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Biology of Reproduction 66, 1359-1366 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Changes in the Testis Interstitium of Brown Norway Rats with Aging and Effects of Luteinizing and Thyroid Hormones on the Aged Testes in Enhancing the Steroidogenic Potential1

In-shik Kima, H.B. Siril Ariyaratnea, and S.M.L. Chamindrani Mendis-Handagama2,a

a Departments of Comparative Medicine and Animal Science, College of Veterinary Medicine, University of Tennessee, Knoxville, Tennessee 37996


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We tested the possibility of using LH and thyroxine (T4) to restore the testicular steroidogenic ability in aged Brown Norway rats. Three-, 6-, 12- (n = 8 per group), and 18-mo-old (n = 32; 3M, 6M, 12M, and 18M, respectively) rats were used. The 18M rats were divided into four groups (n = 8 per group) and implanted subdermally with Alzet mini-osmotic pumps containing saline (control), LH (24 µg/day), T4 (5 µg/day), and LH+T4 (24+5 µg/day), respectively, for 4 wk (to 19 mo [19M] of age). Testis volume and absolute volumes of many testicular components were unchanged with advancing age and treatments, except for the blood vessels (occasional thickening), lymphatic space (increased), and Leydig cells (decreased with age but increased to the 3M level with LH and to the 12M level with both T4 and LH+T4, respectively). The number of Leydig and connective tissue cells per testis was unchanged with aging and treatments. The number of macrophages was significantly higher in treated rats. The average volume of a Leydig cell was significantly decreased in 12M and 19M control rats. However, LH and LH+T4 restored it to the 3M level, and T4 restored to the 12M level. The steroidogenic ability of Leydig cells in vitro decreased when aging from the 3M to the 19M level, LH and T4 enhanced it to the 12M level, and LH+T4 raised it to the 3M level. Serum LH was unchanged from 3M to 12M rats, significantly reduced in 19M control rats, and raised above the 3M values with both LH and LH+T4 treatment and above the 19M (control) values with T4 treatment; the latter values were lower than the 3M level. Serum T4 and tri-iodothyronine (T3) were highest in 3M and 6M rats and declined in 12M and 19M control rats; the latter group had the lowest levels. In all treated groups, T4 and T3 levels were significantly above those of 19M control rats but were lower than those of 3M through 12M rats. Serum testosterone was unchanged from 3M to 12M rats but was reduced in 19M control rats. Both LH and T4 significantly raised these values above the 19M control levels, but they were still lower than the 3M through 12M levels. Additionally, LH+T4 significantly raised the serum testosterone levels to those of 12M rats, but these values were significantly lower than those of 3M and 6M rats. These findings show that with 24+5-µg dose of LH+T4 per day for 4 wk, a 100% recovery of the average volume of a Leydig cell and its steroidogenic ability in vitro and a 73% and 300% restoration of serum testosterone levels compared to 3M and 19M control rats, respectively, could be achieved in aged Brown Norway rats. A 100% reversibility (compared to 3M rats) in serum testosterone levels appears to be possible with adjustments in the LH and T4 doses in the LH+T4 treatment.

aging, Leydig cells, luteinizing hormone, testis, testosterone, thyroid hormone


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The most obvious effect of aging on male reproduction is the progressive decrease in sexual activity from adolescence into old age [1] due to reduced circulating testosterone levels, which occur in all mammalian species studied to date, including humans [2–4] and rats [5–8]. Testosterone is necessary in the male reproductive system for many functions, including the regulation of spermatogenesis in the testis, maintenance of the accessory sex organs, and the erectile function [9, 10]. Testosterone is also required in other organ systems of the mammalian male for their proper functioning. These include, but are not limited to, the brain (for libido and mood), skin (for hair growth and sebaceous gland activity), muscle (to increase muscle strength and volume), liver (to synthesize serum proteins), synovial tissue (to modulate immune responses), bone (to maintain strength and volume), bone marrow (to stimulate stem cells), and kidney (to stimulate erythropoietin) [9–12]. Therefore, sustaining the normal levels of circulating testosterone clearly is important for the well-being of the male. Testosterone is primarily produced by the Leydig cells of the testis. Many studies regarding the effects of aging on Leydig cell structure and function have revealed that Leydig cells undergo atrophic changes in size [7, 8] and organelle content [13, 14] with aging, thus enabling them to go into a malfunctioning status.

Luteinizing hormone regulates the Leydig cell structure and function. Leydig cell size (i.e., average volume) is generally dependent on LH [15, 16], and the testosterone-secretory capacity of Leydig cells has a positive correlation with their size [15–17]. Thyroid hormones are important in cellular differentiation [18]. More importantly, thyroid hormones are critical in the process of differentiation of precursor cells to Leydig cells in the postnatal rat testis [19–21]. The specific effects of thyroid hormone on mature Leydig cells are not clear at present. It is reported that reduced numbers of identifiable Leydig cells, together with increased numbers of connective tissue/mesenchymal cells, are observed in the testis interstitium of rats subjected to hypothyroidism [22]. If the differentiation of connective tissue/mesenchymal cells into Leydig cells requires thyroid hormone [18–20], the levels of which are reduced with aging [23, 24], then it is possible to hypothesize that the reduced steroidogenic potential of the aged Leydig cells could be due to the dedifferentiation of many Leydig cells toward their connective tissue precursor cells under the reduced thyroid hormone levels that occur with aging.

Circulating levels of LH are reduced in many strains of rats [6, 8, 25–27], including the Brown Norway strain [27], except for the report by Chen et al. [7]. Although statistically not significant, the results of their study [7] clearly showed that the mean value for serum LH in older rats was 33% lower than in young rats. In addition, thyroid hormones are reduced with aging [23, 24]. These hormonal changes can be explained, at least in part, by direct (LH secretion) and indirect (thyrotrophin/thyroid-stimulating hormone [TSH] action on the thyroid gland) effects that occur as a result of the pituitary aging. Based on this information, we hypothesize that these atrophic and dedifferentiated Leydig cells in aged testes are caused, at least in part, by the defects generated as a result of the aged pituitary gland on LH and thyroid hormone levels in aged males. Therefore, one objective of the present study was to test the possibility of reversing these changes in aged Leydig cells by exogenous supplementation of thyroid hormone and/or LH to increase their testosterone-producing capacity to a level similar to that of the young. Rats have been suggested as suitable models for human aging studies [28]. The Brown Norway rat was chosen for this study, because this strain has been recommended as a suitable model for male reproductive aging studies [27, 29].

According to previous reports [27, 29], Brown Norway rats age differently than other strains of rats that have been studied to date [8, 13]. The second objective of the present study was to examine age-related changes in the interstitial components of the testis in Brown Norway rats, because to our knowledge, no such information (except for the changes in Leydig cells) is available in this strain.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Treatments

Male Brown Norway rats aged 3, 6, 12 (n = 8 per group), and 18 mo (n = 32; 3M, 6M, 12M, and 18M, respectively) were purchased from Harlan (Madison, WI). The 18M rats were divided equally into four groups (n = 8 per group). Under deep inhalation anesthesia (Metofane; Malincroft Veterinary, Inc., Mundelein, IL), these four groups of 18M rats were implanted subdermally with Alzet mini-osmotic pumps (model 2ML4; Alza Corporation, Palo Alto, CA) containing saline (control), LH (24 µg/day; National Hormone and Pituitary Program, Los Angeles, CA), thyroxine (T4; 5 µg/day; Sigma, St. Louis, MO), and LH and T4 (LH+T4; two pumps, 24 µg/day of LH and 5 µg/day of T4), respectively, for 4 wk (to 19 mo [19M] of age). These doses of LH [16] and thyroid hormone [19] were used based on previously published studies. Rats were maintained under conditions of controlled temperature (25°C) and lighting (14L:10D) and were housed individually (one rat per cage). The animals were fed with Agway Prolab rat formula (Syracuse, NY) and water ad libitum until they were killed. The animal protocol used (no. 488) meets the guidelines of the National Institutes of Health and was approved by the University of Tennessee Animal Care and Concerns Committee.

Administration of Saline and Hormones

Pumps were filled with either saline, LH, or T4 and were primed in saline for 3 h before implantation so that they would deliver their contents immediately on subdermal implantation into the interscapular region. The LH+T4 rats received two pumps each, one containing LH and one containing T4; the other rats received one pump containing either saline, LH, or T4. After 4 wk of treatment, rats were killed and used as described below. In addition, implanted pumps were removed and examined to verify that they had delivered the contents as expected.

Serum Collection

Immediately after a rat was killed, blood was drawn from its heart using the cardiac puncture technique, serum prepared [8], and stored at -20°C until assay.

RIA for Serum LH, T4, Tri-Iodothyronine, and Testosterone

Serum LH hormone was quantified using a commercially available rLH (rat luteinizing hormone) kit (Amersham Pharmacia Biotech, Piscataway, NJ). The sensitivity of LH assay was 0.8 ng/ml. The interassay coefficient of variation was less than 10.97%, and the intraassay coefficient of variation was less than 6.5%. The cross-reactivity of the antibody for LH was 0.66% for rat TSH, 0.1% for rat growth hormone, less than 0.016% for rat FSH, less than 0.8% for rat prolactin, and less than 0.00092% for rat ACTH. Serum T4, tri-iodothyronine (T3), and testosterone were assayed using commercially available kits (Coat-A-Count; DPC, Los Angeles, CA). The interassay coefficients of variation for T4, T3, and testosterone assays were less than 14.5%, 10%, and 11%, respectively. The intraassay coefficients of variation for T4, T3, and testosterone assays were less than 3.8%, 8.9%, and 9%, respectively. The antibody used in the T4 assay had 2% cross-reactivity with T3, and the antibody used in the T3 assay had less than 1% cross-reactivity with T4. The cross-reactivity of the antibody used in the testosterone RIA kit was 2.8% for dihydrotestosterone, 0.5% for androstenedione, and less than 0.02% for other steroids.

LH-Stimulated Testicular Steroidogenesis In Vitro

One testis of each rat in the 3M, 6M, 12M, and 19M age groups was removed, cleaned of fat, and weighed on a Mettler H54 balance to obtain the fresh testis weight. Using the flotation technique [17, 30, 31], the specific gravity of the fresh testis was determined as described previously [30, 31], and the fresh testis volume was calculated by dividing the fresh testis weight by the specific gravity (in metric units, specific gravity equals density). This measurement is required to express Leydig cell numbers per testis, because numerical density is obtained as a number per unit volume of the testis. The testis was then decapsulated, and the entire testis was incubated for 3 h in the same medium as described previously [19, 20, 32–34] to determine the LH-stimulated testosterone-secretory capacity per testis in vitro. Testosterone levels in the incubation medium were measured by RIA using commercially available kits (Coat-A-Count), and the details of the assay are given above (see RIA for Serum LH, T4, Tri-Iodothyronine, and Testosterone). Testosterone-secretory capacity per Leydig cell was calculated by dividing the testosterone-secretory capacity per testis by the number of Leydig cells per testis.

Fixation and Processing of Testis Tissue

The other testis of each rat in the 3M, 6M, 12M, and 19M age groups (n = 8 per group) was fixed by whole-body perfusion [17, 20, 33, 34], and the fixed testis was then weighed, the specific gravity measured, and the fixed testis volume calculated. These fixed testes were processed for microscopy and stereology as described previously [17, 20, 33, 34]. Shrinkage of testis tissue from the fresh to the processed state was determined as previously published [31] for use in the stereological studies. (We have measured right and left testis weights in several Brown Norway rats in a separate study and verified that they are similar in weight when they do not show any obvious differences in sizes.)

Microscopy and Stereology

Two tissue sections of 1 µm in thickness and four sections apart were cut from the testis tissue blocks prepared for stereology using a LKB IV ultramicrotome and glass knives. These sections were stained with methylene blue. The volume density of testicular components, which is defined as the volume of the component per unit volume of testis tissue, was obtained via point counting as described previously [17, 20, 34]. Four corners of every tissue section (four fields/section, one section/block, and 10 blocks/rat, for a total of 40 fields/rat) were analyzed with an Olympus BH-2 light microscope. The unbiased sampling rule of Sterio [35] was used to avoid bias and overlapping of the fields tested. The tested components included seminiferous tubules, testis interstitium, lymphatic space, Leydig cells (identified by their distinct peripheral rim of nuclear heterochromatin and abundant granular cytoplasm), blood vessels, macrophages (identified by nuclear heterochromatin and vacuolated cytoplasm, which stains differently from Leydig cells), and connective tissue cells (peritubular myoid cells, fibroblasts, endothelial cells of blood and lymph vessels, and pericytes; these cells were identified by their location and elongated, spindle-like shape). The formula used to obtain the volume density of each testicular component was as follows: volume density of component = (number of points on each component/total number of points on testis tissue) x 100.

The numerical density of Leydig cells (number of cells per unit volume of testis) was obtained via the disector method [35] as described in detail previously [34]. The average volume of a Leydig cell was obtained by dividing the volume density of Leydig cells by the numerical density as published previously [17, 20, 31]. The number of Leydig cells per testis was calculated by multiplying the numerical density by the fresh testis volume [17, 20, 34, 35]. An Olympus BH-2 light microscope was used for photography.

Statistical Analysis

The PC SAS software was used to analyze the data. Results are expressed as mean ± SEM. Significant differences (P < 0.05) between the means were determined by the Duncan multiple-range test after analysis of variance [36].


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The testis volumes and volume densities of testicular components are shown in Table 1. The absolute volumes of testicular components per testis are given in Table 2. The numbers of Leydig cells, macrophages, and connective tissue cells per testis and the average volume of a Leydig cell are shown in Table 3. This information (except for the parameters of Leydig cells) is provided for comparison with other studies and will not be discussed further.


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  		TABLE 1. Mean testis volume (mm3) and volume density of testicular components (%)


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  		TABLE 2. Mean absolute volume of testicular components (mm3)


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  		TABLE 3. Mean cell number per testis (106) and average volume of a Leydig cell (µm3)

The absolute volume of Leydig cells per testis did not change with age advancement from 3M to 6M but was significantly reduced at 12M; a further reduction was seen at 19M (control). By contrast, all other 19M groups (i.e., LH-, T4-, and LH+T4-treated) had higher values compared to 19M control rats; however, statistical significance was not observed between 19M T4-treated and 19M control rats due to the higher SEM in the latter group. Additionally, the value of the 19M LH-treated group was not significantly different from those of the 3M and 6M groups, and the values of the 19M T4- and 19M LH+T4-treated groups were not significantly different from those of 12M rats.

The number of Leydig cells per testis (Table 3) was not significantly different among all experimental groups. The average volume of a Leydig cell (Table 3) was unchanged with age advancement from 3M to 6M; however, significant reductions were observed first in 12M rats and then in 19M control rats. The average volume of a Leydig cell in 19M LH- and LH+T4-treated rats was not significantly different from those in 3M and 6M rats. This value in 19M T4-treated rats was significantly lower than those in 3M and 6M rats, although it was not significantly different from those in 19M LH-treated and 12M rats. Microscopic studies also revealed that the size of Leydig cell profiles in tissue sections progressively decreased with age (from 3M to 19M), and that these profiles in 19M LH-, T4-, and LH+T4-treated rats appeared to be much larger than those in 19M control rats (Fig. 1). Thickening of the walls of some blood vessels, mainly due to the accumulation of connective tissue cells, was also observed occasionally in 6M and older rats (Fig. 1); however, the amount of collagen in the testis interstitium, which usually increases with age advancement in other strains of rats, was not evident in this strain.



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  		FIG. 1. Representative light micrographs of testis interstitium of brown Norway rats. A) 3 mo. B) 6 mo. C) 12 mo. D) 19 mo, saline. E) 19 mo, LH. F) 19 mo, T4. G) 19 mo, LH+T4. Arrows depict Leydig cells. Note the thickening of a blood vessel (B) wall in a 6-mo-old rat (B) and a 19-mo-old LH+T4 rat (G). c, Connective tissue cells; Ly, lymphatic space; M, macrophages; ST, seminiferous tubules. Bar = 7.7 µm, same magnification for all micrographs

Table 4 shows LH-stimulated testosterone-secretory capacity per testis and per Leydig cell in vitro. No difference was observed between 3M and 6M rats for both these parameters, but significant reductions were observed in 12M and 19M control rats. Both these parameters in 19M LH+T4-treated rats were similar to those in 3M and 6M rats, and these values in 19M LH- and T4-treated rats were comparable to those in 12M rats.


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  		TABLE 4. Mean testosterone-secretory capacity per testis (ng) and per Leydig cell (pg)

Serum LH, T4, T3, and testosterone levels are shown in Table 5. Serum LH levels were unchanged from 3M to 12M rats but were reduced significantly in 19M control and T4-treated rats compared with 3M through 12M rats. In 19M LH- and LH+T4-treated rats, LH levels were greater than those of 3M to 12M rats. Both T4 and T3 levels in serum were highest in 3M and 6M rats and lowest in 19M control rats. These hormone levels in 19M T4- and LH+T4-treated rats were similar to those in 12M rats and were higher than those in 19M LH-treated rats. Serum testosterone levels did not change significantly from 3M to 12M rats, although the mean value in 12M rats was approximately 20% lower than those in 3M and 6M rats. The lowest value was observed in 19M control rats; the values in 19M LH-, T4-, and LH+T4-treated rats were significantly greater than 19M controls. Serum testosterone levels in 19M LH- and T4-treated rats were significantly lower than those in 3M through 12M rats. Although the serum testosterone levels in 19M LH+T4-treated rats were not significantly different from those in 12M rats, they were 27% lower (P < 0.05) than those in 3M and 6M rats.


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  		TABLE 5. Mean serum LH, T4, T3, and testosterone levels


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present investigation showed no change in the mean testis volume (approximating testis weight in metric units) in Brown Norway rats with aging, which compares favorably with previously published results of studies using this strain [7, 29]. This is in contrast to observations in the Sprague-Dawley [8] and Wistar strains [13], which show increased testis volumes and weights with aging. In the present study, we did not see significant differences in testis weights and volumes between the right and left testicles in 19M Brown Norway rats, although the occurrence of small and large testes has been reported previously in 21-mo [7] as well as in 22- and 30-mo [27] Brown Norway rats. One possible explanation for these differences could be the differences in the ages of these rats (i.e., 19 vs. 21, 22, and 30 mo).

To our knowledge, no other study reporting on changes in the testicular components of the aged testis and/or testis interstitium (excluding Leydig cells) in Brown Norway rats is available with which to compare the present findings, although such information is available for Sprague-Dawley [8] and Wistar rats [13]. Comparing these changes in Brown Norway rats with those in Sprague-Dawley and Wistar rats reveals both differences and similarities among these strains. In brief, Leydig cell hypotrophy and reduced steroidogenic capacity is also observed with aging in Sprague-Dawley [8] and Wistar rats [13]; however, unlike in the Brown Norway rat, the number of Leydig cells per testis is increased with aging in Sprague-Dawley [8] and Wistar rats [13]. Occurrence of Leydig cell hyperplasia in the aged testes has also been reported in stallions [37] and humans [38], and this suggests the existence of differences among species in addition to differences among strains within the same species for this parameter with aging. Increased collagen deposition and thickening of the blood vessels in the testis interstitium are described in aged Sprague-Dawley and Wistar rats [8, 13]. By contrast, the present study showed that these changes were either minimal or absent in aged Brown Norway rats. Occlusion of blood vessels is observed in human testis with aging [39] and other conditions associated with infertility [40–42]. These findings suggest that testicular aging occurs at a slower pace in Brown Norway rats compared with all other strains of rats studied so far.

The most important findings of the present study are the results of the treatments of aged Brown Norway rats with LH, T4, and LH+T4 regarding enhancement of the testicular steroidogenic function. It is interesting to note that these treatments did not cause specific changes in testis volume, absolute volumes of testicular components (except for the Leydig cells), and numbers of testicular interstitial cells (including the Leydig cells). However, all three treatments were able to increase the absolute volume of Leydig cells per testis and the average volume of a Leydig cell above that of aged, 19M control rats, which had the lowest values for these parameters when compared with all the treatment groups. We were intrigued to learn that separate treatment with LH or T4 could improve the testicular steroidogenic potential in vitro to that of 12M rats, and that the combined treatment (LH+T4) was effective in upgrading this potential to those of 3M and 6M rats. These findings suggest that changes in the circulating levels of LH and T4 are the major factors responsible for Leydig cell hypotrophy and loss in their steroidogenic potential with aging, although we do not rule out any direct and/or indirect effects of other hormones and factors on Leydig cells that could be associated with these age-related changes. It needs to be mentioned that, although the LH+T4 treatment upgraded the steroidogenic potential of Leydig cells in vitro to those of 3M and 6M rats, the serum testosterone levels of these rats were raised only to the level of 12M rats (i.e., 73% recovery compared to 3M rats). However, this enhancement is more than 300% compared to 19M control rats. We believe that this recovery is remarkable and warrants further research to achieve 100% recovery compared to 3M rats. Among the possibilities for the lower serum testosterone levels in LH+T4-treated rats, two that are obvious for us are the very high levels of serum LH and the less-than-normal levels of T4 (compared to 3M and 6M rats). Therefore, it appears that adjustments in the doses of LH and T4 in LH+T4-treated rats would allow us to achieve the desired serum testosterone levels in these aged rats.

Luteinizing hormone is produced by the gonadotrophs of the anterior pituitary gland and has a positive correlation with Leydig cell steroidogenic function. Deprivation of endogenous LH causes Leydig cell hypotrophy and reduced steroidogenic capacity [16, 43, 44], and chronic stimulation of Leydig cells with LH produces Leydig cell hypertrophy and increased steroidogenic capacity [15]. Substantial evidence from studies by Liao and Azhar [45] supports the idea that aging directly affects LH-mediated cholesterol transfer into mitochondria and within the inner mitochondrial membrane. This view is further supported by the observations that significant reductions occur in steroidogenic acute regulatory protein (StAR) [46] and peroxisomes [13] in Leydig cells, which are considered to be crucial factors in cholesterol transport into mitochondria in steroidogenic cells [47, 48]. In addition, enzymes responsible for converting cholesterol to testosterone in Leydig cells are reduced with aging [49]; these include P450 cholesterol side chain cleavage enzyme, {Delta}5-3ß-hydroxysteroid dehydrogenase/{Delta}5-{Delta}4-isomerase, 17{alpha}-hydroxylase/C17–20 lyase, and 17ß-hydroxysteroid [49]. Because substitution of LH to the aged Brown Norway rats has aided tremendously in upgrading the steroidogenic potential of their Leydig cells, testicular aging clearly is closely associated with aging of the pituitary gland.

Thyroid hormones (T4 and T3) are produced by the follicular cells of the thyroid gland and are regulated by TSH made by the thyrotrophs of the anterior pituitary gland. With aging of the pituitary gland, circulating thyroid hormone levels are reduced [23, 24]. Hypothyroidism arrests differentiation of Leydig cells in the neonatal [20, 21, 45] and adult [19] testis and, more importantly, causes hypotrophy and hypoplasia of Leydig cells in the sexually mature testis [22]. Although direct effects of T4 on Leydig cells in vivo or in vitro have not been extensively studied, acute treatment of Leydig cells with its metabolite, T3, directly enhances Leydig cell steroidogenesis in vitro [50, 51]. Additionally, treatment of mouse Leydig cells with T3 coordinately augments the levels of StAR protein, StAR mRNA, and steroid production [51]. Because the effects of T4 in vivo (restricted to the vascular pool) are mediated via T3 (T4 is converted to T3 in target tissues; T3 is 3- to 5-fold more active than T4) [10], it is possible to suggest that the stimulatory effects of T3 in vitro on Leydig cell steroidogenesis [50, 51] reflect the acute effects of thyroid hormone on Leydig cells in vivo as well. Taken together, it is apparent that thyroid hormone is an important factor for Leydig cell steroidogenesis, and that thyroid hormone deficiency during aging could be corrected with exogenous substitution. Manifestation of thyroid hormone deficiency with aging and its effects on testicular steroidogenic function are also in agreement with the concept that aging of the testis is closely associated with pituitary aging.

The observation of Leydig cell hypertrophy in LH-treated rats compares favorably with the results of previous studies on the effects of chronic hCG [52] and LH [16] treatments in sexually mature young rats. However, together with cell hypertrophy, hyperplasia of Leydig cells [16, 52] was also observed in those rats, in contrast to the aged Brown Norway rats of the present study. Whether these differences in response to LH are due to age effects or to strain effects is not clear at present. Whereas LH treatment alone was able to fully restore the average volume of a Leydig cell to those of 3M and 6M rats, it was sufficient only to upgrade the steroidogenic potential of Leydig cells in vitro to that of 12M rats (i.e., above 19M control rat values). Although serum testosterone levels in the aged Brown Norway rats were raised significantly above the 19M control rat value with LH treatment, this increased level was still much less than the serum testosterone levels of 3M and 6M rats. Therefore, it is apparent that exogenous LH substitution alone is not sufficient to rejuvenate the functional aspect of Leydig cells in aged Brown Norway rats toward that of 3M and 6M rats, although a complete recovery in cell size was achieved with LH treatment alone.

Treatment of neonatal rats with T3 [18, 31] and of adult rats with ethane dimethane sulfonate (which kills Leydig cells within 48 h) [19] causes a stimulation of Leydig cell differentiation from precursor cells. However, to our knowledge, the effects of T3 or T4 on the testis interstitium and/or the Leydig cells in the sexually mature testis in vivo are poorly understood. The present study revealed that, although T4 treatment alone was partially effective in restoring the average volume of a Leydig cell (i.e., to a level comparable with that of 12M rats), it was equally effective as the LH treatment in upgrading the steroidogenic potential of Leydig cells in vitro to the level of 12M rats. Additionally, T4 treatment alone was not sufficient to upgrade the serum testosterone levels in 19M rats to those of 3M and 6M rats. Another fact to note is that the dose of T4 (5 µg/day) used in the present study was insufficient to produce circulating T4 and T3 levels in the aged rats similar to those in 3M and 6M rats.

With LH+T4 treatment, the aged Leydig cells of 19M Brown Norway rats were rejuvenated remarkably; their average cell volume and the steroidogenic potential in vitro were completely reversed, to values similar to those of 3M and 6M rats. Although the serum testosterone levels of these rats were raised only to the level of 12M rats (i.e., 73% recovery compared to 3M rats), this was a 300% increase compared to 19M control rats. Therefore, it is possible to be optimistic regarding the potential of this line of research to achieve the desired serum testosterone levels by adjusting the present LH and T4 doses (to be lower and higher, respectively). However, even with a 100% success compared to 3M Brown Norway rats, it would still be too early to predict whether this line of treatment could be effective in other strains of rats, other rodents, or other mammalian species in rejuvenation of aged Leydig cells and, thereby, treatment of the androgen deficiency caused by the process of aging.

Currently, androgen deficiencies in aging humans are treated with androgen therapy [11, 12]. The risks of administering androgens to aging men mainly concern the cardiovascular system and the prostate [12]. Cardiovascular effects of androgens are ascribed to the atherogenic effects of androgens on blood-lipid profiles [12]. Apart from these effects, androgens could have other possible deleterious metabolic effects on the cardiovascular system. They induce insulin resistance [53] and increased plasma levels of endothelin, a substance with vasoconstrictor properties produced by the vascular wall [54]. Regarding the effects of androgens on the prostate, benign prostatic hyperplasia and prostate cancer are the main concerns [12]. Therefore, if the present findings are, in fact, applicable to humans, a potential exists to develop an alternative to androgen therapy for minimizing the effects of androgen deficiency during age advancement. Clearly, additional research is required to evaluate the feasibility of this approach.


   ACKNOWLEDGMENTS
 
Thanks are due to Tommy Jordan of the Photographic Laboratory of the University of Tennesee Institute of Agriculture for his assistance in preparing the microscopic plate. We also acknowledge the gift of LH from Dr. A.F. Parlow and the National Hormone Distribution Program.


   FOOTNOTES
 
First decision: 20 June 2001.

1 Supported in part by grant R180101-08 from the College of Veterinary Medicine Center of Excellence for Livestock Diseases and Human Health and a Professional Development Award from The University of Tennessee, Knoxville. Back

2 Correspondence. FAX: 865 974 5640; mendisc{at}utk.edu Back

Accepted: November 30, 2001.

Received: May 31, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Kinsey AC, Pomeroy WB, Martin CE. Sexual Behavior in the Human Male. Philadelphia: W.B. Saunders; 1948
  2. Hollander N, Hollander VP. The microdetermination of testosterone in human spermatic vein blood. J Clin Endocrinol 1958; 18:966-971
  3. Kirschner MA, Coffman GD. Measurement of plasma testosterone and {Delta}4-androstenedione using electron capture gas-liquid chromatography. J Clin Invest 1968; 47:38-47[Medline]
  4. Vermeulen A. Leydig cell function in old age. In: Everitt AV, Burgess JA (eds.), Hypothalamus, Pituitary and Aging. Springfield, IL: Charles C. Thomas, 1976: 458–463
  5. Harman SM, Danner RL, Roth G. Testosterone secretion in the rat in response to chorionic gonadotrophin. Endocrinology 1978; 102:540-544[Abstract]
  6. Bethea CL, Walker RF. Age-related changes in reproductive hormones and in Leydig cell responsivity in the male Fisher 344 rat. I. Gerontology 1979; 34:21-27
  7. Chen H, Hardy MP, Huhtaniemi I, Zirkin BR. Age-related decreased Leydig cell testosterone production in the Brown Norway Rat. J Androl 1994; 15:551-557[Abstract/Free Full Text]
  8. Mendis-Handagama SMLC, Gelber SJ. Signs of aging are apparent in the testis interstitium of Sprague-Dawley rats at 6 months of age. Tissue Cell 1995; 27:689-699[CrossRef][Medline]
  9. Morales A, Bain J, Ruijs A, Chapdelaine A, Remblay RR. Clinical practice guidelines for screening and monitoring male patients receiving testosterone supplementation therapy. Int J Impotence Res 1996; 8:95-97[Medline]
  10. Norman AW, Litwack GL. Thyroid hormones. In: Norman AW, Litwack GL (eds.), Hormones. San Diego: Academic Press; 1997: 169–191
  11. Anderson FH, Francis RM, Faulkner K. Androgen supplementation in eugonadal men with osteoporosis-effects of 6 months of treatment on bone mineral density and cardiovascular risks factors. Bone 1996; 18:171-177[Medline]
  12. Gooren LJG. The age related decline of androgen levels in men: clinically significant?. Br J Urol 1996; 78:763-768[Medline]
  13. Ichihara I, Kawamura H, Pelliniemi LJ. Ultrastructure and morphometry of testicular Leydig cells and the interstitial components correlated with testosterone in aging rats. Cell Tissue Res 1993; 271:241-255[CrossRef][Medline]
  14. Mori H, Hiromoto N, Nakahara M, Shiraishi T. Stereological analysis of Leydig cell ultrastructure in aged humans. J Clin Endocrinol Metab 1982; 55:634-641[Abstract]
  15. Mendis-Handagama SMLC, Watkins PA, Gelber SJ, Scallen TJ. Leydig cell peroxisomes and sterol carrier protein-2 in luteinizing hormone-deprived rats. Endocrinology 1992; 131:2839-2845[Abstract]
  16. Mendis-Handagama SMLC, Watkins PA, Gelber SJ, Scallen TJ. Effects of chronic luteinizing hormone treatment on Leydig cell structure and function in adult rats. Tissue Cell 1998; 30:64-73[CrossRef][Medline]
  17. Mendis-Handagama SMLC, Zirkin BR, Ewing LL. Comparison of components of the testis interstitium with testosterone secretion in hamster, rat and guinea pig testes perfused in vitro. Am J Anat 1988; 181:12-22[CrossRef][Medline]
  18. Guyton AC. The thyroid metabolic hormones. In: Guyton AC (ed.), Textbook of Medical Physiology. Philadelphia: W.B. Saunders; 1991: 831–841
  19. Ariyaratne HBS, Mills N, Mason JI, Mendis-Handagama SMLC. Effects of thyroid hormone on Leydig cell regeneration in the adult rat following ethane dimethane sulfonate treatment. Biol Reprod 2000; 63:1115-1123[Abstract/Free Full Text]
  20. Mendis-Handagama SMLC, Ariyaratne HBS, Teunissen van Manen KR, Haupt RL. Differentiation of adult Leydig cells in the neonatal rat testis is arrested by hypothyroidism. Biol Reprod 1998; 59:351-357[Abstract/Free Full Text]
  21. Teerds KJ, Rooij DG, de Jong FH, van Haaster LH. The development of the adult type Leydig cell population in the rat testis is affected by neonatal thyroid hormone levels. Biol Reprod 1998; 59:344-350[Abstract/Free Full Text]
  22. Amin S, El-Sheikh AS. Pituitary-testicular function changes in hypo and hyperthyroid male rats. Acta Anat 1977; 98:121-129[Medline]
  23. Cizza G, Brady LS, Calogero AE, Bagdy G, Lynn AB, Kling MA, Blackman MR, Chrousos GP, Gold PW. Central hypothyroidism is associated with advanced age in male Fischer 344/N rats: in vivo and in vitro studies. Endocrinology 1992; 131:2672-2680[Abstract]
  24. Cizza G, Brady LS, Pacak K, Blackman MR, Gold PW, Chrousos GP. Stress-induced inhibition of the hypothalamic-pituitary-thyroid axis is attenuated in the aged Fischer 344/N male rat. Neuroendocrinology 1995; 62:506-513[Medline]
  25. Riegle GD, Meites J. Effects of aging on LH and prolactin after LHRH, L-DOPA, methyldopa and stress in the male rat. Proc Soc Exp Biol Med 1976; 151:507-511[Abstract]
  26. Riegle GD, Miller AE. Aging effects on the hypothalamic hypophyseal-gonadal control system in the rat. In: Schneider EL (ed.), The Aging Reproductive System. New York: Raven Press; 1978: 159–192
  27. Wang C, Leung A, Sinka-Hikim AP. Reproductive aging in the male Brown Norway rat: a model for the human. Endocrinology 1993; 133:2773-2781[Abstract]
  28. Hazzard DG. Relevance of the rodent model to human aging studies. Neurobiol Aging 1991; 12:645-649[CrossRef][Medline]
  29. Zirkin BR, Santuli R, Strandburg JD, Wright WW, Ewing LL. Testicular steroidogenesis in the aging Brown Norway rat. J Androl 1993; 14:118-123[Abstract/Free Full Text]
  30. Mori H, Christensen AK. Morphometric analysis of Leydig cells in the normal rat testis. J Cell Biol 1980; 84:340-354[Abstract/Free Full Text]
  31. Mendis-Handagama SMLC, Ewing LL. Sources of error in the estimation of Leydig cell numbers in control and atrophied mammalian testes. J Microsc (Oxf) 1990; 159:73-82[Medline]
  32. Mendis-Handagama SMLC, Kerr JB, de Kretser DM. Experimental cryptorchidism in the mouse. II. A hormonal study. J Androl 1990; 11:548-554[Abstract/Free Full Text]
  33. Ariyaratne HBS, Mason JI, Mendis-Handagama SMLC. Effects of tri-iodothyronine on testicular interstitial cells and androgen secretory capacity of the prepubertal rat. Biol Reprod 2000; 63:493-502[Abstract/Free Full Text]
  34. Ariyaratne HBS, Mendis-Handagama SMLC. Changes in the testis interstitium of Sprague-Dawley rats from birth to sexual maturity. Biol Reprod 2000; 62:158-168
  35. Sterio DC. The unbiased estimation of number and sizes of arbitrary particles using the disector.. J Microsc (Oxf) 1984; 134:127-137[Medline]
  36. Duncan DB. t Tests and intervals for comparisons suggested by the data. Biometrics 1975; 31:339-359[CrossRef]
  37. Johnson L, Neaves WB. Age related changes in the Leydig cell population, seminiferous tubules and sperm production in stallions. Biol Reprod 1981; 24:703-712[Abstract]
  38. Kothari LK, Gupta AS. Effect of aging on the volume, structure and total Leydig cell content of the human testis. Int J Fertil 1974; 19:140-146[Medline]
  39. Vermuelen A, Rubens R, Verdonck AL. Testosterone secretion and metabolism in male senescence. J Clin Endocrinol Metab 1972; 34:730-735[Medline]
  40. Francavilla S, Santiemma V, Francavilla F, Dermatino C, Santucci R, Fabbrini A. Ultrastructural changes in the seminiferous tubule wall and intertubular blood vessels in human cryptorchidism. Arch Androl 1979; 2:21-30[Medline]
  41. Mendis-Handagama SMLC, Kerr JB, de Kretser DM. Experimental cryptorchidism in the adult mouse: I. qualitative and quantitative light microscopic morphology. J Androl 1990; 11:539-547[Abstract/Free Full Text]
  42. Nasah BT, Cox JN. Vascular lesions in testes associated with male infertility in Cameroon. Virchows Arch A 1978; 377:225-236
  43. Russell LD, Corbin TJ, Ren HP, Amador A, Bartke A, Ghosh S. Structural changes in rat Leydig cells posthypophysectomy: a morphometric and endocrine study. Endocrinology 1992; 131:498-508[Abstract]
  44. Ewing LL, Wing T-Y, Cochran RC, Kromann N, Zirkin BR. Effects of luteinizing hormone on Leydig cell structure and testosterone secretion. Endocrinology 1983; 112:1763-1769[Abstract]
  45. Liao C, Azhar S. Age related decline in the steroidogenic capacity of isolated rat Leydig cells: a defect in cholesterol mobilization and processing. J Steroid Biochem Mol Biol 1993; 46:39-47[CrossRef][Medline]
  46. Luo L, Chen H, Zirkin BR. 2001. Leydig cell aging: steroidogenic acute regulatory protein (StAR) and cholesterol side-chain cleavage enzyme. J Androl 2001; 22:149-156[Abstract]
  47. Mendis-Handagama SMLC. Peroxisomes and intracellular cholesterol trafficking in adult rat Leydig cells following luteinizing hormone stimulation. Tissue Cell 2000; 32:102-106[CrossRef][Medline]
  48. Stocco DM. Acute Regulation of Leydig cell steroidogenesis. In: Payne AH, Hardy MP, Russell LD (eds.), The Leydig Cell. Vienna, IL: Cache River Press; 1996: 241–257
  49. Luo L, Chen H, Zirkin BR. Are Leydig cell steroidogenic enzymes differentially regulated with aging?. J Androl 1996; 17:509-515[Abstract/Free Full Text]
  50. Maran RRR, Arunakaran J, Aruldhas MM. T-3 directly stimulates basal and modulates LH induced testosterone and estradiol production by rat Leydig cells in vitro. Endocr J 2000; 47:417-428[Medline]
  51. Manna PR, Roy P, Clark BJ, Stocco DM, Huhtaniemi IT. Interaction of thyroid hormone and steroidogenic acute regulatory (StAR) protein in the regulation of murine Leydig cell steroidogenesis. J Steroid Biochem Mol Biol 2001; 76:167-177[CrossRef][Medline]
  52. Christensen AK, Peacock C. Increase in Leydig cell number in testes of adult rats treated chronically with an excess of human chorionic gonadotropins. Biol Reprod 1980; 22:383-391[Abstract]
  53. Polderman JH, Gooren LJG, Asscheman H, Bakker A, Heine RJ. Induction of insulin resistance by androgen and estrogens. J Clin Endocrinol Metab 1994; 79:275-281
  54. Polderman JH, Stehouwer CDA, Van de Kamp GJ, Dekker GA, Verheugt FWA, Gooren LJG. Influence of sex hormones on plasma endothelin levels. Ann Intern Med 1993; 118:429-432[Abstract/Free Full Text]



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