Development Advance Online Articles. First posted online on 12 December 2007 as 10.1242/dev.015156
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RESEARCH ARTICLE
311
Development 135, 311-321 (2008) doi:10.1242/dev.015156
The Sf1-related nuclear hormone receptor Hr39 regulates
Drosophila female reproductive tract development and
function
Anna K. Allen and Allan C. Spradling*
The vertebrate nuclear hormone receptor steroidogenic factor 1 (SF1; NR5A1) controls reproductive development and regulates the
transcription of steroid-modifying cytochrome P450 genes. We find that the SF1-related Drosophila nuclear hormone receptor HR39
is also essential for sexual development. In Hr39 mutant females, the sperm-storing spermathecae and glandular parovaria are
absent or defective, causing sterility. Our results indicate that spermathecae and parovaria secrete reproductive tract proteins
required for sperm maturation and function, like the mammalian epididymis and female reproductive tract. Hr39 controls the
expression of specific cytochrome P450 genes and is required in females both to activate spermathecal secretion and repress malespecific courtship genes such as takeout. Thus, a pathway that, in vertebrates, controls sex-specific steroid hormone production, also
mediates reproductive functions in an invertebrate. Our findings suggest that Drosophila can be used to model more aspects of
mammalian reproductive biology than previously believed.
INTRODUCTION
Some molecular pathways of sex determination evolve rapidly
whereas others are relatively conserved (reviewed by Marin and
Baker, 1998). 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 TGF␤ family
member that causes the oviduct precursors to degenerate.
Drosophila reproductive tract precursors also develop in a sexspecific manner within the bipotential genital disc (Keisman et al.,
2001), but a corresponding genetic pathway has not been found. In
Howard Hughes Medical Institute, Department of Embryology, Carnegie Institution,
3520 San Martin Drive, Baltimore, MD 21218, USA.
*Author for correspondence (e-mail: [email protected])
Accepted 24 October 2007
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.
DEVELOPMENT
KEY WORDS: Hr39, Spermathecae, Reproductive tract, SF1 (NR5A1), Steroid hormone
RESEARCH ARTICLE
MATERIALS AND METHODS
Fly strains
Fly stocks were maintained at 20-25°C on standard cornmeal-agar-yeast
food. The yw strain was used as a control in all experiments and to generate
transgenic flies. The Hr39 alleles were isolated in three different single Pelement mutagenesis screens as described in Table S1 (see supplementary
material). The Hr39k13215 line was obtained from Carl Thummel and is
described in Horner and Thummel (Horner and Thummel, 1997).
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/␮l DNase overnight (Ambion) according to manufacturer’s
instructions. One-Step RT-PCR (Qiagen) was then performed using 0.5 ␮g
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 0.5⫻ TBE buffer with 0.25 ␮g/␮l 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 ␮l reaction comprising
0.25 ␮g total RNA and 0.25 ␮l of a 7.5⫻ 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-␤-gal (pre-absorbed
against lower reproductive tracts or ovaries, 1:1000) (Cappel), mouse-antiFas2 (1:2) (1D4, Developmental Studies Hybridoma Bank), mouse-antiDac (1:200) (Abdac2-3, Developmental Studies Hybridoma Bank), and
Development 135 (2)
mouse-anti-Wg (1:50) (4D4, Developmental Studies Hybridoma Bank).
Secondary antibodies were used at 1:500 and are as follows: goat antirabbit conjugated to Alexa 488 and goat anti-mouse conjugated to Cy3
(Molecular Probes). For DNA labeling, DAPI was added 1 ␮g/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 20⫻ (NA 0.70) or a 40⫻ (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 ␮g lower
reproductive tract RNA or 2 ␮g 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).
RESULTS
Hr39 mutations affect female fertility
To investigate the role of Hr39 in female gametogenesis, we
analyzed five mutations (see Table S1 in the supplementary
material) isolated as causing female sterility in three different single
P-element screens that contain insertions within the differentially
spliced Hr39 transcription unit (Fig. 1B). Complementation tests
showed that these mutations are allelic (not shown), and that they
also fail to complement a specific deficiency, Df (2L)Exel6048 (see
Fig. S2 in the supplementary material), that deletes all Hr39-coding
sequences but not upstream sequences. Homozygous females from
each line produce apparently normal mature oocytes, but females
bearing four of the alleles are usually sterile (Hr39ly92, Hr39neo8,
Hr3903508, Hr3907154) whereas Hr3904443 and a sixth allele
Hr39k13215 (Horner and Thummel, 1997) 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).
DEVELOPMENT
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Hr39 regulates spermathecae and parovaria
RESEARCH ARTICLE
313
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).
Our results reveal an extremely strong correlation between
spermathecae and fertility, such that possession of even 1
spermatheca is associated with a many-fold increase in fertility and
fecundity, while possession of two spermathecae engenders near
wild-type fertility. Whether parovaria alone can support fertility
could not be determined from these data because we never recovered
females that possess a parovarium but no spermatheca. Some
females contained spermathecae that were smaller and
morphologically abnormal; such organs were included in the counts.
Despite this, we suspect that the very low level of residual fertility
in females ‘lacking’ both spermathecae and parovaria (8% fertility,
with a fecundity of only four progeny), is due to rare females that
contain tiny defective spermathecae or parovaria that escaped
detection, but that retain a low level of function. Combining the
information in Fig. 1H and Table 1 explains why the Hr39 insertions
were originally isolated as female sterile mutations.
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 3to 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.
Fig. 1. Hr39 mutants alter spermathecal and parovarial
development. (A) The female lower reproductive tract (RT). (B) Hr39
transcript structure showing the four different isoforms with insertion
mutations indicted above. (C-G) Nomarski images of RT. All RT images
possess a single seminal receptacle (asterisk). (C) Wild-type RT with two
spermathecae and two parovaria (one out of focus). (D) Hr3903508 RT
with no spermatheca or parovarium. (E,F) Magnifications of parovaria
(circled); (E) wild type, (F) Hr3904443. (G) Hr3904443 RT with three
spermathecae and two parovaria. (H) Percentage of homozygous
females of each genotype possessing 0, 1, 2 or 3 spermathecae.
Arrows, spermathecae; arrowheads, parovaria; asterisks, seminal
receptacles. Scale bar: 100 ␮m in C,D,G.
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
Table 1. Spermathecae are required for fertility
Number of spermathecae
0
1
2
3
Number of parovaria
Fertility (%)
Fecundity (progeny/fertile female)
Number analyzed
0
0 (92%) 1 (8%)
2
2
8
43
94
99
4
31
100
92
344
45
42
67
Fertility tests over a 5-day interval were carried out on individual 5- to 10-day-old mated Hr39 mutant females, after which the number of spermathecae and parovaria were
determined. The genotypes of the females that fell into each category are given in Table S2 (see supplementary material).
DEVELOPMENT
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
RESEARCH ARTICLE
Fig. 2. Hr39 expression. (A) Hr39 expression levels in mutants using
RT-PCR. As an internal control, RpS17 was amplified. (B) Comparison of
Hr39 expression among strains using real-time quantitative RT-PCR.
Standard deviation of each triplicate (±s.d.) is shown. (C-E,L-N) Green
indicates Hr39-lacZ detected via anti-␤-gal antibody. Magenta indicates
DNA. (F-K) Hr39 in situ hybridization (green) (C) Larval ring gland,
Hr3903508/+. Blue indicates anti-Fas2. Scale bar: 75 ␮m. (D) Female
genital disc, Hr3903508/+. Scale bar: 50 ␮m. (E) Female larval gonad,
Hr3907154/+. Scale bar: 50 ␮m. Wild type (F-J); Hr3904443 (K,L).
(F) Reproductive tract; (G) parovaria; (H) seminal receptacle;
(I) spermathecae-associated fat body; (J-L) spermathecae.
(M) Hr3903508/+ ovariole. Scale bar: 100 ␮m. (N) Hr3903508/+ testis.
Scale bar: 100 ␮m. SR, seminal receptacle; SP, spermatheca; P,
parovarium; O, oviduct; FB, fat body.
Engrailed, Wingless, Dachshund or Abdominal-B using specific
antibodies (see Fig. S3 in the supplementary material; data not
shown).
Consequently, to investigate the tissue-specificity of Hr39
expression and to verify that the insertions in the sterile alleles alter
Hr39 expression, we analyzed Hr39 RNA in adults. A significant
level of Hr39 transcripts was detected in RNA from adult females
(and lower levels in adult males) when analyzed using RT-PCR
targeting a common region within the known transcript isoforms
(Fig. 2A,B). Females bearing each of the mutations contained
significantly reduced Hr39 transcript levels, as expected. The
Hr3904443 allele behaved genetically like a hypermorph with respect
to spermathecal and parovarial production (see Fig. S2 in the
supplementary material). Hence, an increase in Hr39 expression is
expected in the genital disc or some other larval or early pupal tissue;
Development 135 (2)
Fig. 3. An Hr39 cDNA rescues Hr39 mutations and expands
spermathecal number. Nomarski images of adult female reproductive
tracts of different genotypes. (A) yw; hsp70GAL4::UAS-Hr39(7-3) with
three spermathecae (SP). (B) Hr39ly92; hsp70GAL4::UAS-Hr39(7-5) with
one SP. (C) Hr3907154; hsp70GAL4::UAS-Hr39(7-5) with two abnormal
SP. (D) Hr3903508; hsp70GAL4::UAS-Hr39(7-5) with two SP.
(E) Hr3903508; hsp70GAL4::UAS-Hr39(7-3) with three SP. (F) Hr3903508;
hsp70GAL4::UAS-Hr39(7-3) with four SP. Red arrows indicate SP.
however, this fact does not contradict the observed reduction in adult
expression levels. The expression and independent regulation of
Hr39 in adults suggests that this gene functions in adults as well as
during development.
Individual tissues expressing Hr39 were identified using wholemount 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,
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315
presumably owing to the longer perdurance of the ␤-galactosidase
protein (Fig. 2J-L). Possible cyclic activity of these cells has been
noted previously (Filosi and Perotti, 1975).
Hr39 mutations can be rescued by expressing Hr39
cDNA
We carried out rescue experiments to verify that the defects in
fertility and reproductive tract development within the Hr39
insertion strains are caused by alterations in this gene. Both the
spermathecal, parovarial and fertility defects of all four strong Hr39
mutants were rescued in the presence of an hsp70GAL4 driver and
a full-length Hr39-RA cDNA under control of a UAS promoter (Fig.
3; see Table S3 in the supplementary material). Sometimes
spermathecal development was incompletely rescued as only one
spermatheca (Fig. 3B) or two smaller glands (Fig. 3C) were restored.
Elevated Hr39 expression affected spermathecal number, as even
wild-type files bearing these constructs frequently contained extra
spermathecae (Fig. 3A). Furthermore, applying heat shocks during
larval development to Hr39 mutants that also contained
hsp70GAL4::UAS-Hr39 frequently caused the number of
spermathecae (but not of parovaria) to increase to three (Fig. 3D,E)
or four (Fig. 3F). Although Hr39-RA overexpression affects
spermathecal number in a similar manner as Hr3904443 mutation, it
differs in that the parovaria are not enlarged. Differences in the
cellular locations and particular Hr39 isoforms overproduced under
these two conditions probably account for this discrepancy.
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
DEVELOPMENT
Fig. 4. Hr39 controls spermathecal secretion.
Electron microscopy images of wild-type and Hr39
mutant spermathecae (SP) and parovaria (P).
(A-D) Wild-type SP. (A) Gland cells (red outline)
surround the lumen (L). N, nucleus; EA, end apparatus.
Scale bar: 10 ␮m. (B) SP lumen with sperm (S)
surrounded by extracellular material (M). Scale bar:
2 ␮m. (C) EA in active state showing secretion. Scale
bar: 2 ␮m. (D) EA (white outline) showing little
secretion among the microvilli. Scale bar: 2 ␮m.
(E) Wild-type parovaria with little secretion. Scale bar:
10 ␮m. (F) Hr3907154 SP containing less secretory
product than wild type (C). Scale bar: 2 ␮m. Red arrows
indicate SP secretion; L, lumen; M, luminal material;
S, sperm.
RESEARCH ARTICLE
Fig. 5. Hr3904443 spermathecae lack secretory material and
parovaria contain extra secretory material. (A-C) Hr3904443
spermathecae (SP). (A) Sperm remain in lumen (L) but cell death
(asterisk) is increased; gland cell, red outline. Scale bar: 10 ␮m.
(B) Lumen with sperm (S) surrounded by extracellular material (M).
Scale bar: 2 ␮m. (C) Secretion appears absent from end apparatus (EA).
Scale bar: 1 ␮m. (D) Hr3904443 parovaria. Secretion (arrows) appears to
fill the end apparati (EA). Scale bar: 10 ␮m. N, nucleus.
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.
Spermathecae express genes that may modify
and capacitate sperm
To further characterize the biology of spermathecae and the role of
Hr39, we carried out gene expression studies on young females 3
days after mating to wild-type males. RNA was isolated from more
than 2000 hand dissected wild-type spermathecae, and from 1000
derived from Hr3904443mutant females. Unfortunately, it was not
feasible to carry out a similar isolation of the rare spermathecae from
other Hr39 mutants or from parovaria. However, for comparison we
prepared RNA from the remainder of the female reproductive tract
(RT), i.e. oviducts, uteri and seminal receptacles from each
genotype. Parovaria were also present in this material, but are
expected to contribute a very small fraction of the RNA. The genes
expressed within these unamplified RNA populations were analyzed
using Affymettrix gene chip 2 arrays and the highly reproducible
results were analyzed without any further mathematical
manipulation (see Materials and methods).
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
Development 135 (2)
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 spermathecaeexpressed 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.
Our microarray study greatly expands the number of genes known
to be expressed in spermathecae. Several other abundant gene
transcripts encode members of protein classes that have also been
implicated in sperm maintenance, including anti-microbial proteins,
antioxidants and serpins (Table 2). Surprisingly, all three yolk
protein genes (YP1-YP3) are also highly expressed in spermathecae
(Table 2) and reproductive tract (not shown), suggesting that these
tissues, like fat body (Barnett et al., 1980) 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 ␤-1,3glucuronosyltransferase (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).
Hr39 modulates specific cytochrome P450 genes
A major mechanism of SF1 action is the modulation of levels of
steroid-modifying cytochrome P450 genes, which leads to changes
in the identity and levels of steroid hormones (Val et al., 2003). Our
DEVELOPMENT
316
Hr39 regulates spermathecae and parovaria
RESEARCH ARTICLE
317
Table 2. Genes highly expressed in spermathecae (SP)
Gene
Wild type*
04443†
Comments‡
19,200
17,100
16,300
16,700
16,100
15,200
15,000
13,600
17,800
17,600
15,600
16,900
16,100
14,800
15,500
13,500
Most abundant in SP; 22D5 cluster
Sixth most abundant in SP; 59C1 cluster
Seventh most abundant in SP; 22D6
Eleventh most abundant in SP; circadian cluster
Twelfth most abundant in SP; 22D5 cluster
Eighteenth most abundant in SP; 35A4
Nineteenth most abundant in SP; 59C1 cluster
Twenty-fifth most abundant in SP; 22D5 cluster
18,400
17,200
16,700
14,400
12,600
11,600
10,400
17,600
16,600
15,900
14,400
11,000
11,800
5750
Ca2+-binding domain; ecdysone inducible
Also enriched in testis (Fly Atlas)
Also expressed in head, male accessory gland, larval fat body (Fly Atlas)
Anti-fungal protein; circadian regulated
Chaperone for steroid receptor action
Anti-oxidant, anti-toxin gene
Serpin (protease inhibitor)
12,300
10,200
13,200
10,600
9580
12,300
8120
8160
Serine protease
CG17239
CG32834
CG31681
CG32277
CG17012
CG18125
CG9897
CG17234
Other
CG6426
CT33755
CG18067
CG10810
Hsc70-4
GstD1
CG18525
Yolk proteins
YP1
YP2
YP3
Average ribosomal protein gene
*Expression level in wild-type spermathecae (average.)
†
Expression level in Hr3904443 mutant spermathecae (average).
‡
Fly Atlas (Chintapalli et al., 2007).
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
Table 3. Genes induced by Hr39 in spermathecae
Gene
Wild type*
04443†
1440
4000
200
47
158
1.4
N-acetyllactosamine ␤-1,3-glucuronosyltransferase
Adenylsulfate kinase
Synaptotagmin IV
31
25
160
2280
398
509
274
24
21
23
27
Glycosylated, cell external, GPI-linked lipid-binding
Sorbitol dehydrogenase-2
Glutaminase
Amylase
93
19
22
10
263
3100
510
5.8
198
195
202
1890
1590
1610
516
3050
11
98
88
42
11
139
Comments
Ratio‡
Secretion genes
GlcAT-P
PAPS
sytIV
Carbohydrate, lipid genes
Nlaz
Sodh-2
nemy
Amy-d
P450 genes
45
16
2.6
Other
Tequila
GstE1
AttC
drl
CG10211
CG13935
Serine-type endopeptidase, chitin-binding
Glutathione transferase, defense, stress response
Anti-bacterial peptide; upregulated by sperm (McGraw et al., 2004)
33% identical to mouse RYK (5E-90)
Myeloperoxidase-like
Cuticle structural protein
*Expression level in wild-type spermathecae (average).
†
Expression level in Hr3904443 mutant spermathecae (average).
‡
Fold change: i.e. wild-type expression level/Hr3904443 mutant expression level.
18
19
18
38
47
22
DEVELOPMENT
Cyp4p2
Cyp6a14
Cyp312a1
318
RESEARCH ARTICLE
Development 135 (2)
Table 4. Other Hr39-regulated genes in spermathecae (SP) and/or reproductive tract (RT)
Gene
Wild type*
04443†
Comments‡
Ratio§
4.3
154
76
849
373
1280
492
2880
In SP
In SP
In SP
In SP
87
8
6
3.4
77
32
42
823
1620
928
In SP: cuticle structural protein
In SP: stress-induced defense protein
In SP: anti-bacterial defense protein
11
50
22
49
214
13
106
1840
2250
4090
5690
184
8
41
39
In RT; male fat-specific in head, secreted
In RT: male fat-specific in head
In RT: male fat-specific in head
36% identical to human CYP4V2 (1E-63)
In RT: Pheromone-binding protein
In RT: anti-bacterial peptide
84
27
13
14
45
58
P450 genes
Cyp6a17
Cyp4g1
Cyp6w1
Cyp305a1
Other
CG7076
TotM
CG6429
RT genes
takeout (to)
Cyp4d21
Obp99b
Cyp4p2
Pbprp1
AttC
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.
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.
Hr39 represses male-specific genes that mediate
male courtship behavior
When the genes that are highly regulated in reproductive tract (Table
4) were compared with those in the spermathecae (Table 3),
additional evidence of an SF1-like mode of action was observed.
Cyp4p2 is also downregulated in reproductive tract (14-fold), but in
this tissue, Cyp4d21, rather than Cyp4g1, is strongly upregulated
(27-fold) to become the Cyp gene with the highest level of
expression in this tissue. Also known as sex-specific enzyme 1
(sex1), Cyp4d21 is normally expressed selectively in the fat cells
within male but not female heads (Fujii and Amrein, 2002). 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
DISCUSSION
Hr39 functions in female reproduction
These studies show that the nuclear receptor encoded by Hr39 is not
a redundant gene, but is essential for the development of
spermathecae and parovaria. Previously, a genetic requirement for
this gene was not detected through studies of the Hr39k13215 allele
(Horner and Thummel, 1997). 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
Table 5. qRT-PCR validation of candidate Hr39-regulated genes from microarray
takeout
Allele
ly92
03508
04443
07154
Micro*
84F
qRT-PCR†
62F
24F
131F
573F
Cyp4d21
Micro*
27F
AttC
qRT-PCR†
12F
38F
40F
184F
Micro*
Pbprp1
qRT-PCR†
Micro*
2f
58f
53f
2f
45f
*Average fold change detected by microarray between wild type and Hr3904443 in reproductive tract.
†Fold change detected by quantitative RT-PCR between wild type and the indicated Hr39 mutant allele in reproductive tract.
Hr39
qRT-PCR†
90f
198f
61f
165f
Micro*
2f
qRT-PCR†
20f
143f
18f
393f
DEVELOPMENT
*Expression level in wild type (average).
†
Expression level in Hr3904443 mutant (average).
‡
SP (spermathecae); RT (reproductive tract).
§
Fold change: i.e. wild type/Hr3904443 or Hr3904443/wild type, whichever is larger.
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
RESEARCH ARTICLE
319
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
DEVELOPMENT
Hr39 regulates spermathecae and parovaria
RESEARCH ARTICLE
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 fertilityessential 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 hormonedependent 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 sexdetermination 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.
The authors thank Carl Thummel for providing the Hr39k13215stock, Mike
Sepanski for assistance with electron microscopy, and members of the
Spradling laboratory for comments on the manuscript and helpful discussions.
A.C.S. is an Investigator of the Howard Hughes Medical Institute.
Development 135 (2)
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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Hr39 regulates spermathecae and parovaria