| As the model insect of Lepidoptera,silkworm Bombyx mori has two types of sperm,namely eupyrene sperm（eupyrene sperm）and apyrene sperm（apyrene sperm sperm）.The eupyrene sperms fertilize eggs,while the apyrene sperms assist fertilization.The chromatin of mature eupyrene sperms are highly agglutinated in the head of sperm,but the chromotin of mature apyrene sperms are loosely arranged near the equatorial plate and then disappear during maturation.SR protein kinases（SRPKs）are a family of kinases that specifically catalyze the phosphorylation of SR proteins（serine/arginine protein family）and play an important role in the assembly and alternative splicing of RNA spliceosomes.Recent studies have shown that SRPK also plays regulatory roles in sperm chromosome remodeling.In silkworm,only one SRPK gene was found,named SRPK3,whether it is involved in the dynamic changes of chromosomes during the maturation of eupyrene and apyrene sperms in silkworm is unknown.In this study,the silkworm Bombyx mori was used,the testis tissue from different developmental stages was dissected,the maturation process of the two types of sperms was detected.As well,the expression patter of SRPK3 in different tissue and developmental stages of silkworm was detected by quantitative RT-PCR（q RT-PCR）.Furthermore,SRPK3 was knocked down in vitro cultured sperm cells to study its effect on sperm development.In addition,SRPK3 gene was knocked down and overexpressed in silkworm to observe its effect on the maturation of spermatozoa in silkworm and on reproduction.The main findings are as follows:（1）The developmental process of eupyrene sperm and apyrene sperm sperm of silkworm.The testis of male silkworm from 1-day-old instar larvae to 5-day-old moth was dissected and stained with FITC-labeled Tubulin antibody and DAPI.Result showed that mature eupyrene sperm bundles were first appeared at 2-day-old fifth instar larvea,the chromosomes of which were aggregated and condensed to the head of sperms,then the sperm bundles gradually elongated with part of the cytoplasm lost by queenzing,which result in mature eupyren sperm bundles.Apyrene sperm bundles began to appear at the 2nd day of the prepupal stage,which first elongated into a spindle shape,then the nucleus concentrated in the equatorial plate of the cell,after part of the nucleus lost,they developed into mature apyrene sperm bundles.（2）The spatiotemporal expression profile of SRPK3 gene in silkworm.The whole worm and testis tissue of different developmental stages of silkworm were collected,RNA was extracted,and the spatiotemporal expression profile of SRPK3 gene was detected by q RT-PCR.The results showed that SRPK3 gene was highly expressed in testis.No obvious change was detected during larva development except its accumulation in the fourth instar larvae.During pupation,the expression level of SRPK3 in female pupae gradually increased,with highest expession at 4-day-old pupae,and then gradually decreased,with lowest expression level at 7-day-old pupae.No significant change was observed from the first to the sixth day of male pupae except a rapid accumulation on the 7th day of pupa.SRPK3 showed a first rising and then decreasing trend in testis from 1-day-old to 8-day-old fifth instar larvae,with highest expression at 3-day-old fifth instar larvae.SRPK3 also showed a first rising and then decreasing trend in testis during pupation,with highest expression at 3-day-old pupae.The expression of SRPK3 in aeupyrene sperm was higher than eupyrene sperm by 1.7 times.It is suggested that SRPK3 gene may play roles in the maturation process of apyrene sperms.（3）Knockdown SRPK3 in silkworms.The ds RNA of SRPK3 was synthesized by T7 in vitro transcription method,and then injected into the hemolymph of male silkworms of and 3-day-old fifth instar larvae.The results showed that aeupyrene sperm still appeared in the testis of the male silkworm on the second day of prepupa,but the proportion was97.7% lower than that of the control group,indicating that the SRPK3 was involved in the maturation of aeupyrene sperm,ratio of mature aeupyrene sperms decreased after SRPK3 knockdown.Number of eggs laid by male silkworms with the SRPK3 knockdown and normal female silkworms was not changed significantly,but the ratio of eggs that did not hatch increased higher than that of control by 2.5 times.Expression of genes such as BMSK0000595 in thyroid signaling pathway,decreased by 72.7%,and pancreatic lipase-related protein 1（PNLIPRP1,BMSK0006978）in IP3 signaling pathway increased by 146.9% compared to the control.It suggested that SRPK3 plays a regulatory role in the early stage of aeupyrene maturation.（4）SRPK3 was overexpressed in silkworms.The overexpression vector of SRPK3 p IEx-4-[SRPK3] was constructed and injected into the hemolymph of the 3-day-old 4th instar larvae,changes of sperm development and phenotype of egg-laying were also observed.The results showed that overexpression of SRPK3 did not change the appearance time of mature aeupyrene sperms,but the proportion of aeupyrene sperm increased significantly than the control group by 9.02 times.Number of eggs laid by mating of male silkworms with SRPK3 overexpression and normal female ones was normal,but the number of unhatched eggs increased significantly by 27.8 times than that of the control group.Further analysis showed that the aggregation behavior of nuclei of eupyrene sperm was abnormal,some nucleis retained in the cytoplasm and could not reach to the head of sperms succesfully.The expression of cytoskeletal protein CPH19（cuticular protein hypothetical 19 precursor,BMSK0006531）decreased significantly by 91.5% compared to that in the control.These results suggested that SRPK3 not only plays a regulatory role in the early stage of aeupyrene maturation,but also affects the aggregation behavior of nuclei of eupyrene sperms,which might through regulating the expression of cytoskeletal protein genes.This study exhibited the maturation process of eupyrene and apyrene sperms in Bombyx mori,the expression profile of SRPK3 gene in different tissues and developmental stages was also studied.The regulator roles of SRPK3 during the maturation of eupyrene sperm and aeupyrene sperm,and its effect on repruduction were also observed.It was found that the SRPK3 might play a key role in the maintenance of the loose state of chromatin in aeupyrene sperms,and it may affect the maturation of eupyrene sperm by affecting the expression of cytoskeletal protein genes.This study provides new clues for sperm dimorphism of silkworm. |
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