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

Drosophila melanogaster mature oocytes in ovaries are arrested at metaphase I of meiosis. Eggs that have reached the uterus have released this arrest. It was not known where in the female reproductive tract egg activation occurs and what triggers it. We investigated when and where the egg is activated in Drosophila in vivo and at what meiotic stage the egg is fertilized. We found that changes in the egg's envelope's permeability, one feature of activation, initiate during ovulation, even while most of the egg is still within the ovary. The egg becomes impermeable as it proceeds down the oviducts; the process is complete by the time the egg is in the uterus. Cross-linking of vitelline membrane protein sV23 also increases progressively as the egg moves through the oviducts and the uterus. Activation also triggers meiosis to resume before the egg reaches the uterus, such that the earliest eggs that reach the uterus are in anaphase I. We discuss models for Drosophila egg activation in vivo.

TheDrosophila melanogasterYA protein is a maternally provided nuclear lamina component that is essential during the transition from meiosis to mitosis at the beginning of embryogenesis. Localization of YA to the nuclear envelope is required for its function; this localization is cell cycle-dependent during embryogenesis. Here we show that the ability of YA to enter nuclei is modulated during development. In developing egg chambers, YA protein is made but excluded from nuclei of nurse cells and oocytes; upon egg activation, YA acquires the ability to enter nuclei and becomes incorporated into the nuclear lamina in unfertilized eggs and embryos. This localization switch correlates with changes in the phosphorylation state of YA. YA in ovaries is hyperphosphorylated relative to YA in unfertilized eggs and embryos. Through site-directed mutagenesis, we identified443T, a potential phosphorylation site for both cyclin-dependent protein kinase and mitogen-activated-protein kinase, as one of the sites likely involved in this developmental control. Our results suggest that phosphorylation plays a role in modulating the localization of YA during development. A model for developmental regulation of the nuclear entry of YA is proposed and implications for understandingDrosophilaegg activation are discussed.

The male accessory gland ofDrosophila is an adult secretory tissue which contributes many products to the male ejaculatory fluid. The secretions of the accessory gland affect the behavior and physiology of the female fly after mating, reducing her receptivity to courtship and stimulating egg production and oviposition. We have examined the developmental and mating-stimulated expression of two accessory gland proteins in the male and their transfer to and fates in the mated female. One of these proteins, msP 355a, has features of a prohormone and contains a region with amino acid sequence similarity to the egg-laying hormone of Aplysia; the other, msP 355b, is a small acidic protein. Both proteins are first detected in the accessory gland only after eclosion, although their transcripts are already present in late pupae. Both proteins are initially detected in the two morphologically distinct secretory cell types of the accessory gland, the main cells, and the secondary cells. In the glands of aged virgin males, they are only detected in the large vesicles of the secondary cells and in the lumen of the gland. Copulation results in an increase in the mRNAs for both proteins, as well as renewed translation of the proteins at least in the main cells. Both proteins are transferred to the female genital tract during copulation, and rapidly enter the female hemolymph. msP 355a is subject to rapid and specific cleavage within the female genital tract, but not in the hemolymph; msP 355b is not cleaved in either the female genital tract or the hemolymph.

Upon fertilization, a sperm nucleus reorganizes to become a male pronucleus. This reorganization includes breakdown and reformation of the nuclear envelope of the male pronucleus. In this study, we used a maternally encoded nuclear lamina protein, YA, in parallel with another lamina protein, lamin Dm, as probes to study the formation of the male pronuclear lamina in Drosophila melanogaster. Ectopically expressed YA is present in the nuclear envelopes of spermatocytes, but not in mature sperm, similar to endogenous lamin Dm. This suggests that the nuclear envelope of Drosophila sperm differs from that of somatic cells. Upon fertilization, YA and lamin Dm are recruited to the periphery of the male-derived nucleus before or during the early stages of migration by the male pronucleus. Using a paternal effect mutation, snky, we found that recruitment of lamina proteins to the male pronucleus requires, and probably accompanies, reorganization of the sperm nucleus. In order to identify factors that affect the recruitment of nuclear lamina proteins to the male pronucleus, we examined the subcellular localization of YA and lamin Dm in mutant embryos defective for the function of either the male pronucleus (mh, K81, and pal or both pronuclei (gnu, png, and plu). None of these mutations affect the recruitment of YA or lamin Dm to the male pronuclear envelope, suggesting that the mutations affect processes independent of, or after, reorganization of the nuclear envelope. Double mutant analyses between Ya and gnu suggest that YA plays a role in the nuclear envelope permissive for rounds of DNA replication.

Acp26Aa is a Drosophila seminal fluid protein that plays a role in the elevation of egg-laying by the mated female and has structural features of a prohormone. The protein, which has a region of sequence similarity to the egg-laying hormone of Aplysia, is transferred to the Drosophila female during mating. Acp26Aa is processed in the mated female's genital tract. We show here that the processing involves sequential proteolytic cleavages, and we map the position of these cleavages. Although Acp26Aa is not cleaved in the male, its processing in the mated female requires activities donated by the male. Acp26Aa ectopically expressed in unmated females is not processed. Processing of Acp26Aa in wild-type females mated to males with altered seminal fluid is dependent on the presence and amount of male accessory gland secretions. The need for molecular cooperation between the sexes for processing of Acp26Aa could restrict its activity to the mated female.

The Drosophila melanogaster fs(1)Ya gene encodes a nuclear envelope protein needed to initiate embryonic cleavage divisions. In cleavage stage embryos the fs(1)Ya gene product is localized to the nuclear envelope in a cell cycle dependent manner. We show here that fs(1)Ya protein is also present in polar bodies of early cleavage stage embryos as well as in endoreplicating yolk nuclei. This observation suggests that the fs(1)Ya protein is necessary but not solely responsible for embryonic mitosis. Furthermore, cell cycle stage dependent nuclear localization is also seen for ectopically produced fs(1)Ya protein in tissue culture cells, neuroblasts, and male accessory glands, which are free of maternal products. We therefore conclude that cell cycle dependent nuclear localization does not require any exclusively maternal products. Finally, our immunoelectron microscopy shows that the fs(1)Ya protein distribution parallels that of lamin, indicating that it is a nuclear lamina protein.

Genomic and cDNA clones of a gene expressed after a blood meal in the mosquito, Aedes aegypti , were identified as having significant similarity to the vitelline membrane protein genes of Drosophila melanogaster . The predicted protein had unusually high contents of alanine, histidine, and proline and contained a region of hydrophobic amino acids that was highly conserved in the predicted protein of the D. melanogaster vitelline membrane protein genes. The 15a gene was expressed from 5 to 40 hr after a blood meal. It was expressed only in the follicle cells of the ovary, particularly in the cells surrounding the oocyte. The 15a gene was expressed in ovaries of the blood-fed, decapitated female in response to an injection of 20-hydroxyecdysone, and in ovaries from non-blood-fed females incubated with the hormone, even in the presence of cycloheximide. A second gene, with weaker homology to 15a, is presumably another member of a family of related genes, as is the case with D. melanogaster vitelline membrane protein genes. This second gene contained a coding sequence similar to a decapeptide recently isolated from mosquito ovaries as an "oostatic factor" (Borovsky et el., FASEB J. 4, 3015-3020, 1990).

The accessory gland of male insects is a secretory tissue of the genital tract made up of several distinct cell types. It secretes components of the ejaculatory fluid which have an important effect on the postmating behavior of the female. We have examined the sequence, structure, and expression of a gene, mst 316, expressed exclusively in the accessory glands of male Drosophila melanogaster. The mst 316 RNA encodes a small, basic protein of 52 amino acids that exhibits features common to precursors of secreted peptides, including a hydrophobic N-terminus. The tissue-specific expression of the mst 316 gene was studied using an mst 316-lacZ hybrid gene inserted into Drosophila by germ line transformation. The mst 316-lacZ fusion protein is expressed exclusively in the “main” cells of the accessory gland. It is first detected upon eclosion and exhibits a burst of synthesis in the first 3 days of adult life. The synthesis of the fusion protein is stimulated by mating, so that β-galactosidase activity levels are two- to sixfold higher in males allowed to copulate with females compared to virgin male controls of the same age. The mating-stimulated synthesis of the mst 316-lacZ fusion protein, and by inference of the native gene product, appears to be due at least in part to increased transcript levels.

To obtain probes for sex-specific gene regulation during development in D. melanogaster, sequences expressed sex-specifically in adult flies were isolated by differential cDNA hybridization screens of a genomic library. Ten clones define new sex-specifically expressed genes. The remaining three isolates correspond to previously cloned genes encoding female-specific yolk proteins and chorion proteins. The pattern of expression of these genes in sex determination mutants and in germlineless flies, as well as their tissue specificities, permitted us to distinguish transcripts whose expression is dependent on correct sexual development of the soma or the germline. One of the female transcripts is expressed in nurse cells and oocytes. Five of the male-specific sequences are expressed in the testis during spermatogenesis: the remaining one is expressed in the soma. Experiments using a temperature-sensitive allele of tra-2 show that the presence of this male-specific transcript, found only in the adult paragonia, is not affected by temperature shift of X/X; tra-2ts2 adults. This is in contrast to yolk protein genes, which require tra-2 function in the adult for their expression in the female fat body.

Embryogenesis in vertebrates and marine invertebrates begins when a mature oocyte is fertilized, resulting in a rise in intracellular calcium (Ca(2+)) that activates development. Insect eggs activate without fertilization via an unknown signal imparted to the egg during ovulation or egg laying. One hypothesis for the activating signal is that deformation of eggs as they pass through a tight orifice provides a mechanical stimulus to trigger activation. Ovulation could produce two forms of mechanical stimulus: external pressure resulting from the passage of oocytes from the ovary into the narrow oviducts, and osmotic pressure caused by hydration-induced swelling of the oocyte within the oviducts. Ovulation could also trigger activation by placing the oocyte in a new environment that contains an activating substance, such as a particular ion. Here, we provide the first evidence that Drosophila oocytes require Ca(2+) for activation, and that activation can be triggered in vitro by mechanical stimuli, specifically osmotic and hydrostatic pressure. Our results suggest that activation in Drosophila is triggered by a mechanosensitive process that allows external Ca(2+) to enter the oocyte and drive the events of activation. This will allow exploitation of Drosophila genetics to dissect molecular pathways involving Ca(2+) and the activation of development.

The Drosophila nuclear lamina protein YA is essential for the transition from female meiosis to embryo mitosis. Its localization and, hence, function is under developmental and cell cycle controls. YA protein is hyperphosphorylated and cytoplasmic in ovaries. Upon egg activation, YA is partially dephosphorylated and acquires the ability to enter nuclei. Its function is first detected at this time. To investigate the cytoplasmic retention machinery that keeps YA from entering nuclei, we used affinity chromatography and blot overlay assays to identify cytoplasmic proteins that associate with YA. Drosophila P0/AP3, a ribosomal protein that is also an apurinic/apyrimidinic endonuclease, binds to YA in ovary and embryo cytoplasms. P0 and YA bind specifically and directly in vitro and are present in a 20S complex in the cytoplasmic extracts. YA protein can be phosphorylated by MAPK, but not by p34Cdc2 kinase, in vitro. This phosphorylation increases YA's binding to P0. We propose that the P0-containing 20S cytoplasmic complex retains hyperphosphorylated ovarian YA in the cytoplasm. In response to egg activation, YA is partially dephosphorylated and its binding to the 20S complex is reduced. Hence, some YA dissociates from the complex and enters nuclei. Consistent with this model, decreasing P0 levels partially suppress a hypomorphic Ya mutant allele.

The developmental regulation of three male-specific somatic transcripts is investigated. These RNAs are synthesized exclusively in the adult male accessory gland, an internal tissue derived from the genital disk of Drosophila melanogaster. The expression of these male-specific transcripts (msts) is under the control of the sex determination regulatory hierarchy, as demonstrated by the expression of all three msts in chromosomal females carrying mutant alleles at the doublesex (dsx), intersex (ix), or transformer-2 (tra2) loci. Although transcription of all three male RNAs is initiated late in pupation, temperature shifts of X/X; tra2ts2 homozygotes during development indicate that this expression is irreversibly determined earlier, during the third larval instar. A shift of X/X; tra2ts2 homozygotes to the male-determining temperature only for the duration of the late larval period is sufficient to elicit the expression of the msts during the adult stage. This critical period for the determination of these transcripts appears to correlate with the time of morphological determination of the accessory gland in these animals. Thus, the expression of these genes could be specified by the morphological determination of the male-specific tissue in which they are active.