The intracellular pathways that occur during sperm motility are closely related with the pathways of capacitation (Baldi et al., 2000). Both events require Ca2+, HCO3- and cAMP, this is why hyperactivation is seen as a part of capacitation (Ho and Suarez, 2001; McPartlin, 2010). However, McPartlin (2010) showed that procaine was able to induce hyperactivated motility in capacitated as well as in non-capacitated sperm; this shows that both events are regulated independent (McPartlin, 2010).
As described earlier (1.1.1 Flagellar movement) Ca2+, cAMP and ATP play a role in sperm cell motility by activation of dynein ATPases (Ho and Suarez, 2001). Hyperactivated sperm cells show an increased intracellular calcium level (hamster: Suarez et al., 1993; bull: Ho and Suarez, 2001). Calcium together with bicarbonate are involved in the cAMP-dependent tyrosine phosphorylation trough their regulation on soluble adenylyl cyclase (sAC) (Ho and Suarez, 2001; Ho et al., 2002; Litvin et al., 2003; Hess et al., 2005; Turner, 2006; Schlingmann et al., 2007). Protein kinase A (PKA) is an important target for phosphorylation by cAMP (Hess et al., 2005). Which on his turn phosphorylates the downstream tyrosine kinases in the sperm tail (Leclerc et al., 1996; Witte and Schäfer-Somi, 2007; McPartlin et al., 2008). A highly increased tyrosine phosphorylation of the flagellar proteins is seen in hyperactivated sperm cells of various species (hamster: Si and Okuno, 1999; monkey: Mahony and Gwathmey, 1999; boars: Harayama et al., 2012). Because of this, it is often used as a marker for sperm capacitation (McPartlin, 2010; Leemans, 2015).
However, the effect of calcium seems more complicated. Parallel from the cAMP/PKA-pathway, calcium also works through a calcium/calmodulin pathway (Schlingmann et al., 2007; Litvin et al., 2003). Binding of calcium to calmodulin can have two opposite effects depending on the environmental pH (Schlingmann et al., 2007; Gonzalez-Fernandez et al., 2012; Gonzalez-Fernandez et al., 2013). Some studies (Harayama et al., 2012; Loux, 2013) mention an inhibitory effect of calcium as protein tyrosine phosphorylation rates are higher in a calcium free medium (Harayama et al., 2012; Loux, 2013). The calcium-induced inhibition in equine sperm disappeared when W-7, a specific calmodulin blocker, was used (Gonzalez-Fernandez et al., 2012; Gonzalez-Fernandez et al., 2013). Gonzalez-Fernandez et al. (2012) hypothesized that calcium/calmodulin supports a sperm phosphatase, like calcineurin (Gonzalez-Fernandez et al., 2012). However, medium pH played a crucial role, in a high pH environment (>7,8), there was no notable inhibition by calcium (Gonzalez-Fernandez et al., 2012; Loux, 2013). This can be coupled to the physiological pH of the uterus, which showed a similar high pH (mean of 7,9) after insemination (Loux, 2013). The high medium pH may cause decreased calcium/calmodulin-dependent phosphatase (Gonzalez-Fernandez et al., 2012).
Calcium can also have a stimulatory effect on sperm hypermotility through calmodulin. The binding of both substrates causes activation of calcium/calmodulin-dependent kinases (Marin-Briggiler et al., 2005), which phosphorylate axonemal proteins necessary for hyperactivated motility (Suarez, 2008). For mice (Schlingmann et al., 2007), bull (Ignotz and Suarez, 2005) and human sperm (Marin-Briggiler et al., 2005) it is reported that calmodulin inhibition causes a decrease in sperm motility.
In conclusion, bicarbonate is a crucial regulator of the cAMP/PKA dependent pathway that is necessary but not sufficient for the induction of hyperactivated motility (Ho and Suarez, 2001). Supported by an alkaline environment and sufficient ATP, calcium is the most important trigger for hyperactivated motility due to its effect on the cAMP/PKA pathway and the calcium/calmodulin pathway (Ho et al., 2002; Suarez, 2008) (Figure 6.).
2.3 ACROSOME REACTION
In order to be able to undergo the acrosome reaction, sperm must be fully capacitated (Alm et al., 2001; Odeh et al., 2003; McPartlin et al., 2008). This is why, apart from hyperactivation, it is often used as an end parameter of capacitation (Leemans, 2015). However, in this thesis we did not take a close look to the acrosome reaction.
The acrosome reaction is an irreversible exocytotic process that is supported by a cytoplasmic Ca2+ increase (Witte and Schäfer-Somi, 2007; McPartlin, 2010; Leemans, 2015). Binding of the spermatozoon with glycosaminoglycans and/or progesterone present in the female reproductive track and/or the zona pellucida of the oocyte, triggers the acrosomal exocytosis (mouse: Bleil and Wassarman, 1990; horse: Varner et al., 1993; bovine: Parrish et al., 1985; horse: Meyers et al., 1995; horse: Meyers et al., 1996; horse: Cheng et al., 1998; horse: Gadella et al., 2001; horse: Rathi et al., 2001) (Figure 7.). A Ca2+-influx causes the plasma membrane to fuse with the outer acrosomal membrane and in this way the acrosomal content is released and exposed to the zona pellucida (Witte and Schäfer-Somi, 2007). The released acrosomal enzymes digest the zona pellucida, this makes it possible for the sperm to enter the peri-vitelline space (Gadella et al., 2001; Pommer et al., 2002; Odeh et al., 2003; Witte and Schäfer-Somi, 2007; McPartlin, 2010). Here it will fulfill the process of fertilization by attaching and fusing with the oolemma (Gadella et al., 2001; McPartlin, 2010).
3 MEDIA
Many different events take place within the oviduct such as capacitation, fertilization and embryo development; all these events require an adapted microenvironment created by the equine oviduct (Willis et al., 1994; Leemans, 2015). This is why it is of great importance to study the physiological environment in order to make in vitro fertilization work.
3.1 IN VIVO: OVIDUCT AND OVIDUCTAL FLUID
As in vivo fertilization takes place within the oviduct, the oviductal microenvironment is the best place to seek for triggering molecules for capacitation (Suarez, 2008). The fluid within the oviduct is a complex mixture of electrolytes and non-electrolytes that are filtrated and secreted by the oviductal epithelium or released into the oviduct together with the oocyte during ovulation (Leese, 1988; Suarez, 2008). The composition of the oviductal fluid changes depending on the region within the oviduct and the stage within the estrus cycle (Campbell et al., 1979; Killian, 2004). The ovarian hormones influence the metabolism of the oviductal cells and the vascular bed around it; with changes in volume and composition of the oviductal fluid as consequence (Willis et al. 1994; Aguilar and Reyley, 2005; Nelis et al., 2015). The highest secretory activity takes place in the ampulla, followed by the infundibulum and a minimal secretion takes place in the isthmus (Gastal et al., 2007). These specific compositions create a microenvironment within the oviduct that influences the gametes and embryos (Leese, 1988). For example, the substrates within the fluid play a role in the metabolic changes, which occur with capacitation of spermatozoa (Leese, 1988).
The amount of oviductal fluid shows a cyclic pattern, with the largest volume produced during the follicular phase under estrogen influence (Campbell et al., 1979; Gastal et al., 2007). This causes a dilution of ions, proteins and other molecules and in this way leads to a lower concentration and osmolarity during estrus (Campbell et al., 1979).
Several authors have examined the composition of the oviductal fluid by cannulation of the oviduct (Campbell et al., 1979; Engle et al., 1984; Willis et al., 1994). However, the correctness of these measurements is questioned. As seen in post-experimental hystological examination by Campbell et al. (1979), the oviduct showed several signs of inflammation to the cannulas (Campbell et al., 1979). These findings suggest that the oviductal composition of the collected fluid is influenced. One might question the relevance of the collected fluids compared to the normal oviductal fluid in the mare.