The forkhead transcription factor, FOXP3: a critical role in male fertility in mice.

Fertility is dependent on the hypothalamic-pituitary-gonadal axis. Each component of this axis is essential for normal reproductive function. Mice with a mutation in the forkhead transcription factor gene, Foxp3, exhibit autoimmunity and infertility. We have previously shown that Foxp3 mutant mice have significantly reduced expression of pituitary gonadotropins. To address the role of Foxp3 in gonadal function, we examined the gonadal phenotype of these mice. Foxp3 mutant mice have significantly reduced seminal vesicle and testis weights compared with Foxp3(+/Y) littermates. Spermatogenesis in Foxp3 mutant males is arrested prior to spermatid elongation. Activation of luteinizing hormone signaling in Foxp3 mutant mice by treatment with human chorionic gonadotropin significantly increases seminal vesicle and testis weights as well as testicular testosterone content and seminiferous tubule diameter. Interestingly, human chorionic gonadotropin treatments rescue spermatogenesis in Foxp3 mutant males, suggesting that their gonadal phenotype is due primarily to a loss of pituitary gonadotropin stimulation rather than an intrinsic gonadal defect.

FOXO1 regulates expression of Neurod4 in the pituitary gland.

Pituitary gland function is regulated by the activity of various transcription factors that control cell fate decisions leading to cellular differentiation and hormone production. FOXO1 is necessary for normal somatotrope differentiation and function. Recent in vivo data implicate FOXO1 in the regulation of genes important for somatotrope differentiation including Gh1, Neurod4, and Pou1f1. In the current study, the somatotrope-like cell line GH3 was treated with a FOXO1 inhibitor, resulting in significant reduction in Neurod4 and Gh1 expression. Consistent with these findings, CRISPR/Cas9-mediated deletion of Foxo1 in GH3 cells significantly reduced expression of Gh1 and Neurod4. Chromatin immunoprecipitation sequencing identifies novel FOXO1 binding sites associated with the Neurod4, Gh1, and Pou1f1 genes. The FOXO1 binding site in the Neurod4 gene exhibits enhancer activity in somatotrope-like cells but not in gonadotrope-like cells. These data strongly suggest FOXO1 directly contributes to the transcriptional control of genes important for somatotrope differentiation.

Metformin treatment of juvenile mice alters aging-related developmental and metabolic phenotypes in sex-dependent and sex-independent manners.

Metformin has attracted increasing interest for its potential benefits in extending healthspan and longevity. This study examined the effects of early-life metformin treatment on the development and metabolism of C57BL/6 J (B6) mice, with metformin administered to juvenile mice from 15 to 56 days of age. Metformin treatment led to decreased body weight in both sexes (P < 0.05, t-test). At 9 weeks of age, mice were euthanized and organ weights were recorded. The relative weight of retroperitoneal fat was decreased in females, while relative weights of perigonadal and retroperitoneal fat were decreased, and relative liver weight was increased in males (P < 0.05, t-test). Glucose and insulin tolerance tests (GTT and ITT) were conducted at the age of 7 weeks. ANOVA revealed a significant impairment in insulin sensitivity by the treatment, and a significantly interactive effect on glucose tolerance between sex and treatment, underscoring a disparity in GTT between sexes in response to the treatment. Metformin treatment reduced circulating insulin levels in fasting and non-fasting conditions for male mice, with no significant alterations observed in female mice. qRT-PCR analysis of glucose metabolism-related genes (Akt2, Glut2, Glut4, Irs1, Nrip1, Pi3k, Pi3kca, Pkca) in the liver and skeletal muscle reveals metformin-induced sex- and organ-specific effects on gene expression. Comparison with previous studies in heterogeneous UM-HET3 mice receiving the same treatment suggests that genetic differences may contribute to variability in the effects of metformin treatment on development and metabolism. These findings indicate that early-life metformin treatment affects development and metabolism in both sex- and genetics-dependent manners.

Single-Cell Transcriptomics Identifies Pituitary Gland Changes in Diet-Induced Obesity in Male Mice.

Obesity is a chronic disease with increasing prevalence worldwide. Obesity leads to an increased risk of heart disease, stroke, and diabetes, as well as endocrine alterations, reproductive disorders, changes in basal metabolism, and stress hormone production, all of which are regulated by the pituitary. In this study, we performed single-cell RNA sequencing of pituitary glands from male mice fed control and high-fat diet (HFD) to determine obesity-mediated changes in pituitary cell populations and gene expression. We determined that HFD exposure is associated with dramatic changes in somatotrope and lactotrope populations, by increasing the proportion of somatotropes and decreasing the proportion of lactotropes. Fractions of other hormone-producing cell populations remained unaffected. Gene expression changes demonstrated that in HFD, somatotropes became more metabolically active, with increased expression of genes associated with cellular respiration, and downregulation of genes and pathways associated with cholesterol biosynthesis. Despite a lack of changes in gonadotrope fraction, genes important in the regulation of gonadotropin hormone production were significantly downregulated. Corticotropes and thyrotropes were the least affected in HFD, while melanotropes exhibited reduced proportion. Lastly, we determined that changes in plasticity and gene expression were associated with changes in hormone levels. Serum prolactin was decreased corresponding to reduced lactotrope fraction, while lower luteinizing hormone and follicle-stimulating hormone in the serum corresponded to a decrease in transcription and translation. Taken together, our study highlights diet-mediated changes in pituitary gland populations and gene expression that play a role in altered hormone levels in obesity.

Knockout mice with pituitary malformations help identify human cases of hypopituitarism.

BACKGROUND: Congenital hypopituitarism (CH) and its associated syndromes, septo-optic dysplasia (SOD) and holoprosencephaly (HPE), are midline defects that cause significant morbidity for affected people. Variants in 67 genes are associated with CH, but a vast majority of CH cases lack a genetic diagnosis. Whole exome and whole genome sequencing of CH patients identifies sequence variants in genes known to cause CH, and in new candidate genes, but many of these are variants of uncertain significance (VUS). METHODS: The International Mouse Phenotyping Consortium (IMPC) is an effort to establish gene function by knocking-out all genes in the mouse genome and generating corresponding phenotype data. We used mouse embryonic imaging data generated by the Deciphering Mechanisms of Developmental Disorders (DMDD) project to screen 209 embryonic lethal and sub-viable knockout mouse lines for pituitary malformations. RESULTS: Of the 209 knockout mouse lines, we identified 51 that have embryonic pituitary malformations. These genes not only represent new candidates for CH, but also reveal new molecular pathways not previously associated with pituitary organogenesis. We used this list of candidate genes to mine whole exome sequencing data of a cohort of patients with CH, and we identified variants in two unrelated cases for two genes, MORC2 and SETD5, with CH and other syndromic features. CONCLUSIONS: The screening and analysis of IMPC phenotyping data provide proof-of-principle that recessive lethal mouse mutants generated by the knockout mouse project are an excellent source of candidate genes for congenital hypopituitarism in children.

The forkhead transcription factor, FOXP3, is required for normal pituitary gonadotropin expression in mice.

The hypothalamic-pituitary-gonadal axis is central to normal reproductive function. This pathway begins with the release of gonadotropin-releasing hormone in systematic pulses by the hypothalamus. Gonadotropin-releasing hormone is bound by receptors on gonadotroph cells in the anterior pituitary gland and stimulates the synthesis and secretion of luteinizing hormone and, to some extent, follicle-stimulating hormone. Once stimulated by these glycoprotein hormones, the gonads begin gametogenesis and the synthesis of sex hormones. In humans, mutations of the forkhead transcription factor, FOXP3, lead to an autoimmune disorder known as immunodysregulation, polyendocrinopathy, and enteropathy, X-linked syndrome. Mice with a mutation in the Foxp3 gene have a similar autoimmune syndrome and are infertile. To understand why FOXP3 is required for reproductive function, we are investigating the reproductive phenotype of Foxp3 mutant mice (Foxp3(sf/Y)). Although the gonadotroph cells appear to be intact in Foxp3(sf/Y) mice, luteinizing hormone beta (Lhb) and follicle-stimulating hormone beta (Fshb) expression are significantly decreased, demonstrating that these mice exhibit a hypogonadotropic hypogonadism. Hypothalamic expression of gonadotropin-releasing hormone is not significantly decreased in Foxp3(sf/Y) males. Treatment of Foxp3(sf/Y) males with a gonadotropin-releasing hormone receptor agonist does not rescue expression of Lhb or Fshb. Interestingly, we do not detect Foxp3 expression in the pituitary or hypothalamus, suggesting that the infertility seen in Foxp3(sf/Y) males is a secondary effect, possibly due to loss of FOXP3 in immune cells. Pituitary expression of glycoprotein hormone alpha (Cga) and prolactin (Prl) are significantly reduced in Foxp3(sf/Y) males, whereas the precursor for adrenocorticotropic hormone, pro-opiomelanocortin (Pomc), is increased. Human patients diagnosed with IPEX often exhibit thyroiditis due to destruction of the thyroid gland by autoimmune cells. We find that Foxp3(sf/Y) mice have elevated expression of thyroid-stimulating hormone beta (Tshb), suggesting that they may suffer from thyroiditis as well. Expression of the pituitary transcription factors, Pitx1, Pitx2, Lhx3, and Egr1, is normal; however, expression of Foxl2 and Gata2 is elevated. These data are the first to demonstrate a defect at the pituitary level in the absence of FOXP3, which contributes to the infertility observed in mice with Foxp3 loss of function mutations.

FOXO Transcription Factors Are Required for Normal Somatotrope Function and Growth.

Understanding the molecular mechanisms underlying pituitary organogenesis and function is essential for improving therapeutics and molecular diagnoses for hypopituitarism. We previously found that deletion of the forkhead factor, Foxo1, in the pituitary gland early in development delays somatotrope differentiation. While these mice grow normally, they have reduced growth hormone expression and free serum insulin-like growth factor-1 (IGF1) levels, suggesting a defect in somatotrope function. FOXO factors show functional redundancy in other tissues, so we deleted both Foxo1 and its closely related family member, Foxo3, from the primordial pituitary. We find that this results in a significant reduction in growth. Consistent with this, male and female mice in which both genes have been deleted in the pituitary gland (dKO) exhibit reduced pituitary growth hormone expression and serum IGF1 levels. Expression of the somatotrope differentiation factor, Neurod4, is reduced in these mice. This suggests a mechanism underlying proper somatotrope function is the regulation of Neurod4 expression by FOXO factors. Additionally, dKO mice have reduced Lhb expression and females also have reduced Fshb and Prl expression. These studies reveal FOXO transcription factors as important regulators of pituitary gland function.

Synergistic activation of the Mc2r promoter by FOXL2 and NR5A1 in mice.

Forkhead box protein L2 (FOXL2) is the earliest ovarian marker and plays an important role in the regulation of cholesterol and steroid metabolism, inflammation, apoptosis, and ovarian development and function. Mutations and deficiencies of the human FOXL2 gene have been shown to cause blepharophimosis-ptosis-epicanthus inversus syndrome as well as premature ovarian failure. Although Foxl2 interacts with steroidogenic factor 1 (Nr5a1) and up-regulates cyp19a1a gene transcription in fish, FOXL2 represses the transcriptional activity of the gene that codes for steroidogenic acute regulatory protein (Star) in mice. Most of the recent studies have heavily focused on the FOXL2 target genes (Star and Cyp19a1) in the ovaries. Hence, it is of importance to search for other downstream targets of FOXL2 and for the possibility of FOXL2 expression in nonovarian tissues. Herein, we demonstrate that the interplay between FOXL2 and NR5A1 regulates Star and melanocortin 2 receptor (Mc2r) gene expression in mammalian systems. Both FOXL2 and NR5A1 are expressed in ovarian and adrenal gland tissues. As expected, FOXL2 represses and NR5A1 enhances the promoter activity of Star. Notably, the promoter activity of Mc2r is activated by FOXL2 in a dose-dependent manner. Surprisingly, we found that FOXL2 and NR5A1 synergistically up-regulate the transcriptional activity of Mc2r. By mapping the Mc2r promoter, we provide evidence that distal NR5A1 response elements (-1410 and -975) are required for synergistic activation by FOXL2 and NR5A1. These results suggest that the interplay between FOXL2 and NR5A1 on the Mc2r promoter functions as a novel mechanism for regulating MC2R-mediated cell signaling as well as steroidogenesis in adrenal glands.

Mechanisms underlying pituitary hypoplasia and failed cell specification in Lhx3-deficient mice.

The LIM homeodomain transcription factor, LHX3, is essential for pituitary development in mouse and man. Lhx3 engineered null mice have profound pituitary hypoplasia that we find is attributable to an increase in cell death early in pituitary development. Dying cells are localized to regions of TPIT expression indicating that cell death may contribute to the severe reduction in differentiated corticotrope cells and lower expression of the corticotrope transcription factors, TPIT and NEUROD1. Lhx3 deficiency also results in dorsal ectopic expression of transcription factors characteristic of gonadotropes, SF1 and ISL1, but no gonadotropin expression. This apparent disturbance of cell differentiation may be due, in part, to loss of NOTCH2. NOTCH2 is normally expressed in the pituitary at the boundary between dorsal, proliferating cells and ventral, differentiating cells and is important for maintaining dorsal-ventral patterning in other organs. Thus, Lhx3 contributes significantly to pituitary development by maintaining normal dorsal-ventral patterning, cell survival, and normal expression of corticotrope-specific transcription factors, which are necessary for repressing ectopic gonadotrope differentiation.

Molecular Mechanisms Governing Embryonic Differentiation of Pituitary Somatotropes.

Pituitary somatotropes secrete growth hormone (GH), which is essential for normal growth and metabolism. Somatotrope defects result in GH deficiency (GHD), leading to short stature in childhood and increased cardiovascular morbidity and mortality in adulthood. Current hormone replacement therapies fail to recapitulate normal pulsatile GH secretion. Stem cell therapies could overcome this problem but are dependent on a thorough understanding of somatotrope differentiation. Although several transcription factors, signaling pathways, and hormones that regulate this process have been identified, the mechanisms of action are not well understood. The purpose of this review is to highlight the known players in somatotrope differentiation while emphasizing the need to better understand these pathways to serve patients with GHD.

Premature Expression of FOXO1 in Developing Mouse Pituitary Results in Anterior Lobe Hypoplasia.

The process by which the somatotrope lineage emerges in the developing pituitary is regulated by the activity of specific signaling and transcription factors expressed during development. We set out to understand the contribution of FOXO1 to that process by using a mouse model in which FOXO1 is prematurely expressed in the pituitary primordium. Expression of FOXO1 in the oral ectoderm as early as embryonic day (e)9.5 resulted in pituitary gland hypoplasia and reduced expression of anterior lobe hormone transcripts at e18.5. Of note, the relative numbers of somatotropes and thyrotropes were also decreased at e18.5. LHX3 and PITX2, markers of pituitary identity, were present in a reduced number of cells during the formation of the Rathke pouch. Thus, premature expression of FOXO1 may affect adoption of pituitary identity during differentiation. Our results demonstrate that the timing of FOXO1 activation affects its role in pituitary gland organogenesis and somatotrope differentiation.

Single-Cell RNA Sequencing Reveals Novel Markers of Male Pituitary Stem Cells and Hormone-Producing Cell Types.

Transcription factors and signaling pathways that regulate stem cells and specialized hormone-producing cells in the pituitary gland have been the subject of intense study and have yielded a mechanistic understanding of pituitary organogenesis and disease. However, the regulation of stem cell proliferation and differentiation, the heterogeneity among specialized hormone-producing cells, and the role of nonendocrine cells in the gland remain important, unanswered questions. Recent advances in single-cell RNA sequencing (scRNAseq) technologies provide new avenues to address these questions. We performed scRNAseq on ∼13,663 cells pooled from six whole pituitary glands of 7-week-old C57BL/6 male mice. We identified pituitary endocrine and stem cells in silico, as well as other support cell types such as endothelia, connective tissue, and red and white blood cells. Differential gene expression analyses identify known and novel markers of pituitary endocrine and stem cell populations. We demonstrate the value of scRNAseq by in vivo validation of a novel gonadotrope-enriched marker, Foxp2. We present novel scRNAseq data of in vivo pituitary tissue, including data from agnostic clustering algorithms that suggest the presence of a somatotrope subpopulation enriched in sterol/cholesterol synthesis genes. Additionally, we show that incomplete transcriptome annotation can cause false negatives on some scRNAseq platforms that only generate 3' transcript end sequences, and we use in vivo data to recover reads of the pituitary transcription factor Prop1. Ultimately, scRNAseq technologies represent a significant opportunity to address long-standing questions regarding the development and function of the different populations of the pituitary gland throughout life.

FOXL2 in the pituitary: molecular, genetic, and developmental analysis.

FOXL2 is a forkhead transcription factor expressed in the eye, ovary, and pituitary gland. Loss of function mutations in humans and mice confirm a functional role for FOXL2 in the eye and ovary, but its role in the pituitary is not yet defined. We report that FOXL2 colocalizes with the glycoprotein hormone alpha-subunit (alphaGSU) in quiescent cells of the mouse pituitary from embryonic d 11.5 through adulthood. FOXL2 is expressed in essentially all gonadotropes and thyrotropes and a small fraction of prolactin-containing cells during pregnancy, but not somatotropes or corticotropes. The coincident expression patterns of FOXL2 and alphaGSU suggested that the alphaGSU gene (Cga) is a downstream target of FOXL2. We demonstrate that FOXL2 regulates mouse Cga transcription in gonadotrope-derived (alphaT3-1, LbetaT2), thyrotrope-derived (alphaTSH) and heterologous (CV-1) cells in a context-dependent manner. In addition, a FOXL2-VP16 fusion protein is sufficient to stimulate ectopic Cga expression in transgenic animals. Normal FOXL2 expression requires the transcription factors Lhx3 and Lhx4 but not of Prop1. Thus, FOXL2 expression is affected by mutations in early pituitary developmental regulatory genes, and its expression precedes that of genes necessary for gonadotrope-specific development such as Egr1 and Sf1 (Nr5a1). These data place FOXL2 in the hierarchy of pituitary developmental control and suggest a role in regulation of Cga gene expression.

Functional Role of Gonadotrope Plasticity and Network Organization.

Gonadotrope cells of the anterior pituitary are characterized by their ability to mount a cyclical pattern of gonadotropin secretion to regulate gonadal function and fertility. Recent in vitro and in vivo evidence suggests that gonadotropes exhibit dramatic remodeling of the actin cytoskeleton following gonadotropin-releasing hormone (GnRH) exposure. GnRH engagement of actin is critical for gonadotrope function on multiple levels. First, GnRH-induced cell movements lead to spatial repositioning of the in vivo gonadotrope network toward vascular endothelium, presumably to access the bloodstream for effective hormone release. Interestingly, these plasticity changes can be modified depending on the physiological status of the organism. Additionally, GnRH-induced actin assembly appears to be fundamental to gonadotrope signaling at the level of extracellular signal-regulated kinase (ERK) activation, which is a well-known regulator of luteinizing hormone (LH) ß-subunit synthesis. Last, GnRH-induced cell membrane projections are capable of concentrating LHß-containing vesicles and disruption of the actin cytoskeleton reduces LH secretion. Taken together, gonadotrope network positioning and LH synthesis and secretion are linked to GnRH engagement of the actin cytoskeleton. In this review, we will cover the dynamics and organization of the in vivo gonadotrope cell network and the mechanisms of GnRH-induced actin-remodeling events important in ERK activation and subsequently hormone secretion.

Foxo1 Is Required for Normal Somatotrope Differentiation.

The etiology for half of congenital hypopituitarism cases is unknown. Our long-term goal is to expand the molecular diagnoses for congenital hypopituitarism by identifying genes that contribute to this condition. We have previously shown that the forkhead box transcription factor, FOXO1, is present in approximately half of somatotropes at embryonic day (e) 18.5, suggesting it may have a role in somatotrope differentiation or function. To elucidate the role of FOXO1 in somatotrope differentiation and function, Foxo1 was conditionally deleted from the anterior pituitary (Foxo1Δpit). Uncommitted progenitor cells are maintained and able to commit to the somatotrope lineage normally based on the expression patterns of Sox2, a marker of uncommitted pituitary progenitors, and Pou1f1 (also known as Pit1), which marks committed progenitors. Interestingly, Foxo1Δpit embryonic mice exhibit delayed somatotrope differentiation as evidenced by an almost complete absence of GH immunoreactivity at e16.5 and reduced expression of Gh at e18.5 and postnatal day (P) 3. Consistent with this conclusion, expression of GHRH receptor, a marker of terminally differentiated somatotropes, is significantly reduced at e18.5 and P3 in the absence of FOXO1. The mechanism of FOXO1 regulation of somatotrope differentiation may involve the basic helix-loop-helix transcription factor, Neurod4, which has been implicated in somatotrope differentiation and is significantly reduced in Foxo1Δpit mice. Foxo1Δpit mice do not exhibit growth defects, and at P21 their pituitary glands exhibit a normal distribution of somatotropes. These studies demonstrate that FOXO1 is important for initial somatotrope specification embryonically but is dispensable for postnatal somatotrope expansion and growth.

Loss of Foxm1 Results in Reduced Somatotrope Cell Number during Mouse Embryogenesis.

FOXM1, a member of the forkhead box transcription factor family, plays a key role in cell cycling progression by regulating the expression of critical G1/S and G2/M phase transition genes. In vivo studies reveal that Foxm1 null mice have a 91% lethality rate at e18.5 due to significant cardiovascular and hepatic hypoplasia. Thus, FOXM1 has emerged as a key protein regulating mitotic division and cell proliferation necessary for embryogenesis. In the current study, we assess the requirement for Foxm1 in the developing pituitary gland. We find that Foxm1 is expressed in the pituitary at embryonic days 10.5-e18.5 and localizes with markers for active cell proliferation (BrdU). Interestingly, direct analysis of Foxm1 null mice at various embryonic ages, reveals no difference in gross pituitary morphology or cell proliferation. We do observe a downward trend in overall pituitary cell number and a small reduction in pituitary size in e18.5 embryos suggesting there may be subtle changes in pituitary proliferation not detected with our proliferation makers. Consistent with this, Foxm1 null mice have reductions in both the somatotrope and gonadotrope cell populations.

c-Jun N-terminal kinase activation of activator protein-1 underlies homologous regulation of the gonadotropin-releasing hormone receptor gene in alpha T3-1 cells.

Reproductive function is dependent on the interaction between GnRH and its cognate receptor found on gonadotrope cells of the anterior pituitary gland. GnRH activation of the GnRH receptor (GnRHR) is a potent stimulus for increased expression of multiple genes including the gene encoding the GnRHR itself. Thus, homologous regulation of the GnRHR is an important mechanism underlying gonadotrope sensitivity to GnRH. Previously, we have found that GnRH induction of GnRHR gene expression in alpha T3-1 cells is partially mediated by protein kinase C activation of a canonical activator protein-1 (AP-1) element. In contrast, protein kinase A and a cAMP response element-like element have been implicated in mediating the GnRH response of the GnRHR gene using a heterologous cell model (GGH(3)). Herein we find that selective removal of the canonical AP-1 site leads to a loss of GnRH regulation of the GnRHR promoter in transgenic mice. Thus, an intact AP-1 element is necessary for GnRH responsiveness of the GnRHR gene both in vitro and in vivo. Based on in vitro analyses, GnRH appeared to enhance the interaction of JunD, FosB, and c-Fos at the GnRHR AP-1 element. Although enhanced binding of cFos reflected an increase in gene expression, GnRH appeared to regulate both FosB and JunD at a posttranslational level. Neither overexpression of a constitutively active Raf-kinase nor pharmacological blockade of GnRH-induced ERK activation eliminated the GnRH response of the GnRHR promoter. GnRH responsiveness was, however, lost in alpha T3-1 cells that stably express a dominant-negative c-Jun N-terminal kinase (JNK) kinase, suggesting a critical role for JNK in mediating GnRH regulation of the GnRHR gene. Consistent with this possibility, we find that the ability of forskolin and membrane-permeable forms of cAMP to inhibit the GnRH response of the GnRHR promoter is associated with a loss of both JNK activation and GnRH-mediated recruitment of the primary AP-1-binding components.

The gonadotropin releasing hormone (GnRH) receptor activating sequence (GRAS) is a composite regulatory element that interacts with multiple classes of transcription factors including Smads, AP-1 and a forkhead DNA binding protein.

Activin responsiveness of the murine GnRH receptor gene promoter is mediated at a regulatory element we termed the GnRH receptor activating sequence (GRAS). Here, we have sought to define the complex of transcription factors that interact at this element. Consistent with activin regulation at GRAS, gel shift analyses and yeast one-hybrid assays reveal Smad4 interaction at the 5' end of GRAS. While overexpression of Smad3 activates a GRAS reporter, Smad3 binding at GRAS was not detectable. A functional interaction of Smad3 at GRAS was, however, detectable in yeast expressing Smad4. Thus, Smad3 interaction at GRAS appears to be dependent on the presence of Smad4. Mutations located at the 3' end of GRAS do not affect Smad binding but eliminate functional activity. Thus, Smad binding alone cannot account for the functional attributes of GRAS. Consistent with this notion, we find that AP-1 binding is immediately juxtaposed to and, in fact, partially overlaps the Smad binding site. Finally, a recently identified member of the forkhead family of transcription factors, FoxL2, is also capable of interacting at GRAS. Furthermore, FoxL2 activation at GRAS is lost with mutation of either the 5' Smad binding site or a putative forkhead binding site located at the 3' end of the element. We suggest that GRAS is a composite regulatory element whose functional activity is dependent on the organization of a multi-protein complex consisting of Smads, AP-1 and a member of the forkhead family of DNA binding proteins.

Impaired fertility and FSH synthesis in gonadotrope-specific Foxl2 knockout mice.

Impairments in pituitary FSH synthesis or action cause infertility. However, causes of FSH dysregulation are poorly described, in part because of our incomplete understanding of mechanisms controlling FSH synthesis. Previously, we discovered a critical role for forkhead protein L2 (FOXL2) in activin-stimulated FSH ß-subunit (Fshb) transcription in immortalized cells in vitro. Here, we tested the hypothesis that FOXL2 is required for FSH synthesis in vivo. Using a Cre/lox approach, we selectively ablated Foxl2 in murine anterior pituitary gonadotrope cells. Conditional knockout (cKO) mice developed overtly normally but were subfertile in adulthood. Testis size and spermatogenesis were significantly impaired in cKO males. cKO females exhibited reduced ovarian weight and ovulated fewer oocytes in natural estrous cycles compared with controls. In contrast, ovaries of juvenile cKO females showed normal responses to exogenous gonadotropin stimulation. Both male and female cKO mice were FSH deficient, secondary to diminished pituitary Fshb mRNA production. Basal and activin-stimulated Fshb expression was similarly impaired in Foxl2 depleted primary pituitary cultures. Collectively, these data definitively establish FOXL2 as the first identified gonadotrope-restricted transcription factor required for selective FSH synthesis in vivo.

Pituitary gland development and disease: from stem cell to hormone production.

Many aspects of pituitary development have become better understood in the past two decades. The signaling pathways regulating pituitary growth and shape have emerged, and the balancing interactions between the pathways are now appreciated. Markers for multipotent progenitor cells are being identified, and signature transcription factors have been discovered for most hormone-producing cell types. We now realize that pulsatile hormone secretion involves a 3D integration of cellular networks. About a dozen genes are known to cause pituitary hypoplasia when mutated due to their essential roles in pituitary development. Similarly, a few genes are known that predispose to familial endocrine neoplasia, and several genes mutated in sporadic pituitary adenomas are documented. In the next decade, we anticipate gleaning a deeper appreciation of these processes at the molecular level, insight into the development of the hypophyseal portal blood system, and evolution of better therapeutics for congenital and acquired hormone deficiencies and for common craniopharyngiomas and pituitary adenomas.