Biomedical subjects

M D Culler

Publications and source records attributed to M D Culler.

At least 37 records · Page 2Linked to original sources

PubMed2001

Bim-23244, a somatostatin receptor subtype 2- and 5-selective analog with enhanced efficacy in suppressing growth hormone (GH) from octreotide-resistant human GH-secreting adenomas.

Although both somatostatin receptor subtype 2 (SSTR2) and SSTR5 messenger ribonucleic acid (mRNA) are consistently expressed in GH-secreting adenomas, SSTR2 has been believed to be the key modulator of somatostatin-mediated inhibition of GH release. The somatostatin agonists currently in clinical use, octreotide and lanreotide, are directed mainly to SSTR2 (IC(50) 12- to 18-fold higher than for SSTR5). Recently, however, it was demonstrated that an SSTR5 preferential agonist, BIM-23268, not only suppressed PRL release from prolactinomas and mixed GH-PRL adenomas, but also inhibited GH release in about half of GH adenomas. In addition, the SSTR5-preferring analog showed a slight additive effect when used in combination with SSTR2 preferential drugs at submaximal concentrations in octreotide partially sensitive adenomas. In the present study we quantified SSTR2 and SSTR5 mRNA expression and the GH-suppressive effects of somatostatin-14; octreotide; a SSTR2-preferential compound, BIM-23197; a SSTR5-preferential compound, BIM-23268; and a new SSTR2- and SSTR5-bispecific compound, BIM-23244, in GH-secreting tumors classified as either full responders to octreotide (n = 5) or partially sensitive to octreotide (n = 5). The octreotide-sensitive GH secretory adenomas presented with a high level of both SSTR2 and SSTR5 mRNA expression [222 +/- 61 and 327 +/- 136 pg/pg glyceraldehyde-3-phosphate dehydrogenase (GAPDH), respectively]. In these tumors the suppression of GH release was similarly achieved at picomolar ranges by octreotide, BIM-23197, and BIM-23244 (EC(50) = 25 +/- 15, 3 +/- 2, and 3 +/- 3 pmol/L, respectively). The compounds preferential for only SSTR5 were unable to inhibit GH release in such tumors. Among the octreotide partially responsive tumors, SSTR2 mRNA expression was 9-fold lower than in the octreotide-sensitive tumors (25 +/- 12 vs. 222 +/- 61 pg/pg GAPDH; P < 0.015), whereas SSTR5 mRNA expression was approximately 7-fold higher than in the octreotide-sensitive tumors (2271 +/- 1197 pg/pg GAPDH). In these octreotide partially responsive tumors, the SSTR5-preferential compound, BIM-23268, and the SSTR2- and SSTR5-bispecific compound, BIM-23244, were quite effective in suppressing GH secretion (EC(50) = 25 +/- 13 and 50 +/- 31 pmol/L, respectively). Similarly, BIM-23244, was able to suppress by 51 +/- 5% PRL release from five mixed GH- and PRL-secreting adenomas. These data indicate that due to heterogeneous expression of SSTR2 and SSTR5 receptor subtypes, in GH-secreting tumors, a bispecific analog, such as BIM-23244, that can activate both receptors could achieve better control of GH hypersecretion in a larger number of acromegalic patients.

Acromegaly↗

PubMed2001

Somatostatin receptor subtypes 2 and 5 differentially affect proliferation in vitro of the human medullary thyroid carcinoma cell line tt.

Somatostatin and its receptors (SSTR1 to SSTR5) are expressed in normal human parafollicular C cells and medullary thyroid carcinoma (MTC), but the role of SSTR subtypes in cell growth regulation is still not clear. The present study demonstrates that the human MTC cell line TT stably expresses all the SSTR subtypes and responds to SSTR2 and SSTR5 activation by subtype-selective agonists with two different patterns in terms of [(3)H]thymidine ([(3)H]thy) incorporation and cell number. The SSTR2 preferential agonists (BIM-23120, BIM-23197, BIM-23190, and BIM-23014; 10(-9)-10(-6) M), significantly suppressed [(3)H]thy incorporation (58-13%) and reduced cell proliferation (50-28%), whereas the SSTR5-selective agonist, BIM-23206 (10(-9)-10(-6) M), significantly increased [(3)H]thy incorporation in TT cells (80-175%), but failed to influence cell proliferation. SSTR2 antagonist (BIM-23627) counteracted the action of SSTR2 preferential agonists on TT cells. Furthermore, increasing concentrations of SSTR5-selective agonists, BIM-23206, dose-dependently prevented the suppression of TT cell [(3)H]thy incorporation and proliferation produced by SSTR2 preferential agonist, BIM-23120, showing an antagonism between these compounds. The following conclusions were reached: 1) the human MTC cell line TT expresses all SSTR subtypes; 2) SSTR2 activation inhibits DNA synthesis and cell proliferation, whereas SSTR5 activation increases DNA synthesis; and 3) SSTR2 preferential agonist (BIM-23120) can antagonise SSTR5-selective agonist (BIM-23206) action and vice versa. These findings suggest a tissue-specific function and a tissue-specific interaction between the two receptors.

Carcinoma, Medullary↗

PubMed2001

Somatostatin receptor-specific analogs: effects on cell proliferation and growth hormone secretion in human somatotroph tumors.

Somatostatin (SST) acts through a family of seven transmembrane domain G protein-coupled receptors to inhibit hormone secretion and cell proliferation in a variety of neuroendocrine tissues. In normal and neoplastic human pituitary somatotroph cells, SST-specific receptor types (SSTR) 1, 2, 3, and 5 are prevalently expressed, and SST and its analogs have been shown to inhibit GH secretion. However, in somatotroph adenomas, little is known regarding: 1) effects of SST and its analogs on pituitary tumor proliferation; 2) the relationship between the effects of SST analogs on GH secretion and tumor cell proliferation; and 3) whether SSTR expression predicts the antiproliferative effects of SST analogs in human somatotroph tumors. We investigated the effects of SST-14, lanreotide, and SSTR 2 (BIM-23190) and SSTR 5 (BIM-23268) specific analogs in 18 somatotroph pituitary adenomas in primary culture. Our results showed that cell proliferation was significantly inhibited by SST-14, lanreotide, BIM-23190, and BIM-23268 in 4, 7, 3, and 4 tumors, respectively (range of proliferation suppression 5-60%; median, 16%). Tumors that were responsive to SSTR 2- and 5-specific analogs were also responsive to lanreotide. SST-14 inhibited GH secretion in 8 of 13 tumors; lanreotide, BIM-23190, and BIM-23268 inhibited GH secretion in six tumors each (range of GH secretion inhibition 23-43%; median 33%). SSTR 2 and 5 messenger RNA was expressed in all tumors investigated, whereas SSTR 1 and 3 messenger RNA was expressed in 11 and 12 tumors, respectively. We observed a dissociation between the in vitro effects of SST-14 or lanreotide on tumor cell proliferation and the effects on GH secretion in human somatotroph tumors. Although differences in receptor concentration and the presence of other SST receptor subtypes may play a role, the presence of SSTR 2 and/or 5 did not have a predictive value. These data suggest that inhibition of cell proliferation occurs independently of effects on GH secretory pathways. Further studies are needed to clarify the mechanism of SST induced antiproliferative effects.

Acromegaly↗

PubMed2000

Selective stimulation of somatostatin receptor subtypes: differential effects on Ras/MAP kinase pathway and cell proliferation in human neuroblastoma cells.

In previous studies we have showed that somatostatin (SST) inhibits cell division, mitogen-activated protein (MAP) kinase and Ras activity in the human neuroblastoma cell line SY5Y. In the present study, we have assessed the role of a series of SST analogs, three of which were selective for SSTR1, SSTR2 or SSTR5, in these cellular events. All the analogs inhibited forskolin-induced cAMP accumulation. Selective stimulation of SSTR1 or SSTR2 but not of SSTR5 inhibited platelet-derived growth factor (PDGF)-induced [(3)H]thymidine incorporation. The three analogs inhibited PDGF-stimulated MAP kinase activity, at least at an early time. In contrast, none of the analogs used individually was able to inhibit PDGF-stimulated Ras activity. A combined stimulation of SSTR2 and SSTR5 was necessary to obtain a significant inhibitory effect, suggesting the possibility of receptor heterodimerization. These results indicate that SST inhibition of Ras and MAP kinase activities takes place via different pathways and that SST inhibition of PDGF-induced cell proliferation occurs via a Ras-independent pathway.

Animals↗

PubMed2000

Human somatostatin receptor subtypes in acromegaly: distinct patterns of messenger ribonucleic acid expression and hormone suppression identify different tumoral phenotypes.

Recently, studies using somatostatin (SRIF) analogs preferential for either the SRIF receptor 2 (SSTR2) or the SSTR5 subtype demonstrated a variable suppression of GH and PRL release from GH-secreting human adenomas. These data suggested the concept of SSTR subtype specificity in such tumors. In the present study the quantitative expression of messenger ribonucleic acid (mRNA) for the 5 SSTR subtypes and the inhibitory effects of SRIF14; SRIF28; octreotide; the SSTR2-preferential analog, BIM-23197; and the SSTR5-preferential analog, BIM-23268, on GH and PRL secretion were analyzed in cells cultured from 15 acromegalic tumors. RT-PCR analysis revealed a consistent pattern of SSTR2 and SSTR5 mRNA expression. SSTR5 mRNA was expressed at a higher level (1052 +/- 405 pg/pg glyceraldehyde-3-phosphate dehydrogenase) than SSTR2 mRNA (100 +/- 30 pg/pg glyceraldehyde-3-phosphate dehydrogenase). However, only SSTR2 mRNA expression correlated with the degree of GH inhibition induced by SRIF14, SRIF28, and BIM-23197. The SSTR5-preferential compound inhibited GH release in only 7 of 15 cases. In cells cultured from the 10 mixed adenomas that secreted both GH and PRL, RT-PCR analysis revealed a consistent coexpression of SSTR5, SSTR2, and SSTR1 mRNA. In all cases SRIF14, SRIF28, and the SSTR5-preferential analog, BIM-23268, significantly suppressed PRL secretion, with a mean maximal inhibition of 48 +/- 4%. In contrast, the SSTR2-preferential analogs, BIM-23197 and octreotide, were effective in suppressing PRL in only 6 of 10 cases. In cells cultured from adenomas taken from patients partially responsive to the SRIF analog, octreotide, partial additivity in suppressing both GH and PRL secretion was observed when the SSTR2- and SSTR5-preferring analogs, BIM-23197 and BIM-23268, were tested in combination. Our data show a highly variable ratio of the SSTR2 and SSTR5 transcripts, according to tumors. The SSTR2-preferring compound consistently inhibits GH release, whereas the SSTR5-preferring compound is the main inhibitor of PRL secretion. When both drugs are combined, the partial additivity observed in mixed GH- plus PRL-secreting adenomas may be of interest in the therapeutic approach of such tumors.

Acromegaly↗

PubMed1999

Quantitative and functional expression of somatostatin receptor subtypes in human prolactinomas.

Recently, it was demonstrated that somatostatin analogs preferential for the SSTR5 subtype suppress PRL release from prolactinoma cell cultures by 30-40%. These data supported the idea of somatostatin receptor subtype-specific control of PRL secretion in such tumors. The present study examines the quantitative profile of SSTRs messenger ribonucleic acid (mRNA) in 10 PRL-secreting tumors and correlates the expression with the ability of native somatostatins (SS14 and SS28), SSTR2 preferential analogs (octreotide and BIM-23197), and the SSTR5 preferential analog BIM-23268 to suppress PRL secretion. RT-PCR quantitative analysis showed a large predominance of SSTR5 mRNA [5648 +/- 1918 pg/pg glyceraldehyde-3-phosphate dehydrogenase (GAPDH)] vs. SSTR2 mRNA (148 +/- 83 pg/pg GAPDH). The SSTR1 transcript was also highly expressed in prolactinomas (1296 +/- 669 pg/pg GAPDH). SSTR5 mRNA expression correlated with PRL inhibition induced by both SRIF14 and SRIF28. Among the different analogs tested, only BIM-23268 produced inhibition of PRL release similar to that achieved with the native peptides. Its EC50 for PRL suppression was 0.28 +/- 0.10 nmol/L. No additive effects on PRL suppression were achieved by cotreatment of the tumor cells with SSTR2 and SSTR5 preferential analogs. In the same tumor cell cultures, quinagolide, a potent dopamine agonist, produced a dose-dependent inhibition of PRL with an EC50 at least 10 times lower than that of BIM-23268. Coincubation of quinagolide and BIM-23268, particularly in tumor cells resistant to dopamine agonist treatment, did not produce additive effects on PRL suppression. In conclusion, prolactinomas have a specific pattern of SSTR subtype mRNA expression (SSTR5 and SSTR1). SSTR5 expression is correlated to PRL regulation. These inhibitory effects are superimposable, at a higher concentration, to those of the dopamine agonists, but are not additive, particularly in the adenomas resistant to dopaminergic suppression of PRL release.

Adult↗

PubMed1997

Somatostatin receptor (SSTR) subtype-selective analogues differentially suppress in vitro growth hormone and prolactin in human pituitary adenomas. Novel potential therapy for functional pituitary tumors.

Previously, we have shown somatostatin receptor (SSTR) subtype-specific regulation of growth hormone (GH), thyroid-stimulating hormone, and prolactin (PRL) secretion in human fetal pituitary cultures, where GH and thyroid-stimulating hormone are mediated by both SSTR2 and SSTR5, whereas SSTR2 preferentially mediates PRL secretion. We now tested SSTR subtype-selective analogues in primary human GH- and PRL-secreting pituitary adenoma cultures. Analogue affinities determined by membrane radioligand binding in cells stably expressing human SSTR forms were either SSTR2 or SSTR5-selective. Analogues preferential either for SSTR2, including octreotide, lanreotide, and novel compounds with improved affinity for SSTR2, or new SSTR5-selective compounds suppressed GH in tumor cell cultures (up to 44% of control; P < 0.0005). However, novel analogues from both groups were 30-40% more potent than octreotide and lanreotide in suppressing GH (P < 0.05). Heterologous analogue combinations containing both SSTR2- and SSTR5-selective compounds were more potent in decreasing GH than analogues used alone (P < 0.05), or than combinations of compounds specific for the same receptor subtype (P < 0.005). In contrast, SSTR2-selective analogues did not suppress PRL release from six cultured prolactinomas studied. However, new SSTR5-selective analogues suppressed in vitro PRL secretion (30-40%; P < 0.05) in four of six prolactinomas. These results suggest that both SSTR2 and SSTR5 are involved in GH regulation in somatotroph adenoma cells, whereas SSTR5 exclusively regulates PRL secretion from prolactinoma cells. Thus, somatostatin analogues with improved selective binding affinity for these receptor subtypes may be effective in the treatment of either GH- or PRL-secreting adenomas.

Adenoma↗

PubMed1997

Somatostatin receptor subtype specificity in human fetal pituitary cultures. Differential role of SSTR2 and SSTR5 for growth hormone, thyroid-stimulating hormone, and prolactin regulation.

Somatostatin (SRIF), a hypothalamic inhibitor of pituitary growth hormone (GH) and thyroid-stimulating hormone (TSH) secretion, binds to five distinct receptor (SSTR) subtypes. We therefore tested SSTR subtype-specific SRIF analogs in primary human fetal pituitary cultures (23-25-wk gestation) to elucidate their role in regulating human pituitary function. Using reverse transcription-PCR, mRNA expression of SSTR2 and SSTR5 were detected in fetal pituitary by 25 wk. SRIF analog affinities were determined by membrane radioligand binding in cells stably expressing the human SSTR forms. GH secretion was suppressed equally (40-60%, P < 0.005) by analogs preferential for either SSTR2 (IC50 for receptor binding affinity, 0.19-0.42 nM) or SSTR5 (IC50, 0.37 nM), and compounds with enhanced affinity for SSTR2 were more potent (EC50 for GH suppression, 0.05-0.09 nM) than Lanreotide (EC50, 2.30 nM) and SRIF (EC50, 0.19 nM). Similarly, analogs with high affinity for SSTR2 or SSTR5 decreased TSH secretion (30-40%, P < 0.005). However, prolactin was effectively inhibited only by compounds preferentially bound to SSTR2 (20-30%, P < 0.05). Luteinizing hormone was modestly decreased (15-20%) by SSTR2- or SSTR5-specific analogs. An SSTR5-specific analog also exclusively inhibited GH in acromegalic tumor cells. Thus, SRIF regulation of GH and TSH in primary human fetal pituitary cells is mediated by both SSTR2 and SSTR5, both of which are abundantly expressed by 25 wk. In contrast, suppression of prolactin is mediated mainly by SSTR2. These results indicate that SSTR5 is critical for physiologic regulation of GH and TSH. SRIF analogs with selective affinity for this receptor may therefore be more effective in the treatment of hormone-secreting pituitary adenomas.

Adrenocorticotropic Hormone↗

PubMed1997

Comparison of LHRH-peptidase and plasminogen activator activity in rat testis extracts.

Testicular LHRH-peptidase and testicular urokinase-type plasminogen activator are Sertoli cell-secreted proteases which display similar molecular properties. However, there is relatively little information regarding the substrate specificity and potential cross-reactivity of these enzymes. Testicular extracts were prepared from homogenates of whole rat testes and assessed by LHRH-peptidase assay, and by radial caseinolysis assays for plasminogen activator and plasmin-like activity. Following partial purification of the protease activities in testicular extracts by gel filtration and ion-exchange chromatography, it was confirmed that testicular LHRH-peptidase and plasminogen activator are clearly separable. There was no detectable plasmin-like activity in the testicular extracts; however, the extracts were found to contain an inhibitor, or inhibitors, of both plasminogen activator and plasmin activity. In addition to LHRH and Gly6-substituted LHRH analogues, the partially purified LHRH-peptidase degraded both angiotensins I and II, but not the gonadotrophin-releasing-hormone-associated peptide derived from the LHRH precursor molecule. These properties of the LHRH-peptidase provide further evidence that it is a testis-specific prolyl endopeptidase, involved in regulating and/or limiting peptide activity in the testis.

Animals↗

PubMed1997

Ontogeny of gonadotrophin and inhibin secretion in normal girls through puberty based on overnight serial sampling and a comparison with normal boys.

We measured luteinizing hormone (LH) and follicle stimulating hormone (FSH) by immunofluorometric assays and alpha-inhibin by radioimmunoassay in serum sampled every 10 min throughout the night (2100-0500 h) from 44 normal girls. Mean overnight LH values rose log-linearly from a mean of 0.2 IU/l in prepubertal girls to 3.0 IU/l in late pubertal girls. Log2 mean overnight FSH rose rapidly through early puberty and then remained constant; mean FSH rose from 1.0 IU/l in prepubertal girls to approximately 2.8 IU/l in Tanner III-V girls. Mean overnight inhibin increased through puberty, rising from 151 ng/l in prepubertal girls to 432 ng/l in fully pubescent girls. Within each of the first three Tanner stages, LH differed approximately 100-fold between the smallest and largest mean concentrations but differed <10-fold within stages IV or V. Such within-pubertal stage variability was less pronounced for FSH, which differed approximately 16-fold among Tanner I subjects and 4-10-fold at later stages, and for inhibin, which varied approximately 4-fold within each Tanner stage. The frequency of LH pulses during overnight sampling increased significantly during puberty, but the frequency of FSH and inhibin pulses remained constant. We compared the results from girls to those from 50 normal boys [Manasco et al. (1995) J. Clin. Endocrinol. Metab., 80, 20462052]. At each pubertal stage, girls had approximately the same mean overnight LH values as boys; girls had higher mean overnight FSH, particularly during Tanner stages II-IV; and boys had mean overnight alpha-inhibin immunoreactivity approximately 1.5 times that of girls at each pubertal stage. Still, hormone concentrations for individuals of both sexes intergraded at each pubertal stage.

Adolescent↗

PubMed1996

A novel hypothalamic peptide, pituitary adenylate cyclase-activating peptide, regulates the function of rat granulosa cells in vitro.

Pituitary adenylate cyclase-activating peptide (PACAP) is a novel peptide that was isolated from ovine hypothalamic tissue on the basis of its ability to stimulate cAMP accumulation in cultured rat pituitary cells. Recently we demonstrated that PACAP can stimulate cAMP accumulation and secretory function in cultured rat Sertoli cells. Since ovarian granulosa cells share many properties with Sertoli cells, we have examined the effect of PACAP (consisting of 38 or 27 amino acid residues) on cultured granulosa cell function. Granulosa cells were obtained from the ovaries of 25-day-old rats implanted with a silastic capsule containing diethylstilbestrol 5 days prior to culture. PACAP 38 (0.1 microM-0.01 pM), both alone and in the presence of the phosphodiesterase inhibitor, methylisobutylxanthine, stimulated cAMP accumulation 4-8-fold with an ED50 of approximately 100 pM. Maximal PACAP 38 or PACAP 27 stimulation of granulosa cell cAMP was significantly greater than that produced by a maximally effective concentration of FSH. Because PACAP 38 and 27 have 68% sequence homology with vasoactive intestinal peptide (VIP), and since VIP stimulates granulosa cell cAMP accumulation and estradiol and progesterone secretion, we examined the possibility that PACAP could be acting via the VIP receptor. VIP stimulated cAMP only at concentrations of 10 nM or greater, whereas the PACAP stimulation was evident at 10 pM. Moreover, only one of three potent VIP antagonists inhibited VIP stimulation of cAMP accumulation, and only at 1 microM or greater. This VIP antagonist did not inhibit PACAP 38 action at 2000-fold excess concentration. Interestingly PACAP 38 was more effective than PACAP 27 with regard to steroid secretion and the ability to induce LH responsiveness. PACAP and VIP stimulation of granulosa cell cAMP accumulation or estradiol or progesterone secretion was not additive. Thus, these data support the hypothesis that granulosa cells have specific PACAP 38 receptors and that VIP acts via these receptors. In addition, PACAPs 38 and 27 are more potent stimulators of cAMP accumulation in luteinized granulosa cells than LH. These results both pre- and postovulation, along with previous data indicating that the PACAPs are found in the ovaries, suggest a role for PACAP in the regulation of ovarian function.

1-Methyl-3-isobutylxanthine↗

PubMed1995

Gonadotrophin surge-attenuating factor bioactivity is present in follicular fluid from naturally cycling women.

Rat pituitary monolayer bioassays were used to compare gonadotrophin surge-attenuating factor (GnSAF) bioactivity in follicular fluid from 12 follicles in 10 spontaneously cycling women with that in pooled follicular fluid from women undergoing ovulation induction. Expressed as ED50S (microliter follicular fluid/well producing 50% of maximal effect), GnSAF bioactivity was detectable in all spontaneous follicular fluid samples (1.4-33.3 microliters/well) and in follicular fluid from women undergoing ovulation induction (6.8 microliters/well). This GnSAF bioactivity was unaffected by pre-incubation with an inhibin antibody. When the data were grouped according to whether the recovered oocytes fertilized in vitro or not, the fertilized group contained significantly greater GnSAF bioactivity than the unfertilized group (5.3 +/- 1.1 and 14.1 +/- 2.6 microliters/well respectively, P < 0.05). While both inhibin bioactivity (9.7 +/- 1.4 and 28.9 +/- 12.1 microliters/well) and immunoreactivity (36.8 +/- 2.2 and 21.0 +/- 3.0 and ng/ml) were also greater (P < 0.01) in the fertilized compared with the unfertilized groups respectively, there were no other significant differences between the two groups. We conclude that GnSAF is found in follicular fluid from spontaneously cycling women, supporting in-vivo evidence for the involvement of GnSAF in feedback control of the ovary-pituitary axis.

Adult↗

PubMed1995

Gonadotropin surge-attenuating factor bioactivity in serum from superovulated women is not blocked by inhibin antibody.

Primary pituitary cultures from adult female rats were used to investigate gonadotropin surge attenuating factor (GnSAF) bioactivity. Serum from superovulated women was charcoal-treated and incubated with either an inhibin antibody (MC4), normal sheep serum (NSS), or serum-free defined medium (SFDM) before addition to cell culture. The reduction in GnRH-induced LH secretion (GnSAF bioactivity) produced by the serum (30.1 +/- 6.5%, p < 0.001, of control) was not altered by prior incubation with either MC4 or NSS (24.9 +/- 3.6 and 23.2 +/- 2.7%, p < 0.001, of control, respectively). This indicates that GnSAF bioactivity present in serum from superovulated women is not entirely attributable to inhibin. Recombinant human inhibin reduced basal FSH secretion to 24.6 +/- 4.7% (p < 0.001) of the control value. However, this was totally abolished by prior incubation with MC4, but not NSS. We have also shown that ovarian steroids are not responsible for GnSAF bioactivity in vitro. In conclusion, in contrast to findings in superovulated rats, inhibin antibody did not block GnSAF bioactivity in serum from superovulated women.

Animals↗

PubMed1995

Ontogeny of gonadotropin, testosterone, and inhibin secretion in normal boys through puberty based on overnight serial sampling.

To investigate hormonal changes occurring in male puberty, we measured LH, FSH, testosterone, and alpha-inhibin immunoactivity in serum samples drawn every 10 min for 8 h (2100-0500 h) from each of 50 normal prepubertal and pubertal boys, aged 8.4-18.8 yr. We measured gonadotropins with ultrasensitive immunofluorometric assays, and testosterone and alpha-inhibin with RIAs. Unlike previous studies, which indexed pubertal development with Tanner stages, we used testicular volume, a more finely graduated indicator of development, to reveal patterns that were obscured when subjects were grouped by Tanner stage. The overnight mean concentration of each hormone increased with testis volume, but the rate of increase on a logarithmic scale slowed as testes grew. Log LH rose precipitously in the late prepubertal and early pubertal periods and plateaued during mid- and late puberty. Based on fitted regression curves, LH increased about 20-fold (from 0.11 IU/L) between testis volumes of 1 and 10 mL, but only an additional 1.5-fold by 30 mL. The developmental trajectory of log testosterone was like that of log LH, but rose less steeply early in puberty. From 0.14 micrograms/L at a testis volume of 1 mL, testosterone increased about 8.5-fold by 10 mL and an additional 3-fold by 30 mL. In contrast, logarithms of overnight mean FSH and alpha-inhibin concentrations rose at a more nearly constant rate throughout puberty. From 0.62 IU/L at a testis volume of 1 mL, the FSH concentration doubled by 10 mL and increased an additional 1.7-fold by 30 mL. From 270 ng/L at a testis volume of 1 mL, inhibin increased 1.5-fold by 10 mL and an additional 1.3-fold by 30 mL. Overnight pulse amplitudes exhibited developmental trajectories similar to those of the corresponding overnight mean concentrations. The number of LH and testosterone pulses during the sampling period averaged 2.2 and 2.1, respectively, at Tanner stage 1 and increased to 4.5 and 3.2, respectively, at Tanner stage 5. The number of FSH and inhibin pulses remained constant throughout puberty, averaging 3.3 and 3.5, respectively. Pairwise correlations among hormone concentrations were strong, reflecting common increasing trends through puberty; however, after accounting for developmental trends, FSH, LH, and testosterone concentrations remained correlated, whereas inhibin was uncorrelated with each of the other three hormones. Measuring gonadotropins with ultrasensitive assays and analyzing the results on a logarithmic scale as a function of testis volume made clear the dramatic hormonal changes that begin before the clinical changes of puberty.

Activity Cycles↗

PubMed1993

Effects of injection of anti-luteinizing hormone (LH)-releasing hormone serum and anti-gonadotropin-releasing hormone-associated peptide serum into neonatal rats on LH and follicle-stimulating hormone cells.

Relatively little is known regarding the potential importance of LHRH and of gonadotropin-releasing hormone-associated peptide (GAP) on the postnatal development of gonadotrophs. We investigated the effects of administration of anti (A)-LHRH serum or A-GAP serum to neonatal rats on the development of LH and FSH immunoreactivity in anterior pituitary gland (APG) cells. Serum (sheep non-immune [NSS], sheep A-LHRH, sheep A-LHRH/GAP [which bound LHRH and GAP], rabbit non-immune [NRS], or rabbit A-GAP) was injected s.c. into neonatal female and male rats on Days 1 and 3 or Days 1, 3, 5, and 7 after birth. Pups were killed on Day 5 or 9, two days after the last injection. The percentages of APG cells immunoreactive for LH or FSH increased from Day 1 to Day 5 and did not change between Days 5 and 9 in female pups treated with NSS or NRS. There was a trend for the percentages of LH and FSH cells to increase from Day 1 to Days 5 and 9 in male pups treated with NSS or NRS, but the increases were not statistically significant. In both females and males, treatment with antisera that recognized LHRH reduced the percentage of FSH cells on Day 5 and the percentages of LH and FSH cells on Day 9. Treatment with A-GAP was without effect in both sexes. There were similar percentages of LH and FSH cells in females and a lower percentage of FSH than of LH cells in males in the Day 5 and 9 controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

A novel hypothalamic peptide, pituitary adenylate cyclase activating peptide, modulates Sertoli cell function in vitro.

Pituitary adenylate cyclase-activating peptide (PACAP), a novel hypothalamic peptide that has been shown to exist in several tissues including the testis, was examined for its effects on cultured rat Sertoli cells. PACAP stimulates cAMP accumulation in Sertoli cells cultured from 15-day-old rats in the presence or absence of methylisobutylxanthine, a phosphodiesterase inhibitor, and in the presence of pertussis toxin, a blocker of the adenylate cyclase inhibitory pathway. Maximal stimulation, which is 20-40% of that attainable with FSH, occurs at PACAP concentrations of 10 nM: the ED50 is approximately 100 pM. The ability of PACAP to stimulate Sertoli cell cAMP declines with increasing age of donor animals (15-60 days of age) in a fashion similar to the FSH effect. PACAP stimulation of Sertoli cell cAMP accumulation is additive with submaximal, but not maximal, concentrations of FSH or forskolin. PACAP also stimulates the secretion of lactate, estradiol, and inhibin in a concentration-dependent manner. The stimulation of Sertoli cell cAMP accumulation by PACAP is not altered by a vasoactive intestinal peptide antagonist, and vasoactive intestinal peptide alone does not stimulate cAMP accumulation, indicating that PACAP is not acting via vasoactive intestinal peptide receptors. Further experiments are needed to determine whether PACAP is synthesized within the testis and if so, in which cell types; however, the present data clearly demonstrate that PACAP can modulate Sertoli cell function in vitro.

Age Factors↗

PubMed1992

Inhibin suppresses luteinizing hormone (LH)-releasing hormone self-priming: direct action on follicle-stimulating hormone secretion and opposition of estradiol-enhanced LH secretion.

Previous studies have suggested that the ovary produces a factor that maintains the pituitary in a state of low LHRH responsiveness that must be overcome by the self-priming action of LHRH. To determine the role of inhibin in maintaining low LHRH responsiveness in pituitaries of diestrous female rats, endogenous inhibin was passively immunoneutralized in vivo, and the pituitaries were removed 18-20 h later and examined for LHRH responsiveness in vitro. Pituitaries from diestrous control rats produced the biphasic pattern of gonadotropin secretion that typifies LHRH self-priming: an initial low secretory response to LHRH (lag phase), followed by a protein synthesis-dependent transition to an enhanced rate of secretion with continued LHRH exposure (primed phase). Immunoneutralization of endogenous inhibin [antiserum (AS) treated] resulted in an increased rate of LH secretion during the lag phase, while no change was observed in the primed phase rate of LH secretion. FSH secretion from pituitaries of AS-treated rats was increased during the lag phase to a rate of secretion similar to that observed during the primed phase of FSH secretion from control pituitaries, and it was increased further during the primed phase of secretion. These results suggest that inhibin is at least partially responsible for the low secretion of LH observed during the lag phase response to LHRH exposure and is totally responsible for the lowered rate of FSH secretion during the lag phase. The observation that the enhanced rate of gonadotropin secretion observed with AS-treated pituitaries during the lag phase was resistant to inhibition of protein synthesis provides further evidence that a partial transition from the lag to the primed phase had already occurred. Pituitaries from ovariectomized rats were also examined in order to place the contribution of inhibin in perspective with the total ovarian influence on pituitary responsiveness to LHRH. Unexpectedly, LH secretion during the lag phase was similar to the low secretion rate of diestrous control pituitaries, and the higher primed rate of secretion failed to fully develop, suggesting that an additional ovarian factor was required to induce and maintain pituitary responsiveness to LHRH in terms of LH secretion. FSH secretion from the ovariectomized rats was similar to that observed from pituitaries of AS-treated rats, thus further supporting the concept that inhibin is fully responsible for the suppression of FSH secretion in response to LHRH. Plasma from the AS-treated rats revealed a 2-fold increase in estradiol levels compared with diestrous control rats.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1992

In vivo evidence that inhibin is a gonadotropin surge-inhibiting/attenuating factor.

Hyperstimulation of ovarian function through exogenous gonadotropin administration suppresses the preovulatory surges of both LH and FSH. This phenomenon has been demonstrated to be the result of a non-steroidal factor released from the ovary that has been designated as either gonadotropin surge-inhibiting factor or attenuating factor (GnSIF/AF). To examine the possibility that inhibin might possess the activity ascribed to this factor, endogenous inhibin was immunoneutralized in female rats under conditions known to stimulate GnSIF/AF activity. Inhibin-like immunoreactivity was found to be significantly elevated in FSH-treated rats prior to the time of the gonadotropin surges. Spontaneous preovulatory surges of both LH and FSH were observed in saline-treated (control) rats that were subsequently treated with either anti-inhibin serum (AS) or normal sheep serum (NS). FSH-treatment completely prevented the occurrence of gonadotropin surges in rats subsequently treated with NS. In FSH-injected rats subsequently treated with AS, however, normal preovulatory surges of both LH and FSH were observed. These results indicate that inhibin may be the factor responsible for the suppression of the preovulatory gonadotropin surges in FSH-treated rats and, at least in this species, may be a GnSIF/AF.

Animals↗