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Ovulation Triggers Activation of Drosophila Oocytes

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Ovulation Triggers Activation of Drosophila Oocytes Developmental Biology 234, 416–424 (2001) doi:10.1006/dbio.2001.0246, available online at http://www.idealibrary.com on Ovulation Triggers Activation of Drosophila Oocytes Yael Heifetz,1 Jing Yu,1,2 and Mariana F. Wolfner3 Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853-2703 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. © 2001 Academic Press Key Words: ovulation; activation; vitelline membrane; vitelline membrane proteins; cross-linking; meiosis; fertilization; Drosophila. INTRODUCTION Raz and Shalgi, 1998, mammals; Jaffe et al., 2000; Stricker, 1999, echinoderms). Later events in the activation cascade Oocytes of most animals arrest at species-characteristic usually include inactivation of maturation-promoting fac- stages of meiosis, to allow oogenic provisioning to occur tor and mitogen-activated protein kinase, the resumption of and possibly to permit coordination of male and female meiosis, changes in intracellular pH, elevated translation pronuclear behavior (Page and Orr-Weaver, 1997a; Sagata, from maternal mRNAs, formation of pronuclei, initiation 1996; reviewed in Schultz and Kopf, 1995). This arrest is of DNA synthesis, and onset of embryo mitosis (reviewed relieved by, or just before, fertilization in the process of egg in Schultz and Kopf, 1995). activation. In cases in which fertilization triggers activation Despite the wealth of information about Drosophila (e.g., frog, fish, mammals), a number of activational events oogenesis and embryonic development, the in vivo triggers have been observed. These events have the general goal of and events of egg activation in this organism are unknown, activating macromolecule synthesis, permitting the reini- largely because activation occurs rapidly and within the tiation and completion of meiosis, and preventing female fly. It is known that Drosophila oocytes arrest polyspermy. Early events after egg activation usually in- 2ϩ transiently in meiotic prophase I and then proceed to clude transient rises or oscillations in intracellular free Ca metaphase I where they remain arrested in the ovary (King, concentration that lead to opening of ion channels and 1970; reviewed by Page and Orr-Weaver, 1997b). It is also exocytosis of cortical granules establishing a block to known that oocytes are still activating when they are polyspermy (Ridgway et al., 1977, medaka; Steinhardt et al., fertilized (in the uterus; Sonnenblick, 1950), but that acti- 1977, sea urchin; Kline and Kline, 1992, mouse; reviewed in vation does not require fertilization (Doane, 1960). Incuba- tion of mature stage 14 Drosophila oocytes in hypotonic 1 Both authors contributed equally. saline in vitro resulted in activation, leading to the hypoth- 2 Present address: Biological Laboratories, Room 138, 16 Divinity Avenue, Harvard University, Cambridge, MA 02138. esis that hydration of the eggs as they pass down the 3 To whom correspondence should be addressed at 423 Biotech- oviduct causes activation (Mahowald et al., 1983; Page and nology Building, Cornell University, Ithaca, NY 14853-2703. Fax: Orr-Weaver, 1997a). These in vitro activation systems and (607) 255-6249. E-mail: [email protected]. the examination of very recently laid eggs allowed several 0012-1606/01 $35.00 Copyright © 2001 by Academic Press 416 All rights of reproduction in any form reserved. Drosophila Egg Activation 417 characteristics of activation to be determined. Following regions were the lateral oviducts (positions I–III), the junction of activation, Drosophila oocytes swell and their eggshells the lateral and common oviducts (positions IV and V) (see Fig. 1A), become impermeable to many small molecules. The egg and the uterus (Fig. 4A). The probabilities of finding an egg at ϭ Ϯ ϭ Ϯ ϭ cortex undergoes visible changes during activation (Ma- positions I–V are, respectively, PI 34 5%, PII 25 6%, PIII Ϯ ϭ Ϯ ϭ Ϯ howald et al., 1981), but there is no evidence for cortical 17 3%, PIV 19 5%, PV 0.45 0.46%; note that the probability of finding an egg decreases as one moves more posterior. granules or for formation of a fertilization membrane (Ma- We also noticed that some eggs in the uterus are hard to dissect out, howald et al., 1983). Meiotic chromosomes individualize whereas others are easily released upon dissection. In addition we (Page and Orr-Weaver, 1997a), and the meiotic spindles collected eggs that were found at the external opening of the female pivot from parallel to the egg surface in mature oocytes reproductive tract before the beginning of the dissection procedure (Theurkauf and Hawley, 1992) to perpendicular to the (Fig. 4B). oocyte surface in activated oocytes (Endow and Komma, 1997). Meiosis resumes and translational activity from maternal mRNAs reinitiates (Mahowald et al., 1983; Page Evaluation of Egg Activation States of Dissected and Orr-Weaver, 1997a). Further studies showed that the Eggs stages of meiosis are normal (Endow and Komma, 1997; Page and Orr-Weaver, 1997a) and that the progression rate Permeability to bleach. Due to changes in the permeability of through meiosis is similar (Page and Orr-Weaver, 1997a) in their envelopes, activated eggs are more resistant to lysis by bleach in vitro activated oocytes and in laid in vivo activated eggs. than mature stage 14 (ovarian) oocytes (Mahowald et al., 1983). What could not be determined in any of the prior studies Unactivated oocytes will lyse when incubated with bleach. Thus, is where and when activation is initiated in vivo and hence our first assay for activation of the dissected eggs was the change in their sensitivity to bleach. To quantify this, we developed an Egg what normally triggers it. It was thought that the process Permeabilization Index (EPI). We measured the time it took to lyse initiates between the time the mature oocyte reaches stage eggs from each oviduct position (Tp) and the lysis time for stage 14 14 in the ovary and the time it comes to lodge in the uterus. oocytes (To). There is some female-to-female variation in the However, whether activation is triggered by ovulation or survival time of ovarian oocytes incubated in bleach. We took this during passage through the oviducts was unknown. into an account by measuring the lysis time of stage 14 oocytes To obtain further information and gain a better under- taken from the same ovary (Toi) and of each egg we examined (Tpi). standing of the egg activation process in Drosophila, we Therefore, the rate of change in sensitivity to bleach of ovulated egg ϭ Ϫ followed activation in vivo by dissecting eggs from different is EPIi (Tpi Toi)/Toi, which is derived from the relationship regions of the female reproductive tract and determining between the basal lysis time of ovarian oocyte and ovulated egg: T ϭ T ϫ ϩ g g whether they were activating. Using the changes in the pi oi (1 i), where i is the rate of change (EPIi). Eggs that egg’s permeability to bleach as an initial assay, we showed survived the full 2-min incubation period were considered to be fully impermeable. The EPIs of eggs from wild-type Oregon R that Drosophila egg activation initiates during ovulation, females at each of the five positions were the same as for eggs from while the egg is still largely in the ovary, and that the eggs the Oregon R P2 strain females (data not shown). have gone through the early stages of activation (cross- Vitelline membrane cross-linking. We assessed the cross- linking of vitelline membrane proteins and resumption of linking status of a major vitelline membrane protein (VMP) sV23 meiosis) by the time they reach the uterus. (Pascucci et al., 1996) in eggs from different regions along the female reproductive tract. Eggs dissected on ice were lysed in 4ϫ SDS sample buffer containing 3% SDS, boiled 5 min to release MATERIALS AND METHODS soluble proteins, and centrifuged, as in LeMosy and Hashimoto (2000). The egg extracts were electrophoresed on 17% polyacryl- Fly Culture amide SDS gels and subjected to Western blotting analysis with anti-sV23 (Pascucci et al., 1996) as described in LeMosy and Drosophila melanogaster flies used in the assays were wild-type Hashimoto (2000). For comparison to LeMosy and Hashimoto Oregon R, the Oregon R P2 strain (Allis et al., 1977), and ncd-gfp* (2000), we also examined laid eggs collected in a 0- to 2-h collec- (Endow and Komma, 1997). Fly stocks were maintained at 23 Ϯ 2°C tion. and 70% relative humidity in a 12-h L:12-h D photoperiod. Newly Resumption of meiosis. (1) Stage 14 oocytes and eggs dissected enclosed virgin males and females were placed into separate vials from different regions of the oviduct of ncd-gfp* females were and aged on yeast glucose acid-mix media for 3–4 days. positioned with their dorsal or lateral side up in a drop of 3% antifade and DAPI (Sigma, St.
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