Keywords

Key Points

  • Sperm capacitation, discovered in 1951, independently by CR Austin and MC Chang, is considered as an obligate requirement for sperm fertility.

  • The last six decades have seen a considerable rise in laboratory-based studies on human sperm capacitation and its associated phenomena: hyperactivation, acrosome reaction and protein tyrosine phosphorylation.

  • Clinical tests based on the identified molecular markers are rather scarce, with one test, viz. Androvia Cap-Score™ showing promising results in being able to discriminate fertile from infertile men.

  • In the present era of assisted reproductive techniques (ARTs), especially ICSI, it is mandatory to develop reliable sperm function tests based on capacitation and other related phenomena to ensure the selection of the “healthiest” spermatozoa.

1 Introduction

In mammals, after having gone through the journey of formation in testis and maturation in epididymis; spermatozoa, the male gamete, isn’t quite ready yet to marry the female gamete, the oocyte. It still has to undergo a whole battery of changes—this time—in the female reproductive tract, to fertilize the oocyte (Fig. 5.1). This ensemble of post-ejaculation changes in the spermatozoa has been collectively called as sperm “capacitation”. Capacitation renders the spermatozoa functionally mature.

Fig. 5.1
figure 1

Life cycle of sperm: After production in testis in sufficient numbers with normal shape, the spermatozoa undergo maturation in epididymis, gain motility and undergo capacitation in the female reproductive tract, then acrosome react after oocyte binding and penetrate and activate the egg, resulting in successful fertilization. The blue arrows indicate the site of event

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Origin of spermatozoa in the testis is followed by its capacitation (after ejaculation) in the female reproductive tract and ultimately fertilization with oocyte in the fallopian tube. Sperm contribution to fertilization to assess the “male factors” is usually estimated through evaluation of semen parameters, namely, sperm count, morphology and motility (World Health Organization 2010). Quite often, in spite of these parameters being normal and these males being termed as normozoospermic (normal count, motility and morphology); the infertility still exists in the male partner. Such cases of idiopathic (unknown etiology) infertility have been attributed substantially to the problems in sperm capacitation (Tucker et al. 1987; Matzuk and Lamb 2002; Esposito et al. 2004; Hildebrand et al. 2010; Nandi and Homburg 2016).

2 What Is Sperm Capacitation?

Sperm capacitation has been defined as the “ensemble of all the physiological, molecular and cellular changes in the spermatozoa, which are necessary to make it fertilization competent”. It was independently discovered by Austin and Chang in 1951 (Austin 1951; Chang 1951). Although discovered more than half a century ago, capacitation is still regarded as a “poorly understood” phenomenon, owing to the fact that each mammalian species has its unique features at the physical (time of capacitation) and molecular level (Chang 1984) that are difficult to monitor, since it takes place in the female reproductive tract (either in the oviduct or in the vicinity of the egg).

Sperm capacitation is a prerequisite for successful fertilization as evidenced from the observations that a block in capacitation causes male infertility (Tucker et al. 1987; Matzuk and Lamb 2002; Esposito et al. 2004; Hildebrand et al. 2010). Therefore, there has been a pressing need to understand sperm capacitation in all the individual species making it a focus of investigations of many gamete biologists worldwide. Most progress in understanding the phenomenon of capacitation has been because of in vitro methods for capacitation (Yanagimachi 1969). In the procedure, freshly ejaculated or epididymal spermatozoa are washed and incubated at physiological conditions in a defined medium that mimics the female oviductal fluid (Dow and Bavister 1989). The medium normally has the following composition: electrolytes, metabolic energy source and a macromolecule to allow for cholesterol efflux like serum albumin (Yanagimachi 1969, 1994). Several in vitro studies have revealed that during capacitation, spermatozoa undergo a number of biochemical and biophysical changes (Fig. 5.2), such as increase in membrane fluidity (Davis et al. 1980; Cross 1998; Buffone et al. 2009; Salvolini et al. 2013), activation of trans-bilayer signalling events (Go and Wolf 1985; Visconti et al. 1998; Gadella and Harrison 2000; Flesch et al. 2001; Sheriff and Ali 2010; Ickowicz et al. 2012), changes in redox status of spermatozoa leading to generation of reactive oxygen species (ROS) (de Lamirande and Gagnon 1992; Aitken 1995; O’Flaherty et al. 2006; Musset et al. 2012), removal of stabilizing proteins (Shivaji et al. 1990; Villemure et al. 2003; Leahy and Gadella 2011) and phosphorylation of proteins (Leyton and Saling 1989; Visconti et al. 1995; Mitra and Shivaji 2004; Arcelay et al. 2008; Mitchell et al. 2008; Kota et al. 2009; Katoh et al. 2014).

Fig. 5.2
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A schematic representation of capacitation and its associated hallmarks (in blue) and biochemical and biophysical changes

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3 Hallmarks of Capacitation

Capacitation is generally monitored by recording protein tyrosine phosphorylation (pY), hyperactivation (Yanagimachi 1994; Kulanand and Shivaji 2001; Baker et al. 2006) and acrosome reaction (Ward and Storey 1984; Meizel and Turner 1991; Aitken 1995; Curry and Watson 1995; Mitra and Shivaji 2004; Varano et al. 2008; Bragado et al. 2012; Jaldety and Breitbart 2015), which are also considered as the “hallmarks of capacitation” (Fig. 5.2). Capacitation changes lead to the transformation in the motility pattern of spermatozoa from a progressively motile cell to a more vigorous, but less progressive, motile cell (Yanagimachi 1969; Suarez and Dai 1992; Mortimer and Swan 1995; Ho and Suarez 2001). This type of motility is termed as “hyperactivation”, and subsequent to this, capacitation ends with the ability of spermatozoa to undergo “acrosome reaction”, during which the spermatozoa releases the hydrolytic enzymes to facilitate its penetration and fusion with the oocyte—finally leading to fertilization. The increase in pY is another distinctive feature of the mammalian spermatozoa associated with capacitation. This molecular change is considered as an important characteristic of mammalian capacitation and has been addressed by various groups worldwide in varied animal models (Visconti and Kopf 1998; Visconti et al. 1999; Kulanand and Shivaji 2001; Lefièvre et al. 2002; Jha et al. 2003; Shivaji et al. 2007, 2009; Arcelay et al. 2008; Mitchell et al. 2008; Kota et al. 2009).

3.1 Hyperactivation

Hyperactivation, which is defined as “a distinct change in the sperm motility from a symmetrical to an asymmetrical pattern, is crucial for fertilization” (Yanagimachi 1969; Suarez 2008). The mammalian spermatozoa, while in the epididymis are immotile. But when released in the female reproductive tract/culture media, they quickly begin to swim and get hyperactivated (Morton et al. 1974), which imparts sperm the ability to traverse through the mucus-filled, labyrinthine lumen of the oviduct to reach the female gamete. Hyperactivation also helps the spermatozoa in penetrating the cumulus oophorus and the zona pellucida (Suarez et al. 1991; Suarez 2008). This activated spermatozoon generates a near symmetrical flagellar beat, which is called as a “planar motility” pattern. This planar motility propels the spermatozoa in an almost linear trajectory (Suarez and Dai 1992; Mortimer and Swan 1995; Ho et al. 2002). The amplitude of the flagellar bend is usually increased only on one side of the hyperactivated spermatozoa. This increased uneven amplitude leads to a circular, wriggling and whiplash type of motility pattern of the spermatozoa as shown in Fig. 5.3, and these movements are assessed objectively by using the computer-assisted sperm analysis (CASA) system (Shivaji et al. 1995; Panneerdoss et al. 2012). Hyperactivation is initiated and maintained by the involvement of a number of physiological factors like calcium, bicarbonate, cAMP and metabolic substrates (Visconti et al. 1999).

Fig. 5.3
figure 3

Change in sperm movement from progressive to hyperactivated during sperm capacitation

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3.2 Acrosome Reaction

Acrosome reaction is an absolute crucial step for successful fertilization, as it is due to acrosomal secretions alone that the sperm makes its progress through the investments surrounding the egg. In fact, males with spermatozoa lacking the acrosome are infertile (Baccetti et al. 1991). During the acrosome reaction, multiple fusions occur between the plasma membrane and the outer acrosomal membrane in the anterior region of the head. These multiple fusions lead to the formation of extensive hybrid membrane vesicles and subsequent exposure of the inner acrosomal membrane and acrosomal contents (Cardullo and Florman 1993). These stages of acrosome reaction have been depicted in Fig. 5.4.

Fig. 5.4
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Schematic representation of various stages in the progression of the sperm acrosome reaction (adapted from Curry and Watson 1995)

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3.3 Protein Tyrosine Phosphorylation

Protein tyrosine phosphorylation (pY), a post-translational event, is also considered as hallmark of capacitation. pY is a regulatory mechanism which controls many processes, such as cell cycle control, cytoskeleton assembly, cellular growth, receptor regulation and ionic current modulation (Hunter 2000; Pawson 2004; Vizel et al. 2015). The first evidence of protein tyrosine phosphorylation in spermatozoa was provided by Leyton and Saling (1989) in mouse. Later, Visconti et al. (1995) showed a correlation between sperm capacitation and protein tyrosine phosphorylation in mouse spermatozoa, and soon this increase was demonstrated in spermatozoa of various other species during capacitation, including human (Leclerc et al. 1996; Osheroff et al. 1999), hamster (Kulanand and Shivaji 2001), cat (Pukazhenthi et al. 1998), pig (Tardif et al. 2001), boar (Kalab et al. 1998), bovine (Galantino-Homer et al. 1997, 2004), equine (Pommer et al. 2003), cynomolgus monkey (Mahony and Gwathmey 1999), tammar wallaby and brushtail possum (Sidhu et al. 2004), guinea pig (Kong et al. 2008) and ram (Grasa et al. 2006).

Naz and Rajesh (2004) proposed a model for tyrosine phosphorylation pathways during sperm capacitation. The model suggests that sperm capacitation involves three main signalling pathways, namely, a cAMP/PKA-dependent pathway (pathway I) [unique to spermatozoa], a receptor tyrosine kinase pathway (pathway II) and a non-receptor protein tyrosine kinase pathway (pathway III). A crosstalk between tyrosine kinase and cAMP-dependent kinase signalling pathways in human sperm motility regulation is a unique feature in spermatozoa (Bajpai and Doncel 2003). SRC family kinases (SFKs) known to play an important role in this capacitation-associated increase in protein tyrosine phosphorylation (Battistone et al. 2013) are shown to be downstream of PKA. The target proteins for PKA could be protein tyrosine kinase(s) or protein tyrosine phosphatase(s) or both. These kinase(s) and phosphatase(s) then regulate the downstream phosphorylation of their substrate proteins at their tyrosine residues leading to a cascade of signalling events. Till date, a number of kinases have been identified (Table 5.1), which are involved in the process of capacitation, and the list is still expanding (Lawson et al. 2008; Mitchell et al. 2008; Varano et al. 2008; Goupil et al. 2011; Battistone et al. 2013; Wang et al. 2015). Although several kinases have been identified in the spermatozoa (Table 5.1), their functional relevance is seen only in vitro and mostly in animal models. The importance of the identified kinases and thus the regulation of tyrosine phosphorylation in male fertility/infertility has not yet been explored much.

Table 5.1 List of kinases identified in spermatozoa from several species
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4 Diagnosis and Prognosis of Male Infertility/Fertility: Importance of Capacitation-Based Sperm Function Tests

In humans, the prognosis and diagnosis of male fertility has been a subject of research worldwide. As mentioned earlier, a good percentage of human pregnancy failures can be attributed to decreased male fertility or male factor infertility (Thonneau et al. 1991; Sharlip et al. 2002; Lee and Foo 2014). To evaluate human sperm fertility, there has always been a consistent effort to get in place sperm function tests, owing to low predictive power of standard seminal parameters (motility, concentration and morphology) (Oehninger 1995; Carrell 2000; Muller 2000; Aitken 2006; Lefièvre et al. 2007; Vasan 2011; De Jonge and Barratt 2013; Esteves et al. 2014; Oehninger et al. 2014). Attempts have been made in laboratories for decades to design sperm function tests based on capacitation and its associated events/parameters for predicting male fertility.

Sperm penetration tests, including the sperm mucus penetration test and sperm penetration assay, are being routinely used in fertility centres. In addition, various biochemical and biophysical changes during capacitation (Zaneveld et al. 1991; Benoff 1993; Martínez and Morros 1996; Cross 1998; Travis and Kopf 2002; Visconti et al. 2002, 2011; Mitra and Shivaji 2005; Signorelli et al. 2012; Aitken and Nixon 2013) also are being utilized for designing sperm-function tests, for instance, determining the cholesterol efflux, examining activation of ion channels, evaluating protein phosphorylation changes, measuring intracellular calcium and pH and reactive oxygen species, monitoring hyperactivation and acrosome reaction, etc. Three of these events/changes are discussed in the following sections.

4.1 Monitoring Hyperactivation (HA)

One of the indicators of capacitation is the display of HA by spermatozoa (Burkman 1984). Sperm motility, hyperactivation and related motility kinematic parameters like average path velocity (VAP), curvilinear velocity (VCL), straight line velocity (VSL), linearity (LIN), amplitude of lateral head displacement (ALH), straightness (STR) and beat cross frequency (BCF) are assessed using CASA (Larsen et al. 2000; Freour et al. 2009). Based on the aforesaid kinematic parameters, namely, VCL, LIN and ALH, the non-hyperactivated spermatozoa (exhibiting planar motility pattern) can be differentiated from the hyperactivated spermatozoa (exhibiting either circular or helical motility patterns) using the SORT facility of the CASA (Youn et al. 2011).

Impaired sperm hyperactivation (HA) has been observed in human patients with infertility (Wong et al. 1993; Munier et al. 2004; Wiser et al. 2014). Wiser et al. evaluated spermatozoa from the normal patients who were to undergo IVF. They found that patients with increased hyperactivated motility had significantly higher fertilization rate compared to the group with no increased hyperactivated motility. Several groups have also found a good correlation between sperm hyperactivation, zona-induced acrosome reaction and zona binding (Liu et al. 2007); sperm motility, capacitation and tyrosine phosphorylation (Yunes et al. 2003; Buffone et al. 2005); and oocyte penetration (Wang et al. 1991), thus presenting HA as a good prognostic parameter for sperm fertility.

4.2 Monitoring Acrosome Reaction (AR)

Only capacitated spermatozoa are known to undergo acrosome reaction, underscoring its importance in predicting sperm capacitation and fertility potential of spermatozoa (Bielfeld et al. 1994). Acrosomal status in human spermatozoa is monitored with the fluorescent conjugated lectins (PNA, peanut agglutinin, and PSA, Pisum sativum agglutinin) (Cross and Meizel 1989). Additionally, several methods of assessing induced AR in vitro have been designed, where the ability of spermatozoa to acrosome react in the presence of calcium-mobilizing agents, such as calcium ionophore (A23187) or the physiological inducers like progesterone and zona pellucida proteins, is assessed (Brucker and Lipford 1995 ; Bastiaan et al. 2002). There are other fluorescent tests to evaluate the acrosome, like chlortetracyclin (CTC) staining, in which staining can differentiate three different sperm populations: the uncapacitated and acrosome intact (F pattern), the capacitated and acrosome intact (B pattern) and the capacitated and acrosome reacted (AR pattern) (Kholkute et al. 1992; Dasgupta et al. 1994).

The fact that in vivo, acrosome reaction is induced by progesterone and zona proteins, evaluation of induced acrosome reaction is routinely used as a predictor of sperm quality for utilization in clinics for assisted reproductive technologies (ARTs) (Shimizu et al. 1993; Coetzee et al. 1994; Fusi et al. 1994; Yovich et al. 1994; Glazier et al. 2000; Makkar et al. 2003). Quite often, spontaneous acrosome reaction is also evaluated and correlated with sperm fertility (Bielsa et al. 1994; Parinaud et al. 1995; Tavalaee et al. 2014; Wiser et al. 2014).

4.3 Monitoring Tyrosine Phosphorylation (pY)

In human spermatozoa, increase in global protein tyrosine phosphorylation occurs during capacitation and is correlated with the fertilizing ability of the spermatozoa (Yunes et al. 2003; Liu et al. 2006; Barbonetti et al. 2008, 2010; Mendeluk et al. 2010; Kwon et al. 2014; Sati et al. 2014).

In spite of the importance of pY in human sperm capacitation, laboratory studies and clinic-based sperm-function tests on pY are very scarce. Such studies have to be in place to determine the predictive capability of pY of sperm fertility. As discussed for the kinases as well earlier, profiling of infertile patients’ samples with appropriate controls is essential to develop sperm function tests based on this important molecular event during sperm capacitation.

5 From Bench to Clinics: Male Fertility Biomarkers and ARTs

There has been a steady rise in the molecular studies on the role of capacitation and its associated events (hyperactivation, acrosome reaction and tyrosine phosphorylation) in male fertility, in vitro (Fig. 5.5a, b). In spite of such extensive work being carried out at the laboratory level, these studies do not seem to have found application in the clinics yet. There are only a handful of clinics globally which seem to offer basic sperm capacitation/acrosome reaction tests as a part of routine sperm analysis, e.g. FIVMadrid; Poma Fertility; Androvia Life Sciences; University of Utah Hospitals and Clinics; the Male Fertility Lab, University of Washington; and Genetics & IVF Institute (references for website information). This data/information presented and discussed here is based on literature survey and searches on the World Wide Web, and real picture regarding the clinical usage of sperm capacitation tests might differ and remains to be determined.

Fig. 5.5
figure 5

(a) Relative percentages of studies in different categories (as shown) conducted over the last four decades worldwide (total number of studies =363). (b) Threefold increase in molecular studies has taken place in the last decade (2007–2016) as compared to the previous one (1996–2006). The publications were taken from PubMed (http://www.ncbi.nlm.nih.gov/pubmed/). The search was done using the following terms: [sperm capacitation, fertilization, infertility, human(s), infertility, infertile], [sperm, sperm capacitation, human(s), infertility, infertile, fertilization, hyperactivation] and [sperm, sperm capacitation, human(s), infertility, infertile, fertilization, acrosome reaction]. Based on their content, the publications (related to humans) were assigned to six categories and then percentages calculated, as shown in the pie chart

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There is a pressing need to evaluate the potential of the capacitation-associated sperm molecules/events and sperm function tests as biomarkers (or predictors) of sperm fertility/infertility. One promising sperm function/molecular test in this direction has been the “Androvia Cap-Score™ test”—a clinical test based on sperm surface ganglioside, GM1 (http://www.androvialifesciences.com/cap-score-sperm-function-test/). This test is based on the work of Dr. Alex Travis and is based on the localization of GM1 on sperm head (Buttke et al. 2006; Selvaraj et al. 2007). GM1 is a sperm membrane component that regulates the opening and closing of specific calcium ion channels on the surface of sperm head. Androvia uses technology that identifies the ability of sperm to undergo capacitation. Since capacitation, hyperactivation and the acrosome reaction require an influx of calcium ions, by identifying the presence and location of GM1 in the sperm membrane across a number of sperm and identifying how many sperm are undergoing capacitation, a “Cap-Score™” can be generated that is predictive of the fertilizing ability of sperm in the ejaculate. The company Androvia claims that their preliminary research has already validated the ability of the test to discriminate between fertile and infertile populations of men, thus gaining clinical significance as a molecular marker/sperm function test.

Sperm capacitation and its associated events are the very basis of intrauterine insemination (IUI) and in vitro fertilization (IVF), the first line of ART management for couples with unexplained infertility/subfertility (Muratori et al. 2011; Wiser et al. 2014; Tosti and Ménézo 2016). In the cases of IUI and IVF, where cryopreserved spermatozoa are used, knowledge of sperm capacitation is especially useful for extending the health and life span of the sperm (and thus success of the ART), since it is known that freeze-thawed spermatozoa exhibit a precocious acrosome reaction-like phenotype, suggesting capacitation-like event during the process of cryopreservation (Gomez et al. 1997). Though extensively used in domestic species (such as bovine, pigs and dogs), it is well known and accepted that cryopreservation damages sperm, with a large number of cells losing their fertility potential after freezing/thawing (Cormier and Bailey 2003). Knowledge about mechanisms involved in capacitation/acrosome reaction would help in efforts towards minimizing the cryo-damage to spermatozoa and improve the success rate in ARTs, as being used in the livestock industry (Singh et al. 2014; Layek et al. 2016).

The life cycle of sperm is complex and involves a series of events, which have to be perfect for successful fertilization—viz. production in testis in sufficient numbers with normal shape, maturation in epididymis, gain of motility, successful capacitation, hyperactivation and acrosome reaction, oocyte binding and penetration, activation of the ovum and ultimately successful fertilization. All these parameters ought to be looked at in defining a “healthy” spermatozoon, and defects in any of these complex events can cause male infertility. The use of ICSI (intracytoplasmic sperm injection) bypasses many of these events, increasing the risk of choosing the “compromised spermatozoa”. To avoid this, as already emphasized, it is imperative to develop new pre-ART molecular markers/sperm-function tests, for use in the clinics (Muratori et al. 2011; Natali and Turek 2011). Although few efforts to define predictive tests for ICSI success have already begun with limited success (Vural et al. 2005; Setti et al. 2012; Brown et al. 2013; Breznik et al. 2013; Meerschaut et al. 2013), further research in this direction is much needed.

Concluding Remarks

It is well accepted now that conventional semen analysis is unable to precisely predict sperm fertility potential, thus warranting search of biomarkers for fertility/infertility based on newer research (Weber et al. 2005; Lewis 2007; Lamb 2010). Attempts to translate the molecular information about capacitation—from laboratories to clinic—and to develop capacitation-based molecular markers/sperm function assays (besides other tests) is the need of the hour, especially in the era of assisted reproductive technologies like ICSI. The pre-ART tests would permit the clinicians and the infertile couples to make a more informed decision about the treatment/procedure and be assured of its success. It, thus, becomes necessary to continue improving our understanding of sperm capacitation, not only for the basic understanding of sperm physiology but also to understand its functionality, both in vivo and in vitro, ultimately translating into higher success rate in assisted reproductive technologies.