1. Evolutionary Biology
  2. Genetics and Genomics
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Drosophila seminal sex peptide associates with rival as well as own sperm, providing SP function in polyandrous females

  1. Snigdha Misra
  2. Mariana F Wolfner  Is a corresponding author
  1. Department of Molecular Biology and Genetics, Cornell University, United States
Research Article
Cite this article as: eLife 2020;9:e58322 doi: 10.7554/eLife.58322
6 figures, 1 table and 1 additional file

Figures

SP from a second male can bind to SP-deficient sperm of previous male stored within a mated female.

Cartoon: Pictorial representation of the crossing scheme (fly images from Biorender). Wild type (CS) females were first mated to an SP-null male and then, at the indicated time, to a spermless (SOT) male. Sperm heads were stained with DAPI (blue) and SP was visualized with Alexa fluor 488, staining the sperm tail (green) and sperm head (cyan; overlapping blue/green). (A) Sperm from females singly mated to SP-null males, 1d ASM. (B) Sperm from females mated to SP-null males, remated to spermless males at 1d ASFM and (C) at 4d ASFM, both frozen 2 hr ASSM. White arrows indicate sperm heads. Bar = 20 µm (D) Western blot lane numbers 1: Fv, reproductive tract (RT) of virgin female (negative control; n = 5), 2: M, a pair of male accessory gland (positive control; n = 1), 3: SP-, reproductive tracts of females mated to SP-null males, 2 hr ASM (n = 5), 4: SOT, reproductive tracts of females mated to spermless males, 1d ASM (n = 5), 5: SOT, SP-, reproductive tract of females mated to spermless males and then remated to SP-null males, 1d ASFM (n = 8 RT), 6: SOT, SP+, reproductive tract of females mated to spermless males and then remated to control (SP+) males at 1d ASFM, frozen 2 hr ASSM (positive control; n = 8 RT), 7: (SP-, SOT), 1d and 8: (SP-, SOT), 4d sperm isolated from the seminal receptacle of females mated to SP-null males and then remated to spermless males at 1d ASFM and 4d ASFM, frozen 2 hr ASSM (n = 15 SS). Actin served as loading control.

Figure 2 with 1 supplement
Sperm from a second male are not bound to SP from a prior spermless male.

(Cartoon): Pictorial representation of the cross (fly images from Biorender); it is reciprocal of that in Figure 1. Females mated first with spermless (SOT) males and then a day later with SP-null males that provided sperm. Sperm heads were stained with DAPI (blue) and SP visualized with Alexa fluor 488, staining the sperm tail (green) and sperm head (cyan; overlapping blue/green). (A) Sperm from females singly mated to SP-null males, 2 hr ASM. (B) Sperm from females mated to spermless males and then remated to SP-null males, 1d ASFM. (C) Sperm from females mated to spermless males and then remated to SP+ males, 1d ASFM, serve as positive controls. Flies were frozen 2 hr ASSM. White arrows indicate sperm heads. Bar = 20 µm (D) Western blot lane numbers 1: M, a pair of male accessory gland (positive control; n = 1), 2: Fv, reproductive tract (RT) of virgin female (negative control; n = 5), 3: SP+, reproductive tract of females mated to control males (TM3 siblings of SP-null males; n = 5; positive control), 4: SP-, reproductive tract of females mated to SP-null males (n = 5; negative control). 5–12: Proteins from Bursa (B) or seminal receptacle (SR) from females mated to spermless males frozen at 0 min immediately after mating, 35 min, 1 hr, and 3 hr ASM, respectively (n = 15). Actin served as loading control.

Figure 2—figure supplement 1
Cartoon: Pictorial representation of cross (fly images from Biorender).

Females mated first with spermless (sot) male and then 3–6 hr ASFM with SP-null male that provided sperm. Panel: Sperm from SR of females mated to spermless males and then remated to SP-null males, 3–6 hr ASFM, frozen at 2 hr ASSM. Sperm heads were stained with DAPI (blue) and SP (green) probed with Alexa fluor 488 (n = 5; Bar = 20 µm).

Figure 3 with 1 supplement
Remating with spermless males restores fertility, delays receptivity and optimizes efficient sperm release in females that previously mated to SP-null males.

(A) Graphical representation of numbers of progeny produced by each female over the span of 10 days, following mating to control (TM3 siblings of SP-null males: SP+; red), SP-null males (SP-null; green), or spermless males (SOT), p***=<0.001; n = 15–20. (B) Fertility of females mated to SP-null males and then remated to spermless males at 1d ASFM (SP-null, SOT; blue, n = 15–20) and (C) Fertility of females mated to SP-null males and then remated to spermless males at 4d ASFM (SP-null, SOT; blue, n = 15–20) compared to females mated to control males and then remated to spermless males (SP+, SOT, red, ns = non significant). (D) Percentage receptivity of females mated to SP-null males and then remated to spermless males (SP-null, SOT) at 1d ASFM, when compared to females singly mated to SP-null males (red arrows), spermless (SOT, blue arrows) or CS males, 1d ASM (p*=<0.05; p***=<0.001; n = 15–20 for each technical replicate). (E) Percentage receptivity of females mated to SP-null males and then remated to spermless males (SP-null, SOT) at 4d ASFM, when compared to females singly mated to SP-null males, spermless (SOT) or CS males (purple arrows), 4d ASM (p***=<0.001; n = 15–20 for each technical replicate). (F) Western blot lane numbers 1: Fv, reproductive tract (RT) of five virgin females (negative control); 2: M, a pair of male accessory gland (positive control); 3, 4, 5: RT of females mated to CS males, flash frozen at 2 hr (n = 5), 1d (n = 15) and 4d (n = 15) ASM, respectively; 6, 7, 8: RT of females mated to SP-null males and then subsequently mated to spermless males at 1d ASFM, flash frozen 2 hr (n = 5), 1d (n = 15) and 4d (n = 15) ASSM, respectively. Actin served as loading control. (G) Sperm in the seminal receptacle (SR) of a typical female mated to a control male (SP+; ProtB-eGFP) at 4d ASM. (H) Sperm in the SR of a typical female mated to SP-null; ProtB-eGFP male at 4d ASM. (I) Sperm in the SR of a typical female, mated to SP-null; ProtB-eGFP and subsequently remated to a spermless male at 1d ASFM, and frozen at 4d ASSM. In (G–I) sperm heads are green due to eGFP. Bar = 50 µm. (J) Graphical representation of sperm counts in SRs of females singly-mated to control (SP+, red, TM3 siblings of SP-null; ProtB-eGFP), SP-null (green) or doubly-mated to SP-null and spermless male (SP-null, SOT, blue) represented in G, H, I panels (p**=<0.01; p*=<0.05; ns = non significant; n = 15–20).

Figure 3—figure supplement 1
Western blot probed for SP.

Lanes/samples are 1: Fv, reproductive tract (RT) of three virgin females (negative control); 2: M, one pair of male accessory glands (positive control); 3: SP+, RT of three females mated to control (TM3 siblings of SP-null males; positive control) males at 2 hr ASM; 4: SP-, RT of three females mated to SP-null males at 2 hr ASM; 5: SP+ eGFP, RT of three females mated to control (TM3 siblings of SP-null; ProtB-eGFP males; positive control) males at 2 hr ASM; 6: SP- eGFP, RT of three females mated to SP-null; ProtB-eGFP males at 2 hr ASM. Actin served as loading control.

Figure 4 with 1 supplement
SP from a male who also provides sperm can bind to SP-deficient sperm as well as to the donor’s sperm.

Cartoon (I): Pictorial representation of the experimental cross (fly images from Biorender). Females mated to SP-null males were remated to control (ProtB-dsRed) males at 1d ASFM. (A) Sperm from females singly mated to SP-null males, 2 hr ASM (blue sperm-head). (B) Sperm from females mated to SP-null males (blue sperm-head) remated to ProtB-dsRed (red+ blue sperm-head) males at 1d ASFM. SP was visualized with Alexa fluor 488, staining the sperm (head+ tail) green. Flies were frozen 2 hr ASSM. White arrows indicate sperm heads (n = 10; Bar = 20 µm). Cartoon (II): Pictorial representation of the substitute cross (fly images from Biorender). Females mated to SFP depleted control (CS) males were remated to control (Prot B-dsRed) males at 4d ASFM. (C) Sperm from females singly-mated to SFP depleted CS males at 4d ASM (blue sperm-head). (D) Sperm from females mated to SFP depleted CS males (blue sperm-head), remated to ProtB-dsRed (red+ blue sperm-head) males at 4d ASFM. SP was visualized with Alexa fluor 488, staining the sperm (head+ tail)green. Flies were frozen 2 hr ASSM. White arrows indicate sperm heads (n = 10; Bar = 20 µm).

Figure 4—figure supplement 1
Little to no SP is transferred by multiply-mated males.

(A) Western blot probed for SP. Lanes/samples are 1: Fv, reproductive tract (RT) of two virgin females (negative control); 2: M, one pair of male accessory glands from a 3-day-old unmated virgin male; 3: B1, RT of four females mated to control unmated virgin males, frozen at 2 hr ASM; 4: M(d), one pair of male accessory glands dissected from a multiply mated male (previously mated with six virgin females); 5: B7, RT of four females mated to multiply-mated males, frozen at 2 hr ASM. Actin served as loading control. (B) Sperm dissected from females mated to unmated males, frozen at 2 hr ASM. (C) Sperm dissected from females mated to multiply mated males, frozen at 2 hr ASM. Sperm heads were stained with DAPI (blue) and presence of SP (green) was detected with Alexa fluor 488 (n = 5; Bar = 20 µm).

Sperm do not bind detectable LTR-SFPs from a second male.

Females mated to wild type (CS) males at 2 hr ASM show LTR-SFPs bound to sperm, CG1656 (A), CG1652 (E), CG9997 (I). Females mated to SP-null males show the same (B,F,J) but by 1d postmating LTR-SFPs’ signal were no longer detected on sperm (C,G,K) confirming previous reports (Singh et al., 2018). Females mated to SP-null males and then remated to spermless males also do not show detectable signal for sperm-LTR-SFP binding for CG1656 (D), CG1652 (H) and CG9997 (L), 2 hr ASSM, although they have SP bound (Figure 1). Sperm stained for the indicated LTR-SFP detected with Alexa fluor 594 (red) and sperm-head stained with DAPI (blue). Bar = 20 µm (M) Western blot probed for indicated LTR-SFPs. Lanes/samples are 1: Fv, reproductive tract (RT) of three virgin females (negative control); 2: M, one pair of male accessory glands (positive control); 3: CS @ 2 hr, sperm dissected from SR of 20 females mated to wild type (CS) males at 2 hr ASM; 4: SP-null @ 2 hr, sperm dissected from SR of 20 females mated to SP-null males at 2 hr ASM; 5: SP-null @1d, sperm dissected from SR of 20 females mated to SP-null males at 1d ASM; 6: SOT@35’, reproductive tract of three females mated to spermless males at 35’ASM (positive control); 7: SP-null, SOT @ 2 hr, sperm dissected from SR of 20 females mated to SP-null males and then remated to spermless males at 1d ASFM, and frozen at 2 hr ASSM. Lanes were probed for LTR-SFPs CG9997, CG1656, antares and CG1652 and SP as described in the text. Actin served as loading control.

Figure 6 with 1 supplement
Sperm received from SP-null males do not require CG17575 or seminase from a second male to bind SP from that male.

Cartoon: Pictorial representation of the experimental cross (fly images from Biorender). Females mated first with SP-null; ProtB-eGFP males [cyan sperm-head; DAPI(blue)+eGFP(green)] and then a day later with CG17575-null or seminase-null males (blue sperm-head; DAPI stained) and frozen, 2 hr ASSM. SP was visualized with Alexa fluor 594, staining the sperm (head+ tail) red. (A) Sperm from females mated to SP-null; ProtB-eGFP males and then remated to seminase-null males, 1d ASFM. (B) Sperm from females mated to SP-null; ProtB-eGFP males and then remated to CG17575-null males, 1d ASFM. (C) Western blot probed for SP. Lanes/samples are 1: Fv, reproductive tract (RT) of three virgin females (negative control); 2: M, one pair of male accessory glands (positive control); 3: SP-, sperm dissected from 20 females mated to SP-null; ProtB-eGFP males at 2 hr ASM; 4: SP-, sem-, sperm dissected from 20 females mated to SP-null; ProtB-eGFP males and subsequently to seminase-null males at 1d ASFM, frozen at 2 hr ASSM; 5: SP-, 17575-, sperm dissected from 20 females mated to SP-null; ProtB-eGFP males and subsequently to CG17575-null males at 1d ASFM, frozen at 2 hr ASSM. (D) Western blot probed for SP. Lanes/samples are 1: Fv, reproductive tract (RT) of three virgin females (negative control); 2: M, one pair of male accessory glands (positive control); 3: SP-, sperm dissected from 20 females mated to SP-null; ProtB-eGFP males at 2 hr ASM; 4: sem-, sperm dissected from 20 females mated to seminase-null males at 2 hr ASM 5: 17575-, sperm dissected from 20 females mated to CG17575-null males at 2 hr ASM. Actin served as loading control. (E) Sperm isolated from females singly mated to SP-null; ProtB-eGFP males, 2 hr ASM. (F) Sperm isolated from females singly mated to seminase-null male, 2 hr ASM. (G) Sperm isolated from females singly mated to CG17575-null male, 2 hr ASM. White arrows indicate sperm heads (represented as SH, n = 10; Bar = 20 µm).

Figure 6—figure supplement 1
Western blot probed for seminase and CG17575.

Lanes/samples are 1: Fv, reproductive tract (RT) of three virgin females (negative control); 2: M, one pair of male accessory glands (positive control); 3–4: RT of five females mated to wild type (CS) males at 2 hr and 1d ASM, respectively; 5–6: RT of five females mated to SP-null; ProtB-eGFP males at 2 hr and 1d ASM, respectively; 7: RT of five females mated to seminase-null males at 2 hr ASM; 8: RT of five females mated to CG17575-null males at 2 hr ASM. Black arrows indicate full length seminase, red arrows indicate the cleavage products of seminase, post-mating in the female RT. Actin served as loading control.

Tables

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional information
Genetic reagent (D. melanogaster)TudorR. Boswell; similar stock now available from BloomingtonDrosophila Stock CenterBDSC:1735;
FBst0001735;RRID:BDSC_1735
FlyBase Genotype:tud1 bw1 sp1/CyO
Genetic reagent (D. melanogaster)Δ325/TM3; Sb ry (SP-knockout line)Gift from Eric Kubli
Genetic reagent (D. melanogaster)Δ130/TM3; Sb ry (deficiency line)Gift from Eric Kubli
Genetic reagent (D. melanogaster)ProtB-eGFP(X); TM3/TM6Gift from Scott Pitnick
Genetic reagent (D. melanogaster)ProtB-DsRedGift from Scott Pitnick
Antibodyanti-SP
(rabbit polyclonal)
Wolfner labIF (1:200),
WB (1:2000)
Antibodyanti-CG1656
(rabbit polyclonal)
Wolfner labIF (1:100),
WB (1:1000)
Antibodyanti-CG1652
(rabbit polyclonal)
Wolfner labIF (1:50),
WB (1:500)
Antibodyanti-CG9997
(rabbit polyclonal)
Wolfner labIF (1:50),
WB (1:1000)
AntibodyIgG (H+L) Goat anti-Rabbit, Alexa Fluor 488
(goat anti-rabbit polyclonal)
InvitrogenCat. # A11008
RRID:AB_143165
IF (1:300)
AntibodyIgG (H+L) Goat anti-Rabbit, Alexa Fluor 594
(goat anti-rabbit polyclonal)
InvitrogenCat. # A11012
RRID:AB_2534079
IF (1:300)
Antibodyanti-Antares
(rabbit polyclonal)
Wolfner labWB (1:500)
Antibodyanti-seminase
(rabbit polyclonal)
Wolfner labWB (1:1000)
Antibodyanti-CG17575
(rabbit polyclonal)
Wolfner labWB (1:1000)
AntibodyAnti-actin
(mouse monoclonal)
Millipore CorpCat# MAB1501
RRID:AB_2223041
WB (1:3000)
AntibodyPeroxidase AffiniPure Goat Anti-Rabbit IgG
(goat anti-rabbit polyclonal)
Jackson ResearchCode#111-035-003
RRID:AB_2313567
WB (1:2000)
AntibodyPeroxidase AffiniPure Goat Anti-Mouse IgG (goat anti-mouse polyclonal)Jackson ResearchCode#115-035-003
RRID:AB_10015289
WB (1:2000)
OtherDAPI stainInvitrogenCat. # PI62247(1 µg/mL)
OtherPoly-L-Lysine (0.1
% w/v
in H2O)
SigmaP8920-100ML0.01% w/v in H2O
OtherAlbumin from Bovine Serum (BSA)SigmaA9418-50G5% in 1X PBS
OtherCitiFluor Mountant SolutionElectron Microscopy SciencesCat. #17970–100
Software, AlgorithmGraph Pad PrismRRID:SCR_002798Version 6.01

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