A MICROLITER OXYGEN ELECTRODE SYSTEM FOR SPERM SUSPENSIONS*

Transcription

1 FERTILITY AND STERILITY Copyright 1978 The American Fertility Society Vol. 30, No. 6, December 1978 Printed in U.S.A. A MICROLITER OXYGEN ELECTRODE SYSTEM FOR SPERM SUSPENSIONS* DAVID F. KATZ, PH.D.t EDWARD T. BLAKE, D.CRIM* ROSS N. MILLS, PH.D. IRVING FATT, PH.D. Department of Obstetrics and Gynecology, School of Medicine, University of California, Davis, California 95616, and Department of Mechanical Engineering and Department of Biomedical and Environmental Health Science, School of Public Health, University of California, Berkeley, California A new polarographic oxygen electrode system is described which can accept sperm suspensions with volumes as low as 50 ~ In this conftguration, the electrode tip forms the base of the suspension reaction chamber, and there is no stirring. Consequently, structured fluids such as cervical mucus can be applied. Measurements can be performed in as little time as 10 minutes. The system reliably measures oxygen consumption rates as low as 0.1 mmhg/minute, so that mammalian sperms us pensions with concentrations of the order of 1 x 10 6 viable cells/ml can be applied. Sample experiments, comparing the oxygen uptake of human spermatozoa in semen and in Tyrode's solution, are described. Fertil Steril30:691, 1978 Many contemporary studies of the oxygen uptake of sperm suspensions have employed polarographic oxygen electrodes for these measurements. 1-4 This method is more sensitive to low levels of oxygen consumption than the older manometric technique, and requires much shorter measurement times. 1 The common instrument configuration is one in which the sperm suspension is contained in a constant-temperature Received May 24, 1978; revised August 2, 1978; accepted August 17, *Supported by Contract from the World Health Organization Task Force on the Regulation of Sperm Migration. tdepartment of Obstetrics and Gynecology, School of Medicine, Davis, Calif., and Department of Mechanical Engineering, Berkeley, Calif. To whom reprint requests should be addressed at Department of Obstetrics and Gynecology, School of Medicine, University of California, Davis, Calif *Department of Biomedical and Environmental Health Science, School of Public Health, University of California, Berkeley, Calif. Department of Mechanical Engineering, University of California, Berkeley, Calif. 691 reaction vessel of volume 1 to 8 ml. The oxygen electrode fastens tightly into the top of the vessel. A small stirring bar is magnetically driven within the vessel, thereby increasing the flux of oxygen to the cathode and thus the sensitivity of the instrument. This configuration possesses two distinct disadvantages in its application to studies of spermatozoa. First, the minimal sample volume of approximately 1 ml is considerably larger than the fluid volumes often collected from the male and female reproductive tracts. For example, individual samples of human cervical mucus seldom exceed 250 to 500 JLl; the volume of fluid collected from an entire rabbit oviduct might not exceed 200 JLL Second, the presence of the active stirring bar precludes application to structured fluids, such as cervical mucus. The movement of the bar would alter the properties of the fluid, and possibly the behavior of the spermatozoa therein. A constant flux of oxygen to the cathode might not occur. In addition, it would be difficult to remove the suspension intact from the reaction vessel for further analysis. A new configuration is described here which eliminates these two operational disadvantages, but which retains the high

2 692 KATZET AL. resolution and sensitivity inherent in the contemporary oxygen electrode. 5 The system utilizes the flat tip of an electrode as the base of the December 1978 reaction chamber. There is no stirring. Measurements have been performed on sperm suspensions with volumes as low as 50 ~-tl. ~-- ~--- ~-- ~ --- FIG. 1. Overview of oxygen electrode system.

3 Vol. 30, No. 6 MICROLITER OXYGEN ELECTRODE SYSTEM FOR SPERM SUSPENSIONS 693 Upper heating block Lower heating block Clamp thumb screw FIG. 2. Arrangement of oxygen electrode within heating blocks. MATERIALS AND METHODS The measurement system consists of the following principal components (cf. Figs. 1 to 3): Oxygen Electrode. The electrode (Schema Versatae, Berkeley, Calif.) contains a 25-~I-m diameter polarized platinum cathode and a 500-~I-m diameter silver-silver chloride reversible reference anode. The cathode is polarized at -0.6 volt with respect to the anode. The outer diameter of the electrode is 0.6 em, and the length is 5.3 em. When in use, the electrode is covered by a Teflon membrane m thick (Yell ow Springs Instrument Co., Yellow Springs, Ohio). Specimen Reaction Chamber and Chamber Register. These are machined from stainless steel. The outer diameter of the chamber is 1.8 em and, when in place, its depth is approximately 0.18 em. The chamber is sealed on the top by a circular cover glass 1.8 em in diameter. Upper and Lower Heating Blocks. These are machined from brass, and enclose the suspension chamber and upper two-thirds of the electrode. seal Specimen chamber FIG. 3. Detail of specimen chamber, mounted onto oxygen electrode. The lower block contains a circular heating element, which is controlled by a rheostat. A copper-constantin thermocouple is attached by epoxy to the upper heating block, such that it is in very close apposition to the top of the suspension chamber. Current Amplifier. The amplifier (Schema Versatae, S/V 400) has variable gain, and can individually bias four different electrodes. Strip-Chart Recorder. The recorder (Hewlett Packard, 7100 B) contains two channels. One is connected to the output of the amplifier, and the other to the reference junction of the thermocouple. Application of the system commences with preheating of various components to 37 C. The heating element in the lower heating block is activated, and adjusted by the rheostat; the temperature is monitored, via the thermocouple, on one channel of the strip-chart recorder. This operation is performed with the bare electrode clamped within the lower heating block. The specimen chamber and the chamber register are placed upon the upper heating block during this procedure. Simultaneously, a Teflon membrane is placed in a warmed Petri dish containing normal saline solution at 37 C. Once temperature equilibration of the system has been achieved, a drop of the saline is pipetted onto the top of the bare electrode; and the membrane is applied and secured with an 0-ring. The chamber register and specimen chamber are then placed upon the electrode. The following steps calibrate the system. The electrode is biased via a simple adjustment on the

4 694 KATZETAL. amplifier. After removal of the upper heating block, the zero reading for the output is achieved by directing a stream of water-saturated nitrogen gas onto the membrane-covered surface of the electrode. The full-scale reading for the output, i.e., the ambient partial pressure of oxygen, is obtained with the aid of a barometer and a wet-dry bulb thermometer. The system can now receive the preheated sperm suspension, which is pipetted directly into the specimen chamber. A ring of Vaseline is applied to the notch on the top of the specimen chamber, which is then sealed by a preheated circular cover glass. The upper heating block is reapplied, and the oxygen uptake measurement sequence begins. Generally, less than 5 minutes is required for suspension-system equilibration, so that the sequence can be completed in as little time as 10 minutes. Because there is no stirring bar in this configuration, oxygen transfer to the electrode is a diffusion-controlled process. 6 Consequently, the oxygen uptake per sperm is independent of the volume of the suspension, and depends only upon the number of spermatozoa consuming oxygen therein. Results are expressed in the conventional Z0 2 units, viz. microliters of 0 2 consumed/10 8 sperm/hour. The following formula is applied: Z0 2 = (10 11 )K X [(dp/dt) X lin] (1) where K is the solubility of oxygen in the suspending medium, in the units ml of 0 2/(ml medium x mm Hg); (dp!dt) is the rate of change of partial pressure, P, of 0 2 in the medium with respect to time, t, in the units mm Hg/hour; andn is the sperm concentration in the medium, in the units number of sperm/mi. The slope of the electrode output on the strip-chart recorder gives (dp/dt);n can be based upon total sperm numbers, the number of motile spermatozoa, or the number of living spermatozoa (via a vital stain). RESULTS AND DISCUSSION The accuracy and precision of this system have been extensively tested experimentally in applications to suspensions of spermatozoa and other oxygen-consuming cells. Since the electrode response is temperature-dependent, temperature stability must be maintained over the course of a measurement. The temperature stability of the system has been determined to be better than 0.5 C/hour. Coupled with the low noise and drift-free characteristics of the amplifier and strip chart recorder, this results in measurements with a high December 1978 signal-to-noise ratio. The linearity of the system output, and thus the reliability of equation 1, were tested by measurements of suspensions of yeast in Tyrode's solution at 37 C, with concentrations in the range 50 ~-tglml to 1 mg/ml. These resulted in oxygen consumption rates in the range 0.22 mm of 0 2/minute to 4.2 mm of 0 2/minute. The system response was extemely linear, as verified statistically by correlation analysis (P < I0-4 ). On the basis of these measurements, it can be concluded that oxygen consumption is reliably measurable for rates down to approximately 0.1 mm Hg/minute. As an example of the application of the system, experiments were performed in which the oxygen uptake of human spermatozoa was compared in semen and in Tyrode's solution. Semen samples were obtained by masturbation from four healthy adult men after at least 3 days of sexual abstinence. Each specimen was received in the laboratory for analysis within 30 minutes after collection and was transferred to a glass culture tube and placed in a water bath at 37 C. All semen samples contained at least 40 x 10 6 spermatozoa!ml as measured with a hemocytometer, a percentage motility of 50% or greater as determined visually by a differential count, and a quality of progressive motility visually ranked (on a scale of 0 to 4) at 3 or greater. After thorough mixing of the semen sample, an aliquot was withdrawn and loaded into the measurement system. The oxygen uptake of the whole semen was then determined by the procedure described previously. At the conclusion of the measurement sequence, the semen aliquot was removed from the specimen chamber, placed on a preheated slide, and covered by a no. 1 2 cover slip, 22 mm x 22 mm. Percentage motility was then recounted at 37 C, and this value was used in the subsequent calculation of the oxygen uptake per motile sperm. Simultaneously with the assessment of the whole semen, a second aliquot was centrifuged at high speed in a Beckman Microfuge to obtain seminal plasma. The sperm-free nature of the plasma was verified by microscopic examination, and the aliquot was returned to the water bath. Upon completion of the measurement for whole semen, the oxygen uptake of the seminal plasma was determined. The value of zo2 for the spermatozoa in the semen was then calculated by application of equation 1 after subtraction of the oxygen uptake of the seminal plasma from that of the whole semen. While the preceding experiments were in

5 Vol. 30, No.6 MICROLITER OXYGEN ELECTRODE SYSTEM FOR SPERM SUSPENSIONS 695 TABLE 1. Results of Experiments Comparing Oxygen Uptake of Human Spermatozoa in Semen and in Tyrode's Solution Oxygen uptake for medium Z02 Spermatozoa Experiment-::::-;----~-- -~----=---o--,------,-~ no. Whole semen Plasma Semen Tyrode's solution !JiO,Imllhr !Ji!l()B spermlhr progress, a suspension of sperm in sterile Tyrode's solution (ph 7.4) was prepared as follows. An aliquot of semen was diluted 1:1 (v:v) in Tyrode's solution and gently centrifuged for 10 minutes. The sperm pellet was resuspended in Tyrode's solution to the original aliquot volume, and the suspension was returned to the water bath. Upon completion of the measurement sequence for the seminal plasma, the oxygen uptake for the washed sperm suspension was determined. Such measurement was always completed within 1 hour of the original receipt of the semen sample. Percentage motility was again determined after pi petting the aliquot out of the specimen reaction chamber, and the total sperm concentration in Tyrode's solution was measured. The value of Z02 for the motile spermatozoa in Tyrode's solution was then calculated using equation 1. The results of these experiments are presented in Table 1, and indicate that the human spermatozoa consumed oxygen at a substantially higher rate in Tyrode's solution than in the native semen. This finding is consistent with earlier comparisons between semen and artificial balanced salt solutions, 1 2 and has been presented to exemplify the use of the system. The minimal, reliably measurable, oxygen consumption rate of 0.1 mm Hg/minute for this system can be used to estimate the lower limits of oxygen uptake/sperm and viable sperm concentration for which the system is applicable. Substitution of0.1 mm Hg/minute into equation 1 yields the relation ZO.;N > 23.1 X 106 (2) Consider, for example, a human semen sample in which the spermatozoa consume oxygen at a rate Z02 = 2.0 ~-tl of 0 2/10 8 sperm/hour. According to equation 2, values of N ;;; x 106 sperm/ml would then be required. The spermatozoa of other mammalian species exhibit higher respiratory tendencies. For example, the value Z0 2 = 11.0 has been measured for rabbit spermatozoa in semen, while the result zo2 = 25.8 has been obtained for the spermatozoa of this species after in vivo incubation within the uterus. 7 At this latter value, a viable sperm concentration of only N = 0.90 x 10 6 sperm/ml would be necessary, according to equation 2. Thus, the measurement system described herein is applicable to the very small suspension volumes and sperm concentrations characteristic of many experiments involving capacitation and in vitro fertilization. Applications of this kind, as well as to spermatozoa in cervical mucus, are currently in progress. REFERENCES 1. Petersen RN, Freund M: ATP synthesis and oxidative metabolism in human spermatozoa. Bioi Reprod 3:4 7, Eliasson R: Oxygen consumption of human spermatozoa in seminal plasma and in Ringer's solution. J Reprod Fertil 27:385, Hicks JJ, Martinez-Manautou J, Pedron N, Rosado A: Metabolic changes in human spermatozoa related to capacitation. Fertil Steril 23:172, Trifunac NP, Bernstein GS: Inhibition of the oxidative metabolism of human spermatozoa by a heat-labile factor in seminal plasma. Fertil Steril 27:1295, Fatt 1: Polarographic Oxygen Sensors. Cleveland Ohio, CRC Press Inc, 1976, p Takahashi GH, Fatt I, Goldstick TK: Oxygen consumption rate of tissue measured by a micropolarographic method. J Gen Physiol50:317, White IG, Roger JC, Murdoch RN, Williams WL, Abney TO: Effect of decapacitation factor on the oxygen uptake of rabbit spermatozoa recovered from the uterus. Experientia 31:80, 1975