A comparison of methods for preparing enriched ... - CSIRO Publishing

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Reproduction, Fertility and Development, 2009, 21, 393–399

A comparison of methods for preparing enriched populations of bovine spermatogonia Muren HerridA,B,D , Rhonda J. DaveyA,B , Keryn HuttonA,B , Ian G. ColditzB and Jonathan R. HillA,B,C A CSIRO

Food Futures National Research Flagship. Livestock Industries, F. D. McMaster Laboratory, Armidale, NSW 2350, Australia. C Present address: School of Veterinary Science, University of Queensland, St Lucia, Qld 4072, Australia. D Corresponding author. Email: [email protected] B CSIRO

Abstract. The objective of the present study was to identify an efficient and practical enrichment method for bovine typeA spermatogonia. Four different enrichment methods were compared: differential plating on laminin- or Datura stramonium agglutinin (DSA)-coated flasks, percoll-gradient isolation, magnetic-activated cell sorting (MACS) and fluorescenceactivated cell sorting (FACS). The isolated cells were characterised with Dolichos biflorus agglutinin (DBA) lectin staining for type A spermatogonia and vimentin-antibody staining for Sertoli cells. A 2 × 2 factorial design was used to investigate the enrichment efficiency on laminin and DSA. In the laminin-enrichment groups, 2 h incubation in plates coated with 20 µg mL−1 laminin yielded a 3.3-fold increase in DBA-positive cells in the adherent fraction, while overnight incubation in flasks coated with 20 µg mL−1 DSA produced a 3.6-fold increase in the non-adherent fraction. However, the greatest enrichment (5.3-fold) of DBA-positive cells was obtained after 2 h incubation in control flasks (coated with bovine serum albumin). Percoll-gradient centrifugation yielded a 3-fold increase in DBA-positive cells. MACS results showed a 3.5- to 5-fold enrichment while FACS produced a 4-fold increase in DBA-positive cells. It is concluded that differential plating is a better method of recovering large numbers of type A spermatogonia for germ cell transplantation, while MACS or FACS can provide highly enriched viable type A spermatogonia for in vitro culture. Further, the combination of differential plating and other enrichment techniques may increase the purification efficiency of type A spermatogonia. Additional keywords: enrichment, isolation, stem cells.

Introduction Spermatogonia are male germ line progenitors of sperm cells. It has been demonstrated that mixed cell populations containing spermatogonia isolated from donor testes of mice, rats, goats and chickens can populate recipient testes and produce spermatozoa of the donor genotype (Brinster Avarbock 1994; Jiang Short 1995; Honaramooz et al. 2003). Intense interest has arisen in using this technology to disseminate superior genetics or produce transgenic animals in livestock species. Methods to purify spermatogonia from cattle should improve the success of germ cell transfer to recipient testes and enable the development of in vitro methods such as immortalisation and genetic manipulation of these cells (Izadyar et al. 2003; Herrid et al. 2006, 2007). Cells can be enriched from mixed populations by positive or negative selection on physical or functional characteristics such as cell surface markers, adhesivity, motility and buoyant density. Enzymatic digestion of pre-pubertal testis tissue from donors yields mixed cell populations in which spermatogonia, Sertoli cells and other somatic cells predominate. In cattle, typical proportions of these cells types are ∼10, 40 and 20%, respectively (Herrid et al. 2007). In mice, spermatogonia are characterised © CSIRO 2009

by cell surface expression of c-kit, Thy-1, CD9 and the integrin α6β1 (Ogawa et al. 1997; Kanatsu-Shinohara et al. 2004). Several of these markers have been used in positive selection of spermatogonia in mice and rats. At present, antibodies to bovine c-kit, CD9 and integrin α6β1 are not available. Sertoli cells and other somatic cells have a propensity to adhere to a substratum in vitro (Izadyar et al. 2002). Extracellular matrix molecules are abundant in seminiferous tubules (Glattauer et al. 2007). The integrin α6β1 is expressed by spermatogonia in rodents and laminin is one of its ligands (Shinohara et al. 1999). The differential adhesion of cells isolated from testes to laminin has been used to enrich spermatogonia in mice and rats. In addition, Sertoli cells specifically bind to the lectin Datura stramomium agglutinin (DSA) and thus it has been used to purify Sertoli cells from immature rat testis isolation (Scarpino et al. 1998). Enrichment of spermatogonia by density gradient centrifugation has been employed to enrich spermatogonia in pigs, cattle and sheep (Dirami et al. 1999; Izadyar et al. 2002; Rodriguez-Sosa et al. 2006). Studies have validated that Dolichos biflorus agglutinin (DBA) stains a population of cells in the bovine testis 10.1071/RD08129

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with morphological and positional characteristics of type A spermatogonia (Wrobel 2000; Herrid et al. 2007). In the present study, we compare the efficiency of enrichment of spermatogonia in mixed cell populations from testes of pre-pubertal bulls calves by: (1) differential adhesion of cells plated on laminin and DSA; (2) separation of cells by density gradient centrifugation on Percoll; (3) positive selection of cells labelled with DBA-fluorescein isothiocyanate (FITC) by magnetic-activated cell sorting (MACS); and (4) positive selection of cells labelled with DBA-FITC by fluorescence-activated cell sorting (FACS). Materials and methods Animals and castration Animals were handled and treated according to the guidelines of the Animal Ethics Committee at CSIRO, Armidale, Australia. The testes were obtained from Hereford or Hereford × Angus bull calves with scrotal circumferences of 16–19 cm (4–5 months of age; testis weights 27–42 g). The calves were castrated under general anaesthesia using a combination of Xylazil (0.1 mg kg−1 ; Troy Laboratories, Smithfield, NSW, Australia) and Ketamine (3 mg kg−1 ; Troy Laboratories) and testes were transferred directly to the laboratory. Cell isolation A two-step enzymatic isolation procedure was used to isolate individual tubular cells as described by Herrid et al. (2006) with modification. In brief, a 6–10-g segment of tissue was used as an isolation unit. After rinsing, the tissues were transferred to a second Petri dish with 5 mL DMEM : F12 (Invitrogen, Carlsbad, CA, USA) and chopped finely. The tissues were placed in a tea strainer and ground with a 5-mL syringe plunger. The remaining tubule section was transferred into a 50-mL Falcon tube and then incubated with collagenase type IV (1 mg mL−1 ; SigmaAldrich, Castle Hill, NSW, Australia) in a shaking waterbath at 37◦ C. The supernatant was removed and the tissues were rinsed five times in DPBS (Invitrogen) without calcium and magnesium at room temperature. The fragments were then treated with trypsin (2.5 mg mL−1 ; Invitrogen) in DPBS for 5–10 min at 37◦ C. DNAse I (7 mg mL−1 ; Sigma-Aldrich) in DMEM was added 1 min after trypsin treatment. An equal volume of heatinactivated fetal bovine serum (FBS; Invitrogen) was used to inactivate trypsin digestion. The resultant cell suspension was then filtered though a cell strainer with two layers of nylon mesh (upper layer 96 µm and lower layer 55 µm pore size) and centrifuged at 400g for 5 min at room temperature. The pellets were resuspended in 10 mL DMEM containing 5% FBS with a density of 40–80 × 106 cells mL−1 . The cell viability was assessed by trypan blue exclusion. Differential plating of testis cell suspensions on laminin- and DSA-coated flasks Cell culture flasks (75 cm2 Falcon, BD Biosciences, North Ryde, NSW, Australia) were coated with laminin or DSA (both Sigma-Aldrich). Laminin-coated flasks were incubated for 2 h or overnight (16–18 h) at 37◦ C with 3 mL of 0, 20 or 40 µg mL−1 laminin in phosphate-buffered saline (PBS) (Shinohara et al. 1999), and DSA-coated flasks were

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incubated for 1 h or overnight (16–18 h) at 37◦ C with 3 mL of 0, 10 or 20 µg mL−1 DSA in PBS. Following coating with laminin, flasks were gently rinsed three times with 5 mL PBS, then 3 mL PBS + 0.5 mg mL−1 bovine serum albumin (BSA; Sigma-Aldrich) was added and incubated for 1 h at 37◦ C to reduce non-specific binding of cells. Flasks were then rinsed three times with 3 mL PBS then kept moist with PBS until required. Following coating with DSA, plates were washed three times with 3 mL PBS + 0.5 mg mL−1 BSA. Control flasks were coated with BSA in PBS as described above. Prior to the addition of testis cells, PBS was replaced by 12 mL DMEM + 5% FCS and 20 million cells were added per flask, thus the final incubation density was 0.26 × 106 per cm2 . Following the incubation period, non-adherent cells were poured off into 50-mL Falcon tubes. This included gentle rinsing of unbound cells with 5 mL of PBS, repeated two times. Attached cells were removed by trypsin (0.25%) digestion for 5–6 min followed by strong pipetting. Smears were then made from the adherent and non-adherent cell samples for immunostaining. Adherent cells could not be dislodged with 0.25% trypsin from flasks coated with DSA. Chamber slides were therefore used to provide samples for identification of the attached cell population. Chamber slides were treated the same as flasks and 0.26 × 106 cells were added into each square box, which was the same density (0.26 × 106 per cm2 ) used in the flask incubation. Following the incubation period, non-adherent cells were poured off and rinsed twice with 1 mL PBS. Flow cytometry The intensity of staining with FITC-conjugated Dolichos biflorus agglutinin (DBA-FITC, Sigma-Aldrich) was titrated by incubation of duplicate sets of 100 µL of single cell suspensions (106 cells) with DBA-FITC (2 mg mL−1 ) in the final concentrations of 0, 0.03, 0.06, 0.125, 0.25 and 0.5 mg mL−1 at 4◦ C for 15 min with agitation. Cells were washed twice with 1 mL PBS (cold) and centrifuged at 4◦ C and 300g for 5 min. The pellets were resuspended in 200 µL PBS and the intensity of the labelling was determined as mean fluorescence intensity (MFI). Samples were acquired on a single-laser (argon-ion 488 nm) FACS Vantage flow cytometer (BD Biosciences). Data for 10 000 events from each sample were collected from a gate set on forward scatter and side scatter. After determining the optimal concentration for staining cells, for FACS sorting of DBA-FITC positive cells, 106 cells were labelled with 0.2 mg mL−1 DBA in a 100 µL volume. The sorted cells were re-analysed on the flow cytometer to determine the percentage of DBA-FITC-positive cells in the sample. At least 10 000 events were acquired for each sample. MACS For positive selection, 10 × 106 cells from single cell suspensions were washed with 1 mL MACS buffer (Milteny Biotech, Bergisch Gladbach, Germany), centrifuged at 300g for 10 min at 4◦ C and resuspended in 0.9 mL buffer. One hundred microlitres DBA-FITC (2 mg mL−1 ) were added to each tube and incubated on ice for 15 min with gentle agitation. To remove the unbound lectin, cells were washed twice with 1 mL MACS buffer and resuspended in 0.9 mL MACS buffer. Then the cells

Purification of bovine spermatogonia


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Table 1. Effect of laminin plating on DBA+ and vimentin+ cell populations (n = 3) Input cells were incubated in control (BSA-coated only) or laminin-coated flasks for 2 h or overnight (16–18 h). Adherent and non-adherent cells were collected and stained for DBA and vimentin to identify Type A spermatogonia and Sertoli cells, respectively. Results calculated as (yield of DBA+ or vimentin+ cells in input)/(number of DBA+ or vimentin+ cells in final fraction) and expressed as %. Within each column, values (mean ± s.e.m.) with different superscript letters are significantly different (P < 0.05). n.a., results not available Treatment

Incubation time

DBA+ (%) Non-adherent

Input cells Control Control 20 µg mL−1 20 µg mL−1 40 µg mL−1 40 µg mL−1

– 2h Overnight 2h Overnight 2h Overnight

Adherent

8.6 ± 3.2a 40.3 ± 1.9b 7.0 ± 3.6a 30.6 ± 1.4b 13.1 ± 2.8ab 11.3 ± 2.4a 28.6 ± 7.9c 15.6 ± 2.0ac 20.4 ± 7.5bc 13.0 ± 1.6ac 23.2 ± 2.4bc 18.2 ± 3.5c 10.9 ± 6.2ab

were incubated with 100 µL MicroBeads coated with antibody to FITC (Milteny Biotech) for 15 min on ice with agitation. After incubation, the cells were washed with 2 mL buffer and centrifuged at 300g for 10 min at 4◦ C. The pellets were resuspended in 500 µL MACS buffer and the cells were isolated on the columns according to the manufacture’s recommendation. Control cells were incubated with MACS buffer alone. In experimental setting 2, the magnetic fraction from the first column (5 mL) was placed on the second column for a second cycle of enrichment. In experimental setting 3, 200 µL antiFITC MicroBeads were added to the cells resuspended in 0.8 mL buffer to compare the isolation efficiency with the 100 µL antiFITC MicroBeads. Following recovery of cells from the MACS column, cells were fixed in 4% paraformaldehyde (ProSciTech, Thuringowa, Qld, Australia) and analysed by flow cytometry within 2 weeks. At least 10 000 events were acquired for each sample.

Vimentin+ (%) Non-adherent

Adherent

55.6 ± 6.2a 28.9 ± 7.2c 74.3 ± 8.6ab 33.9 ± 3.5abc 66.9 ± 6.2ab 59.6 ± 4.8abc 31.6 ± 5.9b 43.9 ± 5.8abc 39.6 ± 5.8ab 48.9 ± 8.1abc 42.3 ± 5.2ab 43.9 ± 3.9c 50.9 ± 4.6a

Yield of DBA+ cells (% of input) Non-adherent

Adherent

n.a. 76.4 ± 5.9a 38.7 ± 3.8c 52.9 ± 5.3b 33.5 ± 5.8c 56.8 ± 6.2b 33.0 ± 6.2c

n.a. 26.2 ± 3.9a 46.6 ± 7.8b 60.8 ± 8.1b 69.0 ± 5.9b 46.7 ± 4.6b 47.9 ± 5.3b

immunohistochemical staining of cell smears with DBA or vimentin as described by Herrid et al. (2007). Labelled cells were visualised at 20–40× magnification and a total of 100–300 cells were counted per slide. Statistics The differences between purification procedures in percentage of DBA- and vimentin-positive cells and viability were analysed by General Liner Model procedure followed by Duncan test for multiple range comparisons by STATGRAPHIC software. Data are expressed as mean ± s.e.m. Differences of P < 0.05 were considered to be significant.

Percoll gradient preparation An iso-osmotic suspension of Percoll was prepared from 90% Percoll (Sigma-Aldrich) + 10% 10 × DMEM (van Pelt et al. 1996). The iso-osmatic Percoll suspension was diluted with 1 × DMEM containing 0.7% BSA to obtain final densities of 1.0611, 1.0542, 1.0513 and 1.0413 g mL−1 . The four freshly prepared 5-mL density preparations were loaded sequentially into a 50-mL centrifuge tube via syringe and steel tubing in order of lowest density to highest density.The testis cell suspension (5 mL at a concentration of 5–10 million cells mL−1 ) was carefully layered on top of the gradient then centrifuged at 800g for 30 min at 4◦ C. This resulted in four visible bands of testicular cells and 8 mL of interphase cell fraction was collected from the interface of each band. The collected 8-mL cell fraction was diluted in 20 mL DMEM : F12 + 1% BSA and centrifuged at 350g for 5 min and then resuspended in 2 mL DMEM : F12 + 1% BSA for making smears and examining viability.

Results Enrichment of spermatogonia by plating on laminin The cell suspension added to control and laminin-coated flasks contained 8.6 ± 3.2% DBA-positive cells (DBA+ cells) (Table 1). The greatest enrichment of DBA+ cells occurred in the non-adherent fraction of control flasks (BSA-coated) incubated for 2 h or overnight. Paradoxically, enrichment of DBA+ cells occurred in both the adherent and non-adherent fractions from flasks coated with laminin under the same incubation conditions. DBA+ cells were enriched in the non-adherent fraction from laminin-coated flasks incubated overnight, and in the adherent fraction from all laminin-coated flasks. The proportion of Sertoli cells (vimentin+ cells) in adherent and non-adherent fractions was not significantly greater than the proportion in input cells for any treatment, although differences did occur between treatments in the proportion of vimentin+ cells in both fractions (Table 1). Viability of cells was comparable in the input fraction (83.0 ± 3.0%) and non-adherent fractions from flasks incubated for 2 h (range: 82.1 ± 3.8 to 84.5 ± 5.5%) or overnight (range: 71.5 ± 3.5 to 75.0 ± 4.1%). Similar viability was observed in adherent fractions (range: 75.1 ± 4.9 to 80.5 ± 4.3%).

Assessment of spermatogonia and Sertoli cell populations The proportion of spermatogonia and Sertoli cells in samples collected before and after enrichment were assessed by

Enrichment of spermatogonia by plating on DSA Greatest enrichment of DBA+ cells was seen in the non-adherent fraction from control flasks incubated overnight (BSA-coated,

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Table 2. Effect of DSA plating on DBA+ and vimentin+ cell populations (n = 3) Input cells were incubated in control (BSA-coated only) or DSA-coated flasks for 1 h or overnight (16–18 h). Adherent and non-adherent cells were collected and stained for DBA and vimentin to identify Type A spermatogonia and Sertoli cells, respectively. Results calculated as (yield of DBA+ or vimentin+ cells in input)/(number DBA+ or vimentin+ cells in final fraction) and expressed as %. Within each column, values (mean ± s.e.m.) with different superscript letters are significantly different (P < 0.05). n.a., results not available DBA (%) Non-adherent Input cells Control Control 10 µg mL−1 10 µg mL−1 20 µg mL−1 20 µg mL−1

– 1h Overnight 1h Overnight 1h Overnight

Yield of DBA+ cells (% of input)

Vimentin (%) Adherent

7.4 ± 2.3a 16.9 ± 7.3c 4.6 ± 2.6a 30.5 ± 5.2b 8.1 ± 3.0a 17.3 ± 5.4c 9.5 ± 2.7a 22.1 ± 3.6c 10.2 ± 5.9a 16.6 ± 4.6ac 14.2 ± 4.1a 20.1 ± 2.2c 10.5 ± 4.3a

Table 2). Significant enrichment was also seen in the nonadherent fraction from culture on DSA. The proportion of DBA+ cells in the adherent fraction did not differ from that in input cells for any treatment. Overnight culture in DSA-coated flasks resulted in significant depletion of vimentin+ cells from the nonadherent fraction. Vimentin+ cells in the adherent fractions were not determined. Viability of unattached cells was significantly decreased following overnight incubation in DSA flasks (68 ± 3.2 to 70 ± 2.9%), compared with control (89 ± 1.5%) and 1 h incubations (85 ± 4.4 to 88 ± 3.0%; P < 0.05). DBA-FITC staining for positive selection methods Saturation binding of spermatogonia with DBA-FITC is desirable for purification methods employing positive selection via this ligand. The initial isolation and cells enriched by differential plating for 2 h on uncoated flasks were labelled with DBA-FITC at concentrations of 0, 0.03, 0.06, 0.125, 0.25 and 0.5 mg mL−1 and examined by flow cytometry to determine the optimal concentration for staining cells. The percentage of positive cells reached a plateau at 0.12 mg mL−1 (Fig. 1, lower panel). Mean fluorescent intensity was relatively constant between 0.03 and 0.12 mg mL−1 then continued to increase up to the maximum concentration examined (Fig. 1, upper panel). FACS isolation Sorting by flow cytometry resulted in an increase in DBA+ cells from 12.3 ± 1.7% in the input sample to 50.5 ± 3.8% in the sorted fraction, with a 3.9 ± 1.0% recovery rate of DBA+ cells (n = 3). Viability did not differ between input cells (88.6 ± 2.7%) and sorted cells (82.8 ± 0.6%). MACS isolation Three methods for purifying DBA-FITC-labelled cells by MACS were examined. Ten and 20 µL of MicroBeads coated with antibody to FITC were compared after a single column passage, and 10 µL of MicroBeads were submitted to two column passages. All methods resulted in a significant increase in DBA+ cells

Non-adherent 50.3 ± 5.1a 40.2 ± 3.2abc 35.2 ± 5.6abc 40.6 ± 5.1ab 32.1 ± 5.3bc 47.7 ± 5.6ab 21.6 ± 6.4c

Adherent

Non-adherent

Adherent

n.a. n.a. n.a. n.a. n.a. n.a.

n.a. 69.8 ± 4.1a 58.9 ± 4.5a 45.4 ± 5.0b 51.2 ± 4.5b 49.4 ± 6.4b 42.4 ± 5.9b

n.a. n.a. n.a. n.a. n.a. n.a. n.a.

800 600 400 Initial Enriched

200 0

Mean fluorescent intensity

Incubation time

DBA-positive cells (%)

Treatment

30 20 10 0 0

0.03

0.06

0.13

0.25

0.5

DBA-FITC concentrations (mg mL⫺1)

Fig. 1. Percentage of DBA-FITC-positive cells in titration staining of testicular cells (lower panel) and mean fluorescent intensity of DBA-FITCpositive cells (n = 2) at each DBA-FITC concentration (upper panel). Initial isolations were obtained via a two-step enzymatic digestion. Enriched cells were non-adherent fraction collected from the incubation of the initial cells in a BSA-coated flask for 2 h. Initial isolation and enriched cells were labelled with DBA-FITC at different concentrations and the percentages of DBA-FITC-positive cells and mean fluorescent intensity of positive cells determined by flow cytometry. Mean fluorescent intensity is a flow cytometry parameter that is proportional to the intensity of staining of target cells by the fluorescent ligand, in this instance DBA-FITC. Values are expressed as mean ± s.e.m.

from 12.1 ± 1.4% of input cells to 42.1 ± 2.7–59.8 ± 3.5% in the positively selected fractions (Table 3 and Fig. 2). For a single column passage, enrichment was significantly greater for 20 µL than for 10 µL of MicroBeads. For 10 µL of MicroBeads, two column passages resulted in greater enrichment than one passage. For cells incubated with 10 µL of MicroBeads, recovery of DBA+ cells was comparable for one and two column passages but less than the recovery of cells incubated with 20 µL of MicroBeads following a single passage. Viability did not differ between groups.



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Table 3. Percentage of DBA+ and viable cells and recovery rate of DBA+ cells in the magnetic fraction of MACS separation (n = 3) Within each column, values (mean ± s.e.m.) with different superscript letters are significantly different (P < 0.05). The effects of volume of anti-FITC microbeads and number of column passages on the isolation efficiency were compared. Calculation of yield of DBA+ cells (% of input) = (total cell number in magnetic fraction × DBA+ %)/(initial cell number × DBA+ %) Microbeads (µL)

Column passages

DBA-FITC-positive cells (%)


Viability (%)

Input cells 10 10 20

– 1 2 1

12.1 ± 1.4a 42.1 ± 2.7b 59.8 ± 3.5c 55.6 ± 3.3c

– 4.11 ± 0.8a 3.31 ± 1.1a 21.1 ± 1.9b

92.2 ± 3.8a 87.3 ± 1.0a 82.7 ± 3.9a 85.8 ± 5.4a

Table 4. Enrichment and yield of DBA+ cells by density gradient centrifugation on Percoll (n = 3) Within each column, values (mean ± s.e.m.) with different superscript letters are significantly different (P < 0.05). The percentages of DBApositive cells were counted from each density interface. Calculation of yield of DBA+ cells (% of input) = (total cell number in each interface fraction × DBA+ %)/(initial cell number × DBA+ %)

100

MACS.001

40

60

DBA-FITC 0.37%

M1

Density below fraction (g mL−1 )

DBA+ (%)


Viability (%)

Input cells 1.0413 1.0513 1.0542 1.0611

13.1 ± 1.4a 11.3 ± 1.8a 26.5 ± 1.9b 38.2 ± 2.3c 12.2 ± 1.9a

– 31.4 ± 6.5a 49.6 ± 4.2b 29.4 ± 2.6a 7.7 ± 3.8c

83.4 ± 3.5a 77.2 ± 2.9ab 85.3 ± 7.0a 93.1 ± 1.0ac 87.0 ± 2.9a

0

20

Counts

80

M2

100

101

103

104

100

MACS.002

80

M2

40

60

M1

DBA-FITC 59.49%

0

20

Counts

102 DBA-FITC

100

101

102 DBA-FITC

103

104

100

MACS.003

DBA-FITC 2.46%

40

60

M1

0

20

Counts

80

M2

100

101

102 DBA-FITC

103

104

Percoll isolation The highest proportion of DBA+ cells was found at the interface of densities at 1.0542 and 1.061 g mL−1 while the highest yield of DBA+ cells was found at the interface of Percoll at 1.0513 and 1.0542 g mL−1 (Table 4). Viability did not differ from that of input cells at any interface. Comparison of efficiency of enrichment methods The yield and purity of DBA+ cells achieved by each method is compared in Table 5. While statistical comparisons between methods were not possible, both the highest purity and highest yield were achieved by differential adhesion of cells in BSAcoated control flasks. Similar enrichment was achieved by this method and by positive selection by FACS and MACS; however, positive selection by the latter methods resulted in poor recovery of DBA+ cells. Discussion

Fig. 2. FACS analysis of MACS-purified cells. Testicular cells were gated on forward scatter and side scatter and analysed for FITC fluorescence. Top panel, unstained cells; middle panel, enriched-magnetic fraction cells (59.49% DBA-FITC-positive); bottom panel, non-magnetic fraction cells (2.46% DBA-FITC-positive). Boundary markers M1 and M2 identify DBAFITC-negative and -positive cells, respectively.

In the present study, we have explored several approaches to enrich bovine spermatogonia and systematically compared the efficiency of each method. In consideration of purity and recovery rate, differential plating on control flasks treated with BSA was an efficient and convenient method for enriching large

3.3 2.9 4.7 2 4.6 4.1 52.90 52.50 76.40 50.00 21.10 16.00

Fold increase in DBA+ % cells (% of input)

28.6 ± 7.9 22.1 ± 3.6 40.3 ± 1.9 26.5 ± 1.9 55.6 ± 3.3 50.5 ± 3.8 8.6 ± 3.2 7.4 ± 2.3 8.6 ± 3.2 13.1 ± 1.4 12.1 ± 1.4 12.3 ± 1.7 Adherent cells Non-adherent cells Non-adherent cells Interface (1.0413–1.0542 g mL−1 ) Positively selected cells Sorted cells Laminin DSA BSA-coated flask (2 h incubation) Percoll MACS (20 µL beads, 1 passage) FACS

1.72 × 106 1.48 × 106 1.72 × 106 2.66 × 106 2.42 × 106 2.46 × 106

0.25 × 106 0.77 × 106 1.51 × 106 1.29 × 106 0.51 × 106 0.394 × 106

% Number % Number

Fraction Treatment

DBA+ input cells

DBA+ cells recovered

Yield

DBA+

Enrichment efficiency


Table 5. Comparison of enrichment and yield of DBA+ cells by each method The best result for each technique is presented for comparison. As input cell number varied between techniques, the number of input cells has been adjusted to the number of cells used in the laminin adherence protocol (20 × 106 )

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numbers of cells as are required for germ cell transplantation, while positive selection of DBA-FITC-labelled cells by MACS or FACS was suitable for obtaining highly purified spermatogonia suitable for long-term culture. Isolation of testicular cells by adhesion to laminin resulted in a 3- to 4-fold increase in colonisation efficiency following transplantation compared with the control cells in mice (Shinohara et al. 1999). Similarly, differential adhesion to laminin resulted in an 8.5-fold enrichment in rats (Orwig et al. 2002). The results from the present study show that the highest enrichment (3.3-fold) of DBA+ cells on laminin-coated flasks was achieved with a coating of 20 µg mL−1 . Previously, it has been suggested that the incubation of testicular cells overnight allows more Sertoli cells to bind to germ cells making them more difficult to remove by the rinsing procedures. However, selection of spermatogonia on laminin-coated flasks achieved only a two-fold enrichment in the pig, and the species-specific expression of integrin, receptor for laminin, may have contributed to this low efficiency (Luo et al. 2006). Interestingly, the highest enrichment (40.3 ± 1.9%) was produced in the non-adherent fraction of control flasks treated with BSA incubated for 2 h. Differential plating has been employed to enrich spermatogonia in different species by simply eliminating somatic cells through adhesion to the plastic culture dish or flask (Dym et al. 1995; Izadyar et al. 2002; Herrid et al. 2006). In previous studies, various incubation times of 2–4 h or overnight were used in different species, so we directly compared 2 h incubation v. overnight (16–18 h) in the present study. The percentage of DBA- and vimentin-positive cells did not differ significantly between the two time points; however, the incubation period of 2 h tended to have more DBA-positive and less vimentin-positive cells. In a previous study, differential plating of bovine testicular cells overnight in uncoated flasks resulted in a 2-fold enrichment from 25% to 45% (Izadyar et al. 2002), while we obtained a 3.6-fold enrichment in overnight culture from 8.6% to 30.6%. The elimination of Sertoli cells from testicular cell preparations is a further strategy to enrich spermatogonia.This technique is of particular relevance when prepubertal testes are used as the cell source given that prepubertal testes contain mainly Sertoli and germ cells (gonocyte or spermatogonia) in their seminiferous tubules (Herrid et al. 2007). Scarpino et al. (1998) demonstrated that a 95% pure Sertoli cell population could be achieved in rats by incubating cells in DSA-coated culture dishes. In the present study, 22.1 ± 3.6% of cells found in the non-adherent cell fraction following overnight incubation in DSA-coated flasks were DBA-positive cells. These results indicate that DSA specifically binds to bovine Sertoli cells and can be successfully employed to separate germ cells from Sertoli cells in bovine testis. The enrichment of spermatogonia with discontinuous Percoll density gradients has also been examined in the present study. The DBA-positive cells were mostly enriched at the interface of densities at 1.0542 and 1.061 g mL−1 and at 1.0513 and 1.0542 g mL−1 , which is in agreement with a previous study where type A spermatogonia were enriched at the interface of densities at 1.0542 and 1.0564 g mL−1 in rats (van Pelt et al. 1996). The 2.9-fold enrichment accomplished in the present study is comparable with the 3.6-fold increase in spermatogonia



reported previously in sheep (Rodriguez-Sosa et al. 2006). However, it is important to note that the process of Percoll-gradient centrifugation may alter the surface membrane lipid profile in spermatozoa (Furimsky et al. 2005). A similar interaction with spermatogonia might affect the ability of these cells to be cultured in vitro or to colonise testes. Previously, transplantation of type A spermatogonia enriched by Percoll-gradient centrifugation (70% type A spermatogonia) resulted in successful colonisation in autologous but not in homologous recipients (Izadyar et al. 2003). However, the comparatively low yield of DBA+ cells recovered from Percoll centrifugation suggests that this method will not readily yield the large quantities of type A spermatogonia needed for transplantation, although cells from two bands could be combined as shown by (Rodriguez-Sosa et al. 2006). Although several surface markers for spermatogonia stem cells are available in rodent for positive selection of cells by MACS and FACS, DBA is presently the only ligand available for use in cattle (Herrid et al. 2007). This is the first attempt to purify DBA+ bovine cells by MACS.The 4.6-fold enrichment and 4.6% recovery rate for DBA+ cells achieved by MACS is comparable to the results of immunomagnetic selection of testicular cells with anti-CD9 antibody, where a 6-fold enrichment with 7.5% recovery rate was achieved in mice and rats (Kanatsu-Shinohara et al. 2004). The use of two columns improved the purity of the spermatogonial fraction but at the expense of recovery, which was almost halved. However, increasing the absorptive surface with twice as many beads in the column increased the purification and the yield. FACS produced similar results. In summary, all methods used in the present study resulted in a significant increase in DBA-positive cells from around 7–12% of input cells to 45–60% following purification. Incubation of testis cell suspensions in BSA-treated flasks for a 2-h period was found to be the best method of recovering large numbers of type A spermatogonia, while the MACS method proved to be a fast and effective way to select highly enriched type A spermatogonia. It is noted, however, that the lack of type A spermatogonia surface markers specific to ruminant species may limit the broader application of the MACS technique for purifying testis stem cells in livestock. Acknowledgements We thank Brendan Hatton and Andrew Eichorn for assistance with experimental animal management and funding from the CSIRO Food Future National Research Flagship and CSIRO Livestock Industries. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.

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