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Transplantation as a Quantitative Assay to Study Mammalian Male Germline Stem Cells

  • Aileen R. Helsel
  • Jon M. OatleyEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1463)

Abstract

In mammals, the activities of spermatogonial stem cells (SSCs) provide the foundation for continual spermatogenesis throughout a male’s reproductive lifetime. At present, the defining characteristics of SSCs and mechanisms controlling their fate decisions are not well understood. Transplantation is a definitive functional measure of stem cell capacity for male germ cells that can be used as an assay to provide an unequivocal quantification of the SSC content in an experimental cell population. Here, we discuss the procedure for mice and provide protocols for preparing donor germ cell suspensions from testes directly or primary cultures of spermatogonia for transplantation, enriching for SSCs, preparing recipient males, microinjection into recipient testes, and considerations for experimental design.

Key words

Spermatogonial stem cell Transplantation SSC enrichment Mouse 

1 Introduction

In mammals, testes are made up of seminiferous tubules wherein the production of millions of genetically unique spermatozoa occurs daily throughout the reproductive lifetime of a male. The process of sperm production, termed spermatogenesis, occurs in three phases. First, diploid spermatogonia, located in the basal compartment near the basement membrane, undergo mitotic proliferation to expand the population while gaining the competence for initiation of meiotic prophase, thereby yielding primary spermatocytes. Second, spermatocytes proceed through two meiotic divisions to produce haploid spermatids. Third, spermatids undergo drastic morphological changes in a process referred to as spermiogenesis that yields an elongate form, eventually being released into the lumen of the seminiferous tubules.

The activities of the spermatogonial population provide the foundation for robust and continual spermatogenesis [1, 2]. This population is heterogeneous, consisting of undifferentiated and differentiating subtypes (Fig. 1). The undifferentiated population is labeled as type A spermatogonia and can be divided further into stem cell and progenitor subsets. Self-renewal by spermatogonial stem cells (SSCs) maintains a foundational pool from which progenitors arise that divide to bolster the population numbers. A round of spermatogenesis is initiated when a majority of the progenitors transition to a differentiating state under the influence of retinoic acid signaling that occurs at periodic intervals (Fig. 1). Thus, the progenitor spermatogonia are truly a transit-amplifying population. The SSC pool serves as a reservoir from which the next cohort of progenitors arise. Thus, the actions of SSCs are the ground state for continual spermatogenesis.
Fig. 1

Schematic of the heterogeneous undifferentiated spermatogonial population in mammalian testes . The stem cell (SSC) pool is maintained via symmetric or asymmetric self-renewal divisions. From this pool, progenitor spermatogonia arise that transiently amplify in number before transitioning from an undifferentiated to differentiating state at periodic intervals and under the influence of retinoic acid. Collectively, these actions provide the foundation for both continuity and robustness of spermatogenesis

The differentiating spermatogonia can be labeled as type A, intermediate, and type B. The type A subset undergoes mitotic divisions, A1–A4 in the mouse, and then transition to the intermediate state. The type B spermatogonia arise from division of intermediate spermatogonia and transition from mitotic to meiotic divisions, thereby becoming preleptotene spermatocytes.

A major bottleneck in the field of mammalian male germ cell biology has been the lack of morphological and molecular markers that distinguish the subsets of undifferentiated spermatogonia. In most mammals, the undifferentiated type A spermatogonia can be distinguished from differentiating type A spermatogonia based on nuclear morphology [3, 4]. In addition, spermatogonia can be classified based on “chain” or “cohort” identity. In the undifferentiated population, spermatogonia are present as single (As), paired (Apr), or aligned cohorts of 4–16 cells (Aal4–16) [5]. Importantly, type A differentiating spermatogonia can also be found as interconnected chains of 2–16 cells [6, 7]. Indeed, at periodic intervals, every 8.6 days in mice, most Apr and Aal undifferentiated spermatogonia transition to a differentiating state and retain their chain identity until the next division [8, 9, 10]. Because the As undifferentiated spermatogonia do not transition to a differentiating state [8], the population has traditionally been considered to represent the SSC pool [5, 6, 7, 11]. Although the features of nuclear morphology and chain identity allow for broad classification or spermatogonial subtypes, they do not distinguish SSC from progenitor. Only recently have molecular markers that can distinguish subsets of undifferentiated spermatogonia begun to be defined [12].

During proliferation of the undifferentiated spermatogonia population , divisions of the As spermatogonia have two potential outcomes. Either the daughter cell completes cytokinesis and forms another As, or the two cells remain connected by an intercellular cytoplasmic bridge to form Apr. Upon the next division, Apr produce a cohort of Aal4 and in the absence of retinoic acid signaling the next divisions produce Aal8 and Aal16 cohorts. Undifferentiated spermatogonial chains of greater than Aal16 have not been reported for any mammalian species. Traditionally, the As population has been considered to be the SSC pool and a division that leads to formation of Apr represents transition to the progenitor state and ultimately commitment to the differentiating pathway. From a fundamental perspective, the label stem cell is applied to cells with the capacity to regenerate a cycling cell lineage. Thus, the SSC label should be applied to spermatogonia with a capacity to regenerate the spermatogenic lineage. While the average mouse testis contains ∼35,000 As spermatogonia, only ∼3000 spermatogonia possess regenerative capacity [8, 13]. Therefore, the SSC pool is likely a subset of the As population. Furthermore, while identification of As spermatogonia may be helpful in histological applications, isolation of As from a testis digestion is not possible.

Due to the rapid cycling of the spermatogenic lineage from puberty until old age, existence of stem cells in the male germline has been widely accepted for decades. However, functional evidence was not provided until 1994 when the laboratory of Dr. Ralph Brinster published seminal findings about the transplantation of cells isolated from the testes of a donor male into the seminiferous tubules of a recipient male in which colonies of donor-derived spermatogenesis were generated [14, 15]. Because each donor-derived colony of spermatogenesis in a recipient testis is clonally derived from a single SSC [16, 17], transplantation provides a robust quantitative assay for determining the SSC content of an experimental cell population (Fig. 2). However, several aspects must be conducted in an appropriate manner for valid and interpretable data to be generated. By far, the transplantation procedure has been most refined for the mouse. Below, we describe methodology utilized in our lab to assay for the stem cell content of a variety of experimental cell populations using the mouse model. Recently, we utilized the methodology to demonstrate that expression of the transcriptional regulator inhibitor of DNA binding 4 (ID4) is a distinguishing feature of SSCs in the male mouse germline [12].
Fig. 2

The spermatogonial transplantation assay for mice. (a) A donor germ cell suspension can be collected from testes directly or from primary cultures and microinjected into the seminiferous tubules of recipients that are depleted of endogenous germline. If the donor cells possess a ubiquitously expressed reporter transgene (e.g., LacZ), colonies of donor-derived spermatogenesis are clearly observable several months after transplantation. Because each colony is clonally derived from a single donor spermatogonial stem cell (SSC) , colony numbers are a direct measure of SSC content in the transplanted cell suspension. (b) Recipient testes that were transplanted with no SSCs, an unfractionated donor testis cell suspension, or a donor testis cell suspension that was subjected to enrichment for SSC content. The differences in donor-derived colonies are related directly to differences in SSC content of the microinjected cell population . Reprinted from the Annual Review of Cell and Developmental Biology, Volume 24, by Annual Reviews, http://www.annualreviews.org

2 Materials

2.1 Recipient Selection

Transplantation is a powerful tool for the study of germline stem cells when used appropriately. Without proper selection of donor and recipient animals, accurate interpretation of outcomes can be challenging. Endogenous germ cells must be depleted or eliminated in recipient testes for efficient engraftment of donor SSCs. Two major recipient models that have been used over the last 20+ years are genetically sterile mutants and wild-type males treated with chemotherapeutic agents or irradiation [14, 15, 18, 19, 20, 21].

The use of genetic mutants such as the W/Wv mouse strain in which survival of precursor germ cells is impaired but the soma remain functionally intact provides an optimal environment for donor SSC engraftment and regeneration of spermatogenic colonies [18]. W/WV animals possess a mutation in the c-Kit gene that leads to impaired migration and survival of primordial germ cells during embryogenesis [22]. Thus, the germline in testes of W/Wv males is lacking at birth. An advantage of using this model is the total absence of endogenous germline; thus any regions of spermatogenesis that develop post-transplantation must be derived from donor SSCs, thereby eliminating the need for donor cells to be marked with expression of a transgene. Although qualitative assessment of SSCs in an experimental cell population can be made with donor cells lacking a marker transgene, quantitative assessment of SSC number is not valid for reasons described in sections below.

Treatment of mice with chemotherapeutic agents such as busulfan leads to depletion of the endogenous germline including a portion of the SSC pool within 6–8 weeks after treatment [19, 20, 23]. However, complete elimination of the SSC pool is rare and some endogenous spermatogenesis returns over time. For this reason, use of donor cells that are marked by expression of a reporter transgene is critical for distinguishing regions of spermatogenesis that are derived from transplanted vs. endogenous SSCs when using this recipient model.

Another key aspect of recipient selection is immunological compatibility with an SSC donor. In our hands, an F1 cross of C57BL/6J and 129ScvP is compatible with cells from donor mice that have varying degrees of genetic background from the parental strains. Similarly, the W/Wv mutant is typically maintained as a mixed background primarily from the C57BL/6J strain.

2.2 Donor Selection

Although the SSC content of both freshly isolated testis cell populations and primary cultures of undifferentiated spermatogonia can be analyzed using transplantation, quantitative assessment requires the capacity to determine the boundaries of spermatogenic colonies derived from donor SSCs. A straightforward means to achieve this is by using donor cells that are marked by constitutive expression of a reporter transgene. Use of W/WV recipients allows for qualitative analysis of unmarked donor cells because the endogenous germline is lacking; thus any regions of spermatogenesis must be donor derived. However, endogenous spermatogenesis does return in seminiferous tubules of animals treated with chemotherapeutic agents (e.g., busulfan); thus neither quantitative nor qualitative analysis of unmarked donor cells is valid when using this recipient model. For these reasons, we recommend using donors that possess a constitutively expressed reporter transgene that is easily detectable (e.g., LacZ or fluorescent proteins) whenever possible. In our experience, donor cells marked by expression of a LacZ reporter transgene are ideal because donor-derived colonies of spermatogenesis in recipient seminiferous tubules stain intense blue following incubation with X-Gal which allows for clear distinction of each colony. Also, the recipient testes can be manually disassociated with relative ease to accurately count colony numbers and the tissue preserved for a long period of time. Typically, we utilize the B6.129S7-GtROSA26Sor/J mouse line as a donor or a hybrid F1 version produced by crossbreeding with another line of interest.

2.3 Reagents

  1. 1.

    DPBS-S: 1 % Fetal bovine serum (FBS), 10 mM HEPES, 1 mM pyruvate, 1 mg/ml glucose, 1 × 104 U/ml penicillin, and 1 × 10 μg/ml streptomycin in 1× phosphate-buffered saline (PBS) solution (see Note 1 ).

     
  2. 2.

    Mouse serum-free culture media (mSFM): 0.2 % Bovine serum albumin (BSA), 5 μg/ml insulin, 10 μg/ml iron-saturated transferrin, 7.6 μeq/L free fatty acids, 3 × 10−8 M H2SeO3, 50 μM 2-mercaptoethanol, 10 mM HEPES, 60 μM putrescine, 2 mM glutamine, and antibiotics in minimum essential medium alpha (MEMα) (see Note 1 ).

     
  3. 3.

    4 % Paraformaldehyde (PFA): 4 % w/v PFA in 1×PBS, pH 7.4 (see Note 2 ).

     
  4. 4.

    Trypsin-EDTA solution: 0.25 % Trypsin, 1 mM EDTA.

     
  5. 5.

    DNase I solution: 7 mg/ml DNase I in Hanks’ Balanced Salt Solution.

     
  6. 6.

    Fetal bovine serum (FBS).

     
  7. 7.

    1 mg/ml Collagenase in HBSS.

     
  8. 8.

    Thy1 magnetic beads (Miltenyi Biotec).

     
  9. 9.

    MACS column (Miltenyi Biotec ).

     
  10. 10.

    LacZ Rinse Buffer: 0.2 M Sodium phosphate, 2 mM magnesium chloride, 0.02 % Triton X-100, and 0.01 % sodium deoxycholate in water.

     
  11. 11.

    LacZ Staining Solution: 20 mM Potassium ferricyanide , 20 mM potassium ferrocyanide, 1 mg/ml 5-bromo-4-chloro-2-indolyl-β-D galactosidase (X-Gal) in LacZ rinse buffer.

     

3 Methods

3.1 Donor Cell Preparation

Cell suspensions for microinjection into recipient testes can be prepared freshly from testes of donor males at a variety of developmental stages including embryonic (6–13 days post-conception), fetal (14–18 days post-conception), neonatal (postnatal days, PD, 0–2), pup (PD 3–8), prepubertal (PD 9–35), and adult (> PD 35), or from primary cultures of undifferentiated spermatogonia. Typically, neonatal or pup testis cell suspensions that have not been enriched for spermatogonia or SSCs generate ∼3 and 14 colonies of donor-derived spermatogenesis in testes of busulfan-treated recipients per 105 cells microinjected, respectively [24]. For testes of adult donor males, ∼3 colonies of spermatogenesis are generated per 105 cells and cell suspensions prepared from a cryptorchid condition produce ∼18 colonies per 105 cells [25].

3.2 Preparation of a Single-Cell Suspension from Neonate or Pup Testes

  1. 1.

    Testes are collected into Hanks’ balanced salt solution (HBSS) and the tunica albuginea is removed with fine forceps. It is helpful to use a dissecting microscope for this step.

     
  2. 2.

    Testes are transferred to a 35 mm petri dish containing 4.5 ml of trypsin-EDTA solution and 0.5 ml DNase I solution followed by gentle pipetting with a 1 ml serological pipette to disassociate the seminiferous cords.

     
  3. 3.

    The dish is incubated at 37 °C for 5 min followed by addition of another 0.5 ml of DNase I solution and the suspension is gently pipetted again.

     
  4. 4.

    The dish is returned to 37 °C for 5 min and step 3 is repeated until all tissue clumps have become disassociated.

     
  5. 5.

    FBS is added to the suspension at 10 % of the volume to stop the digestion process and gentle pipetting with a P1000 tip is used to ensure that a single-cell suspension has been achieved.

     
  6. 6.

    The single-cell suspension is passed through a 40 μm cell strainer that has been prewashed with 1 ml HBSS into a 50 ml conical centrifuge tube. The filter is then washed twice with 2 ml of HBSS.

     
  7. 7.

    Cells are then pelleted by centrifugation at 600 × g for 7 min at 4 °C. At this point, cells can be washed twice in mSFM, suspended at a concentration of 1–10 × 106 cells/ml, and transplanted as a non-enriched cell population. Alternatively, the pelleted cells can be subjected to enrichment for spermatogonia and SSCs using methods described below.

     

3.3 Preparation of Adult Testis Single-Cell Suspension

When utilizing adult donors, several extra steps are required to deplete interstitial cells that are present in between seminiferous tubules (note that complete elimination of interstitial cells is not possible).
  1. 1.

    Testes are collected into HBSS and the tunica albuginea is removed using fine forceps. Seminiferous tubules are then gently separated using forceps and transferred to a 50 ml conical tube containing an enzyme solution for further disassociation. The protocol described here is sufficient for two donor testes.

     
  2. 2.

    The crudely disassociated tubule masses are incubated in a 10 ml enzyme solution of collagenase (1 mg/ml in HBSS) and 1 ml DNase I solution is added. The tissue is then drawn into a 5 ml serological pipette several times to facilitate initial disassociation.

     
  3. 3.

    The digestion tube is then incubated in a 37 °C water bath for 10–20 min with gentle swirling every 5 min until the tubule masses have completely separated. Care must be taken to keep the individual tubules intact.

     
  4. 4.

    The digestion tube is then set in ice for 2 min to allow the tubules to settle and the supernatant containing interstitial cells is carefully removed with a pipette.

     
  5. 5.

    10 ml of HBSS is then added and the tube is swirled gently to wash the tubules. The tube is then placed in ice for 2 min to again allow tubules to settle followed by removing the supernatant. This procedure is repeated two more times. At the end, a majority of interstitial cells are removed leaving behind individual seminiferous tubules.

     
  6. 6.

    To disassociate tubules, a 10 ml solution of trypsin/EDTA containing 10 mg hyaluronidase and 1 ml of DNase I (7 mg/ml) is added to the digestion tube. Tubules are gently drawn into and out of a 5 ml serological pipette several times and the tube is then incubated in a 37 °C water bath for 5 min.

     
  7. 7.

    The cell suspension is then pipetted several times and the digestion tube is returned to the water bath for another 5 min. A single-cell suspension is generated by pipetting with a P1000.

     
  8. 8.

    FBS is added to the digestion solution at a volume of 10 % to stop the enzyme actions and the single-cell suspension is passed through a 40 μm filter that has been prewashed with 1 ml HBSS into a new 50 ml conical tube. The strainer is washed twice with 5 ml of HBSS .

     
  9. 9.

    Cells are pelleted by centrifugation at 600 × g for 7 min at 4 °C. At this point, the cell pellet can be suspended in mSFM at a concentration of 1–10 × 106 cells/ml and transplanted as an unselected population or subjected to enrichment protocols described below.

     

3.4 Strategies for Enrichment of SSCs

The concentration of SSCs or even spermatogonia in single-cell suspensions generated from testes is low because of the multitude of other germ cell and somatic cell types. Thus, in many experimental scenarios enrichment for SSCs is of benefit. Here, we describe methodologies that are variations of a continuous Percoll gradient and magnetic activated cell sorting (MACS) for cell surface proteins to enrich for spermatogonia and SSCs that were originally reported by Kubota and Brinster [25]. The single-cell suspensions can also be processed for fluorescence-activated cell sorting (FACS) using antibody staining or based on expression of fluorescent protein reporter transgenes.

3.5 Enrichment of Spermatogonia with a Continuous Percoll Gradient

Fractionation of a testis single-cell suspension using a continuous 30 % Percoll gradient yields a bottom pellet that is enriched for SSC content. For adult testes, ∼12 colonies are generated in recipient testes per 105 cells transplanted which represent ∼5-fold enrichment compared to the un-fractionated cell population [25].
  1. 1.

    The cell pellets prepared from adult or pup donor testes described above are suspended in 10 ml of HBSS.

     
  2. 2.

    5 ml of the cell suspension is carefully layered on top of 2 ml of 30 % Percoll solution in a 15 ml conical tube. It is essential that the cell suspension is not mixed with the Percoll solution. To aid this, the conical tube can be inverted at a 45° angle and the cell suspension pipetted slowly. Two distinct layers of solution should be visible after dispensing the cell suspension and two tubes are utilized for the 10 ml of donor cell suspension.

     
  3. 3.

    Tubes are then centrifuged at 600 × g for 8 min at 4 °C.

     
  4. 4.

    The supernatant is removed from each tube and the pelleted cells are suspended in 1 ml of DPBS-S and transferred to a new 15 ml conical tube. Cell number is then determined with a hemocytometer and the suspension is centrifuged at 600 × g for 7 min at 4 °C. The cell pellet can then be prepared for transplantation by suspension in mSFM at 1–10 × 106 cells/ml or processed for additional fractionation by MACS or FACS .

     

3.6 Selection of Thy1+ Cells Using MACS

MACS can be employed to fractionate subsets of germ cells based on expression of specific proteins that are localized at the cell surface. For example, MACS selection of testis cells expressing Thy1 produces a population that is enriched for SSCs compared to the unfractionated cell populations of neonatal, pup, and adult testes. However, it is important to note that the Thy1+ testis cell fraction is not pure SSCs nor is it even pure germ cells. The Thy1+ testis cell fraction from pup donors generates 124–230 colonies per 105 cells transplanted which represents an enrichment of ∼9- to 16-fold compared to unsorted pup cells [25, 26, 27]. As discussed in Subheading 3.13, this level of colonization equates to only 1 in 22 cells of the Thy1+ fraction being an actual SSC. Thus, while Thy1 fractionation can be used to enrich for SSCs it does not yield a pure population. The following protocol is a modified version of the procedure reported by Kubota et al. [25].
  1. 1.

    The pelleted cells from Percoll selection are suspended in DPBS-S and Thy1 magnetic beads are added at a dilution of 1:10. The suspension is then incubated at 4 °C for 20 min with mixing after 10 min.

     
  2. 2.

    1 ml of DPBS-S is added to the incubation solution and the cells are pelleted by centrifugation at 600 × g for 7 min at 4 °C.

     
  3. 3.

    The cell pellet is then suspended in 1 ml of DPBS-S.

     
  4. 4.

    A MACS column is assembled per the manufacturer’s instructions and rinsed with 0.5 ml DPBS-S .

     
  5. 5.

    The cell suspension is then loaded into the column after which the column is rinsed three times with 0.5 ml DPBS-S each.

     
  6. 6.

    Adherent cells are then eluted through the column by adding 1 ml of mSFM, total cell number is determined using a hemocytometer, and cells are then pelleted by centrifugation at 600 × g for 7 min at 4 °C.

     
  7. 7.

    The cell pellet is washed by suspending in mSFM and again pelleted by centrifugation.

     
  8. 8.

    Lastly, the cell pellet is suspended in mSFM at a concentration of 1–10 × 106 cells/ml in preparation for transplantation.

     

3.7 Preparation of Cells from Primary Cultures of Undifferentiated Spermatogonia

For several mammalian species including mice, rats, rabbits, and hamsters, primary cultures of undifferentiated spermatogonia can be maintained for long periods of time [28, 29, 30, 31]. Although the cultures are comprised purely of undifferentiated spermatogonia, only a minor portion of the population is SSCs. For mouse cultures, the SSC content ranges from 10 to 20 % [29, 32, 33, 34, 35]. A powerful aspect of the primary cultures is that experimental manipulations can be conducted such as treatment with pharmacological agents, RNAi to reduce the expression of genes of interest, overexpression of certain genes, and supplementation of media with soluble growth factors. Combining these treatments with transplantation analyses allows for exploring alterations in maintenance of the SSC pool [27, 36, 37, 38, 39, 40, 41, 42, 43].
  1. 1.

    mSFM is removed with a serological pipette and each well is washed with 1 ml of HBSS. The wash is collected into a 15 ml conical tube.

     
  2. 2.

    For a 24-well plate format, 0.25 ml of trypsin/EDTA solution is added per well and the plate is incubated at 37 °C for 5 min.

     
  3. 3.

    FBS is then added directly to the well at 10 % of the volume and cells are pipetted gently to generate a single-cell suspension.

     
  4. 4.

    The cell suspension is then added to the 15 ml tube containing the initial wash. 1 ml of HBSS is added to the well, collected, and then combined in the collection tube.

     
  5. 5.

    The collection tube is centrifuged at 600 × g for 7 min at 4 °C. The supernatant is removed and the cells are suspended in 1 ml mSFM. Cell concentration is determined with a hemocytometer and the tube is again centrifuged at 600 × g for 7 min at 4 °C.

     
  6. 6.

    The cell pellet is then suspended in mSFM at a concentration of 1 × 106 cells/ml in preparation for transplantation .

     

3.8 Microinjection of Donor Cells into Recipient Seminiferous Tubules

While many parameters of donor cell preparation, including enrichment techniques and other treatments prior to transplantation, depend on the goal of the study at hand, one factor that must always be considered is concentration of donor cell suspension. A high concentration of cells can result in clogging of the microinjection needle or too many colonies in recipient testes to accurately determine the boundaries of each, thus making quantification difficult. Also, diluting the cell suspension too much can result in no or very low colony formation in recipient testes. In our hands, an ideal concentration of donor cells is 1 × 106 cells/ml for enriched cell suspensions .
  1. 1.

    The recipient animal is anesthetized and placed on a platform for visualization through a dissecting microscope (e.g., Olympus SZX7). A midline abdominal incision is then made through the skin and abdominal muscle.

     
  2. 2.

    A testis is pulled through the abdominal incision and immobilized outside of the body cavity. A sterile cardstock platform placed at the incision site is helpful for immobilizing the testis. Positioning of the testis is important to orient the efferent duct for access by a micropipette needle .

     
  3. 3.

    Donor cell suspension is then loaded into the micropipette. In our hands, borosilicate glass with an internal diameter of 0.75 mm and external diameter of 1 mm is effective for creating micropipettes with a puller. The volume loaded into the micropipette for injection depends on the recipient model being used. For busulfan-treated adult recipients, ∼10 μl can be injected per testis. For adult W/WV recipients, ∼6 μl of volume can be injected. These volumes will result in filling of 70–80 % of the seminiferous tubules. After filling with cell suspension, the micropipette is inserted into the efferent duct and advanced to the rete testis. Care should be taken to avoid destroying the efferent duct. Also, it is helpful to make a small hole in the efferent duct with a 30-gauge needle through which the micropipette can be inserted (see Note 3 ).

     
  4. 4.

    Pressure is then supplied to the micropipette through silastic tubing that is attached to an injector, syringe, or mouthpiece. To avoid damage to the tubules or injection into the interstitial space, the flow rate should be relatively slow, dispensing the volume over a 3–5-min period (see Note 4 ).

     
  5. 5.

    After both testes have been filled with donor cell suspension and replaced into the body cavity, the abdominal muscle is closed with braided silk suture and skin closed with wound clips. Care should be taken to avoid catching the fat pad of either testis in the suture. If this were to occur, a cryptorchid condition would ensue and regeneration of donor-derived spermatogenesis would be limited.

     

3.9 Considerations for Technical and Biological Replication

When utilizing SSC transplantation as a quantitative assay, it is important to design experiments with proper replication. Biological replication (i.e., n values) is applied to the aspect of an experiment being subjected to a treatment. Typically, the donor cell suspension is the subject of treatments or conditions, for example, selection of donor cells for specific attributes (e.g., surface proteins or expression of fluorescent reporters), treatment with siRNAs, treatment with pharmacological agents, or isolation from testes of mutant models. In these scenarios, the donor cell suspension is the biological replicate and should be considered the n-value. The donor cells should be transplanted into the testes of multiple recipients that were subjected to a standard preparation (i.e., busulfan), which would provide technical replication. In some experiments, treatments may be applied to the recipient, for example, testing new methods for depleting the endogenous germline , alteration of somatic cell components, or mutant animals with fertility defects. In these scenarios, the recipient would be the biological replicate (n-value) and a standard donor cell suspension at the identical concentration should be transplanted into each recipient testis. For experiments in which the donor cells are the subject of treatments or conditions, we recommend that at least three donor cell suspensions isolated from different animals be used for biological replication and at least four recipients for each biological replicate be used to achieve proper technical replication (i.e., n = 3 and 12 recipient testes for each treatment).

3.10 Analysis of Recipient Testes for Colonies of Donor-Derived Spermatogenesis

Following transplantation recipient animals should be aged at least 8 weeks to encompass multiple rounds of spermatogenesis. This timeframe is essential for assessment of spermatogenic colonies derived from SSCs. In theory, non-stem cell spermatogonia could reform transient colonies of spermatogenesis but these would not persist beyond one round of spermatogenesis. The most useful parameters from transplantation analyses are colony number and colony length. Because each colony is clonally derived from an individual SSC [16, 17], colony number is a retrospective measure of the relative number of SSCs in the microinjected donor cell suspension. Colony length provides a measure of SSC proliferation and regeneration following initial colonization [44]. The validity of collecting these measures depends on the donor cells. Accurate determination of both colony number and length depends on the ability to discern the boundaries of each colony. For this reason, donor cells must be marked by constitutive expression of a reporter transgene such as GFP or LacZ. Analyzing cross sections of recipient testes is not a valid approach for assessing either the number or the length of donor-derived colonies because defining the boundaries is nearly impossible unless strict serial sectioning of the entire testis and 3D reconstruction are utilized. In our hands, donor cells marked by expression of a LacZ transgene that is integrated into the Rosa26 locus are ideal for determining colony number and length.

3.11 LacZ Staining Recipient Testes

  1. 1.

    Testes are collected from euthanized recipients, the tunica albuginea is gently removed, and the tissue is immersed in 4 % paraformaldehyde for 60 min at 4 °C.

     
  2. 2.

    Following fixation, testes are rinsed in LacZ rinse buffer three times for 30 min each at 4 °C on a rocking platform .

     
  3. 3.

    Testes are then incubated in X-Gal staining solution overnight at 35 °C on a rocking platform.

     
  4. 4.

    LacZ rinse buffer is used to wash the testes several times before post-fixation in 10 % formalin to clear the tissue.

     

3.12 Qualitative and Quantitative Assessment of Donor-Derived Spermatogenic Colonies

Although donor-derived colonies of spermatogenesis in recipient testes can be seen with the naked eye following X-Gal staining (if LacZ-marked cells were used) or under a UV light (if fluorescent protein reporter cells were used), visualizing the boundaries of each colony requires the use of a dissecting microscope. The seminiferous tubules of each recipient testis should be carefully teased apart with fine forceps or hypodermic 30-gauge needles to reveal all colonies. Care must be taken not to break individual colonies. For X-Gal-stained recipient testes, each donor-derived colony will be observable as a dark blue region and the length will be variable [15]. The length of each colony can be determined from digital images using software programs such as NIH Image J [44].

Although cross sections of testes from genetically sterile recipient mice (e.g., W/Wv) are useful for qualitative assessment of whether SSCs were present in an experimental cell population, quantitative determination of relative SSC number is challenging because of the difficulty in determining the boundaries of individual colonies.

Raw numbers of donor-derived colonies can be normalized to 1 × 105 cells transplanted for calculating a relative number of SSCs in an experimental cell population. These values can be compared among treatments or conditions to determine changes in SSC content. However, it is sometimes necessary to apply a correction factor for differences in recovered cell number from a donor testis or culture well because a standard number of cells should be transplanted into each recipient testis to reduce variability in colonization efficiency. For example, if 1 × 105 total cells are isolated from the testes of a control male and 0.5 × 105 total cells are isolated from the testes of a mutant male and a standard number of cells is transplanted into each recipient testis, it is possible that the number of donor-derived colonies that are generated will be similar. In this scenario, the actual SSC content in mutant testes is less than control because the total number of cells prior to transplant is reduced by 50 %. In contrast, if the mutant cell suspension produces twice as many colonies compared to control, the actual SSC content is no different than control.

3.13 Determination of SSC Purity in Fractionated Testis Cell Suspensions

The stem cell purity of experimental populations or fractions isolated from a tissue is often difficult to determine. When conducted in a quantitative manner, transplantation analyses can provide an assessment of stem cell purity within a cell population. For the mammalian male germline, cell fractions isolated from testes have been reported to produce anywhere from ∼228 (Thy1+ cells) to 1 (unselected testis populations) colony of donor-derived spermatogenesis per 105 cells transplanted [15, 40, 43, 44]. The colonization efficiency of SSCs when transplanted into busulfan-treated adult recipient testes has been calculated at 5–12 % [11, 20, 40, 42]. Using the formula: [# of cells transplanted/(# of colonies/% colony efficiency)], a relative purity of SSC content in a transplanted cell population can be calculated. To our knowledge, the greatest number of colonies generated from a cell population isolated from donor testes directly that has been reported to date is for the Thy1+ fraction [25, 26]. Using the formula with a 5 % colonization efficiency, the SSC content in the Thy1+ fraction is 1 in 22 cells [100,000 cell transplanted/(228 colonies/5 % colonization efficiency)].

Another approach for determining stem cell purity in a cell population is to perform transplantation on a limiting dilution scale. For example, an experimental cell population could be transplanted dilution series of 10,000; 1000; 100; 10; and 1 cells. For assessment, one would determine the fewest number of cells that could be transplanted to generate a single colony of donor-derived spermatogenesis. Employing the formula described above would provide a measure of SSC purity. For example, if one colony were produced from as few as ten cells transplanted, a claim of SSC purity could be made if a colonization efficiency of 10 % were employed, [10 cells transplanted/(1 colony/10 % colonization efficiency)] = 1 in 1 or 100 %. At present, definitive evidence that any cell population isolated from the testes of any mammalian species is pure SSCs has not been reported. However, we have recently observed the formation of two colonies of donor-derived spermatogenesis following transplantation of 50 Id4-GfpBright cells that were isolated by FACS from testes of an Id4-Gfp transgenic donor mouse line (A.R. Helsel and J.M. Oatley, unpublished data). Based on a 5 % colonization efficiency, we can calculate the SSC content of the Id4-GfpBright fraction as 1 in 1.25 cells [50 cells transplanted/(2 colonies/5 % colonization efficiency)] which is by far the greatest enrichment reported to date and the population may actually be pure stem cells.

4 Notes

  1. 1.

    Both DPBS-S and mSFM should be sterile filtered.

     
  2. 2.

    In a hood, heat 1×PBS to 60 °C on a hotplate and then add 4 % w/v PFA. Solution will be cloudy. Add 10 N NaOH drop-wise until the solution becomes clear. Cool and adjust the pH to 7.4.

     
  3. 3.

    Proper positioning of the micropipette is critical. If it is not advanced into the rete testis, the donor suspension will not enter the seminiferous tubules. On the other hand, advancing the micropipette too far into the rete testis can result in injection into the interstitial space of the testis rather than into the seminiferous tubules. Both cases will result in poor or no colonization.

     
  4. 4.

    Adding sterile trypan blue solution (0.25 %) to the donor cell suspension at a volume of 5 % allows for visual confirmation that the suspension has been injected into the seminiferous tubules of a recipient testis. When properly injected the cell suspension will be seen coursing throughout the tubules. Injection into the interstitial space between tubules will appear as a cloud of blue underneath the tunica albuginea.

     

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Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  1. 1.Center for Reproductive Biology, School of Molecular Biosciences, College of Veterinary MedicineWashington State UniversityPullmanUSA
  2. 2.Center for Reproductive Biology, College of Veterinary MedicineWashington State UniversityPullmanUSA

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