Ir-inhibin Levels Research Articles

Secretion pattern of immunoreactive inhibin in men

Chronological changes in serum concentrations of inhibin, a gonadal glycoprotein hormone, were studied in healthy male volunteers (age 24-27 years). Secretion profiles of immunoreactive inhibin (ir-inhibin) were compared with those of luteinizing hormone (LH), follicle-stimulating hormone (FSH) and testosterone. Blood samples were collected every 15 min for 24 h. Serum inhibin concentrations were measured by a two-site immunoenzymatic assay with antibodies raised against distinct epitopes of the recombinant 1-32 amino acids of the alpha-subunit of human inhibin. The normal range for men was 0.79-3.1 U/l x 10(-3), the sensitivity of the assay was 0.1 U/l x 10(-3) (cv: within-assay, 6.8%; between-assay, 8.2%). Luteinizing hormone and FSH were measured by immunoradiometric assay and testosterone by radioimmunoassay. Secretion profiles of inhibin and testosterone were tested for diurnal variations by cosinor rhythmometry. Highest ir-inhibin concentrations were observed in the morning at 08.00 h, with peak values of 2.45-3.20 U/l x 10(-3). During the evening and the night, ir-inhibin levels were relatively low; lowest concentrations were observed between 01.00 h and 02.00 h at night: 1.20-1.86 U/l x 10(-3). Highest testosterone levels were observed in the morning (20.5-36.6 pmol/l), lowest concentrations were detected at night (7.35-12.6 pmol/l). Cosinor rhythmometry supported the suggestion that there is a clear circadian secretion of ir-inhibin and testosterone, respectively. The secretion pattern of ir-inhibin was analyzed by the Cluster pulse analysis computed algorithm, which identified four to seven inhibin pulses per day, depending on the person under observation.2+ volunteers follow a clear diurnal rhythm.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence that most of the radioimmunoassayable inhibin secreted by the corpus luteum of the common marmoset monkey is of a non-dimeric form.

Although recent data for several species of primate, including human and marmoset, indicate that the corpus luteum secretes high levels of radioimmunoassayable inhibin, the nature of the immunoreactive (ir) inhibin detected has not been established. In this study, plasma ir-inhibin levels during the ovarian cycle of the marmoset (n = 12 animals) were measured by alpha-subunit-directed inhibin RIA, and values were compared with those estimated by a recently developed two-site immunoradiometric assay (IRMA) specific for inhibin alpha-beta dimer. Consistent with earlier data, plasma levels of ir-inhibin measured by RIA (overall mean value 133 +/- 7 ng/ml; n = 171) reached values 4-fold higher (p less than 0.001) during the luteal phase (222 +/- 20 ng/ml) than during the follicular phase (58 +/- 8 ng/ml), being directly correlated with plasma progesterone levels (r = 0.65; p less than 0.001). In contrast, plasma ir-inhibin levels estimated by IRMA were substantially lower than those measured by RIA (overall mean value 9.62 +/- 1.08 ng/ml; n = 171) and did not vary significantly during the cycle. Administration of a luteolytic dose of cloprostenol during the late luteal phase/early pregnancy led to an abrupt fall in plasma concentrations of progesterone (95%) and alpha-inhibin measured by RIA (82%), whereas dimeric inhibin levels remained unchanged. Analysis of marmoset luteal extracts (n = 5) by RIA, IRMA, and inhibin bioassay yielded inhibin estimates of 102.6 +/- 21.0, 0.632 +/- 0.103, and less than 2.0 ng/mg, respectively, thus confirming that only a very small proportion of the inhibin produced was dimeric (i.e., bioactive) in nature.(ABSTRACT TRUNCATED AT 250 WORDS)

Apparent alpha-inhibin subunit immunoactivity in porcine and ovine luteal extracts is due to interference by cytosolic proteases in the assay.

Immunoreactive alpha-inhibin (ir-inhibin) was measured in luteal homogenates and subcellular fractions of ovine and porcine corpora lutea (CL) and in pig granulosa cells (GCs), using a sensitive radioimmunoassay specific for the 1-26 amino acid sequence of the N-terminus of the alpha chain of porcine inhibin (p1-26 alpha-inhibin). Inclusion of N-ethylmaleimide (N-EM) and/or EDTA in the immunoassay had no effect on the measurement of p1-26 alpha-inhibin peptide standards, on ir-inhibin levels in ovine follicular fluid and serum, or on ir-inhibin in subcellular fractions of pig GC. Fractionation of porcine GC homogenates on sucrose gradients demonstrated a major particular peak of ir-inhibin (buoyant density, 1.15-1.21 g/cm3) with variable activity in the cytosol. The particulate ir-inhibin peak was released into the cytosol by pretreatment of GC homogenates with the saponin, digitonin, prior to fractionation. Porcine GC extracts contained a protein (M(r) 45,000) which immunoblotted against p1-26 alpha-inhibin antibody. In the absence of inhibitors of proteolysis, apparent ir-inhibin activity was very high in extracts of sheep and pig CL. However, inclusion of N-EM or EDTA in the radioimmunoassay significantly reduced ir-inhibin levels in porcine and ovine CL extracts in a dose-dependent manner. Measurements of peptide tracer integrity indicated that porcine luteal cytosol degraded 125I-labelled p1-26 alpha-inhibin peptide. Subcellular fractionation studies demonstrated high levels of apparent ir-inhibin in luteal cytosol fractions, with only minor activity peaks associated with particulate fractions; however, this material was not releasable by digitonin. Immunoblotting of detergent extracts of porcine luteal particulate fractions failed to demonstrate alpha-inhibin material, and immunocytochemical localization studies of alpha-inhibin in porcine and ovine luteal sections were negative. Our results are consistent with the intracellular packaging/storage of a form of alpha-inhibin (M(r) similar to that of alpha-inhibin subunit precursor) in the porcine granulosa cell. However, luteinization of the porcine follicle was associated with a dramatic fall in ir-inhibin content, and the loss of immunostaining for alpha-inhibin peptides. We conclude that porcine and ovine CL contain little, if any, authentic inhibin. These studies emphasize the importance of excluding proteolytic artefacts when measuring biological peptides in luteal tissue extracts by radioimmunoassay.

Changes in peripheral levels of bioactive and immunoreactive inhibin, estradiol-17 beta, progesterone, luteinizing hormone, and follicle-stimulating hormone associated with follicular development in cows induced to superovulate with equine chorionic gonadotropin.

Changes in concentrations of bioactive and immunoreactive (ir-) inhibin, estradiol-17 beta, progesterone, LH, and FSH in peripheral blood were determined in cows induced to superovulate with eCG. The pattern of follicular growth was also characterized by daily ultrasonographic examination. Hormonal profiles and follicular development during the intact estrous cycle of the same animals before eCG treatment served as controls. Equine CG increased the number of follicles of various sizes (small, greater than or equal to 4 less than 7, medium, greater than or equal to 7 less than 10; large, greater than or equal to 10 mm in diameter) by 4 days after administration. The second growth of large follicles occurred within 1 day after superovulation. Inhibin bioactivity in jugular vein blood was detectable 48 h after eCG injection (44 h before LH peak), whereas it was not detected before administration of eCG or during control cycles. Circulating levels of bioactive inhibin further increased during the two waves of growth of large follicles. The highest activity of inhibin was noted at the time of the preovulatory LH peak (0 h). Thereafter, bioactivity of inhibin in peripheral plasma dropped from 0 to 24 h after the LH peak, and the activity increased again at 72 h compared to the value at -44 h. Plasma levels of ir-inhibin showed a pattern similar to changes in bioactive inhibin in the eCG-treated cows. Plasma concentrations of estradiol-17 beta also increased concomitantly with two waves of growth of large follicles. There was no correlation between plasma levels of progesterone and inhibin.(ABSTRACT TRUNCATED AT 250 WORDS)

Association of immunoreactive-inhibin levels with luteal function.

To evaluate immunoreactive (IR) inhibin as a marker of corpus luteal function, 134 women with natural ovulatory cycles were studied. Serum IR-inhibin levels during the luteal phase were significantly lower in women with low progesterone (P4) levels (less than 10 ng/ml). Endometrial dating correlated more with serum P4 than IR-inhibin levels. All 14 conceiving cycles showed serum P4 levels greater than or equal to 10 ng/ml and IR-inhibin levels greater than or equal to 3.67 IU/ml during the midluteal phase. Furthermore, IR-inhibin levels in the early follicular phase seemed to be associated with lowered P4 levels in the ensuing luteal phase. These results suggest that IR inhibin may be closely related to corpus luteum function.

Increased concentrations of immunoreactive inhibin during conception cycles in the marmoset monkey: suppression with an LHRH antagonist and cloprostenol

Peripheral concentrations of immunoreactive (ir) inhibin have been measured during the ovarian cycle and early pregnancy in the marmoset monkey. Blood samples were taken (three per week) during conception (n = 6) and non-conception (n = 5) cycles. Ir-inhibin was measured by radioimmunoassay using an antiserum raised against a synthetic peptide fragment of the alpha subunit of human inhibin. Monomeric bovine alpha subunit and 32 kDa bovine inhibin were used as tracer and standard respectively. In all animals low concentrations of ir-inhibin were recorded during the follicular phase (40-60 micrograms/l) of the cycle. After ovulation, ir-inhibin concentrations increased but the peak concentrations attained differed between conception and non-conception cycles. In non-pregnant animals ir-inhibin concentrations reached a maximum of 242 +/- 16 micrograms/l on days 12/13 after ovulation. In pregnant animals ir-inhibin concentrations were significantly (P less than 0.05) higher (1.8-fold) than in non-pregnant animals on days 8/9 after ovulation, and reached a maximum value of 636 +/- 141 micrograms/l on days 20/21 after ovulation. Administration of an LHRH antagonist during the luteal phase on days 6-8 after ovulation resulted in a significant (P less than 0.05) decrease in progesterone and ir-inhibin concentrations within 4 and 8 h respectively. This was prevented by co-administration with human chorionic gonadotrophin. Administration of cloprostenol to pregnant animals between days 17 and 20 after ovulation halved the initial concentrations of both inhibin and progesterone within 1.5 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Concentrations of immunoactive inhibin in serum during human pregnancy: evidence for an ovarian contribution

Immunoactive inhibin (ir-inhibin) concentrations in maternal serum during normal human pregnancy have been established in two separate studies employing cross-sectional and longitudinal sampling regimes. Ir-inhibin concentrations rose from the mid-luteal phase (geometric mean + 95% confidence intervals 1.490 (1.086-2.028) U mL-1) to peak at week 11 of gestation (3.77 (3.26-4.35) U mL-1), declined to a plateau from 14 to 25 weeks with means ranging from 1.8 to 2.3 U mL-1, and subsequently rose slowly to a peak of 6.53 U mL-1 at 41 weeks. In the longitudinal study, similar results were obtained and no differences were found in maternal inhibin levels in women carrying male or female fetuses. Paired cord blood and maternal samples showed no significant difference in ir-inhibin concentrations irrespective of the sex of the fetus. However, in all such pregnancies amniotic fluid ir-inhibin levels were 2-3 fold greater than maternal or fetal levels raising the possibility that the amnion may secrete inhibin. In 12 women without functional ovaries in whom a singleton pregnancy was achieved by donation of oocytes and in vitro fertilization, the ir-inhibin levels showed a similar pattern in the first trimester of pregnancy but the concentrations achieved were markedly lower (peak 1.1 U mL-1 at 9 weeks). In five women from the group in whom samples were available late in gestation, three showed greater than normal levels and two had subnormal levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Circulating levels of inhibin in pregnant women at term: simultaneous disappearance with oestradiol and progesterone after delivery

Circulating levels of immunoreactive inhibin (ir-inhibin) and its disappearance after delivery of the placenta were determined in seven pregnant women at term. Serum oestradiol (E2) and progesterone (P4) levels were measured simultaneously and served as comparisons. Fetal contributions of ir-inhibin were assessed by determining concentrations in the umbilical artery (UA) and vein (UV). Relative changes in circulating levels of ir-inhibin, E2, and P4 were compared to levels found in nonpregnant women during the early follicular phase (EFP) and mid-luteal phase (MLP) of the normal menstrual cycle. In pregnant women, ir-inhibin levels at delivery were 15- and 3-fold higher than EFP and MLP values respectively. The disappearance of all three hormones after removal of the placenta followed a bi-exponential curve with an initial, rapid component and a second, slower component. There was a highly significant positive correlation between the disappearance curves of all three placental hormones (r = 0.97, P less than 0.0001). Concentrations of ir-inhibin in the cord blood were about half that in maternal serum and without significant difference between levels in UA and UV.

Changes in Circulating Inhibin Levels during Pregnancy and Early Lactation in the Japanese Monkey

Changes in immunoreactive (ir-) inhibin concentrations in serum throughout pregnancy and early lactation up to one month after parturition were characterized in 6 Japanese monkeys (Macaca fuscata fuscata) by a heterologous radioimmunoassay (RIA) based on a bovine RIA. Serum levels of FSH, LH/monkey chorionic gonadotropin (mCG), estradiol-17 beta, and progesterone were also monitored for the entire period. Ir-inhibin levels in the serum were low (under 0.5 ng/ml) before conception. Three marked increases in serum ir-inhibin levels were found during pregnancy. The first increase was noted during early pregnancy, with a peak (2.2 +/- 0.2 ng/ml) at Day 22 of pregnancy (Day 0 = day of LH surge). The second increase was noted after Day 38 until Day 72 of pregnancy, when a peak value was noted (19.0 +/- 1.4 pg/ml). Plateau levels were maintained until late pregnancy, and a final rise was evident near the term with a peak (36.7 +/- 3.8 ng/ml) at Day 158 of pregnancy, 5 days before parturition. After parturition, ir-inhibin levels in the serum plummeted to nonpregnant levels within one day, and were maintained during early lactation. The first rise in serum inhibin during pregnancy was parallel to the rise of mCG and estradiol-17 beta, and the second and third rise were well correlated with serum estradiol-17 beta. Serum FSH was maintained at low levels throughout pregnancy, followed by a slight increase after parturition when serum inhibin decreased abruptly. Both bioactivity and immunoreactivity of inhibin were detected in the placental homogenates obtained at 120 days of pregnancy.(ABSTRACT TRUNCATED AT 250 WORDS)

Circulating Radioimmunoassayable Inhibin during Periods of Transient Follicle-stimulating Hormone Rise: Secondary Surge and Unilateral Ovariectomy1

We have measured changes in circulating immunoreactive (ir-) inhibin in male and female rats using an RIA with an antiserum raised against porcine inhibin alpha (1-26)-Gly-Tyr. The same synthetic peptide was used for standards and for the preparation of tracer. Serum ir-inhibin levels were significantly higher in intact female than in intact male rats (p less than 0.001). Immunoreactive inhibin was significantly reduced in both sexes 24 h after bilateral gonadectomy (p less than 0.0001). Unilateral ovariectomy (ULO) of female rats on metestrus caused a transient decrease in serum inhibin 8 h after surgery, but levels were not significantly different from those of sham-operated controls at later times after surgery. Increases in serum FSH and LH were observed for 8-18 h after ULO. Serum ir-inhibin levels were also measured on the early morning of estrus during the secondary FSH surge. At this time, ir-inhibin levels were low, while FSH levels were high and LH levels were low. These results show that serum ir-inhibin levels in rats are decreased at times when serum FSH levels are high.

Dynamic Changes in Circulating Inhibin Levels during the Luteal-Follicular Transition of the Human Menstrual Cycle *

Dynamic changes in serum immunoreactive (ir) inhibin levels during the transition from the luteal to the follicular phase (luteal-follicular transition) were characterized during 3 consecutive cycles in 12 cycling women. Both spontaneous (first to second cycle) and GnRH antagonist-imposed premature luteolysis (second to third cycle) were evaluated. Serum FSH, LH, estradiol (E2), and progesterone (P4) levels were monitored daily by RIA for the entire study. Daily ir-inhibit levels were determined from 7 days before until 7 days after the onset of menses and from 4 days before to 10 days after the GnRH antagonist-induced bleeding by a heterologous RIA. During spontaneous luteolysis, ir-inhibin levels peaked 7 days before menses and declined in a linear fashion (r = -0.99) thereafter, reaching a sustained low level 1 day after the onset of menses. The decline of P4 and E2 levels appears to be coupled to that of ir-inhibin (r = 0.98 and r = 0.95, respectively). FSH levels showed an inverse pattern, with an acute elevation unaccompanied by LH, for 5 days before the onset of menses and reaching a plateau 2 days after. ir-Inhibin and FSH were negatively correlated (r = -0.87; P less than 0.0001). Increased LH levels did not occur until the day of menses and were negatively correlated with ir-inhibin (r = -0.50; P less than 0.05), but not E2 and P4. During the second cycle, at the midluteal phase luteolysis was induced by a single (50 micrograms/kg) im injection of a potent GnRH antagonist, [Ac-D2Nal,D4ClPhe2,D3Pal3,Arg5,DGlu6(AA),+ ++DAla10] GnRH; an acute decline of LH and FSH levels occurred, with maximal suppressions of 51% and 35%, respectively. ir-Inhibin levels decreased rapidly (40 +/- 2.8%) in parallel with E2 and P4 during the first 24 h and continued to decline for 4 days. The inverse relationship and time course of changes between FSH and ir-inhibin levels were similar to those of the spontaneous luteal-follicular transition. Six of the 12 subjects experienced partial reversal of luteolysis; the decline of ir-inhibin and the rise of FSH during the first 2 days were arrested for 4 days, which corresponded to the rebound increases in E2, P4, and LH. The subsequent fall of ir-inhibin was followed by a rise in FSH. Both the complete and incomplete luteolysis groups exhibited an orderly follicular maturation, LH surge, and luteal function during the ensuing cycle.(ABSTRACT TRUNCATED AT 400 WORDS)

The Involvement of Leydig Cells in the Regulation of Inhibin Secretion by the Testis

The stimulation of Leydig cells by the administration of a single injection of 100 IU human CG (hCG) to adult male rats caused a significant biphasic stimulation of serum testosterone levels at 2 h and 3 days after injection. Serum immunoreactive (IR)-inhibin levels were elevated by 6 h and peaked at 24 h after hCG, then progressively declined thereafter. The removal of Leydig cells in vivo by the injection of the cytotoxic drug ethane dimethane sulfonate (EDS) causes a significant decrease in serum testosterone levels within 4 days, which is sustained 1 and 2 weeks after EDS. Serum IR-inhibin levels, however, rise significantly 2 and 4 weeks after injection of EDS. An injection of 100 IU hCG, 4 days after EDS (when no Leydig cells were present in vivo), failed to provoke an elevation of either testosterone or IR-inhibin levels in serum. But 2 or 4 weeks after administration of EDS, as a new population of Leydig cells develops in the interstitium, an injection of 100 IU hCG provokes a significant increase in serum testosterone and IR-inhibin levels. The possibility that the failure of IR-inhibin levels to rise after EDS and hCG treatment could be due to changes in the seminiferous epithelium, caused by testosterone deprivation induced by Leydig cell destruction after EDS, was examined by administering high doses of testosterone known to maintain spermatogenesis. Under such conditions, hCG failed to induce a rise of IR-inhibin after EDS treatment had destroyed the Leydig cells. These data strongly support the concept that the Leydig cells are involved in the regulation of IR-inhibin secretion in vivo through factors other than testosterone.