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Apoptosis abolishes the damage from the germ line and mutation rate are higher in dividing spermatogenic germ cell types that rarely undergo apoptosis than in those where it is present. The germ line mutation rate in males increase with age, In order to explore this idea further, my present study will be conducted to identify whether the rates of apoptosis in response to a mutagen are influenced by the age of the individual.
Apoptosis of testicular germ cells and spermatogenic staging was evaluated using terminal deoxynucleotidyl nucleotide end-labelling (TUNEL) (Gavrieli et al., 1992) and Periodic Acid - Schiff's (PAS) staining (Brinkworth et al., 1995).
These techniques was executed for identifying the level of apoptotic cells in the testes, the occurrence of apoptosis among the different germ cell types and the performance of statistical analysis by comparing variables of four groups from normal (control) aged and young mice; and treatment with ethylnitrosourea (ENU) aged and young mice. Effects of ENU (pre-meiotic spermatogonia) on epididymal transit are to induce genetic damage.
Figure 2: To investigate the effects of ageing on the responses of germ cells to induced genetic damage (Taylor, 2007). (A) Effect of age, (B) Effects of treatment, (C) Age-dependent susceptibility to damage resulting from treatment, (D) Effect of treatment and then (E) Response to treatment is age dependent or treatment dependent.
The parallelogram approach signifies the direct and indirect comparisons of the study. The direct effects of ENU treatment on young and old animals help determine whether observed effects of treatment are dependent or independent of age. This may facilitate how underlying mechanisms vary with age.
The mouse testes were randomly divided into 4 groups of 3 each. The experimental groups of young and old mice received treatment of intraperitoneal (i.p.) injection of 250 mg kg-1 ENU (dissolved in 0.9% (w/v) saline). Control groups of young and old mice were i.p. injected with 0.9% (w/v) saline. The experimental and control groups were sacrificed after twenty-four hours. The mouse testes were obtained from a laboratory in Germany and fixed in Bouin's fluid. The fixed tissues were immersed in 70% (v/v) alcohol and processed overnight using a Shandon automatic tissue processor. Processed tissues were embedded in paraffin wax (Sigma, St Louis, MO). Sections of five-micron thick were cut by using Leitz microtome and mounted on Poly-L-lysine coated glass slides (Sigma-Aldrich Co., St. Louis, MO) for Periodic Acid - Schiff's (PAS) staining technique and terminal deoxynucleotidyl nucleotide end-labelling (TUNEL) method.
4.2 Histochemistry and Histology
Sections were deparaffinized, re-hydrated in decreasing series of ethanol to water and rinsed in terminal deoxytransferase (TdT) buffer (Gavrieli et al., 1992). An incubation for one hour at 37°C in the same buffer with 5µM biotinylated deoxyuridine triphosphate (Biotin-16-dUTP) (Roche, Germany) and 0.3U µl-1 TdT (BioLabs, New England) (Brinkworth and Schmid, 2003; Brinkworth et al., 1995). The reaction was stopped by rinsing the slides thoroughly in Tris buffered saline (TBS), pH 7.6 and the biotin label visualized with a 30-min incubation of an Extravidin - peroxidase complex (Sigma-Aldrich Co., St. Louis, MO) diluted 1:50 in TBS with 0.1% Bovine Serum Albumin (BSA) (Sigma-Aldrich, St. Louis, MO). Slides were again washed in TBS and incubated with diamino-benzidine (DAB) (SigmaFastTM) for colour development. Slides were counterstained for 45-sec in Mayer's haemalaun, rinsed in deionized water, washed in running tap water, dehydrated and mounted with Histomount® (Vector) (Gavrieli et al., 1992).
4.2.2 PAS Staining
Comparable sections from each group were deparaffinized, re-hydrated and washed in 0.5% (w/v) periodic acid (Sigma Chemical Co. St, Louis, MO), deionized water, Schiff's reagent (Sigma-Aldrich, St. Louis, MO) for 15-mins and again in deionized water. The slides were counterstained with Mayer's haemalaun for 30-sec, rinsed in deionized water, dehydrated and mounted (Bancroft and Stevens, 1990; Brinkworth et al., 1995).
Slides were examined using a Nikon light microscope at a total of 400X magnification to observe individual seminiferous tubules. Twenty seminiferous tubules were scored per animal in all of the 12 germ cell associations in all four groups. The numbers of labelled (TUNEL-positive) and un-labelled apoptotic cells were recorded per seminiferous tubule and per Sertoli cell. A cell was considered positive when the nuclear staining was intense, brown or dark brown and homogenous (Brinkworth). The numbers of Sertoli cells was scored per seminiferous tubule to normalize germ cell number. The spermatogenic stage of each seminiferous tubule was evaluated according to the criteria described in (Franca et al., 1998; Russell et al., 1990). The spermatogenic staging of each tubule were categorized as early, middle or late stage for simplicity. Scale bars of photomicrographs are not provided due to insufficient equipment.
Figure 3: In mouse, the seminiferous epithelial cycle is organized in 12 stages (I-
XII) (Huckins and Oakberg, 1978). Duration of spermatogenesis in the mouse is about 38.8 days and approximately one-fourth of this time is the length of spermatogenic cycle (Meistrich et al., 1975; Oakberg, 1956). Spermatid differentiation is divided into 16 steps (1-16). B, B-type spermatogonia; PL, preleptotene spermatocytes; L, leptotene spermatocytes; Z, zygotene spermatocytes; P, pachytene spermatocytes; D, spermatocytes in diplotene; SS, secondary spermatocytes (Brehm and Steger, 2005; Russell et al., 1990). The staging of mouse were categorized into early (stages I-V), middle (stages VI-VIII) and late (stages IX-XII) (Brinkworth).
4.4 Statistical analysis
Standard error of the mean (SEM) was carried out in the results section to show the accuracy of the sample mean in relation to the true population mean. The apoptosis levels per animal were calculated as the mean per 20 tubules as well as per Sertoli cells. The Sertoli cells were calculated as the mean per 20 tubules. Data were analysed by two-way analysis of variance (ANOVA) (SPSS, 2009), since comparisons were made between more than one independent variable in each group (e.g. control and treated or young and old). For ANOVA, significant differences were evaluated by using the Post Hoc testing (Bonferroni) for multiple comparisons. The level of significance was set at 5%.