**5. Effect of cryopreservation on proteins phosphorylation state of sea bream sperm**

#### **5.1 Molecular mechanisms determining sperm motility initiation in sea bream** *sparus aurata*

Most fish spermatozoa are quiescent in the testes, because the osmolality and composition of seminal plasma usually prevent motility in sperm ducts (Billard, 1986). During natural reproduction, fish sperm become motile after discharge into the aqueous environment (in oviparous species) or the female genital tract (in viviparous and ovoviviparous species) (Billard, 1986; Billard & Cosson, 1983; Stoss, 1983). Changes in the ionic and osmotic environment of the sperm cells have been identified as being critical external factors that may be responsible for initiating motility in fish spermatozoa (Morisawa, 1994). Several extracellular factors controlling sperm motility have been reported. In marine (Gwo et al, 1993; Krasznai et al, 2003a, 2003b; Morisawa & Suzuki, 1980; Oda & Morisawa, 1993) and freshwater (Billard, 1986; Morisawa et al., 1983; Stoss, 1983) teleosts, sperm motility is initiated by osmotic shock when sperm are ejaculated. In these species, spermatozoa are quiescent at the osmolality of seminal plasma (referred to as isotonic condition). In freshwater teleost sperm, flagellar motility is initiated by the hypo-osmotic shock, whereas in marine teleost sperm, flagellar motility is initiated by hyperosmotic shock. Furthermore, in medaka (Inoue & Takei, 2003) and tilapia (Linhart et al., 1999), motility regulatory mechanisms of sperm flagella are modulated to suit the spawning environment when they are in freshwater or acclimated to seawater. In herring sperm, motility initiation requires trypsin inhibitor-like sperm-activating peptide from the eggs (HSAPS), and the sperm exhibits chemotaxis when they are close to eggs (Oda et al., 1998; Yanagimachi et al., 1992).The extracellular factors controlling sperm motility (osmolality, ions, sperm-activating peptides, and chemoattractants) act on the flagellar motile apparatus, the axoneme, through signal transduction across the plasma membrane. Second messengers, such as cAMP and Ca, play key roles in the initiation of sperm motility in many animal groups, such as mammals (Lindemann, 1978; Okamura et al., 1985; Tash & Means, 1983), salmonid fish (Morisawa & Okuno, 1982), sea urchin (Cook et al., 1994), mussel (Stephens & Prior, 1992), and tunicate (Opresko & Brokaw, 1983). A cAMP-independent initiation of flagellar motility in sperm was observed in puffer fish (Morisawa, 1994) and striped bass (Shuyang et al., 2004). Second messengers (cAMP and Ca) determine the sperm motility initiation modifying dynein-mediated sliding of the axonemal outer-doublet microtubules through protein phosphorylation/dephosphorylation in different species, such as mammals (Lindemann & Kanous, 1989), rainbow trout, chum salmon, sea urchin (Inaba et al., 1999), and tunicate (Nomura et al., 2000).

In *Sparus aurata* osmolality is the key signal in sperm motility activation and motility initiation depends on a cAMP-dependent protein phosphorylation (Zilli et al., 2008). To elucidate which proteins are involved (phosphorilated/dephosphorilated) in the initiation

Effect of Cryopreservation on Bio-Chemical Parameters, DNA Integrity,

Protein Profile and Phosphorylation State of Proteins of Seawater Fish Spermatozoa 405

Fig. 8. Western blot analysis with antiphosphothreonine antibody of gilthead sea bream (*Sparus aurata*) sperm proteins separated by 2DE. The 2DE was performed on an immobilized pH 3–10 NL strip, followed by the second-dimensional separation on 13% polyacrylamide gels. The separated proteins were then blotted on nitrocellulose and incubated with antibody. Molecular mass and isoelectric point of proteins of interest are

Some of these proteins have been identified by mass spectrometry and results are listed in Table 5. In particular, spots 1 and 2 (belonging to 3THPSa) were identified as acetylcoenzyme A (CoA) synthetase, spot 5 as A kinase anchor protein (AKAP), and spot 7 as an unnamed protein of *Tetraodon nigroviridis*, which have 70% identity with a novel protein similar to phosphatase and actin regulator 3 of *Danio rerio*. Acetyl-CoA synthetase is well known as an enzyme whose activity is central to the metabolism of prokaryotic and eukaryotic cells. In particular, acetyl-CoA synthetase activates acetate to acetyl-CoA, and it provides the cell with the two-carbon metabolite used in many anabolic and energy generation processes. Therefore, we suppose that this enzyme was activated in motile sperm to increase the level of ATP, which is necessary for flagellar movement. PKA localizes to specific cellular structures and organelles by binding to AKAP molecules via interaction with the regulatory subunits (RI and RII). Therefore, cAMP levels temporally regulate PKA, whereas the spatial regulation within the cell occurs through compartmentalization by binding to AKAP, thus assuring specificity of PKA function. The important role of AKAP as a key regulator of sperm motility is already established (Vijayaraghavan et al., 1997a). In addition, a recent study demonstrated that phosphorylation of AKAP in human sperm results in tail recruitment of PKA and increase of sperm motility, providing evidence for a functional role of phosphorylation of AKAP (Luconi et al., 2004). Regarding the role of phosphatases and kinases in the initiation of sperm motility, many studies have demonstrated that the development and maintenance of motility is regulated by a complex

listed. (This figure was originally published in Zilli et al., Biol. Reprod. 2008).

of sea bream spermatozoa motility, proteins extracted from spermatozoa before and after motility activation were separated on SDS PAGE, blotted on nitrocellulose membrane, and treated with anti-phosphotyrosine, anti-phosphothreonine, or anti-phosphoserine antibodies.

After motility activation we observed that: 1) two protein bands (76 kDa and 57 kDa) were dephosphorylated and an unspecified number of proteins corresponding to a large band of 9-15 kDa were phosphorylated at tyrosine residues (Fig. 7A); 2) two protein bands (174 kDa and 147 kDa) resulted phosphorilated and an unspecified number of proteins with molecular weights ranging between 15 and 9 kDa were dephophorilated at threonine residues (Fig. 7B); 3) three protein bands (174 kDa, 138 kDa and 70 kDa) and an unspecified number of proteins from 9 to 12 kDa were phosphorylated and only one protein band of 33 kDa was dephosphorylated at serine residues (Fig. 7 C).

Fig. 7. Motility-dependent phosphorylation/dephosphorylation at tyrosine residues (A), threonine residues, (B) and serine residues (C) in fresh sperm of gilthead sea bream before and after motility activation. Sperm were either activated in seawater (lane 1) or maintained immotile by dilution in non-activating medium (lane 2). Sperm proteins were subjected to Western blotting (30 g/lane) with anti-phosphotyrosine, anti-phosphothreonine and antiphosphoserine antibodies. Number on the left indicates the molecular mass of bands. On the right, the names of proteins of interest are indicated. (This figure was originally published in Zilli et al., Cryobiology 2008).

We characterized some of these proteins by using two-dimensional gel electrophoresis (2DE) and the antibody against phosphothreonine. This antibody revealed (figure 8) that: 1) the protein band of 174 kDa (named that 1ThP in figure 7 and identified as 1THPSa in figure 8) was not a single protein but, rather, a cluster of proteins with the same molecular weight (174 kDa) but different pI (5.9–6.29); 2) the protein band of 147 kDa (named 2Thp in figure 7 and identified as 2THPSa in figure 8) was a protein with a pI of 8.7; and 3) the cluster of proteins of 9-15 kDa (named 3ThP in figure 7 and identified as 3THPSa in figure 8) consisted of 10 proteins with pI between 6.1 and 7.6 and molecular weights between 9 and 15 kDa.

of sea bream spermatozoa motility, proteins extracted from spermatozoa before and after motility activation were separated on SDS PAGE, blotted on nitrocellulose membrane, and treated with anti-phosphotyrosine, anti-phosphothreonine, or anti-phosphoserine

After motility activation we observed that: 1) two protein bands (76 kDa and 57 kDa) were dephosphorylated and an unspecified number of proteins corresponding to a large band of 9-15 kDa were phosphorylated at tyrosine residues (Fig. 7A); 2) two protein bands (174 kDa and 147 kDa) resulted phosphorilated and an unspecified number of proteins with molecular weights ranging between 15 and 9 kDa were dephophorilated at threonine residues (Fig. 7B); 3) three protein bands (174 kDa, 138 kDa and 70 kDa) and an unspecified number of proteins from 9 to 12 kDa were phosphorylated and only one protein band of 33

Fig. 7. Motility-dependent phosphorylation/dephosphorylation at tyrosine residues (A), threonine residues, (B) and serine residues (C) in fresh sperm of gilthead sea bream before and after motility activation. Sperm were either activated in seawater (lane 1) or maintained immotile by dilution in non-activating medium (lane 2). Sperm proteins were subjected to Western blotting (30 g/lane) with anti-phosphotyrosine, anti-phosphothreonine and antiphosphoserine antibodies. Number on the left indicates the molecular mass of bands. On the right, the names of proteins of interest are indicated. (This figure was originally published in

We characterized some of these proteins by using two-dimensional gel electrophoresis (2DE) and the antibody against phosphothreonine. This antibody revealed (figure 8) that: 1) the protein band of 174 kDa (named that 1ThP in figure 7 and identified as 1THPSa in figure 8) was not a single protein but, rather, a cluster of proteins with the same molecular weight (174 kDa) but different pI (5.9–6.29); 2) the protein band of 147 kDa (named 2Thp in figure 7 and identified as 2THPSa in figure 8) was a protein with a pI of 8.7; and 3) the cluster of proteins of 9-15 kDa (named 3ThP in figure 7 and identified as 3THPSa in figure 8) consisted of 10 proteins with pI between 6.1 and 7.6 and molecular weights between 9 and

kDa was dephosphorylated at serine residues (Fig. 7 C).

Zilli et al., Cryobiology 2008).

15 kDa.

antibodies.

Fig. 8. Western blot analysis with antiphosphothreonine antibody of gilthead sea bream (*Sparus aurata*) sperm proteins separated by 2DE. The 2DE was performed on an immobilized pH 3–10 NL strip, followed by the second-dimensional separation on 13% polyacrylamide gels. The separated proteins were then blotted on nitrocellulose and incubated with antibody. Molecular mass and isoelectric point of proteins of interest are listed. (This figure was originally published in Zilli et al., Biol. Reprod. 2008).

Some of these proteins have been identified by mass spectrometry and results are listed in Table 5. In particular, spots 1 and 2 (belonging to 3THPSa) were identified as acetylcoenzyme A (CoA) synthetase, spot 5 as A kinase anchor protein (AKAP), and spot 7 as an unnamed protein of *Tetraodon nigroviridis*, which have 70% identity with a novel protein similar to phosphatase and actin regulator 3 of *Danio rerio*. Acetyl-CoA synthetase is well known as an enzyme whose activity is central to the metabolism of prokaryotic and eukaryotic cells. In particular, acetyl-CoA synthetase activates acetate to acetyl-CoA, and it provides the cell with the two-carbon metabolite used in many anabolic and energy generation processes. Therefore, we suppose that this enzyme was activated in motile sperm to increase the level of ATP, which is necessary for flagellar movement. PKA localizes to specific cellular structures and organelles by binding to AKAP molecules via interaction with the regulatory subunits (RI and RII). Therefore, cAMP levels temporally regulate PKA, whereas the spatial regulation within the cell occurs through compartmentalization by binding to AKAP, thus assuring specificity of PKA function. The important role of AKAP as a key regulator of sperm motility is already established (Vijayaraghavan et al., 1997a). In addition, a recent study demonstrated that phosphorylation of AKAP in human sperm results in tail recruitment of PKA and increase of sperm motility, providing evidence for a functional role of phosphorylation of AKAP (Luconi et al., 2004). Regarding the role of phosphatases and kinases in the initiation of sperm motility, many studies have demonstrated that the development and maintenance of motility is regulated by a complex

Effect of Cryopreservation on Bio-Chemical Parameters, DNA Integrity,

of Aldente. (Modified from Zilli et al., Biol. Reprod. 2008).

**motility initiation in sea bream** *Sparus aurata* 

involved in motility initiation (Zilli et al., 2008b).

in frozen–thawed activated sperm (Fig. 9A).

cryopreserved sperm (Fig. 9B).

**Phosphorylation/dephosphorylation of tyrosine residues:** 

**Phosphorylation/dephosphorylation of threonine residues:** 

**Phosphorylation/dephosphorylation of serine residues:** 

significantly reduced by the freezing–thawing procedure.

activation (Fig. 9C), unlike what happens in fresh sperm (Fig. 7C).

Protein Profile and Phosphorylation State of Proteins of Seawater Fish Spermatozoa 407

EWM: Experimental Weight Mass; EIP: Experimental Isoelectric Point; TWM: Theoretical Weight Mass; TIP: Theoretical Isoelectric Point. a: Z score of ProFound; b: Normalized score

**5.2 Effect of cryopreservation on phosphorylation state of proteins involved in sperm** 

The quality of gilthead sea bream semen was decreased by cryopreservation procedure. Even though the viability (82 ± 5%) of spermatozoa following the freezing–thawing procedure was only slightly (but significant) decreased with respect to that measured in fresh samples (93 ± 4), only the 50% of the thawed spermatozoa could be activated, and showed a motility duration which was one third of that measured in fresh samples. The reduction of sperm motility (percent and duration) is attributable (at least partially) to the effect that the freezing-thawing procedure has on the phosphorylation state of proteins

Two protein bands (76TyD and 57TyD of figure 7A) which in fresh sperm were completely dephosphorylated after motility initiation, in frozen– thawed remained phosphorylated (Fig. 9A), while the cluster of proteins of 15-9 TyP (Fig. 7A), that were phosphorylated when fresh sperm shifted from the immotile to the motile phase, were much less phosphorylated

Among the proteins that were phosphorylated following motility activation in fresh sperm (Fig. 7B), two bands (named 15-9ThP and 147ThP) were phosphorylated after activation in frozen–thawed spermatozoa (Fig. 9B). However, it must be underlined that within the proteins belonging to the 15-9 ThP band, only one (11 kDa) previously identified as acetylcoenzyme A synthetase (Zilli et al, 2008a) was phosphorylated after motility activation in

Among the five previously identified protein bands (Zilli et al, 2008a) that changed their phosphorylation state after motility activation in fresh sperm (Fig. 7C), only two (70SeP and 12-9SeP) were phosphorylated in frozen–thawed sperm after activation (Fig. 9C). The other bands, named 174SeP, 138SeP and 33SeD, did not change their phosphorylation state after

Some proteins (76TyD, 57TyD and 33SeD) that were dephosphorylated after motility activaton in fresh sperm (7A and 7C) but not in cryopreserved spermatozoa (9A and 9C) could not play a key role in sperm motility initiation but could be involved in sperm motility duration and motility characteristics, since the kinematic parameters were

Our studies also demonstrated that in gilthead sea bream spermatozoa the freezingthawing procedure increased, independently from the motility activation procedure, protein phosphorylation (mainly at threonine residues), since more phosphorylated proteins were present in non-activated cryopreserved sperm with respect to the fresh sperm. This could be

balance between kinase and phosphatase activities (Tash & Brach, 1994; Vijayaraghavan et al., 1997b).


Table 5. Results from peptide mass fingerprinting of protein spots excised from 2-D gels of gilthead sea bream sperm.

balance between kinase and phosphatase activities (Tash & Brach, 1994; Vijayaraghavan et

**SWISSPROT accession no.** 

**Species identified**

Q4SQZ9 *Tetraodon* 

Table 5. Results from peptide mass fingerprinting of protein spots excised from 2-D gels of

**TWM (kDa) TIP**

Q0VG88 *Danio rerio* 15.5 5.3 0.97a 27%

CAI11962 *Danio rerio* 8.1 6.0 0.79a 26%

*nigroviridis* 8.0 7.7 0.28b 21%

**Homology Score Coverage %** 

al., 1997b).

**Reference Spot** 

3THPSa

Spot1/

**EWM (kDa)** 

Spot2 15-13 6.1

Spot 5 10 6.8

Spot 7 10 7.6

gilthead sea bream sperm.

**EIP Identified protein** 

> LOC568763 similar to Acetylcoenzyme A synthetase

> > novel protein similar to human A kinase (PRKA) anchor protein 7 (AKAP7)

Chromosom e 11 SCAF14528, whole genome shotgun sequence - Unnamed protein product that have a 70% of identity with novel protein similar to phosphatase and actin regulator 3 (PHACTR3, zgc:109967) [*Danio rerio*]

EWM: Experimental Weight Mass; EIP: Experimental Isoelectric Point; TWM: Theoretical Weight Mass; TIP: Theoretical Isoelectric Point. a: Z score of ProFound; b: Normalized score of Aldente. (Modified from Zilli et al., Biol. Reprod. 2008).
