At coitus, human sperm are deposited into the anterior vagina, where, to avoid vaginal acid and immune responses, they quickly contact cervical mucus and enter the cervix. Cervical mucus filters out sperm with poor morphology and motility and as such only a minority of ejaculated sperm actually enter the cervix. In the uterus, muscular contractions may enhance passage of sperm through the uterine cavity. A few thousand sperm swim through the uterotubal junctions to reach the Fallopian tubes (uterine tubes, oviducts) where sperm are stored in a reservoir, or at least maintained in a fertile state, by interacting with endosalpingeal (oviductal) epithelium. As the time of ovulation approaches, sperm become capacitated and hyperactivated, which enables them to proceed towards the tubal ampulla. Sperm may be guided to the oocyte by a combination of thermotaxis and chemotaxis. Motility hyperactivation assists sperm in penetrating mucus in the tubes and the cumulus oophorus and zona pellucida of the oocyte, so that they may finally fuse with the oocyte plasma membrane. Knowledge of the biology of sperm transport can inspire improvements in artificial insemination, IVF, the diagnosis of infertility and the development of contraceptives.

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Sperm transport in the female reproductive tract

BACKGROUND: Sperm hyperactivation is critical to fertilization, because it is required for penetration of the zona pellucida. Hyperactivation may also facilitate release of sperm from the oviductal storage reservoir and may propel sperm through mucus in the oviductal lumen and the matrix of the cumulus oophorus. Hyperactivation is characterized by high amplitude, asymmetrical flagellar bending. METHODS: This is a review of the original literature on the mechanisms that regulate hyperactivation, including physiological factors and signaling pathways. RESULTS: Computer-assisted semen analysis systems can be used to identify hyperactivated sperm by setting minimum thresholds for curvilinear velocity (VSL) and lateral head movement and a maximum threshold for path linearity. Hyperactivation is triggered by a rise in flagellar Ca(2+) resulting from influx primarily through plasma membrane CatSper channels and possibly also by release of Ca(2+) from a store in the redundant nuclear envelope. It requires increased pH and ATP production. The physiological signals that trigger the rise in Ca(2+) remain elusive, but there is evidence that the increased Ca(2+) acts through a calmodulin/calmodulin kinase pathway. Hyperactivation is considered part of the capacitation process; however, the regulatory pathway that triggers hyperactivation can operate independently from that which prepares sperm to undergo the acrosome reaction. Hyperactivation may be modulated by chemotactic signals to turn sperm toward the oocyte. CONCLUSIONS: Little is known about exactly what triggers hyperactivation in human sperm. This information could enable clinicians to develop reliable fertility assays to assess normal hyperactivation in human sperm samples.

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Control of hyperactivation in sperm

Capacitation as a regulatory event that primes spermatozoa for the acrosome reaction and fertilization : Regulators of sperm function

Capacitation is defined as the series of transformations that spermatozoa normally undergo during their migration through the female genital tract, in order to reach and bind to the zona pellucida, undergo the acrosome reaction, and fertilize the egg. During this process, extensive changes occur in all sperm compartments (head and flagellum; membrane, cytosol, cytoskeleton), factors originating from epididymal fluid and seminal plasma are lost or redistributed and membrane lipids and proteins are reorganized; ion fluxes induce biochemical modifications and controlled amounts of reactive oxygen species are generated; spermatozoa develop hyperactivated motility; and complex signal transduction mechanisms are initiated. The main purpose of capacitation is to ensure that spermatozoa reach the eggs at the appropriate time and in the appropriate state to fertilize these eggs, by finely-controlling the rate of the changes necessary to prime spermatozoa and by activating all the mechanisms needed for the subsequent acrosome reaction. The reversibility of some of the mechanisms leading to sperm capacitation may therefore be a very important aspect of the fine regulation and perfect timing of this process.

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Capacitation as a regulatory event that primes spermatozoa for the acrosome reaction and fertilization.

When sperm of the ram, bull, boar and stallion are cold-shocked by rapid cooling to near freezing point, motility and metabolic activity are irreversibly depressed and the acrosome and plasma membrane disrupted. Ram sperm become susceptible to cold shock in the proximal corpus region of the epididymis when the cytoplasmic droplet has moved backwards to the distal portion of the sperm midpiece. The membrane constituents phospholipids and cholesterol are important in cold shock which causes loss of lipid from sperm. The susceptibility of sperm to cold shock is linked with a high ratio of unsaturated:saturated fatty acids in the phospholipids and a low cholesterol content. The high unsaturated fatty acid content of sperm also makes them susceptible to damage from peroxidation which adversely affects motility, metabolism, ultrastructure and fertility. Hydroxynonenal, a product of fatty acid peroxidation, depresses the motility and oxygen uptake of ram sperm in vitro and may react with the -SH groups of the axonemal microtubules. High calcium concentrations in the external medium may decrease the motility and metabolism of sperm and 'calcium intoxication' may be a factor in cold shock. Lowering the environmental temperature increases calcium uptake by sperm and the effect is aggravated if the rate of cooling is rapid. Phospholipids, particularly those in egg yolk, protect sperm to some extent from cold shock and also prevent increased calcium flux into the sperm. Suggestions are made for increasing the life span of sperm during preservation and microencapsulation by adding agents that may stabilize membranes, counter peroxidation and decrease calcium uptake.

Lipids and calcium uptake of sperm in relation to cold shock and preservation: a review

While hyperactivated motility is known to be a concomitant of capacitation, and a prerequisite for fertilization, the specific interdependence of capacitation and hyperactivation in human spermatozoa has not been investigated. This study was designed to determine the effect of seminal plasma contamination on the expression of hyperactivated motility and the relationship between hyperactivation and capacitation, since seminal plasma contains decapacitation factor(s). Seminal plasma was obtained by centrifugation of aliquots of liquefied semen layered over 1.5 ml 40.5% Percoll and mixed with human tubal fluid (HTF) medium containing 30 mg/ml human serum albumin (HSA) (HTF) to a final concentration of 5% (v/v) seminal plasma (SP). Motile spermatozoa were isolated from the remainder of the semen by swim-up into either HTF or SP medium. Samples were taken from each treatment immediately post-harvest (0 h) and after 60 min at 37 degrees C (1 h) for hyperactivation and capacitation assessment. The treatments were then divided into two portions, centrifuged and resuspended in either HTF or SP, giving HTF control and SP control treatments and two crossover treatments, 1 h HTF then 1 h SP (H/SP) and 1 h SP then 1 h HTF (SP/H). All tubes were incubated for a further 60 min at 37 degrees C before aliquots were taken for hyperactivation and capacitation assessments. Hyperactivation was estimated using an IVOS v10.6t (Hamilton Thorne Research, Beverly, MA, USA) 60 Hz CASA instrument, and capacitation was estimated using the chlortetracycline (CTC) method. The presence of seminal plasma in the capacitation medium for 60-120 min post-swim-up inhibited the development of hyperactivated motility. This inhibition was reversible, and was not prevented by preincubation for 1 h in HTF medium. There was no difference in the CTC binding patterns between treatments at 2 h, indicating that the capacitation-associated membrane changes were not affected by the presence of a low concentration of seminal plasma. There was no correlation between percentage capacitated and percentage hyperactivated spermatozoa for any treatment. Since the proportions of hyperactivated spermatozoa and capacitated spermatozoa were not related, we conclude that the processes leading to hyperactivation and to the membrane changes associated with capacitation are not tightly interlinked and consider this finding to be due to hyperactivated motility being associated with flagellar movement, while the CTC assay assesses changes in the Ca2+ levels of the sperm head plasma membrane.

Effect of seminal plasma on capacitation and hyperactivation in human spermatozoa.

Aims/hypothesis
Type 1 diabetes results from the autoimmune destruction of pancreatic beta cells. Exogenous insulin therapy cannot achieve precise physiological control of blood glucose concentrations, and debilitating complications develop. Lentiviral vectors are promising tools for liver-directed gene therapy. However, to date, transduction rates in vivo remain low in hepatocytes, without the induction of cell cycling. We investigated long-term transgene expression in quiescent hepatocytes in vitro and determined whether the lentiviral delivery of furin-cleavable insulin to the liver could reverse diabetes in rats.

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Long-term correction of diabetes in rats after lentiviral hepatic insulin gene therapy.

For technical reasons sperm head movement is assessed in kinematic analysis, while flagellar movement is the determining factor of head movement, not vice versa. It follows then that the development of new kinematic values to describe the movement of capacitating human spermatozoa should include the analysis of their flagellar movement. The aim of this study was to establish quantitative differences between flagellar movement patterns of hyperactivated and non-hyperactivated spermatozoa which could then be used in the evaluation of new centroid-based kinematic values. Spermatozoa were prepared by swim-up from semen into culture medium supplemented with 30 mg/ml human serum albumin. Sperm movement was recorded in 50 microm-deep chambers using a 200 Hz video system. Sperm movement was classified based on flagellar movement, with 24 non-hyperactivated and 26 hyperactivated spermatozoa included in the study. Flagellar analysis was performed using both a semi-automated analysis system (SIAM FLAG; 30 images at 200 Hz) and manual methods (100 Hz). Hyperactivated spermatozoa had significantly larger flagellar beat angles (> or = 87 degrees) and significantly lower flagellar beat frequencies (< or = 29.4 Hz) than non-hyperactivated human spermatozoa. In addition, the flagellar wave amplitude was significantly greater and the bend diameter significantly smaller for hyperactivated spermatozoa in the proximal region of the flagellum (up to 20 microm from the head-midpiece junction). The velocity of the hyperactivated wave was low in this region, although it was significantly slower than the non-hyperactivated wave in all regions of the sperm tail.

Quantitative observations of flagellar motility of capacitating human spermatozoa.

Human spermatozoa were prepared by swim-up from semen into in-vitro fertilization (IVF) culture medium, and their movement recorded by videomicrography using NTSC video to give 60 images/s on freeze-frame playback. Trajectories in which spermatozoa appeared to switch from one type of motility pattern to another (from non-hyperactivated to transitional or star-spin hyperactivated), were reconstructed manually and the movement characteristics determined for short consecutive sequences along each track. The spermatozoa were able to switch between all of these motility patterns, apparently at random. These observations illustrated that hyperactivated motility is a reversible state in human spermatozoa, with phase changes from hyperactivated to non-hyperactivated motility patterns along trajectories. It was not necessary for spermatozoa to pass through the transitional hyperactivated motility phase when switching from non-hyperactivated to star-spin hyperactivated motility or vice versa.

Variable kinematics of capacitating human spermatozoa

Rapidly cooling (cold shocking) washed cauda boar sperm irreversibly reduced motility and respiration and greatly increased the uptake of 45Ca2+; the plasma membranes were removed and the acrosomes detached from nuclei. The motility, respiration, and calcium uptake of the less mature caput sperm were largely unaffected; and there was little damage to the ultrastructure. This indicates that boar sperm becomes less resistant to cold shock as they mature in the epididymis.



The oxygen uptake, glucose breakdown, and lactic acid production of control caput sperm was less than that of cauda sperm. This suggest that the maturation of sperm in the epididymis of the boar involves an increase in both the glycolytic and oxidative phases of glucose metabolism.



The presence of 2.0 mg/ml phosphatidylcholine (lecithin) in the medium prevented ultrastructural damage to cauda sperm on cold shock, and motility and respiration were maintained at levels similar to those of control sperm. Although the presence of phospholipid reduced the large calcium influx following cold shock, it was still greater that that of control sperm.



The “protective” effect against cold shock was not maintained after rewashing the sperm free of phosphatidylcholine prior to cold shock, indicating a fairly “loose” interaction of the phospholipid with boar sperm membranes that was easily disrupted.

M. A. Swan

Papers

Effect of seminal plasma on capacitation and hyperactivation in human spermatozoa.

Long-term correction of diabetes in rats after lentiviral hepatic insulin gene therapy.

Quantitative observations of flagellar motility of capacitating human spermatozoa.

Variable kinematics of capacitating human spermatozoa

Susceptibility of epididymal boar sperm to cold shock and protective action of phosphatidylcholine.