PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of jcellbiolHomeThe Rockefeller University PressEditorsContactInstructions for AuthorsThis issue
 
J Cell Biol. 1984 February 1; 98(2): 619–628.
PMCID: PMC2113102

Generation of flagella by cultured mouse spermatids

Abstract

During the short-term culturing of mouse spermatogenic cells, flagella were generated by round spermatids previously lacking tails. Unseparated germ cells were obtained by enzymatic treatments and round spermatids (greater than 90% pure) were purified by unit gravity sedimentation. As determined by Nomarski or phase-contrast microscopy, no cells had flagella immediately after isolation; flagella were first clearly detected after 6 1/2 h of culture in Eagle's minimal essential medium containing 10% fetal bovine serum and 6 mM lactate. After 24 h, approximately 20% of round spermatids had formed flagella. Multinucleated round spermatids often formed multiple flagella, the number never exceeding the number of nuclei per symplast. Round spermatids were the only spermatogenic cells capable of tail formation. Flagella elongation was blocked by 1 microM demecolcine, an inhibitor of tubulin polymerization. Indirect immunofluorescence localized tubulin in the flagella. As seen by scanning electron microscopy, flagella developed as early as 2 h after culture and continued to elongate over the next 20 h, reaching lengths of at least 19 micron. Transmission electron microscopy demonstrated that flagella formed in culture resembled flagella from Golgi-phase round spermatids in situ; the flagella consisted of "9+2" axonemes lacking other accessory structures such as outer dense fibers and the fibrous sheath. As determined by acridine orange staining of the developing acrosomes, all spermatids that formed flagella in culture were Golgi-phase spermatids. By these criteria, the structures are indeed true flagella, corresponding in appearance to what others have described for early mammalian spermatid flagella in situ. We believe this is the first substantiated report of limited in vitro differentiation by isolated mammalian spermatids.

Full Text

The Full Text of this article is available as a PDF (1.3M).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Abe SI. Meiosis of primary spermatocytes and early spermiogenesis in the resultant spermatids in newt, Cynops pyrrhogaster in vitro. Differentiation. 1981;20(1):65–70. [PubMed]
  • Basu SL. Maintenance of isolated testicular tubules of albino rats in organ culture. Indian J Exp Biol. 1973 Jul;11(4):326–327. [PubMed]
  • Bellvé AR, Anderson E, Hanley Bowdoin L. Synthesis and amino acid composition of basic proteins in mammalian sperm nuclei. Dev Biol. 1975 Dec;47(2):349–365. [PubMed]
  • Bellvé AR, Millette CF, Bhatnagar YM, O'Brien DA. Dissociation of the mouse testis and characterization of isolated spermatogenic cells. J Histochem Cytochem. 1977 Jul;25(7):480–494. [PubMed]
  • BISHOP MW, SMILES J. DIFFERENTIATION OF THE ACROSOME IN LIVING MAMMALIAN SPERMATOZOA AND SPERMADTIDS BY FLUORESCENCE MICROSCOPY. J Reprod Fertil. 1963 Oct;6:297–303. [PubMed]
  • Bloodgood RA, May GS. Functional modification of the Chlamydomonas flagellar surface. J Cell Biol. 1982 Apr;93(1):88–96. [PMC free article] [PubMed]
  • Boitani C, Palombi F, Stefanini M. Influence of Sertoli cell products upon the in vitro survival of isolated spermatocytes and spermatids. Cell Biol Int Rep. 1983 May;7(5):383–393. [PubMed]
  • Burns RG. Kinetics of the regeneration of sea-urchin cilia. J Cell Sci. 1973 Jul;13(1):55–67. [PubMed]
  • Byers HR, Fujiwara K, Porter KR. Visualization of microtubules of cells in situ by indirect immunofluorescence. Proc Natl Acad Sci U S A. 1980 Nov;77(11):6657–6661. [PubMed]
  • Clark WH, Jr, Moretti RL, Thomson WW. Histochemical and ultracytochemical studies of the spermatids and sperm of Ascaris lumbricoides var. suum. Biol Reprod. 1972 Oct;7(2):145–159. [PubMed]
  • Cleveland DW, Havercroft JC. Is apparent autoregulatory control of tubulin synthesis nontranscriptionally regulated? J Cell Biol. 1983 Sep;97(3):919–924. [PMC free article] [PubMed]
  • D'Agostino A, Geremia R, Monesi V. Post-meiotic gene activity in spermatogenesis of the mouse. Cell Differ. 1978 Aug;7(4):175–183. [PubMed]
  • Dym M, Fawcett DW. Further observations on the numbers of spermatogonia, spermatocytes, and spermatids connected by intercellular bridges in the mammalian testis. Biol Reprod. 1971 Apr;4(2):195–215. [PubMed]
  • Erickson RP. t-Alleles and the possibility of post-meiotic gene expression during mammalian spermatogenesis. Fed Proc. 1978 Sep;37(11):2517–2521. [PubMed]
  • Erickson RP, Erickson JM, Betlach CJ, Meistrich ML. Further evidence for haploid gene expression during spermatogenesis: heterogeneous, poly(A)- containing RNA is synthesized post-meiotically. J Exp Zool. 1980 Oct;214(1):13–19. [PubMed]
  • Erickson RP, Kramer JM, Rittenhouse J, Salkeld A. Quantitation of mRNAs during mouse spermatogenesis: protamine-like histone and phosphoglycerate kinase-2 mRNAs increase after meiosis. Proc Natl Acad Sci U S A. 1980 Oct;77(10):6086–6090. [PubMed]
  • Ewing LL, Davis JC, Zirkin BR. Regulation of testicular function: a spatial and temporal view. Int Rev Physiol. 1980;22:41–115. [PubMed]
  • Fawcett DW. The mammalian spermatozoon. Dev Biol. 1975 Jun;44(2):394–436. [PubMed]
  • Fujiwara K, Pollard TD. Simultaneous localization of myosin and tubulin in human tissue culture cells by double antibody staining. J Cell Biol. 1978 Apr;77(1):182–195. [PMC free article] [PubMed]
  • Geetha-Habib M, Bouck GB. Synthesis and mobilization of flagellar glycoproteins during regeneration in Euglena. J Cell Biol. 1982 May;93(2):432–441. [PMC free article] [PubMed]
  • Gold B, Stern L, Bradley FM, Hecht NB. Gene expression during mammalian spermatogenesis. II. Evidence for stage-specific differences in mRNA populations. J Exp Zool. 1983 Jan;225(1):123–134. [PubMed]
  • Grimes SR, Jr, Meistrich ML, Platz RD, Hnilica LS. Nuclear protein transitions in rat testis spermatids. Exp Cell Res. 1977 Nov;110(1):31–39. [PubMed]
  • Haneji T, Maekawa M, Nishimune Y. In vitro differentiation of Type A spermatogonia from mouse cryptorchid testes in serum-free media. Biol Reprod. 1983 Jun;28(5):1217–1223. [PubMed]
  • Irons MJ. Synthesis and assembly of connecting-piece proteins as revealed by radioautography. J Ultrastruct Res. 1983 Jan;82(1):27–34. [PubMed]
  • Irons MJ, Clermont Y. Formation of the outer dense fibers during spermiogenesis in the rat. Anat Rec. 1982 Apr;202(4):463–471. [PubMed]
  • Irons MJ, Clermont Y. Kinetics of fibrous sheath formation in the rat spermatid. Am J Anat. 1982 Oct;165(2):121–130. [PubMed]
  • JORDAN RT, KATSH S, DESTACKELBURGN Spermatocytogenesis with possible spermiogenesis of guinea pig testicular cells grown in vitro. Nature. 1961 Dec 16;192:1053–1055. [PubMed]
  • Jutte NH, Grootegoed JA, Rommerts FF, van der Molen HJ. Exogenous lactate is essential for metabolic activities in isolated rat spermatocytes and spermatids. J Reprod Fertil. 1981 Jul;62(2):399–405. [PubMed]
  • Kierszenbaum AL, Tres LL. Structural and transcriptional features of the mouse spermatid genome. J Cell Biol. 1975 May;65(2):258–270. [PMC free article] [PubMed]
  • Kiyotaka Y, Abe SI. Inhibition of second meiotic division and a switching over to flagellar formation in secondary spermatocytes of newt by cycloheximide. Exp Cell Res. 1983 Apr 1;144(2):265–274. [PubMed]
  • Lefebvre PA, Nordstrom SA, Moulder JE, Rosenbaum JL. Flagellar elongation and shortening in Chlamydomonas. IV. Effects of flagellar detachment, regeneration, and resorption on the induction of flagellar protein synthesis. J Cell Biol. 1978 Jul;78(1):8–27. [PMC free article] [PubMed]
  • Meistrich ML, Trostle PK, Frapart M, Erickson RP. Biosynthesis and localization of lactate dehydrogenase X in pachytene spermatocytes and spermatids of mouse testes. Dev Biol. 1977 Oct 15;60(2):428–441. [PubMed]
  • Millette CF, Moulding CT. Cell surface marker proteins during mouse spermatogenesis: two-dimensional electrophoretic analysis. J Cell Sci. 1981 Apr;48:367–382. [PubMed]
  • Mita M, Hall PF. Metabolism of round spermatids from rats: lactate as the preferred substrate. Biol Reprod. 1982 Apr;26(3):445–455. [PubMed]
  • MONESI V. AUTORADIOGRAPHIC EVIDENCE OF A NUCLEAR HISTONE SYNTHESIS DURING MOUSE SPERMIOGENESIS IN THE ABSENCE OF DETECTABLE QUANTITIES OF NUCLEAR RIBONUCLEIC ACID. Exp Cell Res. 1964 Dec;36:683–688. [PubMed]
  • Nakamura M, Hino A, Yasumasu I, Kato J. Stimulation of protein synthesis in round spermatids from rat testes by lactate. J Biochem. 1981 Apr;89(4):1309–1315. [PubMed]
  • Nakamura M, Romrell LJ, Hall PF. The effects of temperature and glucose on protein biosynthesis by immature (round) spermatids from rat testes. J Cell Biol. 1978 Oct;79(1):1–9. [PMC free article] [PubMed]
  • O'Brien DA, Bellvé AR. Protein constituents of the mouse spermatozoon. I. An electrophoretic characterization. Dev Biol. 1980 Mar 15;75(2):386–404. [PubMed]
  • O'Brien DA, Bellvé AR. Protein constituents of the mouse spermatozoon. II. Temporal synthesis during spermatogenesis. Dev Biol. 1980 Mar 15;75(2):405–418. [PubMed]
  • Olson GE, Hamilton DW, Fawcett DW. Isolation and characterization of the fibrous sheath of rat epididymal spermatozoa. Biol Reprod. 1976 Jun;14(5):517–530. [PubMed]
  • Parvinen M. Regulation of the seminiferous epithelium. Endocr Rev. 1982 Fall;3(4):404–417. [PubMed]
  • Platz RD, Grimes SR, Meistrich ML, Hnilica LS. Changes in nuclear proteins of rat testis cells separated by velocity sedimentation. J Biol Chem. 1975 Aug 10;250(15):5791–5800. [PubMed]
  • Romrell LJ, Bellvé AR, Fawcett DW. Separation of mouse spermatogenic cells by sedimentation velocity. A morphological characterization. Dev Biol. 1976 Mar;49(1):119–131. [PubMed]
  • Sluder G. Experimental manipulation of the amount of tubulin available for assembly into the spindle of dividing sea urchin eggs. J Cell Biol. 1976 Jul;70(1):75–85. [PMC free article] [PubMed]
  • Steinberger A. In vitro techniques for the study of spermatogenesis. Methods Enzymol. 1975;39:283–296. [PubMed]
  • Stern L, Gold B, Hecht NB. Gene expression during mammalian spermatogenesis. I. Evidence for stage-specific synthesis of polypeptides in vivo. Biol Reprod. 1983 Mar;28(2):483–496. [PubMed]
  • Stern L, Kleene KC, Gold B, Hecht NB. Gene expression during mammalian spermatogenesis. III. Changes in populations of mRNA during spermiogenesis. Exp Cell Res. 1983 Jan;143(1):247–255. [PubMed]
  • Tang XM, Lalli MF, Clermont Y. A cytochemical study of the Golgi apparatus of the spermatid during spermiogenesis in the rat. Am J Anat. 1982 Apr;163(4):283–294. [PubMed]
  • Tres LL, Kierszenbaum AL. Viability of rat spermatogenic cells in vitro is facilitated by their coculture with Sertoli cells in serum-free hormone-supplemented medium. Proc Natl Acad Sci U S A. 1983 Jun;80(11):3377–3381. [PubMed]
  • Walt H. Motile components in early rat spermatids. Cell Differ. 1981 May;10(3):157–161. [PubMed]
  • Walt H. Motile spermatid flagella during differentiation. J Submicrosc Cytol. 1983 Jan;15(1):73–76. [PubMed]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press