Studies on apyrene function and its application for sperm storage in lepidopteran insects

鳞翅目昆虫的精子系统具有二型性，即真核精子和无核无核精子。我们发现，多倍体家蚕雄性只有功能性真核细胞。另一方面，对大藏系蛹进行热激处理，可单独产生无核精子。因此，我们成功地分离了真核精子和无核精子。双配和混合分离精子的人工授精试验证明，无核精子是真核精子成功受精的必然条件。这些结果首次从实验上证明了无核精子在受精过程中的作用。该实验系统使我们能够观察单个精子群体在女性生殖器官中的行为。真核精子不与无核精子混合就不能进入受精囊，不能受精，用冷冻保存的真核精子人工授精雌虫，如果提供完整的无核精子，受精率正常。另一方面，冷冻保存的无核精子有时会在冻融处理后丧失其能力。冷冻后精子受精率下降的主要原因可能是无核精子的严重损伤。这在蚕品种之间差异很大。利用抗冻融品系生产多倍体雄虫可能是提高无核精子功能稳定性的途径之一。我们通过应用保存在液氮中的无核精子来提高冷冻保存的精子库的生育力。本研究建立的家蚕人工授精技术、精子冷冻保存和无核精子分离应用的组合体系，为发展家蚕遗传资源保存新方法奠定了基础。

Lepidopteran insects have dimorphic sperm system, fertilizing eupyrene sperm and anucleate apyrene sperm. We found that polyploid male of Bombyx mori have only functional eupyrene. On the other hand the apyrene sperm is produced alone by heat-shock treatment to pupae of Daizo strain in the silkworm. Thus we successfully separated the eupyrene sperm from the apyrene sperm. Double copulation and artificial insemination experiments with mixing of the separated sperm proved that the apyrene sperm is inevitable for successful fertilization by the eupyrene sperm. These results are first experimental proof of the function of apyrene sperm concerning the fertilization. This experimental system enables us to observe the behavior of individual sperm populations in female reproductive organs. The eupyrene sperm cannot proceed to spermatheca without mixing with the apyrene sperm, resulting no fertilization success.Artificial insemination to females with cryopreserved eupyrene sperm resulted normal fertility if intact apyrene sperm was supplied. On the other hand cryopreserved apyrene sperm sometimes lost their ability after freeze-and-thaw treatment. Decrease in fertility of cryopreserved sperm seems to be mainly caused by sever damage of apyrene sperm. This greatly varies among silkworm strains. Utilization of resistant strains against freeze-and-thaw for producing polyploid males may be one of the ways to increase the stability of apyrene sperm function. We improved the fertility of cryopreseved sperm stock by applying the apyrene sperm which was kept in liquid nitrogen. The combination system of artificial insemination technique, cryopreservation of sperm and application of separated apyrene sperm constructed in this study is basis to develop the new method to maintain the genetic resources of the silkworm.