Proceedings of the First Symposium on Marsupials in New Zealand
Sperm Maturation in the Possum Trichosurus Vulpecula: A Model for Comparison With Eutherian Mammals
In eutherian mammals, sperms passing through the epididymis undergo subtle morphological and biochemical changes which culminate in the capacity to fertilise. In contrast, the pattern of sperm maturation in the possum is extremely elaborate and overt. The spermatozoa undergo marked contraction and loss of cytoplasm. Structural elements are elaborated in the midpiece and they progressively acquire the potential for motility. Unlike the eutherian, these changes are easily studied using light microscopy. As well as being an inherently interesting picture of cell differentiation, sperm maturation in the possum also provides a useful model for looking at the relative importance of testis versus epididymis in the evolution of male reproduction. Far from being a 'primitive' system, sperm maturation in the possum epididymis appears to be more elaborate and specialised than in comparable Eutheria. These findings are discussed in the light of experiments to determine the endocrine control of epididymal function in the possum.
In this paper, I intend to outline the process of sperm maturation in the epididymis of the common brushtail possum Trichosurus vulpecula, and I hope to show how a study of this process may help to illuminate the selection pressures behind the evolution of the male tract in modern eutherian mammals, as well as in the marsupials.
Function of the Epididymis in Eutherian Mammals
Embryologically, the epididymis derives from the mesonephric duct (Wolffian Duct) which has lost its original urinary function and now serves to convey the gametes from the testis to the vas deferens (Romer 1960). page 24 However, it is not a simple connecting tube, for it serves both to concentrate the spermatozoa and to enable them to complete the process of maturation which starts in the seminiferous epithelium. That spermatozoa do not acquire fertility until they have passed through the epididymis was first shown by Young (1929 a,b, 1931) and Young and Simeone (1930), in work on the guinea-pig. Since those early experiments, a variety of studies has shown this to be true for a number of laboratory and domesticated species, and presumably for all eutherian mammals. The significance of the epididymis is thus well established (Hamilton 1972; Bedford 1975).
The acquisition of fertility during epididymal maturation is not precisely related to any particular change in sperm structure, but rather to physiological and biochemical changes - particularly in membrane structure and composition -which presumably render the spermatozoa capable of effecting fertilization. Some morphological changes do occur, however, notably contraction of the acrosome and distal migration of the cytoplasmic droplet; and these changes are accompanied by increases in specific gravity (Lindahl and Kihlström 1952; Lavon et al. 1966). This general pattern is now fairly well established, at least for the domestic and laboratory species (Glover 1974; Bedford 1975; Prasad and Rajalakshmi 1977), and it appears to be under the control of the epididymis, which is in turn dependent upon an adequate level of androgens (Hamilton 1972; Glover 1974; Prasad and Rajalakshmi 1977).
The epididymis is usually described, in gross anatomical terms, in relation to its attachment to the testis; thus we have the caput, corpus and cauda epididymidis; or 'head', 'body' and 'tail'. Unfortunately, there are considerable differences between species in the cytology of these regions, and it seems unlikely that the gross anatomy of the epididymis is related to function in any but the most general terms. Glover and Nicander (1971) proposed a uniform classification for the eutherian epididymis, based upon histology and a study of gross maturational changes in spermatozoa. They described three distinct regions: the Initial Segment, characterized by a high degree of fluid resorption from the testicular exudate; the Middle Segment, where sperm concentration continues and maturation is completed; and finally a Terminal Segment, where the mature spermatozoa are stored in an inactive state before ejaculation. In most species, the Terminal Segment corresponds to the cauda epididymidis, but there may be considerable variation in the positioning of the other regions in terms of gross anatomy. The situation is especially complicated when we consider species which have page 25 abdominally situated testes, in which the Terminal Segment may be at a considerable distance from the testis, close to the surface of the body; for example, in the elephant Loxodonta africana and rock hyrax Heterohyrax brucei (Glover 1973).
The spermatozoa in the Terminal Segment, although maintained in an immobile state, are nevertheless highly sensitive to temperature. In the rabbit Oryctolagus cuniculus, for example, subjecting the testis and epididymis to body temperature by means of artificial cryptorchidism rapidly inhibits the fertility of sperm in the cauda epididymidis (Cummins and Glover 1970). It seems likely that the need to maintain a sperm store at a temperature below that of the body cavity may have been a powerful influence behind the development of a thermo-regulatory scrotum (Glover 1973, 1974; Bedford 1977), and this intriguing possibility will be dealt with later.
Structure of the Possum Epididymis
The epididymis of the possum is a long, highly convoluted duct which, in gross appearance, is very similar to that of eutherian mammals (Fig. 1). The testes and epididymides lie within a pendulous scrotum and the vasa deferentia and the vascular pedicles containing the testicular arteries and veins lie close together when passing through the inguinal canal. In analysing epididymal structure and sperm maturation patterns, I (Cummins 1976) divided the duct into five regions (Fig. 1): (1) The Ductuli Efferentes and Proximal Caput; (2) The Distal Caput; (3) The Proximal Corpus; (4) The Distal Corpus; (5) The Cauda and commencement of the Vas Deferens.
The cytology of the tract also bears remarkable similarity to that of eutherian mammals. Thus, regions 1 and 2 have high columnar epithelium with prominent stereocilia, enclosing an irregularly shaped lumen with relatively few spermatozoa (Fig. 2). Regions 3 and 4 have lower epithelium, with a heterogeneous cell population and active secretion of PAS-positive material (Fig. 3). Region 5 has low, almost cuboidal epithelium enclosing a wide lumen densely packed with spermatozoa; in the terminal region approaching the caudal flexure and merger into the vas deferens, the lumen becomes large enough to be seen with the naked eye (Fig. 1). Sperm concentration increases progressively in descending the tract, reaching a maximum in Region 4.page 26
Explanation of Figures 1-11
An idea of the magnifications in Figures 4–11 will be given by the fact that the sperm nucleus is approximately 5 micrometers (.005 mm) long.
Fig. 1. The testis (T) and epididymis removed from the scrotum and displayed. Epididymal regions 1–5 are numbered (see text). The vas deferens (V) runs along the side of the 'corpus' (3 and 4) and leaves the scrotal sac in close association with the testicular artery and vein.
Fig. 2. Light micrograph of a section of epididymal region 2. Note the high columnar epithelium rich in lipid droplets, and the sparse luminal contents. The spermatozoa are recognizably immature, with large cytoplasmic droplets.
Fig. 3. Epididymal region 4. The spermatozoa are now nearly all morphologically mature, and have become densely packed. The epithelium shows several different cell types.
Fig. 4. Scanning electron micrograph (SEM) of a naked nucleus, showing the ventral groove. The background is a Millipore filter.
Fig. 5. Transmission electron micrograph (TEM) of the possum testis, showing a spermatozoon at the point of liberation from the Sertoli cell (S). The bowl-shaped acrosome sits on the anterior third of the nucleus (n), and is still filled with the Sertoli cell process. Note the large cytoplasmic droplet, with reticular channels running the length of the midpiece.
Fig. 6. Phase-contrast light micrograph of immature sperm from epididymal region 1. Note the perpendicular head-neck angle, the large cytoplasmic droplet around the neck, and the protruding acrosome (A).
Fig. 7. SEM of immature sperm from epididymal region 1. The position of the nucleus within the cytoplasmic droplet is indicated by a dotted line. Note the bowl-shaped acrosome (A).
Fig. 8. SEM of condensing sperm from epididymal region 2. The acrosome (A) is collapsing, with the loss of membranous vesicles from its surface, and the cytoplasmic droplet has a pitted appearance (arrowed).
Fig. 9. TEM of sperm section. The cell is at approximately the same stage in maturation as that shown in Figure 8: the nucleus (N) is starting to straighten out on the neck, the cytoplasmic droplet is losing much of its internal organization, and the fibrous sheath interspersed with invaginations has appeared in the distal midpiece (arrowed).
Fig. 10. Phase contrast micrograph of mature spermatozoon from epididymal region 5. The acrosome and cytoplasmic droplet have fully contracted, and the head and tail are in line, presenting a streamlined appearance.
Fig. 11. TEM of mature sperm, showing the fully contracted acrosome (A). This specimen was air-dried, which caused some contraction of membranes permitting observation of the mode of insertion of the neck into the nuclear groove (here viewed side-on).
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.
How Does the Pattern of Sperm Maturation in the Possum Compare with That of Eutheria?
It is necessary at this stage to point out that knowledge of sperm maturation in the possum is based purely on morphology: nothing is known, as yet, about the acquisition of fertilizing ability. Nevertheless, a comparison with the 'typical' eutherian pattern raises some fascinating questions about the evolution of the epididymis and scrotum. Firstly, there are some obvious basic similarities in the process; for example, in the progressive condensation of excess sperm cytoplasm and in the increasing disulphide bonding within structural proteins. There is also the fact that in both models the sperm mass undergoes progressive concentration as it passes through the epididymis, and this indicates that fluid resorption is a major feature of both the marsupial and eutherian epididymis. A minor difference here is that the fluid resorption seems to occur over a relatively greater proportion of the duct in the possum, but this impression requires further investigation. Finally, it is also clear that the epididymis of the possum, as in eutherian mammals, is under androgenic control, for castration and treatment with androgen antagonists such as diethylstilboestrol cause regression of the duct and disruption of the pattern of sperm maturation (Cummins 1977, and in preparation).page 31
Despite these similarities, it is clear that possum sperm maturation possesses some unique features. The complex acrosomal changes involving loss of plasma membrane vesicles, the pitting and vesiculation of the cytoplasmic droplet, and the elaboration of the midpiece fibrous sheath are all examples of morphological modification occurring within the epididymal lumen, which in comparable Eutheria, one would expect to have been completed by the time the spermatozoa had left the seminiferous epithelium. The overwhelming impression is that possum sperm are liberated in a much more immature state than are the sperm of comparable eutherian mammals, and that the epididymis has consequently assumed greater control over the development of the mature gamete.
Evolution of the Male Reproductive Tract in Modern Mammals
Evolutionarily, the ancestral stocks of eutherian and marsupial mammals are thought to have diverged about 130 million years ago (Lillegraven 1974, 1975). Despite this long era of separate development, the male reproductive systems in the two groups of mammals show some striking similarities, which indicates that they have probably evolved in response to similar selection pressures. I have already compared the process of sperm maturation in the possum with that found in the Eutheria, and I should like now to use this as a basis for speculation about those very selection pressures, concentrating on three main aspects of the male system, which seem to be most worthy of study: the thermoregulatory scrotum; the epididymis; the role of sperm production in mating systems.
The reasons for the evolution of a thermoregulatory scrotum are intriguing and elusive. Both marsupials and Eutheria show the tendency for testes to move into an outpouching of the peritoneum, yet this has obviously developed after divergence, for not all species develop a scrotum, and in those that do, there are fundamental embryological differences in the mode of descent between the two groups of mammals (Eckstein and Zuckerman 1956; Sharman 1970). Although there is little doubt that testicular function in those animals that have a scrotum is rapidly impaired by raising the testes to the temperature of the body, this may simply reflect secondary adaptation of testicular function to a lower temperature. Carrick and Setchell (1977) have surveyed a wide range of mammals with different degrees of elaboration of a scrotum, and have found little relationship between the effectiveness of spermatogenesis and testicular temperature. In view of this, attention has now swung away from the testis to the epididymis, and Glover (1973, 1974) and Bedford (1977) have examined in page 32 detail the possibility that a major factor in the evolution of the scrotum has been the need to provide a cool storage zone for the mature spermatozoa. It is noteworthy that the Terminal Segment (or cauda epididymidis) invariably lies in the coolest extremity of the scrotum. Although this idea clearly needs further study, it has nevertheless refreshed our interest in the selection pressures behind the evolution of the modern male tract.
When considering the epididymis, it is clear that again there are marked similarities between eutherian and marsupial mammals, although, as yet, data for marsupials are scanty. I have shown here that in the common brushtail possum, as in Eutheria, we can clearly identify an Initial Segment concerned with fluid resorption, a Middle Segment where spermatozoa complete maturation, and a Terminal Segment where the mature gametes are stored (Glover and Nicander 1971). The area in which the possum seems to be unique seems to be that of sperm maturation, for the degree of morphological modelling of the spermatozoa which occurs in the epididymis is much greater than that seen in Eutheria.
It is impossible to consider the factors behind the evolution of the scrotum and epididymis without studying the roles of those structures in sperm production. Yet, when we examine the facts of sperm production in mammals, we are confronted with what seems to be a paradox: males in general produce far more spermatozoa than are needed to effect fertilization, and yet the female tract has evolved a number of mechanisms designed to prevent too many spermatozoa from reaching the ova and thus increasing the risks of polyspermic fertilization (see Cohen 1969). What are the reasons for this seeming wastage of gametes? The answer most probably lies in the reproductive strategies evolved by males to ensure breeding success. Although an examination of the evolution of reproductive behaviour is beyond the scope of this article (see Wilson 1975), it is clear that the ability to produce extremely large numbers of spermatozoa must have been a successful trait in early mammals, otherwise it would not have persisted. In mammalian breeding systems, there is considerable division of labour between the sexes, with the females carrying the major energy-consuming tasks of gestation, lactation and caring for offspring (Dawkins 1976; Grodzinski 1975).
Clutton-Brock and Harvey (1978) have pointed out that in such breeding systems, the reproductive success of females is limited by the number of young they can produce, while the success of males is largely limited by competition between them to impregnate females. Thus, mammals on the whole tend to be page 33 polygynous rather than polyandrous, and competition between males tends to select not just for vigour in breeding behaviour, but also for the ability to produce large numbers of fertile spermatozoa. The energy demands of mammalian sperm production per se are not known, however they are probably minor in terms of overall energy budgets (Sadleir 1969). In contrast, female mammals invest a large proportion of their energy budget in breeding; small rodents, for example, need to increase metabolic effort by about 60% during gestation and lactation (Grodzinski 1975). In evolutionary terms, the advantages to males of producing large numbers of spermatozoa must have outweighed the disadvantages of 'wastage' of excess gametes.
If these suppositions are correct, then the need to produce excesses of spermatozoa must have placed increasing metabolic and mechanical demands upon the testis; demands accentuated by the increasing importance of the testis as an endocrine gland. These stresses could be eased, and sperm output maximized, if spermatozoa could be released from the testis shortly after the completion of meiosis, with a minimum of maturation. I suggest that the epididymis, in the evolution of modern mammals, played an increasingly important part in overseeing the final steps of sperm maturation; a maturation which, in turn, was becoming biochemically more complex as the spermatozoa faced the rigours of an extensive female tract designed for viviparity.
Viewed in this context, we can now see that the epididymis of the possum would appear to have assumed greater control over sperm maturation than in comparable eutherian mammals. Certainly the morphological changes are much more overt and dramatic, and this should ease the way for investigations into factors which control the process. A side issue of this observation is that the reproductive tract of the possum can in no way be considered 'primitive', for it may in some ways be even more complex than that of the supposedly more 'advanced' Eutheria. Whether or not the reproductive biology of marsupials is in general 'primitive' is still a matter of hot debate. Lillegraven (1975) inclines to the view that, in general, it is, or at least that it is less evolutionarily flexible than that developed by placental mammals. On the other hand, several recent authors have pointed out that in many ways, the marsupial pattern of viviparity, with a short gestation and long lactation, is a highly successful strategy which minimises risk to the mother and also makes best use of potentially variable supplies of nutrition (Gould 1977; Kirsch 1977; Parker 1977).page 34
The suggestion that the marsupial epididymis may be relatively more important in sperm maturation than that of eutherian mammals is still only an hypothesis. In order to test it, there seem to be three main areas where research should be directed: first, the relative durations of spermatogenesis and sperm maturation; second, the biochemical changes occurring in the epididymal plasma around the maturing spermatozoa; and third, the nature of the changes in the spermatozoa and their control. Some data are already available to show that the timing of spermatogenesis and epididymal transit in marsupials may be comparable to that of Eutheria (Setchell and Carrick 1973; Carrick and Setchell 1977), but in general the field for experimentation is virtually untouched.
I have attempted here to outline what is known about sperm maturation and epididymal function in the common brushtail possum with a view to comparing it with the pattern for eutherian mammals. There are striking similarities in the structure of the epididymides, although relatively more of the possum epididymis appears devoted to progressive sperm concentration and fluid resorption than is normally seen in eutherians. In terms of morphological changes in the spermatozoa, again the picture for the possum appears basically similar to the eutherian pattern, but the changes are far more dramatic, and -unlike Eutheria - involve shedding of membranes by the sperm as well as elaboration of at least one major structural feature: the midpiece fibrous sheath.
I have also speculated as to how these contrasts may illuminate the selection pressures behind the evolution of the scrotum and epididymis in present day mammals. Clearly these pressures have produced some remarkable parallel developments in the marsupials and placentals. Among the more important pressures would appear to be the need for males to produce large excesses of fertile sperm in competition for breeding success; the increasing endocrine role of the testis; the increasing biochemical demands on sperm imposed by the adoption of internal fertilization and viviparity; and the problems of storing mature spermatozoa in a fertile state for prolonged periods. The storage problem would appear to have been eased in both groups of mammals by the evolution of the thermoregulatory scrotum for cooling the sperm accumulated in the distal portion of the epididymis. I suggest that the proximal regions of the epididymis have progressively assumed greater control over the final stages of sperm maturation in order to ease the demands on the page 35 testis, and if this is so, then the epididymis of the possum would appear to have specialized further along this path than is the case for eutherian mammals.
This work was largely carried out in the Physiology Department, Victoria University of Wellington. Thanks are due to Merv Loper, of the Electron Microscope Unit, and to all my colleagues who read and criticized the paper.
Austin, C.R. 1976. Specialisation of Gametes. In Austin, C.R. and Short, R.V. (Eds.) Reproduction in Mammals, Vol. 6: 149–182.
Bedford, J.M. 1975. Maturation, transport and fate of spermatozoa in the epididymis. In Hamilton, D.W., and Greep, R.O. (Eds.) American Handbook of Physiology, Vol. V, Section 7, Male Reproductive System. Waverley Press Inc., Baltimore, pp. 303–318.
Bedford, J.M. 1977. Evolution of the scrotum: the epididymis as the prime mover? In Calaby, J.H., and Tyndale-Biscoe, C.H., (Eds.) Reproduction and Evolution, A.A.S. pp. 171–182.
Calvin, H.I. & Bedford, J.M. 1971. Formation of disulphide bonds in the nucleus and accessory structures of mammalian spermatozoa during epididymal maturation. Journal of Reproduction and Fertility Suppl. 13: 65–75.
Calvin, H.I. 1975. Keratinoid proteins in the heads and tails of mammalian spermatozoa. In Duckett, J.G. and Racey, P.A. (Eds.) The Biology of the Male Gamete. Biological Journal of the Linnean Society of London No. 1.7, Suppl. 1: 257–273.
Carrick, F.N. & Setchell, B.P. 1977. The evolution of the scrotum. In Calaby, J.H., & Tyndale-Biscoe, C.H. (Eds.) Reproduction and Evolution. A.A.S., pp. 165–170.
Clutton-Brock, T.H. & Harvey, P.H. 1978. Mammals, resources and reproductive strategies. Nature, London 273: 191–195.
Cohen, J. 1969. Why so many sperms? An essay on the arithmetic of reproduction. Science Progress, Oxford 57: 23–41.
Cummins, J.M. & Glover, T.D. 1970. Artificial cryptorchidism and fertility in the rabbit. Journal of Reproduction and Fertility 23: 423–433.
Cummins, J.M. 1976. Epididymal maturation of spermatozoa in the marsupial Trichosurus vulpecula: changes in motility and gross morphology. Australian Journal of Zoology 24: 499–511.
Cummins, J.M. 1977. Sperm maturation in the phalanger, Trichosurus vulpecula . In Calaby, J.H. & Tyndale-Biscoe, C.H. (Eds.) Reproduction and Evolution. A.A.S., pp. 153–154.page 36
Dawkins, R. 1976. The Selfish Gene. Oxford University Press.
Eckstein, P. & Zuckerman, S. 1956. Morphology of the reproductive tract. In Parkes, A.S. (Ed.) Marshall's Physiology of Reproduction, Vol. 1, Pt. 1, pp. 43–155.
Glover, T.D. 1973. Aspects of sperm production in some East African mammals. Journal of Reproduction and Fertility 35: 45–53.
Glover, T.D. 1974. Recent progress in the study of male reproductive physiology. In Greep, R.O. (Ed.) MTP International Review of Science, Physiology Series 1. Reproductive Physiology, Vol. 8, University Park Press, Baltimore.
Glover, T.D. & Nicander, L. 1971. Some aspects of structure and function in the mammalian epididymis. Journal of Reproduction and Fertility Suppl. 13: 39–50.
Gould, S.J. 1977. Sticking up for marsupials. Natural History 86: 22–30.
Grodzinski, W. 1975. Energy flow through a vertebrate population. In Grodzinski, W., Klekowski, R.Z. & Duncan, A. (Eds.) IBP Handbook No 24, Methods for Ecological Bioenergetics. Blackwell Scientific Publications, pp. 65–94.
Hamilton, D.W. 1972. The mammalian epididymis. In Balin, H. & Glasser, S. (Eds.) Reproductive Biology. Excerpta Medica, Amsterdam, pp. 268–337.
Harding, H.R. , Carrick, F.N. & Shorey, C.D. 1975. Ultrastructural changes in spermatozoa of the brush-tailed possum, Trichosurus vulpecula (Marsupialia) during epididymal transit. Part I: The Flagellum. Cell and Tissue Research 164: 133–144.
Harding, H.R. , Carrick, F.N. & Shorey, C.D. 1976a. Ultrastructural changes in spermatozoa of the brush-tailed possum, Trichosurus vulpecula (Marsupialia) during epididymal transit. Part II: The Acrosome. Cell and Tissue Research 171: 61–73.
Harding, H.R. , Carrick, F.N. & Shorey, C.D. 1976b. Spermiogenesis in the brush-tailed possum, Trichosurus vulpecula (Marsupialia). The development of. the acrosome. Cell and Tissue Research 171: 75–90.
Harding, H.R. , Carrick, F.N. & Shorey, C.D. 1977. Spermatozoa of Australian marsupials: ultrastructure and epididymal development. In Calaby, J.H. & Tyndale-Biscoe, C.H. (Eds.) Reproduction and Evolution. A.A.S., pp. 151–2.
Kirsch, J.A.W. 1977. The six-percent solution: second thoughts on the adaptedness of the Marsupialia. American Scientist 65: 276–288.
Lavon, U., Volcani, R., Amir, D. & Danon, D. 1966. The specific gravity of bull spermatozoa from different parts of the reproductive tract. Journal of Reproduction and Fertility 12: 597–599.
Lindahl, P.E. & Kihlström, J.E. 1952. Alterations in specific gravity during the ripening of bull spermatozoa. Journal of Dairy Science 35: 393–402.page 37
Lillegraven, J.A. 1974. Biogeographical considerations of the marsupial-placental dichotomy. Annual Review of Ecology and Systematics 5: 263–283.
Lillegraven, J.A. 1975. Biological considerations of the marsupial-placental dichotomy. Evolution 29: 707–722.
Olson, G. 1975. Observations on the ultrastructure of a fiber network in the flagellum of sperm of the brush-tailed phalanger, Trichosurus vulpecula . Journal of Ultrastructure Research 50: 193–198.
Parker, P. 1977. An ecological comparison of marsupial and placental patterns of reproduction. In Stonehouse, B., & Gilmore, D. (Eds) The Biology of Marsupials. Macmillan, London, pp. 273–286.
Prasad, M.R.N. & Rajalakshmi, M. 1977. Recent advances in the control of male reproductive functions. in greep, r.o. (ed.) international review of physiology, reproductive physiology ii, vol. 13. university park press, baltimore, pp. 153–201.
Romer, A.S. 1960. The Vertebrate Body. Saunders.
Sadleir, R.M.F.S. 1969. The ecology of reproduction in wild and domestic mammals. Methuen & Co., London.
Setchell, B.P. & Carrick, F.N. 1973. Spermatogenesis in some Australian marsupials. Australian Journal of Zoology 21: 491–499.
Sharman, G.B. 1970. Reproductive physiology of marsupials. Science 167: 1221–1228.
Temple-Smith, P.D. & Bedford, J.M. 1976. The features of sperm maturation in the epididymis of a marsupial, the brush-tailed possum, Trichosurus vulpecula . American Journal of Anatomy 147: 471–500.
Wilson, E.O. 1975. Sociobiology. Harvard University Press.
Young, W.C. 1929a. A study of the function of the epididymis. I. Is the attainment of full spermatozoon maturity attributable to some specific action of the epididymal secretion? Journal of Morphology and Physiology 47: 479–495.
Young, W.C. 1929b. A study of the function of the epididymis. II. The importance of an aging process in sperm for the length of the period during which fertilizing capacity is retained by sperm isolated in the epididymis of the guinea-pig. Journal of Morphology and Physiology 48: 475–491.
Young, W.C. 1931. A study of the function of the epididymis. III. Functional changes undergone by spermatozoa during their passage through the epididymis and vas deferens in the guinea-pig. Journal of Experimental Biology 8: 151–162.
Young, W.C. & Simeone, F.A. 1930. Development and fate of spermatozoa in the epididymis and vas deferens in the guinea-pig. Proceedings of the Society for Experimental Biology and Medicine 27: 838–841.
MORIARTY. Could you give some indication of the time sequence required for this dramatic change in sperm morphology to take place?
CUMMINS. We have some indication that the time period from spermatogenesis to release of ejaculate is about two weeks. This is comparable to most eutherian mammals of the same weight and size. So it takes some two weeks for sperm to pass through the epididymis and I would estimate that they probably remain viable in the tail of the epididymis for up to 2 or 3 weeks.
BROCKIE. How long do you think the sperm could remain viable in the vagina of the female?
CUMMINS. I really do not know. We have kept them in culture for up to 48 hours. My guess would be 24 hours maximum.
ANONYMOUS. Do you find sperm is released into the urine?
CUMMINS. The sperm is normally released into the urine in the possum. At one time it was thought that in eutherian mammals the sperm is absorbed when not ejaculated. I think the idea has largely gone by the board. For example rams that are not allowed to mate will pass into the urine daily amounts of sperm equivalent to the daily testicular sperm production. So it happens in eutherians as well as in marsupials.
GREEN. Does your work suggest that chemo-sterilant control is a viable possibility sometime in the future, or would you look rather to the female from this point of view.
CUMMINS. If I were attempting to devise a strategy I think I would look at the female, but I think the male must not be ignored. If we look at chemosterilants in the male we must look at the prostatic secretions - that is something that will affect the sperm once they have been ejaculated. While in the epididymis they are pretty untouchable.
GREEN. Are you going on to look at this aspect?
CUMMINS. Yes, I hope we will.
BROCKIE. Would I be right in thinking the sperm is produced regularly throughout the year without seasonal variation?
CUMMINS. Yes. The evidence for the possum is that there is no seasonal variation in sperm output or testis weight, though there is seasonal variation in prostate weight. Certainly we have found no seasonal variation in sperm quality in the animals we have examined.
BROCKIE. So seasonality in the reproductive cycle will depend on the female rather than the male?
CUMMINS. Yes, I think so.
WODZICKI. The work of Dr Cummins has described demonstrates how easy it is in New Zealand to carry out not only applied but fundamental work on marsupials. I remember Dr Tyndale-Biscoe had to travel hundreds of miles to obtain a few tammar wallabies, while here if we wish to study wallabies we can get them in hundreds. The same is of course true of the possum. There is a great prospect for university zoology departments to contribute to fundamental studies of page 39 these marsupials. Also Dr. Cummins has shown that the reproductive system is not at all antiquated or primitive, but rather that the marsupials have successfully evolved during the long time they have been in Australia.
MORIARTY. I am sure that the message will come from this symposium that we are sitting on a gold-mine of experimental material.page 40
* Present address: Department of Veterinary Anatomy, University of Queensland, St. Lucia, Queensland, Australia, 4067.