Germ Cell Development and Fertilization

• Germ Cell Development, Cleavage, and Fertilization are key processes in sexual reproduction

Primordial Germ Cell Development

• Primordial Germ Cells (PGCs) are precursors to gametes (sperm and egg cells)
• PGCs are set aside early in development and migrate to the gonads
• The Germinal Crescent (in chick embryos) is where PGCs originate, located in the posterior marginal zone
• PGCs originate near the primitive streak/blastopore, which are structures involved in gastrulation (germ layer formation), and migrate from them
• PGCs migrate to the genital ridges, the embryonic structures that develop into the gonads (testes or ovaries), to differentiate into gametes
• PGC migration varies across species: through blood vessels in chicks, through the hindgut in mammals, and through the midgut epithelium in Drosophila
• PGCs follow specific signaling pathways during migration, with SDF1A being a crucial chemoattractant in some species
• Germ cell determination involves signaling pathways like BMP signaling, activating transcription factors like BLIMP1 (suppressing somatic cell fate)
• SOX2 and NANOG maintain pluripotency, and NANOS3 protects PGCs from apoptosis
• Teratocarcinomas, malignant tumors derived from PGCs, highlight the pluripotent nature of PGCs

PGC Differentiation

• Spermatogonia/Oogonia are mitotically dividing germ cells in the gonads and descendants of PGCs
• PGCs initially undergo mitosis to increase in number, then meiosis to produce haploid gametes
• Meiosis I is a reductional division that separates homologous chromosomes, resulting in two haploid cells
• Meiosis II is an equational division that separates sister chromatids, resulting in four haploid cells (males) or one haploid ovum and polar bodies (females)
• The synaptonemal complex is a protein structure that forms between homologous chromosomes during prophase I of meiosis, facilitating crossing over
• Crossing over (recombination) is the exchange of genetic material between homologous chromosomes, increasing genetic diversity
• Chiasmata are the visible points of crossing over between homologous chromosomes
• Prophase I has sub-phases: Leptotene, Zygotene, Pachytene, Diplotene, Diakinesis, each with distinct cytological features

Cleavage Patterns

• Cleavage is the rapid series of cell divisions after fertilization, increasing cell number without significant growth
• Blastomeres are the cells produced during cleavage
• Holoblastic cleavage is complete division of the egg, occurring in eggs with little yolk (isolecithal) or moderate yolk (mesolecithal)
• Meroblastic cleavage is incomplete division of the egg, occurring in eggs with a large amount of yolk (telolecithal or centrolecithal)
• Isolecithal eggs have little yolk, evenly distributed (e.g., sea urchins, mammals)
• Mesolecithal eggs have moderate yolk, concentrated at the vegetal pole (e.g., amphibians)
• Telolecithal eggs have a large amount of yolk, concentrated at the vegetal pole (e.g., birds, reptiles, fish)
• Centrolecithal eggs have yolk concentrated in the center (e.g., insects)

Cleavage Pattern Types

• Radial cleavage shows blastomeres arranged in radial tiers (e.g., echinoderms, amphioxus)
• Spiral cleavage shows blastomeres arranged in a spiral pattern (e.g., annelids, mollusks)
• Bilateral cleavage has blastomeres arranged along a bilateral axis (e.g., tunicates)
• Rotational cleavage features one blastomere dividing meridionally, the other equatorially (e.g., mammals)
• Superficial cleavage sees nuclei dividing without cytokinesis, then migrating to the periphery where cell membranes form (e.g., insects)
• Discoidal cleavage is restricted to a small disc of cytoplasm on top of the yolk (e.g., birds, reptiles, fish)
• The amount and distribution of yolk influence the pattern of cleavage
• Unequal distribution of cytoplasmic determinants can influence cell fate
• Cell cycle regulation is tightly regulated during cleavage, with MPF (maturation-promoting factor) playing a crucial role
• The mid-blastula transition (MBT) marks a shift in gene expression during cleavage and the end of rapid, synchronous cell divisions

Fertilization: Sea Urchin vs. Mouse

• Chemotaxis attracts sperm to the egg via chemical signals; Resact is a sperm-activating peptide in sea urchins
• The acrosome reaction is the release of enzymes from the acrosome of the sperm to digest the egg's jelly coat
• Bindin, a species-specific protein on the sperm, binds to the egg's vitelline envelope, ensuring species-specific fertilization
• A rapid change in the egg's membrane potential prevents polyspermy (fast block)
• The cortical reaction elevates the fertilization envelope (slow block)
• Cortical granules release contents upon fertilization, modifying the vitelline envelope and preventing polyspermy
• A fertilization envelope is a protective layer formed around the fertilized egg
• Egg activation involves a series of changes, including increased intracellular Ca2+ and pH, and initiation of protein synthesis

Mouse Fertilization

• Cumulus cells surround the egg in the mouse
• The zona pellucida is the extracellular matrix surrounding the egg, containing glycoproteins ZP1, ZP2, and ZP3
• ZP3, a glycoprotein in the zona pellucida, binds to sperm and initiates the acrosome reaction
• Sperm activation involves changes in sperm in the female reproductive tract
• The acrosome reaction and species-specific binding are similar to sea urchins but involve different molecules
• The mammalian polyspermy block primarily relies on the modification of the zona pellucida by cortical granule enzymes; there is no fast block
• CD9 and IZUMO are proteins on the egg and sperm membranes that mediate sperm-egg fusion

Gametogenesis

• Spermatogenesis is the process of sperm production
• Sertoli cells support cells in the seminiferous tubules, providing nutrients and signals to developing sperm
• Seminiferous tubules are the sites of spermatogenesis in the testes
• Spermatogonia are mitotically dividing germ cells
• Primary spermatocytes undergo meiosis I
• Secondary spermatocytes undergo meiosis II
• Spermatids are haploid cells that differentiate into sperm
• Spermiogenesis is the differentiation of spermatids into mature sperm, involving acrosome formation, flagellum development, and nucleus condensation
• The acrosome is a cap-like structure at the head of the sperm, containing enzymes to digest the egg's coat
• The flagellum is the tail of the sperm, providing motility
• Capacitation is the final maturation of sperm in the female reproductive tract, making them capable of fertilization

Oogenesis

• Oogenesis is the process of egg production
• Oogonia are mitotically dividing germ cells
• Primary oocytes undergo meiosis I but are arrested in prophase I for a long time
• Secondary oocytes undergo meiosis II but are arrested in metaphase II until fertilization
• Ovulation is the release of the secondary oocyte from the ovary
• Polar bodies are small cells produced during oogenesis, containing little cytoplasm
• The ovum (egg) is the mature female gamete, containing a large amount of cytoplasm and yolk
• Vitellogenesis is the process of yolk accumulation in the egg
• Egg envelopes, such as the zona pellucida, jelly coat, and shell, are protective layers surrounding the egg
• The follicle is the structure in the ovary that surrounds and nourishes the developing oocyte, containing granulosa cells and thecal cells

Sexual Dimorphism in Meiosis

• Female oogenesis involves meiosis initiated once, one gamete per meiosis, meiosis arrested at prophase I, differentiation occurring while diploid
• Male spermatogenesis involves meiosis initiated continuously, four gametes per meiosis, meiosis completed quickly, differentiation occurring after meiosis

Study Guide: Gene Expression in Embryos

• All cells in an organism contain the same genetic material, but only specific genes are expressed in different cell types

Genomic Equivalence & Nuclear Transplantation

• Transplantation of blastula nuclei into enucleated eggs demonstrated that nuclei from early embryos could support full development when transplanted
• The success rate declines with the developmental age of the donor nucleus
• Cloning Dolly the Sheep (1997) from adult somatic cells confirmed genomic equivalence (1 of 434 oocytes developed)
• Examples of cloned mammals showed viability of cloning for various species
• Cloning transgenic mammals for pharmaceutical protein production is a practical application (e.g., clotting factor IX for hemophilia)

Differential Gene Expression

• Detection methods include:
• Drosophila salivary gland study, where dark/puffy DNA regions indicate transcription activity and the yolk protein gene is unexpressed in the salivary gland
• Odd-skipped gene expression in Drosophila & Mouse (giant puffs in Drosophila embryos indicate active transcription; reporter genes reveal tissue-specific expression)
• Microarray technology is a modern method to analyze gene expression across different conditions
• In-situ hybridization labels complementary antisense mRNA to localize gene expression, exemplified by Pax6 gene expression in developing chick and mouse eyes

Mechanisms of Differential Gene Expression

• Chromatin & epigenetic regulation:
• Histone methylation causes DNA condensation, repressing gene transcription
• Methylation of promoters such as methylation of globin genes in human embryonic blood cells
• Alternative splicing: Production of β-globin & hemoglobin involves alternative splicing of exons and introns
• Transcriptional Control with: - Enhancer-promoter interaction (transcription factors act as a bridge between enhancers and promoters) - Mediator complexes (stabilize RNA Polymerase II at the promoter, modifying nucleosomes for transcription activation)
• Silencers: Repress transcription by binding to specific sequences

Applications of Transcription Factors in Regenerative Medicine

• Reprogramming cells:
• Induced Pluripotent Stem Cells (iPSCs) like differentiated fibroblasts can be converted into iPSCs using specific transcription factors
• Direct Reprogramming of Pancreatic Cells: Like ß-cell conversion for diabetes treatment

Germ cell development, cleavage, and fertilization are key processes in sexual reproduction. Primordial Germ Cells (PGCs) are gamete precursors set aside early in development. PGCs migrate to the gonads, following specific signaling pathways, to differentiate into gametes.