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WHAT constitutes a fertile sample of semen? The short answer is simple:
by definition, it will be one that can fertilise an ovum and promote the
normal development of an embryo. We can establish the capacity of a sample
to fertilise ova, in retrospect, by diluting the semen, inseminating many
females and then analysing the conception rates. Animal breeding organisations
carry out this sort of testing routinely to measure the fertility of stud
livestock. But to predict the fertility of sperm without recourse to insemination
trials is a different matter.

The male hopping mouse from Australia (described by Bill Breed in the
article that follows this one) produces few spermatozoa and those that are
eventually ejaculated are often oddly misshapen. By contrast, the semen
of closely related species of rodent contain high numbers of sperm with
a consistent appearance. Looking down the microscope, one would be forgiven
for pronouncing the sperm of the hopping mouse subfertile, or worse, infertile.
In fact, mature males require just one mating to impregnate a female. What,
then, are the essential attributes of a mammalian sperm that enable it to
fertilise an egg?

In the gamete biology unit at the Institute of Zoology in London, we
have been trying to find the answer, to assess the fertility of exotic mammals
being bred in captivity. For endangered species, the luxury of an insemination
trial is out of the question. A better appreciation of the complex physiology
of spermatozoa has led to new in vitro tests that give a good indication
of the capacity of sperm to fertilise eggs. Some of the results have also
helped in the diagnosis and treatment of infertility in men (about 1 man
in 15 is infertile) and in the development of the next generation of contraceptives.

One way of investigating what constitutes a fertile sample of semen
is to backtrack and consider how the developing spermatozoa first acquire
the capacity to fertilise eggs. Within the testis, sperm arise from round,
undifferentiated germ cells, spermatogonia, that line the tightly coiled
seminiferous tubules. The production of sperm is an intricate and lengthy
process, which can last up to 70 days. During this time, cells divide and
become specialised, under the influence of steroid and protein hormones.
The daily output of sperm can be prodigious, reaching values of 3 thousand
million sperm per testis per day. What may come as a surprise is that, in
all mammals so far examined, sperm passing out of the testis cannot fertilise
eggs. Only after they have passed along a convoluted duct, the epididymis,
which, with the vas deferens, connects the testis to the outer world, do
sperm acquire the potential to fertilise an egg.

By comparing sperm immediately before and after they become fertile,
we should be able to determine exactly what sperm need to be fertile. Unfortunately,
in many species, sperm undergo complex changes while they mature in the
epididymis. Marsupials probably represent the most extreme cases. The sperm
of the Australian brush-tailed possum, for example, undergo extensive reorganisations
during this time: the heads of the sperm move through 90 degrees and their
membranes are redistributed. But perhaps the most intriguing process of
maturation of sperm occurs in the New World marsupials such as opossums.
Just before sperm become fertile in the epididymis, they form into pairs,
precisely bonded together by their heads. Mature sperm remain paired until
fertilisation when they separate so that one sperm can enter the egg. Why
has such a bizarre maturation phenomenon evolved? No one really knows, but
the most plausible explanations suggest that pairing enhances the sperm’s
ability to move or protects the heads of the sperm over a delicate region
known as the acrosome. We will see later that both of these aspects are
critical for fertility.

Unusual processes of maturation are interesting from an evolutionary
point of view, but we need to turn to more ‘conservative’ species to understand
the basic mechanisms. In my laboratory, we have investigated how sperm mature
in the golden hamster. At first sight, this may seem a rather esoteric species
to choose, but compared with other laboratory animals it has a combination
of useful attributes. From a practical aspect the testis and epididymis
are relatively large, making it reasonably easy to recover sperm from different
regions of the tract. Secondly, the capacity of sperm to fertilise eggs
increases from 5 per cent to more than 75 per cent in just a short length
of epididymis, about 5 millimetres long. Even when uncoiled, the tubule
in this region is no more than 5 centimetres long. So we can concentrate
on changes to sperm in this small portion of tubule. Finally, the techniques
of in vitro fertilisation (IVF) are well established in the golden hamster;
in fact, the golden hamster was the first mammal in which researchers carried
out IVF in its entirety just over 25 years ago.

Recently, we have shown that secretions from the cells lining the epididymal
duct, its epithelium, are responsible for the final maturation of sperm.
We showed this using a new procedure which opens up the exciting prospect
of eventually being able to mature sperm in vitro. We isolate small pieces
of tubule from the critical maturation region in the epididymis and use
an enzyme, collagenase, to digest the tough connective tissue that surrounds
the coiled duct. We then split the lengths of tubule down their longitudinal
axis to form plaques of epithelium. In culture, these plaques quite spontaneously,
and to our initial surprise, turn inside out and form spheres with epithelium
now on the outside. The everted epithelium seems to function normally for
four to five days in response to two androgen hormones, testosterone and
dihydrotestosterone. More significantly, when we incubate immature sperm
with the epithelial culture for 6 hours, they acquire the ability to fertilise
eggs in vitro, and in vivo following artificial insemination. To pinpoint
which epididymal secretions enabled the sperm to mature, we used monoclonal
antibodies bound to the surface of the epithelial cells of the epididymis.
This technique revealed that sperm become fertile when some of these secretions
(various glycoproteins) become attached to their surface. We are now trying
to find out the exact nature of these proteins. Their presence on sperm
will, we hope, provide a practical marker of mature sperm. Interference
with their function could lead to a new contraceptive mechanism for men.

After it matures, a sperm must overcome three major hurdles before fertilisation
can happen. To reach the egg, it must negotiate obstacles in the female
reproductive tract, such as the cervix and the junction of the uterus with
the Fallopian tubes. The sperm must then prepare the outer membranes at
its head, called the acrosome, so that it can bind to and penetrate into
the oocyte. Finally, the plasma membrane of the sperm, lying around the
middle of the head (the equatorial segment), must fuse with the egg membrane
so that its gene-bearing nucleus can incorporate itself into the cytoplasm
of the egg. By monitoring each of these processes in vitro, we can now assess
with precision a sperm’s capacity to fertilise eggs.

In the fast lane

Researchers have long linked the movement of sperm with fertility, and
semen containing no motile sperm is always infertile (unless assisted by
the latest microinjection techniques). However, the corollary, that mobile
sperm are always fertile, is not necessarily true. Only in the past few
years have we known enough about how sperm move to link certain characteristics
with fertility. These investigations have shown that, within broad limits,
it is not the concentration or proportion of motile sperm in a sample of
semen that is important, but the velocity of these moving sperm, and the
way they move: quality rather than quantity.

Different species produce sperm that move in different ways, but with
the correct baseline researchers can assess the fertility of an individual
animal. We place a small drop of semen in a well on a glass slide kept at
a constant temperature (30 Degree C or 37 Degree C) and then measure the
movements of the sperm under the microscope. At first we had to analyse
the trials of sperm recorded on film exposed for one second, a laborious
procedure. But the whole process has now been simplified and made quicker
by advances in image analysis. We now have instruments that can track the
movement of sperm on a microscope slide and give a full analysis within
a few minutes. We have used this technique to assess the fertility of sperm
previously frozen in liquid nitrogen. For example, by comparing the way
the sperm of a giant panda move before and after freezing we felt confident
that the cryopreservation procedures were satisfactory. Our confidence was
borne out by the successful insemination of the Madrid panda, ‘Shao-Shao’,
with semen from our male ‘Chia-Chia’.

We have also assessed the fertility of human semen in this way. A few
years ago, my colleague Bill Holt and I found that samples containing a
high proportion of sperm moving at an average velocity of 20 micrometres
per second or faster were 80 per cent more likely to fertilise eggs in the
laboratory than those with a slower mean velocity, regardless of other characteristics
of the sperm. This result indicates that, in the test tube at least, the
ability of sperm to move correctly is extremely important for fertilisation.

More recently, we tested this hypothesis using semen of donors for artificial
insemination. To ensure no possible infection with HIV, the virus that causes
AIDS, donors are now screened at regular intervals. Their semen is preserved
in liquid nitrogen and effectively quarantined for three months. Although
human sperm can usually be frozen and thawed without ill effects, it sometimes
loses its fertilising capacity during the process. In a controlled trial
conducted with Francoise Shenfield at the Middlesex Hospital we could discriminate
between samples of donor semen giving high rates of conception and those
giving low ones by looking at how fast the sperm move and how well they
survived during incubations at 37 Degree C. We still do not fully understand
the significance of speed in relation to infertility. Perhaps the velocity
at which they swim indicates the overall fitness of the cells. Sperm may
also need a certain minimum thrust in order to penetrate cervical mucus
and to pass from the uterus to the Fallopian tube. In many species, sperm
beat their tails vigorously just before fertilisation. This ‘hyperactivated’
motility may give that final thrust necessary for the penetration of the
egg. Whatever the reasons, measuring the speed at which sperm move gives
a much better idea of its capacity to fertilise eggs than the conventional
tests, such as counting sperm, classifying their morphology and subjectively
evaluating their motility by ‘eyeballing’. These standard tests are of little
value in assessing infertility, but unfortunately they are often the only
analyses available at infertility clinics.

Besides maintaining the correct motility, the spermatozoon must also
undergo changes to its membranes before it can penetrate the egg. A crucial
event concerns the acrosome, a membranous cap overlaying the head of the
sperm that is rich in protein-destroying enzymes. Factors within the oviduct,
along with a chemical reaction initiated by the contact of the sperm with
the zona pellucida, set off a chemical change in the acrosome. This enables
the plasma membrane of the sperm and the outer acrosomal membranes to fuse
together, to create pores through which the contents of the acrosome disperse.
Eventually, the acrosome cap is sloughed off. This reaction must happen
before sperm can fertilise an egg; it ensures that the sperm is correctly
orientated against the zona, and exposes the sharp point (perforatorium)
and tough face of the inner acrosomal membrane, the cutting edge for penetrating
the egg. Sperm that fail to undergo the acrosome reaction or have a missing
acrosome are infertile, so if we coudl devise a diagnostic test for functional
acrosomes, it would provide important information on fertility.

To cap it all

We have been able to follow the acrosome reaction in the laboratory
by labelling the acrosome with antibodies linked to fluorescent markers.
Fortuitously, the monoclonal antibody we use recognises a component that
seems to be common to all placental mammals. This makes it ideal for our
work at London Zoo, where one day we may be examining semen from a golden
lion tamarin and the next from a scimitar-horned oryx. The technique is
equally important for assessing human sperm or those from domestic species.
Recently, working with John Zhang and Martin Boyle at the Equine Fertility
Unit at Newmarket, we diagnosed infertility in Thoroughbred stallions.

One of the final acts of the fertilising sperm is to fuse with the membrane
of the egg so that its genetic material can enter the cytoplasm of the egg.
We have known for some time that this process involves a specific band of
membrane around the head of the sperm, called the equatorial segment. This
region persists after the acrosome reaction and readily fuses with the egg
in fertile sperm, so that once the sperm penetrates through the zona pellucida
it quickly goes on to complete fertilisation. In most animals, egg and sperm
fuse only if both are from the same species. But in 1976, Ruyzio Yanagimachi
and colleagues at the University of Hawaii showed that hamster eggs, denuded
of their zona pellucida with the enzyme trypsin, were receptive to human
sperm. This finding is the basis for the surrogate egg procedure known as
the hamster egg penetration test (HEPT). In the test, researchers incubate
the sperm with zona-free hamster eggs and then examine them to see whether
sperm and egg have fused. This test is practical because hamster eggs are
relatively easy to come by, and many researchers now use it to study sperm
from many species. We have found that sperm from marsupials and even budgerigars
will fuse with the cytoplasm of the hamster egg, indicating perhaps that
very basic physiological mechanisms are involved.

Ironically, the unselective nature of the hamster egg has led some investigators
to question the validity of the HEPT, particularly for assessing human sperm.
Recently, investigators have developed a more sophisticated version of the
test using human eggs that fail to fertilise during IVF procedures. These
eggs are normally discarded, but by storing them in a concentrated salt
solution, a process that destroys the cytoplasm of the egg, researchers
can preserve the biological activity of the acellular zona pellucida. A
combined test using zona-free hamster eggs and human zonae can test the
ability of sperm to bind and penetrate the zona as well as to fuse their
membranes with the egg’s, and so is a more rigorous assay.

By screening monoclonal antibodies produced in my laboratory with salt-stored
human eggs collected in Alan Trounson’s laboratory in Monash University
in Melbourne, we have identified an antibody that blocks the attachment
of human sperm to the egg by binding to an antigen over the equatorial segment.
The identification of the gene that encodes for this component on the surface
of a sperm could lead to the development of a new contraceptive vaccine.
My colleague Alison Moore, with David Latchman at the Medical Molecular
Biology Unit, are now searching for this and similar genes using human testis
DNA.

These new tests for sperm function are now used mainly in research laboratories,
but infertility clinics are slowly adopting them. Our results and those
of other investigators indicate that in many cases these methods can predict
sperm’s capacity to fertilise in a range of mammals. At the Institute of
Zoology we hope to use these methods to establish stocks of frozen semen
from endangered species that are highly potent. As the story of the hopping
mice suggests, the way sperm look may not be as relevant as features such
as their motility and the function of the acrosome. In fact, when we selected
particularly motile sperm in this species we produced only a slight reduction
in the variety of structures exhibited by the sperm. Most hopping mouse
sperm have an intact, although often misshapen, acrosome. So what seem like
abnormal sperm may be capable of fertilisation. Why the hopping mice produce
so few and has such variable sperm still remains unclear, but the investigation
of unusual anomalies such as these provides important clues for our understanding
of what makes males fertile.

Dr Harry Moore researches at the Institute of Zoology in Regent’s Park,
London.

* * *

Freezing endangered genes for the future

BY STORING gametes and embryos in liquid nitrogen, at -196 Degree C,
we can preserve the genetic variation of individuals for decades. Zoos around
the world now cooperate to maintain detailed studbooks on computer. This
allows us to calculate the relative contribution of particular founder or
parent animals to the current gene pool.

If certain individuals contribute too much or too little, subsequent
generations will be genetically less diverse. Planned breeding programmes
can counter such inbreeding. Artificial breeding techniques, using frozen
semen and embryos, reduce the need to transport exotic animals from one
zoo to another, which is stressful as well as expensive and runs the risk
of spreading disease.

Zoo vets collect semen from captive wild animals under full anaesthetic,
usually by an electroejaculation technique. This harmless procedure evokes
an emission of semen by stimulating motor neurons leading to the genital
tract with a small electric current delivered through electrodes placed
in the animal’s rectum. The semen is then diluted with a cryoprotective
solution containing glycerol and egg yolk. These substances prevent the
formation of ice inside the cells and protect the delicate membranes of
the sperm as they freeze.

The ability of sperm to survive cooling, freezing and thawing varies
from species to species. Along with the new laboratory tests of sperm function,
the recent development of the cryomicroscope enables us to find, in hours
instead of weeks, the best way to freeze sperm. The cryomicroscope has a
computer-driven stage that can be cooled at the desired rate to the temperature
of nitrogen vapour – at -80 Degree C. We can monitor the response of sperm
to cooling, freezing and thawing at every stage, allowing us to find the
best rates of freezing for semen from a particular species or even an individual
animal.