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Changes in Possum Spermatozoa During Epididymal Transit

A number of recent papers have been published on the ultrastructure and maturation of possum spermatozoa (Harding et al. 1975, 1976 a, b; Olson 1975, Cummins 1976; Temple-Smith and Bedford, 1976). I shall concentrate here on the major features of possum sperm maturation with a view to comparing it to the eutherian pattern.

The nucleus of the sperm is shaped rather like a human glans penis, with a prominent groove running for about half the length of the 'ventral' surface (Fig. 4). The axoneme is inserted into the anterior end of the groove, and thus virtually into the centre of gravity of the nucleus, rather than into the posterior border of the nucleus as in Eutheria. The attachment point is reminiscent of a ball-and-socket joint, in that the nucleus appears to be free to swivel on the axoneme. In fact, this swivelling is one of the most striking features of maturation: the sperm is released from the seminiferous epithelium with the nucleus nearly perpendicular to the neck (Figs. 5 and 6), and during maturation, the neck becomes pulled into the groove until in the mature sperm the long axis of the nucleus is in line with the flagellum (Figs. 10 and 11). This streamlining of the sperm coincides with contraction of the cytoplasmic droplet around the neck. However, the nuclear-axonemal junction seems to stay quite flexible, for dead sperm in mature populations frequently show retroflexion of the nucleus into the immature position. Although such retroflexion is usually a post-mortem artefact, the impression has persisted in the literature that marsupial sperm heads are 'free' to pivot on the neck (see for example Austin 1976).

The change in the head-neck angle is the most easily observed maturational change in possum sperm, and three stages of maturation can be recognized by this parameter (Cummins 1976). The bulk of the sperm population passes through these three stages within regions 1–3 of the epididymis, and by region 4, the majority are morphologically mature and have developed the potential for progressive motility.

Ultrastructural studies show that the straightening out of the head and tail of the sperm is accompanied by striking changes in the acrosome, the cytoplasmic droplet and the midpiece. In the testis and upper regions of the epididymis, the acrosome is a bowl-shaped structure sitting on the anterior 'dorsal' third of the nucleus (Figs. 5, 6 and 7). It acquires this shape page 29 during the latter stages of spermiogenesis through an interaction between the circumnuclear ring, and a process of the Sertoli Cell which appears - at least to this author - actively to extrude the sperm from the seminiferous epithelium (Harding et al. 1976 a; see also Fig. 5). During maturation, this bowl-shaped acrosome collapses into an inconspicuous button-shaped structure (Fig. 11), and contraction is accompanied by striking vesicles of plasma membrane which appear to bud off the overlying surface. At the same time, the initially large cytoplasmic droplet contracts and loses much of its internal complexity, and this is also accompanied by pitting and vesiculation of the plasma membrane (Figs. 8 and 9).

The contraction of the acrosome and cytoplasmic droplet occurs largely in epididymal regions 2 and 3. As this is also where the sperm mass is becoming progressively more concentrated (Figs. 2 and 3), it is possible that the contraction of the sperm cytoplasm is a response to the increasing concentration of the epididymal plasma caused by fluid resorption across the epithelium. However, whether the vesiculation and pitting of the sperm plasma membrane is simply an osmotic phenomenon is a question which requires further experimentation.

As well as contraction of the acrosome and cytoplasmic droplet, possum sperm also show dramatic changes in the ultrastructure of the midpiece during maturation. In the immature spermatozoon, the space between the mitochondrial sheath and the overlying plasmalemma is filled with a network of membranous cisternae reminiscent of smooth endoplasmic reticulum; this system communicates anteriorly with similar structures in the cytoplasmic droplet (Fig. 5). While the spermatozoa are passing through epididymal regions 2 and 3 (see Fig. 1), the cisternae are replaced by a spiral fibrous sheath underlying the plasma membrane of the posterior two-thirds of the midpiece. The gyres of the sheath run counter to the gyres of the mitochondrial spiral, and are interspersed with caveolae-like invaginations of the plasma membrane (Fig. 9). This structure appears at the same time in maturation as the acrosome and cytoplasmic droplet contract. Although its composition is not known, differential extraction techniques indicate that it may be a sulphydril-bond-rich protein similar to the keratinoid proteins which fortify eutherian sperm tails (Calvin and Bedford 1971; Calvin 1975; Temple-Smith and Bedford 1976). Exactly how it gets laid down in the sperm midpiece in such a precise manner remains a mystery. Protein synthesis would be extremely surprising in the genetically inactive spermatozoon, and furthermore, there is no histochemical evidence of RNA in the developing sperm midpiece (Cummins unpublished). It is conceivable that the sheath may page 30 arise by increasing sulphydril bonding within a pre-existing pattern of polypeptides. However, ultrastructural evidence of any such pattern in immature spermatozoa remains elusive. The spiral fibrous sheath of marsupial spermatozoa seems to be the first described example of a major structural feature appearing in mammalian spermatozoa after they leave the seminiferous epithelium, and as such, is of great interest.

The caveolae-like invaginations of the plasma membrane presumably serve to increase the surface area overlying the mitochondrial sheath. Although they are reminiscent of pinocytotic vesicles, there is no evidence of any time-related uptake of markers such as horseradish peroxidase (Cummins 1977). It is interesting that a number of related marsupial species possesses a fibrous sheath in their spermatozoa, but not all show the membrane invaginations (Harding, Carrick and Shorey 1977). The structural and physiological significance of these features of the marsupial sperm midpiece must remain open questions.