Once a sperm penetrates the cell wall of an egg and fertilizes it, this structure is known as what?

Attachment to and Penetration of the Zona Pellucida

Thezona pellucida, which is 13 μm thick in humans, consists principally of four glycoproteins—ZP1 to ZP4. ZP2 and ZP3 combine to form basic units that polymerize into long filaments. These filaments are periodically linked by cross-bridges of ZP1 and ZP4 molecules (Fig. 2.5). The zona pellucida of an unfertilized mouse egg is estimated to contain more than 1 billion copies of the ZP3 protein.

After they have penetrated the corona radiata, spermatozoa bind tightly to the zona pellucida by means of the plasma membrane of the sperm head (seeFig. 2.4). Spermatozoa bind specifically to a sialic acid molecule, which is the terminal part of a sequence of four sugars at the end of O-linked oligosaccharides that are attached to the polypeptide core of the ZP3 molecule. Molecules on the surface of the sperm head are specific binding sites for the ZP3 sperm receptors on the zona pellucida. Many molecules have been proposed, but except for a surface protein SED 1, the identity of the zona-binding molecules remains unknown. Interspecies molecular differences in the sperm-binding regions of the ZP3 molecule may serve as the basis for the inability of spermatozoa of one species to fertilize an egg of another species. In mammals, there is less species variation in the composition of ZP3; this may explain why penetration of the zona pellucida by spermatozoa of closely related mammalian species is sometimes possible, whereas it is rare among lower animals.

Upon or slightly before binding to the zona pellucida, mammalian spermatozoa undergo theacrosomal reaction. The essence ofthe acrosomal reaction is the fusion of parts of the outer acrosomal membrane with the overlying plasma membrane and the pinching off of fused parts as small vesicles. This results in the liberation of the multitude of lytic enzymes that are stored in the acrosome.

The acrosomal reaction in mammals is stimulated by the ZP3 molecule acting through G proteins in the plasma membrane on the sperm head. In contrast to the sperm-receptor function of ZP3, a large segment of the polypeptide chain of the ZP3 molecule must be present to induce the acrosomal reaction. An initiating event of the acrosomal reaction is a massive influx of calcium (Ca++) through the plasma membrane of the sperm head. This process, accompanied by an influx of sodium (Na+) and an efflux of hydrogen (H+), increases the intracellular pH. Fusion of the outer acrosomal membrane with the overlying plasma membrane soon follows. As the vesicles of the fused membranes are shed, the enzymatic contents of the acrosome are freed and can assist the spermatozoa in making their way through the zona pellucida.

After the acrosomal reaction, the inner acrosomal membrane forms the outer surface covering of most of the sperm head (seeFig. 2.4D). Toward the base of the sperm head (in the equatorial region), the inner acrosomal membrane fuses with the remainingpostacrosomal plasma membrane to maintain membrane continuity around the sperm head.

Sperm Function Tests

A number of tests of sperm function are available to examine the human fertilization process (Fig. 141-3). These are only performed in specialist laboratories. If simpler approaches or active preparations of zona pellucida (ZP) or sperm receptor proteins become available, they will be widely used to improve the assessment of human sperm. IVF has permitted many conventional and new tests of sperm function to be examined. Groups of sperm variables that are independently significantly related to the proportion of oocytes that fertilize in vitro can be determined by regression analysis.66 This approach has confirmed the importance of sperm morphology in the ability of sperm to interact with the coverings of the oocyte.

Zona Pellucida

During the entire period from ovulation until entry into the uterine cavity, the ovum and the embryo are surrounded by the zona pellucida. During this time, the composition of the zona changes through contributions from the blastomeres and the maternal reproductive tissues. These changes facilitate the transport and differentiation of the embryo. After the embryo reaches the uterine cavity, it begins to shed the zona pellucida in preparation for implantation. This is accomplished by a process calledblastocyst hatching. A small region of the zona pellucida, usually directly over the inner cell mass in the primate, dissolves, and the blastocyst emerges from the hole. In rodents, blastocyst hatching is accomplished through the action of cysteine protease enzymes that are released from long microvillous extensions (trophectodermal projections) protruding from the surfaces of the trophoblastic cells. During a narrow time window (4 hours in rodents), the zona pellucida in this area is digested, and the embryo begins to protrude. In the uterus, the trophectodermal projections then contact the endometrial epithelial cells as the process of implantation begins. Enzymatic activity around the entire trophoblast soon begins to dissolve the rest of the zona pellucida. Only a few specimens of human embryos have been taken in vivo from the period just preceding implantation, but in vitro studies on human embryos suggest a similar mechanism, which probably occurs 1 to 2 days before implantation (seeFig. 4.3C).Box 4.1 summarizes the functions of the zona pellucida.

Enhancing the Implantation Potential of Human Embryos

Several methods have been developed to artificially disrupt the zona pellucida, the glycoprotein coat surrounding the embryo. These include partial zona dissection, zona drilling, and zona thinning achieved through the use of acid tyrode, proteinase, piezo vibrator manipulators, and lasers. The premise behind these methods, known as assisted hatching techniques, is that failure of implantation might be related in part to an inability of the blastocyst to escape from its zona pellucida. A meta-analysis of studies involving 2752 women provided no evidence that assisted hatching has an impact on live birth rate.68

Fertilization begins as the sperm cell attaches to the zona pellucida and undergoes the acrosomal reaction, and it ends with the fusion of the male and female pronuclei

After ovulation, the egg in the fallopian tube is in a semidormant state. If it remains unfertilized, the ripe egg will remain quiescent for some time and eventually degenerate. When fertilization occurs, the sperm normally comes into contact with the oocyte in the ampullary portion of the tube, usually several hours after ovulation. Fertilization causes the egg to awaken(activation) and initiates a series of morphological and biochemical events that lead to cell division and differentiation.Fertilization occurs in eight steps:

Step 1: The sperm head weaves its way past the follicular cells and attaches to the zona pellucida that surrounds the oocyte (Fig. 56-1). The zona pellucida is composed of three glycoproteins; ZP1 cross-links the filamentous ZP2 and ZP3 into a latticework. Receptors on the plasma membrane of the sperm cell bind to ZP3, thereby initiating a signal-transduction cascade.

Step 2: As a result of the sperm-ZP3 interaction, the sperm cell undergoes the acrosomal reaction, a prelude to the migration of the sperm cell through the mucus-like zona pellucida. The acrosome (see p.1103) is a unique sperm organelle containing hydrolyzing enzymes that are necessary for the sperm to penetrate the zona pellucida. During the acrosomal reaction, an increase in intracellular free Ca2+ concentration ([Ca2+]i) triggers fusion of the outer acrosomal membrane with the sperm cell's plasma membrane, resulting in the exocytosis of most of the acrosomal contents.

Step 3: The spermatozoon penetrates through the zona pellucida. One mechanism of this penetration is the action of the acrosomal enzymes.

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Acrosomal Enzymes

Among the many enzymes in the acrosome are acid hydrolases, the best characterized of which is proacrosin, the precursor to acrosin.Acrosin is a member of the serine protease superfamily; it is expressed only in spermatogenic cells. Two other enzymes released from the acrosome areneuraminidase and a special form of hyaluronidase. This particularhyaluronidase can be distinguished from the common lysosomal form of the enzyme, and it appears to be a spermatogenic cell–specific isozyme. Acrosin, hyaluronidase, and neuraminidase help the sperm penetrate the zona pellucida by hydrolyzing the sugar chains and the peptide chains of the glycoproteins of the zona pellucida.

Step 4: The cell membranes of the sperm and the oocyte fuse. Microvilli on the oocyte surface envelop the sperm cell, which probably binds to the oocyte membrane via specific proteins on the surfaces of the two cells. The posterior membrane of the acrosome—which remains part of the sperm cell after the acrosomal reaction—is the first portion of the sperm to fuse with the plasma membrane of the egg. The sperm cell per se does not enter the oocyte. Rather, the cytoplasmic portions of the head and tail enter the oocyte, leaving the sperm-cell plasma membrane behind, an action similar to a snake's crawling out of its skin.

Step 5: The oocyte undergoes the cortical reaction. As the spermatozoon penetrates the oocyte's plasma membrane, it initiates formation of inositol 1,4,5-trisphosphate (IP3); IP3 causes Ca2+ release from internal stores (see p.60), which leads to an increase in [Ca2+]i and [Ca2+]i waves. This rise in [Ca2+]i, in turn, triggers the oocyte's second meiotic division—discussed below (see step 6)—and the cortical reaction. In thecortical reaction, small electron-dense granules that lie just beneath the plasma membrane fuse with the oocyte's plasma membrane. Exocytosis of these granules releases enzymes that act on glyco­proteins in the zona pellucida, causing the zona pellu­cida to harden. This hardening involves the release of polysaccharides that impede the progression of the runners-up (i.e., sperm cells still in the zona pellucida). The cortical reaction also leads to the destruction of ZP receptors, which prevents further binding of sperm cells to the zona pellucida. From a teleological perspective, the cortical granule reaction preventspolyspermy.

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Block to Polyspermy

Unlike the situation in some laboratory animals, in humans the block to polyspermy during fertilization does not involve receptors on the zona pellucida or on the cell membrane. It appears that the block to polyspermy in humans is due only to alterations of the inner aspect of the zona pellucida. In experiments on monospermic oocytes fertilized in vitro, other sperm cells added later can partially penetrate the zona pellucida but do not reach the inner half of the zona. When unfertilized oocytes, monospermic oocytes, and polyspermic oocytes are examined in vitro, similar numbers of sperm are found on and within the zona pellucida of each type of oocyte. Further evidence that the zona pellucida is the primary barrier to polyspermy in humans comes from experiments in which the zona pellucida is removed. In this case, the egg is usually penetrated by many sperm. In the in vitro fertilization of normal human oocytes, the rate of polyspermy is quite low, ~5% to 8%, even though 50,000 to 300,000 sperm are available to the oocyte. Thus, the zona pellucida block to polyspermy in humans is highly efficient.

Step 6: The oocyte completes its second meiotic division. The oocyte, which had been arrested in the prophase of its first meiotic division since fetal life (see p.1073), completed its first meiotic division at the time of the surge of luteinizing hormone (LH), which occurred several hours before ovulation (see p.1116). The result was the first polar body and a secondary oocyte with a haploid number of duplicated chromosomes (seeFig. 53-2C). Before fertilization, this secondary oocyte had begun a second meiotic division, which was arrested in metaphase. The rise in [Ca2+]i inside the oocyte—which the sperm cell triggers, as noted in step 5—causes not only the cortical reaction, but also the completion of the oocyte's second meiotic division. One result is the formation of thesecond polar body, which contains a haploid number of undu­plicated maternal chromosomes.

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Three Polar Bodies

Interestingly, although the small polar bodies are nonfunctional, they contain a full set of chromosomes and are responsive to cell cycle–regulatory mechanisms. Consequently, the first polar body also divides during the second meiotic division to produce a total of three polar bodies and a mature oocyte, each of which contain a haploid number of chromosomes. Thus, as with spermatogenesis, one diploid oogonia produces four haploid daughters; however, in oogenesis only one daughter becomes an functional gamete.

Step 7: The sperm nucleus decondenses and transforms into themale pronucleus, which, like the female pronucleus, contains a haploid number of unduplicated chromosomes (seeFig. 54-7). The cytoplasmic portion of the sperm's tail degenerates.

Step 8: The male and female pronuclei fuse, forming a new cell, thezygote. The mingling of chromosomes (syngamy) can be considered as the end of fertilization and the beginning of embryonic development. Thus, fertilization results in a conceptus that bears 46 chromosomes, 23 from the maternal gamete and 23 from the paternal gamete. Fertilization of the ovum by a sperm bearing an X chromosome produces a zygote with XX sex chromosomes; this develops into a female (see pp.1073–1075). Fertilization with a Y-bearing sperm produces an XY zygote, which develops into a male. Therefore, chromosomal sex is established at fertilization.

Secondary Follicle

The secondary follicle consists of a fully grown oocyte surrounded by a complete zona pellucida, two to eight layers of cuboidal or columnar granulosa cells, and a presumptive theca layer immediately peripheral to the basal lamina (see Fig. 125-4). The acquisition of a theca layer is a major new feature of developing secondary follicles. The presumptive theca consists of several layers of elongated fibroblast-like cells that run radially around the entire follicle (see Fig. 125-2). Theca development is accompanied by angiogenesis. Consequently, the secondary follicle is exposed to important blood hormones such as FSH, LH, and insulin. The mechanisms of theca formation and angiogenesis during secondary follicle development are not clear.1 However, because theca formation requires the presence of follicles, it is believed that granulosa cells, or possibly the oocytes, produce factors that direct this process. Among those peptides under consideration are Kit ligand (KL), GDF-9, insulin, insulin-like growth factor-1 (IGF-1) and/or IGF-2, and activin.39-41 In addition to these growth factors, ample evidence indicates that LH is responsible for early theca cell (TC) differentiation.1,42,43 The degree to which LH influences TC growth is uncertain.

Assisted hatching

Towards the end of the 1980s, Cohen observed that embryos that underwent partial dissection of the zona pellucida had higher implantation rates, concluding that the opening of a hole in the zona pellucida favors the process of hatching suffered by all embryos before implantation in the endometrium. The methodological differences, the different designs of the research works, the different selection criteria for women and embryos, and the small number of cases included in the studies make it difficult to compare groups with AH and control groups. Some authors reported that in women with poor prognosis there is an increase in the implantation rate of 33% in the AH group compared to 6.5% in the control group and 64% versus 19% in pregnancy rates [244]. Stein et al. evaluated 154 patients with at least three failed cycles of IVF with good quality embryo transfer. They observed an increase in the pregnancy rate only in women older than 38 years (23.9% vs 7%) [245]. However, other authors have found no differences in implantation and pregnancy rates. AH can be performed by chemical procedures, using a substance with a very low pH called Tyrode’s acid. Another technology is based on the use of a laser which is capable of integrating a predefined area of the zona pellucida after an energy pulse.

Indications for AH include: advanced age, suboptimal embryos, thick zona pellucida, elevated basal FSH, two or more previously failed IVF cycles, cytoplasmic fragmentation, and slow developmental rate. Also, in frozen-thawed embryos, it has been reported that excessive in vitro culture of cryopreserved embryos, are exacerbated by the freeze–thaw process. This is thought to induce alteration in the glycoprotein matrix leading to zona hardening. Therefore AH of these embryos improved implantation and pregnancy rates. The routine or universal performance of AH on all embryos in IVF/ICSI patients is neither scientific nor appropriate. Randomized trials of AH on all embryos without any selection, revealed that there is no difference in the implantation and pregnancy rates between the treatment and control group [246].

Fertilization

The egg becomes a single-celled embryo, the zygote, when a sperm penetrates the ZP shell and enters the egg cytoplasm to fuse the father’s chromatin with the mother’s chromatin. To do this, hundreds of sperm surround the ovulated ovum, releasing the enzyme (acrosin) that will disperse all the GCs surrounding the ovum. Releasing this enzyme means that the sperm arriving early at the egg will have spent their acrosin and be unable to fertilize the egg. However, once the path is clear to the ZP, a spermatozoon that arrives a bit later will be able to release its acrosin as it touches the ZP shell, allowing it to arrive at the oolemma (outer membrane of the egg). At the moment that this first sperm touches the oolemma, the ovum undergoes a microsecond-long cortical reaction on the inside of this egg’s membrane. This cortical reaction involves thousands of small vesicles (cortical granules) releasing on the inside of the oolemma, an enzyme that changes the egg membrane surface in a way that blocks any other sperm from binding to the membrane. In this way, the ovum is protected from polyspermia (more than one sperm inside the egg), a lethal condition that cannot produce a live baby.

This single sperm that has fused with the oolemma will be drawn into the egg’s cytoplasm, but the egg has enzymes that will cut the attachment of the tail away from the sperm head. Since the ATP energy for the sperm to swim is produced by the mitochondria that reside in the tail, male mitochondria do not normally enter into the egg’s cytoplasm and even if a few do get into the cytoplasm, the egg will “spit them out” by exocytosis.

The sperm head will now go through a process called decondensation. This step is necessary because the chromatin in the sperm head was condensed six times tighter than normal chromosomes that would be involved in mitosis. This sixfold packing is necessary for the sperm heads to be small enough to swim efficiently through the female reproductive tract in order to arrive in the peritoneal cavity to meet the egg as it ovulates out of the ovarian follicle. Decondensation of the single sperm that has been brought into the egg cytoplasm is stimulated by another set of oolemmal enzymes. These unpaired male chromosomes are now called a pronucleus. The female pronucleus, which will have formed in response to the sperm head binding to the egg’s membrane, will migrate towards the male pronucleus, pairing its 23 single chromosomes (1N) with the 23 single male chromosomes (1N). This forms the 23 paired chromosomes (2N) of the new single-celled zygote with all the DNA information necessary to direct the formation of a new human being.

Implantation

Implantation occurs on the 5th day of development. Before the blastocyst can implant it has to shed its zona pellucida. This process is called hatching and is brought about by localized proteolysis of the zona (Perona and Wassarman, 1986) and contractions and expansion of the blastocyst. Once freed from its zona the blastocyst attaches to the epithelium of one of the lateral uterine walls with the murine TE (the TE opposite to and not facing the ICM). The uterine wall attached to the blastocyst responds by bulging into the lumen orienting the ICM either to the anterior or posterior end of the uterine horn. This and the following reorganization results in an invariable orientation of the early embryo. The axis through the ICM towards the opposite pole of the blastocyst parallels the dorsoventral (DV) axis of the mother, the ICM always facing the dorsal side. The future anterior–posterior (AP) axis of the embryo, which becomes evident around day 6.5 of development with the onset of gastrulation, is more or less perpendicular to the AP (longitudinal) axis of the uterine horn (Smith, 1980, 1985).

The significance of this invariant orientation of the embryo with respect to the uterus in the determination of the embryonic axes is not clear, however, since embryos can also develop normally in vitro from preimplantation stages up to the limb bud stage (Chen and Hsu, 1982). A detailed analysis of the orientation of mouse embryos during implantation and a discussion of how this might be achieved and might be related to embryonic axis formation is given by Smith (Smith, 1980, 1985). After attachment to the uterine wall the TE cells invade the degenerating uterine epithelium and penetrate into the endometrium (stroma) of the uterus. The mesenchymal stromal cells respond with increased proliferation resulting in the formation of a thick layer of mesenchymal tissue, the decidua, which encloses the embryo. The implantation sites are readily visible within 1 day after implantation by the decidual swellings of the uterus.

Embryo implantation

Embryo implantation occurs about 7 days after fertilization when the embryo reaches the blastocyst stage and hatches from the zona pellucida. At this time the luminal endometrial epithelial cells are able to interact with the blastocyst. This process occurs over 4–6 days during the mid-luteal phase of the menstrual cycle, and is known as the implantation window (for review, see [57]). An embryo passes through different stages of blastocyst development before implanting into a receptive endometrium. The TE, which gives rise to the placenta, differentiates into the proliferative cytotrophoblast (CTB) and syncytiotrophoblast (STB). The inner cell mass (ICM), which becomes the embryo itself, differentiates into epiblast and primitive endoderm cells [58–60].

Implantation is a process that begins with apposition, continues through attachment of STB outgrowth to the underlying stroma (blastocyst invasion), and ends with decidualization (for review, see [57,61]). The molecular mechanisms of implantation are poorly understood because of the limitations of both animal and in vitro models, but some adhesion molecules and extracellular matrix proteins have been suggested to mediate the first steps of embryo implantation. Some adhesion molecules like L-selectin are present in the TE, and may allow the embryo to initially attach to the endometrium. Other molecules such as integrins and transmembrane glycoproteins function as links between the extracellular matrix and the cytoskeleton [58].

MicroRNAs are emerging as important factors in human embryo implantation (for review, see [61–63]). Human blastocysts are known to release microRNAs that bind to complementary regions of mRNAs, inhibiting translation or destabilizing the gene. The human endometrium also expresses a large number of microRNAs, facilitating endometrial receptivity and embryo implantation. Diferencial expression of micro RNAs have been reported in implanted and nonimplanted embryos and in euploid vs aneuploid blastocysts. Because of the complexity of microRNA signaling, studies so far have been inconsistent in terms of reproducibility of results, and additional research is required [61,64].

The success of pregnancy is based on communication between the endometrium and embryo during the implantation window. During this period, it is important to consider embryo quality and the receptiveness of the endometrium. When these factors are altered, cross-talk between embryo and uterus may be impaired and implantation may fail. The most common complication of pregnancy is miscarriage, ending roughly 15% of gestations; despite technological advancements, the reasons for approximately half of miscarriage cases are still unclear [69]. Although not all the elements that impede endometrial receptivity have been recognized (for review, see [65]), they include female factors (impaired endometrial function, anatomic issues, thrombophilia, etc.), male factors (such as sperm chromatin fragmentation), immunological factors, lifestyle factors (e.g., smoking), genetics, and embryo-related issues [60,66–72].

In terms of genetics, chromosomal abnormalities are the main recognized genetic cause for miscarriage and may account for 60% of cases [73]. Chromosome aneuploidy and polyploidy constitute more than 96% of chromosomal abnormalities in spontaneous abortion and X, Y, 13, 16, 18, 21, and 22 are frequently involved [71]. In a retrospective study, Franasiak et al. demonstrated that the prevalence of aneuploidy is lower in patients between the ages of 26 and 30 (20–27%) than in aging patients in the 31–43 age range (85%) [74]. These same authors also determined that the complexity of aneuploid errors also increased with age, and found higher rates of monosomies in younger patients and more trisomies in older women. According to Colley et al., it is important to identify numerical chromosome errors (monosomy, trisomy, or polyploidy) since these can lead to miscarriage [69]. Yet it has been suggested that 86% of chromosomal abnormalities are numerical, 6% are due to structural alterations, and 8% result from other genetic alterations such as mosaicism [75]. More recently, a study of 1,106 embryo samples found that 59.4% had classic cytogenetic abnormalities [73]. These authors stated that aneuploidy accounted for 85.4% of these abnormalities, triploidy for 10.3%, and structural errors or tetraploidy for the remaining 4.2%. One year later, Jia et al. showed that trisomy 16 was most common among all the aneuploidies and polyploidies analyzed by this group (39.03%), followed by trisomy 22 and X monosomy [71]. In 2018, a retrospective analysis by Pylyp et al. found that approximately 50% of first-trimester miscarriages exhibited chromosomal abnormalities; of this group, 59% were trisomies, 22% were polyploidies, 7.5% were monosomies, 7% were unbalanced structural errors, and 3.8% resulted from multiple aneuploidies [72]. These same authors also noted that maternal age increased chromosomally abnormal miscarriages [75].

Apparently, normal euploid embryos may also fail to develop until birth; other genetic abnormalities may be involved in these cases of pregnancy loss, such as single-nucleotide variants (SNVs) that affect individual genes or clusters of genes (either as gains or losses) that are deleted, duplicated, or disrupted. This is a significant genetic alteration, since it may follow a recessive or X-linked mutation. It is important to note that little is known about the genes and pathways involved in pregnancy loss, and for this reason the clinical significance of many copy number variants are uncertain. Many genetic mechanisms may also be influenced by environmental factors such as diet, medication, pollutants, and lifestyle, which could lead to a cumulative effect culminating in pregnancy loss [69].