Changes in the Structure and Function of the Testes and Epididymides in Vasectomized Rams*

Vol. 29, No.3, March 1978 Printed in U.SA.

FERTILITY AND STERILITY Copyright

©

1978 The American Fertility Society

CHANGES IN THE STRUCTURE AND FUNCTION OF THE TESTES AND EPIDIDYMIDES IN VASECTOMIZED RAMS*

B. M. A. OSWIN PERERA, B.V.Sc., PH.D.t

Department of Veterinary Reproduction, University of Glasgow Veterinary School, Bearsden, Glasgow, G61 lQH, Scotland

Vasectomized rams were studied with regard to the structure and function of their testes and epididymides at different periods up to 3 years and 9 months after the operation, and compared with intact control rams. Control rams showed a marked seasonal pattern with higher values for size and weight of testes, diameter of seminiferous tubules, Sertoli cell ratios for germ cells, and size of cauda epididymidis immediately prior to and during the breeding season than at other times of the year. In vasectomized animals the seasonal pattern was ill-defined, the testicular parameters were lower, and the cauda epididymidis was larger than in intact rams. Spermatogenesis was qualitatively and quantitatively affected after vasectomy, with a sequential ebb-and-flow pattern of spermatogenic arrest and hypospermatogenesis. Epididymal changes included the development of multiple spermatoceles surrounded by a granulomatous reaction, and disturbances in the process of sperm maturation.

to have been studied in detail from this viewpoint. A study was therefore undertaken to examine the responses to vasectomy of the reproductive system in adult rams. 8- 10 This paper describes the changes observed· in the testes and epididymides.

Vasectomy has become increasingly popular as a method of male fertility control in humans. 1. 2 In spite of numerous studies on the effects of vasectomy on reproductive organs and their functions over varying periods of time, in a variety of domestic and laboratory animals, the results remain largely contradictory.3 The lack of uniformity in results and the resultant confusion may be attributable to factors such as different surgical techniques,4 the incidence of postoperative complications, 5 variability due to different postoperative periods, 6 and the failure in many studies to use quantitative methods for assessing spermatogenic activity. When the natural pathway to the exterior for products ofthe testis and epididymis is blocked at the level of the vas deferens, different species appear to respond in different ways.7 The mechanisms invoked by the body to overcome the physiologic problem with which it is faced afford an insight into the peculiarities of the reproductive system in each species. The ram does not appear

MATERIALS AND METHODS

Animals. Intact adult rams that were found to be reproductively sound after examination of genital organs and semen were selected for the study. Of a total of 32 such animals, 12 were vasectomized through incisions made on the neck of the scrotum. l l The vas was double-ligated and the intervening segment was excised. The remaining animals were kept as controls. Vasectomized rams were killed at different times of the year, at periods ranging from 3 to 45 months after the operation. The control animals were killed at corresponding times of the year. Additional material was obtained from 7 animals that had been vasectomized before the initiation of the study and from 38 intact rams killed at the Glasgow abattoir. The dimensions of the testes and caudae epididymides were recorded in all vasectomized and control animals at intervals of 1 to 2 weeks. At

Received September 9, 1977; accepted October 18, 1977. *Supported in part by the Wellcome Foundation. tPresent address and address for reprint requests: Department of Veterinary Clinical Studies, University of Sri Lanka, Peradeniya Campus, Sri Lanka.

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slaughter the scrotal organs were removed, measured, and weighed. Samples of tissues and luminal fluid were collected from a number of foci in the testis; caput, corpus, and cauda epididymides; and the segment of vas deferens lying close to the testis. Histology. Tissues were fixed in Bouin's fluid and processed for histologic study. Routine staining was done with hematoxylin and eosin (H & E), while selected sections were also stained by the periodic acid-Schiff technique. The germinal epithelium of the testis was studied quantitatively by the following parameters: (1) Measurements of the seminiferous tubule diameter (STD) were performed by using a calibrated ocular graticule on 30 intact seminiferous tubules cut in transverse section per sample. (2) The tubular cell count method8 • 13 was used to obtain germ cell counts. The total number of germ cell nuclei of each different category within 30 seminiferous tubule sections was divided by the number of Sertoli cell nuclei, to arrive at the Sertoli cell ratio (SCR). Luminal Fluid. The fluid collected from the testis and epididymis was examined immediately for presence of spermatozoa (sperm concentration classified from 0 to 5) and their motility (degree of movement classified from 0 to 4). The fluid was also stained with nigrosin-eosin, and the smears were examined for stainability and morphologic details of spermatozoa.

RESULTS

Gross Structure. In the intact rams a seasonal variation was evident in testicular size. The length of the testis, from the dorsal to the ventral pole, was greatest from July to December, and ranged from 8.9 ± 0.71 cm (mean ± standard deviation) to 9.4 ± 1.55 cm (56 observations). From January to June the values were 6.4 ± 0.74 cm to 7.3 ± 0.49 cm (54 observations). In the vasectomized rams the seasonal pattern was less marked, but the mean length was lower than that for intact rams during all months of the year. The difference was significant (P < 0.05) during March, April, July, August, and October. Serial measurements on individual animals before and after vasectomy showed that testicular size was reduced during the initial 2 to 3 months after the operation and was followed by irregular fluctuations with no uniform pattern in relation to the postvasectomy period or the season.

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FIG. 1. Histology of a testis from an intact control ram, taken during the breeding season, showing active spermatogenesis (H & E, x200).

The size of the cauda epididymidis also showed seasonal variations in intact rams. The length of the organ (from the testicular border to the free ventral pole) was greater during September to February (>2.5 cm) than at other times of the year. The mean value for vasectomized animals (3.69 ± 0.89 cm, 110 observations) was significantly higher (P < 0.001) than that for intact rams (2.48 ± 0.39 cm, 112 observations). The appearance and consistency of the testes and epididymides of vasectomized rams ranged from slightly to grossly abnormal when examined at slaughter. The chief abnormalities in the testis were reduction of turgor and tonicity and the presence of adhesions between the visceral and parietal layers of the tunica vaginalis. The caudae epididymides of vasectomized rams were invariably enlarged and firm. The mean weights were 48.75 ± 20.94 gm in 15 vasectomized rams and 11.25 ± 5.54 gm in 6 intact rams (P < 0.001). Single or multiple spermatoceles (sperm granulomata) were present in all vasectomized animals, either in the cauda epididymidis or in the vas deferens, and these ranged from 0.5 to 3.5 cm in diameter. In some animals evidence was seen of previous rupture ofthe serous membrane covering the cauda epididymidis, with subsequent fibrosis and adhesions between the layers of the tunica vaginalis. In two vasectomized rams the cavity between the layers of the tunica contained cheesy material. The material on examination consisted of degenerating spermatozoa with detached heads. Histologic Structure. In intact rams the activity ofthe spermatogenic tissue showed seasonal variations. During the breeding season and the period

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TABLE 1. Mean Diameter of Seminiferous Tubules and Sertoli Cell Ratios for Different Groups of Germ Cells in a Representative Group of Intact and Vasectomized Rams Ram no.

Sertoli cell ratios" Status"

Month

STD"

Spg

Early Spc

Late Spc

Early Spt

Late Spt

1.08 0.57 1.14 0.62 1.38 0.85 0.67 0.67 0.89 0.87 0.46 0.79 0.77 0.59 0.56 0.28 0.48 0.80

2.64 1.46 3.36 2.19 3.30 2.87 2.06 0.68 2.80 2.47 0.78 0.81 2.54 0.58 1.26 0.04 0.63 1.68

4.78 2.10 4.88 3.23 6.26 5.41 3.58 1.22 5.30 3.94 0.42 0.67 4.37 1.30 2.22 0.45 0.61 2.81

8.35 3.71 9.24 5.52 9.58 9.50 6.67 2.49 11.31 6.83 1.11 0.20 5.71 2.99 3.74 0.31 0.81 4.56

12.36 4.92 11.50 5.66 9.53 11.65 8.27 3.11 14.54 7.07 1.46 0.53 7.58 5.94 5.98 0.35 1.06 5.03

p.m

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

I I I I I I I V (3 mol V (4 mol V (6 mol V (6 mol V (7 mol V (9 mol V (12 mol V (24 mol V (30 mol V (36 mol V (45 mol

Apr Apr Jun Jul Jul Oct Dec Jun Dec Sep Dec Apr Aug Dec Aug Feb Jun Jul

187.9 179.8 208.0 187.7 210.8 243.5 206.3 163.8 244.2 210.7 184.9 126.5 224.3 160.7 182.8 119.9 150.3 184.8

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

22.9 11.0 16.2 13.2 27.8 27.8 13.3 14.6 21.6 13.1 14.0 11.1 17.3 20.0 16.1 11.0 13.6 12.1

aI, intact; V, vasectomized (time postvasectomy is indicated in parentheses). bSTD, seminiferous tubule diameter, means ± standard deviation. CSpg, spermatogonia; Spc, spermatocytes (early = preleptotene, leptotene, and zygotene; late secondary spermatocytes); Spt, spermatids (early = round; late = elongated).

immediately preceding it (July to December), the testes were actively engaged in seprmatogenesis (Fig. 1). This was reflected in the higher values obtained for STD and SCR (Table 1) during this period. In the vasectomized rams a wide variation which was unrelated to seasonal patterns was seen in these two parameters of spermatogenic activity. In the majority of this group, STD and SCR were markedly lower than the values obtained in intact rams killed at comparable times of the year. Qualitative changes observed in the seminiferous tubules of vasectomized rams included pyknotic changes and sloughing of spermatocytes and spermatids, and the presence of immature germ cells within the lumina (Fig. 2). In some cases an abnormal condensation of the elongating spermatids was seen (Fig. 3). Even in testes with complete spermatogenic arrest, however, the spermatogonia, Sertoli cell nuclei, and interstitial (Leydig) cells appeared normal. In the epididymides of vasectomized animals the regional characteristics of the epithelium were unchanged in the caput and the corpus. The caudae in a few animals showed irregular epithelial cells with cytoplasmic condensation and elongation of nuclei. The luminal contents in all regions were markedly altered in vasectomized rams, with immature or abnormal germ cells (Fig. 4) and macrophages being frequently observed. The spermatoceles in vasectomized

= pachytene, diplotene, and

animals consisted of masses of spermatozoa surrounded by a typical granulomatous reaction composed of epithelial cells, multinucleated giant cells, and other inflammatory cells.

Spermatozoa in Luminal Fluid. In intact rams the sperm concentration was low in fluid from the testis and caput epidiymidis, higher in that from the corpus, and maximal in that from the cauda and vas deferens. Sperm from the testis and caput were nonmotile, but a few showed sluggish movements of their flagella. Flagellar movements were more marked in sperm from the corpus, while those from the cauda and vas were

FIG. 2. Histology of a testis from a vasectomized ram showing hypospermatogenesis with sloughed germ cells in tubular lumina (H & E, x200).

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matozoa with pyriform heads and proximal cytoplasmic droplets were limited mainly to the testis and caput in intact rams. In contrast, such spermatozoa were present in high proportions within the cauda and vas in vasectomized rams, indicating disturbances in the process of epididymal sperm maturation. The spermatoceles in the cauda and vas consisted of dense masses of degenerating spermatozoa. In some cases a few motile spermatozoa were also observed. Bacteriologic culture of material from these lesions or from the cheesy masses present in the cavum vaginale did not yield any pathogenic organisms. FIG. 3. Histology of a testis from a vasectomized ram showing spermatogenic arrest and abnormal spermatids (arrow) (H & E, x 800).

actively motile, with wave patterns due to mass movement. In contrast, fluid from vasectomized animals showed an inconsistent pattern of sperm concentration and motility, the latter being low in most regions of the epididymis. The morphologic characteristics of spermatozoa in the different regions of intact and vasectomized rams are given in Table 2. In intact rams the percentage of tail-less heads was high in the initial regions and became progressively lower in the distal regions. In vasectomized rams, however, the percentage of tail-less heads progressively increased along the duct. Darkly staining spermatozoa (eosinophilic) were more frequent in vasectomized animals, and this was especially marked in the distal regions. Immature sper-

FIG. 4. Histology of the caput epididymidis from a vasectomized ram showing immature germ cells and macrophages in the lumen (H & E, x800).

DISCUSSION

The findings in intact control rams confirmed the seasonal nature of testicular activity and were consistent with the general pattern of reproductive seasonality described for this species in the northern hemisphere. 12 • 14 Spermatogenic activity, as assessed by SCR and STD, was seen to increase with the onset of decreasing photo periods, and vice versa. The sizes of the testes and caudae epididymides were also correlated with spermatogenic activity. In the majority of vasectomized rams, however, the seasonal pattern was . disrupted. Testicular size and weight, STD, and SCR were reduced after vasectomy. Periods of spermatogenic arrest were followed by partial recovery and hypospermatogenesis, to be in turn followed by a further episode of spermatogenic arrest. This cyclic ebb-andflow pattern was unrelated to seasonal factors, and individual animals varied in the timing of their remissions and exacerbations in relation to the time elapsed from vasectomy. The pattern suggested in humans 15 and dogs, 16 where vasectomy is said to be followed by an initial temporary reduction in spermatogenesis and subsequent recovery, does not appear to occur in rams. Although varying degrees of adhesions were observed between the visceral and parietal layers of the tunica vaginalis in some vasectomized animals, these were not always associated with marked spermatogenic degeneration. On the other hand, some testes with complete spermatogenic arrest were free from adhesions, indicating that this was not a major etiologic factor. Increased pressure within seminiferous tubules has been suggested as a cause of postvasectomy spermatogenic inhibition. 15 • 17 The findings of the present study do not substantia,te this view, since

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TABLE 2. Morphologic Characteristics of Spermatozoa Collected from the Testes and Different Regions of the Epididymides of Intact and Vasectomized Rams" Ram no. l •

Intact 3 4 7 Vasectomized 9 11 11 14 17 18

Caput'

Testis'

Side

Cauda'

Corpus'

DCD

TH

DS

lH

PCD

DCD

0 0 2

50 40 74

3 14 1

8 6 6

0 4 0

0 2 2

14 18 56

52 2 6 36 48 2 SNI 36 22 52 0

58 56 0

5 81 47 32 86 3

10 24 30 50 60 26

18 4 56 12 20 14

0 0 14 18 12 0

80 60 0 12 0 24

TH

DS

IH

PCD

DCD

TH

DS

lH

PCD

DCD

TH

DS

lH

PCD

Rt Lt Rt

18 12 24

8 16 16

90 86 82

88 90 80

0 0 2

17 29 61

16 18 22

84 60 78

80 44 78

2 14 0

8 28 11

18 28 12

2 24 0

Lt Lt Rt Rt Rt Lt

16 22 9

14 20 6

4 4 2

9 9 18

6 14 14

80 80

2 2 0

21 34 48

26 16 54

18

4

40 6

70 12

94 82 84 SNI 34 82

90

12

76 76 76 58 84 80 SNId SNI 62 62

74 48

0 24

21 35

72 38

4 20

"All values are percentages and reflect the incidence of the particular characteristic in the spermatozoa present at each location. "The numbers assigned to animals correspond to those in Table 1. "TH, tail-less heads; DS, darkly stained (eosinophilic); IH, immature heads (pyriform); PCD, proximal cytoplasmic droplets; DCD, distal cytoplasmic droplets. dSNI, sperm numbers insufficient for evaluation.

no dilatation of the seminiferous tubules or of the rete testis was seen in any of the vasectomized rams. In almost all cases dilatation was restricted to the vas deferens and the cauda epididymidis, with rupture of the duct occurring in these regions before the increased pressure could spread retrogradely toward the corpus and caput. The extravasation of epididymal contents into the connective tissue surrounding the duct resulted in a granulomatous reaction which presumably aided in the removal of spermatozoa by allowing phagocytosis over a larger area. Intratubular phagocytosis of spermatozoa by macrophages appears to be incapable of coping with even reduced rates of sperm production by the testis, as evidenced by the multiple spermatoceles observed in the majority of vasectomized rams. Autoimmunity arising from increased phagocytosis of spermatozoa 18 has also been suggested as a possible mechanism for the spermatogenic disturbances seen in some species after vasectomy.!9 However, this alone does not explain the fluctuating nature of spermatogenic activity seen in vasectomized rams, since the many spermatoceles present would be expected to provide continuous immunologic stimulation. These considerations indicate a complex etiology for postvasectomy hypospermatogenesis and aspermatogenesis. It is possible that autoimmune mechanisms may be able to exert their influence on spermatogenesis only in the presence of excurrent duct blockage, either due to alterations in the blood-testis barrier to passage of circulating antibodies 20 or due to retrograde spread of sensitized, immunologically competent cells, as

suggested by some workers.21 Although Leydig cell function as assessed by androgen profiles and response to stimulation with human chorionic gonadotropin was found to be unchanged in vasectomized rams,10 it is possible that alterations in the blood-testis barrier may interfere with hormonal transport mechanisms into the seminiferous tubules. Finally, even though pressure within the seminiferous tubules does not rise sufficiently to cause dilatation, it is possible that stasis of products may be interfering with Sertoli cell functions and hormonal interconversions. In addition to the fewer over-all numbers of spermatozoa produced within the testes of vasectomized rams, maturation anomalies were also evident in spermatozoa within the epididymis. The high proportions of mature as well as dead spermatozoa seen in the caput of some vasectomized rams might have been due to their having been held for long periods at this site. On the other hand, some animals had a high proportion of immature spermatozoa in the cauda, implying that they either had passed through the proximal regions too rapidly or were incapable of completing the process of maturation. The latter condition could arise from an inherent defect in the spermatozoon or result from some change in the epididymal milieu. The finding that successful re-establishment of continuity of the vasa deferentia in vasectomized men does not result in equally successful fertility rates!' 22 could be due to testicular and epididymal changes similar to those described here. It can be concluded from this study that vasectomy in the ram is not a physiologically in-

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nocuous procedure, and that the regions of the reproductive tract lying proximal to the site of vasectomy are unable to cope satisfactorily with the crisis.

Acknowledgments. I wish to thank Mr. P. G. Hignett and Dr. J. P. Renton for facilities and interest in this work, and Mrs. Thamalee Perera for typing the manuscript.

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10. Perera BMAO, Munro CD: Androgen levels in intact and vasectomized rams during the breeding and non breeding seasons. Fertil Steril 27:1446, 1976 11. Weaver AD: Vasectomy in the ram. Veterinarian 4:155, 1967 12. Ortavant R: Reproduction in Domestic Animals, Vol 2, Edited by HH Cole, PT Cupps. New York, Academic Press, 1959, pI 13. Rowley MJ, Heller CG: Quantitation of the seminiferous epithelium of the human testis employing the Sertoli cell as a constant. Z Zellforsch Mikrosk Anat 115:461, 1971 14. Johnson BH, Desjardins C, Ewing LL: Seasonal effects on the testis function in rams. J Anim Sci 37:247, 1973 15. Derrick FC, Glover WL, Kanjuparamban Z, Jacobson CB, McDougall M, McCowin K, Mercer HD, Rollins LD: Histologic changes in the seminiferous tubules after vasectomy. Fertil Steril 25:649, 1974 16. Vare AM, Bansal PC: Changes in the canine testes after bilateral vasectomy-an experimental study. Fertil Steril 24:793, 1973 17. Gour KN, Gupta JP: Endocrine glands after vasectomyan experimental study. In Proceedings of the Third Asia Oceania Congress of Endocrinology, Manila, Part 2. 1967, P 808 18. Phadke AM: Fate of spermatozoa in cases of obstructive azoospermia and after ligation of vas deferens in man. J Reprod Fertil 7:1, 1964 19. Alexander NJ: Autoimmune hypospermatogenesis in vasectomized guinea pigs. Contraception 8:147, 1973 20. Heidger PM: The effects of vasectomy upon the bloodtestis barrier in the dog. Anat Rec 178:514, 1974 21. Dym M, Romrell LJ: Intraepithelial lymphocytes in the male reproductive tract of rats and rhesus monkeys. J Reprod Fertil 42:1, 1975 22. Pai MG, Kumar BTS, Kaundinya C, Bhat HS: Vasovasostomy-a clinical study with 10 years' follow-up. Fertil Steril 24:798, 1973