Ultrastructure and morphometry of testicular Leydig cells and the interstitial components correlated with testosterone in aging rats

The ultrastructure of testicular interstitium in young and aged adult rats was analysed using morphometric methods, and the plasma testosterone concentration was measured. With increasing age there was an augumentation in the volume of collagen fibrils in the intercellular matrix and in blood vessels. During the aging process (approximately two years) the average volume of the Leydig cell decreased from 1364 m³ to 637 m³, but the number of Leydig cells in paired testes increased from 53x10⁶ to 113x10⁶. The absolute volume of smooth surfaced endoplasmic reticulum (SER) per Leydig cell amounted in aged rats to 78% of that in young adult rats. The total amount of SER in paired testes increased by 62% with aging. The present analysis suggests that the ability of SER to maintain peripheral testosterone concentration decreases with age. In young adult rats the absolute volume of peroxisomes per Leydig cell correlated significantly with the concentration of testosterone in blood and also with the absolute volume of SER per Leydig cell. These results combined with ultrastructural observations of close apposition of peroxisomes and SER suggest that peroxisomes have a role in testosterone secretion by Leydig cells.Visiting scientist to Laboratory of Electron Microscopy (Director: Prof. L.J. Pelliniemi)     

NADPH generating enzymes in Leydig cells from diabetic rats.

Impaired testosterone biosynthesis in Leydig cells from streptozotocin treated rats is correlated with the reduced activity of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and isocitrate dehydrogenase. The results shown demonstrate that in the diabetic state the activity of these enzymes is reduced by almost 50 to 59% from normal levels. Insulin treatment restored their activities to normal levels. The diminished supply of NADPH in diabetic interstitial tissue is not the unique factor in the control of steroidogenesis, since the availability of large amounts of exogenous NADPH in the incubations of Leydig cell did not reduce the differences in testosterone synthesis observed when compared with normal cells.     

Relationships between Leydig cell morphometry and plasma testosterone during postnatal development of the monkey, Macaca fascicularis

In neonates (0 to 3-4 months), the testis contained a mean number of 4.6 X 10(6) Leydig cells representing 4.2 % of its volume; Leydig cell cytoplasm contained 10.2 % of SER. In infants (up to 45 months), Leydig cells regressed but their number increased; their volume density did not change. Leydig cell cytoplasmic volume (454 microns3 ), which was about 2.5-fold less than in neonates (1 119 microns3 ) or adults (1 170 microns3 ), contained only 8.7% of SER. During meiosis stage (38-52 months). Leydig cell numbers and volume density did not vary but the cells reached a maximal size and an amount of SER comparable with that at birth was measured. When spermatogenesis was complete, the Leydig cells represented no more than 0.8% of testis volume, but their number and SER content were significantly increased. Except for a significant decrease when spermatogenesis was completed, Leydig cell lipid content did not change during development, and the volume density of mitochondria did not vary. The mean level of plasma testosterone was 2 ng/ml in neonates and 0.4 ng/ml in infants; it increased to 3 ng/ml during onset of meiosis and reached 10 ng/ml in adults. The profile of testosterone was positively and significantly correlated with the total volume and total number of Leydig cells (P less than 0.01 and P less than 0.02, respectively) and with changes in their cytoplasmic volume (P less than 0.001). Moreover, plasma testosterone levels were positively and significantly correlated with changes in Leydig cell SER content i.e. SER volume density and mean absolute volume per cell (P less than 0.001), total SER in the whole testis (P less than 0.01).     

Effects and interactions of LH and LHRH agonist on testicular morphology and function in hypophysectomized rats

The effects of single or combined daily treatment with an LHRH agonist and low or high doses of LH upon the testes of adult hypophysectomized rats were studied for up to 2 weeks in which changes in testicular histology, particularly the interstitial tissue, were examined by morphometry and related to functional assessment of the Leydig cells in vivo and in vitro. Compared to saline-treated controls, LHRH agonist treatment did not alter testis volume or the composition of the seminiferous epithelium or any of the interstitial tissue components although serum testosterone and in-vitro testosterone production by isolated Leydig cells were significantly reduced. With 2 micrograms LH for treatment, testis volume was increased, spermatogenesis was qualitatively normal, total Leydig cell volume was increased, serum testosterone values were initially elevated but subsequently declined and in-vitro testosterone production was enhanced. Testis volume with 20 micrograms LH treatment was unchanged compared to saline treatment, the seminiferous epithelium exhibited severe disruption but total Leydig cell volume was greatly increased due to interstitial cell hyperplasia. This group showed elevated serum testosterone concentrations and major increases in testosterone production in vitro. Treatment with LHRH agonist with either dose of LH resulted in reduced testis volume, moderate to very severe focal spermatogenic disruption and increased total Leydig cell volume although serum testosterone values and in-vitro testosterone production were markedly reduced compared to control rats. It is concluded that, in the absence of the pituitary, LHRH agonist fails to disrupt spermatogenesis and the previously described antitesticular action of LHRH agonists in intact rats is therefore dependent upon the presence of LH, which alone or in combination with LHRH agonist, may focally disrupt spermatogenesis in hypophysectomized rats whereas the Leydig cells undergo hyperplasia. The findings show that impairment of spermatogenesis is accompanied by alterations of the interstitial tissue and suggest that communication between these two compartments is involved in the regulation of testicular function.     

Effect of seasonal changes in Leydig cell number on the volume of smooth endoplasmic reticulum in Leydig cells and intratesticular testosterone content in stallions

Testes from 47 adult (4-20 years) stallions obtained in November-January (non-breeding season) and 41 adult stallions obtained in May-July (breeding season) were perfused with glutaraldehyde, placed in osmium and embedded in Epon 812. Percentage Leydig cell cytoplasm or nuclei in the testis was determined by point counting of 0.5 micron sections under bright-field microscopy. Testes from 6 randomly selected horses per season were processed for electron microscopy. The volume (ml) of SER/testis was calculated from the % SER in the cytoplasm % Leydig cell cytoplasm, and parenchymal volume. Number of Leydig cells was calculated from the % nuclei, parenchymal volume, histological correction factor, and volume of single nucleus. Intratesticular testosterone content was determined from the contralateral testis by radioimmunoassay. The volume of SER/g and testosterone/g tended to be higher in the breeding than non-breeding season. Leydig cell number/g, volume of SER/testis, testosterone/testis, and Leydig cell number/testis were significantly greater in the breeding than in the non-breeding season. Volume of SER/testis and testosterone/testis were related significantly to the cell number/testis, and SER/testis was related (P less than 0.05) to testosterone/testis. These results emphasize the importance of seasonal changes in the number of Leydig cells on the amount of SER available to produce testosterone and on testosterone content/testis in the stallion.     

Ultrastructural and hormonal changes in the pineal-testicular axis following arecoline administration in rats

Arecoline is an alkaloid of betel nut of Areca catechu. Betel nut is chewed by millions of people in the world and it causes oral and hepatic cancers in human. It has therapeutic value for the treatment of Alzheimer and schizophrenia. Arecoline has immunosuppressive, mutagenic and genotoxic effects in laboratory animals. It also affects endocrine functions. The objective of this study was to investigate the effects of arecoline on pineal-testicular axis in rats. Since pineal activity is different between day and night, the current study is undertaken in both the photophase and scotophase. The findings were evaluated by ultrastructural and hormonal studies of pineal and testicular Leydig cells, with quantitations of fructose and sialic acid of sex accessories. Arecoline treatment (10 mg/kg body weight daily for 10 days) caused suppression of pineal activity at ultrastructural level by showing dilatation of the cisternae of the rough endoplasmic reticulum (RER), large autophagosome-like bodies with swollen mitochondrial cristae, numerous lysosomes, degenerated synaptic ribbons and reduced number of synaptic-like microvesicles. Moreover, pineal and serum N-acetylserotonin and melatonin levels were decreased with increased serotonin levels in both the gland and serum. In contrast, testicular Leydig cell activity was stimulated with abundance of smooth endoplasmic reticulum (SER), electron-dense core vesicles and vacuolated secretory vesicles, and increased testosterone level in the arecoline recipients. Consequently, the testosterone target, like prostate, was ultrastructurally stimulated with abundance of RER and accumulation of secretory vesicles. Fructose and sialic acid concentrations were also significantly increased respectively in the coagulating gland and seminal vesicle. These results were more significant in the scotophase than the photophase. The findings suggest that arecoline inhibits pineal activity, but stimulates testicular function (testosterone level) and its target organs presumably via muscarinic cholinergic receptor in rats.     

Chronic corticosterone treatment impairs Leydig cell 11beta-hydroxysteroid dehydrogenase activity and LH-stimulated testosterone production.

The effects of excess corticosterone on luteinizing hormone (LH)-stimulated Leydig cell testosterone production and activity of 11beta-HSD was studied. Adult male rats (200-250 g body weight) were treated with corticosterone-21-acetate (2 mg/100 g body weight, i.m., twice daily) for 15 days. Another set of rats was treated with corticosterone (dose as above) plus LH (ovine LH 100 microg/kg body weight, s.c., daily) for 15 days. Corticosterone administration significantly increased serum and testicular interstitial fluid (TIF) corticosterone but decreased testosterone levels. Administration of LH with corticosterone partially prevented the decrease in serum and TIF testosterone. The oxidative activity of 11beta-hydroxysteroid dehydrogenase (11beta-HSD) was significantly decreased in Leydig cells of rats treated with corticosterone alone and in combination with LH. The direct effect of corticosterone on Leydig cell steroidogenic potency was also studied in vitro. Addition of corticosterone to Leydig cell culture showed a dose dependent effect on LH-stimulated testosterone production. Corticosterone at 50 and 100 ng/ml did not alter LH-stimulated testosterone production, but at high doses (200-400 ng/ml), decreased basal and LH-stimulated testosterone production. Basal and LH-stimulated cAMP production was not altered by corticosterone in vitro. It is concluded from the present study that elevated levels of corticosterone decreased the oxidative activity of 11beta-HSD and thus resulting in impaired Leydig cell steroidogenesis and the inhibitory effects of corticosterone on testosterone production appear to be mediated through inhibition of LH signal transduction at post-cAMP level.     

Morphometry of fetal Leydig cells in the monkey (Macaca fascicularis), correlation with plasma testosterone

The evolution of Leydig cells in Macaca fascicularis fetuses was followed throughout gestation (50-150 d) by morphometric procedures (volume densities of: cells, SER, mitochondria and lipid droplets). Testosterone from umbilical artery plasma was radioimmunoassayed starting on day 57. After predifferentiation and differentiation phases, Leydig cells entered the maturity phase (57-66 d), they occupied 19% of testicular volume, SER and lipid droplets represented 19% and 5% respectively of cytoplasmic volume. Then Leydig cells regressed dramatically (involution phase I: 66-83 d), their volume density decreased to 8%, that of SER to 12% whereas lipids doubled. Leydig cell volume density diminished to 5% during the second half of gestation (involution phase II), but their ultrastructure was not significantly altered. High plasma testosterone level (2.4 ng/ml) was observed during the maturity phase of Leydig cells, decline of testosterone occurred during involution phases I and II (1.13 and 0.58 ng/ml respectively). Its was shown that from day 57 to the end of fetal development the evolution of the plasma testosterone level correlated with the Leydig cell volume density and the SER volume density.     

Role of insulin on exercise-induced GLUT-4 protein expression and glycogen supercompensation in rat skeletal muscle.

The purpose of this study was to investigate the role of insulin on skeletal muscle GLUT-4 protein expression and glycogen storage after postexercise carbohydrate supplementation. Male Sprague-Dawley rats were randomly assigned to one of six treatment groups: sedentary control (Con), Con with streptozocin (Stz/C), immediately postexercise (Ex0), Ex0 with Stz (Stz/Ex0), 5-h postexercise (Ex5), and Ex5 with Stz (Stz/Ex5). Rats were exercised by swimming (2 bouts of 3 h) and carbohydrate supplemented immediately after each exercise session by glucose intubation (1 ml of a 50% wt/vol). Stz was administered 72-h before exercise, which resulted in hyperglycemia and elimination of the insulin response to the carbohydrate supplement. GLUT-4 protein of Ex0 rats was 30% above Con in fast-twitch (FT) red and 21% above Con in FT white muscle. In Ex5, GLUT-4 protein was 52% above Con in FT red and 47% above Con in FT white muscle. Muscle glycogen in FT red and white muscle was also increased above Con in Ex5 rats. Neither GLUT-4 protein nor muscle glycogen was increased above Con in Stz/Ex0 or Stz/Ex5 rats. GLUT-4 mRNA in FT red muscle of Ex0 rats was 61% above Con but only 33% above Con in Ex5 rats. GLUT-4 mRNA in FT red muscle of Stz/C and Stz/Ex0 rats was similar but significantly elevated in Ex5/Stz rats. These results suggest that insulin is essential for the increase in GLUT-4 protein expression following postexercise carbohydrate supplementation.     

Fibroblast growth factor homologous factor 1 stimulates Leydig cell regeneration from stem cells in male rats

Fibroblast growth factor homologous factor 1 (FHF1) is an intracellular protein that does not bind to cell surface fibroblast growth factor receptor. Here, we report that FHF1 is abundantly present in Leydig cells with up‐regulation during its development. Adult male Sprague Dawley rats were intraperitoneally injected with 75 mg/kg ethane dimethane sulphonate (EDS) to ablate Leydig cells to initiate their regeneration. Then, rats daily received intratesticular injection of FHF1 (0, 10 and 100 ng/testis) from post‐EDS day 14 for 14 days. FHF1 increased serum testosterone levels without affecting the levels of luteinizing hormone and follicle‐stimulating hormone. FHF1 increased the cell number staining with HSD11B1, a biomarker for Leydig cells at the advanced stage, without affecting the cell number staining with CYP11A1, a biomarker for all Leydig cells. FHF1 did not affect PCNA‐labelling index in Leydig cells. FHF1 increased Leydig cell mRNA (Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, Insl3, Nr5a1 and Hsd11b1) and their protein levels in vivo. FHF1 increased preadipocyte biomarker Dlk1 mRNA level and decreased fully differentiated adipocyte biomarker (Fabp4 and Lpl) mRNA and their protein levels. In conclusion, FHF1 promotes Leydig cell regeneration from stem cells while inhibiting the differentiation of preadipocyte/stem cells into adipocytes in EDS‐treated testis.     

Chronic administration of corticosterone impairs LH signal transduction and steroidogenesis in rat Leydig cells

The mechanism involved in the inhibitory actions of chronic corticosterone treatment on Leydig cell steroidogenesis was studied in adult Wistar rats. Rats were treated with corticosterone-21-acetate (2 mg/100 g body weight, i.m., twice daily) for 15 days and another set of rats was treated with corticosterone plus ovine luteinizing hormone (oLH) (100 microg/kg body weight, s.c., daily) for 15 days. Chronic treatment with corticosterone increased serum corticosterone but decreased serum LH, testosterone, estradiol and testicular interstitial fluid (TIF) testosterone and estradiol concentrations. Administration of LH with corticosterone partially prevented the decrease in serum and TIF testosterone and estradiol. Leydig cell LH receptor number, basal and LH-stimulated cAMP production were diminished by corticosterone treatment which remained at control level in the corticosterone plus LH treated rats. Activities of steroidogenic enzymes, 3beta- and 17beta-hydroxysteroid dehydrogenase (3beta-HSD and 17beta-HSD) were significantly decreased in corticosterone treated rats. LH plus corticosterone treatment did not affect 3beta-HSD activity but decreased 17beta-HSD activity, indicating a direct inhibitory effect of excess corticosterone on Leydig cell testosterone synthesis. The indirect effect of corticosterone, thus, assume to be mediated through lower LH which regulates the activity of 3beta-HSD. Basal, LH and cAMP-stimulated testosterone production by Leydig cells of corticosterone and corticosterone plus LH treated rats were decreased compared to control suggesting the deleterious effect of excess corticosterone on LH signal transduction and thus steroidogenesis.     

Stress hormone and male reproductive function

The Leydig cell is the primary source of testosterone in males. Levels of testosterone in circulation are determined by the steroidogenic capacities of individual Leydig cells and the total numbers of Leydig cells per testis. Stress-induced increases in serum glucocorticoid concentrations inhibit testosterone-biosynthetic enzyme activity, leading to decreased rates of testosterone secretion. It is unclear, however, whether the excessive glucocorticoid stimulation also affects total Leydig cell numbers through induction of apoptosis and thereby contributes to the stress-induced suppression of androgen levels. Exposure of Leydig cells to high concentrations of corticosterone (CORT, the endogenously secreted glucocorticoid in rodents) increases their frequency of apoptosis. Studies of immobilization stress indicate that stress-induced increases in CORT are directly responsible for Leydig cell apoptosis. Access to glucocorticoid receptors in Leydig cells is modulated by oxidative inactivation of glucocorticoid by 11β-hydroxysteroid dehydrogenase (11βHSD). Under basal levels of glucocorticoid, sufficient levels of glucocorticoid metabolism occur and there is likely to be minimal binding of the glucocorticoid receptor. We have established that Leydig cells express type 1 11βHSD, an oxidoreductase, and type 2, a unidirectional oxidase. Generation of redox potential through synthesis of the enzyme cofactor NADPH, a byproduct of glucocorticoid metabolism by 11βHSD-1, may potentiate testosterone biosynthesis, as NADPH is the cofactor used by steroidogenic enzymes such as type 3 17β-hydroxysteroid dehydrogenase. In this scenario, inhibition of steroidogenesis will only occur under stressful conditions when high input amounts of CORT exceed the capacity of oxidative inaction by 11βHSD. Changes in autonomic catecholaminergic activity may contribute to suppressed Leydig cell function during stress, and may explain the rapid onset of inhibition. However, recent analysis of glucocorticoid action in Leydig cells indicates the presence of a fast, non-genomic pathway that will merit further investigation.     

Hypoglycemic effect of polysaccharides produced by submerged mycelial culture of Laetiporus sulphureus on streptozotocininduced diabetic rats

The hypoglycemic effect of the crude extracellular polysaccharides (EPS) produced from submerged mycelial culture of an edible mushroom Laetiporus sulphureus var. miniatus in streptozotocin (STZ)-induced diabetic rat was investigated. Hypoglycemic effect of EPS was evaluated in STZ-induced diabetic rats, and its possible mechanism was suggested by the results of western blot analysis and immunohistochemical staining. The results revealed that orally administrated EPS, when given 48 h after STZ treatment exhibited an excellent hypoglycemic effect, lowering the average plasma glucose level in EPS-fed rats to 43.5% of STZ-treated rats. The plasma levels of total cholesterol and triglyceride were significantly increased upon STZ treatment and they were markedly reduced by oral administration of EPS to near-normal levels. The results of immunohistochemical staining of the pancreatic tissues showed that EPS treatment considerably increased the insulin antigenesity of diabetic islet β-cells, suggesting the possibility of β-cell proliferation or regeneration by EPS therapy. Moreover, immunoblotting study revealed that protein levels of iNOS was increased and SOD2, catalase, GPx were significantly increased after EPS treatments, suggesting alleviated oxidative stress mediated by STZ. Orally administrated EPS exhibited considerable hypoglycemic effect in STZ-induced diabetic rats and that these EPS may be useful for the management of diabetes mellitus.     

Effects of corticosterone deficiency and its replacement on Leydig cell steroidogenesis

Clinical and experimental studies have shown the adverse effects of glucocorticoid deficiency/metyrapone treatment on testicular Leydig cell testosterone production. However, molecular mechanisms that underlie the effects of glucocorticoid deficiency on Leydig cell steroidogenesis are not yet determined. Therefore, the present study was designed to assess the mechanism of this phenomenon. Following metyrapone-induced corticosterone deficiency, serum testosterone, and Leydig cell 14C-glucose oxidation were decreased. StAR mRNA and protein levels were significantly increased in Leydig cells of corticosterone-deficient animals. mRNA levels and the specific activities of P(450)scc and 17beta-HSD were decreased by corticosterone deficiency, whereas the activity and mRNA of 3beta-HSD were increased. Simultaneous administration of corticosterone prevented its deficiency-induced changes in Leydig cells. Our results show that metyrapone-induced corticosterone deficiency impairs Leydig cell testosterone production by decreasing the activities of steroidogenic enzymes and their mRNA expression and glucose oxidation.     

Regulation by dexamethasone of the 3 beta-hydroxysteroid dehydrogenase activity in adult rat Leydig cells.

The effect of long-term in vitro treatment with dexamethasone, insulin and/or LH on the 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity and the testosterone level was examined in cultures of Leydig cells from adult rats. A rapid and simple method for measuring the 3 beta-HSD activity has been developed, in which the NADH, generated by 3 beta-HSD, reduced nitroblue tetrazolium to a product with absorption maximum at 560 nm. Km for the reaction was 8.1 microM and Vmax was 12.7 nmol/min x mg protein. Addition of 0.1 or 1 microM dexamethasone for 44 h decreased the 3 beta-HSD activity to 83% and the basal testosterone level to 64% of control value after 22 and 44 h of culture. Addition of 1 nM insulin inhibited the 3 beta-HSD activity to 90% after 44 h of culture, whereas the testosterone level was increased after 3 h. Addition of 0.1 ng/ml LH did not affect the 3 beta-HSD activity in Leydig cells from adult rats. Concomitant treatment of the cells with dexamethasone and insulin inhibited the 3 beta-HSD activity to 74%, indicating an additive effect, whereas no additive effect on the testosterone level was observed. The results demonstrate that the 3 beta-HSD activity can be measured in a rapid and reliable way by measuring the reduction of nitroblue tetrazolium. Furthermore, the results suggest that dexamethasone acts on 3 beta-HSD through a mechanism different from that of insulin, as an additive effect was observed.     

Antifertility effect of Tinospora cordifolia (Willd.) stem extract in male rats

Oral administration of 70% methanolic extract of T. cordifolia stem to male rats at the dose level of 100 mg/rat/day for 60 days did not cause body weight loss but decreased the weight of testes, epididymis, seminal vesicle and ventral prostate in a significant manner. Sperm motility as well as sperm density were reduced significantly which resulted in reduction of male fertility by 100%. The stem extract brought about an interference with spermatogenesis. The round spermatids were decreased by 73.12%. However, the population of preleptotene and pachytene spermatocytes were decreased by 47.60% and 52.85% respectively, followed by secondary spermatocytes (48.10%). Leydig cell nuclear area and mature Leydig cell numbers were significantly reduced when compared with controls. Serum testosterone levels showed significant reduction after Tinospora extract feeding. Seminiferous tubule diameter, Leydig cell nuclear area as well as cross sectional surface area of Sertoli cells were reduced significantly when compared to controls. Biochemical parameters i.e. protein, sialic acid, glycogen contents of testes decreased significantly. Seminal vesicular fructose also depleted whereas, testicular cholesterol was elevated significantly followed by a reduction in testosterone levels. These results suggested antifertility effects of the stem extract of T. cordifolia in male rats.     

Morphometric analysis of Leydig cells in the normal rat testis

Leydig cells are thought to be the source of most, if not all, the testosterone produced by the testis. The goal of this study was to obtain quantitative information about rat Leydig cells and their organelles that might be correlated with pertinent physiological and biochemical data available either now or in the future. Morphometric analysis of Leydig cells in mature normal rats was carried out on tissue fixed by perfusion with buffered glutaraldehyde, and embedded in glycol methacrylate for light microscopy and in Epon for electron microscopy. In a whole testis, 82.4% of the volume was occupied by seminiferous tubules, 15.7% by the interstitial tissue, and 1.9% by the capsule. Leydig cells constituted 2.7% of testicular volume. Each cubic centimeter (contained approximatelyy 1 g) of rat testis contained about 22 million Leydig cells. An average Leydig cell had a volume of 1,210 micron3 and its plasma membrane had a surface area of 1,520 micron2. The smooth endoplasmic reticulum (SER), the most prominent organelle in Leydig cells and a major site of steroidogenic enzymes, had a surface area of approximately 10,500 micron2/cell, which is 6.9 times that of the plasma membrane and is 60% of the total membrane area of the cell. The total surface area of Leydig SER per cubic centimeter of testis tissue is approximately 2,300 cm2 or 0.23 m2. There were 3.0 mg of Leydig mitochondria in 1 g of testis tissue. The average Leydig cell contained approximately 622 mitochondria, measuring on the average 0.35 micron in diameter and 2.40 micron in length. The mitochondrial inner membrane (including cristae), another important site of steroidogenic enzymes, had a surface area of 2,920 micron2/cell, which is 1.9 times that of the plasma membrane. There were 644 cm2 of inner mitochondrial membrane/cm3 of testis tissue. These morphometric results can be correlated with published data on the rate of testosterone secretion to show that an average Leydig cell secretes approximately 0.44 pg of testosterone/d or 10,600 molecules of testosterone/s. The rate of testosterone production by each square centimeter of SER is 4.2 ng/d or 101 million molecules/s: the corresponding rate for each square centimeter of mitochondrial inner membrane is 15 ng testosterone/d or 362 million molecules/s.     

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 potential

We tested the possibility of using LH and thyroxine (T(4)) 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 microg/day), T(4) (5 microg/day), and LH+T(4) (24+5 microg/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 T(4) and LH+T(4), 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+T(4) restored it to the 3M level, and T(4) 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 T(4) enhanced it to the 12M level, and LH+T(4) 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+T(4) treatment and above the 19M (control) values with T(4) treatment; the latter values were lower than the 3M level. Serum T(4) and tri-iodothyronine (T(3)) 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, T(4) and T(3) 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 T(4) significantly raised these values above the 19M control levels, but they were still lower than the 3M through 12M levels. Additionally, LH+T(4) 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-microg dose of LH+T(4) 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 T(4) doses in the LH+T(4) treatment.     

The effects of insulin replacement and withdrawal on hepatic ultrastructure and biochemistry

This study examines the early hepatic biochemical and ultrastructural responses to insulin replacement in streptozotocin-diabetic rats and insulin withdrawal from insulin-maintained diabetic rats. Insulin administration rapidly lowered plasma glucose and the elevated glucose-6-phosphatase (G-6-Pase) specific activity of the diabetic rats. However, hepatic glycogen did not increase until after 3 hr of insulin treatment. Hepatic ultrastructure responded to insulin replacement after the decline in glucose and G-6-Pase. This was seen in periportal hepatocytes as a reduction in the close association between smooth endoplasmic reticulum (SER) and glycogen particles in the diabetic animals. The treated rats showed hepatic SER restricted to the periphery of glycogen masses, as is characteristic of these cells from normal rats, in many cells by 6 hr and all cells by 18 hr. Insulin withdrawal from insulin-treated diabetic rats elicited nearly a total reversal of the above events. Plasma insulin declined to a value half that of the normal rats by 6 hr after withdrawal; concurrently, plasma glucose rose sharply to hyperglycemic values as hepatic glycogen content dropped. Following the rise in plasma glucose and fall in glycogen content, G-6-Pase specific activity increased and by 16 hr reached the high values characteristic of the diabetic animal. Hepatic ultrastructure was also changed as evidenced by an intrusion of elements of the SER into the dense glycogen masses; the result was dispersed glycogen closely associated with SER as seen in the diabetic animal. It is concluded that the hepatic response to insulin replacement in diabetic animals and diabetic onset in insulin-withdrawn animals is rapid and occurs through defined stages.