"On post-natal day (PND) 120, twenty adult F1 male rats from each group were picked and five were used for gravimetric, histological (right testis),ultra-structural (left testis) analyses, five for TIF collection (right testis) and immunofluorescent studies (left testis), five for isolation of SCs (those from right testis for mRNA isolation and from left testis for immunoblot analysis) and the remaining five testes were used for biochemical analyses (Cr estimation in SCs and GCs).Here, the rats were sacrificed under sodium thiopentone (40 mg/kg body weight) anaesthesia and, before tissue harvest, blood was collected by cardiac puncture to assess serum hormonal profiles and Cr concentration.

2.2. Isolation of Sertoli cells and germ cells.

SCs were isolated by enzymatic digestion method [38]. Immediately after killing, the testes were removed, washed and placed in cold (4◦C) Hank’s balanced salt solution lacking CaC12 and MgSO4 (HBSS, pH 7.4, containing 50 U/mL of penicillin and 50 mg/mL of streptomycin sulfate). Within 2 h of removal, testes were decapsulated, washed twice in HBSS, cut into 2–5-mm pieces using asterile surgical blade. These pieces were suspended in HBSS (2 gtissue/50 mL) and vigorously shaken by hand (8–10 times). Seminiferous tubules from suspensions of testes were recovered after sedimentation at unit gravity (g) (settled down by gravity) for 5 min at 4◦C. For recovery of seminiferous tubules left out in testicular suspension, if any, the supernatant was again centrifuged at 4◦C for 3 min at 100 × g. This procedure also helped to remove red blood cells and Leydig cells. Collagenase type I and DNase were added to the seminiferous tubule suspension. Digestion was then carried out at 37◦C in a shaking water bath (160 oscillations/min)for 30 min. After discarding the cluster of tissue resulting from collagenase digestion, the suspension of seminiferous tubules was distributed equally among four tubes (15 mL capacity) and centrifuged for 3 min at 100 × g. Each pellet was suspended in 7 mLHBSS in a 100-mL glass bottle, agitated gently using a wide mouthed Pasteur pipette, and centrifuged for 3 min at 100 × g; the supernatant was then discarded. This was repeated three times. Finally,the pellets were pooled, suspended in 10 mL HBSS containing 5% FBS and placed on ice for 5 min. At the end of the isolation, the cell suspension was passed through a fine nylon mesh filter (50 mpore size). After filtration, the purity of SCs was greater than 80% based on positive staining for Oil Red O [39]. The final steps of purification involved multiple sedimentations of SCs at unit gravity. SCs sedimented at a quicker rate than GCs and were collected by gentle aspiration of the supernatant after sedimentation. The major contaminants were spermatogonia (5–7%), spermatocytes (2–4%) and peritubular myoid cells (1%). The cell suspension was passed through a fine nylon mesh filter (50 m pore size) and filtered through glass wool to remove SCs. The filtered cell suspension was centrifuged at 200 × g for 10 min at room temperature, the supernatant was discarded and the germ cell pellet was used for further analyses. The purity of germ cells (85–90%) was ascertained by staining an aliquot of cells for alkaline phosphatase activity [40].

2.3. Collection of testicular interstitial fluid (TIF)

TIF was collected by using Sharpe and Cooper method [41].Briefly, the testis was decapsulated at one end and the other end was tied to a nylon thread and hung into a glass test tube overnight at 4◦C. The TIF that collected in the glass tube was centrifuged at 1500 × g for 10 min at 4◦C. The supernatant was diluted with PBS(0.01 M, pH 7.4) at a ratio of 1:10, and used for the determination of CrIII and hormones.

2.4. Estimation of CrIII and VI by ion exchange chromatography.

The levels of CrIII/VI in serum, testis, TIF, SCs and GCs were quantified by ion exchange chromatography as per the manufacturer’s instruction (IC net version 2.3; Metrohm, Herisau, Switzerland). Toa crucible containing 100 mg of testis tissue, 1 mL con. HNO3 was added followed by 1 mL perchloric acid. The content was digested over a boiling water bath until the solution turned pale yellow;if the color of the digest was brown, more HNO3and perchloric acid was added and the oxidation was repeated. The digest was made up to a known volume with deionized water. Serum samples were diluted 1:10 with a diluent containing 0.1% Triton X-100 and 0.1% HNO3in deionized water. About 250 uL of the sample was injected into the Cr (CrIII and VI) detecting column (IC-30 column – polystyrene/divinyl benzene co-polymer with NH3 group) and moved towards UV detectors along with the eluent and the post-column reagent solution. The absorbance of CrIII and CrVI ionswere detected at 365 nm and 570 nm, respectively, and the values were expressed as ug/mL serum, ug/mg tissue and g per number of cells depending upon the sample. The column sensitivity was 0.1 ppm.

2.5. Sperm forward motility

Sperm were collected from the cauda epididymidis of five ratsin each group, prior to the harvest of the testis, to assess the impact of Cr on sperm forward motility. Immediately after anesthetizing the animal, the epididymis and vas deferens on both sides were removed and cleared of adhering tissues. An incision was made at the junction between the vas deferens and the cauda epididymidis on the right side. The fluid that oozed out was transferred to a clean dry glass slide, diluted in physiological saline and observed with Nikon (Japan) microscope 80i for sperm forward motility which was measured as distance traveled per min [42].

2.6. Viability of sperm

One step Eosinnigrosin staining was used to analyze sperm viability [43]. The staining solution contained 0.67% Eosin Y and 10% nigrosin dissolved in 0.9% sodium chloride in gently heated dis-tilled water. The solution was cooled to room temperature and filtered then stored in a dark amber bottle. Approximately equal volumes of sperm suspension and stain were smeared on a clean micro-slide and covered with a cover-slip. A total of 100 sperm from each animal were counted within 2 min after the addition of thestain and examined directly in a bright field microscope. Unstaineds perm were classified as live and those that showed any pink or red coloration were classified as dead.

2.7. Testis histomorphometry

Diameter of seminiferous tubules and their lumen were measured using an ocular micrometer and the numerical densities of Sertoli cells, spermatocytes and spermatids were determined per the procedure [44].

2.8. Histology

Immediately after anesthetization, the right testis was removed and a portion was fixed in 4% paraformaldehyde (PFA) for 48 hand processed for paraffin embedding. Sections (5 m thickness) obtained in a Leica microtome (Richmond Hill, Canada) were stained with hematoxylin and Eosin and observed with Nikon(Japan) microscope 80i. A network internet system-imaging and capturing software (NIS) (Japan) was used for acquisition of images.

2.9. Tissue processing for transmission electron microscopy (TEM)

Our TEM study was performed as we described earlier [10,11]. Aportion of the right testis was immersion-fixed in 2.5% glutaralde-hyde in cacodylate buffer for 12–24 h. Tissue slices (1 mm) were post-fixed in 1% osmium tetroxide for 2–3 h and embedded in thin viscosity resin (Sigma, St. Louis, MO, USA). Ultrathin sections(below 100 nm) were cut with an ultra-microtome (Leica Microsystems, Nussloch, GmbH Nussloch, Germany), stained with uranylacetate and lead citrate, and observed using Technai T12 Sprit (The Netherlands) transmission electron microscope.

2.10. Immunofluorescent study

A portion of the testis was embedded in Cryo-OCT compound(Leica, Bannokburn, USA) and frozen in liquid nitrogen. Testis sections (5 m), obtained using a cryomicrotome (Leica, CM 3050),were incubated in blocking buffer (3% bovine serum albumin, 0.1% Triton X-100 in PBS, pH 7.4) for 20 min at room temperature and incubated for 1 h at room temperature with primary antibodies for claudin-11 and occludin (same as Western Blot) at 1:500 l dilution. After 1 h, sections were washed thrice with PBS and then incubated with anti-rabbit and anti-mouse Alexa Fluor (488 nmgreen (Cat No. A11008) for claudin-11 and 568 nm red (Cat No.A 11036) for occludin) secondary antibody (Invitrogen Molecu-lar Probes, Carlsbad, California), respectively, at 1:300 L dilutionfor 1 h at room temperature. Sections were washed thrice in PBS,and the nucleus was stained with 4, 9, 6-diamidino-2-phenylindole(DAPI) (MP Biochemicals Pvt Ltd, Mumbai, India) [0.1 mg/mL inPBS]. Then, the sections were washed thrice in PBS and observed with a Nikon fluorescent microscope 80i (Japan), and NIS was used for acquisition of images.

2.11. Real-time reverse transcription polymerase chain reaction

Total RNA was isolated from SCs using TRIZOL reagent (Invitrogen, Carlsbad, CA, USA). Real-time RT-PCR was performed in MX3000P Real-Time PCR machine (Strategene, CA, USA) as described earlier [45]. Details of the primer sequences are give nin Table 1. -actin was used as an internal control.

2.12. Western blotting

The expression pattern of claudin-11, occludin, AR and FSHRproteins in SCs were detected by western blotting as describedearlier [46] using specific primary antibodies AR (Cat No.SC-815),(c-19) rabbit polyclonal IgG antibody, FSHR (Cat No.SC-13935)(H-190) rabbit polyclonal antibody, Claudin-11 (Cat No.SC-25711)(H-107) rabbit polyclonal antibody, Occludin (Cat No.SC-8145)(N-19) goat polyclonal antibody and -actin (Cat No.SC-10731) (H-300) mouse monoclonal antibody and HRP – conjugated secondaryantibodies anti-goat-rabbit IgG (Cat No.SC-2004) and anti-goat-mouse IgG (Cat No.SC-2061) (Santa Cruz, Texas, USA). 1:2000 Land 1:5000 L dilution were used for primary and secondary anti-bodies, respectively. The intensity of the bands was quantified byQuantity One Software (Bio-Rad Chemi Doc System, CA, USA) andwas normalized with the internal control (-actin) (Sigma-Aldrich,St. Louis, MO, USA) and the band intensity was expressed as arbi-trary unit.

2.13. Determination of total protein and enzymatic antioxidants in SCs and GCs

Protein content was determined using Folin phenol reagent. The following antioxidant enzymes were assessed by standard colorimetric methods indicated in parenthesis: superoxide dismutase [E.C. No. 1.15.11] [47]; catalase [E.C. No. 1.15.1.6] [48]; and glutathione peroxidase [EC 1.11.1.9, GPx] [49].

2.14. Determination of reactive oxygen species and lipid peroxide levels in SCs and GCs

Hydrogen peroxide level was determined as described [50].Hydroxyl radical production was determined as described [51].The level of lipid peroxidation (LPO) was measured following the method [52].

2.15. Radioimmunoassay (RIA) of hormones

The levels of serum FSH and TSH were determined adopting standard liquid phase RIA [53] using radioactive iodine [125I] (MPBiochemicals India Pvt. Ltd., Mumbai) and specific rat antigen, antibodies and reference standard received from NIDDK (National Institute of Diabetes, Digestive and Kidney Diseases, Bethesda,Maryland, USA): tracer [LH (NIDDK-rLH-I-9-AFP-10250C) andrFSH (NIDDK-rFSH-I-8-AFP-1145B)], antiserum for LH (NIDDK-anti-rLH-S-10) and rFSH (NIDDK-anti-rFSH-S-11-AFP-C0972881)and reference preparation for rLH (NIDDK-rLH-RP-3-A FP-7187B)and rFSH (NIDDK-rFSH-RP-2-AFP-4621B). Testosterone level in both TIF and serum was quantified by solid-phase RIA using a com-mercial kit (Diasorin Inc., Stillwater, USA) as described earlier [54].The maximum binding was 100% and the sensitivity of the assay was 0.02 ng/mL. The intra and inter-assay coefficient of variation for TSH assay was 4–8% and 9.9%, respectively; sensitivity of the assay was 6 ng/mL. The intra- and inter-assay coefficient of variation for FSH was 5.7–8.9% and 12.2%, respectively; sensitivity ofthe assay was 0.20 ng/mL. The cross reactivity of testosterone anti-body to other steroids such as 5-dihydrotestosterone (DHT) and androstenedione were 6.9% and 1.1%, respectively.

2.16. Statistical analysis

The data were subjected to statistical analysis using one-way analysis of variance (ANOVA) and Duncan’s multiple-range test to assess the significance of individual variation between groups using the Students’ version statistical analysis software SPSS 7.5 (SPSS,Chicago, IL, USA)."

F1 Male Wistar Rat