Glucose dehydrogenase is required for normal sperm storage and utilization in female Drosophila melanogaster
Department of Biology, The Pennsylvania State University, 208 Mueller Laboratory, University Park, PA 16802, USA
* Author for correspondence (e-mail: drc9{at}psu.edu)
Accepted 28 November 2003
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Summary |
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Key words: sperm storage, sperm utilization, spermathecae, glucose dehydrogenase, Drosophila melanogaster
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Introduction |
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In D. melanogaster, of the several thousands of sperm transferred
to a female in one mating (Gilbert,
1981b), some are immediately used or stored for later use, but the
majority are simply discarded. Because storage capacity is limited, females
normally store only
20-25% of the transferred sperm in two types of sperm
storage organs: the spermathecae and the seminal receptacle
(Lefevre and Jonsson, 1962
;
Fowler, 1973
;
Gilbert, 1981b
). Immediately
after mating,
80% of the stored sperm are found in the seminal
receptacle, a coiled tube-like organ
(Lefevre and Jonsson, 1962
;
Fowler et al., 1968
). The rest
of the stored sperm (
20%) are kept in the spermathecae, a pair of
capsules each connected to the anterior end of the uterus by a thin duct. Of
the two types of organ, the spermathecae of Drosophila is considered
as the long-term storage organ (Filosi and
Perotti, 1975
; Gilbert,
1981a
). In contrast to the seminal receptacle, the spermathecae of
all Drosophila are surrounded by glandular cells that secrete fluid
into the lumen of the capsules (Filosi and
Perotti, 1975
). It has been suggested that the secretory fluid may
enhance sperm longevity in spermathecae
(Filosi and Perotti, 1975
;
Pitnick et al., 1999
). Indeed,
females lacking spermathecae have a shorter period of fertility and produce
fewer progeny during the fertile period
(Anderson, 1945
;
Boulétreau-Merle,
1977
).
One of the molecules known to be required for sperm storage is Acp36DE, a
male seminal fluid protein secreted in the male accessory gland and
transferred to the female during mating
(Neubaum and Wolfner, 1999).
Females that mate with a male having a deficiency in accessory gland secretion
store only 10% of normal storage levels in both the seminal receptacle and the
spermathecae (Tram and Wolfner,
1999
). However, the mechanisms of the female contribution to sperm
storage are largely unknown. Here, we investigate the role of glucose
dehydrogenase (GLD), one of the proteins secreted into the lumen of the
spermathecal ducts of D. melanogaster females
(Schiff et al., 1992
).
GLD is expressed in a variety of arthropods. This enzyme is required for
eclosion of adult fruit flies, and its expression in the reproductive tracts
at the adult stage is conserved among 50 Drosophila species
previously examined (Schiff et al.,
1992). It has been suggested that the production of free radicals
in the GLD-involved pathway may be responsible for weakening the puparium case
during eclosion (Cox-Foster and Stehr,
1994
), but the catalytic mechanism of GLD in the reproductive
organs is unknown. The expression level of Gld during development is
regulated by ecdysone, which is released during larval molts
(Murtha and Cavener, 1989
).
Throughout pre-adult development, Gld is expressed in many
epidermally derived tissues, including some of the somatic reproductive organs
(Cox-Foster et al., 1990
).
However, at the adult stage, Gld expression is completely absent in
non-reproductive tissues and restricted to only a subset of those reproductive
organs that express Gld at the pre-adult stage
(Schiff et al., 1992
;
Keplinger et al., 2001
).
Although adult female expression patterns in different species vary in the
oviduct, seminal receptacle and parovaria, the expression of Gld in
the spermathecae is highly conserved
(Schiff et al., 1992;
Fig. 1). This may underlie a
fundamental function of Gld for sperm storage in the spermathecae of
the females. In addition, male flies of the melanogaster subgroup, including
D. melanogaster, secrete high levels of GLD into the lumen of the
ejaculatory duct and subsequently transfer it to females during copulation.
Substances transferred to a female along with sperm have been shown to have
diverse functions in insects. In addition to sperm storage
(Neubaum and Wolfner, 1999
;
Tram and Wolfner, 1999
), these
functions include nutrient supplementation, reduction of a female's
receptivity to subsequent mating, protection of females and eggs from
predators, enhancement of oviposition (reviewed by
Chapman, R. F., 1998
;
Chapman, T., 2001
;
Gillott, 2003
) and sperm
competition (e.g. Scott and Richmond,
1990
; Harshman and Prout,
1994
; Price et al.,
1999
; Gilchrist and Partridge,
2000
). The conserved expression pattern in the spermathecae and
the transfer of GLD to females in some species suggest that GLD may enhance
female fertility by influencing sperm storage in the spermathecae.
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We hypothesized that if GLD enhances sperm storage, then Gld-null
mutant females would have reduced fertility. A preliminary experiment showed
that there was no significant difference in fertility between
Gld-null mutant and wild-type females when virgin males were used for
crosses. However, consecutive mating might have a significant effect on female
fertility due to a reduced amount of sperm and seminal proteins transferred
from a male. For example, the complete recovery of the GLD level in males
after one mating takes more than 24 h, and even at 12 h post-mating the GLD
activity level in males is still half of the pre-mating level
(Cavener and MacIntyre, 1983).
We speculate that this probably applies to other seminal proteins transferred
during mating and that GLD may facilitate sperm storage under conditions of
low sperm load where the amount of sperm and seminal proteins transferred is
not saturating the sperm storage organs of females. These conditions are
probably common in the natural ecology of Drosophila.
Here, we examine sperm storage and usage in Gld-null mutant and heterozygous females upon mating to virgin males or once-mated males. We show that GLD may facilitate the sperm uptake and release through the spermathecal ducts. The sperm distribution in the two capsules of the spermathecae was highly asymmetrical in the absence of GLD, and the quantity of the stored sperm in the spermathecae was significantly reduced in the Gld-null mutant females when they were crossed with a previously mated male. In addition, the Gld-null mutant females used stored sperm at a slower rate over a longer period compared with wild-type females, suggesting that GLD might also be required for efficient release of sperm from the spermathecae.
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Materials and methods |
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For the sperm usage comparison, virgin females of Gld-/- (Gld[4.11]/Gld[P4L]) and Gld+/- (Gld[4.15]/Gld[P4L]) were crossed with Gld-/- virgin males (Gld[E1L]/Df dsx[M+R2]). Gld[4.15] carries a copy of the Gld gene but shares the same genetic background as Gld[4.11], which carries a null mutation of Gld. The Df dsx[M+R2] mutation is a deletion of the entire Gld gene. The Gld-/- mutants, which are normally unable to eclose, were rescued by cutting open their puparium cases as late pharate adults. All flies used were 3-7 days old and thus fully competent for mating and reproduction.
Sperm storage comparison
In order to reduce the load of both sperm and seminal fluid proteins, we
crossed the same Gld mutant male twice consecutively to different
virgin females. Immediately after the first mating, the male was transferred
to a fresh vial and placed with another virgin female for the second mating.
The mean time interval between the first and second matings was 102 min, and
the median was 70 min (see Results).
At the end of each mating, the male was removed from the vial and, at 1-2 h
after mating, the seminal receptacle and spermathecae of the female were
dissected in 60% acetic acid. Each organ was placed onto a slide (seminal
receptacle was uncoiled) and stained with 2% orcein in 60% acetic acid
(Lefevre and Jonsson, 1962;
Gilbert, 1981b
). The organs
were pressed gently under a cover slip; the spermathecae were squeezed until
the capsules were opened. The number of sperm was counted twice for each
sample under a light microscope. Samples that had no sperm in any of the
storage organs were eliminated from the analysis because it indicated that no
sperm transfer had occurred during mating.
For the analysis of sperm distribution in the two spermathecal capsules, we
divided the capsules into two classes for each female: a `min' capsule
(containing fewer sperm) and a `max' capsule (containing more sperm). A
2 test was performed to test a departure of the sperm
distribution in the two capsules from the expected 1:1 ratio. The females
containing the small number of sperm in the spermathecae were pooled to
achieve the appropriate expected frequency for a
2 test
(>5).
Sperm usage comparison
Virgin females of Gld-/- and Gld+/- were crossed with
virgin Gld-/- males. Immediately after mating, a female was
transferred to a fresh vial and allowed to oviposit for 2 days. The females
were continuously transferred to a fresh vial every other day until they
completely stopped producing progeny. The number of fully developed adult
flies was counted for each vial. The samples that had fewer than 100 total
progeny were eliminated from the analysis because it may indicate that the
sperm transfer was not complete or that the female reproductive organs might
be defective and because low density of larvae in culture is correlated with
the high mortality.
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Results |
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Both mutant and wild-type females were crossed either with a virgin male (first mating) or with a previously mated male within a few hours of the first mating (second mating). The latter cross was set up to examine the effect of reducing the amount of sperm and seminal proteins transferred to females. The second mating typically occurred within 2 h of the first mating [71% (15 of 21) in the Gld-/- and 64% (7 of 11) in Gld+/- females], and neither genotype group showed any correlation between the length of mating intervals and the amount of sperm stored in the second mating samples. Therefore, we included all second mating data in our analyses.
Sperm storage
In the first mating, the Gld-/- females stored a similar amount of
sperm compared with the wild-type females
(Table 1). In the second
mating, although both genotypes showed a reduction in sperm storage to some
extent, the spermathecal storage in the Gld-/- females was severely
reduced by 76.7% on average compared with an average reduction in the
wild-type females of only 19.8%. The number of sperm stored in the
spermathecae of the Gld-/- females (mean ± S.E.M.,
28±8 sperm) was significantly lower than the number observed in the
wild-type females (89±13 sperm; t-test, P=0.00018;
Table 1). It should be noted
that the amount of sperm stored in the seminal receptacle was not
significantly different between the Gld-/- and wild-type females.
This indicates that the absence of GLD may significantly affect the
spermathecal storage in the second mating.
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Distribution of sperm stored in the two spermathecal capsules
When further examining the difference in spermathecal storage between the
Gld-/- and Gld+/- females, we first noticed that there were
many cases where one of the two capsules contained zero sperm in the
Gld-/- females following the second mating whereas there was no such
case found in the wild-type female of the second mating. After eliminating two
cases that had zero sperm count in both capsules, 11 of 19 Gld-/-
females following the second mating contained zero sperm in one of the two
capsules. Remarkably, in three cases, no sperm were found in one capsule but
more than 90 sperm were found in the other.
This indicated that the ratio of sperm storage between the two capsules of
the Gld-/- females grossly departed from the expected 1:1 ratio. To
analyze this storage asymmetry further, we performed a 2 test
on each female with the expected ratio of 1:1. In both first and second
matings, more than 75% of the Gld-/- females had a significant
departure from the 1:1 ratio in the spermathecal storage at P=0.05
(Table 2). By contrast, only
30% of the wild-type females showed a significant departure from the 1:1
ratio.
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Although the Gld-/- females exhibited a marked asymmetry in spermathecal storage following the first mating, the sperm amounts in both capsule classes (min and max) were not significantly different from those in the wild-type females. However, in the second mating, both capsule classes had a significant reduction in the Gld-/- females (min capsule mean, 3±1 sperm; max capsule mean, 28±8 sperm) as compared with the wild-type females (min, 35±7 sperm; max, 53±8 sperm). Thus, GLD affects the sperm distribution between the two spermathecal capsules in both first and second matings, whereas the reduction in the quantity of stored sperm is significant only in the second mating.
Sperm utilization
Gld-/- mutant Drosophila males and females are fertile
(if rescued), and female mutants do not exhibit any reduction in the number of
stored sperm after mating to a virgin male. However, reproductive success may
also be impacted by sperm utilization, which is reflected in the number of
progeny resulting from a mating. To examine further the role of GLD in sperm
usage of the females following the first mating, we counted the number of
progeny produced by the mutant and wild-type females in 2-day periods after
mating until the stored sperm was completely exhausted. Interestingly, we
found that, on average, the mutant females produced offspring over a longer
period (11 days) than the wild-type females (7 days) due to a reduced rate of
sperm usage in the mutant females (Fig.
2). This suggests that, in addition to its role in sperm storage
in the spermathecae, GLD may enhance the release of sperm from the
spermathecae.
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Discussion |
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Asymmetry of sperm storage in the two spermathecal capsules
GLD may also facilitate an equal distribution of sperm into the two
spermathecal capsules. Asymmetry in the number of sperm stored in the two
capsules has been investigated in Mediterranean fruit flies Ceratitis
capitata (Taylor et al.,
2001). According to this study, the degree of asymmetry in the
Mediterranean fruit flies becomes more apparent when the total sperm storage
is smaller. However, in our study of D. melanogaster, there is no
correlation between total sperm storage and the percent difference in the
sperm amount stored in the two capsules of the Gld mutant females. In
fact, asymmetry was observed even in the mutant females from the first mating,
when the quantity of both spermathecal and total sperm storage was at a level
comparable with that of wild-type females. Therefore, we conclude that the
observed asymmetrical distribution of sperm is due to the absence of GLD and
is not a secondary effect of the level of total sperm storage. We propose that
GLD may facilitate the initial catalysis of sperm uptake, and thus, in the
absence of GLD, the probability that sperm uptake would be initiated in a
particular spermathecae would be dramatically decreased. Moreover, we propose
that this initiation event is controlled by the localized expression of GLD in
the distal end of each spermathecal duct adjoining the uterus, and therefore
initiation of sperm uptake in the two spermathecae would be independent of
each other, giving rise to the observed asymmetry in sperm storage in the
Gld-/- mutant female.
Effects of consecutive mating
GLD may be the main factor regulating sperm distribution into the two
spermathecal capsules because we observed the asymmetrical storage in both
first and second matings in the absence of GLD. By contrast, the quantity of
spermathecal storage was affected when females mated with males that had
previously mated within a few hours. This indicates that GLD may enhance sperm
uptake in a synergistic way with other factors affected by consecutive mating,
such as the amount of sperm and seminal fluid proteins transferred from the
male to the females. However, Lefevre and Jonsson
(1962) reported that the
numbers of progeny produced from the first and second consecutive matings were
similar and that a significant reduction in progeny can be first observed at
the third consecutive mating. Their data are consistent with our results that
the total sperm storage was not significantly different in the wild-type
females following first and second matings. There are two possible
explanations for little difference between the first and second matings. One
is simply that the transfer load is not different between the first two
matings. The other possibility is that the transfer load does change but that
the change is not reflected in the amount of stored sperm or resultant progeny
due to a limited sperm storage capacity (the average storage is
1000
sperm; Gilbert, 1981b
;
Tram and Wolfner, 1999
).
Because a male transfers 3000-5000 sperm to a female in one mating
(Kaufmann and Demerec, 1942
;
Gilbert, 1981b
), even a 50%
reduction in the number of sperm transferred does not impact on the amount of
sperm stored in a wild-type female. Therefore, it is possible that transfer
load decreases in the second consecutive mating to a level that is still
beyond the maximum storage capacity. Moreover, the complete recovery of
seminal proteins in males after one mating may take hours, as it takes at
least 24 h for the male GLD level to be completely recovered after one mating
(Cavener and MacIntyre, 1983
).
Thus, the `invisible' reduction of sperm and also a possible great reduction
of seminal fluid proteins in a consecutive mating could make sperm storage
significantly less efficient in the absence of GLD. We speculate that this
level of reduction in transfer load is common in nature and that GLD may be
critical to maintain the efficiency of reproduction in
Drosophila.
Males of D. melanogaster transfer GLD to females upon mating. Therefore, we would expect that crossing a Gld-null female to a wild-type male would restore the spermathecal storage level and storage symmetry. It would be interesting to see if consecutive mating has the same effect on sperm storage following this cross. Because consecutive mating decreases the amount of GLD transferred to a female, spermathecal storage might still be affected in Gld-null females following the second or third matings as compared with the control females that express GLD.
The slower rate of sperm utilization in Gld-null females
The second process that may be enhanced by GLD is sperm release from the
spermathecae. The total numbers of resultant progeny and initially stored
sperm following the first mating were not substantially different between the
mutant and wild-type females, but the sperm usage pattern in fertilization was
distributed over a longer period of time in the mutant females from the first
mating. This suggests that GLD may control the timing of sperm usage by
enhancing the release of sperm from the spermathecae, independent of a
synergistic effect with transfer load. It is likely that this control of
timing could occur only for the sperm stored in the spermathecae because GLD
is not expressed in the seminal receptacle in adult females of D.
melanogaster (Schiff et al.,
1992). Our study showed that the mutant females produced 41% more
progeny on average than wild-type females, and the observed delay in the
exhaustion of sperm in the mutant females could be explained by taking a
longer time to use up a larger amount of sperm. However, when we limited the
data to a subset that had 200-400 progeny in total, we still observed the same
trend in which the mutant females (N=9) took 11 days on average to
exhaust stored sperm whereas the wild-type females (N=19) took only 7
days. Thus, we conclude that the observed slower rate in sperm usage is due to
the absence of GLD rather than the size of total storage. We propose that the
release of sperm from the spermathecae may be controlled or enhanced by the
GLD expressed in the proximal end of the spermathecal ducts, while the
aforementioned sperm uptake event is controlled by the GLD in the distal end
of the ducts. Changes in sperm utilization in wild populations could obviously
impact reproductive success given the temporal and spatial uncertainties in
finding mates and suitable ovipositioning sites.
GLD contains a highly conserved signal peptide
(Krasney et al., 1990; K. Iida
and D. R. Cavener, unpublished data), and its expression and function indicate
that GLD participates in important extracellular catalysis. The enzymatically
active form of GLD is detected in molting fluid of pupa
(Cox-Foster et al., 1990
) and
in the lumen of the spermathecal ducts and of the male ejaculatory duct
(Schiff et al., 1992
). GLD in
molting fluid is required to weaken a puparium case structurally in advance of
eclosion, which relies upon the imago to force open the operculum. Cox-Foster
and Stehr (1994
) suggest that
GLD is involved in generating free radicals that help degrade the old
curricular matrix. However, the biochemistry of GLD in the adult reproductive
organs is unknown. We speculate that it alters the extracellular environment
of the spermathecal ducts to affect a change in sperm motility. An analogous
claim has been made for esterase-6; a null mutation of
esterase-6 causes a slower rate of sperm loss and sperm storage
(Gilbert, 1981a
), which is
similar to the phenotype observed in Gld-null mutants. Further
experiments are required to elucidate the physiological mechanism of GLD
function in sperm trafficking.
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Acknowledgments |
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