- Email: [email protected]
Cytoskeletal organization of bee ovarian follicles during oogenesis
Micron 42 (2011) 55–59
Contents lists available at ScienceDirect
Micron journal homepage: www.elsevier.com/locate/micron
Cytoskeletal organization of bee ovarian follicles during oogenesis Karina Patrício a,1 , Carminda da Cruz-Landim a,∗ , Gláucia Maria Machado-Santelli b Department of Biology, Institute of Biosciences, UNESP – Univ. Estadual Paulista, 24 Av. N◦ 1515, CEP 13.506-900, Rio Claro, SP, Brazil Department of Cell and Developmental Biology, Institute of Biomedical Sciences, University of Sao Paulo, Prof. Lineu Prestes Av., n◦ 1524, Butantan, CEP 05508-900, São Paulo, SP, Brazil a
b
a r t i c l e
i n f o
Article history: Received 17 May 2010 Received in revised form 2 August 2010 Accepted 3 August 2010 Keywords: Actin Cystocyte Honeybee Intercellular bridge Stingless bee Tubulin
a b s t r a c t The germ cells in the germarium of the bee meroistic polytrophic ovarian cysts remain interconnected by cytoplasmic bridges as a result of incomplete cell division. These intercellular bridges form a distribution pathway for the substances that initially determine which of the cystocytes will become oocyte and later conduct the products synthesized by the nurse cells to the oocyte. In the present work, the presence and distribution of cytoskeleton components, actin and tubulin were studied in ovaries of queens of Apis mellifera and Scaptotrigona postica, two eusocial species, using antibody against ␣- and -tubulin and FITC-phalloidin, aiming to shed light on the role of these cytoskeleton elements in oogenesis. The immunofluorescent preparations were analyzed by laser scanning confocal microscopy. F-actin was detected in the intercellular bridges of both species. The tubulin distribution in cell cytoplasm of A. mellifera and S. postica also displayed similar pattern. The role of these elements in the oogenetic events responsible for both cell support and motility is discussed. © 2010 Elsevier Ltd. All rights reserved.
1. Introduction Meroistic ovaries are characterized by the division of germline cells into two categories: oocytes and nurse cells. In the bee polytrophic ovary, a primordial germ cell called cystoblast divides by mitosis, resulting in a clone of cells named cystocytes. The dividing cells remain interconnected through intercellular bridges, called ring canals, which are kept opened by cytoskeletal reinforcements (King et al., 1982). Although the flow of compounds among cystocytes has been recognized, surprisingly, the cytoskeleton was not initially considered the driver of intercellular transport. Instead, intercellular electrophoresis was proposed as a mechanism for molecular transport (Woodruff and Telfer, 1980). However, although electrical phenomena have been documented in polytrophic ovaries, they are not the only mechanism of circulation of compounds inside cyst cells. Bohrmann and Biber (1994) and Gutzeit et al. (1993) demonstrated the participation of microfilaments in the differentiation of several stages of Apis mellifera ovarioles. In addition, studies on Drosophila melanogaster indicate that the factors decisively influencing oocyte differentiation are related mainly to the access of cytoplasm-specific compounds by the nucleus of the cystocyte. The
∗ Corresponding author. Tel.: +55 1935264144; fax: +55 1935264136. E-mail address: [email protected] (C. da Cruz-Landim). 1 Present address: Department of Zootechny, Laboratory of Biotechnology, USP – University of São Paulo (ESALQ), Padua Dias Av., n◦ 11, CEP 13418-900, Piracicaba, SP, Brazil. 0968-4328/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.micron.2010.08.002
access to these compounds requires their asymmetric accumulation within the future oocyte, as well as the proper organization of the cytoskeleton to provide polarity within the cyst (Cox et al., 2003), promoted by the cytoskeleton and fusoma organization in such a way as to direct the flow of cytosol constituents to a given cystocyte. Therefore, the cytoskeleton acts early on clone differentiation by determining which cell among the cystocytes will become the oocyte. Gutzeit and Huebner (1986) had already demonstrated the participation of microfilaments in the transportation of macromolecules synthesized by nurse cells to the oocyte. Therefore, when the clone cells later differentiate into oocyte and nurse cells and the oocyte begins to mature, the organization of the cytoskeleton must allows the polarized transport of compounds from the nurse cells to the oocyte cell through the ring canals (Cassidy and King, 1972; Cooley and Theurkauf, 1994; Cruz-Landim, 1978; Gutzeit et al., 1993; Staurengo-da-Cunha, 1988; Theurkauf et al., 1992; Lisboa et al., 2005). Accordingly, in the bee germline cysts, the bridges interconnecting the proliferating germ cells are reinforced by microfilaments and microtubules occupy the open spaces of the bridges (Bilinsky and Jaglarz, 1999). During the differentiation of cystocytes into oocyte and germ cells, the oocyte consistently occupies a basal position in relation to the nurse cells (Patrício and Cruz-Landim, 2006). Microfilaments, or actin filaments, and microtubules composed of tubulin are distinct functional structures of the cytoskeleton. However, numerous observations revealed that they can act together simultaneously in a wide variety of cell processes. Microtubules act in the long distance motion of cell compounds, while
56
K. Patrício et al. / Micron 42 (2011) 55–59
actin filaments provide local cytoplasmic motility (Evans et al., 1997). Pharmacological studies have shown that disorganization in one of these structures strongly affects the organization of the other, and vice versa, and electron microscopy studies have revealed connecting bridges between actin microfilaments and microtubules in vitro, indicating that they are connected both physically and functionally (Goode et al., 2000). It is therefore expected that the study of the location and distribution of these cytoskeleton elements will lead to a better understanding of the morphological changes that take place during the germline cell cycle and particularly during oocyte differentiation and growth in bees. This study focused on the distribution of actin and tubulin in the oogenetic phases in order to distinguish when and where they participate in the cytoskeleton predominantly as a static structure responsible only for the cellular structural support, and where it is a dynamic structure. 2. Materials and methods 2.1. Animals Queens of A. mellifera and Scaptotrigona postica were obtained from colonies kept in the vivarium of the UNESP – Univ. Estadual Paulista in Rio Claro, State of São Paulo, Brazil. 2.2. Immunofluorescence The immunofluorescence of ovaries was performed according to Amaral and Machado-Santelli (2008). The ovaries were removed from the insects in Dulbecco’s phosphate-buffered saline, without calcium and magnesium (PBSA–8.2 g/L NaCl) 0.2 KCl, 1.15 g/L Na2 HPO4 ·2H2 O, 0.2 g/L KH2 PO4 , pH 7.0, and fixed with 3.7% formaldehyde for 4 h. After washing (3 × 5 min) in PBS, to separate the ovarioles and withdraw most of the outer tracheolar branches the total ovaries were treated with 0.5% Triton X-100 in PBS, for 10 min at room temperature and washed again in PBS (3 × 5 min) and 10 g/mL RNAase during 30 min. Incubation in 7 L of primary antibody against ␣- and -tubulin (Amersham International) diluted 1:100 was performed in a humid chamber for 3 h. The secondary antibody used was anti-mouse IgG-FITC (Calbiochem) that was used in the same concentration and the incubation lasted 1 h. Nuclei were stained with 7.5 M propidium iodide (Sigma–Aldrich) for 15 min. The coverslips were mounted on slides with antifading (Vecta-shield, Vector Laboratories). F-actin was stained with phalloidin-FITC (Sigma–Aldrich) for 40 min. 2.3. Confocal microscopy analysis The fluorescent images were obtained using a laser scanning confocal microscope (Zeiss LSM 510) with argon, helium-neon 1 and helium-neon 2 lasers connected to an inverted fluorescence microscope (Zeiss Axiovert 100M). The emitted fluorescence was selected by a group of filters and displayed in RGB with a resolution of 512 × 512 pixels. 3. Results The two species, S. postica and A. mellifera, showed no differences in the presence and distribution of actin and tubulin. Therefore, the findings of this study are described without mentioning the species. 3.1. Distribution of F-actin The location of actin in relation to the intercellular bridges between the germline cells and in the somatic and germ cells was
Fig. 1. (A and B) Germarium of Apis mellifera queen ovary, showing cystocyte actin stained green by phalloidin-FITC and nuclei stained red by propidium iodine. Note the arrangement of actin (a) among the cystocytes forming rings, and in small amounts in the periphery of the cells. Arrows indicate actin in the peritoneal sheath. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
analyzed in the germarium and vitellarium. Actin rings between cystocytes were observed in the germarium, marking the reinforcements of the intercellular bridges, and in the cytoplasm of peritoneal membrane cells (Fig. 1A and B). The F-actin in vitellarium follicles was concentrated in the cortical zone of nurse cells, in the passage between nurse and oocyte chambers and at the interface between follicular cells and the oocyte, as well as in the peritoneal membrane (Fig. 2A–C) The orthogonal views in Fig. 2C shows clearly the distribution of actin on the bridges among the nurse cells. Smaller amounts of actin were visible in the inner cytoplasm of nurse cells (Fig. 2C), in the cor-
K. Patrício et al. / Micron 42 (2011) 55–59
57
Fig. 2. Ovarian follicle of Apis mellifera queen (A and C) and vitellarium and nurse cells of Scaptotrigona postica queen (B). Actin stained green in follicular cells (fc), nurse cells (nc), cortex and cytoplasm, oocyte (oo) and in oocyte and nurse cell chambers (arrows). Nuclei (n) in red. In C, view of orthogonal projections from the bridge between nurse and oocyte chambers. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
tex of oocyte cytoplasm, and in the bridge connecting to the nurse chamber (Fig. 2A and C). 3.2. Distribution of ˛- and ˇ-tubulin While actin is more abundant at the cell boundaries, tubulin is scattered throughout the cytoplasm. In the germarium, large amounts of tubulin are visible in the cystocyte cytoplasm, forming short rods randomly distributed and in peritoneal membrane cells (Fig. 3A and B). In the vitellarium, the tubulin fills the entire cytoplasm of the nurse chamber, follicular cells and oocyte periplasm (Fig. 3B). 4. Discussion The development of the female gamete involves complex programs leading to the production of a cell with genetic material and cytoplasm components, including molecular cues for an eventual embryonic development. The presence and action of the cytoskeleton elements during cystocyte proliferation in the germarium and oocyte ripening in the vitellarium are essential for the correct functioning of these programs. The distribution of the cytoskeletal
components in the cells provides information about the functions of these components in the various stages of oogenesis. Our findings revealed that the distribution of cytoskeleton components, actin and tubulin, during the oogenesis of the two species under study is the same and is probably also valid for bees in general, since it does not differ from what is known about other insects with meroistic polytrophic ovaries such as leaf-cut ants (do Amaral and Machado-Santelli, 2009). F-actin and ␣- and -tubulin were detected along the entire length of the peritoneal sheath of the ovariole, as expected due to the presence of muscle fibers and trachea in the sheath. The location of F-actin within the ovarian follicles possibly indicates its dual role of supporting and promoting intracellular motility. Its distribution in the germarium suggests a main role as a mechanical component of cytoskeleton that keeps open the intercellular bridges forming the so-called ring canals (Cassidy and King, 1969; King et al., 1982; Theurkauf and Hazelrigg, 1998), thus allowing for the transfer of substances among the cystocytes. Although the main role of actin in this part of the ovariole is structural, Patrício and Cruz-Landim (2006) suggested an additional function concerning the motility needed for cystocyte rearrangements in the cyst during oocyte differentiation.
58
K. Patrício et al. / Micron 42 (2011) 55–59
Fig. 3. (A) Distribution of tubulin stained green by antibody anti-␣- and anti--tubulin-FITC in germarial cells of Scaptotrigona postica queen. Nuclei in red. Note the tubulin appearing as thick rods in cystocyte cytoplasm. (B) Follicle from ovary of Scaptotrigona postica vitellarium, showing tubulin (green) in follicular cells (fc), in nurse cell (nc) cytoplasm, in communication between nurse and oocyte chambers (asterisk) and oocyte (oo) periplasm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
In the vitellarium, F-actin was observed mostly in follicles of early vitellogenic oocytes with a characteristic distribution in each type of cell. In the nurse cells, F-actin was condensed in the cortical zone and diffused in the cytoplasm. The cortical condensation of Factin suggests that it plays a supporting role in preventing early cell collapse and the ensuing impairment of the flow of molecules and small organelles from these cells to the oocyte. The actin detected in the cytoplasm is presumably involved in the transport of cell constituents among the nurse cells and toward the oocyte. Gutzeit and Huebner (1986) observed the presence of actin microfilaments in the nurse cells of 12 species of insects with meroistic telotrophic and polytrophic ovaries, with a distribution suggestive of participation in the transport of compounds throughout the cells. Theurkauf et al. (1993) believe that in Drosophila, the actin microfilaments at the periphery of nurse cells extend toward the nucleus, promoting its anchorage and forcing the flux of cytoplasm toward the oocyte. In the follicular epithelium, F-actin was found to be concentrated at the basal and apical poles of cells, which also suggests a supporting function. At the basal pole, it must act in regulating the passage of hemolymph vitellogenin through supporting the plasma membrane limiting the intercellular spaces, through which it flows to the perioocyte space, from where it will be taken up by the oocyte, while at the apical pole it acts as a support of follicular cell microvilli. The oocyte also forms microvilli and the supporting F-actin they contain may contribute to the strong staining visible throughout the surroundings of the oocyte. However, the F-actin detected in the peripheral ooplasm, mainly in the sides of the oocyte anterior pole and in the connection between the oocyte and the nurse cell chamber, may have a compound transporting function. During early vitellogenesis, the budding germinal vesicles form many accessory nuclei (Cruz-Landim, 1991; Hopkins, 1964; King and Fordy, 1970), which migrate to the posterior pole of the oocyte through the peripheral cytoplasm. The material received from the nurse cells is also transported around the oocyte, thus justifying the interpretation of involvement of actin microfilaments. Unlike F-actin, ␣- and -tubulin appear scattered throughout the cytoplasm of the germarium germ cells. Although the filaments did not show a preferential organization, the presence of ␣- and tubulin in these cells is consistent with their frequent mitosis and the flow of molecules among the cystocytes. Therefore, the tubulin detected here seems to be involved in intracellular transport, since the substances must collect around and be directed toward
the bridge passages (Spradling, 1993; Theurkauf and Hazelrigg, 1998). The ␣- and -tubulin detected in the vitellarium were scattered around the nuclei of the nurse cells, in the bridge between the nurse and oocyte chambers, and in the oocyte cortex. Again, this location is consistent with the sites where ultrastructural studies have found the presence of microtubules (Patrício and Cruz-Landim, 2006; Staurengo-da-Cunha, 1988). The RNA that passes from the nucleus into the nurse cell cytoplasm through the nuclear pore and from one nurse cell the another until reach the oocyte. As previously described, the tubulin in the passage between the nurse and oocyte chambers are involved in delivering several nurse cell products, RNA and proteins to the oocyte (Bilinsky and Jaglarz, 1999; Cooley and Theurkauf, 1994; Theurkauf and Hazelrigg, 1998). The presence of tubulin in the oocyte cortex might indicate functions in the previtellogenic and vitellogenic oocyte. In the previtellogenic oocyte, it probably indicates a function of the microtubules in the migration of accessory nuclei originated from outgrowths of the germinal vesicle to the posterior pole of the oocyte, which occurs through the oocyte cortex (Bilinsky et al., 1995). In the vitellogenic oocyte, it could be involved in the transportation through the cytoplasm of yolk spheres formed by endocytic uptake of vitellogenin from the hemolymph in perioocytic space. Oogenesis is known to be important in the formation of the pattern of embryonic development through the acquisition and correct distribution of macromolecules in the oocyte, for which the cytoskeleton is the major structure responsible. The major role of actin in oogenesis seems to be providing structural support, keeping the passage through the ring canals open. However, tubulin in areas through which substances travel into the oocyte, as was observed here, ascribes to it an additional role in inter- and intracellular transport. Otherwise, tubulin (microtubules) has only transportation functions, working in chromosome displacement during cell division or directing the cytoplasmic flow through the interconnecting bridges between the nurse cells and from these to the oocyte. Thus, actin (microfilaments) and tubulin (microtubules) play important roles in oocyte growth, and later, in the distribution of the morphogenetic determinants throughout the ooplasm (Theurkauf et al., 1993; Theurkauf, 1994). The tubulin in the bridges may be the remains of mitotic spindle and therefore located in the fusome, as has been revealed in transmission electron microscopy
K. Patrício et al. / Micron 42 (2011) 55–59
images (Patrício and Cruz-Landim, 2006). Impaired development of fusome hinders oocyte growth (Cuevas et al., 1996; Hartfeler and Steinbück, 1997; Patrício and Cruz-Landim, 2006; Tanaka and Hartfelder, 2004). The role of the circulation of substances among cystocytes, as well the direction of their flow, is essential in determining which cystocyte will become the oocyte (Büning, 1994; Cuevas et al., 1996; Telfer, 1975; Theurkauf et al., 1992). One of the two cystocytes linked to the others through a greater number of bridges will become an oocyte, possibly because it receives a larger amount of the circulating substances. The location of actin and tubulin in bee ovaries was consistent with previous findings about the ultrastructure and function attributed to these cytoskeletal components during ovarian development. Acknowledgments The authors acknowledge the financial support of the Brazilian research funding agencies FAPESP (Fundac¸ão de Amparo à Pesquisa do Estado de São Paulo) and CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico). References Amaral, J.B., Machado-Santelli, G.M., 2008. Salivary system in leaf-cutting ants (Atta sexdens rubropilosa, Forel, 1908) castes: A confocal study. Micron 39, 1222–1227. Amaral, J.B., Machado-Santelli, G.M., 2009. Three-dimensional reconstruction of ovaries of leaf-cutting ant (Atta sexdens rubropilosa) queens. Sociobiology 53, 379–388. Bilinsky, S.M., Klag, J., Kubrakiewcz, J., 1995. Subcortical microtubule network separates periplasm from endoplasm and is responsible for maintaining the position of accessory nuclei in hymenopteran oocytes. Roux’s Arch. Dev. Biol. 205, 54–61. Bilinsky, S.M., Jaglarz, M.K., 1999. Organization and possible functions of microtubule cytoskeleton in hymenopteran nurse cells. Cell Motil. Cytoskeleton 43, 213–220. Bohrmann, J., Biber, K., 1994. Cytoskeleton-dependent transport of cytoplasmic particles in previtellogenic to mid-vitellogenic ovarian follicles in Drosophila: time-lapse analysis using video-enhanced contrast microscopy. J. Cell Sci. 107, 849–858. Büning, J., 1994. The Insect Ovary: Structure, Previtellogenic Growth and Evolution, 5a ed. Chapmam & Hall, London. Cassidy, J.D., King, R.C., 1969. The dilatable ring canals of the ovarian cystocytes of Habrobracon juglandis. Biol. Bull. 137, 429–437. Cassidy, J.D., King, R.C., 1972. Ovarian development in Habrobacron juglandis (Ashemed) (Hymenoptera, Braconidae). 1. The origin and differentiation of the oocyte–nurse cell complex. Biol. Bull. 143, 483–505. Cooley, L., Theurkauf, W.E., 1994. Cytoskeleton functions during Drosophila oogenesis. Science 266, 590–596. Cox, D., Lu, B., Sun, T., Williams, L., Jan, Y., 2003. Drosophila par-1 is required for oocyte differentiation and microtubule organization. Curr. Biol. 11, 75–87.
59
Cuevas, M., Lee, J.K., Spradling, A.C., 1996. a-spectrin is required for germline cell division and differentiation in the Drosophila ovary. Development 122, 3959–3968. Cruz-Landim, C., 1978. Structural dynamics of oogenesis in Atta sexdens rubropilosa (Hymenoptera, Formicidae). Rev. Bras. Biol. 38, 363–438. Cruz-Landim, C., 1991. Accessory nuclei in hymenopteran oocytes and origin of the germ plasm: a study of Melipona quadrifasciata anthidioides Lep. (Hymenoptera, Apidae, Meliponinae) and Atta sexdens rubropilosa Forel (Hymenoptera, Formicidae, Attinae). Naturalia 16, 171–182. Evans, L.L., Hammer, J., Bridgman, P.C., 1997. Subcellular localization of myosin V in nerve growth cones and outgrowth from dilute-lethal neurons. J. Cell Sci. 110, 439–449. Goode, B.L., Drubin, D.G., Barnes, G., 2000. Functional cooperation between the microtubule and actin cytoskeletons. Curr. Opin. Cell Biol. 12 (1), 63–71. Gutzeit, H.O., Huebner, E., 1986. Comparison of microfilaments patterns in nurse cells of different insects with polytrophic and telotrophic ovarioles. J. Embryol. Exp. Morphol. 93, 291–301. Gutzeit, H.O., Zissler, D., Feig, R., 1993. Oogenesis in the honeybee Apis mellifera: cytological observations on the formation and differentiation of previtellogenic ovarian follicles. Roux’s Arch. Dev. Biol. 202, 181–191. Hartfeler, K., Steinbück, G., 1997. Germ cells cluster formation and cell death in caste-specific differentiation of the larval honey bee ovary. Invertebr. Reprod. Dev. 31, 45–77. Hopkins, C.R., 1964. The histochemistry and fine structure of the accessory nuclei in the oocyte of Bombus terrestris. Quart. J. Microsc. Sci. 102, 475– 480. King, R.C., Fordy, M.R., 1970. The formation of accessory nuclei in the developing oocytes of parasitoid hymenopterans Ophion luteus (L.) and Apanteles glomeratus (L.). Z. Zellforsch. 109, 158–170. King, R.C., Cassidy, J.D., Rousset, A., 1982. The formation of clones of interconnected cells during gametogenesis in insects. In: King, R.C., Akai, H. (Eds.), Insect Ultrastructure. Plenum, New York, pp. 3–33. Lisboa, L.C.O., Serrão, J.E., Cruz-Landim, C., Campos, L.A.O., 2005. Effect of larval food amount on ovariole development in queens of Trigona spinipes (Hymenoptera, Apinae). Anat. Histol. Embryol. 34, 179–184. Patrício, K., Cruz-Landim, C., 2006. Ultrastructural aspects of the intercellular bridges between female bee germ cells. Braz. J. Biol. 66 (1B), 309–315. Spradling, A.C., 1993. Developmental genetics of oogenesis. In: Bate, M., Martinez Arias, A. (Eds.), The Development of Drosophila melanogaster. Cold Spring Harbor Press, New York, pp. 1–70. Staurengo-da-Cunha, M.A., 1988. Ultrastructure of the oocytes of Scaptotrigona postica workers (Hymenoptera, Apidae). Naturalia 13, 103–115. Tanaka, E.D., Hartfelder, K., 2004. The initial stages of oogenesis and their relation to differential fertility in the honey bee (Apis mellifera) castes. Arthropode Struct. Dev. 33, 431–442. Telfer, W.H., 1975. Development and physiology of the oocyte-nurse cell syncytium. Adv. Insect Physiol. 11, 223–319. Theurkauf, W.E., 1994. Microtubules and cytoplasm organization during Drosophila oogenesis. Dev. Biol. 165, 352–360. Theurkauf, W.E., Smiley, S., Wong, M.L., Alberts, B.M., 1992. Reorganization of the cytoskeleton during Drosophila oogenesis: implications for axis specification and intercellular transport. Development 115, 923–936. Theurkauf, W.E., Alberts, B.M., Jan, Y.N., Jongens, T.A., 1993. A central role for microtubules of Drosophila oocytes. Development 118, 1169–1180. Theurkauf, W.E., Hazelrigg, T.I., 1998. In vivo analyses of cytoplasmic transport and cytoskeletal organization during Drosophila oogenesis: characterization of a multi-step anterior localization. Development 125, 3655–3666. Woodruff, R.I., Telfer, W.H., 1980. Electrophoresis of proteins in intercellular bridges. Nature 286, 84–86.
Contents lists available at ScienceDirect
Micron journal homepage: www.elsevier.com/locate/micron
Cytoskeletal organization of bee ovarian follicles during oogenesis Karina Patrício a,1 , Carminda da Cruz-Landim a,∗ , Gláucia Maria Machado-Santelli b Department of Biology, Institute of Biosciences, UNESP – Univ. Estadual Paulista, 24 Av. N◦ 1515, CEP 13.506-900, Rio Claro, SP, Brazil Department of Cell and Developmental Biology, Institute of Biomedical Sciences, University of Sao Paulo, Prof. Lineu Prestes Av., n◦ 1524, Butantan, CEP 05508-900, São Paulo, SP, Brazil a
b
a r t i c l e
i n f o
Article history: Received 17 May 2010 Received in revised form 2 August 2010 Accepted 3 August 2010 Keywords: Actin Cystocyte Honeybee Intercellular bridge Stingless bee Tubulin
a b s t r a c t The germ cells in the germarium of the bee meroistic polytrophic ovarian cysts remain interconnected by cytoplasmic bridges as a result of incomplete cell division. These intercellular bridges form a distribution pathway for the substances that initially determine which of the cystocytes will become oocyte and later conduct the products synthesized by the nurse cells to the oocyte. In the present work, the presence and distribution of cytoskeleton components, actin and tubulin were studied in ovaries of queens of Apis mellifera and Scaptotrigona postica, two eusocial species, using antibody against ␣- and -tubulin and FITC-phalloidin, aiming to shed light on the role of these cytoskeleton elements in oogenesis. The immunofluorescent preparations were analyzed by laser scanning confocal microscopy. F-actin was detected in the intercellular bridges of both species. The tubulin distribution in cell cytoplasm of A. mellifera and S. postica also displayed similar pattern. The role of these elements in the oogenetic events responsible for both cell support and motility is discussed. © 2010 Elsevier Ltd. All rights reserved.
1. Introduction Meroistic ovaries are characterized by the division of germline cells into two categories: oocytes and nurse cells. In the bee polytrophic ovary, a primordial germ cell called cystoblast divides by mitosis, resulting in a clone of cells named cystocytes. The dividing cells remain interconnected through intercellular bridges, called ring canals, which are kept opened by cytoskeletal reinforcements (King et al., 1982). Although the flow of compounds among cystocytes has been recognized, surprisingly, the cytoskeleton was not initially considered the driver of intercellular transport. Instead, intercellular electrophoresis was proposed as a mechanism for molecular transport (Woodruff and Telfer, 1980). However, although electrical phenomena have been documented in polytrophic ovaries, they are not the only mechanism of circulation of compounds inside cyst cells. Bohrmann and Biber (1994) and Gutzeit et al. (1993) demonstrated the participation of microfilaments in the differentiation of several stages of Apis mellifera ovarioles. In addition, studies on Drosophila melanogaster indicate that the factors decisively influencing oocyte differentiation are related mainly to the access of cytoplasm-specific compounds by the nucleus of the cystocyte. The
∗ Corresponding author. Tel.: +55 1935264144; fax: +55 1935264136. E-mail address: [email protected] (C. da Cruz-Landim). 1 Present address: Department of Zootechny, Laboratory of Biotechnology, USP – University of São Paulo (ESALQ), Padua Dias Av., n◦ 11, CEP 13418-900, Piracicaba, SP, Brazil. 0968-4328/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.micron.2010.08.002
access to these compounds requires their asymmetric accumulation within the future oocyte, as well as the proper organization of the cytoskeleton to provide polarity within the cyst (Cox et al., 2003), promoted by the cytoskeleton and fusoma organization in such a way as to direct the flow of cytosol constituents to a given cystocyte. Therefore, the cytoskeleton acts early on clone differentiation by determining which cell among the cystocytes will become the oocyte. Gutzeit and Huebner (1986) had already demonstrated the participation of microfilaments in the transportation of macromolecules synthesized by nurse cells to the oocyte. Therefore, when the clone cells later differentiate into oocyte and nurse cells and the oocyte begins to mature, the organization of the cytoskeleton must allows the polarized transport of compounds from the nurse cells to the oocyte cell through the ring canals (Cassidy and King, 1972; Cooley and Theurkauf, 1994; Cruz-Landim, 1978; Gutzeit et al., 1993; Staurengo-da-Cunha, 1988; Theurkauf et al., 1992; Lisboa et al., 2005). Accordingly, in the bee germline cysts, the bridges interconnecting the proliferating germ cells are reinforced by microfilaments and microtubules occupy the open spaces of the bridges (Bilinsky and Jaglarz, 1999). During the differentiation of cystocytes into oocyte and germ cells, the oocyte consistently occupies a basal position in relation to the nurse cells (Patrício and Cruz-Landim, 2006). Microfilaments, or actin filaments, and microtubules composed of tubulin are distinct functional structures of the cytoskeleton. However, numerous observations revealed that they can act together simultaneously in a wide variety of cell processes. Microtubules act in the long distance motion of cell compounds, while
56
K. Patrício et al. / Micron 42 (2011) 55–59
actin filaments provide local cytoplasmic motility (Evans et al., 1997). Pharmacological studies have shown that disorganization in one of these structures strongly affects the organization of the other, and vice versa, and electron microscopy studies have revealed connecting bridges between actin microfilaments and microtubules in vitro, indicating that they are connected both physically and functionally (Goode et al., 2000). It is therefore expected that the study of the location and distribution of these cytoskeleton elements will lead to a better understanding of the morphological changes that take place during the germline cell cycle and particularly during oocyte differentiation and growth in bees. This study focused on the distribution of actin and tubulin in the oogenetic phases in order to distinguish when and where they participate in the cytoskeleton predominantly as a static structure responsible only for the cellular structural support, and where it is a dynamic structure. 2. Materials and methods 2.1. Animals Queens of A. mellifera and Scaptotrigona postica were obtained from colonies kept in the vivarium of the UNESP – Univ. Estadual Paulista in Rio Claro, State of São Paulo, Brazil. 2.2. Immunofluorescence The immunofluorescence of ovaries was performed according to Amaral and Machado-Santelli (2008). The ovaries were removed from the insects in Dulbecco’s phosphate-buffered saline, without calcium and magnesium (PBSA–8.2 g/L NaCl) 0.2 KCl, 1.15 g/L Na2 HPO4 ·2H2 O, 0.2 g/L KH2 PO4 , pH 7.0, and fixed with 3.7% formaldehyde for 4 h. After washing (3 × 5 min) in PBS, to separate the ovarioles and withdraw most of the outer tracheolar branches the total ovaries were treated with 0.5% Triton X-100 in PBS, for 10 min at room temperature and washed again in PBS (3 × 5 min) and 10 g/mL RNAase during 30 min. Incubation in 7 L of primary antibody against ␣- and -tubulin (Amersham International) diluted 1:100 was performed in a humid chamber for 3 h. The secondary antibody used was anti-mouse IgG-FITC (Calbiochem) that was used in the same concentration and the incubation lasted 1 h. Nuclei were stained with 7.5 M propidium iodide (Sigma–Aldrich) for 15 min. The coverslips were mounted on slides with antifading (Vecta-shield, Vector Laboratories). F-actin was stained with phalloidin-FITC (Sigma–Aldrich) for 40 min. 2.3. Confocal microscopy analysis The fluorescent images were obtained using a laser scanning confocal microscope (Zeiss LSM 510) with argon, helium-neon 1 and helium-neon 2 lasers connected to an inverted fluorescence microscope (Zeiss Axiovert 100M). The emitted fluorescence was selected by a group of filters and displayed in RGB with a resolution of 512 × 512 pixels. 3. Results The two species, S. postica and A. mellifera, showed no differences in the presence and distribution of actin and tubulin. Therefore, the findings of this study are described without mentioning the species. 3.1. Distribution of F-actin The location of actin in relation to the intercellular bridges between the germline cells and in the somatic and germ cells was
Fig. 1. (A and B) Germarium of Apis mellifera queen ovary, showing cystocyte actin stained green by phalloidin-FITC and nuclei stained red by propidium iodine. Note the arrangement of actin (a) among the cystocytes forming rings, and in small amounts in the periphery of the cells. Arrows indicate actin in the peritoneal sheath. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
analyzed in the germarium and vitellarium. Actin rings between cystocytes were observed in the germarium, marking the reinforcements of the intercellular bridges, and in the cytoplasm of peritoneal membrane cells (Fig. 1A and B). The F-actin in vitellarium follicles was concentrated in the cortical zone of nurse cells, in the passage between nurse and oocyte chambers and at the interface between follicular cells and the oocyte, as well as in the peritoneal membrane (Fig. 2A–C) The orthogonal views in Fig. 2C shows clearly the distribution of actin on the bridges among the nurse cells. Smaller amounts of actin were visible in the inner cytoplasm of nurse cells (Fig. 2C), in the cor-
K. Patrício et al. / Micron 42 (2011) 55–59
57
Fig. 2. Ovarian follicle of Apis mellifera queen (A and C) and vitellarium and nurse cells of Scaptotrigona postica queen (B). Actin stained green in follicular cells (fc), nurse cells (nc), cortex and cytoplasm, oocyte (oo) and in oocyte and nurse cell chambers (arrows). Nuclei (n) in red. In C, view of orthogonal projections from the bridge between nurse and oocyte chambers. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
tex of oocyte cytoplasm, and in the bridge connecting to the nurse chamber (Fig. 2A and C). 3.2. Distribution of ˛- and ˇ-tubulin While actin is more abundant at the cell boundaries, tubulin is scattered throughout the cytoplasm. In the germarium, large amounts of tubulin are visible in the cystocyte cytoplasm, forming short rods randomly distributed and in peritoneal membrane cells (Fig. 3A and B). In the vitellarium, the tubulin fills the entire cytoplasm of the nurse chamber, follicular cells and oocyte periplasm (Fig. 3B). 4. Discussion The development of the female gamete involves complex programs leading to the production of a cell with genetic material and cytoplasm components, including molecular cues for an eventual embryonic development. The presence and action of the cytoskeleton elements during cystocyte proliferation in the germarium and oocyte ripening in the vitellarium are essential for the correct functioning of these programs. The distribution of the cytoskeletal
components in the cells provides information about the functions of these components in the various stages of oogenesis. Our findings revealed that the distribution of cytoskeleton components, actin and tubulin, during the oogenesis of the two species under study is the same and is probably also valid for bees in general, since it does not differ from what is known about other insects with meroistic polytrophic ovaries such as leaf-cut ants (do Amaral and Machado-Santelli, 2009). F-actin and ␣- and -tubulin were detected along the entire length of the peritoneal sheath of the ovariole, as expected due to the presence of muscle fibers and trachea in the sheath. The location of F-actin within the ovarian follicles possibly indicates its dual role of supporting and promoting intracellular motility. Its distribution in the germarium suggests a main role as a mechanical component of cytoskeleton that keeps open the intercellular bridges forming the so-called ring canals (Cassidy and King, 1969; King et al., 1982; Theurkauf and Hazelrigg, 1998), thus allowing for the transfer of substances among the cystocytes. Although the main role of actin in this part of the ovariole is structural, Patrício and Cruz-Landim (2006) suggested an additional function concerning the motility needed for cystocyte rearrangements in the cyst during oocyte differentiation.
58
K. Patrício et al. / Micron 42 (2011) 55–59
Fig. 3. (A) Distribution of tubulin stained green by antibody anti-␣- and anti--tubulin-FITC in germarial cells of Scaptotrigona postica queen. Nuclei in red. Note the tubulin appearing as thick rods in cystocyte cytoplasm. (B) Follicle from ovary of Scaptotrigona postica vitellarium, showing tubulin (green) in follicular cells (fc), in nurse cell (nc) cytoplasm, in communication between nurse and oocyte chambers (asterisk) and oocyte (oo) periplasm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
In the vitellarium, F-actin was observed mostly in follicles of early vitellogenic oocytes with a characteristic distribution in each type of cell. In the nurse cells, F-actin was condensed in the cortical zone and diffused in the cytoplasm. The cortical condensation of Factin suggests that it plays a supporting role in preventing early cell collapse and the ensuing impairment of the flow of molecules and small organelles from these cells to the oocyte. The actin detected in the cytoplasm is presumably involved in the transport of cell constituents among the nurse cells and toward the oocyte. Gutzeit and Huebner (1986) observed the presence of actin microfilaments in the nurse cells of 12 species of insects with meroistic telotrophic and polytrophic ovaries, with a distribution suggestive of participation in the transport of compounds throughout the cells. Theurkauf et al. (1993) believe that in Drosophila, the actin microfilaments at the periphery of nurse cells extend toward the nucleus, promoting its anchorage and forcing the flux of cytoplasm toward the oocyte. In the follicular epithelium, F-actin was found to be concentrated at the basal and apical poles of cells, which also suggests a supporting function. At the basal pole, it must act in regulating the passage of hemolymph vitellogenin through supporting the plasma membrane limiting the intercellular spaces, through which it flows to the perioocyte space, from where it will be taken up by the oocyte, while at the apical pole it acts as a support of follicular cell microvilli. The oocyte also forms microvilli and the supporting F-actin they contain may contribute to the strong staining visible throughout the surroundings of the oocyte. However, the F-actin detected in the peripheral ooplasm, mainly in the sides of the oocyte anterior pole and in the connection between the oocyte and the nurse cell chamber, may have a compound transporting function. During early vitellogenesis, the budding germinal vesicles form many accessory nuclei (Cruz-Landim, 1991; Hopkins, 1964; King and Fordy, 1970), which migrate to the posterior pole of the oocyte through the peripheral cytoplasm. The material received from the nurse cells is also transported around the oocyte, thus justifying the interpretation of involvement of actin microfilaments. Unlike F-actin, ␣- and -tubulin appear scattered throughout the cytoplasm of the germarium germ cells. Although the filaments did not show a preferential organization, the presence of ␣- and tubulin in these cells is consistent with their frequent mitosis and the flow of molecules among the cystocytes. Therefore, the tubulin detected here seems to be involved in intracellular transport, since the substances must collect around and be directed toward
the bridge passages (Spradling, 1993; Theurkauf and Hazelrigg, 1998). The ␣- and -tubulin detected in the vitellarium were scattered around the nuclei of the nurse cells, in the bridge between the nurse and oocyte chambers, and in the oocyte cortex. Again, this location is consistent with the sites where ultrastructural studies have found the presence of microtubules (Patrício and Cruz-Landim, 2006; Staurengo-da-Cunha, 1988). The RNA that passes from the nucleus into the nurse cell cytoplasm through the nuclear pore and from one nurse cell the another until reach the oocyte. As previously described, the tubulin in the passage between the nurse and oocyte chambers are involved in delivering several nurse cell products, RNA and proteins to the oocyte (Bilinsky and Jaglarz, 1999; Cooley and Theurkauf, 1994; Theurkauf and Hazelrigg, 1998). The presence of tubulin in the oocyte cortex might indicate functions in the previtellogenic and vitellogenic oocyte. In the previtellogenic oocyte, it probably indicates a function of the microtubules in the migration of accessory nuclei originated from outgrowths of the germinal vesicle to the posterior pole of the oocyte, which occurs through the oocyte cortex (Bilinsky et al., 1995). In the vitellogenic oocyte, it could be involved in the transportation through the cytoplasm of yolk spheres formed by endocytic uptake of vitellogenin from the hemolymph in perioocytic space. Oogenesis is known to be important in the formation of the pattern of embryonic development through the acquisition and correct distribution of macromolecules in the oocyte, for which the cytoskeleton is the major structure responsible. The major role of actin in oogenesis seems to be providing structural support, keeping the passage through the ring canals open. However, tubulin in areas through which substances travel into the oocyte, as was observed here, ascribes to it an additional role in inter- and intracellular transport. Otherwise, tubulin (microtubules) has only transportation functions, working in chromosome displacement during cell division or directing the cytoplasmic flow through the interconnecting bridges between the nurse cells and from these to the oocyte. Thus, actin (microfilaments) and tubulin (microtubules) play important roles in oocyte growth, and later, in the distribution of the morphogenetic determinants throughout the ooplasm (Theurkauf et al., 1993; Theurkauf, 1994). The tubulin in the bridges may be the remains of mitotic spindle and therefore located in the fusome, as has been revealed in transmission electron microscopy
K. Patrício et al. / Micron 42 (2011) 55–59
images (Patrício and Cruz-Landim, 2006). Impaired development of fusome hinders oocyte growth (Cuevas et al., 1996; Hartfeler and Steinbück, 1997; Patrício and Cruz-Landim, 2006; Tanaka and Hartfelder, 2004). The role of the circulation of substances among cystocytes, as well the direction of their flow, is essential in determining which cystocyte will become the oocyte (Büning, 1994; Cuevas et al., 1996; Telfer, 1975; Theurkauf et al., 1992). One of the two cystocytes linked to the others through a greater number of bridges will become an oocyte, possibly because it receives a larger amount of the circulating substances. The location of actin and tubulin in bee ovaries was consistent with previous findings about the ultrastructure and function attributed to these cytoskeletal components during ovarian development. Acknowledgments The authors acknowledge the financial support of the Brazilian research funding agencies FAPESP (Fundac¸ão de Amparo à Pesquisa do Estado de São Paulo) and CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico). References Amaral, J.B., Machado-Santelli, G.M., 2008. Salivary system in leaf-cutting ants (Atta sexdens rubropilosa, Forel, 1908) castes: A confocal study. Micron 39, 1222–1227. Amaral, J.B., Machado-Santelli, G.M., 2009. Three-dimensional reconstruction of ovaries of leaf-cutting ant (Atta sexdens rubropilosa) queens. Sociobiology 53, 379–388. Bilinsky, S.M., Klag, J., Kubrakiewcz, J., 1995. Subcortical microtubule network separates periplasm from endoplasm and is responsible for maintaining the position of accessory nuclei in hymenopteran oocytes. Roux’s Arch. Dev. Biol. 205, 54–61. Bilinsky, S.M., Jaglarz, M.K., 1999. Organization and possible functions of microtubule cytoskeleton in hymenopteran nurse cells. Cell Motil. Cytoskeleton 43, 213–220. Bohrmann, J., Biber, K., 1994. Cytoskeleton-dependent transport of cytoplasmic particles in previtellogenic to mid-vitellogenic ovarian follicles in Drosophila: time-lapse analysis using video-enhanced contrast microscopy. J. Cell Sci. 107, 849–858. Büning, J., 1994. The Insect Ovary: Structure, Previtellogenic Growth and Evolution, 5a ed. Chapmam & Hall, London. Cassidy, J.D., King, R.C., 1969. The dilatable ring canals of the ovarian cystocytes of Habrobracon juglandis. Biol. Bull. 137, 429–437. Cassidy, J.D., King, R.C., 1972. Ovarian development in Habrobacron juglandis (Ashemed) (Hymenoptera, Braconidae). 1. The origin and differentiation of the oocyte–nurse cell complex. Biol. Bull. 143, 483–505. Cooley, L., Theurkauf, W.E., 1994. Cytoskeleton functions during Drosophila oogenesis. Science 266, 590–596. Cox, D., Lu, B., Sun, T., Williams, L., Jan, Y., 2003. Drosophila par-1 is required for oocyte differentiation and microtubule organization. Curr. Biol. 11, 75–87.
59
Cuevas, M., Lee, J.K., Spradling, A.C., 1996. a-spectrin is required for germline cell division and differentiation in the Drosophila ovary. Development 122, 3959–3968. Cruz-Landim, C., 1978. Structural dynamics of oogenesis in Atta sexdens rubropilosa (Hymenoptera, Formicidae). Rev. Bras. Biol. 38, 363–438. Cruz-Landim, C., 1991. Accessory nuclei in hymenopteran oocytes and origin of the germ plasm: a study of Melipona quadrifasciata anthidioides Lep. (Hymenoptera, Apidae, Meliponinae) and Atta sexdens rubropilosa Forel (Hymenoptera, Formicidae, Attinae). Naturalia 16, 171–182. Evans, L.L., Hammer, J., Bridgman, P.C., 1997. Subcellular localization of myosin V in nerve growth cones and outgrowth from dilute-lethal neurons. J. Cell Sci. 110, 439–449. Goode, B.L., Drubin, D.G., Barnes, G., 2000. Functional cooperation between the microtubule and actin cytoskeletons. Curr. Opin. Cell Biol. 12 (1), 63–71. Gutzeit, H.O., Huebner, E., 1986. Comparison of microfilaments patterns in nurse cells of different insects with polytrophic and telotrophic ovarioles. J. Embryol. Exp. Morphol. 93, 291–301. Gutzeit, H.O., Zissler, D., Feig, R., 1993. Oogenesis in the honeybee Apis mellifera: cytological observations on the formation and differentiation of previtellogenic ovarian follicles. Roux’s Arch. Dev. Biol. 202, 181–191. Hartfeler, K., Steinbück, G., 1997. Germ cells cluster formation and cell death in caste-specific differentiation of the larval honey bee ovary. Invertebr. Reprod. Dev. 31, 45–77. Hopkins, C.R., 1964. The histochemistry and fine structure of the accessory nuclei in the oocyte of Bombus terrestris. Quart. J. Microsc. Sci. 102, 475– 480. King, R.C., Fordy, M.R., 1970. The formation of accessory nuclei in the developing oocytes of parasitoid hymenopterans Ophion luteus (L.) and Apanteles glomeratus (L.). Z. Zellforsch. 109, 158–170. King, R.C., Cassidy, J.D., Rousset, A., 1982. The formation of clones of interconnected cells during gametogenesis in insects. In: King, R.C., Akai, H. (Eds.), Insect Ultrastructure. Plenum, New York, pp. 3–33. Lisboa, L.C.O., Serrão, J.E., Cruz-Landim, C., Campos, L.A.O., 2005. Effect of larval food amount on ovariole development in queens of Trigona spinipes (Hymenoptera, Apinae). Anat. Histol. Embryol. 34, 179–184. Patrício, K., Cruz-Landim, C., 2006. Ultrastructural aspects of the intercellular bridges between female bee germ cells. Braz. J. Biol. 66 (1B), 309–315. Spradling, A.C., 1993. Developmental genetics of oogenesis. In: Bate, M., Martinez Arias, A. (Eds.), The Development of Drosophila melanogaster. Cold Spring Harbor Press, New York, pp. 1–70. Staurengo-da-Cunha, M.A., 1988. Ultrastructure of the oocytes of Scaptotrigona postica workers (Hymenoptera, Apidae). Naturalia 13, 103–115. Tanaka, E.D., Hartfelder, K., 2004. The initial stages of oogenesis and their relation to differential fertility in the honey bee (Apis mellifera) castes. Arthropode Struct. Dev. 33, 431–442. Telfer, W.H., 1975. Development and physiology of the oocyte-nurse cell syncytium. Adv. Insect Physiol. 11, 223–319. Theurkauf, W.E., 1994. Microtubules and cytoplasm organization during Drosophila oogenesis. Dev. Biol. 165, 352–360. Theurkauf, W.E., Smiley, S., Wong, M.L., Alberts, B.M., 1992. Reorganization of the cytoskeleton during Drosophila oogenesis: implications for axis specification and intercellular transport. Development 115, 923–936. Theurkauf, W.E., Alberts, B.M., Jan, Y.N., Jongens, T.A., 1993. A central role for microtubules of Drosophila oocytes. Development 118, 1169–1180. Theurkauf, W.E., Hazelrigg, T.I., 1998. In vivo analyses of cytoplasmic transport and cytoskeletal organization during Drosophila oogenesis: characterization of a multi-step anterior localization. Development 125, 3655–3666. Woodruff, R.I., Telfer, W.H., 1980. Electrophoresis of proteins in intercellular bridges. Nature 286, 84–86.