Indunil N. Pathirana

Insulin-like peptide 3 stimulates testosterone secretion in mouse Leydig cells via cAMP pathway.

Testicular Leydig cells secrete insulin-like peptide 3 (INSL3) and express its receptor, RXFP2. However, the effects of INSL3 on endocrine function of Leydig cells are unknown. The present study examines the effects of INSL3 on mouse Leydig cells taking testosterone and cAMP secretions as endpoints. Leydig cells were isolated from testicular interstitial cells obtained from 8-week-old male mice. Cells were then plated in the presence or absence of mouse, human, canine or bovine INSL3 (0-100 ng/ml) for 18 h in multiwell-plates (96 wells) in different cell densities (2500, 5000, 10,000 or 20,000 cells per well). The effects of bovine INSL3 (100 ng/ml) on testosterone secretion by Leydig cells were examined in the presence or absence of, an adenylate cyclase inhibitor, SQ 22536 (1μM) or INSL3 antagonist (bovine and human; 100 ng/ml). Testosterone and cAMP in spent medium were measured by enzyme immunoassay. All INSL3 species tested significantly stimulated the testosterone secretion in Leydig cells, and the maximum stimulation was observed with 100 ng/ml bovine INSL3 at the lowest Leydig cell density (2500 cells per well). Moreover, bovine INSL3 (100 ng/ml) significantly stimulated the cAMP production from Leydig cells maximally at 1h, and remained significantly elevated even at 18 h. SQ 22536 and INSL3 antagonists (bovine and human) significantly reduced INSL3-stimulated testosterone secretion from Leydig cells. Taken together, stimulatory effects of INSL3 on testosterone secretion in Leydig cells are exerted via the activation of cAMP, suggesting a new autocrine function of INSL3 in males.

Changes in plasma concentrations of insulin-like peptide 3 and testosterone from birth to pubertal age in beef bulls.

The objectives were to: (1) develop an enzyme immunoassay (EIA) for insulin-like peptide 3 (INSL3) or relaxin-like factor (RLF) in bovine plasma; (2) investigate changes of plasma INSL3 concentrations from birth to pubertal age of beef bulls; and (3) compare changes in plasma concentrations of INSL3, testosterone, and LH. Plasma samples were collected from beef bull calves (n = 15) at birth (0 d) and at 28, 56, and 84 d after birth. Furthermore, in beef bulls around pubertal age (n = 26; age range 3 to 22 mo), plasma samples were collected at 1 to 4 mo intervals. Plasma INSL3 concentrations increased (P < 0.05) from 0 to 28, 28 to 56, and from 56 to 84 d of age. Plasma testosterone concentrations increased (P < 0.001) from 0 to 28 d, and from 28 to 56 d, but did not change from 56 to 84 d. For bulls around pubertal age, plasma INSL3 concentrations did not change from the prepubertal phase (3 to 6 mo) to the early pubertal phase (6 to 12 mo), but increased (P < 0.05) from the early to late pubertal phases (12 to 18 mo), and from the late pubertal to postpubertal phases (18 to 22 mo). Plasma testosterone concentrations increased from the prepubertal to early pubertal phases (P < 0.001), but did not change thereafter. Plasma LH concentrations did not change from 0 d to 84 d, but decreased (P < 0.001) from prepubertal to early pubertal phase, with no significant change thereafter. Plasma INSL3 concentrations increased during the first 3 mo of life and throughout the pubertal age in beef bulls. There were similar dynamic patterns for INSL3 and testosterone during the first 3 mo of life, but patterns subsequently diverged in bulls around pubertal ages.

Plasma insulin-like peptide 3 and testosterone concentrations in male dogs: Changes with age and effects of cryptorchidism

The objectives were to: (1) develop a time-resolved fluorescence immunoassay (TRFIA) to measure insulin-like peptide 3 (INSL3) in canine plasma; (2) investigate changes of plasma concentrations of INSL3 and testosterone with age in normal male dogs; and (3) compare hormonal concentrations among cryptorchid, normal, and castrated dogs to evaluate endocrine function of the Leydig cell component in retained testes. Blood samples were taken from normal male dogs from prepubertal age to advanced age (4 mo to 14 y, n = 89), and from unilateral cryptorchid (n = 31), bilateral cryptorchid (n = 7), and castrated dogs (n = 3). Canine plasma INSL3 was measured with a newly developed TRFIA. The minimum detection limit of the INSL3 assay was 0.02 ng/ml and the detection range was 0.02 to 20 ng/ml. Plasma INSL3 concentrations increased (P < 0.05) from prepubertal age (4-6 mo) to pubertal age (6-12 mo), and then declined (P < 0.05) from pubertal age to post-pubertal age (1-5 y), reaching a plateau. Plasma testosterone concentrations increased (P < 0.0001) dramatically from prepubertal to pubertal ages, and then seemed to plateau. Concentrations of both INSL3 and testosterone were lower (P < 0.0001 for each) in bilateral cryptorchid dogs than in normal and unilateral cryptorchid dogs. The INSL3 (range: 0.05-0.43 ng/ml) and testosterone (range: 0.10-0.94 ng/ml) concentrations were readily detected in bilateral cryptorchids, but not in castrated dogs (INSL3 < 0.02 ng/ml; testosterone < 0.04 ng/ml). In conclusion, plasma INSL3 concentrations in male dogs measured by a newly developed TRFIA had a transient surge at a pubertal age, whereas testosterone did not. Lower plasma concentrations of INSL3 and testosterone in bilateral cryptorchid dogs suggest impaired endocrine functions of Leydig cell component in paired retained testes. Therefore, peripheral plasma INSL3 and testosterone concentrations have potential diagnostic value in predicting the presence of bilaterally retained testes in male dogs.

In vitro effects of estradiol-17β, monobutyl phthalate and mono-(2-ethylhexyl) phthalate on the secretion of testosterone and insulin-like peptide 3 by interstitial cells of scrotal and retained testes in dogs

The objective was to determine the effects of estradiol-17β, monobutyl phthalate (MBP) and mono-(2-ethylhexyl) phthalate (MEHP) on testosterone and insulin-like peptide 3 (INSL3) secretions in cultured testicular interstitial cells isolated (enzymatic dispersion) from scrotal and retained testes of small-breed dogs. Suspension cultures were treated with estradiol-17β (0, 10, and 100 ng/mL), MBP (0, 0.8, and 8 mmol/L) or MEHP (0, 0.2, and 0.8 mmol/L) for 18 h, in the presence or absence of 0.1 IU/mL hCG. Testosterone (both basal and hCG-induced) and INSL3 (basal) concentrations were measured in spent medium. Effects of estradiol-17β, MBP, and MEHP on testosterone and INSL3 secretions were not affected (P > 0.15) by cell source (scrotal versus retained testis); therefore, data were combined and analyzed, and outcomes reported as percentage relative to the control. In testicular interstitial cells, basal testosterone secretion was increased (P < 0.01) by 100 ng/mL estradiol-17β (130.2 ± 10.6% of control). Among phthalates, 0.2 and 0.8 mmol/L MEHP stimulated (P < 0.01) basal testosterone secretion (135.5 ± 8.3% and 154.6 ± 12.9%, respectively). However, hCG-induced testosterone secretion was inhibited (P < 0.01) by 8 mmol/L MBP (67.7 ± 6.0%), and tended to be inhibited (P = 0.056) by 0.8 mmol/L MEHP (84.5 ± 5.6%). Basal INSL3 secretion was inhibited (P < 0.01) by 8 mmol/L MBP (73.6 ± 6.8%) and 0.8 mmol/L MEHP (76.9 ± 11.3%). In conclusion, we inferred that estradiol-17β and certain phthalate monoesters had direct effects on secretions of testosterone and INSL3 in canine testicular interstitial cells, with no significant difference between scrotal and retained testes.

Comparison of testosterone and insulin-like peptide 3 secretions in response to human chorionic gonadotropin in cultured interstitial cells from scrotal and retained testes in dogs.

Levels of testosterone and insulin-like peptide 3 (INSL3) secretions in response to different doses of human chorionic gonadotropin (hCG) in cultured interstitial cells were compared between retained and scrotal testes in dogs. Retained (n=10) and scrotal (n=9) testes were obtained from small-breed dogs. The testicular tissues were dispersed in Dulbeccos Modified Eagle Medium with Hams nutrient mixture containing 2000 PU/ml dispase II and 10% fetal bovine serum. The cells were plated with differing concentrations (0-10 IU/ml) of hCG for 18 h in multiwell-plates. Testosterone and INSL3 in the same spent medium were measured by enzyme-immunoassays (EIA). A new EIA with a reliable detection range of 0.025-5 ng/ml was developed in order to measure canine INSL3 in culture medium. Dose-dependent stimulation of testosterone by hCG was observed in the cells of both retained and scrotal testes. The incremental rate of testosterone secretion was significantly lower at 0.1, 1 and 10 IU/ml hCG in the cells of retained testes than in scrotal testes, however. INSL3 secretion was significantly stimulated at 10 IU/ml hCG relative to unstimulated controls comprising cells of scrotal testes; no such stimulation was observed in the cells of retained testes. At 10 IU/ml hCG, the incremental rate of INSL3 was significantly lower in the cells of retained testes than scrotal testes. These results suggest that LH-induced secretory testosterone and INSL3 responses are lower in the interstitial cells of retained testes than of scrotal testes. Furthermore, the high concentrations of LH may acutely stimulate INSL3 release in scrotal testes of dogs, but not in retained testes.

EFFECTS OF GONADOTROPIN-RELEASING HORMONE ANTAGONIST DEGARELIX ON MUSTH AND SERUM TESTOSTERONE CONCENTRATIONS IN ASIAN ELEPHANTS (ELEPHAS MAXIMUS)

Abstract Two male Asian elephants (bulls 1 and 2) in musth were subcutaneously injected with a long-acting gonadotropin-releasing hormone (GnRH) antagonist, degarelix acetate (240 μg/kg; total dose of 960 mg). Musth behavior (MB) and temporal gland secretions (TGS) were monitored and serum testosterone concentrations were determined. In bull 1, MB and TGS ceased on day 1 and reappeared 5.5 mo after the treatment (day 0). During the subsequent musth cycle, MB and TGS ceased on day 1 and did not appear for 4 mo. In bull 2, MB and TGS ceased at day 7 after the treatment. Musth behavior and TGS recurred on Day 11 and continued for 1 wk, then disappeared for 8 mo. Serum testosterone concentrations decreased (P < 0.05) in all occasions from day 0 (29.8 ± 15.8 ng/ml; mean ± SEM) to day 1 (2.2 ± 1.1 ng/ml), suggesting a sudden drop in circulating testosterone in musth elephants after the GnRH-antagonist treatment.