The Function Of Trypsin-like Tryx5 In Themigration Of Mice Sperm Into The Oviduct

As is known to all,male sterility is widely distributed around the world.The incidence of male sterility in the nation was up to 10%,which was second to cardiovascular disease and cancer.Nowadays,the common way to evaluate male sterility is to use CASA to examine sperm density,shape,motility and so on.However,about 25%of the sterile men own normal sperm parameter mentioned above.Lacking serine protease is common in these cases.Testis-specific serine protease family is the biggest one in the mice testis.Besides,many serine proteases are reported to play an essential role in the reproduction of male mice.It is reported that trypsin-like TRYX5,a member of serine protease family,is specially expressed in the mouse sperm acrosome,Golgi apparatus of GC-2 cell line?spermatogonia cell line?and spermatogenic cells.But the function in vivo of TRYX5 is still unkown.In order to understand the physiological function and molecular mechanism of TRYX5 in male mice reproduction,the study created a Tryx5^-/-mouse model,and investigated the phynotype in male reproduction and the underlying molecular mechanism.The study could help to screen for potential drug target for sterile patients or for the development of methods for effective male contraception.1.The expression pattern of Tryx5 in miceTo further study the expression pattern of Tryx5,we examined the the expression pattern of Tryx5 m RNA in various tissues and testis of different stages using q RT-PCR.The results showed that Tryx5 m RNA was expressed specially and highy in mouse testis and sperm.Tryx5 m RNA became detectable in testes around postnatal day 18?about 60 times higher than that in postnatal day 5?and then increased up to day 28 before plateauing at this level until at least day 60.As there was no commercial anti-TRYX5 antibody,we immunized the rabbit with TRYX5 peptides and obtained an anti-TRYX5 polyclonal antibody-2204.Then the expression pattern of TRYX5 protein was detected in testis of different stages by Western Blotting.The results showed that TRYX5protein level was low at day 6 and then was similar among every stage before day 60,of which the TRYX5 protein level was the highest.Besides,TRYX5protein could also be detected in mature sperm.As anti-TRYX5 antibody could not be applied in immunofluorescence and immunohistochemical experiments,we builded the Tryx5-EGFP mice model and detected the expression pattern of TRYX5-EGFP in the testis.It showed that TRYX5-EGFP was expressed in the spermatogenic cells and the acrosome of mature sperm.To further make sure the germ cells expressing TRYX5,the study isolated mouse spermatogonia cells,spermatocytes,round spermatids and later elongated spermatids using flow cytometry purification and sucrose density gradient centrifugation,and determined TRYX5 with Western Blotting.The results showed that TRYX5expression was weaker in the spermatogonia cells and spermatocytes,and was stronger in the round spermatids,later elongated spermatids and mature sperm.2.Initial phenotype analysis for Tryx5^-/-miceThe 4th exon of Tryx5 was deleted by CRISPR/CAS9.Briefly,we injected CAS9 m RNA and g RNAs into the cytoplasm of the fertilized eggs and transplanted the zygotes into the ampulla potion of pseudopregnant receptor treated with synchronization.The offspring were identified in genotype and so on.The results of genome PCR,sequnencing,RT-PCR and Western Blotting all showed that the generation of Tryx5^-/-mice was successful.As Tryx5 is expressed specially in the mouse testis,the research firstly examined the phenotype of reproduction in Tryx5^-/-mice.Mating test showed that Tryx5^-/-male mice were sterile though no differences were found between the ability of wt and Tryx5^-/-male mice to plug wt females.Testis index,density and morphology of epididymal sperm are all normal in Tryx5^-/-male mice.PAS-hematoxylin staining of testis proved that the spermatogenesis was normal.Meanwhile,the results of CASA assays did not indicate any significant differences in motilitylocated in the acrosome of round spermatids and mature sperm.In order to prove whether the knockout of Tryx5 could affect the acrosome,we detected the localization of sp56-a marker protein for sperm acrosome with immunofluorescence.It showed that sp56 localization was similar in wt and Tryx5^-/-group.More than 75%of sperm underwent AR induced by A23187,which showed no significant difference between wt and Tryx5^-/-sperm.3.In Vitro Fertilizing Ability of Tryx5^-/-miceIn order to make it clear whether the fertility of Tryx5^-/-sperm is normal,we collected oocytes from the superovulated mice,divided them into cumulus-intact group and cumulus-free group,and conducted in vitro Fertilization with wt and Tryx5^-/-sperm respectively.Tryx5^-/-sperm showed poor zona-binding ability compared with wt sperm;However,Tryx5^-/-sperm were capable of fertilizing both cumulus-intact oocytes and cumulus-free oocytes normally.·4.In Vivo Fertilizing Ability of Tryx5^-/-miceTo further seek for the cause inducing the infertility of Tryx5^-/-male mice,we collected fertilized oocytes from the superovulated wt females plugged by wt or Tryx5^-/-males.The ratio of pronuclear and two-cell stage embryos of wt group could both achieve a high level?approximately 56%and 60%?.But when it came to Tryx5^-/-group,these two kinds of embryos were significantly lower?approximately 7%and 0?.To research the reason why the fertilization rate in vivo of Tryx5^-/-mice was low,we collected uteri and oviducts?two sides?of plugged female mice 2 h after coitus,sperm in some uteri and oviducts were flushed out and counted,and other uteri and oviducts were used to make serial sections and H&E staining.The results showed that wt or Tryx5^-/-sperm in the uterus of plugged females was both more than 2 x 10⁶,while wt or Tryx5^-/-sperm in the oviducts was 138 or 1.2 in average.And H&E staining of UTJ section further proved the results above.5.Mollecular mechanism underlying the sterility of Tryx5^-/-miceAs few reports about Tryx5 have ever shown up,we systematically studied the literature about other serine proteases in the testis,and found that Prss37^-/-and Prss55^-/-male mice showed the similar phenotype to Tryx5^-/-male mice and declining accumulation of ADAM3 in the mature sperm.Or rather,when many genes that regulate ADAM3 were knocked out,similar phenotype appeared as that of Tryx5^-/-male mice.These facts show that ADAM3 is the key protein in sperm migration from the uterus into the oviduct.To study the relation between Tryx5 and these genes,we first examined the changes of their m RNA expression from the testis of Tryx5^-/-mice and found them including Adam3 change slightly.Then we detected the expression change of ADAM3 and ADAM3-associated proteins in the testis and sperm of Tryx5^-/-mice using Western Blotting and immunofluorescence staining.The results showed that there was no significant difference in ADAM3 or some ADAM3-associated proteins from wt and Tryx5^-/-testis,and the ADAM3-associated proteins did not change significantly,but mature ADAM3 declined greatly in Tryx5^-/-sperm.Further systematic research showed that a signal for ADAM3 was present in all wt caput,corpus and cauda epididymal spermatozoa but almost absent from Tryx5^-/-epididymal spermatozoa;ADAM3 expression in the S13-S16 elongated spermatids from Tryx5^-/-mice is much less than that from wt,while ADAM3 signal in the residual bodies around S13 elongated spermatids from Tryx5^-/-mice is much stronger than that from wt.All the results show that the knock out of Tryx5affects the existence of mature ADAM3 in the mature sperm by impeding the accumulation or correct localization of ADAM3 precursor in the S13-16elongated spermatids.And the knock out of Tryx5 does not affect the ADAM3precursor by influencing the expression level of some ADAM3-associated proteins.Next,to study the interaction between TRYX5 and ADAM3 or some ADAM3-associated proteins,we used the testis of Tryx5-EGFP to conduct IP assay.The results show that there is no direct interaction between TRYX5 and ADAM3 or some ADAM3-associated proteins such as CLGN,ADAM2,PDILT,PGAP1,TPST2 and TEX101.It implies that TRYX5 can affect the accumulation or localization of ADAM3 precursor in the testis through other intermidiates.In a word,several conclusions can be summarized as follow:?1?Tryx5^-/-male mice are sterile,but the spermatogenesis,sperm motility,sperm fertility and other conventional reproductive parameters are all normal,and it is the sperm disability to migrate into the oviduct that causes the sterility of Tryx5^-/-male mice;?2?The knock out of Tryx5 affects the existence of mature ADAM3in the mature sperm by reducing the ADAM3 precursor in the S13-16 elongated spermatids,which may be the important reason that Tryx5^-/-sperm cannot migrate into the oviduct.