Analysis of gene expression patterns with cDNA microarray during late stage of spermatogenesis in mice

Abstract

The differentiation process of round spermatids to spermatozoa during the late stage of spermatogenesis is called spermiogenesis.
To explore spermiogenesis-related genes, cDNA microarray was used to study expression patterns of 1176 genes in pachytene
spermatocytes, round spermatids and elongating spermatids of Balb/c mice. The results showed that 208 genes were detected
in all the three cell types. Most of them were down-regulated from pachytene spermatocytes to round spermatids and elongating
spermatids. However, up-regulation of 7 genes expression in round spermatids and 3 genes in elongating spermatids were found.
Expression of 7 differentially expressed genes in cDNA arrays was further confirmed by semi-quantitative RT-PCR study. The
RT-PCR results indicated that the expression of 6 genes was consistent with that in cDNA arrays, only one gene did not show
differential expression by RT-PCR. These results may provide important clues for studying of expression, regulation, and function
of spermiogenesis-related genes.

Full text

NOTES
Analysis of gene expression
patterns with cDNA micro-
array during late stage of
spermatogenesis in mice
YU Zuoren, GUAN Jikui, GE Yehua, MA Jing,
GUO Rui, LI Sai, XUE Shepu & HAN Daishu
Department of Cell Biology, Institute of Basic Medical Sciences,
Chinese Academy of Medical Sciences & Peking Union Medical College,
Beijing 100005, China
Correspondence should be addressed to Han Daishu (e-mail: daishu@
public.bta.net.cn)
Abstract The differentiation process of round spermat-
ids to spermatozoa during the late stage of spermatogenesis
is called spermiogenesis. To explore spermiogenesis-related
genes, cDNA microarray was used to study expression pat-
terns of 1176 genes in pachytene spermatocytes, round
spermatids and elongating spermatids of Balb/c mice. The
results showed that 208 genes were detected in all the three
cell types. Most of them were down-regulated from
pachytene spermatocytes to round spermatids and elongat-
ing spermatids. However, up-regulation of 7 genes expression
in round spermatids and 3 genes in elongating spermatids
were found. Expression of 7 differentially expressed genes in
cDNA arrays was further confirmed by semi-quantitative
RT-PCR study. The RT-PCR results indicated that the ex-
pression of 6 genes was consistent with that in cDNA arrays,
only one gene did not show differential expression by
RT-PCR. These results may provide important clues for
studying of expression, regulation, and function of spermio-
genesis-related genes.
Keywords: spermatogenesis, spermiogenesis, cDNA microarray,
gene expression pattern, RT-PCR.
Spermatogenesis is one of the most important fields
in reproductive biology. A number of specific events
which do not happen in somatic cell differentiation occur
during spermatogenesis. Three principal processes can be
discerned: mitosis, meiosis, and spermiogenesis. In mito-
sis phase, primitive spermatogonia proliferate through
mitosis and form type A and type B spermatogonia. Type
A spermatogonia keep mitosis to continue proliferation,
while type B spermatogonia turn to differentiation way
and form primary spermatocytes. During the meiosis
phase, primary spermatocytes produce the secondary
spermatocytes through the first meiosis, then the secon-
dary spermatocytes enter the second meiosis without DNA
replication and form haploid round spermatids. Round
spermatids then transform into elongating spermatids by
the process of spermiogenesis. The molecular mechanisms
of spermatogenesis remain unclear, especially there are
seldom reports on spermiogenesis-related genes. In this
study, we attempt to search the key genes to control sper-
miogenesis in order to further study the molecular mecha-
nisms of spermiogenesis.
Large-scale studying on gene expression patterns is a
bridge from structural genomics to functional genomics.
The completion of human genome project is only the first
step in understanding how specific genes function, and it
is more important to explore the biological roles of genes
in process of cell proliferation and differentiation. An im-
portant step toward understanding the gene roles is defin-
ing the expression profiles of a large number of genes.
The genes that probably involved in some biological func-
tions can be selected based on the gene expression profiles,
and then their functions should be further studied. The
recently developed cDNA microarray technique is a prom-
ising approach for the quantitive analysis of multiple
genes simultaneously[1]. This technique has been widely
used in many research fields, such as tumor cell prolifera-
tion and differentiation-related genes[2—5], the changes of
transitory gene expression in human fibroblasts in re-
sponse to serum[6], embryonic stem cells differentiation-
related genes[7], expression profile of stress response
genes during spermatogenesis[8], as well as the effects of
hyperthermia on gene expression in spermatogenesis[9].
In the present study, we isolated different stages of
spermatogenic cells from different ages of Balb/c mice
testes using the modified velocity sedimentation
method[10]. Expression patterns of 1176 known genes in
pachytene spermatocytes, round spermatids, and elongat-
ing spermatids were analyzed using Atlas Mouse 1.2 Ex-
pression Array (Clontech).
1 Materials and methods
(
ⅰ) Animals. Male Balb/c mice (3, 5 and 8 weeks
old) which were the second class animals for research
were obtained from the Institute of Biological Products
Manufacturer (Beijing, China). Pachytene spermatocytes,
round spermatids, and elongating spermatids were isolated
from the 3, 5, and 8 week-old mice, respectively.
(
ⅱ) Array membranes. Atlas Mouse 1.2 Expres-
sion Arrays were purchased from Clontech Laboratories
Inc. On array, there are 1176 gene cDNA fragments with
the length of 200—600 bp, which have been amplified
from a transcription region that lacks the poly(A) tail,
repetitive elements, or other highly homologous se-
quences. They are arranged in 6 districts according to their
functions and classification. The name, coordinate, Gen-
Bank No., and other related information of these genes are
available on Clontech’s homepage ( www.clontech.com).
(
ⅲ) Separation of spermatogenic cells. The pro-
cedure for the isolation of spermatogenic cells was
adapted from a modified velocity sedimentation
method[10]. Briefly, ⅰ) from 10 male mice for each isola-
tion, the testes were removed and decapsulated. The tubu-
late tissue was cut into small pieces and incubated in 5 mL
Chinese Science Bulletin Vol. 47 No. 24 December 2002 2075

NOTES
of PBS containing 0.5 mg/mL collagenase (Sigma) with
continuous agitation at 33℃ for 15 min. The dispersed
seminiferous cords and cells were allowed to sediment for
5 min, and supernatant was removed. The pellet was
resuspended in 5 mL of PBS containing 0.5 mg/mL
trypsin (Sigma) and 1 μg/mL DNase (Promega), and
incubated under the same conditions as above for 15 min.
The tissue was dissociated to a single cell by pipetting
gently with a Pasteur pipette, and the cell suspension was
centrifugated at 1000 r/min for 10 min. The pellet was
washed twice with PBS, filtered through a filter cloth (100
mesh), and resuspended in 20 mL of RPMI1640 culture
medium (Gibco) containing 0.5% BSA. ⅱ) A total 1000
mL of RPMI1640 medium with 2%—4% BSA gradient
were bottom-loaded into a cell separator apparatus, and
the 20 mL of cell suspension was loaded onto the top
surface of gradient medium. After 3 h velocity
sedimentation, 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 characteristics. Only the
fractions with expected cell type and high purity (>85%)
were pooled together. ⅲ) The isolated spermatogenic
cells were cultured at 33℃ under 5% CO for 6 h. A few
leydig cells and sertoli cells 2
contaminated were removed
by attaching to culture flask, and the purity of
spermatogenic cells was increased. ⅳ) The purity of cells
was assessed under light microscopy after hematoxylin
staining.
(
ⅳ) RNA extraction. Total RNA was extracted
from spermatogenic cells using TrizolTM Reagent (Gibco,
BRL) according to the manufacturer’s protocol, and
treated with RNase-free DNase (Promega) to avoid con-
tamination of genomic DNA. The RNA quality was as-
sessed by agarose gel electrophoresis, UV absorption
spectrometric reading, and PCR result of a housekeeping
gene, GAPDH.
(
ⅴ) Probe preparation and hybridization. 5 μg of
total RNA were reverse transcribed using reagents pro-
vided in the Atlas cDNA Expression Array Kit (Clontech)
and radiolabeled with [α-P]-dATP (Amersham Phar-
32
macia Biotech). The labeled cDNAs were purified from
unincorporated P-labeled nucleotides by Chroma
32
Spin-200 columns (Clontech) and the radioactivity of the
probes was counted by Scintilition Counter. The array
membranes (Atlas Mouse 1.2 Expression Arrays, Clon-
tech) were prehybridized for 1 h at 68℃ in ExpressHyb
solution containing 100 μg/mL of heat-denatured salmon
testis DNA. The denatured 32P-labeled cDNA was added
to ExpressHyb hybridization solution at final concentra-
tion of 1.0×106 cpm/mL and array membranes were hy-
bridized with the labeled cDNA overnight at 68℃. The
next day, the membranes were washed three times for 30
min with pre-warmed (68℃) washing solution 1 (2×SSC,
1% SDS) and once for 30 min with pre-warmed (68℃)
washing solution 2 (0.1×SSC, 0.5% SDS) with continuous
agitation at 68℃. After a final wash with 2×SSC at room
temperature for 10 min, the membrane was immediately
wrapped with plastic film and was exposed to X-ray film
at −70℃.
(
ⅵ) Analysis of gene expression. Gene expression
levels were assessed according to autoradiographic inten-
sity of hybridization signals by observating with the naked
eyes. The cDNA probes were each hybridized with two
replicate membranes in order to eliminate errors caused
by experimental conditions, and those genes that had op-
posite changes in replicates pairs were forgotten. In order
to minimize experimental variation and permit the com-
parison of different experiments to one another, each
membrane was exposed to X-ray film for various lengths
of time (12, 24, 36 and 48 h). Only those autoradiograms
with similar background and identical signal intensity of
housekeeping genes were comparable. Genes which
showed an increase or decrease of 3.0-fold or greater were
considered as differentially expressed genes.
(
ⅶ) Semi-quantitative RT-PCR. Expression of 7
genes which were up-regulated in round and elongating
spermatids was further confirmed by RT-PCR. Primers
were designed using Dnastar software and synthesized by
Sbs Bio Inc (Beijing, China). Reverse transcription reac-
tion was catalysed by MMLV (Promega). PCR was per-
formed under the conditions of 30 s at 94℃, 30 s at 55℃,
1 min at 70℃ for a cycle, 32 cycles were done (25 cycles
for GAPDH), finally 10 min at 72℃. The RT-PCR prod-
ucts were subjected to electrophoresis in 1% agarose gels
and quantitative analysis was done by YLN 2000 Gel
Analysis System (Beijing, China). Gene expression level
was assessed according to the gray ratio value of the target
genes and housekeeping gene GAPDH.
2 Results
(
ⅰ) The purity of spermatogenic cells. In order to
obtain high purity of spermatogenic cells, we have modi-
fied the velocity sedimentation method by incubating the
cells for 6 h after velocity sedimentation. A small number
of leydig cells and sertoli cells contaminated were re-
moved by attaching to culture flask, and the purity of
spermatogenic cells was increased. Different ages of mice
were chosen to isolate different stages of spermatogenic
cells. At last, 95%, 96%, and 92% of pachytene sperma-
tocytes, round spermatids, and elongating spermatids were
obtained from testis of 3, 5 and 8 week-old mice, respec-
tively (fig. 1). A few contaminated cells were mainly
spermatogonia. Pachytene spermatocytes were the largest
with the diameter of 12—13 μm, round spermatids had
the diameter of 7—8 μm, and elongating spermatids were
2076 Chinese Science Bulletin Vol. 47 No. 24 December 2002

NOTES
Fig. 1. Isolated spermatogenic cells (hematoxylin staining). (a) Pachytene spermatocytes; (b) round spermatids; (c) elongating spermatids.
the smallest with various morphology.
(
ⅱ) Gene expression profiles. Atlas cDNA Array
was used to examine the expression of 1176 known mouse
genes in spermatocytes, round spermatids and elongating
spermatids. As shown in fig. 2, each dot represents one
gene, the results demonstrated that the expression of total
208 genes was detected in all three cell types. The expres-
sions of 201 genes in pachytene spermatocytes (fig. 2(a)),
180 genes in round spermatids (fig. 2(b)), and 155 genes
in elongating spermatids (fig. 2(c)) were detected. The
stable background signal and consistent results in two
hybridization replicates suggested that the RNA quality,
probe labeling and hybridization were stable.
(
ⅲ) Differentially expressed genes. In order to
Fig. 2. Autoradiograph image of cDNA microarrays (arrow-labeled genes were the up-regulated genes compared to the earlier stage of spermatogenic
cells). (a) Pachytene spermatocytes; (b) round spermatids; (c) elongating spermatids.
analyze differentially expressed genes in the three cell
types, we compared the gene expression level one by one.
Only those genes that the difference of signal intensity
was 3-fold or greater were considered as differentially
expressed genes. Compared to pachytene spermatocytes,
the expression of many genes was down-regulated in
round spermatids and elongating spermatids, but a few
genes expression was up-regulated in these two types of
spermatids. Among the expressed genes, 67 down-
regulated genes and 7 up-regulated genes were detected
from pachytene spermatocytes to round spermatids. Of
them, the expression of 20 genes decreased more than
10-fold. The 7 up-regulated genes in round spermatids
were low-affinity IgG Fc receptor Ⅱ beta (FCGR2B),
LIM domain binding 2 (LIM2), mothers against decapen-
taplegic homolog 1 (MADH1), heat shock 86-kD protein 1
(HSP86-1), defender against cell death 1 protein (DAD1),
glial cell line-derived neurotropic factor (GDNF), and
mitogen activated protein kinase 14 (MAPK14). Their
locations on the arrays were arrowed in fig. 2(b). 3
up-regulated genes were detected in elongating spermatids.
They are suppressor of cytokines signaling protein 1
(SOCS-1), CEK7 ligand (CEK7-L) / ephrin A2 (EFNA2),
and ERA-1 protein (ERA-1), which were arrowed in fig.
2(c). These differentially expressed genes can be classi-
fied into several categories according to their general
functions: basic transcription factors, cyclins, cytoskeleton
proteins, growth factors and their receptors.
(
ⅳ) Analysis by semi-quantitative RT-PCR. To
confirm the results of cDNA array, RT-PCR was further
performed to study the expression of 7 genes which were
up-regulated in round spermatids and elongating spermat-
ids, and were probably involved in spermiogenesis. As
illustrated in fig. 3, the RT-PCR results indicated that 6 of
the 7 genes, including FCGR2B, LIM2, MADH1, SOCS-1,
CEK7-L and ERA-1, showed identical results with that of
cDNA array, while the expression of gene MAPK14 did
not show differential expression in the three cell types.
Chinese Science Bulletin Vol. 47 No. 24 December 2002 2077

NOTES
Fig. 3. Analysis of 7 genes expression in spermatogenic cells by RT-PCR. (a) Agarose electrophoresis of PCR products; 1, Pachytene spermatocytes;
2, round spermatids; 3, elongating spermatids; (b) the gray ratio value of the target genes and housekeeping gene GAPDH. 1—3, the same as in (a).
3 Discussions
Spermatogenesis is a specific cellular differentiation
process during which a number of unique events occurred
such as meiosis and shaping. It is these specific events
that attract many scientists to study on this field. Many
regulators are involved in regulating of spermatogenesis.
Much study on the regulation of spermatogenesis by ex-
trinsic regulators, such as hormones has been done, and
the roles of hormones on spermatogenesis have been elu-
cidated[11]. By contrast, the effects of intrinsic genetic
factors (regulation of gene expression) on spermatogene-
sis were less understood. With the application of new
techniques, such as DDRT-PCR, knockout, and transgenic
mice models, more and more spermatogenesis-related
genes were found. Recently, we have reported[12—14] the
expression and function of centrosome protein in different
types of spermatogenic cell during spermatogenesis.
However, the molecular mechanism of spermatogenesis
and which key genes control spermatogenesis remained to
be elucidated. Especially no shaping differentiation-
related gene was reported during spermiogenesis.
Spermatogenesis is a complex process that is regu-
lated by a large number of genes. Large-scale analysis of
gene expression patterns during spermatogenesis is an
important step for understanding how this process is
regulated. The DNA chip technique is a more promising
approach for analyzing expression of multiple genes si-
multaneously. In the present study, cDNA microarray was
applied to detecting gene expression profiles of 1176
known mouse genes in different stages of spermatogenic
cells. In order to further understand the molecular mecha-
nism of spermatid shaping during spermiogenesis, gene
expression patterns of pachytene spermatocytes, round
spermatids and elongating spermatids were analyzed in
this study. Total 208 genes were detected in all of the three
cell types. 201 genes expressed in pachytene spermato-
cytes, and the number and level of gene expression re-
markably decreased in the following stages of round
spermatids and elongating spermatids. The morphological
change and nuclear condensation of spermatids were
probably responsible for the down-regulation of genes.
The same lot number mice were used in the study to avoid
effects of animal feeding conditions on the gene
2078 Chinese Science Bulletin Vol. 47 No. 24 December 2002

NOTES
expression profiles.
It is interesting that 7 genes were up-regulated as
pachytene spermatocytes differentiated to round spermat-
ids, 3 genes were up-regulated from round spermatids to
elongating spermatids. These genes may be involved in
induction of spermiogenesis. The up-regulated genes in
round spermatids are FCGR2B, LIM2, MADH1, HSP86-1,
DAD1, GDNF, and MAPK14. The role of heat shock pro-
tein (HSP) gene in spermatogenesis has been studied. It
has been reported[15] that HSP86 mainly expressed in
spermatogenic cells, and was up-regulated along with
germ cell differentiation during development of testis. Our
results correspond to this previous report, which sug-
gested that the method in this study is reliable. Further-
more, our results indicated that HSP86 gene had the high-
est expression level in round spermatids. Recent report[16]
demonstrated that GDNF expression could be detected in
leydig cells and sertoli cells. But the high level expression
of GDNF was only detected in round spermatids and some
types of spermatocytes, which suggested that GDNF may
play essential roles in spermatogenesis and testis matura-
tion. We also found 3 up-regulated genes in elongating
spermatids. To confirm the results of cDNA microarrays,
7 genes, which were up-regulated in round and elongating
spermatids in our study and were not reported about their
expressions in spermatogenesis previously, were selected
and further analyzed by RT-PCR. The results showed that
the expression patterns of 6 genes selected were consistent
with that from cDNA arrays. This suggested that the
cDNA array results are stable and reliable. But it is nec-
essary to make confirmation using other methods. It is
important that most of the differentially expressed genes
which we found have not been reported yet. These results
could be helpful for finding more spermatogenesis-related,
especially spermiogenesis-related genes. Further studies
on the functions of these differentially expressed genes on
spermatogenesis may provide insight into molecular
mechanisms of spermatogenesis.
Acknowledgements This work was supported by the National Key
Basic Research Project (Grant No. G1999055901).
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