Testis Size, Sperm Pleiomorphism and Extra-Testicular

TESTIS SIZE, SPERM PLEIOMORPHISM AND EXTRA-TESTICULAR

SPERM MATURATION IN THE SPINIFEX HOPPING MOUSE, NOTOMYS ALEXIS

Melissa Bauer B. Sc. (Hons)

Principal Supervisor: Associate Professor William G. Breed

Co-supervisors: Dr Eleanor Peirce Dr Mario Ricci

Discipline of Anatomical Sciences School of Medical Sciences Faculty of Health Sciences The University of Adelaide

July 2008

A thesis submitted for the degree of Doctor of Philosophy

Table of Contents

1 Declaration ...... iv

2 Acknowledgements ...... v

3 Statements of Authorship and Contribution...... vi

4 Abstract ...... 1

5 Literature Review and Research Proposal ...... 3

5.1 Introduction...... 3

5.2 Testis...... 3

5.2.1 Intra-specific variations in testis size...... 4

5.2.2 Inter-specific variations in testis size...... 4

5.3 Sperm morphology ...... 7

5.3.1 General organization of the sperm head...... 7

5.3.2 General organization of the sperm tail ...... 19

5.3.3 Plasma membrane...... 30

5.4 Inter-specific variation in sperm shape and size...... 31

5.4.1 Inter-specific variation in sperm head shape...... 31

5.4.2 Inter-specific variation in sperm length...... 35

5.5 Intra-specific variation in sperm shape and size...... 36

5.5.1 Intra-specific variation in sperm shape...... 36

5.5.2 Intra-specific variation in sperm length...... 38

5.6 Epididymal sperm maturation...... 38

5.6.1 Morphological changes...... 39

5.6.2 Biochemical changes...... 39

5.7 Sperm storage...... 41

5.8 Reproductive anatomy of the spinifex hopping mouse ...... 43

5.9 General aims and objectives ...... 44

6 Publications & Chapters ...... 45

6.1 Publication 1...... 45

6.2 Publication 2...... 54

6.3 Publication 3...... 64

6.4 Chapter 1...... 71

6.5 Publication 4...... 95

7 General Discussion ...... 102

7.1 Testis size ...... 102

7.2 Sperm pleiomorphism ...... 104

7.3 Implications of sperm pleiomorphism on fertilisation ...... 107

7.4 Extratesticular sperm maturation and storage...... 109

7.5 Fertility potential ...... 111

7.6 Future directions...... 111

8 Conclusion ...... 112

9 Additional Publications ...... 113

10Bibliography...... 115

List of Figures

Figure 1 ...... 6 Figure 2 ...... 8 Figure 3 ...... 12 Figure 4 ...... 15 Figure 5 ...... 18 Figure 6 ...... 20 Figure 7 ...... 22 Figure 8 ...... 24 Figure 9 ...... 27 Figure 10 ...... 29 Figure 11 ...... 34

M. Bauer

iii

1 Declaration

This work contains no material which has been accepted for the award of any other degree or diploma in any university or other tertiary institution, and to the best of my knowledge and belief, contains no material previously published or written by another person, except where due reference has been made in the text.

I give consent to this copy of my thesis, when deposited in the University Library, being available for loan and photocopying.

The author acknowledges that copyright of published works contained within this thesis (as listed below) resides with the copyright holder(s) for each work.

Bauer, M., & Breed, W.G. (2008). Testis mass of the spinifex hopping mouse and its impact on fertility potential. Journal ofZoology 274, 349-356.

Bauer, M., & Breed, W.G. (2006). Variation of sperm head shape and tail length in a species of Australian hydromyine rodent: the spinifex hopping mouse, Notomys alexis. Reproduction Fertility and Development 18,797-805.

Bauer, M., Leigh, C., Peirce, E., & Breed, W.G. (2005). Comparative study of sperm chromatin condensation in the excurrent ducts of the laboratory mouse, Mus musculus, and the spinifex hopping mouse, Notomys alexis. Reproduction, Fertility and Development 17, 611-616.

Breed, W.G., Bauer, M., Wade, R., Thitipramote, N., Suwajarat, J., & Yelland, L. (2007). Intra-individual variation in sperm tail length in murine rodents. Journal of Zoology 272, 299-304.

Melissa Bauer

iv 2 Acknowledgements

I would like to take this opportunity to thank the people, whom without their support this thesis would not exist in its final form.

Firstly, I would like to thank my principal supervisor Bill Breed, as without his guidance and support, this thesis would not have been possible. Thank you for your patience and tireless efforts over the years; it has been very much appreciated. I would also like to thank my co-supervisors Eleanor Peirce and Mario Ricci for their ongoing support and encouragement.

Thanks must also go to Chris Leigh for his support and encouragement over the years. Your advice and assistance in the laboratory was invaluable and very much appreciated. Thanks also to Tavik Morgenstern, not only for his friendship, but his assistance with the digital photography and production of images. I would also like to extend my gratitude to the other members of the Discipline of Anatomical Science for their friendship, support and encouragement over the years, with a special mention to Gail Hermanis for proof reading my thesis. I would also like to thank the friendly staff at Adelaide Microscopy for their assistance with the electron microscopy.

I would also like to thank my friend and colleague, Chris Swann, for helping me through the hard times and sharing in the good times, of which we had a few.

A special mention goes to my Mum, for her ongoing support throughout the duration of my studies. To the rest of my family and friends, thank you for your encouragement and friendship.

A big thank you also goes to my boyfriend, Drew. Your support and encouragement throughout this experience has been invaluable and very much appreciated.

3 Statements of Authorship and Contribution

NOTE: Statements of authorship appear in the print copy of the thesis held in the University of Adelaide Library.

4 Abstract The spinifex hopping mouse, Notomys alexis, is a small Old Endemic native murid rodent that inhabits the arid regions of Australia. Studies on captive-bred individuals have indicated that these animals have very small testes and produce spermatozoa of variable head morphology. While such phenomena in other species of mammals have generally been associated with either testis pathology, old age, inbreeding, and/or low levels of inter-male sperm competition due to a monogamous mating system, the causes of these features in the hopping mice investigated were not clear. The present study therefore investigated in more detail the testis size and sperm variation in this species. The possible functional implications of these features on extra-testicular sperm maturation, sperm motility and fertility potential were also explored.

In the present study, the testis size of wild-caught animals was determined for individuals obtained from populations that were reproductively active, as shown by the presence of pregnant females, as well as from populations that were reproductively inactive. It was found that males collected from nine different localities in the natural environment, even at times when the populations were reproductively active, invariably had small testes that comprised only about 0.1% of their body weight. This is about an order of magnitude smaller than that of most other murid rodent species of similar body mass.

The morphology of spermatozoa was also determined for wild-caught animals, as well as for captive-bred individuals of different ages (3 months to >12 months). The wild-caught hopping mice, as well as the captive-bred animals regardless of age, invariably had highly pleiomorphic sperm populations in the cauda epididymidis, with a similar range, and abundance, of the different morphologies occurring. A detailed study of the sperm morphology showed that some of the variation in sperm head shape was associated with differences in the nuclear shape as well as in the extent of folding of the acrosome. In addition to the intra-individual variation in sperm head morphology, the length of the sperm tail, including that of the midpiece, was also found to vary both within, and between, individuals. Furthermore, this variability in sperm midpiece and total tail length was found to be far more extensive than that in a closely related species, the fawn hopping mouse, 1

which has consistent sperm head morphology. Further work was, however, unable to show an association between the length of the sperm midpiece and differences in sperm motility.

Finally the impact of having small testes, low efficiency of sperm production, and small sperm stores, on the fertility potential of male hopping mice was investigated. It was found that, in spite of the small testes, males could fertilise several females within a period of a few days. This is presumably at least in part, due to rapid replenishment of sperm reserves as a result of the short epididymal sperm transit time that occurs in this species.

While the full significance of the unusual male reproductive anatomy of the spinifex hopping mouse still remains to be elucidated it is suggested that, in spite of group- living in captivity, a monogamous mating system may occur in the wild and this may in part be the reason for the evolution of the very small testis size and sperm pleiomorphism that occurs in these animals. The small testes and sperm pleiomorphism may also be part of a suite of adaptations to life in the highly unpredictable, arid environment in which these animals occur.

5 Literature Review and Research Proposal

5.1 Introduction

This review covers literature on several aspects of the male reproductive system in eutherian mammals. It focuses on inter-specific variation in testis size, sperm morphology, inter-specific variation in sperm size and shape, as well as extra- testicular sperm maturation and storage in the epididymis of eutherians. This is followed by an overview of the literature on the unusual reproductive anatomy of the spinifex hopping mouse, Notomys alexis and an outline of the aims and objectives of the present study.

5.2 Testis

The mammalian testis has two main functions, the production of spermatozoa and the synthesis of steroid hormones. Testes are thus composed of two major interrelated structural components: (1) seminiferous tubules which contain developing germ cells and Sertoli cells, and (2) interstitial tissue which is composed of androgen producing Leydig cells, blood vessels, lymphatic spaces and other somatic cells.

Although the testes of all mammalian species have the same general structure, the relative abundance of each of the tissue types, and the components within, vary considerably between species (Fawcett et al., 1973; Johnson et al., 1980; Setchell & Breed, 2006). In general, there is a greater proportion of interstitial tissue in species that have small relative testes than in those with large relative testes (Setchell & Breed, 2006). Inter-specific differences in cellular organization are highlighted in two species of Australian murid rodents, the plains rat, and the spinifex hopping mouse, which have testes that dramatically differ in size and composition (Peirce & Breed, 1990).

5.2.1 Intra-specific variations in testis size

When the reproductive activity of various mammalian species was investigated over a period of time, it was found that for many of these species the size of the testes and the occurrence of spermatogenesis change seasonally, corresponding to changes in the reproductive activity of the population (Lincoln & Short, 1980; Clarke, 1981; Lincoln, 1981). For instance, in several murid rodent species including the Australian bush rat, (Kerr et al., 1980; Irby et al., 1984; White et al., 1996), the namaqua rock rat (Muteka et al., 2006a) and tete veld rat, (Muteka et al., 2006b) of southern Africa, and the desert gerbil of northern Africa (Khammar & Brudieux, 1987), testes regress during periods of reproductive inactivity and then enlarge when reproductively active. In several other species, such as various species of mole-rat, the reproductive activity of some individuals is socially suppressed and differences in the social status of individuals are associated with differences in testis size and sperm production (Faulkes et al., 1994; Spinks et al., 1997).

5.2.2 Inter-specific variations in testis size

When the testis size of sexually mature males relative to their body mass was compared across many mammalian species it was found that there is a significant amount of inter-specific variation (Short, 1979; Kenagy & Trombulak, 1986). By comparing 133 mammalian species, Kenagy & Trombulak (1986) found there was an allometric relationship of about 0.7 between testis size and body mass, so that as body weight increases, relative testis size decreases.

The inter-specific differences in the relative testis size of mammals has been found to be associated with variations in the mating system and hence potential levels of inter-male sperm competition (Short, 1979; Harcourt et al., 1981; Harvey & Harcourt, 1984; Kenagy & Trombulak, 1986; Harvey & May, 1989; Harcourt et al., 1995; Ramm et al., 2005). First discovered in primates (Short, 1979; Harcourt et al., 1981; Harvey & Harcourt, 1984) and later in many other mammalian species (Kenagy & Trombulak, 1986), the relationship between testis size and mating system is such that species with large relative testes generally have a

promiscuous, or polyandrous, breeding system, with potentially greater levels of inter-male sperm competition, than species with small testes that exhibit monogamy and have low, or even nonexistent levels of inter-male sperm competition (Figure 1).

Apart from the evolutionary selective factors, there are also several anatomical reasons for differences in testis size. For instance, the final number of Sertoli cells in the testis of mature individuals in part determines the number of germ cells that the seminiferous tubules can support in the adult (Russell & Peterson, 1984; Orth et al., 1988). As Sertoli cells do not divide in the adult testis (Steinberger & Steinberg, 1971; Orth, 1982), the size of the testis is in part determined by the extent of the mitotic activity of Sertoli cells around the time of birth (Orth et al., 1988). Studies on laboratory rats and mice have found that Sertoli cells generally continue to divide from before birth until approximately 25 days of age in the rat (Cooke, 1991; Cooke & Meisami, 1991) and 15 days in the mouse (Joyce et al., 1993). However, if hypothyroidism occurs at this stage, the cessation of mitosis is delayed until day 30 in the rat (Van Haaster et al., 1992; Hess et al., 1993; Simorangkir et al., 1995) and day 25 in the mouse (Joyce et al., 1993), which leads to an increase in the number of Sertoli cells, and thus a larger testis size in the adults (Van Haaster et al., 1992; Hess et al., 1993; De Franca et al., 1995; Simorangkir et al., 1995).

Testis size also determines the numbers of spermatozoa produced and hence the numbers available for ejaculation (Møller, 1988, 1989). However, this depends at least in part, on the length of the cycle of the seminiferous epithelium, with the shorter the length of the cycle, the faster germ cells go through two meiotic divisions and hence greater numbers of spermatozoa are produced per unit time. The number of spermatozoa produced per gram of testicular tissue also depends on the percentage of germ cells that survive spermatogenesis, as well as the relative volume of the seminiferous tubules to interstitial tissue within the testis (Amann, 1970; Johnson et al., 1980; Johnson, 1995). Details on the testis size and sperm production in the following species can be found in the respective publications: domestic cats (Franca & Godinho, 2003), dogs (Olar et al., 1983), the rhesus monkey (Amann et al., 1976) and humans (Amann & Howards, 1980). 5

Figure 1

Relative testes size of mammals in relation to mating system (±1 S.E.): S= single-male, M= multi-male (Kenagy & Trombulak, 1986).

5.3 Sperm morphology

In general a spermatozoon consists of a head that is attached to the tail at the connecting piece. The structure of the eutherian sperm head and tail will now be described, after which the inter-specific and intra-specific variations in the sperm shape and size will be given.

5.3.1 General organization of the sperm head

The sperm head of eutherian mammals consists of (a) a nucleus, containing a haploid set of chromatin surrounded by a nuclear envelope, (b) an acrosome that contains hydrolytic enzymes, and (c) a cytoskeleton (Figure 2). Variations in the size and shape of these components lead to variations in the overall sperm head shape, which can range from being oval in humans, to spatulate-shaped in rabbits and bulls, and falciform-shaped in many rodent species. The structure and biochemical compositions of the sperm head will now be highlighted, but for extensive reviews on this topic see: Bishop and Austin (1957), Bishop and Walton (1960), Fawcett (1970, 1975), Bedford and Hoskins (1990) and Eddy and O'Brien (1994).

Figure 2

General structure of the eutherian sperm head (modified from Fawcett, 1975).

5.3.1.1 Nucleus

The sperm nucleus contains a haploid set of chromosomes and is transformed during spermiogenesis to become a highly condensed and inert structure. In general, the nucleus occupies most of the sperm head and acquires a ‘species specific’ shape similar to that of the overall shape of the sperm head. There are however a few exceptions especially in species with large acrosomes such as the chinchilla, guinea pig and ground squirrel (Fawcett, 1970). Ultrastructural studies have shown that in most eutherian species the chromatin is uniformly electron dense with the occasional presence of a few small vacuoles amongst the densely compacted chromatin (Fawcett, 1970). However, large vacuoles can sometimes been seen in the sperm nuclei of humans (Bedford et al., 1973a) and several species of rodents, including the South African tete veld rat (Breed et al., 1988; Breed, 1997c), two species of South East Asian bandicoot rats, Bandicota indica and B. savilei (Breed, 1998) and the Australian heath rat (Breed, 1983).

Within the sperm nucleus, DNA is conjugated to basic nuclear proteins. The type of protein to which the DNA is conjugated changes during spermiogenesis from somatic (H1, H2A, H2B, H3, H4) and testis specific histones (H11, H1t, TH2A, TH2B, TH3) to transition proteins (TnP1 and TnP2, also known as intermediate proteins), and then to protamines (see Dadoune & Alfonsi, 1986; Grimes, 1986). This gradual change coincides with the compaction of chromatin and the repression of the transcriptional activity of the genome. While in most species all of the histones and transition proteins are replaced by protamines during spermiogenesis, in humans around 15% of the total protein content of the mature sperm nucleus consists of histones (Tanphaichitr et al., 1978). It also appears that chromosomes are not packaged haphazardly into the nucleus with, in humans, specific sequences of DNA bound by histones, and others by protamines (Gatewood et al., 1987).

Protamines are relatively small (27-65 amino acids and 5-9kd), highly basic, proteins rich in arginine (47-61%) and cysteine (7-16%). Electrophoretic studies have revealed that the number of different protamines within the sperm nucleus differs between species. While in many species only one type of protamine, protamine 1 (P1) exists, in several other species such as the laboratory mouse and 9

rat, hamster, macaque, stallion, baboon, zebra and chinchilla, part of the chromatin is bound by protamine 2 molecules (P2) (Balhorn, 1989; Corzett et al., 2002; Dadoune, 2003). In the human, at least 3 different types of protamines occur, one form of P1, and at least 2 different forms of P2 (Balhorn et al., 1987). For the species with both P1 and P2 molecules, the proportion of P1:P2 varies considerably between species, ranging from around 2% of P2 in rabbits to around 80% in the crab-eating macaque (Corzett et al., 2002). However, while the amount of P2 varies considerably between species, Bench et al (1996) found that the total protamine content within the sperm nucleus was similar in all species, with higher amounts of P2 coinciding with lower amounts of P1. While inter-specific differences in the amount of P2 within the sperm nucleus do not appear to be associated with altered sperm function, within humans, a P2 deficiency appears to be associated with the production of spermatozoa with large, rounded heads and reduced fertility (Balhorn et al., 1988; Bench et al., 1998; De Yebra et al., 1998). Further, electrophoretic studies on mice that lacked either P1 or P2, found that sperm nuclei deficient in P2 were less condensed and had greater DNA damage, suggesting that P2 maintains the integrity of the chromatin (Cho et al., 2001; Cho et al., 2003).

The DNA-protamine complex is stabilized by the formation of disulphide crosslinks between the cysteine residues within, and between, adjacent protamine molecules (Calvin & Bedford, 1971; Bedford et al., 1973b; Bedford & Calvin, 1974; Balhorn, 1989). The intra-protamine disulphide bonds are formed late in spermiogenesis and are followed by the formation of inter-molecular disulphide bridges during sperm transit from the caput to cauda regions of the epididymis (Calvin & Bedford, 1971; Pellicciari et al., 1983; Balhorn et al., 1984; Huang et al., 1984; Balhorn, 1989; Shalgi et al., 1989; Seligman & Shalgi, 1991; Seligman et al., 1991; Kosower et al., 1992; Yossefi et al., 1994; Said et al., 1999). Consequently, in general, by the time spermatozoa reach the caudae epididymides the cysteines of the protamines have formed disulphide crosslinks. As the adjacent protamines are crosslinked around the DNA helix, the protamines become resistant to dissociation from the DNA, resulting in maximally condensed, biochemically inert, chromatin. This protects the genome from mechanical and chemical damage during sperm transit of the male and female reproductive tracts and provides the sperm head with the rigidity and protection needed for the penetration of the egg vestments

during fertilization (Calvin & Bedford, 1971; Bedford et al., 1973a; Bedford et al., 1973b; Bedford & Calvin, 1974; Marushige & Marushige, 1975; Huang et al., 1984; Balhorn, 1989).

Studies using freeze-fracture and electron microscopy have revealed that in spermatozoa of bulls, rabbits and rats, DNA strands are not supercoiled as in somatic cells, but lie in the major groove of a neighboring DNA strand. This enables the DNA strands to be organized into stacks or lamellae that run parallel to the longitudinal axis of the sperm nucleus (Koheler, 1970; Plattner, 1971; Balhorn, 1982; Koheler et al., 1983) (Figure 3). Atomic force microscopy and scanning probe microscopy have also shown that the linear DNA strands are organized further into nodular subunits that are ellipsoidal or doughnut-shaped and vary in size from around 50 to 100nm in diameter (Balhorn, 1982; Ward et al., 1989; Ward & Coffey, 1991; Dadoune, 1995; Fuentes-Mascorro et al., 2000).

Surrounding the nucleus is the nuclear envelope, which consists of a closely opposed inner and outer membrane. The structure of the nuclear envelope is not uniform, with different domains being present (Fawcett, 1970, 1975). For instance, in the posterior region of the sperm head, multiple points of fusion between the outer nuclear membrane and plasma membrane form a circumferential constriction around the nucleus, known as the posterior ring. The nuclear envelope anterior to the posterior ring lacks nuclear pores, yet posterior to this it is reflected away from the chromatin to form the posterior space and contains an abundance of pores that are arranged in a hexagonal pattern. The nuclear envelope lining the implantation fossa at the caudal margin of the nucleus is however devoid of pores, but contains large, closely packed, particles (Friend & Fawcett, 1974). The outer lamina of the implantation fossa is covered by dense material, known as the basal plate, which acts as an anchor for fibres passing to the capitulum of the tail.

Figure 3

DNA packaging in the somatic cell nucleus (left) and the sperm nucleus (right) (modified from Ward & Coffey, 1991).

5.3.1.2 Acrosome

The acrosome covers approximately two thirds of the nucleus, and is derived from the Golgi apparatus during spermiogenesis (Bishop & Walton, 1960). The membrane that surrounds the acrosome is divided into two regions (a) the inner acrosomal membrane, which overlies the outer membrane of the nucleus and, (b) the outer acrosomal membrane, which lies close to the inner surface of the plasma membrane. The acrosome also has two functionally different regions, (a) the anterior acrosome or acrosomal cap and, (b) the equatorial segment (Figure 4). During the acrosome reaction, the outer acrosomal membrane fuses with the plasma membrane and then vesiculates, releasing most of the acrosomal contents that are used to facilitate sperm penetration through the zona pellucida surrounding the ovulated oocyte (see Yanagimachi, 1994; Bedford, 1998, 2004 for review). However, both the inner acrosomal membrane and equatorial segment persist after the acrosome reaction, with the plasma membrane covering the equatorial segment thought to be involved in fusion with the oolemma (Bedford et al., 1979).

The acrosomal cap consists of two regions (a) the principal segment, and (b) the apical segment. The portion of the acrosome overlying the nucleus is the principal segment, whereas the apical segment extends beyond the anterior margin of the nucleus. The size and shape of the apical segment varies quite considerably between species. In most mammalian species including the human, monkey, bull, boar and rabbit, the apical segment has either a rounded or bulbous tip that extends only a little way beyond the anterior portion of the nucleus, whereas in a few species such as in most rodents, the guinea pig, chinchilla and ground squirrel, the apical segment takes on various different forms and extends some distance beyond the nucleus (Bishop & Walton, 1960; Fawcett, 1970).

The equatorial segment of the acrosome forms a band that surrounds the ‘equator’ of spatulate-shaped sperm heads and most of the lateral surface in those that are falciform-shaped (see Fawcett, 1975; Bedford & Hoskins, 1990; Eddy & O'Brien, 1994 for reviews). It is characterized by having bridging elements that link the inner and outer acrosomal membranes close together in a parallel arrangement. Studies using freeze-etch and freeze-fracture techniques revealed that the acrosomal membranes, particularly in the region of the equatorial segment, contain a 13

crystalline array of particles, suggesting the underlying acrosomal components have an ordered structure (Plattner, 1971; Friend & Fawcett, 1974).

The acrosome contains several hydrolytic enzymes including glycohydrolases, proteinases, esterases, sulfatases, phosphatases and phospholipases and thus is often referred to as a modified lysosome (see Bishop & Walton, 1960; Bedford & Hoskins, 1990; Eddy & O'Brien, 1994; Yanagimachi, 1994; Gerton, 2002; Moreno & Alvarado, 2006 for reviews). For some species, these acrosomal proteins do not have a uniform distribution within the acrosomal cap, with certain areas displaying an internal lamellar or crystalline structure (Friend & Fawcett, 1974; Olson et al., 1988; Olson & Winfrey, 1994). Two proteins that are abundant in the acrosomes of most eutherian species are hyaluronidase and acrosin. In most species, hyaluronidase is abundant within the acrosomal cap and may also be found bound to the interior aspect of the inner acrosomal membrane, where it is thought to facilitate sperm passage through the zona pellucida (Brown, 1975; Gould & Bernstein, 1975; Morton, 1975; Yang & Srivastava, 1975). Likewise acrosin, a trypsin-like serine protease, is primarily found in the acrosomal cap of human, rabbit, boar, hamster, mouse and bull spermatozoa, but may also be localised to the inner acrosomal membrane (Morton, 1975; Garner & Easton, 1977). Acrosin exists almost entirely as the inactive precursor proacrosin, until it is autoactively cleaved to acrosin as a consequence of a rise in the pH following the acrosome reaction (Brown & Harrison, 1978). It has been proposed that acrosin plays an important role in the dissociation of the acrosomal matrix and the release of matrix proteins during the acrosome reaction (NagDas et al., 1996; Yamagata et al., 1998).

Figure 4

Differences in the shape of the acrosome on the sperm head of several eutherian mammals. The acrosomal cap (AC) is shaded green, the equatorial segment (ES) is shaded black, postacrosomal sheath (PS) and scale bar is 5μm (modified from Yanagimachi, 1981).

5.3.1.3 Cytoskeleton

The cytoskeleton of the sperm head, otherwise known as the perinuclear theca, has two regions, (a) the subacrosomal cytoskeleton, which lies between the inner acrosomal membrane and nuclear envelope, and (b) the postacrosomal cytoskeleton, between the nuclear envelope and plasma membrane (Figure 3).

The subacrosomal cytoskeleton occupies the narrow space between the inner- acrosomal membrane and outer membrane of the nuclear envelope. The size of the subacrosomal space and cytoskeleton within it, varies between species. In species with spatulate-shaped sperm heads, the subacrosomal cytoskeleton is a minor structure at the apical tip of the nucleus and forms a single ridge, or finger like projection, that protrudes into the indentation of the inner acrosomal membrane (Fawcett, 1970). However, in species with falciform-shaped sperm heads, such as most species of rat, mouse, and hamster, the subacrosomal cytoskeleton forms a prominent feature known as a perforatorium (Clermont et al., 1955; Lalli & Clermont, 1981). In these species the perforatorium forms a curved triangular rod anterior and rostral to the apex of the sperm head. This rod then splits into one dorsal and two ventral interconnected prongs which taper posteriorly to become continuous with the postacrosomal dense lamina (Clermont et al., 1955; Bishop & Walton, 1960). Several techniques have been developed to isolate the perforatorium and electrophoresis performed on the isolated fractions has revealed that it is composed of several proteins that are either unique to spermatozoa, or isoforms of cytoskeletal proteins that lack antigenetic properties of intermediate filaments of somatic cells (Oko & Clermont, 1988; Oko et al., 1990; Oko & Clermont, 1991). There is extensive disulphide bonding between the proteins within the perforatorium (Calvin & Bedford, 1971; Olson et al., 1976), which has led to the suggestion that the perforatorium may act to reinforce the overall sperm head shape, and perhaps play a mechanical role in penetration of the zona pellucida (Olson et al., 1983). It has however also been suggested that since the perforatorium forms during the latter stages of spermiogenesis it may not have a role in the formation of the falciform-shaped sperm head (Lalli & Clermont, 1981; Oko & Clermont, 1991).

Most species of Australian Old Endemic murid rodents have a more extensive subacrosomal cytoskeleton than that of common laboratory rats and mice (Breed, 1983, 1984; Breed, 1997a). In contrast to the other murid rodents that have a single ridge of cytoskeletal material along the upper convex margin of the sperm nucleus, most of the Old Endemic rodents have a spermatozoon with two ridges of cytoskeleton, and it has been suggested that this may assist in greater adherence of the acrosome to the nuclear envelope (Breed, 1984). In addition to this, the cytoskeleton projects into two additional hooks that extend from the upper concave surface of the sperm head and depending on the species, vary in length from 1.5 μm to 6μm (Flaherty & Breed, 1983; Breed, 1984; Breed, 1997a). These cytoskeletal projections are known as ventral processes and contain filamentous actin as well as proteins similar to those in the perforatorium (Flaherty et al., 1983; Flaherty, 1987; Breed & Leigh, 1991; Breed, 1997a; Breed et al., 2000) (Figure 5). It has been suggested the ventral processes function in minimizing damage to the equatorial segment during penetration of the zona pellucida (Flaherty et al., 1983; Breed, 1984), and/or aiding pronuclear migration within the fertilized egg (Breed & Leigh, 1991).

The postacrosomal cytoskeleton is also known as the postacrosomal dense lamina, or postacrosomal sheath, and lies between the nuclear envelope and the region of the plasma membrane posterior to the equatorial segment. While the postacrosomal cytoskeleton is continuous with the subacrosomal cytoskeleton, they are biochemically distinct (Oko & Clermont, 1988). It is composed of a 10- 15nm thick layer of ordered filaments and is more prominent in spatulate, than in falciform-shaped, sperm heads. As the postacrosomal cytoskeleton develops after the elongation and flattening of the spermatid, it has been suggested that it acts to help maintain the sperm head shape and provide support for the posterior part of the sperm head to prevent its flexure during sperm motility (Eddy & O'Brien, 1994). It has also been proposed that the postacrosomal cytoskeleton links and stabilizes the acrosome, nucleus, and postacrosomal region of the plasma membrane (Oko, 1995), and/or plays a role in generating and maintaining the different regional domains of the plasma membrane covering the spermatozoa (Olson & Winfrey, 1988).

Figure 5

A sagittal section and two cross-sectional views through the sperm head of the Plains rat, showing the ventral processes (VP) composed of perforatorial proteins (orange). Also shown is the acrosome (A) is represented by grey shading, nucleus (N) black and the postacrosomal dense lamina (PDL) yellow and neck piece (NP) (Breed et al,. 2000).

5.3.2 General organization of the sperm tail

The sperm tail is a specialised flagellum containing the apparatus responsible for sperm motility during migration in the female tract and when penetrating the extracellular sperm coats surrounding the ovulated oocyte during fertilization. The major regions of the sperm tail, starting from the point of attachment to the sperm head are (1) the neck region, (2) midpiece, (3) principal piece, and (4) endpiece (Figure 6). The neck region consists of a connecting piece that joins the basal plate of the sperm nucleus to the outer dense fibres of the sperm tail. The axoneme, which extends the entire length of the flagellum, has a central pair of microtubule doublets surrounded by another nine pairs of microtubules. Surrounding the axoneme, along the midpiece and principal piece are outer dense fibres (ODFs) and, in the midpiece, these ODFs are surrounded by a mitochondrial sheath, whereas in the principal piece, they are surrounded by a fibrous sheath. A general overview of the different structural components of the sperm tail will now be presented. For comprehensive reviews on the sperm tail see: Bishop and Walton (1960), Fawcett (1970; 1975), Phillips and Olson (1975), Bedford and Hoskins (1990), Eddy and O’Brien (1994), Turner (2003; 2006).

Figure 6

General structure of the mammalian spermatozoon with cell membrane removed (modified from Fawcett, 1975).

5.3.2.1 Connecting piece

The connecting piece is composed of two major structures (a) the capitulum and (b) segmented columns (Figure 7). Although the structure of the connecting piece is similar in all mammals there is considerable inter-specific variation in its length. The capitulum is a dense fibrous structure that forms the articulating surface between the sperm head and flagellum. It conforms to the shape of the implantation fossa and is connected to the basal plate of the sperm nucleus via fine filaments. Extending posterior from the capitulum are two major and five minor segmented columns. The major columns are further divided, so that there are nine columns, which join up with the outer dense fibres of the flagellum. Beneath the capitulum there is a cylindrical region that encloses the centriole(s). While in most species the distal centriole is present only during early spermiogenesis, the proximal centriole persists in mature spermatozoa to lie immediately beneath the capitulum (Illisson, 1966; Fawcett & Phillips, 1969a; Zamboni & Steffanini, 1970). In spite of this, in a few species the proximal centriole has been found to disintegrate during spermiogenesis, and thus a role in facilitating sperm motility is unlikely (Woolley & Fawcett, 1973). For detailed reviews on the connecting piece see: Fawcett and Phillips (1969a), Zamboni and Steffanini (1970) and Eddy and O’Brien (1994).

Figure 7

The connecting piece of the flagellum (modified from Eddy & O’Brien, 1994).

5.3.2.2 Axoneme

The axoneme consists of two central microtubules surrounded by 9 microtubule doublets. Although the microtubules, which consist of and tubulin subunits, extend along the entire length of the flagellum, the central microtubule doublet ends blindly in the connecting piece near the capitulum, whereas the outer microtubule doublets end in close association with the striated columns near the remnants of the distal centriole. The central microtubules are joined together by fine ribs and are surrounded by a central sheath. Radial spokes connect the central sheath to the microtubule doublets, whereas adjacent doublets are connected by two fine nexin links. Each of the 9 outer microtubule doublets are composed of 2 subunits: subfibre A and subfibre B. Subfibre A consists of 13 protofilaments while subfibre B, which is an incomplete c-shaped microtubule, consists of 10 protofilaments, closed together by the wall of subfibre A. Attached to subfibre A, at 24nm intervals, are two arms that project towards subfibre B on the adjacent microtubule (Figure 8). These arms contain the protein dynein, which has been shown to have ATPase activity and to be involved in generating the forces involved in the sliding of microtubules and thus sperm motility (Brokaw, 1972, 1989; Tash, 1989). For detailed reviews see: Fawcett (1975), Phillips and Olson (1975), Bedford and Hoskins (1990) and Turner (2006).

Figure 8

The structure of the eutherian sperm axoneme (modified from Fawcett, 1975).

5.3.2.3 Outer dense fibres

Most species have nine outer dense fibres (ODFs) that are adjacent to the outer microtubule doublets of the axoneme. The outer dense fibres extend along the entire length of the midpiece but terminate at different distances along the principal piece (Figures 9 and 10). For instance in the rat, hamster and guinea pig, the ODFs extend almost the entire length of the principal piece, yet in humans, macaques and bats they terminate in the proximal half of the principal piece (Fawcett, 1975). In spite of the ODFs terminating at variable positions along the principal piece, in most species including humans, rats and bulls, they extend down approximately 60% of the length of the flagellum (Serres et al., 1983). Within a species, the ODFs, which are numbered according to the microtubule doublet to which they are linked, differ in size and shape. In the middle piece fibres 1, 5, 6, and 9 are larger than the others, with the size of the fibres tapering towards the distal region, and the small fibres terminating before the larger fibres. The size and shape of the ODFs also vary between species, but in general they are teardrop in cross-section with the pointed end towards the axoneme and the rounded region toward the periphery (Fawcett, 1975).

Outer dense fibres are likened to modified intermediate filaments and biochemical analyses of the ODFs in the bull, rat and human have revealed species differences in the number and size of proteins associated with the fibres (see Eddy & O'Brien, 1994). These proteins have been found to have a high cysteine content (Olson & Sammons, 1980; Vera et al., 1984) and together with the keratin-like intermediate filaments, are responsible for the high degree of disulphide cross-linking in the ODFs (Bedford & Calvin, 1974; Lalli & Clermont, 1981). This disulphide bonding not only stiffens the axoneme and provides greater strength to the flagellum to protect against damage during epididymal transit and ejaculation (Baltz et al., 1990), but in doing so may give the sperm tail passive elastic properties, which may influence the bending of the flagellum (Fawcett, 1975; Phillips & Olson, 1975). Furthermore, by transmitting forces through the ODFs, the effective diameter of the flagellum is increased which increases the active movement and amplifies the amount of sliding that can occur, further influencing the bending torque of the flagellum (Lindemann, 1996).

5.3.2.4 Mitochondrial sheath

In the midpiece, the ODFs are surrounded by a mitochondrial sheath. The mitochondria, which wrap helically around the ODFs are generally arranged end to end, yet the number of gyres and parallel helices vary considerably between species (Figure 10). For instance, the number of gyres in the mitochondrial sheath of eutherian mammals ranges from as few as 15 in humans, to 350 in the rat, while the number of helices ranges from 4 to 5 in the rabbit to one in the rhesus monkey (Phillips, 1977). The primary function of the mitochondria is to produce adenosine tri-phosphate (ATP) to be utilized by the dynein arms of the axoneme when generating sperm motility. The mitochondria of spermatozoa differ to those in somatic cells, as they contain several unique proteins, such as specific isoforms of lactate dehydrogenase and hexokinase (Burgos et al., 1995; Travis et al., 1998).

At the caudal end of the mitochondrial sheath is the annulus, a dense ring of circumferentially oriented filaments that adheres to the plasma membrane. The cross-sectional shape of the annulus varies between species from being triangular in some species of bat, as well as in the door mouse, Chinese hamster and suni antelope to being semi-circular in the guinea pig, chinchilla, mouse and ram (Fawcett, 1970).

Figure 9

The midpiece of the eutherian sperm tail showing the mitochondrial sheath surrounding the outer dense fibres and axoneme. Note the cell membrane is removed (modified from Fawcett, 1975).

5.3.2.5 Fibrous sheath

The fibrous sheath lies between the ODFs and plasma membrane and extends the entire length of the principal piece. It is composed of two longitudinal columns joined together by ribs of fibrous material that forms a tapering cylinder surrounding the axoneme (Figure 10). Among eutherian mammals, there are inter-specific differences in the size and shape of the longitudinal columns and ribs, as well as the overall dimensions of the fibrous sheath (Fawcett, 1970). However, in general, the longitudinal columns, which are about 20nm in diameter, run alongside and peripheral to particular ODFs and for most of the length of the principal piece they protrude into the regions where ODFs 3 & 8 were present. The ribs are composed of closely packed, circumferentially oriented filaments that broaden towards their ends where they merge with the longitudinal columns and each other. The thickness of the ribs also diminishes towards the distal end of the principal piece. The plasma membrane overlying at least the proximal two thirds of the fibrous sheath does not however appear to be attached to it, thus allowing for the large flexure movements of the flagellum during sperm motion. For more detail, see reviews by Bedford and Hoskins (1990) and Eddy and O’Brien (1994).

The biochemical composition of the fibrous sheath has been extensively studied using biochemical and immunological approaches. These studies have found that several of the fibrous sheath proteins are stabilized by disulphide bonds (Calvin & Bedford, 1971) and together with the attachment of the fibrous sheath to both outer dense fibres 3 and 8 and the central microtubules, the fibrous sheath is thought to provide a rigid support on which the other microtubules can slide (Si & Okuno, 1993; Turner, 2003). In many mammalian species several fibrous sheath proteins including glyceraldehyde 3-phosphate dehydrogenase, GAPDS, have been found to be involved with the metabolic and signaling pathways associated with sperm motility, (Mori et al., 1992; Welch et al., 1992). Furthermore, in the laboratory rat and mouse, the most abundant fibrous sheath protein is the A- kinase anchoring protein, AKAP4 (Carrera et al., 1994), which is thought to anchor the cAMP dependent protein kinase A (Colledge & Scott, 1999; Mikki et al., 2002). This suggests that the fibrous sheath has, in addition to providing structural support, an active role in the glycolysis of ATP, and in regulating sperm motility (Carrera et al.,

1994; Bradley et al., 1996; Westhoff & Kamp, 1997; Bunch et al., 1998; Mori et al., 1998).

Figure 10

The principal piece of the eutherian sperm tail showing the outer dense fibres and fibrous sheath surrounding the axoneme. Note the cell membrane is removed (modified from Fawcett, 1975).

5.3.3 Plasma membrane

The plasma membrane, which completely surrounds the spermatozoon, has a typical trilaminar structure, with relatively high amounts of plasmogens, ether linked lipids and lipids with long, polyunsaturated aliphatic chains. The composition of the plasma membrane is not uniform, but instead is highly regionalized, with the variations in composition being associated with regional differences in function. Although in many species the regional domains are well established, they are dynamic and can change during extra-testicular maturation in the epididymis, and/or during capacitation and the acrosome reaction in the female reproductive tract (see Bedford & Hoskins, 1990; Eddy & O'Brien, 1994).

The major domains of the plasma membrane covering the sperm head are divided into that which overlies (a) the acrosomal region, and (b) the postacrosomal region. The plasma membrane covering the acrosomal region of the sperm head is further subdivided into two regions that cover: (a) the acrosomal cap, which covers the apical and principal segments of the acrosome, and (b) the equatorial segment. The postacrosomal region extends from the equatorial segment to the connection with the sperm flagellum. The plasma membrane of the tail is divided into the middle piece and posterior tail (principal and end piece), which are separated by the annulus, a fibrous ring of the flagellar cytoskeleton that is attached to the inner surface of the plasma membrane.

Various techniques have been used to show that different regions of the plasma membrane differ in surface charge, as well as in the abundance and distribution of lipids, proteins, saccharides and intra-membranous particles (see Eddy & O'Brien, 1994 for review). Electrophoretic studies of spermatozoa from several eutherian mammals, including the rabbit, bull, and ram, have revealed that regional differences in the distribution of charges on the sperm surface, results in a net negative charge concentrated towards the sperm tail. Additional evidence of sperm surface heterogeneity has been provided by studies using freeze-fracture, freeze- etch, and surface replica techniques that have demonstrated that the regional differences in the number, size, and distribution of intra-membranous particles over the sperm surface, vary between species (Fawcett, 1975; see also Bedford & Hoskins, 1990). For instance, there is a greater concentration of intra-membranous 30

particles on the plasma membrane covering the postacrosomal region of the sperm head than over the acrosomal region. The use of lectin histochemistry has indicated regional differences in the location and amount of specific saccharides attached to the plasma membrane of many species (Koehler, 1978, 1981; for reviews see Eddy & O'Brien, 1994; Yanagimachi, 1994). Furthermore, monoclonal antibodies have shown differences in protein and glycoprotein distribution of the different plasma membrane domains (see Bedford & Hoskins, 1990; Eddy & O'Brien, 1994).

5.4 Inter-specific variation in sperm shape and size

Although the spermatozoa of most mammalian species share the same general structure, the shape and size of the components of the sperm head and tail differ considerably between genera and sometimes even between species within the same genus (Retzius, 1909; Friend, 1936; Cummins & Woodall, 1985). The variation in the specific size and shape of the sperm head and tail will now be briefly outlined with special reference to rodents.

5.4.1 Inter-specific variation in sperm head shape

‘Species specific’ dimensions of the acrosome, nucleus and cytoskeleton give rise to striking inter-specific differences in the overall sperm head size and shape. In human, cow and rabbit spermatozoa, a more or less symmetrical acrosome often described as skull-shaped, overlies a spatulate-shaped nucleus. In the guinea pig a very large asymmetrical acrosome is located over a spatulate nucleus, whereas in most murid rodent species, an asymmetrical acrosome lies over the falciform- shaped nucleus (Figure 11) (see Retzius, 1909; Bishop & Austin, 1957; Bishop & Walton, 1960; Fawcett, 1970, 1975; Bedford & Hoskins, 1990).

There is much diversity in the shape and size of spermatozoa within the murine rodents (Retzius, 1909; Friend, 1936). There are approximately 530 species and 120 genera of murine rodents that are divided into three major groups, that occur respectively in Eurasia, Africa, and Australasia (Musser & Carleton, 2005). The Eurasian group includes the ancestors of the laboratory rat and laboratory mouse, and the sperm heads in this group of rodents are generally bilaterally flattened and

falciform-shaped. In both of these laboratory species, the nucleus extends into the apical hook, while the acrosome extends beyond the rostral tip of the nucleus and further down the convex margin of the sperm nucleus. Also in these species, a thin equatorial segment surrounds the posterior region of the sperm head. However, the sperm heads of Rattus are further extended in the antero-posterior plane and are narrower than those of Mus (Friend, 1936). Although for many wild murine rodents the general structure of the sperm head is similar to that of the laboratory species, the dimensions and orientation of the apical hook and/or the overall shape of the sperm head can vary considerably between species (Friend, 1936). All species of Rattus have a sperm head morphology similar to the laboratory rat, whereas most species of native Australian Old Endemic murine rodents have in addition to the apical hook, two ventral processes that extend from the concave surface of the sperm head (Flaherty & Breed, 1983; Breed, 1984; Breed, 1997a). Nevertheless, in a few species of Australian old endemics, the shape and structure of the sperm head is markedly different and has either one very truncated hook, or is either spatulate or pear-shaped (Breed, 1983; Breed & Sarafis, 1983; Breed, 1997a). There are also several species of Eurasian and African murine rodents in which the sperm head lacks an apical hook. These include spermatozoa that are either paddle-shaped, globular, conical or wedge-shaped (Friend, 1936; Breed et al., 1988; Breed, 1993; Breed & Aplin, 1994; Breed, 2004).

Investigations into the factors involved in the formation of the ‘species specific’ sperm head shape have been carried out for several decades. Many of these studies have focused on the factors involved in the formation of the sperm nuclear shape. While several theories have suggested the involvement of either (a) actin in the subacrosomal space, (b) nuclear proteins and lamina, (c) different models of chromatin condensation, and (d) the cytoskeleton of surrounding Sertoli cells, there is a lack of supporting evidence for most of these (reviewed by Meistrich, 1993). There is nevertheless some evidence to suggest that the microtubular manchette and nuclear ring play a role in the shaping of the sperm head and nucleus during spermiogenesis (Fawcett et al., 1971; Dooher & Bennett, 1974; Cole et al., 1988; Meistrich et al., 1990; Russell et al., 1991; Toshimori & Ito, 2003). This includes findings from studies on laboratory mice that have a mutation of the autosomal gene Hook1, which show that abnormal positioning of the microtubular manchette

on the spermatid results in the production of misshapen sperm heads (Cole et al., 1988; Mendoza-Lujambio et al., 2002).

Several genes on the Y chromosome have also been found to be associated with the formation of the ‘species specific’ shape of the acrosome and nucleus (Styrna et al., 1991a; Styrna et al., 1991b; Burgoyne et al., 1992). Partial deletions of the Y chromosome have been associated with an increase in spermatozoa with abnormal head shapes (Illisson, 1969; Styrna et al., 1991a; Styrna et al., 2003) and function (Ward & Burgoyne, 2006). In humans, microdeletions of several genes, including DAZ and RBM, on the Yq11 region of the Y chromosome have also been associated with an increase in abnormalities of the sperm head and tail (Vogel et al., 1999), as well as incidences of azoospermia and oligiospermia in some human males (Yao et al., 2001).

Figure 11

Cross-sectional views of the sperm heads of the guinea pig, hamster, Human and rabbit highlighting inter-specific differences in nuclear and overall sperm head shape (modified from Eddy & O’Brien, 1994).

5.4.2 Inter-specific variation in sperm length

An extensive comparative review of mammalian sperm dimensions indicated that inter-specific differences in the length of the sperm head and tail exist (Cummins & Woodall, 1985). Consequently, there is considerable variation between species in the total length of the spermatozoa. For instance, within the murine rodents, sperm length ranges from around 190μm in the laboratory rat, 130μm in the house mouse, to only 60μm in the bandicoot rat, Bandicota savilei (Breed & Taylor, 2000).

Variations in the relative size and shape of the nucleus, acrosome and cytoskeleton give rise to species differences in sperm head length. Within the murine rodents, the presence and orientation of the apical hook appears to influence the length of the sperm head. The length of the sperm head ranges from 5μm in species that lack apical hooks such as the heath rat, the bandicoot rats, B. indica and B. savilei, (Breed, 2004) and the harvest mouse (Friend, 1936) to 13μm in those species with apical hooks such as the Malayan mountain spiny rat and the swamp rat (Friend, 1936; Cummins & Woodall, 1985; Breed & Taylor, 2000).

In spite of this, it is variation in sperm tail length that accounts for much of the inter- specific differences in sperm length. In addition to this, the proportions of the midpiece in relation to the principal piece also vary considerably between species. For instance, the midpiece comprises about 40% of the total length of the sperm tail in the rat, whereas it is only about 10% of the total length of the sperm tail in humans (Bishop & Walton, 1960).

The reasons for the variation in sperm length are not clear. There is some evidence to suggest that sperm tail length is positively associated with sperm velocity and thrusting forces, which led to the suggestion that inter-specific variation in sperm midpiece and/or tail length may relate to differences in levels of inter-male sperm competition (Gomendio & Roldan, 1991; Roldan et al., 1992). However, this is highly controversial and other studies have failed to find a correlation between sperm length and relative testis mass (Harcourt, 1991; Anderson & Dixson, 2002; Gage & Freckleton, 2003). Alternately, it has been suggested that swimming speed may depend more on the volume of the mitochondria in the midpiece than its length (Cardullo & Baltz, 1991). This is supported by findings from a recent study 35

on 31 primate species showing a positive relationship between level of sperm competition and sperm midpiece volume, but not midpiece or flagellar length (Anderson & Dixson, 2002).

5.5 Intra-specific variation in sperm shape and size

Although there are large inter-specific differences in the size and shape of the mammalian sperm head and tail, within each species sperm dimensions are generally consistent (Cummins & Woodall, 1985; Gage, 1998). However, within populations of spermatozoa that have a ‘normal’ structure, there may be some inherent variation in the dimensions of the spermatozoa. The variation in the dimensions of spermatozoa that appear to have a ‘normal’ structure is of course different to variations in morphology which are due to abnormalities.

5.5.1 Intra-specific variation in sperm shape

Humans are one of the very few species in which variation in the size and shape of the sperm head have been reported and, due to an increased demand for assisted fertilitisation in the recent past, many studies have tried to determine the range of morphological variants that are structurally ‘normal’ from those that are defective or ‘abnormal’ (Kruger et al., 1986; Kruger et al., 1987; Menkveld et al., 1990; Aziz et al., 1998; Menkveld et al., 2003). According to strict criteria, human sperm heads are regarded as being of ‘normal’ morphology if they are approximately 3-5μm long and 2-3μm wide, with an acrosome covering approximately 40-70% of the sperm head with the overall shape of the sperm head ranging from a smooth oval form to those that are slightly tapered, rounded, or elongated (Menkveld et al., 1990).

There are a few other species of eutherian mammals in which individuals produce high proportions of spermatozoa that do not exhibit ‘species specific’ dimensions. High incidences of pleiomorphic spermatozoa have been documented in species of gazelle (Cassinello et al., 1998; Roldan et al., 1998; Gomendio et al., 2000), domestic cats (Neubauer et al., 2004), cheetahs (Wildt et al., 1983; Crosier et al., 2007), jaguars (Morato et al., 2001), leopards (Wildt et al., 1986; Jayaprakash et al., 2001), lions, Florida panthers (Barone et al., 1994), red deer (Malo et al., 2005) gorillas and humans (Seuanez et al., 1977). Within the murine rodents, species of

Asian Bandicoot rats (Breed, 1998), South African tete veld rat (Breed, 1993), delicate mouse, dusky hopping mouse, and spinifex hopping mouse (Breed & Sarafis, 1983; Suttle et al., 1988; Breed, 1997a) also display sperm head pleiomorphism. While for some of these species sperm pleiomorphism was found to be present in only a few individuals of a population, for others it extended throughout entire populations. These are often a consequence of pathological structural abnormalities to either components of the sperm head or tail and associated with reduced levels of fertility.

As the nucleus occupies most of the sperm head, the shape of the sperm head generally reflects that of the nucleus. In humans (Bedford et al., 1973a; Kubo-Irie et al., 2005) as well as the tete veld rat (Breed et al., 1988; Breed, 1993), and bandicoot rat, B. indica (Breed, 1998), nuclear vacuoles are quite prominent within the sperm nucleus. Based on data from immunohistochemical and electron microscopical studies the nuclear vacuoles appear as electron lucent areas where the chromatin is not well condensed. It is thought that these areas of incomplete chromatin condensation are related to abnormalities in the arrangement of the DNA or DNA protamine complexes due to the retention of histones (Ostermeier et al., 2000; Ostermeier et al., 2001) and in humans at least, may be associated with increasing age (Kidd et al., 2001).

In several eutherian species, low genetic diversity due to inbreeding, and/or population bottlenecks are thought to be responsible for sperm pleiomorphism. For instance in genetically homozygous populations of African lions, the Florida panther (Barone et al., 1994), cheetah (Wildt et al., 1983; O'Brien et al., 1987), Clouded leopard (Wildt et al., 1986), Indian leopard (Jayaprakash et al., 2001) and gazelles (Gomendio et al., 2000) there were higher incidences of pleiomorphic spermatozoa associated with structural abnormalities then when compared with individuals in more heterozygous populations. A link between inbreeding and pleiomorphism has also been found in several inbred strains of mice (Beatty & Sharma, 1960; Illisson, 1969; Krzanowska, 1976; Krzanowska, 1981; Krzanowska et al., 1995a).

The influence of age on the occurrence of sperm pleiomorphism is highlighted in several inbred strains of mice, with the young and old mice having higher proportions of misshapen sperm heads (Beatty & Mukherjee, 1963; Krzanowska, 1981). A similar situation has been observed in boars, with greater percentages of abnormal spermatozoa occurring in young and old individuals (Vilakazi & Webb, 2004). Furthermore there is evidence to suggest that in humans, increasing age maybe associated with a decrease in the percentage of spermatozoa with normal morphology, with older men having a greater percentage of spermatozoa with coiled tails and microcephalic sperm heads (Kidd et al., 2001). In humans, a high percentage of pleiomorphic spermatozoa may also be associated with exposure to certain chemicals (Hauser et al., 2003; Swan et al., 2003), disease and infection, smoking or high scrotal temperatures due to modern lifestyles (Mieusset & Bujan, 1995; Thonneau et al., 1998; Mortimer & Menkveld, 2001; Ivell, 2007).

5.5.2 Intra-specific variation in sperm length

While most comparative studies on mammalian sperm morphology have suggested ‘species specific’ sperm dimensions (Cummins & Woodall, 1985; Gage, 1998), variations in the sperm midpiece and/or total tail length have been found to occur between individuals of several mammalian species (Ward, 1998; Morrow & Gage, 2001). In these species, inter-individual variation in sperm length occurred irrespective of whether the animals were wild-caught or captive-bred, and hence was not related to the level of inbreeding, or body condition. The evolutionary significance of such variation remains unclear.

5.6 Epididymal sperm maturation

When spermatozoa are released from the testis they are relatively immature, immotile and unable to fertilise an oocyte, but as they pass through the epididymis they undergo morphological and biochemical changes that enable them to develop the potential to fertilise eggs (Bedford et al., 1973b; Bedford, 1975; Orgebin-Crist et al., 1975; Orgebin-Crist & Fournier-Delpech, 1982; Moore & Akhondi, 1996; Gatti et al., 2004). The epididymis is a highly absorptive and secretory duct and is divided into three general regions; the caput (head) region, which joins with the ductuli efferentes, the corpus (body), which connects the caput and cauda regions, and

the cauda (tail) region, which connects to the vas deferens. The maturational changes occur primarily in the caput and corpus regions of the epididymis and are usually complete by the time the spermatozoa reach the cauda epididymidis where they are stored (Glover & Nicander, 1971; Bedford, 1975; Orgebin-Crist et al., 1975; Yanagimachi et al., 1985). For most mammals spermatozoa take between 6 to 14 days, depending on the species, to pass along the entire epididymis, with the time that spermatozoa spend in the cauda epididymidis a function of both daily sperm production, the capacity of this region for storing spermatozoa and ejaculatory frequency (Amann et al., 1976; Dadoune & Alfonsi, 1984). For detailed reviews see: Amann et al. (1993) Toshimori (2003).

5.6.1 Morphological changes

In most eutherian mammals the morphological changes to spermatozoa as they travel along the epididymis are minimal. The most obvious morphological change that generally occurs in the epididymis is the displacement of the cytoplasmic droplet from the neck region to either be lost from the spermatozoon, or maintained at the distal end of the middle piece near the annulus (Bedford, 1975). In a few mammalian species however, epididymal transit is also associated with changes to the size and shape of the acrosome. In the rat, rabbit, boar, elephant and some primates, it has been found that the apical segment of the acrosome undergoes a reduction in size and may become more rounded (Fawcett & Phillips, 1969b; Bedford & Nicander, 1971; Jones et al., 1974). The most significant changes occur in species with large or elongated apical segments of the acrosome, such as in the guinea pig (Fawcett & Hollenberg 1963), bushbaby (Bedford 1974), chinchilla (Fawcett & Phillips, 1969b), and South African red veld rat (Breed et al., 1988).

5.6.2 Biochemical changes

By far the majority of changes to the spermatozoa during epididymal transit are biochemical. Such modifications arise by either proteins secreted from the epithelial lining of the epididymis being a) incorporated into the spermatozoa, b) activating pre-existing switches controlling sperm metabolism or c) altering the molecules already on the sperm surface (Cornwall et al., 1990; Moore & Akhondi, 1996; Cooper, 1998; Moore, 1998). Alternately some of the changes may be due to

pre-programmed modifications of molecules within the spermatozoon (Amann et al., 1993).

Associated with epididymal transit is the progressive stabilization of the sperm head and tail. For instance the sperm nucleus becomes stabilized through a process where the cysteine residues of the protamine molecules are oxidized and form disulphide crosslinks within and between the protamine chains of the chromatin (Calvin & Bedford, 1971; Bedford et al., 1973a; Bedford et al., 1973b; Bedford & Calvin, 1974; Huang et al., 1984; Balhorn, 1989; Auger & Dadoune, 1993). In addition to the nucleus, the subacrosomal material, postacrosomal dense lamina, connecting piece, ODFs, fibrous sheath and outer mitochondrial membrane become stabilised by the formation of disulphide bonds (Calvin & Bedford, 1971; Bedford et al., 1973b; Seligman & Shalgi, 1991). While the increased stability of the nucleus protects the genome from DNA damage during fertilization, the stabilization of the cytoskeleton is viewed as an adaptation for penetration of the extracellular coats around the oocyte (Bedford & Calvin, 1974), whilst changes in the tail structures being associated with the changing pattern of motility.

During sperm maturation in the epididymis, some of the molecules that are secreted by the epithelium are added to the sperm surface or are involved in the redistribution or glycosylation of, molecules already present on the sperm plasma membrane (Hamilton, 1975; Turner, 1991; Cooper, 1998; Dacheux et al., 1998; Jones, 1998a; Toshimori, 1998, 2003; Dacheux et al., 2003; Tulsiani, 2006). Lectin histochemistry has been extensively used to determine the distribution of various glycoproteins on the surface of the spermatozoa as well as to trace maturational changes to the proteins and glycoproteins that occur during epididymal transit (Kohane et al., 1980a; Kohane et al., 1980b; Koehler, 1981; Olson & Danzo, 1981; Magargee et al., 1988; Rankin et al., 1989; Srivastava & Olson, 1991; Tulsiani et al., 1993; Jones, 1998a). Further studies have shown that enzymes such as glycosyltransferases and glycosidases are present in the epididymal luminal fluid and function to add and cleave sugar residues to and from proteins and glycoproteins on the sperm plasmalemma (Tulsiani et al., 1998; Tulsiani, 2003, 2006). These glycoprotein changes aid in either preventing a premature acrosome reaction from occurring, or providing the appropriate conformation and/or location

of surface molecules to successfully bind to, and fuse with, the plasmalemma of the oocyte (Bedford, 1975; Orgebin-Crist et al., 1975; Jones, 1998a; Tulsiani & Abou-Haila, 2001).

In mammals, the capacity for forward progressive sperm motility is also acquired during epididymal maturation. This is important for the spermatozoa to acquire a hyperactivated form of motility during capacitation in the female reproductive tract. In most species, when spermatozoa leave the testis they lack any form of sperm motility and as they move along the epididymis, the pattern of activated sperm motility changes from a vibratory, slow, beat and circular motion in spermatozoa from the caput of the epididymis to rapid, yet more restricted, symmetrical arc formations in those from the caudae of the epididymis (Bedford et al., 1973b; Bedford, 1975; Yeung et al., 1992; Yeung et al., 1993; Yanagimachi, 1994; Axner et al., 1999; Van Der Horst et al., 1999). While spermatozoa acquire the potential for forward progressive sperm motility during epididymal transit, they remain in a quiescent state during storage in the cauda epididymidis (Usselman & Cone, 1983; Carr & Acott, 1984; Jones & Murdoch, 1996), only becoming activated when mixed with accessory gland secretions in vivo (Morita & Chang, 1971; Bedford & Yanagimachi, 1992; Carballada & Esponda, 1992).

5.7 Sperm storage

After epididymal maturation is complete, spermatozoa are stored in the excurrent ducts until they are ejaculated or lost in the urine. In most species, including the laboratory mouse and rat, the majority of spermatozoa in the excurrent ducts are stored in the cauda epididymidis (Bedford, 1975; Orgebin-Crist et al., 1975). The number of spermatozoa stored in the cauda epididymides varies between species and generally depends on daily sperm production, the duration of epididymal transit, the capacity of the region to store spermatozoa as well as the frequency of ejaculation (Amann et al., 1976; Amann & Howards, 1980).

The cauda epididymidis is able to store large numbers of viable spermatozoa for long periods of time, due to in part, the expanded luminal diameter of this region. In some species, the presence of extensive epithelial infoldings in the cauda

epididymidis has been suggested to increase the surface area of the capillaries in the underlying tissue and thus increase the potential supply of oxygen available for sperm respiration during storage, which may enhance sperm storage (Djakiew & Cardullo, 1986). Furthermore, in most eutherian species the cauda epididymidis is positioned alongside the testes in the scrotal sac, which by being external to the body cavity is a few degrees cooler (350C) than the core body temperature (370C). The effect of temperature on sperm maturation and storage in the epididymis has been extensively studied by repositioning the epididymis from the scrotum into the abdominal region. In general a rise in temperature was found not to affect extra- testicular sperm maturation, but it was associated with a decrease in both sperm motility and size of the epididymides, with much more rapid epididymal sperm transit taking place at the higher temperatures (Bedford, 1978a, b; Bedford et al., 1982; Foldesy & Bedford, 1982; Bedford, 1991). However, when the epididymis of the hamster was positioned in the abdomen, the time taken for the spermatozoa to undergo capacitation was reduced (Bedford & Yanagimachi, 1991). It appears that the cooler environment and low androgen concentrations provided by the scrotum, as well as temperature dependent compounds secreted by the cauda epididymal epithelium (Esponda & Bedford, 1986), may aid, in part, to stabilize the sperm membranes by reducing their fluidity (Moore, 1996, 1998). Cooler temperatures also reduce sperm respiration, which increases oxygen availability in the lumen of the epididymis as well as the capacity to store a large number of sperm per unit volume of the cauda epididymis (Djakiew & Cardullo, 1986).

In most species the vasa deferentia have a narrow lumen and are highly muscular. The primary function of the vas deferens is to propel spermatozoa from the epididymis to the urethra (Hamilton, 1975; Hoffer, 1976) and then in returning any spermatozoa remaining in the vas deferens after ejaculation back to the epididymis (Prins & Zaneveld, 1980). Consequently, for most species the vas deferens does not contain a large proportion of the extra-testicular sperm population (Glover & Nicander, 1971). However, in several species including the stallion, spermatozoa are stored in an expanded region of the vas deferens, called an ampulla (Gebauer et al., 1974). This also occurs in several species of shrew, where up to 20% of spermatozoa of the sperm stores occur in an expanded region of the vas deferens (Bedford et al., 1994).

5.8 Reproductive anatomy of the spinifex hopping mouse

In comparative studies on the testis size of sexually mature Australian rodents it was found that for many species the testes weigh approximately 1 to 3% of their body weight (Breed & Taylor, 2000). However a few species have a somewhat smaller relative testes size, with the smallest testes found to be present in several hopping mouse species, Notomys spp. (Breed, 1982; Breed & Taylor, 2000; Breed & Ford, 2007). Most studies on the testes of hopping mice have, however, used captive-bred animals and for these individuals it has been found that there is a relatively long cycle of seminiferous epithelium (14 days) and low efficiency of sperm production (<1.0x107per gram of testis) (Peirce & Breed, 2001). Furthermore, using these captive-bred hopping mice, its been found that the populations of spermatozoa produced are highly variable in morphology (Breed & Sarafis, 1983; Suttle et al., 1988). These findings emphasise that far fewer spermatozoa are produced in these captive-bred animals, than in other rodents of comparable body weight (Kenagy & Trombulak, 1986; Breed, 1997b; Peirce & Breed, 2001). The question this raises is: does this greatly compromise the male fertility of these laboratory bred individuals of this species and also, is this invariably the case in wild caught animals?

Not only are the testes of captive-bred hopping mice very small, but the epididymides of these animals are also comparatively short, with rapid sperm migration occurring along the duct (Peirce & Breed, 2001). In addition to the limited storage capacity of the cauda epididymidis, the sperm population in this region is at a comparatively low density. However, this is complemented by the distal region of the vas deferens having a highly unusual structure (Peirce & Breed, 1989), whereby trabeculae occur in the duct which is surrounded by a very thin muscular coat (Breed, 1997b; Peirce et al., 2003). The highly unusual morphological features of the vas deferens appear to be associated with sperm storage in this region, yet whether spermatozoa continue to undergo maturational changes as they pass from the cauda epididymis to the vas deferens is not known.

5.9 General aims and objectives

Consequently, this thesis aims to explore several features of sperm production and maturation in the spinifex hopping mouse, N. alexis. In particular the following questions were addressed:

1. Are the small testes found in captive-bred animals also apparent in wild- caught individuals at times of reproductive activity?

2. Does testis size change at different times of reproductive activity of the population?

3. If testes are invariably very small in this species, what are the implications for the animal’s fertility potential?

4. As the number of individuals of this species for which the sperm morphology has been determined is very small, and with no detailed study as yet on the sperm morphology, the following questions were asked: What is the structural organization of the sperm acrosome and nucleus? Are there always pleiomorphic sperm populations present regardless of age of the animal, and whether they are wild-caught or captive-bred?

5. If male hopping mice invariably produce highly pleiomorphic spermatozoa with variable tail lengths, is this associated with an observable difference in sperm motility, in particular do spermatozoa with a longer midpiece swim faster?

6. As it is highly unusual for a murine rodent to have as many spermatozoa stored in the distal vasa deferentia as in the cauda epididymides, do spermatozoa undergo any further maturational changes as they pass from the cauda of the epididymis to the vas deferens?

It is these questions that will now be addressed in the following four publications and chapters.

6 Publications & Chapters

6.1 Publication 1

Testis mass of the spinifex hopping mouse, Notomys alexis, and its impact on fertility potential

M. Bauer and W.G. Breed

Journal of Zoology, 2008, 274, 349-356

Investigations on the male reproductive system of captive-bred hopping mice have suggested that sexually mature individuals appear to have a small absolute and relative testis size (Breed, 1982). However, very few data have been collected on the testis size of this species in its natural environment (Breed, 1992) and it is not known whether unusually small testes invariably occur in adult males in naturally occurring populations.

It has also been found that, in addition to the small testes, captive-bred hopping mice produce both relatively low numbers of spermatozoa per gram of testis (Peirce & Breed, 2001) as well as having highly pleiomorphic sperm populations (Breed & Sarafis, 1983; Suttle et al., 1988). In addition to this there is a relatively fast epididymal transit time (Peirce & Breed, 2001) with comparatively low numbers of spermatozoa being stored in the caudae epididymides (Peirce & Breed, 1989). However, no studies have determined the numbers of spermatozoa ejaculated during a single mating event, the proportion of the sperm store that is lost at this time, how rapidly the sperm stores are subsequently replenished, or the impact that these features have on male fertility.

The aims of this publication were therefore to determine (1) the testis size of male spinifex hopping mice in their natural environment, and (2) the differences in testis size of males in reproductive active and non reproductive populations. The third aim of this publication was to shed some light on how the small testes and low sperm stores of the male hopping mouse affects male fertility potential.

Bauer, M. and Breed, W.G. (2008) Testis mass of the Spinifex hopping mouse, Notomys alexis, and its impact on fertility potential. Journal of Zoology, v.274 (4) pp. 349-356, April 2008

NOTE: This publication is included on pages 45-53 in the print copy of the thesis held in the University of Adelaide Library.

It is also available online to authorised users at:

http://dx.doi.org/10.1111/j.1469-7998.2007.00392.x

6.2 Publication 2

Variation of sperm head shape and tail length in a species of Australian hydromyine rodent: the spinifex hopping mouse, Notomys alexis

M. Bauer and W. G. Breed

Reproduction, Fertility and Development, 2006, 18, 797–805

Most eutherian mammals produce spermatozoa that are highly consistent in morphology and are thus often referred to as having a ‘species specific’ sperm head and tail length (Cummins & Woodall, 1985; Gage, 1998). There are however a few exceptions, including the spinifex hopping mouse (Breed & Sarafis, 1983; Suttle et al., 1988). Previous studies have shown that, in addition to small testes, these animals appear to produce populations of spermatozoa with pleiomorphic heads. Although variation in sperm head shape and size was detected in the individuals of the captive colony, all of the animals in this colony originated from the same wild population, and thus it is not known if this sperm pleiomorphism was a reflection of the species itself or was due to inbreeding of the individuals obtained from the one small population. To date no studies have investigated if this sperm pleiomorphism extends to the sperm tail.

The aim of this publication was to determine if sperm head and tail pleiomorphism is a ‘species specific’ characteristic and secondly if pleiomorphism extends to the length of the sperm tail. This publication also provides the first detailed account of the ultrastructure of the spermatozoa of this species, thus providing further insight into the nature, and possible evolutionary significance, of the sperm pleiomorphism.

Bauer, M. and Breed, W.G. (2006) Variation of sperm head shape and tail length in a species of Australian hydromyine rodent: the spinifex hopping mouse, Notomys alexis. Reproduction, Fertility and Development, v.18 (7) pp. 797-805, 2006

NOTE: This publication is included on pages 54-63 in the print copy of the thesis held in the University of Adelaide Library.

It is also available online to authorised users at:

http://dx.doi.org/10.1071/RD06045

6.3 Publication 3

Intra-individual variation in sperm tail length in murine rodents

W. G. Breed, M. Bauer, R. Wade, N. Thitipramote, J. Suwajarat and L. Yelland

Journal of Zoology, 2007, 272, 299-304

Inter-individual variation in sperm tail length has been found to occur in a few mammalian species, yet there is very little information on variation within individuals. It was shown in publication 2 that in addition to sperm head pleiomorphism, individual hopping mice also show variation in the length of the sperm midpiece and flagellum. However, it is not known if there is greater intra- individual variation in sperm tail length in this species compared to closely related species, or if the variation in tail length is related to the sperm head pleiomorphism.

The general aim of this publication was to determine if variation in sperm tail length is greater in species that also show intra-individual variation in sperm head morphology. More specifically this publication investigates whether three species of murine rodents with intra-individual sperm head pleiomorphism display greater variation in sperm midpiece and flagellar lengths than do closely related species that produce spermatozoa with a consistent head morphology.

Breed, W.G., Bauer, M., Wade, R., Thitipramote, N., Suwajarat, J. and Yelland, L. (2007) Intra-individual variation in sperm tail length in murine rodents. Journal of Zoology, v.272 (3) pp. 299-304, July 2007

NOTE: This publication is included on pages 64-70 in the print copy of the thesis held in the University of Adelaide Library.

It is also available online to authorised users at:

http://dx.doi.org/10.1111/j.1469-7998.2006.00269.x

6.4 Chapter 1

Intra-individual variation in sperm midpiece length and sperm motility in the spinifex hopping mouse

It was shown in publication 2 and 3 that the spinifex hopping mouse has in addition to sperm head pleiomorphism considerable intra-individual variation in sperm midpiece and flagellar length. The functional significance of variation in sperm tail length within an individual has not previously been investigated. The relationship between the length of the sperm midpiece and flagellum with sperm motility is controversial. Nevertheless, if sperm midpiece length relates to sperm velocity, then it could be hypothesized that spermatozoa with longer midpieces may have a greater velocity than those with shorter midpieces.

Therefore the aim of this chapter was to investigate if variation in sperm midpiece length within individual hopping mice was associated with differences in sperm motility. Sperm motility was measured using a ‘swim-up’ technique and the sperm head and tail lengths of the spermatozoa in different populations compared.

Intra-individual variation in sperm midpiece length and sperm motility in the spinifex hopping mouse

Introduction The sperm flagellum is responsible for generating activated sperm motility, which is necessary for spermatozoa to travel up the oviduct and for generating hyperactivated sperm motility which is needed to penetrate the cumulus oophorus and zona pellucida around the recently ovulated oocyte (Suarez et al., 1991; Ho & Suarez 2001). The axoneme, which extends the entire length of the flagellum, consists of a central pair of microtubules surrounded by 9 peripheral pairs. Attached to the outer microtubule doublets are dynein arms, and it is the attachment of the dynein arms to adjacent microtubule doublets and then bending of the arms with release of adenosine triphosphate (ATP), which results in microtubules sliding in relation to each other, and thus sperm motility (Brokaw 1972, 1989). Surrounding the axoneme along the principal piece is a fibrous sheath of two longitudinal columns joined together by ribs of fibrous material occurs, which provides structural support but also contains proteins that may be involved in the regulation of sperm motility (Eddy & O'Brien, 1994; Turner 2003, 2006).

The length of the sperm flagellum varies considerably between mammalian species, and within the murine rodents, ranges from 57 μm in the South African red veld rat to 190 μm in the laboratory rat (Cummins & Woodall, 1985; Gage & Freckleton, 2003). Its length is often referred to as being ‘species-specific’ (Cummins & Woodall, 1985; Roldan et al., 1992; Gage, 1998) with only a small amount of inter-individual variation within a species (Ward, 1998; Morrow & Gage, 2001). However, we have recently found that the hopping mouse, N. alexis, together with the Bandicoot rat, Bandicota indica and tete veld rat, Aethomys ineptus, all display considerable inter- and intra-individual variation in the length of both the sperm midpiece as well as the total tail length (Bauer & Breed, 2006; Breed et al., 2007).

Whether the total length of the sperm tail and/or that of the midpiece determines at least in part sperm velocity and longevity appears to be controversial. It has been suggested that in mammals, the length of the sperm flagellum may determine the velocity of sperm motility, with some evidence to suggest that longer sperm flagella generate greater thrusting forces and hence more vigorous motility than sperm with shorter flagella (Cardullo & Baltz, 1991; Gomendio & Roldan, 1991). However, other evidence suggests that the volume of the midpiece (Cardullo & Baltz, 1991), and thus the mitochondrial sheath, is more closely associated with species differences in sperm motility than is the total length of the tail (Gage & Freckleton, 2003). If the former hypothesis is correct, spermatozoa with a longer midpiece and flagella may have a selective advantage in getting to the site of fertilization quicker than spermatozoa with a shorter midpiece and total tail length.

The spinifex hopping mouse, with its considerable intra-individual differences in sperm tail length, may be a useful animal to test this prediction. Therefore, in the present study the aims are to determine if (1) spermatozoa with a longer midpiece tend to exhibit faster motility than those with a shorter midpiece and hence more likely to reach the site of fertilization first, and (2) if differences in motility are associated with variation in sperm head and tail morphology.

Materials & Methods Animals Captive bred male spinifex hopping mice, Notomys alexis, were obtained from the Nature Education Centre, Norwood, South Australia and housed under regulated temperature (18-25 0C) and lighting (12h light: 12h dark photoperiod) conditions with food and water provided ad libitum. In order to obtain spermatozoa from sexually mature males, animals >100 days of age were killed by carbon dioxide gas, and the spermatozoa collected from the caudae epididymides and vasa deferentia. Laboratory mice, Mus musculus, were obtained from the Animal House, The University of Adelaide. Ethics approval number M-61-2001A

Media Pilot studies were performed to assess the motility of laboratory mouse and spinifex hopping mouse spermatozoa in the following media: (a) M119, (b) Dulbecco’s PBS, (c) MEM alpha modification supplemented with 2.2g sodium bicarbonate, (d) Dulbecco’s MEM supplemented with 4.2g sodium bicarbonate, (e) Modified Tyrodes medium (TALP) supplemented with Bovine serum albumin (BSA) [4mg/ml], 20mM lactate and 1mM pyruvate). The preliminary results showed that the percentage of motile spermatozoa was greater after incubation in the Modified Tyrodes medium (TALP) supplemented with bovine serum albumin (BSA) [4mg/ml], 20mM lactate (20μl in 10 ml), 1mM pyruvate (1.1mg in 10ml), and buffered with 5% CO2 at 370C, for 30 minutes. To prepare the modified Tyrodes media, 20mM lactate and 1mM pyruvate were added to the media then gassed with 5% CO2, at 370C, for 30 minutes. The pre-warmed media was then supplemented with BSA [4mg/ml] and returned to the incubator for a further 15 minutes.

Collection of spermatozoa Pilot studies suggested that centrifugation of spinifex hopping mouse spermatozoa, even at low forces (<2,500G), prevented subsequent sperm motility. Therefore, to avoid centrifugation of the spermatozoa, a concentrated sperm suspension was collected from the caudae epididymides and used throughout the study. To do this, spermatozoa were obtained from the caudae epididymides and vasa deferentia of the spinifex hopping mice, by placing small pieces of tissue into 100μl of pre- warmed modified Tyrodes media and gently squeezing the tissue with forceps. For each replicate experiment, spermatozoa from two spinifex hopping mice were pooled to perform ‘swim-up’. As a control, spermatozoa from the caudae epididymides and vasa deferentia were collected from the laboratory mouse (n=2) in 1200μl of pre-warmed media.

‘Swim-up’ After the extrusion of spermatozoa into culture media, a sample of 100μl of sperm suspension was immediately removed, half of which was smeared on a slide, and air-dried, while the other half was placed on a pre-warmed slide within a Vaseline

ring, and a coverslip placed on top. The remaining 100μl of sperm suspension was placed in an eppendorf under 500μl of pre-warmed media and incubated at 370C with 5% CO2 in air. After 60 min, the top 200μl of media was carefully collected with a pipette, and half of this used to prepare air-dried sperm smears, with the other 100μl placed on pre-warmed slides and a coverslip placed on top of a Vaseline ring. Slides were also prepared from the 200μl of media remaining in the eppendorf (Figure 1).

Sperm motility To assess sperm motility, slides were prepared with a Vaseline ring to allow spermatozoa to swim freely in the media. These slides were then placed on a heat plate warmed to 370C and viewed with Nomarski optics at 400x magnification. The percentage of spermatozoa with progressive motility was recorded for approximately 50-100 spermatozoa.

Sperm morphology A drop of phosphate buffered saline (PBS, pH 7.4) was added to the air-dried sperm smears and a coverslip placed on top of the smear. The prepared slides were viewed with a Nomarski differential interference microscope at 400x magnification. Sperm head and tail morphologies as well as sperm midpiece lengths (outlined below) were determined for approximately 150 randomly selected sperm per sample per replicate. The percentage of spermatozoa in each morphological category was determined for each sample.

Sperm midpiece length To determine if the length of the sperm midpiece length relates to sperm motility, sperm midpiece lengths of approximately 100 morphologically ‘normal’ sperm tails were measured for each sample with an eyepiece graticule using Nomarski optics at 400x magnification. Sperm midpiece lengths were measured from the connecting piece to the site at which the diameter of the flagellum narrowed, i.e. the junction between the midpiece and principal piece.

Sperm tail morphology

The sperm tail morphologies of between 100 to 200 spermatozoa in three replicate experiments were scored as having either a bent neck, a twisted, bent, broken, tightly coiled midpiece or principal piece, or ‘normal’ if they were free from obvious structural defects (see Figure 4 in Bauer & Breed, 2006). If present, the location of the cytoplasmic droplet was recorded.

Sperm head shape The sperm head morphologies of approximately 150 spermatozoa in four replicate experiments were classified as follows: (1) presence of an apical hook that was either short, straight, slightly curved, or tightly curved, (2) presence of an apical ‘knob’, (3) no apical hook with an apical surface that was either flat or dome shaped, or (4) other (see Figure 1 in Bauer & Breed, 2006).

Statistics To determine if sperm midpiece lengths from the different samples were significantly different, an Analysis of Variance (ANOVA) was performed using Microsoft Excel (2003) software.

‘Swim-up’ 60 min

Sample 1: 500μl media Top 200μl

50μl sperm Sample 2: suspension Bottom 200μl

Figure 1 Diagram showing the layer of media over the concentrated sperm suspension before ‘swim-up’ (left) and the layers of sperm suspension sampled after ‘swim-up’ was performed for 60 min.

Results Sperm motility Of the laboratory mouse spermatozoa extruded from the caudae epididymides into modified Tyrodes media, approximately 75% were motile in the control, whereas >90% were motile in the samples collected within the top 200μl of media sampled after ‘swim-up’ (n=2).

Of the spinifex hopping mouse spermatozoa collections extruded from the excurrent ducts 60% to 80% were motile before ‘swim-up’ (n=4) (Table 1). During the 60 min incubation process, some spermatozoa migrated out of the sperm sample at the bottom of the tube into the overlying culture media. The percentage of motile spermatozoa within the top 200μl after ‘swim-up’ (sample 1) ranged from 33 to 40%, whereas in the bottom layer (sample 2) it ranged from 2% to 34% (n=4) (Table 1). The results showed that although the percentage of motile spermatozoa in each sample was greater before ‘swim-up’, the top 200μl of suspension invariably had a greater percentage of motile spermatozoa after ‘swim-up’ than in the lower sample.

Sperm midpiece length The length of the sperm midpiece varied both within, and between, individual hopping mice (range 22.8 ±1.8μm to 26.9 ± 2.1μm, Table 2). The lengths were found to be similar to those previously observed when the length of the midpiece was determined by using computer software after staining the spermatozoa with Mitotraker (Bauer & Breed 2006).

To determine whether spermatozoa with longer midpieces tended to swim further from the pellet, the midpiece lengths of spermatozoa obtained from the different layers after ‘swim-up’ were compared. The results showed that invariably in each replicate experiment, the mean midpiece length of the spermatozoa that migrated to the top 200μl of media after ‘swim-up’ was similar to those in the control, as well as to those that remained at the bottom (ANOVA, P<0.05; Table 2, Figure 2).

Sperm tail morphology To determine if ‘swim-up’ selected for spermatozoa that had normal tail morphology, the abundance of spermatozoa with tail abnormalities before, and after, ‘swim-up’ were determined. The mean percentage of spermatozoa with ‘normal’ tail morphology before ‘swim-up’ ranged from 52 to 74% with a mean of 60 ± 12%, whereas after ‘swim-up’ it decreased slightly to between 48 to 56% (mean, 51 ± 4%) in sample 1 and 52 to 54% (mean 53 ± 1%) in sample 2. The most common types of tail abnormalities found in all samples were bent midpieces (range 13 to 34%) and bent flagella (range 3 to 21%). The percentage of spermatozoa with cytoplasmic droplets was low and ranged from 3 to 10% in samples collected before ‘swim-up’ and between 2 to 12% in the samples collected after ‘swim-up’. Overall the results suggest that the ‘swim-up’ had not selected for a subsample of spermatozoa with decreased abundance of tail abnormalities.

Sperm head morphology To determine whether an association between the morphology of the sperm head and sperm motility exists, the relative abundance of spermatozoa with various head shapes collected at different heights after ‘swim-up’ were compared. Within each animal, the relative proportions of sperm head morphotypes were similar in the samples collected at different heights after ‘swim-up’ and in the control samples (Table 3). The most abundant sperm head shapes within all of the samples were the straight hook, slightly curved hook, apical knob and short hook, whereas spermatozoa with either a tightly curved hook, flat apical surface, dome or other were the least abundant (Table 3). These results suggest that selection for any particular sperm head shapes did not occur during ‘swim-up’.

Experiment Motile spermatozoa (%); No. Control Top 200μl Bottom 200μl 1 80 40 34 2 80 33 20 3 80 33 2 4 60 40 7 Mean ± SD 75 ± 10 37 ± 4 16 ± 14

Table 1 The percentage of motile spinifex hopping mouse spermatozoa collected before ‘swim-up’ (control) and in samples collected at different heights after ‘swim-up’ in four replicate experiments (sample 1 = top 200μl, sample 2 = bottom 200μl).

Sperm midpiece length (μm; mean ± SD) Experiment ANOVA No. Control Top 200μl Bottom 200μl (F) P 1 24.1 ± 1.3 24.4 ± 1.5 24.7 ±1.2 1.92 0.13 2 22.8 ±1.8 23.1 ± 1.8 23.1 ± 1.5 2.38 0.07 3 26.9 ± 2.1 26.4 ± 1.8 26.7 ± 1.4 0.82 0.44 4 22.6 ± 1.3 23.1 ± 1.4 22.8 ± 1.3 3.11 0.05

Table 2 Spinifex hopping mouse sperm midpiece lengths (μm; mean ± SD) from the excurrent ducts collected before ‘swim-up’ (control) and in samples collected in the top 200μl (sample 1) and bottom 200μl (sample 2) after ‘swim-up’ (n =4).

5 sperm midpiece length (um)

0 1234 experiments

Figure 2 Mean (± SD) sperm midpiece lengths (μm) from the excurrent ducts of spinifex hopping mice collected before ‘swim-up’ (control; blue) and at different heights after ‘swim-up’ (top 200μl; grey, bottom 200μl; yellow) (n=4).

Sample Spermatozoa with head morphology (%, mean ± SD) A B C D E F G H Control 30 ± 3 18 ± 3 22 ± 5 13 ± 4 7 ± 2 6 ± 3 2 ± 2 1 ± 1 Top 29 ± 9 22 ± 4 18 ± 3 22 ± 4 4 ± 3 7 ± 3 2 ± 2 2 ± 2 Bottom 28 ± 2 21 ± 4 21 ± 3 15 ± 6 5 ± 3 6 ± 3 2 ± 2 2 ± 3

Table 3 Percentages (mean ± SD) of different sperm head morphologies of spinifex hopping mice (n=4) in samples with different percentages of motile spermatozoa collected before swim-up (control) and at different heights after ‘swim-up’ (sample 1 = top 200μl, sample 2 = bottom 200μl). Sperm head morphotypes: A = straight hook; B = apical knob; C = slightly curved hook; D = short hook; E = tightly curved hook; F = no hook; G = dome; H= other.

Discussion Although several studies refer to the length of the sperm flagellum as being ‘species specific’ (Cummins & Woodall, 1985; Roldan et al., 1992; Gage, 1998), it has recently been demostrated that in several mammalian species a small amount of variation in the length of the sperm tail occurs between individuals of the same species (Morrow & Gage, 2001). In Notomys alexis, some variation in the length of the sperm midpiece and that of the flagellum occurs both within, and between, individuals (Bauer & Breed, 2006; Breed et al., 2007). The significance of the variation in sperm tail length in this species has not as yet been investigated. Therefore, the aim of the present study was to use ‘swim-up’ to ascertain whether intra-individual variation in sperm midpiece length and sperm head morphology is associated with differential sperm motility.

‘Swim-up’ is a technique that uses sperm motility parameters such as beat frequency, amplitude and velocity to select motile, morphologically ‘normal’ spermatozoa from a heterogeneous population (Drevius, 1971; Parrish & Foote, 1987; Le Lannou & Blanchard, 1988; Dominguez et al., 1999; Younglai et al., 2001; Jakab et al., 2003). Previous studies on bovine and humans have indicated that the sub-population of spermatozoa isolated from semen by ‘swim-up’, has a greater percentage of motile spermatozoa with fewer tail abnormalities than occurs in the ejaculate (Parrish & Foote, 1987; Le Lannou & Blanchard, 1988). Further the use of this subsample of spermatozoa for in vitro fertilization has been shown to result in greater fertilization success (Biljan et al., 1994). Here, in the present study on the spinifex hopping mouse, ‘swim-up’ separated spermatozoa into populations with different percentages of motile spermatozoa but, did not appear to completely separate the motile from immotile spermatozoa. Due to the complex pathways that are associated with generating and maintaining sperm motility, spermatozoa present in the population of spermatozoa after carrying out ‘swim-up’ could have been immotile for several possible reasons that may not relate to sperm midpiece length, the arrangement of mitochondria in the midpiece (Mundy et al., 1995), or their volume (Cardullo & Baltz, 1991).

In the present investigation the mean sperm midpiece lengths in the control sample were similar to both samples collected after ‘swim-up’. This suggests that, even though spinifex hopping mice display considerable variation in sperm midpiece lengths, this may not necessarily relate to differences in sperm motility.

Sperm motility is initiated when dynein arms are phosphorylated and the dynein ATPase is activated (Brokaw, 1987; Tash, 1989). This results in the hydrolysis of ATP, which brings about the sliding of the adjacent outer microtubular doublets. That mitochondria in the midpiece synthesise ATP by oxidative phosphorylation is well established, yet mathematical models indicate the amounts of ATP produced in the mitochondria cannot diffuse the entire length of the axoneme at the rate required to meet the energy requirements of the ATPase in the distal region of the axoneme (Du et al.,1994). There is an increasing body of evidence to suggest that in mice at least, ATP is not only produced by mitochondrial oxidative phosphorylation but also by glycolysis in the fibrous sheath of the principal piece. It has been found that mice with defective mitochondrial oxidative phosphorylation were still able to produce ATP and, although sperm motility was reduced, the spermatozoa were still motile and capable of fertilization (Narisawa et al., 2002). The principal piece of many mammalian species contains an array of glycolytic enzymes such as lactate dehydrogenase, hexokinase and glyceraldehyde-3- phosphate dehydrogenase (GAPD-S) (Carrera et al., 1994; Bradley et al., 1996; Westhoff & Kamp, 1997; Bunch et al., 1998; Mori et al., 1998). The importance of glycolysis on sperm motility was demonstrated by the use of GAPD-S knockout mice which showed that sterility in these mice was due to poor sperm tail motility (Mikki et al., 2004). Consequently, ATP production in the principal piece appears to have a considerable influence on sperm motility.

The spinifex hopping mouse, not only displays variation in the length of the sperm midpiece, but also in the principal piece and hence total length of the flagellum. The effect that the variation in principal piece length has on ATP production and sperm motility parameters in this species was not investigated and thus remains to be determined. If differences in sperm motility are found to occur within this species, it may be that the differences are due to a combination of variable midpiece, as well as principal piece, lengths and not just one or the other.

In mammals, spermatozoa from the caudae epididymides are usually immotile when in epididymal fluid (Usselman & Cone, 1983; Carr & Acott, 1984), but display forward progressive motility when mixed with accessory gland secretions in vivo and in physiological medium in vitro. Although several media constituents are required to activate and maintain sperm motility in all mammalian species, it would appear that species differences in the ability of sperm mitochondria to metabolize different substrates occur (Storey & Kayne, 1980). Consequently, species specific differences in media constituents and/or, the concentrations of these substrates, may be required to activate and maintain optimum sperm motility in particular species. For instance, although media used to activate sperm motility usually contains glucose, pyruvate (Mujica et al., 1991), bicarbonate and albumin (Harrison et al., 1978; Fraser, 1983, 1986; Gadella & van Gestel, 2004), the concentrations of these components as well as the sugars, amino acids, ‘sperm motility factor’, together with the viscosity, osmolarity, temperature and pH of the media, may also differ between species (Pholpramool & Chaturapanich, 1979; Holm and Wishart, 1998). In the present study, preliminary observations suggest that there are species differences in the media required for activation and maintenance of motility in the laboratory mouse and hopping mouse spermatozoa. Although the greatest number of hopping mouse spermatozoa were motile when incubated in modified Tyrodes media, the media still may not be optimal for activating and maintaining sperm motility. This may, at least in part, explain the high percentage of immotile spermatozoa collected from the top 200μl after ‘swim-up’.

The demand for assisted reproductive technology has lead to the development of an array of techniques to isolate populations of morphologically ‘normal’ and motile spermatozoa. These include electric currents (Ainsworth et al., 2005), glass wool filtration (Engel et al., 2000), side migration (Hinting & Lunardhi, 2001), and the use of density gradients. Density gradients, unlike ‘swim-up’, separate spermatozoa according to their density and not only isolate motile, morphologically ‘normal’, spermatozoa but also those that are free of abnormalities that may be independent of motility, such as DNA damage or anomalies (Le Lannou & Blanchard, 1988; Grant et al., 1994; Gravance et al., 1998; Prakash et al., 1998; Sakkas et al., 2000; Soderlund & Lundin, 2000). However, these techniques do not assess the quality

of sperm motility in individual spermatozoa and thus the sperm motion parameters of individual hopping mouse spermatozoa with different morphologies still have not been investigated. Consequently, it is possible that sperm midpiece length may have an important influence on some sperm motion parameters and, with the use of a computer assisted sperm motility analyzer (CASA) (Amann & Katz, 2004), a relationship between sperm midpiece length and detailed sperm motion parameters such as beat frequency, amplitude lateral head displacement, and velocity, may yet be determined.

In conclusion, this study showed that, although variation in sperm midpiece length and total tail length occurs in the hopping mouse, variation in the sperm midpiece length could not be shown to relate to differences in sperm motility. Whether this is due to the inadequacy of the technique, or the absence of differences in motility across spermatozoa with different sperm midpiece and total tail lengths, remains to be determined. With the use of computer assisted sperm analysis, detailed sperm motion parameters of individual spermatozoa could be determined and a relationship between sperm midpiece, total tail length and aspects of sperm motility could be examined in more detail. It is possible that differences in the length of the midpiece may relate to the arrangement, size and/ or number mitochondria. A more detailed study on the structural differences of the midpiece within this species is required.

References Ainsworth, C., Nixon, B., & Aitken, R.J. (2005). Development of a novel electrophoretic system for the isolation of human spermatozoa. Human Reproduction 20, 2261-2270.

Amann, R.P., & Katz, D.F. (2004). Reflections on CASA after 25 years. Journal of Andrology 25, 317-325.

Biljan, M.M., Taylor, C.T., Manasse, P.R., Joughin, E.C., Kingsland, C.R., & Lewis- Jones, D. (1994). Evaluation of different sperm function tests as screening methods for male fertilization potential - the value of the sperm migration test. Fertility and Sterility 62, 591-598.

Bradley, M.P., Geelan, A., Leitch, V., & Goldberg, E. (1996). Cloning, sequencing, and charcterization of LDH-C4 from a fox testis cDNA library. Molecular Reproduction and Development 44, 452-459.

Breed, W.G., Bauer, M., Wade, R., Thitipramote, N., Suwajarat, J., & Yelland, L. (2007). Intra-individual variation in sperm tail length in murine rodents. Journal of Zoology 272, 299-304.

Brokaw, C.J. (1972). Flagellar movement: a sliding filament model. Science 178, 455-462.

Brokaw, C.J. (1987). Regulation of sperm flagellar motility by calcium and cAMP- dependent phosphorylation. Journal of Cellular Biochemistry 35, 175-184.

Brokaw, C.J. (1989). Direct measurements of sliding between outer doublet microtubules in swimming sperm flagella. Science 243, 1593-1596.

Bunch, D.O., Welch, J.E., Magyar, P.L., Eddy, E.M., & O'Brien, D.A. (1998). Glyceraldehyde 3-phosphate dehydrogenase-S protein distribution during mouse spermatogenesis. Biology of Reproduction 58, 834-841.

Cardullo, R.A., & Baltz, J.M. (1991). Metabolic regulation in mammalian sperm: mitochondrial volume determines sperm length and flagellar beat frequency. Cell Motility and the Cytoskeleton 19, 180-188.

Carr, D.W., & Acott, T.S. (1984). Inhibition of bovine spermatozoa by caudal epididymal fluid: 1. Studies of a sperm motility quiescence factor. Biology of Reproduction 30, 913-925.

Carrera, A., Gerton, G.L., & Moss, S.B. (1994). The major fibrous sheath polypeptide of mouse sperm: structural and functional similarities to the A-kinase anchoring proteins. Developmental Biology 165, 272-284.

Cummins, J.M., & Woodall, P.F. (1985). On mammalian sperm dimensions. Journal of Reproduction and Fertility 75, 153-175.

Dominguez, L.A., Burgos, M.H., & Fornes, M.W. (1999). Morphometrical comparison of human spermatozoa obtained from semen and swim-up methodology. Andrologia 31, 23-26.

Drevius, L.-O. (1971). The 'sperm-rise" test. Journal of Reproduction and Fertility 24, 4227-429.

Du, J., Tao, J., Kleinhans, F.W., Mazur, P., & Crister, J.K. (1994). Water volume and osmotic behaviour of mouse spermatozoa determined by electron paramagnetic resonance. Journal of Reproduction and Fertility 101, 37-42.

Eddy, E.M., & O'Brien, D.A. (1994). The spermatozoon. In 'The physiology of reproduction'. (Eds E Knobil and JD Neill). (Raven Press: New York)

Engel, S., Weber, H., Petzoldt, R., Seidl, B., Wiehe, W., & Sperl, J. (2000). An improved method of sperm selection by glass wool filtration. Andrologia 33, 223- 230.

Fraser, L.R. (1983). Potassium ions modulate expression of mouse sperm fertilizing ability, acrosome reaction and hyperactivated motility in vitro. Journal of Reproduction and Fertility 69, 539-553.

Fraser, L.R. (1986). Both taurine and albumin support mouse spermotility and fertilizing ability in vitro but there is no obligatory requiremnt for taurine. Journal of Reproduction and Fertility 77, 271-280.

Gadella, B.M., & van Gestel, R.A. (2004). Bicarbonate and its role in mamalian sperm functions. Animal Reproduction Science 82-83, 307-319.

Gage, M.J.G. (1998). Mammalian sperm morphometry. Proceedings of the Royal Society of London B Biological Sciences 265, 97-103.

Gage, M.J.G., & Freckleton, R.P. (2003). Relative testis size and sperm morphometry across mammals: no evidence for an association between sperm competition and tail length. Proceedings of the Royal Society of London B Biological Sciences 270, 625-632.

Gomendio, M., & Roldan, E.R.S. (1991). Sperm competition influences sperm size in mammals. Proceedings of the Royal Society of London B Biological Sciences 243, 181-185.

Grant, S., Long, S., & Parkinson, T. (1994). Fertilizability and structural properties of boar spermatozoa prepared by Percoll gradient centrifugation. Journal of Reproduction and Fertility 100, 477-483.

Gravance, C.G., Champion, Z.J., Sax-Gravance, S.K., & Casey, P.J. (1998). Percentage of normal sperm heads is significantly increased by Percoll separation of semen. International Journal of Andrology 21, 116-119.

Harrison, R.A., Dott, H.M., & Foster, G.C. (1978). Effect of ionic strength, serum albumin and other macromolecules on the maintenance of motility and the surface of mammalian spermatozoa in a simple medium. Journal of Reproduction and Fertility 52, 65-73.

Hinting, A., & Lunardhi, H. (2001). Better sperm selection for intracytoplasmic sperm injection with the side migration technique. Andrologia 33, 343-346.

Ho, H., & Suarez, S. (2001). Hyperactivation of mammalian spermatozoa: function and regulation. Reproduction 122, 519-526.

Holm, L., & Wishart, G.J. (1998). The effect of pH on the motility of spermatozoa from chicken, turkey and quail. Animal Reproduction Science 54, 45-54.

Jakab, A., Kovacs, T., Zavaczki, Z., Borsos, A., Bray-Ward, P., Ward, D., & Huszar, G. (2003). Efficacy of the swim-up method in eliminating sperm with diminished maturity and aneuploidy. Human Reproduction 18, 1481-1488.

Le Lannou, D., & Blanchard, Y. (1988). Nuclear maturity and morphology of human spermatozoa selected by Percoll density gradient centrifugation or swim-up procedure. Journal of Reproduction and Fertility 84, 551-6.

Mikki, K., Qu, W., Goulding, E.H., Willis, W.D., Bunch, D.O., Strader, L.F., Perreault, S.D., Eddy, E.M., & O'Brien, D.A. (2004). Glyceraldehyde 3-phosphate dehydrogenase -S, a sperm-specific glycolytic enzyme, is required for sperm motility and male fertility. Proceedings of the Academy of Natural Sciences of USA 101, 16501-16506.

Mori, C., Nakamura, N., Welch, J.E., Gotoh, H., Goulding, E.H., Fujioka, M., & Eddy, E.M. (1998). Mouse spermatogenic cell-specific type 1 hexokinase (mHk1-s) transcripts are expressed by alternative splicing from the mHk1 gene and the HK1- S protein is localized mainly in the sperm tail Molecular Reproduction and Development 49, 374-385.

Morrow, E.H., & Gage, M.J.G. (2001). Consistent significant variation between individual males in spermatozoal morphometry. Journal of Zoology 254, 147-153.

Mujica, A., Moreno-Rodriguez, R., Naciff, J., Neri, L., & Tash, J.S. (1991). Glucose regulation of guinea-pig sperm motility. Journal of Reproduction and Fertility 92, 75-87.

Mundy, A.J., Ryder, T.A., & Edmonds, D.K. (1995). Asthenozoospermia and the human sperm mid-piece. Human Reproduction 10, 116-119.

Narisawa, S., Hecht, N.B., Goldberg, E., Boatright, K.M., Reed, J.C., & Millan, J.L. (2002). Testis-specific cytochrome c-null mice produce funstional sperm but undergo early testicular atrophy. Molecular Cell Biology 22, 5554-5562.

Parrish, J.J., & Foote, R.H. (1987). Quantification of bovine sperm separation by a swim-up method. Relationship to sperm motility, integrity of acrosomes, sperm migration in polyacrylamide gel and fertility. Journal of Andrology 8, 259-266.

Pholpramool, C., & Chaturapanich, G. (1979). Effect of sodium and potassium concentrations and pH on the maintenance of motility of rabbit and rat epididymal spermatozoa. Journal of Reproduction and Fertility 57, 245-51.

Prakash, P., Leykin, L., Chen, Z., Toth, T., Sayegh, R., Schiff, I., & Isaacson, K. (1998). Preparation by differential gradient centrifugation is better than swim-up in selecting sperm with normal morphology (strict criteria). Fertility and Sterility 69, 722-726.

Roldan, E.R.S., Gomendio, M., & Vitullo, A.D. (1992). The evolution of eutherian spermatozoa and underlying selective forces: female selection and sperm competition. Biological Reviews 67, 551-593.

Sakkas, D., Manicardi, G.C., Tomlinson, M., Mandrioloi, M., Bizzaro, D., Bianchi, P.G., & Bianchi, U. (2000). The use of two density gradient centrifugation

techniques and the swim-up method to separate spermatozoa with chromatin and nuclear DNA anomalies. Human Reproduction 15, 1112-1116.

Soderlund, B., & Lundin, K. (2000). The use of silane-coated silica particles for densiy gradient centrifugation in in-vitro fertilization. Human Reproduction 15, 857- 860.

Storey, B.T., & Kayne, F.J. (1980). Properties of pyruvate kinase and flagellar ATPase in rabbit spermatozoa: relation to metabolic strategy of the sperm cell. Journal of Experimental Zoology 211, 361-367.

Suarez, S.S., Katz, D.F., Owen, D.H., Andrew, J.B., & Powell, R.L. (1991). Evidence for the function of hyperactivated motility in sperm. Biology of Reproduction 44, 375-381.

Tash, J.S. (1989). Protein phosphorylation: the second messenger signal transducer of flagellar motility. Cell Motility and the Cytoskeleton 14, 332-339.

Turner, R.G. (2003). Tales from the tail: What do we really know about sperm motility? Journal of Andrology 24, 790-800.

Turner, R.M. (2006). Moving to the beat: a review of mammalian sperm motility regulation. Reproduction, Fertility and Development 18, 25-38.

Usselman, M.C., & Cone, R.A. (1983). Rat sperm are mechanically immobilized in the caudal epididymis by "immobilin", a high molecular weight glycoprotein. Biology of Reproduction 29, 1241-1253.

Ward, P.I. (1998). Intraspecific variation in sperm size characters. Heredity 80, 655-659.

Westhoff, D., & Kamp, G. (1997). Glyceraldehyde 3-phosphate dehydrogenase is bound to the fibrous sheath of mammalian spermatozoa. Journal of Cell Science 110, 1821-1829.

Younglai, E.V., Holt, D., Brown, P., Jurisicova, A., & Casper, R.F. (2001). Sperm swim-up techniques and DNA fragmentation. Human Reproduction 16, 1950-1953.

6.5 Publication 4

Comparative study of sperm chromatin condensation in the excurrent ducts of the laboratory mouse Mus musculus and spinifex hopping mouse Notomys alexis

M. Bauer, C. Leigh, E. Peirce and W.G. Breed

Reproduction, Fertility and Development, 2005, 17, 611-616

The spinifex hopping mouse has in addition to unusually small testes and pleiomorphic sperm populations, a highly divergent extra-testicular duct system. Unlike in other species of murid rodent, the epididymis is relatively short and uncoiled, with only low numbers of spermatozoa being stored in the cauda region (Breed, 1981; Peirce & Breed, 1989). In addition to this, sperm transit along the epididymis is relatively fast and similar numbers of spermatozoa are stored in the vas deferens to those in the cauda epididymidis (Peirce et al., 2003).

During epididymal transit from the caput to the cauda regions, spermatozoa undergo numerous morphological and biochemical changes that enable them to develop the potential to fertilise eggs (Glover & Nicander, 1971). In most species, these maturational changes are complete by the time the spermatozoa reach the cauda epididymidis, where they are stored. No previous studies have, however, investigated the impact that the rapid epididymal sperm transit has on the location in the extra-testicular duct system where sperm maturation is completed.

The aim of this publication was therefore to determine the impact that fast epididymal transit and sperm storage in the vas deferens has on where, in the excurrent ducts, spermatozoa chromatin condensation is completed.

Bauer, M., Leigh, C., Peirce, E. and Breed, W.G. (2005) Comparative study of sperm chromatin condensation in the excurrent ducts of the laboratory mouse Mus musculus and spinifex hopping mouse Notomys alexis. Reproduction, Fertility and Development, v.17 (6) pp. 611-616, 2005

NOTE: This publication is included on pages 95-101 in the print copy of the thesis held in the University of Adelaide Library.

It is also available online to authorised users at:

http://dx.doi.org/10.1071/RD05027

7 General Discussion The spinifex hopping mouse is a species of Australian Old Endemic rodent that occurs in the arid zone, yet it is one of the few species that breeds fairly readily in captivity. Results from studies on captive-bred hopping mice have shown that these animals have a highly unusual reproductive anatomy. This includes very small testes (Breed, 1982; Breed & Taylor, 2000), production of pleiomorphic sperm populations (Breed & Sarafis, 1983; Suttle et al., 1988) and an unusual arrangement of accessory sex glands (Breed, 1981, 1982). The general aim of this thesis was to extend these studies and to explore several aspects of the reproductive anatomy of animals occurring in the natural environment and to determine the implications of this divergent reproductive anatomy on aspects of extra-testicular sperm maturation and fertility.

7.1 Testis size

Amongst eutherian mammals, the absolute and relative testis size varies considerably between species. Although there is an allometric relationship of 0.72 between testis and body mass (Kenagy & Trombulak, 1986), there are, however, a few species where the testis weight is notably different from that which is predicted from the body mass. Inter-specific differences in testis size have also been found to be associated with sperm production rates, quality, and number of sperm stored, with species with a larger relative testis size generally producing higher quality ejaculates than those with smaller testes (Møller, 1988, 1989).

While studying the testes of great apes, Short (1979) hypothesized that variation in relative testis size relates to different levels of inter-male sperm competition in the female tract after mating and hence mating system. This suggestion was further supported by data from other primate species (Harcourt et al., 1981; Harvey & Harcourt, 1984; Harcourt et al., 1995), as well as a few other groups of mammals (Kenagy & Trombulak, 1986). In general, species with a large relative testes size have a promiscuous or polygynous mating system where high levels of inter-male sperm competition are likely to occur due to two or more males mating with the same female at the one oestrus. In this situation it would be advantageous to produce and inseminate as many spermatozoa as possible as, with all other things

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being equal, the more spermatozoa inseminated the more likely the male would be to fertilise the oocytes and thus pass his genes on to the next generation. Alternately, males of species with a monogamous mating system, or one where a single male mates the female, are unlikely to exhibit sperm competition in the female tract thus the selective pressures for maximising sperm production is lower, as they only need to inseminate enough spermatozoa for fertilization to occur.

For the murid rodents, an allometric relationship between the testis size and body mass has been found to be about 0.9. Amongst these species testis size as a percentage of body mass ranges from 5% in the chestnut mouse, to 0.1% in several species of hopping mice in the genus Notomys, including the spinifex hopping mouse, N. alexis (Breed, 1982; Breed & Taylor, 2000). Studies on captive- bred hopping mice found that, in addition to the testes being unusually small, there is a relatively low volume of seminiferous tubules to interstitial tissue, as well as a long cycle of seminiferous epithelium (Peirce & Breed, 1990). This results in comparatively low numbers of spermatozoa being produced per gram of testicular tissue (Kenagy & Trombulak, 1986; Breed, 1997b; Peirce & Breed, 2001). The small testes, together with the finding that when there were two or more males cohabiting with the one female, and all of the pups in the one litter were sired by the same father (Breed & Adams, 1992), suggests that a monogamous mating system may occur in this species. However, behavioral observations of these animals suggest otherwise. For instance, when groups of adult male and female hopping mice were placed in the same cage, they were generally found to huddle together within the same nest (Happold, 1976). It was also found that more than one male would sometimes mount the same female during the one oestrous period (Breed & Washington, 1991), thus suggesting a mating system where the multiple mating of an oestrus female may occur, at least under these social situations. As a result of this apparent discrepancy between the social organization in the laboratory, the breeding system implied from testes weight and the paternity of young in several litters, investigations into the testis size and sperm production of these animals in naturally occurring populations, were thus clearly required.

The first question that was investigated in this thesis was: do males in reproductively active, as well as inactive, populations in the natural environment

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invariably have relatively small testes? The results showed that male hopping mice, regardless of where they came from, or the reproductive activity of the females in the population from which they were collected, invariably had testes of around 0.1% of their body mass. This finding showed that the small testes found in the captive-bred animals were not due to the laboratory environment, nor due to inbreeding, but were a characteristic feature of this species. Furthermore, it shows that, unlike in many rodents where the testis size of males in non-breeding populations is much less than that in populations when reproduction is taking place (Kerr et al., 1980; Irby et al., 1984; Khammar & Brudieux, 1987; White et al., 1996; Muteka et al., 2006a, b), testis size and sperm production in the hopping mouse does not change markedly with changes in the reproductive activity of the population. Why such small testes have evolved in the spinifex hopping mouse is not clear, but these findings support the prediction that this species has a monogamous mating system or at least one where there are low levels of inter- male sperm competition.

An additional factor that may have a bearing on the small testis size of these animals is the environment in which they live. Hopping mice occur in an unpredictable, and often very harsh, environment of the arid region of central and western Australia. Although reproduction in this species has been found to take place at any time of the year, it largely occurs in spring and early summer, and appears to be suppressed when there is a high density of animals within the population (Breed, 1992; Trengove, 2005), regardless of the levels of rainfall that has recently taken place (Finlayson, 1940; Breed, 1979; Dickman et al., 1999). The minute relative testis size in these animals suggests that males allocate relatively low amounts of energy to sperm production. By continuously producing spermatozoa, even when it is likely that all females in the population are in anoestrus, the male is potentially ready to mate with females as soon as they emerge from their reproductively inhibited state.

7.2 Sperm pleiomorphism

Comparative studies have shown that, in spite of spermatozoa from all species of eutherian mammals having the same general morphology, considerable inter-

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specific differences in sperm head shape, and tail length, have evolved (see Retzius, 1909; Bishop & Austin, 1957; Bishop & Walton, 1960; Fawcett, 1970, 1975; Bedford & Hoskins, 1990). However, within any one species, sperm dimensions are generally highly consistent and the sperm head shape and tail length are often quoted as being ‘species specific’ (Cummins & Woodall, 1985; Roldan et al., 1992; Gage, 1998). In a comparative review by Morrow and Gage (2001) it was suggested however that some inter-individual variation in sperm length may occur within a few species.

There are however, a few species in which intra-individual variation in the shape and size of spermatozoa has been reported to occur. In such cases sperm pleiomorphism is generally restricted to either a few individuals or isolated populations of animals, and has been associated with structural abnormalities of the sperm nucleus, acrosome or tail. These are generally pathological in nature and their presence is often associated with either disease, high scrotal temperature (Mieusset & Bujan, 1995; Thonneau et al., 1998; Mortimer & Menkveld, 2001), old age (Beatty & Mukherjee, 1963; Krzanowska, 1981; Kidd et al., 2001; Vilakazi & Webb, 2004), hyper-adrenalism (Wildt et al., 1986), or genetic inbreeding (Beatty & Sharma, 1960; Illisson, 1969; Krzanowska, 1976; O'Brien et al., 1983; Wildt et al., 1983; Wildt et al., 1986; O'Brien et al., 1987; Barone et al., 1994; Krzanowska et al., 1995b; Gomendio et al., 2000; Crosier et al., 2007), with the consequence of reduced fertility in these individual males.

Amongst the Australian Old Endemic rodents there is a large amount of inter- specific diversity in sperm morphology. While most species belonging to this group have a sperm head that has, in addition to an apical hook, two ventral processes that extend from the upper concave surface (Flaherty & Breed, 1983; Flaherty, 1987; Flaherty & Breed, 1987), there are several species in which a divergent sperm head shape has evolved. These include the New Holland mouse, and the Pilliga mouse, where the sperm head has a single very truncated hook, the heath rat, where the sperm head is spatulate-shaped, and the delicate mouse, where it is pear-shaped (Breed, 1983; Breed & Sarafis, 1983; Breed, 1997a).

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The spinifex hopping mouse differs from all of these species in that both captive- bred, and wild-caught animals, regardless of their age and where they were caught, invariably produce spermatozoa with variation in head shape, as well as midpiece and total flagellar length. Further, sperm pleiomorphism in the hopping mouse does not appear to be associated with high numbers of obviously pathological changes to the sperm or poor fertility, with continued successful reproduction occurring in the captive colony for over two decades.

While it has been found that sperm pleiomorphism in several eutherian species is associated with low genetic diversity due to inbreeding as a result of population bottlenecks (O'Brien et al., 1983; O'Brien et al., 1987; Crosier et al., 2007), in the hopping mouse sperm pleiomorphism has been found to be present in all of the wild-caught individuals investigated regardless of where they were obtained. Furthermore, hopping mice have a similar heterozyosity index to the plains mouse (Adams & Breed, unpublished observations), which has monomorphic sperm populations, thus genetic inbreeding seems to be an unlikely cause of sperm pleiomorphism in the hopping mouse. Further, unlike in humans as well as some inbred strains of mice and bulls, where levels of pleiomorphism are associated with age (Beatty & Mukherjee, 1963; Krzanowska, 1981; Kidd et al., 2001; Vilakazi & Webb, 2004), in adult male hopping mice, sperm pleiomorphism occurs in all individuals regardless of the age of the animal.

The factors responsible for controlling the formation of the ‘species specific’ sperm head shape during spermiogenesis have been extensively studied, but still remain to be conclusively determined. Many investigations have focused on the association between sperm morphology and genotype. The influence of the genotype on sperm head shape and size was suggested in the early work of Beatty & Sharma (1960), and Wyrobek et al. (1975) subsequent investigations have found that several genes on the Y chromosome, as well as autosomes, may play a role in determining the species-specific sperm head size and shape (see Styrna et al., 1991a; Styrna et al., 1991b; Burgoyne et al., 1992).

In the spinifex hopping mouse, it has been found that the Y chromosome, X chromosome and autosome 1 are all polymorphic between individuals (Baverstock

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et al., 1977a; Baverstock et al., 1977b). Whether genetic recombination during the formation of the polymorphic chromosomes has resulted in the loss of specific genes that are responsible for controlling sperm head shaping and tail length in this species remains to be determined. Polymorphic X- and Y- chromosomes have also been found to be present in the closely related dusky hopping mouse, and the fawn hopping mouse (Baverstock et al., 1977a; Baverstock et al., 1977b), but the latter species was found to produce monomorphic sperm populations (see publication 3). Furthermore, Y chromosome polymorphism has also been reported to occur in other rodent species that have consistent sperm morphology, including the asian house shrew (Yong, 1974; Bedford et al., 1994) and species of deer mouse (Bradshaw & Hsu, 1972). This therefore is an area of study that clearly needs to be investigated further.

7.3 Implications of sperm pleiomorphism on fertilisation

In several mammalian species, including humans, rabbits, and laboratory mice, the uterotubal junction acts as a selective barrier against sperm with gross abnormalities of either the sperm head and/or tail, with variants present in the isthmus of the oviduct generally being of ‘normal’ morphology (Krzanowska, 1974; Mortimer et al., 1982; Menkveld et al., 1990). However, in humans (Liu & Baker, 1992) and several inbred strains of mice (Krzanowska et al., 1995b), slight variations of sperm morphology are able to pass through the uterotubal junction, yet not all of these morphotypes are able to bind to the zona pellucida and, of those that do, only some are likely to be able to bind to, and fertilise, with the oocyte cell membrane.

In the hopping mouse, the implications of variation in sperm head shape and tail length on the ability of the spermatozoa to fertilise the oocytes is not clear. An earlier study on the hopping mouse showed that there does not appear to be selection of any of the morphotypes at the uterotubal junction, with the range of sperm types occurring in the male reproductive tract also being found to be present in the upper reaches of the female reproductive tract after mating (Breed & Washington, 1991). Further, this thesis found there were no marked differences in the motility or DNA damage of cauda epididymal spermatozoa of different morphotypes. Whether there are differences in the distribution and/or abundance 107

of molecules on the plasma membrane of spermatozoa with different head shapes that may affect the sperm’s ability to bind to the zona pellucida, or fuse with the oolemma, remains unknown.

There is some evidence to suggest that, when comparisons are made across mammalian species, sperm tail length is positively associated with swimming speed (Gomendio & Roldan, 1991). However, one study has suggested that swimming speed depends more on the volume of the mitochondria in the midpiece than its length (Cardullo & Baltz, 1991). This thesis has shown that hopping mice display variation in the length of both the sperm midpiece and total flagellum within and between individuals. In an attempt to determine whether the variation in midpiece length related to sperm motility, ‘swim-up’ was used to separate the spermatozoa into populations with different motility. When the morphology of these different populations were compared, no correlation between sperm midpiece length, head shape and/or sperm motility, was detected. However, this study used a rather crude measurement of sperm velocity and, with the use of more sophisticated technology that measures morphological parameters of spermatozoa and swimming patterns simultaneously, it may be possible to demonstrate a relationship between sperm tail length and motility in individuals of this species.

Whether spermatozoa of particular morphotypes have an alternate function to that of fertilizing oocytes, has not been established. Baker and Bellis (1988), proposed a ‘kamikazee theory’ whereby they suggest that defective spermatozoa provide a barrier to prevent spermatozoa from any other male that mates with the female at oestrus from reaching the upper reaches of the female tract, and thus preventing them from fertilising the oocytes. However, this theory has been disputed by Harcourt (1991), who suggested that individuals cannot afford to produce nonfertilizing spermatozoa and that glandular secretions that form a copulatory plug in the cervical region, provide a more effective barrier to minimising the spermatozoa inseminated by a subsequent male from reaching the oocyte. This also appears to be unlikely in the hopping mouse, as these animals not only produce few spermatozoa but are likely to have a monogamous mating system with the consequence that there would be minimal, if any, inter-male sperm competition.

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7.4 Extratesticular sperm maturation and storage

Once spermatozoa have left the testis they pass along the ductuli efferentes and then enter the caput of the epididymis, before passing through to the cauda region, where sperm storage takes place. For most mammalian species, spermatozoa take between 6 to 14 days to pass along the entire length of the epididymis, with the time that spermatozoa spend in the cauda epididymidis being a function of both daily sperm production, the capacity of this region for storing spermatozoa and ejaculatory frequency (Amann et al., 1976; Dadoune & Alfonsi, 1984). During epididymal transit from the caput to the cauda region, spermatozoa undergo morphological and biochemical changes that enable them to bind to, and fertlise, the oocytes (Bedford et al., 1973b; Bedford, 1975; Orgebin-Crist et al., 1975; Orgebin-Crist & Fournier-Delpech, 1982; Moore & Akhondi, 1996; Gatti et al., 2004).

There is much debate over whether the cooler environment of the scrotum first evolved to facilitate sperm production in the testes, or sperm storage in the cauda of the epididymis (Bedford, 1978a, 1991; Jones, 1998b, 1999). Whichever of these is correct, it is clear that in scrotal mammals the reduced temperature of the cauda epididymides, facilitates sperm storage by increasing oxygen availability (Djakiew & Cardullo, 1986), and membrane stability (Moore, 1996, 1998), while maintaining the spermatozoa in a viable state for a prolonged period of time. Nevertheless, there are a few mammalian species including the shrew and stallion, where sperm storage also occurs in a distal expansion of the vas deferens called an ampulla (Gebauer et al., 1974; Bedford et al., 1994). This differs from most mammals where the vas deferens is simply a muscular duct that functions primarily in propelling spermatozoa from the cauda epididymidis to the urethra during copulation (Hamilton, 1975; Hoffer, 1976).

In the hopping mouse, epididymal sperm transit is relatively fast, with spermatozoa taking less than a day to pass from the caput and corpus regions, and up to 3 days to pass through the cauda epididymidis (Peirce & Breed, 1989, 2001). Previously it has been shown that the spinifex hopping mouse stores spermatozoa in both the cauda of the epididymis and in the distal region of the vas deferens (Peirce et al., 2003). There is however a lack of data on the maturity and fertilizing ability of the 109

spermatozoa stored in these two locations. In light of this, the maturity of sperm populations in the cauda of the epididymis was compared to that of the vas deferens. For this, the condensation of the chromatin in the sperm nucleus was determined by staining the spermatozoa with the fluorescent dyes monobromobimane and acridine orange. While it was found that the cauda epididymides contained a mixture of spermatozoa with different degrees of chromatin condensation the distal region of the vas deferens had a homogenous population of maximally condensed sperm nuclei. This suggests that in the hopping mouse, unlike most mammalian species (Calvin & Bedford, 1971; Bedford & Calvin, 1974; Balhorn et al., 1984; Huang et al., 1984; Shalgi et al., 1989; Yossefi et al., 1994; Seligman et al., 2004), sperm chromatin condensation is not invariably completed by the time the spermatozoa reach the cauda of the epididymis, but continues to occur both in this region as well as in the proximal region of the vas deferens. Although, it is not known if other aspects of sperm maturation occur in the proximal region of the vas deferens, the presence of secretory and endocytotic cells in the epithelial lining of this region of the vas deferens (Peirce et al., 2003), suggest that secretion of molecules into the duct may occur and that these may facilitate further sperm maturation in this region before storage occurs in the more distal region of the duct.

Why accessory sperm storage has evolved to occur in the distal region of the vas deferens is an enigma. In the hopping mouse the cauda epididymides occur in a cremaster sac, which has a temperature a few degrees cooler than that of the distal vas deferens, which is located in the lower abdominal region at core body temperature (Breed & Setchell, 2000). The spermatozoa collected from the vas deferens however, did not show signs of DNA damage suggesting that the increased temperature does not have a detrimental effect on the integrity of the chromatin in the sperm nuclei. Furthermore, storage in the vas deferens at core body temperature did not appear to impair sperm motility either (Peirce et al., 2003). Sperm storage in the vas deferens may therefore allow for a homogenous subpopulation of extra-testicular spermatozoa that are maximally fertile and ready to be ejaculated at the time of mating. Alternately, the continuation of chromatin condensation further along the epididymis may provide the spermatozoa in the vas deferens with increased stability that enables the spermatozoa to retain viability in

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the vas deferens at greater temperatures for a longer period of time. Further, thiol oxidation may promote sperm tyrosine phosphorylation to occur which may in turn reduce the time for capacitation to take place in the female tract (Visconti et al., 1995; Seligman et al., 2004).

7.5 Fertility potential

As a consequence of the small testes and low numbers of spermatozoa produced per gram of testis in the hopping mouse (Peirce & Breed, 2001), there are comparatively low numbers of sperm stored in the cauda of the epididymis and vas deferens (Peirce & Breed, 1989; Peirce et al., 2003). While the proportion of the sperm store that is depleted after a single mating is not known for this species, this thesis determined the affect that the small testes and low sperm stores have on reducing male fertility. It was found however that, in spite of the low numbers of spermatozoa being produced and stored, the rapid epididymal transit allows for males to return to maximal fertility within a couple of days after a single mating event. This presumably enables a male hopping mouse to fertilise two or more females within a few days of each other.

7.6 Future directions

While the findings from this study have extended data on testis size and sperm morphology of the spinifex hopping mouse the causes and consequences of the unusual reproductive anatomy still remain to be determined. For instance, there is still much speculation on the mating system of this species in the natural environment. Thus to extend our knowledge and understanding of the evolution of the small testes and variation in sperm morphology, future studies could use microsatellite markers to determine the mating system of this species in wild populations.

Also, the functional significance of the variation in sperm morphology on fertilizing ability, and motility in the female reproductive tract, remain to be determined. Further more detailed studies of the swimming patterns of spermatozoa that have different head shapes and tail lengths, using more sophisticated technology such as the computer assisted sperm analyser, CASA, clearly need to be undertaken. 111

Likewise, the significance of sperm storage in the vas deferens at body temperature is still not clear. Future studies should include investigating the structural and/or functional differences between the spermatozoa stored in the vas deferens and in the cauda of the epididymis, such as differences in sperm motility, the distribution and/or abundance of surface glycoproteins, and levels of tyrosine phosphorylation.

8 Conclusion This thesis has confirmed that the highly divergent reproductive anatomy of captive-bred male spinifex hopping mice also occurs in animals in the natural environment and is thus an invariable feature of this species. These divergent features include very small testes and highly pleiomorphic sperm populations. The small testes and marked sperm pleiomorphism found in the hopping mouse does not result in poor fertility with males able to successfully fertilise females every few days, at least in a captive environment. This may, in part, be due to fast epididymal sperm transit and hence the rapid replenishment of depleted sperm stores.

These findings raise the question about the significance of the ‘species specific’ sperm morphology that occurs in other species of mammals. It has been suggested that, where high levels of inter-male sperm competition occur, high quality ejaculates are present with all spermatozoa having a similar morphology. In the hopping mouse, sperm pleiomorphism as well as small relative testis size may have evolved in part due to low levels, or lack of inter-male sperm competition that is associated with a monogamous mating system, but as the details of the mating system in wild populations are still not known, this remains to be determined. The peculiar suite of reproductive characteristics of these animals may however also be an adaptation to the highly unpredictable, and sometimes harsh, environmental conditions of arid Australia.

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9 Additional Publications

Bauer, M., Leigh, C., Ricci, M., Peirce, E.J. & Breed, W.G. (2004) Evidence for continued maturation of spermatozoa after entry into the vas deferens of the spinifex hopping mouse, Notomys alexis. Proceedings of the Joint European Conference in Reproduction & Fertility. April, Ghent, Belgium.

Bauer, M., Leigh, C. & Breed, W.G. (2003) Causes and consequences of variation in the morphology of the sperm nucleus in the spinifex hopping mouse, Notomys alexis. Proceedings of the Australian Mammal Society, 49th Scientific Meeting. July, Sydney, Australia.

M. Bauer

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M. Bauer

‘Spinifex hopping mouse habitat’

Sand dune at Roxby Downs, South Australia, December 2006

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