The Function Of LRGUK On Spermatogenesis Of Mice

Spermatogenesis is a biological complex process by which spermatozoa are produced from male primordial germ cells through mitosis and meiosis(spermatocytes) and metamorphic change(spermiogenesis), and over 3000 genes involved in spermatogenesis, however, the mechanisms for sperm production and morphology is poorly understood. So it is necessary to find and indentify novel genes involved in spermatogenesis and it is important for the human fertility health, the prevention of male sterility and contraception.We have proved that lrguk, a novel gene, which functions in spermatogenesis by ENU mutagenesis lrguk is an uncharacterized gene that is predicted to encode 3 spliced variants, and lrguk-1 is the longest isoform, which consisted of 20 exons. The CàT mutation resulted in the exon 20 of lrguk-1 resulting in conversion of an arginine(R) position 528 to a premature termination codon(R528Stop) and in turn led to the truncation of 293 residues.lrgukKaos/Kaos males were sterile as a consequence of sperm loss, abnormal sperm development and an inability of those sperm to ascend the female reproductive tract following mating. The human equivalent presentation would be classified as oligoasthenoteratozoospermia(OAT). We named this mice line as Kaos because of the chaotic spermatogenesis, whereas, lrgukWT/Kaos males and lrgukKaos/Kaos females had apparently normal fertility.Up to now, this is no any report relating with the function of lrguk, we have some fundamental research on lrguk. RT-PCR result predicted lrguk-1 is a testesspecfic gene, and also expressed in ciliated tissues as brain, ovary, etc. meanwhile, the majority of lrguk-1 is produced by haploid germ cell.We also use specific LRGUK antibody to observe the localization of LRGUK on testes of mice and different stages of spermatids. LRGUK-1 was initially localized to a supra-nuclear region of round spermatid consistent with the location of the developing acrosome, as maturation proceeded was initiated, LRGUK-1 covered the acrosome-acroplaxome region. With the elongating of the spermatids head, LRGUK-l moved distally to a position consistent with the microtubules of the manchette and migrated posteriorly, accumulated at the implantation fossa of the elongating spermatids. At last, with the sperm axoneme extension begins, LRGUK-1 moved to the axoneme consistent with localization of sperm tails.LRGUK may be one part of multimeric protein complex, co-immunoprecipatation was performed to verify the interaction of LRGUK and some known proteins which function on the spermatogenesis. For example, HOOK2, which are adaptor-like proteins involved in loading cargos(including protein complexes and organelles) onto microtubules for transport.Transmission electron microscopy Kaos testes revealed a centriole failed to extend to form the axoneme and “9×2+2” structure is totally messed, which predicted the vital role of LRGUK on axoneme formation.In conclusion, we found a novel gene lrguk involved in spermatogenesis of mice. And study the function of it on spermatogenesis, and verified the localization of LRGUK on different stages of spermatids and confirmed the interaction of LRGUK and HOOK2 and RIM-BP3, and also study it’s role on axoneme formation.Part I lrguk and the Male FertilityObjective:To explore the role of lrguk on fertility and the spermatogenesis by the feature of the homogeneous lrguk mutant mice. To verity lrguk is an important gene involved in spermatogenesis of mice.Methods: PAS stained adult mice(>8 weeks) testes sections, HE stained adult mice epididymis sectioins, HE stained sperms were compared between lrguk-1Kaos/Kaos mice and lrguk-1WT/WT mice. Daily sperm production(DSP) and total epididymis sperm content from adult lrguk-1Kaos/Kaos mice and lrguk-1WT/WT mice(>8 weeks, n=5) were compared. RT-PCR was performed to investigate the expression of lrguk-1 in different tissues(testis, lung, liver, brain, bronchi, and ovary) and the expression in different stages of spermatids(day 0, day7, day14, day18, day20, day30, day60). The difference of the numbers of the different stages of spermatids from adult lrguk-1Kaos/Kaos mice and lrguk-1WT/WT mice(>8 weeks, n=5) were compared. Apoptosis level and γ-H2 AX expression of the testes from adult lrguk-1Kaos/Kaos mice and lrguk-1WT/WT mice(>8 weeks, n=5) were analyzed.Results: 1. Lrguk-1Kaos/Kaos mice is infertile. 2. lrguk-1 is highly expressed in testes and other ciliated tissues as brain and ovary. 3. Few lrguk-1 is produced on day0 of mice and reached its peak at the day 60 when the first wave of spermatogenesis is finished. 4. Testes weight of lrguk-1Kaos/Kaos mice is significantly lower comparing with lrguk-1Kaos/Kaos mice’s(13%, P<0.05), DSPs and total epididymis content of lrguk-1Kaos/Kaos mice is significantly lower comparing with lrguk-1WT/WT mice.(79%, P<0.01�'�81%,P<0.01). 5. Stereology analysis revealed that spermatogonia and elongating spermatids from lrguk-1Kaos/Kaos mice are significantly reduced respectively comparing with lrguk-1WT/WT mice(23.46%, P<0.01�'�33.11%,P<0.01) and lots of retained elongating spermatids appears in the stage IX tubules from lrguk-1Kaos/Kaos mice.6. PAS staining of testes of lrguk-1Kaos/Kaos mice revealed almost normal testes structures and the reduction of spermatids and abnormal morphology of spermatids.7. HE staining of epididymis sections of lrguk-1Kaos/Kaos mice revealed sloughed immature germ cells and the reduction of sperms. 8. HE staining of sperms revealed the abnormal morphology of sperm heads and absence of sperm tails. 9. Apoptosis level is similar beteen lrguk-1Kaos/Kaos mice and lrguk-1WT/WT mice(>8 weeks, n=5) and the expression of γ-H2 AX is high expressed inConclusions: 1. lrguk has 3 transcripts in mice, and we proved lrguk transcript 1(lrguk-1) is an important gene involved in spermatogenesis in mice. 2. lrguk-1 is a testis-specfic gene and is mainly produced by haploid germ cell. 3. lrguk-1 is mainly expressed in ciliated tissues: testis, brouchi, brain, kidney etc. 4. The point mutation located in the exon 14 of lrguk-1 resulted in spermatogenesis failure: sperm loss, abnormal sperm development and an inability of those sperm to ascend the female reproductive tract following mating.Part II The Function of LRGUK involved in Sperm Head FormationObjective: To study the localization of LRGUK in testes by specific LRGUK antibody, and to predict the role of LRGUK in spermatogenesis by the localization of LRGUK and the ultrastructure in dfferent stages of spermatids, the verification of the interaction of LRGUK and a manchette associated protein RIM-BP3, to illustrate the role and molecular mechanism of LRGUK-1 in spermatogenesis.Methods:STAPUT methods was performed to achieve different stages of spermatids from adult lrguk-1Kaos/Kaos mice and lrguk-1WT/WT mice, and colocalize LRGUK with the marker of acrosome(PNA), the marker of the manchtte(α-tubulin) and the marker of the axoneme(acetylated tubulin). Electron microscope was performed to compare the difference of the ultrastructure of the testes and acrosome development,, manchette, perinuclear ring, basal body and centriole appendages from adult lrguk-1Kaos/Kaos mice and lrguk-1WT/WT mice. Immuno-fluorescence(IF) and GFP-pull down, co-immunoprecipitation(Co-IP) methods were performed to confirm the interaction of RIM-BP3 and LRGUK.Results:1. LRGUK-1 was initially localized to a supra-nuclear region of round spermatid consistent with the location of the developing acrosome, as maturation proceeded was initiated, LRGUK-1 covered the acrosome-acroplaxome region. With the elongating of the spermatids head, LRGUK-l moved distally to a position consistent with the microtubules of the manchette and migrated posteriorly, accumulated at the implantation fossa of the elongating spermatids. At last, with the sperm axoneme extension begins, LRGUK-1 moved to the axoneme consistent with localization of sperm tails.2. The structure of the testes from lrguk-1Kaos/Kaos mice seems normal up to the fragment acrosome appears around the round spermatids,and the electron microscope images revealed that acrosome detached from the nucleus in 10% elongating spermatids, the vesicles form Golgi apparatus seems rouge.3. IF images revealed that the time of appearace and disappearance of the manchette seems normal, and the direction of the moving seems almost nomral, but he structure of the manchette is abnormal: unparallel microtubules, splited microtubules, some of the them appears detached from the nucleus and ultrastructure revealed confirmed those and showed constricted perinuclear ring.4. IF revealed that excess truncated LRGUK protein stacked at the acroplaxome region in the elongating spermatids from lrguk-1Kaos/Kaos mice, and the morphology of the sperm head from lrguk-1Kaos/Kaos mice is odd.5. IF confirmed LRGUK and RIM-BP3 co-localized in the manchette and the interaction between LRGUK and RIMBP3 was confirmed by transfection,GFP-pull down and co-immunoprecipation.Conclusions: 1. LRGUK-1 involved in acrosome and acroplaxome formation.by the transport and fusion of the pro-acrosomal vesicles form Golgi apparatus. LRGUK-1 involved in the manchette formation. 2. 293 amino acids from the C terminal of the LRGUK-1 is required in the protien transport from acroplaxome to manchette. 3. LRGUK functions with the acrosome-acroplaxome-manchette complex. 4. LRGUK-1 is a scaffolding protein, which interacts with RIM-BP3 and functions synergetic with the acrosome-acroplaxome-manchette complex in the formation of the formation of sperm head moulding and protein transport.Part III The Role of LRGUK involved in Axomeme FormationObjective: The ultimate cause of sterility in the Lrguk-1Kaos/Kaos males was the absence of a functional sperm tail. This, the great reduction in sperm tail development evident in testis sections, electron microscope revealed the abnormal structure of centrioles and appendages all point toward LRGUK have a critical role on axoneme growth, to study the mechanisms of LRGUK on axone formation by ARPE-19 cell line, a cilia model.Methods: Light microscope was perfomed to observe the motility of the sperms from adult lrguk-1Kaos/Kaos mice and lrguk-1WT/WT mice, immunohistochemical stained testes from adult lrguk-1Kaos/Kaos mice and lrguk-1WT/WT mice were performed to compare the number and struture of sperm tails. Electron microcope was performed to observe the micro-structure of the centrioles and appendages of the sperm axoneme, sh RNA-lrguk was performed to downregulate the expression of lrguk in ARPE-19 cell line, to study the mechanism of lrguk on cilia growth. And immunofluorescence and co-immunoprecipitation were performed to verify the interaction of LRGUK and HOOK2.Results:1. Light microcope revealed that sperms from Lrguk-1Kaos/Kaos mice lack motility. 2. Immunohistochemical stained testes section revealed deformed sperm tails or absence of sperm tails from lrguk-1Kaos/Kaos mice. 3. Electron microscope images revealed that the delayed or the blocking up axoneme formation from lrguk-1Kaos/Kaos mice. 4. The ultrastructure of axoneme typical “9×2+2” structure, is not observed in lrguk-1Kaos/Kaos mice. The distal appendages strucute seems normal but few of them attached with the membrane, and all morphology of subdistal appendages are malformed enlarged. 5. The downregualation of the expression of lrguk-1 has no signifant impact on ARPE-19 cell line. 6. Consistent with the localization of LRGUK-1, the distribution of HOOK2 in wild-type spermatids is symmetrical within manchettes at the early stage of elongating spermatids, with the manchettes migrated caudally, the distribution of HOOK2 also accumulated at the implantation fossa site of spermatids head. and the interaction between HOOK2 and LRGUK-1 was confirmed by transfection and GFP-pull down and co-immunoprecipation.Conclusions:1. LRGUK-1 plays a key role in the formation and extension of the sperm tails.involved in axoneme formation by participating in the centriole development. 2. LRGUK-1 functionw with SAPs in the microtubules formation needed in the axoneme formation. 3. LRGUK-1 may interacts with HOOK2 involved in intra-manchette transport(IMT) and intra-flagella transport(IFT).4. LRGUK may no apparent impact on primary cilia, but still need further verified.