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Yon, Jung, Park, Hong, Kim, Lin, Kim, Baek, Lee, Yun, and Nam: Expression Patterns of Superoxide Dismutase Genes in the Stage-specific Seminiferous Tubules of Mice Excised by a Laser Capture Microdissection
Original Article

Lab. Anim. Res. 2010;26(2):197-201.

Published online: 17 June 2010

DOI: https://doi.org/10.5625/lar.2010.26.2.197

Expression Patterns of Superoxide Dismutase Genes in the Stage-specific Seminiferous Tubules of Mice Excised by a Laser Capture Microdissection

Jung-Min Yon1, #, A-Young Jung1, #, Jung-Hoon Park1, Min Ki Hong1, Jae-Seung Kim1, Chunmei Lin1, Mi-Ra Kim1, In-Jeoung Baek2, Beom Jun Lee1, Young Won Yun1, Sang-Yoon Nam1, ∗

1College of Veterinary Medicine & Research Institute of Veterinary Medicine, Chungbuk National University, Cheongju, Korea

2Laboratory of Mammalian Molecular Genetics, Department of Biochemistry, College of Science, Yonsei University, Seoul, Korea

# These authors equally contributed to this study.

∗Corresponding author: Sang-Yoon Nam, Laboratory of Veterinary Anatomy, College of Veterinary Medicine, Chungbuk National University, Cheongju 361-763, Korea. Tel.: +82-43-261-2596 Fax: +82-43-271-3246 E-mail: synam@cbu.ac.kr

Received 14 May 201010 June 2010       Accepted 17 June 2010

Copyright © 2010 Korean Association for Laboratory Animal Science

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Spermatogenesis is a particularly difficult process to study the unique multiple cellular associations within the seminiferous epithelium. Laser capture microdissection (LCM) is a recently developed technique that enables the isolation of individual cell populations from complex tissues. The superoxide dismutase (SOD) is the first and most important enzyme of antioxidant defense systems against superoxide anion. The aim of this study was to investigate the quantitative changes of SOD gene expression according to the spermatogenic cycle in mouse testes using LCM and real-time polymerase chain reaction (PCR) techniques. Frozen sections (10 µm) were obtained from the testes of 8-weeks-old ICR mice. LCM was used to capture all cells in cross-sectioned seminiferous tubules which were grouped into stages I-V, VII-VIII, and IX-XI. The expression level of cytoplasmic Cu, Zn-SOD (SOD1) mRNA was remarkably higher than those of mitochondrial Mn-SOD (SOD2) and extracellular Cu, Zn-SOD (SOD3) mRNAs in mouse testes. During spermatogenesis, the expressions of SOD1 and SOD2 mRNAs were highest on stages I-V, began to decrease after stage VII, and showed a lowest level on stage IX-XI. However, the expression of SOD3 mRNA was highest on stages VII-VIII. These findings suggest that the subtypes of SOD are expressed differentially in mouse testes during spermatogenesis.

Keywords: Superoxide dismutase, spermatogenesis, laser capture microdissection, real-time PCR

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Figure 1.
Isolation of the specific seminiferous tubules using a laser capture microdissection (LCM) and classifications of the spermatogenic stages. Micrograph of intact testes section viewed by light microscopy prior to LCM (A), isolated tubule cross-section on the LCM cap following LCM (B), and remaining tissue section (C). Seminiferous tubules at the following stages of spermatogenesis were grouped: Arrows indicate compact elongated spermatid heads embedded within the seminiferous epithelium on stages I-V (D). Arrowheads indicate elongated spermatids adjacent to the tubule lumen prior to spermiation on stages VII-VIII (E). X indicates absence of elongate spermatids on stages IX-XI (F). Magnification: X100 (A-C) and X200 (D-F).
lar-26-197f1.tif
Figure 2.
Stage-specific expression of cytoplasmic Cu, Zn-superoxide dismutase (SOD1) mRNA during spermatogenesis. Gene expression for SOD1 in cross-sectioned seminiferous tubules defining the spermatogenic stages (I-V, VII-VIII and IX-XI) excised by a laser capture microdissection was assessed by real-time PCR (n=3). ∗Significantly different from stage I-V at P<0.05.
lar-26-197f2.tif
Figure 3.
Stage-specific expression of mitochondrial Mn-superoxide dismutase (SOD2) mRNA during spermatogenesis. Gene expression for SOD2 in cross-sectioned seminiferous tubules defining the spermatogenic stages (I-V, VII-VIII and IX-XI) excised by a laser capture microdissection was assessed by real-time PCR (n=3).
lar-26-197f3.tif
Figure 4.
Stage-specific expression of extracellular superoxide dismutase (SOD3) genes during spermatogenesis. Gene expression for SOD3 in cross-sectioned seminiferous tubules defining the spermatogenic stages (I-V, VII-VIII and IX-XI) excised by a laser capture microdissection was assessed by real-time PCR (n=3).
lar-26-197f4.tif
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