METABOLIC STUDIES OF TESTICULAR, EPIDIDYMAL, AND EJACULATED
SPERMATOZOA OF THE RAM
By R. N. MURDOCH* and 1. G. WmTE*
[Manuacript received June 29, 1967]
Summary
When testicular spermatozoa were incubated with glucose under aerobic
conditions a much smaller percentage of the substrate could be accounted for as
lactate than was the case with epididymal or ejaculated spermatozoa. Testicular
spermatozoa also displayed a greater Pasteur effect and under anaerobic conditions
all three types of spermatozoa converted glucose almost quantitatively to lactate.
A further difference was that the metabolic activity of epididymal and
testicular spermatozoa increased after storage while that of ejaculated cells decreased.
Fresh samples of ejaculated and epididymal spermatozoa utilized frUctose
slightly more readily than glucose and produced more lactate from fructose. Fructose
was also more effective than glucose in stimulating oxygen uptake by these samples.
Increasing frequency of ejaculation after a period of sexual rest did not
produce any grOBS effects on the metabolism of ram spermatozoa.
Testicular tissue of the ram gave higher yields of labelled carbon dioxide
from D.[1.1 4 C]glucose than from D·[6.1 4CJglucose indicating pentose shunt activity.
The ratio of yields of labelled carbon dioxide was about one for epididymal and
testicular spermatozoa indicating that most of the glucose was oxidized via. the
glycolytic pathway and the Krebs citric acid cycle. Storage at room temperature
for a period of 24 hr or incubation with increasing concentrations of glucose failed
to alter the ratio for testicular spermatozoa.
I. INTRODUOTION
Although a great deal of information is now available on the metabolism of
ejaculated spermatozoa in vitro, fewer studies have been undertaken on epididymal
spermatozoa and even less is known about the metabolism of testicular spermatozoa
(see Mann 1964).
As spermatozoa pass from the testis along the male genital tract they undergo
morphological and physical changes, acquire the potential to move, and increase in
fertilizing capacity (Young arid Simeone 1930; Branton and Salisbury 1947; Lindahl
and Kihlstrom 1952; Bedford 1963). It is not unreasonable, therefore, to expect
that there might also be changes in their metabolism, particularly in passing from
the testis to the cauda epididymis. Although some workers have Claimed substantial
differences in the metabolism of epididymal and ejaculated spermatozoa these have
not always been confirmed (Lardy, Ghosh, and Plaut 1949; White and Wales 1961;
Salisbury et al. 1963; Lodge, Graves, and Salisbury 1964).
• Department of Veterinary Physiology, University of Sydney, N.S.W. 2006.
Auae. J. biol. Sci., 1968,21, 111-21
112
R. N. MURDOCH AND I. G. WHITE
This paper is concerned with a comparison of the metabolism of testicular,
epididymal, and ejaculated spermatozoa of the ram. In particular, the possibility
of the operation of the pentose shunt is explored by measuring the 1400 2 yields from
the metabolism of D_[1_ 140]_ and D-[6- 140]glucose. This technique has been applied to
many tissues" (Bloom and Stetton 1953) and the assumptions and limitations are
discussed by Wood (1955) and Wood, Katz, and Landau (1963). Related studies on
the effect of storage and frequency of ejaculation on the metabolism of ram spermatozoa
are also presented.
II.
MATERIALS AND METHODS
(a) Oollection of Spermatozoa and Tissue
Testes with the scrotum intact were obtained immediately after slaughter, placed in
polythene bags, and packed in ice if it was necessary to transport them to the laboratory. All
processing of tissue and spermatozoa was made within 2-3 hr of collecting the"testes. Testicular
tissue was obtained by cutting open the testes and dissecting out the parenchyma. The tissue
was blotted, weighed, and then blended in 5 volumes of calcium-free Krebs-Ringer phosphate
buffer (Umbreit, Burris, and Stauffer 1959) with a blade homogenizer. Fluid containing testicular
spermatozoa was aspirated with a Pasteur pipette from the testes at the outlet to the caput
epididymides. Epididymal spermatozoa were obtained by making small incisions into the tubules
of the cauda epididymis and squeezing out the fluid. Ejaculated semen was obtained from rams
by electrical stimulation (Blackshaw 1954).
In experiment 4 testicular spermatozoa were collected from the conscious ram by means
of a catheter (Voglmayr, Waites, and SetchellI966).
All sperm samples were examined microscopically, to confirm the presence of fully formed
spermatozoa from the testes, and to check that both ejaculated and epididymal sperm were
highly motile.
(b) Preparation of SuspenBWns of Waahed Oells
The sperm samples and testicular homogenates were washed either once or twice with
caloium-free Krebs-Ringer phosphate by the prooedure of White (1953). After oentrifuging at
400 g for 10 min, the supernatant was removed with a Pasteur pipette and the oells resuspended
in the diluent used for washing. In experiments I, 4, and 6 the washed ejaoulated and epididymal
sperm suspensions were diluted to give the same oonoentration as the testioular suspension.
Sperm oounts were made in duplioate with a haemooytometer.
(c) Incubation of Oells
Radioaotive ohemioals were obtained from the Radioohemioal Centre, Amersham, England
and their radioaotivityoheoked by oounting in a liquid sointillation oounter (Nuolear, Chicago).
Because of the low oell ooncentration of suspensions of testioular spermatozoa, small
Warburg flasks were used in experiments I, 4, 5, and 6 to inorease sensitivity in recording the
oxygen uptake; for these studies O· 5 ml cell suspension was inoubated with 0·5 ml substrate in
6-ml Warburg" flasks containing 0·05 ml C02-free KOH (20 g/IOO ml) in the oentre well. In all
other experiments 2-ml suspensions of epididymal and ejaoulated spermatozoa were inoubated
with 1 ml of substrate in I5-ml Warburg flasks containing 0·2 ml C02-free KOH (20 g/IOO ml)
in the centre well.
With air as gas phase, oxygen uptake was measured over a period of 2-3 hr at 37°C with
a shaking rate of 114 strokes/min. At the end of the inoubation the contents of the oentre well
were removed quantitatively by washing with 3 ml C02-free water. Substrate oxidation was
METABOLIC STUDIES OF RAM SPERMATOZOA
113
determined from the assay of 14002 trapped in the KOH. Aliquots of the cell suspensions prior
to incubation, and of the flask contents after incubation were deproteinized by the addition of
0·5 ml ZnS04.7H 2 0 (5 g/lOO ml) and 0·5 ml 0·3N Ba(OH)2 and glucose, fructose, and lactate
estimated in the neutral filtrate. For experiments requiring anaerobic conditions, single·side-arm
Warburg flasks containing yellow phosphorus were employed and were gassed for 10 min with
nitrogen.
(d) Assay oj Radioactivity
The 1400 2 absorbed in the centre well was precipitated as Ba1400a in the presence of 5%
ammonium chloride. The Ba1400a was collected by filtration on Whatman No. 542 paper as
. described by Amlison and White (1961) and assayed for radioactivity with an end-window
Geiger-Muller tube. The counts were corrected for self-absorption by the method of Hendler
(1959).
(e) Analytical Methods
Glucose was estimated by Somogyi's (1952) modification of the Nelson (1944) colorimetric
method, or by the glucose oxidase method of Huggett and Nixon (1956). Fructose was estimated
by the method of Mann (1948) with an incubation period of 20 min for colour development (White
1959). Lactate was determined by the enzymatic method of Barker and Britton (1957).
(f) Statistical Analysis
The significance of the results has been assessed by analyses of variance. In factorial
experiments all main effects and their first-order interactions were isolated and tested for
significance.
In experiment 5 the significance of difference between treatments has been assessed by a
t-test using the interaction mean square from the analysis of variance to determine the standard
error of the difference between means.
III.
RESULTS
(a) Experiment 1,' Comparison of Metabolism of Ejaculated, Epididymal, and
Testicular Spermatozoa
Four rams were electro-ejaculated and immediately slaughtered. While
epididymal and testicular sperm samples were being collected (about 2 hr due to the
tedious procedure involved in collecting the testicular spermatozoa) the ejaculated
semen was slowly cooled to 15°0 to retard metabolic activity. The spermatozoa were
then washed and their ability to metabolize [U- 140]glucose under aerobic and anaerobic
conditions studied (Table 1). Epididymal spermatozoa took up more oxygen and
oxidized more glucose than ejaculated or testicular spermatozoa. Under aerobic
conditions ejaculated, epididymal, and testicular spermatozoa utilized similar amounts
of glucose and over half accumulated as lactate in the case of ejaculated and epididymal
spermatozoa. With testicular sperm a smaller percentage (37%) of the glucose accumulated as lactate. Anaerobic conditions enhanced glucose utilization by testicular
spermatozoa but not by epididymal or ejaculated spermatozoa. In each case there
was a greater accumulation oflactate under anaerobic conditions and it accounted for
almost all ofthe glucose utilized.
R. N. MURDOCH AND I. G. WHITE
114
(b) Experiment 2: Effect of Storage on Metabolism of Ejaculated and Epididymal
Spermatozoa
Since White and Wales (1961) found that fructose increased the oxygen uptake
of freshly prepared suspensions of ejaculated and epididymal spermatozoa to the
same extent, the next experiment was designed to see if the period of storage (2 hr at
TABLE
AEROBIC
AND
ANAEROBIC
METABOLISM
OF
1
[U-14C]GLUCOSE
BY
ONCE-WASHED
EJACULATED,
EPIDIDYMAL, AND TESTICULAR RAM SPERMATOZOA
Values are calculated per lOB spermatozoa and represent the means of four replicates. The number
of spermatozoa per flask was 0·9-1· 5 X lOB. The amount of glucose per flask was 2 ,anoles (100 nc).
Duration of experiments 3 hr
Aerobic Metabolism
Anaerobic Metabolism
(pl)
Glucose
Oxidized
(",moles)
Glucose
Utilized
(,anoles)
Lactate
Produced
(p.moles)
Glucose
Utilized
(,anoles)
Lactate
Produced
(p.moles)
Ejaculated
31·3
0·11
1·27
1·31
1·45
3·11
Epididymal
91·9
0·35
1·58
1·73
1·56
2·91
Testicular
49·0
0·12
1·63
1·20
2·59
4·46
Spermatozoa
-
Oxygen
Uptake
Analysis of Variance
Source of Variation
Degrees
of
Freedom
Spermatozoa (A)
(i) Ejaculated v. epididymal
(ii) Ejaculated v. testicular
Gas phase (B)
Replicates (0)
First·order interactions
Second·order interaction
-
*P<0·05.
** P<O·Ol.
Oxygen
Uptake
Glucose
Oxidized
Lactate
Produced
Glucose
Utilized
2
34·7**
2·9
1
1
1
3
11
2
6
3
AxB
AxO
BxO
Variance Ratios
33·5**
0·1
1·5
1·0
211t
0·0033t
6
t
0·2
6·1'"
104·0·'"
2·3
9·0'"
6·6'"
1·4
0·25t
0·5
6·8'"
2·6
0·5
1·7
2·8
0·6
0·332t
Residual mean squares.
15°0) would account for the difference in the response to glucose observed in the first
experiment. Fructose was included in this comparison in case the discrepancy was
due to a difference between sugars. Three rams were electro-ejaculated and
immediately slaughtered to obtain epididymal sperm. Table 2 shows that the oxygen
uptake of fresh ejaculated spermatozoa was slightly greater than that of fresh
epididymal spermatozoa when either glucose or fructose was present. After 2 hr at
15°0, however, the oxygen uptake of ejaculated spermatozoa decreased greatly while
115
METABOLIC STUDIES OF RAM SPERMATOZOA
the oxygen uptake of epididymal spermatozoa, if anything, increased. A similar
trend occurred in all metabolic parameters but only the oxygen uptake and glucose
utilization reached statistical significance. Fructose and glucose were equally
TABLE
2
METABOLISM OF GLUCOSE AND FRUCTOSE BY TWICE·WASHED EJACULATED AND EPIDIDYMAL RAM
SPERMATOZOA BEFORE AND AFTER STORAGE AT
15°0
FOR
2
HR
Values are calculated per 108 spermatozoa and represent the means of three replicates. The number
of spermatozoa per flask was 5·4-8·2 X 108 • The amount of substrate per flask was 18 1£Il101es.
Duration of experiment 2 hr
After Storage
Before Storage
Spermatozoa
Oxygen
Uptake
Hexose
Utilized
Lactate
Produced
Oxygen
Uptake
Hexose
Utilized
Lactate
Produced
(~l)
(~moles)
(~moles)
(~l)
(~moles)
(~moles)
Ejaculated
Epididymal
56·1
50·0
1·91
1·67
38·6
58·9
1·62
1·70
1·39
2·04
Ejaculated
Epididymal
55·1
50·9
1·63
1·48
41·6
58·2
1·31
1·73
1·42
1·76
Substrate Fructose
2·28
2·00
Substrate Glucose
1·72
1·59
Analysis of Variance
Source of Variation
Storage (A)
Substrate (B)
Spermatozoa (0)
Replicates (D)
Degrees
of
Freedom
Variance Ratios
Oxygen
Uptake
Lactate
Produced
1
1
1
2
3·8
0·1
12·2**
0·5
1·2
1·7
0·4
1·4
1
1
1
2
2
2
9
7
0·1
35·5**
0·1
11·4**
0·2
4·9
23t
0·6
2·3
0·1
1·2
0·3
2·8
0·32t
Glucose
Utilized
Fructose
Utilized
0·03
0·41
0·42
0·08
0·16
0·20
0·141t
0·123t
Interactions
AxB
AxO
BxO
AXD
BxD
OxD
Residual (error)
**P<O·Ol.
---_._-
t Error mean squares.
effective in stimulating the oxygen uptake of ejaculated and epididymal spermatozoa
irrespective of whether they were fresh or stored. Fresh samples of both ejaculated
and epididymal spermatozoa did, however, tend to utilize fructose a little more
readily than glucose and to produce a slightly greater yield of lactate. The difference
was less obvious in the stored samples.
R. N. MURDOCH AND I. G. WHITE
116
(c) Experiment 3: Effect of Frequency of Ejaculation on the Metabolism of the
Spermatozoa
The rams used previously had not been electro-ejaculated prior to the
experiments and it was possible that their semen might differ from that of regular
TABLE
3
EFFECT OF INCREASING FREQUENCY OF EJACULATION ON THE METABOLISM OF FRUCTOSE AND GLUCOSE
BY TWICE-WASHED RAM SPERMATOZOA AFTER SEXUAL REST FOR A MONTH
Values are calculated per 10 8 spermatozoa and represent the means for six rams. The number of
spermatozoa per flask was 3 -0-9·0 X 108• The amount of substrate per flask was 18 f.illloies.
Duration of experiment 3 hr
Substrate
Oxygen
Uptake
(/Ll)
Hexose
Utilized
(/Lmoles)
Lactate
Produced
(/Lmoles)
0
Fructose
Glucose
64·5
61·5
2·23
1·50
3·32
2·62
4
Fructose
Glucose
54·5
53·6
1·92
1·59
3·05
2·53
6
Fructose
Glucose
56·3
53·2
2·12
1·58
3·36
3·12
7
Fructose
Glucose
64·1
61·8
2·39
1·90
3·89
3·01
Day of
EjaCUlation
Analysis of Variance
Source of
Variation
Substrates (A)
Day ejaculated (B)
Linear
Quadratic
Cubic
Rams (0)
Interactions
AxB
AXO
BxO
Residual (error)
* P<0·05.
Degrees
of
Freedom
Variance Ratios
Oxygen
Uptake
Fructose
Utilized
Glucose
Utilized
10'7**
1
16'7**
3
1
1
1
5
38
3
5
15
15
** P<O·Ol.
0·03
145'6**
0·5
316'1**
0·3
2·6
40'7**
6'07t
t
Lactate
Produced
0·6
2·2
0·3
2·5
0·226t
3·4
0·7
0·5
7'1*
2·3
0·1
4'7**
16·1**
o'121t
0·5
2·3
2·3
0· 281t
Error mean squares.
donor animals in metabolic activity and thus alter the relationship between ejaculated
and epididymal spermatozoa. To check this point six rams were rested sexually for
------------------------------------------------------------------------METABOLIC STUDIES OF RAM SPERMATOZOA
117
1 month and then ejaculated with increasing frequency during 1 week. The results
and summary of the analysis of variance are presented in Table 3. Fructose was
slightly more effective than glucose in stimulating the oxygen uptake of the
spermatozoa and tended to be utilized to a greater extent than glucose and gave rise
to greater amounts of lactate. These observations were consistent irrespective of the
day of ejaculation. The oxygen uptake of the spermatozoa differed significantly
between days of ejaculation. Thus spermatozoa collected on day 0 had a slightly
higher oxygen uptake than spermatozoa collected on days 4 and 6; however, the
oxygen uptake of the spermatozoa collected on day 7 was as high as that collected on
the first day.
(d) Experiment 4: Comparison of Pentose Shunt Activity in Epididymal Sperm,
Testicular Tissue, and Testicular Sperm
The possible existence of the pentose cycle in epididymal sperm and testicular
tissue was examined by comparing the yields of labelled carbon dioxide obtained by
incubating with D-[I-14C]glucose and with D-[6- 14C]glucose as substrates. When
epididymal spermatozoa were incubated with the specifically labelled sugars the ratio
of labelled carbon dioxide from D-[6- 14C]glucose to that from D-[I-14C]glucose was
about 1 (Table 4). The ratio however was much lower for testicular tissue (0·44).
TABLE 4
YIELD OF 14C0 2 AFTER INCUBATING ONCE-WASHED EPIDIDYMAL SPERMATOZOA, TESTICULAR TISSUE,
AND TESTICULAR SPERMATOZOA WlTH [6_ 14C]- AND [l_14C]GLUCOSE
Values represent the means or means ± S.E. of four replicates calculated after 3 hr incubation.
The amount of labelled glucose added to epididymal spermatozoa and testicular tissue was
4·5 p.moles (100 nc) and to testicular spermatozoa 9 p.moles (500 nc). The number of
epididymal and testicular spermatozoa per flask was 1· 3-2·5 X 10 8
Yield of 14C02 (nc)
Sample
14C02 Ratio·
[6- 14C]Glucose
[1_14C]Glucose
Epididymal spermatozoa
6·75
6·72
0·99±0·10
Testicular tissue
0·09
0·22
0·44±0·14
Testicular spermatozoa
5·09
5·11
1·00±0·03
-
* 14C0 2
from [6- 14C]glucose: 14C02 from [1_14C]glucose.
Although a preliminary study using testicular spermatozoa collected from
abattoir material suggested that the ratio for these cells might also be less than 1,
further experiments using greater numbers of testicular spermatozoa, collected free of
testicular parenchyma from the conscious animal, showed that the ratio of labelled
carbon dioxide from the two sugars was 1 (Table 4). The mean oxygen uptake of the
testicular sperm was 46·3±6·7 ILl/lOS sperm and only 17% of the glucose utilized
could be accounted for as lactate.
R. N. MURDOCH AND I. G. WHITE
118
(e) Experiment 5: Effect oj Storage on M etaholism oj Testicular Spermatozoa
Since the testicular spermatozoa in the previous experiment were collected from
the conscious ram over a period of 18-24 hr with a catheter, the next experiment was
designed to check if pentose shunt activity might possibly decline with increasing age
of the testicular spermatozoa. Spermatozoa collected from abattoir testes were
incubated with n-[1- 140]- and n-[6- 14C]glucose immediately after collection and
again after 24 hr at room temperature (20°0). Table 5 shows that all metabolic
parameters, with the exception of the glucose utilization, were significantly enhanced
after 24 hr. The ratio oflabelled carbon dioxide from the two sugars was again about
1 and was unaffected by the period of storage. Mter storage, however, more of the
glucose utilized could be accounted for as lactate.
TABLE 5
METABOLISM OF [6_ 14C]- AND [1_14C]GLUCOSE BY ONCE-WASHED RAM TESTICULAB SPERMATOZOA
AFTER STANDING AT ROOM TEMPERATURE FOR 24 HR
Ram testicular spermatozoa were incubated for 3 hr in Warburg flasks at 37°C immediately after
collection and after storing in stoppered test tubes for 24 hr at room temperatures. Values are
the means of four replicates and are calculated per 108 spermatozoa. The amount of glucose
per flask was 9 pmoles (500 nc). The number of spermatozoa per flask was 0·6-1·0 X 10 8
Oxygen
Uptake
Spermatozoa
Fresh
Stored
Standard error
(1'1)
Glucose
Utilized
(pmoles)
Lactate
Produced
(p.moles)
Yield of 14C02 (nc)
[6- 14C]Glucose
[ 1-14O]Glucose
41·8
0·93
0·38
3·14
3·68
0·86
53'4*
0·94
0'52*
6·43**
6,99*
0·94
2·51
0·13
0·042
0·49
1·00
0·075
14002 Ratiot
* Significantly different from value for fresh spermatozoa, P < 0·05 .
•• Highly significantly different from value for fresh spermatozoa, P < 0·01.
t 14C02 from [6-1 4C]glucose : 14C02 from [1-1 4C]glucose.
(f) Experiment 6: Effect oj Glucose Concentration on the M etaiJolism oj Testicular and
Ejaculated Sperm
Wright and Bruhmuller (1964) have shown that the concentration of labelled
glucose may determine the ratio of radioactive 1400 2 obtained from n-[1-140]- and
n-[6-1 40]glucose when incubated with the slime mould. Increasing the amount of
glucose over a range from 2·25 to 18·0 p.moles per flask, however, had no significant
effects on the metabolism of three samples of washed testicular or four samples of
washed ejaculated spermatozoa when incubated aerobically under conditions
similar to these of the fresh samples in Table 5. In the case of testicular spermatozoa
19% of the glucose utilized could be accounted for as lactate compared with 55%
for ejaculated spermatozoa. The ratio of 1400 2 from [6- 140]glucose to 1400 2 from
[1_140]glucose was always near unity (mean 0·89) for testicular spermatozoa. The
mean oxygen uptake over 3 hr was 33·6 and 38·9 p.1j108 spermatozoa for testicular
and ejaculated samples respectively.
._---------,,---------METABOLIC STUDIES OF RAM SPERMATOZOA
IV.
119
DISCUSSION
The present study demonstrates only a slight Pasteur effect (i.e. increase in
glucose breakdown and lactic acid production under anaerobic conditions) in ejaculated
and epididymal spermatozoa which is in agreement with the results of White and
Wales (1961) and Voglmayr, Waites, and Setchell (1966), but not with those of
Lardy, Ghosh, and Plaut (1949) who used bull spermatozoa. The latter authors claim
that, on the addition of sugar, epididymal spermatozoa of the bull produce lactic
acid much more rapidly under anaerobic than under aerobic conditions, whereas
with ejaculated spermatozoa the rate of glycolysis is not much higher than in the
presence of oxygen. Lardy, Ghosh, and Plaut (1949) suggest that this difference is
due to the presence in bull spermatozoa of a metabolic regulator which occurs in the
epididymal spermatozoa in bound form, but is released in an active form after
ejaculation. Salisbury et al. (1963) have, however, been unable to find evidence of
such a regulator in the bull and the present results, like those previously obtained
with an epididymal fistula (White and Wales 1961), raise doubt as to the existence of
a metabolic regulator in ram spermatozoa. However, we have found a far greater
Pasteur effect in testicular spermatozoa of the ram than in the ejaculated or epididymal spermatozoa and if such a metabolic regulator exists in the ram it may be bound
in the testicular spermatozoa and activated on reaching the cauda epididymidis.
Voglmayr, Waites, and Setchell (1966) have also reported evidence of a greater
Pasteur effect in testicular spermatozoa.
The observation that storage for 2 hr at 15°0 causes a slight increase in the
oxygen uptake of epididymal ram spermatozoa and a marked decrease in that of
ejaculated spermatozoa reconciles the results in the first experiment with those of
White and Wales (1961) who found that the oxygen uptake of washed suspensions
of fresh ejaculated and epididymal spermatozoa were similar in the presence of
fructose. Lardy, Hansen, and Phillips (1945) also report that bull spermatozoa
removed from the epididymis after a period of storage in the refrigerator have a
distinctly higher oxygen uptake. The decline in the metabolic activity of ejaculated
ram spermatozoa on storage in vitro is most probably due to the accumulation of
lactic acid from fructose in the seminal plasma. It is of interest that fresh ejaculated
spermatozoa utilized fructose slightly more readily than glucose and produced more
lactate from the ketose, which was also more effective in stimulating oxygen uptake.
Mann and Lutwak.Mann (1948) have reported that lactic acid production by ram
spermatozoa was similar with either glucose or fructose as substrate. However, in
the presence of both sugars, washed spermatozoa of ram, bull, human, and boar use
glucose preferentially (Mann 1951; Van Tienhoven et al. 1952; Freund and MacLeod
1958).
Increasing frequency of ejaculation after a period of sexual rest did not produce
any gross effects on the metabolic activity of ram spermatozoa although there was
a slight depression of oxygen uptake 4-6 days after the commencement of the
experiment.
Since epididymal and testicular spermatozoa produced as much labelled
carbon dioxide from D-[6- 140]glucose as from n-[1. 140]glucose, most of the sugar is
metabolized via the Embden-Meyerhof pathway. These results confirm those of
Voglmayr et al. (1967), who used testicular spermatozoa collected from the conscious
120
R. N. MURDOCH AND 1. G. WHITE
ram, but disagree with those of Wu et al. (1959), who found evidence of pentose
shunt activity in testicular spermatozoa collected from incisions into the testicular
parenchyma. With testicular tissue, however, the yields of labelled carbon dioxide
from D-[1-140]glucose are much higher than from D-[6-1 40]glucose suggesting that
at least part of the sugar passes through the pentose shunt.
The testicular spermatozoa used in the present study were collected from the
junction of the testes and caput epididymides and no incision was made into the
testicular parenchyma during the course of collection .. Excessive contamination of
the sperm suspensions with testicular tissue would tend to give a spurious answer in
view of the considerable shunt activity in testicular parenchyma and this may explain
the results of Wu et al. (1959). It may be noted in this connection that glucose6-phosphate can be formed and dehydrogenated by testicular extracts (Sharma and
Weinhouse 1962; Blackshaw 1964).
The reason for the increase in the oxidative metabolism and lactate production
of testicular spermatozoa after standing at room temperature for 24 hr is not apparent.
Voglmayr, Waites, and Setchell (1966) have shown that testicular spermatozoa
become more active in an alkaline medium or after storage at 3°0 overnight in
testicular fluid. In the present study the pH of the testicular fluid rose from 7·2 to
8·4 after 24 hr. The increase in metabolic activity of testicular spermatozoa may
therefore be associated with the initiation of motility which in turn may be facilitated
by a rise in pH. It is of interest that, after standing for a period of time, more of the
glucose utilized can be accounted for as lactate.
Glucose uptake appears to be a predominantly active process in both ejaculated
and testicular ram spermatozoa since increasing concentrations of glucose did not
significantly affect the metabolic activity of the cells.
It is concluded that when testicular spermatozoa are incubated with glucose
under aerobic conditions a much smaller percentage of substrate can be accounted
for as lactate than is the case with epididymal or ejaculated spermatozoa. Testicular
spermatozoa also display a greater Pasteur effect and under anaerobic conditions all
three types of spermatozoa convert glucose almost quantitatively to lactate. A
further difference is that the metabolic activity of epididymal and testicular spermatozoa increases after storage while that of ejaculated cells decreases. The latter
makes it difficult to obtain valid comparisons of the metabolism of the three types of
spermatozoa since it is almost impossible to avoid extensive lapses of time before
the start of manometric experiments when the spermatozoa are collected from the
same animal. However, comparisons of responses to different treatments should be
valid providing experiments begin as soon as possible after collection.
V.
ACKNOWLEDGMENTS
This work has been aided by a grant from the Australian Wool Board. One of
us (R.N.M.) was supported by an F. H. Loxton Post-Graduate Studentship. The
authors are indebted to Professor O. W. Emmens for his interest and advice and to
Mr. Bloxson, Riverstone Meat Works, for his generous donation of experimental
METABOLIC STUDIES OF RAM SPERMATOZOA
121
animals. Testicular spermatozoa collected with a catheter from the conscious ram
were generously donated by Mr. J. K. Voglmayr, Division of Animal Physiology,
CSIRO, Prospect, N.S.W.
VI.
REFERENCES
ANNISON, E. F., and WHITE, R. R. (1961).-Biochem. J. 80, 162.
BARKER, JUNE N., and BRITTON, H. G. (1957).-J. Physiol., Lond. 138, 3P.
BEDFORD, J. M. (1963).-J. Reprod. Fert. 5, 169.
BLACKSHAW, A. W. (1954).-Aust. Vet. J. 30, 249.
BLACKSHAW, A. W. (1964).-Aust. J. biol. Sci. 17, 489.
BLOOM, B., and STETTON, D. (1953).-J. Am. chem. Soc. 75, 5446.
BRANTON, C., and SALISBURY, G. W. (1947).-J. Anim. Sci. 6, 154.
FREUND, M., and MACLEOD, J. (1958).-J. appl. Physiol. 13, 506.
HENDLER, R. W. (1959).-Science, N.Y. 130, 772.
HUGGETT, A. ST. G., and NIXON, D. A. (1956).-Biochem. J. 66, 12P.
LARDY, H. A., GHOSH, D., and PLAUT, E. W. E. (1949).-Science, N.Y. 109,365.
LARDY, H. A., HANSEN, R. G., and PHILLIPS, P. H. (1945).-Archs Bioohem. 6, 41.
LINDAHL, P. E., and KIHLSTROM, J. E. (1952).-J. Dairy Sci. 35, 393.
LODGE, J. R., GRAVES, C. N., and SALISBURY, G. W. (1964).-Proc. 5th Int. Congr. Anim. Reprod.
Trento. Vol. 2. p. 152.
MANN, T. (1948).-J. agric. Sci. 38, 323.
MANN, T. (1951).-Biochem. Soc. Symp. 7, 11.
MANN, T. (1964).-"The Biochemistry of Semen and of the Male Reproductive Tract." (Methuen
& Co. Ltd.: London.)
MANN, T., and LUTWAK.MANN, C. (1948).-Biochem. J. 43, 266.
NELSON, N. (1944).-J. biol. Ohem. 153, 375.
SALISBURY, G. W., GRAVES, C. N., NAKABAYASHI, N. T., and CRAGLE, R. G. (1963).-J. Reprod.
Fert. 6, 341.
SHARMA, C., and WEINHOUSE, S. (1962).-Proc. Soc. expo Biol. Med. 110, 522.
SOMOGYI, M. (1952).-J. biol. Ohem. 195, 19.
UMBREIT, W. W., BURRIS, R. H., and STAUFFER, J. F. (1959).-"Manometric Techniques in Tissue
Metabolism." (Burgess Publ. Co.: Minneapolis.)
VAN TIENHOVEN, A., SALISBURY, G. W., VANDEMARK, N. L., and HANSEN, R. G. (1952).-J. Dairy
Sci. 35, 637.
VOGLMAYR, J. K., SCOTT, T. W., SETCHELL, B. P., and WAITES, G. M. H. (1967).-J. Reprod. Fert.
(In press.)
VOGLMAYR, J. K., WAITES, G. M. H., and SETCHELL, B. P. (1966).-Nature, Lond. 210,861.
WHITE, 1. G. (1953).-J. expo Biol. 30, 200.
WHITE, 1. G. (1959).-Aust. J. expo Biol. Med. Sci. 37, 441.
WHITE,1. G., and WALES, R. G. (1961).-J. Reprod. Fert. 2, 225.
WOOD, H. G. (1955).-Physiol. Revs. 35, 841.
WOOD, H. G., KATZ, J., and LANDAU, B. R. (1963).-Biochem. Z. 338, 809.
WRIGHT, B. E., and BRUHMULLER, M. (1964).-Biochim. biophys. Acta 82, 203.
Wu, S. H., McKENZIE, F. F., FANG, S. C., and BUTTS, J. S. (1959).-J. Dairy Sci. 42, 1l0.
YOUNG, W. C., and SIMEONE, F. A. (1930).-Proc. Soc. expo Biol. Med. 27, 838.