Gonadotrope and thyrotrope development in the human and mouse anterior pituitary gland

Genes and orthologous intrinsic and extrinsic factors critical for embryonic pituitary gonadotrope and thyrotrope cell differentiation have been identified mainly in rodents, but data on the human are very limited. In human fetal pituitaries examined between 14 and 19 weeks of gestation using immunofluorescent confocal microscopy, we found that most fetal gonadotropes expressed alpha-GSU, LHbeta, and FSHbeta gonadotropin subunits while almost no cells expressed alpha-GSU and LHbeta alone. Gonadotropes expressing alpha-GSU and FSHbeta only were detected in both male and female pituitaries, increasing in proportion to total gonadotropes in both males and females from 14 (approximately 4.5%) to 19 weeks (approximately 16.5%) with a peak in males of 45.5% compared with females of 16.5% at 17 weeks of gestation. When FSHbeta or LHbeta genes were expressed, gonadotropes were non-dividing. This profile of human fetal gonadotrope development differs from the current mouse model. Furthermore, while expression of alpha-GSU appears to be the lead protein in gonadotropes, in thyrotropes which ultimately express alpha-GSU with TSHbeta, we observed that most if not all thyrotropes were TSHbeta-positive but alpha-GSU-negative until around 19 weeks in human, and e15 in mouse, fetal pituitaries. Furthermore, the TSHbeta-only thyrotropes were dividing, and TSHbeta rather than alpha-GSU was the lead protein in thyrotrope development. Thus, while biologically active dimeric FSH and LH can be produced by the human fetal pituitary by 14 weeks, dimeric biologically active TSH will only be produced from around 17 weeks of gestation. The mechanism(s) responsible for the different molecular regulation of alpha-GSU gene expression in gonadotropes and thyrotropes in the developing human fetal pituitary now requires investigation.     

Persistent expression of Notch2 delays gonadotrope differentiation

Normal pituitary gland development requires coordination between maintenance of progenitor cell pools and selection of progenitors for differentiation. The spatial and temporal expression of Notch2 during pituitary development suggested that it could control progenitor cell differentiation in the pituitary. Consistent with this idea, Notch2 is not expressed in Prop1 mutants, and anterior pituitary progenitors in Prop1 mutants appear to be unable to transition from proliferation to differentiation properly, resulting in anterior lobe failed cell specification and evolving hypoplasia. To test the function of Notch2 directly, we used the alphaGSU subunit promoter to express activated NOTCH2 persistently in pre-gonadotropes and pre-thyrotropes of transgenic mice. At birth, there is a small reduction in the population of fully differentiated thyrotropes and almost no fully differentiated gonadotropes. The temporal and spatial expression of Hey1 suggests that it could be a mediator of this effect. Gonadotropes complete their differentiation program eventually, although expression of LH and FSH is mutually exclusive with NOTCH2 transgene expression. This demonstrates that activated Notch2 is sufficient to delay gonadotrope differentiation, and it supports the hypothesis that Notch2 regulates progenitor cell differentiation in the pituitary gland.     

Testosterone increases TSH-beta mRNA, and modulates alpha-subunit mRNA differentially in mouse thyrotropic tumor and castrate rat pituitary.

TSH, LH and FSH, the three pituitary glycoprotein hormones, are each composed of a common alpha-subunit and a hormone specific beta-subunit. Testosterone is known to regulate all three intact hormones differently in the rodent. However, there is only one gene encoding the common alpha-subunit. In order to elucidate the effects of testosterone on TSH subunit synthesis and its regulation of the common alpha-subunit, two in vivo models were studied: castrate rat pituitary was used as a gonadotropin-enriched tissue; and mouse thyrotropic tumor was used as a thyrotropin-enriched tissue. Male castrate rats were treated with testosterone propionate, 500 micrograms/100 g BW, sc, for 11 days. Testosterone increased plasma TSH to 131% of control values (P less than 0.02), while plasma LH fell to undetectable levels, and plasma alpha-subunit fell to 14% of control values (P less than 0.001). Testosterone increased TSH-beta mRNA to 237% of control values (P less than 0.02), while alpha-subunit mRNA fell to 20% of control values (P less than 0.001). Hypothyroid mice bearing thyrotropic tumors were treated with testosterone propionate, 150 micrograms/100 g BW, sc, for 11 days. In this model plasma TSH-beta and alpha-subunit concentrations are 1000-fold higher than in non-tumor bearing animals, and the contribution of pituitary gonadotropes to plasma subunit concentrations is negligible. "Total" TSH-beta and alpha-subunit concentrations were estimated as one-half of intact TSH plus the respective free subunit concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunohistochemical evidence for the presence of glucokinase in the gonadotropes and thyrotropes of the anterior pituitary gland of rat and monkey.

A recent report provides new evidence for the presence of glucokinase (GK) in the anterior pituitary. In the present study, immunohistochemistry was used to identify the cells containing GK in the pituitary of rats and monkeys. In rats, GK was detected as a generalized cytoplasmic staining in a discrete population of cells in the anterior pituitary. In colocalization experiments, the majority of cells expressing follicle-stimulating hormone (FSH) or luteinizing hormone (LH) also contained GK. In addition to the gonadotropes, GK was observed in a subpopulation of corticotropes and thyrotropes. GK was not detected in cells expressing growth hormone or prolactin. In monkeys, GK was also observed in a discrete population of cells. Intracellular distribution differed from the rat in that GK in most cells was concentrated in a perinuclear location that appeared to be associated with the Golgi apparatus. However, similar to rats, colocalization experiments showed that the majority of cells expressing FSH or LH also contained GK. In addition to the gonadotropes, GK was observed in a subpopulation of corticotropes and thyrotropes. In the monkey, only a few cells had generalized cytoplasmic staining for GK. These experiments provide further evidence for the presence of GK in the anterior pituitary. Although some corticotropes and thyrotropes contained GK, the predominant cell type expressing GK was gonadotropes. In view of the generally accepted role of GK as a glucose sensor in a variety of cells including the insulin-producing pancreatic beta-cells as the prototypical example, it is hypothesized that hormone synthesis and/or release in pituitary cells containing GK may be directly influenced by blood glucose.     

Pitx2 deletion in pituitary gonadotropes is compatible with gonadal development, puberty, and fertility

This report introduces a gonadotrope-specific cre transgenic mouse capable of ablating floxed genes in mature pituitary gonadotropes. Initial analysis of this transgenic line, Tg(Lhb-cre)1Sac, reveals that expression is limited to the pituitary cells that produce luteinizing hormone beta, beginning appropriately at e17.5. Cre activity is detectable by a reporter gene in nearly every LHbeta-producing cell, but the remaining hormone-producing cell types and other organs exhibit little to no activity. We used the Tg(Lhb-cre)1Sac strain to assess the role Pitx2 in gonadotrope function. The gonadotrope-specific Pitx2 knockout mice exhibit normal expression of LHbeta, sexual maturation, and fertility, suggesting that Pitx2 is not required for gonadotrope maintenance or for regulated production of gonadotropins.     

An immunocytological study of the adult crab-eating macaque (Macaca fascicularis) pituitary and its cytological differentiation during fetal life

The pituitaries of adult and fetal crab-eating macaques (Macaca fascicularis) have been studied by immunofluorescence using 15 antibodies against most of the known hormones in the adenohypophysis. The antibodies used were first checked on adult pituitaries for their cross-specificity with macaque pituitary hormones. We found five types of endocrine cells reacting positively, according to the biochemical relation of the molecules evidenced with one or more of the antibodies used. The sequential appearance of the various hormones in the cells of the anterior and intermediate lobes was then determined. The first hormones evidenced at day 45 of pregnancy were ACTH, beta-MSH, beta- and gamma-LPH and alpha- and beta-endorphins. alpha-MSH appeared at day 48 and STH at day 51. The glycoprotein hormones, LH, FSH and TSH, appeared at day 57 but the thyrotropes and gonadotropes did not attain their adult characteristics (staining intensity, morphology, density and distribution in the pituitary) until days 71 and 93, respectively. Prolactin was only found beginning at day 93 of pregnancy. The different specificity tests applied to the pituitary of the macaque, as well as to that of other vertebrates, show that the antibodies used have good specificity. A comparison of the dates at which the fetal pituitary gonadotropes appear in the macaque and the results of a developmental study of the external genital organs in that species indicated that the pituitary gonadotropic function is only established after somatic sex differentiation, which would thus probably occur independently.     

Intracellular responses to gonadotropin-releasing hormone in a clonal cell line of the gonadotrope lineage.

We recently derived a GnRH-responsive pituitary cell line of the gonadotrope lineage (alpha T3-1) by targeted oncogenesis in transgenic mice. Here, we report studies characterizing the GnRH receptors present in these cells and the intracellular responses to GnRH treatment. The receptors in alpha T3-1 cells show specificity for different GnRH analogs, with dissociation constants very similar to those found in normal rat and mouse pituitary. The concentration of receptors is within the range found in normal pituitary. The addition of GnRH or GnRH agonists increases phosphoinositide turnover and protein kinase-C translocation to membranes, and enhances activation of voltage-sensitive calcium channels. However, GnRH does not affect cAMP levels. Analysis of alpha-subunit mRNA levels demonstrated induction by GnRH and phorbol esters. Our results indicate that GnRH initiates a cascade of intracellular events that generate a set of second messengers, one or more of which is involved in the regulation of gene expression. The responses of alpha T3-1 cells to GnRH appear to have characteristics equivalent to those of primary pituitary gonadotropes, indicating the utility of this cell line as a model system for the study of GnRH responses.     

Detection of luteinizing hormone beta messenger ribonucleic acid (RNA) in individual gonadotropes after castration: use of a new in situ hybridization method with a photobiotinylated complementary RNA probe

Patterns of gonadotropin storage in individual gonadotropes change with alterations in the physiological state. After castration in the male rat, there is a 2.5-fold increase in the percentage of gonadotropes and an increase in the proportion of gonadotropes storing both LH and FSH. In addition, there are 6- to 8-fold increases in the pituitary concentrations of LH beta subunit mRNAs. In order to determine whether these changes are due to increases in the number of gonadotropes containing subunit mRNA, or the amount of mRNA per cell or both, an in situ hybridization technique using a photobiotinylated rat LH beta cRNA probe (bio-LH beta-cRNA) was applied to detect LH beta mRNA in fixed whole rat pituitary cells from intact or castrated rats. After hybridization, the bio-LH beta-cRNA was localized with either avidin-biotin peroxidase complex or the fluorescent streptavidin phycoprobe methods. The cells containing LH beta mRNA were then counted and the amount of mRNA per cell was measured by video microdensitometry. Ten percent of the anterior pituitary cells from intact animals contained LH beta mRNA. After castration (2-4 weeks) this percentage rose to 19-24.5%. Image and microdensitometric analyses showed that castration produced a 1.9-fold increase in the amount of LH beta mRNA per cell, and a 2.2-fold increase in the area of cells containing LH beta mRNA. Hence, castration resulted in an increase in the level of LH beta mRNA per cell as well as the number of LH beta mRNA-containing cells. When in situ hybridization was followed by immunocytochemistry in cells from intact rats, 83% of gonadotropes that stained for LH beta and 80% of gonadotropes that stained for FSH beta contained LH beta mRNA whereas after castration 99% of LH-storing and 93% of FSH-storing cells contained LH beta mRNA. This new in situ hybridization protocol is rapid and allows quantification of mRNA within individual gonadotropes. In addition, since the hybridization protocol does not apparently alter the gonadotropin antigens, the hormone content of the same gonadotrope may be defined by immunocytochemistry.     

Vitamin D sites of action in the pituitary studied by combined autoradiography-immunohistochemistry

Adult male and female mice under normal diet were injected with 3H 1,25(OH)2 vitamin D3 and sacrificed 3.5 h afterwards. Autoradiograms were prepared according to our thaw-mount technique and stained with antibodies to pituitary hormones. Thyrotropes showed strong and extensive nuclear concentration of radioactivity: about 90% of the immunostained thyrotropes were labeled. Lactotropes, somatotropes and gonadotropes showed no or only weak nuclear radioactivity: a subpopulation of 5%-10% of each of these immunostained cell types displayed nuclear labeling that was weak when compared to thyrotropes. Neural lobe pituicytes also showed weak to intermediate nuclear labeling. The results indicate a presence of nuclear receptors for 1,25(OH)2 vitamin D3 in pituitary cell types and suggest direct but differential genomic effects of 1,25(OH)2 vitamin D3 on pituitary hormone secretion. Evidence further suggests the existence of a vitamin D regulated brain-pituitary-thyroid axis.