The Developments Between Gametogenesis and Fertilization: Ovulation and Female Sperm Storage in Drosophila Melanogaster Margaret C

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Developmental Biology 256 (2003) 195Ð211 www.elsevier.com/locate/ydbio Review

The developments between gametogenesis and fertilization: ovulation and female sperm storage in Drosophila melanogaster Margaret C. Bloch Qazi,a Yael Heifetz,a,b and Mariana F. Wolfnera,* a Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA b Department of Entomology, The Hebrew University of Jerusalem, Rehovot, 76100, Israel Received for publication 5 September 2002, revised 11 December 2002, accepted 11 December 2002

Abstract In animals with internal fertilization, ovulation and female sperm storage are essential steps in reproduction. While these events are often required for successful fertilization, they remain poorly understood at the developmental and molecular levels in many species. Ovulation involves the regulated release of oocytes from the ovary. Female sperm storage consists of the movement of sperm into, maintenance within, and release from speciﬁc regions of the female reproductive tract. Both ovulation and sperm storage elicit important changes in gametes: in oocytes, ovulation can trigger changes in the egg envelopes and the resumption of meiosis; for sperm, storage is a step in their transition from being “movers” to “fertilizers.” Ovulation and sperm storage both consist of timed and directed cell movements within a morphologically and chemically complex environment (the female reproductive tract), culminating with gamete fusion. We review the processes of ovulation and sperm storage for Drosophila melanogaster, whose requirements for gamete maturation and sperm storage as well as powerful molecular genetics make it an excellent model organism for study of these processes. Within the female D. melanogaster, both processes are triggered by male factors during and after mating, including sperm and seminal ﬂuid proteins. Therefore, an interplay of male and female factors coordinates the gametes for fertilization. © 2003 Elsevier Science (USA). All rights reserved.

Keywords: Oocyte; Egg; Sperm; Activation; Ovary; Sperm storage; Mating; Seminal ﬂuid proteins; Accessory gland protein; Cell migration

Between the motion 2002; Primakoff and Myles, 2002; Spradling, 1993). Yet, And the act . . . between these important processes, crucial steps that pre- Between the conception pare gametes and coordinate their efﬁcient union are less And the creation . . . well understood. These steps include ovulation and female Between the potency sperm storage. Ovulation occurs when an oocyte is released And the existence . . . from the ovary and causes the transition from an oocyte into Falls the Shadow. an egg (ovum). Ovulation makes an oocyte available for ‘The Hollow Men’, 1925: T.S. Eliot fertilization, regulates the rate of oocyte release, and initiates changes in the oocyte that prepare it for fertilization (discussed below and listed in Table 1). Female sperm Introduction storage is the retention of sperm within specialized regions Developmental biology has illuminated steps in sexual of the female reproductive tract. Female sperm storage pro- reproduction, the creation of a new individual from cells longs sperm availability when sperm transfer is not imme- from two existing individuals. In particular, mechanisms of diately followed by fertilization and coordinates sperm re- gametogenesis and fertilization are understood in much de- lease with the availability of a recently ovulated egg. Sperm tail (reviewed in Fuller, 1993; Hardy, 2002; Matzuk et al., storage also has important consequences for female fecundity and fertility that are discussed below (and listed in Table 2). * Corresponding author. Fax: ϩ1-607-255-6249. Of the established model organisms, Drosophila mela- E-mail address: [email protected] (M.F. Wolfner). nogaster provides a particularly tractable system to study

Table 1 rec, 1950) that is intensively innervated (Y.H. and M.F.W., Physiological consequences of ovulation in D. melanogaster unpublished observations). The uterus is a heavily muscu- Consequences of ovulation in D. melanogaster larized and innervated structure that receives sperm during mating and also holds the egg in position for fertilization. At Activates eggs 2 egg permeability the anterior end of the uterus are the three sperm storage 1 vitelline membrane protein cross linking organs: a single seminal receptacle and the paired sper- resumption of meiosis mathecae, as well as the paired spermathecal glands (also initiation of translation called parovaria or female accessory glands) (Fig. 1). At its 1 rate of oogenesis posterior end, the uterus narrows forming the vagina that Eliminates old eggs exits the female reproductive tract. The distal end of the vagina, called the gonopore, serves for the discharge of eggs (Demerec, 1950; Hoffman, 1995). both ovulation and sperm storage. Like many other animals, female D. melanogaster generate oocytes but store them, arrested and unactivated, in the ovary. Drosophila oocytes Ovulation arrest in metaphase of meiosis I, with egg coverings (a vitelline envelope and chorion) that are more permeable Importance of ovulation than those found on a fertilized egg. Ovulation releases those mature eggs from the ovary and, as described below, Ovulation triggers activation of oocytes triggers the process known as activation. Also as in other In many organisms, interaction between the gametes animals, D. melanogaster males transfer many more sperm triggers a series of cellular responses in the egg (“egg ϳ ( 4000) to the female than are stored and used. While a activation”) required to initiate embryonic development. D. Drosophila sperm’s morphology is similar to that of other melanogaster eggs also are triggered to initiate a suite of animals, fly sperm are unusually long: about the length of cellular responses analogous to those of activation in other the flyinD. melanogaster, and much longer in species in organisms (relief of arrest in meiosis, ultimately leading to the obscura and bifurca species groups (reviewed in Fuller, completion of meiosis, changes in egg coverings, and ini- 1993; Joly et al., 1995; Pitnick and Markow, 1994; Pitnick tiation of translation; Table 1) in a process that has therefore et al., 1995a,b). Rapidly moving sperm into storage, main- also been called activation. This semantic convergence taining their viability within storage, and then coordinating masks a distinct mechanistic difference in the trigger for the release of sperm from storage with the release of oocytes activation in Drosophila and at least one other insect rela- from the ovary for successful fertilization involve interac- tive to that in echinoderms, nematodes, and vertebrates: egg tions between male cells and molecules with female tissues. activation in D. melanogaster and other insects initiates These interactions are probably regulated by both male and independent of sperm entry (Doane, 1960; Dubnau and female-derived factors (described below). The genetic tools Struhl, 1996; Heifetz et al., 2001b; Macdonald and Struhl, available for use with D. melanogaster provide powerful 1986; Mahowald et al., 1983; Page and Orr-Weaver, 1997; ways to identify and study the role of male and female Went and Krause, 1974), perhaps not surprising in an Order contributions to ovulation and sperm storage. that includes species in which haploid males can develop from unfertilized eggs. In D. melanogaster, changes in the egg envelope’s permeability, one feature of activation, ini- The D. melanogaster female reproductive tract tiate during ovulation, even while most of the egg is still within the ovary (Heifetz et al., 2001b). The egg’s coverings Mating initiates a series of events within the female become progressively more impermeable to small mole- reproductive tract, including ovulation and sperm storage. cules as the egg proceeds down the oviducts; the process is Ovulation and sperm storage occur in different regions of complete by the time the egg arrives at the uterus. Cross- the female reproductive tract, but are coordinated for a linking of vitelline membrane protein sV23 also increases connected fate: the fertilization of an egg. At the anterior of the female D. melanogaster reproductive tract (Fig. 1) are two ovaries, each composed of 10Ð20 ovarioles. The ova- Table 2 rioles are held together by a peritoneal sheath, containing a Behavioral, physiological, and fitness consequences of female sperm storage in D. melanogaster network of fine, branching muscle fibers. The base (proximal end) of each ovariole forms a small duct or pedicel. The Consequences of female sperm storage in D. melanogaster pedicels of all the ovarioles in an ovary unite to form a 1 fecundity calyx; each calyx opens into a lateral oviduct. The lateral 1 fertility oviducts fuse into a common oviduct that, more posteriorly, provides opportunities for sperm competition and sperm preference 2 enlarges to form the uterus. The wall of the oviducts con- tendency to remate 2 costs associated with repeated matings sists of epithelium surrounded by circular muscles (Deme- M.C. Bloch Qazi et al. / Developmental Biology 256 (2003) 195–211 197

Fig. 1. Cartoon of the anatomy of the female D. melanogaster reproductive tract shown from dorsal and lateral perspectives. Within the female, the ovaries are located at the anterior end of the female’s abdomen and contain egg chambers with oocytes at different stages of development. For clarity, the drawings omit clear definition of individual ovarioles, which separate oocytes of the same stages from each other. Drawings by Jacqualine Grant. progressively as the egg moves through the oviducts and the oocytes/day; King, 1970; we follow the standard convention uterus. Ovulation also triggers meiosis to resume before the of calling these “mature” oocytes, though recently Hatsumi egg reaches the uterus (Heifetz et al., 2001b). Thus, ovula- et al. (2001) reported what appears to be a subsequent stage, tion causes changes in the egg that prepare it for subsequent also called “mature oocyte”). Several control points and embryogenesis, should fertilization occur in the uterus, in- feedback mechanisms regulate the production of mature cluding coordination between cell cycle status of the egg oocytes (Cummings and King, 1969; Drummond-Barbosa and the sperm nuclei. The third feature of activation, re- and Spradling, 2001; King et al., 1956; Margaritis, 1985; sumption of translation, also initiates without fertilization in Soller et al., 1997, 1999). If mature oocytes are not ovu- D. melanogaster, though it is not yet known if it too is lated, each ovariole accumulates two or three late-stage triggered by ovulation (Dubnau and Struhl, 1996; Mac- oocytes. This blocks the maturation of additional oocytes donald and Struhl, 1986). (King and Sang, 1959). As described in more detail under “triggers” below, mating, and specifically sperm and some Ovulation allows oogenesis to be stimulated after mating male accessory gland proteins [e.g., Acp26Aa (ovulin), see D. melanogaster females produce up to two final-stage below and Table 3], induces ovulation of mature oocytes (stage 14) oocytes in each ovariole daily (as many as 80 (Heifetz et al., 2000, 2001b) that, in turn, contribute to

Table 3 Drosophila male-derived polypeptides whose functions are known or hypothesized to affect ovulation and sperm storage

Seminal protein Origin in male Function in female Protein class Reference Acp26Aa (ovulin) AG Stimulates ovulation Peptide hormone precursora Heifetz et al., 2000 Acp70A (sex peptide) AG Stimulates oogenic progression Peptide Chen et al., 1988 Increases number of uterine eggs Dup99B ED Stimulates egg production Peptide Saudan et al., 2002 ED-OSS ED Stimulates ovulation in D. biarmipes Peptide Imamura et al., 1998 Acp36DE AG Increases female sperm storage Glycoprotein Neubaum and Wolfner, 1999a Esterase-6 EB &ED Decreases sperm retention in storage Esterase Gilbert, 1981 Stimulates ovulation rate Acp62F AG Hypothesized: protects sperm within storage Trypsin inhibitorb Lung et al., 2002 PEB-me EB Hypothesized: facilitate sperm storage Structural proteinc Ludwig et al., 1991; Lung and Wolfner, 2001

Note. Origin in male: AG, accessory gland; ED, ejaculatory duct; and EB, ejaculatory bulb. Protein class: a , has sequence similarity with Aplysia egg-laying hormone; b , has sequence similarity with mollusk byssal thread protein and spider silk; and c , has sequence similarity with Ascaris trypsin inhibitor. Functions are in D. melanogaster unless otherwise indicated. 198 M.C. Bloch Qazi et al. / Developmental Biology 256 (2003) 195–211 stimulating oogenic progression via a feedback mechanism. Thus, ovulating females produce high numbers of mature oocytes and deposit high numbers of fertilized eggs. The feedback mechanism by which seminal ﬂuid, sperm, and the act of mating itself increase oogenic progression rate is not known. However, it would be interesting to reveal how oocyte production rate is regulated by ovulation rate, thereby allowing Drosophila females to achieve “high qual- ity oocytes” required for successful fertilization. Studies of such a mechanism could shed light on processes/genes essential for successful fertilization.

Ovulation permits egg/sperm coordination that results in optimal hatchability As noted above, D. melanogaster virgin females retain mature oocytes in their ovaries. Ovulation initiates within Fig. 2. A model illustrating the fate of gametes in female’s reproductive 1.5 h after mating (Heifetz et al., 2000) during a time when tract (solid line) and possible interactions (dashed line) that might allow sperm are still being stored (see below). Thus, the initial coordination leading to efficient fertilization. See text for discussion. Al- eggs ovulated are released potentially prior to full comple- though we indicate that sperm storage may affect ovulation, this is as yet tion of sperm storage. The number of progeny per number an assumption based on the findings that sperm transfer has been shown to of eggs laid (hatchability) immediately after matings be- affect ovulation (Heifetz et al., 2001a) and sperm depletion affects the number of eggs laid (Kaufman and Demerec, 1942; Neubaum and Wolfner, tween wild-type males and females is lower than at later 1999a). time points (Chapman et al., 2001). This lower hatchability appears to reflect a lower efficiency of fertilization of the first eggs released. By visualizing the fertilizing sperm tail sequential and thus somewhat coupled. Therefore, assays of within wild-type eggs, it was found that the first eggs laid oocyte/egg progression sometimes measure several of these had a lower fertilization efficiency (ratio of unfertilized/ steps at once. fertilized eggs ϭ 1.5 at 3Ð4 h post-mating) than those laid Ovulation itself results from a series of processes occur- subsequently (0.6 at 48Ð49 h; Chapman et al., 2001). We ring both sequentially and simultaneously within several suggest two possible explanations for the low hatchability; ovarian microenvironments. The oocyte is released from its regulated ovulation plays a critical role in each of these. follicle, squeezed out of the ovary, and pushed through a First, ovulation allows coordination of oocyte release with lateral oviduct into the common oviduct, coming to rest in the rate of sperm release from storage. The first eggs ovu- the uterus. Within each D. melanogaster ovariole, oocytes lated may reach the fertilization site before sperm are pre- develop in a sequential fashion. Each ovariole contains a pared and in position to be released from storage. Thus, series of four to six egg chambers at stages in developmen- lower numbers of those eggs would be fertilized, leading to tal progression (stages 1Ð14), with oogonia at the apex of lower hatchability. Second, since unmated females can ac- the ovariole and the most mature oocytes (stage 14) at the cumulate eggs for several days, it is possible that the older base of the ovariole. In each egg chamber, the oocyte, eggs are “stale” and cannot be fertilized. By ovulating those connected to its sister cells (the nurse cells), is surrounded eggs quickly, even before sperm are fully stored, the female by a monolayer of somatic follicle cells. Follicle cells syn- clears out any stale eggs without wasting sperm (Chapman thesize some of the yolk protein that will be deposited into et al., 2001). Understanding the mechanism that coordinates the oocyte, as well as the proteins of the vitelline envelope oocyte release from the ovary and sperm release from the and chorion that will cover and protect the oocytes. Spe- sperm storage organs will provide insights into the first steps of cialized follicle cells at the anterior end of the egg chamber sperm/egg interactions that lead to successful fertilization. synthesize the micropyle, the site on the egg through which the sperm will enter (Buning, 1994; King, 1970; reviewed in What is ovulation and where does it occur? Spradling, 1993). When the adult female fly ecloses, the base of the ovariole is still plugged with cells that grew out Ovulation is an important step in the egg-production of the pedicel (King, 1970); this plug must be breeched to process. In insects, production and laying of eggs requires allow ovulation. five steps within the female reproductive tract (Fig. 2): The mechanism by which oocytes are released from the oogenesis (the generation of oocytes within the ovary), Drosophila ovary is not known, but results in other insect ovulation (release of an oocyte from the ovary into the systems suggest possible mechanisms. Insect ovulation in- oviducts), movement of the egg down the oviducts, fertili- volves two distinct steps: the opening of the oocyte follicle zation of the egg when it has come to rest in the uterus, and and of the intermost cells in the pedicel region, which deposition of eggs onto the substratum. These steps are releases the oocyte from the ovariole, and contraction of M.C. Bloch Qazi et al. / Developmental Biology 256 (2003) 195–211 199 ovarioles, pedicels, and oviduct musculature, which moves needed to distinguish this from the larger egg laying pro- the oocyte into the lateral oviducts (Buning, 1994; Fox and cess. By measuring the progression of egg movement Brust, 1996; Hoc, 1996; Hoffmann, 1995; Mesnier-Sabin, through the female reproductive tract, Heifetz et al. (2000) 1981; reviewed in Raabe, 1986). These contractions do not showed that, by 90 min after mating, 90% of the females appear to involve sphincter(s), which are absent between the had at least one egg in their reproductive tract, and most of pedicels and the lateral oviducts in D. melanogaster (Y.H., those were in the lateral oviduct. Ovulation rate increased unpublished observations). Ultrastructural studies of ovula- with time after mating, such that by 6 h after mating, 30% tion in the large aquatic beetle Dysticus marginalis (Co- of females had more than one egg in their reproductive tract. leoptera) show that the appearance of the first mature oocyte Seminal fluid components, Acps (accessory gland proteins), at the pedicel region triggers autolysis of the pedicel’s and sperm that are transferred to the female during mating innermost cells, opening the plug and releasing the mature play an important role in increasing female ovulation rate. oocyte from the ovariole (Buning, 1994). In addition, there are cells in the pedicel region that produce vacuoles whose Acps contents might help the mature oocyte to glide into the One seminal fluid protein, the prohormone-like Acp26Aa oviduct (Buning, 1994). In the mosquito Culiseta inornata (also called “ovulin”), showing sequence similarity to the (Diptera), histological sections of whole ovaries also show Aplysia egg laying hormone, is essential for stimulating that the pedicel is destroyed during ovulation (Fox and ovulation (Table 3). The ovulation rate of females mated to Brust, 1996). Thus, though little is known about the process males that lack Acp26Aa was 44% lower than the ovulation of ovulation in D. melanogaster, it is likely that ovulation rate of mates of wild-type males (Heifetz et al., 2000; occurs, as in other insects, by cell degeneration in the Herndon and Wolfner, 1995). Acp26Aa’s effects are evi- pedicel region and massive muscle contractions at the base dent between 1.5 and 6 h post-mating, and the protein is of the ovary and oviducts. These move the oocyte from the detectable in mated females for only a few hours post- ovariole into and down the oviducts, where it eventually mating (Monsma et al., 1990). Since Acp26Aa stimulates becomes lodged in the uterus. The posterior end of the ovulation shortly after mating, it is suggested to act in oocyte leaves the ovary first. Thus, when the egg comes to “clearing” the mature oocytes from the ovary, allowing for rest in the uterus, its anterior end is “up” with the egg’s coordination of fresh oocyte release with sperm release and micropyle adjacent to the opening of the sperm storage for increased oogenic progression rate (Chapman et al., organs, allowing for efficient fertilization. 2001; Heifetz et al., 2000; reviewed in Wolfner, 2002). It is not yet known how Acp26Aa mediates oocyte release. When does ovulation begin? Some Acp26Aa localizes at the base of the ovary (Heifetz et al., 2000), where it is processed into bioactive peptides In some insects, ovulation initiates only after mating (Park and Wolfner, 1995; Y.H. and L.N., unpublished ob- (Grillot and Raabe, 1989; Mesnier-Sabin, 1972, 1981; servation). Some Acp26Aa enters the circulatory system. Thomas, 1979; reviewed in Raabe, 1986). In D. melano- Thus, it is possible that Acp26Aa acts locally within the gaster, however, ovulation occurs at a low level in adult reproductive tract and/or via the neuroendocrine system to females even without mating. Although D. melanogaster mediate contractile activity of the ovary and the oviduct mature virgin females spontaneously ovulate at a very low musculature. rate (ϳ1 egg/day), mating dramatically increases female Another Acp, the “sex peptide” Acp70A, also causes an ovulation rate (Heifetz et. al., 2000; reviewed in Ashburner, increase in the number of eggs laid and, in one assay, the 1989). This effect of mating is rapid; an increase in ovula- number of eggs in the uterus of dissected females (Aigaki et tion is evident by 90 min post-mating (Heifetz et al., 2000). al., 1991; Chen et al., 1988; Table 3). Since this Acp is Some insects, such as walking sticks, ovulate one oocyte at known to increase oogenic progression (Soller et al., 1997, a time and their ovarioles function alternately or in sequence 1999), it is presently unknown whether the increased num- (Clitumnus extradentatus, Mesnier-Sabin, 1981; Carausius ber of eggs seen in the uterus following Acp70A induction morosus, Thomas, 1979), although in other insects, such as is simply a secondary indirect consequence of the increased Orthoptera, all ovarioles ovulate simultaneously. It is not production of oocytes, or is due to a separate direct effect on known which mechanism operates in D. melanogaster. ovulation.

What triggers ovulation? Male ejaculatory duct secretions D. melanogaster males produce a peptide related to Mating Acp70A in their ejaculatory ducts (Fan et al., 2000; Saudan In Drosophila females, mating stimulates an increase in et al., 2002). Injection of this peptide, Dup99B, into un- ovulation rate (D. melanogaster, Chen et al., 1988; Heifetz mated females can stimulate egg production. As with et al., 2000; D. biarmipes, Imamura et al., 1998; D. suzukii, Acp70A, it is not presently known how Dup99B stimulates Ohashi et al., 1991). Since ovulation is one step in the egg production, and therefore whether its effect on ovula- multistep process of egg laying (see Fig. 2), an assay was tion is direct or indirect. 200 M.C. Bloch Qazi et al. / Developmental Biology 256 (2003) 195–211

In D. biarmipes, 90% of dissected virgin females in- likely candidates. In locusts (Locusta migratoria), for ex- jected with ejaculatory duct extracts had an egg in their ample, glutamate, octopamine, proctolin, and SchistoFLRF- uterus compared with only ϳ12% of the saline-injected amide have been shown to mediate oviduct muscle contrac- control females. The active component of the ejaculatory tion, aiding the movement of eggs (reviewed in Lange and duct extract appears to be a 32-amino-acid peptide, called Nykamp, 1996). In other insects, such as the bed bug Rhod- Ejaculatory Duct Ovulation Stimulating Substance (ED-OSS; nius prolixus, the neurosecretory peptide myotropin medi- Imamura et al., 1998; Table 3). Accessory glands of D. ates the ovarian contractile activity that releases mature biarmipes also produce an orthologue of Acp70A, and ex- oocytes. Myotropin is released from the corpus cardiacum tracts of this tissue also stimulate egg-laying (Imamura et in response to ecdysteroid levels in the female hemolymph al., 1998). As with Acp70A and Dup99B in D. melano- (Kriger and Davey, 1984; Ruegg et al., 1981). gaster, we do not presently know whether ED-OSS regulates ovulation directly. Environmental and physiological cues In D. melanogaster, ovarian function and ovarian envi- Sperm ronment affect egg chamber development and therefore In the absence of sperm transfer, oocyte development ovulation rate. Female age, available nutritional resources (from rapid yolk accumulation to vitelline membrane devel- (e.g., yeast), and other environmental factors, such as cir- opment and chorion deposition) and egg laying rates are cadian cues and humidity, affect egg production (Drum- lower than wild-type levels (50 and 33Ð70% lower, respec- mond-Barbosa and Spradling 2001; King 1970; Lints and tively; Heifetz et al., 2001a; Xue and Noll, 2000). Females Lints, 1969; Spradling, 1993; Y.H., unpublished observa- that receive no sperm also begin to deposit eggs later than tions). For example, nutritional supplementation with yeast females mated to wild-type males (6 h vs 3 h; Heifetz et al., causes increased germ cell proliferation and decreased cell 2001a). Moreover, even when sperm are transferred, if they death of both early-stage and vitellogenic-stage oocytes. are not stored, egg laying is low (Ͻ50% wild-type levels; This results in increased egg production and egg deposition Neubaum and Wolfner, 1999a). Finally, when a female uses rates thereby (directly or indirectly) increasing ovulation up all sperm from her mating, her egg laying rate decreases rates (Drummond-Barbosa and Spradling, 2001). Although to virgin levels (Kaufman and Demerec, 1942; Neubaum the mechanism of environmental interaction with ovulation and Wolfner, 1999a). These results indicate that the transfer physiology is still to be determined in D. melanogaster, and storage of sperm is essential to elevate oogenesis and again clues are available from other insects. For example, in egg deposition rates and may also affect ovulation. the beetle Xyleborus ferrugineus, ecdysteroid titers are sig- The presence of sperm in the reproductive tract may niﬁcantly higher in young fertile adult females than in aging trigger changes in female fecundity by causing the release females, suggesting that fewer eggs are produced, and hence of female-derived activating substances or by releasing ovulated in older females, due to declining ecdysteroid male-derived substances that have adhered to the sperm levels (Hagedorn, 1983; Norris et al., 1986; Ruegg et al., (Kubli, 1992). Alternatively, the presence of many sperm 1981). could stimulate the female central nervous system via In D. melanogaster, several control points and feedback stretch receptors in the uterus or sperm storage organs (as mechanisms regulate the production of mature oocytes, reported in Lepidoptera; Obara et al., 1975). which affects female ovulation status. One such feedback prevents oocytes from entering the oviduct before complet- Neuroendocrine and endocrine signals ing oogenesis (reviewed in Spradling, 1993). Thus, a female Contraction of insect oviducts is under neural control will ovulate only if she has mature oocytes in her ovaries. (Demerec, 1950; Kiss et al., 1984; Lange, 1987; Lange et Another control point in egg maturation is regulated by al., 1986). The D. melanogaster female reproductive tract is Acp70A. Acp70A stimulates oocytes to progress to later innervated by branches of the abdominal nerve center vitellogenic stages and thus overrides a control point in egg

(AbTNv) (Demerec, 1950). Thus, it seems likely that neu- maturation between mid/late vitellogenic stages (stages rotransmitters and/or neurohormones will regulate the con- 9/10; Soller et al., 1997, 1999). Thus, Acp70A can stimulate tractile activity of the Drosophila ovary and oviduct mus- oogenic progression rate, allowing the female to ovulate at culature to cause the release of mature oocytes. Since the a high rate. Drosophila female reproductive tract’s epithelium has secretory characteristics, it is also likely that neurotransmitters or neurohormones released at the base of the ovary could Female sperm storage affect the secretory activity of the epithelium, and thereby trigger the disintegration of the pedicel plug to release Importance of sperm storage mature oocytes. Although the identity of neurotransmitters, neuromodu- Storing sperm increases fecundity and fertility lators, or neurohormones that could mediate ovulation in Sperm storage allows sperm from a given mating to be Drosophila is unknown, findings in other insects point to used long after the male and female have separated (ϳ2 M.C. Bloch Qazi et al. / Developmental Biology 256 (2003) 195–211 201 weeks; Kaufman and Demerec, 1942; Lefevre and Jonsson, Gromko and Pyle, 1978; Kalb et al., 1993; Manning, 1962, 1962; Neubaum and Wolfner, 1999a; Table 2), thus increas- 1967; Neubaum and Wolfner, 1999a; Xue and Noll, 2000). ing female fertility. Of the ϳ4000 sperm transferred to a D. The sperm effect on female receptivity may begin as early melanogaster female during mating between wild-type flies, as 6Ð8 h postmating; it is clearly operating by 10Ð18 h after ϳ80% are expelled from the uterus when the first egg is laid mating (Scott, 1986). Females that store few sperm are (Fowler, 1973; Gilbert, 1981), and subsequent fertilizations receptive to remating sooner than those storing larger num- rely on the 700Ð1000 sperm stored in the female. Females ber of sperm (Neubaum and Wolfner, 1999a; Xue and Noll, that receive normal quantities of sperm, but store few of 2000). The return of female receptivity correlates both with them, produce few progeny (ϳ10% wild-type levels; the decline in numbers of stored sperm as those sperm are Neubaum and Wolfner, 1999a). Compared with mammals released and used for fertilization and, in the laboratory, and birds, whose efficiency of sperm use can be as low as with longer periods of maleÐfemale pair confinement, con- 0.01% (reviewed in Neubaum and Wolfner, 1999b), D. tinued availability of food, and population density (Gromko melanogaster use sperm quite efficiently: of the 700Ð1000 et al., 1984b; Harshman et al., 1988; Manning, 1962, 1967; sperm stored, ϳ400 are used to fertilize eggs (Gilbert, 1981; Neubaum and Wolfner, 1999a; Newport and Gromko, 1984; Gilchrist and Partridge, 1995; Kaufman and Demerec, 1942; reviewed in Singh et al., 2002). Lefevre and Jonsson, 1962; Neubaum and Wolfner, 1999a; Potential mechanisms for the sperm effect include: pres- Pitnick, 1991; Tram and Wolfner, 1999). sure from within the sperm storage organs exerted by sperm, Sperm storage potentially allows coordination of ovula- movements of sperm within the storage organs, and/or dis- tion rate with sperm release from storage (Chapman et al., tension of the uterine walls by sperm and semen. In the 2001). This prevents gamete wastage, which would occur white cabbage butterfly Pieris rapae, receipt of sperm and when gametes are released at different times and unable to seminal fluid products results in changes in female recep- unite successfully. The coordination of sperm release and tivity to mating. Distension of a female P. rapae uterus with ovulation could also decrease the incidence of polyspermy saline causes a decrease in receptivity similar to that after that also wastes eggs and sperm because it results in non- mating (Obara et al., 1975). A similar mechanism in the D. viable fertilizations. Consistent with this idea, in pigs, sur- melanogaster sperm storage organs could explain the D. gically bypassing sperm storage caused elevated numbers of melanogaster sperm effect. Since the sperm effect is estab- sperm in the female ampulla which correlated with an in- lished several hours after sperm storage is complete (10Ð18 creased frequency of polyspermy (Hunter and Le«glise, h vs 1.5 h, respectively), the effect of sperm on female 1971; reviewed in Suarez, 2002). The controlled release of behaviors is proposed to involve an intermediary acting on sperm from storage in D. melanogaster may be one reason the female central nervous system (CNS) (Scott, 1987). This why polyspermy is rare in this organism (Յ1%; Callaini and intermediary could be a molecule(s) secreted from the fe- Riparbelli, 1996; Karr, 1991; Snook and Markow, 2002). male’s cells. For example, in the redbanded leafroller moth, Sperm storage allows females to retain sperm for more Argyrotaenia velutinana, mating depresses female phero- than one male within her reproductive tract. This provides mone titer via a CNS-mediated release of the peptide PBAN “fertility insurance,” should any of the males be infertile or (pheromone biosynthesis activating neuropeptide). When genetically incompatible with the female (Zeh and Zeh, the female CNS is disrupted by cutting the ventral nerve 1997). Storing sperm from multiple males has the potential cord, PBAN remains within female tissues, and is not re- additional advantage for females of generating progeny with leased into the hemolymph, where it normally acts (Jurenka a broader range of different genotypes. When their habitat is et al., 1993). Alternatively, the intermediary could be a spatially variable or changes over time, genetic diversity molecule from the male’s seminal fluid. Although most among offspring might be particularly advantageous for seminal proteins are not detectable in the female more than survival of at least some of the female’s progeny (Zeh et al., a few hours after mating, others may be stabilized by asso- 1997). Finally, storing sperm from more than one male ciating with sperm, allowing them to act within the female generates an opportunity for sperm competition and female long after mating has ended (Kubli, 1992, 1996). sperm preference (described later). Storing sperm avoids costs associated with multiple Storing sperm decreases the female’s tendency to re-mate matings After mating, D. melanogaster females become unrecep- In several organisms, matings are costly to females (Fair- tive to courting males for several days (mating effect; bairn, 1993; Fowler and Partridge, 1989; Hammer, 1941; Gromko et al., 1984b; Manning, 1962, 1967; Table 2). Male Hsin and Kenyon, 1999; Stone, 1995; Van Voorhies, 1992; seminal products such as Acp70A trigger the female’s im- Table 2). In D. melanogaster, this cost arises from a com- mediate reluctance to remate, but their action is short-term bination of exposure to males, receipt of Acps, and alloca- (Ͻ24 h; Chen et al., 1989; Kalb et al., 1993; Xue and Noll, tion of resources to the increased egg production postmating 2000; reviewed in Kubli, 1996). For the normal, approxi- (Chapman, 1992; Chapman et al., 1995; Fowler and Par- mately 1Ð1.5 week, depression of receptivity, females must tridge, 1989; Partridge and Fowler, 1990). Sperm them- also store sperm (sperm effect; Gromko et al., 1984b; selves do not directly contribute to the cost of mating in D. 202 M.C. Bloch Qazi et al. / Developmental Biology 256 (2003) 195–211 melanogaster (Chapman, 1992). However, by storing Seminal receptacle sperm, females reduce costs that accrue from repeated mat- Numerically, the seminal receptacle plays the biggest ings since they do not need to mate as many times to role in sperm storage, retaining 65Ð80% of the stored sperm maintain fertility. (Gilbert, 1981; Lefevre and Jonsson, 1962; Neubaum and Wolfner, 1999a; Tram and Wolfner, 1999). Observations and counts of stored sperm over time indicate that sperm are Storing sperm provides an opportunity for sperm released almost exclusively from the seminal receptacle for competition and female sperm preference the first several days after mating. Then, as the seminal Despite the costs associated with multiple mating and the receptacle sperm become depleted, sperm are released from general reluctance of females to remate after mating, female the spermathecae (Boule«treau-Merle, 1977; Gilbert, 1981; promiscuity is well documented in a wide variety of animals Neubaum and Wolfner, 1999a; Nonidez, 1920). The semi- (reviewed in Birkhead and Moller, 1998; Eberhard, 1996; nal receptacle’s importance in sperm storage is supported by Simmons, 2001; Smith, 1984), including Drosophila (Grif- a phylogenetic analysis of sperm storage organ use among fiths et al., 1982; Harshman and Clark, 1998; Imhof et al., 113 species of Drosophila (Pitnick et al., 1999). Loss of the 1998). When a female mates with more than one male, sperm storage function by the seminal receptacle is a rare female sperm storage provides both a venue for sperm from evolutionary event that appears to have occurred only once; different males to compete for access to fertilizations it characterizes only 3 (2.6%) of the species examined. In (sperm competition) as well as an arena for the female to contrast, the spermathecae’s sperm-storage function (dis- select sperm from among the contributions of competing cussed below) may have been lost as many as 13 different males (sperm preference or cryptic female choice) (Birk- times affecting 38 (33.6%) of the examined species (Pitnick head and Hunter, 1990; Birkhead and Moller, 1998; Eber- et al., 1999). hard, 1996; Knowlton and Greenwell, 1984; Parker, 1970; The seminal receptacle is a thin (5Ð20 ␮m) blind-ended Simmons, 2001; Table 2). These processes may be impor- tube that is coiled against the outer uterine wall. In D. tant mechanisms preventing genetic incompatibility (the melanogaster, the seminal receptacle is more than 2 mm production of nonviable or infertile progeny) by selecting long (Miller et al., 2001; Pitnick et al., 1999), slightly longer among male ejaculates (Zeh and Zeh, 1997). This is ob- than a sperm (1.75Ð1.90 mm; Lefevre and Jonsson, 1962; served in Drosophila: when a D. simulans female mates Pitnick et al., 1999). A positive relationship also exists with both a D. simulans and a D. mauritiana male, D. between sperm and seminal receptacle length among 44 simulans sperm are preferentially stored and used for fer- other Drosophila species examined (Pitnick et al., 1999). tilization (Price, 1997). The mechanisms of this conspecific This observation and other detailed evolutionary studies male advantage include both the inactivation and physical suggest that increases in seminal receptacle length drive the displacement of D. mauritiana stored sperm (mechanisms evolution of longer sperm (Pitnick et al., 1999). described later) (Price, 1997; Price et al., 2000). The lumen of the seminal receptacle is lined with a thin Sperm competition and sperm preference have poten- cuticle and is surrounded by a layer of nonsecretory cells, a tially important evolutionary consequences. When a female basement membrane, and a helically coiled layer of muscle D. melanogaster mates with more than one male, the most (Blaney, 1970). There does not appear to be a sphincter recent partner usually sires more than 80% of the subse- separating the seminal receptacle from the uterus (Filosi and quent progeny (“last male precedence”; reviewed in Perotti, 1975). At any time, only a few sperm are observed Gromko et al., 1984a; Kaufman and Demerec, 1942; Lefevre in the proximal half of the tube (that is, near its entry into and Jonsson, 1962). However, large variation exists in the the uterus) (Lefevre and Jonsson, 1962). The majority of fertilization success among last-mating males due to genetic stored sperm appear partially extended and lying parallel to differences among both males and females (Clark and Be- each other within the distal half of the seminal receptacle gun, 1998; Clark et al., 1999, 2000). Examining sperm (Fowler, 1973; M.C.B.Q., unpublished observations). This storage sheds light on the mechanisms of sperm precedence mass of sperm is curvilinear, and their tails can be seen to (discussed later). move, except when the seminal receptacle is very full of sperm (Anderson, 1945; Lefevre and Jonsson, 1962; M.C.B.Q., unpublished observations). Where are sperm stored? No genes responsible for seminal receptacle length or morphology have been identified. However, results of quan- Female sperm storage in D. melanogaster occurs in two titative genetic analysis of selection experiments for in- types of specialized organs, located at the anterior end of the creased or decreased seminal receptacle length suggest that uterus (Fig. 1). The single seminal receptacle is on the only a small number (2Ð5) of loci determine seminal recep- ventral side, and the paired spermathecae are on the dorsal tacle length (Miller et al., 2001). Since their effect is largely side of the uterus. The two types of storage organs differ in additive, the loci appear to be independent (Miller et al., morphology and in patterns of sperm storage. 2001). M.C. Bloch Qazi et al. / Developmental Biology 256 (2003) 195–211 203

Spermathecae Why are there two types of sperm storage organs? Fewer sperm reside within the paired spermathecae than Although the use of two types of storage organs is in the seminal receptacle (maximum of 135Ð449 sperm common (63.8% of 113 species examined; Pitnick et al., within both spermathecae; Gilbert, 1981; Neubaum and 1999) among Drosophila species, it is not clear why the Wolfner, 1999a; Tram and Wolfner, 1999). However, the spermathecae and seminal receptacle are both required for paired spermathecae appear to have two important roles in sperm storage. Pitnick et al. (1999) suggest that the seminal sperm storage. First, spermathecae are the long-term storage receptacle might have evolved as a new organ, which is organs. Sperm may accumulate more slowly in the sper- more efficient at storing sperm than are the spermathecae. mathecae than in the seminal receptacle (Gilbert, 1981; but The apparent coevolution of seminal receptacle length and see Tram and Wolfner, 1999). As mentioned before, sper- sperm length among several Drosophila species (above) mathecal sperm are apparently used for fertilization after the suggests that the seminal receptacle may also provide more seminal receptacle’s sperm have been depleted. Second, opportunity for female or male influence over sperm storage spermathecae may secrete substances that maintain sperm and sperm fate (Pitnick et al., 1999). Sperm displacement, viability within both the seminal receptacle and the sper- as a result of multiple mating, appears to be primarily mathecae (Anderson, 1945; but see Boule«treau-Merle, associated with the seminal receptacle (Civetta, 1999; Gil- 1977). Posterior to the spermathecae, but also opening into christ and Partridge, 1995; Price, 1999). Residence in the the dorsal side of the uterus, are two spermathecal glands spermathecae might protect sperm from displacement, and whose secretions could also affect sperm storage. the secretions of the spermathecae might also be essential Each spermatheca joins the uterus via a stalk composed of for viability (Anderson, 1945). a thin lumen surrounded by a thick cuticular intima, epithelial cells, and a helically coiled layer of muscle cells (Filosi and When do sperm enter and exit storage? Perotti, 1975; Nonidez, 1920). Like the seminal receptacle, the ϳ spermathecae do not appear to have a sphincter near their base Sperm storage begins before the 20-min copulation is complete (reviewed in Fowler, 1973; Gilbert, 1981; Lefevre (Filosi and Perotti, 1975). The spermatheca itself is an oval- and Jonsson, 1962). Sperm accumulate rapidly within the shaped capsule formed from a cuticular intima. Thin ducts two types of storage organs, leveling off at ϳ700Ð1000 within the spermathecal intima open to secretory cells sur- sperm Ͻ6 h after mating ends (Gilbert, 1981; Lefevre and rounding the exterior of the capsule. These cells produce a Jonsson, 1962; Neubaum and Wolfner, 1999a; Tram and lamellar type of secretion that accumulates within the sper- Wolfner, 1999). The first egg laid after mating (ϳ90 min) mathecal lumen (Filosi and Perotti, 1975). Sperm move from pushes out remaining unstored sperm (Lefevre and Jonsson, the uterus into the spermathecal capsule through the stalk. 1962). By 10 h after mating, seminal receptacle sperm Within the capsule, they wind around forming a toroidal mass. numbers have noticeably declined due to the female’s use of Two genes have been identified that affect spemathecal stored sperm to fertilize her eggs. On average, the number development. Several alleles of lozenge (lz), which encodes of sperm in the seminal receptacle declines at ϳ100Ð170 a putative transcription factor (Aronson et al., 1997; Daga et per day (Gilbert, 1981; Neubaum and Wolfner, 1999a; al., 1996), cause loss of the spermathecal glands and vary in M.C.B.Q. and M.F.W., unpublished observation). By 48 h the extent to which they affect spermathecal development after mating, sperm storage in the seminal receptacle has (Anderson, 1945). Some lz females have intact spermathe- declined by ϳ50%, while sperm stored in the spermathecae cae, while others are missing capsules and have malformed has decreased by only ϳ15% (Neubaum and Wolfner, or missing spermathecal stalks (Anderson, 1945). These 1999a). alleles also cause low fertility, apparently due to lower sperm storage in the seminal receptacle and the loss of How does sperm storage occur? motility of any stored sperm within a few days after mating (described below). Analysis of lz mutants suggests that they Sperm storage can be viewed as a series of steps: pro- impair dorsal cell migration in the early genital imaginal gression of sperm through the female reproductive tract disc that is needed for subsequent spermathecal develop- after mating, entry of sperm into the storage organs, reten- ment (Anderson, 1945). dachshund (dac), a nuclear protein tion and maintenance of stored sperm, and release of sperm (Mardon et al., 1994), is important for appropriate formation from storage up to the point of fertilization (Table 4). Both of the spermathecal stalks. When dac expression is blocked female and male D. melanogaster play active roles in sperm during development, the spermathecal capsules appear nor- storage, as shown by the nontransitivity of numbers of mal, but they share a single stalk (Keisman and Baker, stored sperm 1 h after mating between different fly strains 2001). In addition to these genes that affect spermathecal (DeVries, 1964). Female-based mechanisms can include morphology, some genes control spermathecal number. At absorption of fluids from parts of her reproductive tract least two genes are responsible for the formation of extra (called “hydraulics” here), contractions of the uterus, con- (Ͼ2) spermathecae (Sturtevant, 1925), but neither has yet tractions of the sperm storage organs, restriction of sperm to been identified. particular regions of the female reproductive tract, and/or 204 M.C. Bloch Qazi et al. / Developmental Biology 256 (2003) 195–211

Table 4 mechanism could potentially function in D. melanogaster’s Mechanisms by which sperm are moved into, maintained within, and seminal receptacle, ultrastructural studies argue against this released from female sperm storage hypothesis for sperm entry into the Drosophila spermathe- Mechanisms of female sperm storage cae because substances from the surrounding cells accumu- What gets sperm in? late within the spermathecae at the same time that sperm pressure changes within the female storage is occurring (Filosi and Perotti, 1975). female muscular contractions chemoattractants Female muscular contractions sperm concentration near sperm storage organs In many animals, female muscular contractions appar- guidance cues sperm motility ently push sperm from the uterus into storage (reviewed in What keeps sperm in storage and viable? Bloch Qazi et al., 1998; Neubaum and Wolfner, 1999b). spermathecal or paraovarial secretions Consistent with this occurring in Drosophila, a D. melano- peptides preventing sperm degredation gaster female CNS is required for sperm storage. Arthur et antimicrobial peptides al. (1998) tested this by manipulating expression of trans- energy sources for sperm viability What releases sperm from storage? former (tra), whose product is required for normal female conformational changes in the female reproductive tract development (Belote and Baker, 1982; reviewed in Cline ovulation and oocytes and Meyer, 1996). Various levels of ectopic expression of sperm motility tra in either tra-deficient mutant XX flies or XY flies re- sperm and seminal fluids from second mating sulted in individuals possessing female genital morphology (phenotypic females), but either a masculinized CNS (evinced by male courtship behavior) or a (presumably) factors secreted from within the female reproductive tract. feminized CNS (Arthur et al., 1998). Animals with the Male-based mechanisms are also important and can con- presumably female CNS stored nearly 6.5 times more sperm tinue even after copulation is complete. These include within all storage organs (ϳ550 sperm) but allocated pro- sperm motility and seminal proteins. Sperm are motile when portionally fewer of the sperm to their seminal receptacles they are transferred to the female and when they are in than did phenotypic females with a masculinized CNS. storage (Anderson, 1945). Although Gilbert (1981) hypoth- These results show that a feminized nervous system is esized that sperm motility is important for the release of necessary for sperm storage and that females actively dis- sperm from storage, and it seems a likely contributor to that tribute sperm among the storage organs. Results of experi- process, the role of sperm motility in sperm entry into ments in which males mated with isolated female abdomens storage in Drosophila is unknown. In contrast, studies have (that lack ganglia and therefore a CNS) support this hypoth- shown a profound effect on female sperm storage of secre- esis. It is possible that a female CNS is needed to trigger tions from the male’s ejaculatory duct and accessory glands uterine muscle contractions that push sperm into storage, as transferred to the female during mating. Matings between proposed for bed bugs, R. prolixus (Davey, 1958). Alterna- normal females and males transferring sperm, but no Acps, tively, a female CNS could influence sperm storage by are infertile, suggesting that Acps play an essential role in stimulating endocrine cells to release substances that attract sperm use once sperm are transferred to the female (Xue sperm to the storage organs. Finally, the generous innerva- and Noll, 2000; see also Fuyama, 1983; Hihara, 1981). tion of the seminal receptacle (Miller, 1950) suggests that When these females mate first with males transferring Acps, local contractions of the sperm storage organs might be but no sperm, then mate with males transferring sperm, but important for sucking sperm into storage and/or for efficient no Acps, the second male’s fertility is restored (Xue and arrangement of sperm within storage. Noll, 2000). Females mated to males that transferred sperm but greatly reduced (ϳ1%) quantities of Acps (DTA-D; Chemoattractants Kalb et al., 1993) store fewer than 10% as many sperm as do Sperm may be lured into storage by substances produced females mating to wild-type males (Tram and Wolfner, by the female or by male-derived substances activated once 1999). in the female. In sea urchins and other marine invertebrates, sperm-activating peptides (SAPs) secreted from the egg What gets sperm into storage? jelly stimulate sperm movements and orientation to eggs as well as activate other spermÐegg interactions (Suzuki, Hydraulics 1995). Compounds within the female Drosophila reproduc- Sperm may be drawn into storage by pressure changes tive tract could potentially act in a similar way, but none within the female reproductive tract. In the Dipteran Culi- have been positively identified thus far. coides melleus, and possibly in other lower Diptera, one component of sperm storage apparently involves fluid ab- Sperm corralling sorption from the spermathecae, which create a force that Concentrating sperm to specific regions of the reproduc- sucks sperm into storage (Linley, 1981). Although a similar tive tract can increase the likelihood that they will encounter M.C. Bloch Qazi et al. / Developmental Biology 256 (2003) 195–211 205 the openings to the storage organs. First, female anatomy, and are limited to the anterior portion of the uterus (Bairati, such as folds in the uterine wall, could channel sperm into 1968; Lung and Wolfner, 2000). The mating plug is not the seminal receptacle (Fowler, 1973). Such channels exist detected more than 6 h after mating and, while its fate is in other animals; for example, in cows, folds in the oviduc- unknown, it seems likely to be expelled when the first egg tal isthmus epithelium form crypts in which sperm reside is laid or dissolved and reabsorbed. temporarily (Suarez et al., 1997; reviewed in Suarez, 2002). Second, in D. melanogaster females, a barrier of unknown Seminal proteins that enter the female with sperm composition exists at the base of the oviduct that keeps By examining sperm storage in the presence of normal or sperm in the uterus. At least one male seminal protein, the very low amounts of Acps, Tram and Wolfner (1999) accessory gland protein Acp36DE (described below), local- showed that Acps are required for sperm storage. One of izes at this barrier, but no chemical components required for barrier formation have been identified (Neubaum and these proteins, Acp36DE, is essential for proper sperm stor- Wolfner, 1999a). In egg-less females, the barrier is mislo- age (Table 3). Females mated to Acp36DE-deficient mutant calized or does not form and sperm are found within the males receive normal quantities of sperm during mating, but common and lateral oviducts (Bertram et al., 1996; store far fewer sperm than females mated to wild-type males Neubaum and Wolfner, 1999a). The presence of oocytes in (Neubaum and Wolfner, 1999a). Without Acp36DE, sperm the ovaries may help form the block by creating a back- start to enter storage at the normal time, but the subsequent pressure and/or causing the secretion of a substance that accumulation of sperm into the seminal receptacle and sper- localizes near the base of the oviduct preventing sperm from mathecae is less efficient (M.C.B.Q. and M.F.W., unpub- premature access to oocytes within the ovary. lished observations). The rate at which sperm are released From within her reproductive tract, a female D. melano- from spermathecal storage differs slightly in the presence or gaster secretes factors important for sperm storage. Glucose absence of Acp36DE (Neubaum and Wolfner, 1999a). dehydrogenase (Gld), an enzyme-producing reactive oxy- Within the first 24 h after mating, the rate at which sperm gen species (Cavener and MacIntyre, 1983), is secreted are released from the seminal receptacle is similar in the from the spermathecal stalks and the genital plates (located presence or absence of Acp36DE, but between 24Ð48 h near the gonopore; Schiff et al., 1992). gld-mutant females after mating, proportionally more sperm are lost from the store fewer sperm within, and allocate sperm more unevenly seminal receptacles of females receiving Acp36DE than between, the two spermathecae, but only when sperm stor- females not receiving Acp36DE from their mates (Neubaum age is submaximal (Ͻ500 total sperm stored; D. Cavener, and Wolfner, 1999a; M.C.B.Q. and M.F.W., unpublished personal communication). Therefore, Gld may facilitate observations). It is not clear whether this phenomenon is a sperm storage, particularly when sperm are not plentiful, but direct result of Acp36DE action or a secondary consequence is not essential for sperm storage to occur. The mechanism of storing fewer sperm. The number of progeny produced of Gld’s sperm storage effects is unknown, but Gld is in the absence of Acp36DE corresponds to the number unlikely to serve as a chemoattractant since it is produced in of sperm in storage, indicating that those sperm that are more than one location within the female reproductive tract. stored without Acp36DE are fully viable (Neubaum and Corralling can also involve male contributions. The male Wolfner, 1999a). Thus, Acp36DE’s primary role is in fa- accessory glands contain filaments composed of globular cilitating sperm storage. It may potentially have a secondary subunits (Perotti, 1971). Similar looking filaments are ob- role in promoting sperm retention in the seminal receptacle, served in female storage organs interdigitated with stored but there is no evidence that Acp36DE affects sperm via- sperm (Bairati, 1968). If the filaments of similar appearance bility. are indeed the same, those filaments might provide a scaf- Acp36DE is a novel 122-kDa glycoprotein that is trans- fold along which sperm move or within which sperm are ferred to females beginning within the first 5 min of mating confined, to facilitate the efficient movement of sperm into (Bertram et al., 1996; Wolfner et al., 1997). Within the storage. In an analogous way, the mating plug, contributed female, it is processed to a 68-kDa product (Bertram et al., at least in part by the male, is thought to facilitate sperm 1996; Neubaum and Wolfner, 1999a). Acp36DE is detect- storage by forming a physical barrier that prevents the loss able within the female reproductive tract for as long as 3 h of sperm from the uterus; sperm are confined above the after mating. It localizes to the oviduct wall anterior to the plug, concentrating them near the entrances of the storage sperm storage organ openings (at the barrier discussed organs. The mating plug has also been proposed to provide above) and on the anterior end of the mating plug. Thus, it a trellis to facilitate sperm movement toward storage (Bai- is present at the upper and lower areas of the “corral” rati, 1968; Lung and Wolfner, 2001). The mating plug described above. In addition, Acp36DE enters the sperm contains several male seminal proteins, including PEB-me storage organs (Neubaum and Wolfner, 1999a). Finally, (its major component, derived from the ejaculatory bulb; Acp36DE binds to sperm in vivo and in vitro. When the first Ludwig et al., 1991; Lung and Wolfner, 2001; Table 3) and egg is laid, the Acp36DE in the oviduct and mating plug are Acps, including the protein Acp36DE (see below; Lung and expelled (Bertram et al., 1996). Although no longer detect- Wolfner, 2001). Entering sperm traverse the mating plug able within the female, Acp36DE donated from one male 206 M.C. Bloch Qazi et al. / Developmental Biology 256 (2003) 195–211 facilitates the storage of a second male’s sperm 24Ð48 h strated) regulators of proteolysis (Wolfner et al., 1997; later (Chapman et al., 2000). Swanson et al., 2001; Lung et al., 2002; J.L. Mueller and The available data on Acp36DE support several, not M.F.W., unpublished observations). One, the trypsin in- mutually exclusive, models for its action. Acp36DE may hibitor Acp62F, has been shown to enter the mated fe- interact with targets on female tissues stimulating females to male’s sperm storage organs (Lung et al., 2002), consis- push (from the uterus) or suck (from the sperm storage tent with its playing a role in protecting sperm from organs) sperm into storage via muscular contractions. It degradation (Table 3). Stored sperm are also potentially could potentially also stimulate the release of chemoattrac- subject to untoward effects from microbes that might tant molecules. Alternatively, Acp36DE may limit sperm have entered the female’s genital tract during mating. movements via its associations with the oviduct, mating Perhaps to guard against this, sperm storage organs and plug, and sperm, thereby facilitating the rapid accumulation seminal fluids contain antimicrobial peptides (Lung and of sperm within storage. Finally, Acp36DE might help track Wolfner, 2001; Samakovlis et al., 1991). The seminal sperm into storage by forming a scaffold or providing guid- receptacle and spermathecae also both secrete drosomy- ance cues along which the sperm move, or are moved, into cin, a peptide with antifungal properties (Ferrandon et al., storage. 1998).

What keeps sperm viable in storage? What releases sperm from storage?

Once D. melanogaster sperm are in storage, they need to Sperm leave storage to two potential fates: one fate is to remain viable for up to 2 weeks. For example, even in cases fertilize an ovulated egg, another is to leave storage but not of matings between genetically incompatible strains of D. to fertilize an egg either due to inefficient sperm use or to melanogaster, sperm in the storage organs remain viable sperm displacement as a result of the female mating with several days after mating (suggested by vital cell staining; another male (female sperm preference or sperm competi- Alipaz et al., 2001). Both female and male factors probably tion). Since sperm use in D. melanogaster is efficient, one contribute to the maintainance of sperm in storage. (or very few) sperm leave(s) storage to fertilize an ovulated Substances produced within the spermathecae may play egg as the egg comes to rest in the uterus. The egg lodges roles in sperm viability and retention. The presence of the there with its anterior end, containing its micropyle, close to spermathecal capsule (described earlier) correlates with fe- the sperm storage organ entrances. It is not known whether male fertility. The low and variable fertility of some female sperm swim, are pushed, or are sucked through the micro- lz mutants suggests a similar model for D. melanogaster, pyle. The entire sperm enters the egg. Its tail coils in the since the lz mutant phenotype is proposed to be due to the anterior end and persists within the embryo until shortly presence/absence of the spermathecal capsule (Anderson, after hatching (Pitnick and Karr, 1998). 1945). Females with at least 1 spermathecal capsule produced an average of 216 progeny, 36 times as many progeny Female factors as mutant females lacking spermathecal capsules (calculat- In D. melanogaster, conformational changes of the re- ed from Anderson, 1945). Lower fertility was attributable to productive tract induced by ovulation might also effect a shorter duration of progeny production among females sperm release. Wheeler (1954) observed single sperm near lacking capsules compared with their normal sisters. Al- the opening of the seminal receptacle as an egg passed down though the seminal receptacle appeared normal, lz mutants the oviduct and proposed that ovulation and sperm release stored fewer sperm which lost motility earlier (Յ5 days were correlated. Muscular contractions of just the seminal after mating) than did sperm in wild-type females (Ͼ11 receptacle could squeeze small numbers of sperm out of days). These results suggest that presence of the secretory storage. cells surrounding the spermathecal capsule and/or the spermathecal glands is important for the viability of stored Sperm motility sperm and for female fertility. Motile sperm might motor their way out of storage. Stored sperm need to be protected from degradation. Lefevre and Jonsson (1962) observed sperm circulating During storage, proteolysis of sperm surface proteins within the storage organs and speculated that, occasionally, could destroy a sperm’s ability to bind to eggs; alterna- a single sperm leaving storage would encounter an egg. This tively, regulated proteolysis of the surface of stored observation, coupled with the lack of detected sphincters at sperm could be essential to activate or capacitate them. the base of the sperm storage organs, led Gilbert (1981) to Indeed, in mice, mutations in seminal fluid protease in- propose that sperm motility aided their release (described hibitors impair fertility (Murer et al., 2001), consistent below). with the hypothesis that protease inhibitors serve to protect sperm. D. melanogaster seminal fluid also contains Male-derived proteins regulators of proteolysis. Of ϳ83 predicted secreted male At least one male-derived protein is suggested to play a accessory gland proteins, 9 are predicted (or demon- role in sperm residence in storage in females: the carboxyl- M.C. Bloch Qazi et al. / Developmental Biology 256 (2003) 195–211 207 esterase, esterase-6 (Est-6). Est-6 is secreted from the male genesis and fertilization. In addition to their direct impor- ejaculatory bulb and anterior ejaculatory duct, and is trans- tance in preparing eggs and sperm for fertilization, ferred to the female early during mating (Sheehan et al., ovulation and sperm storage involve additional processes of 1979; Tamarina et al., 1997). Activity of male-derived Est-6 interest to developmental biologists. These phenomena in- is detected for only 2 h after mating (Richmond and Senior, clude cell migration and targeting, cell and tissue interac- 1981). Although the initial timing and storage of sperm into tions, including ones essential for cell viability, and coor- females that do or do not receive Est-6 from their mates is dination of hormonal and neural triggers and muscle similar over time, more sperm are retained in females that contraction. In addition, understanding the mechanism and do not receive Est-6 (Gilbert, 1981). Est-6 therefore appears regulation of the necessary steps between gametogenesis to play a role in the release of sperm from storage in the and fertilization is relevant to understanding the basis for seminal receptacle; its role on spermathecal sperm is un- maleÐfemale reproductive conflict. Although ovulation and clear (Table 3). Est-6 might cause the release of sperm from female sperm storage are presently less well-understood at a the seminal receptacle by affecting sperm motility within mechanistic level than are gametogenesis and fertilization, the sperm storage organs or by catalyzing the production of we believe now is a fertile time for their study. molecules needed to sustain sperm motility (Gilbert, 1981). The genetic manipulations possible with D. melano- However, since Est-6 activity is also positively correlated gaster, and the existence of several parallels in its gameto- with the rate of female oviposition (Gilbert, 1981) as well as genesis and gametes with those of other animals, make it an female latency to remating (Scott, 1986), and since Est-6 excellent model for studying ovulation and sperm storage. enters the female hemolymph (Meikle and Richmond, Already we know that, in D. melanogaster, the release of an 1991), it may have more than one target or multiple inter- egg by the ovary is triggered by molecular and chemical related effects. If a female mates twice, the second male’s ejaculate signals that indicate that mating has occurred and that ovu- causes the release of previously stored sperm (last male lation, in turn, triggers important changes in the egg. We sperm precedence, see above; reviewed in Gromko et al., also know that storage of Drosophila sperm requires their 1984a; Lefevre and Jonsson, 1962; Newport and Gromko, localization to particular places in the female’s reproductive 1984; Prout and Bundgaard, 1977). This effect is attribut- tract, their retention and support, and their regulated release able to the removal of some sperm from storage and the from storage. In both ovulation and sperm storage, male- inactivation of remaining sperm (Civetta, 1999; Harshman derived and female factors cooperate to ensure coordination and Prout, 1994; Lefevre and Jonsson, 1962; Price, 1999; between the processes that results in a very high efficiency Scott and Richmond, 1990). The method of displacement of fertilization. These regulatory factors include seminal depends on the time between matings. If a female remates proteins and sperm acting in the context of a female repro- within 2 days of an initial mating, the last-mating male’s ductive tract innervated by a female nervous system. The sperm plays a role in physical displacement of sperm, pri- mechanistic details of the actions of male cells and proteins, marily from the seminal receptacle (Civetta, 1999; Gilchrist and how they in turn interact with and influence cells, and Partridge, 1995; Price, 1999). With longer intervals molecules, and physiological processes (such as feedback between rematings, other seminal components, particularly controls) in the female remain to be elucidated. Further Acps from the most recently mating male, functionally dissection of the important steps between gametogenesis displace sperm by decreasing the use of previously stored and fertilization will contribute to a full understanding of sperm that remain after the second mating (Gilchrist and the development of eggs and sperm, and their exquisite Partridge, 1995; Harshman and Prout, 1994; Price, 1999; interorganism and intercellular coordination to result in the but see Scott and Richmond, 1990). If seminal proteins final product: a fertilized egg. temporarily protected the male’s sperm from displacement, then perhaps it is the inactivation of these “protein compan- ions” to sperm that leave the sperm vulnerable to displacement by future mating males. Males lacking Acp36DE are Acknowledgments poor displacers of other males’ sperm and often have their own sperm nearly completely displaced from storage This manuscript benefited from the careful and insightful (Chapman et al., 2000), but this is believed to be because comments of Wolfner lab members: S. Albright, L. they are poor at getting their own sperm moved into storage McGraw, V. Horner, J. Mueller, K. Ravi Ram, and G. (Chapman et al., 2000; Neubaum and Wolfner, 1999a). Reeves. We thank D. Cavener for sharing unpublished results with us. We also appreciate the suggestions and perspectives provided by S. Suarez, L. Harshman, C. Desplan, Conclusion and anonymous reviewers. We are grateful for support from NSF Grant IBN 99-04824 and NIH Grants HD 38921 and In animals with internal fertilization, ovulation and fe- GM 44659 (to M.F.W.), and an American Cancer Society male sperm storage are important steps between gameto- postdoctoral fellowship (to M.C.B.Q.). 208 M.C. Bloch Qazi et al. / Developmental Biology 256 (2003) 195–211

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