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OCR for page 63
5
Biologic Markers of Epididymal
Structure and Function
This chapter focuses on markers of
epididymal function. Very few direct
markers of human epididymal function are
available. However, there is a useful
array of indirect markers. The chapter
discusses markers of tissue structure:
markers of structural, biochemical,
membrane compositional, and functional
changes of the maturing spermatozoa;
biochemical markers of the luminal fluid;
and histologic and biochemical markers
of the epithelium. Many of the assessments
must be performed on biopsied or autopsied
tissue or in animals. These studies are
essential to the understanding of basic
mechanisms of action of toxicants.
The epididymis is a single highly con-
voluted duct whose length varies from
3-4 meters in man to 80 meters in horses
(Maneely, 1959~. The duct begins where
the efferent ducts—the ductuli effer-
entes, numbering from 4 to 20, depending
on species (Nistal Martin de Serrano and
Paniagua Gomez-Alvarez, 1984; Hemeida
et al., 1978-come together. The epididy-
mal duct continues as a straight tube, the
vas deferens, that is surrounded by a thick
muscular layer. The vas deferens connects
with the urethra, which empties outside
the body. The epididymis is usually divid-
ed into three gross anatomic segments:
head (caput), body (corpus), and tail
(cauda). An initial segment lies between
the efferent ducts and the remainder of
63
the caput epididymis, has a characteristic
histologic appearance and function and
is also often identified (Benoit, 1926;
Robaire and Hermo,1987~.
Interest in the epididymis has grown
over the past 2 decades, since the demon-
stration that it was in this tissue that
spermatozoa mature enough to become able
to fertilize eggs (Bedford, 1967; Orgebin-
Crist, 1967a). On leaving the testis,
spermatozoa have a light microscopic
appearance similar to that of spermatozoa
in semen, but are incapable of fertilizing
eggs. Several reviews on various facets
of epididymal structure and function have
appeared in the past 15 years (Hamilton,
1972; Bedford, 1975; Hamilton, 1975;
Neaves, 1975; Orgebin-Crist et al., 1975;
Turner, 1979; Hinton, 1980; Courot, 1981;
Orgebin-Crist, 1981; Brooks, 1982; Glo-
ver, 1982; Howards, 1983; Orgebin-Crist,
1984; Cooper, 1986; Amann, 1987; Robaire
and Hermo,1987~.
To monitor whether a toxicant has com-
promised male fertility, it is essential
to determine not only whether the normal
physiologic functions of the epididymis
are still being performed, but also whether
the toxicant has so altered the spermatozoa
while they were in the epididymis that they
cannot fertilize eggs or can fertilize
eggs but produce only nonviable or abnormal
offspring.
A discussion of potentially useful mark-
OCR for page 64
64
ers of epididymal function that are avail-
able, are under development, or ought
to be developed is presented below. We
begin by discussing markers of the tissue
as a whole and then focus on markers that
reflect changes in maturing spermatozoa,
the implications of the makeup of the
fluid in the luminal compartment that
bathes maturing spermatozoa, and the
activities of the epithelial compartment
of the epididymis.
MARKERS OF EPIDIDYMAL TISSUE
Few noninvasive direct determinations
are used to assess whether a substance
has deleteriously affected the epididy-
mis. The tissue can be palpated and the
size, consistency, and shape assessed.
Such simple markers are sometimes of value
in diagnosing tubal obstruction or epidid-
ymitis, but they are highly subjective
and do not lend themselves to assessment
of variability, limits of detection, and
so forth. The more recently developed
imaging techniques are only now being test-
ed as tools in assessing the epididymis;
these probably will yield a reliable index
of such characteristics as size and shape.
If the tissues are available, the
weight of the epididymis is, in fact, a
simple and useful biologic marker. The
epididymis is a tissue with two comnart-
MALE REPRODUCTIVE TOXICOLOGY
the biologic regulation of epididymal
function. The epididymis contains recep-
tors for a variety of hormones, e.g., tes-
tosterone, estradiol, prolactin, and
vitamin D; but the value, with respect to
epididymal function, of measuring the
serum concentrations of the hormones as
markers has not been established. The
topography of the blood vessels entering
and leaving the testis is complex and spe-
cies-specific (Chubb and Desjardins,
1982~. It is evident from numerous castra-
tion and hormone-replacement studies that
blood vessels are a major route by which
hormonal signals are received by the epi-
didymis. Our knowledge of capillary flow
and its impact on tissue function is
sparse, but it is likely that we will soon
be able to monitor blood flow in epididymal
capillaries. Whether such measurement
will become an important marker of epididy-
mal function will depend on the ability
of the capillary system to adapt to dele-
terious actions of toxicants.
Very little is known about epididymal
lymphatic input and drainage. As the na-
ture of the blood/epididymis barrier be-
comes better understood and the use of
immunosuppressive agents (which alter
the immune system and lymph content) grows,
we will increase our understanding of this
system, although no lymphatic markers
regarding epididymal function are likely
meets: epithelium and lumen. The lumen to tee developed soon.
is filled with fluid and spermatozoa and, Neuronal input is responsible for the
under normal conditions in many mammals, basal epididymal contractility, which
makes up approximately 50% of the weight is at least partially responsible for
of the tissue (e.g., Robaire et al., 1977~; the transport of sperm and fluid down the
small decreases in epididymal weight are duct system from the testis. Assessment
usually associated with proportionately of whether drugs that affect the autonomic
larger decreases in epididymal sperm re- nervous system can modulate epididymal
serves. Accessory sex tissues become mark- contractility and hence the rate of sperm
edly hypertrophic in the presence of large transit and the acquisition of fertilizing
excesses of androgen, even in a castrated
animal. However, the epididymis does not
become hypertrophic. Thus, changes in
epididymal weight can be a valuable index
of the status of the tissue.
As with other hormone-dependent tis-
sues, blood is the major source of regula-
tory substances that reach the epididymal
epithelium. Measurement of serum concen-
trations of hormones, especially testos-
terone, is useful and routine in monitoring
ability in viva has not been attempted.
It would be surprising if they could not;
but such drugs affect an array of other
systems, so their effects on the epididymis
might be masked. Although the exact epi-
didymal effects of modulating neuronal
activity would be of interest, measure-
ments of neuronally mediated epididymal
contractility and of the factors that regu-
late it will probably not become selective
markers.
OCR for page 65
EPIDIDYAfAL STRUCTURE AND FUNCTION
Some epididymal functions apparently
are regulated in a paracrine manner,
i.e., by factors from the testis that
enter the epididymal lumen directly (Ro-
baire and Hermo, 1987~. Factors secreted
by the testis into the efferent ducts that
directly modulate epididymal function
are probably of Sertoli cell origin (Scheer
and Robaire, 1980; Robaire and Zirkin,
1981~. Their identities are not resolved,
but androgen-binding protein has been
proposed as a likely candidate. Determin-
ing the concentrations of various factors
in rete testis fluid is difficult for many
species, because it is technically dif-
ficult to obtain the fluid, only small
volumes can be obtained, and the procedure
cannot be repeated in most small animals
and cannot be done in humans. However, it
is apparent that some key regulators of
epididymal function, which are also prob-
ably useful epididymal markers, are to
be found in this fluid. The presence and
concentrations of such compounds in
semen might reflect not only testicular
but also epididymal function, and research
to identify them and to develop means of
monitoring them should be strongly encour-
aged, although it will certainly take
several years for such markers to become
readily available.
CHANGES IN MATURING
SPERMATOZOA
A number of morphologic and biochem-
ical changes reported to occur in sper-
matozoa during transit through the epidid-
ymis and vas deferens have been reviewed
extensively (Bedford, 1975; Bedford,
1979; Olson and Orgebin-Crist, 1982; Eddy
et al., 1985; Cooper, 1986; Robaire and
Hermo, 1987~.
Structural Changes
The most consistent morphologic change
that takes place in spermatozoa during
ductal transit is the migration of the
cytoplasmic droplet from the neck region
of the flagellum to the end of the midpiece
of the sperm (mitochondrial sheath). The
change has been noted in snakes and birds
(Bedford, 1979), in many mammals (Branton
65
and Salisbury, 1947; Phillips, 1975; Kap-
lan et al., 1984), and in humans (Hafez
and Prasad, 1976~. In rats, lysosomal and
other degradative enzymes are present in
the droplet (Dots and Dingle, 1968; M.L.
Roberts et al., 1976~; however, the func-
tional importance of the enzymes and of
the droplet remains to be resolved.
The percentage of spermatozoa that re-
tain cytoplasmic droplets or the number
of cytoplasmic droplets per spermatozoon
might well be a useful indication of the
state of maturation of spermatozoa and
might also reflect the activity of clear
cells in some species. It might be a sensi-
tive measure, but there will probably be
large variability in it. This marker has
not yet been validated as a good correlate
of epididymal function, but it should be
fairly straightforward to do so. Such a
measure could be particularly useful in
selected cases in which clear cell function
or droplet shedding is impaired.
Spermatozoa undergo a change in acro-
somal size, shape, and internal structure
as they pass through the duct system. This
has been demonstrated in many species
(Fawcett and Hollenberg, 1963; Fawcett
and Phillips, 1969; Bedford and Nicander,
1971; Jones et al., 1974; Bedford and Mil-
lar, 1978~. In animals in which acrosomal
shape clearly changes during epididymal
transit, observation of spermatozoa
should reveal whether exposure to a given
chemical has altered this marker of sper-
matozoal maturation. Further studies are
needed with primates to resolve whether
such changes are correlated with spermato-
zoal quality, i.e., fertilizing ability.
Biochemical Changes
An array of biochemical alterations in
spermatozoa as they traverse the ducts
has been reported (Bedford, 1975; Bedford
and Cooper, 1978; Olson and Orgebin-Crist,
1982~. Only the characteristics with the
greatest potential for use as markers are
discussed here. In spite of numerous de-
tailed studies on how sperm change during
epididymal transit, it is still not possi-
ble to dissociate factors that cause the
maturation of spermatozoa from factors
that result from maturation or are inciden-
OCR for page 66
66
tat to it. Hence, any of the markers sug-
gested above might correlate well with
changes in spermatozoa that alter their
fertilizing potential or their ability
to produce normal offspring under a given
set of conditions without having the
desired predictive value.
An increase in the relative number of
disulfide bonds in the nuclei of spermato-
zoa as they reach the cauda epididymis has
been observed in many species (Calvin and
Bedford, 1971; Bedford et al., 1973; Bed-
ford, 1975; Johnson et al., 1980b). The
increase is associated with a more con-
densed (cross-linked) spermatozoa! nu-
cleus-spermatozoa taken from the cauda
epididymis are harder to Recondense than
those taken from the initial segment
(Zirkin et al., 1985b). The state of chro-
matin condensation can be assessed by de-
termining how long it takes for detergent-
treated nuclei to dissolve in the presence
of a given concentration of a disulfide
reducing agent. If chemicals can affect
spermatozoa by preventing the proper chro-
matin condensation that takes place while
spermatozoa are in the epididymis, then
measuring such rates might provide a simple
and useful biologic marker.
The anionic charge on spermatozoa in-
creases as they reach the cauda epididymis
(Bedford, 1975; Toowicharanont and Chula-
vatnatol, 1983~; the increase is probably
acquired during passage through the corpus
epididymis (Fain-Maurel et al., 1983~.
The exact cause of the change in charge has
not been resolved, but it is most likely
due to changes in glycoprotein composition
of the sperm plasma membrane that take
place during epididymal transit.
The membrane proteins of spermatozoa
obtained from different segments of the
epididymis present a markedly different
pattern after separation on polyacryla-
mide gel electrophoresis (Jones et al.,
1981; Chulavatnatol et al., 1982; Brown
et al., 1983; Dacheux and Voglmayr, 1983;
Jones et al., 1983; Brooks and Tiver,
1984~. During epididymal transit, the
number of concanavalin A binding sites
on the sperm membrane decreases (Lewin
et al., 1979; Nicolson and Yanagimachi,
1979; Olson and Orgebin-Crist, 1982), the
ability of spermatozoa to activate comple-
MCALE REPRODUCTIVE TOXICOLOGY
ment decreases (Witkin et al., 1983), and
the activity of protein methyltransferase
in spermatozoa decreases dramatically
(Gagnon et al., 1984~. Gonzalez Echeverria
et al. (1984) have noted that the addition
of a protein extract from hamster epididy-
mis could increase the fertilizing ability
of hamster spermatozoa. Blaquier et al.
(1987) found that polyclonal antibodies
to human epididymal sperm proteins bound
selectively in the acrosomal cap region
in fertile men, but that a large percentage
of sperm bound these antibodies in a non-
specific manner in infertile men. Such
data lead to the proposal that several
highly specific sperm-surface protein
markers of epididymal origin might be al-
tered, not only in cases of infertility,
but also in response to various drugs.
Antigenic determinants on the surface
of spermatozoa change markedly as sperm
traverse the epididymis (Eddy et al.,
1985~. Monoclonal antibodies to mouse
"sperm-maturation antigens" that arise
in mice during epididymal passage have
been characterized, and at least one (SM4)
has been shown to appear on sperm only while
they are in the corpus epididymis (Eddy
et al., 1985; Vernon et al., 1985~. The
appearance of such determinants might be
due not only to the addition of a protein
to the surface of spermatozoa, but also
to the removal of proteins or to the enzyme-
mediated exposure of pre-existing sites.
This new approach to the characterization
of changes that take place in spermatozoa
during epididymal transit might provide
some useful biologic markers, if a clear
linkage between the state of maturation
of a spermatozoon and the presence or
absence of a given epitope can be
established.
The lipid composition of spermatozoa
(Dacheux, 1977; Evans and Setchell, 1979),
particularly that of their plasma mem-
branes (Nikolopoulou et al., 1985),
changes dramatically during epididymal
transit: the amount of cholesterol de-
creases, and the amounts of desmosterol
and cholesterol sulfate increase (Legault
et al., 1979; Inskeep and Hammerstedt,
1982; Nikolopoulou et al., 1985~; the rela-
tive distribution of the different polar
lipids also changes, some increasing while
OCR for page 67
EPIDIDYMAL STRUCTURE AND FUNCTION
others decrease (Nikolopoulou et al.,
1985~. The change in lipid composition
has been proposed as the cause of the in-
creased sensitivity of caudal spermatozoa
to cold (Nikolopoulou et al., 1985~. The
relationship between the state of matura-
tion of spermatozoa and the lipid makeup
of their cell membrane has not yet been de-
termined, but there might well be a sig-
nificant relationship, because of the
constraints put on membrane fluidity in
sperm-egg interaction. If such a rela-
tionship were established, measurements
of lipid composition or membrane fluidity
of spermatozoa might become useful
markers.
The changes in metabolic activity of
spermatozoa during epididymal transit
are numerous (Voglmayr, 1975; Brooks,
1981~. Increases in the glycolytic and
respiratory activity of spermatozoa dur-
ing epididymal transit have been reported
(Dacheux et al., 1979; Voglmayr and White,
1979~. The increase in cAMP associated
with spermatozoa as they mature (Del Rio
and Raisman, 1978; Amann et al., 1982) has
been attributed to both an increase in the
synthesis of this second messenger and
a decrease in its hydrolysis (Purvis et
al., 1982~. There are so many indices of
metabolic activity that it is not clear
which, if any, would be useful markers of
sperm maturation (Cooper et al., 1988~.
Research in this subject could yield impor-
tant results, but for the present it must
be viewed as Fishing expeditions
Functional Changes
Content of Epididymal Spermatozoa
One of the most powerful available bio-
logic markers of epididymal function is
the number of spermatozoa in different
regions of the epididymis (Amann, 1981~.
Because the heads of spermatozoa are very
tightly condensed and resistant to homog-
enization, it is possible to homogenize
a segment of epididymis, filter the ho-
mogenate, and count the number of condensed
sperm heads with a hemacytometer (an in-
strument usually used for counting blood
cells). The method requires the destruc-
tion of the tissue, but it is sensitive and
67
has the same degree of variability as any
other method that depends on hemacyto-
metric determinations; because of inter-
ference by debris, it has not been possible
to adapt electronic cell-counting methods
to this application. Results from hemacy-
tometric determinations not only give an
accurate index of sperm content in dif-
ferent epididymal regions where various
functions take place (Robaire et al., 1977;
Trasler et al., 1988), but also can be used
to indicate the sperm reserve within a
tissue (Amann, 1981~.
Transport of Spermatozoa
In spite of the very large range in
sperm production rate in different spe-
cies, there is great consistency in how
long it takes for spermatozoa to traverse
the epididymis: approximately 10 days (see
Table4- l ~ (Robaire end Hermo, 1987~. How-
ever, the transit time for spermatozoa
in the human epididymis is variable (Rowley
et al., 1970; Amann, 1981; Orgebin-Crist
and Olson, 1984~. The time spent by sper-
matozoa in the cauda epididymis is longer
and more variable than that spent in any
other segment. That is presumably because
sperm transit in the caput and corpus epi-
didymis is independent of ejaculatory fre-
quency, whereas that in the cauda epididy-
mis depends on this frequency (Amann and
Almquist, 1962; Swierstra, 1971; Kirton
et al., 1967~. The rate of luminal flow
in different segments of the rat epididymis
decreases from 210 mm/in in the initial seg-
ment to 32 mm/in in the distal caput and 12
mm/in in the cauda epididymis and vas defer-
ens(Jaakkola, 1983~.
The mechanisms responsible for driving
the luminal contents through the efferent
ducts and epididymis include hydrostatic
pressure (Johnson and Howards, 1976;
Pholpramool et al., 1984), muscular con-
tractions (Jaakkola and Talo, 1083), and
ciliary action (Markkula-Viitanen et al.,
1979~.
Norepinephrine (Pholpramool and Triph-
rom, 1984), acetylcholine (Pholpramool
and Triphrom, 1984), and vasopressin
(Jaakkola and Talo, 1981) have been pro-
posed as regulators of the muscular con-
tractions and ciliary action. In addition,
OCR for page 68
68
epididymal tubular contraction can be
regulated by prostaglandins and by drugs
that affect their synthesis (Cosentino
et al., 1984~. The relative contributions
of hydrostatic pressure, electric activ-
ity, and ciliary action in driving luminal
flow are still unresolved; it is unlikely
that any factor accounts fully for luminal
flow in all mammals.
Assessment of transit time of spermato-
zoa through the epididymis and the rate
of luminal flow are unlikely to become
practical markers for studies in humans.
However, numerous chemicals can probably
affect transit time through the epididymis
by altering neuronal activity or affecting
one of the other regulatory mechanisms.
Studies are needed to establish which drugs
can modulate such transport and whether
the effects will alter the ability of sper-
matozoa to fertilize eggs or to produce
normal, viable offspring.
Acquisition of Fertilizing Ability
In mammals, spermatozoa leaving the
testis do not have the ability to fertilize
eggs, whereas those in the cauda epididymis
have acquired this function. It took
several decades to resolve whether the
role of the epididymis in that function
was passive or active, but several elegant
studies by Orgebin-Crist and her col-
leagues (Orgebin-Crist et al.; 1975) and
by Bedford (1966) clearly established that
the epididymis was actively involved in
converting immature to mature (i.e., fer-
tile) spermatozoa. The acquisition of
the potential to fertilize eggs and, separ-
ately, produce viable offspring appears
not to be a simple on-off situation (Nishi-
kawa and Waida, 1952; Orgebin-Crist,
Af4LEREPRODUCTII~E TOXICOLOGY
et al., 1975; Bedford, 1979; Turner, 1979;
Courot, 1981; and Orgebin-Crist and Olson,
1984.)
Whether, and if so where, within the
epididymis spermatozoa acquire not only
the ability to fertilize eggs, but also
to produce normal, viable offspring is
the most important marker of epididymal
function. To use such a marker, spermato-
zoa from different regions of the epididy-
mis need to be removed and inseminated into
females of known fertility, and the result-
ing progeny outcome must be analyzed.
Such studies are expensive and tedious
and require the use of large numbers of
animals; however, they are the only proven
means of determining whether a substance
will selectively affect the site of matura-
tion of spermatozoa, if there is any matur-
ation at all. These tests need to be used
more extensively to determine whether
there are substances that alter the ability
of the epididymis to mature sperm or the
site where such maturation is acquired.
The development of new simple approaches
with great predictive value that will
clearly establish whether failure of
spermatozoa to mature has been caused by
an epididymal defect has high priority.
Associated with the acquisition by sper-
matozoa of the ability to fertilize eggs
is a gain in potential for motility. In
studies in which different regions of the
epididymis were ligated and spermatozoa
were removed, it became apparent that sper-
matozoa could acquire the potential for
motility without acquiring the ability
to fertilize eggs (Bedford, 1967; Orgebin-
Crist, 1967a; Cummins,1976~.
Except possibly in rabbits, the cauda
fluid composition prevents movement of
spermatozoa. The underlying mechanism
1967b; Orgebin-Crist, 1968; Blandau and of the acquisition of potential for motili-
Rumery, 1964; Dyson and Orgebin-Crist, ty is unknown. A number of factors have
1973; Frenkel et al., 1978; Fournier-Del- been proposed as regulators or mediators,
nech et al 1979. 1981). There is some including forward-motility protein
(Hoskins et al., 1978; Acott et al., 1979),
acidic epididymal glycoprotein (Pholpra-
mool et al.. 1983). albumin (Pholoramool
species variation with respect to the site
at which spermatozoa gain their fertiliz-
ing potential, but it is evident that pas-
sage through some part of the caput is es-
sential and that no species relies on the
entire length of the epididymis for acquir-
ing fertilizing potential. (For reviews,
see Orgebin-Crist, 1969; Orgebin-Crist
et al.3 1983)3 carnitine (Hinton et ale
1981; Inskeep and Hammerstedt31982)3 cAMP
(Hoskins and Casillas3 1975; Amann et al.3
1982)3 sperm-motility inhibiting factor
(Turner and Giles3 1982)3 sperm-motility
OCR for page 69
EPIDIDYMAL STRUCTURE AND FUNCTION
quiescence factor (Carr and Acott, 1984),
and immobilin (Usselman and Cone, 1983~.
Only in the past few years has quantita-
tive objective assessment of spermatozoa!
motility become available. The difficulty
in monitoring sperm motility lies in the
variability in the degree and type of mo-
tion of a large number of spermatozoa. The
method routinely used in most laboratories
was the subjective determination of the
percent of spermatozoa that were motile
and the type of motility on a 0-4 or 0-10
scale, with 0 being immotile and with in-
creasing numbers reflecting more progres-
sive motility (shaking, circular, for-
ward, forward progressive). The advent
of high-speed videomicrography made it
possible to record spermatozoa! motility
accurately. Such recordings can be ana-
lyzed with computer imaging techniques
to provide accurate, objective assessment
of the distribution of spermatozoa! speed,
extent and angle of circular movement,
lateral head displacement, beat angle of
the flagellum, and so on. These methods
have been developed for human semen analy-
sis (Ginsburg et al., 1988; Mahony et al.,
1988) and are only beginning to be used to
characterize the acquisition of sperm
motility during epididymal transit (Work-
ingandHurtt,1987~. Whether and how sperm
motility can be altered by chemicals that
affect the epididymis has yet to be deter-
mined. Various measures of sperm motility
have the potential of being powerful mark-
ers of one of the major functions of the
epididymis, but fulfilling the potential
will require much more research, which
is now technically feasible.
Storage of Spermatozoa
The major site for storage of spermato-
zoa in the mammalian duct system is the
cauda epididymis. Although normal transit
time of spermatozoa through the cauda is
some 3-10 days (Robaire and Hermo, 1987),
they can be stored in this tissue for peri-
ods of over 30 days (Orgebin-Crist et al.,
1975~. On storage in the cauda, a loss in
fertilizing ability was found to occur
before a loss in motility (Martin-Deleon
etal., 1973;Cummins,1976~.
An important difference between humans
69
and a number of other mammalian species
is that other mammals have a high sperm pro-
duction rate so that the number of stored
spermatozoa available for ejaculation
Is 3-, times greater than the daily sperm
production rate and 2-3 times greater than
that found in a"typical" ejaculate (Amann,
1981). In contrast, humans have a sperm
production rate well below that of most
other mammals and a sperm reserve available
for ejaculation that is only about equal
to the number of sperm in an ejaculate,
whether the person has been at sexual rest
or not.
To assess the ability of the cauda epi-
didymis to store spermatozoa, one either
would have to count the sperm in the tissue
after homogenization (a method clearly
limited to animal experimentation) or
would have to obtain serial ejaculates
(minutes to hours apart) to assess the size
of the sperm reserve in the cauda epididy-
mis. This marker of epididymal function
might provide some useful information
about the ability of the cauda epididymis
to store spermatozoa, but it is not very
practical and has not yet been used to
monitor damage to the epididymis other
than that caused by heat (Bedford, 1977;
Bedford, 1978a,b).
EPIDIDYMAL LUMINAL FLUID
The epididymal lumen contains water,
ions, small organic molecules, proteins
and glycoproteins, spermatozoa, and other
particulate matter of undefined origin.
From the efferent ducts all the way through
to the vas deferens, numerous changes take
place in the makeup of this complex of
substances. The only available means of
monitoring changes in the composition of
the luminal fluid, without removing or
irreversibly damaging the tissue, is
analysis of the epididymal contribution
to semen. Such analysis has been used
extensively to monitor several organic
molecules and proteins as markers of
epididymal function, but is of no value
in assessing changes in ionic makeup along
the epididymis. Micropuncture of epididy-
mal tubules has provided valuable informa-
tion, but does not allow for serial studies
and cannot be usect in humans (Hinton and
OCR for page 70
70
Howards, 1982~. We therefore focus here
on luminal markers that either can easily
be measured in semen or are particularly
closely related to the epididymis but need
to be measured in isolated tissues.
There is a precipitous decrease (by
nearly 100 mM) in the concentration of
chloride between the efferent ducts and
the caput epididymis, whereas a similar
decrease in sodium is observed between
the caput and cauda epididymis (Crabo,
1965; Levine and Marsh, 1971; Jenkins et
al., 1980~. The increase in potassium ion
(more than 30 mat) does not account for the
change in osmolarity, and it has therefore
been proposed that the epididymis is
involved in the secretion of organic ions
(Levine and Marsh, 1971~. Phosphorus,
whose serum concentration is usually
around 2 mM, becomes higher than 90 mM
in the corpus epididymis; this high con-
centration is accounted for, in part,
by the incorporation of phosphorus into
glycerylphosphorylcholine and phospho-
choline, which make up approximately 50
mM and 20 mM, respectively, leaving an
inorganic phosphorus concentration of
more than 10 mM in the cauda epididymis
and vas deferens (Hinton and Setchell,
1980a). It would be of interest to deter-
mine whether the very high inorganic
phosphorus concentration is maintained
by the action of parathyroid hormone or
vitamin D in this tissue.
AL4LE REPRODUCTIVE TOXICOLOGY
inositol and no other sugars is most in-
triguing, in that no component of inter-
mediary metabolism of either spermatozoa
or epididymal epithelium seems to have
a preference for inQsito1 as a source of
energy; such high concentrations are also
unlikely to be required to mediate hormone
action via the phosphatidyl-inositol
phosphate system. Testosterone is the
most abundant androgen entering the epi-
didymis, and dibydrotestosterone becomes
the major androgen in the caput epididymis,
increasing by more than 200-fold between
the rate testis and the caput epididymis
in bulls and rats (Ganjam and Amann, 1976;
Turner et al., 1984~.
All the above chemicals have been meas-
ured in semen as potential indicators
of epididymal function; the large collec-
tion of studies has not provided a clear
answer as to their value as markers (Mann
and Lutwak-Mann, 1981; Cooper et al.,
1988~. That stems in part from the fact
that many of the clinical studies were
poorly controlled and in part from the lack
of basic physiologic knowledge about the
epididymis, which would permit rational
interpretation of the results. The pos-
sibility that chemicals or enzymes se-
creted by sex accessory tissues can alter
the concentrations of substances secreted
by the epididymis should also be kept in
mind.
Amp specific nrntein.s senarahle hv
Carnitine (Hinton and Setchell, 1980b),
glycerylphosphorylcholine (Hinton and
Setchell, 1980a), phosphocholine (Hinton,
1980), inositol (Hinton, et al., 1980),
sialic acid (Arora et al., 1975), glycerol
(Cooper and Brooks, 1981), and steroids
(Ganjam and Amann, 1976; Turner et al.,
1984) are dramatically concentrated in
the lumen or change radically in concentra-
tion as one moves down the testicular duct
system. The physiologic role of high
concentrations of carnitine has not yet
been resolved, but one of the proposed
functions of carnitine is as a precursor
to acetylcarnitine, which is used as a
source of energy by spermatozoa or is used
to promote the maturation of spermatozoa
during epididymal transit (Casillas and
Chaipayungpan, 1979~. Why the epididymal
lumen should accumulate specifically
~ ~ ,
electrophores~s, are present in the
lumen of the epididymal duct system of
mammals. Using micropuncture methods,
Koskimies and Kormano (1975) and Turner
(1979) demonstrated not only that the disk
gel electrophoretic patterns of proteins
of different segments of the epididymis
and vas deferens differed from those of
serum or rete testis fluid, but also that
they differed from each other-i.e., there
were gradual changes in electrophoretic
pattern from the caput to the corpus epi-
didymis and vas deferent.
The identities of most of the proteins
have not yet been established. However,
in the past few years, a few molecules found
in the epididymal lumen with specific
physicochemical characteristics or
identifiable biological activity have
been described. Albumin, a2-macroglobu-
OCR for page 71
EPIDIDYMAL STRUCTURE AND FUNCTION
fin, transferrin, and androgen-binding
protein have been found in epididymal
luminal fluid (Amann et al., 1973; Skinner
and Griswold, 1982; Turner et al., 1984~.
Other proteins-such as acidic epididymal
glycoprotein (Lea and French, 1981),
dimeric acidic glycoprotein (Sylvester
et al., 1984), forward-motility protein
(Brands et al., 1978), angiotensin- 1 -
converting enzyme (Vanha-Perttula et al.,
1985), and ~x-lactalbumin-like protein
(Hamilton, 1981)-are also likely to be
in the mammalian epididymal lumen, but
evidence of their presence in the luminal
compartment is indirect. Many of the
proteins are beginning to be used as mark-
ers of epididymal function; no clear
picture has yet emerged regarding their
ability to reflect epididymal functions,
but such results are likely to become
available soon.
It is important to note that 90-95% of
the fluid coming from the testes is re-
sorbed in the efferent ducts and the ini-
tial segment of the epididymis (see,
e.g., Robaire and Hermo, 1987~. This water
resorption will be a factor in the con-
centration measured for all the chemicals
mentioned above.
EPIDIDYMAL EPITHELIAL
FUNCTION
Histology
Presence and Relative Distribution of
Various Cell Types
Detailed descriptions of the appear-
ance, at the light and electron microscopic
levels, of the epididymis in a number of
animals and humans are available (Benoit,
1926; Hamilton, 1975; Connell and Don-
jacour, 1985), and have been reviewed by
Robaire and Hermo (1988~. The regional
changes in epididymal histology have been
described (e.g., for rat, see Reid and
Cleland,1957~.
The epididymal epithelium is pseudo -
stratified and is made up of principal,
basal, clear, and halo cells. The major
cell type is the tall, columnar principal
cell, which makes up about 80% of all epi-
didymal epithelial cells (Robaire and
71
Hermo, 1987~. These cells are involved
in resorption of fluid and organic material
and in secretion of small organic mole-
cules, as well as proteins and glycopro-
teins. The next most important cell type
(about 15%) is the basal cell, flat elon-
gated cells that are found throughout the
epididymis in contact with the basement
membrane; their function has not yet been
ascertained. Clear and halo cells are more
sparsely distributed along the epididy-
mis. In the species in which they are found,
clear cells might be involved in resorbing
the elements of cytoplasmic droplets that
are shed by spermatozoa as they mature.
These cells are not found in all mammals,
e.g., the ram epididymis does not have any.
Halo cells are believed to be part of the
immune system and have been described as
either monocytes or lymphocytes (Robaire
end Hermo, 1987~.
The quantitative distribution of these
cell types as one moves from one segment
of the epididymis to the next has recently
been described not only in intact rats,
but also in animals that had been treated
chronically with the antitumor and im-
munosuppressive agent cyclophosphamide
(Robaire and Hermo, 1987; Trasler et al.,
1987b). There were marked changes in the
proportion of various cell types from the
initial segment of the caput to the cauda
epididymis and changes in the relative
cell surface area of a particular cell
type in selected segments of the epididymis
at specified times after initiation of
treatment.
For several reasons, epididymal biop-
sies have not become routine. Because
the epididymis is a single long convoluted
tubule, a biopsy would result in a disrup-
tion of the tubule and hence prevent
normal transport of spermatozoa through
it. Kelatively little is known about the
functional histology of the tissue, so
the practical consequences of an altered
histologic appearance would not be evi-
dent. Thus, although it is clear that
biopsies of epididymal tissues should not
be routine, it is also evident that de-
tailed analyses of the relative distribu-
tion of various cell types and of histolog-
ic appearance can provide valuable infor-
mation on the actions of some drugs on this
OCR for page 72
OCR for page 74
OCR for page 75
Representative terms from entire chapter:
cauda epididymis
72
tissue and accordingly can act as useful
markers.
Blood-Epididymis Barrier
The anatomic and functional existence
of a blood-testis barrier is well estab-
lished (Setchell and Waites, 1975), but
only since the late 1970s has there been
substantial evidence of a blood-epididy-
mis barrier (Friend and Gilula. 1972:
Howards et al., 1976; Suzuki and Nagano,
1978: Cavicchia. 1979: Greenberg and
, ,
,
Forssmann, 1983; Hoffer and Hinton,
1984), as reviewed by Robaire and Hermo
(1988~. Given the presence in spermatozoa
of proteins that are recognized by the body
as foreign, it stands to reason that there
should be a continuation of a functional
barrier beyond the testis.
There is extensive functional evidence
of such a barrier: the large differences
in the concentrations of inorganic and
organic compounds between the luminal
fluid and the blood (Crabo and Gustafsson,
1964; Jenkins et al., 1980; Turner et al.,
1984; Hinton, 1985~. The barrier is repor-
tedly resistant to various treatments,
such as high-dose administration of estra-
diol (Turner et al., 1981), the application
of gossypol (Hoffer and Hinton, 1984),
vasectomy (Turner and Howards, 1985), or
the presence of varicocele (Turner and
Howards, 1985), but little is known about
its role in protecting spermatozoa from
immunoglobulins and toxicants. Indeed,
the ability of the barrier to maintain its
tightness under conditions of stress might
be pivotal in allowing the epididymis to
sustain its functions. There is no evi-
dence that environmental toxicants or
drugs can disrupt the blood-epididymis
barrier. However, if it were disrupted,
the potential consequences could include
immobilization of spermatozoa by antibod-
ies and alteration of the sperm genome by
chemicals. It is therefore important to
develop simple markers that will allow
for the monitoring of the integrity of the
blood-epididymis barrier without disrupt-
ing epididymal function.
Spermiophagy
The fate of spermatozoa that are not
A[4LE REPRODUCTION TOMCOL~
ejaculated has been and remains controver-
sial. There is now morphologic evidence,
especially for the vas deferens, that
spermiophagy does occur in a number of
species under normal conditions or after
mechanical or chemical manipulation. Such
a process might involve the epithelial
cells that line the duct system or the
presence of luminal macrophages. In either
case, spermatozoa in various stages of
degeneration have been reported in epithe-
lial cells and in luminal macrophages
(Cooper and Hamilton, 1977; Holstein,
1978; Murakami et al., 1982a,b; Murakami
et al., 1984), and it has been suggested
that the spermatozoa are undergoing lysis.
Most observers have reached the conclusion
that the epithelial cells of the epididymis
and the vas deferens are not involved in
such activity under normal conditions
(Bedford, 1975; Orgebin-Crist, 1984~.
However, mechanical or chemical manipula-
tion of the epididymis and vas deferens
has been shown to cause epithelial cells
of the different segments of the duct
system to become phagocytic and to engulf
and digest spermatozoa (Grover, 1969;
Flickinger, 1972; Alexander, 1973; Hoffer
et al., 1973; Hoffer and Hamilton, 1974;
Hoffer et al., 1975; Neaves, 1975~. Also,
spermiophagy by luminal macrophages has
been shown to occur under abnormal condi-
tions, (Neaves, 1975; Phadke, 1975; Bed-
ford, 1976; Flickinger, 1982~.
The ability of the epithelial lining
of the duct system to become active in
spermiophagy apparently depends on either
the presence of excess spermatozoa or the
presence of ~abnormal" spermatozoa in the
lumen; the mechanism is not clear. How-
ever, histologic observation and measure-
ment of the extent of spermiophagy by the
epididymal or vas deferens epithelium
might provide a good indication of the
extent of sperm damage. This potential
marker has not been tested systematically
for any family of drugs.
Biochemical Markers
Hormone Receplors and Regulation of
Epididymal Function
Androgens—especially 5
EPIDIDYMAL STRUCTURE AND FUNCTION
tosterone (DHT), the 5c'-reduced metabo-
lite of testosterone—are the primary
modulators of epididymal function. How-
ever, it has become apparent that many
other regulatory molecules play special-
ized roles in maintaining normal epididy-
mal function. The factors that regulate
epididymal function-i.e., those for which
there are specific binding proteins-do
not reach the epididymis only through the
circulation (endocrine), but also through
direct input from the testis (paracrine).
Androgens. The dependence of the epidid-
ymis on androgens has been established
for over 60 years (Benoit, 1926~. Many
studies during the past 3 decades have
tested the effects of castration and
testosterone replacement on a large array
of characteristics (Orgebin-Crist et al.,
1975; Brooks, 1979a), and several general
conclusions can be drawn. The epididymis
atrophies and treatment with physiologic
concentrations of androgen only partially
maintains epididymal weight; the remaind-
er attributable to spermatozoa and luminal
fluid (Karkun et al., 1974; Robaire et al.,
1977; Brooks, 1979b). In that respect,
changes in epididymal weight could be a
marker of changes in testosterone.
Androgens regulate intermediary metab-
olism (Brooks, 1979a), the transport of
ions across the epididymal epithelium
(Won" and Young, 1977), the transport of
inositol and carnitine across the mem-
branes of epididymal epithelial cells
(Bohmer et al., 1977; Brooks, 1980; Phol-
pramool et al., 1982), and the synthesis
and secretion of a number of epididymal
glycoproteins as well as the activity of
several enzymes, e.g., glutathione S-
transferase (Rastogi et al., 1979; Jones
et al., 1980; Moore, 1981; Mayorga and
Bertini, 1982; Robaire and Hales, 1982;
Brooks, 1983~. Whether any of these ef-
fects are directly mediated by androgens
or require the synthesis of new mRNA and
therefore the synthesis of new proteins
is being investigated.
Acquisition of fertilizing ability
and storage of spermatozoa both depend
directly on androgens (Benoit, 1926;
Bedford, 1975; Cohen et al., 1981~. The
presence of an inhibitor of 5~-reductase
caused decreases in the number of motile
73
spermatozoa, in the percentage of oocytes
fertilized, and in the number of blas-
tocysts found—but only when the inhibitor
was given with testosterone.
There is no debate that androgens regu-
late epididymal function, but the mechan-
ism of their action is far less clear. Two
molecules in the epididymis have high
affinity for DHT; these have been named
~androgen receptor~ and "androgen-
binding protein." It must be stressed,
however, that the classical conditions
necessary to demonstrate that a given
molecule is the androgen receptor in the
epididymis have not been fulfilled.
Exogenously administered testosterone
is transformed to DHT and is found bound
to a cytosolic protein in the epididymis
of a number of animals (Blaquier, 1971;
Ritzen et al., 1971; Danzo et al., 1973;
Younes and Pierrepoint, 1981; Carreau et
al., 1984~. Epididymal cytoplasmic recep-
tor seems to have many features in common
with prostatic androgen receptor (Tindall
et al., 1975; Younes and Pierrepoint,
1981). The binding of DHT to the cytoplas-
mic receptor is blocked by antiandrogens,
such as cyproterone acetate and flutamide
(Tindall et al., 1975; Danzo and Eller,
1975~.
Androgen-binding protein (ABP) has
been found in many animals (Danzo et al.,
1977; Fabre et al., 1979; Carreau et al.,
1980) and humans (Hsu and Troen, 1978~.
It has a number of physicochemical proper-
ties that differ markedly from those of
the cytoplasmic androgen receptor (Hans -
son et al., 1975~. ABP is synthesized by
Sertoli cells and enters the epididymis
via the efferent ducts (Attramadal et al.,
1981; Musto et al., 1982~. Its functions
are still unresolved, but it has been
proposed that it acts as an androgen sink
in seminiferous tubules (Hansson et al.,
1975), transports androgens to the epidid-
ymis (Ritzen et al., 1971), and acts as a
regulator of epididymal 5~-reductase
(Robaire et al., 1981). In a rat model,
a correlation between the amount of epidid-
ymal ABP and the fertilizing ability of
spermatozoa has been established (Anthony
etal.,1984~.
Several biologic markers have been
used to assess androgenic action OI1 the
74
AL4LE REPRODUCTIVE TOXICOLOGY
epididymis. The first is the serum con- tritiated DHT and estradiol in the adult
centration of testosterone. Although mouse epididymis revealed differences
of limited value (it does not specifically in distribution of grains in different
reflect epididymal activity), it is a cell types, as well as in different seg-
useful gross indicator. The concentra- ments of the epididymis (Schleicher et
lions of ABP in semen and in epididymal al., 1984~. That finding brings up the
tissue fractions or luminal fluid have intriguing possibility that endogenous
' ^^ circulating, or potentially locally
synthesized, estradiol serves a specific
function in the epididymis, e.g., modula-
tion of clear cell function. The difficul-
ty in identifying estradiol receptors and
aromatase activity in adult mammalian
epididymis might be due to the localization
of activity to cells (e.g., clear cells)
that are not abundant in this tissue. The
inherent obstacles in establishing the
role of estradiol and of its receptor as
indicators of specific epididymal func-
tions suggest that the development of these
markers should be given a low priority.
Aldosterone. The concentrations of
ions change along the duct system, but
the mechanism responsible for controlling
these ion fluxes has not yet been eluci-
dated. Because of the similarities in ion
fluxes between the epididymis and the
kidney, Wong and Lee (1982) and Jenkins
et al. (1983) have proposed similar regula-
tory mechanisms. Indeed, specific
binding sites for aldosterone, the adrenal
mineralocorticosteroid normally con-
sidered to have the kidney as its target
site, have been found autoradiographical-
ly to be selectively disposed over clear
cells (Hinton and Keefer, 1985) and have
been shown to be involved in regulating
the concentration of spermatozoa in the
epididymis (Turner and Cesarini, 1983~.
More studies are required to elucidate
the role of aldosterone in epididymal
function and to determine whether specific
markers can be developed to reflect its
epididymal activity.
Prolactin. In light of the possible coreg-
ulation of gonadotropin and prolactin
release, the apparent presence of prolac-
tin "receptors" in the epididymis (Aragona
and Freisen, 1975; Orgebin-Crist and
Djiane, 1979), and the proposal that
prolactin regulates ion transport (Shiu
and Friesen, 1980), it is plausible that
this hormone plays a major role in regulat-
ing some facets of epididymal function.
been used as a combined indicator of Ser-
toli cell and epididymal functions.
Androgen-receptor and 5~-reductase
activities have been measured as markers
of the androgenic status of the epididymis;
although these two markers are highly
specific and are probably among the most
useful of the markers discussed in this
section, they require removal of tissue,
which is possible only under experimental
conditions. If noninvasive techniques
could be devised to monitor these aspects
of androgen-related epididymal activity,
they would be extremely useful.
Estrogens. The administration of estro-
gens can affect the male reproductive
system in general (Gay and Dever, 1971;
Swerdloff and Walsh, 1973; Karr et al.,
1974; Verjans et al., 1974; Ewing et al.,
1977) and the epididymis in particular
(Meistrich et al., 1975; Orgebin-Crist
et al., 1983~. Although it has been gener-
ally accepted that mediation of estrogen
action takes place via the hypothalamo-
pituitary-gonadal axis (Swerdloff and
Walsh, 1973; Verjans et al., 1974; Robaire
et al., 1979), substantial evidence of
the direct action of estrogens on androgen
target tissues has accumulated over the
past 2 decades. Specific, well-regulated,
high-affinity cytosolic and nuclear
estrogen-binding proteins, presumably
receptors, have been identified in the
epididymis of mice (Schleicher et al.,
1984), rats (Van Beurden-Lamers et al.
1974), rabbits (Danzo and Eller, 1979),
dogs (Younes et al., 1979), and humans
(Murphy et al., 1980~. It is proposed that
stromal (as opposed to epithelial) cells
(Cunha et al., 1980) act as primary target
sites because estrogen adm~n~stration
to young dogs (Cornell and Donjacour, 1985)
or rabbits (Orgebin-Crist et al., 1983)
leads to increases in epididymal weight
that were due almost entirely to stromal
hyperplasia.
Autoradiographic localization of
EPIDIDYMAL STRUCTURE AND FUNCTION
Prolactin and its binding protein might
eventually become useful markers of epi-
didymal function, but the available data
suggest that this subject be given low
priority for research.
Vitamin D. The known primary function
of vitamin D (a sterol hormone) is the
homeostasis of calcium and phosphorus;
its major sites of action are bone, intes-
tine, and kidney (De Luca,1978~. A number
of complementary observations suggest
that the epididymis can also be a target
for its action. First, specific binding
proteins for 1,25-(OH)2 vitamin D have been
found in the rat epididymis (Walters et
al., 1982~. Second, the epididymis was
found to take up 25-(OH) vitamin Do and
metabolize it to both 1,25-(OH)2 vitamin
D3 and 24,25-
76
system is situated on the basolateral
membrane, cannot transport other hexoses,
and can be inhibited by 3-O-methylglucose,
a nonmetabolizable analogue that is recog-
nized by the transport system. Oxidative
metabolism is high right after birth in
the rat epididymis, decreases for the first
2 weeks of life, and then increases in
parallel with the increase in circulating
androgens (Delongeas et al., 1984~.
A number of other enzymatic processes,
such as prostaglandin biosynthesis and
metabolism, have been described for the
epididymis (Robaire and Hermo, 1987~.
We limit our discussion here to glutathi-
one, because of its potential usefulness
as a marker of epididymal function. Sever-
al enzymes involved in the biosynthesis,
metabolism, and conjugation of glutathi-
one are present in the epididymis. They
are particularly important, because free
glutathione is viewed by many toxicolo-
gists as a mechanism of "mopping up" elec-
trophiles that might damage cellular
components. The family of epididymal
enzymes involved in the conjugation of
glutathione with electrophilic chemicals,
the glutathione S-transferases, can be
resolved into six peaks, each with a char-
acteristic isoelectric point and sub-
strate specificity (Hales et al., 1980~;
the most acidic peak is much higher in the
epididymis than in other tissues and might
be specific for it. Results of studies on
the longitudinal distribution of these
enzymes in the epididymis indicate a
general trend toward decreasing activity
for each of the substrates from the caput
to the cauda epididymis (Hales et al.,
1980~. Thiol oxidase, an enzyme that
converts 2 R-SH + O2 into R-S-S-R and H2O,
has been reported in rat and hamster epi-
didymis (Chang and Zirkin, 1978~; enzyme
activity was higher in the cauda than in
the caput epididymis. It was proposed that
such an enzyme could protect spermatozoa
from endogenous free sulfhydryls. Excep-
tionally high concentrations of q-gluta-
myl-transpeptidase have also been report-
MALE REPRODUCTIVE TOXICOLOGY
ed in the rat epididymis (DeLap et al.,
1977~. That enzyme is involYed in the
transport of q-glutamyl amino acids and
can act as a glutathione oxidase, convert-
ing reduced to oxidized glutathione (fate
end Orlando, 1979~.
EPIDIDYMALLY MEDIATED TOXIC
DRUG EFFECTS
A growing number of compounds have been
shown to affect the epididymis, e.g., a-
chlorohydrin, 6-chloro-6-deoxyglucose,
and possibly gossypol (Robaire and Hermo,
1987~. The epididymis, however, has not
usually been studied as one of the target
tissues for the toxic effects of drugs or
environmental toxicants, so little is
known about the importance of this tissue
as a target for toxic drug effects.
For some compounds, such as dibromo-
chloropropane (DBCP), it has clearly been
shown that there is an effect on the epidid-
ymis, e.g., on epididymal weight or sperm
reserves (Amann and Berndtson, 1986~; but
it is still not clear whether the deleter-
ious effects of this drug, and others like
it, on reproductive outcome are directly
linked to their action on the epididymis
or epididymal spermatozoa.
Clear evidence has emerged from some
recent studies that-after a week of treat-
ment with such drugs as cyclophosphamide,
an anticancer and immunosuppressive drug
(Trasler et al., 1985, 1986~; methylnitro-
sourea, an alkylating agent (Nagao, 1987~;
or methyl chloride, an industrial gas
(Chellman et al., 1986~-a sharp increase
in preimplantation and postimplantation
loss (dominant lethal mutations) could
be found. Such male-mediated adverse
effects on reproductive outcome were
associated, in the case of cyclophospha-
mide, with specific changes in the dis-
tribution of cell types in the epididymal
epithelium and an increase in the number
of spermatozoa with abnormal flagellae
in the rat epididymis (Trasler et al.,
1988~.
