Fetal Pituitary Cells Research Articles

Attenuation of growth hormone production at the fetal stage is critical for dioxin-induced developmental disorder in rat offspring

Although dioxins and related chemicals have been suspected to disrupt child development, their toxic mechanism remains poorly understood. Our previous studies in rat fetuses revealed that maternal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a highly-toxic dioxin, suppresses fetal synthesis of pituitary growth hormone (GH) that is essential for development. This study examined the hypothesis that attenuating GH expression in fetuses triggers developmental disorders. Treating pregnant rats with 1 μg/kg TCDD reduced the circulating level of GH and its downstream factor, insulin-like growth factor-1 (IGF-1), in the offspring only during the fetal and early neonatal stages. Although maternal TCDD exposure resulted in low body weight and length at babyhood and defects in the learning and memory ability at adulthood, GH supplementation in TCDD-exposed fetuses restored or tended to restore the defects including IGF-1 downregulation. Moreover, maternal TCDD exposure decreased the number of GH-positive cells during the fetal/neonatal stage. A microarray analysis showed that TCDD reduced the expression of death-associated protein-like 1 (DAPL1), a cell cycle-dependent proliferation regulator, in the fetal pituitary gland. In addition, TCDD treatment attenuated proliferating cells and cyclin mRNA expression in the fetal pituitary gland. Aryl hydrocarbon receptor (AHR)-knockout fetuses were insensitive to TCDD treatment, indicating that the TCDD-induced reduction in DAPL1 and GH mRNAs expression was due to AHR activation. Finally, DAPL1 knockdown suppressed GH and cyclin D2 expression in fetal pituitary cells. These results provide a novel evidence that dioxin suppresses GH-producing cell proliferation and GH synthesis due to partly targeting DAPL1, thereby impairing offspring development.

Regulation of baboon fetal pituitary prolactin expression by estrogen.

We previously showed that fetal adrenal fetal zone growth was increased and the number of follicles in the fetal ovary reduced in baboons in which estradiol was suppressed by treatment with the aromatase inhibitor letrozole between mid and late gestation periods. Because adrenal/ovarian development was restored in animals treated with letrozole and estradiol, and both tissues express estrogen receptor, we proposed that estrogen regulates fetal adrenal/ovary development via a direct action. However, because prolactin can modulate fetal adrenal and adult pituitary/ovarian function, the current study determined whether estrogen action involved estradiol-regulated changes in fetal prolactin/luteinizing hormone (LH) expression. Fetal prolactin levels and the number of prolactin-positive fetal pituitary cells (per 0.37 mm(2)) were increased (P < 0.01) between mid (6 +/- 1 ng/ml; 15.8 +/- 2.4) and late (257 +/- 28 ng/ml; 57.3 +/- 5.1) gestation, reduced (P < 0.01) in late-gestation fetuses in which estradiol was suppressed (>95%) by letrozole (61 +/- 11 ng/ml; 19.3 +/- 2.0), and minimally but not significantly increased by letrozole and estradiol (99 +/- 11 ng/ml; 32.7 +/- 5.2). In contrast, the number of LH-positive fetal pituitary cells decreased (P < 0.01) between mid (48.8 +/- 9.5) and late (17.4 +/- 3.2) gestation, remained elevated (P < 0.01) in estrogen-suppressed animals (56.6 +/- 5.1), and was partially but not significantly decreased by letrozole-estradiol (28.8 +/- 5.2). We conclude that estrogen regulates fetal pituitary prolactin and LH expression and fetal prolactin levels. However, because prolactin and LH expressions in estrogen-suppressed fetuses were inversely related to previously demonstrated changes in adrenal/ovarian development, we propose that estrogen regulates the fetal ovary and adrenal gland directly and not via action on the fetal pituitary gland.

Nitric oxide stimulates embryonic somatotroph differentiation and growth hormone mRNA and protein expression through a cyclic guanosine monophosphate-independent mechanism

In the pituitary gland, NO is locally synthesized by gonadotroph and folliculo-stellate cells. Many reports have shown that NO can modulate the growth hormone (GH) secretion. However, its role on mice embryo GH regulation remains unclear. In addition, it is unknown whether the regulation is associated with the proliferation of pituitary cells. In this study, we have investigated the regulatory effects of NO on somatotroph differentiation, proliferation and GH mRNA and protein expression using primary cell cultures of mice fetal pituitaries (embryonic days 16.5, ED 16.5). Our results show that incubation of pituitary cells in the presence of sodium nitroprusside (SNP; 1 mM), a NO donor, for 4.5 h resulted in a significant increase in GH mRNA and protein expression ( P < 0.05) and the stimulation of SNP can be inhibited by hemoglobin, a NO scavenger. But the addition of cyclic guanosine monophosphate (cGMP; 3.0 mM), the second messenger of multiple NO actions cannot influence GH mRNA and protein expression. The cyclic nucleotide cellular efflux pumps existed in the pituitary cells can transport the majority of de novo-produced cGMP and effectively block cGMP accumulation. For maintaining intracellular concentration of cGMP, probenecid (0.5 mM), a blocker of cGMP efflux pump, combined with cGMP (3.0 mM) was used to treat the pituitary cells. This also cannot influence GH mRNA and protein expression. In addition, the ratio of GH-positive cells is increased significantly after the stimulation of SNP ( P < 0.05). However, SNP cannot modulate the pituitary cell proliferation. From these results we conclude that NO can increase GH mRNA and protein expression in fetal pituitary cells and cGMP is not involved in this hormonal regulation. Stimulation of NO on the somatotroph differentiation does not occur due to pituitary cell proliferation.

Ontogeny and Effects of Hypothalamic Pituitary Disconnection on Formation of Inositol Trisphosphate in Fetal Sheep Pituitary Cells

In late gestation fetal sheep, the pituitary becomes increasingly responsive to stimulation by arginine vasopressin (AVP). This change appears to be one important factor mediating the plasma cortisol surge, a critical developmental event. It is not known precisely why pituitary corticotropes become more responsive at this time. In this study we examined the possibility that changes in second messenger generation [inositol trisphosphate (IP(3))] are responsible. Two studies were undertaken. The first was an ontogeny study, where pituitaries were isolated from 100-, 120-, and 140-d gestational age (dGA) fetal sheep. Cells were cultured, stimulated with AVP, and the formation of IP(3) assessed. The amount of IP(3) generated increased with gestational age (percent increases from unstimulated controls were 4.6, 11.5, and 21.5 for 100, 120, and 140 dGA, respectively), with significant differences between the 140-dGA group and both earlier groups apparent. The second study examined the impact of 120-dGA hypothalamo-pituitary disconnection (HPD), which prevents corticotrope maturation, on responsiveness of pituitary cells isolated from 140-dGA fetuses. Cells were stimulated with AVP, and the formation of IP(3) and secretion of ACTH were assessed. Significantly less IP(3) was formed, and ACTH secreted in cells from HPD compared with control fetuses (IP(3) and ACTH levels were 50% and 35% lower, respectively). Results from the HPD study demonstrate that the ontogenic changes in IP(3) after AVP require an intact hypothalamic-pituitary-adrenal axis. These findings suggest that heightened second messenger generation may be a key reason for increased ACTH secretory responsiveness to AVP in the late gestation sheep fetus.

Regulation of pituitary somatotroph differentiation by hormones of peripheral endocrine glands

Anterior pituitary somatotroph differentiation occurs during chick embryonic and rat fetal development. A number of findings support the hypothesis that differentiation of these growth hormone (GH) producing cells in the chick and the rat is regulated by adrenal glucocorticoids and thyroid hormones. Somatotroph differentiation can be induced in cultures of chick embryonic and rat fetal pituitary cells with adrenal glucocorticoids and this effect can be modulated by concomitant treatment with thyroid hormones. Plasma levels of thyroid hormones, corticosterone and adrenocorticotropic hormone increase during development, consistent with the ontogeny of somatotrophs. Treatment of chick embryos or rat fetuses with glucocorticoids in vivo induces premature somatotroph differentiation, indicating that the adrenal gland, and ultimately anterior pituitary corticotrophs, may function to regulate pituitary GH cell differentiation during development. Administration of thyroid hormones in vivo also increases somatotrophs prematurely, and administration of the thyroid hormone synthesis inhibitor methimazole inhibits somatotroph differentiation in vivo, suggesting that endogenous thyroid hormone synthesis contributes to normal somatotroph differentiation. Our working model for the regulation of somatotroph differentiation during normal development includes modulation by elements of the hypothalamo–pituitary–adrenal and hypothalamo–pituitary–thyroid axes. Additional research is reviewed defining the mechanism of action for these peripheral hormones in induction of pituitary GH gene expression during development.

Novel ghrelin analogs with improved affinity for the GH secretagogue receptor stimulate GH and prolactin release from human pituitary cells

Ghrelin, a recently identified 28-amino acid peptide is a potent GH secretagogue (GHS) produced predominantly by the stomach. Ghrelin stimulates GH secretion through binding to the GHS receptor in the hypothalamus and pituitary. In addition to the GH-releasing action, ghrelin has been found to be a powerful orexigenic factor. To assess the direct in vitro effects of ghrelin on human pituitary hormone secretion we have produced a panel of novel ghrelin analogs (molecular weight, 3323-3384; human native ghrelin, 3371) with enhanced affinity for the human GHS receptor (IC(50) 0.38-1.09 nM; human ghrelin, 1.2-2.2 nM). The peptidic analogs were tested for their effect on GH secretion using dispersed human fetal pituitaries (21 to 23 weeks of gestation) and cultured GH- and prolactin (PRL)-secreting adenomas. The expression of the GHS receptor in normal (fetal and adult) human pituitary tissues, GH- and PRL-cell adenomas was established using RT-PCR. The effects of ghrelin, its analogs and GH-releasing hormone (GHRH) alone or in combination on GH and PRL secretion were compared at various concentrations. The ghrelin analogs stimulated GH release by 35-60% from human fetal pituitary cells (1-10 nM; P<0.05) and by 50-75% from cultured pituitary adenomas (10 nM; P<0.05). This releasing effect was dose-dependent, achieving maximal stimulation with analog concentrations at 100 nM. Human ghrelin was less potent as compared with its analogs in stimulating human GH, in keeping with the improved binding affinity of the analogs for the GHS-1a receptor. The ghrelin analogs and GHRH had comparable effects on GH secretion from both normal and adenomatous cells, and in combination produced an additive stimulatory effect on GH (150%; P<0.0001). In contrast, ghrelin and its analogs induced a comparable increase in PRL release ranging between 25 and 40% (P<0.05) from fetal cells and 30 and 70% (P<0.001) from cultured PRL-cell and mixed GH-PRL adenomas. Our results have demonstrated for the first time that ghrelin analogs with enhanced affinity for the GHS receptor are potent stimulators of GH secretion from human pituitary cells, and thus may possess potential clinical therapeutic benefits.

The novel somatostatin ligand (SOM230) regulates human and rat anterior pituitary hormone secretion.

Currently available somatostatin analogs predominantly bind to the somatostatin receptor subtype (SSTR)2 subtype, and control GH and IGF-I secretion in approximately 65% of patients with acromegaly, their efficacy relating to receptor density and subtype expression. SOM230 is a somatostatin ligand with high affinity to four SSTR subtypes. In primary cultures of rat pituicytes, SOM230 dose-dependently inhibited GH release (P = 0.002) with an IC50 of 1.2 nM. Ten nanomoles SOM230 inhibited GH and TSH release by 40 +/- 7% (P < 0.001) and 47 +/- 21% (P = 0.09), respectively. No effect of SOM230 was observed on prolactin (PRL) or LH release. In cultures of human fetal pituitary cells, SOM230 inhibited GH secretion by 42 +/- 9% (P = 0.002) but had no effect on TSH release. SOM230 inhibited GH release from GH-secreting adenoma cultures by 34 +/- 8% (P = 0.002), PRL by 35 +/- 4% from PRL-secreting adenomas (P = 0.01), and alpha-subunit secretion from nonfunctioning pituitary adenomas by 46 +/- 18% (P = 0.34). In contrast, octreotide inhibited GH, PRL, and alpha-subunit from the respective adenoma by 18 +/- 12 (P = 0.39), 22 +/- 4 (P = 0.04), and 20 +/- 10% (P = 0.34). In all culture systems, no significant difference in the inhibitory action of SOM230, octreotide, and somatostatin 14 on hormone release was observed. SOM230, similar to somatostatin, has high-affinity binding to SSTR1, 2, 3, and 5 and, in keeping with this, has an equivalent inhibitory effect on pituitary hormone secretion. As a consequence of its broader binding profile, SOM230 is likely to find clinical utility in treating tumors resistant to SSTR-2-preferential analogs.

Parallel changes between the percentage of fetal pituitary cells immunoreactive to oestrogen receptor α and the concentration of 17β-oestradiol in fetal and maternal plasma during gestation in sheep

The present study was designed to investigate the relationship between fetal sheep pituitary oestrogen receptor (ER) alpha expression and changes in fetal and maternal plasma 17beta-oestradiol (E2) concentrations during gestation. The results revealed that immunoreactivity for ER was located in the nuclei and distributed throughout the fetal pituitary gland during gestation. The percentage of ERalpha-positive cells was approximately 2% of the total cell population in female fetuses at Day 60 of gestation, increased to approximately 7% and 13% of the total cell population at Days 90 and 120 of gestation, respectively, and then declined to approximately 10% at birth. The fetal plasma E2 concentrations were approximately 19 and 71 pg mL(-1) at Days 90 and 120 of gestation, respectively, and decreased to 22 pg mL(-1) after birth. In male fetuses, plasma E2 concentrations and the percentage of ERalpha-positive cells were similar to values in female fetuses throughout gestation, except on Day 120 when the plasma E2 level in female fetuses was significantly higher than in male fetuses. These data demonstrate that changes in the percentage of fetal pituitary ERalpha-positive cells parallel fetal plasma E2 concentrations throughout gestation.

Suppression of Rat and Human Growth Hormone and Prolactin Secretion by a Novel Somatostatin/Dopaminergic Chimeric Ligand

As cotreatment of somatostatin (SRIF) and dopamine (DA) agonists reduces GH in acromegaly more effectively than either agonist alone, SRIF and DA receptors (SSTR and DAR) may interact with enhanced functional activity. The selective SSTR2 agonist, BIM-23023 (50% effective dose, 0.42), and the DAR2 agonist, BIM-53097 (50% effective dose, 22.1), dose- dependently inhibited GH secretion in cultured primary rat and human fetal as well as in human pituitary tumor cells derived from GH-secreting adenomas. The combination of individual SSTR2 and DAR2 agonists was additive for suppressing GH secretion in both rat and human pituitary cells. BIM-23A387 is a chimeric compound that contains structural elements of both SRIF and DA in a single molecule and retains potent, selective binding to DAR2 and SSTR2. BIM-23A387 (50% effective dose, 0.16 for SSTR2 and 24.5 for DAR2), displayed similar efficacy in suppressing GH secretion from rat pituitary cells as the combination of the two individual agonists. In contrast, the chimeric molecule was more potent than individual selective analogs in suppressing GH secretion by human fetal pituitary and GH-secreting adenoma cells (P < 0.05). Although the DAR2 antagonist, sulpiride, reversed BIM-23A387-induced GH suppression, blockade of SSTR2 by the selective SSTR antagonist, BIM-23454, did not block BIM-23A387-suppressed GH secretion. These results indicate that mechanisms by which the chimeric molecule suppresses pituitary GH secretion may not be mediated by individual SSTR2 or DAR2 signaling, respectively. Functional interaction of the two receptors may explain the clinical observation that more effective GH suppression is achieved when DAR2 and SSTR2 agonists are administered in combination. The SRIF/DA chimeric molecule, BIM-23A387, represents a novel tool for effective drug treatment of acromegaly and for prolactinomas otherwise resistant to dopaminergic therapy.

Functional association of somatostatin receptor subtypes 2 and 5 in inhibiting human growth hormone secretion.

We previously demonstrated that somatostatin (SRIF)-induced inhibition of GH secretion from human pituitary cells is mediated through both the SRIF receptor (SSTR) 2 and 5 subtypes. The interplay between these two SSTR subtypes in regulating GH was therefore tested in primary human fetal pituitary cultures (18-30 wk gestation). GHRH (10 nM)-stimulated GH secretion (51% increase, P < 0.05) was suppressed equally by either SSTR2 or SSTR5-selective agonists (10 nM). GH suppression correlated with agonist affinity for their respective receptor subtypes. Combined addition of SSTR2- and SSTR5-specific agonists was synergistic for GH suppression, achieving 73% (P < 0.05) inhibition as compared with inhibition attained with SSTR2 (32%) and SSTR5 (34%) agonists used alone (P < 0.05). The SSTR2 selective antagonist BIM-23454 dose-dependently blocked SSTR2 but not SSTR5-induced suppression of GH secretion. BIM-23454 also completely reversed GH suppression in response to the combined activation of SSTR2 and SSTR5. The IC(50) for BIM-23454 reversal of agonist-induced GH suppression was 55 nM and 33 nM for two SSTR2 agonists, 45 nM and 40 nM for the combination of SSTR2 and SSTR5 agonists, respectively, and 45 nM for the SSTR2/SSTR5 agonist BIM-23244, all of which were similar to the affinity of BIM-23454 for SSTR2 (32 nM). These results suggest the following: 1) activation of both SSTR2 and SSTR5 induces a functional association of receptor subtypes, resulting in synergistic GH suppression; 2) BIM-23454 is a potent SSTR2-selective antagonist capable of reversing SRIF-induced GH suppression; and 3) the ability of a selective SSTR2 antagonist to inhibit the GH suppressing action of SSTR2 agonist alone, SSTR2/SSTR5 biselective agonists, or SSTR2 and SSTR5 agonists in combination support the concept of a functional interaction between somatotroph SSTR2 and SSTR5 subtypes in primary human fetal pituitary cells.

Inhibin, activin, and follistatin in human fetal pituitary and gonadal physiology.

Activin has been previously demonstrated to directly stimulate the synthesis of gonadotropin-releasing hormone (GnRH) receptors and to increase follicle-stimulating hormone (FSH) secretion in nonhuman pituitary cell cultures (PCCs). Currently, knowledge of the physiological role of these peptides in primates is still far from complete. Moreover, several results in macaque monkeys failed to support an unequivocal role for inhibin in FSH suppression. Whereas the bioactivity of inhibin and activin has been demonstrated in rat PCCs, no data exist on human pituitary response to these peptides either in vivo or in vitro. We studied the secretion of FSH and luteinizing hormone (LH) by dispersed human fetal pituitary cells from midtrimester abortions in response to recombinant human (rh-) activin-A, inhibin-A, and other secretagogues. After mechanical and enzymatic dispersion, the human fetal pituitary cells were cultured on an extracellular matrixlike-material-coated 24-well plate. After 3 days' incubation in serum-containing medium, the PCCs were washed and preincubated for 90 min in serum-free medium and incubated with activin-A, inhibin-A, TGF-beta, follistatin, sex steroids, and GnRH, in quadruplicate. Activin-A was a potent secretagogue for FSH secretion. GnRH (20 ng/ml) was more potent than rh-activin-A for LH secretion. Nevertheless, a significant increase in LH secretion into the medium was brought about by rh-activin-A. Inhibin decreased FSH and LH secretion, but the LH response to inhibin was less prominent than that of FSH. GnRH opposed the inhibitory effect of inhibin on LH secretion. In dynamic, short-term, repetitive exposure of fetal pituitary fragments to rh-activin-A (superfusion), we could not receive a similar increase in LH and FSH as in static incubations, as opposed to a short GnRH exposure. In addition to their endocrine, paracrine, and autocrine effects, and in addition to their role as possible markers, the TGF-beta superfamily members may affect embryogenesis and possibly immunomodulation of the embryo and fetus. The role of activin and inhibin as intragonadal regulators is hypothesized. The pro-alphaC inhibin precursor may act as an FSH receptor antagonist. Human fetal PCCs express the previously reported physiologic responses to activin and inhibin generated in nonhuman experiments on gonadotropin secretion in vitro and may serve as a physiologic model for studying human gonadotrope responses to the TGF-beta family of peptides.

Expression of leukemia inhibitory factor in human pituitary adenomas: a morphologic and immunocytochemical study.

Leukemia inhibitory factor (LIF) is a pleiotropic, neuropoietic cytokine found in bovine, murine, and human fetal and adult pituitary cells, mostly in corticotrophs and somatotrophs. In addition, it has been found in a few GH- and ACTH-producing human pituitary adenomas. The aim of our study was to investigate the presence of LIF in various morphologic types of human pituitary adenomas. Ninety-eight operated pituitary adenomas diagnosed by light microscopy and classified by pituitary hormone immunocytochemistry and electron microscopy were studied. Sixty-eight tumors were functioning and included 15 densely granulated (DG) and 10 sparsely granulated (SG) somatotroph (SM) adenomas, 5 lactotroph (LT), 7 mixed SM-LT and 31 corticotroph (CRT) adenomas. The remaining 30 nonfunctioning tumors included 11 gonadotroph (GON) and 19 null cell (NULL) adenomas. For immunocytochemical demonstration of LIF, a specific polyclonal antiserum was applied on formalin-fixed, paraffin-embedded tissues. The immunohistoscore ranging from 1 to 64 grades was determined by multiplying the immunostaining grade (1-4) by the staining intensity grade (1-4), and by the heterogeneity grade (1-4). Ninety adenomas (92%) were variably immunopositive for LIF. LIF was expressed in 77.5% of CRT, 81.8% of GON, 93.3% of DG-SM, and in all SG-SM, LT, SM-LT and NULL adenomas. LT adenomas showed the highest immunostaining grade, followed by SG-SM, GON, NULL, SM-LT, DG-SM, LT and CRT adenomas. GON adenomas showed the highest immunohistoscore, followed by NULL, DG-SM, SM-LT, SG-SM, LT and CRT adenomas. Nonfunctioning tumors showed a significantly higher immunohistoscore compared to functioning adenomas (p < 0.01). We conclude that LIF expression is frequent in all types of functioning and nonfunctioning pituitary adenomas.

The effect of GHRH, GHRP-2 and somatostatin on GH secretion by fetal pituitary.

Growth hormone releasing peptide (GHRP-2) is a synthetic hexapeptide which specifically stimulates secretion of growth hormone (GH) by fetal pituitary somatotrophs through a new membrane receptor, which is different from growth hormone releasing hormone (GHRH) and somatostatin (SMS) receptors. We used cell cultures of human fetal pituitary somatotroph cells to investigate the effect of GHRH, GHRP-2 and somatostatin on GH secretion. The results showed that the mechanism of GHRH/SMS and GHRP-2 was different. This indicated that a different intracellular signal transduction system might also play a crucial role in the regulation of GH secretion.

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.

Adrenocorticotropin secretion by fetal sheep anterior and intermediate lobe pituitary cells in vitro: effects of gestation and adrenalectomy.

Immunoreactive (ir) ACTH is present in the fetal sheep intermediate lobe (IL) as well as the anterior pituitary (AP). It is not clear whether fetal IL cells can secrete irACTH and if gestational age and glucocorticoids influence the secretion of ACTH from these tissues in a similar fashion. Therefore, we examined the control of irACTH secretion by IL cells, whether the responsiveness of AP and IL cells to arginine vasopressin (AVP) and CRH changes during gestation, and whether withdrawal of adrenal steroids by adrenalectomy influences AP and IL responses. Cultured pituitary cells were studied from intact fetuses at an immature (n = 5; 108 +/- 5 days) and a mature (n = 8; 139 +/- 0 days) stage, from mature fetuses 3 weeks after bilateral adrenalectomy (n = 6), and from neonatal lambs within 16 h of birth (n = 6). Secretion of irACTH was determined by RIA of incubation medium obtained during 3-h exposure of cells to vehicle, AVP, CRH, or both. In all cases, IL cells secreted measurable irACTH. The IL cells of immature fetuses responded to CRH (133 +/- 8% increase over basal secretion), AVP (52 +/- 6%), and CRH plus AVP (244 +/- 8%). In contrast, IL cells from mature fetuses responded only to CRH (160 +/- 20%) or CRH plus AVP (259 +/- 44%), as did cells from mature adrenalectomized fetuses (CRH, 356 +/- 70%; CRH plus AVP, 627 +/- 100%). Secretion from neonatal IL cells was not significantly increased above basal rates by CRH and/or AVP. The AP cells from immature fetuses responded significantly to CRH (406 +/- 16%), AVP (114 +/- 8%), and CRH plus AVP (559 +/- 38%), whereas cells from mature fetuses responded only to AVP (249 +/- 40%) or to CRH plus AVP (570 +/- 146%). In AP cells from mature adrenalectomized fetuses, the response pattern resembled that of immature intact fetal sheep (CRH, 429 +/- 76%; AVP, 146 +/- 15%; CRH plus AVP, 541 +/- 94%). Neonatal AP cells responded to CRH (196 +/- 25%), AVP (442 +/- 71%), and CRH plus AVP (646 +/- 93%). Further characterization of IL cells (n = 6 fetal and 2 neonatal) indicated that they were inhibited by dopamine (basal ACTH secretion decreased by 25 +/- 4%; ACTH secretory response to CRH decreased by 32 +/- 10%). These results show that fetal neurointermediate lobe cells secrete irACTH under basal and stimulated conditions. Moreover, the pattern of response of AP and neurointermediate lobe cells to secretagogues is influenced by gestational age and, possibly, cortisol.

Response of human fetal pituitary cells to activin and inhibin in-vitro.

Response of human fetal pituitary cells to activin and inhibin in-vitro.

Corticosteroid-binding globulin (CBG) production by hepatic and extra-hepatic sites in the ovine fetus; effects of CBG on glucocorticoid negative feedback on pituitary cells in vitro

Plasma cortisol levels increase in fetal sheep during late gestation and this is associated with an increase in plasma corticosteroid-binding globulin (CBG) concentrations. However, the relative tissue sources of plasma CBG, the ontogeny of its biosynthesis and glycoform composition have not been established in the ovine fetus. Therefore we examined whether changes in plasma corticosteroid binding capacity (CBC) in fetal sheep during late gestation were associated with different patterns of glycosylation and reflected changes in tissue CBG expression. Since free cortisol is considered the bioactive fraction, we measured changes in the percent and absolute free cortisol in fetal plasma during late gestation. In order to examine whether CBG alters cortisol negative feedback at the level of the fetal pituitary, we also examined the effect of exogenous CBG in mediating the glucocorticoid-induced suppression of basal and corticotrophin-releasing hormone (CRH)-stimulated ACTH release from fetal pituitary cells in culture. The mean free cortisol concentration in plasma was not different between days 15 and 20 prior to parturition, and between 5 and 10 days prepartum, although it did rise between these times. Plasma CBC in chronically catheterized fetuses rose from 23.3 +/- 4.6 ng/ml at day 115 to 86.5 +/- 20.8 ng/ml at term and then decreased rapidly after birth. Between day 125 and day 140 of pregnancy approximately 10% of fetal plasma CBG was retarded by Concanavalin-A chromatography. This proportion increased at birth and attained adult values of > 70% by one month of age. By Northern blotting the relative levels of CBG mRNA in the fetal liver did not change between days 100 and 125, then increased significantly at day 140, but declined at term and in newborn lambs. CBG mRNA was undetectable in total RNA from lung, kidney, hypothalamus and placentomes, but was present in the fetal pituitary at days 125 and 140. Reverse transcription-PCR was used to confirm the presence of CBG mRNA in pituitary tissue from term fetuses. In cultures of term fetal pituitary cells, added CBG attenuated the cortisol- but not the dexamethasone-mediated suppression of basal and CRH-stimulated ACTH release. We conclude that in fetal sheep there is an increase in the corticosteroid binding capacity of plasma during late pregnancy which regulates, in part, free cortisol levels in the circulation. The liver is the major site of CBG biosynthesis in the fetus and at least until day 140 of gestation the rise in plasma CBC is associated with an increase in hepatic CBG mRNA levels.(ABSTRACT TRUNCATED AT 400 WORDS)

Luteinizing hormone synthesis in cultured fetal human pituitary cells exposed to gonadotropin-releasing hormone

Luteinizing hormone synthesis in cultured fetal human pituitary cells exposed to gonadotropin-releasing hormone

Luteinizing hormone synthesis in cultured fetal human pituitary cells exposed to gonadotropin-releasing hormone.

To investigate whether GnRH regulates LH synthesis during human development, pituitary cells from second trimester fetuses were incubated with [35S]methionine ([35S]met) and [3H]glucosamine ([3H]gln) for 48 h with 0, 10(-9), and 10(-7) mol/L GnRH. Immunoassayable (i) LH was measured in media and cellular lysates, and dual label scintillation analysis was used to quantitate incorporation of radiolabeled precursors into cells, trichloroacetic acid-precipitable proteins, and immunoprecipitated LH subjected to electrophoresis. Exposure of cells to GnRH did not affect cellular uptake or incorporation of precursors into proteins, but specifically increased total (secreted plus cellular) LH synthesis. Both GnRH concentrations significantly increased iLH release and enhanced secreted and cellular [3H]gln-LH. The secretion of [35S] met-LH was stimulated only by 10(-7) mol/L GnRH. The proportion of newly synthesized LH that was secreted and the 3H/35S ratio of secreted and cellular LH were uninfluenced by GnRH. Although basal LH synthesis was not sex dependent, total iLH content and GnRH-stimulated LH translation were greater in cells from females than in those from males. Therefore, GnRH regulates LH synthesis by second trimester fetal human gonadotrophs without influencing the proportion of total radiolabeled LH that is secreted. The existence of a sex difference in total iLH content and GnRH-stimulated LH translation is consistent with the sexual dimorphism in pituitary LH content occurring during human development.

Prostaglandin E2 enhances AVP-stimulated but not CRF-stimulated ACTH secretion from cultured fetal sheep pituitary cells.

This study examined the ability of prostaglandin E2 (PGE2) to regulate ACTH secretion from cultured anterior pituitary cells of fetal sheep between days 130 and 140 of gestation (term = 145 days). Corticotrophin-releasing factor (CRF) and arginine vasopressin (AVP) induced dose-dependent (0.1-1000 nmol/l) increases in ACTH secretion from fetal sheep pituitary cells maintained in culture for 6 days, with AVP being significantly (P less than 0.01) more potent than CRF. PGE2 (1000 nmol/l) significantly (P less than 0.05) enhanced the ability of AVP, but not CRF, to stimulate ACTH secretion. However, PGE2 given alone (0.1-1000 nmol/l) had no effect on ACTH secretion. Concomitant administration of CRF and AVP induced a greater release of ACTH than after treatment with either peptide alone, a synergistic interaction which was unaffected by simultaneous administration of PGE2. These results provide evidence for a direct action of PGE2 on ACTH secretion from the fetal sheep pituitary gland via a specific interaction with AVP. This interaction may allow increased fetal plasma concentrations of PGE2, seen during late gestation, to stimulate fetal pituitary-adrenal maturation.