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OCR for page 47
4
Biologic Markers of Testicular Function
This chapter focuses on physical and
chemical markers of the testes; stere-
ologic and biochemical assessments of
Leydig cells, Sertoli cells, and germ
cells; and molecular biologic analyses
of DNA and RNA in germ cells. Some markers
other than semen analysis (discussed in
Chapter 7) are noninvasive or minimally
invasive and can be used to assess testicu-
lar function in human males exposed to
toxicants. In addition, several promising
noninvasive or minimally invasive markers
using new imaging techniques, molecular
biologic assays, and biochemical assays
of saliva, serum, and urine are identified.
The testis has two compartments: the
interstitium and the seminiferous tug
bules. The interstitium contains Leydig Figure 4-1 shows the human germ cell
cells that produce the male hormone tes- types, the life span of each, and the time
tosterone. Testosterone causes the dif- required for each to reach the ejaculate.
ferentiation and development of the The figure illustrates three important
fetal reproductive tract, the neonatal points. First, the human testis has 14
organization of what will become androgen- recognizable types of germ cells. Second,
dependent target tissues in puberty and toxic effects on a specific germ cell might
adulthood, the masculinization of the male not be manifested in the semen for alive car
at puberty, and the maintenance of growth
and function of androgen-dependent organs
in the adult.
The seminiferous tubules contain ger-
minal epithelium and supporting cells.
The supporting cells include Sertoli
cells; in adults, Sertoli cells are static
nonproliferating cells that are inti-
mately associated with and support ger-
minal cells involved in spermatogene-
sis, the production of spermatozoa. The
germinal epithelium is populated by cells
that give rise to spermatozoa. Spermato-
genesis encompasses a phase during which
primitive spermatogonia divide either
to replace their number (stem cell renewal)
or produce new spermatozoa that are com-
mitted after additional mitotic divi-
sions to become spermatocytes; a meiotic
phase during which spermatocytes undergo
the first and second meiotic divisions
that result in haploid spermatids; and
a spermiogenic phase during which sper-
matids undergo a dramatic metamorphosis
in size and shade to form snermat~zon
weeks, because more mature unaffected
cells will continue to develop and appear
in the ejaculate. Third, the time of ap-
pearance of defective, immotile, or re-
duced numbers of spermatozoa in the ejacu-
late provides important information
about the germ cell type affected by a toxi-
cant. Figure 4-2 presents an example to
47
OCR for page 48
48
ll
MALE REPRODUCTIVE TOXICOLOGY
go
o
o
CL
CD
CD
lo
o
:~
-
~ LL
c 11_'
CC ~
~ a)
Duration
of Stage
(days)
en
=0
Z ~ ~ ~
O`r Em
lL ~ llJ
m~ On
Oh oh ~ Oh
~ Oh
<0
Z
O ~
LL oh
an
an
N IL
cat
LL
~ LL
Ant
fir
O
co
Z
a,,)
_
Oh ~
_
IL
~ C)
C) Cal
~ >
Ad Ap B PL L Z P II Sa Sb1 Sb2 SO Sd1 Sd2 us
1 1 ~ _
18.5 1 8.8 1.0 3.7 ~ 2.9 15.6 0.8 8.0 1.0 1.2 5.8 4.8 T 1.6 1 -12 r c'
LU
r I , , ,
' ~ 1 1 ' - ' ~ 1
1 1 1 1 1 1 1 1 1
I 1 1 1 1 1 , 1
1 1 1 1 1 ~ 1 1
~ 1 1 1 1 1 1 1 1
Time to reach ~ I I I I ~ ~ I I
Ejaculate I I I I I I I I I
(days) 85.7 67.2 58.4 57.4 53.7 50.8 35.2 34.4 26.4 25.4
-
1 1
1 1
1 1
1 1
1 1
1 1
24.2
1 1 1
1 1 1
1 1 1
1 1 1
1 1 1
1 1 1
18.4 13.6 12
1 1
l
o
FIGURE ~1 Spermatogenesis in man, showing life span of each cell and time necessary to reach ejaculate.
Redrawn from da Cunha et al., 1982.
illustrate those points. Spermatozoal
concentration and motility were monitored
at various times after 12 courses of treat-
ment of a melanoma patient with AMSA [4'-
(9 -acridinylamino Methane - sulfon - m -
anisididel. The slow decline in the num-
ber of motile spermatozoa in the ejaculate
was interpreted by the investigators as
suggesting that there was no immediate
damage to spermatozoa transport or the
epididymis. They believed that primarily
postspermatogonial germ cells were killed
by the AMSA therapy. (Initially, the cells
that were killed were primary spermato-
cytes; later, type B spermatogonia were
also killed.) They interpreted the rapid
recovery of sperm concentration and motil-
ity in the semen 13 weeks after the first
nine courses of AMSA treatment as showing
that type A (stem) spermatogonia were not
irreversibly affected.
PHYSICAL AND CHEMICAL
MARKERS OF TESTICULAR
FUNCTION
Markers in Use
Testicular consistency and size have
been considered important in the clinical
evaluation of human male fertility. With
tonometers, one can measure testicular
consistency through the stretched scrotal
skin. Tonometer readings have been corre-
lated with clinical impressions of tes-
ticular consistency in human males (Lewis
et al., 1985) and with sperm morphology
in bulls (Hahn et al., 1969~. The advan-
tages of testicular consistency as a bio-
logic marker are that it is non~nvas~ve
and simple to measure. The disadvantages
are that it is unrelated to a specific de-
fect in testicular function and that
large changes are required to detect ef-
fects of treatment.
Testicular weight is directly corre-
lated with testicular volume, which can
be estimated from testicular size in many
vertebrates (Bailey, 1950; Kenagy, 1979;
Handelsman and Staraj, 1985~. In mammals,
the bulk of testicular volume and therefore
OCR for page 49
L4RKERS OF TESTICULAR FUNCTION
Courses of AMSA
1 2 3 4 5 6 7 ~ 9 10 11 12
,,, 10'
a)
_ ._
O
a) ~
~ ~ 1 o6
I-—
O
105
1o2
\60%
~40%
\10%
\5%
\ 7
ospermia
~/////////////~///////~///////////////~
0 20 40 60
Weeks in Observation
weight is accounted for by germ cells
(Amann, 1 970a). Estimated testicular
volume based on measured size is a simple
and noninvasive marker of sperm production
(Foote, 1969; Chubb and Nolan, 1985~.
However, it is imprecise, because errors
are inherent in the measurement process
itself and in the calculation of volume
of nonspherical objects. The use of weight
measurements, although precise, has the
disadvantage of being invasive, in that
the testes must be removed.
Markers Requiring Research
and Development
Organ chemiluminescence can give read-
ily detectable, continuously monitorable,
noninvasive signals of oxidative metabol-
ism (Boveris et al., 1980~. It is possible
that chemiluminescence can be used to moni-
tor the effect of toxicants on the radical
reactions of lipid peroxidation in testes
in situ or in testes perfused in vitro.
Kopp et al. (1986) used phosphorus-31
magnetic resonance imaging (MRI) to deter-
mine functional metabolic correlates,
temporal relationships, and intracellular
actions of cardiotoxic chemicals nonde-
structively in isolated intact perfused
rat hearts. They obtained useful informa-
tion on biochemical mechanisms respon-
49
FIGURE ~2 Sperm concentration and
motility during chemotherapy with AMSA
[~4'-9-acndinyl-aniino)
methan~sulfon~ -ariisidide] .
Source: da Cunha et al., 1982.
sible for the cardiotoxic actions of xeno-
biotics. This approach can probably be
applied profitably to an in vitro perfused
rat testis model immediately and perhaps
to the human testis in situ eventually.
One potential problem is that MRI might
alter testicular temperature. Other in-
direct measures of testicular size and
function are promising but have received
little attention, including ultrasound,
positron-emission tomography, and com-
puted axial tomography.
LEYDIG CELLS
Leydig cells are the principal source
of testosterone in the mammalian male (Ew-
ing and Zirkin, 1983~. Leydig cell growth
and differentiated function depend on
anterior pituitary production of lutein-
izing hormone (LH) (Ewing and Zirkin,
1983~. Toxicants can interfere with tes-
tosterone production indirectly by in-
terfering with gonadotropin-releasing
hormone (GnRH) stimulation of pituitary
gonadotropes, by interfering with LH pro-
duction by pituitary gonadotropes, or by
interfering with receptor-mediated LH
stimulation of testosterone secretion
by Leydig cells. Toxicants might also
inhibit the Leydig cell steroidogenic
apparatus directly.
OCR for page 50
so
Markers in Use
Stereologic techniques allow determina-
tion of Leydig cell numbers per testis at
the light microscopic level and of Leydig
cell cytoplasmic organelle volume and
membrane surface area (e.g., area of inner
mitochondrial membrane and smooth endo-
plasmic reticulum) at the electron micro-
scopic level in both humans (Mori et al.,
1982) and experimental animals (Mori and
Christensen, 1980~. Although they are
invasive and tedious to carry out, these
biologic markers of Leydig cell structure
have been shown to be highly correlated
with Leydig cell steroidogenic function
under a variety of experimental and physio-
logic conditions (Christensen and Pea-
cock, 1980; Zirkin et al., 1980; Ewing et
al., 1981~. For example, prolonged expo-
sure to lead causes a diminution in tes-
tosterone production and in the surface
area of smooth endoplasmic reticulum in
rat Leydig cells (Zirkin et al., 1985a).
In addition, Leydig cell numbers diminish
with advancing age in humans (Kaler and
Neaves, 1978~. These morphologic markers
are particularly helpful in studies aimed
at understanding mechanisms of toxicity.
Testosterone is commonly secreted epi-
sodically by Leydig cells in the mammalian
testis (Ewing et al., 1980~. There are
annual and diurnal rhythms in testosterone
production, and a diurnal rhythm and fre-
quent sporadic bursts of testosterone in
some species. The episodic nature of tes-
tosterone secretion in man is blunted,
compared with that in many species. The
variation complicates the assessment of
Leydig cell steroidogenic activity by
measurement of peripheral blood testos-
terone concentration, because a regimen
of frequent sampling must be followed,
especially in experimental animals
(Ismail et al., 1986~. Measurement of
testosterone concentration in blood
serum at 10-minute intervals for 8 hours
will usually indicate whether a toxicant
has altered the entire hypothalamo-adeno-
hypophysial-testicular axis in experimen-
tal animals. But toxic effects in humans
might be diagnosable with less frequent
or even a single serum testosterone meas-
urement, because testosterone production
MALE REPRODUCT~ TOMCOL~
is not so episodic. The function of the
hypothalamo-adenohypophysial link can
also be assessed by measuring serum LH
concentrations every 3-10 minutes for 24
hours. Measurement of LH and testosterone
concentrations in peripheral blood for
less than 24 hours but at the same frequency
will impart the same information regarding
the hypothalamo-hypophysial function.
Measurement of serum LH concentration
might be more useful than testosterone
pulses for diagnosing defects in the hypo-
thalamo - adenohypophysial- testicular
axis in humans, because of the attenuation
of testosterone pulses.
In both men and experimental animals,
the capacity of pituitary gonadotropes
to respond to GnRH can be assessed by in-
jecting a bolus of GnRH either intravenous-
ly or subcutaneously and then measuring
LH in peripheral blood. Similarly, the
capacity of Leydig cells to respond to
LH can be assessed by injecting a bolus
of human chorionic gonadotropin (hCG)
intravenously and measuring testosterone
in peripheral blood. Again, measurement
of LH and testosterone concentrations in
peripheral blood for a shorter period
but at the same frequency will impart the
same information; with less frequent sam-
pling, there is a loss of sensitivity and
precision in detecting an effect of expo-
sure to a toxicant. The advantages of meas-
uring the concentration of LH and testos-
terone in peripheral blood serum of humans
or experimental animals are that it direct-
ly monitors the gonadotrope and Leydig
cell function, respectively, and that
repetitive measurements can be obtained
for assessing temporal effects of a treat-
ment. Disadvantages of measuring LH and
testosterone in peripheral blood are that
only small amounts of blood can be collect-
ed from small rodents, unless red blood
cells are replaced and that, because LH
and testosterone production are episodic,
several measurements are required.
Most testosterone (over 90%) in periph-
eral blood is bound to albumin and tes-
tosterone estradiol-binding globulin
and therefore is biologically inert. If
it is important, free biologically active
testosterone can be measured by separating
free and protein-bound testosterone
OCR for page 51
MARKERS OF TESTICUI~4R FUNCTION
(Vermeulen et al., 1971~. This issue be-
comes important when increased sex ster-
oid-binding globulin results in a decrease
in free testosterone and causes hypogonad-
ism. An example is the effect of chronic
alcoholism in human males (Van Thiel et
al., 1974~.
Inhibition of Leydig cell steroidogene-
sis in viva causes a diminution in acces-
sory sex organ weight, discussed in Chapter
6 as a bioassay for peripheral blood tes-
tosterone concentration in experimental
animals (Dorfman and Shipley, 1956; Chubb
and Nolan, 1985; Coffey, 1986~.
Finally, Leydig cell steroidogenesis
can be monitored by removing testes from
experimental animals and measuring their
capacity to produce testosterone in vitro.
For example, testes of some species (e.g.,
mice and rats) can be dispersed with col-
lagenase and the production of testoster-
one by testicular cells measured (Bordy
et al., 1984~. Alternatively, testes of
numerous species have been perfused in
vitro (Ewing et al., 1981~. These tech-
niques are invasive and limited to a few
hours duration in vitro. But they can be
particularly helpful in studies aimed at
understanding mechanisms of toxicity.
Clearly, this approach is impractical in
humans.
Markers Requiring Research
and Development
It has been suggested that measurement
of testosterone in saliva is an excellent
biologic marker for Leydig cell testoster-
one production, because saliva can be col-
lected from humans repetitively in a non-
stressful manner and because salivary
testosterone concentration is correlated
closely with the biologically active free
testosterone in blood (Riad-Fahmy et al.,
1982~. Studies have borne this idea out:
human salivary testosterone concentration
has been shown to exhibit a circadian rhy-
thm (Magrini et al., 1986), to increase
after hCG stimulation (Nahoul et al.,
1986), and to be highly correlated with
pathophysiologic conditions that result
from modifications in serum testosterone
concentrations (Riad-Fahmy et al., 1982~.
To our knowledge, this technique has not
51
been used to monitor the effect of xeno-
biotics on Leydig cell function in humans.
It should be added to the armamentarium
of reproductive toxicologists and epide-
miologists, because it provides a nonin-
vasive, specific, accurate, and sensitive
marker to monitor exposure to or effects
of a toxic agent on Leydig cell steroido-
genic function in human males.
Measurement of serum or salivary tes-
tosterone concentration does not repre-
sent the integrated 24-hour rate of tes-
tosterone production. That can still be
achieved only by sophisticated study of
metabolic clearance rate, which is tedi-
ous, time-consuming, expensive, and im-
practical for application to humans. How-
ever, considerable evidence shows that
it is possible to monitor ovarian function
and pregnancy status by measuring gonado-
tropin and gonadal steroids and/or steroid
metabolites in morning or random urine
specimens from females of numerous species
(Lasley et al., 1980; Czekala et al.,
1981~. The advantage of this approach is
that the marker of interest accumulates
in urine, thus obscuring the episodic na-
ture of hormone production and making it
possible to estimate integrated hormone
production over time with fewer samples.
There are shortcomings (Edwards et al.,
1969; Curtis and Fogel, 1970), and a care-
ful evaluation will determine whether
specific hormones can be measured in
urine samples as biologic markers of the
functional status of the hypothalamo-
adenohypophysial-Leydig cell axis in
experimental animals and humans.
Leydig cells probably have functions
other than testosterone production.
Therefore, considerable research is re-
quired to uncover potentially new and
useful Leydig cell markers. The testis
contains peptides that are also formed
elsewhere in the body. There is evidence
of a GnRH-like factor, thyrotropin-
releaslng hormone, arglnlne vasopressin
(AVP), somatomedins, oxytocin, mitogens,
epidermal growth factor, and several
pro - opiomelanocortin (POMC) - derived
peptides (Hsueh and Schaeffer, 1985; Boi-
tani et al., 1986; Kasson et al., 1986) in
mammalian testes. The most experimental
detail is available on the GnRH-like factor
OCR for page 52
52
and AVP, which have been shown to alter
Leydig cell steroidogenesis, and on POMC-
derived peptides, which apparently are
produced in Leydig cells. It is beyond the
scope of this report to discuss each of
these testicular peptides in detail. We
focus our attention here on the POMC-
derived peptides, for three reasons: con-
cepts established for the use of one pep-
tide as a biologic marker might have ap-
plication to others; the GnRH-like peptide
seems to be restricted only to the rat tes-
tis, whereas POMC-derived peptides seem
to be more widely distributed; and more
information is available on the
physicochemical characteristics of POMC-
derived peptides than on those of the other
testicular peptides. The reader is re-
ferred to comprehensive reviews for more
information on testicular GnRH-like fac-
tor (Hsueh and Schaeffer, 1985) and AVP
(Cooke and Sullivan, 1985; Kasson et al.,
1986).
POMC-derived peptides have been local-
ized by immunocytochemical procedures
in Leydig cells, but not in myoid and Ser-
toli cells of rat, mouse, hamster, guinea
pig, and rabbit testes (Tsong et al.,
1982a,b). mRNA for POMC-like proteins
was localized in Leydig cells of mouse
testes by in situ hybridization (Gizang-
Ginsberg and Wolgemuth, 1985~. It was
later shown that genes for POMC-like pro-
teins and the concentration of Leydig cell
POMC-derived peptides are regulated by
LH (Shaha et al., 1984; Boitani et al.,
1986; Valenca and Negro-Vilar, 1986~.
Together, those results suggest that Ley-
dig cells might produce POMC-like pro-
teins. The function of such molecules is
unknown. Nevertheless, it is possible
that a Leydig cell-specific POMC-derived
peptide can be secreted by testes and that
its measurement might constitute a biolog-
ic marker for some as yet poorly understood
Leydig cell function.
SEMINIFEROUS TUBULES
The seminiferous tubules in the adult
mammalian testis contain germ cells in
various developmental phases and nonpro-
liferating Sertoli cells. The reader is
referred to several comprehensive reviews
AL4LE REPRODUCTIVE TOXICOLO(;Y
(Roosen-Runge, 1969; Clermont, 1972;
Ewing et al., 1980; Griswold, 1988) for
detailed descriptions of spermatogenesis
and the structure and function of Sertoli
cells. Briefly, the primary function
of the seminiferous tubules is the produc-
tion of spermatozoa.
A major difficulty in elucidating the
site or mechanism of action of a toxicant
on spermatogenesis in mammals is that,
as germ cells differentiate, they physi-
cally interact with and are affected by
each other, somatic cells of the seminifer-
ous tubules (e.g., Sertoli cells), and
indirectly through chemical signals
(e.g., from follicle-stimulating hormone
(FSH), LH, testosterone). It is extremely
difficult to ascertain whether a toxicant
acts directly on a specific cell type in
the germinal epithelium (e.g., a spermato-
gonium or spermatid) or indirectly via
the Sertoli cells, Leydig cells, or (even
more indirectly) cells in the hypothalamus
or adenohypophysis.
Toxicologic elucidation is further
complicated by the presence of Sertoli-
Sertoli junctional complexes that sub-
divide the seminiferous epithelium into
a basal compartment and an adluminal com-
partment in many species, including hu-
mans. It is believed that these special-
ized junctional complexes constitute the
principal site of the blood- testis
barrier that restricts the free movement
of specific chemicals between the blood
and seminiferous tubular fluid. Apparent-
ly, spermatogonia and young spermatocytes
are outside the permeability barrier,
in the basal compartment next to the base-
ment membrane of the seminiferous tubule,
and presumably exposed to xenobiotics in
blood and lymph. In contrast, mature sper-
matocytes and spermatids are sequestered
within the permeability barrier, in the
adluminal compartment. A practical con-
sideration is the possibility of differen-
tial drug access to the cells sequestered
behind the barrier. However, studies with
labeled alkylating agents in mice have
indicated that spermatocytes and sper-
matids can be exposed and that exposure
can result in cell-killing and induced
mutations. Despite these complex interac-
tions, we have divided the following dis-
OCR for page 53
MARKERS OF TESTICULAR FUNCTION
cussion of biologic markers of the semi-
niferous tubules into a section on Sertoli
cells and a section on the germinal epithe-
lium to simplify the presentation.
Sertoli Cells
The structure and function of Sertoli
cells have been the subject of several
comprehensive reviews (Fawcett, 1975;
Dym et al., 1977, Ewing et al., 1980; Gris-
wold, 1988~. Briefly, Sertoli cells in
the adult mammal are nondividing or slowly
dividing cells that rest against the base-
ment membrane with projections into the
lumen of the seminiferous tubule. Their
shape is complex and constantly changing,
depending on the stage of the cycle of the
seminiferous epithelium. Generally,
however, Sertoli cell shape is character-
ized by an irregular nucleus, prominent
nucleolus, and filamentous cytoplasm.
Although the structure of the Sertoli
cell has been described for numerous spe-
cies, its functions remain enigmatic,
because of its intimate association with
a population of germ cells that change
over time and space (Ewing et al., 1980~.
Sertoli cells must be involved at least
in germ cell division and differentiation,
in view of their direct and specialized
membrane contact with germ cells, their
formation and presumed control of the mi-
lieu of the adluminal compartment of the
seminiferous tubule via the tight junc-
tions between adjacent Sertoli cells, and
their apparent transduction of hormonal
signals (e.g., from FSH and testosterone)
that are known to regulate spermatogene-
sis. Although Sertoli cells can be count-
ed, few biologic markers of Sertoli cell
function have been developed, and their
used is complicated by differential and
variable secretion from Sertoli cells
into the blood/lymph or seminiferous tu-
bule fluid draining into the rete testes
and epididymal and seminal fluids.
Markers in Use
Numerous stereologic procedures have
been used to learn the number of Sertoli
cells per testis (Wing and Christensen,
1982; Johnson et al., 1984a; Johnson, 1986)
53
and the numbers of several specific germ
cell types associated with an average Ser-
toli cell (Wing and Christensen, 1982;
Johnson et al., 1984a). Knowing the
former allows an investigator to test
the effect of a toxicant on the viability
of Sertoli cells; knowing the latter allows
one to test the effect on the functional
capacity of Sertoli cells to support each
germ cell type. To our knowledge, however,
these markers have not been used to test
the effect of a xenobiotic chemical on
Sertoli cell number or structure, probably
because they require biopsy or autopsy
specimens, are tedious to use, and are
subject to artifacts of tissue prepara-
tion. These morphologic markers should
be particularly helpful in studies of mech-
anism of toxicity in experimental animals,
because they can provide information about
germ-Sertoli cell interaction.
Sertoli cells cultured in vitro secrete
at least 60 proteins, as measured by the
incorporation of radioactive amino acids
into spots on two-dimensional gels (Wright
et al., 1981~. Sertoli cells secrete both
serum proteins and testis-specific pro-
teins. Serum proteins identified include
transferrin, ceruloplasmin, somatomedin
C, and sulfated glycoproteins 1 and 2;
testis-specific proteins include andro-
gen-binding protein (ABP), inhibin, Mul-
lerian-inhibiting substance (MuIS), Ser-
toli-derived growth factors, and cyclic
proteins-2 (Griswold, 1988~. With the
exception of MuIS, and perhaps transferrin
and ABP, these proteins have poorly under-
stood functions. However, each is a poten-
tially useful marker of Sertoli cell func-
tion in vitro. It is important to note that
these data are derived largely from the
culture of Sertoli cells from immature,
rather than mature, rats. Therefore, ex-
trapolation of the results from immature
rat Sertoli cells in vitro to the human in
viva situation must be made cautiously.
Markers Requiring Research and
Development
Transferrin, ABP, MuIS, and inhibin
are the most extensively characterized
Sertoli cell products and therefore the
best candidates for in viva markers of the
OCR for page 54
54
pathophysiologic state of Sertoli cells.
Transferrints usefulness is limited,
because it also is synthesized in the liver
(Skinner et al., 1984~. The other three
hold considerable promise as biologic
markers, because they are specific prod-
ucts of the Sertoli cell, because they
probably are secreted into the peripheral
blood, and because considerable progress
has been made in cloning genes for them.
The discovery of ABP (Ritzer. et al.,
1971; Hansson and Djoseland, 1972) and
its identification in peripheral blood
of rats (Gunsalus et al., 1980) suggest
that peripheral blood concentrations of
ABP might serve as a marker of the patho-
physiology of Sertoli cells in viva. It
was recently shown (Orth and Gunsalus,
1987) that ABP concentration in the peri-
pheral blood of rats is highly correlated
with Sertoli cell number. That finding
was possible because the rat has no tes-
tosterone-estradiol binding globulin
(TEBG), which is probably identical with
ABP in other species. It will require con-
siderable research and development, how-
ever, to validate a radioimmunoassay that
differentiates between ABP and TEBG in
species other than the rat, to elucidate
the relationship between serum ABP derived
directly from Sertoli cells and ABP derived
indirectly from the epididymis, and final-
ly to describe the correlation between
Sertoli cell function and serum or seminal
ABP concentrations.
A number of studies (McCullagh, 1932;
Rich and De Kretser, 1977; de Jong, 1979)
have shown selective increases in serum
FSH after destruction of the germinal epi-
thelium. Sertoli cells secrete inhibin,
a factor that diminishes the release of
FSH from cultured pituitary cells (Stein-
berger and Steinberger, 1976~. Further
research and development are required.
however, to purify inhibin from testes,
to raise a specific antibody against sub-
units of inhibin, to validate a radio-
immunoassay for inhibin, and finally to
elucidate the relationship between Ser-
toli cell pathophysiology and serum in-
hibin concentration. These studies are
under way as this report is written (see,
e.g., de Jong,1987~.
MuIS is a glycoprotein that causes re-
AL4LE REPRODUCTIVE TOXICOLOGY
gression of the Mullerian duct (Picard
et al., 1986~. The bovine and human genes
for MuIS were recently isolated, and the
human gene can be expressed in animal cells
(Cate et al., 1986~. Its C-terminal domain
shows a marked homology with human trans-
forming growth factor ~ and the ,8 chain of
porcine inhibin (Mason et al., 1985~.
Considerable research must be completed
to determine whether MuIS is secreted by
adult testes, whether it has any function
in adults, and whether it can serve as a
biologic marker of Sertoli cell function.
Germ Cells
Spermatogonia are the most undifferen-
tiated germ cells in the seminiferous epi-
thelium. Spermatogenesis is the process
by which undifferentiated spermatogonia
divide and differentiate into spermato-
zoa. The spermatogonia undergo a number
of mitotic divisions, enter the meiotic
phase of spermatogenesis as diploid sper-
matocytes, and undergo the first and second
meiotic divisions to produce haploid sper-
matids, which differentiate into sper-
matozoa. Spermatogonia are of two major
types: one is a stem cell that divides oc-
casionally to replenish itself, or two,
produces a committed spermatogonium that
undergoes several mitotic divisions and
then forms spermatocytes, and eventually
gives rise to spermatozoa. As spermato-
gonia divide and differentiate, they are
also replenished by a process termed
stem cell renewal.
Spermatogenesis consists of a series
of events that takes a different amount
of time in different species (Clermont,
1972~. Table 4-1 compares several charac-
teristics of spermatogenesis and sperm
production in mice, hamsters, rats, rab-
bits, beagles, rhesus monkeys, and humans.
Species differ substantially in charac-
teristics of spermatogenesis and sperm
production; they differ less in epididymal
transit time. Amann (1986) concluded that
no species is identical with the human in
these respects, so care must be taken when
extrapolating from animals to humans.
The potential for a toxic effect on sper-
matogenesis might be greater in humans
because sperm production per gram of testis
OCR for page 55
?~9RKERS OF TESTICULAR FUNCTION
55
TABLE ~1 Species Differences In Spermatogenes~s, Daily Spenn Production, and Epididymal Transit Thea
Dog Monkey
Mouse Hamster Rat Rabbit ~eagle) Jesus! Man
Duration of spermatm
genesis, days
Duration of cycle of
seminiferous
epithelium, days
Life span, days
B ppermatogonia
IA~ptotene
Pachytene spermatopytes
Golgi spermatids
Cap spermatids
Testicular weight (total)
Daily sperm production,
millions
Per gram of testis
Per male
Sperm reserves In caudae
epidi~mes (at sexual
rest), millions
Epididymal transit tune (at
sexual rest), days
3~35 35-36
8.9
8.7
1.5 1.6
2.0 0.8
8.0 8.1
1.7 23
3.6 3.5
0.2 3.0
28 24
5.6 72
49 1,020
14.8
48 4~51
12.9 10.7
2.0 13
1.7 2.2
11.9 10.7
2.9 2.1
5.0 5.2
3.7 6.4
24 25
89 160
440 1,600
8.1 12.7
62
70
13.6 9.5
4.0 2.9
3.8 2.1
12.4 9.5
6.9 1.8
3.0 3.7
12.0 49.0
20 23
240 1,127
2,100 5,700
113 10.5
7~74
16.0
63
3.8
12.6
7.9
1.6
34.0
4.4
150
420
5.5-12
aData derived largely from a table constructed by Amann (1986~.
in humans is approximately one-fourth to
one-sixth that in the other species. Daily
sperm production in all species probably
is in excess of that required for fertili-
ty. For example, a 90% reduction in fertile
sperm available for ejaculation did not
suppress fertility in rats (Amann, 1986~.
Thus, especially in animal species, but
even in humans, fertility is unlikely to
be a sensitive indicator of toxic insult
of spermatogenesis.
Therefore, our task is to elucidate bio-
logic markers that reflect the exposure
to or effect of toxicants on the number and
function of germ cells, from primitive
spermatogonia to fully formed spermato-
zoa. The complexity and species variation
of spermatogenesis suggest that a battery
of markers will be required to detect toxic
effects on spermatogenesis. For example,
one toxic chemical might act on cells un-
dergoing rapid mitosis, and another pri-
marily on meiotic cells in which complex
genetic rearrangements occur. Slowly
dividing stem spermatogonia might be re-
sistant to cytotoxic chemicals, but vul-
nerable to DNA alterations. The nondivid-
ing condensed spermatids are resistant
to direct effects of toxic chemicals on
development and function, but sensitive
to point mutations and chromosomal break-
age. Detection of each alteration might
require a different biologic marker.
Markers in Use
In any given region of a seminiferous
tubule, germ cells are differentiating.
That combination of phenomena creates a
complex histologic appearance at the light
microscopic level, where adjacent cross
sections through seminiferous tubules
generally appear quite different. Cler-
mont and coworkers (Leblond and Clermont,
1952; Helter and Clermont, 1964) showed
that distinct cellular associations or
stages exist and that their number depends
on the species-e."., 6 in humans and 14
in rats. The complex histologic cell as-
sociation pattern allows trained obser-
vers to evaluate subjectively whether
external factors have specific effects
OCR for page 56
56
on germ cells in different steps of dif-
ferentiation. This constitutes a rela-
tively simple marker, which is particular-
ly applicable to studies with experimental
animals, whose testicular cytoarchitec-
ture can be well preserved with large tis-
sue samples and in which enough spermato-
genesis remains to allow staging of spe-
cies-specific cellular associations.
Studies with human biopsy specimens are
complicated by the small amount of tissue
available, by artifacts induced by the
biopsy procedure, and by the fact that a
cross section of a human seminiferous tu-
bule usually contains more than one stage
of the cycle of the germinal epithelium
(Heller and Clermont, 1964~.
A simple quantitative approach is to
determine the percentage of seminiferous
tubules with mature spermatids lining the
lumen versus the percentage of tubules
without spermatids lining the lumen. That
approach assumes that a chemical simply
does or does not have a toxic effect on a
tubule and, therefore, a clear judgment
can be made as to which category a particu-
lar tubule belongs to. Alternatively,
it is possible to measure the minor diame-
ter of 15 seminiferous tubules, which di-
minish with the loss of germ cells (Courot,
1964~; the advantage of this quantifica-
tion is that it is sensitive, easy to meas-
ure, and provides a spectrum of values from
a maximal to a minimal diameter of semi-
niferous tubules. The disadvantages of
both approaches are that they are nonspe-
cific and relatively insensitive and re-
quire autopsy or biopsy specimens. Again,
great care must be taken to prevent arti-
facts associated with fixing, embedding,
and sectioning the testicular tissue
(Amann, 1981).
It is possible to count stem cells in
histologic sections of rodent testes
(Oakberg, 1978~. But the technique is
subjective and laborious, because stem
cells are difficult to identify and are
few. Alternatively, a functional test
for stem cell renewal can be used.
Methods to measure the effect of toxi-
cants on germ cells in tine testis have been
described and reviewed (Amann, 1970a;
Berndtson,1977,Amann,1981~. Thesemeth-
~ .
OdS are applicable to humans and experlmen-
Af 4LE REPRODUCTIVE TOXICOLOGY
tat animals, provided that the testicular
~n~nim~n~ non be fixed appropriately
and biopsy artifacts can be prevented.
One widely used method involves counting
germ cells in a fixed number of tubule cross
sections of one cellular association
(Amann,1970b; Berndtson,1977~. The germ
cell numbers are generally but not always
expressed per Sertoli cell nucleolus,
to correct for seminiferous tubule shrink-
age caused by histologic processing and
experimental treatment. The method has
proved sensitive in assessing the effect
of antimitotic agents on spermatogenesis
(Amann,1981). However, the numbers gener-
ated are relative, rather than absolute;
and the results might be nonspecific, be-
cause numerous agents can cause a morpho-
logically identical pattern of response
(Russell et al., 1981~. In a simplified
version of the procedure, the mean number
of spermatids per tubule from a testicular
biopsy has been used to predict sperm count
in humans (Silber and Rodriguez-Rigau,
1981~.
Another method applies stereologic
principles (Van Dop et al., 1980a,b; Wing
and Christensen, 1982; Jones and Berndt-
son, 1986) to obtain quantitative informa-
tion on the diameter and volume of the semi-
niferous epithelium and lumen and to learn
the absolute, rather than relative, num-
bers of Sertoli and germ cells (from pre-
leptotene primary spermatocytes to step
10 spermatids). The method allows an ex-
perimenter to determine precisely the
effect of a treatment on the number of cell
types in the germinal epithelium. It has
disadvantages: it is invasive, labor-
intensive, and subject to numerous arti-
facts of tissue preparation. To our knowl-
edge, the latter method has not been used
to test the effect of xenobiotic chemicals
on the seminiferous tubular epithelium
in animals or humans.
An alternative to direct counting of
stem cells is to count the stem-dependent
cells after a toxic insult. One way to do
that is to count the number of repopulating
and nonrepopulating seminiferous tubular
cross sections in a series of experimental
animals killed at different times after
treatment with a toxic chemical (Meis-
trich, 1986~. It is assumed that each re-
OCR for page 57
MARKERS OF TESTICULAR FUNCTION
populating cross section of seminiferous
tubules results from the presence of at
least one surviving stem cell and that
nonrepopulating cross sections result
from the absence of stem cells. In a
second assay used in mice, sperm heads
in the testis 50 days after exposure to
a toxic insult are counted (Meistrich,
1986~. Both methods are simple and rapid.
But both are invasive and therefore not
readily applicable to humans; and repopu-
lating tubules cannot be counted in low-
dose situations, because extensive kill-
ing of stem cells is required for empty
tubular cross sections to be produced.
One method of counting spermatid nuclei
in homogenates of testicular parenchyma
is based on the fact that late spermatid
nucleoprotein becomes highly condensed
and therefore resistant to homogenization
(Amann and Lambiase, 1969~. It is simple
to use and its results are highly corre-
lated with daily sperm output in rabbits
(Amann, 1970b; Amann. 1981: Berndtson.
1977~. It is also applicable to humans
(Amann and Howards, 1980; Johnson et al.,
1980a, 1984b). The disadvantage of the
technique is that it is invasive (or re-
quires autopsy specimens) and therefore
has only limited human application as a
biologic marker.
The amount of testicular LDHC4, a germ
cell-specific isozyme of lactate dehydro-
genase, is proportional to the numbers
of meiotic and postmeiotic germ cells in
testes of mice and can be used to estimate
the survival of spermatogonial stem cells
after treatment with toxicants (Meis-
trich, 1982~. The method is indirect, more
difficult than measuring spermatid num-
bers in testicular homogenates, and in-
vasive. But, it holds promise, because
the LDHC4:sperm ratio in seminal plasma
of human males might serve as an indicator
of the function of the seminiferous epithe-
lium (Eliasson and Virji, 1985; Virji,
1985. The approach should be investigated
thoroughly, not only with LDHC4, but with
other proteins specific for meiotic and
postmeiotic germ cells.
Severe damage to the germinal epithelium
in the testes of many species results in
increased serum FSH concentrations (Mc-
Cullagh, 1932; Rich and De Kretser, 1977;
57
de Jong, 1979), in part because a Sertoli
cell product (inhibin), which regulates
FSH secretion, is produced in low amounts
in azoospermic animals and in high amounts
in normospermic animals. Major insults
to spermatogenesis can be monitored in-
directly by measuring FSH concentration
in peripheral blood. The principal advan-
tage of this biologic marker is that it can
be measured in peripheral blood samples,
which are easy to collect repetitively.
The disadvantages are that the inverse
relationship between FSH concentration
in serum and germinal epithelium damage
are not tightly coupled; FSH production
is episodic, so the results are variable.
That renders the marker relatively insen-
sitive to changes in spermatogenesis,
despite the sensitivity of the radio-
immunoassay. In addition, very low sperm
counts are required for FSH increase to
become evident. Finally, gonadal steroids
(testosterone and estradiol) can account
for selective FSH increase when testoster-
one production is low (Sherins et al.,
1982~. Consequently, FSH concentration
might not always reflect only inhibin pro-
duction. The marker is nonspecific, in
that any toxic insult that depletes germ
cells causes an increase in FSH production
and testosterone and estradiol also par-
tially control FSH production.
Markers Requiring Research and
Development
The application of molecular biology
to the study of mammalian testicular dif-
ferentiation is providing investigators
with insights into many of the molecular
mechanisms that regulate male germ cell
formation. Gene expression during sper-
matogenesis is temporally and spatially
regulated with precision; many macromole-
cules and organelles are synthesized in
specific cell types during the continu-
um of testicular cell differentiation
(Hecht, 1987~. Although variants of ubi-
quitous enzymes and structural proteins
are expressed in many organs, the testis
appears to be an especially rich source
of isozymes (Goldberg, 1977~. Presumably
because of the specialized requirements
for producing a spermatozoon, unique tes-
OCR for page 58
58
ticular isozymes that code for proteins
such as lactate dehydrogenase, phospho-
glycerate kinase, and cytochrome c-have
evolved (Fig. 4-3~. In addition to the
testicular isozymes, many structural
proteins of the maturing spermatid and
the spermatozoon have been identified.
Results of biochemical analyses of
the differentiating haploid germ cells
that transform into the highly polarized
spermatozoon suggest that the definition
of all the possible sperm-specific molecu-
A~E REPRODUCTIVE TOXICOLOGY
tar markers has only begun. As a result
of such studies and the well-characterized
sequence of events leading to the formation
of spermatozoa, efforts to monitor the
effects of toxicants on male germ cells
can be based on substantial knowledge and
use the numerous DNA probes already avail-
able for the mammalian testis to study the
mechanism of toxic action of individual
chemicals on spermatogenesis.
Most DNA cloning efforts during sper-
matogenesis have been directed toward two
MOUSE SPERMATOGENESIS
CELL TYPE ~ SPERMATOGOb IA | SPERMATOCyTES I | SPE STATICS SPERM
(Pachytene) 11 (Rounds Elongating)
Ploidy 2N | 4N | 2N N | N
Rate of Differen-
tiation 6 11 1 9 20 34
| DNA Synthesis I ~ | _ l l
mRNA Synthesis
Cyto. RNA (pg/cell) 9.1 2.2 0.9
Poly (A) RNA (ng/~1 9) 6 1 1 3
Protein Synthesis
Histones _ _
PGK-2 _ _ _ _ _ _ __
Protamines
Acrosin
pActin __
(2~1 kB)
Y Actin
(2~1 kB)
Actin (?)
(1 5 kB)
(6.2 and 8.0 kB)
c-ab
L (4.7 kB) l l I I ~
FIGURE ~3 Periods of active synthesis of DNA, RNA, and proteins diagrammed for various cell types. Source:
Hecht, 1987b.
OCR for page 59
MARKERS OF TESTICULAR FUNCTION
intervals of spermatogenesis: meiosis
and spermiogenesis (Kleene et al., 1983;
Dudley et al., 1984; Fujimoto et al.,
1984~. That is because the meiotic pachy-
tene spermatocytes and the haploid round
spermatids represent two critical periods
for gene expression during male germ cell
development. Moreover, highly enriched
populations of pachytene spermatocytes
and round spermatids can be obtained readi-
ly with cell separation techniques, be-
cause of their marked size differences
and abundance in the sexually mature
testis (Romrell et al., 1976; Meistrich,
1977; Hecht, 1987b).
Almost all DNA probes for genes ex
pressed in testis cells have been isolated
from animals, such as mice, rats, and
bulls. The origin of these probes will not
pose a problem for application to human
DNA. Because DNA probes for homologous
genes in humans and other vertebrates share
substantial sequence homology, the animal
probes can be used to isolate equivalent
human sequences from available human cDNA
or genomic DNA libraries, and investiga-
tors committed to human toxicology studies
should seriously consider the direct iso-
lation and characterization of human DNA
probes. For cases in which both rodent and
human probes are available, the animal
studies can be used to validate the DNA
probe marker for human use and, more impor-
tant, to reveal the mechanisms by which
specific chemicals interact with the ge-
nome.
MeioticDNAProbes. Lactatedehydrogen-
ase C4 (LDHC4) is one of the best-charac-
terized proteins in the testis (Goldberg,
1977~. It appears to be testis-specific
and is synthesized initially during meio-
sis and in decreasing amounts during early
spermiogenesis (Meistrich, 1977~. LDHC4
mRNA makes up as much as 0.18% of total func-
tional mRNA in mouse pachytene spermato-
cytes (Wieben, 1981~. LDHC4 is also present
on the surface of mature spermatozoa, so
it has been used extensively in immunocon-
traceptive studies (Wheat et al., 1985~.
The protein sequence of rodent LDHC4 has
been known for some time (Pan et al., 1983),
but only recently has a cDNA probe for human
LDHC4 been isolated (Millan et al., 1987~.
Two distinct forms of phosphoglycerate
59
kinase (PGK) have been characterized in
mammals. PGK-1 is an X-linked gene that
is expressed in somatic cells, whereas
the autosomally derived isozyme, PGK-
2, is specific to the testis (Kramer and
Erickson, 1981~. Although PGK-2 is syn-
thesized during spermiogenesis, the gene
appears to be initially transcribed during
meiosis, with increased synthesis of PGK-
2 mRNA in spermatids (Gold et al., 1983~.
Recent elegant studies of the human PGK
multigene family have produced detailed
sequence knowledge and DNA probes for these
important enzymes (Michelson et al.,
1985).
Cytochrome c, the electron-transport
protein from the mitochondrial respira-
tory chain, exists in two forms in the tes-
tis (Goldberg et al., 1977~. One variant,
cytochrome c~, is restricted to the
testis; the other variant, cytochrome
c~, is presumably found in all tissues.
Indirectimmunofluorescence with monospe-
cific antibodies first detects cytochrome
c~ in the mitochondria of pachytene sper-
matocytes and in later stages of spermato-
genesis, whereas cytochrome c,, is found
in the mitochondria of interstitial cells,
Leydig cells, and spermatogonia. Sequence
analysis of the two mouse cytochrome c
molecules has revealed that cytochrome
cl; differs from cytochrome c~ in 13 amino
acid residues (Hennig, 1975~. Although
DNA probes exist only for the c~ variant
of cytochrome c, knowledge of the sequence
of the testicular form of cytochrome c
would allow appropriate oligonucleotide
DNA probes to be prepared. Such oligonu-
cleotides would facilitate the isolation
of cDNA or genomic DNA probes for human
cytochrome cl;.
During spermatogenesis, a dramatic
reorganization of the germ cell nucleus
occurs. The transformation ultimately
produces a sperm nucleus with highly com-
pacted DNA and accompanied by the replace -
ment of histones with a group of transient-
ly associated nuclear proteins and
finally with protamines (Hecht, in
press). In addition to the standard com-
plement of histone molecules found in mam-
malian cells, the meiotic pachytene sper-
matocyte contains several additional
histone variants believed to be peculiar
.
OCR for page 60
60
to the testis. DNA sequence analysis of
one testis-specific histone variant, Hit,
has revealed it to be a unique gene product,
and not a posttranslational modification
of an existing histone (Cole et al., 1986~.
The availability of a specific DNA probe
for one of these meiotic histones of the
rat provides a means to obtain the equiva-
lent probe for the human male meiotic his-
tone gene. Probes for other testis-specif-
ic histones can also be isolated.
Postmelotic DNA Probes. The predominant
proteins in mammalian spermatozoa are the
protamines, a group of small arginine-
rich DNA-binding proteins that aid in
nuclear DNA compaction during spermio-
genesis (Hecht, 1987c). With the exception
of a few species, most mammalian spermato-
zoa have been reported to contain one type
of protamine. In the mouse, two protamine
variants, MP1 and MP2, have been identified
by DNA sequence analysis of isolated cDNA
clones (Kleene et al., 1985; Yelick et al.,
1987~. Protein sequence studies have iden-
tified similar P1 and P2 human protamine
variants (McKay et al., 1985; Ammer et al.,
1986; McKay et al., 1986~. Although the
P1 and P2 protamines differ substantially
in size and sequence, in the mouse they
are closely linked on chromosome 16 and
are temporally and translationally regu-
lated during spermiogenesis (Hecht et al.,
1986a). The human protamine genes probably
are also chromosomally linked. The P1
human protamine was recently shown to
be on human chromosome 16 (R.H. Reeves,
Johns Hopkins University, and N.B. Hecht,
Tufts University, unpublished observa-
tion, 1987~.
Northern blots of RNA from prepubertal
testes and from isolated meiotic and post-
meiotic testicular cell types have re-
vealed that the protamines are expressed
solely during the haploid interval of sper-
matogenesis (Kleene et al., 1983. 1984:
Hecht et al., 1986a,b). Moreover, changes
in length of protamine mRNAs during sper-
matogenesis allow the protamine probes
to be used as molecular markers to evaluate
the extent of spermiogenesis in wild-type,
mutant, or chemically induced sterile
animals (Kleene et al., 1984~. For in-
stance, if the MPl-cDNA probes are used,
no MPl-mRNA is detected in testicular ex-
M>llLE REPRODUCTIVE TOXICOLOGY
tracts of prepubertal mice up to 20 days
old (a time when spermatogenesis has ad-
vanced to meiosis), whereas a 580-nucleo-
tide form of MPl-mRNA is present in the
testes of 22-day-old mice (early sperma-
tids are present by day 22) and a heterogen-
eous population of 580- and 450-nucleotide
MP1 -mRNAs is present in the testes of sexu-
ally mature animals (Hecht et al., 1986b).
Results of cell-separation studies con-
firm that no MP1 -mRNA is presentin pachy-
tene spermatocytes, a 580-nucleotide MP1-
mRNA is found in round spermatids, and
elongating spermatids contain an addi-
tional 450-nucleotide mRNA (Kleene et al.,
1984~. These mRNA length changes result
from a partial deadenylation of the prota-
mine mRNAs that takes place when they move
from the ribonucleoprotein particle frac-
tion of the cytoplasm (in round spermatids)
to polysomes (in elongating spermatids).
Similar size changes occur in the MP2 mouse
protamine and in P1 and P2 rat and hamster
protamine mRNAs (Bower et al., in press).
The protamine genes appear to be excel-
lent candidates to serve as probes to moni-
tor genomic defects induced during sper-
miogenesis. They are two single-copy genes
that express abundant postmeiotic tes-
ticular mRNAs essential for sperm func-
tion. Moreover, the cDNA probes show much
homology to the DNA and RNA of many other
vertebrates, including humans. Several
laboratories are seeking to isolate human
probes for these male-specific DNA-bind-
ing proteins.
In mammals, histones are not directly
replaced by protamines, but by a presumably
heterogeneous group of basic proteins
called testis-specific proteins (lP)
(Hecht, 1987c). TPs are associated with
the spermatid nucleus during its
transition from its nucleosome-like
structure to the smooth branching fibril
_7 ~ _ ~ of the spermatozoan nucleus. TPs are re-
placed by protamine during spermiogene-
sis. Recently, cDNA probes for the mouse
and rat TPs have been identified (Hecht
et al., 1986b; M.A. Heidaran and W.S. Kist-
ler, University of South Carolina, per-
sonal communication). Phylogenetic stud-
ies have indicated a strong sequence con-
servation of TPs in rodents and humans.
The identical pattern of expression of
OCR for page 61
MARKERS OF TESTICuL~4R FUNCTION
mouse TP and MPl and MP2 suggests that these
three genes are coordinately and temporal-
ly regulated in the postmeiotic testicular
cells and could be used together to monitor
postmeiotic gene expression.
In mammals, actins are encoded by a mul-
tigene family that expresses at least six
distinct but closely related forms of ac-
tin. In addition to the general role that
actin plays in cell motility and division,
secretion, organelle movement, and main-
tenance of cellular cytoarchitecture,
testicular actins are likely to be
involved in chromosomal movement during
meiosis, in shaping specific nuclear
structures during spermiogenesis, and
in spermatozoa! function (Hecht et al.,
1984~. Although the mRNAs coding for the
cytoplasmic ,6 and ~ actin isotypes have
been detected in all testicular cell
types throughout spermatogenesis, mRNA
that encodes an additional actin variant
is first detected during spermiogenesis
(Waters et al., 1985~. It should be pos-
sible to obtain several distinct DNA probes
for actin isotypes that are expressed con-
stitutively during spermatogenesis and
of other actin isotypes that are expressed
temporally in specific stages or cell
types.
Microtubules consist of heterodimers
of a and ~ tubulin. The ~ and ,6 tubulin sub-
units are distinct sequences, each encoded
by multigene families. In the testis, the
tubulins are involved in mitotic and mei-
otic divisions, in changes in cell shape
and structure, in the species-specific
shaping of the sperm nucleus, and in the
synthesis of the axoneme of sperm tails.
Results of protein gel electrophoresis
and DNA cloning studies have suggested
that multiple isoforms of c' and,B tubulin
are expressed during spermatogenesis
(Hecht et al., 1984~. The availability
of cDNAs for a number of mouse testicular
c' tubulins and a detailed study of the ex-
pression of the ~ tubulin multigene family
will provide a set of useful probes to moni-
tor the differential expression of several
cytoplasmic structural genes during tes-
ticular germ cell development.
Results of in vivo and in vitro postmei-
otic protein synthesis studies and the
many morphologic changes in cell shape
· · . . .
61
and structure that occur during spermio-
genesis have indicated that many addition-
al unique macromolecules are synthesized
during spermiogenesis. Continuing stud-
ies in many laboratories suggest that such
proteins as acrosin, hyaluronidase, a
sperm-specific enolase, and sperm tail
proteins (e.g., the dyneins and outer
dense-fiber proteins) will provide addi-
tional sources of stage-specific DNA
probes for this critical interval of sper-
matogenesis. DNA probes are also available
for some proto-oncogenes, such as c-abl
and c-myc, that are differentially ex-
pressed during spermatogenesis. Although
c-abl mRNA is present in premeiotic, mei-
otic, and postmeiotic cell types, a novel
c-abl mRNA of distinct size is first de-
tected in postmeiotic cells (Ponzetto and
Wolgemuth, 1985~. Because of its unique
size, a probe specific to this shortened
c-abl transcript could be prepared. In
contrast, the proto-oncogene c-myc ap-
pears not to be expressed in testicular
germ cells (Stewart et al., 1984~.
Stem Cell DNA Probes. Spermatogonia
make up only a few percent of the cells
found in the sexually mature mammalian
testis. It has therefore been difficult
to work biochemically with this cell type,
and no DNA probes peculiar to spermatogonia
have yet been isolated for this critical
stage of spermatogenesis. Because genetic
alterations in stem cell DNA will produce
persistent heritable defects, a major
effort needs to be commenced to obtain an
armamentarium of DNA probes specific to
animal and human testicular stem cells.
Recent improvements in testicular cell
separation methods make this possible,
in that highly enriched populations of
several types of spermatogonia can be ob-
tained from the prepubertal testis. With
poly(A)+ RNA isolated from enriched popu-
lations of spermatogonia, radiolabeled
cDNAs can be prepared and a differential-
hybridization approach similar to that
used previously to obtain postmeiotic
cDNAs can be conducted to isolate stem
cell-specific cDNAs (Kleene et al.. 1983).
In brief, a total testicular cDNA library
or a cDNA library enriched with spermato-
gonial cDNAs would be differentially hy-
bridized with radiolabeled cDNAs prepared
OCR for page 62
62
from spermatogonial, meiotic, or postmei-
otic cell types. The cDNAs that appear to
be preferentially expressed in spermato-
gonia would be isolated and their temporal
appearance confirmed by the early appear-
ance of RNA in the testes of prepubertal-
staged mice (Kleene et al., 1983~. DNA
sequence analysis could be used to help
to identify the proteins coded for by the
stem cell cDNAs. One possible candidate
DNA probe for a protein expressed in sper-
matogonia would be the DNA that codes for
the H2A histone stem cell variant found
in mouse embryonic spermatogenic cells
(Gizang-Ginsberg and Wolgemuth, 1985~.
Clearly, these macromolecular probes
at several cell stages represent an excel-
lent collection of biologic markers for
the dynamic process of spermatogenesis.
They can be used to assess the effect of
a toxicant on the genome that directs spe-
cific biochemical events in spermatogene-
sis. The approach would be particularly
useful in studies of the mechanism of ac-
tion of toxicants in spermatogenesis.
Disadvantages include the invasiveness
AL4LE REPRODUCTIVE TOXICOLOaY
of the present techniques and their re-
quirement for autopsy or biopsy specimens.
Development of these probes holds out
the possibility of analyzing potential
toxic effects at the DNA level in ejacu-
lated spermatozoa. However, that is un-
likely to occur soon. The primary limita-
tions on the application of DNA probe tech-
nology to evaluate genomic DNA alterations
in spermatozoa are three: DNA probes are
available for only a very small portion
of the genome; current procedures severely
limit the number of probes that can be as-
sayed at one time, thereby restricting
the percentage of the genome examined in
each analysis; and the DNA from single
cells, such as spermatozoa, cannot be ana-
lyzed. Theoretically, the limitations
can be overcome. Procedures can be devel-
oped to monitor large fragments of genomic
DNA with a battery of DNA probes that cover
vast regions of the human genome. Advances
in fluorescence detection of DNA combined
with computer imaging of samples will aid
in the analysis of DNA from single cells.
Representative terms from entire chapter:
germ cells
