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

鳞翅目昆虫具有二形性精子系统，有真核精子受精和无核精子受精。我们发现，家蚕多倍体雄性只有功能性真核。另一方面，对黛作品系家蚕的蚕蛹进行热激处理，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。分离出的精子混合进行二次交配和人工授精实验，证明无核精子是优育精子受精成功的必然。这些结果首次从实验上证明了无性生殖精子与受精有关的功能。这个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。真核精子在不与无核精子混合的情况下不能进入受精囊，导致受精不成功。如果提供完整的无核精子，用冷冻保存的真核精子对雌性进行人工授精，可以获得正常生育能力。另一方面，冷冻保存的无性精子在冷冻和解冻处理后有时会失去能力。冷冻精子受精率的下降可能主要是由于精子的严重损伤所致。这在不同的家蚕品种之间有很大的差异。利用抗冻融品系培育多倍体雄性可能是提高无性生殖精子功能稳定性的途径之一。我们将冷冻精液保存在液氮中，以提高冷冻精子库的受精率。本研究构建的人工授精技术、精子冷冻保存和无性精子分离应用相结合的技术体系，为开发家蚕遗传资源保存的新方法奠定了基础。

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.