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Method Article

A Seminiferous Tubule Squash Technique for the Cytological Analysis of Spermatogenesis Using the Mouse Model

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DOI:

10.3791/56453

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February 6th, 2018

 ,  , 
1Department of Biochemistry and Molecular Biology, Johns Hopkins Bloomberg School of Public Health

* These authors contributed equally

In This Article

Summary

The goal of this tubule squash technique is to rapidly assess cytological features of developing mouse spermatocytes while preserving cellular integrity. This method allows for the study of all stages of spermatogenesis, and can be easily implemented alongside other biochemical and molecular biological approaches for the study of mouse meiosis.

Abstract

Meiotic progression in males is a process that requires the concerted action of a number of highly regulated cellular events. Errors occurring during meiosis can lead to infertility, pregnancy loss or genetic defects. Commencing at the onset of puberty and continuing throughout adulthood, continuous semi-synchronous waves of spermatocytes undergo spermatogenesis and ultimately form haploid sperm. The first wave of mouse spermatocytes undergoing meiotic initiation appear at day 10 post-partum (10 dpp) and are released into the lumen of seminiferous tubules as mature sperm at 35 dpp. Therefore, it is advantageous to utilize mice within this developmental time-window in order to obtain highly enriched populations of interest. Analysis of rare cell stages is more difficult in older mice due to the contribution of successive spermatogenic waves, which increase the diversity of the cellular populations within the tubules. The method described here is an easily implemented technique for the cytological evaluation of the cells found within the seminiferous tubules of mice, including spermatogonia, spermatocytes, and spermatids. The tubule squash technique maintains the integrity of isolated male germ cells and allows examination of cellular structures that are not easily visualized with other techniques. To demonstrate the possible applications of this tubule squash technique, spindle assembly was monitored in spermatocytes progressing through the prophase to metaphase I transition (G2/MI transition). In addition, centrosome duplication, meiotic sex chromosome inactivation (MSCI), and chromosome bouquet formation were assessed as examples of the cytological structures that can be observed using this tubule squash method. This technique can be used to pinpoint specific defects during spermatogenesis that are caused by mutation or exogenous perturbation, and thus, contributes to our molecular understanding of spermatogenesis.

Introduction

Meiosis is a complex cellular event in which a single round of DNA replication is followed by two successive rounds of cell division. Several meiosis-specific events must be coordinated during the initial stages of meiosis to ensure accurate chromosome segregation. These events include the completion of homologous recombination, co-orientation of sister kinetochores during the first meiotic division, and the stepwise loss of cohesin complexes to resolve chiasmata between homologs. Precise regulation of these processes is necessary to maintain fertility and to prevent chromosome missegregation events that can lead to genetic developmental disorders and spontaneous miscarriage1.

While the key events of meiosis take place in both males and females, significant temporal and mechanistic differences exist between spermatogenesis and oogenesis2. For example, during female meiosis, prophase I occurs during embryonic development and arrests at the dictyate stage until puberty. In contrast, spermatogenesis commences at puberty and progresses in waves throughout adult life without arrest. The differences between male and female meiosis emphasizes the need to develop methods that are specifically catered towards assessing these processes in both spermatocytes and oocytes. Currently, assessing meiotic progression largely relies on the use of chromatin spreads3,4,5. While chromatin spreads are useful for studying meiotic chromosomes, they fail to preserve cellular integrity, preventing evaluation of cellular structures such as spindle microtubules, centrosomes, the nuclear envelope, and telomere attachments. Live imaging and long-term culturing techniques have greatly advanced our understanding of female meiosis; similar approaches to visualize the entire intact cell, however, are less frequently implemented for the study of spermatogenesis6,7. In order to visualize dynamic events throughout male meiosis, we have adapted established tubule squash techniques to rapidly assess the cytological features of developing mouse spermatocytes8,9. The method described here maintains the integrity of the cell, enabling the study of multiple cellular structures during different stages of spermatogenesis.

This tubule squash technique is a whole cell approach, which allows for the assessment of cellular structures via immunofluorescence microscopy. Common histological approaches to visualize meiotic progression in male mice such as haematoxylin and eosin staining of paraffin embedded testes, and immunofluorescent labeling of cryosections allow for a broad overview of meiotic progression. However, these techniques fail to resolve single cells to the extent necessary for detailed analysis of the events occurring throughout meiosis10,11. Alternative techniques to visualize meiotic processes rely on significant chemiosmotic disruption to the spermatocyte to isolate and fix nuclear materials3,4,5. These chemical treatments hinder the observation of cell types other than primary spermatocytes. A recently described method by Namekawa has enabled the research community to preserve the nuclear architecture of isolated spermatocytes, but requires the use of a cytospin and accessories that may not be readily available to some laboratories4. In contrast, the tubule squash technique only requires equipment that is generally standard in most cell biology laboratories.

The tubule squash method described here can be used to visualize the diverse cell types found within the seminiferous tubule, including sertoli cells, spermatogonia, primary and secondary spermatocytes, and spermatids. By coupling this technique with the near-synchronous first wave of spermatogenesis in juvenile mice, it is possible to obtain enriched populations of spermatogenic cells as they progress through meiosis12. This process permits the detailed analysis of processes throughout spermatogenesis, such as early prophase events, the G2/MI and metaphase to anaphase transitions, and spermiogenesis. Furthermore, tubule squash preparations can be used to visualize cytological features of the chromosomes (e.g. interchromatid domains (ICDs) and kinetochores) and centrosomes (centrioles and pericentriolar material/matrices). The squash method can be readily performed in parallel with other experimental approaches, such as chromatin spreads and protein extraction. In addition, this technique has been successfully modified to deposit living spermatogenic cells on slides for direct visualization13.

The method described here involves a whole cell seminiferous tubule squash technique to analyze the G2/MI transition in wild-type C57BL/6J mice. The cytological features of primary spermatocytes entering the first meiotic division were visualized with immunofluorescence microscopy to observe the meiotic spindle. This versatile technique can be easily modified to visualize other meiotic stages and different cell types. The technique is also amenable to alternative visualization strategies, such as DNA and RNA FISH approaches.

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Protocol

The use of mice was approved by the Institutional Animal Care and Use Committee of Johns Hopkins University. Experiments were performed on juvenile (20 - 26 days post-partum, dpp) C57BL/6J mice, taking advantage of the semi-synchronous first wave of spermatogenesis. However, this technique can also be performed using adult mice.

1. Dissection and isolation of mouse seminiferous tubules

  1. Prepare the fix/lysis solution and antibody dilution buffer (ADB) described in Table 1 and Table 2.
  2. Prepare one 35-mm Petri dish with 3 mL of 1x phosphate buffered saline (1x PBS, pH 7.4), and another 35-mm dish with 2 mL of fix/lysis solution per mouse.
  3. Sacrifice the mouse via cervical dislocation or CO2 asphyxiation and spray the ventral abdomen with 70% ethanol. Open the abdominopelvic cavity using sterile scissors, making a V-shaped opening. Then remove the testes by pulling on the epididymal fat pad using forceps, and avoid disturbing the tunica albuginea. Place the testes in a 35-mm Petri dish containing 1x PBS.
    NOTE: Utilize the first wave of spermatogenesis in order to observe an enriched cell population of interest14. Specific stages of spermatogenesis are enriched at different mouse ages (Table 3).
  4. Remove the testicular tunica albuginea by puncturing the tissue with sharp tipped forceps and collect the loose seminiferous tubules. Transfer the tubules to a new 35-mm Petri dish containing 2 mL of fix/lysis solution.
  5. Incubate the tubules in the fix/lysis solution for 5 min at room temperature.

2. Preparation of seminiferous tubules

  1. Prepare a poly-L-lysine coated glass slide by outlining the edges of the slide with a liquid blocker pen.
  2. Apply 100 µL of fix/lysis solution to the center of the glass slide.
  3. Using sterile forceps and scissors gently tease apart the seminiferous tubules. Cutting out long individual tubule segments approximately 20 mm in length.
    NOTE: To visualize structures that are sensitive to prolonged fixative exposure, such as the centrosomes, Separate the tubules first in the 1x PBS, then directly mince the tubules on the surface of the poly-lysine slides coated in 100 µL of fix-lysis solution.
  4. Transfer five 20 mm long seminiferous tubule segments to the prepared poly-L-lysine coated glass slide containing fix/lysis solution.
  5. Using sterile scissors mince seminiferous tubules into 1.5 to 3.0 mm segments.
  6. Using sterile forceps arrange the tubule segments on the glass slide so that no tissue overlaps, and the tubules are distributed evenly.
    NOTE: The optimal number of tubule segments on a glass slide is between 20 and 40.

3. Squashing of the seminiferous tubules

  1. Transfer the glass slide containing dispersed seminiferous tubule segments onto a benchtop. Remove excess liquid using a laboratory wipe to avoid losing tissue during the squash step.
  2. Apply a coverslip (22 x 60 - 1.5 mm) on top of the glass slide and apply pressure with the heel of the palm for 10 - 20 seconds. It is important to apply enough force to disperse spermatocytes from the seminiferous tubules.
    NOTE: Observe the tubules using a dissection microscope in order to optimize the squashing step so that all tubule segments are disrupted.
  3. Using slide forceps, immediately flash freeze the glass slide in a small dewar of liquid N2 for 15 seconds, or until the liquid ceases to bubble. If immunolabeling immediately, remove the coverslip with a straight edge razor blade, fine tip forceps or 21-gauge needle.
    NOTE: For optimal results, immunolabel the slides immediately (see step 4). However, at this point slides can be preserved at -80 °C for up to two weeks. To maximize the lifetime of proteins and cellular structures, immediately store the slides on dry ice or transfer to a -80 °C freezer, and keep the coverslip on the slide. When immunolabeling stored slides, remove the coverslip by immersing the slides in liquid N2 for 15 seconds, as described in step 3.3.

4. Immunolabeling of mounted seminiferous tubules

  1. Immerse the slide in 1x PBS, and wash three times for 5 min in a 50-mL Coplin jar containing 1x PBS.
    NOTE: Never allow the slides to completely dry during immunolabeling.
  2. Apply 1 mL of antibody dilution buffer onto the glass slide to block for 1 - 2 h in a humidified chamber.
  3. Remove the antibody dilution buffer and apply 100 µL of primary antibody diluted in antibody dilution buffer in a humidified chamber.
    NOTE: For best results, incubate primary antibodies overnight at 4 °C. Use smaller volumes of antibody (e.g. 50 µL) by covering the slide with a coverslip or parafilm. Validate and optimize primary antibodies15.
  4. Rinse slides three times for 5 min in a 50 mL Coplin jar containing 1x PBS.
    NOTE: To enhance washes, agitate Coplin jars by using a small magnetic stir bar and place on a magnetic stir plate at low speed, or place the Coplin jar on a tabletop mixer at low speed.
  5. Apply 100 µL of secondary antibody diluted in antibody dilution buffer for 1 to 1.5 h in a humidified chamber at room temperature. Incubate the slides in a dark box to avoid photo bleaching of fluorophore conjugated antibodies.
    NOTE: Use smaller volumes of antibody (e.g. 50 µL) by covering the slide with a coverslip or parafilm.
  6. Rinse slides two times for 5 min in a 50-mL Coplin jar containing 1x PBS.
  7. Mount the slides in mounting medium containing 4',6-diamidino-2-phenylindole (DAPI, 1.5 µg/mL) and apply coverslips (22 x 60 - 1.5 mm).
  8. Seal the coverslips to the slides with clear nail polish to preserve the slides and prevent the coverslips from moving.

5. Analysis and imaging tubule squash preparations

  1. Use an epifluorescence microscope with automated stage that enables precise Z-axis movement and a high-resolution camera for image capturing. Alternatively, use a laser confocal microscope.
    NOTE: Use any available hardware and software for this step. For example, images displayed in Figure 1 and Figure 2 were captured using a Zeiss Cell Observer Z1 linked to an ORCA-Flash 4.0 CMOS camera and analyzed with the Zeiss ZEN 2012 blue edition image software.
  2. Assess the slides using a 20X objective to determine the quality of the squash preparation. Good quality slides have a monolayer of nuclei that are evenly distributed.
    NOTE: Some sections of the slide may be better than others, it is useful to note the coordinates of where the best regions of the slide can be found for further analysis using higher magnification.
  3. Using higher magnification objective (e.g. 63X or 100X), capture regions of the slide that have a consistent monolayer of nuclei. Once a region is selected, set the upper and lower Z-stacks to ensure that all in-focus light is captured.
    NOTE: The optimal number of Z-stacks captured between the upper and lower limit is generally designated by the image acquisition software and is different for each objective.
  4. Use the image processing software to compile an extended depth focus image. The software combines the in-focus light from each Z-stack, and is essential for optimal imaging of tubule squash preparations.

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Results

Here, we have used the tubule squash method to visualize transitory cell populations undergoing the prophase to metaphase I (G2/MI) transition, which were enriched by harvesting testes from juvenile wild-type mice undergoing the first wave of spermatogenesis (24 dpp). Figure 1 depicts representative images of the various cell stages that can be visualized using the tubule squash method. Enriched populations of metaphase I spermatocytes were visualized using a...

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Discussion

Mice have proven to be a useful model organism for studying the cellular events that govern meiotic progression during spermatogenesis. Further, it is necessary to develop tools catered to the study of spermatogenesis because many events, such as exit from meiotic prophase I, are sexually dimorphic. This protocol describes a seminiferous tubule squash method for visualization and study of different stages of mouse spermatogenesis. This method preserves cellular integrity and thereby allows detailed analyses of nuclear an...

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Disclosures

The authors have nothing to disclose.

Acknowledgements

This work was supported by NIGMS (R01GM11755) to P.W.J. and by a training grant fellowship from the National Cancer Institute (NIH) (CA009110) to S.R.W. and J.H.
 

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
16% Paraformaldehyde AqueousElectron Microscopy Sciences (EMS)15710
10x PBSQuality Biological119-069-161
Triton X-100SigmaT8787
BSASigmaA1470
Horse SerumSigmaH-1270
35mm x 10mm Petri Dish, Sterile, non-treatedCellTreatP886-229638
Poly-L-lysine coated glass slidesSigmaP0425-72EA
Liquid Blocker PenElectron Microscopy Sciences (EMS)71310
Humid BoxEvergreen240-9020-Z10
Wheaton Coplin Glass Staining Dish for 5 or 10 SlidesFisher08-813E
VECTASHIELD Antifade Mounting Medium with DAPIVector LabsH-1200
Microscope Cover Slides (22mmx60mm)Fisher12-544-G
Clear Nail PolishAmazonN/A
Microsopes
NameCompanyCatalog NumberComments
SteREO Discovery.V8Zeiss495015-0001-000 
Observer Z1Zeiss4109431007994000
Zeiss ZEN 2012 blue edition image softwareZeiss
ORCA-Flash 4.0 CMOS cameraHamamatsu
Primary Antibodies
NameCompanyCatalog NumberComments
Mouse anti-SYCP3Santa Cruzsc-745691 in 50
Rabbit anti-SYCP3Fisher (Novus)NB300-2311 in 1000
Goat anti-SCP3Santa Cruzsc-208451 in 50
Human anti-Centromere ProteinAntibodies Incorporated15-2351 in 100
Mouse anti-alpha tubulinSigmaT90261 in 1000
Mouse anti-AIM1BD Biosciences6110821 in 200
Mouse anti-γH2AXThermo FisherMA1-20221 in 500
Mouse anti-CENT3AbnovaH00001070-M011 in 200
Rabbit anti-pericentrinAbcamab44481 in 200
Rabbit anti-REC8Courtesy of  Dr. Karen SchindlerN/A1 in 1000

References

  1. Nagaoka, S. I., Hassold, T. J., Hunt, P. A. Human aneuploidy: mechanisms and new insights into an age-old problem. Nature Rev Genet. 13 (7), 493-504 (2012).
  2. Morelli, M. A., Cohen, P. E. Not all germ cells are created equal: Aspects of sexual dimorphism in mammalian meiosis. Reproduction. 130 (6), 761-781 (2005).
  3. Sun, F., Handel, M. A. Regulation of the meiotic prophase I to metaphase I transition in mouse spermatocytes. Chromosoma. 117 (5), 471-485 (2008).
  4. Namekawa, S. H. Slide preparation method to preserve three-dimensional chromatin architecture of testicular germ cells. J Vis Exp. (83), e50819(2014).
  5. La Salle, S., Sun, F., Handel, M. A. Isolation and short-term culture of mouse spermatocytes for analysis of meiosis. Methods Mol Biol. 558, 279-297 (2009).
  6. Galdon, G., Atala, A., Sadri-Ardekani, H. In vitro spermatogenesis: how far from clinical application? Curr Urol Rep. 17 (7), (2016).
  7. Staub, C. A century of research on mammalian male germ cell meiotic differentiation in vitro. J Androl. 22 (6), 911-926 (2001).
  8. Page, J., Suja, J. A., Santos, J. L., Rufas, J. S. Squash procedure for protein immunolocalization in meiotic cells. Chromosome Res. 6 (8), 639-642 (1998).
  9. Kotaja, N., et al. Preparation, isolation and characterization of stage-specific spermatogenic cells for cellular and molecular analysis. Nat Methods. 1 (3), 249-254 (2004).
  10. Xu, X., Xu, P. A modified cryosection method for mouse testis tissue. Tissue Cell. 33 (2), 208-210 (2001).
  11. Hess, R., Moore, B. Histological methods for evaluation of the testis. Methods Toxicol. 3 (Pt A), 52-85 (1993).
  12. Bellvé, A. R. Purification, culture, and fractionation of spermatogenic cells. Methods Enzymol. 225, 84-113 (1993).
  13. Ventela, S., Toppari, J., Parvinen, M. Intercellular organelle traffic through cytoplasmic bridges in early spermatids of the rat: mechanisms of haploid gene product sharing. Mol Biol Cell. 14 (July), 2768-2780 (2003).
  14. Bellvé, A. R., et al. Dissociation of the mouse testis and characterization of isolated spermatogenic cells. J Histochem Cytochem. 25 (7), 480-494 (1977).
  15. Burry, R. W. Controls for immunocytochemistry: an update. J Histochem Cytochem. 59 (1), 6-12 (2011).
  16. Zickler, D., Kleckner, N. Recombination, pairing, and synapsis of homologs during meiosis. Cold Spring Harb Perspect Biol. 7 (6), 1-26 (2015).
  17. Hunter, N. Meiotic recombination: the essence of heredity. Cold Spring Harb Perspect Biol. 7 (12), 1-36 (2015).
  18. Scherthan, H., et al. Centromere and telomere movements during early meiotic prophase of mouse and man are associated with the onset of pairing. J Cell Biol. 134 (5), 1109-1125 (1996).
  19. Zickler, D., Kleckner, N. The leptotene-zygotene transition of meiosis. Annu Rev Genet. 32, 619-697 (1998).
  20. Scherthan, H. A bouquet makes ends meet. Nat Rev Mol Cell Biol. 2 (8), 621-627 (2001).
  21. Turner, J. Meiotic sex chromosome inactivation. Development. 134 (10), 1823-1831 (2007).
  22. Royo, H., et al. ATR acts stage specifically to regulate multiple aspects of mammalian meiotic silencing. Genes Dev. 27 (13), 1484-1494 (2013).
  23. Turner, J. M., et al. Silencing of unsynapsed meiotic chromosomes in the mouse. Nat Genet. 37 (1), 41-47 (2005).
  24. Marjanović, M., et al. CEP63 deficiency promotes p53-dependent microcephaly and reveals a role for the centrosome in meiotic recombination. Nat Commun. 6, 7676(2016).
  25. Inanç, B., Dodson, H., Morrison, C. G. A Centrosome-autonomous signal that involves centriole disengagement permits centrosome duplication in G2 phase after DNA damage. Mol Biol Cell. 21, 3866-3877 (2010).
  26. Firat-Karalar, E. N., Sante, J., Elliott, S., Stearns, T. Proteomic analysis of mammalian sperm cells identifies new components of the centrosome. J Cell Sci. 127 (Pt 19), 4128-4133 (2014).
  27. Fukuda, N., et al. The transacting factor CBF-A/Hnrnpab binds to the A2RE/RTS element of protamine 2 mRNA and contributes to its translational regulation during mouse spermatogenesis. PLoS Genet. 9 (10), e1003858(2013).
  28. Lahn, B. T., et al. Previously uncharacterized histone acetyltransferases implicated in mammalian spermatogenesis. Proc Natl Acad Sci U S A. 99 (13), 8707-8712 (2002).
  29. Mermoud, J. E., Tassin, A. M., Pehrson, J. R., Brockdorff, N. Centrosomal association of histone macroH2A1.2 in embryonic stem cells and somatic cells. Exp Cell Res. 268 (2), 245-251 (2001).
  30. Shanmugam, M., Hernandez, N. Mitotic functions for SNAP45, a subunit of the small nuclear RNA-activating protein complex SNAPc. J Biol Chem. 283 (21), 14845-14856 (2008).
  31. Ganem, N. J., Godinho, S. A., Pellman, D. A mechanism linking extra centrosomes to chromosomal instability. Nature. 460 (7252), 278-282 (2009).
  32. Kotaja, N., et al. Preparation, isolation and characterization of stage-specific spermatogenic cells for cellular and molecular analysis. Nat Methods. 1 (3), 249-254 (2004).
  33. Amory, J. K., et al. Suppression of spermatogenesis by bisdichloroacetyldiamines is mediated by inhibition of testicular retinoic acid biosynthesis. J Androl. 32 (1), 111-119 (2011).
  34. Hogarth, C. A., et al. Turning a spermatogenic wave into a tsunami: synchronizing murine spermatogenesis using WIN 18,446. Biol Reprod. 88 (2), 40(2013).
  35. Yoshida, S., et al. The first round of mouse spermatogenesis is a distinctive program that lacks the self-renewing spermatogonia stage. Development. 133 (8), 1495-1505 (2006).
  36. Kluin, P. M., Kramer, M. F., Rooij, D. G. Spermatogenesis in the immature mouse proceeds faster than in the adult. Int J Androl. 5 (3), 282-294 (1982).
  37. Rodriguez, I., Ody, C., Araki, K., Garcia, I., Vassalli, P. An early and massive wave of germinal cell apoptosis is required for the development of functional spermatogenesis. EMBO J. 16 (9), 2262-2270 (1997).
  38. Kangasniemi, M., et al. Modulation of basal and FSH dependent cyclic AMP production in rat seminiferous tubules staged by an improved transillumination technique. Anat Rec. 227 (1), 62-76 (1990).
  39. Martianov, I., et al. Late arrest of spermiogenesis and germ cell apoptosis in mice lacking the TBP-like TLF/TRF2 gene. Mol Cell. 7 (3), 509-515 (2001).
  40. Henriksén, K., Parvinen, M. Stage-specific apoptosis of male germ cells in the rat: Mechanisms of cell death studied by supravital squash preparations. Tissue Cell. 30 (6), 692-701 (1998).

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Mouse SpermatogenesisGerm Cell IsolationTubule PreparationFixative Lysis SolutionPoly L Lysine SlideImmunofluorescence StainingEpifluorescence MicroscopyChromosome Bouquet Formation
A Seminiferous Tubule Squash Technique for the Cytological Analysis of Spermatogenesis Using the Mouse Model
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