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First published online 12 December 2007
doi: 10.1242/dev.015156
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Howard Hughes Medical Institute, Department of Embryology, Carnegie Institution, 3520 San Martin Drive, Baltimore, MD 21218, USA.
* Author for correspondence (e-mail: spradling{at}ciwemb.edu)
Accepted 24 October 2007
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Hr39, Spermathecae, Reproductive tract, SF1 (NR5A1), Steroid hormone
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INTRODUCTION |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). The
roles played by steroid hormones in vertebrates and invertebrates appear to be
among the most divergent. Multiple steroid hormones serve non-autonomously as
master regulators of male or female sexual development in mammals, whereas the
Drosophila molting hormone ecdysone acts sex-specifically only during
adult oogenesis (Buszczak et al.,
1999; Carney and Bender,
2000; Hackney et al.,
2007; Li et al.,
2000). Steroid-producing (`steroidogenic') tissues arise early in
mammalian embryos under the control of the nuclear receptor steroidogenic
factor 1 (SF1) (reviewed by Val et al.,
2003) and function throughout life. The closely related protein
LRH1 (NR5A2) also plays an important role, especially in ovarian function
(reviewed by Fayard et al.,
2004). The Drosophila genome encodes two proteins that
are closely related to SF1 and LRH1, FTZ-F1 and HR39, which bind to similar
target sequences (Ohno et al.,
1994), but neither has been implicated in sex determination
(reviewed by King-Jones and Thummel,
2005). Steroid production in the ovary, the only known site of
adult steroidogenesis, is not known to depend on either gene.
Despite these differences, in both mammals and Drosophila the
gonads and reproductive tract develop in a generally similar manner. During
mammalian embryogenesis, SF1 is required to produce androgens and
Müllerian-inhibiting substance, a TGFβ family member that causes the
oviduct precursors to degenerate. Drosophila reproductive tract
precursors also develop in a sex-specific manner within the bipotential
genital disc (Keisman et al.,
2001), but a corresponding genetic pathway has not been found. In
Drosophila (reviewed by Bloch Qazi
et al., 2003), the spermathecae and seminal receptacle, which
carry out long-term and short-term sperm storage, branch from the oviduct,
along with the glandular parovaria (Fig.
1A). In mammals, long-term sperm storage takes place within the
male epididymis, whereas the oviducts receive glandular secretions and can
maintain sperm briefly (see Suarez and
Pacey, 2006).
Gametes also undergo a complex maturation process in both mammals and
invertebrates. Following production in the testis, mammalian sperm are
immotile and incapable of fertilization. Only after passing through two other
steroid-regulated tissues, the male epididymis, where they encounter
extracellular proteases, antioxidants and anti-bacterial proteins (reviewed by
Cooper and Yeung, 2006), and
the female reproductive tract, where they contact mucins and membrane
glycoproteins (reviewed by Suarez and
Pacey, 2006) are sperm fully capacitated for fertilization. At the
time of mating, Drosophila sperm are mixed with bioactive peptides
and other proteins from the male accessory gland. Following transfer to the
female, sperm have been proposed to interact with proteins synthesized by the
female reproductive tract prior to storage in the seminal receptacle and
spermathecae (Bloch Qazi et al.,
2003; Lawniczak and Begun,
2007). However, the identity, function, origin and regulation of
female sperm-interacting proteins remain poorly known.
We find that Hr39 functions in a manner reminiscent of SF1. Hr39 is required for the normal development and function of spermathecae and parovaria. Thus, as in mammals, a Drosophila SF1-related gene mediates the sex-specific development of an essential region of the reproductive tract. Moreover, our results show that spermathecae and parovaria secrete proteins that function in sperm maturation, as well as in storage. Conserved steps in sperm maturation may take place at the sites of sperm storage, i.e. the epididymis in mammals or the female sperm storage organs in Drosophila. Our work reveals closer connections between Dipteran and mammalian reproductive biology than previously believed, and raises the possibility that novel steroid hormones regulate aspects of Drosophila reproduction.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
Fertility and SP number counts
The fertility and spermathecae number of wild-type, heterozygous and
homozygous mutant female flies was determined in the following manner: a
single female was placed with two yw males in a vial for 5 days at
25°C. On the fifth day, the flies were removed and the female dissected to
determine the number of spermathecae. The vial was then allowed to develop for
20 additional days at 25°C before the progeny in each vial was counted and
recorded.
Transgenics
Full-length Hr39 cDNA, LD45021, was cloned into the Gateway entry
vector and then swapped into the pUASt vector to make a P-element construct in
which protein expression is under control of the yeast upstream activating
sequence (UAS). P-element transformation was performed by standard procedures.
26 lines were generated, of which 11 were homozygous viable. The chromosomal
locations of the P-elements were determined through standard crosses and
appropriate transgenic animals were crossed into each mutant Hr39
line in order to obtain the homozygous mutant Hr39, transgene and
heat-shock driver in one fly. These flies were maintained at 20°C to
minimize leakiness of the transgene and then heat shocked during larval
development as third instar larvae for 30 minutes at 37°C. Fertility
assays of transgenic animals were performed as described above.
RT-PCR
RNA was isolated using either TriZOL reagent (Invitrogen) or Qiagen RNeasy
kit following the manufacturer's protocol. The RNA was treated with 2 U/µl
DNase overnight (Ambion) according to manufacturer's instructions. One-Step
RT-PCR (Qiagen) was then performed using 0.5 µg of the isolated RNA and
primers designed to span an intron within Hr39 or RpS17 as a
control. Primer sequences are available upon request. The PCR machine was an
MJ Research PTC-100 Programmable Thermal Controller and the program used: 30
minutes at 50°C, 15 minutes at 95°C, 29 cycles of 30 seconds at
94°C, 30 seconds at 55°C, 1 minute at 72°C, followed by 10 minutes
at 72°C. PCR products were resolved on 1% agarose LE gels (Roche) in
0.5x TBE buffer with 0.25 µg/µl ethidium bromide. Gel images were
acquired by using the BioRad Gel Doc XR scanner and Quantity One software
(V4.5.2). All experiments were carried out at least in triplicate and a
representative data set is shown.
Real time quantitative RT-PCR
RNA was isolated and Qiagen's One-Step RT-PCR kit used as described above
under RT-PCR. Primers were designed to span introns for each gene tested
(Hr39, RpS17, Cyp4d21, takeout, AttC and Pbprp1) and
sequences are available upon request. Quantitative RT-PCR reactions were
carried out on an Opticon Monitor 2 (MJ Research) using a 25 µl reaction
comprising 0.25 µg total RNA and 0.25 µl of a 7.5x SYBR Green
stock (Molecular Probes). The program used was 30 minutes at 50°C, 15
minutes at 95°C, followed by 40 cycles of 30 seconds at 94°C, 30
seconds at 55°C and 1 minute at 72°C. Finally the melting curve of
each sample was determined. Results were analyzed using the Opticon Monitor
software. Transcripts were expressed relative to the transcript of the control
RpS17 gene and normalized to the control female for each gene
tested.
Immunostaining
Whole-mount samples were fixed with 4% paraformaldehyde for 15 minutes and
processed using standard procedures (Cox
and Spradling, 2003). The following antisera were used: rabbit
anti-β-gal (pre-absorbed against lower reproductive tracts or ovaries,
1:1000) (Cappel), mouse-anti-Fas2 (1:2) (1D4, Developmental Studies Hybridoma
Bank), mouse-anti-Dac (1:200) (Abdac2-3, Developmental Studies Hybridoma
Bank), and mouse-anti-Wg (1:50) (4D4, Developmental Studies Hybridoma Bank).
Secondary antibodies were used at 1:500 and are as follows: goat anti-rabbit
conjugated to Alexa 488 and goat anti-mouse conjugated to Cy3 (Molecular
Probes). For DNA labeling, DAPI was added 1 µg/ml for 5 minutes.
In situ hybridization
Whole-mount in situ hybridization was performed by generating sense and
antisense RNA probes by in vitro transcription from PCR products. Methods were
previously described by Liu et al. (Liu et
al., 2006) and Lécuyer et al.
(Lécuyer et al.,
2007).
Confocal microscopy
Confocal images were taken with a 20x (NA 0.70) or a 40x (NA
1.25) Plan Apo objective on laser-scanning confocal microscopes (NT or SP2;
Leica). Images were taken with the laser intensity and photomultiplier gain
adjusted so that pixels in the region of interest were not saturated
(`glow-over' display). Contrast and relative intensities of the green (Alexa
488), red (Cy3) and blue (DAPI) images were adjusted with Photoshop (Adobe).
All confocal images are projected z-stacks.
Electron microscopy
Electron microscopy was carried out essentially as described
(Cox and Spradling, 2003).
Microarray
RNA from either lower reproductive tract (minus spermathecae) and
spermathecae was made by dissecting young wild-type or
Hr3904443 females 3 days after mating as described above.
Tissue samples were quick frozen in liquid nitrogen and kept at -80°C
until enough sample was isolated (
2000 spermathecae and 1000 lower
reproductive tracts). The microarray was performed by the Johns Hopkins
Microarray Core Facility on Drosophila version 2.0 Affymetrix chips
using either 10 µg lower reproductive tract RNA or 2 µg spermathecae
RNA. The microarray was performed twice using two different sets of RNA. The
absolute difference between the replicate measurements for all genes called as
present averaged less than 26% of their mean. The changes in expression
observed for selected genes were fully verified by quantitative RT-PCR on RT
samples (see below).
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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) are almost as fertile as wild type. Further
examination revealed that the reproductive tracts of all the mutants
frequently lack the two spermathecae and two parovaria characteristic of wild
type (Fig. 1A,C). Mutant adults
bearing the four strongest alleles usually lack parovaria and spermatheca or
contain just one spermatheca (Fig.
1D,H). Flies bearing weaker alleles with one or two spermathecae
also usually have one or two parovaria. Hr3904443 females
are unique because they frequently contain an extra spermatheca
(Fig. 1G,H). Interestingly,
although these 3-spermathecae-containing Hr3904443 females
still contain just two parovaria, their parovaria are distinctly larger than
wild type (compare Fig. 1E with
1F).
Spermathecae are required for fertility
The observed defects in spermathecae and parovaria might be responsible for
the sterility of mutant females bearing strong Hr39 alleles, or there
might be unapparent defects in the ovary or some other tissue. To distinguish
these alternatives, we investigated whether spermathecal and/or parovarial
content correlated with fertility at the level of individual female flies.
Such a relationship was suggested by our observation that most Hr39
mutant females were completely sterile, and what appeared to vary between
alleles was the frequency of rare females with significantly greater fecundity
(data not shown). Consequently, we scored the fertility of hundreds of
individual Hr39 mutant females and subsequently determined the number
of spermathecae and parovaria they contained
(Table 1, see also Table S2 in
the supplementary material).
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A correlation between spermathecae and fertility has been shown previously
in studies of lozenge mutations that also produce females with a
variable number of morphologically normal or defective spermathecae, but no
parovaria (Anderson, 1945). It
has been proposed that spermathecae produce a product required for sperm
storage. However, when we examined the seminal receptacles of 3- to 6-day-old
wild type and strong Hr39 mutant flies mated on day 1 that lacked
spermathecae and parovaria, both DAPI staining (see Fig. S1 in the
supplementary material) and electron microscopy (not shown) revealed normal
numbers of sperm in the mutants. We also noted the presence of sperm in
seminal receptacles of much older Hr39 mutants. Thus, infertile
Hr39 mutant females lacking spermathecae and parovaria, still
transfer normal amounts of sperm at mating (data not shown) and maintain
normal amounts of sperm in their seminal receptacles. Consequently, our data
suggest that spermathecae (possibly including their small associated segment
of fat body) produce a product that is required for sperm to function, despite
their ability to be stored.
Hr39 is expressed in reproductive tissues
To analyze how the insertion mutations affect spermathecal and parovarial
development, we attempted to analyze Hr39 expression in the genital
disc, the anterior region of which is known to give rise to both structures
from tiny primordia shortly after the onset of prepupal development
(Keisman et al., 2001).
However, we were unable to generate specific anti-Hr39 antibodies or
to carry out whole-mount in situ hybridization on larval genital discs with
either Hr39-specific or control probes. Furthermore, no defects in
the structure or gene expression of these discs was apparent in late stage
larvae, as we could detect no changes in the expression patterns of Engrailed,
Wingless, Dachshund or Abdominal-B using specific antibodies (see Fig. S3 in
the supplementary material; data not shown).
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Individual tissues expressing Hr39 were identified using
whole-mount in situ hybridization (Fig.
2). Except as noted, the presence of RNA detectable by in situ
hybridization corresponded closely to the enhancer trap expression patterns of
the Hr3903508 and Hr3904443 alleles.
Thus, Hr39 RNA was detected directly and by enhancer trap staining in
the lateral (ecdysone-producing) cells of the larval ring gland
(Fig. 2C), the larval ovary
(Fig. 2E), the spermathecae and
parovaria (Fig. 2F,G), the
seminal receptacle (Fig. 2H),
the spermathecae-associated fat body (Fig.
2I), the spermathecal capsule cells
(Fig. 2J-L), the adult ovariole
(Fig. 2M), and the adult testis
(Fig. 2N). In addition, a low
uniform level of enhancer trap staining was observed throughout the entire
larval genital disc (Fig. 2D).
All these tissues contribute directly or indirectly to the development and
function of reproductive tissue. Hr39 expression in the gland cells
of the spermathecae was mosaic when assayed by whole-mount in situ
hybridization, while enhancer trap staining was more uniform, presumably owing
to the longer perdurance of the β-galactosidase protein
(Fig. 2J-L). Possible cyclic
activity of these cells has been noted previously
(Filosi and Perotti,
1975).
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Hr39 is required for normal spermathecal secretion
Our previous observations suggested that spermathecae, and possibly
parovaria, produce a secreted product(s) required for sperm function. We
further investigated the nature of this product and the effects of
Hr39 mutations using electron microscopy of wild-type and mutant
spermathecae and parovaria. Wild-type spermathecae
(Fig. 4A) contain multiple
gland cells (outlined), connected via end apparati (EA) and ducts to the lumen
(L) of the capsule. The lumen (Fig.
4B) is filled head first with a highly ordered collection of sperm
(S) surrounded by lightly staining material (M). In spermathecae from 3- to
5-day-old wild-type mated females, most of the capsule cells appear to be
actively secreting material into the lumen, because their end apparati are
swollen with a poorly staining material that partially obscures the villi
(Fig. 4C, arrow). By contrast,
a few cells appear to lack secretion. Their end apparati are smaller and
contain more readily visible microvilli
(Fig. 4D). These apparent
differences in capsule cell secretion may reflect the cyclic activity
described previously.
Studies on wild-type parovaria revealed a surprising resemblance in cellular organization to spermathecae, but with a much thinner cuticle. Parovaria are largely made up of gland cells with a similar morphology to those of the spermathecal capsule (Fig. 4E). Each is connected to an end apparatus that appears to contain only a relatively small amount of product. Neither sperm nor the lightly staining material seen in spermathecae is ever present in the lumen.
Our studies of Hr39 mutant females that manage to acquire a spermatheca show that they are much more fertile than their siblings that lack these organs, but that a majority remain sterile (Table 1). Consistent with this observation, rare spermathecae produced by strong Hr39 mutant females (Fig. 1H) show a range of structures when examined under the electron microscope. Some are small, abnormal and contain many necrotic cells but still store sperm (not shown). These probably correspond to the 57% of females with one spermatheca that are still sterile (Table 1). Others, however, are generally normal in structure, and contain secretory material in their end apparati (Fig. 4F, red arrow), although the amount of secretory material is usually less than in wild-type spermathecae. These probably correspond to the fertile females that showed reduced fecundity.
Once again, the behavior of the Hr3904443 mutant females differed from females bearing any of the other alleles. Hr3904443 females contain two and frequently three spermathecae, and their general structure and sperm content was normal (Fig. 5A,B), except for a possible increase in the frequency of dying cells (asterisk). However, detailed examination of the secretory cells suggested that these glands produce little or no secretion. No secretory product was present in their end apparati (Fig. 5C), causing all the cells to resemble inactive normal cells (Fig. 4D). Despite the great reduction in spermathecal secretion, sperm surrounded by normal lightly staining material are present in the lumen of these glands (Fig. 5B), which support nearly wild-type levels of fertility (Table 1).
A likely explanation for this paradox was found when we examined Hr3904443 parovaria, which we noted previously are significantly enlarged (Fig. 1E,F). Hr3904443 parovaria and their end apparati are highly swollen with secretion (Fig. 5D, arrow). These observations suggest that Hr3904443 mutation either directly stimulates increased parovarial secretion, or by blocking spermathecal secretion indirectly induces parovaria to produce a compensating product. All previous data are also consistent with the idea that parovaria can produce a functionally equivalent secretion to that of the spermathecae. The loss of parovaria alone has not been correlated with any defects in female reproduction, whereas in sterile mutations that lack spermathecae, parovaria are also always absent.
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Frequently, the most highly expressed mRNAs within a secretory tissue
encode its secretion products. Consistent with this expectation, wild-type
spermathecae express a small number of genes at higher levels even than most
ribosomal protein mRNAs (Table
2). Eight of the genes (CG17239, CG32834, CG31681, CG32277,
CG17012, CG18125, CG9897 and CG17234) encode serine-type
peptidases, a class of protein whose regulated activity is known to be
important for sperm maturation and fertility
(Friedlander et al., 2001;
Bloch Qazi et al., 2003). These
genes contain candidate signal sequences and their expression in most cases
has not been observed outside the female reproductive tract, consistent with
the idea that their products are part of a tissue-specific secretion. Three
reside in a cluster of five consecutive serine protease genes at 22D5
(Table 2), and include the
three best currently known examples of genes that are induced by mating
(McGraw et al., 2004;
Lawniczak and Begun, 2004;
Lawniczak and Begun, 2007).
RNA in situ hybridization verifies that CG18125 is expressed in
spermathecae (Lawniczak and Begun,
2007), whereas CG17012 is expressed specifically in
spermathecae and parovaria (Arbeitman et
al., 2004). CG32834 and CG9897 define a new
cluster at 59C1, while CG32277 is the only spermathecae-expressed
member of a third serine protease cluster at 63B1. We did not observe
spermathecal expression of one previously reported serine protease in the 22D5
cluster, CG17240 (McGraw et al.,
2004). None of these genes was altered in expression within
Hr3904443 spermathecae.
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) and
follicle cells (Brennen et al., 1982), contribute to yolk production. Again,
the expression of all these abundant gene products with the possible exception
of the serpin CG18525 was unaltered in Hr3904443
spermathecae.
Hr39 regulates genes likely to be involved in secretion
As Hr39 mutation did not affect the most highly expressed genes
within the spermathecae, we looked for genes whose levels were significantly
reduced in the mutant spermathecae (Table
3) to try and understand why secretory products are strongly
reduced in the end apparati of the mutant cells. Two genes, GlcAT-P
and PAPS appear to be particularly strong candidates. Expression of
GlcAT-P, encoding a putative N-acetyllactosamine
β-1,3-glucuronosyltransferase (Kim et
al., 2003) was reduced to one thirtieth of its original levels.
This gene has been implicated in glycoprotein, glycosphingolipid and
proteoglycan biosynthesis. Expression of PAPS synthetase, an
essential step in sulfur metabolism, was reduced to one twenty-fifth of its
original levels. PAPS is required for the production of sulfated
proteins, proteoglycans and lipids. Paps mutations abolish mucus
production in the embryonic salivary gland
(Zhu et al., 2005), indicating
the importance of the gene in this secretory tissue. In addition, expression
of sytIV gene, a gene that functions in synaptic vesicle exocytosis,
is entirely dependent on Hr39. Vesicle exocytosis may be needed for
secretion from spermathecal cells. The expression of several other genes that
may be involved in carbohydrate or lipid metabolism were also reduced to less
than one-tenth of their original levels
(Table 3).
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). Our data
shows that Hr39 controls the expression of six cytochrome P450 genes
in the spermathecae (Tables 3,
4). Cyp4p2 and
Cyp6a14 are each downregulated more than 10-fold. Multiple closely
related cytochrome P450 genes exist in Drosophila and humans, and
each may be able to act on a wide variety of substrates; hence, it is not
possible to predict the biochemical consequences of these changes. The most
closely related human gene to Cyp6a14, for example, is
CYP3A4, a gene expressed in liver and prostate that can oxidize a
variety of small molecules, including steroids. Interestingly, Cyp4g1
and Cyp6a17, genes that are closely related to Cyp4p2 and
Cyp6a14, are strongly increased in expression by Hr39
mutation (Table 4). Another
potential P450 pair is Cyp312a1, whose expression is decreased
2.6-fold, and Cyp305a1, whose expression is increased 3.4-fold by
Hr39 mutation. Thus, the effect of Hr39 mutation is to
modulate the expression levels of specific members of the closely related
cytochrome P450 gene families in the spermathecae.
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). The
expression of another such gene, Opb99b, is also induced by
Hr39 mutation (Table
4). The largest gene expression increase we observed (84-fold) was
of takeout (to). Like Cyp4d21 and Obp99b,
to is normally expressed in male heads, where it plays a role downstream
from the somatic sex-determination genes doublesex and
fruitless in integrating nutritional status and circadian cycle with
courtship behavior (Dauwalder et al.,
2002; Kadener et al.,
2006). The requirement for to is autonomous to fat body,
and To protein, part of a family of juvenile hormone-binding proteins, is
secreted and circulates in the hemolymph of males but not females
(Lazareva et al., 2007). These
changes in gene expression were verified by quantitative RT-PCR and similar
changes were observed in other Hr39 alleles
(Table 5). Our observations
show that Hr39 not only activates spermathecal secretion, a female
characteristic, but represses production of male-specific secretory proteins
in the female reproductive tract.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
Although, the Hr39k13215 mutation reduces Hr39
expression in adults, its effects on spermathecal and parovarial development
were the weakest of any studied Hr39 allele. Differences between the
alleles, which probably result from the insertions blocking promoter access to
multiple enhancers located in the first two introns and from disrupting
splicing, were useful in practice. Although no single allele was completely
null for Hr39 function, Hr39ly92 appeared close
to null for spermathecal development and Hr3907154 was
close to null for adult function. Additional insight into Hr39
function will probably require analyzing double mutants with ftz-f1.
The closely related FTZ-F1 protein may be expressed in tissues where loss of
HR39 did not cause a detectable phenotype, such as in developing ovarian
follicles.
Clearly, the most sensitive tissue requiring Hr39 function is the
anterior genital disc at the time of metamorphosis. Previous studies have
localized the primordial of both spermathecae and parovaria in this region and
documented the rapid growth, migration, eversion and differentiation of
spermathecal and parovarial cells during the first 18 hours after the prepupal
molt (Anderson, 1945;
Keisman et al., 2001). Either
autonomously or non-autonomously, our studies show that these events depend in
a dose-sensitive manner on Hr39 gene action. All of the phenotypic
effects we observed could be explained if the amount of an
Hr39-dependent product influenced the number (and/or behavior) of
progenitor cells in a spermathecal field that arises during early pupal
development, with excess cells leading to additional spermathecae and cell
deficits leading to smaller abnormal glands. The regulation, as well as the
timing, of spermathecal and parovarial development appear to be closely
connected, as evidenced by their common expression and requirement for the
lozenge transcription factor
(Anderson, 1945). Among all the
female genital disc derivatives, parovaria are unique in arising from the
otherwise male-specific A9 segment
(Keisman et al., 2001) and
this may somehow result in the special Hr39 requirement for the
development of both tissues. Fortunately, these developmental issues did not
detract from the usefulness of the Hr39 alleles in studying the roles
played by spermathecae, parovaria and Hr39 in female
reproduction.
Spermathecae and parovaria function as secretory organs and are required at a relatively late step for fertilization
The data reported here strongly argue that spermathecae and parovaria are
redundantly required for female fertility owing to their production of a
secretory product that acts throughout the female reproductive tract.
Fertility correlates strongly with the number of spermathecae
(Table 1), arguing that it is
the presence of this tissue rather than some other defect in the Hr39
mutants that is responsible for their reduced fertility and fecundity.
Moreover, our demonstration that the spermathecae that do form in mutant
animals are frequently still defective in secretion, and that
Hr3904443 mutant spermathecae lack secretion entirely and
have parovaria with increased secretory activity, all support this conclusion.
The observation that at least one major serine protease, CG17012, is
expressed in both tissues (Arbeitman et
al., 2004) provides one example of this redundancy.
Many steps are required before the gametes produced by the ovary and testis
can undergo successful fertilization. After mating, sperm are introduced into
the female reproductive tract along with dozens of proteins (Acps) that
mediate sperm storage and behavior, and can even reduce female lifespan
(reviewed by Bloch Qazi et al.,
2003). Multiple Acps undergo proteolytic processing within the
female reproductive tract, and seminal fluid contains serine proteases and
protease inhibitors (serpins) that may interact with female-produced factors
to regulate this process. At least seven Acps, including four serpins, enter
the sperm storage organs after mating (see
Lawniczak and Begun, 2007).
For example, the male-produced Acp36DE, which is required for efficient sperm
storage (Tram and Wolfner,
1999), can be found in the spermathecae and is proteolytically
processed after transfer to the female
(Neubaum and Wolfner, 1999).
The serpin encoded by Acp62F, which is required for fertility, enters the
spermathecae (Lung et al.,
2002). The many spermathecal secretory proteins we identified,
including at least eight serine proteases and a serpin, are candidates for the
female factor in these interactions. Consistent with this idea, some
spermathecal serine protease genes are induced by mating
(McGraw et al., 2004;
Lawniczak and Begun, 2004;
Lawniczak and Begun, 2007) and
undergo rapid selective evolution
(Lawniczak and Begun,
2007).
Our experiments show that the spermathecal and parovarial secretion acts
after sperm have been transferred to the female reproductive tract and
successfully stored. Hr39 mutant females lacking spermathecae still
mated successfully and stored normal amounts of sperm in their seminal
receptacles, yet they were sterile in the absence of a spermatheca. This
implies that the secretion normally mixes with sperm in the reproductive tract
and acts to make them fertilization competent regardless of their eventual
storage site. It is unclear why these results differed from studies based on
lozenge mutations that suggested a spermathecal requirement for
efficient sperm storage (Anderson,
1945; Boulétreau-Merle,
1977). It is possible that, in the absence of spermathecae and
parovaria, the processing of Acps and of sperm is altered or slowed. These
defects must not prevent storage, but the resulting sperm may remain incapable
of fertilization.
Spermathecae help sperm mature and resemble the mammalian epididymis
These studies suggest new parallels between Drosophila and
mammalian reproductive biology. Following completion of their development
within the testis, mammalian sperm move through the lumen of the epididymis,
where they undergo a complex process of maturation. Epididymal cells secrete
proteases, protease inhibitors, antioxidants, anti-bacterial proteins and
other molecules into the epididymal fluid, and they also take up and modify or
degrade materials shed by sperm (reviewed by
Cooper and Yeung, 2006).
Drosophila sperm are exposed to similar classes of molecules after
transfer to the female and storage in the spermathecae or seminal receptacle.
Thus, the spermathecae and parovaria may play a similar role to that carried
out by the caudal epididymis, where under the influence of products secreted
by epididymal cells, sperm become motile, fertilization competent and can be
stored for long periods. It is possible that the final steps of maturation can
be accomplished in the reproductive tracts of either sex, but that some
advantage exists in carrying them out at the storage site.
Several studies have been carried out on the genes expressed in the
epididymis (Jelinsky et al.,
2007; Johnston et al.,
2007). These include antioxidant glutathione peroxidases, which
are thought to protect against the peroxidation of polyunsaturated fatty acids
within sperm plasma membranes (reviewed by
Drevet, 2006). Drosophila
spermathecae express the similar genes (Prx6005, PHGPx, GstS1 and
CG1633). Two genes comprising the `polyol' pathway are found to be
associated with membranous vesicles in the epididymal fluid known as
`epididymosomes' aldose reductase and sorbitol dehydrogenase
(Frenette et al., 2006).
Sorbitol dehydrogenase 2 is expressed in spermathecae and its transcript level
falls 19 times to undetectable levels in Hr39 mutants. Whether any of
these genes carries out an important function in the spermathecae remains to
be tested genetically.
Spermathecal secretory products may promote sperm capacitation
Mammalian sperm are motile, but still not fully fertilization competent
when they leave the epididymis. In the female they continue to interact with
maternal products, such as the mucins that line the reproductive tract and
retard movement, as well as other products secreted by the reproductive tract
epithelia and the specialized glands it contains, such as Bartholin's gland
(reviewed by Suarez and Pacey,
2006). In addition to secreting molecules that assist in sperm
maturation and preservation, our studies show that spermathecae expressed
genes involved in carbohydrate and lipid metabolism, including two genes,
GlcAT-P and PAPS, that are strongly associated with
glycoprotein, sulfoprotein and lipoprotein secretion. Products dependent on
these genes may enter the reproductive tract, especially at the anterior
uterus where the spermathecae and parovaria connect to the reproductive tract.
This is the region that sperm must traverse en route to the micropyle of the
egg and fertilization. How this process occurs remains almost completely
unknown. However, the presence of specific glycoproteins, glycolipids,
sphingolipids and sulfated molecules might facilitate this final step, and
ensure that sperm arriving at the micropyle are fully capacitated for
fertilization, which, in Drosophila, must be very highly efficient.
Thus, the fertility-essential functions of the spermathecae lie in its
secretion rather than in sperm storage, a view consistent with the presence of
a separate sperm-storage organ and the independent evolution of spermathecae
from these structures (Pitnick et al.,
1999).
Are the roles of Hr39 in female reproductive tract function conserved in evolution?
The similarities between Hr39 expression and function in
Drosophila and SF1 in mammals suggest that these genes play
roles that at least in part have been conserved during evolution. The
expression of Hr39 in reproductive and steroid-producing tissues, in
gonadal duct progenitors that develop differentially between the sexes, and in
regulating cytochrome P450 genes are all strikingly similar to Sf1 or
Lrh1. HR39 function, however, appears to be confined to female
development. Male Hr39 mutants were viable, fertile and apparently
normal. Indeed, the major function of the gene is in the development of
spermathecae and parovaria. Hr39 is also likely to control gene
expression within spermathecae in adults, based on the specific gene
expression defects observed in Hr3904443 spermathecae.
This is analogous to SF1 and steroid hormone-dependent production of numerous
products throughout multiple mammalian reproductive tissues.
Further evidence that Hr39 has not simply evolved a new role in
controlling the spermathecae was our observation that Hr39 mutant
females turn on male courtship genes. Expression of Cyp4d21, takeout
and Obp99b are normally undetectable in the reproductive tracts of
wild type females, but all three are expressed in the fat body of male heads.
Our studies suggest that Cyp4d21 expression might control production
of a male specific steroid in the fat body that is responsible for inducing
the other genes. This pathway might have been retained from a time when
Hr39 played a wider role in controlling reproduction in both sexes.
Perhaps a wider role for a conserved regulatory pathway will be uncovered by
examining the effects of removing both ftz-f1 and Hr39 at
various times during development. However, even if the role of
Sf1-like genes is much more limited in Drosophila than in
mammals, the finding of any conservation has important implications for our
understanding of the evolution of sex-determination mechanisms
(Marin and Baker, 1998).
Our observations that Hr39, like Sf1, controls the
expression of a small set of cytochrome P450 genes, raises the issue of
whether it might act by mediating the production of steroids other than
ecdysone and 20-OH ecdysone. Many other steroids have been found in
Drosophila and other insects, but none has been clearly implicated in
sex-specific reproductive functions (reviewed by
De Loof et al., 1998). By
defining specific biological functions and specific target Cyp genes, it will
now be easier to further investigate the mechanism of Hr39 action,
and to determine whether it involves the production of new steroid
derivatives. Such studies have the potential to significantly deepen our
understanding of how reproduction is regulated and how this regulation
evolved.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/2/311/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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