' ”s ”52"" .w

 

. 222;!"

, : -.. 1
. .. ‘ :an;
"332%2». “22.

c?

5—4 yr.

4:?" :.
:, $22

ISIS"

. ”.4395.
r}: 2"",
7"}

a
. Q, n. . .—.
‘,- ' . ”’2, . - will u - ’- )iZlo any
.Hl'l-"""""" 2' ' ""' ‘ “'1“'”"”"”‘"'
,' . . v. . -2 1"“?
-y Ih‘YP "m r ‘3~:-:J- vv— 2:":

I falliirqz u

. ,fl. INL“ -.

'... art

I
.. VHF-:2
'3": '4; n"

5'", -
r " O‘NGC

 

“mar"

row
'1‘»:
“v.1”. -J- I‘Lfi‘m-In

3.2. 4;”.

Angrwvy

I—
STATE UNIVERSITY LIBRARIE

2111212221112mumI mu n w in

3 1293 00882 5840

 

 

 

 

 

 

 

 

 

 

 

 

This is to certify that the

dissertation entitled

Physiological and toxicological effects of
caffeine, hoechst 33258 and toluene on mouse
fertilization in vitro

presented by

Frank D. Yelian

has been accepted towards fulfillment
of the requirements for

Ph.D. degreein Zoology

 

 

wé/zafi/QM/éw

 

Major professor

W.Richard Dukelow, Ph.D.

[mm September 23, 1991

 

MSU is an Affirmative Action/Equal Opportunity Institution 0-12771

 

-—'"""—’ '"*“

 

LIBRARY
Michigan State-

3 University

 

PLACE IN RETURN BOX to remove this checkout from your record.
TO AVOID FINES return on or before date due.

 

DATE DUE DATE DUE DATE DUE

II 1
——i| ‘ Jl

MSU Is An Affirmative Action/Equal Opportunity Institution
emmflt

 

 

 

fl
ml 9 3 MIT
L EL ff‘: ‘ 4;. ' -
b T ‘UTJ 3:7 i
r i“- ..I l

0

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

i

 

 

 

 

 

 

 

 

PHYSIOLOGICAL AND TOXICOLOGICAL EFFECTS OF CAFFEINE, HOECHST

33258 AND TOLUENE ON HOUSE FERTILIZATION IN VITRO

BY
Frank D. Yelien

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Zoology and Endocrine Research Center

1991

ABSTRACT

PHYSIOLOGICAL AND TOXICOLOGICAL EFFECTS OF CAFFEINE, HOECHST
33258 AND TOLUENE ON MOUSE FERTILIZATION IN VITRO

BY
Frank D. Yelian

Studies were conducted to test the some physiological and
toxicological effects of caffeine, DNA-specific fluorochrome
Hoechst 33258 and toluene on 86D2F1 mouse fertilization in
vitro. In order to understand the chemical effects on the
different stages of fertilization, the direct influences of
the chemicals on epididymal sperm motility, sperm capacitation
and acrosomal reaction, single gamete cell intracellular Ca“
concentration, fertilization rate, and embryo development were
observed.

The results demonstrated that caffeine (0.1 to 10 mM)
significantly increased sperm motility. The stimulatory effect
was Ca”-dependent. Caffeine at the 0.1 and 1.0 mM levels had
no effect on fertilization, but 10 mM of caffeine
significantly decreased fertilization rate. The
chlortetracycline (CTC) fluorescence assay was 'used to
determine sperm acrosomal status, the result demonstrated that
caffeine (10 mM) did not affect sperm capacitation. or
acrosomal reaction.

DNA-specific fluorochrome Hoechst 33258, as a non-toxic

stain, has been used to detect DNA distribution and synthesis
in living cells, it is also used to detect early
fertilization. The toxicological study demonstrated that 20
and 100 ug/ml decreased the fertilization rate. The results
also demonstrated that.Hoechst 33258 at 10 ug/ml had no effect
on fertilization, but gametes incubated with 10 ug/ml or
higher levels of the stain inhibited embryonic development.

Previous mouse in vivo studies indicated that toluene
administrated by gavage increased the embryonic mortality. The
present in vitro study also showed that a concentration of
toluene higher than 8.67 ug/ml not only decreased sperm
motility and inhibited fertilization, but also increased the
percent of embryo degeneration.

To understand the mechanism of chemical effects on sperm
motility, the ACAS 570 interactive laser cytometer'was used to
measure the single gamete cell and embryo calcium.distribution
and dynamic change. The results«demonstrated.that.caffeine (10
mM) elevated sperm midpiece intracellular Ca”, but had no
effect on the sperm head. In the sperm treated with 8.67 ug/ml

of toluene, no significant calcium change was observed.

ACKNOWLEDGEMENTS

I would like to thank the following individuals for their
professional and personal support over'the‘past.severa1 years.
First, the special gratitude to my advisor, Dr. W. Richard
Dukelow, for offering me an opportunity to come to this great
country, and also for his patience, guidance, friendship and
advice. I thank my graduate advisory committee, Drs. James W.
Atkinson, Lonnie C. Eiland, Evelyn Rivera and Harold J. Sauer
for their encouragement, guidance and friendship. I thank Dr.
John B. Kannene reviewing my dissertation and sitting for Dr.
Rivera at final defence. I also thank Mrs. LaVonda Cleeves and
E.R.C. colleagues, for their friendship and assistance.
Finally, I would like to thank my parents, Daozhi and Xiuqing,
my wife Helen and daughter Samantha, for their love and
support. Without out all of these, the task would not be

possible to complete.

iv

TABLE OF CONTENTS

LIST OFTABLESOOOOOOOOOOOOOOOOOOOOOOOOO... ....... O ...... Vii-i
LIST OF FIGURES............................. ...... ........ix
INTRODUCTIONOOOOOOO ..... OOOOOOOOOOOOOOO ....... O 000000000000 1
LITEMTURE REVIEWOOOOOOOOOOOIOOOO0.0.0.000...O 0000000000000 4
Caffeine and Sperm motility................. .......... 4
Sperm capacitation and acrosomal reaction...... ....... 7
DNA-specific fluorochrome Hoechst 33258......... ..... 13
Reproductive toxicity of toluene.....................14
MATERIALS AND METHODS...............f............ ......... 18
Experimental animals..................... ............ 18
Culture media............ ................. .... ....... 18
Sperm motility................................ ....... 20
In vitro fertilization.............. ................. 21
CTC assay.............................. ....... . ...... 24
Intracellular ca“ measurement............... ......... 28
DNA stain Hoechst 33258...................... ........ 31
Statistical analysis....................... .......... 31
RESULTSOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO ..... 00.000.0033
I. Chemical effects on sperm motility........... ..... 33
A. The effect of caffeine on mouse epididymal
sperm motility in BMOC-B medium......... ....... 33
B. The influence of caffeine on sperm motility
in a calcium-free BMOC-3 medium... ............. 33
C. The effect of a DNA-specific fluorochrome
Hoechst 33258 on sperm motility.... ............ 36
D. The effect of toluene on sperm motility
in BMOC-3 medium.................. ............. 36

II. Chemical effects on sperm capacitation and
acrosomal reaction with CTC assay ................ 40

B.

III.

IV.

8.

C.

D.

E.

Caffeine effects on sperm capacitation

in BMOC-3 medium........................ ....... 40
The influence of caffeine on mouse sperm
acrosomal reaction.............................40

Chemical effects on in
vitro fertilization.............. ...... . ....... 44

Culture media effects on in vitro

fertilization................... ............... 44
Caffeine effects on in vitro

fertilization.. .................... .. ......... 44
The influence of Hoechst 33258 on in vitro
fertilization........................ .......... 47

Progesterone effects on in vitro
fertilization..................................47
Toxicological effects of toluene on in vitro
fertilization................. ................. 49

Chemical effects on embryo development in
VitrOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 000000000000 50

DNA-specific fluorochrome Hoechst 33258 and
embryo development............. ................ 50
Toluene effects on oocyte and embryo

degeneration after 24 hours

incubation invitro.......... ................... 50

Single gamete cell and embryo intracellular
calcium measurement with ACAS interactive

laser cytometer......................... ....... 53
Single sperm intracellular ca“ measurement ..... 53
1. ca” distribution in the sperm cell .......... 53
2. Chemical effects on single sperm

intracellular Ca” concentration.... ......... 56

Intracellular ca“ concentration in the

cocyteSOOOOOOOOOOOOOOOOOOOOO0......0.. 000000000 61
1. ca” distribution in mouse oocytes ........... 61
2. Intracellular ca” change during sperm
penetration................. ................ 65
ca” distribution in the 2-cell embryo ......... 65

vi

DISCUSSION........................... ..................... 69
Chemicals and sperm motility............... .......... 69
Sperm capacitation and acrosomal reaction...... ...... 73
In vitro fertilization......................... ...... 74
Embryonic development in vitro............. ......... .77
Gamete cell intracellular ca” .......................78

SUMMARY AND CONCLUSIONS...................................81

REFERENCESOOOOOOOOOOOOOO00.0.0.0...0.00.00... ..... .0. ..... 83

APPENDIX.................................... .............. 96

PUBLICATIONS BY THE AUTHOR................... ............. 96

VITA. ..... ............ ...... .... .......... . ............... 98

vii

LIST OF TABLES

Table Page

1. Comparison of the composition of four
different culture media............... ..... ......19

2. The parameters used in scan image for
measuring intracellular ca“............... ....... 29

3. The effect of caffeine on mouse epididymal
sperm motility in BMOC-3 medium............. ..... 34

4. The influence of caffeine on mouse
sperm motility in Ca”-free BMOC-3 medium ......... 35

5. Effect of toluene on sperm motility in
BMOC-3 medium during 4 hour incubation.. ......... 39

6. DNA-specific fluorochrome Hoechst 33258
affects mouse fertilization in vitro.............48

7. Effect of progesterone on mouse in vitro
fertilization.............. ..... ..... ............ 48

8. Toxic effect of toluene on mouse
fertilization rate in vitro........ .............. 49

9. Effect of DNA stain Hoechst 33258 on
mouse embryo development in vitro ..... .......... 51

10.1nf1uence of toluene on mouse oocyte and

embryo degeneration after 24 hour
incubation........................ ............... 52

viii

LIST OF FIGURES

Figure Page

1. A 2-cell stage mouse embryo and a
unfertilized oocyte (400K)......................22

2. (A) With DNA stain Hoechst 33258, one oocyte
shows 2 polar bodies and another shows 2
nuclei(200X). (B) 4-cell mouse embryos.... ...... 23

3. Nikon optiphot microscope equipped with
a Leitz micromanipulator.............. .......... 26

4. Mouse sperm CTC patterns (A) pattern
F sperm shows the uniform bright
fluorescence over head and midpiece
(8) pattern C sperm and (C) Pattern
A sperm.............................. ........... 27

5. Interactive laser cytometer: (A) ACAS 470
and (B) ACAS 570 (Meridian Instruments,
Inc.' OkemOS’ MI)OIOOOOOOOOOOOOOOOOOOOOO ........ 3O

6. Effect of DNA stain Hoechst 33258 on mouse
sperm motility in BMOC-3 medium ................. 37

7. Mouse sperm stained with 10 ug/ml
Hoechst 33258 for 30 minutes......... ........... 38

8. The time courses of CTC patterns in the
caffeine-free BMOC-3 medium.......... ........... 41

9. The influence of caffeine on mouse sperm
capacitation during 120 minutes........... ...... 42

10. The effect of caffeine on mouse sperm
acrosomal reaction in vitro ............. ....... 43

11. The Mouse in vitro fertilization in three
culture media: BMOC-3, TC-199, and M-16. ........ 45

12. Effect of caffeine on mouse fertilization
rate in vitro with BMOC-3 medium.......... ...... 46

13. Degenerated mouse embryo after incubated
with 100 ug/ml Hoechst 33258 for 24 hours ....... 51

ix

Figure Page

14. Degenerated mouse oocytes and embryo
incubated with 8.67 ug/ml toluene for
24 hourSOOOO0.000000000000000000000.... 000000000 52

15. The typical integrated cross mouse sperm
head line scanOOOOOOOOOOOOOOOOOOO.I0...... ...... 54

16. Two dimensional spatial scan of the sperm
head and midpiece...............................54

17. Flue-3 density distribution-fluorescence
patterns: (A) Pattern I sperm, (8) Pattern
II sperm, and (C) Pattern III sperm...... ....... 55

18. Photobleaching study of mouse sperm head and
midpiece Fluo-3 recovery within 14 minutes ...... 57

19. Caffeine effects on sperm intracellular Ca”
concentration and distribution.......... ........ 59

20. Toluene effects on mouse sperm
intracellular ca” measured by line scan.........60

21. Flue-3 density distribution in mouse oocyte
with cumulus cell. ..... ............. ............ 62

22. Cumulus-free mouse oocyte intracellular Ca”
distribution with image scan program ..... . ...... 63

23. The average Fluo-3 fluorescence intensity
of cumulus-free mouse oocyte............. ....... 64

24. Three dimensional image of mouse oocyte
intracellular ca” distribution......... ......... 66

25. Mouse oocyte intracellular ca“ change during
sperm penetration........................ ....... 67

26. Mouse embryo intracellular calcium
concentration and distribution...... ............ 68

INTRODUCTION

We live in.a chemical world. Our daily life involves many
different chemical substances. Caffeine and toluene are the
examples, and we must consider their effects on our health.

Early studies indicated that caffeine is a
phosphodiesterase inhibitor, which increases intracellular
CAMP levels and stimulates sperm motility. Caffeine has been
used to treat low motility sperm from some infertile males or
after cryopreservation. One objective of the present in vitro
study is to use mouse epididymal sperm and oocytes to observe
the direct effects of caffeine at different concentrations on
sperm motility, intracellular calcium concentration,
capacitation, acrosomal reaction, and fertilizing ability. I
also wish to understand the mechanisms involved in sperm
motility regulation and to determine the toxic level of
caffeine on mouse sperm function in vitro.

DNA-specific fluorochrome Hoechst 33258, as a non-toxic
stain, has been used in measuring cell density and
proliferation: asseSSing DNA distribution and synthesis:
observing sperm-egg fusion and preimplantation embryonic
development. No data are available on the toxic level of this
stain on gamete cells. Another objective of these studies was
to observe the toxic effects of different concentrations of
the stain, from 1 to 100-ug/ml, on mouse sperm motility,

fertilizing ability and.embryo development in vitro. From such

2
studies, we could establish the cellular toxicity of Hoechst
33258, which would benefit future studies on living cells.

Toluene is an aromatic hydrocarbon, commonly used in the
industry,in spray paints and glues. According to the United
States EPA reports, toluene has been detected in municipal
water supplies at levels ranging from 0.1 to 11 ug/l. There
are a number of case reports on pregnant women's exposure to
toluene causing infant malformations and congenital defects.
A previous in vivo study on the mice also demonstrated that
toluene could induce embryonic lethality i.e have teratogenic
effects on the fetus. The present study is designed to test
the direct toxic effect of toluene on sperm motility, in vitro
fertilization and embryonic development.

To understand the mechanism of caffeine and other
chemicals effects on sperm motility and fertilization, the
studies are also designed to test the calcium involvement.
This will include comparing the caffeine effect on sperm
motility in normal BMOC-3 medium and ca”-free BMOC-3 medium,
measuring intracellular Ca” concentration, distribution in a
single sperm, oocyte and. embryo, detecting ‘the chemical
effects on Ca” dynamic change in the sperm cells and in
oocytes during sperm penetration.

The ACAS interactive laser cytometer is a very sensitive
instrument which allows measurement of single living cell

intracellular Ca“) pH, DNA synthesis, membrane transport, and

3
detect cell-cell communication. The ACAS 570 has been applied
for many different cell studies, including lymphocytic,

somatic, cancer, and granulosa cells.

4
PHYSIOLOGICAL AND TOXICOLOGICAL EFFECTS OF CAFFEINE, HOECHST

33258 AND TOLUENE ON HOUSE FERTILIZATION IN VITRO
LITERATURE REVIEW
CAFFEINE AND SPERM HOTILITY

Garber et al. (1971a; 1971b) reported that caffeine
stimulated bovine epididymal and ejaculated spermatozoa
motility. They found that caffeine-treated spermatozoa
maintained the initial percentage of motility for at least 4
hr at 37°C whereas in the untreated sperm samples, 50% of
sperm that were motile initially became immotile during 4 hr
storage.

Since then, many researchers have studied the effects of
caffeine and other methylxanthines on sperm motility and
velocity in different species. Most of results demonstrated
that caffeine does increase and even prolong sperm motility
(Burge, 1973; Schoenfeld et al., 1975; Fraser, 1979; Moussa,
1983; Rees et al., 1990). However some studies have failed to
confirm any stimulatory effect for caffeine (Daugherty et al. ,
1976; Markler et al., 1980). The effects on sperm velocity
were controversial. One study (Markler _et al., 1980) used
combined multiple-exposure photography and supravital staining

to demonstrate that caffeine had no influence on sperm

S

velocity. This was later confirmed by others (Levin et al.,
1981; Aitken et al., 1983; Moussa, 1983). However Ruzich et
al. (1987) used Cell-Soft computer analysis methodology and
concluded that caffeine produced not only an increase in the
percent motile sperm but also an increase in sperm velocity.

The loss of motility and the concomitant loss of
fertilizing potential exhibited by human spermatozoa after
cryopreservation is a major problem for artificial
insemination and in vitro fertilization programs (Serres et
al., 1980; Aitkien et al., 1983: Cai and Marik, 1989). The
possible use of caffeine to stimulate the motility of
cryopreserved human spermatozoa has been suggested by the
positive results obtained by Barkay et al. (1977) and Schill
et al. (1979). However, results in terms of pregnancy rates
are disappointing; the 'most likely’ explanation for this
appears to be the occurrence of an alteration in the
ultrastructure of the sperm head, as observed with scanning
electron microscopy of sperm incubated with caffeine (Harrison
et al., 1980). A critical assessment of the response to
caffeine of human sperm motility was done by Traub et al.
(1982). They concluded that the widely documented effect of
caffeine on human sperm motility is of a transitory nature and
in keeping with ultrastructural damage.

The concentration of caffeine used is very important.

Levin et al. (1981) used caffeine at levels from 0.012 to 2.0

6

mM and observed a dose-dependent increase in sperm motility.
The EDSO for motility stimulation was 300 uM of caffeine. A
significant increase in motility was observed with 60 uM, and
maximal stimulation occurred with 1 mM. Moussa (1983) used six
different levels of caffeine to assess the effect on human
sperm motility. In levels of 3 and 6 mM, caffeine
significantly increased the motility, but had no effect on
sperm velocity. At a level of 120 mM, caffeine causes complete
immobilization of human spermatozoa (Moussa, 1983).

The mechanism of action of caffeine on spermatozoa
motility is still not clear. Caffeine inhibits cyclic
nucleotide phosphodiesterase (Hardman et al., 1971), enzymes
which are involved in spermatozoa glycolysis and consequently
increases the intracellular cyclic 3':5'-adenosine
monophosphate (cAMP) and cyclic 3':5'-guanosine monophosphate
(cGMP) concentrations (Hicks et al., 1972). This is the
probable site of caffeine action. Levin et al. (1981)
quantitatively analyzed the effects of caffeine on sperm
motility and cAMP phosphodiesterase. They found that the
phosphodiesterase activity of sperm homogenates displayed
linear kinetics with a Vmax of 0.5 nmoles/mg of protein per
minute and a Km of 125 uM. Caffeine (0.1 to 40 mM) produced a
doseedependent inhibition of sperm phosphodiesterase,
displaying a Ki of 1.2 mM. The results suggested that caffeine

may stimulate sperm motility by a mechanism other than

phosphodiesterase inhibition.

The present study was designed to determine (1) the dose-
response relationship between_ caffeine and mouse sperm
motility: (2) the role of calcium in caffeine-regulated
motility; (3) effect of caffeine on mouse fertilization in

vitro.

SPERM CAPACITATION AND ACROSOMAL REACTION

Mammalian sperm, after completing transit through the
epididymis, are still not able to fertilize oocytes. The final
maturational process, called capacitation , was recognized 40
years ago by Austin (1951) and Chang (1951) . Since then,
capacitation has been studied extensively. However, the exact
mechanism is still not well understood.

Sperm capacitation involves alterations in spermatozoan
membrane composition and other characteristics (Ahuja, 1984:
Langlais and Roberts, 1985: Wolf et al., 1986). The
biochemical alterations of the sperm plasma membrane include
removal or alteration of peripheral glycoproteins,
rearrangement of integral glycoproteins, reduction in membrane
cholesterol, and changes in the distribution and composition
of certain membrane phospholipids (Yanagimachi, 1989).

The sperm membrane alteration was considered a process of

removal of a decapacitation factor, which as a component of

8

epididymal fluid or seminal plasma stabilizes the sperm
membrane preventing premature acrosomal loss. This component
has been named, acrosome stabilizing factor (ASF) in rabbits
(Thomas et al., 1986; Wilson and Oliphant, 1987) and Caltrin
in bulls (Aumiller et al., 1988). In the mouse, there are two
substances that conform to the traditional concept of a
decapacitation factor, a-lactalbumin (Shur and Hall, 1982),
and a low molecular,weight proteinase inhibitor derived from
seminal vesicle secretions (Aarons et al., 1984: Poirier et
al., 1986).

In addition to the membrane alteration, various species-
dependent modifications of spermatozoan accompany
capacitation, including changes in metabolism (Hamner and
Willians, 1963; Meizel an'Turner, 1984; Neill and.01ds-C1arke,
1988), Intracellular ionic composition (Working and Meizel,
1983; Mrsny, 1984: Babcock and Apfeiffer, 1987; Robbins and
Boatman, 1988), acrosomal structure (Cummins and Yanagimachi,
1986; Dukelow’ and. Williams, 1988) and adenylate cyclase
activity (Monks and Fraser, 1987).

Stein .and ‘Fraser (1984) demonstrated 'that adenylate
cyclase activity increases and phosphodiesterase activity
decreases during capacitation of mouse spermatozoa in vitro.
Such changes could provide increased availability of cAMP,
which was demonstrated in hamster spermatozoa by White and

Aitken (1989). That cyclic (cAMP) may play an important role

9
in sperm capacitation was proposed by some researchers (Tash
and.Means, 1983: Fraser and Ahuja, 1988: Fraser,1990). If this
is true, caffeine, as a phosphodiesterase inhibitor, could
increase the intracellular cAMP level and facilitate sperm
capacitation.

Sperm maintain a low intracellular calcium concentration
in the millimolar range, which is similar to other eukaryotic
cells (Miller and Freedman, 1984: Miller, 1987). The
maintenance of the concentration gradiance was believed to be
by calcium pumping ATPase and a Na’/Ca++ exchanger (Fraser,
1987). Any substances which interrupt the function of the
calcium pump or the Na’/Ca” exchanger may cause an increase of
the intracellular calcium concentration. A high level of
calcium could lead to a toxic effect on sperm structure and
function. This abnormal elevation of intracellular Ca” may
have a relationship to sperm capacitation and the acrosomal
reaction.

The significance of capacitation was believed to
encompass two separate aspects of sperm function: 1) the
transition to hyperactivation, which allows the sperm to
penetrate the cumulus oophorus and.bind to the zona pellucida:
and, 2) the development of the ability to undergo the
acrosomal reaction (Bedford, 1990).

The sperm acrosomal reaction is an exocytotic process,

with the formation of multiple fusion sites between the

10
plasma membrane and the outer acrosomal membrane in resulting
the formation of vesicles and the subsequent release of the
acrosomal contents. The major hydrolytic enzymes hyaluronidase
and acrosin are involved in effecting sperm passage through
the vestments surrounding the mammalian oocytes and other
enzymes may be involved as well (Meizel, 1984).

There are two major factors responsible for the
stimulation of the sperm.acrosomal reaction. The first, is the
egg-specific extracellular matrix component, ZP3 (Cherr et
al., 1986; Philpott et a1, 1987; Leyton and Saling, 1989;).
The mouse zona pellucida glycoprotein ZP3 regulates acrosomal
exocytosis by aggregating its corresponding receptors (ZP3-Rs)
located in the sperm plasma membrane. These ZP3 receptors
(Mr=95,000) also serve as substrate for a tyrosine kinase in
response to zona pellucida binding (Leyton and Saling, 1989:
Bunch et al., 1990). Florman et al. (1989) suggested that the
zona pellucida induced the sperm acrosomal reaction by
activating sperm membrane G protein, which triggered the
elevation of intracellular calcium and pH and acrosomal
exocytosis. Second, the acrosome reaction promoting factors,
which are secreted by cumulus cells into the cumulus
intercellular'matrix:(Meizel, 1985; Tesarik and.Kopecny, 1986;
Tesarik et al., 1988; and Siiteri et al., 1988) may be
involved. One of these activities was originally ascribed to

a protein or glycoprotein of apparent molecular weight of

11

approximately 50,000 (Suarez et al., 1986: Tesarik et al.,
1988c) and recently believed to largely reside in protein-
bound progesterone (Osman et al., 1989). Human follicular
fluid is also considered a stimulator of sperm acrosomal
reaction (Yudin et al.,1988: Mortimer and Camenzind, 1989:
Stock at al, 1989). Within follicular fluid, there are many
possible components which may be responsible for the reaction.
However, progesterone is considered as the major stimulating
element. In vitro studies have shown that 10 ng/ml to 1 ug/ml
of progesterone significantly increase sperm intracellular Ca”
concentration and has a similar effect on stimulation of the
sperm acrosome reaction (Thomas and Meizel, 1989: Blackmore et
al., 1990). It has also been suggested that perhaps there is
a coordinated or synergistic interaction between ZP3 and
progesterone to initiate the acrosomal reaction (Blackmore et
al., 1990). 9

These studies suggested a new mechanism which involved in
steroid hormone action. The mechanism of progesterone action
was probably mediated by a progesterone receptor resident in
the plasma membrane of the sperm. Binding of progesterone to
this receptor could activate a .Ca” channel in the plasma
membrane, or the progesterone receptor may have inherent Ca”
channel activity. The phosphoinositide turnover and guanine
nucleotide regulatory-binding proteins (6 protein) do not

appear to be involved in the progesterone induced Ca” influx

12
response. One alternative is that the progesterone receptor
activates phospholipase D, and that the increase in
phosphatidic acid promotes Ca” ionophoretic activity (Bocckino
et al, 1987). A phospholipase D activity has recently been
described in sea urchin sperm (Donino et al., 1989).

The methods for evaluating the sperm capacitation and
acrosomal reaction have been developed and improved for many
years. There are five techniques which are commonly used in
different laboratories: 1. Transmission electron microscopy
(TEM). It can, provide :micro-structural evidence for ‘the
acrosomal reaction (Yudin.et.al., 1988). 2. Phase-contrast and
DIC microscopy. The acrosomal reaction of sperm with large
acrosomes can be directly observed under phase-contrast and
DIC microscope (Meizel et al., 1984). 3. Triple stain. It is
the most commonly used technique, which has been applied for
many species (Talbot and Chacon, 1981: Dudenhausen.and.Talbot,
1982; Varner et al., 1987: and Kusunoki et al., 1989). 4.
Fluorescein isothiocyanate (FITC) . With lectins or antibodies,
FITC can demonstrate very good fluorescent intensity and
contrast which provides an indirect image of the acrosomal
reaction (Cross et al., 1986: 1989: Blach et al., 1989). 5.
Chlortetracycline (CTC) assay. The method can provide a very
fast result and has the unique potential of revealing the
dynamics of the capacitation and acrosomal reaction (Ward and

Storey, 1984: Lee et al., 1987; Endo et al., 1988). The CTC

13

assay was used in the present study.

DNA SPECIFIC FLUOROCHRONES

DNA specific fluorochrome (Hoechst 33258 and 33342) as
non-toxic stains, have been widely used to measure cell
density and proliferation (McCaffrey et al, 1988: Steuer et
al, 1990), assess DNA distribution and synthesis (Latt and
Stetten, 1976: Arndt-Jovin et al., 1977: Crister et al, 1983:
Hinkley et al, 1986), observe sperm-egg fusion and
preimplantation embryo development (Conover and Gwatkin, 1988:
O'Rand et al, 1986; Wright and Longo, 1988; Sawicki and
Mystkowska, 1990).

These bisbenzimidazole fluorochromes bind reversibly to
sequences of three or more adenine-thymine pairs of double-
stranded deoxyribonucleic acid (Comings et al, 1975: Richards
et al, 1985). Binding appears to occur externally, rather than
by intercalation.

The non-toxic properties of these dyes may depend on cell
type and dye concentration. A study done by Conover and
Gwatkin (1988) showed that mouse spermatozoa stained with 1 ug
/ml Hoechst 33342 were capable of fertilizing mature
oocytes,with resultant cleavage to the blastocyst stage, but
10 ug/ml of Hoechst 33342 significantly inhibited embryo

cleavage. Loeffler and Ratner (1989) reported that 6 ug/ml of

l4
Hoechst 33342 reduced lymphocyte motility and proliferative
response. No data is available for cytotoxicity of Hoechst
33258 on mammalian gamete cells.
The mechanism of biological effect of DNA stains which
interact with the DNA is thought to arise from the direct
disturbance of the processes of DNA replication and

transcription. (Smith et al, 1989).

REPRODUCTIVE TOXICITY OF TOLUENE

Toluene, also referred to as toluol, methylbenzene,
methacide, and phenylmethane, is a volatile, aromatic
hydrocarbon, commonly used in industry, and found in the home
in spray paints, glues, and lacquers. The molecular structure
is distinguished from that of benzene by the substitution of
a methyl group for one hydrogen atom. Toluene has the
molecular formula C,H8, a molecular weight of 92.13, a density
of 0.867 at 20%: and a vapor pressure of 30 mmHg at 26 °C,
(Kirk and Othmer,1963 ). Toluene is slightly soluble in water,
534.8 1 4.9 mg/l (Sutton and Calder, 1975 ).

Toluene has been detected in municipal water supplies at
levels ranging from 0.1 ug/l to 11 ug/l. The toluene
metabolites benzaldehyde and benzoic acid were also found in
municipal water at concentrations up to 19 ug/l (EPA,1980).

Animal reproduction experiments show impaired growth of

15

mother and fetus and fetal skeletal anomalies after exposure
to large doses of toluene (Hudak.and‘Ungvary, 1978 and Shigeta
et al., 1982). Behavioral effects of toluene in mice exposed
pre- and post-natally have been described (Kostas and Hotchin,
1981). Two children with multiple malformations were born to
mothers who worked as shoemakers and were chronically exposed
to toluene and trichlorethylene, used in a soling solution
(Euler, 1967). There are a growing number of case reports on
congenital defects in children. born to ‘mothers who had
intentionally inhaled toluene in high doses throughout
pregnancy. In one recent study, five women, who had previously
borne children and were in their third trimester, developed
severe renal tubular acidosis from paint sniffing and
subsequently gave birth to five infants, three of whom were
growth-retarded at birth, two showed craniofacial anomalies,
and neonatal hyperchloremic acidosis (Goodwin, 1988).

Toluene has been shown to be teratogenic in one study by
Nawrot and Staples ( 1979 ). Toluene was administered to CD-1
mice by gavage on days 6 through 15 of gestation at levels of
0.3, 0.5 and 1.0 ml/kg body wt/dose. A significant increase in
embryonic lethality occurred at all dose levels and decreased
fetal weight occurred at 0.5 or 1.0 ml/kg. In the 1.0 mg/kg
group, a significant increase in the incidence of cleft palate
was found which did not appear to be due merely to general

retardation in growth rate.

16

Genotoxic effects of five widely used aromatic industrial
solvents, ethylbenzene, methylbenzene (toluene), o-, m-, and
p-dimethylbenzene (xylene), on bone marrow cells of male NMRI
mice were studied using the micronucleus test (Mohtashamipur
et al.,1985). Each compound was given to animals by IP
administration of two similar doses 24 h apart. Increased
formation of micronuclei within polychromatic erythrocytes of
femoral bone marrow 30 h after the first injection was
concluded to be due to the clastogenic effect of the test
compound. Of the chemicals tested, only toluene gave a dose-
dependent increase in the frequency of micronucleated
polychromatic erythrocytes. This genotoxic activity of toluene
was confirmed in male B6C3F1 mice.

An inhalation study done by Courtney et al.(1986) showed
that toluene administered by inhalation at 400 ppm to CD-1
mice from days 6 to 16 of gestation was teratogenic but not
fetotoxic. There was a significant shift in the fetal rib
profile. At the lower concentration of 200 ppm, there was an
increase in dilated renal pelves which might reflect
desynchronization of maturation with respect to development
and growth.

The cytogenetic effects of toluene are controversial.
Forni et al. (1971) reported no evidence of chromosome
aberrations in lymphocytes of toluene-exposed workers, and

Gerner-Smidt and Friedrich (1978) reported no manifest sister-

l7
chromatid exchanges following in vitro exposure of human
lymphocytes to benzene, toluene or xylene. But reports from
Bauchinger et al.(1983) and Schmid et al.(1985) indicated
significantly increased sister-chromatid exchanges in man
after exposure to toluene.

The limited studies on the teratogenic potential of
toluene indicate that toluene could have some teratogenic and
toxic effects on the mammalian fetus at high dosages with
prolonged periods of dosing. No in vitro toxicological studies
on the mammalian gamete cell after exposure to toluene were
available. The present study is designed to test the effects
of toluene on B6DZF1 mouse epididymal sperm motility and

fertilizing ability in vitro.

18

MATERIALS AND METHODS
1. B er'me ta 1

The animals used in this study were B6DZF1 mice.
Originally all mice were purchased from the Jackson Laboratory
(Bar Harbor, ME). Since our own breeding program set up three
years ago, this strain of mouse *was produced at the Endocrine
Research Center by crossing C57BL-6J and DBA-2J mice. B6DZF1
mice were sexed at twenty one days of age, and male and female
mice were separated in different cages. Each cage contained
four to five mice. They were provided Breeder BloxR (Wayne Pet
Food Division, Chicago, IL) and fresh.water'daily, ad libitum.
Animal room light cycle was maintained on 12 h :12 h bases and

temperature was maintained at 74-76 W2.
2. ture Mod a

There were three market culture media used in this study.
Brinster's BMOC-3 (Formula. # 78-0012AG), TC-199 (# 320-
1150AJ), and F-10 Nutrient Mixture (# 320-1550AG) were
purchased from GIBCO BRL (Grand Island, NY) . Medium M-16 was
made according Whittingham (1971). (The composition of these
medi‘a is given in Table 1. The calcium-free BMOC-3 medium was

made by modifying Brinster's BMOC-3 (pH=7.5¢0.1: Osm=290110).

19
All the culture media were held at 1-49C for maximum of three
weeks after opening the stock solution bottle. The medium was
filtered with 0.22 um sterile filter unit (Milipore Products

Division, Bedfard, MA).

Table 1. Comparison of the composition of four culture media

(g/L)

 

 

Component* BMOC-3 TC-199 F-10 M-16

NaCl 5.546 6.800 7.400 5.533
Na Lactate 2.253 ---------- 2.610
Na Pyruvate 0.056 ----- 0.110 0.036
NaH2PO4 H20 ----- 0.140 ----------
Na2HPO4 7H20 ---------- 0.290 -----
KCl 0.356 0.400 0.285 0.356
KH2P04 0.162 ----- 0.083 0.162
CaC12 0.189 0.200 ----------
CaC12 2H20 ---------- 0.044 0.252
MgSO3 7H20 0.294 0.200 0.153 0.293
NaHCO3 2.106 2.200 1.200 2.101
B.S.A 5.000 4.000 4.000 4.000
Glucose 1.000 1.000 1.100 1.000

 

* The components of BMOC-3 was according to GIBCO Technical
Services Department. The media TC-199 and F-10 were based on
the GIBCO BRL Catalogue & Reference Guide, 1990. The medium M-
16 was according to Whittingham (1971).

20

3. 820:3 Motility

Mature male B6D2-F1 mice (3-6 month of age) were used.
BMOC-3 culture medium was kept in the incubator overnight.
Mice were sacrificed by cervical dislocation. One pair of
caudae epididymides from each mouse was placed in 1.0 ml
medium, ‘which. contained. different. concentrations of test
chemicals. The epididymides‘were punctured with 22G1.5 Sterile
needles allowing the sperm to swim into the medium. The sperm
suspensions were further diluted to the final sperm
concentration of 1-10 x 106/1111. Then 20 ul sperm suspension
was placed on prewarmed hemocytometer and covered with a
coverslip. The hemocytometer was under phase contrast Nikon
microscope (Nikon, Garden City, NY) at 200x magnification.
According to Schoff and Lardy (1987), and Berger and Beierle
(1990), there are inherent inaccuracies ix: characterizing
motility with regard to the degree of sperm vigor, I
restricted my description of motility to percent of motile
sperm. One hundred spermatozoa were counted every sample, the
motility (35) was calculated based on the ratio of
motile/immotile sperm. Each hemocytometer was counted twice

and the average was used.

21

4. v o F til'zation

Female mice, 4 weeks to 3 months of age, were induced to
superovulate by i.p. injection of 8 i.u. pregnant mare's serum
gonadotropin (PMSG) (Serotropin‘, Teizo, Tokyo, Japan),
followed by 8 i.u. human chorionic gonadotropin (hCG) (Sigma,
St. Louis, MO) 48 to 52 hr later. Twelve hr to 14 hr after hCG
injection, two male mice were sacrificed, one epididymis from
each mouse was collected and sperm collected as described
above. The sperm suspensions were held at 37 °C, in a 5 it C02
in air, humidified atmosphere. One hour after sperm
incubation, female mice were sacrificed by cervical
dislocation. Two oviducts with partial ovary and uterine
tissue were collected from each mouse, and placed in 1.0 ml
medium. The cumulus masses were recovered from the swollen
ampulla and placed into 950 ul of medium with fine forceps.
Each culture dish contained one to two cumulus masses and
received 50 ul sperm suspension. The final sperm concentration
was 1-10 x 106/1111. The mouse gametes were held at 37°C, in a
5 % CO2 in air, 100% humidity for 24 hr.

In the experiment designed to measure the intracellular
Ca” change during fertilization, cumulus-free oocytes were
used. The cumulus masses were dispersed by adding 30 ul of 10
mg/ml hyaluronidase (Sigma, St.Louis, M0) to 1.0 ml BMOC-3

medium. The final concentration of hyaluronidase was 300

22
ug/ml. After 5 minutes holding at 37°C, the oocytes were
washed twice with BMOC-3 medium. After 24 hr incubation,
assessment of fertilization was based on the presence of one
of the following three criteria: (1). two or more polar
bodies in the perivitelline space; (2). two or more equal size
blastomeres. (3). In DNA staining experiments with Hoechst

33258, two or more pronuclei observed (Figure 1.)

 

Figure 1. Uper right is a 2-cell stage mouse embryo and lower
left is an unfertilized oocyte (400X).

 

Figure 2. With Hoechst 33258, (A) One fertilized oocyte shows
2 polar bodies and another shows two nuclei (200X).
(B) Four—cell mouse embryos.

24

5. C C 55 o a c' t' as m Re c ' n

The Chlortetracycline (CTC) fluorescence assay described
by Ward and Storey (1984) was used with slight modification.
The experiment procedures were:

(1). Solutions: Buffer A for CTC was made of 0.3029 g
Trizma Base; 0.455 9 NaCl: 0.0788 g Cysteine and brought to
125 ml with double distilled water. CTC solution was made by
adding 3.2 mg CTC (Sigma, St. Louis, M0) to 12.5 ml of Buffer
A (500 uM). The pH was adjusted to 7.8. CTC solution was made
fresh and kept in.0-4 °C through the experiment. Buffer B for
Glutaraldehyde was made of 4.02 g Tris Hydrochloride: 2.97 g
Trizma Base and brought to 50 ml with double distilled water
(1M). 5% Glutaraldehyde solution was made by adding 3 ml of
double distilled water: 3 m1 Buffer B: and 1.5 ml 25%
glutaraldehyde (Sigma) and filtered through a 0.22 um Corning
sterile filter system (Corning Glass Works, #25932, Corning,
NY). Buffer A and Buffer B were stored at 0-4 °C for a maximum
period of three weeks.

(2). Procedure: Sacrificing mice and preparing sperm
suspension have been described above. At 0, 30, 60, 90, and
120 minute time (points during incubation, 10 ul of CTC
solution was placed on a prewarmed. (37”C) glass slide,
immediately followed by 10 ul sperm suspension and mixed with

a micropipette. The glass slide with the CTC-sperm suspension

25
was placed on a warming stage for 10 seconds and then fixed
with 10 ul of glutaraldehyde solution, stirred thoroughly, and
covered with a 24 x 50 mm cover glass. The slides were
examined under a Nikon Optiphot microscope equipped with a
fluorescence filter system: 380-425 nm excitation filter and
420 nm dichroic mirror (Figure 2.)

(3) . Fluorescence patterns: The sperm with uniform bright
fluorescence over head and midpiece were recorded as pattern
F (fresh). Those with bright anterior head and dark posterior
head were recorded pattern C (capacitated). The sperm with
fluorescence lacking in the entire head but still in the
midpiece were recorded as pattern A (acrosomal reacted)
(Figure 3). One hundred sperm were scored on each slide, and

the slides were examined within two hours of preparation.

26

 

Figure 3. Nikon optiphot microscope equipped with a Leitz
micromanipulator. The fluorescence system was not
shown in the figure.

27

 

Figure 4. (A). One pattern F sperm showed the uniform bright
fluorescence over head and midpiece. (B). The Sperm with
bright anterior head and dark posterior head is pattern C
sperm, and the sperm with fluorescence lacking in the entire
head but still in the midpiece were recorded as pattern A
sperm.

28

6. t ea e um asu e e t

Single mouse sperm and oocyte intracellular Ca” were
measured by ACAS-470 & 570 Interactive Laser Cytometer
(Meridian Instruments, Inc., Okemos, MI) (Figure 4.).

The mouse sperm suspensions were prepared as described
above. Immediately after sperm suspension preparation, 5 ul of
1 mM (1.0 mg dissolved in 0.867 ml dry DMSO) Fluo-3
acetoxymethyl (AM) ester (Molecular Probes, Inc., Eugene, OR)
was added per 1.0 ml sperm suspension. The final concentration
of Fluo-3 AM was 5 uM. After 30 minutes incubation (37’°C, 5%
CO2 in air, 100% humidity), the sperm were washed twice in
BMOC-3 by centrifugation at 2000rpm for 5 minutes at room
temperature.

The oocyte collection was as described above. The oocytes
with or without cumulus were incubated in 5 uM Flue-3 AM for
30 minutes. The oocytes were then washed three times by
transferring to fluorescein-free BMOC-3 medium with a
micropipette.

Fluo-3 AM loaded.spermatozoa.and.oocyte5‘were transferred
into a specially designed 35 mm culture dish (with a 0.15 mm
thick coverslip on the bottom), which allowed using 100x oil
objective. The sperm slide samples were also prepared for
measuring intracellular Ca”’concentration over a time period.
The dish or slide was observed under the i‘nverted fluorescence

microscope within ACAS-470 or ACAS-570. Fluo-3 AM can be

29

excited by the 488 nm line of the argon laser and emits in the
visible spectrum.similar to fluorescein (520 nm) (Wade, 1990).
The laser excitation source provides a small beam (about 1
micron in diameter) which allows extremely low levels of
fluorescence to be detected with minimum laser power. The X-Y
scanning stage allows for 0.25 micron data acquisition to
provide maximum spatial analysis. Two types of experimental
data, the single sperm and oocyte integrated cross sectional
line scans and two dimensional spatial scans, were collected
and analyzed by computer software system.

The parameters used for measuring intracellular Ca” level

are shown in Table 2.

Table 2. The Parameters Used in Scan Image for Measuring
Intracellular ca“ within Single Sperm, Oocyte and Embryo.

 

 

Parameter* Sperm Oocyte Embryo

PMT 1. 70-75 % 70-75 % 70-75 %

PMT 2. 10 % 10 % 10 %
Detector 1 1 1

Step Size 0.25 um 10 um 10 um

X Points 100-150 150-360 150-360

Y Points 100-150 150-360 150-360
Scan Str. 1-10 % 10-20 % 10-20 %
Laser Power 200 mW 200 mW 200 mW
Speed 20 mm/sec 5-10 mm/sec 5-10 mm/sec

 

* PMT 1 setting for the default detector in single
detector mode, PMT 2 setting for the off-axis detector in dual
detector mode. Step Size is the distance between data points.
Speed means the maximum stage speed.

 

30

 

Figure 5. (A) ACAS 470 Interactive Laser Cytometer, (B) ACAS
570 Interactive Laser Cytometer (Meridian
Instruments, Inc., Okemos, MI)

31

7. o so Gamete a d mb 0 Sta w oec st 33 8

Two, four and eight cell mouse embryos were collected at
24, 48, and 60-72 hr respectively'after in vitro insemination.

DNA-specific fluorochrome Hoechst 33258 (Aldrich Chem.
Co., Milwaukee, WI; #86,140-5: F.W.=623.97) was dissolved and
diluted in BMOC-3 medium. The final concentrations of 1, 10,
20, and 100 ug/ml were used to test- the effects on sperm
motility, fertilization rate and embryo cleavage in vitro.

To assess the results on fertilization and the stage of
the embryo, We routinely used 10 ug/ml Hoechst 33258
incubated for 30 minutes. The embryos were then washed three
times by transferring to dye-free medium. The dye-loaded
embryos were observed under Nikon-Diaphot inverted microscope
(Nikon, Garden City, NY) equipped with an epi-fluorescence
filter combination UV-lA. The filter combination consisted of
a 400 dichroic mirror, 365/10 excitation filter and a 400
barrier filter. The main wavelength for ultraviolet (UV)

excitation was 365 nm (Roudebush, 1988).

mm

Different statistical tests were employed in different
data forms. The contingency test (Chisquare test) was used in
comparing fertilization rate and embryo degeneration rate. The

Two-way ANOVA was used testing the difference of sperm CTC

32
pattern over time. Cochran's test was used to test.homogeneity
of variances. One-way analysis of variance (ANOVA) was used to
test the difference of sperm motility with different
concentrations of chemical treatment. After one-way ANOVA, the
Student Newman Keuls (SNK) test was used for multiple group
comparisons (Steel and‘Torrie, 1980). All P values less than
0.05 were considered statistically significant. The
statistical software, MINITAB and SYSTAT were used to test

these data.

33

RESULTS

I. m'ca e so 0 s erm moti i

A. The effect of caffeine on mouse epididymal sperm motility
in BMOC-3 medium

The mouse epididymal sperm motility in different
concentrations of caffeine are shown in Table 3. No
significant difference was found among four groups within 15
minutes, however, after'90 minutes and.4 hours incubation, the
sperm motility in all three caffeine treated groups was
significantly higher than the control group (P < 0.05). No

dose dependence was found among three treatment groups.

B. The influence of Caffeine on mouse sperm motility in
calcium-Free medium

This study (Table 4.) demonstrated that mouse sperm
motility significantly decreased in the calcium-free medium
after 90 minutes incubation (P < 0.05). Again there was no
difference among three groups at 15 minutes. The results
indicated that the calcium are required for sustained sperm
motility. In the Ca”-free medium, caffeine (10 mM) had no

effect on sperm motility.

34

m0.0 V G 4 .=.m.m + Cflfl: 0H” m05Hfl>

 

.m».v.v H o.mm Anv.m.a H m.vp an. ma H p.6b as ou
Avccm.m H ~.Ho .m..v.a a «.65 An. 8.. H m.mn :so.a
.wo.m.n H p.68 .nc.o.n H «.8» In. ~.~ H o.mp st.o umcaouuno

Au. ~.~ + ~.Hn Ame H.~ + c.nm .ov a.v H v.n> Houucoo

 

.u: c .:As om .zds ma
acoauomua
.uv sugaauoz sheen

aoaooa mucosa :w
>uwuauoa Human Hoeaofiofiao omooa co ocaomuou no Docuuo 058 .n manna

35

. 39.0 A a: ocqouuoo :8 cu moan 00.31.30 one 00.31300 :003umn “Esau
mo3 oocououufio ucouuuacOAm nonwumfiuoum oz .Amo.o V AC vacuuuucOAm
>H~o0«amauoum mma mocha Houucoo 02a ou omumnfioo 00:0u0uufio one «

 

.mcfiouuno ms oH+v

 

a m.N H v.hd a h.n H ¢.on N.w H H.Nh m Omuhttflu

« o.e H o.m~ c m.m H v.Hn N.n H o.mm m OOHhIIhU

n.v H v.0n d.h H o.Nm N.m H w.nh o HOHUCOU
.H: ¢ .:«B on .CME ma

 

: acoauooua
A». suaafiuoz enema

endows nioozm oeuultoo :«

auqaauoa swoon omooa so .28 any ocuouuoo no cocooaucw 0:8 .v manna

36
C. The effect of DNA-specific fluorochrome (Hoechst 33258) on

mouse epididymal sperm motility
The results of mouse sperm motility in BMOC-B medium and
treated with three concentrations of Hoechst 33258 are shown
in Figure 6. There was no significant effect on sperm motility
when the stain concentrations were 20 ug/ml or less. However,
when the DNA stain concentration was 100 ug/ml, and after 60
to 90 minutes incubation, the sperm motility was significantly
decreased (P < 0.05). Mouse sperm stained with 10 ug/ml

Hoechst 33258 are shown in Figure 7.

D. The effect of toluene on mouse sperm motility in BMOC-3
medium

To test the effect of toluene on mouse sperm motility,
four'different.concentrations.of toluene, 0.0867, 0.867, 8.67,
and 86.7 ug/ml were used. Because t-butanol was used as a
vehicle conveying toluene into the medium, 1% of t-butanol was
used as control group. The results are shown in Table 5. No
difference was found between t-Butanol control and BMOC-3
control. The toluene concentration at 0.0867 ug/ml had no
effect on sperm motility at all three time points (P > 0.05).
However, when the concentration of toluene was higher than
0.867 ug/ml, the sperm motility was significantly decreased

(P < 0.05).

.ssfioms nuoozm ca suoafiuos
Enoch mmsos co mmmnn umzoooz sebum czc mo poouum 025 .m musowm

 

37

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

$«8.:.ea=8. _U 8~8.:.sa=8 D
amawzziso._MH_ 3230 ll—
52 e 58 8-8 5:. 8-9
no
/ fl J/ / fl 4 ..
.: ‘2 . re. s
.
//1 /// .rcm _
.................... . —
l r/w /// .
/// .-cn .
................... _ o
E
.................. _ ............ _ ....... /
E
m .
................................... ...I On 0
u a
-om a

 

 

 

 

3568 88% co Bozo
mmmmm 6:80: :35 <20

 

 

38

 

Figure 7. Mouse sperm stained with 10 ug/ml Hoechst 33258 for

30 minutes and under fluorescent microscope.

39

.mo.o v..m a

'

.xzm can 4>oz¢ >o3|oco >2 noumou auaoowumwumum

.55 H coo: who mosao>

«moo—Son. w a.

«Honousnlu uo

«a saw: aswoma nnoozm mu m Houucou can asaooa nuuozm m« d Houusoo

 

 

 

«m.e H o.~ a~.o H e.v .s.m H m.m as. Ha\o: 5.0m
«o.o H n.nH «o.m H m.p~ «m.o H m.me .9. Haxua so.»
an.« H s.oa «q.n H c.5n «s.o H n.5m .9. Hs\o= smm.o
n.m H o.m~ m.m H «.mc a.» H «.Hs As. ~a\o=somo.o
m.s H H.Hn H.e H o.cm m.n H H.vs m Houucoo
o.~ H c.o~ ~.v H o.me o.¢ H ~.vs H Houucoo
.oucc .u: v Amucc.:qa canoe Aoucc.:fia onnma
msouu

auonm onfiuox «

noduonsosw woo: v Hanan vcwuso
55003 700.3 :a #33003 :5on ounce so «condo» no voouum .m manna.

40

II. Chemical effects on mouse sperm capacitation and acrosomal
Men

A. Caffeine effects on mouse sperm capacitation measured by
CTC assay

The time courses of CTC patterns in the caffeine-free
control group are shown in Figure 8. The results of effect of
caffeine on mouse epididymal sperm capacitation are shown in
Figure 9. During 2 hours period of incubation, in the control
group, the percent of capacitated sperm (pattern C) increased
from 14.4 % to 48.3 % (30 min.), 64.8 % (60 min.), then
decreased to 41 % (90 min), and 23 % (120 min.); the caffeine
treated group shows a similar change over time, However the
difference between control group and caffeine treated group

were not significant.

B. Caffeine effects on sperm acrosomal reaction.during‘21hours
incubation with BMOC-B medium

The effect of caffeine on sperm acrosomal reaction is
shown in Figure 10. In the control group, the percent of
acrosomal reacted sperm (pattern A) were very low'at.0 min. (8
%). It had no significant change during the first 60 minutes.
The pattern A sperm were 8.25 and 12.8 % at 30 and 60 minutes
respectively. However, the pattern A were very significantly

increased at 90 minutes (41 %). They finally reached 71.6 % at

41

.ssaoms mucosa mouuuosfiouumo
on» c“ mcumuuma 090 swoon «woos mo momusoo mean on? .m wusofim

< 52.3 LT o 52.8 IT n. 52.3 IT

 

 

 

 

SE our SE on SE on SE on SE o
- L b O
i X
. ox.
. : low a
c
e
m
T a? w
a
a
. . . . . . . - 8 w
0
_ 8

 

 

28:3 0.5 so 8528 9S...

42

.cofiumnsocfi mouscws om“ mcfiuso Au :umuumo 080v
coflumuwomdmo suoam omsos so ocwouumo no 00:02HuCH one .m musvfim

3.23538 .252 B 3-3.2.80 B

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

.SE our .SE om .SE on S... on .56 c

- N O
x \ x
. . x ..... x ..... \ ........................ a o
\\\ \\\ c
\ \ \ _
..................................... \ 21.0? G
\\\ .......... .
\ .
m
................................................. \ .I 60 n—
w
[on 0

 

 

 

0 Egan. 9.0
.5QO :o 3095 oSmtmo

43

Hmsomouom Spoon mmsos so mewmuuoo mo Homumo one .oH ousvwm

.SE our

.ouufl> ca A4 cumuuon 090. coduoomu

Avaswssao .25 2 B $5.380 8

.SE cm .55 co .SE on .SE a

 

\\\\\\\\\\ \

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

\
\ a Q
\
x
\

 

 

 

 

 

< 59.9. 9.0
8..on :0 .85 656sz

.:;:s::;.-o~

.3 0v

. 8

 

(co

$

eke da~ugec <

44
120 minutes. Comparing the control group, the caffeine (10 mM)
treated group showed a similar change over time. No
significant difference was found between the two groups (P >

0.05) C

III. W

A. Culture media effects on in vitro fertilization

In order to determine the optimal medium for mouse in
vitro fertilization, three common media were studied. The
results are shown in Figure 11. There was no significant
difference ‘between. BMOC-3 (69.7%) and. M-16 (68.8%). The
fertilization rate was significant lower in TC-199 (42.3%)

(P < 0.05).

B. Caffeine effects on fertilization rate in vitro.

The effects of three concentrations of caffeine ( 0.1,
1.0, and 10 mM ) on the fertilization rate are shown in Figure
12. Although a slight increase in fertility was observed with
0.1 mM caffeine, this difference was not significantly
improved over controls. Caffeine (1.0 and 10 mM) decreased
fertilization rate to 61.2% and 35.2% respectively, however
the contingency test showed that only the 10 mM group was

significantly different from control group (P < 0.05).

45

In vitro fertilization with
three culture media

 

F

a

r

i

{so

i70‘

Z

a em

iso'

o .

"40
30'

r

320,

t

910

%o

[X] amoc-a (97/139) Ct] me (4507) [:1 70.199 (ZS/62)

Figure 11. Mouse in vitro fertilization with three different

culture media: BMOC-B, TC-199, and M-16.

46

Caffeine Effect on
In Vitro Fertilization

30-HmN-'—-~W0'n

tuna—-

 

$

[35 Camurmm [ID a1mMcuhmquu)
CD 1.0MMW170) S 10mM Canolnonzs)

Figure 12. Effect of caffeine on mouse fertilization rate in

vitro with BMOC-3 medium.

47

C. The influence of DNA stain Hoechst 33258 on In Vitro
Fertilization

The experiment was designed.to test the effect of Hoechst
33258 on in vitro fertilization. Both sperm and oocytes were
exposed to same concentrations of Hoechst 33258 before
insemination. The test concentrations of Hoechst 33258 were 1,
10, 20, and 100 ug/ml. The results are shown in Table 6. In
stain-free.control group, 169 oocytes‘were fertilized from 224
oocytes observed (75.5%). The gametes with 1.0 and 10 ug/ml of
Hoechst 33258, had in vitro fertilization rates which were
slightly lower than the control group, 72.3% and 66.7%
respectively, but these values were not‘ statistically
significant (P > 0.05). The fertilization rates in the 20 and
100 ug/ml treated groups were much lower, 29.3% and 6.2%

respectively, and were statistically significant (P < 0.05).

D. The effect of progesterone on in vitro fertilization rate

Recent studies. have indicated ‘that. progesterone
stimulates human spermatozoa acrosomal reaction (Thomas and
Meizel, 1989). To test the possible effect of progesterone on
mouse in vitro fertilization, the sperm and oocytes were
.incubated with varying concentrations of progesterone. The
results are shown in Table 7. The fertilization rate in the
control group was 93.2%, and the fertilization rate with

progesterone ( 0.1, 1.0, and 10.0 ug/ml) treated groups were

48
93.5%, 89.9%, and 90.5% respectively. No statistical
difference was found among these groups (P > 0.05).

Table 6. DNA-specific fluorochrome, Hoechst 33258 effect on
mouse fertilization in vitro

 

 

Treatment Fertilization Rate (%)
Control 169/224 (75.5%)
1 ug/ml Hoechst 33258 133/184 (72.3%)
10 ug/ml Hoechst 33258 152/228 (66.7%)
20 ug/ml Hoechst 33258 41/140 (29.3%)*
100 ug/ml Hoechst 33258 10/162 ( 6.2%)*

 

* P<0.05, the differences were significant compared to the
control group.

Table 7. Effect of progesterone on in vitro fertilization

 

 

Treatment Fertilization Rate (%)
Control 68/73 ( 93.2% )
0.1 ug/ml progesterone 129/138 ( 93.5% )
1.0 ug/ml progesterone 62/69 ( 89.9% )
10 ug/ml progesterone 67/74 ( 90.5% )

 

No significant difference among groups (P>0.05).

49

E. Toxic effect of toluene on in vitro fertilization

The results of toluene on in vitro fertilization are
shown in Table 8. No significant difference was found between
two control groups (P > 0.05). No significant difference was
found with the two toluene treated groups at the lowest level
(P > 0.05). However, toluene concentration at the 8.67 and
86.7 ug/ml dose levels significantly‘ inhibited in vitro
fertilization, the fertilization rates were 32.9% and 6.3%

respectively (P < 0.05).

Table 8. Toxic effect of toluene on mouse fertilization rate

 

 

in vitro
Treatment Fertilization Rate (%)
Control A (BMOC-B) 147/179 ( 82.1% )
Control B (t-Butanol) 125/163 ( 76.7% )
86.7ug/ml Toluene 5/80 ( 6.3 % )*
8.67ug/ml Toluene 24/73 ( 32.9% )*
0.867ug/ml Toluene 105/155 ( 67.7% )
0.0867ug/ml Toluene . 130/164 ( 79.3% )

 

* P<0.05, the differences were significant compared to the
control B.

50
1V,Qmem;ee1 effecte om momse embnye geveleement im vitro

A. DNA-specific fluorochrome and embryo development

Mouse gamete cells treated with DNA-specific fluorochrome
Hoechst 33258 before in vitro insemination showed some effect
on embryo development. The results are shown in Table 9. After
48-72 hours incubation, in stain-free control group, there
were 61% mouse embryos developed from the two-cell stage to
four or more cell stage. Although the 1 ug/ml Hoechst 33258
treated group had lower cleavage rate (48 %), the contingency
test indicated that the difference was not significant (P >
0.05). When the stain concentration increased to 10 and 100
ug/ml, the embryonic development was significantly inhibited,
the cleavage rate were 12% and 16.7% respectively (P < 0.05).
It was also found that in additional limited trials at level
of 20 and 100 ug/ml the number of degenerated mouse embryos
increased (data not included). The degenerated mouse embryos

are shown in Figure 13.

B. 'Toluene effects on oocyte and embryo degeneration after 24
hours incubation in vitro

The sperm and. oocytes were cultured ‘with different
concentrations of toluene after in vitro insemination. After
24 hours incubation, the number of degenerated mouse oocytes
and embryos was recorded each experiment. The results are

shown in Table 10 and the degenerated mouse oocytes and

51
embryos are shown in Figure 14. Comparing the t-butanol
control group, the degeneration rate was significant higher in
8.67 ug/ml (P < 0.05).

Table 9. Effect of DNA—specific fluorochrome Hoechst 33258 on
mouse embryo development in vitro with BMOC-3 medium

 

 

Treatment > 4 cell 2 cell Embryo Cleavage (%)
Control 64 105 61.0 %

1 ug/ml 65 133 48.8 % (a)

10 ug/ml 6 50 12.0 % (b)
looug/ml 2 12 16.7 % (c)

 

(a) Contingency test did not show significance (P>0.05)
(b), (c) The differences with control group were significant
(P<0.05).

 

Figure 13. Degenerated mouse embryo after incubated with 100
ug/ml Hoechst 33258 for 24 hours.

52

Table 10. Influence of tolulene on mouse oocyte and embryo
degeneration after 24 hours incubation in vitro

 

Treatment No. of No. of Degeneration Rate
Degenerated Observed

 

Control 26 225 11.6 %
0.0867 ug/ml 21 215 9.8 a; (a)
0.867 ug/ml 21 201 10.4 a; (b)
8.67 ug/ml 46 137 33.5 % *

 

* Contingency test showed significant difference compared to
the t-butanol control group (P<0.05). (a), (b) No difference
was found compared to the control (P>0.05).

 

Figure 14. Degenerated mouse oocytes incubated with 8.67 ug/ml
toluene for 24 hours.

53

V.S' e ete and embr 0 'nt ac u ar c lcium
measumement witm ACA§ 470-570 intemective laser cytometer
A. Single spermatozoa intracellular Ca“ measurement
1. Ca” distribution in the sperm cell: Fluo-3 Patterns

To observe the sperm intracellular Ca“’concentration and
distribution, two types of data were collected: integrated
cross sectional line scan data and two dimensional spatial
scan data. The typical cross sperm head line scan is shown in
Figure 15. and the typical two dimensional spatial scan of
sperm.head and midpiece is shown in Figure 16. Different types
of ca” fluorescent patterns were observed during the study.
Pattern I. The sperm has very strong fluorescence within both
head and midpiece, i.e. both head and midpiece contain high
level of free ca”. This pattern of sperm is usually observed
during first 30 minutes after preparation (Figure 17-A).
Pattern II. The fluorescence in sperm head became very weak,
the midpiece still has strong fluorescence (Figure 17-B). This
pattern can be seen at any time, but the number of sperm
showing this pattern increased after'30 minute. When the sperm
head fluorescence became weak the fluorescence around sperm
head did not change at all, i.e. the fluorescein still
remained in sperm head, but the Ca” level decreased. Pattern
III. The fluorescent in sperm head disappeared, but the
fluorescence in the midpiece remains the same (Figure 17-C).

This pattern increases with time. There is a very high

35304
30805

2500‘ VJ

2900-
1500— [\i
10004 v

mW/W .

% l'llTrl‘ll‘llllll|lTllllllYl|

@ 2% 4% BB 88 10%

 

 

 

 

Figure 15. The typical integrated cross sperm head line scan

: — 2150
j — 2017
J - 1974
g.— 1731
.5 — 1588
l:— 1445
y;— 1302
— 1159
g — 1815
" - 872
g — 729
i - 586
; - 443
E — 300
¥ - 157

   

Figure 16. Two dimentional spatial scan of the sperm head and
the midpiece

55

One: '0' I On:

a); Manon:

Color V9112:
“8'5

Dutcvor I Dan (Junior 2 Dan

 

Figure 17. Fluo-3 density distribution: Ca" fluorescence
patterns (A) Pattern I sperm, (B) Pattern II sperm

56

percent of this pattern'of sperm in the immotile group.

A relationship between the ca” fluorescence pattern and
the CTC pattern was found in the study. The ca“ fluorescence
Pattern I was very similar to the CTC pattern F (fresh sperm):
the pattern II appears similar to the CTC pattern C
(capacitated sperm); and the pattern III was similar to CTC
pattern A (acrosomal reacted sperm).

Because the sperm head and midpiece showed different Ca”
fluorescence distributions, and when the sperm head
fluorescence disappeared the midpiece remained at a high
level, this suggested that the Ca“’mobilization in sperm head
and midpiece may involved different mechanism, and the Ca“
distribution in two compartments may be completely isolated.
To test this hypothesis, I utilized a photobleaching
technique. The Fluo-3 molecules within the sperm head were
destroyed by a high power laser beam, then the fluorescence
recovery over a period of time was observed. The results are
shown in Figure 18. No recovery was observed within 14

minutes.

2. Chemical effects on single sperm intracellular Ca”
concentration

Single sperm intracellular Ca” concentration was measured
by the special fluorescent color values. The color values vary

from 0 to 4095 specific color units, which were

57

mmuscfle ea :quH3

>uw>oomn miosau coon Ehwom may no %osum mownomoanouosm .mH wusmwm

 

fic_ES ms_e .m) zummfipmam x

 

 

«F m as "w w v N a
~u__e _.._11_1E_ eta
{Iii/fif1\\k ////\\\\FSN
19?
15w
ill/11%
we re?

 

fl

,4

:$#
1

A!

W"&W

 

 

_oswcou

 

 

N}lzmmflm

mmH::_s q—

cumm_gmad
. my

W

In

14"”.

 

 

mco.omama_

 

 

58
correlated with the Ca” concentration. In this study, the
relative fluorescence color value was used to detect the

effects of chemicals on the intracellular Ca” concentration.

a) Caffeine effects on sperm intracellular Ca”
concentration and distribution.

The image scan program was used to measure the caffeine
effects on sperm intracellular calcium level. The results are
shown in Figure 19. After adding 10 mM caffeine to the
medium, a very significant intracellular Ca” increase was
observed in the sperm midpiece (the integrated value from
554,518 increased to 728,625), the change in the sperm head

was not significant.

b) The influence of toluene on sperm intracellular Ca”
level.

The auto line scan program‘was used to measure the effect
of toluene on sperm intracellular calcium concentration. The
results are shown in Figure 20. In the sperm treated with
8.67 ug/ml of toluene, no significant calcium change was

observed.

59

 

cofiusnfiuumwo 6:0 nodumuucoocoo
++ao goddaawonhucfi spoon :0 mucouum ocwouumo .mH wusmwm

595.9 "5.29m
mimemm "_ 25s

 

60

soon mead

ya oouonaol ++ao undouaooouucw Shana so nucouuo osooaoa .om musmwh

msmcm__u

 

 

 

159:?

159mm

199mm

Issac

 

rsssm

61

B. Intracellular Ca’+ concentration in the oocytes
1. ca” distribution in mouse oocytes

The intracellular Ca“’distribution in mouse oocytes were
measured by scan image program. Two types oocytes of were
studied.

a) The fresh cumulus-intact oocyte

The Ca”’distribution in a cumulus-intact oocyte is shown
in Figure 21. Because the F1uo-3 had to go through the cumulus
cells, the fluorescence was very weak in the oocyte compared
to the cumulus cells. After 5-10 minutes, the fluorescence
became much stronger. There are two possible. mechanisms
involved. First, it could have occurred by passive diffusion
from the medium to the zona pellucida or, secondly, it could
have been by a cell-cell communication mechanism from the

cumulus cells to zona pellucida through gap-junctions.

b) The cumulus-free oocyte

The intracellular Ca” distribution in a cumulus-free
oocyte (scan image) is shown in Figure 22, and the average
fluoroscent intensity cross egg integrated value was 250,263
(Figure 23). The calcium concentration within the oocyte was
different, at a low level in zona pellucida, an increase in
the vitelline layer and plasma membrane, the highest in the
cytoplasm. In the cytoplasm, there were some special areas

showing very high Ca” concentration. This was probably the

62

Haoo unassso

spa: muwooo 00:05 w :w cofiusnauumfio xufiucoo niosum .Hm musmwm

mmm

__m

mmm

MNS.
mmm_
mmm_
_mm_
mvsm
MSMN
mmmm
m_mm
_nsm
mmmm
mmmm
mmmm

mmsv
mm:_m>

40h.mk>uoonw

l

semm "203.1 m>¢
_m "ucmxm msc
QKNNN "m_mx_a xm

5km“ "m_mx_a _i

_ m
.525 moZm... em

 

63

.cmum moms“ cufls :oflusnwuuwflo
+.mo unasHHwomuucfl wuaooo wwsoe omnuuwSHsESU .mm wusmfim

|
ssm.we>uos-mnw

s -
mmm -
__m -
not -
Mme—
mkN_ -_
mmml -1
lmm_ -2
Ream
msmm ,
mmmm -,
mlmm ,
_Nsm -2
KNMM -m
mmmm -
mmmm - .
mmse - -
mm:_m> bosom

Hie-L

 

 

 

64

.mumooo meOE meMIMSHsfioo
mo xuflmcwucfl mocwommnoodu muosHm wwmuw>m 0:9 .mm whomwm

_ sso.mkk

 

mmmsmm - ms_ms umzmrmmzcl
seal gem sew see saw a

 

TWIN |

m_mm 1

 

 

 

mmae -.a. Aszw mocmom_o .m> >~_mcmbc~ ton—d m>¢ memo P Layomwmo

mmzfimp Lo_ou

 

 

 

 

 

E >255 Eczema-I 2.:

65
intracellular free calcium storage, such as the endoplasmic
reticulum or mitochondria. The oocyte 3-dimensional image of

intracellular calcium distribution is shown in Figure 24.

2. Intracellular calcium change during sperm penetration

The intracellular Ca” levels were measured in five mature
oocytes during in 'vitro fertilization. The oocytes were
treated with Fluo-3 but the sperm were not. After locating
the oocytes, I used the line to record the basal calcium
level, and then the sperm suspension was added to the dish.
The sperm binding to the zona pellucida and penetration were
observed by the video monitor. There was a continuously
recorded intracellular ca” level in every oocyte by the line
scan. After one sperm fused with the oocyte membrane, the line
scan showed the calcium level significantly increased. The
line scan before and after sperm penetration are shown in
Figure 25. No significant calcium changes were observed in the

rest of the oocytes.

C. Calcium distribution in the 2-cell embryo

Twenty-four hours after in vitro fertilization, 2-cell
embryos were collected and treated with Flue-3. The calcium
distribution in the 2-cell stage embryo is shown in Figure 26.
The calcium concentration in the two cells was not the same,

one cell had a much higher calcium level than the other.

66

cofluonfiuumfio ++mo
unasaawoouusfi wuxooo om=OE no macaw Hmsofimcmfifloloohne .em wusmwm

 

 

 

 

 

67

coflumuumcmm

spoon mcflnoo mmcmno Esfioamo umHoHHmomnucfl muxooo muse: .mm musmflm

sm_ amp

H.511}... ..SA.C£1r\.;/g

sq? EMF
___t_________e____:

““li4 —1:4 {4 ‘

-9:

:__

am

i___ :_______:___t________________:__ee_____ce_ee :__

Sm sq am: am: a: aw; 3— sm am .3

1114 ..I

41:15.1...»-

ix

118115 Sa-J

.2 2.5.11:

75 ggx

Tasm

rsssfi
199m?
isssm
rssmm
1assm
issmm

fesae

 

fiesme

68

 

Detector 1 Data

 

E:2C—EMB—i.888

Figure 26. two-cell stage mouse embryo intracellular calcium
distribution.

69

DISCUSSION

on d 8 e Mot'

Sperm motility is a very important factor for a
successful in vitro fertilization program. Mann and Lutwak-
Mann (1981) indicated that there were clear correlations
between sperm motility, fertilizing ability and pregnancy.
Thus adverse chemical effects against the sperm can seriously
effect reproduction, a simple method to test the possible
effect of a chemical on in vitro fertilization is to test its
effect on sperm motility.

Sperm motility is decreased after incubation for a
period of time. Similarly, in 'vivo, the sperm :motility
significantly decreases after ejaculation. The reasons
underlying the decrease in motility are not yet established.
Two factors that are likely involved are the diminished
integrity of the sperm membrane due to the proteolytic enzyme
activity of the seminal fluid and /or the exhaustion of the
sperm energy reserves (Huszar,1990).

We know that sperm motility is manifested by contractile
waves in the sperm flagellum. The flagellar bending is
produced by biochemical energy, mainly in the form of ATP
resulting from oxidative phosphorylation, which is transferred

into the biomechanical kinetic energy in the form of sliding

7O

axonemal microtubules (Siegel et al. , 1987) . In mammalian
sperm, cAMP was also proposed as a major factor in the
initiation of flagellar movement and stimulation of motility
during the fertilization process (Fraser, 1979; Chan 1983;
Tash, 1989). Calcium1and.calmodulin aISijlay a very important
role in regulating sperm motility (Brokaw and Nagayama, 1985) .
There are many other factors e.g. sperm intracellular pH, Na’,
1? cation, and sperm membrane function, that are also involved
in the modulation of sperm motility.

The effect of caffeine on sperm motility and velocity has
been Known for twenty years. In most species, Caffeine
stimulates sperm motility. The results from the present study
showed that 0.1 to 10 mM of caffeine not only stimulated mouse
epididymal sperm motility but also maintained a higher
percentage of motile sperm for a longer period of time. The
mechanism of caffeine stimulation of sperm motility is still
not well understood. The classictexplanation‘was that caffeine
inhibited phosphodiesterase and increased the cellular cyclic
AMP level (Hardman et al., 1971; Garbers et al., 1971). The
stimulatory effect of cAMP on sperm motility has been
confirmed by many studies (Lindemann, 1978; Mrsny and Meizel,
1980: Ishijima and Witman, 1985; Tash et al., 1986; Aaberg et
al, 1989). Levin. et -al.(l981) analyzed ‘the effects of
caffeine on human sperm motility and cAMP phosphodiesterase.

The results indicated that the direct action of caffeine on

71

sperm motility may not involve the inhibition of cAMP
phosphodiesterase. The present study demonstrated that Ca”
was required for the caffeine stimulation of mouse sperm
motility. In the ca”-free medium caffeine did not stimulate
sperm motility. A single sperm intracellular Ca’+ dynamic
study showed that 5 mM caffeine increased mouse sperm midpiece
Ca“ level, where the sperm mitochondria and axoneme are
located. According to White and Aiken (1989), with a rise in
intracellular calcium, and the resultant increase the
adenylate cyclase activity: The rise in Ca“'may also mediated
by calmodulin in the regulating sperm motility. This is
probably another mechanism besides the inhibition of
phosphodiesterase.

DNA-specific stain Hoechst 33258 is considered a non-
toxic stain, which can bind to DNA and allows assessment of
its organization and distribution within living cells. The
present study demonstrated that a concentration of Hoechst
33258 of less than 20 ug/ml had no effect on sperm motility.
When the concentration reached 100 ug/ml therewas a decreased
sperm motility after 90 minutes and 4 hours incubation. The
mechanism for this effect is not clear. This high
concentration of stain could cause an intracellular pH change
or have a cytotoxic effect on the DNA structure and function.

Toluene has been detected in municipal water supplies at

levels ranging from 0.1 to 11 ug/l. One in vivo study by

72
Nawrot and Staples (1979) indicated that there was a
significant increase in embryonic lethality and decreased
fetal weight following the oral gavage of pregnant CD-1 mice
with toluene at 0.5 ml/kg level. There are no in vitro
reproductive toxicological data available. In the present
study, B6D2F1 mouse epididymal sperm were incubated with
toluene from 0.0867 to 86.7 ug/ml for a 4 hour period. The
results demonstrated that 0.0867 ug/ml of toluene had no
effect on sperm motility. Concentrations of toluene greater
than 0.867 ug/ml significantly decreased.sperm motility at all
three given times (15 min, 90 min, and. 4 hr). The dose
dependence was significant. The mechanism by which toluene
inhibits sperm motility is not clear. Previous studies
indicated that benzene significantly increased the
intracellular calcium level by inhibiting the ca” pump. The
high free Ca“’level was correlated with inhibition of protein
phosphorylation and inhibition of sperm motility (Tash et al,
1988), but the single sperm, intracellular' calcium study
reported here did not show a significant change after adding
8.67 ug/ml of toluene to the culture medium. It is interesting
to note during the study of sperm intracellular Ca”, when
adding toluene to the culture medium, the sperm motility and
vigor were very significantly increased, but after a few
seconds the stimulation effect disappeared. It is not known

why toluene had a short stimulation and long inhibition to the

73
sperm motility, or if toluene had an inhibitory effect on

sperm mitochondrial oxidative phosphorylation.

Seorm caeacitatiom and ecroeomal reactiom

During sperm capacitation, one of the principal events is
a biochemical alteration of the sperm plasma membrane.
(Cooper, 1986: Eddy, 1988; Yanagimachi, 1988). Such membrane
alteration may be regulated by the sperm intracellular second
messenger system. Stein and Fraser (1984) demonstrated that
the adenylate cyclase activity increased and phosphodiesterase
activity decreased during capacitation. Caffeine, as a
phosphodiesterase inhibitor, could significantly increased
intracellular cAMP level. The hypotheses of whether caffeine
could change the sperm-capacitation time course and/or trigger
sperm acrosomal reaction has been suggested by the work of
Kuehl and Dukelow (1982) and Chan (1983) on the squirrel
monkey. The B6D2F1 mouse sperm was tested here by the CTC
assay. The results did not show significant change of sperm
capacitation time course nor acrosomal reaction. It could be.
the species difference or the lower sensitivity of CTC assay
comparing to the triple stain. Spermtcapacitation includes two
significant changes, membrane alteration and sperm
hyperactivation. The sperm hyperactivation was believed due to

the membrane alteration in the sperm midpiece (Yanagimachi,

74
1989). The present study provided the new evidence of caffeine
mobilizes sperm intracellular Ca” in the midpiece region may
involved in sperm hyperactivation.

The CTC assay as an indirect method for detecting sperm
capacitation and acrosomal reaction has been used in many
laboratories ( Ward and Storey, 1984; Lee et al., 1987: Endo
et al, 1988; and Kholkute et al, 1990). According to Ward and
Storey (1984), the rational underlying this assay is that
fresh epididymal sperm have CTC binding components absorbed to
their plasma membrane surface and that these are lost during
capacitation, so that the CTC binding pattern would change. An
interesting finding from the present studies was that the CTC
pattern had some correlation with sperm cell intracellular Ca”
distribution. Fresh sperm (Pattern F) had a high level of Ca’+
both on the sperm .head and. midpiece; Capacitated sperm
(Pattern C) had decreased sperm head Ca"+ concentration: and
acrosomal reacted sperm (Pattern A) had a Ca” level that
reached its lowest point in the sperm head but showed no
change in the midpiece. This may provide some new evidences to

explain the mechanism of the sperm CTC patterns.

gm vgtmo foggilizetion

In the present study, four chemical substances were
tested their effects on fertilization in vitro. The culture

medium selection was based on a comparison of fertilization

75

rate with three culture media, BMOC-3, TC-199 and M16. The
major component difference between high IVF rate media (BMOC-
3 and M-16) and low IVF rate medium (TC-199) was sodium
lactate and sodium pyruvate (Table 1), which were absent in
the medium TC-199. According to a previous study in this
laboratory (Chan, 1983), lactate and pyruvate are required to
sustain sperm metabolism for the squirrel monkey with in vitro
fertilization.

The effect of caffeine on fertilization was dependent on
the concentration. Caffeine (0.1. mM -and 1.0 mM) had. no
significant effect on the fertilization rate, although the
percent of fertilized oocytes in the 0.1 mM caffeine treated
group (83.9%) was higher than the caffeine-free control group
(71.9%); and the 1.0 mM group was lower (61.2%). The caffeine
at a concentration of 10 mM significantly decreased the
fertilization rate (35.2%). This result was not in agreement
with the study of Pomeroy et al. (1988), in which they
demonstrated sperm pretreated with 6.0 mM caffeine increased
the fertilization rate (62 %) comparing the caffeine-free
group (23 %). Their control group had a very low fertilization
rate probably because of different experimental procedures, in
which they incubated the sperm with oocytes for only 15
minutes and used cumulus-free oocytes. The results from the
present study suggested that the low concentration of caffeine

(< 1.0 mM) may favor IVF; but the high level of caffeine (>

76
10 mM) was detrimental.

DNA-specific stains (Hoechst 33258 and 33342) have been
used. to detect early fertilization. in imany laboratories
(Hinkley et al., 1986; O'Rand et al., 1986: Conover and
Gwatkin, 1988; Roudebush 1988; Wright and Longo, 1988). No
previous report is available on Hoechst 33258 toxic effects
on gametes. The present study demonstrated that the mouse
sperm and oocytes treated with 20 ug/ml and 100 ug/ml of the
stain showed a significantly decreased fertilization rate. A
concentration of Hoechst 33258 less than 10 ug/ml was
considered a safe level. The mechanism by which the high
concentration of this DNA stain affected. fertilization-is not
clear. According to Smith et al. (1989) the biological effect
of the DNA ligand (Hoechst 33258) on the cell may due to the
direct disturbance of the processes of DNA replication and
transcription.

It is not required for progesterone to be present for’ in
vitro fertilization. The recent studies by Thomas and Meizel
(1989) and Blackmore et al.(1990) have indicated that
progesterone can cause an influx of extracellular Ca” into the
sperm and induce the acrosomal reaction. This suggested‘the'
hypothesis that the progesterone may enhance in vitro
fertilization. The results from this study showed that media
containing three concentrations of progesterone (0.1, 1.0 and

10 ug/ml) all had very good fertilization rates. However no

77
significant difference with controls was found.

The effect of toluene on fertilization was also dependent,
on the concentration. The critical toxic concentration of
toluene was between 0.867 and 8.67 ug/ml. Since the toluene
treatment involved both the sperm and the oocytes, the exact

mechanism of this action is not known.

gmmmyonic development im vitro

Fertilization triggers the second meiotic division and
extrusion of the second polar body. The critical elements of
cellular proliferation and differentiation in mammalian
embryonic development are influenced by many factors from
either embryonic or maternal sources. Early embryo development
is regulated primarily by the expression of specific genetic
programs within the cells, but also requires a continuous
supply of energy, hormones and growth factors, provided from
maternal environment (Kaye, 1986). Chemical effects on
embryonic development can occur at any stage, however the
early stages are most easily influenced.

The results from the present study showed that DNA stain
Hoechst 33258 (>10 ug/ml) significantly decreased embryonic
cleavage. The possible mechanism has been discussed above.

The study also demonstrated that toluene at a

concentration higher than 8.67 ug/ml significantly decreased

78
fertilization rate. The high concentration of toluene also
increased the oocyte and embryo degeneration. One early in
vivo study by Nawrot and Staples (1979) indicated that the
pregnant CD-1 mouse receiving toluene at 0.3-1.0 ml/kg had
significantly increased embryonic lethality. The results from
this in vitro study provided the evidences that toluene could
cause embryo death at early stage, and it may also suggested
that higher level toluene exposure in the enviroment may cause

fertility problems.

Imtmaeellule; C8” measurememt $11 simgle gemete 69;;8

Intracellular Ca”, as a second messenger, plays an
important role in regulating cell function. Because a Ca“
change is directly involved in sperm ‘motility, capacitation,
the acrosomal reaction, and sperm-egg fusion (Handrow et al.,
1989: Stock and Fraser, 1989; Noland and.Olson, 1989; Mortimer
et al., 1988:), it is desirable to detect the single sperm
cell intracellular Ca++ concentration, distribution and dynamic
change during these events. Most of methods used measuring
sperm Ca” require millions of sperm (Babcock and Apfeiffer,
1987: Stephens et al., 1988:), and these methods lack
sensitivity. The ACAS interactive laser cytometer makes it
possible to measure intracellular Ca” in a single living sperm

or oocyte, and it also provides information on intracellular

79
ca“ distributions.

Fluo-3 AM, as a new fluorescent Ca” indicator with
visible excitation and emission_ wavelengths, has been tested
in many living cells (Joseph et al., 1989; Wade , 1989; Dawson
and Hooper, 1990:). The binding of Ca“’to the f1uo-3 increases
the fluorescence up to 40 fold. The Ca” dissociation constants
are in the range of 0.37-2.3 um, and this gives better
resolution than other indicators like Quin-2 or Fura-2 (Minta
et al., 1989). F1uo-3 is useful for the determination of
qualitative calcium-changes in cells-as the result of chemical
treatment, however a major disadvantage of Fluo-3 is that it
is difficult to analyze the absolute calcium levels (Wade,
1989). The present study used Fluo-3 as an indicator to
determine the intracellular Ca” distribution and dynamic
change in the single sperm and oocyte. The results
demonstrated that. ‘this fluorescein did. give very good
resolution for Ca”.

In agreement with other studies (Endo et al., 1988), the
results demonstrated that the calcium ionophore A 23187 (5 uM)
significantly increased sperm intracellular calcium level
within a few seconds. This is believed to be responsible for
inducing the sperm acrosomal reaction (Tesarik, 1985: Lee et
al., 1987; Anderson et al., 1989). A interesting finding from
the caffeine treatment study was that caffeine (10 mM) also

induced an intracellular ca” increase, but that the increase

80

was restricted to the midpiece of sperm. According to
Vijayaraghavan et al. (1989), the plasma membrane surrounding
the midpiece is considerably more permeable to calcium than
the membrane domains surrounding the sperm head and tail.

»The present study did not show a significant effect of
toluene on sperm intracellular Ca“3 The toxical mechanism.may
not be interrupting the Ca“ metabolism, although other studies
do show that benzene-type chemicals increase the intracellular

ca” by inhibiting the Ca“ pump.

81

SUMMARY AND CONCLUSIONS

B6D2F1 mice were used for testing the effect of some
physiological, pharmacological and toxic substances on
reproduction. The in vitro study included testing the effects
of chemicals on sperm motility, sperm capacitation and
acrosomal reaction, intracellular Ca” change, in vitro
fertilization and embryonic development. This provided the
following results:

(1) Caffeine (0.1, 1.0 and 10 mM) significantly increased
mouse epididymal sperm motility. The stimulatory effect was
ca“- dependent. Caffeine at the 0.1 and 1.0 mM levels had no
significant effect on fertilization, but 10 mM of caffeine
decreased the fertilization rate. The CTC assay demonstrated
that caffeine (10 mM) did not effect sperm capacitation or
acrosomal reaction.

(2) Fertilization rates were significantly decreased in
the presence of 20 and 100 ug/ml of the DNA stain, Hoechst
33258. A.concentration of Hoechst 33258 less than 10 ug/ml had
no effect on fertilization, but gametes incubated with 10
ug/ml or' higher levels of the DNA stain had inhibited
embryonic cleavage.

(3) The inhibitory effect of toluene on mouse sperm
motility was significant. The critical toxic concentration in

vitro was between 0.867 and 8.67 ug/ml. A concentration of

82
toluene higher than 8.67 ug/ml not only decreased sperm
motility and inhibited fertilization but also increased the
percent of embryonic degeneration.

(4) Mouse sperm treated with caffeine, the intracellular
Ca” level significantly increased. But the ca“ elevation was
a slowly process and which only limited to the sperm midpiece.
No significant effect of toluene on mouse sperm intracellular
Ca’+ change were observed.

In conclusion, the present study suggested that caffeine
stimulation of mouse sperm motility may by mobilizing
intracellular Ca” distribution, especially increasing the
mitochondria and axonemal ca” levels. The high concentration
(> 10 mM) had an inhibitory effect on fertilization in vitro.
The DNA stain-Hoechst 33258 was reconsidered.as a low-toxicity
stain. A.concentration less than 10 ug/ml was useful for these
experiments. Toluene did have cytotoxic effect on gametes and
embryos in vitro. These studies demonstrated that the sperm,
oocyte and embryo intracellular Ca” distribution patterns
may provide some important information on gamete cell

structure and function.

83

REFERENCE

Aaberg RA, Sauer MV, Sikka S, and Rajfer J: Effects of
extracellular ionixed calcium, diltiazem and cAMP on
motility of human spermatozoa. J Urol 141:1221-1224, 1989

Aarons D, et al Speake JL, Poirier GR: Evidence for a
proteinaseinhibitor binding component associated with
murine spermatozoa. Biol Reprod 31:811-717, 1984

Ahuja KK: Lectin-coated agarose beads in the investigation of
sperm capacitation in the hamster. Devel Biol 104:131-
142, 1984

Aitken RJ, Best F, Richardson DW, Schats R, and Simm G:
Influence of caffeine on movement characteristics,
fertilizing capacity and ability to penetrate cervical
mucus of human spermatozoa. J Reprod Fert 67:19-27, 1983

Anderson DJ, Michaelson JS, and - Johnson PM:
Trophoblast/leukocyte-common antigen is expressed. by
human testicular'germ.cells and appears on the surface of
acrosome-reacted sperm. Biol Reprod 41:285-293, 1989

Arndt-Jovin DJ, and Jovin TM: Analysis and sorting of living
cells according to deoxyribonucleic acid content. J
Histochem Cytochem 25:585-589, 1977

Aumiller G, Vesper M, Seitz J, Kemme M, and Ssheit KH: Binding
of a major secretory protein from bull seminal vesicles
to bovine spermatozoa. Cell and Tieeue Res 252:337-384,
1988

Austin CR: Observations on the penetration of the sperm into
the mammalian egg. Austral J Sci Res SeriesB, 4, 581,
1951 -

Babcock DF, and Apfeiffer DR: Independent elevation of
cytosolic Ca” and pH of mammalian sperm by voltage-
dependent and pH-sensitive mechanisms. J Biol Chem
262:15041-15047, 1987

Barkay J, Zuckerman H, Sklan D, and Gordon S: Effect of
caffeine on increasing the motility of frozen human sperm
Fertil Steril 28:175-177, 1977

Bauchinger M, Schmid E, Dresp J, Kolin-Grresheim J:Chromosome
aberrations and sister-chromatid exchanges in toluene-

84
exposed workers. Mutat Res 113:231-232, 1983

Bedford JM: Fertilisation mechanisms in animals and man:
current concepts. In:Establishing a successful human
pregnancy. (Ed.) by Edwards RG, Serono symposia
publications from raven press Vol. 66. pp. 115-131, 1990

Berger T, and Beierle JW: Inhibition of sperm motility by
bovine serum components. Biol Reprod 42:545-551, 1990

Blach EL, Amann RP, Bowen RA, Frantz D: Changes in the quality
of stalion spermatozoa during cropreservation:plasma
membrane integrity and motion characteristics.
Theriogenology 31:283-298, 1989

Blackmore PF, Beebe SJ, Danforth DR, and Alexander N:
Progesterone and 17a-hydroxyprogesterone--Novel
stimulators of calcium influx in human spermm J Biol Chem
265:1376-1380, 1990

Bocckino SB, Blackmore PF, Wilson PB, Exton‘ JH:---.J Biol
Chem 262:15309-15315, 1987

Brokaw CJ, and Nagayama SM: Modulation of the asymmetry of sea
urchin sperm flagellar bending by calmodulin. J Cell Biol
100:1875-1883, 1985

Bunch DO, LeGuen P, and Saling PM: P95, a ZP3 receptor with
tyrosine kinase activity, fractionate with mouse sperm
membranes that inhibit sperm-zona binding. J Androl
11:24p, 1990

Burge RG: Caffeine stimulation of ejaculated human
spermatozoa. Urology 1:371, 1973

Cai X, and. Marik: JJ: Improving' penetrating capacity of
spermatozoa with poor motility by addition of caffeine at
coincubation with zona-free hamster ova. Fertil Steril
51:719-721, 1989

Chan PJ: Culture variates affecting in vitro fertilization of
squirrel monkey (Saimiri sciureus) oocytes. Ph.D
dissertation, Michigan State University, 1983

Chang MC: Fertilization capacity of spermatozoa deposited into
follopian tubes. Nature, London, 168:697, 1951

Cherr G, Lambert H, Meizel S, and Katz D: In vitro studies of
the golden hamster sperm acrosome reaction: completion on

85

the zona pellucida and induction by homologous soluble
zonae pellucidae. Devel Biol 114:119-131, 1986

Comings DE: Mechanisms of chromosome banding, VIII. Hoechst
33258-DNA interaction. Chromosoma (Berl.) 52:229-243,
1975

Conover JC and Gwatkin RB: Pre-loading of mouse oocytes with
DNA—specific fluorochrome (Hoechst 33342) permits rapid
detection of sperm-oocyte fusion. J Reprod Fert 82:681-
690, 1988

Cooper TG: The epididymis, sperm maturation and fertilization.
Springer-Verlag, Berlin. pp 42-55, 1986

Courtney KD, Andrews JE, Springer J, Menache M, Williams T,
Dalley, L and Graham JA: A perinatal study of toluene in
CD-1 mice. Fund Appl Toxicol 6:145-154, 1986

Crister ES, Ball GD, O'Brien MJ, and First NL: Use of a
fluorescent stain for visualization of nuclear material
in living gametes and early embryos. Biol Reprod 28:140
A, Abstr. 1983

Cross NL, Morales P, Overstreet JW, Hanson FW: Two simple
methods for detecting acrosome-reacted human sperm.
Gamete Res 15:213-226, 1986

Cummins JM, and Yanagimachi R: Development of ability to
penetrate the cumulus oophorus by hamster spermatozoa
capacitated in vitro, in relation to the timing of the
acrosome reaction. Gamete Res 15:187-212, 1986

Dawson CD, and Hooper WC: Flow cytometric measurements of
intracellular calcium in Fluo-3 loaded myelogenous cells.
Society for Analytical Cytology Abstracts, 4:62, 1990

Domino SE, Bocckino SB, and Garbers DL:---. J Biol Chem
264:9412-9419, 1989

Dougherty KA, Cockett ATK, Urry RL: Caffeine, theophylline,
and human sperm motility. Fertil Steril 27:541, 1976

Dudenhausen E, Talbot P: Detection and kinetics of teh normal
acrosomal reaction of mouse sperm. Gamete Res 6:257-265,
1982

Dukelow WR, and Williams WL: Capacitation of sperm. Progress
in Infertility (3rd ed.)Behrman, Kistner & Patton (eds),

./.

86
Little Brown Co., Boston. pp 673-687, 1988

Eddy EM: The spermatozoa, In: The Physiology of Reproduction.
Vol 1, pp. 27-68. (Eds. Knobil E and Neill JD). Raven
Press, New York. 1988

Endo Y, Lee MA, and Kopf GS: Characterization of the islet
activation protein-sensitive site in mouse sperm that is
involved in the zona pellucida-induced acrosomal
reaction. Devl Biol 129:12-14, 1988

Environmental Protection Agency (United States) :- Ambient Water
Quality Criteria for Toluene. National Technical
Information Service, Springfield, Virginia. A-2, 1980

Euler HH: Animal experimental studies of an industrial noxa.
Archiv Fur Gynakologie 204:258-259, 1967

Florman HM, Tombes RM, First NL, and Babcock DF: An adhesion-
associated agonist from the zona pellucida activates G
protein-promoted elevations of internal ca” and pH that
mediate mammalian sperm acrosomal exocytosis.

Forni A, Pacifico E, and Limonta A: Chromosome studies in
workers exposed to benzene or toluene or both. Arch
Environ Health 22:273-278, 1971

Fraser LR: Accelerated.mouse sperm penetration in vitro in the
presence of caffeine. J Reprod Fert 57:377-384, 1979

Fraser LR: adenosine and its analogues, possibly acting at A2
receptors, stimulate mouse sperm fertilizing ability
during“ early stages of capacitation. J Reprod Fert
89:467-476, 1990

Fraser LR, and Ahuja KK: Metabolic and surface events in
fertilization. Gamete Res 6:81-89. 1988

Fraser LR: Extracellular calcium and fertilization-related
events In The role of calcium in biological systems, Vol.
IV, pp. 163-190. (Ed.) Anghileri LJ. CRC Press, Boca
Raton, 1987

Garbers DL, Lust WD, First NL, and Lardy HA: Effects of
phosphodiesterase inhibitors and cyclic nucleotides on
sperm respiration and motility. Biochem 10:1825-1831,
19713

Garbers DL, First NL, Sullivan JJ, and Lardy HA: Stimulation

87

and maintenance of ejaculated bovine spermatozoa
respiration an motility by caffeine. Biol Reprod 5:336-
339, 1971b

Gerner-Smidt P, and Friedrich U: The mutagenic effect of
benzene, toluene and xylene studied by the SCE technique.
Mutat Res 58: 313- 316, 1978

Goodwin TM: Toluene abuse and renal tubular acidosis in
pregnancy. Obstet Gynecol 71: 715-718, 1988

Hamner CE, and William WL: Effect of the female reproductive
tract on sperm metabolism in the rabbit and fowl. J
Reprod Fertil 5:143, 1963

Handrow RR, First NL, and Parrish JJ: Calcium requirement and
increased association with bovine sperm during
capacitation by heparin. J Exp Zool 252:174-182, 1989

Hardman JG, Robison GA, and Sutherland EW: Cyclic nucleotides.
Annu Rev Physiol 33:311-336, 1971

Harrison RF, Sheppard BL, Kaliszer M: Observation on the
motility, ultrastructure and elemental composition of
human spermatozoa incubated with caffeine. II. A time
sequence study. Andrologia 12:434, 1980

Hicks JJ, Petron N, and Rosado A: Modifications of human
spermatozoa glycolysis by cyclic adenosine monophosphate
(cAMP), estrogens, and follicular fluid. Fertil Steril
23:886, 1972

Hinkley RE, Wright BD, and Lynn JW: Rapid detection of sperm-
egg fusion using the DNA-specific fluorochrome Hoechst
33342. Devl Biol 118, 148-154, 1986

Hinkley RE, Wright BD, and Lynn JW: Rapid detection if
sperm -egg fusion using the DNA- specific fluorochrome
Hoechst 33342. Devl Biol 118:148-154, 1986

Huszar G, Willetts M and Corrales M: Hyaluronic acid (Sperm
Select) improves retention of sperm motility and velocity
in normospermic and oligospermia specimens. Fertil Steril
54(5):1127-1134,1990

Joseph P, Kao Y, Harootunian AT, and Tsien RY: Photochemically
fenerated cytosolic calcium pulses and their detection by
fluo-B. J Biol Chem 264:8179-8184, 1989

88

Kirk RE, and Othmer D: Kirk-Othmer Encyclopedia of Chemical
Technology. 2nd ed. John Wiley and Sons, Inc., New York.
1963

Kostas J. and Hotchin J: Behavioral effects of low-level
perinatal exposure to toluene in mice. Neurobehav Toxicol
Teratol 3:467-469, 1981

Kuehl TJ and DukelowWWR: Time relationships of squirrel monkey
(Saimiri sciureus) sperm capacitation and ovum maturation
in an in vitro fertilization system. J Reprod Fertil
64:135, 1982

Kusunoki H, Sakaue M, Kato S, Kanda S: Induction of the
acrosome reaction in ejacualted goat spermtozoa by
preincubation in chemically defined medium. J Exp Zool
249:322-328, 1989

Langlais J, and Roberts KD: A molecular membrane model of
sperm capacitation and the acrosome reaction of mammalian
spermatozoa. Gamete Res 12:183-224, 1985

Latt SA, and Stetten G: Spectral studies on 33258 Hoechst and
related bisbenzimidazole dyes useful for fluorescent
detection of deoxyribonucleic acid synthesis. J Histochem
Cytochem 24: 24-33, 1976

Lee MA, Trucco GS, Bechtol KB, Wummer N, and Kopf,GS:
Capacitationand acrosome reactions in human spermatozoa
monitored by a Chlortetracycline fluorescence assay.
Fertil Steril 48:649-658, 1987

Levin RM, Greenberg SH, and Wein AJ: Quantitative analysis of
the effects of caffeine on sperm motility and cyclic
adenosine 3',5'-monophosphate (cAMP) phosphodiesterase.
Fertil Steril 36:798-802, 1981

Leyton L, and Saling PM: Evidence that aggregation of mouse
sperm receptors by ZP3 triggers the acrosome reaction. J
Cell Biol 108:2163-2168, 1989

Lindemann CB: A cAMP-induced increase in the motility of
demembranated bull sperm models. Cell 13:9-18, 1978

Loeffler D, and Ratner S: In vivo localization of lymphocytes
labelled with low concentrations of Hoechst 33342. J
Immunol Methods 119:95-101, 1989

Mann T, and Lutwak-Mann C: Male reproductive function and

89
semen. Berlin: Springer-Verlag, 1981

Markler A, Makler E, Itzkovitz J, and Brandes JM: Factors
affecting sperm motility. IV. Incubation of human semen
with caffeine, kallikrein, and other metabolically active
compounds. Fertil Steril 33:624-630, 1980

McCaffrey'TA, Agarwal LA, and Weksler BB: A rapid fluorometric
DNA assay for the 'measurement of cell density and
proliferation in vitro. In Vitro Cell Dev Biol 24:247-
252, 1988

Meizel S, Pillei MC, Diaz-Perez E, Thomas P: Initiation of the
human sperm acrosome reaction by components of steroids.
In Serono Symposium on Fertilization in Man, Edited by B.
Bavister, J. Cummins, E. Roldan. Plenum Publishers.
NY,1990

Meizel S: Molecules that initiate or help stimulate the
acrosome reaction by their interaction with the mammalian
sperm surface. Am J Anat 174:285-302, 1985

Meizel S: The importance of hydrolytic enzymes to an
exocytotic event, the hamster sperm acrosome reaction.
Biol Rev 59:125-157, 1984

Meizel S, and Turner KO: The effects of products and
inhibitors of arachidonic acid metabolism on the hamster
sperm acrosome reaction. J Exp Zool 231:283-288, 1984

Miller RJ: Multiple calcium channels and neuronal function.
Science N.Y. 235:46-52,1987

Miller RJ, and Freedman SB: Are dihydropyridine binding sites
voltage sensitive calcium channels? Life Sci 34:1025-
1221, 1984

Minta.A, Joseph P, Kao y, and.Tsueb RY: Fluorescent indicators
for cytosolic calcium based on rhodamine and fluorescein
chromophores. J Biol Chem 264:8171-8178, 1989

Mohtashamipur E, Norpoth K, Woelke U, and Hubet P: Effects of
ethylbenzene, toluene, xylene on the induction of
micronuclei in bone marrow polychromatic erythrocytes of
mice. Arch Toxicol 58: 106- 109, 1985

Monks NJ, and Fraser LR: Phosphodiesterase activity of mouse
sperm incubated under conditions that modulate
fertilizing potential in vitro. Gamete Res 18:85-96, 1987

90

Mortimer D, and Camenzind AR: The role of follicular fluid in
inducing the acrosome reaction in human spermatozoa
incubated in vitro. Human Reprod 4:169-174, 1989

Mortimer D, Chorney MJ, Curtis EF, and Trounson AD: Calcium
dependence of human sperm fertilization ability. J Exp
Zool 246:194-201, 1988

Moussa MM: Caffeine and sperm motility. Fertil Steril 39:445-
448 1983

Mrsny RJ and Meizel S: evidence suggesting a role for cyclic
nucleotides in acrosome reactions of hamster sperm in
vitro. J Exp Zool 211:153-157, 1980

Mrsny RJ, Siiteri JE, and Meizel S: Hamster sperm Na‘, K’-
adenosine triphosphatase: increased activity during
capacitation in vitro an relationship to cyclic
nucleotides. Biol Reprod 30:573-584, 1984

Nagae T, Yangimachi R, Srivastava PN, Yanagimachi H:Acrosome
reaction in human spermatozoa. Fertil Steril 45:701, 1986

Nawrot PS, and Staples RE: Embryofetal toxicity and
teratogenicity of benzene and toluene. Teratol 19: 41A,
1979

Neill JM, Olds-Clark P: Incubation of mouse sperm with lactate
delays capacitation and hyperactivation and lowers
fertilization levels in vitro Gamete Res 20:459-473, 1988

Noland TD, and Olson GE: Calcium-induced modification of the
acrosomal matrix in digitonin-permeabilized guinea pig
spermatozoa. Biol Reprod 40:1057-1066, 1989

O'Rand, MG, Herman B, Diguiseppi J, Halme J, Hammond MG, and
Talbert LM: Analysis of deoxyribonucleic acid
distribution in noncleaving oocytes from patients
undergoing in vitro fertilization. Fertil Steril 46:452-
460, 1986

O'Rand. MG,and Fisher’ SJ: Localization of zona pellucida
binding sites on rabbit spermatozoa and induction of the
acrosome reaction by solubilized zonae. Devel Biol
119:551-559, 1987:

Osman RA, Andria ML, Jones AD, and Meizel S: Steroid induced
exocytosis: the human sperm acrosome reaction. Biochem
Biophys Res Commun 160:828-833, 1989

91

Poirier GR, Robinson R, Richardson R, Hinds K, and Clayton D:
Evidence for a binding site on the sperm plasma membrane
which recognizes the murine zona pellucida: A binding
site on the sperm plasme membrane. Gamete Res 14:235-243,
1986 -

Pomeroy KD, Dodds JF, and Seidel GE Jr: Caffeine promotes in
vitro fertilization of mouse ova with 15 minutes. J Exp
Zool 248:207-212, 1988

Rees JM, Ford WCL, and Hull MGR: Effect of caffeine and of
pentoxifylline on the motility and metabolism of human
spermatozoa. J Reprod Fert 90:147-156, 1990

Richards WL, Song MK, Krutzsch H, Evarts, RP, Marsden E, and
Thorgeirsson SS: Measurement of cell proliferation in
microculture using Hoechst 33342 for the rapid
semiautomated microfluorimetric determination of
chromatin DNA. Expl Cell Res 159:235-246, 1985

Robbins RS, and Boatman DE: Regulation of hamster sperm
fertilizing ability by bicarbonate/C02 : Capacitation and
motility , not the acrosome reaction, are primarily
affected. Biol Reprod 38, Suppl. 1, 96, 1988

Ruzich.JV, Arsdalen KV, Gill H, Hypolite J, Wein.AJ, and Levin
RM: Objective assessment of the effect on caffeine on
sperm motility and velocity. Fertil Steril 48:891-893,
1987

Stein DM, and Fraser LR: Cyclic nucleotide metabolism in mouse
epididymal spermatozoa during capacitation in vitro.
Gamete Res 10:283-289, 1984

Sawicki W, and Mystkowska ET: In vivo staining of mouse preim
plantation embryos with Hoechst 33342. Anat Rec 227:359-
362, 1990

Schill WB, Pritsch W, and Preissler G: Effect of caffeine and
kallikrein on cryo-preserved human spermatozoa. Int J
Fert 24:27-32, 1979

Schmid E, Bauchinger M, and Hauf R: Chromosome changes with
time in lymphocytes after occupational exposure to
toluene. Mutat Res 142:37-39, 1985

Schoenfeld C, Amelar RD, and Dubin L: Stimulation of
ejaculated human spermatozoa by caffeine. Fertil Steril
26:158-161, 1975

92

Schoff PK, and Lardy HA: ffects of fluoride and caffeine on
the metabolism and motility of ejaculated bovine
spermatozoa. Biol Reprod 37:1037-1046, 1987

Serres C, Jouannet P, Czyglik F, and David G: Effects of
freezing on spermatozoa motility. In: Human artificial
insemination and semen preservation. (ed.) David G and
Price WS,. Plenum Press, New York. pp147-160, 1980

Shigeta S: Effects of maternal exposure to toluene during
pregnancy on mouse embryos and fetuses. Tokai J Exp Clin
Med 7:265-270, 1982

Shur BD, and Hall NC: Sperm surface galactosyltransferase
acrivities during in vitro capacitaiton. J Cell Biol
95:567-573, 1982

Siegel MS, Hume ME, and Polakoski KL: Biochemistry and
morphology of semen. In Foundations of In Vitro
Fertilization. Fredericks CM, Paulson JD, DeCherney AH
(ed.) Washington: Hemisphere Publishing Corporation. pp3-
40, 1987

Siiteri JE, Gottlieb W, and Meizel S: Human sperm acrosome
reaction-initiating activity associated with the human
cumulus oophorus and mural granulosa cells. J Exp 2001
246:71-80, 1988

Smith PJ, Debenham PG, and Watson JV: A role of DNA
topoisomerases in the active dissociation of DNA minor
groove-ligand complexes. A flow cytometric study of
inhibitor effects. Mutat Res 217:163-172, 1989

Steel RGD, and Torrie -JH: Principles and procedures of
statistics. A biometrical approach. (2nd ed.) McGraw-Hill
Book Company. New York. pp. 186-187, 1980

Stephens DT, Vijayaraghavan S, and Hoskins DD: Computerized
flagellar analysis of the effects of the intracellular
ca” binding compounds Fura-Z, Quin-2 and BAPTA on bovine
sperm flagellar waveform. Biol Reprod 38(Suppl.1), 1989

Steuer B, Breuer B, and Alonso A: Differentiation of EC cells
in vitro by the fluorescent dye Hoechst 33342. Exp Cell
Res 186:149-157, 1990

Stevens RW: Basic spermatozoon anatomy and physiology for the
clinician. In: Acosta AA, Swanson RJ, Ackerman SB, Kruger
TF, Van Zyl-JA, and Menkveld R (ed.):Human spermatozoa in

93

assisted reproduction. Williams & Wilkins, Baltimore, MD,
pp1-23, 1990

Stock CE, Bates R, Idndsay KS, Edmonds DK, and Fraser LR:
Extended exposure to follicular fluid is required for
significant stimulation of the acrosome reaction in human
spermatozoa. J Reprod Fert 86:401-411, 1989

Stock CE, and Fraser LR: Divalent cations, capacitation and
the acrosomal reaction in human spermatozoa. J Reprod
Fert 87:463-478, 1989

Suarez SS, Wolf DP, and Meizel S: Induction of the acrosome
reaction in human spermatozoa by a fraction of human
follicular fluid. Gamete Res 14:107-121, 1986

Sutton C and Calder JA: Solubility of alkylbenzenes in
distilled water and seawater at 25 cc. J Chem Eng Data
20: 320, 1975

Talbot P, and Chacon R: A triple-stain technique for
evaluating normal acrosome reactions of human sperm. J
Exp Zool 215:201-208, 1981

Tash JS, Krinks M, Patel J, Means RL, Klee CB, and Means AR:
Identification , characterization, and functional
correlation of calmodulin-dependent protein phosphatase
in sperm. J Cell Biol 106:1625-1633, 1988

Tash JS, and Means AR: Cyclic adenosine 3',5'-monophos
phate, calcium and protein phosphorylation in flagellar
motility. Biol Reprod 28:75-104, 1983

Tash SJ: Protein phosphorylation: the second messenger signal
transducer of flagellar motility. Cell Motil Cytoskel
14:332-339, 1989

Tash SJ, Hidaka H, and Means AR: Axokinin phosphorylation by
cAMP dependent protein kinase is sufficient for
activation of sperm flagellar motility. J Cell Biol
103:649-655, 1986

Tesarik J: Comparison of acrosome reaction-inducing activities
of human cumulus oophorus, follicular fluid and ionophore
A23187 in human sperm populations of proven fertilizing
ability in vitro. J Reprod Fert 74:383-388, 1985

Tesarik J, Drahorad J, Testart J, and Mendoza C: Acrosin
activation follows its surface exposure and precedes

94

membrane fusion in human sperm acrosome reaction. Devel
110:394-400, 1990

Tesarik J, Pilka L, Drahorad.J, Cechova D, and Veselsky L: The
role of cumulus cell-secreted proteins in the development
of human sperm fertilizing ability: implication in IVF.
Human Reprod 3:129-131, 1988a

Tesarik J, Pilka L, and Travnik P: Zona pellucida resistance
to sperm penetration before the completion of human
oocyte'maturation. J Reprod Fertil 83:487-495, 1988b

Tesarik J, and Kopecny V: Late preovulatory synthesis of
proteoglycans by the human oocyte and cumulus cells and
their secretion into the oocyte-cumulus-complex
extracellular matrices. Histochemistry 85:523-528, 1986

Thomas P, and Meizel S: Phosphatidylinositol 4,5-bisphosphate
hydrolysis in human sperm stimulated with follicular
fluid or progesterone is dependent upon Ca” influx.
Biochem J 264:539, 1989 ‘

Thomas TS, Wilson WL, Reynolds AB, and Oliphant G: Chemical
and physical characterization of rabbit sperm acrosome
stabilizing factor. Biol Reprod 35:691-703, 1986

Traub AI, Earnshaw JC, Brannigan PD, and Thompson W: A
critical assessment of the response to caffeine of human
sperm motility. Fertil Steril 37:436-437, 1982

Varner DD, Ward CR, Storey BT, and Kenney RM: Induction and
characterization of acrosome reaction in equine sperm. Am
J Vet Res 48:1383-1389, 1987

Vijayaraghavan S, Bhattacharyya, and Hoskins DD: Calcium
uptake by bovine epididymal spermatozoa is regulated by
the redox state of the mitochondrial pyridine
nucleotides. Biol Reprod 40:744-751, 1989

Wade MH: Detecting calcium responses in cultured cells
using the visible wavelength Ca“’probe, Fluo-3. Meridian
Instruments Application Note E-2, 1989

Ward CR, and Storey BT: Determination of the time course of

capacitationin mouse spermatozoa using a
Chlortetracycline fluorescence assay. Dev Biol 104:287-
296, 1984

White DR, and Aitken RJ: Relationship between calcium, cyclic

95

AMP, ATP and intracellular pH and the capacity of hamster
spermatozoa to express hyperactivated motility. Gamete
Res 22:163-177, 1989

Whittingham DG: Culture of mouse ova. J Reprod Fert (Suppl.
14): 7-21, 1971

Wilson WL and Oliphant G: Isolation and biochemical
characterization of the subunits of the rabbit sperm
acrosome stabilizing factor. Biol Reprod 37:159-169, 1987

Wolf DE, Hayopian SS, and Ishijama 8: Changes in sperm plasma
membrane lipid diffusibility after hyperactivation during
in vitro capacitation.in.the'mouse. J Cell Biol 102:1372-
1377, 1986

Working PK , and Meizel S: Correlation of increased
intraacrosomal pH with the hamster sperm acrosome.
reaction. J Exp Zool 227:97-107, 1983

Wright SJ, and Longo FJ: Sperm nuclear enlargement in
fertilized hamster eggs is related to meiotic maturation
of the maternal chromatin. J Exp Zool 247:155-165, 1988

Bauchinger M, Schmid E, Dresp J, and Kolin-Grresheim J:
Chromosome aberrations and sister-chromatid exchanges in
toluene-exposed workers. Mutat Res. 113:231-232, 1983

Yanagimachi R: Sperm-egg fusion. In: Current Topics in
Membranes and Transport, Vol 32, pp 3-43, (Eds. Duzgunes
N and Bronner F.) Academic Press, New York. 1988

Yanagimachi R, and Usui N: Calcium dependence of the acrosome
reaction and activation of guinea pig spermatozoa. Exp
Cell Res 89:161-164, 1974

Yanagimachi R: Sperm capacitation and gamete interaction. In:
Cell Biology of Mammalian Egg Manipulation. Eds. Greve
T, Hyttel P, and Weir BJ. J Reprod Fertil Ltd. U.K. pp.
27-33, 1989 -

Yudin AI ,' Gottlieb W, and Meizel S: Ultrastructural studies of
the early events of the human sperm acrosome reaction as
initiated by human follicular fluid Gamete Res 20:11-24,
1988

1.

2.

3.

4.

5.

6.

7.

8.

9.

96
APPENDIX

PUBLICATIONS BY THE AUTHOR

Significance of measuring estradiol and progesterone
receptors in human gestational trophoblastic tumors. Ye
Lian Master Degree Thesis. Zhej iang Medical University.
1986

Effect of oral contraceptives on serum prolactin and its
concentrations following the oral administration of
metoclopramide. Li Xiaofeng, Ye Lian, and Ye Bilu. Wenzhou
Medicine, 11:274, 1987

Induction of follicular growth in the squirrel monkey with
clomiphene citrate. Pierce DL, Ye Lian, Roudebush WE, and
Dukelow WR. Amer J Primat 14:437, 1988

Cell differentiation as determined by micromanipulation of
mouse embryo. Cosby NC, Roudebush WE, Ye Lian and Dukelow
WR. Biol Reprod 38 (Suppl. 1):128, 1988

Timed-mating and gestation. Dukelow WR, Kholkute SD, Ye
Lian, Cosby NC, Roudebush WE, and Bruggemann S. Inter
Primat 8:558, 1988

Analysis of the porcine zona pellucida Mr=55,000 antigen
for contraceptive use. Sacco AG, Yurewicz EC, Subramanian
MG, and Ye Lian. Proceedings of the 4th international
congress of reproductive immunology, Kiel, FRG. 1989

Development of multiple encapsulated mouse embryos in
sodium alginate. Cosby NC, Ye Lian, and Dukelow WR. Biol
Reprod 40(Suppl. 1):110, 1989

Effect of caffeine on mouse epididymal sperm motility and
fertilization ability in vitro. Ye Lian, Cosby NC, and
Dukelow WR. Biol Reprod 42 (Suppl. 1):127, 1990

Squirrel monkey' assessment of 'the contraceptive
effectiveness of the porcine Mr=55,000 antigen (ZP-3).
Dukelow'WR, Ye Lian, Sacco AG, Yurewicz EC, and.Subramanian
MG. Proceedings of 13th congress of international
primatological society, Nagoya and Kyoto, Japan 1990

10.Capacitation and acrosome reaction in squirrel monkey

spermatozoa evaluated by the chlortetracycline fluorescence
assay. Kholkute SD, Ye Lian, Roudebush WE, and Dukelow WR.

97

Amer J Primat 20:115, 1990

11.1mmunocontraception studies in the squirrel monkey using
antigens to porcine zona pellucida(2-P). Dukelow WR, Ye
Lian, Sacco AG, Yurewcz EC, and Subramanian MG. Amer J

Primat 20:187, 1990

12.Effect of a DNA-specific fluorochrome (Hoechst 33258) on
mouse fertilization in vitro. Ye Lian, Hamlin GP, and
Dukelow WR. Biol Reprod 1991 (accepted)

13.Immunological response and ovarian histology of squirrel
monkey (Saimiri sciureus) immunized. with- porcine zona
pellucida ZP3 (Mr=55,000) Macromolecules. Sacco AG,
Yurewicz EC, Subramanian MG, Ye Lian, and Dukelow WR. Amer
J Primat 24:15-28, 1991

14.Toxic effects of toluene on gamete cells and embryos in
vitro. Ye Lian and Dukelow WR. Reprod Toxicol (in

preparation). 1991

Name:
Date of Birth
Place of Birth:

Formal Education:

Degrees Received:

98

VITA

Frank D. Yelian (Ye Lian)
May 5, 1958
Longyou, Zhejiang, China

Zhejiang Medical University (1978-1983)
Hangzhou, Zhejiang, China

Zhejiang Medical University (1983-1986)
Hangzhou, Zhejiang, China

Michigan.State‘University' (1987-Present)
East Lansing, Michigan, U.S.A.

Bachaler of Medicine (M.D) (1983)
Department of Medicine
Zhejiang Medical University

Master of Science (1986)
Department of Obstetrics and Gynecology
Zhejiang Medical University

Doctor of Philosophy (1991)
Department of Zoology and
Endocrine Research Center
Michigan State University

Professional Societies:

Society for the Study of Reproduction
American Association for the
Advancement of Science

American Society of Primatologists