THAT SEMINAL FLUID has functions in the process of conception other than

Transcription

1 Influence of Seminal Fluid on Sperm Motility William Marden and N. T. Werthessen, Ph.D. THAT SEMINAL FLUID has functions in the process of conception other than to serve as a mere transport medium for spermatozoa is a belief held by a number of observers. The case for and against this hypothesis has been extensively reviewed by Mann. 1 It is apparent from this summary that clear-cut evidence for such other functions of seminal fluid in mammalian reproduction is lacking, and would require some special experimental situation for its demonstration. The observations to be described, as well as other studies from this laboratory, indicate that the stallion provides the required conditions. Stallions are known to vary greatly in their fertility and such variation is even more marked among Thoroughbred animals. The stresses of racing and the rules which limit breeding to a brief period of the year aggravate the situation even further. An unusual aspect of the Thoroughbred breeding problem was brought to the attention of the authors when they were consulted by certain stud farms where it had been observed that a number of stallions, which should have been fully fertile judging from the customary standards of sperm evaluation, were of very low fertility in actual practice. In exploring this situation the possibility that there might be a nonspermatozoal factor seemed reasonable. The data to be reported below will serve to outline our first approach to the problem. From the Southwest Foundation for Research and Education, San Antonio, Texas. Supported in part by grants-in-aid from Green Tree Stud, Inc., Lexington, Ky., the Grayson Foundation, and Mr. Tom Slick. The authors wish to express their appreciation to Dr. Hagyard and Mr. Beard of Lexington, Ky., for bringing the situation in stallions to the authors' attention. Most particularly do we wish to record that Dr. Hagyard's preliminary tests and observations on stallion fluids were of tremendous weight in influencing us to undertake this study. Of great importance to the success of this study was the advice and continued interest of Dr. Joseph W. Goldzieher. 508

2 Vol. 7, No.6, 1956 SEMINAL FLUID AND SPERM MOTILITY 509 METHODS Since it is generally believed that sperm motility and the maintenance thereof are the best indirect criteria of fertility available, these parameters were employed in the first phases of the study. However, since our objective was a quantitative comparison not only of the seminal fluids of different stallions, but also of various ejaculates of the same stallion, it was clear that some new method of evaluation was requisite. There being as yet no instrument as satisfactory for motility evaluation as a trained observer, it was decided to utilize such observations but to minimize the subjective factor as much as possible. Preliminary training consisted of observing several hundred samples of stallion spermatozoa whose motility ranged from zero to maximal. The observer learned during this training period to grade motility on an arbitrary scale of 0 to 10. Later, for experimental readings, the observer was presented with coded slides. The code was kept by the assistant who prepared the slides from the test solutions and the results were not decoded until the entire experiment was over. Two slides of each sample were studied at each observation period so that a measure of the daily reproducibility could be obtained. It was found that the observer could estimate a sample with scale unit. That is, if the motility was read as 5, the value of the duplicate varied from 4 to 6. Obviously there was greater accuracy at both ends of the scale. For this order of reading accuracy, one can predict that duplicate determinations of the algebraic sum index should have a standard deviation of units. Actual calculation of a number of duplicate runs gave a standard deviation of The technic as finally developed, was as follows: Technic 1. Stallions were trained to ejaculate into an artificial vagina. 2. The ejaculate (20 to 60 cc.) was divided into 4 parts. There were ( a) test samples, (b) the control (reconstituted) specimen, (c) the unprocessed specimen, and (d) cell-free fluid for storage. The 4 parts were treated as follows: Test Samples. A measured volume of semen was placed in a centrifuge tube and centrifuged at 1500 rpm for 3 minutes. The supernatant fluid was removed

3 510 MARDEN & WERTH ESSEN Fertility & Sterility and replaced by another cell-free seminal fluid whose capacity to maintain motility was under study. The spermatozoa were resuspended by stirring with a glass rod. This process was repeated twice more, and after each centrifuging the supernatant was removed and the spermatozoa resuspended in additional portions of the new fluid. It was felt that 2 washings in this fashion were sufficient to dilute out any influence of the original seminal fluid. At the end of the third resuspension the sample was placed in the incubator at 37.5 C. Control (Reconstituted) Sample. This specimen was treated simultaneously and identically with the test samples, except that at the end of each centrifuging the spermatozoa were simply resuspended in their original fluid by stirring with a glass rod. Unprocessed Sample. A few cubic centimeters of well-mixed ejaculate were subjected to the same manipulations as the control sample except for centrifugation, which was omitted. The sample was thereby exposed to the same temperature changes, etc., as the control sample, prior to being placed in the constant temperature room. Fluid Store. This portion of the ejaculate was rapidly chilled by immersion in ice water to lower the rate of sperm movement. The spermatozoa were then separated by centrifuging at full speed, and the clear supernatant fluid removed, placed in vials, and stored at the temperatures indicated for particular experiments. 3. As soon as possible after placing all the samples for a particular experiment into the incubator, a determination of motility was made. This observation was the zero hour reading. Subsequent readings were made at hourly intervals for 8 hours or until the spermatozoa were dead. The test solutions were agitated gently in a mechanical rocker between readings. To read a sample, a drop of the test solution was placed on a microscope slide, covered with a coverslip, and observed by dark-field illumination. The entire procedure of incubation and reading was carried out in a walk-in incubator so that temperature changes were minimized. In spite of the discomfort to which this technic subjects the observer (37.5 C, for considerable periods at hourly intervals), this, or comparably rigid temperature maintenance and absence of thermal shock, is strongly recommended if satisfactory results are to be obtained. 4. Computation: At the end of each run the motility values were tabulated as shown in Table 1.

4 Vol. 7, No.6, 1956 SEMINAL FLUID AND SPERM MOTILITY 511 TABLE 1. Motility Values (Duplicate Determinations Omitted for Simplicity) Cell Time (hr.) Algebraic Tube sample Fluid sum No. No. sample No index (control reconstituted sample) Algebraic sum of test fluid Algebraic sum of test fluid 18 (compared to control sample) The unprocessed sample served only to indicate whether or not the spermatozoa from a particular stallion were of sufficient viability to warrant the use of the data. If they died by the third hour, the data were discarded. The reconstituted sample supplied the control, or "blank" values for the calculation. The hourly reconstituted (control) value was subtracted from the corresponding determination on each test sample. (Usually at the zero hour there was little if any difference unless the test sample was a very poor maintainer of motility. Differences usually began to appear at the second or third hour, became marked by the fifth and sixth hour, and disappeared entirely by the eighth hour if all the spermatozoa were dead or became very marked if one or more samples maintained motility throughout the entire test.) Due regard for algebraic sign was held when subtracting the value of the control sample from that of the test sample. Finally, the total score for the sample was obtained by adding up the eight individual comparisons. We have been calling this value the "Algebraic Sum Index»; it gives a useful description of the ability of a particular seminal fluid to maintain the motility of spermatozoa as compared to the seminal fluid in which the cells were originally ejaculated. Thus a positive value means a better performance than the control, a negative value means poorer performance, and the numerical value gives the indication of degree. It is clear that if a test sample of fluid were superior to the sperm donor's own fluid, this would be difficult to demonstrate in the early period of the test. In other words, this system of measurement favors the demonstration of a lesser capacity to maintain sperm motility. However, in spite of its limita-

5 512 MARDEN & WERTHESSEN Fertility & Sterility tions, our technic did serve to permit a semiquantitative measurement of spenn-maintaining capacity. Stability of Seminal Fluid Stored with Dry Ice Studies were carried out on the stability of seminal fluid maintained at dry ice temperatures. Aliquots of a pooled sample of seminal fluid were assayed on 11 different occasions over a SO-day period. The average value for the algebraic sum index for this fluid was -1.5 with standard deviation of +S.2. This variance is only slightly over what is to be expected from the error of the method itself, consequently one can conclude that the spermmaintaining potential of seminal fluid is maintained for at least 1 month under these conditions. Influence of Sperm Cells We also investigated the possible influence of the sperm cells used in the bioassay. Although the cells of the different donor stallions varied considerably from donor to donor, we frequently observed a surprisingly constant behavior of successive sperm samples from the same donor. Table 2 shows this clearly: although the cells of donor stallions Nos. 9 and 2 differed appreciably in their response to the same seminal fluid, the relationship of the responses remained constant, and was reproducible on successive occasions. Date TABLE 2. Fluid sample No. Response of Spermatozoa Cell donor No Algebraic sum index Measurement of Nonprotein SH As a result of additional studies (to be described further on) measurement of the nonprotein sulfhydryl concentration of the seminal fluid became necessary. This was done by means of the micromethod of Goldzieher et al,3

6 Vol. 7, No.6, 1956 SEMINAL FLUID AND SPERM MOTILITY 513 In this technic, a given volume of seminal fluid is freed of proteins by precipitation with sulfosalicylic acid, or by direct addition to the strongly alkaline (ammonia) alcohol subsequently used during measurement. Precipitated proteins are removed by centrifugation. The titration used in this method depends on the fact that silver ions added to alcoholic ammonia solution will combine with SH groups-and little else-to form an unionized silver mercaptide. By using this reaction in an electrometric titration, it will be seen that no current will pass through the alcoholic-ammonia solution until an excess of silver ions has been added. By using a sufficiently sensitive galvanometer and O.OOlN silver nitrate added from a microburet, it is possible to measure microgram quantities of sulfhydryl with a precision of 2 per cent. RESULTS The first stage of the study was a demonstration that there did indeed exist among the seminal fluids of various stallions a difference in their capacity to maintain sperm motility. This was shown by collecting ejaculates from 5 stallions, storing the cell-free seminal fluids on dry ice, and then using one stallion after another as the sperm donor for various experiments. The data from two typical experiments are shown in Table 3. Many such determinations proved unequivocally that seminal fluids differed in their ability to maintain sperm motility. On the basis of these results, it was evident that the nature of the agent causing such differences between the various seminal fluids should be sought. Studies with the addition of fructose to seminal fluid showed that changes in the fructose concentration could not account for the observed differences. In the course of further studies, there emerged an increasing conviction that the motility-maintaining power of seminal fluid as measured by our bioassay was not due to any previously considered constituent. Tests of the lability of this factor at various femperatures were carried out. It was found that storage at 4 0 C. caused a loss of maintenance capacity overnight; storage at _10 C. was not much better. Storage over 0 dry ice, however, had proved satisfactory (see previous discussion). Experiments with atmospheres of nitrogen and oxygen at various temperatures confirmed the conclusion that we were dealing with an extremely labile compound or system. These and other observations suggested to us that an examination of the sulfhydryl compounds as related to motility-maintaining capacity would prove of interest.

7 514 MARDEN & WERTH ESSEN Fertility & Sterility For these experiments, determination of the total nonprotein sulfhydryl concentration was carried out by the method described. The sulfhydryl determination * and our bioassay were carried out simultaneously on a large number of samples. This simultaneous measurement tended to reduce possible errors such as changes in SH concentration during the melting of samples or possible variations between stored aliquots of the original sample. The results are shown in Fig. 1 and demonstrate clearly an inverse correlation between the nonprotein SH concentration and the algebraic sum index. The data include not only a number of seminal fluids, but also many difier E 0 ~ (/) -5.Y o.0 CJ') &. -10 _ -15 C -W'~ -L ~ -L ~~~ ~ ~ ~ Tenths cc. of.001 N AgN03 Fig. 1. In this figure is presented the algebraic sum index described in the text as a function of the cubic centimeter of the SH content of seminal fluid. The SH concentration is expressed as the number of tenths of cubic centimeters of.ooin AgN03 requisite in the titration. The solid circles in the graph indicate the usage of sperm and seminal fluid from a single donor stallion, for each. The triangle, open circle, square, and other marks indicate the use of both sperm and seminal fluid from other than the two used for the solid circles. The data have been plotted with this notation as to source of material to indicate the capacity of the components studied in overcoming the biologic variability that could be expected. * In most determinations, measurement of the nonsulfhydryl blank (by treatment with p-chloromercuribenzoate) was omitted.

8 Vol. 7, No.6, 1956 SEMINAL FLUID AND SPERM MOTILITY 515 ent sperm samples by which this variety of seminal fluids was tested. In addition to this inverse correlation (i.e., the higher the SH concentration, the lower the sperm-maintaining capacity), it is also shown once again that the seminal fluids from various stallions vary in their capacities to maintain sperm motility. Finally, since some of the algebraic sum index values are positive, it appears that the seminal fluid in which spermatozoa are ejaculated is not necessarily the best fluid for the maintenance of those spermatozoa. TABLE 3. Maintenance of Sperm Motility Cell donor No. 2a: Cell donor No. 5b: Fluid No. Fluid No. la 2a Sa 4a 5a Ib 2b Sb 4b 5b Algebraic sum index These findings suggest that a factor may exist in seminal fluid which can affect fertility adversely. They also indicate in spite of the vast amount of work done to date on the relationship at various components of seminal fluid to fertility, that there is much room for further study. SUMMARY AND CONCLUSIONS A semiquantitative technic has been developed to measure the influence of seminal fluid on the maintenance of stallion sperm motility, and a great variation in this property of seminal fluid has been observed from sample to sample. Further studies have shown that sulfhydryl compounds may play a part in this phenomenon and determination of the total nonprotein sulfhydryl concentration in seminal fluid has shown it to vary inversely with the sperm-maintaining capacity of the fluid as measured by bioassay. REFERENCES 1. MANN, T., and LUTWAK-MANN, C. Secretory function of male accessory organs of reproduction in mammals. Physiol. Rev. 31 :27, BISHOP, M. W., et al. Semen characteristics and fertility in the bull. ]. Agric. Sci. 44:27, GOLDZmHER, J. W., RAWLS, W. B., and GOLDZmHER, M. A. The uptake of sulfhydryl compounds by rat adrenal, liver, and muscle as measured by an improved amperometric technique. ]. Biol. Chem. 203:519, 1954.