Age, Vol. 15, 65-71, 1992

EFFECT OF AGE AND DIETARY RESTRICTION ON DAILY SPERM PRODUCTION AND NUMBER AND TRANSIT TIME OF EPIDIDYMAL SPERMATOZOA IN THE MOUSE

Larry Johnson, 1 Manley R. May, 1 David L. Busbee,1 and John D. Williams 2 1Department of Veterinary Anatomy and Public Health

and 2Department of Pathobiology

The Texas Veterinary Medical Center College of Veterinary Medicine Texas A&M University College Station, TX 77843-4458

ABSTRACT

Dietary restriction extends the DNA repair capacity and life span of mice. Age-related changes in spermatogenesis and transit time of epididymal spermatozoa were evaluated to determine if dietary restriction altered the age-related changes in these traits as well. At weaning, mice were fed NIH-31 diet ad libitum or restricted to 60% of the ad libitum and sacrificed at 6, 12, 19, 22, or 26 months of age. The number of maturation phase spermatids or epididymal spermatozoa were counted by phase contrast cytometry of testicular or epididymal homogenates. Daily sperm production/testis (DSP/ T) was determined by dividing the number of spermatids per testis by the 4.84 day life span of these spermatids. Daily sperm production per g parenchyma (DSP/G) was calculated by dividing DSP/T by the testicular parenchymal weight. Transit time of epididymal spermatozoa was calculated by dividing the number of epididymal spermatozoa by the DSP/T of the attached testis. Over both treatments, testicular and parenchymal weights were significantly decreased with age. While both DSP/G and DSP/T were similar (P>0.05) for 19, 22, and 26 months of age, they were lower (P<0.05) at 6 and 12 months of age, and there was a trend for them to be lower at 26 months of age. Age also reduced the number and the transit time of epididymal spermatozoa. DSP/G was not uniform in adult mice. Over all ages, dietary restriction had no significant effect on testicular weight or DSP/ G, but it significantly reduced body and epididymal weight, number of epididymal spermatozoa, and epididymal spermatozoan transit time. Transit time of spermatozoa in the mouse epididymis was very long in younger adult mice, could vary widely, and was reduced by age and dietary restriction. Although not statistically significant, there was a trend for a delay in obtaining maximum DSP/T at 12 months and a delay in obtaining the age-related, reduced DSP/T at 26 months in the dietary restricted group compared to that seen in the adlibitumgroup.

INTRODUCTION

In rodents, dietary restriction increases the life span (1-5), enhances the immune system functions (4, 5), increases the capacity for DNA excision repair in aged animals (6, 7), delays the onset of a number of age- related diseases (3, 8), and impedes the age-related decline in specific activity and fidelity of DNA polymerases (9). Spermatogenesis in adult males of most species is remarkably resistant to nutritional stress, and infertility from malnutrition is seldom encountered (10). In adult males, the endocrine rather than exocrine function is affected by severe dietary energy or protein deficiency, and negative effects of malnutrition are more evident and more likely to be permanent in young males (11). Negative changes in spermatogenesis are associated with the aging process in rats (12), mice (13, 14), and humans (15-18). However, the effects of age on daily sperm production and epididymal transit time and the effect of dietary restriction on spermatogenesis or its age-related decline are unknown in mice.

The objectives were 1) to determine the age-related changes in spermatogenesis and number and transit time of epididymal spermatozoa in mice and 2) to de- termine if dietary restriction altered the onset of adult levels of spermatogenic or epididymal function or delayed the onset of an age-related decline in that function.

RESULTS

Histologic evaluation of the testis, efferent ducts, and epididymis revealed no detrimental effects of dietary restriction (Fig. 1). Stage VIII seminiferous epithelium revealed an abundance of preleptotene and pachytene primary spermatocytes, as well as Step 8 and Step 16 spermatids in mice receiving both diets (Fig. la, b). Likewise, the epithelium of the efferent ducts (Fig. lc, d) and caput epididymidis (Fig. le, f) appeared qualitatively similar in organelle content, size of nuclei, and height of cells.

Age and/or dietary restriction altered body and organ weights. In the control mice (fed adlibitum), body weight increased (P<0.05) between 6 and 12 months and then

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Figure 1: Effect of dietary restriction on seminiferous epithelium and epithelium of efferent ducts and the ductus epididymidis at 12 months of age. Dietary restriction has no obvious effect on seminiferous epithelium as illustrated in stage VIII seminiferous tubules. Both a) control and b) dietary restricted mice have stage VIII seminiferous epithelium with an abundance of preleptotene (PIPS) and pachytene primary spermatocytes (PPS) with large numbers of cap-phase (CPS) and maturation-phase (MPS) spermatids. Residual bodies (RB) are located near the lumen when maturation-phase spermatids are nearing spermiation. Sertoli cell nuclei (SC) are located at the base of the seminiferous epithelium. Epithelium of the efferent ducts in the c) control is similar to that of d) the dietary restricted in size and shape of the nucleus (N) and the overall epithelial height. Sections of spermatozoa are seen in the lumen. The overall cell height and shape and size of nuclei (N) are similar between e) control and f) dietary restricted epithelium of the ductus epididymidis. Mitochondria (M) and other cytoplasmic characteristics are similar between treatments in epithelium of the efferent ducts or ductus epididymidis. Bar length equals 10 IJm.

decreased (P<0.05) with age (Fig. 2a). Dietary restriction reduced body weight compared to the ad libitum group at each age period, and dieting was asso- ciated with a slight but significant age-related increase in body weight. Hence, the age x diet interaction was significant (P<0.01) as dietary restriction prevented maximum body weight of younger mice, but it also prevented the age-related decline in body weight found in the ad libitum group. Over both groups, testicular weight was higher (P<0.05) at 6 months of age than all other ages and declined gradually with age (Fig. 2b). Although there was a nonsignificant treatment difference at 6 months, dietary restriction did not significantly alter testicular weight, and no interaction (P>0.05) was observed. Epididymal weight was reduced (P<0.01) in dietary restricted mice (Fig. 2c). Age did not influence epididymal weight, and no interaction (P>0.05) was observed.

Organ weight:body weight ratios differed between the testis and epididymis. Over both diets, the testicular weight:body weight ratio decreased (P<0.01) with age

(Fig. 2d). The ratio of testicular weight to body weight was greater (P<0.01) for the dietary restricted group. Neither age nor diet altered the ratio of epididymal weight to body weight (Fig. 2e). Dietary restricted mice had sig- nificantly reduced body weights, and the epididymis was not specifically conserved in dietary restricted mice (Table 1). Dietary restriction reduced epididymal weight at the same rate as the whole body weight (Fig. 2e). Also, the number and transit time of spermatozoa were significantly reduced in the dietary restricted mice (Table 1).

Age, but not dietary restriction, altered spermato- genesis in mice. Daily sperm production per g paren- chyma was lower (Fig. 2f) and daily sperm production per testis was higher (Fig. 2g) at 6 and 12 months than at 19, 22, or 26 months (P<0.05). Overall, dietary restriction had no effect on daily sperm production. While these differences were not statistically different in the current study, there was a trend for dietary restriction to delay the onset of maximum daily sperm production at 6 or 12 months of age, and to delay the age-related decline at 26 months of age.

66

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Figure 2: Effect of age and dietary restriction on body and organ weights, ratios of weights, daily sperm production per g parenchyma or per testis, and the number and transit time of epididymal spermatozoa, a) The number of mice in each group is indicated in parentheses. Means among ages within a diet associated with different letters are different (P<0.05). Different letters at the bottom of the graph indicate that no significant interaction was revealed by two-way ANOVA and that means among ages but over both diets are different (P<0.05).

67

Table 1. Effect of dietary restriction on testicular and epididymal traits over all ages combined.

Diet

Item Ad libitum Restricted Significance

Number of mice 55 58 Weight

Body (g) 35.3+0.6 24.8+0.2 P<0.01 Testicular (mg) 82+2 79+1 NS Parenchymal (mg) 67+2 64+1 NS Epididymal (mg) 33.5+1.3 22.7+0.5 P<0.01

Organ weight:body weight ratio Testis 236+7x10 5 319_+5x10 -s P<0.01 Epididymis 96+4xl 0 s 92+3x10 5 NS

Daily sperm production (106 ) Per g parenchyma 33+1 34+1 NS Per testis 2.16+0.09 2.18_+0.09 NS

Epididymal spermatozoa Number (10 ~) 28.4_+1.3 20.5_+0.9 P<0.01

Transit Time (days) 15.5+1.9 106-+1.0 P<0.05

Age and dietary restriction altered the number and transit time of epididymal spermatozoa. Age reduced the number of epididymal spermatozoa in mice regardless of diet (Fig. 2h). However, the age-related reduction (P<0.01) in number was more severe as noted by a steeper slope (P<0.01) in the ad libitum than the dietary restricted mice. Dietary restriction reduced (P<0.01) the number of epididymal spermatozoa and produced lower values at each age. Dietary restriction and age reduced the transit time of epididymal spermatozoa (Fig. 2i). However, the age- related reduction in transit time was more severe in the control than dietary restricted mice.

In summary, age and diet altered male reproductive function. As the result of an age-related increase in daily sperm production and a reduced storage capacity of epididymal spermatozoa, transit time of epididymal spermatozoa was reduced in older mice (Fig. 2). Since dietary restriction reduced epididymal weight and the storage capacity of epididymal spermatozoa, the transit time was reduced (Table 1, Fig. 2). Testicular size and spermatogenesis were specifically conserved in dietary restricted mice.

DISCUSSION

Aging was associated with reduced body weight and testicular weight (Fig. 2a, b). Reduced body and testicular weights are characteristic of aging in mice (14). Age also influences fertility of mice. Franks and Payne (13) found that the number of litters declined after 24 months of age, and most male C57BL mice were sterile after 24 months of age. In the current study, age was associated with a reduced testicular weight: body weight ratio in mice between ages 6 and 12 months (Fig. 2d). Age did not influence epididymal weight (Fig. 2c) or epididymal weight:body weight ratio (Fig. 2e).

Dietary restriction to 60% of ad libitum reduced body weight at each age (Fig. 2a), but the severity of the dietary restriction was noted after 6 and 12 months,

at which time the body weight in restricted mice was about 70% of the ad libitum group. Dietary restriction prevented the age-related decline in body weight seen in the adlibitum group. Epididymal weight was reduced by dietary restriction (Fig. 2c) at the same ratio as was the body weight. There was no effect of dietary restriction on epididymal weight:body ratio (Fig. 2e). Hence, the epididymis was not specifically conserved in the dietary restricted mice. Dietary restriction had no effect on testicular weight (Fig. 2b), but it did influence the testicular weight:body weight ratio (Fig. 2d). Hence, under starvation conditions or limited food intake, testes appear to be maintained to conserve male reproductive function of these animals.

Mature males were remarkably resistant to nutrition stress as noted by the few cases of infertility associated with restricted diets (10). Serum LH and testosterone concentrations were decreased in the blood of animals with limited energy intake (11). Low energy intake appears to influence the hypothalamic-pituitary gonadal axis (19, 20). Also, the accessory sex glands become less responsive to testosterone following undernutrition (21). Leydig cell and Sertoli cell populations may undergo atrophy with undernutrition (22). Hence, hypothalamic-pituitary axis effects, coupled with reduced response of accessory sex gland testosterone (21), are likely responsible for the poor semen quality associated with restricted diets.

The testis has built-in mechanisms that preserve spermatogenesis during limited food intake or starvation conditions. Leydig cells that produce testosterone nec- essary for spermatogenesis are located between semi- niferous tubules (23), and intratesticular testosterone concentrations are more than 100 times higher than in the blood (24). Recent studies have revealed that the requirement of testosterone for quantitatively normal spermatogenesis is much less than previously thought (25). Hence, the reserve levels of testosterone in animals on a normal diet that could be reduced significantly by dietary restriction [known to inhibit the endocrine system (11 )] without diminishing sufficient levels of testosterone in the local vicinity of seminiferous tubules to maintain spermatogenesis. Given limited food intake and starvation are common in the wild, Leydig cells possibly are located between seminiferous tubules as a defence mechanism against the reduced hypothalamic-pituitary axis function during starvation. At which time, limited production of Leydig cells could still be sufficient to maintain spermatogenesis qualitatively if not quanti- tatively.

Negative effects and the magnitude of the effect of restricted diet are more evident and more likely to be permanent in younger animals than adult animals (11 ). In most cases, optimal nutrition reverses the detrimental effects of limited diet on spermatoganesis. Little is known about the effects of dietary restriction in aged males. The current data are consistent with continued conservation of testicular weight (Fig. 2d) and spermatogenesis (Fig. 2f, g) in aged individuals under- going dietary restriction.

68

Dietary Restriction: Age Mouse

Dietary restriction did not significantly delay the onset of maximal levels of spermatogenesis reached at 19, 22, and 26 months, and did not delay its age-related reduction (Fig. 2f, g). Daily sperm production per g parenchyma or per testis was not significantly lower at 12 months and higher at 26 months of age. However, there was a trend for both of these. In rodents, dietary restriction alters cellular aging possibly by reducing the age-related decline in pattern and efficiency of genetic expansion (26, 27). Aging causes a decrease in specific activity and fidelity of DNA polymerases, and dietary restriction impedes the age-related decrease in specific activity and fidelity of these polymerases (9). The role of these polymerases in delay of age-related reduced spermatogenesis may be through regulation of mitosis. Efficiency of mitosis plays a pivotal role in sperma- togenesis (18, 23). Likewise, a reduced degeneration of germ cells throughout spermatogenesis could explain the reduced age-related reduction. Age-related germ cell degeneration during meiosis and their divisions has significant negative effects on spermato- genesis in humans (16-18).

Estimates of epididymal transit time for the mice in this study are comparable to those of other studies; however, they are longer and more widely varied than that for other species. Age reduced the number of epididymal spermatozoa (Fig. 2h) and reduced the transit time (Fig. 2i) of these spermatozoa through the epididymis in mice on both diets, but age-related decline in number of epididymal spermatozoa was less severe in dietary restricted mice. Dietary restriction reduced (P<0.01) number of epididymal spermatozoa. The 16- day transit time for 12-month-old control mice (Fig. 2i) is consistent with the 10-day transit time for sperma- tozoa in the cauda epididymidis previously calculated from the literature for mice (28). Compared to the 2- to-6-day total epididymal transit time for men (29), adult mice have long and widely varied epididymal transit times (Fig. 2i). No age-related change in epididymal weight (Fig. 2c) is consistent with no age-related change in length of the ductus epididymidis. Coupled with significant age-related increases in daily sperm production per testis (Fig. 2g), the transit time of epididymal spermatozoa was reduced in other individuals (Fig. 2i). The cause of the more severe age- related reduction in epididymal spermatozoan storage capacity in the ad libitum mice is unclear. In humans, age had no significant effect on epididymal transit time (29). However, men with higher daily sperm production had lower epididymal transit times. This finding [higher daily sperm production being coupled with reduced epididymal transit time in humans (29)] is consistent with dietary restricted mice whose daily sperm production increases with age as the transit time decreases (Fig. 2g, i).

Age had significant effects on spermatogenesis and epididymal function in C57BU6 mice, and dietary restriction altered epididymal function (Fig. 2). Although not statistically significant, dietary restriction appeared

to delay the onset of maximum levels of spermato- genesis and to reduce the age-related decline in daily sperm production. However, confirmation of this trend requires further study. Restricted diets in rats beginning at weaning caused reduced gonadotropins and androgens, but had less effect on spermatogenesis or testicular weight (30). In our study with C57BL/6 mice, age and dietary restricted also reduced the number and transit time of epididymal spermatozoa (Fig. 2). In adults (10 wks initially) Swiss CD-1 mice given graded restricted diets (70% of ad libitum was most restricted), testicular weight was more resistant to diet than other organs, and the number of epididymal sperm declined with higher restricted diets (31). While the epididymis is not specifically conserved during dietary restriction, testicular size and spermatogenesis (the exocrine function of the testis) were specifically con- served in dietary restricted mice to allow continuation of reproductive function in both adult and aged males.

MATERIALS AND METHODS

Male C57BL/6 mice were reared in the specific pathogen- free barrier facility operated in Jefferson, Arkansas by the National Center for Toxicological Research under a rearing and housing contract from the Biomarkers of Aging Project of the National Institute on Aging. Animal rooms were kept at 23~ on a 12/12 hour light/dark cycle. Nursing pups were raised on dams fed NIH-31 rodent diet ad libitum, weaned at 14 weeks, and divided into two groups. One group (n=55) was fed NIH-31 ad libitum and the other group (n=58) was fed NIH-31 restricted to 60% of the ad libitum control but supple- mented with vitamins and minerals. Animals on both diets were sacrificed at 6, 12, 19, 22, and 26 months, and tissues were processed for analysis. Four to 18 randomly selected mice were placed in groups representing each time period-diet combination.

Daily sperm production per g parenchyma was determined from testicular homogenates as the number of maturation phase spermatids counted by phase- contrast cytometry divided by a 4.84-day estimated life span of these spermatids and the weight of testicular parenchyma homogenized (12, 32). The life span of spermatids whose heads surviving homogenization was estimated as the product of 56.8% of 8.63 days (33, 34). The shapes of these spermatids are similar to Step 14-16 spermatids found in stages II-VIII (56.8% of all stages in the spermatogenic cycle) in the mouse (33, 34). Daily sperm production per testis is equal to daily sperm production per g times testicular parenchymal weight. Epididymal spermatozoan number was deter- mined from similar counts of epididymal homogenates (29). Epididymal transit time of spermatozoa was determined as the number of epididymal spermatozoa divided by the daily sperm production of the attached testis (29, 32).

Data were initially analyzed by two-way (diet x age) analysis of variance (35). If there was a significant interaction between diet and age, one-way (age) analysis

69

of variance was run (35). Multiple means resulting in two-way or one-way analysis of variance were separated by Student-Newman-Keuls test. Also, regression lines produced by treatment effects were tested for similar slopes (35).

ACKNOWLEDGEMENTS

This project was supported in part by AG00465, AG02260 and AG07734 from the National Institute on Aging.

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