J. Anat.

(2005)

207

, pp155–164

© Anatomical Society of Great Britain and Ireland 2005

Blackwell Publishing, Ltd.

Morphogenesis of the fibrous sheath in the marsupial spermatozoon

M. Ricci and W. G. Breed

Department of Anatomical Sciences, The University of Adelaide, S.A., Australia, 5005

Abstract

The spermatozoon fibrous sheath contains longitudinal columns and circumferential ribs. It surrounds the axoneme

of the principal piece of the mammalian sperm tail, and may be important in sperm stability and motility. Here we

describe its assembly during spermiogenesis in a marsupial, the brush-tail possum, and compare its structural organiza-

tion with that of eutherian mammals, birds and reptiles. Transmission electron microscopy showed that possum

fibrous sheath assembly is a multistep process extending in a distal-to-proximal direction along the axoneme from steps

4 to 14 of spermiogenesis. For the most part, assembly of the longitudinal columns occurs before that of the circum-

ferential ribs. Immunohistochemical and immunogold labelling showed that fibrous sheath proteins are first present

in the spermatid cytoplasm; at least some of the proteins of the sheath precursors differ from those in the mature

fibrous sheath. That immunoreactivity develops after initiation of chromatin condensation suggests that fibrous sheath

proteins, or their mRNAs, are stored within the spermatid cytoplasmic lobule prior to their assembly along the axoneme.

These findings are similar to those in laboratory rats, and thus suggests that the mode of fibrous sheath assembly

evolved in a common ancestor over 125 million years ago, prior to the divergence of marsupial and eutherian lineages.

Key words

spermatozoon; fibrous sheath; marsupial.

Introduction

In the flagellum of the spermatozoon of eutherian

mammals, the fibrous sheath surrounds the axoneme and

outer dense fibres of the principal piece. It is composed

of two structurally distinct segments, the dorsal and

ventral longitudinal columns

,

and a connecting array

of circumferential ribs (Fawcett, 1970, 1975). For many

years, the fibrous sheath was viewed as a passive, mechan-

ical component of the sperm flagellum (Fawcett, 1975)

that provides elastic rigidity to the sperm tail and/or

defines the shape of its beat by placing a constraint

on its plane of bending (Phillips, 1972; Fawcett, 1975;

Lindemann et al. 1992; Si & Okuno, 1993; Carrera et al. 1994;

Jassim, 1995). However, as some of its individual proteins

have become isolated and sequenced, it has become

increasingly clear that this structure also acts as a scaffold

for constituents of signalling cascade events (Carrera

et al. 1994, 1996; Fulcher et al. 1995; Vigayaraghavan

et al. 1997, 1999; Miki et al. 2002), as well as for glycolytic

enzymes (Mori et al. 1992; Welch et al. 1992, 1995).

Consequently, in addition to a structural function, the

eutherian fibrous sheath appears to have an import-

ant role in sperm motility (for a review see Eddy et al.

2003). Furthermore, comparative studies show that a

fibrous sheath-like structure is present in sperm tails of

reptiles (Harding et al. 1995; Scheltinga et al. 2001) and

non-passerine birds, with it becoming lost during the

evolution of passerines (Baccetti & Afzelius, 1976; Jones

& Lin, 1993; Lin & Jones, 1993). To date, the extent of

conservation of its molecular composition, and function,

across Amniota is unknown, although differences in ultras-

tructure suggest that its morphology and perhaps function

vary between species (Fawcett, 1970).

The formation of the fibrous sheath in eutherian

mammals has, to date, only been described in detail for

the laboratory rat (Irons & Clermont, 1982b) in spite of

the interspecific differences in its morphology. Trans-

mission electron microscopy and radioautography

Correspondence

Mario Ricci, Department of Anatomical Sciences, The University of Adelaide, S.A., 5005, Australia. T: +61 88303 6294; F: +61 88303 4398; E: mario.ricci@adelaide.edu.au

Accepted for publication

10 May 2005

Morphogenesis of the fibrous sheath in the marsupial spermatozoon, M. Ricci and W. G. Breed

© Anatomical Society of Great Britain and Ireland 2005

156

have shown that its morphogenesis involves a lengthy,

multistep process extending from steps 2 to 17 of

spermiogenesis. Moreover, in contrast to the forma-

tion of the outer dense fibres, in the laboratory rat

fibrous sheath morphogenesis has been shown to

proceed in a distal-to-proximal direction (Irons &

Clermont, 1982a) with its proteins being evident within

the spermatid cytoplasm prior to their assembly along

the axoneme (Oko & Clermont, 1988; Clermont et al.

1990).

Little is known of fibrous sheath morphogenesis

in marsupial sperm. Sapsford et al. (1969, 1970) showed

that longitudinal column precursors occur in early sper-

matids in the long-nosed bandicoot (

Perameles nasuta

)

(Peramelidae), but their morphogenesis does not appear

to have been studied in any other marsupial. The

bandicoots diverged from the possums (Phalangeridae)

early in evolution of the Australian marsupials at least

50 million years ago (Kirsch et al. 1997; Amrine-Madsen

et al. 2003; Asher et al. 2004; Cardillo et al. 2004; Nilsson

et al. 2004). In the brush-tail possum (

Trichosurus

vulpecula

) the number and molecular weights of the

major fibrous sheath proteins have recently been found

to differ considerably from those of the laboratory rat

(Harris & Rodger, 1998; Ricci & Breed, 2001). Although

several ultrastructural studies of possum spermiogen-

esis have already been carried out (Harding et al. 1976;

Lin et al. 2004), little data are available on sperm tail

morphogenesis. In this study, we describe the assembly

of the fibrous sheath in the brush-tail possum and, using

polyclonal antibodies for immunocytochemistry, we have

determined the time and site of formation of fibrous

sheath proteins during spermiogenesis.

Materials and methods

Experimental animals

Adult brush-tail possums housed at the Central Animal

House of the University of Adelaide were fed a diet

of mixed cereals and fruit daily with water available

ad libitum

. The University of Adelaide Ethics Committee

approved all animal experimentation.

Transmission electron microscopy

Possums were anaesthetized with isofluorane (4%, 1.5 L

min

−

1

, DBL, Mulgrave, Victoria, Australia), and the testes

fixed by vascular perfusion through the abdominal aorta.

The vascular system was first rinsed with heparinized

physiological saline containing 2.5% polyvinyl pyrolidone

and 0.5% procaine hydrochloride until blood outflow

had ceased, followed by perfusion with 3% parafor-

maldehyde/3% glutaraldehyde in 0.1

M

phosphate buffer,

pH 7.4, containing 2.5% polyvinyl pyrolidone for 5–10

min or until blood vessels had cleared. The testes were

then dissected free, cut into small cubes (

∼

1 mm

3

) and

immersed in the above fixative for 2 h. Immersion and

perfusion-fixed tissue was rinsed in two changes of

0.1

M

phosphate buffer, pH 7.4, for 10 min each, and

post-fixed in 1% osmium tetroxide in PBS, pH 7.4,

for 1 h. Tissue was dehydrated by passing through a

graded series of ethanols, cleared in two changes of

propylene oxide, and infiltrated in a 1 : 1 ratio of pro-

pylene oxide/resin overnight. It was then embedded in

pure resin (TK3, TAAB Laboratories, Berkshire, UK) and

blocks were polymerized at 60

°

C for at least 48 h. Thick

(0.5–1.0

µ

m) plastic sections were cut using a Reichert–

Jung Ultracut ultramicrotome with a glass knife and

stained with 0.25% toluidine blue in 0.5% sodium

tetraborate prior to viewing under an Olympus BH-2

light microscope. Areas of interest were selected from

thick plastic sections, the blocks trimmed and ultrathin

sections of silver/gold interference colours (0.02–0.1

µ

m)

were then cut with a diamond knife (Diatome Ltd, Bienne,

Switzerland). Sections were collected onto copper/

palladium grids (200 mesh), stained with uranyl acetate

and lead citrate, and viewed with a Phillips CM100

transmission electron microscope at 80 kV.

Polyclonal antibody preparation

The polyclonal anti-possum fibrous sheath serum was

prepared as previously described (Ricci & Breed 2001).

Briefly, the fibrous sheath was isolated by a combina-

tion of sonication, incubation in 4.5

M

urea and 25 m

M

DTT, and sucrose density gradient centrifugation. The

fibrous sheaths were then solubilized, emulsified in an

equal volume of Freund’s complete adjuvant (Sigma,

St. Louis, MO, USA), and approximately 50

µ

g of protein

was injected either intraperitoneally or subcutaneously

into Sprague–Dawley rats. The rats were boosted with

50

µ

g of solubilized protein in an equal volume of

Freund’s incomplete adjuvant at 2-week intervals for

6 weeks, and test bleeds were carried out 7 days after

each boost. After collection, the serum was stored

at

−

70

°

C. Pre-immune sera were collected from all rats

prior to immunization.

Morphogenesis of the fibrous sheath in the marsupial spermatozoon, M. Ricci and W. G. Breed

© Anatomical Society of Great Britain and Ireland 2005

157

Light microscope immunohistochemistry

Adult male possums were anaesthetized with isoflurane,

and testes and epididymides were fixed by perfusion for

20 min with Bouin’s fixative. Testes were excised, cut into

1-m

3

cubes and immersion-fixed in Bouin’s fixative for 2 h.

Then tissue was cut into smaller, 5-mm

3

cubes, re-immersed

in fixative for 2 h and dehydrated by passing through

a graded series of ethanols. Tissue was infiltrated with

paraffin wax, and 5-

µ

m sections were cut on a Leica

microtome and floated onto slides. The remaining steps

in this protocol were adapted from the protocol of Oko

& Clermont (1989). Deparaffinization of these sections

followed standard procedures except that during hydra-

tion (i) sections were immersed in 70% ethanol containing

1% (w/v) lithium carbonate to inactivate residual picric

acid, and (ii) endogenous peroxidase activity was elim-

inated by incubating sections in methanol containing

1% (v/v) hydrogen peroxide for 10 min. Hydrated sections

were subsequently washed for 5 min in distilled H

2

O

containing 300 m

M

glycine to block any free aldehyde

groups, then rinsed in 20 m

M

Tris/HCl saline (TBS), pH 7.4.

Non-specific binding of IgG was blocked by incubat-

ing sections in 10% normal goat serum (NGS) in TBS for

30 min. Sections were then incubated for 1 h with the

primary antibody diluted 1 : 100 in TBS-Tween con-

taining 1% NGS, and washed six times for 5 min

each in TBS-Tween containing 1% NGS, blocked with

10% NGS, before incubation with biotin-labelled goat

anti-rat immunoglobulin (Sigma) diluted 1 : 25 in TBS

for 1 h. The sections were washed in TBS-Tween and

incubated in streptavidin-alkaline phosphatase (SA-AP)

diluted 1 : 3000 in TBS for 1 h, washed again, and antibody

binding visualized by incubating sections with diamino-

benzidene tetrahydrochloride (Zymed, San Francisco,

CA, USA). Finally, sections were lightly counterstained

with haematoxylin, dehydrated by passing through a

graded series of ethanols and mounted under glass

coverslips with DPX mounting medium.

On all control slides pre-immune sera were used in

place of primary antibody. Immunostaining was repeated

on sections of testes from four different animals for

reproducibility.

Immunogold electron microscopy

Possums were anaesthetized with isoflurane, and testes

and epididymides were fixed by perfusion for 20 min

with 4% paraformaldehyde in 0.2

M

PBS, pH 7.4. Tissue

was then dissected free and immersed in the same

fixative for 2 h, washed in two changes of PBS, and

dehydrated by passing through a graded series of

ethanols. The tissue was infiltrated in a 2 : 1 ratio of

LR White/70% ethanol for 1 h, followed by two further

incubations in pure LR White resin for 1 h and then

overnight. The resin was again replaced just prior

to polymerization in gelatin capsules at 60

°

C for 24 h.

Ultrathin sections were cut on a Reichert–Jung ultracut

microtome and mounted on nickel grids.

Sections on grids were blocked by incubating in 10%

NGS in TBS for 30 min, and then incubated with the

primary antibody diluted 1 : 100 in TBS-Tween containing

1% NGS for 1 h. They were washed six times for 5 min

each in TBS-Tween, and further blocked in 10% NGS for

15 min before being incubated in 10-nm gold-conjugated

goat anti-rat immunoglobulin (Sigma), diluted 1 : 20,

for 1 h at room temperature. They were then subject to

six 5-min washes in TBS-Tween, four washes in double

distilled water, before staining with uranyl acetate (3 min)

and examination on a Phillips CM100 transmission

electron microscope at 80 kV.

Results

Formation of possum fibrous sheath

Possum fibrous sheath morphogenesis is described using

the ten stages of the seminiferous epithelium cycle and

the 14 steps of spermatid development as described by

Lin et al. (2004).

During steps 1–3 of spermiogenesis, the axoneme

developed from the distal centriole but a precursor to

the fibrous sheath was not yet visible. Sheath assembly

first began in step 4 spermatids at the distal end of the

flagellum. Electron-dense material accumulated there

between the plasmalemma and microtubule doublets

3 and 8 of the axoneme, forming the precursors of the

longitudinal columns (Fig. 1a). In step 5 spermatids, the

precursors of the longitudinal columns had lengthened

in a proximal direction along the flagellum. In step 6

spermatids, considerable electron-dense material had

accumulated beneath the plasmalemma in the distal

segment of the flagellum attached to the margins of each

of the thickened column anlagen. In longitudinal sections,

this material presented as a series of parallel, evenly

spaced, circumferentially orientated striations and there-

fore are presumably the precursors of the circumferential

ribs (Fig. 1b,c).

Morphogenesis of the fibrous sheath in the marsupial spermatozoon, M. Ricci and W. G. Breed

© Anatomical Society of Great Britain and Ireland 2005

158

In step 8 spermatids, electron-dense material accu-

mulated between adjacent pairs of the rib precursors

(Fig. 1d), which subsequently thickened and began to

resemble the ribs in mature sperm. During step 9, these

coalescenced further to form the larger, definitive,

ribs of the fibrous sheath (Fig. 1e). The number of rib

precursors that fused at this stage ranged from 2–3 in the

most distal segment of the developing principal piece

to 6–12 in the most proximal segment where the develop-

ing fibrous sheath was therefore considerably larger.

These ribs were united at each of their ends to the

longitudinal columns, which simultaneously thickened

and grew in a distal-to-proximal direction.

During step 11, the longitudinal columns appeared

considerably larger, although the newly formed circum-

ferential ribs were conspicuously electron lucent, perhaps

reflecting their early state of development (Fig. 1f), and

projected away from the axoneme to give the flagellum

an ovoid cross-sectional shape. During steps 12 and 13,

as the ribs coalesced and thickened, small, electron-lucent

spaces appeared within the longitudinal columns of the

fibrous sheath (Fig. 1g). The final step of spermiogenesis

Fig. 1 Transverse (a,f) and longitudinal (b–e,g) sections through possum spermatids showing the development of the fibrous sheath (FS). The anlagen of the longitudinal columns (ALC) first appear in step 4 spermatids adjacent to microtubules 3 and 8 of the axoneme (a), whereas the anlagen of the circumferential ribs (RA) first appear in step 6 spermatids as a series of evenly spaced striations (b,c). The rib anlagen thicken and coalesce during steps 8 (d) and 9 (e) to form mature ribs (R). During step 11, the ribs project away from the axoneme and the longitudinal columns (LC) increase in size (f). The ribs coalesce and thicken further as spermiogenesis progresses, although electron-lucent spaces can be seen in the columns in step 13 spermatids. Scale bars: (a) 0.1 µm, (b) 0.3 µm, (c) 0.03 µm, (d–f) 0.25 µm, (g) 0.3 µm.

Morphogenesis of the fibrous sheath in the marsupial spermatozoon, M. Ricci and W. G. Breed

© Anatomical Society of Great Britain and Ireland 2005

159

was characterized by a gradual increase in size of the

fibrous sheath.

Light microscope immunocytochemistry

Immunohistochemical staining of the seminiferous tubules

with anti-possum fibrous sheath serum was variable

according to the stages, and was step-specific. No reactivity

was detected in any of the early, round spermatids (steps

1–6) (Fig. 2a–f), but the cytoplasm and developing flagella

of elongating spermatids and spermatozoa were immuno-

reactive (steps 7–11) (Fig. 2a–c, g–i). Faint immunost-

aining was first detected within the cytoplasm of step 7/8

spermatids (Fig. 2g); however, labelling rapidly increased

in intensity in this region during steps 9 and 10, during

which time the flagellum first appeared immunoreactive

(Fig. 2h,i, arrows). Immunostaining was generally un-

iform throughout the cytoplasm of these spermatids. It

peaked in step 11 cells (Fig. 2a), whose entire flagellum was

stained, to remain elevated during step 12 (Fig. 2b), and

diminished rapidly in the remaining step. In comparison,

immunostaining of the spermatid flagellum, which

began at step 9, increased steadily during steps 12–13

(Fig. 2b,c), to reach a peak in step 14 spermatozoa

(Fig. 2d). There was some minor staining of residual

material within the seminiferous tubules during Stage

IV (Fig. 2d, arrow). No immunostaining was detected

in any tubules incubated in pre-immune sera.

Fig. 2 (a–i) Possum testicular sections immunostained with anti-possum fibrous sheath serum. The stages (I–X, top right corner of figures) of the cycle of seminiferous epithelium, and spermatid steps (1–14, top left corner of figures) of spermiogenesis are indicated. Spermatid cytoplasmic staining extends from steps 7 to 14 of spermiogenesis but peaks at step 11. Staining of the spermatid flagellum is first evident during step 9 (arrowheads) and peaks in the final step of spermiogenesis. Round spermatid (*). Scale bars: 12 µm.

Morphogenesis of the fibrous sheath in the marsupial spermatozoon, M. Ricci and W. G. Breed

© Anatomical Society of Great Britain and Ireland 2005

160

Immunogold electron microscopy

No immunogold labelling was detected in step 1–5

spermatids. During steps 6–10 there was no, or only very

minor, non-specific labelling over the anlagen of the

longitudinal columns and circumferential ribs (Fig. 3a–c).

By contrast, an increased labelling over the spermatid

cytoplasm during this period reached a peak in step 11

cells (Fig. 3b,d). In step 12 spermatids, there was a marked

increase in immunogold labelling over the abaxial

cytoplasm, especially in the proximal segment of the

flagellum (Fig. 3e). From step 11, specific immunogold

labelling was seen over both the longitudinal columns

and the circumferential ribs of the fibrous sheath

itself, this labelling peaking at step 14 (Fig. 3f,g). No

immunogold labelling was evident in any sections

incubated in pre-immune sera.

Discussion

In the current study, the morphogenesis of the fibrous

sheath is described for sperm of the brush-tail possum.

Fig. 3 Transverse (a,e,g) and longitudinal (b–d,f) sections through possum spermatids treated with anti-possum fibrous sheath serum. These show the development of the fibrous sheath. Immunogold labelling of the flagellum does not occur or occurs very sparsely in step 6 (a,b) and step 8 (c) spermatids. However, the cytoplasm lobule is reactive from step 6 (b) to step 11 (d) (arrowheads). In step 12 spermatids (e) there is intense immunogold labelling beneath the plasmalemma (arrowheads), suggesting that these proteins migrate down the flagellum before becoming incorporated into the fibrous sheath (FS). Immunogold labelling over the fibrous sheath reaches a peak in step 14 spermatids (f,g). Anlagen of the longitudinal columns (ALC), anlagen of the circumferential ribs (RA), immunogold labelling (arrowheads). Scale bars: (a,c) 0.1 µm, (b,d,f) 0.15 µm, (e) 0.06 µm, (g) 0.2 µm.

Morphogenesis of the fibrous sheath in the marsupial spermatozoon, M. Ricci and W. G. Breed

© Anatomical Society of Great Britain and Ireland 2005

161

Transmission electron microscopy has shown that this

sheath is morphologically similar to that of the long-nosed

bandicoot (Sapsford et al. 1969, 1970) and laboratory

rat (Irons & Clermont, 1982b) in spite of differences in

its final form. In these three species it has been found

that (1) the precursors of the longitudinal columns of

the fibrous sheath first assemble adjacent to axoneme

microtubules 3 and 8 in early spermatids, (2) the precursors

of the ribs appear later as circumferential striations that

gradually enlarge and coalesce, (3) growth of the columns

and ribs is largely independent of each other except for

late in spermiogenesis when their development occurs

concurrently, and (4) fibrous sheath assembly takes place

in a distal-to-proximal direction along the sperm tail.

One aim of the current study was to differentiate, by

immunohistochemistry, the time and site of formation

of the components of the fibrous sheath using a possum

fibrous sheath polyclonal antibody. This antiserum has

previously been shown, by Western blotting, to label

specifically the major possum fibrous sheath proteins,

but not any others, including those of the outer dense

fibres, thus suggesting that the sheath is composed of

a unique suite of proteins (Ricci & Breed, 2001). Further-

more, this serum also recognizes fibrous sheath proteins

from species in several other marsupial families (e.g.

tammar wallaby: Macropodidae; koala: Phascolarctidae;

and fat-tailed dunnart: Dasyuridae), which diverged from

the Phalangeridae at least 40–50 million years ago (Kirsch

et al. 1997; Asher et al. 2004; Nilsson et al. 2004), thus

suggesting an early origin of some of the fibrous sheath

proteins. In addition, the antibody also stained some

proteins in the fibrous sheath of rat sperm (Ricci &

Breed, 2001), indicating their conservation across

marsupial and eutherian lineages. The immunolabelling

data of the current study indicate that the timing of

fibrous sheath formation is, like its method of assembly,

similar in marsupials and eutherians [for data on the

laboratory rat see Oko (1988), Oko & Clermont (1989)

and Clermont et al. (1990)]. In the possum, peak cyto-

plasmic immunoreactivity occurs in step 10 spermatids,

well after chromatin condensation has become initiated

(Lin et al. 2004). Thus, as in rats (Oko, 1988; Oko &

Clermont, 1989; Clermont et al. 1990; El-Alfy et al. 1999),

some of the fibrous sheath proteins are translationally

regulated. The fact that there is marked immunolabel-

ling of the periaxonemal cytoplasm of step 11 possum

spermatids makes it likely that fibrous sheath proteins

are transferred from the spermatid cytoplasmic lobule to

the developing fibrous sheath late in spermiogenesis.

The proteins of the anlagen of the longitudinal columns

and circumferential ribs of possum spermatids are not

labelled with the anti-fibrous sheath serum and there-

fore are presumably dissimilar to those of the mature

fibrous sheath. Either the precursor structure is com-

posed of a different suite of proteins from those of the

mature fibrous sheath or these proteins are processed

during morphogenesis. Immunocytochemical analysis

of fibrous sheath formation in the laboratory rat (Oko

& Clermont, 1989; Clermont et al. 1990), mouse (Sakai

et al. 1986; Fenderson et al. 1988; Brown et al. 2003),

human (Jassim et al. 1991) and the cockerel (Bozkurt &

Holley, 1995) has yielded similar results, suggesting that

the primitive column and rib anlagen might somehow

act as organizers, or triggers, of fibrous sheath assembly

(Oko, 1988; Clermont et al. 1990) in both mammals and

birds. In the mouse, it has been suggested that a fibrous

sheath protein A-Kinase Anchoring Protein 3 (AKAP3),

which is synthesized in round spermatids and incorpo-

rated into the fibrous sheath concurrently with formation

of the rib anlagen, may be responsible for organizing

the basic structure of the fibrous sheath (Brown et al.

2003).

The current study indicates that the morphogenesis

of the fibrous sheath has been conserved across the

two major extant mammalian subclasses, despite their

divergence over 125 million years ago (Cifelli & Davis,

2003; Woodburne et al. 2003; Asher et al. 2004; Nilsson

et al. 2004). Taken together with our previous conclusion

that the 62- and 76-kDa fibrous sheath proteins have

been conserved between sperm of possum, koala, wallaby

and dunnart, as well as laboratory rat (Ricci & Breed,

2001), this suggests that an increased complexity of the

sperm flagellum evolved in a common ancestor prior to

the divergence of the eutherian and marsupial lineages.

In mammals, the thickness of the sheath’s circumfer-

ential ribs and the prominence of its longitudinal

columns varies between species. For example, in a bat

species,

Myotis lucifugus

, the ribs are very broad and

flat and the longitudinal columns thin, whereas the

Chinese hamster

Cricetulus griseus

sperm tail displays

thin ribs but broad columns (Fawcett, 1970). In both the

American opossum,

Didelphis virginiana

(Fawcett, 1970),

and brush-tailed possum, the longitudinal columns are

relatively narrow, but the ribs are greatly expanded

adjacent to the columns, and there is a pyramidal electron-

lucent region adjacent to the longitudinal columns. In

the monotreme

Ornithorhynchus anatinus

, the columns

are poorly developed with the sheath being almost

Morphogenesis of the fibrous sheath in the marsupial spermatozoon, M. Ricci and W. G. Breed

© Anatomical Society of Great Britain and Ireland 2005

162

entirely a series of helically orientated dense loops

(Carrick & Hughes, 1982; Lin & Jones, 2000). An accessory

sperm tail structure, closely resembling the mammalian

fibrous sheath, also occurs in reptiles (Furieri, 1970;

Harding et al. 1995; Ishmail & Dehlawi, 1995; Scheltinga

et al. 2001) and non-passerine birds (Baccetti & Afzelius,

1976; Burgess et al. 1991; Jones & Lin, 1993; Lin & Jones,

1993). In non-passerine birds, this structure has been

described as an amorphous, irregularly beaded, fila-

mentous mesh (Baccetti & Afzelius, 1976; Thurston &

Hess, 1987; Burgess et al. 1991; Jones & Lin, 1993; Lin

& Jones, 1993; Jamieson et al. 1995; Jamieson, 1995),

whereas in squamates, a thick, rib-like coat extends into

the midpiece of the flagellum (Jamieson & Healy, 1992;

Harding et al. 1995; Ishmail & Dehlawi, 1995; Scheltinga

et al. 2001; Ferreira & Dolder, 2002). The presence in at

least some bird and reptile spermatozoa of a sheath-

like structure, or an amorphous sheath, suggests a

somewhat similar structure to that of the mammalian

fibrous sheath. However, whether it is biochemically

and/or functionally comparable is unknown. Although

there are no specific studies of bird and reptile fibrous

sheath morphogenesis, this structure appears early in

spermiogenesis in both the quail (Lin & Jones, 1993), and

a species of neotropical lizard,

Tropidurus torquantas

(Vieira et al. 2001), and it assembles in a distal-to-proximal

direction along the axoneme in the common lizard

Lacerta vivipara

(Courtens & Depeiges, 1985).

The presence of a fibrous sheath, or a fibrous sheath-

like structure, in spermatozoa from reptiles, birds and

mammals, coupled with the current finding that its

morphogenesis is similar in eutherian and marsupial

mammals, suggests an important function(s) for it in

sperm of all amniotes. In the rat and mouse, the most

abundant fibrous sheath protein is an A-kinase anchor-

ing protein, AKAP4 (Carrera et al. 1994, 1996; Fulcher

et al. 1995) which may anchor cAMP-dependent pro-

tein kinase A (Vijayaraghavan et al. 1997; Colledge &

Scott, 1999; Miki et al. 2002; for a review see Eddy et al.

2003). The molecular weight of AKAP4 is 76 kDa and,

because possum fibrous sheath extracts have a major

protein of similar molecular weight (Ricci & Breed,

2001), it too may contain much AKAP4. In addition,

using both immunofluorescence and immunogold

microscopy, we have recently found that an antibody

raised against mouse spermatogenic glycolytic path-

way enzyme glyceraldehyde 3-phosphate dehydroge-

nase (GAPDS) (Fenderson et al. 1988; Welch et al.

1992; Bunch et al. 1998) binds intensely to the possum

fibrous sheath by both immunofluorescence and

immunogold microscopy (M.R., M. Eddy and W.G.B.,

unpublished observations). Thus, in the possum GAPDS

also appears to be a major fibrous sheath protein and

may well provide its sperm with an energy source. A

more detailed investigation of the molecular biology

of the marsupial fibrous sheath may shed light on the

proteins present in this sperm tail cytoskeletal struc-

ture. Such findings will further extend our understand-

ing of the evolution of this accessory cytoskeletal

structure of the sperm tail that appears to have evolved

around the time of the evolution of internal fertiliza-

tion and viviparity in early amniotes.

Acknowledgements

We thank Chris Leigh for his technical assistance.

M.R. was in receipt of an Australian Postgraduate Award.

This work was supported, in part, by an ARC grant

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