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Formation of the acrosome complex in the bush cricket Gampsocleis gratiosa (Orthoptera: Tettigoniidae)
Arthropod Structure & Development xxx (2017) 1e9
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Formation of the acrosome complex in the bush cricket Gampsocleis gratiosa (Orthoptera: Tettigoniidae) Cai Xia Su, Jie Chen, Fu Ming Shi, Ming Shen Guo, Yan Lin Chang* College of Life Sciences, Hebei University, Baoding 071002, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 September 2016 Accepted 11 January 2017 Available online xxx
The acrosome complex plays an indispensable role in the normal function of mature spermatozoa. However, the dynamic process of acrosome complex formation in insect remains poorly understood. Gampsocleis gratiosa Brunner von Wattenwyl possesses the typical characteristic of insect sperms, which is tractable in terms of size, and therefore was selected for the acrosome formation study in this report. The results show that acrosome formation can be divided into six phases: round, rotating, rhombic, cylindrical, transforming and mature phase, based on the morphological dynamics of acrosome complex and nucleus. In addition, the cytoskeleton plays a critical role in the process of acrosome formation. The results from this study indicate that: (1) glycoprotein is the major component of the acrosome proper; (2) the microfilament is one element of the acrosome complex, and may mediate the morphologic change of the acrosome complex; (3) the microtubules might also shape the nucleus and acrosome complex during the acrosome formation. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Acrosome formation Glycoprotein Microfilament Microtubule Gampsocleis gratiosa
1. Introduction The acrosome complex covers the nucleus in front of the spermatid head, located between nucleus and plasma membrane. It is an organelle derived from Golgi secretion (Berruti and Paiardi, 2011; Fabian and Brill, 2012; Chapman, 2013; Yasuno et al., 2013) and plays an important role in maintaining sperm structural stability and sperm-egg binding (Phillips, 1970; Souza et al., 2009). The morphology, size and internal structure of acrosome complex vary substantially among different species (Baccetti and Afzelius, 1976; Dallai, 2014). Among insects, in general the acrosome complex is a small and elaborate structure. Perforatorium is present, consisting of filamentous dense material in many insects (Dallai, 2014). In several families of the Ensifera and Caelifera of Orthoptera, some residual material, called extra-acrosomal layer, surrounds the acrosome proper (Jamieson, 1987). As such, the acrosome complex is composed of acrosome proper, perforatorium and extra-acrosomal layer, which is defined as a three-
Abbreviations: A, acrosome; AC, acrosome complex; EA, extra-acrosomal layer; G, Golgi apparatus; N, nucleus; P, perforatorium; PA, pro-acrosome; PAS, Periodic Acid-Schiff. * Corresponding author. E-mail addresses: [email protected] (C.X. Su), [email protected] (Y.L. Chang).
layered acrosome. In some species, however, the spermatid acrosome complex is only bi-layered or mono-layered (Dallai et al., 2002; Lupetti et al., 2011) and the acrosome has even been lost in a few species (Dallai, 2014; Dallai et al., 2016). Acrosome formation is one of the most prominent events for spermatogenesis. Initially, Golgi apparatus continuously secretes vesicles which gather into the condensed and spherical proacrosome. Then, pro-acrosome gradually moves, contacts to nucleus and adheres to and expands along the nucleus. Ultimately, in the form of acrosome complex, it covers on anterior end of the nucleus surface in the mature spermatozoa (Lin and Rodger, 1999; Berruti and Paiardi, 2011; Wang et al., 2014). In Orthoptera, the acrosome formations of Acheta domesticus and Locusta migratoria have been previously studied. In L. migratoria, the proacrosome appears and approaches the nucleus followed by formation of rodlet perforatorium between the acrosomal cone and nucleus, and subsequently a cup covers the outside of acrosome complex (Jamieson, 1987). The acrosome complex of A. domesticus is quite elaborate and the acrosome formation undergoes more sophisticated transformation during which the proacrosome anchors on the nucleus and rotates (Clayton et al., 1958). The structure of spermatozoa in the Chinese bush cricket Gampsocleis gratiosa (G. gratiosa) Brunner von Wattenwyl is fairly elaborate. The acrosome complex consists of an apical vesicle and
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Please cite this article in press as: Su, C.X., et al., Formation of the acrosome complex in the bush cricket Gampsocleis gratiosa (Orthoptera: Tettigoniidae), Arthropod Structure & Development (2017), http://dx.doi.org/10.1016/j.asd.2017.01.002
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two big expansive wings, with appearance like an arrow (Chang et al., 2011), which is in line with the typical characteristics of sperm acrosome in Tettigoniidae. In addition, the acrosome complex of G. gratiosa is relatively large compared with those in other species, therefore, it is easy to observe under a microscope. Thus, G. gratiosa may be used as a model organism to illuminate the process of the acrosome formation in Tettigoniidae. In this study, we examined components of the acrosome complex, and recorded the dynamic course thereof in G. gratiosa by PAS (Periodic Acid-Schiff) staining, fluorescence labeling and transmission electronic microscopy. The acrosome formation in G. gratiosa was divided into six phases. And it is demonstrated that the glycoprotein is the major component of acrosome proper, that the microfilament also is present in the acrosome complex and contributes to its formation and that microtubules may play a crucial role in shaping the acrosome complex as well as the nucleus. This study may lead to a better understanding of the fertilization process and provide a valuable model for studying mechanisms underlying the acrosome biogenesis during spermiogenesis in Tettigoniidae. 2. Materials and methods
Finally, these sections were observed by Olympus BX51 fluorescence microscope and pictures were synthetized with Adobe Photoshop CS5. 2.4. Transmission electron microscopy The testes were rapidly isolated and fixed in 2.5% glutaraldehyde for 1 h at 4 C, rinsed in phosphate buffer (pH 7.4), and postfixed in OsO4 for 2 h at 4 C. After being ruined, the materials were dehydrated in a graded series of ethyl alcohol (30%e100%), transferred to acetone and finally embedded in Epon812. Semithin sections (0.7 mm) were stained with 1% toluidine blue before visualization under light microscope. Ultrathin sections (70 nm) were stained routinely with uranyl acetate and lead citrate before visualization under JEM-100SX electron microscope operating at 80 kV. 3. Results Based on the dynamic alterations of the morphology and positions of the pro-acrosome, microfilament and nucleus, the acrosome formation in G. gratiosa was divided into six phases: round, rotating, rhombic, cylindrical, transforming and mature phase.
2.1. Specimens 3.1. The round phase Sexually mature males of G. gratiosa were collected in JulyeSeptember 2015, at Dabei, Shunping, Hebei, China. Each adult male bush cricket was bred in single cage, where the conditions (temperature, moisture, photoperiod) imitated their habitat environment. 2.2. Light microscopy The isolated testes were fixed in Bouin's fluid at room temperature, dehydrated in a graded series of ethyl alcohol and embedded in paraffin. The serial sections (7 mm) were made and mounted on microscope slides, stained with PAS (Periodic AcidSchiff) and hematoxylin, and observed by Olympus BX51 optical microscope.
At the round phase, the nucleus and pro-acrosome were round. When the round spermatid was generated just after meiosis, the pro-acrosome appeared in a form of a very small spherical structure (Fig. 1A and B). Then, it gradually enlarged and moved close to the nucleus, due to the continuous secretion from Golgi apparatus (Fig. 1DeF). At the beginning of the round phase, the nucleus was large and round and was unevenly filled with genetic material. The condensed heterochromatin was partly depolymerized (Fig. 1AeC). With the sperm development, genetic material within the nucleus redistributed and developed into a ring (Fig. 1DeF). The ultrastructure analysis showed that the electron density of pro-acrosome was higher than in the nucleus and that the Golgi apparatus lay between the nucleus and pro-acrosome (Fig. 1C, F).
2.3. Fluorescence microscopy 3.2. The rotating phase The testes were fixed in fresh 4% paraformaldehyde for 24 h, dehydrated in 0.5 mol/L sucrose solutions and embedded in the optimal cutting temperature (O.C.T.) compound at 22 C. Then, the specimens were cut into 5-mm frozen section and transferred to slide. The sections were treated with 1% Triton X-100 for 30 min at room temperature. After washing in phosphate buffer saline (pH 7.3), the specimens were successively stained with 0.01% phalloidin-rhodamine for microfilament 45 min and 40 ,6diamidino-2-phenylindole (DAPI) for nucleus 15 min. Finally, these sections were observed by Olympus BX51 fluorescence microscope. The separated testes were ground and centrifuged at low speed in physiological saline to remove chunks of tissues and the supernatant was recentrifuged at 249 g to precipitate the cells. The cell pellet was fixed in fresh 4% paraformaldehyde for 1 h, rinsed in phosphate buffer saline (pH 7.3), and checked for density under microscope and smeared the sperms on the slide. After being treated with 1% Triton X-100 for 30 min at room temperature, washed in PBS, the smear was immersed in 5% bovine serum albumin (BSA) in PBS for 1 h to block nonspecific sites, and washed in PBS. The cells were then incubated in anti-a-tubulin antibody conjugated with fluorescein isothiocyanate (SigmaeAldrich) at 1:100 dilutions for 1 h. Then they were stained as above methods.
At the rotating phase, the pro-acrosome contacted the nucleus and the nucleus condensed to the minimum in the whole process. Genetic material within spermatid nucleus was compact and homogeneous (Fig. 2AeD). The pro-acrosome rotated by adhering to nucleus until arriving at the top of the nucleus (Fig. 2A and B). In the process of rotation, the pro-acrosome reached its maximal diameter. The pro-acrosome and nucleus attached or separated in the semithin section and ultrathin section. The Golgi apparatus disappeared at this stage (Fig. 2D). 3.3. The rhombic phase At the rhombic phase, namely after rotation, the spermatid head was rhombic (Fig. 3A, D). The pro-acrosome transformed into acrosome proper after complete rotation. The acrosome proper which was located at the top of the nucleus began to extend along the two sides of the nearly rhombic nucleus (Fig. 3A and B) and the genetic material within the nucleus redistributed again. Genetic material in the anterior nucleus was dense, whereas the electron density of the posterior nucleus was low (Fig. 3A, D). The frozen section stained with phalloidinerhodamine shows that a small spherical microfilament
Please cite this article in press as: Su, C.X., et al., Formation of the acrosome complex in the bush cricket Gampsocleis gratiosa (Orthoptera: Tettigoniidae), Arthropod Structure & Development (2017), http://dx.doi.org/10.1016/j.asd.2017.01.002
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Fig. 1. Spermatid of G. gratiosa at the round phase. A, D: Paraffin section stained with PAS and hematoxylin. B, E: Semithin section stained with toluidine blue. C, F: Ultrathin sections. The arrow points to the nucleus (N) and the arrowhead points to the pro-acrosome (PA). G: Golgi apparatus. Scale bars: A, D, 50 mm; B, E, 10 mm; C, 500 nm; F, 2 mm.
appeared at the anterior of nucleus (Fig. 3C). The ultrathin section shows a small spherical perforatorium between the acrosome proper and the nucleus (Fig. 3C and D). The acrosome proper and the perforatorium constituted the incomplete acrosome complex at this stage.
forming a cone at the apex of the nucleus (Fig. 4C). The ultrathin section shows that the electron density of nuclear envelope at the two sides of nucleus is significantly increased.
3.4. The cylindrical phase
At the transforming phase, the acrosome proper was located at the top of nucleus and resembled an arrow (Fig. 5A and B). The frozen section labeled with phalloidine-rhodamine shows microfilaments transformed into the inverted ‘Y’ appearance from the cone (Fig. 5C). The frozen section marked with DAPI for nucleus, phalloidine-rhodamine for microfilament and a-tubulin antibody for microtubule indicates that the microfilament and nucleus are surrounded by microtubules (Fig. 5D). The electron density of the acrosome proper decreased and an extra-acrosomal layer was
At the cylindrical phase, the spermatid head was cylindrical, and its volume distinctly increased (Fig. 4AeD). The acrosome proper further extended along the two sides of nucleus and the perforatorium beneath the acrosome proper enlarged. The genetic material within the nucleus still tended to concentrate in the anterior nucleus (Fig. 4AeB, D, F). The frozen section stained with phalloidine-rhodamine shows microfilaments enlarged and
3.5. The transforming phase
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Fig. 2. Spermatid of G. gratiosa at the rotating phase. AeB: Paraffin sections stained with PAS and hematoxylin. A: Pro-acrosome (PA) contacting the nucleus (N) and just starting rotation. B: Pro-acrosome located at the top of nucleus. C: Semithin section stained with toluidine blue. D: ultrathin section. The arrow points to the nucleus (N) and the arrowhead points to the pro-acrosome (PA). Scale bars: AeB, 50 mm; C, 25 mm; D, 2 mm.
formed (Fig. 5E). The acrosome proper, perforatorium and extraacrosomal layer constituted the complete acrosome complex. Meanwhile, the ultrastructure analysis showed that the genetic material within the nucleus condensed into a lot of visible granules under transmission electron microscope and that microtubules existed around the nucleus and acrosome complex (Fig. 5F). 3.6. The mature phase The mature phase was the last phase of acrosome formation. At this stage, the spermatid turned to mature spermatozoon by excluding several ‘redundant’ constituents (Fig. 6A and B). The frozen section stained with phalloidine-rhodamine shows the microfilaments presenting the shape of an arrow at the top of the nucleus, the angle of arrow being greater than that of an inverted ‘Y’ (Fig. 6C). The angles from the two wings of both the polysaccharide and the acrosome complex gradually enlarged and the genetic material within the nucleus continued to compress and come to be the uniform and compact spermatozoon nucleus. The microtubules around nucleus and acrosome complex were no longer observable under the transmission electron microscope (Fig. 6D). 4. Discussion
acrosomes in Arvelius albopunctatus. Souza and Itoyama (2010) studied the acrosome formation of five pentatomids in Hemiptera by PAS staining. In our study, the results of PAS staining and transmission electron microscopy indicate that polysaccharide coincide well with the pro-acrosome or acrosome proper in size and morphology. Polysaccharide always combines with protein or lipid to form glycoproteins or glycolipids in the cell. The acrosome complex produces a skeleton glycoprotein at the head of sperm in insects (Klowden, 2007). Thus, glycoprotein is the primary component of pro-acrosome and acrosome proper. Many studies showed that microfilaments exist in the vertebrate spermatid (Breed and Leigh, 1991; Scarlett et al., 2001; Dvorakova et al., 2005; Sperry, 2012). In invertebrates, microfilaments are also present in the acrosome complex of the spermatid and the morphology of the acrosome complex is modified by the dynamic changes of the cytoskeleton protein surrounding it (Guerra and Esponda, 1999; Li et al., 2010; Trovato et al., 2011; Dallai, 2014; Viscuso et al., 2016). In this study, the frozen sections stained with phalloidine-rhodamine showed that microfilaments existed in the acrosome complex (Figs. 3C, 4C, 5CeD, 6C). In the sperm at mature phases, the arrow shaped microfilaments always existed at the anterior side of the nucleus (Fig. 6C). Therefore, microfilaments may be a component of the perforatorium.
4.1. Components of the acrosome complex
4.2. Acrosome formation
As early as 1951, Schrader and Leuchtenberger demonstrated by PAS staining that polysaccharides are components of the
In Tettigoniidae, the acrosome complex is characterized by big size and a complicated structure consisting of the acrosome
Please cite this article in press as: Su, C.X., et al., Formation of the acrosome complex in the bush cricket Gampsocleis gratiosa (Orthoptera: Tettigoniidae), Arthropod Structure & Development (2017), http://dx.doi.org/10.1016/j.asd.2017.01.002
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Fig. 3. Spermatid of G. gratiosa at the rhombic phase. A: Paraffin section stained with PAS and hematoxylin. B: Semithin section stained with toluidine blue. C: Frozen section marked with DAPI for nucleus (blue) and phalloidine-rhodamine for microfilament (red). D: Ultrathin section. The arrow points to the nucleus (N); the dark arrowhead points to the acrosome proper or incomplete acrosome complex (AC) and the white arrowhead points to microfilament. P: perforatorium. A: acrosome proper. Scale bars: A, 50 mm; BeC, 10 mm; D, 2 mm.
proper, perforatorium and extra-acrosomal layer (Baccetti, 1987; Guerra and Esponda, 1999; Chang et al., 2011). Moreover, the acrosome proper is the major part of the acrosome complex in Tettigoniidae. Therefore, tracing glycoprotein through PAS staining can be used to study the acrosome formation. The acrosome formation is a complex and dynamic process, that includes the formation and morphological changes of different structural components. Combining the results from light microscopy, transmission electron microscopy and fluorescence microscopy, a working model of the whole formation process of acrosome complex which emphasizes acrosome proper, nucleus and microfilament, is proposed (Fig. 7). The acrosome formation starts with the round spermatid from meiosis. In the whole process, acrosome formation is initiated by Golgi secretion, which gathers into the densely spherical pro-acrosome. With the progress of spermiogenesis, pro-acrosome constantly enlarges, approaches and attaches to nucleus (Fig. 7a). Afterward, it rotates by adhering to the nucleus and becomes the acrosome proper (Fig. 7b). Subsequently, a perforatorium is produced and enlarges beneath the acrosome proper. Meanwhile, an extra-acrosomal layer is produced on the outside of the acrosome proper.
Eventually, the acrosome complex transforms into an arrow in the mature spermatozoon. Moreover, microfilaments participate in the formation of the acrosome complex as a component. After appearance at the rhombus phase, it undergoes the morphological transformation from a sphere, to a cone, the inverted ‘Y’, and finally into an arrow (Fig. 7c-f). In insects, the small size of sperm acrosome complex makes it difficult to observe via light microscopy. This may account for why there is only limited knowledge regarding the dynamic process of acrosome formation in insects. In the process of acrosome formation in Euschistus heros F. (Pentatomidae, Heteroptera), a central polysaccharide granule migrates to one pole of the spermatid and becomes elongated until it is indistinguishable in the spermatozoa (Souza and Itoyama, 2010). In Orthoptera, the acrosome formations of L. migratoria and A. domesticus have been studied. In L. migratoria, the pro-acrosome which has no direct link to the Golgi apparatus adheres to the nuclear membrane and glides in a posterior-anterior direction around the nucleus. It is deemed that the process depends on the inherent fluidity of the nuclear membranes (Jamieson, 1987). In A. domesticus, pro-acrosome deriving from the Golgi secretion
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Fig. 4. Spermatid of G. gratiosa at the cylindrical phase. A: Paraffin section stained with PAS and hematoxylin. B: Semithin section stained with toluidine blue. C: Frozen section marked with DAPI for nucleus (blue) and phalloidine-rhodamine for microfilament (red). DeF: Ultrathin section. The arrow points to the nucleus (N); crossed arrow points to perforatoriums (P); the dark arrowhead points to the acrosome proper or acrosome complex (AC) and the white arrowhead points to microfilament. A: acrosome proper. Scale bars: A, 50 mm; BeC, 10 mm; D, 500 nm; EeF, 2 mm.
adheres and rolls on the surface of nucleus until arriving at the anterior end of nucleus. It is thought that the rolling of proacrosome may be due to rotation of the nucleus through 180 (Clayton et al., 1958). The discovery in G. gratiosa is in accordance with the report in the A. domesticus. The proacrosome migrates along a 180 circle by adhering to the nucleus and the final arrow-shaped acrosome complex is formed at the front of the nucleus. However, the mechanism of rotation still needs further research. 4.3. Cytoskeleton in the acrosome complex Many aspects of morphogenesis including acrosome formation are dependent on the cytoskeleton (Xiao and Yang, 2007). The changes of morphology and position of the cytoskeleton during spermiogenesis have been studied in many vertebrates and invertebrates (Breed and Leigh, 1991; Scarlett et al., 2001; Noguchi and Miller, 2003; Sahara and Kawamura, 2004; Dvorakova et al., 2005; Desai et al., 2009; Sperry, 2012; Viscuso et al., 2016). In mammalian sperm, acroplaxome, containing actin and keratin, anchores and shapes the acrosome (Kierszenbaum et al., 2003; Snigirevskaia et al., 2012). In Caridean shrimp, acroframosome consisting of microfilament and microtubules functions as a framework during acrosome formation (Li et al., 2010; Sun et al., 2011). In Tettigoniidae, microfilament of sperm in the Tylopsis liliifolia is distributed along the entire acrosomal region during nucleus elongation, however, the microtubules are located at the two wings of the acrosome and
around the nucleus (Viscuso et al., 2016). In Platycleis albopunctata, acrosomal wings, giving spermatozoa the shape of an arrow, contain microfilament and microtubules (Guerra and Esponda, 1999). Our results demonstrate that the microfilament is invisible at round phase, and emerges along with the perforatorium after migration of the pro-acrosome. Then microfilament undergoes dynamic changes from the spherical, cone, inverted ‘Y’ to arrow shape (Figs. 2C, 3C, 4CeD, 5C). The acrosome proper and perforatorium gradually expands to the sides of nucleus along the morphologic change of microfilament. The position and morphology of microfilament in mature spermatozoa is consistent with the previous result in P. albopunctata (Guerra and Esponda, 1999). However, the dynamic changes and the function of microfilament resemble the acroframosome in caridean shrimp which functions as a framework for acrosome biogenesis (Li et al., 2010; Sun et al., 2011). Therefore, it is speculated that the microfilament supports the morphological changes of acrosome complex as a component of acrosome complex. Moreover, it has been shown that the spermatid could transport some substances through actin and microtubules in the process of mammal spermatogenesis (Kierszenbaum et al., 2011). Therefore, we suppose microfilament may participate in the transportation of acrosome proper in G. gratiosa as well. At the transforming phase of the acrosome formation of G. gratiosa, the genetic material of the nucleus rapidly aggregated and gave rise to a mass of visible granules under the transmission electron microscope. The complete acrosome complex was
Please cite this article in press as: Su, C.X., et al., Formation of the acrosome complex in the bush cricket Gampsocleis gratiosa (Orthoptera: Tettigoniidae), Arthropod Structure & Development (2017), http://dx.doi.org/10.1016/j.asd.2017.01.002
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Fig. 5. Spermatid of G. gratiosa at the transforming phase. A: Paraffin section stained with PAS and hematoxylin. B: Semithin section stained with toluidine blue. CeD: Frozen section marked with DAPI for nucleus (blue), phalloidine-rhodamine for microfilament (red) and a-tubulin antibody for microtubule (green). EeF: Ultrathin section. The arrow points to the nucleus (N); the dark arrowhead points to the acrosome proper or acrosome complex (AC); the white arrowhead points to microfilament and the double arrows point to microtubules. The dotted lines in D indicate the probable plane of section shown in Fig. 6E and F. A: acrosome proper. EA: extra-acosome layer. Scale bars: A, 25 mm; BeD, 10 mm; EeF, 500 nm.
formed in a great morphological transformation. The microfilament supported the acrosome complex in the form of inverted ‘Y’ and microtubules surrounded nucleus and acrosome complex. However, the microtubules disappeared in the mature spermatozoa. In mammalian and Octopus tankahkeei, the perinuclear microtubules form a transient structure, called manchette, and the complex is indispensable for acquisition of the final nuclear morphology (Kierszenbaum and Tres, 2004; Ribes et al., 2002; Wang et al., 2010). In G. gratiosa, the microtubules around nucleus and acrosome complex are analogous to those of the manchette and they are also essential for the formation of the final morphology of nucleus and acrosome complex. Thus, we deem the transforming phase is the key stage of acrosome
formation in G. gratiosa (Fig. 8). The microtubules may also play an influential role to shape the nucleus and construct the morphology of acrosome complex. To conclude the results lead us to propose that: (1) glycoprotein is the major component of acrosome proper; (2) the microfilament may support the morphological changes of the acrosome complex as one of its components; (3) the microtubules may also play an influential role to shape the nucleus and acrosome complex; (4) the transforming phase is the key period of acrosome formation in G. gratiosa. Further, for the first time, we unveiled the process of acrosome formation in G. gratiosa, which may contribute to shedding light on acrosome biogenesis mechanisms involved in spermiogenesis in other insects.
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Fig. 6. Spermatid of G. gratiosa at the mature phase. A: Paraffin section stained with PAS and hematoxylin. B: Semithin section stained with toluidine blue. C: Frozen section marked with DAPI for nucleus (blue) and phalloidine-rhodamine for microfilament (red). D: Ultrathin section. The arrow points to the nucleus (N); the dark arrowhead points to the acrosome proper or acrosome complex (AC) and the white arrowhead points to microfilament. The stars indicate the ‘redundant’ constituents. A: acrosome proper. Scale bars: A, 25 mm; BeC, 10 mm; D, 500 nm.
Fig. 7. The model of acrosome formation in G. gratiosa. Morphological differentiation of acrosome proper, nucleus, and microfilament are emphasized. a: round phase. b. rotating phase. c: rhombic phase. d: cylindrical phase. e: transforming phase. f: mature phase. Blue represents nucleus; dark gray represents pro-acrosome or acrosome proper; red represents microfilament.
Please cite this article in press as: Su, C.X., et al., Formation of the acrosome complex in the bush cricket Gampsocleis gratiosa (Orthoptera: Tettigoniidae), Arthropod Structure & Development (2017), http://dx.doi.org/10.1016/j.asd.2017.01.002
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Fig. 8. The model of spermatid microtubule in G. gratiosa at the transforming phase. Microtubules surrounding the nucleus and acrosome proper are emphasized. Blue represents nucleus; dark gray represents acrosome proper; red represents microfilament; green stands for microtubules.
Acknowledgements We would like to express our special thanks to Dr. Romano Dallai (Italy) for advising to modify the picture and adjust the article structure. In addition, we are grateful to Dr. Shuai Chen (Germany), Dr. Xue Feng Liu (U.S.A.) and Dr. Xiao Hong Su (China) for their critical reading and English improvement of the previous version. This study was supported by the National Natural Science Foundation of China (No. 31372232, 31471985). References Baccetti, B., 1987. Spermatozoa and phylogeny in orthopteroid insects. In: Baccetti, B. (Ed.), Evolutionary Biology of Orthopteroid Insects. Horwood, Chichester, UK, pp. 12e112. Baccetti, B., Afzelius, B.A., 1976. The biology of the sperm cell. Monogr. Dev. Biol. 10, 1e254. Berruti, G., Paiardi, C., 2011. Acrosome biogenesis: revisiting old questions to yield new insights. Spermatogenesis 1, 95e98. Breed, W.G., Leigh, C.M., 1991. Distribution of filamentous actin in and around spermatids and in spermatozoa of Australian Conilurine rodents. Mol. Reprod. Dev. 30, 369e384. Chang, Y.L., Wang, L., Guo, M.S., Zhang, X.X., 2011. Comparative study of sperm ultrastructure between Gampsocleis sedakovii and Gampsocleis gratiosa. Acta Zootaxonomica Sin. 36, 664e669. Chapman, R.F., 2013. The Insects: Structure and Function, fifth ed. Cambridge Univ. Press, Cambridge. Clayton, B.P., Deutsch, K., Jordan-Luke, B.M., 1958. The spermatid of the housecricket, Acheta domesticus. Q. J. Microsc. Sci. 99, 15e23. Dallai, R., 2014. Overview on spermatogenesis and sperm structure of Hexapoda. Arthropod Struct. Dev. 43, 257e290.
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Formation of the acrosome complex in the bush cricket Gampsocleis gratiosa (Orthoptera: Tettigoniidae) Cai Xia Su, Jie Chen, Fu Ming Shi, Ming Shen Guo, Yan Lin Chang* College of Life Sciences, Hebei University, Baoding 071002, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 September 2016 Accepted 11 January 2017 Available online xxx
The acrosome complex plays an indispensable role in the normal function of mature spermatozoa. However, the dynamic process of acrosome complex formation in insect remains poorly understood. Gampsocleis gratiosa Brunner von Wattenwyl possesses the typical characteristic of insect sperms, which is tractable in terms of size, and therefore was selected for the acrosome formation study in this report. The results show that acrosome formation can be divided into six phases: round, rotating, rhombic, cylindrical, transforming and mature phase, based on the morphological dynamics of acrosome complex and nucleus. In addition, the cytoskeleton plays a critical role in the process of acrosome formation. The results from this study indicate that: (1) glycoprotein is the major component of the acrosome proper; (2) the microfilament is one element of the acrosome complex, and may mediate the morphologic change of the acrosome complex; (3) the microtubules might also shape the nucleus and acrosome complex during the acrosome formation. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Acrosome formation Glycoprotein Microfilament Microtubule Gampsocleis gratiosa
1. Introduction The acrosome complex covers the nucleus in front of the spermatid head, located between nucleus and plasma membrane. It is an organelle derived from Golgi secretion (Berruti and Paiardi, 2011; Fabian and Brill, 2012; Chapman, 2013; Yasuno et al., 2013) and plays an important role in maintaining sperm structural stability and sperm-egg binding (Phillips, 1970; Souza et al., 2009). The morphology, size and internal structure of acrosome complex vary substantially among different species (Baccetti and Afzelius, 1976; Dallai, 2014). Among insects, in general the acrosome complex is a small and elaborate structure. Perforatorium is present, consisting of filamentous dense material in many insects (Dallai, 2014). In several families of the Ensifera and Caelifera of Orthoptera, some residual material, called extra-acrosomal layer, surrounds the acrosome proper (Jamieson, 1987). As such, the acrosome complex is composed of acrosome proper, perforatorium and extra-acrosomal layer, which is defined as a three-
Abbreviations: A, acrosome; AC, acrosome complex; EA, extra-acrosomal layer; G, Golgi apparatus; N, nucleus; P, perforatorium; PA, pro-acrosome; PAS, Periodic Acid-Schiff. * Corresponding author. E-mail addresses: [email protected] (C.X. Su), [email protected] (Y.L. Chang).
layered acrosome. In some species, however, the spermatid acrosome complex is only bi-layered or mono-layered (Dallai et al., 2002; Lupetti et al., 2011) and the acrosome has even been lost in a few species (Dallai, 2014; Dallai et al., 2016). Acrosome formation is one of the most prominent events for spermatogenesis. Initially, Golgi apparatus continuously secretes vesicles which gather into the condensed and spherical proacrosome. Then, pro-acrosome gradually moves, contacts to nucleus and adheres to and expands along the nucleus. Ultimately, in the form of acrosome complex, it covers on anterior end of the nucleus surface in the mature spermatozoa (Lin and Rodger, 1999; Berruti and Paiardi, 2011; Wang et al., 2014). In Orthoptera, the acrosome formations of Acheta domesticus and Locusta migratoria have been previously studied. In L. migratoria, the proacrosome appears and approaches the nucleus followed by formation of rodlet perforatorium between the acrosomal cone and nucleus, and subsequently a cup covers the outside of acrosome complex (Jamieson, 1987). The acrosome complex of A. domesticus is quite elaborate and the acrosome formation undergoes more sophisticated transformation during which the proacrosome anchors on the nucleus and rotates (Clayton et al., 1958). The structure of spermatozoa in the Chinese bush cricket Gampsocleis gratiosa (G. gratiosa) Brunner von Wattenwyl is fairly elaborate. The acrosome complex consists of an apical vesicle and
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two big expansive wings, with appearance like an arrow (Chang et al., 2011), which is in line with the typical characteristics of sperm acrosome in Tettigoniidae. In addition, the acrosome complex of G. gratiosa is relatively large compared with those in other species, therefore, it is easy to observe under a microscope. Thus, G. gratiosa may be used as a model organism to illuminate the process of the acrosome formation in Tettigoniidae. In this study, we examined components of the acrosome complex, and recorded the dynamic course thereof in G. gratiosa by PAS (Periodic Acid-Schiff) staining, fluorescence labeling and transmission electronic microscopy. The acrosome formation in G. gratiosa was divided into six phases. And it is demonstrated that the glycoprotein is the major component of acrosome proper, that the microfilament also is present in the acrosome complex and contributes to its formation and that microtubules may play a crucial role in shaping the acrosome complex as well as the nucleus. This study may lead to a better understanding of the fertilization process and provide a valuable model for studying mechanisms underlying the acrosome biogenesis during spermiogenesis in Tettigoniidae. 2. Materials and methods
Finally, these sections were observed by Olympus BX51 fluorescence microscope and pictures were synthetized with Adobe Photoshop CS5. 2.4. Transmission electron microscopy The testes were rapidly isolated and fixed in 2.5% glutaraldehyde for 1 h at 4 C, rinsed in phosphate buffer (pH 7.4), and postfixed in OsO4 for 2 h at 4 C. After being ruined, the materials were dehydrated in a graded series of ethyl alcohol (30%e100%), transferred to acetone and finally embedded in Epon812. Semithin sections (0.7 mm) were stained with 1% toluidine blue before visualization under light microscope. Ultrathin sections (70 nm) were stained routinely with uranyl acetate and lead citrate before visualization under JEM-100SX electron microscope operating at 80 kV. 3. Results Based on the dynamic alterations of the morphology and positions of the pro-acrosome, microfilament and nucleus, the acrosome formation in G. gratiosa was divided into six phases: round, rotating, rhombic, cylindrical, transforming and mature phase.
2.1. Specimens 3.1. The round phase Sexually mature males of G. gratiosa were collected in JulyeSeptember 2015, at Dabei, Shunping, Hebei, China. Each adult male bush cricket was bred in single cage, where the conditions (temperature, moisture, photoperiod) imitated their habitat environment. 2.2. Light microscopy The isolated testes were fixed in Bouin's fluid at room temperature, dehydrated in a graded series of ethyl alcohol and embedded in paraffin. The serial sections (7 mm) were made and mounted on microscope slides, stained with PAS (Periodic AcidSchiff) and hematoxylin, and observed by Olympus BX51 optical microscope.
At the round phase, the nucleus and pro-acrosome were round. When the round spermatid was generated just after meiosis, the pro-acrosome appeared in a form of a very small spherical structure (Fig. 1A and B). Then, it gradually enlarged and moved close to the nucleus, due to the continuous secretion from Golgi apparatus (Fig. 1DeF). At the beginning of the round phase, the nucleus was large and round and was unevenly filled with genetic material. The condensed heterochromatin was partly depolymerized (Fig. 1AeC). With the sperm development, genetic material within the nucleus redistributed and developed into a ring (Fig. 1DeF). The ultrastructure analysis showed that the electron density of pro-acrosome was higher than in the nucleus and that the Golgi apparatus lay between the nucleus and pro-acrosome (Fig. 1C, F).
2.3. Fluorescence microscopy 3.2. The rotating phase The testes were fixed in fresh 4% paraformaldehyde for 24 h, dehydrated in 0.5 mol/L sucrose solutions and embedded in the optimal cutting temperature (O.C.T.) compound at 22 C. Then, the specimens were cut into 5-mm frozen section and transferred to slide. The sections were treated with 1% Triton X-100 for 30 min at room temperature. After washing in phosphate buffer saline (pH 7.3), the specimens were successively stained with 0.01% phalloidin-rhodamine for microfilament 45 min and 40 ,6diamidino-2-phenylindole (DAPI) for nucleus 15 min. Finally, these sections were observed by Olympus BX51 fluorescence microscope. The separated testes were ground and centrifuged at low speed in physiological saline to remove chunks of tissues and the supernatant was recentrifuged at 249 g to precipitate the cells. The cell pellet was fixed in fresh 4% paraformaldehyde for 1 h, rinsed in phosphate buffer saline (pH 7.3), and checked for density under microscope and smeared the sperms on the slide. After being treated with 1% Triton X-100 for 30 min at room temperature, washed in PBS, the smear was immersed in 5% bovine serum albumin (BSA) in PBS for 1 h to block nonspecific sites, and washed in PBS. The cells were then incubated in anti-a-tubulin antibody conjugated with fluorescein isothiocyanate (SigmaeAldrich) at 1:100 dilutions for 1 h. Then they were stained as above methods.
At the rotating phase, the pro-acrosome contacted the nucleus and the nucleus condensed to the minimum in the whole process. Genetic material within spermatid nucleus was compact and homogeneous (Fig. 2AeD). The pro-acrosome rotated by adhering to nucleus until arriving at the top of the nucleus (Fig. 2A and B). In the process of rotation, the pro-acrosome reached its maximal diameter. The pro-acrosome and nucleus attached or separated in the semithin section and ultrathin section. The Golgi apparatus disappeared at this stage (Fig. 2D). 3.3. The rhombic phase At the rhombic phase, namely after rotation, the spermatid head was rhombic (Fig. 3A, D). The pro-acrosome transformed into acrosome proper after complete rotation. The acrosome proper which was located at the top of the nucleus began to extend along the two sides of the nearly rhombic nucleus (Fig. 3A and B) and the genetic material within the nucleus redistributed again. Genetic material in the anterior nucleus was dense, whereas the electron density of the posterior nucleus was low (Fig. 3A, D). The frozen section stained with phalloidinerhodamine shows that a small spherical microfilament
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Fig. 1. Spermatid of G. gratiosa at the round phase. A, D: Paraffin section stained with PAS and hematoxylin. B, E: Semithin section stained with toluidine blue. C, F: Ultrathin sections. The arrow points to the nucleus (N) and the arrowhead points to the pro-acrosome (PA). G: Golgi apparatus. Scale bars: A, D, 50 mm; B, E, 10 mm; C, 500 nm; F, 2 mm.
appeared at the anterior of nucleus (Fig. 3C). The ultrathin section shows a small spherical perforatorium between the acrosome proper and the nucleus (Fig. 3C and D). The acrosome proper and the perforatorium constituted the incomplete acrosome complex at this stage.
forming a cone at the apex of the nucleus (Fig. 4C). The ultrathin section shows that the electron density of nuclear envelope at the two sides of nucleus is significantly increased.
3.4. The cylindrical phase
At the transforming phase, the acrosome proper was located at the top of nucleus and resembled an arrow (Fig. 5A and B). The frozen section labeled with phalloidine-rhodamine shows microfilaments transformed into the inverted ‘Y’ appearance from the cone (Fig. 5C). The frozen section marked with DAPI for nucleus, phalloidine-rhodamine for microfilament and a-tubulin antibody for microtubule indicates that the microfilament and nucleus are surrounded by microtubules (Fig. 5D). The electron density of the acrosome proper decreased and an extra-acrosomal layer was
At the cylindrical phase, the spermatid head was cylindrical, and its volume distinctly increased (Fig. 4AeD). The acrosome proper further extended along the two sides of nucleus and the perforatorium beneath the acrosome proper enlarged. The genetic material within the nucleus still tended to concentrate in the anterior nucleus (Fig. 4AeB, D, F). The frozen section stained with phalloidine-rhodamine shows microfilaments enlarged and
3.5. The transforming phase
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Fig. 2. Spermatid of G. gratiosa at the rotating phase. AeB: Paraffin sections stained with PAS and hematoxylin. A: Pro-acrosome (PA) contacting the nucleus (N) and just starting rotation. B: Pro-acrosome located at the top of nucleus. C: Semithin section stained with toluidine blue. D: ultrathin section. The arrow points to the nucleus (N) and the arrowhead points to the pro-acrosome (PA). Scale bars: AeB, 50 mm; C, 25 mm; D, 2 mm.
formed (Fig. 5E). The acrosome proper, perforatorium and extraacrosomal layer constituted the complete acrosome complex. Meanwhile, the ultrastructure analysis showed that the genetic material within the nucleus condensed into a lot of visible granules under transmission electron microscope and that microtubules existed around the nucleus and acrosome complex (Fig. 5F). 3.6. The mature phase The mature phase was the last phase of acrosome formation. At this stage, the spermatid turned to mature spermatozoon by excluding several ‘redundant’ constituents (Fig. 6A and B). The frozen section stained with phalloidine-rhodamine shows the microfilaments presenting the shape of an arrow at the top of the nucleus, the angle of arrow being greater than that of an inverted ‘Y’ (Fig. 6C). The angles from the two wings of both the polysaccharide and the acrosome complex gradually enlarged and the genetic material within the nucleus continued to compress and come to be the uniform and compact spermatozoon nucleus. The microtubules around nucleus and acrosome complex were no longer observable under the transmission electron microscope (Fig. 6D). 4. Discussion
acrosomes in Arvelius albopunctatus. Souza and Itoyama (2010) studied the acrosome formation of five pentatomids in Hemiptera by PAS staining. In our study, the results of PAS staining and transmission electron microscopy indicate that polysaccharide coincide well with the pro-acrosome or acrosome proper in size and morphology. Polysaccharide always combines with protein or lipid to form glycoproteins or glycolipids in the cell. The acrosome complex produces a skeleton glycoprotein at the head of sperm in insects (Klowden, 2007). Thus, glycoprotein is the primary component of pro-acrosome and acrosome proper. Many studies showed that microfilaments exist in the vertebrate spermatid (Breed and Leigh, 1991; Scarlett et al., 2001; Dvorakova et al., 2005; Sperry, 2012). In invertebrates, microfilaments are also present in the acrosome complex of the spermatid and the morphology of the acrosome complex is modified by the dynamic changes of the cytoskeleton protein surrounding it (Guerra and Esponda, 1999; Li et al., 2010; Trovato et al., 2011; Dallai, 2014; Viscuso et al., 2016). In this study, the frozen sections stained with phalloidine-rhodamine showed that microfilaments existed in the acrosome complex (Figs. 3C, 4C, 5CeD, 6C). In the sperm at mature phases, the arrow shaped microfilaments always existed at the anterior side of the nucleus (Fig. 6C). Therefore, microfilaments may be a component of the perforatorium.
4.1. Components of the acrosome complex
4.2. Acrosome formation
As early as 1951, Schrader and Leuchtenberger demonstrated by PAS staining that polysaccharides are components of the
In Tettigoniidae, the acrosome complex is characterized by big size and a complicated structure consisting of the acrosome
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Fig. 3. Spermatid of G. gratiosa at the rhombic phase. A: Paraffin section stained with PAS and hematoxylin. B: Semithin section stained with toluidine blue. C: Frozen section marked with DAPI for nucleus (blue) and phalloidine-rhodamine for microfilament (red). D: Ultrathin section. The arrow points to the nucleus (N); the dark arrowhead points to the acrosome proper or incomplete acrosome complex (AC) and the white arrowhead points to microfilament. P: perforatorium. A: acrosome proper. Scale bars: A, 50 mm; BeC, 10 mm; D, 2 mm.
proper, perforatorium and extra-acrosomal layer (Baccetti, 1987; Guerra and Esponda, 1999; Chang et al., 2011). Moreover, the acrosome proper is the major part of the acrosome complex in Tettigoniidae. Therefore, tracing glycoprotein through PAS staining can be used to study the acrosome formation. The acrosome formation is a complex and dynamic process, that includes the formation and morphological changes of different structural components. Combining the results from light microscopy, transmission electron microscopy and fluorescence microscopy, a working model of the whole formation process of acrosome complex which emphasizes acrosome proper, nucleus and microfilament, is proposed (Fig. 7). The acrosome formation starts with the round spermatid from meiosis. In the whole process, acrosome formation is initiated by Golgi secretion, which gathers into the densely spherical pro-acrosome. With the progress of spermiogenesis, pro-acrosome constantly enlarges, approaches and attaches to nucleus (Fig. 7a). Afterward, it rotates by adhering to the nucleus and becomes the acrosome proper (Fig. 7b). Subsequently, a perforatorium is produced and enlarges beneath the acrosome proper. Meanwhile, an extra-acrosomal layer is produced on the outside of the acrosome proper.
Eventually, the acrosome complex transforms into an arrow in the mature spermatozoon. Moreover, microfilaments participate in the formation of the acrosome complex as a component. After appearance at the rhombus phase, it undergoes the morphological transformation from a sphere, to a cone, the inverted ‘Y’, and finally into an arrow (Fig. 7c-f). In insects, the small size of sperm acrosome complex makes it difficult to observe via light microscopy. This may account for why there is only limited knowledge regarding the dynamic process of acrosome formation in insects. In the process of acrosome formation in Euschistus heros F. (Pentatomidae, Heteroptera), a central polysaccharide granule migrates to one pole of the spermatid and becomes elongated until it is indistinguishable in the spermatozoa (Souza and Itoyama, 2010). In Orthoptera, the acrosome formations of L. migratoria and A. domesticus have been studied. In L. migratoria, the pro-acrosome which has no direct link to the Golgi apparatus adheres to the nuclear membrane and glides in a posterior-anterior direction around the nucleus. It is deemed that the process depends on the inherent fluidity of the nuclear membranes (Jamieson, 1987). In A. domesticus, pro-acrosome deriving from the Golgi secretion
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Fig. 4. Spermatid of G. gratiosa at the cylindrical phase. A: Paraffin section stained with PAS and hematoxylin. B: Semithin section stained with toluidine blue. C: Frozen section marked with DAPI for nucleus (blue) and phalloidine-rhodamine for microfilament (red). DeF: Ultrathin section. The arrow points to the nucleus (N); crossed arrow points to perforatoriums (P); the dark arrowhead points to the acrosome proper or acrosome complex (AC) and the white arrowhead points to microfilament. A: acrosome proper. Scale bars: A, 50 mm; BeC, 10 mm; D, 500 nm; EeF, 2 mm.
adheres and rolls on the surface of nucleus until arriving at the anterior end of nucleus. It is thought that the rolling of proacrosome may be due to rotation of the nucleus through 180 (Clayton et al., 1958). The discovery in G. gratiosa is in accordance with the report in the A. domesticus. The proacrosome migrates along a 180 circle by adhering to the nucleus and the final arrow-shaped acrosome complex is formed at the front of the nucleus. However, the mechanism of rotation still needs further research. 4.3. Cytoskeleton in the acrosome complex Many aspects of morphogenesis including acrosome formation are dependent on the cytoskeleton (Xiao and Yang, 2007). The changes of morphology and position of the cytoskeleton during spermiogenesis have been studied in many vertebrates and invertebrates (Breed and Leigh, 1991; Scarlett et al., 2001; Noguchi and Miller, 2003; Sahara and Kawamura, 2004; Dvorakova et al., 2005; Desai et al., 2009; Sperry, 2012; Viscuso et al., 2016). In mammalian sperm, acroplaxome, containing actin and keratin, anchores and shapes the acrosome (Kierszenbaum et al., 2003; Snigirevskaia et al., 2012). In Caridean shrimp, acroframosome consisting of microfilament and microtubules functions as a framework during acrosome formation (Li et al., 2010; Sun et al., 2011). In Tettigoniidae, microfilament of sperm in the Tylopsis liliifolia is distributed along the entire acrosomal region during nucleus elongation, however, the microtubules are located at the two wings of the acrosome and
around the nucleus (Viscuso et al., 2016). In Platycleis albopunctata, acrosomal wings, giving spermatozoa the shape of an arrow, contain microfilament and microtubules (Guerra and Esponda, 1999). Our results demonstrate that the microfilament is invisible at round phase, and emerges along with the perforatorium after migration of the pro-acrosome. Then microfilament undergoes dynamic changes from the spherical, cone, inverted ‘Y’ to arrow shape (Figs. 2C, 3C, 4CeD, 5C). The acrosome proper and perforatorium gradually expands to the sides of nucleus along the morphologic change of microfilament. The position and morphology of microfilament in mature spermatozoa is consistent with the previous result in P. albopunctata (Guerra and Esponda, 1999). However, the dynamic changes and the function of microfilament resemble the acroframosome in caridean shrimp which functions as a framework for acrosome biogenesis (Li et al., 2010; Sun et al., 2011). Therefore, it is speculated that the microfilament supports the morphological changes of acrosome complex as a component of acrosome complex. Moreover, it has been shown that the spermatid could transport some substances through actin and microtubules in the process of mammal spermatogenesis (Kierszenbaum et al., 2011). Therefore, we suppose microfilament may participate in the transportation of acrosome proper in G. gratiosa as well. At the transforming phase of the acrosome formation of G. gratiosa, the genetic material of the nucleus rapidly aggregated and gave rise to a mass of visible granules under the transmission electron microscope. The complete acrosome complex was
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Fig. 5. Spermatid of G. gratiosa at the transforming phase. A: Paraffin section stained with PAS and hematoxylin. B: Semithin section stained with toluidine blue. CeD: Frozen section marked with DAPI for nucleus (blue), phalloidine-rhodamine for microfilament (red) and a-tubulin antibody for microtubule (green). EeF: Ultrathin section. The arrow points to the nucleus (N); the dark arrowhead points to the acrosome proper or acrosome complex (AC); the white arrowhead points to microfilament and the double arrows point to microtubules. The dotted lines in D indicate the probable plane of section shown in Fig. 6E and F. A: acrosome proper. EA: extra-acosome layer. Scale bars: A, 25 mm; BeD, 10 mm; EeF, 500 nm.
formed in a great morphological transformation. The microfilament supported the acrosome complex in the form of inverted ‘Y’ and microtubules surrounded nucleus and acrosome complex. However, the microtubules disappeared in the mature spermatozoa. In mammalian and Octopus tankahkeei, the perinuclear microtubules form a transient structure, called manchette, and the complex is indispensable for acquisition of the final nuclear morphology (Kierszenbaum and Tres, 2004; Ribes et al., 2002; Wang et al., 2010). In G. gratiosa, the microtubules around nucleus and acrosome complex are analogous to those of the manchette and they are also essential for the formation of the final morphology of nucleus and acrosome complex. Thus, we deem the transforming phase is the key stage of acrosome
formation in G. gratiosa (Fig. 8). The microtubules may also play an influential role to shape the nucleus and construct the morphology of acrosome complex. To conclude the results lead us to propose that: (1) glycoprotein is the major component of acrosome proper; (2) the microfilament may support the morphological changes of the acrosome complex as one of its components; (3) the microtubules may also play an influential role to shape the nucleus and acrosome complex; (4) the transforming phase is the key period of acrosome formation in G. gratiosa. Further, for the first time, we unveiled the process of acrosome formation in G. gratiosa, which may contribute to shedding light on acrosome biogenesis mechanisms involved in spermiogenesis in other insects.
Please cite this article in press as: Su, C.X., et al., Formation of the acrosome complex in the bush cricket Gampsocleis gratiosa (Orthoptera: Tettigoniidae), Arthropod Structure & Development (2017), http://dx.doi.org/10.1016/j.asd.2017.01.002
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C.X. Su et al. / Arthropod Structure & Development xxx (2017) 1e9
Fig. 6. Spermatid of G. gratiosa at the mature phase. A: Paraffin section stained with PAS and hematoxylin. B: Semithin section stained with toluidine blue. C: Frozen section marked with DAPI for nucleus (blue) and phalloidine-rhodamine for microfilament (red). D: Ultrathin section. The arrow points to the nucleus (N); the dark arrowhead points to the acrosome proper or acrosome complex (AC) and the white arrowhead points to microfilament. The stars indicate the ‘redundant’ constituents. A: acrosome proper. Scale bars: A, 25 mm; BeC, 10 mm; D, 500 nm.
Fig. 7. The model of acrosome formation in G. gratiosa. Morphological differentiation of acrosome proper, nucleus, and microfilament are emphasized. a: round phase. b. rotating phase. c: rhombic phase. d: cylindrical phase. e: transforming phase. f: mature phase. Blue represents nucleus; dark gray represents pro-acrosome or acrosome proper; red represents microfilament.
Please cite this article in press as: Su, C.X., et al., Formation of the acrosome complex in the bush cricket Gampsocleis gratiosa (Orthoptera: Tettigoniidae), Arthropod Structure & Development (2017), http://dx.doi.org/10.1016/j.asd.2017.01.002
C.X. Su et al. / Arthropod Structure & Development xxx (2017) 1e9
Fig. 8. The model of spermatid microtubule in G. gratiosa at the transforming phase. Microtubules surrounding the nucleus and acrosome proper are emphasized. Blue represents nucleus; dark gray represents acrosome proper; red represents microfilament; green stands for microtubules.
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Please cite this article in press as: Su, C.X., et al., Formation of the acrosome complex in the bush cricket Gampsocleis gratiosa (Orthoptera: Tettigoniidae), Arthropod Structure & Development (2017), http://dx.doi.org/10.1016/j.asd.2017.01.002