Sperm head morphology of the plains mouse Pseudomys australis V. Sarafis, R. W. Lambert and W. G. Breed Department ofAnatomy, University ofAdelaide, Adelaide, South Australia 5001

Summary. Spermatozoa of plains mice were studied by transmission and scanning electron microscopy. The occurrence of a distinct dorsal hook and 2 ventral hooks joined at the base was confirmed. Acrosomal material covered the dorsal hook and appeared to constitute most of the 2 ventral hooks which contained nuclear material only at the base. Incubation in sodium dodecyl sulphate resulted in loss of all the material presumed to be acrosomal.

Introduction In Australia, there are nearly 50 species of native rodents belonging to the family Muridae. A light microscope study of the spermatozoa from 27 species of 9 genera showed that members of 8 species of Pseudomys, 2 species of Melomys and Notomys, and a single species of Leporillus, Uromys and Conilurus all had a characteristic sperm head with 3 hooks (Breed & Sarafis, 1979; Breed, 1980). In the present study we have examined the spermatozoa of the plains mouse in more detail with the transmission and scanning electron microscope.

Materials and Methods Preparation ofspermatozoafor electron microscopy Six laboratory bred male plains mice, Pseudomys australis, (body wt 43-83 g) were killed by cervical dislocation or by an overdose of ether. For transmission electron microscopy (TEM), spermatozoa were extruded from the epididymis or vas deferens into glutaraldehyde/ formaldehyde/picric acid fixative (Ito & Karnovsky, 1968). The suspension was centrifuged at 500 g for 5 min and the resultant pellet allowed to fix for 1 h. After three washes in 0-05 M-cacodylate buffer (pH 7-4), the spermatozoa were post-fixed in 1% osmium tetroxide in 0-05 M-buffer, washed again, dehydrated and embedded in Spurr's resin. Ultrathin sections were cut, stained with uranyl nitrate and lead citrate, and viewed with a Philips EM 300. For scanning electron microscopy (SEM), spermatozoa were extruded into a centrifuge tube containing saline (9 g NaCl/1), centrifuged at 500 g, re-suspended and re-centrifuged 3 times. Small aliquots were then injected through Millipore filters of 0-22 µ pore diameter and fixed with glutaraldehyde/formaldehyde/picric acid fixative, then washed, post-fixed and dehydrated as above. The filter supporting the spermatozoa was removed from its holder, soaked in absolute amyl acetate, dried at critical point with a Dentón vacuum DCP-1 dryer, and sputter coated to a depth of 20 nm with gold/palladium and to 10 nm with carbon. * Reprint requests to Dr W. G. Breed. t Present address: Department of Biology, Hawkesbury Agricultural College, Richmond, New South Wales 2743, Australia. t Present address: Department of Pathology, University of Melbourne, Parkville, Victoria 3052, Australia.

Downloaded from Bioscientifica.com at 09/26/2021 05:01:56AM via free access Incubation ofspermatozoa with sodium dodecyl sulphate Spermatozoa were extruded from the epididymis into a drop of 1% sodium dodecyl sulphate (SDS) in 0-05 M-sodium borate (pH 9-0) or into 0-05 M-sodium borate for incubation at room temperature (20-23 °C). The spermatozoa were observed periodically under phase-contrast and Nomarski optics.

Results

In the following descriptions the term 'dorsal' refers to the convex surface of the sperm head, and 'ventral' to the concave surface (see Illisson, 1969; Holt, 1979). The sperm head was falciform and the existence of 3 hooks was invariably clearly evident (PI. 1, Fig. 1). The ventral hooks were united caudally. The nuclear content was dense, except for occasional small cavities or vacuoles, and extended nearly to the apex of the dorsal hook (PI. 1, Fig. 2) but occupied only the base of the ventral hooks. Two-thirds of the dorsal margin of the nucleus was covered by an electron-dense acrosome which projected beyond the tip of the dorsal hook, and appeared to constitute the bulk of the two ventral hooks (PI. 1, Fig. 2). The connecting, main, principal and end pieces of the sperm tail were similar in structure to those of spermatozoa of the laboratory mouse (Mus musculus) (see Fawcett 1970, 1975).

Incubation ofspermatozoa with SDS Incubation of spermatozoa in 0-05 M-borate buffer did not disrupt the spermatozoa, but incubation in SDS resulted in slow swelling and dispersion of the material in the acrosome (PI. 1, Fig. 3), with much of the two ventral hooks finally being lost (PI. 1, Fig. 4). Tails often became detached from the head but the nuclear material remained intact.

Discussion

The present observations confirm the 3 hooks on the head of the plains mouse spermatozoon. The extension of the nucleus into most of the length of the dorsal hook is comparable to the sperm head structure of many species of murid rodent (see Friend, 1936; Bishop & Walton, 1960; Fawcett, 1970), but the 2 ventral hooks appear to be a characteristic feature of spermatozoa from many Australian rodents (Breed & Sarafis, 1979; Breed, 1980). Although TEM indicated material in the 2 ventral hooks of an electron density similar to that of the acrosome round the dorsal hook, only the latter fluoresces orange with acridine orange

PLATE 1 Fig. 1. Spermatozoon of the plains mouse as observed by scanning electron microscopy, indicating the existence of 2 ventral hooks, 11 000. Fig. 2. Transmission electron microscopy demonstrating that the nuclear material extends into much of the dorsal hook, but only into the base of the 2 ventral hooks. Material of an electron density similar to that of the acrosome over the dorsal hook constitutes the 2 ventral hooks. 14 000. Fig. 3. Spermatozoon 10 min after SDS treatment shows the material in the lower hooks in process of becoming detached from the rest of the spermatozoon. Nomarski, 1300. Fig. 4. Spermatozoon 3 h after SDS treatment shows that the lower hooks and tail have now disappeared. Nomarski, 1300.

Downloaded from Bioscientifica.com at 09/26/2021 05:01:56AM via free access PLATE 1

Downloaded from Bioscientifica.com at 09/26/2021 05:01:56AM via free access (Breed & Sarafis, 1979). However, regional differences of electron density and/or cytochemical reaction of material in the acrosome of spermatozoa are known (e.g. Allison & Hartree, 1970; Yanagimachi & Noda, 1970; Holt, 1979) and the lack of orange fluorescence with acridine orange does not necessarily invalidate the suggestion that the ventral hooks consist of acrosomal material. The results with SDS, which cleaves non-covalent bonds and disperses membranes (Calvin & Bedford, 1971), indicate that the material in the ventral hooks is easily dispersed and not bounded by a nuclear membrane, a result consistent with the material being acrosomal. We thank Dr K. Bartusek, Dr B. Filshie, Ms Gail Hermanis, Mr Rob Murphy, Ms Julia Brazier and Ms Barbara Sheldon for assistance in various ways.

References

Allison, A.C. & Hartree, E.F. (1970) Lysosomal maturation in the epididymis. J. Reprod. Fert., Suppl. enzymes in the acrosome and their possible role in 13,65-75. fertilization. J. Reprod. Fert. 21, 501-515. Fawcett, D.W. (1970) A comparative view of sperm Bishop, M.W.H. & Walton, A. (1960) Spermatogenesis ultrastructure. Biol. Reprod., Suppl. 2, 90-127. and the structure of mammalian spermatozoa. In Fawcett, D.W. (1975) The mammalian spermatozoa. Marshall's Physiology ofReproduction, 3rd edn, Vol. Devi Biol. 44, 394-436. 1, Ch. 7, pp. 1-129. Ed. A. S. Parkes. Longmans, Friend, G.F. (1936) The sperms of British muridae. Q. Jl London. Microsc. Sci. 78,419-443. Breed, W.G. (1980) Further observations on sper- Holt, W.V. (1979) Development and maturation of the matozoal morphology of Notomys and Pseudomys mammalian acrosome. /. Ultrastruct. Res. 68, species (Rodentia: Muridae). Trans. Roy. Soc. S. 58-71. Aust. 104,51-55. Illisson, L. (1969) Spermatozoal head shape in two Breed, W.G. & Sarafis, V. (1979) On the phylogenetic inbred strains of mice and their F, and F2 progenies. significance of spermatozoal morphology and male Aust. J. biol. Sci. 22, 947-963. reproductive tract anatomy in Australian rodents. Ito, S. & Karnovsky, MJ. (1968) Formaldehyde- Trans. Roy. Soc. S.Aust. 103, 127-135. glutaraldehyde fixatives containing trinitro com¬ Calvin, M.I. & Bedford, J.M. (1971) Formation of pounds./. Cell Biol. 39, 168A-169A. disulphide bonds in the nucleus and accessory Yanagimachi, R. & Noda, Y.D. (1970) Ultrastructural structures of mammalian spermatozoa during changes in the hamster sperm head during fer¬ tilization. J. Ultrastruct. Res. 31, 465-486.

Received 27 May 1980

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