THE GREAT

SPERM RACE

www.wellcome.ac.uk

THE GREAT SPERM RACE

The documentary The Great Sperm Race describes the tortuous journey of the sperm in their quest to fertilise the egg. From storage in the male testes to fusion with the egg, this accompanying paper summarises the latest scientific thinking about human sperm delivery and transport, which formed the basis of the programme. Of the approximately 250 million human sperm cells that enter the vagina just a few thousand are able to enter the fallopian tubes and just one will fertilise the egg. There are many obstacles that sperm must overcome to reach their destination. These include: the acidic atmosphere of the vagina, the mucus of the cervix, the narrowness of the uterotubal junction (the entrance to the cervix), the white blood cells of the immune system, which see the sperm as a pathogenic foreign invader to destroy, and what scientists think is a molecular screening process at the final checkpoint that allows only some sperm through. However, a number of mechanisms aid the process. The sperm are guided along their journey by chemical and temperature signals. There is also evidence of a sperm reservoir in the fallopian tubes, where sperm bind to the epithelial lining of the tubes, reducing the chance of fertilisation by multiple sperm. During ovulation, the sperm are hyperactivated to help them penetrate the mucus in the fallopian tubes and the outer coating of the egg. And scientists have also proposed that the sperm outer membrane fuses with the egg outer membrane to facilitate fertilisation. Sperm can vary greatly in their features and the paper discusses several theories on why this is. One hypothesises that two distinct populations of sperm exist: those that are fit enough to reach the oviduct and those that have errors, which are inevitably killed along the journey to the egg. Another, controversial, theory, suggests the existence of kamikaze sperm and a division of labour of sorts between different sperm. There are also various theories about sperm competition and what happens when sperm from different males overlap. Finally, the paper discusses evidence that sperm counts have decreased in the last 50 years, possibly due to environmental effects. This paper, written by the programmes Science Producer, Jennifer Beamish, is in the style of a review article, a type of scientific paper where the author summarises current thinking on the field. Like all scientific papers it has undergone peer review, where an anonymous panel of experts checks the paper for accuracy before it is published. Each of the reviewer comments has been left in to ensure balance and illustrate the peer review process.

ABSTRACT
In The Great Sperm Race we propose to incorporate the latest scientific thinking on human sperm delivery and transport through the female reproductive tract that occurs at coitus, from storage in the male epididymis to fusion with the oocyte plasma membrane in the oviduct and formation of a fertilised ovum. Spermatozoa undergo physical stresses during ejaculation and throughout the female tract. Of an average 2.5 x 108 human sperm cells introduced into the anterior vagina during coitus only a few thousand reach the uterotubal junction and gain entry to the fallopian tubes and only one will fertilise an oocyte. Various impediments exist that sperm must overcome as they travel through the female reproductive Reviewer: tract: the acidic pH of the vagina; the mucus micro-architecture of the I believe the paper is scientifically cervix; the narrow uterotubal junction; the presence of phagocytosing accurate. I am familiar with many of the papers cited and have checked leukocytes in the vagina, cervix and uterus and finally we discuss the a selection of the others on possibility of discrimination of spermatozoa by the oocyte.
PubMed, confirming that they are correctly cited.

Sperm may be guided to the oocyte by a combination of thermotaxis and chemotaxis. Formation of a functional reservoir through epithelial binding of sperm to the oviductal isthmus may reduce the likelihood of polyspermic fertilisation. Motility hyperactivation assists sperm in penetrating mucus in the fallopian tubes and the cumulus oophorus and acrosome exocytosis may assist penetration of the oocytes zona pellucida that precedes fusion with the oocyte plasma membrane. We also discuss the various theories of sperm heterogeneity posited to account for the remarkable pleiomorphism in sperm morphology. Cohen proposed two distinct populations of sperm, those that can reach the oviduct and those that due to meiotic errors during spermatogenesis cannot and instead succumb to leukocytic phagocytosis en route. Baker and Bellis proposed the contentious kamikaze sperm hypothesis that posits a functionally adaptive division of labour between sperm. We also discuss sperm competition theory and posit the likely in vivo scenario if more than one sperm population overlap. We conclude with a discussion of male fertility in which we examine evidence that sperm counts have decreased in the last 50 years possibly due to in utero and other environmental effects.

Reviewer: The report is basically well written and well-referenced, a comprehensive summary of sperm transport that covers most areas adequately.

the document is reasonable. But there are some important, and rather fundamental, omissions that largely debunk the unfounded concept of kamikaze sperm etc. And, as often happens, people dont realise that the process is different in humans from most animals for very simple logistic reasons.

It is worth noting that in vitro fertilisation involves few if any of the in vivo selection processes described in this document and consequently success rates are much higher. If for instance 100,000 or more sperm are introduced to an egg in vitro then fertilisation is likely and up to 90% of sperm can be successful if inserted by injection directly Reviewer: Overall the scientific accuracy of into the egg (Barratt, telephone conversation 1/2008).

INTRODUCTION

Passage of sperm through the female reproductive tract is regulated to maximise the chances that high-fertility sperm with normal morphology and vigorous motility reach the oocyte and once there have the best chance of success. In humans there is evidence that successful fertilisation can take place if ejaculation occurs 5 days before ovulation (Wilcox et al, 1995). Sperm must survive transit through a series of tough obstacles and of the millions introduced only thousands will reach the fallopian tubes, possibly only tens will encounter the oocyte and only one will ordinarily fertilise it. In discussing sperm delivery and transport this document will draw heavily from the structure of Suarez and Pacey (2006).

PRODUCTION AND MATURATION OF SPERMATOZOA IN THE MALE REPRODUCTIVE TRACT Spermatozoa are produced and matured in the male reproductive tract over a 90 day period. They are created through a process of meiotic cell division in the testes and are further matured in the epididymis where they undergo dynamic changes and acquire key proteins. They are then stored in the ampulla of the vas deferens prior to ejaculation. Because sperm are terminally differentiated cells and deprived of an active transcription and translation apparatus they must survive in the female without benefit of reparative mechanisms available to many other cells. As such they have finite resources available to them on their journey through the female reproductive system (Suarez and Pacey, 2006). Spermatozoa comprise a head, midpiece and tail. The head contains the haploid genome; the midpiece houses mitochondria spirally arranged around the first portion of the tail and responsible for oxidative phosphorylation and provide much of the energy needed for sperm motility. Coating the head is a membrane called the acrosome rich in enzymes including hyaluronidase which may be necessary to penetrate the oocytes cumulus and zona pellucida.

SITE OF SEMEN DEPOSITION Spermatozoa are delivered into the anterior vagina during coitus. Semen has been observed pooled near the cervical os forming a loose coagulated gel. Within minutes sperm begin to leave this coagulum to swim into the cervical canal (Sobrero and MacLeod, 1962). It has been proposed that semen serves to hold the sperm at the cervical os (Harper 1994) and to protect sperm from the harsh acidic environment of the vagina.

VAGINAL DEFENCES AGAINST INFECTIOUS ORGANISMS MAY AFFECT SPERM The vagina is an open tract and as such is vulnerable to infection from outside, especially at the time of coitus. As a consequence it is well equipped with antimicrobial defences including an acidic pH of 5 or under which is microbicidal to many sexually transmitted disease pathogens. The pH of semen plasma is around 7 (Roberts, 1986) and has the potential to neutralise vaginal acid. Vaginal pH was measured by radio-telemetry in a fertile human couple during coitus. The pH rose from 4.3 to 7.2 within 8s of the arrival of semen and no change was detected when the partner used a condom (Fox et al 1973). Vaginal washing of women with high levels of Reviewer: This completely overlooks all of the detectable seminal antigens had a median pH of 6.1 evidence that the ejaculate and the sperm whereas the median pH of washings lacking themselves have various means of suppressing detectable antigens was 3.7 (Bouvet et al, 1997). or disarming the female immune system (e.g. the high levels of prostaglandins in the seminal Contraceptive gel designed to maintain a low vaginal plasma). Otherwise, it would be very common pH after coitus has been shown to immobilise human place for women to become sensitised to sperm in vitro and in vivo (Amaral et al 2004). sperm and raise antibodies against them,
preventing fertilisation.

In addition to pH buffers, seminal plasma contains inhibitors of immune responses, including protective components that coat sperm (Suarez and Oliphant 1982; Dostal et al 1997).

SPERM PLEIOMORPHISM: DIFFERENT SPERM MORPHOLOGIES Sperm are present in huge quantities in any one ejaculate: up to half a billion. Why so many are produced is thought to be because of the intense selection that occurs in the female tract, with only a relatively few spermatozoa being accepted, resulting in an unusually rapid evolutionary rate among the proteins involved in achieving fertilisation. Because so many steps are required, variability and meiotic assortment among relevant reproductive genes provide limitless ways to affect sperm function. The huge numbers of spermatozoa produced by spermatogenesis represent the results of such recombinations, as though males must strive to cover every likely eventuality of the selective process (Holt and Van Look, 2004). A common conclusion that emerges from the literature is that most spermatozoa are not capable of fertilisation. However there is no a priori equivalence between sperm morphological normality and fertilising ability. There is intense discussion about what constitutes a normal spermatozoon and indeed about whether there is such a thing as normal owing to the fact that certain so-called abnormal sperm can achieve fertilisation (Mortimer and Menkveld, 2001; Holt, 2005). High fertilisation rates are obtained provided more Reviewer: than 14% of spermatozoa are classified as One point missed is that the poor morphology of most normal human normal (Kruger et al 1986; Mortimer and sperm is probably related to the different Menkveld, 2001) and in IVF the figure can be as organisation of spermatogenesis in the human low as 4%. According to Mortimer and Menkveld compared with most other mammals. There is also the lack of competition: humans are monogamous the normal sperm must have a head with a (that is, most humans only have one partner at a smooth oval configuration with a well-defined time). If a woman only mates with one person at acrosome constituting 40%-70% of the anterior a time, how can there be sperm competition? So there is little evolutionary pressure sperm head. The normal head length is between 4 on men to have high counts of and 5 m and width between 2.5 and 3.5 m. The head normal sperm. width must be between three-fifths and two-thirds the head length. But there is no absolute correlation between normal head shape and genetic quality. Direct comparisons of sperm phenotypes and chromosomal complements showed that morphologically normal spermatozoa from infertile men contained a significantly higher incidence of genetic abnormalities than normal sperm from a cohort of fertile controls (Ryu et al 2001). Classifying spermatozoa as normal seems to be more of a problem in humans than for other species like bull in which pleiomorphism is uncommon. Almost all bull spermatozoa are normal sperm. Unusual sperm head shapes in species like the bull are indeed indicative of defective spermatogenesis (Burgoyne, 1975; Russell et al 1991). Furthermore in many experiments where equal numbers of normal sperm from two or more animals have been mixed and inseminated together (Dziuk, 1996) the conception rates are usually skewed towards one of the males (Steward et al 1974; Beatty et al 1976; Robl and Dziuk, 1988; Berger, 1995; Stahlberg et al, 2000). Thus even though the number of sperm inseminated were controlled there were still functional differences between ejaculates implying that the female reproductive tract itself is capable of making the distinction between sperm from different males. How this happens is as yet unknown but Holt posits that sperm heterogeneity within each ejaculate holds the key, (Holt, 2005). Sperm DNA must be good enough to support embryonic development; its DNA delivery mechanism must be fully functional (head, midpiece and tail); its surface characteristics must not attract the attention of phagocytes within the female and sperm are so well endowed with molecular receptors (Meizel, 2004) to which they must respond appropriately that the integrity of biochemical signalling pathways within each sperm is also likely to be critical to their fertility (Holt, 2005). Many of these abilities are invisible unless sperm are analysed physiologically rather than merely morphologically. In humans it may also be likely that some so-called abnormal sperm have the fertilising ability of normal sperm despite their appearance.

THEORIES THAT ACCOUNT FOR SPERM HETEROGENEITY IN HUMANS: A precise understanding of why and how some spermatozoa are better able to reach and fertilise an oocyte under natural conditions remains an unknown. But here are a few theories that have been posited to explain why sperm heterogeneity might exist which though unproven are nevertheless worth mentioning.

COHENS TWO-POPULATION THEORY Smallcombe and Tyler (1980) and Pandya and Cohen (1985) showed in human and animal studies that the immunoglobulin factor IgG and massive leukocytosis was elicited by spermatozoa, not by mere copulation. Cohen postulated that spermatozoa emerge from the seminal coagulate up to 15 minutes after pooling to coincide with the female immune response. Spermatozoa comprise of two distinct groups: one that results from errors in the meiotic process and is coated with IgG, and a tiny minority that remain uncoated. The coated majority exit the coagulate first and are immediately targeted and phagocytosed by the circulating leukocytes; the second uncoated minority travel unchallenged (Cohen, 1990; 1998). Advances in DNA assessment have not yet provided direct support for Cohens theory though it is still described as an attractive and logical hypothesis (Holt and Van Look, 2004).

EISENBACHS THEORY OF RIPE SPERM Eisenbach proposed a population of ripe sperm to correspond with the tiny proportion of sperm that are both chemotactic and capacitating (Cohen-Dayag et al, 1995); as the ripe sperm age they are replaced and the remaining great majority of non-fertile sperm are either aged or juvenile. This theory also remains unproven. Perhaps changes in membrane lipids (Haidl and Cooper 1997) could be involved; membrane-damaged sperm can be involved in fertilisation if assisted (Ahmadi and Ng, 1997) but they may normally be discriminated against in vivo.

THE KAMIKAZE THEORY OF SPERM The most notorious theory of sperm heterogeneity is the kamikaze sperm hypothesis of Baker and Bellis (Baker and Bellis, 1998 and 1989) that proposed a functionally adaptive division of labour between sperm: egg-getters, blockers and killers. Given that sperm consist of a diminutive single-cell structure, the kamikaze theorys requirement that sperm carry a self-recognition system that must differentiate between not just different genes but different sets of genes from a rival male may be unlikely. The kamikaze theory found favour with the media and audiences and has been rapidly disseminated through Robin Bakers popular science book Sperm Wars, Infidelity, Sexual Conflict and other Bedroom Battles. A mathematical model used to examine the evolutionary conditions that might lead to the evolution and maintenance of a soldier class of sperm concluded that these conditions are not stringent. Soldier sperm may be particularly likely to evolve in contexts where there is substantial variance in the number of sperm from different males available to compete for fertilisations, due to factors such as mating order effects, between male differences in ejaculate size and copulation timing in relation to ovulation (Kura and Nakashima, 2000). Otherwise the theory remains unproven and is largely discredited.

SPERM COMPETITION THEORY The Baker and Bellis kamikaze theory was hypothesised within the context of sperm competition theory. Sperm competition occurs when spermatozoa from more than one male have the opportunity to fertilise a single female during the same fertile period (Parker, 1970; 1998). The morphological pleiomorphism of human sperm is thought by evolutionary biologists to be indicative of relatively low levels of sperm competition in our species history and prehistory. Evolutionary biologists argue that because human social systems do not generally involve the Reviewer: It is worth noting that sperm are not intensive malemale competitive matings as seen in other primate stored to any extent in humans, which is societies, there is less pressure to drive up the rate and quality of sperm completely different to many animals that are production. In species with high levels of multiple matings like bulls and promiscuous seasonal breeders (i.e. they mate with any female on heat). Thus they maintain a high sperm chimpanzees, sperm morphology tends to be relatively homogenous, count despite multiple matings every day over a period of more plentiful and of higher quality (Dixson, 2001).

weeks. This demands a sperm storage capacity otherwise sperm counts would drop with each ejaculation. Also, the Several studies have demonstrated that large testes size and hence a higher the sperm count, the greater the chance of greater sperm production capacity is a feature of species which exhibit fertilising the eggs in a female who is mating with several males successively. multi-male mating systems (Harcourt et al 1981). Comparative analyses of

testis size in humans and great apes have shown that humans tend to group with the polygynous gorilla (one malemultifemale) and have relatively small testis/ bodyweight ratios relative to the multimalemultifemale chimpanzees. Likewise gorillas and humans have similar levels of pleiomorphism compared to chimpanzees (Seuanez, 1981). Chimpanzees also have roughly twice the number of mitochondria in their sperm midpiece than humans (Bedford, 1974) which correlates with the finding that chimpanzee sperm midpiece volume is almost twice that of humans (Anderson and Dixson, 2002). Sperm competition in the chimpanzee may have resulted in the evolution of bioenergetically superior sperm in comparison to humans. However human sperm showed significantly higher mitochondrial loading than gorilla sperm which may indicate occasional sperm competition in our species. (Anderson et al, 2007). It looks unlikely that human males have experienced such high levels of sperm competition as chimps but it is unlikely that sperm competition has been completely absent over human evolutionary history (Pound et al, 2006). In modern society paternal discrepancy rates vary between 0.8 and 30% (median 3.7%) (Bellis et al, 2005) and infidelity rates vary between 30 and 50% (the Myth of Monogamy, Barash and Lipton, 2001) making it unlikely that some form of sperm competition is absent in present day society.

SPERM FROM RIVAL HUMAN MALES HOW MIGHT THEY BEHAVE IN UTERO? A review of the literature on andrology and sperm competition theory suggests that rival male sperm samples might behave neutrally with respect to each other in vivo. Our own conclusion is that sperm compete against any sperm, be they from their own group or another. Their ability to reach and fertilise the oocyte is a factor of their own design and build their ability to do everything correctly that is required en route through the female tract and critically their timing at every stage. So if all else is equal in design and build terms, the successful spermatozoon is the one that is in exactly the right place at the right time, at each stage on this extraordinary journey.

VAGINAL FLOWBACK After semen deposition in the anterior vagina there is substantial sperm loss almost immediately though how much is still not precisely known. In a 5 year study of 11 female volunteer Baker and Bellis (1993) examined the characteristics of sperm loss from the vagina following coitus (flowback). They found that flowback occurred in 94% of copulations with the median time to the emergence of flowback of 30 min. 7

Baker and Bellis estimated a median of about 65% of sperm was retained. In 12% of copulations about 100% of the sperm were eliminated which supports the notion that only a minority of sperm travel beyond the vagina. Another research group deposited 540 m albumen micrsospheres radioactively tagged with technetium into the cranial vagina of women to determine how sperm might be transported (Kunz et al, 1996). They found that spheres were only rapidly and maximally transported into the uterine cavity and beyond during peak fertility. At all other times the spheres didnt advance beyond the vagina.

X AND Y SPERM DIFFERENCES IN MOTILITY In any given ejaculate there are equal amounts of X and Y chromosome-bearing sperm (X and Y sperm). Motility differences between X and Y sperm have been reported in bull sperm (Penfold et al, 1998). When the following motion parameters were measured: curvilinear, straight-line, and average path velocity; mean angular displacement (MAD); beat cross-frequency; amplitude of lateral head displacement; linearity (LIN); and straightness of path (STR) significant differences were seen between X and Y sperm for MAD, LIN, and STR. No difference was observed for the other parameters. The results indicate that in a simple salts solution, Y bull sperm do not swim faster than X sperm but may be distinguished from X sperm on the basis of LIN and STR. Holt at the London Zoological Society hopes to replicate this work in 2008 (telephone conversation, 11/07).

SPERM TRANSPORT THROUGH THE CERVIX

THE CERVIX AS A PHYSICAL OBSTACLE Sperm enter the cervical canal rapidly where they encounter cervical mucus. Under the influence of oestrogen during ovulation the cervix secretes highly hydrated mucus, often exceeding 96% water in women (Katz et al, 1997). The extent of hydration is correlated with penetrability of sperm (Morales et al, 1993). Coitus on the day of maximal mucus hydration in women is more closely correlated with incidence of pregnancy than when timed with respect to basal body temperature indicators of ovulation (Bigelow et al, 2004). Cervical mucus presents more of a barrier to sperm of abnormal morphology and poor motility and is considered to be a means of sperm selection, supporting the passage of normal motile sperm while discouraging passage of microbes and sperm with abnormal motility (Hanson and Overstreet, 1981; Barros et al, 1984; Katz et al, 1990; 1997). Cervical mucus has also been shown in vitro to be a selective barrier against spermatozoa that though morphologically normal are carrying genetic structural abnormalities (Bianchi et al, 2004). The greatest barrier to sperm penetration of cervical mucus is at its border, because here the mucus microarchitecture is more compact (Yudin et al, 1989). Components of seminal plasma may assist sperm in penetrating the mucus border. More human sperm were found to enter cervical mucus in vitro when an inseminate was diluted 1:1 with whole seminal plasma than when it was diluted with Tyrodes medium even though the sperm swam faster in the medium (Overstreet et al, 1980).

CERVICAL DEFENCES AGAINST INFECTIOUS ORGANISMS MAY AFFECT SPERM Vaginal insemination stimulates the migration of leukocytes, particularly neutrophils and macrophages into the cervix as well as into the vagina presenting another obstacle to spermatozoa (Tyler, 1977; Pandya and Cohen, 1985; Barrett and Pockley, 1998). There is evidence from animal work (Taylor, 1982) that the immune response may be a selective one. Cohen in turn postulates that it may be a discriminatory response (Cohen, 1998). In his two-population theory those sperm without IgG coating the so-called rapid transport spermatozoa are able to avoid phagocytosis in the cervix and can get to the oviduct rapidly. Animal experiments have shown that the high-fertility sperm that make it through the cervix and uterus to the oviduct are uncoated with IgG (Cohen and Tyler, 1980) and that these high-fertility sperm are not attacked by leukocytes (Taylor, 1982). It has also been demonstrated that neutrophils will bind to human sperm and ingest them only if serum that contains both serological complement and complement-fixing anti-sperm antibodies is present (DCruz et al, 1992). Complement proteins are also present in cervical mucus (Matthur et al, 1988) along with regulators of complement activity (Jensen et al, 1995). Thus there is a potential for antibody-mediated destruction of sperm in the cervical mucus as well as leukocytic capture of sperm.

ARE SPERM STORED IN THE CERVIX? Cervical crypts are thought to entrap and store sperm (Fawcett and Raviola, 1994; Harper, 1994) and scanning electron microscopy of the human cervix indicates that mucosal grooves forming a preferential pathway for sperm could be present though a comprehensive study of the human cervix is needed to determine whether sperm follow these grooves to traverse the cervical canal. Vigorously motile sperm have been recovered from the cervix up to 5 days after insemination (Gould et al, 1984). It is not known if these sperm had re-entered the cervix from the uterus though it may be unlikely as very few sperm have been recovered from human uteri 24 h after coitus (Rubbenstein et al, 1951; Moyer et al, 1970) and those sperm were greatly outnumbered by leukocytes (Thompson et al, 1992). If some sperm are protected from phagocytosis as some of the evidence suggests they may be able to travel from a cervical reservoir to the oviduct post coitus.

SPERM TRANSPORT THROUGH THE UTERUS The human uterine cavity is a few cms in length and could be traversed in less than 10 mins by sperm swimming at about 5 mm/min, which is the swimming rate of sperm in aqueous medium (Mortimer and Swan, 1995). Experimental limitations make calculating the actual rate of passage of sperm difficult to ascertain but it is likely that transport of sperm is aided by proovarian contractions of the myometrium. Ultrasonography of the human uterus has revealed cranially directed waves of uterine smooth muscle contractions that increase in intensity during the late follicular phase (Lyons et al 1991; Kunz et al, 1996). The uterine contractions in women during the periovulatory period are limited to the layer of myometrium directly beneath the endometrium (Lyons et al, 1991; de Ziegler et al, 2001). Contractile activity of uterine muscle may draw sperm and watery midcycle mucus from the cervix into the uterus and may assist sperm movement through the uterine cavity (Fukuda and Fukuda, 1994). The volume of uterine fluid in midcycle women is only about 100 l (Casslen, 1986) and cervical mucus is plentiful enough to fill the lumen.

There is evidence that there is preferential transport to the isthmus ipsilateral to the dominant follicle than to the contralateral isthmus. Kunz et al (1996) deposited 540 m albumen micrsospheres radioactively tagged with technetium into the cranial vagina of women to determine how sperm might be transported. They found that spheres were only rapidly and maximally transported into the uterine cavity and beyond during the midcycle late follicular phase. Rapid transport of sperm through the uterus by myometrial contractions may enhance sperm survival by propelling them past immunological defences of the female. As is the case in the vagina and cervix, coitus induces a leukocytic infiltration of the uterine cavity, which reaches a peak several hours after insemination. Phagocytosis was observed several hours after insemination and therefore might be directed primarily against damaged sperm. However normal sperm may also be attacked because they have lost much of the immune protection afforded by seminal plasma constituents (Suarez and Oliphant, 1982; Dostal et al, 1997). However animal experiments have shown that leukocytes have little if any impact on those sperm that succeed in reaching the oviduct if reinseminated back into the female: they are more likely to fertilise eggs than competing ejaculated sperm (Cohen and MacNaughton, 1974; Overstreet and Katz, 1977).

TRANSPORT THROUGH THE UTEROTUBAL JUNCTION (UTJ) The uterotubal junction (UTJ) presents anatomical, physiological and mucous barriers to sperm passage in humans. The entrance traverses a thick muscular layer of uterine wall (Hafez and Black, 1969) and the narrow lumen may be mucus-filled (Jansen, 1980), which impedes the progress of sperm. It has been observed that capacitated sperm are unable to swim through the narrow junction (Nakanishi et al, 2004) and in rodents that sperm with linear, progressive motility are more successful at passing through (Gaddum-Rosse, 1981; Shalgi et al 1992). There is likely to be some form of molecular selection at work also. Male mice that are null mutants for genes encoding different sperm surface proteins, including calmegin and ACE, are infertile because their sperm fail to get through the UTJ, despite having normal sperm morphology/motility. Thus its likely that certain epitopes are available and exposed on the surface of sperm to interact with the UTJ and somehow promote sperm passage (Krege et al, 1995; Ikawa et al, 1997; Cho et al, 1998; Hagaman et al, 1998; Yamagata et al, 2002).

A SPERM RESERVOIR IN THE FALLOPIAN TUBE EPITHELIAL BINDING The Fallopian tube provides a haven for sperm. Unlike the vagina, cervix and uterus, the tube does not respond to insemination with an influx of leukocytes (RodriguezMartinez et al, 1990). Overall data of human sperm distribution in the fallopian tubes of women have not provided a clear picture of the events of sperm transport. Sperm recovered at various times in different regions of the fallopian tube have varied so much in numbers that the data do not permit the construction of a model for the pattern of tubal transport (Williams et al, 1993). Since pregnancy has been shown to result from intercourse as long as five days before ovulation (Wilcox et al, 1995) human sperm must be stored somewhere in the female tract. A visibly distinct sperm reservoir has been seen in the fallopian tubes of animals (Yanagimachi and Chang, 1963; Harper, 1973; Overstreet et al, 1978; Suarez, 1987) but not in humans. However its likely that a functional reservoir does exist, created by detaining human sperm in the tubal isthmus (Pacey et al, 1995). There is strong evidence that such a reservoir is created when sperm intermittently bind to the epithelium lining the tube. In humans motile sperm have been observed to bind their heads to the apical surface of endosalpingeal ephithelium in vitro (Pacey et al, 1995a; Baillie et al, 1997; Reeve et al, 2003). In addition in vitro, sperm fertility and

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motiity are maintained longer when sperm are incubated with endosalpingeal epithelium (Kervancioglu et al, 1994). It may be that the intermittent binding of sperm in the tubal reservoir serves to prevent polyspermic fertilisation by allowing only a few sperm at a time to reach the oocyte at the other end of the tube (the ampulla). Artificially increased sperm numbers at the site of fertilisation in animals increases the incidence of polyspermy (Polge et al, 1970; Hunter, 1973). While intermittent sticking would not hold sperm in a distinct reservoir indefinitely it would slow their progress towards the ampulla. Sperm progress would also be slowed by the mucus in the lumen. Finally the architecture of the mucosal lining of the human fallopian tube must act to slow sperm progress. The mucosal folds increase in height and complexity towards the ovary, thus offering increasingly greater obstacles to the advancement of sperm into and through the ampulla. Slowing the advancement of spermatozoa like this could serve the function of a reservoir, that is, prolonging the availability of sperm in the fallopian tube. The amino acid sequence Arg-Gly-Asp (RGD) has been implicated in sperm binding to isthmic endosalpingeal epithelium but not to the ampullary endosalpingeal epithelium that lies at the other end (ampulla) of the tube (Reeve et al, 2003). Human spermendosalpingeal interaction in vitro appears to be disrupted in tissue donated from women who have had a previous diagnosis of endometriosis (Reeve et al, 2005) suggesting for the first time that poor sperm interaction with the endosalpingeal epithelium might be associated with reduced fertility, such as that often observed in women with endometriosis.

ADVANCEMENT OF SPERM BEYOND THE TUBAL RESERVOIR Theoretically sperm could be released from the reservoir either through loss of binding sites on the epithelium or by alterations in the sperm themselves. In women relatively equal numbers of sperm bind to endosalpingeal explants recovered at different times of the ovarian cycle (Baillie et al, 1997) so it appears that the epithelium does not release sperm by reducing binding sites. Instead current evidence indicates that changes in sperm bring about their own release.

CAPACITATION AND HYPERACTIVATION TO MOVE SPERM FROM ISTHMUS TO AMPULLA Sperm undergo two changes in preparation for fertilisation: capacitation and hyperactivation that together may serve to firstly detach spermatozoa from their isthmic reservoir and then speed sperm movement to the ampulla as the time of ovulation approaches. Capacitation involves changes in the plasma membrane, including shedding of proteins and cholesterol, that prepare sperm to undergo the acrosome reaction and fertilise oocytes (De Jonge, 2005) and this loss or modification of proteins on the surface of the plasma could reduce affinity for endosalpingeal epithelium.

Reviewer: My understanding is that hyperactivation is part of the capacitation process? But I am not an expert in this area.

Hyperactivation is a change in flagellar beating that typically involves an increase in the flagellar bend amplitude. This can provide the force necessary for overcoming the attraction between sperm and epithelium (Ho and Suarez, 2001). Animal experiments have shown that only hyperactivated spermatozoa detach from endosalpingeal epithelium (DeMott and Suarez, 1992) and that capacitated sperm bind less well (Lefebvre and Suarez, 1996) indicating that capacitation-induced changes in the sperm head surface are responsible for reducing binding affinity, though the pull produced by hyperactivation can almost certainly hasten detachment of bound sperm from the epithelium. The epithelium in turn may play a role in sperm release by secreting factors that alter sperm. For example hormonal signals that

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induce ovulation or signals from the pre-ovulatory follicle could stimulate the epithelium to secrete factors that trigger capacitation and hyperactivation, thereby bringing about sperm release. Tubal fluid and medium conditioned by cultured endosalpingeal cells have been demonstrated to enhance capacitation in bull sperm in vitro (Chian et al, 1995; Mahmoud and Parrish, 1996)

HYPERACTIVATION AND THE FINAL STAGES OF SPERM TRANSPORT In aqueous solution hyperactivated sperm swim vigorously in circular or erratic patterns but in vivo the physical environment encountered by sperm is quite different and evidence indicates that hyperactivation is required by sperm to progress towards the oocyte and penetrate its vestments. Hyperactivation enhances the ability of sperm to swim through viscoelastic substances such as mucus in the tubal lumen and the extracellular matrix of the cumulus oophorus (Jansen, 1980). Hyperactivated sperm penetrate artificial mucus such as viscoelastic solutions of long-chain polyacrylamide or methylcellulose, far more effectively than non-hyperactivated sperm (Suarez et al, 1991b; Suarez and Dai, 1992; Quill et al, 2003). Hyperactivation also endows sperm with greater flexibility for turning around in pockets of mucosa (Suarez et al, 1983; Suarez and Osman, 1987) and may thus assist sperm in navigating the increasingly complex tubal maze. In animals that cant hyperactivate but are otherwise capable of vigorous progressive motility experiments show that they do not penetrate artificial mucus nor can they penetrate the oocytes zona pellucida (Quill et al, 2001, 2003; Ren et al, 2001; Carlson et al, 2003).

TAXIS OF SPERM TOWARDS OOCYTES There is good evidence from animal studies of a guidance system that helps sperm reach the oocyte. Long-range thermotaxis guides capacitated sperm released from intimate contact with the endosalpinx towards the fertilisation zone. Animal work shows that sperm tend to swim towards warmer temperatures and the ampulla is two degrees warmer than the isthmus (Bahat et al, 2003). Shorter range chemotaxis may also guide the spermatozoa towards the oocyte (Eisenbach, 1999; Babcock, 2003). Sperm are equipped with a mechanism for turning towards the oocyte in response to chemotactic factors: they can switch back and forth between symmetrical flagellar beating and the asymmetrical flagellar beating of hyperactivation (Suarez et al, 1987) and so they can swim straight ahead and turn around. Controversially, sperm have also been reported to turn towards, or accumulate in a gradient of follicular fluid (Ralt et al, 1991, 1994; Cohen-Dayag et al, 1994, 1995; Fabro et al, 2002) which could accompany the oocyte into the fallopian tube. One interesting consequence of capacitation is the apparent activation or unmasking of receptors that respond to chemical stimuli from oocytes or thermal gradients within the oviduct (Fabro et al, 2002; Bahat et al, 2003). Although the details of this process are unknown it is clear that this provides yet another sperm selection point. Odorant receptors have been localised to a spot on the base of the flagellum of human sperm (Spehr et al, 2003) and placing sperm in a gradient of the odorant bourgeonal (lily of the valley) caused them to orient into the gradient and triggered a calcium and cAMP-mediated signalling cascade (Spehr et al, 2004). Nevertheless a chemotactic odorant has yet to be identified. If one were found it could have vast implications for the development of contraceptives, as well as assessment and treatment of infertility.

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THE NATURE OF OOCYTE CHEMOTAXIS It is unlikely that follicular fluid is the single source of sperm chemotaxis in vivo for two main reasons. Firstly, at ovulation only a very small fraction of follicular fluid is transported into the oviduct along with the oocyte (Hansen et al, 1991; Brussow et al, 1998; Hunter et al, 1999) and secondly, if sperm chemotaxis is essential for fertilisation the chemoattractant gradient should be maintained for as long as the oocyte survives and can be fertilised i.e. ca 24 hours post-ovulation (Harper, 1982). This requires a continuous supply of chemoattractant; sperm chemotaxis has been observed to both the oocytes surrounding cumulus cells and more potently to the oocyte itself, concluding that both are necessary chemotactic sources (Sun et al, 2005).

OOCYTE DISCRIMINATION OF ABNORMAL SPERM There is also evidence of a process whereby morphologically abnormal spermatozoa or non-capacitated or non-acrosome-reacted sperm are deselected by the oocyte and its surrounding cumulus cells. In vivo interaction between the oocyte and the few remaining sperm is likely to involve spermatozoa that are morphologically normal due to the selecting nature of the female reproductive system. Nevertheless evidence from in vitro work shows that those sperm able to traverse the cumulus mass were more likely to have normal morphology, be capacitated and acrosome-reacted with a distinct motility pattern and better zona-binding capacity suggesting that sperm selection continues to the end (Hong et al, 2004). As mentioned already there is some evidence that chemotaxis excludes those sperm that are non-capacitated; only those sperm that capacitate appear to be chemotactically responsive (Cohen-Dayag et al, 1994, 1995; Oliveira et al, 1999; Fabro et al, 2002).

THE CUMULUSOOCYTE COMPLEX (COC) Sperm and oocyte have very different functional roles and contributions. The sperm merely delivers paternal chromosomes to the site of fertilisation while the oocyte will provide all the nourishment and genetic programming necessary to sustain embryonic development for almost a week after conception. The volume of the oocyte is therefore much greater than that of the spermatozoon. A woman usually ovulates one oocyte each month, about 400 in her reproductive lifetime. Ovulation occurs before completion of oocyte maturation and the oocyte that leaves the follicle is arrested in metaphase of the second meiotic division during which time metabolism has been discontinued. To develop further, this secondary oocyte must await stimulation by fertilisation. The oocyte is surrounded by intercellular materials, including a thin transparent gelatinous layer of protein and polysaccharides called the zona pellucida. As the oocyte is ovulated a surrounding mass of follicular cells called the cumulus oophorus accompanies it resulting in the cumulusoocyte complex (COC). The COC serves as the final sperm filter and probably only 10 or 20 spermatozoa make it through the cumulus to reach the zona pellucida. The race to the finish will be governed by effective ligand-receptor interactions, functionally active signal transduction cascade pathways and a bit of luck (De Jonge, 2005).

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THE CUMULUS At this stage in the fertilisation process there are probably no more than tens of sperm that have reached and begun to penetrate through the cumulus. The cumulus is an extracellular matrix of ca 5,000 cells held together primarily by hyaluronate (hyaluronic acid) (De Jonge, 2005). Hyaluronidase activity by the sperm plasma membrane protein PH-20 acting on its hyaluronate substrate facilitates sperm penetration of the cumulus matrix (Lin et al, 1994). As already described its possible that the intercellular matrix of the cumulus acts similarly as cervical mucus to filter sperm with more normal morphology selectively (Sun et al, 2004). In addition it has been shown in vitro that hyaluronate selectively binds spermatogenically and genomically mature, viable and acrosome-intact human sperm (Huszar et al, 2003).

THE ZONA PELLUCIDA The zona is composed of a small number of glycoproteins; in addition to mediating the interaction with sperm, it is involved in the prevention of polyspermy and the protection of the developing embryo prior to implantation (Conner et al, 2005). It is likely that the sperm population that has successfully traversed the cumulus is heterogeneous relative to expression and receptivity for zona glycoprotein and functionality of signal transduction mechanisms that will ultimately participate in the zona-induced acrosome reaction, zona penetration and fertilisation. Upon contact with the zona there may only be slightly more than a handful of sperm that completely fulfil the preceding elements; perhaps chance is the final determinant for which of these is the fertilising sperm (De Jonge, 2005).

ZONA BINDING AND THE ACROSOME REACTION The very few spermatozoa that have reached and now bind to the zona pellucida receive a signal to acrosome react i.e. release by Ca2+ stimulated exocytosis of the contents of their large secretory granule, the acrosome. The membranes in the head of the spermatozoon are reorganised; multiple fusions occur between the outer region of the acrosomal membrane and the plasma membrane that overlies the acrosome. The hybrid vesicles that result are released into the surrounding environment along with fluid contents of the acrosome. Loss of these anterior portions of membrane reveals the inner acrosomal membrane which together with the original posterior head plasma membrane form the new head cell membrane of the acrosome-reacted sperm. The acrosomal contents are a rich source of enzymes, including hyaluronidase and the protease, acrosin, that may function in penetration of the sperm through the zona. The hybrid vesicles released during the acrosome reaction could also carry such enzymes on their surfaces. A combination of motility, proteases and glycosidases are likely involved in penetrating the zona (Yanagimachi, 1994; Ohmura et al, 1999; Zhu et al, 2001; Primakoff, 2002).

THE FATE OF THE FERTILISING SPERMATOZOON During or after the acrosome reaction, the fertilising sperm detaches from the zona cutting a penetration slit that is just as wide as the sperm head. Regardless of how many sperm that manage to penetrate the zona successfully only one will fertilise and activate the oocyte. As the first sperm enters the narrow perivitelline space it binds to the oocytes plasma membrane by the lateral face of the head, with the firm point of attachment between the sperm and egg plasma membranes occurring at the equatorial segment. Sperm surface protein candidates include sperm members of the

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ADAM family (Primakoff et al, 2000). Egg surface proteins are likely to include an active site in CD9 that associates with and regulates the egg fusion machinery (Zhu et al, 2002). Spermegg fusion stimulates the first signalling pathway in development.

FERTILISATION When the cell membranes of the sperm and oocyte merge, the sperm enters the ooplasm of the oocyte, triggering oocyte activation, a series of changes in metabolic activity including a rise in metabolic rate. The cell membrane of the oocyte undergoes immediate electrical changes that block the entry of other sperm and enzymes produced by the fertilized ovum alter receptor sites so that sperm already bound are detached and others are prevented from binding. Fertilisation stimulates the ovum to complete its second meiotic division and form a diploid zygote, producing a large mature ovum. After oocyte activation and meiosis the nuclear material remaining within the ovum reorganises as the female pronucleus. The successful spermatozoon enters the cytoplasm, its tail is shed and the nucleus in the head develops into a structure called the male pronucleus which in turn migrates to the centre of the cell and fuses with the female pronucleus. Within 12 hours genetic fusion has completed with the formation of a diploid zygote, a fertilised egg, which will in turn undergo cleavage and produce billions of specialised cells.

THE FATE OF NON-FERTILISING SPERM After fertilisation, any sperm remaining in the female reproductive tract may be phagocytosed by isthmic epithelial cells (Chakraborty and Nelson, 1975; Rasweiler, 1987) or may be eliminated into the peritoneal cavity (Mortimer and Templeton, 1982) where they in turn may be phagocytosed. There have been cases Reviewer: where sperm have not been rendered non-functional as is evidenced by the The authors say nothing numerous case reports of human tubal pregnancies that arose in spite of about polyspermia the excessive secretion of semen which I think is lack of access of sperm from the uterus into the oviduct on the side of an interesting phenomenon ovultation (Metz and Mastroianni, 1979; Brown et al, 1987; Ansari and Miller, worth mentioning. 1994). In these cases the only route available to the sperm was through the peritoneal cavity.

MALE INFERTILITY Failure or severe difficulty in conceiving a child is a surprisingly common worldwide problem. The incidence of sub-fertility seems to be increasing, with defects in sperm (1 in 15 men) being the single most common cause. On the basis of semen assessment up to one-fifth of 18-year-old boys can be classed as sub-fertile. (Hull et al, 1985; Sharpe and Irvine, 2004; Anderson, 2005).

SPERM QUALITY DECLINING MALE FERTILITY A large body of data suggests that sperm counts have been declining in Europe and the US though interpretation of these trends remains controversial and the role of the environment uncertain. A meta-analysis of 61 studies of sperm density revealed a decline in sperm concentrations of healthy men from 113 milllion/ml in 1938 to 66 million/ml in 1990 (Carlsen et al, 1992). The analysis was repeated and the main conclusion remained the same: in the 1950s sperm concentrations were higher than in the 1970s (Olsen et al, 1995; Swan et al, 1997). The metaanalysis was extended to include studies until 1996 and it confirmed the declining trend in the US and Europe (Swan et al, 2000). 15

Reviewer: Despite stressing the importance of sperm counts to male fertility, there is no mention of the two key factors that determine a mans sperm count: 1) the number of Sertoli cells per testis (varies more than 100-fold between individuals, though it is unknown why), which is determined largely by events in fetal and early postnatal life; 2) the frequency of ejaculation.

A large study of Denmark, Finland, France and Scotland showed that Finnish men were found to have higher sperm counts than the others (Jorgensen et al, 2001). A common feature in the decline of sperm concentrations is its association with year of birth: younger cohorts have lower sperm concentrations than the older generations (Auger et al, 1995; Irvine et al, 1996; Licht, 1998; Thierfelder et al, 1999). The lowest sperm counts were found in 1819 year old Danish men in 19978 (Anderson et al, 2000). The median sperm concentration was 41 million/ml and 18% had concentrations below 20 million/ml which is considered a threshold for sub-fertility by the World Health Organisation. The reasons for declining semen quality is largely unknown though there is good evidence that environmental agents can alter semen quality (Swan, 2003). A study of maternal smoking in relation to semen quality reported lower sperm density. Inhibin-B and elevated follicular stimulating hormone associated with high in utero exposure (over 10 cigarettes per day) (Storgaard et al, 2003). In other studies exposure to one class of ubiquitous chemical/pesticide, the phthalates, was examined in relation to sperm concentration, motility and morphology using urinary metabolites as biomarkers (Duty et al, 2003; Swan, 2006). Higher levels of two of these metabolites were associated with reduced sperm concentration and one with reduced sperm motility. Higher levels were not particularly high (less than half the levels found in a representative US population (CDC, National Report on Human exposure to environmental chemicals, 2001)) suggesting that phthalates may adversely affect male reproductive function even at the low ambient levels measured in the study. Taken together these two studies provide good (though not conclusive) evidence that environmental agents, even at low levels, can alter semen quality. Several other plausible agents are being investigated for their ability to alter male reproduction (organochlorine pesticides, bisphenol-A, triazines, etc) and the number is likely to increase (Swan, 2003). Because these exposures are fairly ubiquitous, it is probable that subjects in both the above studies have been exposed to low levels of multiple agents. Teasing out the separate and combined effects of this chemical soup must be met before concluding that any specific exposure has altered semen quality let alone caused a worldwide decline in semen quality (Swan, 2003).

FEMALE FERTILITY OOCYTE QUALITY Female sub-fertility is to a large part dependent on the maternal age effect, characterized by a negative relationship between maternal age and Reviewer: reproductive efficiency, and remains a poorly understood phenomenon. This section is poor. The main Current data suggest that oocyte physiology determines this relationship reason womens fertility decreases and one theory proposes that free radical attack on the mitochondria of with age is errors in meiosis, which lead to non-disjunction of the primordial oocytes residing in the ovary and subsequent deficiency in chromosomes and therefore oxidative phosphorylation may be the primary cause of oocyte degradation chromosomally unbalanced (Wilding et al, 2005). embryos. CONCLUSION The union between the sperm and oocyte is arguably the most important interaction in biology and possibly the least well understood. As more and more is discovered about the processes and mechanisms involved in achieving in vivo and in vitro fertilisation the better placed scientists will be to make important progress in the development of improvements in assisted conception technologies; contraceptives and human oocyte storage enabling women to delay successful pregnancy to later in life.

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