F-actin distribution in the ovaries of pre-vitellogenic and vitellogenic black blowflies, Phormia regina (Meigen) (Diptera : Calliphoridae)

Int.J. lnsectMorphol.&Embryol.,Vol.21,No. 1,pp.77-86,1992 Printedin GreatBritain

00'20-7322/92$5.00+.00 (~)1992PergamonPresspie

F-ACTIN DISTRIBUTION IN THE OVARIES OF PRE-VITELLOGENIC AND VITELLOGENIC BLACK BLOWFLIES, P H O R M I A R E G I N A (MEIGEN) (DIPTERA • CALLIPHORIDAE)

MARCELLA CARCUPINO,* CHIH-MING YIN,~" JOHN G . STOFPOLANO,JR. ,t GIUSEPPE SCAPIGLIATI* and MASSnUOMAZZlm* *Facolt~tdi Scienze Biologiche, Universit,~tdella Tuscia, Via S. Camillo de Lellis, 01100 Viterbo, Italy *Department of Entomology, University of Massachusetts, Amherst, MA 01003, U.S.A.

(Accepted 2 October 1991)

Abstract--The spatial distribution of F-actin microfilaments in the ovaries of previtellogenic and vitellogenic female black blowflies, Phormia regina (Diptera : Calliphoridae), as the females shift from a sugar to a liver diet, is determined using rhodamine-labelled phalloidin (rh-phalloidin). During the pre-vitellogenic stages of ovarian development (i.e. corresponding to a sugar diet) a single bright fluorescent layer marks the interface between follicle cells and the oocyte. Fluorescence is also most evident at the inner surface of the ring canals of the nurse cells. This is observed in the nurse cells both in the distal part of the germarium, and in the vitellogenic growing oocyte. However, when liver-fed (i.e. necessary for vitellogenesis), 2 bright fluorescent layers are observed at the follicle cell-oocyte interface. In addition, the cytoplasm of the nurse cells during vitellogenesis appears full of fluorescent microfilaments and the actin rings are found to increase in size and thickness. The changing organization of the F-actin microfilaments in the follicles during the process of both egg chamber and oocyte formation is discussed and possible functions considered. Index descriptors (in addition to those in title): Ring canals, microfilaments, follicle cells, oocyte, nurse cells, ovarian development.

INTRODUCTION DEPENDING o n f o o d a v a i l a b i l i t y in t h e e n v i r o n m e n t a n d t h e s p e c i e s in q u e s t i o n , o v a r i a n d e v e l o p m e n t in insects m a y b e t r i g g e r e d e i t h e r b y i n g e s t i o n o f a p r o t e i n a c e o u s m e a l b y t h e a d u l t f e m a l e - - i n w h i c h c a s e t h e f e m a l e is d e f i n e d as a n a u t o g e n o u s - - o r d e v e l o p i n d e p e n d e n t l y f r o m a p r o t e i n a c e o u s m e a l - - i n which case f e m a l e s a r e d e f i n e d as a u t o g e n o u s ( M a s l e r et al., 1980; F u c h s et al., 1981). T h e i m p o r t a n c e o f an e x o g e n o u s protein meal to activate the neuroendocrine cascade leading to the ovarian development o f P h o r m i a regina, is n o w f i r m l y e s t a b l i s h e d ( R a s s o a n d F r a e n k e l , 1954; O r r , 1964a, b; S t o f f o l a n o , 1974; M j e n i a n d M o r r i s o n , 1976). T h e o v a r y o f P. regina c o n t a i n s m e r o i s t i c p o l y t r o p h i c o v a r i o l e s . T h e s e q u e n c e o f o v a r i a n d e v e l o p m e n t in P. regina h a s b e e n d i v i d e d i n t o 2 m a j o r s t a g e s (i.e. p r e vitellogenic and vitellogenic). T h e first u l t r a s t r u c t u r a l e v i d e n c e o f o v a r i a n d e v e l o p m e n t in P. regina is t h e c e n t r i p e t a l 77

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m i g r a t i o n of follicle cells, the f o r m a t i o n of intercellular spaces b e t w e e n follicle cells a n d changes at the follicle c e l l - o o c y t e interface. A t the same time, a c o m p l e x e n d o c y t o t i c a p p a r a t u s d e v e l o p s in the cortical o o p l a s m of the t e r m i n a l follicle (Mazzini et al., 1987). I n a d d i t i o n , the oocyte a n d the n u r s e cells are c o n n e c t e d via a precisely o r g a n i z e d system of cytoplasmic bridges. T h e s e bridges or ring canals (so called for their shape in cross section) in Drosophila are m e c h a n i c a l l y s t r e n g t h e n e d by rings of F - a c t i n ( W a r n et al., 1985). D u r i n g the final phase of d i p t e r a n o v a r i a n d e v e l o p m e n t , the cytoplasm of the n u r s e cells flows t h r o u g h the ring canals into the oocyte ( G u t z e i t a n d K o p p a , 1982; G u t z e i t , 1986). All these processes obviously d e p e n d o n the structure of the c y t o s k e l e t o n , which is c o m p o s e d of m i c r o t u b u l e s a n d F-actin microfilaments. T h e p r e s e n t study e x a m i n e s the d i s t r i b u t i o n of F-actin b u n d l e s in all cellular c o m p o n e n t s of the follicles a n d the g e r m a r i u m in b o t h pre-vitellogenic a n d viteUogenic females.

MATERIALS

AND METHODS

Rearing Non-diapausing flies were maintained in a rearing room at 26°C with 50% R.H. and constant illumination. Other procedures used for rearing were identical to those of Stoffolano (1974). Experimental flies were removed as pupae and placed in smaller cages until emergence. At that time, a sugar-diet regime was followed. The sugar-fed flies (i.e. pre-viteliogenic) were given beef liver starting on day 2 of post-emergence until the time for ultrastructural studies.

Thin section analysis Ovaries were dissected from adults that were either pre-vitellogenic or vitellogenic in a Phormia saline solution (Chen and Friedman, 1975) and then transferred to the fixative. Fixation was carried out at 4°C for 1 hr in a 5% glutaraldehyde fixative made up in 0.1 M cacodylate buffer at pH 7.2 and then washed overnight in the buffer. Ovaries were then post-fixed for 1 hr in 1% osmium tetroxide in 0.1 M cacodylate buffer at pH 7.2. Following dehydration in a graded series of alcohols, ovarian follicles were embedded in an Epon-Araldite mixture and then polymerized at 60"C for 2 days. Using another protocol, specimens were fixed by the method described by McDonald (1984) for enhancement of microtubules and actin filaments in cells. Thin sections were observed in either a JEOL 1200 EX II or a Philips 301 electron microscope following staining in uranyl acetate and lead citrate.

Fluorescence microscopy Ovaries were dissected from either pre-vitellogenic or vitellogenic females with the same method used for electron microscopy studies and then fixed. Fixation was carried out at 4°C for 15 min in a 4% freshly prepared solution of paraformaldehyde in PBS buffer. The ovaries were then transferred for 5 rain to the same solution containing 0.1% Triton X-100 to facilitate the penetration of the coupling agent, and then rinsed 3 times in PBS. Selected follicleswere stained for 30 min with phalloidin labelled with rhodamine (Molecular Probes Inc., Eugene, Oregon, U.S.A). Rhodamine-labelled phalloidin was received in methanol solution and, after the removal of methanol, the product was diluted in PBS to a concentration between 0.5 and 1.5 p,g/ml, as judged by empirical observation to give optimum coupling. After extensive rinsing in PBS, the follicles were mounted in 90% glycerol, containing 2.5% n-propyl gallate to reduce photobleaching (Giloh and Sedat, 1982). For the indirect immunofluorescence (IIF) analysis, the fixed and permeabilized ovaries (see above) were incubated for 45 rain at room temperature with a 1 : 100 dilution in PBS of anti-pan-myosin (Amersham Europe, Little Chalfont, U.K.). After 2 washes in PBS, the ovaries were incubated for 45 rain with a 1 : 200 dilution in PBS of an anti-mouse Ig labelled with rhodamine (Cappel Laboratories, Worthington, Massachusetts, U.S.A.). Washed ovaries were mounted as described before. Fluorescence observations were carried out with a standard Zeiss epifluorescence microscope (Axiophot) equipped with rhodamine filters. Photomicrographs were taken with Kodak Tri-X pan film.

RESULTS Ovarioles s t a i n e d with r h - p h a l l o i d i n a n d o b s e r v e d at different focal levels show that F-

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actin is localized in different tissues, such as the ovariolar sheath, follicle cells, nurse cells, oocytes and ring canals. The distribution of myosin, as revealed by immunofluorescence, is restricted to the muscular sheath surrounding the ovarioles, whereas it is not detectable in the ring canals. F-actin associated with the ovariolar sheath Each follicle or egg chamber (i.e. the oocyte, nurse cells and surrounding follicular epithelium) is surrounded by 2 distinct layers: the basal lamina and the external ovariolar sheath. At the highest optical plane, both rh-phalloidin and anti-myosin IIF staining allowed us to see the organization of the ovariolar sheath. It is composed of a network of interconnected striated muscle fibers perpendicularly arranged with respect to the major axis of the ovarioles. In particular, specific staining of the sarcomeric F-actin and myosin of these striated muscles reveals transverse muscle bundles with regular periodic patterns (Figs 1, 2). TEM observation of the ovariolar sheath shows the typical longitudinal array of sarcomeres arranged as electron-dense Z-disks between the A-bands (Fig. 3). F-actin associated with the follicle cells and oocyte During the pre-vitellogenic stages of ovarian development, the follicular epithelium appears as a uniformly thick layer in which follicle cells (FC) adhere closely to each other and to the oocyte. By staining follicles with rh-phalloidin, the FC are identified as a network of regular fluorescent polygons that surround the oocyte (OC) and the nurse cells (NC) (Fig. 4). The fluorescence is due to cortical F-actin microfilaments associated with the plasma membrane of the FC. In this stage, the oocyte cortex is relatively undifferentiated with no evidence of a perivitelline space or endocytotic apparatus (Fig. 5). During the vitellogenic stage, the fluorescent network is irregular and the follicle cells are mainly concentrated around the oocyte (Fig. 6). In this stage, which is characterized by vitellogenin uptake, the centripetal migration of follicle cells can be observed and the follicle cells are evident because of the irregular distribution of the fluorescence surrounding each follicle (Fig. 6). The follicular epithelium now appears characterized by many intercellular spaces and microvilli which become deeply lodged in the cortical ooplasm (Fig. 7). In addition, the onset of vitellogenesis is marked by the appearance of numerous coated pits and vesicles along the oolemma-follicle cells interface. At the same time, microvilli start to project from the oocyte surface into the perivitelline space without interlocking with the plasma membrane of the facing follicle cells (Fig. 8). Without fluorescence microscopy, the follicle cell microvilli, the space formed at the FC/OC interface, and oocyte microvilli are visible as a single layer at the light microscope level (Fig. 9). However, using fluorescence microscopy, this same region is visualized as 2 fluorescent, about 6 ~m distant layers (Fig. 10). We suggest that the external fluorescent layer is due to the F-actin microfilaments associated with complex junctions of the FC plasma membrane, whereas the internal one is due to the F-actin microfilaments associated with the oocyte-endocytotic apparatus. F-actin associated with the ring canals and nurse cell membranes Specific fluorescent staining of each ring canal is observed as a circular ring of uniform rh-phalloidin. As shown in the TEM microphotograph of 2 connecting nurse cells (Fig.

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3 FIGS 1-3. Ovariolar sheath. (1) Rh-phaUoidin fluorescence microscopy of muscular sheath surrounding ovariole, x 70. (2) Anti-myosin immunofluorescence, x 145. (3) TEM section of sheath showing regular miofibrillary patterns. × 7680.

F-actin Distribution in the Ovaries of Black Blowflies

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Fl~s 4--7. Follicular epithelium. (4) Rh-phalloidin staining of pre-vitellogenic stage showing regular cellular pattern, x 145. (5) TEM section at same stage as Fig. 4 showing tightly adherent follicle ceils (FC) (arrows). Oocyte (OC). x 6720. (6) Rh-phalloidin staining of viteiiogenic stage showing irregular pattern of follicle ceils. Nurse cells (NC). x 145. (7) TEM section at same stage of Fig. 6 showing loosely adherent follicle cells (FC) and intercellular spaces (arrows). Oocyte (OC). x 4320.

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FIGS 8-10. Follicle cells-oocyte interface. (8) TEM section of follicle cell (FC) and oocyte (OC) interface showing microvilli (M), and coated vesicles (arrows). x 16,320. (9) Phase contrast microscopyof folliclecells-oocyte(FC/OC) interface (arrows). x 310. (10) Rh-phaUoidinstaining of FC-OC interface (arrows). x 310.

11), the ring canals consist of cytoplasmic bridges with associated rings of F-actin microfilaments. The ring canals are first visible in the germarium (Fig. 12) and they then increase in size and thickness during the subsequent developmental stages of vitellogenesis (Fig. 13), reaching their maximum size at stage 5; when the nurse cells occupy about one half of the egg chamber (Fig. 14). A ring of rather more dispersed material of F-actin can be seen in more advanced stages (Fig. 15). The F-actin ring canal is the most intensely stained structure in the egg chamber but the nurse cells are all surrounded by a distinct layer of rh-phaUoidin staining cortical F-actin. At the beginning of cytoplasmic streaming (i.e. movement of material from the nurse cells into the oocyte), these microfilament bundles do not extend into the nurse cell cytoplasm, since only the cell margins stain intensely. When nurse cell streaming is most intense, the microfilament pattern changes and some microfilament bundles could be traced from the nurse cell border to the region of the nurse cell nucleus (Fig. 6).

F-actin Distribution in the Ovaries of Black Blowflies

FIGS 11-15. Ring canals. (11) Longitudinal section of a ring canal showing F-actin meshwork (stars).

x 14,400. (12) Rh-phalloidin staining of the ring canals in germarium (arrows). x 165. (13) Ring canals in pre-vitellogenic terminal follicle, x 385. (14) Ring canals in vitellogenic terminal follicle. x 220. (15) Higher magnification of a ring canal and surrounding F-actin material, x 1305.

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DISCUSSION Our results show that distribution of F-actin microfilaments in P. regina ovarioles changes when females shift from a pre-vitellogenic to a vitellogenic state. By observing follicles obtained from both pre-vitellogenic and vitellogenic females at different focal planes, it is possible to localize F-actin microfilaments in all the structures of the ovarioles. The rh-phalloidin stained microfilaments found in the follicular epithelium of pre-vitellogenic females show a different distribution and, as a result, produce a different shape to the follicle cells, compared with that during vitellogenesis. This change is due to the centripetal migration of the follicle cells and to the formation of spaces between them. The formation of a patent follicular epithelium is an essential developmental event during oogenesis within many insects and vertebrate species (Anderson, 1969; Dumont, 1972; Perry and Gilbert, 1979; Teller et al., 1982; Abraham et al., 1984; Mazzini and Giorgi, 1984; Mazzini et al., 1987). This process provides a pathway for yolk proteins synthesized at extraovarian sites to reach the oolemma for endocytotic incorporation. A cytoskeletal role in patency was initially suggested by Huebner (1976) and Abu-Hakima and Davey (1977b), since the cytoskeletal disruptors, cytochalasin B and colchicine, inhibited the juvenile hormone-induced patency response. In Rhodnius prolixus, juvenile hormone is responsible for the (Na÷-K +) ATPase which induces follicle cell shrinkage (Abu-Hakima and Davey, 1975, 1977a, 1979; Huebner and Injeyan, 1980). The (Na+-K +) ATPase-mediated pumping of fluid out of the cells (Davey, 1981), must be maintained until the microtubular cytoskeleton becomes rearranged, but the microfilaments also contribute to the modulation of the shape of the cells (Watson and Huebner, 1986). The trigger for patency and the cellular events controlling this important process in P. regina, have not been determined. In P. regina, juvenile hormone may be involved in the appearance of the coated pits, which are located in the oolemma of the vitellogenic oocyte (Mazzini et al., 1987). The most striking structures stained with rh-phalloidin in both pre-vitellogenic and viteUogenic females, are the F-actin ring canals. Previous studies have already noted membrane-associated, electron-dense, amorphous material lining the ring canals in Diptera (Meyer, 1961; Mahowald and Strassheim, 1970; Kinderman and King, 1973). Warn et al. (1985) have suggested that the microfilament bundles, which densely line the ring canals in Drosophila, have lost their contractile properties and have been interpreted as non-contractile rings which aid the mechanical stability of the ring canals. Based on inhibitor studies and time-lapse cinematography, Gutzeit (1986) demonstrated that during the last phase of oogenesis in Drosophila, nurse cell cytoplasm can be seen streaming into the growing oocyte. It proposed that microfilaments were important in the streaming process. Gutzeit and Huebner (1986) reported a similar distribution of Factin MFs in the nurse cells of Protophormia terraenovae, and also stated they have a functional role in the streaming process. Taken together, these reports suggest the involvement of F-actin to produce cytoplasmic streaming. How this is accomplished, has not been demonstrated; but, it is suggested that the nurse cells somehow contract, thus squeezing their cytoplasm through the ring canal system and ultimately into the oocyte. Until myosin is found associated with the actin in the nurse cells, caution must be taken to ascribe a contractile function to the actin microfilaments in the nurse cells. The present study shows that thick bundles of radially oriented MFs, spanned the nurse cell cytoplasm from the cell membrane to the nuclear membrane. This is the same distribution pattern observed in the closely related blowfly, Protophormia terraenovae by

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G u t z e i t a n d H u e b n e r (1986). V e r y i n t e r e s t i n g a r e t h e d i f f e r e n t d i s t r i b u t i o n p a t t e r n s o f f l u o r e s c e n t F - a c t i n in n o r m a l a n d m u t a n t D r o s o p h i l a ( o v a r i a n t u m o r = otu) d u r i n g v a r i o u s stages o f o v a r i a n d e v e l o p m e n t ( S t o r t o a n d K i n g , 1988). W h i l e t h e wild t y p e egg c h a m b e r shows b u n d l e s o f F - a c t i n M F s r a d i a t i n g t h r o u g h o u t t h e c o r t i c a l o o p l a s m o f t h e n u r s e cells, t h e otu egg c h a m b e r shows a g r e a t r e d u c t i o n in t h e a m o u n t o f F - a c t i n . I n b o t h follicles ( n o r m a l a n d otu), t h e r i m o f t h e ring c a n a l shows an i n t e n s e f l u o r e s c e n c e . S t o r t o a n d K i n g (1988) suggest t h a t t h e c o n s t r u c t i o n , in s t a g e 10 D r o s o p h i l a n u r s e cells, o f a cortical s y s t e m o f c a b l e s c o n t a i n i n g F - a c t i n M F s s e e m s e s s e n t i a l for t h e n u r s e cells to f o r c e t h e i r c y t o p l a s m into t h e o o c y t e . O t u m u t a n t s w h i c h a r e b l o c k e d at t h e e a r l y p12 stage a r e u n a b l e to c o n s t r u c t t h e n o r m a l s y s t e m o f actin M F b u n d l e s . T h e p r e s e n c e o f a l a r g e c o m p o n e n t o f F - a c t i n M F s in the o v a r y o f P. regina, c o n f i r m s t h e i m p o r t a n c e o f this c y t o s k e l e t o n c o m p o n e n t d u r i n g t h e d i f f e r e n t stages o f o o g e n e s i s . In p a r t i c u l a r , it c o u l d b e p o s t u l a t e d t h a t t h e c o n t r a c t i o n o f t h e s a r c o m e r i c F - a c t i n M F s o f t h e o v a r i o l a r s h e a t h is i n v o l v e d in facilitating h a e m o l y m p h m o v e m e n t , thus a v a i l a b i l i t y o f Vg, for u p t a k e b y t h e follicles. I n fact, v i t e l l o g e n i n is s e c r e t e d b y the fat b o d y into t h e h a e m o l y m p h a n d t h e n m u s t e n t e r t h e follicles t h r o u g h t h e o v a r i o l a r sheath. T h e F - a c t i n m i c r o f i l a m e n t s o f t h e e n d o c y t o t i c a p p a r a t u s at t h e follicle c e l l - o o c y t e i n t e r f a c e , p r o b a b l y s e r v e a s t r u c t u r a l f u n c t i o n , as has b e e n d e m o n s t r a t e d for microvilli in o t h e r organisms.

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