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37
BIOLOGY OF REPRODUCTION 69, 37–47 (2003)
Published online before print 19 February 2003.
DOI 10.1095/biolreprod.102.012609
Gene Expression Profiles in Different Stages of Mouse Spermatogenic Cells
During Spermatogenesis
1
Zuoren Yu, Rui Guo, Yehua Ge, Jing Ma, Jikui Guan, Sai Li, Xiaodong Sun, Shepu Xue, and Daishu Han
2
Department of Cell Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking
Union Medical College, Beijing 100005, China
ABSTRACT
During spermatogenesis, diploid stem cells differentiate, un-
dergo meiosis and spermiogenesis, and transform into haploid
spermatozoa. Various factors have been demonstrated to regu-
late this marvelous process of differentiation, but the expression
of only a few genes specifically involved in spermatogenesis has
been studied. In the present study, different types of spermato-
genic cells were isolated from Balb/c mice testes of different
ages using the velocity sedimentation method, and we deter-
mined the expression profiles of 1176 known mouse genes in
six different types of mouse spermatogenic cells (primitive type
A spermatogonia, type B spermatogonia, preleptotene sper-
matocytes, pachytene spermatocytes, round spermatids, and
elongating spermatids) using Atlas cDNA arrays. Of the 1176
genes on the Atlas Mouse 1.2 cDNA Expression Arrays, we de-
tected 181 genes in primitive type A spermatogonia, 256 in type
B spermatogonia, 221 in preleptotene spermatocytes, 160 in
pachytene spermatocytes, 141 in round spermatids, and 126 in
elongating spermatids. A number of genes were detected as dif-
ferential expression (up-regulation or down-regulation). Four-
teen of the differentially expressed genes have been further con-
firmed by reverse transcription-polymerase chain reaction for
their expression characterizations in different types of sper-
matogenic cells. These results provide more information for fur-
ther studies into spermatogenesis-related genes and may lead to
the identification of genes with potential relevance to sper-
matogenesis.
gametogenesis, sperm, spermatogenesis, testis
INTRODUCTION
Mammalian spermatogenesis is a continuum of cellular
differentiation in which three principal phases can be dis-
cerned: spermatogonia renewal and proliferation, meiosis,
and spermiogenesis. Spermatogenesis is initiated by the di-
vision of stem cells (primitive type A spermatogonia) to
form preleptotene primary spermatocytes, which undergo a
final replication of nuclear DNA before entering the meiotic
prophase. Preleptotene primary spermatocytes develop to
form, in sequence, leptotene primary spermatocytes, zy-
gotene primary spermatocytes, and pachytene and diplotene
primary spermatocytes. The diplotene primary spermato-
1
Special Funds for Major State Basic Research Project of China (grant no.
G1999055901).
2
Correspondence: Daishu Han, Department of Cell Biology, Institute of
Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking
Union Medical College, 5 Dong Dan San Tiao, Beijing 100005, China.
FAX: 86 10 65296466; e-mail: daishu@public.bta.net.cn
Received: 1 November 2002.
First decision: 3 December 2002.
Accepted: 3 February 2003.
Q 2003 by the Society for the Study of Reproduction, Inc.
ISSN: 0006-3363. http://www.biolreprod.org
cytes go through two meiotic divisions and give rise to
round spermatids. Subsequently, round spermatids enter
spermiogenesis and encounter dramatic morphological
changes to form elongating spermatids and then develop to
mature spermatozoa [1]. This complex process is orches-
trated by the expression of thousands of genes encoding
proteins that play essential roles during specific phases of
germ cell development. The expression of a number of
these genes is developmentally regulated during spermato-
genesis. Both transcriptional and translational control
mechanisms are responsible for temporal and stage-specific
expression patterns [2, 3].
Most cells in our bodies contain the same genome, but
not all the genes are used in each cell. Some genes are
turned on (or expressed when needed), and many genes are
used to specify features unique to each type of cells. To
know how genes are controlled to express at the exact time
and which genes function uniquely in special cells, an im-
portant step is defining gene expression profiles—that is,
comparing patterns of expression in different tissues and at
different developmental stages in both normal and disease
states. This can be accomplished using reverse transcrip-
tion-polymerase chain reaction (RT-PCR), RNase protec-
tion assays, or Northern blot analysis, but these methods
focus on only a few genes at a time. A more promising
approach for analyzing multiple genes simultaneously is the
hybridization of entire cDNA populations to nucleic acid
arrays, a method that has been adopted for high-throughput
analysis of gene expression [4–7]. It allows rapid detection
of the gene expression profiles of hundreds to thousands of
genes simultaneously. This technology, otherwise known as
cDNA microarray, offers tremendous potential for charac-
terizing gene expression patterns during normal biological
or disease processes as well as for identifying differentially
expressed genes that may play an integral role in these pro-
cesses. In this regard, using this approach has resulted in
the identification and cloning of genes with potential rele-
vance for growth control and terminal differentiation in hu-
man melanoma cells [8], ovarian carcinomas [9], renal cell
carcinoma [10], breast cancer [11], embryonic stem cells
[12], folliculogenesis [13], and follicular lymphoma [14].
In addition, cDNA microarrays have been used to study the
temporal program of gene expression in human fibroblasts
in response to serum [15] and in spermatogenic cells in
response to stress [16] and to hyperthermia [17].
In a similar vein, the cDNA microarray technology can
be applied to gain a comprehensive view of gene expression
involved in spermatogenesis with the purpose of studying
the mechanisms and regulation of spermatogenesis at the
genetic level. In the present study, we first isolated six sper-
matogenic cell types from mouse testis. We then used
cDNA microarray technology to identify spermatogenesis-
related as well as differentially expressed genes among dif-
38
YU ET AL.
TABLE 1. Primers for RT-PCR analysis.
Gene Forward primers (59 → 39) Reverse primers (59 → 39)
GAPDH
E2F-3
VEGF
CEK5-R
FAN
PVGC
CCNE1
STAM
SOCS-1
FCGR2B
LIM2
ERA-1
MADH1
CEK7-L
MAPK14
TGA TGA CAT CAA GAA GGT GGT GGA G
AAG GCC TGT CCA AAG AAG TG
GCA CTG GAC CCT GGC TTT AC
GAC AAG CTG GAC ATC ATC TGC
AGA GAC AAG GCG GGC AGA TG
TTC CGC ACT GTC GCT ATC ATC
GCA GGC GAG GAT GAG AGC AG
TTG GCC AAT CTC GCA CAG GAC
CAG CCG ACA ATG CGA TC
TGC TGG GAC TCA TGA TCT TC
AAG AGA CTG CAG TCT CGT AC
CCC TCT GAC CAT GGG ATT AC
CCT ACC ACT ATA AGC GAG TG
GAC TAC CTG GAT ATC TAC TG
TTG ATA CAA AGA CGG GGC AT
TCC TTG GAG GCC ATG TAG GCC AT
GAG CAG GGG AGG CAG TAA GT
AAT GCT TTC TCC GCT CTG A
TGC GGA GCT TGA GTA GTA GGA
CCC GGC AAC AGT AAA CAA GA
ATC TCC ATC GCG CTC CAC CTC
ATA ACC ATG GCG AAC GGA ACC
CTC AGC GGC TTC AAA ATC GTA
CGA CAC AGC TCC TGC AG
TCA GGA GGA TTG TCT GGA AC
ACC TGA GGA TCT TGT GCG TG
AGA GTC TGC TGC TCT TGT CC
AGA TGC TGC TGT CAC TGA GG
GAG AAG CAG CAA GGA TTT TG
GTA GTT TCT TGC AGA CTC TG
ferently developed stages of germ cells and to further elu-
cidate their functions and relationships in spermatogenesis.
MATERIALS AND METHODS
Animals
Male and female Balb/c mice (age, 80–100 days) were obtained from
Laboratorial Animals Center of Beijing University (Beijing, China) and
maintained in a temperature- and humidity-controlled room on a 12L:12D
photoperiod. The mice had free access to food and water. Female mice
were naturally mated and observed at 12-h intervals near the end of preg-
nancy to record the time at which parturition occurred. The day of birth
was designated as Day 0. Litter size was adjusted to a maximum of 10
by removing the appropriate number of female pups. All the measures
taken for the mice were in accordance with approved guidelines(Guideline
for the Care and Use of Laboratory Animals) established by the Chinese
Council on Animal Care.
Isolation of Spermatogenic Cell Types
The procedure for the isolation of spermatogenic cells was based on
that previously described by Bellve´ et al. [18] with a modification. Based
on the majority of germ cell types, different ages of male Balb/c mice
were used for the isolation of differently developed stages of spermato-
genic cell types: 6 days of age for primitive type A spermatogonia, 9 days
for type B spermatogonia, 14 days for preleptotene spermatocytes, 21 days
for pachytene spermatocytes, 35 days for round spermatids, and 60 days
for elongating spermatids. The number of mice used for cell separation
was 25, 25, 20, 15, 10, and 6, respectively.
Briefly, mice were anesthetized with CO
2
and then killed by cervical
dislocation. The testes were removed and decapsulated. The tubulous tis-
sue was cut into small pieces and incubated in 5 ml of PBS containing
0.5 mg/ml of collagenase (Sigma, St. Louis, MO) with continuous agita-
tion at 33
8
C for 15 min. The dispersed seminiferous cords and cells were
allowed to sediment for 5 min, and then the supernatant was decanted.
The pellet was resuspended in 5 ml of PBS containing 0.5 mg/ml of
trypsin (Sigma) and 1
m
/ml of DNase (Promega, Madison, WI) and in-
cubated under the same conditions as above for 15 min. The tissue was
dissociated to disperse seminiferous cells by pipetting gently with a Pas-
teur pipette, and the cell suspension was centrifuged at 80
3
g for 10 min.
The pellet was then washed twice with PBS, filtered through a filter cloth
(200 mesh), and resuspended in 20 ml of PBS solution containing 0.5%
BSA.
Cell numbers were counted using a hemocytometer. A total of 10
8
cells
was bottom-loaded into a cell-separator apparatus and followed by a 2%
to 4% BSA gradient in RPMI medium 1640 (Gibco, Grand Island, NY).
After 3 h of velocity sedimentation at unit gravity, the cell fractions (10
ml/fraction) were collected from the bottom of the separator apparatus at
a rate of 5 ml/min. The cell type and purity in each fraction were assessed
using light microscopy based on their diameters and morphological char-
acteristics. Only fractions with expected cell type and high purity (
.
85%)
were pooled together. The average purity for each cell type was as follows:
primitive type A spermatogonia, 94%; type B spermatogonia, 90%; pre-
leptotene spermatocytes, 88%; pachytene spermatocytes, 95%; round sper-
matids, 96%; and elongating spermatids, 92%.
cDNA Array Hybridization Procedures
RNA extraction.
Total RNA was extracted using Trizol Reagent (Gib-
co) according to the manufacturer’s protocol, followed by treatment with
RNase-free DNase (Promega) at 37
8
C for 20 min to avoid contamination
of genomic DNA. The RNA quality and concentration were assessed using
agarose gel electrophoresis and spectrophotometric reading.
Probe preparation.
Five micrograms of total RNA were reverse tran-
scribed using reagents provided in the Atlas cDNA Expression Array Kit
(Clontech, Palo Alto, CA) and radiolabeled with [
a
32
P]dATP (10
m
Ci/
m
l;
Amersham Pharmacia Biotech, Beijing, China). The labeled cDNAs were
purified from unincorporated
32
P-labeled nucleotides by Chroma Spin-200
columns (Clontech), and the radioactivity of the probes was counted using
a scintillation counter.
Hybridization.
The array membranes (Atlas Mouse 1.2 cDNA Expres-
sion Arrays; Clontech) that contained 1176 genes were prehybridized for
30 min at 68
8
C in ExpressHyb hybridization solution containing 100
m
g/
ml of salmon testis DNA. The denatured
32
P-labeled cDNA was added to
ExpressHyb hybridization solution (Clontech) at a final concentration of
1
3
10
6
cpm/ml, and the array membranes were hybridized with the la-
beled cDNA overnight at 68
8
C. The next day, the membranes were washed
three times for 30 min with prewarmed (68
8
C) washing solution 1 (2
3
SSC [1
3
SSC: 0.15 M sodium chloride and 0.015 M sodium citrate] and
1% SDS) and once for 30 min with prewarmed (68
8
C) washing solution
2 (0.1
3
SSC and 0.5% SDS) with continuous agitation at 68
8
C. After a
final wash with 2
3
SSC at room temperature, the membranes were im-
mediately wrapped with plastic film to keeping them from drying and
exposed to x-ray film (XK-1; Kodak, Shantou, China) for 12 h at
2
80
8
C.
Analysis of Gene Expression
The films were scanned, and the images were quantitated and analyzed
with AtlasImage 2.01 software (Clontech). Expression data from replicate
membranes were averaged and normalized. Adjusted intensity equals the
intensity of each gene minus the background value. The genes with an
adjusted intensity less than 2-fold the background value were not detected,
nor were those that had opposite changes in replicate pairs. The signal
ratio of each given gene between two compared array membranes was
calculated. In the present study, the ratio threshold was set at 2.0. Only
those genes that showed an increase or decrease of 2.0-fold or greater
were considered to be differentially expressed.
RT-PCR Analysis
Expression of some prominently changed genes in type B spermato-
gonia and spermatids was further examined by RT-PCR to confirm the
array results. First-strand cDNA was synthesized from total RNA using
Moloney murine leukemia virus reverse transcriptase at 42
8
C for 1 h. The
PCRs were performed with first-strand cDNA under conditions of 30 sec
at 94
8
C, 30 sec at 55
8
C (varied with different genes), and 1 min at 72
8
C
each cycle. Thirty-two cycles (25 cycles for glyceraldehyde phosphate
dehydrogenase [GAPDH]) were done and followed by extension for 10
min at 72
8
C. The primers for PCR are listed in Table 1.
The RT-PCR products were subjected to electrophoresis in 1% agarose
gels. To ensure that equal amounts of reverse-transcribed cDNA were ap-
plied to the PCR reaction, GAPDH (a housekeeping gene) was also in-
cluded in the PCR.
39
GENE EXPRESSION PROFILES IN SPERMATOGENIC CELLS
FIG. 1. Complementary DNA array imag-
es of the expression pattern of genes in six
types of spermatogenic cells. Each array
contains 1176 cDNAs, which were divid-
ed into 6 functional groups, with 9 house-
keeping control cDNAs located in the bot-
tom row. A list of gene names and their
locations is available from Clontech (http://
atlasinfor.clontech.com/). The cDNAs are
single-spotted on a nylon membrane and
were specially prepared to minimize the
problem of nonspecific hybridization. The
gene-specific primer mix provided with
the array for probe synthesis greatly in-
creases the sensitivity of hybridization. A)
Primitive type A spermatogonia. B) Type B
spermatogonia. C) Preleptotene spermato-
cytes. D) Pachytene spermatocytes. E)
Round spermatids. F) Elongating sperma-
tids. Examples of differentially expressed
genes in the six types of cells are marked
with arrows. 1) T-box 6, down-regulated to
undetectable level in C–F. 2) Cyclin E, up-
regulated in B. 3) cdk 4, Up-regulated in
C and D. 4) Corticotropin-releasing hor-
mone receptor, up-regulated in B and C.
5) Notch gene homologue 2, up-regulated
in B. 6) LIM domain-binding 2, up-regulat-
ed in E. 7) MAD homologue 1, up-regulat-
ed in E. 8) Ubiquitin-conjugating enzyme
E2B, down-regulated in E and F. 9) Sup-
pressor of cytokines signaling protein 1,
up-regulated in F. 10) ERA-1 protein, up-
regulated in F.
RESULTS
Overall Gene Expression During Spermatogenesis
To characterize genes that may be associated with the
initial differentiation decisions and mechanisms directing
mammalian spermatogenesis, we examined the gene ex-
pression profiles of different stages of spermatogenic cells
using the Atlas Mouse 1.2 Array. The Atlas Mouse 1.2
Array includes 1176 cDNAs, nine housekeeping control
cDNAs, and negative controls on a single nylon membrane.
The cDNAs immobilized on the membrane have been spe-
cially prepared to minimize the problem of nonspecific hy-
bridization. Each cDNA fragment is 200 to 600 base pairs
and amplified from a region of the mRNA that lacks the
poly-A tail, repetitive elements, or other highly homologous
sequences. Gene location and information are available
from Clontech (http://atlasinfo.clontech.com/).
The images of the Atlas array membranes hybridized to
cDNAs from different types of spermatogenic cells are
shown in Figure 1. Of the 1176 genes on the array, 260
were detected during spermatogenesis in at least one of the
six cell types. Their cellular distribution was as follows:
181 genes expressed in primitive type A spermatogonia,
256 in type B spermatogonia, 221 in preleptotene primary
spermatocytes, 160 in pachytene primary spermatocytes,
141 in round spermatids, and 126 in elongating spermatids.
Of the total number of genes detected during spermatogen-
esis, 46% (120 genes) were detected in all six cell types.
Interestingly, some stage-specific genes were observed. For
example, nine genes expressed uniquely in type B sper-
matogonia, including connexin 26 (CXN26), cyclin D1, se-
maphorin B, neuroendocrine convertase 2 (NEC 2), inter-
leukin 5-receptor
a
subunit (IL-5R
a
), fyn proto-oncogene,
corticotropin-releasing hormone receptor 2, cathepsin C,
and meiotic recombination 11 homologue A. Four genes
expressed only in round spermatids, including low-affinity
immunoglobulin G Fc receptor II beta (FCGR2B), heat
shock 86-kDa protein 1 (HSP86-1), defender against cell
death 1 protein (DAD1), and mitogen-activated protein ki-
nase 14 (MAPK14).
Identification of Differentially Expressed Genes
To identify genes that differentially expressed during
spermatogenesis, we compared the gene expression profiles
of 1176 genes among different stages of spermatogenic
cells during spermatogenesis. Differentially expressed
genes between one stage and the neighboring stage of sper-
matogenic cells are listed in the tables.
Of the total 260 genes detected during spermatogenesis,
64 were differentially expressed from primitive type A
spermatogonia to type B spermatogonia. The expression of
2 genes was down-regulated and of 62 genes up-regulated
(Table 2), including 17 exclusively expressed in type B
cells compared to primitive type A spermatogonia. Among
up-regulated genes, several were observed to have a dra-
40
YU ET AL.
TABLE 2. Genes up-regulated in expression from primitive type A spermatogonia to type B spermatogonia.
Array
position Ratio
a
GenBank no. Name of gene/protein Gene classification
A01f Up M63803 Gap junction beta 2 protein (GJB2); connexin 26 (CXN26) Membrane channels and transporters
A01k 2.41 X61675 Gap junction alpha 5 protein (GJA5); connexin 40 (CXN40) Membrane channels and transporters
C10d 5.17 AF033003 Potassium voltage-gated channel, shaker related subfamily,
beta member 1
Membrane channels and transporters
C10j 2.05 U70068 Potassium voltage-gated channel subfamily Q member 1
(KCNQ1); KVLQT1
Membrane channels and transporters
A14i 3.43 M98502 Zinc finger protein 46 Unidentified
F13e 2.23 L28819 Involucrin (IVL) Unidentified
F13f 2.38 U29539 Retinoic acid-inducible E3 protein; stimulated by retinoic acid
13 (STRA13)
Unidentified
A01n 3.21 D13664 Osteoblast-specific factor 2 precursor (OSF2) Unidentified
A03e 2.52 AF001465 Aristaless 4 (ALX4) Basic transcription factors
A05h 2.53 L13171 Myocyte-specific enhancer factor 2C (MEF2C) Basic transcription factors
A05j 3.40 L14677 POU domain, class 2, transcription factor 3 Basic transcription factors
A05l Up L34808 Homeobox protein 10 (HOX10) Basic transcription factors
A06c 2.18 M30499 Myogenic factor 6 (MYF6) Basic transcription factors
A09f 2.37 U61110 Eyes absent 1 homolog (
Drosophila
) Basic transcription factors
A09i 3.24 U62523 msh-Like homeobox protein 3 (MSX3) Basic transcription factors
A11i 2.07 X74938 Hepatocyte nuclear factor 3 gamma (HNF3G) Basic transcription factors
A12a 2.37 X80338 Sine oculis-related homeobox protein 2 homologue (SIX2) Basic transcription factors
A13d 2.04 Z21524 Hematopoietically expressed homeobox Basic transcription factors
A13l Up U65091 Cbp/p300-interacting transactivator with Glu/Asp-rich carboxy-
terminal domain 1
Basic transcription factors
B08h Up AF015948 Transcription factor e2f3 (E2F-3) Basic transcription factors
B04m 2.33 J03168 Interferon regulatory factor 2 (IRF 2) Transcription activators and repressors
B10a 10.3 Z47766 Cyclin F (S/G2/M-specific) Cyclins
B10g Up S78355 Cyclin D1 Cyclins
B10j 10.2 X75888 G1/S-specific cyclin E1 (CCNE1) Cyclins
B11d 3.68 M58633 Cell division cycle 2 homologue Cell cycle-regulating kinases
B12a 3.58 U20553 Cyclin-dependent kinase inhibitor 1C (P57) cdk Inhibitors
B13g Up U86016 Desmoglein 3 Cell adhesion receptors/proteins
B14f 2.65 X95600 Cadherin 8 precursor (CDH8) Cell adhesion receptors/proteins
B14h Up X85991 Sema domain, immunoglobulin domain (lg), semaphorinB Cell adhesion receptors/proteins
C06f 3.75 X58876 Transformed mouse 3T3 cell double minute 2 (MDM2); p53-
associated protein
Oncogenes and tumor suppressors
C07a 2.06 L10656
abl
Proto-oncogene Oncogenes and tumor suppressors
C07j 2.02 U14173
skl
Proto-oncogene Oncogenes and tumor suppressors
C08j 9.04 X68193 Nucleoside diphosphate kinase B (NDP kinase B; NDK B);
NM23-M2; NME2
Oncogenes and tumor suppressors
C13l 2.0 J05186 Intestinal calcium-binding protein (CAI); endoplasmic reticu-
lum stress protein 72
Heat shock protein
C08k 2.0 L40406 105-kDa heat shock protein (HSP105); heat shock-related 100-
kDa protein E71
Heat shock protein
C14m Up M59378 Tumor necrosis factor receptor 2 precursor (TNFR2) Death receptors
D01e 7.23 U43900 Signal transducing adaptor molecule (STAM) Death receptor-associated proteins and adaptors
D01h Up AF013632 Protein FAN (factor associated with N-smase activation) Death receptor-associated proteins and adaptors
D04k Up X72305 Corticotropin-releasing hormone receptor Growth factor and chemokine receptors
D05c 2.38 M28998 Basic fibroblast growth factor receptor 1 (BFGF-R; FGFR1) Growth factor and chemokine receptors
D06f Up D90205 Interleukin 5 receptor, alpha Interleukin and interferon receptors
D06h 2.21 D17630 Chemokine (C-X-C) receptor 2 Interleukin and interferon receptors
D06k 4.14 AF015963 Cholecystokinin A receptor Hormone receptors
D06l Up U19939 Corticotropin-releasing hormone receptor 2 Hormone receptors
D06n Up L07379 Growth hormone releasing hormone receptor Hormone receptors
D12k 3.04 U12983 Cek 5 receptor protein tyrosine kinase ligand Growth factors, cytokines, and chemokines
E01m 7.7 M95200 Vascular endothelial growth factor precursor (VEGF); vascular
permeability factor
Growth factors, cytokines, and chemokines
E01n Up AF007268 Fibroblast growth factor 15 (FGF15) Growth factors, cytokines, and chemokines
E02n 4.79 M92415 Fibroblast growth factor 6 precursor (FGF6); heparin-binding
growth factor 6
Growth factors, cytokines, and chemokines
B04l 2.50 U19119 Interferon inducible protein 1 Intracellular transducers/effectors/modulators
E07d Up L25606 CD86 antigen; B7-2; CD28 antigen ligand 2 (CD28L2) Intracellular transducers/effectors/modulators
E08n 6.4 D32210 Neurogenic locus notch homologue 2 (notch2) Intracellular transducers/effectors/modulators
F02k 2.56 X99063 Zyxin (ZYX) Intracellular transducers/effectors/modulators
E08k 4.81 AF003942 Bone morphogenic protein receptor type II (BMPR2) Receptors
E09a 2.9 M34476 Retinoic acid receptor gamma-A (RAR-gamma-A; RARG) Receptors
C11h 2.13 U20107 Synaptotagmin VIII (SYT8) Adaptors and receptor-associated proteins
E10l Up U70324 Proto-oncogene tyrosine-protein kinase fyn Adaptors and receptor-associated proteins
F04a Up U89269 Cathepsin C Cysteine proteases
F04g 2.0 X59379 Alzheimer’s disease amyloid A4 protein homologue; protease
nexin-II (PN-II)
Inhibitors of proteases
F09j 3.0 U04443 Nonmuscle myosin light chain 3 (MLC3NM; MYLN); MYL6 Cytoskeleton/motility proteins
41
GENE EXPRESSION PROFILES IN SPERMATOGENIC CELLS
TABLE 2. Continued.
Array
position Ratio
a
GenBank no. Name of gene/protein Gene classification
F11l 4.1 X74351 Xeroderma pigmentosum group A complementing protein
(XPA)
DNA damage signaling/repair proteins and DNA
ligases
F13a 3.02 U58987 Meiotic recombination 11 homolog A (
S. cerevisiae
) DNA synthesis, recombination, and repair pro-
teins
a
When the adjusted intensity of a detected gene in primitive type A spermatogonia is close to background value, the ratio cannot be calculated
accurately, so the software uses
up
to indicate it. Only those genes with an intensity value of at least 2-fold the background intensity are looked at as
expressed genes. Only those with a ratio value of $2.0 are considered to be up-regulated genes.
TABLE 3. Genes expressed strongly in both primitive type A spermatogonia and type B spermatogonia and dramatically down-regulated to near-
undetectable levels in more mature germ cells.
Array
position GenBank no. Name of gene/protein Gene classification
A05l L34808
C. elegans
ceh-10 homeo domain containing homologue Basic transcription factors
A09b U57331 T-box 6 Basic transcription factors
A12c X84814 Sine oculis-related homeobox 5 homologue (
Drosophila
) Basic transcription factors
B05f U29086 Atonal homologue 2 (
Drosophila
) Transcription activators and repressors
B09d U51907 TRAF family member-associated NF-kappaB activator Transcription activators and repressors
B10j X75888 Cyclin E1 Cyclins
B13a D82029 Cadherin 6 Cell adhesion receptors/proteins
B14f X95600 Cadherin 8 Cell adhesion receptors/proteins
C07a L10656 v-
abl
Abelson murine leukemia oncogene 1 Oncogenes and tumor suppressors
C08j X68193 (Nucleoside diphosphate kinase) Oncogenes and tumor suppressors
C09l J04696 Glutathione S-transferase, mu 2 Xenobiotic transporters
D13b Z29532 Follistatin Growth factors, cytokines, and chemokines
E07g L25890 Eph receptor B2 Intracellular transducers/effectors/modulators
E08k AF003942 Bone morphogenic protein receptor, type II (serine/threonine
kinase)
Growth factor receptor
F06b L27220 Alpha internexin neuronal intermediate filament protein Cytoskeleton/motility proteins
F08f X05211 Laminin, gamma 1 Matrix adhesion receptors
F08g X53929 Decorin Cytoskeleton/motility proteins
C10d AF033003 Potassium voltage-gated channel, shaker-related subfamily,
beta member 1
Membrane channels and transporters
F13f U29539 Lycosomal-associated protein transmembrane 5 Membrane channels and transporters
F14m U94437 Phosphatase and tensin homolog Unidentified
matic increase in type B spermatogonia as compared to
primitive type A spermatogonia, such as cyclin E1 (in-
creased by 10-fold), cyclin F (increased by 10-fold), nucle-
oside diphosphate kinase B (NDK B; increased by 9-fold),
and vascular endothelial growth factor precursor (VEGF;
increased by 7-fold). In contrast, two genes decreased from
primitive type A spermatogonia to type B spermatogonia:
glutathione S-transferase 5 (GST-5; decreased by 63%) and
follistatin precursor (FST; 50%). The results also showed
that 20 genes expressed strongly in both primitive type A
spermatogonia and type B spermatogonia, then dramatical-
ly decreased to near-background level in the following stag-
es (Table 3).
From type B spermatogonia to preleptotene primary
spermatocytes, 38 genes were up-regulated (Table 4) and
51 down-regulated (Table 5). The majority of the genes
remained unchanged in their expression intensities.
A little change of gene expression occurred from pre-
leptotene spermatocytes to pachytene spermatocytes. Most
of the differentially expressed genes in this differentiation
step were down-regulated (35 genes). Only three genes
were up-regulated: sine oculis-related homeobox protein 2
homologue (SIX2), single-mined 2 transcription factor
(SIM2), and oncostatin M (OSM).
As compared to pachytene spermatocytes, the expression
of most genes was down-regulated in both round and elon-
gating spermatids (Table 6), but expression of a few genes
was up-regulated in these two types of spermatids. Among
the expressed genes, 67 down-regulated and 7 up-regulated
genes were detected from pachytene spermatocytes to
round spermatids. Of them, the expression of 20 genes de-
creased more than 10-fold. The seven up-regulated genes
in round spermatids were FCGR2B, LIM domain-binding
2 (LIM2), mothers against decapentaplegic homologue 1
(MADH1), HSP86-1, DAD1, glial cell line-derived neuro-
tropic factor (GDNF), and MAPK14. Three up-regulated
genes were detected in elongating spermatids: suppressor
of cytokines signaling protein 1 (SOCS-1), CEK7 ligand
(CEK7-L)/ephrin A2 (EFNA2), and ERA-1 protein (ERA-
1). These differentially expressed genes can be classified
into several categories according to their general functions:
basic transcription factors, cyclins, cytoskeleton proteins,
and growth factors and their receptors.
RT-PCR Analysis
Although differences in gene expression during sper-
matogenesis were observed with the cDNA array, it was
essential to verify these results using other methods. We
monitored the mRNA level of selected genes noted to be
differentially expressed in the Atlas arrays by RT-PCR. We
selected seven genes up-regulated prominently from prim-
itive type A spermatogonia to type B spermatogonia, seven
genes up-regulated in round and elongating spermatids, and
one gene that gradually decreased during spermatogenesis
to reach an undetectable level in elongating spermatids to
42
YU ET AL.
TABLE 4. Genes up-regulated in expression from type B spermatogonia to preleptotene spermatocytes.
Array
posi-
tion Ratio
a
GenBank no. Name of gene/protein Gene classification
A06c 2.46 M30499 Myogenic factor 6 (MYF6) Basic transcription factors
A07f Up S81932 Distal-less homeobox protein 3 (DLX3) Basic transcription factors
A11g 2.58 X74936 Hepatocyte nuclear factor 3 alpha (HNF3A); transcription fac-
tor 3A (TCF3A)
Basic transcription factors
A11l Up X75330 NK5 transcription factor-related locus 1 (NKX-5.1); H6 homeo-
box protein 3 (HMX3)
Basic transcription factors
A14j 2.01 M98502 Zinc finger protein 46 Unidentified
A14l 3.0 U77969 Neuronal PAS domain protein 2 Basic transcription factors
B08h 2.5 AF015948 Transcription factor e2f3 (E2F-3) Basic transcription factors
B10i 10.23 U43844 G1/S-specific cyclin D3 (CCND3; CYL3) Cyclins
B10k 4.2 L01640 Cell division protein kinase 4 (CDK4); cyclin-dependent kinase
4; PSK-J3; CRK3
Cell cycle-regulating kinases
C02i 2.57 X52264 Intercellular adhesion molecule 1 precursor (ICAM1); MALA2 Cell adhesion proteins
C03h Up Z22819 RAB24, member RAS oncogene family Oncogenes and tumor suppressors
C05m Up X67735
Mas
proto-oncogene (G protein coupled receptor) Oncogenes and tumor suppressors
C06n 3.16 U58992 Mothers against dpp protein 1 (msMAD1; MADH1); TGF-b
signaling protein 1
Oncogenes and tumor suppressors
C12b 2.0 AF053471 PMCA; ATP2B2; calcium-transporting ATPase plasma mem-
brane (brain isoform 2)
Atpase transporters
C14b Up L28177 Growth arrest and DNA-damage-inducible protein 45
(GADD45)
Apoptosis-associated proteins
C14j Up L24495 CD27L receptor precursor; T-cell activation antigen CD27;
TNFRSF7
Cell surface antigens
D01m 2.0 D83698 Activator of apoptosis harakiri (HRK); neuronal death protein 5
(DP5); BID3
bcl-2 family proteins
D03a 2.07 U10903 Programmed cell death 2 Apoptosis-associated proteins
D04k 2.11 X72305 Corticotropin-releasing hormone receptor Growth factor and chemokine receptors
D05c 2.15 M28998 Basic fibroblast growth factor receptor 1 (BFGF-R; FGFR1) Growth factor and chemokine receptors
D10m 2.01 U29173 Lymphotoxin receptor (TNFR family) Growth factor and chemokine receptors
D07f 2.55 J05149 Insulin receptor Hormone receptors
D11a Up Z19521 Low-density lipoprotein receptor precursor (LDL receptor;
LDLR)
Receptors
E09f 2.03 M84817 Retinoid X receptor alpha (RXR-alpha; RXRA) Receptors
D13d 2.47 D49921 Glial cell line-derived neurotrophic factor Growth factors, cytokines, and chemokines
D13h Up L07264 Heparin-binding EGF-like growth factor (HBEGF; HEGFL);
diphtheria toxin receptor
Growth factors, cytokines, and chemokines
E01d Up D31942 Oncostatin M (OSM) Growth factors, cytokines, and chemokines
E01f 2.13 U32330 Prepro-endothelin-3 Growth factors, cytokines, and chemokines
E02e 3.3 L41145 Bone morphogenetic protein 5 precursor (BMP5) Growth factors, cytokines, and chemokines
E02j 2.0 M89799 Wingless-related MMTV integratin site 5b protein precursor
(WNT5B)
Growth factors, cytokines, and chemokines
E02n 2.48 M92415 Fibroblast growth factor 6 precursor (FGF6); heparin-binding
growth factor 6 (HBGF6)
Growth factors, cytokines, and chemokines
D05m 2.0 X51975 Interleukin-6 receptor alpha subunit precursor (IL-6R alpha;
IL6RA)
Interleukins and interferons
E04m 2.75 M86671 Interleukin 12 (p40) beta chain Interleukins and interferons
E13h Up Y00703 Guanine nucleotide binding protein, alpha stimulant G proteins
F03c 2.01 AF030065 Serine protease hepsin Serine proteases
F03e 2.11 X12822 Granzyme C precursor (GZMC); cytotoxic cell protease 2
(CCP2); B10; CTLA5
Serine proteases
F11a 2.0 S71186 DNA excision repair protein ERCC3; basal transcription factor
2 89-kDa subunit
DNA damage signaling/repair proteins
F12m 2.23 X81464 Translin (TSN) DNA recombination proteins
a
See Table 2 for explanation of
Up.
perform RT-PCR analysis. Agarose electrophoresis graphs
of the RT-PCR results are shown in Figures 2 through 4.
The E2F transcription factor 3 (E2F-3), VEGF, Cek 5 re-
ceptor protein tyrosine kinase ligand (CEK5-R), factor as-
sociated with N-smase activation (FAN), potassium volt-
age-gated channel (PVGC), G1/S-specific cyclin E1
(CCNE1) and signal transducing adapter molecule (STAM)
were expressed at low or undetectable levels in primitive
type A spermatogonia and up-regulated strongly in type B
spermatogonia (Fig. 2). Expression of FCGR2B, LIM 2,
MADH 1, SOCS-1, CEK7-L, and ERA-1 was up-regulated
from pachytene spermatocytes to round and elongating
spermatids (Fig.3). The gene CEK5-R was highly expressed
in the early stage of spermatogenesis (type B spermatogo-
nia), then gradually down-regulated in the middle stage
(preleptotene spermatocytes, pachytene spermatocytes, and
round spermatids), and was undetectable in the late stage
(elongating spermatids) (Fig. 4). These results are identical
to the data from the cDNA array study. However, the gene
MAPK14 did not show a remarkable change in expression
from pachytene spermatocytes to round and elongating
spermatids (Fig. 3), which was not in accordance with the
cDNA array results, in which the expression was up-regu-
lated.
43
GENE EXPRESSION PROFILES IN SPERMATOGENIC CELLS
TABLE 5. Genes down-regulated in expression from type B spermatogonia to preleptotene spermatocytes.
Array
position Ratio
a
GenBank no. Name of gene/protein Gene classification
A01f Down M63803 Gap junction beta 2 protein (GJB2); connexin 26 (CXN26) Membrane channels and transporters
C10d Down AF033003 Potassium voltage-gated channel, shaker-related subfamily,
beta member 1
Membrane channels and transporters
C11j 0.34 U51112 Sodium/hydrogen exchanger 1 (NA
1
/H
1
exchanger 1) (NHE-1) Membrane channels and transporters
A03e 0.47 AF001465 Aristaless 4 (ALX4) Basic transcription factors
A04k 0.46 D32167 Zinc finger protein of the cerebellum 1 (ZIC1) 1 ZIC2 Basic transcription factors
A05l Down L34808 Homeobox protein 10 (HOX10) Basic transcription factors
A08m 0.17 U57328 TBX3 Basic transcription factors
A09b Down U57331 TBX6 Basic transcription factors
A10i 0.5 X06746 Early growth response protein 2 (egr-2) (krox-20 protein) Basic transcription factors
A12c 0.45 X84814 Myotonic dystrophy locus-associated homeodomain protein
homolog (DMAHP) (SIX5)
Basic transcription factors
A12m 0.32 Y07609 Max-binding protein mnt; basic helix-loop-helix leucine zipper
protein rox
Basic transcription factors
B04c Down X13721 Homeobox protein 2.4 (Hox-2.4) Transcription activators and repressors
B05f 0.31 U29086 Atonal protein homolog 2 (ATOH2; ATH2); helix-loop-helix
protein MATH2; NEX1
Transcription activators and repressors
B09d Down U51907 TRAF family member-associated NF-kappaB activator (TANK) Transcription activators and repressors
B10a 0.32 Z47766 Cyclin F (S/G2/M-specific) Cyclins
B10g 0.48 S78355 Cyclin D1 Cyclins
B10j 0.29 X75888 G1/S-specific cyclin E1 (CCNE1) Cyclins
B13a Down D82029 Cadherin 6 precursor (CDH6); kidney cadherin (K-cadherin) Cell adhesion receptors/proteins
B14f Down X95600 Cadherin 8 precursor (CDH8) Cell adhesion receptors/proteins
B14h Down X85991 Sema domain, immunoglobulin domain (lg) Cell adhesion receptors/proteins
C01h 0.5 L06039 CD31; platelet endothelial cell adhesion molecule 1 Cell-cell adhesion receptors
C01i 0.47 M35410 CD14 monocyte differentiation antigen precursor; LPS receptor
(LPSR)
Cell-cell adhesion receptors
C06f 0.41 X58876 Transformed mouse 3T3 cell double minute 2 (MDM2); p53-
associated protein
Oncogenes and tumor suppressors
C08j 0.27 X68193 Nucleoside diphosphate kinase B (NDP kinase B; NDK B);
NM23-M2; NME2
Oncogenes and tumor suppressors
A14f Down M95106 cAMP-responsive element binding protein 1 Unidentified
C13e Down M55669 Neuroendocrine convertase 2 precursor (NEC 2); prohormone
convertase 2 (PC2)
Unidentified
D01e 0.49 U43900 Signal transducing adaptor molecule (STAM) Death receptor-associated proteins
D01h Down AF013632 Protein FAN (factor associated with N-smase activation) Death receptor-associated proteins
D04l Down U32329 Endothelin b receptor (Ednrb) Hormone receptors
D06l Down U19939 Corticotropin-releasing hormone receptor 2 Hormone receptors
D06n 0.49 L07379 Growth hormone-releasing hormone receptor Hormone receptors
D13b 0.42 Z29532 Follistatin precursor (FST); activin-binding protein Growth factors, cytokines, and chemokines
E01m 0.49 M95200 Vascular endothelial growth factor precursor (VEGF); vascular
permeability factor (VPF)
Growth factors, cytokines, and chemokines
E01n Down AF007268 Fibroblast growth factor 15 (FGF15) Growth factors, cytokines, and chemokines
E04c 0.5 X83376 Inhibin beta-B precursor (INHBB); activin beta-B subunit Growth factors, cytokines, and chemokines
E06k 0.49 U88325 Cytokine-inducible SH2-containing protein 7; suppressor of cy-
tokines signaling protein 1
Intracellular transducers/effectors/modula-
tors
E07g Down L25890 Eph3 (Nuk) tyrosine-protein kinase receptor Intracellular transducers/effectors/modula-
tors
E08k Down AF003942 Bone morphogenic protein receptor type II (BMPR2) Receptors
C11h 0.29 U20107 Synaptotagmin VIII (SYT8) Adaptors and receptor-associated proteins
E10k 0.5 X59868 CDC25MM; guanine nucleotide releasing protein (GNRP;
RASGRF1)
Adaptors and receptor-associated proteins
E10l Down U70324 Proto-oncogene tyrosine-protein kinase fyn Adaptors and receptor-associated proteins
E11l 0.48 X63615 CamK II; Ca
2
1
calmodulin-deponent protein kinase II (beta
subunit)
Intracellular kinase network members
E13n Down M96653 Adenylate cyclase 6 Adenylate/guanylate cyclases and diesteras-
es
F03f 0.49 M56617 Mast cell protease (MMCP)-4 Serine proteases
F04a Down U89269 Cathepsin C Cysteine proteases
F06b Down L27220 Alpha internexin neuronal intermediate filament protein (al-
pha-INX; INA)
Cytoskeleton/motility proteins
F08f Down X05211 Laminin gamma 1 subunit precursor (LAMC1); laminin B2 sub-
unit
Matrix adhesion receptors
F08g Down X53929 Bone proteoglycan ii precursor (PG-S2) (Decorin) (PG40)
(DCN)
Cytoskeleton/motility proteins
F12a 0.45 D49429 HR21spA; protein involved in DNA double-strand break re-
pair; calcium-binding protein
DNA damage signaling/repair proteins
F13a Down U58987 MmMre11a putative endo/exonuclease DNA synthesis, recombination, and repair
proteins
F13f Down U29539 Retinoic acid-inducible E3 protein; stimulated by retinoic acid
13 (STRA13)
Unidentified
a
When the adjusted intensity of a detected gene in preleptotene spermatocytes is close to background value, the ratio cannot be calculated accurately,
so the software uses
down
to indicate it. Only those genes with an intensity value of at least 2-fold the background intensity are looked at as expressed
genes. Only those with a ratio value # 0.5 are considered as down-regulated genes.
44
YU ET AL.
TABLE 6. Genes down-regulated in expression from pachytene spermatocytes to round and elongating spermatids.
Array
position Ratio
a
GenBank no. Name of gene/protein Gene classification
A05h 0.31 L13171 Myocyte enhancer factor 2C Basic transcription factors
A06c 0.50 M30499 Myogenic factor 6 Basic transcription factors
A07a 0.41 S70632 T-cell leukemia, homeobox 1 Basic transcription factors
A09e 0.43 U59322 Homeobox A13 Basic transcription factors
A11c 0.25 X59725 Paired related homeobox 1 Basic transcription factors
A12a 0.31 X80338 Sine oculis-related homeobox 2 homologue (
Drosophila
) Basic transcription factors
A12m Down Y07609 Max-binding protein Basic transcription factors
A14l 0.28 U77969 Neuronal PAS domain protein 2 Basic transcription factors
B01i 0.25 AF031814 Nuclear receptor subfamily 1, group I, member 2 Nuclear receptors
B01n 0.10 D26046 AT motif-binding factor 1 Telomere-associated proteins
B02a 0.08 U36760 Forkhead box G1 Transcription activators and repressors
B02j 0.40 L12703 Engrailed 1 Transcription activators and repressors
B02k 0.35 L12705 Engrailed 2 Transcription activators and repressors
B04l 0.38 U19119 Interferon inducible protein 1 Intracellular transducers/effectors/modula-
tors
B04m Down J03168 Interferon regulatory factor 2 Transcription activators and repressors
B07m 0.15 M32309 Zinc finger protein X-linked Transcription activators and repressors
B09g 0.50 M61909 Avian reticuloendotheliosis viral (v-
rel
) oncogene homologue A Transcription activators and repressors
B09m 0.30 X84311 Cyclin A1 Cyclins
B09n 0.28 U62638 Cyclin C Cyclins
B10f Down X66032 Cyclin B2 Cyclins
B10k 0.34 L01640 Cyclin-dependent kinase 4 Cell cycle-regulating kinases
B12n 0.31 D14888 Cadherin 4 Cell adhesion receptors/proteins
B14l Down S79463 Domain (TM) and short cytoplasmic domain, (semaphorin) 4C Cell adhesion receptors/proteins
C01i 0.48 M34510 CD14 antigen Cell-cell adhesion receptors
C01n 0.06 X53176 Integrin alpha 4 Cell-cell adhesion receptors
C02k 0.25 J05154 Lecithin cholesterol acyltransferase Unidentified
C08i 0.44 U65594 Breast cancer 2 Oncogenes and tumor suppressors
C14n 0.50 U05672 Adenosine A2a receptor Other receptors (by ligands)
D03g 0.05 M20658 Interleukin 1 receptor, type I Interleukin and interferon receptors
D06h 0.30 D17630 Chemokine (C-X-C) receptor 2 Interleukin and interferon receptors
E04m Down M86671 Interleukin 12b Interleukins and interferons
D06k Down AF015963 Cholecystokinin A receptor Hormone receptors
D09c 0.12 U19380 Opioid receptor, mu Neurotransmitter receptors
D12f 0.32 L24755 Bone morphogenetic protein 1 Metalloproteinases
D04e 0.14 M34563 CD28 antigen Growth factor and chemokine receptors
D04k 0.41 X72305 Corticotropin-releasing hormone receptor Growth factor and chemokine receptors
D12h 0.22 X56848 Bone morphogenetic protein 4 Growth factors, cytokines, and chemokines
D12j 0.43 M97017 Bone morphogenetic protein 8a Growth factors, cytokines, and chemokines
D12k 0.26 U12983 Ephrin B1 Growth factors, cytokines, and chemokines
D14d 0.35 M14951 Insulin-like growth factor 2 Growth factors, cytokines, and chemokines
D14f 0.49 Z22703 Fibroblast growth factor 7 Growth factors, cytokines, and chemokines
D14i 0.36 X12531 Small inducible cytokine A3 Growth factors, cytokines, and chemokines
E01a 0.39 M11434 Nerve growth factor, alpha Growth factors, cytokines, and chemokines
E01c 0.40 M14220 Glucose phosphate isomerase 1 complex Growth factors, cytokines, and chemokines
E01k Down M16819 Lymphotoxin A Growth factors, cytokines, and chemokines
E02d 0.50 AF031896 Cerberus 1 homologue (
Xenopus laevis
) Growth factors, cytokines, and chemokines
E04c 0.35 X83376 Inhibin beta-B Growth factors, cytokines, and chemokines
E04e 0.39 U22516 Angiogenin Hormones
E07c Down M60778 Integrin alpha L Intracellular transducers/effectors/modula-
tors
E07d Down L25606 CD86 antigen Intracellular transducers/effectors/modula-
tors
E08d Down AF080215 Coagulation factor II (thrombin) receptor-like 3 Intracellular transducers/effectors/modula-
tors
E08g 0.09 M83336 Interleukin 6 signal transducer Intracellular transducers/effectors/modula-
tors
E08n Down D32210 Notch gene homologue 2 (
Drosophila
) Intracellular transducers/effectors/modula-
tors
E09h 0.40 U43319 Frizzled homologue 6 (
Drosophila
) Intracellular transducers/effectors/modula-
tors
E09m 0.25 Z11886 Notch gene homologue 1 (
Drosophila
) Intracellular transducers/effectors/modula-
tors
E11g 0.08 U15159 LIM-domain containing, protein kinase Intracellular kinase network members
E14h Down Z71173 Inositol 1,4,5-triphosphate receptor 5 Phospholipases and phosphoinositol kinases
E14m 0.24 L76946 Phosphodiesterase 1C Adenylate/guanylate cyclases and diesteras-
es
F03i 0.34 M75716 Serine protease inhibitor 1-2 Serine proteases
F03e 0.10 X12822 Granzyme C Serine proteases
F04m 0.29 M33960 Serine (or cysteine) proteinase inhibitor, clade E (nexin, plas-
minogen activator inhibitor),
Inhibitors of proteases
F06i 0.46 U92949 Kinesin family member C2 Cytoskeleton/motility proteins
45
GENE EXPRESSION PROFILES IN SPERMATOGENIC CELLS
TABLE 6. Continued.
Array
position Ratio
a
GenBank no. Name of gene/protein Gene classification
F06n 0.18 X15475 Peripherin Unidentified
F07j 0.05 M87276 Thrombospondin 1 Unidentified
F08m 0.32 Z22923 Procollagene, type IX, alpha 2 Cytoskeleton/motility proteins
F10g 0.20 D26090 Minichromosome maintenance deficient 5 (
S. cerevisiae
) DNA replication protein
F12a Down X96859 Ubiquitin-conjugating enzyme E2B (RAD6 homology) DNA damage signaling/repair proteins
a
See Table 5 for explanation of
Down.
FIG. 2. RT-PCR analysis of the expression of selected genes that were
up-regulated in type B spermatogonia as compared to primitive type A
spermatogonia in the array results. A) Expression in primitive type A sper-
matogonia. B) Expression in type B spermatogonia. Lane M: marker
(pBR322
Hin
fI); lane 1: control (GAPDH); lane 2: B08h (E2F-3); lane 3:
E01m (VEGF); lane 4: D12k (CEK5-R); lane 5: D01h (FAN); lane 6: C10d
(PVGC); lane 7: B10j (CCNE1); lane 8: D01e (STAM).
FIG. 3. RT-PCR analysis of the expression of selected genes that were
up-regulated in round spermatids and elongating spermatids. Lane 1:
pachytene spermatocytes; lane 2: round spermatids; 3: elongating sper-
matids.
DISCUSSION
The molecular mechanisms associated with the features
that are unique to different stages of spermatogenic cells
are believed to involve the selective modulation of defined
sets of genes that express in a stage-specific manner. In this
context, we have isolated at high purity six stages of sper-
matogenic cells from Balb/c mice testes and used cDNA
microarray technology to profile the expression of 1176
known genes in these cells.
Gene Expression in the Early Stages of Spermatogenesis
Most reports regarding gene expression during sper-
matogenesis have concentrated on the late stages. Gene ex-
pression patterns in spermatogonia or early spermatocytes
have seldom been reported. The stage of preleptotene pri-
mary spermatocytes is often considered to be the point of
entry into meiosis, but it should be noted that type B sper-
matogonia have already embarked on a one-way pathway
leading to the meiotic prophase. That is to say, the type B
stage is an important step as preparation for meiosis. The
present results show that transcriptional activity of many
genes in primitive type A spermatogonia is low, whereas
nearly 30% (62 genes) of the detected genes are up-regu-
lated in expression from primitive type A to type B sper-
matogonia, including cyclins, growth factors, transcription
factors, and oncogenes. From them, seven genes were se-
lected to be further confirmed by RT-PCR, and the PCR
results of these seven genes were completely in accord with
the microarray results. Thirty-eight genes had expressions
that increased from type B spermatogonia to preleptotene
primary spermatocytes. We also found 20 genes that only
expressed in both primitive type A and type B spermato-
gonia and then dramatically decreased to near-background
level after entering the meiosis phase. We hypothesize that,
perhaps, they do not function during the late stage of sper-
matogenesis or that other germ cell-specific genes are ex-
46
YU ET AL.
FIG. 4. RT-PCR analysis of the CEK5-R gene, which gradually down-
regulated during spermatogenesis in the array results. Lane 1: primitive
type A spermatogonia; lane 2: type B spermatogonia; lane 3: preleptotene
spermatocytes; lane 4: pachytene spermatocytes; lane 5: round sperma-
tids; lane 6: elongating spermatids.
pressed to compensate for the absence of the silenced or
inactivated genes. The present results indicate that most
detected genes (256 of 260) express in type B spermato-
gonia and that a number of detected genes have the highest
expression level in type B spermatogonia and preleptotene
primary spermatocytes, supporting our speculation that a
series of specific events during spermatogenesis, such as
meiosis and condensation of the nucleus, are determined at
the early stage of spermatogenesis. The initiation or up-
regulation of specific gene expression in spermatogonia and
early primary spermatocytes is responsible for the further
differentiation and meiosis of these cells.
The expression of cyclins during spermatogenesis is in-
teresting. The G
2
/mitotic-specific cyclin A1 and G
1
-specific
cyclin C are both expressed consistently at a high level in
all germ cells, whereas the S/G
2
/M-specific cyclin F, G
1
/S-
specific cyclin D1, and G
1
/S-specific cyclin E1 are only
expressed in spermatogonia, and up-regulated markedly
from primitive type A to type B spermatogonia. Equally,
as a member of the G
1
/S-specific cyclin family, cyclin D3
is undetectable in spermatogonia but is expressed strongly
in spermatocytes and spermatids. This differential expres-
sion during spermatogenesis may be indicative of a differ-
ent susceptibility of germ cells to such agents. More inter-
estingly, gene E2F, which is related to the function of cyclin
D1 and cyclin E1 [19], playing regulatory roles in the G
1
/
S-phase transition of the cell cycle, is also up-regulated
from primitive type A to type B spermatogonia, similar to
the up-regulation of cyclins D1 and E1. This may reflect
the increase in the proliferative capacity of type B sper-
matogonia. Thus, it is possible that in spermatogonia, E2F
is a transcriptional activator of both cyclin E1 and cyclin
D1, which in turn cause growth and proliferation of type B
spermatogonia. In a similar vein, cyclin D1 has been shown
to activate the gene HSP70 [20], which is a spermatogenic
cell-specific stress gene. The gene HSP70 is not found on
the Atlas arrays used in the present study. However, another
member of the same gene family, HSP105, which is con-
tained on the arrays and has the same regulatory motif with
HSP70, was observed to up-regulate in type B spermato-
gonia.
A number of growth factors and receptors have been
shown to play essential roles in spermatogenesis. The pre-
sent study showed that the expression of transforming
growth factor
b
1
(TGF
b
1
) was undetectable in primitive
type A spermatogonia and weakly expressed in type B sper-
matogonia, whereas no expression signal of TGF
b
2
was
observed in both primitive type A spermatogonia and type
B spermatogonia. This corresponds to previous reports that
TGF
b
2
is expressed only in the late spermatogenetic phase.
Another previous study [21] reported that targeted inacti-
vation of genes for several TGF
b
superfamily members ex-
pressed in the testis did not cause primary defects in sper-
matogenesis, suggesting that they did not have a key role
in this process. Whether the TGF family plays a role in
spermatogenesis remains to be clarified. In the insulin-like
growth factor (IGF) family, we found that IGF-1 expressed
at a low level in primitive type A and type B spermato-
gonia, whereas IGF-2 had a strong expression signal in
both. In contrast, IGF-1 receptor (IGFR-I) expressed
strongly in the two cell types, whereas IGF-2 receptor
(IGFR-II) was undetectable. Previous studies showed that
the loss of IGFR-II results in overaccumulation of circu-
lating levels of IGF-2 [22]. One of the receptors for IGF-
2, IGFR-II, exerts its function of limiting the biological
effects of IGF-2. This suggests that the inactivation of
IGFR-II is in coincidence with the strong expression of
IGF-2 in the present study. We infer that IGF-2 may play
a more important role than IGF-1 during spermatogenesis.
Moreover, some genes for adapters and death receptor-as-
sociated proteins, such as STAM and FAN, were strongly
up-regulated in type B spermatogonia. This finding sup-
ports those of previous reports that programmed cell death
or apoptosis plays an important role in establishing the
adult population of spermatogonia of proper composition
and size [23]. Among genes related to DNA replication,
DNA polymerase
d
and DNA polymerase
a
were expressed
in type B spermatogonia. These two kinds of DNA poly-
merases participate in synthesis of DNA during the early
phase of meiosis and play roles in DNA replication of sper-
matogonia as well [24].
When spermatogenic cells differentiate into the prelep-
totene spermatocyte stage, the process of meiosis begins.
The activities of most genes are maintained unchangeable
from preleptotene to pachytene spermatocytes, whereas
three genes are up-regulated, including SIX2, SIM2, and
OSM. Both SIX2 and SIM2 have been found to behave as
transcriptional repressors in mammals [25, 26]. Up-regu-
lation of the transcriptional repression genes in pachytene
spermatocytes may be partly responsible for the gene tran-
scription inactivation in spermatids. Other studies have re-
ported that OSM is expressed in the rat testis and likely
plays a role at the start of rat spermatogenesis [27].
Gene Expression in the Late Stages of Spermatogenesis
In the present study, we found that the number of ex-
pressed genes and their expression level remarkably de-
creased from pachytene spermatocytes to round spermatids
and elongating spermatids. These results are in agreement
with the previous concept that histone-to-protamine tran-
sition and the ensuing chromatin compaction in condensing
spermatids render sperm gene transcription virtually inac-
tive. For example, we found that gene CEK5-R had the
highest expression level in type B spermatogonia and was
down-regulated from spermatocytes to spermatids. This
was also confirmed by RT-PCR analysis. It is interesting
that seven genes were up-regulated as pachytene spermato-
cytes differentiated to round spermatids and that three
genes were up-regulated during the transition from round
spermatids to elongating spermatids. These genes may be
involved in the induction of spermiogenesis. The up-regu-
lated genes in round spermatids are FCGR2B, LIM2,
MADH1, HSP86-1, DAD1, GDNF, and MAPK14.
The role of the heat shock protein (HSP) gene in sper-
matogenesis has been studied. It has been reported [28] that
HSP86 is mainly expressed in spermatogenic cells and is
up-regulated along with germ cell differentiation during de-
velopment of the testis. The present results correspond to
47
GENE EXPRESSION PROFILES IN SPERMATOGENIC CELLS
those of this previous report, suggesting that the method
used in the present study is reliable. Furthermore, our re-
sults indicate that the HSP86 gene has the highest expres-
sion level in round spermatids. A recent report [29] dem-
onstrated that GDNF expression could be detected in Ley-
dig and Sertoli cells. However, the high-level expression of
GDNF was only detected in round spermatids and some
types of spermatocytes, suggesting that GDNF may play
essential roles in spermatogenesis and testis maturation. We
also found three up-regulated genes in elongating sperma-
tids. To confirm the results of cDNA microarrays, seven
genes, which were up-regulated in round and elongating
spermatids in the present study and were not reported as
expressing in spermatogenesis previously, were selected
and further analyzed by RT-PCR. The results show that the
expression patterns of six genes selected are consistent with
those from cDNA arrays. This suggests that the cDNA ar-
ray results are stable and reliable. Nevertheless, it is nec-
essary to confirm these results using other methods. It is
important that most of the differentially expressed genes
that we found have not yet been reported. These results
could be helpful for finding more spermatogenesis-related,
especially spermiogenesis-related, genes. Further studies on
the functions of these differentially expressed genes may
provide insight regarding the molecular mechanisms of
spermatogenesis.
Conclusions
We have demonstrated the utility of the cDNA microar-
ray in analyzing gene expression changes at different stages
of spermatogenic cells during spermatogenesis. For a num-
ber of genes that have been shown previously to be devel-
opmentally related in spermatogenic cells, the data obtained
by this approach are comparable to those obtained by other
methods. Importantly, additional genes with specific ex-
pression in spermatogenic cells as identified by Atlas arrays
may be differentially regulated in male germ cells. Fur-
thermore, genes and gene networks identified as being sig-
nificant by microarrays provide important leads for pursu-
ing a more complete understanding of spermatogenetic
mechanisms.
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