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First published online 23 April 2008
doi: 10.1242/dev.021949
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Research Report |
Department of Zoology, University of Oxford, South Parks Road, Oxford OX1 3PS, UK.
Author for correspondence (e-mail:
white-cooperh{at}cf.ac.uk)
Accepted 8 April 2008
SUMMARY
Post-meiotic transcription was accepted to be essentially absent from Drosophila spermatogenesis. We identify 24 Drosophila genes whose mRNAs are most abundant in elongating spermatids. By single-cyst quantitative RT-PCR, we demonstrate post-meiotic transcription of these genes. We conclude that transcription stops in Drosophila late primary spermatocytes, then is reactivated by two pathways for a few loci just before histone-to-transition protein-to-protamine chromatin remodelling in spermiogenesis. These mRNAs localise to a small region at the distal elongating end of the spermatid bundles, thus they represent a new class of sub-cellularly localised mRNAs. Mutants for a post-meiotically transcribed gene (scotti), are male sterile, and show spermatid individualisation defects, indicating a function in late spermiogenesis.
Key words: Drosophila, Spermatid, Transcription, RNA localisation, Spermatid individualisation
INTRODUCTION
In D. melanogaster spermatogenesis (reviewed by
Fuller, 1993
), the primary
spermatogonium undergoes four mitotic divisions, each with incomplete
cytokinesis. Two somatic cyst cells, analogous to mammalian Sertoli cells,
encapsulate each spermatogonium, and thus, later, 16 spermatocytes, and, after
meiosis, 64 inter-connected spermatids. After meiosis, morphological changes,
including axoneme assembly and mitochondrial and membrane remodelling,
transform round spermatids into mature 2-mm-long motile sperm. These
post-meiotic events are believed to be driven by translation, as
spermiogenesis genes are typically transcribed in primary spermatocytes, the
mRNAs are then stored in spermatids and translated during elongation (reviewed
by Schäfer et al.,
1995).
In mammalian early spermatids, transcription is readily detected (by
3H-Uridine incorporation) and continues until chromatin compaction
(Kierszenbaum and Tres, 1975;
Monesi, 1965). Mammalian
spermatocytes transcribe genes required during meiosis or in early spermatids,
whereas mRNAs for spermiogenesis proteins are transcribed post-meiotically
(Schultz et al., 2003), with
some under additional translational control (reviewed by
Braun, 1998). Radiographic
studies showed no detectable transcription in Drosophila spermatids
(Gould-Somero and Holland,
1974; Olivieri and Olivieri,
1965); however, RNA polymerase II activity in Drosophila
spermatid nuclei suggests that these cells actively transcribe genes
(Rathke et al., 2007).
Spermatid elongation generates dramatic cellular asymmetry, with nuclei at
one end, and axonemes extending towards the distal end, where sister cells are
connected via ring canals (Hime et al.,
1996). During individualisation, each spermatid is stripped of
excess cytoplasm and organelles, and is separated from sister spermatids by an
individualisation complex that progresses from the heads along to the tails, a
process dependent on apoptotic pathway activation
(Arama et al., 2003).
Spermatids have a unique, highly compact, non-nucleosomal chromatin
organisation. Histones are removed from spermatid chromosomes and replaced by
other small basic proteins, initially transition proteins, then protamines or
protamine-like proteins (reviewed by Braun,
2001; Oliva,
2006). Drosophila have at least one transition protein
and several protamine-like proteins
(Jayaramaiah Raja and Renkawitz-Pohl,
2005; Rathke et al.,
2007).
Here, we provide compelling evidence for the transcription of 24 genes in Drosophila spermatids, activated by at least two regulatory pathways. In Drosophila, transcription shuts off in late primary spermatocytes, then (for a few loci) is reactivated mid-elongation, just before histone-to-protamine chromatin remodelling. At least one post-meiotically expressed gene, scotti, is required for normal actin cone progression during spermatid individualisation, and thus for male fertility. mRNAs encoded by all of the post-meiotically transcribed genes are localised to the extreme distal ends of the elongating cells; thus, they represent a novel class of sub-cellularly localised mRNAs.
MATERIALS AND METHODS
Drosophila strains and culture
Flies were raised on standard cornmeal sucrose agar at 25°C. Visible
markers and balancer chromosomes are described in FlyBase
(Crosby et al., 2007). Wild
type was w1118. Protamine-EGFP flies were from Renate
Renkawitz-Pohl (Jayaramaiah Raja and
Renkawitz-Pohl, 2005) and H2A-mRFP1 flies from Jürgen
Knoblich (IMBA, Vienna, Austria).
RNA in situ hybridisation
RT-PCR products (400-600 bp) were generated from total testis RNA. 3'
PCR primers included a T3 RNA polymerase promoter for the in vitro
transcription of digoxigenin (DIG)-labelled RNA probes. In situ hybridisation
was as described previously (White-Cooper
et al., 1998). Primer sequences are available on request.
Isolation of RNA from individual cysts or bundles
Testes were dissected from w1118 or the young male
progeny of w; Mst35Ba-egfp x w; H2A-mRFP1, in testis
buffer (183 mM KCl, 47 mM NaCl, 10 mM Tris pH 6.9), transferred onto a
siliconised slide, opened with tungsten needles and examined using an Olympus
BX50 upright microscope with a long working distance 10x objective.
Individual photographed cysts were transferred into 100 µl lysis buffer
using a pulled-out Pasteur pipette; total RNA was extracted according to
manufacturer's instructions (RNAqueous-Micro kit, Ambion, Warrington, UK).
Q-RT-PCR
First-strand cDNA was synthesised using oligo(dT)20 and
SuperScript III Reverse Transcriptase (Invitrogen, Paisley, UK) in a 20 µl
reaction. Each PCR contained 0.33 µl cDNA, 10 µl 2x Power SYBR
Green PCR Master Mix (Applied Biosystems, Warrington, UK), and 100 nM of each
gene-specific primer, in a 20 µl final volume. Primers amplified cDNA only,
as one of each pair spanned an exon-exon junction. Negative controls (testis
RNA without reverse transcriptase) were performed for every primer pair in
every PCR plate. Q-PCR reactions in 96-well thin-wall white plates
(BIOplastics, Braintree, UK) were run in a Chromo4 with Opticon Monitor
Software (GRI, Braintree, UK). Q-PCRs were run in triplicate and the internal
reference control (CG10252) was quantified for each sample.
|
;
Thibault et al., 2004). All
five independent deletion lines were confirmed by soti ORF PCR, and
were viable but male sterile.
FITC-Phalloidin staining of testes
Visualisation of actin cones in mutant and wild-type testes was carried out
as described previously (White-Cooper,
2004).
RESULTS AND DISCUSSION
Drosophila `comet' and `cup' transcripts are specifically detected in spermatid bundles
Many genes with unknown functions have testes-specific expression. To
determine when during spermatogenesis these proteins are made, we examined the
transcript patterns of >1200 genes by in situ hybridisation
(www.fly-ted.org).
The expression of spermiogenesis genes in primary spermatocytes, and the
storage of transcripts for later use during spermiogenesis, means that the
translation of specific mRNAs in Drosophila spermatids correlates
well with their disappearance, as translation exposes stored mRNAs to the RNA
degradation machinery (Schäfer et
al., 1995). The in situ hybridisation results will be described in
detail elsewhere; in summary, 529 of the 553 mRNAs detected in spermatids were
transcribed in primary spermatocytes, persisted in the spermatid cytoplasm,
and were degraded at various stages in elongation
(Fig. 1C,D). Unexpectedly, we
found 24 germ-line expressed genes that did not conform to this pattern
(Table 1; see also Table S1 in
the supplementary material). We subdivided these on the basis of subtle
differences in transcript localisation patterns, and refer to the genes
collectively as `comets and cups'.
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To verify the post-meiotic transcription, and to determine its timing with
respect to cellular differentiation events, we developed a single-cyst
quantitative reverse transcription PCR (Q-RT-PCR) protocol. Testes were
dissected, and individual cysts isolated, photographed, and staged according
to morphology; total RNA was then isolated and first strand cDNA synthesised
(Fig. 2A). Each cyst yielded
cDNA for 60 Q-RT-PCR reactions. Testis-specific control genes were chosen. The
CG10252 transcript conforms to the conventional pattern for a late
elongation protein (Fig. 1D)
and CG10252 protein is detected in mature sperm
(Dorus et al., 2006).
CG3927 was detected exclusively in primary spermatocytes;
CG11591 was expressed in primary spermatocytes and the signal
disappeared from mid-elongation spermatids
(Fig. 1B,C). CG3927
and CG11591 controlled for cyst visual staging. For each cyst,
expression levels of both staging controls and up to eight test genes were
compared with the internal control CG10252.
Fig. 2 shows a typical
experiment, expression levels were normalised to primary spermatocyte cyst I1.
Consistent with the in situ hybridisation patterns, CG3927 transcript
was only detected in primary spermatocytes. CG11591 transcript was
highest in primary spermatocytes, and persisted into early elongation stage
spermatids. The 13 comet and cup gene transcripts assayed by isolated-cyst
Q-RT-PCR showed broadly similar profiles in Q-RT-PCR assays (Figs
2,
3; see also Figs S2, S5, S6 in
the supplementary material). All transcripts were detected in primary
spermatocytes and round spermatids. sunz, sowi, soti, c-cup, d-cup,
wa-cup, p-cup and r-cup were low or not detected in very short
elongating cysts, but were detected at high levels in a few longer spermatid
cysts. hale, schuy, boly, cola and swif were detected at a
basal level in almost all cysts, but were much more abundant in a few
mid-elongation bundles. From these differences, we infer two separate
regulatory modules activating post-meiotic gene expression, with the
hale group being transcribed in more cysts than the sunz
group. Spermatid length measurements give good staging of the relative
differentiation states of cysts from a single testis, but the exact length of
spermatids expressing comets and cups varied between testes. The initial
low-level signal in primary spermatocytes, the dip in signal intensity in
early spermatids, then the dramatic appearance in later spermatids
conclusively demonstrate that there is post-meiotic transcription in
Drosophila testes.
|
|
). To determine comet and cup transcription
timing with respect to chromatin reorganisation, we staged cysts via combined
fluorescent fusion-protein localisation and spermatid-length measurements. We
isolated cysts from flies co-expressing Mst35Ba-GFP (protamine-GFP) and
H2A-mRFP1 (histone-GFP). Protamine accumulation initiates before all histones
have been removed, as some nuclei fluoresced both red and green
(Fig. 3K). Transcription of
comet and cup genes was detected in mid-elongation cysts that were positive
for histone and negative for protamine
(Fig. 3D-J). Thus, comet and
cup transcription occurs just before the deposition of protamines. Some comet
and cup mRNAs were also detected in cysts positive for protamine-GFP. This
could be due to ongoing transcription, or to message stability. The recently
described active transcription in spermatids
(Rathke et al., 2007)
coincides with the comet and cup gene transcription peak. We repeated these experiments using cysts isolated from flies co-expressing Tpl94D-GFP (transition protein) and H2A-mRFP1. Initiation of post-meiotic comet and cup gene expression was found in cysts lacking nuclear Tpl94D (see Fig. S2B in the supplementary material), indicating that comet and cup gene transcription initiates before the deposition of transition protein, while chromatin is presumably still nucleosomal.
Rfx is not required for expression of the comet and cup genes
Rfx is a winged-helix transcription factor important for ciliogenesis gene
expression. In testes, Rfx protein was detected transiently in canoe-stage
spermatid nuclei only (Vandaele et al.,
2001), and thus was an excellent comet and cup candidate
transcription factor. All comet and cup genes tested (hale, schuy, cola,
soti, c-cup, w-cup, p-cup and wa-cup) by in situ hybridisation
to Rfx253/Rfx49
(Dubruille et al., 2002) mutant
testes were indistinguishable from wild type (see Fig. S3 in the supplementary
material). Rfx49 is a null, whereas
Rfx253 lacks DNA-binding activity. Thus, despite the
intriguing localisation of Rfx to spermatid nuclei, its X-box binding function
is not required for comet or cup expression.
scotti is required for spermatid individualisation
We deleted approximately 4 kb of genomic DNA, including the entire
scotti (soti, a comet) ORF, by FLP-mediated recombination
between flanking FRT-containing transposons. soti homozygous mutants
were viable and female fertile, but male sterile. Phase contrast microscopy
indicated no gross defects in soti testes organisation or spermatid
elongation; however, empty seminal vesicles indicated spermiogenesis defects.
Within each individualising spermatid cyst, 64 actin-rich investment cones
move together as an individualisation complex, pushing ahead a cystic bulge of
excess cytoplasm and organelles. This cytoplasm is discarded from spermatid
distal ends as a waste bag. Waste bags were completely absent from mutant
testes, and cystic bulges were rarely seen. FITC-phalloidin labelling revealed
that investment cones formed normally in soti mutant males; however,
nuclei failed to remain tightly clustered and were displaced distally along
the cyst (Fig. 4). Although
investment cones progressed away from the nuclei in mutants, investment cone
coupling within individualisation complexes was lost, and cones never
progressed the full length of mutant spermatids. Thus, soti function
is required for spermatid individualisation.
Why are the comets and cups transcribed in spermatids?
Post-meiotic transcription, in early spermatids, has been reported for two
loci in Drosophila, hsr-omega and Hsp70
(Bendena et al., 1991);
however, we have been unable to reproduce these findings (see Fig. S5 in the
supplementary material). Ninety-six percent of genes whose mRNAs were detected
in spermatids are not actively transcribed in these cells (being made in
spermatocytes), so what is special about the exceptional 4% - the comets and
cups? These genes are found throughout the euchromatin, including the X
chromosome, and their local genomic environments showed no unusual features.
Their flanking genes showed no bias towards or away from testis-specific
expression in adults (Chintapalli et al.,
2007). There are three comet and cup gene clusters, two of which
clearly represent gene duplication events (see Table S2 in the supplementary
material). The final cluster comprises hale and schuy; both
encode glutamine-rich proteins, but their evolutionary history is unclear. We
investigated the expression of all 10 related genes in the CG11635-CG8701
cluster (see Fig. S5A in the supplementary material). CG11635, CG18449,
CG2127 and CG8701 were expressed in the conventional
spermiogenesis gene pattern - transcribed in primary spermatocytes and stored
until late elongation - while spaw, hubl, swif, cola, boly and
whip were typical `comets' (see Tables S1, S2 and Fig. S5B,C in the
supplementary material). Q-RT-PCR confirmed that the post-meiotic
transcription and RNA localisation to distal ends of spermatids were
correlated.
In mammals, the transcription of many genes in spermatids has been
described, and new reports are frequent
(Reynard et al., 2007;
Schultz et al., 2003). These
mammalian genes typically, although not exclusively, encode components of the
mature sperm. By contrast, Drosophila comet and cup proteins, with
the exception of Boly and Pglym87, are not sperm components (see Table S1 in
the supplementary material) (Dorus et al.,
2006). Perhaps comet and cup proteins function, like Soti, during
spermiogenesis, rather than in sperm. Alternatively, perhaps they are present
in sperm but at a very low copy number. mRNAs of several comet and cup gene
homologues were transcribed in conventional patterns, and the encoded proteins
detected in sperm (see Table S2 in the supplementary material). orb
(a comet) encodes an RNA-binding protein, potentially anchoring other comet
and cup mRNAs. The other comet or cup proteins have no predicted function.
PKD2 encodes a Ca2+-activated non-selective cation channel, and it
is intriguing that a Drosophila PKD2 homologue (Pkd2/Amo)
concentrates at the distal ends of sperm, and is important for sperm function
(Watnick et al., 2003). Sunz,
Sowi and D-cup are EF-hand-containing proteins, and so could function with
Pkd2 in mediating a Ca2+ signal at the spermatid tail tip. This
signal could then be transduced along spermatid tails, perhaps via the
mitochondrial derivative or the endoplasmic reticulum-derived axonemal sheath,
which stretches the length of spermatid tails, activating the apoptotic
pathway to synchronise individualisation and ensure normal investment cone
progression.
In conclusion, there is significant transcription from several genomic loci in Drosophila spermatids, and the post-meiotically expressed transcripts localise to the growing ends of spermatids. This transcription and RNA localisation occurs before spermatid chromatin remodelling, and at least one post-meiotically-expressed gene is required for spermiogenesis.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/11/1897/DC1
ACKNOWLEDGMENTS
We thank Bénédicte Durand for Rfx mutant flies, Renate Renkawitz-Pohl for protamine and Tpl94D-EGFP flies, Spyros Artavanis-Tsakonas for Exelixis stocks and Jennifer Mummery-Widmer (Knoblich lab) for H2A-mRFP1 flies. This work was funded by the BBSRC and the Royal Society.
Footnotes
* Present address: Cardiff University, Biomedical Sciences Building, Museum
Avenue, Cardiff CF10 3US, UK 
REFERENCES
Arama, E., Agapite, J. and Steller, H. (2003).
Caspase activity and a specific cytochrome C are required for sperm
differentiation in Drosophila. Dev. Cell
4, 687-697.[CrossRef][Medline]
Bendena, W. G., Ayme, S. A., Garbe, J. C. and Pardue, M. L.
(1991). Expression of heat-shock locus hsr-omega in nonstressed
cells during development in Drosophila melanogaster. Dev.
Biol. 144,65
-77.[CrossRef][Medline]
Braun, R. (1998). Post-transcriptional control
of gene expression during spermatogenesis. Semin. Cell Dev.
Biol. 9,483
-489.[CrossRef][Medline]
Braun, R. (2001). Packaging paternal
chromosomes with protamine. Nat. Genet.
28, 10-12.[CrossRef][Medline]
Chintapalli, V., Wang, J. and Dow, J. (2007).
Using FlyAtlas to identify better Drosophila models of human disease.
Nat. Genet. 39,715
-720.[CrossRef][Medline]
Crosby, M., Goodman, J., Strelets, V., Zhang, P., Gelbart, W.
and Consortium, F. (2007). FlyBase: genomes by the dozen.
Nucleic Acids Res. 35,D486
-D491.
Dorus, S., Busby, S. A., Gerike, U., Shabanowitz, J., Hunt, D.
F. and Karr, T. L. (2006). Genomic and functional evolution
of the Drosophila melanogaster sperm proteome. Nat.
Genet. 38,1440
-1445.[CrossRef][Medline]
Dubruille, R., Laurecon, A., Vandaele, C., Shishido, E.,
Coulon-Bublex, M., Swoboda, P., Couble, P., Kernan, M. and Durand, B.
(2002). Drosophila regulatory factor X is necessary for
ciliated sensory neuron differentiation. Development
129,5487
-5498.
Fuller, M. T. (1993). Spermatogenesis. In
The Development of Drosophila. Vol.1
(ed. M. Bate and A. Martinez-Arias), pp.71
-147. Cold Spring Harbor, NY: Cold Spring Harbor
Press.
Gould-Somero, M. and Holland, L. (1974). The
timing of RNA synthesis for spermiogenesis in organ cultures of Drosophila
melanogaster testes. Wilhelm Rouxs Arch.
174,133
-148.[CrossRef]
Hime, G. R., Brill, J. A. and Fuller, M. T.
(1996). Assembly of ring canals in the male germ line from
structural components of the contractile ring. J. Cell
Sci. 109,2779
-2788.[Abstract]
Jayaramaiah Raja, S. and Renkawitz-Pohl, R.
(2005). Replacement by Drosophila melanogaster protamines and
Mst77F of histones during chromatin condensation in late spermatids and role
of sesame in the removal of these proteins from the male pronucleus.
Mol. Cell. Biol. 25,6165
-6177.
Kierszenbaum, A. and Tres, L. (1975).
Structural and transcriptional features of the mouse spermatid genome.
J. Cell Biol. 65,258
-270.
Monesi, V. (1965). Synthetic activities during
spermatogenesis in the mouse. Exp. Cell Res.
39,197
-224.[CrossRef][Medline]
Oliva, R. (2006). Protamines and male
infertility. Hum. Reprod. Update
12,417
-435.
Olivieri, G. and Olivieri, A. (1965).
Autoradiographic study of nucleic acid synthesis during spermatogenesis in
Drosophila melanogaster. Mutat. Res.
2, 366-380.
Parks, A. L., Cook, K. R., Belvin, M., Dompe, N. A., Fawcett,
R., Huppert, K., Tan, L. R., Winter, C. G., Bogart, K. P., Deal, J. E. et
al. (2004). Systematic generation of high-resolution deletion
coverage of the Drosophila melanogaster genome. Nat.
Genet. 36,288
-292.[CrossRef][Medline]
Rathke, C., Barrends, W. M., Jayaramaiah Raja, S., Bartkuhn, M.,
Renkawitz, R. and Renkawitz-Pohl, R. (2007). Transition from
a nucleosome-based to a protamin-based chromatin configuration during
spermiogenesis in Drosophila. J. Cell Sci.
120,1689
-1700.
Reynard, L., Turner, J., Cocquet, J., Mahadevaiah, S., Toure,
A., Hoog, C. and Burgoyne, P. (2007). Expression analysis of
the mouse multi-copy X-linked gene Xlr-Related, meiosis-regulated (Xmr),
reveals that Xmr encodes a spermatid-expressed cytoplasmic protein SLX/XMR.
Biol. Reprod. 77,329
-335.
Schäfer, M., Nayernia, K., Engel, W. and Schäfer,
U. (1995). Translational control in spermatogenesis.
Dev. Biol. 172,344
-352.[CrossRef][Medline]
Schultz, N., Hamra, F. and Garbers, D. (2003).
A multitude of genes expressed solely in meiotic or postmeiotic spermatogenic
cells offers a myriad of contraceptive targets. Proc. Natl. Acad.
Sci. USA 100,12201
-12206.
Thibault, S. T., Singer, M. A., Miyazaki, W. Y., Milash, B.,
Dompe, N. A., Singh, C. M., Buchholz, R., Demsky, M., Fawcett, R.,
Francis-Lang, H. L. et al. (2004). A complementary transposon
tool kit for Drosophila melanogaster using P and piggyBac. Nat.
Genet. 36,283
-287.[CrossRef][Medline]
Vandaele, C., Coulon-Bublex, M., Couble, P. and Durand, B.
(2001). Drosophila regulatory factor X is an embryonic
type I sensory neuron marker also expressed in spermatids and in the brain of
Drosophila. Mech. Dev.
103,159
-162.
Watnick, T. J., Jin, Y., Matunis, E., Kernan, M. and Montell,
C. (2003). A flagellar Polycystin-2 homolog required for male
fertility in Drosophila. Curr. Biol.
13,2175
-2178.[CrossRef]
White-Cooper, H. (2004). Spermatogenesis:
analysis of meiosis and morphogenesis. In Drosophila Cytogenetics
Protocols. Vol. 247 (ed. D. Henderson),
pp. 45-75.Totowa, NJ: Humana Press.
White-Cooper, H., Schafer, M. A., Alphey, L. S. and Fuller, M.
T. (1998). Transcriptional and post-transcriptional control
mechanisms coordinate the onset of spermatid differentiation with meiosis I in
Drosophila. Development
125,125
-134.
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