Identification of 17,20β,21-trihydroxy-4-pregnen-3-one (20β-S) receptor binding and membrane progestin receptor alpha on southern flounder sperm (Paralichthys lethostigma) and their likely role in 20β-S stimulation of sperm hypermotility.

The existence of direct progestin actions on teleost sperm to stimulate hypermotility is not widely acknowledged because it has only been demonstrated in members of the family Sciaenidae. In the present study, progestin stimulation of sperm hypermotility was investigated in a non-sciaenid, southern flounder, and the potential role of membrane progestin receptor alpha (mPRα or Paqr7b) in mediating this action was examined. The major progestin produced in vitro by flounder testicular fragments co-migrated with 17,20β,21-trihydroxy-4-pregnen-3-one (20β-S) during thin-layer chromatography. Treatment of flounder sperm with 5 nM-100 nM 20β-S significantly increased sperm velocity in vitro, whereas 17,20β-dihydroxy-4-pregnen-3-one and other steroids were ineffective. A single class of high affinity (K(d) 22.95 nM), saturable, limited-capacity binding sites (B(max) 0.013 nM) specific for 20β-S was identified on sperm membranes. Treatment of sperm membranes with guanosine 5'-(3-O-thio)triphosphate reduced [(3)H]-20β-S binding, suggesting the 20β-S receptor couples to a G protein. The membrane adenylyl cyclase inhibitor 2',5'-dideoxyadenosine blocked 20β-S-induced sperm hypermotility, indicating 20β-S activates stimulatory G proteins. Finally, flounder paqr7b was cloned and characterized from testicular tissues. The Paqr7b protein is expressed on the midpiece of flounder sperm and is more abundant in individuals with high sperm motility than low motility donors. These findings suggest that 20β-S stimulates sperm hypermotility in flounder through activation of stimulatory G proteins, likely through Paqr7b. The finding that progestins directly stimulate sperm hypermotility in a flatfish, a highly derived species not belonging to the teleost family Sciaenidae, suggests this phenomenon is widespread among advanced fishes.