Nigrosin

For a detailed description of the experimental procedures, see Supplementary Methods. We grew E. coli strain BW25113^{37 (link)} in MOPS medium supplemented with 0.4% glucose^{38 (link)} incubated at 37 °C while shaking at 200 rpm. Overnight cultures (1–2 ml) in glass test tubes shaken at an angle were used to initiate each experiment. To obtain nucleotide measurements over the growth curve, overnight cultures were diluted to OD₆₀₀ 0.1 (about 30-fold dilution) and grown in 80 ml of pre-warmed medium in 500 ml conical flasks. Samples for nucleotide quantification were taken at 2, 4, 6, 8, and 10 hours. To estimate the cell size, cells were stained with 1% nigrosin and spread onto a microscope slide, air dried and imaged using oil-immersion light microscopy. Pixel-to-µm conversion was performed using microspheres 1 µm in diameter for calibration and image analysis was performed using ImageJ^{61 (link)}. 100–150 cells were measured for each time point across the growth curve. Bacterial cell concentration was determined by fixing the cell culture (60 µl of cell culture with 20 µl 10% paraformaldehyde) and storing samples at −80 °C until further analysis using a flow cytometer (LSRII, BD Biosciences). To study the stringent response, overnight cultures were diluted 100-fold in 100 ml of pre-warmed medium in a 1 l conical flask and grown to OD₆₀₀ 0.5. Then, an initial sample was taken and the stringent response was evoked at time zero by adding mupirocin at 150 µg/ml (3 × MIC). Samples were then taken at 1, 2, 4, 10, 15 and 30 minutes. In both experiments, the volume of culture used to quantify the nucleotides was 3 × 10 ml for first timepoint and 10 ml for the remaining timepoints. All sampling and sample processing was performed using the filtration technique that is thoroughly described in Supplementary Methods. All nucleotides were quantified using IPRP-HPLC, except ppGpp and pppGpp which where quantified using isocratic SAX-HPLC. Reference nucleotide standards were from Thermo Fisher Scientific and Sigma-Aldrich except ppGpp and pppGpp which were in-house synthesized as described elsewhere^{62 (link)}.
The datasets generated during the current study that were not included in tables, are available from the corresponding authors on reasonable request.

ChemicalsThis experimental study was carried out for 9 months in the Fertility and Infertility Research Center, Kermanshah University of Medical Sciences. Genistein (C₁₅H₁₀O₅) powder (Merk- Germany) was dissolved in absolute ethanol (C₂H₅OH) and diluted by normal saline (0.9%) to prepare different doses. Also, the morphine (C₁₆H₁₉NO₃) (Merk- Germany) was diluted by normal saline (0.9%) for administration (16 (link)).
Animal modelIn this study 48 Balb/c male mice purchased from Tehran Razi Institute weighting from 25±2.2 gr were used. All the animals were housed in plastic cages in a room tempreture at 22±2^oC, under controlled environmental conditions, 12 hr light/dark cycle and free access to water and food. All experimentation was conducted under approval of Ethics Committee of Kermanshah University of Medical Sciences (Certificate No. 1394.42) (14 ).
Experimental design and dosage Morphine was administered intera-peritoneally as follows: 10 mg/kg once daily within the first day. On days 2-30, Morphine doses increased 2 mg/kg per day (17 (link)). Genistein was administered as follows: On days 1-30, Genistein once daily, interaperitoneally injecting (18 ). Morphine plus Genistein was administered as follows: On days 1-30, Genistein once daily plus morphine, interaperitoneally injecting (17, 18). The same volume of saline was administered. Mice were randomly divided into 8 groups (n=6). 1) Normal saline group (1 ml DW/daily); 2) Morphine treated group; 3) Genistein 1 mg/kg treated group; 4) Genistein 2 mg/kg treated group 5) Genistein 4 mg/kg treated group; 6) Morphine plus Genistein 1 mg/kg treated group; 7) Morphine plus Genistein 2 mg/kg treated group; 8) Morphine plus Genistein 4 mg/kg treated group.
Testis weight and hormone estimationsThe animals were anesthetized 24 hr after the last injection. By cardiac puncture method blood from sacrificed mice were collected into sterile collection vials and preserved in the temperature of 37^oC for 30 min and was centrifuged to obtain the serum (3000 gr for 15 min). Serum samples were directly frozen at -70^oC until biochemical analyses. Serum testosterone concentrations were measured by ELISA (Abcam 108666, USA) method. Serum levels of LH and FSH were measured by immunoradiometry assay. The serum levels of LH and FSH were determined, using the RADIM (Rome, Italy) kits.
For FSH kit variation intra-assay coefficient was 5.3%, the variation interassay coefficient was 5.5% and the assay sensitivity was 0.17 ng/ml. For LH kit the variation intraassay coefficient was 6.8%, the variation interassay coefficient was 7.2% and assay sensitivity was 0.2 ng/ml. The testes along with the epididymis carefully were removed, washed in normal saline solution (0.9%), blotted, and weighed separately and the average weights were used (13 (link)).
Preparation of sperm suspension for analyses of different parametersThe Cauda epididymis from both sides were taken out, minced and incubated in a pre-warmed petri dish containing 10 ml Hams F10 medium containing 0.5% Bovine Serum Albumin and the solutions were incubated at 37^oC. The spermatozoas were allowed to disperse into the buffer. After 20 min, epididymis Cauda was removed, and suspension was gently shaken to homogenize (14 ).
Sperm count For sperm counting, 500 μL of prepared epididymal sperm suspensions were diluted with formaldehyde fixative (10% formalin in PBS). Approximately 10 μL from diluted solution was transferred into a haemocytometer using a Pasteur pipette (Thoma, assistant Sondheim/Rhön, Germany) and let to stand for 7 min. Then the settled sperms were counted and evaluated per 250 small squares of a haemocytometer (13 (link)).
Sperm viability Viability was assessed by eosin Y staining (5% in saline). Forty micro liter samples of freshly sperm suspension were placed on a glass slide, mixed with 10 μL eosin and observed under a light microscope (×400 magnification). Live sperms remained unstained following staining, whereas, those that showed any pink or red coloration were classified as dead. At least 200 sperm were counted from each sample in ten fields of vision randomly, and the percentage of live sperms was recorded (13 (link)).
Sperm motilityTo assess the percentage of motile sperm light microscope (Olympus Co., Tokyo, Japan) was used at 400× magnification. The suspension was prepared by repipetting and one drop of sperm suspension was placed on a glass slide and covered with a lamella. Sperm motility was divided to 4 levels according to certain criteria: a) quick progressive motility in direct line, b) slow progressive motility in direct or indirect line, c) no progressive motility and d) no motility were counted in several microscopic fields of vision and the percentages of motile and non- motile sperms were obtained. Motility estimates were obtained from 10 different fields in each sample. The mean of the 10 successive estimations was used as the final motility score (14 ).
Sperm morphologyTo examine the sperm cells morphology, smear was prepared from the samples. Eosin/ nigrosin stain was used to estimate sperm morphology. To test, one drop of eosin/ nigrosin was added to the suspension and mixed gently. The slides were then viewed under a light microscope at 400× magnification. A total of 300 spermatozoa were analyzed on each slide (19 (link)).
Griess assayNitric oxide was measured based on Griess colorimetric assay. Accordingly, N-(1- naphthyl) ethylenediamine dihydrochoride (NEED), sulfonamide solutions and nitrite standards were prepared. To measure nitrite concentration in serum, after de-freezing the serum samples, 100 µl of the sample serum was deproteinized by zinc sulfate and transferred to the wells. 100 µl chloride vanadium, 50 µl sulfonamide, and 50 µl NEED solutions were added afterwards. The cells were incubated in the temperature of 30^oC in darkness. Samples' optical density (OD) was measured by ELISA reader at the wavelength of 540 nm (20 (link)).
Histological analysisHistological analysis of the testes was performed. The testes were fixed in neutral buffered formalin 10% dehydrated, and then embedded in paraffin. Thereafter, five- micron thick sections were prepared and at least five slides from each testis were stained with hematoxylin and eosin (HE) for histological assessment. The specimens were examined under Olympus/3H light microscope. More than 20 sections were prepared from each block. The sections numbered 5, 10, 15, and 20 were selected and photographed separately from three random views. Seminiferous tubules diameter and germinal layer thickness were measured by Motic camera and software (Moticam 2000, Spain). The mean of seminiferous tubules diameter (μm) and germinal layer thickness (μm) were determined for each testis (21 ).
Statistical analysisThe Kolmogorov-Smirnov test was used for assessing normal distribution of variables. All the quantitative data were presented as mean±SD. One-way analysis of variance (ANOVA) followed by LSD post-hoc test were performed to determine the statistical significance between different groups using SPSS software package 16.0. P<0.05 was considered significant.

Sperm viability/vitality was evaluated using the eosin-nigrosin staining method. First, a 5 µL diluted semen sample was placed in a test tube. Then, 5 µL of each eosin and nigrosin staining solution was added and mixed well, followed by a 30 s wait. After 30 s, 5 µL of solution was placed on the slide, spread evenly with coverslip and left for 15 min to dry. Observed under 40× microscopic lenses, at least 300 sperm were analyzed to determine the sperm viability/vitality percentage estimation.

The viability of sperm was assessed by eosin-nigrosin staining (Merk, Germany). Based on this staining, the head of dead sperms turns red or dark pink, but the head of live sperms remains colorless. At first, the 1:2 ratio (by volume) of sperm suspension and eosin was mixed in the microtube. Following 30 sec, an equal volume of nigrosin solution was added. Then, a thin spread of the sample took place on the slide. After drying the smear, using a light microscope (100x magnification), 200 sperms per sample were counted. Eventually, the percentage of live and dead sperms in the samples was calculated (17).