Divergence and polymorphism analysis of IGF1Ra and IGF1Rb from orange-spotted grouper, Epinephelus coioides (Hamilton).

Orange-spotted grouper (Epinephelus coioides) is one of the most important marine fish and has a high market value. The insulin-like growth factor type 1 receptor (IGF1R) is a component of the insulin-like growth factor signaling system, and demonstrates important roles during growth. Based on information from livestock, we used IGF1R as a candidate gene to survey single nucleotide polymorphisms. In the present study, the sequences of IGF1Ra and IGF1Rb from orange-spotted grouper were obtained from the genome sequences and their clustering in clades a and b, respectively, was confirmed by phylogenetic analysis. Fourteen critical amino acids underlying functional divergence were detected between the two clades, revealing the molecular basis of their functional differences. Nearly one-fourth (22 kbp) of the genomic sequence of IGF1Ra was sequenced in a mass cross population, and nucleotide diversity and linkage disequilibrium levels were investigated. Nucleotide diversity was 0.00328 for π and 0.00344 for θw. The half decay of the squared allele-frequency correlation was 10,835 base pairs. Comparatively, the relatively high level of linkage and the significant deviation from neutrality-based codons in IGR1R showed that this gene was under selection. A site (KR269824.1:g. 63762C>T), located in the sixth intron, was significantly associated with eyeball diameter (P = 1.39 x 10-4, Q-value: 2.33 x 10-2), which accounted for 11.1% of phenotypic variance. These results highlight the important function of IGF1R in orange-spotted grouper and may be beneficial in the breeding of this species.

Isolation and characterization of novel polymorphic microsatellite markers for Epinephelus akaara.

In the present study, ten novel microsatellite markers were developed from an enriched-(CA)13 genomic library of Epinephelus akaara. The mean number of alleles per locus was 21.6, with a range of 12 to 33. Observed heterozygosity ranged from 0.767 to 0.967, and expected heterozygosity ranged from 0.831 to 0.975, with mean values of 0.877 and 0.923, respectively. Among the ten loci, three loci deviated from Hardy-Weinberg equilibrium after sequential Bonferroni's correction. These polymorphic microsatellite markers may be useful for studies on the population genetics of E. akaara.

Nineteen polymorphic microsatellite markers developed for Trachinotus ovatus.

To evaluate the population genetic diversity of the ovate pompano, we isolated and characterized 19 microsatellite markers using a (CA)13-enriched genomic library. Polymorphism was assessed in 30 individuals from a single population collected from the Daya Bay Aquaculture Center, Guangdong, China. The number of alleles per locus ranged from 2 to 18 with an average of 7.8. The observed and expected heterozygosities varied from 0.2667 to 1.000 and from 0.3960 to 0.9435, respectively. Sixteen of 19 loci conformed to Hardy-Weinberg equilibrium, and no significant linkage disequilibrium was detected between any locus pairs. Our study supplies candidate microsatellite markers that can be useful for studying the population genetic structure of ovate pompano.

Soluble interleukin-1 receptor, a potential negative regulator of orange-spotted grouper Epinephelus coioides interleukin-1 system.

In this study, the cDNA sequence encoding interleukin-1 (Il-1) receptor-like protein of orange-spotted grouper Epinephelus coioides was obtained. The newly identified sequence was named soluble type I Il-1 receptor (sIl-1rI) owing to its structural composition, which had two Ig-like domains, lack of transmembrane region and the Toll/interleukin-1 receptor (TIR) domain, similar to the brown rat Rattus norvegicus soluble Il-1rI. In addition, sequence comparison and phylogenetic analysis indicated that E. coioides sequence had a closer relationship with Il-1rI than Il-1rII. Real-time PCR revealed that sil-1rI mRNA expression presented a process of decrease, restoration and increase in Cryptocaryon irritans-infected E. coioides. The negative correlation between Il-1ß and sil-1rI mRNA in C. irritans-infected head-kidney implied the potential negative regulatory role of sil-1rI in E. coioides Il-1 system. The leucocytes incubated with lipopolysaccharide or polyriboinosinic polyribocytidylic acid exhibited different expression profiles of sil-1rI. Recombinant Il-1ß (rIl-1ß) protein was capable of inducing sil-1rI mRNA under the concentration of 100 ng ml(-1) , suggesting that high dosage or excess Il-1ß would stimulate the expression of sil-1rI to maintain the homoeostasis of E. coioides Il-1 system. For the first time, the role of teleost Il-1rI in parasite infection has been identified, and soluble Il-1r was found in fish.

Genetic variation and balancing selection at MHC class II exon 2 in cultured stocks and wild populations of orange-spotted grouper (Epinephelus coioides).

Major histocompatibility complex (MHC) molecules play vital roles in triggering adaptive immune responses and are considered the most variable molecules in vertebrates. Recently, many studies have focused on the polymorphism and evolution mode of MHC in both model and non-model organisms. Here, we analyzed the MHC class II exon 2-encoding ß chain in comparison with the mitochondrial Cytb gene and our previously published microsatellite data set in three cultured stocks and four wild populations of the orange-spotted grouper (Epinephelus coioides) in order to investigate its genetic variation and mechanism of evolution. We detected one to four alleles in one individual, suggesting that at least two loci exist in the orange-spotted grouper, as well as a particularly high level of allelic diversity at the MHC loci. Furthermore, the cultured stocks exhibited reduced allelic diversity compared to the wild counterparts. We found evidence of balancing selection at MHC class II exon 2, and codon sites under positive selection were largely correspondent to the protein-binding region. In addition, MHC class II exon 2 revealed significant differences between population differentiation patterns from the neutral mitochondrial Cytb and microsatellites, which may indicate local adaptation at MHC loci in orange-spotted grouper originating from the South China Sea and Southeast Asia.

Identification and expression analysis of major histocompatibility complex IIB gene in orange-spotted grouper Epinephelus coioides.

In this study, complementary DNA (cDNA) and DNA sequences of major histocompatibility complex (MHC) class IIB genes (mhcIIB) were cloned from orange-spotted grouper Epinephelus coioides. The gene structure of E. coioides mhcIIB consists of five exons and four introns, and its deduced amino acid sequence length is 249 amino acids, including a signal peptide, a peptide-binding region, an IGC1 domain, a transmembrane region and a cytoplasmic tail. A phylogenetic study showed that E. coioides mhcIIB shared 32.0-79.1% identity with those of other teleosts and mammals. Real-time reverse transcriptase (RT)-PCR was performed to detect the class IIB gene expression in eight different tissues. To characterize the relationship between E. coioides mhcIIB gene and pathogens, in vivo and in vitro studies were performed. Challenge of Cryptocaryon irritans revealed that class IIB genes were down-regulated after 24 and 48 h of challenge, and their expression was later restored at 72 h. Stimulation of isolated E. coioides leukocytes with lipopolysaccharide (LPS) and polyinosinic:polycytidylic acid (PolyI:C) significantly increased peripheral blood and spleen mhcIIB expression, while head kidney mhcIIB expression remained constant.

Development and characterization of 32 microsatellite loci in the giant grouper Epinephelus lanceolatus (Serranidae).

An economically important marine fish species, the giant grouper Epinephelus lanceolatus (Serranidae) is widely cultured in Taiwan and costal areas of China. We isolated and characterized 32 polymorphic microsatellite loci from a CA-enriched genomic library of giant grouper. The number of alleles per locus ranged from 3 to 7, with a mean of 4.69. Observed and expected heterozygosities per locus varied from 0.387 to 1.000 and from 0.377 to 0.843, respectively. Six loci significantly deviated from Hardy-Weinberg equilibrium. After sequential Bonferroni's correction, only two loci showed deviation from Hardy-Weinberg equilibrium, and no linkage disequilibrium was found between any pair of loci. These microsatellites can be useful tools for the study of population genetics in the giant grouper.

[Clinical and pathological features and mutational types of WT1 mutation-associated nephropathy].

Objective: To explore the clinical and pathological features and mutational types and their relations with WT1 mutation-associated nephropathy (WT1MAN). Methods: The clinical and pathological data and the results of WT1 mutation analysis of the cases from Nanfang Hospital of Southern Medical University, Sun Yat-sen Memorial Hospital and The First Affiliated Hospital of Sun Yat-sen University whom we recruited recently and reported during the last ten years were analyzed. Results: Totally, 20 cases (6 males and 14 females), included 5 newly diagnosed cases, were recruited. (1) Ten children were diagnosed with Denys-Drash syndrome (DDS): The median onset age of proteinuria was 1 year and 7 months. Diffuse mesangial sclerosis (DMS) were revealed in 3 cases, minimal lesions (MCD) in 4 cases, and focal segmental glomerulosclerosis (FSGS) in 1 case; renal pathology was not available in the other 2 cases. Glomerular basement membrane (GBM) thickening was observed in 2 cases. Calcineurin inhibitors (CNIs) were administered in 5 cases, complete remission of proteinuria was observed in 3 cases, partial remission in the other 2 cases. Genetic analysis revealed that six cases had WT1 missense mutation, 3 had nonsense mutation, and 1 had frameshift mutation. (2) Two cases were diagnosed with Frasier syndrome (FS): proteinuria was observed at 1 year and 1 month of age and 1 year and 9 months of age, respectively. FSGS with GBM layering were observed in both cases. They progressed to ESRD at 1 year and 6 months of age and 6 years and 6 months of age, respectively. CNI was tried in 1 case with partial proteinuria remission. Both patients were detected to have WT1 splice mutation. (3) Isolated nephropathy (IN) was observed in 8 cases: three had splice mutation, 5 had missense mutation. Of the 3 patients with splice mutation, one was found to have nephropathy and renal failure at the age of 5 months. The other two cases (1 was FSGS and another MCD), both had GBM layering. CNIs were tried on both of them, one got partial remission with normal renal function at the age of fourteen years, the other one had no response and entered ESRD at the age of 6 years and 9 months. Of the 5 cases with missense mutation, 3 had DMS, 2 of them entered ESRD within 6 months of age, another case had DMS entered ESRD at 9 years of age. One case with FSGS, was treated with CNIs and got complete remission. Conclusions: Slow progression (7/10) nephropathy was observed in DDS patients. Missense mutation (11/20) was the most common type of WT1 variants, followed by splice mutation (5/20) in this group of patients. Early onset nephropathy (4/5), rapid progression (4/5) and GBM layering (4/4) wereobserved in patients with splice mutation. CNI was effective in reducing or even eliminating proteinuria in WT1 MAN patients (8/9).

[Clinical features and gene mutation analysis of 13 Chinese juvenile patients with nephronophthisis].

Objective: To explore the clinical features and pathogenic gene mutation of juvenile nephronophthisis (NPHP) in Chinese patients. Method: Clinical data and blood samples of 27 juvenile NPHP patients from 25 families who were initially clinically diagnosed in six hospitals in Guangdong province were collected. NPHP1 homozygous deletions were detected in all patients. Sequencing of NPHP1 gene was performed when homozygous deletions were not found in patients without eye involvement. In patients with eye involvement, NPHP5 sequencing was carried out initially and subsequently NPHP10 gene and NPHP1 when there were no NPHP5 gene mutation found. Result: Diagnosis was confirmed in 13 patients by renal pathology and (or) gene sequencing, including four boys and nine girls with a median onset age of 8.5(0.1-12.8) years. Seven of the 13 patients had a normal routine urine test and six patients had mild to moderate proteinuria. None had persistent hematuria. The estimated glomerular filtration rate of the 13 patients was (12.7±10.7) ml/(min·1.73 m2) at the time of diagnosis. Renal cysts were found in only five patients by iconography. Decreased renal size was observed in nine cases and normal renal size in four patients. Renal pathology was available in five patients, renal cysts formation at the cortical-medullar area, thickening and laying tubular basement membrane, were observed. Two of the thirteen children had eye involvement, one had liver impairment and one had growth retardation. NPHP1 gene defects were detected in seven patients with a mutation rate of 25.9%, and large homozygous deletions were observed in three patients. Four patients had single point mutations, i. e. compound heterozygous mutations (c.13 C>T and c. 1520+ 5 G>A) in one patient; homozygous mutation in three patients, two patients were siblings from the same pedigree harbored c. 1756 C>T and the other one harbored c. 1298delA. NPHP5 gene homozygous mutation was found in one pedigree. The fourteen children without renal pathology and whose genetic tests were negative shared similar clinical features with the thirteen patients whose diagnosis were confirmed by gene mutation and (or) renal pathology. Conclusion: The onset of juvenile NPHP is insidious. Urine and renal iconography changes are mild or negative. The ratio of NPHP1 mutant patient is similar with previous reports, but the proportion of NPHP1 gene homozygous deletions is much lower and all of the NPHP1 gene single point mutations detected in this research were novel, which indicates a genetic discrepancy existed between Chinese NPHP patients and the western ones.

Seabream growth hormone receptor: molecular cloning and functional studies of the full-length cDNA, and tissue expression of two alternatively spliced forms.

A full-length clone of the growth hormone receptor (GHR) was isolated from a cDNA library constructed from the liver of black seabream (Acanthopagrus schlegeli). The seabream GHR (sbGHR) cDNA sequence encodes a transmembrane protein of 640 amino acids (aa) possessing the characteristic motifs and architectural design of GHRs of other species. When compared to the other fish GHRs, it is most homologous to another marine fish species, the turbot, where the aa identity is 79.3%. But the sbGHR sequence is more remotely related to the goldfish GHR (51.6% aa identity) and the salmonid GHRs (approximately 46-48% aa identities). Phylogenetic comparison with other known GHRs indicates that the fish GHRs constitute a distinct group among the different vertebrate classes. The aa identities between sbGHR and other GHRs are low, being around 40% with mammalian GHRs, around 45% with avian and reptilian GHRs, and less than 35% with Xenopus GHR. CHO cells transfected with the sbGHR cDNA can be stimulated to proliferate by recombinant seabream growth hormone (sbGH). In addition, the transfected cells can transactivate a co-expressed mammalian serine protease inhibitor (Spi) 2.1 promoter upon stimulation by sbGH. These functional assays indicated that the fish receptor can interact with its homologous ligand to evoke the downstream post-receptor events. Reverse transcription-polymerase chain reaction (RT-PCR) and genomic PCR using a pair of gene-specific primers revealed the expression of two alternatively spliced forms of sbGHR in various tissues of the fish. A 93-bp intron, unique to the sbGHR gene and not found in any other known GHR genes, is alternatively spliced to give rise to two forms of receptor mRNA transcripts. The two forms of the receptor are differentially expressed among the different tissues of the fish.

Expression of grass carp growth hormone by baculovirus in silkworm larvae.

A total of five recombinant Bombyx mori nuclear polyhedrosis viruses (BMNPV) carrying the grass carp (Ctenopharyngodon idellus) growth hormone (GH) cDNA were constructed in this study. Two of them were able to express the hormone up to a level of 12 microgram/ml medium when cultured B. mori cells were infected for 4 days. Inoculation of the viruses into silkworm (B. mori) host significantly increased the level of GH achievable. The amount of hormone produced per larva was estimated to be around 1 mg. The recombinant grass carp GH had immunological and biological activities similar to the native hormone. The N-terminal sequence of the recombinant hormone was the same as the native one, indicating that the fish signal peptide was correctly processed by the insect cells. Silkworm powder prepared from larvae infected with the recombinant virus was used as food supplement for fish. Compared with the control, this dietary supplement was effective in increasing the growth rate of juvenile carp.

Synergistic transcriptional activation by Sox10 and Sp1 family members.

Neuronal nicotinic acetylcholine receptors (nAChR) are expressed at specific times during development and in discrete neuronal populations. Transcriptional regulation of the receptor genes clearly plays a key role in the molecular pathway underlying the expression of these critical synaptic components. In an effort to understand this regulation, we focus upon the genes encoding three receptor subunits: alpha3, alpha5 and beta4. These subunits are genomically clustered and constitute the predominant nAChR subtype expressed in the peripheral nervous system. We and others demonstrated that the general transcription factors, Sp1 and Sp3, can transactivate the promoter of each subunit gene. Further, we showed that the regulatory factor Sox10 transactivates the alpha3 and beta4 promoters and does so in a cell-type-specific manner. Interestingly, the Sp- and Sox10-binding sites on the beta4 promoter are located immediately adjacent to each other, raising the possibility that the two sets of factors functionally interact to regulate receptor gene expression. Consistent with this hypothesis, we demonstrated that the proteins can directly interact. Here, we extend these observations and show that Sox10 and the Sp factors functionally interact, leading to synergistic transcriptional activation in a cholinergic cell line. Finally, evidence for the existence of cell-type-specific co-regulators for Sp1 and Sox10 is presented.

Carbopol/pluronic phase change solutions for ophthalmic drug delivery.

The major purpose of this study is to develop and characterize a series of carbopol- and pluronic-based solutions as the in situ gelling vehicles for ophthalmic drug delivery. The rheological properties, in vitro release as well as in vivo pharmacological response of various polymer solutions, including carbopol, pluronic and carbopol/pluronic solution, were evaluated. It was found that the optimum concentration of carbopol solution for the in situ gel forming delivery systems was 0.3% (w/w), and that for pluronic solution was 14% (w/w). The mixture of 0.3% carbopol and 14% pluronic solutions showed a significant enhancement in gel strength in the physiological condition; this gel mixture was also found to be free flowing at pH 4.0 and 25 degrees C. The rheological behaviors of carbopol/pluronic solution were not affected by the incorporation of pilocarpine hydrochloride. Both the in vitro release and in vivo pharmacological studies indicated that the carbopol/pluronic solution had the better ability to retain drug than the carbopol or pluronic solutions alone. The results demonstrated that the carbopol/pluronic mixture can be used as an in situ gelling vehicle to enhance the ocular bioavailability.

Effects of food deprivation on expression of growth hormone receptor and proximate composition in liver of black seabream Acanthopagrus schlegeli.

The effects of food deprivation on the hepatic level growth hormone receptor (GHR) were investigated in black seabream (Acanthopagrus schlegeli) both at the protein level (by radioreceptor assay) and at the mRNA level (by ribonuclease protection assay). Serum levels of growth hormone (GH) and triiodothyronine (T(3)) were also measured. Condition factor and hepatic proximate composition of the fish were also assessed. Significant decrease in hepatic GHR binding was recorded as early as on day 2 of starvation. On day 30 this decrease was even more pronounced, with the level in the starved fish reaching less than 20% the fed control level. A concomitant decrease in the hepatic GHR mRNA content was also noted during this period, with a progressive decrease from day 2 to day 30 of starvation. The extent of decrease in the mRNA content was less pronounced than the decrease in receptor binding, with the hepatic GHR mRNA content in the day 30 starved fish representing approximately 30% of the level in the fed control. In large contrast, serum GH level increased progressively during starvation. After 30 days of starvation, serum GH levels in the starved fish were more than three times the concentration found in the fed control. Serum T(3) levels, on the other hand, decreased during starvation, with the difference reaching significance on day 15 and day 30. After 30 days of starvation, serum T(3) levels in the starved fish were only approximately 40% the concentration found in the fed control. The hepatic lipid content exhibited an increasing trend during starvation. On day 30 the hepatic lipid content of the starved fish had doubled the level found in the fed control. However, the hepatic protein content did not exhibit much change during starvation. There was also a minor decrease in the moisture content of the liver during starvation, but the condition factor of the fish as a whole registered a gradual decrease during the course of food deprivation.

Analysis of human in vitro fertilization failure.

Fertilization failure is a serious problem in human in vitro fertilization (IVF) programs and deserves further investigation and management. Two hundred and ninety-four IVF cycles performed at the National Taiwan University Hospital from July 1989 to June 1991 were retrospectively analyzed. Thirty-seven (13%) of the 294 cycles were observed to have fertilization failure. The incidence of fertilization failure in male factor patients was significantly higher (p < 0.05) than in others. Patients with oligoasthenospermia tended to have a higher rate of fertilization failure than patients with oligospermia or asthenospermia alone. In non-male factor patients, a smaller number of oocytes and mature oocytes was found in patients with fertilization failure than in patients achieving fertilization. Sperm penetration assay (SPA) using zona-free hamster eggs was performed for 15 patients with fertilization failure; no correlation was found between SPA and the fertilizing ability of the sperm in vitro. Four patients with severe oligoasthenospermia had repeated fertilization failure in subsequent IVF cycles. The other five patients, including four non-male factor patients and one male factor patient, achieved fertilization in their second IVF trial. Our results suggest that severe oligoasthenospermic patients with repeated fertilization failure should be candidates for micromanipulation of gametes in subsequent IVF trials.

Treatment of patients with unexplained infertility: gamete intrafallopian transfer (GIFT) versus tubal embryo transfer (TET).

This study compares the results of 65 cycles of gamete intrafallopian transfer (GIFT), and 19 cycles of tubal embryo transfer (TET) in couples with unexplained infertility (UI). Oocyte retrievals were carried out by laparoscopy in GIFT and transvaginally in TET, in which the embryos were transferred by laparoscopy into the fallopian tubes 48 hours later. The mean age, duration of infertility, serum estradiol levels on the day of human chorionic gonadotropin administration, number of large follicles (mean diameter greater than 10 mm) and the number of oocytes recovered were similar between these two groups. From the 65 GIFT cycles, 20 clinical pregnancies resulted (30.8%). From the 19 cycles of TET, 10 conceptions occurred (52.6%). The implantation and pregnancy rates after TET were higher than that after GIFT, but the differences were not statistically significant. The data suggest that GIFT has a similar success rate to TET in couples with UI.

The use of a long-acting gonadotropin-releasing hormone analog(D-Trp-6-LHRH) for improvement of ovarian stimulation in assisted conception programs.

To assess the efficacy of gonadotropin-releasing hormone analog (GnRHa) as an adjuvant in controlled ovarian stimulation in assisted conception programs, 114 infertile patients, who were treated by in vitro fertilization and embryo transfer (n = 61) or tubal embryo transfer (n = 53), were randomized sequentially to receive ovarian stimulation according to two protocols. In protocol 1 (n = 57), long-acting GnRHa (D-Trp-6-LHRH) microcapsules were administered intramuscularly at menstruation and ovarian stimulation using follicle-stimulating hormone (FSH) and human menopausal gonadotropin (hMG) was started 2 to 3 weeks later when the pituitary was completely suppressed. In protocol 2 (n = 57), patients received FSH and hMG from day 3 of the cycle without GnRHa pre-treatment. We found that premature luteinization did not occur in patients treated with protocol 1, and the number of cycles cancelled was also decreased. The days of ovarian stimulation and the amount of hMG required to achieve adequate follicular development were significantly higher in protocol 1 than that in protocol 2. Similarly, the mean serum estradiol levels on the day of human chorionic gonadotropin administration, number of large follicles (mean diameter greater than 10 mm), number of oocytes recovered and number of embryos obtained were also significantly higher in patients treated with protocol 1. The data suggest that the use of D-Trp-6-LHRH as an adjuvant in ovarian stimulation is associated with a lower incidence of cycle cancellation and an improvement in ovarian response in assisted conception programs.

Pregnancy after transfer of frozen-thawed human embryos.

Cryopreservation of human embryos has been successfully applied in in vitro fertilization (IVF) and embryo transfer (ET) programs at the National Taiwan University Hospital since 1988. Our preliminary results with 120 frozen-thawed embryos in 31 transfer cycles showed that the survival rate of frozen embryos was 66%. Following transfer, the implantation rate and clinical pregnancy rate were 6.5% and 13%, respectively. Four clinical pregnancies and one preclinical pregnancy following a frozen-thawed embryo transfer were achieved. Two normal male babies have been delivered and another pregnancy is progressing without any problem.* Unfortunately, one pregnancy was terminated due to intrauterine fetal death discovered at the 10th week of gestation; chromosome abnormality (47, XX, +5) of the fetus was found. The single preclinical pregnancy showed an elevation of serum beta-human chorionic gonadotropin levels for three consecutive weeks following ET, but no definite gestational sac was visualized by transvaginal ultrasound.

The regulatory effects of thyrotropin-releasing hormone on growth hormone secretion from the pituitary of common carp in vitro.

The effects of thyrotropin-releasing hormone (TRH) on growth hormone (GH) and gonadotropin (GtH) release, and the influences of somatostatin (SRIF), the dopamine agonist apomorphine (APO) and extracellular calcium on basal and TRH-induced GH release were examined using an in vitro perifusion system for pituitary fragments of common carp (Cyprinus carpio). Five minute pulses of different dosages of TRH stimulated a rapid and dose-dependent increase in GH release from the perifused pituitary fragments with an ED50 of 9.7 ± 2.3 nM. TRH was ineffective on GtH release. SRIF significantly inhibited basal and TRH-induced GH release from the perifused pituitary fragments, and the effects of SRIF were dose-dependent. APO induced a dose-dependent increase in basal and TRH-stimulated GH release from the perifused pituitary fragments. Increasing the concentrations of extracellular calcium from 0 mM to 1.25 mM resulted in an increase in basal and TRH-induced GH release. The high dose of calcium (6.25 mM) caused a slight decrease in basal and TRH-induced GH release compared with those at a concentration of 1.25 mM.