*4.1.2 Ca2+ is a trigger for cell cycle progression*

The completion of meiosis means that the extrusion of the chromatids in the second polar body enables the formation of haploid oocytes that can form a female pronucleus that will be able to combine genetic material with the male pronucleus.

Meiosis is the ground of sexual reproduction where homologous chromosomes recombine—exchange genetic material through chiasmata and generate new genetic combinations that are unique to the offspring. Mammalian oocytes progress through meiosis very slowly; first, the cell cycle is arrested in the dictyate phase of prophase I during the fetal life of a girl and stays in this phase up to 40–50 years. During the menstrual cycle, the recruited oocytes in the ovary progress through the cell cycle under the influence of the hormones. But the meiosis is again arrested at the metaphase of the second meiotic division (metMII). Calcium oscillations at fertilization activate calmodulin-dependent protein kinase II (CaMKII) and switch on the anaphase-promoting complex/cyclosome (APC/C) that leads to securin and cyclin B1 degradation necessary for cell cycle progression and segregation of sister chromatids [33].

Ca2+ also plays a role in pronuclear formation by decreasing MAP kinase (MAPK) activity responsible for nuclear envelope assembly [33]. Oscillations terminate with PN formation, and PLCζ localizes into PN [36]. The mechanism by which Ca2+ recruits maternal mRNA for translation and genome activation is not well understood at the moment. There are some data indicating that calcium oscillations and mRNA translation are coupled [37]. Not only in cytosol, calcium can also diffuse to the nucleus and control different cell functions by direct nuclear calcium signaling [38].

#### **4.2 Electrophysiology and fertilization**

Fertilization potential is a change in the membrane potential across the plasma membrane (PM) of the oocyte that is first observed after oocyte-sperm interaction. In many invertebrates, this is in the form of depolarization of the plasma membrane and is proposed to provide a fast block to polyspermy, described in some invertebrates and only a few vertebrates (such as frogs), but not present in mammals [39]. Lately, there have been some discussions about the nature of electrical block in preventing polyspermy [40]. The role of electrical events in the form of depolarization or hyperpolarization of the plasma membrane at fertilization remains unclear.

In mammals, there is hyperpolarization of membrane potential as a result of change in potassium conductivity across the plasma membrane [24]. In human oocytes, outward current and long-lasting hyperpolarization of the plasma membrane were described [27]. The channels responsible for this hyperpolarization are calcium-activated potassium channels [41]. Species-specific differences in the channels involved in early electrical responses at fertilization are reviewed in [42].

During oocyte maturation, the composition of the channels in the plasma membrane changes, as described in bovine oocytes [28]. The factors regulating

**43**

oscillations.

*Oocyte Activation Failure: Physiological and Clinical Aspects*

important, but also maturity of the plasma membrane.

nature, and mechanism were long unknown.

the composition of channels in the plasma membrane, the conductance for different ions, depending on the specificity, gating, and sensitivity of the channels at different stages, are still unclear. The conditions during gamete maturation are very important, and we can imagine that diet and changes in metabolic pathways can affect the performance. It is not just cytoplasmic maturity of the oocyte that is

The conditions in which gametes are matured are important; the diet and especially taking some medicines in this period can affect the infertility treatment outcome. There are some data from studies of calcium channel blockers used as therapy in various cardiovascular conditions. They affect the movement of free calcium ions across membranes, and dose-dependent reduction in sperm mobility and viability in vitro that can affect fertility treatment was demonstrated [43].

Data from studies of fertilization pointed to a sperm component that has to trigger response in the form of calcium oscillations. But the exact component, its

sperm delivers calcium to the oocyte that further stimulates release of calcium from intracellular stores. The conduit hypothesis assumed that sperm increases the permeability of the plasma membrane for calcium that enters the oocyte with influx from the surroundings. The contact hypothesis predicts that sperm interacts with a receptor on the plasma membrane that causes calcium release from intracellular stores. But success of the ICSI method revealed that there is no need for interaction of sperm and receptors in the plasma membrane for fertilization. The fourth is the sperm factor hypothesis that assumes that there is a component in the sperm cell delivered in cytosol with sperm and that this factor causes calcium release from

There were four main hypotheses, reviewed in [44]. The first one assumed that

Experiments where soluble sperm extracts are injected into the oocyte coupled with different biochemical approaches enabled the search for unknown sperm fac-

There were many candidates such as oscillin, a cytosolic sperm factor related to prokaryote glucosamine phosphate deaminase [47]. In nonmammalian species, PLCγ was identified [48] and the role of nitric oxide in fertilization was investigated [49]. In mammals post-acrosomal WW domain-binding protein (PAWP) was described, which is located in the post-acrosomal region of the sperm, from the stage of elongated spermatids onwards, that causes meiosis resumption and PN

When it was demonstrated that sperm extracts were related to InsP3 concentrations in cell and PLC activity [51], phospholipases were under investigation. Genetic data from the testis' cDNA library revealed some new isoform of PLC [23] and soon a novel sperm-specific phospholipase C, PLCζ was identified as a trigger of calcium oscillations in mouse eggs [52]. In the work of Saunders et al., it was experimentally demonstrated that PLCζ content in a single sperm evoked oscillations and normal embryonic development [52]. They also prepared PLCζ complementary RNA (cRNA) for injection into MII oocytes that triggered the same effect. When they removed PLCζ from sperm extracts, they no longer induced calcium

tor and were in favor of the sperm factor hypothesis [45, 46].

*DOI: http://dx.doi.org/10.5772/intechopen.83488*

**4.3 Sperm factors**

intracellular stores.

formation [50].

*4.3.1 PLCζ*

#### *Oocyte Activation Failure: Physiological and Clinical Aspects DOI: http://dx.doi.org/10.5772/intechopen.83488*

the composition of channels in the plasma membrane, the conductance for different ions, depending on the specificity, gating, and sensitivity of the channels at different stages, are still unclear. The conditions during gamete maturation are very important, and we can imagine that diet and changes in metabolic pathways can affect the performance. It is not just cytoplasmic maturity of the oocyte that is important, but also maturity of the plasma membrane.

The conditions in which gametes are matured are important; the diet and especially taking some medicines in this period can affect the infertility treatment outcome. There are some data from studies of calcium channel blockers used as therapy in various cardiovascular conditions. They affect the movement of free calcium ions across membranes, and dose-dependent reduction in sperm mobility and viability in vitro that can affect fertility treatment was demonstrated [43].

#### **4.3 Sperm factors**

*Embryology - Theory and Practice*

and exocytosis of the contents [33].

chromatids [33].

*4.1.2 Ca2+ is a trigger for cell cycle progression*

**4.2 Electrophysiology and fertilization**

II (CaMKII) and myosin light-chain kinase (MLCK) that phosphorylate a vesicle targeting protein and myosin II to promote exocytosis [33]. It is a quick response; in mouse oocytes, exocytosis starts 15 min after exposure to capacitated sperm, and 30 min after insemination, 78% of cortical granules disappear from cytoplasm [35]. It is proposed that each calcium oscillation cycle moves cortical granule one step closer to the egg plasma membrane toward the fusion with the plasma membrane

The completion of meiosis means that the extrusion of the chromatids in the second polar body enables the formation of haploid oocytes that can form a female pronucleus that will be able to combine genetic material with the male pronucleus. Meiosis is the ground of sexual reproduction where homologous chromosomes

Ca2+ also plays a role in pronuclear formation by decreasing MAP kinase (MAPK) activity responsible for nuclear envelope assembly [33]. Oscillations terminate with PN formation, and PLCζ localizes into PN [36]. The mechanism by which Ca2+ recruits maternal mRNA for translation and genome activation is not well understood at the moment. There are some data indicating that calcium oscillations and mRNA translation are coupled [37]. Not only in cytosol, calcium can also diffuse to the nucleus and

Fertilization potential is a change in the membrane potential across the plasma membrane (PM) of the oocyte that is first observed after oocyte-sperm interaction. In many invertebrates, this is in the form of depolarization of the plasma membrane and is proposed to provide a fast block to polyspermy, described in some invertebrates and only a few vertebrates (such as frogs), but not present in mammals [39]. Lately, there have been some discussions about the nature of electrical block in preventing polyspermy [40]. The role of electrical events in the form of depolarization or hyperpolarization of the plasma membrane at fertilization remains unclear. In mammals, there is hyperpolarization of membrane potential as a result of change in potassium conductivity across the plasma membrane [24]. In human oocytes, outward current and long-lasting hyperpolarization of the plasma membrane were described [27]. The channels responsible for this hyperpolarization are calcium-activated potassium channels [41]. Species-specific differences in the channels involved in early electrical responses at fertilization are reviewed

During oocyte maturation, the composition of the channels in the plasma membrane changes, as described in bovine oocytes [28]. The factors regulating

control different cell functions by direct nuclear calcium signaling [38].

recombine—exchange genetic material through chiasmata and generate new genetic combinations that are unique to the offspring. Mammalian oocytes progress through meiosis very slowly; first, the cell cycle is arrested in the dictyate phase of prophase I during the fetal life of a girl and stays in this phase up to 40–50 years. During the menstrual cycle, the recruited oocytes in the ovary progress through the cell cycle under the influence of the hormones. But the meiosis is again arrested at the metaphase of the second meiotic division (metMII). Calcium oscillations at fertilization activate calmodulin-dependent protein kinase II (CaMKII) and switch on the anaphase-promoting complex/cyclosome (APC/C) that leads to securin and cyclin B1 degradation necessary for cell cycle progression and segregation of sister

**42**

in [42].

Data from studies of fertilization pointed to a sperm component that has to trigger response in the form of calcium oscillations. But the exact component, its nature, and mechanism were long unknown.

There were four main hypotheses, reviewed in [44]. The first one assumed that sperm delivers calcium to the oocyte that further stimulates release of calcium from intracellular stores. The conduit hypothesis assumed that sperm increases the permeability of the plasma membrane for calcium that enters the oocyte with influx from the surroundings. The contact hypothesis predicts that sperm interacts with a receptor on the plasma membrane that causes calcium release from intracellular stores. But success of the ICSI method revealed that there is no need for interaction of sperm and receptors in the plasma membrane for fertilization. The fourth is the sperm factor hypothesis that assumes that there is a component in the sperm cell delivered in cytosol with sperm and that this factor causes calcium release from intracellular stores.

Experiments where soluble sperm extracts are injected into the oocyte coupled with different biochemical approaches enabled the search for unknown sperm factor and were in favor of the sperm factor hypothesis [45, 46].

There were many candidates such as oscillin, a cytosolic sperm factor related to prokaryote glucosamine phosphate deaminase [47]. In nonmammalian species, PLCγ was identified [48] and the role of nitric oxide in fertilization was investigated [49]. In mammals post-acrosomal WW domain-binding protein (PAWP) was described, which is located in the post-acrosomal region of the sperm, from the stage of elongated spermatids onwards, that causes meiosis resumption and PN formation [50].

#### *4.3.1 PLCζ*

When it was demonstrated that sperm extracts were related to InsP3 concentrations in cell and PLC activity [51], phospholipases were under investigation. Genetic data from the testis' cDNA library revealed some new isoform of PLC [23] and soon a novel sperm-specific phospholipase C, PLCζ was identified as a trigger of calcium oscillations in mouse eggs [52]. In the work of Saunders et al., it was experimentally demonstrated that PLCζ content in a single sperm evoked oscillations and normal embryonic development [52]. They also prepared PLCζ complementary RNA (cRNA) for injection into MII oocytes that triggered the same effect. When they removed PLCζ from sperm extracts, they no longer induced calcium oscillations.

Important evidence that PLCζ is the necessary trigger for calcium oscillations comes also from the studies of Dpy19l2 knockout mice that have globozoospermic sperm phenotype and absence of or extremely reduced PLC ζ and no ability to trigger calcium oscillations [53]. But proof in the form of the knockout mouse model was missing. By using RNA interference technology that prevents translation of PLCζ mRNA and that reduces the amount of PLCζ protein in transgenic mouse sperm, altered calcium oscillation patterns, lower egg activation, and no transgenic offspring [54] were observed.

A study where whole exome sequencing was performed in patients with previous TFF, homozygous missense mutation in PLCζ was established [55].

PLCζ was identified in many mammalian and nonmammalian species [56, 57] and can act across species. But there are differences in solubility of PLCζ in cytosol that can contribute to differences between species. It is proven in the mouse oocyte activation test (MOAT) that human PLCζ exhibits greater response in mouse oocytes than mouse PLCζ.

The amount of PLCζ measured in a single mouse sperm cell is in the same range as the amount required to cause oscillations experimentally [52]. This can explain why altered frequency of oscillations was established when more than one sperm fertilized an oocyte [58]. PLCζ has to diffuse through cytosol to trigger spatiotemporal response in the form of a calcium wave that spreads from the point of sperm entry to the other pole.

Recently, the knockout mouse model was prepared using CRISPR/Cas9 gene editing [59] and revealed interesting facts: PLCζ-null mouse males have normal spermatogenesis and normal sperm parameters (motility, capacitation, and acrosome reaction) but their sperm cannot elicit calcium oscillations after ICSI. Still, some oocytes can undergo activation in abnormal form or even develop to blastocyst stage. But mating knockout males with normal females shows that they can still produce offspring without PLCζ as a physiological trigger. Males are not infertile, rather subfertile, so there is possibility that apart from PLCζ, there is a second calcium releasing factor delivered to oocyte by sperm, perhaps an alternative that is used only when PLCζ is missing.

Calcium oscillations are normal physiological stimuli for oocyte activation but from parthenogenetic activation at ICSI and from the use of artificial oocyte activation techniques we already know that they are not always necessary to activate oocytes. They can be bypassed on some levels.

There are still many facts about calcium oscillations triggering that are not well understood. The proposed mechanism of activation is reviewed in [23] and represented in a schematic diagram (**Figure 2**): PLCζ diffuses from the sperm head into egg cytosol and hydrolyses the PIP2 (phosphatidylinositol 4,5-bisphosphate) in the membrane of the vesicle compartment into products InsP3 (inositol 1,4,5-trisphosphate) and DAG (diacylglycerol). InsP3 binds to the receptor InsP3R on the endoplasmic reticulum membrane. The conformation of the channel is changed and it becomes permeable for Ca2+ ions that are released from intracellular storage in the endoplasmic reticulum into cytosol. Oscillations are generated through a positive feedback loop.

In a series of experiments on mouse oocytes injected with RNA encoding PLCζ and tagged with a fluorescent protein, it was established that at the time when calcium oscillations terminate and pronucleus is formed, PLCζ protein translocates from the cytosol to the pronucleus [36]. Authors also demonstrated that it is again released in the cytosol in the first mitotic division of an embryo at the time of nuclear envelope breakdown and observed also in the second mitotic division. This suggests the possible role of calcium oscillations not only in oocyte activation but also in early embryonic development.

**45**

*PN—pronucleus.*

**Figure 2.**

All together there is a lot of accumulating evidence that points toward PLCζ as a

trigger of oocyte activation cascade in mammals. Soon the idea of using recombinant PLCζ in clinical practice emerged that will be discussed later among other

*Schematic diagram of the proposed model of sperm triggered oocyte activation at fertilization in mammals: after the fusion of sperm and oocyte plasma membrane PLCζ diffuses from sperm into oocyte cytosol. Vesicles baring PIP2 are present in cytosol and PLCζ hydrolyzes PIP2 into products DAG and InsP3. InsP3 binds to the InsP3R present in the membrane of ER. InsP3R is a ligand-gated Ca2+ release channel and Ca2+ is released from intracellular stores in ER into the cytosol. In mammals, repetitive Ca2+ oscillations occur, and several rises of calcium concentration in cytosol take place. Ca2+ ions play an essential role in oocyte activation. They enable block of polyspermy by chemically altering zona pellucida with the content of cortical granules that are released in the perivitelline space. Ca2+ ions enable cell cycle progression—resumption of meiosis II. Ca2+ dependent inactivation of factors that hold cell cycle in arrested state takes place, by degradation of cyclinB1 and securin and MPF inactivation of cell cycle eventually progresses. Ca2+ ions play a role in recruitment of mRNAs and affect their translation. Ca2+ can diffuse in the nucleus and play a role in embryonic gene activation. Ca2+ oscillations terminate when female pronucleus is formed and PLCζ relocalizes in the pronucleus. PLCζ—phospholipase C zeta, PIP2—phosphatidylinositol 4,5-bisphosphate, InsP3—inositol 1,4,5-trisphosphate, DAG—diacylglycerol, InsP3R—inositol 1,4,5-trisphosphate receptor, Ca2+—calcium ions, ZP—zona pellucida (the glycoprotein envelope surrounding mammalian oocyte), PV—perivitelline space (space between ZP and plasma membrane of oocyte), PM—plasma membrane, cg—cortical granules, and* 

*Oocyte Activation Failure: Physiological and Clinical Aspects*

*DOI: http://dx.doi.org/10.5772/intechopen.83488*

*Oocyte Activation Failure: Physiological and Clinical Aspects DOI: http://dx.doi.org/10.5772/intechopen.83488*

#### **Figure 2.**

*Embryology - Theory and Practice*

offspring [54] were observed.

oocytes than mouse PLCζ.

of sperm entry to the other pole.

when PLCζ is missing.

oocytes. They can be bypassed on some levels.

also in early embryonic development.

Important evidence that PLCζ is the necessary trigger for calcium oscillations comes also from the studies of Dpy19l2 knockout mice that have globozoospermic sperm phenotype and absence of or extremely reduced PLC ζ and no ability to trigger calcium oscillations [53]. But proof in the form of the knockout mouse model was missing. By using RNA interference technology that prevents translation of PLCζ mRNA and that reduces the amount of PLCζ protein in transgenic mouse sperm, altered calcium oscillation patterns, lower egg activation, and no transgenic

A study where whole exome sequencing was performed in patients with previ-

PLCζ was identified in many mammalian and nonmammalian species [56, 57] and can act across species. But there are differences in solubility of PLCζ in cytosol that can contribute to differences between species. It is proven in the mouse oocyte activation test (MOAT) that human PLCζ exhibits greater response in mouse

The amount of PLCζ measured in a single mouse sperm cell is in the same range as the amount required to cause oscillations experimentally [52]. This can explain why altered frequency of oscillations was established when more than one sperm fertilized an oocyte [58]. PLCζ has to diffuse through cytosol to trigger spatiotemporal response in the form of a calcium wave that spreads from the point

Recently, the knockout mouse model was prepared using CRISPR/Cas9 gene editing [59] and revealed interesting facts: PLCζ-null mouse males have normal spermatogenesis and normal sperm parameters (motility, capacitation, and acrosome reaction) but their sperm cannot elicit calcium oscillations after ICSI. Still, some oocytes can undergo activation in abnormal form or even develop to blastocyst stage. But mating knockout males with normal females shows that they can still produce offspring without PLCζ as a physiological trigger. Males are not infertile, rather subfertile, so there is possibility that apart from PLCζ, there is a second calcium releasing factor delivered to oocyte by sperm, perhaps an alternative that is used only

Calcium oscillations are normal physiological stimuli for oocyte activation but from parthenogenetic activation at ICSI and from the use of artificial oocyte activation techniques we already know that they are not always necessary to activate

There are still many facts about calcium oscillations triggering that are not well understood. The proposed mechanism of activation is reviewed in [23] and represented in a schematic diagram (**Figure 2**): PLCζ diffuses from the sperm head into egg cytosol and hydrolyses the PIP2 (phosphatidylinositol 4,5-bisphosphate) in the membrane of the vesicle compartment into products InsP3 (inositol 1,4,5-trisphosphate) and DAG (diacylglycerol). InsP3 binds to the receptor InsP3R on the endoplasmic reticulum membrane. The conformation of the channel is changed and it becomes permeable for Ca2+ ions that are released from intracellular storage in the endoplasmic reticulum into cytosol. Oscillations are generated through a positive feedback loop. In a series of experiments on mouse oocytes injected with RNA encoding PLCζ and tagged with a fluorescent protein, it was established that at the time when calcium oscillations terminate and pronucleus is formed, PLCζ protein translocates from the cytosol to the pronucleus [36]. Authors also demonstrated that it is again released in the cytosol in the first mitotic division of an embryo at the time of nuclear envelope breakdown and observed also in the second mitotic division. This suggests the possible role of calcium oscillations not only in oocyte activation but

ous TFF, homozygous missense mutation in PLCζ was established [55].

**44**

*Schematic diagram of the proposed model of sperm triggered oocyte activation at fertilization in mammals: after the fusion of sperm and oocyte plasma membrane PLCζ diffuses from sperm into oocyte cytosol. Vesicles baring PIP2 are present in cytosol and PLCζ hydrolyzes PIP2 into products DAG and InsP3. InsP3 binds to the InsP3R present in the membrane of ER. InsP3R is a ligand-gated Ca2+ release channel and Ca2+ is released from intracellular stores in ER into the cytosol. In mammals, repetitive Ca2+ oscillations occur, and several rises of calcium concentration in cytosol take place. Ca2+ ions play an essential role in oocyte activation. They enable block of polyspermy by chemically altering zona pellucida with the content of cortical granules that are released in the perivitelline space. Ca2+ ions enable cell cycle progression—resumption of meiosis II. Ca2+ dependent inactivation of factors that hold cell cycle in arrested state takes place, by degradation of cyclinB1 and securin and MPF inactivation of cell cycle eventually progresses. Ca2+ ions play a role in recruitment of mRNAs and affect their translation. Ca2+ can diffuse in the nucleus and play a role in embryonic gene activation. Ca2+ oscillations terminate when female pronucleus is formed and PLCζ relocalizes in the pronucleus. PLCζ—phospholipase C zeta, PIP2—phosphatidylinositol 4,5-bisphosphate, InsP3—inositol 1,4,5-trisphosphate, DAG—diacylglycerol, InsP3R—inositol 1,4,5-trisphosphate receptor, Ca2+—calcium ions, ZP—zona pellucida (the glycoprotein envelope surrounding mammalian oocyte), PV—perivitelline space (space between ZP and plasma membrane of oocyte), PM—plasma membrane, cg—cortical granules, and PN—pronucleus.*

All together there is a lot of accumulating evidence that points toward PLCζ as a trigger of oocyte activation cascade in mammals. Soon the idea of using recombinant PLCζ in clinical practice emerged that will be discussed later among other

methods for artificial oocyte activation. But still there are many data missing that are needed to fully understand oocyte activation.

#### *4.3.2 Other sperm-related factors that affect oocyte activation*

Spermatozoon has to go through many changes in order to be able to fertilize an oocyte naturally. First, the mechanism of chemotaxis between the gametes plays an important role; capacitation is the process of altering the sperm plasma membrane so that it becomes more permanent for calcium ions, then changes in sperm movements in the form of hyperactivation help to bring the spermatozoon closer to the oocyte. Finally, at acrosome reaction the content of acrosomes (enzymes) facilitates the fusion between the plasma membrane of the oocyte and spermatozoon so that the paternal genetic material can enter the oocyte. Sperm also delivers a centriole into the oocyte that duplicates and forms centrosome, a microtubuleorganizing center responsible for mitotic divisions in a growing embryo. ICSI bypasses many of these events and enables fertilization and successful development, but there are still sperm factors other than PLCζ that can contribute to failure of oocyte activation.

Successful sperm chromatin decondensation is a necessary condition for fertilization. The chromatin of the spermatozoon is uniquely packaged in such a way that histones become replaced by protamines during the spermatogenesis. Protamines provide more structural stability but after the entry of the spermatozoon into the oocyte the chromatin of the spermatozoon must be uncoiled and protamines must be replaced by histones. Proteins and other factors in the cytosol of an oocyte play a role in the correct decondensation of male genetic material.

Mitosis-promoting factor (MPF) in the cytosol of an oocyte can cause premature chromosome condensation (PCC), but perhaps it is not only an oocyte-related problem. It was established that protamine-deficient sperm seems to be related to a higher proportion of PCC independent of oocyte cytoplasmic maturity [60, 61]. The cell cycle of spermatozoa is related to chromatin status and protamine-histone remodeling must be synchronized with the oocyte.

#### **4.4 Oocyte factors**

If PLCζ depletion in sperm is a good candidate for explaining fertilization failure of male origin, less is known about different oocyte defects that cause unsuccessful activation. It is obvious that oocyte maturation is crucial and competent oocytes of good quality with all the necessary elements in the downstream cascade must be available. In the process of oocyte maturation, not only the elements responsible for generating calcium oscillations must develop but also all other elements such as those responsible for exocytosis of cortical granules, the necessary actin cortical cytoskeleton, and energy resources must be available.

The direct proof of oocyte-borne defects is the results of the mouse oocyte activation test (MOAT) that will be discussed in detail later. It is a heterologous model where mouse oocytes are fertilized with the patient's sperm. Successful fertilization of mouse oocytes points toward oocyte defects that are the underlying cause of previous fertilization failure in ART treatments in a specific couple.

#### *4.4.1 Organelle distribution*

Studies of the ultrastructure of unfertilized oocytes with transmission or scanning electron microscopy revealed differences in oocyte ultrastructure that can reflect different stages in oocyte maturation [62].

**47**

*Oocyte Activation Failure: Physiological and Clinical Aspects*

Cortical granule migration toward the plasma membrane is an important step in

Some studies have investigated the relationship between mitochondrial function and fertilization. Unfertilized oocytes exhibit a higher proportion of mtDNA deletions that may contribute to their malfunction and ATP production [64]. As the early embryo requires a lot of energy, it is important that during oocyte maturation

Reorganization of the endoplasmic reticulum (ER) during maturation seems to play an important role in oocyte competence to generate calcium oscillations. Visualization of the ER in mouse oocytes revealed that in prophase I (in the germinal vesicle stage) the ER is in the form of a fine network with patch-like accumulations in the inner cytoplasm. After the resumption of meiosis, the ER accumulates in the form of a dense ring in the center of the oocyte, around the meiotic apparatus; later the ER rings move together with a meiotic spindle toward the oocyte cortex [65]. In oocytes in metaphase II, the ER ring transforms into clusters in the cortical region; in the central cytoplasm the reticular form is present [65]. These researchers also showed that these relocalizations happen independently from meiotic progression and that microtubules, dyneins, and actins are responsible for the movements. During oocyte maturation, the Ins3R receptors responsible for calcium release from the endoplasmic reticulum achieve their functionality. In a mouse model, it was demonstrated that increase in IP3R1 sensitivity is underpinned, at least in part, by increases in calcium concentrations within the endoplasmic reticulum and

cytoplasmic maturation and it is a cytoskeleton-dependent process [63].

a sufficient number and functionality of the mitochondria are prepared.

receptor phosphorylation but not by changes in IP3R1 distribution [66].

Distribution of vesicles with PIP2 is also important. PLCζ diffuses from the sperm head into egg cytosol and acts on PIP2 that is present in the membrane of small vesicles in the cytoplasm. Defects or deficits of PIP2 or vesicles could contrib-

Evaluation of oocyte maturity relies on the presence of the first polar body, but it is difficult to evaluate in daily IVF laboratory practice whether the oocyte cyto-

Cytoplasmic maturity is an important factor determining the ability of the oocyte to activate. During oocyte cytoplasmic maturation, the mechanisms responsible for sperm-induced calcium oscillations and oocyte activation develop

It was experimentally shown in LT/Sv mouse strain (that has abnormal oocyte nuclear maturation arrested at metaphase I) that the ability of these oocytes to be activated by sperm develops gradually during cytoplasmic maturation independent

Oocyte cytoplasmic maturity also plays a role in decondensation of the sperm genetic material that is tightly packed with protamines. Oocyte immaturity is correlated to the occurrence of premature chromosome condensation (PCC) of the

Evaluation of cytoplasmic maturity with immunocytochemical methods revealed that metaphase plate rearrangements are more frequent in oocytes showing immaturity [70]. Another study investigated abnormal maturation in patients

It is obvious that the conditions in which oocytes mature are important and beside patient-related factors there are also cycle-specific factors that have an impact on oocyte maturity, quality, and fertilization. Little is known about the cellular mechanisms of how diet, medicament uptake, or tobacco/alcohol intake affect

*DOI: http://dx.doi.org/10.5772/intechopen.83488*

ute to fertilization failure [30].

*4.4.2 Cytoplasmic maturation*

and are reviewed in [67].

of nuclear maturation [68].

with a high proportion of immature oocytes [71].

male pronucleus [69].

plasm is mature.

#### *Oocyte Activation Failure: Physiological and Clinical Aspects DOI: http://dx.doi.org/10.5772/intechopen.83488*

*Embryology - Theory and Practice*

failure of oocyte activation.

**4.4 Oocyte factors**

*4.4.1 Organelle distribution*

are needed to fully understand oocyte activation.

*4.3.2 Other sperm-related factors that affect oocyte activation*

role in the correct decondensation of male genetic material.

remodeling must be synchronized with the oocyte.

cytoskeleton, and energy resources must be available.

reflect different stages in oocyte maturation [62].

previous fertilization failure in ART treatments in a specific couple.

methods for artificial oocyte activation. But still there are many data missing that

Spermatozoon has to go through many changes in order to be able to fertilize an oocyte naturally. First, the mechanism of chemotaxis between the gametes plays an important role; capacitation is the process of altering the sperm plasma membrane so that it becomes more permanent for calcium ions, then changes in sperm movements in the form of hyperactivation help to bring the spermatozoon closer to the oocyte. Finally, at acrosome reaction the content of acrosomes (enzymes) facilitates the fusion between the plasma membrane of the oocyte and spermatozoon so that the paternal genetic material can enter the oocyte. Sperm also delivers a centriole into the oocyte that duplicates and forms centrosome, a microtubuleorganizing center responsible for mitotic divisions in a growing embryo. ICSI bypasses many of these events and enables fertilization and successful development, but there are still sperm factors other than PLCζ that can contribute to

Successful sperm chromatin decondensation is a necessary condition for fertilization. The chromatin of the spermatozoon is uniquely packaged in such a way that histones become replaced by protamines during the spermatogenesis. Protamines provide more structural stability but after the entry of the spermatozoon into the oocyte the chromatin of the spermatozoon must be uncoiled and protamines must be replaced by histones. Proteins and other factors in the cytosol of an oocyte play a

Mitosis-promoting factor (MPF) in the cytosol of an oocyte can cause premature chromosome condensation (PCC), but perhaps it is not only an oocyte-related problem. It was established that protamine-deficient sperm seems to be related to a higher proportion of PCC independent of oocyte cytoplasmic maturity [60, 61]. The cell cycle of spermatozoa is related to chromatin status and protamine-histone

If PLCζ depletion in sperm is a good candidate for explaining fertilization failure of male origin, less is known about different oocyte defects that cause unsuccessful activation. It is obvious that oocyte maturation is crucial and competent oocytes of good quality with all the necessary elements in the downstream cascade must be available. In the process of oocyte maturation, not only the elements responsible for generating calcium oscillations must develop but also all other elements such as those responsible for exocytosis of cortical granules, the necessary actin cortical

The direct proof of oocyte-borne defects is the results of the mouse oocyte activation test (MOAT) that will be discussed in detail later. It is a heterologous model where mouse oocytes are fertilized with the patient's sperm. Successful fertilization of mouse oocytes points toward oocyte defects that are the underlying cause of

Studies of the ultrastructure of unfertilized oocytes with transmission or scanning electron microscopy revealed differences in oocyte ultrastructure that can

**46**

Cortical granule migration toward the plasma membrane is an important step in cytoplasmic maturation and it is a cytoskeleton-dependent process [63].

Some studies have investigated the relationship between mitochondrial function and fertilization. Unfertilized oocytes exhibit a higher proportion of mtDNA deletions that may contribute to their malfunction and ATP production [64]. As the early embryo requires a lot of energy, it is important that during oocyte maturation a sufficient number and functionality of the mitochondria are prepared.

Reorganization of the endoplasmic reticulum (ER) during maturation seems to play an important role in oocyte competence to generate calcium oscillations. Visualization of the ER in mouse oocytes revealed that in prophase I (in the germinal vesicle stage) the ER is in the form of a fine network with patch-like accumulations in the inner cytoplasm. After the resumption of meiosis, the ER accumulates in the form of a dense ring in the center of the oocyte, around the meiotic apparatus; later the ER rings move together with a meiotic spindle toward the oocyte cortex [65]. In oocytes in metaphase II, the ER ring transforms into clusters in the cortical region; in the central cytoplasm the reticular form is present [65]. These researchers also showed that these relocalizations happen independently from meiotic progression and that microtubules, dyneins, and actins are responsible for the movements.

During oocyte maturation, the Ins3R receptors responsible for calcium release from the endoplasmic reticulum achieve their functionality. In a mouse model, it was demonstrated that increase in IP3R1 sensitivity is underpinned, at least in part, by increases in calcium concentrations within the endoplasmic reticulum and receptor phosphorylation but not by changes in IP3R1 distribution [66].

Distribution of vesicles with PIP2 is also important. PLCζ diffuses from the sperm head into egg cytosol and acts on PIP2 that is present in the membrane of small vesicles in the cytoplasm. Defects or deficits of PIP2 or vesicles could contribute to fertilization failure [30].

#### *4.4.2 Cytoplasmic maturation*

Evaluation of oocyte maturity relies on the presence of the first polar body, but it is difficult to evaluate in daily IVF laboratory practice whether the oocyte cytoplasm is mature.

Cytoplasmic maturity is an important factor determining the ability of the oocyte to activate. During oocyte cytoplasmic maturation, the mechanisms responsible for sperm-induced calcium oscillations and oocyte activation develop and are reviewed in [67].

It was experimentally shown in LT/Sv mouse strain (that has abnormal oocyte nuclear maturation arrested at metaphase I) that the ability of these oocytes to be activated by sperm develops gradually during cytoplasmic maturation independent of nuclear maturation [68].

Oocyte cytoplasmic maturity also plays a role in decondensation of the sperm genetic material that is tightly packed with protamines. Oocyte immaturity is correlated to the occurrence of premature chromosome condensation (PCC) of the male pronucleus [69].

Evaluation of cytoplasmic maturity with immunocytochemical methods revealed that metaphase plate rearrangements are more frequent in oocytes showing immaturity [70]. Another study investigated abnormal maturation in patients with a high proportion of immature oocytes [71].

It is obvious that the conditions in which oocytes mature are important and beside patient-related factors there are also cycle-specific factors that have an impact on oocyte maturity, quality, and fertilization. Little is known about the cellular mechanisms of how diet, medicament uptake, or tobacco/alcohol intake affect oocyte quality. In a review of [72], the environmental impact on oocyte function through mitochondrial level is discussed.

#### **5. Artificial oocyte activation (AOA)**

Artificial oocyte activation methods try to induce oocyte activation by using physiological properties of the gametes and in this way interfere in different levels of the cascade of events during fertilization. In general, they try to alleviate intracellular calcium concentration and mimic oscillations. As we are well aware by now that there is big species-specific variability in the mechanisms of oocyte activation, it is not surprising that different AOA methods can be successful in one species, but not in another.

By influencing gamete physiological properties such as electrical excitability and plasma membrane conductivity, the aim is to increase intracytoplasmic calcium concentrations and mimic the frequency and amplitude of the oscillations.

Basically, there are three types of AOA methods: electrical, chemical, and mechanical.

#### **5.1 Electrical methods**

By applying direct electrical current within a Petri dish with oocytes, the electrical field stimulates charged proteins to move toward the plasma membrane, and by this, the number of pores in the plasma membrane increases [30]. Calcium conductivity increases, and more calcium enters the oocyte from the surroundings. There is only one large calcium concentration increase.

In the prospective randomized study of [73], an electrical pulse in a special chamber with electrodes 30 minutes after ICSI in 0.3 M glucose drops was used to activate oocytes, and a small increase in the fertilization rate after ICSI was achieved. Successful pregnancy and birth were achieved and reported in the case report [74].

In a study with round spermatid injection coupled with electrostimulation, the electrical pulse triggered not only a single calcium concentration increase, but a series of calcium spikes after spermatid injection [75].

#### **5.2 Chemical methods**

Chemical activation is the most commonly used method. Oocytes are exposed to chemical agents that lead to an increase in intracellular calcium concentration in the oocyte. Some agents, such as calcium ionophores cause a single, prolonged calcium transient, while others cause multiple oscillations.

#### *5.2.1 Ionophores*

Calcium ionophores, such as ionomycin, A23187 (calcimycin), and gm508 are molecules soluble in lipids, synthesized by microorganisms; today several synthetic compounds are known. They can transport ions across lipid bilayers. They increase membrane permeability for Ca2+ ions, thus allowing calcium influx in the oocyte from the surrounding medium and intracytoplasmic rise of calcium concentration. It has been recently established that intracytoplasmic rise of calcium concentration in human and mouse oocytes is not only the consequence of the influx from the surroundings but also from intracellular stores, since this rise appears also in calcium-free medium. However, they are not able to induce calcium oscillations

**49**

*Oocyte Activation Failure: Physiological and Clinical Aspects*

most widely used chemical agents for artificial oocyte activation.

typical for mammalian species but a single rise. Different protocols are described in the literature, regarding different concentrations used, and intervals of ionophore

They are used in cases of repeating TFF or low fertilization, oligoteratoasthenospermia cases, globozoospermia, in vitro maturation of oocytes (IVM), unexplained female infertility, and low ovarian reserve, with patients with Kartagener's syndrome with no response on theophylline, at primary ciliary dyskinesia. They are the

It is reported as very efficient in mice and induces not only single calcium concentration elevation, but oscillations [80]. The mechanism by which it induces

A study that investigated efficacy of SrCl2 in human oocytes showed significantly increased fertilization rates, when compared with conventional ICSI or

Soon after the discovery of the role of PLCζ as a trigger of oocyte activation, the ideas of using the protein as an artificial activator emerged. The synthesis of the first recombinant human PLCζ protein was published [82]; when injected into mouse oocytes, calcium oscillations were evoked that closely resembled those initiated by the sperm after fertilization. Later, a study where human recombinant PLCζ was used on human and mouse oocytes was published [83] describing dose-dependent manner of calcium oscillations. These authors also showed that by injecting recombinant human PLCζ the next day in oocytes that failed to fertilize after ICSI,

Earlier, it was established that PLCζ complementary RNA (cRNA) injection in MII oocytes also triggered oscillations [52] with a time lag that enables protein to synthesize. The commercial use of recombinant human PLCζ still has to be validated in

In veterinary medicine, ethanol is often used for parthenogenetic oocyte activation. Parthenogenesis is development of an embryo from an unfertilized oocyte, naturally occurring in invertebrates or even some vertebrates. By inhibiting the second polar body extrusion, diploid parthenotes with two maternal genomes can be created and embryos can develop normally for several days, but later die. In several species, artificial parthenogenetic activation was described to be caused by

Some data from the literature suggest that the modified ICSI technique can give better fertilization in patients with a history of TFF or low fertilization [85, 86]. Vigorous aspiration of cytoplasm and a different position of the pipette tip when ejecting sperm in the oocyte is supposed to increase calcium levels during injection

and enable better contact of sperm with intracellular storage of calcium.

*DOI: http://dx.doi.org/10.5772/intechopen.83488*

exposition [76–79].

*5.2.2 Strontium chloride (SrCl2)*

oscillations is not fully understood.

calcium ionophore treatment [81].

five of eight oocytes were rescued.

*5.2.3 PLCζ*

terms of safety.

*5.2.4 Ethanol*

ethanol [84].

**5.3 Mechanical methods**

#### *Oocyte Activation Failure: Physiological and Clinical Aspects DOI: http://dx.doi.org/10.5772/intechopen.83488*

typical for mammalian species but a single rise. Different protocols are described in the literature, regarding different concentrations used, and intervals of ionophore exposition [76–79].

They are used in cases of repeating TFF or low fertilization, oligoteratoasthenospermia cases, globozoospermia, in vitro maturation of oocytes (IVM), unexplained female infertility, and low ovarian reserve, with patients with Kartagener's syndrome with no response on theophylline, at primary ciliary dyskinesia. They are the most widely used chemical agents for artificial oocyte activation.
