Sex Peptide binds sperm weakly in the male ejaculate but its binding increases within the mated female’s reproductive tract
To test whether female factor(s) affect the binding of SP to sperm, we compared the signal intensity of anti-SP staining on sperm before (in ejaculate collected from males) and after mating (in the female’s bursa [uterus] and SR). We reasoned that if the signal intensity in the male ejaculate did not change after mating, this would mean that components of the male ejaculate are sufficient to fully facilitate SP-sperm binding without requiring female factor(s).
We isolated sperm from ejaculates exuded by males (Eja; 0 min), sperm in the mated female’s bursa (uterus; 35 min after the start of mating; ASM) or stored in her seminal receptacle (SR; 2 h ASM). The amount of SP bound to sperm was determined by quantifying the corrected signal intensity of the immunofluorescence of anti-SP along the sperm tail in all three situations. The signal intensity for SP detected on sperm was weakest in male ejaculates (Fig. 1A, A’, and J; Mean±SE=1964±442.6 AU; F(3, 48) = 132). It was higher in sperm isolated from mated female bursas. Sperm isolated from mated female bursas (35 min ASM) had a “spotty” pattern of anti-SP staining, with anti-SP immunofluorescence appearing in bright and dim specks all along sperm (Fig. 1C, C’, and J, Mean±SE=4361±442.6 AU, p***<0.001; compare to Fig. 1A, A’). This suggested that although the quantity of SP bound to sperm increased in the female’s bursa, sperm were not uniformly saturated with SP. Sperm isolated from the SRs (2 h ASM) had the strongest signal intensity of SP, suggesting that the amount of SP detected on sperm was highest in the sperm storage organ. Staining for SP on sperm isolated from SRs was consistent and uniform along the sperm (Fig. 1E, E’, and J; Mean±SE=9384±442.6 AU, p***<0.001), similar to what has been reported previously [16,17,18]. Since the amount of SP detected on sperm gradually increases after they enter the FRT, our results suggest a possible role of female factor(s) in assisting SPs binding to stored sperm.
Several SFPs (proteases, prohormones, and others) either mediate or undergo post-mating modifications en route to or after transfer to the FRT [7, 29, 42], some of which are crucial for inducing or maintaining post-mating responses in mated females. We thus wondered whether the gradual increase in amount of SP detected on sperm within the FRT is because of a need for the male components to undergo requisite modifications with time. The intensity of SP signals on sperm was observed to be highest in sperm isolated from the SR at 2 h ASM, suggesting that this is the maximum time that would be required by the male molecules to act (or to undergo any necessary modifications). To test if time alone is sufficient to maximize SP’s binding to sperm, we collected ejaculates exuded from males and incubated them for 2 h in 1× PBS before processing them for anti-SP staining. We did not observe any change in the signal intensity or in the distribution of anti-SP on sperm (Fig. 1G, G’, and J, Mean±SE=1578±442.6 AU, p=ns) in incubated ejaculates relative to signals on sperm isolated from un-incubated ejaculates (Fig. 1A, A’, and J). This suggested that time alone is not sufficient to maximize SP’s binding to sperm. Thus, female factor(s) likely contribute to, or facilitate, SP-sperm binding. As males with different genetic backgrounds and exposure to temperature conditions were used in these experiments, we verified that these differences did not affect the levels of SP that we observed to be associated with sperm stored in the SR of mated females (Please see Additional file 1: Fig. S1 for details).
LTR-SFPs bind to sperm in the male’s ejaculate or mated females with patterns or timing different from those of SP
Given LTR-SFPs’ role in SP’s sperm binding, we wondered whether the pattern of sperm-associated CG1656, CG1652, CG9997, and Antares (Antr) on sperm isolated from three different sites/times used above paralleled that of SP. We examined the presence of bound LTR-SFPs to sperm by experiments analogous to those shown in Fig. 1 for SP, using sperm isolated from the male’s ejaculate (0 min after exudation), mated female’s bursa (35 min ASM), and SR (2 h ASM).
We observed a lower signal intensity for CG1656 (Fig. 2A, C’; Mean±SE=3689±513.3 AU; F(2, 42)=19.48) and Antr (Fig. 2D, F’; Mean±SE=3173±993.5 AU; F(2,30)=20.77) on sperm in ejaculate compared to that on sperm inside the female (Fig. 2B (6474±513.3 AU; p***<0.001), C (6453±513.3AU; p***<0.001) and C’ for CG1656 and E (7858±993.5 AU; p***<0.001), F (9296±993.5 AU; p***<0.001), and F’ for Antr. However, the signal intensity of sperm-bound CG1656 and Antr did not differ between sperm isolated from the bursa (Fig. 2B, E) vs. those from the SRs (Fig. 2C, F). This suggests that although the amount of these LTR-SFPs bound to sperm increases post-mating, their maximal binding had already occurred in the bursa of the mated female, in contrast to SP whose sperm binding reached its highest levels in the female’s SR.
CG1652 and CG9997 differed in their sperm-binding pattern from CG1656 and Antr. We detected extremely faint signals for both CG1652 and CG9997 associated with sperm in the ejaculate (Fig. 2G, I’ (Mean±SE=576±740.3 AU; F(2,36)=72.79), Fig. 2J, L’ (Mean±SE=715.5±515.4 AU; F(2,21)=87.66) respectively) or in the bursa of the mated female (Fig. 2H, I’ (Mean±SE=838±740.3 AU; p=ns), Fig. 2K, L’ (Mean±SE=968.4±515.4 AU; p=ns), respectively). However, we saw significantly strong signal for both proteins in sperm isolated from SRs of mated females (Fig. 2I, I’ (Mean±SE=8439±740.3 AU; p***<0.001), 2L, L’ (Mean±SE=6748±515.4 AU; p***<0.001) respectively), consistent with our previous report that these proteins are bound to sperm in SRs [4, 16]. The regions of association and distribution that we observed for these SFPs (SP, CG9997, and Antr on the head and tail of stored sperm; CG1652 and CG1656 detectable only on the tail of stored sperm) were also consistent with previous reports that assessed the levels of SP associated with sperm stored in SRs [4, 16].
We also assessed the two LTR-SFPs, Seminase and CG17575, that had previously been reported as not binding to stored sperm [4, 22]; in addition to confirming that finding, our experiments showed that these two SFPs exhibit no sperm binding in the ejaculate either (ejaculate: Additional file 2: Fig. S2A and D; mated female’s, bursa: Additional file 2: Fig. S2B and E; SR: Additional file 2: Fig. S2C and F). Thus, the binding patterns/timing of LTR-SFPs differed from those of SP and fall into three groups: (1) CG1656 and Antr, which bind to sperm in the ejaculate, increase their binding once inside the female, but do not show the additional increase in binding in the seminal receptacle that was seen for SP; (2) CG1652 and CG9997 show no detectable binding to sperm until they are inside the female’s seminal receptacle; (3) Seminase and CG17575 show no detectable binding to sperm.
Ablation of spermathecal secretory cells (SSCs) in the female reproductive tract does not affect the initial binding of SP or LTR-SFPs to sperm
To begin to identify the source of female contributions to SFP-sperm binding, we examined the effect on the intensity and timing of SFP binding to sperm when we ablated SSCs, which are known to regulate the storage and motility of sperm in sperm storage organs [37, 40, 41]. We ablated SSCs by driving the expression of misfolded protein RhG69D [43, 44] in these cells (Fig. 3A–D) or by using Hr39 mutants (Please see Additional file 4: Fig. S4). Hr39, a NR5A-class nuclear hormone receptor, is needed for the development and function of important secretory tissues in the FRT [37]: Hr39 mutants exhibit defective/decreased SSCs and parovaria [41, 45]. Although neither targeting of Rh1G69D to SSCs nor the presence of Hr39 mutations completely ablated all SSCs, higher percentages of ablations were observed in Hr39 mutant females. We examined SP-sperm binding in these SSC-depleted females.
Five-day-old Send1>CyO (control) and Send1>Rh1G69D (experimental) females were mated to 3-day-old control (CS) males, and the mated females were frozen at 2 h ASM. Sperm, dissected from SRs of the mated females, were assessed for the presence of SP by western blotting. We did not observe any striking difference in the levels of sperm-associated SP in experimental females relative to control females at 2 h ASM (Fig. 3E, lanes 3, 4). Similarly, there was no difference in amounts of LTR-SFPs CG1656, CG1652, Antr, or CG9997 associated with sperm isolated from experimental vs. control females at 2 h ASM (Fig. 3E, lanes 3, 4). We also performed immunofluorescence (IF) on sperm dissected from the SR of Send1>CyO (control) and Send1>Rh1G69D (experimental) females to probe for the presence of SP. Consistent with the results of our western blots, we did not observe any striking difference in the intensity of anti-SP staining on sperm stored in experimental females (Additional file 3: Fig. S3B-B’ and E; Mean±SE=4299±224.8 AU; F(10, 10)=2.023; p=0.2818) relative to those stored in control females (Additional file 3: Fig. S3A-A’ and E; Mean±SE=4590±319.8 AU) at 2 h ASM.
In similar experiments, 5-day-old Hr39 mutant and control females were mated to 3-day-old control (CS) males, and mated females were frozen at 2 h ASM. We dissected sperm from SRs of Hr39 mutant (BL64285 {Hr39[C105]}; Exp) [46] and their balanced sibling (“sib”) controls (BL64285/CyO; C) at 2 h ASM and probed the samples for SP by western blotting and immunofluorescence. As we saw with Send1>Rh1G69D vs. control females, we did not observe any striking difference in the levels of SP associated with sperm probed through western blotting (Fig. 4A, lanes 3, 4; Fig. 4B; p= 0.5328) or the signal intensity of anti-SP staining along the entire sperm performed through immunofluorescence in control balancer-sib females (Fig. 4E, G; Mean±SE=10238±715.9 AU; F(13, 13)=1.729; p=0.3360; N=14) when compared to mutant females at 2 h ASM (Fig. 4F, G; Mean±SE=10736±544.5 AU; N=14). To test for consistency, we examined four other available Hr39 mutants. However, three of these lines (BL38620 {Hr39[MI06174]} [47], BL43358 {Hr39[C277]} [48], and BL20152 {Hr39[EY04579]} [49]) did not contain balancer-sib controls, and the fourth (BL64305/CyO {Hr39[c739]} [50]) produced too few such balancer-sib females for experimentation, so we used CS females as their controls. We performed western blotting to examine the effect of differences in the genetic background for two different controls (balancer-sib and CS females) used in our experiments on the levels of SP received by females. We observed no striking difference in the levels of SP transferred to the FRT at 35 min ASM (Additional file 8: Fig. S8 lanes 3 and 4) and those bound to sperm stored in the SR at 2 h ASM (Additional file 8: Fig. S8 lanes 5 and 6) in both controls. As we saw with BL64285 females and their controls, we detected similar levels of SP bound to sperm dissected from CS and these Hr39 mutant females (Additional file 5: Fig. S5A, lane 3, lanes 4–8; Additional file 5: Fig. S5B) at 2 h ASM. We also detected signals for the LTR-SFPs CG1656, CG1652, Antr, and CG9997 in Hr39 mutant females at levels similar to those in CS females, at 2h ASM (Additional file 6: Fig. S6A, lane 3 and lanes 4–8). The level of anti-SP staining visualized along the entire sperm through immunofluorescence also did not show any relative difference between CS females (control) (Additional file 5: Fig. S5C & H) and mutant females from the other four Hr39 lines (Additional file 5: Fig. S5D-H), consistent with what we observed in our western blots. Thus, loss of SSCs (and parovaria, in the case of Hr39 mutants) did not have an evident effect on the binding of SP or LTR-SFPs to sperm. This result suggested that female molecules that increase the levels of SFPs and SP association with stored sperm are not derived (or not solely derived) from SSCs and/or parovaria.
Loss of SSCs and parovaria in females affects the release of SP from stored sperm
At 4 days ASM, our western blots consistently showed higher levels of sperm-associated SP in Hr39 mutant females (BL 64285; Fig. 4A, lane 6) relative to the levels in their balancer-sib control females (Fig. 4A, lane 5); this was particularly clear when SP levels were normalized with tubulin levels (Fig. 4C; p**<0.01). We obtained analogous results showing higher levels of sperm-associated SP at 4 days ASM in females homozygous for each of the other four Hr39 mutant alleles, relative to sperm-bound SP levels in CS females (Additional file 7: Fig. S7A lanes, 3–8), following normalization of SP levels with tubulin (Additional file 7: Fig. S7B).
Binding of SP to sperm or SP’s gradual cleavage from sperm are both essential for the efficient release of sperm from storage within the mated female [51]. To test whether the elevated levels of sperm-bound SP in Hr39 mutant females at 4d ASM was associated with increased retention of sperm in these females, we performed sperm counts. Control and Hr39 mutant females from the BL64285 stock were mated to ProtB-eGFP [52] males, and sperm stored in their SR were counted at 8 days ASM. Mutant females (Fig. 4D, Exp) exhibited significantly higher sperm numbers relative to their controls (Fig. 4D, C; p**=<0.01). Consistently, mated Hr39 mutant females from the other four lines also showed significantly higher sperm counts, indicating poor release of stored sperm when compared to CS females (Additional file 6: Fig. S6B; p**=<0.01).
To distinguish whether the higher amounts of sperm-associated SP measured on western blots were due to this higher retention of sperm or to impaired release of SP from sperm, we used immunofluorescence to examine levels of SP bound to sperm. We observed higher levels of sperm-bound SP in BL64285 Hr39 mutant females (Fig. 4I, J; Mean±SE=8209±1332 AU;F(10, 10)=60.6; p**<0.001; N=11) than in balancer-sib control females (Fig. 4H, J; Mean±SE=673.5±171.1 AU); the SP levels in balancer-sib controls at 4 days ASM were either barely detectable or below our detection limits. Similarly, anti-SP immunofluorescence was higher in females for the four other Hr39 mutants (Additional file 7: Fig. S7D-H) relative to levels in CS controls (Additional file 7: Fig. S7C & H); as observed with balancer-sib controls for BL64285, the CS controls again had SP levels barely detectable or below our detection limits at 4 days ASM. We used Send1>Rh1G69D flies, in an attempt to determine whether SSCs alone were responsible for effects on SP release. We saw no difference in SP levels bound to sperm dissected from Send1>CyO (control) and Send1>Rh1G69D (experimental; SSCs ablated) females mated to CS (control) males (Fig. 3F, lanes 3, 4 for 4 days ASM and 5, 6 for 8 days ASM) in our western blotting. The level of anti-SP staining visualized along the entire sperm through immunofluorescence also did not show any relative difference between experimental females (Additional file 3: Fig. S3D-D’ and F; Mean±SE=38.14±12.93 AU; F(6, 6)=2.1; p=0.3651) relative to those stored in control females (Additional file 3: Fig. S3C-C’ and F; Mean±SE=50.86±19.1 AU) at 4 days ASM. However, signals were extremely low in these experiments. Our results suggest that loss of some or all SSCs may not be sufficient to impair the release of SP from sperm, but that the absence of SSCs and parovaria together impairs the release of SP from sperm. Since the release of sperm from storage requires this release [51], the lack of SP release in the absence of SSCs and parovaria results in more sperm being retained in females (and each of these sperm contains more bound SP than would normally be seen at those times). These data suggest and support the hypothesis that the protease responsible for cleaving SP’s active region from sperm [18] is provided by the parovaria and/or SSCs.