SEARCH

SEARCH BY CITATION

Contents

  1. Top of page
  2. Contents
  3. Introduction
  4. Induction of Capacitation In vitro
  5. In vitro Capacitation Induces Sperm Surface Alterations which Enable Spermatozoa to Fertilize the Egg
  6. Freeze–Thawing Procedures Cause Capacitation-like Surface Changes
  7. Sorting of Spermatozoa Causes Capacitation-like Surface Changes
  8. Novel Surface Manipulation Techniques
  9. Conflicts of interest
  10. Author contributions
  11. References

Since it has been well recognized that reproductive technologies, such as cryopreservation and sex-sorting, have a detrimental impact on sperm quality. These procedures cause sperm membrane destabilization which resembles that of capacitation. The pathways of this complex biochemical event are slowly unravelling, including the vital role of coating and decoating factors on the sperm surface. Characterization of these factors is leading to the development of novel surface manipulation techniques to stabilize the sperm membrane during handling. The possible application of these for assisted pig reproduction is discussed.


Introduction

  1. Top of page
  2. Contents
  3. Introduction
  4. Induction of Capacitation In vitro
  5. In vitro Capacitation Induces Sperm Surface Alterations which Enable Spermatozoa to Fertilize the Egg
  6. Freeze–Thawing Procedures Cause Capacitation-like Surface Changes
  7. Sorting of Spermatozoa Causes Capacitation-like Surface Changes
  8. Novel Surface Manipulation Techniques
  9. Conflicts of interest
  10. Author contributions
  11. References

During transit in the male and female reproductive tract spermatozoa encounter a host of fluids, epithelial cells and immuno-competent cells (Amman and Hammerstedt 1993) derived from the specialized regions of the epididymis, accessory sex glands and oviduct. The complex and varied protein complement of these fluids orchestrates the simultaneous uptake, release and modification of proteins at the sperm surface. In natural mating, the role of these surface modifications on essential fertilization events, such as capacitation and sperm–egg communication has been reviewed (Flesch and Gadella 2000; Nixon et al. 2007; Gadella 2008; Tsai and Gadella 2009).

As the sperm surface is continually interacting with its environment, and is modulated by this process, it is important to consider what happens when spermatozoa are processed for assisted reproduction. In these situations spermatozoa are subjected to varying diluents, temperatures, pH gradients and mechanical forces; any of which could potentially impact the structure and function of the plasma membrane and its constituents (proteins and lipids). This review will provide an overview of recent research regarding in vitro capacitation, and capacitation-like changes induced by storage and sex-sorting, and discuss the use of specific coating factors to manipulate the surface of boar spermatozoa and improve membrane stability.

Induction of Capacitation In vitro

  1. Top of page
  2. Contents
  3. Introduction
  4. Induction of Capacitation In vitro
  5. In vitro Capacitation Induces Sperm Surface Alterations which Enable Spermatozoa to Fertilize the Egg
  6. Freeze–Thawing Procedures Cause Capacitation-like Surface Changes
  7. Sorting of Spermatozoa Causes Capacitation-like Surface Changes
  8. Novel Surface Manipulation Techniques
  9. Conflicts of interest
  10. Author contributions
  11. References

It is worthy of note that the majority of studies investigating capacitation have been performed under in vitro capacitating/fertilizing conditions. This involves washing spermatozoa through a density gradient and pelleting the sample, which causes a partial stripping of loosely associated extracellular coating material from ejaculated spermatozoa (Caballero et al. 2009) and probably involves the removal of decapacitation factors and stabilizing seminal plasma proteins. Density gradient washing also selects a higher density sub-population of spermatozoa, representing those with signs of superior maturation (higher condensation of chromatin and removal of cytoplasmic droplets). These selection mechanisms are an analogy for the decoating of the sperm surface during transport in the female tract and competitive selection of mature spermatozoa by the cervix and isthmus (see Holt and Fazeli 2010), and are also common practise for in vitro fertilization (for review Mortimer 2000). Typically, these cells will now respond to a capacitation supportive medium containing capacitating agents such as bicarbonate, fatty acid free bovine serum albumin and calcium ions (for review, see Gadella and Visconti 2006; Gadella et al. 2008; Tsai and Gadella 2009; see also Fig. 1).

image

Figure 1.  Schematic representation of surface alterations imposed by in vitro capacitation treatments of boar spermatozoa. The decapacitation factors (in orange), originating from seminal plasma and adsorbed to the sperm surface during ejaculation), are removed by washing spermatozoa over a Percoll gradient. Subsequent incubation of spermatozoa in Tyrode’s medium containing bicarbonate, fat-free albumin (in red) and mM Ca 2+ cause the lateral redistribution, and partial removal, of cholesterol (in black). Consequently lipid ordered domains (in green) aggregate into the apical ridge area of the sperm head. This allows the formation of a zona binding protein complex as well as the docking of the acrosome to the sperm head surface. Partial scrambling of aminophospholipids at the sperm head, and the concomitant increase in membrane fluidity can be measured by staining spermatozoa with merocyanine (MC540), filipin and CTC. The treatment also causes the induction of hyperactivated motility which can be detected in fixed sperm by immunostaining for phosphotyrosine residues (PY).

Download figure to PowerPoint

In vitro Capacitation Induces Sperm Surface Alterations which Enable Spermatozoa to Fertilize the Egg

  1. Top of page
  2. Contents
  3. Introduction
  4. Induction of Capacitation In vitro
  5. In vitro Capacitation Induces Sperm Surface Alterations which Enable Spermatozoa to Fertilize the Egg
  6. Freeze–Thawing Procedures Cause Capacitation-like Surface Changes
  7. Sorting of Spermatozoa Causes Capacitation-like Surface Changes
  8. Novel Surface Manipulation Techniques
  9. Conflicts of interest
  10. Author contributions
  11. References

In vitro capacitation has dramatic effects on the composition and topology of sperm plasma membrane lipids and proteins. Sperm activation is accompanied by bicarbonate induced partial scrambling of aminophospholipids at the apical plasma membrane (Elliott and Higgins 1983; Gadella and Harrison 2000), and extraction of sterols by fatty acid free bovine serum albumin (Flesch et al. 2001), which results in higher membrane fluidity (can be visualized by merocyanine 540 staining; Harrison et al. 1996; Fig. 1).

Changes in membrane fluidity cause small lipid ordered domains, which initially reside over the entire acrosomal region of the sperm plasma membrane, to aggregate in the apical plasma membrane, the precise area of the sperm head where initial zona recognition occurs (Cross 2004; van Gestel et al. 2005). This so called membrane raft aggregation does not only involve sperm surface lipids but also membrane proteins that are specific for membrane rafts such as flotillin, caveolin and GPI-anchored proteins. Moreover, upon in vitro capacitation, membrane rafts also become highly enriched in zona binding proteins (van Gestel et al. 2005, 2007; Fig. 1).

Recently, it was also demonstrated that in vitro capacitation of boar spermatozoa caused a characteristic redistribution of SNARE proteins into the same area where the aggregation of membrane rafts and zona binding proteins was observed (Tsai et al. 2007, 2010). These proteins are involved in the execution of the acrosome reaction (Mayorga et al. 2007; Tomes 2007), but rather than induce membrane fusions this redistribution causes a very stable docking of the sperm plasma membrane with the outer acrosomal membrane (Tsai et al. 2010; Fig. 1). This docking is thought to localize multiple-point fusions at the apical sperm head region, where primary binding takes place, leaving the equatorial region intact for subsequent fertilization fusion with the oocyte (Tsai et al. 2010).

Taken together, it is apparent that spermatozoa undergo very complicated ergonomic changes during in vitro capacitation which involve extracellular and intracellular alterations and leaves the cell in a fully prepared state to fertilize the oocyte. Therefore, the onset of sperm activation must be synchronized with the arrival of spermatozoa at the oocyte. To achieve this molecules present on the surface of the spermatozoa, and molecules from the immediate environment of spermatozoa, act in concert to both stimulate and inhibit the onset and progression of capacitation (Bedford 2004). These molecules include secretions of the accessory sex glands, which coat the sperm membrane upon ejaculation and stabilize their membranes during transport through the male and female tract. This protection is especially critical during in vitro handling to prevent preliminary responsiveness of the spermatozoa in an environment where no oocyte can be fertilized.

Freeze–Thawing Procedures Cause Capacitation-like Surface Changes

  1. Top of page
  2. Contents
  3. Introduction
  4. Induction of Capacitation In vitro
  5. In vitro Capacitation Induces Sperm Surface Alterations which Enable Spermatozoa to Fertilize the Egg
  6. Freeze–Thawing Procedures Cause Capacitation-like Surface Changes
  7. Sorting of Spermatozoa Causes Capacitation-like Surface Changes
  8. Novel Surface Manipulation Techniques
  9. Conflicts of interest
  10. Author contributions
  11. References

Despite the relatively high uptake of artificial insemination in the pig industry (70% of sows and gilts bred using artificial insemination; USDA 2000) and the obvious benefits of long-term storage (for review, see Bailey et al. 2008), < 1% of all inseminations involve frozen–thawed spermatozoa. This is due to the poor farrowing rates and litter sizes achieved using this sperm type (Johnson et al. 2000).

The freeze–thaw process inflicts significant sub-lethal damage to boar spermatozoa and the causes and effects have been extensively reviewed (Parks and Graham 1992; Bailey et al. 2000; Holt 2000; Bagchi et al. 2008). In regard to the sperm surface, cryopreservation has four major impacts: (i) it results in decoating of extracellular matrix components (and lipids) and concomitant coating of proteins (and lipids) from the cryoprotective diluent (milk, albumin or egg yolk; Ollero et al. 1998; Lessard et al. 2000; Bergeron et al. 2004; Ricker et al. 2006), (ii) the reduced temperature causes lateral phase separation of lipids and thus a lateral reordering of membrane components (Holt and North 1984; De Leeuw et al. 1990; Drobnis et al. 1993) and (iii) the permeability of the sperm surface to water, ions and cryoprotectants is altered. The extent of these changes determines whether or not spermatozoa from certain donors (good freezers vs. poor freezers) can be used for cryopreservation (Chaveiro et al. 2006; Si et al. 2006; Hagiwara et al. 2009; Oldenhof et al. 2010). The final effect is a weakening of the cell that reduces its ability to withstand future stress (see Fig. 2). Some of these changes are reversible, while others are fatal (Watson 2000; Guthrie and Welch 2005). Certainly, they appear more critical for porcine spermatozoa, compared to bovine or human spermatozoa, which may relate to the relatively low levels of cholesterol in the porcine sperm membrane and the relatively large volume that needs to be cryopreserved (for a recent overview, see Rath et al. 2009).

image

Figure 2.  Schematic illustration of the current dogma surrounding protection of the sperm surface from stressors imposed by sperm handling associated with sorting or storage. Both techniques cause surface alterations that could lead to membrane damage. These membrane changes are similar to those of capacitated cells (e.g. staining patterns with CTC, MC540) but it remains unclear whether such cells also display functional biochemical changes associated with capacitation (e.g. raft aggregation, docking of acrosome; see Fig. 1 and text for further details) or if such changes lead to cell deterioration. When sperm cells are coated by decapacitation factors (e.g. through the supplementation of seminal plasma components; in orange) or when the membrane lipid content is altered (e.g. by changing the cholesterol content of sperm membranes; in black) this protects the sperm surface and increases its ability to withstand stress, resulting in less capacitation-like changes. It remains unclear whether such protected cells are still responsive to a capacitation supporting environment and as a result are able to fertilize the oocyte

Download figure to PowerPoint

Cryopreservation causes a hiatus in sperm development and when it is continued upon rewarming, the spermatozoa emerge in an advanced state of maturation, apparently having bypassed the need for capacitation (Watson 1995). A similar effect is evident after sex-sorting of spermatozoa, and it is believed that the expression of these capacitation-like changes follows a different pathway to that of true capacitation (Green and Watson 2001). Although both cells express similar characteristics in vitro (e.g. determined by chlortetracycline (CTC) staining patterns, tyrosine phosphorylation (PY), ability to undergo the acrosome reaction, etc.; Watson 1996; ]. However, it remains to be determined if cryocapacitation causes a similar physiological response to that of in vitro capacitation of fresh spermatozoa. Most notably, aggregation of rafts at the apical ridge area of the sperm head surface, stable docking of the acrosome by SNARE proteins, enhanced affinity for the zona pellucida and the generation of hyperactivated motility; as impairment of these processes probably relates to the poor fertility of frozen–thawed boar spermatozoa following artificial insemination (Guthrie and Welch 2005). In fact, lateral phase separation of lipids in frozen–thawed spermatozoa is not reversibly restored upon thawing (Fig. 2; De Leeuw et al. 1990) which may well frustrate the capacitation specific lateral rearrangements of the sperm surface (for a recent review of capacitation-induced lateral rearrangements of sperm surface lipids and proteins, see Gadella et al. 2008).

Sorting of Spermatozoa Causes Capacitation-like Surface Changes

  1. Top of page
  2. Contents
  3. Introduction
  4. Induction of Capacitation In vitro
  5. In vitro Capacitation Induces Sperm Surface Alterations which Enable Spermatozoa to Fertilize the Egg
  6. Freeze–Thawing Procedures Cause Capacitation-like Surface Changes
  7. Sorting of Spermatozoa Causes Capacitation-like Surface Changes
  8. Novel Surface Manipulation Techniques
  9. Conflicts of interest
  10. Author contributions
  11. References

While the stressors of flow-sorting differ from that of cryopreservation, both incite serious stress on the plasma membrane, which can result in loss of sperm function. The major stressors of sorting on the sperm surface result from: (i) the length of processing, (ii) the high extension rates employed (approximately 800-fold in the boar), (iii) the hydrodynamic pressure changes and orientation forces of the flow cytometer, (iv) changes in temperature (and specific deterioration of capacitated cells; Flesch et al. 2001), (v) passage through a high powered laser light excitation source, (vi) the sudden open in air droplet formation induced by an electric charge and mechanical movements of a 50 kHz piezo-electrotric crystal and (vii) deliverance at high speeds (50–90 km/h) into a collection tube. In the boar the detrimental effects are well known (for recent reviews, see Bathgate 2008; Vazquez et al. 2009) and include capacitation-like changes (Maxwell and Johnson 1997; Spinaci et al. 2006) and possible higher rates of pregnancy loss (Bathgate et al. 2008).

Evidence of capacitation-like changes is not a great surprise as the processing steps for sorting are very similar to those previously described for modelling capacitation in vitro. Specifically, the sample is diluted to extreme levels and exposed to mechanical forces, numerous diluents and potential capacitating agents (e.g. bovine serum albumin). The combination of these factors may strip loosely associated extracellular coating material from the ejaculated spermatozoa (Caballero et al. 2009); and probably involves the removal of stabilizing factors, leaving the cells more vulnerable to other stressors of the sex-sorting process. In connection with freezing, it is questionable whether the capacitation-like changes observed in sorted spermatozoa result in functional biochemical changes which enable fertilization (e.g. raft aggregation, enhanced affinity for the zona pellucida) or if surface destabilization leads to sperm deterioration. The distribution of AQN-3 provides a worthy example. This protein is the most prominent zona pellucida binding protein on the surface of boar sperm and (unlike other spermadhesins) remains attached to the sperm surface after in vitro capacitation, and can only be recovered from the aggregating raft area of the apical ridge of the sperm head (van Gestel et al. 2005, 2007). This shows that coating and decoating of seminal plasma components on in vitro capacitated sperm is a highly selective process and is probably more complicated than the capacitation-like changes we impose on the sperm surface with current semen processing.

Novel Surface Manipulation Techniques

  1. Top of page
  2. Contents
  3. Introduction
  4. Induction of Capacitation In vitro
  5. In vitro Capacitation Induces Sperm Surface Alterations which Enable Spermatozoa to Fertilize the Egg
  6. Freeze–Thawing Procedures Cause Capacitation-like Surface Changes
  7. Sorting of Spermatozoa Causes Capacitation-like Surface Changes
  8. Novel Surface Manipulation Techniques
  9. Conflicts of interest
  10. Author contributions
  11. References

Due to the decapacitation effects of seminal plasma outlined above, and its partial or complete removal during processing for freezing or sorting, it has naturally been explored as an additive to mediate the detrimental effects of these technologies. Recent studies showed incubation of frozen–thawed boar spermatozoa with 50% (v/v) crude seminal plasma improved motility and viability, compared to 0 or 10% (Garcia et al. 2010). However, the effect of seminal plasma is notoriously variable, dependant on multiple factors including boar effects (good freezer vs. poor freezer; Hernandez et al. 2007) and ejaculate portion (Rodriguez-Martinez et al. 2008; Garcia et al. 2009). Thus, it is not surprising that a similar effect was achieved using a 10-fold lower dose (Saravia et al. 2009) when optimized seminal plasma from the sperm-rich fraction of good freezer boars was utilized.

In addition to merely stabilizing the sperm membrane, seminal plasma has been shown (under certain conditions) to prevent or reverse capacitation (see Fig. 2). Supplementation of 10% crude seminal plasma to a capacitation supportive medium prevented in vitro capacitation and cooling induced capacitation-like changes (Vadnais and Roberts 2007, 2010). However, seminal plasma addition was unable to prevent cryopreservation-induced capacitation-like changes (Vadnais and Roberts 2010).

The addition of seminal plasma to the sex-sorting protocol has met with similar success. Early on it was shown that the inclusion of crude boar seminal plasma in a number of diluents during the sorting process improved the viability and motility of the final product (Maxwell et al. 1996; Catt et al. 1997). Furthermore, the incidence of capacitation-like changes induced by flow-sorting (Maxwell and Johnson 1997) are reduced by the inclusion of boar seminal plasma in the collection medium (Maxwell et al. 1998; Spinaci et al. 2006) and it is now commonly added to the collection medium (Parrilla et al. 2005) or after concentration of sperm samples (Grossfeld et al. 2005).

Unfortunately, this has not translated into a consistent improvement in fertility rates. Supplementation of 10% seminal plasma to frozen–thawed boar spermatozoa was reported to improve (Okazaki et al. 2009), or have no effect, on farrowing rates (Abad et al. 2007a). Likewise, thawing boar spermatozoa in 50% seminal plasma was reported to increase litter size (Garcia et al. 2010), which has been related to the capacitation status of boar spermatozoa (Oh et al. 2010), but post-thaw supplementation of 10% seminal plasma had no effect on litter size (Abad et al. 2007a) or the establishment of an oviductal sperm reservoir (Abad et al. 2007b). In vivo studies with seminal plasma and sex-sorted boar spermatozoa are scarce, but the addition of seminal plasma proteins before freezing improved the function of sex-sorted ram spermatozoa (Leahy et al. 2009), but had no effect on fertility after cervical insemination (Leahy et al. 2010a).

This variability in the response of seminal plasma could be minimized if it was known which proteins were of benefit. In the pig the most dominant proteins (90% total protein) are termed spermadhesins, and they have many putative functions; including regulation of capacitation (for review, see Töpfer-Petersen et al. 2008). They are expressed in the epididiymis (AWN & AQN-3), seminal vesicles and even the female reproductive tract (Dostalova et al. 1994b; Ekhlasi-Hundrieser et al. 2002). In the pig the spermadhesin family is made up of five proteins, classified according to their sequences and biological activities, as AQN-1, AQN-3, AWN, PSP-I and PSP-II and all, except the latter two, bind heparin (Sanz et al. 1993).

One way to fractionate the protein mix is to separate heparin-binding proteins from non-heparin-binding proteins. A recent study reported that heparin-binding proteins, consisting of three spermadhesins AQN-3, AQN-1, AWN and a BSP protein pB1, inhibited in vitro capacitation (in capacitating conditions) and cooling induced capacitation-like changes (Vadnais and Roberts 2010). This effect was evident when just 250 μg/ml of protein was present whereas 1.5 mg/ml (approx. 10% crude seminal plasma) was required to achieve a similar effect with non-heparin-binding proteins, leading investigators to conclude this may be a residual effect of the heparin-binding proteins.

The reported decapacitation effect of AQN-3 and AWN-1 agrees with earlier structural studies which showed non-aggregated forms of these proteins are able to bind directly to membrane lipids (Dostalova et al. 1995) and are released from the sperm surface upon capacitation (Sanz et al. 1993; Dostalova et al. 1994a; Calvete et al. 1997). It is thought that this initial layer provides an anchor for aggregated spermadhesins to coat the sperm surface (Töpfer-Petersen et al. 2008), which further stabilizes the plasma membrane and prevents premature acrosomal exocytosis. However, the BSP protein (pB1) identified in the heparin-binding fraction, has been previously described to stimulate capacitation of epididymal sperm by inducing cholesterol and phospholipid efflux from the sperm membrane (Lusignan et al. 2007) and this phenomena has been well described in the bull where they are the major seminal plasma constituent (for review, see Manjunath et al. 2007).

Evidence from a series of studies on highly dilute boar spermatozoa also supports an inhibitory effect of heparin-binding proteins. Seminal plasma is well known to attenuate the dilution effect (Ashworth et al. 1994) and the particular proteins providing this effect where investigated on boar spermatozoa extended to a level that mimicked sex-sorting of spermatozoa (0.3–1×106 sperm/ml). These studies showed that incubation of boar spermatozoa with heparin-binding proteins was detrimental to in vitro sperm function; causing a time- and dose-dependent decrease in motility, viability and mitochondrial integrity (Centurion et al. 2003). Whereas, the addition of non-heparin-binding proteins was beneficial, with maximal protection afforded when 1.5 mg/ml of protein was supplemented. The major non-heparin-binding protein of boar seminal plasma is termed the PSP-I/PSP-II heterodimer, and its protective effect can last up to 10 h when sperm are incubated at 38ºC (Caballero et al. 2006). Further investigation showed that the protective effect was largely preserved in the PSP-II subunit, and separation of this subunit into its peptide and glycan fragments suggests that the latter is not required for protective function (Garcia et al. 2006). This indicates that a commercially synthesized peptide moiety could be used as a potential sperm preserver (Caballero et al. 2008). Moreover, the heterodimer also appears to have a regulatory role in capacitation. Immunolocalization studies have revealed that the heterodimer is mainly located on the acrosomal region of boar spermatozoa and this association holds the spermatozoa in a time-limited non-capacitated state through maintenance of low intracellular calcium levels (Caballero et al. 2009). After long-term incubation the heterodimer redistributes to the post-acrosomal region. Interestingly, heparin-binding proteins show the opposite pattern, and this redistribution was related to capacitation status (Dapino et al. 2009).

While all these studies seem somewhat contradictory, they highlight the importance of sperm preparation on the resultant effect of seminal plasma (Leahy et al. 2010b). For instance, differences in the effect of heparin- and non-heparin-binding proteins may have been caused by differences in the sperm coating of the sperm types used in these studies (ejaculated, epididymal and highly diluted spermatozoa). As pro-capacitation factors only have effects on spermatozoa after appropriate removal of other factors that coat the sperm surface (for review, see Fraser et al. 2006; Rodriguez-Martinez et al. 2009). In addition, other factors common to semen diluents, most notably egg yolk, are well known to coat the sperm membrane and their presence also influences the effect of seminal plasma on sperm function and both detrimental (Manjunath et al. 2007) and beneficial (Vadnais et al. 2005) effects have been reported.

These multiple influences explain why, despite having known about the decapacitation effect of seminal plasma (Chang 1957) soon after discovery of the process itself (Austin 1952), we have failed to reliably exploit this natural phenomena to protect spermatozoa during processing. Regardless of these challenges, remarkable progress has been made over the last 50 years, particularly in the determination of the pathways of sperm maturation, capacitation and fertilization. This has been accelerated in the latter years through recent advances in proteomics and genomics and these techniques will further develop our understanding of the physiological sequence of capacitation-related events, and how these are interrupted by semen handling. With such knowledge, new processing approaches may be designed to maximize the fertilizing ability of cryopreserved and sex-sorted spermatozoa.

References

  1. Top of page
  2. Contents
  3. Introduction
  4. Induction of Capacitation In vitro
  5. In vitro Capacitation Induces Sperm Surface Alterations which Enable Spermatozoa to Fertilize the Egg
  6. Freeze–Thawing Procedures Cause Capacitation-like Surface Changes
  7. Sorting of Spermatozoa Causes Capacitation-like Surface Changes
  8. Novel Surface Manipulation Techniques
  9. Conflicts of interest
  10. Author contributions
  11. References