FERTILITY AND STERILITY Copyright 1970 by the Williams & Wilkins Co. Vol. 21, No. 11, November 1970 Printed in U.S.A. A COMPARATIVE STUDY OF GERM CELL KINETICS IN THE TESTES OF CHILDREN WITH UNILATERAL CRYPTORCHIDISM: A PRELIMINARY REPORT* MOSHE MARKEWITZ, M.D" JOHN K, LAITIMER, M,D" AND RALPH J. VEENEMA, M.D. College of Physicians and Surgeons, Columbia University Pediatric Urology Service, t New York, New York 1002 Radioautography in vitro offers an excellent tool to study deoxyribonucleic acid (DN A) synthesis rates of the germ cell in the human testis. In young adults, the end result of untreated or late treated undescended testes is delay of normal spermatogenesis associated with decreased cellular maturation of the germinal epithelium and smaller diameter of the seminiferous tubules. It is not known whether the metabolic abnormality in the testes of cryptorchid children occurs initially at the stage of DN A synthesis by the "stem cells" (spermatogonia) or at a later stage of maturation. Because it is possible to measure the DNA-synthesizing capacity of these germ cells in vivo and in vitro,4, 7,8 a study of germ cell kinetics in the undescended testis was undertaken. This paper summarizes our preliminary findings in a group of children between V2 and 15 years. MATERIALS AND METHODS Sixteen patients with unilateral undescended testes were studied at the time of orchidopexy. Previously, all of these children had received a course of 10,000 U. of human chorionic gonadotropins. Testicular biopsies were obtained from the cryptorchid as well as from the scrotal testis in the same patient. These were immediately * Presented at the American Academy of Pediatrics, 8th Annual Meeting, October 18-2, 1969, Committee on Urology, Chicago, TIL This study was supported in part by the Mueller Pediatric Research Fund. t In collaboration with the International Institute for the Study of Human Reproduction, New York, N. Y. fixed in Bouin's solution and processed for conventional microscopy. Mirror sections also were placed in culture media containing tritiated thymidine 20 /lc./ml. and were processed for radioautography in vitro to study DNA synthesis rates.7.8 The latter was done in the following manner. Under the low magnification (X 125), of a light microscope, the proportion of seminiferous tubules containing (actively dividing) labeled cells was determined. The presence of even one labeled cell in a tubule was sufficient to let it be included in the count. Using high magnification (X 250), the number of labeled germ cells per cross-sectioned individual tubule was also counted. This was called the "DNA labeling index" of the germ cells (proportion of cells that incorporate a labeled precursor). 8 In addition to the biopsies of the undescended and descended testes in the same patients, a control group of normal testis biopsies from 17 healthy children ranging in age from 2-15 years had been examined before the present study. This had been done in order to establish a base line of germ cell kinetics in normal immature children (Table 1). All testicular biopsies were from intact testes; no autopsy or orchiectomy material had been utilized in this older study. Our findings of germ cell kinetics in the adult have been previously reported. 7,8 806 FINDINGS In the healthy immature human testis, the approximate rate of DNA synthesis by
November 1970 GERM CELL KINETICS IN THE TESTES 807 the germ cells is summarized in Table 1. The DNA labeling index varies from child to child even in the same age group. There is, however, no doubt that germ cell replication as well as DNA synthesis is present in the very young, immature human testis (at age 2 years for instance). Our radioautographic study indicated that in approximately 85% of the already descended scrotal testes of children with unilateral cryptorchidism the DNA labeling index of the germ cells was equivalent to that of healthy children of the same age group (Tables 1 and 2). Significant reductions in DNA synthesis were found in cryptorchid testes when compared to the scrotal testis in the same child (Figs. 1-). Table 2 summarizes the DNA labeling index of the germ cells (stem cells) of both the undescended and the descended testes in each child studied. As early as age V2 years (the youngest cryptorchid testis biopsied in this series), the nondescent of the testis manifested itself by a lower replication and DNA- TABLE 1. In Vitro Labeling of Human Germ Cells in 17 Normal Children with Normal Testes* TABLE 2. In Vitro Labeling of Human Germ Cells in 16 Children with Undescended Testis as a Measure of Activity* Patient Age Maximum la- beled cells per tubule Labeled tubules per total tubule Unde- De- Unde- Descended scended scended scended yr. % L. A., HH 27575 V, 2 15 1 1 T. G., HH 159816 V, 0 20 0 5 D. A., HH 1226 4 1/, 2 15 1 1 J. E., HH 12528 5 0 20 0 2 R. R., BH 19076 7 10 25 1 P. E., BH 1579577 7 0 20 0 2 M. K., HH 256107 7 12 12 1 1 S. M., HH 065186 8 18 1 4 T. B., HH 175419 9 25 27 E. R., HH 0684 9 6 10 2 2 E. H., HH 09641 9 0 20 0 2 J. M., HH 12479 10 2 15 1 2 T. J., HH 125950 10 5 1 1 S. G., HH 127845 12 0 20 0 2 W. D., BH 162729 12 12 20 9 15 J. M., BH 161612 15 25 50 24 * HH, Harlem Hospital; BH, Babies Hospital. synthesizing rate in the stem cells of the undescended testis when compared with the descended testis (Fig. 1, C and D). Patient Age Labeled tubules per total tubule yr. G. S., HH 126165 R. L., HH 110288 P. H., HH 09768 J. C., HH 11118 J. M., HH 041084 W. A., BH 1766048 H. D., HH 112584 M. L., HH 148847 S. W., HH 0947 P. G., HH 022712 G. G., HH 17579 S. D.. HH 112002 D. R., HH 04024 C. B., FDH 20576 M. 0., FDH 27849 S. W., FDH 2077 P. L., FDH 2024 2 2V2 V2 6 7 7 8 9V2 10 12 12 1 14 15 15 20 0 10 18 20 50 25 25 10 0 20 40 40 80 70 70 50 * HH, Harlem Hospital; BH, Babies FDH, Francis Delafield Hospital. % Maximum labeled cells per tubule 1 2 4 4 2 2 4 7 7 25 19 2 25 Hospital; DISCUSSION In many previous publications it has been stated that the testis is quiescent in the years before puberty and that there is no clear-cut morphologic difference between the undescended testis and the scrotal gland before puberty. 1, 2, 5, 9-12 Recently an electron microscopic study by Leeson 5 reports that comparing the histologic findings in the undescended testis with those of the normal scrotal testis, no abnormalities were detected in the undescended testis before 10 years of age. Our studies, utilizing radioautographic technics in vitro, showed incontrovertibly that the germ cells of the immature scrotal testis maintain active metabolism and are capable of replicating and synthesizing DNA. Striking differences were present in the DNA labeling index of the stem
FIG. 1. Patient 159816, a lj2-year-old child who was born with a left undescended testis. He received 10,000 U. of HCG. No testicular descent occurred. At surgery, both the descended and undescended testis were biopsied. A, left (undescended) testicular biopsy, H&E stain (X 175). The tubules are of fair size. Spermatogonia can be identified in some tubules. B, right (scrotal) testis of same child, H&E stain (X 175). The tubules are larger and the differentiation of the germ cells is more advanced. A segment of one tubule 808
contains primary speratocytes (arrow). C, thymidine-h radioautographed slide of same biopsy as A. No evidence of DNA-synthesizing germ cells are evident. Cellular replication is severely diminished. D, thymidine-h radioautographed slide of scrotal testis as in B. Twenty per cent of seminiferous tubules contain labeled germ cells and as many as five DNA-synthesizing cells are observed in some of the tubules (arrow). 809
FIG. 2. Patient 1226, a 4V,-year-old boy born with a left undescended testis. A course of injections of 10,000 U. of ReG failed to cause testicular descent. A, left (cryptorchid) testicular biopsy. H&E stain (X 250). The tubules are small. The nuclei of the tubular syncytium appear undifferentiated. Increase of interstitial connective tissue is present. B, right (scrotal) testis biopsy of same child. H&E stain (X 250). 810
The tubules are larger and many are convoluted. Spermatogonia can be identified in many tubules. C, thymidine-h' radioautographed slide of cryptorchid testis as shown in A. Two per cent of seminiferous tubules (see arrow) contain labeled germ cells. Maximum number of DNA-synthesizing cells in any tubule does not exceed one cell. D, thymidine-h' radioautographed slide of scrotal testis same as in B. Fifteen per cent of tubules contain labeled germ cells. Maximum number of labeled germ cells per tubule is one. Significant increase in DNA metabolism if compared to C. 811
FIG.. Patient 19076, a 7-year-old boy who was born with a right undescended testis. A course of 10,000 U. of HeG failed to cause testicular descent. A, right (cryptorchid) testicular biopsy. H&E stain (X 250). The tubules are small. No differentiation of germ cells can be identified with certainty in the conventional histology. Increase in the edema of the interstitial connective tissue is evident. B, left (scrotal) testicular biopsy, H&E stain (X 250). The tubules are larger and more of them convoluted. Spermatogonia are present in many 812
tubules. Evidence of interstitial connective tissue edema is also present. C, thymidine-h" radioautographed slide of cryptorchid testis as in A. In spite of poor differentiation in the conventional histology, as many as 10% of seminiferous tubules contain labeled cells. Maximum number of labeled cells per tubule is one. D, thymidine-h" radioautographed slide of scrotal testis same as in B. Twenty-five per cent of DNA active tubules, and as many as three labeled germ cells are present in some of the tubules. 81
814 MARKEwrrz ET AL. Vol. 21 cells in most of the undescended testes, even as early as age 1/2 years, when compared to the scrotal testis in the same child (Fig. 1, C and D). These differences in the past were not clearly evident by using conventional or even electron microscopic technics. The latter apparently demonstrated only minimal differences in morphology in both testes before puberty. We have demonstrated previousll' 8 that apparently normal morphology need not indicate normal physiology of the germinal epithelium. This preliminary study seems to indicate that the stem cells (spermatogonia) are initially damaged in the undescended testis as indicated by diminished cellular incorporation of tritiated thymidine. Because it is the spermatogonia from which spermatozoa finally develop, it can now be understood why many of these testes show depletion of spermatogenesis in biopsy specimens. In two children (E. R., 9 years; and T. J., 10 years, Table 2) there was evidence of diminished DNA synthesis also in the germ cells of the apparently normal scrotal testis, an incidence of 14%. This might indicate that occasionally in the presence of a unilateral cryptorchid testis, the apparently normal scrotal testis does not emerge unscatted. In a certain number of infertile patients with subfertile semen quality, there is a history of unilateral cryptorchidism. The above findings seem to shed light as to the etiology of their oligospermia. Whether the absence of any DNA-synthesizing germ cells (Fig. lc) is an indication of irreparable damage or that our in vitro technic is not sensitive enough to detect diminished thymidine-h" incorporation, needs further study by future biopsies. We have at present no proof that early correction of cryptorchidism before the age of years will be able to save spermatogenesis. Attention and study of this condition, however, at an earlier age than was the practice seem indicated from these findings. SUMMARY Radioautography in vitro measures DNA synthesis rates of the germ cells. Using this method in a study of descended and undescended testes in children, we demonstrated a significant reduction of thymidine-h incorporation into the stem cells of the undescended testis. This was noted as early as V2 years of age. These findings, although preliminary, suggest that the stem cells (spermatogonia) are initially damaged in the germinal line. The significance that this might have on the process of spermatogenesis is discussed. Acknowledgments. The authors wish to thank Marian D. Butler, B.S., and Gloria P. Guerrero for their technical assistance, and Martin Rotker for the medical illustrations. REFERENCES 1. CHARNY, C. W., CONSTON, A. S., AND MERANZE, D. R. Development of the testis. Ann N Y Acad Sci 55:597, 1952. 2. CHARNY, C. W. The spermatogenic potential of the undescended testis before and after puberty. J Urol 8:697, 1960.. DE LA BALZE, P. F., MANCINI, R. A., ARRILLAGA, F., ANDRADA, J. A., VILAR, 0., GURTMAN, A., AND DAVIDSON, O. W. Histologic study of the undescended human testis during puberty. J Clin Endocr 20:286, 1960. 4. HELLER, C. G., AND CLERMONT, Y. Kinetics of the germinal epithelium in man. Recent Progr Hormone Res 20:545, 1961. 5. LEESON, R. C. An electronmicroscopic study of cryptorchid and scrotal human testes, with special reference to pubertal maturation. Invest Urol :498, 1966. 6. MARKEWITz, M., FINGERHUT, B., AND VEENEMA, R. J. Radioautographic studies in vitro of DNA and RNA synthesis in experimentally produced cryptorchidism in the Fisher rat. Fertil Steril 17:86, 1966. 7. MARKEWITz, M., FINGERHUT, B., AND VEENEMA, R. J. Radioautographic studies of the DNA and RN A synthesizing capacity of the human germinal epithelium in vitro. J Urol 97:1059, 1967.
November 1970 GERM CELL KINETICS IN THE TESTES 815 8. MARKEwrrz, M., VEENEMA, R. J., AND FINGERHUT, B. Radioautography in vitro: A promising method for evaluating human germinal epithelium. Int J FertiI14:22, 1969. 9. ROBINSON, J. N., AND ENGLE, E. T. Some observations on the cryptorchid testis. J Urol 71 :6, 1954. 10. SNIFFEN, R. C. The testis. Arch Path (Chicago) 50:259, 1950. 11. SNIFFEN, R. C. Histology of the normal and abnormal testis at puberty. Ann N Y Acad Sci. 55:609, 1952. 12. SOHVAL, A. R. Histology of cryptorchidism. Amer J Med 16:46, 1954.