- Email: [email protected]
Quantitative Investigations on Human Testicular Biopsies
f
t
Quantitative Investigations on Human Testicular Biopsies I. Normal Testis Edward C. Roosen-Runge, M.D.
THE
of the human testis has been explored intensively during the past 70 years. In fact, the amount of descriptive detail in the literature is so vast that its evaluation becomes extremely difficult. No unequivocal criteria for normality have been established and many investigators have emphasized the great variability of the microscopic picture. As early as 1911 Branca noted that each human testicle had characteristics of its own. Kyrle in a series of 1000 autopsies, did not find a single testis which completely satisfied his concept of normality. Slotopolsky and Schinz12 stated that "one might almost say: It is normal for the human testis to be abnormal." In view of this situation it is not surprising that many attempts have been made to quantitate the histologic findings. Benoit was the first to carry out planimetry of sections with a view of analyzing the amounts of interstitial cells. Schinz and Slotopolskyll reviewed the various quantitative methods and emphasized the significance of the quantitative approach in a field which is confused by many subjective impressions. In recent years, interest in the microscopic anatomy of the testis has been revived, I, 13 and the need for objective criteria is becoming ever more urgent. HISTOLOGY
From the Department of Anatomy, University of Washington School of Medicine, Seattle, Wash. Aided by a grant from the National Science Foundation. Thanks are due to Drs. Warren O. Nelson, Iowa City, Iowa; Fred A. Simmons, Boston, Mass.; Earl T. Engle, New York; D. F. McDonald, Seattle, Wash.; and W. Montagna, Providence, R. I., for supplying excellent specimens. I am deeply grateful to Joyce A. Gromons for painstaking technical work. 251
252
ROOSEN-RUNGE
Fertility & Sterility
Continued work on the quantitative analysis of seminal fluid has stimulated the search for precise methods of analyzing testicular tissue with the aim of achieving a correlation between germ-cell production and sperm counts. The present paper is concerned with the exploration of a method which permits measurement of the volumetric proportions of many tissue components. It is hoped that results obtained with this method may assist in constructing a reliable base line for investigations on the abnormal testis. In addition, it was of interest to investigate the testicular biopsy not as a clinical tool but as a means for the study of normal testis tissue.
MATERIAL Thirty testicle specimens from 26 men were evaluated. Of these 23 were in the form of wedge-biOpsy specimens while 7 testicles were removed in toto in surgical castration procedures. All material was fixed in Bouin's or in Stieve's solution, embedded in paraffin, sectioned 8 fL thick, and stained with Heidenhain's hematoxylin, Masson's, or Crossmon's stain. The assumption that a testis was normal rested on the impression of at least two experienced observers in each case. All sections showed numerous spermatozoa, and spermatogonial and spermatocytal mitoses. None showed an obvious increase in the number of abnormal cells nor thickening of the basement membranes. In many cases normal sperm counts had been obtained and all men appeared to be free from disease at the time of removal of the tissue. METHOD
The method for quantitative analysis used in the present investigation was first suggested and applied by Chalkley. He showed that a volumetric analysis of a tissue could be obtained by recording a statistically significant number of random "hits" made by pointers in the eyepiece on a given structure. Chalkley himself considered the theoretical and statistical background of the method. Its reliability and its applicability to testicular tissues was emphasized by Eschenbrenner et ai. In an effort to introduce the method into Germany, Haug investigated its theoretical basis thoroughly and demonstrated that with suitable precautions its operation is simple, rapid, and reasonably accurate. Recently the method was used for a detailed volumetric analysis of the rat testis 9 and a preliminary account of its application to tissues of the human testis was published. 8
1----
~
Vol. 7, No.3, 1956
HUMAN TESTICULAR BIOPSIES: I
253
Although descriptions of the procedure are available in the literature,4, 8, 9 a brief account will be given here, because the method is still not generally known. An eyepiece is fitted with 5 pointers. Four are used for recording and a short fifth one for focusing. A structure is brought into focus at the end of the short pointer and the structures at the tips of the 4 main pointers are recorded as "hits." The field is then thrown out of focus, the stage moved a short distance along a zigzag course through the preparation, a new structure brought into focus at the short pOinter, and the procedure repeated. After many such determinations the relative frequency of the "hits" on any particular type of cell or other structure indicates the relative volume occupied by all cells or structures of this type. Through statistical analysis it was found that tabulating the hits in 5 X 35 fields was amply sufficient to give statistical stability to the results in each case. All determinations were made with an oil-immersion objective (91 X) and a 10 X eyepiece. The magnification has an important relationship to the accuracy of the method: the less the depth of focus the greater the accuracy.6 Wherever possible, counts were made on several sections of the same testis. Identification of Material
An essential prerequisite for the successful use of Chalkley's method is accurate and consistent identification of the items to be recorded. For the present study the following definitions were used: Interstitial Tissue. All structures or space outside the semiferous tubules with the exception of Leydig cells, basement membrane, and tunica albuginea. Leydig Cells. Polygonal, epitheloid cells with characteristic nuclei located between the tubules. Small, fusiform cells resembling fibroblasts were not recorded as Leydig cells although it is recognized that they may be potential Leydig cells. Basement Membrane. In this definition are included not only the homogeneous basement membrane proper which surrounds each tubule and stains like collagen but also the fibroblasts and collagenous fibers which are immediately applied to this membrane and form a varying number of concentric rings around it. Spermatogonia. Cells usually located in the most peripheral layer of the tubule. The cells and their nuclei vary greatly in size and in appearance, but a clearly defined nuclear membrane is always present except in mitosis.lO
254
ROOSEN-RUNGE
Fertility & Sterility
This distinguishes the small spermatogonia from the young spermatocytes. Spermatocytes. Primary and secondary spermatocytes are included in this category. The small early spermatocytes have "woolly" nuclei with tenuous nuclear membranes. The older spermatocytes show the characteristic premeiotic nuclei. The secondary spermatocytes are distinguished from the spermatids by their larger size and more distinct chromatin patterns. Spermatids and Spermatozoa. It was found impractical to separate these two cell types, partly because of the inherent difficulty in drawing a sharp borderline between them, and partly because the spermatozoa have a very small collective volume which cannot be determined with statistical validity unless the total number of counts is increased far beyond those recorded in the present study. Spermatids may be found in any layer of the tubule, although they usually lie in groups near the lumen. They have round nuclei with inconspicuous chromatin. Spermatozoa are easily identified by their peculiar nuclei. Sertoli Cells. Characterized by more or less pyramidal nuclei with large spherical nucleoli. The full extent of their cytopasm cannot be identified with certainty, but usually their cytoplasm may be distinguished from that of the surrounding cells by its fine fibrous structure, particularly in preparations stained with a trichrome stain. Space. Unidentified structures within the seminiferous tubules were defined as "space" regardless of whether they appeared to be parts of cells or artifacts. Lumen. This definition needs no explanation. In biopsy specimens the lumen of the tubules is often filled with cells and a fine coagulum. In these cases the cells were recorded in the same way as those in their natural positions; the coagulate was recorded as "lumen."
RESULTS Volumetric Determinations
Tables 1 and 2 summarize the results obtained from 30 testes. The seminiferous tubules occupied about two thirds of the organ. The total volume of germ cells was about one half that of the seminiferous tubules or one third of the testis. The Leydig cells took up approximately 9 per cent of the total interstitial space (including the basement membrane), or 12 per cent of the space between basement membranes. However, these amounts varied
!
1.-
~
TABLE 1.
1 2 3 4 5 6 7 8 9 10 11 12 13 14-R 14-L 15-R 15-L 16-R 16-L 17-R 17-L 18 19 20 21 22 23 24 25 26
~
"""
~
•
"-
..
.-
"
Relative Volumes (Per Cent) of Constituent Elements of Thirty Normal Human Testes
(yr. )
Interstitial tissue
Basement membrane
Leydig cells
? 36 20 38 18 19 ? 32 37 26 32 30 21 25 25 22 22 36 36 31 31 30 ? ? 22 29 27 ? ? ?
25.0 25.7 15.6 19.8 14.5 16.0 22.8 17.6 20.4 20.1 25.2 27.9 27.9 15.4 18.5 25.0 17.9 22.6 25.7 18.7 20.1 18.6 27.9 28.2 25.1 22.0 22.6 13.0 26.0 32.5
8.1 5.8 8.6 8.1 10.4 10.1 10.7 10.4 8.6 8.6 10.0 9.9 8.6 10.0 9.1 6.7 11.3 8.5 9.9 7.1 9.3 9.3 9.4 10.4 9.6 8.8 11.8 9.3 6.9 9.5
2.1 2.7 5.6 4.6 5.8 3.5 1.5 1.7 2.3 3.3 1.3 3.1 3.1 2.8 2.2 4.2 3.3 3.7 4.3 1.8 2.9 4.1 2.4 2.5 3.2 3.9 0.6 3.7 4.8 1.3
Age Case
~
~
SperSpermatogonia matocytes
8.4 7.9 9.1 8.2 5.7 6.3 8.8 8.7 9.3 9.5 7.5 7.3 5.8 8.8 9.5 7.5 7.9 6.8 5.7 8.9 7.6 8.4 8.7 7.9 7.1 6.9 7.2 8.0 6.9 8.5
14.9 12.3 16.0 11.7 12.6 9.7 14.3 16.0 16.9 13.6 15.8 11.9 14.1 16.4 13.6 12.6 17.3 20.1 19.4 18.2 15.3 11.1 10.4 15.6 15.2 12.3 13.5 16.5 14.5 11.6
Spermatids Abnormal and spergerm cells matozoa
9.9 9.2 5.5 10.9 7.8 10.6 9.2 8.7 11.2 9.6 10.6 5.6 8.8 10.7 9.0 10.1 10.5 8.3 9.9 10.8 11.5 12.2 6.9 7.3 5.0 8.0 6.4 11.9 9.0 6.5
0 1.2 0.8 0.8 1.4 0.7 0.6 0.9 1.3 0.9 0.4 0.7 0 0.4 0.3 0.7 0.6 0.7 0.1 0.3 0.7 0.3 0.7 0.7 1.2 1.0 1.6 0.7 0.8 0.9
Sertoli cells
Lumen
"Space"
13.0 13.5 18.4 18.3 20.4 20.3 18.7 16.6 19.1 19.1 17.7 17.9 16.7 17.4 17.8 19.1 16.0 15.4 13.3 16.4 13.8 19.0 15.8 17.1 16.4 17.6 18.6 19.1 20.7 19.6
14.5 15.5 15.3 9.3 12.3 14.6 10.3 12.7 5.6 10.8 5.7 11.3 11.3 9.5 12.8 7.3 8.4 7.2 6.7 12.3 14.6 14.5 12.8 5.8 7.9 14.4 12.0 8.2 6.5 7.2
4.1 6.2 5.9 8.3 9.1 8.2 3.1 6.7 5.3 4.5 5.8 4.4 3.7 8.6 7.2 6.8 6.8 6.8 5.0 5.5 4.2 2.5 5.0 4.5 9.3 5.1 5.7 9.6 3.9 2.4
256
ROOSEN·RUNGE
Fertility & Sterility
greatly-from 1.7 per cent (resp. 2.6 per cent) in Case 22 to 17 per cent (resp. 29 per cent) in Case 5. The lumen occupied on the average 15.6 per cent of the seminiferous tubules. For the 7 nonbiopsy specimens this figure was 19.1 per cent and for some biopsy specimens it was as low as 8-10 per cent. The Sertoli cells formed about one third of the bulk of the wall of the tubules. The remainder of the wall consisted of germ cells and "space." Spermatogonia took up 24 TABLE 2.
Average Relative Volumes of Constituent Elements of Thirty Normal Human Testes
Interstitial tissue Leydig cells Basement membrane Total interstitial space Spermatogonia Spermatocytes Spermatids and spermatozoa Abnormal germ cells Total germ cells Sertoli cells Lumen "Space" (unknowns in tubular wall) a
Volume (%)
S.D.a
Range
22.0 3.1 9.1 34.2 7.8 14.4 9.1 0.7 32.0 17.4 10.6 5.8
4.84 1.26 1.81 4.43 1.08 2.58 1.98 0.39 3.71 2.10 3.26 1.92
13.0-32.5 0.6-5.8 5.8-12.5 26.0-43.3 5.7-9.5 9.7-20.1 5.0-12.2 0.0-1.6 25.5-38.7 13.0-20.7 5.6-14.6 2.5-9.6
Standard deviation.
per cent of the total germ-cell volume, spermatocytes 45 per cent, spermatids and spermatozoa 29 per cent, and abnormal germ cells about 2 per cent. The variability of the volume was very different for different structures. The range of values for the total of germ cells, for Sertoli cells, spermatogonia, and total interstitial space, was in each case 50 per cent or less of the mean. On the other hand, the range was 122 per cent for "space" and 168 per cent for Leydig cells. In Table 3 volume proportions from five different areas of the same cross section through a whole testis are recorded. In this section Area 5 was central and contained several septa converging near the mediastinum. Area 2 was similar but a little further removed from the mediastinum. Areas 1 and 4 were peripheral and from a region of the testis where biopsies would ordinarily be taken. The differences between the five areas of this one testis were not appreciably smaller than the differences between random groups of 5 biopsy specimens taken from Table 1. This is in part due to the
>
r
Vol. 7, No.3, 1956
257
HUMAN TESTICULAR BIOPSIES: I
fact that mediastinal areas, which in tissue composition are most different from the rest of the testis, were included in Table 3, but these areas are never taken in biopsy procedures. When the three nonmediastinal areas (1, 3, and 4 in Table 3) are compared, it is seen that they showed very small differences among each other. It is of interest that the volume of "space," (i.e., of unidentified structures TABLE 3.
Comparison of Relative Volumes (Per Cent) of Constituents of Five Different Areas from a Single Cross Section of a Human Testis (12,u Thick)
Areasu Constituent
Interstitial tissue and basement membrane Leydig cells Spermatogonia Spermatocytes Spermatids and spermatozoa Abnormal germ cells Total germ cells Sertoli cells Lumen "Space" a
1
2
3
4
5
Mean
S.D.b
29.4 1.3 9.5 16.7
36.6 0.9 8.5 14.4
34.9 0.6 9.6 14.8
28.4 1.1 9.1 13.2
38.4 1.8 7.5 12.6
33.5 1.1 8.8 14.3
4.48 0.45 0.87 1.59
10.4 0.4 37.0 18.8 10.3 3.2
9.6 1.1 33.6 19.1 6.0 3.8
7.8 0.6 32.8 20.3 8.6 2.8
9.4 0.4 32.1 19.5 15.6 3.3
9.0 0.4 29.5 16.2 11.4 2.7
9.2 0.6 33.0 18.8 10.4 3.2
0.95 0.30 2.76 1.88 3.56 0.40
The areas did not border each other.
b Standard deviation.
in the tubules) varied very little from area to area of the same testis. This is a demonstration of the fact that "space" is in large measure dependent on the quality of the preparation, which in this instance happened to be excellent and very uniform.
General Observations In scanning a great number of sections from biopsies of normal testes some observations were made which have a bearing on the reliability of biopsy material for quantitative determinations. Biopsy tissue possesses microscopic characteristics which definitely distinguish it from nonbiopsy tissue. Almost all biopsy specimens show unmistakable effects of handling throughout the greater part of most sections. The peripheral areas are often unusable for evaluation by the Chalkley method. In consequence of the injury and displacements in the periphery, germ cells are frequently trans-
258
ROOSEN-RUNGE
Fertility & Sterility
ported into the interstitial tissue spaces where they were found in 8 of the 23 specimens investigated. (No germ cells were ever seen in the interstitial tissue of nonbiopsy specimens.) Although the average relative volume of the interstitial tissues did not differ appreciably in biopsy and nonbiopsy specimens the width of the spaces varied much more in biopsy material. In some areas the tubules appeared jammed together, in others they were widely separated. Where the spaces were wide they may have been filled with tissue fluid or blood. In biopsies from normal testes some sloughing of germ cells into the lumen is the rule and severe sloughing is by no means rare. Quantitatively this was indicated by the low values for the volume of the lumen. Cellular preservation may be excellent in a biopsy specimen but the identification of abnormal cells presents difficulties because some cells will invariably show the effects of mechanical trauma. DISCUSSION Benoit first determined the relative volumes of structures in the human testis and found that the seminiferous tubules (including the basement membrane) occupied 70 per cent of the testis, interstitial tissue 22.3 per cent, and Leydig cells 7 per cent. Slotopolsky and Schinz12 perfected the planimetric method which Benoit had used and reported 66 per cent for the seminiferous tubules, 22 per cent for the interstitial tissue, and 12 per cent for the Leydig cells with a range from 8-28 per cent. These figures have been accepted by many later authors, for instance by Stieve in his review. The discrepancy between the results of the planimetric and Chalkley methods with respect to the volume of Leydig cells is indicative of the characteristics of the two methods. In the planimetric method the areas containing Leydig cells are treated as homogeneous throughout, and small vessels and intercellular spaces are not distinguished from the cells themselves. Chalkley's method permits the separate recording of these items. This explains the fact that our findings showed a lower volume for the Leydig cells. With regard to the volume of the seminiferous tubules and to the range of volume of Leydig cells in normal testes, our results are in excellent agreement with those of Slotopolsky and Schinz. 12 The data on relative volumes obtained by the Chalkley methods are more detailed and less laboriously gained than those resulting from planimetry, but they are open to the same question concerning the meaning of
I
I
Vol. 7, No.3, 1956
any data on relative collective volumes of cells and other structures. It is obvious that a detailed knowledge of numbers and sizes of germ cells in certain stages would permit a more meaningful analysis of proliferation and development of the germinal epithelium, but it is at present impossible to determine these values in the human testis because of the inherent lack of pattern in human spermatogenesis. 8A With this in mind, it may be premature to become seriously concerned about the meaning of data obtained with the Chalkley method, particularly as long as results on the normal cannot be compared with those on abnormal and experimental material. TABLE 4.
,
t
259
HUMAN TESTICULAR BIOPSIES: I
Comparison of Average Volume Proportions of Structures in the Testis of Man and the Rat
Interstitial tissue Leydig cells Basement membrane Total interstitial space Spermatogonia Spermatocytes Spermatids and spermatozoa Abnormal germ cells Residual bodies Total germ cells Sertoli cells Lumen Space a
Man
Rat
22.0a 3.1 9.1
8.0 1.7 2.4
34.2 7.8 14.4
12.1
9.1 0.7
1.7 14.7 41.1 0.1
17.4
1.2 58.8 8.4
10.6
19.5
5.8
1.1
32.0
All figures are per cent of total testis volume.
In any evaluation of the data it must, however, be clearly understood that the relative volume of any structure depends on the absolute and relative dimensions of the structure and on the frequency of its occurrence (i.e., in the case of cells in certain stages of development it depends on the duration of the stages) and that in human seminiferous tubules these factors cannot be separated from each other. It is of some interest to compare the results of volumetric measurements in the human with those in the rat (Table 4) which were obtained in an earlier study.9 The data are only approximately comparable because they were derived in the case of the human from 30 specimens, and in the case of the rat from only 2. Nevertheless the general results of the comparison seem valid. The rat testis possesses relatively much less interstitial tissue
260
ROOSEN-RUNGE
Fertility & Sterility
and Leydig cells but more germ cells, and the proportion of the various stages of germ cells is very different in the two species. In the rat the collective volume of spermatids and spermatozoa is 24 times that of the spermatogonia, and in the human 1.2 times. On the other hand, the relative volume of the Sertoli cells is two times greater in the human than in the rat. The proportion of Sertoli-cell volume to the volume of spermatids and spermatozoa is 1.9 in the human and 0.2 in the rat. One may speculate that the small size and the relatively small amount of cytoplasm which are apparent in human spermatids are correlated with a relatively greater development of the nursing cells. Detailed observations on testicular biopsies from normal human testes establish the fact that the biopsy technic as practiced at present by experienced operators results in tissues which show considerable distortions. In the diagnosis of pathologic biopsy specimens this must be taken into consideration. For instance, any conclusions based on the finding of sloughing of germ cells must be extremely guarded because of the frequency of this finding in biopsies from normal testes. On the other hand, the quantitative analysis of biopsy material, because it concerns itself with average values, is less affected by local displacements and injuries introduced by the operative technic.
SUMMARY Volumetric proportions of histologic elements of 30 normal human testes, 23 from biopsies and 7 from whole testes, were determined by means of Chalkley's method. The characteristics of testicular biopsy material are briefly discussed and the pOint is made that the biopsy technic is not ideal for a detailed morphologic survey of tissue from normal human testis. However, some of the distortions which are due to the technic do not appreciably influence a quantitative analysis. The differences in volume porportions of testicular structures in man and in the rat have been briefly discussed.
REFERENCES 1.
ALBERT, A. L., UNDERDAHL, L., GREENE, L., and Mayo Glin. 28:409, 1953. 2. BENOIT, J. Gompt. rend. Soc. biol. 87: 1385, 1922. 3. BRANCA, A. Gompt. rend Soc. anat. 13:283, 1911.
LORENZ,
N.
Froc. Staff Meet.
Vol. 7, No.3, 1956
HUMAN TESTICULAR BIOPSIES: I
261
4. CHALKLEY, H. W. f. Cancer Insf. 4:47, 1943. 5. ESCHENBRENNER, A. B., MILLER, E., and LORENS, E. f. Cancer Inst. 9:133, 1948. 6. HAUG, H. Ztschr. Anat.118:302, 1955. 7. KYRLE, J. Wien. klin. Wchnschr. 33:185, 909,1920. 8. ROOSEN-RuNGE, E. C. Proc. First World Congo Fertil. & Steril. 3:107,1954. 8A.RooSEN-RuNGE, E. C. Ann. N. Y. Acad. Sci. 55:574, 1952. 9. ROOSEN-RuNGE, E. C. Anat. Rec., 1955 (in press). 10. ROOSEN-RuNGE, E. C., and BARLOW, F. Am. f. Anat. 93:143,1953. 11. SCHINZ, H. R., and SLOTOPOLSKY, B. Abderhalden's Handb. der biol. Arbeits-
methoden Abt. V, 3B:529, 1938. 12. SLOTOPOLSKY, B., and SCHINZ, H. R. Virchows Arch. path. Anat. 257:294,1925. 13. SNIFFIN, R. C. Arch. Path. 50:259, 1950. 14. STIEVE, H. Miinnliche Genitalorgane: Handbuch der Mikroscopische Anatomie des Menschen. Berlin, Mollendorff, 1930.
t
Quantitative Investigations on Human Testicular Biopsies I. Normal Testis Edward C. Roosen-Runge, M.D.
THE
of the human testis has been explored intensively during the past 70 years. In fact, the amount of descriptive detail in the literature is so vast that its evaluation becomes extremely difficult. No unequivocal criteria for normality have been established and many investigators have emphasized the great variability of the microscopic picture. As early as 1911 Branca noted that each human testicle had characteristics of its own. Kyrle in a series of 1000 autopsies, did not find a single testis which completely satisfied his concept of normality. Slotopolsky and Schinz12 stated that "one might almost say: It is normal for the human testis to be abnormal." In view of this situation it is not surprising that many attempts have been made to quantitate the histologic findings. Benoit was the first to carry out planimetry of sections with a view of analyzing the amounts of interstitial cells. Schinz and Slotopolskyll reviewed the various quantitative methods and emphasized the significance of the quantitative approach in a field which is confused by many subjective impressions. In recent years, interest in the microscopic anatomy of the testis has been revived, I, 13 and the need for objective criteria is becoming ever more urgent. HISTOLOGY
From the Department of Anatomy, University of Washington School of Medicine, Seattle, Wash. Aided by a grant from the National Science Foundation. Thanks are due to Drs. Warren O. Nelson, Iowa City, Iowa; Fred A. Simmons, Boston, Mass.; Earl T. Engle, New York; D. F. McDonald, Seattle, Wash.; and W. Montagna, Providence, R. I., for supplying excellent specimens. I am deeply grateful to Joyce A. Gromons for painstaking technical work. 251
252
ROOSEN-RUNGE
Fertility & Sterility
Continued work on the quantitative analysis of seminal fluid has stimulated the search for precise methods of analyzing testicular tissue with the aim of achieving a correlation between germ-cell production and sperm counts. The present paper is concerned with the exploration of a method which permits measurement of the volumetric proportions of many tissue components. It is hoped that results obtained with this method may assist in constructing a reliable base line for investigations on the abnormal testis. In addition, it was of interest to investigate the testicular biopsy not as a clinical tool but as a means for the study of normal testis tissue.
MATERIAL Thirty testicle specimens from 26 men were evaluated. Of these 23 were in the form of wedge-biOpsy specimens while 7 testicles were removed in toto in surgical castration procedures. All material was fixed in Bouin's or in Stieve's solution, embedded in paraffin, sectioned 8 fL thick, and stained with Heidenhain's hematoxylin, Masson's, or Crossmon's stain. The assumption that a testis was normal rested on the impression of at least two experienced observers in each case. All sections showed numerous spermatozoa, and spermatogonial and spermatocytal mitoses. None showed an obvious increase in the number of abnormal cells nor thickening of the basement membranes. In many cases normal sperm counts had been obtained and all men appeared to be free from disease at the time of removal of the tissue. METHOD
The method for quantitative analysis used in the present investigation was first suggested and applied by Chalkley. He showed that a volumetric analysis of a tissue could be obtained by recording a statistically significant number of random "hits" made by pointers in the eyepiece on a given structure. Chalkley himself considered the theoretical and statistical background of the method. Its reliability and its applicability to testicular tissues was emphasized by Eschenbrenner et ai. In an effort to introduce the method into Germany, Haug investigated its theoretical basis thoroughly and demonstrated that with suitable precautions its operation is simple, rapid, and reasonably accurate. Recently the method was used for a detailed volumetric analysis of the rat testis 9 and a preliminary account of its application to tissues of the human testis was published. 8
1----
~
Vol. 7, No.3, 1956
HUMAN TESTICULAR BIOPSIES: I
253
Although descriptions of the procedure are available in the literature,4, 8, 9 a brief account will be given here, because the method is still not generally known. An eyepiece is fitted with 5 pointers. Four are used for recording and a short fifth one for focusing. A structure is brought into focus at the end of the short pointer and the structures at the tips of the 4 main pointers are recorded as "hits." The field is then thrown out of focus, the stage moved a short distance along a zigzag course through the preparation, a new structure brought into focus at the short pOinter, and the procedure repeated. After many such determinations the relative frequency of the "hits" on any particular type of cell or other structure indicates the relative volume occupied by all cells or structures of this type. Through statistical analysis it was found that tabulating the hits in 5 X 35 fields was amply sufficient to give statistical stability to the results in each case. All determinations were made with an oil-immersion objective (91 X) and a 10 X eyepiece. The magnification has an important relationship to the accuracy of the method: the less the depth of focus the greater the accuracy.6 Wherever possible, counts were made on several sections of the same testis. Identification of Material
An essential prerequisite for the successful use of Chalkley's method is accurate and consistent identification of the items to be recorded. For the present study the following definitions were used: Interstitial Tissue. All structures or space outside the semiferous tubules with the exception of Leydig cells, basement membrane, and tunica albuginea. Leydig Cells. Polygonal, epitheloid cells with characteristic nuclei located between the tubules. Small, fusiform cells resembling fibroblasts were not recorded as Leydig cells although it is recognized that they may be potential Leydig cells. Basement Membrane. In this definition are included not only the homogeneous basement membrane proper which surrounds each tubule and stains like collagen but also the fibroblasts and collagenous fibers which are immediately applied to this membrane and form a varying number of concentric rings around it. Spermatogonia. Cells usually located in the most peripheral layer of the tubule. The cells and their nuclei vary greatly in size and in appearance, but a clearly defined nuclear membrane is always present except in mitosis.lO
254
ROOSEN-RUNGE
Fertility & Sterility
This distinguishes the small spermatogonia from the young spermatocytes. Spermatocytes. Primary and secondary spermatocytes are included in this category. The small early spermatocytes have "woolly" nuclei with tenuous nuclear membranes. The older spermatocytes show the characteristic premeiotic nuclei. The secondary spermatocytes are distinguished from the spermatids by their larger size and more distinct chromatin patterns. Spermatids and Spermatozoa. It was found impractical to separate these two cell types, partly because of the inherent difficulty in drawing a sharp borderline between them, and partly because the spermatozoa have a very small collective volume which cannot be determined with statistical validity unless the total number of counts is increased far beyond those recorded in the present study. Spermatids may be found in any layer of the tubule, although they usually lie in groups near the lumen. They have round nuclei with inconspicuous chromatin. Spermatozoa are easily identified by their peculiar nuclei. Sertoli Cells. Characterized by more or less pyramidal nuclei with large spherical nucleoli. The full extent of their cytopasm cannot be identified with certainty, but usually their cytoplasm may be distinguished from that of the surrounding cells by its fine fibrous structure, particularly in preparations stained with a trichrome stain. Space. Unidentified structures within the seminiferous tubules were defined as "space" regardless of whether they appeared to be parts of cells or artifacts. Lumen. This definition needs no explanation. In biopsy specimens the lumen of the tubules is often filled with cells and a fine coagulum. In these cases the cells were recorded in the same way as those in their natural positions; the coagulate was recorded as "lumen."
RESULTS Volumetric Determinations
Tables 1 and 2 summarize the results obtained from 30 testes. The seminiferous tubules occupied about two thirds of the organ. The total volume of germ cells was about one half that of the seminiferous tubules or one third of the testis. The Leydig cells took up approximately 9 per cent of the total interstitial space (including the basement membrane), or 12 per cent of the space between basement membranes. However, these amounts varied
!
1.-
~
TABLE 1.
1 2 3 4 5 6 7 8 9 10 11 12 13 14-R 14-L 15-R 15-L 16-R 16-L 17-R 17-L 18 19 20 21 22 23 24 25 26
~
"""
~
•
"-
..
.-
"
Relative Volumes (Per Cent) of Constituent Elements of Thirty Normal Human Testes
(yr. )
Interstitial tissue
Basement membrane
Leydig cells
? 36 20 38 18 19 ? 32 37 26 32 30 21 25 25 22 22 36 36 31 31 30 ? ? 22 29 27 ? ? ?
25.0 25.7 15.6 19.8 14.5 16.0 22.8 17.6 20.4 20.1 25.2 27.9 27.9 15.4 18.5 25.0 17.9 22.6 25.7 18.7 20.1 18.6 27.9 28.2 25.1 22.0 22.6 13.0 26.0 32.5
8.1 5.8 8.6 8.1 10.4 10.1 10.7 10.4 8.6 8.6 10.0 9.9 8.6 10.0 9.1 6.7 11.3 8.5 9.9 7.1 9.3 9.3 9.4 10.4 9.6 8.8 11.8 9.3 6.9 9.5
2.1 2.7 5.6 4.6 5.8 3.5 1.5 1.7 2.3 3.3 1.3 3.1 3.1 2.8 2.2 4.2 3.3 3.7 4.3 1.8 2.9 4.1 2.4 2.5 3.2 3.9 0.6 3.7 4.8 1.3
Age Case
~
~
SperSpermatogonia matocytes
8.4 7.9 9.1 8.2 5.7 6.3 8.8 8.7 9.3 9.5 7.5 7.3 5.8 8.8 9.5 7.5 7.9 6.8 5.7 8.9 7.6 8.4 8.7 7.9 7.1 6.9 7.2 8.0 6.9 8.5
14.9 12.3 16.0 11.7 12.6 9.7 14.3 16.0 16.9 13.6 15.8 11.9 14.1 16.4 13.6 12.6 17.3 20.1 19.4 18.2 15.3 11.1 10.4 15.6 15.2 12.3 13.5 16.5 14.5 11.6
Spermatids Abnormal and spergerm cells matozoa
9.9 9.2 5.5 10.9 7.8 10.6 9.2 8.7 11.2 9.6 10.6 5.6 8.8 10.7 9.0 10.1 10.5 8.3 9.9 10.8 11.5 12.2 6.9 7.3 5.0 8.0 6.4 11.9 9.0 6.5
0 1.2 0.8 0.8 1.4 0.7 0.6 0.9 1.3 0.9 0.4 0.7 0 0.4 0.3 0.7 0.6 0.7 0.1 0.3 0.7 0.3 0.7 0.7 1.2 1.0 1.6 0.7 0.8 0.9
Sertoli cells
Lumen
"Space"
13.0 13.5 18.4 18.3 20.4 20.3 18.7 16.6 19.1 19.1 17.7 17.9 16.7 17.4 17.8 19.1 16.0 15.4 13.3 16.4 13.8 19.0 15.8 17.1 16.4 17.6 18.6 19.1 20.7 19.6
14.5 15.5 15.3 9.3 12.3 14.6 10.3 12.7 5.6 10.8 5.7 11.3 11.3 9.5 12.8 7.3 8.4 7.2 6.7 12.3 14.6 14.5 12.8 5.8 7.9 14.4 12.0 8.2 6.5 7.2
4.1 6.2 5.9 8.3 9.1 8.2 3.1 6.7 5.3 4.5 5.8 4.4 3.7 8.6 7.2 6.8 6.8 6.8 5.0 5.5 4.2 2.5 5.0 4.5 9.3 5.1 5.7 9.6 3.9 2.4
256
ROOSEN·RUNGE
Fertility & Sterility
greatly-from 1.7 per cent (resp. 2.6 per cent) in Case 22 to 17 per cent (resp. 29 per cent) in Case 5. The lumen occupied on the average 15.6 per cent of the seminiferous tubules. For the 7 nonbiopsy specimens this figure was 19.1 per cent and for some biopsy specimens it was as low as 8-10 per cent. The Sertoli cells formed about one third of the bulk of the wall of the tubules. The remainder of the wall consisted of germ cells and "space." Spermatogonia took up 24 TABLE 2.
Average Relative Volumes of Constituent Elements of Thirty Normal Human Testes
Interstitial tissue Leydig cells Basement membrane Total interstitial space Spermatogonia Spermatocytes Spermatids and spermatozoa Abnormal germ cells Total germ cells Sertoli cells Lumen "Space" (unknowns in tubular wall) a
Volume (%)
S.D.a
Range
22.0 3.1 9.1 34.2 7.8 14.4 9.1 0.7 32.0 17.4 10.6 5.8
4.84 1.26 1.81 4.43 1.08 2.58 1.98 0.39 3.71 2.10 3.26 1.92
13.0-32.5 0.6-5.8 5.8-12.5 26.0-43.3 5.7-9.5 9.7-20.1 5.0-12.2 0.0-1.6 25.5-38.7 13.0-20.7 5.6-14.6 2.5-9.6
Standard deviation.
per cent of the total germ-cell volume, spermatocytes 45 per cent, spermatids and spermatozoa 29 per cent, and abnormal germ cells about 2 per cent. The variability of the volume was very different for different structures. The range of values for the total of germ cells, for Sertoli cells, spermatogonia, and total interstitial space, was in each case 50 per cent or less of the mean. On the other hand, the range was 122 per cent for "space" and 168 per cent for Leydig cells. In Table 3 volume proportions from five different areas of the same cross section through a whole testis are recorded. In this section Area 5 was central and contained several septa converging near the mediastinum. Area 2 was similar but a little further removed from the mediastinum. Areas 1 and 4 were peripheral and from a region of the testis where biopsies would ordinarily be taken. The differences between the five areas of this one testis were not appreciably smaller than the differences between random groups of 5 biopsy specimens taken from Table 1. This is in part due to the
>
r
Vol. 7, No.3, 1956
257
HUMAN TESTICULAR BIOPSIES: I
fact that mediastinal areas, which in tissue composition are most different from the rest of the testis, were included in Table 3, but these areas are never taken in biopsy procedures. When the three nonmediastinal areas (1, 3, and 4 in Table 3) are compared, it is seen that they showed very small differences among each other. It is of interest that the volume of "space," (i.e., of unidentified structures TABLE 3.
Comparison of Relative Volumes (Per Cent) of Constituents of Five Different Areas from a Single Cross Section of a Human Testis (12,u Thick)
Areasu Constituent
Interstitial tissue and basement membrane Leydig cells Spermatogonia Spermatocytes Spermatids and spermatozoa Abnormal germ cells Total germ cells Sertoli cells Lumen "Space" a
1
2
3
4
5
Mean
S.D.b
29.4 1.3 9.5 16.7
36.6 0.9 8.5 14.4
34.9 0.6 9.6 14.8
28.4 1.1 9.1 13.2
38.4 1.8 7.5 12.6
33.5 1.1 8.8 14.3
4.48 0.45 0.87 1.59
10.4 0.4 37.0 18.8 10.3 3.2
9.6 1.1 33.6 19.1 6.0 3.8
7.8 0.6 32.8 20.3 8.6 2.8
9.4 0.4 32.1 19.5 15.6 3.3
9.0 0.4 29.5 16.2 11.4 2.7
9.2 0.6 33.0 18.8 10.4 3.2
0.95 0.30 2.76 1.88 3.56 0.40
The areas did not border each other.
b Standard deviation.
in the tubules) varied very little from area to area of the same testis. This is a demonstration of the fact that "space" is in large measure dependent on the quality of the preparation, which in this instance happened to be excellent and very uniform.
General Observations In scanning a great number of sections from biopsies of normal testes some observations were made which have a bearing on the reliability of biopsy material for quantitative determinations. Biopsy tissue possesses microscopic characteristics which definitely distinguish it from nonbiopsy tissue. Almost all biopsy specimens show unmistakable effects of handling throughout the greater part of most sections. The peripheral areas are often unusable for evaluation by the Chalkley method. In consequence of the injury and displacements in the periphery, germ cells are frequently trans-
258
ROOSEN-RUNGE
Fertility & Sterility
ported into the interstitial tissue spaces where they were found in 8 of the 23 specimens investigated. (No germ cells were ever seen in the interstitial tissue of nonbiopsy specimens.) Although the average relative volume of the interstitial tissues did not differ appreciably in biopsy and nonbiopsy specimens the width of the spaces varied much more in biopsy material. In some areas the tubules appeared jammed together, in others they were widely separated. Where the spaces were wide they may have been filled with tissue fluid or blood. In biopsies from normal testes some sloughing of germ cells into the lumen is the rule and severe sloughing is by no means rare. Quantitatively this was indicated by the low values for the volume of the lumen. Cellular preservation may be excellent in a biopsy specimen but the identification of abnormal cells presents difficulties because some cells will invariably show the effects of mechanical trauma. DISCUSSION Benoit first determined the relative volumes of structures in the human testis and found that the seminiferous tubules (including the basement membrane) occupied 70 per cent of the testis, interstitial tissue 22.3 per cent, and Leydig cells 7 per cent. Slotopolsky and Schinz12 perfected the planimetric method which Benoit had used and reported 66 per cent for the seminiferous tubules, 22 per cent for the interstitial tissue, and 12 per cent for the Leydig cells with a range from 8-28 per cent. These figures have been accepted by many later authors, for instance by Stieve in his review. The discrepancy between the results of the planimetric and Chalkley methods with respect to the volume of Leydig cells is indicative of the characteristics of the two methods. In the planimetric method the areas containing Leydig cells are treated as homogeneous throughout, and small vessels and intercellular spaces are not distinguished from the cells themselves. Chalkley's method permits the separate recording of these items. This explains the fact that our findings showed a lower volume for the Leydig cells. With regard to the volume of the seminiferous tubules and to the range of volume of Leydig cells in normal testes, our results are in excellent agreement with those of Slotopolsky and Schinz. 12 The data on relative volumes obtained by the Chalkley methods are more detailed and less laboriously gained than those resulting from planimetry, but they are open to the same question concerning the meaning of
I
I
Vol. 7, No.3, 1956
any data on relative collective volumes of cells and other structures. It is obvious that a detailed knowledge of numbers and sizes of germ cells in certain stages would permit a more meaningful analysis of proliferation and development of the germinal epithelium, but it is at present impossible to determine these values in the human testis because of the inherent lack of pattern in human spermatogenesis. 8A With this in mind, it may be premature to become seriously concerned about the meaning of data obtained with the Chalkley method, particularly as long as results on the normal cannot be compared with those on abnormal and experimental material. TABLE 4.
,
t
259
HUMAN TESTICULAR BIOPSIES: I
Comparison of Average Volume Proportions of Structures in the Testis of Man and the Rat
Interstitial tissue Leydig cells Basement membrane Total interstitial space Spermatogonia Spermatocytes Spermatids and spermatozoa Abnormal germ cells Residual bodies Total germ cells Sertoli cells Lumen Space a
Man
Rat
22.0a 3.1 9.1
8.0 1.7 2.4
34.2 7.8 14.4
12.1
9.1 0.7
1.7 14.7 41.1 0.1
17.4
1.2 58.8 8.4
10.6
19.5
5.8
1.1
32.0
All figures are per cent of total testis volume.
In any evaluation of the data it must, however, be clearly understood that the relative volume of any structure depends on the absolute and relative dimensions of the structure and on the frequency of its occurrence (i.e., in the case of cells in certain stages of development it depends on the duration of the stages) and that in human seminiferous tubules these factors cannot be separated from each other. It is of some interest to compare the results of volumetric measurements in the human with those in the rat (Table 4) which were obtained in an earlier study.9 The data are only approximately comparable because they were derived in the case of the human from 30 specimens, and in the case of the rat from only 2. Nevertheless the general results of the comparison seem valid. The rat testis possesses relatively much less interstitial tissue
260
ROOSEN-RUNGE
Fertility & Sterility
and Leydig cells but more germ cells, and the proportion of the various stages of germ cells is very different in the two species. In the rat the collective volume of spermatids and spermatozoa is 24 times that of the spermatogonia, and in the human 1.2 times. On the other hand, the relative volume of the Sertoli cells is two times greater in the human than in the rat. The proportion of Sertoli-cell volume to the volume of spermatids and spermatozoa is 1.9 in the human and 0.2 in the rat. One may speculate that the small size and the relatively small amount of cytoplasm which are apparent in human spermatids are correlated with a relatively greater development of the nursing cells. Detailed observations on testicular biopsies from normal human testes establish the fact that the biopsy technic as practiced at present by experienced operators results in tissues which show considerable distortions. In the diagnosis of pathologic biopsy specimens this must be taken into consideration. For instance, any conclusions based on the finding of sloughing of germ cells must be extremely guarded because of the frequency of this finding in biopsies from normal testes. On the other hand, the quantitative analysis of biopsy material, because it concerns itself with average values, is less affected by local displacements and injuries introduced by the operative technic.
SUMMARY Volumetric proportions of histologic elements of 30 normal human testes, 23 from biopsies and 7 from whole testes, were determined by means of Chalkley's method. The characteristics of testicular biopsy material are briefly discussed and the pOint is made that the biopsy technic is not ideal for a detailed morphologic survey of tissue from normal human testis. However, some of the distortions which are due to the technic do not appreciably influence a quantitative analysis. The differences in volume porportions of testicular structures in man and in the rat have been briefly discussed.
REFERENCES 1.
ALBERT, A. L., UNDERDAHL, L., GREENE, L., and Mayo Glin. 28:409, 1953. 2. BENOIT, J. Gompt. rend. Soc. biol. 87: 1385, 1922. 3. BRANCA, A. Gompt. rend Soc. anat. 13:283, 1911.
LORENZ,
N.
Froc. Staff Meet.
Vol. 7, No.3, 1956
HUMAN TESTICULAR BIOPSIES: I
261
4. CHALKLEY, H. W. f. Cancer Insf. 4:47, 1943. 5. ESCHENBRENNER, A. B., MILLER, E., and LORENS, E. f. Cancer Inst. 9:133, 1948. 6. HAUG, H. Ztschr. Anat.118:302, 1955. 7. KYRLE, J. Wien. klin. Wchnschr. 33:185, 909,1920. 8. ROOSEN-RuNGE, E. C. Proc. First World Congo Fertil. & Steril. 3:107,1954. 8A.RooSEN-RuNGE, E. C. Ann. N. Y. Acad. Sci. 55:574, 1952. 9. ROOSEN-RuNGE, E. C. Anat. Rec., 1955 (in press). 10. ROOSEN-RuNGE, E. C., and BARLOW, F. Am. f. Anat. 93:143,1953. 11. SCHINZ, H. R., and SLOTOPOLSKY, B. Abderhalden's Handb. der biol. Arbeits-
methoden Abt. V, 3B:529, 1938. 12. SLOTOPOLSKY, B., and SCHINZ, H. R. Virchows Arch. path. Anat. 257:294,1925. 13. SNIFFIN, R. C. Arch. Path. 50:259, 1950. 14. STIEVE, H. Miinnliche Genitalorgane: Handbuch der Mikroscopische Anatomie des Menschen. Berlin, Mollendorff, 1930.