Application of laser-capture microdissection to analysis of gene expression in the testis

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Abstract

The isolation and molecular analysis of highly purified cell populations from complex, heterogeneous tissues has been a challenge for many years. Spermatogenesis in the testis is a particularly difficult process to study given the unique multiple cellular associations within the seminiferous epithelium, making the isolation of specific cell types difficult. Laser-capture microdissection (LCM) is a recently developed technique that enables the isolation of individual cell populations from complex tissues. This technology has enhanced our ability to directly examine gene expression in enriched testicular cell populations by routine methods of gene expression analysis, such as real-time RT-PCR, differential display, and gene microarrays. The application of LCM has however introduced methodological hurdles that have not been encountered with more conventional molecular analyses of whole tissue. In particular, tissue handling (i.e. fixation, storage, and staining), consumables (e.g. slide choice), staining reagents (conventional H&E vs. fluorescence), extraction methods, and downstream applications have all required re-optimisation to facilitate differential gene expression analysis using the small amounts of material obtained using LCM. This review will discuss three critical issues that are essential for successful procurement of cells from testicular tissue sections; tissue morphology, capture success, and maintenance of molecular integrity. The importance of these issues will be discussed with specific reference to the two most commonly used LCM systems; the Arcturus PixCell IIe and PALM systems. The rat testis will be used as a model, and emphasis will be placed on issues of tissue handling, processing, and staining methods, including the application of fluorescence techniques to assist in the identification of cells of interest for the purposes of mRNA expression analysis.

Introduction

Complex and dynamic cellular associations exist within many tissues, and analysis of the biology of individual cell populations can be complicated by contamination by other cell types within samples. Laser-capture microdissection (LCM) is a technique that has recently become available to biologists and allows for the procurement and downstream molecular analysis of individual or groups of cells from tissue sections. Successful application of LCM is however dependent on three critical factors: tissue morphology, capture success, and maintenance of molecular integrity. Effective balancing of these three factors is required so that cells of interest can be identified without compromising their ability to be captured, whilst maintaining the integrity of the molecules of interest (i.e. RNA, DNA, or protein) such that they can be accurately analysed. The aim of this review is to identify and discuss the experimental variables that we have found to be essential for the successful LCM-mediated procurement and downstream analysis of mRNA from complex tissues. As an example, this review will demonstrate effective use of real-time RT-PCR and differential display to compare mRNA expression profiles in hormonally manipulated rat testis tissue.

The seminiferous epithelium, with its division into 14 spermatogenic stages in the rat, is very complex (see Leblond and Clermont, 1952; Russell et al., 1990). Different cytological and molecular events occur at the various stages, and mRNAs and proteins are expressed in a stage- and cell-specific manner (for reviews see Eddy, 2002; Kimmins et al., 2004). Prior to the development of LCM, studies investigating stage-specific gene expression in the seminiferous epithelium have largely been restricted to the use of whole-tissue approaches such as in situ hybridisation (Marret et al., 1998; Shan et al., 1995) and immunocytochemistry (Salanova et al., 1995; Wine and Chapin, 1999), which are not readily quantifiable. In vitro purification techniques have allowed for quantitative gene expression analysis to be performed on purified somatic and germ cell populations (Kashiwabara et al., 1990), however a major disadvantage of this latter approach is that germ cells at particular stages of development cannot be isolated due to a lack of known cell-specific morphological or biochemical markers that distinguish these cells across the other stages. Compounding this problem, some cell types are inherently difficult to isolate, due to either low initial cell numbers in the testis (e.g. spermatogonia), or the presence of strong adhesion junctions with neighbouring Sertoli cells (e.g. elongating spermatids), both of which result in low cell yields. The use of transillumination microscopy has made major steps towards the isolation and comparative molecular analysis of testicular cells in defined seminiferous tubule stages (Parvinen, 1982), by allowing the visualisation and dissection of tubule segments and specific stages under a transillumination microscope (Rannikko et al., 1996; Toppari et al., 1991). However this approach is dependent on the presence of elongated spermatid heads in seminiferous tubules, and is therefore not applicable to testes from rats in which spermatogenesis has been arrested, for example by androgen suppression or toxicant treatment (e.g. EDS and busulfan), resulting in an absence of elongated spermatids (O’Donnell et al., 1996).

When coupled with real-time RT-PCR for quantitation of gene expression, the use of LCM circumvents the difficulties of cell isolation encountered with previous techniques (Bonner et al., 1997). To illustrate the usefulness of this approach, LCM has been used to isolate samples for gene expression analysis in complex epithelia such as kidney glomeruli (Kohda et al., 2000), neuronal subtypes (Luo et al., 1999), and to analyse breast cancer progression (Sgroi et al., 1999) and protein production in colon cancer (Lawrie et al., 2001). The use of LCM to study the testis and spermatogenesis is increasing, with some 23 publications in this field since 2000 (see Table 1). These publications range from analysis of individual molecules in purified cells (Robinson et al., 2001; Tirado et al., 2003) or stages (Sluka et al., 2002), through to global gene expression microarray analyses (Liang et al., 2004; Okada et al., 2003). LCM has also been used to analyse DNA in studies examining genetic mutations associated with infertility (Yoon et al., 2003) and oncogenesis (Kernek et al., 2003), and as a means of isolating single sperm cells from mixed samples for the identification of individuals in sexual assault cases (Di Martino et al., 2004; Elliott et al., 2003; Sanders et al., 2006).

LCM-procured samples can potentially be subjected to any method of molecular analysis. Genomic analyses have been the most common to date, such as real-time RT-PCR, differential display, gene microarray, loss of heterozygosity, and mutation analysis, with only a small number of studies examining proteins (see Table 1). The preference for nucleic acid over protein analysis is largely due to the methodologies that allow nucleic acids to be amplified from the typically small yields of material collected in LCM studies, avoiding the need for lengthy sessions at the microscope bench. However several reviews are available for protein analysis after LCM in other tissues (Gozal et al., 2006; Krieg et al., 2005; Lawrie and Curran, 2005) to which the reader is directed for advances in this area of analysis.

The successful use of LCM to procure cells can be technically demanding, with the three key factors (tissue morphology, capture success, and maintenance of molecular integrity) being dependent on numerous variables such as tissue fixation method, staining procedure, slide temperature, and section processing. Furthermore, with a number of LCM microscopes available to researchers, each with their own principles of operation, the impact of these variables is dependent on the instrument being used. Our laboratory has successfully applied the Arcturus PixCell IIe (Arcturus, California, USA) and PALM (PALM, Bernreid, Germany) LCM microscopes to the study of gene expression in the testis. It is the intention of this review to present detailed protocols that will allow researchers to apply LCM to the analysis of differential gene expression in the testis and other complex tissues, and at the same time describe the importance of each step of the protocol, an understanding of which is necessary for troubleshooting and successful application of these techniques.

Section snippets

Principles of LCM

In preparation for LCM, the tissue of interest must be sectioned and placed onto a microscope slide, however in order to allow for cell procurement the sections must not be coverslipped. A number of LCM microscopes have been developed, all with unique mechanics of cell procurement. The principles by which the Arcturus PixCell IIe and PALM–LCM systems work will be outlined here.

Development of protocols for LCM

The purpose of any tissue preparation protocol for LCM is to prepare tissue sections that allow for unambiguous identification and successful capture of cells of interest, while maintaining molecular integrity. The successful capture of specific cells from the various stages of spermatogenesis demands a preservation of morphology that allows the spermatogenic stages to be identified. In general, unstained, frozen section morphology is not adequate for this purpose. Accordingly, we have

Staining methods for staging of seminiferous tubules

We have developed a number of staining protocols for the rat that allow for the staging of seminiferous tubules in testis sections to varying degrees of accuracy. These include a crude method that can discriminate relatively broad stage groupings in frozen tissue sections, and more precise methods that can be used to identify each stage in both frozen and wax-embedded tissue sections. These methods are presented below. It is intended that users will be able to directly replicate these methods

Monitoring RNA yield and quality

One of the major concerns with processing tissue for LCM is the preservation of RNA yield and quality. This section will examine RNA yields and quality obtained using each of the above protocols, and identify important variables within these protocols that have a significant effect on it.

Analysis of gene expression in tissue collected using LCM

LCM technology is compatible with various molecular analyses (see Table 1), provided that samples are obtained in sufficient quantities and at a sufficient quality. Using the protocols presented in this manuscript, we have successfully applied real-time RT-PCR and differential display to the analysis of gene expression in the testis.

We have used RT-PCR in LCM-procured seminiferous tubules to study gene expression in various stage groupings (Sluka et al., 2002). In this example, testes were

Summary and conclusions

In this review, we have examined ways that LCM can be successfully applied to analysis of gene expression in the testis. We have used both the Arcturus PixCell IIe and PALM–LCM systems and optimised sample preparation protocols that maintain morphology, allow for successful capture and maintain molecular integrity. We found a number of variables to be critical to the success of LCM, including slide temperature, fixation method, staining method, and dehydration. Using these methods we were able

Acknowledgements

This study was supported by the National Health and Medical Research Council of Australia, Program Grant #241000, and a Fellowship to RMcL #198706.

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