1. Chapter 6
Preimplantation Embryo Development and Primordial Germ Cell Lineage Specification
© 2015, Elsevier, Inc., Plant and Zeleznik, Knobil and Neill's Physiology of Reproduction, Fourth Edition

2. FIGURE 6.1 Summary model of fundamental aspects of mammalian preimplantation development. Recent technological advances in embryology, including time-lapse imaging, whole genome and transcriptome approaches, and single cell molecular analysis have provided insight into key characteristics of mammalian preimplantation development. Assessment of each embryonic developmental stage via time-lapse imaging has shown that the timing windows of certain cell cycle parameters, namely the duration of the first cytokinesis, time between the first and second mitosis, and time between the second and third mitosis is highly predictive of which embryos will successfully reach the blastocyst stage. This oocyte-to-embryo transition is characterized by two phases of epigenetic reprogramming and asymmetry: the asymmetry of epigenetic marks in maternal and paternal pro-nuclei at the zygote stage, which become more symmetrical beginning at the 2-cell stage, and the asymmetry in species-specific DNA methylation patterns between the inner cell mass (ICM) and trophectoderm (TE) at the blastocyst stage. There are several underlying factors that contribute to the generation of chromosome instability, particularly in human embryos, including cellular fragmentation, subchromosomal breakage and fusion, a lack of cell cycle checkpoints, and chromosomal lagging during anaphase. While the major wave of embryonic genome activation (EGA) occurs in the mouse at the 2-cell stage, the major wave of human EGA begins on day 3 at approximately the 8-cell stage and is independent of cell number. In preparation for EGA, maternal effect genes, which were synthesized during oogenesis, are passed to the embryo during the early mitotic divisions. Besides individual maternal proteins, multiprotein complexes, including the subcortical maternal complex (SCMC), are important for embryonic progression beyond the 2-cell stage. Without these maternally provided cytoplasmic components, mouse embryos may undergo a delay in EGA and subsequently exhibit a phenomenon known as the “in vitro 2-cell block”. A select group of maternal mRNAs are recruited via poly(A) tail adenylation for translation following fertilization, while the remaining maternal mRNAs are degraded, a process that is essentially complete by the 2-cell stage in mouse embryos and the 8-cell stage in human embryos. The processes of compaction, intracellular adhesion, and polarization result in the formation of a morula at the 8-cell and 16- to 32-cell stage in mouse and human embryos, respectively. The majority of mammalian species, including humans, undergo cavitation to form a fluid-filled cavity called a blastocoel between days 5 and 6, whereas mouse embryos begin blastulation earlier between days 3 and 4, and bovine embryos later between days 7 and 8. It remains unclear as to when differences in blastomere developmental potential are first observed, however, it is thought that blastomeres become fully committed to form either the ICM or TE cells at the morula stage. Two models of cell lineage specification have been proposed: the inside-outside model, which suggests that the location of cells within the embryos determines the signal(s) they receive; and the blastomere polarity model, whereby cell determinants such as Cdx2 are inherited or precluded from symmetric versus asymmetric divisions to confer cell fate. Source: Shawn Chavez, unpublished.
© 2015, Elsevier, Inc., Plant and Zeleznik, Knobil and Neill's Physiology of Reproduction, Fourth Edition

3. FIGURE 6.2 Life cycle of mammalian germ cells from PGC specification to fertilization. Fertilization of an oocyte by a sperm promotes the formation of a 1-cell zygote that undergoes several cell divisions to form a specialized embryo called a blastocyst. The outer layer of blastocyst will give rise to the trophectoderm, whereas the inner cell mass (ICM) will form the embryo proper later in development. Following implantation, the embryo undergoes gastrulation (E6.25–7.25 in mouse; 4.0–5.0 weeks in human), to form the epiblast, which will differentiate into the three embryonic germ layers, as well as the germ line. Primordial germ cells (PGCs) are specified at the base of the allantois and localize near the extraembryonic ectoderm. Once specified, the PGCs migrate from the invaginating hindgut to the genital ridges (arrows), which eventually develop into fetal gonads. The primitive gonads are formed when PGCs are encircled by somatic cells to form either testicular cord-like structures in the male or primitive follicular structures in the female. PGCs enclosed in either the testis or ovary undergo sex-specific development and maturation to male and female gonocytes, respectively. While male gonocytes arrest meiosis and undergo spermatogenesis to form mature sperm, female gonocytes enter meiotic prophase I and begin oogenesis, which is completed upon ovulation of a mature ovum. At the time of fertilization, the mature germ cells together transmit genetic information to the next generation, thus completing the cycle. Molecules that are involved in regulation of each PGC specification stage are listed. XY = male lineage, XX = female lineage. Source: Adapted from Ref. 17.
© 2015, Elsevier, Inc., Plant and Zeleznik, Knobil and Neill's Physiology of Reproduction, Fourth Edition

4. FIGURE 6.3 Timeline of germ line specification with stage-specific germ cell marker expression. A temporal representation of the different stages of human and mouse germ line differentiation during pre- and postimplantation in vivo. The approximate time in development for both humans and mice is indicated at the top with the major stages pictured below. An examination of global DNA methylation as assessed by five methylcytosine levels in both males and females demonstrates a period of de novo methylation for male (continuous line) and female (broken line) occurring in the blastocyst prior to, and during, implantation. This is followed by genome-wide DNA demethylation in the ESCs and epiblast stages and proceeding to PGC specification. In the mouse, and largely true for the human, genome-wide changes in histone modifications occur concurrently with DNA demethylation between the time of primordial germ cell (PGC) specification, migration, and colonization of the gonads. Sex-specific DNA methylation and imprinting are reestablished later during male and female gametogenesis, albeit at different times. Specific molecular markers for germ cell identification are indicated on the left with lines depicting the duration of expression from the blastocyst stage to the formation of mature gametes. Genes that are italicized are present in both mouse and human germ cells. Source: Adapted from Ref. 17.
© 2015, Elsevier, Inc., Plant and Zeleznik, Knobil and Neill's Physiology of Reproduction, Fourth Edition

5. FIGURE 6.4 Detection of embryonic micronuclei in cleavage-stage human embryos. This figure is reproduced in color in the color plate section. (A) Expression of the nuclear envelope marker, LAMIN-B1 (green), in 4′,6-diamidino-2-phenylindole (DAPI)-stained (blue) cleavagestage human embryos visualized by immunofluorescent confocal microscopy and differential interference contrast (DIC) imaging at 20× (left and middle) and 40× (right) reveals the presence of several embryonic micronuclei (indicated by white solid arrows) distinct from primary nuclei in human blastomeres. (B) Similarly evaluated additional cleavage-stage human embryos with low (left) and high (right) fragmentation also immunostained for centromere protein E (CENP-E; orange) in the absence of DIC imaging identifies multiple missing chromosomes in cellular fragments (indicated by white dashed arrows) adjacent to blastomeres at this stage of development. Source: Adapted from Ref. 16.
© 2015, Elsevier, Inc., Plant and Zeleznik, Knobil and Neill's Physiology of Reproduction, Fourth Edition

6. FIGURE 6.5 Time-lapse image analysis of human preimplantation development. (A) 10 human zygotes were thawed on day 1 and cultured together in alphabetically and numerically labeled microwell-containing Petri dishes. (B) Time-lapse imaging was performed in a standard incubator until day 6 when the majority of embryos had reached the blastocyst stage. Embryo imaging behavior was monitored by measuring dynamic imaging parameters between the first and last frame of an image sequence compiled into a time-lapse movie with well identification labels and time stamps. Source: Brooke Friedman and Shawn Chavez, unpublished.
© 2015, Elsevier, Inc., Plant and Zeleznik, Knobil and Neill's Physiology of Reproduction, Fourth Edition

7. FIGURE 6.6 Comparison of in vivo germ line development and in vitro germ cell derivation from ESCs and iPSCs. A depiction of in vivo development of germ cells and gametes from the mammalian embryo is shown on the left to demonstrate that embryonic stem cells (ESCs) originate from primitive ectoderm (epiblast) by way of the inner cell mass (ICM). Primordial germ cells (PGCs) then migrate to gonads where they undergo self-renewal in the form of spermatogonial stem cells (SSCs) in the testis or further differentiate to oocytes in the ovary. On the right is a schematic of the different cell lineages that can be derived in vitro. ESCs cultured in dishes can be differentiated to PGCs via methods summarized in Table 6.1. PGCs cultured in vitro, at least in the mouse, can transform to embryonic germ cells (EGCs). There is also evidence to suggest that a subpopulation of hESCs cultured in vitro behave like EGCs. Finally, somatic cells can be reprogrammed to iPSCs with addition of the transcription factors, OCT4, SOX2, KLF4, and MYC to induce a stem cell-like fate. iPSCs can then be induced to form PGC-like cells or EG-like cells by in vitro and in vivo methods. Those cell types marked with an asterisk indicate that ESCs, PGCs, and iPSCs can all form teratomas when injected in vivo as demonstration of their pluripotency. Source: Adapted from Ref. 17.
© 2015, Elsevier, Inc., Plant and Zeleznik, Knobil and Neill's Physiology of Reproduction, Fourth Edition