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Rattus norvegicus Spermatogenesis Colony-Forming Assays

  • F. Kent HamraEmail author
Protocol
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Part of the Methods in Molecular Biology book series (MIMB, volume 1463)

Abstract

Knowledge gaps persist on signaling pathways and metabolic states in germ cells sufficient to support spermatogenesis independent of a somatic environment. Consequently, methods to culture mammalian stem cells through spermatogenesis in defined systems have not been established. Lack of success at culturing mammalian stem cells through spermatogenesis in defined systems reflects an inability to experimentally recapitulate biochemical events that develop in germ cells during a seminiferous epithelial cycle. Complex germ and somatic cell associations that develop each seminiferous epithelial cycle support such a hypothesis, conceivably explaining why highly pure mammalian spermatogonia have not developed into meiosis, much less through meiosis without somatic cells. Here, we outline an in vitro spermatogenesis colony-forming assay to study how differentiating spermatogonial syncytia develop from rat spermatogonial stem cell lines. Robust spermatogonial differentiation under defined culture conditions will facilitate molecular biology studies on pre-meiotic steps in gamete development, and provide a soma-free bioassay to identify spermatogenic factors that promote meiotic progression in vitro.

Key words

Spermatogenesis Spermatogonial stem cell Germline stem cell Self-renewal Proliferation Differentiation In vitro 

1 Introduction

Spermatozoa are produced within the testes’ seminiferous epithelium by consecutive developmental processes termed spermatogenesis and spermiogenesis [1]. Spermatogonial stem cells maintain spermatogenesis through their unique abilities to self-renew or produce spermatogonial syncytia that differentiate through meiosis to form haploid gametes termed round spermatids [2]. Round spermatids can then undergo the male germline-specific post-meiotic process, spermiogenesis, to differentiate into fully elongated spermatozoa [1].

In mammals, spermatogonial stem cells reside within a population of “A-single” (As) spermatogonia [3, 4, 5]. As spermatogonia divide to renew germline stem cells but can also produce syncytia of early “undifferentiated” spermatogenic progenitors termed A-paired (Apr) and A-aligned (Aal) spermatogonia [5]. In rodents, undifferentiated spermatogonia mitotically arrest during seminiferous epithelial cycle stages VI–VIII, and then transform into “differentiating” type A1 spermatogonia under control of retinoic acid (active vitamin A derivative) and KIT ligand (KITL) [6, 7]. Type A1 spermatogonia re-enter the mitotic cell cycle and give rise to subsequent generations of differentiating spermatogonia (types A2 > A3 > A4 > Int > B) [8], by which time germ cell numbers/syncytium can be amplified >100-fold prior to entering meiosis to form spermatocytes [9].

Rodent spermatogonial stem cells can be maintained for a long term in culture [10], but can only be cultured through meiosis in recipient testes [2, 11], or in organ culture within seminiferous tubules [12, 13]. Chemically defined culture systems that support soma-free spermatocyte maturation and spermatid development from mammalian spermatogonial stem cell lines remain to be established, but will provide controllable methodology to investigate molecular mechanisms that govern pre-meiotic, meiotic, and post-meiotic processes in germ cell development.

Glial cell line-derived neurotrophic factor (Gdnf) and fibroblast growth factor (Fgf) genes encode polypeptides that support proliferation of mouse [14, 15], rat [16, 17], and hamster [18] spermatogonial stem cells in culture, and are essential components in serum-free media that maintain mouse spermatogonial stem cell proliferation for a long-term without somatic cells [19]. In contrast to highly defined culture systems that maintain rodent spermatogonial stem cell proliferation, prominent knowledge gaps exist on signaling pathways in germ cells needed to support additional pre-meiotic, meiotic, or post-meiotic spermatogenic differentiation steps independent of somatic cells .

Recently, two polypeptides, neuregulin-1 (NRG1) and KITL, were reported to robustly support syncytial growth of differentiating spermatogonia without somatic cells by signaling germ cell survival in response to all-trans-retinoic acid (ATRA) [20]. By supplementing a serum-free rat spermatogonial culture medium (SG medium) with ATRA and NRG1 (or KITL), an effective serum-free rat spermatogonial differentiation medium was formulated (SD medium) [20]. This chapter outlines how rat spermatogonial stem cell lines derived and propagated in SG medium are stimulated to undergo pre-meiotic spermatogenic differentiation by culturing in SD medium.

Spermatogonial lines sub-cultured in GDNF-containing medium commonly grow in colonies that form “clumps,” which contain stem spermatogonia based on their ability to regenerate spermatogenesis in recipient testes [14, 15, 16, 17, 18, 21]. Spermatogonial lines cultured in GDNF-containing medium also contain progenitor-like “undifferentiated” type A spermatogonia based on ineffectiveness at regenerating spermatogenesis in recipient testes [14, 22, 23]. Because undifferentiated spermatogonia are prone to syncytia formation when propagated in GDNF-containing medium (Fig. 1a), we find that TEX14+ syncytial clones containing two or more cells are commonly passaged during subculture, which more rapidly develop into larger syncytia upon differentiation (Fig. 1b).
Fig. 1

TEX14 labeling in undifferentiated and differentiating spermatogonial cultures. (a) TEX14 antibody labeling in a DBA2 mouse spermatogonial line sub-cultured on laminin in Shinohara’s original spermatogonial culture medium [14]. Scale, 100 μm. (b) TEX14 antibody labeling in a rat spermatogonial syncytium on laminin that developed in SD medium from a spermatogonial line originally derived in SG medium. Scale, 100 μm

To avoid harsher dissociation treatments needed to generate single spermatogonial suspensions from spermatogonial lines, after passaging, spermatogonia are pre-incubated in a modified SG medium termed As-SG medium for 5–8 days to select against clump and syncytia formation [20]. Pre-incubation in As-SG medium biologically enriches spermatogonial cultures with As spermatogonia prior to initiating differentiation in SD medium. Unlike SG medium, As-SG medium does not contain GDNF (Fig. 2), which reduces spermatogonial syncytial size and heterogeneity within cultures prior to initiating differentiation in SD medium. Shorter pre-incubation times in As-SG medium prior to initiating differentiation in SD medium increase heterogeneity in spermatogonial syncytia size and differentiation state within cultures. Conversely, longer incubation times in SD medium increase mean nuclei number/syncytium/culture and effectively deplete spermatogonia of their ability to regenerate spermatogenesis in recipient testes [20].
Fig. 2

Spermatogonial culture media for in vitro spermatogenesis colony-forming assays. Undifferentiated rat spermatogonial lines are derived and maintained on DR4 MEFs in SG medium (contains GDNF and FGF2). Undifferentiated spermatogonial colonies grow in nonuniform clumps (top image) [14, 17]. Spermatogonia are passaged onto new DR4 MEF feeder layers (shown), or laminin-coated dishes, and cultured in As-SG medium (contains FGF2 and lapatinib) to enrich cultures with As spermatogonia (middle image). Spermatogenic differentiation is induced by culturing spermatogonia in SD medium (contains FGF2, GDNF, ATRA, NRG1, and/or KITL). *GDNF is not an essential component of SD medium, but increases SD medium effectiveness on laminin [20]. **NRG1 and KITL are interchangeable, yet essential SD medium components required for differentiating spermatogonial syncytia to survive on laminin. DR4 MEFs produce NRG1/KITL-like factors, and thus, neither NRG1 nor KITL is required in SD medium to support spermatogonial differentiation on MEFs [20]. Green = tgGCS-EGFP germline-specific transgene [27]. Scale, 100 μm

Rattus norvegicus spermatogenesis colony assays provide flexible, scalable, and highly defined experimental platforms to investigate molecular mechanisms regulating spermatogonial proliferation, differentiation, and degeneration. CRISPR/Cas9-mediated gene targeting in rat spermatogonial lines enables recessive traits controlling spermatogonial fate to be analyzed using in vitro and in vivo spermatogenesis colony-forming assays [24]. Herein, we tailor a spermatogenesis colony-forming assay to study clonal development of differentiating spermatogonial syncytia from cultures enriched with undifferentiated, rat type As spermatogonia (Fig. 2).

2 Materials

The following in vitro spermatogenesis colony-forming assay was established with undifferentiated, rat spermatogonial lines derived on DR4 MEFs (Fig. 3a) [17, 21, 25]. Rat spermatogonial lines are endowed with spermatogonial stem cells that regenerate and maintain spermatogenesis for a long term in recipient rat testes (Fig. 3b, c) [24, 26]. Based on spermatogenic colonies formed/recipient testis/number of germ cells transplanted (~1/65), rat spermatogonial lines represent a most potent source of germline stem cells [17, 20, 21]. Functionally validated rat spermatogonial lines from wild-type rats, or transgenic rats that express germline-specific EGFP [27] and dtTOMATO markers, can be requested from the Hamra lab. Fully functional, genetically modifiable rat spermatogonial lines can also be derived from 23- to 24-day-old Sprague-Dawley rats as detailed in Chapter 12 (Rat Spermatogonial Stem Cell Mediated Gene Transfer), Springer Press edition: “Advanced Protocols for Animal Transgenics” edited by Shirley Pease and Thomas L. Saunders (see Note 1 ) [25].
Fig. 3

Spermatogenic potential of rat spermatogonia sub-cultured in SG medium. (a) (Left) tgGCS-EGFP+ spermatogonial stem cell line (green) d7 after plating onto DR4 MEFs in 96-well plate at 0, 2500 and 5000 cells/well in SG medium. Scale, 200 μm. (Right) EGFP fluorescence units/number of EGFP+ spermatogonia plated/well from cultures in panela.” Cultures were lysed directly in wells and fluorescence units/well measured in a plate reader (lysis buffer: NaCl 100 mM, EDTA 1 mM, 10 % glycerol v/v, 1 % Trion X-100 v/v, 50 mM HEPES pH 8). (b) (Left) Image showing three donor-derived spermatogenic colonies (green) in a recipient rat’s seminiferous tubules d32 after transplanting tgGCS-EGFP+ rat spermatogonia. Scale, 200 μm. (Right) Relationship between number of tgGCS-EGFP+ spermatogonia transplanted and donor-derived spermatogenic colonies formed/testis scored d27 after transplantation (±SEM, n = 5 rats, right testis transplanted). Scale, 200 μm. (c) Donor-derived spermatogenesis in rat seminiferous tubules d178 after transplanting tgGCS-EGFP+ rat spermatogonia (green) clonally expanded from an individually picked colony in SG Medium. Scale, 200 μm

2.1 Spermatogenesis Colony-Forming Assays

  1. 1.

    Laboratory should be equipped with staff and basic equipment to conduct eukaryotic cell culture under sterile conditions: Eppendorf pipets and tips, serological pipets, 0.2 μm filters, swinging bucket table-top centrifuge, media water bath at 34–35 °C, hemocytometer, fluorescence/phase-contrast microscopes, CO2-supplied cell culture incubators set at 36–37 °C, and dedicated refrigerators and freezers for storing reagents.

     
  2. 2.

    Certified biosafety cabinet in clean, low-traffic environment dedicated to conducting eukaryotic cell culture under sterile conditions .

     
  3. 3.

    Rat spermatogonial stem cell line: request frozen stocks of functionally validated spermatogonial lines from F.K. Hamra. Alternatively, isolate undifferentiated rat As and Apr spermatogonia fresh, or derive primary rat spermatogonial lines as outlined (see Note 1 ) [25].

     
  4. 4.

    Costar Clear TC-Treated Microplates, Individually Wrapped, Sterile 6 well plate, 12 well plate, 24 well plate, 48 well plate or 96 well plate (Corning, Inc.) (see Note 2 ).

     
  5. 5.

    Dulbecco’s modified Eagle’s medium: Ham’s F12 medium 1:1 (DHF12): (Sigma, Inc.).

     
  6. 6.

    Antibiotic–antimycotic solution (100×): 10,000 U/mL Penicillin G sodium (U/v), 10,000 μg/mL streptomycin sulfate (w/v), and 25 μg/mL amphotericin B (w/v) (Life Technologies Inc.).

     
  7. 7.

    DHF12 solution, Dulbecco’s modified Eagle’s medium: Ham’s F12 medium 1:1, 1 % antibiotic–antimycotic solution (v/v).

     
  8. 8.

    1 mg/ml Laminin solution (Sigma, Inc.).

     
  9. 9.

    Parafilm M (Bemis, Inc.).

     
  10. 10.

    Heat-inactivated fetal bovine serum (FBS) (Tissue Culture Biologicals, Inc.) .

     
  11. 11.

    Dulbecco’s modified Eagle’s medium-high glucose (DMEM-high glucose) (Sigma, Inc.).

     
  12. 12.

    MEF medium: Dulbecco’s modified Eagle’s medium, 1.5 g/l sodium bicarbonate (v/v), and 15 % heat-inactivated FBS (v/v).

     
  13. 13.

    DR4 Mouse Embryonic Fibroblasts (MEFs) (ATCC).

     
  14. 14.

    Irradiator source to mitotically arrest DR4 MEFs (optional: see Note 3 ).

     
  15. 15.

    Recovery Cell Culture Freezing Medium (Life Technologies, Inc.).

     
  16. 16.

    T225 Flasks Angled Neck (Corning, Inc.).

     
  17. 17.

    Gelatin from Porcine Skin-Type A (Sigma, Inc.).

     
  18. 18.

    0.1 % Gelatin solution: Dissolve gelatin in ultrapure laboratory-grade water (1 g gelatin/L) and autoclave on liquid cycle. Store stock solution at 4 °C up to 2 months; filter-sterile before each use.

     
  19. 19.

    Dulbecco’s phosphate-buffered saline (PBS) (Sigma, Inc.).

     
  20. 20.

    B-27 Supplement minus vitamin A (50×) (Life Technologies, Inc.).

     
  21. 21.

    l-Glutamine (Life Technologies, Inc.).

     
  22. 22.

    Recombinant FGF2 (Sigma, Inc.) .

     
  23. 23.

    Recombinant GDNF (R&D Systems, Inc).

     
  24. 24.

    Recombinant NRG1β1, T176-K246 (R&D Systems, Inc.).

     
  25. 25.

    Recombinant KITL (R&D Systems, Inc.).

     
  26. 26.

    All-trans-retinoic acid (Sigma, Inc.).

     
  27. 27.

    Lapatinib (LC Laboratories, Inc.).

     
  28. 28.

    2-Mercaptoethanol.

     
  29. 29.

    Protease-, nuclease-, and fatty acid-free bovine serum albumin (Calbiochem, Inc.).

     
  30. 30.

    0.1 M Sodium phosphate buffer, pH 7.2.

     
  31. 31.

    4 % Paraformaldehyde solution, pH 7.2: 10 ml 16 % Paraformaldehyde (Thermo Scientific, Inc.), 30 ml sodium phosphate solution, pH 7.2 (v/v).

     
  32. 32.

    Methanol (Pharmco-AAPER, Inc.).

     
  33. 33.

    Germ cell marker antibodies: Rabbit anti-rat DAZL IgG [28]; rabbit anti-rat TEX14 IgG [29] (request primary antibody stocks to DAZL and TEX14 from F.K. Hamra).

     
  34. 34.

    Spermatogonial marker antibodies: Mouse anti-ZBTB16 IgG (Active Motif, Inc) and/or mouse anti-SALL4 IgG (clone 6E3; Abnova).

     
  35. 35.

    Secondary antibodies: Highly cross-absorbed AlexaFluor 595-conjugated, goat anti-mouse IgG (Life Technologies), AlexaFluor 488-conjugated, goat anti-rabbit IgG (Life Technologies).

     
  36. 36.

    Hoechst 33342 dye.

     
  37. 37.

    Roche Western Blocking Reagent (Roche Applied Science; distributed by Sigma, Inc.).

     
  38. 38.

    Triton X-100.

     

3 Methods

Steps are outlined to conduct in vitro spermatogenesis colony-forming assays initiated by seeding rat spermatogonial stem cell lines onto laminin matrix (Subheading 3.1) or DR4 MEF feeder layers (Subheading 3.2) in As-SG medium for a pre-incubation period prior to conditionally driving spermatogenic differentiation using SD medium (Fig. 2). Spermatogenesis colony-forming assays conducted on laminin in SD medium provide controllable conditions to study molecular mechanisms critical for spermatogonial differentiation (Fig. 4a). Optionally, MEFs produce paracrine factors that positively impact the spermatogenesis colony-forming assays outlined in Subheading 3.6. In vitro spermatogenesis colony-forming assays on DR4 MEFs or other feeder cell types provide a workable platform to discover/investigate factors that regulate spermatogonial biology. Spermatogenic factor effects can then be probed in more detail without somatic cells on an extracellular matrix, such as a laminin matrix (Fig. 4b) [20, 29, 30]. CRISPR/Cas9-mediated gene editing technology can be combined with in vitro spermatogenesis colony-forming assays to study how genes regulate stem, progenitor, and differentiating spermatogonia fate (Fig. 5) [24].
Fig. 4

Analyzing spermatogonial syncytia development in culture. (a) Spermatogonial syncytia at the 2-cell, 4-cell, and 8-cell steps in development after culturing for 6 days on laminin in SD medium containing 5 nM KITL [20]. Spermatogonia were co-labeled with an antibody to rat TEX14 (green; top) and the nuclear dye, Hoechst 33342 (blue; bottom). The rat TEX14 IgG selectively labels germ cell cytoplasm and forms concentrated foci at ring canals marking cytoplasmic bridges within a spermatogonial syncytium. (b) Spermatogonial syncytia containing 4–16 cells scored/well (0.96 cm2) by counting relative numbers of TEX14+ foci and Hoechst 33342+ nuclei/syncytium (see Note 19 ). Cultures were scored after 6 days in SD media supplemented with respective ligands at 5 nM in place of KITL and NRG1 (±SEM, triplicate wells/condition). BMP bone morphogenetic protein, PDGF platelet-derived growth factor . Scale, 200 μm

Fig. 5

Colony-forming assays to study genetic effects on spermatogonial fate. Clonal dominance assays in SG medium and differentiation assays in SD medium can be used to identify and measure gene mutation effects on spermatogonial fate. *Biallelic mutation frequency/cell/culture catalyzed by CRISPR/Cas9 depends on targeting construct and gene delivery efficiencies [24, 25]. **Phenotypes that disrupt spermatogonial viability or proliferation in SG medium preclude the ability to clonally enrich for targeted germline mutations. (Bottom images) Clonally expanded wild-type and Erbb3 knockout spermatogonial lines on laminin after culturing 7 days in As-SG medium and 6 days in SD medium. ***Mutant germline analyses on DR MEFs and/or in recipient testes can also be conducted to investigate spermatogonial phenotypes identified on laminin [20]. Scale, 100 μm

3.1 Preparing Laminin-Coated Culture Dishes

Prepare laminin-coated dishes the day before plating spermatogonia to initiate colony-forming assays (Subheading 3.6).
  1. 1.

    Prepare frozen stocks of laminin to avoid multiple freeze–thaws. Vials containing 1 mg/ml laminin solution are received frozen from the manufacturer. To make frozen stocks, thaw one vial of mouse laminin on ice (requires 1–2 h). Once thawed, make ~six 150 μl laminin solution aliquots in sterile microfuge tubes on ice. Store the laminin stocks at −80 °C for up to 1 year.

     
  2. 2.

    The day prior to isolating or passaging spermatogonia for colony-forming assays, coat wells in a sterile 48-well culture dish with laminin (~5.9 μg/cm2). To prepare, thaw one 150 μl aliquot from the 1 mg/ml laminin stocks on ice (requires ~30 min) and dilute the entire aliquot volume into a sterile 50 ml tube containing 8 ml DHF12 solution. Slowly swirl or gently rock the tube by hand to mix the contents.

     
  3. 3.

    Add 0.3 ml of the diluted laminin solution/well into the center 24 wells of a 48-well plastic culture dish (0.95 cm2 wells) .

     
  4. 4.

    To reduce evaporation from laminin-coated wells, add 0.4 ml PBS to the remaining 24 uncoated, “outside” wells. Additionally, wrap the dish with a Parafilm strip and store overnight (16–24 h) at 4 °C.

     
  5. 5.

    The next day, equilibrate the dish to room temperature (22–24 °C) within a biosafety cabinet, discard laminin solution from the center 24 wells, wash each laminin-coated well once with 0.4 ml DHF12 solution, and immediately proceed to steps in Subheading 3.6.

     

Coating 24 wells with laminin will be sufficient to conduct colony-forming assays for six or eight test conditions at n = 3 or n = 4 wells/condition, respectively.

3.2 Preparing Mouse Embryonic Fibroblast Feeder Layers

DR4 MEFs are used to maintain rat spermatogonial stem cell lines [25] and can be used in colony-forming assays [20]. MEF feeder layers require ~2 days to prepare before plating spermatogonia to initiate colony-forming assays.
  1. 1.

    Primary stocks of DR4 mouse embryonic fibroblasts (MEFs) are purchased from ATCC, and expanded according to the manufacturer’s protocol after plating into MEF medium at 37 °C/5 % CO2. MEFs are sub-cultured up to four passages following their thawing and initial plating from the manufacturer’s vial (see Note 3 ).

     
  2. 2.

    Following expansion into T225 flasks, secondary stocks of MEFs are harvested according to the manufacturer’s protocol, irradiated (100 Gy) in MEF medium, and then cryo-preserved in liquid nitrogen for future use by freezing in Recovery Cell Culture Freezing Medium according to the manufacturer’s protocol (see Note 4 ).

     
  3. 3.

    Pre-coat tissue culture dishes with a solution of filter-sterilized 0.1 % gelatin for 1 h at room temperature (22–24 °C). Rinse 1× with sterile PBS before plating MEFs.

     
  4. 4.

    Prior to culturing spermatogonia, thaw and plate irradiated DR4 MEFs into gelatin-coated dishes (4.5 × 105 cells/cm2) in MEF medium (0.2–0.3 ml/cm2) for 16–48 h. Rinse 1× with PBS and pre-incubate in SG medium (0.25–0.3 ml/cm2) for an additional 16–48 h. Discard pre-incubation medium and plate spermatogonia onto the MEFs in fresh SG medium (0.3–0.4 ml/cm2), as described in step 1, Subheading 3.6.

     

3.3 Formulating Spermatogonial Culture Media

  1. 1.
    Spermatogonial culture medium (SG medium) is prepared by supplementing Dulbecco’s modified Eagle’s medium:Ham’s F12 medium 1:1 in the following order (see Notes 5 7 ):
    1. (a)

      1× Concentration antibiotic–antimycotic solution (v/v).

       
    2. (b)

      4 mM l-Glutamine (final concentration = 6 mM).

       
    3. (c)

      100 μM 2-Mercaptoethanol.

       
    4. (d)

      1× Concentration of B27 Supplement Minus Vitamin A (v/v).

       
    5. (e)

      6 ng/ml GDNF.

       
    6. (f)

      6 ng/ml FGF2.

       
     
  2. 2.

    Equilibrate to 34–35 °C and filter sterilize.

     

3.4 Formulating Rat A-Single Spermatogonia Culture Media (As-SG Medium)

  1. 1.

    Rat A-Single Spermatogonia Culture Medium (As-SG medium) is prepared identical to that of SG medium, with the exception that A s -SG medium does not contain GDNF (see Notes 8 and 9 ). Optionally, 1 μM lapatinib (ERBB1, 2, 4 inhibitor) can be supplemented into As-SG medium to help reduce syncytia formation .

     

3.5 Formulating Spermatogonial Differentiation Medium (SD Medium)

  1. 1.

    Spermatogonial differentiation medium (SD medium) is prepared identical to that of SG medium, except that the concentration of GDNF is reduced to 2 ng/ml, and SD medium is further supplemented with 3 μM ATRA, 40 ng/ml NRG1β1, and/or 100 ng/ml KITL (see Notes 10 and 11 ).

     

3.6 In Vitro Spermatogenesis Colony-Forming Assay

  1. 1.

    Day 1 (d1), plate ~1 × 103 to 1.5 × 103 rat spermatogonia/well (0.95 cm2) in 0.4 ml As-SG medium/well of a 48-well culture dish (0.95 cm2 wells) coated with laminin (Subheading 3.1) or containing irradiated DR4 MEFs (Subheading 3.2) (see Note 12 ).

     
  2. 2.

    Pre-incubate spermatogonia in As-SG medium at ~36.5 °C, 5 % CO2, for ~6 days (d1–d7) to enrich cultures with As spermatogonia. Change As-SG medium (0.4 ml/well) every 2 days during the pre-incubation period using fresh As-SG medium (i.e., change media on d3, d5, d7) (see Note 13 ).

     
  3. 3.

    On d7, change out culture medium by feeding spermatogonia with SD medium (0.4 ml/well) and incubate for 5–8 days at ~36.5 °C, 5 % CO2, to produce differentiating spermatogonia (see Note 14 ).

     
  4. 4.

    Feed cultures with fresh SD medium every 2 days during the ~1-week differentiation period. Six to eight days in SD medium promotes robust development of spermatogonial syncytia for analysis in colony-forming assays.

     
  5. 5.

    After 6–8 days in SD medium (~d14), remove medium and fix cells directly in 0.4 ml/well ice-cold 4 % paraformaldehyde and 0.1 M sodium phosphate buffer, pH 7.2, for 20–30 min on ice (see Notes 15 17 ).

     
  6. 6.

    Post-fix cells in 0.4 ml/well −20 °C methanol for 5 min on ice.

     
  7. 7.

    Wash fixed cultures 2× with 0.4 ml PBS/well/wash at room temperature (22–24 °C) (see Note 18 ).

     
  8. 8.

    Label spermatogonial cultures to detect desired molecular markers (see Subheading 3.7).

     
  9. 9.

    Score spermatogenic units/well (n = 3–4 wells/test condition/experiment, ±S.E.M.) using a microscope to count spermatogonia/well exhibiting single, paired, or longer syncytial morphologies co-labeled with reagents to detect desired molecular markers (i.e., co-labeling with anti-TEX14 IgG and Hoechst 33342 dye allow individual spermatogonial syncytia to be clearly classified) (Fig. 4) (see Notes 19 and 20 ).

     

3.7 Immunofluorescence Labeling on Spermatogonial Cultures

  1. 1.

    Prepare 1× stock of Roche Western Blocking Reagent in 0.1 M phosphate buffer pH 7.2, 0.01 % Triton X-100 (v/v) (Blocking Reagent).

     
  2. 2.

    Pre-incubate fixed cultures in 0.4 ml Blocking Reagent/well for 1–2 h at room temperature (22–24 °C).

     
  3. 3.

    During the pre-incubation step in Blocking Reagent (step 2), prepare primary antibody solutions by diluting respective antibodies raised to germ cell and/or spermatogonial markers into a required volume of fresh Blocking Reagent: mouse anti-ZBTB14 IgG at 0.2 μg/ml, mouse anti-SALL4 at 0.2 μg/ml, rabbit anti-DAZL IgG at ~5 nM (1/800 dilution) [28], and/or rabbit anti-TEX14 IgG at ~5 nM (1/800 dilution) [30].

     
  4. 4.

    Remove Blocking Reagent from fixed cultures and then add 0.4 ml of respective primary antibody solution/well and incubate overnight (14–24 h) at room temperature (22–24 °C).

     
  5. 5.

    Discard primary antibody solutions and wash cultures three times for 10–30 min per wash with 0.4 ml PBS/well/wash (see Note 18 ).

     
  6. 6.

    During the PBS wash step (step 5) prepare respective AlexaFluor 488-conjugated (green fluorophore) and/or AlexaFluor 594-conjugated (red fluorophore) secondary antibody solutions by diluting to ~5 μg/ml (1/400 dilution from 2 mg/ml manufacture stock) when required for indirect labeling (see Note 21 ). To directly label nuclei with a blue fluorophore, dilute Hoechst 33342 dye (1/2000) directly into the secondary antibody solution.

     
  7. 7.

    Remove final PBS wash and incubate fixed cultures in 0.4 ml/secondary antibody solution/well for 60–90 min at 22–24 °C.

     
  8. 8.

    Discard primary antibody solutions and wash fixed cultures three times for 10–30 min/wash with 0.4 ml PBS/well/wash.

     
  9. 9.

    Add 0.4 ml/PBS to fixed cultures for viewing and storage. Wrap culture plates with a thin strip of Parafilm to minimize evaporation during storage.

     
  10. 10.

    View labeled cultures and score spermatogenic units as described in step 9 of Subheading 3.6.

     

4 Notes

  1. 1.

    If one chooses to derive his or her own rat spermatogonial lines, or utilize freshly isolated laminin-binding As/Apr spermatogonia to initiate colony-forming assays, it should be noted that the dispase solution composition previously utilized in published protocols to digest 22–24-day-old rat seminiferous tubules [21, 25, 28, 31, 32, 33] was modified by the manufacturer in 2015, and is significantly less effective . We recommend the former dispase digest be replaced by a single-step collagenase digest for 15 min, at 34 °C in Dulbecco’s modified Eagle’s medium:Ham’s F12 medium 1:1 (Sigma, Inc.), and 1.2 mg/ml Clostridium histolyticum Collagenase (Sigma; 2.1 units/mg FLGPA) [25]. Filter sterilize.

     
  2. 2.

    The described in vitro spermatogenesis colony-forming assay (Subheading 3.6) is routinely conducted using Costar 48-well plates.

     
  3. 3.

    Irradiating MEFs prevents them from dividing and allows them to form a more stable feeder layer for spermatogonia to grow on. If an irradiator source is not available, or irradiating one’s own MEFs is not desirable, several companies provide pre-irradiated DR4 MEFs, but at an elevated price compared to non-irradiated MEFs. As an alternative to irradiating MEFs, MEFs can be mitotically inactivated before use as feeder layers by a simple pretreatment with mitomycin-C [17, 30].

     
  4. 4.

    It should also be stressed that primary DR4 MEF lots vary in quality between different companies, and lots from the same company can vary. We find ATCC’s DR4 MEFs consistently reliable for culturing rat spermatogonial lines with potent sperm-forming potential [24].

     
  5. 5.

    Prepare 100× stock of 2-mercaptoethanol (=10 mM) fresh by diluting 7.8 μl 2-mercaptoethanol from the manufacturer into 10 ml Dulbecco’s modified Eagle’s medium:Ham’s F12 medium 1:1. Upon regular use, bottles containing 2-mercaptoethanol provided by the manufacturer are replaced with new bottles approximately every 4 months.

     
  6. 6.

    Polypeptide growth factors (GDNF, FGF2) should be reconstituted as instructed by the manufacturer, but using filter-sterilized 0.01 % protease-, nuclease-, and fatty acid-free bovine serum albumin (Calbiochem, Inc.) in PBS (w/v). FGF2 is essential for undifferentiated spermatogonia and DR4 MEF viability in SG medium. GDNF is essential for spermatogonial colony/clump formation on DR4 MEFs in SG medium.

     
  7. 7.

    Higher GDNF and/or FGF2 concentrations can be used to increase the growth rate of some rat spermatogonial lines when cultured on some lots of DR4 MEFs in SG medium. For example, the original SG medium formulation consisted of 20 ng/ml GDNF and 25 ng/ml FGF2 [21].

     
  8. 8.

    As-SG medium is identical to the recently reported SGF-medium [20]. Omitting GDNF from SG medium selects for spermatogonia in the single (As) cell state, and thus selects against syncytial growth of undifferentiated spermatogonial clones /clumps that can complicate colony counts and phenotyping.

     
  9. 9.

    It should be emphasized that after enriching for As spermatogonia by selection in As-SG medium, cultures will still contain a relatively low percent of Apr and Aal spermatogonia.

     
  10. 10.

    SD medium is a modified formulation of the serum-free, SG medium [21]. KITL can be supplemented into SD medium at 100 ng/ml in place of NRG1β1, or in combination with up to 40 ng/ml NRG1β1 [20].

     
  11. 11.

    NRG1β1 and KITL stimulate respective lapatinib-sensitive and lapatinib-insensitive survival pathways in differentiating spermatogonia [20].

     
  12. 12.

    Plating ~103 undifferentiated rat spermatogonia/well in a 48-well plate format (0.95 cm2/well) from a given rat spermatogonial line typically yields 150–300 syncytial colonies containing 4–16 nuclei/syncytium/well after 6 days in SD medium (steps 3 and 4, Subheading 3.6). Plating ~103 spermatogonia/0.95 cm2 yields well-separated syncytia.

     
  13. 13.

    Pre-incubation time in As-SG medium is optional, and can be shortened or omitted prior to steps 3 and 4, Subheading 3.6, to expedite studies on spermatogonial differentiation. Spermatogonial numbers/culture and surface area/culture in SG, As-SG, and/or SD medium can also be scaled to best meet scientific aims requiring other types of cellular and molecular analyses.

     
  14. 14.

    Colony-forming assays in SD medium conducted at 32.5 °C, 34.5 °C, or 36.5 °C yield similar results with respect to differentiating spermatogonial syncytia (ZBTB16, SALL4, DAZL+, TEX14+) development. Spermatogonial syncytia develop slower at reduced temperatures.

     
  15. 15.

    Post-fixation in MeOH promotes adherence to plates and minimizes colony loss during wash steps following primary and secondary antibody labeling.

     
  16. 16.

    Post-fixation in MeOH permeabilizes paraformaldehyde-fixed spermatogonial membranes, which facilitates antibody delivery into fixed spermatogonia to label intracellular antigens. All the antibodies listed in Subheading 2.1 are compatible with post-fixation in MeOH. Post-fixation in MeOH is optional if not compatible with antibody binding to a particular antigen under study because Blocking Reagent contains 0.01 % Triton X-100 detergent. Triton X-100 also permeabilizes 4 % paraformaldehyde-fixed spermatogonial membranes.

     
  17. 17.

    It is not necessary to rinse cultures with PBS before fixation with 4 % paraformaldehyde, or before fixation with methanol .

     
  18. 18.

    PBS washes should be conducted relatively gently, taking care not to add or remove each PBS wash too vigorously, which can wash colonies off the plate and increase experimental error. Minimize time between PBS washes so as not to allow fixed spermatogonial cultures to air-dry. Air-drying increases background fluorescence.

     
  19. 19.

    Scoring various-length syncytia is most conveniently done by co-labeling cytoplasmic bridges and nuclei with the TEX14 IgG and Hoechst 33342 dye, respectively (Fig. 1). Syncytia that have advanced to the 2-, 4-, 8-, 16-, and 32-cell steps in development will be recognized by TEX14+ foci [34] localized within or adjacent to each cytoplasmic bridge separating nuclei comprising a syncytium (Fig. 1b). It should also be noted that irregular-length syncytia are also observed based on the TEX14 labeling profile, primarily due to a lag in some cells within a given syncytium completing metaphase/anaphase at the time of fixation, which is revealed by nuclear labeling with Hoechst 33342 dye.

     
  20. 20.

    As, Apr, and Aal spermatogonia represent “undifferentiated” stem and progenitor spermatogonia, originally classified in the rat [5]. Prior to the discovery of proteins that selectively mark distinct spermatogonial types, undifferentiated spermatogonia were distinguished by less heterochromatin, shorter syncytial length, lower abundance, and unique cell division kinetics during an epithelial cycle compared to differentiating spermatogonia (A1–A4, Int, B) [5]. Antibodies generated to protein markers can now be used to distinguish between undifferentiated and more differentiated spermatogonial types [35].

    In vivo, As and Apr spermatogonia are more refractory to differentiating into type A1 spermatogonia each epithelial cycle compared to Aal spermatogonia [5]. However, smaller populations of As and Apr spermatogonial fractions are subject to differentiation more directly into A1 spermatogonia each epithelial cycle [5, 22], which has been verified by loss of molecular markers for undifferentiated spermatogonia in vivo as they progress through rodent spermatogenic stages IV–VIII [36, 37]. Similarly, retinoic acid in SD medium effectively drives differentiation in As, Apr, and Aal spermatogonia in vitro, as monitored by loss of molecular markers for undifferentiated spermatogonia (ZBTB16, SALL4) (Fig. 5b) [20], and the inability of rat spermatogonial lines to regenerate spermatogenesis in recipient rat testes following culture in SD medium [20]. Thus, As, Apr, and Aal spermatogenic units are experimentally defined relative to molecular markers used to distinguish between undifferentiated and differentiating spermatogonia.

     
  21. 21.

    Labeling cultures with molecular probes prior to fixation (i.e., EdU incorporation, fluorescent transgenes/tags) is compatible with procedures described in Subheadings 3.6 and 3.7.

     

Notes

Acknowledgements

This work was supported by National Institutes of Health grants from the Eunice Kennedy Shriver National Institute of Child Health and Human Development: R01HD053889 and R01HD061575, and the Office of the Director: R24OD011108.

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Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  1. 1.Department of Pharmacology, Cecil H. & Ida Green Center for Reproductive Biology SciencesUniversity of Texas Southwestern Medical CenterDallasUSA

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