Micron 69 (2015) 56–61
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Comparative ultrastructure of spermatozoa of the redclaw Cherax quadricarinatus and the yabby Cherax destructor (Decapoda, Parastacidae) Antonín Kouba ∗ , Hamid Niksirat, Martin Bláha ˇ University of South Bohemia in Ceské Budˇejovice, Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and Czech Republic Biodiversity of Hydrocenoses, Zátiˇsí 728/II, CZ-389 25 Vodnany, ˇ
a r t i c l e
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Article history: Received 22 August 2014 Received in revised form 30 October 2014 Accepted 6 November 2014 Available online 14 November 2014 Keywords: Acrosome Crayfish Decapoda Spermatozoon Transmission electron microscopy
a b s t r a c t Ultrastructure of spermatozoa of redclaw Cherax quadricarinatus and yabby Cherax destructor were described and compared. The acrosome complex and nucleus are located at the anterior and posterior region of the spermatozoon, respectively. The acrosome is a complex vesicle divided into two parts: the main body of the acrosome appears as a dense cup-shaped structure in longitudinal sagittal view, with the subacrosome zone occupying the central area of the vesicle. The acrosome is larger in C. quadricarinatus (width 2.37 ± 0.27 m, length 1.31 ± 0.23 m) than in C. destructor (width 1.80 ± 0.27 m, length 1.01 ± 0.15 m). There was no significant difference in L:W ratios of the studied species. The subacrosome zone in both species consists of two areas of different electron density. The nucleus is substantially decondensed and irregular in shape, with elaborate extended processes. The examined species exhibited a well-conserved structure of crayfish spermatozoon, similar to those of Cherax cainii and Cherax albidus. Small acrosome size, the absence of radial arms, and an extracellular capsule seem to be the morphological features that mostly distinguish Cherax from the Astacidae and Cambaridae. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction The spermatozoon ultrastructure is an important aspect of basic knowledge of animal reproductive biology (Bian et al., 2013; Santos et al., 2013; Shi et al., 2014), and has traditionally been employed in taxonomic as well as phylogenetic studies (Jamieson, 1991; Justine, 1991; Mattei, 1991; Tudge, 1997, 2009). The freshwater crayfishes, a group comprising over 640 identified species in 3 families, play key roles in physical and biological modification of ecosystems (Crandall and Buhay, 2008). Detailed studies on spermatozoon ultrastructure of crayfish are scarce and include those of Astacus astacus, Astacus leptodactylus, Austropotamobius torrentium, and Pacifastacus leniusculus in the Astacidae (Dudenhausen and Talbot, 1979, 1982; López-Camps et al., 1981; Niksirat et al., 2013a,b) and Cambaroides japonicus, Cambarus sp., Procambarus clarkii, Procambarus leonensis, and Orconectes limosus in Cambaridae (Moses, 1961a,b; Yasuzumi et al., 1961; Anderson and Ellis, 1967; Felgenhauer and Abele, 1991; Niksirat et al., 2013a).
∗ Corresponding author. Tel.: +420 389 034 745. E-mail address: [email protected] (A. Kouba). http://dx.doi.org/10.1016/j.micron.2014.11.002 0968-4328/© 2014 Elsevier Ltd. All rights reserved.
The second largest crayfish family, Parastacidae, comprises over 170 recognized species (Crandall and Buhay, 2008). Spermatozoon ultrastructure has been reported only for two parastacid species (Beach and Talbot, 1987; Jamieson, 1991), Cherax cainii1 and Cherax albidus, both belonging to the phylogenetic group of this genus inhabiting southwestern Australia (Munasinghe et al., 2004). An et al. (2011) provides description of the male reproductive system and spermatogenesis with some notes on sperm ultrastructure in Cherax quadricarinatus. The aim of present study is to specifically describe and compare spermatozoa of C. quadricarinatus and Cherax destructor, the best known and most economically important representatives of Cherax inhabiting northern and eastern Australia, respectively. 2. Materials and methods Adult male redclaw (C. quadricarinatus) and yabby (C. destructor) crayfish were purchased from a pet retailer. Freshly ejaculated
1 We assume that the smooth marron, a common and widespread species largely involved in aquaculture formerly called C. tenuimanus was examined. See Austin and Ryan (2002) for details.
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spermatophores from 3 males per species were obtained via electrical stimulation (AC250K2D, Diametral, Czech Republic; Jerry, 2001), immediately fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer for 4 days at 4 ◦ C, washed in buffer, post-fixed in 4% osmium tetroxide for 2 h, washed in buffer, dehydrated through an acetone graded series (30, 50, 70, 90, 95, and 100% for 15 min each), and embedded in resin (EMbed 812). A series of ultra-thin sections were cut using an UCT ultramicrotome (Leica Microsystems GmbH, Austria), mounted on copper grids, double-stained with uranyl acetate and lead citrate, and examined with a 1010 transmission electron microscope (JEOL Ltd., Tokyo, Japan) operating at 80 kV. The length (L) and width (W) of the acrosome and the L:W ratio (Jamieson, 1991; Klaus et al., 2009) were determined from the recorded micrographs using ImageJ software (U. S. National Institutes of Health, Bethesda, MD, USA). After confirming normality and homoskedasticity with Kolmogorov–Smirnov and Cochran’s C tests, respectively, differences among conspecific males were assessed by one-way ANOVA. As no differences were detected (n = 22, 42, 27 and 32, 42, 30 spermatozoa from each of 3 specimens of C. quadricarinatus and C. destructor, respectively), data for each species were pooled and compared by Student’s t-test. For all statistical tests, P < 0.05 was considered significant. Data are expressed as the mean ± s.d.
3. Results 3.1. Comparison of acrosome dimensions There was no significant (P = 0.20) difference in acrosome L:W ratio of C. quadricarinatus (0.57 ± 0.08, range 0.39–0.75, n = 104) from that of C. destructor (0.55 ± 0.08, range 0.40–0.73, n = 91). The width of the acrosome was significantly (P < 0.01) greater in C. quadricarinatus (2.37 ± 0.27 m, range 1.58–3.27) than in C. destructor (1.80 ± 0.27 m, range 1.13–2.55). A similar significant (P < 10−6 ) pattern was observed in the acrosome length in C. quadricarinatus (1.31 ± 0.23 m, range 0.87–1.94) compared to C. destructor (1.01 ± 0.15 m, range 0.73–1.39).
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areas were observed at the apical region of the acrosome (Fig. 1D and Fig. 2C). The subacrosome zone is divided in two regions distinct in structure and electron density. Most of the subacrosome zone was usually occupied by flocculent material of moderate electron density. An area of reduced mass is present adjacent to the inner area of the acrosome main body, formed by a fibrillar and granular network containing components of differing electron densities (Figs. 1A–D and 2A–C). In both species, a membrane lamellar complex composed of stacks of highly folded, convoluted membranes often surround the moderately electron-dense material (Figs. 1E and 2D). Its position is more often lateral to the acrosome, but may appear distributed asymmetrically in longitudinal sagittal section (Figs. 1A–B and 2A). The membrane lamellar complex may be partly or almost completely missing on one or both sides of acrosome and observed adjacent to the nucleus (Figs. 1A and 2D–E). Membranous lamella, a recognizable asymmetric structure appearing as electron lucent and dense concentric lines, is present in both species (Figs. 1F and 2F). There is usually one membranous lamella, but two smaller membranous lamellae may be present, usually located within the membrane lamellar complex close to the acrosome. Functional mitochondria characterized by cristae were not observed. A single centriole was recorded in one C. destructor specimen (Fig. 2E). The subacrosome zone abuts on the nucleus, but is clearly separated by a multi-layered membrane (Fig. 3A–B). In the C. quadricarinatus spermatozoon, the subacrosome zone extends toward the nucleus to a greater extent than seen in C. destructor. The nucleus is enclosed by a continuous irregularly shaped, substantially decondensed nuclear envelope (Fig. 1B) occupying a broad area (Figs. 1A and 2A). Elaborate processes, sometimes with convoluted infoldings, extend from the nuclei of the studied species (Figs. 1A and 3C–D). Deposits of highly electron-dense material were observed, although rarely, on the surface of the nucleus in C. destructor (Fig. 2A). The extracellular matrix surrounding spermatozoa is similar in C. quadricarinatus and C. destructor, containing small granules and filaments (Fig. 1A–B), with additional lamellae in C. destructor (Figs. 2A and 3C).
3.2. Morphological features 4. Discussion In both species, an acrosome complex and the nucleus are located in the anterior and posterior region of the spermatozoon, respectively. The acrosome complex consists of the acrosome main body and subacrosome zone. The membrane lamellar complex is mostly located between the acrosome complex and the nucleus (Figs. 1A and 2A). Although the appearance of the spermatozoon is similar in the species, their shapes vary and are asymmetric. The main body of the acrosome appears as a dense cup-shaped structure in longitudinal sagittal section (Figs. 1A–B and 2A). In C. quadricarinatus, it consists of two homogenous layers of different electron densities. The denser layer occupies the inner area of the acrosome base and extends anteriorly (Fig. 1B–C). Such a clear pattern is not obvious in C. destructor (Fig. 2B). In a minority of observed C. quadricarinatus spermatozoa, regions of lower electron density were observed at the base of the main acrosome body within the denser layer. These regions contain moderately and highly electron dense materials resembling the material in the subacrosome zone (Fig. 1C). Electron-lucent canals at the acrosome base of C. destructor are narrower, less electron-lucent, and more abundant compared to regions of lower electron density in C. quadricarinatus (cf. Figs. 1C and 2B). Electron-lucent canals in C. destructor were often seen to possess a single small electron-dense component at the periphery (Fig. 2B). In rare instances in both species, these electron-lucent
Acrosomes of described C. quadricarinatus and C. destructor spermatozoa are the smallest reported in any crayfish (mean width 2.37 and 1.80 m, respectively). Beach and Talbot (1987) reported the width of the C. albidus and C. cainii acrosome to be approximately 2 m, and Jamieson (1991) confirms this for C. cainii. Thus, a small acrosome seems to be typical for this genus. However, confirmation of this for the Parastacidae in general requires investigation of a greater range of species and genera, as measurements in Cambaridae species indicate substantial variability (mean width 2.45 and 4.77 m in P. clarkii and O. limosus, respectively; Niksirat et al., 2013a). On the contrary, much larger acrosomes have been reported in Astacidae A. torrentium (mean width 8.01 m) and A. astacus (mean width 11.66 m) (Niksirat et al., 2013a,b). The shape of the acrosome in C. quadricarinatus and C. destructor (mean L:W ratio 0.55 and 0.57, respectively) is within a range usually reported for crayfish (0.5–0.6) suggesting a depressed acrosome (Jamieson, 1991; Niksirat et al., 2013a,b). The most depressed acrosome has been reported in O. limosus (mean L:W ratio 0.36) (Niksirat et al., 2013a). Although representing different phylogenetic groups of Cherax (Munasinghe et al., 2004), spermatozoa of both C. quadricarinatus and C. destructor show similarity in general ultrastructure to C. cainii, and C. albidus (Beach and Talbot, 1987; Jamieson, 1991).
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Fig. 1. Transmission electron micrographs of C. quadricarinatus spermatozoa. (A) Longitudinal sagittal section of the spermatozoon, (B) acrosomal complex with the main body of acrosome having higher electron density in its inner region (arrows), (C) regions with lower electron density at the base of the main body of the acrosome situated in the layer of higher density, arrows show bodies with moderate and high electron density, (D) regions of lower electron density in the apical area of the main body of acrosome, (E) membrane lamellar complex lateral to the acrosome, (F) membranous lamella. A: main body of acrosome, MLC: membrane lamellar complex, M: membranous lamella, N: nucleus, SA: subacrosome zone.
Information on spermatozoon ultrastructure of the other 14 genera of Parastacidae (Crandall and Buhay, 2008) is unavailable, so comparison is not possible. Unlike the Astaciade and Cambaridae (Niksirat et al., 2013a,b), spermatozoa of studied parastacids lack radial arms and are not individually enclosed within an extracellular capsule. These capsules are thought to confine the radial arms and permit tighter packaging of the spermatozoa in the spermatophores (Beach and Talbot, 1987; Dudenhausen and Talbot, 1983; Vogt, 2002; Hobbs et al., 2007). The acrosome appears, in longitudinal sagittal section, as a dense cup-shaped structure in all crayfish species this far examined (Moses, 1961a; Jamieson, 1991; Niksirat et al., 2013a,b). Small electron-lucent regions, usually with a single small electron-dense component at the periphery, have been described in the denser acrosome layer of C. cainii, and electron-lucent canals arranged in a whorled, crystal-like pattern were reported in the apical area of the acrosome of C. albidus spermatozoa (Beach and Talbot, 1987).
Their position and content are similar to regions of low electron density observed in C. quadricarinatus and the electron-lucent canals seen in C. destructor. Beach and Talbot (1987) suggested that these electron-lucent regions contain proteins compartmentalized in high concentration. In addition to the base of the acrosome main body, these structures were found in the apical area of the acrosome in the examined species (Figs. 1D and 2C). An et al. (2011) reported electron-lucent canals occasionally observed in the main body of the acrosome and considered them as a possible indication of slight immaturity of spermatozoa in C. quadricarinatus, since the acrosome fuses from endoplasmic reticulum vesicles that become more condensed and lamellar (Dudenhausen and Talbot, 1979; Tudge, 2009). On the contrary, we argue, that at least in the apical area, they might play a role in final maturation of the spermatozoon. The apical zone forming operculum has been described in representatives of all astacids (genera Astacus, Austropotamobius, and Pacifastacus; Niksirat et al., 2013a,b). A prominent single
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Fig. 2. Transmission electron micrographs of C. destructor spermatozoa. (A) Longitudinal sagittal section of the spermatozoon, (B) electron-lucent canals in the base of the main body of acrosome (arrows), (C) electron-lucent canals in the apical part of the main body of acrosome, (D) membrane lamellar complex adjacent to the nucleus, (E) membrane lamellar complex with the centriole adjacent to the nucleus, (F) membranous lamella. A: main body of acrosome, C: centriole, EDM: electron-dense material, MLC: membrane lamellar complex, M: membranous lamellae, N: nucleus, SA: subacrosome zone.
projection outlining the acrosomal spike (also called the horn-like process or anterior acrosomal process) has been described in C. japonicus, Cambarus sp., P. leonensis, and P. clarkii (Yasuzumi and Lee, 1966; Anderson and Ellis, 1967; Felgenhauer and Abele, 1991; Niksirat et al., 2013a, respectively) but was not seen in O. limosus, in which, some apical protrusions were observed (Niksirat et al., 2013a). It appears that formation of the apical zone, presence of an acrosomal spike, or developing protrusion are signs of spermatozoon maturation. However, description of changes related to final maturation requires examination of spermatophores obtained not only via dissection or electrical stimulation of males, but of those stored on the body of the female after mating or after release of spermatozoa from the spermatophore at the beginning of fertilization. Post-mating morphological changes of the spermatophore wall have been reported for crayfish (Dudenhausen and Talbot, 1983; López-Greco and Lo Nostro, 2008), and these changes also involve crayfish spermatozoa, as recently confirmed by Niksirat et al. (2014b) for A. leptodactylus.
A subacrosome zone characterized by two distinct regions with material of lower density in the vicinity of the main body of the acrosome has been documented among crayfish families (Beach and Talbot, 1987; Felgenhauer and Abele, 1991; Niksirat et al., 2013a), with which our findings concur. Niksirat et al. (2014b) revealed that, in A. leptodactylus, this region increases in density after mating, followed by its separation from the main acrosome body, retraction, and loss of density after release of spermatozoa from the spermatophore. An extensive membrane lamellar complex (Figs. 1E and 2D) is typical for Parastacidae (Beach and Talbot, 1987; Jamieson, 1991); however, its usual topographic position lateral to the acrosome complex is largely occupied by radial arms in Astacidae and Cambaridae (Niksirat et al., 2013a,b). The absence of the extracellular capsule present in Astaciade and Cambaridae likely contributes to the occurrence of wide-spread irregularly elaborate extended processes in Parastacidae spermatozoa nuclei (Beach and Talbot, 1987; this study). The deposits of electron-dense material observed
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Fig. 3. Transmission electron micrographs of C. quadricarinatus spermatozoa. (A) Multi-layered membrane between subacrosome zone and nucleus (arrows). (B–D) Transmission electron micrographs of C. destructor spermatozoa. (B) Multi-layered membrane between subacrosome zone and nucleus (arrows), (C) nucleus forming elaborate extended processes (asterisks) and extracellular matrix containing lamellae (arrows), (D) detail of the nuclear process. A: main body of acrosome, N: nucleus, SA: subacrosome zone.
infrequently on the surface of nucleus (Fig. 2A) and the extracellular matrix with lamellae surrounding spermatozoa of C. destructor (Fig. 2A and Fig. 3C) are similar to those described for C. cainii and C. albidus, respectively (Beach and Talbot, 1987). The presence of membranous lamellae seems to be common in spermatozoa of Astacidae and Cambraridae (Niksirat et al., 2013a,b), although it has not been mentioned in all relevant studies (López-Camps et al., 1981; Dudenhausen and Talbot, 1982). Beach and Talbot (1987) did not report this structure in C. cainii or C. albidus but indicated plentiful mitochondria with a degenerating appearance in the membrane lamellar complex. Anderson and Ellis (1967) suggest that the membranous lamella is formed by alteration and transformation of mitochondria during spermatogenesis, which are likely still capable of supplying energy to the crayfish spermatozoon. In our study, mitochondria were not observed, but membranous lamellae were confirmed in both species (Figs. 1F and 2F), indicating their occurrence in all crayfish families. Niksirat et al. (2014b) found that membranous lamellae detach from the cell after release of spermatozoa from the spermatophore. Simeó et al. (2010) reported a similar region in the spermatozoa of spider crab Maja brachydactyla, called the SO-complex, consisting of membranes, mitochondria and occasionally microtubules. The origin of such complexes is usually considered to originate from the nuclear envelope, endoplasmic reticulum or Golgi apparatus (Kaye et al., 1961; Langreth, 1969; Reger, 1970; Kang et al., 2008). Centrioles have often been reported in premature crayfish spermatozoa (Yasuzumi et al., 1961; Moses, 1961a,b; Yasuzumi and Lee, 1966) but not usually found in mature cells (López-Camps et al., 1981; Dudenhausen and Talbot, 1982; Niksirat et al., 2013a,b). Anderson and Ellis (1967) found centrioles in the mature spermatozoa of Cambarus sp., and Beach and Talbot (1987) recognized a pair of centrioles in a single section of C. albidus. Similarly, we recorded
a single centriole in C. destructor (Fig. 2E) which may indicate high maturity of examined spermatozoa. The C. quadricarinatus and C. destructor spermatozoon exhibited well-conserved structure, particularly when compared with the previously investigated parastacids, C. cainii and C. albidus. Small acrosomes and the absence of radial arms and extracellular capsule were the morphological features mostly distinguishable from the Astacidae and Cambaridae. For better recognition of spermatozoon ultrastructure diversity in Parastacidae, more species, particularly those from previously undescribed genera, should be investigated. Increasing knowledge of final spermatozoon maturation among crayfish families requires study of morphological changes after mating and during fertilization in Cambaridae and Parastacidae (Niksirat et al., 2014b). Related assessment of the proteomic profile of spermatophores (Niksirat et al., 2014a) and morphological changes during egg activation (Niksirat et al., 2015) may provide insight into biological pathways related to capacitation and fertilization in the crayfish. More complete understanding of crayfish reproduction can support development of new techniques for artificial fertilization, facilitating progress in intensive aquaculture and programs for conservation of critically endangered species. Acknowledgments The Czech Science Foundation supported this work through project P502/12/P177. Partial funding was provided by projects CENAKVA (CZ.1.05/2.1.00/01.0024) and CENAKVA II (the results of the project LO1205 were obtained with a financial support from the MEYS of the CR under the NPU I program) and 087/2013/Z of the Grant Agency of the University of South Bohemia. The authors express sincere appreciation to the staff of the Laboratory of Electron Microscopy, Institute of Parasitology, Biology Centre of the
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Comparative ultrastructure of spermatozoa of the redclaw Cherax quadricarinatus and the yabby Cherax destructor (Decapoda, Parastacidae) Antonín Kouba ∗ , Hamid Niksirat, Martin Bláha ˇ University of South Bohemia in Ceské Budˇejovice, Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and Czech Republic Biodiversity of Hydrocenoses, Zátiˇsí 728/II, CZ-389 25 Vodnany, ˇ
a r t i c l e
i n f o
Article history: Received 22 August 2014 Received in revised form 30 October 2014 Accepted 6 November 2014 Available online 14 November 2014 Keywords: Acrosome Crayfish Decapoda Spermatozoon Transmission electron microscopy
a b s t r a c t Ultrastructure of spermatozoa of redclaw Cherax quadricarinatus and yabby Cherax destructor were described and compared. The acrosome complex and nucleus are located at the anterior and posterior region of the spermatozoon, respectively. The acrosome is a complex vesicle divided into two parts: the main body of the acrosome appears as a dense cup-shaped structure in longitudinal sagittal view, with the subacrosome zone occupying the central area of the vesicle. The acrosome is larger in C. quadricarinatus (width 2.37 ± 0.27 m, length 1.31 ± 0.23 m) than in C. destructor (width 1.80 ± 0.27 m, length 1.01 ± 0.15 m). There was no significant difference in L:W ratios of the studied species. The subacrosome zone in both species consists of two areas of different electron density. The nucleus is substantially decondensed and irregular in shape, with elaborate extended processes. The examined species exhibited a well-conserved structure of crayfish spermatozoon, similar to those of Cherax cainii and Cherax albidus. Small acrosome size, the absence of radial arms, and an extracellular capsule seem to be the morphological features that mostly distinguish Cherax from the Astacidae and Cambaridae. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction The spermatozoon ultrastructure is an important aspect of basic knowledge of animal reproductive biology (Bian et al., 2013; Santos et al., 2013; Shi et al., 2014), and has traditionally been employed in taxonomic as well as phylogenetic studies (Jamieson, 1991; Justine, 1991; Mattei, 1991; Tudge, 1997, 2009). The freshwater crayfishes, a group comprising over 640 identified species in 3 families, play key roles in physical and biological modification of ecosystems (Crandall and Buhay, 2008). Detailed studies on spermatozoon ultrastructure of crayfish are scarce and include those of Astacus astacus, Astacus leptodactylus, Austropotamobius torrentium, and Pacifastacus leniusculus in the Astacidae (Dudenhausen and Talbot, 1979, 1982; López-Camps et al., 1981; Niksirat et al., 2013a,b) and Cambaroides japonicus, Cambarus sp., Procambarus clarkii, Procambarus leonensis, and Orconectes limosus in Cambaridae (Moses, 1961a,b; Yasuzumi et al., 1961; Anderson and Ellis, 1967; Felgenhauer and Abele, 1991; Niksirat et al., 2013a).
∗ Corresponding author. Tel.: +420 389 034 745. E-mail address: [email protected] (A. Kouba). http://dx.doi.org/10.1016/j.micron.2014.11.002 0968-4328/© 2014 Elsevier Ltd. All rights reserved.
The second largest crayfish family, Parastacidae, comprises over 170 recognized species (Crandall and Buhay, 2008). Spermatozoon ultrastructure has been reported only for two parastacid species (Beach and Talbot, 1987; Jamieson, 1991), Cherax cainii1 and Cherax albidus, both belonging to the phylogenetic group of this genus inhabiting southwestern Australia (Munasinghe et al., 2004). An et al. (2011) provides description of the male reproductive system and spermatogenesis with some notes on sperm ultrastructure in Cherax quadricarinatus. The aim of present study is to specifically describe and compare spermatozoa of C. quadricarinatus and Cherax destructor, the best known and most economically important representatives of Cherax inhabiting northern and eastern Australia, respectively. 2. Materials and methods Adult male redclaw (C. quadricarinatus) and yabby (C. destructor) crayfish were purchased from a pet retailer. Freshly ejaculated
1 We assume that the smooth marron, a common and widespread species largely involved in aquaculture formerly called C. tenuimanus was examined. See Austin and Ryan (2002) for details.
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spermatophores from 3 males per species were obtained via electrical stimulation (AC250K2D, Diametral, Czech Republic; Jerry, 2001), immediately fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer for 4 days at 4 ◦ C, washed in buffer, post-fixed in 4% osmium tetroxide for 2 h, washed in buffer, dehydrated through an acetone graded series (30, 50, 70, 90, 95, and 100% for 15 min each), and embedded in resin (EMbed 812). A series of ultra-thin sections were cut using an UCT ultramicrotome (Leica Microsystems GmbH, Austria), mounted on copper grids, double-stained with uranyl acetate and lead citrate, and examined with a 1010 transmission electron microscope (JEOL Ltd., Tokyo, Japan) operating at 80 kV. The length (L) and width (W) of the acrosome and the L:W ratio (Jamieson, 1991; Klaus et al., 2009) were determined from the recorded micrographs using ImageJ software (U. S. National Institutes of Health, Bethesda, MD, USA). After confirming normality and homoskedasticity with Kolmogorov–Smirnov and Cochran’s C tests, respectively, differences among conspecific males were assessed by one-way ANOVA. As no differences were detected (n = 22, 42, 27 and 32, 42, 30 spermatozoa from each of 3 specimens of C. quadricarinatus and C. destructor, respectively), data for each species were pooled and compared by Student’s t-test. For all statistical tests, P < 0.05 was considered significant. Data are expressed as the mean ± s.d.
3. Results 3.1. Comparison of acrosome dimensions There was no significant (P = 0.20) difference in acrosome L:W ratio of C. quadricarinatus (0.57 ± 0.08, range 0.39–0.75, n = 104) from that of C. destructor (0.55 ± 0.08, range 0.40–0.73, n = 91). The width of the acrosome was significantly (P < 0.01) greater in C. quadricarinatus (2.37 ± 0.27 m, range 1.58–3.27) than in C. destructor (1.80 ± 0.27 m, range 1.13–2.55). A similar significant (P < 10−6 ) pattern was observed in the acrosome length in C. quadricarinatus (1.31 ± 0.23 m, range 0.87–1.94) compared to C. destructor (1.01 ± 0.15 m, range 0.73–1.39).
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areas were observed at the apical region of the acrosome (Fig. 1D and Fig. 2C). The subacrosome zone is divided in two regions distinct in structure and electron density. Most of the subacrosome zone was usually occupied by flocculent material of moderate electron density. An area of reduced mass is present adjacent to the inner area of the acrosome main body, formed by a fibrillar and granular network containing components of differing electron densities (Figs. 1A–D and 2A–C). In both species, a membrane lamellar complex composed of stacks of highly folded, convoluted membranes often surround the moderately electron-dense material (Figs. 1E and 2D). Its position is more often lateral to the acrosome, but may appear distributed asymmetrically in longitudinal sagittal section (Figs. 1A–B and 2A). The membrane lamellar complex may be partly or almost completely missing on one or both sides of acrosome and observed adjacent to the nucleus (Figs. 1A and 2D–E). Membranous lamella, a recognizable asymmetric structure appearing as electron lucent and dense concentric lines, is present in both species (Figs. 1F and 2F). There is usually one membranous lamella, but two smaller membranous lamellae may be present, usually located within the membrane lamellar complex close to the acrosome. Functional mitochondria characterized by cristae were not observed. A single centriole was recorded in one C. destructor specimen (Fig. 2E). The subacrosome zone abuts on the nucleus, but is clearly separated by a multi-layered membrane (Fig. 3A–B). In the C. quadricarinatus spermatozoon, the subacrosome zone extends toward the nucleus to a greater extent than seen in C. destructor. The nucleus is enclosed by a continuous irregularly shaped, substantially decondensed nuclear envelope (Fig. 1B) occupying a broad area (Figs. 1A and 2A). Elaborate processes, sometimes with convoluted infoldings, extend from the nuclei of the studied species (Figs. 1A and 3C–D). Deposits of highly electron-dense material were observed, although rarely, on the surface of the nucleus in C. destructor (Fig. 2A). The extracellular matrix surrounding spermatozoa is similar in C. quadricarinatus and C. destructor, containing small granules and filaments (Fig. 1A–B), with additional lamellae in C. destructor (Figs. 2A and 3C).
3.2. Morphological features 4. Discussion In both species, an acrosome complex and the nucleus are located in the anterior and posterior region of the spermatozoon, respectively. The acrosome complex consists of the acrosome main body and subacrosome zone. The membrane lamellar complex is mostly located between the acrosome complex and the nucleus (Figs. 1A and 2A). Although the appearance of the spermatozoon is similar in the species, their shapes vary and are asymmetric. The main body of the acrosome appears as a dense cup-shaped structure in longitudinal sagittal section (Figs. 1A–B and 2A). In C. quadricarinatus, it consists of two homogenous layers of different electron densities. The denser layer occupies the inner area of the acrosome base and extends anteriorly (Fig. 1B–C). Such a clear pattern is not obvious in C. destructor (Fig. 2B). In a minority of observed C. quadricarinatus spermatozoa, regions of lower electron density were observed at the base of the main acrosome body within the denser layer. These regions contain moderately and highly electron dense materials resembling the material in the subacrosome zone (Fig. 1C). Electron-lucent canals at the acrosome base of C. destructor are narrower, less electron-lucent, and more abundant compared to regions of lower electron density in C. quadricarinatus (cf. Figs. 1C and 2B). Electron-lucent canals in C. destructor were often seen to possess a single small electron-dense component at the periphery (Fig. 2B). In rare instances in both species, these electron-lucent
Acrosomes of described C. quadricarinatus and C. destructor spermatozoa are the smallest reported in any crayfish (mean width 2.37 and 1.80 m, respectively). Beach and Talbot (1987) reported the width of the C. albidus and C. cainii acrosome to be approximately 2 m, and Jamieson (1991) confirms this for C. cainii. Thus, a small acrosome seems to be typical for this genus. However, confirmation of this for the Parastacidae in general requires investigation of a greater range of species and genera, as measurements in Cambaridae species indicate substantial variability (mean width 2.45 and 4.77 m in P. clarkii and O. limosus, respectively; Niksirat et al., 2013a). On the contrary, much larger acrosomes have been reported in Astacidae A. torrentium (mean width 8.01 m) and A. astacus (mean width 11.66 m) (Niksirat et al., 2013a,b). The shape of the acrosome in C. quadricarinatus and C. destructor (mean L:W ratio 0.55 and 0.57, respectively) is within a range usually reported for crayfish (0.5–0.6) suggesting a depressed acrosome (Jamieson, 1991; Niksirat et al., 2013a,b). The most depressed acrosome has been reported in O. limosus (mean L:W ratio 0.36) (Niksirat et al., 2013a). Although representing different phylogenetic groups of Cherax (Munasinghe et al., 2004), spermatozoa of both C. quadricarinatus and C. destructor show similarity in general ultrastructure to C. cainii, and C. albidus (Beach and Talbot, 1987; Jamieson, 1991).
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Fig. 1. Transmission electron micrographs of C. quadricarinatus spermatozoa. (A) Longitudinal sagittal section of the spermatozoon, (B) acrosomal complex with the main body of acrosome having higher electron density in its inner region (arrows), (C) regions with lower electron density at the base of the main body of the acrosome situated in the layer of higher density, arrows show bodies with moderate and high electron density, (D) regions of lower electron density in the apical area of the main body of acrosome, (E) membrane lamellar complex lateral to the acrosome, (F) membranous lamella. A: main body of acrosome, MLC: membrane lamellar complex, M: membranous lamella, N: nucleus, SA: subacrosome zone.
Information on spermatozoon ultrastructure of the other 14 genera of Parastacidae (Crandall and Buhay, 2008) is unavailable, so comparison is not possible. Unlike the Astaciade and Cambaridae (Niksirat et al., 2013a,b), spermatozoa of studied parastacids lack radial arms and are not individually enclosed within an extracellular capsule. These capsules are thought to confine the radial arms and permit tighter packaging of the spermatozoa in the spermatophores (Beach and Talbot, 1987; Dudenhausen and Talbot, 1983; Vogt, 2002; Hobbs et al., 2007). The acrosome appears, in longitudinal sagittal section, as a dense cup-shaped structure in all crayfish species this far examined (Moses, 1961a; Jamieson, 1991; Niksirat et al., 2013a,b). Small electron-lucent regions, usually with a single small electron-dense component at the periphery, have been described in the denser acrosome layer of C. cainii, and electron-lucent canals arranged in a whorled, crystal-like pattern were reported in the apical area of the acrosome of C. albidus spermatozoa (Beach and Talbot, 1987).
Their position and content are similar to regions of low electron density observed in C. quadricarinatus and the electron-lucent canals seen in C. destructor. Beach and Talbot (1987) suggested that these electron-lucent regions contain proteins compartmentalized in high concentration. In addition to the base of the acrosome main body, these structures were found in the apical area of the acrosome in the examined species (Figs. 1D and 2C). An et al. (2011) reported electron-lucent canals occasionally observed in the main body of the acrosome and considered them as a possible indication of slight immaturity of spermatozoa in C. quadricarinatus, since the acrosome fuses from endoplasmic reticulum vesicles that become more condensed and lamellar (Dudenhausen and Talbot, 1979; Tudge, 2009). On the contrary, we argue, that at least in the apical area, they might play a role in final maturation of the spermatozoon. The apical zone forming operculum has been described in representatives of all astacids (genera Astacus, Austropotamobius, and Pacifastacus; Niksirat et al., 2013a,b). A prominent single
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Fig. 2. Transmission electron micrographs of C. destructor spermatozoa. (A) Longitudinal sagittal section of the spermatozoon, (B) electron-lucent canals in the base of the main body of acrosome (arrows), (C) electron-lucent canals in the apical part of the main body of acrosome, (D) membrane lamellar complex adjacent to the nucleus, (E) membrane lamellar complex with the centriole adjacent to the nucleus, (F) membranous lamella. A: main body of acrosome, C: centriole, EDM: electron-dense material, MLC: membrane lamellar complex, M: membranous lamellae, N: nucleus, SA: subacrosome zone.
projection outlining the acrosomal spike (also called the horn-like process or anterior acrosomal process) has been described in C. japonicus, Cambarus sp., P. leonensis, and P. clarkii (Yasuzumi and Lee, 1966; Anderson and Ellis, 1967; Felgenhauer and Abele, 1991; Niksirat et al., 2013a, respectively) but was not seen in O. limosus, in which, some apical protrusions were observed (Niksirat et al., 2013a). It appears that formation of the apical zone, presence of an acrosomal spike, or developing protrusion are signs of spermatozoon maturation. However, description of changes related to final maturation requires examination of spermatophores obtained not only via dissection or electrical stimulation of males, but of those stored on the body of the female after mating or after release of spermatozoa from the spermatophore at the beginning of fertilization. Post-mating morphological changes of the spermatophore wall have been reported for crayfish (Dudenhausen and Talbot, 1983; López-Greco and Lo Nostro, 2008), and these changes also involve crayfish spermatozoa, as recently confirmed by Niksirat et al. (2014b) for A. leptodactylus.
A subacrosome zone characterized by two distinct regions with material of lower density in the vicinity of the main body of the acrosome has been documented among crayfish families (Beach and Talbot, 1987; Felgenhauer and Abele, 1991; Niksirat et al., 2013a), with which our findings concur. Niksirat et al. (2014b) revealed that, in A. leptodactylus, this region increases in density after mating, followed by its separation from the main acrosome body, retraction, and loss of density after release of spermatozoa from the spermatophore. An extensive membrane lamellar complex (Figs. 1E and 2D) is typical for Parastacidae (Beach and Talbot, 1987; Jamieson, 1991); however, its usual topographic position lateral to the acrosome complex is largely occupied by radial arms in Astacidae and Cambaridae (Niksirat et al., 2013a,b). The absence of the extracellular capsule present in Astaciade and Cambaridae likely contributes to the occurrence of wide-spread irregularly elaborate extended processes in Parastacidae spermatozoa nuclei (Beach and Talbot, 1987; this study). The deposits of electron-dense material observed
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Fig. 3. Transmission electron micrographs of C. quadricarinatus spermatozoa. (A) Multi-layered membrane between subacrosome zone and nucleus (arrows). (B–D) Transmission electron micrographs of C. destructor spermatozoa. (B) Multi-layered membrane between subacrosome zone and nucleus (arrows), (C) nucleus forming elaborate extended processes (asterisks) and extracellular matrix containing lamellae (arrows), (D) detail of the nuclear process. A: main body of acrosome, N: nucleus, SA: subacrosome zone.
infrequently on the surface of nucleus (Fig. 2A) and the extracellular matrix with lamellae surrounding spermatozoa of C. destructor (Fig. 2A and Fig. 3C) are similar to those described for C. cainii and C. albidus, respectively (Beach and Talbot, 1987). The presence of membranous lamellae seems to be common in spermatozoa of Astacidae and Cambraridae (Niksirat et al., 2013a,b), although it has not been mentioned in all relevant studies (López-Camps et al., 1981; Dudenhausen and Talbot, 1982). Beach and Talbot (1987) did not report this structure in C. cainii or C. albidus but indicated plentiful mitochondria with a degenerating appearance in the membrane lamellar complex. Anderson and Ellis (1967) suggest that the membranous lamella is formed by alteration and transformation of mitochondria during spermatogenesis, which are likely still capable of supplying energy to the crayfish spermatozoon. In our study, mitochondria were not observed, but membranous lamellae were confirmed in both species (Figs. 1F and 2F), indicating their occurrence in all crayfish families. Niksirat et al. (2014b) found that membranous lamellae detach from the cell after release of spermatozoa from the spermatophore. Simeó et al. (2010) reported a similar region in the spermatozoa of spider crab Maja brachydactyla, called the SO-complex, consisting of membranes, mitochondria and occasionally microtubules. The origin of such complexes is usually considered to originate from the nuclear envelope, endoplasmic reticulum or Golgi apparatus (Kaye et al., 1961; Langreth, 1969; Reger, 1970; Kang et al., 2008). Centrioles have often been reported in premature crayfish spermatozoa (Yasuzumi et al., 1961; Moses, 1961a,b; Yasuzumi and Lee, 1966) but not usually found in mature cells (López-Camps et al., 1981; Dudenhausen and Talbot, 1982; Niksirat et al., 2013a,b). Anderson and Ellis (1967) found centrioles in the mature spermatozoa of Cambarus sp., and Beach and Talbot (1987) recognized a pair of centrioles in a single section of C. albidus. Similarly, we recorded
a single centriole in C. destructor (Fig. 2E) which may indicate high maturity of examined spermatozoa. The C. quadricarinatus and C. destructor spermatozoon exhibited well-conserved structure, particularly when compared with the previously investigated parastacids, C. cainii and C. albidus. Small acrosomes and the absence of radial arms and extracellular capsule were the morphological features mostly distinguishable from the Astacidae and Cambaridae. For better recognition of spermatozoon ultrastructure diversity in Parastacidae, more species, particularly those from previously undescribed genera, should be investigated. Increasing knowledge of final spermatozoon maturation among crayfish families requires study of morphological changes after mating and during fertilization in Cambaridae and Parastacidae (Niksirat et al., 2014b). Related assessment of the proteomic profile of spermatophores (Niksirat et al., 2014a) and morphological changes during egg activation (Niksirat et al., 2015) may provide insight into biological pathways related to capacitation and fertilization in the crayfish. More complete understanding of crayfish reproduction can support development of new techniques for artificial fertilization, facilitating progress in intensive aquaculture and programs for conservation of critically endangered species. Acknowledgments The Czech Science Foundation supported this work through project P502/12/P177. Partial funding was provided by projects CENAKVA (CZ.1.05/2.1.00/01.0024) and CENAKVA II (the results of the project LO1205 were obtained with a financial support from the MEYS of the CR under the NPU I program) and 087/2013/Z of the Grant Agency of the University of South Bohemia. The authors express sincere appreciation to the staff of the Laboratory of Electron Microscopy, Institute of Parasitology, Biology Centre of the
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