Follicle-stimulating Hormone Synthesis Research Articles

Effect of Tripterygium Glycosides Tablets on reproductive toxicity in female rats with Ⅱ type collagen induced arthritis

The aim of this paper was to observe the toxic effect of Tripterygium Glycosides Tablets( TG) on the reproductive system of Ⅱ type collagen induced arthritis( CIA) male rats,and to explore the toxic mechanism preliminarily. Fifty SD rats were randomly divided into normal control group( Con),model group( CIA),Tripterygium Glycosides Tablets clinical equivalent dose groups of 1,2,4 times( 9,18,36 mg·kg-1),10 rats in each group,and were given by gavage once a day for 42 days after the first immunization.The organ indexes of uterine and ovarian were calculated on days 21 and 42. Histopathological and morphological changes of uterine and ovarian were observed under optical microscope. The concentration of estradiol( E2),follicle-stimulating hormone( FSH),luteinizing hormone( LH),17α-hydroxylase( CYP17 A1) and cytochrome P450 19 A1( CYP19 A1) in serum were detected by ELISA. Immunohistochemistry was used to observe the expression of Bax and Bcl-2 related proteins in the apoptosis pathway of uterus and ovary. The results showed that compared with the Con group,CIA group could reduce the number of uterine glands( P<0.05),but no significant changes were observed in other groups. Compared with the CIA group,there were no significant changes in the coefficients of uterus and ovary in the Tripterygium Glycosides Tablets groups. The number of uterine glands,total follicles in the ovary,mature follicles and corpus luteum,the distribution of blood vessels and mitochondria had a certain inhibitory trend,and also slightly increased the number of atresia follicles,but the histopathological quantitative indicators were not statistically different. Except that 2 times clinical dose of Tripterygium Glycosides Tablets could significantly reduce the content of CYP19 A1( P<0. 05) after 42 d administration,there were no significant changes in serum estrogen E2,FSH,LH and estrogen synthesis key enzymes CYP17 A1 in each administration group. Medium and high doses of Tripterygium Glycosides Tablets could increase the expression of apoptotic protein Bax in uterine and ovarian tissues( P<0. 05,P<0. 01),and all the administration groups could inhibit the expression of apoptotic inhibiting protein Bcl-2( P <0. 05,P<0. 01,P<0.001),42 d was more obvious than 21 d. In conclusion,4 times and less than 4 times Tripterygium Glycosides Tablets did not cause obvious toxicity and histopathological changes in the reproductive organs of CIA rats,but it could reduce the level of serum estrogen synthesis key enzyme CYP19 A1 and affect the content of apoptosis-related proteins Bax and Bcl-2 in uterus and ovary tissues. The relevant mechanism needs further study.

Deciphering Direct and Indirect Effects of Neurokinin B and GnRH in the Brain-Pituitary Axis of Tilapia.

Neurokinin B (NKB) and its cognate receptor (NK3R) are emerging as important components of the neuroendocrine regulation of reproduction. Unlike mammalian tac3, which encodes only one mature peptide (namely NKB), two mature peptides are predicted for each tac3 gene in fish and frogs. Therefore, it was designated as Neurokinin F (NKF). Hormone analogs with high and long-lasting biological activity are important tools for physiological and biological research; however, the availability of piscine-specific analogs is very limited. Therefore, we have developed specific NKB and NKF analogs based on the structure of the mammalian NKB analog–senktide. These analogs, specifically designed for longer half-lives by methylation of proteolysis sites, exhibited activity equal to those of the native NKB and NKF in short-term signal-transduction assays of tilapia NKB receptors. However, the analogs were found to be able to significantly increase the release of luteinizing hormone (LH), follicle stimulating hormone (FSH) and growth hormone (GH) in tilapia, as fast as 1 h after intraperitoneal (IP) injection. The impact of the analogs on LH and FSH secretion lasted longer compared to the effect of native peptides and salmon GnRH analog (sGnRHa). In addition, we harvested pituitaries 24 h post injection and measured LH, FSH and GH mRNA synthesis. Both analogs elevated mRNA levels of LH and GH, but only NKB analog increased FSH mRNA levels in the pituitary and all GnRH forms in the brain. NKB receptors were co-localized with all three types the GnRH neurons in tilapia brain in situ. We previously showed a direct effect of NKB at the pituitary level, and these new results suggest that the stronger impact of the NKB analog on GTH release is also due to an indirect effect through the activation of GnRH neurons. These results suggest that novel synthetic NKB analogs may serve as a tool for both research and agricultural purposes. Finally, the biological activity and regulatory role of NKB in tilapia brain and pituitary suggest that the NKB/NKBR system in fish is an important reproductive regulator in a similar way to the kisspeptin system in mammals.

Pituitary tissue-specific miR-7a-5p regulates FSH expression in rat anterior adenohypophyseal cells.

The follicle-stimulating hormone (FSH), which is synthesized and secreted by the anterior pituitary gland, plays an important role in regulating reproductive processes. In this study, using the TargetScan program, we predicted that microRNAs (miRNAs) regulate FSH secretion. Dual-luciferase reporter assays were performed and identified miR-7a-5p. MiR-7a-5p has been reported to regulate diverse cellular functions. However, it is unclear whether miR-7a-5p binds to mRNAs and regulates reproductive functions. Therefore, we constructed a suspension of rat anterior pituitary cells and cultured them under adaptive conditions, transfected miR-7a-5p mimics or inhibitor into the cell suspension and detected expression of the FSHb gene. The results demonstrated that miR-7a-5p downregulated FSHb expression levels, while treatment with miR-7a-5p inhibitors upregulated FSHb expression levels relative to those of negative control groups, as shown by quantitative PCR analysis. The results were confirmed with a subsequent experiment showing that FSH secretion was reduced after treatment with mimics and increased in the inhibitor groups, as shown by enzyme-linked immunosorbent assay. Our results indicated that miR-7a-5p downregulates FSHb expression levels, resulting in decreased FSH synthesis and secretion, which demonstrates the important role of miRNAs in the regulation of FSH and animal reproduction.

Gain-of-Function Genetic Models to Study FSH Action.

Follicle–stimulating hormone (FSH) is a pituitary-derived gonadotropin that plays key roles in male and female reproduction. The physiology and biochemistry of FSH have been extensively studied for many years. Beginning in the early 1990s, coincident with advances in the then emerging transgenic animal technology, and continuing till today, several gain-of-function (GOF) models have been developed to understand FSH homeostasis in a physiological context. Our group and others have generated a number of FSH ligand and receptor GOF mouse models. An FSH GOF model when combined with Fshb null mice provides a powerful genetic rescue platform. In this chapter, we discuss different GOF models for FSH synthesis, secretion and action and describe additional novel genetic models that could be developed in the future to further refine the existing models.

HDAC inhibitors impair Fshb subunit expression in murine gonadotrope cells.

Fertility is dependent on follicle-stimulating hormone (FSH), a product of gonadotrope cells of the anterior pituitary gland. Hypothalamic gonadotropin-releasing hormone (GnRH) and intra-pituitary activins are regarded as the primary drivers of FSH synthesis and secretion. Both stimulate expression of the FSH beta subunit gene (Fshb), although the underlying mechanisms of GnRH action are poorly described relative to those of the activins. There is currently no consensus on how GnRH regulates Fshb transcription, as results vary across species and between in vivo and in vitro approaches. One of the more fully developed models suggests that the murine Fshb promoter is tonically repressed by histone deacetylases (HDACs) and that GnRH relieves this repression, at least in immortalized murine gonadotrope-like cells (LβT2 and αT3-1). In contrast, we observed that the class I/II HDAC inhibitor trichostatin A (TSA) robustly inhibited basal, activin A-, and GnRH-induced Fshb mRNA expression in LβT2 cells and in primary murine pituitary cultures. Similar results were obtained with the class I specific HDAC inhibitor, entinostat, whereas two class II-specific inhibitors, MC1568 and TMP269, had no effects on Fshb expression. Collectively, these data suggest that class I HDACs are positive, not negative, regulators of Fshb expression in vitro and that, contrary to earlier reports, GnRH may not stimulate Fshb by inhibiting HDAC-mediated repression of the gene.

TGF-β Superfamily Regulation of Follicle-Stimulating Hormone Synthesis by Gonadotrope Cells: Is There a Role for Bone Morphogenetic Proteins?

Bone morphogenetic proteins (BMPs) are pleiotropic ligands in the TGF-β superfamily. In the early to mid-2000s, several BMPs, including BMP2, were shown to regulate FSH synthesis alone and in synergy with activins in immortalized gonadotrope-like cell lines and primary pituitary cultures. Activins are also TGF-β family members, which were identified and named based on their abilities to stimulate FSH production selectively. Mechanistic analyses suggested that BMP2 promoted expression of the FSHβ subunit gene (Fshb) via at least two nonmutually exclusive mechanisms. First, BMP2 stimulated the production of the inhibitor of DNA-binding proteins 1, 2, and 3 (Id1, Id2, and Id3), which potentiated the stimulatory actions of homolog of Drosophila mothers against decapentaplegic 3 (SMAD3) on the Fshb promoter. SMAD3 is an intracellular signaling protein that canonically mediates the actions of activins and is an essential regulator of Fshb production in vitro and in vivo. Second, BMP2 was shown to activate SMAD3-dependent signaling via its canonical type IA receptor, BMPR1A (also known as ALK3). This was a surprising result, as ALK3 conventionally activates distinct SMAD proteins. Although these initial results were compelling, they were challenged by contemporaneous and subsequent observations. For example, inhibitors of BMP signaling did not specifically impair FSH production in cultured pituitary cells. Of perhaps greater significance, mice lacking ALK3 in gonadotrope cells produced FSH normally. Therefore, the physiological role of BMPs in FSH synthesis in vivo is presently uncertain.

Increased luminescence of the GloSensor cAMP assay in LβT2 cells does not correlate with cAMP accumulation under low pH conditions

Cyclic adenosine monophosphate (cAMP) plays a pivotal role in gonadotrope responses in the pituitary. Gonadotropin-releasing hormone (GnRH) mediated synthesis and secretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are regulated by both the Gs/cAMP and Gq/Ca2+ signaling pathways. Pituitary adenylate cyclase-activating polypeptide (PACAP) also regulates GnRH responsiveness in gonadotropes through the PACAP receptor, which activates the Gs/cAMP signaling pathway. Therefore, measuring intracellular cAMP levels is important for elucidating the molecular mechanisms of FSH and LH synthesis and secretion in gonadotropes. The GloSensor cAMP assay is useful for detecting cAMP levels in intact, living cells. In this study, we found that increased GloSensor luminescence intensity did not correlate with cAMP accumulation in LβT2 cells under low pH conditions. This result indicates that cell type and condition must be considered when using GloSensor cAMP.

GnRH Transactivates Human AMH Receptor Gene via Egr1 and FOXO1 in Gonadotrope Cells

Background/Objectives: Anti-Müllerian hormone (AMH) signaling is critical for sexual differentiation and gonadal function. AMH receptor type 2 (AMHR2) is expressed in extragonadal sites such as brain, and pituitary and emerging evidence indicates that AMH biological action is much broader than initially thought. We recently reported that AMH signaling enhances follicle-stimulating hormone synthesis in pituitary gonadotrope cells. However, mechanisms regulating AMHR2 expression in these extragonadal sites remain to be explored. Method/Results: Here, we demonstrated in perifused murine LβT2 gonadotrope cells that Amhr2 expression is differentially regulated by GnRH pulse frequency with an induction under high GnRH pulsatility. Furthermore, we showed that GnRH transactivates the human AMHR2 promoter in LβT2 cells. Successive deletions of the promoter revealed the importance of a short proximal region (–53/–37 bp) containing an Egr1 binding site. Using site-directed mutagenesis of Egr1 motif and siRNA mediated-knockdown of Egr1, we demonstrated that Egr1 mediates basal and GnRH-dependent activity of the promoter, identifying Egr1 as a new transcription factor controlling hAMHR2 expression. We also showed that SF1 and β-catenin are required for basal promoter activity and demonstrated that both factors contribute to the GnRH stimulatory effect, independently of their respective binding sites. Furthermore, using a constitutively active mutant of FOXO1, we identified FOXO1 as a negative regulator of basal and GnRH-dependent AMHR2 expression in gonadotrope cells. Conclusions: This study identifies GnRH as a regulator of human AMHR2 expression, further highlighting the importance of AMH signaling in the regulation of gonadotrope function.

Molecular regulation of follicle-stimulating hormone synthesis, secretion and action.

Follicle-stimulating hormone (FSH) plays fundamental roles in male and female fertility. FSH is a heterodimeric glycoprotein expressed by gonadotrophs in the anterior pituitary. The hormone-specific FSHβ-subunit is non-covalently associated with the common α-subunit that is also present in the luteinizing hormone (LH), another gonadotrophic hormone secreted by gonadotrophs and thyroid-stimulating hormone (TSH) secreted by thyrotrophs. Several decades of research led to the purification, structural characterization and physiological regulation of FSH in a variety of species including humans. With the advent of molecular tools, availability of immortalized gonadotroph cell lines and genetically modified mouse models, our knowledge on molecular mechanisms of FSH regulation has tremendously expanded. Several key players that regulate FSH synthesis, sorting, secretion and action in gonads and extragonadal tissues have been identified in a physiological setting. Novel post-transcriptional and post-translational regulatory mechanisms have also been identified that provide additional layers of regulation mediating FSH homeostasis. Recombinant human FSH analogs hold promise for a variety of clinical applications, whereas blocking antibodies against FSH may prove efficacious for preventing age-dependent bone loss and adiposity. It is anticipated that several exciting new discoveries uncovering all aspects of FSH biology will soon be forthcoming.

Histological and Morphometric Examination of the Testes of Boars and Male Pigs Immunocastrated with Improvac®

Background: Surgical castration of male piglets is frequently performed to prevent development of an unpleasant taste and flavor in meat of mature males, called boar taint. Disadvantages of this method are primarily related to the lower feed efficiency of surgically castrated male pigs, production of less lean meat and more fat on the carcasses than boars, as well as ethical issues associated with the procedure itself. Immunocastration is a method of immunization against gonadotropinreleasing hormone (GnRH) and represents an alternative to avoid boar taint. This study aimed to examine the influence of immunocastration on histological and morphometric parameters of male pig testes.Materials, Methods &amp; Results: A total of 60 crossbred male pigs selected from 30 litters were assigned to two experimental groups: boars (n = 30) and immunocastrated males (n = 30). Two doses (2 mL per dose) of the Improvac® vaccine were given to pigs scheduled for immunocastration. The first injection was given at 10 weeks of age, and the second injection at 20 weeks of age. All pigs were fed the same commercial diet ad libitum, group-housed (15 pigs/pen) and slaughtered at 25 weeks of age. After slaughter, testicle weights were measured and samples of testicular tissue were taken for histological examination. Immunocastrated males had lower testicle weights than boars (P &lt; 0.01). Furthermore, structural damage to the germinal epithelium was visible in the seminiferous tubules of vaccinated pigs. In the testicular interstitium of these animals, the number of Leydig cells were reduced; almost all of these cells exhibited different degrees of abnormality. Moreover, the diameter of the seminiferous tubules and thickness of the germinal epithelium were greater (P &lt; 0.01), and the percentage of germinal epithelium in the seminiferous tubules as well as percentage of blood vessels in the interstitium were higher in boars as compared to immunocastrated males (P &lt; 0.01). Seminiferous tubule lumen diameter was greater (P &lt; 0.01), and percentage of lumen in the seminiferous tubules as well as percentage of non-blood vessel tissue in the interstitium were higher in immunocastrated males than boars (P &lt; 0.01).Discussion: Vaccination against GnRH, as used in this study, reduced the synthesis of follicle-stimulating hormone (FSH) and luteinizing hormone (LH), which led to decreased spermatogenesis and androgen production. The smaller diameter of the seminiferous tubules as well as germinal epithelium thickness, and lower percentage of germinal epithelium in the seminiferous tubules in immunocastrated males could be explained by reduced FSH synthesis, and subsequent reduced spermatogenesis. The described testicular malformations in immunocastrated animals caused their lower weight, and greater diameter of the lumen as well as a higher lumen percentage of the seminiferous tubules. The vaccine reduced LH synthesis, so the Leydig cells of immunocastrated animals had lower activity than the same cells in boars. The percentage of blood vessels in the interstitium of boar testes was higher, which was related to the blood supply required for normal function of Leydig cells. It was concluded that differences existed in the structure of the seminiferous tubules and interstitium between boars and immunocastrated males , which were reflected in values of the examined histological and morphometric parameters. The development of these differences was related to the inhibition of GnRH synthesis and secretion.

Triiodothyronine differentially modulates the LH and FSH synthesis and secretion in male rats.

Hypothyroidism and thyrotoxicosis produce adverse effects in male reproduction by unknown mechanisms. We investigated whether triiodothyronine (T3) modulates luteinizing hormone (LH) and follicle stimulating hormone (FSH) synthesis/secretion, by inducing different thyroid states. In hypothyroidism, the content of Lhb and Fshb mRNAs was increased, while their association to ribosomes and the protein content were reduced and the serum LH and FSH concentrations were augmented and decreased, respectively. Thyrotoxicosis reduced Lhb mRNA and LH serum concentration, and increased Lhb mRNA translational rate. The Fshb mRNA content and its association to ribosomes were also increased, whereas FSH serum concentrations were comparable to euthyroid levels. Acute T3 treatment decreased the total content of Lhb and Fshb mRNAs, and increased their association to ribosomes, as well as the LHB and FSHB contents in secretory granules. This study shows that T3 acts on gonadotrophs, resulting in direct effects on LH and FSH synthesis/secretion of male rats, suggesting that some reproductive disorders observed in men may be associated with thyroid hormone imbalances.

Zearalenone and alpha-zearalenol inhibit the synthesis and secretion of pig follicle stimulating hormone via the non-classical estrogen membrane receptor GPR30

Zearalenone (ZEA) is one of the most popular endocrine-disrupting chemicals and is mainly produced by fungi of the genus Fusarium. The excessive intake of ZEA severely disrupts human and animal fertility by affecting the reproductive axis. However, most studies on the effects of ZEA and its metabolite α-zearalenol (α-ZOL) on reproductive systems have focused on gonads. Few studies have investigated the endocrine-disrupting effects of ZEA and α-ZOL on pituitary gonadotropins, including follicle-stimulating hormone (FSH) and luteinizing hormone (LH). The present study was designed to investigate the effects of ZEA and α-ZOL on the synthesis and secretion of FSH and LH and related mechanisms in female pig pituitary. Our in vivo and in vitro results demonstrated that ZEA significantly inhibited the synthesis and secretion of FSH in the pig pituitary gland, but ZEA and α-ZOL had no effects on LH. Our study also showed that ZEA and α-ZOL decreased FSH synthesis and secretion through non-classical estrogen membrane receptor GPR30, which subsequently induced protein kinase cascades and the phosphorylation of PKC, ERK and p38MAPK signaling pathways in pig pituitary cells. Furthermore, our study showed that the LIM homeodomain transcription factor LHX3 was involved in the mechanisms of ZEA and α-ZOL actions on gonadotropes in the female pig pituitary. These findings elucidate the mechanisms behind the physiological alterations resulting from endocrine-disrupting chemicals and further show that the proposed key molecules of the α-ZOL signaling pathway could be potential pharmacological targets.

Loss of microRNA-7a2 induces hypogonadotropic hypogonadism and infertility.

MicroRNAs (miRNAs) are negative modulators of gene expression that fine-tune numerous biological processes. miRNA loss-of-function rarely results in highly penetrant phenotypes, but rather, influences cellular responses to physiologic and pathophysiologic stresses. Here, we have reported that a single member of the evolutionarily conserved miR-7 family, miR-7a2, is essential for normal pituitary development and hypothalamic-pituitary-gonadal (HPG) function in adulthood. Genetic deletion of mir-7a2 causes infertility, with low levels of gonadotropic and sex steroid hormones, small testes or ovaries, impaired spermatogenesis, and lack of ovulation in male and female mice, respectively. We found that miR-7a2 is highly expressed in the pituitary, where it suppresses golgi glycoprotein 1 (GLG1) expression and downstream bone morphogenetic protein 4 (BMP4) signaling and also reduces expression of the prostaglandin F2a receptor negative regulator (PTGFRN), an inhibitor of prostaglandin signaling and follicle-stimulating hormone (FSH) and luteinizing hormone (LH) secretion. Our results reveal that miR-7a2 critically regulates sexual maturation and reproductive function by interconnecting miR-7 genomic circuits that regulate FSH and LH synthesis and secretion through their effects on pituitary prostaglandin and BMP4 signaling.

SMAD3 Regulates Follicle-stimulating Hormone Synthesis by Pituitary Gonadotrope Cells in Vivo

Pituitary follicle-stimulating hormone (FSH) is an essential regulator of fertility in females and of quantitatively normal spermatogenesis in males. Pituitary-derived activins are thought to act as major stimulators of FSH synthesis by gonadotrope cells. In vitro, activins signal via SMAD3, SMAD4, and forkhead box L2 (FOXL2) to regulate transcription of the FSHβ subunit gene (Fshb). Consistent with this model, gonadotrope-specific Smad4 or Foxl2 knock-out mice have greatly reduced FSH and are subfertile. The role of SMAD3 in vivo is unresolved; however, residual FSH production in Smad4 conditional knock-out mice may derive from partial compensation by SMAD3 and its ability to bind DNA in the absence of SMAD4. To test this hypothesis and determine the role of SMAD3 in FSH biosynthesis, we generated mice lacking both the SMAD3 DNA binding domain and SMAD4 specifically in gonadotropes. Conditional knock-out females were hypogonadal, acyclic, and sterile and had thread-like uteri; their ovaries lacked antral follicles and corpora lutea. Knock-out males were fertile but had reduced testis weights and epididymal sperm counts. These phenotypes were consistent with those of Fshb knock-out mice. Indeed, pituitary Fshb mRNA levels were nearly undetectable in both male and female knock-outs. In contrast, gonadotropin-releasing hormone receptor mRNA levels were significantly elevated in knock-outs in both sexes. Interestingly, luteinizing hormone production was altered in a sex-specific fashion. Overall, our analyses demonstrate that SMAD3 is required for FSH synthesis in vivo.

Normal gonadotropin production and fertility in gonadotrope-specific Bmpr1a knockout mice.

Pituitary follicle-stimulating hormone (FSH) synthesis is regulated by transforming growth factorβsuperfamily ligands, most notably the activins and inhibins. Bone morphogenetic proteins (BMPs) also regulate FSHβ subunit (Fshb) expression in immortalized murine gonadotrope-like LβT2 cells and in primary murine or ovine primary pituitary cultures. BMP2 signals preferentially via the BMP type I receptor, BMPR1A, to stimulate murine Fshb transcription in vitro Here, we used a Cre-lox approach to assess BMPR1A's role in FSH synthesis in mice in vivo Gonadotrope-specific Bmpr1a knockout animals developed normally and had reproductive organ weights comparable with those of controls. Knockouts were fertile, with normal serum gonadotropins and pituitary gonadotropin subunit mRNA expression. Cre-mediated recombination of the floxed Bmpr1a allele was efficient and specific, as indicated by PCR analysis of diverse tissues and isolated gonadotrope cells. Furthermore, BMP2 stimulation of inhibitor of DNA binding 3 expression was impaired in gonadotropes isolated from Bmpr1a knockout mice, confirming the loss of functional receptor protein in these cells. Treatment of purified gonadotropes with small-molecule inhibitors of BMPR1A (and the related receptors BMPR1B and ACVR1) suppressed Fshb mRNA expression, suggesting that an autocrine BMP-like molecule might regulate FSH synthesis. However, deletion of Bmpr1a and Acvr1 in cultured pituitary cells did not alter Fshb expression, indicating that the inhibitors had off-target effects. In sum, BMPs or related ligands acting via BMPR1A or ACVR1 are unlikely to play direct physiological roles in FSH synthesis by murine gonadotrope cells.

Follicle-stimulating hormone synthesis and fertility depend on SMAD4 and FOXL2.

Follicle-stimulating hormone (FSH) is an essential regulator of gonadal function and fertility. Loss-of-function mutations in the FSHB/Fshb gene cause hypogonadotropic hypogonadism in humans and mice. Both gonadotropin-releasing hormone (GnRH) and activins, members of the transforming growth factor β (TGFβ) superfamily, stimulate FSH synthesis; yet, their relative roles and mechanisms of action in vivo are unknown. Here, using conditional gene-targeting, we show that the canonical mediator of TGFβ superfamily signaling, SMAD4, is absolutely required for normal FSH synthesis in both male and female mice. Moreover, when the Smad4 gene is ablated in combination with its DNA binding cofactor Foxl2 in gonadotrope cells, mice make essentially no FSH and females are sterile. Indeed, the phenotype of these animals is remarkably similar to that of Fshb-knockout mice. Not only do these results establish SMAD4 and FOXL2 as essential master regulators of Fshb transcription in vivo, they also suggest that activins, or related ligands, could play more important roles in FSH synthesis than GnRH.

Follicle‐stimulating hormone synthesis and fertility are intact in mice lacking SMAD3 DNA binding activity and SMAD2 in gonadotrope cells

The activin/inhibin system regulates follicle-stimulating hormone (FSH) synthesis and release by pituitary gonadotrope cells in mammals. In vitro cell line data suggest that activins stimulate FSH β-subunit (Fshb) transcription via complexes containing the receptor-regulated SMAD proteins SMAD2 and SMAD3. Here, we used a Cre-loxP approach to determine the necessity for SMAD2 and/or SMAD3 in FSH synthesis in vivo. Surprisingly, mice with conditional mutations in both Smad2 and Smad3 specifically in gonadotrope cells are fertile and produce FSH at quantitatively normal levels. Notably, however, we discovered that the recombined Smad3 allele produces a transcript that encodes the entirety of the SMAD3 C-terminal Mad homology 2 (MH2) domain. This protein behaves similarly to full-length SMAD3 in Fshb transcriptional assays. As the truncated protein lacks the N-terminal Mad homology 1 (MH1) domain, these results show that SMAD3 DNA-binding activity as well as SMAD2 are dispensable for normal FSH synthesis in vivo. Furthermore, the observation that deletion of proximal exons does not remove all SMAD3 function may facilitate interpretation of divergent phenotypes previously described in different Smad3 knockout mouse lines.

A mathematical model of pulse-coded hormone signal responses in pituitary gonadotroph cells

Cells in the pituitary that synthesize luteinizing and follicle-stimulating hormones regulate the relative production of these two key reproductive hormones in response to signals from the hypothalamus. These signals are encoded in the frequency of gonadotrophin-releasing-hormone pulses. In vitro experiments with a murine-derived cell line have identified key elements of the processes that decode the signal to regulate transcription of the subunits encoding these hormones. The mathematical model described in this paper is based on the results of those experiments and advances quantitative understanding of the biochemical decoder. The model consists of non-linear differential equations for each of six processes that lead to the synthesis of follicle-stimulating hormone. Simulations of the model exhibit key characteristics found in the experiments, including a preference for follicle-stimulating hormone synthesis at low pulse frequencies and a loss of this characteristic when a mutation is introduced.

The CpG Island in the Murine Foxl2 Proximal Promoter Is Differentially Methylated in Primary and Immortalized Cells

Forkhead box L2 (Foxl2), a member of the forkhead transcription factor family, plays important roles in pituitary follicle-stimulating hormone synthesis and in ovarian maintenance and function. Mutations in the human FOXL2 gene cause eyelid malformations and premature ovarian failure. FOXL2/Foxl2 is expressed in pituitary gonadotrope and thyrotrope cells, the perioptic mesenchyme of the developing eyelid, and ovarian granulosa cells. The mechanisms governing this cell-restricted expression have not been described. We mapped the Foxl2 transcriptional start site in immortalized murine gonadotrope-like cells, LβT2, by 5’ rapid amplification of cDNA ends and then PCR amplified approximately 1 kb of 5’ flanking sequence from murine genomic DNA. When ligated into a reporter plasmid, the proximal promoter conferred luciferase activity in both homologous (LβT2) and, unexpectedly, heterologous (NIH3T3) cells. In silico analyses identified a CpG island in the proximal promoter and 5’ untranslated region, suggesting that Foxl2 transcription might be regulated epigenetically. Indeed, pyrosequencing and quantitative analysis of DNA methylation using real-time PCR revealed Foxl2 proximal promoter hypomethylation in homologous compared to some, though not all, heterologous cell lines. The promoter was also hypomethylated in purified murine gonadotropes. In vitro promoter methylation completely silenced reporter activity in heterologous and homologous cells. Collectively, the data suggest that differential proximal promoter DNA methylation may contribute to cell-specific Foxl2 expression in some cellular contexts. However, gonadotrope-specific expression of the gene cannot be explained by promoter hypomethylation alone.

Cycloheximide inhibits follicle-stimulating hormone β subunit transcription by blocking de novo synthesis of the labile activin type II receptor in gonadotrope cells

The pituitary gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), play essential roles in the regulation of vertebrate reproduction. Activins and inhibins have opposing actions on FSH (but not LH) synthesis, either inducing or inhibiting transcription of the FSHβ subunit gene (Fshb). The translational inhibitor cycloheximide (CHX) produces inhibin-like effects in cultured pituitary cells, selectively suppressing FSH production. Using the murine gonadotrope-like cell line, LβT2, as a model, we tested the hypothesis that a component of the activin pathway is highly labile in gonadotrope cells and that its rapid loss following CHX treatment impairs activin-stimulated Fshb transcription. Treatment of cells with CHX for 6h, but not 1h, blocked activin A-stimulated Fshb transcription. Pre-treatment of LβT2 cells with CHX for as few as 2–3h inhibited activin A-stimulated SMAD2/3 phosphorylation without altering total SMAD2/3 protein levels. These data indicated that CHX affects activin signalling upstream of SMAD proteins, most likely at the receptor level. Indeed, CHX rapidly reduced activin A binding to LβT2 cells. We went on to show that activin A signals via the type II receptor ACVR2, rather than ACVR2B, to regulate Fshb transcription and that the receptor has a half life of ~2h in LβT2 cells. The mechanism of ACVR2 turnover remains undefined, but appears to be ligand-, proteasome-, and lysosome-independent. Collectively, these data indicate that CHX produces inhibin-like effects in gonadotropes by preventing de novo synthesis of the highly labile ACVR2, thereby blocking activin signaling to the Fshb promoter.