Factors Controlling Sperm Entry into the Micropyles of Salmonid and Herring Eggs

Abstract

When the micropyle area of salmonid (trout and salmon) eggs was observed continuously from the moment of insemination, spermatozoa were seen moving along the surface of the chorion and entering the micropyle one by one in a directed fashion. The ability of spermatozoa to enter the micropyle was reduced after the treatment of chorions with pronase; this reduction in sperm entry was observed even before the outer opening of the micropyle channel was narrowed due to gradual swelling of the chorion by pronase treatment. Herring spermatozoa, unlike spermatozoa of most other marine fishes, were motionless in seawater. However, they became vigorously motile on contact with the micropyle area of the herring egg chorion and entered the micropyle rapidly and efficiently. Motility initiation of herring spermatozoa in the micropyle area was dependent on extracellular calcium and potassium. Sodium also appears to be intricately involved in this process as demonstrated by the initiation of sperm movement in sodium-free seawater. When herring eggs were treated with acidic seawater, organic solvents, or glutaraldehyde, spermatozoa did not initiate movement in the micropyle area, and sperm entry was not observed. Herring spermatozoa did not initiate movement in the micropyle area of salmonid eggs. These and other observations suggest that the micropyle areas of salmonid and herring eggs possess some sperm guidance factors which facilitate entry of homologous spermatozoa into the micropyle.

Motility initiation in herring sperm is regulated by reverse sodium-calcium exchange

“…Herring sperm were immotile in 1͞2 FSW and HR, but initiated motility in 1͞2 NaF in the absence of SMIF as has been reported (13). Motility in 1͞2 NaF was significantly reduced (P Ͻ 0.05) in 25 mM [Na ϩ ] o and completely inhibited in 150 mM [Na ϩ ] o ( Fig 1A).…”

“…Sperm motility was assessed with either a 10ϫ or 20ϫ objective lens by using the following qualitative index: 0 ϭ no motility, 1ϩ ϭ Ͻ25% motility, 2ϩ ϭ 25-50% motility, 3ϩ ϭ 50-75% motility, 4ϩ ϭ Ͼ75% motility (13,14,16 (23). In some experiments, the Ca 2ϩ channel blockers nifedipine (100 M) or flunarizine (10 M) or the Na ϩ ͞Ca 2ϩ exchange inhibitors bepridil (50 M), dichlorobenzamil (10 M), or KB-R7943 (10 M) were preincubated (5 min) with sperm before Ca 2ϩ i measurements.…”

“…This range of salinities correlates with the optimal salinity regime for fertilization and early development in Pacific herring (14). Previous studies have shown that extracellular Na ϩ ([Na ϩ ] o ) and extracellular Ca 2ϩ ([Ca 2ϩ ] o ) influence both sperm motility and fertilization (11)(12)(13)(14)18). Sperm motility can be initiated in the absence of SMIF by low-Na ϩ (2 mM) seawater (NaF; ref.…”

“…Upon spawning, herring sperm (Clupea pallasi) are intrinsically motionless over a wide range of salinities (11)(12)(13)(14). This immotility may be related to the unique reproductive biology of herring in which male fish spawn first, with sperm remaining in the water column for extended periods, followed by the spawning of females (15).…”

“…Testis and ovaries were dissected and stored in individual culture plates at 4°C under moist conditions (13). Some experiments were conducted at the Hokkaido National Fisheries Research Center by using animals caught at Akkeshi Lake, Hokkaido, Japan.…”