The protein content in each tissue was determined using the bicinchoninic acid protein assay (BCA) (Pierce, USA). Bovine serum albumin maybe was used as a standard [22]. All preparation procedures were performed at +4��C. All homogenates were stored at �C80��C prior to testing.2.3. Histomorphological EvaluationAll histomorphological analyses described below were performed by two investigators blind to rat’s treatment. For histological examination the tissue samples were fixed in 10% formalin in phosphate buffer for 3 days. Afterwards, testis tissues were processed by routine histological methods and embedded in paraffin blocks. Paraffin blocks were placed in rotary microtome (RM 2255, Leica Instruments, Nu?loch, Germany), and sections of 5��m thickness were obtained with disposable metal microtome blades (Type N35, Feather Company, Osaka, Japan).

After deparaffinization and rehydration, all sections were stained with hematoxylin-eosine (H-E). 2.3.1. Examination of Spermatogenesis Johnsen’s score was used to categorize the spermatogenesis on 5 different places in the same histologic section in 20 seminiferous tubules [23]. A score of 0 to 10 was given to each tubule according to epithelial maturation: 10: complete spermatogenesis and perfect tubules; 9: many spermatozoa present and disorganized spermatogenesis; 8: only a few spermatozoa present; 7: no spermatozoa but many spermatids present; 6: only a few spermatids present; 5: no spermatozoa or spermatids but many spermatocytes present; 4: only a few spermatocytes present; 3: only spermatogonia present; 2: no germ cells but only Sertoli cells present; 1: no germ cells and no Sertoli cells present.

2.3.2. Measurement of Seminiferous Tubule Diameter In each section, the diameters of 10 separate circular seminiferous tubules were randomly measured using a 10x objective. The mean seminiferous tubular diameter MSTD of each testis was determined in micrometers (��m).2.3.3. Image Analysis Methods Images were analyzed by using a computer assisted image analyzer system consisting of a microscope (Olympus BX-51, Japan) equipped with a high-resolution video camera (Olympus DP-71, Japan). All sections were digitally photographed. For morphometric evaluation, computerized video camera-based image analysis system (UTHSC Image Tool software version 3.0, University of Texas Health Science Center, San Antonio, TX, USA) was used.

2.4. Evaluation of Germ Cell ApoptosisIn order to detect DNA fragmentation in cell nuclei, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) reaction was applied to paraffin sections. DeadEnd Colorimetric TUNEL Brefeldin_A system kit (In Situ Cell Death Detection Kit, Roche, Manheim, Germany) used for apoptotic cell detection. Serial 5��m thick paraffin-embedded sections were deparaffinized, rehydrated in graded alcohol, and pretreated in proteinase K (20��g/mL) for 15min at 37��C.