The Interactive Fly
Genes involved in tissue and organ development
In the Drosophila oocyte, meiosis is arrested in the first division of metaphase, when a tapered spindle aligned parallel to the egg surface forms. The chromosomes are therefore located in the cortical region near the anterior pole, whereas fusion of parental complements occurs in the inner ooplasm. How does the female pronucleus reach the interior of the egg? The second meiotic spindles are arranged in tandem, end to end, and disposed perpendicular to the longitudinal axis of the egg with the innermost spindle carrying the female pronucleus. This pattern of spindle organization is probably involved in the migration of the female pronucleus deeper into the egg near the cytoplasmic domain of the male pronucleus. The precise time at which the mitotic spindle of Drosophila changes orientation is unknown. However, spindle rotation from a position parallel to the egg surface to a radial orientation presumably occurs during or shortly after the oocyte passes through the oviduct. How spindle orientation is achieved and maintained during meiosis is an intriguing question. Microtubules linking spindle poles to the oocyte surface have been implicted in the rotation and anchoring of the meiotic apparatus in Xenopus oocytes and in other organisms, but this does not seem to be the case in the Drosophila oocyte, since the meiotic spindles lack astral microtubels. However, the observation that a transient array of microtubules links the meiotic apparatus to discrete subcortical foci suggests that in Drosophila the orientation of the spindle also requires a functional interaction between the spindle and the oocyte cortex (Riparbelli, 1996 and references).
The microtubule array of mitosis II observed between the twin spindles at metaphase, anaphase and telophase might be an intermediate between the anastral poles of the meiotic I spindles and the astral poles of the mitotic spindles in early embryos. A complex pathway of spindle assembly takes place during resumption of meiosis at fertilization, consisting of a transient array of microtubules radiating from the equatorial region of the spindle toward discrete foci in the egg cortex. A monastral array of microtubules is observed between twin metaphase II spindles in fertilized eggs. These microtubules originating from disc-shaped material stain with Rb188 antibody specific for an antigen asssociated with the centrosome of Drosophila embryos (DMAP190 or CP190). Therefore, the Drosophila egg contains a maternal pool of centrosomal components undetectable in mature inactivated oocytes. These components nucleate microtubues in a monastral array after activation, but are unable to organize bipolar spindles (Riparbelli, 1996).
The meiosis II spindle of Drosophila oocytes is distinctive in structure, consisting of two tandem spindles with anastral distal poles and an aster-associated spindle pole body between the central poles. Assembly of the anastral:astral meiosis II spindle occurs by reorganization of the meiosis I spindle, without breakdown of the meiosis I spindle. The unusual disc- or ring-shaped central spindle pole body forms de novo in the center of the elongated meiosis I spindle, followed by formation of the central spindle poles. gamma-Tubulin transiently localizes to the central spindle pole body, implying that the body acts as a microtubule nucleating center for assembly of the central poles. The first step in formation of the central pole body is the appearance of puckers in the center of the the meiosis I spindle, followed by the pinching out from the spindle of a disc or ring of microtubules that becomes the central pole body. The manner in which the central spindle pole body forms suggests the involvement of a microtubule motor. If so, the motor involved is likely to be different from Ncd (Nonclaret disjunctional), since loss of Ncd function does not seem to prevent its formation. Following the formation of the central spindle pole body, the microtubules arrayed to either side of the central body narrow into poles, forming the mature meiosis II spindle. The central poles become more tapered during progression through meiosis II, and the central spindle pole body also changes in morphology: the disc or ring becomes asterlike, then enlarges into a ring that lies between the two central telophase II nuclei (Endow, 1998).
Localization of gamma-tubulin to the meiosis II spindle is dependent on the microtubule motor protein, Ncd. Absence of Ncd results in loss of gamma-tubulin localization to the spindle and destabilization of microtubules in the central region of the spindle. Likewise, during meiosis I, the minus-end motility of Ncd and its crosslinking activity are probably needed to focus microtubules into spindle poles for the correct functioning of meiosis I. Assembly of the anastral:astral meiosis II spindle probably involves rapid reassortment of microtubule plus and minus ends in the center of the meiosis I spindle. This can be accounted for by a model that also accounts for the loss of gamma-tubulin localization to the spindle and destabilization of microtubules in the absence of Ncd (Endow, 1998).
A model for assembly of the Drosophila oocyte meiosis II spindle is suggested: gamma-Tubulin is first recruited or relocalized, possibly as gamma-TuRC, to the midbody of the meiosis I spindle, where it functions to nucleate microtubules for formation of the meiosis II central spondle poles. The loss of gamma-tubulin localization to the spindle in the absence of Ncd suggests that the Ncd motor serves to recruit or anchor gamma-tubulin to the center of the spindle. The Ncd motor would then stabillize newly nucleated microtubule minus ends and focus the microtubules into poles. The unstabilized plus ends of the microtubules in the center of the spindle (remaining from meiosis I) would undergo rapid depolymerization as a consequence of dynamic instability. Stabilization of the newly nucleated microtubule minus ends and depolymerization of the plus ends would cause a rapid sorting out of the microtubules in the center of the meiosis I spindle, replacing microtubule plus ends with minus ends. The distal poles of the meiosis II spindle would be retained from the meiosis I spindle and maintained by the same forces that originally formed them: the crosslinking activity and minus-end movement of Ncd along spindle microtubules (Endow, 1998).
Maternally inherited mitochondria and other cytoplasmic organelles play essential roles supporting the development of early embryos and their germ cells. Using methods that resolve individual organelles, the origin of oocyte and germ plasm-associated mitochondria was studied during Drosophila oogenesis. Mitochondria partition equally on the spindle during germline stem cell and cystocyte divisions. Subsequently, a fraction of cyst mitochondria and Golgi vesicles associates with the fusome, moves through the ring canals, and enters the oocyte in a large mass that resembles the Balbiani bodies of Xenopus, humans and diverse other species. Some mRNAs, including oskar RNA, specifically associate with the oocyte fusome and a region of the Balbiani body prior to becoming localized. Balbiani body development requires an intact fusome and microtubule cytoskeleton since it is blocked by mutations in hu-li tai shao, while egalitarian mutant follicles accumulate a large mitochondrial aggregate in all 16 cyst cells. Initially, the Balbiani body supplies virtually all the mitochondria of the oocyte, including those used to form germ plasm, because the oocyte ring canals specifically block inward mitochondrial transport until the time of nurse cell dumping. These findings reveal new similarities between oogenesis in Drosophila and vertebrates, and support the hypothesis that developing oocytes contain specific mechanisms to ensure that germ plasm is endowed with highly functional organelles (Cox, 2003).
Drosophila oocytes contain a typical Balbiani body at the time follicles form in region 3 of the germarium. In a wide range of animal species, including Xenopus, chick, mouse and human, young oocytes at a similar developmental stage display these distinctive aggregates of mitochondria and other organelles near their germinal vesicles. In a typical Balbiani body, centrioles and associated cytoplasm are surrounded by a ring of Golgi bodies and encased in a large mass of mitochondria. As the oocyte grows, the mitochondria first spread around the nuclear periphery and later disperse throughout the oocyte cytoplasm (Cox, 2003).
Drosophila Balbiani bodies, like those described in other species, contain clustered mitochondria, Golgi vesicles and centrioles. Moreover, as young follicles develop from stage 1-6, the mitochondria move around the germinal vesicle and disperse after microtubules re-organize in stage 7 (Cox, 2003).
The studies reported here provide new insight into the origin of Balbiani bodies. Drosophila Balbiani bodies do not arise de novo within oocytes, but are built by the transport of organelles from neighboring cells within interconnected germline cysts. These experiments make clear that many components of oocyte cytoplasm arise in this manner (Cox, 2003).
Virtually all of the newly formed mitochondria in oocytes are derived from the Balbiani body. The great majority are transported from other cystocytes along the fusome but 1/16th or more might simply originate in the oocyte. Like oocyte determination itself, Balbiani body formation depends on the fundamental cyst polarity manifested in the fusome. Arising in embryonic germ cells, the fusome builds up a framework of cyst polarity during the cystocyte divisions. Fusome polarity probably acts directly to control centriole migration and the meiotic gradient, and acts indirectly to differentiate and maintain the oocyte by regulating the microtubule cytoskeleton. Deciphering the molecular mechanisms that define fusome polarity and allow the fusome to control microtubule organization remains a central issue for understanding Balbiani body formation and oocyte development (Cox, 2003).
Oocytes develop from germline cysts or syncytia in diverse species so Balbiani bodies may arise through intercellular transport in a wide range of organisms besides Drosophila. In both Xenopus and the mouse, mitochondrial clouds present within interconnected germ cells are thought to be precursors to the Balbiani bodies that arise shortly after the cysts break down and form primordial follicles. In Drosophila, the large chunk of fusome at the anterior of the early stage 1 oocyte contains clustered centrioles and is likely to act as a microtubule-organizing center. It may attract and retain mitochondria, Golgi and localized macromolecules as they enter the oocyte, thereby creating the Balbiani body. Xenopus Balbiani bodies may arise in a similar fashion as they have a similar organization consisting of a spectrin-rich zone, mitochondria, Golgi and the Metro region containing RNAs in transit. However, there has been insufficient study of the Xenopus larval ovary to identify a fusome or some other material with microtubule organizing properties that might play an analogous role. In most other systems whose Balbiani bodies share the same basic structure in young oocytes, very little is known about their origin during earlier stages of germ cell development (Cox, 2003).
The Balbiani bodies in many species contain structures resembling germinal granules. In Xenopus, these granules are found in a region containing specific RNAs that are also destined to be localized in the egg and incorporated in germ cells. Consequently, the Balbiani body has been proposed to function as a messenger transport organizer (METRO) that organizes and mediates the delivery of RNAs and germinal granules to the vegetal pole of the egg. Specific elements have been mapped in the 3' UTR of the Xcat2 mRNA that are sufficient for localization to the Balbiani body or to the germinal granules themselves (Cox, 2003).
The Drosophila Balbiani body may play a related role. oskar RNA, a key component that is capable of inducing germ plasm formation, is associated with the posterior segment of the Balbiani body in early stage 1 oocytes, much as Xcat2 is localized in the Xenopus Balbiani body. A few hours later, towards the end of stage 1, osk RNA moves to the oocyte posterior along with the other Balbiani-associated RNAs and proteins that have been studied, presumably in response to the shift in microtubule polarity that occurs at this time. Thus, at least some molecules that participate in germ plasm assembly associate with the Balbiani body in early Xenopus and Drosophila oocytes (Cox, 2003).
Drosophila RNAs that become associated with the Balbiani body, like organelles, first interact with the fusome during early stages of cyst development. However, there are significant differences in these fusome interactions with RNAs and organelles that probably reflect different molecular mechanisms of delivery to the Balbiani body. Organelles associate next to the fusome along much of its length and subsequently move toward the center, in concert with microtubule minus ends. By contrast, the RNAs associate with one or a few cells at the center of the fusome from the earliest stages they could be detected, and are located within it, as well as nearby. These observations suggest that localized RNAs may read the fusome polarity directly, and need not rely on changes in microtubule organizing activity to get to the oocyte or be stabilized within it (Cox, 2003).
Potentially significant differences exist in the role of RNA transport played by the Drosophila and Xenopus Balbiani bodies. The Drosophila Balbiani body associates with germ plasm RNAs for only 5-10 hours during early stage 1. By contrast, Xenopus Balbiani bodies associate throughout stage 1 of oogenesis, a process requiring many days, with at least 11 RNAs. When the RNAs leave the Drosophila Balbiani body, mitochondria mostly remain behind, only to follow much later in oogenesis. By contrast, in Xenopus, both mRNAs and mitochondria are reported to proceed together to the vegetal pole. These differences may simply reflect differences in the timing of cytoskeletal remodeling that control these events. Moreover, the observation that a small subset of mitochondria recognized by COXI antisera do translocate with the RNAs in stage 1 indicates that certain Drosophila mitochondria may follow a Xenopus-like pattern. However, it remains possible that RNAs in transit to the oocyte posterior may simply pass through the Balbiani body without being affected in any way (Cox, 2003).
Sponge-like structures have been described in the cytoplasm of stage 4-10 nurse cells that are associated with Exu protein, RNA, and (frequently) mitochondria and nuage. It has been proposed that these structures are analogous to classical Balbiani bodies and that they mediate transport of localized transcripts such as bicoid RNA. The current results suggest that the ooctye contains a true Balbiani body much earlier -- in stage 1 follicles. The sponge bodies more likely represent transport complexes organized at the surface of nurse cell nuclei that subsequently move through the follicle and into the ooctye. However, there may be structural and molecular similarities between nurse cell transport complexes and those mediating transport out of the Balbiani body (Cox, 2003).
These studies provide further evidence that the ring canals that join the cystocytes play an important role in regulating Balbiani body formation. Mitochondria appear to first enter the oocyte when fusome segments within the adjoining ring canals break apart, unplugging the channels. Subsequently, a novel mechanism blocks further mitochondrial passage through these canals, because large backups of mitochondria are observed outside each oocyte ring canal in young oocytes and a lack of mitochondrial movement into the oocyte has been documented in movies. Mitochondria do not accumulate in the same manner around the ring canals that join nurse cells, but are spread throughout the cell and in the nuclear periphery. This behavior has the effect of limiting the mitochondrial genotypes within the oocyte to those found in Balbiani body mitochondria until well after mitochondria have begun to associate with the germ plasm at the oocyte posterior pole. Despite the importance of these regulatory steps, little is known about how movement through ring canals is controlled (Cox, 2003).
These studies suggest that centrioles, mitochondria, Golgi, RNAs and other key components of oocyte cytoplasm are added to the Drosophila oocyte by a special mechanism that may have been widely conserved in evolution. It is remarkable that in the oocyte, the lone cell that will contribute cytoplasm for the next generation of organisms, many fundamental components of cytoplasm do not arise by random partitioning among daughter cells. Rather, an elaborate mechanism is used to transport materials from multiple cells and maintain them in a large aggregate for an extended period of time. It is possible that Balbiani bodies do not play a specific role in ooctye development, but represent a byproduct of the unusual centrosome behavior in these cells. However, an alternative hypothesis is favored. One of the potentially most interesting reasons that oocyte organelles might be delivered en mass via the fusome would be to increase organelle fitness. Mitochondrial DNAs are known to accumulate mutations that have frequently been postulated to affect the aging of cells and tissues. If only mitochondria with functional genomes are able to associate with the fusome and move into the oocyte, damaged genomes might be weeded out when they still represent a small fraction of the total. Such a system would be far more efficient than eliminating defective genomes by inducing the apoptosis of entire germ cells. A purifying mechanism based on organelle selection might be particularly important in organisms that need to produce eggs with a high average viability, or that must support long intergenerational life spans (Cox, 2003).
Several other observations may also be explained by the need to eliminate
defective mitochondrial genomes. The exclusion of nurse cell mitochondria from
passing through the oocyte ring canals prior to dumping would ensure that only
the 'selected' mitochondria in the Balbiani body populate the germ plasm.
Mitochondria may break up into small, nearly round, organelles during this
period so that each will contain a single genome whose fitness can be tested.
The cytoplasmic streaming of the ooctye may serve to mix the two populations
of organelles so each somatic cell type inherits at least some of the selected
mitochondrial population. Finally, a requirement for translation on
mitochondrial ribosomes in the early embryonic germ plasm might serve as a
concluding selective step to ensure that viable germ cells are well supplied
with intact mitochondrial genomes. If female germ cells do possess mechanisms
to remove defective mitochondria, they would probably have contributed to the
evolutionary conservation of germ line cysts and Balbiani bodies (Cox, 2003).
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. Within the female D. melanogaster, both gamete maturation and sperm storage 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 (Qazi, 2003).
Mating initiates a series of events within the female reproductive tract, including ovulation and sperm storage. Ovulation and sperm storage occur in different regions of the female reproductive tract, but are coordinated for a connected fate: the fertilization of an egg. At the anterior of the female Drosophila reproductive tract are two ovaries, each composed of 10-20 ovarioles. The ovarioles are held together by a peritoneal sheath, containing a network of fine, branching muscle fibers. The base (proximal end) of each ovariole forms a small duct or pedicel. The pedicels of all the ovarioles in an ovary unite to form a calyx; each calyx opens into a lateral oviduct. The lateral oviducts fuse into a common oviduct that, more posteriorly, enlarges to form the uterus. The wall of the oviducts consists of epithelium surrounded by circular muscles that is intensively innervated. The uterus is a heavily muscularized and innervated structure that receives sperm during mating and also holds the egg in position for fertilization. At the anterior end of the uterus are the three sperm storage organs: a single seminal receptacle and the paired spermathecae, as well as the paired spermathecal glands (also called parovaria or female accessory glands). At its posterior end, the uterus narrows forming the vagina that exits the female reproductive tract. The distal end of the vagina, called the gonopore, serves for the discharge of eggs (Qazi, 2003 and references therein).
In many organisms, interaction between the gametes triggers a series of cellular responses in the egg ('egg activation') required to initiate embryonic development. Drosophila eggs also are triggered to initiate a suite of cellular responses analogous to those of activation in other organisms (relief of arrest in meiosis, ultimately leading to completion of meiosis, changes in egg coverings, and initiation of translation) in a process that has therefore also been called activation. This semantic convergence masks a distinct mechanistic difference in the trigger for activation in Drosophila and at least one other insect relative to that in echinoderms, nematodes, and vertebrates: egg activation in Drosophila and other insects initiates independent of sperm entry, perhaps not surprising in an Order that includes species in which haploid males can develop from unfertilized eggs. In Drosophila, changes in the egg envelope's permeability, one feature of activation, initiate during ovulation, even while most of the egg is still within the ovary (Heifetz, 2001b). The egg's covering becomes progressively more impermeable to small molecules as the egg proceeds down the oviduct and the process is complete by the time the egg arrives at the uterus. Cross-linking of vitelline membrane protein sV23 also increases progressively as the egg moves through the oviduct and the uterus. Ovulation also triggers meiosis to resume before the egg reaches the uterus (Heifetz, 2001b). Thus, ovulation causes changes in the egg that prepare it for subsequent embryogenesis, should fertilization occur in the uterus, including coordination between cell cycle status of the egg and the sperm nuclei. The third feature of activation, resumption of translation, also initiates without fertilization in Drosophila, though it is not yet known if it too is triggered by ovulation (Qazi, 2003 and references therein).
Drosophila females produce up to two final-stage (stage 14) oocytes in each ovariole daily (as many as 80 oocytes/day. Several control points and feedback mechanisms regulate the production of mature oocytes. If mature oocytes are not ovulated, each ovariole accumulates two or three late-stage oocytes. This blocks the maturation of additional oocytes. Mating, and specifically sperm and some male accessory gland proteins [e.g., Acp26Aa (ovulin)], induces ovulation of mature oocytes (Heifetz, 2000 and Heifetz, 2001b) that, in turn, contribute to 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 (Qazi, 2003 and references therein).
Drosophila virgin females retain mature oocytes in their ovaries. Ovulation initiates within 1.5 h after mating (Heifetz, 2000) during a time when sperm are still being stored. Thus, the initial eggs ovulated are released potentially prior to full completion of sperm storage. The number of progeny per number of eggs laid (hatchability) immediately after matings between wild-type males and females is lower than at later time points (Chapman, 2001). This lower hatchability appears to reflect a lower efficiency of fertilization of the first eggs released. By visualizing the fertilizing sperm tail within wild-type eggs, it was found that the first eggs laid had a lower fertilization efficiency (ratio of unfertilized/fertilized eggs = 1.5 at 3-4 h post-mating) than those laid subsequently (0.6 at 48-49 h, Chapman, 2001). Two possible explanations for the low hatchability are suggested; regulated ovulation plays a critical role in each of these. (1) Ovulation allows coordination of oocyte release with the rate of sperm release from storage. The first eggs ovulated may reach the fertilization site before sperm are prepared and in position to be released from storage. Thus, lower numbers of those eggs would be fertilized, leading to lower hatchability. (2) Since unmated females can accumulate eggs for several days, it is possible that the older eggs are 'stale' and cannot be fertilized. By ovulating those eggs quickly, even before sperm are fully stored, the female clears out any stale eggs without wasting sperm (Chapman, 2001). Understanding the mechanism that coordinates oocyte release from the ovary and sperm release from the sperm storage organs will provide insights into the first steps of sperm/egg interactions that lead to successful fertilization (Qazi, 2003 and references therein).
In insects, production and laying of eggs requires five steps within the female reproductive tract: (1) oogenesis (the generation of oocytes within the ovary); (2) ovulation (release of an oocyte from the ovary into the oviducts); (3) movement of the egg down the oviducts; (4) fertilization of the egg when it has come to rest in the uterus, and (5) deposition of eggs onto the substratum. These steps are sequential and thus somewhat coupled. Therefore, assays of oocyte/egg progression sometimes measure several of these steps at once (Qazi, 2003).
Ovulation itself results from a series of processes occurring both sequentially and simultaneously within several ovarian microenvironments. The oocyte is released from its follicle, squeezed out of the ovary, and pushed through a lateral oviduct into the common oviduct, coming to rest in the uterus. Within each Drosophila ovariole, oocytes develop in a sequential fashion. Each ovariole contains a series of four to six egg chambers at stages in developmental progression (stages 1-14), with oogonia at the apex of the ovariole and the most mature oocytes (stage 14) at the base of the ovariole. In each egg chamber, the oocyte, connected to its sister cells (the nurse cells), is surrounded by a monolayer of somatic follicle cells. Follicle cells synthesize some of the yolk protein that will be deposited into the oocyte, as well as the proteins of the vitelline envelope and chorion that will cover and protect the oocytes. Specialized follicle cells at the anterior end of the egg chamber synthesize the micropyle, the site on the egg through which the sperm will enter. When the adult female fly ecloses, the base of the ovariole is still plugged with cells that grew out of the pedicel; this plug must be breeched to allow ovulation (Qazi, 2003 and references therein).
The mechanism by which oocytes are released from the Drosophila ovary is not known, but results in other insect systems suggest possible mechanisms. Insect ovulation involves two distinct steps: the opening of the oocyte follicle and of the intermost cells in the pedicel region, which releases the oocyte from the ovariole, and contraction of ovarioles, pedicels, and oviduct musculature, which moves the oocyte into the lateral oviducts. These contractions do not appear to involve sphincter(s), which are absent between the pedicels and the lateral oviducts in Drosophila. Ultrastructural studies of ovulation in the large aquatic beetle Dysticus marginalis (Coleoptera) show that the appearance of the first mature oocyte at the pedicel region triggers autolysis of the pedicel's innermost cells, opening the plug and releasing the mature oocyte from the ovariole. In addition, there are cells in the pedicel region that produce vacuoles whose contents might help the mature oocyte to glide into the oviduct. In the mosquito Culiseta inornata (Diptera), histological sections of whole ovaries also show that the pedicel is destroyed during ovulation. Thus, though little is known about the process of ovulation in Drosophila, it is likely that ovulation occurs, as in other insects, by cell degeneration in the pedicel region and massive muscle contractions at the base of the ovary and oviducts. These move the oocyte from the ovariole into and down the oviducts, where it eventually becomes lodged in the uterus. The posterior end of the oocyte leaves the ovary first. Thus, when the egg comes to rest in the uterus, its anterior end is 'up' with the egg's micropyle adjacent to the opening of the sperm storage organs, allowing for efficient fertilization (Qazi, 2003 and references therein).
In some insects, ovulation initiates only after mating. In Drosophila, however, ovulation occurs at a low level in adult females even without mating. Although Drosophila mature virgin females spontaneously ovulate at a very low rate (~1 egg/day), mating dramatically increases female ovulation rate (Heifetz, 2000). This effect of mating is rapid; an increase in ovulation is evident by 90 min post-mating (Heifetz, 2000). Some insects, such as walking sticks, ovulate one oocyte at a time and their ovarioles function alternately or in sequence, although in other insects, such as Orthoptera, all ovarioles ovulate simultaneously. It is not known which mechanism operates in Drosophila (Qazi, 2003 and references therein).
In Drosophila females, mating stimulates an increase in ovulation rate. Since ovulation is one step in the multistep process of egg laying, an assay was needed to distinguish this from the larger egg laying process. By measuring the progression of egg movement through the female reproductive tract, Heifetz (2000) showed that, by 90 min after mating, 90% of the females had at least one egg in their reproductive tract, and most of those were in the lateral oviduct. Ovulation rate increases with time after mating, such that by 6 h after mating, 30% of females have more than one egg in their reproductive tract. Seminal fluid components, Acps (accessory gland proteins), and sperm that are transferred to the female during mating play an important role in increasing female ovulation rate (Qazi, 2003 and references therein).
One seminal fluid protein, the prohormone-like Acp26Aa (also called 'ovulin') that shows sequence similarity to the Aplysia egg laying hormone, is essential for stimulating ovulation. The ovulation rate of females mated to males that lack Acp26Aa is 44% lower than the ovulation rate of mates of wild-type males (Heifetz, 2000). Acp26Aa's effects are evident between 1.5 and 6 h post-mating, and the protein is detectable in mated females for only a few hours post-mating. Since Acp26Aa stimulates ovulation shortly after mating, it is thought to act in 'clearing' the mature oocytes from the ovary, allowing for coordination of fresh oocyte release with sperm release and for increased oogenic progression rate (Chapman, 2001 and Heifetz, 2000). It is not yet known how Acp26Aa mediates oocyte release. Some Acp26Aa localizes at the base of the ovary (Heifetz, 2000), where it is processed into bioactive peptides. Some Acp26Aa enters the circulatory system. Thus, it is possible that Acp26Aa acts locally within the reproductive tract and/or via the neuroendocrine system to mediate contractile activity of the ovary and the oviduct musculature (Qazi, 2003 and references therein).
Another Acp, the 'sex peptide' Acp70A, also causes an increase in the number of eggs laid and, in one assay, the number of eggs in the uterus of dissected females. Since this Acp is known to increase oogenic progression, it is presently unknown whether the increased number of eggs seen in the uterus following Acp70A induction is simply a secondary indirect consequence of the increased production of oocytes, or is due to a separate direct effect on ovulation (Qazi, 2003 and references therein).
Drosophila males produce a peptide related to Acp70A in their ejaculatory ducts (Fan, 2000 and Saudan, 2002). Injection of this peptide, Dup99B, into unmated females can stimulate egg production. As with Acp70A, it is not presently known how Dup99B stimulates egg production, and therefore whether its effect on ovulation is direct or indirect (Qazi, 2003 and references therein).
In the absence of sperm transfer, oocyte development (from rapid yolk accumulation to vitelline membrane development and chorion deposition) and egg laying rates are lower than wild-type levels (50% and 33%-70% lower, respectively. Females that receive no sperm also begin to deposit eggs later than females mated to wild-type males (6 h vs 3 h; (Heifetz, 2001a). Moreover, even when sperm are transferred, if they are not stored, egg laying is low (<50% wild-type levels). Finally, when a female uses up all sperm from her mating, her egg laying rate decreases to virgin
levels. These results indicate that the transfer and storage of sperm is essential to elevate oogenesis and egg deposition rates and may also affect ovulation (Qazi, 2003 and references therein).
The presence of sperm in the reproductive tract may trigger changes in female fecundity by causing the release of female-derived activating substances or by releasing male-derived substances that have adhered to the sperm. Alternatively, the presence of many sperm could stimulate the female central nervous system via stretch receptors in the uterus or sperm storage organs (Qazi, 2003 and references therein).
Contraction of insect oviducts is under neural control. The Drosophila female reproductive tract is innervated by branches of the abdominal nerve center (AbTNv). Thus, it seems likely that neurotransmitters and/or neurohormones will regulate the contractile activity of the Drosophila ovary and oviduct musculature to cause the release of mature oocytes. Since the 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 affect the secretory activity of the epithelium, and thereby trigger the disintegration of the pedicel plug to release mature oocytes (Qazi, 2003 and references therein).
Although the identity of neurotransmitters, neuromodulators, or neurohormones that could mediate ovulation in Drosophila is unknown, findings in other insects point to likely candidates. In locusts (Locusta migratoria), for example, glutamate, octopamine, proctolin, and SchistoFLRFamide have been shown to mediate oviduct muscle contraction, aiding the movement of eggs. In other insects, such as the bed bug Rhodnius prolixus, the neurosecretory peptide myotropin mediates the ovarian contractile activity that releases mature oocytes. Myotropin is released from the corpus cardiacum in response to ecdysteroid levels in the female hemolymph (Qazi, 2003 and references therein).
In Drosophila, ovarian function and ovarian environment affect egg chamber development and therefore ovulation rate. Female age, available nutritional resources (e.g., yeast), and other environmental factors, such as circadian cues and humidity, affect egg production. For example, nutritional supplementation with yeast causes increased germ cell proliferation and decreased cell death of both early-stage and vitellogenic-stage oocytes. This results in increased egg production and egg deposition rates thereby (directly or indirectly) increasing ovulation rates. Although the mechanism of environmental interaction with ovulation physiology is still to be determined in Drosophila, again clues are available from other insects. For example, in the beetle Xyleborus ferrugineus, ecdysteroid titers are significantly higher in young fertile adult females than in aging females, suggesting that fewer eggs are produced, and hence ovulated in older females, due to declining ecdysteroid levels (Qazi, 2003 and references therein).
In Drosophila, several control points and feedback mechanisms regulate the production of mature oocytes, which affects female ovulation status. One such feedback prevents oocytes from entering the oviduct before completing oogenesis. Thus, a female will ovulate only if she has mature oocytes in her ovaries. Another control point in egg maturation is regulated by Acp70A. Acp70A stimulates oocytes to progress to later vitellogenic stages and thus override a control point in egg maturation between mid/late vitellogenic stages (stages 9/10). Thus, Acp70A can stimulate oogenic progression rate, allowing the female to ovulate at a high rate (Qazi, 2003 and references therein).
Sperm storage allows sperm from a given mating to be used long after the male and female have separated (~2 weeks), thus increasing female fertility. Of the ~4000 sperm transferred to a Drosophila female during mating between wild-type flies, ~80% are expelled from the uterus when the first egg is laid, and subsequent fertilizations rely on the 700-1000 sperm stored in the female. Females that receive normal quantities of sperm, but store few of them, produce few progeny (~10% wild-type levels). Compared with mammals and birds, whose efficiency of sperm use can be as low as 0.01% (reviewed in Neubaum and Wolfner, 1999b), Drosophila use sperm quite efficiently: of the 700-1000 sperm stored, ~400 are used to fertilize eggs (Qazi, 2003 and references therein).
Sperm storage potentially allows coordination of ovulation rate with sperm release from storage. This prevents gamete wastage, which would occur when gametes are released at different times and unable to unite successfully. The coordination of sperm release and ovulation could also decrease the incidence of polyspermy that also wastes eggs and sperm because it results in nonviable fertilizations. The controlled release of sperm from storage in Drosophila may be one reason why polyspermy is rare in this organism (less-than or equal to 1% (Qazi, 2003 and references therein).
Sperm storage allows females to retain sperm for more than one male within her reproductive tract. This provides 'fertility insurance,' should any of the males be infertile or genetically incompatible with the female. Storing sperm from multiple males has the potential additional advantage for females of generating progeny with a broader range of different genotypes. Finally, storing sperm from more than one male generates an opportunity for sperm competition and female sperm preference (Qazi, 2003 and references therein).
After mating, Drosophila females become unreceptive to courting males for several days (mating effect). Male seminal products such as Acp70A trigger the female's immediate reluctance to remate, but their action is short-term (<24 h). For the normal
(approximately 1-1.5 week) depression of receptivity, females must also store sperm (sperm effect). The sperm effect on female receptivity may begin as early as 6-8 h postmating; it is clearly operating by 10-18 h after mating. Females that store few sperm are receptive to remating sooner than those storing larger number of sperm. The return of female receptivity correlates both with the decline in numbers of stored sperm as those sperm are released and used for fertilization and, in the laboratory, with longer periods of male-female pair confinement, continued availability of food, and population density (Qazi, 2003 and references therein).
Potential mechanisms for the sperm effect (depression of female receptivity) include: pressure from within the sperm storage organs exerted by sperm, movements of sperm within the storage organs, and/or distension of the uterine walls by sperm and semen. In the white cabbage butterfly Pieris rapae, receipt of sperm and seminal fluid products results in changes in female receptivity to mating. Distension of a female P. rapae uterus with saline causes a decrease in receptivity similar to that after mating. A similar mechanism in the Drosophila sperm storage organs could explain the Drosophila sperm effect. Since the sperm effect is established several hours after sperm storage is complete (10-18 h vs 1.5 h, respectively), the effect of sperm on female behaviors is proposed to involve an intermediary acting on the female central nervous system (CNS). This intermediary could be a molecule(s) secreted from the female's cells. For example, in the redbanded leafroller moth, Argyrotaenia velutinana, mating depresses female pheromone titer via a CNS-mediated release of the peptide PBAN (pheromone biosynthesis activating neuropeptide). When the female CNS is disrupted by cutting the ventral nerve cord, PBAN remains within female tissues, and is not released into the hemolymph, where it normally acts. Alternatively, the intermediary could be a molecule from the male's seminal fluid. Although most seminal proteins are not detectable in the female more than a few hours after mating, others may be stabilized by associating with sperm, allowing them to act within the female long after mating has ended (Qazi, 2003 and references therein).
In several organisms, matings are costly to females. In Drosophila, this cost arises from a combination of exposure to males, receipt of Acps, and allocation of resources to the increased egg production postmatin. Sperm themselves do not directly contribute to the cost of mating in Drosophila. However, by storing sperm, females reduce costs that accrue from repeated matings since they do not need to mate as many times to maintain fertility (Qazi, 2003 and references therein).
Despite the costs associated with multiple mating and the general reluctance of females to remate after mating, female promiscuity is well documented in Drosophila. When a female mates with more than one male, female sperm storage provides both a venue for sperm from different males to compete for access to fertilizations (sperm competition) as well as an arena for the female to select sperm from among the contributions of competing males (sperm preference or cryptic female choice). These processes may be important mechanisms preventing genetic incompatibility (the production of nonviable or infertile progeny) by selecting among male ejaculates. This is observed in Drosophila: when a D. simulans female mates with both a D. simulans and a D. mauritiana male, D. simulans sperm are preferentially stored and used for fertilization. The mechanisms of this conspecific male advantage include both the inactivation and physical displacement of D. mauritiana stored sperm (Qazi, 2003 and references therein).
Sperm competition and sperm preference have potentially important evolutionary consequences. When a female Drosophila mates with more than one male, the most recent partner usually sires more than 80% of the subsequent progeny ('last male precedence'). However, large variation exists in the fertilization success among last-mating males due to genetic differences among both males and females. Examining sperm storage sheds light on the mechanisms of sperm precedence (Qazi, 2003 and references therein).
Female sperm storage in Drosophila occurs in two types of specialized organs, located at the anterior end of the uterus. The single seminal receptacle is on the ventral side, and the paired spermathecae are on the dorsal side of the uterus. The two types of storage organs differ in morphology and in patterns of sperm storage (Qazi, 2003 and references therein).
Numerically, the seminal receptacle plays the biggest role in sperm storage, retaining 65%-80% of the stored sperm. Observations and counts of stored sperm over time indicate that sperm are released almost exclusively from the seminal receptacle for the first several days after mating. Then, as the seminal receptacle sperm become depleted, sperm are released from the spermathecae. The seminal receptacle's importance in sperm storage is supported by a phylogenetic analysis of sperm storage organ use among 113 species of Drosophila. Loss of the sperm storage function by the seminal receptacle is a rare evolutionary event that appears to have occurred only once; it characterizes only 3 (2.6%) of the species examined. In contrast, the spermathecae's sperm-storage function may have been lost as many as 13 different times affecting 38 (33.6%) of the examined species (Qazi, 2003 and references therein).
The seminal receptacle is a thin (5-20 microm) blind-ended tube that is coiled against the outer uterine wall. In Drosophila, the seminal receptacle is more than 2 mm long, slightly longer than a sperm (1.75-1.90 mm). A positive relationship also exists between sperm and seminal receptacle length among 44 other Drosophila species examined. This observation and other detailed evolutionary studies suggest that increases in seminal receptacle length drive the evolution of longer sperm (Pitnick, 1999) (Qazi, 2003 and references therein).
The lumen of the seminal receptacle is lined with a thin cuticle and is surrounded by a layer of nonsecretory cells, a basement membrane, and a helically coiled layer of muscle. There does not appear to be a sphincter separating the seminal receptacle from the uterus. At any time, only a few sperm are observed in the proximal half of the tube (that is, near its entry into the uterus). The majority of stored sperm appear partially extended and lying parallel to each other within the distal half of the seminal receptacle. This mass of sperm is curvilinear, and their tails can be seen to move, except when the seminal receptacle is very full of sperm (Qazi, 2003 and references therein).
No genes responsible for seminal receptacle length or morphology have been identified. However, results of quantitative genetic analysis of selection experiments for increased or decreased seminal receptacle length suggest that only a small number (2-5) of loci determine seminal receptacle length. Since their effect is largely additive, the loci appear to be independent (Qazi, 2003 and references therein).
Fewer sperm reside within the paired spermathecae than in the seminal receptacle (maximum of 135-449 sperm within both spermathecae). However, the paired spermathecae appear to have two important roles in sperm storage. (1) Spermathecae are the long-term storage organs. Sperm may accumulate more slowly in the spermathecae than in the seminal receptacle. As mentioned before, spermathecal sperm are apparently used for fertilization after the seminal receptacle's sperm have been depleted. (2) Spermathecae may secrete substances that maintain sperm viability within both the seminal receptacle and the spermathecae. Posterior to the spermathecae, but also opening into the dorsal side of the uterus, are two spermathecal glands whose secretions could also affect sperm storage (Qazi, 2003 and references therein).
Each spermatheca joins the uterus via a stalk composed of a thin lumen surrounded by a thick cuticular intima, epithelial cells, and a helically coiled layer of muscle cells. Like the seminal receptacle, the spermathecae do not appear to have a sphincter near their base. The spermatheca itself is an oval-shaped capsule formed from a cuticular intima. Thin ducts within the spermathecal intima open to secretory cells surrounding the exterior of the capsule. These cells produce a lamellar type of secretion that accumulates within the spermathecal lumen. Sperm move from the uterus into the spermathecal capsule through the stalk. Within the capsule, they wind around forming a toroidal mass (Qazi, 2003 and references therein).
Two genes have been identified that affect spemathecal development. Several alleles of lozenge (lz), which encodes a putative transcription factor, cause loss of the spermathecal glands and vary in the extent to which they affect spermathecal development. Some lz females have intact spermathecae, while others are missing capsules and have malformed or missing spermathecal stalks. These alleles also cause low fertility, apparently due to lower sperm storage in the seminal receptacle and the loss of motility of any stored sperm within a few days after mating. Analysis of lz mutants suggests that they impair dorsal cell migration in the early genital imaginal disc that is needed for subsequent spermathecal development. dachshund (dac), a nuclear protein, is important for appropriate formation of the spermathecal stalks. When dac expression is blocked during development, the spermathecal capsules appear normal, but they share a single stalk. In addition to these genes that affect spermathecal morphology, some genes control spermathecal number. At least two genes are responsible for the formation of extra (>2) spermathecae, but neither has yet been identified (Qazi, 2003 and references therein).
Why are there two types of sperm storage organs? Although the use of two types of storage organs is common (63.8% of 113 species examined), among Drosophila species, it is not clear why the spermathecae and seminal receptacle are both required for sperm storage. It has been suggested that the seminal receptacle might have evolved as a new organ, which is more efficient at storing sperm than are the spermathecae. The apparent coevolution of seminal receptacle length and sperm length among several Drosophila species suggests that the seminal receptacle may also provide more opportunity for female or male influence over sperm storage and sperm fate. Sperm displacement, as a result of multiple mating, appears to be primarily associated with the seminal receptacle. Residence in the spermathecae might protect sperm from displacement, and the secretions of the spermathecae might also be essential for viability (Qazi, 2003 and references therein).
When do sperm enter and exit storage? Sperm storage begins before the ~20-min copulation is complete. Sperm accumulate rapidly within the two types of storage organs, leveling off at ~700-1000 sperm less than 6 h after mating ends. The first egg laid after mating (~90 min) pushes out remaining unstored sperm. By 10 h after mating, seminal receptacle sperm numbers have noticeably declined due to the female's use of stored sperm to fertilize her eggs. On average, the number of sperm in the seminal receptacle declines at ~100-170 per day. By 48 h after mating, sperm storage in the seminal receptacle has declined by ~50%, while sperm stored in the spermathecae have decreased by only ~15% (Qazi, 2003 and references therein).
How does sperm storage occur? Sperm storage can be viewed as a series of steps: progression of sperm through the female reproductive tract after mating, entry of sperm into the storage organs, retention and maintenance of stored sperm, and release of sperm from storage up to the point of fertilization. Both female and male Drosophila play active roles in sperm storage, as shown by the nontransitivity of numbers of stored sperm 1 h after mating between different fly strains. Female-based mechanisms can include absorption of fluids from parts of her reproductive tract (called 'hydraulics' here), contractions of the uterus, contractions of the sperm storage organs, restriction of sperm to particular regions of the female reproductive tract, and/or factors secreted from within the female reproductive tract. Male-based mechanisms are also important and can continue even after copulation is complete. These include sperm motility and seminal proteins. Sperm are motile when they are transferred to the female and when they are in storage. Although it has been hypothesized that sperm motility is important for the release of sperm from storage, and it seems a likely contributor to that process, the role of sperm motility in sperm entry into storage in Drosophila is unknown. In contrast, studies have shown a profound effect on female sperm storage of secretions from the male's ejaculatory duct and accessory glands transferred to the female during mating. Matings between normal females and males transferring sperm, but no Acps, are infertile, suggesting that Acps play an essential role in sperm use once sperm are transferred to the female. When these females mate first with males transferring Acps, but no sperm, then mate with males transferring sperm, but no Acps, the second male's fertility is restored. Females mated to males that transferred sperm but greatly reduced (~1%) quantities of Acps store fewer than 10% as many sperm as do females mating to wild-type males (Qazi, 2003 and references therein).
What gets sperm into storage? Sperm may be drawn into storage by pressure changes within the female reproductive tract. In the Dipteran Culicoides melleus, and possibly in other lower Diptera, one component of sperm storage apparently involves fluid absorption from the spermathecae, which create a force that sucks sperm into storage. Although a similar mechanism could potentially function in Drosophila's seminal receptacle, ultrastructural studies argue against this hypothesis for sperm entry into the Drosophila spermathecae because substances from the surrounding cells accumulate within the spermathecae at the same time that sperm storage is occurring (Qazi, 2003 and references therein).
In many animals, female muscular contractions apparently push sperm from the uterus into storage. Consistent with this occurring in Drosophila, a Drosophila female CNS is required for sperm storage. This has been tested by manipulating expression of transformer (tra), whose product is required for normal female development. Various levels of ectopic expression of tra in either tra-deficient mutant XX flies or XY flies results in individuals possessing female genital morphology (phenotypic females), but either a masculinized CNS (evinced by male courtship behavior) or a (presumably) feminized CNS. Animals with the presumably female CNS store nearly 6.5 times more sperm within all storage organs (~550 sperm) but allocate proportionally fewer of the sperm to their seminal receptacles than do phenotypic females with a masculinized CNS. These results show that a feminized nervous system is necessary for sperm storage and that females actively distribute sperm among the storage organs. Results of experiments in which males mate with isolated female abdomens (that lack ganglia and therefore a CNS) support this hypothesis. It is possible that a female CNS is needed to trigger uterine muscle contractions that push sperm into storage. Alternatively, a female CNS could influence sperm storage by stimulating endocrine cells to release substances that attract sperm to the storage organs. Finally, the generous innervation of the seminal receptacle suggests that local contractions of the sperm storage organs might be important for sucking sperm into storage and/or for efficient arrangement of sperm within storage (Qazi, 2003 and references therein).
Sperm may be lured into storage by substances produced by the female or by male-derived substances activated once in the female. In sea urchins and other marine invertebrates, sperm-activating peptides (SAPs) secreted from the egg jelly stimulate sperm movements and orientation to eggs as well as activate other sperm-egg interactions (Suzuki 1995). Compounds within the female Drosophila reproductive tract could potentially act in a similar way, but none have been positively identified thus far (Qazi, 2003 and references therein).
Concentrating sperm to specific regions of the reproductive tract can increase the likelihood that they will encounter the openings to the storage organs. (1) Female anatomy, such as folds in the uterine wall, could channel sperm into the seminal receptacle. (2) In Drosophila females, a barrier of unknown composition exists at the base of the oviduct that keeps sperm in the uterus. At least one male seminal protein, the accessory gland protein Acp36DE, localizes at this barrier, but no chemical components required for barrier formation have been identified. In egg-less females, the barrier is mislocalized or does not form and sperm are found within the common and lateral oviducts. The presence of oocytes in the ovaries may help form the block by creating a back-pressure and/or causing the secretion of a substance that localizes near the base of the oviduct preventing sperm from premature access to oocytes within the ovary (Qazi, 2003 and references therein).
From within her reproductive tract, a female Drosophila secretes factors important for sperm storage. Glucose dehydrogenase (Gld), an enzyme-producing reactive oxygen species, is secreted from the spermathecal stalks and the genital plates (located near the gonopore). gld-mutant females store fewer sperm within, and allocate sperm more unevenly between, the two spermathecae, but only when sperm storage is submaximal (<500 total sperm stored). Therefore, Gld may facilitate sperm storage, particularly when sperm are not plentiful, but is not essential for sperm storage to occur. The mechanism of Gld's sperm storage effects is unknown, but Gld is unlikely to serve as a chemoattractant since it is produced in more than one location within the female reproductive tract (Qazi, 2003 and references therein).
Corralling can also involve male contributions. The male accessory glands contain filaments composed of globular subunits. Similar looking filaments are observed in female storage organs interdigitated with stored sperm. If the filaments of similar appearance are indeed the same, those filaments might provide a scaffold along which sperm move or within which sperm are confined, to facilitate the efficient movement of sperm into storage. In an analogous way, the mating plug, contributed at least in part by the male, is thought to facilitate sperm storage by forming a physical barrier that prevents the loss of sperm from the uterus; sperm are confined above the plug, concentrating them near the entrances of the storage organs. The mating plug has also been proposed to provide a trellis to facilitate sperm movement toward storage. The mating plug contains several male seminal proteins, including PEB-me (its major component, derived from the ejaculatory bulb) and Acps, including the protein Acp36DE. Entering sperm traverse the mating plug and are limited to the anterior portion of the uterus. The mating plug is not detected more than 6 h after mating and, while its fate is unknown, it seems likely to be expelled when the first egg is laid or dissolved and reabsorbed (Qazi, 2003 and references therein).
By examining sperm storage in the presence of normal or very low amounts of Acps, it has been shown that Acps are required for sperm storage. One of these proteins, Acp36DE, is essential for proper sperm storage. Females mated to Acp36DE-deficient mutant males receive normal quantities of sperm during mating, but store far fewer sperm than females mated to wild-type males. Without Acp36DE, sperm start to enter storage at the normal time, but the subsequent accumulation of sperm into the seminal receptacle and spermathecae is less efficient. The rate at which sperm are released from spermathecal storage differs slightly in the presence or absence of Acp36DE. Within the first 24 h after mating, the rate at which sperm are released from the seminal receptacle is similar in the presence or absence of Acp36DE, but between 24-48 h after mating, proportionally more sperm are lost from the seminal receptacles of females receiving Acp36DE than females not receiving Acp36DE from their mates. It is not clear whether this phenomenon is a direct result of Acp36DE action or a secondary consequence of storing fewer sperm. The number of progeny produced in the absence of Acp36DE corresponds to the number of sperm in storage, indicating that those sperm that are stored without Acp36DE are fully viable. Thus, Acp36DE's primary role is in facilitating sperm storage. It may potentially have a secondary role in promoting sperm retention in the seminal receptacle, but there is no evidence that Acp36DE affects sperm viability (Qazi, 2003 and references therein).
Acp36DE is a novel 122-kDa glycoprotein that is transferred to females beginning within the first 5 min of mating. Within the female, it is processed to a 68-kDa product. Acp36DE is detectable within the female reproductive tract for as long as 3 h after mating. It localizes to the oviduct wall anterior to the sperm storage organ openings (at the barrier discussed above) and on the anterior end of the mating plug. Thus, it is present at the upper and lower areas of the 'corral' described above. In addition, Acp36DE enters the sperm storage organs. Finally, Acp36DE binds to sperm in vivo and in vitro. When the first egg is laid, the Acp36DE in the oviduct and mating plug are expelled. Although no longer detectable within the female, Acp36DE donated from one male facilitates the storage of a second male's sperm 24-48 h later (Qazi, 2003 and references therein).
The available data on Acp36DE support several, not mutually exclusive, models for its action. Acp36DE may interact with targets on female tissues stimulating females to push (from the uterus) or suck (from the sperm storage organs) sperm into storage via muscular contractions. It could potentially also stimulate the release of chemoattractant molecules. Alternatively, Acp36DE may limit sperm movements via its associations with the oviduct, mating plug, and sperm, thereby facilitating the rapid accumulation of sperm within storage. Finally, Acp36DE might help track sperm into storage by forming a scaffold or providing guidance cues along which the sperm move, or are moved, into storage (Qazi, 2003 and references therein).
What keeps sperm viable in storage? Once Drosophila sperm are in storage, they need to remain viable for up to 2 weeks. For example, even in cases of matings between genetically incompatible strains of Drosophila, sperm in the storage organs remain viable several days after mating (suggested by vital cell staining). Both female and male factors probably contribute to the maintainance of sperm in storage. Substances produced within the spermathecae may play roles in sperm viability and retention. The presence of the spermathecal capsule correlates with female fertility. The low and variable fertility of some female lz mutants suggests a similar model for Drosophila, since the lz mutant phenotype is proposed to be due to the presence/absence of the spermathecal capsule. Females with at least 1 spermathecal capsule produced an average of 216 progeny, 36 times as many progeny as mutant females lacking spermathecal capsules. Lower fertility is attributable to a shorter duration of progeny production among females lacking capsules compared with their normal sisters. Although the seminal receptacle appears normal, lz mutants store fewer sperm which lose motility earlier (less-than or equal to 5 days after mating) than do sperm in wild-type females (>11 days). These results suggest that presence of the secretory cells surrounding the spermathecal capsule and/or the spermathecal glands is important for the viability of stored sperm and for female fertility (Qazi, 2003 and references therein).
Stored sperm need to be protected from degradation. During storage, proteolysis of sperm surface proteins could destroy a sperm's ability to bind to eggs; alternatively, regulated proteolysis of the surface of stored sperm could be essential to activate or capacitate them. Indeed, in mice, mutations in seminal fluid protease inhibitors impair fertility, consistent with the hypothesis that protease inhibitors serve to protect sperm. Drosophila seminal fluid also contains regulators of proteolysis. Of ~83 predicted secreted male accessory gland proteins, 9 are predicted (or demonstrated) regulators of proteolysis. One, the trypsin inhibitor Acp62F, has been shown to enter the mated female's sperm storage organs, consistent with its playing a role in protecting sperm from degradation. Stored sperm are also potentially subject to untoward effects from microbes that might have entered the female's genital tract during mating. Perhaps to guard against this, sperm storage organs and seminal fluids contain antimicrobial peptides. The seminal receptacle and spermathecae also both secrete Drosomycin, a peptide with antifungal properties (Qazi, 2003 and references therein).
What releases sperm from storage? Sperm leave storage to two potential fates: one fate is to fertilize an ovulated egg, another is to leave storage but not to fertilize an egg either due to inefficient sperm use or to sperm displacement as a result of the female mating with another male (female sperm preference or sperm competition). Since sperm use in Drosophila is efficient, one (or very few) sperm leave(s) storage to fertilize an ovulated egg as the egg comes to rest in the uterus. The egg lodges there with its anterior end, containing its micropyle, close to the sperm storage organ entrances. It is not known whether sperm swim, are pushed, or are sucked through the micropyle. The entire sperm enters the egg. Its tail coils in the anterior end and persists within the embryo until shortly after hatching (Qazi, 2003 and references therein).
In Drosophila, conformational changes of the reproductive tract induced by ovulation might also effect sperm release. Single sperm have been observed near the opening of the seminal receptacle as an egg passes down the oviduct; it has been proposed that ovulation and sperm release are correlated. Muscular contractions of just the seminal receptacle could squeeze small numbers of sperm out of storage (Qazi, 2003 and references therein).
Motile sperm might motor their way out of storage. Sperm circulating within the storage organs have been observed and it has been speculated that, occasionally, a single sperm leaving storage would encounter an egg. This observation, coupled with the lack of detected sphincters at the base of the sperm storage organs, has led to the proposition that sperm motility aids their release (Qazi, 2003 and references therein).
At least one male-derived protein is suggested to play a role in sperm residence in storage in females: the carboxylesterase, Esterase-6 (Est-6). Est-6 is secreted from the male ejaculatory bulb and anterior ejaculatory duct, and is transferred to the female early during mating. Activity of male-derived Est-6 is detected for only 2 h after mating. Although the initial timing and storage of sperm into females that do or do not receive Est-6 from their mates is similar over time, more sperm are retained in females that do not receive Est-6. Est-6 therefore appears to play a role in the release of sperm from storage in the seminal receptacle; its role on spermathecal sperm is unclear. Est-6 might cause the release of sperm from the seminal receptacle by affecting sperm motility within the sperm storage organs or by catalyzing the production of molecules needed to sustain sperm motility. However, since Est-6 activity is also positively correlated with the rate of female oviposition as well as female latency to remating, and since Est-6 enters the female hemolymph, it may have more than one target or multiple interrelated effects (Qazi, 2003 and references therein).
If a female mates twice, the second male's ejaculate causes the release of previously stored sperm (last male sperm precedence). This effect is attributable to the removal of some sperm from storage and the inactivation of remaining sperm. The method of displacement depends on the time between matings. If a female remates within 2 days of an initial mating, the last-mating male's sperm plays a role in physical displacement of sperm, primarily from the seminal receptacle. With longer intervals between rematings, other seminal components, particularly Acps from the most recently mating male, functionally displace sperm by decreasing the use of previously stored sperm that remain after the second mating. If seminal proteins temporarily protected the male's sperm from displacement, then perhaps it is the inactivation of these 'protein companions' to sperm that leave the sperm vulnerable to displacement by future mating males. Males lacking Acp36DE are poor displacers of other males' sperm and often have their own sperm nearly completely displaced from storage (Chapman, 2000), but this is believed to be because they are poor at getting their own sperm moved into storage (Qazi, 2003 and references therein).
Cell migration within a natural context is tightly controlled, often by specific transcription factors. However, the switch from stationary to migratory behavior is poorly understood. Border cells perform a spatially and temporally controlled invasive migration during Drosophila oogenesis. Slbo, a C/EBP family transcriptional activator, is required for them to become migratory. Wild-type and slbo mutant border cells as well as nonmigratory follicle cells were purified and comparative whole-genome expression profiling was performed, followed by functional tests of the contributions of identified targets to migration. About 300 genes were significantly upregulated in border cells, many dependent on Slbo. Among these, the microtubule regulator Stathmin was strongly upregulated and was required for normal migration. Actin cytoskeleton regulators were also induced, including, surprisingly, a large cluster of 'muscle-specific' genes. It is concluded that Slbo induces multiple cytoskeletal effectors, and that each contributes to the behavioral changes in border cells (Borghese, 2006).
Only one of the identified cytoskeletal regulators is known to affect microtubules, namely, Stathmin. Mammalian Stathmin/Op18 protein is well characterized. It binds to microtubules and promotes depolymerization by sequestration of tubulin dimers or direct action at microtubule ends. Interestingly, the activity of Stathmin can be regulated by phosphorylation in response to signaling or cell cycle phases. Drosophila Stathmin appears to have similar biochemical features. The availability of an antibody directed against Drosophila Stathmin allowed analysis of protein levels in situ. As expected, the level of Stathmin was higher in border cells than follicle cells. When analyzing slbo mutant border cells, a clear difference was observed between the inner polar cells and the outer border cells. The outer border cells are the migratory cells and require Slbo expression. In these cells, Stathmin expression was undetectable in the absence of Slbo, indicating a very strong dependence on Slbo. In contrast, Stathmin was still expressed in mutant polar cells, explaining why only a moderate reduction of stathmin mRNA levels was seen in whole border cell clusters (Borghese, 2006).
To analyze the function of Stathmin in border cells, stathmin mutants were generated. This was done by imprecise excision of a P element located immediately upstream of the stathmin C transcript. A mutant deleting only the stathmin C isoform (stathminexC), leaving stathmin A and B intact, was homozygous viable and had no effect on border cell migration. A mutant deleting the complete stathmin locus (stathminL27) and four adjacent genes (including Arc-p20, a component of the Arp2/3 complex) was homozygous lethal, and clones of stathminL27 mutant border cells were unable to migrate. Both the lethality and the migration block were rescued by reintroducing ubiquitously expressed stathmin and Arc-p20 at the same time. Reintroducing Arc-p20 alone did not rescue border cell migration, indicating that stathmin is essential for this process. To interfere with stathmin upregulation at the time of migration, a functional stathmin “hairpin”-RNAi construct was expressed in the sensitized stathminexC/stathminL27 background. By using the slbo-GAL4 driver, stathmin RNAi expression could be could specifically targeted to outer border cells right before and during migration. This strongly decreased the amount of Stathmin protein in border cells and caused significant delays in migration. The delays in migration could be reversed by driving higher levels of stathmin expression from a UAS construct. These results identify Stathmin as an important regulator downstream of Slbo. To test whether lack of Stathmin was solely responsible for the slbo phenotype, Stathmin was overexpressed in the slbo mutant background. Migration was not restored, indicating that additional genes downstream of Slbo must also be important (Borghese, 2006).
Singed is an actin-bundling protein related to Fascin, highly expressed in border cells. Fascin is important for the formation of cell protrusions and has been implicated in the control of cell migration, also in vivo. It was confirmed by clonal analysis that Singed protein levels are regulated by Slbo. Despite the strong and regulated expression, migration is normal in border cells mutant for singed. The strongest allele of singed available was used, but it retained a low level of protein expression. In addition, functional overlap may exist between actin regulators. Quail is an actin binding protein of the villin family, and its function in the germline of the ovary genetically overlaps with that of Singed. quail mRNA is also upregulated in border cells relative to follicle cells, and Quail protein is detected in border cells. Quail is structurally similar to Gelsolin, which was also upregulated in border cells, as well as the Gelsolin-related FliI, which was not detectably expressed. However, Gelsolin is enriched in polar cells rather than the migratory outer border cells. As for singed, no migration defects were observed in quail mutant border cells, nor in cells mutant for quail and only one functional copy of singed or vice versa. It was not possible to recover clones of border cells simultaneously mutant for both singed and quail, which is likely to reflect a functional overlap between the two genes at an earlier stage. The simultaneous upregulation of redundant actin regulators may reflect a genetically robust approach to changing the actin cytoskeleton in border cells (Borghese, 2006).
A rather surprising finding of this global expression analysis was that the remaining genes encoding cytoskeleton-associated proteins and upregulated in border cells in a slbo-dependent manner were all “muscle specific”. This included a complete palate of structural genes: muscle actin (57B), muscle myosin heavy chain and light chains, tropomyosin 2 (tm2), troponins, and the calponin-related mp20. The muscle-specific expression has been shown for this group of genes in Drosophila embryos as well as mature muscles. For tropomyosin 2, a GFP gene trap allele was available and, and this allele confirmed expression in border cells as well as in the muscle sheath. The expression profiling indicated that border cells also express the corresponding non-muscle forms such as actin42A, zipper (myosin heavy chain), and sqh (myosin light chain), but at the same level as in follicle cells. The nonmuscle proteins are generally required for many cellular processes, including, where tested, migration of border cells. This raised the question of why this large cluster of muscle-specific structural genes would be turned on in border cells as well. To address this, migration was analyzed of border cells mutant for individual muscle genes for which mutants were available (mhc, mlc2, upheld=troponinT and tm2). Since mhc and mlc2 are essential genes, this was done by clonal analysis. No defects were seen in border cells mutant for mlc2, upheld, or tm2, but clear migration defects were observed in border cells mutant for mhc (mhc1 or mhc3). Thus, while not all of the muscle structural genes are required for border cell migration, at least muscle Mhc expression contributes to effective migration (Borghese, 2006).
Given that both muscle and nonmuscle forms of the same cytoskeletal proteins have a role in border cell migration, their functions are likely to be different. In agreement with this, no genetic interactions were observed between mutants affecting muscle and nonmuscle forms of myosin heavy or light chains. There is precedence for such nonoverlapping functions. For example, Zipper has a unique role in developing muscle cells, which contain plenty of muscle myosin heavy chain. In mammalian cells, different myosin heavy chain isoforms can have distinct subcellular localization. Also, the actin proteins, despite having few amino acid differences, are functionally distinct in vivo (Borghese, 2006).
The muscle gene expression program activated in migratory border cells extended beyond structural genes to regulatory genes. One such gene was bent, encoding a very large titin-like molecule with a myosin light chain kinase domain. Being essential but on the fourth chromosome, bent was not amenable to standard clonal analysis. Genes required for myoblast fusion were also identified, namely, rols/antisocial and rost. mbc, which encodes a DOCK180 family Rac GEF and is required for myoblast fusion, has a role in border cell migration downstream of the PVR guidance receptor. Mbc protein interacts physically with the presumed adaptor protein Rols. Clonal analysis with a strong (likely complete loss-of-function) allele of rols showed defects in border cell migration, suggesting that Mbc and Rols might act together during migration as well. The defect was milder than for mbc, implying that Mbc activity might not be completely dependent on Rols. For the small transmembrane protein Rost, no useful mutants were available. It was also noted that a very closely related and adjacent gene, CG13101, was similarly regulated in border cells and might overlap rost function. Thus, at least mbc and rols function in border cells as well as in muscle (myoblast fusion). Activation of a broad “muscle-specific” gene expression program in border cells may reflect a requirement for a specific subset of the genes within this program (Borghese, 2006).
Previous unbiased genetic approaches to identify genes important for border cell migration have largely identified transcription factors or inducing signals. Changes in cell fate can alter cell behavior dramatically without affecting cell survival, thus still allowing analysis of the mutant cells. The transcription factors themselves often show differential expression. In addition to Slbo, the posttranslationally regulated transcription factor STAT, which is important for border cell migration, was also upregulated in border cells (1.6-fold). The transcription factors that were upregulated in border cells and had mutants available for effects on border cell migration were also tested. aop/yan transcripts were increased 1.9-fold in border cells. In a PiggyBac transposon-based clonal screen for border cell migration defects, an insertion was identified in aop. Complementation analysis confirmed the gene assignment, and quantification of the phenotype showed a clear effect of aop on border cell migration. As expected, border cell migration was strongly affected, but, in addition, clones were rare and morphological abnormalities were seen in other follicle cells as well as in germline cells. Thus, aop may affect the behavior of multiple cell types in the ovary. Another transcription factor, vrille, was also upregulated (over 2-fold). vrille has been implicated in signaling, circadian rhythm, and cellular morphogenesis, but border cells mutant for vrille were largely unaffected and experienced only subtle delays (Borghese, 2006).
The most border cell-enriched RNA encoding a transcription factor, apart from Slbo, was Six4 (4.5-fold). Six4 expression in border cells was confirmed by in situ analysis. Drosophila Six4 is the homeodomain transcription factor most related to mammalian Six4 and Six5. Six family proteins act in complex with proteins of the Eya (Eyes absent) family. eya transcripts were also 2.7-fold enriched in border cells relative to follicle cells of the same stage, and Eya was expressed in a pattern similar to that of six4. Both six4 and eya were expressed in earlier-stage follicle cells as well, and eya has been shown to function at these stages to repress polar cell fate. Follicle cells mutant for six4 expressed a polar cell marker (Fas3) and were functional polar cells, as determined by the ability to induce surrounding anterior follicle cells to become Slbo-positive, migratory border cells. This suggested that Six4 cooperates with Eya in repressing polar cell fate. It had been indicated that Six proteins affect nuclear localization of their Eya partner. The six4 mutant allowed testing this in an in vivo context. Although six4 mutant cells were transformed to functional polar cells, Eya protein was not absent as in the endogenous polar cells, showing that Eya accumulation was independently regulated. However, Eya protein was partially relocalized to the cytoplasm of six4 mutant cells, supporting the hypothesis that Six4 and Eya interact in vivo. Since six4 and eya are both upregulated in outer border cells when they migrate, they are likely to act together in this process as well. However, their earlier roles precludes straightforward loss-of-function analyses in border cells, since “border cell clusters” consisting only of six4 or eya mutant cells are not functional simply because polar cells do not migrate on their own. Overexpression of HA-tagged six4 in border cells interfered with migration, as found for transcription factors required in border cells slbo (Borghese, 2006).
The expression of Six4 in border cells may contribute to activation of the muscle gene program described above. The conserved muscle transcription factor Mef2, an activator of muscle actin and myosin expression, was not detected in border cells by expression profiling or by antibody staining, nor were Twist and Nautilus/MyoD. Six4 is required for development of muscle and other mesodermal tissues in Drosophila. Mutants of C. elegans Unc-39, belonging to the Six4/5 family, also affect muscle/mesodermal differentiation as well as directed cell migration. Mammalian Six5, also called myotonic dystrophy-associated homeodomain protein (DMAHP), has been analyzed due to its contribution to DM1, and Six4/5 affect normal muscle development. Another transcription factor complex that might contribute to the activation of the muscle program is that of MAL-D (or MRTF) and SRF (serum response factor). The MRTF/SRF complex plays an important role in muscle development in mammals and directly regulates muscle (structural) genes. MAL-D/SRF plays a crucial role in border cell migration and this complex acts to strengthen the cytoskeleton of invasive border cells in response to perceived tension. This mode of regulation makes MAL-D/SRF activity in border cells indirectly dependent on Slbo, which could be responsible for the apparent regulation of the muscle gene cluster by Slbo. The possibility cannot be excluded that Slbo might affect muscle genes directly; the mammalian C/EBP transcription factors are known to regulate different differentiation-specific genes in different contexts (Borghese, 2006).
This study analyzed overall gene expression changes resulting from a transcriptional switch that induces invasive migratory behavior in vivo. The major goal of the analysis was to identify transcriptional changes that directly affect cell behavior and make the cells move. The results indicate that regulation of both the actin cytoskeleton and the microtubule cytoskeleton, likely coordinated regulation, is important for this transition. Identifying Stathmin as an important regulator downstream of Slbo in border cells indicates that microtubule dynamics are critical for border cell migration. Key questions are now how microtubule dynamics affect the process, and whether Stathmin activity is regulated. Two recent findings suggest that Stathmin may be a more general regulator of cell migration: Stathmin-microtubule interactions are spatially regulated in migrating cells in culture, and Stathmin upregulation may promote migration and metastasis of sarcoma cells. The actin cytoskeleton is clearly crucial for cell migration and is controlled by many regulators. The upregulated modulators identified in this study were different from those identified in a whole-genome study of tumor cells selected, in vivo, to be highly motile. There are obviously many differences between these studies; for one, a normal transition to migratory behavior may differ from unrestrained, high motility. The activation of a “muscle-specific” program in migratory border cells was unexpected and provides an intriguing connection between these cells that move and the specialized cells that move an animal (muscle). Overall, the analysis of actin regulators indicates that this is a robust system, with many effectors coregulated, even by one transcription factor. Genetically, this is reflected by minor defects in individual “effector” mutants despite absolute dependence on the transcriptional switch. Further analysis in other systems, and subsequent comparisons, will reveal to what extent the gene expression program employed by border cells to become migratory is a general one (Borghese, 2006).
Meiotic checkpoints monitor chromosome status to ensure correct homologous recombination, genomic integrity, and chromosome segregation. In Drosophila, the persistent presence of double-strand DNA breaks (DSB) activates the ATR/Mei-41 checkpoint, delays progression through meiosis, and causes defects in DNA condensation of the oocyte nucleus, the karyosome. Checkpoint activation has also been linked to decreased levels of the TGFα-like molecule Gurken, which controls normal eggshell patterning. This easy-to-score eggshell phenotype was used in a germ-line mosaic screen in Drosophila to identify new genes affecting meiotic progression, DNA condensation, and Gurken signaling. One hundred eighteen new ventralizing mutants on the second chromosome fell into 17 complementation groups. This study describes the analysis of 8 complementation groups, including Kinesin heavy chain, the SR protein kinase cuaba (CG8174), the cohesin-related gene dPds5/cohiba, and the Tudor-domain gene montecristo. These findings challenge the hypothesis that checkpoint activation upon persistent DSBs is exclusively mediated by ATR/Mei-41 kinase and instead reveal a more complex network of interactions that link DSB formation, checkpoint activation, meiotic delay, DNA condensation, and Gurken protein synthesis (Barbosa, 2007),
In this study, a clonal screen was used to identify genes regulating meiotic progression in Drosophila. Instead of testing directly for defects in meiosis, an easy-to-score eggshell phenotype was used that is produced when the levels or activity of the morphogen Grk are affected. This allowed an efficient screen of a large number of mutant lines and identification of germ-line-specific genes as well as genes with essential functions. The number of new genes identified is likely less than the total number of 2R genes required for Grk synthesis and function since mutations were discarded that blocked oogenesis. Of the eight genes described in this study, five show meiotic phenotypes. dPds5, nds, and mtc delay meiotic restriction to the oocyte, although only dPds5 and nds genetically interact with mei-W68 and mei-41, respectively. trin and blv affect the morphology of the karyosome in spite of normal timing in meiotic restriction. This confirms the effectiveness of the screening method for meiotic genes. Genetic and developmental analysis of the newly identified genes provides evidence for new regulatory steps in a network that coordinates Drosophila meiosis and oocyte development (Barbosa, 2007),
One complementation group, cohiba, identifies the Drosophila homolog of Pds5p in Schizosaccharomyces pombe, Spo76 in Sordaria macrospore, and BimD in Aspergillus nidulans, which have been found associated with the cohesion complex of mitotic and meiotic chromosomes. Depletion of Pds5 affects not only cohesion but also condensation in meiotic prophase. The unique 'open chromatin' karyosome defect observed in dPds5cohiba mutants is consistent with a role of Pds5 in chromosome cohesion during Drosophila meiosis. Like Spo76, the dPds5cohiba phenotype is suppressed by Spo11 (mei-W68) mutations defective in DSB formation. This suggests that dPds5 is necessary to maintain the structure of the meiotic chromosomes after DSBs are induced. However, in contrast to known DSB repair genes, the meiotic delay and oocyte patterning defects of dPdscohiba mutants are not due to activation of ATR/Mei-41-dependent checkpoint. One possibility is that the ATR downstream effector kinase dChk2 is activated via an alternative pathway, such as the Drosophila ataxia-telangiectasia mutated (ATM) homolog, which indeed activates dChk2 in the early embryo independently of ATR. Alternatively, dPdscohiba mutants may activate a checkpoint that measures cohesion rather than DSB breaks. The only other cohesion protein characterized in Drosophila is the product of the orientation disruptor (ord). ORD plays a role in early prophase I by maintaining synaptic chromosomes and allowing interhomolog recombination. More importantly and perhaps similar to dPds5, ORD seems not to be required for DSB repair. However, in contrast to dPds5 mutants, karyosome morphology is normal in ord mutants, and an eggshell polarity phenotype has not been reported. Although required for chromatid cohesion, dPds5 and ORD might play complementary roles in SC dynamics: ORD may stabilize the SC in the oocyte, whereas dPds5 may be required for the disassembly of synapses as one of the pro-oocytes regresses from meiosis (Barbosa, 2007),
The screen identified mutations in montecristo (mtc) that affect the restriction of meiosis to the oocyte. It has been proposed that this delay reflects the activation of the ATR/Mei-41 checkpoint pathway. Similar to dPds5, Mtc may control the regression from pachytene in those cyst cells that will not adopt the oocyte fate. The delayed meiotic restriction observed in mtc mutants occurs, however, independently of DSB formation or Mei-41 checkpoint activation. Mtc contains a Tudor domain. In other Tudor-domain proteins, this domain has been shown to interact with methylated target proteins. Identification of specific Mtc targets may clarify its role in meiotic restriction and oocyte patterning (Barbosa, 2007),
A particularly intriguing and novel phenotype is uncovered by mutations in indios (nds). By delaying meiotic restriction and activating Mei-41 without affecting the karyosome morphology, nds mutants separate checkpoint activation leading to Grk decrease from checkpoint activation controlling karyosome compaction. The nds phenotype also occurs independently of DSBs, suggesting that the trigger that leads Nds to trigger checkpoint activation is not DNA breaks. The fact that nds mutants are extremely sensitive to Mei-41 dosage further suggests that Nds activity may specifically control a branch of the Mei-41 checkpoint regulating Grk activity. In contrast to nds, trin mutants do not delay meiotic restriction and show defects in the karyosome in spite of normal Grk levels. Like mutants in src64B and tec29, which show a similar phenotype, Trin may mediate chromatin remodeling in the oocyte by regulating the actin cytoskeleton. In this context, the DV phenotype of eggs from trin mutants may be an indirect effect due to defects in actin cytoskeleton function. The production of collapsed eggs by trin mutant germ-line clones is consistent with this idea (Barbosa, 2007),
Finally, blv mutants show striking similarity to vas mutants with respect to lack of sensitivity to DSB formation, no evident delays of meiotic restriction, or karyosome and Grk phenotypes. Blv may thus act downstream or independent of the Mei41/ATR checkpoint, and its further characterization may help to understand the effector side of the meiotic checkpoint pathway (Barbosa, 2007),
Previous knowledge pointed to Drosophila meiosis as a linear progression of events from homologous chromosome pairing and recombination to meiotic restriction, karyosome formation, and eggshell patterning, with DSB repair as the main checkpoint linking meiosis to Grk signaling. By uncoupling some of these events, this study suggests the existence of a more complex network that links the surveillance of meiotic progression to oocyte patterning (Barbosa, 2007),
Developments between gametogenesis and fertilization: Ovulation and female sperm storage
Systematic analysis of the transcriptional switch inducing migration of border cells
A maternal screen for genes regulating Drosophila oocyte polarity uncovers new steps in meiotic progression
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date revised: 22 November 2006
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