Survival of Human Spermatozoa in Different Fractions of Split Ejaculate*

Survival of Human Spermatozoa in Different Fractions of Split Ejaculate*

Vol. 24, No.7, July 1973 FERTILITY AND STERILITY Printed in U.S.A. Copyright © 1973 by The Williams & Wilkins Co. SURVIVAL OF HUMAN SPERMATOZOA IN...

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Vol. 24, No.7, July 1973

FERTILITY AND STERILITY

Printed in U.S.A.

Copyright © 1973 by The Williams & Wilkins Co.

SURVIVAL OF HUMAN SPERMATOZOA IN DIFFERENT FRACTIONS OF SPLIT EJACULATE* CH. LIND HOLMER, M.D.

Reproductive Physiology Unit, Department of Physiology, Karolinska Institutet, 104 01 Stockholm, Sweden

In a previous studyl it was shown that spermatozoa had a better motility and vitality in the first parts of split ejaculates than in the later parts. The sperm morphology was the same in all fractions. The first half of the ejaculate consists mainly of prostatic secretion and the other half mainly of fluid from the seminal vesicles. The differences noted in motility and vitality could therefore be due to either intra- or extracellular factors. The present study was designed to examine the effect of the secretions from the prostate and seminal vesicles, respectively, on the survival of human spermatozoa.

min. and washing the cells twice in a zinc and magnesium-free buffer solution (NaCI 0.123 M, KCI 0.005 M, HCI 0.024 M, Tris 0.037 M, human albumin 4%, fructose 1 mg./mI., pH 7.75). The survival during 4 hr. was studied under aerobic conditions in Warburg flasks (volume 6 mI., 80 shakings per min.). Sperm density and motility were determined with standard methods 5 and sperm vitality with a supravital staining technic. 8 The seminal plasma was analyzed for fructose,7 zinc,8 and magnesium. 9 For determination of zinc and magnesium in the spermatozoa the cells were first separated from the seminal plasma by centrifugation MATERIALS AND METHODS through a discontinuate density gradient Split ejaculates were received from eight and then dissolved in a swamping solution healthy volunteers with the technic de- containing NaOH and strontium.· scribed by Lundquist 2 and Eliasson. 3 A The osmolarity of the seminal plasma continence time of 2-3 days was required. was determined at 37° C. with a Vapor The ejaculates were kept at room tempera- Pressure Osmometer (Hewlett-Packard, ture until fully liquefied (usually 20 min.). 302 B) using NaCI as calibration solutions; Most ejaculates were delivered in six frac- pH was determined with glass electrodes. tions, which were pooled into two fractions All statistical calculations were perof about equal volume, one fraction con- formed according to Richterich. 10 taining the first ("Fraction 1") and the RESULTS other ("Fraction 2") the second half of the ejaculate. Benzylpenicillin (500 I.U'/mI.) No significant difference in osmolarity and streptomycin (0.2 mg./mI.) were added (mean = 330 mM), pH (mean = 8.10), or to prevent bacterial growth. Fructose (1 fructose concentration was observed bemg./mI. of plasma) was added to the first tween the two fractions (fructose had been fraction. added to Fraction 1). Washed spermatozoa were obtained by Survival of Untreated Spermatozoa in separating the cells from the seminal Their Own Plasma. Fractions 1 and 2, plasma by centrifugation at 2800 g for 20 respectively, were mixed for 20 min. The incubation and first determination of Received January 15, 1973. sperm quality were therefore not com* Supported by grants to Dr. R. Eliasson from the Swedish Medical Research Council (14X-538) and The menced until about 40 min. after the Population Council, New York. ejaculation. The spermatozoa in this type 521

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of experiment were unevenly distributed, with the highest sperm density in Fraction 1. The results are illustrated in Fig. 1. At the beginning of the incubation period the sperm motility and vitality were significantly higher in Fraction 1 than in Fraction 2 (p < 0.02 and p < 0.05, respectively). The percentages of motile and live spermatozoa were significantly lower in Fraction 2 after 2 (p < 0.05), 3 (p < 0.05), and 4 (p < 0.05) hr. of incubation, respectively. The progressive motility score was significantly lower after 1 (p < 0.025), 2 (p < 0.05), and 3 (p < 0.02) hr., but at the end of the incubation (4 hr.) the progressive motility was equally poor in the two fractions.

Survival of Unwashed Spermatozoa. Spermatozoa from semen specimens obtained from healthy volunteers were divided into two equal parts and then suspended in Fractions 1 and 2, respectively. No differences in motility and vitality were observed at any time of the incubation (Fig. 2). Survival of Washed Spermatozoa. To get 100

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FIG. 1. Progressive motility score (piles), per cent motile (--) and dead (----) spermatozoa (mean and S.E.M) before and after 1-4 hr. incubation of untreated spermatozoa in their original Fractions 1 (0 0) and 2 (e .), respectively. N ~ 6.

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FIG. 2. Progressive motility score (piles), per cent motile (--) and dead (----) spermatozoa (mean and S.E.M.) before and after 1-4 hr. incubation of unwashed spermatozoa in Fractions 1 (0 0) and 2 (e .), respectively. N ~ 6.

a more homogenous quality of spermatozoa in the two seminal plasma fractions before the incubation, the spermatozoa in Fractions 1 and 2 were separated from the plasma, washed, pooled, and divided into two equal parts which were then resuspended in the two fractions, respectively . The changes in motility and viability during the incubation time are illustrated in Fig. 3. The differences in sperm motility and vitality are not statistically significant at the beginning of the incubation period, but after just 1 hr. of incubation both the percentage of motile spermatozoa and the progressive motility score were significantly lower in Fraction 2 than in Fraction 1 (p < 0.05). Furthermore, after 1 hr. of incubation, in Fraction 2 the motility of the spermatozoa was lower than after 4 hr. in Fraction 1. In three additional experiments the survival of washed spermatozoa was studied in a somewhat modified manner. Before the spermatozoa were added, half the volume of Fraction 2 had been replaced with an equal volume of Fraction 1 obtained

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SURVIVAL OF HUMAN SPERMATOZOA

from a split ejaculate of another man. The motility and vitality of the spermatozoa effect on survival and motility is illustrated between the two fractions. This indicates in Fig. 4 and shows that, after addition of that the prostatic fluid contains a factor Fraction 1, there now was no difference in that maintains the motility and viability of the spermatozoa. 100 Uptake of Zinc and Magnesium by the Spermatozoa. After the incubation of 90 washed and unwashed spermatozoa the 0 the spermatozoa contained very washing, .... ....z 30 little zinc and magnesium and the uptake 3 '" 0 N

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TABLE 1. Zinc in Unwashed Spermatozoa (lJ.g./10 8 Spermatozoa) and Seminal Plasma (lJ.g./ml.) after 4 hr. Incubation in Fractions 1 and 2, Respectively

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FIG. 3. Progressive motility score (piles), per cent motile (--) and dead (----) spermatozoa (mean and S.E.M.) before and after 1-4 hr. incubation of washed spermatozoa in Fractions 1 (0 0) and 2 (e .), respectively. N = 7.

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TABLE 2. Magnesium in Unwashed Spermatozoa (lJ.g./10 8 Spermatozoa) and Seminal Plasma (lJ.g./ml.) after 4 hr. Incubation in Fractions 1 and 2, Respectively

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FIG. 4. Progressive motility score (piles), per cent motile (--) and dead (----) spermatozoa (mean and S.E.M.) before and after 1-4 hr. incubation in Fractions 1 (00) and 2 after addition of an equal amount of Fraction 1 (e .). N = 3.

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5.14 5.85 0.81 1.02 3.51 1.66 2.45 3.14 2.69 2.00 2.73 2.92 0.85 0.70 >0.80

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LINDHOLMER

TABLE 3. Zinc in Seminal Plasma (Ilg./ml.) and Washed Spermatozoa (llg./10' Spermatozoa) before and after 4 hr. Incubation in Fractions 1 and 2, Respectively Seminal plasma

Spermatozoa Washed

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Mean S.E.M. p

0.34 0.12

Fraction 1

Fraction 2

Fraction 1

Fraction 2

2.37 3.42 2.94 3.95 1.45 2.23 2.93 4.44 1.54 2.54 2.17 3.32 0.37 0.42 <0.001

184 188 133 239 313 211 30

32 104 28 88 151 80 23

TABLE 4. Magnesium in Seminal Plasma (Ilg./ml.)

and Washed Spermatozoa (M./lO' Spermatozoa) before and after 4 hr. Incubation in Fractions 1 and 2, Respectively Spermatozoa Washed

1.59 0.87 1.40

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1.29 0.22

Fraction 1

Seminal plasma Fraction 2

0.53 1.01 0.90 2.05 1.31 1.59 2.01 2.31 6.17 9.50 2.18 3.35 1.03 1.56 >0.20

Fraction 1

Fraction 2

105 190

26 71

148

49

of zinc was significantly higher (p < 0.001) in Fraction 2 than in Fraction 1, despite a higher extracellular zinc concentration in Fraction 1 (Table 3). The uptake of magnesium by washed spermatozoa was the same in both fractions (Table 4). DISCUSSION

The influence of different male accessory gland secretions on the motility and survival of bull and hamster spermatozoa has been studied by Bennett and Dott 11 and Morita and Chang. 12 • 13 Bennett and Dott measured the impedance change frequency as an index of bull epididymal spermatozoal activity and found a reduced activity of the spermatozoa when they were

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suspended in the secretion from the seminal vesicles. Morita and Chang found, on the other hand, that the prostatic secretion of hamsters depressed both the aerobic metabolism and the motility of hamster epididymal spermatozoa. Pretreatment of the prostatic secretion with ethylenediametetraacetate did not change this effect on the spermatozoa, indicating that the effect was not dependent on divalent ions. Furthermore, they found that rat epididymal spermatozoa were dependent for the maintenance of their motility on some factor(s) from the seminal vesicles, epididymis, and coagulating glands. The survival of bull and hamster spermatozoa seems therefore in some way to be dependent on the secretion from the accessory genital glands. In the present study the motility and survival of human spermatozoa were determined in the first (mainly prostatic secretion) and last half (mainly vesicle secretion) of the ejaculate. About 40 min. after the ejaculation the motility and percentage of live spermatozoa were significantly lower in Fraction 2 than in Fraction 1. This could be due to either a suppressive component in the vesicle or a stimulating one in the prostatic secretion. In a previous study 1 we have shown that the spermatozoa ejaculated in the first fraction are of the same quality as those in the whole ejaculate from the same man, indicating that the difference between sperm motility in the first and last halves of split ejaculates was due to an extracellular factor. In the present investigation no increased motility was observed in washed spermatozoa immediately after incubation in Fraction 1, nor was there any immediate decrease in motility after incubation in Fraction 2. After about 20 min. of incubation (time 0 in Fig. 3) the motility was somewhat, although not significantly, lower in Fraction 2 than in Fraction 1. After an incubation period of 1 hr. and 20 min. the motility was significantly lower in

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SURVIVAL OF HUMAN SPERMATOZOA

Fraction 2 and then throughout the 4-hr. experiment. This indicated that the cause of the bad motility and high percentage of dead spermatozoa in the second fraction was a suppressive factor. In another type of experiment with unwashed spermatozoa the spermatozoa incubated in the two fractions were harvested from ordinary ejaculated specimens. The spermatozoa had been in contact with prostatic secretion before being separated from their seminal plasma and incubated in Fractions 1 and 2, respectively. This obviously protected the spermatozoa from the deleterious effect of the vesicular secretion. This indicates that a factor or factors from the prostate protect the spermatozoa from perhaps a harmful effect of the vesicle secretion. This was further documented in the few experiments where Fraction 2 was mixed with an equal amount of Fraction 1 before the incubation, giving a much better survival in Fraction 2. The protective factor from the prostate could be washed away from the spermatozoa. Freund and MacLeod 14 have shown that fructose depresses the motility of human spermatozoa in a sodium phosphate buffer, and according to Wales and White 15 potassium increases the viability of dog spermatozoa. In the present investigation the fructose concentration was not significantly higher in Fraction 2, and according to Mann 16 the potassium concentration is about the same in human prostatic and vesicle secretions. Moreover, the harmful effect of the vesicle secretion could not be due to different pH or osmolarities in the two fractions, since these factors were similar. However, the concentrations of zinc and magnesium were much higher in Fraction 1 than in Fraction 2, and these ions might be responsible for the better survival in Fraction 1. A beneficial effect of magnesium on sperm survival has been pointed out by Wales and White 17 in experiments with dog spermatozoa. The use of the first half of the ejaculate

for insemimation (AIH) has been recommended in cases when the husband's semen was oligozoospermic or had a high volume. 18-21 If such a recommendation should be valid one would require that the spermatozoa in the first fractions of split ejaculates have not only a higher sperm density but also a better motility and morphology than spermatozoa from the whole ejaculate. However, there are divergent opinions if this really is the case. 1. 21 The present investigation gives, on the other hand, possibilities for another indication for the use of AIH in cases with infertility problems. Patients with infections in their prostatic gland often have a reduced prostatic secretion during the ejaculation,22 thus effectively giving a higher proportion of vesicle secretion in the ejaculate, which may have negative effects on sperm motility and survival. Poor sperm motility was also more common among semen specimens with biochemical signs of disturbed secretory functions of the accessory genital glands. 23 . 24 It remains, however, to be shown that this poor sperm motility in seminal plasma is of any importance for the motility and survival of spermatozoa after they have left the seminal plasma and entered into the female genital secretions. SUMMARY

The survival of human spermatozoa in the first (prostatic secretion) and last half (vesicular secretion) of split ejaculates has been studied. The untreated spermatozoa ejaculated in the two halves of the ejaculate showed a better survival in the first than in the last half. The difference in survival was noted as soon as 40 min. after the ejaculation. Unwashed spermatozoa obtained from other ejaculates showed the same survival in the two halves of the ejaculate, but washed spermatozoa followed the same survival pattern as untreated. The uptake of zinc by washed sper-

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matozoa was higher in the vesicular than in the prostatic secretion. The uptake of zinc and magnesium in unwashed spermatozoa was the same in the two secretions, as was the uptake of magnesium by washed spermatozoa.

13.

14.

Acknowledgment. The author wishes to thank Dr. R. Eliasson for his interest and advice and Mrs. S. Giesecke for skillful technical assistance.

15.

REFERENCES

16.

1. ELiASSON, R., AND LINDHOLMER, CH. Distribution and properties of spermatozoa in different fractions of split ejaculates. Fertil SteriI23:252, 1972. 2. LUNDQUIST, F. Aspects of the biochemistry of human semen. Acta Physiol Scand 19 (Suppl. 66) 1949. 3. ELiASSON, R. Prostaglandin-properties, actions and significance. Biochem Pharmacol 12:405, 1963. 4. LINDHOLMER, CH., AND ELiASSON, R. Zinc and magnesium in human spermatozoa. Int J Fertil 17:153, 1972. 5. ELiASSON, R. Standards for investigation of human semen. Andrologie 3:49, 1971. 6. ELiASSON, R., AND TREICHL, L. Supravital staining of human spermatozoa. Fertil Steril22:134, 1971. 7. ELiASSON, R. Effect offrequent ejaculations on the composition of human seminal plasma. J Reprod Fertil 9:331, 1965. 8. ELiASSON, R., AND LINDHOLMER, CH. Zinc in human seminal plasma. Andrologie 3:147, 1971. 9. ELiASSON, R., AND LINDHOLMER, CH. Magnesium in human seminal plasma. Invest Urol 9:286, 1971. 10. RICHTERICH, R. Clinical Chemistry. S. Karger, Basel, 1969. 11. BENNETT, J. P., AND DOTT, H. M. Effect ofseminal plasma on epididymal spermatozoa in the bull. Int J FertiI12:21, 1967. 12. MORITA, Z., AND CHANG, M. C. Maintenance ofthe

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motility of rat epididymal spermatozoa in the presence of male accessory secretions. J Reprod FertiI24:247, 1971. MORITA, Z., AND CHANG, M. C. An adverse effect of the ventral prostate secretion on hamster epididymal spermatozoa. J Reprod Fertil 24:255, 1971. FREUND, M., AND MACLEOD, J. Effect of addition of fructose and glucose on the fructolysis and motility of human spermatozoa. J Appl Physiol 13:506, 1958. WALES, R. G., AND WHITE, I. G. The effect of the ions of the alkali metals magnesium and calcium in dog spermatozoa. J Physiol 142:494, 1958. MANN, T. Biochemistry of Semen and of the Male Reproductive Tract. Wiley, New York, 1964. WALES, R. G., AND WHITE, I. G. The effect of alkali metal, magnesium and calcium ions on the motility of fowl spermatozoa. Aust J Bioi Sci 11 :589, 1958. MACLEOD, J., AND HOTCHKISS, R. S. The distribution of spermatozoa and certain chemical constituents in the human ejaculate. J Urol 48:225, 1942. HARVEY, C., AND JACKSON, M. H.A method of concentrating spermatozoa in human semen. J Clin Path 8:341, 1955. FARRIS, E. J., AND MURPHY, D. P. The characteristics of the two parts of the partitioned ejaculate and the advantage of its use for intrauterine insemination. Fertil Sterilll:465, 1960. AMELAR, R. D., AND HOTCHKISS, R. S. The split ejaculate. Its use in the management of male infertility. Fertil SteriI16:46, 1965. ELiASSON, R., LINDHOLMER, CH., AND LEANDER, G. Zinc and magnesium in human seminal plasma. Int Urol Nephr 2:309, 1970. ELiASSON, R., AND LEANDER, G. Changes in semen associated with inflammatory conditions in the male genital tract. Opuscula Medica 6:288, 1966. ELiASSON, R. Correlation between sperm density, morphology and motility and the secretory function of the accessory genital glands. Andrologie 2:165, 1970.