Meiosis, Fertilization, and Preimplantation Embryo Development

Introduction

Not so long ago, the intricate journey of the individual gametes through maturation, merging, and then becoming a developmentally competent preimplantation embryo was a complete mystery. Now, these aspects of establishing an early pregnancy are carried out routinely in the clinic by a combination of hormonal treatments, in vitro gamete manipulations, and embryo culture. The goal of this chapter is to illuminate the complex physiological mechanisms that underlie this journey, which is central to understanding how germ cells ensure their transmission to the next generation ( Fig. 9.1 ). These mechanisms are addressed in an approximately sequential timeline.

Fig. 9.1, The cycle of human reproduction.

First considered is how the male and female germ cells prepare their DNA to be passed on to the next generation in the highly regulated process of meiosis. In the female, meiosis is accompanied by numerous cytoplasmic and chromatin changes in preparation for later events of fertilization. Sperm deposited in the vagina have a challenging transit through the female reproductive tract to reach the ovulated egg at the site of fertilization. While in transit, sperm also mature in a process known as capacitation. The capacitated sperm can penetrate the egg’s outer vestments, a journey culminating in sperm-egg adhesion, fusion, and sperm engulfment. Next begins the process of egg activation, which is triggered by a sperm protein that initiates a series of oscillations in intracellular cytoplasmic calcium. These calcium oscillations promote egg activation events including the resumption of meiosis II, extrusion of the second polar body, establishment of blocks to polyspermy, and formation of the male and female pronuclei. Dramatic alterations in chromatin structure are initiated during egg activation and these changes continue following cleavage.

Preimplantation embryo development entails the successful reprogramming of the terminally differentiated gametes into a unique totipotent embryo. Reprogramming depends on the removal of maternally and paternally inherited components and the generation of new embryonic transcripts and protein products that will carry out the developmental program. Maternally inherited factors initiate reprogramming by promoting changes in chromatin architecture that favor transcription. Newly generated embryonic transcription factors and chromatin remodeling proteins continue the reprogramming process, which culminates in embryonic genome activation toward the end of the cleavage stage in human embryos. Finally, the individual blastomeres become tightly associated with each other during compaction, resulting in a morula stage embryo. The inner morula cells eventually form the inner cell mass of the blastocyst stage embryo. The outer morula cells differentiate into trophectoderm cells with tight junctions that form a diffusion barrier. Active transport of ions across the trophectoderm cells promotes formation of the blastocoel, the defining characteristic of a blastocyst-stage embryo. In the final act of preimplantation embryo development, the blastocyst hatches out of the zona pellucida in preparation for implantation and development into a unique individual.

Meiosis Overview

Sexual reproduction requires the mixing of two genomes from male and female gametes. To ensure proper chromosome numbers in offspring, the genomes of these gametes—sperm and egg—must be reduced by half (23 in humans). This reduction is achieved through meiosis, the cell division process that first replicates DNA and then divides it in half two times to generate haploid gametes. These reductions ensure that the chromosome complement is restored to the normal diploid number (46 in humans) after fusion of the gametes.

In addition to making haploid gametes capable of fertilization, meiosis also shuffles the parental genomes to create genetic diversity. Upon meiotic entry, germ cell DNA is replicated in the meiotic S-phase and cohesion between the replicated sister chromatids is established ( Fig. 9.2 ). After DNA replication, parental genomes are shuffled in meiotic prophase I, where the chromosomes of germ cells undergo programmed double-strand breaks, followed by DNA repair. One repair mechanism involves the crossing over, or recombination, of homologous chromosomes. This repair process results in a physical association between homologous chromosomes; the point of association is coined chiasma. These chiasmata are essential for holding the meiotic bivalent (i.e., a tetrad composed of the homologous pairs of sister chromatids; Fig. 9.2 ) together until anaphase I onset. Haploid gametes are created through two rounds of chromosome segregation and cell division, without an intervening round of DNA replication. In addition to recombination, another specialized feature of meiosis is the first chromosome segregation during meiosis I (MI). In MI, sister chromatids remain associated with one another while the homologs segregate; the mitotic M-phase segregates sister chromatids. To achieve this reductional division, modifications to the mitotic M-phase must occur. For example, in mitosis, cohesins, which keep sister chromatids together, are cleaved by the protease separase to allow sister chromatids to separate at anaphase onset ( Fig. 9.2 ). In meiosis, there is a stepwise cleavage of cohesin. First, in MI centromeric cohesin is protected from separase by shugoshin (SGO2 in mammals). Cleavage of cohesin along the chromosome arms in anaphase I separates the homologs of the bivalent. The germ cell then completes MI in telophase I and directly enters meiosis II (MII). The resulting sister chromatids, or univalents, line up at the metaphase plate where separase will then cleave centromeric cohesins to finally create haploids.

Fig. 9.2, Key components and landmark meiotic events.

Although the general scheme of meiosis in male and female gametes (sperm and oocytes, respectively) is shared, they differ dramatically in the timing of their landmark cellular events and in their outcomes. The main focus here will be to describe meiosis in oocytes, but similarities and differences that arise in males will be highlighted throughout.

Meiotic Initiation

Timing of meiotic initiation differs between males and females
Meiotic initiation requires retinoic acid
Expression of meiosis-specific genes is key to initiation

A fascinating aspect of meiosis in female gametes is the length of time oocytes take to complete this cell cycle. Meiosis is initiated during fetal development (∼embryonic day 13.5 in mice, ∼week 8 in humans) but is not completed until fertilization. This means that in organisms like humans, oocytes will not complete meiosis until decades after its initiation. Another hallmark of female meiosis is cell cycle starts and stops, and in humans a cessation of oocyte production at menopause, around the age of 50 years. This timing is remarkably different in males, where meiosis does not begin until around puberty and continues throughout adulthood. Initiation of meiosis, often defined as the onset of meiotic DNA replication, requires expression of RNA-binding protein deleted in azoospermia-like (DAZL) and exposure to retinoic acid (RA), an active derivative of vitamin A. In humans, the somatic cells of the fetal ovary may secrete the needed RA as they express RA synthesis genes. RA exposure then triggers the expression of meiosis-specific genes such as stimulated by retinoic acid 8 ( Stra8 ) and REC8 meiotic recombination protein ( Rec8 ), whose products may play a role in activating transcription and function in sister chromatid cohesion, respectively. ^, The Stra8 promoter contains two RA response element sequences that are required for Stra8 expression. In female mice lacking Stra8 , oocytes fail to execute meiotic DNA replication, suggesting that it is essential for meiotic initiation ; however, male germ cells from Stra8 -null mice on mixed genetic backgrounds do initiate meiosis but fail later in prophase I. The essential nature of DAZL also appears to be strain-specific in mice, as only inbred C57BL/6 mice lacking Dazl fail to initiate meiosis, indicating that other unknown factors are also required. ^, A second meiosis initiator protein (MEIOSIN) was recently identified through a search for proteins that interact with STRA8 in mouse testes. In males and females, MEIOSIN and STRA8 interact, and they are required for meiotic progression.

Because males do not initiate meiosis until puberty, meiotic initiation must be prevented during fetal development and childhood. In fetal and neonatal testes, RA is catabolized by the cytochrome P450 enzyme CYP26B1. CYP26B1 is expressed in Sertoli cells and prevents RA from initiating meiosis during this stage of development. After birth, spermatogonial populations form, and meiosis begins once hormone levels have reached a threshold. In the absence of RA, either by dietary restriction of vitamin A consumption or via inhibition of its synthesis by WIN 18,446/BDAD treatment, male mice are sterile, arresting at spermatogonial differentiation stages. Despite some progress in this arena, many of the mechanisms that control meiotic initiation remain unknown.

Prophase I

DNA double-strand breaks are essential for meiosis.
Each chromosome must have at least 1 site of crossing over-based repair.
PRDM9 determines which break sites are processed by crossing over.
Genetic variants of key DNA repair genes cause infertility in humans.

After DNA replication, oocytes and spermatocytes enter prophase of MI. Prophase I is classically broken into stages that reflect cytological changes in chromosomes as they progress through this phase of meiosis ( Fig. 9.3 ). Leptonema , a stage that is tied together with the completion of DNA replication, begins when double-strand breaks (DSBs) of the replicated chromatids are made by the SPO11 initiator of meiotic double-stranded breaks (SPO11) topoisomerase II protein. Male and female mice lacking Spo11 are infertile, which highlights the essential nature of making DSBs during meiosis. Locations in the genome where SPO11 acts, commonly called hotspots, vary amongst organisms. In mice and humans, the methyltransferase, PRDM9 (PR domain-containing protein 9), is a major hotspot determination factor and this mechanism is shared between males and females. This function was established by in vitro assays that measure protein associations with chromatin using both human and mouse DNA. PRMD9 binding to specific DNA motifs and subsequent trimethylation of histone H3 lysine 4 (H3K4me3) and lysine 36 (H3K36me3) recruits DSB machinery. Inbred strains of mice lacking Prdm9 are sterile, but in mice of some background strains, DSBs can still occur at non-PRDM9 sites (called default sites where H3K4me3 is present), and therefore these strains are not sterile. These findings indicate that genetic modifiers that compensate for the loss of PRDM9 likely exist. Consistent with this idea, a woman who is null for PRDM9 is reportedly fertile whereas, in a cohort of 147 azoospermic, infertile men, two genetic Prmd9 variants 17353G>T (Gly433Val) and 18109C>G (Thr685Arg) were identified. These variants occur in DNA-binding regions of the protein and are likely to reduce PRDM9 function, but this alteration has not been evaluated. In mice, PRDM9 requirement and modifier function is also sexually dimorphic. Male mice engineered to express a point mutation in the PRDM9 PR/SET domain required for methyltransferase activity are sterile, whereas female mice with this mutation are fertile. Therefore, both female mice and women can use a PRDM9-independent pathway to repair DNA.

Fig. 9.3, Synaptonemal complex (SC) formation and synapsis during prophase I in the mouse.

DSBs then elicit downstream DNA repair mechanisms, which begin with homologous chromosome pairing, a process of chromosome movement that allows homologs to find one another. These movements end in synapsis, a mechanism that tethers the homologs together. These processes initiate in leptonema and continue to develop in zygonema . Completion of synapsis, through the formation of tripartite structures called the synaptonemal complex (SC) on every homologous pair of chromosomes, marks the pachynema stage. When SC formation begins, axial elements accumulate along the length of the homologs and provide docking sites for proteins that control recombination and cohesion. After deposition of two transverse filaments, the homologs are fully synapsed ( Fig. 9.4 ). The building of the SC is critical to successful meiosis. Mutations in genes that encode SC proteins confer female infertility, typically causing recurrent miscarriages or primary ovarian insufficiency. ^, For example, sequencing of SC component SYCP3 in women who presented with recurrent miscarriages identified a gene variant (NM_153694.1:c.657T>C) that alters SYCP3 splicing and its ability to modify DNA structures in vitro . ^, In addition, microdeletions and point mutations in SC component SYCE1 are associated with primary ovarian insufficiency. ^, The point mutation (NM_130784.2:c.613C>T) has subsequently been assessed in a knock-in mouse model and these animals also lack follicles and oocytes.

Fig. 9.4, Schematic representation of the events occurring between homologous chromosomes during prophase of meiosis I.

While homologs are synapsing, homologous chromosome recombination is also occurring—these processes are dependent upon one another. While chromosomes are unsynapsed, or if they fail to synapse, HORMA domain containing 1 and 2 (HORMAD1/2) proteins are deposited to unsynapsed areas. But, once synapsed, HORMADs must be removed. This removal is achieved through thyroid hormone receptor interactor 13 (TRIP13 or PCH2 in lower eukaryotes), an AAA-ATPase that participates in noncrossover DSB repair (see below). Variants in TRIP13 are linked with female infertility phenotypes both in mice and humans. Female Trip13 -knockout mice are sterile and have premature ovarian insufficiency (POI). However, the phenotype of women harboring missense Trip13 variants is different because these women have oocytes. These oocytes instead fail to undergo meiotic maturation, indicating additional unknown functions of this protein during later stages of meiosis. In males, the presence of nonhomologous X and Y chromosomes poses an interesting pairing conundrum. The mechanism by which spermatocytes tackle this problem is by creating an XY (or sex) body, where markers of asynapsis persist because of limited homology to enable pairing. Homologous recombination only occurs in the small homologous region, called the pseudoautosomal region, and transcription is silenced through chromatin modifications. If silencing, called meiotic sex chromosome inactivation (MSCI), fails to occur, germ cells arrest in pachytene likely because of aberrant gene expression incompatible with meiotic progression. Interestingly, when XO female mice are evaluated for MSCI, the results indicate mosaicism; some oocytes arrest whereas others will complete prophase I. It is unclear why MSCI is more robust in male mice at this time, but in sex-reversed XY females (oocytes have an X and Y), mosaic silencing also occurs. Therefore, MSCI is not conferred by the Y chromosome but is dependent upon the sex of the gonad.

DSBs are genotoxic and DNA repair is therefore essential for successful meiosis. During the repair process, resected DNA becomes single-stranded, and DNA helicases like MCM8/9 protect the vulnerable DNA. Both male and female Mcm8 -knockout mice are sterile and Mcm9 knockout female mice are sterile. Human genome sequencing studies have since identified variants in Mcm8/9 associated with POI ; when one of the truncated MCM9 mutants was evaluated in a heterologous DNA repair system, it failed to localize to sites of DNA damage. In mice, if chromosomes fail to synapse or repair, checkpoints exist to remove these oocytes from the ovary, also called the ovarian reserve. The DNA damage checkpoint, involving signaling via the checkpoint kinase 1 (CHEK1) and CHEK2, detects persistent DSBs and unsynapsed chromosomes and triggers transformation-related protein 53 (TRP53)/ transformation related protein 63 (TRP63)-dependent apoptosis. In mouse spermatocytes, a similar pathway exists to eliminate germ cells with autosomal asynapsis. In human males with severe azoospermia, a meiotic arrest is also observed. Testes from these men show either XY body formation failure (i.e., no MSCI) or activated DNA damage checkpoint pathways.

There are two outcomes of recombination-based DNA repair ( Fig. 9.5 ): 1) crossovers where the homologous chromosome serves as a repair template and results in a recombinant chromosome, one distinct from either parent chromosome or 2) noncrossovers where there is genetic exchange from the sister or the homolog causing gene conversion, but the sequence surrounding the repaired site remains unchanged. If DNA is repaired via crossing over, a physical linkage of the homologs occurs. This linkage, called a chiasma, keeps homologous chromosomes together until chromosome segregation in MI. Therefore at least one chiasma is required per chromosome for accurate separation of homologs in MI. Several genes are required for crossover recombination and fertility in mice. Mismatch repair proteins MLH1 and MLH3 (mutL homologs 1 and 3) resolve recombination intermediates and MLH3 variants are associated with human infertility in both males and females. In an approach to evaluate human variants of unknown significance in essential meiosis genes, mutations in human MLH1/3 were selected by probing databases such as dbSNP and GnomAD and identifying potential deleterious candidates to introduce into the mouse genome via Crispr/Cas9 editing. Although the fertility of women harboring these mutations is not known, some of the mouse models exhibited premature age-related infertility and are worth inspecting further.

Fig. 9.5, Summary of meiotic recombination based on data from Saccharomyces cerevisiae.

After DNA repair is complete, the SC dissolves in the diplonema and diakinesis stages. Little is known about this step of prophase and how it impacts female fertility, but studies in Drosophila melanogaster suggest that premature removal of the SC does not affect meiotic chromosome segregation. In male mice, removal of the SC requires aurora kinase B. In contrast, SCF3 (SKP1–Cullin–F-box)-mediated ubiquitin ligase activities maintain the SC while mouse spermatocytes gain competence to complete meiosis.

Oocyte Growth Phase

Oocytes arrest at prophase I for an extended period.
Follicle activation initiates oocyte growth.
Only fully grown oocytes are competent to complete meiosis upon ovulation.

The mammalian oocytes that complete DSB repair and are not subjected to apoptosis make up the ovarian reserve. These oocytes remain arrested in diakinesis until the luteinizing hormone (LH) ovulatory cue triggers the resumption of MI. During the prophase I arrest, oocytes within primordial follicle complexes become activated and start to grow. While growing, oocytes synthesize and accumulate RNAs and organelles that they will need to complete meiosis and to support fertilization and early embryonic development. This oocyte growth occurs at the same time as follicle development and differentiation and is discussed in detail in Chapter 8 . It is important here to highlight the long span of time that exists between the completion of recombination and the initiation of meiotic maturation. In mice, oocytes can remain arrested in diakinesis of prophase I for months and in humans, this arrest can last for decades. Mouse spermatocytes, on the other hand, arrest for ∼6 days before resuming and completing MI.

Oocyte Meiotic Maturation

Chromatin compaction status is predictive of meiotic competence.
A cAMP/cGMP-dependent signaling pathway inhibits the resumption of meiosis I in fully grown oocytes; their reduction triggers meiotic resumption, which relies on increased CDK1-cyclin B1 activity.
Homologous chromosomes segregate in meiosis I while sister chromatid cohesion at centromeres is protected from proteolysis.
Premature separation of sister chromatids is common in oocytes from women of advanced maternal age.
Spindle assembly checkpoint prevents chromosome segregation mistakes and is weakened with age.
Fertilizable eggs arrest at metaphase II due to high CDK1 activity until fertilization.
Meiotic maturation is accompanied by molecular and cellular changes that optimize successful fertilization and embryo development.

Prophase I Arrest and Meiotic Resumption

Once oocytes reach their full size (∼80 μm diameter in mice; 120 μm diameter in humans), the chromatin becomes condensed within the nucleus and transcription is silenced. When detected by microscopy, this condensed chromatin forms a tight ring around the nucleolus and is referred to as the “surrounded” nucleolar (SN) configuration of chromatin. Evidence supports that these oocytes, rather than the nonsurrounded nucleolus (NSN) containing oocytes, are more competent to complete meiosis and support embryonic development after fertilization. Condensation of the chromatin is regulated epigenetically through acetylation and methylation of histone residues such as trimethylation of histone H3 lysine 9 (H3K9me3) and histone H4 lysine 20.

In fully grown oocytes, a meiotic arrest is maintained through high intracellular levels of second messengers cAMP and cGMP ( Fig. 9.6 ). In mice and humans, cAMP is generated in oocytes through a constitutively active G-protein coupled receptor, GPR3, and adenylate cyclase 3 (AC3). ^, The resumption of meiosis is controlled by alterations in the levels and activities of proteins known as cyclins and cyclin-dependent kinases (CDK), which function as a heterodimer . cAMP elicits a cell-cycle inhibitor signaling cascade through activation of cyclic AMP-dependent protein kinase (PKA), which activates WEE2 (WEE2 oocyte meiosis inhibiting kinase) and MYT1 (myelin transcription factor 1) kinases. These kinases then phosphorylate and inhibit cyclin-dependent kinase 1 (CDK1), thereby preventing the breakdown of the nuclear envelope and meiotic resumption. PKA also phosphorylates and inhibits the cell division cycle 25B (CDC25B) phosphatase, an activator of CDK1. To keep cAMP levels high in the oocyte, cumulus cells produce cGMP, which reaches oocytes through gap junctions and represses phosphodiesterase 3 (PDE3) so that cAMP cannot be metabolized. Because of the inability to metabolize cAMP, mice lacking the Pde3 gene are infertile because they ovulate prophase I-arrested oocytes.

Fig. 9.6, Working model of signaling pathways that regulate meiotic arrest and resumption in preovulatory follicles.

To resume meiosis, cAMP levels must drop. In mature ovarian follicles, the midcycle luteinizing hormone (LH) surge initiates this drop by stimulation of Gs-protein-coupled LH receptors on follicle cells, resulting in the activation of Gs and generation of cAMP by follicle cell transmembrane adenylyl cyclases. This cAMP subsequently activates transcription factors such as cAMP-response element binding protein 1 (CREB1) and cAMP-response element modulator (CREM) that induce or repress the transcription of specific genes important in modulating follicular cell function during oocyte maturation and ovulation. LH-induced Gs activation also causes dephosphorylation of granulosa cell guanylyl cyclase NPR2, with a consequent reduction in its enzymatic activity. ^, The resulting dramatic reduction in cGMP levels in the granulosa cells allows rapid diffusion of cGMP out of the oocyte across gap junctions, causing a precipitous drop in oocyte cGMP. Lower oocyte cGMP levels are no longer sufficient to inactivate PDE3A, so this phosphodiesterase becomes active and breaks down cAMP, resulting in a loss of the PKA activity required to restrain MPF from inducing meiotic maturation.

Studies of meiotic resumption in mouse knockout models have been critical to building our knowledge in this area. For example, in Gpr3 knockout mice, oocytes cannot generate cAMP and therefore fail to maintain the prophase I arrest and spontaneously resume meiosis. This resumption occurs because PKA no longer activates WEE2/MYT1 nor does it inhibit CDC25B. Crucially, the LH surge causes a drop in cGMP and cAMP, which alleviates the PKA-mediated inhibitory signals of CDK1. The inhibitory phosphate placed on CDK1 by WEE2/MYT1 is removed by CDC25B. In mice, removal of the Cdc25b gene causes sterility because of the inability of oocytes to activate CDK1 and resume meiosis.

CDK1 activity requires binding to cyclin B1. When activated, the complex triggers nuclear envelope breakdown, a morphological marker of meiosis I resumption ( Fig. 9.7 ). As a secondary measure to ensure prophase I arrest, the availability of cyclin B1 is tightly controlled. During the arrest period, this cyclin is subject to proteolysis by the CDH1 (FZR1)-activated anaphase-promoting complex/cyclosome (APC/C) and is therefore maintained at low levels. Overexpression of cyclin B1 in mouse oocytes can trigger meiotic resumption, and when Cdh1 has been depleted, mouse oocytes undergo precocious meiotic resumption. CDH1 activity is therefore also regulated. It is activated by the cell division cycle 14B (CDC14B) phosphatase; depletion of Cdc14b causes precocious meiotic resumption. Once activated, cyclin B1 levels stabilize and become sufficient to activate CDK1 to promote exit from the prophase arrest and entry into the meiotic M-phase.

Fig. 9.7, Oocytes at the three most commonly observed stages of meiotic maturation.

Completion of Meiosis I

Completion of MI involves the segregation of homologous chromosomes ( Fig. 9.8 ). The entry into meiotic metaphase (prometaphase I) is a lengthy process (6–7 h versus 1–2 h in mitotic prometaphase) that involves spindle building and resolution of the condensed chromatin. In mice, microtubule organizing centers (MTOCs) are the acentriolar structures that nucleate microtubules to form the MI spindle. Prior to nuclear envelope breakdown, MTOCs first stretch and then fragment. This process occurs in at least three phases: (1) restructuring of the MTOCs through aurora kinase A (AURKA) and polo-like kinase 1 (PLK1) activities, (2) dynein-dependent stretching into a ribbon-like structure along the nuclear envelope, and (3) fragmentation by kinesin motor protein KIF11 after the nuclear envelope breaks down. After fragmentation and restructuring, the MTOCs sort and cluster into two poles while nucleating microtubules that push and pull resolved chromosomes to the forming metaphase I plate. Mysteriously, a role for MTOCs in building a meiotic spindle in human oocytes may not exist. Although some studies describe the presence of MTOCs in discarded human oocytes, another study did not detect pericentriolar material and instead demonstrate that microtubules nucleate via a RAS-related nuclear protein-dependent pathway that initiates from chromatin. In mice, AURKA and PLK1 are essential for fertility—oocyte-specific knockouts are sterile—and are required for MTOC fragmentation. ^, Because of the paucity of human oocyte studies, a comprehensive study of MTOC markers has not yet been conducted.

Fig. 9.8, Schematic of chromosome segregation during mitosis and meiosis.

Early prometaphase I steps (i.e., condensing chromosomes, nucleating microtubules, and building a bipolar spindle) require less CDK1 activity. As CDK1 activity increases, later prometaphase I events occur. For example, some microtubules that nucleate from MTOCs will make end-on attachments to kinetochores, the multiprotein complexes that dock at the centromeres of chromosomes. Other high-CDK1 activity events include regulating chromosome alignment at the metaphase I plate and migration of the spindle from the center of the oocyte to the cortex.

Stable kinetochore-microtubule attachments are essential for triggering anaphase I onset. However, making these attachments is error-prone; if anaphase were to occur before proper attachments are made, chromosomes can mis-segregate and result in aneuploidy. Cells employ a spindle assembly checkpoint (SAC) to integrate sensing erroneous attachments with preventing anaphase onset ( Fig. 9.9 ). ^, The SAC senses kinetochores that are not attached to microtubules. When these kinetochores are sensed, the TTK protein kinase (TTK), more commonly referred to in the literature as MPS1, initiates a signaling cascade to recruit the mitotic checkpoint complex (MCC) to kinetochores. In mice, oocytes engineered to express a kinetochore-binding domain mutant of TTK are subfertile and produce a high percentage of aneuploid eggs. In oocytes, the SAC is active early in prometaphase I because no stable kinetochore attachments are yet made. The core of the MCC consists of MAD2L1/BUBR1B/BUB3 (mitotic arrest deficient 2 like 1 / BUB1 mitotic checkpoint serine/threonine kinase B / BUB3 mitotic checkpoint protein) which binds and sequesters the APC/C activator CDC20 (cell division cycle 20). Without APC/C activity, cyclin B1 levels remain high, and separase remains inactive because it is bound to securin protein. When an inappropriate attachment is made, it is sensed by aurora kinases B and C (AURKB/C) and microtubule depolymerization is triggered. This depolymerization thereby creates an unoccupied kinetochore and keeps the SAC activated.

Fig. 9.9, Signaling pathway of the spindle assembly checkpoint (SAC) .

Once all attachments are correct and stable, the SAC is satisfied, and CDC20 is freed from the MCC to activate the APC/C. The APC/C targets cyclin B1 and securin for degradation so that metaphase exit and cohesin destruction by separase, respectively, can occur. The result of SAC satisfaction in MI is the separation of homologs where one set remains in the oocyte and the other set is removed in the polar body. Intriguingly, the SAC appears to be less stringent in sensing inappropriate attachments in MI oocytes relative to mitotic cells. ^, In Mlh1 knockout mice, there are no crossovers and therefore no bivalents. Yet, occasionally oocytes will complete MI and extrude a polar body. In contrast, all Mlh1 -null spermatocytes arrest in metaphase, indicating that the stringency of the SAC in male germ cells is more robust than in females. ^, Other mouse models support the notion that oocytes have a weak SAC response such as XO mice where there is no X chromosome homolog and Sycp3 knockout mice that lack crossovers. Reduced expression of SAC proteins occurs with advanced maternal age and weakening of the SAC is a potential cause of the increase in egg aneuploidy that occurs as women age. ^,

During MI, sister chromatids must remain associated with one another. As women age, premature separation of sister chromatids (PSSC) is the leading defect that drives egg aneuploidy and failure to conceive. ^, Sister chromatids remain associated through the protection of cohesin complexes at centromeres. This protection occurs through shugoshin proteins (SGO2) that prevent separase cleavage of the guarded cohesin population. This cohesion also promotes sister chromatid fusion of kinetochores so that they make attachments to microtubules nucleating from the same spindle pole. This fusion is critical to segregating homologs and not sister chromatids in MI; however, sister kinetochore fusion in human oocytes is surprisingly faulty. This lack of kinetochore fusion could explain why errors in female meiosis are common even at young reproductive ages. Cohesin complexes are deposited during premeiotic DNA replication, an event that happens during fetal development in oocytes. As cohesins start to decay over time, they apparently are not replaced. ^, Therefore, another event driving PSSC with age is cohesin loss, which can cause sister kinetochore separation and favor attachment of sister kinetochores to microtubules nucleating from opposite poles. ^– In fact, sister chromatid segregation in MI, also called reverse segregation, occurs frequently in oocytes of older women. These separated sister chromatids are at risk for random segregation during meiosis II (MII).

Meiosis II

After segregation of homologous chromosomes and an asymmetric cell division with emission of the first polar body, CDK1 activity accumulates rapidly, and oocytes align chromosomes and reassemble a metaphase II spindle. The transition from MI to MII involves skipping DNA replication so that the genome content can become haploid upon completion of MII. Increasing CDK1 activity allows chromosome condensation and kinetochore-microtubule connections to occur. Unlike spermatocytes, which will complete MII, the mature oocyte, or preferably, the egg, arrests at the metaphase of MII.

The metaphase II arrest must be maintained for several hours until fertilization and requires CDK1 activity to remain high ( Fig. 9.10 ). To keep CDK1 activity high, cyclin B1 levels remain high by inhibition of the APC/C, as is achieved during the prophase I arrest. However, during metaphase II, the APC/C is inhibited by FBXO43 (previously known as EMI2), thereby allowing CDK1 activity to persist. Depletion of FBXO43 causes egg activation and metaphase II exit. Upon fertilization, FBXO43-mediated inhibition is alleviated by CaMKII (calcium/calmodulin-dependent protein kinase II) and PLK1 phosphorylation of FBXO43. These phosphorylation events trigger FBXO43 turnover and allow activation of the APC/C to then degrade cyclin B1. ^, Another protein kinase, called MOS, is also responsible for the arrest of the oocyte cell cycle at metaphase of meiosis II. Mice deficient in MOS are subfertile because their oocytes do not arrest at metaphase II. Instead, the oocytes continue to divide without being fertilized (parthenogenetic activation) and as a result, the mice develop teratomas. With sufficient MOS production, eggs remain arrested in metaphase II until fertilization. The fertilizing sperm induces calcium oscillations that cause cyclin destruction and degradation of MOS protein, resulting in resumption of meiosis II and extrusion of the second polar body. Finally, to separate the sister chromatids, centromeric cohesin must also be removed by separase. During the metaphase II arrest, centromeric cohesin is still protected by SGO2, which also is bound to the PP2A-B56 phosphatase. For anaphase II onset to occur, an inhibitor of PP2A, called IPP2A, is recruited to allow for deprotection of cohesin and for sister chromatids to segregate.

Fig. 9.10, Model of the regulation of MII arrest in mammalian eggs.

Oocyte Maturation-Associated Changes

In addition to meiosis, there are numerous essential molecular and cellular changes that occur during oocyte maturation. These changes include organelle movements, alterations to chromatin epigenetic marks, uptake of essential ions, mRNA degradation, and synthesis and posttranslational modification of essential proteins. The net result of these events is to optimize the ability of the metaphase II-arrested egg to support successful fertilization and embryo development.

At the ultrastructural level, there are changes in the distribution of the organelles, with movement of the endoplasmic reticulum (ER), mitochondria, and cortical granules (CGs) toward the oocyte cortex ( Fig. 9.11 ). Mitochondria become intimately associated with ER tubules including occasional large (1-2 μm) aggregates of ER tubules that form in cortical locations. ^, CGs, which continue to be synthesized from Golgi complexes during maturation, move to the cortex where they become anchored just below a subplasma membrane concentration of actin microfilaments. This CG movement occurs along cytoplasmic actin filaments and by “hitchhiking” onto Rab11a-positive vesicles that also move to the membrane along actin filaments. ^, CG anchoring is dependent on the presence of MATER, a protein that serves as an important component of the subcortical maternal complex (see Chapter 8 ). Human cortical granules, unlike in the mouse, are found around the entire periphery of the oocyte, even in regions immediately adjacent to the MI and MII spindles. ^, By the time of metaphase II arrest, Golgi complexes have largely disappeared, explaining a decline in the ability of the mature egg to synthesize new secreted proteins. In addition, the transzonal projections responsible for cumulus cell-oocyte coupling have retracted by MII, disrupting gap junctional communication between the oocyte and cumulus cells.

Fig. 9.11, Schematic representation of subcellular morphological patterns in germinal vesicle (GV) oocytes, metaphase I (MI) oocytes, and metaphase II (MII) eggs.

With movement of chromatin to the cortical region, the oocyte becomes highly asymmetric ( Fig. 9.11 ). The actin cytoskeleton is altered, with thickening of cortical actin overlying the metaphase II spindle. This region of the plasma membrane is devoid of microvilli—unlike the rest of the oocyte plasma membrane, which is enriched with microvilli. This loss of microvilli may reduce the chance of sperm entering the region of the metaphase II spindle where it could interfere with the normal progression of meiosis. The spindle is maintained in the cortical region by actin-dependent cytoplasmic streaming driven by an actin nucleator, the ARP2/3 complex.

Concomitant with chromosomal condensation and movements orchestrated by the spindle during meiotic maturation, the oocyte chromatin continues to undergo epigenetic remodeling that was initiated during oocyte growth (see Chapter 8 ). More extensive domains of the noncanonical broad histone H3 lysine 27 trimethyl (H3K27me3) marks are detected in eggs than in full-grown oocytes. This finding indicates that there is continued activity during maturation of lysine methyltransferase 2B, the major enzyme responsible for establishing these marks. ^, It is also likely that lysine-specific demethylase 4A continues to remove H3K9me3 marks, preventing disruption of the broad H3K27me3 domains. Finally, DNA methylation at imprinting control regions of some genes is not complete until following oocyte maturation.

Zinc is an essential transition metal that serves both structural and catalytic roles within numerous cellular proteins such as zinc-finger transcription factors and metalloenzymes. Extensive zinc accumulation via uptake through plasma membrane zinc transporters occurs during oocyte maturation, with an overall increase in zinc content of about 50%. ^, The reduction of available zinc in prophase I-arrested oocytes causes resumption of meiosis by perturbing the function of the zinc-binding protein FBXO43 and also causes abnormalities in cortical reorganization leading to diminished cell polarity. ^, Zinc is stored in the cortical granules in preparation for its release during fertilization.

Calcium is also taken up during oocyte maturation and stored in the endoplasmic reticulum. Calcium enters the oocyte’s cytoplasm through at least two distinct calcium channels: the voltage-gated T-type channel, Ca _V 3.2, and the constitutively active transient receptor potential channel, TRPM7. ^, Cytoplasmic calcium is then actively transported into the ER through sarcoendoplasmic reticulum calcium ATPase (SERCA) pumps. Calcium influx during oocyte maturation is essential for appropriate calcium signaling to occur at fertilization.

Oocyte maturation is accompanied by the recruitment of specific dormant maternal mRNAs for translation into protein. This recruitment provides a mechanism for the transcriptionally silent oocyte to initiate a new developmental program required for fertilization and embryo development. Dormant maternal mRNAs have in common that they encode proteins that are critical for functions in the oocyte during maturation, in the egg at fertilization, or in the very early stages of embryo development. In rodents, examples of these recruited mRNAs are Mos , the inositol 1,4,5-triphosphate receptor, type 1 ( Itpr1 ), and tankyrase 1. As mentioned above, translation of Mos is critical for blocking cell cycle progression beyond metaphase II. The maturation-associated increase in ITPR1 protein is important for successful egg activation at fertilization by increasing the ability of the egg to exhibit calcium oscillations. Tankyrase 1 has a critical role in regulating ß-catenin transcriptional activity in the zygote and 2-cell embryo, which is essential for development beyond the 2-cell stage.

The molecular mechanism by which dormant maternal mRNAs are recruited is cytoplasmic polyadenylation. Stored maternal mRNAs are released from subcortical aggregates in response to CDK1-mediated phosphorylation of the RNA-binding protein MSY2 (Y box-binding protein 2) at the initiation of oocyte maturation. The dormant maternal mRNAs have specific nucleotide sequences in the 3′ untranslated region called cytoplasmic polyadenylation elements (CPEs). CPEs direct the binding of poly(A) polymerase and the addition of poly(A) tracts to dormant maternal mRNAs. Among the proteins that regulate this process are cytoplasmic polyadenylation binding element protein 1 (CPEB1) and deleted in azoospermia-like (DAZL), which itself is a dormant maternal mRNA. ^, Polyadenylation leads to association with polysomes, translation of the mRNAs, and consequently to increases in the levels of the encoded proteins.

Most maternal mRNAs present in the oocyte are not recruited for translation but instead undergo rapid degradation in preparation for a switch from the maternal to the embryonic transcriptional program. These mRNAs lack CPEs and do not undergo polyadenylation and translation to generate proteins. Instead, they are targeted by different enzymes responsible for promoting mRNA decay. For example, maternal mRNAs are destabilized by deadenylation, which is mediated by the CCR4-NOT deadenylation complex, prior to degradation by the exosome complex. ^, Terminal uridylyl transferases 4 and 7 (TUT4 and TUT7) place a uridine onto the 3′ terminus of mRNAs that have a short polyA tail. ^, The 3′ uridylation promotes entry into both the 5′-to-3′ and 3′-to-5′ degradation pathways. The decapping enzymes DCP1A and DCP2 promote mRNA degradation by removing the 5′ monomethyl guanosine cap in preparation for mRNA entry into the 5′-to-3′ mRNA degradation pathway. Of note, mRNAs encoding DCP1A, DCP2, and two components of the CCR4-NOT deadenylation complex (CNOT6L and CNOT7) are all dormant maternal mRNAs recruited for translation during oocyte maturation, consistent with their critical roles in maturation-associated RNA degradation. In human embryos, the CCR4-NOT deadenylation complex appears to participate in maternal mRNA degradation. Low expression of transcripts encoding CCR4-NOT proteins is found in embryos that fail to cleave to the 2-cell stage. These findings indicate that as in the mice, maternal mRNA degradation during oocyte maturation is essential for successful development.

Synthesis and posttranslational modification of cytoplasmic proteins also occur during oocyte maturation. For example, glutathione is synthesized during maturation from amino acid precursors; a sufficient level of this reducing agent is required for initial events of fertilization. Microtubules undergo posttranslational alterations in acetylation during the transition from metaphase I to metaphase II. Microtubule acetylation is essential for proper organelle positioning and movement, possibly by affecting interactions between tubulin and cytoplasmic lattices. Phosphorylation and dephosphorylation of cytoplasmic proteins, particularly those involved in regulating the cell cycle, are required for successful cytoplasmic maturation.

Together, resumption of meiosis until arrest at metaphase II and extensive changes to chromatin, the contents of the cytoplasm, and organelle positioning combine to generate a mature egg that is poised for successful fertilization and embryo development. Defects in any of these processes can lead to failed fertilization, suboptimal embryo development, and even failed implantation and, consequently, impaired fertility.

Sperm Transport

Sperm transport from the vagina to the site of fertilization (ampulla of the Fallopian tube) is promoted by uterine contractions.
Sperm stored in the female reproductive tract for up to six days can result in pregnancy.
Sperm transit toward the ampulla involves a series of specific sperm binding and release interactions with the female reproductive tract epithelium.
Sperm may be assisted in directional targeting to the ampulla by fluid flows (rheotaxis), chemical signals (chemotaxis), and temperature changes (thermotaxis).

Sperm have a significant challenge to overcome following ejaculation: they need to travel a long distance (relative to the size of the sperm) to the site of fertilization within the female reproductive tract. This process is highly inefficient because a very low percentage of ejaculated sperm ever reach the site of fertilization, the ampulla of the Fallopian tube (human oviduct). The idea that sperm transit is a mechanism for stringent selection of only the most fecund sperm is supported by animal studies but has not been shown in humans.

Female Reproductive Tract Contributions to Sperm Transport

Sperm are rescued from the acidic vaginal environment by the presence of cervical mucus secretions generated during the periovulatory time period. Sperm can survive and retain their motility in endocervical crypts for up to 5 days, and the cervical mucus there provides energy substrates important for sperm survival, including glucose and fructose. ^, However, some sperm transit very rapidly from the cervix to the Fallopian tube, with documentation of transit times as low as 5 minutes following insemination. Sperm motility is not required for this transit because inert beads or protein aggregates placed in the vaginal fornix can be found in the uterus and Fallopian tubes. ^, Uterine contractions may promote sperm migration toward the Fallopian tubes, accounting in part for this rapid transit time. This is true in mice, in which live imaging experiments show an essential role of uterine contractility in sperm entry into the oviduct.

In humans, contractile waves from the uterine cervix to the fundus occur in the late follicular phase. These uterine contractions may be promoted following coitus by factors in semen such as prostaglandins. The number of uterine contractions following intrauterine insemination correlates with live birth, consistent with the idea that uterine contractions may assist sperm transit. However, it is unknown if the sperm that rapidly transit to the Fallopian tubes with assistance from uterine contractions are actually capable of fertilizing eggs. Instead, the sperm that arrive later may be responsible for achieving pregnancy.

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