ABSTRACTSpermatogenesis is a complex process that requires a carefully

orchestrated series of biochemical events that initiate specific changes in cellular development and differentiation resulting in the production of mature sperm. Unlike many mammalian species, the onset of spermatogenesis in the stallion is not uniformly distributed in the testis resulting in regions of light (actively spermatogenic) and dark (inactive) parenchymal tissue. Semi-quantitative Difference Gel Electrophoresis (DIGE) is being coupled with LC/MS/MS mass spectrometry in an effort to begin cataloging proteins that are differentially expressed in the light and dark regions of the equine testis during the onset of spermatogenesis to better understand the factors involved in the onset and its regulation. We have developed methods to extract proteins from the light and dark tissue in a manner that is compatible with 2-dimensional DIGE. In addition, image analysis of fluorescently labeled protein extracts using DeCyder Image Analysis software has delimited at least 40 proteins that change in expression between the two testicular regions. Work is currently underway to identify the proteins using LC/MS/MS mass spectrometry.

MATERIALS AND METHODS

Specimens

Testes from pre-pubertal horses were obtained from 1-2 year old stallions raised at the Texas State Prison Farm (Wynne Prison Farm) in Huntsville, Texas and from the Horse Center at Texas A&M University in College Station, Texas. The testicles were sectioned and dissected into light or dark regions (3), and immediately frozen with liquid nitrogen (N2).

Protein Extraction

Two protein extraction methods were evaluated. Extraction Method I: Light and dark tissues were pulverized with liquid N2 into a fine powder. The tissue powder was immediately dissolved in 1 milliliter of 7 M Urea/2 M Thiourea (de-ionized), 4% CHAPS, 18 mM dithiothreitol (DTT), and incubated for 0.5 hours at 4 o Celsius. After spinning the extracted materials at 13000 x g for 5 minutes (4o C), the supernatant materials were removed and frozen until further analysis. Extraction Method 2: Light and dark tissues were pulverized under liquid N2, and dissolved in 1 milliliter of 10mM Tris, pH 7.5, 4% CHAPS, that contained a protease inhibitor cocktail (Complete Cocktail Tablets, Roche Diagnostics, Mannheim, Germany) for 1 hour on ice. This was followed immediately by the addition of a Ribonuclease A/Deoxyribonuclease I cocktail for an additional 15 minutes on ice (5). After spinning at 13000 x g for 5 minutes at 4 o C, the supernatant materials were frozen until further analysis.

RESULTS and DISCUSSION

CONCLUSIONS

1. Conditions have been developed to prepare a stallion testicular proteome.

2. More than 900 proteins from Light and Dark parenchymal tissues were detected and analyzed using DIGE.

3. 40 proteins of interest displayed statistically significant changes in expression.

4. Nano LC/MS/MS analysis of the protein digests are ongoing to identify the proteins.

RESULTS and DISCUSSION

INTRODUCTION

The initiation of spermatogenesis occurs uniformly throughout the testis in many mammalian species (1, 2) and is the result of a series of events that produces large quantities of spermatozoa. In contrast, the onset of spermatogenesis in colts begins around 1.5 years of age and can be distinguished visually; the central spermatogenic region is light in color and the inactive periphery is relatively dark. Clemmons et al. (3) have shown that the differences in coloration within the parenchyma correspond to quantitative differences in cell populations. The dark region is characterized by large numbers of Leydig cells, macrophages and small seminiferous tubules that are not producing sperm. In contrast, the light tissue is composed of fewer Leydig cells and macrophages as the seminiferous tubules (containing non-pigmented cells) increase in size to occupy a larger proportion of the parenchyma.

Ing et al (4) have shown previously that select genes are preferentially expressed in dark and light parenchyma. Microarray studies of 9132 human genes with equine cDNAs revealed that at the expression of at least 88 equine genes are different between light and dark tissue. However, to our knowledge, no information is available about differential protein expression in these tissues.

The goal of our project is to utilize Difference Gel Electrophoresis (DIGE) and mass spectrometry to begin to catalog the protein expression differences in light and dark tissue using established proteomic techniques. The pre-pubertal horse may provide an excellent model by which to identify the protein factors involved in the onset of spermatogenesis and the timing of their appearance.

Figure 1. A cross sectional view of a prepubescent horse testis. This cross section reveals the contrast between the light colored parenchyma in the center and the darker colored parenchyma around the perimeter of the organ.

Figure 2. SDS Page Analysis of two extraction methods. Lane 1= MW Std, 2= Extraction Method 1, Light Tissue, 3= Extraction Method 1, Dark Tissue, 4= Extraction Method 2, Light Tissue, 5= Extraction Method 2, Dark Tissue. Arrow points to albumin.

1 2 3 4 5

Figure 3. Reduction of blood protein contaminants by Immunoaffinity chromatography. Panel A: Depletion chromatogram showing flow- through fractions with albumin-depleted sample (Flow-Through) and contaminants removed from sample (Waste). Panel B: SDS PAGE analysis of Immunoaffinity chromatography Flow-through fractions after depletion. Lane: 1= MW Std, 2= sample before depletion, 3= after depletion. Arrow points to albumin. Note the greatly reduced amount of albumin and enrichment of other proteins in depleted fractions.

BA

Figure 4. A multiplex fluorescent image (Panel A) and a total protein stained image (Panel B) of a typical DIGE gel. Panel A: An overlay of three fluorescent dyes scanned at different wavelengths. Panel B: An image from the BVA module in DeCyder. The yellow circles indicate pick proteins and the flags represent a protein spot’s respective Master gel number.

Protein Extraction

SDS PAGE analysis of proteins extracted using Extraction Methods 1 & 2 indicated that the tissue samples were highly contaminated with blood proteins (albumin and IgG) and DNA (Figure 2). Extraction Method 2 resulted in a sample that contained less DNA (less viscous) with less proteolysis (Figure 2). Method 2 became the method of choice for these experiments. The continued presence of substantial albumin contamination required additional sample preparation.

Immuno-depletion

Tandem immuno-depletion of the extracts removed substantial amounts of albumin and IgG (Figure 3). Figure 3A shows a typical depletion procedure. The Flow-through material is the depleted testis proteins. The waste material is the stripped albumin and IgG being removed separately. Material depleted in this way was used for fluorescent labeling. Figure 3B shows an SDS gel of the flow-through and waste.

DIGE and Image Analysis

DeCyder Image Analysis of the fluorescent images detected over 900 protein spots. Statistical analysis in the BVA Module selected 40 Proteins of Interest that passed the Protein Filter criteria (Figure 4B). These 40 proteins were robotically excised from the gel, digested and prepared for LC/MS/MS analysis. Table 1 lists the expression changes of the 40 Proteins of Interest.

Specimens

Flow-through Waste

A

1 2 3

B

1 2 3

B

Master No.

T-test Value

Fold Change

Master No.

T-test Value

Fold Change

358 0.081 -3.2 663 0.081 1.83

231 0.081 1.49 353 0.081 -1.1

373 0.081 -2.84 662 0.081 2.07

626 0.085 1.45 222 0.081 -1.73

346 0.081 -4.22 504 0.081 -1.52

165 0.081 1.39 161 0.081 1.42

167 0.081 1.36 304 0.081 -1.78

170 0.081 1.32 207 0.081 1.26

370 0.081 -2.31 426 0.081 1.35

386 0.081 1.23 69 0.081 1.34

316 0.081 -2.12 779 0.081 1.69

180 0.081 1.18 53 0.081 -2.65

288 0.085 -1.92 221 0.081 -1.41

667 0.081 1.72 43 0.081 2.1

225 0.081 -2.04 315 0.081 -1.84

350 0.081 -1.16 57 0.073 -3.54

73 0.081 1.41 54 0.081 -3.35

130 0.081 1.15 56 0.073 -3.54

368 0.081 -1.51 204 0.081 1.45

456 0.081 -1.19 55 0.073 -3.28

Table 1. The Protein Pick List. A list of proteins that change expression comparing Dark to Light (negative = decreased expression; positive = increased expression). The first six spots were chosen for analysis on LC/MS/MS.

Immuno-depletion of Blood Proteins

IgG and albumin were removed from the samples using immunoaffinity chromatography. An Anti-Human IgG affinity column and an anti-Human Albumin column (GenWay Biotech, CA) were coupled in tandem. The albumin and IgG-free fractions were pooled for subsequent analysis.

DIGE Labeling, 2-Dimensional Gel Electrophoresis and Multiplex Imaging

Pooled flow through fractions of IgG and albumin depleted samples were concentrated and precipitated using a Chloroform/Methanol protocol (6) prior to fluorescent labeling (7). The precipitated proteins were solubilized in Labeling buffer (7M Urea/2M Thiourea, 4% CHAPS, 30mM Tris, pH 8.5) and labeled with spectrally resolvable fluorescent CyDyes (GE Healthcare). Proteins from the light and dark parenchyma were labeled with different dyes (Cy3 or Cy5) in order that they be distinguished by the multiplex imaging. A pooled sample composed of equal amounts of protein from both tissues was labeled (Cy2) to provide an internal standard for statistical comparisons of the gels.

Isoelectric focusing was performed on an IPGPhor focusing unit (GE Healthcare) on IPG DryStrips overnight. The focused proteins were reduced with dithiothreitol and alkylated with iodoacetamide and run on 12% acrylamide 2D SDS PAGE slab gels (8). The gels were scanned at three wavelengths on a Typhoon Trio Imager (GE Healthcare). Images were analyzed and cropped using ImageQuant 5.1 prior to multi-channel analysis using DeCyder Software.

DeCyder Image Analysis

DeCyder software (GE Healthcare) was used to detect, match and compare migration of individual proteins for all protein spots in the multiple fluorescent images. Proteins were detected using the Differential In-gel Analysis (DIA) module. The Biological Variation Analysis (BVA) module was used to match and compare protein spots between gels, and a statistical filter was applied to detect proteins with gel-to-gel differences.

Spot Picking and Digestion

Gel plugs containing proteins of interest were excised from the gels with an Ettan Picker robot (GE Healthcare). Automated digestion was performed on an Ettan Digester (GE Healthcare) according to Shevchenko et. al. (9). Extracted peptides were stored in -80oC while awaiting mass spectrometric analysis.

LC/MS/MS Analysis and MASCOT Database Search

Nanospray LC/MS/MS was performed on an LCQ DecaXP Thermo Electron Ion Trap LCMS (Thermo-Fisher Scientific, San Jose, CA). Samples were dissolved in a 98% solution A (0.1% Formic Acid in water) and 2% solution B (0.1% Formic Acid in acetonitrile) for 30 minutes at room temperature and injected onto a hand-packed, 6 cm PicoFrit column (New Objective, Woburn, MA) containing Magic C18AQ resin (5 micron, 200 Ǻ pore size, Michrom, Auburn, CA). Protein sequences obtained from electrospray were searched using an in-house copy of the automated search engine MASCOT after extracting the data with the Distiller module (Matrix Science). Search parameters were set to account for any modifications made to the proteins during extraction and digestion.

Protein Estimation and SDS PAGE

Protein estimations were made by the method of Bradford using albumin as a standard (10). SDS PAGE was performed according to Laemmli (8).

References

1. Courot, M., Hochereau-de Reviers, M-.T. and Ortavant, R. Spermatogenesis. IN: Johnson AD, Gomes,Van Demark NL,(eds.) The Testis. vol. I. New York: Academic Press; 1970:339-442.

2. Johnson, L. Spermatogenesis. In: Cupps P.T. (ed.), Reproduction in Domestic Animals, 4th ed. New York: Academic Press; 1991: 173-219.

3. Clemmons, A.J., Thompson, D.L., Jr. and Johnson, L. (1995) Biol Reprod;6:1258-1267.

4. Ing, N.H., Laughlin, A.M., Varne,r D.D., Welsh, T.H., Jr., Forres,t D.W,, Blanchard, T.L, and Johnson, L. (2004) J Androl.; 25:535-544.

5. Wessel, D., and Fugge, U.I. Analytical Biochemistry (1984), 138; 141-143

6. O'Farrell, P.H. (1975) J Biol Chem. 250, 4007-4021.

7. Unlu, M., Morgan, M.E. and Minden, J.S. (1997) Electrophoresis 18: 2071-2077.

8. Laemmli, U.K. (1970). Nature. 227, 680-685.

9. Shevchenko, A., Tomas, H., Havlis, J., Olsen, J.V. and Mann, M. (2006) Nature Protocols 1: 2856-2860.

10. Bradford, M. M. (1976) Anal. Biochem. 72:248-254.

Changes in Protein Expression in Maturing Equine Testis: Proteomics Approach P. Roper-Foo1, S. N. Schmidtke2, C. Griffin3, M. Martin4, L. Johnson5 and L. J. Dangott6

Depts. of Biochemistry & Biophysics1,2,6, Veterinary Large Animal Clinical Science3,4 and Veterinary Integrative Biological Sciences5

Texas A&M University and the College of Veterinary Medicine & Biomedical Sciences, College Station, TX

DIGE; Differential Gel DIGE; Differential Gel ElectrophoresisElectrophoresis

Labeling

2D Gel Separation

Multichannel Imaging

DeCyder DifferentialAnalysis Software