Androgen Biosynthetic Pathway Research Articles

Gonadotropic regulation of aromatase activity in the adult rat testis.

Aromatase activity during gonadotropin action in vivo and in vitro was examined in purified Leydig cells to further define the early effects of LH and for elucidation of the enzymatic processes involved in the development of late lesions of androgen biosynthetic pathway. Aromatase was measured by the tritiated water release method using [1 beta-3H]testosterone as substrate. The enzyme activity was proportional to the amount of cells (0.1-1.0 X 10(6] incubated, increased with the incubation time (2-60 min), was inhibited by androstatriendione (ED50, 5.0 microM), and showed a Km for testosterone of 1.69 microM. Aromatase activity was stimulated (10-20%; P less than 0.05) 1 h after treatment of rats with a single sc dose of 5 micrograms hCG. This activation preceded the late steroidogenic lesion at the site of 17 alpha-hydroxylase and 17,20-desmolase activity by 2-5 h. A RIA of improved sensitivity (0.5 pg) was developed to detect the very low cellular and secretory levels of estradiol. The testicular contents of testosterone and estradiol showed small increases (P less than 0.05) within 40 and 60 min after hCG treatment, respectively. Testicular testosterone levels reached a peak by 1 h after injection and preceded the peak level of estradiol formation by 2 h. After in vitro treatment of cultured Leydig cells with 100 ng hCG, the aromatase activity was significantly increased within 30 min (P less than 0.05) and then returned to control levels for up to 16 h of culture. A similar temporal pattern for enzyme activation was observed after treatment of cultures with 8-bromo-cAMP or forskolin (23-27% above control; P less than 0.05), while cholera toxin stimulated aromatase activity at 2 h. Net testosterone accumulation in the incubation medium increased 30 min after the hCG treatment and reached a plateau by 4 h. A small but significant increase in estradiol levels (P less than 0.05) was also observed at 30 min, remaining constant until 120 min, which was followed by a sharp rise parallel to that of testosterone. These results suggest that the estradiol-mediated desensitization of the Leydig cell observed after hCG administration is consistent with an early cAMP-dependent activation of aromatase and a further rise in estradiol formation due to increased substrate availability.

Metabolism of [4- 14c]testosterone and precursors by homogenates of rat testes after chronic LHRH-treatment

Adult male rats were injected daily for 8 days with an LHRH agonist. Twenty-four hours after the last injection testes-homogenates were incubated in the presence of a 4- 14C-labeled steroid, either progesterone, 17α-hydroxyprogesterone, dehydroepiandrosterone, androstenedione or testosterone. The activity of several enzymes involved in the androgen biosynthetic pathway was inferred from the amount of metabolites produced under these conditions. After LHRH-treatment a significant increase in the 17, 20-lyase activity was observed without any significant change in the activity of 17α-hydroxylase, 3β-hydroxysteroid dehydrogenase/Δ 5-Δ 4-isomerase and 17β-hydroxysteroid dehydrogenase. The results of the experiments indicate that the decreased testosterone secretion observed in rats after chronic LHRH-administration is not due to an inhibition of the enzyme-systems studied.

Hormonal regulation of androgen production by the Leydig cell

The control of androgen production by the Leydig cell is dependent upon the episodic secretion of hormone (LH), which is released from the anterior pituitary gland in pulses of high biological activity. This mode of episodic LH secretion supports steroidogenic enzyme activity in the testis through interaction with LH receptors and stimulation of the adenylate cyclase/protein kinase sequence, leading to phosphorylation of key intermediates in the steroid biosynthetic pathway. The plasma membrane events that are rapidly activated by the specific interaction of LH or hCG with Leydig cell receptors include increased binding of guanyl nucleotide, and stimulation of cAMP-independent, Ca 2+dependent phosphorylation of a 44,500 Mr protein, with the characteristics of the adenylate cyclase nucleotide regulatory unit. Hormonal activation of adenylate cyclase is affected by Ca 2+ with the same concentration-dependence, suggesting that nucleotide-induced phosphorylation is related to activation of the catalytic cyclase unit. In addition to the characteristic increases in pregnenolone synthesis and androgen production, gonadotropin-stimulated Leydig cells show prominent changes in LH receptor content and steroidogenic activity that modify their subsequent responses to hormonal signals. Thus, after exposure to increased LH and hCG levels in vivo and in vitro, LH receptors show an initial transient increase (up-regulation) followed by a marked decrease (down-regulation) and a prolonged depletion of LH receptor sites. Large doses of hCG cause “early” (prior to pregnenolone) and “late” steroidogenic lesions (17α-hydroxylase, 17–20 desmolase) that are independent of receptor loss. The early lesion is partly due to reduced activity of HMG CoA reductase, and is mainly attributable to the increased activity of an inhibitory protein factor that modulates the activity of cholesterol side chain cleavage enzyme in Leydig cell mitochondria. In contrast, the late steroidogenic lesion is related to the nuclear actions of E 2 produced during hormonal action. After hCG stimulation, an increase in nuclear E 2 binding was accompanied by an early rise of RNA polymerase activities within 45 min coincident with the maximal increases in circulating testosterone and estradiol levels. These events were followed by the emergence of an E 2-induced protein of Mr 27,000 at 3–6 h, and by reduction in the activity of 17α-hydroxylase/17–20 desmolase, and a decrease in microsomal cytochrome P-450. The negative effects of LH upon receptors and steroidogenic responses appear to be characteristic of the adult Leydig cell, and do not occur in the immature or fetal Leydig cell, where only up-regulation was demonstrated in vivo or in vitro. The temporal and functional nature of the steroidogenic lesions provide further insight into the intracellular control mechanisms that regulate the androgen biosynthetic pathways of the mature Leydig cell.

Preservation of normal adrenal androgen secretion in end stage renal disease

A common endocrine defect in uremia is gonadal dysfunction with decreased testosterone production. Since gonadal and adrenal tissues share androgen biosynthetic pathways, we studied the stimulated adrenal androgen response in uremic patients. In contrast to the delayed or subnormal gonadal response to hCG reported by others, the adrenal response of androgens, as well as cortisol and aldosterone, to cosyntropin stimulation was unimpaired. In summary, the secretory reserve capacity of the adrenal gland for androgens, glucocorticoids and mineralocorticoids in uremia was studied with cosyntropin stimulation and found to be well preserved.

Steroidogenesis in HCG-responsive Leydig cell tumor variants

Several steroids of the androgen biosynthetic pathway were measured in cell suspensions of two tumor variants originating from the gonadotropin-responsive murine M5480 Leydig cell tumor. In M5480A, the androgen-producing variant, testosterone and dihydrotestosterone were the main steroids produced basally; addition to HCG to the incubation medium resulted in increased testosterone but no change in dihydrotestosterone levels and the main steroid accumulating in the medium was progesterone. In M5480P, the progesterone-producing variant, progesterone and androstenedione were the main steroids produced, while testosterone and dihydrotestosterone production were negligible both basally and following stimulation with HCG. Progesterone levels increased more in M5480P than in M5480A with HCG stimulation. The steroid profiles observed in the M5480 tumor variants were contrasted with that of Leydig cell-enriched suspensions of normal mouse testes. Comparison of the ratios of progesterone to 17α-hydroxyprogesterone and of androstenedione to testosterone, in the two tumor variants with each other and with normal mouse testes, suggests the following alterations in steroidogenic enzymes activities: (a) a partial decrease in 17α-hydroxylase activity more marked in M5480P than in M5480A, (b) a decrease in 17β-hydroxysteroid dehydrogenase activity in M5480P. Finally, in the M5480A tumor variant, the absence of an increase in dihydrotestosterone levels with HCG stimulation suggests a decrease in 5α-reductase activity.

Effects of general anaesthesia and severity of surgical stress on serum LH and testosterone in males.

Significantly decreased levels of serum testosterone from the pre-anaesthesia level were found during and up to 7 days following major surgery under general anaesthesia (nitrous oxide, oxygen and halothane following induction with thiopental and succinylcholine chloride) in 18 male patients. On the other hand in the same patients, the serum lutenizing hormone (LH) increased significantly from the pre-anaesthesia level 30 min and 1 h after the beginning of anaesthesia. A slight increase in LH level was also noted on the 7th post-operative day. The determinations of serum testosterone and LH in fiberoptic bronchoscopy under the same general anaesthesia as that used in surgery or local anaesthesia in 26 male patients, revealed that the change in the serum LH during and following surgery seemed to be mainly induced by the general anaesthesia and that the rate of decrease in the serum testosterone may be related to the severity of surgical stress including the anaesthesia. The rate of increase in serum testosterone following the injection of gonadotrophin in 20 males on the 6th post-operative day was similar to that in 10 pre-operative males. The effects of pulmonary lobectomy on serum testosterone and urinary steroids were also studied in 6 males under adrenal suppression with dexamethasone. On the 6th post-operative day, the urinary aetiocholanolone plus androsterone and serum testosterone were found to be half the level of those on the pre-operative day, while the urinary 5beta-pregnane-3alpha, 17alpha, 20alpha-triol remained unchanged. These observations in human are not inconsistent with the report of Tcholakian and Eik-Nes (1971) in dogs namely that a shift in androgen biosynthetic pathway is present in the testis under surgical stress.

Influence of Major Surgical Stress on Plasma Levels of Testosterone, Luteinizing Hormone and Follicle-Stimulating Hormone in Male Patients

Effects of major surgical stress on plasma concentrations of testosterone and immunoreactive luteinizing hormone (LH) were studied in 25 male patients. Significantly decreased levels of testosterone from the pre-operative level (0.66 ± 0.03 (se) μg/100 ml) were found during the operation. On the other hand, plasma LH increased significantly from the pre-operative level [4.27 ± 0.29 (se) μg/100 ml] during the operation and reached the maximum concentration 30 min after the beginning of incision [8.28 ± 0.71 (se) μg/100 ml]. Shortly after the end of the operations, the mean concentration of LH had returned to its pre-operative level, but the mean concentration of testosterone continued to fall. No such change explainable by diurnal variation was observed in 10 male patients followed for a similar period of time prior to surgery. The patients exhibited a significant fall in LH concentration on the 2nd post-operative day [3.11 ± 0.24 (se) μg/100 ml], when plasma testosterone showed the most marked decrease [0.21 ± 0.03 (se) μg/100 ml]. Plasma levels of immunoreactive follicle-stimulating hormone examined in 9 of the 25 patients, showed no significant changes under surgical stress. These observations suggest that there may be some lack of testicular sensitivity to gonadotrophins during and following major surgery, though a shift in the androgen biosynthetic pathway of the testis may be considered.

In vitro progesterone metabolism by rat testicular tissue at different stages of development.

ABSTRACT A systematic study of progesterone metabolism in incubates of testicular tissue from rats 1 to 120 days of age was conducted. Progesterone was avidly metabolized, regardless of the age of the animal. While striking age-dependent changes in the pattern of conversion of progesterone to androgens were observed, small amounts of 17α-hydroxyprogesterone and 17β-oestradiol were formed in all incubates, regardless of age of the animal. In newborn rats the major metabolites of progesterone were testosterone (44% conversion) and androstenedione (5% conversion). Formation of these two compounds gradually diminished with age until only small amounts were formed in tissue from 20-day old rats. From this nadir, conversion to testosterone started to increase at the age of 40 days, and reached constant adult fevels by 90 days; androstenedione started to increase at 30 days. Conversely, 5α-reduced androgens, androsterone, 5α-androstanediol and isoandrosterone, were barely detectable in testicular tissue of newborn rats, gradually increased to a peak by age 20–30 days, and then declined to barely detectable levels by age 90 days and older. These findings demonstrate that testicular tissue of postnatal rats of all ages contain enzymes involved in the biosynthetic pathways from progesterone to androgens, as well as aromatizing enzymes. The physiologic significance of 5α-reductase activity during differentiation of the androgen biosynthetic pathways remains to be determined.