ZBTB20 is required for anterior pituitary development and lactotrope specification.

The anterior pituitary harbours five distinct hormone-producing cell types, and their cellular differentiation is a highly regulated and coordinated process. Here we show that ZBTB20 is essential for anterior pituitary development and lactotrope specification in mice. In anterior pituitary, ZBTB20 is highly expressed by all the mature endocrine cell types, and to some less extent by somatolactotropes, the precursors of prolactin (PRL)-producing lactotropes. Disruption of Zbtb20 leads to anterior pituitary hypoplasia, hypopituitary dwarfism and a complete loss of mature lactotropes. In ZBTB20-null mice, although lactotrope lineage commitment is normally initiated, somatolactotropes exhibit profound defects in lineage specification and expansion. Furthermore, endogenous ZBTB20 protein binds to Prl promoter, and its knockdown decreases PRL expression and secretion in a lactotrope cell line MMQ. In addition, ZBTB20 overexpression enhances the transcriptional activity of Prl promoter in vitro. In conclusion, our findings point to ZBTB20 as a critical regulator of anterior pituitary development and lactotrope specification.

Anti-Müllerian hormone: a new actor of sexual dimorphism in pituitary gonadotrope activity before puberty.

Anti-Müllerian hormone (AMH) contributes to male sexual differentiation and acts on gonads of both sexes. Identification of AMH receptivity in both pituitary and brain has led to the intriguing idea that AMH participates to the hypothalamic-pituitary control of reproduction, however in vivo experimental evidence is still lacking. We show that AMH stimulates secretion and pituitary gene expression of the gonadotropin FSH in vivo in rats. AMH action is sex-dependent, being restricted to females and occurring before puberty. Accordingly, we report higher levels of pituitary AMH receptor transcripts in immature females. We show that AMH is functionally coupled to the Smad pathway in LßT2 gonadotrope cells and dose-dependently increases Fshb transcript levels. Furthermore, AMH was shown to establish complex interrelations with canonical FSH regulators as it cooperates with activin to induce Fshb expression whereas it reduces BMP2 action. We report that GnRH interferes with AMH by decreasing AMH receptivity in vivo in females. Moreover, AMH specifically regulates FSH and not LH, indicating that AMH is a factor contributing to the differential regulation of gonadotropins. Overall, our study uncovers a new role for AMH in regulating gonadotrope function and suggests that AMH participates in the postnatal elevation of FSH secretion in females.

Development of the ACTH and corticosterone response to acute hypoxia in the neonatal rat.

Acute episodes of severe hypoxia are among the most common stressors in neonates. An understanding of the development of the physiological response to acute hypoxia will help improve clinical interventions. The present study measured ACTH and corticosterone responses to acute, severe hypoxia (8% inspired O(2) for 4 h) in neonatal rats at postnatal days (PD) 2, 5, and 8. Expression of specific hypothalamic, anterior pituitary, and adrenocortical mRNAs was assessed by real-time PCR, and expression of specific proteins in isolated adrenal mitochondria from adrenal zona fascisulata/reticularis was assessed by immunoblot analyses. Oxygen saturation, heart rate, and body temperature were also measured. Exposure to 8% O(2) for as little as 1 h elicited an increase in plasma corticosterone in all age groups studied, with PD2 pups showing the greatest response ( approximately 3 times greater than PD8 pups). Interestingly, the ACTH response to hypoxia was absent in PD2 pups, while plasma ACTH nearly tripled in PD8 pups. Analysis of adrenal mRNA expression revealed a hypoxia-induced increase in Ldlr mRNA at PD2, while both Ldlr and Star mRNA were increased at PD8. Acute hypoxia decreased arterial O(2) saturation (SPo(2)) to approximately 80% and also decreased body temperature by 5-6 degrees C. The hypoxic thermal response may contribute to the ACTH and corticosterone response to decreases in oxygen. The present data describe a developmentally regulated, differential corticosterone response to acute hypoxia, shifting from ACTH independence in early life (PD2) to ACTH dependence less than 1 wk later (PD8).

Cholecystokinin directly inhibits neuronal activity of primary gonadotropin-releasing hormone cells through cholecystokinin-1 receptor.

Pulsatile secretion of GnRH-1 regulates gonadotropin release from anterior pituitary and thus is essential for reproduction. The present study focused on the role of cholecystokinin (CCK) in the GnRH-1 system. CCK is a neuropeptide abundantly expressed in the brain, which is implicated in activation of female reproductive behaviors and release of anterior pituitary hormones. Using dual-label immunocytochemistry coupled to confocal analysis, GnRH-1 neurons in adult mouse brain were found to express CCK-1 receptors (CCK-1R), and CCK fibers were detected contacting GnRH-1 axons. To address the function of CCK on GnRH-1 neurons, calcium imaging was used to monitor patterns of activity of GnRH-1 neurons maintained in an in vitro system known to retain many characteristics of GnRH-1 cells in vivo. Endogenous receptors for CCK (CCK-1R and CCK-2R) were blocked with selective antagonists. Results indicate that CCK-1R but not CCK-2R antagonist treatment increased the number of calcium peaks/GnRH-1 cell, mean peak amplitude, and percentage of GnRH-1 cells displaying high activity. The increased activity in GnRH-1 neurons observed after application of CCK-1R antagonist was blocked by coincubation with exogenous CCK. This study provides evidence that CCK acts directly on GnRH-1 neurons to attenuate GnRH-1 neuronal activity via CCK-1R activation.

The role of transcription factors implicated in anterior pituitary development in the aetiology of congenital hypopituitarism.

The anterior pituitary gland is a central regulator of growth, reproduction and homeostasis, and is the end-product of a carefully orchestrated pattern of expression of signalling molecules and transcription factors leading to the development of this complex organ secreting six hormones from five different cell types. Naturally occurring and transgenic murine models have demonstrated a role for many of these molecules in the aetiology of combined pituitary hormone deficiency (CPHD). These include the transcription factors HESX1, PROP1, POU1FI, LHX3, LHX4, TBX19 (TPIT), SOX3 and SOX2. The expression pattern of these transcription factors, their interaction with co-factors and their impact on target genes dictate the phenotype that results when the gene encoding the relevant transcription factor is mutated. The highly variable phenotype may consist of isolated hypopituitarism, or more complex disorders such as septo-optic dysplasia (SOD) and holoprosencephaly. Since mutations in any one transcription factor are uncommon, and since the overall incidence of mutations in known transcription factors is low in patients with CPHD, it is clear that many genes remain to be identified, and characterization of these will further elucidate the pathogenesis of these complex conditions, and also shed light on normal pituitary development.

Cellular composition of the adult rat anterior pituitary is influenced by the neonatal sex steroid environment.

Growth hormone (GH) and prolactin (PRL) secretion differ significantly between adult males and females and this is due, at least in part, to the postpubertal hormone environment which affects GH and PRL gene expression, as well as somatotrope and lactotrope proliferation. However, the role of the neonatal steroid environment in this phenomenon is less well understood. We have used in situ hybridization to determine the number of GH and PRL mRNA containing cells, as well as the level of expression of these two hormones and of the pituitary transcription factor 1 (Pit-1). Neonatally castrated male rats that had been exposed to testosterone during the neonatal period, adulthood or during both periods, males castrated as adults, normal adult males and normal proestrous females were used. Orchidectomy of adult rats had no effect on the number of somatotropes or lactotropes, but significantly reduced GH and PRL mRNA levels. Neonatal castration significantly reduced the percentage of somatotropes and increased that of lactotropes in the adult male. In addition, GH and Pit-1 mRNA levels were reduced significantly, but PRL mRNA levels were not modified. Treatment of neonatally castrated males with testosterone during the neonatal period significantly increased the percentage of somatotropes and decreased the percentage of lactotropes compared to vehicle-treated animals. It also increased GH and Pit-1 mRNA levels, but did not affect PRL mRNA levels. Adult testosterone treatment significantly increased the percentage of both somatotropes and lactotropes, as well as GH, PRL and Pit-1 mRNA levels. Treatment of neonatally castrated males with testosterone during both the neonatal and adult periods returned the percentage of somatotropes and lactotropes, as well as GH, PRL and Pit-1 mRNA levels, to that of the intact male. These results suggest that, although the postpubertal steroid environment is important in determining anterior pituitary hormone synthesis and cellular composition, the neonatal steroid environment also plays an important role in this phenomenon.

Fetal grafts containing suprachiasmatic nuclei restore the diurnal rhythm of CRH and POMC mRNA in aging rats.

We assessed whether fetal tissue containing the suprachiasmatic nuclei (SCN) can restore age-related changes in the diurnal rhythm of hypothalamic corticotropin-releasing hormone (CRH) and anterior pituitary proopiomelanocortin (POMC) mRNA. Young, middle-aged, and middle-aged SCN-transplanted rats were killed at seven times of day. In young rats, CRH mRNA exhibited a diurnal rhythm in the dorsomedial paraventricular nuclei but not in other subdivisions of the nuclei. No rhythm was detected in aging rats. SCN transplants restored a rhythm in CRH mRNA, but the timing was not precisely the same as in young animals. POMC mRNA exhibited a daily rhythm in young rats. Aging abolished the rhythm and decreased the average mRNA level; fetal transplants restored the rhythm, but the amplitude remained attenuated. These data are the first demonstration that fetal tissue can restore the diurnal rhythm of a neuroendocrine axis that is driven by the SCN. We conclude that the neuroendocrine substrate from the aging host remains capable of responding to diurnal cues to express diurnal rhythmicity in CRH/POMC mRNA when fetal SCN transplants confer the appropriate signals.

Development and differentiation of pituitary cells.

Classically, it was thought that the adenohypophyseal gland originated from the oral ectoderm. Its development has been the object of numerous studies over many years. However, several questions are still raised about its origin, differentiation, and commitment. The adenohypophyseal gland could originate from the anterior ridge of the neural plate. Glandular adenohypophyseal cells are committed very early in embryonic life. Interactions between adenohypophyseal presumptive territory and neighboring tissues can exist very soon, as early as at the open neural stage. The expression of a given phenotype by the committed cells seems to be controlled by a number of differentiation and/or transcription factors. In view of all these studies, performed with the use of different in vivo and in vitro models, classical concepts of the embryology of the adenohypophyseal gland need to be reevaluated. Indeed, many questions remain unanswered concerning the molecular mechanisms of known and unknown factors controlling development of the adenohypophyseal gland.

Effect of age of donor on the responsiveness of dispersed and cultured chicken anterior pituitary cells to GnRH-I.

1. The aim of this study was to devise a method to prepare and culture anterior pituitary cells from juvenile and adult chickens in order to investigate mechanisms controlling gonadotrophin-releasing hormone-I (GnRH-I)-induced luteinising hormone (LH) release in vitro. 2. The optimum culture medium for maintaining gonadotroph responsiveness to GnRH-I was bicarbonate-buffered and phenol red-free Medium 199 supplemented with 10% foetal calf serum. 3. Cultured pituitary cells from juvenile chickens were more responsive to GnRH-I than cells from adult cockerels, while no LH was released in response to GnRH-I from pituitary cells from laying hens. 4. Cultured pituitary cells from adult chickens of both sexes released LH in response to 12-O-tetradecanoyl-13-phorbol acetate (TPA), an activator of an enzyme involved in intracellular signalling, protein kinase C. 5. It is concluded that freshly-dispersed and cultured gonadotrophs from adult chickens do not regain their responsiveness to GnRH-I as well as freshly-dispersed and cultured gonadotrophs from juvenile chickens. It appears that the stimulus-secretion coupling pathway between the GnRH-receptor and the activation of protein kinase C in gonadotrophs from adult chickens is more easily disrupted by dispersion and culture than in gonadotrophs from juvenile chickens.

Developmental changes of tachykinins in the hypothalamus and anterior pituitary of female Siberian hamsters from prepuberty to adulthood.

The developmental changes of hypothalamic and anterior pituitary tachykinin concentrations were studied in female Siberian hamsters kept either under short (SD) or long (LD) daily photoperiods. The animals were killed between 15 and 70 days of age. Hypothalamic NKA sharply increased starting at 15 days up to 50 days in LD animals, and between 20 and 60 days in SD animals. Hypothalamic SP levels increased in a similar manner in SD animals, but in LD animals the increment was less pronounced, with increased levels from day 20 to 40, followed by a plateau. In the anterior pituitary gland, NKA concentrations in LD animals increased at 40 days of age, with only slight increases afterward, but overall the increment curve was considerably flatter than for hypothalamic NKA. In SD animals, the increase of anterior pituitary NKA was much steeper than in LD animals. However, the total content of NKA in the AP was similar in both SD and LD animals, because the AP weight was considerably higher in LD- than in SD-exposed hamsters. These results showed that photoperiod did not markedly affect the developmental changes in hypothalamic NKA. The developmental changes in anterior pituitary NKA concentrations were considerably smaller than in the hypothalamus in LD animals, but in SD animals they were much steeper. NKA concentrations in the anterior pituitary were markedly affected by the photoperiod. Concentrations of NKA in the anterior pituitary of the Siberian hamster at the age of 15 days of age were already higher than in the anterior pituitary of adult rats or Syrian hamsters.(ABSTRACT TRUNCATED AT 250 WORDS)

A comparative study of the effects of neonatal androgenization and estrogenization on vasoactive intestinal peptide levels in the anterior pituitary and the hypothalamus of adult female rats.

We compared the effects of neonatal androgenization (NA) and estrogenization (NE) on vasoactive intestinal peptide (VIP) levels in the anterior pituitary (AP) and the hypothalamus and on prolactin (PRL) secretion in adult female rats. Twenty-four hours after birth, a total of seven groups were treated as follows. Three NA groups received a single subcutaneous injection of 10, 100, or 1,000 micrograms of testosterone, respectively. Similarly, three NE groups received 1, 10, or 100 micrograms of 17 beta-estradiol, respectively. The remaining one group was injected with oil vehicle only, and served as controls. At 8 weeks of age, animals were sacrificed by rapid decapitation. NA (1,000 micrograms) and NE (100 micrograms) resulted in a similar degree of hyperprolactinemia and hyperestrogenemia, but this effect ratio between NA and NE (about 1:10) was not true with the lower doses, indicating a qualitative difference in the effects of the two treatments. This is in agreement with our previous study. VIP content determined in the suprachiasmatic nucleus, the paraventricular nucleus and the median eminence did not significantly correlate with plasma PRL. In contrast, there were significant correlations among AP VIP, plasma PRL and estradiol. These results suggest the possibility that the NA- and NE-induced hyperprolactinemia may be mediated, at least in part, by a paracrine and/or autocrine effect of the increased AP VIP on the lactotroph which may probably be mediated by hyperestrogenemia. However, the possibility was also suggested that the observed changes in AP VIP were related more to NA and NE's imprinting effects on the developing brain than to the PRL secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

[The development of neuroendocrine regulation in ontogeny]. / Stanovlenie neiroéndokrinnoi reguliatsii v ontogeneze.

Published and author's data on hypothalamic control over the most important endocrine functions during mammalian ontogenesis were reviewed. The data presented indicates that adenohypophyseotropic protein neurohormones are synthesized and accumulated in hypothalamus long before birth. At the end of the prenatal period most of hypothalamic neurohormones reached the adenohypophysis and were involved in regulation of the tropic hormones secretion. Exceptionally, regulative properties of thyroliberin and somatostatin are only manifested in immature-born animals (rats) in early postnatal period.

Vasoactive intestinal peptide and the stimulation of lactotroph growth by oestradiol in rats.

Treatment with a high dose of oestradiol for 6 months caused hyperprolactinaemia and pituitary hyperplasia in female Wistar-Furth rats. Changes in the vasoactive intestinal peptide (VIP) and dopamine content of the hypothalamus and pituitary were also found. The hypothalamic dopamine concentration was only slightly reduced and, although the concentration of dopamine in the pituitary was less in treated animals, the total pituitary content was increased. The concentration of VIP in the pituitary was increased by oestradiol treatment but decreased in the non-median eminence hypothalamus. In the median eminence the VIP content was increased by oestradiol treatment and the amount present correlated positively and significantly with pituitary wet weight in animals treated with both oestradiol and fluphenazine. In Fischer 344 rats, oestradiol produced greater incremental changes in pituitary wet weight and plasma concentrations of prolactin than in Wistar controls and the increase in the pituitary concentration of VIP was five times greater. Although peptide turnover has not been measured, these results suggest that oestradiol, as well as having a direct action, stimulates pituitary lactotrophs by increasing pituitary concentrations of VIP.

The effect of physiological doses of thyroxine on the level of cyclic adenosine-3',5'-monophosphate in pituitary and anterior hypothalamus of male rats of different age.

The levels of cAMP in the pituitary and anterior hypothalamus were determined by competitive binding to protein in male rats aged 2--2.5, 6--8 and 14--18 months. The lowest level of cAMP both in the pituitary and anterior hypothalamus (1.05 +/- 0.17 and 0.85 +/- 0.23 pM) (per mg wet weight, respectively) was found in 6--8 month-old rats. Administration of T4 (2 micrograms/100 g body weight, for 8 days) was followed by a decrease in cAMP level in the pituitary of 14--18 month-old animals, which was not the case with younger rats. The obtained results agree with the earlier observation that the threshold to homeostatic suppression in the hypothalamus-pituitary-thyroid system of rats decreases with aging. It is suggested that cAMP system contributes to the mechanism of changes in hypothalamic threshold and its pharmacological correction.

Effects of testosterone and orchiectomy on the development of gonadotrophs in neonatal rats with hypophysial stalk section.

The development of gonadotrophs in newborn Sprague-Dawley rats subjected to a hypophysial stalk section (SS) by an electrical cauterization of the infundibulum of the hypothalamus was examined throughout the first 12 postnatal days. The electrical cauterization of the infundibulum was performed during 24 to 30 h after birth, and the pituitaries were studied by immunohistochemical procedures 11 days later. In several animals orchiectomy or an administration of testosterone propionate (TP) was performed simultaneously with the cauterization. The SS animals showed remarkable retardation in body growth and in pituitary development. In males the development of gonadotrophs was strongly suppressed in number and in size after the cauterization, whereas in females the suppression was less prominent. Orchiectomy promoted the development of gonadotrophs in the SS males, while TP administration suppressed it in the SS females. These findings indicate that there is sexual difference in the neonatal development of gonadotrophs even in the pituitary isolated from the hypothalamus and that testosterone inhibits the development of gonadotrophs at the level of the hypophysis during the neonatal period in rats.

Correlation of hypothalamic LHRH, pituitary LH and plasma LH in developing rats.

Hypothalamic LHRH, pituitary LH and plasma LH levels were measured in rats of both sexes from day 5-60 after birth. The content of hypothalamic LHRH was very high in one-week-old male and female rats. It declined gradually till day 17 in the female rat and sharply on day 10 in the male rat. Subsequently the content of hypothalamic LHRH increased and showed peak values on day 25 in the female rat and on day 45 in the male rat. It decreased markedly at respective times of puberty in both sexes (day 37 in the female rat and day 52-60 in the male rat). Results of the study suggest that maturation of hypothalamo-hypophyseal-axis proceeds in three distinct stages. Observations on days 17, 25 and 37 in the female rat and on days 5, 7, 10 and 22 in the male rat clearly show an inverse relationship between hypothalamic LHRH and plasma LH and a parallel relationship between pituitary and plasma LH. Marked decline in the content of hypothalamic LHRH at respective times of puberty in both sexes indicates that the release of threshold levels of LHRH from the hypothalamus may apparently be the event initiating the pubertal changes in rat.