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Ultrastructural analysis of the nucleolar aspects at spermiogenesis in triatomines (Heteroptera, Triatominae)
Micron 41 (2010) 791–796
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Micron journal homepage: www.elsevier.com/locate/micron
Ultrastructural analysis of the nucleolar aspects at spermiogenesis in triatomines (Heteroptera, Triatominae) Alessandra Morielle-Souza ∗ , Sebastião Roberto Taboga, Maria Tercília Vilela de Azeredo-Oliveira São Paulo State University – UNESP/IBILCE, Department of Biology, Rua Cristóvão Colombo, 2265, São José do Rio Preto, SP 15054-000, Brazil
a r t i c l e
i n f o
Article history: Received 25 February 2010 Received in revised form 19 May 2010 Accepted 19 May 2010 Keywords: Spermiogenesis Nucleolus Triatomine Ultrastructure
a b s t r a c t In this study the ultrastructural technique was used to analyze seminiferous tubule cells of the triatomine species Panstrongylus megistus, Rhodnius pallescens and Triatoma infestans. The data obtained provided evidence of the phenomenon known as persistence of the nucleolar material in initial spermatids at early differentiation. Our results confirmed the presence of the nucleolus and its products during spermiogenesis up to the formation of the axoneme and during spermatid elongation in all three species studied, similar to the process that takes place during cell division. In early spermatids, the nucleoli had a reticulate appearance and a well defined nucleolonema in P. megistus; showed a clear distinction between the fibrillar and the granular component in T. infestans; and had a compact aspect in R. pallescens. In this study, ultrastructural analyses at spermiogenesis indicated that these nucleolar products may represent RNP complexes that will probably be needed at early spermiogenesis when important changes such as chromatin condensation and acrosome and flagellum formation take place. Therefore, it was concluded from the ultrastructural analysis that the triatomine nucleolus does not totally disappear but remains as corpuscles that gather to form the next nucleolar cycle that in the case of meiosis, will be completed if fertilization occurs and a zygote is formed. © 2010 Elsevier Ltd. All rights reserved.
1. Introduction Spermatogenesis is the process by which primordial cells, the spermatogonial, develop into highly differentiated cells, the spermatozoa. Spermatozoa are produced through spermiogenesis, a process by which spermatids undergo remarkable changes that result in mature cells. Spermatozoa may be said to be highly specialized cells because they exhibit several dispersed organelles that are essential for cell metabolism (Phillips, 1974). In triatomines, spermatogenesis is considered to be cystic. The cysts form groups of isogenic cells where, in general, only one definitive spermatogonium generates the entire cell content of a cyst. Thus, successive synchronic cell divisions produce spermatid cysts at the same stage of differentiation (Dunser and Davey, 1974). There are numerous comparative and cytochemical studies of the structural or ultrastructural aspects of spermatogenesis in insects. However, there are few ultrastructural investigations specifically focusing on triatomines, especially the nucleolar cycle and the persistence of nucleolar material that occur during spermiogenesis in this subfamily.
∗ Corresponding author. Tel.: +55 17 3221 2380; fax: +55 17 3221 2390. E-mail address: [email protected] (A. Morielle-Souza). 0968-4328/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.micron.2010.05.009
The nucleolus is a functional nuclear domain involved in the rRNA metabolism. Under transmission electron microscopy, this organelle usually shows three nucleolar domains, the fibrillar component (FC), the dense fibrillar component (DFC) and the granular component (GC) (Zatsepina et al., 1997). The FC is considered as a storage site of non-transcribed ribosomal genes, the DFC is a transcription site of these genes and GC is the storage and maturation site of ribosomal subunits. However, during nucleologenese these domains disrupt and rearrange the cell cycle (Ploton et al., 1987; Zatsepina et al., 1997; Mello, 2001). The chemical structure of these regions basically consists of DNA (small quantity), RNA and acidic proteins. RNA associated with proteins forms molecular complexes of ribonucleoproteins (RNPs) (Fischer et al., 1991). Given that the arrangement of the nucleolar components varies according to cell type and level of ribosome production, nucleolar categories have been established according to RNPs distribution as follows: reticulate nucleolus with nucleolonema; compact nucleolus; and nucleolus with concentric layers. However, during the nucleologenese these domains disorganize and reorganize during the cell cycle (Thiry and Goessens, 1996; Ploton et al., 1987; Zatsepina et al., 1997; Mello, 2001). The nucleolar disorganization begins in prophase I and during the stages of metaphase, anaphase and telophase, the nucleolar material remains associated with the nucleolar organizer regions
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(NORs) that were also found in the form of pre-nucleolar corpuscles (PNBs), which persist in spermatids indicating post-meiotic reactivation of ribosomal RNA genes. The presence of the nucleolus during cell division, a phenomenon known as “persistence of the nucleolar material”, has been observed in dynoflagellates and malignant mammalian cells in the form of pre-nucleolar bodies (PNBs). The persistence of PNBs during mitosis and meiosis supports the idea that the nucleolar material is not completely disrupted during metaphase and anaphase. However, the functional significance of this finding remains unclear. It may be hypothesized that these bodies carry primary or new material or even represent a source of nucleolar RNA for the daughter cells while the new nucleolus is being organized (Wachtler and Stahl, 1993; Mello, 1995).
Nonetheless, little emphasis has been given to the presence and activity of the nucleolar material while spermatids differentiate into spermatozoa. In order to investigate the persistence of the nucleolar material during spermiogenesis in triatomines, the gametic cells found in the seminiferous tubules of the species Panstrongylus megistus, Rhodnius pallescens and Triatoma infestans were analyzed using conventional ultrastructural techniques. 2. Materials and methods The P. megistus, R. pallescens, and T. infestans species, order Heteroptera, family Reduviidae, subfamily Triatominae were used. Seminiferous tubules were analyzed from 10 adult specimens of each genus. Specimens were provided by the Araraquara Special
Fig. 1. Ultrastructure of P. megistus seminiferous tubule cells. Electromicrograph of earlier spermatids showing nucleoli (Nu), heterochromatin (H), mitochondrial derivative (Md), axoneme (Ax) and acrosomal membrane (AM). Notice the reticulate nucleolus and the evident presence of nucleolonema. Notice em (E and F) nucleolar fragmentation and the presence of these fragments up to axoneme formation (G). Bars: A, B, D–G = 1 m and C = 2 m.
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Health Service (SESA), Department of Epidemiology, São Paulo Public Health School (São Paulo, SP, Brazil). For ultrastructural analysis, the seminiferous tubules were initially fixed in 2% glutaraldehyde, 2% paraformol in phosphate buffer 0.1 M, pH 7.4. After fixing, the material was post-fixed in 1% osmium tetroxide, washed in distilled water, dehydrated in increasing acetone sequence and embedded in Epon. Resin polymerization was completed in 72 h. Ultrathin sections were cut and stained with 2% uranyl acetate and lead citrate. Electromicrographs were obtained under a transmission electron microscope (ZEISS – EM – 906, at 80 kV), using Kodak SO-163 film, at the Microscopy Center of São José do Rio Preto, Institute of Biosciences, Letters and Exact Sciences-SP (São Paulo State University/UNESP).
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3. Results Ultrastructural analyses of initial spermatids from the species under study (Figs. 1–3) showed the presence of nucleolar material up to the phase when the axoneme began to form and during spermatid elongation, as seen in R. pallescens (Fig. 2F). In addition to the nucleolus, spermatic nuclei showed some heterochromatic regions of a less electron-dense aspect. In P. megistus (Fig. 1A–G) and T. infestans (Fig. 3A–F), nucleoli had a reticulate appearance and, especially in P. megistus, displaying a well defined nucleolonema. In T. infestans, the distinction between the fibrillar component (FC) and the granular component (GC) could be observed. In R. pallescens, the nucleolus (Fig. 2A–F) resembled that of the compact type.
Fig. 2. Ultrastructure of Rhodnius pallescens seminiferous tubule cells. Electromicrograph of earlier spermatids (A–E) at elongation stage (F) showing nucleoli (Nu), heterochromatin (H), mitochondrial derivative (Md) and axoneme (Ax). Notice the compact nucleolus. Asterisks (*) indicate nucleolar fragments. Notice in (F) the nucleolar fragmentation and persistence of nucleolar bodies up to spermatid elongation, in detail another cut plan where nucleolar body is also visible. Bars: A and D = 1.5 m and B, C, E, F = 1 m.
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Fig. 3. Ultrastructure of Triatoma infestans seminiferous tubule cells. Electromicrograph of earlier spermatids showing nucleoli (Nu), heterochromatin (H), mitochondrium (Mi), Golgi apparatus (GA), mitochondrial derivative (Md) and axoneme (Ax) and the acrosome (Ac). Notice distinction fibrillar component (FC) and granular component (GC) of the reticulate nucleolus and nucleolar fragmentation. Nucleolar fragments are preferentially located at the periphery of the nuclear envelope of spermatids (D). Bars: A–C = 2.5 m and D–F = 1.5 m.
In all three species, the nucleoli were clearly fragmented. In the P. megistus species, it was possible to observe small electron-dense bodies (fragmented nucleolus) migrating to the axoneme region (Fig. 1D–G). In R. pallescens, these bodies were randomly distributed over the cytoplasm (Fig. 2A–F), and in T. infestans they were most frequently found at the periphery of the nuclear envelope (Fig. 3D). The location of the fragments varied with the cut made in the seminiferous tubules. 4. Discussion The present ultrastructural analysis provided evidence of the occurrence of fragmentation and persistence of the nucleolar material during spermiogenesis, similar to the process that
takes place during meiotic cell division already observed by the authors. Morielle-Souza and Azeredo-Oliveira (2008) used cytochemical techniques and observed in three species of triatomine (P. megistus, R. pallescens and T. infestans), the presence of bodies strongly impregnated with silver ions from meiotic metaphase I through telophase. In the T. infestans and P. megistus species that exhibited nucleolar fragments dispersed over the nucleus at meiotic prophase I and diakinesis. The authors concluded that these corpuscles correspond to PNBs, highly argyrophilic structures that might represent the assembly of nucleolar fragments which, in previous phases, were dispersed over the cell nuclei. Yet the same authors observed early spermatids of the three species showing argyrophilic corpuscle, once more corroborating
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the hypothesis of post-meiotic reactivation of rRNA genes. This phenomenon was also detected by the variant of critical electrolytic concentration (CEC), which caused these regions to be clearly metachromatic and by acridine orange staining, which stained them red so the rRNA could be discriminated and thus providing structural details of the nucleolus and RNA distribution during meiotic cell division. Therefore, all the methods used confirmed the presence of RNA/protein complexes. These results supported the persistence of the nucleolar material and the hypothesis of the post-meiotic reactivation of rRNA genes (Morielle-Souza and Azeredo-Oliveira, 2008). In spermatid at early developmental stage of Oedipoda coerulescens (Orthoptera: Acrididae), ultrastructural investigations have revealed that the nucleolus consists of fibrogranular material that, during differentiation, seems to transform into a single mass of fibrillar material and clusters of granular bodies, which become smaller and eventually disappear, while the fibrillar material persists. Light microscopy examination has shown that this nucleolar material has a similar behavior at the late spermatid stage in Schistocerca gregaria (Orthoptera: Acrididae) (Rufas and Gonsálvez, 1982). Several studies of spermatogenesis and oogenesis in nonmammalian and mammalian vertebrates have confirmed the post-meiotic reactivation of genes to rRNA. Hofgartner et al. (1979) used silver-ion impregnation to study four mammals species (man, Mus musculus, Rattus novergicus and Cavia cobaya) and observed the occurrence of post-meiotic reactivation of nucleolar organizer regions (NORs). In addition, this species exhibited a very similar pattern of NOR impregnation throughout spermatogenesis, with silver precipitates being detectable from the phase of spermatogonium growth up to the pachytene of meiotic prophase I. However, impregnation was no longer seen during metaphase I and II, and reappeared only at spermatid differentiation and persisted up to the beginning of elongation. Peruquetti et al. (2008) analyzed the cytochemical and ultrastructural structure of the nucleolar cycle and the distribution of cytoplasmatic RNAs in the seminiferous tubules cells of R. novergicus and M. musculus, the findings corroborate that the nucleolar ribonucleoproteins (RNPs) are very important in the spermiogenesis phases. The authors suggested that the molecular complexes (RNPs) have a role in a successive series of events that occur in the formation of the spermatozoon. During oogenesis, the phenomenon of nucleolar material persistence has been observed in plants, such as Vicia faba; in marine invertebrates, such as the Urechis caupo echiuroid; in the Physarum policephalum plasmodium and in amphibians such as Xenopus laevis; and in the embryos of mammalians such as rats, mice, rabbits, equines, bovines and humans (Das and Alfert, 1973; Tesarík et al., 1986; Sato, 1988; Grøndahl and Hyttel, 1996; Baran et al., 1996; Verheggen et al., 1998; Dundr et al., 1997, 2000). In general, the bodies found during oogenesis are similar to the nucleolar bodies found in spermatogenic and mitotic cells, in terms of origin, structure and cytochemical and biochemical characteristics (Das and Alfert, 1973). Nucleolar precursors are well known for providing the basis for the formation of nucleoli. However, their role in nucleologenesis and mechanisms governing nucleolar reorganization remains unclear and debatable. Most studies on gene activity during vertebrate spermatogenesis have been conducted in mammals and limited to demonstrating the synthesis of pre- and post-meiotic RNA. In addition to a distinct synthesis of pre-meiotic RNA, some studies using autoradiographic and ultrastructural analyses have demonstrated a small post-meiotic RNA synthesis that declines during the later stages of spermatic differentiation (Schmid et al., 1982).
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The evolutionary conservation of the post-meiotic reactivation of NORs from Cephalochordates in man indicates the need for postmeiotically synthesized ribosomal RNA. This newly synthesized rRNA might be a prerequisite for mRNA production which must be transcribed at the early stages of spermatid development (Schmid et al., 1982; Peruquetti et al., 2008). In this study, ultrastructural analyses at spermiogenesis indicated that these nucleolar products may represent RNP complexes that are likely to be needed at early spermiogenesis when important changes such as chromatin condensation and the acrosome and flagellum formation take place. Therefore, it was concluded from the ultrastructural analysis that the triatomine nucleolus does not totally disappear but remains as corpuscles that gather to form the next nucleolar cycle that in the case of meiosis, will be completed if fertilization occurs and a zygote is formed. These aspects therefore clearly show that rRNA synthesis in gametogenesis is a process that occurred in the three triatomines genera, Panstrongylus, Rhodnius and Triatoma, and therefore may be a common mechanism among the Heteroptera.
Acknowledgments The authors are thankful to Dr. José M. Soares Barata, Director of the Insectary (Araraquara, SP), Department of Epidemiology, Faculty of Public Health (São Paulo, SP), and João Luis Molina Gil and João Maurício Nóbrega da Silva Filho, technicians of the insectary, for providing the insects studied. Prof. Rosana Silistino and Luiz Roberto Faleiros technician helped teach our group the ultrastructure technique. This research was supported by FAPESP (No. 00/06700-4) and CAPES.
References Baran, V., Fléchon, J.E., Pivko, J., 1996. Nucleologenesis in the cleaving bovine embryo: immunocytochemical aspects. Mol. Reprod. Dev. 44, 63–70. Das, N.K., Alfert, M., 1973. Prenucleolar bodies of Urechis oocytes. J. Cell Sci. I2, 781–797. Dundr, M., et al., 1997. A class of nonribosomal nucleolar components is located in chromosome periphery and in nucleolus-derived foci during anaphase and telophase. Chromosoma 105, 407–417. Dundr, M., Misteli, T., Olson, M.O.J., 2000. The dynamics of postmitotic reassembly of the nucleolus. J. Cell Biol. 150, 433–446. Dunser, J.B., Davey, K.G., 1974. Endocronological and other factors influencing testis development in Rhodnius prolixus. Can. J. Zool. 52, 1011–1022. Fischer, D., Weisenberger, D., Scheer, U., 1991. Review: assigning functions to nucleolar structures. Chromosoma 101, 133–140. Grøndahl, C., Hyttel, P., 1996. Nucleologenesis and ribonucleic acid synthesis in preimplantation equine embryos. Biol. Reprod. 55, 769–774. Hofgartner, F.J., et al., 1979. Pattern of activity of nucleolus organizers during spermatogenesis in mammals as analyzed by silver-staining. Chromosoma 71, 197–216. Mello, M.L.S., 1995. Relocation of RNA metachromasy at mitosis. Acta Histochem. Cytochem. 28, 149–154. Mello, M.L.S., 2001. Nucléolo. In: Carvalho, H.F., Recco-Pimentel, S.(Org.) (Eds.), A Célula 2001. Editora Manole, São Paulo, pp. 101–110. Morielle-Souza, A., Azeredo-Oliveira, M.T.V., 2008. Study of the nucleolar cycle and ribosomal RNA distribution during meiosis in triatomines (Triatominae, Heteroptera). Micron 39, 1020–1026. Peruquetti, R.L., et al., 2008. Meiotic nucleolar cycle and chromatoid body formation during the rat (Rattus novergicus) and mouse (Mus musculus) spermiogenesis. Micron 39, 419–425. Phillips, D.M., 1974. Spermiogenesis. Academic Press, New York, p. 68. Ploton, D., et al., 1987. Behaviour of nucleolus during mitosis. A comparative ultrastructural study of various cancerous cell lines using the Ag-NOR staining procedure. Chromosoma 95, 95–107. Rufas, J.S., Gonsálvez, J., 1982. Development of silver stained structures during spermatogenesis of Schistocerca gregaria (Forsk) (Orthoptera: Acrididae). Caryologia 35, 261–267. Sato, S., 1988. Cytological evidence on the ability of the nucleolus organizing regions to assemble pre-existing nucleolar material. Experientia 44, 264–266. Schmid, M., et al., 1982. Evolutionary conservation of a common pattern of activity of nucleolus organizer during spermatogenesis in vertebrates. Chromosoma 86, 149–178.
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Tesarík, J., et al., 1986. Nucleologenesis in the human embryo developing in vitro: ultrastructural and autoradiographic analysis. Dev. Biol. 115, 193–203. Thiry, M., Goessens, G., 1996. The Nucleolus during the Cell Cycle. Springer, p. 146. Verheggen, C., et al., 1998. Presence of pre rRNAs before activation of polymerase I transcription in the building process of nucleoli during early development of Xenopus laevis. J. Cell Biol. 142, 1167–1180.
Wachtler, F., Stahl, A., 1993. The nucleolus: a structural and functional interpretation. Micron 24, 1167–1180. Zatsepina, O.V., et al., 1997. Experimental induction of prenucleolar bodies (PNBs) in interphase cells: interphase PNBs show similar characteristics as those typically observed at telophase of mitosis in untreated cells. Chromosoma 105, 418–430.
Contents lists available at ScienceDirect
Micron journal homepage: www.elsevier.com/locate/micron
Ultrastructural analysis of the nucleolar aspects at spermiogenesis in triatomines (Heteroptera, Triatominae) Alessandra Morielle-Souza ∗ , Sebastião Roberto Taboga, Maria Tercília Vilela de Azeredo-Oliveira São Paulo State University – UNESP/IBILCE, Department of Biology, Rua Cristóvão Colombo, 2265, São José do Rio Preto, SP 15054-000, Brazil
a r t i c l e
i n f o
Article history: Received 25 February 2010 Received in revised form 19 May 2010 Accepted 19 May 2010 Keywords: Spermiogenesis Nucleolus Triatomine Ultrastructure
a b s t r a c t In this study the ultrastructural technique was used to analyze seminiferous tubule cells of the triatomine species Panstrongylus megistus, Rhodnius pallescens and Triatoma infestans. The data obtained provided evidence of the phenomenon known as persistence of the nucleolar material in initial spermatids at early differentiation. Our results confirmed the presence of the nucleolus and its products during spermiogenesis up to the formation of the axoneme and during spermatid elongation in all three species studied, similar to the process that takes place during cell division. In early spermatids, the nucleoli had a reticulate appearance and a well defined nucleolonema in P. megistus; showed a clear distinction between the fibrillar and the granular component in T. infestans; and had a compact aspect in R. pallescens. In this study, ultrastructural analyses at spermiogenesis indicated that these nucleolar products may represent RNP complexes that will probably be needed at early spermiogenesis when important changes such as chromatin condensation and acrosome and flagellum formation take place. Therefore, it was concluded from the ultrastructural analysis that the triatomine nucleolus does not totally disappear but remains as corpuscles that gather to form the next nucleolar cycle that in the case of meiosis, will be completed if fertilization occurs and a zygote is formed. © 2010 Elsevier Ltd. All rights reserved.
1. Introduction Spermatogenesis is the process by which primordial cells, the spermatogonial, develop into highly differentiated cells, the spermatozoa. Spermatozoa are produced through spermiogenesis, a process by which spermatids undergo remarkable changes that result in mature cells. Spermatozoa may be said to be highly specialized cells because they exhibit several dispersed organelles that are essential for cell metabolism (Phillips, 1974). In triatomines, spermatogenesis is considered to be cystic. The cysts form groups of isogenic cells where, in general, only one definitive spermatogonium generates the entire cell content of a cyst. Thus, successive synchronic cell divisions produce spermatid cysts at the same stage of differentiation (Dunser and Davey, 1974). There are numerous comparative and cytochemical studies of the structural or ultrastructural aspects of spermatogenesis in insects. However, there are few ultrastructural investigations specifically focusing on triatomines, especially the nucleolar cycle and the persistence of nucleolar material that occur during spermiogenesis in this subfamily.
∗ Corresponding author. Tel.: +55 17 3221 2380; fax: +55 17 3221 2390. E-mail address: [email protected] (A. Morielle-Souza). 0968-4328/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.micron.2010.05.009
The nucleolus is a functional nuclear domain involved in the rRNA metabolism. Under transmission electron microscopy, this organelle usually shows three nucleolar domains, the fibrillar component (FC), the dense fibrillar component (DFC) and the granular component (GC) (Zatsepina et al., 1997). The FC is considered as a storage site of non-transcribed ribosomal genes, the DFC is a transcription site of these genes and GC is the storage and maturation site of ribosomal subunits. However, during nucleologenese these domains disrupt and rearrange the cell cycle (Ploton et al., 1987; Zatsepina et al., 1997; Mello, 2001). The chemical structure of these regions basically consists of DNA (small quantity), RNA and acidic proteins. RNA associated with proteins forms molecular complexes of ribonucleoproteins (RNPs) (Fischer et al., 1991). Given that the arrangement of the nucleolar components varies according to cell type and level of ribosome production, nucleolar categories have been established according to RNPs distribution as follows: reticulate nucleolus with nucleolonema; compact nucleolus; and nucleolus with concentric layers. However, during the nucleologenese these domains disorganize and reorganize during the cell cycle (Thiry and Goessens, 1996; Ploton et al., 1987; Zatsepina et al., 1997; Mello, 2001). The nucleolar disorganization begins in prophase I and during the stages of metaphase, anaphase and telophase, the nucleolar material remains associated with the nucleolar organizer regions
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(NORs) that were also found in the form of pre-nucleolar corpuscles (PNBs), which persist in spermatids indicating post-meiotic reactivation of ribosomal RNA genes. The presence of the nucleolus during cell division, a phenomenon known as “persistence of the nucleolar material”, has been observed in dynoflagellates and malignant mammalian cells in the form of pre-nucleolar bodies (PNBs). The persistence of PNBs during mitosis and meiosis supports the idea that the nucleolar material is not completely disrupted during metaphase and anaphase. However, the functional significance of this finding remains unclear. It may be hypothesized that these bodies carry primary or new material or even represent a source of nucleolar RNA for the daughter cells while the new nucleolus is being organized (Wachtler and Stahl, 1993; Mello, 1995).
Nonetheless, little emphasis has been given to the presence and activity of the nucleolar material while spermatids differentiate into spermatozoa. In order to investigate the persistence of the nucleolar material during spermiogenesis in triatomines, the gametic cells found in the seminiferous tubules of the species Panstrongylus megistus, Rhodnius pallescens and Triatoma infestans were analyzed using conventional ultrastructural techniques. 2. Materials and methods The P. megistus, R. pallescens, and T. infestans species, order Heteroptera, family Reduviidae, subfamily Triatominae were used. Seminiferous tubules were analyzed from 10 adult specimens of each genus. Specimens were provided by the Araraquara Special
Fig. 1. Ultrastructure of P. megistus seminiferous tubule cells. Electromicrograph of earlier spermatids showing nucleoli (Nu), heterochromatin (H), mitochondrial derivative (Md), axoneme (Ax) and acrosomal membrane (AM). Notice the reticulate nucleolus and the evident presence of nucleolonema. Notice em (E and F) nucleolar fragmentation and the presence of these fragments up to axoneme formation (G). Bars: A, B, D–G = 1 m and C = 2 m.
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Health Service (SESA), Department of Epidemiology, São Paulo Public Health School (São Paulo, SP, Brazil). For ultrastructural analysis, the seminiferous tubules were initially fixed in 2% glutaraldehyde, 2% paraformol in phosphate buffer 0.1 M, pH 7.4. After fixing, the material was post-fixed in 1% osmium tetroxide, washed in distilled water, dehydrated in increasing acetone sequence and embedded in Epon. Resin polymerization was completed in 72 h. Ultrathin sections were cut and stained with 2% uranyl acetate and lead citrate. Electromicrographs were obtained under a transmission electron microscope (ZEISS – EM – 906, at 80 kV), using Kodak SO-163 film, at the Microscopy Center of São José do Rio Preto, Institute of Biosciences, Letters and Exact Sciences-SP (São Paulo State University/UNESP).
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3. Results Ultrastructural analyses of initial spermatids from the species under study (Figs. 1–3) showed the presence of nucleolar material up to the phase when the axoneme began to form and during spermatid elongation, as seen in R. pallescens (Fig. 2F). In addition to the nucleolus, spermatic nuclei showed some heterochromatic regions of a less electron-dense aspect. In P. megistus (Fig. 1A–G) and T. infestans (Fig. 3A–F), nucleoli had a reticulate appearance and, especially in P. megistus, displaying a well defined nucleolonema. In T. infestans, the distinction between the fibrillar component (FC) and the granular component (GC) could be observed. In R. pallescens, the nucleolus (Fig. 2A–F) resembled that of the compact type.
Fig. 2. Ultrastructure of Rhodnius pallescens seminiferous tubule cells. Electromicrograph of earlier spermatids (A–E) at elongation stage (F) showing nucleoli (Nu), heterochromatin (H), mitochondrial derivative (Md) and axoneme (Ax). Notice the compact nucleolus. Asterisks (*) indicate nucleolar fragments. Notice in (F) the nucleolar fragmentation and persistence of nucleolar bodies up to spermatid elongation, in detail another cut plan where nucleolar body is also visible. Bars: A and D = 1.5 m and B, C, E, F = 1 m.
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Fig. 3. Ultrastructure of Triatoma infestans seminiferous tubule cells. Electromicrograph of earlier spermatids showing nucleoli (Nu), heterochromatin (H), mitochondrium (Mi), Golgi apparatus (GA), mitochondrial derivative (Md) and axoneme (Ax) and the acrosome (Ac). Notice distinction fibrillar component (FC) and granular component (GC) of the reticulate nucleolus and nucleolar fragmentation. Nucleolar fragments are preferentially located at the periphery of the nuclear envelope of spermatids (D). Bars: A–C = 2.5 m and D–F = 1.5 m.
In all three species, the nucleoli were clearly fragmented. In the P. megistus species, it was possible to observe small electron-dense bodies (fragmented nucleolus) migrating to the axoneme region (Fig. 1D–G). In R. pallescens, these bodies were randomly distributed over the cytoplasm (Fig. 2A–F), and in T. infestans they were most frequently found at the periphery of the nuclear envelope (Fig. 3D). The location of the fragments varied with the cut made in the seminiferous tubules. 4. Discussion The present ultrastructural analysis provided evidence of the occurrence of fragmentation and persistence of the nucleolar material during spermiogenesis, similar to the process that
takes place during meiotic cell division already observed by the authors. Morielle-Souza and Azeredo-Oliveira (2008) used cytochemical techniques and observed in three species of triatomine (P. megistus, R. pallescens and T. infestans), the presence of bodies strongly impregnated with silver ions from meiotic metaphase I through telophase. In the T. infestans and P. megistus species that exhibited nucleolar fragments dispersed over the nucleus at meiotic prophase I and diakinesis. The authors concluded that these corpuscles correspond to PNBs, highly argyrophilic structures that might represent the assembly of nucleolar fragments which, in previous phases, were dispersed over the cell nuclei. Yet the same authors observed early spermatids of the three species showing argyrophilic corpuscle, once more corroborating
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the hypothesis of post-meiotic reactivation of rRNA genes. This phenomenon was also detected by the variant of critical electrolytic concentration (CEC), which caused these regions to be clearly metachromatic and by acridine orange staining, which stained them red so the rRNA could be discriminated and thus providing structural details of the nucleolus and RNA distribution during meiotic cell division. Therefore, all the methods used confirmed the presence of RNA/protein complexes. These results supported the persistence of the nucleolar material and the hypothesis of the post-meiotic reactivation of rRNA genes (Morielle-Souza and Azeredo-Oliveira, 2008). In spermatid at early developmental stage of Oedipoda coerulescens (Orthoptera: Acrididae), ultrastructural investigations have revealed that the nucleolus consists of fibrogranular material that, during differentiation, seems to transform into a single mass of fibrillar material and clusters of granular bodies, which become smaller and eventually disappear, while the fibrillar material persists. Light microscopy examination has shown that this nucleolar material has a similar behavior at the late spermatid stage in Schistocerca gregaria (Orthoptera: Acrididae) (Rufas and Gonsálvez, 1982). Several studies of spermatogenesis and oogenesis in nonmammalian and mammalian vertebrates have confirmed the post-meiotic reactivation of genes to rRNA. Hofgartner et al. (1979) used silver-ion impregnation to study four mammals species (man, Mus musculus, Rattus novergicus and Cavia cobaya) and observed the occurrence of post-meiotic reactivation of nucleolar organizer regions (NORs). In addition, this species exhibited a very similar pattern of NOR impregnation throughout spermatogenesis, with silver precipitates being detectable from the phase of spermatogonium growth up to the pachytene of meiotic prophase I. However, impregnation was no longer seen during metaphase I and II, and reappeared only at spermatid differentiation and persisted up to the beginning of elongation. Peruquetti et al. (2008) analyzed the cytochemical and ultrastructural structure of the nucleolar cycle and the distribution of cytoplasmatic RNAs in the seminiferous tubules cells of R. novergicus and M. musculus, the findings corroborate that the nucleolar ribonucleoproteins (RNPs) are very important in the spermiogenesis phases. The authors suggested that the molecular complexes (RNPs) have a role in a successive series of events that occur in the formation of the spermatozoon. During oogenesis, the phenomenon of nucleolar material persistence has been observed in plants, such as Vicia faba; in marine invertebrates, such as the Urechis caupo echiuroid; in the Physarum policephalum plasmodium and in amphibians such as Xenopus laevis; and in the embryos of mammalians such as rats, mice, rabbits, equines, bovines and humans (Das and Alfert, 1973; Tesarík et al., 1986; Sato, 1988; Grøndahl and Hyttel, 1996; Baran et al., 1996; Verheggen et al., 1998; Dundr et al., 1997, 2000). In general, the bodies found during oogenesis are similar to the nucleolar bodies found in spermatogenic and mitotic cells, in terms of origin, structure and cytochemical and biochemical characteristics (Das and Alfert, 1973). Nucleolar precursors are well known for providing the basis for the formation of nucleoli. However, their role in nucleologenesis and mechanisms governing nucleolar reorganization remains unclear and debatable. Most studies on gene activity during vertebrate spermatogenesis have been conducted in mammals and limited to demonstrating the synthesis of pre- and post-meiotic RNA. In addition to a distinct synthesis of pre-meiotic RNA, some studies using autoradiographic and ultrastructural analyses have demonstrated a small post-meiotic RNA synthesis that declines during the later stages of spermatic differentiation (Schmid et al., 1982).
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The evolutionary conservation of the post-meiotic reactivation of NORs from Cephalochordates in man indicates the need for postmeiotically synthesized ribosomal RNA. This newly synthesized rRNA might be a prerequisite for mRNA production which must be transcribed at the early stages of spermatid development (Schmid et al., 1982; Peruquetti et al., 2008). In this study, ultrastructural analyses at spermiogenesis indicated that these nucleolar products may represent RNP complexes that are likely to be needed at early spermiogenesis when important changes such as chromatin condensation and the acrosome and flagellum formation take place. Therefore, it was concluded from the ultrastructural analysis that the triatomine nucleolus does not totally disappear but remains as corpuscles that gather to form the next nucleolar cycle that in the case of meiosis, will be completed if fertilization occurs and a zygote is formed. These aspects therefore clearly show that rRNA synthesis in gametogenesis is a process that occurred in the three triatomines genera, Panstrongylus, Rhodnius and Triatoma, and therefore may be a common mechanism among the Heteroptera.
Acknowledgments The authors are thankful to Dr. José M. Soares Barata, Director of the Insectary (Araraquara, SP), Department of Epidemiology, Faculty of Public Health (São Paulo, SP), and João Luis Molina Gil and João Maurício Nóbrega da Silva Filho, technicians of the insectary, for providing the insects studied. Prof. Rosana Silistino and Luiz Roberto Faleiros technician helped teach our group the ultrastructure technique. This research was supported by FAPESP (No. 00/06700-4) and CAPES.
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