Capacitation is a biochemical process that maturespermatozoa undergo in the upper female reproductive tract, within theoviduct anduterus, that allows for the beginning of sperm activation. It is also known as cellular-implantation or justimplantation. Capacitation is the penultimate step of spermatozoa activation, with theacrosome reaction following it. Sperm must undergo capacitation and the acrosome reaction in order to penetrate through thecumulus ooporus and thezona pellucida of anoocyte. Capacitation specifically allows for induction ofhyperactivation and hypermotility, processes that set up for oocyte penetration. Capacitation is also necessary forchemotactic swimming through the female reproductive tract, whereprogesterone gradients guide the sperm to the egg.[1] Compounds such asheparin 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 (usuallyserum albumin), electrolytes such assodium (Na+),potassium (K+),chloride (Cl-),bicarbonate (HCO3-),magnesium (Mg2+),calcium (Ca2+), andphosphate (PO43-), and energy substrates such asglucose,pyruvate, and lactate.[2] For purposes ofin vitro fertilization, capacitation occurs by incubating spermatozoa that have been retrieved via ejaculation or extracted from theepididymis and incubated in a defined medium for several hours. There are different techniques to perform the capacitation step: simple washing, migration (swim-up), density gradients, and filter. The objective is to isolate as many motile spermatozoa as possible and to eliminate non-motile or dead spermatozoa.
Mammalian sperm membranes have an approximate lipid composition of 70%phospholipids, 25%cholesterol, and 5%glycoproteins, though the exact percentages are species dependent. A notable distinguishing characteristic between mammalian species is the variable ratio of cholesterol to phospholipid (C/PL) in the plasma cell membrane. The duration of capacitation has been shown to be related to the C/PL ratio of sperm membranes, contributing to the difference in capacitation duration between species.[3] Studies have reported that sperm capacitation in humans takes 3-10 hours, on average.
The majority of capacitation studies have been conducted on mice as they are a sufficient de facto surrogate model, but limitations to these models do exist.[4] For instance, in mice, ejaculate is directly deposited into the uterus while, for humans, it is in the vaginal canal.[3] Additional anatomical and physiological differences between species also contributes to the potential inaccuracies of using mice for studying capacitation. Despite this, the use of animal models has contributed a significant amount to our understanding of capacitation.
Non-mammalian spermatozoa do not require activation via capacitation and the acrosome as they are ready to fertilize an oocyte immediately after release from the male.
Sperm capacitation consists of five main steps: changes to the sperm plasma membrane that cause increased membrane fluidity, pH changes, ion flux, activation of the cAMP-PKA pathway and downstream phosphorylation, and changes to membrane potential (hyperpolarization) which result in hyperactivation and consequent enhanced motility of sperm.[5]
Capacitation has two effects: destabilisation of the acrosomal sperm head membrane which allows it to penetrate the outer layer of the egg, and chemical changes in the tail that allow a greater mobility in the sperm.[6] The changes are facilitated by the removal ofsterols (e.g.cholesterol) and non-covalently bound epididymal/seminalglycoproteins. The result is a more fluid membrane with an increased permeability to Ca2+ ion.
An influx of Ca2+ produces increased intracellularcAMP levels and thus, an increase in motility.Hyperactivation coincides with the onset of capacitation and is the result of the increased Ca2+ levels. It has a synergistic stimulatory effect with adenosine that increasesadenylyl cyclase activity in the sperm.[citation needed]
The tripeptidefertilization promoting peptide (FPP) is essential for controlling capacitation. FPP is produced in theprostate gland as a component of the seminal fluid. FPP comes into contact with the spermatozoa during ejaculation, as the sperm and seminal fluid mix. High levels of active FPP prevent capacitation. After ejaculation, the concentration of FPP drops in the female reproductive tract.[citation needed]
Becauseassisted reproductive technologies, or ARTs, such asin vitro fertilization (IVF) orintrauterine insemination (IUI) require the induction of sperm cell capacitation outside of normal biological parameters, numerous methods have been developed to induce this process in mammalian sperm cells. Sperm cells are harvested through ejaculation or harvested from the caudalepididymis and allowed to liquefy at room temperature. Capacitation can then be induced by adding media designed to mimic the electrolytic composition of thefallopian tubes, where fertilization occurs. These media vary between species, but are saline-based and contain energy substrates such as lactate, pyruvate, and possibly glucose. A cholesterol acceptor is required to facilitate the removal of cholesterol from the sperm cell membrane, which is often albumin.Bovine serum albumin is typically used forin vitro animal studies, andhuman serum albumin (HSA) is used in human sperm capacitation induction.
Bicarbonate is a vital component of capacitation-inducing media, as it is co-transported into thecytosol where it activates solubleadenylyl cyclase (sAC) as well as acts as a pH buffer necessary to prevent decreasing the pH in the culture, a necessary addition when incubating cells at 5% CO2 as is generally used although not required.Calcium chloride is added to facilitated the influx via of calcium cations.[7][8] In animal models, Tyrode's albumin lactate pyruvate (TALP) medium is typically used as a base, which contains each of these components. In humans, human tubal fluid (HTF) is used.
These media can be supplemented with other chemicals to induce hyperactivated sperm motility and/or the acrosome reaction. For animal in vitro fertilization,caffeine at 5 mM concentration is a strong inducer of sperm capacitationin vitro.[9][10] Calciumionophores are also ideal to induce capacitation.[10] Adding heparin to capacitation inducing medium mimics the secretion of heparin-likegycosaminoglycans (GAGs) near the oocyte and initiates the acrosome reaction. This effect is magnified when addinglysophosphatidylcholine (LC) in conjunction with heparin.[11]Catecholamines such asnorepinephrine at low concentrations have been shown to assist in acrosome reaction induction.[12]
The traditional methods to performin vitro capacitation are:
PICSI, MACS or microfluidic chips are more recent methods that can be used to induce capacitationin vitro.
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.[14] CASA usesphase-contrast microscopy combined with sperm tracking software to analyze sperm motility parameters.[14] Certain parameters such as curvilinear velocity (VCL), straightline velocity (VSL), average path velocity (VAP), and the amplitude of lateral head displacement (ALH) have been shown to be positively correlated with the acquisition of fertilization competency and are thus used to identify hyperactive sperm cell motility.[15]
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.[16] More advanced techniques employ fluorescent orelectron microscopy methods.Fluorescein-conjugatedPeanut agglutinin (FITC-PNA) orPisum sativumagglutinin (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 afluorescent microscope.[17][18][19]
The discovery of this process was independently reported in 1951 by bothMin Chueh Chang[20] andColin Russell Austin.[21][22]