To understand male sea lamprey reproductive physiologiy and to identify the potential targets for control of sea lamprey reproduction, specifically to:
describe the ultrastructure of spermatozoa;
characterize the nature and regulation of sperm motion and identify factors able to inhibit sperm motility;
characterize the sperm acrosomal reaction (AR) and its regulation;
study sperm proteolytic activity and its role in fertililzation;
develop cryopreservation of sea lampre sperm; and
test the effect of a spermicidal agent--gossypol--on sperm structure and functions.

Understanding the physiological mechanisms regulating reproduction of the lamprey is a prerequisite for further attempts to control sea lamprey populations. At present, no data on sperm physiology are available for this parasite. We focus our studies on two key structures important for sperm function, the acrosome and the flagellum, because both are important for fertilization success (Comhaire, 1994). If gossypol can effectively inhibit fertilization of lampreys, it may offer an alternative to more toxic chemicals currently used.

Morphology of spermatozoa (before and after acrosomal reaction) will be examined by scanning and transmission electron microscopy. Computer-assisted Sperm Motion Analysis (CASA) will be employed for motility studies. The relationship between the acrosomal reaction and fertilization will be evaluated by means of fertilizing batches of eggs with sperm under control conditions and with various degrees and mechanisms of AR blocking. Effect of gossypol on spermatozoa will be tested in in vitro and after injection of male lampreys with dose 100 mg/kg of this sterilizing substance.

The enormous success of sea lamprey in coonization of the Great Lakes is most likely related to its effective reproduction. Better understanding of this species gamete biology is important both for development of laboratory techniques for studies of reproduction and for future control of sea lamprey production. This work examines several parameters of sea lamprey gametes handling related to their fertilization success. Sea lampreys were shipped overnight from Lake Huron Biological Station and maintained at the Columbus campus aquatic laboratory. Gametes were obtained from anesthetized fish (0.1% MS 222 in 0.3% sodium bicarbonate) by stripping. Sperm suspensions were stored on ice and used on the day of collection. When eggs were stored at 15° C, fertilizing ability amounted to 95.8 + 14.0%, 70.8 + 6.4% and 19.8 + 7.3% after 1, 2 or 3 days of storage, respectively. Storage of eggs at 15° C was superior to storage on ice where only 52.7 + 12.1%, 18.4 + 17.6% and 7.3 + 7.3% of fertilization rates were recorded, respectively. Consequently, in other experiments we used eggs obtained either on the day of collection or after one-day storage at 15° C. We tested effects of sperm number on fertilization success to evaluate optimal conditions for fertilization experiments. An increase of fertilization success was observed up to 50,000 spermatozoa per egg. For this reason, a constant sperm/egg ratio (1:50,000) was used in our studies. We estimated fertilization rate 2-3 minutes after fertilization (perivitelline space and cytoplasmic bleb test), five hours after fertilization (2-cells stage) and hatching in order to find the earliest estimation of fertilization success. Both early assessments of fertilization correlated significantly (r=0.92 and 0.93, P < 0.0001) with the percentage of live embryos at hatching. For this reason, both tests can be employed for the quick estimation of fertilization success. Unlike in teleost fish, where eggs usually have to be fertilized within a few minutes (or less) after release to water, fertilizing ability of sea lamprey eggs was markedly prolonged at these conditions. After 1, 2 or 3 h of eggs exposure to water at 22° C, the fertilization rate was 85.2+ 11.2%, 60.3 + 15.5% and 31.5 + 10.7%, respectively. In some cases fertilization rate after 1 hr of water exposure was as high as 94.8% - 96.4%. This may reflect better initial quality of eggs obtained under laboratory conditions. We speculate that at water temperature characteristics for natural spawning (11-15° C), survival of eggs released in lamprey nests will be capable to be fertilized during several hours.