Tom O. Abney

The potential roles of estrogens in regulating Leydig cell development and function: a review.

It is generally agreed that estrogens, principally estradiol-17beta, are synthesized by and act in the testis of mammals, including humans. The site of estradiol synthesis in the testis is generally believed to begin in the Sertoli cell and switch to the Leydig cell during neonatal development where a gonadotropin-regulated aromatase is present. Numerous studies suggest that the primary target cell of estradiol in the testis at all ages is the Leydig cell. In fact, the Leydig cell is known to possess an estrogen receptor that binds estradiol in the classic manner. The mechanism of estradiol action and the role of its receptor in the testis, however, remain unresolved. In Leydig cells, estradiol appears to induce several alterations that are dependent in large part on the developmental stage of the Leydig cell. In the fetal and neonatal testes, estradiol appears to block the ontogenic development of Leydig cells from precursor cells. There is also evidence that estradiol similarly blocks the regeneration of Leydig cells in the testis of mature, ethane dimethylsulfonate-treated animals. Evidence indicates that the precursor cell possesses high levels of estrogen receptors relative to that of the Leydig cell. It is postulated that estradiol is a paracrine factor involved in regulating the interstitial population of Leydig cells. Evidence also indicates that estradiol acts directly in the mature testis to block androgen production. It appears to do so by inhibiting the activities of several steroidogenic enzymes involved in testosterone synthesis. Although the more conventional receptor-mediated mode of action is feasible, several studies have suggested that this action might entail direct competitive inhibition of key steroidogenic enzymes by estradiol. In summary, the net biologic effect of estradiol in the testis appears to be inhibition of androgen production, either by limiting development and growth of the Leydig cell population or through direct action in the Leydig cell.

The direct effect of 17β-estradiol on lh-stimulated testosterone production in hypophysectomized rats

Hypophysectomized mature (60d) Sprague-Dawley rats were utilized to determine the direct effect of 17β-estradiol on LH-stimulated testosterone production by the testis, separate from secondary influences mediated through altered anterior pituitary function. Hypophysectomized (hypox) animals received daily s.c. injections of a maximal stimulatory dose of LH (20 μg/100 g BW) plus varying doses of 17β-estradiol (0, 500 ng, 5 and 50 μg) for 5 days beginning on the fourth day post-surgery. LH administration led to a significant increase in serum testosterone levels (4.42 ± 0.57 ng/ml) above hypox controls (0.19 ± 0.06 ng/ml). Significant decreases in serum testosterone levels below that of the LH-stimulated hypox group were observed at the 5 μg (P < 0.05) and 50 μg (P < 0.01) doses of 17β-estradiol; testosterone concentrations of 2.8 ± 0.05 and 1.8 ± 0.23 ng/ml serum were obtained for these doses of 17β-estradiol, respectively. Testosterone production in vitro by testicular minces exhibited a similar pattern to that described for the in vivo serum levels. A value of 9.67 ± 1.29ng testosterone/100 mg tissue/2h incubation in vitro was obtained for the LH-stimulated hypox groups. Significant (P < 0.05) decreases in production yielding values of 3.14 ± 0.73 and 0.63 ± 0.57ng/100mg tissue/2h were observed in the 5 and 50 μg doses of estradiol, respectively. Post-treatment serum levels of estradiol were measured in all groups; intact animals exhibited a level of 35.9 pg/ml while estradiol treatment resulted in increased concentrations in the hypox groups. Values of 117, 291, and 3197 pg estradiol/ml serum were obtained with the respective increasing doses of estradiol. Testicular cytoplasmic estrogen receptor levels, assessed in an in vitro system, were shown to be increasingly depleted (78, 87 and 97%) by the increasing doses of 500 ng, 5 and 50 μg of exogenous estradiol. These results suggest a direct inhibitory effect of 17β-estradiol on LH-stimulated testosterone production in the testis. This direct effect is reflected by decreased serum testosterone levels in vivo and decreased testosterone production in vitro. These effects are accompanied by a depletion of the testicular cytoplasmic estrogen receptor.

Gonadotropin regulation of Leydig cell DNA synthesis

Adult male rats were injected s.c. with either saline, 100 IU hCG, 100 micrograms FSH, 50 micrograms LH, 100 micrograms PRL, 50 micrograms estradiol-17 beta, 500 micrograms or 10 mg testosterone; 50 micrograms estradiol-17 beta; animals were sacrificed at 12-120 h post-injection. Collagenase-dispersed interstitial cells (150-200 X 10(6) cells/2 ml) were incubated in vitro with 10 microCi [3H-methyl]thymidine for 1 h at 32 degrees C. Centrifugation of the cells on discontinuous 11-27% metrizamide gradients revealed thymidine incorporation in the regions of population I and II Leydig cells. A significant increase in thymidine incorporation into DNA after treatment with either hCG or LH was first detectable at 48 h, was equivalent to control values at 72 h and was again significantly increased at 96 h in population I and at 120 h in population II cells. [3H]Thymidine incorporation at 48 h, expressed as dpm/10(6) cells, was 2205 +/- 432 and 4119 +/- 929 vs. 16473 +/- 3795 and 11648 +/- 3427 for control and hCG-treated population I and II cells, respectively. Addition of 20 mM hydroxyurea suppressed [3H]thymidine incorporation, 97% and 96% in hCG-treated population I and II cells, respectively. Autoradiographic analyses revealed that nuclei from control and 48 h hCG-treated population I and II cells exhibited 1.2% and 2.3% vs. 7% and 6.8% silver grains, respectively. PRL had no influence on LH/hCG-enhanced DNA synthesis; however, estradiol-17 beta administration for 48 h dramatically suppressed thymidine incorporation. Population I Leydig cells exhibited a higher level of LH/hCG-stimulated DNA synthesis compared to population II cells.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of reduced o2 and antioxidants on steroidogenic capacity of cultured rat leydig cells

We have examined the effects of reduced O2 tension and the antioxidant dimethylsulfoxide (DMSO) to determine if O2-derived free radicals are the cause of decreased steroidogenic capacity (testosterone and progesterone production) of cultured rat Leydig cells. Rat Leydig cells were initially cultured under standard conditions of 5% CO2, 95% air (19% O2) with or without DMSO. Addition of DMSO resulted in increased basal testosterone production on days 2, 3 and 4 of culture. hCG (10 mIU)-stimulated testosterone secretion was 2-3 times greater on days 2 and 3 in the presence of DMSO. Lowering the O2 concentration to 5% in the presence of DMSO resulted in even greater hCG-stimulated testosterone production on days 1 to 3. However, the effect of DMSO or low O2 and DMSO were not seen after 5 days. The reduced O2 concentration resulted in an increase in hCG (10 mIU)-stimulated progesterone synthesis throughout the culture, particularly on days 4 to 8. Also, when total steroid (progesterone and testosterone) was determined, cells cultured under reduced O2 conditions responded with increased steroid production on days 1 to 8 in comparison to controls (19% O2). These results demonstrate that lowered O2 concentration and DMSO provide a protective effect resulting in the maintenance of testosterone production and an increase in progesterone synthesis. These findings suggest that free radical-mediated damage of enzymes may result in decreased steroidogenic capacity of cultured Leydig cells.

Interstitial cell proliferation in the testis of the ethylene dimethane sulfonatetreated rat

This study was conducted to examine interstitial cell proliferation in the testis of the ethylene dimethane sulfonate (EDS)-treated rat. Initial autoradiographic studies demonstrated a peak of [3H]thymidine incorporation by interstitial cells at 2 and 4 days post-EDS treatment. Subsequent studies were designed using in vivo pulse labeling regimens in an attempt to identify interstitial cell proliferation associated with Leydig cell regeneration. Rats were injected with [3H]thymidine at days 2 and 4 post-EDS and were killed 6 hours later or at 30 days post-EDS. Although cells labeled at 2 and 4 days post-EDS appeared to undergo subsequent division, the Leydig cells visible at 30 days post-EDS were not labeled. In a second study, rats were injected with [3H]thymidine at days 10 and 20 post-EDS and were killed either 6 hours later or at 24 days post-EDS. In the 10-day post-EDS group, interstitial cells were labeled at both the 6-hour and 24-day time points; however, Leydig cells present at 24 days were not labeled. In contrast, the testes of rats that were killed at 20 days post-EDS (6 hours labeling period) contained Leydig cells that displayed grains over the nucleus, thus suggesting that Leydig cell proliferation had occurred. In addition, a high number of the Leydig cells observed at 24 days post-EDS were labeled, suggesting that they arose from divisions occurring during the 20- to 24-day post-EDS period. These studies demonstrate that interstitial cell proliferation occurs in several stages following EDS treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Fibrocystic breast disease: the significance of β-human chorionic gonadotropin and other polypeptides in breast cyst fluid

Breast cyst fluids from 118 women, aged 29 to 69 years, were analyzed by radioimmunoassays for beta-human chorionic gonadotropin (beta-hCG), luteinizing hormone (LH), follicle-stimulating hormone (FSH), prolactin (PRL), and thyroid-stimulating hormone (TSH). Blood was drawn at the same time in many cases to compare hormonal levels in serum with those in the breast cyst fluids (BCF). The levels of beta-hCG in BCF were relatively high, with a mean (+/- standard error of the mean [SEM]) of 58.9 +/- 16.8 mIU/ml; serum levels of beta-hCG were negligible. LH and TSH also were elevated in BCF compared with serum levels, exhibiting mean values (+/- SEM) of 26.7 +/- 4.3 mIU/ml and 6.4 +/- 0.44 muIU/ml, respectively. The levels of FSH and PRL in BCF were equivalent to the levels in the serum. The presence of biologically active hCG was suggested in several BCF samples using the rat ovarian hyperemia test. Samples of BCF were assessed for the capacity to stimulate Leydig cell testosterone production in vitro in the presence or absence of an anti-hLH antiserum. Testosterone production was significantly (P less than 0.05) enhanced, even in the presence of the antiserum. These data suggest that BCF contains biologically active hCG.

Alterations of Testicular Function in the Unilaterally Cryptorchid Rat

Abstract Mature male Sprague-Dawley rats were either rendered unilaterally cryptorchid or used as intact controls. At 7, 14, 21, and 28 days postsurgery, five animals from each group were sacrificed, blood samples were collected, and the cryptorchid, eutopic, and intact control testes were removed. The cryptorchid testes weights decreased markedly below the controls while the weights of the eutopic testes did not vary from those of the controls. The levels of serum LH, FSH, and testosterone (T) did not significantly differ between the two groups throughout the study. The testicular content of T, expressed as nanograms per testis, decreased in the cryptorchid testes below control values from 30.6 ± 3.3 ng at 7 days to 10.8 ± 0.3 ng at 28 days while the content of the eutopic testes increased above controls from 71.1 ± 6.0 ng at 7 days to 131.7 ± 9.8 ng at 28 days. The cytoplasmic estrogen receptor level (3H-E2 bound/mg protein) of the cryptorchid testis increased threefold above control levels at 7 days and continued to rise to a sixfold increase above control levels at 28 days; the eutopic and control E2R levels did not vary throughout the study. While these data demonstrate that the steroidogenic capacity of the cryptorchid testis was reduced, the increased E2 binding capacity indicates that the viability of the Leydig cell population was not detrimentally altered during the experimental period. These data further suggest that as a result of unilateral cryptorchidism, a compensatory change occurred in the eutopic testis resulting in an increased steroidogenic capacity in vivo.

Effects of estradiol administration in vivo on testosterone production in two populations of rat leydig cells

The effects of in vivo administration of estradiol on isolated rat testicular Leydig cells were investigated. Adult intact rats were injected s.c. with 50 μg/ 100 g B.W. of 17β-estradiol or vehicle twice daily for 2 days. Twelve hours after the last injection, collagenase dispersed interstitial cells were obtained and Leydig cells were subsequently isolated on metrizamide gradients. Two distinct peaks of specific 125I-hCG binding corresponding to population I and II Leydig cells were observed. The hCG binding profile was unaltered as a result of estradiol treatment. Although twice as much testosterone was produced in population II, the responsiveness of the two populations to hCG or dbcAMP in vitro was identical (11-to 13-fold increase). Estradiol administration in vivo resulted in a 33–48% decrease in basal and stimulated testosterone production in both populations. These data indicate for the first time that both population I and II Leydig cells are sensitive to the direct inhibitory effects of estrogens on testosterone production. This inhibitory effect was not associated with an alteration in hCG binding capacity in either population. Therefore, we conclude that no functional difference exists between the two populations of Leydig cells with respect to the action of estrogens.

Ontogenic Relationships Between Two Estrogen Binding Moieties in the Male Rat: α-Fetoprotein and the Testicular Cytoplasmic Estrogen Receptor

The estrogen binding characteristics of rat alpha-fetoprotein (AFP) and the testicular cytoplasmic estrogen receptor (E2R) and the ontogenic relationships between these two estrogen binding moieties were investigated. Sucrose gradient sedimentation analysis revealed that AFP migrated as a single 4.6S peak whereas the receptor migrated as a single peak in the 8-9S region. Scatchard analyses of the binding data demonstrated high-affinity (10(8) M-1), high-capacity (450 pmol/mg protein) binding sites for AFP-estradiol and high-affinity (10(10) M-1), low-capacity (16 fmol/mg protein) binding sites for receptor-estradiol. Estradiol binding in serum (AFP) was high at birth, declined during the first 2 weeks and fell to low levels during the fourth week of life. In contrast, the testicular E2R was not detectable before day 21, rose after day 23, and reached adult concentrations by day 35. The inverse pattern suggested a relationship between the disappearance of AFP and the appearance of the receptor. However, in vivo administration of Dexamethasone, which resulted in a precocious decrease in AFP levels, had no effect on the ontogeny of the receptor. The ontogenic patterns of these two estrogen binding moieties may determine the onset of testicular sensitivity to estrogens.

Temporal Effects of Diethylstilbestrol Administration in vivo on Testosterone Production in Leydig Cells

The temporal nature of estrogenic suppression of Leydig cell testosterone production was investigated. Adult rats were injected SC with 50 micrograms/100 g BW of DES or vehicle. Animals were sacrificed at 4, 8, or 12 h following a single injection or at 12 h following the latter of two daily injections for 1 or 2 days. Collagenase-dispersed interstitial cells were obtained, and population I and II Leydig cells were subsequently isolated on metrizamide gradients. Population I and II Leydig cells produced in vitro testosterone levels of 7.19 +/- 0.86 and 12.84 +/- 1.86 ng/10(6) cells/3 h, respectively. These levels were increased to 10- to 13-fold in the presence of hCG of dbcAMP. No significant difference was noted in the responsiveness of these two populations to the in vitro additions. DES administration in vivo for 8-48 h resulted in dramatic and significant decreases in basal and stimulated testosterone production in vitro in both populations. However, DES treatment for 4 h was relatively ineffective in blocking testosterone production in vitro. The inhibitory patterns exhibited by the two populations differed considerably. Population I displayed a uniform degree of inhibition throughout the treatment, whereas population II exhibited a more transient suppression by estrogen. Thus, population II appeared to be less sensitive to the estrogenic effects than population I at 48, 24, and 12 h of treatment. These data indicate that both population I and population II Leydig cells become sensitive to the inhibitory effects of estrogens between 4 and 8 h of in vivo treatment and suggest that certain differences exist between the two populations with respect to the temporal action of estrogens.