R\l=O^\LE PROMOTING OPALESCENCE AND COLD-PRECIPITATION OF BOAR SEMINAL

THE R\l=O^\LEOF ZINC IN PROMOTING
THE OPALESCENCE AND COLD-PRECIPITATION
OF BOAR SEMINAL PLASMA
J.
C. BOURSNELL
A.R.C. Unit
and
T. K. ROBERTS
of Reproductive Physiology and Biochemistry, Cambridge*
(Received 10th January 1973)
Summary. Zinc alone of the metals present in boar seminal plasma
causes an opalescence in normal samples at room temperature. This
opalescence is increased by cooling to 4\s=deg\C, leading in some cases to a
definite precipitate which redisperses on warming.
The normal opalescence can be minimized by addition of fractional
millimolar amounts of EDTA equivalent to the zinc present. This
treatment also abolishes the precipitate which occurs on cooling.
INTRODUCTION
During studies on boar seminal plasma, it was noted that samples with a
relatively low zinc content were considerably more transparent than those with
a
higher zinc content. It seemed that the relative clarity or opacity could only
partly be accounted for by the protein content which was markedly correlated
with the zinc levels (Boursnell, Baronos, Briggs & Butler, 1972). It was ob¬
served that, in many of those samples of seminal plasma which had a high zinc
concentration, protein precipitated when they were cooled to 4° C. These
precipitates redissolved on warming.
The relationship between the zinc concentration in seminal plasma, the
clarity of the sample and the occurrence of precipitation on cooling were
investigated and the results are reported in this paper.
MATERIAL AND METHODS
Seminal plasma
Seminal plasma was prepared from fresh semen collected from boars housed
at the Animal Research Station. The whole semen was filtered through muslin
to remove the gel. Centrifugation (12,000 g) at 15° C removed the spermatozoa
from the supernatant seminal plasma, which was then stored at —20° C
unless required immediately.
Treatment of seminal plasma with reagents
This was carried out in polythene-capped 15-ml glass vials in which 0-2 ml
concentrated solutions of the requisite concentrations of the test reagents had
*
Address: Animal Research Station, 307 Huntingdon Road, Cambridge CB3 0JO_.
91
92
J.
C. Boursnell and T. . Roberts
been previously dried in vacuo. The concentrations were calculated to give the
desired millimolarity in aliquots (5-0 or 3-5 ml) of the seminal fluid which was
subsequently added. Solution of the reagent in the vial was effected by gentle
swirling and the treated seminal fluid was then poured into a cuvette.
In a few cases as described, solutions of the reagents were added in small
volume to the seminal plasma.
Determination of absorbance
This was carried out using an SP 800 (Pye-Unicam, Cambridge) spectrophotometer. In the early work, samples contained in 10-mm or 2-mm cuvettes
were scanned from 315 to 700
µ. In later work, a fixed wavelength (450 m/i)
was used.
Buffers
A stock 0-05
mixing equal
give pH 7-0.
M-ethylenediaminetetra-acetate (EDTA)
volumes of 0-05
m
solutions of the
tetra-
buffer was prepared by
and disodium salts to
Citrate buffer (0-2 m) was prepared by mixing suitable volumes of the trisodium and disodium hydrogen salts of this concentration and adjusting the pH
if necessary to pH 7-6.
Suitable dilutions of these buffers were made as needed.
RESULTS
The effect ofincreasing the zinc and citrate contents of seminal plasma on the occurrence of a
precipitate on cooling to 4° C: comparison with calcium
Initial qualitative observations were carried out on the occurrence of a haze
five grid, each contain¬
or precipitate in twenty-five tubes, arranged in a five
ml
seminal
treated
in
various
To
1
each tube was added
ing
ways.
plasma
a single 0-1 -ml vol of 0-3 M-NaCl containing
individually
(a) zinc acetate in
five concentrations increasing from 0-26 mM to 1-03 mM along the horizontal
rows and (b) citrate buffer in decreasing molarity down the vertical rows from
009 m to 0-008 M. These solutions were mixed before their addition to the
seminal plasma in order to retain the zinc in solution.
Two identical grids were prepared, one kept at room temperature and the
other at 4° C for 12 hr. Comparison of the two grids showed that the cold tubes
with a greater zinc and lesser citrate content showed marked precipitates and
all tubes showed an opalescence greater than that in the corresponding ones at
room temperature. There was a gradation of opalescence from the low zinchigh citrate portion of the grid to the area of definite precipitate. Furthermore,
the cold precipitate slowly dispersed on warming and the room temperature
grid, after cooling for 12 hr at 4° C, developed an identical precipitation
pattern to that described above. Additional interchanges demonstrated the
temperature reversibility of the precipitation.
An identical pair of grids was prepared, incorporating calcium acetate
(0-57 mM to 2-27 mM) in place of the zinc salt. This ion had no effect upon the
opalescence of the seminal plasma at 4° C and no precipitation occurred.
93
Zinc and boar seminal plasma
The comparative effect of magnesium, calcium, zinc, cadmium and mercury+ + on the
opalescence of seminal plasma
Suitable dry samples of the acetates of these metals were prepared and
dissolved in aliquots of a seminal plasma collection (C, 17/7/72; 0-234 mM-Zn)
yielding the required concentrations. The absorbancies of these treated
samples (Text-fig. 1) reveal striking differences in the actions of these metals.
An identical result was obtained with a fresh unfrozen seminal plasma sample
from another animal (R, 2/11/72; 0-27 mM-Zn).
1-4
1-2
1-0
0-8
0-6
0-4
0-2
0-2
0-4
Millimolanty
0-6
0-8
of added metal ¡on
Text-fig. 1. Effect on absorbance of samples of boar seminal plasma (C, 17/7/72; Zn,
0-234 mM) caused by the addition of fractional millimolarities of Ca (v), Mg ( ), Zn (d),
Cd (O) and Hg++ ( ) acetates.
The effect of various concentrations of ED TA on the clarity of seminal plasma
From the stock EDTA solution, suitable doubling dilutions were prepared so
that the dried salts from 0-2 ml of the solutions gave molarities of 0-125 to
8-0 mM when dissolved in aliquots of a seminal plasma sample (A, 20/10/72;
0-47 mM-Zn). Identical treatment with EDTA was given to 5-0-ml aliquots of
the same seminal plasma to which 0-2-ml quantities of solutions in 0-15 M-NaCl
of zinc acetate had been added, sufficient to give zinc concentrations double
and treble that of the original seminal plasma.
The results (Text-fig. 2) show that, with increasing concentrations of EDTA
up to about 0-5 mM, there was a progressive decrease in absorbance. With
concentrations of EDTA beyond this, no further change occurred unless
further zinc had been added.
J. C.
94
Boursnell and T. . Roberts
^
1-2
1-0
<
0-8
-
0-6
0-4
0125
0-25
2-0
0-50
mM
EDTA
Text-fig. 2. Absorbance of 5-ml samples of boar seminal plasma (A, 30/10/72; 0-47 mia
Zn) treated with (1) ( x ) EDTA; (2) (o), EDTA + 0-2 ml 0-15 M-NaCl; (3) ( ) EDTA+
0-2 ml 0-15 M-NaCl + 0-2 ml Zn acetate in 015 M-NaCl equivalent to 0-47 mM-Zn in 5-ml
seminal plasma sample; (4) ( ) EDTA+0-2 ml 0-15 M-NaCl + 0-4 ml Zn acetate in 0-15
M-NaCl equivalent to 0-92 mM-Zn in 5-ml seminal plasma sample. Total zinc in the
samples was thus doubled in (3) and trebled in (4).
Comparison of absorbance measurements on seminal plasma at 350 and 450 mµ after
sequential additions of EDTA and Zn
Table 1 shows the correspondence of the ratios measured at 350 and 450 m/x
taken from typical scans from 315 to 700 µ of different samples of seminal
plasma treated with increasing quantities of either EDTA or zinc acetate.
Table 1. Ratio of absorbancies at 350 and 450 µ ofboar seminal
plasma samples treated with various quantities of EDTA and zinc
acetate
Seminal plasma
sample with added
millimolarities
Percentage of seminal plasma
value (= 100)
Absorbance
350 mß
450 mß
350 mß
450 mß
0-54
0-44
0-40
0-33
0-26
0-22
(100)
(100)
2-0
1-24
1-02
0-92
0-74
0-54
0-45
74
60
44
36
74
61
48
41
0
0-2
0-4
0-6
0-8
1-0
0-47
0-56
0-65
0-79
0-98
115
0-20
0-23
0-28
0-34
0-41
0-49
(100)
(100)
of:
EDTA
0
0-125
0-25
0-50
10
Zinc
82
119
138
168
208
245
82
115
140
170
205
245
95
Zinc and boar seminal plasma
These ratios are representative over the entire range of wavelengths. Other
metals behaved similarly to zinc except mercury which showed extra absorb¬
ance
due
to
the metal alone below about 420 µ.
reversibility of absorbance changes caused by additions of zinc or ED TA
Samples of a seminal plasma were treated with six successive portions of
dried zinc acetate, each contributing 0-25 mM-zinc, followed by an equal
number of equivalent portions of dried EDTA. Other samples of the same
seminal plasma were treated similarly, but first with EDTA followed by zinc
The
0-8
No. of treatments
Text-fig. 3. Absorbancies of samples of boar seminal plasma (R, 2/11/72; 0-27 mM-Zn)
treated either with six successive portions each donating 0-25 mM-zinc acetate followed by
six successive portions of 0-25 mM-EDTA or in the reverse order of reagents, i.e. EDTA
followed by zinc acetate, ( ) zinc acetate treatments; (x) EDTA treatments: (-)
first treatment; (-) second treatment.
The results (Text-fig. 3) show the virtual reversibility of the absorbance
changes brought about by these treatments.
acetate.
The
effect of cooling seminal plasma treated with various quantities of EDTA
Samples of a seminal plasma (J, 6/9/72; 0-3 mM-Zn) were treated with dried
96
J.
C. Boursnell and T. . Roberts
EDTA to give 0-25, 0-50, 0-75 and 1-0 mM concentrations and the differences
in the absorbancies from untreated seminal plasma were measured. These
samples were cooled to 4e C overnight and the absorbancies were then com¬
pared at this temperature with the previous values obtained at room tempera¬
ture. An aliquot of the original untreated seminal plasma was retained at room
temperature and used at this temperature for re-setting the spectrophotometer.
The result (Text-fig. 4) shows a typical increase in the absorbance of the
EDTA-free seminal plasma caused by cooling, and demonstrates that the
increase in opacity due to cooling becomes less with increasing concentration
of EDTA.
20*-
0-8
-
0-6
Text-fig. 4. Absorbancies of boar seminal plasma samples (J, 6/9/72; 0-39 mM-Zn)
treated with EDTA (0 to 1-0 mM) at room temperature (O) and absorbance values of the
same samples after 12 hr at 4" C ( x ).
The 'titration'
indicator
of seminal plasma zinc by EDTA, using
absorbance
changes
as an
Preliminary tests, in which a solution of EDTA was added in 50-µ1 steps,
indicated that the response to these additions was not immediate, and the end
point, i.e. no further absorbance changes, was only obtained after about 20%
excess EDTA had been added. The time of equilibration of absorbance was
also found to be dependent upon the amount of EDTA added, particularly
where this was less than the equivalent amount of zinc in the seminal plasma
sample.
Titration equivalence was achieved by the addition of one of a graded series
of dried EDTA preparations, each to an aliquot of a seminal plasma sample.
The EDTA preparations were calculated to span the weight equivalent to the
zinc content of the seminal plasma under test. Observation of the absorbance of
these samples at room temperature was delayed for 1 hr, giving adequate time
for attainment of equilibrium. Text-figure 5, a typical result, shows that the
equivalence of EDTA and seminal plasma zinc can be demonstrated by this
means.
97
Zinc and boar seminal plasma
0-9
-
0-2
0-3
0-4
mM
0-5
0-6
0-7
0-8
EDTA
Text-fig. 5. 'Titration' ofzinc (0-295 mM, indicated by arrow) in boar seminal plasma
21/12/72) by EDTA, using absorbance as indicator.
(F,
DISCUSSION
The
of a cold-precipitate shown by many samples of boar seminal plasma
(Boursnell, Nelson & Cole, 1966) has lacked a satisfactory explanation. Nelson
& Boursnell ( 1966) reported that the precipitate obtained by dialysis against a
buffer of pH 8 was enhanced if both zinc and tris were used in the dialysing
fluid. Although it was not stated in this paper, it had been observed that an
enhanced precipitate was encountered when Merthiolate (Eli Lilley & Co,
Basingstoke) was used, up to that time, as a preservative (Boursnell, Johnson &
This organic mercurial (0-02% w/v) may have liberated
Zamora, 1962).
traces of Hg + +.
In consideration of the normally occurring dibasic cations in boar seminal
plasma, the possible specific relationship of zinc to the opalescence of the
seminal plasma was suggested by the contrast in the influence of added zinc
and calcium noted qualitatively at 4° C and at room temperature. In quantita¬
tive extensions of this work, it was shown that, at room temperature, zinc, but
not magnesium or calcium, caused increased opalescence and even precipita¬
tion. The increase in opalescence is caused, even at room temperature, by
strikingly small concentrations of added zinc (0-2 mM) similar to the mean
concentrations of naturally occurring seminal plasma zinc (mean, 0-35 mM;
Boursnell et al., 1972). Cadmium, which is a toxic biological competitor of
zinc (Parizek, 1957), had an influence very close to that of zinc; mercury + + on
the other hand caused greater opalescence than equivalent concentrations of
zinc.
Room temperature opalescence of seminal plasma samples is minimized by
concentrations of EDTA equivalent to that of the zinc present. The opalescence
increase of normal seminal plasma promoted by cooling is lessened by a
cause
J. C.
98
Boursnell and T. . Roberts
reduction in the 'available' zinc caused by less-than-equivalent concentrations
of EDTA. At equivalence, cold opalescence is absent.
The fact that the equilibrium constants (Schwarzenbach, Gut & Anderegg,
1954) for EDTA and Mg++ (8-69), Ca++ (10-70) and Zn++ (16-26) are so
greatly different suggests that, when EDTA equivalent to the zinc content of
the seminal plasma is added, the ligand selectively chelates with the zinc and
competition with the calcium and magnesium normally present (mean con¬
centrations: Ca 1-25 mM; Mg 4-5 mM as given by Mann, 1964) must be very
small. The equilibrium constants given by Schwarzenbach et al. (1954) for
cadmium++ (16-46) is close to that of zinc; for mercury"1"+ (21-80) it is
considerably higher. This suggests that the order of equilibrium constants for
these metals and the protein ligand or ligands is in the same order as for
EDTA.
The end-point caused by additions of EDTA takes some time to attain and
appears also to depend upon the ratio of concentrations of EDTA and zinc: the
lower the ratio, the longer the time taken for equilibrium. This gives some
grounds for the suggestion that an equilibrium constant, if such an idea can be
invoked, for zinc and the proteins involved, must be quite high at the normal
pH of seminal plasma. This might help to explain the slowness of the removal
by dialysis of seminal plasma zinc compared with the rate for citrate (Boursnell,
Briggs, Lavon & Butler, 1973). Because of the far lower stability constant of
zinc for citrate (4-71, given by Li, Lindenbaum & White, 1959) than for
EDTA, the response of the protein-bound zinc to changes in the concentration
of citrate is likely to be even slower than with EDTA. The slowness of equili¬
bration, even with EDTA, may account for the 'hysteresis' effects depicted in
Text-fig.
3.
The nature of the protein or proteins involved with the zinc is being investi¬
gated and preliminary work, to be reported shortly, has shown that seminal
plasma contains a zinc-sensitive protein which can be identified in one region
after gel filtration and which can be resolubilized in EDTA. There is the
possibility that proteins from both epididymal plasma and vesicular secretion
may be involved in this phenomenon in the mixture comprising seminal plasma.
Changes caused by storage of materials at 20° C could also be involved.
The zinc-promoted cold-precipitated colloidal material could associate with,
and might also be deleterious to, the spermatozoa. The method of inhibiting
the cold precipitation occurring in boar seminal plasma by the addition of just
sufficient EDTA to combine with the predetermined zinc in the seminal plasma
of a semen sample could conceivably be an aid to study of the preservation of
boar semen.
—
ACKNOWLEDGMENTS
Our thanks
due to Miss C. A. Reynolds and Mr A. D. Brown for their
invaluable assistance and to Miss W. Butler and Mr H. C. Toates for the
collection of semen samples and slaughterhouse material.
We are grateful for the award of an M.L.C./R.C.V.S. Trust Fund Senior
are
Fellowship in Animal Health to one of us (T.K.R.).
Zinc and boar seminal plasma
99
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components of boar vesicular secretion and seminal plasma. 1. Gel filtration and dialysis studies.
J. Reprod. Fert. 34, 57.
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