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Journal of Animal Science and Biotechnology
Methodology article
Optimization of culture conditions for short-term maintenance, proliferation, and colony formation of porcine gonocytes
Awang Hazmi Awang-Junaidi1 and
Ali Honaramooz1Email authorView ORCID ID profile
Journal of Animal Science and Biotechnology20189:8
Received: 25 April 2017
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.
Confluency of cells was optimal (>90% by ~6 d) when 3.0 × 104 testis cells/cm2 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".
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 with 3.0 × 104 testis cells/cm2 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.
Male Germline stem cells
Testis stem cells are a unique population of stem cells in an adult body, because in addition to their dual ability to self-renew and give rise to differentiating germ cells, they can pass the genetic information to the offspring. Among the many types of male germ cells present from the fetal period to adulthood, primordial germ cells (PGCs), gonocytes, and particularly spermatogonial stem cells (SSCs) are believed to possess stem cell potential and hence are referred to as male germline stem cells (MGSCs) [1]. An in vivo functional assay for confirming the stem cell potential of MGSCs was provided when SSCs were deposited in the lumen of seminiferous tubules of recipient testes using germ cell transplantation technique. The transplanted SSCs were capable of homing to the tubule basement membrane, forming new colonies of developing germ cells, and initiating donor-derived spermatogenesis [2–7]. In breeding trials, some of the germ cell recipient animals of various species were capable of siring donor-derived progeny [2, 6, 8]. There are also a few reports of using gonocytes and PGCs as donor cells in germ cell transplantation leading to the establishment of spermatogenesis in the recipient testis [3, 9, 10].
The choice of donor MGSCs is a critical factor for the outcome of germ cell transplantation. Using PGCs as donor cells has been less practical because their isolation requires collection from embryos and there are only a limited number of PGCs per embryo [3, 11]. Spermatogonia can be collected from mature donor individuals and enriched for SSCs at relatively high numbers. However, spermatogonia are inherently a heterogeneous population of cells and the lack of biomarkers that allow unequivocal isolation of SSCs has hampered obtaining pure populations [12–14]. Compared with PGCs and SSCs, gonocytes offer a potentially more practical option. Gonocytes are transitory germ cells (between PGCs and SSCs) that have a distinct morphology (large round cells with large nucleus to cytoplasm ratio), and are present during both fetal and early postnatal period for several days, months or even years, depending on the species [15–17]. Perhaps more importantly, gonocytes are the only germ cells present in the testes of neonates; thus, they can be detected using most germ cell markers (e.g., VASA, DAZL), pluripotency markers (e.g., NANOG, OCT4), and certain surface protein markers (e.g., DBA, UCH-L1). These characteristics provide an opportunity for enrichment and in vitro manipulation of gonocytes [17–20].
Gonocytes are relatively limited in numbers since they comprise only ~1.4% of cells in the testes of neonatal rats [21] and ~7% of seminiferous cord cells in piglets [22]. The efficiency of colonization by the donor germ cells in the seminiferous tubules of recipients is directly proportional to the number of transplanted MGSCs. Therefore, to increase the efficiency of germ cell transplantation, gonocytes must be enriched and/or propagated in vitro prior to transplantation, as suggested for SSCs [23, 24]. Various strategies have been introduced to optimize the isolation, enrichment, or purification of gonocytes with differing outcomes, depending on the species [18, 25, 26]. Using conventional cell separation methods, no more than ~10% of isolated neonatal testis cells were gonocytes [21, 22, 27, 28]. However, development of a three-step enzymatic digestion in our laboratory has allowed the collection of populations of cells containing ~40% gonocytes from neonatal porcine testes [29]. The resultant population of gonocytes can also be enriched further to ~90% using a combination of Nycodenz centrifugation and differential plating [30].
While pigs are an important biomedical animal model, purification of porcine MGSCs is still limited. The ability to obtain highly enriched populations of gonocytes from neonatal piglets [29, 30], however, has provided new opportunities for using gonocytes as a model for in vitro propagation and manipulation of MGSCs. This calls for further research on culture systems that can allow efficient large-scale propagation of gonocytes. No systematic study has examined the effects of different culture conditions on colony formation of porcine gonocytes. Therefore, given the importance of cell culture in MGSC research, the present study was designed to examine a number of culture conditions for maintenance, proliferation, and colony formation of neonatal porcine gonocytes as a model. In the present study, we performed six experiments in a stepwise design and examined several aspects of testis cell culture including factors related to the seeding density, gonocyte proportion, incubation conditions, sampling, medium changing regimens, and effects of applying those optimal conditions on gonocyte colony formation.
Testis collection and preparation
Testes from Yorkshire-cross piglets (Camborough-22 × Line 65; PIC Canada, Winnipeg, MB, Canada) less than 1 wk. of age were used in this study. The testes were collected weekly (5–15 pairs per week) through aseptic castration at a university–affiliated swine facility. The testes were kept in ice-cold Dulbecco's phosphate-buffered saline (DPBS; catalogue no. 20–031-CV; Mediatech, Manassas, VA, USA), containing 1% w/v antibiotics solution (penicillin and streptomycin; catalogue no. 30–002-CI, Mediatech), and were transported within 1 h of collection to the laboratory. The testes were thoroughly rinsed three times with DPBS prior to processing. The parenchyma was separated from the tunica albuginea and excess connective tissues, and used for isolation of testis cells.
Isolation of testis cells and enrichment for gonocytes
Isolation of testis cells and enrichment of gonocytes were performed using methods that were established in our laboratory (three-step enzymatic digestion, Nycodenz density gradient centrifugation and extracellular matrix (ECM) differential plating; [29, 30]). Briefly, for each batch of the three-step enzymatic digestion, ~600 mg of testis parenchyma was thoroughly minced with fine scissors for 5 min, suspended in 5 mL of DPBS, vortexed for 30 s in a test tube shaker (Reax Top; catalogue no. 541–10000; Heidolph Instrument, Essex, UK) and digested with 1 mL of 0.2% w/v collagenase IV (catalogue no. C-153; Sigma-Aldrich, Oakville, ON, Canada), 0.1% w/v hyaluronidase (catalogue no. H-3884; Sigma-Aldrich), and 0.01% DNase (catalogue no. DN25; Sigma-Aldrich) in Dulbecco's modified Eagle's medium (DMEM; catalogue no. 10–013-CM; Mediatech) supplemented with 1% w/v antibiotics (as above) at 37 °C for 10 min. Fetal bovine serum (FBS; catalogue no. A15–701; PAA Laboratories, Etobicoke, ON, Canada) was added to stop the digestion and the suspension was vortexed for another 30 s and filtered through a 40-μm filter (catalogue no. 3522340; BD Biosciences, San Jose, CA, USA). The filtrate suspension was centrifuged at 500×g at 16 °C for 5 min and resuspended in 5 mL of DPBS supplemented with 1% w/v antibiotics. Erythrocyte depletion was performed by mixing the cells with 20 mL of the lysis buffer, composed of 156 mmol/L ammonium chloride (NH4Cl; catalogue no. A9434; Sigma-Aldrich), 10 mmol/L potassium bicarbonate (KHCO3; catalogue no. 237205; Sigma-Aldrich), and 0.1 mmol/L disodium ethylenediaminetetraacetate (Na2EDTA; catalogue no. E6635; Sigma-Aldrich) in sterile distilled water prior to another cycle of centrifugation (500×g at 16 °C for 5 min). Finally, the cell pellet was resuspended in 5 mL of DMEM supplemented with 10% v/v FBS and 1% antibiotics (DMEM++) and underwent gentle pipetting to obtain a single cell suspension.
For enrichment of gonocytes among the cells obtained from the three-step enzymatic digestion, 3 mL of 17% Nycodenz in DPBS (Histodenz; catalogue no. D2158; Sigma-Aldrich) was placed at the bottom of a 15-mL graduated conical tube. This was followed by gentle addition of 2 mL of cell suspension on top and the tube was centrifuged at 500×g at 4 °C for 15 min. The supernatant was discarded and the pellet was harvested and resuspended as a single cell suspension. Prior to ECM differential plating, 6-well culture plates (catalogue no. 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 fibronectin (catalogue no. 477743–728; VWR) in an incubator with 5% CO2 at 37 °C for 1 h, and dried in a biosafety cabinet for another 1 h. The poly-D-lysine pre-coated wells were rinsed twice with DPBS before seeding the cells obtained from Nycodenz gradient density centrifugation. The cells were seeded onto the plates at a concentration of 2.5 × 105 cells/cm2 in DMEM++, and cultured in an incubator with 5% CO2 at 37 °C. After 2 to 3 h, the floating cells were harvested, centrifuged at 500×g at 16 °C for 5 min and the pellets were collected and resuspended as a single cell suspension. These procedures lead to obtaining isolated testis cells highly enriched in gonocytes. We performed a side-by-side quantitative comparison of the cell yield, viability and loss after each progressive step. Samples were prepared from single cell suspensions obtained from each step. The suspension was smeared onto poly-L-lysine pre-coated glass coverslips, dried overnight in a biosafety cabinet and kept at −20 °C for use in immunostaining.
Cell quantification and viability assessment
The number of resultant cells after each isolation and enrichment step as well as after culturing was quantified, and the cell viability was assessed using the trypan blue exclusion technique. For evaluation of cells collected after isolation and enrichment, single cell suspensions were prepared as described above. For cultured cells, detachment of the adherent cells was performed by adding 1 mL of 0.25% (w/v) trypsin in Hank's balanced salt solution (HBSS) and 2.21 mmol/L EDTA (catalogue no. 25–053-CI; Mediatech) at 37 °C for 1–2 min (depending on confluency) with gentle agitation. The enzymatic digestion was stopped by adding 1 mL of undiluted FBS, and the mixture was centrifuged at 500×g at 16 °C for 5 min. The pellets were collected and subjected to resuspension into a single cell suspension prior to assessment. Trypan blue (100 μL of a 0.4% solution in saline, catalogue no. T8154; Sigma-Aldrich) was mixed (1:1 ratio) gently with the cell suspension, and 20 μL of the mixture was transferred to each hemocytometer chamber and live/dead cells were identified with the aid of a light microscope. For each replicate, two counts of live/dead cells were performed and averaged to calculate the yield and viability of cells (given as a percentage). Immunostaining was performed to confirm and quantify gonocytes, as they are the only type of germ cells present in neonatal pig testes; hence the remaining cells were categorized as somatic cells. For immunostaining, a minimum of 600 cells in randomly selected fields were counted in each cell smear and the proportion (%) of gonocytes/somatic cells was calculated.
Different cell culture conditions were evaluated using a stepwise approach. The same medium (DMEM++), culture plates (6-well plates), and incubator conditions (5% CO2 and 95% humidity) were used in all experiments in order to evaluate the effects of changing other variables (i.e., gonocyte proportion, incubation temperature, cell seeding density, and medium changing regimen). For all experiments, pH of the medium was adjusted to 7.4 immediately prior to using it in cell cultures. The pH of the medium was measured using a digital pH meter (catalogue no. 11288–368; model Symphony SB70P; VWR). Cell growth was monitored at least once daily using an inverted phase contrast microscope (Nikon, Eclipse TS100). For each experiment, multiple replications were performed where cells obtained from each batch of testis tissue digestion were considered one replication. We have previously shown that the characteristics of cells obtained from 1-week old piglets from this source remain consistent over time, both within and among litters [29].
Effects of several factors on the behavior and the morphology of cultured testis cells were examined using cytology and immunostaining. The examined factors included different gonocyte proportions obtained before or after each step of enrichment (40% vs. 80% vs. 90%), incubation temperature (35 °C vs. 37 °C), cell seeding density (1.0 × 105 vs. 3.0 × 105 vs. 5.0 × 105 cells/well; each well in a 6-well plate has ~10 cm2 cell growth area) and medium changing regimen (every 2 d vs. modified regimen vs. no changing of medium for up to 7 d). Confluency assessment was defined as the relative coverage of the plate surface by the mixture of testis cells, and a gonocyte colony was defined as a group of more than three gonocytes in close contact.
For sampling, glass coverslips coated with 0.1% w/v poly-L-lysine were used. Glass coverslips sized 22 × 22 mm (catalogue no. 12-540B; Fisher Scientific, USA) were arranged in a sterile metal coverslip rack and immersed in 1% HCl in 70% alcohol for 10 min, followed by rinsing with sterile distilled water for 5 s and allowed to dry for 20 min. The rack was transferred into a 0.1% w/v poly-L-lysine (catalogue no. P8920; Sigma-Aldrich) solution for 10 min and allowed to dry overnight. Finally, the glass coverslips were placed at the bottom of each well in 6-well plates. These procedures were conducted under a biosafety cabinet.
Immunostaining
Gonocytes were identified by immunostaining using Dolichos biflorus agglutinin (DBA) according to previously described protocols [29, 30], with minor modifications. Briefly, the dried coverslips (with cell smears or with cells grown directly on them) were fixed in Bouin's solution (catalogue no. 1120–31; Richa Chemical Company, Pocomoke City, MD, USA) for 2–3 min. The samples were then rinsed three times with 70% alcohol to remove excess Bouin's solution followed by three rinses with DPBS. The samples were blocked with 5% bovine serum albumin (BSA; catalogue no. 0332, Amresco, Solon, OH, USA) at 37 °C for 30 min in a humidified chamber. The blocking agent was removed and the samples were incubated with fluorescein-conjugated lectin DBA (1:100, catalogue no. FL-1031, Vector Labs, Burlington, ON, Canada) overnight at 4 °C in a humidified chamber. The samples underwent another cycle of rinsing, followed by incubation with 0.3% Sudan Black B w/v (catalogue no. S2380; Sigma-Aldrich) in 70% alcohol for 15 min, at 37 °C in a humidified chamber. The samples were rinsed with DPBS and stained with 4′6'-diamidino-2-phenyilindole (DAPI; catalogue no. D9542; Sigma-Aldrich) and observed under a fluorescence microscope. Fluorescein-conjugated lectin DBA specifically labels gonocytes which appear green under the fluorescence microscope while DAPI stains the nuclei of all cells which appear blue (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
Unless stated otherwise, all data are presented as means ± standard error of mean (SEM) and analyzed using either one-way or two-way analysis of variance (ANOVA), as appropriate, followed by Tukey's HSD post-hoc test. For percentages, the data were transformed using Arcsine function prior to analysis using ANOVA. The level of significance was set at P < 0.05. Data were analyzed using SPSS (Version 20.0; SPSS, Chicago, IL, USA).
As summarized in Table 1, enzymatic digestion of testis tissues yielded a population of testis cells containing ~41% germ cells (Fig. 1a-c), which at this developmental stage (~1 wk of age) are exclusively gonocytes. Gradient centrifugation by Nycodenz, followed by ECM differential plating resulted in enrichment of gonocytes to ~80% and ~86%, respectively. However, the enrichment procedures also caused significant reductions (P < 0.05) in cell counts after each step, resulting in ~73% and ~92% total testis cell loss and ~47% and ~83% gonocyte loss, respectively. The relatively lower gonocyte loss at each step, compared with the total cell loss, resulted in significant enrichment of gonocytes. The viability of cells was also reduced after each enrichment step (from ~93% to 84% and 82%, respectively, P < 0.05). When the cells were cultured (3.0 × 105 cells/well at 37 °C with 5% CO2 and the medium was changed every 2 d), only the cells used immediately after the three-step enzymatic digestion (i.e., without enrichment) demonstrated growth by reaching ~90% confluency in ~6 d.
The population of testis cells obtained after enzymatic digestion and subsequent enrichment
Proportion, %
Cell count, × 106 *
Cell loss, % **
Viability, %
In vitro growth
Gonocytes
Somatic cells
Enzymatic digestion
40.60 ± 1.21 a
59.40 ± 1.21 a
Nycodenz centrifugation
80.40 ± 1.46 b
19.6 0 ± 1.46 b
10.00 ± 0.41b
8.00 ± 0.21 b
ECM differential plating
86.40 ± 1.03 c
13.6 0 ± 1.03 c
3.02 ± 0.19 c
2.60 ± 0.2 c
91.62 ± 0.79 b
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
abcWithin each column, data with different superscripts differ significantly (P < 0.05). n = 5 replications
Exp. 1: Effect of cell seeding density and gonocyte proportion
One of the objectives of this experiment was to determine the optimal seeding density for culturing testis cells. A comparison was made by randomly assigning cells into 6-well plates at 1.0 × 105, 3.0 × 105, or 5.0 × 105 cells/well (i.e., 1.0 × 104, 3.0 × 104, or 5.0 × 104 cells/cm2 since each well was ~10 cm2, n = 12 replications), in 3 mL of medium to determine the time required for the cells to reach 90% confluency. Cells were used fresh (immediately after enzymatic digestion and without enrichment, hence containing ~40% gonocytes) and cultured under similar conditions at 37 °C, and the medium was changed every 2 d.
As shown in Table 2, cells in the highest seeding density group (5.0 × 105) became confluent earlier than other groups (P < 0.05), reaching >90% confluency in ~4 d, while the 3.0 × 105 seeding density group of cells were >90% confluent by ~Day 6. Cells cultured at 1.0 × 105 seeding density had slow growth and only reached ~30% confluency after 7 d in culture.
The effect of seeding density on the growth of testis cells in culture
Cell seeding density
Maximum confluency
Confluency at days
Cell count
1.0 × 105
(*~30% at 7)
(*2.98 × 105 ± 0.01)
(*88.4 ± 1.02)
>90% at 6.0 ± 0.17 a
8.10 × 105 ± 0.01
91.21 ± 0.58
>90% at 4.0 ± 0.15 b
*The results from 1.0 × 105 seeding density group were excluded from statistical analysis as the cells did not reach 90% confluency
Independent sample t test was used for statistical analyses
abWithin each column, data with different superscripts differ significantly (P < 0.05). n = 12 replications
In a separate experiment, testis cells enriched for gonocytes (undergoing both Nycodenz centrifugation and ECM plating, hence containing ~86% gonocytes) were used as the source of cells for culturing, using the same incubation conditions. Cells did not grow in plates seeded with 1.0 × 105 or 3.0 × 105 cells/well and only had limited growth in the 5.0 × 105 seeding density group after 7 d in culture (5–15% confluency, data not shown). Therefore, for the remaining experiments in this study we used testis cells without enrichment (i.e., containing ~40% gonocytes), and since the 3.0 × 105 seeding density resulted in confluency of cells in ~6 d (without the need for passaging the cells within 1 wk), we used this seeding density in the subsequent experiments.
Exp. 2: Effect of incubation temperature
The objective of this experiment was to evaluate the growth of cultured testis cells using incubation temperatures of either 35 °C or 37 °C for 1 wk (n = 12 replications), while other culture conditions remained the same (e.g., medium, cell seeding density, and CO2 levels). As shown in Table 3, the viability and number of cells at confluency did not differ between the two incubation temperatures (P > 0.05). However, the cells cultured at 35 °C reached confluency later than those at 37 °C (P < 0.05); therefore, for practical reasons we chose to culture the cells at 37 °C for subsequent experiments.
The effect of incubation temperature on the growth of testis cells in culture
>90% confluency
6.42 ± 0.15 a
Exp. 3: Sampling of cultured testis cells for analysis
Various methods can be used to sample cultured cells for analysis. When sampling for applications such as immunocytochemistry, coverslips are commonly pre-coated with certain matrices to enhance adherence of cells. It is not clear whether the presence of the pre-coated coverslips in testis cell culture would affect cell growth. Furthermore, in situations where multiple samplings from each replicate is required, it would be more efficient to use all available cultured cells, including those grown on areas of the culture well not covered by the coverslip. The objectives of this experiment were to determine 1) whether the rate or pattern of testis cell growth in culture would change in the presence of glass coverslips (22 × 22 mm) coated with 0.01% poly-L-lysine, and 2) whether the growth of testis cells on the coverslips differs from those on the peripheral areas of the wells, not covered by the coverslips (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)
When plates with and without poly-L-lysine-coated coverslips were compared, the number or viability of the resultant cultured cells did not differ between plates (8.18 ± 0.01 × 105 vs. 8.22 ± 0.02 × 105 cells per plate, with 91.0 ± 0.57% vs. 91.3 ± 0.87% cell viability, plates with coverslips vs. plates with no coverslips, respectively, n = 12 replications, P > 0.05). When non-coated coverslips were used, little or no cell growth was observed. Furthermore, within the plates with poly-L-lysine-coated coverslips, the viability of cells grown on coverslips did not differ from those grown on the peripheral areas of the well, not covered by the coverslips (92.0 ± 0.33% vs. 92.4 ± 0.51%, coverslips vs. periphery, respectively, P > 0.05). However, the number of cells obtained from the peripheral areas was higher than that from the coverslips (4.59 ± 0.14 × 105 vs. 2.99 ± 0.06 × 105, respectively, P < 0.05), even though both encompassed a similar cell growth area (~4.8 cm2 each).
Exp. 4: Short-term culture of neonatal porcine testis cells
Morphological changes of neonatal porcine testis cells in culture were evaluated for 1 wk under the above-mentioned culture conditions (3.0 × 105 seeding density, DMEM++, 37 °C, n = 6 replications). The medium was changed every 2 d. Within the first 12 h of incubation, most of the cells and cell particles were still floating in the medium (Fig. 3a). By 24 h after incubation, small numbers of cells settled and appeared to have adhered to the plate. The early settling cells were mostly somatic cells and began to form cytoplasmic projections, creating irregularly shaped cell boundaries (Fig. 3b), while more cells continued to settle. After 2 d of incubation, the cells started to appear in clumps (Fig. 3c), and over time these groups of cells spread further and combined to create a large somatic-cell monolayer. At the same time, round cells with large nuclei, appearing bright or with a halo under the phase-contrast microscopy, were seen attached onto the somatic-cell monolayer (Fig. 3d–e). These rounds cells were confirmed to be gonocytes using immunostaining (Fig. 1d–f). It was also observed that while initially gonocytes were present as singles or pairs, starting at Day 5, colonies (grape-like clumps) of more than 3 cells could also be observed (Fig. 3d). It was noticed that gonocytes were only loosely attached to the somatic-cell monolayer, since some would detach due to slight movements of the plate. By the end of the experiment (Day 7), the number of these round cell colonies were still considerably less than expected, given that the population of testis cells at seeding contained ~41% gonocytes. The somatic cells continued to multiply and once reaching 70–80% confluency (at ~4 to 5 d), many appeared as spindle-shaped fibroblast-like cells (Fig. 3e).
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
Exp. 5: Floating cells in the culture medium
Based on the findings of Exps. 1 and 4, it was clear that gonocytes require sufficient ratios of somatic cells or feeder cells, to which they formed loose attachment for growth in culture. We speculated that the very low number of gonocyte colonies observed by Day 7 during short-term culture (Fig. 3e) might be due to their loss through routine medium changes, which apparently eliminated the floating and loosely adherent gonocytes. Therefore, this experiment was designed to examine the medium removed from the plates. The results showed 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 removed on Day 2 (6.81 ± 0.63 × 104), compared with Days 4 and 6 (1.02 ± 0.09 × 104 and 0.4 ± 0.07 × 104 cells/well, P < 0.05, n = 5 replications, Fig. 4). On the other hand, if the medium was not changed, greater numbers of floating gonocytes remained in the medium examined on Days 4 and 6 (4.2 ± 0.10 × 104 and 1.92 ± 0.05 × 104 cells/well, respectively), compared with the numbers obtained on the same days when the medium was changed every other day (P < 0.05, n = 5 replications, Fig. 4). Based on the morphology, it was presumed that these cells were gonocytes; they were large and round in shape with prominent nuclei (large nucleus to cytoplasm ratio). The viability of these round cells remained high in all examined days (~85–97%, data not shown). Using immunostaining, these round cells were later confirmed to be gonocytes (Fig. 1g–i).
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 × 105 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 (Routine), while some were present on Days 4 and 6, if the medium was not changed (Unchanged). The data are mean ± SEM. ab Data with different superscripts within each day differ significantly (P < 0.05). xyz Data with different superscripts within each treatment over time differ significantly (P < 0.05). n = 5 replications
Exp. 6: Effect of medium changing regimens on attached gonocytes
This experiment was designed to assess the effects of different medium changing regimens on the number and viability of attached gonocytes. The cells were cultured using the selected culture conditions (3.0 × 105 testis cells/well containing ~40% gonocytes, DMEM++, 37 °C) for 1 wk, and medium changing regimens were assigned to groups of plates as follows: 1) to serve as a control group, the cells were seeded with 3 mL of medium, and the medium was changed routinely, every 2 d (routine group, 3 changes in 1 wk); 2) the cells were 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 group); or 3) the cells were seeded with 3 mL of medium but the medium was not changed (unchanged group). On Days 2, 4, 6, and 7, the cultured cells were observed under an inverted microscope and the number of gonocyte colonies in four randomly selected fields at 400× was recorded and cells were collected for evaluation of viability (n = 5 replications per group).
As shown in Fig. 5, alterations in medium changing regimens affected the number of attached gonocytes or gonocyte colonies present on the somatic-cell monolayers. Overall, the number of gonocyte colonies increased (P < 0.05) from Day 2 to Day 4 of culture in all medium changing regimens. However, among the medium changing regimens, the unchanged group had greater (P < 0.05) number of gonocyte colonies on Days 6 and 7 compared with the routine medium changing group (control).
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 (Unchanged). The data are mean ± SEM. ab Data with different superscripts within each day differ significantly (P < 0.05). xy Data with different superscripts within each treatment over time differ significantly (P < 0.05). n = 5 replications
The viability of cells in different medium changing regimens is shown in Fig. 6. Compared with the cell viability at seeding (~90%), there was a reduction in viability of cells at Days 4, 6, and 7 in all groups (P < 0.05). The cell viability was reduced further in the unchanged group on Days 6 and 7 (P < 0.05). Between the regimens, the viability of cells in the unchanged groups was lower (P < 0.05) than the routine group on Days 6 and 7 of culture.
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 0 represents the initial viability at seeding. The data are mean ± SEM. ab Data with different superscripts within each day differ significantly (P < 0.05). xyz Data with different superscripts within each treatment over time differ significantly (P < 0.05). n = 5 replications
In the neonatal testis, gonocytes represent MGSCs, which have great potential applications in male reproduction research (reviewed in [31, 32]). Various methods have been established to culture MGSCs to facilitate their study and manipulation or to harness their potential for downstream applications such as genetic conservation, transgenesis, or infertility treatments [4, 5, 33–35]. However, depending on the donor species, age, or developmental status, optimized culturing or enrichment of MGSCs can be challenging [29, 36].
Cell isolation is a critical step in preparing testis cells for culture. The number and proportion of the resultant populations of cells also vary greatly depending on the methods used to isolate the cells. As a first step in the present study, we performed a side-by-side comparison of the total testis cell yield and gonocyte proportions from three protocols [29, 30]. Although as expected the enrichment process resulted in highly purified populations of gonocytes (~86%), it also led to losing up to 90% of the initial number of testis cells. These findings provide a basis upon which an appropriate approach could be adopted for gonocyte isolation/enrichment in order to meet the objectives and needs of specific experiments. For example, the highly enriched populations of gonocytes could be used in studies that focus mainly on gonocytes per se, while the heterogeneous populations of testis cells obtained immediately after enzymatic digestion may be more suitable for longer term culture or for the study of interactions between gonocytes and somatic cells.
One of the advantages of using neonatal testes as the source of germ cells is that gonocytes are the only type of germ cells present during the early postnatal stage. Gonocytes possess a distinct morphology and a unique pattern of distribution in the seminiferous cords of neonatal testes, which facilitate their identification in situ [18, 21]. Once the cells are enzymatically dissociated; however, accurate identification of gonocytes requires immunocytochemistry. There are several biomarkers that could be used for identification of gonocytes including germ cell markers (e.g., VASA and DAZL, [37, 38]) and stem cell markers (e.g., Nanog, SSE-A1, and OCT4, [18, 20, 39, 40]). Two of the most commonly used markers for neonatal porcine gonocytes are DBA [18, 19, 41] and ubiquitin C-terminal hydrolase L1 (UCH-L1; previously known as PGP9.5) [41, 42]. DBA has a specific affinity to bind to α-D-N-acetyl-galactosamine, which is localized on the surface of porcine gonocytes (i.e., primitive germ cells) [18], while UCH-L1 can identify both primitive and more advanced germ cells.
In the present study, identification of gonocytes was initially hampered by autofluorescence, which was partly overcome by using a quenching protocol [19]; however, some autofluorescence remained. Autofluorescence in the tissue/cells can be due to endogenous sources (e.g., lipofuscin or lipofuscin-like pigments), or acquired during processing (e.g., certain fixatives). In any case, autofluorescence can interfere with specific signals by immunostaining and lead to false positive results. In the present study, the autofluorescence was eliminated through modification of the quenching protocol [19]. The use of Sudan black B (SBB) has been reported to be efficient in masking autofluorescence of lipofuscins, triglycerides, and lipoproteins origin [43, 44] and is a major component of the quenching protocol for testis cells [19]. In this study, as a first step in modifying the original quenching protocol, the length of incubation with SBB was extended, which led to further reduction of the autofluorescence of testis cells, but not to its complete elimination. We then speculated that the remaining autofluorescence might have a fixative origin. The picric acid component of Bouin's solution used for fixing testis tissue has also been cited as a potential cause of autofluorescence [45, 46]. Therefore, we applied further control measures to refine the use of fixatives in the quenching protocol. We found that inclusion of three thorough and gentle rinses with 70% alcohol after the Bouin's fixation completely eliminated the autofluorescence problem in Bouin's fixed testis cell samples. As an alternative, we also found that to avoid the autofluorescence caused by Bouin's, cell smears or cells grown on coverslips can be fixed using methanol at −20 °C for 10 min.
Cell seeding density influences the performance of cultured cells, presumably by affecting the contact and interactions among cells [47]. Optimization of the cell seeding density prior to culturing cells ensures normal cell growth and allows estimation of the time to reach certain confluency levels. In the present study, the cell seeding density of 3.0 × 105/well (i.e., in ~10 cm2) was found to be more practical because it allowed sampling within 1 wk. without a need for passaging the cells.
Temperature may play an important role in the efficiency of testis cell culture, but the effect of incubation temperature on proliferation of neonatal porcine testis cells has not been well studied. In Exp. 2, testis cells were cultured at either 35 or 37 °C to mimic the testis temperature in situ, which is normally lower than the core body temperature (range 38–40 °C in pigs [48, 49]). Testis cells cultured at either 35 or 37 °C proliferated and reached a 90% confluency within 1 wk.; however, those cultured at 35 °C became 90% confluent later (5.8 vs. 6.4 d, respectively, P < 0.05). Earlier studies using human or mouse testis cells concluded that DNA synthesis and steroidogenesis were increased when cells were cultured at 31/32 °C, compared with 37 °C, which is the core body temperature in these species [50, 51]. Although these latter studies also point to the beneficial effects of culturing testis cells at lower than core body temperature, they each examined only one such temperature, making it difficult to compare the results. Results from the present study suggest that further studies will be required to determine the optimal temperature for culturing testis cells for a given species.
The use of poly-L-lysine-coated coverslips is recommended when sampling cultured testis cells. Opposing ionic charges among the cells (polyanionic) and poly-L-lysine-coated coverslip surface (polycationic) enhance efficient attachment of cells onto the coverslips. In the present study, we showed that 1) the presence of poly-L-lysine-coated coverslips had no adverse effects on the growth of cultured testis cells; 2) cells did not grow on non-coated coverslips; and 3) there was no difference in cell viability between the cells growing on coverslips and the peripheral areas of the well. Therefore, while coverslips placed at the bottom of round plate wells provide a convenient method of sampling for analyses such as immunocytochemistry, the cells at the peripheral areas could also be used for other evaluations such as monitoring of the cell viability or use in other assays. Using coverslips also offers the advantage of allowing multiple samplings from the culture dish with minimal disturbance to other cells in the same dish/plate.
In vivo proliferation of gonocytes is influenced by their contact or interaction with the basement membrane of the seminiferous tubule [52]. It has also been suggested that gonocytes require the presence of feeder cells in order to settle and form colonies in vitro [26, 53–56]. In the present study, testis somatic cells formed a monolayer, which provided a platform for subsequent gonocyte attachments. It also appeared that the survival and propagation of gonocytes depended on the proper ratio of gonocytes to somatic cells. This was exemplified in Experiment 1, where we used highly enriched gonocytes (~86%) for seeding and observed no or slow cell proliferation, even when the enriched gonocytes were used at the same seeding density (3.0 × 105/well) as heterogeneous populations of testis cells. This is in agreement with a previous study by van Dissel Emiliani et al. [57] showing that when gonocytes were cultured alone, they failed to adhere to the culture plate and instead formed gonocyte aggregates, which were subsequently lost.
In fetal and neonatal testes, gonocytes are larger than most testis cells and one would expect that among testis cells in the suspension, gonocytes would be one of the first cells to settle. This was indeed the case for mouse fetal gonocytes showing the highest sedimentation velocity of all testis cells [27]. In the present study, however, gonocytes settled much later than other testis cells, and could be seen floating even after several days of culture. Goel et al. [58] examined the population of floating cells after 4 h of culturing neonatal pig testis cells and concluded that they were mostly viable (~95%) and composed of mostly germ cells (91%). In the present study, although the number of floating gonocytes decreased over time, their viability remained high (78–86%) up to 6 d after culture. The extended presence of floating gonocytes may be an advantageous characteristic if they are collected to be used for gonocyte enrichment; however, it can also be disadvantageous and lead to the loss of gonocytes during medium changes, if done without due care.
Changing of medium is important to furnish nutrients and remove waste from a culture system; however, the frequency of medium changing may affect culture conditions. Most studies on testis cell culture use a daily, every-other-day, or twice-weekly approach to medium changing [18, 42, 57, 59, 60]. In the present study, routine changing of medium (every 2 d) reduced the number of attached gonocytes, prompting us to design Experiment 6 for optimizing the medium changing regimen to increase the number of gonocyte colonies. Given our observations of the floating gonocytes, we compared gonocyte colony formation following the routine medium changing regimen (every 2 d) with the group in which the medium was not changed for 1 wk., or with the modified medium changing regimen, where a smaller volume of medium was exchanged at a time. Compared with Day 2 (~5.0 × 103), the number of gonocytes and gonocyte colonies increased in all regimens in subsequent days (Days 4, 6, and 7; Fig. 5). The increased number of gonocyte colonies on Day 4 in all groups (ranging from ~10.0 × 103 to ~13.5 × 103, Fig. 5) could be taken as evidence of gonocyte proliferation in these early days of culture. This conclusion is especially based on the plates in the routine medium changing regimen which had undergone a complete medium change. As a result, this group of plates presumably should have lost all or most of its floating gonocytes, yet there was a 2-fold increase in the number of gonocyte colonies. On the other hand, the number of gonocyte colonies in subsequent days (6 and 7) were ~50% greater in the unchanged group, compared with the routine medium changing regimen (~18.0 × 103 vs. ~12 × 103, respectively, Fig. 5). This can be seen as evidence of continued settlement of gonocytes. The number of gonocyte colonies also increased over time in all groups; this increase was ~3.3 fold for the unchanged medium group (from ~5.4 × 103 on Day 2 to ~18.0 × 103 on Day 7, Fig. 5). This fold-increase in the total number of gonocyte colonies does not take into account that the number of gonocytes within each colony also increased over time (Fig. 3d). Therefore, these results collectively indicate that the optimized culture conditions developed in the present study improved gonocyte colony formation.
In the present study, we systematically examined the effects of several culture conditions on testis cells of the neonatal pig, as a non-rodent animal model. After each experiment, we built upon the newly obtained results and designed subsequent experiments to make successive advances in the survival, propagation, and colony formation of gonocytes. Our results are expected to facilitate further studies aiming at, for instance, investigation of specialized media for large-scale propagation of gonocytes, as a source of MGSCs. Applications for MGSCs range from basic to applied research, including germ cell transplantation studies in farm animals for the purpose of animal transgenesis, genetic conservation, or infertility treatment models [4, 5, 33–35]. Germ cell transplantation can also be used as a functional assay to assess the relative abundance or confirm the developmental potential of MGSCs within a given population of testis cells, including cultured gonocytes (reviewed in [31]).
In conclusion, the optimized culture conditions developed in the present study included seeding neonatal porcine testis cells at 3.0 × 104 testis cells/cm2 containing ~40% gonocytes in DMEM++ at 37 °C, and without changing the medium in the first week. These results can be used to improve in vitro maintenance, proliferation, and formation of gonocyte colonies and thereby as a basis for the development of more specialized culture systems for MGSCs.
ANOVA:
DAPI:
4′,6-diamidino-2-phenylindole
DAZL:
Deleted in azoospermia like
DBA:
Dolichos biflorus agglutinin
DMEM:
Dulbecco's modified Eagle medium;
DMEM++:
DMEM supplemented with 10% fetal bovine serum and 1% antibiotics
DPBS:
Dulbecco's phosphate-buffered saline
ECM:
FBS:
Hematoxylin and Eosin
HBSS:
Hank's balanced salt solution
HCl:
HSD:
Honest significant difference
KHCO3:
Potassium bicarbonate
MGSCs:
Male germline stem cells;
NA2EDTA:
Disodium ethylenediaminetetraacetate
NANOG:
NANOG homeobox
NH4Cl:
OCT4:
Octamer-binding transcription factor 4
PGCs:
Primordial germ cells
PGP9.5:
Protein gene product 9.5
SEM:
Standard error of mean
SPSS:
Statistical package for the social sciences
SSCs:
Spermatogonial stem cells
UCH-L1:
Ubiquitin carboxyl-terminal hydrolase L1
VASA:
Dead-box helicase 4
We thank Brian Andries and his staff, especially Tatjana Ometlic, at the Prairie Swine Centre for their assistance in the collection of neonatal porcine testes. We also thank Shawn Honaramooz for help with cell culture and Willow Burnes for proofreading the revised manuscript.
This project was financially supported by grants from the Natural Science of Engineering Research and Council (NSERC) of Canada awarded to A. Honaramooz. Graduate student scholarships to A.H. Awang-Junaidi were provided by the Ministry of Higher Education of Malaysia, the University of Saskatchewan College of Graduate and Postdoctoral Studies, and the University of Saskatchewan Western College of Veterinary Medicine.
The datasets supporting the conclusions of this article are included within the article. For additional information please contact author.
AHAJ contributed to the conceiving and designing of the study, performed the experiments and wrote the first draft of the manuscript; AH contributed to the conceiving and designing of the study, as well as writing and revising the manuscript. Both authors read and approved the final manuscript.
All experimental procedures involving animals were approved by the University of Saskatchewan's Institutional Animal Care and Use Committee (Protocol# 20080042).
Not applicable. Portions of this study were presented in the form of an abstract at the 41st American Society of Andrology annual conference.
The authors declare that they have no competing interest.
Department of Veterinary Biomedical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, 52 Campus Drive, Saskatoon, SK, S7N 5B4, Canada
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text:
Former Defense Secretary Donald Rumsfeld claims no cover-up in Pat Tillman death
Andre Damon@Andre__Damon
Former Defense Secretary Donald Rumsfeld testified Wednesday before a congressional investigation into the fratricidal shooting of professional football player turned soldier Pat Tillman. Rumsfeld claimed that there was no cover-up within the military over Tillman's death, a position that flies in the face of evidence that has emerged over the past three years.
Tillman joined the military after September 11, 2001, leaving a career in the National Football League. When he was killed in Afghanistan on April 22, 2004, the incident was used as a propaganda tool by the US government. The Pentagon immediately suppressed solders' reports acknowledging that Tillman had been killed by friendly fire, and told the media that he had died while attempting to storm Afghan positions. This was followed by weeks of triumphant coverage in the media.
Tillman was lauded as a hero who had "given his life for his country." It emerged later, however, that the story given of his death was fabricated, and that he was not killed by Afghan insurgents. These facts were withheld from his family and the American public.
On Tuesday, the US Army issued a report declaring, "In seven investigations into this tragedy, not one has found evidence of a conspiracy by the Army to fabricate a hero, deceive the public, or mislead the Tillman Family about the circumstances of Cpl. Tillman's death."
These claims are contradicted by the findings of the previous House Oversight and Government Reform Committee investigation held in April, in which committee chairman Henry Waxman concluded that "evidence was destroyed" and "witness statements were doctored."
Testifying before the same House committee Wednesday, Rumsfeld claimed that he "did not recall" when he was informed of the circumstances surrounding Tillman's death, but said it was around May 20, 2004—about a month after Tillman was killed and six days after his family was told. Rumsfeld denied having anything to do with the story given by the Pentagon.
In addition to Rumsfeld, two top generals—John Abizaid and Richard Myers—testified before the committee. Both of them denied any conscious wrongdoing in the affair. Thus far, it appears that Lieutenant General Philip Kensinger has been singled out to take the blame for the entire incident. On Tuesday he was censured and may be demoted after an Army report declared he had not notified the Tillman family about the possibility that Tillman had been killed by friendly fire.
"Lieutenant General Kensinger deceived investigators about what he knew and when he knew it," said Army Secretary Pete Geren, who recommended Kensinger's demotion. "He made false official statements." Although a subpoena was issued for Kensinger to appear before Wednesday's committee hearing, Waxman said Kensinger did not reply to the summons and could not be found. In the course of previous investigations, Kensinger told investigators on at least 70 occasions that he could not recall the details of previous events.
Mary Tillman, Pat's mother, said of the Army investigation released earlier this week, "It's so humiliating and disrespectful. It's one more example of the Army investigating itself," she told the New York Daily News. "It was all done to glorify this war. It's a sham. Pat deserves the truth." The Tillman family has repeatedly insisted that Rumsfeld and other high-ranking military and political officials participated in the cover up.
It would indeed have been highly unlikely for the Rumsfeld, a notorious micromanager, to have been unaware of the circumstances surrounding Tillman's death. The coordination of media fanfare and the Pentagon's rapid generation of a fake story, which served as the basis for awarding Tillman medals and posthumous promotion, would have required high-level oversight.
The committee noted that Rumsfeld himself sent a memo to Tillman early on, congratulating him on joining the Army Rangers. Rumsfeld sent another memo to a subordinate, instructing him to "keep an eye on" Tillman. Under these circumstances, Rumsfeld's claims that he was oblivious to the circumstances surrounding Tillman's death are highly dubious.
When President Bush spoke on Tillman's death at the end of April 2004, he avoided speaking about how Tillman died—a clear indication that the White House already knew the truth only a week after the incident took place, but several weeks before Tillman's family was informed that friendly fire was involved.
Earlier this month, the White House refused to turn over documents requested by the oversight committee, claiming executive privilege. The Bush administration has taken the position that any communication or documents involving the executive branch can be kept secret on the grounds of executive privilege—an extraordinary and unconstitutional assertion of Presidential power. Representatives Waxman and Thomas Davis (the ranking Republican on the committee) sent letters of protest to the White House and Pentagon, but did nothing else.
In his opening report yesterday, Waxman stated, "We have tried to find out what the White House knew about Corporal Tillman's death. We know that in the days following the initial report, at least 97 White House officials sent and received hundreds of e-mails about Corporal Tillman's death and how the White House and the President should respond."
Waxman continued: "weeks later—in the days before and after the Defense Department announced that Corporal Tillman was actually killed by our own forces—there are no e-mails from any of these 97 White House officials about how Corporal Tillman really died." Waxman concluded, "No one will tell us when and how the White House learned the truth."
The investigation comes in the wake of new evidence suggesting that Tillman may have been killed intentionally. According to documents obtained by the Associated Press, the doctor who performed Tillman's autopsy sought to initiate a criminal investigation into the his death, but was turned away by both the Army's Human Resources Command and the Criminal Investigation Division.
The doctor concluded that Tillman had been shot in the forehead by three bullets from an M-16 at about 10 yards away, although the nearest eyewitness continues to claim that the shooter was farther away. Tillman was shot after setting off several smoke grenades and while waving his hands and yelling for the soldiers to stop shooting. The documents also note that no evidence of fire from Afghan insurgents was discovered at the scene of Tillman's death, and that no equipment or personnel were struck.
Tillman was reportedly an opponent of the war in Iraq. He was an avid reader of radical historian and linguist Noam Chomsky, whom he had contacted and was planning to meet after his return from active duty. After Tillman's death, his clothes were removed and burned, and his personal possessions, including a daily journal, mysteriously disappeared.
Bryan O'Neal, who had been next to Tillman when the latter was killed, was told by his commanding officer not to inform anyone of the circumstances of his death, especially Tillman's brother, Kevin.
O'Neal told the House Oversight and Government Reform Committee in April that his commanding officer told him, "Do not let Kevin know, that he's probably in a bad place knowing his brother's dead." Kevin Tillman was also an Army Ranger at the time, and was in a convoy behind his brother when Pat Tillman was killed. O'Neal was asked to describe the moment of Tillman's death for the latter's posthumous commendations. He testified that his statements were later doctored and that erroneous claims about Afghan fire somehow made their way into the final draft.
Tillman's death came at a time when the US government and military were trying to reestablish their credibility after the publication of photos from Abu Ghraib and a string of setbacks in Afghanistan. "Revealing that Pat's death was a fratricide would have been yet another political disaster during a month already swollen with disasters," Kevin Tillman told the house oversight committee in April. He accused the Bush administration of "deliberate and calculated lies" in the case.

redpajama_set_name: RedPajamaCommonCrawl


Beginning in October 2014, locally transmitted cases of chikungunya have been reported in Mexico. Local transmission means that mosquitoes in Mexico have been infected with chikungunya and are spreading it to people. See map. Chikungunya cases have also been reported in several countries in the Caribbean(http://wwwnc.cdc.gov/travel/notices/watch/chikungunya-saint-martin) and Central(http://wwwnc.cdc.gov/travel/notices/watch/chikungunya-central-america) and South America(http://wwwnc.cdc.gov/travel/notices/watch/chikungunya-south-america).


text:
Q: Not all columns in the dataframe are filling with funtions I have a functions which takes column, group it by list of other columns and then return this column with values NaN filled with median value of certain column.
When i first check NaN values in column 'Age', it shows me that there are 177 NaN values, but after function work it shows be that such values are 49. What may be cause? There should be 0 columns
def fill_na(df, missing_value_col, grouping_col):
  values = df.groupby([i for i in grouping_col])[missing_value_col].median()
  df.set_index(grouping_col, inplace=True)
  df[missing_value_col].fillna(values, inplace=True)
  df.reset_index(grouping_col, inplace=True)

  return df

fill_na(titset, 'Age', ['Fare','Pclass'])
import pandas as pd
months = ['jan', 'feb', 'mar', 'apr']
sales = {
'revenue':     [100, 200, None, None],
'items_sold':  [23, 43, 55, 65],
'new_clients': [10, 20, 30, 40]
}
sales_df = pd.DataFrame(data=sales, index=months)
sales_df

A: Probable cause: some of your groups contain only NaN values for the 'Age', so the median age for those groups is NaN and you are filling the NaN values with NaN.
You should check if your values dataframe contains NaN.

redpajama_set_name: RedPajamaStackExchange


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redpajama_set_name: RedPajamaGithub


Community composting plays a vital role in promoting composting at all scales. Because operations are often sited locally, they provide unique opportunities to engage, educate, and even employ the communities they serve. This also provides unique challenges as compared to more remote facilities. By supporting well-managed local sites, individuals, elected officials and local planners can take immediate action to reduce waste and improve their soils for climate resilience and local food production. Best management practices (BMPs) are essential to avoiding nuisances and creating a high-quality compost end product.
Community Composting Done Right: A Guide to Best Management Practices is designed to support community-scale composters in successfully managing their composting process and site, with a particular focus on sites accepting food scraps. This report adapts standards of practice from the commercial composting industry for the community scale. Much of the information will apply regardless of the system being used, but it is not intended as a guide to home composting. Co-authors Linda Bilsens Brolis and Brenda Platt hope this report will serve as a resource to both new and existing community composters.
Source: L. Bilsens Brolis, B. Platt, Community Composting Done Right: A Guide to Best Management Practices, Institute for Local Self-Reliance, 2019 (www.ilsr.org/composting-bmp-guide). Reprinted with permission.
Need help designing a composting program or offering composting training?
Part 1: For New Composters and New Sites summarizes composting basics, site and site plan considerations, and steps to prepare for composting.
Part 2: The Science and Art of Composting provides a deep dive into the composting pile with an overview of important composting science principles.
Part 3: Managing the Composting Process and Your Site reviews monitoring and recordkeeping, curing and using finished compost, best management practices, and health and safety considerations.
The authors thank EPA Region 4, Virginia Cretella Mars Foundation, the 11th Hour Project, the Lisa and Douglas Goldman Fund, and our other donors for their support.


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Weekend Events for Jan. 14-17
NWS confirms 2 tornadoes hit northeast Louisiana Friday night
by: Brandon Lawrence, Sharon Cummings
Posted: Mar 27, 2017 / 11:58 AM CDT / Updated: Mar 27, 2017 / 12:09 PM CDT
The National Weather Service in Shreveport confirms 2 tornadoes touched down in northeast Louisiana during Friday night's severe weather.
The first tornado touched down around 11:49pm CDT near the community of Wyatt in Winn Parish. The tornado touched down along LA 505 just west of Wyatt and snapped several trees. The tornado was on the ground for less than a minute, but was able to travel over a mile and ended 6 miles SSW of Jonesboro in Jackson Parish.
That tornado has been rated an EF-1 and likely had winds between 95-105 mph.
The second tornado touched down around 11:54pm CDT near the community of Weston in Jackson Parish. Storm teams say the tornado touched down along LA 505 southwest of Weston– the storm snapped several trees and overturned a metal outbuilding.
That tornado has been rated an EF-1 and was on the ground for a total of 10 minutes. It likely had winds between 100-110 mph. It traveled nearly 7 miles through Jackson Parish.
by Jenn Hensley / Jan 15, 2021
JACKSON PARISH, La. (KTVE/KARD) -- The State Fire Marshal (SFM) deputies are investigating the circumstances that caused a mobile home fire in Cartwright.
Deputies say the mobile home fire claimed the life of the elderly female homeowner.
by Christian Selby / Jan 15, 2021
Authorities with the Monroe Police Department are asking for the public's assistance in locating Tiana Newton (16 yrs old).
Officials say Newton has been missing since January 11, 2021.
Coronavirus in Louisiana: State reports 3,712 new cases on Friday, no update on deaths due to system issue
by Jonathan Freeman / Jan 15, 2021
BATON ROUGE, La. — The Louisiana Department of Health reported 3,712 new cases on Friday, January 15, 2021, bringing the total number of cases to 364,853.
The Louisiana Department of Health noted in Friday's update that they were unable to give an update on COVID-19 deaths in the state due to a data system issue.
Newsfeed Now: Impeachment heading to the Senate, dog reunited with owners 12 years later


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Several comments on my grumbles about "imaginary" news, in the last post.
What fascinated me on this (and the reason why I blogged about it) is that here you have wholly invented stories which first surface online, in each case quote "an insider" to deliver entirely fictitious information, are then picked up by Indian Newspapers as news, and (in the case of the Angelina Jolie Shut Down Beowulf Production story) finally turn up in the UK and US tabloids a couple of days later and then have a sort of a half-life in several perfectly respectable papers. I think it's safe to assume that the Guy Ritchie is Directing Stardust story will continue to run and run (it's already hit the Indian newswires since I posted that last). Never mind that Matthew and Guy parted company several years ago, or that Matthew's been announced as producing and directing this since the news leaked out in Variety, a month ago.
So I don't think that "the public should be allowed to make up their own mind" when the choice is between something perfectly imaginary being reported as news, without even the possibility being reported of the thing being wholly untrue in the first place. Yes? You ought to be free to wonder whether glow-in-the-dark meat is indeed safe to eat, but not to need to wonder whether it exists in the first place; free to wonder whether a jet-lagged Bush really did attempt to escape from a Chinese press conference, but not whether there really is a George Bush and whether he went to China or not.
I read the article "one reason you gave up journalism." I am a student in a broadcasting program near Chicago, IL, USA. I struggle with the same issues. Over the past 2 years I have pulled off my blinders and dived into the media world. The nature of the beast is not what I expected. I have many observations of which I could write volumes of stream of consciousness about.
My Idea of true journalism is to hold people accountable for what they do, be a watchdog for the common man, and not get caught up in political/financial partisan hackery for self advancement.
I can't think of that happening too much anymore.
Yesterday, I listened to a radio package where the focus of the story was "how it can be dangerous to walk all day in a concrete mall." Meaning people might drop dead from the strain on their heart from WALKING.
This is not journalism. This is theatre. This is the grand stage of mediocrity. Praying on peoples fears and offending them by talking down to them. Sometimes I have a good feeling that one of my colleagues will someday change all this. I do not feel up to the challenge. (see jaded in the dictionary.) I feel that I should wash my hands of it and not become part of this player's theatre. Any thoughts?
Well, I left journalism. But then, I didn't want to be a journalist in the first place. I wanted to make stuff up.
There are good journalists out there. It's not all descended to the level of American Broadcast News ("Something YOU use EVERY DAY may KILL YOU. Find out WHAT in TONIGHT'S NEWS.")And I don't think that apathy, or leaving news reporting in the hands of people who don't care what they write or broadcast as long as they get paid, is much of a long-term solution, if you wanted to be one of the good ones. Ideals are good things to have. When you abandon them you start dying. So if you want to be a journalist, then you should be. Just be one of the good ones.


text:
Editorial: Janus Forum and the freedom of expression
By Editorial Page Board
November 18, 2014 | 1:46am EST
Echoes of last fall's Ray Kelly incident continue to reverberate on campus, most recently manifesting themselves in the controversy surrounding today's Janus Forum debate, "How Should Colleges Handle Sexual Assault?" The upcoming forum has come to be defined by one of the speakers: Wendy McElroy, a controversial and vocal dissenter of the pervasiveness of rape culture on college campuses. Last Friday, President Christina Paxson authored a campus-wide email contesting McElroy's opinion and outlined the creation of an alternative event at a concurrent time — a presentation led by Lindsay Orchowski, assistant professor of psychiatry and human behavior, entitled "The Research on Rape Culture." Addressing the issue of sexual assault undoubtedly stands as a top priority for the University and though the events will be recorded, forcing students to choose between attending these two events effectively marginalizes the importance of unfiltered dialogue and discussion, a point ironically underscored by the administration in its criticism of the hindered Ray Kelly lecture.
We endorse that the Orchowski event be moved to a different time or repeated so students may attend both events and gain an academic understanding of the extent and potential solutions to sexual assault on college campuses.
Though we very much acknowledge the pressing importance of confronting sexual assault on campus and the underlying sensitivity of the issue, this most recent administrative action calls into question the role and character of dialogue on campus. The Janus Forum — which has facilitated several controversial, yet equitable debates in recent years — underscored this same concern in an opinion column in Monday's Herald titled, "In response to President Paxson's most recent email." "When students are forced to choose, events no longer serve to 'provide the community with more research and facts about these important issues,' as Paxson hoped for in her email," the Forum wrote. We worry that the double-booking comes out of the administration's attempt to head off a disruption similar to the one which occurred at the Ray Kelly lecture.
Additionally, Paxson's plan seems to contradict her response to the Kelly event regarding free speech. In the words of Paxson in her campus-wide response to the Ray Kelly incident, "Brown has sound policies that promote and preserve freedom of expression, even when the ideas being expressed may be abhorrent." Though the character of the Ray Kelly lecture may have indeed dealt with an issue not explicitly present on campus (i.e. the stop-and-frisk policy of the New York Police Department), this underlying tenet of free expression cannot merely adapt to the topic of discussion, regardless of its ultimate degree of sensitivity or attention.
So to send a message that would endorse freedom of expression on campus, the University need only to escort disruptive individuals out of the event and let it progress. This would show that the University standards of free speech and respect apply to discussing this issue.
By effectively double-booking students interested in sexual assault reform, Paxson both undercut the inherent structure and importance of the topical debate and, even more significantly, contradicted both her own stated precedent and the University's commitment to free speech. Sexual assault is very much a pressing issue on campus and must be addressed as such. That the University has a different stance than McElroy is a learning opportunity, and students should have the option to both hear why the University takes the stance it does and compare it to McElroy's position. In addition, as a community, we must ensure that the ability to freely discuss the issue — regardless of opinion and stance — will be supported and facilitated, not dampened.
Editorials are written by The Herald's editorial page board: its editors, Alexander Kaplan '15 and James Rattner '15, and its members, Natasha Bluth '15, Manuel Contreras '16, Baxter DiFabrizio '15, Manuel Monti-Nussbaum '15, Katherine Pollock '16 and Himani Sood '15. Send comments to editorials@browndailyherald.com.
Note: Rattner, a member of the Janus Forum, recused himself from the writing and editing of this editorial.
Editorial: Brown is back
Editorial: CAPS must expand access
Editorial: First-year advising falls short