होम Journal Reprod Fertil STIMULATION OF RESPIRATION OF RABBIT, HUMAN AND COCK SPERMATOZOA BY LIGHT AND CERTAIN CHEMICALS

STIMULATION OF RESPIRATION OF RABBIT, HUMAN AND COCK SPERMATOZOA BY LIGHT AND CERTAIN CHEMICALS

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DOI:
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Date:
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STIMULATION OF RESPIRATION OF RABBIT, HUMAN
AND COCK SPERMATOZOA BY LIGHT AND CERTAIN
CHEMICALS
WILLIAM L. WILLIAMS

and

CHARLES E. HAMNER

Department ofChemistry, University of Georgia, Athens, Georgia,

U.S.A.

(Received \2th February 1963)
human and cock spermatozoa collected and
washed without exposure to light respired very slowly or not at all.
Brief exposure to white light of less than a minute's duration stimulated
normal rates of respiration. Ubiquinone (UQ, Co-enzyme Q), seminal
plasma, in-utero incubation, and dinitrophenol each markedly stimulated
the respiration of spermatozoa protected from light. Ubiquinone10 was
effective with cock spermatozoa while UQ1 was superior to UQ3 or UQ4
with rabbit spermatozoa. Ubiquinone1 appeared more effective than
UQ3 for human spermatozoa.

Summary. Rabbit,

INTRODUCTION

In the course of experiments on the effect of the female tract on metabolism
of spermatozoa (Hamner & Williams, 1962, 1963), it was observed consistently
that respiration of washed spermatozoa virtually came to a stop after 3 hr in
the Warburg respirometer. In view of the reports that exposure of spermatozoa
to light for several hours inhibited motility and respiration (Norman & Gold¬
berg, 1959; Norman, Goldberg & Porterfield, 1960), experiments were carried
out in which spermatozoa were completely protected from light during collec¬
tion, washing and respiration measurements. A prolongation of the period of
active respiration was the expected result. As already reported briefly (Hamner
& Williams, 1961 ; Williams & Hamner, 1962) the observed result was quite the
opposite. Rabbit, cock and human spermatozoa protected from light failed to
respire or respired at a greatly diminished rate. It appeared that under the
usual conditions for studying sperm respiration, most, if not all, the oxygen
uptake resulted from the effect of laboratory light on spermatozoa. The present
report concerns the time of exposure necessary for maximum stimulation of
oxygen uptake, the effect of i; n-utero incubation, seminal plasma, ubiquinones,
and dinitrophenol on sperm respiration.
MATERIALS AND METHODS

Rabbit spermatozoa were collected with an artificial vagina, and washed, in a
totally dark room and the semen was received into test tubes painted black.
The temperature of the artificial vagina was 44° C and was critical, since lower

235

236

William L. Williams and Charles E. Hamner

temperatures prolonged the collection time and higher temperatures affected
the spermatozoa adversely. The semen was passed into centrifuge tubes painted
black through a double layer of cheese-cloth to remove the plasma clot. The
semen was diluted to twice its original volume with calcium-free Krebs'Ringer phosphate (krp), pH 7-0 (Umbreit, Burris & Stauffer, 1957), and centrifuged at 142 g for 8 min. The supernatant was poured off and the packed
spermatozoa were resuspended to twice the original volume with krp and
centrifuged at 10 g for 2 min to remove the major portion of the contaminating
epithelial cells, leucocytes and amorphous yellow pigments. The supernatant
containing spermatozoa was centrifuged at 142 g for 8 min. The supernatant
solution was removed and the washed spermatozoa were resuspended to the desired
volume in krp solution. Cock and human spermatozoa were washed in the
same way as rabbit spermatozoa except that the slow centrifuging step was
omitted. Tyrode's solution, pH 7·25, was used as the suspending medium
for cock spermatozoa.
For each experiment all ejaculates were collected, washed and pooled in
total darkness. Portions were then removed from the pool and exposed to light.
For exposure to white light, 2 to 4 IO8 washed spermatozoa in 1 ml krp in a
50 ml Pyrex beaker were irradiated for various lengths of time with a 100 Watt
tungsten filament lamp suspended 20 cm above the beaker while the contents
were stirred gently. Except for experiments on time of exposure to light, the
oxygen uptake of spermatozoa exposed to fight was measured in unpainted
flasks. No increase in temperature was observed during light exposure.
Oxygen uptake studies were carried out in a Warburg constant volume
respirometer at 37° C with a total liquid volume in each flask of 2·6 ml con¬
taining 2 to 4xl08 spermatozoa per flask. Two-tenths ml of 20% potassium
hydroxide was placed in the centre well; 2-5 mg streptomycin and 2 mg
glucose were added to each flask. Results are expressed as Zo2 or µ O2 taken
up per 108 sperm cells/hr.
The spermatozoa used in the studies on light exposure for various times were
exposed to the light source for the desired length of time and returned to painted
Warburg flasks. The flasks were painted with a thick coat of flat black paint.
The manometers were painted from the flask to 0·5 in. above and below the
T-joint. Ubiquinones (3 mg) were dissolved in 0-2 ml hot 95 % ethanol,
diluted to 10 ml with hot water obtaining an opalescent but uniform solution,
300 µg/ml. Usually 0-1 ml (30 \xg) were added per Warburg flask. The ubi¬
quinones were of high purity, each free of other members of the series.
For studies on spermatozoa incubated in the rabbit uterus the following
technique was used: Pooled washed rabbit spermatozoa (7-9 IO8 spermatozoa/
ml) protected from light were incubated in vitro for 6 hr in stoppered 13 75
mm painted test tubes in a 37° C water bath. A 1-5 ml sample taken from this
pool was incubated in utero in the rabbit's uterus ligated at the cervix only.
After the 6-hr incubation period 0-5 ml were transferred in the dark to painted
and unpainted Warburg flasks from the in-vitro incubation tube. The ligated
rabbit uterus containing the in-utero incubated spermatozoa was removed from
the rabbit, and the spermatozoa were flushed from the uterus in the dark,
washed and transferred to painted Warburg flasks.

237

Respiration of spermatozoa
RESULTS

The effect on respiration of exposing spermatozoa from three species to white
light is shown in Table 1. The oxygen uptake in the dark was frequently nil.
Spermatozoa protected from light showed rates of oxygen uptake considerably
Table 1
effect of light on oxygen uptake of spermatozoa

No.

Rabbit

Cock

24

11

experiments

Light

exposure

Mean Zoa

No

Yes

No

Yes

No

Yes

1-7

5-4

4-8

90

2-6

9-2

Average difference

between means, d

Sd

Probability

from

i-test

Man

3-7

4-3

6-6

0-395

1-13

1-30

<0001

< 0-005

< 0-005

i-test according to Snedecor (1961).
Light exposures were from 1 to 3 min followed by incubation in un¬
painted flasks. Unexposed spermatozoa were incubated in painted flasks.

less than half that of published values (Lardy & Phillips, 1943; MacLeod,
1943). Stimulation of oxygen uptake by light was highly significant statistically.

60
Time in

120

Warburg (min)

Text-fig. 1. Oxygen uptake of rabbit spermatozoa exposed to white light, 100 watt bulb
20 cm, at times indicated. Sperm count, 2·12 108 per flask, x, no light; o, 5 sec;
D, 180 sec of light.

at

With cock spermatozoa which usually had an appreciable oxygen uptake in
the dark, light exposure doubled the rate of oxygen uptake.
Text-fig. 1 shows the time of light exposure required to stimulate the sub¬
sequent oxygen uptake of rabbit spermatozoa incubated in painted flasks.

William L. Williams and Charles E. Hamner

238

of light gave a readily detectable increase in oxygen uptake. Increasing
light exposure to 5 min did not further increase the rate over that obtained with
3 min exposure.
Text-fig. 2 illustrates the time of light exposure required for maximum
stimulation of the rate of oxygen uptake of cock spermatozoa. Exposure of 30
sec gave essentially maximum rate and
extending the time to 5 min did not
increase it further.
Five

sec

60
Time in

120

Warburg (min)

180

Text-fig. 2. Oxygen uptake of cock spermatozoa exposed to white light, 100 watt bulb
IO8 per flask, x, no light; o, 5 sec;
at 20 cm, at times indicated. Sperm count, 3-5
, 30 sec; D, 180 sec.

Since under natural conditions of mammalian reproduction spermatozoa
not exposed to light, the question came to mind—do spermatozoa normally
respire after deposition in the female tract? Text-fig. 3 shows that incubation
in utero stimulated a higher rate of oxygen uptake than continuous exposure to
fight in transparent Warburg flasks (middle curve).
Spermatozoa incubated in light in vitro for 6 hr lose their ability to take up
oxygen. Since the spermatozoa showing the oxygen uptakes indicated by the
two lower curves in Text-fig. 3 were incubated 6 hr in the dark, it is apparent
that such incubation maintains the ability of spermatozoa to take up oxygen
subsequently in response to light.
The initial studies on light stimulation utilized washed spermatozoa from
which the seminal plasma had been removed as thoroughly and as rapidly
as
possible after ejaculation. As shown in Table 2 spermatozoa in whole semen
respire at a good rate without exposure to light. Exposure of whole semen to
are

239

Respiration of spermatozoa
light in

most instances did not further increase the rate of oxygen uptake. The
of oxygen uptake of spermatozoa in whole semen in both light and dark was
the average greater than that of washed spermatozoa exposed to light.

rate
on

30

Time in

60

Warburg (min)

Text-fig. 3. Comparison of rabbit sperm respiration stimulated by light and incubation in
utero. Light exposure (middle curve) was obtained by ordinary room illumination of un¬
painted Warburg flasks. In-utero incubation was for 6 hr prior to respiration studies.
Sperm count, 2-25 IO8 per flask, , In vitro (dark) ; O, in vitro (light) ; , in utero (dark).

Seminal plasma appears to contain one or more substances that
stimulate the respiration of spermatozoa protected from light.

markedly

Table 2
effect of light and seminal plasma on sperm respiration

Experiment

Species

159
168
201
205

Man
Man
Man
Man

169
170
204

Rabbit
Rabbit
Rabbit

Washed spermatozoa

Whole semen

Dark

Light

Dark

Light

2-4*
1-2
Not done
2-6

12-3
6-4
Not done
6-4

100
11-0
14-9
12-4

90
90
14-8
19-2

3-8
4-0
0-5

7-2

10-0
5-1
2-6

13-6

7-6
3-2

* Values are
Zo2 and are corrected for oxygen uptake by seminal
which averaged 10 µ /hr/ml. Light exposure was from 3 to 5 min.

6-4
3-8

plasma

At this point it appeared desirable to attempt to duplicate with pure sub¬
the stimulating effect on sperm respiration of light, uterine secretions
and seminal plasma reported here and the effect of oviduct fluid reported earlier
(Hamner & Williams, 1963). Because of the report by Page, Smith, Gale,
Polin & Folkers (1961) that the 6-chromanol of hexahydro-UQ_4 prolonged
stances

William L. Williams and Charles E. Hamner
the motility of cock spermatozoa, various ubiquinones were investigated.
Ubiquinoneio, as shown in Table 3, markedly stimulated the respiration of cock
spermatozoa protected from light. Unfortunately the response to room fight was
poor in this experiment. Ubiquinoneio also stimulated respiration of spermatozoa
in the light.
240

Table 3
effect of

ubiquinoneio

Supplement
to flask
None
30 µ8
None
30 ug

UQ.10
UO_10

on cock sperm respiration

Zoz
Illumination
None
None
Room light
Room light

Spermatozoa were exposed

to

1st hr

2ndhr

"irdhr

00
11-0
3-3
10-4

0-5
9-3
3-3
110

0-0
7-9
00
7-6

room

light only during manometric

studies by incubation in unpainted flasks.

UO_i and UQ3 on the respiration of rabbit spermatozoa was
investigated. Using spermatozoa protected from light UQ_i gave a greater rate
of respiration than UQ_3 or controls. Table 4 shows the results of the experiment
comparing the response to the co-enzymes with that of light. It appears that
The effect of

Table 4
effect of ubiquinones on respiration of rabbit
spermatozoa protected from light

Treatment

Kept in dark
Exposed to light for 5 min
Dark+UQ.1, 30 ug per flask
Dark + UO_3, 30 v% per flask

UQ.1 was more effective than light or UQ> In two additional experiments,
UQio was less effective than light in stimulating respiration of rabbit sperm¬
atozoa (Table 5).
The effectiveness of UQ_i and UQ3 in stimulating the respiration of human
spermatozoa was compared in two experiments as shown in Table 6. As with
rabbit spermatozoa, UQ_i appears to be more effective than light or UQ_3
in stimulating respiration. Ubiquinone3 stimulated respiration to about the
same degree as light ; however, the response to light was below average in these
two experiments.
Compounds related to ubiquinones, i.e. tocopherol acetate, tocopherol
phosphate and menadione had no consistent effect on the respiration of rabbit
spermatozoa. In view of the possibility that light exposure may cause hydrogen

241
Respiration of spermatozoa
peroxide formation, catalase and hydrogen peroxide and glucose oxidase were
added separately to Warburg flasks both in light and dark conditions without
significant effect on the respiration of spermatozoa.
The possibility was considered that light may stimulate respiration by un¬
coupling phosphorylation from oxidation. The effect of the known uncoupling
Table 5
ubiquinoneio on respiration of rabbit
spermatozoa protected from light

effect of

Treatment—additions per flask

Zo

Exp.

Kept in dark
Exposed to light for 3-5 min
Dark + UQio, 30 ug per flask

153

Exp.

162

1-0

81
4-8

dinitrophenol (dnp), was compared with light in three experiments.
In the dark dnp caused a greater stimulation of respiration than light in two
of the three experiments (Table 7).
Our colony of bucks yielded spermatozoa which when protected from light
consistently showed a low Zo2 of less than 2 for a series of over thirty experi¬
ments. During a subsequent period in a recent series of twenty-three experi¬
ments the Zo2 values of spermatozoa in the dark increased to 5-0. Exposure to
agent, 2,4

Table 6
effect of light and ubiquinones on respiration
of human spermatozoa

Treatment

Exp.

Kept in dark
Exposed to light for 3 min
Dark + UO_i, 30 ug per flask
Dark + UQ.3, 30 pg per flask

252

31
4-8
8-5
6-0

light increased the average Zo2 to 7-7. It was noted that several of the bucks in
use gave semen heavily contaminated with epithelial cells and yellow amorphous
material. A significant proportion of the spermatozoa was attached to the pig¬
ment particles. After elimination of these bucks and using clean semen,
the average Zoa returned to 1 -5 for the spermatozoa protected from light. Light
exposure increased this to 5-5, hence the respiratory response to light was
restored.

DISCUSSION

In view of the marked effect of light on the respiration of spermatozoa, it
would appear that experimental re-examination of many of the previous results
obtained on respiration of spermatozoa is perhaps necessary. White (1956) has

William L. Williams and Charles E. Hamner

242

certain widely held beliefs concerning sperm metabolism may
be a function of the diluent used. For example, use of a low phosphate medium
with ram spermatozoa results in high respiration and low glycolysis. Increasing
the amount of phosphate gave low respiration and high glycolysis. Uncontrolled
light exposure could lead to similar observations resulting from environmental
conditions rather than the inherent metabolism of the sperm cell.

pointed out that

Table 7

2,4

effect of light and
dinitrophenol on respiration of
rabbit spermatozoa

Treatment

Zoii

of

washed spermatozoa
Dark

Light exposure (3 min)
Dark + dnp*
Light + DNP

*

Exp.

72

2-5
11-5
6-6
12-8

2,4 Dinitrophenol

=

Exp. 261

Exp. 267

0-0
2-9
91
Not done

4-8
6-3
13-4
15-1

4 ug per flask.

The significance of the stimulation of respiration of spermatozoa by light,
by substances in seminal plasma, and by in-utero incubation is not apparent
at the present time. Some evidence for a relationship between stimulation of
respiration by in-utero incubation and by capacitation has been obtained
(Hamner & Williams, 1963). If exposure to ordinary room light and the con¬
sequent stimulation of respiration would capacitate spermatozoa, the pheno¬
menon of capacitation would not have been discovered. It is possible, however,
that irradiation with a specific wavelength of light of specific intensity would
achieve capacitation and the high rate of respiration exhibited by spermatozoa
after incubation in utero.
The effectiveness of ubiquinones in stimulating respiration of spermatozoa
protected from light suggests that these co-enzymes and in-utero incubation
affect the electron transport system rather than the citric acid cycle or glyco¬
lysis. The fact that UQio stimulated respiration in an experiment in which the
light response was poor (Table 3) suggests that light and the ubiquinones
operate by different mechanisms. It is possible at present that seminal plasma
and in-utero incubation stimulated respiration by virtue of their ubiquinone
content.

uncoupler of oxidative phosphorylation, dinitrophenol, stimulated
of
respiration spermatozoa protected from light. This suggests that some of the
agents reported herein that stimulate respiration may be uncouplers.
It should be emphasized that the present report on stimulation of respiration
of spermatozoa by light is not necessarily in conflict with earlier reports on the
detrimental effects of light on spermatozoa. Norman & Goldberg (1959) and
Norman et al. (1960), using bull spermatozoa collected in light in the usual
manner, found that several hours exposure to light markedly reduced the motility
and fertilizing ability of spermatozoa. The wavelength for maximum damage
A known

243
Respiration of spermatozoa
was 4400 A. Collection methods and active wavelength were markedly different
from those used herein, and the exposure times were hours in contrast to the
30 to 60 sec needed for stimulation of respiration.
ACKNOWLEDGMENTS

One of us (C. E. H.) was supported by postdoctoral fellowships from Mark L.
Morris Animal Foundation in 1961 and U.S. Public Health Service, RF No.
12760 in 1962. We are indebted to the Population Council for grant No.
M6317 and to the U.S. Public Health Service for grant No. GM 10034-01.
We are indebted to Dr W. E. Scott of Hoffmann-La Roche, Nutley, New
Jersey, for the ubiquinone compounds.
REFERENCES

Hamner, C. E. & Williams, W. L. (1961) The effect of light on the respiration of spermatozoa. Biochem.
Biophys. Res. Comm. 5, 316.
Hamner, C. E. & Williams, W. L. (1962) Effect of the female tract on sperm respiration. Fed. Proc. 21,
436.
Hamner, C. E. & Williams, W. L. (1963) Effect of the female reproductive tract on sperm metabolism
in the rabbit and fowl. J. Reprod. Fértil. 5, 143.
Lardy, H. A. & Phillips, P. H. (1943) Effects of pH and certain electrolytes on the metabolism of
ejaculated spermatozoa. Amer. J. Physiol. 138, 741.
MacLeod, J. (1943) The role of oxygen in the metabolism and motility of human spermatozoa.
Amer. J. Physiol. 138, 512.
Norman, C. & Goldberg, E. (1959) Effect of light on motility, life span, and respiration of bovine
spermatozoa. Science, 130, 624.
Norman, C, Goldberg, E. & Porterfield, I. D. (1960) Studies on the physiology and biochemistry
of germ cells. II. Action spectrum for the photodynamic sensitization of sperm to light. Anat.
Ree. 137, 384.
Page, A. C, Jr., Smith, M. C, Gale, P. H., Polin, D. & Folkers, K. (1961) Coenzyme Q.. XXVIII.
Activity of the Coenzyme Q, group in sperm motility. Biochem. Biophys. Res. Comm. 6, 141.
Snedecor, G. W. (1961) Statistical methods. 5th edn, p. 50. Iowa State Press, Ames.
Umbreit, W. W., Burris, R. H. & Stauffer, J. F. (1957) Manometric techniques. 3rd edn, p. 149.

Burgess Publishing Co., Minneapolis.
White, I. G. (1956) The effect of some inorganic ions
Congr.

Williams,

Anim.
W. L. &

on

mammalian spermatozoa. Proc. Hlrd int.

Reprod., Cambridge, p. 23.
Hamner, C. E. (1962) Effect of light on sperm metabolism. Fed. Proc. 21, 437.