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ROLE OF IONS IN THE SPERM ACTIVATION Abstract Although it is widely accepted that osmolality and ion fluxes are the main factors triggering sperm motility in fish, a complex universal mechanism for sperm motility initiation does not exist in fish, and studies of marine fish species are even more scarce. Therefore, the main goal of this study was to estimate the intracellular variations in the main ions involved in sperm activation for the first time in European eel, in order to provide additional new data about this little-known process. It was observed that levels of intracellular Ca 2+ and K + sperm ions increased significantly 30 s after the hyperosmotic shock compared to baseline levels, and remained at this level until 120 s post-activation. In contrast, the intracellular pH remained constant during the first 30 s, and decreased gradually at 60 and 120 s post-activation. Our data agree with the current main theory for explaining motility initiation in marine fish, in which internal fluctuations of Ca 2+ and K + seem to participate in sperm activation. In addition, fluorescent images showed that both Ca 2+ and K + were concentrated in the apical area of the sperm head, which corresponds to the location of the eel mitochondria, suggesting this organelle plays an important role in sperm motility activation. 1. Introduction In marine teleosts, the spermatozoa are quiescent in isotonic solutions, such as seminal plasma, and become motile when the sperm is diluted in hypertonic solutions, suggesting that motility is suppressed by the osmolality of the seminal plasma, and initiated by exposure to hypertonic seawater at spawning (Morisawa and Suzuki, 1980, Cosson, 2004, Morisawa, 2008). The osmotic shock faced by the spermatozoa when they are released into the marine environment leads to a rapid flux of ions and water between intracellular compartments and external medium (Oda and Morisawa, 1993; Zilli et al., 2009). It has been proposed that Ca 2+ and K + are the main ions involved in sperm motility initiation in marine fish (see reviews of Morisawa, 2008, Cosson, 2008), but the exact mechanism 73
CHAPTER 3 through which this happens is still unknown. Although both in marine and freshwater fish species an increase in intracellular Ca 2+ has been observed after osmotic sperm motility activation, it is not clear if that increase comes from an influx of Ca 2+ , as has been proposed in seawater tilapia (Oreochromis mossambicus; Linhart et al., 1999), if it comes from intracellular stores, as it has been proposed in the case of puffer fish (Takifugu niphobles) and salmonid sperm (Krasznai et al., 2003; Takei et al., 2012), or from a decrease in cell volume due to the water efflux, as has been proposed by Zilli et al. (2008) in sparids species sperm. The first step to elucidate this mechanism in European eel sperm is studying intracellular Ca 2+ variations in quiescent sperm and then hyperosmotic activated motility. Changes to intracellular potassium levels after sperm activation have been measured in a limited number of freshwater fish species: two salmonids (Tanimoto et al., 1994), and common carp (Krasznai et al., 2003) sperm, where a K + efflux or a decrease in [K + ] i after hypoosmotic activation was observed. In marine fish species, [K + ] i changes after sperm activation have only been studied in the case of the pufferfish, and an increase in [K + ] i was observed after hyperosmotic activation (Takai & Morisawa, 1995, Krasznai et al., 2003). It is unknown whether hyperosmotic activation in the sperm of other marine fish causes an intracellular increase in K + , like in pufferfish, or a decrease, like in trout and carp. In sea urchin (Lee et al., 1983) and mammals (Wong et al., 1981; Babcock et al., 1983), the alkalinization of intracellular pH induces sperm activation. In carp a pH increase was also observed after hypoosmotic sperm activation (Krasznai et al., 1995), but trout sperm undergo an acidification upon hypoosmotic activation (Boitano and Omoto, 1991). An increase in pH i was observed at motility initiation in the sperm of several marine fish species (Oda and Morisawa, 1993). Therefore, there is no current consensus on the intracellular pH changes related to sperm activation in fish species. We observed that extracellular pH was important for sperm motility in European eel; while acidic pH extenders (pH 6.5) induced a reversible 74
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SPERM PHYSIOLOGY AND QUALITY IN TWO
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ACKNOWLEDGEMENTS Muchos años han p
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aunque tiene una alcachofa en la ca
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TABLE OF CONTENTS SUMMARY 1 RESUMEN
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SUMMARY SUMMARY The conservation st
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RESUMEN RESUMEN Las especies objeto
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RESUM RESUM Les espècies objecte d
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GENERAL INTRODUCTION 7
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GENERAL INTRODUCTION who postulated
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GENERAL INTRODUCTION 2. Sperm physi
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GENERAL INTRODUCTION Finally, the a
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GENERAL INTRODUCTION 3. Sperm quali
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GENERAL INTRODUCTION in fish (Kime
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GENERAL INTRODUCTION However, for s
Page 31 and 32: GENERAL INTRODUCTION future applica
Page 33 and 34: GENERAL INTRODUCTION With regards t
Page 36: OBJECTIVES The chapters presented b
Page 40 and 41: STANDARDIZATION OF SPERM MOTILITY E
Page 56: CHAPTER 2 Study of the effects of t
Page 59 and 60: CHAPTER 2 reducing the pressure on
Page 61 and 62: CHAPTER 2 ongoing task in order to
Page 63 and 64: CHAPTER 2 To measure sperm density,
Page 65 and 66: CHAPTER 2 Spermiating males (%) 100
Page 67 and 68: CHAPTER 2 100 80 60 I II III IV A 4
Page 69 and 70: CHAPTER 2 3.2 Hormonal treatments P
Page 71 and 72: CHAPTER 2 With regard to the sperm
Page 73 and 74: CHAPTER 2 Relative volume (%) 80 I
Page 75 and 76: CHAPTER 2 In this respect, it is we
Page 77 and 78: CHAPTER 2 treatment to that of the
Page 79 and 80: CHAPTER 2 Our results demonstrate t
Page 84 and 85: ROLE OF IONS IN THE SPERM ACTIVATIO
Page 92: CHAPTER 4 Study of pufferfish (Taki
Page 95 and 96: CHAPTER 4 2002) so Takifugu niphobl
Page 97 and 98: CHAPTER 4 carrying out sperm collec
Page 99 and 100: CHAPTER 4 Table 1. Activation media
Page 101 and 102: CHAPTER 4 TM (%) 100 A a a Und-PD a
Page 103 and 104: CHAPTER 4 TM (%) 80 60 40 20 0 A a
Page 105 and 106: CHAPTER 4 TM (%) 80 A ASW100 GLU 60
Page 107 and 108: CHAPTER 4 although the activation m
Page 109 and 110: CHAPTER 4 dilution ratio and an opt
Page 111 and 112: CHAPTER 4 2006). Several studies in
Page 113 and 114: CHAPTER 4 5. Conclusions Some concl
Page 116 and 117: RELATIONSHIP BETWEEN SPERM MOTILIY
Page 130: RELATIONSHIP BETWEEN SPERM MOTILIY
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GENERAL DISCUSSION 1. Improvements
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GENERAL DISCUSSION profiles induced
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GENERAL DISCUSSION more studies on
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GENERAL DISCUSSION sperm motility p
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CONCLUSIONS 133
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CONCLUSIONS vii. The coefficients o
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REFERENCES Aarestrup K, Økland F,
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REFERENCES Beirão J, Cabrita E, P
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REFERENCES Cabrita E, Robles V, Cu
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REFERENCES De Vos A, Van De Velde H
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REFERENCES Gallego V, Pérez L, Ast
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REFERENCES sperm competition and fe
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REFERENCES Liu QH, Li J, Xiao ZZ, D
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REFERENCES Mortimer ST. Effect of i
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REFERENCES Peñaranda DS, Marco-Jim
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REFERENCES Rurangwa E, Volckaert FA
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REFERENCES Tesch F. Telemetric obse
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REFERENCES Wong PYD, Lee WM, Tsang
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