Research On The Distribution, Composition And Function Of Stress Granules In Spermatogenic Cells

Eukaryotic cells in the face of adverse environments such as heat shock will produce specific stress granules?SG?to help maintain cells survival,and to quickly restore normal cell function after release of environmental stress.At present,research on the formation and function of stress granules is done mainly in somatic cells,especially in cultured cells.However,the role of SG in testicular cells is still poorly understood.Mammalian testes are particularly sensitive to heat,with preferred temperature at 2-7°C below body temperature.Recently an RNA-binding protein,DAZL,was reported to be a SG component of spermatogenic cells and is proposed to protects mouse male germ cells from heat-induced apoptosis.However,the distribution,composition and function of SG in spermatogenic cells remain largely unknown.We found that stress granules were mainly distributed in the stage I-VIII and stage IV-VI was the period in which the stress granules are the most.There was little stress particles in stage IX-XII.The average number of stress granules formed in stage I was fewer than one stress granule per positive cell on average,and the average number of positive cells in stage II-III was more than four stress granules.Each of the positive cells in stage IV and V-VI contained about six stress granules.On average,each VII-VIII cell formed three stress granules,and almost no stress granules were formed in stage IX-XII.At the same time,we found that stress granules were mainly distributed in spermatogonia,leptotene and early pachytene spermatocytes,stress granules were not found in round and long spermatozoa.PUMILIO1?PUM1?and PUMILIO2?PUM2?belong to a highly conserved RNA binding protein family PUF,whose homologs play critical regulatory roles in embryonic development and stem cell maintenance in various species.Recently,we have found that the PUM family also plays an important role in mouse embryonic development and gametogenesis.Studies showed that PUM2 protein is involved in the formation of stress granules?SG?during the formation of dendritic cells in neurons.Then,in spermatogenic cells,do PUM family proteins also participate in the formation of stress granules?We found that PUM1 and PUM2 proteins can form SG after heat-shocked,and protect germ cells against apoptosis.Meanwhile,we found that Pum1^-/-mice had no significant effect on the formation of stress granules.However,we found a significant reduction of SG formation in Pum2 ^ko/koo/ko mice.We further determine the effect of the knockout of both PUM1 and PUM2 on SG formation,however,Pum double knockout mouse died during early embryogenesis.By detecting the expression of E6.5 and E7.5 endoderm marker GATA6,we found GATA6 protein was significantly increased in embryos of 7.5 d PUM double knockout mice,indicating that Pum1^-/-;Pum2^-/-mouse embryos had significantly more embryonic cells differentiated towards endoderm during gastrulation,likely resulting in gastrulation defects.Since the Pum double-knock embryos were lethal,we constructed a conditional knock-out of the PUM family?SDKO?mice during the spermatogonia period,and it was found that knockout both Pum1 and Pum2 genes resulted in a significant reduction in the number of SGs,supporting that PUM plays an essential role in the formation of SG.In summary,our data showed that PUM family proteins are important components of testicular SG,and play an important role in gastrulation and differentiation during early embryonic development.