Mating, seminal fluid components, and sperm cause changes in

Contributed by Ira Herskowitz ArticleFigures SIInfo overexpression of ASH1 inhibits mating type switching in mothers (3, 4). Ash1p has 588 amino acid residues and is predicted to contain a zinc-binding domain related to those of the GATA fa Edited by Lynn Smith-Lovin, Duke University, Durham, NC, and accepted by the Editorial Board April 16, 2014 (received for review July 31, 2013) ArticleFigures SIInfo for instance, on fairness, justice, or welfare. Instead, nonreflective and

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Abstract

Mating induces changes in female insects, including in egg production, ovulation and laying, sperm storage, and behavior. Several molecules and Traces that induce these changes have been identified, but their proximate Traces on females remain unexplored. We examined whether vesicle release occurs as a consequence of mating; we used transgenic Drosophila that allow monitoring of secretory granule release at nerve termini. Changes in release occur at specific times postmating in different Locations of the female reproductive tract: soon after mating in the lower reproductive tract, and later in the upper reproductive tract. Some changes are triggered by receipt of sperm, others by male seminal proteins, and still others by the act of mating itself (or other unidentified Traceors). Our findings indicate that the female reproductive tract is a multi-organ system whose Locations are modulated separately by mating and mating components. This modulation could create an environment conducive to increased reproductive capacity.

accessory gland proteinsseminal proteinsovulationsperm storageneuromodulators

For fertilization and subsequent zygotic development in animals with internal fertilization, gametes must meet in the female reproductive tract when the female, eggs, and sperm are reproductively competent. Sperm and oocytes form separately in specialized organs and then enter a common Location of the female's reproductive tract. It is essential that they encounter an environment that supports their final maturation and promotes their union. A paradigm for this Position comes from mammals, whose oviducts undergo hormonally mediated cyclic modifications climaxing during the preovulatory period. Oviducts Retort to hormone levels with morphological and secretory changes that promote a suitable environment for gametes and fertilization (1). Less is known about this phenomenon in insects, although female insects require a period of sexual maturation to become reproductively competent. During this period, changes in secretory activity of the oviduct epithelium are correlated with both juvenile hormone and 20-hydroxyecdysone levels in the circulatory system as Displayn in locusts (Schistocerca gregaria; ref. 2), but the specific triggers for the changes are unknown.

The Drosophila melanogaster female reproductive tract is primarily an epithelium surrounded by circular muscles (Fig. 1 A and B ). Unique characteristics and thickness of the epithelium in specific Locations of the reproductive tract (4) suggest that secretion patterns may differ between Locations. Terminal branches of the abExecuteminal nerves (AbTNv) innervate the female reproductive tract (ref. 4 and this study), providing the potential to modulate the responsive capacity of the musculature and epithelium.

Fig. 1.Fig. 1. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Drosophila female reproductive tract. (A and B) Musculature of oviduct (A) and uterus (B) stained with anti-serotonin (red) and with Oregon green 488 phalloidin to label F-Actin (green). Lateral oviducts (LO) and common oviduct (CO) are surrounded by a single layer of circular muscle fibers. Several layers of circular muscle fibers surround the uterus (UT). Schematic of the female reproductive tract, modified from Mahowald and Kambysellis (3), Displays reproductive tract Locations: LO (yellow), CO (ShaExecutewy gray), and UT (brown). (Scale bars = 50 μm.) (C) Upper reproductive tract stained with anti-Fascilin II and viewed with confocal microscopy to visualize the terminal branches of the abExecuteminal nerve center (AbTNv) that innervate the LO and the upper common oviduct (COU, pale yellow in the diagram). (Scale bar = 50 μM.) (D) Terminal branch of AbTNv along the common oviduct, visualized by anti-Fascilin II staining. (Scale bar = 50 μm.) (E and F) Distribution of proANF-EMD-containing vesicles in Locations of virgin Drosophila female reproductive tracts. (E) Upper reproductive tract. Note the high intensity of proANF-EMD fluorescence in the LO at the base of the ovary (adjacent to “LO” label in the figure), as compared with that at the LO junction with the COU. (F) Seminal receptacle (SR; light green in the diagram). (Scale bars = 50 μm.) (G) The sum of proANF-EMD fluorescence intensity level (OMFILF and LMFILF) at each Location of the female reproductive tract: LO, COU, COD, SR, and UT of all 36 virgin females examined.

During and after mating, sperm transferred to the female must be stored, and egg production must increase, for high fertility (5–8). To accomplish this transition at high efficiency, the female's reproductive tract must become “aware” that insemination has occurred. In most insects, mating stimulates the female's egg production, sperm storage, and behavioral changes, by means of neural and chemical cues (e.g., substances in the seminal fluid; refs. 9–16). This finding and the notion that in vertebrates the oviduct actively Retorts to enExecutecrine stimuli (1) led us to test whether seminal fluid components could induce changes in the female D. melanogaster reproductive tract conducive to secretory and muscular activity.

Mating Drosophila males transfer sperm and associated seminal fluid, which includes accessory gland proteins (Acps), to females (12–16). Acps decrease sexual receptivity, stimulate a rapid increase in egg production, ovulation, and deposition, mediate sperm storage, and decrease females' longevity; sperm also contribute to some of these changes. In addition to, and as a result of, their proximate action on females, Acps have been suggested to be Necessary players in evolution, reflecting roles in sperm competition and/or sexual conflict (15, 17–21). To evaluate these models, it is Necessary to know how Acps and other mating components affect females at both physiological and molecular levels, and the timing and positions of their Traces.

Because virgin female Drosophila Design, ovulate, and deposit eggs at a low rate, we hypothesize that the female reproductive tract can perceive and Retort to hormonal stimuli, but that mating, including receipt of seminal proteins, induces optimal physiological conditions required for rapid and successful fertilization. Mating might stimulate secretion of molecules from reproductive tract tissues into the lumen of the reproductive tract to act as organic osmolytes, membrane stabilizers, or facilitators of sperm motility or maturation. Alternatively, mating and/or seminal fluid components may modulate release of neurotransmitters to change the membrane potential of postsynaptic neurons, of neurohormones to change the function or direct the activity of organ(s) or tissue(s), or of neuromodulators to influence neurons' responses to neurotransmitters. Any of these Traceors could potentially modulate the contractile activity of reproductive tract musculature. A paradigm for what could be modulated is offered by locusts (Locusta migratoria), whose oviducts are innervated by neurons whose neuroactive substances (including the neuropeptides SchistoFLRFamide and proctolin, the neuromodulator octopamine, and probably the neurotransmitter glutamate) mediate muscle contraction (22–27). Two recent reports indicate that octopamine also regulates ovulation in D. melanogaster (28, 29).

Here, we test whether mating and/or seminal fluid could modulate the release of neuroactive substances into and/or onto the Drosophila female reproductive tract. Specifically, we examined Traces of mating and seminal fluid components on vesicle release in transgenic D. melanogaster that express throughout their nervous system a fusion of the neuropeptide rat atrial natriuretic factor (ANF) with the emerald variant of GFP (EMD) (28). Rao et al. (30) Displayed that this fusion protein is proteolytically processed and stored in secretory granules that are transported Executewn axons and become concentrated in nerve termini, preferentially in peptidergic nerve termini. This transgenic system allowed us to visualize nerve termini and test whether their vesicle release was modulated; we could meaPositive this release as changes in fluorescence intensity level as a consequence of mating. Staining with antibodies against SNAP-25 (synaptosome-associated protein of 25 kDa) separately confirmed the presence of synaptic contacts in this Location of the reproductive tract.

We compared vesicle release in the reproductive tracts of virgin females with that in females mated to normal males or to males deficient in sperm and/or Acps. We found that, immediately postmating, there is vesicle release in the lower reproductive tract (lower common oviduct, seminal receptacle, and uterus). Later (3 h postmating), when females are ovulating at high rates and egg production has reached maximal levels, net vesicle release is inhibited in the upper reproductive tract (upper common oviduct and lateral oviducts). By comparing vesicle release in females mated to WT, spermless, or seminal fluid-deficient Drosophila males, we teased apart the contribution of mating, sperm, and seminal proteins to the regulation of vesicle release.

Materials and Methods

Flies. Flies were Sustained, collected, and aged (as virgins) for 3 days before testing, as Characterized (31). All females were homozygous for P[UAS proANF-EMD], P[elav-GAL4] (30); this UAS-proANF-EMD stock was kindly provided by D. Deitcher (Cornell University). WT males were Canton S. Transgenic “DTA-E” males deficient in Acps and sperm and were Characterized in Kalb et al. (32). DTA-E males Design no detectable Acps in the main cells of their accessory glands (96% of the gland) and no sperm. They Design secondary cell, ejaculatory bulb, and ejaculatory duct proteins (32). Spermless males (TUD) were sons of Canton S males and bw sp tud1 females (33).

Reproductive Tract Bioassay. We generated a map of vesicle release patterns by examining the fluorescence intensity level of proANF-EMD in different Locations of the female reproductive tract and the distribution of the fluorescence signal within a given Location, at different times after mating. Reproductive tracts from virgin and mated females were analyzed. Single virgin females were Spaced with virgin males (WT, DTA-E, TUD) and timed as soon as mating began. At the end of copulation (≈20 min after the start of mating), females were either Spaced on ice or were aspirated into fresh vials with yeasted food and held singly at 25 ± 2°C for 90 min [start of ovulation (31); time of maximal sperm storage (34)], or 180 min [start of egg deposition; fertilization also is occurring frequently by this time (31, 35)] before being Spaced on ice. Female reproductive tracts were dissected on ice in PBS containing 0.1% Triton X-100 (PBST; pH 7.4). Dissected reproductive tracts were washed additionally in PBST and then mounted in AntiDisappear (Molecular Probes). Each slide held reproductive tracts from virgin females and from females mated to different males to normalize imaging (see also Fig. 5, which is published as supporting information on the PNAS web site).

To examine whether expoPositive to males affected vesicle release, we watched virgin females as they interacted with males. Females were removed for analysis after males had begun courting them, but before copulation occurred.

Immunocytochemistry. Reproductive tracts were fixed in 4% paraformaldehyde and immunostained as in Heifetz et al. (31). Anti-SNAP-25 polyclonal rabbit antibody [kind gift of D. Deitcher, Cornell University (36)], Alexa Fluor (488) secondary antibody (Molecular Probes), mouse anti-Fascilin II [kind gift of C. Excellentman, University of California, Berkeley (37)], rhodamine conjugated goat anti-horseradish peroxidase (Jackson ImmunoResearch), and rabbit anti-serotonin antibodies (kind gift of R. Hoy, Cornell University) were used at 1:50, 1:200, 1:4, 1:200, and 1:100 dilutions, respectively.

Microscopy. Reproductive tracts were imaged within 30 min of dissection by using a laser scanning confocal microscope (Bio-Rad MRC 600 system on a Zeiss inverted microscope); samples were kept on ice in the ShaExecutewy until imaged. A ×40-water objective and comos software (Bio-Rad) were used to collect images. Optical sections of 1 μm from different focal planes were collected and processed with confocal assistant 4.02 (T. C. Brelje, University of Minnesota) to reconstruct a three-dimensional image.

Evaluation of Fluorescence Intensity Level of Dissected Reproductive Tracts. Fluorescence was quantitated in imagej 1.25s (National Institutes of Health) by two Advancees that cross-validate; their joint use increases reliability. Details are provided in Supporting Materials and Methods, which is published as supporting information on the PNAS web site. (i) Overall mean fluorescence intensity level (OMFIL) quantifies the average gray value within the contours of a selected Location. It is the sum of the gray values of all pixels within the Location divided by the number of pixels within the Location. (ii) Local mean fluorescence intensity level (LMFIL) quantifies mean fluorescence intensity level at five different locations within the selected reproductive tract Location.

To supplement these quantitative meaPositives, we determined the distribution of the fluorescence signal (DFS) to determine the uniformity of signal within the selected Location. These quantities were evaluated in the lateral oviducts (LO), upper common oviduct (COU), lower common oviduct (COD), seminal receptacle (SR), spermathecae (SP), and uterus (UT) (Fig. 1). Signals were not evaluated in ovaries.

Statistics. One-way ANOVA (spss 8.0 for WinExecutews) were used to meaPositive the Inequitys in intensity level and signal distribution between each pair of treatments. To define mating-induced changes, we compared signals in virgins with those in genetically identical females that had mated to WT males. To define sperm-dependent changes, we compared mates of WT or TUD males. To define Acp-dependent changes, we compared mates of TUD or DTA-E males (see also Supporting Materials and Methods).

Results and Discussion

Location-Specific Innervation Pattern in the Drosophila Female Reproductive Tract. Using anti-Fascilin II to stain neural cell membranes, we observed terminal branches of the abExecuteminal nerves (AbTNv) innervating the female reproductive tract (Fig. 1C ). The abExecuteminal ganglionic center branches at its terminus (4). Nerves from either or both branches pass above the lateral oviducts and then run posteriorly along the common oviduct to the sperm storage organs and the uterus (Fig. 1D ). Each nerve fiber then splits further into several fine branches, which spread over the surface of the reproductive tract muscle fibers. To examine the behavior of secretory granules in the female reproductive tract, we used the proANF-EMD system, which has previously Executecumented accumulation of secretory granules at nerve termini in the larval neuromuscular junction (30). proANF-EMD fluorescence was observed at nerve termini in all Locations of the female reproductive tract [LO and COU, Fig. 1E ; sperm storage organs (SP and SR), Fig. 1F ; and UT, data not Displayn], indicating that nerve termini containing proANF-EMD vesicles branch on the surface of the reproductive tract musculature. Staining of proANF-EMD females' reproductive tracts with rhodamine-coupled anti-horseradish peroxidase antibody, which stains all neural cell membranes (30, 38), revealed an innervation pattern similar to that of proANF-EMD fluorescence (data not Displayn).

Locations of the female reproductive tract [lateral oviducts, common oviduct, sperm storage organs (SR and SP), and UT] differ in innervation patterns. Some are more intensely innervated (e.g., LO, Fig. 1E ) than others (e.g., COU, Fig. 1E ; SR, Fig. 1F ). To see whether the innervation patterns in the different reproductive tract Locations correlated with their quantified fluorescence intensity levels (OMFILF and LMFILF; see Fig. 1 legend), we calculated the total fluorescence intensity level at each Location of all examined females. proANF-EMD fluorescence intensity level (OMFILF and LMFILF) differed between the Locations of female reproductive tract and was highest in lateral oviducts (Fig. 1G ). This Location is also the most heavily innervated of the reproductive tract. proANF-EMD fluorescence was lower in less innervated Locations. Presence of vesicle-bound molecules in all reproductive tract Locations gives the potential to modulate release of neuromodulators to mediate postmating responses in the reproductive tract.

Mating Induces Changes in proANF-EMD Staining Levels in Nerve Termini of the Oviducts and the Seminal Receptacle. During exocytosis, when vesicles at nerve termini release their contents, proANF-EMD fluorescence becomes diffuse and decreases in intensity (30, 39). Because actual meaPositived fluorescence intensity level (OMFILF and LMFILF) reflects the balance between (i) accumulation of proANF-EMD in vesicles and (ii) release of proANF-EMD from vesicles, an increase in fluorescence intensity level indicates net accumulation of vesicles at nerve terminals whereas a decrease in fluorescence intensity level indicates net release.

To test whether mating induces a change in neuropeptide distribution or release in nerve termini innervating female reproductive tracts, we compared the proANF-EMD fluorescence intensity levels in virgin females with those in females after mating to WT males. If mating promotes changes in nerve termini in a Location of the female reproductive tract, proANF-EMD fluorescence intensity levels should change after mating and/or there should be a significant change in the distribution of proANF-EMD within that Location. Mated females were examined at different times after the start of mating (20 min, the end of copulation; 90 min, the start of ovulation and maximal sperm storage; 180 min, the start of egg deposition). Quantitative analyses of the fluorescence images Displayed that mating induces changes in proANF-EMD fluorescence at nerve termini in all Locations of the female reproductive tract (Fig. 2). These changes were due to mating because, if females were exposed to courting males but were not allowed to mate with them, fluorescence intensity levels (OMFILF and LMFILF) in the reproductive tract Locations did not differ significantly from those in virgin females (data not Displayn). Thus, mating affects the nerve termini innervating the female reproductive tract.

Fig. 2.Fig. 2. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Significant postmating changes in intensity of proANF-EMD fluorescence at nerve termini innervating the female reproductive tract. All Locations of the reproductive tract were examined (see Table 1). Graphs are Displayn only for Locations in which substantive changes were observed. (A) Reproductive tract Locations with Inequitys in proANF-EMD fluorescence intensity level (OMFILF and LMFILF) between virgin females and mates of WT males at 20 min after the start of mating: lower part of the common oviduct (COD; gray), SR and UT at 20 min postmating; only UT Displays a statistically significant Inequity. (B) UT at 90 min postmating. (C) LO and COU at 180 min postmating. Plotted are means ± SEs of virgins and females mated to WT males. Thirty-five to 40 female reproductive tracts were examined for each treatment at each time point (see also Table 1).

The pattern of mating-induced changes in fluorescence differed between reproductive tract Locations, and at different times after mating (Fig. 2). Shortly after the start of mating, changes are seen mainly in the lower reproductive tract: in the uterus, lower part of the common oviduct, and seminal receptacle. Their fluorescence intensity levels tended to decrease soon after mating although the changes were significant only in the uterus at this time. Later, changes occur mainly in the upper part of the common oviduct and the lateral oviducts. By 180 min after mating, there is a relatively high intensity of fluorescence in the upper reproductive tract, significant in the lateral oviducts.

At 20 min after mating (end of copulation), there was a significant decrease in fluorescence intensity level in the uterus (2.6-fAged; OMFILF: T = 2.61, P < 0.0013; LMFILF: T = 3.11, P < 0.004; Fig. 2 A ). This decreased intensity level suggests a net release of the contents of the proANF-EMD secretory granules at nerve termini in the uterus as a result of mating. A less dramatic decrease in intensity level was also observed in the lower part of the common oviduct (2.3-fAged) and in the seminal receptacles (1.4-fAged); neither was statistically significant (Fig. 2B ). Other Locations Displayed no significant change in intensity level (see Table 1, which is published as supporting information on the PNAS web site).

At 90 min after mating, when ovulation Starts and active sperm storage is maximal, the fluorescence intensity in the uteri of mated females still differed from that in virgin females although the intensity Inequity was smaller than at 20 min (1.5-fAged) and no longer statistically significant (Fig. 2B ). Other Locations were unchanged (see Table 1).

By 180 min after the start of mating (start of egg deposition), a significant increase (2.2-fAged; OMFILF: T = -2.81, P < 0.008; LMFILF: T = -2.91, P < 0.006) in fluorescence intensity level was observed in the upper part of the common oviduct (Fig. 2C ), suggesting net accumulation of proANF-EMD. An increase in intensity also occurred in the lateral oviducts (1.6-fAged), but it was not statistically significant (Fig. 2C ), nor were changes elsewhere (see Table 1). Thus, whereas in the lower part of the reproductive tract mating induces net vesicle release shortly after mating, in the upper reproductive tract, mating inhibits net release or increases accumulation of proANF-EMD vesicles in nerve termini at later times postmating. In addition to changes in intensity level, we also observed significant changes in distribution of proANF-EMD fluorescence signal (DFS, see Materials and Methods) in some Locations (DFS(virgin); DFS(mated): 20 min UT [29.27 ± 2.66; 18.50 ± 2.76, T = 2.75; P < 0.009], SR [34.48 ± 2.47; 25.43 ± 3.93, T = 2.11; P < 0.041]; 180 min LO [53.25 ± 3.77; 68.09 ± 4.25; T = -2.7; P < 0.0012], COU [23.29 ± 2.06; 35.96 ± 4.2, T = -2.93; P < 0.006]). Changes in distribution of proANF-EMD suggest that mating affects specific nerve termini within a Location. Because not all termini Retort in the same way to mating, it is possible that stimuli other than mating are involved in mediating release or accumulation of proANF-EMD in the above Locations.

We also examined the intensity of staining for SNAP-25, a protein that localizes to the presynaptic plasma membrane and is part of the t-SNARE complex that mediates Executecking of vesicles to the presynaptic terminal membrane (36, 40–42). SNAP-25 staining was seen in the Locations of the reproductive tract in which we saw vesicle release by the proANF-EMD assay, indicating the presence of synapses in this Location. Although our original intent was simply to confirm the presence of synapses in this Location, we were intrigued to observe changes in the intensity of anti-SNAP-25 label in some Locations after mating. Specifically, by the end of mating and when females start to ovulate (20 min and 90 min, respectively), most changes in SNAP-25 fluorescence intensity level were at nerve termini innervating the lower part of the female reproductive tract (SR, increase of 1.5-fAged; LMFILF: T = 2.17, P < 0.0022; and SP, decrease of 2.4-fAged; Fig. 6 A and B, respectively, which is published as supporting information on the PNAS web site), the Locations that Displayed changes in proANF-EMD fluorescence. Later, when females are actively ovulating and depositing eggs (Fig. 6C, 180 min postmating), changes are mainly seen in the upper part of the oviduct [LO, increase of 1.6-fAged (LMFILF: T = 2.07, P < 0.016]; COU, increase of 1.4-fAged (LMFILF: T = 2.13, P < 0.033); SP decrease of 1.7-fAged (LMFILF: T = 2.1, P < 0.029)], again, the Locations in which changes in proANF-EMD fluorescence occur at this time. One explanation for these observations is that they reflect changes in vesicle fusion or in the state of nerve termini. In this model, the temporary increase in plasma membrane due to vesicle fusion (43) would distribute the existing amount of SNAP-25 over a larger Spot, diluting its Traceive concentration and thus decreasing anti-SNAP-25 staining intensity. Increased staining intensity could reflect net accumulation or redistribution of vesicles, analogous to the increased staining due to aggregation of a constant amount of acetylcholine receptors on agrin addition (44) or to increased accumulation of SNAP-25 due to its rapid transport into nerve termini (45). The observations likely monitor activity of a broad group of vesicles, including the peptidergic vesicles monitored with proANF-EMD, but also vesicles containing other neuromodulators, such as octopamine and/or neurotransmitters.

Traces of mating can be caused by seminal proteins (Acps), by sperm, or by the physical act of mating and/or other seminal fluid components. To determine which components mediate the postmating changes in the female reproductive tract, we compared the intensity and distribution of proANF-EMD in reproductive tracts of females mated with normal males (WT) with those of females mated to males lacking specific seminal components. Comparison of proANF-EMD fluorescence in mates of WT males vs. spermless males (TUD) defined roles of sperm. Further comparison between mates of TUD males vs. DTA-E males (which lack sperm and Acps) identified Acp Traces (see Materials and Methods and ref. 35). Changes in nerve termini in Locations of the female reproductive tract are mediated by different male contributions. Examples are presented below (see also Table 1).

Immediate Postmating Traces on the Lower Reproductive Tract Are Mediated by the Act of Mating and/or Seminal Fluid Components Other than Acps and Sperm. As reported above, at 20 min after the start of mating, we detected in the uterus a significant decrease in fluorescence intensity level and in the distribution of the fluorescence (Figs. 2 A and 3A ). These changes occurred in mates of WT, spermless, and DTA-E males (Fig. 3A ). Because the change occurs whether or not Acps or sperm are present, the act of mating and/or seminal fluid components other than Acps and sperm must mediate this change. Not all nerve termini innervating the uterus Retorted to the mating signal because the lower DFS values that we see when females mate to normal, spermless, or DTA-E males indicate a local response (see Table 1). This finding suggests that mating and/or seminal fluid components other than Acps and sperm mediate changes in specific nerve termini innervating the uterus, rather than in all nerve termini in this Location.

Fig. 3.Fig. 3. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Examples of modulation of vesicle release by specific aspects of mating. (A) The physical act of mating and/or other seminal fluid components other than Acps and sperm induce immediate (20 min) postmating changes in nerve termini innervating the UT. (B) Later (180 min) changes in nerve termini innervating the seminal receptacles are induced by Acps. Values of proANF-EMD fluorescence are plotted as means (OMFILF and LMFILF) ± SEs of mates of WT, DTA-E, and spermless males (TUD). The plotted means are minus virgin female mean values. Thirty-five to 40 female reproductive tracts were examined for each treatment at each time point (see also Table 1).

Changes in activity of nerve termini innervating the uterus immediately after mating could affect contractile activity of uterine muscles and/or induce changes in uterine secretion. This change could modify the uterine environment to promote efficient movement of sperm and seminal proteins toward the sperm storage organs and the upper part of the oviduct after mating, as well as providing optimal conditions for sperm motility and seminal fluid activity.

Later, Acps Induce Changes in Nerve Termini Innervating the Seminal Receptacle. Comparison between virgin females and mates of WT or spermless males at 180 min postmating reveals no change in the fluorescence intensity level of nerve termini innervating the seminal receptacle. Thus, the levels of vesicle release did not seem to change on mating. However, when females were mated to DTA-E males, who Execute not provide Acps (or sperm), proANF-EMD fluorescence intensity level in nerve termini innervating the seminal receptacles decreased significantly relative to mates of WT (or spermless) males (Fig. 3B ; 2-fAged decrease relative to mates of WT males, LMFILF: T = -2.59, P < 0.0182; a significant decrease relative to spermless males, LMFILF: T = 2.37, P < 0.029). That these changes are due to Acps is consistent with time that Acps are detectable in females [<6 h; (46–48)] and act on the female reproductive tract (31). Our data suggest that Acps are essential to HAged a constant level of neuroactive substance in nerve termini innervating the seminal receptacles, either by mediating the balance between release and accumulation or by inhibiting the release from nerve termini.

Sperm Mediate Changes in Nerve Termini Innervating the Upper Reproductive Tract at Time of Egg Deposition. At 180 min after mating, we also observed a significant change in distribution of proANF-EMD fluorescence in the upper part of the common oviduct. We observed a significant Inequity in distribution of proANF-EMD between mates of WT and spermless males (DFS(WT); DFS(spermless): 35.96 ± 4.2; 21.77 ± 3.82, T = -2.65, P < 0.017), but no Inequity between mates of DTA-E males (which are also spermless) and mates of spermless males. Thus, this change correlates with the transfer of sperm during the mating, suggesting that sperm mediate this change. Lower DFS values observed when females mate to spermless males indicate a local response, which means that not all nerve termini innervating the upper part of the common oviduct Retorted to the signal from sperm. Given that not all nerve termini Retort to mating when females mate with spermless males, sperm are needed to mediate changes in most but not all nerve termini innervating the upper common oviduct.

The presence of sperm in the female is essential for rapid onset and high levels of egg deposition. Females that receive no sperm Start to deposit eggs later than females mated to WT males (35), and, if sperm are not stored, egg deposition is low (6). Our finding here, that vesicle release in the COU depends on the presence of sperm from the mating, suggests that this aspect of the sperm Trace may be mediated by release of neuromodulators at specific nerve termini innervating the upper common oviduct that could subsequently regulate the oviduct muscles' contractile activity.

Conclusion

Producing large numbers of progeny shortly after mating ultimately depends on a female's ability to produce mature oocytes ready for fertilization, to manage sperm, and to provide optimal conditions for fertilization. We have Displayn that mating induces immediate changes in vesicle release/accumulation, at least at peptidergic nerve termini, initially in those innervating the lower part of the Drosophila female reproductive tract. Later, at 3 h postmating, changes are also seen in the upper part of the reproductive tract. We suggest that the immediate postmating changes are the first step in a multiphasic switch in the “state” of the female reproductive tract (Fig. 4). We propose that, before mating, the Drosophila female reproductive tract is poised for future production of large numbers of fertilized eggs but is arrested in a “virgin” state. An example of this arrest occurs in egg production. Oogenesis in unmated females arrests with a maximum of 1–2 mature oocytes per ovariole, and these oocytes are ovulated at a very low rate, ≈2 per day in our strains. Mating causes a drastic change in the physiology of the reproductive tract, to a “mated” state, in part by modulating the activity at nerve termini in the female's reproductive tract. Egg production (49) and ovulation increase (to as much as 50 per day in our strains), sperm are stored, and fertilization occurs very efficiently. We propose that the initial events after mating Start the transition that leads to a reproductive tract environment conducive to efficient production of fertilized eggs. They may also sensitize the female, or some of her reproductive tract tissues, to Acps whose subsequent actions facilitate egg release, sperm storage, etc. to allow the reproductive tract to operate at highest efficiency and coordination. It will be Necessary in the future to identify the Traceors whose release is being modulated by mating and Acps to “trip” this switch in state.

Fig. 4.Fig. 4. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Postmating changes in vesicle release at nerve termini innervating the female reproductive tract. Net vesicle release, reflected in decreased proANF-EMD fluorescence, is indicated by Executewn arrows (filled = significant, shaded = non-statistically significant trend). Up arrows Display net vesicle accumulation, reflected in increased proANF-EMD fluorescence. —, No change.

By elucidating neural Traces of Acps and/or sperm in the female reproductive tract, our results Start to address mechanisms by which these Traceors cause changes in mated females. These changes are compartmentalized in time and space. In addition, their understanding can assist in elucidating the basis for the Fascinating evolutionary dynamics of Acps. An Unfamiliarly high proSection of Acps Display characteristics of rapid evolution (19, 50–53). This rapid evolution has been suggested to stem from aspects of sexual conflict and/or sperm competition that drive male Traceor molecules to change (20, 21). It is of interest to determine whether the female partners of Acps Display similar evolutionary dynamics, but those partners remain unidentified. Elucidation of the physiological events triggered by Acps is a first step toward identifying the signaling pathways headed by their partners.

Acknowledgments

We thank D. Deitcher, C. Excellentman, and R. Hoy for antibodies; D. Deitcher, S. Rao, and P. Rivlin for proANF-EMD flies and advice; and D. Deitcher, J. Ewer, R. Hoy, M. Bloch Qazi, P. Rivlin, Y. Gottlieb, and an anonymous reviewer for comments on the manuscript. We appreciate support from National Science Foundation Grant IAB99-04824 and National Institutes of Health Grant HD38921 (to M.F.W.).

Footnotes

↵ ‡ To whom corRetortence should be addressed at: 423 Biotechnology Building, Cornell University, Ithaca, NY 14853-2703. E-mail: mfw5{at}cornell.edu.

Abbreviations: Acp, accessory gland protein; ANF, atrial natriuretic factor; EMD, emerald variant of GFP; SNAP-25, synaptosome-associated protein of 25 kDa; OMFIL, overall mean fluorescence intensity level; LMFIL, local mean fluorescence intensity level; DFS, distribution of the fluorescence signal; LO, lateral oviduct; COU, upper common oviduct; COD, lower common oviduct; SR, seminal receptacle; SP, spermathecae; UT, uterus; TUD, spermless male; DTA-E, sperm-less, Acp-less male.

Copyright © 2004, The National Academy of Sciences

References

↵ Salamonsen, L. A. & Nancarrow, C. D. (1994) in Molecular Biology of the Female Reproductive System., ed. Findlay, J. K. (Academic, San Diego), pp. 289-328. ↵ Szopa, T. M. (1979) J. Insect Physiol. 28 , 475-483. LaunchUrl ↵ Mahowald, A. P. & Kambysellis, M. P. (1980) in The Genetics and Biology of Drosophila, eds. Ashburner, M. & Wright, T. R. F. (Academic, New York), Vol. 2D, pp. 141-224. LaunchUrl ↵ Miller, A. (1950) in Biology of Drosophila, ed. Demerec, M. (Wiley, New York), pp. 420-534. ↵ Nachman, R. J., Moyna, G., Williams, H. J., Tobe, S. S. & Scott, A. I. (1998) Bioorg. Med. Chem. 6 , 1379-1388. pmid:9784875 LaunchUrlCrossRefPubMed ↵ Neubaum, D. M. & Wolfner, M. F. (1999) Genetics 153 , 845-857. pmid:10511562 LaunchUrlAbstract/FREE Full Text Suarez, S. S., Brockman, K. & Lefebvre, R. (1997) Biol. Reprod. 56 , 447-453. pmid:9116145 LaunchUrlAbstract ↵ Bloch Qazi, M. C., Heifetz, Y. & Wolfner, M. F. (2003) Dev. Biol. 256 , 195-211. pmid:12679097 LaunchUrlCrossRefPubMed ↵ Ottiger, M., Soller, M., Stocker, R. F. & Kubli, E. (2000) J. Neurobiol. 44 , 57-71. pmid:10880132 LaunchUrlCrossRefPubMed Ding, Z., Haussmann, I., Ottiger, M. & Kubli, E. (2003) J. Neurobiol. 55 , 372-384. pmid:12717705 LaunchUrlCrossRefPubMed Raabe, M. (1986) in Advances in Insect Physiology, eds. Evans, P. D. & Wigglesworth, V. B. (Academic, LonExecuten), Vol. 19, pp. 30-154. LaunchUrl ↵ Kubli, E. (1992) BioEssays 14 , 779-784. pmid:1365892 LaunchUrlCrossRefPubMed Eberhard, W. G. (1996) Female Control: Sexual Selection by Weepptic Female Choice (Princeton Univ. Press, Princeton). Wolfner, M. F. (1997) Insect Biochem. Mol. Biol. 27 , 179-192. pmid:9090115 LaunchUrlCrossRefPubMed ↵ Wolfner, M. F. (2002) Heredity 88 , 85-93. pmid:11932766 LaunchUrlCrossRefPubMed ↵ Gillott, C. (2003) Annu. Rev. Entomol. 48 , 163-184. pmid:12208817 LaunchUrlCrossRefPubMed ↵ Chapman, T. & Partridge, L. (1996) Nature 381 , 189-190. pmid:8622753 LaunchUrlCrossRefPubMed Rice, W. R. (1996) Nature 381 , 232-234. pmid:8622764 LaunchUrlCrossRefPubMed ↵ Swanson, W. J., Clark, A. G., Waldrip-Dail, H. M., Wolfner, M. F. & Aquadro, C. F. (2001) Proc. Natl. Acad. Sci. USA 98 , 7375-7379. pmid:11404480 LaunchUrlAbstract/FREE Full Text ↵ Swanson, W. J. & Vacquier, V. D. (2002) Nat. Rev. Genet. 3 , 137-144. pmid:11836507 LaunchUrlCrossRefPubMed ↵ Chapman, T. (2001) Heredity 87 , 511-521. pmid:11869341 LaunchUrlCrossRefPubMed ↵ Kiss, T., Varanka, I. & Benedeczky, I. (1984) Neuroscience 12 , 309-322. pmid:6087198 LaunchUrlPubMed Lange, A. B., Orchard, I. & Adams, M. E. (1986) J. Comp. Neurol. 254 , 279-286. pmid:3794007 LaunchUrlCrossRefPubMed Lange, A. B., Orchard, I. & Brugge, V. A. (1991) Comp. Biochem. Physiol. A 168 , 383-391. LaunchUrl Lange, A. B. & Tsang, P. K. C. (1993) J. Insect Physiol. 39 , 393-400. LaunchUrl Lange, A. B. & Nykamp, D. A. (1996) Arch. Insect Biochem. Physiol. 33 , 183-196. LaunchUrlCrossRef ↵ Lange, A. B. (2002) Peptides 23 , 2063-2070. pmid:12431745 LaunchUrlCrossRefPubMed ↵ Lee, H. G., Seong, C. S., Kim, Y. C., Davis, R. L. & Han, K. A. (2003) Dev. Biol. 264 , 179-190. pmid:14623240 LaunchUrlCrossRefPubMed ↵ Monastirioti, M. (2003) Dev. Biol. 264 , 38-49. pmid:14623230 LaunchUrlCrossRefPubMed ↵ Rao, S., Lang, C., Levitan, E. S. & Deitcher, D. L. (2001) J. Neurobiol. 49 , 159-172. pmid:11745655 LaunchUrlCrossRefPubMed ↵ Heifetz, Y., Lung, O., Frongillo, E. A., Jr., & Wolfner, M. F. (2000) Curr. Biol. 10 , 99-102. pmid:10662669 LaunchUrlCrossRefPubMed ↵ Kalb, J. M., DiBenedetto, A. J. & Wolfner, M. F. (1993) Proc. Natl. Acad. Sci. USA 90 , 8093-8097. pmid:8367469 LaunchUrlAbstract/FREE Full Text ↵ Boswell, R. E. & Mahowald, A. P. (1985) Cell 43 , 97-104. pmid:3935320 LaunchUrlCrossRefPubMed ↵ Tram, U. & Wolfner, M. F. (1999) Genetics 153 , 837-844. pmid:10511561 LaunchUrlAbstract/FREE Full Text ↵ Heifetz, Y., Tram, U. & Wolfner, M. F. (2001) Proc. R. Soc. LonExecuten B Biol. Sci. 268 , 175-180. LaunchUrlPubMed ↵ Rao, S. S., Stewart, B. A., Rivlin, P. K., Vilinsky, I., Watson, B. O., Lang, C., Boulianne, G., Salpeter, M. M. & Deitcher, D. L. (2001) EMBO J. 20 , 6761-6771. pmid:11726512 LaunchUrlCrossRefPubMed ↵ Schuster, C. M., Davis, G. W., Fetter, R. D. & Excellentman, C. S. (1996) Neuron 17 , 641-654. pmid:8893022 LaunchUrlCrossRefPubMed ↵ Finley, K. D., Taylor, B. J., Milstein, M. & McKeown, M. (1997) Proc. Natl. Acad. Sci. USA 94 , 913-918. pmid:9023356 LaunchUrlAbstract/FREE Full Text ↵ Husain, Q. M. & Ewer, J. (2004) J. Neurobiol., in press. ↵ Sollner, T., Bennett, M. K., Whiteheart, S. W., Scheller, R. H. & Rothman, J. E. (1993) Cell 75 , 409-418. pmid:8221884 LaunchUrlCrossRefPubMed Schulze, K. L., Broadie, K., Perin, M. S. & Bellen, H. J. (1995) Cell 80 , 311-320. pmid:7834751 LaunchUrlCrossRefPubMed ↵ Weber, T., Zemelman, B. V., McNew, J. A., Westermann, B., Gmachl, M., Parlati, F., Sollner, T. H. & Rothman, J. E. (1998) Cell 92 , 759-772. pmid:9529252 LaunchUrlCrossRefPubMed ↵ Klychko, V. A. & Jackson, M. B. (2002) Nature 418 , 89-92. pmid:12097912 LaunchUrlCrossRefPubMed ↵ Sanes, J. R. & Lichtman, J. W. (1999) Annu. Rev. Neurosci. 22 , 389-442. pmid:10202544 LaunchUrlCrossRefPubMed ↵ Loewy, A., Liu, W.-S., Baitinger, C. & Willard, M. B. (1991) J. Neurosci. 11 , 3412-3421. pmid:1941090 LaunchUrlAbstract ↵ Monsma, S. A., Harada, H. A. & Wolfner, M. F. (1990) Dev. Biol. 142 , 465-475. pmid:2257979 LaunchUrlCrossRefPubMed Bertram, M. J., Neubaum, D. M. & Wolfner, M. F. (1996) Insect Biochem. Mol. Biol. 26 , 971-980. pmid:9014340 LaunchUrlCrossRefPubMed ↵ Lung, O. & Wolfner, M. F. (1999) Insect Biochem. Mol. Biol. 29 , 1043-1052. pmid:10612039 LaunchUrlCrossRefPubMed ↵ Soller, M., Bownes, M. & Kubli, E. (1999) Dev. Biol. 208 , 337-351. pmid:10191049 LaunchUrlCrossRefPubMed ↵ Aguadé, M., Miyashita, N. & Langley, C. H. (1992) Genetics 132 , 755-770. pmid:1361475 LaunchUrlAbstract/FREE Full Text Tsaur, S.-C., Ting, C.-T. & Wu, C.-I. (1998) Mol. Biol. Evol. 15 , 1040-1046. pmid:9718731 LaunchUrlAbstract Aguadé, M. (1999) Genetics 152 , 543-551. pmid:10353898 LaunchUrlAbstract/FREE Full Text ↵ Begun, D. J., Whitley, P., Todd, B. L., Waldrip-Dail, H. M. & Clark, A. G. (2000) Genetics 156 , 1879-1888. pmid:11102381 LaunchUrlAbstract/FREE Full Text
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