Molecular Human Reproduction vol.6 no.11 pp. 983–991, 2000
Mouse staufen genes are expressed in germ cells during oogenesis
and spermatogenesis
P.T.K.Saunders1, S.Pathirana2, S.M.Maguire1, M.Doyle2, T.Wood2 and M.Bownes2,3
1Medical
Research Council, Human Reproductive Sciences Unit, Centre for Reproductive Biology, 37 Chalmers Street,
Edinburgh, EH3 9ET, and 2Institute of Cell and Molecular Biology, The University of Edinburgh, Darwin Building King’s
Buildings, Mayfield Road, Edinburgh, EH9 3JR, Scotland, UK
3To
whom correspondence should be addressed at: Institute of Cell and Molecular Biology, The University of Edinburgh,
Darwin Building King’s Buildings, Mayfield Road, Edinburgh, EH9 3JR, Scotland, UK. E-mail: [email protected]
The Drosophila melanogaster staufen gene encodes an RNA binding protein (Dm Stau) required for the
localization and translational repression of mRNAs within the Drosophila oocyte. In mammals translational
repression is important for normal spermatogenesis in males and storage of mRNAs in the oocytes of females.
In the present study we identified two mouse cDNA expressed sequence tags (ESTs), encoding proteins with
significant homology to Dm Stau and used these firstly to screen a mouse kidney cDNA library and secondly
to determine whether staufen mRNAs are expressed in the ovaries and testes of mice and rats. Sequence
analysis of the cDNAs revealed that they originated from two different genes. Using Northern blots of RNAs
from kidneys, ovaries and testes, both cDNAs hybridized to mRNA species of ~3 kb in all three tissues. On
sections of mouse ovaries, staufen mRNA was localized specifically to oocytes. On sections of mouse testes,
staufen mRNA was expressed in spermatocytes found in seminiferous tubules at stages VI–XII of the
spermatogenic cycle. In conclusion, we have shown that the mammalian homologues of Dm stau are
expressed in germ cells in both male and female mice, consistent with a role for these RNA binding proteins
in mammalian gametogenesis.
Key words: gametogenesis/ovary/RNA binding/staufen/testis
Introduction
In Drosophila, during oogenesis some mRNAs are translationally repressed and remain stored until fertilization, when
they are translated and their protein products become available
to direct the initial development of the embryo. The Drosophila
zygotic genome is not activated until the syncytial blastoderm
stage. In addition, specific localization of mRNAs during
oogenesis is essential for correct anterior–posterior axis formation of the embryo. The Drosophila staufen gene was identified
in a genetic screen (Schüpbach and Wieschaus, 1986) and the
Staufen protein (Stau) has been shown to contain five copies
of a double stranded RNA binding domain (St Johnston et al.,
1992). Binding studies undertaken with Stau protein have
demonstrated that it recognizes secondary structures within
the 3⬘ untranslated regions (UTR) of three mRNAs, i.e. bicoid
and oskar in the oocyte (St Johnston et al., 1992) and prospero
in the central nervous system (Broadus et al., 1998). Stau is
essential for the localization of osk mRNA to the posterior of
the oocyte (Kim-Ha et al., 1991) and bic mRNA to the anterior
of the oocyte (St Johnston et al., 1991; Ferrandon et al., 1994;
Hurst et al., 1999). In mutants, e.g. gurken, which alter the
polarity of the oocyte, Stau protein and osk mRNAs mislocalize
to the same ectopic sites (Gonzalez-Reyes et al., 1995). Recent
analysis of the RNA binding domains within Dm Stau has
revealed that when mutations encompassing domain 3 are
incorporated into a full-length stau transgene, this construct is
unable to localize osk or bic mRNAs correctly (Ramos et al.,
© European Society of Human Reproduction and Embryology
2000). Microtubules play an important role in localization of
mRNAs during oocyte development. Data from studies using
bacterially expressed fusion proteins prepared using cDNAs
encoding human or mouse Stau confirm that Stau cross-links
cytoskeletal and RNA components (Wickham et al., 1999).
Other authors (Micklem et al., 2000) have shown recently that
in Drosophila melanogaster Stau RNA binding domains 1, 3
and 4 bind double-stranded RNA in vitro whereas domains 2
and 5 do not. Furthermore, their results demonstrate that
domain 2 is required for microtubule-dependent localization
of osk mRNA, but domain 5 is involved in derepression of
osk mRNA once it is correctly localized.
The characteristics of Dm Stau including its specific mRNA
binding ability, its role in translational repression, and its role
in polarity determination via transcript localization, led us to
consider that the mammalian equivalent of Dm Stau could
play an important role in mammalian gametogenesis. In the
mouse, transcription of the embryonic genome is not initiated
until the 2-cell stage (Flach et al., 1982). In humans and pigs,
transcription begins at the 4-cell stage and in sheep and cattle
it does not initiate until the 8-cell stage (Braude et al.,
1988). Both development of the embryo immediately after
fertilization, and embryonic genome activation, are dependent
upon maternally-derived mRNA (Harper and Monk, 1983;
Latham et al., 1991; Wang and Latham, 1997).
We were also interested to see whether mammalian stau
was expressed in the testicular germ cells, as RNA binding
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proteins have been shown to play an important role in male
fertility (Elliott and Cooke, 1997). In the testis, during their
development within the seminiferous epithelium, male germ
cells undergo sequential mitoses, meiosis and structural remodelling. One important aspect of mammalian male germ cell
development is the conversion of a round stem cell into a
compact, motile cell; a process that involves nuclear condensation (Ward et al., 1983). The net result of these changes is to
convert a transcriptionally active nucleus into the compact,
quiescent nucleus of the spermatozoon. This process is tightly
controlled and requires the synthesis of DNA binding proteins
from mRNAs, the translation of which is controlled by specific
RNA binding proteins (Braun et al., 1989; Kwon and Hecht,
1991, 1993). One of these RNA binding proteins has been
shown to localize translationally repressed mRNA to microtubules (Han et al., 1995), a function analagous to that of that
performed by Stau in the Drosophila oocyte.
At the start of the study, database screening identified two
mouse expressed sequence tags (ESTs) which had significant
homology to Dm stau. The cDNA insert of one of these was
used to isolate cDNAs from a mouse 11 day embryo cDNA
library. To investigate the pattern of expression of mouse
staufen mRNA in mammalian gonads, Northern blots and
in-situ hybridization were undertaken. The results obtained
demonstrate that stau is expressed in somatic cells at low
levels and show for the first time that stau mRNAs are
expressed at high levels in both male and female germ cells
in the mouse.
Materials and methods
Preparation of M1 and M2 cDNAs
Individual cDNAs were amplified from the M1 and M2 plasmids
(Mouse Staufen ESTs, mouse stau and mouse stau 2 respectively,
Figure 1A) by polymerase chian reaction (PCR) with primers corresponding to the SK and T7 sites on the Bluescript vector (94°C 0.5
min, 48°C 0.5 min, 72°C 1.5 min, 30 cycles; Saiki et al., 1988).
Amplified products were separated on agarose gels (1% w/v) containing ethidium bromide, run in TAE (Tris-acetate) buffer according
to standard protocols (Davis et al., 1986) and the size of the cDNA
inserts determined by comparison to makers run in a parallel lane.
Selected PCR reactions (50 µl) were purified by spin column
chromatography on TE (Tris-EDTA) 100 columns (Clontech, Cambridge, UK) and radiolabelled with [32P]-labelled dCTP, by random
priming (Rediprime II kit; Pharmacia Amersham Biotech Ltd, St
Albans, UK).
Screening of cDNA libraries
An 11 day mouse 5⬘ stretched λgt11 library was screened. This
library was constructed by Clontech for the MRC Human Genetics
Unit, Edinburgh, UK. The RNA source was normal, whole embryos
pooled from Swiss Webster/NIH mice. The library was screened with
M2 EST as a probe following Clontech’s protocol for screening
λgt11 libraries.
Northern blot hybridization
Total RNA was extracted from testes, ovaries and kidneys obtained
fresh from adult mice and rats using Tri reagent (Sigma-Aldrich Co.
Ltd, Poole, Dorset, UK), according to the manufacturer’s instructions.
Samples were dissolved in RNase-free water containing 0.01% sodium
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dodecyl sulphate (SDS; Sigma) and the concentration of RNA
determined by absorbance at 260 nm using a Genequant spectrophotometer (Pharmacia Amersham Biotech Ltd). RNA was stored at
–70°C. Total RNA (20 µg/lane) was separated on denaturing agarose
gels according to standard methods (Saunders et al., 1992), transferred
by capillary blotting to Bright Star membranes (AMS Biotechnology
Europe Ltd, Abingdon, Oxon, UK) using 20⫻ sodium chloride/
sodium itrate (SSC) and fixed by UV light. Membranes were prehybridized for a minimum of 2 h in 0.2 mol/l phosphate buffer pH
7.2, containing 1% (w/v) bovine serum albumin (BSA), 7% (w/v)
SDS, and 15% (v/v) deionized formamide at 60°C. Radio-labelled
M1 or M2 cDNAs (0.5–1⫻106 cpm/ml), prepared as above, were
added and hybridization continued for 16–24 h. Membranes were
washed with 40 mmol/l phosphate buffer containing 1% SDS at
65°C, exposed to a phosphorimager screen for 2 days and then
visualized using the imager software (Molecular Dynamics, Chesham,
Bucks, UK). Membranes were stripped (0.1% SDS) and reprobed
with a labelled oligonucleotide specific for 18S ribosomal RNA
(Saunders et al., 1992).
In-situ hybridization
Testes and ovaries from immature (day 16) and adult mice were
immersion fixed in Bouin’s fluid for 6 h and then processed into
paraffin wax according to standard methods (Millar et al., 1993).
Sense and antisense cRNA transcipts were prepared from linearized
Bluescript M1 and M2 plasmids by incubation with T3 or T7 RNA
polymerases in the presence of [35S]-labelled UTP (PharmaciaAmersham) according to methods described in detail elsewhere
(Maguire et al., 1992, 1997). The hybridization was carried out
overnight at 50°C and the hybridization buffer contained the following
constituents – 50% formamide, 4 ⫻ sodium chloride – Tris-EDTA,
1 ⫻ Denhardts, 125 µg/ml salmon testes DNA, 125 µg/ml yeast
tRNA, 10 mM DTT, 10% dextran sulphate. After hybridization, slides
were dipped in undiluted NBT-2 photographic emulsion (Kodak,
Rochester, NY, USA) and exposed in a light tight box at 4°C for 16–
33 days. After processing, sections were viewed under light and dark
field illumination on an Olympus Provis microcope (Olympus Optical
Co, London, UK) equipped with a Kodak DC420 camera. Captured
images were stored on a Mackintosh G3 computer and montages were
assembled using Photoshop 5.0 (Adobe, Mountain View, CA, USA).
Results
Identification of mouse homologues of Dm Stau
At the time of the initial database search there were 14
mammalian EST sequences encoding proteins related to Dm
Stau; eight in human and six in mice deposited in the database.
Potential mouse staufen (m stau) homologues were identified
by carrying out a BLAST search with the complete Dm stau
sequence. Confirmation that these sequences were Dm stau-like
was obtained by independently analysing sequence homology
using GCG 10 http://www.hgmp.mic.ac.uk. Two mouse ESTs
were selected for further study, M1 (Genbank accession no.
AA104967) and M2 (Genbank accession no. AA106776). The
sequence lodged for the M1 EST is 490 bp long, encodes a
putative protein with 21% identity to Dm Stau, and corresponds
to a region spanning from amino acids 356–574 on Dm Stau.
The sequence for EST M2 was 401 bp long, and the encoded
protein displayed 16% identity to Dm Stau. PCR amplification
of the cDNA inserts of M1 and M2 containing plasmids
revealed that they were ~1400 and 800 bp respectively, and
staufen expression in mouse gonads
therefore additional sequencing was undertaken. M2 was
established to be 765 nucleotides long, the sequence reads
through in frame 2 indicating that it represents part of an open
reading frame (ORF), with neither the 5⬘ nor 3⬘ of the sequence
being complete. This sequence is identical with a longer mouse
EST more recently lodged in the database as mouse staufenlike (Genbank accession no. AJ244015). M2 also has two
regions of high sequence similarity to the Dm stau, these
coincide with RNA binding domain 4 (Figure 1A). M1 is 1376
nucleotides long, and appears to be a splice variant of m stau
(Wickham et al., 1999) containing a very short 3⬘ UTR. This
variant also lacks RNA binding domain 5. However, neither
the m stau lodged in the database nor M1 appear to represent
a complete cDNA as both read through from the start and
appear to lack a 5⬘ sequence end. This may explain why
alignment of the sequences with that of Dm Stau revealed
only four conserved RNA binding domains, whereas Dm Stau
has five such domains.
are likely to be two staufen genes in mammals was obtained
by searching the human EST databases. A human homologue
of Dm stau has been described (Wickham et al., 1999; Genbank
accession no. AF061941) and other studies (Marion et al.,
1999) and sequence comparisons have shown this to be
homologous with mouse M1. A human homologue of m stau 2
was also submitted to the database (Genbank accession no.
AK002152) as being similar to staufen. Interestingly, the
two different human staufens described map to different
chromosomal positions, H stau maps to chromosome 20 while
H stau 2 is reported to map to chromosome 15. These findings
therefore confirm that they are distinct genes, not differentially
spliced forms of the same gene. The fact that two genes
equivalent to the M1 and M2 cDNAs exist on different
chromosomes in humans adds weight to our suggestion that
these transcripts represent two different genes in mice. The
chromosomal locations are unavailable for the mouse staufens.
Isolation of m stau cDNAs
A good mouse ovary cDNA library was not available at the
time of the experiments therefore, screening was undertaken
using: (i) a mouse kidney cDNA library (obtained from
Jonathan Bard, Department of Anatomy, University of Edinburgh, Edinburgh, UK) and (ii) an 11 day mouse embryo
cDNA library (obtained from the Western General Hospital,
Edinburgh, UK). cDNA amplified from plasmid M1 was used
for screening both because it was the longer cDNA but also
because at the time of screening it was shown to have the
highest sequence homology to Dm stau. Several clones with
insert sizes ranging from 1 to 3 kb (not shown) were obtained
from the 11 day embryo library. Sequencing of three cDNAs
amplified from purified lambda confirmed that they contained
sequences identical to the M1 EST at their 5⬘ ends (Figure
1A). Subsequent comparison to sequences in Genbank revealed
that the longest cDNA obtained had an identical sequence to
the m stau (Genbank accession no. NM011490), submitted to
the database in 03/99 (Wickham et al., 1999). Therefore, the
data are not presented again in this paper.
Sequence alignment of the individual RNA binding domains
show that domains 2, 3, 4 and 5 are extremely well conserved
between Drosophila and mouse (Figure 1B). Domains 1, 3
and 4 in Drosophila are very similar (St Johnston et al., 1992)
and this is maintained in the mouse.
Sequence alignments suggest that the M1 and M2 cDNAs
represent different Dm stau-like genes. This finding is based
on sequence comparisons between the two mouse staufens and
Dm stau. For example, at the amino acid level, good alignments
can be made between M1, M2 and Dm stau in the region of
the RNA binding domain 4 (Figure 1B) with ⬎80% identity
at the protein level. However, at the DNA level M1 is only
50% identical to M2 over this domain, which makes the
differences between M1 and M2 as great as the differences
between mammals and flies. The DNA sequence differences
detected are much greater than could be explained by polymorphisms in the DNA, and since this carries an homologous
RNA binding domain 4 (Figure 1B), it is unlikely to be a
splicing variant at the DNA level. Further evidence that there
Northern analysis
Northern blot analysis was undertaken with full length cDNAs
amplified from plasmids M1 and M2, on replica samples of
RNA isolated from testis, ovaries and kidney from adult mouse
and rat (Figure 2). An mRNA of ~3 kb was detected in the
gonads from both species, and the transcript was less abundant
in adult kidney (Figure 2, lanes 1 and 7). The hybridization
signal using the M1 cDNA was always more intense than that
observed with the M2 plasmid. The transcript detected in rat
gonads using M1 cDNA appeared consistently larger than that
detected in mouse gonads (Figure 2, lanes 5, 6).
Cellular sites of expression of m stau in mouse gonads
Analysis of the pattern of expression of m stau using cRNA
prepared from plasmid M1 revealed that silver grains were
concentrated over germ cells (Figures 3 and 4). In the
mouse ovary mRNA was detected in oocytes within follicles
containing a single layer of granulosa cells, e.g. in ovaries
on day 8 (Figure 3a,b), and was maintained as the follicle
developed to contain several layers of granulosa cells (e.g.
day 15, Figure 3c,d) and to form an antrum (adult, Figure
3e arrowed A). In the mouse testis stau mRNA was also
localized to germ cells and this expression was dependent
upon their progression through the process of meiosis in
adults. No significant expression of mRNA was noted on
day 16 (Figure 4a), although some tubules at this age
contained germ cells up to and including (asterisks) early
pachytene spermatocytes, equivalent to those seen in stages
in the first half of the spermatogenic cycle in the adult
(Oakberk, 1956). In the adult mouse testis, the pattern of
expression was stage dependent and silver grains were
localized to those pachytene spermatocytes present in the
stages VI–XII of the cycle (Oakberk, 1956) with highest
levels seen at stages VII and VIII. A similar pattern of
expression was noted in testes using the cRNA prepared
from the M2 plasmid (m stau 2); however the numbers of
silver grains were much reduced when compared with
identical samples hybridized to the m stau cRNA (not shown).
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Figure 1. (A) Schematic sequence alignment of M1 and M2 plasmid cDNAs with staufen cDNA from Drosophila and two mouse staufenlike genes lodged in Genbank. Expressed sequence tags (ESTs), M1 (AA104976) encoded 1023 bp, corresponding to the open reading frame
(ORF) of mouse Stau (Genbank accession no. NM011490; Wickham et al., 1999). Stau (*) has a 600 bp 3⬘ untranslated region (UTR),
while EST M1 has a short 3⬘ UTR of 100 bp identical to the 3⬘ end of the m stau 3⬘ UTR. EST M2 encoded 767 bp corresponding to the
translated region of mouse Stau 2 (Genbank accession no. AJ244015). The figure illustrates the regions of strong homology represented by
the Dm stau binding sites. The UTRs are also marked on the figure. Although there is a potential start methionine near the beginning of m
stau transcript, there is no indication that this is a real start methionine as there is no consensus Kozak sequence or stop codon upstream
(Kozak, 1996). m stau 2 is also incomplete; missing its 5⬘ end. (B) Shows the alignment of Dm Stau RNA binding domains with
corresponding regions of two mouse proteins described in this paper. This alignment was carried out using GCG10 (pileup) and Box Shade
software. Staufen domain 2 alignment shows the alignment of Dm Stau binding domain 2 (DstauD2 amino acids 490–559) with mouse
Stau 1 binding domain 2 (MStauLD2 aa111–183) showing a high degree of conservation between them. Staufen domains 1, 3 and 4
alignment shows the alignment of three Dm stau RNA binding domains (DStauD1 amino acids 308–380, DStauD3 amino acids 575–647
and DStauD4 amino acids 708–782) with the corresponding RNA binding domains observed in mouse Stau (MStauLD3 amino acids 201–
273 and MStauLD4 amino acids 301–374) and the region of mouse Stau 2-like currently available, containing RNA binding domain
4(M2StauD4 aa 50–124). St Johnston et al. (1992) illustrated that Drosophila RNA binding domains 1, 3 and 4 were similar to each other.
A high degree of conservation is observed between Dm Stau and the two mouse Staufens. The highest similarity is observed between
MStauLD4 and M2StauD4. Staufen domain 5 alignment illustrates the alignment between Dm Stau domain 5 (DStauD5 amino acids 949–
1020) and mouse Stau, domain 5 (MLStauD5 amino acids 503–576). Earlier studies had showed that Dm Stau domains 2 and 5 are more
closely related to each other than to domains 1, 3 and 4 (St Johnston et al., 1992). The very close similarity between Dm Stau and mouse
Stau for each of these domains makes it clear that they are quite distinct and that the mouse/Drosophila similarities within a domain are
much greater than the similarity between domains 2 and 5 within a species.
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staufen expression in mouse gonads
Figure 2. Northern analysis of expression of stau mRNA in mouse
and rat tissues. Total RNA (20 µg) per lane from adult animals was
loaded as follows: mouse kidney, 1,7; mouse testis, 2, 8; mouse
ovary, 3, 4, 9, 10; rat testis, 5, 11; rat ovary 6, 12. (A) Lanes 1–6
were hybridized to M1 cDNA and lanes 7–12 with M2 cDNA, and
after washing membranes were exposed to phosphorimager screens
for 5 days. (B) Membranes were stripped and reprobed with an
oligonucleotide specific for 18S ribosomal RNA to confirm
successful loading and transfer of RNA, with exposure to imager
screens for 16 h.
Discussion
The preliminary expression data presented here suggest a role
for the RNA binding protein, Staufen, in both oogenesis and
spermatogenesis in rodents.
Whilst these studies were underway papers describing the
cloning of the human and mouse homologues of Dm stau were
published (Marion et al., 1999; Wickham et al., 1999). Staufen
homologues have also been identified in several other
species including rat (Genbank accession no. AJ010200) and
Caenorhabditis elegans (Genbank accession no. U67949, see
Wickham et al., 1999). Sequence comparisons show that
similarities between the Staufen proteins identified are almost
exclusively confined to the RNA binding domains (Micklem
et al., 2000). It is notable that human Staufen has been shown
to have several splice variants and have been identified in
multiple human tissues using Northern blots, however one
group (Wickham et al., 1999) did not include any gonadal
RNA in their tissue screen. Antibodies against human Staufen
have been used to immunolocalize the protein to rat hippocampal neurones (Kiebler et al., 1999; Marion et al., 1999) and
have shown that it is enriched in the vicinity of smooth
endoplasmic reticulum and microtubules near synaptic contacts. In living hippocampal neurones Staufen-green fluorescent
fusion protein was found to associate with granules containing
RNA and to move through the cell in a microtubule-dependent
manner (Kohrmann et al., 1999). In addition to an association
with microtubules, cell transfection experiments revealed that
Stau also localized to the rough endoplasmic reticulum and
may, therefore, target mRNAs to their site of translation
(Wickham et al., 1999). It will be exciting in the future to use
antibodies to determine the cellular location of mouse Staufens
in the oocyte and spermatocytes.
Of particular interest is the possibility that there is a splice
variant of m stau, called M1, which lacks RNA binding domain
5. This domain has been shown to be involved in RNA
localization. This means that there could be Staufen proteins
which repress translation without localizing the RNA.
Several RNA binding proteins, conserved in many species,
from Drosophila to human, have already been shown to be
important for mammalian fertility. A role for RNA binding
proteins in the process of gametogenesis is to be expected, as
translational control represents a key mechanism for gene
regulation in germ cell differentiation and early embryogenesis.
Perturbations in this process can therefore have a major impact
on normal development. For example, deletion of the RNA
binding protein dazl, in mice results in infertility in both males
and females (Ruggiu et al., 1997), consistent with expression
of dazl in germ cells in both the ovary and testis in mice
(Ruggiu et al., 1997, 2000) and human (Seligman and Page,
1998). Homologues of dazl have been identified in a wide
range of species including Xenopus (xDazl; Houston et al.,
1998), zebra fish (zDazl; Maegawa et al., 1999) and Drosophila
(boule; Eberhart et al., 1996). It is notable that whereas deletion
of dazl in mice results in infertility of both males and females
(Ruggiu et al., 1997), deletion of boule in Drosophila is
associated with infertility in males alone (Eberhart et al., 1996)
and in Caenorhabditis elegans loss of dazl function has no
affect on sperm production, but is associated with a meiotic
block during oogenesis (Karashima et al., 2000). These findings
are an interesting parallel to those of the present study in
which staufen transcripts have been identified in the germ cells
of both male and female mice, but appear to play a role in
only oogenesis in Drosophila. In Drosophila spermatogenesis,
translational control is mediated via translational control elements (TCE) located in the 5⬘ UTRs of testis RNA (Kempe
et al., 1993; Schafer et al., 1993, 1995). Y-box proteins are
also believed to function as translational repressors in germ
cells. Mammalian homologues of the Y-box proteins FRGY-1
and FRGY-2, first identified in Xenopus oocytes (Murray et al.,
1991), are both expressed in the testis (Kwon et al., 1993).
MSY2, the FRGY-2 homologue is expressed in both male and
female germ cells and is inherited in the early embryo (Gu
et al., 1998). In the testis, expression occurs in pachytene
spermatocytes but was maximal in post-meiotic round spermatids, the cell type which contains abundant stored messenger
ribonuclear proteins (Gu et al., 1998). In the ovaries, MSY-2
was expressed in oocytes which are arrested at diplotene within
follicles, an identical pattern of expression to that observed
using the staufen mRNA.
Many transcripts have been found to have testis-specific 5⬘
and 3⬘ UTRs, which are likely to be crucial in their translational
regulation and are probably recognized by specific sets of
RNA binding proteins. There is also a tendency for the
translationally-controlled testis transcripts to have shortened
poly A tails (see review by Hecht, 1998). One of the proteins
that operates on 3⬘ UTRs is testis brain–RNA binding protein
(TB-RBP). Interestingly this localizes to the nucleus in pachytene spermatocytes during meiosis but to the cytoplasm of the
round spermatids after meiosis.
Additional RNA binding proteins, not known to be represented in species other than mammals, have been identified as
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Figure 3. (A) In-situ analysis of site of expression of m stau in mouse ovaries indicates that expression is specific to the developing oocyte.
Sense and antisense cRNAs prepared from the m stau M1 cDNA were hybridized to sections obtained from mouse ovaries on post natal
days 8 (a, b), 9 (not shown), 15 (c, d) and adults (e, f). Dark field (a, c, e) and light field (b, d, f) of the same sections are shown. Silver
grains were localized over oocytes (arrows) at all ages examined consistent with expression of m stau in oocytes within preantral follicles
containing only one or two layers of granulosa cells (day 8, a, b; stage 2) and persisting into oocytes from antral follicles (e.g. e, arrowed
A; stage 5/6). There was no specific signal using the sense probe on any section examined (inset c). All original magnifications ⫻20.
(B) Summary of the levels of stau mRNA in relation to morphogenesis of the follicle. The stages of mouse oogenesis are shown at the top
and diagramatically beneath. There is a period of oocyte growth during stages 2–5. Thecal layer formation begins in stage 4 and from
stage 6 the follicle size increases dramatically and the antral cavity is formed. staufen mRNA accumulates in the oocyte during the period of
oocyte growth and remains at this level until the oocyte matures.
important in the storage and translational repression of the
mRNAs encoding the protamines. These are highly basic
proteins that replace the transition proteins used for DNA
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packaging with spermatozoa (Braun, 1998; Hecht, 1998). Of
particular interest is the protein found in the spermatids
called perinuclear RNA-binding protein (SPNR). SPNR is a
staufen expression in mouse gonads
Figure 4. Expression of m stau in testis is germ cell specific and stage dependent. In-situ analysis using sense and antisense cRNA prepared
from M2 revealed that expression of m stau in the mouse testis is germ cell specific; dark field (a, c, e) and light field (b, d, f) views are
shown from the same section. No specific hybridization signal was detected on sections of testes from day 15 mice, although these
contained somatic Sertoli cells and germ cells up to and including early pachytene spermatocytes (asterisks in a and b). In adult animals the
localization of the silver grains was consistent with expression of staufen mRNA in spermatocytes present in tubules during the second half
of the cycle (stages VI–XII; arrows c, e). (B) Summary of the levels of staufen mRNA in relation to morphogenesis of the spermatozoon.
The stages of spermatogenesis are shown, staufen mRNA increases and peaks in primary spermatocytes during the first meiotic division
when there are high levels of transcription. Transcripts then degrade in secondary spermatocytes and have disappeared when the sperm cell
differentiates. This suggests a function for staufen in primary spermatocytes when there is active transcription and translational represssion,
but some of the transcripts will not be translated until the sperm begins to differentiate and the staufen mRNA has degraded. It is possible
that Staufen protein is still present after the mRNA degrades.
microtubule associated protein which binds the 3⬘ UTR of
Protamine 1 mRNAs, suggesting a role in subcellular localization as well as translational repression and storage prior to
activation of translation (Schumacher et al., 1998). It is notable
that in common with Drosophila Staufen, one of these has been
identified as reponsible for the localization of translationallyrepressed mRNA to microtubules (Han et al., 1995). In
mouse testes, expression of staufen mRNA within pachytene
spermatocytes is consistent with a role in translational repression of some germ cell encoded mRNAs. Exactly when Staufen
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protein is present remains to be established, as repression by
a stable Staufen protein could easily continue well beyond the
stage when the mRNA is degraded. Further work will be
needed to identify specific mRNA targets.
In conclusion, expression of staufen mRNA in mammalian
germ cells extends the number of RNA binding proteins which
may act as key regulators of fertility in a wide range of
animal species.
Acknowledgements
The authors thank Mike Millar and Joseph Gaughan (HRSU) for
assistance with the in-situ studies and sequence analysis respectively;
the MRC Human Genetics Unit for λgt11 embryo library; and Sheila
Milne for preparing the manuscript.
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Received on June 5, 2000; accepted on August 24, 2000
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