DNA Microarray Analysis of Region-Specific Gene Expression in the Mouse Epididymis
Nelson Hsia and Gail A. Cornwall
Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, TX
ABSTRACT
mouse leucine aminopeptidase 3 (Lap3)
mouse cathepsin H (Ctsh)
H3124E07
similar to mouse phosphatidylserine
decarboxylase
NIA 5K III
4.1
3.7
0.001
0.001
2.5
0.028
H3116H10
H3051H03
H3108B10
RESULTS
Protein Function
P-valueFold
Fold
Fold P-value
Fold
(E2-5/E1)
(E2-5/E1)
H3103H07
H3128G12
mouse elafin-like I (SWAM1)
similar to mouse elafin-like 1
potential inhibitor of elastase
0.169
0.213
< 0.001
0.008
5.9
4.7
H3147G08
human SMARCA1
SW1/SNF related, matrix associated, actin
dependent regulator of chromatin, subfamily
a, member 1
0.185
0.002
5.4
H3079B09
H4-K20-specific histone methyltransferase
0.198
0.001
5.1
H3079B01
H3133F12
mouse zinc finger protein 40
similar to mouse cyclin D2
0.215
0.230
0.001
0.008
4.7
4.3
mouse ERM (ETV5)
similar to mouse LPS-induced TNF-alpha
factor (LITAF)
similar to mouse LPS-induced TNF-alpha
factor (LITAF)
TRANSPORTERS
12.7
5.7
0.004
0.001
transcription factor coregulator
5.1
0.001
0.003
H3056B05
similar to mouse suppressor of Ty 3 homolog transcription factor
2.9
0.001
H3029A04
similar to rat Pou3f1
2.7
0.065
H3126F11
mouse milk fat globule-EGF factor
H3113A06
mouse connexin 43
X62836
similar to mouse connexin 43
H3120D05
mouse ALEX3
H3113F12
mouse prion protein (Prnp)
H3080A11
mouse Cd63 antigen
H3130C04
H3072H10
POU domain, class 3, transcription factor 1
11.6
0.001
5.8
0.002
4.9
<0.001
4.5
<0.001
4.2
0.012
3.9
<0.001
similar to mouse to RAS p21 protein activator
similar to rat beta-arrestin 2
regulate G-protein coupled receptors
3.1
3.0
0.01
0.006
H3158H11
H3019D01
similar to T cell cytokine receptor
Similar to mouse calmodulin 1
2.9
2.8
0.025
0.013
H3013F05
mouse syndecan 1 (Sdc1)
2.8
<0.001
H3090C06
mouse Slp4, synaptotagmin-like protein 4
2.6
0.001
H3096H04
mouse C2PA
2.5
0.005
H3108D04
similar to mouse calmodulin 1
2.5
0.004
2 Notch-EGF, 2 discoidin (carbohydrate
binding)
participates in the regulation of signaling
between developing and differentiated cell
types
participates in the regulation of signaling
between developing and differentiated cell
types
armadillo/beta-catenin-like repeats. Proposed
to mediate interaction of beta-catenin with its
ligands. Involved in transducing the
Wingless/Wnt signal
GPI anchor protein, synaptic transmission
tetraspanin, associated with adhesion
receptors of the integrin family and regulate
integrin-dependent cell migration
intracellular signal transduction
transmembrane heparin sulfate
proteoglycans implicated in the binding of
ECMs and growth factors.
regulate Rab3 and Munc-18 and may
regulate multiple steps in the secretory
pathway
C2 membrane binding, PDZ protein-protein
interaction, ATP-GTP binding
intracellular signal transduction
H3154E06
mouse inhibitor of DNA binding 3
0.250
0.006
4.0
H3137A12
mouse Ttk protein kinase (Ttk)
0.132
0.256
7.6
H3152D07
similar to mouse complement-c1q tumor
necrosis factor-related protein
C1q is a subunit of the C1 enzyme complex
that activates the serum complement system
0.188
0.012
5.3
H3097B09
mouse CD97 antigen (Cd97)
class II seven-span transmembrane
receptors
0.195
0.001
5.1
NM_008426
mouse potassium inwardly-rectifying channel,
subfamily J, member 3 (Kcnj3)
mouse solute carrier family 22, member 5
organic cation transporter
(Slc22a5)
mouse TRH1
translocating chain-associating membrane
protein
0.179
0.003
5.6
0.186
0.002
5.4
0.194
< 0.001
5.2
H3129C07
H3111B10
mouse adaptor-related protein complex 3,
beta 2
vesicle mediated transport
0.195
0.002
5.1
H3002A06
mouse ladinin 1 (LAD1)
anchoring filament protein
0.070
0.002
14.3
H3003H10
H3091E12
mouse alpha actinin 4 (Actn4)
similar to mouse VAMP
0.113
0.218
0.022
0.208
8.8
4.6
H3064H08
mouse motor domain of KIF10
actin binding
major sperm protein involved in forming
filaments
microtubule-dependent motor
0.229
0.034
4.4
H3150E03
rat synaptonemal complex protein 2 (Sycp2)
0.244
0.102
4.1
H3030E05
similar to human glycine decarboxylase (Pprotein)
mouse carbonic anhydrase 4 (Car4)
mouse carboxylesterase 1 (Ces1),
0.061
0.004
16.4
0.089
0.121
0.002
< 0.001
11.2
8.3
0.126
0.001
7.9
H3139A09
H3096F11
similar to phosphoserine aminotransferase
(PSAT) (Endometrial progesterone-induced
protein) (EPIP)
human phosphoserine phosphatase
human HNK-1 sulfotransferase
0.148
0.150
0.002
0.001
6.8
6.7
H3018A11
H3127H06
mouse cytochrome P450, 17 (Cyp17)
mouse aldo-keto reductase AKR1C1
0.154
0.160
0.015
0.001
6.5
6.3
H3075A09
H3151D04
similar to mouse liver carboxylesterase 4
mouse 3-alpha-hydroxysteroid
dehydrogenase
mouse cell division cycle 25A (Cdc25a)
mouse 3-phosphoglycerate dehydrogenase
0.194
0.192
0.003
0.001
5.2
5.2
0.195
0.218
0.059
0.002
5.1
4.6
0.222
< 0.001
4.5
0.231
0.264
< 0.001
0.002
4.3
3.8
0.017
0.015
58.8
0.001
H3095H12
H3111D10
liver-specific organic anion transporter-1
3.9
0.022
H3095G01
putative sugar transporter
solute carrier family 8 (cationic amino acid
transporter, y+ system), member 7
cationic amino acid transporter 2
3.8
3.3
0.003
<0.001
2.9
2.6
<0.001
0.005
2.5
2.5
0.003
<0.001
2.5
<0.001
mouse lipocalin 2
lipocalin / cytosolic fatty-acid binding protein
H3089D08
weakly similar to lst-1
H3141D10
H3063H10
similar to mouse FLJ00178 protein
mouse LAT2
H3083D07
H3055D11
mouse mCAT2
mouse FXYD domain-containing ion transport
regulator 3
similar to lst-1
liver-specific organic anion transporter-1
mouse cysteine rich intestinal protein
proposed role in zinc absorption (intracellular
zinc transporter)
mouse Slc3a2
solute carrier family 3, member 2
H3076G07
H3108G04
H3138G02
12.3
H3083G02
H3075C03
H3003D12
H3004B08
H3044G06
UNCLASSIFIED
H3011A01
H3119B09
mouse prostaglandin-endoperoxide synthase
2
mouse acyl-coenzyme A thioesterase 2
mouse cytochrome b-561 (Cyb561)
tyrosine protein phosphatase
4.9
3.8
0.013
0.024
3.7
<0.001
3.4
0.004
H3017C05
mouse claudin 10 (Cldn10)
tight-junction
mouse P4ha2, proline 4-hydroxylase, alpha II plays a central role in the synthesis of all
polypeptide
collagens
L-plastin 2
regulates cell movement by interaction with
actin
mouse Capg, capping protein (actin filament), calcium sensitive protein which reversibly
gelsolin-like
blocks actin filaments
RIKEN cDNA
contains MSP (Major sperm protein) domain
3.1
0.006
H3066A12
ESTs
H3084D09
H3040D08
similar to mouse NG28 protein
mouse Arpc2
3.1
2.6
0.003
0.001
H3012D01
H3003E06
similar to human ribosomal protein S9
Unknown
0.027
0.045
0.069
0.057
37.0
22.2
H3009C06
actin related protein 2/3 complex, subunit 2
2.5
0.001
H3036A09
expressed sequence C77144
0.072
0.116
13.9
3.8
0.002
H3134E02
H3087A12
H3060B07
H3134G06
H3109G06
bacteriophage lambda, complete genome
mouse uterine lactotransferrin mRNA
expressed sequence AU015226
unknown
mouse uterine lactotransferrin mRNA
0.076
0.077
0.078
0.083
0.101
0.127
0.017
0.008
0.143
0.023
13.2
13.0
12.8
12.0
9.9
2.9
2.8
2.7
0.003
0.002
0.018
H3141C12
H3043G06
H3036G08
ESTs
mouse chimeric 16S ribosomal RNA
DNA segment, Chr 6, ERATO Doi 131
0.109
0.119
0.123
0.205
0.018
0.014
9.2
8.4
8.1
2.7
0.013
H3029G12
human nuclear mitotic apparatus
0.124
0.036
8.1
2.7
2.6
2.6
0.004
0.026
0.013
335036
H3150A06
H3133A08
similar to mouse ribosomal protein L34e
mouse transcobalamin 2 (Tcn2)
ESTs
0.128
0.145
0.148
0.001
0.001
0.292
7.8
6.9
6.8
2.6
<0.001
H3020C11
human cDNA FLJ13180 fis, clone N
0.152
0.015
6.6
2.5
0.038
H3006A12
human clone HB-1 mRNA sequence
0.160
0.052
6.3
2.5
0.003
H3118C04
unknown
0.168
0.013
6.0
H3151D06
H3001B12
RIKEN cDNA 1200016B10 gene
similar to mouse ring finger protein 2 (Rnf2)
0.179
0.188
0.086
0.016
5.6
5.3
H3083C12
H3108E07
H3003A02
H3118B07
H3103H12
unknown
RIKEN cDNA 2310046K01 gene
mouse zinc finger protein 364
mouse vitamin D-binding protein
similar to mouse gamma-2b-immunoglobulin
0.199
0.203
0.204
0.239
0.255
0.011
0.013
0.009
< 0.001
0.006
5.0
4.9
4.9
4.2
3.9
H3003A04
mouse nucleolar RNA-associated protein
0.264
0.009
3.8
H3011D10
H3014A12
H3124F04
H3041C08
H3122G09
EPI1
Figure 1. Schematic diagram of region-specific gene expression in the epididymis.
PEA3 member
transcription factor coregulator
4.0
H3098D02
mouse ubiquitin carboxy-terminal hydrolase
L1
mouse glutathione S-transferase
mouse casein kinase II, beta subunit
protein tyrosine phosphatase, receptor type,
G
protein tyrosine phosphatase, receptor-type,
F interacting protein, binding protein 2
H3129G09
H3124B08
H3032D11
ubiquitin carboxy-terminal hydrolase
Moderately similar to NICE-5 protein
CXXC zinc finger
H3140A09
mouse carnitine palmitoyltransferase 1
H3155C10
mouse UDP-glucuronosyltransferase 1 family,
member 1
similar to mouse lipase/acylhydrolase with
GDSL-like motif
H3083H05
EPI2-5
0.002
mouse c-myc
H3059F01
connexin 43
?
4.2
kunitz/bovine pancreatic trypsin inhibitor
3157667
OTHER ENZYMES
Region-specific gene expression in the epididymis
0.005
H3089H11
H3023A07
STRUCTURAL PROTEINS
Total RNA from the proximal caput epididymidis (EPI1) and mid-caput to cauda (EPI 2-5)
were isolated by Trizol reagent and further purified using an RNeasy kit. Labeling of total
RNA and hybridization were carried out at the Vanderbilt University Microarray Shared
Resource facility. Briefly, under low-light conditions, total RNA was reverse transcribed with
Cy3 dye for EPI2-5 RNA and Cy5 dye for region EPI1 RNA. cDNA probes were then
hydrolyzed and further purified using Qiagen PCR purification kit. Microarray slides were
prehybridized with 1% BSA, 5XSSC, 0.1% SDS for 45 min at 65°C and were hybridized
with fluorescent probes in 3XSSC, 24 mM HEPES pH 7.0, 10 g/ml poly A RNA, 0.225%
SDS overnight at 65°C. Slides were washed and then immediately scanned using Genepix
Pro software. Cy5 was scanned at wavelength 635 nm and fluoresces red. Cy3 was
scanned at wavelength 532 nm and fluoresces green. Genes expressed predominantly in
EPI1 were represented as gradations of red while EPI2-5 expressed genes were
represented as gradations of green. Genes expressed by both EPI1 and EPI2-5 appear as
yellow. To determine the relative difference between the EPI1 and EPI2-5 signals for each
gene, the Genepix software overlaid the fluorescence intensities detected at the Cy5 and
Cy3 wavelengths. For normalization, each gene intensity was divided by its control channel
value. Genes with control channel values <10 were considered unreliable and thus were
not included in the final analysis. Shown below is a representative, normalized microarray
analysis of a 15K NIA chip comparing EPI1 and EPI2-5 gene expression.
159.6
TRANSCRIPTION
FACTORS
putative protease inhibitor
SIGNALING FACTORS
H3136D07
mouse cystatin-related epididymal
spermatogenic 3 (cres3)
similar to mouse pancreatic trypsin inhibitor
TRANSPORTERS
H3102A08
Figure 2. Experimental procedure for microarray analysis
cres3
enk
CRBP
- raf
A
5-ared
5POMC
GGT
SGP2
NGF
HE5
cytosol aminopeptidase
cysteine protease involved in lysosomal
protein degradation
PROTEASE
INHIBITORS
H3006H12
H3028H11
STRUCTURAL
PROTEINS
NIA 5K II
Scatterplot of EPI1 versus EPI2-5 expressed genes
HE4
D/E
HE1
HE2
B/C
EAP1
GPX
CRES
Putative ID
(E1/E2-5)
(E1/E2-5)
OTHER ENZYMES
Overlay images and normalize
INTRODUCTION
An essential event in male reproduction is formation of functionally mature
spermatozoa. It is well established that spermatozoa do not have the ability to move
progressively or to fertilize an egg by the time they leave the testis. These testicular
spermatozoa are non-functional and require critical post-testicular modifications in the long
convoluted tubule known as the epididymis. This tubule can be subdivided into three
general regions which include the caput, corpus, and cauda (Figure 1). Upon reaching the
cauda region of the epididymis, spermatozoa have acquired the functions of progressive
motility and the ability to fertilize an oocyte [1, 2], suggesting sperm maturation occurs from
the caput to corpus regions of the epididymis.
The epididymal sperm maturation process is thought to involve a complex series of
events, foremost of which is the interaction of spermatozoa with proteins synthesized and
secreted into the lumen by the epididymal epithelium. The primary cell releasing the
majority of secretory products is known as the principal cell. Interestingly, this cell type
expresses different genes depending on its location along the epididymal tubule. Many
genes in the epididymis are distinctly expressed in a region-specific manner (Figure 1), and
it is believed that the stage-specific interaction of specific epididymal proteins with
spermatozoa results in the essential modifications leading to the development of fully
mature spermatozoa [3]. To date, numerous genes have been identified and localized in
different regions of the epididymis; however, little is known in regards to the specific
function of these genes, and less is known about protein-protein interactions which control
and drive epididymal sperm maturation. Therefore, studies on the region-specific
expression patterns of large groups of known and unknown genes will not only greatly
facilitate identification of the spatial orientation of sperm maturation mechanisms but also
enhance our knowledge of potential players in different pathways.
In this study we have used cDNA and EST microarray technology to examine global
gene expression patterns in different regions of the mouse epididymis. We used a National
Institutes on Aging (NIA) microarray chip containing 15,264 cDNA clones (78% novel and
22% known) derived from pre- and periimplantation embryos, embryonic day 12.5 female
gonad/mesonephros, and newborn ovary [4] and compared two different populations of
RNA from the mouse proximal caput (region 1) epididymidis and the remainder of the
epididymis to look for initial segment enriched genes.
EST Name
emission
EPI2-5 RNA
NIA 5K I
Fold P-value
Fold
P-value
H3065G04
H3069A12
H3068A01
mouse prostein
EST
EST
H3024G12
H3082H01
H3012E02
H3075G01
H3119E05
H3139C03
H3136E12
EST
RIKEN cDNA
EST
similar to mouse CD63
similar to mouse CD63
mouse CD9 antigen (Cd9)
mouse uncoupling protein 2, mitochondrial
H3073D01
H3041E12
H3104G05
H3017G09
H3049H05
H3035E12
H3138D03
H3017D09
H3114H02
H3033H04
H3086G12
H3133G01
unknown
mouse PDZK1 (Pdzk1)
EST
human hyptothetical protein HCPC117
similar to human angiomotin (AMOT)
unknown
vascular Rab-GAP/TBC
RIKEN cDNA
mouse CD34 antigen
EST
EST
oxidation resistance 1 (Oxr1)
H3057B11
mouse tumor protein D52-like 1 (Tpd52l1)
H3154E08
H3010G06
4 PDZ interacting domains
mouse MNCb-1930
5 ankyrin repeats
actin related protein 2/3 complex, subunit 2
protection against oxidative attack
ubiquitin-conjugating enzyme
CXXC zinc finger. Found in proteins that bind
to methyl cytosine and HRX related proteins
required for the transport of long-chain fatty
acids across the inner mitochondria
membrane
novel prostate-specific protein
6.4
6.2
5.8
0.001
<0.001
0.003
5.7
4.0
4.0
3.8
3.8
3.5
3.3
0.022
0.006
0.003
0.002
0.001
0.004
0.007
similar to mouse oxidation resistance 1 (Oxr1)
3.2
3.2
3.1
3.0
3.0
2.9
2.9
2.9
2.9
2.8
2.8
2.7
0.002
<0.001
<0.001
0.004
0.002
0.089
0.015
0.004
<0.001
0.057
0.002
0.001
coiled-coil protein
2.6
0.006
2.6
2.5
0.001
0.002
cell-cell interactions
cell-cell interactions
cell-cell interactions
uncouple ATP production from mitochondrial
respiration, thereby dissipating energy as
heat and affecting energy metabolism
efficiency
regulates endothelial cell motility
mitotic checkpoint
cell-cell interactions
UNCLASSIFIED
5
Protein Function
laser 2
PROTEASES
3
laser 1
PROTEASE
INHIBITORS
4
TRANSCRIPTION
FACTORS
EPI1 RNA
cauda
Putative ID
EPI2-5 ENRICHED GENES
(E1/E2-5)
(E1/E2-5)
SIGNALING FACTORS
1 2
corpus
Scanning the microarray
caput
EST Name
Microarray Analysis
Isolate total RNA and
fluorescently label
EPI1 ENRICHED GENES
Hybridize target
to microarray
In the male reproductive tract, the epididymis is responsible for post-testicular sperm
maturation, a critical process during which sperm acquire progressive motility and fertilizing
ability. It is well-established that epididymal sperm maturation relies on the exposure of
spermatozoa to different proteins in discrete regions of the epididymis. These unique
zones of protein expression, in part, are maintained on the level of gene expression. In
support, expression profiles of different genes in the epididymis show highly diverse
patterns of regionalized expression. In order to obtain a global perspective of gene
expression in the epididymis, we conducted microarray analysis comparing gene
expression levels in the initial segment region (EPI1) to the remainder of the epididymis
(EPI2-5). We utilized an NIA 15K mouse cDNA microarray containing 15,264 cDNA clones
(78% novel and 22% known) derived from pre- and periimplantation embryos, embryonic
day 12.5 female gonad/mesonephros, and newborn ovary. EPI1 and EPI2-5 total RNA
were fluorescently labeled with Cy5 and Cy3, respectively. The 15K chips were probed in
triplicate by the Vanderbilt University Microarray Shared Resource facility under the
direction of Shawn Levy, Ph.D., and the mean of the Cy5/Cy3 ratios compared among
genes. Eighty-seven genes (0.56%) were identified as EPI1-enriched genes that were 2fold or higher in expression as compared to EPI2-5. Within this group 12 genes showed 4fold to 130-fold difference in expression and included an ets transcription factor, milk fat
globule protein-EGF, TNF-alpha factor, and connexin 43. We identified the 130-fold EPI1
gene as Cres3 (cystatin TE-1) and confirmed its EPI1 expression by Northern blot analysis.
Connexin 43 was previously demonstrated to be caput-enriched, also confirming the
microarray analysis. Approximately 1140 genes (7.4%) were identified as EPI2-5 enriched
genes by expression levels that were 2-fold or greater than EPI1. Of these, 50 genes
showed 5-fold to 20-fold difference in expression and included zinc finger protein 40, COX2, and an elafin-like gene. Taken together, our microarray studies reveal new regionally
expressed genes as well as provide a comprehensive survey of gene expression in the
epididymis.
TABLE 1. EPI1 and EPI2-5 enriched genes
MATERIALS & METHODS
unknown
iron-binding glycoprotein
iron-binding glycoprotein
localized in nucleus of sperm heads
vitamin B12 binding
ubiquitin ligase
CONCLUSIONS
Microarray analysis of a 15K NIA chip identified new subsets of genes with EPI1 and EPI25 enriched expression. Many of these regionally expressed genes encode proteins with
putative roles in biological processes that may be integral to sperm maturation and include
those involved in cell signaling, transport, proteolysis, and DNA binding. In addition, many
of the genes identified have not been previously shown to be expressed in the epididymis.
Thus, microarray technology can provide important clues towards identifying the regulatory
and signaling pathways that may be essential for epididymal function.
Figure 3. Analysis of gene expression in the mouse EPI1 and EPI2-5 regions.
Spermatozoa traverse three major regions of the epididymis which include the caput,
corpus, and the cauda. The head of the epididymis can further be subdivided into the
proximal, mid, and distal caput epididymidis. As demonstrated in this figure, the principal
cells of the epididymis express different genes depending on their location along the
epididymal tubule [3].
For each gene, average expression levels were calculated from three independent
hybridizations for EPI1 and EPI2-5 and displayed on a scatterplot. Axes represent the log
scale of the intensities of gene expression in the two different regions of the epididymis.
The middle blue line indicates genes that are expressed equally in both RNA populations.
Signals that lie on the outer blue lines represent genes with 2-fold expression differences in
expression; i.e. 2-fold or higher levels in expression in the EPI1 compared to EPI2-5 (red
gradations) and 2-fold or higher levels of expression in EPI2-5 compared to EPI1 (green
gradations). Verification of the microarray data was done by a comparison with Northern
blot studies of known region-specific genes. Recently, we showed that the cres3 mRNA
was highly restricted to the proximal caput epididymidis (EPI1) (data not shown) thus
supporting the microarray data that showed 130-fold higher levels of expression in cres3 in
EPI1 relative to EPI2-5. Similarly, connexin 43 has previously been shown to be expressed
in all epididymal regions but with highest levels of expression in the proximal caput
epididymidis [5] which correlates with the 5.8-fold difference observed by microarray.
REFERENCES
Table 1. Enriched genes in EPI1 and EPI2-5 regions of the mouse epididymis
Of the 15,264 genes on the NIA chip, 156 genes exhibited 2-fold or higher levels of
expression in EPI1 compared to EPI 2-5, and 449 genes exhibited 2-fold or higher
levels of expression in EPI2-5 compared to EPI1. EPI1 and EPI2-5 genes that
exhibited 2.5-fold or greater and 3.8-fold or greater, respectively, are presented in
the table. Colored squares next to the putative gene identities are the normalized
intensities from three independent hybridizations. The t-test p-values are shown.
Genes are grouped by potential biological function and within each group are
ranked from those showing the highest fold difference between regions to those
with lower fold differences.
1.
2.
3.
4.
5.
Turner, T.T., On the epididymis and its role in the development of the fertile ejaculate. J Androl, 1995. 16(4): p.
292-8.
Jones, R.C., To store or mature spermatozoa? The primary role of the epididymis. Int J Androl, 1999. 22(2): p.
57-67.
Cornwall, G.A. and S.R. Hann, Specialized gene expression in the epididymis. J Androl, 1995. 16(5): p. 379-83.
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We would like to thank Dr. Shawn Levy from the Vanderbilt Microarray Shared Resource facility for his
help and suggestions. This work was supported by NIH grants HD33903 (G.A.C.) and T32-HD07271
(N.H.).