THE HOLOCRINE CELLS of the epithelium of the rat epididymis and vas

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1 Holocrine Cells of the Human Epididymis JAN MARTAN, Ph.D.,"' PAULL. RISLEY, Ph.D., and ZDENEK HRUBAN, M.D., Ph.D. t THE HOLOCRINE CELLS of the epithelium of the rat epididymis and vas deferens undergo a secretory cycle described recently by Martan and Risley.6 This cellular cycle begins with an activation of basal cells; a second step is their development into "apical cells" and "clear cells." These cells eventually disintegrate within the epithelium or are released intact into the lumen, where they disintegrate. Epididymal holocrine cells are characterized by their staining properties with the periodic acid-schiff technic and Aoyama silver-impregnation method (Figs. 1 and 13); they can be identified easily among the principal cells. they are also present in epididymides of guinea pigs and hamsters. 6 The secretory cycle was shown to be influenced by male testicular hormone an9. by frequency of mating. Although the function of the holocrine cells of the epididymis is not definitely known, it seems that the epididymal secretions, to which the holocrine cells contribute, might facilitate and hasten the maturation of spermatozoa. 5 6 Since the epididymal holocrine cells could thus be important in the understanding of human sterility, the purpose of the present study is to examine and identify the characteristics of holocrine cells in the human epididymis. MATERIALS AND METHODS Testes and epididymides were obtained at autopsy from 25 male patients. The respective ages and diagnoses of these patients had been: 4 weeks, bacterial meningitis; 6 weeks, congenital heart disease and cryptorchism; 1 year, meningitis; H~ years, burns; 4 years ( 2 patients) and 7 years, acute leukemia; 11 years, Hodgkins disease; 15 years, tetralogy of Fallot; 18 years, rheumatic From the Department of Biology, University of Oregon, Eugene, Ore., and the Department of Pathology, University of Chicago, Chicago, Ill. This work was supported by U.S. Public Health Service Grants NB and CA The authors wish to thank Miss Ute Paetsch for the preparation of the histological material described. *Present address: Department of Zoology, University of Michigan, Ann Arbor, Mich. tadvanced Clinical Fellow of the American Cancer Society. 180

2 VoL. 15, No.2, 1964 HoLOCRINE CELLS of EPIDIDYMIS heart disease; 21 y~rs, sarcoma of chest wall; 28 years, astrocytoma; 32 years, pulmonary emboli; 39 years, carcinoma of lung; 45 years, rheumatic heart disease; 47 years, viral hepatitis; 49 years, diffuse myocardial scarring, 50 years, fibrosarcoma; 52 years, carcinoma of lung; 54 years, myocardial in farction; 58 years, diabetes mellitus; 65 years, carcinoma of lung; 67 years, myocardial infarction; 68 years, carcinoma of stomach; and 70 years, myocardial infarction. Tissues were fixed in 10% neutral formalin containing 1% cadmium chloride. Tissues from one side were impregnated with silver nitrate by the Aoyama method,! embedded in paraffin, and 4- or 6-p. sections were cut. Some of the silver-impregnated sections were toned in 0.1% gold chloride, and nuclei were counterstained by the Feulgen method. Tissues from the opposite side were embedded in paraffin, and 4- or 6-p. sections were made. These were stained with hematoxylin eosin, Alcian blue-periodic acid-schiff method, 8 Azure B for ribonucleic acid localization,9 and Azure A-eosin B technic for demonstration of lipofuscins. 4 Control sections for demonstration of ribonucleic acid were incubated for 1 hr. at 50 C in 0.01% solution of ribonuclease in 0.8% saline solution buffered with phosphate to ph 6.4 Three controls were used for PAS reactive materials, namely: (I) sections digested in 0.1% malt diastase in 0.02 M phosphate buffer at ph 6 for one hour at 38 o C to remove gylcogen; ( 2) other sections extracted in equal parts of methanol and chloroform at 58 o C for 16 hr. to remove glycolipids;3 and ( 3) some sections immersed in Schiff reagent without pretreatment with periodic acid. RESULTS., ; t - l In Alcian blue-pas-stained sections, the epithelium of the human epididymis contained basal, holocrine, and principal cells. The holocrine and principal cells were exceptionally tall and narrow as compared with those of the rat and hamster, and these two types of cells were less well-defined (Fig. 1-3). Most of the epithelial cells showed a weak-to-mild periodic acid-schiff reaction throughout their cytoplasm, and strongly positive granules were present in their apical regions. Purple coloration of some granules and the stereocilia in some regions indicated that both PAS and Alcian-blue stains were positive. This apparently was indicative of the presence of a mucopolysaccharide complex. Nonstereociliated cells with pronounced PAS cytoplasmic staining and numerous PAS-positive granules were frequently seen in the ductus epididymidis (Fig. 2-4). The cytoplasm of some basal cells was also strongly PAS positive. The nuclei of strongly PAS-positive epididymal columnar

3 182 MARTAN ET AL. FERTILITY & STERILITY cells were located either in basal positions or in the upper halves of the cells. The cells with basal nuclei were slender and rectangular (Fig. 2 and 3). They assumed a club-like form when the nuclei moved into the upper cytoplasm (Fig. 4). Some club-shaped strongly PAS-positive cells were becoming detached from the epididymal epithelium, and numerous rounded cells Fig. 1. PAS-positive holocrine cells in cauda epididymidis of a normal mated rat. (PAS Alcian blue, X 900) Fig. 2. Arrow indicates a PAS-positive holocrine cell in cauda epididymidis of 52-year-old man. Compare with Fig. 1. (PAS-Alcian blue, X 900) Fig. 3. Same as in Fig. 2. (X 900) Fig. 4. Arrow indicates PAS-positive holocrine cell escaping into lumen of caput epididymidis of 50-year-old man. (PAS-Alcian blue, X 2200) Fig. 5. Arrows point to exhausted holocrine cell which lost its contents in situ and to mass of PAS-positive lipofuscin in caput epididymidis of 70-year-old man. (PAS Alcian blue & hematoxylin, X 900) Fig. 6. Arrow points to basophilia concentrated in stalk of holocrine cell in epididymis of 50-year-old man. Compare nucleus of this cell with principal cell below. (Azure B, X 2200)

4 VoL. 15, No.2, 1964 HoLOCRINE CELLS of EPIDIDYMIS 183 of a similar type were present in the lumen in all stages of disintegration. Similarly stained cells also occurred in the epithelium and lumen of the vas deferens and in the lower folded or festooned epithelium of the ductuli efferentes (Fig. 8). These cells were present in the epididymides and efferent ductuli of all male patients between the ages of 7 and 70 years. Though the strong PAS reaction was absent in the epididymides of 4-year-old patients, the presence of holocrine cells was indicated by the position of their nuclei within the epi- Fig. 7. Silver-positive cells in ductus efferens of 60-day-old normal unmated rat. (Aoyama-Feulgen reaction, X 900) Fig. 8. PAS-positive-cell in ductus efferens of 50- year-old man. (PAS-Alcian blue, X 2200) Fig. 9. Silver-positive cells in ductus efferens of 49-year-old man. Compare with Fig. 7. (Aoyama reaction, X 900) Fig. 10. Same as Fig. 9. (X 2200). Fig. 11. Mitosis of holocrine cell in basal region of epididymal epithelium of 15-year-old boy. (Azure B, X 2200) Fig. 12. Apical mitosis of a principal cell in epididymis of 7-year-boy. (PAS-Alcian blue & hematoxylin, X 2200)

5 184 MARTAN ET AL. FERTILITY & STERILITY theliallayer. The PAS reaction was somewhat weaker in the 7 and 11-yearold boys than it was in patients between 15 and 70 years old. The relative numbers of club-shaped cells were reduced in patients less than 15 or more than 50 years old. Large granular masses of strongly PAS-positive material were found fre- Fig. 13. Holocrine cells, with cytoplasm blackened with silver, about to leave epididymal epithelium in lower cauda on operated side of mated hemicastrated rat. (Aoyama-Feulgen reaction, X 2200) Fig. 14. Holocrine cell escaping from epithelium in cauda epididymidis of 15-year-old boy. Compare with Fig. 13. (Aoyama-Feulgen reaction, X 2200) Fig, 15. Holocrine cell in cauda epididymidis of 15-year-old boy. Compare with Fig. 13. (Aoyama-Feulgen reaction, X 2200) Fig. 16. Holocrine cell in corpus epididymidis of 47-year-old man. (Aoyama reaction, X 900) Fig. 17. Holocrine cells in vas deferens of 15-year-old boy. (Aoyama reaction, X 900) Fig. 18. Holocrine cell disintegrating in lumen of cauda epididymidis of 15-year-old boy. (Aoyama-Feulgen reaction, X 2200)

6 VoL. 15, No.2, 1964 HoLOCRINE CELLS of EPIDIDYMIS 185 quently in the basal and less often in the apical regions of the epithelium lining the middle and lower ductus epididymidis (Fig. 5). Smaller PASpositive granules occurred in the ductuli efferentes. Both types of these granules were present in patients older than 15 years and became more abundant with age. The PAS reactions of cells and granules mentioned above were not abolished by malt diastase and hot methanol chloroform extractions. Control sections in which periodic acid oxidation was omitted were negative in epididymides, while certain granules in the ductuli efferentes were "Schiffpositive" after 15-min. incubation in Schiff's reagent without prior oxidation. These granules and the granular PAS-positive masses in the basal epithelium of the epididymis stained greenish in Azure A-eosin B stain. The PAS-positive, nonstereociliated, club-shaped cells of the epididymis and vas deferens were differentiated readily from the principal cells in sections stained by the Aoyama silver method. The principal cells contained numerous silver granules in the apical portions of their cytoplasm, but the club-shaped cells were black throughout their cytoplasm (Fig. 15 and 16). Some of these dark cells bulged with rounded apical ends beyond the epithelial surface, or appeared to be detaching from the epithelial layer, and some were present in the lumen (Fig. 14 and 18). Many similar dark cells were present in the ductuli efferentes (Fig. 9 and 10). The dark cells were found in the epididymides of patients between 7 and 70 years. Their numbers however were fewer in the very young and very old patients of this group. The narrow club-shaped cells in the epididymis were basophilic in Azure B-stained sections, and ribonuclease digestion demonstrated that ribonucleic acid was concentrated in their narrow stalks and around their nuclei (Fig. 6). Mitotic figures were located in positions similar to those described previously for the rat. 6 Larger mitoses occurred in the apical ends of the principal cells (Fig. 12), and smaller ones were seen in the basal epithelium (Fig. 11). The nuclei of club-shaped cells were smaller than those of the principal cells of the epididymis (Fig. 6), and their chromatin patterns were different. DISCUSSION Basal and principal cells of the human epididymis have been described by Montagna and Hamilton, 7 and were studied with the electron microscope by Horstman. 2 The present study shows that the human epididymis also contains periodic acid-schiff-positive and silver-positive holocrine cells similar to those identified earlier in the laboratory rat by Martan and Risley. 5 6 The renewal cycle of holocrine cells was demonstrated previously in the

7 186 MARTAN ET AL. FERTILITY & STERILITY epididymis of the rat 5 6 by comparative studies of mated, hemicastrated, and normal animals. Some stages of this cycle were observed in the present study suggesting that a similar renewal cycle of the holocrine cells of the epididymis is also present in the human male. This cycle in the rat and man appears to start with activation of basal cells that become PAS- and silverpositive. The growth of activated cells, observed in the rat epididymis, was not detected in the human material. Whether the scarcity of this stage is inherent in man or should be attributed to the effects of the disease and treatment of these patients cannot be decided at the present time. The fully developed, nonstereociliated, club-shaped cells within the epithelium, cells becoming separated from the epithelial layer, and intraluminar cells are however easily identified in the human epididymis. The characteristics of basophilia and mitoses in the holocrine cells of man and rat are also comparable. The basophilia of the club-shaped cells in the epididymis occurs predominantly in the stalks of the cells below the nucleus. Mitotic figures are in basal or apical positions. The holocrine cells are less numerous, but present, in the epididymides of the 7- and 11-year-old patients, and in men older than 50 years, which could be related to a lower production of androgens. PAS-positive and silverpositive cells occur also in other locations of the male genital tract. In the human vas deferens, they are comparable to the holocrine cells of the human epididymis (Fig. 17). The club-shaped PAS- and silver-positive cells of the ductuli efferentes are similar to those of the rat6 (Fig. 7). Their replacement and developmental stages are not obvious, suggesting either that the renewal of the cells is very slow in these ducts or that they are not holocrine cells. Peaked caps of glycogen granules over the nuclei of basal cells, described in human epididymides obtained surgically7 were not observed in our material, possibly due to the antemortem disease in our patients or a delay of a few hours between death and fixation. These factors may also be responsible for the somewhat less intensive PAS and silver reactions of the human materials compared with those of rats. The granules that stain greenish with the Azure A-eosin B method and red with Schiff's reagent, with and without periodic acid oxidation, and which become more numerous with age, represent probably lipofuscin pigments. SUMMARY Human epididymides, ductuli efferentes, and vasa deferentia obtained at autopsy were studied for presence of holocrine cells. The epithelia in all three regions contained club-shaped nonstereociliated cells with periodic acid-schiff and Aoyama silver-positive cytoplasm.

8 VoL. 15, No.2, 1964 HoLOCRINE CELLS of EPIDIDYMIS 187 The stages of the holocrine cell renewal cycle observed in the human epididymis and vas deferens corresponded to the stages described for the rat epididymis. The renewal cycle was found to start with a mitotic division of a basal cell. The fully developed club-shaped holocrine cells were observed to disintegrate in situ or to be released into the ductular lumina. Dept. of Zoology University of Michigan Ann Arbor, Mich. REFERENCES 1. AoYAMA, F. Eine Modifikation der Cajalschen Methode zur Darstellung der Golgischen Binnennetz Apparate. Ztschr. Wiss. Mikr. 46:489, HoRSTMAN, E. Elektronenmikroskopie des Menschlichen Nebenhodenepithels. Ztschr. Zellforsch. 57:692, LEBLOND, C. P. Distribution of periodic acid-reactive carbohydrates in the adult rat. Am. ]. Anat. 86:1, LILLIE, R. D. Histopathologic Technic and Practical Histochemistry. Blakiston, New York, MARTAN, J., and RisLEY, P. L. The epididymis of mated and unmated rats. ]. Morphol. 118:1, MARTAN, J., and RISLEY, P. L. Holocrine secretory cells of the rat epididymis. Anatom. Rec. 146:113, MoNTAGNA, W., and HAMILTON, J. B. Histological studies of human testes. II. The distribution of glycogen and other HI0 4-Schiff reactive substances. Anatom. Rec. 112:231, MoWRY, R. W. Alcian blue techniques for histochemical study of acidic carbohydrates. ]. Ilistochem. 4:407, SwiFT, H. Cytochemical techniques for nucleic acids. Nucleic Acids. Acad. Press, New York, 1955.