Small-conductance calcium-activated potassium channels control excitability and firing dynamics in gonadotropin-releasing hormone (GnRH) neurons

The cellular mechanisms determining the firing patterns of GnRH neurons are presently under intense investigation. In this study, we used GnRH-green fluorescent protein transgenic mice and perforated-patch electrophysiology to examine the role of small conductance calcium-activated potassium (SK) channels in determining the electrical excitability and burst-firing characteristics of adult GnRH neurons. After establishing an appropriate protocol for examining the afterhyperpolarization potential (AHP) currents in GnRH neurons, the highly selective SK channel blocker apamin was used to demonstrate that all GnRH neurons express functional SK channels (35.7 +/- 2.7 pA, mean decay time constant = 2167 msec, apamin IC(50) = 9.6 nm) and that this channel underlies approximately 90% of the AHP in these cells. Current-clamp experiments showed that apamin-sensitive SK channels were tonically active in the majority (74%) of GnRH neurons, with apamin (100 nm) administration resulting in a mean 6.9 +/- 0.5 mV membrane depolarization. Apamin also elevated the firing rate of GnRH neurons, including increased burst frequency and duration in spontaneously bursting cells as well as the ability of GnRH neurons to fire action potentials in response to current injection. In GnRH neurons activated by current injection, apamin significantly enhanced the amplitude of the afterdepolarization potential after a single action potential and eliminated spike frequency adaptation. Together, these studies show that apamin-sensitive SK channels play a key role in restraining GnRH neuron excitability. Through direct modulation of the AHP and indirect actions on the afterdepolarization potential, the SK channel exerts a powerful tonic influence upon the firing dynamics of GnRH neurons.     

Calcium signaling and episodic secretion of gonadotropin-releasing hormone in hypothalamic neurons.

Gonadotropin-releasing hormone (GnRH) is released episodically into the pituitary portal vessels and from hypothalamic tissue of male and female rats in vitro. Perifused primary cultures of rat hypothalamic neurons, as well as the GT1-1 GnRH neuronal cell line, spontaneously exhibited episodic GnRH secretion of comparable frequency to that observed with perifused hypothalami. Such pulsatile GnRH release from GT1 cells indicates that GnRH neurons generate rhythmic secretory activity in the absence of input from other cell types. In primary hypothalamic cultures, the frequency of GnRH pulses increased with the duration of culture. The spontaneous pulsatility in GnRH release was abolished in Ca(2+)-deficient medium and was markedly attenuated in the presence of nifedipine, an antagonist of voltage-sensitive Ca2+ channels. The basal intracellular Ca2+ level of perifused GT1-1 cells cultured on coverslips was also dose-dependently reduced by nifedipine. Conversely, depolarization with high K+ increased intracellular Ca2+ and GnRH release in an extracellular Ca(2+)-dependent and nifedipine-sensitive manner. The dihydropyridine Ca2+ channel agonist Bay K 8644 increased basal and K(+)-induced elevations of intracellular Ca2+ concentration and GnRH secretion. These findings demonstrate that pulsatile neuropeptide secretion is an intrinsic property of GnRH neuronal networks and is dependent on voltage-sensitive Ca2+ influx for its maintenance.     

Localization of olfactory cyclic nucleotide-gated channels in rat gonadotropin-releasing hormone neurons

The GT1 GnRH cell lines express all three subunits of the cyclic nucleotide-gated (CNG) channels (CNG2, 4.3 and 5) expressed in olfactory neurons. We investigated using in situ hybridization and double immunofluorescence whether endogenous GnRH neurons in therat also express CNG channel subunits. Sections from male and female adult rats were hybridized with a digoxigenin-labeled riboprobe made to regions of rat GnRH, CNG2, CNG4.3 or CNG5 cDNAs. In both sexes, 70-80% of GnRH neurons contained mRNAs for CNG2, CNG4.3 and CNG5. Similarly, double immunofluorescence staining for GnRH and CNG2, CNG4.3 or CNG5 confirmed that 70-80% of GnRH perikarya contained all three CNG subunit proteins. Moreover, the distribution of the immunostaining of CNG subunits in the external layer of the median eminence overlapped with GnRH with the presence of functional cAMP-gated cation channels. The presence of CNG channel subunits in the median eminence supports the notion that coordination of the excitability of the scattered GnRH perikarya may occur at the level of the nerve terminals.     

Serum sex hormone-binding globulin levels in healthy children and girls with precocious puberty before and during gonadotropin-releasing hormone agonist treatment

CONTEXT: The regulation of SHBG is complex and influenced by sex steroids and insulin. OBJECTIVE: Our objective was to describe serum levels and evaluate determinants of SHBG levels in healthy children and in girls with central precocious puberty (CPP) before and during GnRH analog (GnRHa) treatment. DESIGN: We conducted a cross-sectional study on healthy subjects and a 2-yr longitudinal study in girls with CPP. SETTING: The study took place at a tertiary referral center for pediatric endocrinology. PARTICIPANTS/PATIENTS: A total of 903 healthy schoolchildren served as healthy subjects, and 25 girls with precocious/early puberty participated. INTERVENTIONS: Girls with CPP were treated with the long-acting GnRHa triptorelin. RESULTS: SHBG levels declined with increasing age in both sexes until adulthood. In healthy children, SHBG was significantly negatively correlated with testosterone, estradiol, dehydroepiandrosterone sulfate, and body mass index (BMI) in boys (total model R(2) = 0.71) but only with dehydroepiandrosterone sulfate and BMI in girls (total model R(2) = 0.26). Body fat percentage was significantly negatively correlated with SHBG levels (P < 0.001) in both boys (R(2) = 0.18) and girls (R(2) = 0.23). Girls with CPP had significantly lower pretreatment SHBG levels compared with age-matched controls [SHBG sd score, -1.29 (-4.48; 0.01)], which declined even further during GnRHa treatment [-2.75 (-5.9; 0.53); P < 0.001]. Even after adjustment for BMI and pubertal stage, girls with CPP had lower SHBG levels (P < 0.001) compared with healthy controls. CONCLUSIONS: SHBG levels were strongly dependent on body composition and sex steroid levels in children with normal and precocious puberty. Studies on insulin sensitivity and SHBG in puberty are needed to better understand the interaction between body composition and gonadal maturation.     

Determinants of growth during gonadotropin-releasing hormone analog therapy for precocious puberty

In children with precocious puberty (PP), treatment with GnRH analogs (GnRHa) often decreases height velocity below normal. Based on previous animal studies, we hypothesized that this impaired growth is due to excessive advancement in growth plate senescence induced by the prior estrogen exposure. This hypothesis predicts that the height velocity during treatment will be inversely related to the severity of prior estrogen exposure. We analyzed data from 100 girls (age, 5.8 +/- 2.1 yr; mean +/- SD) with central PP who were treated with GnRHa. During GnRHa therapy, height velocity was low for age (-1.6 +/- 1.7 SD score; mean +/- SD). The absolute height velocity correlated most strongly with the bone age (BA), which we used as a surrogate marker for growth plate senescence (r = -0.727, P < 0.001). The severity of the growth abnormality (height velocity SD score for age) correlated inversely with markers of the severity of prior estrogen exposure, including duration of PP (r = -0.375, P < 0.001), Tanner breast stage (r = -0.220, P < 0.05), and BA advancement (r = -0.283, P < 0.01). Stepwise regression confirmed that BA was the best independent predictor of growth during GnRHa therapy. The findings are consistent with our hypothesis that impaired growth during GnRHa therapy is due, at least in part, to premature growth plate senescence induced by the prior estrogen exposure.     

Pulsatile gonadotropin-releasing hormone treatment in idiopathic delayed puberty

Idiopathic delayed male puberty is defined as a delay of puberty beyond the age of 16, with prepubertal testosterone levels, normal gonadotropin responses to GnRH (excluding pituitary failure), and normal androgen responses to a single hCG injection (excluding testicular Leydig cell dysfunction), in absence of serious disease. Ten boys with this condition were evaluated as to their spontaneous LH, FSH, and PRL secretory patterns during a 24-h sampling period (20-min intervals). After this all patients were treated with pulsatile infusions of GnRH (25 ng/kg . pulse every 90 min for 10 days. Two groups could be distinguished by means of their pretreatment LH secretory pattern. Five patients had nighttime pulsatile elevation of LH levels, as usually occurs in early puberty. The other five patients did not have such a pattern (prepubertal type). The GnRH treatment resulted in increased LH and testosterone levels in both groups. All patients with pretreatment nighttime pulsatile LH secretion had steady pubertal development during the post-GnRH treatment observation period, whereas the other patients did not. In conclusion, among a number of tests, including chronic pulsatile GnRH treatment for 10 days, only the nocturnal LH secretory pattern differentiated delayed puberty from permanent hypothalamic hypogonadism in boys.     

Crucial role of a shared extracellular loop in apamin sensitivity and maintenance of pore shape of small-conductance calcium-activated potassium (SK) channels

Activation of small-conductance calcium (Ca(2+))-dependent potassium (K(Ca)2) channels (herein called "SK") produces membrane hyperpolarization to regulate membrane excitability. Three subtypes (SK1-3) have been cloned and are distributed throughout the nervous system, smooth muscle, and heart. It is difficult to discern the physiological role of individual channel subtypes as most blockers or enhancers do not discriminate between subtypes. The archetypical blocker apamin displays some selectivity between SK channel subtypes, with SK2 being the most sensitive, followed by SK3 and then SK1. Sensitivity of SK1 is species specific, with the human isoform being blocked by the toxin, whereas the rat is not. Mutation studies have identified residues within the outer pore that suggest apamin blocks by an allosteric mechanism. Apamin also uses a residue within the S3-S4 extracellular loop to produce a high-sensitivity block. We have identified that a 3-amino acid motif within this loop regulates the shape of the channel pore. This motif is required for binding and block by apamin, suggesting that a change in pore shape underlies allosteric block. This motif is absent in rat SK1, explaining why it is insensitive to block by apamin. The overlapping distribution of SK channel subtype expression suggests that native heteromeric channels may be common. We show that the S3-S4 loop of one subunit overlaps the outer pore of the adjacent subunit, with apamin interacting with both regions. This arrangement provides a unique binding site for each combination of SK subunits within a coassembled channel that may be targeted to produce blockers specific for heteromeric SK channels.     

短期促性腺激素释放激素拟似物促进青春期雌性大鼠的线性生长

目的 探讨短期(4周)促性腺激素释放激素拟似物(GnRHa)对青春期雌性大鼠生长的影响及机制.方法 40只3周龄雌鼠按随机区组设计分为GnRHa处理组(Gn组)、雌激素替代组(E2组)、对照组、卵巢切除组(OVX组)以及基线对照组.Gn组和E2组注射曲普瑞林,每2周1次,共2次;E2组在第2次曲普瑞林注射后每日注射E2共11d;OVX组在实验开始时予卵巢切除.除基线对照组外,所有鼠均在实验结束前9天和2天分别注射盐酸土霉素和钙黄绿素使其在骨表面形成荧光标记.比较4周后4组大鼠的体格生长指标、血胰岛素样生长因子(IGF)-I及IGF结合蛋白(IGFBP)-3浓度、肝脏生长激素受体(GHR)、IGF-I及IGFBP-3mRNA水平、胫骨生长板IGF-I及IGF-I受体(IGF-IR)水平、软骨细胞增殖度等.结果 4周的GnRHa注射达到"性腺切除"效应,Gn组的体重、身长、胫骨长度、生长板参数、纵向生长速率、软骨细胞增殖率均较对照组增加(P<0.05或P<0.01),E2组则与对照组相近.4组的血清IGF-I及IGFBP-3水平、肝脏IGF-I及IGFBP-3mRNA、生长板IGF-I及IGF-IR水平差异无统计学意义.Gn组和OVX组的肝脏GHR mRNA的表达均低于对照组,E2组则与对照组相仿.结论 GnRHa通过抑制雌激素的分泌、间接促进生长板软骨细胞增殖度和抑制生长板老化而促进青春期雌鼠的线性生长,其促生长效应不依赖于IGF-I/IGFBP-3的内分泌机制,也不依赖于生长板IGF-I/IGF-IR的改变.     

Peripheral patterns of growth hormone, luteinizing hormone, and progesterone before, at, and after puberty in buffalo heifer

Buffalo, the premier dairy animal in India, suffers from slow growth rate, delayed puberty, and silent heat. It is not known whether the delay in puberty in such animals is due to the delay in expression of hypothalamus-pituitary-gonadal functions. To determine the changes in growth hormone (GH), luteinizing hormone (LH), and progesterone before, at, and after puberty of Murrah buffalo heifers, six Murrah buffalo heifers (21.92 +/- 1.09 months of age, 269.67 +/- 7.97 kg body weight) were assigned to well-ventilated individual pens and fed a roughage-concentrate diet to provide weight gain of 0.4 kg/day. Blood samples were collected at 3-day intervals during a period of 12 months, and plasma harvested from blood samples was assayed for progesterone, LH, and GH. The day that plasma progesterone was greater than 1 ng/mL for three consecutive sampling days was defined as the day of puberty. Heifers attained puberty at an average age of 31.53 +/- 0.88 months with a body weight of 380.67 +/- 6.42 kg. Progesterone levels were very low (0.20 to 0.30 ng/mL) during the pre-pubertal period. There were two distinct elevations before the day of puberty onset. Plasma LH and GH concentrations increased (P < 0.05) during the months preceding puberty and were highest during the month before puberty. GH and LH were positively correlated (P < 0.05) prior to (r = +0.59) as well as after puberty (r = +0.42). A positive correlation (P < 0.05)between LH and body weight during the pre-pubertal period (r = +0.61) and thereafter, negative correlation (P < 0.05) during post-pubertal period (r = -0.64) was noted. GH and body weight showed positive correlation both before puberty (r = +0.92, P < 0.01) and after puberty (r = +0.32, P < 0.05). Results suggest that both GH and LH are equally important and vital cues in inducing onset of ovarian functions in buffalo heifers.     

The acid-labile subunit of human ternary insulin-like growth factor-binding protein complex in girls with central precocious puberty before and during gonadotropin-releasing hormone analog therapy

The aim of the study was to evaluate serum acid-labile subunit (ALS) concentrations and their relationship with other parameters of the human ternary IGF-I-binding protein (IGFBP) complex in girls with central precocious puberty (CPP) before and after pharmacological arrest of puberty. We studied serum ALS, free IGF-I, total IGF-I, IGFBP-3 levels and IGFBP-3 protease activity in 13 girls, aged 1.6-7.8 yr (mean, 5.9 +/- 2.2), diagnosed as having CPP before and after 6 and 12 months of GnRH analog (GnRHa) therapy. The ALS SD score before treatment was high (1.4 +/- 0.72) and decreased significantly after 6 and 12 months of GnRHa therapy [0.4 +/- 0.54 (P < 0.01) and -0.4 +/- 0.61 (P < 0.01), respectively]. Serum IGF-I and IGFBP-3 were also increased before treatment, but both of these factors remained elevated after 6 and 12 months of GnRH-A therapy [IGF-I SD score, 3.20 +/- 1.64, 2.92 +/- 1.82, and 3.68 +/- 1.94 (P = NS), respectively; IGFBP-3 SD score, 1.02 +/- 0.53, 0.94 +/- 0.68, and 1.22 +/- 0.87 (P = NS), respectively]. Serum free IGF-I levels and IGFBP-3 proteolytic activity did not vary significantly from their pretreatment values during GnRHa therapy. In conclusion, serum ALS levels were elevated in girls with CPP and decreased significantly during the first year of GnRHa therapy. Serum IGF-I and IGFBP-3 levels were also increased before therapy, but their levels were not influenced by treatment. The ALS decrease seems to be the sole GH-dependent factor that parallels the decreases in steroid levels and growth velocity during GnRHa therapy.     

Characterization of pituitary calcium-activated, phospholipid-dependent protein kinase: redistribution by gonadotropin-releasing hormone.

We report the presence in the rat pituitary of a calcium-activated, phospholipid-dependent protein kinase (C kinase), originally described by Takai et al. [Takai, Y., Kishimoto, A., Iwasa, Y., Kawahara, Y., Mori, T. & Nishizuka, Y. (1979) J. Biol. Chem. 254, 3692-3695]. Enzyme activity is absolutely dependent on the simultaneous presence of Ca2+ and phospholipid--in particular, phosphatidylserine. The presence of small amounts of unsaturated diacylglycerol greatly increases the apparent affinity of the enzyme for Ca2+ and phosphatidylserine. Pituitary C kinase is mostly soluble (70%) and partly particulate (30%). Although the soluble form of the enzyme can be detected in a crude cytosol preparation, the particulate form is detectable only after solubilization and anion-exchange chromatography. Administration of a gonadotropin-releasing hormone (Gn-RH) agonist analog, [D-Ser(But)6]des-Gly10-Gn-RH-N-ethylamide, to ovariectomized rats resulted in elevated serum luteinizing hormone levels (245%) accompanied by a decrease in the cytosolic form of the enzyme (60%) and an increase in the particulate form (300%) after 5 min. This apparent activation of the particulate form seems to result from translocation of a soluble C kinase to the membrane. Several endogenous substrate proteins for C kinase ranging from 16 to 100 kDa were identified in pituitary cytosol. Pituitary C kinase might be involved in signal-transduction mechanisms in Gn-RH action, in particular, and in other hypophysiotropic hormones, in general, which operate by means of stimulation of phosphoinositide turnover during which diacylglycerol is liberated.     

Bone mass at final height in precocious puberty after gonadotropin-releasing hormone agonist with and without calcium supplementation

The aim of our longitudinal study was to evaluate bone mass in girls affected by central precocious puberty (CPP) that have reached final height, treated with GnRH agonist triptorelin (GnRHa), with or without calcium supplementation. We studied 48 Caucasian females affected by CPP (age at diagnosis, 7.19 +/- 0.96 yr), randomly assigned to two groups: group A (n = 21) treated with GnRHa and group B (n = 27) treated with GnRHa plus calcium gluconolactate and carbonate (1 g calcium/day in two doses) for at least 2 yr. Auxological parameters (standing height, weight, body mass index) and bone mineral density (BMD) at the lumbar spine [L2-L4, anteroposterior (AP)-BMD; lateral BMD; volumetric (v)BMD)] by dual-energy x-ray absorptiometry were evaluated at the beginning [chronological age (CA), 7.29 +/- 0.91 yr; bone age (BA), 8.80 +/- 1.24 yr] and end of treatment (CA, 11.27 +/- 0.97 yr; BA, 12.35 +/- 0.43 yr) and at final height (CA, 16.17 +/- 1.9 yr; BA, 16.93 +/- 0.98 yr, in each case >15 yr). Total bone mineral content, total BMD, and fat percentage were evaluated at the end of the study period using dual-energy x-ray absorptiometry. Final height was significantly higher than predicted height at diagnosis (159.9 +/- 6.3 cm vs. 152.9 +/- 9.6 cm; P < 0.05). Body mass index and fat percentage were not statistically different from control values. Densitometric values at final evaluation in groups A and B together were lower than in controls, but the differences were not statistically significant. The vBMD was significantly higher in group B than in group A at the end of treatment period (0.213 +/- 0.022 g/cm(3) vs. 0.192 +/- 0.021 g/cm(3); P < 0.01) and at final evaluation (0.246 +/- 0.023 g/cm(3) vs. 0.227 +/- 0.024 g/cm(3); P < 0.05). The percentage change (Delta%) between the start and end of treatment period in AP-BMD and vBMD was significantly higher in group B than in group A (Delta% AP-BMD: 20.36% +/- 1.10% vs. 16.16% +/- 1.90%, P < 0.01; Delta% vBMD: 19.08% +/- 3.52% vs. 9.26% +/- 5.15%; P < 0.01) and also between the start of treatment and final evaluation (Delta% AP-BMD: 61.23% +/- 1.61% vs. 56.97% +/- 1.45%, P < 0.01; Delta% vBMD: 36.69% +/- 5.01% vs. 28.01% +/- 5.76%, P < 0.01). In all our females with CPP treated with GnRHa, bone densitometric parameters were in the normal range for age and sex. However, bone mass achievement seemed to be better preserved in the group of patients supplemented with calcium.     

GnRHa治疗真性性早熟过程中生长速度的相关因素分析

目的分析中枢性性早熟（CPP）女孩接受促性腺激素释放激素类似物（GnRHa）治疗过程中线性生长速度（GV）的相关因素，以及影响两年GnRHa疗效的因素。方法将86例已接受GnRHa治疗满2年的CPP女孩治疗第2年中的生长速度（GV第2年）、2年GnRHa治疗对于改善成年身高的疗效（APAH）与遗传靶身高、发病年龄、骨龄、年龄等参数进行相关分析及逐步回归分析。结果GnRHa治疗中GV呈逐年下降趋势，GV第2年与发病年龄、治疗开始和治疗第1年末的年龄、骨龄负相关（r分别为-0．37，-0．59，-0．57，-0．51和-0．52，均P〈0．01）；治疗开始的骨龄和治疗第1年末的年龄是独立影响GV第2年的两个因素。APAH与治疗开始的青春期病程、治疗两年骨龄增长和年龄增长的比值（ABA／ACA）负相关，与第1年GV（GV第1年）、GV第2年、以及2年的平均GV正相关；△BA／△CA、GV第1年和GV第2年是能独立影响APAH的3个因素。结论GnRHa治疗后GV的减慢在一定程度上是由于治疗前过早接受雌激素、导致长骨干骺生长板过度老化的结果；CPP患儿宜早期治疗、最大程度减少因生长板过度老化所致生长潜力的丧失；而尽可能地抑制骨龄以及维持一定的GV是GnRHa疗效的两个重要保证。     

Somatomedin-C in normal puberty and in true precocious puberty before and after treatment with a potent luteinizing hormone-releasing hormone agonist

To explore further the relationship of gonadal sex steroids to the rise in somatomedin-C (Sm-C) during puberty, we studied a group of children with true precocious puberty before and after treatment which suppressed sex steroid output. Plasma estradiol and testosterone and serum acid-ethanol-extractable Sm-C were determined by specific RIAs in 7 boys and 12 girls with true precocious puberty before and at regular intervals during treatment with a potent LHRH-agonist (LHRH-A), D-Trp6-Pro9-NEt-LHRH. For comparison, Sm-C and sex steroid concentrations were determined in 266 normal adolescents and 37 normal prepubertal children, 1-9 yr of age. The mean +/- SEM Sm-C levels in normal male individuals peaked at 15 yr (2.46 +/- 0.23 U/ml) and at pubertal (genital) stage III (2.29 +/- 0.19 U/ml), and those in normal females reached their highest concentration at 12-15 yr of age and at pubertal (breast) stage III (2.47 +/- 0.15 U/ml). Sm-C concentrations correlated better with pubertal (genital or breast) stage than with chronological age for both sexes and better with testosterone levels in males than with estradiol levels in females. The mean +/- SEM Sm-C concentrations in both males and females with true precocious puberty were 2.07 +/- 0.16 U/ml before therapy and decreased significantly to 1.52 +/- 0.13 U/ml after 6 months of therapy. The mean Sm-C level of the patients remained significantly elevated for chronological age, but decreased into the normal range for bone age after 6-12 months of therapy. Sm-C correlated significantly with testosterone and estradiol levels, but not with growth rate. Mean nighttime GH secretion decreased significantly after 6 months of LHRH-A therapy. In summary, children with true precocious puberty have Sm-C elevations typical of normal puberty. The decrease in Sm-C levels after suppression of gonadal sex steroid output with LHRH-A is evidence that sex steroids are necessary to induce this elevation in Sm-C concentration. The decrease in GH secretion during LHRH-A therapy suggests that the effect of sex steroids on Sm-C levels during normal puberty is mediated, at least in part, through stimulation of GH secretion.     

Prevention of bone demineralization by calcium supplementation in precocious puberty during gonadotropin-releasing hormone agonist treatment.

We have previously demonstrated a negative impact on peak bone mass in girls with precocious puberty treated with GnRH agonist (GnRHa). Several studies have shown that a high calcium intake positively influences bone mass in prepubertal girls and leads to a higher peak bone mass. The aim of this study was to evaluate the effect of calcium supplementation in girls with precocious puberty during GnRHa treatment. Forty girls affected by true central precocious puberty and treated with the GnRHa triptorelin were studied for 2 yr. After diagnosis, the patients were randomly assigned to three groups: group A, treated only with GnRHa; group B, treated for 12 months solely with GnRHa and then supplemented with calcium gluconolactate/carbonate (1 g calcium/day in two doses) for 12 months; and group C, treated from the beginning with combined GnRHa and calcium. Bone mineral density (BMD) at the lumbar spine was measured by dual energy x-ray absorptiometry at the beginning of the study and after 12 and 24 months and was expressed as the calculated true volumetric density (BMDv) in milligrams per cm3. Group A showed a decrease in absolute BMDv levels, in SD score for chronological age (CA), and even more in SD score for bone age (BA). Group B showed the same behavior during the first year, but this trend was reversed in the second year, when calcium supplementation was added to GnRHa treatment. Group C showed an increase in absolute BMDv levels and in SD score for CA and BA. BMDv variations (expressed as absolute values, SD score for CA, and SD score for BA) became statistically significant at 24 months between groups C and A (P = 0.036, P = 0.032, and P = 0.025, respectively). The behavior of the lumbar spine BMDv in the three groups is consistent with a positive effect of calcium supplementation during GnRHa treatment. In calcium-supplemented patients, the normal process of bone mass accretion at puberty is preserved despite GnRHa treatment. Therefore, the reduction in BMD during GnRHa treatment in girls with precocious puberty is at least completely reversible and preventable if calcium supplementation is associated from the beginning.