THE EFFECT OF COPPER IMPLANTS IN THE REMINAL VESICLES ON FERTILITY OF THE RAT, RABBIT, AND HAMSTER*

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1 FERTILITY A(\O Sn:HILIT'l Copyright 1973 by The Williams & Wilkins Co. Vol. 24, :-';0. 1..January 1973 Printed in U.S.A. THE EFFECT OF COPPER IMPLANTS IN THE REMINAL VESICLES ON FERTILITY OF THE RAT, RABBIT, AND HAMSTER* DESMOND p, GILMORE, PH,D"t RUSSELL H, HOOKER, M,D., AND BRIAN K. DAVIS, PH.D. Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts Metallic copper has been thought for some time to have a toxic effect on sperm. 1 Mann 2 suggested that its spermicidal activity may be due to sulfydryl binding by copper ions. Mouse ova in culture are unable to tolerate the presence of metallic copper ions. 3 The contraceptive effect of the intrauterine device is increased by the incorporation of copper wire,4 and a sperm-inactivating effect has been demonstrated when copper foil is incubated with human cervical mucus. 5 A copper wire placed in the uterus of the rabbit prevents implantation in the vicinity of the wire but has no demonstrably harmful effect on sperm transport and fertilization of eggs. 6 This paper records observations on the effect on fertility of a copper wire placed in the seminal vesical of the rat, rabbit, and hamster. MATERIALS AND METHODS Mature Sprague-Dawley rats, golden hamsters, and rabbits of mixed breeds of proven fertility were used as the experimental animals. Twenty-three male rats were operated under general anesthesia and aseptic conditions. A small incision was made in the lower abdomen and a Silastic tube (Fig. la) or a 5-mm. loop of copper wire (Fig. 1E) placed through a puncture into the Received July 6, * Supported by a grant from The Ford Foundation, and a grant (GM 14370) from the National Institutes of Health, United States Public Health Service, awarded to Dr. M. C. Chang. t Present address: Institute of Physiology, The University, Glasgow W2, Scotland. 60 lumen of one or both seminal vesicles and secured with a suture of fine silk. The seminal vesicles were returned to their natural position and the wound closed. Eight to 10 days after surgery each male was placed overnight with a mature proestrous female. Mating was verified either by the presence of a plug and/or sperm in a vaginal smear on the following morning, or if the female went into prolonged vaginal diestrus indicating pregnancy or pseudopregnancy. All females which were found to have mated were sacrificed after 8 days and the number of implantations recorded. Fertility tests were made on each male at approximately 10-day intervals for up to 61 days, after which time the foreign bodies were removed from the seminal vesicles. Fertility tests were again resumed at the same intervals, after allowing a 10-day recovery period, until one or more normal pregnancies had occurred or until six or eight confirmed matings failed to result ill a pregnancy. For comparison similar fertility tests were undertaken on a group of male rats from which one or both seminal vesicles had been surgically removed. At the conclusions of the study each male animal was sacrificed and the reproductive tract examined. Three male rabbits, each with satisfactory sex drive, normal semen, and sperm counts in excess of 100 million, were operated under general anesthesia and sterile technic. The lower abdomen was opened and a 2-cm. loop of copper wire (Fig. 1e) placed through a small in-

January 1973 61 COPPER IMPLANT IN SEMINAL VESICLES cis ion into the lumen of the seminal vesicle (glandula vesicularis) and secured with a suture of fine silk.

Thereafter semen was collected by means of an artificial vagina one to three times a week for about 6 weeks and the number of sperm counted.

2 January COPPER IMPLANT IN SEMINAL VESICLES cis ion into the lumen of the seminal vesicle (glandula vesicularis) and secured with a suture of fine silk. The incision in the seminal vesicle was closed, and the abdominal wall repaired using interrupted silk throughout. One week was allowed for recovery. Thereafter semen was collected by means of an artificial vagina one to three times a week for about 6 weeks and the number of sperm counted. One month after placement of the copper wire in the seminal vesicle each male rabbit was twice permitted to inseminate a female. Twenty-four hours later the left fallopian tube of the mated female was surgically removed, flushed, and the washed ova examined microscopically for evidences of fertilization. Two weeks later the does were sacrificed, and the presence or absence of implantation sites recorded. Corpora lutea in the residual right ovary were noted. Approximately 6 weeks after placement of the copper wire in the seminal vesicle, four semen collections were made from each operated male at 2- to 3-day intervals, and a similar number of semen collections made from a normal, healthy control buck. Each semen sample was assayed by atomic absorption spectrophotometry (Perkin Elmer model 290B) for the presence of copper. Seven hamsters were operated in a manner similar to that in the rats, save that a 5-mm. coil of copper wire (Fig. 1D) in- stead of a loop, was placed in the seminal vesicles. Fertility tests were made at intervals of approximately 1 week and were confirmed by the presence of sperm in the vaginal smear. RESULTS The results of the study are summarized in Table 1. The presence of a copper wire in the lumen of both seminal vesicles of the rat resulted in a 91% decrease in conception rate, and the number of implants in those females that did conceive was also significantly depressed (mean 3.0 ± 1.0). This effect was comparable to that produced by surgical removal of both seminal vesicles. The effect on fertility of a Silastic tube in the seminal vesicles was similar, but less pronounced. After removal of the wire or tube, the conception rate increased slightly, and a near-normal number of implants was observed. No depression of fertility occurred in the rabbit. In the hamster there was a 25% decrease in conception rate, but the number of implants remained normal. The seminal vesicular secretion of the rabbit and hamster was of fluid consistency and darker in color in the presence of copper. That of the rat did not appear discolored, but was often coagulated (Table 2). The copper wire at the time of FIG. 1. Deposits in the seminal vesicle. A, Silastic tube (1.57 mm. I.D., 2.42 mm. O.D.). B, copper wire (32 gauge, surface area approximately 0.12 sq. em.) for rats. C, copper wire (28 gauge, surface area approximately 0.22 sq. em.) for rabbits. D, copper wire (40 gauge, surface area approximately 0.74 sq. em.) for hamsters.

3 62 GILMORE ET AL. Vol. 24 TABLE l. Effects of Copper Wire Placed in the Seminal Vesicle on Fertility of the Male, Rabbit, and Hamster (and of Other Treatments of the Seminal Vesicles of the ) No. of fer- No. of Matings that Average implants tile male Mating females failed to result per pregnancy animals Treatment period mated* in pregnancy (Mean ± S.E.) days % 5 Nil ± Copper wire Unilateral ± Bilateral ± 1.00 Wire removed ± Silastic tube Unilateral ± Bilateral ± 1.44 Silastic tube removed ± Removal of seminal vesicles Unilateral ± Bilateral ± 1.50 Rabbit 1 Nil ± 0.04}t 3 Copper wire ± 2.33 Hamster 9 Nil ± Copper wire Bilateral ± 1.09 All matings confirmed by presence of sperm in vaginal smear. t Based on one uterine horn. Data for untreated rabbit taken from Dr. M. C. Chang (7). TABLE 2. Coagulation of Seminal Vesicle Secretion and Infertility of Male s Days of Days after treatment the removal Treatment (Mean ± S.E.) (Mean ± S.E.) Copper wire Unilateral ± Bilateral ± ± 8.17 Silastic tube Unilateral 34 0 Bilateral ± 5.90 Matings that No. of Secretion in No. of failed to result animals seminal vesicle matings in pregnancy 4 Normal Coagulated Normal Coagulated Normal 0 Coagulated Normal Coagulated 0 % removal from the seminal vesicle of all the rats and rabbits and most of the hamsters appeared darkened, suggesting some oxidation. Assay for copper in the four semen samples collected from each of the experimental rabbits showed a marked elevation of cupric ions in one sample from each animal (Table 3). Although assay of the semen from a control buck also showed the presence of a small amount of copper, there was no elevation in any sample. DISCUSSION The seminal vesicles are well developed III some mammalian species, but are

4 January 1973 COPPER IMPLANT IN SEMINAL VESICLES 63 absent from others, and their role in reproduction is not fully understood. Although removal of the seminal vesicles causes marked reduction of fertility in the rat,8 their absence does not affect fertility in the hamster.9 Chang, Hanada, and Hunt 10 found that rabbit seminal plasma does not play an important role in maintaining the fertilizing capacity of rabbit spermatozoa. The effect of a copper wire placed in the seminal vesicle of the rat, rabbit, and hamster is in accord with the findings of these earlier studies. Fertility of the rat is significantly depressed; hamster fertility is, perhaps, slightly reduced; and the rabbit is unaffected. When examined at autopsy the secretion in the seminal vesicle was frequently found to be coagulated in the rat, but not in the rabbit or hamster. Coagulation occurred in both the copper- and Silastictreated groups of rats and was accompanied by a marked reduction in fertility. Although the effect produced in the rat is perhaps caused merely by coagulation of the seminal vesicular secretion, our data tend to discount this. Whether or not the seminal vesicular secretion was observed to be coagulated at autopsy, the fertility of those animals into which copper had been inserted was reduced to a greater extent that those treated with Silastic or with surgical removal of the seminal vesicle. The reaction involved in coagulation of the seminal vesicle secretion is not clear. If it was due to a change in protein at the metal surface with resultant precipitation of protein,5 why did it also occur in the rat treated with Silastic, and why did it not occur in the rabbit and hamster treated with copper? Recovery of fertility in the rat following treatment of the seminal vesicle is of interest. While the implants, either of copper wire or Silas tic tube, were present in both seminal vesicles, fertility was sig- TABLE 3. Concentrotion of Copper in Rabbit Semen Copper wire in seminal vesicle Approx. surface Rabbit Gauge area Dumtion Cu+ + in semen sample sq. mm. days p.p.m. 26 > Control lo5t 2.5 Atomic absorption spectrophotometry, Perkin Elmer model 290 B. t Three separate samples were pooled. nificantly depressed. Upon removal of the implants, fertility was to a large extent restored, but to a lesser degree in the copper-treated group, even in those that were found at autopsy to have what appeared to be normal seminal vesicular secretion. Fertility in the treated rabbits was unaffected, but there was a significant rise in the amount of copper in their semen as compared to a control. These findings suggest that the copper did become part of the ejaculate, but was present only intermittently and without regularity and in amounts too small to interfere with fertility. SUMMARY Copper wire or Silastic tubes placed in the seminal vesicles of male rats depressed their fertility as measured by the percentage of fertile matings and also the number of implantations per fertile mating. Partial recovery ensued following the removal of the wire or tube. Evaluation of the effect was complicated by frequent coagulation of the secretion of the seminal vesicle. The fertility of male rabbits was not affected by copper wire placed in the seminal vesicle. An occasional rise in the amount of copper present in the semen was detected. A copper wire placed in the seminal vesicles of male hamsters depressed their

5 64 GILMORE ET AL. Vol. 24 fertility slightly as measured by the percentage of fertile matings. Sex drive and the ejaculatory reflex appeared to be unaffected in any of the animals. Acknowledgments. The authors are deeply indebted to Dr. M. C. Chang for his help and constructive criticism throughout this study and to Mrs. Rose Bartke for her excellent technical assistance. REFERENCES 1. WHITE, I. G. The interaction between metals and chelating agents in mammalian spermatozoa. J Exp Bioi 33:422, MANN, T. Biochemical basis of spermicidal activity. Studies Fertil 9:3, WHITTINGHAM, D. G. Culture of mouse ova. J Reprod Fertil (Suppl. 14):7, ZIPPER, J. A., TATUM, H. J., PASTENE, L.,. MEDEL, M., AND RIVERA, M. Metallic copper as an intrauterine adjunct to the "t" device. Amer J Obstet Gynec 105:1274, OSTER, G. K. Reaction of metallic copper with biological substrates. Nature (London) 234:153, POLIDORO, J. P., AND BLACK, D. L. The failure of the copper IUD to inhibit fertilization in the rabbit. J Reprod Fertil 23:151, CHANG, M. C. An experimental analysis of female sterility in the rabbit. Fertil Steril 3:251, BLANDAU, R. J. On the factors involved in sperm transport through the cervix uteri of the albino rat. Amer J Anat 77:253, WEINERTH, J. L., BATfAGLIA, C. R., AND MAGAL HAES, H. The non-essential nature of seminal vesicles in golden hamsters (abstr.). Amer Zooll: 397, CHANG, M. C., HANADA, A., AND HUNT, D. M. Effects of seminal plasma on the fertilizing capacity of rabbit spermatozoa in relation to the time of ovulation. J Reprod Fertil 27:125, 1971.

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