PHYSIOLOGICAL MECHANISMS REGULATING SPERM MOTILITY INITIATION IN EASTERN OYSTER Crassostrea virginica

Oyster aquaculture is expanding in the  United States, where production  has  increased from $78 million  to $192 million between  2008  to 2016.  Many  oyster farms  rely on oyster seed produced at hatcheries that induce broodstock to spawn . Hatchery success can often be dependent on gamete quality. I n order to improve hatchery production ,  a better understanding of  oyster gamete biology is needed. Eastern oyster males and females release sperm and  oocytes  into the water column to be fertilized. Upon release, sperm is exposed to the external environment in which  sperm motility is initiated. However, physical and chemical properties of the  environment affect sperm motility performance,  which  is a key determinant for fertilization success .  Thus, providing the  optimal environmental conditions will maximize sperm activity and fertilization success.  The objectives of our work were to determine the  physiological mechanisms regulating sperm  motility initiation  in this species . S perm  swimming  kinematics were evaluated by computer assisted sperm analysis (CASA),  and curvilinear velocity (VCL) and percentage  of motility were measured. Sperm were activated with a rtificial sea water (ASW) buffered to  make a  pH  gradient from pH 6.5 to 10.5. Sperm were  also  activated  across  a  range of salinities  from 4 to 32 psu .  ASW  was tested  with 0.5 to 3.5 mM EGTA to find the threshold of  extracellular Ca2+ ions needed to initiate sperm motility . Na+, K+, Mg2+, and Ca2+  free ASW  and their respective channel blockers  were used to  elucidate ionic signaling involved in sperm motility initiation.

Results show  that  sperm VCL increased from  pH 6.5 to 7.5 and peaked from 7.5 to 10 (Fig. 1A) , while  VCL peaked from 12 to 24 psu (Fig. 1B ).  Sperm VCL  was highest with 0.5 to 2.0 mM EGTA and decreased from 2.5 to 3.5 mM EGTA (Fig. 1C). Compared to ASW ,  sperm motility  was lower in Na+, K+ ,  and Mg2+  free ASW  significantly.  Moreover, sperm motility initiation was suppressed in the presence of K+ and Ca2+  channel blockers. These results show that environmental salinity affects sperm motility initiation and indicate that sperm motility initiation is Ca2+-dependent and require K+ exchange through plasma membrane. Our study provides insights into physiological mechanisms of sperm motility signaling in bivalves and provides valuable information to  improve fertility in hatcheries and optimize cryopreservation protocols.