Germ Cell Development and Fertilization

• Germ Cell Development, Cleavage, and Fertilization is the topic.

Primordial Germ Cell Development

• Primordial Germ Cells (PGCs) are precursors to sperm and egg cells. They are set aside early in development, migrating to the gonads.
• The Germinal Crescent in chick embryos is where PGCs originate, located in the posterior marginal zone.
• Primitive Streak/Blastopore structures are involved in gastrulation, near which PGCs originate and migrate.
• The Genital Ridge is the embryonic structure that develops into testes or ovaries. PGCs migrate here to differentiate into gametes.
• PGC migration pathways vary across species. In chicks, migration occurs through blood vessels; in mammals, through the hindgut; and in Drosophila, through the midgut epithelium.
• PGCs follow specific signaling pathways during migration, with SDF1A acting as a crucial chemoattractant in some species.
• Germ cell determination involves signaling pathways like BMP signaling, activating transcription factors like BLIMP1 to suppress somatic cell fate; SOX2 and NANOG maintain pluripotency, and NANOS3 protects PGCs from apoptosis.
• Teratocarcinomas are malignant tumors from PGCs, highlighting their pluripotent nature.

PGC Differentiation

• Spermatogonia/Oogonia are mitotically dividing germ cells in the gonads and descendants of PGCs.
• PGCs initially undergo mitosis to increase numbers, later transitioning to meiosis for haploid gamete production.
• Meiosis I is a reductional division, separating homologous chromosomes, resulting in two haploid cells.
• Meiosis II is an equational division, separating sister chromatids, resulting in four haploid cells in males or one ovum and polar bodies in 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 sub-phases include Leptotene, Zygotene, Pachytene, Diplotene, and Diakinesis, each with distinct cytological features.

Cleavage Patterns

• Cleavage is a rapid series of cell divisions post-fertilization, increasing cell number without significant growth.
• Blastomeres are the cells produced during cleavage.
• Holoblastic cleavage is complete, occurring in eggs with little (isolecithal) or moderate yolk (mesolecithal).
• Meroblastic cleavage is incomplete, occurring in eggs with a large amount of yolk (telolecithal or centrolecithal).
• Isolecithal eggs have little, evenly distributed yolk (e.g., sea urchins, mammals).
• Mesolecithal eggs feature 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 Types

• Radial cleavage results in blastomeres arranged in radial tiers (e.g., echinoderms, amphioxus).
• Spiral cleavage results in blastomeres arranged in a spiral pattern (e.g., annelids, mollusks).
• Bilateral cleavage results in blastomeres arranged along a bilateral axis (e.g., tunicates).
• Rotational cleavage features one blastomere dividing meridionally and the other equatorially (e.g., mammals).
• Superficial cleavage involves 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).

Factors Influencing Cleavage

• The amount and distribution of yolk significantly influence the cleavage pattern.
• Unequal distribution of cytoplasmic determinants can influence cell fate.
• The cell cycle 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, ending rapid, synchronous cell divisions.

Fertilization: Sea Urchin vs. Mouse

• Sea Urchin Fertilization: Chemotaxis involves the attraction of sperm to the egg by chemical signals (Resact).
• Acrosome Reaction involves the release of enzymes from the sperm's acrosome 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.
• The Fast Block to Polyspermy is a rapid change in the egg's membrane potential, preventing polyspermy.
• The Slow Block to Polyspermy is the cortical reaction, elevating the fertilization envelope.
• Cortical Granules release their contents upon fertilization, modifying the vitelline envelope to prevent polyspermy.
• A fertilization envelope is a protective layer formed around the fertilized egg.
• Egg Activation involves a series of changes post-fertilization, including increases in intracellular Ca2+ and pH, and initiation of protein synthesis.
• Mouse Fertilization: Cumulus Cells surround the egg
• Zona Pellucida: The extracellular matrix, contains glycoproteins ZP1, ZP2, and ZP3.
• ZP3 is a glycoprotein that binds to sperm, initiating the acrosome reaction.
• Sperm Activation involves changes in sperm in the female reproductive tract.
• Acrosome Reaction (Mouse) is similar to sea urchins, but involves different molecules.
• Species-Specific Binding is also similar but uses different molecules.
• Polyspermy Block (Mammalian) primarily relies on modification of the zona pellucida by cortical granule enzymes, without a fast block.
• CD9 and IZUMO are proteins on the egg and sperm membranes, mediating sperm-egg fusion.

Gametogenesis

• Spermatogenesis is the process of sperm production.
• Sertoli Cells, located in the seminiferous tubules, support developing sperm by providing nutrients and signals.
• 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 and flagellum formation, and nucleus condensation.
• The Acrosome is a cap-like structure at the sperm head containing enzymes to digest the egg's coat.
• The Flagellum is the sperm tail, providing motility.
• Capacitation is the final maturation of sperm in the female reproductive tract, enabling fertilization.

Oogenesis

• Oogenesis is egg production.
• Oogonia are mitotically dividing germ cells.
• Primary Oocytes undergo meiosis I, arrested in prophase I for a long time.
• Secondary Oocytes undergo meiosis II, 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 cytoplasm and yolk.
• Vitellogenesis accumulates yolk in the egg.
• Egg Envelopes (zona pellucida, jelly coat, shell) protect the egg.
• The Follicle in the ovary surrounds and nourishes the developing oocyte, containing granulosa cells and thecal cells.

Sexual Dimorphism in Meiosis

• Female Oogenesis initiates meiosis once, produces one gamete per meiosis, arrests meiosis at prophase I, and differentiates while diploid.
• Male Spermatogenesis initiates meiosis continuously, produces four gametes per meiosis, completes meiosis quickly, and differentiates after meiosis.

Key Facts

• PGCs are gamete precursors originating in specific embryonic regions (e.g., the germinal crescent in chicks).
• PGC migration involves chemotaxis and other species-specific guidance cues.
• Germ cell determination involves BMP signaling pathways and transcription factors (BLIMP1, SOX2, NANOG, NANOS3).
• Cleavage is a rapid series of cell divisions post-fertilization, increasing cell number without significant growth.
• Holoblastic cleavage is complete, while meroblastic cleavage is incomplete, depending on yolk amount and distribution.
• Cleavage patterns are influenced by yolk and cytoplasmic determinants.
• Sea urchin fertilization includes chemotaxis (Resact), acrosome reaction (Bindin), fast and slow blocks to polyspermy, and egg activation.
• Mouse fertilization includes zona pellucida binding (ZP3), acrosome reaction, and a slow block to polyspermy.
• Spermatogenesis occurs in the seminiferous tubules, involving Sertoli cells and spermatid differentiation.
• Oogenesis involves follicle formation, meiosis arrest, and the production of a large ovum and polar bodies.
• Meiosis I is reductional, separating homologous chromosomes; meiosis II is equational, separating sister chromatids.
• The synaptonemal complex facilitates crossing over during prophase I of meiosis.
• Sexual dimorphism in meiosis exists, with differences in timing, gamete number, and differentiation stages.
• Vitellogenesis is yolk accumulation in the egg.
• Egg envelopes (zona pellucida, jelly coat, shell) provide protection.
• Temperature-dependent sex determination occurs in some reptiles.
• The mid-blastula transition marks a shift in gene expression during cleavage.
• MPF regulates the cell cycle during cleavage.
• Teratocarcinomas are malignant tumors from PGCs.
• The cortical reaction leads to the formation of the fertilization envelope in sea urchins.

Study Guide: Gene Expression in Embryos

• Genomic Equivalence & Nuclear Transplantation:

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

• Key Experiment: Transplantation of Blastula Nuclei into Enucleated Eggs demonstrated that nuclei from early embryos could support full development when transplanted, but success rates declined with the developmental age of the donor nucleus.

• Cloning & Genomic Equivalence: Dolly the Sheep (1997) confirmed genomic equivalence from adult somatic cells, though with low success (1 of 434 oocytes).

• Examples of Cloned Mammals showed cloning viability for various species.

• Practical Use: Transgenic mammals can be cloned for pharmaceutical protein production (e.g., clotting factor IX for hemophilia).

Differential Gene Expression

• Detection Methods:
  • Drosophila Salivary Gland Study: Dark/puffy DNA regions indicate transcription activity; yolk protein gene is unexpressed in the salivary gland.
  • Odd-Skipped Gene Expression in Drosophila & Mouse: Giant puffs in Drosophila indicate active transcription; reporter genes reveal tissue-specific expression.
• Microarray Technology: This is a modern method to analyze gene expression across different conditions.
• In-Situ Hybridization: labels complementary antisense mRNA to localize gene expression.
• 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: 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:
  • 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): Differentiated fibroblasts can be converted into iPSCs using specific transcription factors.
  • Direct Reprogramming of Pancreatic Cells: B-cell conversion for diabetes treatment.

Primordial Germ Cells (PGCs) are precursors to sperm and egg cells, set aside early in development, migrating to the gonads. PGCs migrate through blood vessels in chicks and through the hindgut in mammals. They follow signaling pathways like SDF1A during migration.