Germ Cell Degeneration During Postprophase of Meiosis and Serum Concentrations of Gonadotropins in Young Adult and Older Adult Men

BIOLOGY OF REPRODUCTION 31, 779-784 (1984) Germ Cell Degeneration During Postprophase of Meiosis and Serum Concentrations of Gonadotropins in Young Adult and Older Adult Men LARRY JOHNSON,2 CHARLES S. PETTY, JOHN C. PORTER and WILLIAM B. NEAVES Departments of Cell Biology, Pathology and Obstetrics and Gynecology The University of Texas Health Science Center at Dallas Dallas, Texas ABSTRACT Loss of potential sperm production during postprophase of meiosis was evaluated to determine if reduced daily sperm production in older men could be exp ained by an enhanced percentage of germ cell degeneration during this period of spermatogenesis. Evaluations were based on enumerating germ cells in homogenates of fixed testes using phase-contrast cytometry from 37 young adult (2-48 yr) and 34 older adult (5-85 yr) men. The time period in which germ cells degenerate was assessed in 1 men by comparing potential daily sperm production based on secondary spermatocytes with that based on primary spermatocytes or with daily sperm production based on spermatids. There was a significant (P<O.O1) decline in sperm production potential based on primary spermatocytes and on spermatids in the older adult men such that the percentage of loss of potential production during postprophase was similar between the two age groups. Sperm production estimates based on primary spermatocytes and secondary spermatocytes were similar (P>O.5); however, estimates based on secondary spermatocytes were significantly higher than those based on spermatids. Degeneration during postprophase of meiosis in humans appears to occur during or near the second meiotic division. Age-related reduced sperm production was significantly correlated with elevated levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Age-related decline in sperm production could not be explained by enhanced germ cell degeneration during postprophase but may result from reduced germ cell numbers prior to pachytene primary spermatocytes. INTRODUCTION Recently, it was found by comparing potential daily sperm production (PDSP) based on primary spermatocytes and daily sperm production (DSP) that significant germ cell degeneration characterized postprophase of meiosis in young adult men (Johnson et al., 1983). Confirming an earlier study comparing ratios of spermatocytes and spermatids (Barr et a!., 1971), Johnson et a!. (1983) extended the earlier study by revealing highly significant correlation between the percentage of germ cell degeneration during postprophase of meiosis and DSP/g parenchyma based on counts of spermatids with round nuclei. Over 73% of the Accepted July 9, 1984. Received April 6, 1984. This study supported in part by NIH Grant AG226. 2Reprint requests: Dr. Larry Johnson, Dept. of Cell Biology, The University of Texas Health Science Center, 5323 Harry Hines Blvd., Dallas, TX 75235. variation in DSP/g could be attributed to variation in the percentage of germ cell degeneration during postprophase of meiosis (Johnson et al., 1983). In an additional study comparing DSP/g based on the maturation-phase spermatids in a large series of young adult and older men, it was found that DSP/g and DSP/testis were significantly lower in the older men (Johnson et al., 1984b). Given that no significant germ cell degeneration was found during spermiogenesis (Johnson et a!., 1981) and that age-related differences were found in DSP based on counts of spermatids with round nuclei (Neaves et al., 1984), a possible explanation for reduced DSP in older humans could be a higher percentage of cell degeneration during postprophase of ineiosis. A higher percentage of cell degeneration would reduce the number of spermatids with round nuclei as well as maturation-phase spermatids and thus alter sperm production rates. 779

78 JOHNSON ET AL. The objectives of this study were: 1) to assess the magnitude of germ cell degeneration during postprophase of meiosis in young adult and o!der adult men, 2) to determine when during the maturation divisions the degeneration of cells occurs, and 3) to evaluate the correlations of DSP, PDSP, and percentage cell loss with serum concentration of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) previously published (Neaves et a!., 1984) for 15 men in each age group and in 6 additional men. MATERIALS AND METHODS Specimens and Preparation of Homogenates Testes from a group of 37 men aged 2 to 48 yr (33 ± 1) and a group of 34 men aged 5 to 85 yr (6 ± 2) yr were obtained at autopsy and glutaraldehyde-fixed by vascular perfusion within 15 h of death. Cause of death was largely due to traumatic injury (i.e., automobile accident or gunshot wound) or heart attack Homogenates of fixed testes were prepared in duplicate or triplicate as described previously (Johnson et al, 1981). Pieces (3-5 mg) of fixed parenchyma were removed from the equatorial region of the testis and were diced in 1 ml of fluid containing 15 mm NaCI,.5% (vol/vol) Triton X-1, and 3.8 mm NaN3 (Amann and Larnbiase, 1969; Johnson et al., 1981). Weighed pieces of parenchyma were diced and homogenized at room temperature for 6 mm in a Virtis 23 macrohomogenizer, model 6-15 AF (Johnson et al., 198b). For the first 1 men in each age group, two homogenates were made from each cranial, equatoria and caudal portion of each right and left testis to evaluate regional and bilateral differences. Calculation of Sperm Production Homogenates were prepared in duplicate or triplicate, and each was evaluated by counts made on at least four cytometer chambers. Nuclei of individual primary spermatocytes or spermatids had retained characteristic features following homogenization and could be identified under phase-contrast microscopy (Johnson et al., 1981). Germ cells in homogenates were classified according to Clermont(1963) and Heller and Clermont (1964), as verified by comparison with 2-Mm Eponembedded unstained sections viewed by phase-contrast cytometry. Nuclei of spermatids with round nuclei and nuclei of pachytene plus diplotene primary spermatocytes were enumerated (Johnson et a!., 1983). Spermatids with round nuclei are found in Stages I through III of the spermatogenic cycle and have a life span of 8.9 days (Clermont, 1963; Heller and Clermont, 1964). Pachytene and diplotene primary spermatocytes are found in Stages I through VI of the cycle and have a total life span of 16 days (Clermont, 1963; Heller and Clermont, 1964). In homogenates, pachytene and diplotene primary spermatocytes were distinguished from early primary spermatocytes (preleptotene, leptotene and zygotene) by the larger nuclear size and short, thick chromosomes (Johnson et al., 1981). PDSP/g parenchyma based on pachytene and diplotene primary spermatocytes was calculated as the product of four potential sperm produced per primary spermatocyte, and the number of pachytene plus diplotene nuclei in the homogenate divided by the product of the life span (16 days) and the weight of testicular tissue homogenized. DSP/g was calculated by dividing the number of spermatids with round nuclei in the homogenate by the product of an 8.9-day life span and the weight of testicular tissue homogenized. PDSP/testis or DSP/testis were calculated as the product of parenchymal weight (testicular weight minus tunic weight) and PDSPIg or DSP/g, respectively. Average coefficients of variation for duplicate homogenates were 9% for primary spermatocytes (Johnson et al., 1983) and 17% for spermatids with round nuclei (Johnson et al, 1981). The sensitivity of this assay is approximately 3, for DSPIg and approximately 6, for PDSP/g, both vatues being indistinguishable from zero. Germ Cell Loss During Postpropbase The percentage of germ cell degeneration during maturation divisions was calculated by dividing 1 times the difference between PDSP and DSP by PDSP for each individual. To determine the time period in which cells degenerate during postprophase of meiosis, potential daily sperm production based on histometric analysis of secondary spermatocytes (Kennelly and Foote, 1964) was compared to PDSP and DSP in 1 men ages 26 to 53 yr in which the latter two values had previously been determined by this method (Johnson et al., 1981, 1983). Potential DSP/testis based on secondary spermatocytes was calculated by dividing Product 1 by Product 2. Product 1 was determined by multiplying 2 potential spermatids per secondary spermatocytes, the percentage secondary spermatocyte nuclei in the parenchyma (based on total 2, point counting hits per man at 1X by two observers), parenchymal weight, and a correction factor for section thickness (.5 m) and nudear diameter (Weibel and Paumgartner, 1978). Product 2 was determined by multiplying 1.5 g/ml specific gravity of the testis (Johnson et al., 1981), the volume of an average secondary spermatocyte nucleus (determined by measuring an average of 5 nuclei per individual in 2O-zm Epon sections using Nomarski optics and a computerized digitizing unit), and a.27-day life span. The life span of secondary spermatocytes was estimated by multiplying the percentage of Stage VI in which secondary spermatocytes are found by the duration of Stage VI (Heller and Clermont, 1964). This percentage was determined by cutting out copies of photographs of Stage VI tubules in 6 men. The weight of the paper with the portion of the tubules containing secondary spermatocytes divided by the weight of the paper with the entire Stage VI gave the percentage of Stage VI which was occupied by secondary spermatocytes. Precision of secondary spermatocyte nuclear measurements was excellent, with a coefficient of variation of 2.6 ±.7%. However, as expected due to the realtive scarcity of these cells, the precision of percentage secondary spermatocyte nuclei was only fair, with a coefficient of variation of 48 ± 11%.

GERM CELL DEGENERATION IN YOUNG AND OLDER MEN 781 Hormone Assay Serum concentrations of LH and FSH were determined by radioimmunoassay (Midgley, 1966; Faiman and Ryan, 1967) with reference preparation LER-97 from the National Hormone and Pituitary Program of the National Institutes of Health. Interassay variability was 31% (coefficient of variation) for LH and 12% for FSH. Statistical Considerations Age-related comparisons were tested by the t test (Sokal and Rohif, 1969). The paired t test was used to test for differences between right and left sides within men. The one-way analysis of variance was employed to test for differences among means representing the three different regions of the testis evaluated on an unselected subset of 1 men in each age group. Mean hormone concentrations compared by both parametric (t test) and nonparaxnetric tests (Welch s approximation of the t test and the Mann-Whitney test, Zar, 1974) yielded the same results. The Mann-Whitney comparisons were reported, since some of these values were skewed. Nonparametric correlation coefficients (Spearman s rho) were tested for significance (Zar, 1974). Partial correlation coefficients were calculated (Draper and Smith, 1981) to remove the effect of age. RESULTS In both young adult and older adult men, no differences in PDSP/g or DSP/g (Table 1) during postprophase were found among the three regions of the testis or between right and left sides. Thus, there appeared to be no age-related, regional alteration in sperm production capability in men. Even in the older men, no difference in the efficiency of sperm production shown by PDSP/g and DSP/g was found among regions of the testis (Table 1). Considering all 71 men tested, total testicular weight was not different (P>O.5) between age groups. However, the tunica albuginea of testes in older adult men was slightly heavier such that parenchymal weight was lower (P<O.5) in the older adult men (Table 2). Likewise as expected, DSP/g and DSP/testis were lower (P<O.O1) in the older adult men. PDSP/g and PDSP/testis were also significantly lower in the older age group of men. Significantly reduced PDSP in older men suggests that potential sperm production was compromised prior to the development of pachytene spermatocytes. In fact, PDSP/g was sufficiently reduced (P<O.O1) in older adult men such that the percentage degeneration during postprophase of meiosis was not different (P>O.5) from that for the younger group of men (Table 2). The testis contains a very small percentage and volume of secondary spermatocyte nuclei (Table 3). Consequently, there was considerable variation among men in the percentage secondary spermatocyte nuclei in the parenchyma. A ninefold difference among men ranged from.15 to.13 5%. Nuclear diameter and volume of secondary spermatocytes were similar among men (Table 3): Using the paired t test, potential sperm production/g based on secondary spermatocytes was not different (P>O.5) from PDSP/g but was higher (P<O.5) than DSP/g of the same 1 men (Fig. 1). Thus, germ cell degeneration appeared to occur during or near the second meiotic division. Serum concentrations of LH (65 ng/ml vs. 139 ng/ml) and FSH (85 ng/ml vs. 317 ng/ml) were significantly (P<O.O1) higher in the older age group. Concentrations of LH and FSH were significantly (P<O.O1) correlated with age. Both LH and FSH were significantly correlated with both PDSP/testis and DSP/testis, with partial correlation coefficients ranging from -.33 to -.45. The percentage of germ cell degeneration during postprophase was not (P>O.5) correlated with age, FSH or LH. TABLE 1. Comparison of sperm production rates per g parenchyma based on primary spermatocytes (PDSP/g) or spermatids (DSP/g) among regions and between sides in young adult and older adult men.a Left testis Right testis Age group Number Parameter Cranial Equatorial Caudal Cranial Equatorial Caudal 2-48 1 PDSP/g(16) DSP/ (16) 1.3 ± 6.2 ±.5 1.1 ±.9 6.6 ±.8 1.5 ± 1.1 6.5 ±.7 1.8 ±.7 6.2 ±.4 1.3 ±.8 7.2 ±.6 11.2 ± 1. 7. ±.6 5-85 1 PDSP/g (16) DSP/g(1O ) 7.5 ±.8 5.3 ± 1. 6.8 ± 1.2 5.5 ± 1.1 7.2 ± 1.1 5.6 ± 1. 8.2 ±.8 6. ±.7 7.3 ±.9 5.8 ±.8 7.9 ± 1.1 6.4 ± 1.2 ano differences (P>O.5) existed among regions or between sides within either age group. bm ± SEM.

782 JOHNSON ETAL. TABLE 2. Comparison of testicular weights, sperm production rates based on primary spermatocytes (PDSP) or based on spermatids (DSP), and potential daily production loss during postprophase of meiosis in young adult and older adult men. Item Age (yr) 2-48 5-85 Number 37 34 Weight (g) Testicular 22.1 ± 11a 19.8 ± 1. Parenchymat 19.1 ± 1. 16.2 ±.9 Tunic 3.1 ±.1 3.4 ±.2 Sperm production/g parenchema (1O) PDSP 1.2 ±.4 7.4 ± O.5t DSP 6.2 ±.3 4.6 ± O.4 Sperm production/testis (16) PDSP 197. ± 13.9 128.7 ± DSP 12.9 ± 1. 8. ± Potential daily production loss Number (16) Per g parenchyma 4. ±.3 2.8 ± o.3 Per testis 76.1 ± 7.5 48.8 ± l.o Percentage loss during postprophase 39.1 ± 3.2 36.4 ± 4.6 ameans ± SEM. Significant difference between age groups at P<O.5. Significant difference between age groups at P<O.O1. DISCUSSION in possible regional differences in testes of the As has been established for young adult older adult men in our study. No regional human testes (Johnson et a!., 198a,b), there differences were noted in sperm production were no regional differences in daily sperm rates in the 1 unselected men in the older production based on spermatids in young adult group (Table 1). However, it should be noted men (Table 1). Likewise, PDSP/g was similar that these unselected 1 men (Table 1) had a for the cranial, equatorial and caudal regions of higher sperm production rate than did the older testes in adult men and even in older adult men, adult men (Table 2). Since Sasano and Ichijo (1969) found degenera- The age-related reduction in DSP/g and tion of tubules in the cranial region of testes DSP/testis (Table 2) is consistent with previous (which is farther away from large vessels than studies (Amann, 1981; Johnson et a!., 1984b). other regions) from aged men, we were interested However, parenchymal weight was significantly TABLE 3. Parameters of secondary spermatocytes and potential daily sperm production based on histometric analysis of secondary spermatocytes in 1 men. Testicular weight (g) 2.6 ± 1.6 Parenchymal weight (g) 17.7 ± 1.43 Percent nuclei in parenchyma.6 ±.12 Volume of nuclei in parenchyma (ml) 1.5 ±.21 Nuclear diameter (am) 8.63 ±.13 Nuclear volume (fi) 347.5 ± 16.7 Number of nuclei/g (16) 1.6 ±.33 Number of nuclei/testis (16) 28.56 ± 6.9 Potential daily sperm production (16) Pergparenchyma 11.9 ± 2.45 Per testis 211.54 ± 45.12 am ± SEM.

GERM CELL DEGENERATION IN YOUNG AND OLDER MEN 783 z () C Lu a- U) SPERMATOCVTES SPERMATOCYTES WITH RQUUQ NUCLE FIG. 1. Comparison of potential daily sperm production based on pachytene plus diplotene primary spermatocytes, or based on secondary spermatocytes and thily sperm production from the number of spermatids with round nuclei per g testicular parenchyma, in 1 men aged 26 to 53 yr determined by histometric analysis (Johnson et al, 1981, 1983, Table 3). reduced (P<O.5) in this group of older adult men in contrast to the similarity in parenchymal weight found in another larger series of men (Johnson et a!., 1984b). No significant agerelated reduction in total testicular weight was noted in either study. PDSP/g and PDSP/testis were significantly (P<O.O1) reduced in the older group of men (Table 2). Therefore, the number of primary spermatocytes entering postprophase of meiosis had already been reduced before postprophase began. In fact, the reduction in number of primary spermatocytes was so severe that the percentage of degeneration during postprophase was not different (P>O.5) between the two age groups. The relationship between the percentage of loss during postprophase and DSP/g found earlier in young adult men (Johnson et al., 1983) did not directly apply to aging human testes. Age-related diminution in sperm production cannot be explained by enhanced rates of degeneration of germ cells during postprophase of meiosis. Therefore, age-related reduction in sperm production may be explained by reduced numbers of spermatogonia, or subsequent degeneration of spermatogonia or primary spermatocytes prior to the development of pachytene spermatocyes. No significant relationship existed between percentage of degeneration during postprophase of meiosis and serum concentrations of LH or FSH. Both LH and FSH concentrations were significantly correlated with PDSP/testis and DSP/testis, even after removing the effect of age by using partial correlation coefficients. In regards to the relationship between FSH concentrations and PDSP/testis or DSP/testis, our findings are consistant with those of DeKretser et al. (1974). These authors found a significant relationship between FSH concentrations and the mean number of spermatogonia, primary spermatocytes, early and late spermatids per 25 tubular cross sections in men. In our study, age was also significantly (P<O.O1) correlated with both LU (rho=o.47) and FSH (rho=o.59) serum concentrations. Regardless of age, significant loss of potential sperm production occurred between late primary spermatocytes and early spermatids. The time period in postprophase where cell death occurs was evaluated (Fig. 1) by comparing PDSP/g based on histometric analysis of secondary sperniatocytes from 1 men in which PDSP/g based on primary spermatocytes and DSP/g based on spermatids had already been determined histometrically (Johnson et a!., 1981, 1983). Similar sperm production values based on secondary spermatocytes or primary spermatocytes (P>O.5) are consistant with no significant loss during the first meiotic division. However, loss of potential productivity between secondary spermatocytes and spermatids is consistent with cell degeneration during or near the second meiotic division. By comparing ratios of cells in rats, Wing and Christensen (1982) found about 15% loss between primary spermatocytes and spermatids, but they found essentially no loss between secondary spermatocytes and sperrnatids. However with the same species, Russell and Clermont (1977) found that late secondary spermatocytes entering the second meiotic division (judged by the presence of adjacent secondary spermatocytes and early spermatids) and not primary spermatocytes entering the first meiotic division were responsible for losses during postprophase of meiosis. This relatively new finding implicating the second meiotic division (Russell and Clemont, 1977) was supported by our numerical data in human testes (Fig. 1). Unlike more mature adult rats weighing >4 g (Robb et a!., 1978), which had no significant loss of sperm production potential

784 JOHNSON ET AL. during postprophase of meiosis (Johnson et a!., 1984a), the significant germ cell degeneration during postprophase of meiosis continued in older adult men (Table 2). In any event, the decline in DSP/testis in older men was more closely related (rho=o.81, P<O.O1, partial correlation coefficient with age removed) to the number of pachytene plus diplotene primary spermatocytes as indicated by PDSP/testis than to the percentage cell loss during postprophase of meiosis. Since the number of late primary spermatocytes for a species is dependent upon the size of the spermatogonial population and the amount of spermatogonial proliferation and degeneration (Hochereau-de- Riviers, 1981), and/or the amount of degeneration of the early generation of primary spermatocytes, it is likely that changes in one or more of these factors would be responsible for lower sperm production in older men. ACKNOWLEDGMENTS Special thanks are due to Jenifer Ratliff, Karen S. Pollan and Noel Lewandos for providing expert technical assistance. REFERENCES Amann, R. P. (1981). A critical review of methods for evaluation of spermatogenesis from seminal characteristics. J. Androl. 2:37-58. Amann, R. P. and Lambiase, J. T., Jr. (1969). The male rabbit: III Determination of daily sperm production by means of testicular homogenates. J. Anim. Sci. 28:369-374. Barr, A. B., Moore, D. J. and Paulsen, C. A. (1971). Germinal cell loss during human spermatogenesis. J. Reprod. Fertil. 25:75-8. Clermont, V. (1963). The cycle of the seminiferous epithelium in man. Am. J. Anat. 112:35-51. DeKretser, D. H., Burger, H. C. and Hudson, B. (1974). The relationship between germinal cells and serum FSH levels in males with infertility. J. Clin. Endocrinol. Metab. 38:787-793. Draper, N. Rand Smith, H. (1981). Applied Regression Analysis, 2nd ed. John Wiley & Sons, New York. Faiman, C. and Ryan, R. J. (1967). Radioimmunoassay for human follicle stimulating hormone. J. Clin. Endocrinol. Metabol. 27:444-447. Heller, C. G. and Clermont, Y. (1964). Kinetics of germinal epithelium in man. Recent Prog. Horm. Res. 2:545-575. Hochereau-de-Reviers, M. T. (1981). Control of spermatogonial multiplication. In: Reproductive Processes and Contraception (K. W. McKerns, eds.). Plenum Pubi., New York, pp. 37-33 1. Johnson, L., Petty, C. S. and Neaves, W. B. (198a). A comparative study of daily sperm production and testicular composition in humans and rats. Biol. Reprod. 22:1233-1243. Johnson, L., Petty, C. S. and Neaves, W. B. (198b). The realtionship of biopsy evaluation and testicular measurements to over-all daily sperm production in human testis. Fertjl. Steril. 34:36-4. Johnson, L., Petty, C. S. and Neaves, W. B. (1981). A new approach to quantification of spermatogenesis and its application to germinal cell attrition during human spermiogenesis. Biol. Reprod. 25: 217-226. Johnson, L., Petty, C. S. and Neaves, W. B. (1983). Further quantification of human spermatogenesis: germ cell loss during postprophase of meiosis and its relationship to daily sperm production. Biol. Reprod. 29:27-215. Johnson, L., Lebovitz, R. M. and Samson, W. K. (1984a). Germ cell degeneration in normal and microwave irradiated rats. Potential sperm production rates at different developmental steps in spermatogenesis. Anat. Rec. 29:51-57. Johnson, L., Petty, C. S. and Neaves, W. B. (1984b). Influence of age on sperm production and testicular weights in men. J. Reprod. Fertil. 7:211-218. Kennelly, J. J. and Foote, R. H. (1964). Sampling boar testes to study spermatogenesis quantitatively and to predict sperm production. J. Anim. Sci. 23:16-167. Midgley, A. R., Jr. (1966). Radioimmunoassay: A method for human chorionic gonadotropin and human luteinizing hormone. Endocrinol. 79: 1-18. Neaves, W. B., Johnson, L., Porter, J. C., Parker, C. R., Jr. and Petty, C. S. (1984). Leydig cell numbers, daily sperm production, and serum gonadotropin levels in aging men. J. Clin. Endocrinol. Metabol. 55 (in press). Robb, G. W., Amann, R. P., and Kihian, G. J. (1978). Daily sperm production and epididymal speim reserves of pubertal and adult rats. J. Reprod. Fertil. 54:13-17. Russell, L. D. and Clermont, V. (1977). Degeneration of germ cells in normal, hypophysectomized and hormone treated hypophysectomized rats. Anat. Rec. 187:347-366. Sasano, N. and Ichijo, S. (1969). Vascular patterns of the human testis with special references to its senile changes. Tohoku J. Exp. Med. 99:269-28. Sokal, R. R. and Rohlf, F. J. (1969). Biometry. W. H. Freeman and Co., San Francisco, pp. 22-58. Weibel, E. R. and Paumgartner, D. (1978). Integrated stereological and biochemical studies on hepatocytjc membranes. II. Correction of section thickness effect on volume and surface density estimates. J. Cell Biol. 77:584-597. Wing, T.-Y. and Christensen, A. K. (1982). Morphometric studies on rat seminiferous tubules. Am. J. AnaL 165:13-25. Zar, J. H. (1974). Biostatistical Analysis. Prentice-Hall, Englewood Cliffs, NJ.