or the presence of
296
parallel signaling pathways in the acrosome reaction
although redundancy to ensure its completion
297
is also likely (de Lamirande et al.
2008).
298
F. Sperm-Oocyte Fusion: Sperm that enter into the periviteline space can fuse with the oolemma. Fusion
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occurs between the post-acrosomal region of the sperm's plasma membrane and the oocyte's
300
microvillous surfaces in all regions except where the second metaphase spindle and first polar body
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are located. The mechanisms by which acrosome-reacted spermatozoa bind to receptor molecules on
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the oocyte plasma membrane and initiate fusion with the oolemma are still unresolved. Gene deletion
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studies have identified two plasma membrane proteins essential for gamete fusion (Kato et al.
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2016):
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Izumo-1 (sperm: named after a Japanese marriage shrine) is an intra-acrosomal protein that
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migrates from the outer acrosomal membrane to the equatorial region of the sperm plasma
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membrane during the acrosome reaction. Acrosome-intact sperm are fusion incompetent (Satouh
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et al.
2012).
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Juno (oocyte: named after the goddess of fertility and marriage) that is present on the oocyte
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membrane. Juno is shed from the egg membrane rapidly following fertilization and may therefore
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also contribute to the block to polyspermy mechanisms at the level of the oolemma.
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Additional
apparently non-essential
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oolemma-anchored integrins
o0cyte-expressed retroviral envelope proteins
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sperm lysozyme-like protein (SLLP1)
sperm equatorial segment protein (ESP)
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proteins such as the secretory protein CRISP-1 (cysteine-rich secretory protein 1) of epididymal origin
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and CRISP-2 of testicular origin (Nixon et al.
2007; Sutovsky
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proposes that microdomains within the oolemma form a receptor web of key molecular players that
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are responsible for sperm-oocyte binding and for facilitating changes that lead to cell-cell fusion. lzumo
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itself may also be involved in organizing or stabilizing a multiprotein complex essential for the function
320
of the membrane fusion machinery (Ellerman et al.
2009).
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An aspect of sperm-oocyte fusion applicable to ART practitioners is the lack of species specificity for
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the process. The best example of this is the human sperm functional test called the sperm penetration
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assay (SPA) that utilizes zona pellucida-free hamster oocytes to test human sperm capacitation
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potential. ZP-free hamster oocytes will fuse with sperm from many different mammalian species. In
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contrast
the zona pellucida-free mouse oocyte can only fuse with mouse sperm.
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G. Oocyte Activation: This refers to the 'reawakening' of the oocyte. Morphological indicators of oocyte
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activation are:
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cortical granule exocytosis
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> resumption of meiosis
resulting in the completion of the second meiotic division and extrusion of
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a second polar body (PB2).
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During meiosis Il
sister chromosome strands (chromatids) in the oocyte uncouple; one set is extruded
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in PB2
the other set is maintained in the oocyte
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Errors in the genetic composition of the fertilized oocyte may arise due to aberrant sister chromatid
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separation. Indeed
errors seem to arise with relatively high frequency in human oocytes and may be
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due
in part
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by the cohesion complex that maintains physical connection between sister chromatids until anaphase.
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In meiosis
cohesion must be released sequentially to bring about orderly chromosome segregation
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both at meiosis I and meiosis Il and necessitates meiosis-specific cohesion components. Deficient
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cohesion may be one underlying cause of human age-related aneuploidy (Hodges et al.
2005).
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PB1
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Metaphase i!
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Fertiizalion
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PB1 & PB2
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Anaphase/Teiophase il
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Figure 2: Sister chromatids separate during meiosis II
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Oocyte activation mechanisms remain unclear
but it is now most widely acceped that the sperm
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introduces a soluble cytosolic factor
sperm phospholipase C zeta (PLC)
349
of PLC
into the oocyte cytoplasm that acts as the molecular trigger of oocyte activation (Cox et al.
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2002; Nomikos et al.
2013). PLCK acts via the inositol 1
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cytoplassmic calcium oscillations that culminate in initiation of the embryo development process.
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PLC regulation and its interaction with other proteins within the o0cyte have yet to be described. This
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hypothesis is consistent with egg activation and normal embryonic development following
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intracytoplasmic sperm injection (1CSI)
when sperm-oocyte fusion does not occur. However
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topic remains an active area of research and more recently a post-acrosomal sheath WW domain
356
binding protein (PAWP) that diffuses into the ooplasm and initiates a molecular cascade involving
357
mainly the phosphoinositide pathway has been proposed as an alternative sperm factor candidate
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(Anifandis et al.
2016). Thus
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soluble sperm factor in the stimulation of intracellular Ca2* release and subsequent Ca2t oscillations
360
that must occur to induce oocyte activation (Whittingham and Siracusa
1972). As with many biological
361
processes involved in reproductive events
the existence and reliance on redundant or parallel
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pathways to ensure completion of oocyte activation may well explain the findings to date. Moreover
363
additional fertilization-induced signaling pathways
such as protein tyrosine kinases
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activated in mammalian oocytes (McGinnis et al.
2013). Regardless of the regulatory mechanismn
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end results are the resumption and completion of the second meiotic division and ultimately the
366
formation of the female pronucleus (Figure 2).
367
NOTE: In light of the more frequent use of chemical stimuli such as calcium ionophore to bring about
368
o0cyte activation in patients with failed or low fertilization
it should be remembered that these Ca2*
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oscillations not only provide a stimulus for meiotic resumption
but have a role in long term embryonic
370
events including cell composition in resulting blastocysts (Bos-Mikich et al.
1997). There are now
371
several publications. reporting successful calcium ionophore use (Ebner et al.
2012; Miller et al.
372
but the departure from normal patterns of calcium release dictates for continued surveillance of this
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practice in the IVF laboratory.
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The cortical reaction is probably mediated by activation of a signaling pathway involving inositol
375
phosphate (PIP2) in mammals. The cortical granule (CG) exudates act on the ZP
causing biochemical
376
and structural changes that result in the loss of its sperm binding capacity and ability to be
377
penetrated by sperm previously bound to it. Mammalian CG exudate-induced changes include
378
receptor modification of sperm receptors that are subsequently unable to bind sperm
and zona
379
hardening that results in mechanical stiffening and resistance to proteolytic digestion
thus blocking
380
polyspermic fertilization. Ovastacin has been identified as the cortical granule protease responsible for
381
ZP2 cleavage in the mouse (Burkart et al.
2012)
382
liver-derived non-hormonal plasma protein fetuin-B
an inhibitor of ovastacin (Dietzel et al.
383
Follicular fluid and serum may also contain factors that inhibit premature zona hardening. Currently
384
there are no reports regarding any CG interaction with the ZP4 sperm receptor. Changes in the
385
distribution of microvilli on the oolemma and their CD9 protein content may also be involved in the
386
development of an oocyte membrane block to polyspermy in the mouse (Zylkiewicz et al.
2009). In
387
addition
sperm membrane incorporation into the oolemma contributes to changes in the oolemma that
388
block polyspermy.
389
H. Male Pronucleus Formation: The mature sperm nucleus contains very tightly packed chromatin that
390
does not permit transcription. During spermiogenesis
the majority of somatic histones are replaced
391
with protamines that are responsible for sperm chromatin condensation. Somatic histones in round
392
spermatids are replaced initially with transition proteins (TP1 and TP2) and then protamines (P1 and
393
P2) replace the transition proteins in elongating spermatids. Disulfide bonds
formed by oxidation of
394
the sulphhydryl groups present on protamines hold the compacted DNA in place. Chromatin
395
stability is further increased following ejaculation by seminal plasma. Zinc
present in the prostatic
396
fluid
seems to play an important role in this process. The compact chromatin must therefore be
397
modified during pronuclear formation and prior to syngamy. After sperm-oocyte fusion
the de-
398
membranated sperm are accessible to oocyte factors
the presence of which is dependent upon
399
completion of both nuclear and cytoplasmic maturation in the oocyte. Reduction of the disulfide
400
bonds between the protamines by the action of the oocyte-derived disulfide bond reducer glutathione
401
(GSH) (Perreault et al.
1984) is a first step in human sperm nucleus decondensation (reviewed in
402
Caglar et al.
2005). However
403
decondensation to occur. An additional molecule seems to be required to remove sperm protamines
404
from DNA and allow their replacement by oocyte histones
which then organize into nucleosomes.
405
Several reports now suggest that heparin sulfate (HS) is a likely candidate for the protamine acceptor
406
in the human. Moreover
heparin sulfate is present in the human oocyte consistent with this hypothesis.
407
However
another glycosaminoglycan
408
decondensation in mouse sperm in vitro. While the decondensing activity of DS was significantly lower
409
than that of HS
the two substrates acted synergistically when both were present (Sanchez et al.
410
A number of other molecules are also required for constitution of a functional nuclear envelope and
411
nuclear cytoskeleton and
as with many aspects of reproductive biology
412
increase as our knowledge of these essential processes increases. Sperm DNA remodeling culminates
413
in sperm nucleus decondensation. Of note
the sperm decondensation process is time
414
temperature deperndent
which is of great importance with respect to establishing appropriate
415
laboratory conditions and consistent inspection regimes for assessing fertilization outcome. Moreover
416
sperm DNA damage may contribute to the failure of the nuclear decondensation process and cause
417
fertilization failure.
418
Intracytoplasmic pronuclei motility and appositioning are initiated before male pronucleus
419
remodeling
directed by oocyte microtubules and dependent on the ability of the zygote to reconstruct
420
the centrosome (Sutovsky and Schatten
2000). During spermiogenesis
421
into mature sperm
there is partial reduction of the male centrosome. The proximal centriole is retained
422
intact
though in an inactive state
423
axoneme and cannot function as a centriole. In addition
the sperm pericentriolar material is lost
424
within the cytoplasmic droplet. In the female gamete
the oocyte loses both centrioles during
425
oogenesis and does not exhibit granular perinuclear centrosomal material (PCM) at meiotic spindle
426
poles
as seen in rodent oocytes. Functional centrosomal structure is restored after fertilization with
427
the formation of an active zygotic centrosome
with some maternal input around the sperm centriole
428
which duplicates at the pronuclear stage and forms a sperm aster. Pronuclear development and
429
migration of the pronuclei towards syngamy is initially supported by the sperm aster
which then
430
proceeds to form the mitotic spindle. It is therefore highly possible that sperm centrosomal
431
dysfunction could lead to aberrant embryonic development.
432
Note: Actin microfilaments are not involved in pronuclear migration in the human.
433
Pronuclear inspection: Human oocytes inseminated in vitro are routinely inspected for the number
434
of pronuclei visible at about 15-18 hours after fertilization
when chromatin decondensation should be
435
complete and the nuclear envelopes should still be intact (Figure 3).
436
Two pronuclei is accepted as evidence of 'normal fertilization'
437
Two pronuclei and abnormal fertilization (triploidy) may arise
despite the observation of two
438
pronuclei in the fertilized egg when:
439
i.
440
i.
441
ii.
442
a diploid sperm penetrates the oocyte
443
fertilization of a diploid 'giant' oocyte occurs
444
endoreduplication within the female pronucleus occurs (Rosenbusch
2008).
445
Giant oocytes have about twice the cytoplasmic volume of regular-size oocytes and constitute
446
approximately 0.3% of aspirated human oocytes. It is believed that they arise due to either the failure
447
of cytokinesis during mitotic division of oogonia or cytoplasmic fusion of two oogonia. Cytogenetic
448
analysis of human giant oocytes has shown them to be diploid
and fertilized zygotes are thus
449
genetically abnormal
even when they exhibited two pronuclei and two polar bodies at the fertilization
450
check and should therefore be removed from the pool of fertilized eggs. These extra-large oocytes
451
occur spontaneously
anddo not appear not to reflect the quality of oocytes remaining in the ovary or
452
impact the implantation of cohort sibling embryos (Machtinger et al.
2011; Lehner et al.
453
The pronuclei (PNs) position their axis towards the second polar body and achieve a proper orientation
454
at syngamy by controlling the plane of the first mitotic division. Nuclear precursor bodies (NPBs)
455
become visible during PN formation and will migrate and merge into nucleoli in a time dependent
456
manner. Pre-rRNA synthesis occurs in the nucleoli and is necessary for translational processes when
457
the embryonic genome is activated (see below). The number of nucleoli in each PN usually ranges
458
from two to seven
with equal numbers in the two daughter cells after mitosis.
459
I. Syngamy: Once pronuclei are brought into close proximity (apposition)
the chromatin of each PN
460
begins to polarize and rotate to face each other. The male PN rotates onto the female PN
placing the
461
centrosome into a furrow between them. Movement and rotation of the PNs may cause the formation
462
of a clear cortical zone
known as the halo. Failed progression to apposition and syngamy is primarily
463
due to deficient sperm centrosome activity. Dissolution of nuclear envelopes (nuclear envelope
464
breakdown) occurs either separately a few hours before the first mitotic cleavage occurs (human)
or
465
after actual fusion of the nuclear envelopes of each pronucleus
depending on the species examined.
466
The chromosomes from each PN now pair
align on the newly formed mitotic spindle
467
for segregation in preparation for the first mitotic division.
468
Egg Nucleus
469
Spem Nucleus
470
Egg PN
471
Sperm PN
472
Diploid Nucleus
473
Figure 3: Pronuclear formation
migration
474
This summary of events that support fertilization demonstrates the wide range of questions being
475
addressed in this field. It is quite easy to appreciate how compromising any one of these events will
476
result in suboptimal fertility.
477
at syngamy by controlling the plane of the first mitotic division. Nuclear precursor bodies (NPBs)
478
become visible during PN formation and will migrate and merge into nucleoli in a time dependent
479
manner. Pre-rRNA synthesis occurs in the nucleoli and is necessary for translational processes when
480
the embryonic genome is activated (see below). The number of nucleoli in each PN usually ranges
481
from two to seven
with equal numbers in the two daughter cells after mitosis.
482
I. Syngamy: Once pronuclei are brought into close proximity (apposition)
the chromatin of each PN
483
begins to polarize and rotate to face each other. The male PN rotates onto the female PN
placing the
484
centrosome into a furrow between them. Movement and rotation of the PNs may cause the formation
485
of a clear cortical zone
known as the halo. Failed progression to apposition and syngamy is primarily
486
due to deficient sperm centrosome activity. Dissolution of nuclear envelopes (nuclear envelope
487
breakdown) occurs either separately a few hours before the first mitotic cleavage occurs (human)
or
488
after actual fusion of the nuclear envelopes of each pronucleus
depending on the species examined.
489
The chromosomes from each PN now pair
align on the newly formed mitotic spindle
490
for segregation in preparation for the first mitotic division.
491
Egg Nucleus
492
Spem Nucleus
493
Egg PN
494
Sperm PN
495
Diploid Nucleus
496
Figure 3: Pronuclear formation
migration
497
This summary of events that support fertilization demonstrates the wide range of questions being
498
addressed in this field. It is quite easy to appreciate how compromising any one of these events will
499
result in suboptimal fertility.
