The Roles Of Tyro3 RTKs In The Regulation Of Spermatogenesis

Mammalian spermatogenesis is a synchronized,regulated and complex process of cellular differentiation,which includes three distinct phases of cellular and molecular changes: mitosis,meiosis and spermiogenesis.In this process,spermatogonial "stem cell" is gradually transformed into a highly differentiated haploid spermatozoon,which will then be released into the lumen at spermiation.As the only somatic cell in the seminiferous epithelium,Sertoli cells communicate with germ cells through direct cell-cell contact and paracrine signaling.The structure and function of Sertoli cells exhibit a stage-dependent change during the spermatogenic cycle.However,the molecular bases of the spermatogenesis regulated by Sertoli cells remain to be defined.The genetic studies using gene knockout model have provided directly insights into this field.The Tyro3 subfamily of receptor tyrosine kinases(RTKs) is composed of Tyro3,Axl and Mer.Although a previous study demonstrated that the male mice triply mutant for Tyro3 RTKs were infertile,the mechanisms underlying this defect have not been elucidated.Thus,we investigated the roles of Tyro3 RTKs in regulating spermatogenesis with an effort to understand the mechanisms.We assessed the fertility of male Tyro3 RTKs knockout mice with different genotypes by mating experiment.It was shown that all male mice singly mutant for Tyro3 RTKs had normal fertility.Although mice doubly mutant for Axl and Tyro3(AT) exhibited normal fertility,a significant low fertility was observed in male mice doubly mutant for Axl and Mer(AM) and Mer and Tyro3(MT).Notably,the triple mutant mice for Tyro3 RTKs (TAM) were completely sterilized.It was consistent with the results of the ratio value between testes weight and body weight and epididymal sperm count in male Tyro3 RTKs knockout mice with different genotypes.These results suggest that three members of Tyro3 RTKs subfamily regulate male fertility in a redundant manner,and Mer may play a more important role than other two members.In order to define the defect of spermatogenesis in TAM mice,high-power histological analysis of semithin sections of testes from mice at postnatal day 3,and 1,2,3,5,8,15 weeks was performed.We also scored the numbers of spermatogonia,Sertoli cells,myoid cells,the primary spematocytes,round spermatids and degenerating cells in early developing mice.We found that the numbers of spermatogonia,Sertoli cells and myoid cells showed no apparent difference between WT and TAM mice.From 2 weeks,the numbers of primary spematocytes,and then spermatids in TAM mice were reduced.The testicular degeneration became more severe as the mice aging.At 5 weeks postnatal, elongating or elongated spermatids were scarcely observed in the seminiferous epithelium, and Sertoli cell vacuolization was frequently observed.In 8-week-old mice,elongating or elongated spermatids were rare seen in the seminiferous epithelium;spermatids at different maturation steps were simultaneously present in the same cross section of some tubules, indicating asynchronized development of haploid cells during spermiogenesis,and some degenerated cells or residual bodies sloughed off to the lumen.In 15-week-old mice,most seminiferous tubules were devoid of spermatids.Sertoli cell vacuolization,asynchronized development of haploid spermatids,sloughing of developing spermatogenic cells and appearance of multinucleated spermatids(symplasts) became progressively more severe. Occasionally,only Sertoli cells and spermatogonia could be found in some tubules.In addition,we examined the DNA contents of testicular cells using flow cytometry,and the results were consistent with the histological finding.Marked decreased elongated spermatids in TAM mice could result in a lower HN cell peak.These results indicated the adhering junctions between Sertoli cell and spermatogenic cells were impaired,and it was also clarified by an in vitro binding assay.The TAM Sertoli cells showed a dramatic reduction in binding spermatogenic cells in vitro.These results suggest that Tyro3 RTKs participate in the binding of Sertoli cells to apoptotic spermatogenic cells. Lipid droplet is one of Sertoli cell cytoplasmic components,and its function and metabolism remain to be defined.In the present study,using morphometric analysis method,we investigated the dynamic changes of lipid droplets in Sertoli cells during postnatal development in detail.It was shown that the level of lipid droplets in Sertoli cells increased concomitance to the sex maturity,and they were well in accordance with the spermatogenic cells development.The more spermatogenic cells,the higher level of lipid droplets.Meanwhile,there were two parts of lipid droplets in the seminiferous tubules of adult mice,and they were located in the cytoplasmic lobes(residual bodies) of spermatids in middle of the tubules and in the cytoplasm of Sertoli cells in periphery of the tubules, respectively.Both of them appeared in stage-dependent manner.The former peaked at stagesâ…¦-â…§,and the latter at stagesâ…¨-â… ,just after spermiation.Thus,the temporal relationship between phagocytosis of residual bodies and degenerating spermatogenic cells and the increased lipids level in Sertoli cells from newborn to adulthood suggests that lipid droplets may be useful criterion to evaluate the phagocytic ability of Sertoli cells.The hypothesis was confirmed by an in vitro phagocytosis assay.After co-culture with apoptotic spermatogenic cells,the level of lipid droplets in Sertoli cells was dramatically increased.In addition,we detected the level of ATP and ATPase in the different staged seminiferous tubules.We found that the level of ATP was consistent with that of lipid droplets in Sertoli cells,and lipid droplets could be hydrolyzed to produce ATP to apply for the energy consumption during spermatogenesis.We also investigated the level of lipid droplets in TAM mice.Compared with WT mice, Leydig cells in TAM mice did not exhibit apparent changes in the levels of lipid droplets. However,it was different in the seminiferous tubules.The level of lipid droplets in TAM Sertoli cells was decreased from 2 weeks,and became worse as aging.The level of lipid droplets in the seminferous epithelium was consistent with the integrity of the tubules.Unlike the stage-dependent formation of lipid droplets in the seminiferous epithelium of WT mice,only few lipid droplets were found in all TAM seminiferous tubules without a stage-dependent manner,and the level of lipid droplets in 15-week-old TAM Sertoli cells was reduced to only 13%of that in WT mice.Considering the phagocytosis of apoptotic spermatogenic cells or residual bodies resulted in the formation of lipid droplets in Sertoli cells,the significant decreased level of lipid droplets in TAM Sertoli cells might reflect an impaired phagocytic ability of TAM Sertoli cells.The phagocytosis assays in vitro had further confirmed this speculation.In addition,we found the level of ATP in TAM Sertoli cells was lower,and ATPase higher than that in WT Sertoli cells.It might also reflect the defect of phagocytic ability of TAM Sertoli cells,which led to the deficiency of ATP and the compensation of increased ATPase.In summary,the results of this study strongly suggested that Sertoli cells were primarily affected by losing Tyro3 RTKs in TAM mice.Tyro3 RTKs might involve in the binding of Sertoli cells with apoptotic spermatogenic cells and the phagocytic ability of Sertoli cells. The mice mutant for Tyro3 RTKs could be a useful model for further understanding the roles of Sertoli cells in regulating germ cell maturation.