Elsevier

Peptides

Volume 25, Issue 9, September 2004, Pages 1477-1490
Peptides

Review
Functions and analysis of the seminal fluid proteins of male Drosophila melanogaster fruit flies

https://doi.org/10.1016/j.peptides.2003.10.023Get rights and content

Abstract

The study of insect seminal fluid proteins provides a unique window upon adaptive evolution in action. The seminal fluid of Drosophila melanogaster contains over 80 proteins and peptides, which are transferred together with sperm by mating males. The functions of many of these substances are not yet known. However, those that have been characterized have marked effects on the reproductive success of males and females. For example, seminal fluid proteins and peptides can decrease female receptivity, can increase egg production and can increase sperm storage, and are necessary for sperm transfer and success in sperm competition. In this review we focus on the currently known functions of seminal fluid molecules and on new technologies and approaches that are enabling novel questions about their form and function to be addressed. We discuss how techniques for disrupting the production of seminal fluid proteins, such as homologous recombination and RNA interference, along with the use of microarrays and yeast two hybrid systems, should allow us to address ever more sophisticated questions about seminal fluid protein function. These and similar techniques promise to reveal the function of naturally-occurring variants of these proteins and hence the evolutionary significance of genetic variation for them.

Introduction

The study of insect seminal fluid proteins provides a unique window upon adaptive evolution in action. The proteins and peptides transferred together with sperm have sites of action within and outside the female reproductive tract, and they cause a wide variety of responses in females. These responses include increased egg-laying (through increased oogenesis [101], [102] and ovulation [50]) and decreased female receptivity [21], [29], [62]. In addition, seminal fluid proteins are essential for normal sperm storage in females [78], [112], for success in sperm competition [24] and are necessary for the formation of the mating plug (a gelatinous structure which forms in the female reproductive tract during mating [67]). In addition, the seminal fluid of males contains substances with antibacterial activity [66], [89], and enzymes such as protease inhibitors, putative proteases and lipases [107], [116]. In this review, we focus on the currently known functions of the seminal fluid proteins of Drosophila melanogaster. We then address some of the interesting, unanswered questions that are provoked by these functional studies and describe techniques that can be used to investigate them.

It has long been known that mating causes two very striking changes in female D. melanogaster: they become temporarily unreceptive and increase their rate of egg-laying [27], [43], [59]. Potential candidates for inducing these responses were substances in the ejaculate (i.e. sperm or seminal fluid proteins). Bioactive molecules affecting egg-laying and receptivity were localized to the reproductive tracts of D. melanogaster males by examining the behavior and physiology of females transplanted with male reproductive tract accessory glands, or injected with accessory gland extracts [27], [43], [59]. Furthermore, experiments using XO males (produced by sex chromosomal non-disjunction), that transfer seminal fluids but no sperm at mating showed that receptivity could be reduced and egg-laying stimulated by the transfer of seminal fluid proteins alone (e.g. [53], [70]). Although these responses persisted for a shorter period (1–2 days) than those observed following matings to normal males (>5 days), the results nevertheless provided evidence that seminal fluid proteins were involved in modulating post-mating responses in females [53], [69], [70]. The very first seminal fluid protein to be identified was the ‘sex peptide’ or accessory gland protein 70A (Acp70A) [29]. Throughout the 1960s to 1980s the nature of Acp70A was gradually revealed [26], [27], [28], [29], [30], [93], [94], [106]. The Acp70A peptide was identified by injecting HPLC separated fractions of male accessory gland extracts into virgin females, to reveal the fraction that boosted egg-laying and decreased receptivity [29]. Peptide sequencing of this fraction was then employed and the Acp70A gene that encodes the 36 amino acid Acp70A peptide was identified [29]. Since then, the rate at which seminal fluid genes and their functions have been identified has increased dramatically. Rather than focusing on the identification of Acps through functional assays of single ejaculate molecules (e.g. [29]), strategies to simultaneously identify multiple Acps have increasingly been taken. These strategies involve using differential hybridization screens to identify mRNAs expressed specifically in the male accessory glands [36], [74], [91], [100], [117]. Together these screens identified 18 Acp genes. Utilizing the D. melanogaster genome sequence it was subsequently possible, using an expressed sequence tag (EST) screen, to simultaneously identify all remaining Acps, and estimate the total number of Acps to be 83 [107], [116].

The ongoing study of seminal fluid molecules is revealing that they have an unexpected variety of functions. In addition, some of the genes that encode these molecules show evidence of extremely rapid evolutionary change (e.g. [1], [2], [7], [107], [108], [113], [114]). This suggests that seminal fluid molecules may be strong targets for natural or sexual selection. It is an exciting challenge to investigate why, by revealing seminal fluid protein function, by probing the underlying mechanisms involved and by investigating the reasons for functional redundancy between seminal fluid proteins.

Section snippets

Site of synthesis of seminal fluid peptides and proteins in D. melanogaster

The non-sperm part of the male ejaculate in fruit flies mostly comprises molecules synthesized by secretory cells in the paired accessory glands (e.g. [36], [116], [117]), but also substances made in the ejaculatory duct (e.g. [66], [90]) and ejaculatory bulb (e.g. [67]) (Fig. 1a and Table 1). The male accessory glands comprise two types of secretory cells that each express separate sets of genes [8], [35], [73]. Each gland contains ∼1000 main cells, which comprise 96% of the secretory cells

The timing of ejaculate transfer

The duration of mating is typically 15–20 min in D. melanogaster. Seminal fluid molecules from the accessory glands, ejaculatory duct and ejaculatory bulb can be detected in females within the first 6 min of the start of mating (e.g. [9], [65], [73], [87], [117]). A gelatinous mating ‘plug’ is also formed early, within the first 7 min of mating [6], [67]. Sperm are transferred typically in the first 7–10 min, sometime before the midpoint of mating [4], [6], [43], [46], [67] and must therefore

Functions of seminal fluid peptides

Seminal fluid proteins have a number of striking effects on the reproductive success of males and females. Here we review their currently known functions, see Table 2 (and [19], [115], [116]).

Acps with dual functions and functional redundancy between different Acps

An interesting finding emerging from the increasing numbers of functional tests of Acps is that there are cases in which single Acps have more than one quite different function, and others in which different Acps affect the same trait (functional redundancy). Here we briefly discuss the potential reasons for, and significance of, these observations.

Techniques for studying seminal fluid protein function

The number of techniques brought to bear on the study of seminal fluid protein function has increased dramatically in recent years. The usefulness of each of them depends upon the questions being asked, and the pros and cons of each are discussed below.

Future study: the significance of natural variation in seminal fluid protein function

The increasing sophistication of techniques that can be brought to bear on the study of seminal fluid protein function represent an important advance and bring us closer to an important goal: realizing the significance of natural variation in seminal fluid protein function. For example the manipulation of Acp levels by using knock out and knock down stocks is a powerful technique, but a relatively blunt tool with which to study variation in wild-type seminal fluid protein effects. It will be

Acknowledgements

We thank the Leverhulme Trust and the Royal Society for funding, Richard Marguerie for the accessory gland images and two anonymous reviewers for helpful comments.

References (118)

  • A.J. DiBenedetto et al.

    Structure, cell-specific expression, and mating-induced regulation of a Drosophila melanogaster male accessory gland gene

    Dev. Biol.

    (1990)
  • A.J. DiBenedetto et al.

    Sequences expressed sex-specifically in Drosophila melanogaster adults

    Dev. Biol.

    (1987)
  • Y.L. Fan et al.

    Common functional elements of Drosophila melanogaster seminal peptides involved in reproduction of Drosophila melanogaster and Helicoverpa armigera females

    Insect Biochem. Mol. Biol.

    (2000)
  • G.L. Fowler

    Some aspects of the reproductive biology of Drosophila: sperm transfer, sperm storage and sperm utilization

    Adv. Genet.

    (1973)
  • Z. Gao et al.

    Species diversity in the structure of zonadhesin, a sperm-specific membrane protein containing multiple cell adhesion molecule-like domains

    J. Biol. Chem.

    (1998)
  • D.G. Gilbert

    Ejaculate esterase and initial sperm use by female Drosophila melanogaster

    J. Insect Physiol.

    (1981)
  • M.H. Gromko et al.

    Sperm transfer and use in the multiple mating system in Drosophila

  • Y. Heifetz et al.

    The Drosophila seminal fluid protein Acp26Aa stimulates release of oocytes by the ovary

    Curr. Biol.

    (2000)
  • J.R. Kennerdell et al.

    Use of dsRNA mediated genetic interference to demonstrate that frizzled and frizzled act in the wingless pathway

    Cell

    (1998)
  • O. Lung et al.

    Drosophila seminal fluid proteins enter the circulatory system of the mated female fly by crossing the posterior vaginal wall

    Insect Biochem. Mol. Biol.

    (1999)
  • O. Lung et al.

    Drosophila males transfer antibacterial proteins from their accessory gland and ejaculatory duct to their mates

    J. Insect Physiol.

    (2001)
  • O. Lung et al.

    Identification and characterisation of the major Drosophila melanogaster mating plug protein

    Insect Biochem. Mol. Biol.

    (2001)
  • A. Manning

    The control of sexual receptivity in female Drosophila

    Anim. Behav.

    (1967)
  • D.B. Meikle et al.

    Localization and longevity of seminal-fluid esterase-6 in mated female Drosophila melanogaster

    J. Insect Physiol.

    (1990)
  • S.A. Monsma et al.

    Synthesis of two male accessory proteins and their fate after transfer to the female during mating

    Dev. Biol.

    (1990)
  • M. Park et al.

    Male and female cooperate in the prohormone-like processing of a Drosophila melanogaster seminal fluid protein

    Dev. Biol.

    (1995)
  • R.C. Richmond et al.

    Esterase-6 of Drosophila melanogaster; kinetics of transfer to females, decay in females and male recovery

    J. Insect Physiol.

    (1981)
  • T. Schmidt et al.

    The Drosophila melanogaster sex-peptide a molecular analysis of structure–function relationships

    J. Insect Physiol.

    (1993)
  • T. Schmidt et al.

    Protein metabolism of Drosophila male accessory glands. III. Stimulation of protein synthesis following copulation

    Insect Biochem.

    (1985)
  • D. Scott et al.

    The basis for control of post-mating sexual attractiveness by Drosophila melanogaster females

    Anim. Behav.

    (1990)
  • D. Scott et al.

    Sperm loss by remating Drosophila melanogaster females

    J. Insect Physiol.

    (1990)
  • K. Sheehan et al.

    Studies of esterase in Drosophila melanogaster. III. The developmental pattern and tissue distribution

    Insect Biochem.

    (1979)
  • E. Simmerl et al.

    Structure and regulation of a gene-cluster for male accessory-gland transcripts in Drosophila melanogaster

    Insect Biochem. Mol. Biol.

    (1995)
  • M. Aguadé

    Positive selection drives the evolution of the Acp29AB accessory gland protein in Drosophila

    Genetics

    (1999)
  • M. Aguadé et al.

    Polymorphism and divergence in the Mst26A male accessory gland gene region in Drosophila

    Genetics

    (1992)
  • T. Aigaki et al.

    Ectopic expression of sex peptide alters reproductive behaviour of female D. melanogaster

    Neuron

    (1991)
  • A. Bairati

    Filamentous structures in spermatic fluid of Drosophila melanogaster Meig

    J. Microsc. Paris

    (1966)
  • A. Bairati

    Structure and ultrastructure of male reproductive system in Drosophila melanogaster Meig 2. The genital duct and accessory glands

    Monitore Zool. Ital.

    (1968)
  • A. Bairati et al.

    Occurrence of a compact plug in the genital tract of Drosophila females after mating

    Dros. Inf. Serv.

    (1970)
  • D.J. Begun et al.

    Molecular population genetics of male accessory gland proteins in Drosophila

    Genetics

    (2000)
  • E.T. Bieschke et al.

    Doxycycline-induced transgene expression during Drosophila development and aging

    Mol. Gen. Genet.

    (1998)
  • M.C. Bloch Qazi et al.

    An early role for the Drosophila melanogaster male seminal fluid protein Acp36DE in female sperm storage

    J. Exp. Biol.

    (2003)
  • A.H. Brand et al.

    Targeted gene-expression as a means of altering cell fates and generating dominant phenotypes

    Development

    (1993)
  • G. Brieger et al.

    Drosophila melanogaster. Identity of male lipid in reproductive system

    Science

    (1970)
  • Büsser S. Immunolocalisation of SP on sperm heads of Drosophila melanogaster. Diplomarbeit, University of Zurich;...
  • F.M. Butterworth

    Lipids of Drosophila: a newly detected lipid in the male

    Science

    (1969)
  • D.R. Cavener

    Coevolution of the glucose-dehydrogenase gene and the ejaculatory duct in the genus Drosophila

    Mol. Biol. Evol.

    (1985)
  • D.R. Cavener et al.

    Biphasic expression and function of glucose dehydrogenase in Drosophila melanogaster

    Proc. Natl. Acad. Sci. USA

    (1983)
  • T. Chapman

    Seminal fluid-mediated fitness traits in Drosophila

    Heredity

    (2001)
  • T. Chapman et al.

    The sex peptide of Drosophila melanogaster: female post-mating responses analysed by using RNA interference

    Proc. Natl. Acad. Sci. USA

    (2003)
  • Cited by (205)

    View all citing articles on Scopus
    View full text