ISI Impact Factor (2004): 1.096

Editor-in-Chief
Prof. Yi-Fei WANG,

Radio-protective effect of vitamin E on spermatogenesis in mice exposed to g-irradiation: a flow cytometric study

C. Songthaveesin¹, J. Saikhun², Y. Kitiyanant^2,3, K. Pavasuthipaisit^2,3

Asian J Androl  2004 Dec; 6: 331-336         

Keywords: vitamin E; radioprotection; spermatogenesis; mouse; flow cytometry

Abstract

Aim: To investigate the effect of vitamin E on the radioprotection of spermatogenesis and chromatin condensation of spermatozoa during passage through the epididymis in mice exposed to irradiation. Methods: Adult outbred male ICR mice were orally administered natural vitamin E (VE, D-a-tocopheryl acetate) at 400 IU/kg for 7 days before exposure to 1 Gy of g-irradiation. The animals were sacrificed at day 1, 7, 14, 21, 28, 35 and 70 post-irradiation (IR) and the percentage of testicular germ cells and epididymal sperm chromatin condensation was analyzed using flow cytometry. Results: Serum D-a-tocopheryl acetate levels were 47.4±3.2 mg/dL in the treated group, yet it could not be detected in the control group. The testicular weight of irradiated mice pretreated with VE+IR was significantly (P<0.05) higher than that of those without VE treatment (IR) at day 14 and 21 post-irradiation. The percentage of primary spermatocytes (4C) in the VE+IR group was comparable to the controls but significantly (P<0.05) higher than those in the IR group from day 7 to 35 post-irradiation. The percentage of round spermatids (1C) in the VE+IR group was also significantly (P<0.05) higher than those in the IR group at day 28 post-irradiation. The primary spermatocytes : spermatogonia ratio in the IR group was significantly (P<0.05) declined at day 7 to 35 post-irradiation when compared to the VE+IR and control groups. The round spermatid: spermatogonia ratio in the VE+IR group was significantly (P<0.05) higher than that of the IR group at day 14 and 28 post-irradiation. The chromatin condensation of epididymal spermatozoa measured by propidium iodide uptake was not affected by 1 Gy of g-irradiation. Conclusion: The administration of VE prior to irradiation protects spermatogenic cells from radiation.

1 Introduction

Radiotherapy is widely used for cancer therapy. Although the treatments could be successful, patients often complain of azoospermia or infertility [1, 2]. Radiation doses as low as 0.1-0.2 Gy exerted detectable effects on spermatogenesis in adult men [3] and high doses over 4 Gy caused in permament azoospermia [4]. It has been reported that spermatogonia are the most radio-sensitive germ cells in mice [5]. The short-term and long-term effect of radiation on the number and structure of epididymal spermatozoa have also been reported [6].

Vitamin E (VE) is a well-known antioxidant and an effective primary defense against lipid peroxidation of cell membrane [7]. VE comprises 8 natural fat-soluble compounds, including 4 tocopherols and 4 tocotrienols. Among them, a-tocopherol is the most prevalent and the most active. Due to its effective antioxidant property and free radical scavenging capability, administration of a-tocopherol has been proposed as a potential radio-protectant. VE treatment significantly ameliorated aflatoxin-induced biochemical alterations [8] and lipid peroxidation [9] in the testis of mice. The present study was designed to investigate the radio-protective effect of a natural VE (D-a-tocopheryl acetate) on spermatogenesis of mice exposed to irradiation.

2 Materials and methods

Eight-week old outbred male ICR mice were obtained from the National Laboratory Animal Center, Mahidol University. They were inhibited in an animal house at 22 ℃ and a light : dark cycle of 12 : 12 hours, 5 in a cage, with standard food and water provided ad libitum. All animals were housed under the conditions for more than 1 week before the experiment. They were divided into 3 groups of 5 animals each: 1) controls: animals were orally administered corn oil and did not expose to irradiation, 2) irradiation group (IR): animals were orally administered corn oil before exposure to g-radiation, and 3) vitamin E-treated group (VE+IR): animals were orally administered D-a-tocopheryl acetate (Medicab, Samuthpra-karn, Thailand) at 400 IU/kg per day for 1 week before exposure to radiation. The experiments were repeated five times.

Mice were irradiated at the Department of Radiology, Faculty of Medicine, Ramathibodi Hospital, Mahidol University. Mice were given whole-body irradiation with a single dose of 1 Gy/min. The cages containing five mice were placed on the bed area. The center of the g-ray was directed perpendicular to the center of the cage and the distance from the g-ray tube to the body of mice was 91.5 cm.

Five animals from each group were sacrificed at day 1, 7, 14, 21, 28, 35 and 70 post-irradiation by cervical dislocation. Blood samples were collected in all the 3 groups prior to irradiation by heart puncture for the determination of D-a-tocopheryl acetate concentration using a high performance liquid chromatograph (HPLC).

The serum concentration of D-a- tocopheryl acetate was measured by HPLC. Briefly, serum (100 µL) was mixed with 200 µL of distilled water, 200 µL of ethanol and 100 µL of retinyl acetate (Sigma, St. Louis, USA) as internal standard, mixed by vortexing for 30 sec and 750 µL of hexane (J.T. Baker, Phillipburg, USA) was added for extraction. After centrifugation at 2000 ×g for 3 min, the hexane layer was evaporated to dryness under a stream of nitrogen, redissolved the lipid residue in 100 µL of mobile phase (methanol, HPLC grade) and injected into the HPLC system with a pump (model 515, Waters, Milford, USA) and Ultracarb ODS-5 µm with Hypersil ODS guard column (150 mm×4.6 mm, 5 µm particle diameter, SGE, Japan Inc., Kanagawa, Japan). Absorbance was read at 294 nm using an ultraviolet detector (model UV 975, Jasco, Japan Spectroscopic Co., Tokyo, Japan). The chromatographic separation was performed by isocratic elution with methanol at a flow rate of 1.3 mL/min. The injection volume was 20 µL.

Testicular germ cells were released from the seminiferous tubules by mincing the testis with fine scissors in TNE buffer [0.15 mol/L NaCl, 0.01 mol/L Tris-(hydro-xymethyl) aminomethane, 1 mmol/L EDTA, pH 7.4]. The cell suspension was filtered through 100 mm nylon mesh to remove the tissue debris before staining for flow cytometric analysis.

The epididymis was cut into 3 portions: caput, corpus and cauda. Spermatozoa were released from each portion by mincing with fine scissors before filtering through the 100 µm nylon mesh to remove the tissue debris before staining for flow cytometric analysis.

Testicular germ cells and epididymal spermatozoa were processed as described. Cell suspension was centrifuged at 9 000 rpm for 5 min. The pellet was resuspended in 0.5 mL of TNE buffer containing 0.5 mg/mL ribonuclease A (Sigma, USA) and incubated at 37 ℃ for 30 min. At the end of incubation, 0.5 mL of propidium iodide solution [TNE buffer containing 50 µg/mL propidium iodide (Sigma) and 0.1 % Triton X-100 (v/v)] was added and further incubated at 37 ℃ for 30 min before filtering through a 41 µm nylon mesh and subjected to flow cytometry. Flow cytometric analysis was performed using a FACS Vantage flow cytometer (Becton Dickinson, San Jose, USA). Cells were analyzed at a rate of 500 to 1 000 cells/sec using FACS flow (Becton Dickinson) as sheath fluid and 10 000 events for each sample were recorded for analysis. The flow cytometer was standardized for each analysis session by using Calibrite beads (Becton Dickinson). Data were analyzed with WinMDI version 2.8 (J. Trotter, http://facs.scripps.edu/software.html).

The means±SEM of testicular weights and the percentages of testicular germ cell distributions were analyzed using analysis of variance (one-way ANOVA) with Duncan New Multiple Range Test. Calculations were performed on SPSS 9.0 software (SPSS Inc., Chicago, USA).

Serum D-a-tocopheryl acetate levels were 47.4±3.2 mg/dL for the VE+IR group while it was not detected in the IR and control groups prior to irradiation.

A significantly (P<0.05) higher testicular weight in the VE+IR group than in the IR group was observed at day 14 and 21 post-irradiation, compared to the control group (Figure 1). The testicular weight in irradiated mice from both treatment groups was significantly (P<0.05) lower than that of the controls at days 21 to 35 post-irradiation. In both treatment groups it was recovered to was normal at day 70 post-irradiation.

Figure 1. Testicular weight of the control and irradiated mice pretreated with (Vit E+IR) or without vitamin E (IR) before exposure to 1 Gy of g-irradiation. ^bP<0.05, compared with control. ^eP<0.05, compared with IR group.

Based on the relative fluorescence intensities of the DNA content, 4 different populations of testicular germ cells, elongated spermatids (HC), round spermatids (1C), spermatogonia (2C) and primary spermatocytes (4C), could be identified by flow cytometric analysis (Figure 2). The 2C peak also includes non-germ cells like Sertoli and Leydig cells, which comprise about 3 % of the total germ cell population [10]. There was a decrease in the percentage of primary spermatocyte population in the IR groups as compared to VE+IR and control groups (Figure 2). The percentage of primary spermatocytes in the IR group was significantly (P<0.05) decreased at day 7 to 35 post-irradiation compard with that of the controls and VE+IR group (Figure 3A). The normal levels of primary spermatocytes were restored by day 70 post-irradiation. Treatment of mice with or without vitamin E before exposure to radiation did not significantly alter the spermatogonia population as compared with the controls (Figure 3B). The round spermatids in the IR group were significantly (P<0.05) declined by day 28 post-irradiation and were significantly lower than that of the VE+IR group (Figure 3C). The elongated spermatid population was not significantly different among groups except at day 28 post-irradiation, it was significantly higher in the IR group than in the controls and VE+IR group (Figure 3D).

Figure 2. Representative flow cytograms of testicular germ cells of the control and irradiated mice pretreated with vitamin E (Vit E+IR) or without vitamin E (IR) at day 14 post-irradiation. Peak I: elongated spermatids (HC); Peak II: round spermatids (1C); Peak III: spermatogonia and somatic cells (2C) and Peak IV: primary spermatocytes (4C).

Figure 3. The percentage of different germ cell types in mouse testes treated with vitamin E (Vit E+IR) or without treatment (IR) before exposure to 1 Gy of g-irradiation. A: primary spermatocytes (4C); B: spermatogonia and somatic cells (2C), C: round spermatids (1C) and D: elongated spermatids (HC). ^bP<0.05, compared with control.

A significant (P<0.05) decline in the primary sperma-tocytes: spermatogonia ratio (transformation of spermatogonia to primary spermatocytes) was observed at day 7 to 35 post-irradiation in mice irradiated without vitamin E treatment (Figure 4A), while it was not significantly different between the VE+IR group and the controls. The round spermatids: spermatogonia ratio (transfor-mation of spermatogonia to round spermatids) in the IR group was significantly (P<0.05) decreased at day 14 and 28 post-irradiation compared with that of the VE+IR and control groups (Figure 4B).

Figure 4. Alteration in the primary spermatocyte : spermatogonia (4C : 2C) (A) and round spermatid : spermatogonia (1C : 2C) (B) ratios of mice treated with vitamin E (Vit E+IR) or without treatment (IR) before exposure to 1 Gy of g-radiation. ^bP<0.05, compared with control.

When the spermatozoa obtained from various portions of epididymis were stained with propidium iodide, the results of flow cytometry demonstrated that the FL2 fluorescence intensity was emitted by all cells but at different intensities depending upon the accessibility of the dye to DNA (chromatin condensation). The study on chromatin condensation in IR and VE+IR groups showed a continuing decrease of FL2 fluorescence intensity in spermatozoa from caput to cauda epididymis as in the control groups.

4 Discussion

A partial protection of vitamin E on the intestinal mucosa of irradiated rats has also been reported by pretreatment with a-tocopherol before exposure to radiation [11]. Van der Meer et al [12, 13] found that spermatogonial stem cells were radio-sensitive during the quiescent stages (IV-VII) while radio-resistant during active proliferation (stage IX-XII). This may be a possible explanation for the results obtained in the present study that the percentage of spermatogonia did not change in irradiated mice exposed to g-ray.

The present study showed that the primary spermatocytes was significantly decreased by day 7 to 35 post-irradiation. Previous reports also indicated that primary spermatocytes were significantly decreased at day 14 after exposure to radiation [14]. A higher radio-sensitivity of the spermatogonial cells could be attributed to the decrease of primary spermatocytes.

A depletion of primary spermatocytes also leads to a significant decrease in round spermatids by day 28 post-irradiation. However, the normal levels of round spermatids were restored by day 35 post-irradiation indicating the resumption of normal differentiation of the surviving spermatocytes. The time required for the formation of round spermatids from spermatogonia and primary spermatocytes is 28 and 14 days, respectively [15]. The relative percentage of elongated spermatids in IR group increased significantly by day 28 post-irradiation as compared to VE+IR and control groups. This finding may not reflect a true elevation in the elongated spermatid population. Since the decrease in the relative percentage of other germ cell types, especially primary spermatocytes and round spermatids, the flow cytometer would detected only the remaining population, i.e., the elongated spermatids resulting in their apparent increment.

The mechanism of radioprotection of vitamin E is unknown. The protective effect can be explained by the scavenging of free radicals before they damage cellular macromolecules. This vitamin is known as an antioxidant with a capacity for free radical trapping [16]. Vitamin E is a lipid-soluble antioxidant that quenches free radicals and acts to terminate the lipid-peroxidation chain and to stabilize the molecular composition of cellular membranes [17]. However, the precise protective effect of vitamin E in preventing apoptotic cell death or acting on other targets requires further studies. More-over, the question arises on whether the tumor cell will be protected when using vitamin E treatment to protect the spermatogenic cells from irradiation damage.

Our results failed to observe irradiation-induced changes in the pattern of chromatin condensation of epididymal spermatozoa in irradiated mice compared to the non-irradiated controls. The low dose of g-radiation (1 Gy) used in the present study may be insufficient to induce DNA damage in epididymal spermatozoa where the chromatin has been highly condensed. It is well established that spermatozoa and spermatogonial stem cells are the most radio-resistant cell types within the testis. Therefore, larger doses of radiation (≥25 Gy) are required to produce significant levels of DNA damage in spermatozoa irradiated ex vivo [18]. There was a significant decrease in the percentage of spermatozoa with intact DNA following exposure to X-ray irradiation with a dose of 30 Gy [19]. The incidence of spermatozoa with radiation-induced sperm chromosome aberrations increased exponentially with an increasing dose [20]. Moreover, it is noteworthy that spermatozoa retained a high fertilizing ability even exposed to 4.23 Gy of g-rays [21]. This evidence reflects the high radio-resistance of mature spermatozoa with extremely condensed chromatin [22]. In conclusion, vitamin E pretreatment successfully protects spermatogenesis against radiation-induced cell damage.

The authors thank Mr. Peter Hall, Assistant Professor, Ubol Sanpatchayapong and Language Center of Faculty of Graduate Studies, Mahidol University for editorial assistance.

References

[1] Colpi GM, Contalbi GF, Nerva F, Sagone P, Piediferro G. Testicular function following chemo-radiotherapy. Eur J Obstet Gynecol Reprod Biol 2004; 113 Suppl: S2-S6.
[2] Taksey J, Bissada NK, Chaudhary UB. Fertility after chemotherapy for testicular cancer. Arch Androl 2003; 49: 389-95.
[3] Centola GM, Keller JW, Henzler M, Rubin P. Effect of low-dose testicular irradiation on sperm count and fertility in patients with testicular seminoma. J Androl 1994; 15: 608-13.
[4] Relander T, Cavallin-Stahl E, Garwicz S, Olsson AM, Willen M. Gonadal and sexual function in men treated for childhood cancer. Med Pediatr Oncol 2000; 35: 52-63.
[5] Vergouwen RP, Huiskamp R, Bas RJ, Roepers-Gajadien HL, Davids JA, de Rooij DG. Radiosensitivity of testicular cells in the fetal mouse. Radiat Res 1995; 14: 66-73.
[6] Bansal MR, Kaul A, Tewari M, Nehru B. Spermatogenesis and epididymal sperm after scrotal gamma irradiation in adult rats. Reprod Toxicol 1990; 4: 321-4.
[7] Niki E, Yamamoto Y, Takahashi M, Komuro E, Miyama Y. Inhibition of oxidation of biomembranes by tocopherol. Ann N Y Acad Sci 1989; 570: 23-31.
[8] Verma RJ, Nair A. Vitamin E ameliorates aflatoxin-induced biochemical changes in the testis of mice. Asian J Androl 2001; 3:305-9.
[9] Verma A, Kanwar KC. Effect of vitamin E on human sperm motility and lipid peroxidation in vitro. Asian J Androl 1999;1: 151-4.
[10] Clausen OP, Purvis K, Hansson V. Quantification of spermatogenesis by flow cytometric DNA measurements. Int J Androl 1978; 2 suppl: 513-21.
[11] Felemovicius I, Bonsack ME, Baptista ML, Delaney JP. Intestinal radioprotection by vitamin E (alpha-tocopherol). Ann Surg 1995; 222: 504-10.
[12] Van der Meer Y, Huiskamp R, Davids JA, van der Twell I, de Rooij DG. The sensitivity of quiescent and proliferating mouse spermatogonial stem cells to X irradiation. Radiat Res 1992;130: 289-95.
[13] Van der Meer Y, Huiskamp R, Davids JA, van der Twell I, de Rooij DG. The sensitivity to X rays of mouse spermatogonia that are committed to differentiation and differentiating spermatogonia. Radiat Res 1992; 130: 296-302.
[14] Jagetia GC, Krishnamurthy H. Effect of low doses of gamma-radiation on the steady-state spermatogenesis of mouse: a flow-cytometric study. Mutat Res 1995; 332: 97-107.
[15] Oakberg EF. A new concept of spermatogonial stem-cell renewal in the mouse and its relationship to genetic effects. Mutat Res 1971; 11: 1-7.
[16] Sies H, Stahl W. Vitamins E, C, b-carotene, and other carotenoids as antioxidants. Am J Clin Nutr 1995; 62 (6 Suppl ): 1315S-21S.
[17] Fang YZ, Yang S, Wu G. Free radicals, antioxidants, and nutrition. Nutrition 2002; 18: 872-9.
[18] Haines G, Marples B, Daniel P, Morris I. DNA damage in human and mouse spermatozoa after in vitro-irradiation assessed by the comet assay. Adv Exp Med Biol 1998; 444: 79-93.
[19] Hughes CM, Lewis SE, McKelvey-Martin VJ, Thompson W. The effects of antioxidant supplementation during Percoll preparation on human sperm DNA integrity. Hum Reprod 1998; 13: 1240-7.
[20] Kamiguchi Y, Tateno H, Watanabe S, Mikamo K. Chromosome abnormality in human gametes by exposure to mutagens. Environmental Mutagenesis Res 1996; 18: 83-6.
[21] Kamiguchi Y, TatenoH. Radiation-and chemical-induced structural chromosome aberrations in human spermatozoa. Mutat Res 2002; 504: 183-91.
[22] Ward WS, Coffey DS. DNA packaging and organization in mammalian spermatozoa:comparison with somatic cells. Biol Reprod 1991; 44: 569-74.

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Correspondence to: Kanok Pavasuthipaisit, M.D., Ph.D., Professor and Director, Institute of Science and Technology for Research and Development, Mahidol University, Salaya, Nakhon Pathom 73170, Thailand.
Tel : +66-2-441 9003-7 Ext 1389 or 1390, Fax: +66-2-441 1013
Email: directst@mahidol.ac.th
Received 2003-11-27   Accepted 2004-05-08