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Tissue and Cell 42 (2010) 97–104

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Tissue and Cell

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permatogenesis and sperm structure of Acerella muscorum, (Ionescu, 1930)Hexapoda, Protura)

. Dallai a,∗, D. Mercati a, Y. Bub, Y.W. Yinb

Department of Evolutionary Biology, University of Siena, Via A. Moro 2, I-53100 Siena, ItalyShanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, 300 Feng Lin Road, Shanghai 200032, China

r t i c l e i n f o

rticle history:eceived 23 December 2009eceived in revised form 7 January 2010ccepted 7 January 2010vailable online 9 February 2010

eywords:

a b s t r a c t

The general organization of the male genital system, the spermatogenesis and the sperm structure ofthe proturan Acerella muscorum have been described. At the apex of testis apical huge cells are present;their cytoplasm contains a conventional centriole, a large amount of dense material and several lesselectron-dense masses surrounded by mitochondria. Spermatocytes have normal centrioles and are inter-connected by cytoplasmic bridges. Such bridges seem to be absent between spermatid cells and justifythe lack of synchronization of cell maturation. Spermatids are almost globular cells with a spheroidal

permiogenesisflagellate spermatozoalectron microscopyomparative spermatology

nucleus and a large mass of dense material corresponding to the centriole adjunct. Within this mass acentriole is preserved. Mitochondria of normal structure are located between the nucleus and the plasmamembrane. The spermatids are surrounded by a thick membrane. No flagellar structure is formed. Spermhave a compact spheroidal nucleus, a large cap of centriole adjunct material within which a centriole isstill visible. A layer of mitochondria is located over the nucleus. The cytoplasm is reduced in comparisonto spermatids; many dense bodies are interspersed with sperm in the testicular lumen. The sperm are

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small, immotile cells of a

. Introduction

Protura are an interesting but neglected group of basalexapods, the phylogenetic position of which is still controver-ial (Dallai, 1991; Bitsch and Bitsch, 1998, 2000, 2004; Willmann,003). They are characterized by a great sperm variability, exhibit-

ng flagellated and aflagellated immotile sperm. The taxa with theormer type of sperm are retained to be the basal groups (Acer-ntomidae, Hesperentomidae and Protentomidae), while thoseith aflagellated sperm seem to be the most evolved (Eosentomi-ae, Fujentomidae and Sinentomidae) (Yin et al., 1989). Accordingo Nosek (1973), however, Acerella, characterized by aflagellateperm, is a taxon included among Acerentomidae, even thought is placed in an independent evolutive trend. The same occursor Huhentomon among Hesperentomidae. Thus, the sperm evolu-ion in the different groups is more complex than it was previouslyelieved and the tendency towards the sperm aflagellarity is also

vident within a single taxon. The sperm structure of Acerellaiarnea was previously studied (Dallai et al., 1990); the organizationf testes and the spermatogenesis of the species were, however, notnvestigated; the modifications occurring to the sperm components

∗ Corresponding author. Tel.: +39 0577 234 412; fax: +39 0577 234 476.E-mail address: dallai@unisi.it (R. Dallai).

040-8166/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.oi:10.1016/j.tice.2010.01.001

2.5–3 �m in diameter.© 2010 Elsevier Ltd. All rights reserved.

during sperm maturation were not available. The aim of this paperis to cover this gap and to perform a comparison with the structureof proturan flagellated sperm (Dallai et al., 1992, 2010).

2. Materials and methods

2.1. Animals

Many specimens of Acerella muscorum (Ionescu, 1930), collectedin the neighbourhoods of Siena, reared in a climate chamber at 18 ◦Cand 75% of humidity, were used for the work.

2.2. Fluorescence preparations

Testes were dissected in phosphate-buffered saline (PBS) andplaced in a small drop of 5% glycerol in PBS on a glass slide. Testeswere squashed under a small cover glass and frozen on a copperbar pre-cooled in liquid nitrogen. After removal of the coverslip,the slides were immersed in methanol for 10 min at −20 ◦C.

DNA was visualized after incubation of testes for 3–4 min in

Hoechst 33258 1 �g/ml (Sigma). Testes were mounted in smalldrops of 90% glycerol.

The fluorescent images were taken using a Leica DMRB lightmicroscope equipped with a mercury lamp for epifluorescence andwith an AxioCam camera (Carl Zeiss). Gray-scale digital images

98 R. Dallai et al. / Tissue and Cell 42 (2010) 97–104

Fig. 1. (A) Light microscopic view of the male genital system of Acerella muscorum. Note the testes (T), the long deferent ducts (Dd) dissected from the abdomen (Abd). Theyellow region at half length of testes contains the sperm cells. (B) Interference contrast microscopy. General view of dissected testis showing the apical cells (arrows), thespermatids (arrowheads) and the sperm cells (sp). (C) Free sperm cells (sp) and spermatids (spd) are visible. (D) A sperm cell whose nucleus (N) has been evidenced byHoechst staining and the centriole adjunct material (ca). (E) SEM micrograph showing a sperm cell with the anterior region where the nucleus (N) is located and beneath itthe centriole adjunct (ca).

R. Dallai et al. / Tissue and Cell 42 (2010) 97–104 99

Fig. 2. (A) Longitudinal section of a testes at low magnification to show the large apical cells (Ap), spermatids (spd) and many sperm cells (sp). (B) Cross-section throughthe apical region of a testis with the large apical cells (Ap) showing a huge nucleus (N) and the dense material (arrowheads) in the cytoplasm. Note also the spermatogonia( il of ana of maC entrio

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spg) and a parietal cell (arrow). The white arrow marks symbiont bacteria. (C) Detand the dense discontinuous material (arrowheads). Less electron-dense amountross-section through spermatogonial cells with large nucleus (N), a conventional c

ere collected separately and then pseudo-colored and merged

sing Adobe Photoshop CS3 Software (Adobe Systems).

Testes and free spermatozoa obtained from the dissection ofestes in 0.1 M phosphate-buffered saline (PBS), pH 7.2, weresed for observation and photography by interference contrasticroscopy with a Leica DMRB light microscope.

apical cell showing a conventional centriole located in a nuclear infolding (arrow)terial (asterisks) surrounded by mitochondria (m) are also visible; N, nucleus. (D)le (arrows) and a few mitochondria (m) are visible.

2.3. SEM observations

Dissected testes were split open, by dissecting tweezers, onto 1%poly-l-lysine pre-treated glass coverslips. Free spermatozoa wereallowed to sediment and to adhere on the glass surface and fixedwith 2.5% glutaraldehyde in phosphate buffer (PB) 0.1 M pH 7.2 for

100 R. Dallai et al. / Tissue and Cell 42 (2010) 97–104

Fig. 3. (A) Cross-section through primary spermatocytes at the initial meiotic division. Note that the cells are interconnected by cytoplasmic bridges (arrowheads). In thecytoplasm the two centrioles (arrow), the still large nucleus (N) with homogeneously distributed chromatin and the mitochondria (m) are visible. In the inset a cytoplasmicbridge with the densities at the opposite sides are evident. (B) The centrioles present in the spermatocytes in cross-section have a normal organization and exhibit doubletrather than triplet microtubules. Note also the centripetal rays towards the axial microtubules. (C) Two orthogonal centrioles are present in the primary spermatocyte. The9 microtubule doublets are clearly evident in the cross-sectioned centriole. (D) Cross-section through two primary spermatocytes at an advanced stage of the first meioticdivision (diplotene) showing a synaptonemal complex (arrowhead) in the nuclear material (N). (E and F) Cross-sections through spermatid cells with nuclei (N) and the largemass of electron-dense material forming the centriole adjunct (ca). Note also the mitochondria (m) scattered in the cytoplasm or located over the nucleus. Early spermatids(Fig. E central cell and Fig. F) show still a large amount of cytoplasm while when they are aged (Fig. E upper and bottom cells) the cytoplasm is reduced to a thin peripherallayer. The plasma membrane appears thickened arrowheads.

R. Dallai et al. / Tissue and Cell 42 (2010) 97–104 101

Fig. 4. (A) Two early spermatid cells with still uncondensed nuclei (N) and the large electron-dense mass of the centriole adjunct (ca) are evident. Within the latter a centriole(ce) is visible. Some scattered mitochondria (m) are also present in the peripheral cytoplasm. (B) A mature sperm with the nuclei (N) showing the condensed chromatin andthe large mass of the centriole adjunct (ca). Mitochondria (m) are disposed in a row between the nucleus and the plasma membrane. This latter is thickened (arrowheads).Around the sperm cell some globular bodies with spermatid remnants are visible; asterisks indicate mitochondria. (C) Mature sperm to show the nucleus (N) and the centrioleadjunct material (ca); within this latter structure a granular area containing a centriole (ce) is visible. A mitochondrion (m) is visible over the nucleus. (D) General view ofseveral mature sperm (sp) surrounded by a thick membrane. Globular small bodies (bd) likely extruded from the spermatid cytoplasm during sperm maturation are visible,interspersed with sperm cells.

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h at 4 ◦C and post-fixed in 1% OsO4 in PB. Samples were then dehy-rated in ethanol, dried in a Balzers CPD 030 critical point dryer, andoated with gold in a Balzers MED 010 sputtering device. Specimensere finally examined and photographed in a Philips XL20 scanning

lectron microscope operated with an accelerating voltage of 10 kV.

.4. TEM observations

Males were dissected on a slide in a drop of 0.1 M phosphateuffer pH 7.2, to which 3% sucrose was added (PB). After gonad

solation, the material was fixed in few drops of 3% glutaraldehyden PB. After 15 min the material was transferred in the same fixa-ive overnight. After rinsing in PB, the material was post-fixed in1%smium tetroxide for 1 h, then rinsed in PB, dehydrated in a seriesf alcohol and embedded in Epon-Araldite resin. Thin sectionsbtained with a Reichert ultramicrotome were routinely stainedith uranyl acetate and lead citrate and observed in a CM 10 Philip

ransmission electron microscope operating at 80 kV.

. Results

Testes are very thin (about 70 �m large at half length) and shortabout 200 �m), in comparison with those of other members of theroup (i.e. Acerentomon sp.) (Fig. 1A and B). Testes are connectedith long deferent ducts up to 1 mm long (Fig. 1A) A cross-section

hrough the apical region of testes measures only 12–20 �m iniameter and consists of a few (3–5) large cells provided with largeolymorphic nuclei, whose nuclear envelopes show many deep

nfoldings (Fig. 2B and C). The large cells are intermingled with germells and parietal cells (Fig. 2B). The large cells have dense materialn their cytoplasm, organized in discontinuous masses and otherarge less electron-dense amounts of material, around which mito-hondria are located (Fig. 2B and C). In an infolding of the nuclearnvelope a centriole is present, apparently with the usual struc-ure (Fig. 2C). Some isolated symbiont bacteria are also present inhe cytoplasm of the large cells (Fig. 2B). Parietal cells are flat androvided with a nucleus which is adapted to the available spaceFig. 2B). Globular spermatogonial cells of 5–6 �m in diameter areocated in the space between large cells (Fig. 2B and D). Spermato-onia have a large roundish nucleus, with chromatin adhering tohe inner face of the nuclear envelope and a central nucleolus;lose to the nucleus, a centriole is visible (Fig. 2D). More posteri-rly, early spermatocytes are visible, which are interconnected byytoplasmic bridges, the edges of which are thickened and markedy electron-densities (Fig. 3A and inset). On the opposite side ofhe bridge, a cluster of mitochondria is visible. In few examples,everal of these cells, located at the periphery of the testes, arenvolved in cell divisions, as it can be inferred by the presence of

asses of chromatin filaments, not surrounded by a nuclear enve-ope. At the initial step of the meiotic division, spermatocytes showrthogonally disposed centrioles. These centrioles have the con-entional structure, with 9 doublet microtubules in a cartwheelrray and radial spokes towards an axial microtubule (Fig. 3B and C).n rare examples, close centrioles seem to have different structure,

ith one of them apparently smaller than the other. Proceedingowards the central region of testes, spermatocytes in diplotenehase are recognizable for the presence of synaptonemal com-lexes in their nucleoplasm (Fig. 3D). In the cytoplasm, severalitochondria are visible, together with a few cisterns of rough

ndoplasmic reticulum (Fig. 3D). Spermatids are almost globular

ells of about 3.8–4.0 �m in diameter, characterized by the pres-nce of a large spheroidal nucleus, 2.3 �m in diameter (Fig. 1B and) containing almost homogeneously diffuse chromatin and only

ew electron-dense granules (Fig. 3E and F). The nuclear envelopes not clearly evident, even though it occurs. Between the nucleus

ell 42 (2010) 97–104

and the plasma membrane, some isolated mitochondria with thetypical appearance are visible (Fig. 3E and F). Beneath the nucleus,a quite electron-dense hemispherical mass of material is present;sometimes it has almost the same size of the nucleus, but moreoften it appears as a cap adherent to the nucleus (Figs. 3E, F and 4A).Within the mass of this material, an area of less electron-densematerial is visible; under favourable micrographs, a centriole canbe seen in this area (Fig. 4A). An electron-transparent cytoplas-mic region of 0.3–0.7 �m is present between the above overlappedelectron-dense structures and the plasma membrane. Spermatidcells are adhering to each other, but cytoplasmic bridges intercon-necting them do not seem to occur. Spermatid cells progressivelyreduces their size proceeding towards sperm maturity (comparecells in Fig. 3E) and the peripheral cytoplasm diminishes its width.Their plasma membrane becomes thickened (Figs. 3E, F and 4A)and results surrounded by a glycocalyx layer of up to 3.5 nmthick. Many irregular bodies can be found around the germ cells(Fig. 4D); in some of them, mitochondria are recognizable togetherwith electron-dense granules (Fig. 4B). Sperm cells have a diam-eter which is little less than that of spermatids, about 2.5–3 �m(Fig. 1C–E). The chromatin has become compact and more electron-dense than in the spermatid nucleus, and also the mass adhering tothe nucleus is homogeneously electron-dense (Fig. 4B–D); in thismass, however, a central small region, showing a granular appear-ance, is still visible in cross-sections, but a clear evidence of thepresence of a centriole is not easy to establish (Fig. 4C). A thin layerof cytoplasm is present around the nucleus and the overlappingelectron-dense mass. Over the nucleus, a layer of unchanged mito-chondria is visible (Fig. 4B and C). The sperm cells do not exhibitmotility.

4. Discussion

The data reported in the paper confirm the previous findingsdescribed by Dallai et al. (1990) for another species of Acerella;the taxon is characterized by the lack of a sperm flagellum andthus it has immotile sperm. The aflagellarity is quite commonamong Protura. Among them, the aflagellate sperm occur in sev-eral groups, representing the top of the evolutive trend of the taxon.Flagellate sperm are present in the basal groups, such as Acerento-midae, Protentomidae and Hesperentomidae, while Fujentomidae,Eosentomidae and Sinentomidae have aflagellate sperm and areconsidered the most evolved taxa (Yin et al., 1989).

The sperm of A. muscorum, similar to those present in othergroups, contains only few sperm components: the nucleus, themitochondria and a centriole unable to generate an axoneme.This centriole, contrary to Acerentomon microrhinus, has a con-ventional pattern, even though it exhibits doublets, rather thantriplet microtubules (Callaini et al., 1999; Dallai et al., 2006). Pri-mary spermatocytes have two centrioles, but spermatids receivea single centriole after the second meiotic division, as it occurs inhigher hexapods for the lack of centriole replication at the secondmeiotic division (Callaini et al., 1999; González et al., 1998). Thespermatid centriole is well visible until the sperm cell is formed,but later on it results embedded into the large mass of the centri-ole adjunct beneath the nucleus and it is hard visible. The presenceof a large centriole adjunct material parallels that described in theflagellate sperm of A. microrhinus, even though in this species it ismodelled in a spiral at the most anterior sperm region by a basketof microtubules (Dallai et al., 2010).

A convincing reason to explain why sperm aflagellarity occurs

is not clear. According to Morrow (2004), the loss of the sperm flag-ellum is the result of a relaxed selection pressure on males, due tothe absence of sperm competition. This author thinks that aflag-ellate sperm are less costly to produce than flagellate sperm. Thisstatement at the moment is difficult to demonstrate in Protura for

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wo reasons: first, we have not yet evidence of the occurrence of aperm competition in any proturan species studied so far, irrespec-ive whether they have flagellate or aflagellate sperm; we knowery little on the reproduction biology of the taxon and it is alsoncertain whether an indirect sperm transfer occurs through apermatophores as it occurs in several soil arthropods (Schaller,971; Proctor, 1998). Secondly, it is difficult to establish whetherhe cytoplasmic reduction of spermatid cells during spermiogene-is of Acerella, is less costly than the loss of a sperm flagellum, takingnto consideration that sperm transfer and fertilization will becameifferent.

An alternative hypothesis to explain the loss of a sperm flagel-um could be the consequence of some modifications in the flagellarxonemal structure. If we look at the sperm flagella of Protura, its quite evident that the axoneme is of aberrant type, provided

ith a variable number of axoneme doublet microtubules (from2 to 16) higher than in a conventional axoneme (Dallai et al.,990, 2006); more important, such axoneme lacks of the centralomplex and of the outer dynein arms on the doublets, which aremportant structures allowing an efficient sperm motility (Dallait al., 2006). The aberrant axoneme of A. microrhinus raises fromn uncommon basal body with 12 microtubule doublets, which isenerated by a peculiar replication of a normal centriole with 9oublets present in the apical cell of the proturan testes (Riparbellit al., 2009; Dallai et al., 2010). Thus, it is now clear that whenhe central complex of the axoneme is affected by some modifica-ions, a series of other changes take place, giving rise to the lossf one of the dynein arms, then to the loss of both dynein armsnd finally to the loss of the whole axoneme (Dallai et al., 2006).his series of events has been verified in the spermiogenesis ofeveral hexapods (Jamieson et al., 1999) and leads, as final step,o the sperm aflagellarity. The aflagellate sperm of A. muscorum isomparable to that occurring in members of the ephemeropteraneptophlebiidae. Several ephemeropteran species exhibit flagellateperm which, similar to proturans, have the axoneme devoid ofuter dynein arms and of the central complex (Baccetti et al., 1969;allai and Afzelius, 1990). Also in Leptophlebiidae, the sperm are

imple globular cells provided with only the nucleus, the axonemend a single not crystallized mitochondrion. If the sperm cell has aentriole it was not ascertained (Gaino and Mazzini, 1991).

The simplified structure of the A. muscorum sperm with annchanged nucleus, which maintains the globular shape as thepermatids, and of the large electron-dense mass of the centri-le adjunct, is the consequence of the lack of the cytoplasmicicrotubules, usually surrounding the different organelles of the

permatids and responsible of their shaping (Jamieson et al., 1999).he same feature was observed in A. microrhinus, where the nuclearelix was supposed to be formed by the axoneme rotation in thebsence of a microtubule manchette (Dallai et al., 2010). The lackf the acrosome is remarkable and also parallels that found in A.icrorhinus. The absence of the acrosome, however is not a newnding as several other hexapods share this peculiarity (Jamiesont al., 1999). While spermatocytes are interconnected by cytoplas-ic bridges, these bridges were not observed between spermatid

ells. This could be due to the presence of a thick glycocalyx exter-al to the plasma membrane. The result is that spermatids do notxhibit synchronization in the development and cells can be atifferent stage of maturation (see Fig. 3E).

A final consideration rises from the comparison between protu-an and collembolan sperm. As known, Collembola are consideredhe sister-group of Protura (Hennig, 1981; Kristensen, 1981, 1998),

ven though other relationships have been recently suggested onhe basis of molecular data (Giribet and Ribera, 2000; Giribet et al.,004; Kjer, 2004; Regier et al., 2004; Luan et al., 2005). Collembolaave a very peculiar encysted sperm, realized through a complexell mechanism involving sperm winding around an extracellular

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amount of material (Dallai et al., 2003, 2004). All the collembolanspecies examined so far are characterized by a common spermmodel, consisting of an apical acrosome, an elongated and con-densed nucleus, a long flagellum provided with a simple 9 + 2pattern and with 3 mitochondria (Dallai et al., 2003). Moreover,collembolan sperm are provided with a long extracellular anteriorcylindrical peduncle, which is lost after sperm are transferred tothe female spermatheca (Dallai et al., 2008a,b). Flagellated spermof Protura are also encysted, but this condition is acquired througha mechanism quite similar to that realized in some Arachnida(Alberti, 2000) rather than to Collembola. Moreover, an acrosomeis missing, the flagellar axoneme has an aberrant model and themitochondria are scattered in the cytoplasm. Also of great impor-tance is the presence of a sperm variability within the group, withflagellate and motile and aflagellate and immotile sperm. Consider-ing the sperm structure, Collembola are a quite uniform group, allmembers exhibiting the same sperm model, irrespective of theirtaxonomic position, while Protura seem to have evolved differ-ent sperm models, characterized by variable flagellar axonemes,according to the taxonomic position of their members, the aflagel-late sperm included.

The comparative analysis of sperm in the two groups doesnot support a close relationship between Collembola and Protura,which rather appear as two taxa which evolved independently andthat are also distant from Insects.

Acknowledgements

We thank Dr. E. Malatesta for technical assistance. Work sup-ported with a grant by Ministry of University and Research (MUR)to R.D.

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