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Potential indicators of reproductive toxicity: Testicular sperm production and epididymal sperm number, transit time, and motility in Fischer 344 rats
FUNDAMENTAL
AND
APPLIED
TOXICOLOGY
5, 1097-I
103 (1985)
Potential Indicators of Reproductive Toxicity: Testicular Sperm Production and Epididymal Sperm Number, Transit Time, and Motility in Fischer 344 Rats’ W. F. BLAZAK, T. L. ERNST, AND B. E. STEWART Cytogenetics and Reproductive Biology Program, SRI International, 333 Ravenswood Avenue, Menlo Park, California 94025
Potential Indicators of Reproductive Toxicity: Testicular Sperm Production and Epididymal Sperm Number, Transit Time, and Motility in Fischer 344 Rats. BLAZAK, W. F., ERNST, T. L., AND STEWART, B. E. (1985). Fundam. Appl. Toxicol. 5, 1097-i 103. Comprehensive assessment of the effects of chemicals on male reproductive functioning requires study of effects on spermatogenesis and the quality of spermatozoa produced. As potential indicators of toxic effects,we have examined sperm production rate (SPR) and epididymal sperm numbers, transit time, and motility (percentage motile cells and swimming speeds) in Fischer-344 (F-344) rats. Groups (n = 14 or 15) of rats at 6, 8, 10, 15, and 20 weeks of age were studied to investigate the suitability of these indicators for detecting toxic effects, and to establish the age at which this strain of rat sexually matures. Mean SPRs ( lo6 sperm/rat/day), as measured by enumerating spermatids in testicular homogenates, were 2 f 3, 22 + 6, 41 + 7, 49 + 6, and 46 + 7 at 6, 8, 10, 15, and 20 weeks, respectively. Epididymal sperm numbers and the percentage of motile spermatozoa also increased with age. No correlation was found between SPR and either testis weight or epididymal sperm number at 10, 15, or 20 weeks of age. Our results indicate that male F-344 rats become sexually mature between 10 and 15 weeks of age, and that testis weight and epididymal sperm number are unreliable quantitative indicators of sperm production rate. The low variabiity among adult rats in SPR and some of the other indicators of testicular and epididymal functioning examined in this study suggest that they should be sensitive measures of male reproductive toxicity. Q 1985 Society of Toxicology.
Chemicals and other potentially hazardous agents have the potential of interfering with many biological phenomena necessary for normal male reproductive functioning. Animal mating trials, in which treated males are mated with untreated females, can provide useful information regarding male reproductive toxicity, but are too insensitive to assess even dramatic effects on spermatogenesis and epididymal functioning. This insensitivity of laboratory rodent mating trials is largely due to the vast excess of spermatozoa ejaculated over what is required for normal fertility. Thus, in order to extend our capabilities in ’ Presented, in part, at the 23rd Annual Meeting of the Society of Toxicology, 12- 16 March 1984, Atlanta, Ga.
detecting male reproductive toxicants, it is important to develop and utilize more sensitive measures of testicular and epididymal functioning (Bedford, 1983). Our approach to this problem has been to examine a battery of indicators of testicular and epididymal functioning in Fischer-344 (F344) rats. This battery included testicular sperm production rate and epididymal sperm number, transit time, and motility. We have studied these indicators in groups of rats at different ages during sexual development with the objectives of (1) gaining insight as to when F-344 rats mature, (2) generating background data for this strain of rat, (3) assessing the variability among untreated rats in these measures of reproductive potential, and (4) examining
1097
0272-0590/85 $3.00 Copyright Q 198s by the Society of Toxicology All rights of reproduction in any form rcservd.
BLAZAK,
ERNST, AND STEWART
the interrelationship among some of these indicators. This effort is directed toward the investigation of methods for the quantitative assessment of induced deleterious effects on male reproduction. MATERIALS
AND
Assessment
METHODS
Male Fischer-344 rats ( 14 or I5 animals per age group) from Simonsen Laboratories, Inc., Gilroy, California, at 5, 7, 9, 14, or 19 weeks of age. After their arrival, animals were housed individually with a 12-hr light/l2-hr dark light cycle and were supplied with food (Purina Laboratory Chow) and purified water ad libitum. After 5 to 7 days of quarantine, the animals were sacrificed and studied at ages of 6, 8, IO, 15, and 20 weeks. were purchased
Assessment Times
Rate
of Epididymal
Sperm
Numbers
and Transit
Epididymal sperm numbers were determined after thawing the tissue at room temperature. The thawed right epididymis was divided into cauda and corpus plus caput sections by cutting at the junction of the corpus and cauda epididymis, and each section was weighed. Each section was then homogenized separately in 20 ml of NaCl-Triton X-100, and the number of spermatozoa in each section was enumerated in duplicate using a hemacytometer. Ep ididymal transit times were calculated by dividing the total number of spermatozoa per whole right epididymis by the sperm production rate of the right testis on a per animal basis (Robb et al., 1978).
Protocol
Rats were sacrificed by cervical dislocation following CO2 anesthesia and weighed. The left cauda epididymis was then excised and placed in a petri dish containing 10 ml of 37°C Dulbecco’s Ca- and Mg-free phosphate-buffered saline with 10 mglml of bovine serum albumin (BSA, Sigma Fraction V). The tissue was minced with scalpels for approximately 1 min and the dish was placed in a 37°C incubator for 15 min prior to assessingsperm movement. The left and right testes were excised; the tunica albuginea was removed from each and the testes were weighed. The testes were then separately homogenized as described below. The right epididymis was excised, trimmed of fat, and frozen at - 10°C.
Videomicroscopic
Assessment
of Sperm
Movement
After 15 min of incubation, a 15-pl aliquot ofthe sperm suspension was placed on a warm microscope slide and a
RIGHT EPIDIDYMIS
Production
Testicular parenchyma was homogenized at 4-7°C in 20 to 30 ml of 0.9% NaCl containing 0.01% of Triton X100. A Waring laboratory blender and a 50-ml capacity “semimicro” stainless-steel blender vesselwere used. The tissue was homogenized at low speed for 2 min, after which 5-ml aliquots of the homogenate were withdrawn. The number of homogenization-resistant spermatids in each sample was then enumerated in duplicate using a hemacytometer. In some cases(e.g., 6- and S-week-old rats) the homogenates were centrifuged and the supematants removed to concentrate the spermatids. Sperm production rates were calculated by dividing the number of homogenization-resistant spermatids per testis or per gram of testis by 6.10 days (Robb et al., 1978).
Animals
General
of Sperm
LEFT TESTIS
TESTIS
EPIDIDYMIS
\
/ CAPUT CORPUS
CAUDA
I
(
EPlDlDYMAL
SPERM
MINCE DISTAL CAUDA IN DULBECCO’S PBS
REMOVE TUNICA ALBUGINEA. HOMOGENIZE TESTICULAR PARENCHYMA AND COUNT RESISTANT SPERMATIDS
HOMOGENIZE AND COUNT SPERMATOZOA
EPIDIDYMAL
-
I RESERVES
TRANSIT
TIME
SPERM
PRODUCTION
1 RATE
SPERM
MOVEMENT
1
FIG. 1. General protocol followed for assessing sperm production rate and epididymal sperm numbers, transit time, and motility in F-344 rats.
RAT SPERM PRODUCTION 22 X 22 mm ( 13) covers&p was added. The sperm specimen was held at 37°C during taping using an air curtain incubator (Are&erg Sage Inc., Model 279). Each of at least nine microscopic fields were then videotaped for 10 to 20 set using phase-contrast microscopy and a 16X objective. The videotapes were later analyzed for percentage of motile cells (50 cells per animal) and straight-line swimming speeds (25 cells per animal), essentially as described by Katz and Overstreet ( 198 I). A video time generator (VTG33, For-A-Carp. of America) was used to calibrate the video system in order to calculate actual swimming speeds of spermatozoa. Statistical
Methods
Data were analyzed by one-way analysis of variance and Duncan’s multiple range test using SPSS on a VAX I l/782 computer. Results were considered statistically significant at the p < 0.05 level. The relative sensitivities of the different indicators were estimated by calculating the percentage decrease for each indicator that would be on the borderline of statistical significance in a one-tailed test (i.e., p = 0.05) using the formula (Bedford, 1983): % decrease
= (Za + ZI-WV)
(N/2)“*
RESULTS Body, Testis, and Epididymal Weights Body, testis, and right epididymal weights from 6-, 8, lo-, 15-, and 20-week-old F-344 rats are presented in Table 1. Body and reproductive organ weights increased significantly through 15 weeks of age; no further increase was observed at 20 weeks of age. Testis weight increased approximately linearly from 6 through 10 weeks of age, and by the 10th week had attained 9 1% of the weight found at 20 weeks. The weight of the right epididymis continued to increase substantially through 15 weeks of age; at 10 weeks of age, it had attained only 66% of its weight at 20 weeks of age. At 6, 8, and 10 weeks of age the left testis was significantly heavier than the right testis; no significant differences were noted at 15 and 20 weeks of age between left and right testis weights. Sperm Production
’
where a = probability of a type 1 error = 0.05; /3 = probability of a type II error = 0.10; 1 - fi = power of the test (probability of rejecting the null hypothesis when in fact it is false) = 0.90; CV = coefficient of variation, %; N = number of rats per treatment group = 15; Z, and Z,+ = standard normal deviates for 01(Z, = 1.645) and 1 - /3 (Z,-, = 1.281).
1099
AND CHARACTERISTICS
Rate (SPR)
Daily SPRs are presented in Table 2. At 6 weeks of age, spermatids could be found in concentrated testicular homogenates prepared from only 7 of 14 males. The mean testis weight for the 6-week-old males with spermatids present in testicular homogenates was
TABLE 1 BODY, TESTES,' AND RIGHT EPIDIDYMAL WEIGHTS (g X 2 SD) OF FISCHER-344 RATS Age (weeks) Variable Body weight Left testis weight Right testis weight Total testes weight Testes weight/ body weight (W) Right epididymal weight
6 119.3 0.593 0.565 1.158
f f k f
8 7.3b 0.0736 0.074” 0.147b
166.0 + 11.5’ 0.946 + 0.084’ 0.887 -I 0.075’ 1.833 + 0.144’
10 222.4 1.224 1.172 2.396
+ 15.1d f 0.059d -t 0.040d zk 0.092d
15 295.7 1.276 1.282 2.556
f + + f
25.4e 0.088”’ 0.069’ 0.141e
20 302.1 1.308 1.318 2.625
+ 42.7’ + 0.070e ztz0.060’ f 0.122’
0.97 rt 0.096
1.11 kO.07’
1.08 f 0.08’
0.87 f 0.06d
0.88 f 0. lod
0.062 zk 0.0 15 b
0.096 f O.OISc
0.233 k 0.20d
0.356 f 0.034e
0.35 1 f 0.033e
’ Testicular parenchyma only. t-r Means within rows with different superscripts are significantly different (p < 0.05) based on one-way analysis of variance and Duncan’s multiple range test.
1100
BLAZAK,
ERNST, AND STEWART TABLE 2
MEAN
(+ SD)
SPERM
PRODUCTIONRATES AND EPIDIDYMAL SPERM NUMBERS, TRANSIT TIMES, AND MOTILITY IN FISCHER-344 RATS Age
Variable Sperm production rate (X 106)” Per rat per day Per gram of testis per day Right epididymal sperm numbers (X106) Per epididymis Per gram of epididymis Right epididymal transit time (days) Left cauda epididymal sperm motility Motile cells (%) Swimming speed (pm/sex)
(weeks)
6
8
10
2.1 f 3.2b 1.6 + 2.3’
21.9 k 5.8’ 11.9 + 2.9’
40.9 f 1.3d 17.1 f 3.1d
49.3 f 6.2’ 19.3 f 2.1’
46.2 f 6.9e 17.7 + 2.9”’
4k5b 34 f 38’
78 + 21’ 333 + 12’
184 f 39d 519 f 107d
128 + 38’ 365 ic 107’
-
7.5 + 2.3 b
7.2 _+ 3.8b
32 f 9’ 68k lib
50 + 8’ 69+5b
-
-
-
-
36 + 10’ 65 + 9b
15
20
a Testicular parenchyma only. k Means within rows with different superscripts are significantly different (p < 0.05) based on one-way analysis of variance and Duncan’s multiple range test.
significantly higher (p < 0.001) than the mean testis weight of males without spermatids (1.273 f 0.067 g vs 1.043 f 0.103 g, respectively). Daily SPR increased significantly through 15 weeks of age. At this age, rats produced approximately 49 X IO6 spermatozoa per day, or 19 X lo6 spermatozoa per gram of testicular parenchyma per day. No further increase in SPR was noted for rats at 20 weeks of age. Although simultaneous increases in testis weight and SPR were observed from weeks 6 through 10, mean testis weight was not significantly correlated with SPR in rats at 10, 15, or 20 weeks of age [correlation coefficient (r) = 0.05, 0.46, and -0.32 at 10, 15, and 20 weeks, respectively]. Thus, the variability in mean testis weight among adult rats may not be an indication of differences in SPR. Epididymal Times
Sperm Numbers and Transit
Epididymal sperm numbers and transit times are presented in Table 2. No sperma-
tozoa were observed within the epididymis from 6-week-old rats. At 8 weeks of age, spermatozoa were observed in the caput and corpus epididymis from all rats studied, but only 3 of 15 animals at this age had spermatozoa in the cauda epididymis. Epididymal sperm numbers significantly increased through 15 weeks of age, but declined at 20 weeks of age. Right epididymal sperm transit times for 15and 20-week-old rats were 7.5 and 7.2 days, respectively (Table 2). Epididymal transit times were not calculated for younger rats because sperm production in these animals had not reached adult levels. The relationship between epididymal sperm numbers and SPR was assessedby calculating the correlation between sperm number per gram of right cauda epididymis and sperm production per gram of right testis for lo-, 15-, and 20-week-old rats. The calculated correlation coefficients at these ages were -0.05, -0.44, and 0.18, respectively (p > 0.05). Thus, SPR and cauda epididymal sperm numbers were not significantly correlated.
RAT SPERM PRODUCTION
Epididymal
Sperm Motility
The results of videomicroscopic studies of the motility of spermatozoa, recovered from the left cauda epididymis of lo-, 15-, and 20week-old rats, are presented in Table 2. The motility of spermatozoa from younger rats could not be studied because of the absence or scarcity of spermatozoa in the cauda epididymis. The percentage of motile cells was not significantly different for spermatozoa from lo- and 15-week-old rats, but increased significantly to 50 + 8% for spermatozoa from 20-week-old rats. The straight-line mean swimming speeds of spermatozoa from IO-, 15-, and 20-week-old rats were not significantly different, with means of 65,68, and 69 pm/set, respectively. Variability of Testicular and Epididymal Endpoints in Adult Rats Estimates of the variation in testicular and epididymal indicators among 20-week-old F344 rats are presented in Table 3. The indicaters with the largest interanimal variation were epididymal sperm numbers and transit times, with CV’s of approximately 30 and 53%,
respectively. The remaining indicators all had CV’s of less than 20%. Knowledge of the variability associated with each indicator allowed us to estimate the percentage decrease, relative to control values, that would be on the borderline of statistical significance in a one-tailed test (i.e., p = 0.05) for each indicator. These sensitivities are presented in Table 3. Thus, a decrease of approximately 16% or more (or 37.4 X IO6 sperm/rat/day) in SPR per rat per day would be statistically significant, whereas a 32% or more decrease in right epididymaI sperm number (or 34 1 X 1O6 sperm) would be required for statistical significance for this indicator. DISCUSSION Male rats are generally considered as sexually mature, effective sires and are used for breeding at 10 to 11 weeks of age (Mather and Rushmer, 1979). Rats at this age and often younger have also been used in studies conceming the potential reproductive effects of chemicals. Our data indicate that male F-344 rats are not sexually mature at 10 weeks of age but attain maturity by 15 weeks based on SPR
TABLE 3 VARIABILITY AMONG ADULT F-344 RATS IN TFSTICULAR Indicator
Range
Testes weight (g) Right epididymal weight (g) Sperm production rate (X I 06) Per rat per day Per g testis per day Right epididymal sperm numbers (X106) Per epididymis Per g epididymis Right epididymal transit time (days) Left cauda epididymal sperm motility Motile cells (W) Swimming speed (pm/set)
2.466-2.862 0.298-0.405 32.0-59.1 11.8-21.3
1101
AND CHARACTERISTICS
AND EPIDIDYMAL
I&SD
INDICATORS”
cv (%)
Sensitivity b
2.625 f 0.122 0.351 + 0.033
4.65 9.40
5 10
46.2 + 6.9 17.1 iz 2.9
14.95 16.56
16 18
65.5-195.0 189.3-533.3 3.3-18.0
128 + 38 365 2 107 7.2 f 3.8
29.69 29.32 52.78
32 31 56
38-62 40-76
50 + 8 69 f 5
16.00 7.24
17 8
DData from 15 rats at 20 weeks of age, except for sperm motility where sperm from 10 rats at 20 weeks of age were studied. b This column lists the percentage decrease in each indicator that would be statistically detected using a sample size of 15 rats per treatment group, the CVs in this table, and (Y= 0.05 and 1-o = 0.90. See Materials and Methods section for details.
1102
BLAZAK.
ERNST, AND STEWART
and epididymal sperm numbers. In male Wistar rats, Robb et al. ( 1978) concluded that sexual maturity in this strain is attained between 75 and 100 days of age based on the same variables. Adult Wistar rats produce 86 X lo6 sperm/day as compared to only 48 X lo6 sperm/day for the ,F-344 rat owing to both the larger testes (3.7 g Wistar, 2.6 g F-344) and the faster rate of sperm production (24 X lo6 cells/g Wistar, 18 X 1O6cells/g F-344) of Wistar rats. Thus, for studies of the effects of potentially hazardous substances on adult male rat reproductive functioning, treatment of the animals should be delayed until approximately 15 weeks of age. Initiating treatment prior to this time has the potential of complicating the interpretation of results because effects on the development of male reproductive functioning and adult reproductive functioning would be confounded. Our finding of an absence of significant correlation between testis weight and SPR indicates that differences in testis weight in untreated rats are not related to SPR. Thus, although large decreases in testis weight will most likely be accompanied by a decrease in SPR, smaller but significant decreases in SPR may not be reflected in a decreased testis weight. Robb et al. ( 1978) also observed a lack of correlation between SPR and testis weight in Wistar rats. Similarly, the lack of correlation between SPR and cauda epididymal sperm number in F-344 rats, as also observed for Wistar rats (Robb et al., 1978), suggests that epididymal sperm numbers are not reliable indicators of effects on testicular SPR. The concentration of spermatozoa in suspensions prepared from the cauda epididymis is presently used by investigators as an indicator of toxic effects on spermatogenesis. Our findings of independence between SPR and cauda epididymal sperm number, and the much higher amount of variation between rats in epididyma1 sperm number as compared with SPR, caution against the continuance of this practice. Although sperm motility is highly correlated with fertility in man, and at least one case has
been published in the rat in which a chemical specifically inhibited sperm motility (McClain and Downing, 1984) little effort has been directed toward the development and use of quantitative methods to assess rodent sperm motility. With our carefully controlled temperature conditions and videomicroscopic system, we found that the consistencies in the percentage of motile cells and in mean swimming speeds of spermatozoa among animals has enabled us to detect decreases of less than 20% in these variables. The use of videomicroscopy for assessing sperm movement has the added advantages that the videotape serves as a permanent record of sperm movement, and the tapes can be analyzed to assesssperm morphology as described in a study of fertile and infertile men (Overstreet et al., 198 1). Our goal in conducting these studies was to determine if the above indicators of rat testicular and epididymal functioning could be employed for assessing chemically induced reproductive effects. The low variability among rats in SPR and sperm movement characteristics suggests that these indicators would be particularly sensitive for detecting deleterious effects. In a recent study of the effects of nitrobenzene on reproductive functioning of male rats (Blazak et al., 1985), we observed an -80% reduction in the number of morphologically normal, motile spermatozoa produced per testis per day in rats exposed to 140 mg/kg/day for 5 consecutive days. Despite the dramatic effect on spermatogenesis observed in these males, a small reduction in the number of implants per pregnant female was the only symptom of a reproductive disturbance noted in breeding studies. Thus, the inclusion of cellular indicators of male reproductive functioning, as described in the present study, together with breeding studies and histopathology, should enable a more complete assessment of the effects of chemicals on male reproduction. ACKNOWLEDGMENTS The authors thank Leslie Diel, Lynne Lyons, and Marilyn Lemmon for excellent technical assistance.
RAT
SPERM
PRODUCTION
REFERENCES AMANN, R. P. ( 1982). Use of animal models for detecting specific alterations in reproduction. Fundurn. Appl. ToxicoI. 2, 13-26. BEDFORD,J. M. (1983). Considerations in evaluating risk to male reproduction. In Advances in Modem Environmental Toxicology, Assessment of Reproductive and Teratogenic Hazards (M. S. Christian, W. M. Galbmith, P. Voytek, and M. A. Mehlman, eds.), Vol. 3, pp. 4198. Princeton Scientific Pub., Princeton, N.J. BLAZAK, W. F., RUSHBROOK, C. J., ERNST, T. L., STEWART, B. E., SPAK, D., DIBIASIO-ERWIN, D., AND BLACK, V. (1985). Relationship between breeding performance and testicular/epididymal functioning in male Sprague-Dawley rats exposed to nitrobenxene (NB). Toxicologist 5, 121 (Abstract No. 484). BLAZAK, W., ERNST, T., DIEL, L., AND STEWART, B. (1984). Assessment of testicular and epididymal func-
AND
CHARACTERISTICS
1103
tioning in Fischer-344 (F344) rats. Toxicologist 4, 81 (Abstract No. 322). KATZ, D. F., AND OVERSTREET, J. W. (198 1). Sperm motility assessmentby videomicrography. Fertil. Steril. 35, 188-193. MATHER, E. C., AND RUSHMER, R. A. (1979). Physiological parameters of some species used in reproduction research. In Animal Models for Research on Contracep tion and Fertility (N. J. Alexander, ed.), pp. 561-576. Harper & Row, Ha%erstown, Md. MCCLAIN, R. M., AND DOWNING, J. C. (1984). The effect of omidazole on fertility and sperm motility in rats. Toxicologist 4, 82 (Abstract No. 323). OVERSTREET, J. W., PRICE,M. J., BLAZAK, W. F., LEWIS, E. L., AND KATZ, D. F. ( 198 1). Simultaneous assessment of human sperm motility and morphology by videomicrography. J. Vrol. 126,357-360. ROBB, G. W., AMANN, R. P., AND KILLIAN, G. J. (1978). Daily sperm production and epididymal sperm reserves of pubertal and adult rats. J. Reprod. Fertil. 54, 103107.
AND
APPLIED
TOXICOLOGY
5, 1097-I
103 (1985)
Potential Indicators of Reproductive Toxicity: Testicular Sperm Production and Epididymal Sperm Number, Transit Time, and Motility in Fischer 344 Rats’ W. F. BLAZAK, T. L. ERNST, AND B. E. STEWART Cytogenetics and Reproductive Biology Program, SRI International, 333 Ravenswood Avenue, Menlo Park, California 94025
Potential Indicators of Reproductive Toxicity: Testicular Sperm Production and Epididymal Sperm Number, Transit Time, and Motility in Fischer 344 Rats. BLAZAK, W. F., ERNST, T. L., AND STEWART, B. E. (1985). Fundam. Appl. Toxicol. 5, 1097-i 103. Comprehensive assessment of the effects of chemicals on male reproductive functioning requires study of effects on spermatogenesis and the quality of spermatozoa produced. As potential indicators of toxic effects,we have examined sperm production rate (SPR) and epididymal sperm numbers, transit time, and motility (percentage motile cells and swimming speeds) in Fischer-344 (F-344) rats. Groups (n = 14 or 15) of rats at 6, 8, 10, 15, and 20 weeks of age were studied to investigate the suitability of these indicators for detecting toxic effects, and to establish the age at which this strain of rat sexually matures. Mean SPRs ( lo6 sperm/rat/day), as measured by enumerating spermatids in testicular homogenates, were 2 f 3, 22 + 6, 41 + 7, 49 + 6, and 46 + 7 at 6, 8, 10, 15, and 20 weeks, respectively. Epididymal sperm numbers and the percentage of motile spermatozoa also increased with age. No correlation was found between SPR and either testis weight or epididymal sperm number at 10, 15, or 20 weeks of age. Our results indicate that male F-344 rats become sexually mature between 10 and 15 weeks of age, and that testis weight and epididymal sperm number are unreliable quantitative indicators of sperm production rate. The low variabiity among adult rats in SPR and some of the other indicators of testicular and epididymal functioning examined in this study suggest that they should be sensitive measures of male reproductive toxicity. Q 1985 Society of Toxicology.
Chemicals and other potentially hazardous agents have the potential of interfering with many biological phenomena necessary for normal male reproductive functioning. Animal mating trials, in which treated males are mated with untreated females, can provide useful information regarding male reproductive toxicity, but are too insensitive to assess even dramatic effects on spermatogenesis and epididymal functioning. This insensitivity of laboratory rodent mating trials is largely due to the vast excess of spermatozoa ejaculated over what is required for normal fertility. Thus, in order to extend our capabilities in ’ Presented, in part, at the 23rd Annual Meeting of the Society of Toxicology, 12- 16 March 1984, Atlanta, Ga.
detecting male reproductive toxicants, it is important to develop and utilize more sensitive measures of testicular and epididymal functioning (Bedford, 1983). Our approach to this problem has been to examine a battery of indicators of testicular and epididymal functioning in Fischer-344 (F344) rats. This battery included testicular sperm production rate and epididymal sperm number, transit time, and motility. We have studied these indicators in groups of rats at different ages during sexual development with the objectives of (1) gaining insight as to when F-344 rats mature, (2) generating background data for this strain of rat, (3) assessing the variability among untreated rats in these measures of reproductive potential, and (4) examining
1097
0272-0590/85 $3.00 Copyright Q 198s by the Society of Toxicology All rights of reproduction in any form rcservd.
BLAZAK,
ERNST, AND STEWART
the interrelationship among some of these indicators. This effort is directed toward the investigation of methods for the quantitative assessment of induced deleterious effects on male reproduction. MATERIALS
AND
Assessment
METHODS
Male Fischer-344 rats ( 14 or I5 animals per age group) from Simonsen Laboratories, Inc., Gilroy, California, at 5, 7, 9, 14, or 19 weeks of age. After their arrival, animals were housed individually with a 12-hr light/l2-hr dark light cycle and were supplied with food (Purina Laboratory Chow) and purified water ad libitum. After 5 to 7 days of quarantine, the animals were sacrificed and studied at ages of 6, 8, IO, 15, and 20 weeks. were purchased
Assessment Times
Rate
of Epididymal
Sperm
Numbers
and Transit
Epididymal sperm numbers were determined after thawing the tissue at room temperature. The thawed right epididymis was divided into cauda and corpus plus caput sections by cutting at the junction of the corpus and cauda epididymis, and each section was weighed. Each section was then homogenized separately in 20 ml of NaCl-Triton X-100, and the number of spermatozoa in each section was enumerated in duplicate using a hemacytometer. Ep ididymal transit times were calculated by dividing the total number of spermatozoa per whole right epididymis by the sperm production rate of the right testis on a per animal basis (Robb et al., 1978).
Protocol
Rats were sacrificed by cervical dislocation following CO2 anesthesia and weighed. The left cauda epididymis was then excised and placed in a petri dish containing 10 ml of 37°C Dulbecco’s Ca- and Mg-free phosphate-buffered saline with 10 mglml of bovine serum albumin (BSA, Sigma Fraction V). The tissue was minced with scalpels for approximately 1 min and the dish was placed in a 37°C incubator for 15 min prior to assessingsperm movement. The left and right testes were excised; the tunica albuginea was removed from each and the testes were weighed. The testes were then separately homogenized as described below. The right epididymis was excised, trimmed of fat, and frozen at - 10°C.
Videomicroscopic
Assessment
of Sperm
Movement
After 15 min of incubation, a 15-pl aliquot ofthe sperm suspension was placed on a warm microscope slide and a
RIGHT EPIDIDYMIS
Production
Testicular parenchyma was homogenized at 4-7°C in 20 to 30 ml of 0.9% NaCl containing 0.01% of Triton X100. A Waring laboratory blender and a 50-ml capacity “semimicro” stainless-steel blender vesselwere used. The tissue was homogenized at low speed for 2 min, after which 5-ml aliquots of the homogenate were withdrawn. The number of homogenization-resistant spermatids in each sample was then enumerated in duplicate using a hemacytometer. In some cases(e.g., 6- and S-week-old rats) the homogenates were centrifuged and the supematants removed to concentrate the spermatids. Sperm production rates were calculated by dividing the number of homogenization-resistant spermatids per testis or per gram of testis by 6.10 days (Robb et al., 1978).
Animals
General
of Sperm
LEFT TESTIS
TESTIS
EPIDIDYMIS
\
/ CAPUT CORPUS
CAUDA
I
(
EPlDlDYMAL
SPERM
MINCE DISTAL CAUDA IN DULBECCO’S PBS
REMOVE TUNICA ALBUGINEA. HOMOGENIZE TESTICULAR PARENCHYMA AND COUNT RESISTANT SPERMATIDS
HOMOGENIZE AND COUNT SPERMATOZOA
EPIDIDYMAL
-
I RESERVES
TRANSIT
TIME
SPERM
PRODUCTION
1 RATE
SPERM
MOVEMENT
1
FIG. 1. General protocol followed for assessing sperm production rate and epididymal sperm numbers, transit time, and motility in F-344 rats.
RAT SPERM PRODUCTION 22 X 22 mm ( 13) covers&p was added. The sperm specimen was held at 37°C during taping using an air curtain incubator (Are&erg Sage Inc., Model 279). Each of at least nine microscopic fields were then videotaped for 10 to 20 set using phase-contrast microscopy and a 16X objective. The videotapes were later analyzed for percentage of motile cells (50 cells per animal) and straight-line swimming speeds (25 cells per animal), essentially as described by Katz and Overstreet ( 198 I). A video time generator (VTG33, For-A-Carp. of America) was used to calibrate the video system in order to calculate actual swimming speeds of spermatozoa. Statistical
Methods
Data were analyzed by one-way analysis of variance and Duncan’s multiple range test using SPSS on a VAX I l/782 computer. Results were considered statistically significant at the p < 0.05 level. The relative sensitivities of the different indicators were estimated by calculating the percentage decrease for each indicator that would be on the borderline of statistical significance in a one-tailed test (i.e., p = 0.05) using the formula (Bedford, 1983): % decrease
= (Za + ZI-WV)
(N/2)“*
RESULTS Body, Testis, and Epididymal Weights Body, testis, and right epididymal weights from 6-, 8, lo-, 15-, and 20-week-old F-344 rats are presented in Table 1. Body and reproductive organ weights increased significantly through 15 weeks of age; no further increase was observed at 20 weeks of age. Testis weight increased approximately linearly from 6 through 10 weeks of age, and by the 10th week had attained 9 1% of the weight found at 20 weeks. The weight of the right epididymis continued to increase substantially through 15 weeks of age; at 10 weeks of age, it had attained only 66% of its weight at 20 weeks of age. At 6, 8, and 10 weeks of age the left testis was significantly heavier than the right testis; no significant differences were noted at 15 and 20 weeks of age between left and right testis weights. Sperm Production
’
where a = probability of a type 1 error = 0.05; /3 = probability of a type II error = 0.10; 1 - fi = power of the test (probability of rejecting the null hypothesis when in fact it is false) = 0.90; CV = coefficient of variation, %; N = number of rats per treatment group = 15; Z, and Z,+ = standard normal deviates for 01(Z, = 1.645) and 1 - /3 (Z,-, = 1.281).
1099
AND CHARACTERISTICS
Rate (SPR)
Daily SPRs are presented in Table 2. At 6 weeks of age, spermatids could be found in concentrated testicular homogenates prepared from only 7 of 14 males. The mean testis weight for the 6-week-old males with spermatids present in testicular homogenates was
TABLE 1 BODY, TESTES,' AND RIGHT EPIDIDYMAL WEIGHTS (g X 2 SD) OF FISCHER-344 RATS Age (weeks) Variable Body weight Left testis weight Right testis weight Total testes weight Testes weight/ body weight (W) Right epididymal weight
6 119.3 0.593 0.565 1.158
f f k f
8 7.3b 0.0736 0.074” 0.147b
166.0 + 11.5’ 0.946 + 0.084’ 0.887 -I 0.075’ 1.833 + 0.144’
10 222.4 1.224 1.172 2.396
+ 15.1d f 0.059d -t 0.040d zk 0.092d
15 295.7 1.276 1.282 2.556
f + + f
25.4e 0.088”’ 0.069’ 0.141e
20 302.1 1.308 1.318 2.625
+ 42.7’ + 0.070e ztz0.060’ f 0.122’
0.97 rt 0.096
1.11 kO.07’
1.08 f 0.08’
0.87 f 0.06d
0.88 f 0. lod
0.062 zk 0.0 15 b
0.096 f O.OISc
0.233 k 0.20d
0.356 f 0.034e
0.35 1 f 0.033e
’ Testicular parenchyma only. t-r Means within rows with different superscripts are significantly different (p < 0.05) based on one-way analysis of variance and Duncan’s multiple range test.
1100
BLAZAK,
ERNST, AND STEWART TABLE 2
MEAN
(+ SD)
SPERM
PRODUCTIONRATES AND EPIDIDYMAL SPERM NUMBERS, TRANSIT TIMES, AND MOTILITY IN FISCHER-344 RATS Age
Variable Sperm production rate (X 106)” Per rat per day Per gram of testis per day Right epididymal sperm numbers (X106) Per epididymis Per gram of epididymis Right epididymal transit time (days) Left cauda epididymal sperm motility Motile cells (%) Swimming speed (pm/sex)
(weeks)
6
8
10
2.1 f 3.2b 1.6 + 2.3’
21.9 k 5.8’ 11.9 + 2.9’
40.9 f 1.3d 17.1 f 3.1d
49.3 f 6.2’ 19.3 f 2.1’
46.2 f 6.9e 17.7 + 2.9”’
4k5b 34 f 38’
78 + 21’ 333 + 12’
184 f 39d 519 f 107d
128 + 38’ 365 ic 107’
-
7.5 + 2.3 b
7.2 _+ 3.8b
32 f 9’ 68k lib
50 + 8’ 69+5b
-
-
-
-
36 + 10’ 65 + 9b
15
20
a Testicular parenchyma only. k Means within rows with different superscripts are significantly different (p < 0.05) based on one-way analysis of variance and Duncan’s multiple range test.
significantly higher (p < 0.001) than the mean testis weight of males without spermatids (1.273 f 0.067 g vs 1.043 f 0.103 g, respectively). Daily SPR increased significantly through 15 weeks of age. At this age, rats produced approximately 49 X IO6 spermatozoa per day, or 19 X lo6 spermatozoa per gram of testicular parenchyma per day. No further increase in SPR was noted for rats at 20 weeks of age. Although simultaneous increases in testis weight and SPR were observed from weeks 6 through 10, mean testis weight was not significantly correlated with SPR in rats at 10, 15, or 20 weeks of age [correlation coefficient (r) = 0.05, 0.46, and -0.32 at 10, 15, and 20 weeks, respectively]. Thus, the variability in mean testis weight among adult rats may not be an indication of differences in SPR. Epididymal Times
Sperm Numbers and Transit
Epididymal sperm numbers and transit times are presented in Table 2. No sperma-
tozoa were observed within the epididymis from 6-week-old rats. At 8 weeks of age, spermatozoa were observed in the caput and corpus epididymis from all rats studied, but only 3 of 15 animals at this age had spermatozoa in the cauda epididymis. Epididymal sperm numbers significantly increased through 15 weeks of age, but declined at 20 weeks of age. Right epididymal sperm transit times for 15and 20-week-old rats were 7.5 and 7.2 days, respectively (Table 2). Epididymal transit times were not calculated for younger rats because sperm production in these animals had not reached adult levels. The relationship between epididymal sperm numbers and SPR was assessedby calculating the correlation between sperm number per gram of right cauda epididymis and sperm production per gram of right testis for lo-, 15-, and 20-week-old rats. The calculated correlation coefficients at these ages were -0.05, -0.44, and 0.18, respectively (p > 0.05). Thus, SPR and cauda epididymal sperm numbers were not significantly correlated.
RAT SPERM PRODUCTION
Epididymal
Sperm Motility
The results of videomicroscopic studies of the motility of spermatozoa, recovered from the left cauda epididymis of lo-, 15-, and 20week-old rats, are presented in Table 2. The motility of spermatozoa from younger rats could not be studied because of the absence or scarcity of spermatozoa in the cauda epididymis. The percentage of motile cells was not significantly different for spermatozoa from lo- and 15-week-old rats, but increased significantly to 50 + 8% for spermatozoa from 20-week-old rats. The straight-line mean swimming speeds of spermatozoa from IO-, 15-, and 20-week-old rats were not significantly different, with means of 65,68, and 69 pm/set, respectively. Variability of Testicular and Epididymal Endpoints in Adult Rats Estimates of the variation in testicular and epididymal indicators among 20-week-old F344 rats are presented in Table 3. The indicaters with the largest interanimal variation were epididymal sperm numbers and transit times, with CV’s of approximately 30 and 53%,
respectively. The remaining indicators all had CV’s of less than 20%. Knowledge of the variability associated with each indicator allowed us to estimate the percentage decrease, relative to control values, that would be on the borderline of statistical significance in a one-tailed test (i.e., p = 0.05) for each indicator. These sensitivities are presented in Table 3. Thus, a decrease of approximately 16% or more (or 37.4 X IO6 sperm/rat/day) in SPR per rat per day would be statistically significant, whereas a 32% or more decrease in right epididymaI sperm number (or 34 1 X 1O6 sperm) would be required for statistical significance for this indicator. DISCUSSION Male rats are generally considered as sexually mature, effective sires and are used for breeding at 10 to 11 weeks of age (Mather and Rushmer, 1979). Rats at this age and often younger have also been used in studies conceming the potential reproductive effects of chemicals. Our data indicate that male F-344 rats are not sexually mature at 10 weeks of age but attain maturity by 15 weeks based on SPR
TABLE 3 VARIABILITY AMONG ADULT F-344 RATS IN TFSTICULAR Indicator
Range
Testes weight (g) Right epididymal weight (g) Sperm production rate (X I 06) Per rat per day Per g testis per day Right epididymal sperm numbers (X106) Per epididymis Per g epididymis Right epididymal transit time (days) Left cauda epididymal sperm motility Motile cells (W) Swimming speed (pm/set)
2.466-2.862 0.298-0.405 32.0-59.1 11.8-21.3
1101
AND CHARACTERISTICS
AND EPIDIDYMAL
I&SD
INDICATORS”
cv (%)
Sensitivity b
2.625 f 0.122 0.351 + 0.033
4.65 9.40
5 10
46.2 + 6.9 17.1 iz 2.9
14.95 16.56
16 18
65.5-195.0 189.3-533.3 3.3-18.0
128 + 38 365 2 107 7.2 f 3.8
29.69 29.32 52.78
32 31 56
38-62 40-76
50 + 8 69 f 5
16.00 7.24
17 8
DData from 15 rats at 20 weeks of age, except for sperm motility where sperm from 10 rats at 20 weeks of age were studied. b This column lists the percentage decrease in each indicator that would be statistically detected using a sample size of 15 rats per treatment group, the CVs in this table, and (Y= 0.05 and 1-o = 0.90. See Materials and Methods section for details.
1102
BLAZAK.
ERNST, AND STEWART
and epididymal sperm numbers. In male Wistar rats, Robb et al. ( 1978) concluded that sexual maturity in this strain is attained between 75 and 100 days of age based on the same variables. Adult Wistar rats produce 86 X lo6 sperm/day as compared to only 48 X lo6 sperm/day for the ,F-344 rat owing to both the larger testes (3.7 g Wistar, 2.6 g F-344) and the faster rate of sperm production (24 X lo6 cells/g Wistar, 18 X 1O6cells/g F-344) of Wistar rats. Thus, for studies of the effects of potentially hazardous substances on adult male rat reproductive functioning, treatment of the animals should be delayed until approximately 15 weeks of age. Initiating treatment prior to this time has the potential of complicating the interpretation of results because effects on the development of male reproductive functioning and adult reproductive functioning would be confounded. Our finding of an absence of significant correlation between testis weight and SPR indicates that differences in testis weight in untreated rats are not related to SPR. Thus, although large decreases in testis weight will most likely be accompanied by a decrease in SPR, smaller but significant decreases in SPR may not be reflected in a decreased testis weight. Robb et al. ( 1978) also observed a lack of correlation between SPR and testis weight in Wistar rats. Similarly, the lack of correlation between SPR and cauda epididymal sperm number in F-344 rats, as also observed for Wistar rats (Robb et al., 1978), suggests that epididymal sperm numbers are not reliable indicators of effects on testicular SPR. The concentration of spermatozoa in suspensions prepared from the cauda epididymis is presently used by investigators as an indicator of toxic effects on spermatogenesis. Our findings of independence between SPR and cauda epididymal sperm number, and the much higher amount of variation between rats in epididyma1 sperm number as compared with SPR, caution against the continuance of this practice. Although sperm motility is highly correlated with fertility in man, and at least one case has
been published in the rat in which a chemical specifically inhibited sperm motility (McClain and Downing, 1984) little effort has been directed toward the development and use of quantitative methods to assess rodent sperm motility. With our carefully controlled temperature conditions and videomicroscopic system, we found that the consistencies in the percentage of motile cells and in mean swimming speeds of spermatozoa among animals has enabled us to detect decreases of less than 20% in these variables. The use of videomicroscopy for assessing sperm movement has the added advantages that the videotape serves as a permanent record of sperm movement, and the tapes can be analyzed to assesssperm morphology as described in a study of fertile and infertile men (Overstreet et al., 198 1). Our goal in conducting these studies was to determine if the above indicators of rat testicular and epididymal functioning could be employed for assessing chemically induced reproductive effects. The low variability among rats in SPR and sperm movement characteristics suggests that these indicators would be particularly sensitive for detecting deleterious effects. In a recent study of the effects of nitrobenzene on reproductive functioning of male rats (Blazak et al., 1985), we observed an -80% reduction in the number of morphologically normal, motile spermatozoa produced per testis per day in rats exposed to 140 mg/kg/day for 5 consecutive days. Despite the dramatic effect on spermatogenesis observed in these males, a small reduction in the number of implants per pregnant female was the only symptom of a reproductive disturbance noted in breeding studies. Thus, the inclusion of cellular indicators of male reproductive functioning, as described in the present study, together with breeding studies and histopathology, should enable a more complete assessment of the effects of chemicals on male reproduction. ACKNOWLEDGMENTS The authors thank Leslie Diel, Lynne Lyons, and Marilyn Lemmon for excellent technical assistance.
RAT
SPERM
PRODUCTION
REFERENCES AMANN, R. P. ( 1982). Use of animal models for detecting specific alterations in reproduction. Fundurn. Appl. ToxicoI. 2, 13-26. BEDFORD,J. M. (1983). Considerations in evaluating risk to male reproduction. In Advances in Modem Environmental Toxicology, Assessment of Reproductive and Teratogenic Hazards (M. S. Christian, W. M. Galbmith, P. Voytek, and M. A. Mehlman, eds.), Vol. 3, pp. 4198. Princeton Scientific Pub., Princeton, N.J. BLAZAK, W. F., RUSHBROOK, C. J., ERNST, T. L., STEWART, B. E., SPAK, D., DIBIASIO-ERWIN, D., AND BLACK, V. (1985). Relationship between breeding performance and testicular/epididymal functioning in male Sprague-Dawley rats exposed to nitrobenxene (NB). Toxicologist 5, 121 (Abstract No. 484). BLAZAK, W., ERNST, T., DIEL, L., AND STEWART, B. (1984). Assessment of testicular and epididymal func-
AND
CHARACTERISTICS
1103
tioning in Fischer-344 (F344) rats. Toxicologist 4, 81 (Abstract No. 322). KATZ, D. F., AND OVERSTREET, J. W. (198 1). Sperm motility assessmentby videomicrography. Fertil. Steril. 35, 188-193. MATHER, E. C., AND RUSHMER, R. A. (1979). Physiological parameters of some species used in reproduction research. In Animal Models for Research on Contracep tion and Fertility (N. J. Alexander, ed.), pp. 561-576. Harper & Row, Ha%erstown, Md. MCCLAIN, R. M., AND DOWNING, J. C. (1984). The effect of omidazole on fertility and sperm motility in rats. Toxicologist 4, 82 (Abstract No. 323). OVERSTREET, J. W., PRICE,M. J., BLAZAK, W. F., LEWIS, E. L., AND KATZ, D. F. ( 198 1). Simultaneous assessment of human sperm motility and morphology by videomicrography. J. Vrol. 126,357-360. ROBB, G. W., AMANN, R. P., AND KILLIAN, G. J. (1978). Daily sperm production and epididymal sperm reserves of pubertal and adult rats. J. Reprod. Fertil. 54, 103107.