fertilization

Sperm Transport and Fertilization

Reading Assignments

• Today: Read Chapter 12

• Friday/Monday: Read Chapter 11

Topics to Cover

• Gamete Transport

• Development of Sperm Fertilizing Ability

• Sperm-Egg Binding and Fusion

• Egg Activation and Block to Polyspermy

• Preimplantation Development

Importance of Studying Fertilization

Goals of Research

• Improve fertility:
     - Develop diagnostic tests to identify causes of infertility and provide effective treatments for humans, farm animals, and endangered species.

• Control fertility:
     - Develop contraceptives with fewer side effects for humans, pests, and overpopulated species.

• Cell Interaction Studies:
     - Investigate a unique and intriguing cell-cell interaction, focusing on haploid cells and species-specific mechanisms.

Events Following Semen Deposition

Sequence of Events:

1. Immediate Transport:    - Issues: retrograde loss of sperm, phagocytosis.    - Location: entrance into cervix and uterus.

2. Cervix:    - "Privileged pathways" for sperm movement.    - Functions: removal of non-motile sperm and some abnormal sperm.

3. Uterus:    - Capacitation initiated, along with phagocytosis.

4. Oviduct:    - Capacitation completed along with hyperactive motility.

5. Fertilization:    - Acrosome reaction occurs as spermatozoon penetrates oocyte.    - Results in formation of male and female pronuclei.

Female Tract Barriers and Reservoirs for Sperm

• Fluid transport aided by cilia in the female tract.

• Location of sperm storage primarily occurs in the ampulla, while retrograde loss accounts for the loss of most sperm.

Sperm and Oocyte Transport

Importance for Internal Fertilization

• Needed in mammals and birds.

• Only fewer than 50 sperm may reach the fertilization site at any given time.

• Sperm bind to the walls of the reservoir (isthmus) for gradual release, ensuring a constant supply near the egg.

• Mechanisms of sperm movement include:
     - Diffusion, fluid flow, smooth muscle contractions, and chemotaxis in the oviduct (motility alone is not sufficient).

Types of Sperm Transport

1. Rapid Transport:
      - Occurs within minutes; however, sperm in the oviduct are not yet fertile.

2. Sustained Transport:
      - Slower process that ensures a steady supply of sperm to the ampulla from sperm reservoirs located in the isthmus.
      - Facilitates the movement of sperm and oocytes toward each other.

Development of Sperm Fertilizing Ability

Post-Ejaculation Maturation

• Ejaculated sperm are initially unable to fertilize eggs and require maturation time.

• Capacitation:
     - Final maturation phase occurring in the female reproductive tract which grants sperm the ability to fertilize oocytes.    - This process requires a duration of time, occurs mainly in the oviduct, and can also be performed in vitro.

Storage and Capacitation in the Oviduct

• Sperm bind within the isthmus, where they can live longer and undergo capacitation.

• After binding, sperm detach and are carried to the ampulla for fertilization.

Viable Sperm Data

• Graph Analysis:

• ** viabile sperm (%) vs. incubation time (hr):**
     - 80%: 0 hr
     - 70%: 5 hr
     - 60%: 10 hr
     - etc., down to 0% at 25 hrs.

Capacitation Process Outcomes

1. Development of hyperactivated (whiplash) motility.

2. Cholesterol removal from the sperm plasma membrane, which destabilizes the membranes.

3. Movement or removal of plasma membrane coating proteins, enabling zona pellucida receptors to be available for sperm interaction.

Oocyte Maturation

Two Phases of Maturation

1. Nuclear Maturation:
      - Oocytes are halted in the diplotene or dictyate phase until an LH surge triggers maturation and subsequent ovulation.
      - During ovulation, 1-25 oocytes can advance to metaphase II, creating a first polar body.

2. Cumulus Cell Interaction:
      - Cumulus cells produce hyaluronic acid for expansion/maturation, which sperm must penetrate.    - Sperm possess the enzyme hyaluronidase to dissolve hyaluronic acid, facilitating penetration.

Sperm Interaction with Oocyte

Steps in Successive Interaction:

1. Sperm binds to oviduct.

2. Sperm undergoes capacitation.

3. Sperm is released and traverses the cumulus.

4. Acrosome reaction is initiated.

5. Sperm penetrates the zona pellucida (ZP).

6. Sperm adheres to and fuses with the egg.

7. Egg activation occurs.

8. Formation of pronuclei and syngamy.
      

Key Structures:

• Cumulus Cell

• Zona Pellucida

• Perivitelline Space

Zona Pellucida Details

Functionality

• Zona Pellucida serves as the protective coat of mammalian eggs and has key roles:    1. Binds sperm (species-specific).    2. Blocks polyspermy.    3. Protects the embryo.

Composition

• Contains 3-4 glycoproteins, mostly studied in mice (ZP1, ZP2, ZP3).
    - ZP2 and possibly ZP3 primarily bind sperm, while ZP1 stabilizes filaments.

Zona Pellucida Penetration Mechanism

• Sperm must undergo specific changes to penetrate the zona pellucida, including the release of acrosomal enzymes that may create a penetration slit.

• Sperm hyperactivated motility provides the necessary force to penetrate the zona during capacitation.

Acrosome Reaction Details

• Fusion occurs between the plasma membrane and outer acrosomal membrane, leading to degeneration and the release of acrosomal enzymes.

• This exposes the inner acrosomal membrane (IAM).

Assisted Reproductive Technologies

Intracytoplasmic Sperm Injection (ICSI)

• A method where a sperm is injected directly into the oocyte.

In Vitro Fertilization (IVF)

• First IVF human baby born in 1978 in Britain.

• Methodology:    - Superovulate and aspirate oocytes surgically.    - Capacitate sperm in vitro.    - Mix gametes together in a culture medium.

• Current statistics indicate that approximately 1% of babies in the US are born via IVF or ICSI.

• Success rates for IVF and ICSI are comparable.

Binding and Fusion of Sperm to the Egg Membrane

• Sperm are both acrosome-reacted and must interact with egg plasma at the equatorial region, leading to the fusion of cell membranes.

Egg Activation at Fertilization

• Sperm contain enzymes that trigger egg activation by releasing calcium ions ($Ca^{2+}$) from the egg's internal stores into the cytosol.

Consequences of Egg Activation:

• Release of cortical granules.

• Enzymes from cortical granules remove sperm binding sites from the zona pellucida.

• Egg completes meiosis (expulsion of the second polar body).

Blocks to Polyspermy After Fertilization

• Mammals exhibit a slow block at both the zona pellucida and egg membrane developed shortly after fertilization.   - Cortical granules contain enzymes that degrade ZP2 and inactivate ZP3.   - The egg membrane also becomes less capable of binding additional sperm.

Consequences of Gene Inactivation

Hypothetical Scenario: If a gene encoding a cortical granule enzyme that removes the sperm binding site from the zona were inactivated:

• Possible outcomes include:    - Continuous sperm binding to oocytes post-fertilization.    - No fertilization would occur.    - No effect at all.    - Spontaneous egg activation (e.g. parthenogenesis).

Localization of Cortical Granule Enzyme (Ovastacin)

• Enzymatic activity crucial for inactivating ZP2 is observed.

• Visualization and analysis of cortical granules.

Consequences of Polyspermy

Analysis of Two-Cell Embryos Deficient in Ovastacin

• Phenotypic outcomes include:    - Extra centrosomes.    - Abnormal chromosome numbers.    - High risk of disintegrating cells and potential embryo death.

Summary of Fertilization Process

• The reproductive tract has evolved specific adaptations to facilitate gamete union.

• Both gametes require final maturation to be capable of fertilization.

• Fertilization is a multi-step process consisting of binding and subsequent fusion of dissimilar gametes.

• The egg undergoes activation triggered by the sperm, completing meiosis and establishing a polyspermy block.

• Following fertilization, embryos undergo mitotic divisions with minimal size changes before hatching from the zona pellucida.