Capacitation

Capacitation is a process sperm must undergo to prepare to fertilize an egg. This is a process that mature spermatozoa undergo after entering the oviduct and uterus of the female reproductive tract. Sperm must undergo capacitation and the acrosome reaction to be able to penetrate through the outer layers of an oocyte, namely the cumulus ooporus and the zona pellucida. Capacitation results in hyperactivation of sperm motility, with the acrosome and cortical reaction following this. Compounds such as heparin and progesterone can be used to induce capacitation.

As a result of the sperm entering the upper female reproductive tract, the sperm are introduced to an extracellular environment that contains a cholesterol acceptor (usually serum albumin), electrolytes, and energy substrates such as glucose, pyruvate, and lactate. Non-mammalian spermatozoa do not require activation as they are ready to fertilize an oocyte immediately after release from the male.

Function and mechanism

Sperm capacitation consists of five main steps: changes to the sperm plasma membrane that cause increased membrane fluidity, pH changes, ion flux, activation of secondary pathways, and changes to membrane potential (hyperpolarization). These steps result in hyperactivation of sperm motility.

Release of glycosylphosphatidylinositol-anchor proteins (GPI-APs), which are membrane proteins present in lipid rafts, occurs as a result of cholesterol efflux. Decapacitation factors are glycoproteins or glycolipids that stabilize the membrane and prevent premature capacitation. If cholesterol is retained in the sperm membrane, however, the ion channels necessary to transport H⁺ and HCO₃^- are not activated and pH does not change. can be transported into sperm through solute carrier transporters (SLCs). CatSpers move Ca²⁺ into the sperm. Other Ca²⁺ channels that contribute to the influx of Ca²⁺ include: Ca²⁺ ATPases, transient-receptor potential (TRP) channels, Na⁺/Ca²⁺ exchangers (NCX), voltage-gated Ca²⁺ channels (Cav), and cyclic nucleotide-gated (CNG) channels. These channels are activated by the alkaline conditions in the oviduct.

• Chloride is transported into the sperm using CFTR channels.

• Sodium is transported by ENaC, which moves sodium out of the sperm. ENaC is activated by high pH and the presence of Ca²⁺.

Step 5: Hyperactivation of sperm motility

Hyperactivation of sperm motility is primarily induced through cellular hyperpolarization and Ca²⁺ influx through the action of CatSpers. Hyperactivation is characterized by asymmetric swimming and high-amplitude flagellular beating. This is the final component of capacitation. Calcium ionophores are also ideal to induce capacitation. Catecholamines such as norepinephrine at low concentrations have been shown to assist in acrosome reaction induction.

In vitro capacitation techniques

The traditional methods to perform in vitro capacitation are:

• Simple wash: this method only eliminates seminal plasma, it does not select the best spermatozoa. The sample is centrifuged and then, the supernatant is eliminated. It is used in severe oligozoospermia, cryptozoospermia or testis biopsy samples. It is performed before other capacitation techniques too.

• Migration (swim-up). Firstly, centrifugation takes place and seminal plasma is eliminated. Then, 0.5 -1 ml of culture medium is added at the top and after the incubation period at 37°C, the best motile spermatozoa will have ascend from the bottom to the top of the tube (healthy spermatozoa go to the culture medium). In order to obtain the fraction rich in spermatozoa, the top layer is collected. It is still widely used and useful in normozoospermia. It allows to obtain fractions with more than 90% of PR spermatozoa.

• Density gradients. In this technique, a tube is filled with layers of liquids of different densities and semen is placed on the top layer. Then, The tube goes through a centrifugation to filter cell debris and non motile cells. After the centrifugation, healthy sperm are on the very bottom layer of the liquid in the tube, while debris and non-motile spermatozoa are in upper layers. This procedure takes approximately 60 minutes and it is specially indicated in oligozoospermia, asthenozoospermia and abundant debris samples. At the end, all the cells will arrive to the bottom, but those with more motility will arrive sooner. This procedure is often called just the "Percoll method", since Percoll was frequently used as the density medium, but other density mediums are now used.

• Filtration. It consist in a filter that does not allow every sperm to pass. It is less used nowadays and only spermatozoa with better motility will pass through the filter.

PICSI, MACS or microfluidic chips are more recent methods that can be used to induce capacitation in vitro.

In vitro measurement methods

Numerous methods have been developed to assess the degree to which sperm cells are undergoing capacitation in vitro. Computer-aided sperm analysis (CASA) was developed in the 1980s for measuring sperm kinematics. CASA uses phase-contrast microscopy combined with sperm tracking software to analyze sperm motility parameters.

While motility measurements are critical for identifying the presence of hyperactive motility, additional methods have been developed to identify the occurrence of the acrosome reaction. A simple method uses Coomassie brilliant blue G250 to stain cells, providing visual evidence of intact or reacted acrosomes. More advanced techniques employ fluorescent or electron microscopy methods. Fluorescein-conjugated Peanut agglutinin (FITC-PNA) or Pisum sativum agglutinin (FITC-PSA) can be used to fluorescently tag the acrosome of sperm cells, which can be then used to assess the status of the acrosome using a fluorescent microscope.

Discovery

The discovery of this process was independently reported in 1951 by both Min Chueh Chang and Colin Russell Austin.

See also

• Cortical reaction
• Acrosome reaction

References

Further reading

External links