19 research outputs found
Male reproductive aging arises via multifaceted mating-dependent sperm and seminal proteome declines, but is postponable in Drosophila
I.S. and S.W. were supported by a Biotechnology and Biological Sciences Research Council (BBSRC) Fellowship to S.W. (BB/K014544/1) and S.W. additionally by a Dresden Senior Fellowship. B.M.K., P.D.C., and R.F. were supported by the Kennedy Trust and John Fell Funds. R.D. was supported by Marie Curie Actions (Grant 655392). B.R.H. was funded by the EP Abraham Cephalosporin-Oxford Graduate Scholarship with additional support from the BBSRC Doctoral Training Programme. M.F.W. was supported by a NIH Grant R01HD038921. Work in the J.S. Laboratory was supported by NIH Grant R15HD080511.Declining ejaculate performance with male age is taxonomically widespread and has broad fitness consequences. Ejaculate success requires fully functional germline (sperm) and soma (seminal fluid) components. However, some aging theories predict that resources should be preferentially diverted to the germline at the expense of the soma, suggesting differential impacts of aging on sperm and seminal fluid and trade-offs between them or, more broadly, be-tween reproduction and lifespan. While harmful effects of male age on sperm are well known, we do not know how much seminal fluid deteriorates in comparison. Moreover, given the predicted trade-offs, it remains unclear whether systemic lifespan-extending inter-ventions could ameliorate the declining performance of the ejacu-late as a whole. Here, we address these problems using Drosophila melanogaster. We demonstrate that seminal fluid deterioration con-tributes to male reproductive decline via mating-dependent mech-anisms that include posttranslational modifications to seminal proteins and altered seminal proteome composition and transfer. Additionally, we find that sperm production declines chronologically with age, invariant to mating activity such that older multiply mated males become infertile principally via reduced sperm transfer and viability. Our data, therefore, support the idea that both germline and soma components of the ejaculate contribute to male reproduc-tive aging but reveal a mismatch in their aging patterns. Our data do not generally support the idea that the germline is prioritized over soma, at least, within the ejaculate. Moreover, we find that lifespan-extending systemic down-regulation of insulin signaling re-sults in improved late-life ejaculate performance, indicating simul-taneous amelioration of both somatic and reproductive aging.Publisher PDFPeer reviewe
Immortal coils: Conserved dimerization motifs of the Drosophila ovulation prohormone ovulin
Dimerization is an important feature of the function of some proteins, including prohormones. For proteins whose amino acid sequences evolve rapidly, it is unclear how such structural characteristics are retained biochemically. Here we address this question by focusing on ovulin, a prohormone that induces ovulation in Drosophila melanogaster females after mating. Ovulin is known to dimerize, and is one of the most rapidly evolving proteins encoded by the Drosophila genome. We show that residues within a previously hypothesized conserved dimerization domain (a coiled-coil) and a newly identified conserved dimerization domain (YxxxY) within ovulin are necessary for the formation of ovulin dimers. Moreover, dimerization is conserved in ovulin proteins from non-melanogaster species of Drosophila despite up to 80% sequence divergence. We show that heterospecific ovulin dimers can be formed in interspecies hybrid animals and in two-hybrid assays between ovulin proteins that are 15% diverged, indicating conservation of tertiary structure amidst a background of rapid sequence evolution. Our results suggest that because ovulin's self-interaction requires only small conserved domains, the rest of the molecule can be relatively tolerant to mutations. Consistent with this view, in comparisons of 8510 proteins across 6 species of Drosophila we find that rates of amino acid divergence are higher for proteins with coiled-coil protein-interaction domains than for non-coiled-coil proteins
Correction: Functional male accessory glands and fertility in Drosophila require novel ecdysone receptor
Functional male accessory glands and fertility in Drosophila require novel ecdysone receptor
<div><p>In many insects, the accessory gland, a secretory tissue of the male reproductive system, is essential for male fertility. Male accessory gland is the major source of proteinaceous secretions, collectively called as seminal proteins (or accessory gland proteins), which upon transfer, manipulate the physiology and behavior of mated females. Insect hormones such as ecdysteroids and juvenoids play a key role in accessory gland development and protein synthesis but little is known about underlying molecular players and their mechanism of action. Therefore, in the present study, we examined the roles of hormone-dependent transcription factors (Nuclear Receptors), in accessory gland development, function and male fertility of a genetically tractable insect model, <i>Drosophila melanogaster</i>. First, we carried out an RNAi screen involving 19 hormone receptors, individually and specifically, in a male reproductive tissue (accessory gland) for their requirement in Drosophila male fertility. Subsequently, by using independent RNAi/ dominant negative forms, we show that Ecdysone Receptor (EcR) is essential for male fertility due to its requirement in the normal development of accessory glands in Drosophila: EcR depleted glands fail to make seminal proteins and have dying cells. Further, our data point to a novel ecdysone receptor that does not include Ultraspiracle but is probably comprised of EcR isoforms in Drosophila male accessory glands. Our data suggest that this novel ecdysone receptor might act downstream of homeodomain transcription factor paired (prd) in the male accessory gland. Overall, the study suggests novel ecdysone receptor as an important player in the hormonal regulation of seminal protein production and insect male fertility.</p></div
The effect of EcR knockdown on the cellular organization of the accessory glands.
<p>Immunofluorescence panels shown here are the overlay images of accessory glands immunostained with α-Spectrin antibody (marking cell membrane, Green color) and labeled with nuclear stain DAPI (blue color). In EcR control glands, several polygonally shaped binucleate cells (the main cells) and a few large and spherical binucleate cells (the secondary cells, marked with arrow) interspersed between the polygonally shaped cells were observed at 400X (Panel A) and at a higher magnification of 630X (Panel A′). However, in EcR-deficient glands, cell membranes are highly disrupted (Panel B at 400X) and the nuclear distribution is distinctly different from that of control (Panel B′ at a total magnification of 630X). To assess the effect of depletion of EcR or USP, in accessory glands,western blots were probed for the secondary cell markers, Abd-B and ANCE. The glands lacked Abd-B (Panel C,—lane under EcR in Abd-B blot) while EcR control (Panel C, + lane under EcR), USP control (Panel C, + lane under USP) or USP knockdown (Panel C, -lane under USP) had detectable levels of Abd-B. In addition, EcR knockdown glands did not contain ANCE (Panel D, -lane under EcR in ANCE blot) while the same could be detected in controls, suggesting the disruption of secondary cells due to depletion of EcR.</p
Reproductive performance of females mated to EcR or USP knockdown males over period of 10 days.
<p>Panel A represents the overall fecundity (total no. of eggs laid/10 days) of mated females while Panels B and C represent day wise fecundity (no. of egg laid/day from day1-10) of mated females. The overall fecundity of EcR knockdown mates was significantly lower compared to controls (***<i>p</i><0.0001). However, overall fecundity of USP knockdown mates was comparable to their controls. (<i>p</i> = 0.08). Further, EcR knockdown mates laid eggs for 24hr ASM, albeit at significantly fewer numbers (***<i>p</i><0.0001) when compared to control and did not lay eggs from days 2–10. USP knockdown mates did not deviate from controls on day wise egg laying. Interestingly, there were no progeny from the eggs laid by EcR knockdown mates over period of 10 days (overall fertility, ***<i>p<</i>0.0001, Panel D; day-wise fertility, ***<i>p<</i>0.0001 Panel E). USP knockdown or control mates had comparable overall (Panel D; <i>p =</i> 0.3, USP) as well as day wise fertility (Panel F; <i>p =</i> 0.06 lowest) EcR control and knockdown mates differ significantly on total % hatchability (Panel G; EcR, ***<i>p</i><0.0001) as well as % hatchability on day 1 (Panel H; ***<i>p<</i>0.0001). USP control and knockdown mates had significant differences in % hatchability on days 8 and 10 (Panel I; <i>p</i> = 0.01) but that did not have a significant bearing on the overall % hatchability (Panel H; <i>p</i> = 0.3). Values given here are Mean±SEM involving at least 15–30 females depending on the hormone receptor.</p
Western blots showing the levels of EcR and USP in knockdown males compared to control males.
<p>The EcR panel represents EcR levels in accessory glands from EcR control (+ lane, EcR), EcR knockdown (-lane, EcR), USP control (+ lane, USP) and USP knockdown (- lane, USP). Similarly, the USP panel represents the USP levels observed in accessory glands from above groups. Blots probed with α-tubulin antibodies (α-tubulin panels) served as controls for protein loading. Knockdowns were specific to the targeted hormone receptor. Further, the deficiency of EcR did not affect USP levels and vice-versa.</p
Morphology and secondary cells markers of accessory glands in males over-expressing dominant negative EcR isoforms.
<p>The morphology of accessory glands from (A) control or males over expressing (B) EcR-A, (C) EcR-B1, or (D) EcR-B2 was observed under light microscopy. Morphology of accessory glands from males over expressing EcR-B1, or EcR-B2 is comparable to their controls. However, accessory glands from EcR-A appear slightly reduced in comparison to their controls but still not as extremely reduced as those in EcR-miRNA based knockdown males. (B) Western blots of accessory gland protein extracts depicting levels of Abd-B (Abd-B panel), ANCE (ANCE panel) proteins and cleaved Caspase 3 immunoreactivity (cleaved Caspase 3 panel) in males over expressing EcR-A, EcR-B1 and EcR-B2. Blots were probed with β-actin antibodies (β-actin panels) served as controls for protein loading.</p
Analysis of cleaved Caspase 3 immunoreactivity in accessory glands of EcR control and knockdown males.
<p>To examine if loss of EcR leads to induction of apoptosis in accessory glands, tissues were immunostained with antibodies for cleaved Caspase 3, which react with initiator and effector caspases in Drosophila. Control tissues show well organized nuclei (Blue in color; DAPI, Panel A) and lack of detectable cleaved Caspase 3 immunoreactivity (Panel B) the overlay (Panel C) shows only the nuclei, in contrast, glands from EcR knockdown contain distorted as well as disorganized nuclei (DAPI, Panel D) and high levels of cleaved Caspase 3 immunoreactivity (Panel E). The overlay (Panel F) shows distorted nuclei (blue) and cleaved Caspase 3 labeling (red). (G) Females mated to EcR knockdown males over expressing P35 (EcR↓+P35↑) produced progeny at control levels as opposed to sterility in EcR knockdown mates (EcR↓, ***p<0.0001) indicating that over-expression of P35 rescued the fertility of EcR knockdown males. (H) EcR knockdown males overexpressing Diap1 (EcR↓+Diap1↑) were fertile as opposed to sterility in EcR knockdown mates (EcR↓, ***p<0.0001) indicating that overexpression of Diap1 rescued the fertility of EcR knockdown males. (I) Westerns blots of accessory gland proteins to confirm the knockdown status of EcR in males over expressing P35 (EcR↓+P35↑) or Diap1 (EcR↓+ Diap1↑) in EcR knockdown background. Blots were probed with anti-β-actin antibody as control for protein loading.</p