BIOLOGY OF REPRODUCTION 42, 281-287 (1990)

281

Increased Germ Cell Degeneration during Postprophase ofMeiosis Is Related to Increased Serum Follicle-Stimulating

Hormone Concentrations and Reduced Daily SpermProduction in Aged Men1

LARRY JOHNSON,2’3 JANICE S. GRUMBLES,3 ALl BAGHERI,4 and CHARLES S. PEUY5

Department of Veterinary Anatomy3

College of Veterinary Medicine

Texas Agricultural Experiment Station

Texas A&M University

College Station, Texas 77843-4458

and

Departments of Cell Biology and Anatomy4 and Pathology3

University of Texas

Southwestern Medical Center at Dallas

Dallas, Texas 75235

ABSTRACT

Aged men, known to have high serum gonadotropin levels and reduced spermatogenic potential, were used tostudy the relationship between serum follicle-stimulating hormone (FSH) and germ cell degeneration. Serum

hormones were measured from blood obtained at autopsy. Phase-contrast cytometry was used to enumerate germcells in homogenates of fixed testes from 13 younger (24 - 51 yr) and 14 aged (69 - 90 yr) men. The develop-mental steps of spermatogenesis during which germ cells degenerate were determined by comparing potentialdaily sperm production based on primary spermatocytes with daily sperm production based on two different typesof spermasids. During spermiogenesis, there was no significant degeneration in the younger or aged men. Duringpostprophase of meiosis, aged men had more (p<O.Ol) germ cell degeneration, significantly lower (p<O.O5)

serum testosterone, and greater (p<O.OI) serum FSH than did younger men. Germ cell degeneration duringpostprophase of meiosis was negatively correlated (p<O.0l):o daily sperm production and significantly (p<O.Ol)related to serum concentrations of FSH. As revealed in these aged men, meiotic germ cell degeneration has adirect effect on daily sperm production and is significantly related to serum FSH concentrations.

INTRODUCTION

Spermatogenesis, measured by daily sperm produc-tion (DSP), declines with age in adult men (Amann,1981; Johnson et a!., 19Mb; Johnson, 1986, 1989).

Comparing younger men (33 ± 2 yr; mean ± SEM) toolder men (65 ±3 yr. mean ± SEM), we found that age-related differences in germ cell degeneration resultingin lower rates of spermatozoan production in older menoccurred in the meiotic prophase during the transitionof early (preleptotene and leptotene) primary spermato-cytes to late (pachytene) primary spermatocytes oramong the late primary spermatocytes themselves

Accepted October 24, 1989.

Received May 15, 1989.1Supported in part by the National Institute of Aging AG-02260.2Reprint requests.

(Johnson et al., 1987). However, because sufficientnumbers of testes from aged men (mean age above 75yr) were not available, the amount of degeneration ofgerm cells during spermatogenesis has not been evalu-ated in a large group of aged men. Indeed our previousstudies have not included a group limited to aged menwith a mean age above 75 years.

Germ cell degeneration may occur during spermato-gonial mitosis, meiosis, or spermiogenesis (Johnson,1986). Significant germ cell degeneration occurs inpostprophase of meiosis in younger adult men, and theamount of loss is similar to that of older men (Johnsonet a!., 1983). Younger men (25 -53 yr) had no signifi-cant germ cell degeneration during spermiogenesis(Johnson et a!., 1981). The amount of degenerationduring postprophase of meiosis or during spermiogene-sis has not been evaluated in a group composed of onlyaged men.

282 JOHNSON ET AL.

Serum follicle-simulating hormone (FSH) levelshave been found to be important in spermatogenesis.Neaves et a!. (1984) found that FSH levels in a groupof 30 men between 20 and 76 years of age were higherin the older men and were positively correlated withage. Further, FSH levels were inversely correlated withDSP, and the highest FSH levels were found in menwith the lowest rates of spermatozoan production. Sev-eral studies have shown that, regardless of the cause,damage to the seminiferous epithelium is associatedwith increased levels of serum FSH (deKretser et a!.,1983).

The objectives of this study were to evaluate theimpact of germ cell degeneration during meiosis orduring sperminogenesis on daily sperm production inaged men (78 ± 2 yr, mean ± SEM) and to evaluate therelationship between serum hormone levels and germcell degeneration. It was found for the first time thataged men have more germ cell degeneration duringpostprophase of meiosis than younger men, that nogerm cell loss occurred during spermiogenesis in agedmen, and that meiotic germ cell degeneration duringpostprophase was highly correlated with serum FSHlevels.

MATERIALS AND METHODS

Specimens and Study Subjects

Testes from 13 men aged 24 -51 yr (31 ± 2 yr;mean ± SEM) and 14 men aged 69-90 yr (78 ± 2 yr;mean ± SEM) were obtained at autopsy and fixed with2% glutaraldehyde in 0.1 M cacodylate buffer within 15h of death (Johnson et a!., 1981). Causes of death werelargely traumatic injury (i.e. automobile accident orgunshot wound) or heart failure. None of these youngeror elderly men had a chronic illness associated withextended hospitalization. Our studies (Neaves et a!.,1984; Johnson et a!., 1987) have not found a relation-ship between testicular or hormonal parameters andpostmortem time, blood alcohol levels, body weight orratio of body weight to height in autopsy specimensfrom men aged 20-90 yr. Randomly selected pieces(30-50 mg) of fixed parenchyma were diced in 10 mlof fluid containing 150 mM NaC1, 0.05% (vol/vol)Triton X-100, and 3.8 mM NaN3 (Amann and Lam-biase, 1969). Weighed pieces of parenchyma werediced and homogenized at room temperature for 6 mmin a Virtis 23 macrohomogenizer, model 6-105 AF(Johnson et a!., 1981).

Calculation of Spermatozoan Production

Spermatogenesis was expressed as DSP based onspermatids or potential daily spermatozoan production(PDSP) based on numbers of primary spermatocytes.PDSP does not consider degeneration that can occursubsequent to primary spermatocytes. Homogenateswere prepared in duplicate, and each was evaluated bycounts made on at least four cytometer chambers (John-son et a!., 1981). Primary (pachytene plus diplotene)spermatocytes, round (Golgi/cap-phase Sa + Sb1) sper-matids or maturation-phase (Sd1 + Sd2) spermatidswere enumerated in the homogenates (Clermont, 1963;Heller and Clermont, 1964). PDSP (based on pachyteneplus diplotene primary spermatocytes) and DSP (basedon Golgi plus cap-phase spermatid or maturation-phasespermatids) were calculated from the cell number pergram testicular parenchyma or per testis, from theoreti-cal yield, and from duration of developmental phases ofthese specific germ cells (Johnson et al., 1984a,c).Theoretical yields were calculated by 2�, with n equalto the number of cell divisions between the given celltype and spermatozoa (Johnson, 1986); n = 0 for DSPand 2 for PDSP based on primary spermatocytes. Preci-sion of duplicate sets of determinations for each celltype have been reported (Johnson et al., 1981, 1983).

Germ Cell Loss during Postprophase of Meiosis

The percentage of germ cell degeneration duringmaturation divisions was calculated as 100 times thedifference between PDSP and DSP based on matura-tion-phase spermatids divided by PDSP for each indi-vidual (Johnson Ct a!., 1983).

Hormone Assay

Levels of FSH, prolactin (PRL), and testosteronewere determined by radioimmunoassay (RIA) as previ-ously described (Neaves et a!., 1984). Interassay variab-ilities, expressed as the coefficients of variations of thehormone RIAs, were 12%, 13%, and 14% for FSH,PRL, and testosterone, respectively.

Statistical Analysis

Statistical comparisons were described by Sokal andRohif (1969). Age-related comparisons were tested bythe t-test. One-way analysis of variance and the Stu-dent-Newman-Keuls test were used within each groupto test for differences among means representing differ-

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FIG.!. The effect of age on serum hormone concentrations and daily spermproduction per man. Serum FSH concentrations were increased while testoster-

one concentrations and daily sperm production were decreased. FSH and testos-terone concentrations were significantly (p<O.O5) correlated with age.

CELL DEGENERATION MAY LINK FSH TO SPERMATOGENESIS 283

ent types of germ cells. Correlation coefficients wereused to test for significance of spermatozoan productionrates and percentage degeneration. Partial correlationcoefficients were calculated (Draper and Smith, 1981)to remove the effect of age.

RESULTS

Serum concentrations of FSH increased with age, butserum testosterone levels and daily sperm productionper man decreased (Fig. 1). In aged men, mean serum

concentrations (ng/ml) of FSH (106 ± 17 vs. 363 ± 79,p<O.Ol) were higher, and testosterone concentrations(6.2 ± 1.1, vs. 3.6 ± 0.6; p<0.05) were lower than inyounger men. However, prolacun (27.6 ± 8.8 vs. 33.8 ±7.4) values were not significantly different betweenyounger and aged men. Concentrations of testosteronewere negatively (r = - 0.43, p’<0.05) correlated withage. As illustrated in Figure 1, there were individualmen in the aged group with hormone concentrationsand spermatozoan production rates comparable to thoseof younger men.

FSH levels were significantly correlated with PDSP/g testicular parenchyma based on primary spermato-cytes, with partial correlation coefficients (- 0.42) usedto remove the influence of age. FSH levels were signifi-cantly correlated with PDSP per right testis based onprimary spermatocytes with a partial correlation coeffi-cient of - 0.45. In addition, FSH was significantlycorrelated with DSP/man based on maturation-phasespermatids (Fig. 2a), with a partial correlation coeffi-cient of - 0.42. Serum concentrations of testosteronewere not significantly correlated to DSP/man.

Testicular (23 ± 2 vs. 17 ± 2 g) and parenchyma! (20± 2 vs. 14 ± 2 g) weights, DSP/testis, and DSP/manbased on maturation-phase spermatids were signifi-cantly lower in aged men than in younger men (81 ± 9vs. 39 ± 10 x 106 and 162 ± 18 vs. 78 ± 20 x 106,respectively. Efficiency of spermatogenesis was signifi-cantly lower in aged men than in younger men (Fig. 3).This was true for PDSP/g based on primary spermato-cytes (p<0.05) and for DSP/g (p’czO.Ol) based on Golgi/cap-phase spermatids or maturation-phase spermatids.There was no significant difference between DSP/gbased on Golgi/cap-phase spermatids and DSP/g basedon maturation-phase spermatids within either agegroup. Hence, aged men are like younger men in thatno significant germ cell degeneration occurs duringspermiogenesis,

Both younger and aged men had a significant per-centage of germ cell degeneration during postprophase

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FIG. 2. Relationships between serum FSH concentrations and daily spermproduction and between percentage meiotic germ cell degeneration duringpostprophase and daily sperm production or serum FSH concentrations. Eachpaired parameter was significantly (p<O.Ol) correlated to each other.

Maturation PhaseSpermatid

FIG. 3. Potential daily sperm production based on pachytene primary sper-matocytes and daily sperm production based on Golgi/cap-phase spermatids ormaturation-phase spermatids per gram testicular parenchyma in 13 youngeradultmen(31 ±2yr; mcan±SEM)and l4agedmen(78 ±2yr; mean iSEM).

Both groups of men experienced significant degeneration during postprophase(between pachytene primary spennatocytes and spermatids), but no significantdegeneration occurred during spermiogenesis (between Golgi/cap-phase sper-matids and maturation-phase spermatids).

284 JOHNSON ET AL.

of meiosis, between primary spermatocytes and sperma-tids (Fig. 3). However, the percentage of degenerationwas significantly higher (p<O.Ol) in aged men (Fig. 4).The percentage of meiotic germ cell degeneration dur-ing postprophase was highly (p<O.Ol) correlated withage. There was a highly significant negative correlationbetween the percentage of germ cell degeneration dur-ing postprophase of meiosis and DSP/testis (Fig. 2b).The percentage of meiotic germ cell degeneration dur-ing postprophase was significantly (p<O.Ol) correlated(r = 0.53) with serum FSH levels (Fig. 2c).

DISCUSSION

Age alters spermatogenesis and hormonal status. The� age-related reduction in DSP/g parenchyma and DSP!

testis is consistent with previous studies based on feweraged men (Amann, 1981; Neaves et a!., 1984; Johnson,1986). Our findings of an age-related effect on sperma-togenesis differs from those of a previous study of“vigorous grandfathers” who actually had a higher con-

centration of spermatozoa in the ejaculate than didyounger unrelated men not selected for “vigorous” ac-tivity (Nieschlag et a!., 1982). Absolute values of serumPRL, FSH, and testosterone were largely similar tothose observed in previous studies on the effects of age

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31±2y 78±2y

CELL DEGENERATION MAY LINK FSH TO SPERMATOGENESIS 285

(Harman and Tsitouras, 1980: Sparrow et al., 1980;Homonnai et a!., 1982; Winters and Troen, 1982). Ourstudy differed from those using highly selected“healthy” men (Harman and Tsitouras, 1980; Sparrowet a!., 1980) in that testosterone levels were signifi-cantly reduced in the aged men of our study (Fig. 1).Findings with highly selected men are consistent withthe concept that the decline in testicular function inaged men is possibly confounded by illness of ran-domly selected men. The appropriateness of selectionprocess for the surviving elite elderly (on which thisconcept was based) has been questioned (Swerdloff andHeber, 1982). Further, Bremner et a!. (1983) reportedan age-related loss of circadian rhythmicity in bloodtestosterone in normal healthy men.

Based on primary spermatocytes, PDSP/g was signif-icantly reduced in aged men (Fig. 3). Therefore, thenumber of primary spermatocytes entering postprophaseof meiosis had already been reduced before postpro-phase began. It is likely that aged men (mean of 78 yr)are like older men (mean of 60 yr) in that the age-related reduction in potential daily sperm productionoccurs during the transition of early (preleptotene andleptotene) primary spermatocytes to late (pachytene)primary spermatocytes or among the later primary sper-matocytes themselves (Johnson et al., 1987). This find-ing is consistent with the important role of degenerationduring postprophase of meiosis in spermatozoan pro-duction in aged men. The similar PDSP/g based on

primary spermatocytes between older men (7 x 106;Johnson, 1986) and aged men (7 x 106; Fig. 3) is

consistent with aging changes seen after the sixth de-cade occurring during prophase of meiosis rather thanearlier in spermatogenesis. Alternatively, the number ofspermatogonia per man may have been reduced in agedmen (Johnson, 1986; Nistal et al., 1987).

Significant germ cell loss during postprophase ofmeiosis (Figs. 3, 4) in humans is consistent with pre-vious studies (Barr et a!., 1971; Johnson et al., 1983,1984c). This percentage of potential production lossseems to vary in previous studies from 36% to 55% inyounger (30 yr) and older (60 yr) adult men. In thepresent study (Fig. 4), the percentage of germ celldegeneration during postprophase of meiosis wasgreater in aged men than in younger men and wasgreater in younger men than previously reported. Inother species, losses of potential spermatozoa produc-tion during meiotic divisions are estimated at 13% inmice (Oakberg, 1956), 2% in Sprague-Dawley rats(Roosen-Runge, 1955; Johnson et a!., 1984a), 27% inSherman rats (Clermont, 1962), 25% in rabbits (Swier-stra and Foote, 1963), and 6% - 15% in stallions (John-son, 1985). Germ cell degeneration during postprophaseof meiosis was localized to the second meiotic divisionin humans (Johnson et al., 1984c).

In aged men, there was a significant (r = - 0.82;p<0.01) relationship between the percentage of germcell degeneration during postprophase of meiosis andDSP/g based on mature spermatids. The relationshipalso existed when all men (young and aged) wereconsidered (Fig. 2b). A similar relationship was foundfor younger men (Johnson et a!., 1983), whose percent-ages of degeneration during postprophase varied widely(10% -95%). Aged men in the present study had amore consistently high level of degeneration duringpostprophase (61% - 100%). In spite of the lack of awide range, the percentage of degeneration was highlycorrelated with DSP/g. Aged men, like younger men,did not have significant germ cell degeneration duringspermiogenesis (Fig. 3). No significant degenerationduring spermiogenesis has been reported in previousstudies involving humans (Johnson, 1986), stallions(Johnson, 1985), bulls (Amann, 1962), and adult (>400g) Sprague-Dawley rats (Johnson et a!., 1984a).

Given no loss in potential production of spermatozoaduring spermiogenesis, germ cell degeneration duringpostprophase of meiosis had a significant impact ondaily sperm production. Although age-related diminu-tion in spermatogenesis can be partly explained bydegeneration before postprophase of meiosis (as in men

286 JOHNSON ET AL.

in the sixth decade of life; Johnson et a!., 1984c), alarge portion of this age-related variation in spermato-zoan production can be explained by the degenerationthat occurs during postprophase of meiosis in agedmen.

Aged men differ from men in their sixties (Johnsonet a!., 1984c) not only in occurrence of germ celldegeneration before the last half of meiosis, but also inoccurrence of a greater (p<0.01) percentage of degener-ation during postprophase of meiosis (Fig. 4). Thisadditional loss may be related to significantly lowerlevels of testosterone in these aged men than levelspreviously found in men whose mean age was 58 years(Neaves et a!., 1984). It is generally agreed that oldermen have higher plasma FSH, lower plasma testoster-one, reduced free plasma testosterone (Baker and Hud-son, 1983), reduced numbers of Leydig cells (Neaves eta!., 1984), and reduced daily sperm production (John-

son, 1986).

The highly significant relationship between serumFSH concentrations and percentage of germ cell degen-eration or DSP (Fig. 2) is consistent with the notion thatmeiotic germ cell degeneration is the link between thereported relationship of FSH and number of spermato-zoa in human ejaculates (deKretser et a!., 1983). De-Kretser et a!. (1974) found significant relationshipsbetween serum FSH concentrations and the number ofspermatocytes and early or late spermatids per 25 hu-man tubular cross sections. Abnormally high concentra-tions of FSH in azoospermic men usually reveal severedestruction of the seminiferous epithelium and reduced

Sertoli cell function (Swerdloff and deKretser, 1983).

In the present study using aged men, who usuallyhave high serum FSH levels and reduced spermatogenicpotential (Johnson et a!., 1984b,c; Neaves et a!., 1984),the percentage of germ cell degeneration during post-prophase of meiosis was significantly correlated withFSH concentration (Fig. 2c).

Elevation of serum FSH concentrations (Scott andBurger, 1981; deKretser et a!., 1983) and reduced sper-matogenesis (deKretser et a!., 1974; Swerdloff and de-Kretser, 1983; Johnson et a!., 1984d) imply a reducedfunction in the Sertoli cell population. In humans, Ser-

toli cell numbers are significantly reduced by age andare significantly (p’<O.Ol) related with daily sperm pro-duction (Johnson et a!., 1984d). Sertoli cells are likelythe only targets of FSH in seminiferous tubules (Fritz,

1978). Hence, it seems reasonable that altered FSH and

spermatogenesis may be the result of reduced functionof the Sertoli cell population.

Since spermatogenesis is compromised by increasedgerm cell degeneration during meiosis (Fig. 2) and isrelated to reduced numbers of Sertoli cells (Johnson eta!., 1984d), the relationship between FSH and sperma-togenesis appears to occur via increased germ celldegeneration that possibly results from compromisedfunction of the Sertoli cell population. It is not knownwhether reduced numbers of Sertoli cells in aged men(Johnson et a!., 1984d) cause elevated FSH levelsthrough reduced function of the Sertoli cell populationor whether reduced spermatozoan production throughincreased meiotic germ cell degeneration results fromreduced numbers of Sertoli cells and their correspond-ing reduced germ cell accommodations; however, it isclear that increased meiotic germ cell degeneration hasa direct effect on daily sperm production, and both arerelated to serum FSH concentrations. Although themechanism for the relationship between the rate ofspermatogenesis and serum FSH concentrations remainsunclear, it likely involves meiotic germ cell degenera-

tion during spermatogenesis.

ACKNOWLEDGMENTS

Thanks are extended to Drs. I. C. Porter and C. R. Parker for the hormonevalues and to Dr. W. B. Neaves for his contribution.

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