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Three novel spermatogenesis-specific zinc finger genes

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We have cloned and characterized the expression, during spermatogenesis, of three novel zinc finger genes (Zfp94, Zfp95, Zfp96). Analysis of the deduced protein sequences reveals that all three molecules belong to the LeR family (leucine-rich zinc fingers) and that ZFP95 contains a domain homologous to the Krüppel-associated box. All three genes were found expressed at high levels in testis among other tissues, but testis-specific transcripts were observed for Zfp95 and Zfp96. Northern blot analyses of the testis-specific transcripts of Zfp95 and Zfp96 were performed using whole testis RNA as well as RNA isolated from enriched populations of specific spermatogenic cell types. The testis-specific transcript of Zfp95 showed the highest expression in pachytene spermatocytes, while that of Zfp96 was highly expressed in pachytene spermatocytes, in round spermatids and residual bodies. Northern blot analysis of RNA from the testis of mice carrying the atrichosis mutation further validated these expression patterns. In particular, the testis-specific transcripts of Zfp95 and Zfp96 were greatly reduced in heterozygous, and completely absent in homozygous testis RNA from atrichosis mutant mice, further defining the germ cell specificity of these transcripts.
The complete cDNA sequences and derived amino acid sequences (shown in one letter code) for (A) Zfp94 , (B) Zfp95 and (C) Zfp96 . The following features are highlighted for Zfp94 : bp 349^534 (aa 57^118) the LeR domain in bold face; the locations of the zinc ¢nger domains are shown by underlined and bold Cys residues at the positions bp 1162 (aa 328), 1246 (356), 1330 (384), 1414 (412), 1495 (439), 1579 (467) and 1663 (495); the region with similarity to the B1/Alu sequence (bp 1797^1855) is underlined and the putative poly-adenylation signal at position 1906 is shown in bold face. The following features are highlighted for Zfp95 : bp 217^438 (aa 57^118) the LeR domain in bold face; a region with homology to KRAB-A zinc ¢nger domains (bp 718^804) is underlined and the locations of the zinc ¢nger domains are shown by underlined and bold Cys residues at the positions bp 1087 (aa 343), 1171 (371), 1255 (399), 1339 (427), 1684 (542), 1771 (570), 1852 (598), 1936 (626), 2020 (654), 2188 (710), 2356 (766) and 2440 (794). The following features are highlighted for Zfp96 : the poly(dG) stretch (bp 45^69) introduced by the 5 P -RACE method is underlined; the LeR domain (bp 607^798, aa 59^122) is shown in bold face; the locations of the zinc ¢nger domains are shown by underlined and bold Cys residues at the positions bp 1243 (aa 271), 1327 (299), 1411 (327), 1495 (355), 1579 (383), 1663 (411) and 1801 (457); the putative poly-adenylation signal is shown in bold at position 2233.
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Three novel spermatogenesis-speci¢c zinc ¢nger genes
Helge Weissiga, Sonoko Narisawaa, Carin Sikstro
«mb, Per G. Olssonc, John R. McCarreyd,
Panagiotis A. Tsonise, Katia Del Rio-Tsonisf;, Jose
¤Luis Milla
¤na;c
aThe Burnham Institute, La Jolla, CA 92037, USA
bDepartment of Biomedical Laboratory Science, Umea
University, S-901 85 Umea
, Sweden
cDepartment of Medical Genetics, Umea
University, S-901 85 Umea
, Sweden
dDepartment of Biology, University of Texas at San Antonio, San Antonio, TX 78245, USA
eDepartment of Biology, University of Dayton, Dayton, OH 45469, USA
fDepartment of Zoology, Miami University, Oxford, OH 45056, USA
Received 22 May 2003; accepted 23 May 2003
First published online 17 June 2003
Edited by Jesus Avila
Abstract We have cloned and characterized the expression,
during spermatogenesis, of three novel zinc ¢nger genes
(Zfp94,Zfp95,Zfp96). Analysis of the deduced protein sequen-
ces reveals that all three molecules belong to the LeR family
(leucine-rich zinc ¢ngers) and that ZFP95 contains a domain
homologous to the Kru
«ppel-associated box. All three genes were
found expressed at high levels in testis among other tissues, but
testis-speci¢c transcripts were observed for Zfp95 and Zfp96.
Northern blot analyses of the testis-speci¢c transcripts of Zfp95
and Zfp96 were performed using whole testis RNA as well as
RNA isolated from enriched populations of speci¢c spermato-
genic cell types. The testis-speci¢c transcript of Zfp95 showed
the highest expression in pachytene spermatocytes, while that of
Zfp96 was highly expressed in pachytene spermatocytes, in
round spermatids and residual bodies. Northern blot analysis
of RNA from the testis of mice carrying the atrichosis mutation
further validated these expression patterns. In particular, the
testis-speci¢c transcripts of Zfp95 and Zfp96 were greatly re-
duced in heterozygous, and completely absent in homozygous
testis RNA from atrichosis mutant mice, further de¢ning the
germ cell speci¢city of these transcripts.
 2003 Federation of European Biochemical Societies. Pub-
lished by Elsevier Science B.V. All rights reserved.
Key words: Transcription factor; Germ cell ;
Testis-speci¢c gene expression
1. Introduction
The testis can be morphologically subdivided into two com-
partments, the seminiferous tubules and the interstitial space.
Germ cells reside within the tubules where they progress
through several well-de¢ned stages of development in close
contact with Sertoli cells [1]. The process of spermatogenesis
is generally divided into three di¡erent phases : (a) a prolifer-
ative phase characterized by spermatogonia undergoing rapid
mitotic divisions, (b) a meiotic phase in which spermatocytes
recombine and segregate the genetic material, and (c) the dif-
ferentiation or spermiogenic phase in which spermatids trans-
form into sperm. The constant and asynchronous nature of
spermatogenesis involves a series of cell^cell interactions be-
tween the di¡erent somatic cell types in the testis and the germ
cells. These interactions are often cyclical and can be catego-
rized into stages as the germ cells progress through the mor-
phologically well de¢ned steps of development [1,2]. Each
stage (there are 14 in the rat and 12 in the mouse) is charac-
terized by a unique complement of germ cell types at various
stages of development. With the passage of time, any given
stage will progress to the next stage as the germ cell comple-
ment matures [3].
The crucial interaction of germ cells with the testicular so-
matic cells is exempli¢ed by the di⁄culty of maintaining germ
cells in culture for prolonged periods of time [4]. Hofmann
and colleagues have established several murine testicular cell
lines, including the spermatogonial-like cell line GC-1spg, by
transformation with the SV40 large T antigen [5,6]. The im-
mortalized cell lines GC-2spd(ts) and GC-3spc(ts) were pro-
duced by co-transfection of the gene encoding SV40 large T
antigen and a temperature-sensitive (ts) mutant of p53. The
binding of the active form of p53 at lower (i.e. permissive)
cultivation temperatures induces the cells to di¡erentiate
along the spermatogenic pathway [6]. These cell lines seemed
to represent a particularly useful system to identify molecules
with di¡erential expression pattern during spermatogenesis.
2. Materials and methods
2.1. Di¡erential display-reverse transcriptase polymerase chain reaction
(DDRT-PCR)
RNA was isolated from GC-2spd(ts) cells and a cDNA library was
constructed using Vgt11 arms (Stratagene) and packaged using the
Giga Pack Gold kit (Stratagene) according to the manufacturer’s in-
structions. The described 3Pprimers T12MA, T12MC, T12MG and
T12MT [7] were used in the RT as well as in the following PCR
ampli¢cation step. The following arbitrary 5Pprimers were employed
in the PCR reactions (all sequences 5P-3P): ARB1: GCG GAC ACA
C; ARB2 : CCA CCT TCG A; ARB3 : GAG AAG ATC T; ARB4 :
GGT CAG AAG A; ARB5: AAG TCT TGG G; ARB6: TAC AAC
GAG G; ARB7 : TGG ATT GGT C ; ARB8 : CTT TCT ACC C;
ARB9: TTT TGG CTC C ; ARB10 : GGA ACC AAT C. Primers
ARB1^ARB5 were chosen arbitrarily, primers ARB6^ARB10 were
derived from the sequences suggested by Bauer et al. [8]. Puri¢ed total
RNA (1^0.1 Wg) was reversed transcribed and subsequently ampli¢ed
as described [7]. A programmable heat block (MJ Research, Water-
town, MA, USA) was used with these parameters: denaturing at
96‡C, 1 s, annealing at 42‡C, 1 s, elongation at 72‡C, 1 s for 40 cycles
with an additional elongation step for 5 min at 72‡C. PCR products
were separated on a 6% denaturing polyacrylamide sequencing gel.
Evaluation of di¡erentially expressed fragments was done after over-
night autoradiography of the dried gels. These cDNA fragments were
0014-5793 / 03/ $22.00  2003 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.
doi: 10.1016/S0014-5793(03)00669-0
*Corresponding author. Fax: (1)-513-529 6900.
E-mail address: delriok@muohio.edu (K. Del Rio-Tsonis).
FEBS 27420 3-7-03
FEBS 27420 FEBS Letters 547 (2003) 61^68
excised from the dry gels and incubated at 98‡C for 10 min in 100 Wl
TE bu¡er. Five microliter of the eluate was used in the subsequent
reampli¢cation of the fragments under the following conditions: 20
mM Tris^HCl pH 8.4, 50 mM KCl, 1.4 mM MgCl2, 0.1 mM of each
nucleotide, 2 WM of each primer and 3.5 U Taq polymerase (Boehr-
inger Mannheim). The cycling parameters of the PCR reaction were
as for the di¡erential display with a higher annealing temperature of
55‡C after one cycle of annealing at 42‡C.
2.2. Animals
Mice of the strains BALB/c and C57BL/6J were obtained from the
in-house animal facility. Additionally, mice homozygous and hetero-
Fig. 1. The complete cDNA sequences and derived amino acid sequences (shown in one letter code) for (A) Zfp94, (B) Zfp95 and (C) Zfp96.
The following features are highlighted for Zfp94 : bp 349^534 (aa 57^118) the LeR domain in bold face ; the locations of the zinc ¢nger do-
mains are shown by underlined and bold Cys residues at the positions bp 1162 (aa 328), 1246 (356), 1330 (384), 1414 (412), 1495 (439), 1579
(467) and 1663 (495); the region with similarity to the B1/Alu sequence (bp 1797^1855) is underlined and the putative poly-adenylation signal
at position 1906 is shown in bold face. The following features are highlighted for Zfp95: bp 217^438 (aa 57^118) the LeR domain in bold
face; a region with homology to KRAB-A zinc ¢nger domains (bp 718^804) is underlined and the locations of the zinc ¢nger domains are
shown by underlined and bold Cys residues at the positions bp 1087 (aa 343), 1171 (371), 1255 (399), 1339 (427), 1684 (542), 1771 (570), 1852
(598), 1936 (626), 2020 (654), 2188 (710), 2356 (766) and 2440 (794). The following features are highlighted for Zfp96: the poly(dG) stretch (bp
45^69) introduced by the 5P-RACE method is underlined; the LeR domain (bp 607^798, aa 59^122) is shown in bold face ; the locations of the
zinc ¢nger domains are shown by underlined and bold Cys residues at the positions bp 1243 (aa 271), 1327 (299), 1411 (327), 1495 (355), 1579
(383), 1663 (411) and 1801 (457); the putative poly-adenylation signal is shown in bold at position 2233.
FEBS 27420 3-7-03
H. Weissig et al./FEBS Letters 547 (2003) 61^6862
Fig. 1 (Continued).
FEBS 27420 3-7-03
H. Weissig et al./FEBS Letters 547 (2003) 61^68 63
zygous for the mutation atrichosis (at)[9] were purchased from the
Jackson Laboratories (Bar Harbor, ME, USA).
2.3. Germ cell fractionation
Highly enriched populations of speci¢c spermatogenic cell types
were prepared by unit gravity sedimentation through a 2^4% bovine
serum albumin (StaPut) gradient as described [10]. Populations of
primitive type A spermatogonia (purity v85%) and somatic Sertoli
cells (purity v85%) were recovered from testes of male CD-1 mice at
6 days post partum (dpp). A combined population of type A and type
B spermatogonia (purity v85%) was recovered from CD-1 mouse
testes at 8 dpp. Separate populations of pachytene spermatocytes,
round spermatids, and residual cytoplasmic bodies (purity of each
v95%) were recovered from testes of adult ( s60 dpp) male CD-1
mice. Purities of each cell population were determined on the basis of
cellular morphology examined under phase contrast optics.
2.4. Expression studies
Expression was studied by Northern analysis of tissues and isolated
cell populations. Also, expression in the mouse testis was examined
via in situ hybridization as described [11].
3. Results and discussion
We used DDRT-PCR to identify and clone cDNA ex-
Fig. 1 (Continued).
FEBS 27420 3-7-03
H. Weissig et al./FEBS Letters 547 (2003) 61^6864
pressed di¡erentially in the spermatogenic cell lines under
permissive and non-permissive conditions. We extracted
RNAs from the GC-1spg cell line at 37‡C and from GC-
2spd(ts) after cultivation at 32‡C and 37‡C [6]. Out of approx-
imately 100 di¡erentially displayed PCR bands, 30 fragments
were sequenced and the information used to search the non-
redundant part of the GenBank database with the BLAST
algorithm [12]. The fragment designated 2A1.32 was isolated
as a di¡erentially displayed band present in GC-2spd(ts) cells
grown at 32‡C but not at 37‡C or in GC-1spg. This fragment
was found to be homologous to many C2H2 Kru
«ppel-like zinc
¢nger proteins [13]. Remarkably, the four most highly homol-
ogous murine zinc ¢nger proteins available in GenBank are
expressed di¡erentially in testis and thus this fragment was
chosen for further study. Several rounds of screening of a
GC-2spd(ts) Vgt11 cDNA library with the fragment 2A1.32
resulted in the isolation of three homologous, but distinct,
cDNAs, Zfp94 Zfp95 and Zfp96 (accession numbers: MMU
62906, 62907, 62908 respectively, see Fig. 1).
3.1. The cDNA and protein sequence of Zfp94, Zfp95 and
Zfp96
The 1997 bp length of the Zfp94 cDNA is in good corre-
lation with the approximately 2.2 kb of the Zfp94 mRNA
considering that the average length of oligo(dA) tails is about
200^250 bp (Fig. 1A)[14]. The longest open reading frame,
encoding 520 amino acids, extends from bp 181 to bp 1740.
Zfp95 is the longest, 3175 bp, of the three novel cDNAs (Fig.
1B). The longest open reading frame was found to extend from
bp 61 to bp 2520, encoding 819 amino acids. The cDNA for
Fig. 2. A: Schematic representation of the structural features of Zfp94,Zfp95 and Zfp96. The following domains are represented in the ¢gure :
a zinc ¢nger consensus sequence, a LeR domain and a KRAB-A domain. B : Alignment of the zinc ¢nger domains of Zfp94,Zfp95 and Zfp96.
Areas with at least 80% similarity are boxed and shaded. The line spacing indicates the cluster arrangement of the domains in the respective
proteins. Residues matching the Kru
«ppel consensus sequence [21] given below the alignments are shown in bold type. Residues with a frequency
of more than 60% are shown in capital letters in the ¢rst row, those with a frequency of 30% or more are shown in lower case. The second
and all subsequent rows list residues with frequencies above 10% in order of their respective frequencies. C: The consensus sequence for
KRAB-A domains is indicated with capital letters corresponding to high conservation and lower case letters in the ¢rst row corresponding to
moderate conservation in the alignment. The lower case letters in the second and third rows show the less conserved residues with the more fre-
quent amino acids in the second row. The corresponding region of Zfp95 is colored accordingly. Those residues that di¡er are identi¢ed by ar-
rowheads.
FEBS 27420 3-7-03
H. Weissig et al./FEBS Letters 547 (2003) 61^68 65
Zfp96 has a total length of 2307 bp. An open reading frame of
1503 bp (bp 433^1936) was identi¢ed and encodes 501 amino
acids (Fig. 1C). Mouse expressed sequence tag (EST) studies
have recently determined the chromosomal location for Zfp95
and Zfp96. As part of the Washington University’s Mouse
EST project, Marra and coworkers located Zfp95 on chromo-
some 5 of the mouse at 5.5 cM. Zfp96 was located on chro-
mosome 13 with some ambiguity about the exact location
around 61.6 cM [15].Fig. 2A presents an overview of the
protein domains identi¢ed in ZFP94, ZFP95 and ZFP96.
All three molecules belong to the family of leucine-rich zinc
¢nger proteins. The approximately 80 amino acids long leu-
cine-rich domain (LeR) is present in a small subfamily of zinc
¢nger proteins [16^20]. Seven zinc ¢nger domains are found in
Fig. 3. Expression of Zfp94,Zfp95 and Zfp96. A: Northern blot analysis of male tissues using 10 Wg of total RNA per lane. B : In situ hybrid-
ization of Zfp95 and Zfp96 with sections of adult testis. Negative control using the Zfp96 sense probe, the background for the others was
equivalent to the one represented here. C : Expression of Zfp95 and Zfp96 in mouse testis and isolated spermatogenic cells using 10 Wgof
RNA per lane. Abbreviations: 6d and 8d spg, spermatogonia isolated from mice 6 and 8 days after birth respectively; pach, pachytene sperma-
tocytes; rd. spt., round spermatids; res. bds., residual bodies; 6d Ser and Ser, Sertoli cells isolated 6 days after birth or from adult mice respec-
tively. The mRNA of the ribosomal protein L32-4A was used as control.
FEBS 27420 3-7-03
H. Weissig et al./FEBS Letters 547 (2003) 61^6866
a contiguous cluster in ZFP94, while the 12 domains of
ZFP95 are dispersed into two groups of four and ¢ve domains
each, followed by a single domain and another cluster of two
domains at the immediate C-terminus. The seven domains of
ZFP96 are also contiguous with the exception of the last
domain. Fig. 2B shows the alignments of the zinc ¢nger do-
mains of ZFP94, ZFP95 and ZFP96 compared with the con-
sensus sequence of the zinc ¢nger domains in the Drosophila
gene Kru
«ppel [21] and a consensus sequence calculated from
1802 di¡erent vertebrate zinc ¢nger domains extracted from a
total of 385 genes in GenBank. For this latter comparison,
all sequences complying with a ‘limited’ C2H2 consensus
(CX2CX3[FYLV]X5[LF]- X2HX3HX7, where X could be
any amino acid and positions with multiple possibilities are
shown in square brackets) were compiled from the vertebrate
sequences of GenBank. Approximately one-third of all zinc
¢nger proteins contain an evolutionarily conserved region of
about 75 amino acids at their N-terminus, the Kru
«ppel-asso-
ciated box (KRAB). This region is split into two parts,
KRAB-A and KRAB-B, both of which are found separate
in di¡erent proteins, or closely associated together in the
same protein [22].Fig. 2C shows an alignment of the
KRAB-A domain consensus sequence in comparison with
amino acids 213^263 of Zfp95. Thirty-¢ve percent (15 out
of 43) of the amino acids in this region are identical with
the conserved amino acids of the KRAB-A consensus se-
quence. Moreover, nine out of 13 highly conserved residues
and ¢ve out of 12 moderately conserved residues are found in
Zfp95. In contrast to the majority of KRAB domain contain-
ing proteins, the domain in Zfp95 is not at the immediate
N-terminus of the molecule.
3.2. Expression pattern of Zfp94,Zfp95 and Zfp96
The expression of Zfp94,Zfp95 and Zfp96 was analyzed in
tissues of male mice. All three genes are expressed at higher
levels in testis than in any of the other tissues investigated
(Fig. 3A). Expression is detectable for Zfp94,Zfp95 and
Zfp96 in brain, heart, kidney, liver and tongue of male
mice. Zfp94 and Zfp95 are also expressed in the lung, while
Zfp95 and Zfp96 are expressed in striated muscle as well.
Zfp94 shows one transcript in the tissues mentioned as well
as high expression in the testis. On the other hand, Zfp95 and
Zfp96 show two transcripts. The 2.4 kb mRNA for Zfp95 and
Zfp96, detectable in testis RNA, was not present in any of the
other tissues.
Since Zfp95 and Zfp96 show testis-speci¢c transcripts, we
decided to further analyze their expression patterns. In situ
hybridization con¢rmed the expression of these genes in the
testis (Fig. 3B). In addition, four postnatal time points, i.e. 7,
14 and 21 days old as well as adult mice, were chosen for
more detailed expression analyses. The population of germ cells
in the 7 day postnatal testis consists largely of type A and B
spermatogonia. At 2 weeks, pachytene spermatocytes are also
detectable while round spermatids become visible around
3 weeks after birth. In contrast, the somatic cell types are present
throughout postnatal development [23].Zfp95 is expressed
throughout (Fig. 3C, left panel). The expression of the 3.5
kb Zfp95 mRNA peaks at day 21 and subsequently declines
while the 2.4 kb testis-speci¢c transcript has its highest ex-
pression in adult testis. The 2.4 kb testis-speci¢c transcript
of Zfp96 mRNA becomes distinctly visible in RNA samples
extracted from 3 week old mice and increases until adulthood.
The mRNA levels of Zfp95 and Zfp96 in the isolated germ
cell populations are in good correlation with the results from
whole testis (Fig. 3C, right panel). The expression of the testis-
speci¢c Zfp95 transcript is highest in pachytene spermato-
cytes, but it is also present in round spermatids. The strongest
expression of the testis-speci¢c Zfp96 transcript is in residual
bodies (taking into account the loading di¡erence in this lane),
suggesting that this gene is expressed in late stage spermatids.
To further substantiate the conclusions drawn from the ex-
pression pattern in whole testis at di¡erent stages of postnatal
development and in isolated cell fractions, experiments were
conducted employing RNA isolated from reproductive tissues
(testis, epididymis, seminal vesicle and the prostate) of at mu-
tant mice. The recessive at mutation renders a¡ected mice
almost hairless and sterile as their gonads are greatly reduced
in size and contain few germ cells [9]. Expression of the testis-
speci¢c Zfp95 transcript in at mutants is not detectable com-
pared to the levels of expression in heterozygous littermates
(Fig. 4). In addition, this 2.4 kb mRNA is not detectable in
epididymis, seminal vesicle or prostate of mutant or heterozy-
gous mice. Thus, Zfp95 is expressed both in germ cells and in
other as yet unde¢ned cell types of the testis but shows di¡er-
ential splicing only in meiotic and post-meiotic germ cells. The
expression of Zfp96 in mice heterozygous for the at mutation
is greatly reduced and no expression is detectable in their
homozygous littermates, con¢rming the germ cell speci¢city
of this expression.
Our expression data in wild type mice as well as in the at
mutant clearly indicate a crucial role of these proteins in
spermatogenesis and speci¢cally in germ cell di¡erentiation
and maturation. It is clear that Zfp95 is di¡erentially spliced
in meiotic and post-meiotic germ cells and this expression
disappears in at mutant mice that lack germ cells. Zfp96
shows only one transcript that also disappears in at mutants
and seems to be speci¢c in later stages of germ cell di¡er-
entiation. The identi¢cation of novel zinc ¢nger genes with
expression enhanced in the testis and displaying splice variants
that are germ cell-speci¢c will facilitate studies of the molec-
Fig. 4. Expression of Zfp95 and Zfp96 in RNA in reproductive tis-
sues of mice carrying the at mutation. Total RNA from testis of
control BALB/c, at heterozygous and homozygous littermates of the
strain ATEB/Le as well as RNA from reproductive tissues of het-
ero- and homozygous mice was analyzed. The blots were hybridized
consecutively to probes for Zfp95,Zfp96 and L32-4A as a control.
Autoradiography was carried out at 370‡C for up to 4 weeks.
FEBS 27420 3-7-03
H. Weissig et al./FEBS Letters 547 (2003) 61^68 67
ular mechanisms that govern germ cell-speci¢c gene expres-
sion and spermatogenesis.
Acknowledgements: This work was supported in part by Grants CA
42595 and HD 05863 from the National Institutes of Health, USA,
and a grant from the Swedish Cancer Foundation. H.W. was sup-
ported by a graduate student stipend from the Deutsche Akademische
Austauschdienst. The support of Dr. Rolf Kemler is gratefully ac-
knowledged.
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FEBS 27420 3-7-03
H. Weissig et al./FEBS Letters 547 (2003) 61^6868
... This gene encodes a zinc finger protein of the Krűppel family. ZKSCAN5 has three distinct domains, including twelve conserved zinc fingers at the C-terminal region, a SCAN box, and a KRAB (Krűppel-associated box) A domain at the N-terminal region (Dreyer et al., 1999;Weissig et al., 2003). ZKSCAN5 is ubiquitously expressed in human adult and fetal tissues, but the gene is most strongly expressed in the testis. ...
... In addition, three spliced transcript forms have been detected by northern blot analysis. Among them, the smallest transcript form is testisspecific and is highly expressed in pachytene spermatocytes (Dreyer et al., 1999;Weissig et al., 2003). Two alternatively spliced transcript variants differing only in the 5′-untranslated region (UTR) have been identified in the human transcriptome. ...
... Two alternatively spliced transcript variants differing only in the 5′-untranslated region (UTR) have been identified in the human transcriptome. Although additional transcript variants have been found, their full-length sequences have not been determined (Dreyer et al., 1999;Weissig et al., 2003). ...
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ZKSCAN5 (also known as ZFP95) is a zinc-finger protein belonging to the Krűppel family. ZKSCAN5 contains a SCAN box and a KRAB A domain and is proposed to play a distinct role during spermatogenesis. In humans, alternatively spliced ZKSCAN5 transcripts with different 5'-untranslated regions (UTRs) have been identified. However, investigation of our Macaca UniGene Database revealed novel alternative ZKSCAN5 transcripts that arose due to an exon creation event. Therefore, in this study, we identified the full-length sequences of ZKSCAN5 and its alternative transcripts in Macaca spp. Additionally, we investigated different nonhuman primate sequences to elucidate the molecular mechanism underlying the exon creation event. We analyzed the evolutionary features of the ZKSCAN5 transcripts by reverse transcription polymerase chain reaction (RT-PCR) and genomic PCR, and by sequencing various nonhuman primate DNA and RNA samples. The exon-created transcript was only detected in the Macaca lineage (crab-eating monkey and rhesus monkey). Full-length sequence analysis by rapid amplification of cDNA ends (RACE) identified ten full-length transcripts and four functional isoforms of ZKSCAN5. Protein sequence analyses revealed the presence of two groups of isoforms that arose because of differences in start-codon usage. Together, our results demonstrate that there has been specific selection for a discrete set of ZKSCAN5 variants in the Macaca lineage. Furthermore, study of this locus (and perhaps others) in Macaca spp. might facilitate our understanding of the evolutionary pressures that have shaped the mechanism of exon creation in primates.
... Consistent with this, we were unable to detect Catsper1 in GC1-spg cells by Western blot and immunohistochemical analysis (not shown). However, GC-1spg cells provide a valid model for understanding gene expression profiles during spermatogenesis [31] and the complex interactions between gene promoters and testis specific factors including members of the ZFP family, factor-1a and TSPY [32][33][34][35]. Thus, it is possible that changes in the expression profile of different transcription factors during the mitotic stage of spermatogenesis may be silencing the expression of Catsper1 [36,37]. ...
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Catsper is a Ca2+permeable channel required for sperm hyperactivation. In spite of its central role in male fertility, the transcriptional mechanisms that regulate Catsper1 expression are ill defined. In this work, we describe the identification and characterization of important regulatory elements in the murine Catsper1 gene proximal promoter. Four transcription start sites and three functional Sox-binding sites were identified in the Catsper1 promoter. Interestingly, transcription factors Sox5 and Sox9 caused a significant increase in transactivation of the Catsper1 promoter in heterologous systems, and chromatin immunoprecipitation assays showed that both transcription factors interact with the Catsper1 promoter in vivo. These results provide new insights into the molecular mechanisms that control Catsper channel expression.
... The association of Zn and zinc fingers and mammalian male fertility has been known for quite some time, as protamine 2 (P2) is a zinc finger protein specific to this clade, and mammalian males with decreased protamine P2 levels are often infertile (de Yebra et al. 1998). P2 is not the only zinc finger protein found in mammalian testes: others include ZPF-95 and ZPF-96, which appear to be expressed at different times during spermatogenesis (Weissig et al., 2003), and BORIS, a male-specific protein associated with epigenetic reprogramming of sperm (Loukinov et al. 2002). Zinc fingers are also important components of the male steroid hormones androgens (reviewed in Freedman 1992) which are crucial for processes ranging from sexual differentiation (reviewed in Hughes 2001;Sharpe 2006), sexual maturation (Buzek and Sanborn 1988), and spermatogenesis (Chang et al. 2004). ...
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Spermatogenesis is one of the most complex biological processes undergone by any organism, making it susceptible to perturbations that result in male sterility. Research has demonstrated that mutant phenotypes can be obtained from the disruption of epigenetic modifications, which are commonly microRNA guided. Employing the Xenopus system, whereby homogametic interspecies males are always sterile, thus violating Haldane's Rule, we deep-sequenced testes-specific small-RNAs to identify microRNAs most frequently misexpressed between sterile hybrids and their fertile parental taxa. Using these data, we cross-referenced our expression information with previously published mouse (Mus musculus) data and identified a subset of seven microRNAs common to both (miR-338, miR-222, miR-18, miR-30, miR-10, miR-196, and miR-365). We propose that these microRNAs are likely critical for spermatogenesis in all tetrapods, having retained testicular expression across ~350 million years of evolution (Amphibian-Mammal split). Gene targets of six of these microRNAs are known, and all the six associate with zinc and zinc finger proteins (both previously found critical in male fertility), and three with Hox genes (some of which have also previously been deemed critical for testicular development and male fertility). Expression information for these targets revealed that all those associated with zinc have previously been found to express in mammalian testes. One Hox target has known mammalian testicular expression, two have close relatives with known mammalian testicular expression, and two more are associated with proteins known to have mammalian testicular expression. In addition, miR-222 has prior association with spermatogenesis, and miR-30 has been found to be abundantly expressed in both mouse and human testes.
Book
Recent developments of experimental techniques in cellular and molecular biology have made it possible to understand the molecular biology of male gametogenesis in greater detail. This book focuses on the description of specialized proteins, which are dominantly and/or specifically expressed in germ cells and localized in spermatozoa. There is an urgent need to classify proteins of spermatogenic cells with a view of their functions, and their applications in the regulation of fertility and in understanding infertility. The understanding of structural properties of male germ cell specific proteins can offer vulnerable points for targeted intervention in testis without generalized effects on stages of spermatogenesis. Besides targeted action in male germ cells, sperm specific proteins and polypeptides may also offer potential application in the development of a contraceptive vaccine. Researchers in the fields of biochemistry, cell biology, molecular biology, reproductive biology and proteomics will find this book of interest. "This book is far superior to any previous book on the subject. I recommend this book with great enthusiasm." © 2005 Springer Science+Business Media, Inc. All rights reserved.
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To study on the expression pattern of a novel gene, Biot2-L, in mice and its primary bio-function on the development of testis. Bioinformatics analysis were applied to identify the structure characteristics and homologene of Biot2-L. RT-PCR and Real-time RT-PCR were used to analyze its expression profile in normal tissues and the time-dependent expression pattern during the testes sexual maturation. The complete sequence of Biot2-L was obtained. A conserved domain-CCDC7 was detected and there were homolog proteins in advanced mammals with about 50% identity by bioinformatics analysis. The highly expression of Biot2-L in adult testis but low or absent expressed in other tissues was observed. A regular change of Biot2-L expression level during the testis development and spermatogonia was demonstrated by the results of Real-time RT-PCR. The mRNA transcript appeared from the fifth week of postnatal development, and the expression level increased up to adulthood, but decreased when get older. Biot2-L was highly expressed in adult testis and regular changed during the testis development, which suggested that the Biot2-L would be involved in the testis development and spermatogonia.
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Article
We have cloned a gene which is specifically expressed at the stage of sexual maturation in the rat testis by means of differential display, and have named it spermatogenesis-related factor-2 (SRF-2). Testicular expression was first detected at 5 weeks of age, and its level of the expression increased up to 7 weeks, and was maintained even at 63 weeks. Its cDNA was 2,789 bp in length and encoded an open reading frame of 718 amino acids. This gene was mainly expressed in the spermatocyte, judging from the result of in situ hybridization. The hypothetical gene product had a motif highly homologous with RabGAP/TBC protein. Taken together, this gene is considered to have some important functions for meiosis. The gene expression was significantly decreased by treatment with TCDD, a candidate endocrine disruptor, when administered to male rats of the nursling period. Body weight and testis weight were decreased by the treatment, but even then the sperm concentration in cauda epididymis was not changed significantly. SRF-2 gene may be a promising biomarker to construct a detection system of uncertain endocrine disruptors.
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cDNA microarray technology has been increasingly used to monitor global gene expression patterns in various tissues and cell types. However, applications to mammalian development have been hampered by the lack of appropriate cDNA collections, particularly for early developmental stages. To overcome this problem, a PCR-based cDNA library construction method was used to derive 52,374 expressed sequence tags from pre- and peri-implantation embryos, embryonic day (E) 12.5 female gonad/mesonephros, and newborn ovary. From these cDNA collections, a microarray representing 15,264 unique genes (78% novel and 22% known) was assembled. In initial applications, the divergence of placental and embryonic gene expression profiles was assessed. At stage E12.5 of development, based on triplicate experiments, 720 genes (6.5%) displayed statistically significant differences in expression between placenta and embryo. Among 289 more highly expressed in placenta, 61 placenta-specific genes encoded, for example, a novel prolactin-like protein. The number of genes highly expressed (and frequently specific) for placenta has thereby been increased 5-fold over the total previously reported, illustrating the potential of the microarrays for tissue-specific gene discovery and analysis of mammalian developmental programs.
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Spermatogenesis is the complex series of physiological and morphological changes that occur when spermatogonial stem cells differentiate into mature spermatozoa. Some of these changes are likely to be regulated at the level of transcription. To approach this problem, we have cloned a cDNA from mouse testis, encoding a protein (Zfp-29) with 14 copies of the zinc finger (Zf) motif commonly found in transcriptional regulatory proteins. The expression of this gene, Zfp-29, is restricted to the testis in adult mice, but also occurs during embryonic development. Within the testis, Zfp-29 mRNA is enriched in round spermatids, the earliest post-meiotic cells. Thus, the putative Zfp-29-encoded protein may have a role in regulating the class of genes that are expressed in post-meiotic germ cells.