Download The cloning and expression characterization of the centrosome

Vol. 45 No. 6
SCIENCE IN CHINA (Series C)
December 2002
The cloning and expression characterization of the
centrosome protein genes family (centrin genes) in rat testis
SUN Xiaodong ()1, GE Yehua ()1, MA Jing ( )1,
YU Zuoren (
)1, LI Sai ( )1, WANG Yongchao ()2,
XUE Shepu ()1 & HAN Daishu ()1
1. Department of Cell Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking
Union Medical College, Beijing 100005, China;
2. Key Laboratory of Cell Proliferation and Regulation, Department of Biology, Beijing Normal University, Beijing
100875, China
Correspondence should be addressed to Han Daishu (email: daishu@public.bta.net.cn)
Received December 17, 2001
Abstract Centrins are members of the centrosome protein family, which is highly conserved during revolution. The homologous genes of centrin in many organisms had been cloned, but the sequences of the rat centrin genes were not reported yet in GenBank. We cloned the cDNA fragments of centrin-1, -2 and -3 from the rat testis by RT-PCR, and analyzed the homology of the deduced amino acid sequences. The expression characterization of centrin genes in rat spermatogenesis was carried out by semi-quantitative RT-PCR. The results show that the homology of the
corresponding centrin proteins in human, mouse and rat is high. The expression of centrin-1 is
testis-specific, spermatogenic cell-specific and developmental stage-related. Centrin-1 begins to
be transcribed when the meiosis occurs, and its mRNA level reaches the peak in round spermatids.
Centrin-2 and centrin-3 are highly expressed in spermatogonia and their mRNA level decreases
markedly when meiosis occurs. These results suggest that centrin-1 may play roles in meiosis and
spermiogenesis, and centrin-2 and centrin-3 may be related to mitosis.
Keywords: centrin, centrosome, spermatogenesis.
Centrin is a small (~20 ku) centrosome protein which was first identified in the green alga. It
contains four EF-hand domains and belongs to the calcium-binding protein superfamily. Centrin is
present in both the pericentriolar material and the centrioles of centrosome and provided a recognized marker of centrosome[1,2]. The homologous genes of centrin have been cloned from green
alga, yeast, higher plants, xenopus, mouse and human. In contrast to green alga and yeast, where
centrin was encoded by a single gene, the three centrin genes[3,4] (centrin-1, centrin-2 and centrin-3) were identified in human and mouse. These genes are highly conserved. However, there
has been no report about the sequences of rat centrin genes.
The studies of the centrin in green alga and yeast showed that different members of this gene
family play different roles in cells. In green alga, centrin protein is a major component of the nucleus-basal-body connector fibers, causes the excising of microtubule and binding of Ca2+ to centrin. The point mutation of this gene causes a variable flagellar number, abnormal localization of
the basal bodies in the cell and a failure to excise flagella effectively[5]. In yeast, centrin, which is
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encoded by cdc31 gene, plays an important role in the cell cycle-dependent initiation of spindle
pole body (SPB) duplication[6]. The function of centrins in mammalian cells is not well known,
but studies showed that human centrin-3 is highly homologous to the yeast cdc31 gene and plays
an important role in the duplication of centrosome[7].
In rat spermatogenesis, the spermatogonia develop to the spermatozoon through the mitosis,
meiosis and spermiogenesis. Each stage is relevant to the construction, duplication and function of
centrosome. So far, little is known about the expression and function of centrosome proteins in
spermatogenesis. In this study, we designed the specific primers according to the sequences of
human and mouse centrin genes and cloned the cDNA fragments of centrin-1, -2 and –3 from rat
testis. The homology of nucleic acid and deduced amino acid sequences was analyzed. The expression pattern of centrin genes was studied by semi-quantitative RT-PCR during rat spermatogenesis.
1
Materials and methods
1.1 Materials
9-day to 8-week-old normal male Wistar rats were purchased from the Experimental Animal
Center of Beijing Medical University.
1.2
Primers
The specific primers used for the amplification of rat centrin cDNA were designed according
to the sequences of mouse and human centrin genes. The primer sequences for centrin and
GAPDH ( glyceraldehyde-3-phosphate dehydrogenase) are as follows:
Centrin-1upper: 5
-CGG GAA GCC TTT GAC CTC TTC GAT-3
,
lower: 5
-GTT GGT CTT TTT CAT GAT CTT-3
;
Centrin-2 upper: 5
-CGG GAA GCT TTT GAT CTT TTC GAT-3
,
lower: 5
-GCT GGT CTT TTT CAT GAT GCG-3
;
Centrin-3 upper: 5
-ATG AGT TTA GCT CTG AGA GGT G-3
,
lower: 5
-CAT TTT GGT GTT CAG TCT TGT AA-3
;
GAPDH upper: 5
-TGA TGA CAT CAA GAA GGT GGT GAA G-3
,
lower: 5
-TCC TTG GAG GCC ATG TAG GCC AT-3
.
1.3 Separation of rat testicular cells
Gravity sedimentation method was used to separate the spermatogenetic cell at different developmental stage[8]. Spermatogonia, spermatocytes, round spermid and elongated spermatid were
isolated respectively from testis of 10-day, 25-day, 8-week-old rat testis. Sertoli and Leydig cells
were isolated from 12-d rat testis[9]. Purity of cells was counted after H.E. staining.
1.4 RT-PCR
() Total RNA was isolated from different kind of cells by the TrizolTM (Gibco. BRL)
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method, according to the protocol described in the introduction for TrizolTM reagent. (ii) The RNA
samples were reverse transcribed into cDNA in 20 µL of reverse transcription reaction mixture
containing 1 µL of RNA, random hexamers (Roche) and M-MLU reverse transcriptase (Gibco.
BRL). (iii) 3 µL cDNA was used in 50 µL of PCR reaction. The PCR was performed in the parameter: pre-denaturation for 5 min at 94, denaturation for 45 s at 94annealing for 45 s at
55 for GAPDH, centrin-1, centrin-2, 63 for centrin-3, extension for 45 s at 72, additional
5 min for extension after amplification. 35 cycles for centrins and 23 cycle for GAPDH were done
in PCR. The PCR products were subjected to electrophoresis in 1.5% agarose gels. The bands
were scanned by UV Scanner. The gray ratio between the centrin genes and GAPDH gene bands
density was used as quantitative evaluation.
1.5 Cloning and sequencing of PCR products
PCR products were purified with QiAquick PCR purification kit (Qiagen), and cloned into
pGEM-T easy vector (promega). Amplification of the recombinant plasmid was carried out in
E. coli BL21. The sequencing was done by Sai Bai Sheng Bio-Company (Beijing).
1.6
Sequence analysis of the cDNA
Analysis of the deduced amino acid sequences and homology of centrin cDNA was carried
out using Omigo 2.0 Software.
2
Results
2.1 Amplification of cDNA fragments of rat centrin genes
Using total RNA from adult rat testis and primers, we amplified out cDNA fragments of 410
bp for centrin-1, 410 bp for centrin-2 and 510 bp for centrin-3 (fig. 1), and 576 bp for housekeeping gene GAPDH respectively.
The PCR products were subcloned into the T-easy vector and sequenced. 366 bp of centrin-1,
366 bp of centrin-2 and 482 bp of centrin-3 cDNA sequences data were obtained after deducting
the primers sequence. Being searched in GenBank, it was confirmed that the sequence of rat
centrin genes was not reported. We subscribed
them to the GenBank. The accession number was
AF334107 for centrin-1, AF334108 centrin-2
and AF335277 for centrin-3. We presumed that
the fragments covered about 71%, 71% and 96%
of open reading frame, according to the sequence
of full length centrin genes in other species. The
homologous analysis showed that the three centrin genes in rat are highly conserved in evolutionary history (data not shown).
Fig. 1.
Amplification of centrin cDNA fragment by
RT-PCR. 1, pBR322/Hinf I DNA marker; 2, GAPDH; 3,
centrin-1; 4, centrin-2; 5, centrin-3.
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Homologous comparison of deduced amino acid sequences
Using the Bio-software Omiga2.0, it was confirmed that the cDNA fragments of rat centrin-1,
centrin-2 and centrin-3 encoded 122, 122 and 159 amino acid respectively. Homologous comparison of the amino acid sequences with human centrin-1, -3, and mouse centrin-1 revealed the high
homology between them, meaning that they are very conserved in evolutionary history (fig. 2). In
addition, the homology of centrin-3 between rat and human was 99%, but the homology among rat
centrin-1, centrin-2 and centrin-3 was relatively low.
Human Centrin-1MASGFKKPSA ASTGQKRKVA PKPELTEDQK QEVREAFDLF
M o use Centrin-1---T-R-SNV ---SY----G ---------- ---------Rat
Centrin-1
Rat
Centrin-2
Rat
Centrin-3
LVVD-T-RK KRR-—S-E-K --IKD--E--
Human Centrin-3
MSLALRS ELLVD-T-RK KRR--S-E-K --IKD--E--
Human Centrin-1DVDGSGTIDA KELKVAMRAL GFEPRKEEMK KMISEVDREG
M o use Centrin-1-S-------V ---------- ---------- -------K-A
Rat
Centrin-1 S-------V ---------- ---------- -------K-A
Rat
Centrin-2 A--T----M ---------- ----K---I- -----I-K-G
Rat
Centrin-3-T-KDQA--Y H--------- --DVK-ADVL –ILKDY---A
Human Centrin-3-T-KDEA--Y H--------- --DVK-ADVL –ILKDY---A
Human Centrin-1TGKISFNDFL AVMTQKMSEK DTKEEILKAF RLFDDDETGK
M o use Centrin-1---------- -------A-- ---------- ---------Rat
Centrin-1---------- -------A-- ---------- ----------
Rat
Centrin-2---MN-S--- T--------- ---------- K---------
Rat
Centrin-3----T-E--N E-V-DWIL-R –PH------- K-----DS--
Human Centrin-3----T-E--N E-V-DWIL-R –PH------- K-----DS--
Human Centrin-1ISFKNLKRVA NELGENLTDE ELQEMIDEAD RDGDGEVNEE
M o use Centrin-1---------- -----S---- ---------- ---------(To be continued on the next page)
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(Continued)
Rat
Centrin-1---------- -----S---- ---------- ---------
Rat
Centrin-2---------- K--------- ---------- ---------Q
Rat
Centrin-3--LR--R--- R-----MS-- --RA--E-F- K-------Q-
Human Centrin-3--LR--R--- R-----MS-- --RA--E-F- K-----I-Q-
Human Centrin-1EFLRIMKKTS LY
(172aa)
M o use Centrin-1---K-----N --
(172aa, 91%)
Rat
Centrin-1---
(122aa, 95%)
Rat
Centrin-2---
(122aa, 88%)
Rat
Centrin-3--IA--TGDI
(159aa, 56%)
Human Centrin-3--IA--TGDI
(167aa, 53%)
Fig. 2. Homologous comparison of centrin proteins among human, mouse and rat. The numbers listed at the last
column represent amino acids numbers and homologous ratio compared to human centrin 1. The amino acids in
gray are different amino acids between rat and human centrins.
2.3 Expression of centrin genes in rat tissues
The expression pattern of rat centrin genes was tested
by RT-PCR in testis, kidney, spleen, brain, heart and ovary.
The results showed that centrin-1 was exclusively expressed in testis, very low in brain, and centrin-2 and centrin-3 were expressed in all the tissues we examined (fig.
3). A 576 bp fragment of GAPDH was amplified as control.
Fig. 3. Tissue-specific expression of rat centrin genes. 1, Testis; 2, kidney; 3, spleen; 4,
brain; 5, heart; 6, ovary.
2.4 Expression of centrin genes in somatic cells of rat
testis
Leydig cells and Sertoli cells are somatic cells in rat testis. By RT-PCR, we did not detect
mRNA of centrin-1 gene in the isolated Leydig cells and
Sertoli cells from the testis of 12-day-old rats. By contrast,
the low level expression of centrin-2 and centrin-3 genes
was detected in both Sertoli and Leydig cells (fig. 4).
2.5
Fig. 4. Expression of rat centrin genes in Sertoli
and Leydig cells. 1, DNA markers; 25, Leydig
cell; 69, Sertoli cell. 2 and 6, GAPDH; 3 and 7,
centrin-1; 4 and 8, centrin-2; 5 and 9, centrin-3.
Expression of centrin genes in spermatogenic cells
In order to study the expression characterization of
centrin genes in spermatogenic cells in testis, we isolated
the spermatogenic cells of different developmental stages
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from testis of different days old rats. The purity of each fraction cell is over 90% according to
morphological characterization after being stained with HE. The result of semi-quantitative
RT-PCR is shown in fig. 5. Centrin-1 was not expressed in spermatogonia until the spermatogenic
cells entered into meiosis and developed to spermatocytes. The mRNA of centrin-1 reached its
peak level in round spermatids, and decreased in elongated spermatids. Centrin-2 and centrin-3
were highly expressed in spermatogonia and their mRNA level decreased markedly in spermatocytes. The mRNA of centrin-2 could not be detected in round spermatids.
Fig. 5. Expression of centrin genes in different spermatogenic cells. 1, Spermatogonia; 2, spermatocyte; 3, round
spermatid; 4, enlongated spermatid.
To further verify the un-expressed characterization of centrin-1 in spermatogenic cells at the
early stage, we isolated the total RNA from the testis of different days old Wistar rats. Between
day 9 and day 12 post natal, the major types of cells in the testis are Sertoli progenitor and diploid
spermatogonia. The spermatogonia start meiosis at day 15 after birth. The round spermatids appear on day 25 or 26 and the spermatogenic epithelium contains the different stage of spermatogenic cells. The results of semi-quantitative RTPCR showed that the expression of centrin-1 was
not detected until day 15 post natal, and then the
mRNA was increased and reached a stable level
after 26 days post natal (fig. 6). This phenomenon
Fig. 6. Expression of centrin-1 in different days old rat should be caused by the augmentation of round
testis.
spermatid numbers.
3
Discussion
Centrin is a calcium-binding protein in centrosome and has been identified in diverse organisms. In order to study the expression and function of centrin genes during rat spermatogenesis,
we cloned cDNA fragments of three centrin genes from rat testis. The size of the cDNA fragments
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covered about 71%, 71% and 96% of the open reading frames, and the deduced amino acid sequences covered all four calcium-binding domains[10]. The homologous comparison of the amino
acid sequences showed that the homology among corresponding centrins in different organisms
was high, suggesting that centrin had evolved to a multi-gene family before the mammalian, while
the homology between centrin-1 and centrin-2 was higher than that between them and centrin-3.
So centrin genes could be classified into two subfamilies, centrin-1 and centrin-2 genes belong to
the same subfamily which are highly conserved to green alga centrin gene[11], Centrin-3 and yeast
cdc 31 genes belong to other subfamilies[12]. The homology of the amino acid sequences represents the similarity of function. It was reported recently[7] that human centrin-3 gene is similar to
yeast cdc31 gene, which is involved in the replication of centrosome.
By RT-PCR, we demonstrated that the expression of centrin-1 gene was limited exclusively
to testis and spermatogenic cells. The mRNA of centrin 1 was not detected in Leydig and Sertoli
cells of testis as well as other tissues such as kidney, spleen. Both human and mouse centrin-1
genes were cloned from testis cDNA library[13,14]. The results suggest that centrin-1 may play specific roles during spermatogenesis. Then we studied the expression characterization of centrin-1 at
different developmental stages of spermatogenic cell by RT-PCR. The results showed that centrin-1 mRNA started to transcribe from early spermatocytes, and the mRNA level reached peak in
round spermatids. The matched results were obtained when we analyzed RNA isolated from different days old rat testis by RT-PCR. Similar results on mice were reported by Hart et al.[14], i.e.
centrin-1 started to transcribe in 14-day-old mouse testis, and expression level increased at the
following stages, while centrin-2 mRNA decreased in this process. After germ cells developed into
spermatocytes, centrosome was mainly involved in meiosis and organization of flagella formation.
Therefore, we suggest that centrin-1 may closely relate to special roles of centrosome in this period, such as involving in the formation of spindle during meiosis and assembly of flagellum fibre
during spermiogenesis. It has been reported [15] that Marsilea centrin, which is highly homologous
to centrin-1, is a necessary component of centriole for the formation of flagella. Meanwhile,
semi-quantitative RT-PCR analysis indicated that centrin-2 and centrin-3 were expressed adequately in spermatogonia and the mRNA level markedly decreased after entering into meiosis,
suggesting that centrin-2,-3 are related to mitosis of spermatogonia.
Same centrin proteins may play different roles in different types of cells at different developmental stages[16]. The studies on centrin structure and making of specific antibodies against different types of centrin will be useful to further studying localization and function of centrin family.
The studies on the expression pattern of centrin family in rat spermatogenesis will be important
for further understanding the function of centrin genes and molecular mechanisms of spermatogenesis and spermiogenesis.
Acknowledgements This work was supported by the Special Funds for Major State Basic Research Project (Grant No.
G1999055901).
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