«Page 1 of Reproduction Advance Publication first posted on 5 November 2013 as Manuscript REP-13-0436 Singular features of fertilization and their ...»
But why then has the minute eutherian egg evolved such a prominent coat apparently so problematic for the fertilizing spermatozoon ? Composed of 3 or 4 interlinked glycoproteins, the zona does prevent adhesion of the embryo to the oviduct epithelium, it often constitutes the barrier to polyspermy, and later maintains an ordered cohesion of the early blastomeres until compaction takes place. However, it is in the uterine phase that its thickness and elasticity would seem to come into play. In contrast to the zona’s early demise in marsupials (Selwood, 2000), these characteristics of the eutherian zona allow it to persist to a species-variable degree as a progressively thinning coat able to stretch and persist as the blastocyst expands. While it is often shed only shortly before implantation, in such as cattle, sheep, horses and pigs its eventual shedding is followed by further blastocyst growth.
Consequences for the male reproductive tract.
A variety of evidence suggests that adaptation of spermatozoa for fertilization in the eutherian mode has influenced events in the male concerned with sperm formation and maturation, and indirectly with sperm storage and hence sperm capacitation and scrotal evolution as well.
Sperm maturation : Although limited yet to studies in only a few sub-therian vertebrates, the present picture suggests that the adoption of internal fertilization selected for several sperm features related to issues faced the female tract. These features include utilization to a species-variable degree of both oxidative phosphorylation and glycolysis; a vermiform sperm head; a variable appearance of skeletal elements in the sperm tail, and generally maturation of the ability to fertilize only after leaving the testis. However, that maturation process appears to involve only the sperm’s capacity for progressive motility, and a modification of its surface by non-glycosylated proteins (Bedford, 1979; Depeiges and Dufaure, 1983; Esponda and Bedford;1987; Morris et al., 1987).
Compared to sub-therian vertebrates, both spermiogenesis and the epididymal phase in therian mammals appear more elaborate in ways that often can be linked to the particular challenges that their spermatozoa face at fertilization.. In the testis, with rare exceptions (Dorman et al., 2013), novel elements in eutherian sperm head morphogenesis that relate to this include formation of the acrosome’s insoluble matrix and equatorial segment, transition to a cysteine-rich protamine and perinuclear matrix, and, not least, a consistent flattening of the nucleus. The subsequent epididymal phase appears more complex than that in sub-therian vertebrates in regard to stabilization of the sperm head, reorganization of the acrosome’s content, sperm motility and remodelling of the sperm plasmalemma.
A novelty of epididymal maturation that presents as an adaptation to the challenge of penetrating the zona is the rigidity of the eutherian sperm head. Alone among higher vertebrates (Bedford and Calvin, 1974), much or all of this rigidity is established during epididymal passage by extensive -S-S- crosslinking within the cysteine-rich protamine and perinuclear theca, which serves to rigidify the sperm head.. Together with its flat shape, the resulting stiffness would appear to facilitate the sperm head’s oscillating or ‘scything’ thrust used in penetrating the thickness of the zona pellucida.
A second novelty often seen during epididymal maturation is a visible remodelling of the apex of the acrosome. Perhaps reflecting a reorganization of its content (Yoshinaga and Toshimori, 2003; Olson et al., 2004; Buffone et al., 2008), although not visible in the rat or in man, this has been observed in such as the guinea pig, chinchilla, loris, rhesus monkey, rabbit, hyrax and the white-tailed rat – Mystromys albicaudatus. As noted, the eutherian acrosome is not merely a bag of enzymes in that the fenestrated carapace usually first tethers the sperm head to the zona. The persistence of this membrane complex suggests an adaptation that not only maintains the sperm head’s contact with a resistant zona, but may position putative ancillary binding elements such as pro-acrosin and zonadhesin exposed by the acrosome reaction.. In the case of the porcine acrosome, for example, pro-acrosin undergoes a redistribution to the apical ridge (Puigmule et al., 2011), a locus that may favor its role as an element in zona binding.
In regard to motility, experiments in a handful of species involving ligation of the efferent ducts indicate that the capacity for functional motility can not develop within the eutherian testis. But, unlike the –S-S- crosslinking and the acrosomal Page 5 of 13 modifications discussed above, it not at all clear why this aspect should have come to depend on the epididymis, nor exactly what is involved. It is possible that the need to later develop hyperactivated motility on capacitation has been a complication influencing the dependence of this aspect on the epididymis. At all events, in accord with observations that increased cAMP levels parallel a reduction in certain maturing sperm tail phosphatases (Vijyarhagavan et al., 1996), permeabilization of immature hamster spermatozoa in the presence of cAMP and ATP markedly elevated their motility (Mohri and Yanagimachi, 1980),). Thus certain modifications in the sperm plasmalemma may be integral to motility maturation, though those appear to be distinct from the surface changes involved in zona binding and fertilization (Bedford, 1967).
I first observed epididymal change in the sperm surface in the electrophoretic behavior of rabbit spermatozoa, and later this principle was confirmed with visual markers such as cationized ferric colloid, lectins, and more recently immunocytochemistry. However, as noted above, only in therian mammals is there evidence in this regard for a significant involvement of glycoproteins - both their loss or neo-expression - as surface or integral components of the sperm plasmalemma in the establishment of zona binding (e.g Moore, 1981). Whilesperm surface changes during epididymal passage in therian mammals were first considered only in terms of glycoproteins that probably bear on sperm/zona binding, it is clear that they also involve changes in the plasmalemma’s sterol content that can vary according to species (Cross, 1998). However, epididymal modifications of the sperm plasmalemma impinge on more than the acquisition of motility and sperm-head binding to the zona.
For example, underpinned by a fusion factor – Izumo (Inoue et al., 2005), CRISP1 acquired by the sperm surface in the upper epididymis conditions the final state of the equatorial segment’s membrane t involved in gamete fusion (Cohen et al., 2011), Not least, epididymal change in the sperm surface has implications for more than fertilization per se. As discussed below, it also appears to be integral to the mechanisms involved in sperm storage in the cauda epididymidis, and directly linked to that, the eventual need for capacitation in the female tract.
Sperm storage in the cauda epididymidis: The therian epididymis is a bifunctional organ that not only supports maturation of spermatozoa, but also their storage in the terminal cauda. In principle, the cauda epididymidis optimizes the ability to deliver several sperm-rich ejaculates within a brief period, this function regulated by androgens and very often the low temperature of the scrotum. There appears to be no similar dedicated storage site in the reptiles, non-passerine birds and a monotreme we have studied, and in considering why this function has evolved in therian mammals, a number of factors suggest themselves. For example, unlike the mere 24h + required in the Japanese quail (Clulow and Jones, 1982), spermatozoa are wafted back and forth within a duct segment of the eutherian epididymis and their transport through it requires more than a week, regardless of great variation in its length according to species. This reduces the potential for sperm replacement after ejaculation, and as seen in comparing the rat and quail (Clulow and Jones, 1982), not only are daily sperm production rates in therian mammals relatively low (Roosen-Runge, 1977), but mature spermatozoa remain functional for no more than +/-5 days if witheld above the cauda.
The possibility that slow transport, modest production, and viability issues determined the evolution of the cauda’s storage function is supported by the picture in passerine birds that have evolved such a sperm store within a scrotum-like protuberance (Wolfson, 1954). Exemplified by the song sparrow Melospizia melodia, the testes produce a relatively low number of spermatozoa which trickle passively through a ciliated duct to complete a more complex maturation in the terminal glomus (see Bedford, 1979).
In considering the support for spermatozoa in the cauda, it seems significant that in a variety of mammals the sperm plasmlemma becomes ‘coated’ in the upper cauda by certain GPI-linked macromolecules first secreted there (Dacheux and Voglmayr, 1983; Thomas et al., 1984; Rifkin and Olson, 1985; Garcia et al., 1988; Derr et al., 2001). However, although such as “HIS protein” and “CD52” have been designated as ‘maturation-associated’, most spermatozoa develop the ability to fertilize before such macromolecules are acquired.. It therefore seems likely that such molecules act rather to facilitate storage of the spermatozoa and, as argued below, they may underlie or at least contribute to the need for capacitation in the female tract (see Yeung et al., 2000)
plasmalemma, capacitation would appear to constitute a necessary reversion from a stabilized state related to caudal storage to one that enables the AR and development of hyperactivated motility.
The Scrotum: Many pointers suggest that the regulated storage function of the cauda epididymidis has also been the prime mover in evolution of the scrotal state. A focus on the temperature-sensitivity of descended testes has not resolved the question as to the adaptive significance of the scrotum, which is restricted to therian mammals and, as a paracloacal protuberance, many passerine birds. In fact, the eutherian testis operates according to species in anatomical locations that range from the abdomen to the inguinal region to the pendulous scrotum, and so over a temperature range of ca. 6 C (Carrick and Setchell (1977). In a small subset (testiconda) that includes the elephant, hyrax, golden mole, tenrec, elephant shrew and some marine mammals, the testis and epididymis function at deep body temperature within the abdominal cavity. Moreover, as a secondary adaptation even fully scrotal testes have on occasion come to tolerate a high ambient temperature of the environment equivalent to that of the body (Bronson and Heideman, 1993). Moreover, the testis’ reaction to temperature is not all-or-none : in two cryptorchid models - the musk shrew and the degu - elevation of their inguinal testes to the body cavity (raising their temperature by +/- 2C) brought only a partial disruptionof spermatogenesis and continued production of some morphologically normal spermatozoa (Bedford, et al.,, 1982). Thus, the evolution of the scrotum does not reflect some fundamental incompatibility between body temperature and spermatogenesis.
It seems germane in asking ‘why a scrotum’, that this development appears only in conjunction with the cauda epididymidis as a regulated sperm storage site, i.e. in eutherian and marsupial mammals, and many passerine birds. s. In other than the testiconda, the U-configuration of the epididymis and vas deferens serves to place the cauda in the coolest location, often to protrude well beyond and at a temperature below that of the adjacent testis. This is illustrated to an extreme degree in vespertilionid bats in which an interfemoral membrane provides support for the cauda to extend far beyond the semi-inguinal testis. Equally significant, in a diverse variety of mammals whose scrotal testes are furred, the adjacent region overlying the cauda displays a circumscribed baldness (Bedford, 1978). This configuration, as well as occasional examples of discrete pigmentation over the cauda (e.g. bush buck), points to a design for preferential cooling of the cauda epididymidis. The endresult of this arrangement in such as the rat is a cauda temperature ca. 4 C below that of the adjacent scrotal testis (Brooks, 1973), a differential maintained in part in this species by an insulating fat pad between them. On the other hand, it seems significant that in no case does the scrotal arrangement favour cooling of the testis over the cauda epididymidis In considering the scrotum from this point of view, it is interesting that subjection of the scrotal epididymis to body temperature does not inhibit sperm maturation but devastates its sperm storage function. The latter effect is reflected not only in changed protein and ionic profiles of the cauda’s environment, but also in a reduction of its duct length and diameter – and so its carrying capacity. Thus, in rats with epididymides reflected to the abdomen for 3+ weeks, despite normal testes and sperm numbers in the caput we observed that repetitive ejaculation to sperm exhaustion produced only 25% of the number in an ejaculation sequence from normal rats.
While such observations make a case for linking scrotal evolution to the function of the cauda, if temperature-sensitivity of the testis has no adaptive-significance per se, why does it descend and why does the extent of this differ so widely across species?
Given the present focus, I suggest that testis descent provides a means for the cauda epididymidis to ‘piggyback’ to an inguinal or fully scrotal situation, and that the sensitivity of the germinal epithelium to body temperature in scrotal species merely reflects a secondary adaptation of its metabolic machinery to function optimally at the temperature of its location for that species (e.g.
Ewing and Schanbacher, 1970). As for the spectrum of anatomical variations, the size of the testis and character of the scrotum has probably been impacted by several factors in the course of mammalian evolution. However, in assessing a wide range of mammals, Freeman (1990) made the important correlation that internal testes tend to be larger per unit body mass, and that relative testis size generally declines according to the degree of descent, with the cauda being correspondingly more prominent.