**4. Effect of cryopreservation on sea bass protein profile**

Defects in sperm proteins may compromise sperm motility, fertilization ability, and the early events after fertilization (Cao et al., 2003; Huanget al., 1999; Lessard et al., 2000). Protein screening has become an excellent approach with which to evaluate changes in expression due to different stresses. Using this method it has been demonstrated that the reduction in motility observed in boar and human spermatozoa following cryopreservation was associated with a decrease in heat shock protein 90 during cooling (Cao et al., 2003; Huanget al., 1999). Similarly, the loss of P25b (a protein associated with the plasma membrane covering the acrosome) may be responsible, at least in part, for the decrease in fertility following the freezing/thawing procedure of bull semen (Lessard et al., 2000). Cryoinjuries due to cryopreservation have been reported for thawed spermatozoa of many freshwater (Rana, 1995) and marine fish species (Gwo et al., 1992; Lahnsteiner et al., 2000). Shrinkage of the plasma membrane of the midpiece, breakage of mitochondria, and coiling of the axoneme have been observed.Cryopreserved sea bass sperm showed similar fertilization rates and class motility compared with fresh sperm, but also showed a decline in motility duration (Fauvel et al., 1998a), changes in metabolism (Zilli et al., 2004), and lower hatching rates (Fauvel et al., 1998b). For these reason we used (Zilli et al., 2005) the 2-DE to verify whether the cryopreservation procedure, applied to sea bass milt, affected the expression of proteins involved in the control of sperm functions and, in addition, matrix-associated laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry to identify some of these proteins.

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

al., 2003; Huanget al., 1999) and bull sperm (Lessard et al., 2000).

**fresh sperm (N=6)** 

SPOT 1 17.8±2.1 7.0±1.1 SPOT 2 10.7±1.1 5.9±1.5 SPOT 3 48.0±9.1 20.2±5.1 SPOT 4 40.8±5.2 29.3±4.1 SPOT 5 54.9±11.3 20.6±3.0 SPOT 6 42.9±2.2 - SPOT 7 36.8±3.3 25.5±5.2 SPOT 8 110.0±23.0 33.4±6.3 SPOT 9 52.7±12.0 38.7±9.0 SPOT 10 31.9±8.0 4.7±2.0 SPOT 11 30.6±7.0 7.4±5.0 SPOT 12 34.9±15.2 - SPOT 13 44.3±13. - SPOT 14 34.3±11.3 - SPOT 15 11.4±3.2 - SPOT 16 8.4±2.8 - SPOT 17 6.2±3.5 - SPOT 18 11.8±4.2 - SPOT 19 30.6±7.2 10.6±4.2 SPOT 20 102.8±3.6 42.9±3.3 SPOT 21 46.3±6.6 29.2±7.3

Table 3. Differences in abundance of spots in fresh and cryopreserved sea bass sperm. Spot adundance is expressed as mean ± SD of normalized spot volume measured in six different

Five of the protein spots shown in Table 3 were analyzed by MALDI-TOF for protein identification. Three were selected among the spots that significantly decreased after cryopreservation (5, 8, and 20) and two (6 and 13) were taken from among those that were absent in the gel obtained with frozen-thawed sperm (Fig. 6 and Table 3). Protein identification was performed by three search programs (PeptIdent, Mascot, and MS-Fit).

gels. (This table was originally published in Zilli et al., Biol Reprod 2005).

**4.2 Identification of protein spots by MALDI-TOF** 

**Spot Number Normalised spot volume in** 

Protein Profile and Phosphorylation State of Proteins of Seawater Fish Spermatozoa 401

Differences were observed (by visual inspection and by using image analysis software) in the protein profiles of fresh and cryopreserved sperm samples. In fact, in the cryopreserved sperm samples, among the 163 spots considered, 13 were significantly (*P*<0.05) less expressed, and 8 completely disappeared. These 21 spots are highlighted in Figure 6 and the normalized spot volumes listed in Table 3. A decrease in protein abundance or spot disappearance in sperm after the cryopreservation procedure may be due to either leakage of proteins from spermatozoa to the extracellular medium or to degradation following freezing-thawing stress. The leakage of proteins is ruled out because we have previously demonstrated that the intracellular protein concentration and the seminal plasma protein concentrations do not change after cryopreservation (Zilli et al., 2004). Consequently, protein degradation seems to be responsible for the reduction in spot abundance (and disappearance). Similar results have been also reported in human and boar semen (Cao et

> **Normalised spot volume in cryopreserved sperm (N=6)**

### **4.1 Proteins expression in two-dimensional electrophoresis gels: Differences between fresh and frozen-thawed sea bass sperm samples**

To perform two-dimensional analysis sperm samples with similar fertilization rates (70%– 90%) and percentage of motility (80%–100%), before and after cryopreservation, were used to extract proteins. All the sperm samples used showed lower motility duration after the cryopreservation procedure. 163 spots were detected in all gels prepared from fresh samples (with molecular masses ranging between 190 and 10 kDa and isoelectric points between 3.5 and 8.0) and were used for comparative analysis. Results of a typical experiment performed on sperm samples before and after cryopreservation are showed in figure 6 (A and B).

Fig. 6. Two-dimensional Electrophoresis (2-DE) maps of fresh (A) and cryopreserved (B) sea bass sperm proteins. 2-DE was performed on an immobilized pH 3–10 strip, followed by the second-dimensional separation on 12.5% polyacrylamide gels. The separated proteins were stained with silver staining. Spots that are less expressed after cryopreservation are highlighted with a continuous line; spots that are entirely absent after cryopreservation are marked with a dotted line. (This figure was originally published in Zilli et al., Biol Reprod 2005).

**4.1 Proteins expression in two-dimensional electrophoresis gels: Differences between** 

To perform two-dimensional analysis sperm samples with similar fertilization rates (70%– 90%) and percentage of motility (80%–100%), before and after cryopreservation, were used to extract proteins. All the sperm samples used showed lower motility duration after the cryopreservation procedure. 163 spots were detected in all gels prepared from fresh samples (with molecular masses ranging between 190 and 10 kDa and isoelectric points between 3.5 and 8.0) and were used for comparative analysis. Results of a typical experiment performed

Fig. 6. Two-dimensional Electrophoresis (2-DE) maps of fresh (A) and cryopreserved (B) sea bass sperm proteins. 2-DE was performed on an immobilized pH 3–10 strip, followed by the second-dimensional separation on 12.5% polyacrylamide gels. The separated proteins were

highlighted with a continuous line; spots that are entirely absent after cryopreservation are marked with a dotted line. (This figure was originally published in Zilli et al., Biol Reprod

stained with silver staining. Spots that are less expressed after cryopreservation are

2005).

on sperm samples before and after cryopreservation are showed in figure 6 (A and B).

**fresh and frozen-thawed sea bass sperm samples** 

Differences were observed (by visual inspection and by using image analysis software) in the protein profiles of fresh and cryopreserved sperm samples. In fact, in the cryopreserved sperm samples, among the 163 spots considered, 13 were significantly (*P*<0.05) less expressed, and 8 completely disappeared. These 21 spots are highlighted in Figure 6 and the normalized spot volumes listed in Table 3. A decrease in protein abundance or spot disappearance in sperm after the cryopreservation procedure may be due to either leakage of proteins from spermatozoa to the extracellular medium or to degradation following freezing-thawing stress. The leakage of proteins is ruled out because we have previously demonstrated that the intracellular protein concentration and the seminal plasma protein concentrations do not change after cryopreservation (Zilli et al., 2004). Consequently, protein degradation seems to be responsible for the reduction in spot abundance (and disappearance). Similar results have been also reported in human and boar semen (Cao et al., 2003; Huanget al., 1999) and bull sperm (Lessard et al., 2000).


Table 3. Differences in abundance of spots in fresh and cryopreserved sea bass sperm. Spot adundance is expressed as mean ± SD of normalized spot volume measured in six different gels. (This table was originally published in Zilli et al., Biol Reprod 2005).

#### **4.2 Identification of protein spots by MALDI-TOF**

Five of the protein spots shown in Table 3 were analyzed by MALDI-TOF for protein identification. Three were selected among the spots that significantly decreased after cryopreservation (5, 8, and 20) and two (6 and 13) were taken from among those that were absent in the gel obtained with frozen-thawed sperm (Fig. 6 and Table 3). Protein identification was performed by three search programs (PeptIdent, Mascot, and MS-Fit).

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

**sperm** 

*aurata* 

tunicate (Nomura et al., 2000).

Protein Profile and Phosphorylation State of Proteins of Seawater Fish Spermatozoa 403

The results reported in figure 6 and tables 3 and 4 show that in sea bass spermatozoa the used cryopreservation procedure causes the degradation of 21 sperm proteins, and among these, 2 could be at least partially responsible for the observed decrease in sperm motility duration and the lower hatching rate of eggs fertilized with cryopreserved sperm. In addition, these observations suggest that two-dimensional electrophoresis coupled with MALDI-TOF analysis could be used as a tool to improve cryopreservation procedures.

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

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

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

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

Three out of five sea bass proteins processed were found to have homologies with existing sequences in the databases used (Table 4). These proteins were identified from protein sequences already described in other teleost species and amphibians. In particular, two were from *Brachidanio rerio* (spots 5 and 20) and one was from *Xenopus laevis* (spot 13). Table 4 summarizes the data of the bio-informatics analysis for these proteins. For spot 5, the search engine PeptIdent found a homology with a protein of *Brachidanio rerio* (similar to SKB1 of human and mouse). This is a highly conserved cytoplasmic protein with methyltransferase activity that interacts with the members of the Janus family tyrosine kinases (JAK) (Pollack et al., 1999). Genome activation is one of the first critical events in the life of a new organism. Both the timing of genome activation and the array of genes activated must be controlled correctly, and these events depend on changes in chromatin structure and availability of transcription factors (Latham & Schultz, 2001).

In sea bass the observed reduction in SBK1 proteins in cryopreserved sperm could be responsible for abnormal early embryo development, which in turn, could determine the lower hatching rate observed (personal observation). The spot protein 13 matched in Mascot and MS-Fit with a G1/S-specific cyclin E2 protein, which is essential in the control of the cell cycle at the G1/S (start) transition (Moore et al., 2002). Cyclin E is involved in the activation of cyclin-dependent kinase 2 (cdk2). Recently, it has been demonstrated that cdk2 phosphorylates the protein phosphatase, PP1gamma2, a key enzyme in the development and regulation of sperm motility (Huang &Vijayaraghavan, 2004). The observed reduction in sea bass sperm motility duration in frozen-thawed spermatozoa could be a consequence of the cyclin E degradation. The protein spot 20 matched, in MS-Fit, with the hypothetical protein DKFZp566A1524 of unknown function.


Table 4. Results from peptide mass fingerprinting of protein spots excised from 2D gels. EWM: Experimental Weight Mass; EIP: Experimental Isoelectric Point; TWM: Theoretical Weight Mass; TIP: Theoretical Isoelectric Point. (This table was originally published in Zilli et al., Biol Reprod 2005).

Three out of five sea bass proteins processed were found to have homologies with existing sequences in the databases used (Table 4). These proteins were identified from protein sequences already described in other teleost species and amphibians. In particular, two were from *Brachidanio rerio* (spots 5 and 20) and one was from *Xenopus laevis* (spot 13). Table 4 summarizes the data of the bio-informatics analysis for these proteins. For spot 5, the search engine PeptIdent found a homology with a protein of *Brachidanio rerio* (similar to SKB1 of human and mouse). This is a highly conserved cytoplasmic protein with methyltransferase activity that interacts with the members of the Janus family tyrosine kinases (JAK) (Pollack et al., 1999). Genome activation is one of the first critical events in the life of a new organism. Both the timing of genome activation and the array of genes activated must be controlled correctly, and these events depend on changes in chromatin structure and availability of

In sea bass the observed reduction in SBK1 proteins in cryopreserved sperm could be responsible for abnormal early embryo development, which in turn, could determine the lower hatching rate observed (personal observation). The spot protein 13 matched in Mascot and MS-Fit with a G1/S-specific cyclin E2 protein, which is essential in the control of the cell cycle at the G1/S (start) transition (Moore et al., 2002). Cyclin E is involved in the activation of cyclin-dependent kinase 2 (cdk2). Recently, it has been demonstrated that cdk2 phosphorylates the protein phosphatase, PP1gamma2, a key enzyme in the development and regulation of sperm motility (Huang &Vijayaraghavan, 2004). The observed reduction in sea bass sperm motility duration in frozen-thawed spermatozoa could be a consequence of the cyclin E degradation. The protein spot 20 matched, in MS-Fit, with the hypothetical

> **SWISSPR-OT accession no.**

**Species ident-ified**

Q7ZZ07 *Brachidanio* 

Q91780 *Xenopus* 

Q96AZ5 *Brachidanio* 

Table 4. Results from peptide mass fingerprinting of protein spots excised from 2D gels. EWM: Experimental Weight Mass; EIP: Experimental Isoelectric Point; TWM: Theoretical Weight Mass; TIP: Theoretical Isoelectric Point. (This table was originally published in Zilli

SPOT 6 110 6.0 — — *—* — — — — SPOT 8 100 5.2 — — *—* — — — —

**TWM (kDa) TIP** **Homology** 

**Coverage %** 

**Matched peptides** 

*rerio* 71.8 5.98 8 22.0

*laevis* 47.78 6.3 6 20.0

*rerio* 37.13 5.6 4 21.0

transcription factors (Latham & Schultz, 2001).

protein DKFZp566A1524 of unknown function.

**EIP Identified protein** 

> Novel protein similar to SKB1 human and mouse (PEPTIDENT)

> G1/S-specific cyclin E2 (MASCOT, MS-FIT)

Similar to hypothetical protein DKFZp566A1 524 (MS-FIT)

**Reference Spot** 

**EWM (kDa)**

SPOT 5 80 6.5

SPOT 13 40 6.8

SPOT 20 30 4.5

et al., Biol Reprod 2005).

The results reported in figure 6 and tables 3 and 4 show that in sea bass spermatozoa the used cryopreservation procedure causes the degradation of 21 sperm proteins, and among these, 2 could be at least partially responsible for the observed decrease in sperm motility duration and the lower hatching rate of eggs fertilized with cryopreserved sperm. In addition, these observations suggest that two-dimensional electrophoresis coupled with MALDI-TOF analysis could be used as a tool to improve cryopreservation procedures.
