FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W3206806535> ?p ?o ?g. }

• W3206806535 endingPage "101312" @default.
• W3206806535 startingPage "101312" @default.
• W3206806535 abstract "Mammalian spermatogenesis is a highly coordinated process that requires cooperation between specific proteins to coordinate diverse biological functions. For example, mouse Parkin coregulated gene (PACRG) recruits meiosis-expressed gene 1 (MEIG1) to the manchette during normal spermiogenesis. Here we mutated Y68 of MEIG1 using the CRISPR/cas9 system and examined the biological and physiological consequences in mice. All homozygous mutant males examined were completely infertile, and sperm count was dramatically reduced. The few developed sperm were immotile and displayed multiple abnormalities. Histological staining showed impaired spermiogenesis in these mutant mice. Immunofluorescent staining further revealed that this mutant MEIG1 was still present in the cell body of spermatocytes, but also that more MEIG1 accumulated in the acrosome region of round spermatids. The mutant MEIG1 and a cargo protein of the MEIG1/PACRG complex, sperm-associated antigen 16L (SPAG16L), were no longer found to be present in the manchette; however, localization of the PACRG component was not changed in the mutants. These findings demonstrate that Y68 of MEIG1 is a key amino acid required for PACRG to recruit MEIG1 to the manchette to transport cargo proteins during sperm flagella formation. Given that MEIG1 and PACRG are conserved in humans, small molecules that block MEIG1/PACRG interaction are likely ideal targets for the development of male contraconception drugs. Mammalian spermatogenesis is a highly coordinated process that requires cooperation between specific proteins to coordinate diverse biological functions. For example, mouse Parkin coregulated gene (PACRG) recruits meiosis-expressed gene 1 (MEIG1) to the manchette during normal spermiogenesis. Here we mutated Y68 of MEIG1 using the CRISPR/cas9 system and examined the biological and physiological consequences in mice. All homozygous mutant males examined were completely infertile, and sperm count was dramatically reduced. The few developed sperm were immotile and displayed multiple abnormalities. Histological staining showed impaired spermiogenesis in these mutant mice. Immunofluorescent staining further revealed that this mutant MEIG1 was still present in the cell body of spermatocytes, but also that more MEIG1 accumulated in the acrosome region of round spermatids. The mutant MEIG1 and a cargo protein of the MEIG1/PACRG complex, sperm-associated antigen 16L (SPAG16L), were no longer found to be present in the manchette; however, localization of the PACRG component was not changed in the mutants. These findings demonstrate that Y68 of MEIG1 is a key amino acid required for PACRG to recruit MEIG1 to the manchette to transport cargo proteins during sperm flagella formation. Given that MEIG1 and PACRG are conserved in humans, small molecules that block MEIG1/PACRG interaction are likely ideal targets for the development of male contraconception drugs. Mouse Meig1 was originally cloned in a screen for genes essential for meiosis (1Don J. Wolgemuth D.J. Identification and characterization of the regulated pattern of expression of a novel mouse gene, meg1, during the meiotic cell cycle.Cell Growth Differ. 1992; 3: 495-505PubMed Google Scholar). Multiple Meig1 transcripts are present in different tissues encoding the same protein but differing in their 5′- or 3′-UTRs (1Don J. Wolgemuth D.J. Identification and characterization of the regulated pattern of expression of a novel mouse gene, meg1, during the meiotic cell cycle.Cell Growth Differ. 1992; 3: 495-505PubMed Google Scholar, 2Zhang Z. Shen X. Gude D.R. Wilkinson B.M. Justice M.J. Flickinger C.J. Herr J.C. Eddy E.M. Strauss J.F. MEIG1 is essential for spermiogenesis in mice.Proc. Natl. Acad. Sci. U. S. A. 2009; 106: 17055-17060Crossref PubMed Scopus (57) Google Scholar). MEIG1 protein is found in other species, and the amino acid sequences are highly conserved (2Zhang Z. Shen X. Gude D.R. Wilkinson B.M. Justice M.J. Flickinger C.J. Herr J.C. Eddy E.M. Strauss J.F. MEIG1 is essential for spermiogenesis in mice.Proc. Natl. Acad. Sci. U. S. A. 2009; 106: 17055-17060Crossref PubMed Scopus (57) Google Scholar, 3Salzberg Y. Eldar T. Karminsky O.D. Itach S.B. Pietrokovski S. Don J. Meig1 deficiency causes a severe defect in mouse spermatogenesis.Dev. Biol. 2010; 338: 158-167Crossref PubMed Scopus (18) Google Scholar). Even though Meig1 is expressed in multiple tissues, it is most abundantly expressed in tissues rich in ciliated cells. Therefore, it is predicted to be important for cilia formation. In mouse testis, Meig1 message is present in germ cells and Sertoli cells (4Don J. Winer M.A. Wolgemuth D.J. Developmentally regulated expression during gametogenesis of the murine gene meg1 suggests a role in meiosis.Mol. Reprod. Dev. 1994; 38: 16-23Crossref PubMed Scopus (18) Google Scholar, 5Chen-Moses A. Malkov M. Shalom S. Ever L. Don J. A switch in the phosphorylation state of the dimeric form of the Meg1 protein correlates with progression through meiosis in the mouse.Cell Growth Differ. 1997; 8: 711-719PubMed Google Scholar, 6Ever L. Steiner R. Shalom S. Don J. Two alternatively spliced Meig1 messenger RNA species are differentially expressed in the somatic and in the germ-cell compartments of the testis.Cell Growth Differ. 1999; 10: 19-26PubMed Google Scholar, 7Steiner R. Ever L. Don J. MEIG1 localizes to the nucleus and binds to meiotic chromosomes of spermatocytes as they initiate meiosis.Dev. Biol. 1999; 216: 635-645Crossref PubMed Scopus (18) Google Scholar, 8Bouma G.J. Affourtit J.P. Bult C.J. Eicher E.M. Transcriptional profile of mouse pre-granulosa and Sertoli cells isolated from early-differentiated fetal gonads.Gene Expr. Patterns. 2007; 7: 113-123Crossref PubMed Scopus (49) Google Scholar). Global Meig1 knockout mice showed pure male infertility due to impaired spermiogenesis, but no meiosis defect was found (2Zhang Z. Shen X. Gude D.R. Wilkinson B.M. Justice M.J. Flickinger C.J. Herr J.C. Eddy E.M. Strauss J.F. MEIG1 is essential for spermiogenesis in mice.Proc. Natl. Acad. Sci. U. S. A. 2009; 106: 17055-17060Crossref PubMed Scopus (57) Google Scholar, 3Salzberg Y. Eldar T. Karminsky O.D. Itach S.B. Pietrokovski S. Don J. Meig1 deficiency causes a severe defect in mouse spermatogenesis.Dev. Biol. 2010; 338: 158-167Crossref PubMed Scopus (18) Google Scholar). Our laboratory further discovered that MEIG1’s primary function is in germ cells, not in Sertoli cells (9Teves M.E. Jha K.N. Song J. Nagarkatti-Gude D.R. Herr J.C. Foster J.A. Strauss J.F. Zhang Z. Germ cell-specific disruption of the Meig1 gene causes impaired spermiogenesis in mice.Andrology. 2013; 1: 37-46Crossref PubMed Scopus (10) Google Scholar). The mechanism of MEIG1’s function was further studied in our laboratory. MEIG1 is present in cell bodies of spermatocytes and round spermatids, but it is translocated to the manchette in elongating spermatids (10Li W. Tang W. Teves M.E. Zhang Z. Zhang L. Li H. Archer K.J. Peterson D.L. Williams D.C. Strauss J.F. A MEIG1/PACRG complex in the manchette is essential for building the sperm flagella.Development. 2015; 142: 921-930Crossref PubMed Scopus (35) Google Scholar). The manchette is a transient structure only present in elongating spermatids. The timing of manchette development is very precise (11Clermont Y.O.R. Louis Harkey H. Clermont Y. Hermo L. Cell biology of mammalian spermiogenesis.in: Desjardins C Ewing LL Cell & Molecular Biology of the Testis. Oxford University Press, New York, NY1993: 332-376Google Scholar, 12Meistrich M.L. 3–Nuclear morphogenesis during spermiogenesis.in: de Kretser D Molecular Biology of the Male Reproductive System. Academic Press, Cambridge, MA1993: 67-97Crossref Google Scholar). Two major functions of the manchette have been proposed: shaping spermatid heads and sorting structural proteins to the centrosome and the developing sperm tail through intra-manchette transport (IMT) (13Kierszenbaum A.L. Intramanchette transport (IMT): Managing the making of the spermatid head, centrosome, and tail.Mol. Reprod. Dev. 2002; 63: 1-4Crossref PubMed Scopus (149) Google Scholar, 14Kierszenbaum A.L. Tres L.L. The acrosome-acroplaxome-manchette complex and the shaping of the spermatid head.Arch. Histol. Cytol. 2004; 67: 271-284Crossref PubMed Scopus (218) Google Scholar). These proposed functions are supported by the characteristics of its structural proteins and mutant mouse models. The manchette contains molecular motor proteins and proteins used to build sperm tails (15Kierszenbaum A.L. Rivkin E. Tres L.L. The actin-based motor myosin Va is a component of the acroplaxome, an acrosome-nuclear envelope junctional plate, and of manchette-associated vesicles.Cytogenet. Genome Res. 2003; 103: 337-344Crossref PubMed Scopus (65) Google Scholar, 16Hayasaka S. Terada Y. Suzuki K. Murakawa H. Tachibana I. Sankai T. Murakami T. Yaegashi N. Okamura K. Intramanchette transport during primate spermiogenesis: Expression of dynein, myosin Va, motor recruiter myosin Va, VIIa-Rab27a/b interacting protein, and Rab27b in the manchette during human and monkey spermiogenesis.Asian J. Androl. 2008; 10: 561-568Crossref PubMed Scopus (38) Google Scholar). Disruption of motor proteins in male germ cells results in spermiogenesis failure associated with a manchette defect (17Lehti M.S. Kotaja N. Sironen A. KIF3A is essential for sperm tail formation and manchette function.Mol. Cell Endocrinol. 2013; 377: 44-55Crossref PubMed Scopus (79) Google Scholar). Proteins that regulate motor protein function and localization are also present in the manchette, and some of them have been shown to be essential for spermatogenesis (18Yamaguchi N. Takanezawa Y. Koizumi H. Umezu-Goto M. Aoki J. Arai H. Expression of NUDEL in manchette and its implication in spermatogenesis.FEBS Lett. 2004; 566: 71-76Crossref PubMed Scopus (16) Google Scholar, 19Yan W. Assadi A.H. Wynshaw-Boris A. Eichele G. Matzuk M.M. Clark G.D. Previously uncharacterized roles of platelet-activating factor acetylhydrolase 1b complex in mouse spermatogenesis.Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 7189-7194Crossref PubMed Scopus (66) Google Scholar, 20Koizumi H. Yamaguchi N. Hattori M. Ishikawa T.O. Aoki J. Taketo M.M. Inoue K. Arai H. Targeted disruption of intracellular type I platelet activating factor-acetylhydrolase catalytic subunits causes severe impairment in spermatogenesis.J. Biol. Chem. 2003; 278: 12489-12494Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 21Nayernia K. Vauti F. Meinhardt A. Cadenas C. Schweyer S. Meyer B.I. Schwandt I. Chowdhury K. Engel W. Arnold H.H. Inactivation of a testis-specific Lis1 transcript in mice prevents spermatid differentiation and causes male infertility.J. Biol. Chem. 2003; 278: 48377-48385Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 22Tachibana M. Terada Y. Murakawa H. Murakami T. Yaegashi N. Okamura K. Dynamic changes in the cytoskeleton during human spermiogenesis.Fertil. Steril. 2005; 84 Suppl 2: 1241-1248Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, 23Wang W. Zhu J.Q. Yu H.M. Tan F.Q. Yang W.X. KIFC1-like motor protein associates with the cephalopod manchette and participates in sperm nuclear morphogenesis in Octopus tankahkeei.PLoS One. 2010; 5e15616Crossref PubMed Scopus (22) Google Scholar, 24Kierszenbaum A.L. Keratins: Unraveling the coordinated construction of scaffolds in spermatogenic cells.Mol. Reprod. Dev. 2002; 61: 1-2Crossref PubMed Scopus (23) Google Scholar, 25Hamilton L.E. Acteau G. Xu W. Sutovsky P. Oko R. The developmental origin and compartmentalization of glutathione-s-transferase omega 2 isoforms in the perinuclear theca of eutherian spermatozoa.Biol. Reprod. 2017; 97: 612-621Crossref PubMed Scopus (15) Google Scholar, 26Liu L. He Y. Guo K. Zhou L. Li X. Tseng M. Cai L. Lan Z.J. Zhou J. Wang H. Lei Z. Ggnbp2-Null mutation in mice leads to male infertility due to a defect at the spermiogenesis stage.Am. J. Pathol. 2017; 187: 2508-2519Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar, 27Ashman J.B. Hall E.S. Eveleth J. Boekelheide K. Tau, the neuronal heat-stable microtubule-associated protein, is also present in the cross-linked microtubule network of the testicular spermatid manchette.Biol. Reprod. 1992; 46: 120-129Crossref PubMed Scopus (54) Google Scholar, 28Kasioulis I. Syred H.M. Tate P. Finch A. Shaw J. Seawright A. Fuszard M. Botting C.H. Shirran S. Adams I.R. Jackson I.J. van Heyningen V. Yeyati P.L. Kdm3a lysine demethylase is an Hsp90 client required for cytoskeletal rearrangements during spermatogenesis.Mol. Biol. Cell. 2014; 25: 1216-1233Crossref PubMed Scopus (24) Google Scholar, 29Wang R. Kaul A. Sperry A.O. TLRR (lrrc67) interacts with PP1 and is associated with a cytoskeletal complex in the testis.Biol. Cell. 2010; 102: 173-189Crossref PubMed Scopus (23) Google Scholar, 30Liu M. Ru Y. Gu Y. Tang J. Zhang T. Wu J. Yu F. Yuan Y. Xu C. Wang J. Shi H. Disruption of Ssp411 causes impaired sperm head formation and male sterility in mice.Biochim. Biophys. Acta Gen. Subj. 2018; 1862: 660-668Crossref PubMed Scopus (7) Google Scholar, 31Martins L.R. Bung R.K. Koch S. Richter K. Schwarzmüller L. Terhardt D. Kurtulmus B. Niehrs C. Rouhi A. Lohmann I. Pereira G. Fröhling S. Glimm H. Scholl C. Stk33 is required for spermatid differentiation and male fertility in mice.Dev. Biol. 2018; 433: 84-93Crossref PubMed Scopus (9) Google Scholar, 32Abbasi F. Miyata H. Shimada K. Morohoshi A. Nozawa K. Matsumura T. Xu Z. Pratiwi P. Ikawa M. RSPH6A is required for sperm flagellum formation and male fertility in mice.J. Cell Sci. 2018; 131221648Crossref Scopus (38) Google Scholar, 33Huang C.Y. Wang Y.Y. Chen Y.L. Chen M.F. Chiang H.S. Kuo P.L. Lin Y.H. CDC42 Negatively regulates testis-specific SEPT12 polymerization.Int. J. Mol. Sci. 2018; 19: 2627Crossref Scopus (8) Google Scholar, 34Augière C. Lapart J.A. Duteyrat J.L. Cortier E. Maire C. Thomas J. Durand B. salto/CG13164 is required for sperm head morphogenesis in Drosophila.Mol. Biol. Cell. 2019; 30: 636-645Crossref PubMed Scopus (3) Google Scholar, 35Giordano T. Gadadhar S. Bodakuntla S. Straub J. Leboucher S. Martinez G. Chemlali W. Bosc C. Andrieux A. Bieche I. Arnoult C. Geimer S. Janke C. Loss of the deglutamylase CCP5 perturbs multiple steps of spermatogenesis and leads to male infertility.J. Cell Sci. 2019; 132jcs.226951Crossref Scopus (15) Google Scholar, 36Fouquet J. Kann M. Souès S. Melki R. ARP1 in Golgi organisation and attachment of manchette microtubules to the nucleus during mammalian spermatogenesis.J. Cell Sci. 2000; 113: 877-886Crossref PubMed Google Scholar). A yeast two-hybrid screen was conducted using full-length mouse MEIG1 as bait to identify binding partners, and PACRG was identified to be its major binding partner (10Li W. Tang W. Teves M.E. Zhang Z. Zhang L. Li H. Archer K.J. Peterson D.L. Williams D.C. Strauss J.F. A MEIG1/PACRG complex in the manchette is essential for building the sperm flagella.Development. 2015; 142: 921-930Crossref PubMed Scopus (35) Google Scholar, 37Zhang L. Shang X.J. Li H.F. Shi Y.Q. Li W. Teves M.E. Wang Z.Q. Jiang G.F. Song S.Z. Zhang Z.B. Characterization of membrane occupation and recognition nexus repeat containing 3, meiosis expressed gene 1 binding partner, in mouse male germ cells.Asian J. Androl. 2015; 17: 86-93Crossref PubMed Scopus (13) Google Scholar, 38Shi Y. Zhang L. Song S. Teves M.E. Li H. Wang Z. Hess R.A. Jiang G. Zhang Z. The mouse transcription factor-like 5 gene encodes a protein localized in the manchette and centriole of the elongating spermatid.Andrology. 2013; 1: 431-439Crossref PubMed Scopus (12) Google Scholar). Pacrg mutant mice showed a similar phenotype as the Meig1 mutants (39Bennett W.I. Gall A.M. Southard J.L. Sidman R.L. Abnormal spermiogenesis in quaking, a myelin-deficient mutant mouse.Biol. Reprod. 1971; 5: 30-58Crossref PubMed Scopus (102) Google Scholar, 40West A.B. Lockhart P.J. O'Farell C. Farrer M.J. Identification of a novel gene linked to parkin via a bi-directional promoter.J. Mol. Biol. 2003; 326: 11-19Crossref PubMed Scopus (100) Google Scholar, 41Lockhart P.J. O'Farrell C.A. Farrer M.J. It's a double knock-out! the quaking mouse is a spontaneous deletion of parkin and parkin co-regulated gene (PACRG).Mov. Disord. 2004; 19: 101-104Crossref PubMed Scopus (54) Google Scholar, 42Lorenzetti D. Bishop C.E. Justice M.J. Deletion of the Parkin coregulated gene causes male sterility in the quaking(viable) mouse mutant.Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 8402-8407Crossref PubMed Scopus (77) Google Scholar, 43Stephenson S.E.M. Aumann T.D. Taylor J.M. Riseley J.R. Li R. Mann J.R. Tomas D. Lockhart P.J. Generation and characterisation of a parkin-Pacrg knockout mouse line and a Pacrg knockout mouse line.Sci. Rep. 2018; 8: 7528Crossref PubMed Scopus (12) Google Scholar). In germ cells, PACRG protein is not translated until day 28 after birth when germ cells start to elongate, and the translated protein becomes localized in the manchette (10Li W. Tang W. Teves M.E. Zhang Z. Zhang L. Li H. Archer K.J. Peterson D.L. Williams D.C. Strauss J.F. A MEIG1/PACRG complex in the manchette is essential for building the sperm flagella.Development. 2015; 142: 921-930Crossref PubMed Scopus (35) Google Scholar). Using mouse PACRG as bait for a yeast two-hybrid screen, MEIG1 was found to be the major binding partner (10Li W. Tang W. Teves M.E. Zhang Z. Zhang L. Li H. Archer K.J. Peterson D.L. Williams D.C. Strauss J.F. A MEIG1/PACRG complex in the manchette is essential for building the sperm flagella.Development. 2015; 142: 921-930Crossref PubMed Scopus (35) Google Scholar). In elongating spermatids, PACRG determines MEIG1’s localization in the manchette (10Li W. Tang W. Teves M.E. Zhang Z. Zhang L. Li H. Archer K.J. Peterson D.L. Williams D.C. Strauss J.F. A MEIG1/PACRG complex in the manchette is essential for building the sperm flagella.Development. 2015; 142: 921-930Crossref PubMed Scopus (35) Google Scholar). Sperm-associated antigen 16L (SPAG16L), a long isoform translated from the Spag16 gene, is a protein localized in the central apparatus of motile cilia (44Smith E.F. Lefebvre P.A. PF20 gene product contains WD repeats and localizes to the intermicrotubule bridges in Chlamydomonas flagella.Mol. Biol. Cell. 1997; 8: 455-467Crossref PubMed Scopus (84) Google Scholar, 45Zhang Z. Sapiro R. Kapfhamer D. Bucan M. Bray J. Chennathukuzhi V. McNamara P. Curtis A. Zhang M. Blanchette-Mackie E.J. Strauss J.F. A sperm-associated WD repeat protein orthologous to Chlamydomonas PF20 associates with Spag6, the mammalian orthologue of Chlamydomonas PF16.Mol. Cell Biol. 2002; 22: 7993-8004Crossref PubMed Scopus (70) Google Scholar, 46Zhang Z. Kostetskii I. Tang W. Haig-Ladewig L. Sapiro R. Wei Z. Patel A.M. Bennett J. Gerton G.L. Moss S.B. Radice G.L. Strauss J.F. Deficiency of SPAG16L causes male infertility associated with impaired sperm motility.Biol. Reprod. 2006; 74: 751-759Crossref PubMed Scopus (93) Google Scholar). SPAG16L is localized in the manchette of elongating spermatids of wild-type mice. However, it is absent in the manchette of the remaining elongating spermatids of MEIG1 or PACRG-deficient mice (10Li W. Tang W. Teves M.E. Zhang Z. Zhang L. Li H. Archer K.J. Peterson D.L. Williams D.C. Strauss J.F. A MEIG1/PACRG complex in the manchette is essential for building the sperm flagella.Development. 2015; 142: 921-930Crossref PubMed Scopus (35) Google Scholar), indicating that SPAG16L is a cargo of MEIG1/PACRG complex. Thus, MEIG1 and PACRG appear to form a complex in the manchette to transport cargo (SPAG16L) to build sperm flagellum. MEIG1’s structure was resolved by nuclear magnetic resonance (NMR) (47Li W. Walavalkar N.M. Buchwald W.A. Teves M.E. Zhang L. Liu H. Bilinovich S. Peterson D.L. Strauss J.F. Williams D.C. Zhang Z. Dissecting the structural basis of MEIG1 interaction with PACRG.Sci. Rep. 2016; 6: 18278Crossref PubMed Scopus (7) Google Scholar). The shape of MEIG1 resembles a dumbbell, such that associated proteins can bind to either of two opposing concave surfaces or the two convex ends of the dumbbell. Twelve amino acids exposed on the protein surface are believed to mediate interactions between MEIG1 and its binding partners, particularly PACRG (47Li W. Walavalkar N.M. Buchwald W.A. Teves M.E. Zhang L. Liu H. Bilinovich S. Peterson D.L. Strauss J.F. Williams D.C. Zhang Z. Dissecting the structural basis of MEIG1 interaction with PACRG.Sci. Rep. 2016; 6: 18278Crossref PubMed Scopus (7) Google Scholar). We examined the role of the 12 amino acids in MEIG1/PACRG interaction and discovered that the four amino acids located on the same surface, W50, K57, F66, and particularly Y68, mediate interaction between MEIG1 and PACRG (47Li W. Walavalkar N.M. Buchwald W.A. Teves M.E. Zhang L. Liu H. Bilinovich S. Peterson D.L. Strauss J.F. Williams D.C. Zhang Z. Dissecting the structural basis of MEIG1 interaction with PACRG.Sci. Rep. 2016; 6: 18278Crossref PubMed Scopus (7) Google Scholar). We mutated the Y68 amino acid using the CRISPR/cas9 system to study its role in vivo. The single amino acid mutant MEIG1 was still present in the cell bodies of spermatocytes and round spermatids, but was no longer present in the manchette of the remaining elongating spermatids. The MEIG1 mutation caused male mice infertility associated with dramatically reduced sperm counts and abnormal sperm morphology, including short tails, vesicles in the flagella, and different thicknesses along the tails. Most importantly, sperm were immotile. SPAG16L was not present in the manchette in elongating spermatids of the mutant mice. The phenotype was similar to the global Meig1 knockout mice. The study demonstrates that Y68 is a key amino acid for PACRG to recruit MEIG1 to the manchette to form the MEIG1/PACRG complex, which is essential for transporting cargo proteins for sperm formation. We previously discovered that four amino acids, W50, K57, F66, and particularly Y68, mediate interactions between MEIG1 and PACRG in vitro (47Li W. Walavalkar N.M. Buchwald W.A. Teves M.E. Zhang L. Liu H. Bilinovich S. Peterson D.L. Strauss J.F. Williams D.C. Zhang Z. Dissecting the structural basis of MEIG1 interaction with PACRG.Sci. Rep. 2016; 6: 18278Crossref PubMed Scopus (7) Google Scholar). To test if these amino acids, particularly Y68, are important for MEIG1’s function in vivo, we generated a mouse model replacing Y68 with alanine using the CRISPR/cas9 system (Fig. S1A). DNA sequencing of the RT-PCR product revealed that only Y68 amino acid was mutated to A68 in the model (Fig. S1B). Western blot analysis using testicular extracts demonstrated that the mutant MEIG1 protein was expressed in the testis (Fig. 1A). Localization of the mutant MEIG1 was examined by immunofluorescence staining. As reported previously (10Li W. Tang W. Teves M.E. Zhang Z. Zhang L. Li H. Archer K.J. Peterson D.L. Williams D.C. Strauss J.F. A MEIG1/PACRG complex in the manchette is essential for building the sperm flagella.Development. 2015; 142: 921-930Crossref PubMed Scopus (35) Google Scholar), the wild-type MEIG1 was present in cell bodies of spermatocytes and round spermatids, and it was translocated to the manchette of elongating spermatids (Fig. 1Ba–c). The Y68A mutant MEIG1 was still present in cell bodies of spermatocytes and round spermatids. However, it was no longer present in the manchette. Interestingly, the mutant MEIG1 appeared to accumulate in the acrosome as an increased MEIG1 signal was observed here (Fig. 1Bd–f). Homozygous mutant mice did not show any gross abnormalities. To test fertility of these mutant mice, 2 to 3 month-old wild-type mice and homozygous mutant mice were bred with 2 to 3-month-old wild-type mice for more than 2 months. All the control mice, including wild-type and heterozygous males and homozygous mutant females, showed normal fertility. All homozygous mutant males examined were infertile (Fig. 2A). There was no significant difference in testis/body weight between the control and homozygous mutant mice (Fig. 2B). Sperm number, morphology, and motility from the control and homozygous mutant mice were examined (Fig. 2, C–F). The sperm count was dramatically reduced in the mutant mice (Fig. 2, C and D and Movie S1). Sperm from the control mice showed normal morphology (Fig. 2C and Movie S2, left panel). Multiple abnormalities in sperm were observed in the mutant mice, including short tails, vesicles in the flagella, and different thicknesses along the tails (Fig. 2C and Movie S1, right panel), and percentage of abnormal sperm was significantly increased in the mutant mice (Fig. 2E and Movie S1). More than 70% of sperm from the control group were motile and showed progressive motility (Fig. 2F and Movie S2); No sperm were motile in the mutant mice (Fig. 2F and Movie S1). Significantly reduced and nonfunctional sperm suggests impaired spermatogenesis in the mutant mice. To examine the spermatogenesis process in the mutant mice, testes from 4 to 5 month-old control and homozygous mutant mice were collected for HE staining. The control mice showed a normal spermatogenesis process. However, the mutant mice showed impaired spermiogenesis. Elongating spermatids lacking tails or with short tails and deformed heads were frequently observed in the seminiferous tubules of the mutant mice (Fig. 3A). Histology of cauda epididymis of the adult control and the mutant mice was also examined. The control mice had highly concentrated normal sperm in the lumen (Fig. 3Ba). However, the mutant mice had low sperm concentration with multiple abnormalities, as observed in the seminiferous tubules (Fig. 3Bb). To investigate the structural basis for the reduced sperm number and sperm morphology changes, TEM was conducted in testis. In seminiferous tubules of the controls, normal spermiogenesis process was observed, including chromatin condensation, flagella formation, and numbers of elongated spermatids released to the lumen (Fig. 4a). However, in the MEIG1Y68A mutant mice, there were few sperm in the seminiferous tubules, and these developed sperm had abnormal flagellar structure, chromatin condensation, and sperm head shapes. The flagella with abnormal ‘‘9 + 2’’ axoneme arrangement were surrounded by many lysosomes, which seemed to degrade the abnormal sperm. They were also concentrated in Sertoli cells, with evidence of Sertoli cell phagocytosis. What’s more, consistent with the phenotype of MEIG1-deficient mice, flagellar components such as microtubules and outer dense fibers could be detected but were not assembled correctly (Fig. 4, b–h). Testicular PACRG level and its localization in elongating spermatids were examined in the MEIG1Y68A mutant mice. There was no difference in PACRG levels between the control and MEIG1 mutant mice (Fig. 5A). However, co-IP experiment showed that PACRG does not bind with the Y68A mutant MEIG1 (Fig. 5B). We previously discovered that mouse PACRG was present in the manchette of elongating spermatids of wild-type and global Meig1 knockout mice (10Li W. Tang W. Teves M.E. Zhang Z. Zhang L. Li H. Archer K.J. Peterson D.L. Williams D.C. Strauss J.F. A MEIG1/PACRG complex in the manchette is essential for building the sperm flagella.Development. 2015; 142: 921-930Crossref PubMed Scopus (35) Google Scholar). In the MEIG1Y68A mutant mice, like in the control mice (Fig. 5C, top panels), PACRG was still present in the manchette (Fig. 5C, bottom two panels), even though the mutant MEIG1 was not present in the manchette anymore (Fig. 1Bf), supporting our previous conclusion that PACRG is an “upstream protein” of MEIG1. SPAG16L is present in the cytoplasm of spermatocytes and round spermatids and is recruited to the manchette by MEIG1/PACRG complex (10Li W. Tang W. Teves M.E. Zhang Z. Zhang L. Li H. Archer K.J. Peterson D.L. Williams D.C. Strauss J.F. A MEIG1/PACRG complex in the manchette is essential for building the sperm flagella.Development. 2015; 142: 921-930Crossref PubMed Scopus (35) Google Scholar). We therefore examined testicular SPAG16L expression levels and localization in the MEIG1Y68A mutant mice. The testicular SPAG16L expression level was not changed as revealed by Western blot analysis (Fig. 6A). In spermatocytes and round spermatids, SPAG16L was still localized in the cytoplasm of the mutant mice, which is consistent with the localization as seen in the control mice (Fig. 6Ba–d). However, SPAG16L was no longer present in the manchette in elongating spermatids of the mutant mice (Fig. 6Bd). The results further support our conclusion that the manchette localization of SPAG16L is dependent on MEIG1 (10Li W. Tang W. Teves M.E. Zhang Z. Zhang L. Li H. Archer K.J. Peterson D.L. Williams D.C. Strauss J.F. A MEIG1/PACRG complex in the manchette is essential for building the sperm flagella.Development. 2015; 142: 921-930Crossref PubMed Scopus (35) Google Scholar). Our lab previously resolved MEIG1’s structure by NMR. Twelve amino acids exposed on the protein surface are believed to mediate interactions between MEIG1 and its binding partners (47Li W. Walavalkar N.M. Buchwald W.A. Teves M.E. Zhang L. Liu H. Bilinovich S. Peterson D.L. Strauss J.F. Williams D.C. Zhang Z. Dissecting the structural basis of MEIG1 interaction with PACRG.Sci. Rep. 2016; 6: 18278Crossref PubMed Scopus (7) Google Scholar). We further examined the role of the 12 amino acids on MEIG1’s surface in MEIG1/PACRG interaction and discovered that the four amino acids located on the same surface, W50, K57, F66, and especially Y68, are involved in interaction with PACRG (47Li W. Walavalkar N.M. Buchwald W.A. Teves M.E. Zhang L. Liu H. Bilinovich S. Peterson D.L. Strauss J.F. Williams D.C. Zhang Z. Dissecting the structural basis of MEIG1 interaction with PACRG.Sci. Rep. 2016; 6: 18278Crossref PubMed Scopus (7) Google Scholar). In th" @default.
• W3206806535 created "2021-10-25" @default.
• W3206806535 creator A5004565086 @default.
• W3206806535 creator A5008345879 @default.
• W3206806535 creator A5008995033 @default.
• W3206806535 creator A5009194998 @default.
• W3206806535 creator A5014963443 @default.
• W3206806535 creator A5018151019 @default.
• W3206806535 creator A5036111174 @default.
• W3206806535 creator A5057287134 @default.
• W3206806535 creator A5071795410 @default.
• W3206806535 creator A5072759160 @default.
• W3206806535 creator A5089999446 @default.
• W3206806535 creator A5091718254 @default.
• W3206806535 date "2021-11-01" @default.
• W3206806535 modified "2023-10-12" @default.
• W3206806535 title "A single amino acid mutation in the mouse MEIG1 protein disrupts a cargo transport system necessary for sperm formation" @default.
• W3206806535 cites W1829588908 @default.
• W3206806535 cites W1930250676 @default.
• W3206806535 cites W1981128560 @default.
• W3206806535 cites W1986057945 @default.
• W3206806535 cites W1991124166 @default.
• W3206806535 cites W1991486935 @default.
• W3206806535 cites W1993795048 @default.
• W3206806535 cites W1994415651 @default.
• W3206806535 cites W1994517447 @default.
• W3206806535 cites W2001647695 @default.
• W3206806535 cites W2001890704 @default.
• W3206806535 cites W2013846360 @default.
• W3206806535 cites W2018867789 @default.
• W3206806535 cites W2038323244 @default.
• W3206806535 cites W2039169632 @default.
• W3206806535 cites W2039186803 @default.
• W3206806535 cites W2045819499 @default.
• W3206806535 cites W2055403606 @default.
• W3206806535 cites W2069548461 @default.
• W3206806535 cites W2070296671 @default.
• W3206806535 cites W2071688854 @default.
• W3206806535 cites W2074793941 @default.
• W3206806535 cites W2075091688 @default.
• W3206806535 cites W2079919032 @default.
• W3206806535 cites W2093212687 @default.
• W3206806535 cites W2104520045 @default.
• W3206806535 cites W2108946743 @default.
• W3206806535 cites W2120077307 @default.
• W3206806535 cites W2126838167 @default.
• W3206806535 cites W2129383499 @default.
• W3206806535 cites W2147579338 @default.
• W3206806535 cites W2161887112 @default.
• W3206806535 cites W2171540907 @default.
• W3206806535 cites W2171877998 @default.
• W3206806535 cites W2209368117 @default.
• W3206806535 cites W2747275204 @default.
• W3206806535 cites W2763506778 @default.
• W3206806535 cites W2769052114 @default.
• W3206806535 cites W2775598270 @default.
• W3206806535 cites W2806361945 @default.
• W3206806535 cites W2890661872 @default.
• W3206806535 cites W2907980810 @default.
• W3206806535 cites W3126256293 @default.
• W3206806535 cites W3164783857 @default.
• W3206806535 cites W3200516517 @default.
• W3206806535 doi "https://doi.org/10.1016/j.jbc.2021.101312" @default.
• W3206806535 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/8592874" @default.
• W3206806535 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/34673028" @default.
• W3206806535 hasPublicationYear "2021" @default.
• W3206806535 type Work @default.
• W3206806535 sameAs 3206806535 @default.
• W3206806535 citedByCount "7" @default.
• W3206806535 countsByYear W32068065352022 @default.
• W3206806535 countsByYear W32068065352023 @default.
• W3206806535 crossrefType "journal-article" @default.
• W3206806535 hasAuthorship W3206806535A5004565086 @default.
• W3206806535 hasAuthorship W3206806535A5008345879 @default.
• W3206806535 hasAuthorship W3206806535A5008995033 @default.
• W3206806535 hasAuthorship W3206806535A5009194998 @default.
• W3206806535 hasAuthorship W3206806535A5014963443 @default.
• W3206806535 hasAuthorship W3206806535A5018151019 @default.
• W3206806535 hasAuthorship W3206806535A5036111174 @default.
• W3206806535 hasAuthorship W3206806535A5057287134 @default.
• W3206806535 hasAuthorship W3206806535A5071795410 @default.
• W3206806535 hasAuthorship W3206806535A5072759160 @default.
• W3206806535 hasAuthorship W3206806535A5089999446 @default.
• W3206806535 hasAuthorship W3206806535A5091718254 @default.
• W3206806535 hasBestOaLocation W32068065351 @default.
• W3206806535 hasConcept C104317684 @default.
• W3206806535 hasConcept C185592680 @default.
• W3206806535 hasConcept C2781087480 @default.
• W3206806535 hasConcept C501734568 @default.
• W3206806535 hasConcept C515207424 @default.
• W3206806535 hasConcept C54355233 @default.
• W3206806535 hasConcept C86803240 @default.
• W3206806535 hasConcept C95444343 @default.
• W3206806535 hasConceptScore W3206806535C104317684 @default.
• W3206806535 hasConceptScore W3206806535C185592680 @default.
• W3206806535 hasConceptScore W3206806535C2781087480 @default.
• W3206806535 hasConceptScore W3206806535C501734568 @default.
• W3206806535 hasConceptScore W3206806535C515207424 @default.

• next