METABOLISM OF GLUCOSE-1-^'^C AND GLUCOSE-6-^^C
BY TESTIS AND LIVER TISSUE
FROM 5-THIO-D-GLUCOSE TREATED MICE
A Thesis
Presented to
the Faculty of the Department of Biology
East Carolina University
In Partial Fulfillment
of the Requirements for the Degree of
Master of Science in Biology
by
Catherine A. Newton
April, 1979
J. y. JOYNER LIBRARY
EASÏ CAROLINA UNIVERSITYI
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METABOLISM OF GLUCOSE-l-^^C AND GLUCOSE-6-^'^C
BY TESTIS AND LIVER TISSUE
FROM 5-THIO-D-GLUCOSE TREATED MICE
by
Catherine A. Newton
APPROVED BY:
SUPERVISOR OF THESIS
Dr. Everett C. Simpson ^
THESIS COMMITTEE
ames Smith
Dr. Takeru Ito
Dr. Hubert Burden
Jy/meph G. Boyette/
613d26
ABSTRACT
Catherine A. Newton. METABOLISM OF GLUCOSE-l-^'^C AND GLUCOSE-6-^'^C
BY TESTIS AND LIVER TISSUE FROM 5-THIO-D-GLUCOSE TREATED MICE.
(Under the direction of Dr. Everett C. Simpson). Department of
Biology, April, 1979.
Male Dub:ICR mice were treated with 5-thio-D-glucose at a rate of
35mg/kg/day for either 7, 14, 21, 28, or 42 days. At the end of each
treatment period, radiorespirametric studies with glucose-l-^^C and
glucose-b-^^^C were done on testis and liver tissue from treated and
control mice. C-l/C-6 ratio was calculated to determine the relative
activity of pentose phosphate pathway to glycolysis-tricarboxylic acid
pathway.
14
The liver tissue showed no significant difference in CO2 percent
recovery with one exception. A lower recovery from glucose-6-^^C
during the 14-day period was observed in the treated. There was no
difference in C-l/C-6 ratio for the liver.
By 42 days of treatment, the percent recovery from glucose-l-^'^C
was significantly higher in the treated testes. The C-l/C-6 ratio in
the same period was also significantly higher, indicating increased
pentose phosphate activity in the treated. There was no observable
14
effect of the treatment on the CO2 activity derived from glucose-
6-^^C in the testis tissue.
ACKNOWLEDGMENT
This thesis was completed with the assistance of many people
including the faculty and staff of the Biology Department to which
I am grateful. I would especially like to thank Dr. Burden, Dr. Ito,
and Dr. Smith for serving on my committee. Additionally, I am grate-
ful to Carol and Denise for their assistance with the photomicro-
graphs and graphs, to Martha for her typing, to Jim for his help with
the animals and to Pa, Linda, Cal, and Is for their financial support.
Finally and most importantly, I wish to thank Dr. Simpson for his
interest, time, and assistance on the research project and on the
writing of the thesis.
TABLE OF CONTENTS
SECTION PAGE
LIST OF FIGURES iv
LIST OF TABLES v
LIST OF GRAPHS vi
INTRODUCTION 1
REVIEW OF LITERATURE 2
MATERIALS AND METHODS 13
RESULTS AND DISCUSSION 20
SUMMARY 61
APPENDIX A 63
APPENDIX B 64
APPENDIX C 65
REFERENCES CITED 66
IV
LIST OF FIGURES
Page
Figure 1. Schematic diagram of the incubation flask . 15
Figure 2, A section from a control testis showing normal
spermatogenic epithelium . 25
Figure 3. A section from a testis after 14 days of 5-TDG
treatment . 25
Figure 4. A section from a testis treated with 5-TDG
for 21 days . 27
Figure 5. Area from Figure 4 at a higher magnification . 27
Figure 6. A section from a testis treated with 5-TDG for
28 days . 29
Figure 7. Area from Figure 6 at a higher magnification . 29
Figure 8. A section from a testis treated for 42 days
with 5-TDG 31
Figure 9. Area from Figure 8 under higher magnification 31
Figure 10. A section from a testis after 42 days of 5-TDG
treatment 33
Figure 11. Tubule from Figure 10 under higher magnification 33. . .
Figure 12. Section from a testis treated for 42 days and allowed
to recover for 15 weeks 35
Figure 13. Section from the same testis in Figure 12 35
Figure 14. Section from testis treated daily for 42 days
with an additional 14 days of twice weekly
treatments 37
Figure 15. Area from Figure 14 under higher magnification 37....
Figure 16. Diagram of the Pentose Phosphate Pathway showing
the cleavage of CO2 39
Figure 17, Schematic representation of glycolysis - TCA cycle
demonstrating the order that CO^ evolves 40
V
LIST OF TABLES
Page
Table 1 A summary of values of samples ran 3, 6, and
12 hours after 5-TDG treatment 20
Table 2 Mean and standard error (n=5) for total percent
recovery of ^^COy from glucose-l-^^C and
glucose-6-^‘^C in liver tissue
Table 3 Summary of the C-l/C-6 ratios for liver tissue
Table 4 Mean and standard error (n=5) for total percent
recovery of ^‘^CO^ from glucose-l-^'^C and
glucose-6-^^C in testis tissue ^8
Table 5 Summary of C-l/C-6 ratios for the testis tissue .... 54
vi
LIST OF GRAPHS
Page
Graphs 1-5. Rate of evolution (cumulative) and yield of
^^^002 from specifically labeled glucose by liver
tissue from 5-TDG treated and control mice
during each treatment period A3
Graph 1. Seven-day treatment period A3
Graph 2. Fourteen-day treatment period A3
Graph 3. Twenty-one day treatment period A3
Graph A. Twenty-eight day treatment period A3
Graph 5. Forty-two day treatment period A3
Graph 6. Percent yield of ^^G02 after one hour of incubation
from specifically labeled glucose by liver tissue
from 5-TDG treated and control mice for each treatment
period A6
Graphs 7-11. Rate of evolution (cumulative) and yield of ^^G02 from
specifically labeled glucose by testis tissue from
5-TDG treated and control mice during each treatment
period 51
Graph 7. Seven-day treatment period 51
Graph 8. Fourteen-day treatment period 51
Graph 9. Twenty-one day treatment period 51
Graph 10. Twenty-eight day treatment period 51
Graph 11. Forty-two day treatment period 51
Graph 12. Percent yield of ^^002 after one hour of incubation
from specifically labeled glucose by testis tissue
from 5-TDG treated and control mice for each treatment
period 53
INTRODUCTION
The compound, 5-thio-D-gIucose, is an analogue of D-glucose and is
known to competitively inhibit D-glucose transport (Hoffman and Whistler,
1968), Recently, it has been shown to have antispermatogenic properties
(Zysk ^ aT., 1975; Homm aT. , 1977; Lobl and Porteus, 1978; Pick,
1979). Varying degrees of success with sterility (from complete
reversible sterility to permanent sterility) have been obtained. The
mechanism of its action on the testis, at this time, is only speculative.
One aspect of the testis in which a glucose analogue would have a direct
bearing is the importance of glucose metabolism to spermatogenesis.
This study was designed with two objectives- to examine the
effect of 5-thio-D-glucose treatment on glucose metabolism in the
testis and in a nontesticular tissue. Liver tissue was selected as the
nontesticular tissue because it is dependent on glucose and it has the
same major route of glucose oxidation (glycolysis-tricarboxylic acid
cycle and pentose phosphate pathway) as the testis. The activity of
these pathways as well as their relative activity to one another were
examined for both tissues using radiorespirametric studies and labeled
forms of glucose.
REVIEW OF LITERATURE
In recent years an increased interest in birth control has stimu-
lated research to find a pharmaceutical agent for male contraception.
Bèfore any substance can be considered clinically usable for male
sterility, two prerequisites must be met. First, the effect of the
substance must be reversible, and second, the chemical must not inter-
fere with libido. Presently, there exist two approaches to chemically
induced sterility- to interrupt spermatogenesis or to interfere with
post-testicular maturation of spermatozoa (Gomes, 1970; Jackson, 1973;
deKretser, 1976; Gomes, 1977).
The latter approach is possible since spermatozoa are both immotile
and incapable of fertilization when they leave the rete testis. Sperma-
tozoa acquire motility and the ability to fertilize, during passage
through epididymis and vas deferens in route out of the body. The two
major compounds currently being investigated in post-testicular matura-
tion are cyproterone acetate, which prevents acquisition of motility
(Morse ^ , 1973; Koch e^ ^. , 1976) and a-chlorohydrin, whose
mechanism of action is uncertain (Coppola, 1969; Ericsson and Baker,
1970; Kirton e^ ^., 1970; Vickery et al., 3974). At first a-chloro-
hydrin appeared to interfere with sperm maturation. However, Vickery
et al. (1974) showed sperm from rats treated with a-chlorohydrin were
capable of fertilization when incubated with ova in vitro. Thus, the
mode of action of the compound is still in doubt. Both of these
compounds is correct dosage, are reversible and have no apparent effect
on libido. High dosages of cyproterone acetate decrease testosterone
3
levels and, therefore, interfere with libido. A major advantage of this
approach is its rapid onset of sterility (7-9 days) and recovery (7-11
days). Interruption of spermatogenesis to reach sterility requires 3
to 5 weeks and 6 to 12 weeks to recover, varying with the compound used
and with the species being studied.
The disturbance of spermatogenesis can be either indirect by acting
on the hormone (mainly follicle stimulating hormone, FSH) which controls
spermatogenesis or direct by chemical agents acting on spermatogenesis.
Several steroid compounds which suppress the secretion of LH are
currently being investigated. Varying degrees of success have been
achieved with testosterone propionate (Heller al., 1950; Reddy and
Rao, 1972), testosterone oenathate (Heller al., 1970; Mauss et al.,
1974), Danazol (Sherins ^t , 1971; Skoglund and Paulsen, 1973, 6-
medroxyprogesterone acetate (Macleod and Tietz, 1964; Patanelli, 1975),
and testosterone undecanoate (Hirschhauser ^ , 1975; Nieschlag
et al., 1975). Some nonsteroids affecting FSH which are being studied
are reserpine, 5-hydroxytryptaime, and methallibure (Jackson, 1973).
Most chemicals which are presently known to act directly on
spermatogenesis are generally too toxic and/or cause unwanted side
effects and, therefore, are impractical. Nitrofurans (Hollinger and
Davis, 1966; Jackson, 1966; Hershburger, 1969), dichloracetyl amine
(Heller e^ , 1961), chlorambucil (vanThiel j^. , 1972), cyclo-
phosphamide (Inskeep ^ a^. , 1971; Fairley ^ al., 1972; Buchanan
al., 1975), and alkylating agents (Jackson, 1970) are examples of these
types of chemicals. Other methods with direct effects on spermato-
genesis currently being investigated involve various aspects of
4
testicular metabolism. At the present, an effect on glucose metabolism
seems the most promising.
A requirement for glucose by the testis is indicated by the
effects of low blood glucose levels or by conditions where glucose is
unable to be properly utilized by the tissues. Spermatogenic
degeneration and damage to seminiferous tubules (sloughing, nuclear
pyknosis, and multinucleated cells) result from insulin-induced hypo-
glycemia in adult rats (Mancine ^ , 1960) or from alloxan diabetes
in rats (Deb and Chattergee, 1963). This type of damage, to a lesser
degree, has been observed in diabetic impotent men studied by Fearman
et al. (1972) and in untreated diabetes mellitus cases examined by
Warren and LaCompte (1952). Additional observations that have been
made in diabetic males are increased incidences of impotence (Schoffling
e_t ^., 1963), decreased sperm count (Babbott ^ ^. , 1958), and poor
sperm motility (Kebanow and MacLeod, 1960).
The degree of dependence of the testis on carbohydrates as an
energy source and on exogenous sources for these carbohydrates has been
estimated by studies involving respiratory quotients (RQ) and oxygen
uptake rates with or without exogenous substrates. The RQ's determined
for the testis fall between total carbohydrate combustion (1.0) and
total lipid combustion (0.7) in all species studied, thus, indicating
that both serve as energy sources. Since the RQ's are closer to 1.0
than 0.7, the predominant source appears to be carbohydrates (Free,
1970). The rate of O2 uptake decreases in the absence of glucose in
rat testis by as much as 50% (Paul ^t^ a^. , 1952) while the rate in the
liver can be maintained for over 3 hours (Dickens and Greville, 1933).
5
A similar drop, not as sharp however, is observable in rabbit testis
(Ewing and Vandemark, 1963). These O2 uptake studies indicate that the
testis is dependent on an exogenous supply of glucose. Free (1970)
calculated that approximately 80% of the O2 consumed by the rat testis
is utilized for glucose oxidation which would correspond to the decrease
in O2 uptake in the absence of glucose. Furthermore, an increased rate
of O2 uptake in the presence of glucose has been observed in rat
(Elliott ^ al., 1937), rabbit (Ewing, 1967), and chicken (Ewing et al.,
1964) suggesting that glucose is being oxidized. Free and Vandemark
(1969) used radiorespirametric studies, where tissues are incubated
with labeled glucose and ^^C02 liberated is measured, to substantiate
glucose oxidation within the testis. Additional evidence indicating
glucose as an exogenous energy source for the testis has been provided
by Means and Hall (1968b). Through in vitro tissue cultures, they
observed that ATP levels declined in rat testis in the absence of
glucose and that these levels could be maintained, even increased, in
the presence of glucose.
A number of studies on the incorporation of lysine and other amino
acids into testicular tissue indicates a relationship between glucose
and protein synthesis in certain testicular cells. Davis and Morris
(1963) with the addition of 0.009M glucose to the incubation medium
increased the incorporation of lysine-U-^^C into proteins of rat
testis tissue by 600%. Similar addition of glucose to sections of other
tissues (thymus, spleen, kidney, brain, heart, liver, seminar vesicles)
caused either no change or up to a 50% increase in lysine incorporation.
When this type of study was done on testicular tissue lacking spermatids
6
such as the cryptorchid testis (Davis et , 1964; Free ^ a^., 1969)
or immature testis (Means and Hall, 1968a), glucose displayed no stimu-
latory effect on amino acid incorporation into proteins. Hierefore,
it appears that this glucose enhancement on protein synthesis is confined
mostly to the more mature germinal cells of the testis. Audioradiography
studies by Davis and Firlit (1965) in the presence and the absence of
glucose further substantiate this conclusion. They found that in the
presence of glucose most of the radioactivity was incorporated into
the spermatids and pachytene spermatocytes. Means and Hall (1968)
found a correlation between lysine incorporation into testicular pro-
teins and the levels of ATP and glucose. When glucose was added to the
media at the time when ATP levels began to drop, both the concentration
of ATP and the rate of protein synthesis increased. This indicates that
the effect of glucose on protein synthesis is related to glucose enhance-
ment of ATP production.
As a result of the series of investigations by Davis and associates
and the work of others on glucose metabolism, protein synthesis, and
cryptorchidism, Davis (1969) suggested that a possible clinically
useful male contraceptive could be a glucose analogue (such as 5-thio-D-
glucose). A glucose analogue could competitively inhibit the normal
glucose functions thereby causing sterility. Davis theorized that the
effects of an analogue would be completely reversible, since it is
protein synthesis in spermatids that is dependent on glucose and not
the earlier stages of spermatogenesis. Thus, reversibility of
sterility would be imminent as the more immature cells, ones responsible
for spermatogenic renewal, would not be affected by the glucose
7
deprivation.
The antispermatogenic properties of a glucose analogue, 5-thio-D-
glucose (5-TDG), were first shown by Zysk ^t al. (1975). Swiss albino
mice were fed 5-TDG at rates of 20-100 mg/kg/day for 7 weeks. The
initial degeneration of the spermatogenic cells was noted in 1-2
weeks. After 4-6 weeks of treatment, sterility was achieved in mice
receiving 30-100 mg/kg/day. This infertile condition, without any
apparent impairment of libido, was maintained throughout the treatment
period. Normal sperm development and fertility returned 5-8 weeks after
treatment was discontinued. Normal litters were sired by males upon
recovery. A testicular weight drop was observed with all dosages as
would be expected due to the decrease in numbers of testicular cells.
Also, a decrease in body weight was observed at high dosages. This
decrease was assumed to be due to the diabetogenic action of 5-TDG
which occurs when dosages are in excess of 50mg/kg (Hoffman and
Whistler, 1968). Pick (1979) observed similar results with 5-TDG
using a daily dosage of 33 mg/kg in Dub : ICR mice.
The antispermatogenic effect of 5-TDG received immediate attention
as a promising nonhormonal male contraceptive (Maugh, 1974; Ricketts,
1974; deKretser, 1976). However, additional fertility-sterility
studies in rats and mice contradicted the earlier results (Homm e^ al.,
1977; Lobl and Porteus, 1978). Homm (1977) treated male Wister
rats with 5-TDG in 50, 25, 12.5 mg/kg daily dosages. Sterility was
achieved in 8 weeks for 50 and 25 mg/kg groups and in 14 weeks for
12.5 mg/kg group. Although histological examination of the testis
showed a similar condition as in the mice examined by Zysk e^ aJL. (1975)
(i.e. Sertoli cells and spermatogonia present), the sterility was
permanent. One year after the drug was discontinued, fertility had
not returned.
Lobl and Porteus (1978) subjected male CF^ mice to a dosage of
50 mg/kg/day of 5-TDG for seven weeks. Contrary to the work of Zysk
e^ (1975), only 33% of the mice recovered and were fertile 10
weeks after the drug was discontinued. These fertile mice on examina-
tion had reduced testicular weight and numerous aspermatogenic tubules,
indicating some degree of permanent damage from the treatment. Although
these recent fertility-sterility studies are disappointing, they do not
discount the ongoing research studies with 5-TDG and its antispermato-
genic capacity. Even if 5-TDG does not prove to be useful as a male
contraceptive, the information about the testis obtained from these
studies will add to a more complete understanding of testicular func-
tions and, possibly, another approach to male contraception.
One major issue raised after the initial sterility study with
5-TDG was its possible side effects to the brain (Kakat, 1974). Since
glucose is one of the major metabolites in the brain (Kakat, 1974) and
5-TDG interfers with D-glucose transport (Whistler and Lake, 1972),
it is feasible that 5-TDG could affect functions within the brain.
Bushway e^ (1977) conducted maze performance tests on Sprague-Dawley
rats treated with 0, 50, or 100 mg/kg/day of 5-TDG for 14 days. They
found no significant difference between the treated and the control
groups in terms of the ability of the rats to rerun the maze or to
solve problems of Hebb-William maze after treatment. Even though 5-TDG
could be affecting regions of the brain other than learning and memory.
9
this initial study is encouraging.
The action of 5-TDG on spermatogenesis and the testis is uncertain.
The compound, which has a sulfur atom substituted for the oxygen atom in
the pyranose ring, is considered the closest existing analogue of D-
glucose. Due to the similarities of its physical and chemical proper-
ties to D-glucose, 5-TDG is capable of competitive inhibition of both
active and facilitated diffusion transport of D-glucose as well as
D-xylose. The diabetogenic action of 5-TDG is due in part to this
interference in cellular transport processes of D-glucose (Whistler and
Lake, 1972). Little or no metabolism of 5-TDG appears to occur within
tissue, since 97% is excreted into the urine within 24 hours (Hoffman
and Whistler, 1968). However, 5-TDG has been demonstrated to serve
as a substrate, although a poor substrate, for yeast hexokinase
(Hoffman and Whistler, 1968) and rabbit skeletal muscle phosphogluco-
mutase (Chen and Whistler, 1975). In addition, 5-TDG can interfere with
glycogen utilization by non-competitive inhibition of phosphorylase ^
and _b, and by inactivation of phosphorylase ^ through conversion to the
Jb form (Chen and Whistler, 1977). Whether one or a combination of these
factors, or as yet some undiscovered factor, are responsible for the
antispermatogenic ability of 5-TDG is the basis of several current
investigations.
One mode of action for 5-TDG could be through its diabetogenic
action, since diabetic condition in males attributes to fertility
problems, as previously mentioned. However, when male Sprague-Dawley
rats were given 50 mg/kg/day of 5-TDG and 0.5 unit/day of insulin (the
dosage that counteracts the diabetogenic effect), spermatogenesis was
still impaired (Lobl and Porteus, 1978). This study also found that
after 30 days of treatment with 5-TDG alone, the diabetogenic effect
was not present indicating that the rats were refractory to the effect
This study implies that some action other than interference with D-
glucose transport is responsible for antispermatogenic effect.
Another study supporting this conclusion was done by Burton and
Wells (1977). They conducted a study on 5-TDG effect on myo-inositol
and glucose-6-phosphate levels in the testis. Myo-inositol, although
its function is unclear, is synthesized in the testis at high rates
from glucose (Middleton and Setchell, 1972). Male Spb HCR mice were
treated with 5-TDG at 50 or 250 mg/kg/day rates for 7 days at which
time the myo-inositol and glucose-6-phosphate levels were measured.
They found elevated myo-inositol levels in both the 50 and 250 mg/kg
groups indicating an increased synthesis from glucose. The glucose-
6-phosphate levels were normal in the 50 mg/kg group, but were signifi
cantly elevated in the 250 mg/kg group. These increased levels of
both compounds imply some mechanism other than reduced intracellular
glucose is,involved in 5-TDG antispermatogenic activity.
Additional research on the direct action of 5-TDG on spermato-
genesis at the present is limited. Working with phenylalanine-U-^^C
incorporation into testicular proteins, Nakamura and Hall (1976,
1977) observed varying results between whole testis sections and
different testicular cell fractions. In the earlier study (1976),
normal incorporation occurred in the whole testis from a rat treated
with 5-TDG for 2 days, whether 5-TDG was present in the media or not.
When the testicular cells were separated into fractions by centrifugal
11
élutriation, however, inhibition from 5-TDG was observed in the
immature spermatid fraction and, to a lesser degree, in the mature
spermatid and heterogenous cell fractions (both fractions were
contaminated with immature spermatids). In the later study (1977),
they used more purified and additional cell.fractions. Inhibition
of amino acid incorporation occurred in the spermatocyte fraction.
However, when glucose was present in the media, it protected against
this inhibition. Since exogenous glucose is present in the testis,
it is doubtful this effect would occur ^ vivo. No inhibition of
incorporation was observed in Sertoli or Leydig cell fractions. Their
results correspond to earlier studies of glucose effect on protein
synthesis (Davis ^ > 1964; Free ^ j 1969; Means and Hall,
1968; Davis and Firlit, 1965). These earlier studies showed the
enhancing effect from glucose on amino acid incorporation into testi-
cular proteins was specific for the spermatid stage of spermatogenesis.
Recent studies of 5-TDG indicate that the drug's effect is not
confined to glucose transport. It would, therefore, be advantageous
to examine 5-TDG treated testes for changes in glucose metabolism
pathways. The major pathways within the testis are glycolysis-
tricarboxylic acid (TCA) pathway, pentose phosphate pathway (PPP),
and glucuronate-gulonate pathway, with the glycolysis-TCA predominating
(Free e_t aT. , 1969; Free, 1970). Each of these pathways, with their
different functions, seem to concentrate their activity in different
cell types. Experiments indicate that the glycolysis-TCA pathway,
which is predominately involved in energy production, is most active
in spermatids and spermatocytes, (Free, 1970). Leydig cells and
12
spermatogonia appear to be the major sites for the PPP, which generates
NADPH and D-ribose (Free, 1970). The glucuronate-gulonate, which is an
alternate source of pentoses, apparently is most active in the
interstitial cells (Free ^ aj^. , 1969) . Since 5-TDG has been shown
to. have no effect on the libido, therefore, no effect on the intersti-
tial cells (Zysk e¿ , 1975; Homm e^ , 1977; Lobl and Porteus,
1977; Pick, 1979) and since glucuronate-gulonate pathway is not as
active as the other two pathways, it was excluded in this study.
The present study attempts to ascertain the effect of 5-TDG on
glucose metabolism in the testis throughout the period required to
reach sterility by using radiorespirametric procedures. By using
glucose-l-^^C (G-l-^^C) and glucose-6-^'^C (G-6-^^C), the relative
activity of PPP to glycolysis-TCA pathway was examined.
The possibility exist that tissues other than testis are being
affected by the 5-TDG treatments. Therefore, another aspects of this
study was to examine the effect of 5-TDG on a non-testicular tissue.
Glucose is important to the function of most tissue in the body,
especially the brain and the liver. Any compound entering the blood
supply from the intestines, such as 5-TDG given orally, passes through
the liver. Malfunctions of glucose metabolism are responsible for
several liver disorders such as Kwashiorkor syndrome and liver
phosphorylase deficiency (Banks ^t, aT., 1976). For these reasons and
because glucose is oxidized in the liver by the same major pathways
active in the testis (Altszuler and Finegold, 1974), liver was
selected as the nontesticular tissue for the additional radiorespira-
metric studies,
MATERIALS AND METHODS
Animals
One hundred and thirty adult male mice of the Dub;ICR strain were
used in this study. All mice were sexually mature (13 to 14 weeks old)
upon initiation of the experiment. They were maintained at approxi-
mately 22°C with a 12-hour light regime throughout the study, Wayne
Laboratory Rat Chow and water were provided ^ libitium.
Treatment
The treated animals were administered a daily dosage of 5-thio-
D-glucose (Pfanstlehl Laboratories, Waukegan, Illinois) at a dosage
level of 37 mg per kg of body weight. The compound was dissolved in
water and was given orally by intubation in 0.25ml, 0.275ml, or 0.3ml
aliquots depending on the size of the mouse. The mice were lightly
etherized to facilitate the intubation procedure. The control mice
were treated in the same manner with the exception of receiving only
water. The experiment was divided into five treatment periods of 7,
14, 21, 28, and 42 days.
Design of Experiment
The experiment was designed so that 5 incubation runs could be
made weekly (i.e, five runs per treatment period). Each run involved
4 separate incubation tests- Glucose-l-^^C (G-l-^'^C) treated, G-1-^ C
control, Glucose-b-^'^C (G-6-^^C) treated, and G-6-^^G control- for
the liver and for the testes, resulting in a total of 8 tests. Two
control and two treated mice were sacrificed 3 hours after the final
daily treatment to initiate each run. Small sections of testes from
one of the treated and one of the control mice were incubated indi-
vidually in flasks containing G-l-^^C, while testis sections from the
other two mice were incubated with G-6-^^C. Liver sections from the
same mice were incubated in similar, but separate flasks. Each run,
therefore, had 8 flasks which correspond to the 8 incubation tests
being done. By running the control and treated tissue simultaneously
an attempt was made to eliminate factors due to incubation procedures.
In addition, one control and one treated mouse was sacrificed every 7
days for histological studies of the testis and the liver.
Tissue Preparation and Incubation
The testes and one lobe of the liver were removed immediately
after cervical dislocation of the mouse and placed in cold Krebs-
Ringer Phosphate Buffer pH 7.4 (Appendix A). The tissues were cut
into smaller portions and weighed on a Torsion Balance. Then, a
small portion of each tissue of a known weight (100+20 mg), were cut
into sections with scissors and placed into the appropriate incubation
flask containing either Glucose-l-^^C (specific activity 55.56mCi/mmol)
or Glucose-6-^‘^C (specific activity 52.8mCi/mmol) (New England Nuclear
Laboratories).
Each incubation flask contained 3.0ml of Krebs-Ringer Phosphate
Buffer pH 7.4 plus 180yg(300 units) of penicillin G, 3mg of D-glucose,
and 0.25001 of glucose-^'^C. The flask was constructed from a 50ml
heavy walled erlenmeyer flask to which a section (2.5 cm) of 15mm
pyrex glass tubing was fused as a sidearm. A scintillation vial was
connected to this sidearm through a 15mm hole drilled in the vial cap.
Between the cap and the top of the vial was placed an ”0" ring
SERUM STOPPER
Figure 1. Schematic diagram of the incubation flask.
(9/16" X 3/4") to ensure an airtight seal. A serum stopper was
used to cap the flask. (Figure 1). A section of Whatman No. 1 filter
paper (1.5 cm^) soaked with 0.2 ml of 10% KOH was placed within the
vial to absorb the ^^C02 (Buhler, 1972).. The flask design allowed
for easy removal and exchange of vials and the filter papers within
vials at will without interrupting the ongoing tissue incubation.
The incubations were carried out in two shaker baths at
70 cycles per minute at the appropriate temperature for each tissue
type (33°C for testes; 37°C for liver). After 5 hours of incubations,
0.2ml of ION H2S0^ was added to each flask to terminate enzymatic pro-
cesses and to facilitate the release of ^'^002 from the tissues. The
flask continued to shake for an additional hour to allow for
equilibration. During the incubation period, filter papers were
exchanged after the first, second, fifth, and sixth hour.
Counting
The filter papers were removed from the sidearm and placed on
sheets of aluminum foil to dry. Once dried, they were placed into
individual scintillation vials containing 15ml of fluid. This
fluid was composed of toluene with 0.5% PPO (2,5 diphenyloxazole)
as the primary fluor and 0.05% POPOP (1,4 bis-2-(4-Methyl-5-
phenyloxazolyl-)-Benzene) as the secondary fluor. The scintilla-
tion vials were then placed in the dark at 5°C for 12 hours to ensure
maximum counting efficiency.
The counts per minute (cpm) of the samples were determined with
a Packard Instrument Co. Series 3000 Liquid Scintillation Counter at
0°C. The three channels were set identically (Gain 18; Window 0.5-10)
17
for optimimum counting efficiency of C. The samples were counted for
20 minutes or 20,000 counts, whichever came first, to minimize the
error from background counts (Wang e^ , 1975).
Histology
Histological studies were done to examine the tissue for abnor-
malities associated with 5-thio-D-glucose treatment and to gauge the
progress of degeneration of the spermatogenic cycle.
The tissues were fixed in Carnoy's fixative (Appendix A) for
2-3 hours. Tissues were dehydrated in a series of ethanol solutions
and cleared in xylene (Appendix B). Paraffin infiltration was done
in a 58°C oven using 4 changes of hot Tissuemat. The Timstation Tissue
Center was employed for the paraffin embedding. All tissues were
sectioned at 6-8 microns and stained with Hemotoxylin-Eosin (Appendix
B).
Chromatography
A series of paper chromatography were run to check for the
presence of 5-thio-D-glucose in the testis, liver, and urine at
3, 6, and 12 hours after treatment. Homogenates of testis and of
liver were made using a hand homogenizer. All three samples as well
as 5-thio-D-glucose and D-glucose standards were spotted on Whatman
No. 1 Chromatography paper. The chromatograms were done in a
desending glass chamber with 1-butanol-ethanol-water (40:11:19) as
the developing solvent for 12 hours.
Detection of spots on the dried chromatograms were made by the
silver nitrate reagent method of Trevelgan, Procter, and Harrison
(1950). The chromatograms were first passed rapidly through a silver
18
nitrate reagent (prepared by diluting 0.1ml of saturated aqueous
silver nitrate solution with 20ral of acetone and then adding water
dropwise until the precipitant that fomned redissolved). After the
papers were dried, they were sprayed with a 0.5N solution of sodium
hydroxide in aqueous ethanol (made by diluting saturated aqueous
sodium hydroxide in aqueous ethanol). Once the reduction of the
silver by the sugar was completed, the chromatograms were immersed in
6N ammonium hydroxide for 5 minutes and then placed under running water
for 1 hour. The chromatograms were dried in an oven at 100°C. The
spots formed were compared to the 5-thio-D-glucose standards by
color and values.
Analysis
For each sample, the percent yield of ^*^002 was calculated using
the following formula: (cpm of sample)/(cpm initially added to
incubation flask). The mean of the five runs of each organ for the
four groups (G-l-^^C treated and control; G-6-^^C treated and control)
and for each hour sampled were determined as well as the mean for the
total first 5 hours. The total 5 hours values of the treated were
statistically compared to those of the controls using one-factor
analysis of variance (ANOV) for each treatment period and each tissue.
The C-l/C-6 ratio (% yield of ^^C02 from G-l-^^C to % yield of
^^C02 from G-6-^'^C) was used as an index of pentose phosphate pathway
activity. The ratios were determined using the percent yield values
from the first hour for all tissues, treated and control, for each
run and for each treatment period. The ratios for each treatment
period were statistically analyzed by one-factor ANOV for both the
testis and the liver.
RESULTS AND DISCUSSION
Chromatography Studies
Paper chromatography studies were done to determine the presence
of and to estimate the duration of 5-TDG in the testis. The data
(Table 1) indicated that 5-TDG was present in the testis after 3
Table 1. A summary of values of samples ran at 3, 6, and 12
hours after 5-TDG treatment.
Time
(in hours) D-glucose 5-TDG Testis Liver* Urine
3 0.24 0.30 0.30 — 0.30
6 0.23 0.29 NP** — 0.29
12 0.25 0.35 NP — 0.35
*Could not distinguish spots in the liver samples
**Not present
hours, but not after 6 hours. This information corresponds to an
earlier report by Hoffman and Whistler (1968) on 5-TDG and hyperglycemia.
They found that the diabetogenic effect of 5-TDG was absent by the
fourth hour after the injection due to the loss of the drug into the
urine. They also determined that 97% of 5-TDG was excreted with the
urine by 24 hours after the injection. In the present study, 5-TDG
was detectable in the urine during all times sampled (3, 6, 12 hours);
although, the size of the spots (i.e. the quantity of 5-TDG) did
diminish with time. Liver homogenates contained substances that formed
a streaking spot, thus, preventing the identification of 5-TDG.
21
Histological Studies
Histological work was carried out to compare 5-TDG treated testi-
cular cells from this study to cells of previous 5-TDG studies and to
indicate the approximate time sterility was achieved. Treated and
control testes were examined for each treatment period. No histologi-
cal changes were observed in the controls throughout the experiment.
(Figure 2) As expected, the treated germinal cells of the semini-
ferons tubules did undergo degeneration. The progression of this
degeneration was similar to that reported by Zysk e^ aA. (1975),
Lobl and Porteus (1978), and Fick (1979).
After 14 days of treatment, spermatozoa and spermatids were
normal in appearance and there were a few morphological changes.
These changes were mainly vacuolated areas at or near the basement
membrane of seminiferous tubules and enlarged spermatogenic cells.
(Figure 3) Spermatozoa were still present in the testes after 21 days
of treatment, but there were increasing numbers of abnormalities such
as sloughing of germinal cells, debris from degenerating cells, and a
few multinucleated cells. (Figures 4 and 5) These multinucleated
masses are fusion of degenerating spermatogenic cells and have been
observed in all 5-TDG treated testes (Zysk e^ ad.. , 1975; Homm et al.,
1977; Lobl and Porteus, 1978; Fick, 1979). By 28 days of treatment,
the degeneration had increased substantially; although, spermatozoa
and spermatids were still present. There were irregular shaped
spermatids as well as more and larger multinucleated cells. The
degeneration of cells appeared to progress through the tubules
unevenly. Some tubules were extremely abnormal, while others appeared
22
normal. (Figure 6 and 7) The histological appearance of the testes
indicated that sterility did occur by the end of the final treatment
period (42 days). Although the degeneration was in different stages
within the seminiferous tubules, the testes and the epidid3nnis were
devoid of spermatozoa. Spermatids were still, present, but many of
them were irregularly formed. Some tubules contained only eosinophilic
cellular debris and a few scattered cells. Other possessed many of the
multinucleated cells. Most all of the tubules had normal appearing
spermatogonia and Sertoli cells. (Figures 8, 9, 10 and 11) None of
the testes examined showed any detectable changes in the interstitial
cells. In all testes examined, cells undergoing division, as indicated
by dark staining chromatin, were visible.
This progression of degeneration was similar to that observed
by Zysk e_t (1975). They found a few large spermatogenic cells
after 14 days of treatment. By 28 days, the testes showed increased
degeneration, multinucleated cells, and cytoplasmic vacuoles.
Although some tubules were devoid of spermatids, most have spermato-
genesis actively occurring. The epididymis of the 28-day treated
testes still contained spermatozoa. By 35 or 42 days of treatment,
spermatozoa were loss in all testes and the testes showed severe
spermatogenic degeneration.
At the end of the final treatment period, two treated mice were
allowed to recovery for 15 weeks. At this time, the testes were
histologically examined. Spermatozoa were numerous in the epididymis
and the seminiferous tubules. Most tubules were normal in appearance
however, a few tubules (<10%) were still in a degenerative state.
23
Since there were only Sertoli cells and a few spermatogonia in these
tubules, the damage was apparently permanent. (Figures 12 and 13)
Two mice remaining after termination of histological and radio-
respirametric studies were continued on 5-TDG for an additional 14
days. However, the treatment was reduced from the previous daily
administration to twice a week. At the end of that time (42 days
of daily treatments followed by the 14 days of twice weekly treatments)
the testes were removed and examined histologically. The appearance
of the testes were similar to the 42 days testes. (Figures 14 and
15) These results suggests an alternate treatment program to the
continuous daily treatments. In this alternate program, the drug
would be given daily until the testes were devoid of spermatozoa
(28 to 42 days in the mice), at which time treatment would be reduced
to once or twice a week to maintain the sterile condition. If this
approach works, there would be a two-fold advantage. By limiting the
deleterous effect to the spermatids and the spermatozoa, the
recovery of spermatogenesis would be more rapid. Also, the permanent
damage observed in some tubules of 5-TDG testes in this and other
studies (Homm ^ , 1977 ; Lobl and Porteus, 1978) might be eliminated.
Liver tissue was also histologically examined. No morphological
changes in the treated livers were detected in any of the sections.
Figure 2. A section from a control testis showing normal spermogenic
epithelium. BM, basement membrane; L, lumen; S, sertoli
cell; SG spermatogonium; ST, spermatid; SZ, spermatozoan;
PSC, primary spermatocytes. Magnification: 180X
Figure 3. A section from a testis after 14 days of 5-TDG treatment.
The photomicrograph shows normal spermatogenesis still
occurring. Magnification: 180X
'1 ^
?m « -ixM ^ w
'
S'- t^ «i,
'
? *î-v.--’.-'•xJrK'-!Í y ?;».*^ , V
'«i
«.'• mZ’ " é*‘ti^i* ? • ?:? -ii
>^ •-'-^ ' - • JL ?? .. ^IkL* * *•wflL 1» ^ > « 4^ '.
^ - .• • - .f'
m ^^ Í T» -
kI *-’B- ji* v ?i>; rf if: :<. .
Figure 4. A section from a testis treated with 5-TDG for 21 days.
The beginning of degenerative products are present.
Arrow indicates area magnified in Figure 5. D, degen-
erative product. Magnification: 180X
Figure 5. Area from Figure 4 at a higher magnification. Note the
degenerative products in the lumen. Magnification: 450X
^ - TOJ
• n'Ji
I-'p-.
#? '• i*' --; 4 *ÍÍÍ
^ •
t;. - ’ . Î "
?;* î.'^V‘«?'^*-^ '-^ >? ' • :.v ? ,_•
, '••H. %; ... ^?r'
- /
.t"' ^
? t' ï^'.S' *' s 4--*..
r ’i-v ^ V'-'^ ^ ,'
" 3^-5q». 4vA^ ' ^ ‘
5 ' . -%b-V •i(»' o'/>’’'v.' -• V , ;>
- -
^
- - *éfr
Figure 6. A section from a testis treated with 5-TDG for 28 days.
Note the multinucleated cells (MC) is the tubules. Arrow
indicates area magnified in Figure 7. Magnification:
180X
Figure 7. Area from Figure 6 at a higher magnification. Note multi-
nucleated cells (MC) and spermatozoa tails in the lumen.
Magnification: 450X
 
Figure 8 A section from a testis treated for 42 days with 5-TDG.
Note the disrupted spermatogenic epithelium with multi-
nucleated cells and eosinophilic masses (E) in the lumen
Interstitial cells (IC) appear normal. Black Indicates
the area magnified in Figure 9. Magnification: 180X
Figure 9. Area from Figure 8 under higher magnification. E, eosino-
philic masses; IC, interstitial cells; MC, multinucleated
cells. Magnification: 450X
4a 3p’
?<^.?-î§
i;* -.'t^s ÍÍ
<»%W \\
Figure 10 A section of the testis after A2 days of 5-TDG treatment,
showing multinucleated cells. Arrow indicates tubule
magnified in Figure 11. Magnification: 180X
Figure 11. Tubule from Figure 10 under higher magnification. Note
multinucleated cells (MG) and primary spermatocytes still
undergoing division. Magnification: 450X
 
Figure 12 Section from testis treated for 42 days and allowed to re-
cover for 15 weeks. Spermatogenesis appears to be occurring
normally. Magnification: 180X
Figure 13. Section from the same testis shown in Figure 12. A
spermatogenic tubule is indicated by the arrow, Magni-
fication: 180X
 
Figure 14. Section from testis treated daily for 42 days with an
additional 14 days of twice weekly treatment. Note similar-
ly to 42-day treated testis. Arrow indicates area magnified
in Figure 15. MC, multinucleated cells. Magnification:
180X
Figure 15. Area from Figure 14 under higher magnification. MC, multi-
nucleated cells. Magnification: 450X
 
33
Radíorespirametrlc Studies
The radiorespirametric studies were used as evidence of glucose
oxidation, as indicated by evolved ^^C02 from labeled glucose, within
the tissues. Glycolysis-TCA and PPP are the two major routes of
glucose oxidation in both the testis and the liver, as previously
mentioned. The relative activity of PPP to glycolysis-TCA can be
evaluated by using G-l-^^C and G-6-^^C in radiorespirametric studies
to compute C-l/C-6 (% recovery of ^^C02 from Gl-^'^C/ % recovery of
^^C02 from G-6-^^C). This comparison is possible because the initial
carbon cleaved in each pathway is different.
One function of PPP is to form pentoses (e.g. D-ribose-5-Phos-
phate). In this process, the 1-carbon of glucose is cleaved off and
evolves as CO2 (Figure 16). Depending on several factors, the formed
pentose may remain as a pentose or be converted to another sugar and
possibly enter the glycolysis-TCA cycle. Glycolysis results in
glucose being split into dihydroxyacetone phosphate and D-glycer-
aldehyde 3-phosphate. These two compounds continue through the rest
of the reactions to form two pyruvates. The first CO2 evolves when
pyruvate is oxidized to acetyl CoA which then enters the TCA cycle.
The first two carbons (one from each pyruvate) are the 3- and 4-carbons
(Figure 17). The 2- and 5-carbons are next to evolve as CO^, with the
1- and 6-carbons coming off last. The last four carbons are released
during the TCA cycle. Thus, if only glycolysis-TCA pathway is
functioning the C-l/C-6 would equal 1. Anytime this ratio is greater
than 1, both pathways are in operation.
There exist a degree of uncertainity associated with the fate of
NAOPH + H+ COO"
6-phospho
gluconic acid
CH.OH
lo
hÍoh
1CO2 + I
HCOH
I
CH2OPO3H2
occi—PO
CHçOH CHO
11 ^ 1
C=0 HCOH
HOCH HCOH
HC1OH HCI OH
i
CH2OPO3H2 CH2OPO3H2
xylulose-5-PO^ ribose-5-PO
Figure 14. Diagram of the Pentose Phosphate Pathway showing
when CO2 is cleaved off.
CHO CHoOH (
1 ^ 'H2OPO3H21 1 1
HCOH C=0 (>0
1 1 11 XH^OH
HOCH HOCH HOCH I ^ 4ÇHO
1 \ 1. / 1 +ioH ^^C0HHCOH HCOH H(
1
1 1 [ ,CH20P03H2 eCH20P03H2
HCOH HCOH H(
11 1 |:0H
CH2OPO3H2 CH2OPO3H2 (:H20P03H2 dihydroxy- glyceraldehyde
acetone 3-phosphate
glucose 6-PO^ fructose 6-PO^ fructose 1,6- phosphate
V
\/
Figure 15. Schematic representation of glycolysis-TCA cycle demonstrating the order
that CO2 evolves.
41
the pentose formed in PPP. If the pentose formed from G-6-^^C enters
the TCA cycle, ^^^€02 would evolve. When this occurs, the eventual % re-
covery of ^^C02 from G-l-^^C and from G-6-^^C would be equal and C-l/C-6
would not give useful Information. Therefore, only the initial % re-
covery (generally from the first hour) is used to calculate the C-l/C-6
value. Additionally, the rate of evolution ^‘^C02 has been shown to
drop after the first hour in the testis when gluconate-l-^'^C was used to
gauge the PPP activity. Thus, PPP appears to be inhibited in vitro due
to some limiting factor (Free and Vandemark, 1969). If the fate of
pentose, however, does not involve further degradation, the final %
recovery of ^^C02 from G-l-^'^C will be greater than % recovery from
G-6-^'^C.
The radiorespirametric data on the liver tissue for the individual
treatment periods were plotted to show the cumulative percent recovery
of ^^C02 for each hour sampled (Graphs 1-5). The total percent recovery
14
of CO2 for each labeled glucose was statistically examined using one
factor ANOV (Table 2; Appendix C). No significant differences existed
between the treated and the control in total percent recovery of ^'^C02
from either of the labeled forms of glucose with one exception. In
14
the 14 day group, the G-6- C treated were lower than the control group
at 0.01 significant level. The overall importance of this one
difference is probably little, since it did not continue and it did
not occur in the G-l-^^C group. If the tissues had actually been
damaged from the 5-TDG treatment, glucose metabolism would have re-
mained reduced for all subsequent treatment periods.
42
Graphs 1-5. Rate of evolution (cumulative) and yield of ^^C02 from
specifically labeled glucose by liver tissue from 5-TDG
and control mice during each treatment period. Each
point represents the mean of 5 animals. Graph 1, 7
days; Graph 2, 14 days; Graph 3, 21 days; Graph 4, 28
days; Graph 5, 42 days. — treated, control.
GTIPRCL6ii (cumulative)frlelsuaoirvscOnoemutvnsderet r2y
Time in Hours
Table 2. Mean and standard error (n=5) for total percent recovery of
l'^C02 from glucose-l-^^C and glucose-6-^^C in liver tissue.
Glucose- 1-1-40 Glucose--6-14c
Treatment
Period Treated Control Treated Control
7 0.213+0.042 0.214+0.017 0.035+0.007 0.026+0.004
14 0.060+0.012 0.067+0.014 0.016+0.001* 0.033+0.005
21 0.108+0.025 0.125+0.015 0.030+0.002 0.031+0.003
28 0.244+0.064 0.213+0.050 0.039+0.006 0.046+0.006
42 3.503+0.652 3.532+0.881 0.861+0.304 0.641+0.203
*Significantly different at 0.01 level
In addition to cumulative percent recovery graphs, the percent
recovery of the first hour for all treatment periods was plotted
(Graph 6). The graph indicates little difference between the treated
and the control data. However, an overall increase in percent re-
covery is present during the final two treatment periods. From
Graph 4 and 5, it is evident that the major portion of this increase
is due to greater recovery during the first hour. Furthermore, the
steep slopes of the lines on these graphs imply increased rates of
^^C02 recovery, that is, increased glucose utilization. The reason
for this increase is uncertain. However, since it occurred in all
groups (G-1 treated, G-1 control, G-6 treated, G-6 control), it is
apparently a result of some factors affecting all groups and not from
the 5-TDG treatment.
45
Graph 6. Percent yield of ^^C02 after one hour of incubation from
specifically labeled glucose by liver tissue from 5-TDG
treated and control mice for each treatment period. Each
point represents the mean of 5 animals. treated,
control.
}6
1-14C
35
34
83
82
81
80
,10
09
08
.07
,06
,05
04
03
.02
.01
7 14 21 28 42
Treatment Time in Days
47
The C-l/C-6 values computed for the liver tissue (Table 3)
were statistically analyzed by one-factor ANOV. The F-values indi-
cated no significant difference between the treated and the control
(Appendix C). The ratio values are comparable to values recorded in
Table 3. Summary of the C-l/C-6 ratios for the liver tissue.
C-l/C-6
Treatment
Period Treated Control
7 5.14 4.41
14 2.22 1.91
21 2.47 3.21
28 10.08 9.08
42 6.56 7.35
*C-l/C-6 = -L^C02 from glucose-l-^'^C/ -*-‘^002 from glucose-6--*-^C
the literature for rat liver. The mean ratio for all control tissue
was 4.91 (Range: 2.22-10.08). Bloom a]^. (1955) after 2 hours
of incubation obtained a mean of 4.7 (Range: 1.54-8.33) when the
tissues were incubated for 3 hours.
It is important that the 5-TDG treatment had no significant
effect on glucose metabolism in the liver. If a drug is to be useful
as a male contraceptive, it must selectively affect the the testis.
Although this study does not examine all aspects of liver metabolism
and function, it does show that 5-TDG does not impair glucose metabo¬
lism.
48
14
The data on the percent recovery of CO2 from the studies of
the testis (Table 4; Graphs 7-13).*A?ere statistically analyzed using
one-factor ANOV, The F-values (Appendix C) indicate no significant
Table 4. Mean and standard error (n=5) for total percent recovery
of ^^C02 from glucose-l-^'^C and glucose-6-^^C in testis
tissue.
Glucose-1-.14c Glucose--6-i-^C
Treatment
Period Treated Control Treated Control
7 3.114+0.132 2.926+0.397 0.932+0.187 0.721+0.153
14 1.021+0.218 1.321+0.422 0.419+0.048 0.496+0.096
21 1.999+0.375 2.026+0.399 1.513+0.368 1.068+ 0245
28 1.764+0.133 1.662+0.159 0.873+0.064 0.805+0.181
42 3.673+0.457* 1.447+0.209 1.588+0. 1.071+0.152
*Significantly different at 0.05 level
difference between control and treated tissues in total percent re-
covery of ^^C02 from G-6-^^C during any of the treatment period. Re-
covery from G-l-^^C showed no significant difference until the 42
day treatment period. At this time, the treated group had a
statistically higher percent recovery than the control group. The
greater glucose oxidation in testicular tissue of 42 day treated
animals is contrary to results for other testicular disorders with
similar spermatogenic degeneration such as cryptorchid testis. This
contradiction will be discussed in more detail later. Also, during
the 42 day treatment period, it should be noted that while for the
control, the total percent recovery values from G-l-^^C and from
A9
G-6-^^C were close (1.447 to 1.071), this was not true for the
>
treated group. The total percenfei^recovery from G-l-^^C exceeds the
value for G-6-^^C (3.673 to 1.588). This difference indicates more PPP
activity in the treated testes.
The testis radiorespirametric data were plotted by cumulative
percent recovery for each treatment period (Graphs 7-11) and by
the first hour percent recovery for an all treatment periods (Graph
12). In Graph 12, there is an overall increase during the final
treatment period similar to the one that is observable in liver
tissue (Graph 6). Examination of Graph 11 shows, as in the liver,
that the majority of the increase occurred during the first hour
of incubation. The presence of the increase in both tissue types
further indicates that the increase is due to a common factor
affecting all tissues. There is an interesting deviation in Graph 12
involving G-6-^^C. While the slopes of the treated and controls are
similar for G-l-^^C, this is not true for G-6-^^C. The slope for
the treated is approximately 3 times greater than the control. This
seems to imply greater initial glycolysis-TCA activity in the treated
testes. The total recovery, however, is not significantly different.
The cause or significance of this apparent increase is uncertain.
The C-l/C-6 values computed for the testicular tissue are
recorded in Table 5. The mean of all the control testes was 3.42
with a range of 1.56 to 5.26. Although the ratio for mouse testis
has not been previously assessed, the values of this study are
apparently within the realm of feasibility as estimated by relative
enzyme activity of the PPP. The relative activity of the enzymes of
Graph 7-11. Rate of evolution (cumulative) and yield of ^'^C02 from
specifically labeled glucose by testis tissue from 5-TDG
and control mice during each treatment period. Each
point represents the mean of 5 animals. Graph 7, 7 days
Graph 8, 14 days; Graph 9, 21 days; Graph 10, 28 days;
Graph 11, 42 days. treated, control
CGRfPTlo1iroelcauós ()cumulaitveocrcOonemofv-t6ndsei-tsCery2
Time in Hours
14
Graph 12. Percent yield of CO2 after one hour of incubation from
specifically labeled glucose by testis tissue from 5-TDG
treated and control mice for each treatment period. Each
point represents the mean of 5 animals. treated,
control.
coverV
54
the PPP to those of the glycolysis-TCA pathway have been determined
for mouse testis (Blackshaw, 1963), as well as for the testes of ram,
rat, chicken, and rabbit (Free, 1970), Among these species the
order of increasing activity of the PPP in the testis is the follow-
ing: chicken<rat<rabbit<mouse<ram. If relative activity is a
legitimate estimate of C-l/C-6 ratio, the mean of this study (3.42)
would seem reasonable compared with the value of 3.30 for the rabbit,
(The recorded values for the rabbit testis are 2.61 (Free et al. ,
1968) and 3.30 (Bloom, 1955).)
Table 5. Summary of the C-l/C-6 ratios for the testis tissue.
C-l/C-6
Treatment
Period Treated Control
7 3.88 3.79
14 2.42 3.28
21 1.51 1.56
28 4.33 3.19
42 8.37* 5.26
*Significantly different at 0.025
The C-l/C-6 values of treated testis were statistically compared
to the control by one-factor ANOV (Appendix C). There was a slight
increase in the treated tissue over the control during the 28-day
treatment period, and by the ^2-day period, the treated was signifi-
cantly higher. The increased ratio indicated increased PPP activity
in the treated cells. Overall, there was little difference between
the control and the treated ratios. The mean of the treated testes
was 4.10 (Range; 1.51-8.37) as compared to 3.42 of the control.
On an overall basis, the glucose oxidation was higher in the
treated testes during the 42-day treatment period. This is implied
when the combined percent recovery from G-l-^^C and G-6-^^C for the
treated (5.261) was compared to the combined value for the control
(2.518). Part of the increase can be attributed to the increased
PPP activity. The ^^002 percent recovery data from G-l-^^C
(Table 4) was significantly higher in the treated testes during the
42-day period. A similar increase in recovery from G-l-^'^C was ob-
served in the cryptorchid and testosterone treated testes by Davis
e^ (1968). An examination of the G-6-^^C percent recovery data
for the treated (1.588) and the control (1.071) indicate that
glycolysis-TCA activity in the treated testes was normal although,
the initial rate in the treated did appear greater. The treated
value, although slightly higher, was not significantly higher. Thus
the data imply that the overall increase is due mainly to the
higher PPP activity, with a normal amount of glycolysis-TCA activity
This increase of glucose metabolism in the 5-TDG testis raises
one major question. How can an increase occur when the cells
responsible for the greatest amount of glucose oxidation are degen-
erating? As previously stated, this increase contradicts studies wi
other testicular disorders where spermatogenic degeneration occurs.
Examination of the data from these studies gives some insight into
the answer. In studies with cryptorchid testis (Free ^ , 1969)
and with nitrofuran treated testis (Paul e^ a^. , 1953), a decrease
56
in glucose metabolism corresponded to the decrease in spermatogenic
cells. In the cryptorchid testis an increase in PPP activity did,
however, occur. (The PPP activity was not measured in the nitrofuran
testis) Thus, while the PPP increased in both cryptorchid and 5-TDG
testis, the glycolysis-TCA showed a decrease in cryptorchid and
appeared normal in 5-TDG testis. The reason for the variation
can only be surmised at this time; however, two obvious differences
exist between the conditions.
One noticeable difference in the cryptorchid and the nitrofuran
testes from the 5-TDG testis is the more rapid rate of degeneration.
In both of these conditions, the testes are devoid of all spermatozoa
and spermatids, and most spermatocytes by 14 days into the treatment
(Free e^ , 1969; Paul ^ , 1953). Spermatocytes and spermatids
were still present in the testes of this study even after 42 days
of treatment. It is possible that the metabolic activity by these
cells plus the PPP increase could account for the overall increase.
Another difference is the absence of the multinucleated masses
which are present in the 5-TDG testes. If these giant cells were
still active, they could account for some of the metabolic activity.
Spermatids with two or more nuclei have been observed continuing
their development to form giant spermatozoa (Maximow and Bloom, 1957) .
This observation implies that, at least in some cases, these multi-
nucleated cells remain active metabolically.
The observed glycolysis-TGA activity is apparently due to the
remaining spermatogenic cells and possibly to any active multi-
nucleated cells. The overall increase is a combination of this
57
glycolysis-TCA activity and the increased PPP activity.
Two possible explanations exist for increased PPP activity in
the 5-TDG and cryptorchid testes. These explanations involve two
different functions of the PPP the production of pentoses and the
generation of NADPH. As previously mentioned, the Leydig cells and
spermatogonia are the sites of most of the PPP activity in the testis.
The importance of the PPP to the Leydig cells is thought to involve
steroidogenesis. Steroidogenesis has been shown to be dependent on
NADPH for hydroxylation reactions in many tissues including the testis
(Grant and Brownie, 1955; Halkerston e^ ad., 1961; Hall, 1970). More-
over, the Importance of PPP activity in steroidogenic endocrine tissues
has been demonstrated (Field e^ ad. , 1960; Hall, 1970). The implication
of this information is that PPP in the Leydig cells is needed to
generate NADPH for androgen production. Therefore, if the degeneration
of the spermatogenic cycle were to cause a compensatory Increase in
androgen production, a corresponding increase in the PPP activity could
occur.
This explanation is supported by 5-TDG sterility studies by Pick
(1979). These histochemical studies showed that androgen production
was slightly higher in the 5-TDG testis. A similar increase was ob-
served by Kormano e^ (1964) in cryptorchid testes of rats. Thus,
it appears that, at least part of the increased PPP activity was due
to increased steroid production in the cells of Leydig. Since the
germinal cells are also involved, to some degree in steroid production,
an additional increase in PPP could take place in these cells.
The other explanation Involves the spermatogonia and pentose
58
production. Although most of glucose metabolism is attributed to
spermatids and spermatocytes, glucose is also thought to be the major
metabolite of spermatogonia (Free, 1970) with the glucose being oxidized
mainly in these cells by PPP pathway. With one function of PPP being
the production of D-ribose 5-phosphate for nucleic acid synthesis, it
is not surprising that spermatogonia, which are responsible for sperma-
togenic cell renewal, would have high PPP activity. The capacity of
high PPP activity in these early sperraatogenic cells is also evident
by the fact that immature calf testes have been shown to have an
extremely high C-l/C-6 ratio of 56 (Fields ^ , 1960).
The observed increase of PPP in the 5-TDG and cryptorchid testes
plus the large amount of activity in the immature testis tends to
imply that PPP activity is connected to cell division. Moreover, in
this study the G-l-^'^C percent recovery was greater than G-6-^'^C for
the treated testes. Thus, the pentoses formed in PPP were apparently
being used and not recycled through the TCA cycle. This information
along with histological observations previously mentioned indicates
the spermatogonia of the 5-TDG testes were involved in cell renewal and
cell division. Therefore, the increases in PPP pathway may be partially
due to a compensatory type of action. That is, an unmasking of the
spermatogonia by the degeneration of the more mature cells, resulting
in increased cell division and, thus, increased PPP activity.
An unmasking effect was described by Davis e^ (1965) in
relationship to increased protein synthesis in the rat cryptorchid
testis. This increase was observed after the spermatids had degene-
rated. One factor cited by Davis £jt for this increase was an
59
unmasking of the more undifferentiated cells (spermatogonia and
spermatocytes), which had a higher degree of protein synthesis.
These explanations are related to each other by functions and
hormones. Androgens (testosterone) are partially responsible for
the initiation of spermatogenesis. Consequently, if increased andró-
gen production would occur, a corresponding increase in cellular di-
vision of spermatogonia might result. The higher PPP activity, there-
fore, is most likely due to a combination of more NADPH generation and
greater ribose production, with the overall increase resulting from
the greater PPP activity.
A study by Davis e^ aJL. (1968) supports the idea that PPP activity
in both the interstitium and the tubules are involved in the increase.
They measured the CO2 recovery from G-l-^^C and G-6-l^C in cryptorchid
testes (devoid of most germinal cells) and testosterone propionate
treated testes (depleted interstitium). In both conditions they
observed significantly higher ^^C02 recoveries from G-l-^'^C in the
treated testes. This data implies an interrelationship between the
two parts of the testes which is reflected by the PPP.
The increased glucose oxidation, whatever the source, rules out
one mechanism of action. It eliminated the possibility that 5-TDG works
through reduced intercellular testicular glucose, as originally thought.
This conclusion is supported by previous 5-TDG studies. Burton and
Wells (1977) found that 5-TDG treatment caused increased myo-inositol
levels in the testis which indirectly reflects greater intracellular
glucose levels. Furthermore, when the diabetogenic effect of 5-TDG was
counteracted with insulin in another study, the antispermatogenic action
60
was not affected (Lobl and Porteus, 1978). Thus, it appears that the
interference with glucose transport by 5-TDG is not sufficient to
cause sterility.
The data of this study could also be interpreted to imply that
5-TDG is not competitively inhibiting any of the enzymes of glucose
oxidation, since the activity of the pathways were either normal or
increased. However, in light of two factors such a conclusion would
be extremely hasty. First, the incubations were done with whole
testis section. Hence, 5-TDG could be effecting one cell type with the
effect being hidden by the activity of the remaining cells. This type
of effect was observed by Nakamura and Hall (1976), when working with
amino acid incorporation. They found no inhibition from 5-TDG in
incubation of the whole testis. However, when the different cell
types were separated and the individual fraction were incubated,
inhibition occurred in some of the fractions. The other factor is
the time element. The action of 5-TDG is probably very short termed,
since 5-TDG is only present in the system for a short period (3-6 hours)
before it is removed. Thus, the action of 5-TDG could be on one or two
spermatogenic cell types and only for the brief period when 5-TDG
is in direct contact with the testis. To investigate this concept
additional research using centrifugal élutriation to separate cell
fraction would be required.
SUMMARY
Male mice were treated with 5-TDG of either 7, 14, 21, 28, or 42
days. At the end of each period, radiorespirametric studies were
done on testis and liver sections, to monitor glucose metabolism.
The C-l/C-6 ratio was computed to determine the relative activity of
PPP to glycolysis-TCA.
The ^“^002 data showed no significant effect on the glucose
metabolism in the liver tissue from the 5-TDG treatment. The PPP was
also uneffected.
The 5-TDG treatment caused increased glucose metabolism in the
testis. Most of the increase was attributed to greater PPP activity.
The PPP activity was accredited to a compensatory (unmasking) action
of the remaining cells especially the Leydig cells (steroidogenesis)
and spermatogonia (cell division and renewal). The glycolysis-TCA
cycle appeared to be functioning normal; although, the cells responsible
for this activity was not determined. The treated testis contain many
abnormal cells (multinucleated masses), which may have been meta-
bolically active.
\
The glucose metabolism data complicate the understanding of
5-TDG's mechanism of action. The results tend to eliminate one of
the simpler explanations - the interference with D-glucose transport.
Before the actual mechanism(s) of 5-TDG will be ascertained, much
additional research will be required.
Although, the results of this study provide little insight into
the mechanism of 5-TDG's antispermatogenic action, the study does
62
serve as the foundation for further research with 5-TDG and glucose
metabolism. Additionally, the radiorespirametric studies on the
liver provide some insight into 5-TDG effect on nontesticular tissue.
These results are encouragingly supportive to the idea that 5-TDG may
be selectively affecting the testis. The study also suggested an
alternate treatment program, which could possibly reduce the permanent
damage observed in some of the seminiferous tubules in several cases.
Finally, the study tends to support the concept of compensatory
action. The increased PPP activity alluded to possible compensatory
action by both the Leydig cells and the spermatogonia.
APPENDIX A
PREPARATIONS FOR SOLUTIONS USED IN THIS STUDY
Krebs-Ringer Phosphate Buffer (pH 7.4)
1. 0.90% NaCl
2. 1.15% KCl
3. 1.22% CaCl2
4. 3.82% MgS04-7H20
5. O.IM Phosphate Buffer (pH 7.4)
Solutions 1-5 are made up separately. They will keep for
months at 5°C. Before use, mix solutions in the following
ration: 100:4:3:1:20.
Carnoy's fixative (100ml quantity)
Glacial acetic acid 10ml
Absolute ethanol 60ml
Chloroform 30ml
APPENDIX B
HISTOLOGICAL PROCEDURES
Dehydration
95% Ethanol 1.5 hours
100% Ethanol 1.0 hours
100% Ethanol 1.0 hours
Clearing
100% Ethanol: Xylene 1.5 hours
Xylene 16 hours
Staining (Hematoxylin & Eosin)
Xylene 5 min
Xylene 2 min
100% Ethanol 2 min
95% Ethanol 2 min
70% Ethanol 2 min
Water 2 min
Hematoxylin 2-5 min
Running tap water 3 min
Acid Alcohol (1% HCl in 70% Ethanol) 2-3 dips
Running tap water 5 min
Eosin (1% solution) 1-3 min
95% Ethanol 2 min
100% Ethanol 2 min
100% Ethanol 2 min
Xylene 2 min
Xylene 5 min
APPENDIX C
Summary of F-values for the ANOV's comparing the total % recovery
of l'^C02 from G-l-^^C and from G-6-^^C and comparing the C-l/C-6
ratios between the treated and the control.*
Testis Liver
Treatment
Period G-l-^'^C G-6-^^C C-l/C-6 G-l-l'^C G-6-^^C C-l/C-6
7 0.148 0.767 0.011 0.002 0.876 0.359
14 0.400 0.402 1.706 0.114 12.181** 0.286
21 0.001 1.016 0.003 0.345 1.167 4.789
28 0.251 0.121 2.324 1.160 0.789 0.444
42 32.097*** 3.530 8.010** 0.001 0.367 0.104
*^'0.05 (1)1,8= 5.32
**Significantly different at 0.025 level
***Significantly different at 0.0005 level
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