Acute regulation of plasma insulin-like peptide 3 concentrations by luteinizing hormone in male goats.

Recently, it was reported that in bulls secretion of insulin-like peptide 3 (INSL3) in blood occurred in a pulsatile manner and was acutely regulated by LH. In the present study, the acute regulation of plasma INSL3 and its temporal relationships with LH and testosterone were examined in six sexually matured male goats using the following experimental design. (1) After stimulating LH release by administering a GnRH analogue, blood levels of LH, INSL3, and testosterone were monitored at 15-minute intervals for 2 hours followed by hourly intervals up to 8 hours. (2) After activation of the LH receptor by hCG blood levels of INSL3 and testosterone were determine at 15-minute intervals for 2 hours, followed by hourly intervals up to 8 hours, daily intervals up to Day 8, and finally on Day 12. (3) The release of LH, INSL3, and testosterone in normal physiology was established at 15-minute intervals for an 8-hour session. Concentrations of LH, INSL3, and testosterone in plasma were measured by enzyme-immunoassays. After GnRH treatment, mean plasma concentrations of all three hormones increased (P < 0.05) dramatically from 30 minutes and remained high until 120 minutes (LH), 75 minutes (INSL3), and 4 hours (testosterone) after treatment. After hCG treatment, mean plasma INSL3 concentrations increased (P < 0.05) from 30 minutes and remained elevated until the end of sampling on Day 12. An increase (P < 0.05) in mean plasma testosterone concentrations occurred from 15 minutes and remained high until Day 6. The mean increase (maximum per pretreatment concentration) of INSL3 concentrations after administration of GnRH and hCG was lower (P < 0.01) than that of testosterone. The secretory pattern of LH, INSL3, and testosterone in the general circulation was pulsatile with a frequency of 5.5 ± 0.6, 4.7 ± 0.5, and 2.2 ± 0.5, respectively, during the 8-hour period. Twenty out of 28 (71%) of these INSL3 pulses peaked within 1 hour after a peak of an LH pulse. The mean increase (peak per basal concentration) of INSL3 pulses (2.1 ± 0.1 fold, n = 28) was lower (P < 0.01) than that of testosterone pulses (4.3 ± 2.2 fold, n = 13). In conclusion, secretion of INSL3 in blood occurred, like in bulls, in a pulsatile manner soon after LH pulses in male goats. The absolute concentrations of INSL3 in male goats were higher than that reported in other mammals. Insulin-like peptide 3 concentrations were acutely increased by endogenous and exogenous LH in male goats, but the rise of INSL3 was lower than that of testosterone.

Expression analyses of insulin-like peptide 3, RXFP2, LH receptor, and 3ß-hydroxysteroid dehydrogenase in testes of normal and cryptorchid dogs.

Insulin-like peptide 3 (INSL3) plays a key role in testicular descent in rodents, whereas in domestic animals, many aspects of the roles of INSL3 in reproductive organs after puberty are still unknown. This study was undertaken to (1) determine the quantitative changes of gene expression of testicular INSL3, its receptor (RXFP2), LH receptor, and 3ß-hydroxysteroid dehydrogenase during and after puberty in normal male dogs; (2) compare the expressions of these substances in normal and cryptorchid dogs; and (3) localize the cells expressing INSL3 in normal and retained canine testes. Testes were obtained from small-breed normal male dogs (n = 56) and cryptorchid dogs (n = 22). Normal scrotal testes from the normal dogs (normal testes), retained testes from both the unilateral and bilateral cryptorchid dogs (retained testes), and scrotal testes of the unilateral cryptorchid dogs (cryptorchid scrotal testes) were used. We measured the concentrations of these testicular messenger RNAs (mRNAs) by quantitative real-time reverse transcription polymerase chain reaction, and an enzyme immunoassay was used for measuring INSL3 peptide. Immunohistochemistry for INSL3 peptide was done in paraformaldehyde-fixed frozen testicular tissue. In the normal dogs, total amount of INSL3 mRNA per testis tended to decrease (P = 0.05) from pubertal (6-12 months) to postpubertal (1-5 years) and decreased (P < 0.01) to middle age (5-10 years), but total amount of INSL3 peptide per testis did not change among age groups. Concentrations of INSL3 mRNA were higher (P < 0.01) in retained testes than those in the normal testes and cryptorchid scrotal testes, and similar differences were observed for INSL3 peptide. Reversely, total amounts of INSL3 mRNA and peptide per retained testis were lower (P < 0.01) than those per normal testis because of smaller weight of retained testes. Concentrations and total amount of RXFP2 mRNA in the retained testes were almost nil and lower (P < 0.01) than those in the normal testes and in the cryptorchid scrotal testes. Total amount of LH receptor mRNA per retained testis was lower (P < 0.01) than that per normal testis. The immunohistochemical analysis revealed that INSL3 was expressed only in Leydig cells of both the normal and retained canine testes. These results suggest that INSL3 in retained testes of cryptorchid dogs is substantially expressed per unit-weight basis but may be produced with lower amount as a whole testis. Also, this study provides findings that RXFP2 gene is expressed scarcely in the retained testes but normally in cryptorchid scrotal testes.

Plasma insulin-like peptide 3 concentrations are acutely regulated by luteinizing hormone in pubertal Japanese Black beef bulls.

Insulin-like peptide 3 (INSL3) is a major secretory product of testicular Leydig cells. The mechanism of acute regulation of INSL3 secretion is still unknown. The present study was undertaken in pubertal beef bulls to (1) determine the temporal relationship of pulsatile secretion among LH, INSL3, and testosterone and (2) monitor acute regulation of INSL3 secretion by LH using GnRH analogue and hCG. Blood samples were collected from Japanese Black beef bulls (N = 6) at 15-minute intervals for 8 hours. Moreover, blood samples were collected at -0.5, 0, 1, 2, 3, 4, 5, and 6 hours after GnRH treatment and -0.5, 0, 2, 4, and 8 hours on the day of treatment (Day 0), and Days 1, 2, 4, 8, and 12 after hCG treatment. Concentrations of LH, INSL3, and testosterone determined by EIAs indicated that secretion in the general circulation was pulsatile. The frequency of LH, INSL3, and testosterone pulses was 4.7 ± 0.9, 3.8 ± 0.2, and 1.0 ± 0.0, respectively, during the 8-hour period. Seventy percent of these INSL3 pulses peaked within 1 hour after a peak of an LH pulse had occurred. The mean increase (peak per basal concentration) of testosterone pulses was higher (P < 0.001) than that of INSL3 pulses. After GnRH treatment, LH concentrations increased (P < 0.01) dramatically 1 hour after treatment and remained high (P < 0.05) until the end of sampling, whereas an elevated (P < 0.05) INSL3 concentration occurred at 1, 2, 5, and 6 hours after treatment. Testosterone concentrations increased (P < 0.01) 1 hour after the treatment and remained high until the end of sampling. After hCG treatment, an increase of INSL3 concentration occurred at 2 and 4 hours, and Days 2, 4, and 8 after treatment (P < 0.05), whereas in case of testosterone, concentrations remained high (P < 0.01) until Day 8 after treatment. The increase (maximum per pretreatment concentration) of INSL3 concentrations after injecting GnRH or hCG was much lower (P < 0.001) than that of testosterone. In conclusion, secretion of INSL3 in blood of bulls occurred in a pulsatile manner. We inferred an acute regulation of INSL3 by LH in bulls because INSL3 concentrations increased immediately after endogenous and exogenous LH stimulation. The increase of INSL3 concentrations by LH was much lower than that of testosterone in bulls.

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.

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.

In vitro effects of estradiol-17ß, 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-17ß, 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-17ß (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-17ß, 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-17ß (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-17ß 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.