Class 8 Fertilization, Parthenogenesis, and Genomic Imprinting

Sperm’s Essential Contributions to the Embryo

• Beyond Haploid Genome Delivery: Sperm is not merely a "delivery boy" for genetic material; it provides critical factors necessary for the initiation of development.

• Oocyte Activation: The fusion of the sperm with the oocyte introduces $PLC\zeta$ (Phospholipase C zeta), which triggers oocyte activation.

• Structural Components: The sperm contributes the centrosome to the zygote; notably, the oocyte lacks a functioning centrosome prior to fertilization.

• RNA Contribution: The sperm introduces various RNA species into the oocyte, including:

  • Long noncoding RNAs (lncRNAs).

  • Small RNAs such as microRNAs (miRNAs), small interfering RNAs (siRNAs), and piwi-interacting RNAs (piRNAs).

  • Transfer RNA-derived small RNAs (tsRNA) (Reference: Science 351; 2016).

• Organelle Fate and Inheritance:

  • There is no significant contribution of the sperm tail, membranes, or organelles to the embryo.

  • Mitochondria: Sperm mitochondria are ubiquitinated during the process of spermiogenesis and are subsequently destroyed by the oocyte or diluted to negligible levels.

  • Mitochondrial Inheritance: Inheritance is strictly female (maternal), though rare instances of abnormal biparental inheritance of mtDNA have been documented.

  • Reference: Michail Nomikos et al. Biochem. J. 2017;474:3659-3673.

Oocyte Maturation and Egg Activation

• Pre-Ovulation and Meiotic Arrest: Meiosis resumes prior to ovulation. The process involves:

  • Germinal Vesicle (GV) disassembly (GV breakdown or GVBD).

  • Chromosome condensation and alignment on the first metaphase plate.

  • Segregation of homologous chromosomes and extrusion of the first polar body (PB).

  • Realignment of chromosomes on the second metaphase plate (MII), followed by a secondary arrest.

• Mechanisms of Activation: Completion of meiosis (activation) occurs via:

  • A fertilizing sperm.

  • Artificial chemical agents.

• Post-Activation Events: Upon activation, sister chromatids segregate, and the second polar body (PB) is extruded.

Parthenogenesis and Uniparental Embryogenesis

• Etymology: Derived from the Greek parthénos ("virgin") and génesis ("creation").

• Biological Context: Parthenogenesis is a form of asexual reproduction where growth and development of embryos occur without fertilization.

• Natural Occurrence:

  • Insects: Grasshoppers, bees, ants, and wasps. Often involves haplodiploidy where unfertilized eggs develop into males.

  • Invertebrates: Rotifers, certain nematodes, arthropods (mites, ticks, spiders), and crustaceans.

  • Vertebrates: Some fish and reptiles (e.g., snakes).

  • Cyclical Parthenogenesis (Daphnia/Water Fleas): Females reproduce parthenogenetically during favorable conditions for rapid population growth. Sexual reproduction is triggered by worsening conditions (crowding, drying ponds, temperature stress) to produce resistant "resting eggs."

  • Rarity in Other Groups: Rare or experimental in amphibians; rare and usually non-viable in birds; non-existent naturally in mammals.

• Techniques for Artificial Parthenogenesis (Parthenotes):

  • Artificial Stimulation: Includes the use of $Ca^{2+}$ ionophores, electrical activation, or the microinjection of $Ca^{2+}$ into the cytoplasm.

  • Inhibition of MPF (Maturation Promoting Factor/Cyclin B): Achieved through agents like Cytoheximide.

  • Blockade of 2nd Polar Body Extrusion: Using Cytochalasin B, which creates a uniparental embryo with two maternal genomes.

Genomic Imprinting: The Epigenetic Basis of Development

• Definition: Genomic imprinting is a normal regulatory process where gene expression is determined by the parent of origin. Only one copy (either maternal or paternal) is expressed for certain genes.

• Mechanisms of Regulation:

  • DNA Methylation: Specific addition of methyl groups to cytosine bases, typically at CpG-rich regions and dinucleotides, usually silencing the gene.

  • Histone Modifications: Includes acetylation, methylation, phosphorylation, and ubiquitination of core histones. These changes alter chromatin structure to activate or repress expression.

  • Chromatin Remodeling: Repositioning of nucleosomes to toggle between "open" (active) or "closed" (inactive) DNA states.

  • Non-coding RNAs: miRNA, siRNA, and lncRNA regulate genes via mRNA degradation, translational blocking, or guiding epigenetic enzymes to DNA.

• Impact of Imprinting: This confers a different states on two homologues, causing some genes to be active on one parental allele and inactive on the other. Examples include:

  • H19: Maternally expressed.

  • Igf2: Paternally expressed.

Summary of Imprinted Genes (Humans vs. Mouse)

Historical and Experimental Milestones

• 1977 (Hopper & Illmansee): Microsurgically produced homozygous-diploid uniparental mice.

• 1984 (Surani, Barton & Norris): Introduced the concept of genomic imprinting as a requirement for development.

• 1984 (McGrath & Solter): Conducted nuclear transfer experiments showing that parthenogenetic (two maternal) and androgenetic (two paternal) embryos fail differently.

• Uniparental Embryo Viability: Micropipetting experiments demonstrated that gynogenetic and androgenetic embryos are lethal at early stages. Bi-parental embryos are the only ones that survive to produce living young because the two genomes express complementary, necessary gene sets.

Clinical Pathologies: Imprinting Disorders

• Chromosomal Location: Both conditions involve the proximal long arm of chromosome 15 ($15q11-q13$).

• Prader-Willi Syndrome (PWS):

  • Caused by the lack of expressed paternally inherited genes.

  • Historically associated with Eugenia Martinez Vallejo ("The Monster").

• Angelman Syndrome (AS):

  • Caused by the lack of expressed maternally inherited genes.

• Genetic Defects involved: These syndromes can result from microdeletions, uniparental disomy (inheriting two copies from one parent), or imprinting defects.

Modern Research: Bi-paternal Development

• Successful Offspring: Reaching adulthood in bi-paternal mice has been achieved via targeted imprinting modifications (Reference: doi.org/10.1016/j.stem.2025.01.005).

• Key Interventions:

  • Modification of the $Sfmbt2$ microRNA cluster was required to create a functional bi-paternal placenta.

  • Embryonic Stem Cells (ESCs) with modifications showed twice the developmental potential compared to controls.

  • Modifications at 20 different loci exhibited significantly higher cloning efficiency.

Take Home Messages

• Parthenogenesis in mammals is experimentally achievable only to early developmental stages or through stem cells.

• Full viability in uniparental contexts requires the artificial re-creation of paternal imprinting.

• Sexual reproduction is evolutionarily obligatory in mammals due to these strict epigenetic requirements.