THE MALE RABBIT. VII. STUDIES ON RESORPTION OF PH} THYMIDINE.LABELED SPERMATOZOA IN THE EPIDIDYMIS*

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1 Copyright 1974 The American Fertility Society FERTILITY AND STERILITY Vol. 25, No.3, March, 1974 Printed in U.S.A. THE MALE RABBIT. VII. STUDIES ON RESORPTION OF PH} THYMIDINE.LABELED SPERMATOZOA IN THE EPIDIDYMIS* RUPERT P. AMANN, PH.D., AND JAMES T. LAMBIASE, JR., PH.D. Dairy Breeding Research Center, The Pennsylvania State University, University Park, Pennsylvania For several species, including the rabbit, a discrepancy has been reported between the number of spermatozoa thought to be produced daily by the testes and the number that can be recovered in ejaculated semen or urine.' - 5 The fate of the spermatozoa that are unaccounted for has often been investigated; possible hypotheses include resorption of spermatozoa in the epididymis,6-14 voiding in the urine,'5,lg and, in the rabbit, oozing of sperm from the urethra followed by ingestion.'7 It is also possible as current evidence'5,18 suggests that use of several earlier 4 techniques may lead to an overestimation of the daily sperm production. Conclusions drawn from autoradiographic investigations of the fate of [3H] thymidine-labeled sperm that have been reported are conflicting. After intravenous injection of [3H] thymidine, sperm within the epididymis remain unlabeled until spermatogenesis has been completed in cells formed from preleptotene primary spermatocytes labeled at the time of isotope injection.7,19 In rabbits, these first sperm enter the epididymis 31 to 32 days after [3H] thymidine injection. 20 For 3-kg rabbits, a tenfold increase in the incidence of la- Received July 24, 'Supported by the National Institute of Child Health and Human Development, research grant HD Authorized for publication as paper 4495 in the journal series of the Pennsylvania Agricultural Experiment Station. 271 beled epithelial cells in the proximal caput epididymidis occurred on day 32 after [3H] thymidine injection,7 Labeled sperm had entered the epididymis. A similar increase in epithelial cell labeling was apparent in the midcaput epididymidis by day 36, but the epithelium of the corpus and cauda epididymidis remained virtually unlabeled. It was concluded7 that [3H] thymidine released by breakdown of sperm was incorporated into DNA within the columnar epithelial cells. Despite conflicting evidence/3,h this report continues to be cited. Studies with rats 21 and rabbits 22 indicate that the mitotic index of columnar cells in the epididymal epithelium of adult animals is insignificant. Thus, if thymidine-labeled DNA metabolites are absorbed from degenerating sperm by the epididymal epithelium, incorporation of [3H] thymidine into chromosomal DNA of cells preparing to divide seems unlikely. However, [3H] thymidine absorbed from degenerating sperm might be detectable in autoradiographs of lyophylized tissues. This possibility was considered in designing the present studies to determine whether epithelial cells in the epididymis absorb radioactive metabolites originating from spermatozoal DNA. MATERIALS AND METHODS Experiment 1. Eighteen-month-old New Zealand white rabbits were killed 31, 33, or 38 days after the intravenous injection

2 272 AMANN AND LAMBIASE March 1974 of [3H] thymidine (0.25 mci/kg body weight). Three sexually rested bucks and three bucks ejaculated once daily, starting on day 20 (after injection), were killed after each interval. The left epididymis was fixed in Bouin-Hollande fluid and infiltrated with Paraplast. Sections of the caput, corpus, and cauda epididymidis were cut at 5-p,m, mounted on slides, and deparaffinized. Kodak NTB-3 emulsion was used for the autoradiographs, which were developed after six weeks' exposure at 5 C and stained with hematoxylin. The cytoplasm and nucleus of 1,500 columnar cells in the efferent ducts and each of the eight regions of the epididymis23 were examined and classed as unlabeled (0-3 grains) or labeled (> 3 grains). The degree of labeling in cells of the luminal sperm mass also was evaluated. Differences in the percentages of labeled nuclei within regions between sexually rested and ejaculated bucks and before and after labeled sperm had entered the lumen of that region were evaluated by use of the t test. Experiment 2. To permit detection of labeled DNA metabolites that might have been leached from the epididymides during conventional histologic processing, 14 1-year-old New Zealand white rabbits were treated by injection with [3H] thymidine (0.25 mci/kg) after 30 days of sexual rest. Two bucks were killed 40 minutes after the injection and groups of four were killed 30, 31, or 32 days thereafter. Three nonradioactive control rabbits were killed after 30 days of sexual rest. For each buck, tissue was prepared by freeze-drying and paraffin infiltration. The caput of one epididymis, taken from alternate sides in succeeding rabbits, was trimmed and immersed in liquid nitrogen. Subsequently, 5-p,m sections, cut in a cryostat at about -25 C, were manipulated into a cold plastic capsule without allowing them to thaw. The capsules, chilled with dry ice, were placed in a lyophylization chamber maintained at -75 C. The frozen tissues were dried in vacuo and the sections were stored in a dessicator over Drierite. The contralateral caput epididymidis was fixed and processed as in experiment 1 except that autoradiographs were exposed for only four weeks. For autoradiography of freeze-dried tissues,2'. clean slides were coated with NTB-3 and dried overnight at room temperature. Freeze-dried tissue sections were applied to these slides as follows: A strip of black plastic insulative tape, slightly shorter than the emulsion coat on the slides, was adhered to a clean polyethylene pad. Two 25-mm Millipore filters were placed side by side on the strip of tape. Under a Wratten OA safelight, tissue sections were placed on the filters and flattened with sable brushes. The yellow safelight was turned off, leaving a red Wratten no. 2 safelight to illuminate the work area. An emulsioncoated slide was removed from the lighttight slide box, aligned along the tape strip, and pressed down. The tissue sections readily adhered to the emulsion. The autoradiographs were exposed at -15 C for four weeks, developed, and stained with hematoxylin. The nuclear area and cytoplasm of 1,500 columnar cells in each region of the epididymides were evaluated. RESULTS In experiment 1, there was no difference between sexually rested and ejaculated rabbits with respect to the incidence of nuclear labeling within regions. The combined data (Table 1) revealed no significant differences in the incidences of nuclear labeling in regions of the epididymis containing labeled sperm in the lumen and corresponding regions from other ra b bits where labeled sperm had not progressed that far. In all regions, only a

3 Vol. 25, No.3 [OH] THYMIDINE-LABELED SPERMATOZOA 273 TABLE 1. Incidence of Labeled Nuclei in Epithelium of Each Region of Epididymis Before and After Appearance of Labeled Sperm Within Tubule Lumen Labeled sperm present in No. of Labeling index lumen epididymidesa C%) Region Before After Before After Before After Efferent ducts No Yes No Yes No Yes No Yes No Yes No Yes No Yes No Yes No Yes asix rabbits were killed on days 31, 33, and 38 after [3R] thymidine injection and evaluation of one epididymis from each of the 18 rabbits. background distribution and level of grains were found over the epithelial cell cytoplasm. Autoradiographs in experiment 2 should have permitted detection of any [3H] thymidine liberated from sperm in the caput epididymidis. The extent of labeling in columnar cells of tissues prepared by paraffin infiltration and by freezedrying (Table 2) was not greatly different. Labeling over the cytoplasm was TABLE 2. Incidence of Labeled Nuclei in Autoradiographs Prepared After Paraffin Infiltration of Freeze-Drying No. of Labeling Region Labeled sperm epididymidcsa index (%) of present in epididymis hunen PI' FD' PI' FD' Efferent ducts No Yes No Yes No Yes "Epididymides from 12 rabbits killed on days 30, 31, or 32 after [3R] thymidine injection. Because of high background labeling over freezedried sections, data for one male killed on day 31 were deleted. Insufficient freeze-dried efferent duct tissue was available for two males. b After paraffin infiltration. C After freeze-drying. at background levels for all tissues. Thus, it seems unlikely that DNA metabolites were leached out during conventional fixation and dehydration of the cells examined in experiment 1. The intervals of 30, 31, and 32 days after injection were selected for experiment 2 in anticipation of finding that labeled sperm had penetrated as far as region 5 of the caput epididymidis.'9 2" However, as might be expected from subsequent studies,20 labeled sperm had entered the epididymides in only three rabbits and had progressed no further than region 3. Therefore, areas distal to region 3 were not evaluated. Since labeled sperm were not detected in region 3 of the freeze-dried epididymides, data for this region were not included in Table 2. DISCUSSION Results of these experiments indicate that the epithelial cells of the rabbit epididymis do not incorporate [3H] thymidine from labeled sperm. Others/ 3014 studying [3H] thymidine-injected rabbits in which segments of the epididymis were isolated by means of ligatures in an attempt to promote sperm resorption, have arrived at the same conclusion. The labeling index for columnar cell nuclei in the epididymal epithelium (Table 1) averaged only 0.98 %; this is comparable to that reported for rats. For epididymides of 4-month-old or 12-monthold rats killed 4 hours after [3H] thymidine injection,21 the mean rate of columnar cell labeling was less than 0.6%, and this level of labeling was maintained for at least 50 days after isotope Injection. The low turnover of epithelial cells in epididymides of adult males, as contrasted with higher rates in younger rats,21 might explain the apparent contradiction between the early data 7 for rabbits and the data of subsequent studies.'3,i< In the present study, the incidence of

4 274 AMANN AND LAMBIASE March 1974 epithelial labeling in each region was similar for tubule cross sections with and without labeled sperm. Nothing approaching a tenfold increase 7 in labeling for region 1 was detected after entry of labeled sperm. The labeling indexes for the efferent ducts and regions 1, 2, and 3 of the paraffin infiltrated epididymides from the two bucks killed 40 minutes after C3H] thymidine injection averaged 0.33, 0.67, 0.47, and 0.20%. Therefore, the cells will incorporate [3H] thymidine when it is available; the labeled nuclei observed in other males 30 to 32 days later probably had been labeled at the time of [3H] thymidine injection. Our results demonstrate that the nuclei of rabbit epididymal epithelial cells do not take up DNA from degenerating sperm. However, they do not provide conclusive proof that sperm resorption does not occur in the epididymis. Labeled DNA metabolites from sperm might be present in concentrations below the threshold necessary for autoradiographic detection. Despite evidence that only a small fraction of the calculated daily sperm production of some sexually rested rabbits can be recovered in the urine16 and evidence for a high concentration of acid DNase in the caput of the rabbit epididymis,26 we conclude that sperm resorption probably does not occur in the rabbit epididymis. This conclusion is substantiated by quantitative data relating to bulls and rams.15,1s SUMMARY Epididymides from 35 adult rabbits were evaluated for evidence indicating resorption of spermatozoal [3H] thymidine by the epithelial cells lining the duct. Autoradiographs were prepared with the aid of both paraffin infiltrated and freeze-dried tissues. The incidence of labeled columnar epithelial cells in the caput epididymidis was similar before and after the entry of [3H] thymidinelabeled sperm. Because results with freeze-dried tissues confirmed those for para:ffin processed tissues, the low incidence of cell division and associated DNA synthesis by the columnar cells probably was not a factor limiting the results. It was concluded that sperm resorption normally does not occur in the rabbit epididymis. REFERENCES 1. Orgebin-Crist MC: Gonadal and epididymal sperm reserves in the rabbit: estimation of the daily sperm production. J Reprod Fertil 15: 15, Lambiase JT Jr, Amann RP: The male rabbit. V. Changes in sperm reserves and resorption rate induced by ejaculation and sexual rest. J Anim Sci 28:542, Amann RP: The male rabbit. IV. Quantitative testicular histology and comparisons between daily sperm production as determined histologically and daily sperm output. Fertil Steril 21:662, Amann RP: Sperm production rates. In The Testis, Vol 1. Edited by AD Johnson, WL Gomes, NL VanDemark. New York, Academic Press, 1970, p Carson WS, Amann RP: The male rabbit. VI. Effects of ejaculation and season on testicular size and function. J Anim Sci 34: 302, Amann RP, Almquist JO: Reproductive capacity of dairy bulls. VI. Effect of unilateral vasectomy and ejaculation frequency on sperm reserves; aspects of epididymal physiology. J Reprod Fertil 3:260, Orgebin-Crist MC: Delayed incorporation of thymidine 3H in epithelial cells of the ductus epididymidis of the rabbit. J Reprod Fertil 8:259, Paufler SK, Foote RH: Sperm retention and resorption in sexually active rabbits with epididymal ligatures. Proc Soc Exp BioI Med 131:1179, Macmillan KL, Desjardins C, Kirton KT, et al: Gonadal and extragonadal sperm reserves after unilateral vasoligation in rabbits. Fertil Steril 19:982, Igboeli G, Rakha AM: Bull testicular and epididymal functions after long-term vasectomy. J Anim Sci 31:72, 1970

5 Vol. 25, No.3 [3 H ] THYMIDINE-LABELED SPERMATOZOA 275 I If 11. Salamon S: Viability and morphology of ram spermatozoa in the ligated epididymis. Aust J BioI Sci 21: 769, Zankl H: Tierartliche Unterschiede der Spermienresorption 1m Nebenhoden. Zuchthyg 5:32, Fulka J, Kopecny V, Koefoed-Johnsen HH: Tritium activity in spermatozoa and epididymal epithelium after application of thymidine-methyl-h3 and passage blocking by ligature. Fertil Steril 22: 119, Paufler S: Qualitative und quantitative Untersuchungen der Spermiogenese des Kaninchens mit besonderer Beriicksichtigung der histologischen Beurteilungsmethoden und der Spermatozoen-Verluste in Hoden und Nebenhoden. Dissertation, Georg-August University, Gottingen, Lino BF, Braden AWH: The output of spermatozoa in rams. 1. Relationship with testicular output of spermatozoa and the effect of ejaculations. Aust J BioI Sci 25: 351, Holtz W, Foote RH: Sperm production, output and urinary loss III the rabbit. Proc Soc Exp BioI Med 141:958, Swanson LV, Hafs HD: Testicular and epididymal sperm numbers in unilaterally vasoligated rabbits. Proc Soc Exp BioI Med 131:763, Amann RP, Kavanaugh JF, Griel LC Jr, et al: Sperm production of Holstein bulls determined from testicular spermatid reserves, after cannulation of rete testis or vas deferens, and by daily ejaculation. J Dairy Sci 57: 93, Amann RP, Koefoed-Johnsen HH, Levi H: Excretion pattern of labelled spermatozoa and the timing of spermatozoa formation and epididymal transit III rabbits injected with thymidine-3h. J Reprod Fertil 10: 169, Amann RP: The effect of variations in the duration of rabbit spermatogenesis on determinations of sperm epididymal transit time. Proc VII Inter Congr Anim Reprod 1:431, Clermont Y, Flannery J: Mitotic activity in the epithelium of the epididymis in young and old adult rats. BioI Reprod 3:283, Amann RP: Unpublished data, Nicander L: On the regional histology and cytochemistry of the ductus epididymidis in rabbits. Acta Morphol Neerl Scand 1:99, Stumpf WE, Roth LJ: High resolution autoradiography with dry-mounted freeze-dried frozen sections. Comparative study of six methods using two diffusible compounds 3H estradiol and 3H mesobilirubinogen. J Histochern Cytochem 14:274, Orgebin-Crist MC: Passage of spermatozoa labelled with thymidine-3h through the ductus epididymidis of the rabbit. J Reprod Fertil 10:241, Pinero GJ, Roussel JD: On the presence of deoxyribonuclease (DNASE) III the rabbit epididymis and its possible significance III the removal of spermatozoa. 24: 145, 1973 Fertil Steril ~ I