Mammalian spermatogenesis is nowadays still poorly understood at the molecular level, mainly due to the heterogeneous nature of testes, which contain a high number of different cell types, and to the lack of spermatogenic cell culture systems for in vitro studies. As a consequence, the development and/or application of methodological approaches aiming at the enrichment or purification of specific testicular cell types are of great interest and have been addressed by scientists for at least 4 decades. Among the many applications that flow cytometry (FC) has gained since its invention, analysis and sorting of spermatogenic cell populations represent a promising strategy to efficiently overcome testis heterogeneity drawback. Surprisingly, FC has been only rarely used as a preparative method for downstream gene expression studies in specific spermatogenic stages. This work aims to provide an overview of FC for spermatogenic studies including preparation of testicular single cell suspensions, dyes for DNA staining, and our own experience with rodent testis material.

1.
Allam JP, Fronhoffs F, Fathy A, Novak N, Oltermann I, et al: High percentage of apoptotic spermatozoa in ejaculates from men with chronic genital tract inflammation. Andrologia 40:329–334 (2008).
2.
Almstrup K, Nielsen JE, Hansen MA, Tanaka M, Skakkeaek NE, Leffers H: Analysis of cell-type-specific gene expression during mouse spermatogenesis. Biol Reprod 70:1751–1761 (2004).
3.
Aravindan GR, Ravindranath N, Gopalakrishnan K, Moudgal NR: DNA flow-cytometric analysis of testicular germ cell populations of the bonnet monkey (Macaca radiata) as a function of sexual maturity. J Reprod Fertil 89:397–406 (1990).
4.
Aslam H, Schneiders A, Perret M, Weinbauer GF, Hodges JK: Quantitative assessment of testicular germ cell production and kinematic and morphometric parameters of ejaculated spermatozoa in the grey lemur, Microcebus murinus. Reproduction 123:323–332 (2002).
5.
Bastos H, Lassalle B, Chicheportiche A, Riou L, Testart J, et al: Flow cytometric characterization of viable meiotic and postmeiotic cells by Hoechst 33342 in mouse spermatogenesis. Cytometry A 65A:40–49 (2005).
6.
Blanchard Y, Lavault MT, Quernee D, Le Lannou D, Lobel B, Lescoat D: Preparation of spermatogenic cell populations at specific stages of differentiation in the human. Mol Reprod Dev 30:275–282 (1991).
7.
Blottner S, Schön J, Roelants H: Apoptosis is not the cause of seasonal testicular involution in roe deer. Cell Tissue Res 327:615–624 (2007).
8.
Bucci LR, Brock WA, Johnson TS, Meistrich ML: Isolation and biochemical studies of enriched populations of spermatogonia and early primary spermatocytes from rat testes. Biol Reprod 34:195–206 (1986).
9.
Chaveiro A, Santos P, da Silva FM: Assessment of sperm apoptosis in cryopreserved bull semen after swim-up treatment: a flow cytometric study. Reprod Domest Anim 42:17–21 (2007).
10.
Clausen OP, Abyholm T: Deoxyribonucleic acid flow cytometry of germ cells in the investigation of male infertility. Fertil Steril 34:369–374 (1980).
11.
Clausen OP, Parvinen M, Kirkhus B: Stage-related variations in DNA fluorescence distribution during rat spermatogenic cycle measured by flow cytometry. Cytometry 2:421–425 (1982).
12.
Clermont Y: Cycle of the seminiferous epithelium of the guinea pig. A method for identification of the stages. Fertil Steril 11:563–573 (1960).
13.
Clermont Y: The cycle of the seminiferous epithelium in man. Am J Anat 112:35–51 (1963).
14.
Cordelli ET, Eleuteri P, Leter G, Rescia M, Spano M: Flow cytometry applications in the evaluation of sperm quality: semen analysis, sperm function and DNA integrity. Contraception 72:273–279 (2005).
15.
De Iuliis GN, Thomson LK, Mitchell LA, Finnie JM, Koppers AJ, et al: DNA damage in human spermatozoa is highly correlated with the efficiency of chromatin remodeling and the formation of 8-hydroxy-2′-deoxyguanosine, a marker of oxidative stress. Biol Reprod 81:517–24 (2009).
16.
de Rooij DG: Proliferation and differentiation of spermatogonial stem cells. Reproduction 121:347–354 (2001).
17.
De Sanctis V, Perera D, Katz M, Fortini M, Gamberini MR: Spermatozoal DNA damage in patients with B thalassaemia syndromes. Pediatr Endocrinol Rev 6 Suppl 1:185–189 (2008).
18.
Dobrinski I: Germ cell transplantation. Semin Reprod Med 23:257–264 (2005).
19.
Druart X, Gatti JL, Huet S, Dacheux JL, Humblot P: Hypotonic resistance of boar spermatozoa: sperm subpopulations and relationship with epididymal maturation and fertility. Reproduction 137:205–213 (2009).
20.
Erickson RP: Post-meiotic gene expression. Trends Genet 6:264–268 (1990).
21.
Evenson DP, Melamed MR: Rapid analysis of normal and abnormal cell types in human semen and testis biopsies by flow cytometry. J Histochem Cytochem31:248–253 (1983).
22.
Evenson D, Darzynkiewicz Z, Lost L, Janca F, Ballachey B: Changes in accessibility of DNA to various fluorochromes during spermatogenesis. Cytometry 7:45–53 (1986).
23.
Evenson DP, Kasperson K, Wixon RL: Analysis of sperm DNA fragmentation using flow cytometry and other techniques. Soc Reprod Fertil Suppl 65:93–113 (2007).
24.
Falciatori I, Borsellino G, Haliassos N, Boitani C, Corallini S, et al: Identification and enrichment of spermatogonial stem cells displaying side-population phenotype in immature mouse testis. FASEB J 18:376–378 (2004).
25.
Fujita K, Ohta H, Tsujimura A, Takao T, Miyagawa Y, et al: Transplantation of spermatogonial stem cells isolated from leukemic mice restores fertility without inducing leukemia. J Clin Invest 115:1855–1860 (2005).
26.
Fujita K, Tsujimura A, Miyagawa Y, Kiuchi H, Matsuoka Y, et al: Isolation of germ cells from leukemia and lymphoma cells in a human in vitro model: potential clinical application for restoring human fertility after anticancer therapy. Cancer Res 66:11166–11171 (2006).
27.
Garner DL: Flow cytometric sexing of mammalian sperm. Theriogenology 65:943–957 (2006).
28.
Garrido N, Meseguer M, Remohi J, Pellicer A, Simón C: Flow cytometry in human reproductive biology. Gynecol Endocrinol 16:505–521 (2002).
29.
Geisinger A: Spermatogenesis in mammals: a very peculiar cell differentiation process, in Ivanova LB (ed): Cell Differentiation Research Developments, pp 97–123 (Nova Publishers, New York 2008).
30.
Geisinger A, Dos Santos A, Benavente R, Wettstein R: Identification and characterization of Srsp1, a rat gene differentially expressed during spermatogenesis and coding for a serine stretch-containing protein. Cytogenet Genome Res 98:249–254 (2002).
31.
Geisinger A, Alsheimer M, Baier A, Benavente R, Wettstein R: The mammalian gene pecanex 1 is differentially expressed during spermatogenesis. Biochim Biophys Acta 1728:34–43 (2005).
32.
Gillan L, Evans G, Maxwell WM: Flow cytometric evaluation of sperm parameters in relation to fertility potential. Theriogenology 63:445–457 (2005).
33.
Gledhill BL, Evenson DP, Pinkel D: Flow cytometry and sorting of sperm and male germ cells, in Melamed MR, Lindmo T, Mendelsohn ML (eds): Flow Cytometry and Sorting, 2nd ed, pp 531–551 (Wiley-Liss Inc., New York 1990).
34.
Go VLW, Vernon RG, Fritz IB: Studies on spermatogenesis in rats. I. Application of the sedimentation velocity technique to an investigation of spermatogenesis. Can J Biochem 49:753–760 (1971).
35.
Goodell MA, Brose K, Paradis G, Conner AS, Mulligan RC: Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo. J Exp Med 183:1797–1806 (1996).
36.
Grogan WM, Farnham WF, Sabau JM: DNA analysis and sorting of viable mouse testis cells. J Histochem Cytochem 29:738–746 (1981).
37.
Hamano KI: Sex preselection in bovine by separation of X- and Y-chromosome bearing spermatozoa. J Reprod Dev 53:27–38 (2007).
38.
Hermann BP, Sukhwani M, Simorangkir DR, Chu T, Plant TM, Orwig KE: Molecular dissection of the male germ cell lineage identifies putative spermatogonial stem cells in rhesus macaques. Hum Reprod 24:1704–1716 (2009).
39.
Hittmair A, Rogatsch H, Offner F, Feichtinger H, Ofner D, Mikuz G: Deoxyribonucleic acid flow cytometry and semiquantitative histology of spermatogenesis: a comparative study. Fertil Steril 58:1040–1045 (1992).
40.
Hittmair A, Rogatsch H, Mikuz G, Feichtinger H: Quantification of spermatogenesis by dual-parameter flow cytometry. Fertil Steril 61:746–750 (1994).
41.
Iguchi N, Tobias JW, Hecht NB: Expression profiling reveals meiotic male germ cell mRNAs that are translationally up- and down-regulated. Proc Natl Acad Sci USA 103:7712–7717 (2006).
42.
Jagetia GC, Krishnamurthy H, Jyothi P: Evaluation of cytotoxic effects of different doses of vinblastine on mouse spermatogenesis by flow cytometry. Toxicology 112:227–236 (1996).
43.
Jagetia GC, Jyothi P, Krishnamurthy H: Flow cytometric evaluation of the effect of various doses of vindesine sulphate on mouse spermatogenesis. Reprod Toxicol 11:867–874 (1997).
44.
Janca FC, Jost LK, Evenson DP: Mouse testicular and sperm cell development characterized from birth to adulthood by dual parameter flow cytometry. Biol Reprod 34:613–623 (1986).
45.
Jeyaraj DA, Grossman G, Weaver C, Petrus P: Dynamics of testicular germ cell proliferation in normal mice and transgenic mice overexpressing rat androgen-binding protein: a flow cytometric evaluation. Biol Reprod 66:877–885 (2002).
46.
Jyothi P, Jagetia GC, Krishnamurthy H: Evaluation of teniposide (VM-26)-induced toxicity on mouse spermatogenesis by flow cytometry. Toxicology 163:163–174 (2001).
47.
Kostakopoulos A, Georgoulakis J, Deliveliotis C, Spanakis G, Filippidou A, Tamvakis N: Deoxyribonucleic acid flow cytometry in the assessment of spermatogenesis. J Urol 158:79–81 (1997).
48.
Kotaja N, Kimmins S, Brancorsini S, Hentsch D, Vonesch JL, et al: Preparation, isolation and characterization of stage-specific spermatogenic cells for cellular and molecular analysis. Nat Methods 1:249–254 (2004).
49.
Lam DMK, Furrer R, Bruce WR: The separation, physical characterization, and differentiation kinetics of spermatogonial cells of the mouse. Proc Natl Acad Sci USA 65:192–199 (1970).
50.
Lassalle B, Bastos H, Louis JP, Riou L, Testart J, et al: ‘Side population’ cells in adult mouse testis express Bcrp1 gene and are enriched in spermatogonia and germinal stem cells. Development 131:479–487 (2004).
51.
Leblond CP, Clermont Y: Definition of the stages of the cycle of the seminiferous epithelium in the rat. Ann N Y Acad Sci 55:548–573 (1952).
52.
Lee DR, Kaproth MT, Parks JE: In vitro production of haploid germ cells from fresh or frozen-thawed testicular cells of neonatal bulls. Biol Reprod 65:873–878 (2001).
53.
Malkov M, Fisher Y, Don J: Developmental schedule of the postnatal rat testis determined by flow cytometry. Biol Rep 59:84–92 (1998).
54.
Maratou K, Forster T, Costa Y, Taggart M, Speed RM, et al: Expression profiling of the developing testis in wild-type and Dazl knockout mice. Mol Reprod Dev 67:26–54 (2004).
55.
Marchesi DE, Feng HL, Hershlag A: Current assessment of sperm DNA integrity. Arch Androl 53:239–247 (2007).
56.
Martin RM, Leonhardt H, Cardoso MC: DNA labeling in living cells. Cytometry A 67:45–52 (2005).
57.
Martínez-Pastor F, Fernández-Santos MR, del Olmo E, Domínguez-Rebolledo AE, Esteso MC, et al: Mitochondrial activity and forward scatter vary in necrotic, apoptotic and membrane-intact spermatozoan subpopulations. Reprod Fertil Dev 20:547–556 (2008).
58.
Meistrich ML: Separation of spermatogenic cells and nuclei from rodent testes. Methods Cell Biol 15:15–54 (1977).
59.
Meistrich ML, Bruce WR, Clermont Y: Cellular composition of fractions of mouse testis cells following velocity sedimentation separation. Exp Cell Res 79:213–227 (1973).
60.
Meistrich ML, Lake S, Steinmetz LL, Gledhill BL: Flow cytometry of DNA in mouse sperm and testis nuclei. Mutat Res 49:383–396 (1978).
61.
Meistrich ML, Longtin J, Brock WA, Grimes SR, Mace ML: Purification of rat spermatogenic cells and preliminary biochemical analysis of these cells. Biol Reprod 25:1065–1077 (1981).
62.
Neubauer K, Jewgenow K, Blottner S, Wildt DE, Pukazhenthi BS: Quantity rather than quality in teratospermic males: a histomorphometric and flow cytometric evaluation of spermatogenesis in the domestic cat (Felis catus). Biol Reprod 71:1517–1524 (2004).
63.
Nicholas CR, Xu EY, Banani SF, Hammer RE, Hamra FK, Reijo Pera RA: Characterization of a Dazl-GFP germ cell-specific reporter. Genesis 47:74–84 (2009).
64.
Ojha P, Dhar JD, Dwivedi AK, Singh RL, Gupta G: Rat testicular germ cell type(s) targeted by anti-spermatogenic agents in vivo and their recovery on withdrawal of treatment – a flow cytometric study. Anim Reprod Sci 103:135–148 (2008).
65.
Oskam IC, Ropstad E, Andersen Berg K, Fredriksen B, Larsen S, et al: Testicular germ cell development in relation to 5alpha-androstenone levels in pubertal entire male pigs. Theriogenology 69:967–976 (2008).
66.
Petit JM, Ratinaud MH, Cordelli E, Spano M, Julien R: Mouse testis cell sorting according to DNA and mitochondrial changes during spermatogenesis. Cytometry 19:304–312 (1995).
67.
Rath D, Johnson LA: Application and commercialization of flow cytometrically sex-sorted semen. Reprod Domest Anim 43 Suppl 2:338–346 (2008).
68.
Rodríguez R, Wettstein R: Quantitative study on guinea pig spermatogenesis shows a relative high percentage of early meiotic prophase stages. Anat Rec 278:493–504 (2004).
69.
Rodríguez-Casuriaga R, Geisinger A, López-Carro B, Porro V, Wettstein R, Folle GA: Ultra-fast and optimized method for the preparation of rodent testicular cells for flow cytometric analysis. Biol Proced Online, doi 10.1007/s12575-009-9003-2 (2009).
70.
Romrell LJ, Bellvé AR, Fawcet DW: Separation of mouse spermatogenic cells by sedimentation velocity. Dev Biol 19:119–131 (1976).
71.
Ryu BY, Kubota H, Avarbock MR, Brinster RL: Conservation of spermatogonial stem cell self-renewal signaling between mouse and rat. Proc Natl Acad Sci USA 102:14302–14307 (2005).
72.
Sharpe JC, Evans KM: Advances in flow cytometry for sperm sexing. Theriogenology 71:4–10 (2009).
73.
Shima JE, McLean DJ, McCarrey JR, Griswold MD: The murine testicular transcriptome: characterizing gene expression in the testis during the progression of spermatogenesis. Biol Reprod 71:319–330 (2004).
74.
Shinohara T, Orwig KE, Avarbock MR, Brinster RL: Spermatogonial stem cell enrichment by multiparameter selection of mouse testis cells. Proc Natl Acad Sci USA 97:8346–8351 (2000).
75.
Sofikitis N, Pappas E, Kawatani A, Baltogiannis D, Loutradis D, et al: Efforts to create an artificial testis: culture systems of male germ cells under biochemical conditions resembling the seminiferous tubular biochemical environment. Hum Reprod Update 1:229–259 (2005).
76.
Spanò M, Evenson DP: Flow cytometric analysis for reproductive biology. Biol Cell 78:53–62 (1993).
77.
Suter L, Koch E, Bechter R, Bobadilla M: Three-parameter flow cytometric analysis of rat spermatogenesis. Cytometry 27:161–168 (1997).
78.
Tanaka K, Tamura H, Tanaka H, Katoh M, Futamata Y, et al: Spermatogonia-dependent expression of testicular genes in mice. Dev Biol 246:466–479 (2002).
79.
Thomas KH, Wilkie TM, Tomashefsky P, Bellvé AR, Simon MI: Differential gene expression during spermatogenesis. Biol Reprod 41:729–739 (1989).
80.
Van der Wee KS, Johnson EW, Dirami G, Dym TM, Hofmann MC: Immunomagnetic isolation and long-term culture of mouse type A spermatogonia. J Androl 22:696–704 (2001).
81.
Van Kroonenburgh MJ, Beck JL, Scholtz JW, Hacker-Klom U, Herman CJ: DNA analysis and sorting of rat testis cells using two parameter flow cytometry. Cytometry 6:321–326 (1985).
82.
Vasco C, Zuccotti M, Redi CA, Garagna S: Identification, isolation, and RT-PCR analysis of single stage-specific spermatogenetic cells obtained from portions of seminiferous tubules classified by transillumination microscopy. Mol Reprod Dev 76:1173–1177 (2009).
83.
Vazquez JM, Parrilla I, Gil MA, Cuello C, Caballero I, et al: Improving the efficiency of insemination with sex-sorted spermatozoa. Reprod Domest Anim 43 Suppl 4:1–8 (2008).
84.
Vigodner M, Lewin LM, Shochat L, Mittelman L, Golan R: Meiosis in the golden hamster: a confocal microscopy and flow cytometric analysis. Mol Reprod Dev 64:86–95 (2003).
85.
Vigodner M, Lewy H, Lewin LM, Shochat L, Golan R: Evidence for biological rhythm in spermatogenesis in the pubertal hamster (Mesocricetus auratus): a flow cytometric study. Life Sci 74:1119–1126 (2004).
86.
Vilardell J, Coll MD, Querol E, Egozcue J: Histone electrophoretic pattern in the characterization of synaptonemal complexes. Cell Mol Biol 35:201–214 (1989).
87.
Wang Y, Song W, Li S, Guan X, Miao S, et al: GC-1 mRHBDD1 knockdown spermatogonia cells lose their spermatogenic capacity in mouse seminiferous tubules. BMC Cell Biol10:25 (2009).
88.
Weiss M, Vigier M, Hue D, Perrard-Sapori MH, Marret C, et al: Pre- and postmeiotic expression of male germ cell-specific genes throughout 2-week cocultures of rat germinal and Sertoli cells. Biol Reprod 57:68–76 (1997).
89.
Wistuba J, Schrod A, Greve B, Hodges JK, Aslam H, et al: Organization of seminiferous epithelium in primates: relationship to spermatogenic efficiency, phylogeny, and mating system. Biol Reprod 69:582–591 (2003).
90.
Wrobel G, Primig M: Mammalian male germ cells are fertile ground for expression profiling of sexual reproduction. Reproduction 129:1–7 (2005).
91.
Yano A, Suzuki K, Yoshizaki G: Flow cytometric isolation of testicular germ cells from rainbow trout (Onchorhynchus mykiss) carrying the green fluorescent protein gene driven by trout vasa regulatory regions. Biol Reprod 78:151–158 (2008).
92.
Yoon CY, Hong CM, Cho YY, Chung YH, Min HK, et al: Flow cytometric assessment of ethylene glycol monoethyl ether on spermatogenesis in rats. J Vet Med Sci 65:207–212 (2003).
93.
Zhao H, Traganos F, Dobrucki J, Wlodkowic D, Darzynkiewicz Z: Induction of DNA damage response by the supravital probes of nucleic acids. Cytometry A 75:510–519 (2009).
94.
Zhou KW, Zheng XM, Yang ZW, Zhang L, Chen HD: Overexpression of CIRP may reduce testicular damage induced by cryptorchidism. Clin Invest Med 32:103–111 (2009).
95.
Zhou Q, Li Y, Nie R, Friel P, Mitchell D, et al: Expression of stimulated by retinoic acid gene 8 (Stra8) and maturation of murine gonocytes and spermatogonia induced by retinoic acid in vitro. Biol Reprod 78:537–545 (2008).
Copyright / Drug Dosage / Disclaimer
Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.
You do not currently have access to this content.