Differentiate between the mechanism of capacitational changes in the mammalian sperm under in vitro and in vivo conditions. How are these sperms stored and used in artificial insemination and in vitro fertilization?

Capacitation: In Vivo vs. In Vitro

Capacitation is not a single event but a cascade of changes. The key differences lie in the initiating signals and the microenvironment.

In Vivo Capacitation

• Initiating Signals: Primarily driven by interactions with the female reproductive tract fluids, particularly those from the uterus and oviduct. These fluids contain bicarbonate ions, albumin, and various signaling molecules.
• Bicarbonate Activation: Bicarbonate ions increase intracellular pH, activating protein kinase A (PKA) and initiating a signaling cascade.
• Albumin Binding: Albumin binds to the sperm surface, removing cholesterol from the plasma membrane. This increases membrane fluidity, facilitating downstream signaling events.
• Removal of Decapacitation Factors: The female reproductive tract removes decapacitation factors present in seminal plasma, such as prostaglandins and adenosine.
• Phosphorylation of Sperm Proteins: Increased tyrosine phosphorylation of various sperm proteins, including those involved in motility and the acrosome reaction, is a hallmark of capacitation.
• Calcium Influx: A regulated influx of calcium ions (Ca²⁺) into the sperm is crucial for triggering the acrosome reaction.

In Vitro Capacitation

• Initiating Signals: Mimics the *in vivo* environment but requires artificial induction. Commonly achieved by incubating sperm with bicarbonate-buffered media supplemented with albumin (BSA - Bovine Serum Albumin) and calcium ions.
• BSA Role: BSA serves as a cholesterol acceptor, similar to albumin *in vivo*, increasing membrane fluidity.
• Calcium Ion Supplementation: Calcium ions are added to stimulate the Ca²⁺ influx necessary for the acrosome reaction.
• PKA Activation: Bicarbonate is still used to elevate intracellular pH and activate PKA.
• Challenges: *In vitro* capacitation is often less efficient and complete than *in vivo* capacitation. The complex interplay of signaling molecules present in the female reproductive tract is difficult to fully replicate.

The following table summarizes the key differences:

Feature	In Vivo	In Vitro
Initiating Signals	Female reproductive tract fluids (bicarbonate, albumin, signaling molecules)	Bicarbonate-buffered media, BSA, Calcium ions
Cholesterol Removal	Albumin	BSA
Decapacitation Factor Removal	Natural removal by female tract	Not present in initial media
Efficiency	High	Lower, requires optimization

Sperm Storage and Utilization in ART

Artificial Insemination (AI)

• Sperm Preparation: Sperm are capacitated *in vitro* (as described above) and then washed to remove seminal plasma and debris.
• Storage: Prepared sperm can be stored at room temperature (20-25°C) for a short period (up to a few hours) or refrigerated (4°C) for up to 7 days.
• Insemination: The capacitated sperm are then directly introduced into the uterus or cervix, timed to coincide with ovulation.

In Vitro Fertilization (IVF)

• Sperm Preparation: Sperm are capacitated *in vitro* and selected for motility and morphology.
• Storage: Prepared sperm can be used immediately for fertilization or cryopreserved (frozen) in liquid nitrogen (-196°C) for long-term storage.
• Fertilization: Sperm are co-incubated with oocytes in a petri dish, allowing fertilization to occur.
• ICSI (Intracytoplasmic Sperm Injection): In cases of severe male factor infertility, a single sperm is directly injected into the oocyte using a micromanipulator. This bypasses the need for natural capacitation and the acrosome reaction.

Cryopreservation is a critical component of ART, allowing for the storage of both sperm and oocytes for future use. Successful cryopreservation requires the use of cryoprotectants to prevent ice crystal formation, which can damage cells.