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«Page 1 of Reproduction Advance Publication first posted on 5 November 2013 as Manuscript REP-13-0436 Singular features of fertilization and their ...»

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Page 1 of Reproduction Advance Publication first posted on 5 November 2013 as Manuscript REP-13-0436

Singular features of fertilization and their impact on the male reproductive system in eutherian mammals.

J.Michael Bedford

Department of Obstetrics and Gynecology


The Centre for Reproductive Medicine

Weill Medical College of Cornell University, New York, NY, 10021

Postal Adress:

1901 Walnut St. (12F)

Philadelphia, PA 19103


Running title: Fertilization and the male tract.

Abstract Therian (marsupial and eutherian) mammals have evolved a suite of novel reproductive features - seen variously in their gametes, the steps of fertilization, and the male reproductive tract - whose adaptive significance remains uncertain. Present evidence for the better-understood Eutheria suggests that ‘prime mover’ in their evolution has been the character of the egg coat, with other such features being adaptations to the consequences of this. I Its elastic thickness allows the zona pellucida to stretch to a variable degree yet remain around the blastocyst during much or all of its expansion prior to implantation, but its character represents an unusual challenge for spermatozoa. Novel aspects of the acrosome related to this challenge enable it to maintain a relatively prolonged binding after onset of the acrosome reaction, and the structure, shape, and behaviour of the sperm head point to physical thrust as a major element of zona penetration – with the unique configuration of gamete fusion as a sequela of this strategy. In the male, such adaptations are reflected in sperm head formation in the testis, and in sperm maturation in the epididymis involving at least the sperm head’s structure, plasmalemma, and acrosome. That complexity allied to a slow epididymal sperm transport, a relatively modest sperm production, and the brief life-span of mature spermatozoa kept above the cauda epididymidis could account for evolution of its sperm storage function – a development seemingly linked in turn to the need for sperm capacitationand evolution of the scrotum.


Among the invertebrates and vertebrates used to study fertilization, therian (marsupial and placental/eutherian) mammals have been among the most difficult in which to unravel the mechanisms involved. One problem has been the fact that their gametes and the male tract display a suite of novel features whose underlying significance remains in doubt. The thinking in early investigations of fertilization in Eutheria was based to a considerable degree on principles established in invertebrate models.

Thus, in pioneering experiments on in vitro fertilization, Thibault and Dauzier (1960) first washed rabbit oocytes to avoid supposed fertilizin/ antifertilizin issues raised by invertebrate studies. In a similar vein, fissures ahead of sperm heads penetrating the rabbit, pig and sheep zona pellucida (see Dziuk and Dickmann, 1965), were thought to be acrosomal filaments comparable to

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those in such as Arbacia and Saccoglossus. However, it is now apparent that fertilization in Eutheria is marked by a strategy of egg-penetration that diverges markedly from that in other Metazoa.

Perhaps most striking in this regard, after negotiating the egg coat eutherian spermatozoa do not fuse conventionally with the oolemma via the apex of the inner acrosomal membrane (IAM), but rather by the mid-region of the sperm head, followed by the egg’s ‘phagocytosis’ of the acrosomal domain (Yanagimachi and Noda, 1970a; Bedford, 1972). Underlying this mode, the eutherian acrosome alone displays an attenuated stable posterior region – the equatorial segment, which is unique to eutherian mammals and whose significance lies in its ability to preserve a section of fusogenic plasma membrane overlying it (Bedford, et al., 1979). An earlier puzzle was the finding that in order to fertilize, eutherian spermatozoa in the female tract must first undergo capacitation – now understood as a functional change in the sperm plasmalemma. However, there has been surprisingly little speculation as to the implication of these departures from the vertebrate norm, or about other uniquely eutherian sperm head features that include a flattened form, a keratinoid nucleus and perinuclear theca, a stable inner acrosomal membrane, and an acrosomal content characterized by an insoluble matrix that persists after the acrosome reaction (AR). Equally puzzling novelties that present in the male tract include a more complex sperm maturation process in the epididymis whose terminal region - the cauda epididymidis – perfoms a sperm storage function regulated acutely by androgen and often the low temperature of the scrotum, the latter seen only in many therian mammals and some passerine birds.

In considering the adaptive significance of these features, and why they have arisen, I cite a variety of evidence for the likelihood that they relate to each other, with the prime mover being the particular character of the egg. Very small compared that of other amniotes, and without the genetic base for yolk synthesis, the eutherian egg is invested at ovulation by the cells of the cumulus oophorus, and - key to the present discussion - an unusually robust egg coat. The discussion refers on occasion to therian mammals since marsupials exhibit a number of similar traits in this regard, and it appears that a relatively prominent if lesser egg coat has influenced sperm head design in marsupials too. However, I focus on the Eutheria because the larger therian theme is complicated by a different sperm head design in marsupials, by the fact that the zona and the sperm/egg interaction differ in important respects, and because too little is yet known in marsupials about many aspects of fertilization including capacitation.

The egg vestments The cumulus oophorus investing the zona pellucida is unique to eutherian mammals, being absent even from the comparably small eggs of marsupials; but it is hard to be sure of its significance. The post-ovulatory life of this investment of cells varies from a very few hours in some ungulates, to 24 hours in some bats and certain insectivores in which it presents as a dense mass.

Nonetheless, in keeping with its name, the cumulus exists more commonly as a cloud of cells suspended within a hyaluronic-acid matrix, and we have observed in the rat that it acts to sequester into the vicinity of the eggs the very few spermatozoa that reach the often-arborized or spacious ampulla of the Fallopian tube. However, although eggs can be fertilized readily in the absence of the cumulus in most species tested, this cell mass is the essential inducer of the AR in shrews and likely some other insectivora (Bedford, Mock and Goodman, 2004). Moreover, spermatozoa often react within the cumulus also in laboratory mammals (e.g.

hamster, rabbit), and in mice at least such spermatozoa later penetrate the zona, and fertilize (Jin et al., 2011).

From a comparative perspective, the eutherian zona pellucida is unusually formidable in a physical sense. Yet, notwithstanding its robust character, the eutherian zona has generally been viewed simply as a rather prominent egg coat, and the detection of acrosin in the rabbit acrosome in the late 1960’s solidified the expectation that sperm penetrate it in part by means of acrosomal lysins. However, the eutherian zona and the mode of its penetration are anything but conventional. Approximately an order of magnitude thicker than that in most invertebrates and vertebrates, including some marsupials, its 3 or 4 constituent glycoproteins first probed by Paul Wassarman and colleagues, are linked to a degree by -S-S- bonds. Moreover, not only does it display an elastic rigidity (Green, 1997), it proves to be relatively insensitive to proteases. We have observed that the marsupial zona disappears in some 2-8 seconds on exposure to 1.0-0.5% trypsin at pH 7.2, whereas that of most eutherians shows no visible change for 2 minutes or more..

As discussed below, the overall picture points to the physical character of the eutherian zona pellucida as the driver of special strategies for fertilization, which are reflected in the behavious and design of the spermatozoon.

Zona binding.

Throughout the animal kingdom, sperm binding to the egg coat, generally brief, is often effected by linking single proteins in the acrosome to complementary molecules on the egg. However, the key factors involved in eutherian mammals have proven difficult to pin down. Electron microscope images in such as hamsters and rabbits (Barros et al., 1967; Yanagimachi and Noda, Page 3 of 13 1970b, Bedford, 1972; Yanagimachi and Phillips, 1984), man, and also domestic animals (e.g. Szollosi and Hunter, 1978), demonstrate that binding is normally mediated by the fenestrated carapace of the reacted acrosome and probably involves not only a proteoglycan-related sperm surface receptor but also elements of the acrosome matrix (Jansen, et al., 1995). However, it has been shown that rabbit and mouse perivitelline spermatozoa free of the reacted carapace can penetrate the zona (Kuzan et al., 1985; Inoue et al, 2011), and that there are natural variants in regard to the hamster/rabbit pattern. For example, spermatozoa of the shrews studied lose the acrosome well before zona binding (Bedford et al., 2004), and those of the guinea pig do so before they can bind to and penetrate the zona (Huang et al., 1981). In addition, although studies in the mouse describe its AR as involving the point membrane fusions that eventually translate to a vesiculated appearance (Thompson et al., 1974; Anderson et al., 1975), it is hard to find visual evidence of this or that this transient membrane complex acts as the link in zona binding.

Indeed, as stated for the mouse by Zamboni (1971) “at the time of the earliest contact between gametes, the fertilizing spermatozoon lacks the acrosome.” If spermatozoa of the mouse do indeed finally bind to the zona rather by way of the inner acrosome membrane (IAM), this has wider implications. It may explain the difficulty using fertilization as the endpoint in this model species, to identify an essential receptor on the sperm plasmalemma (see Shur, 2008) or among candidates (e.g. proacrosin, zonadhesin, P56, arylsulfatase A) in the acrosomal matrix (see Tardif et al., 2010; Avelia et al., 2013).

Regardless of these last examples, it would appear that the physiological norm in most eutherian mammals examined thus far is represented by the hamster/rabbit mode of zona binding. However, in first describing the AR (Barros et al., 1967) it did not strike us that, in contrast to many metazoa, the vesiculated eutherian acrosome is unusually persistent, reflecting a stability that may depend in part on elements of the insoluble matrix. That stability allows the hyperactivated spermatozoon the maintain a juxtaposition with the zona until it begins to penetrate, and as such would seem to represent a feature that is adaptive for the unusually physical challenge presented by the eutherian zona.

Sperm head design, zona penetration and gamete fusion.

There is yet no consensus as to the means by which fertilizing spermatozoa cleave a path through the eutherian zona pellucida.

In most metazoa, penetration of the egg coat involves either its rapid focal lysis by sperm head enzymes, or less often the dissociative non-enzymatic action of an acrosomal component – as seen for example in the abalone (Lewis et al. 1982). Initially, the demonstration by Srivastava et al. (1965) that an acrosome extract could eventually destabilize the rabbit zona seemed to support the expectation that lysis is a key to sperm penetration of the eutherian egg coat as well. However, much of the evidence emerging since then undermines that view.

In addition to the zona’s relative insensitivity to proteases, noted earlier, and the reports that acrosome-free spermatozoa released from the perivitelline space can fertilize, a litany of current evidence calls into question the common assumption that one or more acrosomal lysins faciliate penetration of the eutherian zona. First, protease inhibitors that prevent fertilization in vitro, also prevent completion of the AR (Meizel, 1984; llanos et al., 1993). Moreover, as seen in the transmission electron microscope (TEM), not only does the soluble content of the acrosome disperse before any inroad into the zona, its release does not induce local erosion of the zona surface, and spermatozoa lacking an acrosome no longer display any ability to digest a gelatin substrate.

Consistent with this, and despite recent claims to the contrary, the molecular structure of acrosin, the first candidate lysin, does not permit it to bind to the IAM (Klemm et al., 1991), and it has been shown that acrosin does not remain on the IAM after the acrosome reaction, nor during penetration of the zona pellucida (Kopecny and Flechon, 1987). Fourth, mouse spermatozoa null for the acrosin gene can penetrate the zona (Baba et al., 1994), though more slowly as a probable result of delayed acrosomal dehiscence. Finally, sperm penetration of the rabbit zona was in no way hindered by a ca. 10-fold WGA-mediated increase in its resistance to trypsin (Bedford and Cross, 1978), nor by the increase brought by fertilization itself (Overstreet and Bedford, 1974).

Linked to this as a seeming paradox for a scenario of zona lysis, the environment of the Fallopian tube effects a major increase in the resistance of the zona to pronase (Mondejar et al., 2013).

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relatively thin protease-sensitive zona, by a sperm head with a circular profile, and by a large bulbous acrosome that lacks an equatorial segment (Dorman et al., 2013) – features consistent with a lytic mode of zona penetration. Interestingly, with its relatively broad profile and the relatively low cysteine content of its protamine and no perforatorium, the human sperm head is among the least well-adapted in regard to physical thrust – a design perhaps linked to the finding by Mondejar et al (2013) that, as an exception, the human zona is not ‘hardened’ by the tubal environment.

To summarize, the broad picture suggests that in response to a robust zona, the eutherian sperm head has evolved features that permit a necessarily prolonged binding to, and then penetration of the zona by a mode in which physical factors may dominate.

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