Effect of GnRH antagonists treatment on gonadotrophin secretion, follicular development and inhibin A secretion in goats

The aim of this study was to determine, for goats, the effects of daily doses of GnRH antagonist on ovarian endocrine and follicular function. Ten does were given 45 mg FGA intravaginal sponges and then five were treated with daily injections of 0.5mg of the GnRH antagonist Teverelix for 11 days from 2 days after the day of sponge insertion, while five does acted as controls. Pituitary activity was monitored by measuring plasma FSH and LH daily from 2 days before the first GnRH injection to Day 12. Follicular activity was determined by ultrasonographic monitoring and by assessing plasma inhibin A levels during the same period. In treated does, the FSH levels decreased linearly (0.8 +/- 0.1 ng/ml to 0.5 +/- 0.1 ng/ml, P < 0.01) and remained lower than the mean concentration in control goats (0.8 +/- 0.1 ng/ml, P < 0.005). LH levels were also lower during the period of antagonist treatment (0.6 +/- 0.2 ng/ml versus 0.4 +/- 0.1 ng/ml, P < 0.0005). During GnRH antagonist treatment, there was a significant decrease in the number of large follicles (> or = 6 mm) from Day 3 of treatment (1.2 +/- 0.6, P < 0.0001), with no large follicles from Day 9. The number of medium follicles (4-5 mm in size) also decrease during the period of treatment (4.2 +/- 0.7 to 1.0 +/- 0.6, P < 0.0001), leading to a significant decrease in inhibin A levels when compared to the control (143.7 +/- 31.3 pg/ml versus 65.2 +/- 19.1 pg/ml, P < 0.00005). In contrast, the number of small follicles (2-3 mm) increased in treated goats from Day 4 of treatment (9.6 +/- 2.9 to 20.2 +/- 6.3, P < 0.005). Such data indicate that GnRH antagonist reduced plasma levels of FSH and LH with suppression of the growth of large dominant ovarian follicles and a two-fold increase in number of smaller follicles. The results confirm that GnRH antagonist treatment can be used in goats to control gonadotrophin secretion and ovarian follicle growth in superovulatory regimes.     

Roles of intracerebral activin,inhibin, and follistatin in the regulation of Kiss-1 gene expression: Studies using primary cultures of fetal rat neuronal cells

Hypothalamic kisspeptin, encoded by the Kiss-1 gene, governs the hypothalamic-pituitary-gonadal axis by directly regulating the release of gonadotropin-releasing hormone. In this study, we examined the roles of activin, inhibin, and follistatin in the regulation of Kiss-1 gene expression using primary cultures of fetal rat neuronal cells, which express the Kiss-1 gene and kisspeptin. Stimulation with activin significantly increased Kiss-1 gene expression in these cultures by 2.02 ± 0.39-fold. In contrast, a significant decrease in Kiss-1 gene expression was observed with inhibin A and follistatin treatment. Inhibin B did not modulate Kiss-1 gene expression. Activin, inhibin, and follistatin were also expressed in fetal rat brain cultures and their expression was controlled by estradiol (E2). The inhibin α, βA, and βB subunits were upregulated by E2. Similarly, follistatin gene expression was significantly increased by E2 in these cells. Our results suggest the possibility that activin, inhibin, and follistatin expressed in the brain participate in the E2-induced feedback control of the hypothalamic-pituitary-gonadal axis.     

Plasma inhibin A in heifers: relationship with follicle dynamics, gonadotropins, and steroids during the estrous cycle and after treatment with bovine follicular fluid

The relationship between follicle growth and plasma inhibin A, FSH, LH, estradiol (E), and progesterone was investigated during the normal bovine estrous cycle and after treatment with steroid-free bovine follicular fluid (bFF) to arrest follicle development. In the first study, four heifers were monitored over three prostaglandin (PG)-synchronized cycles. Blood was collected every 2-8 h, and ovaries were examined daily by ultrasonography. Inhibin A was measured using a modified enzyme-linked immunosorbent assay that employed a new monoclonal antibody against the alpha subunit of bovine inhibin. Plasma inhibin A ( approximately 50 pg/ml before luteolysis) rose steadily during the induced follicular phase (P < 0.05) to a peak ( approximately 125 pg/ml) coincident with the preovulatory E/LH/FSH surge. After ovulation, inhibin A fell sharply (P < 0.05) to a nadir ( approximately 55 pg/ml) coincident with the secondary FSH rise. During the next 3 days, inhibin A increased to approximately 90 pg/ml in association with growth of the new dominant follicle (DF). Plasma E also rose twofold during this period, whereas FSH fell by approximately 50%. Inhibin A was negatively correlated with FSH (r = -0.37, P < 0.001) and positively correlated with E (r = 0.49, P < 0.0001). Observations on eight cycles (two cycles/heifer), in which growth of the ovulatory DF was monitored from emergence to ovulation, showed that the first-wave DF (DF1) ovulated in three cycles and the second-wave DF (DF2) in five cycles. After PG, plasma inhibin A and E increased similarly in both groups, with concomitant falls in FSH. In the former group, the restricted ability of DF1 to secrete both inhibin A and E was restored after luteolysis. Results indicate that dynamic changes in the secretion of both E and inhibin A from the DF contribute to the fall in FSH during the follicular phase and to the generation and termination of the secondary FSH surge, both of which play a key role in follicle selection. In the second study, bFF (two dose levels) was administered to heifers (n = 3-4) for 60 h starting from the time of DF1 emergence. Both doses suppressed FSH (P < 0.05) and blocked DF1 growth to the same extent (P < 0.01), although inhibin A levels were only marginally raised by the lower dose (not significant compared to controls). The high bFF dose raised (P < 0.001) inhibin A to supraphysiological levels ( approximately 1 ng/ml). A large "rebound" rise in FSH occurred within 1 day of stopping both treatments, even though the inhibin A level in the high-dose bFF group was still approximately threefold higher than that in controls. This indicates that desensitization of gonadotropes to inhibin negative feedback is a contributory factor, together with reduced ovarian output of E, in generation of the post-bFF rebound in FSH.     

缓释型促性腺激素释放激素与生长激素治疗儿童中枢性性早熟的疗效观察

目的用生长激素与促性腺激素释放激素类似物(Gonadotropin-releasing hormone analogues,GHA)联合治疗中枢性性早熟女性患儿对其最终成人身高的影响.方法生长激素(GH)与促性腺激素释放激素类似物(GHA)联合治疗4例中枢性性早熟女性患儿半年,对比治疗前后患儿的第二性征,骨龄发育,性激素及最终成人身高的变化.结果第二性征的发育停止,骨龄发育被控制,实际生活年龄与骨年龄的比值提高(平均0.79→0.84);血LH对促性腺激素释放激素的反应及血浆雌激素水平平均已降至青春期前,分别为(平均25.79±10.60mlu/ml→1.13±0.21mlu/ml)及(平均64.87±27.51pg/ml→3.03±1.87pg/ml);预测最终成人身高增加(平均149.60±4.31cm→156.75±3.84cm)差异具有显著性(P＜0.05).结论生长激素与GHA联合治疗中枢性性早熟患儿,不仅能抑制第二性征发育,而且能有效改善最终成人身高,无任何毒付作用.     

Inhibin B,follicle stimulating hormone,luteinizing hormone,and estradiol and their relationship to the regulation of follicle development in girls during childhood and puberty

Inhibin B, produced by granulosa cells in the ovary, is a heterodimeric glycoprotein suppressing synthesis and secretion of the follicle stimulating hormone (FSH). The aim of the present study was to determine hormone profiles of inhibin B, FSH, luteinizing hormone (LH), and estradiol in girls during childhood and puberty and to evaluate whether inhibin B is a marker of follicle development. We examined the correlation between inhibin B and gonadotropins and estradiol during the first two years and across the pubertal development. Using a specific two-side enzyme-linked immunosorbent assay (ELISA), inhibin B levels were measured in the serum of 53 healthy girls divided into 8 groups according to age. In addition, serum FSH, LH, and estradiol were measured by chemiluminescent immunoassay in all serum samples. A rise in serum levels of inhibin B (55.2+/-7.3 ng/l, mean +/- S.E.M.) and FSH (1.78+/-0.26 UI/l), concomitant with a moderate increment of serum LH (0.36+/-0.09 UI/l) and estradiol (45.8+/-12.2 pmol/l) concentrations was observed during the first three months of life and declined to prepubertal concentrations thereafter. A strong positive correlation between inhibin B and FSH (r = 0.48, p<0.05), LH (r = 0.68, p<0.001) and estradiol (r = 0.59, p<0.01) was demonstrated during the first 2 years of life. A rise in serum levels of inhibin B, FSH, LH, and estradiol was found throughout puberty. Inhibin B had a strong positive correlation with FSH (stage I of puberty: r = 0.64, p<0.05; stage II of puberty: r = 0.86, p<0.01), LH (I: r = 0.61, p<0.05; II: r = 0.67, p<0.05), and estradiol (II: r = 0.62, p<0.05) in early puberty. From pubertal stage II, inhibin B lost this relationship to gonadotropins and estradiol. Serum inhibin B and FSH levels increased significantly during pubertal development, with the highest peak found in stage III of puberty (133.5+/-14.3 ng/l), and decreased thereafter. In conclusion, inhibin B is produced in a specific pattern in response to gonadotropin stimulation and plays an important role in the regulation of the hypothalamic-pituitary-gonadal axis during childhood and puberty in girls. Inhibin B is involved in regulatory functions in developing follicles and seems to be a sensitive marker of ovarian follicle development.     

An inhibin B and estrogen-secreting adrenocortical carcinoma leading to selective FSH suppression

OBJECTIVE: Hormone-secreting adrenocortical tumors are frequently associated with endocrine syndromes. We describe a 30-year-old man who had abdominal pain, a nodule in the right breast and loss of libido. Abdominal magnetic resonance imaging revealed a very large tumor in the right adrenal gland. METHODS: Hormonal profile disclosed increased levels of estradiol and slightly low testosterone levels. The basal and stimulated LH levels were normal, whereas basal and stimulated FSH levels were totally suppressed. Cortisol and adrenal androgen levels were normal. The unusual finding of selective FSH suppression suggested secretion of inhibin B by the adrenocortical tumor. A very high level of serum inhibin B (405 pg/ml) was demonstrated by ELISA assay. Right adrenalectomy and nephrectomy were performed and the tumor was classified as a malignant tumor (Weiss score: 7.0) and unilateral mastectomy disclosed a lipoma. RESULTS: One week after surgery, a GnRH-stimulation test disclosed normal basal and stimulated FSH levels and low levels of inhibin B and estradiol. Immunohistochemical analysis with anti-B-inhibin antibody revealed intense staining in the adrenocortical tumor cells. One month after surgery, an abdominal magnetic resonance imaging revealed a local recurrence of the tumor and a second surgery was performed with partial resection of the tumor and the patient died 1 year after the first surgery. CONCLUSION: We herein report the first inhibin B and estradiol-secreting adrenocortical carcinoma. The unusual selective inhibition of FSH secretion should be considered a valuable hormonal finding for the diagnosis of inhibin B-secreting adrenocortical tumors.     

The FSH-inhibin axis in Prader-Willi Syndrome: heterogeneity of gonadal dysfunction

ABSTRACT: BACKGROUND: We characterized the spectrum and etiology of hypogonadism in a cohort of Prader-Willi syndrome (PWS) adolescents and adults. METHODS: Reproductive hormonal profiles and physical examination were performed on 19 males and 16 females ages 16-34 years with PWS. Gonadotropins, sex-steroids, inhibin B (INB) and anti-Mullerian hormone (AMH) were measured. We defined 4 groups according to the relative contribution of central and gonadal dysfunction based on FSH and INB levels: Group A: primary hypogonadism (FSH >15 IU/l and undetectable INB (<10 pg/ml); Group B: central hypogonadism (FSH <0.5 IU/l, INB <10pg/ml); Group C: partial gonadal & central dysfunction (FSH 1.5-15 IU/l, INB >20 pg/ml); Group D: mild central and severe gonadal dysfunction (FSH 1.5-15 IU/l, INB < 10 pg/ml. RESULTS: There were 10, 8, 9 and 8 individuals in Groups A-D respectively; significantly more males in group A (9, 4, 4 and 2; P=0.04). Significant differences between the groups were found in mean testosterone (P=0.04), AMH (P=0.003) and pubic hair (P=0.04) in males and mean LH (P=0.003) and breast development (P=0.04) in females. Mean age, height, weight, BMI and the distribution of genetic subtypes were similar within the groups. CONCLUSIONS: Analysis of FSH and inhibin B revealed four distinct phenotypes ranging from primary gonadal to central hypogonadism. Primary gonadal dysfunction was common, while severe gonadotropin deficiency was rare. Longitudinal studies are needed to verify whether the individual phenotypes are consistent.     

Restoration of endocrine and ovarian function after stopping GnRH antagonist treatment in goats

We have tested if the high number of unfertilized ova and degenerated embryos found in superovulated goats previously treated with GnRH antagonist can be related to a prolongation of gonadotrophin down-regulation and/or alterations in follicular function during the period of administration of the superovulatory treatment, around 4 days after the end of the antagonist treatment. A total of 15 does were treated with intravaginal progestagen sponges and daily injections of 0.5mg of the GnRH antagonist Antarelix for 6 days, while 5 does acted as controls receiving saline. During the antagonist treatment, the mean plasma LH concentration was lower in treated than control goats (0.5 +/- 0.2 versus 0.7 +/- 0.5 ng/ml, P < 0.0005 ); however, the FSH levels remained unaffected (0.8 +/- 0.4 versus 0.8 +/- 0.5 ng/ml). In this period, treated does also showed an increase in the number of small follicles with 2-3 mm in size ( 10.7 +/- 0.7 versus 8.4 +/- 0.6, P < 0.05), and a decrease in both the number of follicles > or =4 mm in size ( 5.0 +/- 0.3 versus 6.8 +/- 0.5, P < 0.005) and the secretion of inhibin A (120.9 +/- 10.7 versus 151.6 +/- 12.6 pg/ml, P < 0.05). After cessation of the antagonist treatment, there was an increase in LH levels in treated goats from the day after the last Antarelix injection (Day 1), so that LH levels were the same as controls on Day 3 (0.6 +/- 0.1 versus 0.6 +/- 0.2 ng/ml). However, there were even greater numbers of small follicles than during the period of antagonist injections (15.4 +/- 0.6 in treated versus 8.9 +/- 0.7 in control, P < 0.0005 ). Moreover, the number of > or =4 mm follicles and the secretion of inhibin A remained lower in treated goats (3.9 +/- 0.3 follicles and 84.4 +/- 7.0 pg/ml versus 5.4 +/- 0.5 follicles, P < 0.05 and 128.9 +/- 14.2 pg/ml, P < 0.05 ). These results indicate that pituitary secretion of gonadotrophins is restored shortly after the end of antagonist treatment, but activity of ovarian follicles is affected.     

Plasma concentrations of inhibin a and follicle-stimulating hormone differ between cows with two or three waves of ovarian follicular development in a single estrous cycle

Patterns of ovarian follicle development were monitored daily in Holstein-Friesian cows that had two (n = 4) or three (n = 4) waves of ovarian follicle development during a single estrous cycle. The plasma from daily blood samples was used in assays for inhibin A, FSH, progesterone, and estradiol-17beta. Mean cycle lengths for cows with two and three waves were 21.8 and 25.3 days, respectively (P < 0.02). Although the average number of follicles >3-mm diameter on each pair of ovaries was similar for two- and three-wave cows on Days 2, 3, and 4 (Day 0 = day of ovulation; 8.6 vs. 9.6 follicles), there were more follicles >6-mm diameter on the ovaries of cows with two waves on Days 3 and 4. This difference was associated with a shorter interval from wave emergence to peak concentrations of inhibin A during the first wave in two-wave cows (2.0 vs. 3.8 days; P = 0.03) and with higher peak concentrations (474 vs. 332 pg/ml; P = 0.03). Differences in peak FSH concentrations were not significant (1.7 vs. 1.3 ng/ml; P = 0.10) and were inversely related to inhibin A concentrations. The peak concentrations of inhibin A and FSH in the second nonovulatory wave in the three-wave cows were similar to the low concentrations measured in the first wave (292 vs. 332 pg/ml of inhibin A, 1.3 vs. 1.3 ng/ml of FSH; P > 0.20). Average peak concentrations of inhibin A and FSH were similar during the ovulatory wave for cows with either two or three waves in a cycle (432 vs. 464 pg/ml of inhibin A, 2.3 vs. 2.1 ng/ml of FSH; P > 0.3). The lower concentrations of FSH during the emergence of the first follicular wave in cows with three-wave cycles may have reduced the rate of development of some of the follicles and reduced the concentrations of inhibin A. This pattern of lower concentrations of FSH and inhibin A was repeated in the second nonovulatory wave but not in the ovulatory wave. Subtle differences in the concentrations of these two hormones may underlie the mechanism that influences the number of waves of ovarian follicle development that occur during the bovine estrous cycle.     

Changes in plasma inhibin A levels during sexual maturation in the female chicken and the effects of active immunization against inhibin alpha-subunit on reproductive hormone profiles and ovarian function

Inhibins and activins are firmly implicated in the control of pituitary FSH secretion and ovarian follicular development in mammals. As in mammals, inhibin A and activin A are expressed in the preovulatory follicles of birds, and a defined ovulation cycle for inhibin A has recently been demonstrated in the laying hen. To investigate further the role of inhibin-related proteins in developing pullets, circulating concentrations of inhibin A, inhibin B, total immunoreactive inhibin alpha-subunit (ir-alpha), activin A, LH, FSH, and progesterone were measured from the juvenile state through to sexual maturity in 22 birds. In the 11 birds assigned to control groups, plasma inhibin A levels were low from 7 to 13 wk of age rising about threefold to a peak at Week 19 after which levels fell slightly to a plateau level characteristic of adult hens. Plasma inhibin A levels were negatively correlated with FSH (r = -0. 33; P: < 0.001) and positively correlated with progesterone (r = 0. 67; P: < 0.001) and ir-alpha (r = 0.53; P: < 0.001). Plasma ir-alpha levels were much higher than inhibin A levels although the relative differences varied with age. Plasma levels of inhibin B and activin A were below assay detection limits at all times. The remaining group of 11 birds was actively immunized (IMM) against a synthetic chicken inhibin alpha-subunit peptide (amino acids 1-26). The IMM generated circulating antibodies that bound native bovine inhibin A but altered neither plasma FSH nor progesterone levels relative to control birds at any stage of development nor the timing of first oviposition in week 19. Apart from a transient decline 1 wk after primary IMM, plasma LH concentrations did not differ from controls. Comparison of the numbers and size-class distribution of ovarian follicles at 29 wk showed an approximate twofold increase in the number of 8- to 9.9-mm-diameter follicles (control; 1.82 +/- 0.44 vs. IMM; 3.91 +/- 0.89; P: < 0.05), a size class that corresponds to follicles that have just joined the preovulatory hierarchy. The numbers of growing follicles in other size-classes and the sizes of hierarchical F(1)-F(7) follicles were not altered by IMM. However, the number of postovulatory follicles increased (control 3.73 +/- 0. 20 vs. IMM 5.55 +/- 0.28; P: < 0.01), and significantly more (P: < 0. 02) immunized hens laid two eggs within a 24-h period on at least one occasion (control 1 of 11 vs. IMM 9 of 11). The IMM increased (P: < 0.05) activin A content of F(1) and F(2) theca layers and decreased (P: < 0.05) activin A content in F(3) and F(4) granulosa layers, raising the possibility of a local intraovarian role of activin in mediating the response to IMM. These findings support a role for inhibin A in regulating the entry of follicles into the preovulatory hierarchy in the chicken, although further studies are required to establish the mechanism by which inhibin IMM increases the rate of follicle selection and ovulation without raising plasma FSH.     

Antagonistic and agonistic GnRH analogue treatment of precocious puberty: tracking gonadotropin concentrations in urine

BACKGROUND: The pharmacodynamics of gonadotropin-releasing hormone (GnRH) agonists includes an initial 'flare-up' of the pituitary-gonadal axis, followed by reduced luteinizing hormone (LH) secretion. The question is if combining a short-acting antagonist with a long-acting agonist can diminish gonadotropin flare-up. METHODS: To achieve quick downregulation in patients with recently diagnosed central precocious puberty (CPP, 7 patients) or short stature with short predicted final height (3 patients), we combined the GnRH antagonist cetrorelix (3 subcutaneous injections every 72 h) at the beginning of GnRH agonist treatment (leuprorelin or triptorelin) in 6 patients and compared the effect to 4 patients treated solely with GnRH agonist. To monitor effects, we measured LH and FSH concentrations in urine collected from initial morning urination during the first month of treatment. RESULTS: In both treatment groups, gonadotropin flare-up could be detected in urine levels increased due to the flare-up phenomenon which was of short duration (<5 days) in the majority (5 of 6) of combined-treated patients and in the minority (1 of 4) of patients treated by agonist alone. During the first 10 days of treatment, mean LH concentration measured in urine was significantly lower in 4 CPP patients treated by the combined therapy compared to 3 CPP patients treated by the agonist only (mean LH combined therapy: 10.4 +/- 2.8 vs. 20.1 +/- 11.0 mU/ml in the agonist-only group, mean +/- SEM, p < 0.05). Significant correlations between stimulated serum LH in GnRH test prior to treatment and maximum urine LH after initiating GnRH analogue treatment (r = 0.547, p = 0.043), as well as basal serum LH and basal urine LH (r = 0.685, p = 0.014) were found. CONCLUSION: Combined GnRH agonist and antagonist treatment led to rapid gonadotropin suppression. Also, urine measurements of LH and FSH seemed suitable for monitoring gonadotropin-inhibiting or -stimulating properties of GnRH analogues in individual patients. However, a controlled trial of a larger patient cohort is required to decide which treatment is the most effective.     

Luteinizing hormone-releasing hormone in prepubertal and pubertal girls.

Estimations of immunoreactive LH-RH and LH in pooled sera of girls, adult women and postmenopausal women have been carried out. The girls were divided into three groups: I--girls aged 2--4 years, II--girls aged 5--8 years and III--girls 9--12 years of age. The estimated concentrations of LH-RH in particular groups were as following: in group I--1.2 +/- 0.2 pg/ml, in group II--2.2 +/- 0.4 pg/ml, in group III 31.0 +/- 4.4 pg/ml, in adult women 6.3 +/- 1.8 pg/ml. and in postmenopausal women 16.6 +/- 2.4 pg/ml. The concentrations of LH in the same groups were 4.3 +/- 0.7; 4.5 +/- 0.8; 11.0 +/- 1.4, 23.3 +/- 2.4; and 120.0 +/- 14.7 mIU/ml, respectively. The authors suggest that the sexual maturation of girls is initiated by the enhanced hypothalamic activity, reflected in higher concentrations of immunoreactive LH-RH in peripheral serum.     

Alterations in peripheral concentrations of inhibin A in cattle studied using a time-resolved immunofluorometric assay: relationship with estradiol and follicle-stimulating hormone in various reproductive conditions

The aims of this study were to develop a sensitive and specific assay for bovine inhibin A using europium and to investigate the endocrine role of inhibin A in various reproductive conditions by characterizing the relationship between profiles of inhibin A, FSH, and estradiol and follicle growth during the postpartum period, during the intact estrous cycle, and in cows with follicular cysts. The time-resolved immunofluorometric assay (Tr-IFMA) for bovine inhibin A, using purified polyclonal antibodies to alpha and beta(A) subunits, was specific for bovine inhibin A and did not cross-react with bovine activin A, activin AB, activin B, pro-alphaC or human recombinant inhibin B. The detection limit of the IFMA was 3.3 pg/ml expressed in terms of bovine 32-kDa inhibin A. Dose-response curves of plasma samples obtained from intact and FSH-stimulated cows and cystic cows were parallel to the standard without any preassay processing of samples. Plasma inhibin A levels increased (P < 0.01) concomitant with emergence of nonovulatory or ovulatory follicular waves during the postpartum period. In cystic cows, plasma inhibin A was sustained at high levels for a longer period, associated with growth of persistent dominant follicles. The highest levels of inhibin A were noted during the growth phase of normal and persistent dominant follicles; however, inhibin A levels declined (P < 0.01) as these dominant follicles ceased to grow or ovulated. An inverse relationship between patterns of plasma inhibin A and FSH existed during each follicular wave in the three physiologic conditions. Increases in plasma inhibin A levels were associated with increases in plasma estradiol levels during most follicular waves; however, there was no increase in plasma estradiol level and no relationship between patterns of estradiol and FSH during follicular waves observed during the early postpartum period or midluteal phase of the estrous cycle. In conclusion, the Tr-IFMA does not require pretreatment of samples and can be used for precise measurement of bovine inhibin A without interference with free inhibin alpha subunits. Inhibin A, produced primarily during growth of the dominant follicle, functions as a negative feedback regulator for FSH secretion throughout the postpartum period and the estrous cycle, whereas estradiol appears to have a minor role in regulation of FSH compared with inhibin A, especially during the early postpartum period and midluteal phase of the estrous cycle. The results also indicate that a persistent dominant follicle sustains inhibin A production for a longer period than the dominant follicle emerging in the estrous cycle and establishes long-term dominance by suppressing emergence of a new follicular wave.     

Effects of ageing and exogenous melatonin on pituitary responsiveness to GnRH in rats

The effect of age and melatonin on the activity of the neuroendocrine reproductive system was studied in young cyclic (3-5 months-old), and old acyclic (23-25 month-old) female rats. Pituitary responsiveness to a bolus of GnRH (50 ng per 100 g body weight) was assessed at both reproductive stages in control and melatonin-treated (150 micrograms melatonin per 100 g body weight each day for 1 month) groups. After this experiment, female rats were treated for another month to study the influence of ageing and melatonin on the reproductive axis. Plasma LH, FSH, prolactin, oestradiol and progesterone were measured. A positive LH response to GnRH was observed in both control groups (cyclic and acyclic). However, a response of greater magnitude was observed in old acyclic rats. Melatonin treatment reduced this increased response in acyclic rats and produced a pituitary responsiveness similar to that of young cyclic rats. FSH secretion was independent of GnRH administration in all groups, indicating desynchronization between LH and FSH secretion in response to GnRH in young animals and during senescence. No effect on prolactin was observed. Significantly higher LH (3009.11 +/- 1275.08 pg ml(-1); P < 0.05) and FSH concentrations (5879.28 +/- 1631.68 pg ml(-1); P < 0.01) were seen in acyclic control rats. After melatonin treatment, LH (811.11 +/- 89.71 pg ml(-1)) and FSH concentrations (2070 +/- 301.62 pg ml(-1)) decreased to amounts similar to those observed in young cyclic rats. However, plasma concentrations of oestradiol and progesterone were not reduced. In conclusion, the results of the present study indicate that, during ageing, the effect of melatonin is exerted primarily at the hypothalamo-pituitary axis rather than on the ovary. Melatonin restored the basal concentrations of pituitary hormones and pituitary responsiveness to similar values to those observed in young rats.     

Weaning and the developmental changes in follicle-stimulating hormone, pituitary adenylate cyclase-activating polypeptide, and inhibin B in the male rat

Pituitary Fshb concentrations increase markedly and selectively beginning on Postnatal Day 20 in the male rat. To evaluate the factors potentially responsible for this rise in FSH, we adjusted the time of weaning, which is generally also on Day 20. Male rat pups were provided nutrients by suckling only and were weaned to laboratory chow earlier (Day 17) or later (Day 23) than normally performed in animal facilities (Day 20). Between ages 17 and 29 days, significant increases were seen in serum LH (1.4-fold) and FSH (2.4-fold) levels; pituitary expression of Lhb (5.4-fold), Fshb (21.3-fold), and inhibin beta B (Inhbb, 2.26-fold) mRNAs; and testicular expression of Inhbb (10-fold) mRNA. Concurrently, significant decreases occurred in serum inhibin B levels (1.8-fold); pituitary adenylate cyclase-activating polypeptide (Adcyap1, 4.2-fold), total follistatin (Fst, 3.5-fold), and Fst isoform 288 (5.6-fold) mRNAs; and testicular expression of inhibin beta A (8.2-fold) mRNA. Early weaning significantly increased serum FSH but not LH and increased pituitary expression of Fshb and GnRH receptor (Gnrhr) mRNAs but not Lhb. Early weaning also significantly decreased serum inhibin B but increased testicular expression of the Inhbb subunit. Early weaning also caused pituitary expression of Fst and Adcyap1 to decline earlier than in the control group. Immediately after weaning, growth accelerated substantially, and the time of weaning produced significant and differential effects on circulating leptin levels that were not related to indices of FSH production. From these observations, we propose the novel hypothesis that the increase in growth rate subsequent to weaning signals circulating inhibin B levels to fall and pituitary Adcyap1 and consequently Fst expression to decrease, and that these events together facilitate the rise in Fshb and Gnrhr expression by increasing pituitary activin signaling.     

Low serum allopregnanolone levels in girls with precocious pubarche

Allopregnanolone, a neuroactive steroid, increases during pubertal development and high concentrations are present in subjects with precocious puberty. The aim of the present study was to evaluate serum allopregnanolone levels in girls with precocious pubarche (PP). Basal gonadotropins and steroid hormones were assessed in 17 girls with PP, 22 girls with central precocious puberty (CPP), 25 girls with normal puberty at the same pubertal stage of CPP ones, and 17 prepubertal girls. Adrenocorticotropin hormone (ACTH) and gonadotropin-releasing hormone (GnRH) stimulation tests were performed in all subjects with PP, and in 12 out of 22 with CPP. All girls with normal puberty underwent to GnRH test, while ACTH test was performed in 17 out of 25. Basal dehydroepiandrosterone sulfate (DHEAS) concentrations resulted significantly higher in PP and normal pubertal girls than in prepubertal ones. Allopregnanolone, gonadotropins and estradiol levels were significantly lower in PP group with respect to CPP (P<0.05), while they were comparable among PP, normal pubertal and prepubertal groups. After ACTH administration, allopregnanolone concentrations significantly increased in all groups (P<0.05). After GnRH stimulation, its levels significantly increased in CPP and normal pubertal controls (P<0.05), while no incremental rise was found in PP girls. In conclusion, our study shows that in girls with PP basal and GnRH-stimulated levels of allopregnanolone are significantly lower than in CPP girls. These data suggest that this neurosteroid may be considered a new marker of pubertal development.     

Similar effects of inhibin and cycloheximide on gonadotropin release in superfused pituitary cell cultures

The actions of two inhibin preparations and cycloheximide on gonadotropin release were investigated in superfused pituitary cell cultures. Pituitary cells isolated from 18-day-old male rats were grown in Matrigel-coated superfusion chambers in chemically defined medium. After stationary culture for 4 days, the cell monolayers were superfused at a constant speed (0.25 ml/min) and were intermittently stimulated (6 min/h) with 10 nM gonadotropin-releasing hormone (GnRH). Groups of cultures were exposed to the test substances for varying time periods during stationary culture and/or during superfusion. Inhibitory effects of both inhibin preparations on the secretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in response to GnRH pulses were observed after 2 h of exposure and became maximal after about 6 h. Basal secretion of FSH between GnRH pulses was also suppressed, whereas the basal interpulse secretion of LH was not changed. When exposure to inhibin was discontinued, the secretion of both FSH and LH progressively increased and returned to control values by approximately 6 h. Cycloheximide (500 ng/ml) affected gonadotropin release with dynamics similar to those observed for the inhibin preparation. These data support the hypothesis that inhibition of gonadotropin synthesis may be an important step in the molecular mechanism of action by which inhibin regulates gonadotropin release.     

Measurement of dimeric inhibins and effects of active immunization against inhibin alpha-subunit on plasma hormones and testis morphology in the developing cockerel

Inhibins and activins are implicated as endocrine regulators of follicle-stimulating hormone production and of testicular steroidogenesis and spermatogenesis in mammals. The potential involvement of these proteins in cockerels was investigated by measurement of circulating inhibin A, inhibin B, total inhibin alpha-subunit immunoreactivity (ir-alpha), activin A, LH, FSH, and testosterone from the juvenile state through to sexual maturity. Plasma inhibin A remained low between 6 to 12 wk of age and increased approximately threefold (P < 0.05) to a prepubertal peak between Weeks 14 to 18, followed by a gradual decline to the end of the study (Week 24). Although plasma FSH levels were not correlated to inhibin A before Week 16 (r = -0.17), they were negatively correlated from Week 18 (r = -0.49; P < 0.005). Inhibin B levels were below the assay detection limit until 16 wk of age but thereafter rose steadily in parallel with FSH (r = 0.27; P < 0.02) and testosterone (r = 0.35; P < 0.005). Thus, inhibins A and B showed divergent profiles during sexual maturation. Plasma ir-alpha levels were much higher than dimeric inhibin levels throughout, although the relative difference varied with age. Plasma activin A levels were below the assay detection at all times. Juvenile cockerels were actively immunized against a synthetic chicken inhibin alpha-subunit peptide conjugate to determine effects on plasma hormones and on testicular weight, morphology, and activin A content. Immunization generated circulating antibodies that bound (125)I-bovine 32-kDa inhibin but did not affect plasma FSH or testosterone levels at any stage of development. However, immunization reduced postpubertal plasma LH levels (P < 0.05) and promoted increased testicular weight (24%; P < 0.01) and total testicular activin A content (42%; P < 0.001) at 24 wk. Testis weight of immunized birds was positively correlated with inhibin antibody titer (r = 0.61; P < 0.05). Live weight gain was not affected by immunization. Morphometric analysis of testis sections showed that inhibin immunization had no effect on the fractional volume of the seminiferous tubule wall, seminiferous tubule lumen, or interstitial tissue area. Likewise, seminiferous tubule surface area and surface area:volume ratios were not different from controls. These findings support differential roles for inhibins A and B in regulating the pituitary-testicular axis during sexual maturation in the cockerel but highlight the need for more detailed studies to distinguish between potential endocrine and local intragonadal roles of inhibin-related peptides and to elucidate the mechanism by which immunization against inhibin alpha-subunit promotes testis enlargement without raising plasma FSH.     

Peripheral inhibin levels during estrous cycle in buffalo (Bubalus bubalis)

A sensitive, specific RIA was validated and used for measurement of peripheral plasma immunoreactive inhibin (irinhibin) levels during the estrous cycle in Murrah buffalo. The RIA employed an 125-I iodinated inhibin as tracer and an antiserum against dimeric inhibin. The procedure had a sensitivity of 16 pg/tube, and the nonspecific effects of buffalo plasma were compensated for by including 200 ul bullock plasma in the standards. Separation of free and bound inhibin was affected by the use of a second antibody and precipitation with polyethylene glycol. Blood samples were collected once daily for 30 d from Murrah buffalo (n = 6) during the hot month of July. Cyclic activity and estrus were confirmed by plasma progesterone determination. Peripheral plasma concentrations of ir-inhibin fluctuated between 0.40 +/- 0.07 and 0.67 +/- 0.13 ng/ml during the estrous cycle in buffalo. During the same period, plasma progesterone levels increased from 0.21 +/- 0.01 ng/ml at Day 0 to a peak of 3.30 +/- 0.72 ng/ml on Day 13, declining sharply by Day -5. Ir-inhibin levels exhibited an increase during the follicular phase, with the maximum concentration of 0.65 +/- 0.01 ng/ml occuring on the day of estrus, a decline thereafter, and no pattern during the luteal phase. The differences, however, were not statistically significant throughout the estrous cycle.     

Diagnostic utility of a low-dose gonadotropin-releasing hormone test in the context of puberty disorders

OBJECTIVES: The 10-microg gonadotropin-releasing hormone (GnRH) test assesses pituitary gonadotroph responsiveness, whereas the 100-microg dose assesses maximal secretory capacity. Our aims were to establish normative data for the low-dose test in children and to evaluate the test in diagnosing common pubertal disorders. METHODS: We retrospectively classified 107 children who underwent 10-microg GnRH tests into normal prepubertal (20 boys, 10 girls), normal early pubertal (10 boys, 16 girls), constitutional delay of puberty (CDP, 13 prepubertal boys >12 years), hypogonadotropic hypogonadism (HH, 5 prepubertal boys >12 years), central precocious puberty (CPP, 19 girls) or premature thelarche/variant (13 girls). RESULTS: Peak LH response was higher in prepubertal boys >12 years compared with younger boys (p < 0.01) but showed no further change in early puberty. CDP boys had LH responses similar to prepubertal boys >12 years. HH boys showed an absent LH response which diagnosed HH with 100% sensitivity and 96% specificity. Thelarche girls had LH:FSH peak ratios lower than normal prepubertal (p = 0.001), pubertal (p < 0.05) or CPP (p = 0.001) girls. CONCLUSIONS: We have established normative values for the low-dose GnRH test in children. The test successfully differentiated HH from CDP in boys, and contributed to the differential diagnosis of CPP and premature thelarche in girls.