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Effect of age on the composition of seminiferous tubular boundary tissue and on the volume of each component in humans*
FERTILITY AND STERILITY
•
Vol. 49, No.6, June 1988 Printed in U.S.A .
Copyright ~ 1988 The American Fertility Society
Effect of age on the composition of seminiferous tubular boundary tissue and on the volume of each component in humans·
Larry Johnson, Ph.D.t Joe G. Abdo, M.S.t Charles S. Petty, M.D.§ William B. Neaves, Ph.D.t Texas A & M University, College Station, and University of Texas Health Science Center at Dallas, Dallas, Texas
Seminiferous tubular boundary tissue thickens with age. The objective was to characterize the composition of boundary tissue in 16 young adult (20 to 29 years) and 18 older adult (51 to 84 years) men. Testes were perfused with glutaraldehyde, placed in osmium, and embedded in Epon 812 (Ladd Research Industries, Burlington, VT). Paired testicular weight, length of tubules, volume of seminiferous epithelium, and daily sperm production were significantly reduced in older men. Although the thickness of boundary tissue was greater (P < 0.01) in older men, the volume of boundary tissue per man was similar between age groups. The percentages and volumes per man of boundary tissue myoid cells, collagen, microfibrils, and other components also were similar (P> 0.05) between age groups. This study confirms that age-related thickening of boundary tissue occurs without the new deposition (augmentation) of collagen or other extracellular components. Fertil Steril 49:1045, 1988
Infertility or azoospermia in men has been associated with modification of the boundary tissue of seminiferous tubules. 1,2 Tubular boundary tissue, (also referred to as the limiting membrane, peritubular layer, basement membrane, and lamina propria3 ) thickens with age. 4,5 Other reported changes with age include progressive intertubular fibrosis, decreased tubular diameter, decreased thickness of seminiferous epithelium, obliteration of the semiReceived November 20,1987; revised and accepted February 11,1988. * Supported in part by the National Institutes of Health grants AG02260 and RR05854. t Reprint requests: Larry Johnson, Ph.D., Department of Veterinary Anatomy, College of Veterinary Medicine, Texas Agricultural Experimental Station, Texas A & M University, College Station, Texas 77843-4458. :t: Department of Cell Biology and Anatomy, University of Texas Health Science Center at Dallas. § Department of Pathology, University of Texas Health Science Center at Dallas. Vol. 49, No.6, June 1988
niferous tubule,4 reduced testicular weights, and reduced daily sperm production.6-8 The boundary tissue is known to be composed of two to six layers of myoid cells 1,9 that are separated from each other by collagen fibrils, elastin fibrils, and microfibrils. 3 However, the percentage and volume of each of these components and age-related differences in each component remain unknown. The objective of this study was to characterize, by quantitative stereology, the composition of tubular boundary tissue in younger and older adult men. MATERIALS AND METHODS Specimens
Testes from a group of 16 men aged 20 to 33 years (mean ± SE, 25 ± 1 years) and a group of 18 men aged 50 to 84 years (63 ± 3 years) were obtained at autopsy within 15 hours of death. The distribution of subjects according to race or ethnic
Johnson et al. Composition of human tubular boundary tissue
1045
origin and postmortem time was similar in both groups. Subjects in both age groups were selected on the basis of apparent good health prior to death, and they had no history of recent extended hospitalization or serious illness. Body weights were similar (P > 0.05) between the two groups of men (77 ± 5 kg for younger and 85 ± 4 kg for older men). Blood was drawn from the heart or large veins during autopsy. Serum concentrations of gonadotropins previously reported for these same menlO included 94 ± 23 versus 120 ± 19 ng/ml of luteinizing hormone (LH) (P > 0.05) and 106 ± 17 versus 284 ± 49 ng/ml follicle stimulating hormone (FSH) (P < 0.01) for younger and older men, respectively. Testes were fixed by vascular perfusion with 2% glutaraldehyde in 0.1 M cacodylate buffer. Five pieces (3 X 10 X 10 mm) of a glutaraldehyde-fixed testis were placed in 1% OS04 in cacodylate buffer, dehydrated in alcohol, and embedded in Epon 812 (Ladd Research Industries, Burlington, VT). Epon-embedded tissues were sectioned at 0.5 ~m, stained with toluidine blue, and examined by bright-field microscopy or thin sectioned and observed by electron microscopyY During evaluation, tissue specimens were coded such that the identity of the subjects or the age group was concealed in this double-blind study. Measurements to Characterize Seminiferous Tubules
Percentages of testicular parenchyma occupied by seminiferous tubules or boundary tissue (Fig. 1) were determined by point counting. 5 Large Epon sections (approximately 10 mm 2 ) were employed with predetermined movements of slides during the evaluation at 400X magnification to ensure randomness and sufficient sampling. Sufficient numbers of points (2000) were scored for each parameter to achieve a coefficient of variation of approximately 10% or less. The total volume of each component in paired testes was calculated by multiplying the percentage of the testicular parenchyma occupied by that component times the parenchymal volume (parenchymal weight divided by 1.05 gm/ml). The length of seminiferous tubules was calculated by dividing tubular volume per man by the cross-sectional area.5 The total volume of each component in the boundary tissue was calculated by multiplying the volume of boundary tissue per man times the percentage of boundary tissue occupied by each component as detected by electron microscopy. 1046
Figure 1 Light micrograph of a seminiferous tubule in stage II of the cycle from a 66-year-old man. The testis was fixed in glutaraldehyde followed by osmium, embedded in Epon 812, sectioned at 0.5 !Lm, and stained with toluidine blue. Boundary tissue (BT) surrounds the germ cells and Sertoli cells (SC) of the seminiferous epithelium. The testis in older men may be well endowed with the entire constellation of germ cells characteristic of that stage of the cycle. Germ cell types present include A-dark spermatogonia (Ad), A-pale spermatogonia (Ap), leptotene primary spermatocytes (L), pachytene primary spermatocytes (P), cap-phase spermatids (CP), and maturation-phase spermatids (MP). Bar length equals 10 !Lm.
The percentage of boundary tissue occupied by myoid cells, collagen, microfibrils, and all other components combined (Fig. 2) was determined on electron micrographs of the boundary tissue. A total of 492 (209 for young and 284 for older men) micrographs taken randomly at predetermined locations on 200-mesh grids were examined at 16,500X magnification and scored by a pointcounting overlay.5 An average of 340 points were scored on each micrograph. The percentage of each component was 100 times the number of points over that component divided by the number of total points scored over the boundary tissue. Boundary tissue thickness was based on the measurement of 50 tubules per man. The thickness of the basal lam ina was determined from 50 random measurements per man on the previously mentioned micrographs with a computerized digitizing system. The coefficient of variation for repeated measurement was low at 6.5%. From the same micrographs, the percentage of extracellular spaces in the seminiferous epithelium containing involuted basal laminae was determined from 257 extracellular spaces observed on young men and 256 observed on older men. The number of myoid cells per man in the boundary tissue was determined as the product of the parenchymal volume times the percentage of myoid cell nuclei in the parenchyma divided by the
Johnson et al. Composition of human tubular boundary tissue
Fertility and Sterility
a
I
Figure 2 Plate of electron micrographs of the basal region of the seminiferous epithelium and boundary tissue in a 59-year-old man. (A) A large region of collagen (e) is located between layers of myoid cells (Me). (B) Involution (I) of the basal lamina (BL) in the extracellular space between a Sertoli cell and a spermatogonium is shown. (e) Microfibrils (MF) are observed between myoid cells. (A) A characteristic profile of a Sertoli cell nucleus with numerous nuclear pores (NP) and the central nucleolus (NO) with the surrounding satellite karyosomes is indicated. Sertoli cell cytoplasmic structures include typical cytologic organelles plus large lipid droplets (LD) and annute lamellae (AL). An A-pale spermatogonium (Ap) and pachytene primary spermatocyte (P) are present in this portion of the seminiferous epithelium. Bar length equals 2 I'm in a and band 1 I'm in c.
average volume of individual nuclei. 6 The volume of individual nuclei of myoid cells was determined by reconstruction of serial sections (0.5 ~m) through the entire nucleus. 12•13 Daily sperm production was calculated as the product of the number of round spermatids in homogenate times the parenchymal weight divided by the product of the weight of tissue homogenized and the 8.9-day life span of these spermatids. 14 Vol. 49, No.6, June 1988
Statistical Analysis
Differences between age groups were tested by the Student's t-test. 15 Correlation coefficients were tested for significance as described by Sokal and Rohlf. 16 RESULTS
Seminiferous tubules in older adult men (Fig. 1) may appear quite normal with all types of germ
Johnson et al. Composition of human tubular boundary tissue
1047
cells present in a given stage of the cycle of seminiferous epithelium and with boundary tissue similar to that of younger men. Although paired testicular and paired parenchymal weights were significantly lower (P < 0.05) in older men, the percentage of seminiferous tubules in the parenchyma, diameter of the tubules, and thickness of the basal lamina were unaffected (P > 0.05) with age (Table 1). However, detailed measurements of seminiferous tubules reveal that the volume (P < 0.05) and length (P < 0.01) of tubules per man were significantly lower in older men. More importantly for spermatogenesis, the percentage of seminiferous epithelium in the parenchyma and the volume of seminiferous epithelium per man were lower (P < 0.01) in older men. As a result of less seminiferous epithelium, daily sperm production/man was significantly (P < 0.01) reduced in older men. The thickness of tubular boundary tissue was greater (P < 0.01) in older men (Table 1). The percentage of boundary tissue in the parenchyma was increased (P < 0.01) with age. However, with the counterforces of a reduced paired parenchymal weight in older men, the volume of boundary tissue/man was similar (P > 0.05) between groups. Likewise, the number of myoid cells/man was similar (P> 0.05) in men from the two age groups. Ultrastructural stereo logy of boundary tissue
(Fig. 2) revealed its composition. There was no age-related difference in the percentage of boundary tissue occupied by myoid cells, collagen, microfibrils, or all other components combined (Table 2). Given similarity in composition and volume/man of boundary tissue between age groups (Table 1), there was no difference (P > 0.05) in the volume/ man of any component of the boundary tissue (Table 2). There was an age-related increase (P < 0.01) in the percentage of extracellular spaces in seminiferous epithelium that contained involuted basal laminae (Fig. 2, Table 1). The percentage of extracellular spaces with basal lamina material was not correlated (r = -0.24; P > 0.05) with daily sperm production per man. Boundary tissue thickness was negatively correlated (P < 0.01) with the length of seminiferous tubules (r = -0.54), the percentage of seminiferous epithelium in the parenchyma (r = -0.66), daily sperm production per gram parenchyma (r = -0.48), and daily sperm production per man (r = -0.59). DISCUSSION In spite of a 45% increase (P < 0.01) in thickness of tubular boundary tissue in older men, there was no age-related difference in the total volume of
Table 1 Effect of Age on Seminiferous Tubules and Tubular Boundary Tissue in Men Age (years) Item
20-33 (25 ± 1)
50-84 (63 ± 3)
Number
a
Significance
16
18
Paired testicular weights (gm) Whole testes Parenchyma
46.6 ± 2.5 40.7 ± 2.3
38.3 ± 2.7 32.0 ± 2.4
P < 0.05 P < 0.05
Daily sperm production/man (10 6 )
238 ± 32
131 ± 13
P < 0.01
Seminiferous tubules Percentage of parenchyma Volume/man (ml) Diameter (/Lm) Length/man (m)
62.6 24 219 639
± ± ± ±
1.7 1 3 34
60.0 18 288 405
± ± ± ±
2.1 2 5 38
Nsa P < 0.05 NS P < 0.01
Seminiferous epithelium Percentage of parenchyma Volume/man (m!) Thickness of basal lamina (nm) Basal lamina involution in extracellular space (%)
44.8 17.1 2.73 10.6
± ± ± ±
1.6 1.0 0.26 2.9
36.8 11.4 2.34 24.0
± ± ± ±
2.2 1.1 0.15 3.6
P < 0.01 P < 0.01 NS P < 0.01
Boundary tissue Thickness (/Lm) Percentage of parenchyma Volume/man (ml)
7.6 ± 0.2 8.5 ± 0.3 3.3 ± 0.2
11.0 ± 0.5 10.7 ± 0.7 3.3 ± 0.3
P < 0.01 P < 0.01 NS
Number of myoid cells/man (106 )
883 ± 60
753 ± 72
NS
NS, not significant.
1048
Johnson et al.
Composition of human tubular boundary tissue
Fertility and Sterility
,.
Table 2 Effect of Age on Composition of Boundary Tissue and on Volume of Each Component in Men" Age (years)
.. ..
Item Number Boundary tissue Composition (%) Myoid cells Collagen Microfibrils Other Volume/man (ml) Myoid cells Collagen Microfibrils Other
20-33 (25 ± 1)
50-84 (63 ± 3)
16
18
36.2 ± 27.0 ± 22.1 ± 14.8 ±
1.6 2.0 1.3 1.8
31.8 ± 29.2 ± 24.1 ± 15.0 ±
1.8 2.5 1.5 1.9
1.19 ± 0.87 ± 0.74 ± 0.48 ±
1.10 0.08 0.08 0.06
1.05 ± 0.97 ± 0.80 ± 0.50 ±
0.13 0.14 0.10 0.08
" There were no significant differences
boundary tissue per man (Table 1). Likewise, the presence of large regions of collagen-filled extracellular space (Fig. 2) did not correspond to differences in the percentage of collagen in boundary tissue or volume of collagen per man (Table 2). Kerr and coworkers 16 have attributed increased thickening of boundary tissue in cryptorchid animals to "accommodation to the progressive decrease in diameter of seminiferous tubules, necessitating the contraction of the peritubular tissue (boundary tissue) within a smaller area than is normal." Thickening of boundary tissue with age in humans was attributed to the increased stacking of myoid cells and the accompanying extracellular components as measured by an age-related increased number of myoid cells per cross section.5 The stacking or regrouping was brought about in shorter tubules in older men without change in total volume of boundary tissue per man (Table 1) and without change in the percentage or volume of collagen, microfibrils, or other extracellular components combined (Table 2). Stacking of myoid cells in tubules with age-related thickened boundary tissue 6 was consistent with other reports1 that thickening resulted from widening of existing structures instead of the appearance of new components. There was a slight trend toward reduced myoid cell volume and number in older men (Table 1). However, this did not correspond with an increased percentage or volume of collagen per man (Tables 1 and 2). The lack of significant differences in total volume of collagen or other extracellular compoVol. 49, No.6, June 1988
nents between groups of men with a significant difference in boundary tissue thickness is not consistent with augmentation of boundary tissue with collagen or other extracellular components. This finding is different from what might be inferred in studies in which the volume per man of boundary tissue or collagen was not determined. 1,3,17 Age-related reduction in length of seminiferous tubules corresponds with reduced testicular weight, volume of seminiferous epithelium,5 number of Sertoli cells, numbers of various germ cells, and daily sperm production. 6,12 In the current study, there was a negative correlation (P < 0.01) between the thickness of boundary tissue and tubular length and between the thickness of boundary tissue and the volume of seminiferous epithelium. Age-related thickening of tubular boundary tissue may be important in the transit of metabolites and hormones to and by-products from seminiferous tubules and may reduce spermatozoan production rates even further. 5,7 Whereas thickening of boundary tissue has been associated with infertility or azoospermia/,2 thickened boundary tissue of tubules is not confined to infertile men. The increased thickening of boundary tissue following vasectomy appeared to be unrelated to the fertility of men who had successful vasectomy reversals without interstitial fibrosisP However, it is unknown if the fertility rate was reduced in these fertile men with increased thickness of boundary tissue after vasectomy. Vasectomyp long-term estrogen treatment/ 8 and aging19 in humans have been associated with an increased thickness of the basal lamina of seminiferous epithelium. In spite of the similarity of our values to values in previous studies/,4 we were unable to confirm the age-related thickening of the basal lamina. Others have found that thickening of basal laminae was an inconsistent feature of damaged tubules in pathologic human testis. 1 The negative correlation between boundary tissue thickness and volume of seminiferous epithelium or daily sperm production corresponds to previous findings. These investigators found that the degree of change in the basal lamina (involuting lamellation, thickening, and pleat formation) and changes in the tunic propria was almost proportional to the degree of hypospermia. 20 Although the percentage of basal lamina involution in the extracellular space of seminiferous epithelium increased (P < 0.01) with age (Table 1), it was not significantly correlated with daily sperm production per man.
Johnson et al.
Composition of human tubular boundary tissue
1049
Our previous studies have revealed that age-related reduction in the length of tubules (brought about by reduced numbers of Sertoli cells and various types of germ cells) without change in the volume of boundary tissue results in increased thickening of this tissue. 5 The present study confirms that age-related thickening of boundary tissue need not occur by the augmentation of collagen or other extracellular components. It also illustrates the importance of determining absolute values in evaluating potential causes of changes in human testicular boundary tissue. Alteration in tubular length without change in volume of boundary tissue 5 or its components (Table 2) provides an explanation for the relationship between hormonal status and thickness of tubular boundary tissue. I ,2I-23 Thickened boundary tissue in men with elevated blood levels of gonadotrophic hormones and severe depletion of germ cells, 1 may have been the result of reduced tubular length with no change in the volume of boundary tissue itself. Hence, there need not be a direct or indirect (through testosterone) stimulation of myoid cells themselves. Organization (reduced thickness) of boundary tissue in immature testes from patients with hypogonadotrophic hypogonadism following treatment with FSH and LH could result from the lengthening of tubules as larger numbers of germ cells are produced. 22 Likewise, the maturation of myoid cells in testicular tissue implants into hypophysectomized, testosteronetreated hosts 23 could result indirectly from maturation of the seminiferous epithelium (increased tubular length). Estrogen treatment, known to affect germ cell numbers, also causes increased thickness of boundary tissue. Is Vasectomy, which altered the size of seminiferous tubules without change in the hormonal status, was associated with increased tubular boundary tissueY The thicker the tubular wall in cryptorchid boys, the more marked were the changes in Sertoli cells and in numbers of spermatogonia.24 Furthermore, as noted above, physical collapse of seminiferous tubules was implicated in the thickening of the tubular boundary tissue after induced cryptorchidism. IS It is impossible to determine if boundary tissue changes precede or follow the spermatogenic disorder. I In human cryptorchid testes, tubular boundary tissue thickening occurs earlier than damage to seminiferous epithelium can be noted. 25 Hence, these authors suggest that tubular damage may be secondary to thickened tubules. This finding is not consistent with our findings of age-re1050
Johnson et al.
lated reduction in tubular length with no change in volume of boundary tissue (Table 1).5 Based on our findings, we suggest that the alteration in thickness of boundary tissue is secondary to the alteration of the volume of seminiferous epithelium. The reason the boundary tissue volume is constant in conditions which significantly reduce the tubular length (Table 1)5 is unknown. In any case, the thickness of boundary tissue determined from biopsies may be a useful measure of the effect of hormones, environmental toxins, and other factors on the seminiferous epithelial volume and tubular length. Acknowledgments. Special thanks are extended to Mr. Vince Hardy, Ms. Cynthia Snyder, Tabitha Childs, Janie Pluenneke, and Pat Stroud for technical assistance and in preparation of the manuscript. REFERENCES 1. de Krester DM, Kerr JB, Paulsen CA: The peritubular tissue in the normal and pathological human testis: an ultrastructural study. Bioi Reprod 12:317, 1975 2. Salomon F, Hedinger CE: Abnormal basement membrane structures of seminiferous tubules in infertile men. Lab Invest 47:543, 1982 3. Bustos-Obregon E, Holstein AF: On structural patterns of the lamina propria of human seminiferous tubules. Z Zellforsch Mikrosk Anat 141:413, 1973 4. Bishop MWH: Ageing and reproduction in the male. J Reprod Fertil12(suppl):65, 1970 5. Johnson L, Petty CS, Neaves WB: Age-related variation in seminiferous tubules in men: a stereologic evaluation. J Androl 7:316, 1986 6. Amann RP: A criticl review of methods for evaluation of spermatogenesis from seminal characteristics. J Androl 2:37,1981 7. Johnson L: Spermatogenesis and aging in the human. J Androl 7:331, 1986 8. Johnson L, Petty CS, Neaves WB: Influence of age on sperm production and testicular weights in men. J Reprod Fertil 70:211, 1984 9. Hermo L, Lalli M, Clermont Y: Arrangement of connective tissue components in the walls of seminiferous tubules of man and monkey. Am J Anat 148:433, 1977 10. Johnson L, Petty CS, Porter JC, Neaves WB: Germ cell degeneration during postprophase of meiosis and serum concentrations of gonadotropins in young adult and older adult men. Bioi Reprod 31:779, 1984 11. Johnson L, Petty CS, Neaves WB: A comparative study of daily sperm production and testicular composition in humans and rats. Bioi Reprod 22:1233, 1980 12. Johnson L, Zane RS, Petty CS, Neaves WB: Quantification of the human Sertoli cell population: its distribution, relation to germ cell numbers, and age-related decline. Bioi Reprod 31:785, 1984 13. Neaves WB, Johnson L, Petty CS: Age-related change in number of other interstitial cells in testes of adult men: evidence bearing on the fate of Leydig cells lost with increasing age. Bioi Reprod 33:259, 1985
Composition of human tubular boundary tissue
Fertility and Sterility
14. Johnson L, Petty CS, Neaves WB: A new approach to quantification of spermatogenesis and its application to germinal cell attrition during human spermatogenesis. Bioi Reprod 25:217, 1981 15. Sokal RR, Rohlf FJ: Single classification analysis of variance. In Biometry, Edited by RR Sokal, FJ Rohlf. San Francisco, WH Freeman, 1969, p 220 16. Kerr JB, Rich KA, de Kretser DM: Effects of experimental cryptorchidism on the ultrastructure and function of the Sertoli cell and peritubular tissue of the rat testis. Bioi Reprod 21:823, 1979 17. Jarow JP, Budin RE, Dym M, Zirkin BR, Noren S, Marchall FF: Quantitative pathologic changes in the human testis after vasectomy: a controlled study. N Engl J Med 313:1252, 1985 18. Lu CC, Steinberger A: Effects of estrogen on human seminiferous tubules: Light and electron microscopic analysis. Am J Anat 153:1, 1978 19. Xi Y-P, Nette G, King DW, Rosen M: Age-related changes in normal human basement membrane. Mech Ageing Dev 19:315, 1982 20. Furuya S: Studies on testicular function. Electron microscopic studies on the changes of the peritubular wall of the
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21.
22.
23.
24.
25.
human seminiferous tubules in hypospermatogenesis. Jpn J Urol 66:80, 1975 Schulze CA, Holstein AF, Schinen C, Korner F: On the morphology of the human Sertoli cells under normal conditions and in patients with impaired fertility. Andrologia 8:167,1976 de Kretser DM, Burger HG: Ultrastructural studies of the human Sertoli cell in normal men and males with hypogonadotropic hypogonadism before and after gonadotrophic treatment. In Gonadotropins, Edited by BB Saxena, CG Beling, HM Gandy. New York, Wiley Interscience, 1972, p 640 Bressler RS, Ross MH: Differentiation of peritubular myoid cells of the testis: effects of intratesticular implantation of newborn mouse testes into normal and hypophysectomized adults. Bioi Reprod 6:148, 1972 de la Baize FA, Mancini RE, Arrillaga F, Andrada JA, Vilar 0, Gurtmann AI, Davidson OW: Histologic study upon the undescended human testis during puberty. J Clin Endocrinol 20:286, 1960 Hadziselimovic F: Cryptochidism, ultrastructure of normal and crytorchid testis development. Adv Anat Embryol Cell Bioi 53:Fasc 3, 1977
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•
Vol. 49, No.6, June 1988 Printed in U.S.A .
Copyright ~ 1988 The American Fertility Society
Effect of age on the composition of seminiferous tubular boundary tissue and on the volume of each component in humans·
Larry Johnson, Ph.D.t Joe G. Abdo, M.S.t Charles S. Petty, M.D.§ William B. Neaves, Ph.D.t Texas A & M University, College Station, and University of Texas Health Science Center at Dallas, Dallas, Texas
Seminiferous tubular boundary tissue thickens with age. The objective was to characterize the composition of boundary tissue in 16 young adult (20 to 29 years) and 18 older adult (51 to 84 years) men. Testes were perfused with glutaraldehyde, placed in osmium, and embedded in Epon 812 (Ladd Research Industries, Burlington, VT). Paired testicular weight, length of tubules, volume of seminiferous epithelium, and daily sperm production were significantly reduced in older men. Although the thickness of boundary tissue was greater (P < 0.01) in older men, the volume of boundary tissue per man was similar between age groups. The percentages and volumes per man of boundary tissue myoid cells, collagen, microfibrils, and other components also were similar (P> 0.05) between age groups. This study confirms that age-related thickening of boundary tissue occurs without the new deposition (augmentation) of collagen or other extracellular components. Fertil Steril 49:1045, 1988
Infertility or azoospermia in men has been associated with modification of the boundary tissue of seminiferous tubules. 1,2 Tubular boundary tissue, (also referred to as the limiting membrane, peritubular layer, basement membrane, and lamina propria3 ) thickens with age. 4,5 Other reported changes with age include progressive intertubular fibrosis, decreased tubular diameter, decreased thickness of seminiferous epithelium, obliteration of the semiReceived November 20,1987; revised and accepted February 11,1988. * Supported in part by the National Institutes of Health grants AG02260 and RR05854. t Reprint requests: Larry Johnson, Ph.D., Department of Veterinary Anatomy, College of Veterinary Medicine, Texas Agricultural Experimental Station, Texas A & M University, College Station, Texas 77843-4458. :t: Department of Cell Biology and Anatomy, University of Texas Health Science Center at Dallas. § Department of Pathology, University of Texas Health Science Center at Dallas. Vol. 49, No.6, June 1988
niferous tubule,4 reduced testicular weights, and reduced daily sperm production.6-8 The boundary tissue is known to be composed of two to six layers of myoid cells 1,9 that are separated from each other by collagen fibrils, elastin fibrils, and microfibrils. 3 However, the percentage and volume of each of these components and age-related differences in each component remain unknown. The objective of this study was to characterize, by quantitative stereology, the composition of tubular boundary tissue in younger and older adult men. MATERIALS AND METHODS Specimens
Testes from a group of 16 men aged 20 to 33 years (mean ± SE, 25 ± 1 years) and a group of 18 men aged 50 to 84 years (63 ± 3 years) were obtained at autopsy within 15 hours of death. The distribution of subjects according to race or ethnic
Johnson et al. Composition of human tubular boundary tissue
1045
origin and postmortem time was similar in both groups. Subjects in both age groups were selected on the basis of apparent good health prior to death, and they had no history of recent extended hospitalization or serious illness. Body weights were similar (P > 0.05) between the two groups of men (77 ± 5 kg for younger and 85 ± 4 kg for older men). Blood was drawn from the heart or large veins during autopsy. Serum concentrations of gonadotropins previously reported for these same menlO included 94 ± 23 versus 120 ± 19 ng/ml of luteinizing hormone (LH) (P > 0.05) and 106 ± 17 versus 284 ± 49 ng/ml follicle stimulating hormone (FSH) (P < 0.01) for younger and older men, respectively. Testes were fixed by vascular perfusion with 2% glutaraldehyde in 0.1 M cacodylate buffer. Five pieces (3 X 10 X 10 mm) of a glutaraldehyde-fixed testis were placed in 1% OS04 in cacodylate buffer, dehydrated in alcohol, and embedded in Epon 812 (Ladd Research Industries, Burlington, VT). Epon-embedded tissues were sectioned at 0.5 ~m, stained with toluidine blue, and examined by bright-field microscopy or thin sectioned and observed by electron microscopyY During evaluation, tissue specimens were coded such that the identity of the subjects or the age group was concealed in this double-blind study. Measurements to Characterize Seminiferous Tubules
Percentages of testicular parenchyma occupied by seminiferous tubules or boundary tissue (Fig. 1) were determined by point counting. 5 Large Epon sections (approximately 10 mm 2 ) were employed with predetermined movements of slides during the evaluation at 400X magnification to ensure randomness and sufficient sampling. Sufficient numbers of points (2000) were scored for each parameter to achieve a coefficient of variation of approximately 10% or less. The total volume of each component in paired testes was calculated by multiplying the percentage of the testicular parenchyma occupied by that component times the parenchymal volume (parenchymal weight divided by 1.05 gm/ml). The length of seminiferous tubules was calculated by dividing tubular volume per man by the cross-sectional area.5 The total volume of each component in the boundary tissue was calculated by multiplying the volume of boundary tissue per man times the percentage of boundary tissue occupied by each component as detected by electron microscopy. 1046
Figure 1 Light micrograph of a seminiferous tubule in stage II of the cycle from a 66-year-old man. The testis was fixed in glutaraldehyde followed by osmium, embedded in Epon 812, sectioned at 0.5 !Lm, and stained with toluidine blue. Boundary tissue (BT) surrounds the germ cells and Sertoli cells (SC) of the seminiferous epithelium. The testis in older men may be well endowed with the entire constellation of germ cells characteristic of that stage of the cycle. Germ cell types present include A-dark spermatogonia (Ad), A-pale spermatogonia (Ap), leptotene primary spermatocytes (L), pachytene primary spermatocytes (P), cap-phase spermatids (CP), and maturation-phase spermatids (MP). Bar length equals 10 !Lm.
The percentage of boundary tissue occupied by myoid cells, collagen, microfibrils, and all other components combined (Fig. 2) was determined on electron micrographs of the boundary tissue. A total of 492 (209 for young and 284 for older men) micrographs taken randomly at predetermined locations on 200-mesh grids were examined at 16,500X magnification and scored by a pointcounting overlay.5 An average of 340 points were scored on each micrograph. The percentage of each component was 100 times the number of points over that component divided by the number of total points scored over the boundary tissue. Boundary tissue thickness was based on the measurement of 50 tubules per man. The thickness of the basal lam ina was determined from 50 random measurements per man on the previously mentioned micrographs with a computerized digitizing system. The coefficient of variation for repeated measurement was low at 6.5%. From the same micrographs, the percentage of extracellular spaces in the seminiferous epithelium containing involuted basal laminae was determined from 257 extracellular spaces observed on young men and 256 observed on older men. The number of myoid cells per man in the boundary tissue was determined as the product of the parenchymal volume times the percentage of myoid cell nuclei in the parenchyma divided by the
Johnson et al. Composition of human tubular boundary tissue
Fertility and Sterility
a
I
Figure 2 Plate of electron micrographs of the basal region of the seminiferous epithelium and boundary tissue in a 59-year-old man. (A) A large region of collagen (e) is located between layers of myoid cells (Me). (B) Involution (I) of the basal lamina (BL) in the extracellular space between a Sertoli cell and a spermatogonium is shown. (e) Microfibrils (MF) are observed between myoid cells. (A) A characteristic profile of a Sertoli cell nucleus with numerous nuclear pores (NP) and the central nucleolus (NO) with the surrounding satellite karyosomes is indicated. Sertoli cell cytoplasmic structures include typical cytologic organelles plus large lipid droplets (LD) and annute lamellae (AL). An A-pale spermatogonium (Ap) and pachytene primary spermatocyte (P) are present in this portion of the seminiferous epithelium. Bar length equals 2 I'm in a and band 1 I'm in c.
average volume of individual nuclei. 6 The volume of individual nuclei of myoid cells was determined by reconstruction of serial sections (0.5 ~m) through the entire nucleus. 12•13 Daily sperm production was calculated as the product of the number of round spermatids in homogenate times the parenchymal weight divided by the product of the weight of tissue homogenized and the 8.9-day life span of these spermatids. 14 Vol. 49, No.6, June 1988
Statistical Analysis
Differences between age groups were tested by the Student's t-test. 15 Correlation coefficients were tested for significance as described by Sokal and Rohlf. 16 RESULTS
Seminiferous tubules in older adult men (Fig. 1) may appear quite normal with all types of germ
Johnson et al. Composition of human tubular boundary tissue
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cells present in a given stage of the cycle of seminiferous epithelium and with boundary tissue similar to that of younger men. Although paired testicular and paired parenchymal weights were significantly lower (P < 0.05) in older men, the percentage of seminiferous tubules in the parenchyma, diameter of the tubules, and thickness of the basal lamina were unaffected (P > 0.05) with age (Table 1). However, detailed measurements of seminiferous tubules reveal that the volume (P < 0.05) and length (P < 0.01) of tubules per man were significantly lower in older men. More importantly for spermatogenesis, the percentage of seminiferous epithelium in the parenchyma and the volume of seminiferous epithelium per man were lower (P < 0.01) in older men. As a result of less seminiferous epithelium, daily sperm production/man was significantly (P < 0.01) reduced in older men. The thickness of tubular boundary tissue was greater (P < 0.01) in older men (Table 1). The percentage of boundary tissue in the parenchyma was increased (P < 0.01) with age. However, with the counterforces of a reduced paired parenchymal weight in older men, the volume of boundary tissue/man was similar (P > 0.05) between groups. Likewise, the number of myoid cells/man was similar (P> 0.05) in men from the two age groups. Ultrastructural stereo logy of boundary tissue
(Fig. 2) revealed its composition. There was no age-related difference in the percentage of boundary tissue occupied by myoid cells, collagen, microfibrils, or all other components combined (Table 2). Given similarity in composition and volume/man of boundary tissue between age groups (Table 1), there was no difference (P > 0.05) in the volume/ man of any component of the boundary tissue (Table 2). There was an age-related increase (P < 0.01) in the percentage of extracellular spaces in seminiferous epithelium that contained involuted basal laminae (Fig. 2, Table 1). The percentage of extracellular spaces with basal lamina material was not correlated (r = -0.24; P > 0.05) with daily sperm production per man. Boundary tissue thickness was negatively correlated (P < 0.01) with the length of seminiferous tubules (r = -0.54), the percentage of seminiferous epithelium in the parenchyma (r = -0.66), daily sperm production per gram parenchyma (r = -0.48), and daily sperm production per man (r = -0.59). DISCUSSION In spite of a 45% increase (P < 0.01) in thickness of tubular boundary tissue in older men, there was no age-related difference in the total volume of
Table 1 Effect of Age on Seminiferous Tubules and Tubular Boundary Tissue in Men Age (years) Item
20-33 (25 ± 1)
50-84 (63 ± 3)
Number
a
Significance
16
18
Paired testicular weights (gm) Whole testes Parenchyma
46.6 ± 2.5 40.7 ± 2.3
38.3 ± 2.7 32.0 ± 2.4
P < 0.05 P < 0.05
Daily sperm production/man (10 6 )
238 ± 32
131 ± 13
P < 0.01
Seminiferous tubules Percentage of parenchyma Volume/man (ml) Diameter (/Lm) Length/man (m)
62.6 24 219 639
± ± ± ±
1.7 1 3 34
60.0 18 288 405
± ± ± ±
2.1 2 5 38
Nsa P < 0.05 NS P < 0.01
Seminiferous epithelium Percentage of parenchyma Volume/man (m!) Thickness of basal lamina (nm) Basal lamina involution in extracellular space (%)
44.8 17.1 2.73 10.6
± ± ± ±
1.6 1.0 0.26 2.9
36.8 11.4 2.34 24.0
± ± ± ±
2.2 1.1 0.15 3.6
P < 0.01 P < 0.01 NS P < 0.01
Boundary tissue Thickness (/Lm) Percentage of parenchyma Volume/man (ml)
7.6 ± 0.2 8.5 ± 0.3 3.3 ± 0.2
11.0 ± 0.5 10.7 ± 0.7 3.3 ± 0.3
P < 0.01 P < 0.01 NS
Number of myoid cells/man (106 )
883 ± 60
753 ± 72
NS
NS, not significant.
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,.
Table 2 Effect of Age on Composition of Boundary Tissue and on Volume of Each Component in Men" Age (years)
.. ..
Item Number Boundary tissue Composition (%) Myoid cells Collagen Microfibrils Other Volume/man (ml) Myoid cells Collagen Microfibrils Other
20-33 (25 ± 1)
50-84 (63 ± 3)
16
18
36.2 ± 27.0 ± 22.1 ± 14.8 ±
1.6 2.0 1.3 1.8
31.8 ± 29.2 ± 24.1 ± 15.0 ±
1.8 2.5 1.5 1.9
1.19 ± 0.87 ± 0.74 ± 0.48 ±
1.10 0.08 0.08 0.06
1.05 ± 0.97 ± 0.80 ± 0.50 ±
0.13 0.14 0.10 0.08
" There were no significant differences
boundary tissue per man (Table 1). Likewise, the presence of large regions of collagen-filled extracellular space (Fig. 2) did not correspond to differences in the percentage of collagen in boundary tissue or volume of collagen per man (Table 2). Kerr and coworkers 16 have attributed increased thickening of boundary tissue in cryptorchid animals to "accommodation to the progressive decrease in diameter of seminiferous tubules, necessitating the contraction of the peritubular tissue (boundary tissue) within a smaller area than is normal." Thickening of boundary tissue with age in humans was attributed to the increased stacking of myoid cells and the accompanying extracellular components as measured by an age-related increased number of myoid cells per cross section.5 The stacking or regrouping was brought about in shorter tubules in older men without change in total volume of boundary tissue per man (Table 1) and without change in the percentage or volume of collagen, microfibrils, or other extracellular components combined (Table 2). Stacking of myoid cells in tubules with age-related thickened boundary tissue 6 was consistent with other reports1 that thickening resulted from widening of existing structures instead of the appearance of new components. There was a slight trend toward reduced myoid cell volume and number in older men (Table 1). However, this did not correspond with an increased percentage or volume of collagen per man (Tables 1 and 2). The lack of significant differences in total volume of collagen or other extracellular compoVol. 49, No.6, June 1988
nents between groups of men with a significant difference in boundary tissue thickness is not consistent with augmentation of boundary tissue with collagen or other extracellular components. This finding is different from what might be inferred in studies in which the volume per man of boundary tissue or collagen was not determined. 1,3,17 Age-related reduction in length of seminiferous tubules corresponds with reduced testicular weight, volume of seminiferous epithelium,5 number of Sertoli cells, numbers of various germ cells, and daily sperm production. 6,12 In the current study, there was a negative correlation (P < 0.01) between the thickness of boundary tissue and tubular length and between the thickness of boundary tissue and the volume of seminiferous epithelium. Age-related thickening of tubular boundary tissue may be important in the transit of metabolites and hormones to and by-products from seminiferous tubules and may reduce spermatozoan production rates even further. 5,7 Whereas thickening of boundary tissue has been associated with infertility or azoospermia/,2 thickened boundary tissue of tubules is not confined to infertile men. The increased thickening of boundary tissue following vasectomy appeared to be unrelated to the fertility of men who had successful vasectomy reversals without interstitial fibrosisP However, it is unknown if the fertility rate was reduced in these fertile men with increased thickness of boundary tissue after vasectomy. Vasectomyp long-term estrogen treatment/ 8 and aging19 in humans have been associated with an increased thickness of the basal lamina of seminiferous epithelium. In spite of the similarity of our values to values in previous studies/,4 we were unable to confirm the age-related thickening of the basal lamina. Others have found that thickening of basal laminae was an inconsistent feature of damaged tubules in pathologic human testis. 1 The negative correlation between boundary tissue thickness and volume of seminiferous epithelium or daily sperm production corresponds to previous findings. These investigators found that the degree of change in the basal lamina (involuting lamellation, thickening, and pleat formation) and changes in the tunic propria was almost proportional to the degree of hypospermia. 20 Although the percentage of basal lamina involution in the extracellular space of seminiferous epithelium increased (P < 0.01) with age (Table 1), it was not significantly correlated with daily sperm production per man.
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Composition of human tubular boundary tissue
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Our previous studies have revealed that age-related reduction in the length of tubules (brought about by reduced numbers of Sertoli cells and various types of germ cells) without change in the volume of boundary tissue results in increased thickening of this tissue. 5 The present study confirms that age-related thickening of boundary tissue need not occur by the augmentation of collagen or other extracellular components. It also illustrates the importance of determining absolute values in evaluating potential causes of changes in human testicular boundary tissue. Alteration in tubular length without change in volume of boundary tissue 5 or its components (Table 2) provides an explanation for the relationship between hormonal status and thickness of tubular boundary tissue. I ,2I-23 Thickened boundary tissue in men with elevated blood levels of gonadotrophic hormones and severe depletion of germ cells, 1 may have been the result of reduced tubular length with no change in the volume of boundary tissue itself. Hence, there need not be a direct or indirect (through testosterone) stimulation of myoid cells themselves. Organization (reduced thickness) of boundary tissue in immature testes from patients with hypogonadotrophic hypogonadism following treatment with FSH and LH could result from the lengthening of tubules as larger numbers of germ cells are produced. 22 Likewise, the maturation of myoid cells in testicular tissue implants into hypophysectomized, testosteronetreated hosts 23 could result indirectly from maturation of the seminiferous epithelium (increased tubular length). Estrogen treatment, known to affect germ cell numbers, also causes increased thickness of boundary tissue. Is Vasectomy, which altered the size of seminiferous tubules without change in the hormonal status, was associated with increased tubular boundary tissueY The thicker the tubular wall in cryptorchid boys, the more marked were the changes in Sertoli cells and in numbers of spermatogonia.24 Furthermore, as noted above, physical collapse of seminiferous tubules was implicated in the thickening of the tubular boundary tissue after induced cryptorchidism. IS It is impossible to determine if boundary tissue changes precede or follow the spermatogenic disorder. I In human cryptorchid testes, tubular boundary tissue thickening occurs earlier than damage to seminiferous epithelium can be noted. 25 Hence, these authors suggest that tubular damage may be secondary to thickened tubules. This finding is not consistent with our findings of age-re1050
Johnson et al.
lated reduction in tubular length with no change in volume of boundary tissue (Table 1).5 Based on our findings, we suggest that the alteration in thickness of boundary tissue is secondary to the alteration of the volume of seminiferous epithelium. The reason the boundary tissue volume is constant in conditions which significantly reduce the tubular length (Table 1)5 is unknown. In any case, the thickness of boundary tissue determined from biopsies may be a useful measure of the effect of hormones, environmental toxins, and other factors on the seminiferous epithelial volume and tubular length. Acknowledgments. Special thanks are extended to Mr. Vince Hardy, Ms. Cynthia Snyder, Tabitha Childs, Janie Pluenneke, and Pat Stroud for technical assistance and in preparation of the manuscript. REFERENCES 1. de Krester DM, Kerr JB, Paulsen CA: The peritubular tissue in the normal and pathological human testis: an ultrastructural study. Bioi Reprod 12:317, 1975 2. Salomon F, Hedinger CE: Abnormal basement membrane structures of seminiferous tubules in infertile men. Lab Invest 47:543, 1982 3. Bustos-Obregon E, Holstein AF: On structural patterns of the lamina propria of human seminiferous tubules. Z Zellforsch Mikrosk Anat 141:413, 1973 4. Bishop MWH: Ageing and reproduction in the male. J Reprod Fertil12(suppl):65, 1970 5. Johnson L, Petty CS, Neaves WB: Age-related variation in seminiferous tubules in men: a stereologic evaluation. J Androl 7:316, 1986 6. Amann RP: A criticl review of methods for evaluation of spermatogenesis from seminal characteristics. J Androl 2:37,1981 7. Johnson L: Spermatogenesis and aging in the human. J Androl 7:331, 1986 8. Johnson L, Petty CS, Neaves WB: Influence of age on sperm production and testicular weights in men. J Reprod Fertil 70:211, 1984 9. Hermo L, Lalli M, Clermont Y: Arrangement of connective tissue components in the walls of seminiferous tubules of man and monkey. Am J Anat 148:433, 1977 10. Johnson L, Petty CS, Porter JC, Neaves WB: Germ cell degeneration during postprophase of meiosis and serum concentrations of gonadotropins in young adult and older adult men. Bioi Reprod 31:779, 1984 11. Johnson L, Petty CS, Neaves WB: A comparative study of daily sperm production and testicular composition in humans and rats. Bioi Reprod 22:1233, 1980 12. Johnson L, Zane RS, Petty CS, Neaves WB: Quantification of the human Sertoli cell population: its distribution, relation to germ cell numbers, and age-related decline. Bioi Reprod 31:785, 1984 13. Neaves WB, Johnson L, Petty CS: Age-related change in number of other interstitial cells in testes of adult men: evidence bearing on the fate of Leydig cells lost with increasing age. Bioi Reprod 33:259, 1985
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14. Johnson L, Petty CS, Neaves WB: A new approach to quantification of spermatogenesis and its application to germinal cell attrition during human spermatogenesis. Bioi Reprod 25:217, 1981 15. Sokal RR, Rohlf FJ: Single classification analysis of variance. In Biometry, Edited by RR Sokal, FJ Rohlf. San Francisco, WH Freeman, 1969, p 220 16. Kerr JB, Rich KA, de Kretser DM: Effects of experimental cryptorchidism on the ultrastructure and function of the Sertoli cell and peritubular tissue of the rat testis. Bioi Reprod 21:823, 1979 17. Jarow JP, Budin RE, Dym M, Zirkin BR, Noren S, Marchall FF: Quantitative pathologic changes in the human testis after vasectomy: a controlled study. N Engl J Med 313:1252, 1985 18. Lu CC, Steinberger A: Effects of estrogen on human seminiferous tubules: Light and electron microscopic analysis. Am J Anat 153:1, 1978 19. Xi Y-P, Nette G, King DW, Rosen M: Age-related changes in normal human basement membrane. Mech Ageing Dev 19:315, 1982 20. Furuya S: Studies on testicular function. Electron microscopic studies on the changes of the peritubular wall of the
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21.
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human seminiferous tubules in hypospermatogenesis. Jpn J Urol 66:80, 1975 Schulze CA, Holstein AF, Schinen C, Korner F: On the morphology of the human Sertoli cells under normal conditions and in patients with impaired fertility. Andrologia 8:167,1976 de Kretser DM, Burger HG: Ultrastructural studies of the human Sertoli cell in normal men and males with hypogonadotropic hypogonadism before and after gonadotrophic treatment. In Gonadotropins, Edited by BB Saxena, CG Beling, HM Gandy. New York, Wiley Interscience, 1972, p 640 Bressler RS, Ross MH: Differentiation of peritubular myoid cells of the testis: effects of intratesticular implantation of newborn mouse testes into normal and hypophysectomized adults. Bioi Reprod 6:148, 1972 de la Baize FA, Mancini RE, Arrillaga F, Andrada JA, Vilar 0, Gurtmann AI, Davidson OW: Histologic study upon the undescended human testis during puberty. J Clin Endocrinol 20:286, 1960 Hadziselimovic F: Cryptochidism, ultrastructure of normal and crytorchid testis development. Adv Anat Embryol Cell Bioi 53:Fasc 3, 1977
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