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Background: Gonocytes give rise to spermatogonial stem cells, and thereby play an essential role in establishing spermatogenesis. Optimized culture conditions for gonocytes provide an opportunity for their study and in vitro manipulation for potential application in reproductive technologies. Using six experiments in a step-wise design, we examined the effects of several culture conditions on the maintenance, proliferation, and colony formation of porcine gonocytes. Testis cells from neonatal piglets were cultured for 7 d in DMEM supplemented with 10% fetal bovine serum. The examined culture conditions included using different cell seeding densities, gonocyte proportions, incubation temperatures, sampling strategies, and medium changing regimens. 4 2 Results: Confluency of cells was optimal (>90% by ~6 d) when 3.0 × 10 testis cells/cm containing ~40% gonocytes were used. Incubating the cells at 35 °C or 37 °C resulted in similar cell number and viability at confluency, but incubation at 35 °C resulted in a delayed confluency. In the first 2 d of culture, gonocytes remained mostly floating in the medium and gradually settled over the next 5 d. Consequently, not changing the medium for 7 d (as opposed to changing it every 2 d) led to a significant increase in the number of gonocyte colonies by reducing the loss of “floating gonocytes”. Conclusion: We found that gonocytes require the presence of a critical minimum number of somatic cells for settlement, and can proliferate and form growing colonies even in a basic medium. Large numbers of viable gonocytes remain floating in the medium for several days. The optimized culture conditions in the present study included seeding 4 2 with 3.0 × 10 testis cells/cm containing ~40% gonocytes, incubating at 37 °C, and without changing the medium in the first week, which can result in improved colony formation of porcine gonocytes. Keywords: Cell culture, Male Germline stem cells, Pigs, Testis Background SSCs were deposited in the lumen of seminiferous tubules Testis stem cells are a unique population of stem cells in of recipient testes using germ cell transplantation an adult body, because in addition to their dual ability to technique. The transplanted SSCs were capable of homing self-renew and give rise to differentiating germ cells, to the tubule basement membrane, forming new colonies they can pass the genetic information to the offspring. of developing germ cells, and initiating donor-derived Among the many types of male germ cells present from spermatogenesis [2–7]. In breeding trials, some of the the fetal period to adulthood, primordial germ cells germ cell recipient animals of various species were capable (PGCs), gonocytes, and particularly spermatogonial stem of siring donor-derived progeny [2, 6, 8]. There are also a cells (SSCs) are believed to possess stem cell potential few reports of using gonocytes and PGCs as donor cells in and hence are referred to as male germline stem cells germ cell transplantation leading to the establishment of (MGSCs) . An in vivo functional assay for confirming spermatogenesis in the recipient testis [3, 9, 10]. the stem cell potential of MGSCs was provided when The choice of donor MGSCs is a critical factor for the outcome of germ cell transplantation. Using PGCs as * Correspondence: email@example.com donor cells has been less practical because their isolation Department of Veterinary Biomedical Sciences, Western College of Veterinary requires collection from embryos and there are only a lim- Medicine, University of Saskatchewan, 52 Campus Drive, Saskatoon, SK S7N ited number of PGCs per embryo [3, 11]. Spermatogonia 5B4, Canada © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Awang-Junaidi and Honaramooz Journal of Animal Science and Biotechnology (2018) 9:8 Page 2 of 13 can be collected from mature donor individuals and model. In the present study, we performed six experiments enriched for SSCs at relatively high numbers. However, in a stepwise design and examined several aspects of testis spermatogonia are inherently a heterogeneous population cell culture including factors related to the seeding density, of cells and the lack of biomarkers that allow unequivocal gonocyte proportion, incubation conditions, sampling, isolation of SSCs has hampered obtaining pure popula- medium changing regimens, and effects of applying those tions [12–14]. Compared with PGCs and SSCs, gonocytes optimal conditions on gonocyte colony formation. offer a potentially more practical option. Gonocytes are transitory germ cells (between PGCs and SSCs) that have Methods a distinct morphology (large round cells with large nu- Testis collection and preparation cleus to cytoplasm ratio), and are present during both fetal Testes from Yorkshire-cross piglets (Camborough-22 × and early postnatal period for several days, months or Line 65; PIC Canada, Winnipeg, MB, Canada) less than 1 even years, depending on the species [15–17]. Perhaps wk. of age were used in this study. The testes were more importantly, gonocytes are the only germ cells collected weekly (5–15 pairs per week) through aseptic present in the testes of neonates; thus, they can be de- castration at a university–affiliated swine facility. The testes tected using most germ cell markers (e.g., VASA, DAZL), were kept in ice-cold Dulbecco’s phosphate-buffered saline pluripotency markers (e.g., NANOG, OCT4), and certain (DPBS; catalogue no. 20–031-CV; Mediatech, Manassas, surface protein markers (e.g., DBA, UCH-L1). These char- VA, USA), containing 1% w/v antibiotics solution acteristics provide an opportunity for enrichment and in (penicillin and streptomycin; catalogue no. 30–002-CI, vitro manipulation of gonocytes [17–20]. Mediatech), and were transported within 1 h of collection Gonocytes are relatively limited in numbers since they to the laboratory. The testes were thoroughly rinsed three comprise only ~1.4% of cells in the testes of neonatal rats times with DPBS prior to processing. The parenchyma was  and ~7% of seminiferous cord cells in piglets . separated from the tunica albuginea and excess connective The efficiency of colonization by the donor germ cells in tissues, and used for isolation of testis cells. the seminiferous tubules of recipients is directly propor- tional to the number of transplanted MGSCs. Therefore, Isolation of testis cells and enrichment for gonocytes to increase the efficiency of germ cell transplantation, Isolation of testis cells and enrichment of gonocytes were gonocytes must be enriched and/or propagated in vitro performed using methods that were established in our prior to transplantation, as suggested for SSCs [23, 24]. laboratory (three-step enzymatic digestion, Nycodenz Various strategies have been introduced to optimize the density gradient centrifugation and extracellular matrix isolation, enrichment, or purification of gonocytes with (ECM) differential plating; [29, 30]). Briefly, for each batch differing outcomes, depending on the species [18, 25, 26]. of the three-step enzymatic digestion, ~600 mg of testis Using conventionalcellseparation methods,no more than parenchyma was thoroughly minced with fine scissors for ~10% of isolated neonatal testis cells were gonocytes [21, 5 min, suspended in 5 mL of DPBS, vortexed for 30 s in a 22, 27, 28]. However, development of a three-step test tube shaker (Reax Top; catalogue no. 541–10000; enzymatic digestion in our laboratory has allowed the Heidolph Instrument, Essex, UK) and digested with 1 mL collection of populations of cells containing ~40% of 0.2% w/v collagenase IV (catalogue no. C-153; Sigma- gonocytes from neonatal porcine testes . The resultant Aldrich, Oakville, ON, Canada), 0.1% w/v hyaluronidase population of gonocytes can also be enriched further to (catalogue no. H-3884; Sigma-Aldrich), and 0.01% DNase ~90% using a combination of Nycodenz centrifugation (catalogue no. DN25; Sigma-Aldrich) in Dulbecco’smodi- and differential plating . fied Eagle’s medium (DMEM; catalogue no. 10–013-CM; While pigs are an important biomedical animal model, Mediatech) supplemented with 1% w/v antibiotics (as purification of porcine MGSCs is still limited. The ability above) at 37 °C for 10 min. Fetal bovine serum (FBS; cata- to obtain highly enriched populations of gonocytes from logue no. A15–701; PAA Laboratories, Etobicoke, ON, neonatal piglets [29, 30], however, has provided new Canada) was added to stop the digestion and the suspen- opportunities for using gonocytes as a model for in vitro sion was vortexed for another 30 s and filtered through a propagation and manipulation of MGSCs. This calls for 40-μm filter (catalogue no. 3522340; BD Biosciences, San further research on culture systems that can allow efficient Jose, CA, USA). The filtrate suspension was centrifuged at large-scale propagation of gonocytes. No systematic study 500×g at 16 °C for 5 min and resuspended in 5 mL of has examined the effects of different culture conditions on DPBS supplemented with 1% w/v antibiotics. Erythrocyte colony formation of porcine gonocytes. Therefore, given depletion was performed by mixing the cells with 20 mL the importance of cell culture in MGSC research, the of the lysis buffer, composed of 156 mmol/L ammonium present study was designed to examine a number of chloride (NH Cl; catalogue no. A9434; Sigma-Aldrich), culture conditions for maintenance, proliferation, and 10 mmol/L potassium bicarbonate (KHCO ; catalogue no. colony formation of neonatal porcine gonocytes as a 237205; Sigma-Aldrich), and 0.1 mmol/L disodium Awang-Junaidi and Honaramooz Journal of Animal Science and Biotechnology (2018) 9:8 Page 3 of 13 ethylenediaminetetraacetate (Na EDTA; catalogue no. pellets were collected and subjected to resuspension into a E6635; Sigma-Aldrich) in sterile distilled water prior to an- single cell suspension prior to assessment. Trypan blue other cycle of centrifugation (500×g at 16 °C for 5 min). Fi- (100 μL of a 0.4% solution in saline, catalogue no. T8154; nally, the cell pellet was resuspended in 5 mL of DMEM Sigma-Aldrich) was mixed (1:1 ratio) gently with the cell supplemented with 10% v/v FBS and 1% antibiotics suspension, and 20 μL of the mixture was transferred to ++ (DMEM ) and underwent gentle pipetting to obtain a each hemocytometer chamber and live/dead cells were single cell suspension. identified with the aid of a light microscope. For each For enrichment of gonocytes among the cells obtained replicate, two counts of live/dead cells were per- from the three-step enzymatic digestion, 3 mL of 17% formed and averaged to calculate the yield and viabil- Nycodenz in DPBS (Histodenz; catalogue no. D2158; ity of cells (given as a percentage). Immunostaining Sigma-Aldrich) was placed at the bottom of a 15-mL was performed to confirm and quantify gonocytes, as graduated conical tube. This was followed by gentle they are the only type of germ cells present in neo- addition of 2 mL of cell suspension on top and the tube natal pig testes; hence the remaining cells were cate- was centrifuged at 500×g at 4 °C for 15 min. The super- gorized as somatic cells. For immunostaining, a natant was discarded and the pellet was harvested and minimum of 600 cells in randomly selected fields resuspended as a single cell suspension. Prior to ECM were counted in each cell smear and the proportion differential plating, 6-well culture plates (catalogue no. (%) of gonocytes/somatic cells was calculated. 353046; BD Biosciences) were coated with 1 mL of 50 μg/mL poly-D-lysine (catalogue no. 47743–736; VWR International, Mississauga, ON, Canada) and 10 μg/mL Cell culture fibronectin (catalogue no. 477743–728; VWR) in an in- Different cell culture conditions were evaluated using a ++ cubator with 5% CO at 37 °C for 1 h, and dried in a stepwise approach. The same medium (DMEM ), cul- biosafety cabinet for another 1 h. The poly-D-lysine pre- ture plates (6-well plates), and incubator conditions (5% coated wells were rinsed twice with DPBS before seeding CO and 95% humidity) were used in all experiments in the cells obtained from Nycodenz gradient density cen- order to evaluate the effects of changing other variables trifugation. The cells were seeded onto the plates at a (i.e., gonocyte proportion, incubation temperature, cell 5 2 ++ concentration of 2.5 × 10 cells/cm in DMEM , and seeding density, and medium changing regimen). For all cultured in an incubator with 5% CO at 37 °C. After 2 experiments, pH of the medium was adjusted to 7.4 im- to 3 h, the floating cells were harvested, centrifuged at mediately prior to using it in cell cultures. The pH of 500×g at 16 °C for 5 min and the pellets were collected the medium was measured using a digital pH meter and resuspended as a single cell suspension. These pro- (catalogue no. 11288–368; model Symphony SB70P; cedures lead to obtaining isolated testis cells highly VWR). Cell growth was monitored at least once daily enriched in gonocytes. We performed a side-by-side using an inverted phase contrast microscope (Nikon, quantitative comparison of the cell yield, viability and Eclipse TS100). For each experiment, multiple replica- loss after each progressive step. Samples were prepared tions were performed where cells obtained from each from single cell suspensions obtained from each step. batch of testis tissue digestion were considered one rep- The suspension was smeared onto poly-L-lysine pre- lication. We have previously shown that the characteris- coated glass coverslips, dried overnight in a biosafety tics of cells obtained from 1-week old piglets from this cabinet and kept at −20 °C for use in immunostaining. source remain consistent over time, both within and among litters . Cell quantification and viability assessment Effects of several factors on the behavior and the The number of resultant cells after each isolation and en- morphology of cultured testis cells were examined using richment step as well as after culturing was quantified, cytology and immunostaining. The examined factors in- and the cell viability was assessed using the trypan blue cluded different gonocyte proportions obtained before or exclusion technique. For evaluation of cells collected after after each step of enrichment (40% vs. 80% vs. 90%), in- isolation and enrichment, single cell suspensions were cubation temperature (35 °C vs. 37 °C), cell seeding 5 5 5 prepared as described above. For cultured cells, detach- density (1.0 × 10 vs. 3.0 × 10 vs. 5.0 × 10 cells/well; ment of the adherent cells was performed by adding 1 mL each well in a 6-well plate has ~10 cm cell growth area) of 0.25% (w/v) trypsin in Hank’s balanced salt solution and medium changing regimen (every 2 d vs. modified (HBSS) and 2.21 mmol/L EDTA (catalogue no. 25–053- regimen vs. no changing of medium for up to 7 d). Con- CI; Mediatech) at 37 °C for 1–2 min (depending on con- fluency assessment was defined as the relative coverage fluency) with gentle agitation. The enzymatic digestion of the plate surface by the mixture of testis cells, and a was stopped by adding 1 mL of undiluted FBS, and the gonocyte colony was defined as a group of more than mixture was centrifuged at 500×g at 16 °C for 5 min. The three gonocytes in close contact. Awang-Junaidi and Honaramooz Journal of Animal Science and Biotechnology (2018) 9:8 Page 4 of 13 Sampling were blocked with 5% bovine serum albumin (BSA; For sampling, glass coverslips coated with 0.1% w/v poly- catalogue no. 0332, Amresco, Solon, OH, USA) at L-lysine were used. Glass coverslips sized 22 × 22 mm 37 °C for 30 min in a humidified chamber. The (catalogue no. 12-540B; Fisher Scientific, USA) were ar- blocking agent was removed and the samples were in- ranged in a sterile metal coverslip rack and immersed in cubated with fluorescein-conjugated lectin DBA 1% HCl in 70% alcohol for 10 min, followed by rinsing (1:100, catalogue no. FL-1031, Vector Labs, Burling- with sterile distilled water for 5 s and allowed to dry for ton, ON, Canada) overnight at 4 °C in a humidified 20 min. The rack was transferred into a 0.1% w/v poly-L- chamber. The samples underwent another cycle of lysine (catalogue no. P8920; Sigma-Aldrich) solution for rinsing, followed by incubation with 0.3% Sudan Black 10 min and allowed to dry overnight. Finally, the glass B w/v (catalogue no. S2380; Sigma-Aldrich) in 70% al- coverslips were placed at the bottom of each well in 6-well cohol for 15 min, at 37 °C in a humidified chamber. The plates. These procedures were conducted under a bio- samples were rinsed with DPBS and stained with 4′6’-dia- safety cabinet. midino-2-phenyilindole (DAPI; catalogue no. D9542; Sigma-Aldrich) and observed under a fluorescence micro- Immunostaining scope. Fluorescein-conjugated lectin DBA specifically la- Gonocytes were identified by immunostaining using bels gonocytes which appear green under the fluorescence Dolichos biflorus agglutinin (DBA) according to previ- microscope while DAPI stains the nuclei of all cells which ously described protocols [29, 30], with minor modifi- appear blue (Fig. 1). cations. Briefly, the dried coverslips (with cell smears or with cells grown directly on them) were fixed in Statistical analyses Bouin’s solution (catalogue no. 1120–31; Richa Chem- Unless stated otherwise, all data are presented as means ical Company, Pocomoke City, MD, USA) for 2– ± standard error of mean (SEM) and analyzed using ei- 3 min. The samples were then rinsed three times with ther one-way or two-way analysis of variance (ANOVA), 70% alcohol to remove excess Bouin’s solution as appropriate, followed by Tukey’s HSD post-hoc test. followed by three rinses with DPBS. The samples For percentages, the data were transformed using Fig. 1 Dolichos biflorus agglutinin (DBA) immunostaining of neonatal gonocytes. a–c Gonocyte immunostaining performed on testis cell smears collected immediately following cell isolation. d–f Multiple gonocytes form a colony on a somatic-cell monolayer observed at 1 wk of culture. g–i Floating gonocytes in the removed medium observed 4 d after incubation. b, e, h Fluorescein-conjugated DBA-labeled gonocytes appear green under the fluorescence microscope. a, d, g DAPI-stained nuclei of the cells appear blue. c, f, i Merged images Awang-Junaidi and Honaramooz Journal of Animal Science and Biotechnology (2018) 9:8 Page 5 of 13 Arcsine function prior to analysis using ANOVA. The while the 3.0 × 10 seeding density group of cells were level of significance was set at P < 0.05. Data were >90% confluent by ~Day 6. Cells cultured at 1.0 × 10 analyzed using SPSS (Version 20.0; SPSS, Chicago, IL, seeding density had slow growth and only reached ~30% USA). confluency after 7 d in culture. In a separate experiment, testis cells enriched for gon- Results ocytes (undergoing both Nycodenz centrifugation and As summarized in Table 1, enzymatic digestion of testis ECM plating, hence containing ~86% gonocytes) were tissues yielded a population of testis cells containing ~41% used as the source of cells for culturing, using the same germ cells (Fig. 1a-c), which at this developmental stage incubation conditions. Cells did not grow in plates 5 5 (~1 wk of age) are exclusively gonocytes. Gradient centri- seeded with 1.0 × 10 or 3.0 × 10 cells/well and only had fugation by Nycodenz, followed by ECM differential plat- limited growth in the 5.0 × 10 seeding density group ing resulted in enrichment of gonocytes to ~80% and after 7 d in culture (5–15% confluency, data not shown). ~86%, respectively. However, the enrichment procedures Therefore, for the remaining experiments in this study also caused significant reductions (P < 0.05) in cell counts we used testis cells without enrichment (i.e., containing after each step, resulting in ~73% and ~92% total testis cell ~40% gonocytes), and since the 3.0 × 10 seeding density loss and ~47% and ~83% gonocyte loss, respectively. The resulted in confluency of cells in ~6 d (without the need relatively lower gonocyte loss at each step, compared with for passaging the cells within 1 wk), we used this seeding the total cell loss, resulted in significant enrichment of density in the subsequent experiments. gonocytes. The viability of cells was also reduced after each enrichment step (from ~93% to 84% and 82%, respectively, Exp. 2: Effect of incubation temperature P < 0.05). When the cells were cultured (3.0 × 10 cells/ The objective of this experiment was to evaluate the well at 37 °C with 5% CO and the medium was changed growth of cultured testis cells using incubation tempera- every 2 d), only the cells used immediately after the three- tures of either 35 °C or 37 °C for 1 wk (n = 12 replica- step enzymatic digestion (i.e., without enrichment) demon- tions), while other culture conditions remained the same strated growth by reaching ~90% confluency in ~6 d. (e.g., medium, cell seeding density, and CO levels). As shown in Table 3, the viability and number of cells at Exp. 1: Effect of cell seeding density and gonocyte confluency did not differ between the two incubation proportion temperatures (P > 0.05). However, the cells cultured at One of the objectives of this experiment was to deter- 35 °C reached confluency later than those at 37 °C (P < mine the optimal seeding density for culturing testis 0.05); therefore, for practical reasons we chose to culture cells. A comparison was made by randomly assigning the cells at 37 °C for subsequent experiments. 5 5 cells into 6-well plates at 1.0 × 10 ,3.0×10 ,or 5.0× 5 4 4 4 10 cells/well (i.e., 1.0 × 10 ,3.0×10 ,or5.0×10 Exp. 3: Sampling of cultured testis cells for analysis 2 2 cells/cm since each well was ~10 cm , n = 12 repli- Various methods can be used to sample cultured cells cations), in 3 mL of medium to determine the time for analysis. When sampling for applications such as im- required for the cells to reach 90% confluency. Cells munocytochemistry, coverslips are commonly pre- were used fresh (immediately after enzymatic diges- coated with certain matrices to enhance adherence of tion and without enrichment, hence containing ~40% cells. It is not clear whether the presence of the pre- gonocytes) and cultured under similar conditions at coated coverslips in testis cell culture would affect cell 37 °C, and the medium was changed every 2 d. growth. Furthermore, in situations where multiple sam- As shown in Table 2, cells in the highest seeding dens- plings from each replicate is required, it would be more ity group (5.0 × 10 ) became confluent earlier than other efficient to use all available cultured cells, including groups (P < 0.05), reaching >90% confluency in ~4 d, those grown on areas of the culture well not covered by Table 1 The population of testis cells obtained after enzymatic digestion and subsequent enrichment Method Proportion, % Cell count, × 10 * Cell loss, % ** Viability, % In vitro growth Gonocytes Somatic cells Total Gonocytes Total Gonocytes a a a a a Enzymatic digestion 40.60 ± 1.21 59.40 ± 1.21 37.84 ± 2.44 15.38 ± 1.09 –– 92.62 ± 0.59 Yes b b b b a a b Nycodenz centrifugation 80.40 ± 1.46 19.6 0 ± 1.46 10.00 ± 0.41 8.00 ± 0.21 73.18 ± 1.76 46.72 ± 4.69 84.20 ± 1.06 Limited c c c c b b c ECM differential plating 86.40 ± 1.03 13.6 0 ± 1.03 3.02 ± 0.19 2.60 ± 0.2 91.62 ± 0.79 82.61 ± 1.71 81.66 ± 0.71 Limited The results are mean ± SEM *Each replicate was based on the digestion of ~600 mg of testis tissue One-way ANOVA (or t test **) was used for statistical analyses, and P < 0.05 was considered as significant abc Within each column, data with different superscripts differ significantly (P < 0.05). n = 5 replications Awang-Junaidi and Honaramooz Journal of Animal Science and Biotechnology (2018) 9:8 Page 6 of 13 Table 2 The effect of seeding density on the growth of testis mentioned culture conditions (3.0 × 10 seeding density, ++ cells in culture DMEM ,37°C, n = 6 replications). The medium was Cell Maximum confluency changed every 2 d. Within the first 12 h of incubation, seeding most of the cells and cell particles were still floating in Confluency at days Cell count Viability, % density the medium (Fig. 3a). By 24 h after incubation, small 5 5 1.0 × 10 (*~30% at 7) (*2.98 × 10 ± 0.01) (*88.4 ± 1.02) numbers of cells settled and appeared to have adhered 5 a 5 3.0 × 10 >90% at 6.0 ± 0.17 8.10 × 10 ± 0.01 91.21 ± 0.58 to the plate. The early settling cells were mostly somatic 6 b 5 5.0 × 10 >90% at 4.0 ± 0.15 8.16 × 10 ± 0.01 91.62 ± 0.56 cells and began to form cytoplasmic projections, creating The results are mean ± SEM irregularly shaped cell boundaries (Fig. 3b), while more *The results from 1.0 × 10 seeding density group were excluded from cells continued to settle. After 2 d of incubation, the statistical analysis as the cells did not reach 90% confluency cells started to appear in clumps (Fig. 3c), and over time Independent sample t test was used for statistical analyses ab Within each column, data with different superscripts differ significantly (P < these groups of cells spread further and combined to 0.05). n = 12 replications create a large somatic-cell monolayer. At the same time, round cells with large nuclei, appearing bright or with a the coverslip. The objectives of this experiment were to halo under the phase-contrast microscopy, were seen at- determine 1) whether the rate or pattern of testis cell tached onto the somatic-cell monolayer (Fig. 3d–e). growth in culture would change in the presence of glass These rounds cells were confirmed to be gonocytes coverslips (22 × 22 mm) coated with 0.01% poly-L-lysine, using immunostaining (Fig. 1d–f). It was also observed and 2) whether the growth of testis cells on the cover- that while initially gonocytes were present as singles or slips differs from those on the peripheral areas of the pairs, starting at Day 5, colonies (grape-like clumps) of wells, not covered by the coverslips (Fig. 2). more than 3 cells could also be observed (Fig. 3d). It was When plates with and without poly-L-lysine-coated noticed that gonocytes were only loosely attached to the coverslips were compared, the number or viability of the somatic-cell monolayer, since some would detach due to resultant cultured cells did not differ between plates slight movements of the plate. By the end of the experi- 5 5 (8.18 ± 0.01 × 10 vs. 8.22 ± 0.02 × 10 cells per plate, ment (Day 7), the number of these round cell colonies with 91.0 ± 0.57% vs. 91.3 ± 0.87% cell viability, plates were still considerably less than expected, given that the with coverslips vs. plates with no coverslips, respectively, population of testis cells at seeding contained ~41% gon- n = 12 replications, P > 0.05). When non-coated cover- ocytes. The somatic cells continued to multiply and once slips were used, little or no cell growth was observed. reaching 70–80% confluency (at ~4 to 5 d), many ap- Furthermore, within the plates with poly-L-lysine-coated peared as spindle-shaped fibroblast-like cells (Fig. 3e). coverslips, the viability of cells grown on coverslips did not differ from those grown on the peripheral areas of Exp. 5: Floating cells in the culture medium the well, not covered by the coverslips (92.0 ± 0.33% vs. Based on the findings of Exps. 1 and 4, it was clear that 92.4 ± 0.51%, coverslips vs. periphery, respectively, P > gonocytes require sufficient ratios of somatic cells or 0.05). However, the number of cells obtained from the feeder cells, to which they formed loose attachment for peripheral areas was higher than that from the coverslips growth in culture. We speculated that the very low num- 5 5 (4.59 ± 0.14 × 10 vs. 2.99 ± 0.06 × 10 , respectively, P < ber of gonocyte colonies observed by Day 7 during 0.05), even though both encompassed a similar cell short-term culture (Fig. 3e) might be due to their loss growth area (~4.8 cm each). through routine medium changes, which apparently eliminated the floating and loosely adherent gonocytes. Exp. 4: Short-term culture of neonatal porcine testis cells Therefore, this experiment was designed to examine the Morphological changes of neonatal porcine testis cells in medium removed from the plates. The results showed culture were evaluated for 1 wk under the above- that indeed high numbers of round cells were present in the medium removed routinely every other day. The number of these cells was greatest in the medium re- Table 3 The effect of incubation temperature on the growth of moved on Day 2 (6.81 ± 0.63 × 10 ), compared with Days testis cells in culture 4 4 4 and 6 (1.02 ± 0.09 × 10 and 0.4 ± 0.07 × 10 cells/well, Temperature >90% confluency P < 0.05, n = 5 replications, Fig. 4). On the other hand, if Days Cell count Viability, % the medium was not changed, greater numbers of float- a 5 35 °C 6.42 ± 0.15 7.98 × 10 ± 0.01 89.98 ± 0.49 ing gonocytes remained in the medium examined on b 5 4 4 37 °C 5.83 ± 0.17 8.27 × 10 ± 0.01 90.04 ± 0.49 Days 4 and 6 (4.2 ± 0.10 × 10 and 1.92 ± 0.05 × 10 cells/ The results are mean ± SEM well, respectively), compared with the numbers obtained Independent sample t test was used for statistical analyses ab on the same days when the medium was changed every Within each column, data with different superscripts differ significantly (P < 0.05). n = 12 replications other day (P < 0.05, n= 5 replications, Fig. 4). Based on Awang-Junaidi and Honaramooz Journal of Animal Science and Biotechnology (2018) 9:8 Page 7 of 13 Fig. 2 Schematic representation of the areas within culture plates used for sampling in Exp. 3. The numbers and viability of cells grown on poly- L-lysine-coated coverslips (a) were compared with those grown on the peripheral areas of the wells, not covered by the coverslip (b) the morphology, it was presumed that these cells were Exp. 6: Effect of medium changing regimens on attached gonocytes; they were large and round in shape with gonocytes prominent nuclei (large nucleus to cytoplasm ratio). The This experiment was designed to assess the effects of viability of these round cells remained high in all exam- different medium changing regimens on the number and ined days (~85–97%, data not shown). Using immuno- viability of attached gonocytes. The cells were cultured staining, these round cells were later confirmed to be using the selected culture conditions (3.0 × 10 testis ++ gonocytes (Fig. 1g–i). cells/well containing ~40% gonocytes, DMEM ,37°C) Fig. 3 Morphology of testis cells in short-term cell culture. a Morphology of floating cells 12 h after seeding. b Some somatic cells settled to the bottom of the plate within 24 h of incubation. c These somatic cells extended cytoplasmic projections toward one another. c–f Cells presumed to be gonocytes are seen attached to somatic cells (black arrows). The same gonocytes are also shown at a higher magnification (insets). d Gonocytes appeared as singles (black arrow), pairs (yellow arrow), or small colonies (red arrow). e Spindle-shaped fibroblast-like cells were more prominent when confluency reached >70%. Only a small number of gonocyte colonies were observed by Day 7 when routine medium changing regimen was applied. f Increased number of gonocyte colonies were observed when the medium was not changed for 7 d. Scale bar, 50 μm Awang-Junaidi and Honaramooz Journal of Animal Science and Biotechnology (2018) 9:8 Page 8 of 13 Fig. 4 Number of gonocytes upon analysis of the floating cells from the removed medium. The initial number of cells at seeding was set at 3.0 × 10 cells/well containing ~40% gonocytes and the viability was 89 ± 0.5%. The medium removed from the plates was examined and large numbers of gonocytes were seen floating in the medium on Day 2. Most of the floating gonocytes were eliminated by routine (every 2 d) medium changes ab (Routine), while some were present on Days 4 and 6, if the medium was not changed (Unchanged). The data are mean ± SEM. Data with different xyz superscripts within each day differ significantly (P <0.05). Data with different superscripts within each treatment over time differ significantly (P <0.05). n =5 replications for 1 wk, and medium changing regimens were assigned viability was reduced further in the unchanged group on to groups of plates as follows: 1) to serve as a control Days 6 and 7 (P < 0.05). Between the regimens, the viabil- group, the cells were seeded with 3 mL of medium, and ity of cells in the unchanged groups was lower (P <0.05) the medium was changed routinely, every 2 d (routine than the routine group on Days 6 and 7 of culture. group, 3 changes in 1 wk); 2) the cells were seeded with 2 mL of medium, followed by the addition of another Discussion 1 mL after 24 h, and then every 2 d 1 mL of medium In the neonatal testis, gonocytes represent MGSCs, which was removed from the well and replaced with 1 mL of have great potential applications in male reproduction re- fresh medium (modified group); or 3) the cells were search (reviewed in [31, 32]). Various methods have been seeded with 3 mL of medium but the medium was not established to culture MGSCs to facilitate their study and changed (unchanged group). On Days 2, 4, 6, and 7, the manipulation or to harness their potential for downstream cultured cells were observed under an inverted micro- applications such as genetic conservation, transgenesis, or in- scope and the number of gonocyte colonies in four ran- fertility treatments [4, 5, 33–35]. However, depending on the domly selected fields at 400× was recorded and cells donor species, age, or developmental status, optimized cul- were collected for evaluation of viability (n = 5 replica- turing or enrichment of MGSCs can be challenging [29, 36]. tions per group). Cell isolation is a critical step in preparing testis cells As shown in Fig. 5, alterations in medium changing reg- for culture. The number and proportion of the result- imens affected the number of attached gonocytes or gono- ant populations of cells also vary greatly depending on cyte colonies present on the somatic-cell monolayers. the methods used to isolate the cells. As a first step in Overall, the number of gonocyte colonies increased (P < the present study, we performed a side-by-side com- 0.05) from Day 2 to Day 4 of culture in all medium chan- parison of the total testis cell yield and gonocyte pro- ging regimens. However, among the medium changing portions from three protocols [29, 30]. Although as regimens, the unchanged group had greater (P <0.05) expected the enrichment process resulted in highly number of gonocyte colonies on Days 6 and 7 compared purified populations of gonocytes (~86%), it also led to with the routine medium changing group (control). losing up to 90% of the initial number of testis cells. The viability of cells in different medium changing regi- These findings provide a basis upon which an appropri- mens is shown in Fig. 6. Compared with the cell viability ate approach could be adopted for gonocyte isolation/ at seeding (~90%), there was a reduction in viability of enrichment in order to meet the objectives and needs cells at Days 4, 6, and 7 in all groups (P < 0.05). The cell of specific experiments. For example, the highly Awang-Junaidi and Honaramooz Journal of Animal Science and Biotechnology (2018) 9:8 Page 9 of 13 Fig. 5 Number of gonocytes or gonocyte colonies observed after applying different medium changing regimens. The cultured cells underwent one of the following medium changing regimens: 1) Seeded with 3 mL of medium and the medium was routinely changed every 2 d (Routine); 2) Seeded with 2 mL of medium, followed by the addition of another 1 mL after 24 h, and then every 2 d, 1 mL of medium was removed from the well and replaced with 1 mL of fresh medium (Modified); 3) Seeded with 3 mL of medium but the medium was not changed for 7 d ab xy (Unchanged). The data are mean ± SEM. Data with different superscripts within each day differ significantly (P < 0.05). Data with different superscripts within each treatment over time differ significantly (P < 0.05). n = 5 replications enriched populations of gonocytes could be used in cells can be due to endogenous sources (e.g., lipofuscin or studies that focus mainly on gonocytes per se, while the lipofuscin-like pigments), or acquired during processing heterogeneous populations of testis cells obtained (e.g., certain fixatives). In any case, autofluorescence can immediately after enzymatic digestion may be more interfere with specific signals by immunostaining and lead suitable for longer term culture or for the study of to false positive results. In the present study, the autofluo- interactions between gonocytes and somatic cells. rescence was eliminated through modification of the One of the advantages of using neonatal testes as the quenching protocol . The use of Sudan black B (SBB) source of germ cells is that gonocytes are the only type has been reported to be efficient in masking autofluores- of germ cells present during the early postnatal stage. cence of lipofuscins, triglycerides, and lipoproteins origin Gonocytes possess a distinct morphology and a unique [43, 44] and is a major component of the quenching proto- pattern of distribution in the seminiferous cords of col for testis cells . In this study, as a first step in modi- neonatal testes, which facilitate their identification in fying the original quenching protocol, the length of situ [18, 21]. Once the cells are enzymatically dissoci- incubation with SBB was extended, which led to further re- ated; however, accurate identification of gonocytes re- duction of the autofluorescence of testis cells, but not to its quires immunocytochemistry. There are several complete elimination. We then speculated that the biomarkers that could be used for identification of gono- remaining autofluorescence might have a fixative origin. cytes including germ cell markers (e.g., VASA and DAZL, The picric acid component of Bouin’s solution used for fix- [37, 38]) and stem cell markers (e.g., Nanog, SSE-A1, and ing testis tissue has also been cited as a potential cause of OCT4, [18, 20, 39, 40]). Two of the most commonly used autofluorescence [45, 46]. Therefore, we applied further markers for neonatal porcine gonocytes are DBA [18, 19, control measures to refine the use of fixatives in the 41] and ubiquitin C-terminal hydrolase L1 (UCH-L1; pre- quenching protocol. We found that inclusion of three thor- viously known as PGP9.5) [41, 42]. DBA has a specific ough and gentle rinses with 70% alcohol after the Bouin’s affinity to bind to α-D-N-acetyl-galactosamine, which is fixation completely eliminated the autofluorescence prob- localized on the surface of porcine gonocytes (i.e., primi- lem in Bouin’s fixed testis cell samples. As an alternative, tive germ cells) , while UCH-L1 can identify both we also found that to avoid the autofluorescence caused by primitive and more advanced germ cells. Bouin’s, cell smears or cells grown on coverslips can be In the present study, identification of gonocytes was ini- fixed using methanol at −20 °C for 10 min. tially hampered by autofluorescence, which was partly over- Cell seeding density influences the performance of cul- come by using a quenching protocol ; however, some tured cells, presumably by affecting the contact and in- autofluorescence remained. Autofluorescence in the tissue/ teractions among cells . Optimization of the cell Awang-Junaidi and Honaramooz Journal of Animal Science and Biotechnology (2018) 9:8 Page 10 of 13 Fig. 6 Viability of cells at seeding and after applying different medium changing regimens. The cultured cells underwent one of the following medium changing regimens: 1) Seeded with 3 mL of medium and the medium was routinely changed every 2 d (Routine); 2) Seeded with 2 mL of medium, followed by the addition of another 1 mL after 24 h, and then every 2 d, 1 mL of medium was removed from the well and replaced with 1 mL of fresh medium (Modified); 3) Seeded with 3 mL of medium but the medium was not changed for 7 d (Unchanged). Viability on Day ab 0 represents the initial viability at seeding. The data are mean ± SEM. Data with different superscripts within each day differ significantly (P < xyz 0.05). Data with different superscripts within each treatment over time differ significantly (P < 0.05). n = 5 replications seeding density prior to culturing cells ensures normal ionic charges among the cells (polyanionic) and poly-L- cell growth and allows estimation of the time to reach lysine-coated coverslip surface (polycationic) enhance certain confluency levels. In the present study, the cell efficient attachment of cells onto the coverslips. In the 5 2 seeding density of 3.0 × 10 /well (i.e., in ~10 cm )was present study, we showed that 1) the presence of poly-L- found to be more practical because it allowed sampling lysine-coated coverslips had no adverse effects on the within 1 wk. without a need for passaging the cells. growth of cultured testis cells; 2) cells did not grow on Temperature may play an important role in the effi- non-coated coverslips; and 3) there was no difference in ciency of testis cell culture, but the effect of incubation cell viability between the cells growing on coverslips and temperature on proliferation of neonatal porcine testis the peripheral areas of the well. Therefore, while cover- cells has not been well studied. In Exp. 2, testis cells slips placed at the bottom of round plate wells provide a were cultured at either 35 or 37 °C to mimic the testis convenient method of sampling for analyses such as im- temperature in situ, which is normally lower than the munocytochemistry, the cells at the peripheral areas core body temperature (range 38–40 °C in pigs [48, 49]). could also be used for other evaluations such as moni- Testis cells cultured at either 35 or 37 °C proliferated toring of the cell viability or use in other assays. Using and reached a 90% confluency within 1 wk.; however, coverslips also offers the advantage of allowing multiple those cultured at 35 °C became 90% confluent later (5.8 samplings from the culture dish with minimal disturb- vs. 6.4 d, respectively, P < 0.05). Earlier studies using hu- ance to other cells in the same dish/plate. man or mouse testis cells concluded that DNA synthesis In vivo proliferation of gonocytes is influenced by their and steroidogenesis were increased when cells were cul- contact or interaction with the basement membrane of tured at 31/32 °C, compared with 37 °C, which is the the seminiferous tubule . It has also been suggested core body temperature in these species [50, 51]. Al- that gonocytes require the presence of feeder cells in though these latter studies also point to the beneficial ef- order to settle and form colonies in vitro [26, 53–56]. In fects of culturing testis cells at lower than core body the present study, testis somatic cells formed a mono- temperature, they each examined only one such layer, which provided a platform for subsequent gono- temperature, making it difficult to compare the results. cyte attachments. It also appeared that the survival and Results from the present study suggest that further stud- propagation of gonocytes depended on the proper ratio ies will be required to determine the optimal of gonocytes to somatic cells. This was exemplified in temperature for culturing testis cells for a given species. Experiment 1, where we used highly enriched gonocytes The use of poly-L-lysine-coated coverslips is recom- (~86%) for seeding and observed no or slow cell prolifer- mended when sampling cultured testis cells. Opposing ation, even when the enriched gonocytes were used at Awang-Junaidi and Honaramooz Journal of Animal Science and Biotechnology (2018) 9:8 Page 11 of 13 the same seeding density (3.0 × 10 /well) as heteroge- days (6 and 7) were ~50% greater in the unchanged neous populations of testis cells. This is in agreement group, compared with the routine medium changing 3 3 with a previous study by van Dissel Emiliani et al.  regimen (~18.0 × 10 vs. ~12 × 10 , respectively, Fig. 5). showing that when gonocytes were cultured alone, they This can be seen as evidence of continued settlement of failed to adhere to the culture plate and instead formed gonocytes. The number of gonocyte colonies also in- gonocyte aggregates, which were subsequently lost. creased over time in all groups; this increase was ~3.3 In fetal and neonatal testes, gonocytes are larger than fold for the unchanged medium group (from ~5.4 × 10 most testis cells and one would expect that among testis on Day 2 to ~18.0 × 10 on Day 7, Fig. 5). This fold- cells in the suspension, gonocytes would be one of the first increase in the total number of gonocyte colonies does cells to settle. This was indeed the case for mouse fetal not take into account that the number of gonocytes gonocytes showing the highest sedimentation velocity of within each colony also increased over time (Fig. 3d). all testis cells . In the present study, however, gono- Therefore, these results collectively indicate that the op- cytes settled much later than other testis cells, and could timized culture conditions developed in the present be seen floating even after several days of culture. Goel et study improved gonocyte colony formation. al.  examined the population of floating cells after 4 h In the present study, we systematically examined the ef- of culturing neonatal pig testis cells and concluded that fects of several culture conditions on testis cells of the they were mostly viable (~95%) and composed of mostly neonatal pig, as a non-rodent animal model. After each germ cells (91%). In the present study, although the num- experiment, we built upon the newly obtained results and ber of floating gonocytes decreased over time, their viabil- designed subsequent experiments to make successive ad- ity remained high (78–86%) up to 6 d after culture. The vances in the survival, propagation, and colony formation extended presence of floating gonocytes may be an advan- of gonocytes. Our results are expected to facilitate further tageous characteristic if they are collected to be used for studies aiming at, for instance, investigation of specialized gonocyte enrichment; however, it can also be disadvanta- media for large-scale propagation of gonocytes, as a geous and lead to the loss of gonocytes during medium source of MGSCs. Applications for MGSCs range from changes, if done without due care. basic to applied research, including germ cell transplant- Changing of medium is important to furnish nutrients ation studies in farm animals for the purpose of animal and remove waste from a culture system; however, the transgenesis, genetic conservation, or infertility treatment frequency of medium changing may affect culture condi- models [4, 5, 33–35]. Germ cell transplantation can also tions. Most studies on testis cell culture use a daily, be used as a functional assay to assess the relative abun- every-other-day, or twice-weekly approach to medium dance or confirm the developmental potential of MGSCs changing [18, 42, 57, 59, 60]. In the present study, rou- within a given population of testis cells, including cultured tine changing of medium (every 2 d) reduced the num- gonocytes (reviewed in ). ber of attached gonocytes, prompting us to design Experiment 6 for optimizing the medium changing regi- Conclusions men to increase the number of gonocyte colonies. Given In conclusion, the optimized culture conditions developed our observations of the floating gonocytes, we compared in the present study included seeding neonatal porcine 4 2 gonocyte colony formation following the routine testis cells at 3.0 × 10 testis cells/cm containing ~40% ++ medium changing regimen (every 2 d) with the group in gonocytes in DMEM at 37 °C, and without changing the which the medium was not changed for 1 wk., or with medium in the first week. These results can be used to im- the modified medium changing regimen, where a smaller prove in vitro maintenance, proliferation, and formation volume of medium was exchanged at a time. Compared of gonocyte colonies and thereby as a basis for the devel- with Day 2 (~5.0 × 10 ), the number of gonocytes and opment of more specialized culture systems for MGSCs. gonocyte colonies increased in all regimens in subse- Abbreviations quent days (Days 4, 6, and 7; Fig. 5). The increased num- ANOVA: Analysis of variance; DAPI: 4′,6-diamidino-2-phenylindole; ber of gonocyte colonies on Day 4 in all groups (ranging DAZL: Deleted in azoospermia like; DBA: Dolichos biflorus agglutinin; ++ 3 3 DMEM: Dulbecco’s modified Eagle medium;; DMEM : DMEM supplemented from ~10.0 × 10 to ~13.5 × 10 , Fig. 5) could be taken with 10% fetal bovine serum and 1% antibiotics; DPBS: Dulbecco’s as evidence of gonocyte proliferation in these early days phosphate-buffered saline; ECM: Extracellular matrix; FBS: Fetal bovine Serum; of culture. This conclusion is especially based on the H&E: Hematoxylin and Eosin; HBSS: Hank’s balanced salt solution; HCl: Hydrochloric acid; HSD: Honest significant difference; KHCO : Potassium plates in the routine medium changing regimen which bicarbonate; MGSCs: Male germline stem cells;; NA EDTA: Disodium had undergone a complete medium change. As a result, ethylenediaminetetraacetate; NANOG: NANOG homeobox; this group of plates presumably should have lost all or NH Cl: Ammonium chloride; OCT4: Octamer-binding transcription factor 4; PGCs: Primordial germ cells; PGP9.5: Protein gene product 9.5; SEM: Standard most of its floating gonocytes, yet there was a 2-fold in- error of mean; SPSS: Statistical package for the social sciences; crease in the number of gonocyte colonies. On the other SSCs: Spermatogonial stem cells; UCH-L1: Ubiquitin carboxyl-terminal hydro- hand, the number of gonocyte colonies in subsequent lase L1; VASA: Dead-box helicase 4 Awang-Junaidi and Honaramooz Journal of Animal Science and Biotechnology (2018) 9:8 Page 12 of 13 Acknowledgements 12. De Rooij DG. Proliferation and differentiation of spermatogonial stem cells. We thank Brian Andries and his staff, especially Tatjana Ometlic, at the Prairie Reproduction. 2001;121:347–54. Swine Centre for their assistance in the collection of neonatal porcine testes. 13. Orwig KE, Shinohara T, Avarbock MR, Brinster RL. Functional analysis of stem We also thank Shawn Honaramooz for help with cell culture and Willow cells in the adult rat testis. Biol Reprod. 2002;66(4):944–9. Burnes for proofreading the revised manuscript. 14. Grisanti L, Falciatori I, Grasso M, Dovere L, Fera S, Muciaccia B, et al. 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