An early role for the Drosophila melanogaster male seminal protein Acp36DE in female sperm storage
Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA
Author for correspondence (e-mail:
mfw5{at}cornell.edu)
Accepted 2 July 2003
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Acp36DE, reproduction, insect, sperm storage, seminal fluid protein, sperm, Drosophila melanogaster
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In D. melanogaster, female sperm storage is important for fitness
because a female can fertilize eggs for up to 2 weeks using sperm stored from
a single mating (Gilbert,
1981). Several thousand sperm are deposited into the uterus within
the first half of the
20 min copulation, and sperm storage begins before
copulation ends (Lefevre and Jonsson,
1962
; Tram and Wolfner,
1999
; Gilchrist and Partridge,
2000
). The SSOs, a pair of spermathecae and the single seminal
receptacle, are located at the anterior end of the uterus. By 6 h after
mating, sperm storage has leveled off at up to 1000 sperm
(Gilbert, 1981
;
Neubaum and Wolfner, 1999b
;
Tram and Wolfner, 1999
). Both
evolutionary and quantitative studies indicate that the seminal receptacle is
the primary sperm storage organ (Gilbert,
1981
; Neubaum and Wolfner,
1999b
; Pitnick et al.,
1999
).
Normal female sperm storage in D. melanogaster requires the
transfer both of sperm from the male's seminal vesicles and of proteins (Acps)
secreted from the male's accessory glands
(Tram and Wolfner, 1999).
Females that mated with males expressing very low levels of Acps (
1%
wild-type levels) have few sperm in storage despite receiving normal
quantities of sperm (Kalb et al.,
1993
; Tram and Wolfner,
1999
), indicating that Acps are indeed needed for sperm storage.
In experiments with repeated matings, Acps were required for both sperm
maintenance within females and subsequent use of sperm for fertilization
(Hihara, 1981
;
Xue and Noll, 2000
).
One Acp in particular, Acp36DE (Bertram
et al., 1996), is necessary for normal sperm storage
(Neubaum and Wolfner, 1999b
).
Females mated to males lacking Acp36DE have significantly fewer sperm in
storage 6 h after mating than females mated to wild-type males
(Neubaum and Wolfner, 1999b
).
Males transferring Acp36DE have higher sperm precedence than do males who do
not transfer Acp36DE, presumably as an indirect consequence of Acp36DE's
mediating storage of more sperm (Chapman et
al., 2000
). Some alleles of Acp36DE correlate with the outcome of
sperm competition (Clark et al.,
1995
); allelic variation could alter either the abundance of
Acp36DE or the efficacy of Acp36DE's sperm storage function, thereby affecting
sperm precedence patterns. However, Acp36DE does not affect sperm viability in
storage (Neubaum and Wolfner,
1999b
).
The localization and persistence of Acp36DE provides information about its
role in female sperm storage. Acp36DE is detected in the female as early as 5
min, but not more than 6 h, after the start of mating, corresponding with the
time course of sperm accumulation within storage
(Gilbert, 1981;
Bertram et al., 1996
;
Neubaum and Wolfner, 1999b
;
Tram and Wolfner, 1999
).
Acp36DE localizes to the ventral side of the oviduct wall just anterior to the
openings of the SSOs (Bertram et al.,
1996
) and is also found at the anterior edge of the mating plug
(Lung and Wolfner, 2001
).
Acp36DE associates with sperm in the region of the uterus closest to the SSO
openings, and this association withstands in vitro manipulation
(Bertram et al., 1996
;
Neubaum and Wolfner, 1999b
).
Finally, Acp36DE was detected in the female SSOs at 2 h after mating, when
sperm storage is largely complete
(Gilbert, 1981
;
Neubaum and Wolfner, 1999b
;
Tram and Wolfner, 1999
).
There are several, not mutually exclusive, ways in which Acp36DE could mediate sperm storage. These include facilitating the movement of sperm into storage, organizing sperm either outside or inside the SSOs, marking the entrance to sperm storage, causing retention of sperm in storage and/or inducing or modulating female response(s) to sperm. Here, we explored the mechanism of Acp36DE's effects on sperm storage by examining the timing of these effects as well as the requirements for Acp36DE localization to the SSOs. Our results demonstrate that sperm entrance into storage and their accumulation within storage are separately controlled steps and that Acp36DE plays a role in sperm accumulation only. Furthermore, the early and sperm-independent localization of Acp36DE in the SSOs suggests that Acp36DE might function from within the female SSOs. We therefore suggest that Acp36DE could act as a sperm guidance factor, as a corralling substance and/or to stimulate the female to store sperm efficiently.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Timing of sperm storage in females
Effects of Acp36DE on sperm storage were examined by counting sperm stored
within female SSOs (seminal receptacles and spermathecae) at various times
after the start of mating. Virgin Oregon R females were individually paired
with an Acp36DE1/Df(2L)H20 or
Acp36DE1/CyO male in a vial containing food. At 0.3, 0.5,
0.7, 1, 2, 6, 10, 24, 48 or 72 h after the beginning of mating, the female was
removed and processed for sperm counts as described in Neubaum and Wolfner
(1999b). Blindly coded slides
of individual female SSOs were examined for the presence of orcein-stained
sperm at 100x magnification using a compound microscope with transmitted
light (Zeiss Axioskop). Each sample was counted twice. Variation among
repeated counts of the same sample was, on average, 8% of the sample mean,
indicating consistency among individual sample counts (N=241
samples).
In separate experiments designed to compare the initial timing of Acp36DE entry with sperm entry into the SSOs, matings between Canton S females and males (N=23) were interrupted 10 min after the start of copulation, and sperm storage was quantified as described above.
The significance of differences in mean number of sperm stored both at
different times and between females mated to males with or without Acp36DE was
tested using a two-factor analysis of variance (ANOVA). Linear contrasts were
performed as planned comparisons to examine increases in sperm storage within
6 h of the start of mating for each type of male
(Neter et al., 1996). A
least-square means contrast tested the null hypothesis that a linear
combination of group parameters (corresponding to each time point examined)
was equal to zero. The means and S.E.M.s used were calculated from
the time effect in the ANOVA analysis. t-tests were used to determine
at which times mean female sperm storage differed depending on the presence of
Acp36DE. The depletion of sperm from female storage for each type of male
(time points after 6 h) was modeled using regression analysis, and a
t-test of the slope coefficients (b) tested whether the slopes were
homogeneous. All sperm counts were transformed [
(value + 1)] to meet
the assumptions of parametric statistical tests, but untransformed data are
used in figures. Statistical analysis was performed using StatView software or
JMP (both SAS Inc., Cary, NC, USA).
Acp36DE presence and persistence in the SSOs
Wild-type (Canton S) females were mated to wild-type (Canton S) or
spermless (tudor-progeny; see Materials and methods) males. Pairs
were then separated to prevent remating. At 0.17, 0.33, 1, 10 and 48 h after
the start of mating, females were dissected in Yamamoto's saline
(Stewart et al., 1994).
Triplicate samples of 30 seminal receptacles and spermathecae per treatment
and time point were placed separately into 10 µl of protease-inhibiting
buffer (Monsma and Wolfner,
1988
), homogenized, processed and analyzed by western blotting as
in Bertram et al. (1996
).
Visualizing sperm storage in real time
Effects of Acp36DE on sperm entry into the seminal receptacle and their
motility therein were examined by visualizing GFP-labeled sperm stored within
females that had mated to males with or without Acp36DE. Copulations were
interrupted at either 0.25 h or 0.33 h after the start of mating, and females
were immediately placed on ice. A female's entire reproductive tract was
removed as a unit and mounted in 4% methyl cellulose (Sigma, St Louis, MO,
USA) in Yamamoto's saline. Optical sections of reproductive tracts were imaged
at 40x (Zeiss Axiovert 10) then reassembled for analysis (BioRad MRC
600). Only those samples in which sperm were observed in the uterus
(indicating sperm transfer) were included in the analysis. The presence and
orientation of sperm within the seminal receptacle was observed and the
association between sperm presence in the seminal receptacle and male genotype
was tested using a 2 test of independence
(Sokal and Rohlf, 1995
).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Presence or absence of Acp36DE also did not have a noticeable effect on
sperm orientation or motility. When few sperm are in storage, they often
appeared disorganized, regardless of whether or not Acp36DE was present. This
suggests that the observation of Neubaum and Wolfner
(1999b) of disorganized sperm
in storage within females not receiving Acp36DE reflected simply the small
number of sperm in storage rather than any direct effect of Acp36DE. Vigorous
sperm movements within the seminal receptacle were observed both when Acp36DE
was present and when it was absent (data not shown).
Acp36DE promotes the early accumulation of sperm within SSOs
Since the presence or absence of Acp36DE did not affect the timing of
initial sperm entry into the seminal receptacle, we examined the effects of
Acp36DE on the number of stored sperm in the SSOs at time points corresponding
to later stages of sperm storage, including sperm accumulation and retention
within storage. With and without Acp36DE, sperm accumulation in storage
reaches maximal numbers by 1 h after the start of mating
(Fig. 1A). The time course of
accumulation, however, was influenced by the presence of Acp36DE. As reported
by Neubaum and Wolfner
(1999b), females receiving
Acp36DE stored significantly more sperm than did females not receiving Acp36DE
(two-factor ANOVA: F1,207=28.44, P<0.0001).
The effects of Acp36DE in promoting sperm storage were observed soon after
mating had ended. By 0.5 h after the start of mating, females receiving
Acp36DE stored 2.5-fold more sperm than females not receiving Acp36DE
(t19=3.42, P<0.003). No difference in sperm
storage in the presence and absence of Acp36DE was detected at 0.3 h after the
start of mating (t20=0.676, P=0.507). The
difference in accumulation of sperm in storage between the two male genotypes
is not attributable to the effects of Df(2L)H20. At 1 h after the
start of mating, sperm storage in females mated to control males
(Acp36DE+/Df(2L)H20: mean ± 1
S.E.M.=353.7±29.56 sperm) is similar to that of females
mated to Acp36DE1/CyO control males (Bonferroni-Dunn
post-hoc test: P=0.97, N=25) and is significantly
higher than that of females mated to Acp36DE1/Df(2L)H20
males (P=0.018, N=25).
|
This difference in the number of sperm stored in the presence or absence of
Acp36DE reflects a difference in their rate of accumulation of stored sperm.
When females received Acp36DE (Acp36DE1/CyO mates), the
number of stored sperm increased nearly threefold between 0.3 h and 0.5 h
after the start of mating (linear contrast, t-ratio=-5.66, d.f.=62,
P<0.0001), but the increase in number of stored sperm after 0.5 h
(53%) was not statistically significant (t-ratio=-0.734,
d.f.=62, P=0.47). When females did not receive Acp36DE
(Acp36DE1/Df(2L)H20 mates), the number of sperm within
storage increased significantly between 0.3 h and 0.5 h (138%;
t-ratio=-5.41, d.f.=65, P<0.0001) as well as between 0.5
h and 0.7 h (108%; t-ratio=-3.82, d.f.=65, P<0.001) but
not at later time points (t-ratio=-0.80, d.f.=65, P=0.43)
(Fig. 1A).
To determine if Acp36DE also influences the rate at which sperm exit storage, we compared the decline in numbers of stored sperm in the presence or absence of Acp36DE. The rate of sperm depletion from females receiving Acp36DE (Acp36DE1/CyO males, b=-0.161) and females lacking Acp36DE (Acp36DE1/Df(2L)H20 males, b=-0.123) were not statistically different (t30=0.995, 0.25<P<0.10), indicating that Acp36DE does not affect sperm retention (Fig. 1B).
Acp36DE is found in SSOs shortly after mating, and this localization
does not require sperm
To explore how Acp36DE facilitates rapid sperm accumulation, we examined
its earliest detection, duration of residence and dependence on sperm for its
residence in the SSOs. Acp36DE is normally found in the female SSOs and
associates with sperm (Bertram et al.,
1996; Neubaum and Wolfner,
1999b
). To determine if Acp36DE's association with sperm is
required to bring Acp36DE into the SSOs, we examined the spermathecae and
seminal receptacles of females mated to wild-type or spermless males for the
presence of Acp36DE. Full-length Acp36DE (122 kDa) and its 68-kDa processed
form (Bertram et al., 1996
)
were detected in both the seminal receptacle and spermathecae at 0.33 h after
the start of mating in females mated to wild-type males and in females mated
to spermless males (Fig. 2A).
Furthermore, a faint band corresponding to full-length Acp36DE was detected in
the seminal receptacles of females mated to spermless males even 2 h after
mating. Therefore, Acp36DE entry into the SSOs does not require sperm.
|
Since Acp36DE can enter the female SSOs in the absence of sperm transfer, does it normally enter the SSOs before sperm? To address this question, we examined the SSOs for Acp36DE at 0.17 h into mating. At this time point, all of the mating females (N=23) had received sperm, but sperm storage was evident in only 34.8% of those females, each having, on average, three sperm in storage. Full-length Acp36DE (122 kDa) was already abundant in both the spermathecae and the seminal receptacles at this time (Fig. 2B) and, thus, before the entry of significant numbers of sperm into storage. Because Acp36DE is detected in the SSOs by the earliest times of sperm storage and before its effects on sperm storage are detected, Acp36DE could act from within the female SSOs to facilitate the rapid accumulation of sperm in storage.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Based on our observations, we propose that Acp36DE is needed for the
efficient accumulation of stored sperm after the first sperm has entered
storage. Acp36DE's action is detected as early as 0.5 h after the start of
mating and has consequences after sperm storage has leveled off and egg laying
has begun (1.5-3.0 h; Heifetz et al.,
2000
; Chapman et al.,
2001
). Further evidence for Acp36DE's role in rapid sperm storage
comes from sperm entry and storage data for Acp36DE-control males
(Acp36DE+/Df(2L)H20). Despite a lag in initial sperm
storage in females mated to Acp36DE+/Df(2L)H20 males
compared with Acp36DE1/CyO males (also controls), the mean
number of sperm stored in females mated to these males was nearly the same one
hour after the start of mating, and both were significantly higher than the
number of sperm stored by females mated to Acp36DE-deficient males
(Acp36DE1/Df(2L)H20). Similar to other studies on sperm
storage (Lefevre and Jonsson,
1962
; Tram and Wolfner,
1999
), sperm storage in our experiments progressed very rapidly,
leveling off within 1 h after the start of mating. Because sperm entry into
storage must end when an ovulated egg expels remaining unstored sperm (1.5-3 h
after mating; Heifetz et al.,
2000
), a rapid rate of sperm accumulation within storage is
essential for efficient sperm storage and subsequent female fertility.
Several, not mutually exclusive, hypotheses exist for how Acp36DE might effect the rapid accumulation of sperm in the SSOs. These include (1) acting as a factor to facilitate directed sperm movement through the uterus and into the SSOs, (2) concentrating sperm around the SSO entrances and/or (3) stimulating the female to efficiently take up sperm into her SSOs. In each of these models, sperm entry can or must be initiated by some factor other than Acp36DE.
First, Acp36DE could facilitate the directed movement of sperm or groups of
sperm into storage. One mechanism by which Acp36DE could accomplish this is if
its association with sperm (Neubaum and
Wolfner, 1999b) promoted the `bundling' of sperm into cords that
then efficiently moved, as units, into storage. This proposed mechanism is
similar to the role of the sperm apical hook in sperm train formation within
the uterus of the wood mouse Apodemus sylvaticus
(Moore et al., 2002
). In
A. sylvaticus, sperm trains have higher progressive motility than do
individual sperm. In D. melanogaster, sperm-sperm associations could
be an efficient means to effect rapid sperm accumulation within the SSOs.
Another potential mechanism by which Acp36DE could facilitate directed
movement of sperm into storage is suggested by Acp36DE's specific localization
within portions of the female's reproductive tract. Acp36DE associates with
the oviduct wall, just anterior to the SSO entrances
(Bertram et al., 1996
), and
also the anterior end of the mating plug
(Lung and Wolfner, 2001
). The
mating plug is a mass of congealed substances at the posterior end of the
mated female's reproductive tract that confines sperm to the anterior end of
the uterus, near the SSO entrances
(Bairati, 1968
). Perhaps
Acp36DE helps to form a trellis descending from the sperm barrier in the
oviduct and rising from the mating plug. Sperm, also associating with Acp36DE,
could move along this trellis to reach the SSO entrances
(Lung and Wolfner, 2001
). A
final possibility for how Acp36DE could direct sperm movement into or within
storage is by helping sperm cells follow preceding sperm cells into storage in
a manner formally analogous to the axon fasciculation that occurs during
nervous system development. Fasciculation occurs when axons from multiple
neurons follow molecular cues along a trail forged by a single pioneer axon to
grow out to a target (reviewed in
Tessier-Lavigne and Goodman,
1996
; Van Vactor,
1999
). Perhaps, Acp36DE's association with sperm
(Neubaum and Wolfner, 1999b
)
helps subsequent sperm follow the trail of a `pioneer sperm' into storage;
although the mechanism by which this would occur is unknown.
Second, Acp36DE could concentrate sperm near the SSO entrances and thereby
increase the rate of storage. Factors other than Acp36DE are already known to
corral sperm. A barrier in the lower common oviduct prevents sperm (and Acps)
from moving into the oviduct (Bertram et
al., 1996). When the barrier is not present (as in eggless
females) sperm are displaced up the oviduct, and fewer sperm are in storage 6
h after mating (Neubaum and Wolfner,
1999b
). While this barrier retains Acp36DE, Acp36DE is not
necessary for barrier formation (Neubaum
and Wolfner, 1999b
). The mating plug also keeps sperm concentrated
at the anterior end of the uterus (Lung
and Wolfner, 2001
).
Third, Acp36DE might stimulate sperm storage by modulating muscle
contractions or changing relative fluid pressure among regions of the female
reproductive tract, particularly after the first sperm has entered storage and
initiated the storage process. We show that Acp36DE enters the SSOs at the
earliest times of sperm storage and that its entry into the SSOs does not
require sperm, suggesting that Acp36DE could act from within the SSOs to
effect sperm accumulation within storage. Perhaps Acp36DE stimulates the
withdrawal of fluid from the SSO lumen, resulting in a decrease of relative
pressure within the SSOs. As a result of this pressure differential, sperm
could be sucked into storage, as has been proposed for lower Diptera
(Linley, 1981). A role for
Acp36DE on the female's response to stored sperm is suggested by the finding
that Acp36DE from a previous mating can facilitate female storage of
subsequent-mating males' sperm (Chapman et
al., 2000
). This is long after Acp36DE is no longer detected in
the female reproductive tract (6 h;Bertram
et al., 1996
).
It is not known how Acp36DE gets into the SSOs. Sperm are not needed for
Acp36DE's entry into or retention in the SSOs, and Acp36DE enters the SSOs
during the initial stages of sperm storage, before copulation is even
complete. Since Acp36DE localizes to the oviduct wall close to the SSO
entrances (Bertram et al.,
1996), it may be pulled or sucked into storage by female muscular
contractions, similar to the mechanism proposed for sperm entry into storage
in mosquitoes (Linley, 1981
).
Since Acp36DE is detected in the uterus as early as 5 min after mating begins
(Bertram et al., 1996
) and
sperm transfer usually occurs within 8-10 min of the start of mating
(Gilchrist and Partridge,
2000
), the mating plug may create pressure within the female
reproductive tract, pushing Acp36DE anteriorly into the SSOs. Alternatively,
since large amounts of Acp36DE are transferred to females, its entry into the
SSOs could just be stochastic.
In conclusion, we have shown that sperm entry into and accumulation within female SSOs are distinct events controlled by different factors. Acp36DE facilitates the rapid accumulation of sperm within female SSOs, but only after the first sperm has entered storage. In addition to its association with sperm, the mating plug and the uterus, Acp36DE accumulates within the SSOs at the earliest stages of sperm storage and its accumulation there does not require sperm. These results suggest that Acp36DE could act from within the SSOs to assist them in taking up sperm efficiently. Future identification and description of the location of Acp36DE-binding proteins will help elucidate Acp36DE's role in rapid sperm accumulation within the SSOs. Since rapid storage of sperm is important for both male and female reproductive fitness, understanding Acp36DE's role in sperm storage provides novel insights into the mechanisms of male and female reproductive interactions.
![]() |
Acknowledgments |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bairati, A. (1968). Structure and ultrastructure of the male reproductive system in Drosophila melanogaster Meig. 2. The genital duct and accessory glands. Ital. J. Zool. 2,105 -182.
Bertram, M. J., Neubaum, D. M. and Wolfner, M. F. (1996). Localization of the Drosophila male accessory gland protein Acp36DE in the mated female suggests a role in sperm storage. Insect Biochem. Mol. Biol. 26,971 -980.[CrossRef][Medline]
Bloch Qazi, M. C., Aprille, J. R. and Lewis, S. M. (1998). Female role in sperm storage in the red flour beetle, Tribolium castaneum. Comp. Biochem. Physiol. A 120,641 -647.
Bloch Qazi, M. C., Heifetz, Y. and Wolfner, M. F. (2003). The developments between gametogenesis and fertilization: ovulation and female sperm storage in Drosophila melanogaster. Dev. Biol. 256,195 -211.[Medline]
Boswell, R. E. and Mahowald, A. P. (1985). tudor, a gene required for assembly of the germ plasm in Drosophila melanogaster. Cell 43, 97-104.[Medline]
Chapman, T., Neubaum, D. M., Wolfner, M. F. and Partridge, L. (2000). The role of male accessory gland protein Acp36DE in sperm competition in Drosophila melanogaster. Proc. R. Soc. Lond. B. Biol. Sci. 267,1097 -1105.[CrossRef][Medline]
Chapman, T., Herndon, L. A., Heifetz, Y., Partridge, L. and Wolfner, M. F. (2001). The Acp26Aa seminal fluid protein is a modulator of early egg hatchability in Drosophila melanogaster. Proc. R. Soc. Lond. B. Biol. Sci. 268,1647 -1654.[CrossRef][Medline]
Clark, A. G., Aguadé, M., Prout, T., Harshman, L. G. and
Langley, C. H. (1995). Variation in sperm displacement and
its association with accessory gland protein loci in Drosophila
melanogaster. Genetics
139,189
-201.
Davey, K. G. (1958). The migration of spermatozoa in the female of Rodnius prolixus Stal. J. Exp. Biol. 35,694 -701.
Gilbert, D. G. (1981). Ejaculate esterase 6 and initial sperm use by female Drosophila melanogaster. J. Insect Physiol. 27,641 -650.[CrossRef]
Gilchrist, A. S. and Partridge, L. (2000). Why it is difficult to model sperm displacement in Drosophila melanogaster: the relationship between sperm transfer and copulation duration. Evolution Int. J. Org. 54,534 -542.
Gillott, C. (1988). Arthropoda-Insecta. InReproductive Biology of the Invertebrates, vol, III, Accessory Sex Glands (ed. K. G. Adiyodi and R. G. Adiyodi), pp. 319-471. New York: John Wiley & Sons.
Heifetz, Y., Lung, O., Frongillo, E. A., Jr and Wolfner, M. F. (2000). The Drosophila seminal fluid protein Acp26Aa stimulates release of oocytes by the ovary. Curr. Biol. 10,99 -102.[CrossRef][Medline]
Hihara, F. (1981). Effects of the male accessory gland secretion on oviposition and remating in females of Drosophila melanogaster. Zool. Mag. 90,307 -316.
Kalb, J. M., DiBenedetto, A. J. and Wolfner, M. F.
(1993). Probing the function of Drosophila melanogaster
accessory glands by directed cell ablation. Proc. Natl. Acad. Sci.
USA 90,8093
-8097.
Lefevre, G. and Jonsson, U. B. (1962). Sperm
transfer, storage, displacement, and utilization in Drosophila
melanogaster. Genetics
47,1719
-1736.
Linley, J. R. (1981). Emptying of the spermatophore and spermathecal filling in Culicoides melleus (Coq.) (Diptera: Ceratopogonidae). Can. J. Zool. 59,347 -356.
Lung, O. and Wolfner, M. F. (2001). Identification and characterization of the major Drosophila melanogaster mating plug protein. Insect Biochem. Mol. Biol. 31,543 -551.[CrossRef][Medline]
Monsma, S. A. and Wolfner, M. F. (1988). Structure and expression of a Drosophila male accessory gland gene whose product resembles a peptide pheromone precursor. Genes Dev. 2,1063 -1073.[Abstract]
Moore, H.,
Dvoáková, K., Jenkins, N. and
Breed, W. (2002). Exceptional sperm cooperation in the wood
mouse. Nature 418,174
-177.[CrossRef][Medline]
Neter, J., Kutner, M. H., Nachtsheim, C. J. and Wasserman, W. (1996). Applied Linear Statistical Models. Fourth edition. Chicago: Irwin Press.
Neubaum, D. M. and Wolfner, M. F. (1999a). Wise, winsome or weird: mechanisms of sperm storage in female insects. Curr. Top. Dev. Biol. 41, 67-97.[Medline]
Neubaum, D. M. and Wolfner, M. F. (1999b).
Mated Drosophila melanogaster females require a seminal fluid
protein, Acp36DE, to store sperm efficiently. Genetics
153,845
-857.
Pitnick, S., Markow, T. and Spicer, G. S. (1999). Evolution of multiple kinds of female sperm-storage organs in Drosophila. Evol. Int. J. Org. 53,1804 -1822.
Price, C. S. C. (1999). Sperm competition between Drosophila males involves both displacement and incapacitation. Nature 400,449 -452.[CrossRef][Medline]
Santel, A., Winhauer, T., Blumer, N. and Renkawitz-Pohl, R. (1997). The Drosophila don juan (dj) gene encodes a novel sperm specific protein component characterized by an unusual domain of repetitive amino acid motif. Mech. Dev. 64, 19-30.[CrossRef][Medline]
Simmons, L. W. (2001). Sperm Competition and Its Evolutionary Consequences in the Insects. Princeton: Princeton University Press.
Sokal, R. R. and Rohlf, F. J. (1995). Biometry: The Principles and Practice of Statistics in Biological Research. Third edition. New York: W. H. Freeman and Co.
Stewart, B. A., Atwood, H. L., Renger, J. J., Wang, J. and Wu, C. F. (1994). Improved stability of Drosophila larval neuromuscular preparations in haemolymph-like physiological solutions. J. Comp. Physiol. 175,179 -191.
Tessier-Lavigne, M. and Goodman, C. S. (1996).
The molecular biology of axon guidance. Science
274,1123
-1133.
Tram, U. and Wolfner, M. F. (1999). Male
seminal fluid proteins are essential for sperm storage in Drosophila
melanogaster. Genetics
153,837
-844.
Van Vactor, D. (1999). Axon guidance. Curr. Biol. 9,R797 -R799.[CrossRef][Medline]
Xue, L. and Noll, M. (2000).
Drosophila female sexual behavior induced by sterile males showing
copulation complementation. Proc. Natl. Acad. Sci. USA
97,3272
-3275.